Universidade Federal do Rio de Janeiro BÁSLAVI MARISBEL ...
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Universidade Federal do Rio de Janeiro
BÁSLAVI MARISBEL CÓNDOR LUJÁN
BIODIVERSITY AND CONNECTIVITY OF CALCAREOUS SPONGES
(PORIFERA: CALCAREA) IN THE WESTERN TROPICAL ATLANTIC
Rio de Janeiro
2017
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Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western
Tropical Atlantic
Báslavi Marisbel Cóndor Luján
Tese apresentada ao Programa de Pós-Graduação em
Biodiversidade e Biologia Evolutiva, Instituto de
Biologia, Universidade Federal do Rio de Janeiro,
como parte dos requisitos necessários à obtenção do
título de Doutor em Ciências Biológicas.
Orientadora: Dra. Michelle Klautau
Rio de Janeiro
Fevereiro/2017
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Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western
Tropical Atlantic
Báslavi Marisbel Cóndor Luján
Orientadora: Dra. Michelle Klautau
Banca examinadora:
Prof. Dra. Carla Zilberberg, IB/UFRJ
______________________________________________
Prof. Dr. Cristiano Valentim da Silva Lazoski, IB/UFRJ
_______________________________________________
Prof. Dr. Eduardo Carlos Meduna Hajdu, MN/UFRJ
_______________________________________________
Prof. Dr. Guilherme Ramos da Silva Muricy, MN/UFRJ
_______________________________________________
Prof. Dr. Marcelo Weksler, MN/UFRJ
_______________________________________________
Dr. Fernando Moraes, JBRJ (Suplente)
_______________________________________________
Prof. Dr. Paulo Cesar de Paiva, IB/UFRJ (Suplente)
_______________________________________________
Rio de Janeiro
Fevereiro/2017
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Trabalho realizado no Laboratório de Biologia de Porifera (LaBiPor)
Departamento de Zoologia, Instituto de Biologia
Universidade Federal do Rio de Janeiro – UFRJ
Orientadora: Profa. Dra. Michelle Klautau
Capa: Centro: Rede filogeográfica de Leucetta floridana (modificada). Sentido horário:
Leucetta floridana, Leucilla uter, Leucascus luteoatlanticus sp. nov., Ascandra torquata sp.
nov., Sycon conulosum sp. nov., Nicola tetela, triactina âncora de Amphoriscus hirstutus sp.
nov., triactina atrial de Grantessa tumida sp. nov., esqueleto de Leucandrilla pseudosagittata sp.
nov., esqueleto de Ernstia citrea. Créditos: A. Padua, E. Hajdu, F. Azevedo e T. Pérez.
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Ficha catalográfica
CÓNDOR LUJÁN, Báslavi Marisbel
Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western
Tropical Atlantic / Báslavi Marisbel Cóndor Luján. Rio de Janeiro: UFRJ, IB, 2017.
xiii, 259f. il; 29.7 (cm)
Orientador: Michelle Klautau.
Tese (Doutorado). UFRJ/IB/Programa de Pós-graduação em Biodiversidade e Biologia
Evolutiva, 2017.
Referências bibliográficas: 236-256f.
1. Esponjas Calcareas. 2. Mar do Caribe. 3. Costa do Brasil. 4. Conectividade genética. I.
Klautau, Michelle. II. Universidade Federal do Rio de Janeiro, Instituto de Biologia, Programa
de Pós-graduação em Biodiversidade e Biologia Evolutiva. III. Tese.
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Dedico esta tesis a mis queridos padres
quienes, estando lejos, están siempre a mi lado.
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Huk llactapi maypi llapan maskhay runakuna mamaqocha hatun yachaypi, Llapan maskhay
runakuna ruanku anchoveta challway, ñoqa huk punchay ruwayramuni qochayuyo sasa
llachayta, soqta huata llanqay tukukuy manan llakikamunichu (In Quechua by E. Pezo Zegarra)
Translation: In a country where almost all marine science is focused on the fishery of
Engraulins ringens (Peruvian anchoveta), I took the risky decision to study sponges. After six
carioca years, I do not regret.
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AGRADECIMENTOS
Esta tesis es el resultado de un maravilloso viaje a través de las primeras descripciones de las
esponjas del Mar del Caribe y del Brasil, colectas en lugares en los que nunca imaginé bucear,
interesantes diálogos con especialistas en Porifera y muchas horas dedicadas al microscopio, a
la amplificación de secuencias de ADN y a las simulaciones bayesianas. Todo esto sólo fue
posible gracias a la colaboración, apoyo e incentivo de muchísimas personas e instituciones.
Muchas gracias a todos y en especial a:
A minha orientadora Michelle Klautau por ter aceito a minha proposta de doutorado e ter me
mostrado o caminho para ser uma boa pesquisadora.
Aos membros do projeto MARRIO, em especial ao Eduardo Hajdu e ao Thierry Pérez por
terem organisado as maravilhosas expedições que geraram os resultados apresentados nesta tese.
Aos membros do LaBiPor, Andrézinho, Bernardo, Bárbara, Gabi, Fernanda, Mattheus,
Tayara, Taynara, Pedro e Raissa pela boa disposição para trabalhar em équipe e me auxiliar com
os pedidos de último momento pelo whatsapp (rsrs). Às meninas que alguma vez fizeram parte
do LabiPor, a Carol e Malena pelo trabalho de bancada e a Natalia e Vivi pela agradável
companhia.
Aos pesquisadores (alemḿ do LaBiPor) que dedicaram um tempo durante os mergulhos para
procurar e coletar Calcarea: A. Bispo, A. Ereskovsky, B. Thacker, C. Leal, C. Ruiz, C. Castello-
Branco, C. Diaz, G. Lôbo-Hajdu, H. Fortunato, J. García-Hernández, J. Vacelet, J. Carraro, L.
Van Bostal, M. Carvalho, O. Thomas, P. Chevaldonné, S. Chenesseau, S. Zea and S. Salani.
Aos pesquisadores espongólogos brasileiros e estrangeiros que conheci ao longo destes anos
(Simpósio de Porifera na Bahia, “La Martinique” Sponge Course, Pacotilles Expedition,
MARRIO Workshops). Todos eles, certamente, contribuiram na minha formação como
pesquisadora.
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Aos profesores do Museu Nacional e do Instituto de Biologia. Um especial “gracias” à
Ghennie Rodriguez pela orientação nas ańalises demográficas da Leucetta floridana.
Às agencias de fomento CAPES, CNPQ, CNRS, COFECUB, FAPERJ e Grupo Boticário de
Proteção à Natureza pelo financiamento das expedicçõs de coleta e os projetos do laboratório.
Ao programa PEC-PG e à CAPES pela concessão da bolsa de doutorado.
A mi mamá, papá y hermano por el apoyo incondicional y la confianza depositada en mi.
A Helmunt quien no sólo ha sabido darme su amor a lo largo de estos años, si no, también
me ha ayudado en momentos computacionales muy críticos.
Muchas Gracias! Muito obrigada!
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ABSTRACT
BIODIVERSITY AND CONNECTIVITY OF CALCAREOUS SPONGES (PORIFERA:
CALCAREA) IN THE WESTERN TROPICAL ATLANTIC
Báslavi Marisbel Cóndor Luján
Supervisor: Dr. Michelle Klautau
Abstract of the thesis submitted to the Graduate Program Biodiversity and Evolutionary
Biology, Institute of Biology, Federal University of Rio de Janeiro – UFRJ, as part of the
requirements to obtain the title of Doctor in Biological Sciences.
The Western Tropical Atlantic (WTA) is a wide region that comprises the Tropical
Northwestern Atlantic (TNA), the North Brazil Shelf (NBS) and the Tropical Southwestern
Atlantic (TSA) provinces. Although it harbours a high diversity of marine species, sponges of
the class Calcarea have been poorly studied. This lack of knowledge hinders our understanding
of the biogeographical affinities of the calcareous sponges in the WTA and the role of the
Amazon River as an effective barrier to dispersal of these sponges. In the present study, the
diversity of the class Calcarea in the WTA was investigated using morphological and molecular
approaches and the population connectivity of a calcareous sponge, Leucetta floridana, was
assessed. With this contribution, the number of calcareous sponges from the WTA raised from
67 to 86, including the descriptions of 15 new species and four new records. Among them, 21
species are endemic to the TNA, three to the NBS and 22 to the TSA. The 14 species shared
between the TNA and TSA support a Caribbean-Brazilian affinity for calcareous sponges.
Genetic analyses evidenced five structured populations in L. floridana: one widespread
population maintaining gene flow between the TNA and TSA provinces and four other isolated
populations within the Caribbean Sea. The panmitic population suggested that the outflow of the
Amazon River in the Atlantic is not an effective barrier to the maintenance of gene flow among
trans-Amazonian populations of L. floridana.
Keywords: Caribbean Sea, NE Brazilian coast, phylogeography, demography, Amazon River.
Rio de Janeiro
February, 2017
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RESUMO
BIODIVERSIDADE E CONECTIVIDADE DE ESPONJAS CALCAREAS (PORIFERA,
CALCAREA) NO ATLÂNTICO TROPICAL OCIDENTAL
Báslavi Marisbel Cóndor Luján
Orientadora: Dra. Michelle Klautau
Resumo da Tese submetida ao Programa de Pós-Graduação em Biodiversidade e Biologia
Evolutiva, Instituto de Biologia, Universidade Federal do Rio de Janeiro – UFRJ, como parte
dos requisitos necessários à obtenção do título de Doutor em Ciências Biológicas.
O Atlântico Tropical Ocidental (WTA) é uma vasta região que compreende as províncias
Atlântico Noroeste Tropical (TNA), Plataforma Norte do Brasil (NBS) e Atlântico Tropical
Sudoeste (TSA). Embora abrigue uma grande diversidade de espécies marinhas, as esponjas da
classe Calcarea têm sido pouco estudadas. Essa falta de conhecimento dificulta nossa
compreensão das afinidades biogeográficas das esponjas Calcarea no WTA e do papel do Rio
Amazonas como uma barreira efetiva para a dispersão dessas esponjas. Neste estudo, foi
investigada a diversidade da classe Calcarea no WTA por meio de abordagens morfológicas e
moleculares e também foi avaliada a conectividade populacional da esponja Calcarea Leucetta
floridana. Com a presente contribuição, o número de esponjas da classe Calcarea no WTA
aumentou de 67 para 86 espécies, incluindo descrições de 15 espécies novas e 4 novos registros.
Entre elas, 21 espécies são endêmicas do TNA, três ocorrem só NBS e 22 são exclusivas do
TSA. As 14 espécies compartilhadas entre o TNA e o TSA sugerem uma afinidade Caribe-Brasil
para as esponjas calcareas. As análises genéticas evidenciaram cinco populações estruturadas
em L. floridana: uma população amplamente distribuida e mantendo fluxo gênico entre as
províncias TNA e TSA e quatro outras populações isoladas no Mar do Caribe. A população
panmítica sugeriu que a descarga do Rio Amazonas no Atlântico não é uma barreira efetiva à
manutenção do fluxo gênico entre populações trans-amazônicas de L. floridana.
Palavras-chave: Mar do Caribe, costa NE do Brasil, filogeografia, demographia, Rio
Amazonas.
Rio de Janeiro
Fevereiro de 2017
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INDEX
1. INTRODUCTION …............................................................................................................. 1
1.1 General introduction …........................................................................................................... 2
1.2 General Aim …........................................................................................................................ 5
1.3 Specific Aims …...................................................................................................................... 5
2. LITERATURE REVIEW …................................................................................................... 6
2.1. Geographic Scenario ….......................................................................................................... 7
2.1.1 The Western Tropical Atlantic …......................................................................................... 7
2.1.2 The Caribbean Sea …........................................................................................................... 8
2.1.3. The Tropical Brazilian Shelf and the Oceanic Islands ….................................................. 11
2.2. Connectivity within the Western Tropical Atlantic ….......................................................... 13
2.2.1 Estimating connectivity …................................................................................................. 13
2.2.2 General patterns of connectivity ….................................................................................... 14
2.2.3 Connectivity patterns in the Western Tropical Atlantic …................................................. 15
2.3. Porifera in the Western Tropical Atlantic …........................................................................ 16
2.3.1 Generalities of the Phylum Porifera ….............................................................................. 16
2.3.2 Generalities of the Class Calcarea …................................................................................. 17
2.3.3 Diversity of Calcarea in the Western Tropical Atlantic …................................................. 20
3. RESULTS ….......................................................................................................................... 21
3.1 Nicola gen. nov. with redescription of Nicola tetela (Borojevic & Peixinho, 1976) (Porifera:
Calcarea: Calcinea: Clathrinida) …............................................................................................. 22
3.2 Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared species
and phylogenetic remarks …...................................................................................................... 34
3.3 New records of calcareous sponges (Porifera: Calcarea) from the Northeastern Brazilian
coast including three new species …......................................................................................... 132
3.4 Evolutionary history of the calcareous sponge Leucetta floridana in the Western Tropical
Atlantic ….................................................................................................................................. 176
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4. GENERAL DISCUSSION …............................................................................................. 215
5. CONCLUSIONS AND PERSPECTIVES ….................................................................... 233
6. REFERENCES …............................................................................................................... 236
APPENDIX ….......................................................................................................................... 257
Appendix 1 …........................................................................................................................... 257
Appendix 2 …........................................................................................................................... 258
Appendix 3 …........................................................................................................................... 259
Chapter 1
INTRODUCTION
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1.1 General Introduction
For many decades, the zoogeographical affinities within the Western Tropical Atlantic,
specifically between the Caribbean and the Brazilian coast, have been discussed and several
biogeographic divisions have been proposed. Both areas were once considered a single
biogeographic province, the Caribbean Province, which extended from the coast of Florida
(United States of America) down to Cabo Frio in Rio de Janeiro (Brazil) (Ekman, 1953). Some
years later, based on the Brazilian endemism of corals, hydrozoans, molluscs and fishes, Briggs
(1974) proposed a division in Caribbean, West Indian (Lesser Antilles) and Brazilian provinces.
The latter included also the Brazilian Oceanic Islands. The discovery of “Caribbean” reef fishes
under the Amazon freshwater plume (Collete & Rützler, 1977) made Helfman et al., (1997)
reconsider the presence of a unique province in this region. Palacio (1982) did not refuse this
but restricted the Tropical Province (Caribbean) down to the 23°C isotherm, between Espírito
Santo and Rio de Janeiro. Nonetheless, as knowledge on Brazilian endemic species increased,
the division in Caribbean and Brazilian provinces became more accepted (Briggs, 1995; Floeter
& Gasparini, 2000; Boschi, 2000).
In 2007, Spalding et al. introduced a new biogeographic classification, the Marine
Ecoregions of the World (MEOW) integrating previous biogeographic divisions,
geomorphological and oceanographic features and new data of species distribution and
dominant habitats. In that classification, the Western Tropical Atlantic (WTA) comprised three
biogeographic provinces: the Tropical Northwestern Atlantic (TNA), the North Brazil Shelf
(NBS) and the Tropical Southwestern Atlantic (TSA). More recently, modifications to those
provinces have been proposed by Floeter et al., (2008) and Briggs & Bowen (2012) based on
new studies on fish endemism and phylogeography.
The existence of different biogeographic provinces in the WTA relies on the possibility of
barriers that can prevent the dispersal of organisms within this broad area, such as the Amazon
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or Orinoco rivers. According to Rocha (2003), the freshwater and sediment outflow from the
Amazon river would act as a strong barrier to some reef fishes and might be responsible for the
endemism observed in the Brazilian coast. Nonetheless, the effectiveness of this barrier might
be restricted to shallow-water organisms and highly influenced by the sea-level fluctuations.
Therefore, larval dispersal (gene flow) between Caribbean and Brazilian populations of
widespread species would occur during interglacial periods and would be facilitated by the
sponge assemblages found underneath the Amazon river that would act as a connectivity
corridor.
Sponges are exclusively aquatic, sessile, filter-feeding, multicellular organisms. They are the
oldest extant metazoan group (Antcliffe et al., 2014) and present many adaptations to different
habitats, including marine and freshwater environments. They play an important ecological role
in marine ecosystems as they contribute to the recycling of nutrients from the water column into
the benthic communities (de Goeij et al., 2013). Moreover, they constitute one of the principal
marine invertebrate sources for the isolation of bioactive compounds of pharmaceutical interest
(Blunt, 2006, Perdicaris et al., 2013)
As all sponges have lecithotrophic larvae (Ereskovsky, 2010), low dispersal capabilities and
restricted geographic distribution ranges are expected. However, some studies revealed that
widespread species do occur along the WTA (Lazoski et al., 2001; Valderrama et al., 2009,
Zilberberg, 2006). These species constitute good models to evaluate the effectiveness of the
Amazon River as a real barrier to gene flow, nonetheless, up to date not such study was
performed. The knowledge on sponge connectivity within the WTA is fragmentary or restricted
to some species within one single biogeographic province (De Biasse et al., 2010, 2016;
Chaves-Fonnegra et al., 2015; López-Legentil & Pawlik, 2009; Richards et al., 2016; de Bakker
et al., 2016; Padua, 2012).
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Among the four extant classes of Porifera (Demospongiae, Homoscleromorpha,
Hexactinellida and Calcarea), Calcarea is the only with skeleton exclusively composed of
calcium carbonate). The knowledge on the diversity of calcareous sponges is somehow
restricted to geographical areas to which specialized taxonomists have access. For instance, the
Mediterranean Sea and the Caribbean Sea have comparable coastal extension (Coll et al., 2010;
Miloslavich et al, 2011); however, the number of calcareous sponges reported in the former is
ca. 65 whereas in the latter, it is only 23. This possibly reflects the lack of taxonomic effort in
the Caribbean Sea, a region considered a diversity hotspot for many other taxa.
Within this context, this study is intended to present the diversity and population
connectivity of calcareous sponges in the Western Tropical Atlantic, with a major emphasis on
the Caribbean Sea and the Northeastern Brazilian coast.
This thesis is organised in five chapters. The first chapter includes this general
introduction and the aims of this work. The second chapter sets the theoretical framework of this
study through a brief review on the characterization of the WTA, population connectivity and
calcareous sponges. The third chapter includes the results of this study and it is divided in four
articles. The first article proposes the new Calcinean genus Nicola based on the redescription of
the type material of “Guancha” tetela and the description of new specimens collected in
Curaçao. In the second and third articles, I described the calcareous sponges from Curaçao and
from the NE Brazilian coast, respectively, using morphological and molecular approaches. The
fourth article is the first study on population connectivity and demography of a calcareous
sponge (Leucetta floridana) in the WTA. The fourth chapter comprises a general discussion. The
fifth chapter presents the main conclusions and perspectives of this study.
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1.2 General Aim
To know the biodiversity and connectivity of the class Calcarea in the Western Tropical Atlantic
1.3 Specific Aims
1.3.1 To identify and describe the sponges of the class Calcarea from the Caribbean Sea.
1.3.2 To identify and describe the sponges of the class Calcarea from the Northeastern Brazilian
coast.
1.3.4 To assess the genetic structure of calcareous sponges in the Western Tropical Atlantic.
1.3.3 To test if the Amazon River is an effective barrier to connectivity among populations of
calcareous sponges.
Chapter 2
LITERATURE REVIEW
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2.1. Geographic Scenario
2.1.2 The Western Tropical Atlantic
The Atlantic Ocean emerged after the break-up of the supercontinent Pangaea in the late
Jurassic Period (163.5 -145 million years BP). Two important geological events occurred: (1)
the separation of the Pangaea in Laurasia and Gondwana, which resulted in the formation of the
Central Atlantic Ocean, and (2) the gradual south-to-north split of West Gondwana in South
America and Africa during the Early Cretaceous (145-100.5 million years BP) that originated
the South Atlantic Ocean (Seton et al., 2012). During the next million years, the Atlantic Ocean
continued spreading until its current configuration.
According to the latest marine biogeographic classification system (MEOW - Spalding et al.,
2007), the Tropical Atlantic Realm (TA, Figure 1A) comprises six biogeographic provinces
(Figure 1B): Tropical Northwestern Atlantic (TNA, n°12), North Brazil Shelf (NBS, n°13),
Tropical Southwestern Atlantic (TSA, n°14), St. Helena and Ascension Islands (n°15), West
African Transition (n°16) and Gulf of Guinea (n°17).
Figure 1. Biogeographical regionalization according to Spalding et al. (2007). A. Realms in theAtlantic Ocean. B. Biogeographic provinces in the Tropical Atlantic Realm. C. Ecoregions inthe Western Tropical Atlantic.
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The Western Tropical Atlantic (WTA) comprises the TNA, NBS and TSA biogeographic
provinces and includes 16 ecoregions (Figure 1C). The TNA comprises nine ecoregions:
Southern Gulf of Mexico (n°69), Western Caribbean (n°68), Southwestern Caribbean (n°67),
Southern Caribbean (n°66), Greater Antilles (n°65), Eastern Caribbean (n°64), Bahamiam
(n°63), Bermuda (n°62), and Floridian (n°63). The NBS includes the Guianian (n°71) and
Amazonia ecoregions (n°72). The TSA Province comprises São Pedro and São Paulo Islands
(n°73), Fernando de Noronha and Atoll das Rocas (n°74), Northeastern Brazil (n°75), Eastern
Brazil (n°76) and Trindade and Martin Vaz Islands (n°77). This study is focused on the TNA and
TSA provinces, specifically, the Caribbean Sea and the northeastern Brazilian coastal shelf.
2.1.2 The Caribbean Sea
The Caribbean Sea is a semienclosed basin within the TNA Province. It is bounded by Central
America on the west, Greater Antilles on the north, Lesser Antilles on the east and the northern
coast of South America on the south. It includes five ecoregions: Western Caribbean,
Southwestern Caribbean, Southern Caribbean, Greater Antilles and Eastern Caribbean. The
Caribbean basin comprises an area of approximately 2,754,000 km2, a volume of about 6.5x106
km3 and its coastline has an extension of more than 13,500 km (Miloslavich et al., 2011).
The geological age of the Caribbean Sea is related to the formation of the Caribbean Plate. In
the Middle Cretaceous (100.5–80 million years BP), a flood basalt event produced an oceanic
crust with a thickness of about 15-20 km (Burke, 1978). According to the model of Meschede &
Frisch (1998), this event took place somewhere between North and South America. An
alternative hypothesis (Pindell, 1994) suggested that the flood basalt occurred in the Pacific
Ocean, in the Galapagos hotspot, which afterwards, drifted eastwards (Figure 2).
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Figure 2. Hypotheses to explain the origin of the Caribbean plate. A. Origin in an inter-American position. B. Pacific origin. Taken from Meschede & Frisch (1998).
The Caribbean Sea is divided into five deep water regions: the Yucatan Basin, the Cayman
Trough, the Colombian Basin, the Venezuelan Basin and the Grenada Basin, which are
separated by underwater ridges - the Cayman Ridge, the Nicaraguan Rise, the Beata Ridge and
the Aves Ridge, respectively (Draper et al., 1994, Figure 3). The average seafloor depth is
approximately 2,400 m and the deepest area (more than 7,500) is comprised between Cuba and
Jamaica, in the Cayman Trough (Matthews & Holcombe, 1985).
Figure 3. General geography and topography of the Caribbean Sea indicating the politicaldivisions and basins mentioned in the text. Source: Encyclopædia Britannica, Inc.
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The present oceanographic configuration of the Caribbean Sea was established only after the
uplift of the Isthmus of Panama (2.8 million years BP - O'dea et al., 2016). Previously, the
Caribbean Sea experienced increased upwelling as it was connected to the Pacific Ocean (O'dea
et al., 2007). Nowadays, the Caribbean waters are clear, oligotrophic and with warm
temperatures ranging from 22 to 29°C (Kinder et al., 1985).
The Caribbean Sea receives the Atlantic inflow from the North Brazil current through several
passages located in the Lesser Antilles (Windward Islands Passages and Leeward Islands
Passages) and the Greater Antilles (Johns et al., 2002). The Caribbean current is formed mainly
by the masses of water that pass through the Windward Island Passages, specially through the
Grenada Passage located at the south of Grenade (near 11.51oN, Cherub, 2007). Within the
Caribbean basin, this current flows westward at 13–16°N and between Nicaragua and Jamaica it
turns northwest into the Gulf of Mexico through the Yucatan Channel. In the southernmost part
of the Caribbean Sea, one of the branches of the Caribbean Current forms the counterclockwise
Panama Gyre (Figure 4, Molinari et al., 1981).
Figure 4. Currents in the Caribbean Sea (as represented by the Mariano Global Surface VelocityAnalysis). Downloaded from http://oceancurrents.rsmas.miami.edu. Abbreviations: P=passage.
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Although the Caribbean waters are mainly clear, they are influenced by the freshwater
outflow from major rivers. The freshwater and sediments from the Amazon (Brazil) and
Orinoco (Venezuela) plumes enter through the North Brazil Current and the discharge of the
Magdalena river (Santa Marta, Colombia) directly interacts with the Caribbean current in the
Southwestern Caribbean (Cherub, 2007).
The Caribbean Sea is considered a global-scale hotspot for marine biodiversity (Roberts et
al., 2002). It encompasses a high diversity of flora and fauna distributed in different ecosystems
including coral reefs, mangroves and seagrasses (Miloslavich et al., 2010).
2.1.3 The Tropical Brazilian Shelf and the Oceanic Islands
The vast extension of the Brazilian coast (7,500 km) includes two major biogeographic realms,
the Tropical Atlantic (TA) and the Temperate South America (Spalding et al., 2007, Figure 1A).
In the present work only the TA was studied.
The Tropical Brazilian Shelf and the Oceanic Islands comprise seven ecoregions, one in the
NBS and six in the TSA (Figure 1C) . This area has been shaped by the Quaternary sea-level
changes over the past 5000 years, with the exposure of coastal sediments, river load, and coastal
drift (Dominguez et al., 1983).
The northern Brazilian coast, which corresponds to the Amazonian ecoregion of the NBS
(Figure 1C, n°72), is characterised by a combination of freshwater, estuarine and marine
ecosystems as a consequence of the outflow of the Amazon river. Benthic environments
comprise muddy-bank-shorelines (Anthony et al., 2010), rhodolith beds (Moura et al., 2016),
coral and sponge reefs (Colette & Rützler, 1976; Moura et al., 1999; 2016).
The northeastern and part of the southern Brazilian Coast (3°S - 20°S), which correspond to
the Northeastern Brazil (n°75), Eastern Brazil (n°76) and Trindade and Martin Vaz Islands
(n°77) ecoregions of the TSA, have an oligotrophic environment with little influence of river
runoff and only subjected to local upwelling (Soares et al., 2016). This area is characterised by a
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variety of ecosystems, including mangrove forests, seagrass beds, coral reefs, sandy beaches,
rocky shores, lagoons and estuaries (Miloslavich et al., 2011; Moura et al., 2013).
The Brazilian oceanic islands include Fernando de Noronha Archipelago, Rocas Atoll (the
only atoll in the South Atlantic) and São Pedro and São Paulo Archipelago (ecoregions n° 73
and 74). These islands harbour tropical environments similar to those of the Caribbean Sea and
constitute very important hotspots of biodiversity (Soares et al., 2016). Mesophotic reefs and
rhodolith beds are also common within these islands (Magalhães et al., 2015; Amado-Filho et
al., 2016).
The entire coast of Brazil is under the influence of warm and temperate marine currents,
freshwater discharge from several rivers and upwelling events (Coelho-Souza et al., 2012;
Soares et al., 2016). In the Tropical Brazilian continental margin, the principal surface currents
are the Brazilian Current (BC) and the North Brazilian Current (NBC), originated from the
South Equatorial Current at about 5–6°S (Stramma, 1991; Silveira et al., 1994). The BC flows
to the south whereas the NBC runs to the north and northwest (Figure 5).
Figure 5. Surface currents in the Tropical Brazilian coast (as represented by the Mariano GlobalSurface Velocity Analysis): A. North Brazil Current (NBC) and B. Brazilian Current (BC).Downloaded from http://oceancurrents.rsmas.miami.
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2.2 Connectivity within the Western Tropical Atlantic
2.2.1 Estimating connectivity
Population connectivity is understood as the exchange of individuals among populations or
subpopulations (Gagnaire et al., 2015) and it can be divided into genetic and demographic
connectivity. Genetic connectivity refers to the degree to which gene flow affects evolutionary
processes within populations, whereas demographic connectivity refers to the degree to which
population growth and vital rates (survival, reproduction, emigration) are affected by dispersal
(Lowe & Allendorf, 2010).
The estimation of connectivity among marine populations is based on the assessment of
larval dispersal through indirect methods. These methods include the use of geochemical tags
from calcified structures (otoliths, statoliths and shells) or from artificial sources (fluorescent
compounds inserted in larvae (Thorrold et al., 2007), coupled biophysical models which
integrate oceanographic factors and larvae biological traits (e.g. Cowen et al., 2006; Paris et al.,
2007) and genetic approaches that allow the calculation of migration rates (e. g. Selkoe et al.
2010), among other parameters,
Molecular approaches include the amplification of neutral “frequency” or “sequence”
markers. Frequency markers such as microsatellites are adequate to answer questions in more
ecological timescales, whereas sequence markers such the cytochrome oxidase I (COI) aid to
resolve the history of divergence among populations. However, both types of markers present
some disadvantages. On the one hand, the isolation of microsatellites can be time-consuming
and consistent results may require large samplings and sophisticated analyses. In some cases, it
is even necessary to test the Mendelian condition of the loci before genotyping (Hellberg, 2009).
On the other hand, duplication or insertions from nuclear genome (pseudogenes) can alter
mtDNA patterns (Williams & Knowlton, 2001; Schizas, 2012).
14
Once chosen the adequate marker, further analyses to estimate connectivity comprise the
computation of indirect estimators (indexes) as the fixation index (FST) and the application of
direct methods such as parentage assignment or clustering analyses.
2.2.2 General patterns of connectivity
Marine population connectivity is driven by two main integrated components: (1) physical
processes and (2) biological traits (Cowen & Sponaugle, 2009). The former includes the
particular oceanographic processes occurring in the connectivity area (currents, tides, upwelling,
hurricanes), whereas, the latter corresponds to the life history of the target species including its
larval behaviour (mechanisms of defense against predators, vertical migrations, horizontal
swimming), chemical signals for settlement and adult spawning (Cowen et al., 2000; Cowen et
al, 2005). Other dispersal factors non-mediated by larvae such as adult rafting also influence
population connectivity (Kinlan et al., 2005; Cowen & Sponaugle, 2009).
In the last 20 years, several studies have notably contributed to the understanding of marine
population connectivity. Herein, I point out which I consider the most relevant conclusions:
1. Not all marine populations are open. Coastal marine populations were previously considered
open populations which would exchange individuals from very distant localities as a
consequence of the passive larval dispersal driven by water currents. Using Eulerian and
Lagrangian flow models, Cowen et al. (2000) found higher levels of larvae retention near the
natal localities than in distant areas. These results were also observed in further studies (Swearer
et al. 2002, Jones et al., 2005). Nowadays, studies suggest that populations range from fully
open to fully closed (Cowen & Sponaugle, 2009).
2. Larval dispersal capability may not be a good indicator of connectivity. Species with low
larval dispersal capabilities are believed to present structured populations or high rates of self-
recruitment (local replenishment); however, several studies on connectivity of reef fishes did not
evidence this. Bowen et al., (2006) found that Myripristis jacobus, a fish with short lifespan had
15
a population much less structured than Holocentrus ascensionis, a species with longer larval
duration. Almany et al. (2007) evidenced that fishes with different larval duration (orange
clownfish - Amphiprion percula and vagabond butterflyfish – Chaetodon vagabundus)
presented equal high levels of self-recruitment (60%). These studies indicated that larval
duration was a poor predictor of connectivity.
3. Genetic analyses should include oceanographic input. As traditional genetic analyses based
on the correlation between geographic distance (Eclidean distance) and genetic dissimilarity
failed to explain the connectivity patterns observed in several studies (e.g. Brandbury &
Bentzen, 2007), environmental information, such as ocean currents, started to be incorporated in
genetic calculations (White et al., 2010). That approach greatly improved the predictive value of
population genetics studies on small spatial scales (Selkoe et al., 2010). Within this context, two
adjacent localities are not necessarily connected nor two distant localities are genetically
isolated (White, 2010).
4. Still dealing with limited knowledge. Despite the considerable advances on marine
connectivity due to studies integrating different approaches, our knowledge is still fragmented
or restricted to specific taxa (mainly, fish and coral species). As pointed out by White (2010),
even with perfect simulations, the obtained results may refer to potential population
connectivity.
2.2.3 Connectivity patterns in the Western Tropical Atlantic
Our knowledge of population connectivity in the Tropical Western Atlantic is mainly based on
studies of reef fish connectivity using mitochondrial DNA sequences and with focus on the
Caribbean Sea and the Northeastern Brazilian coast.
Rocha et al. (2002) evaluated the role of the Amazon river as an effective barrier to the
dispersal of surgeonfishes (Acanthurus) and found levels of connectivity that correlated with
habitat preference. The species A. bahianus, which rarely settles outside shallow reefs, revealed
16
highly structured Brazilian and Caribbean populations, whereas A. chirurgus, collected on soft
sponge bottoms under the mouth of the Amazon River, presented genetically homogeneous
populations. This indicated that the Amazon outflow acted as a semi-permeable barrier. Rocha
(2003) suggested that the sponge assemblages discovered under the mouth of the Amazon river
(Colette & Rützler, 1976) would act as a corridor for species that require clear waters with
normal salinity but not for species strictly dependent on shallow reef habitats.
In the Caribbean Sea, Cowen et al. (2006) defined four broad regions of connectivity: (1) the
eastern Caribbean (Puerto Rico to Aruba); (2) the western Caribbean (Cuba to Nicaragua); (3)
Bahamas, Turks and Caicos Islands; and (4) the peripheral area of the Colombia-Panama Gyre.
Within these regions, population isolation and connectivity would be related to oceanographic
conditions such as the strong currents at the Mona Passage between Cuba and Puerto Rico and
the Colombia-Panama counter-gyre or to topographical features e.g. the shallow shelves that
remained exposed during the Pleistocene sea-level fluctuations.
2.3. Porifera in the Western Tropical Atlantic
2.3.1 Generalities of the Phylum Porifera
The phylum Porifera is composed of exclusively aquatic, sessile and multicellular organisms,
commonly known as sponges (Hooper et al., 2002). They are the oldest metazoan group still
extant and they present many ecological adaptations to different habitats including marine and
freshwater environments.
These animals are also characterised by the presence of a skeleton composed of spicules
(mineral aggregations of calcium carbonate or silica) or spongin (a protein from the family of
the collagen) and of an aquiferous system, principally used for filtering-feeding (Hooper et al.,
2002).1
1 Porifera also includes species without any type of skeleton (e.g. Hexadella and Oscarella spp.) or aquiferous system (carnivorous sponges).
17
Sponges present cells with high mobility and totipotency, which facilitate cellular
differentiation and transdifferentiation (Klautau, 2016). Except in species of the class
Homoscleromorpha, these cells do not form true tissues, however, the choanoderm (composed
of choancytes) and the pinacoderm (formed by pinacocytes), important components of the
aquiferous system, act as functional epithelia (Leys & Riesgo, 2012).
Sponges have short-lived lecithotrophic larvae (Ereskovsky, 2010) with a maximum lifespan
of two weeks (Fry, 1971; Maldonado, 2006). This may constrain their dispersion and
consequently, restrict their geographic distribution. Nonetheless, they present asexual processes
such as budding, gemmulation and fragmentation (Maldonado & Riesgo, 2008) that may
contribute to expand their distribution range.
According to the World Porifera Database (Van Soest et al., 2016), the phylum Porifera
currently comprises 8781 species distributed in four classes: Calcarea Bowerbank, 1862,
Demospongiae Sollas, 1885, Hexactinellida Schmidt, 1870 and Homoscleromorpha Gazave et
al, 2012. Among them, Calcarea is the only class that reunites the species whose skeleton is
exclusively composed of spicules made of calcium carbonate.
2.3.2 Generalities of the class Calcarea
The class Calcarea comprises marine sponges with a skeleton exclusively composed of calcium
carbonate, consisting of free, rarely linked or cemented spicules, to which a solid calcitic
skeleton can be added. They are viviparous and present blastula larvae (Manuel et al., 2002). In
this class, all the known types of aquiferous system in Porifera (asconoid, syconoid, sylleibid,
solenoid and leuconoid) are present (Figure 6, Cavalcanti & Klautau, 2011).
18
Figura 6. Aquiferous system of Porifera: asconoid (a), syconoid (b), sylleibid (c), leuconoid (d)and solenoide (e). Grey lines represent the choanoderm. Taken from Cavalcanti & Klautau(2011).
Calcarean sponges commonly reach small sizes (measured in mm or a few cm), however,
species of more than 20 cm in length were reported e.g. Leucandra multiformis, Leucetta
avocado, Pericharax heteroraphis, Sycon ciliatum (Poléjaeff, 1883; Koechlin, 1977; Van Soest
et al., 2012). Although most of the known species are white or beige; some yellow, red and pink
species have also been described e.g. Clathrina clathrus, C. rubra, Leucascus roseus. They can
be found in shallow tropical waters as well as in very deep waters in the Antarctic (Janussen &
Rapp, 2011; Rapp et al., 2011) and North Atlantic (Greenland, Rapp et al., 2013), in cryptic
environments protected of light such as overhangs, roofs of caves, crevices or underneath
boulders. Associated microorganisms have been found within the mesohyl of calcareous
sponges (Fromont et al., 2016) as well as benthic fauna (Padua et al., 2013) among the tubes or
atrium of these sponges.
The class Calcarea is divided into two monophyletic subclasses Calcinea and Calcaronea
Bidder, 1898(Figure 7). Calcaroneans are characterised by presenting sagittal spicules,
apinucleated choanocytes, amphiblastula larvae and diactines as the first spicules to be
produced, whereas calcineans have a skeleton mainly composed of regular spicules,
basinucleated choanocytes, calciblastula larvae and triactines as the first spicules to be secreted
(Manuel et al. 2002).
19
Figure 7. Calcaronea: A. Amphiblastula larvae (Lanna & Klautau, 2016), B. Apinucleatedchoanocyte (Borojevic et al., 2000), C. Sagittal spicules and diactines (Van Soest et al., 2012).Calcinea: D. Calciblastula larvae (Ereskovsky & Willenz, 2008). E. Basinucleated choanocytes(Borojevic et al, 1990). F. Regular spicules and diactines (Van Soest et al., 2012).
Phylogenetic relationships within these two subclasses remain unclear as many orders,
families and even genera are paraphyletic or polyphyletic (Voigt et al., 2012; Voigt & Wörheide,
2016). However, in the subclass Calcinea, certain skeletal traits have evidenced phylogenetic
signal in some genera, e.g. Clathrina and Borojevia (Rossi et al., 2011; Klautau et al., 2013).
The total number of described calcareous species (ca. 680) represents only about 8% of all
described extant sponges (Van Soest et al, 2012) and most of these records are restricted to areas
where the taxonomic effort on calcareous sponges has been intense (e. g. Australia, Japan and
Mediterranean Sea). Furthermore, the plasticity of a few morphological characters in some
Calcarean taxa difficults the species identification and sometimes require integrative approaches
(e.g. Valderrama et al., 2009; Imeseck et al., 2014; Azevedo et al., 2015; Klautau et al., 2016).
20
2.3.3 Diversity of Calcarea in the Western Tropical Atlantic
In general terms, the sponge diversity (including all the classes) is concentrated in the tropics
(Soares et al., 2016). However, the first attempt to elucidate the distribution patterns within the
class Calcarea (Van Soest et al., 2012) revealed very low values of diversity in that area. As
pointed out in the same study, those results did not reflect the real diversity but the need of
describing the sponge fauna from poorly explored areas.
The number of valid species known for the Western Tropical Atlantic, compiled from the
World Porifera Database (Van Soest et al., 2016; Van Soest, 2017), unpublished literature
(Azevedo, 2013) and submitted manuscripts (Azevedo et al., submitted) is 67 species. Some
species are shared between two or three biogeographic provinces and other species are endemic
to a province and in some cases, to an ecoregion.
Chapter 3
RESULTS
22
Nicola gen. nov. with redescription of Nicola tetela (Borojevic & Peixinho, 1976) (Porifera:
Calcarea: Calcinea: Clathrinida)
CÓNDOR-LUJÁN, B. & KLAUTAU, M.
Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.
Carlos Chagas Filho, 373, 21941-902, Rio de Janeiro, RJ, Brasil. [email protected];
Corresponding author: [email protected]
Article published in ZOOTAXA 4103(3): 230-238
Abstract
Guancha tetela was originally described as a species having a peduncle and a skeleton
exclusively composed of sagittal triactines. Therefore, according to the most recent phylogeny
of Clathrinida, it should be placed in the genus Clathrina. This species was collected on the
Northeastern Brazilian coast in 1968 and it was not collected again until 2011 in Curaçao. In
this study, we reanalyzed the type material and the new specimens from Curaçao under a
morphological-molecular approach. Morphological analysis revealed the presence of tetractines
in the skeleton of all the studied specimens, including a slide of the holotype. In the molecular
phylogeny G. tetela grouped with genera containing tetractines, but as an independent new
lineage, different from all the other genera of Clathrinida. Based on these results, we propose
the erection of a new genus, Nicola gen. nov., to include species whose body is composed of
tubes without anastomosis nor branches but that run in parallel and coalesce at the apical and
basal regions. Moreover, the skeleton is exclusively composed of sagittal triactines and
tetractines.
Key words: Atlantic Ocean, Northeastern Brazilian Coast, Caribbean Sea, Curaçao, molecular
systematics, new genus, Guancha tetela.
Introduction
The genus Guancha was originally proposed by Miklucho-Maclay (1868) in order to describe
Guancha blanca, a species from the Canary Islands (Lanzarote) that presented a clathroid
cormus with a stalk. Although Miklucho-Maclay (1868) considered that the presence of a stalk
was sufficient to separate this species in a new genus, subsequent authors did not agree with this
point of view and placed G. blanca in different genera: Ascetta (Haeckel 1872; Vosmaer 1881;
Lendenfeld 1891), Leucosolenia (Lackschewitsch 1886; Vosmaer 1887; Breitfuss 1896, 1898;
23
Dendy & Row 1913; Arndt 1928; Hôzawa 1929; Burton 1930; Brøndsted 1931; Breitfuss 1932,
1935; Topsent 1936; Arndt 1941; Tanita 1943), and Clathrina (Minchin 1896; Jenkin 1908).
Only in 1976 the genus Guancha became accepted, with the description of G. tetela by
Borojevic and Peixinho (1976), and had its first diagnosis:
"Clathrinidae à cormus constitué d'un pédoncule et d'un corps clathroïde. Spicules
réguliers et parassagittaux, ou uniquement parasagittaux, orientés parallèlement dans les
parois des tubes, au moins dans la partie basale de l'éponge avec l'actine impaire basipète"
(Borojevic & Peixinho 1976) (Translation: Clathrinidae with a cormus composed of a stalk and
a clathroid body. Spicules are regular and parasagittal or only parasagittal, organised in parallel
in the tubes wall, at least at the basal part of the sponge with the unpaired actine basipetally
oriented).
Later on, in the Systema Porifera (Borojevic et al. 2002), the diagnosis proposed was:
"Clathrinidae with a cormus composed of a peduncle and a clathroid body. The peduncle
may be formed by true tubes with a normal choanoderm, or may be solid with a special
skeleton. The skeleton is composed of regular (equiangular and equiradiate) spicules to which
parasagittal spicules are added, at least in the peduncle. In some species only parasagittal
spicules are present. The unpaired actine of parasagittal spicules is always basipetally oriented."
Since the publication of the Systema Porifera and this new diagnosis of Guancha, four new
species were described within this genus: Guancha arnesenae Rapp, 2006, Guancha camura
Rapp, 2006, Guancha pellucida Rapp, 2006, and Guancha ramosa Azevedo et al., 2009. More
recently, however, molecular studies showed that Guancha was not a monophyletic genus, and
the authors proposed its synonymisation with Clathrina (Rossi et al. 2011; Klautau et al. 2013).
This synonymisation was confirmed when the type species of the genus (G. blanca) was
included in a molecular tree (Imešek et al. 2014). Currently, we follow what was proposed by
Klautau et al. (2013), that all Guancha species with only triactine spicules should be transferred
to Clathrina, and that Guancha species with triactines and tetractines should be assigned to any
of the other genera proposed or rediagnosed in the same article (namely Arthuria, Ascandra,
Borojevia, Brattegardia, and Ernstia).
According to this proposal, the species Guancha tetela, originally described as having a
skeleton exclusively composed of sagittal triactines, should be placed in Clathrina. However, a
more detailed revision of this species revealed the presence of tetractines in its skeleton, which
precludes its classification as a Clathrina. As G. tetela presents more triactines than tetractines,
it should then be considered Arthuria, however, the organisation of the cormus of G. tetela is
24
different of that of other Arthuria. Hence, molecular and detailed morphological analyses were
performed in the present work to verify the correct classification of G. tetela.
Material and Methods
The holotype of Guancha tetela is deposited in the Muséum Nationale d'Histoire Naturelle de
Paris under the number MNHN-LBIM-C-1975-1 and there are also two slides from the holotype
(one spicule slide and one section slide) deposited in the collection of the Museu Nacional do
Rio de Janeiro (MNRJ 40). In the present work we analysed the slides MNRJ 40. The holotype
of G. tetela was collected by dredging during a survey of the oceanographic vessel “Almirante
Saldanha” along the Northeastern Brazilian Continental Shelf. Recently (2011), five specimens
were collected from Curaçao by SCUBA diving and were deposited in the Porifera Collection of
the Biology Institute of the Universidade Federal do Rio de Janeiro (UFRJPOR 6714,
UFRJPOR 6723, UFRJPOR
6724, UFRJPOR 6746, and UFRJPOR 6767). Species names, voucher numbers, and GenBank
accession numbers of the DNA sequences used for a phylogenetic analysis are listed in Table 1.
Morphological analyses
For the preparation of spicule slides, fragments of the sponge were dissolved in sodium
hypochlorite (commercial bleach) in a test tube. After digestion, the spicules were washed five
times in distilled water and three times in absolute ethanol. They were then transferred to slides
and the ethanol was heat-evaporated. The mounting medium used was Entellan (Merck).
For the scanning electron microscopy (SEM), the spicules were placed on a cover-slip
mounted on a stub with double-sided carbon tape and sputter-coated with gold. The analysis was
performed in a JSM-6510 scanning electron microscope at the Institute of Biology of the
Universidade Federal de Rio de Janeiro.
For the preparation of the slides sections, small fragments of the sponge were stained with a
5% acid Fuchsin alcoholic solution for 10 min. The excess of Fuchsin was removed with
absolute ethanol for 5 min and the fragments were transferred to the slides, covered with some
drops of xylene and mounted with Entellan.
Spicules measurements were made using an ocular micrometer. The length and the width at
the base of each actine were measured for every spicule category. The results are presented in
tabular form, featuring length and width (minimum, mean, standard deviation [s], and
maximum), and sample size (n). Photographs were taken with a Zeiss AxioCam ERc5s coupled
to a ZEISS Stemi 2000C stereoscope and with a digital camera connected to a Zeiss Axioscop
microscope.
25
Molecular phylogenetic analyses
Genomic DNA was extracted with the guanidine/phenol-chloroform protocol (Sambrook et al.
1989) and stored at –20°C until amplification. The region comprising the partial 18S and 28S,
the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA was amplified by PCR with the
following primers: 18S (5`-TCATTTAGAGGAAGTAAAAGTCG-3`) and 28S (5`-
GTTAGTTTCTTTTCCTC CGCTT -3`) (Lobo-Hajdu et al. 2004). Each PCR amplification
reaction mixture contained: 1X buffer (5X GoTaq R Green Reaction Buffer Flexi, PROMEGA),
0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM of each
primer, one unit of Taq DNA polymerase (Fermentas) and 1 µL of DNA, summing up to 15 µL
with Milli-Q water. PCR steps included one first cycle of 4 min at 94°C, 1 min at 50°C and 1
min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and one minute at 72°C, and a final
cycle of 6 min at 72°C. Forward and reverse strands were automatically sequenced in an ABI
3500 sequencer (Applied Biosystems). Sequences were aligned through the online version of
the program MAFFT v.7 (Katoh & Standley 2013) using the strategy Q-INS-i (Katoh & Toh
2008).
Phylogenetic analyses were performed under maximum likelihood (ML) and Bayesian
inference (BI). The ML analysis was conducted online on PhyML 3.0 (Guindon et al. 2010;
available at http://www.atgc-montpellier.fr/phyml). The model for the ML analysis was selected
using Modeltest 3.7 and the Akaike information criterion (AIC) (Posada & Crandall, 1998),
which indicated GTR (General Time Reversible). One thousand bootstrap pseudo-replicates
were performed (Felsenstein 1985). Bayesian inference reconstructions were obtained with
MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) under 106
generations and a burn-in of 1000 sampled trees, yielding a consensus tree of majority.
Table 1. Analyzed specimens with voucher numbers and GenBank accession numbers.
Species Voucher number Genbank accessionnumber
Ascaltis reticulum UFRJPOR6258 HQ588973Ascandra falcata UFRJPOR5856 HQ588962Ascandra contorta UFRJPOR6327 HQ588970Arthuria hirsuta ZMAPOR07061 KC843431Arthuria spiralatta MNRJ12860 KC985139Borojevia cerebrum UFRJPOR6322 HQ588964Borojevia brasiliensis UFRJPOR5214 HQ588978Brattegardia nanseni UFRJPOR6332 HQ588982Clathrina antofagastensis MNRJ 9289 HQ588985Clathrina blanca PMR-14307 KC479087
26
Clathrina clathrus UFRJPOR6315 HQ588974Clathrina lacunosa UFRJPOR6334 HQ588991Clathrina ramosa MNRJ 10313 HQ588990Ernstia tetractina UFRJPOR5183 HQ589000Ernstia sp. UFRJPOR6621 KC843433Guancha tetela UFRJPOR 6723 KU568492
Leucaltis clathria UFRJPOR 6944 KU568493Leucaltis nodusgordii QMG316050 AJ633857Leucettusa nuda MNRJ 10804 KC843453Leucascus simplex BMOO16283 KC843454Leucetta floridana PTL09.P100 KC843456Leucetta potiguar UFPEPOR547 EU781986
Results
Class Calcarea Bowerbank, 1864
Subclass Calcinea Bidder, 1898
Order Clathrinida Hartman, 1958 emend.
Nicola gen. nov.
Etymology: For Nicole Boury-Esnault in recognition of her dedicated work on the taxonomy of
sponges, including calcareous sponges.
Type species: Nicola tetela (Borojevic & Peixinho, 1976)
Diagnosis: Clathrinida with a globular to ovoid body composed of parallel tubes that coalesce at
the apical and basal regions. They do not anastomose nor ramify. The skeleton exclusively
contains sagittal spicules: triactines and tetractines. The paired actines are rudimentary and they
form a straight angle (180°s). The unpaired actine is always the longest actine. Diactines
including trichoxeas may be added. The aquiferous system is asconoid.
Nicola tetela comb. nov.
Synonyms: Guancha tetela, Borojevic & Peixinho 1976: 998
Material examined: Slide of the holotype (MNRJ 40), Station 1966, Northeastern Brazilian
Continental Shelf (Southern coast of Bahia State) (17°55'S, 37°30'W), collected by dredging by
the “Almirante Saldanha” (SAL) vessel, 17th August 1968, 47 m deep; UFRJPOR 6714,
UFRJPOR 6723, UFRJPOR 6724, Playa Kalki, Westpunt, Curaçao
(12°22'29.86 N,ʺ 69°09'30.63 W), collected by E. Hajdu and B. Cóndor-Luján, 21ʺ st August
2011, 5.6 m deep; UFRJPOR 6746, Sunset Waters, Soto, Curaçao
(12°16'01.58 N,ʺ 69°07'44.85 W), collected by E. Hajdu, 20ʺ th August 2011, 8.9 m deep;
27
UFRJPOR 6767, Sunset Waters, Soto, Curaçao (12°16'01.58 N,ʺ 69°07'44.85 W); collected byʺ
B. Cóndor-Luján; 22nd August 2011, 9–12 m deep.
Colour: bright orange in life and white in ethanol.
Description The specimens have a globular (Figure 1A) to ovoid body (Figure 1B), with apical
osculum and a peduncle at the base. The peduncle is formed by coalescent tubes with
choanoderm. Above the stalk, each tube is divided into two tubes which do not anastomose nor
ramify; instead, they run in parallel and then converge to form the osculum (Figure 1C). The
aquiferous system is asconoid, with choanocytes, intercalated by porocytes, covering the interior
of the tubes (Figure 1D). The surface is smooth and bright and the consistency is fragile.
The skeleton is composed of triactines and tetractines arranged in parallel, the triactines
being more abundant than the tetractines. The unpaired actine of the spicules is always
basipetally oriented (Figure 1E). The apical actine of the tetractines is projected into the lumen
of the tubes (Figure 1F).
Triactines are equally distributed all over the sponge body, whereas tetractines seem to be
more concentrated in the apical region, near the osculum (at least in UFRJPOR 6723). The size
of the spicules is very variable and although the unpaired actine is frequently much longer than
the paired ones (Figures 1G, H), it sometimes can be only a little longer (Figure 1I).
Spicules (Table 2, Figures 1G-K).
Triactines. Sagittal. Actines are straight, conical, with sharp tips. The unpaired actine presents a
constriction near its base. They present very variable size and are the most abundant spicules
(Figures 1G-I). Size: 75.0-440.0/5.0-10.0 µm (unpaired actine); 17.5-60.0/5.0-7.5 µm (paired
actine).
Tetractines. Sagittal. Actines are straight, conical with sharp tips. The unpaired actine has a
constriction near its base (Figures 1G, J). The apical actine is smooth and can be straight or
curved. It is longer and, generally, narrower than the paired actines (Figure 1K). They present
very variable size. Size: 80.0-370.0/5.0-10.0 µm (unpaired actine); 12.5-45.0/5.0-8.7 µm (paired
actine); 17.5-75.0/2.5-6.2 µm (apical actine).
Ecology: The Brazilian specimen was collected at 47 m deep in a calcareous-algae bottom,
whereas the specimens from Curaçao were found underneath broken corals in shallow waters
down to 12 m. No organisms were found on the surface or among the tubes of the studied
specimens.
28
Figure 1. Nicola tetela comb. nov. A-B: Live specimens: UFRJPOR 6714 (A) and UFRJPOR 6746 (B)(photos taken by E. Hajdu). C. Specimens after fixation (UFRJPOR 6714, 6723, 6724). D. Tangentialsection of the skeleton showing choanocytes and porocytes (pc). E. Detail of the apical region of the body.F. Apical actines projected into the lumen of a tube (arrow pointing to one apical actine). G. Spicules:Triactine (left) and tetractine (right). H – K: SEM images of spicules: H. Large triactine. I. Small triactine.J. Tetractine. I. Apical actine of a tetractine. All skeleton and spicule images were taken from thespecimen UFRJPOR 6723.
29
Table 2. Spicule measurements of Nicola tetela comb. nov., including the originalmeasurements of the holotype (MNRJ 40) by Borojevic & Peixinho (1976).
Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR Triactine Unpaired 75.0 223.6 91.7 440.0 5.0 7.5 0.9 10 306723 Paired 27.5 31.3 4.3 47.5 5.0 6.4 1.0 7.5 30
Tetractine Unpaired 102.5 202.2 54.9 305.0 5.0 8.0 1.3 10 30Paired 25.0 31.4 3.2 37.5 5.0 6.1 1.2 8.7 30Apical 30.0 50.4 10.4 62.5 2.5 3.9 1.0 5 20
UFRJPOR Triactine Unpaired 75.0 231.6 95.3 435.0 5.0 6.5 1.2 8.7 306746 Paired 17.5 29.6 5.0 40.0 5.0 5.3 0.7 7.5 30
Tetractine Unpaired 80.0 215.0 110.5 370.0 5.0 6.7 1.0 7.5 6Paired 25.0 32.5 8.1 45.0 5.0 5.2 0.5 6.3 6Apical 35.0 58.5 15.5 75.0 2.5 3.3 1.1 5 5
UFRJPOR Triactine Unpaired 102.5 213.5 69.2 375.0 5.0 7.1 1.1 8.7 306767 Paired 20.0 33.4 6.7 50.0 5.0 6.3 1.2 7.5 30
Tetractine Unpaired 100.0 173.7 40.4 255.0 5.0 7.0 1.1 8.7 30Paired 12.5 29.8 7.1 40.0 5.0 5.6 1.0 7.5 30Apical 17.5 37.5 8.9 47.5 2.5 2.7 0.7 5 12
MNRJ 40 Triactine Unpaired 150.0 - - 400 7.0 - - 10 -(original) Paired 30.0 - - 60 - - - - -MNRJ 40(presentwork)
Triactine Unpaired 100.0 229.3 64.2 375.0 6.3 7.4 0.4 7.5 20Paired 32.5 36.9 3.0 42.5 5.0 6.4 1.0 7.5 20
Tetractine Unpaired 145.0 222.3 51.3 315.0 5.0 7.4 0.7 8.7 20Paired 30.0 35.0 5.0 45.0 5.0 7.1 0.8 8.7 20Apical 32.5 32.5 0.0 32.5 5.0 5.6 0.9 6.2 2
Remarks: Borojevic & Peixinho (1976) originally described the skeleton of this species as
being exclusively composed of triactines. Reanalysing the type material, we also found
tetractines, although those spicules were outnumbered by triactines (Figures 2A, B). Because of
the scarce quantity of tetractines in the holotype slide, they might have neglected them.
Moreover, Borojevic & Peixinho (1976) characterised the spicules as parasagittal, pointing out
the straight angle (90°s) formed by the unpaired and paired actines. We do recognize the
referred angle, however, we consider that it would be more correct to characterise these spicules
as sagittal, according to Boury-Esnault & Rützler (1997).
The molecular analysis produced the same tree topology with both phylogenetic methods
(ML and BI) and recovered the lineages found by Klautau et al. (2013) (Figure 3). Nicola tetela
comb. nov. did not cluster with any of the already known genera, not even Arthuria, confirming
that it is a new genus.
30
Figure 2. Photographs taken from the original slide of the holotype (MNRJ 40). A. Apicalregion. B. Basal region. In each photo a tetractine is indicated by an arrow.
Figure 3. Bayesian 50% majority rule consensus tree (106 trees sampled; burn-in =1000 trees)inferred from the ITS rDNA sequences under the GTR model. Bayesian posterior probabilities(BI) and bootstrap (ML) are given on the branches.
31
Discussion
The anastomosis of the cormus is an important taxonomic character in the order Clathrinida. For
example, all species of the genus Ascandra show a different anastomosis, with tubes free at least
at the apical region, while Arthuria, Borojevia, Brattegardia, Clathrina, and Ernstia have well
anastomosed tubes. In the new genus Nicola, tubes are not anastomosed but run in parallel and
are reconnected at the base and at the osculum. Another remarkable morphological character of
Nicola gen. nov. is its skeleton composed exclusively of sagittal spicules, which has not been
found in any other Calcinean genus. The triactines with reduced paired actines present in Nicola
gen. nov. are even similar to the “nail-spicules” found in the Calcaronean genera Kebira and
Grantiopsis (Calcaronea: Lelapiidae), most probably as a result of convergence. Our results
point that spicule shape and composition together with the organisation of the body are very
important characters for the taxonomy of Calcinea. Considering only spicule composition we
would expect to find a closer proximity of Nicola gen. nov. with Arthuria, however, we
observed in our phylogenetic tree that they are not sister genera. This means that the presence of
only sagittal spicules and the differentiated organisation of the cormus in Nicola gen. nov. are
strong characters that define well this new genus as separated from all the others known up to
date.
Acknowledgments
We are indebted to E. Hajdu and G. Lôbo-Hajdu for assistance and photographing during the
sample collections. Mark Vermeij and CARMABI are acknowledged for providing logistical
support in Curaçao. B. C. L. received scholarship from the Brazilian National Research Council
(CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). M.K.
is funded by fellowships and research grants from the CNPq, CAPES, and the Rio de Janeiro
State Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do
Rio de Janeiro - FAPERJ). This paper is part of the DSc. requirements of Báslavi Cóndor Luján
at the Biodiversity and Evolutionary Biology Program of the Federal University of Rio de
Janeiro.
REFERENCES
Arndt, W. (1928) Porifera, Schwämme, Spongien. Tierwelt Deutschlands, 4, 1–94.Arndt, W. (1941) Lebendbeobachtungen an Kiesel-und Hornschwämmen des Berliner
Aquariums. Zoologische Garten (N. F.), 13, 140–166.Azevedo, F., Hajdu, E., Willenz, P. & Klautau, M. (2009) New records of calcareous sponges
(Porifera, Calcarea) from the Chilean coast. Zootaxa, 2072, 1–30.
32
Bidder, G.P. (1898) The skeleton and the classification of calcareous sponges. Proceedings ofthe Royal Society of London, 64, 61–76.
Borojevic, R. & Peixinho, S. (1976) Éponge calcaires du nord-nord-est du Brésil. Bulletin duMuséum National d´Histoire Naturelle, 3(402), 988–1036.
Borojevic, R., Boury-Esnault, N., Manuel, M. & Vacelet, J. (2002) Order Clathrinida Hartman,1958. In: Hooper, J.N.A. & van Soest, R.W.M. (Eds.), Systema Porifera: A Guide to theClassification of Sponges, © Kluwer Academic/Plenum Publishers, New York, pp. 1141–1152
Boury-Esnault, N. & Rützler, K. (1997) Thesaurus of Sponge Morphology. SmithsonianInstitution Press, Washington, D.C., 55 pp.
Bowerbank, J.S. (1864) A monograph of the British Spongiadae. Vol. 1. Ray Society, London,290 pp.
Breitfuss, L. (1896) Kalkschwämme der Bremer-Expedition nach Ost-Spitzbergen.Zoologischer Anzeiger, 19, 426–432.
Breitfuss, L. (1898) Kalkschwammfauna des Westküste Portugals. Zoologische Jahrbücher, 2,89–102.
Breitfuss, L. (1932) Die Kalkschwammfauna des arktischen Gebietes. Fauna Arctica 6, 235–252.
Breitfuss, L. (1935) La Spugne calcarea dell’Adriatico con riflesso a tutto il Mediterraneo.Memorie Reale Comitato Talassographico Italiano, 223, 1–45.
Brøndsted, H.V. (1931) Die Kalkschwämme. Deutschen Südpolar Expedition 1901–3, 20: 1–47.Burton, M. (1930) The Porifera of the Siboga expedition III. Calcarea. In: Weber, M. (Ed.),
Siboga-Expeditie, Leiden: E. J. Brill. 1–18.Dendy, A. & Row, H. (1913) The classification and phylogeny of the calcareous sponges, with a
reference list of all the described species, systematically arranged. Proceedings of theZoological Society of London, 47, 704–813.
Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap.Evolution, 39, 783–791.
Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010) NewAlgorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing thePerformance of PhyML 3.0. Systematic Biology, 59(3), 307–21.
Hartman, W. (1958) A re-examination of Bidder’s classification of the Calcarea. SystematicZoology, 7, 97–110.
Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogeny.Bioinformatics, 17, 754–755.
Haeckel, E. (1872) Die Kalkschwämme, eine Monographie. G. Reimer, Berlin, 418 pp. Hôzawa, S. (1929) Studies on the calcareous sponges of Japan. Journal of the Faculty of
Science of the University of Tokyo, Zoology, 1, 277–389.Imešek, M., Pleše, B., Pfannkuchen, M., Godrijan, J., Pfannkuchen, D.M., Klautau, M. &
Ćetković, H. (2014) Integrative taxonomy of four Clathrina species of the Adriatic Sea,with the first formal description of Clathrina rubra Sarà, 1958. Organisms, Diversity andEvolution, 14, 21–29.
Jenkin, C.F. (1908) The Calcarea of the National Antarctic Expedition. Natural History Reports,4, 182–311.
Katoh, K. & Toh, T. (2008) Recent developments in the MAFFT multiple sequence alignmentprogram. Briefings in Bioinformatics, 9(4), 286–298.
Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7:improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780.
33
Klautau, M. & Valentine, C. (2003) Revision of the Genus Clathrina (Porifera, Calcarea).Zoological Journal of the Linnean Society, 139:1–62.
Klautau, M., Azevedo, F., Cóndor-Luján, B., Rapp H.T., Collins, A & Russo, C. (2013) Amolecular phylogeny for the order Clathrinida rekindles and refines Haeckel’s taxonomicproposal for calcareous sponges. Integrative and Comparative Biology, 53, 447–461.
Lackschewitsch, P. (1886) Über die Kalkschwämme Menorcas. Zoologische Jahrbücher, 1,297–310.
von Lendenfeld, R. (1891) Die Spongien der Adria. I. Die Kalkschwämme. WilhelmEngelmann, Leipzig, 212 pp.
Lôbo-Hajdu, G., Guimarães, A.C.R., Salgado, A. Lamarão, F.R.M., Vieiralves, T., Mansure, J.J.& Albano, R.M. (2004) Intragenomic, Intra – and interspecific variation in the rDNA ITS ofPorifera revealed by PCR-single-strand conformation polymorphism (PCR-SSCP).Bolletino dei Musei e Degli Istitui Biologicci dell´Università di Genova, 68, 413–423.
Miklucho-Maclay, N. (1868) Beiträge zur Kenntniss der Spongien I. Jenaische Zeitschrift fürMedicin und Naturwissenschaft, 4, 221–240.
Minchin, E.A. (1896) Suggestions for a natural classification of the Asconidae. Annals andMagazine of Natural History, 18, 349–362.
Posada, D. & Crandall, K.A. (1998) Modeltest: testing the model of DNA substitution.Bioinformatics, 14(9), 817–818.
Rapp, H.T. (2006) Calcareous sponges of the genera Clathrina and Guancha (Calcinea,Calcarea, Porifera) of Norway (north-east Atlantic) with the description of five new species.Zoological Journal of the Linnean Society, 147, 331–365.
Ronquist, F. & Huelsenback, J.P. (2003) MrBAYES 3: Bayesian phylogenetic inference undermixed models. Bioinformatics, 19, 1572–1574.
Rossi, A., Russo, C.A.M., Solé-Cava, A., Rapp, H. & Klautau, M. (2011) Phylogenetic signal inthe evolution of body colour and spicule skeleton in calcareous sponges. ZoologicalJournal of the Linnean Society, 163, 1026–1034.
Sambrook, J., Fritsch, E.F. & Maniatis, T. (1987) Molecular Cloning: a laboratory manual. 2nd
Edition. Cold Spring Harbor Laboratory Press, New York, 1659 pp .Tanita, S. (1943) Studies on the Calcarea of Japan. Science Reports of the Tohoku Imperial
University, 17, 353–490.Topsent, E. (1936) Étude sur les Leucosolenia. Bulletin de l’Institut Océanographique de
Monaco, 711, 1–47.Vosmaer, G.C. (1881) Vorloopig Berigt omtrent het onderzoek door den ondergeteekende aan
de nederlandsche Werktafel in het Zoölogisch Station te Napels verrigt, 20 Nov. 1880–20Feb. 1881. La Haye.
Vosmaer, G.C. (1887) Klassen und Ordnungen der Spongien (Porifera). In: Bronn, H. G. (Ed.),Die Klassen und Ordnungen des Thierreichs, Leipzig und Heidelberg, 1–496.
34
Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared
species and phylogenetic remarks
Báslavi Cóndor-Luján1, Taynara Louzada1,2, Fernanda Azevedo1, André Padua1, Eduardo Hajdu3
& Michelle Klautau1
1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.
Carlos Chagas Filho, 373, CEP 21941-902, Rio de Janeiro, RJ, Brasil. 2 Universidade Federal do Estado do Rio de Janeiro, Instituto de Biociências, Av. Pasteur, 458,
Urca, CEP 22290-240, Rio de Janeiro, RJ, Brasil.3Universidade Federal do Rio de Janeiro, Museu Nacional, Departamento de Invertebrados,
Quinta da Boa Vista, São Cristóvão, CEP 20940-040, Rio de Janeiro, RJ, Brasil.
Corresponding author: M. Klautau, [email protected]
Journal: Journal of the Marine Biological Association of the United Kingdom.
ABSTRACT
This is the first inventory of calcareous sponges from Curaçao, Caribbean Sea. The
studied material comprised specimens sampled along the Curaçaoan Island as well as some
Brazilian specimens previously collected. They were analysed using morphological and
molecular (ITS and C-LSU) approaches. A total of 18 species were found and are described
here: 11 calcineans and seven calcaroneans. Eleven of these species are new to science; nine
are provisionally endemic to Curaçao, Amphoriscus micropilosus sp. nov., Arthuria vansoesti
sp. nov., Clathrina curaçaoensis sp. nov., Grantessa tumida sp. nov., Leucandra caribea sp. nov.,
Leucandrilla pseudosagittata sp. nov., Leucilla antillana sp. nov., Sycon conulosum sp. nov.,
Sycon magniapicalis sp. nov., and two are shared between Curaçao and Brazil, Ascandra
torquata sp. nov. and Clathrina aspera sp. nov. The formerly Brazilian endemic species C. lutea,
C. insularis, C. mutabilis and Borojevia tenuispinata have their distribution widen to the
Caribbean Sea. Clathrina cf. blanca, C. hondurensis and Leucetta floridana are new records for
Curaçaoan waters. The new phylogenetic affinities in Calcaronea as well as already reported
Calcinean relationships recovered in the molecular analyses are discussed.
Key words: Biodiversity, Calcinea, Calcaronea, Caribbean Sea, Southern Caribbean ecoregion,
Systematics, Western Tropical Atlantic.
35
INTRODUCTION
The Caribbean Sea is considered a global-scale hotspot of marine biodiversity (Roberts et al.,
2002). In its 2,754,000 km2 and over 13,500 km of coastline, it encompasses a high diversity of
flora and fauna distributed in different ecosystems including coral reefs, mangroves, seagrasses
and other environments (Miloslavich et al., 2010).
The sponges (Phylum Porifera) constitute one of the most diverse benthic faunal groups in
the subtidal habitats of coral reefs and mangroves (Diaz & Rützler, 2011), however, the studies
on sponge diversity are mostly restricted to the conspicuous species of the class Demospongiae.
As species of the class Calcarea are usually small and devoid of colour (Wörheide & Hooper,
1999; Rapp, 2006) and inhabit light protected or cryptic environments (e.g. overhangs, caves,
crevices), they are easily neglected in sponge fauna inventories. Furthermore, the plasticity of a
few morphological characters within some Calcarean taxa makes the identification of these
species difficult, sometimes requiring complementary approaches such as molecular analyses
(e.g. Valderrama et al., 2009; Imešek et al., 2014; Azevedo et al., 2015; Klautau et al., 2016).
The class Calcarea comprises species with skeleton composed exclusively of calcium
carbonate. It is divided in two monophyletic subclasses, Calcinea and Calcaronea Bidder, 1898.
Phylogenetic relationships within these two subclasses are still not well-understood as many
orders, families and even genera are polyphyletic (Voigt et al., 2012; Voigt & Wörheide, 2016).
However, certain skeletal traits have evidenced phylogenetic signal in some Calcinean genera
(Rossi et al., 2011; Klautau et al., 2013).
Up to date, only 24 calcareous sponges have been reported from the Caribbean Sea (Cóndor-
Luján & Klautau, 2016; Van Soest et al., 2016;) including representatives of both subclasses. In
a similar geographical range such as the Mediterranean Sea, whose basin comprises 2,969,000
km2 (Coll et al., 2010), the number of recorded calcareous species is more than doubled;
including ca. 65 species (Van Soest et al., 2016).
Among the Caribbean coral reefs, the Curaçaoan reefs are considered some of the healthiest
reefs, harbouring a great diversity of organisms including endemic species (Vermeij, 2012).
Although the sponge diversity within this island has been intensively assessed for many years,
very few calcareous species were reported for Curaçao. The pioneering study of Arndt (1927)
included the first record of the subclass Calcarea, Leucilla amphora Haeckel, 1872. Further
studies compelling shallow-water sponges (Van Soest, 1978, 1980, 1981, 1984; Hajdu & Van
Soest, 1992; Alvarez et al., 1998; De Weerdt, 2000), sponges from cryptic habitats (Van Soest,
2009) and deep-water sponges (Van Soest, 2014) did not report any other Calcarean species,
although one of them did mention the presence of calcareous sponges (Van Soest, 1981). More
36
recently, a former Brazilian endemic species, Nicola tetela (Borojevic & Peixinho, 1976) was
recorded for this island (Cóndor-Luján & Klautau, 2016). This very low number of records may
just reflect the lack of taxomomic expertise in this region, reinforcing the necessity of
faunistical studies on Calcarea.
The present work aimed to start filling the gap on the knowledge of Calcarea from the
Caribbean Sea through the description of the calcareous sponge fauna of Curaçao, integrating
morphological and molecular information. We also discuss the phylogenetic relationships within
Calcarea considering the species described here.
MATERIALS AND METHODS
Abbreviations
BMNH = The Natural History Museum, London, United Kingdom
GW = Gert Wörheide
IRB = Institut Ruđer Bošković, Zagreb, Croatia
MM = Michel Manuel
MNRJ = Museu Nacional do Rio de Janeiro, Brazil
PMJ = Phyletisches Museum Jena, Germany
PMR = Prirodoslovni Muzej Rijeka, Croatia
QM = Queensland Museum, Australia
SAM = South Australian Museum, Australia
UFRJPOR = Porifera collection of the Biology Institute of Universidade Federal do Rio de Ja-
neiro (UFRJ), Brazil
WAMZ = Zoological collection of the Western Australian Museum, Perth, Australia
ZMAPOR = Zoölogisch Museum, Instituut voor Systematiek en Populatiebiologie, Amsterdam,
The Netherlands
Study area: Curaçao
Curaçao is a volcanic island located in the Leeward Antilles Ridge, a boundary zone between
the Caribbean and the South American Tectonic Plates. It was originated in the Cretaceous
Period and received different Miocenic sediment depositions. Its current geology has been
shaped during the Quaternary (Hyppolyte & Mann, 2011).
It is localised in the southern Caribbean Basin, at 60 km north from Venezuela (Figure1A)
and it is included in the Southern Caribbean ecoregion of the Tropical Northwestern Atlantic
37
Province (TNA - Spalding et al., 2007). This island has a total area of 444 km2 and it is
surrounded by a fringing reef with 7.85 km2 situated 20-250 m from the coast (Vermeij, 2012).
Fig. 1. Study area. (A) location of Curaçao in the Caribbean Sea; (B) localities sampled along the coast of Curaçao.
Analysed Material
In this study, a total of 50 specimens were analysed. The material included specimens collected
in Curaçao in 2011 as well as Brazilian specimens already deposited in the Porifera Collection
of the Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR) and that were conspecific
with the Curaçaoan specimens.
In Curaçao, eight localities along the western coast were surveyed (Figure 1B). The
collections were performed by SCUBA down to 20 m of depth. The specimens were
photographed in situ and carefully removed from the substrate with the aid of forceps and small
knives. At the CARMABI (Caribbean Research and Management of Biodiversity) Marine
Research Station, they were fixed in 96% ethanol.
Brazilian specimens were previously collected by SCUBA at depths ranging from four to 15
m, in localities within the southeastern coast (Rio de Janeiro State) and in a southern island
(Arvoredo Island in the Santa Catarina State).
All the samples are preserved in 96% ethanol and deposited in the UFRJPOR Collection.
Morphological analyses
The external morphology was examined through the observation of macroscopic characters on
the fixed specimens and complemented with information from the in situ pictures. The anatomy
was assessed through the analysis of the skeleton composition obtained from microscopy slides.
The preparation of section and spicule slides as well as the spicule measurements followed
38
standard procedures (Wörheide & Hooper, 1999; Klautau & Valentine, 2003; Cóndor-Luján &
Klautau, 2016). The spicule measurements are presented in tabular form, featuring length and
width (minimum, mean, standard deviation [SD] and maximum). Spicule measurements of the
species used for comparative purposes were obtained from the original descriptions or from
more detailed recent descriptions of the type material.
The species identifications followed the Systema Porifera (Hooper & Van Soest, 2002) and
additional literature (Klautau & Valentine, 2003; Klautau et al., 2013; Azevedo et al.,
submitted).
To illustrate the species descriptions, photographs were taken with a digital Canon camera
coupled to a Zeiss Axioscop microscope. Scanning electron microscopy (SEM) micrographs of
particular spicule ornamentations were taken at the Biology Institute of the UFRJ using a JSM-
6510 SEM microscope. The spicule preparation for SEM images followed Azevedo et al.
(2015).
Molecular analyses
The total genomic DNA was extracted using the guanidine/phenol-chloroform protocol
(Sambrook et al., 1989) or with a QIAamp DNA MiniKit (Qiagen) and stored at –20°C until
amplification. Two DNA regions were amplified. The C-region of the 28S (C-LSU) was
amplified using the primers fwd: 5'GAAAAGCACTTTGAAAAGAGA-3' (Voigt & Worheide,
2015) and rv: 5'-TCCGTGTTTCAAGACGGG-3' (Chombard et al., 1998) and the region
containing the partial genes 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal
DNA (named herein as ITS) was amplified with the primers: fwd: 5`-
TCATTTAGAGGAAGTAAAAGTCG-3` and rv: 5`-GTTAGTTTCTTTTCCTCCGCTT-3`)
(Lôbo-Hajdu et al., 2004). The ITS region was only amplified for calcinean species.
The PCR mixture included 1x buffer (5x GoTaq® Green Reaction Buffer Flexi,
PROMEGA), 0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM
of each primer, one unit of Taq DNA polymerase (Fermentas or PROMEGA) and 1 µL of DNA
in a volume of 15 µL. The PCR amplification comprised one first cycle of 4 min at 94°C, 1 min
at 50°C and 1 min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 48°C, 50°C or 52 °C and one
minute at 72°C, and a final cycle of 6 min at 72°C. Forward and reverse strands were
automatically sequenced in an ABI 3500 (Applied Biosystems) at the Biology Institute (UFRJ).
Sequences used in recent phylogenies (Klautau et al., 2013; Klautau et al., 2016; Voigt &
Wörheide, 2016) were retrieved from the Genbank database and are listed in Table 1 as well as
the ones generated in this study. These sequences were aligned through the MAFFT v.7 online
39
platform (Katoh & Standley, 2013) using the strategy Q-INS-i (Katoh & Toh, 2008) which
provides a better alignment as it considers the secondary structure of the amplified region. The
nucleotide substitution model that best fit the alignment was GTR+G+I for both DNA regions,
as indicated by the Bayesian Information Criterion in MEGA 6 (Nei & Kumar, 2000; Tamura et
al., 2013).
Phylogenetic reconstructions were performed under Maximum Likelihood (ML) and
Bayesian Inference (BI) approaches. The ML analyses were conducted on MEGA 6 using an
initial NJ tree (BIONJ) and a 1000 pseudo-replicates bootstrap. The BI reconstructions were
obtained with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003)
under 3.5 x 106 generations and a burn-in of 3,500 sampled trees, yielding a consensus tree of
majority.
Table 1. Species used in the phylogenetic analyses with locality, voucher number and GenBank(GB) accession number. *Sequences generated in the present study.
Species Locality Voucher number GB accession numberCALCINEA LSU ITSAscandra contorta Mediterranean UFRJPOR6327 - HQ588970Ascandra coralicolla Norway UFRJPOR6329 - HQ588994Ascandra falcata Mediterranean UFRJPOR5856 - HQ588962Ascandra sp. Polynesia BMOO16290 - KC843446Ascandra spalatensis Adriatic Sea UFRJPOR7540 - KP740024Ascandra torquata sp. nov.* Brazil UFRJPOR6080 - This studyAscandra torquata sp. nov. Brazil UFRJPOR6084 - KC843448Ascandra torquata sp. nov.* Curaçao UFRJPOR6738 - This studyBorojevia aff. aspina Brazil UFRJPOR5245 - HQ588998Borojevia aff. aspina Brazil UFRJPOR5495 HQ589017 -Borojevia brasiliensis Brazil UFRJPOR5214 HQ589015 HQ588978Borojevia cerebrum Mediterranean UFRJPOR6322 HQ589008 HQ588964Borojevia croatica Adriatic Sea IRB-CLB6 - KP740023Borojevia sp. QMG313824 JQ272287 -Borojevia tenuispinata Brazil UFRJPOR6484 - KX548916Borojevia tenuispinata Brazil UFRJPOR6492 - KX548917Borojevia tenuispinata* Curaçao UFRJPOR6700 This study This studyBorojevia trispinata Brazil UFRJPOR6487 - KX548919Clathrina aspera* Brazil UFRJPOR5531(P) - This studyClathrina aspera* Brazil UFRJPOR6346 This studyClathrina aspera* Brazil UFRJPOR6472 This studyClathrina aspera* Brazil UFRJPOR6510 This studyClathrina aspera* Curaçao UFRJPOR 6758 This study -Clathrina aurea Brazil MNRJ 8998 HQ589005 HQ588968Clathrina clathrus Mediterranean UFRJPOR6315 HQ589009 HQ588974
Clathrina conifera Brazil UFRJPOR8991 HQ589010 HQ588959Clathrina coriacea Norway UFRJPOR6330 HQ589001 HQ588986
40
Clathrina curaçaoensis sp. nov.*
Curaçao UFRJPOR6734 This study This study
Clathrina cylindractina Brazil UFRJPOR 5206 HQ589007 HQ588979Clathrina fjordica Chile MNRJ8143 HQ588984Clathrina fjordica Chile MNRJ9964 HQ589016 -Clathrina helveola Australia QMG313680 JQ272291 HQ588988 Clathrina insularis Brazil UFRJPOR6527 - KX548920Clathrina insularis* Brazil UFRJPOR6530 - This studyClathrina insularis Brazil UFRJPOR6532 - KX548921Clathrina insularis* Brazil UFRJPOR6533 - This studyClathrina insularis Brazil UFRJPOR6536 - KX548922Clathrina insularis* Brazil UFRJPOR6537 - This studyClathrina insularis* Curaçao UFRJPOR6737 This study KC843435Clathrina lutea Brazil UFRJPOR5172 HQ589004 HQ588961Clathrina lutea Brazil UFRJPOR5173 - HQ588976Clathrina lutea Brazil UFRJPOR 6543 - KX548923Clathrina lutea Brazil UFRJPOR 6545 - KC843442Clathrina lutea* Curaçao UFRJPOR6761 - KC843445Clathrina lutea Virgin Islands ZMAPOR08344 - KC843444Clathrina lutea Florida, USA UFRJPOR5818 - KC843443Clathrina luteoculcitella Australia QMG313684 JQ272283 HQ588989Clathrina mutabilis* Brazil UFRJPOR6525 - This studyClathrina mutabilis Brazil UFRJPOR6526 - KX548925Clathrina mutabilis Brazil UFRJPOR6528 - KX548926Clathrina mutabilis* Brazil UFRJPOR6539 - This studyClathrina mutabilis* Brazil UFRJPOR6540 - This studyClathrina mutabilis* Curaçao UFRJPOR6704 - This studyClathrina mutabilis* Curaçao UFRJPOR6719 - This studyClathrina mutabilis Curaçao UFRJPOR6733 - KC843436Clathrina mutabilis* Curaçao UFRJPOR 6735 - This studyClathrina mutabilis* Curaçao UFRJPOR6740 - This studyClathrina mutabilis* Curaçao UFRJPOR6741 This study KC843437Clathrina mutabilis* Curaçao UFRJPOR6743 - This studyClathrina mutabilis* Curaçao UFRJPOR6744 - This studyClathrina mutabilis* Curaçao UFRJPOR6745 - This studyClathrina mutabilis* Curaçao UFRJPOR6747 - This studyClathrina sp. Polynesia UF:Porifera:1600 - KC843438Clathrina sp. Polynesia UFRJPOR6461 - KC843439Clathrina wistariensis Australia QMG313663 JQ272303 HQ588987Clathrina zelinhae* Brazil UFRJPOR 6627 This study -Leucetta chagosensis - BMOO16210 -- -Leucetta floridana Panama PTL09.P100 - KC843456Leucetta floridana* Curaçao UFRJPOR6726 - This studyLeucetta floridana* Curaçao UFRJPOR6765 - This studyLeucetta antarctica Antarctic MNRJ13798 - KC849700Leucetta microraphis Australia QMG313659 - AJ633874Leucetta pyriformis Antarctic MNRJ13843 - KC843457Leucetta potiguar Brazil UFPEPOR569 - EU781987Nicola tetela Curaçao UFRJPOR6723 KU568492 -
41
CALCARONEA LSUAmphoriscus micropilosus sp. nov. * Curaçao UFRJPOR6755(P) This study -Anamixilla torresi - - AY563636 -Aphroceras sp. SAM-PS0349 JQ272273 -Eilhardia schulzei QMG316071 JQ272256 -Grantessa tumida sp. nov.* Curaçao UFRJPOR6701(P) This study -Grantessa tumida sp. nov.* Curaçao UFRJPOR6695(P) This study -Grantessa aff. intusarticulata
GW979 JQ272278 -
Grantia compressa - - AY563538 -Grantiopsis heroni Australia QMG313670 JQ272261 -Grantiopsis cylindrica Australia GW973 JQ272261 -Leucandra aspera - - AY563535 -Leucandra falakra Adriatic Sea UFRJPOR8349 KT447560 -Leucandra nicolae QMG313672 JQ272268 -Leucandra sp. QMG316285 JQ272265 -Leucandra spinifera Adriatic Sea UFRJPOR8348 KT447561Leucandrilla pseudosagittata sp. nov.* Curaçao UFRJPOR6696 This study -L. pseudosagittata sp. nov.* Curaçao UFRJPOR6705 This study -Leucascandra caveolata QMG316057 JQ272259 -Leucilla antillana sp. nov.* Curaçao UFRJPOR6768 This study -Leuconia nivea - - AY563534 -Paraleucilla dalmatica Adriatic Sea UFRJPOR8346 KT447566 -Paraleucilla magna - GW824 KT447564 -Petrobiona massiliana - GW1729 JQ272307 -Sycettusa aff. hastifera Red Sea GW893 JQ272267 -Sycettusa cf. simplex Western India ZMAPOR11566 JQ272279 -Sycettusa sp. - MM-2004 AY563530 -Sycettusa tenuis Australia QMG313685 JQ272281 -Sycon ancora Adriatic Sea UFRJPOR8347 KT447568 -Sycon capricorn - QMG316187 JQ272272 -Sycon carteri Australia SAM-PS0143 JQ272260 -Sycon ciliatum - - AY563532 -Sycon conulosum sp. nov.* Curaçao UFRJPOR 6707 This study -Sycon cf. villosum - GW51115 KR052809 -Sycon magniapicalis sp. nov.*
Curaçao UFRJPOR6748 This study -
Sycon magniapicalis sp. nov.*
Curaçao UFRJPOR6763 This study -
Sycon raphanus - - AY563537 -Syconessa panicula Australia QMG313672 AM181007 -Synute pulchella - WAMZ1404 JQ272274 -Teichonopsis labyrinthica - SAMPS0228 JQ272264 -Utte aff. syconoides - QMG323233 JQ272269 -Utte aff. syconoides - QMG313694 JQ272271 -Ute ampullacea - QMG313669 JQ272266 -Vosmaeropsis sp. - MM-2004 AY026372 -
42
RESULTS
Taxonomy
Integrating traditional morphological examination with molecular phylogenetic analyses, we
recognised 18 species including 11 calcineans and seven calcaroneans. Within the subclass
Calcinea, the richest genus was Clathrina with seven species: C. aspera sp. nov., Clathrina cf.
blanca, C. curaçaoensis sp. nov., C. hondurensis, C. insularis, C. lutea, and C. mutabilis. In
Calcaronea, Sycon was the richest genera with two species: Sycon conulosum sp. nov and Sycon
magnapicalis sp. nov. The calcinean genera Ascandra and Borojevia as well as the calcaronean
Leucandrilla and Grantessa constitute not only new records for Curaçao, but also for the
Caribbean Sea.
Systematic Index
Class CALCAREA Bowerbank, 1862
Subclass CALCINEA Bidder, 1898
Order CLATHRINIDA Hartman, 1958
Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013
Arthuria vansoesti sp. nov.
Genus Ascandra Haeckel, 1872
Ascandra torquata sp. nov.
Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013
Borojevia tenuispinata Azevedo et al., submitted
Genus Clathrina Gray, 1867 sensu Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo,
2013
Clathrina aspera sp. nov.
Clathrina cf. blanca Miklucho-Maclay, 1868
Clathrina curaçaoensis sp. nov.
Clathrina hondurensis Klautau & Valentine, 2003
Clathrina insularis Azevedo et al., submitted
Clathrina lutea Azevedo et al., submitted
Clathrina mutabilis Azevedo et al., submitted
Genus Leucetta Haeckel, 1872
Leucetta floridana Haeckel, 1872
Subclass CALCARONEA Bidder, 1898
Order LEUCOSOLENIIDA Hartman, 1958
43
Family AMPHORISCIDAE Dendy, 1893
Genus Amphoriscus Haeckel, 1872
Amphoriscus micropilosus sp. nov.
Genus Leucilla Haeckel, 1872
Leucilla antillana sp. nov.
Family GRANTIIDAE Dendy, 1893
Genus Leucandra Haeckel, 1872
Leucandra caribea sp. nov.
Genus Leucandrilla Borojevic, Boury-Esnault & Vacelet, 2000
Leucandrilla pseudosagittata sp. nov.
Family HETEROPIIDAE Dendy, 1893
Genus Grantessa Lendenfeld, 1885
Grantessa tumida sp. nov.
Family SYCETTIDAE Dendy, 1892
Genus Sycon Risso, 1827
Sycon conulosum sp. nov.
Sycon magniapicalis sp. nov.
Description of taxa
Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013
TYPE SPECIES
Arthuria hirsuta (Klautau & Valentine, 2003).
DIAGNOSIS
"Calcinea in which the cormus comprises a typical clathroid body. A stalk may be present. The
skeleton contains regular (equiangular and equiradiate) triactines and tetractines. However,
tetractines are more rare. Diactines may be added. Asconoid aquiferous system" (Klautau et al.
2013).
Arthuria vansoesti sp. nov. (Figure 2, Table 2)
ETIMOLOGY
Named after Rob van Soest in recognition of his dedicated work on the taxonomy of the
sponges, including those from Curaçao.
44
TYPE LOCALITY
Daai Booi, St. Willibrordus, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6720 (specimen in ethanol and slides); Daai Booi, St. Willibrordus;
12°12'43.12"N, 69°05'8.42''W; 5.2 m deep; coll. B. Cóndor-Luján, 19 August 2011.
COLOUR
Yellow in life and white to beige in ethanol.
MORPHOLOGY AND ANATOMY
This species has a massive and smooth cormus. The holotype is 0.7 x 0.6 x 0.2 cm (Figures 2A-
B). The cormus is composed of irregular and loosely anastomosed tubes. A water-collecting tube
(2 x 1 mm) was present in the centre of the cormus of the holotype (arrow in Figure 2A). The
aquiferous system is asconoid. The skeleton has no special organization (Figure 2C) and it is
composed of abundant triactines (two shape categories) and rare tetractines.
SPICULES
Triactines I. Regular (equiangular and equiradiate). Most abundant spicules. Actines are
cylindrical, slightly undulated at the distal part and with rounded tips (Figure 2D). They
resemble the triactines of Clathrina aurea. Size: 72.5-87.5/3.8-5 µm.
Triactines II. Regular (equiangular and equiradiate). Rare. Actines are straight, slightly conical
with blunt to sharp tips (Figure 2E). Size: 52.5-82.5/2.5-5 µm.
Tetractines. Regular (equiangular and equiradiate). Basal actines are straight, cylindrical, with
rounded to blunt tips (Figure 2F). The apical actine is the shortest actine. It is straight and
smooth; however, some curved actines were found (Figure 2G). It has sharp or blunt tip. Size:
60-87.5/3.8-5 µm (basal actine) and 25/3.8-5 µm (apical actine).
ECOLOGY
This sponge was found in a cryptic habitat, underneath coral boulders.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
The genus Arthuria now comprises 11 valid species, A. africana (Klautau & Valentine, 2003),
A. alcatraziensis (Lanna et al., 2007); A. darwinii (Haeckel, 1870); A. dubia (Dendy, 1891); A.
hirsuta (Klautau & Valentine, 2003); A. spirallata Azevedo et al., 2015; A. sueziana (Klautau &
Valentine, 2003); A. tenuipilosa (Dendy, 1905); A. trindadensis Azevedo et al., submitted, A. tu-
buloreticulosa Van Soest & de Voogd, 2015 and A. vansoesti sp. nov. Most of them possess a
skeleton mainly composed of triactines with conical actines except for A. dubia, A. sueziana, A.
45
tubuloreticulata and A. vansoesti sp. nov. whose skeletons also include triactines with
cylindrical actines.
Arthuria vansoesti sp. nov. can be easily distinguished from A. dubia and A. sueziana
because they have different spicule width (Table 2), being thinner in the new species (2.5–5.0
µm) than in A. dubia (13.7 – 17.0 µm) and A. sueziana (8.0 – 11.8 µm). Besides, C. dubia has
granular cells which are absent in the new species. The species whose skeleton more resembles
A. vansoesti sp. nov. is the Indonesian A. tubuloreticulata, however, they present some
differences. A. tuboloreticulata is orange in live and its cormus has several oscula whereas the
new species is yellow in life and presents water-collecting tubes. Moreover, the spicules of the
Curaçaoan species have rounded tips and in the Indonesian species, they are sharp or blunt.
Table 2. Spicule measurements of Arthuria vansoesti sp. nov. (UFRJPOR 6720), A. dubia(BMNH 1891.9.19.2) taken from Klautau & Valentine (2003), A. sueziana (BMNH 1912.2.1.3)
and A. tuboreticulata (RMNH Por. 5547). H=holotype, L=lectotype.. B=basal and A=apicalactines.
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6720 (H)
Triactine I 72.5 79.8 4.9 87.5 3.8 4.6 0.6 5.0 30Triactine II 52.5 70.4 7.8 82.5 2.5 3.9 0.8 5.0 30Tetractine B 60.0 75.3 8.1 87.5 3.8 4.8 0.5 5.0 15
A 25.0 25.0 0.0 25.0 3.8 4.4 0.7 5.0 6BMNH 1891.9.19.2 (L)*
Diactine 77.5 256.0 97.0 418.2 - 13.7 5.0 - 30Triactine 92.5 151.5 14.0 170.0 - 15.5 1.8 - 30Tetractine B 120.0 140.3 9.5 155.0 - 16.0 1.0 - 9
A 100.0 110.0 6.8 117.5 - 9.8 0.5 -BMNH 1912.2.1.3 (H)
Trichoxea 250.0 - - - <0.3 - - -Triactine 75.0 91.3 10.9 137.5 - 10.3 1.5 - 30Tetractine B 70.0 86.0 6.1 97.5 - 9.4 1.4 - 30
A 50.0 56.3 4.5 62.5 - 5.0 0.0 - 4RMNH Por. 5547 (H)
Triactine 63 112.1 - 138 4 5.3 - 6.5 -Tetractine B 62 119.5 - 156 4 5.4 - 6 -
A 39 - - 132 3.5 - - 6.5 -
46
Fig. 2. Arthuria vansoesti sp. nov. (UFRJPOR 6720): (A) specimen in vivo; (B) specimen afterfixation; (C) tangential section of the body (cormus); (D) triactine I; (E) triactine II; (F)tetractine; (G) apical actine of tetractine.
47
Genus Ascandra Haeckel, 1872
TYPE SPECIES
Ascandra falcata Haeckel 1872
DIAGNOSIS
" Calcinea with loosely anastomosed tubes. Tubes are free, at least in the apical region. The
skeleton contains regular (equiangular and equiradiate) or sagittal triactines and tetractines. The
apical actine is very thin (needle-like) or very thick at the base. Diactines may be added.
Asconoid aquiferous system" (Klautau et al., 2016, emend).
Ascandra torquata sp. nov. (Figures 3 & 4, Table 3)
ETIMOLOGY
From the Latin torquere (= twist), for the twisted tip of the apical actine of the tetractines.
TYPE LOCALITY
Ponta do Vidal, Arvoredo Island, Reserva Biológica Marinha (REBIOMAR) do Arvoredo, Santa
Catarina, Brazil.
TYPE MATERIAL
Holotype (specimen in ethanol and slides) UFRJPOR 6084, Ponta do Vidal, REBIOMAR
Arvoredo, Santa Catarina, Brazil; 27°17’52.3’’S, 48°21’33.4’’W; 10-15 m deep; coll. F.
Azevedo, J. Carraro and A. Padua, 11 December 2009.
Paratypes (specimens in ethanol and slides) UFRJPOR 6080 and UFRJPOR 6082, Ponta do
Vidal, REBIOMAR Arvoredo, Santa Catarina, Brazil; 27°17'52.3"S, 48°21'33.4"W; 10-15 m
deep; coll. F. Azevedo, J. Carraro and A. Padua, 11 December 2009. UFRJPOR 6738, Playa
Jeremi, Soto, Curaçao; 12°19'43.73"N, 69°09'07.80"W; 15 m deep; coll. B. Cóndor-Luján and
E. Hajdu, 22 August 2011.
COLOUR
White in life and beige to light brown in ethanol.
MORPHOLOGY AND ANATOMY
The holotype (UFRJPOR 6084) measures 4.0 x 1.5 x 1.5 cm (Figure 3A). Cormus varying from
encrusting to massive, fragile in consistency and composed of irregular and loosely
anastomosed tubes at the base and free at the apical region. In fact, at the apical region, there is
no anastomosis as large vertical tubes arise and connect themselves culminating in oscula with
variable diameters (1-3 mm, Figure 3B). At the base, the surface is smooth while at the apical
region, it is very hispid due to diactines protruding the surface. Few diactines were also found in
the basal region of some specimens. The aquiferous system is asconoid. The skeleton has no
48
special organization and it is composed of abundant tetractines differentiated in two size
categories and of less frequent triactines and diactines (Figure 3C-D).
The specimen from Curaçao (UFRJPOR 6738, Figure 4A) is partitioned as shown in Figure 4B.
The largest fragment is 5 mm long and the free tubes can reach 5 mm high. In this specimen,
diactines were not observed (Figure 4C).
SPICULES (Table 3)
Diactines. Slightly sinuous with sharp tips (Figure 3E). Very variable size (length/width): 100.0-
395.0/5.0-17.5 µm.
Triactines. Regular (equiangular and equiradiate). Actines are conical, straight, with blunt to
sharp tips (Figures 3F-G and 4D). Subregular triactines were also found. Size (length/width):
45.0-197.5/7.5-15.0 µm.
Tetractines I. Regular (equiangular and equiradiate). The basal actines are slightly conical,
straight, with blunt to sharp tips (Figures 3H and 4E). The apical actine is smooth and conical.
Most apical actines are characteristically twisted, however, some straight ones with sharp tips
were also observed. Size (length/width): 57.0-225.0/5.0-22.5 µm (basal actine) and 50.0-
167.5/6.2-14.0 µm (apical actine).
Tetractines II. Regular (equiangular and equiradiate). Larger than tetractines I. The basal actines
are conical, straight, with blunt to sharp tips (Figure 3I and 4F). The apical actine is thinner than
the basal ones. It is smooth, straight, conical, with sharp tips. Some twisted apical actines were
also observed (Figure 3J). Size (length/width): 170.0-487.5/10.0-37.5 µm (basal actine) and
52.5-205.0/12.5-32.5 µm (apical actine).
ECOLOGY
Specimens inhabited sunlight protected environments. Brazilian specimens were collected in
vertical walls of large boulders while the Curaçaoan specimen was found underneath small
boulders. Amphipods, bryozoans, ophiuroids, polychaets and hydrozoans were found living in
association with the Brazilian specimens.
GEOGRAPHIC DISTRIBUTION
Disjunct distribution in the Western Atlantic: Southern Caribbean (Curaçao) and Southern Brazil
ecoregion of the Warm Temperate Southwestern Atlantic Province (Santa Catarina State).
REMARKS
Ascandra is currently composed of 15 species. Ascandra torquata sp. nov. can be easily distin-
guished from all them by the twisted apical actine of the tetractines which is absent in all the
other 14 known species. This new species is the third Ascandra registered for the Atlantic Ocean
49
- the other two are A. ascandroides (Borojević, 1971) from Brazil and A. corallicola (Rapp,
2006) from Norway.
A previous study evidenced that trichoxeas were not good taxonomic characters for
identifying Clathrina species as they could be present or not in C. mutabilis (Azevedo et al.,
submitted). The same seems to be valid for the diactines of A. torquata sp. nov. as they were
present in the specimens from Brazil but not in the one from Curaçao. Despite having this
morphological difference, the molecular analysis confirmed their conspecificity (Figure 31).
Therefore, at least for A. torquata sp. nov., diactines are not a reliable taxonomic character.
The clade of Ascandra was well supported in our ITS phylogenetic tree (pp=1, b=99, Figure
31), confirming the validity of this genus with the present diagnosis. In that tree, A. torquata sp.
nov. is a sister species of the type species of the genus A. falcata and of A corallicola. The ITS
sequence of A. torquata sp. nov. (UFRJPOR 6084) had already been published by Klautau et al.
(2013) as Clathrina sp. nov. 11.
Table 3. Spicule measurements of Ascandra torquata sp. nov. H=holotype; P=paratype.
Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR6084 (H)
Diactine - 100.0 208.9 90.7 382.5 7.5 12.0 3.0 17.5 30Triactine - 45.0 138.5 40.4 197.5 7.5 11.2 1.9 15 30Tetractine I basal 120.0 163.2 22.5 212.5 12.5 15.2 1.4 17.5 30
apical 72.5 112.0 30.8 167.5 7.5 8.8 1.7 14 30Tetractine II basal 195.0 256.1 38.8 337.5 10 26.6 6.6 37.5 22
apical 52.5 101.2 31.8 150.0 12.5 19.6 2.8 22.5 12UFRJPOR6080 (P)
Diactine - 115 245.5 53.5 335.0 5 10.8 2.4 15 30Triactine - 67.5 116.5 24.2 157.5 7.5 9.8 1.5 12.5 30Tetractine I basal 57.5 124.5 28.4 165.0 5 12.7 3.5 22.5 30
apical 62.5 112.2 26.5 162.5 7.5 9.6 1.8 12.5 25Tetractine II basal 207.5 360.7 81.6 487.5 21.2 32.1 6.3 32.1 30
apical 80.0 139.9 32.3 190.0 17.5 25.2 3.5 32.5 30UFRJPOR6082 (P)
Diactine - 142.5 255.8 73.1 395.0 5.0 14.3 2.8 15.0 17Triactine - 97.5 150.3 23.9 192.5 7.5 12.3 2.1 12.5 30Tetractine I basal 100.0 159.8 27.9 225.0 5.0 13.7 2.5 22.5 30
apical 65.0 118.5 19.9 150.0 7.5 10.5 2.2 12.5 30Tetractine II basal 170.0 272.5 47.7 375.0 21.3 29.6 3.8 45.0 30
apical 87.5 141.8 30.6 205.0 17.5 25.7 2.8 32.5 30UFRJPOR6738 (P)
Triactine - 75.0 116.4 20.0 150.0 8.8 11.6 1.6 15.0 30Tetractine I basal 85.0 122.5 22.0 192.5 10.0 12.4 2.1 17.5 30
apical 50.0 66.5 25.3 107.5 6.2 7.8 1.4 10.0 5Tetractine II basal 205.0 261.8 36.7 325.0 25 27.3 2.7 35 20
apical - 125.0 - - - 15.0 - - 1
50
Fig. 3. Holotype of Ascandra torquata sp. nov. (UFRJPOR 6084). (A-B) specimens afterfixation; (C-D) tangential section of the body indicating diactines (white arrows); (E) diactine;(F-G) triactines; (H) tetractine I; (I) tetractine II; (J); apical actines of tetractines I.
51
Fig. 4. Paratype of Ascandra torquata sp. nov. (UFRJPOR 6738). (A) specimen in vivo; (B)specimen after fixation; (C) tangential section of the body (cormus); (D) triactine; (E) tetractineI; (F) tetractine II.
52
Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013
TYPE SPECIES
Borojevia cerebrum (Haeckel, 1872)
DIAGNOSIS
"Calcinea in which the cormus comprises tightly anastomosed tubes. The skeleton contains
regular (equiangular and equiradiate) triactines, tetractines, and tripods. The apical actine of the
tetractines has spines. Aquiferous system asconoid" (Klautau et al., 2013).
Borojevia tenuispinata Azevedo et al., submitted (Figure 5, Table 4)
SYNONYMS
Borojevia tenuispinata: Azevedo et al., submitted
TYPE LOCALITY
Cabeço da Tartaruga, São Pedro e São Paulo Archipelago, Brazil.
MATERIAL EXAMINED
UFRJPOR 6700 and UFRJPOR 6708 (specimens in ethanol and slides); Daai Booi, St.
Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18
August 2011.
COMPARATIVE MATERIAL EXAMINED
Borojevia tenuispinata. Holotype (specimen in ethanol and slides) UFRJPOR 6484; Cabeço da
Tartaruga, São Pedro e São Paulo Archipelago, Brazil; 0o54'57''N, 29o20'46''W; coll. G.
Rodríguez & F. Azevedo, 16 June 2011.
COLOUR
White in life and ethanol.
MORPHOLOGY AND ANATOMY
The largest specimen (UFRJPOR 6700) measures 0.7 x 0.4 x 0.3 mm. Cormus massive, rough
and compressible, composed of irregular and (mostly) tightly anastomosed tubes (Figure 5A).
The tubes in contact with the substrate are less tightly anastomosed than the most superficial
ones. The aquiferous system is asconoid. No water-collecting tubes were observed. The skeleton
has no special organization and it comprises triactines, tripods and tetractines (Figure 5B).
SPICULES
Triactines. Regular (equiangular and equiradiate). Actines are straight, conical, with blunt tips
(Figure 5C). They are the most abundant spicules. Size: 62.5-100/6.3-11.3µm.
53
Tripods. Regular (equiangular and equiradiate). Actines are straight, conical, with blunt tips.
They do not have the raised centre of typical tripods, instead they look like stout conical
triactines (Figure 5D). Size: 72.5-162.5/12.5-22.5 µm.
Tetractines. Regular (equiangular and equiradiate). The basal actines are straight, conical, with
blunt tips (Figure 5E). The apical actine is shorter and thinner than the basal ones and present
spines. The most common pattern of spine distribution observed consisted of spines arranged in
rows spreading from about two-thirds of the actine up to the tip (Figure 5F). Some few
tetractines with curved paired actines were also found. Size: 62.5-95.0/7.5-11.3 µm (basal
actine), 25-40/5-7.5 µm (apical actine).
ECOLOGY
This sponge was found in a cryptic habitat, underneath boulders.
GEOGRAPHIC DISTRIBUTION
São Pedro and São Paulo Islands ecoregion of the Tropical Southwestern Atlantic (São Pedro e
São Paulo Archipelago, Azevedo et al., submitted) and Southern Caribbean (Curaçao, this
study).
REMARKS
The genus Borojevia comprises eight species. Among them, six have a skeleton composition
(one category of tripods, triactines and tetractines) similar to the specimens from Curaçao.
These species are the Brazilians B. aspina (Klautau et al., 1994), B. brasiliensis (Solé-Cava et
al., 1991), B. tenuispinata and B. trispinata Azevedo et al., submitted, B. cerebrum (Haeckel,
1872) from the Mediterranean Sea and B. croatica Klautau et al., 2016 from the Adriatic Sea.
However, only B. brasiliensis, B. croatica and B. tenuispinata, have tetractines with apical
actines bearing a spine distribution pattern similar to the Curaçaoan specimens and share
comparable spicule size range (Table 4). Different from the species B. brasiliensis and B.
croatica, whose skeletons present more triactines than tetractines, in B. tenuispinata and in the
specimens from Curaçao, triactines and tetractines occur in the same proportion. The only
difference observed between the Caribbean specimens and the holotype of B. tenuispinata is
that the spicules of the former can attain larger sizes. This difference, however, can be perhaps
attributed to plasticity. Besides, in the ITS phylogenetic tree (Figure 31), the specimen
UFRJPOR 6700 clustered within the clade of B. tenuispinata (pp=0.98, b=100), indicating its
co-specificity.
54
Table 4. Spicule measurements of Borojevia tenuispinata from Curaçao (UFRJPOR6700 andUFRJPOR6708) and of the holotype (UFRJPOR 8464), B. brasiliensis (MNHN-LBIM.C.
1989.2) taken from Klautau & Valentine (2003) and B. croatica (UFRJPOR6865). H=holotype.
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6700
Triactine 62.5 79.5 9.7 100.0 6.3 8.8 1.6 11.3 36Tripod 87.5 109.4 16.6 162.5 12.5 13.9 2.9 22.5 27Tetractine basal 67.5 78.8 7.1 92.5 7.5 8.5 1.0 10.0 30
apical 25.0 33.6 4.5 40.0 5.0 7.2 0.8 7.5 9UFRJPOR6708
Triactine 62.5 75.3 5.9 85.0 7.5 9.8 1.1 11.3 30Tripod 72.5 99.5 12.4 125.0 12.5 13.4 1.1 15.0 30Tetractine basal 62.5 75.7 7.9 95.0 7.5 9.3 1.2 11.3 30
apical 25.0 - - - - 7.5 - - 1UFRJPOR8464 (H)
Triactine 59.4 65.3 4.9 75.6 5.4 7.2 0.7 8.1 30Tripod 56.7 80.9 11.9 102.6 8.1 10.1 1.4 12.2 30Tetractine basal 51.3 64.1 5.3 72.9 5.4 7.2 0.8 8.1 30
apical 27.0 40.0 8.6 56.7 4.1 4.9 0.8 6.8 20MNHN-LBIM.C. 1989.2 (H)
Triactine 60.9 78.2 10.6 102.2 - 10.8 1.5 - 20Tripod 67 81 8.2 95.7 - 11 1.7 - 20Tetractine basal 56.5 75.3 10.0 91.3 - 10.4 1.3 - 20
apical 17.4 36.4 9.1 50.0 - 8-0 2.2 - 20UFRJPOR6865 (H)
Triactine 57.5 66.6 6.7 82.5 7.5 7.5 0.0 7.5 20Tripod 85.0 102.6 10.0 115.0 10.0 11.9 1.5 15.0 20Tetractine basal 60.0 70.0 6.3 77.5 7.5 8.3 1.2 11.3 10
apical - 20 . . . 5.0 - - 1
55
Fig. 5. Borojevia tenuispinata (UFRJPOR 6700): (A) specimen after fixation; (B) tangentialsection of the body (cormus); (C) triactine; (D) tripod; (E) tetractine; (F) detail of the spinedapical actine of a tetractine.
56
Genus Clathrina Gray, 1867
TYPE SPECIES
Clathrina clathrus (Schmidt, 1864)
DIAGNOSIS
"Calcinea in which the cormus comprises anastomosed tubes. A stalk may be present. The
skeleton ontains regular (equiangular and equiradiate) and/or parasagittal triactines, to which
diactines and tripods may be added. Asconoid aquiferous system" (Klautau et al., 2013).
Clathrina aspera sp. nov. (Figures 6 & 7, Table 5)
ETIMOLOGY
From the Latin asper (= rough), after the species rough surface.
TYPE LOCALITY
Water Factory, Willemstadt, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6758 (specimen in ethanol and slides); Water Factory, Willemstadt,
Curaçao; 12°06'30.88"N, 68°57'13.53"W; 13.2 m deep; coll. B. Cóndor-Luján and E. Hajdu, 23
August 2011.
Paratypes: UFRJPOR 5487 (specimen in ethanol and slides); Ilhas Botinas, Angra dos Reis, Rio
de Janeiro, Brazil; 23º03'19.36''S, 44º19'44.98''W; 1-3 m deep; coll. F. Azevedo & M. Klautau,
25 May 2007 and UFRJPOR 5531 (specimen in ethanol and slides); Praia do Bonfim, Angra dos
Reis, Rio de Janeiro, Brazil; 23°01'14.26''S, 44°19'48.18''W; 1-2 m deep; coll. M. Klautau, 27
May 2007.
ADDITIONAL ANALYSED MATERIAL
UFRJPOR 6345 (specimen in ethanol and slides); UFRJPOR 6472 (specimen in ethanol and
slides); Enseada dos Cardeiros, Arraial do Cabo, Rio de Janeiro, Brazil; 7m deep; coll. F.
Azevedo and G. Rodríguez; 22 May 2011 and UFRJPOR 6510 (specimen in ethanol and slides);
Praia do Forno, Arraial do Cabo, Rio de Janeiro, Brazil; 1.5 m deep; coll. E. Lanna and A.
Padua, April 2011.
COLOUR
White in life and beige to light brown in ethanol.
MORPHOLOGY AND ANATOMY
Cormus massive (Figure 6A) to almost spherical (Figure 7A), composed of thin, irregular to
regular and tightly anastomosed tubes finishing in large apical oscula. The holotype measures
0.8 x 0.6 x 0.2 cm (Figure 6B). It has rough surface due to the presence of large triactines on the
57
external tubes. Water-collecting tubes were not observed. The aquiferous system is asconoid.
The skeleton has no special organization and it is composed of two categories of triactines
(Figures 6C and 7B). The smaller triactines are the most abundant spicules whereas the large
triactines are less frequent and located on the surface. Near the oscular region, the triactines
become more sagittal.
SPICULES (Table 5)
Triactines I. Regular (equiangular and equiradiate). Actines are slightly conical to conical, with
blunt to sharp tips (Figures 6D, 6F and 7C-D). Some subregular triactines were also found
(Figure 7E). Size: 50.0-152.5/6.8-12.5 µm.
Triactines II: Regular (equiangular and equiradiate). Actines are conical and robust with sharp
tips (Figure 6E). Size: 127.5-325.0/17.5-37.5 µm.
ECOLOGY
The specimens inhabited shaded environments, underneath boulders or attached to an oyster
farming rope. They were found associated to macroalgae and to other invertebrates (octocorals,
bryozoans) or growing on oyster shells.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (Curaçao) and Eastern Brazil ecoregion of the Tropical Southwestern
Atlantic Province (Rio de Janeiro, Southern Brazilian Coast).
REMARKS
Within Clathrina, six species whose colour alive is white (or unknown) present two or more
spicule categories (Klautau & Valentine, 2003; Klautau et al. 2016; Azevedo et al., submitted);
however, only three have a cormus composed of tightly anastomosed tubes and large triactines
(including tripods) in the external tubes: C. clara Klautau & Valentine, 2003 from Christmas
Islands, C. laminoclathrata Carter 1886 from southern Australia and C. rotunda Klautau &
Valentine, 2003 from South Africa. Clathrina aspera sp. nov. can be easily distinguished from
those species as its skeleton comprises triactines whose actines are slightly conical to conical
and with blunt to sharp tips whereas the skeletons of the other three species only comprise
triactines whose actines are conical with sharp tips. Moreover, they differ in spicule size (Table
5) and external morphology.
Clathrina clara presents slightly thinner triactines II (21.8±3.5 µm) than those of C. aspera
sp. nov. (28.5±5.6 µm) and its cormus have water-collecting tubes which are absent in the new
Curaçaoan species. Clathrina laminoclathrata has three categories of triactines and even the
largest category (triactine III) is much thinner (18.0±3.0 µm). In this species, the large triactines
are present in the external tubes and can also be found at the base of the sponge (“basal lamina”
58
according to Carter, 1886) whereas in C. aspera sp. nov, they are restricted to the external tubes.
Clathrina rotunda has smaller triactines (triactines I: 52.6±3.6/5.8±0.7 µm and tripods:
59.0±9.8/9.6±2.4 µm) compared to C. aspera sp. nov. (triactine I: 115.4±12.9/9.7±1.1 µm and
triactine II: 227.5±50.8/28.5±5.6 µm – holotype measurements) and their actines are slightly
undulated whereas in the new Curaçaoan species, they are straight. Besides, water-collecting
tubes are also present in C. rotunda.
In the ITS phylogenetic tree, the sequences of C. aspera obtained from several specimens
(UFRJPOR 5531, UFRJPOR 6346, UFRJPOR 6472 and UFRJPOR 6510) clustered together.
However, as sequences of C. clara, C. laminoclathrata and C. rotunda were not available in the
Genbank databse, it was not possible to infer the phylogenetic affinities among them.
Fig. 6. Holotype of Clathrina aspera sp. nov. (UFRJPOR 6758): (A) specimen in vivo; (B)specimen after fixation; (C) tangential section of the body (cormus); (D and E) triactine I; (F)triactine II.
59
Fig. 7. Paratype of Clathrina aspera sp. nov. (UFRJPOR 5487): (A) specimen after fixation; (B)tangential section of the body (cormus) indicating triactines II (white arrows); (C-E) triactine I.
Table 5. Spicule measurements of Clathrina aspera sp. nov. (UFRJPOR 6758, UFRJPOR 5487and UFJPOR 5531), C clara (BMNH 1927.2.14.152), C. laminoclathrata (BMNH
1887.7.12.42) taken from Klautau & Valentine (2003) and C. rotunda (BMNH 1935.10.21.50).H=holotype, P=paratype, L=lectotype.
Specimen Spicule Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR 6758 (H)
Triactine I 75.0 115.4 12.9 152.5 7.5 9.7 1.1 12.5 40Triactine II 132.5 227.2 50.8 325.0 17.5 28.5 5.6 37.5 30
UFRJPOR5487 (P)
Triactine I 65.0 91.1 13.5 117.5 6.3 7.2 0.9 10 40Triactine II 232.5 242.5 9.0 250.0 27.5 31.7 3.8 35.0 3
UFRJPOR5531 (P)
Triactine I 57.5 95.0 15.7 127.5 6.3 7.6 1.2 10 40Triactine II 212.5 235.0 31.8 257.5 27.5 28.7 1.8 30 2
BMNH 1927.2.14.152 (H)
Triactine I 67.5 84.5 8.8 102.5 - 9.8 0.8 - 30Triactine II 102.5 164.5 34.3 245.0 - 21.8 3.5 - 30
BMNH 1887.7.12.42 (L)
Triactine I 50.0 72.0 15.0 113.0 - 8.0 2.0 - 30Triactine II 88.0 132.0 16.0 168.0 - 13.0 2.0 - 30Triactine III 125.0 188.0 31.0 235.0 18.0 3.0 30
BMNH 1935.10.21.50 (H)
Triactine I 45.6 52.6 3.6 57.6 - 5.8 0.7 - 20Tripod 45.6 59.0 9.8 79.6 - 9.6 2.4 - 20
60
Clathrina cf. blanca (Miklucho-Maclay, 1868) (Figure 8, Table 6)
SYNONYMS
Ascetta blanca, Haeckel, 1872: 38; Hansen, 1885: 20; Lendenfeld, 1891: 34; Arnesen, 1901: 9;
Mello-Leitão et al., 1961: 3.
Clathrina blanca: Minchin, 1896: 359; Jenkin, 1908: 438; Borojevic, 1971: 525; Ereskovsky,
1995: 730; Imešek et al., 2014: 23 Klautau et al., 2016: 37,38.
Clathrina cf. blanca: Rapp, 2015: 9.
Guancha blanca: Miklucho-Maclay, 1868: 221; Borojevic & Boury-Esnault, 1987: 14; Barthel
& Tendal, 1993: 84; Janussen et al., 2003: 17; Rapp, 2006: 352.
Leucosolenia blanca: Lackschewitsch, 1886: 300; Breitfuss, 1896: 426; Breitfuss, 1898a: 13;
Breitfuss, 1898: 105; Breitfuss, 1911: 224; Derjugin, 1915: 289; Breitfuss, 1927: 27; Arndt,
1928: 19; Hôzawa, 1929: 282; Brøndsted, 1931: 12; Breitfuss, 1930: 275; Breitfuss, 1932: 240;
Breitfuss, 1935: 7; Breitfuss, 1936: 5; Topsent, 1936: 9; Arndt, 1941: 45; Tanita, 1942b: 75.
TYPE LOCALITY
Lanzarote Islands, Canary Islands, Atlantic Ocean.
MATERIAL EXAMINED
UFRJPOR 6753 and UFRJPOR 6759 (specimens in ethanol and slides); Tug Boat, Caracasbaai,
Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 10 m deep; coll. B. Cóndor-Luján, 23
August 2011.
COLOUR
White in life and in ethanol.
MORPHOLOGY AND ANATOMY
This species has a globular clathroid body with an apical osculum and a stalk (Figure 8A). The
surface is smooth and the texture is soft. The consistency is compressible, even the stalk. In the
largest specimen (UFRJPOR 6759), the clathroid body measures 0.5 x 0.5 x 0.1 cm and the stalk
is 0.5 x 0.1 x 0.1 cm. The cormus is formed by irregular and tightly anastomosed tubes, which
converge at the centre of the sponge forming a single apical osculum. The stalk is formed by
true tubes with choanoderm (arrow in Figure 8A), except for the portion that attaches it to the
substrate which is solid. The aquiferous system is asconoid. The skeleton of the clathroid body
has no special organisation and it is composed of two categories of regular triactines (Figure
8B). The skeleton of the stalk is composed exclusively of parasagittal triactines, whose unpaired
actine is basipetally oriented. These spicules are more numerous and more closely disposed in
the median part of the stalk.
61
SPICULES
Triactines I of the clathroid body. Regular (equiangular and equiradiate) or subregular. Most
frequent spicules. Actines are cylindrical with blunt tips (Figure 8C). Some of them are slightly
undulated at the distal part. Size: 67.5-108.0/4.1-5.4 µm.
Triactines II of the clathroid body. Regular (equiangular and equiradiate). Actines are conical
and straight with sharp tips. Shorter and thicker than triactine I (Figure 8D). Size: 45.9-62.1/5.4-
8.1 µm.
Triactines of the stalk. Parasagittal (equiangular). The paired actines are slightly conical and
very short (sometimes they seem to be rudimentary). The unpaired actine is straight and
cylindrical to slightly conical with sharp tips (Figure 8F). Size: 40.5-67.5/5.4-8.1 µm (paired
actine) and 94.5-199.8/5.4-8.1 µm (unpaired actine).
ECOLOGY
This sponge was collected in a light protected environment, underneath coral boulder.
GEOGRAPHIC DISTRIBUTION
This species has an allegedly cosmopolitan distribution (Van Soest et al., 2016) including the
Caribbean Sea (Southen Caribbean, Curaçao, present study).
REMARKS
The external morphology of the specimens from Curaçao, a clathroid body attached to a stalk,
resembles the species formerly considered guanchas and now allocated in Clathrina (Klautau et
al., 2013). Among them, only two species have a clathroid body composed of tightly
anastomosed tubes, a stalk formed of true tubes and a skeleton composed of regular and
parasagittal triactines, as observed in the Curaçaoan specimens. These species are Clathrina
blanca (Miklucho-Maclay, 1868) from the Lanzarote Islands and C. pellucida (Rapp 2006) from
Norway.
Clathrina pellucida has a short stalk (less than 1/3 of the body) and a skeleton exclusively
composed of triactines with undulated actines whereas the Curaçaoan specimens have a longer
stalk (half the length of the body) and its skeleton also comprises triactines with straight actines.
Besides, the smaller triactines observed in the Curaçaoan specimens (Triactines II of the
clathroid body) are absent in C. pellucida.
Clathrina blanca is the species that most resembles the specimens from Curaçao. As spicule
sizes were not provided in the original description (Miklucho-Maclay, 1868), we used the
measurements provided by Haeckel (1872) for the parasagittal triactines (length/width): 50-
70/3-4 µm (paired actines) and 80-100/3-4 (unpaired actine). Compared to them, the analised
Curaçaoan specimens have thicker actines (5.4-8.1 µm). Compared to more detailed
62
descriptions of C. blanca reported from different localities, namely, Brazil (Borojevic, 1971),
Bay of Biscaye (Borojevic & Boury-Esnault, 1987), Norway (Rapp, 2006) and Adriactic Sea
(Imešek et al., 2014), we observed certain dissimilarities. In none of the referred descriptions,
spicules comparable to the small triactines observed in the specimens from Curaçao were
mentioned or illustrated. Moreover, the parasagittal triactines described for C. blanca sensu
Borojevic (1971) and C. blanca sensu Borojevic & Boury-Esnault (1987) are different to the
ones observed in our material (the paired actines are shorter in the Curaçaoan specimens). The
cormus of C. blanca sensu Imešek et al. (2014) presents several water-collecting tubes whereas
in our specimens, the tubes gather together in a single water-colllecting tube as also described
for C. blanca sensu Rapp (2006). As shown above, the specimens named after C. blanca show
a great range of morphological variability and it is possible that they constitute a species
complex, as already suggested by some authors (Muricy et al., 2011; Rapp, 2015).
The DNA amplification of the Curaçaoan specimens was not successful and its relationship
with the only available sequence of C. blanca from the Adriatic Sea (Imešek et al., 2014)
remains unknown. We name the specimens collected in Curaçao and analised in this study as
Clathrina cf. blanca until a molecular study considering the putative populations of Clathrina
“blanca” is done.
63
Fig. 8. Clathrina cf. blanca (UFRJPOR 6759): (A) specimen after fixation (arrow indicates thestalk); (B) tangential section of the clathroid body; (C) triactine I; (D) triactine II; (E) triactineof the stalk.
Table 6. Spicule measurements of Clathrina cf. blanca from Curaçao (UFRJPOR 6759).
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6759
Triactine I (body -
67.5 87.2 11.6 108.0 4.1 5.3 0.4 5.4 30
Triactine II (body) -
45.9 54.8 4.6 62.1 5.4 6.4 0.9 8.1 30
Triactine (stalk)
Unpaired 94.5 119.8 21.6 151.2 5.4 7.9 1.8 9.4 7Paired 40.5 51.8 5.7 62.1 8.1 8.1 0.0 8.1 10
Clathrina curaçaoensis sp. nov. (Figure 9, Table 7)
ETIMOLOGY
Named after the country of the type locality.
TYPE LOCALITY
Sunset Waters, Soto, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6734 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao;
12°16'01.58''N, 69°07'44.85''W; 3-10 m deep; coll. B. Cóndor-Luján, 20 August 2011.
COLOUR
Yellow in life and light beige in ethanol.
MORPHOLOGY AND ANATOMY
The analysed specimen has a massive cormus (0.7 x 0.4 x 0.3 mm) with a smooth and delicate
surface. The cormus is composed of irregular and loosely anastomosed tubes (Figure 9A). No
water-collecting tubes were observed. The aquiferous system is asconoid. The skeleton has no
special organization (Figure 9B) and it is exclusively composed of triactines. The tubes that
attach the sponge to the substrate are composed of parasagittal triactines (Figure 9C).
SPICULES (Table 7)
Triactines I. Regular (equiangular and equiradiate). Actines are slightly conical with blunt to
sharp tips (Figure 9D). Size: 87.5-130.0/7.5-10.0 µm.
Triactines II. Parasagittal (equiangular). Actines are slightly conical with blunt to sharp tips
(Figure 9E). Size: 57.5-80/7.5 µm (paired actine) and 99.9-137.7/8.1-8.8 µm (unpaired actine).
ECOLOGY
The specimen was found underneath boulders.
64
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
Within the genus Clathrina, seven species are yellow as C. curaçaoensis sp. nov.: C. aurea
Solé-Cava et al., 1991; C. chrysea Borojevic & Klautau, 2000, C. clathrus (Schmidt, 1864), C.
insularis Azevedo et al., submitted, C. lutea Azevedo et al., submitted, C. luteoculcitella
Wörheide & Hooper, 1999 and C. mutabilis Azevedo et al., submitted. However, compared to
C. curaçaoensis sp. nov, these species do not possess tubes differentiated for substrate
attachment supported by parasagittal triactines (triactines II) nor the same external morphology .
Among the non-yellow clathrinas with stalk (former “guanchas”), the species whose external
morphology most resembles that of C. curaçaoensis sp. nov. is C. arnesenae (Rapp, 2006).
Nonetheless, its skeleton does not comprise regular triactines as is the case of the new species;
instead, it is only composed of parasagittal triactines with cylindrical actines.
Regarding the triactines of the body of C. curaçaoensis sp. nov. (triactines I), they resemble
those of C. lutea. However, both species differ in their external morphology (as already
mentioned above). The cormus of C. lutea is formed by regular and tightly anastomosed tubes
while the cormus of C. curaçaoensis sp. nov. is composed of irregular and loosely anastomosed
tubes. Furthermore, in the ITS and C-LSU phylogenetic trees (Figures 31 and 32), C.
curaçaoensis sp. nov. (UFRJPOR 6734) did not cluster with C. lutea nor with any other yellow
clathrina. It appeared as a different lineage. Thus, based in morphological and DNA evidence,
we recognise it as a new species of Clathrina.
Table 7. Spicule measurements of Clathrina curaçaoensis sp. nov. (Holotype=UFRJPOR6734).
Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxTriactine I 87.5 102.8 13.6 130.0 7.5 8.5 1.0 10.0 20Triactine II Unpaired 99.9 119.9 13.1 137.7 8.1 8.2 0.3 8.8 10
Paired 57.5 68.5 6.3 80.0 7.5 7.5 0 7.5 10
65
Fig. 9. Clathrina curaçaoensis sp. nov. (UFRJPOR 6734): (A) specimen after fixation; (B)tangential section of the body (cormus); (C) tangential section of an attachment tube; (D)triactine I; (E) triactine II (parasagittal).
Clathrina hondurensis Klautau & Valentine, 2003 (Figure 10, Table 8)
SYNONYMS
Clathrina hondurensis: Klautau & Valentine, 2003: 46;
Non Clathrina hondurensis: Rützler et al., 2014:101
Non Clathrina cf. hondurensis: Imešek et al., 2014:25; Klautau et al., 2016: 21.
TYPE LOCALITY
Turneffe, British Honduras, Caribbean Sea.
MATERIAL EXAMINED
UFRJPOR 6732 (specimens in ethanol and slides); Porto Mari, St. Willibrordus; 12°13'6.62"N,
69°05'13.26"W; 7.9 m deep; coll. B. Cóndor-Luján, 20 August 2011.
66
COMPARATIVE MATERIAL EXAMINED
Clathrina hondurensis. Holotype (slide) BMNH 1938.3.28.4; Turneffe, British Honduras,
Caribbean Sea; coll. J.H. Borley, 20–22 March 1935.
COLOUR
White in life and light brown in ethanol.
MORPHOLOGY AND ANATOMY
The specimen is massive and it measures 1.4 x 1.0 x 0.2 cm (Figure 10A). The surface is
smooth and the consistency is compressible. The cormus is composed of irregular and tightly
anastomosed tubes (Figure 10B). No water-collecting tubes were observed. The aquiferous
system is asconoid. The skeleton has no special organization (Figure 10C) and it is composed of
triactines and rare trichoxeas (Figure 10C, arrow).
SPICULES (Table 8)
Trichoxeas. Straight and very slender (Figure 10D). Mostly broken. The size of the unique
whole trichoxea found is 325/2.5 µm.
Triactines. Regular (equiangular and equiradiate). Actines are conical with sharp tips (Figure
10E). Size: 100.0-212.5/13.6-25 µm.
ECOLOGY
This specimen was collected underneath boulders.
GEOGRAPHIC DISTRIBUTION
Southwestern Caribbean (Belize, Klautau & Valentine, 2003) and Southern Caribbean (Curaçao,
present study) ecoregions.
REMARKS
The external morphology and the shape of the triactines observed in the specimen from Curaçao
are similar to C. hondurensis. However, in the original description of that species, trichoxeas
were not reported and after the re-examination of the slides of the holotype, we did not find
them. As seen in other species, the presence of trichoxeas may not constitute a diagnostic
character, as they apparently constitute plastic characters. Regarding the spicule size, although
the triactines of the specimen from Curaçao can attain slightly larger sizes compared to the
specimen from Turneffe (Table 11), they are in the same size range. It is important to point out
that C. hondurensis was originally described based on one single specimen. Considering this,
the presence of trichoxeas and of slightly larger triactines in the specimen from Curaçao can be
attributed to intraspecific variation.
Rützler et al. (2014) recorded C. hondurensis from another Belizean locality (Belize barrier
reef near Carrie Bow), however, the skeleton of their specimen was composed of triactines
67
whose size range was 85-100 μm in length and 8-12 μm in width. Based on the original
description of C. hondurensis and the specimens from Curaçao, we believe that the specimen
reported by Rützler et al. (2014) does not correspond to C. hondurensis.
Recently, Klautau et al. (2016) considered the possibility of synonymy between C.
hondurensis and C. primordialis (Haeckel, 1872). In fact, both species present similar
morphology and their spicule size range do overlap, however, it is possible to recognize thicker
spicules in C. hondurensis (Table 6). As we could not successfully amplify any DNA region
from UFRJPOR 6732, we cannot affirm that the difference in width reflects plasticity and that
C. hondurensis and C. primordialis are synonyms. Therefore, we decided to maintain C.
hondurensis as a valid species and to identify the Curaçaoan specimen as C. hondurensis.
Fig. 10. Clathrina hondurensis (UFRJPOR 6732): (A) specimen in vivo; (B) specimen afterfixation; (C) tangential section of the body (cormus) skeleton; (D) detail of trichoxea indicatedby an arrow; (E) triactine.
68
Table 8. Spicule measurements of Clathrina hondurensis from Curaçao (UFRJPOR 6732) andof the holotype (BMNH 1938.3.28.4) and of C. primordialis taken from * Klautau & Valentine
(2003) and ** Haeckel (1872).
Specimen SpiculeLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR 6732
Triactine 100.0 149.6 30.0 212.5 13.6 19.1 3.1 25 30Trichoxea 325.0 - - - 2.5 - - 1
BMNH 1938. 3.28.4
Triactine 120.0 142.9 15.5 175.0 12.5 16.8 2.4 20 20
BMNH 1938. 3.28.4*
Triactine 105.6 133.4 17.0 156.0 - 15.6 1.7 - 20
C. primordialis**
Triactine 100.0 - - 150.0 8 - - 12.0 -
Clathrina insularis Azevedo et al, submitted (Figure 11, Table 9)
SYNONYMS
Clathrina insularis: Azevedo et al, submitted
Clathrina sp. nov. 3 - UFRJPOR 6737a=UFRJPOR 6737: Klautau et al., 2013, 449 and 451.
TYPE LOCALITY
Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil
MATERIAL EXAMINED
UFRJPOR 6737 (specimen in ethanol and slides); Playa Jeremi, Soto;
12°19'43.73''N,69°09'07.80''W; 14.9 m; coll. B. Cóndor-Luján, 22 August 2011.
ADDITIONAL MATERIAL RE-ANALYZED
Specimens in ethanol and slides: UFRJPOR 6533; Cagarras, Fernando de Noronha Archipelago,
Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m; coll. F. Azevedo and G. Rodríguez,
27 June 2011. UFRJPOR 6530 and 6537; Ilha do Meio, Fernando de Noronha Archipelago,
Pernambuco, Brazil; 03°49'5.88''S,32°23'36.6''W;15 m ; coll. F. Azevedo and G. Rodríguez, 27
June 2011.
MATERIAL STUDIED FOR COMPARISON
Clathrina insularis. Holotype (specimen in ethanol and slides) UFRJPOR 6532; Cagarras,
Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m;
coll. F. Azevedo and G. Rodríguez, 27 June 2011.
COLOUR
Pale yellow in life and beige in ethanol.
69
MORPHOLOGY AND ANATOMY
The specimen is finely encrusting (0.4 x 0.4 x 0.1 mm, Figure 11A). The surface is smooth and
the texture is soft. The cormus is composed of irregular and loosely anastomosed tubes. Water-
collecting tubes are not present. The aquiferous system is asconoid. The skeleton has no
organization (Figure 11B) and it is composed of two categories of triactines.
SPICULES
Triactines I. Regular (equiangular and equiradiate). Actines are conical with sharp tips (Figure
9C). Size: 52.5-77.5/3.8-6.3 µm.
Triactines II. Regular (equiangular and equiradiate) or subregular (equiangular). Most abundant
spicules. Actines are cylindrical to slightly conical, distally undulated and with sharp tips
(Figure 9D-E). Size: 102.7-145.0/3.7-6.6 µm.
ECOLOGY
This sponge was found in a cryptic habitat, underneath boulder.
GEOGRAPHIC DISTRIBUTION
Fernando de Noronha and Atoll das Rocas ecoregion of the Tropical Southwestern Atlantic
Province (Fernando de Noronha Archipelago, Azevedo et al., submitted) and Southern
Caribbean (Curaçao, present study).
REMARKS
The external morphology and the skeleton composition of the Curaçaoan specimen match the
description of C. insularis. In the ITS phylogenetic tree (Figure 31), it clustered in the clade of
that species (pp=1, b=100), corroborating its morphological identification. The specimens
UFRJPOR 6530, UFRJPOR 6533 and UFRJPOR 6537 were already analysed in Azevedo et al.,
submitted, and herein, we provide their ITS sequences.
Table 9. Spicule measurements of Clathrina insularis (UFRJPOR 6737) from Curaçao and ofthe holotype (UFRJPOR 6532).
Specimen SpiculeLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR 6737
Triactine I 52.5 62.6 6.7 77.5 3.8 5.2 0.5 6.3 30Triactine II 102.7 119.0 8.6 145.0 4.1 5.4 0.7 6.8 50
UFRJPOR 6532
Triactine I 47.5 76.4 12.4 97.5 5.0 6.1 0.7 7.5 30Triactine II 98.8 121.1 11.0 145.0 5.7 6.3 0.8 8.2 60
70
Fig. 11. Clathrina insularis (UFRJPOR 6737): (A) specimen in vivo; (B) tangential section ofthe body (cormus), (C) triactine I, (D-E) triactine II.
Clathrina lutea Azevedo et al., submitted (Figure 12, Table 10)
SYNONYMS
Clathrina primordialis: Lehner & Van Soest, 1998: 97?
Clathrina sp. nov. 8 - UFRJPOR 6761 and ZMAPOR 08344: Klautau et al., 2013, 449 and 451.
TYPE LOCALITY
Pedra Lixa, Abrolhos Archipelago, Caravelas, Bahia, Brazil.
MATERIAL EXAMINED
UFRJPOR 6761 (specimen in ethanol and slides); Porto Mari, St. Willibrordus, Curaçao;
12°13'6.62"N, 69°05'13.26"W; 10.6 m deep; coll. Giselle Lôbo-Hajdu, 20 August 2011.
COMPARATIVE MATERIAL EXAMINED
Clathrina lutea. Holotype (specimen in ethanol and slides) UFRJPOR 5173; Pedra Lixa,
Abrolhos Archipelago, Caravelas, Bahia, Brazil; 17°41'S, 38°59'W; 7 m deep; coll. C.
Zilberberg & L. Monteiro, 21 March 2005.
71
COLOUR
Dark yellow in life and white in ethanol.
MORPHOLOGY AND ANATOMY
This specimen has a rough massive cormus (1.0 x 0.4 x 0.1 cm, Figure 12A) composed of
regular and tightly anastomosed tubes (Figure 12B). Water-collecting tubes were observed. The
aquiferous system is asconoid. The skeleton has no organization and it is composed of one
category of triactines (Figure 12C). Some broken trichoxeas were found in the spicule slide.
SPICULES
Triactines. Regular. Actines are cylindrical to slightly conical, slightly undulated and with blunt
tips (Figure 12D). Size: 75.0-97.5/7.5-10 µm.
ECOLOGY
This sponge was collected in a light protected environment, inside a small crevice.
GEOGRAPHIC DISTRIBUTION
Within the Tropical Southwestern Atlantic includes the Floridian (Florida, United States of
America: UFRJPOR 5818, Klautau et al., 2013), Eastern Caribbean (Virgin Islands: ZMAPOR
08344, Klautau et al., 2013) and Southern Caribbean (Curaçao, present study) ecoregions. In the
Tropical Northwestern Atlantic includes Eastern Brazil (Abrolhos Archipelago) and Fernando de
Noronha and Atoll das Rocas (Rocas Atoll) ecoregions (Azevedo et al., submitted).
REMARKS
Compared to the type material of C. lutea, our specimen has slightly larger triactines and actines
are not very undulated. Besides, some broken trichoxeas were found in the spicule slide of the
Curaçaoan material whereas in the Brazilian specimens they were not observed. Despite these
slight differences, the Curaçaoan specimen grouped in the clade of C. lutea (pp=1, b=100,
Figure 31), which included Brazilian and Caribbean specimens.
In 1998, Lehner & Van Soest reported C. primordialis to Jamaica. After revising the
description of that record, including the in vivo figure (Figure 23, page 97), we rather consider it
as C. lutea based principally on its external morphology.
Table 10. Spicule measurements of Clathrina lutea from Curaçao (UFRJPOR 6761) and of theholotype (UFRJPOR 5173).
Specimen SpiculeLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR 6761 Triactine 75.0 88.8 5.6 97.5 7.5 9.4 0.7 10.0 30UFRJPOR 5173 Triactine 69.3 78.5 3.8 84.0 6.5 7.5 0.4 8.3 30
72
Fig. 12. Clathrina lutea (UFRJPOR 6761): (A) specimen in vivo (photo taken by G. Lôbo-Hajdu); (B) specimen after fixation; (C) tangential section of the body (cormus); (D) triactine.
Clathrina mutabilis Azevedo et al., submitted (Figure 13, Table 11)
SYNONYMS
Clathrina mutabilis: Azevedo et al., submitted
Clathrina sp. nov. 4 - UFRJPOR 6733 and UFRJPOR 6741: Klautau et al., 2013, p: 449 and
451.
TYPE LOCALITY
Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil.
MATERIAL EXAMINED
Specimens in ethanol and slides: UFRJPOR 6704 and UFRJPOR 6719; Playa Kalki, Westpunt,
Curaçao; 12°22'29.86"N, 69°09'30.63"W; 6.7 m deep; coll. E. Hajdu and B. Cóndor-Luján, 21
August 2011; UFRJPOR 6717; Water Factory, Willemstadt, Curaçao; 12°06'30.88"N,
73
68°57'13.53"W; coll. B. Cóndor-Luján, 19 August 2011; UFRJPOR 6733 and UFRJPOR 6735,
Porto Mari, St. Willibrordus, Curaçao; 12°13'6.62"N, 69°05'13.26"W; 7.9 m deep; coll. B.
Cóndor-Luján, 20 August 2011; UFRJPOR 6740; Sunset Waters, Soto, Curaçao; 12°16'01.58"N,
69°07'44.85"W, 9-12 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6741,
UFRJPOR 6743 and UFRJPOR 6744; Sunset Waters, Soto, Curaçao; 12°16'01.58"N,
69°07'44.85"W, 8.9 m, 9.8 m and 7.2 m deep, respectively; coll. B. Cóndor-Luján, 22 August
2011. Slides: UFRJPOR 6699; Hook’s Hut, Willemstadt, Curaçao; 12°07'18.94"N,
68°58'11.46"W; < 10 m deep; coll. B. Cóndor Luján and G. Lôbo-Hajdu, 17 August 2011;
UFRJPOR 6712; Daai Booi, St. Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 4.9 m
deep; coll. B. Cóndor-Luján, deep, 18 August 2011; UFRJPOR 6736 (specimens in ethanol and
slides); Playa Jeremi, Soto, Curaçao; 12°19'43.73 N, 69°09'07.80 W; 4.9 m deep; coll. B.ʺ ʺ
Cóndor-Luján, 22 August 2011; UFRJPOR 6747; Sunset Waters, Soto, Curaçao; 12°16'01.58"N,
69°07'44.85"W, 4.9 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6750; Tug Boat,
Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W, coll. B. Cóndor-Luján, 23
August 2011.
COMPARATIVE MATERIAL EXAMINED
Clathrina mutabilis. Holotype (specimen in ethanol and slides) UFRJPOR 6526; Cagarras,
Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m;
coll. F. Azevedo and G. Rodríguez, 27 June 2011.
COLOUR
Pale yellow in life and white to beige in ethanol.
MORPHOLOGY AND ANATOMY
The specimens have a massive cormus composed of irregular and loosely anastomosed tubes
(Figure 11A) which form water-collecting tubes. When tubes are contracted, this sponge has a
more encrusting growth form (UFRJPOR 6743, Figure 11B). The largest fragment deposited in
our collection belongs to the specimen UFRJPOR 6735 (Figure 11C) which measures 0.8 x 0.6
x 0.2 cm. The aquiferous system is asconoid. The skeleton has no organization (Figure 11D) and
it is composed of two categories of triactines.
SPICULES (Table 11)
Triactines I. Regular (equiangular and equiradiate) but some subregular spicules (equiangular
but not equiradiate) were also found. Actines are conical, straight, with sharp tips (Figure 13E).
Smaller than triactines II. Size: 60-112.5/7.5-10 µm.
74
Triactines II. Regular (equiangular and equiradiate), subregular (equiangular but not
equiradiate) (Figure 13F) or parasagittal (Figure 13G). Frequent. Actines are slightly conical to
cylindrical, slightly undulated, with blunt tips. Size: 100.0-195.0/5-11.3 µm.
ECOLOGY
The specimens were found underneath boulders and broken corals.
GEOGRAPHIC DISTRIBUTION
Fernando de Noronha and Atoll das Rocas (Fernando de Noronha Archipelago , Azevedo et al.,
submitted) and Southern Caribbean (Curaçao , present study) ecoregions. Clathrina mutabilis
was found in all Curaçaoan sampled localities.
REMARKS
In the ITS phylogenetic tree, these specimens clustered within the clade of C. mutabilis
(pp=0.81, b=91%, Figure 31) and they match its morphological description. However, some
slight differences were found. Compared to the holotype, the skeleton of the Curaçaoan
specimens has more parasagittal triactines II and does not present trichoxeas (or at least, they
were not observed). This may be the result of plasticity. It is important to point out that in the
analised specimens, it was possible to observe water-collecting tubes, and thus, we complement
the original description of this species providing one more diagnostic character.
Table 11. Spicule measurements of Clathrina mutabilis from Curaçao (UFRJPOR 6741,UFRJPOR 6704 and UFRJPOR 6745) and of the holotype (UFRJPOR 6526). U=unpaired and
P=paired actines.
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6741
Triactine I 100.0 123.8 10.4 145.0 7.5 8.9 0.9 10 30ParasagittalTriactine I
U 125.0 166.0 17.3 195.0 5 8.2 1.1 10 30P 100.0 116.1 11.2 132.5 5 7.5 1.1 10 30
Triactine II 67.5 82.4 8.5 95.0 6.3 8.0 0.8 8.8 30UFRJPOR6704
Triactine I 107.5 135.5 15.9 162.5 7.5 10.3 1.0 11.3 20ParasagittalTriactine I
U 122.5 142.4 15.6 177.5 7.5 8.3 1.0 10 30P 75.0 98.7 12.8 127.5 6.3 7.6 0.5 8.8 30
Triactine II 60.0 95.5 12.6 112.5 7.5 8.8 1.2 10 20UFRJPOR6745
Triactine I 100.0 126.0 18.1 160.0 7.5 9.0 1.1 10 20ParasagittalTriactine I
U 150.0 169.5 13.1 195.0 7.5 9.1 1.0 10 30P 97.5 121.0 12.9 140.0 7.5 8.3 0.9 10 30
Triactine II 60.0 81.6 13.4 102.5 7.5 8.6 1.1 10 20UFRJPOR6526
Triactine I 94.5 124.7 14.4 148.5 6.8 7.9 0.6 9.5 40Triactine II 56.7 69.8 7.9 91.8 8.1 8.4 0.6 9.5 20
75
Fig. 13. Clathrina mutabilis: (A) specimen UFRJPOR 6704 in vivo; (B) specimen UFRJPOR6743 in vivo; (C) specimen UFRJPOR 6735 after fixation; (D) tangential section of the body(cormus); (E) triactine I; (F) triactine II; (G) parasagittal triactine II. Skeleton images were takenfrom slides of the specimen UFRJPOR 6741.
76
Genus Leucetta Haeckel, 1872
TYPE SPECIES
Leucetta primigenia Haeckel, 1872.
DIAGNOSIS
"Leucettidae with a homogeneous organisation of the wall and a typical leuconoid aquiferous
system. There is neither a clear distinction between the cortex and the choanoskeleton, nor the
presence of a distinct layer of subcortical inhalant cavities. The atrium is frequently reduced to a
system of exhalant canals that open directly into the osculum" (Borojevic et al., 2002).
Leucetta floridana Haeckel, 1872 (Figure 14, Table 12)
SYNONYMS
Amphoriscus floridanus: Haeckel, 1872: 144.
Dyssycus floridanus: Haeckel, 1872: 144.
Leucaltis floridana: Haeckel, 1872: 144.
Leucaltis impura: Haeckel, 1872: 144.
Leucaltis pura: Haeckel, 1872: 144.
Leucetta aff. floridana: Lehnert & van Soest, 1998: 99.
Leucetta floridana: de Laubenfels, 1950: 146; Moraes et al., 2006: 167; Muricy et al., 2008:
132; Valderrama et al., 2009: 9; Lanna et al., 2009: 7; Muricy et al., 2011: 36-37; Rützler et al.,
2014: 102; Azevedo et al, submitted.
Leucetta microraphis: Tanita, 1942: 111; Borojevic & Peixinho, 1976: 1003.
Leucetta sp. : Moraes et al., 2003: 17.
Leucilla floridana: Jenkin, 1908: 453.
Lipostomella floridana: Haeckel, 1872: 144.
TYPE LOCALITY
Coast of Florida, United States of America.
MATERIAL EXAMINED
UFRJPOR 6726 (specimen in ethanol and slides); Water Factory, Willemstadt; 12°06'30.88"N,
68°57'13.53"W; 17.8 m deep; coll. E. Hajdu, 19 August 2011; UFRJPOR 6757 (specimen in
ethanol and slides); Tug Boat, Caracasbaai, Willemstadt; 12°04'08.20"N, 68°51'44.40"W; 6.2
m deep; coll. B. Cóndor-Luján, 23 August 2011; UFRJPOR 6765 (specimen in ethanol); Hook’s
Hut, Willemstadt, Curaçao; 12°07'18.94"N, 68°58'11.46"W; 13.3 m deep; coll. E. Hajdu, 18
August 2011.
77
COLOUR
White to light blue in life and grayish white to brown in ethanol.
MORPHOLOGY AND ANATOMY
This species has a massive growth form (Figure 14A). The largest specimen measures 1.6 x 2.6
cm (Figure 14B). The body is ridged and hispid. The three analised specimens presented a
single apical osculum (largest diameter=0.5 cm). In the specimen UFRJPOR 6757, the osculum
is particularly elongated and bears a very delicate margin. The atrial cavity is wide and also very
hispid. The aquiferous system is leuconoid. The consistency is very rough and incompressible.
SKELETON
The skeleton is typical of the genus. It does not have a special organization and it is composed
of two size categories of triactines (I and II) and tetractines (I and II). The cortex and atrial wall
are thin whereas the choanosome is thick. Triactines II and tetractines II, which are the largest
spicules, are found in in the cortex and in the choanosome, tangentially disposed. Tetractines II
are rare. Triactines I and tetractines I are spread in the choanosome and in the atrium. The apical
actine of tetractines I penetrates the exhalant canals and the atrial cavity. Near the atrium,
triactines I and tetractines I become sagittal.
SPICULES
Triactines I. Regular. Actines are conical, straight, with blunt tips (Figure 14C). Frequent.
Sagittal triactines I were also observed. Size: 87.5-175.0/10.0-22.5 µm.
Triactines II. Regular. Actines are conical, straight, with blunt tips (Figure 14D). Very variable
size: 378.4-2378.4/54.1-389.2 µm.
Tetractines I. Regular. Actines are slightly conical, straight, with blunt to sharp tips (Figure
14E). The apical actine is smooth, thinner than the basal actines and has a sharp tip. Sagittal
tetractines I were also observed. Size: 102.5-200.0/11.2-20.0 µm (basal actine) and 25-50/7.5-10
µm (apical actine).
Tetractines II. Regular. Rare. Actines are conical, straight, with blunt tips (Figure 14F). Very
variable size: 464.9-2162.2/108.1-270.3 µm.
ECOLOGY
This species was found underneath boulders close to some incrusting and massive (cf. Clathria)
demospongias. No associated organism was found on the surface of the analised specimens.
GEOGRAPHIC DISTRIBUTION
Widespread in the Western Atlantic: Tropical Southwestern Atlantic Province including the
Floridian (Haeckel, 1872), Bermuda (Bermudas, de Laubenfels, 1950), Greater Antilles
(Jamaica, Lehner & van Soest, 1998) and the Southern Caribbean (Urabá and San Andrés,
78
Valderrama et al., 2009 and Curaçao, present study) ecoregions, North Brazil Shelf Province
(Pará, Borojevic & Peixinho, 1976) and Tropical Northwestern Atlantic Province comprising the
Northeastern Brazil, Eastern Brazil, Fernando de Noronha and Atoll das Rocas ecoregions
(Borojevic & Peixinho, 1976; Valderrama et al., 2009).
REMARKS
Our specimens match the original description of L. floridana as well as the redescription
provided by Valderrama et al. (2009). Haeckel's measurements are provided here: Triactines and
tetractines I: 150-250/10-15 µm and triactines and tetractines II: 700-1500/100-150 µm and
those of Valderrama et al., (2009) are presented in Table 12. Leucetta floridana is not only one
of the few species of Calcarea already reported for the Caribbean Sea but it is also one of the
most widespread species along the Western Tropical Atlantic.
Table 12. Spicule measurements of Leucetta floridana from Curaçao (UFRJPOR 6726 andUFRJPOR 6757) and of its redescription (UFRJPOR 5360. Valderrama et al., 2009).
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6726
Triactine I - 121.5 143.6 14.6 172.8 10.8 13.9 1.6 16.2 20Triactine II - 972.0 1431.3 354.9 2378.4 129.6 214.5 52.5 345.6 21Tetractine I B 105.0 139.1 23.3 200.0 12.5 15.2 2.2 20.0 20
A 25.0 33.4 8.4 45 7.5 8.1 1.2 10 8Tetractine II B 675.7 1228.2 293.6 1621.6 108.1 171.8 49.7 216.2 9
UFRJPOR6757
Triactine I - 87.5 126.7 22.2 175.0 10.0 15.5 3.2 22.5 30Triactine II - 378.4 1383.3 673.9 2378.4 54.05 207.7 98.3 389.2 24Tetractine I B 102.5 138.3 19.6 200.0 11.2 15.3 2.8 20.0 30
A 40.0 44.4 4.3 50.0 10 10.0 0.0 10 4Tetractine II B 464.9 1294.9 568.7 2162.2 108.1 172.0 59.1 270.3 9
UFRJPOR5360
Triactine I - 105.6 143.3 28.7 217.8 9.9 17.1 4.9 33,0 30Triactine II - 257.4 696.2 279.7 1181.5 33.0 102.1 46.2 194.6 30Tetractine I - 105.6 137.4 24.1 224.4 9.9 15.4 3.6 26.4 30Tetractine II - 278.0 665.5 301.0 1042.5 48.7 102.5 51.3 180.7 8
79
Fig. 14. Leucetta floridana (UFRJPOR 6757): (A) specimen in vivo; (B) specimen afterfixation; (C) triactine I; (D) triactine II; (E) tetractine I; (F) tetractine II.
Genus Amphoriscus Haeckel, 1872TYPE SPECIES
Amphoriscus chrysalis (Schmidt, 1864)
DIAGNOSIS
"Amphoriscidae with syconoid organization of the aquiferous system. Scattered spicules in the
choanosome are always absent" (Borojevic et al., 2002).
80
Amphoriscus micropilosus sp. nov. (Figures 15 & 16, Table 13)
ETIMOLOGY
From the Latin pilosus (= hairy), for the presence of microdiactines crossing the cortex.
TYPE LOCALITY
Sunset Waters, Soto, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6739 (specimens in ethanol and slides); Sunset Waters, Soto, Curaçao;
12°16'01.58"N, 69°07'44.85"W; 13.1 m deep; coll. B. Cóndor-Luján, 22 August 2011.
Paratypes: UFRJPOR 6755 and UFRJPOR 6756 (specimens in ethanol and slides); Tug Boat,
Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 8.6 m deep; coll. B.
Cóndor-Luján, 23 August 2011.
COLOUR
White in life and in ethanol. Transparent and bright.
MORPHOLOGY AND ANATOMY
This sponge has a very variable external morphology (Figure 15A-F), which can be tubular
(Figures 15A and 15D) to flatened sac-shaped (Figures 15C and 15F) but always with an apical
osculum. The surface is smooth, although microdiactines protrude through the surface. The
consistency is rough. The largest specimen (UFRJPOR 6756) measures 1.2 x 0.4 cm and it
presents a short peduncle (arrow in Figure 15D). The osculum has a margin sustained by T-
shaped triactines and it is surrounded by short trichoxeas. The aquiferous system is syconoid.
SKELETON
The skeleton is typical of the genus (Figure 15G). The cortical skeleton is composed of
perpendicular microdiactines spread through the surface (Figure 15H) or organized in tufts (only
in UFRJPOR 6739, Figure 15I), triactines and the paired actines of the subcortical tetractines.
The triactines are distributed tangentially to the surface (arrow in Figure 15J). The choanosomal
skeleton is inarticulated, composed of the apical actines of the subcortical tetractines and by rare
subatrial triactines. The apical actine of the tetractines crosses the choanosome and occasionally
reaches the atrium (black arrow in Figure 15K). The unpaired actine of the subatrial triactines
points toward the cortex (white arrow in Figure 15K). The atrial skeleton is exclusively
composed of tetractines with their apical actine projected into the atrial cavity (asterisk in Figure
15K).
SPICULES
Microdiactines. Fusiform, straight, with sharp tips. Size: 27.0-94.5/1.1-1.4 µm
81
Cortical triactines. Sagittal. Actines are smooth, conical, with sharp tips (Figure 16A).
Sometimes the paired actines are curved. Size: 102.6-310.5/8.1-18.9 µm (paired actines), 89.1-
310.5/6.7-18.9 µm (unpaired actine).
Subcortical tetractines. Sagittal. Actines are straight, smooth, conical, with sharp tips (Figure
16B). The apical actine is very large. They are the largest spicules in this species. Size: 172.8-
572.4/21.6-64.8 µm (paired actine), 183.6-540/30-64.8 µm (unpaired actine), 162.0-
1036.8/21.6-64.8 µm (apical actine).
Subatrial triactines. Sagittal. Actines are conical, smooth, straigth, with sharp tips (Figure 16C).
The paired actines are shorter than the unpaired ones and some are slightly curved. Sometimes,
one paired actine is longer than the other one. Size: 81.0-240.3/8.1-21.6 µm (paired actine),
91.8-610/8.1-18.9 µm (unpaired actine).
Atrial tetractines. Sagittal. Actines are conical, straight, smooth and have sharp tips. The
unpaired actine is slightly longer than the paired ones (Figure 16D). The apical actine is the
shortest actine. Size (length/width): 100.0-264.6/10-24.3 µm (paired actine), 83.7-297/10-21.6
µm (unpaired actine), 27-115/8.1-13.5 µm (apical).
ECOLOGY
Specimens were found underneath broken coral boulders. No associated organism was found.
Some balls of sediment were found inside the atrial cavity of the holotype.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao present study).
REMARKS
The genus Amphoriscus comprises 16 species (Van Soest et al., 2016, Van Soest, 2017). Among
them, four species have been reported for the Caribbean Sea: Amphoriscus oviparus (Haeckel,
1872), A. perforatus (Haeckel, 1872), A. testiparus (Haeckel, 1872), and A. urna Haeckel, 1872,
however, none of them present a skeleton composition similar to A. micropilosus sp. nov.
Different from the new species, whose subatrial skeleton is exclusively composed of triactines,
all the referred species have subatrial tetractines and lack cortical microdiactines. Moreover, A.
perforatus presents atrial triactines, which are absent in A. micropilosus sp. nov., and A. urna
lacks the cortical triactines present in the Curaçaoan species.
Considering the species recorded out of the Caribbean Sea, the species that most resembles
the new species is A. elongatus (Poléjaeff, 1883), originally described from the Indian Ocean.
Nonetheless, these two species differ in spicule size (Table 13), mainly in the width of the apical
actine of the atrial tetractines. which is thinner in the new species (8.1-13.5 µm) than in A.
elongatus (16-20 µm). Besides, in A. elongatus the radial tubes “meet in threes, in fours, or in
82
larger numbers around the same shallow invagination of the gastric cavity” (Poléjaeff, 1883),
which was not observed in the new species.
83
Fig. 15. Amphoriscus micropilosus sp. nov.: (A-C) UFRJPOR 6756, UFRJPOR 6739 andUFRJPOR 6755 in vivo; (D-F) UFRJPOR 6756, UFRJPOR 6739 and UFRJPOR 6755 afterfixation; (G) cross section of the skeleton; (H) trichoxeas along the surface in UFRJPOR 6755;(I) tufts of trichoxeas in UFRJPOR 6739; (J) cortical skeleton showing triactines (arrow); (K)choanosomal and atrial skeletons and the apical actine of cortical tetractines (black arrow),unpaired actine of a subatrial triactine (white arrow) and apical actine of an atrial tetractine (*).Abbreviations: cx=cortex; at=atrium. All photos were taken from the holotype slide (UFRJPOR6739) except when indicated.
Fig. 16. Spicules of Amphoriscus micropilosus sp. nov. (Holotype=UFRJPOR 6739): (A)cortical triactine; (B) subcortical tetractine; (C) subatrial triactine; (D) atrial tetractine.
Table 13. Spicule measurements of Amphoriscus micropilosus sp. nov. (UFRJPOR 6739,UFRJPOR 6755 and UFRJPOR 6756) and of A. elongatus . H=holotype. P=paired, U=unpaired,
A=apical and B=basal actines.
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD Max
UFRJPOR6739 (H)
Micro-diactine - 51.3 75.6 16.8 94.5 1.1 1.3 0.1 1.4 7Cortical Triactine
P 102.6 204.0 51.7 310.5 8.1 14.4 2.8 18.9 14U 110.7 229.5 54.5 283.5 6.7 14.1 3.0 18.9 15
SubcorticalTetractine
P 356.4 475.2 71.1 572.4 32.4 52.9 9.2 64.8 20U 216.0 392.0 127.2 540.0 48.6 54.5 4.7 64.8 10A 162.0 449.6 158.8 658.8 21.6 49.7 12.4 64.8 19
Subatrial Triactine
P 81.0 177.5 46.7 240.3 8.1 13.7 3.7 21.6 20U 91.8 186.3 50.5 243.0 8.1 12.4 2.6 16.2 20
84
Atrial tetractine
P 167.4 205.2 29.8 264.6 12.2 17.1 3.6 24.3 9U 140.4 228.6 45.2 297.0 12.2 15.6 3.0 21.6 9A 51.3 65.1 11.4 81.0 8.1 11.5 1.9 13.5 8
UFRJPOR6755
Micro-diactine - 27.0 51.3 22.7 81.0 1.1 1.2 0.2 1.4 4Cortical triactine
P 113.4 174.2 32.3 240.3 8.1 14.0 2.6 18.9 20U 89.1 221.1 64.9 310.5 8.1 14.4 3.2 18.9 20
SubcorticalTetractine
P 172.8 223.2 62.6 367.2 21.6 30.5 7.5 43.2 14U 183.6 229.0 54.8 324.0 32.4 40.0 9.0 54.0 5A 291.6 491.8 217.8 1036.8 21.6 35.7 11.4 54.0 13
Subatrial triactine
P 86.4 165.4 41.8 232.2 8.1 14.0 2.5 18.9 20U 135.0 242.2 61.6 351.0 9.5 13.7 2.4 18.9 20
Atrial tetractine
P 113.4 173.3 34.1 229.5 10.8 14.0 1.7 16.2 20U 83.7 200.0 56.6 283.5 10.8 15.6 2.5 21.6 19A 27.0 49.7 16.2 78.3 8.1 11.1 1.8 13.5 19
UFRJPOR6756
Micro-diactine - 45.0 61.7 16.7 87.5 1.0 1.2 0.1 1.2 6Cortical triactine
P 107.5 157.3 23.6 212.5 10 12.0 1.5 17.5 27U 157.5 195.4 23.0 230.0 10.0 12.2 0.8 12.5 26
SubcorticalTetractine
P 250.0 363.9 67.9 510.0 25.0 39.3 5.9 50 23U 250.0 279.0 23.3 315.0 30.0 37.0 4.5 40.0 5A 325.0 555.0 110.4 740.0 30.0 42.3 7.0 55.0 30
Subatrial triactine
P 125.0 160.8 23.5 207.5 12.5 12.9 0.9 15 9U 255.0 346.3 110.2 610.0 10.0 13.8 2.4 18.8 15
Atrial tetractine
P 100.0 142.3 25.4 212.5 10.0 11.9 1.7 15 27U 125.0 184.7 43.4 250.0 10.0 12.4 1.8 17.5 22A 55.0 78.8 17.8 115.0 10.0 10.3 0.8 12.5 10
A.elongatus
Micro-diactine - - 100 - - - 2.5 - - -Cortical triactine
P - - - 250 - 15 - - -U - - - 450 - 15 - - -
Cortical tetractine
B - 600 - - - 70 - - -A 600 - - - - - - -
Atrial tetractine
P - 250 - - 16 - - 20 -U - - - 450 16 - - 20 -
Genus Leucilla Haeckel, 1872
TYPE SPECIES
Leucilla amphora Haeckel, 1872
DIAGNOSIS
"Amphoriscidae with sylleibid or leuconoid organization. The choanoskeleton is formed
primarily by the apical actines of giant cortical tetractines and the unpaired actines of subatrial
triactines or tetractines. It may contain dispersed spicules, but a typical articulated
choanoskeleton is always absent" (Borojevic et al., 2002, emend).
85
Leucilla antillana sp. nov. (Figures 17 & 18, Table 14)
ETIMOLOGY
From its distribution in Curaçao located in the Leeward Antilles Ridge.
TYPE LOCALITY
Water Factory, Willemstadt, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6768 (specimen in ethanol and slides); Water Factory, Willemstadt,
Curaçao; 12°06'30.88"N, 68°57'13.53"W; 9.9 m deep; coll. B. Cóndor-Luján, 23 August 2011.
COLOUR
White in life and in ethanol.
MORPHOLOGY AND ANATOMY
This species has an irregular tubular shape being wider at the base (Figure 17A). It measures 1.0
x 0.8 x 0.1 cm. The surface is slightly hispid due to some protruding spicules. Its texture is
rough. The osculum is apical and has a delicate crown of trichoxeas (arrown in the Figure 17B).
The aquiferous system is leuconoid.
SKELETON
The skeleton is characteristic of the genus (Figure 17C). The cortical skeleton is exclusively
formed by tetractines (Figure 17D) with the basal actines tangentially disposed on the surface.
The choanosomal skeleton is inarticulated, composed of the apical actine of the cortical
tetractines (arrow in Figure 16E), which occasionally crosses the atrial skeleton, and of the
unpaired actine of the subatrial triactines (white arrow in Figure 16F). The subatrial triactines do
not form a continuous layer, instead, they are irregularly scattered at this region. The atrial
skeleton is composed of tetractines with the apical actine projected into the atrium (black arrow
in Figure 16F).
SPICULES (Table 14)
Cortical tetractines. Sagittal. Actines are conical with sharp tips. The paired actines are
frequently curved. The apical actine is straight and it is the longest actine (Figure 17A). Some
undulated apical actines were also observed. Size: 350-485/35-60 μm (paired actine), 75-
200/35-60 μm (unpaired actine) and 285-550/35-55 μm (apical actine).
Subatrial triactines. Sagittal. Actines are conical with sharp tips. The paired actines are straight
and smaller than the unpaired one (Figure 17B-C). Some slightly curved paired actines were
also observed. Very variable size: 175-460/15-60 μm (paired actine) and 225-490/15-55 μm
(unpaired actine).
86
Atrial tetractines. Sagittal. Actines are conical with very sharp tips. The unpaired actine is
slightly longer than the paired ones (Figure 17D). The apical actine is the thinnest and shortest
actine. Size: 120-270/10-12.5 μm (paired actine), 185-355/10-12.5 μm (unpaired actine) and 15-
50/5-10 μm (apical actine).
ECOLOGY
This species was found underneath coral boulders.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
The genus Leucilla comprises 13 valid species distributed in all oceans (Van Soest et al., 2016).
Among these species, none of them has the same skeleton composition as Leucilla antillana sp.
nov. The species that most resemble our new species are Leucilla uter Poléjaeff, 1883 (type
locality: Bermudas) and L. sacculata Carter, 1890 (type locality: Fernando de Noronha
Archipelago, Brazil). However, their skeletons include subatrial tetractines, which are absent in
L. antillana sp. nov. Besides, the size range of the other spicule categories does not match our
new species (Table 14). The apical actine of the cortical tetractines of L. uter is almost twice
longer (400--1200 μm) than that of L. antillana sp. nov. (285-550 μm) and its atrial tetractines
are thicker as well (20 μm against 10-12.5 μm). Comparing to L. sacculata, L. antillana sp. nov.
has thinner spicules (84.7 μm against 15-60 μm). Leucilla sacculata and L. uter also differ from
L. antillana sp. nov. in the ornamentation of the osculum. In L. antillana sp. nov., the osculum is
surrounded by a crown of trichoxeas whereas in L. sacculata it is surrounded by a crown of
microdiactines and in L. uter it is naked. However, this last difference should be taken with
caution as the presence of a crown of trichoxeas is not consistent among individuals of the same
species within some calcaronean genera.
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Fig. 17. Leucilla antillana sp. nov. (UFRJPOR 6768): (A) specimen in vivo; (B) specimen afterfixation with the crown of trichoxea (arrow); (C) cross section of the skeleton; (D) tangentialsection of the cortex; (E) choanoskeleton with the apical actine of a tetractine (arrow); (F) atrialskeleton with a subatrial triactine (white arrow) and an apical actine of an atrial tetractine (blackarrow). Abbreviations: ct=cortex, at=atrium.
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Fig. 18. Spicules of Leucilla antillana sp. nov. (UFRJPOR 6768): (A) cortical tetractine; (B)large subatrial triactine; (C) small subatrial triactine; (D) atrial tetractine.
Table 14. Spicule measurements of Leucilla antillana sp. nov. (H=holotype=UFRJPOR 6768),L. uter and L. sacculata. P=paired, U=unpaired, A=apical and B=basal actines.
Species Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6768 (H)
Cortical Tetractine
P 305.0 404.4 62.0 485.0 35 50.1 6.4 60.0 17U 75.0 131.0 45.3 200.0 35 48.8 7.4 60.0 10A 285.0 405.0 64.6 550.0 35 49.2 7.1 55.0 19
Subatrial Triactine
P 175.0 276.0 91.3 460 15.0 32.8 13.1 60.0 20U 225.0 389.4 71.2 490.0 15.0 34.4 13.4 55.0 18
Atrial Tetractine
P 120.0 224.0 34.8 270.0 10 10.3 0.8 12.5 20U 185.0 265.0 41.9 355.0 10 10.5 1.0 12.5 20A 15.0 30.0 8.9 50.0 5 8.0 1.7 10.0 20
Leucilla uter
Micro-diactine - - 400 - - - 2.5 - - -Cortical Tetractine
B 400.0 - - 600.0 - - - 50 -A 400.0 - - 1200.
0- - - 50 -
Subatrial Triactine
U - - - 600.0 30 - - 50 -P - - - 420.0 21 - - 35 -
89
Atrial Tetractine
P - - - 400.0 - 20 - - -U 250.0 - - 350.0 - 20 - - -A - - - 200.0 - 20 - - -
Leucilla sacculata
Micro-diactine - - 84.7 - - - - - - -Cortical Tetractine
- - 564.4 - - - - - 84.7 -
Subatrial Triactine
- - 564.4 - - - - - 84.7 -
Subatrial Tetractine
- - 564.4 - - - - - 84.7 -
Genus Leucandra Haeckel, 1872
TYPE SPECIES
Leucandra egedii (Schmidt, 1870)
DIAGNOSIS
"Grantiidae with sylleibid or leuconoid organization. Longitudinal large diactines, if present, are
not restricted to the cortex, but lie obliquely across the external part of the sponge wall and
protrude from the surface of the sponge" (Borojevic et al., 2002).
Leucandra caribea sp. nov. (Figures 19-21, Table 17)
ETIMOLOGY
Named after its distribution in the Caribbean Sea.
TYPE LOCALITY
Tug Boat, Caracasbaai, Willemstadt, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6754 (specimen in ethanol and slides); Tug Boat, Caracasbaai,
Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 13.9 m deep; coll. B. Cóndor-Luján, 23
August 2011.
COLOUR
White in life and beige in ethanol.
MORPHOLOGY AND ANATOMY
This species has a sac-shaped external morphology: it is wide at the base and becomes narrower
near the apical osculum (Figure 19A). It measures 0.7 cm length and 0.3 cm width (Figure 19B).
This sponge is quite smooth and compressible. Near the suboscular region, scattered short
trichoxeas protrude through the surface (Figure 19C). Although some trichoxeas do protrude the
90
surface, it is not very hispid. The osculum is supported by triactines, tetractines and has a
discrete crown of trichoxeas (Figures 19D and 19E). The aquiferous system is leuconoid with
subspherical choanocytary chambers ranging from 28 to 34 µm (Figure 19F).
SKELETON
The skeleton is typical of the genus (Figure 20A). As mentioned before, the oscular margin has
a differentiated skeleton. It is composed of T-shaped triactines and tetractines tangentially
disposed and short trichoxeas perpendicular to the cortex. The cortical skeleton is composed of
tangential triactines (Figure 20B). The unpaired actine can point either to the surface or to the
choanosome. The choanosomal skeleton does not have a special organization and it is formed by
triactines of variable size (as shown in Figures 18F and 19A). Several exhalant choanosomal
canals with a diameter varying from 140 to 300 µm were observed within this region. They are
surrounded by tetractines with the apical actine projected inside them (Figure 20C). Some
triactines lining the canals were also found (Figure 20D). No subatrial skeleton was observed.
The atrial skeleton is formed by triactines (Figures 20E) and rare tetractines (Figure 20F). The
apical actine of the tetractines protrudes into the atrial cavity (arrow in Figure 19F).
SPICULES
Cortical triactines. Subregular or sagittal. Actines are slightly conical with sharp tips. The
paired actines are frequently curved and slightly longer than the unpaired one which is always
straight (Figure 21A). Compared to the atrial triactines, the cortical triactines are thicker. Size:
110-340/7.5-16.3 μm (paired actine) and 105-325/7.5-16.3 μm (unpaired actine).
Choanosomal triactines. Regular or subregular. Actines are conical with sharp tips (Figure
21B). They are largest spicules in L. caribea sp. nov. Very variable size: 370-960/25-75 μm.
Triactines and tetractines of the canals. Sagittal. Actines are conical with sharp tips. The paired
actines are curved, following the shape of the choanosomal canals (Figure 21C). The apical
actine of the tetractines is smooth and it is thinner than the basal ones (as shown in Figures 20C-
D). The size of the triactines is similar to that of the tetractines. Size of tetractines: 112.5-
220.0/7.5-12.5 μm (paired), 62.5-210.0/7.5-12.5 μm (unpaired) and 45.0-110.0/5.0-10.0 μm
(apical).
Atrial triactines (shown in Figure 20E): Sagittal. Actines are conical with sharp tips. Compared
to the cortical triactines, the angle formed by the paired actines of the atrial triactines is more
open. Size: 132.3-253.8/8.1-13.5 μm (paired actine) and 164.7-253.8/5.4-13.5 μm (unpaired
actine).
91
Atrial tetractines. Sagittal. Actines are conical with sharp tips (Figure 21D). The apical actine is
smooth. Size: 150-262.5/7.5-15.0 μm (paired actine), 162.0-297.0/8.1-16.2 μm (unpaired actine)
and 35.0-132.5/7.5-12.5 μm (apical actine).
ECOLOGY
This specimen was found underneath a coral boulder.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
Among the species of Leucandra reported from the Caribbean Sea, namely L. crustacea
(Haeckel, 1872), L. barbata (Duchassaing & Michelotti, 1864), L. curva (Schuffner, 1877), L.
multiformis Poléjaeff, 1883, L. rudifera Poléjaeff, 1883, and L. typica (Poléjaeff, 1883) (Van
Soest et al., 2016), only L. typica possess a similar skeleton composition as that of L. caribea
sp. nov. The skeletons of the other Caribbean species include cortical tetractines (L. crustacea
and L. curva), diactines (L. barbata, L. multiformis and L. rudifera) and atrial grapnel spicules
(also in L. rudifera), all of which are not present in L. caribea sp. nov.
Leucandra caribea sp. nov. can be differentiated from L. typica as the former possess an
atrial skeleton mainly composed of triactines and few tetractines whereas in the latter triactines
and tetractines are in the same proportion (or at least, Poléjaeff did not indicate the opposite).
Besides, in the new species, the choanosomal canals are lined by tetractines and triactines and in
L. typica, they are only lined by tetractines. In L. typica, trichoxeas (<300/1 μm) are scattered in
the choanosome and spindle-shaped microdiactines (100/4 μm) are concentrated in the
suboscular region whereas in L. caribea sp. nov., trichoxeas (>100/1.2) were found only in the
suboscular region.
Within the other species of Leucandra with similar skeleton composition and external
morphology, L. falakra Klautau et al., 2016 from the Adriactic Sea is the one that most
resembles L. caribea sp. nov. Nonetheless, they have some important differences. The
choanosomal skeleton of the Curaçaoan species is composed of one single type of triactine
(370-960/25-75) while in the Adriatic species, it is composed of small (paired actine: 94.5-
180.9/8.1-13.5 μm and unpaired actine: 70-143.1/8.1-16.2) and giant triactines (342-
1047.6/48.6-118.8 μm). In addition, although almost all the spicule categories have similar size
(Table 17), the unpaired actine of the atrial tetractines is shorter in L. falakra (59.4-126.9 µm)
than in the new species (162-297 µm).
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Table 17. Spicule measurements of Leucandra caribea sp. nov. (H=holotype: UFRJPOR 6754)and Leucandra falakra (H=holotype: UFRJPOR 8349). P=paired, U=unpaired and A=apical
actines.
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6754
Trichoxea (crown)
- - - - >105 1.1 1.2 0.2 1.6 15
Trichoxea (cortex)
- - - - >108 1.1 1.2 0.2 1.6 10
Cortical triactine
P 110-.0 218.4 65.2 340.0 7.5 11.6 2.4 16.3 25U 105.0 219.3 63.7 325.0 7.5 12.6 2.4 16.3 25
Choanosomaltriactine
- 370.0 576.3 188.8 960.0 25.0 41.3 10.8 75.0 24
Canal tetractine
P 112.5 179.3 32.0 220.0 7.5 9.8 1.8 12.5 10U 62.5 140.6 60.4 210.0 7.5 10.0 2.0 12.5 4A 45.0 70.9 19.0 110.0 5.0 7.9 1.3 10.0 14
Atrial triactine
P 132.3 209.4 41.8 253.8 8.1 11.2 1.9 13.5 12U 164.7 210.6 35.0 253.8 5.4 10.1 3.4 13.5 6
Atrial tetractine
P 150.0 206.9 28.7 262.5 7.5 11.6 1.9 15.0 20U 162.0 237.6 35.1 297.0 8.1 12.9 2.5 16.2 20A 35.0 69.0 28.3 132.5 7.5 9.6 1.2 12.5 20
UFRJPOR8349
Cortical triactine
P 94.5 136.4 24.0 180.9 8.1 11.1 1.9 13.5 20U 70.2 106.0 18.8 143.1 8.1 11.4 2.4 16.2 20
Cortical and choanosomal triactine
- 342.0 624.5 192.3 1047.6
48.6 81.5 20.6 118.8
23
Choanosomaltriactine
P 162.0 214.2 39.8 288.9 13.5 18.3 4.0 27.0 20U 108.0 189.7 58.9 351.0 13.5 19.8 4.1 29.7 20
Canal tetractine
P 99.9 154.0 26.4 199.8 8.1 12.4 2.4 16.2 19U 45.9 143.0 56.5 288.9 9.5 12.4 1.9 16.2 19A 50 80.6 24.4 137.5 7.5 9.6 1.5 12.5 20
Atrial triactine
P 140.4 222.7 33.7 294.3 9.5 15.1 2.5 20.3 30U 78.3 111.2 24.4 159.3 8.1 12.3 1.7 16.2 30
Atrial tetractine
P 145.8 191.4 26.0 256.5 10.8 14.9 2.6 18.9 30U 59.4 92.0 22.1 126.9 10.8 13.1 1.7 16.2 16A 67.5 110.3 30.3 162.0 8.1 11.9 2.8 16.2 15
93
Fig. 19. Leucandra caribea sp. nov.: (A) specimen in vivo; (B) specimen after fixation; (C)detail of trichoxeas (arrow) along the surface; (D) tangential section of the oscular region; (E)detail of the oscular trichoxeas (arrow); (F) cross section of the skeleton. Abbreviations:c=choanosomal canals, cc=choanocytary chambers.
94
Fig. 20. Skeleton of Leucandra caribea sp. nov.: (A) cross section of the skeleton; (B) cortex;(C) tetractine of a choanosomal canal; (D) triactine of a choanosomal canal; (E) atrial triactine;(F) atrial tetractine with the apical actine projected into the atrial cavity (arrow). Abbreviations:cx=cortex, c=choanosomal canal, ct=cortical triactines, at=atrium.
95
Fig. 21. Spicules of Leucandra caribea sp. nov: (A) cortical triactine; (B) choanosomaltriactine; (C) tetractine of choanosomal canals; (D) atrial tetractine.
Genus Leucandrilla Bojorevic, Boury-Esnault & Vacelet, 2000
TYPE SPECIES
Leucilla wasinensis Jenkin, 1908
DIAGNOSIS
"Grantiidae with leuconoid organization. In addition to triactines the cortex contains tetractines,
with the apical actines turned into the choanoderm. The articulated choanoskeleton is supported
by subatrial triactine spicules, and numerous rows of choanosomal triactines and/or tetractines,
with apical actines of cortical tetractines in the distal region" (Borojevic et al., 2002).
Leucandrilla pseudosagittata sp. nov. (Figures 22-24, Table 18)
ETIMOLOGY
Derived from the presence of subcortical tetractines with pseudosagittal shape.
96
TYPE MATERIAL
Holotype: UFRJPOR 6752 (specimen in ethanol and slides); Tug Boat, Caracasbaai,
Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 15.2 m deep; coll. E. Hajdu, 23 August
2011.
Paratypes: UFRJPOR 6696 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao;
12°07'18.94"N, 68°58'11.46"W; <10 m deep; coll. B. Cóndor-Luján and G. Lôbo-Hajdu, 17
August 2011 and UFRJPOR 6705 (specimen in ethanol and slides); Water Factory, Willemstadt,
Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011.
TYPE LOCALITY
Tug Boat, Caracasbaai, Willemstadt, Curaçao.
COLOUR
White in life and in ethanol.
MORPHOLOGY AND ANATOMY
This species has a cylindrical massive body (Figure 22A-E). The holotype is the largest
specimen and it measures 3.8 cm x 2.0 cm (Figures 22A and 22C). In this specimen, the
choanosomal wall is thinner at the apical region (0.2 cm) and thicker at the basal region (0.6
cm).The surface is slightly rough and the consistency is hard. The osculum is apical and its
diameter is 1.2 mm. The atrial cavity is not hispid and measures 0.4 cm. The aquiferous system
is leuconoid with spherical choanocytary chambers.
SKELETON
In the three analised specimens the oscular margin is composed of T-shaped spicules including
triactines and rare tetractines (Figure 23A). The skeleton is not typical of the genus (Figure 23B)
as it presents rare tetractines with pseudosagittal shape. The cortical skeleton is composed of
triactines (Figure 23B and 23C) tangentially disposed on the surface. The subcortical skeleton is
formed by large sagittal tetractines. Some of them have pseudosagittal shape (white arrow in
Figure 23D). The longer paired actine of the pseudosagittal tetractines and the apical actine of
the sagittal tetractines cross the choanosome and sometimes reach the atrial skeleton. The
choanosomal skeleton has no special organization and some subcortical tetractines invade this
region. The choanosomal canals (average diameter: 108-238 µm) are surrounded by small
tetractines whose apical actine is projected into them (Figure 23E). The poorly developed
subatrial skeleton is formed only by tetractines (black arrow in Figure 23D). The atrial skeleton
is exclusively composed of tetractines with the apical actine projected into the atrial cavity
(Figure 23F).
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SPICULES
Cortical triactines. Sagittal. Actines are conical with blunt to sharp tips. Some paired actines are
slightly curved (Figure 24A). Very variable size: 97.2-421.2/8.1-21.6 µm (paired actine), 75.6-
421.2/5.4-21.6 µm (unpaired actine).
Subcortical tetractines I. Sagittal. Actines are conical with sharp tips. The paired actines are
slightly curved (Figure 24B). The apical actine is generally longer than the paired ones. Size:
248.4-1188.0/27.0-75.6 µm (paired actine), 162.0-564.0/27.0-64.8 µm (unpaired actine) and
345.6-1122.0/21.6-75.6 µm (apical actine).
Subcortical tetractines II (indicated in Figure 24D). Pseudosagittal. Rare. Actines are conical
with sharp tips. Size: 270.0-529.2/37.8-54.0 µm (shorter paired actine), 237.6-1134.0/32.4-75.6
µm (longer paired actine), 270.0-1036.8/32.4-75.6 µm (unpaired actine) and 162.0-540.0/21.6-
64.8 µm (apical actine).
Canal tetractines. Sagittal. Actines are conical with sharp tips. The paired actines are curved
following the shape of the canals and they are longer than the other actines (Figure 24C). Size:
118.8-264.6/8.1-17.6 µm (paired actine), 70.2-213.3/8.1-17.6 µm (unpaired actine) and 40.5-
132.3/8.1-13.5 µm (apical actine).
Subatrial tetractines. Sagittal. Actines are conical with sharp tips (Figure 24D). The apical
actine is shorter than the basal ones. Size: 205.2-993.6/21.6-75.6 µm (paired), 205.2-766.8/21.6-
70.2 µm (unpaired) and 129.6-432.0/16.2-54.0 µm (apical actine).
Atrial tetractines. Sagittal. Actines are conical with blunt to sharp tips (Figure 24E). Sometimes
the paired actines are slightly curved. The apical actine is conical, shorter than the basal ones
and has sharp tips. Size: 67.5-391.5/8.1-21.6 µm (paired actine), 59.4-270.0/8.1-21.6 µm
(unpaired actine) and 21.6-108.0/6.4-13.5 µm (apical actine).
ECOLOGY
The three specimens were collected in light protected habitats. The holotype and one paratype
(UFRJPOR 6696) were collected underneath boulders. A polychaete was found inside the
holotype.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
Although the skeleton of the analysed specimens from Curaçao presents rare subcortical
tetractines with pseudosagittal shape, which would suggest their allocation in one of the genera
of the family Heteropiidae, we placed them in the genus Leucandrilla following Borojevic et
al., 2000: "In any calcaronean sponge with a strong cortex, some subcortical spicules may be in
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the position and have the shape of pseudosagittal spicules, due to the restriction of their growth
by the rigidity of the cortical skeleton. They should not be interpreted as an indication that the
sponge belongs to the family Heteropiidae."
Three species are considered within the genus Leucandrilla: L. intermedia (Row, 1909)
from the Red Sea, L. lanceolata (Row & Hôzawa, 1931) from Southwestern Australia and L.
wasinensis (Jenkin, 1908) from East Africa. Leucandrilla pseudosagittata sp. nov. can be easily
differentiated from them by the absence of diactines and choanosomal triactines and by the
presence of pseudosagittal subcortical tetractines.
The two sequences of L. pseudosagittata sp. nov. formed a monophyletic clade (pp=1,
bb=100) within the large clade LEUCII (which did not include any Heteropiidae species, see
Phylogenetic section). In both BI and ML phylogenetic trees, L. pseudosagittata nested with
other Grantiidae and Amphoriscidae species but with low support. In the BI tree (Figure 30), it
grouped with Amphoriscus micropilosus sp. nov., Leucilla antillana sp. nov., Sycon conulosum
sp. nov. and S. megapicalis sp. nov. (pp=0.57) and in the ML tree (data not shown), it clustered
with Leucandra nicolae, Paraleucilla magna and P. dalmatica (b=44.7).
Fig. 21. Leucandrilla pseudosagittata sp. nov.: (A-B) Specimens in vivo; (C-E) specimens afterfixation.
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Fig. 22. Skeleton composition of Leucandrilla pseudosagittata sp. nov. (paratype UFRJPOR6705): (A) osculum; (B) cross section of the skeleton; (C) cortex; (D) choanoskeletonindicating the subcortical tetractine (white arrow) and the subatrial tetractine (black arrow); (E)choanosomal canals (c); (F) atrial skeleton. Abbreviations: ct=cortex, at=atrium.
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Fig. 23. Spicules of Leucandrilla pseudosagittata sp. nov. (paratype UFRJPOR 6705): (A)cortical triactine; (B) subcortical tetractine; (C) tetractine of a canal; (D) subatrial tetractine; (E)atrial tetractine.
Table 18. Spicule measurements of Leucandrilla pseudosagittata sp. nov. (UFRJPOR 6696,UFRJPOR 6705 and UFRJPOR 6752). H=holotype. P=paired, U=unpaired and A=apical
actines.
Specimen Spicule ActineLength Width N
Min Mean SD Max Min Mean SD MaxUFRJPOR6696 (H)
Cortical Triactine
P 97.2 273.2 77.4 399.6 8.1 14.9 3.6 21.6 20U 97.2 207.4 70.3 345.6 5.4 14.1 4.3 21.6 20
SubcorticalTetractine I
P 248.4 543.8 161.5 810.0 27.0 43.5 10.3 64.8 20U 162.0 271.1 90.0 399.6 32.4 37.8 5.7 43.2 10A 367.2 541.8 104.2 702.0 21.6 38.4 10.9 64.8 17
SubcorticalTetractine II
P + 356.4 574.4 154.7 896.4 32.4 53.0 11.3 64.8 11P - 313.2 402.8 71.8 529.2 37.8 46.4 5.8 54.0 10U 324.0 479.5 105.7 648.0 32.4 49.7 8.0 59.4 10A 162.0 261.4 78.7 378.0 21.6 32.9 6.5 43.4 10
Canal Tetractine
P 118.8 200.7 39.9 256.5 8.1 12.6 2.8 17.6 20U 97.2 135.8 25.4 189.0 8.1 12.4 2.6 17.6 20A 54.0 89.4 23.1 124.2 8.1 10.2 1.8 13.5 20
Subatrial P 205.2 522.7 172.1 993.6 21.6 44.8 12.3 75.6 20
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Tetractines U 205.2 388.3 113.8 637.2 21.6 35.4 7.9 48.6 20A 129.6 276.9 87.8 432.0 16.2 33.2 8.2 43.2 14
Atrial Tetractine
P 67.5 201.1 57.5 297.0 8.1 13.1 3.2 18.9 19U 59.4 187.5 54.3 240.3 8.1 12.6 3.4 18.9 11A 32.4 62.9 23.0 105.3 6.4 9.3 2.1 14.9 11
UFRJPOR 6705
Cortical Triactine
P 129.6 307.3 85.0 410.4 9.5 13.8 3.2 21.6 21U 129.6 261.4 65.3 367.2 9.5 14.0 3.4 21.6 12
SubcorticalTetractine I
P 259.2 639.4 211.0 972.0 27.0 54.3 13.5 70.0 20U 162.0 350.1 103.3 464.4 27.0 39.6 11.4 64.8 12A 345.6 511.2 147.7 864.0 37.8 51.3 8.7 70.2 20
SubcorticalTetractine II
P + 453.6 680.4 162.5 1134.0
43.2 58.4 7.9 70.2 16
P - 237.6 460.1 145.5 648.0 43.2 52.4 3.6 54.0 10U 270.0 583.2 145.9 756.0 48.6 53.3 4.0 64.8 15A 270.0 359.1 78.3 486.0 43.2 52.0 7.0 64.8 8
Canal Tetractine
P 143.1 210.7 32.1 264.6 8.1 12.5 2.5 17.6 20U 97.2 143.3 29.1 205.2 9.5 13.1 2.3 17.6 18A 62.1 86.4 16.7 124.2 8.1 9.6 1.5 13.5 20
Subatrial Tetractines
P 259.2 489.8 207.9 864.0 32.4 48.3 13.2 70.2 20U 216.0 405.0 131.6 669.6 32.4 48.3 11.8 70.2 20A 129.6 279.0 99.5 432.0 21.6 35.1 9.4 54.0 12
Atrial Tetractine
P 135.0 230.0 69.4 391.5 8.1 13.8 3.5 21.6 20U 94.5 164.4 43.8 264.6 9.5 13.4 3.1 21.6 20A 27.0 46.4 14.0 64.8 8.1 9.5 2.3 13.5 19
UFRJPOR 6752
Cortical Triactine
P 118.8 276.5 89.3 421.2 10.8 15.0 2.7 20.3 20U 75.6 242.5 84.9 421.2 12.2 15.1 2.3 18.9 20
SubcorticalTetractine I
P 313.2 689.6 222.4 1188.0
32.4 52.9 11.0 75.6 20
U 194.4 360.7 134.3 594.0 32.4 36.7 7.1 54.0 10A 367.2 693.6 232.2 1122.
032.4 51.3 11.0 75.6 20
SubcorticalTetractine II
P + 237.6 539.2 197.1 810.0 32.4 45.5 11.2 75.6 14P - 270.0 459.0 121.5 594.0 37.8 47.0 6.3 54.0 10U 313.2 548.1 258.1 1036.
832.4 48.6 12.6 75.6 12
A 216.0 346.8 93.1 540.0 32.4 39.0 5.2 43.2 9Canal Tetractine
P 118.8 186.4 46.5 245.7 8.1 12.0 2.4 16.2 20U 70.2 128.5 41.2 213.3 8.1 10.9 2.6 16.2 20A 40.5 81.7 23.5 132.3 8.1 10.2 1.7 13.5 20
Subatrial Tetractine
P 280.8 518.4 191.1 853.2 32.4 46.5 10.7 59.4 13U 302.4 502.7 167.1 766.8 32.4 44.7 9.4 54.0 11A 216.0 332.6 66.1 432.0 27.0 40.0 10.6 54.0 10
Atrial Tetractine
P 153.9 205.9 48.0 332.1 9.5 13.8 3.0 21.6 29U 126.9 202.8 40.9 270.0 9.5 13.8 3.1 21.6 24A 21.6 60.5 23.0 108.0 6.7 9.8 1.9 14.9 30
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Genus Grantessa Lendenfeld, 1885TYPE SPECIES
Grantessa sacca Lendenfeld, 1885
DIAGNOSIS
"Heteropiidae with syconoid organization and an articulated choanoskeleton. A thin cortex is
formed by triactines but lacks longitudinal large diactines. The distal part of the radial tubes is
frequently decorated by tufts of radially arranged diactines, indicating a close relationship to the
genus Syconessa" (Borojevic et al., 2002).
Grantessa tumida sp. nov. (Figures 24 & 25, Table 19)
ETIMOLOGY
From the Latin tumidus (=swollen), for the presence of subatrial and atrial spicules with distally
swollen unpaired actines.
TYPE LOCALITY
Sunset Waters, Soto, Curaçao
TYPE MATERIAL
Holotype: UFRJPOR 6766 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao;
12°16'01.58"N, 69°07'44.85"W; 13.1 m deep; coll. B. Cóndor-Luján, 23 August 2011.
Paratypes: UFRJPOR 6701 (specimen in ethanol and slides); Daai Booi, St. Willibrordus,
Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011
and UFRJPOR 6695 (specimen in ethanol and slides); Hook’s Hut, Willemstadt, Curaçao;
12°07'18.94"N, 68°58'11.46"W; 6.3 m deep; coll. B. Cóndor-Luján & G. Lôbo-Hajdu, 17
August 2011.
COLOUR
Beige to light brownish in life and beige in ethanol.
MORPHOLOGY AND ANATOMY
This species has a tubular to sac-shaped body with an apical osculum surrounded by a crown of
trichoxeas. The holotype (UFRJPOR 6766) is the largest specimen. It measures 0.5 x 0.2 cm
(Figure 24A). The surface is smooth to the touch although diactines protrude through the
surface. The consistency is compressible. The aquiferous system is syconoid.
SKELETON
The osculum is surrounded by a crown of trichoxeas supported by T-shaped triactines and rare
tetractines (Figure 24B). The skeleton is typical of the genus. The cortical skeleton is composed
of diactines and triactines (Figure 24C). The diactines are perpendicularly arranged in tufts and
103
do not penetrate the choanosome. The triactines are tangentially disposed with the paired actines
laying tangentially to the subcortical region (black arrow in Figure 24C). The subcortical
skeleton is composed of pseudosagittal triactines with the longest paired actine (actine 1)
penetrating the choanosome (white arrow in Figure 24C). The tubar skeleton is articulated
(Figure 24D), composed of several rows of triactines with the unpaired actine pointing to the
cortex. The subatrial skeleton is composed of triactines with the unpaired actine pointing to the
cortex (Figure 24E). The atrial skeleton is composed of triactines and tetractines with the
unpaired actine adjacent to the atrium (in the common position of the paired actines). Some
atrial tetractines with the unpaired actine disposed in the traditional position were also found.
The apical actine of the tetractines penetrates the atrial cavity (Figure 24F).
SPICULES
Diactines. Fusiform, straight, with tips usually sharp (Figures 25A-B). Size: 108.0-637.2/5.4-
16.2 µm.
Cortical triactines. Sagittal. Actines are conical with sharp tips. The paired actines are less
straight than the unpaired one (Figure 25C). Size: 48.6-102.6/4.1-8.1 µm (paired actine), 51.3-
175.5/4.1-8.1 µm (unpaired actine).
Subcortical triactines. Pseudosagittal. Actines are conical with sharp tips. One of the paired
actines is shorter and more curved than the other. The unpaired actine is straight (Figure 25D).
Size: 67.5-145.8/4.1-5.4 µm (paired actine 1), 51.3-113.4/2.7-5.4 µm (paired actine 2), 54.0-
129.6/4.1-8.1 µm (unpaired actine).
Tubar triactines I. Sagittal. Actines are conical with sharp tips. One of the paired actines is
shorter and more curved than the other. The unpaired actine is straight and usually slightly
longer than the paired ones (Figure 25E). Size: 62.1-140.4/5.4-8.1 µm (paired actine), 21.6-
72.9/4.1-8.1 µm (short paired actine), 99.9-202.5/5.4-8.1µm (unpaired actine).
Tubar triactines II. Sagittal. Actines are conical with sharp tips. The paired actines have almost
the same size and are slightly curved. The unpaired actine is straight and generally longer than
the paired ones (Figure 25F). Size: 48.6-121.5/5.4-8.1 µm (paired actine), 72.9-205.5/5.4-8.1
µm (unpaired actine).
Subatrial triactine. Sagittal. Actines are slightly conical with sharp tips. The unpaired actine is
straight and longer than the paired ones. The paired actines are inwardly curved (Figure 25G).
Some paired actines with different lengths in the same spicule were also found. Size: 40.5-
108.8/4.1-6.8 µm (paired actine), 35.1-243.0/4.1-8.1 µm (unpaired actine).
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Atrial triactine. Sagittal. Actines are conical with sharp tips. The unpaired actine is elongated
and swollen at the distal part (Figures 25H-I). Size: 89.1-162.0/4.1-8.1µm (paired actine),
159.3-283.5/5.4-8.1 µm (unpaired actine).
Atrial tetractine I: Sagittal. Actines are conical with sharp tips. The unpaired actine is longer
than the paired ones (Figure 25J). Some apical actines are curved. Size: 78.3-148.5/4.1-5.4 µm
(paired actine), 108.0-280.8/5.4-8.1 µm (unpaired actine), 13.5-108.0/4.1-6.8 µm (apical actine).
Atrial tetractine II. Sagittal. Actines are conical with sharp tips. The unpaired actine is elongated
in tetractines II and, unlike the unpaired actine of the tetractines I, it is swollen at the distal part
(Figure 25H). The apical actine is curved and it is the shortest actine of this species. Size: 94.5-
151.2/4.1-8.1 µm (paired actine), 162.0-283.5/5.1-8.1 µm (unpaired actine), 13.5-43.2/4.1-5.4
µm (apical actine).
ECOLOGY
The specimens were found underneath coral boulders. One of the paratypes (UFRJPOR 6695)
was covered with sediment when collected.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
Among the 28 valid species of the genus Grantessa, only G. ramosa sensu Borojevic (1967)
from South Africa and G. tenhoveni Van Soest & de Voogd, 2015 from Indonesia present atrial
spicules with swollen unpaired actine. Grantessa ramosa sensu Borojevic (1967) is very similar
to the Curaçaoan species, however, they present some dissimilarities. In G. tumida sp. nov. all
the spicules (but the diactines) are thinner (Table 19) and the apical actine of the atrial
tetractines is straight whereas in G. ramosa sensu Borojevic (1967) it is curved. Differently
from G. tenhoveni, the skeleton of our new species does not bear subatrial tetractines nor atrial
tetractines with a very long apical actine (660-960 µm) as described for the Indonensian
species.
It is important to point out here that G. ramosa sensu Borojevic (1967) differs greatly from
the original description of G. ramosa (Haeckel, 1872), also from South Africa, in spicule size
and shape (Table 22). Based on the written descriptions and drawings, they probably belong to
two different species.
The C-LSU sequences of G. tumida sp. nov. (UFRJPOR6701 and UFRJPOR6695) grouped
together in the same cluster (pp=1, b=100) within the large clade LEUCI (which included
species of the Grantiidae and Heteropiidae families). Nonetheless, G. tumida sp. nov. did not
group with the other Grantessa species, G. aff. intusarticulata (Figure 30).
105
Fig. 24. Grantessa tumida sp. nov. (Holotype=UFRJPOR 6766): (A) specimen after fixation;(B) osculum; (C) distal part of the skeleton indicating a cortical (black arrow) andpseudosagittal subcortical triactine (white arrow); (D) tubar skeleton; (E) subatrial skeleton; (F)atrial skeleton.
106
Fig. 25. Spicules of Grantessa tumida sp. nov (Holotype=UFRJPOR 6766): (A, B) diactine; (C)cortical triactine; (D) subcortical triactine; (E) tubar triactine I; (F) tubar triactine II; (G)subatrial triactine; (H) atrial triactine; (I) detail of the swollen tip of the atrial triactine; (J) atrialtetractine II.
107
Table 19. Spicule measurements of Grantessa tumida sp. nov. (UFRJPOR 6695, UFRJPOR6701 and UFRJPOR 6766), G. ramosa (Haeckel, 1872) and G. ramosa sensu Borojevic (1967).
H=holotype, P=Paired, U=unpaired, A=apical, CP=cortical paired and IP=internal pairedactines.
Specimen Spicule Actine Length (µm) Width (µm) N
Min Mean S Max Min Mean S Max
UFRJPOR6695
Diactine 108.0 250.6 99.6 432.0 5.4 9.7 2.8 16.2 20Cortical triactine
P 48.6 62.1 9.8 81.0 5.4 5.4 0.0 5.4 20U 51.3 93.9 21.7 121.5 5.4 5.6 0.5 6.8 20
Pseudosagittal Triactine
P1 67.5 106.7 27.3 140.4 4.1 5.1 0.6 5.4 15P2 51.3 73.7 17.9 108.0 4.1 5.1 0.6 5.4 13U 54.0 93.4 24.5 124.2 4.1 5.1 0.6 5.4 15
Tubar Triactine 1
P1 72.9 110.0 22.2 140.4 5.4 6.4 1.1 8.1 20P2 29.7 50.1 14.3 72.9 5.4 5.8 0.6 6.8 20U 99.9 138.4 21.4 164.7 5.4 6.0 0.8 8.1 20
Tubar Triactine
P 51.3 91.8 20.8 121.5 5.4 6.4 1.1 8.1 20U 99.9 153.4 35.3 207.9 5.4 6.0 0.8 8.1 20
Subatrial Triactine
P 43.2 83.5 21.8 108.0 4.1 5.2 0.9 6.8 15U 70.2 156.2 60.8 229.5 4.1 5.8 0.9 6.8 14
Atrial triactine
P 94.5 119.8 17.4 148.5 4.1 5.1 0.6 5.4 14U 159.3 223.3 32.9 283.5 5.4 6.1 1.0 8.1 20
Atrial Tetractine I
P 78.3 117.5 22.7 148.5 4.1 5.1 0.6 5.4 12U 121.5 196.9 53.9 270.0 5.4 6.4 1.2 8.1 12A 24.3 56.2 17.8 86.4 4.1 4.9 0.7 5.4 10
Atrial Tetractine II
P 94.5 119.2 14.0 148.5 4.1 5.3 0.6 6.8 14U 162.0 222.3 29.5 270.0 5.1 5.6 0.5 6.8 18A 24.3 30.2 5.2 37.8 4.1 5.0 0.7 5.4 6
UFRJPOR6701
Diactine - 108.0 360.7 110.0 615.6 5.4 10.8 3.5 16.2 20Cortical triactine
P 56.7 78.4 10.7 102.6 5.4 5.5 0.6 8.1 20U 86.4 117.5 15.8 140.4 5.4 6.0 0.8 8.1 20
Pseudosagittal Triactine
P1 78.3 125.1 17.5 145.8 5.4 5.4 0.0 5.4 20P2 56.7 92.2 15.6 108.0 4.1 5.2 0.5 5.4 20U 78.3 105.3 13.9 129.6 4.1 5.3 0.8 8.1 20
Tubar Triactine 1
P1 62.1 89.4 9.1 105.3 5.4 6.2 1.2 8.1 20P2 27.0 45.6 12.3 72.9 4.1 5.6 1.6 8.1 20U 105.3 129.9 20.5 194.4 5.4 6.1 0.9 8.1 20
Tubar Triactine 2
P 78.3 88.0 7.9 105.3 5.4 6.1 1.1 8.1 20U 72.9 129.2 27.7 189.0 5.4 6.2 1.2 8.1 20
Subatrial Triactine
P 48.6 74.9 11.8 89.1 4.1 5.1 0.6 5.4 16U 35.1 151.6 59.2 243.0 4.1 5.6 1.0 8.1 16
Atrial triactine
P 89.1 120.4 19.8 162.0 4.1 5.2 0.5 5.4 20U 162.0 223.4 27.2 270.0 5.4 6.3 1.2 8.1 20
Atrial Tetractine I
P 83.7 115.0 25.9 145.8 4.1 5.1 0.6 5.4 10U 129.6 179.6 49.3 280.8 5.4 5.9 1.0 8.1 8A 21.6 46.5 24.4 86.4 4.1 5.1 0.6 5.4 9
Atrial P 94.5 125.3 18.6 151.2 4.1 5.1 0.6 5.4 10
108
Tetractine II
U 189.0 230.6 28.3 270.0 5.4 6.3 1.3 8.1 12A 13.5 29.2 11.7 43.2 4.1 5.1 0.6 5.4 5
UFRJPOR6766 (H)
Diactine 194.4 381.2 138.2 637.2 5.4 9.7 2.8 16.2 20Cortical Triactine
P 51.3 79.4 12.7 102.6 4.1 5.3 0.4 5.4 20U 81.0 128.9 22.3 175.5 4.1 5.5 0.8 8.1 20
Pseudosagittal Triactine
P1 78.3 110.7 18.1 137.7 4.1 5.3 0.3 5.4 15P2 62.1 84.6 14.2 113.4 2.7 5.0 0.8 5.4 15U 56.7 101.8 26.9 126.9 4.1 5.3 0.4 5.4 10
Tubar Triactine 1
P1 62.1 95.9 15.2 113.4 5.4 6.3 1.2 8.1 20P2 21.6 50.2 11.5 72.9 5.4 5.6 1.5 8.1 20U 124.2 162.9 21.2 202.5 5.4 5.9 0.7 6.8 20
Tubar Triactine 2
P 48.6 95.6 13.3 118.8 5.4 6.0 1.0 8.1 20U 83.7 145.9 27.9 205.2 5.4 5.9 0.8 8.1 20
Subatrial Triactine
P 40.5 80.3 17.3 99.9 4.1 5.3 0.6 6.8 16U 67.5 162.0 47.6 224.1 4.1 5.7 1.0 6.8 16
Atrial triactine
P 97.2 123.1 13.2 148.5 4.1 5.7 1.1 8.1 20U 189.0 233.1 26.7 283.5 5.4 6.1 1.0 8.1 20
Atrial Tetractine I
P 97.2 118.8 17.5 137.7 4.1 5.1 0.7 5.4 4U 108.0 160.7 53.4 234.9 5.4 6.4 1.3 8.1 4A 13.5 83.4 27.6 108.0 5.4 5.7 0.6 6.8 10
Atrial Tetractine II
P 94.5 121.5 16.9 148.5 5.4 6.2 1.1 8.1 10U 189.0 240.5 28.7 283.5 5.4 6.1 1.1 8.1 11A 13.5 21.2 8.4 35.1 5.4 5.4 0.0 5.4 6
Grantessaramosa (Haeckel, 1872)
Diactine - 60.0 - - 80.0 - - - -Tubar Triactine
P 40.0 - - 80.0 6.0 - - 8.0U 80.0 - - 120.0 6.0 - - 8.0
Subatrial Triactine
P 40.0 - - 80.0 6.0 - - 8.0U 160.0 - - 200.0 6.0 - - 8.0
Atrial Tetractine
P 50.0 - - 80.0 6.0 - - 8.0U 100.0 - - 120.0 6.0 - - 8.0A 20.0 - - 30.0 - 8.0 - -
Grantessaramosa sensu Borojevic (1967)
Diactine - 150.0 - - 380.0 6.0. - - 12.0Cortical Triactine*
U 70.0 - - 150.0 8.0 - - 12.0
Subcortical Triactine
CP 80.0 - - 180.0 15.0 - - 18.0IP 150.0 - - 240.0 15.0 - - 18.0U 100.0 - - 200.0 14.0 - - 16.0
Tubar Triactine
U 150.0 - - 230.0 14.0 - - 18.0
Subatrial Triactine
P 80.0 - - 120.0 9.0 - - 11.0U 180.0 - - 250.0 12.0 - - 14.0
AtrialTriactine
P 60.0 - - 180.0 8.0 - - 10.0U 200.0 - - 400.0 8.0 - - 10.0
Atrial Tetractine
P 60.0 - - 180.0 - 25.0 - -U <200 - - <400 - 25.0 - -A - - - 140.0 - 25.0 - -
* Paired actines slightly longer than unpaired actine.
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Genus Sycon Risso, 1827
TYPE SPECIES
Sycon humboldti Risso, 1827
DIAGNOSIS
"Sycettidae with radial tubes partially or fully coalescent; distal cones are decorated by tufts of
diactines. The inhalant canals are generally well defined between the radial tubes and are often
closed at the distal end by a membrane that is perforated by an ostium, devoid of a skeleton.
There is no continuous cortex covering the distal ends of the radial tubes. Skeleton of the atrium
and of the tubes composed of triactines and/or tetractines" (Borojevic et al., 2002).
Sycon conulosum sp. nov. (Figures 26 & 27, Tables 20 & 21)
ETIMOLOGY
Derived from the conulose appearance of the surface.
TYPE LOCALITY
Daai Booi, St. Willibrordus, Curaçao.
TYPE MATERIAL
Holotype: UFRJPOR 6707 (specimen in ethanol and slides); Daai Booi, St. Willibrordus,
Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5m; coll. B. Cóndor-Luján, 18 August 2011.
COLOUR
White in life and in ethanol.
MORPHOLOGY AND ANATOMY
This species has a ficiform shape (Figure 26A). It measures 0.9 cm long and 0.7 cm wide. The
surface is hispid with tufts of diactines protruding through the body. It has a conulose
appearance because of the distal cones, which are very separated. The osculum (diameter=0.9
mm) is apical with no crown and it is supported by sagittal triactines (Figura 26B-C). The
atrium measures 2.4 mm. The radial tubes are fully coalescent (Figure 26D). The aquiferous
system is syconoid.
SKELETON
The skeleton is typical of the genus. The skeleton of the distal cones is composed of short
diactines, rare trichoxeas and triactines (white arrow in Figure 26E). The tubar skeleton is
articulated, composed of several rows of triactines with the unpaired actine pointing towards the
distal cones (black arrow in Figure 26E). The subatrial skeleton is composed of triactines whose
unpaired actine points to the surface (arrow in Figure 26F). The atrial skeleton is composed of
110
tangential triactines (black arrow in Figure 26G) and fewer tetractines (white arrow in Figure
26G). The apical actine of the tetractines penetrates the atrial cavity.
SPICULES (Table 20)
Diactines. Fusiform with tips usually sharp (Figure 27A). Size: 100-275/6.3-11.3 µm.
Triactines of the distal cones. Sagittal. Actines are conical with sharp tips. The paired actines are
frequently curved (Figure 27B). In the distal part of the cone, the unpaired actine is very long.
Size: 50-102.5/5-10 µm (paired actine) and 65-175/5-8.8 µm (unpaired actine).
Tubar triactines I. Sagittal. Actines are conical with sharp tips. One of the paired actines is
shorter and more curved than the other. The unpaired actine is straight and it is generally slightly
longer than the paired ones (Figure 27C, left). Size: 32.5-60/5-10 µm (short paired actine), 67.5-
95/5-11.3 µm (long paired actine) and 82.5-125/5-10 µm (unpaired actine).
Tubar triactines II. Sagittal. Actines are conical with sharp tips. The paired actines are slightly
curved and have equal sizes. The unpaired actine is straight and generally longer than the paired
ones (Figure 27C, right). Size: 40-80/3.8-10 (paired actine) and 62.5-155/3.8-10 µm (unpaired
actine).
Subatrial triactines. Sagittal. Actines are slightly conical with sharp tips. The paired actines are
inwardly curved. Some paired actines have different lengths. The unpaired actine is straight and
longer than the paired ones (Figure 27D). Size: 42.5-80/5-8.8 µm (paired actine) and 88.5-
172.5/5-10 µm (unpaired actine).
Atrial triactines. Sagittal. Actines are conical, straight and have sharp tips. The unpaired actine
is usually longer than the paired ones (Figure 27E). Size: 80-130/7.1-14.3 µm (paired actine)
and 80-172.5/5-10 µm (unpaired actine).
Atrial tetractines. Sagittal. Actines are conical, straight with sharp tips (Figure 27F). The apical
actine is the shortest actine of this species. Size: 62.5-90/7.5-11.3 µm (paired actines), 92.5-
130/7.5-10 µm (unpaired actine) and 15-40/5-10 µm (apical actine).
ECOLOGY
This species was found in a light protected environment. No associated organisms were found.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
Within the 88 accepted species of Sycon (Van Soest et al., 2016), 15 have the same skeleton
composition as the analised specimen from Curaçao: S. abyssale Borojevic & Graat-Kleeton,
1965, S. ampulla (Haeckel, 1870), S. arcticum (Haeckel, 1870), S. barbadensis (Schuffner,
1877), S. brasiliense Borojevic, 1971, S. boreal (Schuffner, 1877), S. dunstervillia (Haeckel,
111
1872), S. formosum (Haeckel, 1870), S. humboldt Risso, 1827, S. raphanus Schmidt, 1862), S.
scaldiense (Van Koolwijk, 1982), S. schmidti (Haeckel, 1872), S. setosum Schmidt, 1862, , S.
tuba Lendenfeld, 1891 and S. villosum (Haeckel, 1870). Sycon conulosum sp. nov. can be easily
differentiated from S. abyssale, S. brasiliense, S. dunstervilla and S. formosum because of the
shape of their diactines. In the new species, diactines are fusiform whereas in S. abyssale and S.
brasiliense, one of the tips is lanceolated and in S. dunstervilla and S. formosum, diactines have
distally swollen tips (originally described as “keulen” or “kolb”). The remaining 11 species
differ from the new species in spicule size (Table 21).
112
Fig. 26. Sycon conulosum sp. nov. (UFRJPOR 6707): (A) specimen after fixation; (B) osculum;(C) detail of the oscular T-shaped triactines; (D) cross section of the skeleton; (E) skeleton witha tuft of diactines, a triactine of the distal cone (white arrow) and a tubar triactine (black arrow);(F) subatrial skeleton indicating a subatrial triactine (arrow); (G) atrial skeleton indicating atrialtriactine (black arrow) and atrial tetractine (white arrow). Abbreviations: at=atrium.
Fig. 27. Spicules of Sycon conulosum sp. nov. (UFRJPOR 6707): (A) diactine of the distal cone;(B) triactine of the distal cone; (C) tubar triactine I (left) and tubar triactine II (right); (D)subatrial triactine; (E) atrial triactine; (F) atrial tetractine.
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Table 20. Spicule measurements of Sycon conulosum sp. nov. (UFRJPOR 6707).
Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxDiactine 100.0 216.5 48.0 275.0 6.3 8.4 1.3 11.3 20Triactine(distal cone)
Paired 50.0 77.4 16.4 112.5 5.0 7.2 1.6 10.0 20Unpaired 65.0 124.1 28.9 175.0 5.0 6.4 1.3 8.8 20
Tubar Triactine 1
Paired (>) 67.5 78.3 7.7 95.0 5.0 8.1 1.6 11.3 20Paired (<) 32.5 48.6 6.5 60.0 5.0 8.3 2.2 10.0 20Unpaired 82.5 64.6 1.6 125.0 5.0 8.3 1.6 10.0 20
Tubar Triactine 2
Paired 40.0 63.5 10.5 80.0 3.8 7.4 1.7 10.0 20Unpaired 62.5 106.4 21.2 155.0 3.8 7.3 1.7 10.0 20
Subatrial Triactine
Paired 42.5 65.1 9.9 80.0 5.0 5.8 1.2 8.8 20Unpaired 82.5 135.1 20.2 172.5 5.0 7.1 1.6 10.0 20
Atrial Triactine
Paired 80.0 103.0 11.8 130.0 7.1 10.0 2.2 14.3 14Unpaired 80.0 126.6 23.7 172.5 5.0 7.9 1.9 10.0 14
Atrial Tetractine
Paired 62.5 77.5 11.4 90.0 7.5 9.1 1.9 11.3 4Unpaired 92.5 111.3 20.3 130.0 7.5 8.8 1.4 10.0 4Apical 15.0 27.9 9.6 40.0 5.0 7.3 1.7 10.0 12
Table 21. Original measurements of Sycon ampulla, S. arcticum, S. barbadense, S. boreale, S.humboldti, S. raphanus, S. scaldiense, S. schmidti, S. setosum, S. tuba and S. villosum. Values in
micrometers (µm). *Taken from Haeckel (1872).
Species - typelocality
Distal cone Tubar triactine
Subatrialtriactine
Atrial triactine
Atrial tetractine
S. ampulla – Atlantic coast of South America
Diactine: 100-500/5
Paired: 50-80/5Unpaired: 100-150/5
- 60-80/5 Unpaired: 60-80/5Apical: 40-60/5(rarely 100)
S. arcticum– Arctic Ocean
Diactine: 1000-3000/10-40Trichoxea: 100-300/1-5Triactine:smaller than tubar
Paired: 100-200/10Unpaired: 200-300/10
- Paired: 50-150/10Unpaired: 200-250/10
Paired: 50-150/10Unpaired: 200-250/10Apical: 20-40/10
S. barbadense– Barbados
Diactine: 400/9 Unpaired: ≤ 150
- ≤ 200/9 Paired: ≤ 150/9Unpaired: ≤ 200/9Apical: ≤ 80/13
Sycon boreale - Norway
Diactine: 500/9 Paired: 500Unpaired: 180
- Paired: shorterthan unpaired.Unpaired:180/6.8
Apical: 50/13
114
S. humboldti* – Western Mediterranean
Diactine I:500–2000/20-40Diactines II: 200-400/5-20Triactine:Paired: 30-60/ 10-15Unpaired: 150-250/20-30
80-120/8-12 - Paired: 50-120/8Unpaired: 50-200/5-8
Paired: 50-20/8Unpaired: 50-200/5-8Apical: 100-120
S. raphanus* – Adriatic Sea
Diactine: 1000–3000/20 -24
Paired: 100-180/10 – 12Unpaired: 150 – 250/10 – 12
?/5-8 (Subregular to sagittal)150 – 250/8 – 10
Subregular to sagittalApical: 60 – 120
S. scaldiense –Netherlands
Diactine: 500–700/5-10Triactine: 30-60/5-10
80-150/9-11 Paired:80-200/5-10Unpaired:180-350/5-10
Paired: 100 - 280/5-10Unpaired: 200– 300/5-10
Paired:100-280/5-10Unpaired: 200–300/5-10Apical: 180–350/5-10
S. schmidti –Adriatic Sea
Diactine I:100 – 300/10Diactine II: 100–500/20 - 30
Paired: 100-150/10Unpaired: 200-300/10
- Subregular200-400/10-15
SubregularBasal:200-400/10-15Apical:40-50/12-16
S. setosum* –Adriatic Sea
Diactine:1000-3000/20Diactine: 100-200/1
Paired: 80-160/5-8Unpaired:120-200/5-8
- Subregular100-115/5
SubregularApical: 300-600/5
S. tuba– Adriatic Sea
Diactine: 300/10
Paired: 280/7Unpaired: 220/7
- Paired: 280/7Unpaired: 320/7
Paired: 280/7Unpaired: 320/7Apical: 200-260/7
S. villosum– North Atlantic
Diactine:1000-3000/10-40Triactine:100-200/5-8
100-300/30 SagittalPaired: 100/30Unpaired:300/10-30
SagittalPaired: 100/30Unpaired: 300/10-30
Paired: 100-150/5-8Unpaired: 300-400/5-8Apical: 500-800/≤10
Sycon magniapicalis sp. nov. (Figures 28 & 29, Table 22)
ETIMOLOGY
From the Latin magna (=large), for the long apical actine of the atrial tetractine.
TYPE LOCALITY
Tug Boat, Caracasbaai, Willemstadt, Curaçao.
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TYPE MATERIAL
Holotype: UFRJPOR 6748 (specimen in ethanol and slides); Tug Boat, Caracasbaai,
Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 14.9 m deep; coll. B. Cóndor-Luján, 23
August 2011.
Paratype: UFRJPOR 6763 (specimen in ethanol and slides); Water Factory, Willemstadt,
Curaçao; 12°06'30.88"N, 68°57'13.53"W; 8.4 m deep; coll. E. Hajdu, 23 August 2011.
COLOUR
White to beige in life and yellowish in ethanol.
MORPHOLOGY AND ANATOMY
This species has a globular body with an apical osculum (Figure 28A). The consistency is hard,
although compressible. The surface is very hispid with long diactines and trichoxeas protruding
through the surface. The holotype measures 6.5 cm length x 2.5 cm width (Figure 28B). The
osculum has a neck with a delicate crown of trichoxeas (arrows in Figure 28B-C). The radial
tubes are fully coalescent. The atrial cavity is hispid and the aquiferous system is syconoid.
SKELETON
The osculum has a neck composed of T-shaped triactines and tetractines and a crown of
trichoxeas (Figure 28D). The triactines and tetractines are arranged parallel to each other
(Figure 28E). In the paratype, the crown is more conspicuous as shown in Figure 28C. The
skeleton of the body is typical of the genus (Figure 28F). The distal cones have diactines,
triactines and rare trichoxeas tangentially disposed (Figure 28G). The diactines comprise two
size categories. In the larger diactines, the proximal part crosses the choanosome and
occasionally, reaches the atrium. The tubar skeleton is articulated, exclusively formed by
triactines with the unpaired actine pointing to the distal cones (Figure 28H). The subatrial
skeleton is composed of tetractines (white arrow in Figure 28I) and rare triactines (black arrow
in Figure 28I) with the unpaired actine pointing to the surface. The atrial skeleton is formed by
tetractines. The basal actines lay tangentially and the very long apical actine is projected into the
atrium (Figure 28I).
SPICULES
Trichoxeas. Straight with tips always broken. Ticker than usually. Size: >1690.2/1.4-5.4 μm.
Diactines I. Fusiform and straight. The proximal tip is lanceolated while the distal tip is usually
abruptly sharp (Figure 29A). Size: 100.0-864.0/5.4-21.6 μm.
Diactines II. Fusiform and straight. The proximal tip is sharp and the distal tip was always
found broken. They are the largest diactines in this species. Size: > 2970.0/23.0-32.4 μm.
116
Triactines of the distal cones. Sagittal. Actines are conical with sharp tips. Some paired actines
can be slightly longer than the unpaired one (Figure 29B). Size: 54.0-153.9/5.4-10.8 μm (paired
actine) and 40.5-175.5/5.4-10.8 μm (unpaired actine).
Tubar triactines. Sagittal. Actines are conical with sharp tips (Figure 29C). Some paired actines
are curved. Size: 54.0-135.0/5.4-10.8 μm (paired actine) and 35.1-189.0/ 5.4-10.8 μm (unpaired
actine).
Subatrial triactines. Sagittal. Actines are conical with sharp tips (Figure 29D). The unpaired
actine is straight and longer than the paired ones. The paired actines are inwardly curved. In
some triactines, one paired actine can be longer than the other. Size: 67.5-126.9/2.7-6.8 μm
(paired actine), 27.0-99.9/2.7- 6.8 μm (smaller paired actine) and 67.5-245.7/4.1- 8.1 μm
(unpaired actine).
Subatrial tetractines. Sagittal. Actines are conical with sharp tips. The unpaired actine is straight
and longer than the basal actines. It can be slightly conical. The paired actines are inwardly
curved (Figure 29E). The apical actine is the shortest actine in this species Size: 40.0-143.1/4.1-
8.1 µm (paired actine), 143.1-237.6/5.4-10.8 μm (unpaired actine) and 10.8-75.6/2.7-6.8 μm
(apical actine).
Atrial tetractines. Sagittal. The basal actines are conical with sharp tips (Figure 29F). The apical
actine is slightly conical to cylindrical, straight, smooth and very long. It can be straight or
curved. Size: 110.7-237.6/5.4-13.5 μm (paired actine), 29.7-151.2/5.4-16.2 μm (unpaired actine)
and 124.2 - 496.0/5.4-16.2 μm (apical actine).
ECOLOGY
Specimens were collected underneath coral boulders, partially covered with sediment.
GEOGRAPHIC DISTRIBUTION
Southern Caribbean (provisionally endemic to Curaçao, present study).
REMARKS
Surprisingly, Sycon plumosum Tanita, 1943 from Palau (Carolinas Islands) is the only species of
Sycon that presents the same skeleton composition as that of the specimens from Curaçao.
However, they greatly differ in spicule size as almost all the spicule categories are smaller in S.
magniapicalis sp. nov. (only the atrial tetractines have similar size in both species).
Additionally, S. plumosum lacks the oscular neck observed in both Curaçaoan specimens.
In the C-LSU phylogenetic tree (Figure 30), the sequences of S. magniapicalis sp. nov.
(UFRJPOR6748 and UFRJPOR6763) grouped together with high support (pp=1, b=99.9),
confirming their co-specificity. Nonetheless, they did not cluster with the other species of Sycon
(S. ancora, S. capricorn, S. cf. vilosum, S. ciliatum, S. conulosum sp. nov. and S. raphanus).
117
Only in the BI tree, S. magniapicalis sp. nov. appeared as a sister taxon (pp=0.99) of the clade
formed by Sycon conulosum sp. nov. + L. antillana (pp=1, b=99.9).
Fig. 28. Sycon magniapicalis sp. nov.: (A) specimen in vivo; (B) specimen after fixation; (C)paratype after fixation; (D) skeleton of the osculum indicating the neck (asterisk) and the crown;(E) detail of the oscular T-shaped triactine (white arrow) and tetractine (black arrow); (F) cross
118
section of the skeleton; (G) distal cones with diactines II (black arrow) and triactines (whitearrow); (H) tubar skeleton; (I) subatrial skeleton with tetractine (white arrow) and triactine(black arrow); (J) atrial skeleton with tetractines. Abbreviations: at=atrium. All picturescorrespond to the holotype except when indicated.
Fig. 29. Spicules of Sycon magniapicalis sp. nov. (UFRJPOR 6748): (A) diactine I; (B) triactineof the distal cone; (C) tubar triactine; (D) subatrial triactine; (E) subatrial tetractine; (F) atrialtetractine.
119
Table 22. Spicule measurements of Sycon magniapicalis sp. nov. (UFRJPOR6748 andUFRJPOR6763) and of S. plumosum from Tanita, 1943. P=paired, U=unpaired and A=apical.
Specimen Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxUFRJPOR6748
Trichoxea 864.0 - - - 1.3 3.6 1.2 5..4 20Diactine I 100.0 285.7 121.9 486.0 5.4 12.1 3.6 18.9 16Diactine II >2835 - - - 23.0 26.4 1.3 27.0 12Triactine (cone)
P 64.8 110.6 17.9 145.8 5.4 9.7 1.6 10.8 20U 40.5 97.7 34.5 175.5 5.4 8.8 1.8 10.8 20
Tubar Triactine
P 54.0 94.0 26.9 135.0 5.4 7.2 1.6 10.8 19U 35.1 65.5 25.2 118.8 5.4 6.3 1.2 8.1 20
Subatrial Triactine
P 67.5 88.2 16.6 126.9 4.1 5.5 0.7 6.8 20P- 29.7 53.2 16.8 78.3 4.1 5.4 0.6 6.8 13U 121.5 184.4 29.6 245.7 5.4 6.1 0.9 8.1 20
Subatrial Tetractine
P 75.6 106.0 20.2 143.1 4.1 5.5 1.0 8.1 20U 143.1 194.5 23.5 237.6 5.4 7.4 1.1 8.1 20A 16.2 28.9 15.1 75.6 2.7 4.7 0.9 6.8 19
Atrial Tetractine
P 116.1 157.0 19.4 183.6 5.4 8.3 1.3 10.8 15U 29.7 67.5 34.3 151.2 5.4 7.5 1.5 10.8 13A 221.4 313.6 84.4 496.0 5.4 8.8 1.7 10.8 11
UFRJPOR6763
Trichoxea - 216.0 671.7 386.0 1690.2 1.4 3.2 2.0 5.4 20Diactine I - 100.0 432.3 178.0 864.0 10.8 15.3 3.0 21.6 22Diactine II - >2970 - - - 24.3 27.4 1.7 32.4 17Triactine (cone)
P 54.0 89.1 24.3 153.9 5.4 6.8 1.2 8.1 20U 40.5 64.4 21.5 121.5 5.4 5.9 1.0 8.1 20
Tubar Triactine
P 54.0 104.1 23.2 129.6 5.4 8.5 1.3 10.8 20U 94.5 156.6 25.7 189.0 5.4 8.8 1.5 10.8 20
Subatrial Triactine
P + 72.9 91.0 13.0 113.4 2.7 4.9 1.0 6.8 20P - 27.0 57.8 22.0 99.9 2.7 4.8 0.9 5.4 20U 67.5 169.0 37.6 234.9 4.1 6.1 1.3 8.1 20
Subatrial Tetractine
P 75.6 103.8 14.6 132.3 5.4 5.7 0.8 8.1 20U 148.5 191.3 24.9 232.2 5.4 7.1 1.5 10.8 20A 10.8 20.9 9.6 51.3 2.7 4.2 1.2 5.4 20
Atrial Tetractine
P 110.7 148.4 25.4 237.6 6.8 9.7 1.8 13.5 20U 32.4 63.5 25.9 113.4 5.4 9.3 2.6 16.2 20A 124.2 227.0 56.5 345.0 8.1 10.8 2.5 16.2 19
Sycon plumosum
Diactine - 800 - - 3000 30.0 - - 35 -Triactine (cone)
P 200 - - 240 - 16 - - -U 140 - - 200 16 - - -
Tubar Triactine
P 170 - - 240 15 - - 18 -U 270 - - 360 15 - - 18 -
Subatrial Triactine
P 120 - - 180 8 - - 10 -U 250 - - 360 8 - - 10 -
Subatrial Tetractine
P 120 - - 180 8 - - 10 -U 250 - - 360 8 - - 10 -A 70 - - 100 8 - - 10 -
Atrial Tetractine
P 170 - - 200 12 - - 16 -U 200 - - 280 12 - - 16 -A 130 - - 350 12 - - 16 -
120
Phylogenetic analyses
Calcaronean phylogeny
C-LSU sequences of six new Calcaronean species, Amphoriscus micropilosus sp. nov. (n=1),
Grantessa tumida sp. nov. (n=2), Leucilla antillana sp. nov. (n=1), Leucandrilla
pseudosagittata sp. nov. (n=2), Sycon conulosum sp. nov. (n=1) and Sycon magniapicalis sp.
nov. (n=2) were produced and provided herein.
The aligned C-LSU sequences had a total length of 458 bp including gaps. BI and ML
approaches recovered similar tree topologies including monophyletic Calcaronean ( pp=1,
b=79) and Calcinean with high BI posterior probability (pp:1) and ML bootstrap (b=100)
support. The complete C-LSU phylogenetic tree obtained by BI is shown in Figure
S1(Supplementary Material).
Within the Calcaronean cluster, we recovered two major clades (Figure 30). One of them
included all Heteropiidae and some Grantiidae species and it was previously referred as LEUC I
(pp=0.89, b=73) by Voigt et al, (2012). The other clade BAE + LEUC II (pp=1, b=90) nested
the Baerida species (BAE: Eilhardia schulzei, Leuconia nivea and Petrobiona massiliana) and
almost all the other Leucosolenida species but Leucosolenia sp. (LEUC II).
In the LEUC I clade, Grantessa tumida sp. nov. did not cluster with G. aff. intusarticulata,
instead it clustered with the Grantiidae species, Synute pulchella (pp=0.93, b=58), suggesting
the non-monophyly of the genus Grantessa. Interestingly, G. tumida sp. nov. as well as S.
pulchella do not present tetractines in the tubar skeleton whereas the specimens identified as G.
aff intusarticulata do have this spicule category.
Sycon was recovered again as a polyphyletic genus (Voigt et al., 2012; Klautau et al., 2016).
The sequences of the new species, S. conulosum sp. nov. and S. magniapicalis sp. nov., as well
as S. ancora, S. carteri, S. cf. ciliatum and S. raphanus appeared in different clades within the
clade BAE + LEUC II whereas S. capricorn and S. carteri nested in the LEUC I clade (pp=0.89.
b=73).
The Amphoriscidae species (A. micropilosus sp. nov., L. antillana sp. nov., Paraleucilla
dalmatica and P. magna) did not cluster together. Contrary to expected, L. antillana sp. nov.
evidenced a high affinity (pp=1, b= 99.9) with S. conulosum sp. nov. These two species are very
different, however, their skeletons do not have choanosomal tetractines (tubar or subatrial).
Two relationships found by Klautau et al. (2016) using the whole 28S LSU were also
recovered in our phylogeny: (1) the genus Paraleucilla (including P. dalmatica and P. magna)
formed a highly supported clade with Leucandra nicolae in the BI analyses (pp=1) and (2)
Leucandra spinifera and L. aspera clustered together as sister species (pp=0.97, b=54).
121
Calcinean phylogeny
We provided ITS and C-LSU sequences for three new species, Ascandra torquata sp. nov. (2
ITS), Clathrina aspera sp. nov. (4 ITS and 1 C-LSU) and C. curaçaoensis sp. nov. (1 ITS and 1
C-LSU), and for five already known calcinean species, Borojevia tenuispinata (1 ITS and 1 C-
LSU), C. lutea (1 ITS), C. insularis (1 ITS and 1 C-LSU), C. mutabilis (8 ITS and 1 C-LSU), C.
zelinhae (1 C-LSU) and Leucetta floridana (2 ITS).
The aligned ITS sequences had a total length of 1200 bp including gaps. BI and ML
approaches recovered similar tree topologies. The BI phylogenetic tree is shown in Figure 31.
The genera Ascandra (pp=1, b=99), Borojevia (pp=0.7, b=65) and Clathrina (pp=1, b=100)
were recovered as monophyletic clades including the type species of each genus and the
sequences of the new species described herein.
The Calcinean C-LSU clade recovered in the general Bayesian tree is shown in Figure 32.
Two important phylogenetic relationships were recovered in both, the ITS and C-LSU
phylogenies: (1) the yellow C. aurea, C. clathrus, C. curaçaoensis sp. nov, C. lutea and C.
luteoculcitella clustered together in one clade (ITS: pp=1, b=55; C-LSU: pp=0.86, b=65)
whereas the other yellow clathrinas C. mutabilis and C. insularis, appeared in separate clades
and (2) Clathrina aspera sp. nov. clustered together with the geographically distant Australian
species C. helveola and C. wistariensis (ITS and C-LSU: pp=1, b=100).
Although several clathrinas from the Tropical Atlantic Ocean were included in the ITS
phylogenetic analysis, C. mutabilis appeared closer to a clathrina from the French Polynesia
(pp=1, bb=100). Another interesting grouping was observed within the clade of L. floridana.
The Curaçaoan specimens evidenced a higher affinity with the Brazilian specimen than with the
Panamanian one despite their geographic distance.
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Figure 30. Calcaronean cluster from the Bayesian phylogenetic tree inferred from the C-LSUsequences. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap).BAE (Baerida), LEUC I (Leucosolenida I) and LEUC II (Leucosolenida II) refer to clades foundby Voigt et al, 2012. *Sequences generated in this study.
123
Figure 31. Bayesian phylogenetic tree inferred from the ITS sequences of the Calcineanspecies. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap).*Sequences generated in this study.
124
Figure 32. Calcinean cluster from the Bayesian phylogenetic tree inferred from the C-LSUsequences. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap).*Sequences generated in this study.
DISCUSSION
Biodiversity and Distribution
With the present work we increased from two to 20 the number of known species of
calcareous sponges from Curaçao. We found a total of 12 representants of the subclass Calcinea
and eight of the subclass Calcaronea. With the addition of these Curaçaoan records, the number
of calcareous sponges reported for the Caribbean Sea rose from 24 to 40 species, an increase of
66.7% in our knowledge of the biodiversity of Caribbean calcareous sponges.
The finding of nine provisionally Curaçaoan endemic species (45%; 9/20) - Amphoriscus
micropilosus sp. nov., Arthuria vansoesti sp. nov., Clathrina curaçaoensis sp. nov., Grantessa
tumida sp. nov., Leucandra caribea sp. nov., Leucandrilla pseudosagittata sp. nov. Leucilla
antillana sp. nov., Sycon conulosum sp. nov. and Sycon magniapicalis sp. nov. - may indicate a
high endemism of Calcarea in this island, however, it is also possible that it is just showing how
understudied this class is in the Caribbean Sea.
Considering the geographic distribution of calcareous sponges species, only six species
were previously reported occurring in both, the Brazilian Coast and the Caribbean Sea, Leucetta
125
floridana, Leucaltis clathria, Leucandra barbata, Leucandra rudifera, Nicola tetela and Sycon
ampulla (Muricy et al., 2011; Cóndor-Luján & Klautau, 2016). In this study, we expanded the
geographical distribution of four former Brazilian endemic species (B. tenuispinata, C.
insularis, C. lutea and C. mutabilis, Azevedo et al., submitted) to the Caribbean Sea (Curaçao)
and reported two new species (Ascandra torquata sp. nov. and Clathrina aspera sp. nov.)
present in both regions. Therefore, 12 species are currently shared between the Brazilian coast
and the Caribbean Sea. This result suggests a Brazilian-Caribbean sponge affinity for Calcarea
and questions the role of the Amazon and Orinoco Rivers as effective barriers to the dispersal
between these two regions.
Taxonomy and Phylogeny
Important phylogenetic traits with systematic implications can be discussed based on our
results. The phylogenetic importance of the skeleton composition in Calcinean systematics has
already been demonstrated (Rossi et al., 2011; Klautau et al., 2013), nonetheless, in Calcaronea
this has not been tested so far. In this study, we found that the presence of subatrial tetractines
clustered Leucilla antillana sp. nov. with Sycon conulosum sp. nov. and the absence of tubar
tetractines separated Grantessa tumida sp. nov. from Grantessa aff. intusarticulata. Whether
choanosomal tetractines (subatrial or tubar) do have phylogenetical signal across other species
within Calcaronea should be analysed in further studies with a larger number of species.
The close affinity between Heteropiidae and Grantiidae with large longitudinal diactines
was firstly observed in a 28S-phylogeny (Voigt et al., 2012) and it was recently recovered with
the shorter C-LSU sequences (Voigt & Worheide, 2016). This relationship was based on two
highly supported clades: (1) Sycettusa spp. + Grantessa aff. intusarticulata and Aphroceras sp.
and (2) Vosmaeropsis sp. + Sycettusa spp. and Sycon ciliatum. Herein, we recovered that
relationship with a third pair of species: G. tumida sp. nov. and Synute pulchella.
Azevedo et al., (submitted) suggested that trichoxeas were not reliable characters to
differentiate species in Clathrina (as specimens of C. mutabilis with and without trichoxeas
clustered together). We not only recovered the same pattern in another Clathrina species
(Clathrina lutea) but also in Ascandra as diactines can be present or not in A. torquata sp. nov.
Perhaps in the future, not only trichoxeas but also diactines will not be considered as diagnostic
characters for all Calcinean sponges.
The obtained ITS and C-LSU phylogenies suggested again that the yellow colour appeared
more than once in Calcinea (Rossi et al., 2011; Klautau et al., 2013) and that affinities found
between Atlantic and Indo-Pacific species (C. aspera sp. nov. and C. wistariensis; C. mutabilis
and Clathrina sp. from French Polynesia) should receive attention in further studies.
126
ACKNOWLEDGEMENTS
We would like to thank Giselle Lôbo-Hajdu for assistance during the sample collections. Mark
Vermeij and CARMABI are acknowledged for providing logistical support in Curaçao. We are
thankful to Marcelo Soares for the assistance with the SEM procedures.
FINANCIAL SUPPORT
This study was funded by the Brazilian National Research Council (CNPq), Coordination for
the Improvement of Higher Education Personnel (CAPES), Foundation Grupo Boticário de
Proteção à Natureza and Rio de Janeiro State Research Foundation (FAPERJ). B. C. L. received
a scholarship from CAPES. T. L. received a scholarship from FAPERJ. F.A. and A.P. are granted
with post-doc scholarships from FAPERJ and CAPES. E.H. and M.K. have fellowships from
CNPq.
REFERENCES
Alvarez B., Van Soest R.W.M. and Rützler K. (1998) A Revision of Axinellidae (Porifera:Demospongiae) in the Central West Atlantic Region. Smithsonian Contributions toZoology 598, 1-47
Arndt W. (1927) Kalk- und Kieselschwämme von Curaçao. Bijdragen tot de Dierkunde 25,133-158.
Azevedo F., Hajdu E., Willenz P. and Klautau M. (2009). New Records of CalcareousSponges (Porifera, Calcarea) from the Chilean coast. Zootaxa 2072, 1-30
Azevedo F., Cóndor-Luján B., Willenz P., Hajdu E., Hooker Y and Klautau M. (2015)Integrative taxonomy of calcareous sponges (subclass Calcinea) from the Peruviancoast: morphology, molecules, and biogeography. Zoological Journal of the LinneanSociety 173, 787-817.
Borojevic R. (1967) Spongiaires d’Afrique du Sud. (2) Calcarea.Transactions of the RoyalSociety of South Africa 37(3): 183-226.
Borojevic R. (1971). Eponges calcaires des côtes du Sud-Est du Brésil, épibiontessur Laminaria brasiliensis et Sargassum cymosum. Revista Brasileira de Biologia 31,525-530.
Borojević R. and Graat-Kleeton G. (1965). Sur une nouvelle espèce de Sycon et quelquesDémosponges récoltés par le 'Cirrus' dans l'Atlantique Nord. Beaufortia 13,154-81.
Borojević R. and Peixinho S. (1976) Éponge calcaires du nord-nord-est du Brésil. Bulletin duMuséum National d´Histoire Naturelle 3(402), 988–1036.
Borojević R. and Boury-Esnault N. (1987) Calcareous sponges coll. N.O. Thalassa on thecontinental margin of the Bay of Biscaye: I. Calcinea. In: Vacelet J. and Boury-EsnaultN. (eds) Taxonomy of Porifera from the NE Atlantic and Mediterranean Sea: 1–27.NATO Asi Series G13.
Borojević R., Boury-Esnault N., Manuel M. and Vacelet J. (2002) Order ClathrinidaHartman, 1958. In Hooper JNA and Van Soest RWM (eds) Systema Porifera: a guide tothe classification of sponges. New York: Kluwer Academic/Plenum Publishers, pp.1141–52.
127
Breitfuss L. (1896) Kalkschwämme der Bremer Expedition nach Ost-Spitzbergen im Jahre1889 (Prof. W. Kükenthal und Dr. A. Walter). Zoologischer Anzeiger 19(513), 426-432.
Brøndsted, H.V. (1931) Die Kalkschwämme. Deutschen Südpolar Expedition 1901-3, 20, 1-47.Carter H.J. (1890) Porifera. In: Ridley, H.N.(ed.) Notes on the Zoology of Fernando do
Noronha. Journal of the Linnean Society (The Natural History of the island of Fernandode Noronha based on the collections made by the British Museum Expedition in 1887),pp. 564-569.
Chombard C., Boury-Esnault N. and Tillier S. (1998) Reassessment of homology ofmorphological characters in tetractinellid sponges based on molecular data. SystematicBiology 47(3), 351–366.
Coll M., Piroddi C., Steenbeek J., Kaschner K., Ben Rais Lasram F., Aguzzi1 J.,Ballesteros E., Carlo Nike Bianchi, Corbera J., Dailianis T., Danovaro R., EstradaM., Froglia C., Bella S. Galil, Gasol J.M., Gertwagen R., Gil J., Guilhaumon F.,Kesner-Reyes K., Kitsos M-S, Koukouras A., Lampadariou N., Laxamana E.,López-Fé de la Cuadra C.M., Heike K. Lotze, Martin D., Mouillot D., Oro D.,Raicevich S., Rius-Barile J., Saiz-Salinas J.I., San Vicente C., Somot S., TempladoJ., Turon X., Vafidis D., Villanueva R. and Voultsiadou E. (year). The Biodiversity ofthe Mediterranean Sea: Estimates, Patterns, and Threats. PLoS ONE 5(8): e11842.
Cóndor-Luján B. and Klautau M. (2016). Nicola gen. nov. with redescription of Nicola tetela(Borojevic & Peixinho, 1976) (Porifera: Calcarea: Calcinea: Clathrinida). Zootaxa 4103(3), 230–238.
de Laubenfels M.W. (1936) A discussion of the sponge fauna of the Dry Tortugas in particularand the West Indies in general, with material for a revision of the families and orders ofthe Porifera. Tortugas Laboratory Occasional Papers, no. 467, 225 pp.
de Laubenfels M. W. (1950). The Porifera of the Bermuda Archipelago. Transactions of theZoological Society of London 27(1): 1-154.
Dendy A. (1891) A monograph of the Victorian sponges, I. The organisation and classificationof the Calcarea Homocoela, with descriptions of the Victorian Species. Transactions ofthe Royal Society of Victoria 3: 1-81.
Dendy A. (1892) Synopsis of the Australian Calcarea Heterocoela, with a proposedclassification of the group and descriptions of some new genera and species.Proceedings of the Royal Society of Victoria, 5, 69–116.
Dendy A. (1893) On a New Species of Leucosolenia from the neighbourhoodof Port PhillipHeads. Proceedings of the Royal Society of Victoria(New Series) 5: 178-180.
Dendy A. (1905) Report on the sponges collected by Professor Herdman,at Ceylon, in 1902. Pp.57-246, pls I-XVI. In: Herdman, W.A. (Ed.), Report to the Government of Ceylon onthe Pearl Oyster Fisheries of the Gulf of Manaar. 3 (Supplement 18). (Royal Society:London).
De Weerdt W.H. (2000) A monograph of the shallow-water Chalinidae (Porifera,Haplosclerida) of the Caribbean. Beaufortia 50(1): 1-67
Diaz M. C. and Rützler K. (2011) Biodiversity of sponges: Belize and beyond, to the greaterCaribbean. In: Too Precious to Drill: the Marine Biodiversity of Belize. FisheriesCentre Research Reports 19(6), 57-65.
Guindon S., Dufayard J.F., Lefort V., Anisimova M., Hordijk W. and Gascuel O. (2010)New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies:Assessing the Performance of PhyML 3.0. Systematic Biology 59(3), 307–21.
Haeckel E. (1870) Prodromus of a system of the Calcareous sponges. Annals and Magazine ofNatural History 4 (5), 176-191.
Haeckel E. (1872). Die Kalkschwämme, eine Monographie. G. Reimer, Berlin, 418 pp.
128
Hajdu E. and Van Soest R.W.M. (1992) A revison of Atlantic Asteropus Sollas, 1888(Demospongiae), including a description of three new species, with a review of thefamily Coppatiidae Topsent, 1898. Bijdragen tot de Dierkunde 62(1), 3-19.
Hooper J. and Van Soest R.W.M. (2002) Systema Porifera: A Guide to the Classification ofSponges. Kluwer Academic/Plenum Publishers, New York.
Hôzawa S. (1929) Studies on the calcareous sponges of Japan. Journal of the Faculty ofSciences, Imperial University of Tokyo 1: 277-389.
Hyppolyte, J.-C- and Mann, P. (2011) Neogene-Quaternary tectonic evolution of the LeewardAntilles islands (Aruba, Bonaire, Curaçao) from fault kinematic analysis. Marine andPetroleum Geology, Elsevier, 28(1), 259-277.
Imešek M., Pleše B., Pfannkuchen M., Godrijan J., Pfannkuchen D.M., Klautau M. andĆetković H. (2014) Integrative taxonomy of four Clathrina species of the Adriatic Sea,with the first formal description of Clathrina rubra Sarà, 1958. Organisms Diversity &Evolution 14(1), 21–29.
Jenkin C.F. (1908) The Marine Fauna of Zanzibar and British East Africa, from Collectionsmade by Cyril Crossland, M.A., in the Years 1901 & 1902. The Calcareous Sponges.Proceedings of the Zoological Society of London 1908, 434-456.
Johnston G. (1842) A History of British Sponges and Lithophytes. (W.H. Lizars: Edinburgh): i-xii, 1-264.
Katoh K. and Standley D.M. (2013) MAFFT multiple sequence alignment software version 7:improvements in performance and usability. Molecular Biology and Evolution 30, 772–780.
Katoh K and Toh H (2008) Improved accuracy of multiple ncRNA alignment by incorporatingstructural information into a MAFFT-based framework. BMC Bioinformatics 9, 212.
Klautau M. and Valentine C. (2003) Revision of the Genus Clathrina (Porifera, Calcarea).Zoological Journal of the Linnean Society 139:1-62.
Klautau M., Azevedo F., Cóndor-Luján, B., Rapp H.T., Collins, A. and Russo C.A.M.(2013) A molecular phylogeny for the Order Clathrinida rekindles and refines Haeckel’staxonomic proposal for calcareous sponges. Integrative and Comparative Biology 53(3):447-461
Klautau M., Imešek M., Azevedo F., Pleše B., Nikolić V. and Ćetković H. (2016) Adriaticcalcarean sponges (Porifera, Calcarea), with the description of six new species and arichness analysis. European Journal of Taxonomy 178, 1–52
Klautau M., Solé-Cava A.M. and Borojević R. (1994) Biochemical systematics of siblingsympatric species of Clathrina (Porifera: Calcarea). Biochemical Systematics andEcology 22, 367–375.
Koolwijk T. Van (1982) Calcareous sponges of the Netherlands (Porifera, Calcarea). BulletinZoölogisch Museum Universiteit van Amsterdam 8 (12), 89-98.
Lanna E., Cavalcanti F. F., Cardoso L., Muricy G., and Klautau M. (2009) Taxonomy ofcalcareous sponges (Porifera, Calcarea) from Potiguar Basin, NE Brazil. Zootaxa 1973,1-27.
Lanna, E., Rossi, A.L., Cavalcanti, F.F., Hajdu, E. and Klautau, M. (2007) Calcareoussponges from São Paulo state, Brazil (Porifera: Calcarea: Calcinea) with the descriptionof two new species. Journal of the Marine Biological Association of the UnitedKingdom 87, 1553–1561.
Lehnert H. and Van Soest R.W.M. (1998) Shallow water sponges of Jamaica. Beaufortia 48,71–103.
Lendenfeld R. Von (1885). A Monograph of the Australian Sponges (Continued). Part III.Preliminary description and classification of the Australian Calcispongiae. Proceedingsof the Linnean Society of New South Wales 9, 1083-1150.
129
Lendenfeld R. Von (1891) Die Spongien der Adria, I. Die Kalkschwämme. Zeitschrift fürwissenschaftliche Zoologie 53(2): 185-321, (3): 361-463.
Lôbo-Hajdu G.; Guimarães A.; Salgado A.; Lamarão F.; Vieiralves T; Mansure J. andAlbano R. 2004. Intragenomic, Intra– and interspecific variation in the rDNA ITS of aporífera revealed by PCR-single-strand conformation polymorphism (PCR-SSCP).Bolletino dei Musei e Degli Istitui Biologicci de Genova 68, 413-423.
Miklucho-Maclay N. (1868). Beiträge zur Kenntniss der Spongien I. Jenaische Zeitschrift fürMedicin und Naturwissenschaft 4, 221-240.
Miloslavich P., Díaz J.M., Klein E., Alvarado J.J., Díaz C., Gobin J., Escobar-Briones E.,Cruz-Motta J.J., Weil E., Cortés J., Bastidas A.C., Robertson R., Zapata F., MartínA., Castillo J., Kazandjian, A. and Ortiz M. (2010) Marine Biodiversity in theCaribbean: Regional Estimates and Distribution Patterns. PLoS ONE 5(8), e11916.
Moraes F. (2011) Esponjas das Ilhas Oceânicas Brasileiras. Série Livros 44. Rio de Janeiro:Museu Nacional.
Muricy G., Lopes D.A., Hajdu E., Carvalho M.S., Moraes F.C., Klautau M., Menegola C.and Pinheiro U. (2011) Catalogue of Brazilian Porifera. Rio de Janeiro: MuseuNacional, Série Livros 46.
Poléjaeff N. (1883) Report on the Calcarea dredged by H.M.S.‘Challenger’, during the years1873-1876. Report on the Scientific Results of the Voyage of H.M.S. ‘Challenger’,1873-1876. Zoology 8(2), 1-76.
Rapp H. T. (2015) A monograph of the calcareous sponges (Porifera, Calcarea) of Greenland.Journal of the Marine Biological Association of the United Kingdom 95(7), 1395-1459.
Rapp H.T. (2006) Calcareous sponges of the genera Clathrina and Guancha (Calcinea,Calcarea, Porifera) of Norway (north-east Atlantic) with the description of five newspecies. Zoological Journal of the Linnean Society 147(3), 331-365.
Rapp H.T.; Klautau M. and Valentine C. (2001). Two new species of Clathrina (Porifera,Calcarea) from the Norwegian coast. Sarsia 86: 69–74.
Risso A. (1827) Histoire naturelle des principales productions de l'Europe Méridionale etparticulièrement de celles des environs de Nice et des Alpes Maritimes, volume 5(i-vii),403 pp.
Roberts C., Mcclean C., Veron J., Hawkins J. and Allen G. (2002) Marine biodiversityhotspots and conservation priorities for tropical reefs. Science 295: 1280-1284.
Rossi A.L., Russo C.A.M., Solé-Cava A.M., Rapp H.T. and Klautau M. (2011). Phylogeneticsignal in the evolution of body colour and spicule skeleton in calcareous sponges.Zoological Journal of the Linnean Society 163, 1026–34.
Row R.W.H. (1909) Reports on the marine biology of the Sudanese Red Sea. XIX. Report onthe Sponges coll. Mr Cyril Crossland in 1904–05. Journal of the Linnean Society ofLondon, Zoology 31, 182–214.
Row R.W.H. and Hôzawa S. (1931) Report on the Calcarea obtained by the Hamburg South-West Australian Expedition of 1905. Science Reports of the Tôhoku University 6, 727–809.
Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning. A Laboratory Manual.Cold Spring: Harbor Laboratory Press.
Schmidt O. (1862) Die Spongien des Adriatischen Meeres, enthaltend die Histologie undsystematiche Ergänzungen. Wilhelm Engelmann, Leipzig.
Schuffner O. (1877) Beschreibung einiger neuer Kalkschwämme. Jenaische Zeitschrift fürNaturwissenschaft 11, 403-433.
Solé-Cava A.M., Klautau M., Boury-Esnault N., Borojević R. and Thorpe J.P. (1991)Genetic evidence for cryptic speciation in allopatric populations of two cosmopolitanspecies of the calcareous sponge genus Clathrina. Marine Biology 111(3), 381–386.
130
Spalding M.D., Fox H.E., Allen G.R., Davidson N., Ferdaña Z.A., Finlayson M., HalpernB.S., Jorge MA, Lombana A., Lourie S.A., Martin K.D., McManus E., Molnar J.,Recchia C.A. and Robertson J. (2007) Marine ecoregions of the world: abioregionalization of coastal and shelf areas. Bioscience 57, 573–583.
Tanita S. (1943) Studies on the Calcarea of Japan. Science Reports of the Tôhoku ImperialUniversity 17(4), 353–490.
Valderrama D., Rossi A.L., Solé-Cava A.M., Rapp HT and Klautau M. (2009) Revalidationof Leucetta floridana (Haeckel, 1872) (Porifera, Calcarea): a widespread species in thetropical western Atlantic. Zoological Journal of the Linnean Society 157: 1–16.
Van Soest, R.W.M. (1978) Marine sponges from Curaçao and other Caribbean localities. Part I.Keratosa. In: Hummelinck, P.W. and Van der Steen, L.J. (eds) Uitgaven van deNatuurwetenschappelijke Studiekring voor Suriname en de Nederlandse Antillen. No.94. Studies on the Fauna of Curaçao and other Caribbean Islands 56 (179), 1–94.
Van Soest R.W.M. (1980) Marine sponges from Curaçao and other Caribbean localities. Part II.Haplosclerida. In: Hummelinck, P.W. and Van der Steen, L.J. (eds) Uitgaven van deNatuurwetenschappelijke Studiekring voor Suriname en de Nederlandse Antillen. No.104. Studies on the Fauna of Curaçao and other Caribbean Islands 62 (191), 1–173.
Van Soest R.W.M. (1981) A checklist of the Curaçao sponges (Porifera Demospongiae)including a pictorial key to the more common reef-forms. Verslagen en TechnischeGegevens Instituut voor Taxonomische Zoölogie (Zoölogisch Museum) Universiteit vanAmsterdam 31, 1–39.
Van Soest RWM. (1984) Marine sponges from Curaçao and other Caribbean localities. Part III.Poecilosclerida. In: Hummelinck P.W. and Van der Steen LJ (eds) Uitgaven van deNatuurwetenschappelijke Studiekring voor Suriname en de Nederlandse Antillen. No.112. Studies on the Fauna of Curaçao and other Caribbean Islands 66, 1–167.
Van Soest R.W.M. (2009). New sciophilous sponges from the Caribbean (Porifera:Demospongiae). Zootaxa. 2107, 1–40.
Van Soest R.W.M., Boury-Esnault N., Hooper J.N.A., Rützler K., de Voogd N.J., Alvarezde Glasby B., Hajdu E., Pisera A.B., Manconi R., Schoenberg C., Janussen D.,Tabachnick K.R., Klautau M.,Picton B., Kelly M., Vacelet J., Dohrmann M., DíazM.C. and Cárdenas P. (2016) World Porifera database. Available athttp://www.marinespecies.org/porifera.
Van Soest R.W.M. and De Weerdt W. H. (2001) New records of Xestospongia species(Haplosclerida: Petrosiidae) from the Curaçao reefs, with a description of a new species.Beaufortia 51 (7), 109-117.
Vermeij M. J.A. (2012). The current state of Curacao´s Coral Reefs. CarmabiFoundation/University of Amsterdam.
Van Soest R.W.M., Meesters E.H. and Becking L.E. (2014). Deep-water sponges (Porifera)from Bonaire and Klein Curaçao, Southern Caribbean. Zootaxa 3878(5), 401-443.
Van Soest R. W. M. and De Voogd, N. J. (2015). Calcareous sponges of Indonesia. Zootaxa3951(1), 1-105
Voigt O. and Worheide, G. (2016) A short LSU rRNA fragment as a standardmarker forintegrative taxonomy in calcareous sponges (Porifera: Calcarea). Organisms Diversityand Evolution 16 (1), 53-64.
Voigt O. Wülfingl E. and Wörheide G. (2012). Molecular Phylogenetic Evaluation ofClassification and Scenarios of Character Evolution in Calcareous Sponges (Porifera,Class Calcarea). PLoS ONE 7(3): e33417.
Wörheide G. and Hooper J.N.A (1999) Calcarea from the Great Barrier Reef. 1: CrypticCalcinea from Heron Island and Wistari Reef (Capricorn-Bunker Group). Memoirs ofthe Queensland Museum, 43 (2), 859–892.
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SUPPLEMENTARY MATERIAL
Figure S1. Bayesian phylogenetic tree inferred from the C-LSU sequences of the Calcaroneanand Calcinean species. Posterior probabilities and bootstrap values are given on the branches(pp/bootstrap). BAE (Baerida), LEUC I (Leucosolenida I) and LEUC II (Leucosolenida II) referto clades found by Voigt et al, 2012. *Sequences generated in this study.
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New records of calcareous sponges (Porifera: Calcarea) from the Northeastern Brazilian
coast including three new species
BÁSLAVI CÓNDOR-LUJÁN1, FERNANDA AZEVEDO1, ANDRÉ PADUA1, EDUARDO
HAJDU2 & MICHELLE KLAUTAU1*
1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.
Carlos Chagas Filho, 373, Rio de Janeiro, RJ, Brasil, 21941-902. E-mail:
[email protected]; [email protected]; [email protected];
[email protected] Federal do Rio de Janeiro, Museu Nacional, Departamento de Invertebrados,
Quinta da Boa Vista, São Cristóvão, Rio de Janeiro, RJ, Brasil, 20940-040. E-mail:
*Corresponding author: [email protected]
Journal to be submitted: ZOOTAXA
ABSTRACT
Despite the efforts done in order to know the diversity of calcareous sponges along the Brazilian
coast, several areas still remain unexplored or poorly investigated. In this study, we examined
the material collected in recent expeditions along the NE Brazilian coast using morphological
and molecular approaches. Sampled localities included three protected areas: Área de Proteção
Ambiental dos Recifes de Corais de Maracajaú in Rio Grande do Norte State, Fernando de
Noronha Archipelago in Pernambuco State and Abrolhos National Marine Park in Bahia State
and other localities in Ceará State and Rio Grande do Norte. Collections were performed by
snorkeling and SCUBA down to 20 m deep. A total of 14 species were identified including three
new species: Amphoriscus hirsutus sp. nov., Grantia grandisapicalis sp. nov. and Leucascus
luteoatlanticus sp. nov. Arthuria vansoesti Borojevia brasiliensis, Clathrina aspera and C.
luteoculcitella constitute new records for the NE Brazil. Clathrina mutabilis, Ernstia citrea and
E. rocasensis are recorded for the first time from Rio Grande do Norte, Bahia and Pernambuco,
respectively. Clathrina aurea, C. lutea, Leucascus roseus and Leucilla uter are recorded from
new localities within the Abrolhos Marine National Park.
Keywords: Brazilian continental shelf, Fernando de Noronha and Atoll das Rocas, NE Brazil,
Eastern Brazil, Calcaronea, Calcinea.
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Introduction
The Northeastern Brazilian coast comprises a large area of more than 3,300 km, representing
almost 45% of the Brazilian coast. It is located in the Tropical Southwestern Atlantic Province
and covers three entire marine ecoregions, São Pedro and São Paulo Islands, Fernando de
Noronha and Atoll das Rocas and Northeastern Brazil, and part of the Eastern Brazil ecoregion
(Spalding et al. 2007). It also includes nine Brazilian political-administrative divisions (States):
Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe and
Bahia. This vast region encompasses a variety of ecosystems, coral and algal reefs, estuaries,
mangroves, rocky shores, sandy beaches, seagrasses, tide pools and coastal and oceanic islands
(Moraes 2011; Longo & Amado-Filho 2014; Araújo & Amaral 2016), housing a great diversity
of marine taxa, including sponges (Muricy et al. 2011). However, due to its huge dimension and
habitat heterogeneity, several areas are still unexplored or badly known concerning its marine
biodiversity, especially for some faunistic groups, such as sponges of the class Calcarea.
The class Calcarea (Porifera) is composed of sponges whose skeleton is built exclusively of
calcium carbonate. They are divided into two monophyletic subclasses: Calcaronea and
Calcinea. Calcaroneans are characterised by presenting sagittal spicules, apinucleated
choanocytes and amphiblastula larvae and diactines are the first spicules to be produced,
whereas calcineans have a skeleton mainly composed of regular spicules, basinucleated
choanocytes, calciblastula larvae and triactines are the first spicules to be secreted (Manuel et al.
2002). Within the subclasses of Calcarea, molecular phylogenetic studies have demostrated that
high taxonomical categories such as orders, families and even some calcaronean genera are
polyphyletic (Voigt et al. 2012; Voigt & Wörheide 2015; Klautau et al. 2016), whereas other
studies have evidenced monophyletic genera in Calcinea (Rossi et al. 2011; Klautau et al.
2013).
According to the catalogue of Brazilian Porifera, among the 47 species of calcareous sponges
recorded from the Brazilian coast, 26 species (55.3%) were reported from the Northeastern
region (Muricy et al. 2011). This might suggest that the diversity of Calcarea within this region
is well investigated, however, recent studies in this area revealed new species to science
(Cavalcanti et al. 2014, 2015; Azevedo et al. submitted), which shows how the diversity of this
class is still poorly known.
The first records of calcareous sponges from the NE Brazil were published in the 19th
century: Amphoriscus synaptum (Schmidt in Haeckel, 1872) and Leucilla sacculata (Carter,
1890). Almost one century later, Borojevic & Peixinho (1976) reported 24 species including
four new species to science at that time. In the last decade, the knowledge of calcareous sponges
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from the NE Brazil increased by studies focusing on specific localities such as the Potiguar
Basin (Rio Grande do Norte, Lanna et al. 2009), Bahia State (Hajdu et al. 2011) and oceanic
and mid-shelf islands (Azevedo et al. submitted), or in genera as Paraleucilla and Vosmaeropsis
(Cavalcanti et al. 2014, 2015, respectively). Despite this considerable increase of descriptions in
the last years, the diversity and distribution of calcareous sponges in the NE Brazil is still
underestimated.
In the present work, using morphological and molecular approaches, we studied material
collected in recent expeditions conducted along the NE Brazilian coast including three Protected
Areas (Corais de Maracajaú, Abrolhos Marine National Park and Fernando de Noronha
Archipelago) and two other localities in Ceará and Rio Grande do Norte States.
Material and Methods
Analysed material
In this study, we examined the calcareous sponges collected in three expeditions carried out in
the NE Brazilian Coast during 2014 and 2016: ExpoCERN (Expedição Poriferos do Ceará e
Rio Grande do Norte, April 2014), Abrolhos Expedition (December 2015) and TAXPOmol
Biodescoberta 2016 Expedition (April 2016). As some localities are within protected areas,
namely, Abrolhos Marine National Park, Fernando de Noronha and Área de Proteção Ambiental
dos Recifes de Corais de Maracajaú (APARC- Maracajaú), these expeditions received the
adequate support and permissions from the ICMBio (Instituto Chico Mendes de Conservação da
Biodiversidade). The sampled localities where calcarean specimens were found are detailed in
Table 1 and Figure 1.
The collections were conducted by snorkeling and SCUBA diving down to 20 m deep. The
specimens were photographed in situ and fixed in 96% ethanol. All the samples are preserved in
96% ethanol and deposited in the Porifera Collection of the Biology Institute of the
Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR) or in the Museu Nacional do Rio de
Janeiro (MNRJ/UFRJ).
Morphological procedures
The external morphology and internal anatomy were assessed through the observation of the
fixed specimens and the examination of microscopy slides. Sections and spicule slides
preparations followed standard protocols (Wörheide & Hooper 1999; Klautau & Valentine
2003). The spicule measurements including length and width (minimum, mean, standard
deviation [SD] and maximum) are presented in tabular form. The species identifications
followed the Systema Porifera (Hooper &Van Soest 2002) and additional appropriate literature
135
for calcineans (Klautau & Valentine 2003; Klautau et al. 2013; Cavalcanti et al. 2013). To
illustrate the species descriptions, photographs were taken with a digital AxioCam MRC5
coupled to a Zeiss Imager A2 microscope. Additionally, spicules were placed on a cover-slip,
mounted on a stub with double-sided carbon tape and sputter-coated with gold for scanning
electron microscopy. Microphotographs were taken with a JSM-6510 SEM at the Institute of
Biology of the Universidade Federal de Rio de Janeiro (Brazil).
Table 1. Sampled localities, ecoregions sensu Spalding et al. (2007) and geographiccoordinates. Brazilian States: BA=Bahia, CE=Ceará, PE=Pernambuco, RN=Rio Grande doNorte.
L. Locality Ecoregion Geographic Coordinates1 Porto do Pecém, São Gonçalo do
Amarante, CENortheastern Brazil 03º32.106'S, 38º47.893'W
2 Ressurreta, Fernando de Noronha Archipelago, PE
Fernando de Noronha andAtoll das Rocas
03°48.817'S, 32°23.483'W
3 Farol Tereza Pança, Área de Proteção Ambiental dos Recifes de Corais de Maracajaú, Maracajaú, RN
Northeastern Brazil 05º24.133'S, 35º17.849'W
4 Batente das Agulhas, Natal, RN Northeastern Brazil 05°33.841'S, 35°04.367'W5 Parcel das Paredes, Abrolhos Marine
National Park, BAEastern Brazil 17°54.319'S, 38°56.658'W
6 Mato Verde, Ilha Santa Bárbara, Abrolhos Marine National Park, BA
Eastern Brazil 17°57.847'S, 38°41.979'W
7 Portinho Sul, Ilha Santa Bárbara, Abrolhos Marine National Park, BA
Eastern Brazil 17°57.876'S, 38°41.877'W
8 Ilha Guarita, Abrolhos Marine National Park, BA
Eastern Brazil 17°57.583'S, 38°41.550'W
9 Chapeirão 1, Abrolhos Marine National Park, BA
Eastern Brazil 17°58.765'S, 38°41.670'W
10 Chapeirão 2, Abrolhos Marine National Park, BA
Eastern Brazil 17°58.244'S, 38°40.263'W
Molecular procedures
The total genomic DNA was extracted with the guanidine/phenol-chloroform protocol
(Sambrook et al. 1989) or with the QIAamp_DNA MiniKit (Qiagen) and stored at –20°C. For
calcinean species, the region containing the partial 18S and 28S, the spacers ITS1 and ITS2 and
the 5.8S ribosomal DNA (ITS) was amplified with the primers: fwd: 5`-
TCATTTAGAGGAAGTAAAA GTCG-3` and rv: 5`-GTTAGTTTCTTTTCCTCCGCTT-3`)
(Lôbo-Hajdu et al. 2004). For calcaroneans, the C-region of the 28S (C-LSU) was amplified
using the primers fwd: 5'GAAAAGCACTTTGAAAAGAGA-3' (Voigt & Wörheide 2015) and
rv: 5'-TCCGTGTTTCAAGACGGG-3' (Chombard et al. 1998).
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Figure 1. Map of South America highlighting the Northeastern Brazilian coast and the sampledlocalities in the present study. (A) Ceará State; (B) Fernando de Noronha Archipelago; (C) RioGrande do Norte State, and (D) Abrolhos Marine National Park. The numbers refers to thesampled localities in Table 1.
The PCR mixture included 1x buffer (5x GoTaq® Green Reaction Buffer Flexi,
PROMEGA), 0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM
of each primer, one unit of Taq DNA polymerase (Fermentas or PROMEGA) and 1 µL of DNA
in a volume of 15 µL. The PCR amplification comprised one first cycle of 4 min at 94°C, 1 min
at 50°C and 1 min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and one minute at 72°C,
and a final cycle of 6 min at 72°C. Forward and reverse strands were sequenced in an ABI 3500
(Applied Biosystems). The new sequences and the ones retrieved from the GenBank database
are listed in Table 2. The set of sequences was aligned through the MAFFT v.7 online platform
(Katoh & Standley 2013) using the strategy Q-INS-i (Katoh & Toh 2008). The nucleotide
substitution models that best fit the alignment were GTR+G for C-LSU and TN93+G for ITS
sequences, as indicated by the Bayesian Information Criterion in MEGA 6 (Nei & Kumar 2000;
Tamura et al. 2013).
Phylogenetic reconstructions were obtained using Bayesian Inference (BI) and Maximum
Likelihood (ML) methods. The BI reconstructions were conducted in MrBayes 3.1.2
(Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) under 106 generations and a
burn-in of 1000 sampled trees, yielding a consensus tree of majority. As the TN93 model is not
implemented in Mr.Bayes, we used the GTR model. The ML analyses were performed on
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MEGA 6 using an initial NJ tree (BIONJ) and a bootstrap of 1000 pseudo-replicates, yielding a
mid-point rooted tree. BI posterior probabilities (pp) values as well as ML bootstrap values (b)
are indicated on the branches of the inferred trees. In order to know the genetic intraspecific
variability within the sequenced species, we calculated the uncorrected p distance considering
complete deletion in MEGA 6.
Table 2. Species used in the phylogenetic analyses with locality, voucher number and GenBank(GB) accession number for the ITS (Calcinea) and C-LSU (mostly Calcaronea) regions.*Sequences generated in the present study. H=holotype, P=paratype.
Species Locality Voucher number GenBank NumberCALCARONEAAmphoriscus micropilosus Curaçao UFRJPOR6755 (H) Curaçao paperAmphoriscus hirsutus sp. nov.* NE Brazil UFRJPOR7570 (H) This studyAnamixilla torresi - - AY563636Aphroceras sp. - SAM-PS0349 JQ272273Grantessa tumida Curaçao UFRJPOR6701 (P) Curaçao paperGrantessa aff. intusarticulata - GW979 JQ272278Grantia compressa - - AY563538Grantia grandisapicalis sp. nov.* NE Brazil UFRJPOR7567 (H) This studyLeucandra aspera - - AY563535Leucandra falakra Adriatic Sea UFRJPOR8349 (H) KT447560Leucandra nicolae - - JQ272268Leucandra sp. - QMG316285 JQ272265Leucandra spinifera Adriatic Sea UFRJPOR8348 (H) KT447561Leucandrilla pseudosagittata Curaçao UFRJPOR 6705 (P) Curaçao paperLeucascandra caveolata - QMG316057 JQ272259Leucilla antillana Curaçao UFRJPOR 6768 (H) Curaçao paperLeuconia nivea - - AY563534Paraleucilla dalmatica Adriatic Sea UFRJPOR8346 (H) KT447566Paraleucilla magna Adriatic Sea IRB-P14 KT447564Sycettusa aff. hastifera Red Sea GW 893 JQ272282Sycettusa cf. simplex Western India ZMAPOR11566 JQ272279Sycettusa tenuis Australia QMG313685 JQ272281Sycettusa sp. - - AY563530Sycon ancora Adriatic Sea UFRJPOR8347 (P) KT447568Sycon carteri Australia SAM-PS0143 JQ272260Sycon ciliatum - - AY563532Sycon conulosum Curaçao UFRJPOR6707 (H) Curaçao paperSycon cf. villosum - GW51115 KR052809Sycon magniapicalis Curaçao UFRJPOR 6748 (H) Curaçao paperSycon raphanus - - AY563537Syconessa panicula Australia QM G313672 AM181007Utte aff. syconoides - QMG323233 JQ272269Utte aff. syconoides - QMG313694 JQ272271Ute ampullacea - QMG313669 JQ272266Vosmaeropsis sp. - MM-2004 AY026372
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CALCINEAAscandra falcata Mediterranean UFRJPOR5856 HQ588962Ascaltis reticulum Mediterranean UFRJPOR 6260 HQ588977Borojevia aff. aspina Brazil UFRJPOR 5245 HQ588998Borojevia brasiliensis SE Brazil UFRJPOR5214 HQ588978Borojevia brasiliensis SE Brazil UFRJPOR5230 HQ588999Borojevia brasiliensis SE Brazil UFRJPOR 5406 KX548909Borojevia brasiliensis* NE Brazil UFRJPOR 7384 This studyBorojevia cerebrum Mediterranean UFRJPOR6322 HQ588964Borojevia croatica Adriatic Sea IRB-CLB6 KP740023Borojevia tenuispinata Brazil UFRJPOR6484 (H) KX548916Borojevia trispinata Brazil UFRJPOR6419 (H) KX548918Clathrina antofagastensis Chile MNRJ 9289 HQ588985Clathrina aphrodita Peru MNRJ 12994 KC985138Clathrina aspera SE Brazil UFRJPOR 5531 (P) Curaçao paperClathrina aspera Curaçao UFRJPOR 6758 (H) Curaçao paperClathrina aspera* NE Brazil UFRJPOR 7390 This studyClathrina aspera* NE Brazil UFRJPOR 7391 This studyClathrina aurea SE Brazil MNRJ 8998 HQ588968Clathrina aurea* NE Brazil UFRJPOR7544 This studyClathrina aurea* NE Brazil UFRJPOR7571 This studyClathrina aurea* NE Brazil UFRJPOR7574 This studyClathrina aurea* NE Brazil UFRJPOR7584 This studyClathrina blanca Adriatic Sea PMR-14307 KC479087Clathrina clathrus Mediterranean UFRJPOR6315 HQ589009 (C-LSU)
HQ588974 (ITS)Clathrina conifera Brazil MNRJ8991 HQ588959Clathrina coriacea Norway UFRJPOR6330 HQ588986 Clathrina curaçaoensis Curaçao UFRJPOR6734 Curaçao paperClathrina cylindractina Brazil UFRJPOR5206 HQ588979Clathrina fjordica Chile MNRJ 8143 HQ588984Clathrina hispanica Mediterranean UFRJPOR 6305 KC843432Clathrina insularis Brazil UFRJPOR6532 (H) KX548921Clathrina insularis Brazil UFRJPOR6536 (P) KX548922Clathrina helveola Australia QMG313680 HQ588988 Clathrina lacunosa Norway UFRJPOR 6334 HQ588991Clathrina lutea NE Brazil UFRJPOR5172 HQ588961Clathrina lutea NE Brazil UFRJPOR5173 (H) HQ588976Clathrina lutea NE Brazil UFRJPOR 6543 KX548923Clathrina lutea NE Brazil UFRJPOR 6545 KC843442Clathrina lutea* NE Brazil UFRJPOR 7549 This studyClathrina lutea* NE Brazil UFRJPOR 7561 This studyClathrina lutea* NE Brazil UFRJPOR 7579 This studyClathrina lutea* NEBrazil UFRJPOR 7591 This studyClathrina lutea* NE Brazil UFRJPOR 7592 This studyClathrina lutea* NE Brazil MNRJ 18900 This studyClathrina luteoculcitella Australia QMG313684 HQ588989Clathrina luteoculcitella Indonesia ZMAPOR08657 KX548906Clathrina luteoculcitella* NE Brazil UFRJPOR 7551 This study
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Clathrina luteoculcitella* NE Brazil UFRJPOR 7569 This studyClathrina mutabilis NE Brazil UFRJPOR6526 (H) KX548925Clathrina mutabilis NE Brazil UFRJPOR 6528 (P) KX548926Clathrina mutabilis* NE Brazil UFRJPOR 7386 This studyClathrina mutabilis Curaçao UFRJPOR 6741 KC843437Clathrina nuroensis Peru MNRJ 13032 KC985136Clathrina peruana Peru MNRJ 12839 (H) KC985135Clathrina primordialis Adriatic Sea UFRJPOR 6863 KP740016Clathrina ramosa Chile MNRJ 10313 HQ588990Clathrina rubra Adriatic Sea PMR 14306 KC479088Clathrina wistariensis Australia QMG313663 HQ588987Ernstia tetractina Brazil UFRJPOR 5183 HQ589000Leucascus luteoatlanticus sp. nov.* NE Brazil UFRJPOR 7582 (H) This studyLeucascus simplex Polynesia BMOO16283 KC843454
RESULTS
Taxonomy
From a total of 69 specimens analysed, we identified three calcaronean and 11 calcinean species
including three new species to science: Amphoriscus hirsutus sp. nov., Grantia grandisapicalis
sp. nov. and Leucascus luteoatlanticus sp. nov. Among the already known species, four are
reported for the first time from the NE Brazil: Borojevia brasiliensis (Solé- Cava et al., 1991),
Clathrina aspera Cóndor-Luján et al., in press, C. luteoculcitella Worheide & Hooper, 1999 and
Arthuria vansoesti Cóndor-Luján et al., in press.
Seven species already recorded from the NE were also found in this study. Clathrina
mutablis Azevedo et al., submitted was originally described from Fernando de Noronha
Archipelago and herein, we expand its distribution to Maracajaú. Ernstia citrea Azevedo et al.,
submitted, was only recorded from Rocas Atoll (Rio Grande do Norte) and now it was found in
the Abrolhos Marine National Park. Ernstia rocasensis Azevedo et al., submitted, previously
reported from Rocas Atoll and Abrolhos Archipelago, is being reported from Fernando de
Noronha Archipelago now. Clathrina aurea Solé- Cava et al., 1991, C. lutea Azevedo et al.,
submitted, Leucascus roseus Lanna et al., 2007 and Leucilla uter Poléjaeff, 1883 were found in
new localities within the Abrolhos Marine National Park.
In the next section, the new records from the NE Brazil as well as the allegedly cosmopolitan
Leucilla uter are broadly described and illustrated. Calcineans already reported from the NE
region and found in the studied material are listed in Table 11.
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Description of species
Class CALCAREA Bowerbank, 1862
Subclass CALCARONEA Bidder, 1898
Order LEUCOSOLENIIDA Hartman, 1958
Family AMPHORISCIDAE Dendy, 1893
Genus Amphoriscus Haeckel, 1872
Amphoriscus hirsutus sp. nov. (Figures 2-3, Table 3)
Etimology. From the Latin hirsutus (= full of bristles), for the presence of diactines protruding
along the body.
Type locality. Ilha Guarita, Abrolhos National Marine Park, Bahia State.
Type material. Holotype (ethanol): UFRJPOR 7570; Ilha Guarita, Abrolhos Marine National
Park; 10.3 m deep, collected by A. Padua & F. Azevedo, 4/XII/2014.
Colour. White in life and yellowish in ethanol.
Description. This sponge has a friable, tubular body with an apical osculum (Figure 2A). The
holotype measures 22 x 6 mm. The osculum has a margin composed of sagittal tetractines and it
is surrounded by a crown of trichoxeas (Figure 2B). The surface is very hispid due to diactines
and anchor-like triactines which protrude through the sponge surface. The diactines are
distributed along the body (arrows in Figure 2B) and the anchor-like triactines are present at the
basal region of the sponge (arrowheads in Figure 2B-C). The aquiferous system is syconoid.
Skeleton. The skeleton is typical of the genus (Figure 2D). Diactines cross perpendicularly the
cortex (arrow in Figure 2D), which is composed of tetractines. The basal actines of the cortical
tetractines lay tangentially to the surface (black arrow in Figure 2E). The apical actine of the
tetractines and the diactines cross the choanosome, eventually reaching the atrium. The
choanosomal skeleton is inarticulated and it is formed by the apical actines of the cortical
tetractines (white arrow in Figures 2E-F) and the unpaired actine of subatrial triactines (black
arrows in Figure 2F). Subatrial-like triactines (arrowhead in Figure 2F) and tetractines (arrow in
Figure 2G) were found scattered in this region. The atrial skeleton is composed of tetractines
with the apical actine projected into the atrial cavity (Figure 2H). The anchor-like triactines are
organised in tufts (Figure 2I) with the unpaired actine located in the choanosome and the paired
actines protruding the sponge surface.
Spicules
Diactines (Figure 3A). One tip is sharp and the other, which protrudes the cortex, is lanceolated.
Very variable size: 462.5-2150.0/12.5-20.0 µm.
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Cortical tetractines (Figure 3B). Sagittal. Actines are conical, straight, smooth and have sharp
tips. The paired actines can be curved. The apical actine is the largest actine and can be slightly
undulated. Size: 150.0-165.0/12.5-17.5 µm (unpaired actine), 200-350/7.5-20.0 µm (paired
actine) and 150-450/10-20 µm (apical actine).
Choanosomal tetractines (Figure 3C). Very rare. Sagittal. Subatrial-like. Actines are slightly
conical and with sharp tips. The paired actines are curved and can have different length.
Subatrial triactines (Figure 3D-F). Sagittal. Actines are conical, straight to slightly curved and
with sharp tips. The paired actines can have different length. Size: 315.0–565.0/10–15 µm
(unpaired actine) and 165.0–365.0/7.5–12.5 µm (paired actine).
Atrial tetractines (Figure 3G). Sagittal. Actines are slightly conical, straight, smooth and with
sharp tips. The paired actines can be curved. The apical actine is the shortest one. Size: 80-
285/7.5-12.5 µm (unpaired actine), 165.0-400.0/5-12.5 µm (paired actine) and 50-150/5-8.7 µm
(apical actine).
Anchor-like triactines (Figures 3H-I). Sagittal. The unpaired actine is very long. It is curved at
the base and very straight from the median region to the distal part (Figure 3H). The paired
actines are rudimentary and centripetally curved (Figure 3I). Size: 1650->1750/10 µm (unpaired
actine) and 25.0–40/6.2–7.5 µm.
Ecology. This specimen was found covered with sediment, surrounded by algae.
Geographic Distribution. Provisionally endemic to the NE Brazil (present study).
Remarks. Amphoriscus hirsutus sp. nov. is the second Amphoriscus species bearing anchor-like
triactines. The other species is A. ancora Van Soest, 2017 from the Guyana Shelf. Although both
species have similar skeletons, they present relevant differences. In A. ancora, diactines and
anchor-like triactines are restricted to the base of the sponge, whereas in A. hirsutus sp. nov. the
diactines are present along the whole body and the anchor-like triactines are present not only in
the base but also in adjacent (basal) regions. The new Brazilian species present triactines and
tetractines scattered in the choanosomal skeleton which are absent in A. ancora. Besides, the
size (mean length/width) of the atrial tetractines of A. hirsutus (paired actine: 293.2/8.6 µm,
unpaired actine: 190.0/9.4 µm and apical actine: 88.4/7.2 µm) exceeds that of A. anconra
(paired actine: 99.0/7.7 µm, unpaired actine: 87/8.6 µm and apical actine: 26/5.3 µm).
Additionaly, the osculum of A. hirsutus presents a crown of trichoxeas whereas in A. ancora, it
is naked, however, as this is a plastic character, it must be treated with caution.
Another species of Amphoriscus recorded from the Brazilian coast is Amphoriscus synaptum
(Schmidt in Haeckel, 1872), however, it can be easily differentiated from A. hirsutus sp. nov. by
the presence of anchor-like tetractines in the former.
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Not considering the anchor-like spicules, the species that more resemble A. hirsutus sp. nov.
in skeleton composition is A. buccichii Ebner, 1887 from the Adriatic Sea, however, the
skeleton of the latter also comprises small subcortical tetractines (length: 80-120 µm) which are
absent in the new species. Moreover, they differ in spicule size. A. buccichii has smaller
diactines (60-200/3-5 µm) and thicker cortical tetractines (width: 30–40 µm) compared to A.
hirsutus sp. nov. (size of diactines: 462.5-2150/12-20 µm and width of cortical tetractines: 7.5-2
µm). For additional comparisons, all the measurements of A. ancora and A. buccichii are
presented in Table 3.
Table 3. Measurements of Amphoriscus hirsutus sp. nov. (UFRJPOR 7570), A. ancora and A.buccichii. *Taken from the original descriptions. H=holotype. P=paired, U=unpaired andA=apical actines.
Species Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR7570(H)
Diactine 462.5 2150.0 510.3 2150.0 12.5 16.9 2.6 20.0 14Cortical Tetractine
P 200.0 280.4 46.1 350.0 7.5 12.6 3.4 20-0 25U 150.0 157.5 10.6 165.0 12.5 15.0 3.5 17.5 2A 150.0 324.8 85.6 500.0 10.0 14.0 2.9 20.0 26
Subatrial Triactine
P 165.0 266.8 51.6 365.0 7.5 10.1 7.5 12.5 22U 315.0 443.8 70.9 565.0 10.0 11.8 1.7 15.0 21
AtrialTetractine
P 165.0 293.2 64.0 400.0 5.0 8.6 1.6 12.5 24U 80.0 190.0 65.9 285.0 7.5 9.4 1.8 12.5 17A 50.0 88.4 31.4 150.0 5.0 7.2 1.0 8.7 20
Anchor-liketriactine
P 25.0 32.1 5.8 40.0 6.2 7.3 0.5 7.5 7U 1650.0 - - >1750.0 - 10 - -
A.ancora*
Diactine 600.0 1017.0 - 1230.0 3.0 4.2 - 6.5 -Cortical Tetractine
P 135.0 239.0 - 320.0 8.0 15.4 - 21.0 -U 151.0 216.0 - 330..0 12.0 16.1 - 21.0 -A 258.0 311.0 - 366.0 8.0 17.8 - 20.0 -
Subatrial Triactine
P 129.0 218.0 - 324.0 7.0 8.6 - 10.5 -U 201.0 367.0 - 468.0 7.0 10.1 - 12.0 -
AtrialTetractine
P 57.0 99.0 - 165.0 3.0 7.7 - 11.0 -U 42.0 87.0 - 186.0 3.0 8.6 - 12.0 -A 9.0 26.0 - 60.0 3.0 5.3 - 15.0 -
Anchor-liketriactine
P 18.0 24.8.0 - 29.0 5.0 6.8 - 9.0 -U 96.0 386.0 - 660 7.0 8.6 - 10.0 -
A.buccichii*
Diactine 60.0 - - 200 3.0 - - 5.0 -Cortical Tetractine
P 360.0 - - 420 30.0 - - 40.0 -U 360.0 - - 540 30.0 - - 40.0 -A 300 - - 420 30.0 - - 40.0 -
Subatrial Triactine
P 100 - - 120 6.0 - - 7.0 -U 200 - - 260 6.0 - - 7.0 -
AtrialTetractine
P 150 - - 200 7.0. - - 10.0 -U 300 - - 400 7.0 - - 10.0 -A 100 - - 150 6.0 - - 12.0 -
143
Figure 2. Amphoriscus hirsutus sp. nov. (UFRJPOR 7570). A. Specimen in vivo. B. Specimenafter fixation showing diactines (arrows) and anchor-like triactine (arrowhead). C. Anchor-liketriactine. D. Cross section of skeleton with diactines (black arrow). D. Cortex with the pairedactines (black arrow) and apical actine (white arrow) of a tetractine. F. Choanosome showing anapical actine of a cortical tetractine (white arrow), unpaired actines of subatrial triactines (black
144
arrows) and a choanosomal triactine (arrowhead). G. Choanosome with subatrial-like tetractine.H. Atrial skeleton. I. Tuft of anchor-like triactines. Abbreviations: cx=cortex; at=atrium.
Figure 3. Spicules of Amphoriscus hirsutus sp. nov. (UFRJPOR 7570). A. Diactines. B. Corticaltetractines. C. Choanosomal subatrial-like tetractine. D-F. Subatrial triactines. G. Atrialtetractine. H. Anchor-like tetractine. I. Paired actines of the anchor-like tetractine.
145
Genus Leucilla Haeckel, 1872
Leucilla uter Poléjaeff, 1883 (Figures 4-5, Table 4)
Synonyms.
Amphoriscus chrysalis Burton, 1963: 545.
Leucilla australiensis, Borojevic, 1967: 221; Borojevic & Peixinho, 1976: 1031
Leucilla uter Poléjaeff, 1883: 53; Dendy & Row, 1913: 784; Borojevic & Boury-Esnault, 1987:
35; Muricy & Silva, 1999: 160, Muricy et al., 2011: 25
Leucilla? uter, Muricy et al., 1991: 1187.
Type locality. Poléjaeff described specimens from Bermudas and Philippines, however the
lectotype deposited in the Natural History Museum (London) is from Bermudas.
Type specimens. Lectotype (ethanol and dried) BMNH 1884.4.22.21, paralectotypes (slides):
BMNH.4.22.30 and BMNH 1884.4.22.31.
Material examined. UFRJPOR 7545 (ethanol), Mato Verde, Ilha Santa Bárbara, Abrolhos
Marine National Park, 7.1 m deep, collected by A. Padua & F. Azevedo, 3/XII/2014.
Colour. Bright white in life and in ethanol.
Description. The analysed specimen has a sac-shaped body with an apical osculum (Figure
4A). It measures 9.4 x 3.3 mm (Figure 4B). The osculum has a crown of trichoxeas (2.6 x 1.7
mm) supported by sagittal T-shaped tetractines. In the apical part of the body, diactines protrude
through the surface (arrow in Figure 4B). The surface is rough and the consistency is friable.
The aquiferous system is leuconoid.
Skeleton. The skeleton is typical of the genus (Figure 4C). The cortical skeleton is formed by
tetractines and very rare triactines (arrow in Figure 4D). The basal actines of the tetractines are
tangentially disposed and the apical actine penetrates the choanosome and can reach the atrium.
Some diactines were also observed in this region. The choanosomal skeleton is inarticulated,
formed by the apical actine of the cortical tetractines (white arrow in Figure 4E) and by the
unpaired actine of the subatrial triactines (black arrow in Figure 4E) and rare subatrial
tetractines. Rare tetractines and triactines were found scattered in the choanosome, with their
unpaired actine pointing to the surface (as shown in Figure 4C). The atrial skeleton is
exclusively composed of tetractines with the apical actine projected into the atrium (Figure 4F).
Spicules
Diactines (Figure 5A). Fusiform (sharp tips). Size (length/width): >1250/6-10 μm.
Cortical triactines (Figures 5B). Sagittal. Very rare. Actines are straight, slightly conical with
sharp tips. The paired actines can be slightly curved and longer than the unpaired one. Size:
200.0-205.0/10.0-11.2 μm (paired actine) and 122.5/10.0-12.5 μm (unpaired actine).
146
Cortical tetractines (Figure 5C-D). Sagittal. Actines are conical with sharp tips. The paired
actines are frequently inwardly curved. The apical actine is the longest actine. Size: 225.0-
530.0/22.5-40.0 μm (paired actine), 265.0-500.0/30-42.5 μm (unpaired actine) and 255-
1000/22.5-40.0 μm (apical actine).
Choanosomal triactines (Figure 5E) and tetractines (Figure 5F-G). Sagittal. Rare. Actines are
conical with sharp tips. Different from the cortical tetractines, the actines are always straight.
Size of triactines: 250.0/22.5 μm (paired actine) and 237.5/22.5-25 μm (unpaired actine). Size of
tetractines: 335-400/15-40 μm (paired actine), 227.5-425.0/27.5-37.5 μm (unpaired actine) and
87.5/25.0 μm (apical actine).
Subatrial triactines (Figure 5H). Sagittal. Actines are conical with sharp tips. The paired
actines are smaller than the unpaired one and can be slightly curved. Very variable size: 75-
320.0/7.5-22.5 μm (paired actine) and 150-540/7.5-20.0 μm (unpaired actine).
Subatrial tetractines (Figure 5I). Sagittal. Rare. Actines are conical with sharp tips, similar to
subatrial triactines. The apical actine is shorter and thinner than the basal ones. Size: 112.5-
285.0/7.5-15 μm (paired actine), 175.0-350.0/7.5-20 (unpaired actine) and 50.0-87.5/7.5 μm
(apical actine).
Atrial tetractines (Figure 5J). Sagittal. Actines are conical with very sharp tips. The paired
actines are longer than the unpaired one. The apical actine is the thinnest and shortest actine.
Size: 115.0-285.0/5.0-12.5 μm (paired actines), 137.5-187.5/7.5-12.5 μm (unpaired actine) and
62.5-150.0/7.4-10.0 μm (apical actine).
Ecology. This specimen was collected underneath boulders.
Geographic distribution. Leucilla uter is an allegedly cosmopolitan species. It was originally
described from Bermudas and Philippines (Poléjaeff, 1883) and then, recorded from Brazil
where it is widely distributed, from Alagoas to Rio de Janeiro States (Borojevic & Peixinho
1976; Muricy et al. 2011).
Remarks. The analysed specimen in the present study mostly matches the original description
of Leucilla uter (Poléjaeff 1833) as well as the description of its lectotype (Borojevic & Boury-
Esnault, 1987). The only difference is the presence of rare diactines and cortical triactines in our
specimen. As the validity of those spicules as diagnostic characters within Leucilla species has
been questioned (Borojevic & Boury-Esnault, 1987), we decided not to differentiate the studied
specimen as a new species and to identify it as L. uter until an update revision of this genus is
done.
It is worth to mention that the revised specimen slightly differs from the specimens
misidentified as L. australiensis by Borojevic & Peixinho (1976) and re-identified as L. uter in
147
Muricy et al. (2011) in the size of the cortical triactines and of the atrial tetractines. The
unpaired actines of both spicule categories are larger in the specimens described by Borojevic &
Peixinho (cortical triactine=200-400/10-16 μm and atrial tetractine=270-400/10-12 μm) than in
our specimen (cortical triactine=122.5/10-12.5 μm and atrial tetractine=137.5-187.5/7.5-12.5
μm).
This is the first record of L. uter occurring in very shallow waters (<10 m). Previously, it was
reported from depths ranging from 10 to 61 m (Muricy et al. 2011).
Figure 4. Leucilla uter (UFRJPOR 7545). A. Specimen in vivo. B. Specimen after fixation. C.Cross section of the skeleton. D. Cortex with cortical triactines (arrow). E. Inarticulated
148
choanosomal skeleton with the apical actine of a cortical tetractine (white arrow) and theunpaired actine of a subatrial triactine (arrow). F. Atrial skeleton. Abbreviations: cx=cortex;at=atrium.
Figure 5. Spicules of Leucilla uter (UFRJPOR 7545). A. Diactines. B. Cortical triactine. C-D.Cortical tetractines. E. Choanosomal triactine. F-G Choanosomal tetractines. H. Subatrialtriactine. I. Subatrial tetractine. J. Atrial tetractine.
149
Table 4. Measurements of Leucilla uter of the specimen UFRJPOR 7545, a Brazilian specimenby Borojevic & Peixinho (1976) and of the type specimen by Poléjaeff (1893).
Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR 7545
Trichoxea 2250 - - - - 2.5 - - 2Diactine 1250 - - - 6 8 - 10 3Corticaltriactine
Paired 200.0 201.7 2.9 205.0 10.0 10.4 0.7 11.2 3Unpaired - 122.5 - - 10.0 11.2 1.8 12.5 2
Cortical tetractine
Paired 225.0 368.5 85.4 530 22.5 32.5 6.1 40.0 20Unpaired 265.0 381.7 66.5 500.0 30.0 36.1 4.3 42.5 20Apical 255.0 535.0 171.3 1000 22.5 31.3 5.7 40.0 26
ChoanosomalTriactine
Paired - 250.0 0.0 - - 22.5 0.0 2Unpaired - 237.5 0.0 - 22.5 23.8 1.8 25.0 2
ChoanosomalTetractine
Paired 335.0 365.7 35.4 400.0 15.0 30.7 8.7 40.0 7Unpaired 227.5 370.4 71.8 425 27.5 33.8 4.9 37.5 6Apical - 87.5 - - - 25 - - 1
Subatrial Triactine
Paired 75.0 186.9 77.7 320.0 7.5 11.6 4.8 22.5 20Unpaired 150.0 370.8 124.5 540.0 7.5 13.9 4.9 20.0 20
SubatrialTetractine
Paired 112.5 222.3 50.4 285.0 7.5 10.1 2.5 15.0 13Unpaired 175.0 275.9 45.7 350.0 7.5 10.8 3.2 20.0 14Apical 50.0 73.1 17.2 87.5 - 7.5 0.0 - 4
AtrialTetractine
Paired 115.0 204.8 50.6 285.0 5.0 7.9 1.7 12.5 15Unpaired 137.5 158.1 21.2 187.5 7.5 10.3 2.5 12.5 4Apical 62.5 95.2 22.3 150.0 5.0 7.4 1.1 10.0 21
Leucilla uter sensu Borojevic &Peixinho, 1976
Corticaltriactine
Paired 170.0 - - 400.0 10.0 - - 16.0 -Unpaired 200.0 - - 400.0 10.0 - - 16.0 -
CorticalTetractine
Basal 300.0 - - 700.0 20.0 - - 60.0 -Apical 180.0 - - 600.0 20.0 - - 60.0 -
ChoanosomalTriactine
300.0 - - 600.0 30.0 - - 50.0 -
ChoanosomalTetractine
Basal 300.0 - - 600.0 30.0 - - 50.0 -Apical - - 200.0 - - - - -
Subatrialtriactine
Paired 170.0 - - 250.0 10.0 - - 16.0 -Unpaired 300.0 - - 400.0 - 16.0 - - -
Atrialtetractine
Paired 200.0 - - 350.0 10.0 - - 12.0 -Unpaired 270.0 - - 400.0 10.0 - - 12.0 -Apical 40.0 - - 80.0 - - - - -
Originaldescription
Diactine - - 400.0 - 2.5 - - -Corticaltetractine
Basal 400.0 - - 600.0 - - - 50.0 -Apical 400.0 - - 1200 - - - 50.0 -
Choanosomaltetractine
Basal 400.0 - - 600.0 - - - 50.0 -Apical 400.0 - - 1200 - - - 50.0 -
Subatrialtriactine
Unpaired - - - 600.0 30.0 - - 50.0 -Paired - - - 420.0 21.0 - - 35.0 -
Subatrialtetractine
Unpaired - - - 600.0 30.0 - - 50.0 -Paired - - - 420.0 21.0 - - 35.0 -
Atrial tetractine*
Paired - - - 400.0 12.5 20.0 - - -Unpaired 250.0 - - 350.0 12.5 20.0 - - -Apical - - - 200.0 12.5 20.0 - - -
150
Family Grantiidae Dendy, 1893
Genus Grantia Fleming, 1828
Grantia grandisapicalis sp. nov. (Figure 6 and 7, Table 5)
Etimology. From the Latin grandis (= large) for the long apical actine of the atrial tetractines.
Type locality. Chapeirão 2, Abrolhos Marine National Park, Bahia State, Brazil.
Type material. Holotype (ethanol): UFRJPOR 7567, Chapeirão 2, Abrolhos Marine National
Park, 15.2 m deep, collected by A. Padua & F. Azevedo, 04/XII/2014.
Colour. White in life and beige in ethanol.
Description. This sponge has a globular body with an apical osculum (Figures 6A-B). The
holotype measures 14 x 6 mm. The surface is very hispid because of diactines protruding
through the cortex (arrow in Figure 6B). The osculum (diameter=2.5 mm) is sustained by a
margin composed of T-shaped triactines and tetractines and it is surrounded by a crown of
trichoxeas (arrowhead in Figure 6B). The atrium is hispid due to the long apical actines of the
atrial tetractines. The aquiferous system is syconoid with elongated chambers arranged side by
side.
Skeleton. The skeleton is typical of the genus (Figure 6C). The cortical skeleton is composed of
perpendicular diactines (white arrow in Figure 6D) and tangential triactines (black arrow in
Figure 6D). Diactines do not penetrate the choanosome. The tubar skeleton is articulated,
composed of several rows of triactines and tetractines (in less proportion) with the unpaired
actine pointing to the cortex (Figure 6E). The subatrial skeleton is composed of triactines with
the unpaired actine pointing to the choanosome. The atrial skeleton is exclusively composed of
tetractines with the apical actine projected into the atrial cavity (Figure 6F).
Spicules
Trichoxea of the crown (Figure 7A). Slender. Size: More than 500 μm long.
Diactines I (Figure 7B). Fusiform and small. Size: 300-580/22.5-30.0 μm.
Diactines II (Figure 7C). Fusiform and large. Very variable size: 1000.0 to > 3125.0/35.0-55.0
μm.
Cortical triactines (Figures 7D-F). Sagittal. Actines are conical, straight and have sharp tips.
The paired actines are frequently longer than the unpaired one and can be slightly curved. Size:
75.0-142.5/7.5-11.3 μm (paired actine) and 47.5-107.5/7.5-12.5 μm (unpaired actine).
Tubar triactines (Figures 7G-I). Sagittal. Actines are conical, straight with sharp tips (Figure
7G). The paired actines have different sizes (Figure 7H) and one of them can be curved (Figure
7I). Size: 32.5-67.5/5-10 μm (paired actine 1), 62.5-120.0/5-7.5 μm (paired actine 2) and 119.4-
140/5-8.8 μm (unpaired actine).
151
Tubar tetractines (Figures 7J-K). Sagittal. Actines are conical, straight with sharp tips (Figure
7J). Some paired actines have different sizes and one of them can be curved. The unpaired
actine can be longer than the paired ones (Figure 7K). Size: 67.5-142.5/5.0-7.5 μm (paired
actines), 80.0-197.5/6.2-8.7 μm (unpaired actine) and 17.5-35.0/5-7.5 μm (apical actine).
Subatrial triactines (Figure 7L). Sagittal. Actines are slightly conical, straight with sharp tips.
The paired actines can be curved. Size: 50-110/5-7.5 μm (paired actines) and 127.5-207.5/7.5
μm (unpaired actine).
Atrial tetractines (Figures 7M-N). Sagittal. Actines are conical with sharp tips (Figure 7M).
The apical actine is long, thick and distally curved (Figure 7N). Size: 80.0-175.0/5-8.7 μm
(paired actines), 80.0-112.5/5-6.2 μm (unpaired actine) and 100-172.5/5-8.7 μm (apical actine).
Ecology. The surface of the analysed specimen was found covered with sediment.
Geographic distribution: This species is provisionally endemic to the NE Brazil (present
study).
Remarks. The genus Grantia comprises 40 valid species (Van Soest et al. 2016). Among them,
only G. aculeata Urban, 1908 from the Mediterranean Sea, G. infrequens Carter, 1886 from
Australia, G. intermedia Thacker, 1908 from Cape Verde and G. kempfi Borojevic & Peixinho,
1976 from Brazil present triactines and tetractines in the tubar skeleton. Grantia kempfi is the
species that more resembles G. grandisapicalis sp. nov., however, they can be distinguished by
the choanocytary chambers which are ramified near the atrium in the former and elongated in
the latter. Besides, the skeleton of G. kempfi comprises comprises subatrial tetractines and atrial
triactines, which are absent in the new species. The other species of Grantia reported from
Brazil is G. atlantica Ridley, 1881 (Muricy et al. 2011), which does not present subatrial
triactines nor tubar tetractines (Borojevic & Peixinho 1976).
152
Figure 6. Grantia grandisapicalis sp. nov. (UFRJPOR 7567). A. Specimen in vivo. B.Specimen after fixation indicating crown (arrowhead) and diactines (arrow). C. Cross section ofthe skeleton. D. Cortical skeleton with diactine (white arrow) and cortical triactine (blackarrow). E. Tubar skeleton with triactine (white arrow) and tetractine (black arrow). F. Atrialskeleton. Abbreviations: cx=cortex; at=atrium.
153
Figure 7. Spicules of Grantia grandisapicalis sp. nov. (UFRJPOR 7567). A. Broken trichoxea.B. Diactine I. C. Broken diactine II. D-F. Cortical triactines. G-I. Tubar triactines. J-K. Tubartetractines. L. Subatrial triactine. M. Atrial tetractine. N. Apical actine of an atrial tetractine.
Table 5. Spicule measurements of Grantia grandisapicalis sp. nov. (UFRJPOR 7567).
Spicule ActineLength (µm) Width (µm) N
Min Mean SD Max Min Mean SD MaxDiactine I - 300.0 440.0 140.0 580.0 22.5 27.5 4.3 30.0 3
Diactine II - 1000.0 >3125.0 35 42.0 6.9 55.0 7
Corticaltriactine
Paired 75.0 112.1 18.0 142.5 7.5 9.0 1.2 11.3 21Unpaired 47.5 74.5 14.2 107.5 7.5 10.3 1.4 12.5 20
Tubartriactine
Paired - 32.5 48.4 9.8 67.5 5.0 6.2 1.3 10 20Paired+ 62.5 87.7 14.1 120.0 5.0 6.4 1.2 7.5 21Unpaired 119.4 90.0 14.3 140.0 5.0 7.3 1.1 8.8 20
154
Tubartetractine
Paired 67.5 94.6 17.8 142.5 5.0 7.0 1.0 7.5 30
Unpaired 80.0 141.6 28.8 197.5 6.2 7.7 0.5 8.7 30
Apical 17.5 26.4 5.0 35.0 5.0 5.4 0.8 7.5 16
Subatrialtriactine
Paired 50.0 80.5 19.3 110.0 5.0 5.9 1.2 7.5 10
Unpaired 127.5 160.8 27.3 207.5 7.5 7.5 0.0 7.5 10Atrialtetractine
Paired 80.0 125.0 29.5 175.0 5.0 6.6 1.1 8.7 20Unpaired 80.0 99.4 13.9 112.5 5.0 5.6 0.7 6.2 4Apical 100 132.2 18.1 172.5 5.0 7.2 0.9 8.7 23
Subclass Calcinea Bidder, 1898
Family Clathrinidae Minchin, 1900
Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013
Arthuria vansoesti Cóndor-Luján, Azevedo, Padua, Hajdu, Klautau, in press (Figure 8, Table 6)
Synonyms
Arthuria vansoesti Cóndor-Luján et al., in press
Type locality. Daai Booi, St. Willibrordus, Curaçao, Caribbean Sea.
Type specimens. Holotype (ethanol): UFRJPOR 6720 (holotype); Daai Booi, St. Willibrordus;
12°12'43.12"N, 69°05'8.42W; 5.2 m deep; collected by B. Cóndor-Luján, 19/VIII/2011.
Material examined. UFRJPOR 7557 (ethanol), Chapeirão2, Abrolhos Marine National Park;
15.2 m deep; collected by F. Azevedo & A. Padua, 4/XII/2014.
Colour. Light yellow in life and beige (or yellowish white) in ethanol.
Description. The analysed specimen has a massive cormus (6 x 5 x 3 mm) formed by irregular
and loosely anastomosed tubes and presents water-collecting tubes (Figures 8A-B). The
aquiferous system is asconoid. No granular cells were observed. The skeleton has no special
organization and it is composed of abundant triactines (distinguished in two shape categories)
and rare tetractines.
Spicules (Table 6)
Triactines I (Figure 8C). Regular (equiangular and equiradiate). Very frequent. Actines are
cylindrical, slightly undulated at the distal part and with rounded tips. Size: 70.0-102.5/2.5-5.0
μm.
Triactines II (Figure 8D). Regular (equiangular and equiradiate). Actines are straight, slightly
conical with blunt to sharp tips. Size: 52.5-92.5/2.5-5.0 μm
Tetractines (Figures 8E-F). Regular (equiangular and equiradiate). Rare. Basal actines are
cylindrical, slightly undulated at the distal part and with rounded to blunt tips (Figure 11E). The
155
apical actine is straight, smooth and bear sharp tips (Figure 11F). Size: 80.0-85.0/3.8-5 μm
(basal actine) and 25.0-67.5/2.5-5.0 μm (apical actine).
Geographic distribution. Curaçao (Cóndor-Luján et al. in press) and Abrolhos Marine
National Park (present study)
Remarks: This species was originally described from the Caribbean Sea (Cóndor-Luján et al.
in press) and we are expanding its distribution to the NE Brazil. The other Arthuria occurring in
the NE is A. trindadensis Azevedo et al., submitted. The new species can be easily differentiated
from A. trindadensis by the in vivo colour, which is yellow in the former and white in the latter.
Besides, the skeleton of A. trindadensis comprises triactines whose actines only have sharp tips
whereas A. vansoesti has triactines with rounded (Triactines I) and sharp (Triactines II) tips.
Table 6. Spicule measurements of Arthuria vansoesti from Abrolhos (UFRJPOR 7557) and ofthe holotype . *Taken from Cóndor-Luján et al. in press.
Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR 7557
Triactine I - 70.0 85.6 7.4 102.5 2.5 3.5 0.7 5.0 25Triactine II - 52.5 76.5 9.9 92.5 2.5 3.4 0.9 5.0 20Tetractine basal 80.0 81.7 2.9 85.0 3.8 4.6 0.7 5.0 3
apical 25.0 46.3 30.0 67.5 2.5 3.8 1.8 5.0 2Holotype* Triactine I 72.5 79.8 4.9 87.5 3.8 4.6 0.6 5.0 30
Triactine II 52.5 70.4 7.8 82.5 2.5 3.9 0.8 5.0 30Tetractine basal 60.0 75.3 8.1 87.5 3.8 4.8 0.5 5.0 15
apical 25.0 25.0 0.0 25.0 3.8 4.4 0.7 5.0 6
156
Figure 8. Arthuria vansoesti (UFRJPOR 7557). A. Specimen in vivo. B. Specimen afterfixation. C. Triactine I. D. Triactine II. E. Tetractine. F. Apical actine of a tetractine.
Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013
Borojevia brasiliensis (Solé-Cava, Klautau, Boury-Esnault, Borojevic & Thorpe, 1991) (Figure
9, Table 7)
Synonyms
Clathrina cerebrum Borojevic 1971: 526
Clathrina brasiliensis Solé-Cava et al. 1991: 382; Klautau et al. 1994: 372; Muricy & Silva
1999: 160; Klautau & Borojevic 2001: 403; Klautau & Valentine 2003: 11; Muricy et al. 2011:
33.
Borojevia brasiliensis Klautau et al. 2013: 459
Type locality. Arraial do Cabo, Brazil
Type specimens. Holotype (ethanol): MNHN-LBIM.C. 1989.2, Arraial do Cabo (Enseada), Rio
de Janeiro, Brazil. Collected by G. Muricy, 16/XII/ 1986.
157
Material examined. UFRJPOR 7384 (ethanol), Porto do Pecém, São Gonçalo do Amarante,
Ceará State, collected by E. Hajdu & S. Salani, 2/IV/2014.
Colour. White in life and ethanol.
Description. This species has a massive, rough and slightly compressible cormus (17 x 6 x 3
mm) composed of irregular and tightly anastomosed tubes (Figures 9A-B). The aquiferous
system is asconoid and the oscula are spread through the cormus. No cells with granules were
observed. The skeleton has no special organization and it comprises tripods that are in fact large
triactines, triactines and tetractines. Triactines are more abundant than tetractines.
Spicules (Table 7)
Tripods. (Figures 9C-D). Equiangular, subregular or parasagittal. Actines are straight, slightly
conical with blunt tips. They do not have the centre raised as in typical tripods. Size: 121.5-
153.9/9.4-12.1 µm.
Triactines.. Regular (equiangular and equiradiate). Very abundant. Actines are straight, conical
with blunt tips (Figure 9E). Sagittal triactines with curved paired actines were also observed
(Figure 8F). Size: 62.1-72.6/8.1-10.8 µm.
Tetractines.. Regular (equiangular and equiradiate). Actines are straight, conical with blunt tips
(Figure 9G-H). Sagittal tetractines with curved paired actines were also observed (Figure 8I).
The apical actine is shorter and thinner than the basal ones and has spines. Spines are located
near the tip of the actine (Figure 9J). Some tetractines with no spines were also observed. Size:
62.1-83.7/8.1-10.8 µm (basal actine) and 37.5-65.0/5.0-7.5 µm (apical actine).
Geographic distribution. SE Brazil (Klautau & Valentine 2003; Muricy et al. 2011) and NE
Brazil (this study).
Remarks. Four Borojevia species are known from Brazilian waters: B. aspina (Klautau, Solé-
Cava, & Borojevic, 1994), B. brasiliensis, B. tenuispinata and B. trispinata Azevedo et al.,
submitted. Among these, our specimen more resembles B. brasiliensis as it presents a skeleton
with more triactines than tetractines and similar spines distribution along the apical actine of the
tetractines, however, the tripods are different. Compared to previous descriptions of B.
brasiliensis (Solé-Cava et al. 1991; Klautau & Borojevic 2001; Klautau & Valentine 2003) the
specimen from Ceará have tripods with longer and less conical actines (Table 7). Nonetheless,
as the variability in the shape of tripods in Borojevia has been assigned to polymorphism or
plasticity (Klautau et al. 2016), the observed difference in the analysed specimen does not
constitute a diagnostic character. Therefore, we identified UFRJPOR 7384 as B. brasiliensis.
Furthermore, in the phylogenetic tree, it clustered within the B. brasiliensis clade with a high
support (pp=0.98, b=95, Figure 15), corroborating the morphological identification.
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Figure 9. Borojevia brasiliensis (UFRJPOR 7384). A. Specimen in vivo. B. Specimen afterfixation. C-D. Tripods. E-F. Triactines. G-I. Tetractines. J. Apical actine of a tetractine.
Table 7. Spicule measurements of Borojevia brasiliensis from Ceará (UFRJPOR 7384) and of the holotype. *Taken from Klautau & Valentine (2003).
Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR7384
Tripod 121.5 138.0 9.4 153.9 9.4 11.2 0.8 12.1 20Triactine 62.1 68.8 3.8 72.6 8.1 8.7 0.8 10.8 20Tetractine basal 62.1 71.4 4.8 83.7 8.1 9.2 0.9 10.8 20
apical 37.5 50.0 7.0 65.0 5.0 6.7 0.8 7.5 20Holotype* Tripod 67 81 8.2 95.7 - 11 1.7 - 20
Triactine 60.9 78.2 10.6 102.2 - 10.8 1.5 - 20Tetractine basal 56.5 75.3 10.0 91.3 - 10.4 1.3 - 20
apical 17.4 36.4 9.1 50.0 - 8.0 2.2 - 20
159
Genus Clathrina Gray, 1867 sensu Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo,
2013
Clathrina aspera Cóndor-Luján, Azevedo, Padua, Hajdu, Klautau, in press (Figure 10, Table 8).
Synonyms
Clathrina aspera Cóndor-Luján et al., in press.
Type locality. Water Factory, Willemstadt, Curaçao, Caribbean Sea.
Type specimens. Holotype (ethanol): UFRJPOR 6758, Water Factory, Willemstadt, Curaçao,
12°06'30.88"N, 68°57'13.53"W, 13.2 m deep, collected by B. Cóndor-Luján and E. Hajdu,
23/VIII/2011. Paratypes (ethanol): UFRJPOR 5487, Ilhas Botinas, Angra dos Reis, Rio de
Janeiro, Brazil; 23º03'19.36''S, 44º19'44.98''W; 1-3 m deep; collected by F. Azevedo and M.
Klautau, 25/V/2007 and UFRJPOR 5531; Praia do Bonfim, Angra dos Reis, Rio de Janeiro,
Brazil; 23°01'14.26''S, 44°19'48.18''W; 1-2 m deep; collected by M. Klautau, 27/V/2007.
Material examined. UFRJPOR 7390 and UFRJPOR 7391 (ethanol), Farol da Praia de
Maracajaú, Área de Proteção Ambiental dos Recifes de Corais de Maracajaú (APARC-
Maracajaú), Rio Grande do Norte State, collected by B. Cóndor-Luján, 2 m deep, 8/IV/2014.
Colour. White in life and in ethanol.
Description. The largest specimen (UFRJPOR 7390) measures 13 x 11 x 4 mm. The cormus is
massive and composed of irregular and tightly anastomosed tubes (Figures 10A-B). Because of
the presence of large triactines located on the external tubes, the surface is rough. The
aquiferous system is asconoid with oscula widespread on the surface. No water-collecting tubes
were observed. The skeleton has no special organization and it comprises two categories of
triactines.
Spicules.
Triactines I (Figure 10C). Regular (equiangular and equiradiate). Large and tripod-like. Actines
are straight, conical with sharp tip. Size: 225.0-390.0/20.0-35.0 µm.
Triactines II (Figure 10D). Regular (equiangular and equiradiate). Actines are straght,
cylindrical to slightly conical with sharp tips. Size: 45.0-125.0/5.0-8.7 µm.
Ecology. This sponge was found underneath boulders. Adjacent individuals to UFRJPOR 7391
were eaten by fish (field observation).
Geographic distribution. Brazil, including the NE (this study) and SE regions, and Curaçao
(Cóndor-Luján et al. in press).
Remarks. The present record of C. aspera from Rio Grande do Norte contributes to the
understanding of its distribution and genetic connectivity in the Western Tropical Atlantic as it
160
was previously reported from two very distant localities, Curaçao (Caribbean Sea) and Rio de
Janeiro (Cóndor-Luján et al. in press). In both ITS and C-LSU phylogenetic reconstructions
(Figures 14 and 15), the NE specimens clustered with sequences of the type material of
C.aspera (UFRJPOR5531 and UFRJPOR6758) with high support (ITS: pp=0.99, b=99 and C-
LSU: pp=1, b=91) in concordance with the morphological identification.
Figure 10. Clathrina aspera (UFRJPOR 7390). A. Specimen in vivo. B. Specimen afterfixation. C. Triactine I. D. Triactines II.
Table 8. Spicule measurements of Clathrina aspera from Rio Grande do Norte (UFRJPOR 7390 and UFRJPOR 7391) and from the holotype. *Taken from Cóndor-Luján et al. in press
Specimen Spicule Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR Triactine I - 245.0 - - - 20 - - 17390 Triactine II 50.0 78.9 24.0 112.5 5.0 7.0 0.8 8.7 40
UFRJPOR Triactine I 225.0 317.5 50.0 390.0 20.0 25.6 3.6 35.0 127391 Triactine II 45.0 78.6 24.5 125.0 5.0 6.7 1.0 7.5 40
Holotype* Triactine I 132.5 227.2 50.8 325.0 17.5 28.5 5.6 37.5 30Triactine II 75.0 115.4 12.9 152.5 7.5 9.7 1.1 12.5 40
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Clathrina luteoculcitella Wörheide & Hooper, 1999 (Figure 11, Table 9)
Synonyms
Clathrina luteoculcitella Wörheide & Hooper 1999: 868; Klautau & Valentine 2003: 30,
Klautau et al. 2013: 12.
Clathrina aff. luteoculcitella: Van Soest & de Voogd 2015:13.
Type locality. Great Barrier Reef, Australia
Type specimen. Holotype (ethanol): QMG 313684, ‘The Patch’, at the N end of the channel
between Heron Island and Wistari Reef, Great Barrier Reef, 23°26.6'S, 151°53.4'E, 25 m deep.
Material examined. UFRJPOR 7551 (ethanol); Chapeirão1, Parque Nacional Marinho dos
Abrolhos; 16.2 m; collected by A. Padua & F. Azevedo, 04/XII/2014; UFRJPOR 7569 (ethanol);
Chapeirão2, Parque Nacional Marinho dos Abrolhos; 15.2 m; collected by A. Padua & F.
Azevedo, 04/XII/2014.
Colour. Beige in life and white in ethanol.
Description. The largest specimen measures 21 x 13 x 5 mm. The cormus is massive, rough and
slightly compressible. It is composed of thin, regular and tightly anastomosed tubes which form
folds (giving an apparent spherical shape, Figure 11A). The aquiferous system is asconoid. The
oscula are spread along the surface (Figure 11B), however, in UFRJPOR 7551, structures
similar to water-collecting tubes (laterally located) were observed. The skeleton has no special
organization and it comprises one single category of triactines and very rare diactines. Diactines
were only found in UFRJPOR 7551 and most of them were located tangentially to the surface
(only one diactine perpendicularly inserted was observed).
Spicules
Triactines (Figure 11C). Regular (equiangular and equiradiate). Actines are slightly conical to
conical, distally undulated and with sharp tips. Size: 59.5-90.0/5.4-10 µm.
Diactines (Figure 11D). Very slender, trichoxea-like. Most of them were broken but it was
possible to distinguish two tips: one sharp tip and another lanceolated (wider). Size: >162.5/2.5
µm.
Ecology. The specimen UFRJPOR 7551 was found in a crevice, on a red calcareous algae. Balls
of sediment were found inside the tubes of UFRJPOR 7569.
Geographic distribution: Australia (Wörheide & Hooper 1999), Indonesia (Van Soest & de
Voogd 2016) and NE Brazil (present study).
Remarks. The analysed specimens resemble the Brazilian endemic species Clathrina
angraensis Azevedo et al., 2007 in external morphology and spicule size (48-100/6.8±10 µm),
162
however, the cormus of the latter does not form folds and its triactines are less conical than
those observed in the specimens from Abrolhos.
The in vivo colour of the studied specimens is beige, nonetheless, in the ITS phylogenetic
analysis, they grouped (pp=1, b=99, Figure 15) with the yellow C. luteoculcitella from
Australia, indicating its conspecificity. Different from the Australian specimens whose skeleton
comprise abundant diactines perpendicularly disposed to the surface of the cormus (Wörheide &
Hooper 1999; Klautau & Valentine 2003), in the Brazilian specimens, (thinner) diactines were
rarely found and, when observed, they were tangential to the surface. This indicates that
diactines may constitute plastic morphological characters in Clathrina, as recently suggested by
Azevedo et al. submitted. Clathrina luteoculcitella constitutes the first confirmed record of an
Indo-Pacific species present in the Western Atlantic Ocean. This disjunct distribution is puzzling
and should receive attention in further studies as it is possible that this species was introduced in
the Brazilian coast by anthropogenic means (M. Klautau, pers. Obs.).
Figure 11. Clathrina luteoculcitella (UFRJPOR 7551). A. Specimen in vivo. B. Specimen afterfixation. C. Triactines. D. Broken diactine.
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Table 9. Spicule measurements of Clathrina luteoculcitella from Abrolhos (UFRJPOR 7551and UFRJPOR 7569) and from the original description (Wörheide & Hooper 1999).
Specimen Spicule Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR 7551 Diactine >87.5 - - >162.5 2.5 2.5 0 2.5 6Triactine 72.5 81.4 5.0 90.0 7.5 7.8 0.7 10 20
UFRJPOR 7569Original description
Triactine 59.5 75.3 8.6 86.5 5.4 6.3 1.2 8.1 20Diactine 90.0 164.4 - 220.0 2.0 3.1 - 6.0Triactine 68.0 77.7 - 84.0 8.0 9.4 - 12.0
Family Leucascidae Dendy, 1892
Genus Leucascus Dendy, 1892
Leucascus luteoatlanticus sp. nov. (Figures 12 and 13, Table 10)
Etimology. From the Latin lutea (=yellow) for the yellow colour of the cormus and the type
locality in the Atlantic Ocean.
Type locality. Parcel das Paredes, Abrolhos Marine National Park, Bahia State, Brazil
Type material. Holotype: UFRJPOR 7582; Parcel das Paredes, Abrolhos Marine National Park,
Bahia State, Brazil, 14. 6 m deep; collected by A. Padua & F. Azevedo, 5/XII/2014.
Material used for comparison: Holotype of L. flavus (ethanol): ZMAPOR 13145, Sulawesi,
Bone Baku, Indonesia, station BB/NV/120597, collected by N. J. de Voogd, 16/V/1997.
Colour. Yellow in life and beige in ethanol.
Description: The analysed specimen measured 18 x 10 x 3 mm. This species has a rough, firm
and massive cormus composed of regular and tightly anastomosed tubes (Figure 12A). It is
possible to recognize thin and delicate cortical and atrial membranes delimiting the cormus and
the atrium, respectively. The cortical membrane is perforated by inhalant apertures. The
aquiferous system is solenoid. The oscula are located on the top of elevations and do not have
ornamentation (Figure 12B).
Skeleton. The skeleton comprises triactines and tetractines (Figure 12C). In the cortex, there
are more triactines than tetractines (Figure 12D), whereas in the tubes and in the atrium,
triactines and tetractines seem to be present in equal proportions (Figure 12F). Most
choanocytary tubes are hispid because of the apical actine projected inside of them (Figure
12E).
Spicules
Triactines (Figure 13A). Regular (equiangular and equiradiate). Actines are slightly conical to
conical, straight with sharp tips. Size: 80.0-125.0/7.5-11.3 µm.
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Tetractines (Figures 13B-C). Regular (equiangular and equiradiate). Actines are slightly
conical, straight with sharp tips (Figure 13B). The apical actine is slightly conical, thinner than
the basal ones and bear spines. The spines are distributed along the apical actine (Figure 13C).
Size: 75-105.0/7.5-10 µm (basal actines) and 40.0-85.0/5-7,5 µm (apical actine).
Ecology. A crustacean and a polychaeta were found inside the atrium.
Geographic distribution. Provisionally endemic to the NE Brazil (present study).
Remarks. The specimen analysed resembles Leucascus flavus Cavalcanti et al., 2013 from
Indonesia. Both species are yellow in vivo, present a cortical skeleton mainly composed of
triactines and the skeleton of their choanocytary tubes have triactines and tetractines in equal
proportions. However, they can be differentiated by their atrial skeletons which present more
tetractines than triactines in L. flavus, whereas in the Brazilian specimen, triactines and
tetractines are present in similar proportions. As this character (proportion of spicules in the
atrial skeleton) has been validated as a diagnostic character in the revision of Leucascus
(Cavalcanti et al. 2013), we propose to name the analysed specimen herein as a new species, L.
luteoatlanticus sp. nov. Additionally, as Van Soest & de Voogd (2015) provided in situ pictures
of L. flavus, it was possible to recognize a wider atrium in L. flavus when compared to L.
luteoatlanticus sp. nov.
Before this study, only two species of Leucascus were reported for the Brazilian coast, L.
roseus Lanna et al., 2007 and L. albus Cavalcanti et al., 2013. Different from L. luteoatlanticus
sp. nov., which is yellow in vivo and have spicules with conical actines, L. roseus is pink and
possesses spicules with cylindrical actines whereas L. albus is white and its skeleton bears
microdiactines.
Table 10. Spicule measurements of Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582) and ofthe holotype of L. flavus. *Taken from Cavalcanti et al. (2013).
Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max
UFRJPOR 7582
Triactine 80.0 100.3 11.9 125.0 7.5 9l4 1.1 11.3 20Tetractine basal 75.0 92.4 9,2 105.0 7.5 8l4 0.9 10 20
apical 40.0 61.9 13.6 85.0 5.0 5l5 1.0 7.5 16Leucascus flavus*
Triactine 70.0 100.9 9.5 120.0 7.5 9l7 1.1 12.5 30Tetractine basal 75.0 98,7 7.4 115.0 7.5 8l9 1.3 10.0 30
apical 41.3 49.6 5.3 60.7 3.6 4l2 0.6 4.9 30
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Figure 12. Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582). A. Specimen in vivo. B.Specimen after fixation. C. Cross section of the skeleton. D. Cortical membrane. E. Atrialmembrane. F. Choanocytary tube with projected apical actines (arrow). Abbreviations:cx=cortex; ct=choanocytary tube.
166
Figure 13. Spicules of Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582). A. Triactines. B.Tetractine. C. Apical actine of a tetractine covered with spines.
167
Table 11. New locality records of calcareous sponges from the NE coast. Species - Type Locality
Material examined
New Record Locality, depth, collectors, collection date
Clathrina aurea -Arraial do Cabo, Rio de Janeiro, Brazil.
UFRJPOR 7544 L6, 7.1 m, coll. A Padua & F. Azevedo, 03/XII/2014.UFRJPOR 7548 L9, 16.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014.UFRJPOR 7552UFRJPOR 7553UFRJPOR 7554
L9, 16.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014.
UFRJPOR 7572 L5, 11.7 m, coll. A. Padua & F. Azevedo, XII/2014.UFRJPOR 7571UFRJPOR 7574UFRJPOR 7576
L5, 14.3 m, coll. A. Padua & F. Azevedo, XII/2014.
UFRJPOR 7580UFRJPOR 7584UFRJPOR 7585
L5, 14.6 m, coll. A. Padua & F. Azevedo, XII/2014.
MNRJ 18965 L10, 13.1 m, coll. A. Bispo & J. Carraro, 04/XII/2014.Clathrina lutea - Pedra Lixa, Abrolhos Archipelago, Bahia, Brazil.
UFRJPOR 7549UFRJPOR 7550UFRJPOR 7555
L9, 16.2 m, coll. A Padua & F. Azevedo, 4/XII/2016.
UFRJPOR 7561 UFRJPOR 7563 UFRJPOR 7564 UFRJPOR 7565UFRJPOR 7566 UFRJPOR 7568
L10, 15.2 m, coll. A. Padua & F. Azevedo, 4/XII/2016.
UFRJPOR 7577UFRJPOR 7578
L5, 14.3 m, coll. T. Pérez & O. Thomas, 05/XII/2014.
UFRJPOR 7579 UFRJPOR 7583UFRJPOR 7589UFRJPOR 7590
L5, 14.6 m, coll. A. Padua & F. Azevedo, 05/ XII /2014.
UFRJPOR 7591 L10, 9-15 m, coll. A. Bispo & J. Carraro, 04/ XII /2014.UFRJPOR 7592UFRJPOR 7593 UFRJPOR 7594UFRJPOR 7595 UFRJPOR 7596UFRJPOR 7597UFRJPOR 7598UFRJPOR 7599UFRJPOR 7600UFRJPOR 7601UFRJPOR 7602
Abrolhos Marine National Park, T. Pérez & O. Thomas, 04/XII/2014.
MNRJ 18844 L10, 11.6 m, coll. E. Hajdu, 04/XII/2014.MNRJ 18900 L5, 5 – 16 m, coll. A. Bispo & J. Carraro, , 04/XII/2014.
Clathrina mutabilis - Fernando de Noronha Archipelago, Brazil
UFRJOR 7386 L4, 20 m, coll. B. Cóndor-Luján, 07/IV/2014.UFRJPOR 7388 L3, 2 m, coll. B. Cóndor-Luján, 08/IV/2014.
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Ernstia citrea - Rocas Atoll, Rio Grande do Norte, Brazil
UFRJPOR 7547 L7, 7.1 m, coll. A. Padua & F. Azevedo, 03/XII/2014.UFRJPOR 7556UFRJPOR 7560
L10, 15.2, coll. A. Padua & F. Azevedo, 04/XII/2014.
UFRJPOR 7573 L5, 14.3 m, coll. A. Padua & F. Azevedo, 05/XII/2014.
Ernstia rocansensis - Rocas Atoll, Rio Grande do Norte, Brazil
UFRJPOR 8547 L2, 4 m, coll. A. Bispo & S. Salani, 23/IV/2016.
Leucascus roseus - Alcatrazes Archipelago, São Sebastião, São Paulo, Brazil.
UFRJPOR 7558UFRJPOR 7559
L10, 15.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014.
UFRJPOR 7575 L5, 14.3 m, coll. A. Padua & F. Azevedo, 04/XII/2014.UFRJPOR 7581 L5, 14.6 m, coll. A. Padua & F. Azevedo, 04/XII/2014.UFRJPOR 7586UFRJPOR 7587UFRJPOR 7588
L5, 14.6 m, coll. A. Padua & F. Azevedo, 05/XII/2014.
MNRJ18884 MNRJ 18885
L10, 10 – 15 m, coll. A. Bispo & J. Carraro, 04/XII/2014.
Molecular analyses
In this study, 19 DNA sequences were generated. We provided sequences for the new species
described herein: C-LSU for Amphoriscus hirsutus sp. nov. and Grantia grandisapicalis and
ITS for Leucascus luteoatlanticus sp. nov. Furthermore, new ITS sequences were generated for
Clathrina aspera, C. aurea, C. lutea, C. luteoculcitella, C. mutabilis and Borojevia brasiliensis.
An additional C-LSU sequence of C. aspera was presented as well.
The C-LSU sequences produced an alignment of 457 bp including gaps. Both phylogenetetic
methods (BI and ML) recovered the same tree topology (BI tree is shown in Figure 14). The
phylogenetic tree yielded similar results to those previously obtained (Voigt & Wörheide 2016;
Cóndor-Luján et al. in press). With the addition of the new sequences, Amphoriscus and
Grantia appeared as non monophyletic genera. Grantita grandispicalis sp. nov. nested within
the clade of Heteropiidae and Grantiidae, instead of clustering with Grantia compressa, which is
the type species of the genus. Interestingly, the skeleton of G. grandisapicalis sp. nov. comprises
tubar tetractines whereas that of G. compressa does not. Furthermore, A. hirsutus sp. nov. whose
choanosomal skeleton is mainly composed of triactines clustered with Sycon conulosum +
Leucilla antillana (pp=0.99, b=90) whose tubar and subatrial skeletons (respectively) are
exclusively composed of triactines (Cóndor-Luján et al. in press).
The intraspecific variation (estimated by the uncorrected p distance) of the ITS Calcinean
sequences ranged between 0 and 1.4%. Borojevia brasiliensis, C. lutea and C. aspera showed
169
no intraspecific variation (0%), The values ranged from 0 to 0.5% in C. mutabilis and from 0-
0.7% in C. luteoculcitella. The highest variation was observed in C. aurea (0-1.4%).
Figure 14. Bayesian phylogenetic tree inferred from the C-LSU sequences of the studiedspecies. Bayesian posterior probabilities and bootstrap values (pp/b) are given on the branches.* Sequences generated in this study.
170
The alignment of the ITS sequences had a total length of 1349 bp including gaps. Both the
BI and ML methods yielded similar tree topologies (ML tree is shown in Figure 15). The
phylogenetic tree (1) recovered the monophyletic genera Clathrina (pp=0.96, b=71) and
Borojevia (pp=1 b=100) obtained in previous studies using a similar set of sequences (Klautau
et al. 2013, 2016; Azevedo et al. submitted; Cóndor-Luján et al. in press) and (2) revealed a
close affinity between Leucascus (L. simplex + L. luteoatlanticus sp. nov.) and Ascaltis (A.
reticulum) supported by high values (pp=0.1, b=99).
DISCUSSION
With the results of the present study, the list of calcareous sponges from the NE Brazil increased
from 42 (Azevedo et al. submitted; Van Soest et al. 2016) to 49 species. Among these, 42.9%
(18) are endemic from this region, 23.8% (10) also occur in the Caribbean Sea and 19% (8)
have an amphi-Atlantic distribution (being present in South Africa or in the Mediterranean Sea).
Whether this apparent high endemism in the NE Brazil reflects a true distribution pattern for
Calcarea remains uncertain as formerly NE Brazilian endemic species have been recently
recorded from other areas, e.g. Grantia kempfi in the Guyana Shelf (Van Soest 2017) and
Borojevia tenuispinata, Clathrina mutabilis, C. insularis and Nicola tetela from Curaçao
(Cóndor-Luján & Klautau 2016; Cóndor-Luján et al. in press). Further samplings from adjacent
localities are necessary to validate this observed endemic pattern.
The new records provided herein bring new insights to understand the distribution patterns of
certain Brazilian calcareous sponges and highlight interesting affinities among the northeastern
localities. The new record of Ernstia rocasensis from Fernando de Noronha suggests an affinity
among three isolated areas: Rocas Atoll, Abrolhos and Fernando de Noronha Archipelagos.
Apart from this species, Leucetta floridana is the only species previously reported from these
localities (although it has a broader distribution in the Western Tropical Atlantic, Valderrama et
al. 2009; Muricy et al. 2011). The occurrence of C. mutabilis in Maracajaú reinforces the
calcarean affinity between the Brazilian continental shelf and the oceanic islands, supported by
the previous records of C. aurea, Leucaltis clathria, Leucascus roseus, and Leucetta floridana
(Muricy et al. 2011). The finding of C. aspera in the NE Brazil (Maracajaú) constitutes an
important intermediate record as previously, the known distribution of this species was disjunct:
Curaçao and Rio de Janeiro (Cóndor-Luján et al. in press) and therefore, we point out the
importance of continuing to preserve this protected area.
The new molecular affinities found in this study suggested an apparent phylogenetic signal
in the skeleton composition (choanosomal triactines) of some calcaronean species, as already
171
pointed out (Cóndor-Luján et al. in press). However, more species with different skeleton
characteristics should be included in the trees to verify this hypothesis. Besides, it is necessary
that more detailed morphological descriptions are made to allow mapping the characters on
phylogenetic trees.
ACKNOWLEDGEMENTS
We are indebted to Marcelo Soares and Ana Luisa Pires Moreira for providing technical support
with collecting licenses, Olivier Thomas, Thierry Pérez, Mariana Carvalho, André Bispo, Sula
Salani, Cristiana Castello-Branco, Camille Leal, João Carraro and Humberto Fortunato for field
assistance and sponge collection, Carol Leite for some slides preparations and the staff of the
Laboratório de Protistologia of Prof. Inácio da Silva Neto for providing assistance with
microscopy images. The dive centres Atlantis Divers, Mar de Noronha Divers and Natal Divers
are acknowledged for proving technical diving assistance. We thank the Brazilian government
agencies that authorized the sampling licenses in MPAs; Instituto Chico Mendes de
Conservação da Biodiversidade (ICMBio), Instituto de Desenvolvimento Sustentável e Meio
Ambiente do Rio Grande do Norte (IDEMA), Reserva Biológica Marinha do Atol das Rocas,
Parque Nacional Marinho de Fernando de Noronha, and the Brazilian Navy (Terminal Portuário
do Pecém). This work was funded by the Brazilian National Research Council (CNPq),
Coordination for the Improvement of Higher Education Personnel (CAPES), Foundation Grupo
Boticário de Proteção à Natureza and Rio de Janeiro State Research Foundation (FAPERJ).
B.C.L. received a scholarship from CAPES, F.A. has a post-doc fellowship by CAPES and A.P.
has a post-doc fellowship by FAPERJ. E.H. and M.K. have fellowships by CNPq.
172
Figure 15. Maximum Likelihood phylogenetic tree inferred from the ITS sequences of theCalcinean species. Bayesian posterior probabilities and bootstrap values (pp/b) are given on thebranches. * Sequences generated in this study. FN: Fernando de Noronha. RN: Rio Grande doNorte.
173
REFERENCES
Araújo, P. V. &Amaral,R. (2016) Mapping of coral reefs in the continental shelf of BrazilianNortheast through remote sensing. Journal of Integrated Coastal Zone Management, 16(1),5-20.
Azevedo, F., Padua, A., Moraes, F., Rossi, A., Muricy, G. & Klautau, M. (submitted). Taxonomyand phylogeny of calcareous sponges (Porifera: Calcarea: Calcinea) from Brazilian mid-shelf and oceanic islands. Zootaxa.
Azevedo, F. & Klautau, M. (2007) Calcareous sponges (Porifera, Calcarea) from Ilha GrandeBay, Brazil, with descriptions of three new species. Zootaxa, 1402: 1-22.
Borojevic, R. (1971) Eponges calcaires des côtes du Sud-Est du Brésil, épibiontes surLaminaria brasiliensis et Sargassum cymosum. Revista Brasileira de Biologia, 31: 525-530.
Borojević, R. & Peixinho, S. (1976) Éponge calcaires du nord-nord-est du Brésil. Bulletin duMuséum National d´Histoire Naturelle, 3(402), 988–1036.
Carter, H.J. (1886) Descriptions of Sponges from the Neighbourhood of Port Phillip Heads,South Australia, continued. Annals and Magazine of Natural History, 5, 18, 34-55, 126-149.
Carter H.J. (1890) Porifera. In: Ridley, H.N.(ed.) Notes on the Zoology of Fernando doNoronha. Journal of the Linnean Society (The Natural History of the island of Fernando deNoronha based on the collections made by the British Museum Expedition in 1887), pp.564-569.
Cavalcanti, F.F., Bastos, N. & Lanna, E. (2015). Two new species of the genus VosmaeropsisDendy, 1892 (Porifera, Calcarea), with comments on the distribution of V. sericata (Ridley,1881) along the Southwestern Atlantic Ocean. Zootaxa, 3956 (4), 476–490.
Cavalcanti, F.F., Menegola, C. & Lanna, E. (2014) Three new species of the genus ParaleucillaDendy, 1892 (Porifera, Calcarea) from the coast of Bahia State, Northeastern Brazil.Zootaxa, 3764(5), 537–554.
Cavalcanti, F.F., Rapp, H.T. & Klautau, M. (2013) Taxonomic revision of Leucascus Dendy,1892 (Porifera: Calcarea) with revalidation of Ascoleucetta Dendy & Frederick, 1924 anddescription of three new species. Zootaxa, 3619 (3), 275–314.
Chombard C., Boury-Esnault N. & Tillier S. (1998) Reassessment of homology ofmorphological characters in tetractinellid sponges based on molecular data. SystematicBiology, 47(3), 351–366.
Cóndor-Luján, B. & Klautau, M. (2016) Nicola gen. nov. with redescription of Nicola tetela(Borojevic & Peixinho, 1976) (Porifera: Calcarea: Calcinea: Clathrinida). Zootaxa, 4103,230–238.
Cóndor-Luján, B., Louzada, T, Azevedo, F., Padua, A., Hajdu, E., Klautau, M. (to be submitted)Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared speciesand phylogenetic remarks on Calcarea. Journal of the Marine Biological Association of theUnited Kingdom.
Ebner, V. Von. (1887). Amphoriscus buccuchii n.sp. Zoologische Jahrbücher, 2, 981-982. Haeckel E. (1872). Die Kalkschwämme, eine Monographie. G. Reimer, Berlin, 418 pp. Hajdu, E., Peixinho, S. & Fernandez, J. (2011). Esponjas Marinhas da Bahia. Guia de Campo e
Laboratório. Rio de Janeiro: Museu Nacional, Série Livros 45. 276 pp. Hooper, J. & Van Soest, R.W.M. (2002) Systema Porifera: A Guide to the Classification of
Sponges. Kluwer Academic/Plenum Publishers, New York.Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogeny.
Bioinformatics, 17, 754-755. Katoh, K. & Standley, D.M. 2013. MAFFT multiple sequence alignment software version 7:
improvements in performance and usability. Molecular Biology and Evolution, 30: 772–780.
174
Katoh, K & Toh, H. 2008. Improved accuracy of multiple ncRNA alignment by incorporatingstructural information into a MAFFT-based framework. BMC Bioinformatics, 9:212.
Klautau, M. & Valentine, C. (2003) Revision of the Genus Clathrina (Porifera, Calcarea).Zoological Journal of the Linnean Society, 139, 1-62.
Klautau, M., Azevedo, F., Cóndor-Luján, B., Rapp, H.T., Collins, A. & Russo C.A.M. (2013) Amolecular phylogeny for the Order Clathrinida rekindles and refines Haeckel’s taxonomicproposal for calcareous sponges. Integrative and Comparative Biology, 53(3), 447-461.
Klautau, M., Imešek, M., Azevedo F., Pleše B., Nikolić V. & Ćetković H. (2016) Adriaticcalcarean sponges (Porifera, Calcarea), with the description of six new species and arichness analysis. European Journal of Taxonomy, 178, 1–52.
Klautau M., Solé-Cava A.M. & Borojević R. (1994) Biochemical systematics of siblingsympatric species of Clathrina (Porifera: Calcarea). Biochemical Systematics and Ecology,22, 367–375.
Klautau, M. & Borojevic, R. (2001) Sponges of the genus Clathrina Gray, 1867 from Arraial doCabo, Brazil. Zoosystema, 23 (3), 395–410.
Lanna, E., Rossi, A.L., Cavalcanti, F.F., Hajdu, E. & Klautau, M. (2007) Calcareous spongesfrom São Paulo State, Brazil (Porifera: Calcarea: Calcinea), with the description of two newspecies. Journal of the Marine Biological Association of the United Kingdom, 87, 1553–1561.
Lanna E., Cavalcanti F. F., Cardoso L., Muricy G., & Klautau M. (2009) Taxonomy ofcalcareous sponges (Porifera, Calcarea) from Potiguar Basin, NE Brazil. Zootaxa, 1973, 1-27.
Lôbo-Hajdu, G., Guimarães, A., Salgado, A., Lamarão, F., Vieiralves, T., Mansure, J. & Albano,R. 2004. Intragenomic, Intra– and interspecific variation in the rDNA ITS of a poríferarevealed by PCR-single-strand conformation polymorphism (PCR-SSCP). Bolletino deiMusei e Degli Istitui Biologicci de Genova, 68, 413-423.
Longo, L.L. & Amado-Filho, G.M. (2014) Knowledge of Brazilian benthic marine faunathroughout time. História, Ciências, Saúde-Manguinhos, 21, 995–1010.
Manuel, M., Borojevic, R., Boury-Esnault, N. & Vacelet, J. (2002) Class Calcarea Bowerbank,1864. In: Hooper, J. N. A. & Van Soest, R. W. M. (Eds.), Systema Porifera. A guide to theclassification of sponges. Kluwer Academic/Plenum Publishers, New York, pp. 1103-1110.
Moraes, F.C. (2011) Esponjas das Ilhas Oceânicas Brasileiras. Museu Nacional, Série Livros44, Rio de Janeiro, 252 pp.
Muricy G., Lopes D.A., Hajdu E., Carvalho M.S., Moraes F.C., Klautau M., Menegola C. &Pinheiro U. (2011) Catalogue of Brazilian Porifera. Rio de Janeiro: Museu Nacional, SérieLivros 46. 299 pp.
Muricy, G. & Silva, O.C. (1999) Esponjas marinhas do Estado do Rio de Janeiro: um recursorenovável inexplorado. In: Ecologia dos Ambientes Costeiros do Estado do Rio de Janeiro,Série Oecologia Brasiliensis, 7:155 -178.
Poléjaeff, N. (1883) Report on the Calcarea dredged by H.M.S.‘Challenger’, during the years1873-1876. In: Report on the Scientific Results of the Voyage of H.M.S. ‘Challenger’,1873-1876. Zoology 8(2), 1-76.
Ronquist, F. & Huelsenback, J.P. 2003. MRBAYES 3: Bayesian phylogenetic inference undermixed models. Bioinformatics, 19: 1572–1574.
Rossi, A.L., Russo, C.A.M., Solé-Cava, A.M., Rapp, H.T. & Klautau, M. (2011). Phylogeneticsignal in the evolution of body colour and spicule skeleton in calcareous sponges.Zoological Journal of the Linnean Society 163, 1026–34.
Solé-Cava A.M., Klautau M., Boury-Esnault N., Borojević R. & Thorpe J.P. (1991) Geneticevidence for cryptic speciation in allopatric populations of two cosmopolitan species of thecalcareous sponge genus Clathrina. Marine Biology, 111(3), 381–386.
175
Spalding, M.D., Fox, H.E., Allen, G.R., Davidson, N., Ferdaña, Z.A., Finlayson, M., Halpern,B.S., Jorge, MA, Lombana, A., Lourie, S.A., Martin K.D., McManus, E., Molnar, J.,Recchia, C.A. & Robertson, J. (2007) Marine ecoregions of the world: a bioregionalizationof coastal and shelf areas. Bioscience 57, 573–583.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Sudhir Kumar. 2013. MEGA6:Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution,30: 2725-2729.
Valderrama, D., Rossi, A.L., Solé-Cava, A.M., Rapp, H.T. & Klautau, M. (2009) Revalidation ofLeucetta floridana (Haeckel, 1872) (Porifera, Calcarea): a widespread species in thetropical western Atlantic. Zoological Journal of the Linnean Society, 157: 1–16.
Van Soest, R. W. M. (2017). Sponges of the Guyana Shelf. Zootaxa. 4217: 1-225.Van Soest R. W. M. & De Voogd, N. J. (2015). Calcareous sponges of Indonesia. Zootaxa,
3951(1): 1-105. Van Soest, R.W.M, Boury-Esnault, N., Hooper, J.N.A., Rützler, K., de Voogd, N.J., Alvarez de
Glasby, B., Hajdu, E., Pisera, A.B., Manconi, R., Schoenberg, C., Janussen, D., Tabachnick,K.R., Klautau, M., Picton, B., Kelly, M., Vacelet, J., Dohrmann, M., Díaz, M.C. &Cárdenas, P. (2016) World Porifera database. Available from:http://www.marinespecies.org/porifera (accessed on November 2016).
Voigt O., Wülfingl E. & Wörheide G. (2012). Molecular Phylogenetic Evaluation ofClassification and Scenarios of Character Evolution in Calcareous Sponges (Porifera, ClassCalcarea). PLoS ONE 7(3): e33417.
Voigt, O. & Worheide, G. (2016) A short LSU rRNA fragment as a standardmarker forintegrative taxonomy in calcareous sponges (Porifera: Calcarea). Organisms Diversity andEvolution, 16(1), 53-64.
Wörheide, G. & Hooper, J.N.A (1999) Calcarea from the Great Barrier Reef. 1: CrypticCalcinea from Heron Island and Wistari Reef (Capricorn-Bunker Group). Memoirs of theQueensland Museum, 43 (2), 859–892.
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Evolutionary history of the calcareous sponge Leucetta floridana in the Western Tropical
Atlantic
Cóndor-Luján, B. 1; García-Hernández, J.2; Schizas, N.2; Pérez, T.3; Zea, S4. Klautau, M. 1
1 Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.
Carlos Chagas Filho 373, CCS, Bloco A, A0-100, 21941-902, Rio de Janeiro, RJ, Brasil.2 Caribbean Laboratory of Marine Genomics, University of Puerto Rico-Mayagüez, P.O. Box
9000, Mayagüez, PR, 00681, United States of America. 3Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale, CNRS, Aix
Marseille Univ, IRD, Avignon Univ. Station Marine d’Endoume, rue de la Batterie des Lions,
13007 Marseille, França.4Centro de Estudios en Ciencias del Mar – CECIMAR, Universidad Nacional de Colombia,
Sede Caribe; c/o INVEMAR, Calle 25 2-55, Rodadero Sur - Playa Salguero, Santa Marta,
Colombia. *Corresponding author: [email protected]
Running title: Evolutionary history of Leucetta floridana
ABSTRACT
Sponges have short-lived lecithotrophic larvae, which constrain their dispersion and
consequently restrict their geographic distribution. However, some calcareous species as
Leucetta floridana are widespread in the Western Tropical Atlantic (WTA). In order to elucidate
the historical processes that originated its current distribution and to evaluate the role of the
Amazon River as a barrier to the gene flow between Caribbean and Brazilian sponge
populations, several genetic analyses using ITS sequences were performed. A total of 162
specimens from different localities (Puerto Rico, Lesser Antilles, Panama, Colombia, Curaçao
and Brazil) was studied. Genetic variation was assessed through sequence-type and nucleotidic
diversity indexes. Phylogenetic trees and a MJ network were constructed to explore the
genealogical relationships among individuals. To determine population structure, FST
comparisons and AMOVA were performed. Neutrality tests, mismatch distributions and
Bayesian skyline plots were conducted to infer demography patterns. The phylogenetic trees as
well as the networks showed high structured populations consistent with FST and AMOVA
results: four populations restricted to the Caribbean Sea and one population widespread in the
WTA. The presence of a large widespread population in the WTA rejects the role of the Amazon
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River as an effective barrier to gene flow between Caribbean and Brazilian localities.
Demography analyses indicated an event of population expansion in the Caribbean Sea during
the Last Maximum Glaciation (20 millions year BP). The patterns of population connectivity
and demography within the WTA are discussed and a hypothetical evolutionary scenario for L.
floridana is presented.
Key words: Amazon River, Brazilian Coast, Caribbean Sea, Greater and Lesser Antilles,
Population connectivity, Porifera
INTRODUCTION
In the marine environment, organisms with short-lived larvae and consequent low dispersal
capabilities, such as sponges, are not expected to attain widespread geographic distributions.
Some morphological and molecular studies on sponges have rejected the putative
cosmopolitanism of some species (Solé-Cava et al., 1991; Boury-Esnault et al., 1992; Hajdu &
van Soest, 1992; Klautau et al., 1994; Muricy et al., 1996; Klautau et al., 1999; Klautau &
Valentine, 2003; Valderrama et al., 2009), while others revealed that species with widespread
distribution do occur in the Western Tropical Atlantic Ocean (WTA) (Lazoski et al., 2001;
Valderrama et al., 2009).
The occurrence of species with this wide distribution represents a good opportunity to assess
the species dispersal across biogeographic barriers such as the Amazon River. Up to date, no
study on the effectiveness of the Amazon River as a barrier to gene flow was performed within
Porifera. Some recent studies evaluated the genetic connectivity of sponges only within the
Tropical Northwestern Atlantic (TNA), e.g. Callyspongia vaginalis (De Biasse et al., 2010,
2016), Cliona delitrix (Chaves-Fonnegra et al., 2015) and Xestospongia muta (López-Legentil
& Pawlik, 2009; Richards et al., 2016; de Bakker et al., 2016), or exclusively in the Tropical
Southwestern Atlantic (TSA) (Padua et al., in prep.: Clathrina aurea) but none considered
populations along the entire Western Tropical Atlantic.
Different biochemical and molecular approaches have been used to unravel the genetic
structure of sponge populations (isozymes, nuclear and mitochondrial DNA regions). In
calcareous sponges, as the traditional mitochondrial DNA marker (COI) was not appropriate for
phylogeography inferences or population structure studies (Worheide et al., 2000), nuclear
intron regions (ITS and ATPSb-Iii) were employed (Wörheide et al., 2002; Bentlage and
Wörheide, 2007, Wörheide et al., 2008). Among them, the ITS proved to be the most suitable
one (Wörheide et al., 2008). Recently, the mitochondrial gene cox3 was proposed as an
alternative phylogeography marker since it evidenced a variability similar to that of nuclear
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intron data (Voigt et al., 2012). However, due to the substantial variation at both intra- and inter-
specific levels found in mitochondrial markers (Lavrov et al., 2013), the use of cox3 remains
uncertain. Therefore, for the class Calcarea, the ITS region is still a useful marker at population
level.
The calcareous sponge Leucetta floridana (Calcarea: Calcinea: Leucettidae) has a patchy
distribution within reefs and inhabits environments protected from light such as small crevices,
overhangs, and vertical slopes (Valderrama et al., 2009). It has been found from -2 to -90 m of
depth (Muricy et al., 2011). As a calcinean species, it is assumed to have a lecithotrophic larvae
(calciblastula) (Borojevic et al., 1990; Maldonado & Berquist, 2002), however, the reproduction
of this species has not been investigated yet.
Leucetta floridana is one of the most widespread calcareous sponges in the TWA and thus,
constitute a good model for evaluating population connectivity within the Western Tropical
Atlantic. In this study, we assessed the genetic structure of this species at phylogeographic,
population and demographic levels in order to (1) understand the historical processes that
culminated in its current geographic distribution and to (2) evaluate the role of the Amazon
River as a barrier to the gene flow between Caribbean and Brazilian populations of sponges.
MATERIALS AND METHODS
Analysed material
The analysed material comprised 162 specimens preliminary identified as Leucetta floridana
from 18 localities within the Tropical Northwestern Atlantic (TNA, n=147) and the Tropical
Southwestern Atlantic (TSA, n=15), at depths varying from -2 to -70 m.
Localities in the TNA included four ecoregions, Greater Antilles - Puerto Rico (n=12),
Eastern Caribbean – Anguilla (n=1), Saint Martin (n=52), Antigua (n=12), Les Saintes (n=8),
Guadeloupe (n=1), Martinique (n=23), and Saba (n=3), Southwestern Caribbean - San Andrés
(n=4), Panama (n=19), Urabá (n=3) and Santa Marta (n=7) and Southern Caribbean - Curaçao
(n=2), whereas in the TSA comprised three ecoregions, Northeastern Brazil – Ceará (n=5) and
Rio Grande do Norte (RN, n=4), Fernando de Noronha and Atoll das Rocas – Fernando de
Noronha Archipelago (n=4) and Rocas Atoll (n=1) and Eastern Brazil – Abrolhos Archipelago
(n=1) (Table 1, Figure 1).
Most of the Caribbean specimens (131 specimens) were collected during several campaigns:
Expedition PaCoTilles (Patterns of Connectivity among the Lesser Antilles, 2015, n=87), Santa
Marta Expedition (2014, n=7), Martinique Expedition (2013, n=10), Puerto Rico Expedition
(2015, n=12) and PorToL Project (2012, n=15). After collection, specimens were fixed and
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stored in ethanol 93 or 96% until DNA extraction. All voucher specimens are deposited in the
Porifera Collection of the Universidade Federal of Rio de Janeiro (UFRJPOR) in Brazil,
Universidad de Puerto Rico or INVEMAR (Instituto de Investigaciones Marinas y Costeras) in
Colombia.
The other 31 specimens were already deposited in the Porifera Collection of the UFRJPOR
and were analysed in previous studies (Valderrama et al., 2009; Klautau et al., 2013; Cóndor-
Luján et al., in press). Detailed information of any specimen can be accessed in Table S1 of the
Supplementary Material.
Additional sequences of Leucetta species were retrieved from Genbank (Table 2) for
comparative phylogenetic analyses described in the corresponding section.
Figure 1. Map indicating the localities considered in this study. Coloured circles representdifferent ecoregions according to the MEOW (Spalding et al., 2007). Blue: Eastern Caribbean,green: Southwestern Caribbean, yellow: Southern Caribbean, red: Northeast Brazil, orange:Fernando de Noronha and Atoll das Rocas, and pink= Eastern Brazil. The Amazon River isindicated.
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Table 1. Locality, geographic coordinates, depth, and Genbank accession numbers of thespecimens of Leucetta floridana analysed in this study. CE=Ceará, PE=Pernambuco, RN=RioGrande do Norte, FN=Fernando de Noronha Archipelago. *Sequences generated in the presentstudy.
Locality Geographic Coordinates Depth (m) Genbank Number
Greater Antilles Ecoregion – Puerto RicoCayoTurrumote 17°56'20.13''N, 67° 2'43.95''W 12.9 - 17.4 N=2* Cayo Mario 17°57'16.89''N, 67° 3'53.42''W 09.0 - 17.7 N=4* Cayo Conserva 17°55'56.91''N, 67° 5'35.01''W 12.6 - 15.6 N=2* Veril – Fallen Rock 17°54'5.04''N, 66°55'27.59''W 31.5 N=1*Pinnacles 17°56'3.96''N, 67° 1'49.80''W 9.9 - 17.1 N=2*Veril - El Hoyo 17°52.529'N, 67° 2.671'W 31.5 N=1*Eastern Caribbean Ecoregion – Lesser AntillesLittle Scrub 2, Anguilla 18°17.903'N, 62°57.294'W 23.5 N=1* Trou David, Terres Basses, SaintMartin
18°04.402'N, 63°07.149'W 6.1 N=4*
Rocher Créole, Saint Martin 18°07.038'N, 63°03.419'W 8.0 – 10.0 N=1* Les Arches1, Saint Martin 18°07.588'N, 62°58.248'W 10 - 20 N=10* Basse Espagnoles, Saint Martin 18°07.821'N, 63°00.270'W 6 - 10 N=16* Chico2, Saint Martin 18°06.501'N, 62°59.005'W < 22 N=11*Les Arches2, Saint Martin 18°07'32.81''N, 62°58'21.49''W < 16 N=7*Southeastern Coast, Saba 17°37.066'N, 63°13.580'W < 27 N=3*Nanton Point, Saint Paul, Antigua and Barbuda
16°59'51.60''N, 61°45'37.22''W < 20 N=9*
Five Islands, Antigua and Barbuda
17°04.990'N, 61°54.840'W 4 N=1*
Diamond Bank, Saint Paul, Antigua and Barbuda
17°12.000'N, 61°52.800'W 7 – 8 N=2*
Cave Cathédrale, Guadeloupe 16°27.740' N, 61°31.837'W 13.7 N=1*Cave 1, Les Saintes, Guadeloupe
15°52.984'N, 61°34.25'W 8 - 10.7 N=9*
Grottes des couleurs, Pointe Burgos, Grande Anse, Anses d'Arlet, Martinique
14°29.787'N, 61°05.351'W <10 KX355573KX355574N=7*
Anses d´Arlet, Martinique 14°30.377'N, 61°05.850'W? <10 KX355575N=1*
Anse de Fortune, Anses d'Arlet, Martinique
14°30.377'N, 61°05.850'W 3 – 6 N=9*
Southwestern Caribbean EcoregionBocas del Toro, Panama 09º20.914'N, 082º09.394'W? < 17 EU78189
EU78190EU78191N=8*
Las Cuevas, Bocas del Toro, Panamá
09º20.914'N, 082º09.394'W <20? N=7*
Cayo Zapatilla, Bocas del Toro, Panamá
09º14.881'N, 82º01.952'W <20? N=2*
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La Piscinita, San Andrés, Colombia
12°33'N, 81°43'W? 2- 5 EU781972 EU781973 EU781974
West View, Leward-reef, San Andrés, Colombia
12°33'N, 81°43'W? 5 EU781971
Bajo Agua Viva, Urabá, Colombia
07°53''N, 76°38'W? 15 EU781968 EU781969EU781970
El Morro, Santa Marta Bay, Colombia
11°14'57.50"N, 74°13'54.50"W 11 - 21 N=4*
Punta Venado, Ensenada de Taganga, Santa Marta, Colombia
11°16'25.00"N, 74°12'24.00"W 18 – 18.6 N=2*
Punta Gaira, Gaira Bay, Santa Marta, Colombia
11°13'08.00"N, 74°14'30.00"W 14 N=1*
Southern Caribbean Ecoregion - CuraçaoWater Factory, Willemstadt 12°06'30.88"N, 68°57'13.53"W 17.8 N=1Hook’s Hut, Willemstadt 12°07'18.94"N, 68°58'11.46"W 13.3 N=1Northeastern Brazil Ecoregion- BrazilPotiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 EU781985
N=2*Risca das Bicudas, RN 04º57'00.9"S, 36º07'49.7"W 10 EU781978Urca do Tubarão, RN 04º50'52.7"S, 36º27'02.1"W N=1*Station 30 - CENPES, CE 2º52'S, 39 º10"W 33 EU781980
EU781982EU781983EU781984N=1*
Fernando de Noronha and Atoll das Rocas Ecoregion - BrazilBarretinha, Rocas Atoll, RN 3°51'36'S, 33°49'04''W 12 EU781975Sela Gineta Island, FN, PE 3°48'49''S, 32°23'29''W 7 EU781976Ressurreta, FN, PE 3°48'49''S, 32°23'29''W 4 – 7.3 EU781977
N=2*Eastern Brazil EcoregionAbrolhos Marine National Park, Bahia - BrazilParcel das Paredes 17°58'S, 38°40'W 8 EU781979
Table 2. Species names, voucher numbers, locality and Genbank accession numbers of thesequences used in the phylogenetic analysis.
Species Voucher Number Locality Genbank NumberLeucetta antarctica MNRJ 13798 Antarctic KC849700Leucetta chagosensis BMOO1612 French Polynesia KC843455Leucetta microraphis QMG313659 Australia AJ633874Leucetta pyriformis MNRJ13843 Antarctic KC843457Leucetta potiguar UFPEPOR 547 Brazil EU781986Leucetta potiguar MNRJ 8474 Brazil EU781981Leucetta potiguar UFPEPOR 569 Brazil EU781987Leucetta potiguar UFPEPOR 588 Brazil EU781988
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DNA extraction, amplification, sequencing and alignment
The DNA of 136 specimens was extracted following the guanidine/phenol-chloroform protocol
(Sambrook et al., 1993) and stored at –20°C until amplification. The region comprising the
partial 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA was amplified by
PCR with the primers: 18S (5`-TCATTTAGAGGAAGTAAAAGTCG-3`) and 8S (5`-
GTTAGTTTCTTTTCCTCCGCTT-3`) (Lôbo-Hajdu et al., 2004). Each PCR amplification
reaction mixture contained: 1X buffer (5X GoTaq R Green Reaction Buffer Flexi, PROMEGA),
0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM of each
primer, one unit of Taq DNA polymerase (Fermentas) and 1 µL of DNA, summing up to 15 µL
with Milli-Q water. PCR steps included one first cycle of 4 min at 94°C, 1 min at 50°C and 1
min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and 1 min at 72°C, and a final cycle of 6
min at 72°C. Successful amplification was visualized in an 1% agarose electrophoresis gel and
purified with the GE Kit. Forward and reverse strands were automatically sequenced in an ABI
3500 (Applied Biosystems).
The electropherograms of the sequences generated in this study as well as the 25 sequences
of L. floridana retrieved from the Genbank database were visually edited using Chromas Lite.
Electropherograms with dubious base attribution (double-peaks) were not considered in any
further analyses and are not presented here. All sequences generated in this work were deposited
in the GenBank database (www.ncbi.nlm.nih.gov).
Phylogenetic analyses
In order to verify the identity of the specimens attributed to L. floridana, phylogenetic
reconstructions, including sequences from other Leucetta species (Table 2), were performed
under maximum-likelihood (ML) and Bayesian Inference (BI).
Sequences were aligned through the MAFFT v.7 online version (Katoh & Standley, 2013)
using the method Q which considers the secondary structure of the amplified region (Katoh et
al., 2005) and consequently, guarantees a better alignment. The nucleotide substitution model
that best fit the alignment was determined using jModelTest2 (Darriba et al., 2012; Guindo &
Gascuel, 2003) and corresponded to GTR + G.
The ML analyses were conducted on MEGA 6 (Tamura et al., 2013) using an initial NJ tree
(BIONJ) and 1000 bootstrap pseudo-replicates. The BI reconstructions were obtained with
MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) under 106
generations and a burn-in of 1 000 sampled trees, yielding a consensus tree of majority.
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The genetic divergence among all the species and the genetic variability among individuals
were assessed by the calculus of the uncorrected p distance, considering complete deletion in
MEGA 6.
Phylogeography analyses
Considering only the individuals of L. floridana, sequences were re-aligned as described in the
above section. Gaps were considered as informative characters. The ITS sequence-types
(referred herein as ST) were calculated by DNASP v. 5.10.01 (Librados & Rozas, 2009). The
nucleotide (π) and the ITS sequence-type (STh) diversities were determined using ARLEQUIN
v. 3.5.1 (Schneider et al., 2000).
To assess the evolutionary and geographical relationships, two analyses were considered: (1)
a tree including the STs within each ecoregion and additional Leucetta sequences, rooted on the
most distant lineage(s) and (2) an unrooted tree (network) considering only the specimens of L.
floridana. Phylogenetic analyses for the rooted tree followed the procedures detailed in the
above section (ML and BI approaches). Networks were calculated using the Median Joining
algorithm (Bandelt et al., 1999) within the program NETWORK v. 4.6 (Forster et al., 2007). ST-
groups (STG) were defined by an analysis of molecular variance (AMOVA) as it maximizes
variation among the groups and minimizes variation within the groups.
Population structure
The population structure across different geographical partitions (provinces, ecoregions and
localities) was assessed through pairwise FST comparisons (Weir & Cockerham, 1984) and an
analysis of molecular variance (AMOVA) as implemented in ARLEQUIN. The statistical
significance of estimates was assessed by 10 000 permutations.
Demographic structure
In order to test for population expansion across geographical (provinces, ecoregions and
localities) and phylogenetic (clades and STGs) groups, Tajima´s D (D) and Fu´s Fs (Fs)
neutrality tests were performed. Moreover, the SSD and raggedness index (r) values based on
mismatch distribution were calculated. All calculations considered 104 simulations and were
computed in ARLEQUIN.
To explore the historical demography of L. floridana, Bayesian skyline analyses (Drummond
et al., 2005) were performed in BEAST 1.8 (Drummond et al., 2012) for each structured
population (n≥10) as suggested by the AMOVA and network results. The nucleotide substitution
model was determined using the Bayesian Information Criterion (BIC) implemented in MEGA
6. The chosen model was Jukes Cantor but as it is not implemented in BEAUti 1.8 (Drummond
et al., 2012), we used the closest model with the lowest BIC: the HKY model. As the ITS
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substitution rate within Calcarea is unknown, we used the 1.0% per MY estimated for the
demospongiae Prosuberites laughlini (Worheide et al., 2004) which was also employed to
estimate phylogeography patterns in other Leucetta species (Worheide et al., 2008). The
analyses assumed a strict molecular clock model and the numbers of groups were set to six
(previous exploratory tests with the same sequence set showed no difference in the Bayesian
Skyline Plot when using six or 10 groups). A MCMC analysis of 108 generations was run with
10% burn-in. The results were summarized in piecewise-constant Bayesian Skyline Plots using
TRACER 1.5 (Rambaut & Drummond, 2009).
RESULTS
Genetic Diversity
P distance
The length of the alignment including all the Leucetta sequences was 764 pb, including 136
variables sites, 74 parsimony-informative sites and 61 singletons. All specimens preliminarily
identified as L. floridana clustered in a clade supported by high ML bootstrap and BI posterior
probability (b=99, p=1) values, corroborating the initial identification (Figure 2).
The genetic divergences among the species of Leucetta were as follows: L. floridana–L.
potiguar: 2.5-3.4%, L. floridana–L. microraphis: 4.7-5.3%, L. floridana–L. chagosensis: 7.1-
7.7% , L. floridana–L. antarctica: 8.0-8.6%, and L. floridana–L. pyriformis: 8.5-9.1%. Leucetta
potiguar, a species restricted to the TSA Province, appeared as the sister taxon of L. floridana as
already reported in previous studies (Valderrama et al., 2009; Klautau et al., 2013).
The length of the alignment considering only L. floridana individuals was 733 pb with 708
invariable sites, 23 variable sites and two sites with alignment gaps. The intraspecific variability
inferred by the p-distance ranged from 0 to 1.8%. Interestingly, this same range value (0-1.8%)
was found (1) between Caribbean and Brazilian individuals and (2) among Caribbean
individuals. Among Brazilian individuals, the range varied between 0 and 0.1%.
Sequence-type richness
A total of 24 STs were found when considering gaps as informative characters. Among them,
two STs were shared by the TNA and TSA (ST1 and ST2), 16 were exclusive to the TNA and
one to the TSA (Table 3). ST1 was the most frequent ST, found in 98 individuals (60.5%) and
present in all the studied ecoregions except the Southern Caribbean (Curaçao). ST2 was
restricted to the Southwestern Caribbean and Fernando de Noronha and Atoll das Rocas
ecoregions. Besides ST1 and ST2, other five STs were shared among different ecoregions and
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localities (ST12, ST15, ST18, ST19 and ST14). Seventeen STs were private to one locality or
island (ST3, ST5, ST6, ST7, ST8, ST9, ST10, ST11, ST13, ST14, ST16, ST17, ST20, ST21,
ST22, ST23, ST24).
The ecoregion with more STs was Eastern Caribbean (K=13) followed by Southwestern
Caribbean (K=10, Table 3). Greater Antilles, Northeastern Brazil and Fernando de Noronha and
Atoll das Rocas presented the same ST contribution (K=2) whereas the Southern Caribbean and
Eastern Brazil presented only one ST. Within the Eastern Caribbean, the island with more STs
was Saint Martin (K=8) followed by Martinique with five STs. Both localities presented the
same number of private STs (P=4). In the Southwestern Caribbean, the localities with more STs
were Panama and San Andrés, both with four STs. Within the Brazilian localities, Fernando de
Noronha and Ceará were the most diverse localities presenting two STS.
Sequence-type (STd) and nucleotidic (π) diversities
The overall STd was 0.62±0.04 and the total π was 0.003±0.002. Similar values were obtained
for the TNA whereas slightly lower values were observed for the TSA (Table 3). Among the
ecoregions, the Southwestern Caribbean presented the highest values: STd=0.83±0.04 and
π=0.0078±0.0039. Within this ecoregion, San Andrés had the highest values: STd=1±0.18 and
π=0.0075±0.0054, being followed by Panama with STd=0.71± 0.07 and π =0.005±0.003.
Greater Antilles (Puerto Rico) presented the lowest values in STd=0.17 ± 0.133 and in π=
0.0002 ± 0.0004.
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Figure 2. Maximum Likelihood phylogenetic tree inferred from the ITS sequences of Leucettafloridana and other Leucetta species. ML bootstrap and BI posterior probability values areindicated on the branches.
Table 3. Genetic Diversity. N=number of sequences, S/T=Segregating/Total number of sites, K=number of STs, P=number of private STs, STd= ST diversity, π=nucleotide diversity, SD=standard deviation. RN=Rio Grande do Norte State, FN=Fernando de Noronha Archipelago.
Ecoregion Locality N S/N K P STd (SD) π(SD)Tropical Northern Atlantic 147 24/733 23 16 0.638 (0.045) 0.0038 (0.0022)Greater Antilles
Puerto Rico 12 1/732 2 1 0.167 (0.134) 0.0002 (0.0004)Eastern Caribbean 100 19/733 13 8 0.558 (0.056) 0.0022 (0.0014)
Anguilla 1 0/732 1 0 - -Saint Martin 52 16/733 8 3 0.3167 (0.084) 0.0015 (0.0011)Saba 3 0/732 1 0 0 0Antigua 12 13/732 4 2 0.454 ( 0.17) 0.003 (0.002)Guadeloupe 1 0/732 1 0 - -Les Saintes 8 1/732 2 0 0.25 (0.18) 0.0003 (0.0005)Martinique 23 4/733 5 3 0.613 (0.104) 0.0016 (0.0012)
Southwestern Caribbean 33 14/732 10 7 0.835 (0.042) 0.007 (0.0039)San Andrés 4 10/731 4 3 1.0 (0.177) 0.0075 (0.0055)Panama 19 9/732 4 2 0.713 (0.074) 0.0055 (0.0032)Urabá 3 1/732 2 1 0.667 (0.314) 0.0009 (0.0011)Santa Marta 7 8/732 2 1 0.476 (0.171) 0.0052 (0.0034)
Southern CaribbeanCuraçao 2 0/732 1 0 0 0
Tropical Southern Atlantic 15 2/732 3 1 0.457 (0.141) 0.0007 (0.0007)Northeastern Brazil 9 1/732 2 1 0.389 (0.164) 0.0005 (0.0006)
Ceará 5 1/732 2 1 0.6 ( 0.175) 0.0008 (0.0009)RN 4 0/732 1 0 0 0
FN and Atoll das Rocas 5 1/732 2 0 0.6 (0.175) 0.0008 (0.0009)FN 4 1/732 2 0 0.5 (0.265) 0.0007 (0.0008)Rocas Atoll 1 0/731 1 0 - -
Eastern BrazilAbrolhos 1 0/732 1 0 - -
Clade 1= STG1+STG2 144 15/733 17 - 0.527 (0.0497) 0.0014 (0.0011)
Clade 2= STG3 9 2/733 3 - 0.556 (0.165) 0.0008 (0.0008)
Clade 3 9 4/732 4 - 0.694 (0.147) 0.0022 (0.0016)
STG1 139 12/733 15 - 0.492 (0.051) 0.0012 (0.0009)
STG2 5 1/733 2 - 0.4 (0.237) 0.0005 (0.0007)
STG4 6 1/732 2 - 0.333 (0.215) 0.0004 (0.0006)
STG5 3 1/732 2 - 0.667 (0.314) 0.0009 (0.0011)
All 162 25/733 24 - 0.624 (0.044) 0.0035 (0.0021)
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Phylogenetic and phylogeographic inferences
The ML phylogenetic tree obtained from the STs of L. floridana observed within each
ecoregion and the other leucettas is shown in Figure 3A. In both ML and Bayesian analysis, the
clade of L. floridana was subdivided into three clusters with high values of ML bootstrap (b)
and Bayesian posterior probability (pp): clade 1 (b=91 and pp=0.99, in light green), clade 2
(b=94 and pp=0.99, in green) and clade 3 (b=97 and pp=0.96, dark green).
Figure 3. A. Maximum-Likelihood phylogenetic tree inferred from the ITS sequences ofLeucetta floridana and other Leucetta species indicating the sequence-type (STs) found withineach ecoregion. Bootstrap and posterior probability values (ML/BI) are indicated on thebranches. B. Parsimony Median-Joining tree obtained from the ITS sequences of Leucettafloridana. The size of the circles is proportional to the frequency of the STs. Colours representdifferent localities. Numbers between STs indicate mutational steps and when not indicated, itrefers to one-mutational step.
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Clade 1 included representatives from all studied ecoregions in the TSA (Greater Antilles,
Eastern Caribbean, Southwestern Caribbean and Southern Caribbean, in black) and in the TNA
(Northeastern Brazil, Fernando de Noronha and Atoll das Rocas and Eastern Brazil, in red) and
from depths varying from -4 to -70 m. This clade clustered 144 individuals grouped in 17 STs.
Clades 2 and 3 comprised exclusively Caribbean specimens. Clade 2 included nine individuals
of the Southwestern Caribbean representing three STs whereas Clade 3 grouped nine specimens
from four STs.
The parsimony MJ network yielded additional information to the phylogenetic ML and IB
trees, unraveling five ST-groups (Figure 3B) supported by the AMOVA analyses (Tables S2 and
S3, Supplementary Material). Clade 1 was divided into STG1 and STG2, Clade 2 corresponded
to STG3 and Clade 3 was separated in STG4 and STG5. (Reference to Supplementary Material:
STG1 = STGA+ STGB + STGC + STGD, STG2 = STGE, STG3 = STGF, STG4 = STGG and
STG5=STGH). STG1 and STG2 are separated by a three-mutational-step distance. STG1 has a
star-like shape in which rare STs are one-mutational-step close to the common central ST1,
indicating a population expansion pattern. STG4 and STG5 are situated in the opposite side of
the network and are separated by two-mutational steps. SGT3 has apparent central position,
however, it is composed of few individuals (n=9). Determined STGs have certain geographic
correspondence: STG1 is distributed along the TSA and TNA. STG2, STG3, and STG4 are
restricted to the Southwestern Caribbean whereas STG5 occurs only in the Eastern Caribbean.
Population Structure
AMOVA
In the analyses of molecular variance (AMOVA), two hypothetical scenarios of population
structure yielded the maximum variation among groups (ɸCT=0.549). The first hypothetical
scenario (H13) comprised eight groups: (1) Puerto Rico + Anguilla + Saint Martin + Saba +
Antigua + Les Saintes + Guadeloupe, (2) Martinique, (3) San Andrés, (4) Panama, (5) Uraba,
(6) Santa Marta, (7) Curaçao, and (8) Brazil. The second scenario (H19) split the Brazilian
group into (1) Coastal Brazil (Rio Grande do Norte + Ceará + Abrolhos) and (2) Insular Brazil
(Rocas Atoll + Fernando de Noronha). Interestingly, in both hypothesis, the only highly
structured locality within the Eastern Caribbean was Martinique, whereas all localities in the
Southwestern Caribbean represented separated populations. Additionally, Curaçao is well
differentiated from the others despite its low sample size. All tested groups and the F-statistics
obtained are presented in Table S4.
FST Comparisons
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Significant FST indexes ranged from moderate (0.14<FST<0.25) to high (0.25≤FST≤1) values
(Table 4). High values were observed when comparing localities within the Southwestern
Caribbean (except for Panama) with the other Caribbean (0.47≤FST≤1) and Brazilian localities
(0.35≤FST≤0.96). Despite the small number of sequences analysed from Curaçao, high FST values
were observed between this island and other Caribbean localities (Les Saintes and Puerto Rico).
Within the Lesser Antilles, high pairwise FST values (0.47≤<FST<≤0.57) were obtained between
Martinique and some of the other islands (Saint Martin, Saba, Antigua and Les Saintes). Among
ecoregions, pairwise FST comparisons yielded significant high values in Greater Antilles x
Southwestern Caribbean (FST=0.86) and Southern Caribbean x Northeastern Brazil (FST=0.73)
whereas the lowest value was found in Fernando de Noronha and Atoll das Rocas x
Southwestern Caribbean (FST=0.19) (Table 5).
When sequences were grouped according to the clades recovered in the phylogenetic
analyses, all pairwise comparisons were significant (Table 6). FST values ranged from 0.82
(Clade 2 x Clade 3) to 0.88 (Clade 1 x Clade 3). When the same set of sequences was grouped
considering the ST groups, the FST values obtained were even higher (0.85≤FST≤ 0.96) in almost
all cases except in STG1 x STG2 (FST=0.779).
Demography Structure
Sequence-type (ST) and nucleotidic (π) diversity
According to Grant and Bowen (1998), historical processes can be inferred for species or
populations based on haplotipic and nucleotidic diversity indexes. Herein, we suggest some
demographic events in L. floridana based on the observed ST (sequence-type) and π
(nucleotidic) diversity indexes.
The overall values of STd>0.5 and π <0.005 suggest the occurrence of a recent bottle-neck
event within the populations of L. floridana. In the TNA, this must have been followed by a
rapid population growth. Within the Eastern Caribbean, the values observed in Martinique
(STd=0.61±0.1 and π=0.002 ±0.001) indicate a rapid growth within this island whereas Antigua,
Saint Martin and Les Saintes may represent founder populations (STd<0.5 and π <0.005). The
high values observed in the Southwestern Caribbean (STd>0.5 and π >0.005) suggest the
presence of a large stable population or a secondary contact among different populations in this
area or at least in Panama and San Andrés. Differently, Santa Marta, given its diversity indexes
(H<0.5 and π >0.005) may have suffered an ancient bottleneck or may represent divergent
populations geographically subdivided. In the TNA, the low values of STd and π of the
Brazilian population may indicate that they have also experienced a recent bottleneck or
represent founder populations.
Table 4. Pairwise FST values among sampled localities or islands. Caribbean localities are PR: Puerto Rico, AG: Anguilla, SN: Saint Martin, SAB: Saba,AN: Antigua, GU: Guadeloupe, LS: Les Saintes, MT: Martinique, PAN: Panama, SAN: San Andrés, URA: Uraba, SM: Santa Marta and CUR: Curaçao.Brazilian localities: CE: Ceará, RN: Rio Grande do Norte, RAT: Rocas Atoll and ABR: Abrolhos Archipelago. Significant values are in bold.
PR AG SN SAB AN GU LS MT PAN SAN URA SM CUR CE RN FN RAT ABRAG -1SN -0.02 -0.96SAB -0.19 0 -0.18AN 0 -1 -0.02 -0.19GU -1 0 -0.96 0 -1LS 0.01 -1 -0.04 -0.17 -0.03 -1MT 0.61 0.38 0.52 0.51 0.47 0.38 0.57PAN 0.25 -0.36 0.31 0.09 0.14 -0.36 0.21 0.48SAN 0.57 -0.38 0.53 0.23 0.29 -0.37 0.45 0.60 0.16URA 0.97 0.91 0.85 0.95 0.75 0.91 0.95 0.88 0.39 0.53SM 0.73 0.333 0.71 0.54 0.51 0.33 0.67 0.75 0.34 0.29 0.52CUR 0.86 1 0.37 1.0 0.20 1 0.81 0.22 0.21 0.18 0.95 1.0CE 0.31 -0.5 0.05 0.12 0.001 -0.5 0.23 0.53 0.18 0.36 0.92 0.6 0.67RN -0.13 0 -0.13 0 -0.13 0 -0.11 0.56 0.13 0.31 0.96 0.58 1 0.19FN 0.11 -1 -0.06 -0.09 -0.09 -1 0.05 0.53 0.14 0.2 0.92 0.57 0.71 0.15 0RAT 0.85 1 0.28 1 -0.04 1 0.78 0.59 -0.02 -0.83 0.91 0.43 1 0.57 1 0.33ABR -1 0 -0.96 0 -1 .0 -1 0.38 -0.36 -0.37 0.91 0.33 1 -0.5 0 -1 1 0
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Table 5. Pairwise FST values among ecoregions and provinces. Ecoregions within the TNA(Tropical Northwestern Atlantic): GA= Greater Antilles, EC=Eastern Caribbean,SWC=Southwestern Caribbean and SC=Southern Caribbean. Ecoregions within the TSA(Tropical Southwestern Atlantic): NB=Northeastern Brazil, FA=Fernando de Noronha and Atolldas Rocas and EB=Eastern Brazil. Significant values (p<0.05) are in bold.
GA EC SWC SC NB FA EB TSAEC 0.025
SWC 0.863 0.367SC 0.263 0.138 0.196NB 0.106 0.036 0.243 0.732FA 0.310 0.057 0.192 0.674 0.231EB -1.0 -0.797 -0.258 1.0 -0.75 -0.5
TNA - - - - - - - 0.035
Table 6. Pairwise FST values among Clades and ST-Groups (STG). All values were significant.
Clade 1 = STG1+STG2
Clade2= STG3
Clade 3= ST4+STG5
STG1 STG2 STG4
Clade 2 = STG3 0.871Clade 3 0.883 0.824STG1 - 0.892 0.901STG2 - 0.928 0.87 0.779STG4 0.884 0.908 - 0.904 0.956STG5 0.907 0.922 - 0.923 0.955 0.847
Neutrality Tests
The values obtained in the neutrality tests considering localities, ecoregions, clades and ST-
groups are shown in Table 7. In the overall sample, both tests indicated a deviation from
neutrality (Tajima's D=-1.21 and Fu's Fs=-9.56), however, Tajima's D test was not significant
(p=0.09). Significant Tajima's D and Fu´s Fs values were obtained for the Eastern Caribbean
ecoregion (D=-1.78, p=0.012 and Fs=-4.26, p= 0.047), Clade 1 (D=-1.73, p=0.013 and Fs=-
11.96, p=0.0) and STG1 (D=-1.97, p=0.002 and Fs=-13.32, p=0) indicating population
expansion within these groups (Aris-Brosou & Excoffier, 1996).
Congruent significant tests were not observed among the other groups tested. Tajima's D
values were significant for Saint Martin (D=-2.04, p=0.004) and Antigua (D=-2.1, p=0.002)
whereas the Fu´s Fs test was significant for the TNA (Fs=-8.068, p=0.012).
Mismatch Distribution
Most geographical partitions analysed herein evidenced a multimodal mismatch distribution,
except for Martinique which had an approximate unimodal distribution (Figures 4A-F). Among
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the genetic groups tested, unimodal-like mismatch distributions were observed for Clade1 and
STG1 (Figures 4G-H). These unimodal distributions (Poisson-like) are the signature of
demographic or spatial expansions (Rogers and Harpending 1992; Bunje and Wirth, 2008). In
none of the cases, the SSD and the Harpending's raggedness index were significant (Figure 4,
Table S5) and consequently, it was not possible to reject the sudden expansion model.
As expected due to the low variability found in Puerto Rico, Northeast Brazil and Fernando de
Noronha and Rocas Atoll, Clade 2, Clade 3, STG3, STG4 and STG5, no clear distribution
pattern was observed for them.
Table 7. Demography statistics of Leucetta floridana. TNA=Tropical Northern Atlantic,TSA=Tropical Southern Atlantic, RN=Rio Grande do Norte, p=p value.
Province/Ecoregion Locality Tajima´s D (p) Fu´s FS (p)TNA Caribbean Sea -1.0404 (0.1416) -8.0681 (0.0119)Greater Antilles Puerto Rico -1.1405 (0.1683) -0.4757 (0.1334)Eastern Caribbean Lesser Antilles -1.7835 (0.0124) -4.2641 (0.0472)
Saint Martin -2.0454 (0.0037) -2.4497 (0.09)Antigua -2.1039 (0.0023) 1.11937 (0.7496)Les Saintes -1.0548 (0.2096) -0.1820 (0.2007)Les Saintes + GuadeloupeMartinique -0.0204 (0.4933) -0.4473 (0.3819)
Southwestern Caribbean 1.6576 (0.9596) 0.7867 (0.6623)San Andrés 0.0834 (0.6645) -0.3992 (0.2231)Panama 1.9623 (0.9809) 4.3061 (0.9612)Urabá 0.0 (1.0) 0.2007 (0.3965)Santa Marta 0.8755 (0.8333) 5.1781 (0.9822)
TSA Brazil -0.3988 (0.2884) -0.4474 (0.2619)Northeastern Brazil Ceará + RN 0.15647 (0.7512) 0.47744 (0.3977)
Ceará 1.2247 (0.9435) 0.6261 (0.5025)Fernando de Noronha (FN) and Atoll das Rocas
FN Archipelago + Rocas Atoll
0.0 (1.0) 0.6261 (0.4986)
FN Archipelago 0.0 (1.0) 0.1718 (0.3388)Clade 1= STG1+STG2 -1.7347 (0.0133) -11.9645 (0.0)
Clade 2= STG3 0.15647 (0.7615) -0.5321 (0.139)Clade 3 0.9867 (0.8481) -0.0611 (0.45050)STG1 (1 A) -1.6636 (0.0177) -11.0736 (0.0001)STG2 (1 B) 0.0 (1.0) 0.0902 (0.2959)STG4 (3 A) 0.0 (1.0) -0.0027 (0.2612)STG5 (3 B) 0.0 (0.9873) 0.2007 (0.3867)All -1.2085 (0.099) -9.5358 (0.0056)
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Bayesian Skyline Plot
Bayesian Skyline Plots (BSP) evidenced a population expansion pattern when considered (1) the
complete set of L. floridana sequences (Figure 5A), (2) STG1+STG2 = Clade 2 (Figure 5B), (3)
STG1 (Figure 5C) and (4) AMOVA-G1: Puerto Rico + Anguilla + Saint Martin + Saba +
Antigua + Les Saintes + Guadeloupe (Figure 5D). In the other groups tested, the BSP did not
detect any demographic event probably due to the small number of individuals per group
(n<10).
DISCUSSION
Genetic Structure
Although the AMOVA results indicated some genetic structure when geographical partitions
were considered, the highest FST values were observed among clades (inferred by ML and BI
methods) and ST-Groups (obtained in the MJ network). Therefore, we recognize the occurrence
of five structured populations of L. floridana within the Tropical Atlantic Ocean: four
populations restricted to the Caribbean Sea and a widespread population in the Western Tropical
Atlantic.
Caribbean restricted populations
Cowen et al. (2006) defined four broad regions of connectivity within the Caribbean: (1) the
eastern Caribbean (Puerto Rico to Aruba); (2) the western Caribbean (Cuba to Nicaragua); (3)
the Bahamas and the Turks and Caicos Islands; and (4) the peripheral area of the Colombia-
Panama Gyre. They also considered smaller areas of isolation within each region. In this study,
the four Caribbean-restricted populations of L. floridana matched two of the referred
connectivity regions and evidenced isolation patterns within them.
In the peripheral area of the Colombian-Panama Gyre, which roughly coincides with the
Southwestern ecoregion (Spalding et al., 2007), we found three structured populations: SGT3 in
the area of direct influence of the Colombian-Panama Gyre (Panama and Urabá), STG4 in
distant localities (San Andrés and Santa Marta) and STG2 in closer localities (Panama and San
Andrés)
Isolation of STG3 from the Caribbean Sea (FST= 0.89 – 0.93) and connectivity among the
individuals within this population can be explained by the cyclonic circulation of the
Colombian-Panama Countercurrent (counter-clockwise to the Central Caribbean Current) which
may act as a larval retention current.
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Figure 4. Mismatch distribution of Leucetta floridana for (A) Antigua, (B) Saint Martin, (C)Panama, (D) Martinique, (E) Southwestern Caribbean, (F) Eastern Caribbean, (G) Clade 1 and(H) STG1. Bars show the observed frequency distribution for the number of pairwisedifferences among all individuals sampled. The solid lines show the expected distribution undereach population expansion model. SSD: Sum of Squared Deviations, r: Harpending's raggednessindex.
195
Figure 5. Bayesian Skyline Plots for (A) all ITS sequences, (B) Clade1= ST-Group1 + ST-Group2 of Leucetta floridana, (C) ST-Group1 and (D) AMOVA Group 1: Puerto Rico +Anguilla + Saint Martin + Saba + Antigua + Les Saintes + Guadeloupe of Leucetta floridana.The maximum time is the upper 95% HPD of the root height. The median estimate (black solidline) and 95% HPD limits (blue line) are indicated.
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As the coast of Santa Marta is directly influenced by the Central Caribbean Current (Diaz et
al., 2001), this can explain why individuals from this locality are isolated from Urabá and
Panama, presenting a private ST (ST6). However, they are somewhat connected with some
individuals from San Andrés (FST<0.25 but not significant), as observed in STG4. This can be
attributed to the presence of the Colombian-Panama Countercurrent in Santa Marta during
raining periods (Diaz et al., 2001). This latitudinal connectivity between San Andrés and Santa
Marta (12.6°N and 11.2°N, respectively) has already been reported in other marine taxa e.g. the
reef fish Stegastes partitus (Ospina-Guerrero et al., 2008).
Different to STG4, STG2 reunites individuals from longitudinally close (82.1 and 81.7°W)
but latitudinally distant (9.3 and 12.6°N) localities (Panama and San Andrés) that may be
warranting the gene flow through the Colombian-Panama Countercurrent. This scenario has also
been suggested for another Caribbean sponge (Cliona delitrix, Chaves-Fonnegra et al., 2015)
Besides the barriers cited above, the whole genetic structure observed within this large area
(peripheral area of the Colombia-Panama Gyre) suggests the presence of a strong barrier
between San Andrés and Urabá as no STG including both localities was found (FST>0.5 but not
significant). An alternative explanation to this could relay on past geological events that
occurred within this area isolating populations.
The last structured population is located in the Eastern Caribbean connectivity region of
Cowen et al., (2006). STG5 included individuals from two islands located in the northern part of
the Lesser Antilles arc (Saint Martin and Antigua) and no individuals from the southern islands
(Martinique nor Guadeloupe). Whether this corresponds to a split of the Eastern Antilles
population in northern and southern populations remains uncertain based on our results,
however, this should receive more attention in further studies compelling the Lesser Antilles.
A Western Tropical Atlantic widespread population
The occurrence of a large population of L. floridana with intense genetic exchange between
Caribbean and Brazilian localities (STG1) does not only reject the role of the Amazon river as
an effective barrier to gene flow in the Western Tropical Atlantic but also suggests the presence
of biological and/or physical mechanisms that would facilitate the observed connectivity
pattern.
In a broad sense, the population connectivity observed in L. floridana may be mediated by
(1) the North Brazilian Current enabling the flow of migrants from the Brazilian coast to the
Caribbean Sea and (2) the sponge assemblages under the mouth of the Amazon river acting as a
connectivity corridor (as proposed by Rocha et al., 2003 for fish species).
197
Within this context, it is suggested that the larva of L. floridana can pass underneath the
Amazon freshwater outflow (-30 m; Curtin, 1986). Although knowledge on the ultrastructure
and behaviour of calciblastula larva is scarce, we know that this larva is hollow, oval and
surrounded by ciliated cells (Amano & Hori, 2001; Ereskovsky & Willenz, 2008), which may
be responsible for its motility. In some Demospongiae, the cilia are used for larval rotation along
the longitudinal axis, apparently allowing the larvae to re-adjust its depth while drifting
(Maldonado et al., 2003). These ciliated cells may play the same role in L. floridana, facilitating
its sinking under the Amazon mouth. Alternatively, the larva of L. floridana can undergo a
passive mechanism, being dragged to the bottom by masses of water with more salinity
(Maldonado, 2006).
Similar patterns of high genetic connectivity along the Western Tropical Atlantic had been
attributed to efficient larval dispersal in other marine taxa (e.g. Rocha et al., 2002; Rodriguez et
al., 2013). In Porifera, this is paradoxal since sponge larvae are lecithotrophic (Ereskovsky,
2010) and remain in the water column for minutes to a few days (usually <2 weeks -
Maldonado, 2006; Padua et al., 2013), which most probably restrict their dispersal and
colonization capabilities.
Some sponge larvae exhibit complex mechanisms that contribute to expand their pelagic
lifespan and consequently increase their dispersal capability such as (1) the incorporation of
dissolved compounds by pinocytotic activity in Tedania ignis (Jaeckle, 1995), (2) the capacity to
phagocyte and digest bacteria and small (<4 μm) unicellular organisms by the ciliated cells of
Halichondria parenchymella (Ivanova, 1999) or (3) just being unpalatable to predators (fish),
observed in some Caribbean sponges (Lindquist & Hay, 1996). Whether this occurs in L.
floridana or in any other calcareous sponge is unknown but should not be discarded.
Another mechanism that improve the dispersal capacity of some sponge larvae is the
fragmentation of reproductive sponges. A fragment containing larvae can travel for a long time
before the larvae are released and this may contribute to maximise the dispersal capacity
(Maldonado & Uriz, 1999).
Demographic history
Our results evidenced a population expansion pattern for L. floridana, specifically within the
widespread population STG1. Diversity indexes, neutrality tests and mismatch distributions
indicated that this occurred in the Caribbean Sea. Complementary information provided by the
bayesian skyline plot situated the onset of this event ca. 20.000 years BP. This estimated date
198
coincides with the final period of the Pleistocene: the Last Glaciation Maximum (LGM, 30.000
– 19.000 years BP, Lambeck et al., 2001).
During the Pleistocene (2.6 million –11.700 years BP), the glacial-interglacial cycles
changed the sea level, superficial sea temperatures, current patterns, upwelling intensity and
coastal habitats (Rohling et al., 1998; Lambeck et al., 2002). Global sea levels were 115–130 m
lower than today (Fairbank, 1989; Lambeck et al., 2002) and tropical sea-surface temperature
oscillated between 1 °C and 3 °C (Herbert et al., 2010). During the LGM, the area of coastal
habitats was reduced by approximately 92% in the Gulf of Mexico and Caribbean Sea
(Bellwood & Wainwright, 2002). Under those conditions, some species became extinct and
several others remained as fragmented populations that nowadays are highly genetically
structured (Ludt & Rocha, 2015).
It is feasible that the shallow overhangs and vertical slopes inhabited by L. floridana during
the Pleistocene ended up being exposed due to the low sea levels and consequently, some
populations were decimated. We suggest that L. floridana suffered a bottle-neck event during
the late Pleistocene and that its populations remained isolated for a long period of time, which is
reflected in the high genetic population structure observed in this study. However, after the
LGM, one of the isolated population (STG1) expanded within the Caribbean.
In the Western Tropical Atlantic, demographic events have been reported as a result of the
fluctuating conditions during the Pleistocene. In the Tropical Northwestern Atlantic, population
bottlenecks have been evidenced in invertebrates including gastropods (Johnston et al., 2012),
decapods shrimps (Cook et al., 2010) and blenny fish (Eytan & Hellberg, 2010) whereas
population expansion was observed in penaeid shrimps (McMillen-Jackson & Bert, 2003),
spiny lobster (Naro-Maciel et al., 2011) and West Indian manatee (Vianna et al. 2006). Although
most of these events have been recorded in the TNA, population expansion has also been
reported in the Tropical Southwestern Atlantic (e.g. Rodriguez-Rey et al., 2013).
Within sponges, genetic structure due to interglacial-glacial cycles have been observed in
the demosponges Hymeniacidon flavia and H. sinapium (Hoshino et al., 2008) from the
Northwest Pacific and the Indo-Pacific calcarean Leucetta chagosensis (Wörheide et al., 2002;
2008) as well as population expansion of Pericharax heteroraphis (Bentlage & Worheide, 2007)
within the Great Barrier Reef in Australia.
Evolutionary history of L. floridana
Based on the phylogenetic, genetic structure and the demographic results obtained in this study
and additional (palaeo)geological information, we propose the following evolutionary scenario
199
for L. floridana. Ancestral populations of this species were broadly distributed in the Western
Tropical Atlantic. Once the outflow of the Amazon River started to be deposited in the Atlantic
in the late Miocene (10.4 million years BP - Hoorn, 1996), populations were isolated and L.
floridana must have been restricted to the Tropical Northwestern Atlantic.
During the Pleistocene, important changes in marine taxa occurred (Lundt & Rocha, 2015).
In L. floridana, we suggest both genetic isolation and connection due to glacial-interglacial
cycles. The glacial periods isolated some populations, as evidenced by the high genetic structure
observed within the Caribbean-restricted ST-groups (STG2, STG3, STG4 and STG5).
According to the demographic bayesian analyses, these ST-groups share a more ancient history
when compared to STG1.
During the most recent interglacial periods, the distribution of one population of L. floridana
(STG1) was expanded to the northeastern Brazilian coast and maintained connectivity
(evidenced by the common ST1) through the sponge corridor, as suggested for other trans-
Amazonian species (Rocha, 2003). This southward dispersal from the Caribbean to Brazilian
localities has been also observed in reef fish (Lima et al., 2005; Rocha et al., 2008) and
although it conflicts with today's current patterns, it must respond to past oceanographic
conditions (Iturralde-Vinent & MacPhee, 1999). According to Bowen et al., 2001, populations
can disperse and potentially coalesce after periods of isolation. Therefore, even if trans-
Amazonian individuals of L. floridana underwent restricted genetic flow for some period of
time as a result of the fluctuating conditions of the Pleistocene, they were able to restore
connectivity later. This must have been facilitated by the large population size of this
widespread species. By the end of the last glaciation maximum, the widespread population
(STG1) suffered a bottleneck and a posterior sudden expansion event in the Lesser Antilles
(most probably, in Martinique). Whether the Lesser Antilles acted as a Pleistocean refugia is
uncertain as not palaeogeological information is available, however, the presence of many
private ST may support this (Maggs et al., 2008).
Acknowledgements
The authors thank Adeline Pouget-Cuvelier, Amandine Vaslet, Julien Chalifour and Philippe
(Filipo) Thélamon for providing relevant information on sampling localities in the Lesser
Antilles and also Alexander Ereskovsky, Bob Thacker?, César Ruiz, Cristina Diaz, Eduardo
Hajdu, Fernanda Azevedo, Jean Vacelet, Laurent Van Bostal, Pedro Leocorny, Pierre
Chevaldonné and Sandrine Chenesseau for collecting L. floridana in the Caribbean Sea.
Ghennie Rodriguez is acknowledged for providing invaluable support for the demography
200
analyses. This work was partly supported by the Associated International Laboratory
‘MARRIO’. B.C.L. received a scholarship from the Brazilian Coordination for the Improvement
of Higher Education Personnel (CAPES). J.E.G.H. received grants from the American Museum
of National History Lerner-Gray Fund for Marine Research (sponge collection) and the Sea-
Grant Puerto Rico (DNA sequencing). M.K. is funded by fellowships and research grants from
the Brazilian National Research Council (CNPq), CAPES, and the Rio de Janeiro State
Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio
de Janeiro - FAPERJ). T.P. is funded by grants from The French National Center for Scientific
Research (CNRS).
REFERENCES
Amano, S. & Hori, A.I. 2001. Metamorphosis of coeloblastula performed by multipotentiallarval flagellated cells in the calcareous sponge Leucosolenia laxa. The BiologicalBullutin, 200: 20 –32.
Aris-Brosou, S., & Excoffier, L. 1996. The impact of population expansion and mutation rateheterogeneity on DNA sequence polymorphism. Molecular Biology and Evolution,13:494–504.
Bandelt, H-J., Forster, P. & Röhl, A. (1999) Median-joining networks for inferring intraspecificphylogenies. Molecular Biology and Evolution, 16:37-48.
Bellwood, D.R. & Wainwright, P.C. (2002) The history and biogeography of fishes on coralreefs. In: Coral reef fishes: dynamics and diversity in a complex ecosystem (ed. by P.F.Sale), pp. 5–32. Academic Press, San Diego, CA.
Bentlage, B. & Wörheide, G. 2007. Low genetic structuring among Pericharax heteroraphis(Porifera: Calcarea) populations from the Great Barrier Reef (Australia), revealed byanalysis of nrDNA and nuclear intron sequences. Coral Reefs, 26: 807–816.
Borojevic, R.; Boury-Esnault, N. & Vacelet, J. A revision of the supraspecific classification ofthe subclass Calcinea (Porifera, class Calcarea). Bulletin du Muséum national d'histoirenaturelle. Paris, 4° sér. 12, section A, n°2: 243-273.
Bowen, B.W., Bass, A.L., Rocha, L.A., Grant, W.S. & Robertson, D.R. 2001. Phylogeographyof the trumpetfish (Aulostomus spp.): ring species complex on a global scale. Evolution,55: 1029–1039.
Bowen, B. W.; Bass, L. A.; Muss, A.; Carlin, J. & Robertson, D. R. 2006. Phylogeography oftwo Atlantic squirrelfishes (Family Holocentridae): exploring links between pelagic larvalduration and population connectivity. Marine Biology, 149: 899-913.
Chaves-Fonnegra, A.; Feldheim, K.A.; Secord, J. & Lopez, J. V. 2015. Population structure anddispersal of the coral excavating sponge Cliona delitrix. Molecular Ecology, 24: 1447-1466.
Collette, B.B. & Rützler, K. 1977. Reef fishes over sponge bottoms off the mouth of theAmazon River. Third International Coral Reef Symposium: 305–310.
Cook, B.D.; Pringle, C.M. & Hughes, J.M. 2010. Immigration history of amphidromousspecies on a Greater Antillean island. Journal of Biogeography, 37: 270–277.
Cowen, R. K.; Paris, C. B. & Srinivasan, A. 2005. Scaling of Connectivity in MarinePopulations. Science, 311(5760): 522-527.
Curtin, T.B. 1986. Physical observations in the plume region of the Amazon River during peakdischarge – II. Water masses. Continental Shelf Research, 6: 53–71.
201
Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. 2012. jModelTest 2: more models, newheuristics and parallel computing. Nature Methods, 9(8): 772.
de Bakker, D.M., Meesters. E.H.W.G, van Bleijswijk, J.D.L, Luttikhuizen, P.C., Breeuwer,H.J.A.J., Becking, L.E. 2016 Population genetic structure, abundance, and health status oftwo dominant benthic species in the Saba Bank National Park, Caribbean Netherlands:Montastraea cavernosa and Xestospongia muta. PLoS ONE, 11(5): e0155969.
DeBiasse, M. B.; Richards, V. P.; Shivji, M. S. 2010. Genetic assessment of connectivity in thecommon reef sponge, Callyspongia vaginalis (Demospongiae: Haplosclerida) reveals highpopulation structure along the Florida reef tract. Coral Reefs, 29: 47–55.
DeBiasse, M. B.; Richards, V. P.; Shivji, M. S. & Hellberg, M. E. 2016. Sharedphylogeographical breaks in a Caribbean coral reef sponge and its invertebratecommensals. Journal of Biogeography.43: 2136–2146.
Drummond, A. J.; Rambaut, A.; Shapiro, B. & Pybus, O. G. 2005. Bayesian coalescentinference of past population dynamics from molecular sequences. Molecular Biologyand Evolution, 22: 1185–1192.
Drummond A.J.; Suchard, M.A.; Xie, D. & Rambaut, A. 2012. Bayesian phylogenetics withBEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29: 1969-1973 -> for theprogram BEAST.
Ereskovsky, A.V. 2010. The Comparative Embryology of Sponges. Springer-Verlag,Dordrecht Heidelberg London.
Fairbanks, R.G. 1989. A 17,000 year glacio-eustatic sea-level record: influence of glacialmelting rates on the Younger Dryas event and deep-ocean circulation. Nature, 342: 637–642.
Ereskovsky, A.V. & Willenz, P. 2008. Larval development in Guancha arnesenae (Porifera,Calcispongiae, Calcinea). Zoomorphology, 127: 175-187.
Floeter, S.R.; Rocha, L. A.; Robertson, D. R.; Joyeux, J. C.; Smith-Vaniz, W. F; Wirtz, P.;Edwards, A. J.; Barreiros, J. P.; Ferreira, C. E. L.; Gasparini, J. L.; Brito, A.; Falcón, J. M.;Bowen, B. W. & Bernardi, G. 2008. Atlantic reef fish biogeography and evolution. Journalof Biogeography, 35: 22–47.
Forster M.; Forster P.; Watson J. 2007. Network version 4.6: A software for population geneticsdata analysis. 4.2.0.1 ed: Fluxus Technology Ltd 1999.
Guindon, S. & Gascuel, O. 2003. A simple, fast and accurate method to estimate largephylogenies by maximum-likelihood". Systematic Biology, 52: 696-704.
Herbert, T.D., Peterson, L.C., Lawrence, K.T. & Liu, Z. 2010. Tropical ocean temperatures overthe past 3.5 million years. Science, 328: 1530-1534.
Hoshino, S., Saito, D.S. & Fujita, T. 2008. Contrasting genetic structure of two PacificHymeniacidon species. Hydrobiologia, 603, 313–326. Huelsenbeck, J.P. & Ronquist, F.2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics, 17: 754-755.
Iturralde-Vinent, M. A., & MacPhee, R. D. E. 1999. Paleogeography of the Caribbean Region:implications for Cenozoic biogeography. Bulletin of the American Museum of NaturalHistory, 238: 1–95.
Ivanova, L.V. 1999. New data about morphology and feeding patterns of Barentz SeaHalichondria panicea Pallas. Memoirs of the Queensland Museum, 44: 262.
Jaeckle, W.B. 1995. Transport and metabolism of alanine and palmitic acid by field-collectedlarvae of Tedania ignis (Porifera, Demospongiae): estimated consequences of limited labeltranslocation. Biological Bulletin, 189: 159–167.
Johnston, L., Miller, M.W. & Baums, I.B. 2012. Assesment of host-associated geneticdifferentiation among phenotypically divergent populations of coral-eating gastropodsacross the Caribbean. PLoS ONE, 7: e47630.
202
Katoh, K. & Standley, D.M. 2013. MAFFT multiple sequence alignment software version 7:improvements in performance and usability. Molecular Biology and Evolution, 30: 772–780.
Katoh, K & Toh, H. 2008. Improved accuracy of multiple ncRNA alignment by incorporatingstructural information into a MAFFT-based framework. BMC Bioinformatics, 9:212.
Klautau, M.; Solé-Cava, A. & Borojevic, R. 1994. Biochemical Systematics of SiblingSympatric Species of Clathrina (Porifera: Calcarea). Biochemical Systematics Ecology,22(4): 367-375.
Klautau, M.; Russo, C.; Lazoski, C.; Boury-Esnault, N.; Thorpe, J. & Sole-Cava, A. M. 1999.Does cosmpolitanism result from overconservative systematics?. A case study using themarine sponge Chondrilla nucula. Evolution, 53: 1414–1422.
Klautau, M. & Valentine, C. 2003. Revision of the Genus Clathrina (Porifera, Calcarea).Zoological Journal of the Linnean Society, 139:1-62.
Klautau, M., Azevedo, F., Cóndor-Luján, B., Rapp H.T., Collins, A & Russo, C. 2013. Amolecular phylogeny for the order Clathrinida rekindles and refines Haeckel’s taxonomicproposal for calcareous sponges. Integrative and Comparative Biology, 53, 447–461.
Lambeck, K.; Esat, T. M. & Potter, E.K. 2002. Links between climate and sea levels for the pastthree million years. Nature, 419: 199-206.
Lavery, S., Moritz, C., and Fielder,Lazoski, C.; Solé-Cava, A.M.; Boury-Esnault, N.; Klautau,M. & Russo, C. 2001. Cryptic speciation in hight gene flow scenario in the oviparousmarine sponge Chondrosia reniformis. Marine Biology, 139: 421-429.
Lavrov, D.L.; Pett, W.; Voigt, O.; Wörheide, G.; Forget, L.; Lang, B.F. & Kayal, E. 2013.Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes,fragmented rRNAs, tRNA editing, and a novel genetic code. Molecular Biology andEvolution, 30(4):865-880.
Librado, P. & RozaS, J. 2009. DnaSP v5: A software for comprehensive analysis of DNApolymorphism data. Bioinformatics, 25: 1451-1452.
Lima, D., Freitas, J.E.P.; Araujo, M.E. & Solé-Cava, A. M. 2005. Genetic detection of crypticspecies in the frillfin goby Bathygobius soporator. Journal of Experimental MarineBiology and Ecology, 320:211-223.
Lôbo-Hajdu, G.; Guimarães, A.; Salgado, A.; Lamarão, F.; Vieiralves, T; Mansure, J. & Albano,R. 2004. Intragenomic, Intra- and interspecific variation in the rDNA ITS of a poríferarevealed by PCR-single-strand conformation polymorphism (PCR-SSCP). Bolletino deiMusei e Degli Istitui Biologicci de Genova, 68:413-423.
López-Legentil, S. & Pawlik, J.R. 2009. Genetic structure of the Caribbean giant barrelsponge Xestospongia muta using the I3-M11 partition of COI. Coral Reefs, 28:157–165.
Ludt, W.B. & Rocha, L.A. 2015. Shifting seas: the impacts of Pleistocene sea-level fluctuationson the evolution of tropical marine taxa. Journal of Biogeography 42, 25-38.
Maldonado, M. 2006. The ecology of the sponge larva. Canadian Journal of Zoology, 84:175–194.
Maldonado, M. & Bergquist, P.R. 2002. Phylum Porifera. In: Atlas of marine invertebratelarvae. Edited by C.M. Young, M.A. Sewell & M.E. Rice. Academic Press, San Diego. pp.21–50.
Maldonado, M., & Uriz, M.J. 1999. Sexual propagation by sponge fragments. Nature, 398: 476.Maldonado, M., Durfort, M., McCarthy, D., & Young, C.M. 2003. The cellular basis of
photobehavior in the tufted parenchymella larva of demosponges. Marine Biology, 143:427–441.
Maggs, C. A.; Castilho, R.; Foltz, D.; Henzler, C.; Jolly, M. T.; Kellt, J.; Olsen, J.; Perez, K. E.;Stam, W., Väinölä, R., Viard, F. & Wares, J. 2008. Evaluating signatures of glacial refugiafor north Atlantic benthic marine taxa. Ecology, 89: S108–S122.
203
McMillen-Jackson, A. L., & Bert, T. M. 2003. Disparate patterns of population geneticstructure and population history in two sympatric penaeid species in the southeasternUnited States. Molecular Ecology, 12: 2895–2905.
Muricy, G.; Lopes, D.A.; Hajdu, E.; Carvalho, M.S.; Moraes, F.C.; Klautau, M.; Menegola, C.& Pinheiro, U. 2011. Catalogue of Brazilian Porifera. Série Livros 46, Museu Nacional,Rio de Janeiro, 300 pp.
Naro-Maciel, E., Reid, B., Holmes, K. E., Brumbaugh, D. R., Martin, M. & DeSalle, R. (2011).Mitochondrial DNA sequence variation in spiny lobsters: population expansion, panmixia,and divergence. Marine Biology, 158: 2027–2041.
Ospina-Guerrero, S.P.; Landinez-García, R.M.; Rodríguez-Castro, D.J.; Arango, R. & Márquez,E. 2008. Genetic connectivity of Stegastes partitus in the South Caribbean evidenced bymicrosatellite analysis. Ciencias Marinas, 34(2): 155–163.
Padua, A., Lanna, E., Zilberberg, C., Paiva, C. & Klautau, M. 2013 Recruitment, habitatselection and larval photoresponse of Paraleucilla magna (Porifera, Calcarea) in Rio deJaneiro, Brazil. Marine Ecology, 34(1):56-61.
Rambaut, A., & Drummond, A. J. 2009. Tracer version 1.5 [computer program]. Available at:http://beast.bio.ed.ac.uk/Tracer.
Richards, V.P.; Bernard, A.; Feldheim, K & Shivji, M.S. 2016. Patterns of population structureand dispersal in the long-lived “redwood” of the coral reef, the giant barrel sponge(Xestospongia muta). Coral Reefs, 35(3):1097–1107.
Rocha, L.A.; Bass, A.L., Robertson, R. & Bowen, B.W. 2002. Adult habitat preferences, larvaldispersal, and the comparative phylogeography of three Atlantic surgeonfishes(Teleostei: Acanthuridae). Molecular Ecology, 11:243-252.
Rocha, L.A. 2003. Patterns of distribution and processes of speciation in Brazilian reef fishes.Journal of Biogeography, 30: 1161–1171.
Rodríguez-Lopez, M.A., Mignucci-Giannoni, A.A., Powell, J.A. & Santos, R.R. 2006.Phylogeography, phylogeny, and hybridization in trichechid sirenians: implications formanatee conservation. Molecular Ecology, 15:433–447.
Rodriguez-Rey, G.; Solé-Cava, A.M. & Lazoski, C. 2013. Genetic homogeneity and historicalexpansions of the slipper lobster, Scyllarides brasiliensis, in the south-west Atlantic.Marine and Freshwater Research, 65: 59-69.
Rohling, E. J., Fenton, M., Jorissen, F. J., Bertrand, P., Ganssen, G. & Caulet, J. P. 1998.Magnitudes of sea-level lowstands of the past 500,000 years. Nature, 394, 162–165.
Ronquist, F. & Huelsenback, J.P. 2003. MRBAYES 3: Bayesian phylogenetic inference undermixed models. Bioinformatics, 19: 1572–1574.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1993. Molecular Cloning: a laboratory manual.Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Schneider, S.; Roessli, D. & Excoffier, L. 2000. Arlequin: a software for population geneticsdata analysis User manual version 2.000. Genetics and Biometry Laboratory, Departmentof Anthropology, University of Geneva; Geneva.
Spalding, M. D.; Fox, H. E.; Allen, G.R.; Davidson, N.; Ferdaña, Z. A.; FINLAYSON, M.;Halpern, B. S.; Jorge, M. A.; Lombana, A.; Lourie, S. A.; Martin, K.D.; McManus, E.;Molnar, J.; Recchia, C. A. & Robertson, J. 2007. Marine ecoregions of the world: Abioregionalization of coast and shelf areas. BioScience, 57: 573–583.
Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A. & Sudhir Kumar. 2013. MEGA6:Molecular Evolutionary Genetics Analysis version6.0. Molecular Biology and Evolution, 30: 2725-2729.
Valderrama, D.; Rossi, A.L.; Solé-cava, A.M.; Rapp, H.T. & Klautau, M. 2009. Revalidationof Leucetta floridana (Haeckel, 1872) (Porifera, Calcarea): a widespread species in thetropical western Atlantic. Zoological Journal of the Linnean Society, 157: 1-16.
204
Vianna, J.A., Bonde, R.K., Caballero, S., Giraldo, J.P., Lima, R.P., Clark, A., Marmontel, M.,Morales-Vela, B., José deSouze, M., Parr, L.,Weir, B.S. & Cockerham, C.C. 1984.Estimating F-statistics for the analysis of population structure. Evolution, 38: 1358-1370.
Wörheide, G; Degnan, B.M. & Hooper J.N.A. 2000. Population phylogenetics of the commoncoral reef sponges Leucetta spp. and Pericharax spp. (Porifera: Calcarea) from the GreatBarrier Reef and Vanuatu. Abstracts. 9th International Coral Reef Symposium, Bali,October 2000, p. 23.
Wörheide, G.; Hooper, J. & Degnan, B. 2002. Phylogeography of western Pacific Leucetta‘chagosensis’ (Porifera: Calcarea) from ribosomal DNA sequences: implications forpopulation history and conservative of the Great Barrier Reef World Heritage Area(Australia). Molecular Ecology, 11: 1753-1768.
Wörheide, G.; Nichols, S. & Goldberg, J. 2004. Intragenomic variation of the rDNA internaltranscribed spacers in sponges (Phylum Porifera): implications for phylogenetic studies.Molecular Phylogenetics and Evolution, 33: 816-830.
Wörheide, G.; Epp, L.S. & Macis, L. 2008. Deep genetic divergences among Indo-Pacificpopulations of the coral reef sponge Leucetta chagosensis (Leucettidae): Founder effects,vicariance, or both? BMC Evolutionary Biology, 8:24.
SUPPLEMENTARY MATERIAL
Table S1. Voucher number, locality, geographic coordinates and Genbank accession number of the specimens of Leucetta floridana analysed in this study.Abbreviations: Brazilian States: BA=Bahia, CE=Ceará, RN= Rio Grande do Norte, PE=Pernambuco. SM-MNR=Saint Martin Marine Natural Reserve.
Voucher Number Sample Locality Geographic Coordinates Depth (m) Genbank Number
Tropical Northwestern AtlanticGreater Antilles ecoregion: Puerto Rico (n=12)PR52 Cayo Mario 17°57'16.89''N, 67°03'53.42''W 9.0 This studyPR81 Cayo Mario 17°57'16.89''N, 67°03'53.42''W 10.8 This studyPR97 Veril – Fallen Rock (Guanica) 17°54'05.04''N, 66°55'27.59''W 31.5 This studyPR99 Cayo Conserva 17°55'56.91''N, 67°05'35.01''W 12.6 This studyPR105 Cayo Conserva 17°55'56.91''N, 67°05'35.01''W 15.6 This studyPR112 Pinnacles 17°56'03.96''N, 67°01'49.80''W 9.9 This studyPR117 Veril - El Hoyo 17°52.0529'N, 67°02.671'W 31.5 This studyPR120 Pinnacles 17°56'03.96''N, 67°1'49.80''W 17.1 This studyPR136 Turromote 17°56'20.13''N, 67°2'43.95''W 17.4 This studyPR141 Turromote 17°56'20.13''N, 67°2'43.95''W 12.9 This studyPR147 Cayo Mario 17°57'16.89''N, 67°3'53.42''W 13.2 This studyPR153 Cayo Mario 17°57'16.89''N, 67°3'53.42''W 17.7 This studyEastern Caribbean ecoregion: Lesser Antilles (n=100)Anguilla (n=1) This studyUFRJPOR 8278 Little Scrub2 18°17.903'N, 62°57.294'W 23.5 This studySaint Martin (n=52)UFRJPOR 7789 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W <20? This studyUFRJPOR 7968 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7970 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7971 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7972 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7974 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7985 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7986 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7990 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7992 Les Arches 2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7993 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 8000 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 8001 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study
UFRJPOR 8022 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8025 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8028 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8029 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8030 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 9.8 This studyUFRJPOR 8031 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 9.8 This studyUFRJPOR 8098 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 8 - 10 This studyUFRJPOR 8142 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 8 - 10 This studyUFRJPOR 8150 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8151 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8152 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8153 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8155 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8157 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8158 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8181 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8182 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8192 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8193 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8194 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8195 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8196 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8197 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8198 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8199 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8216 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8217 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8218 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8219 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8221 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8222 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8223 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8224 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8231 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 20 This studyUFRJPOR 8232 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13.8 This studyUFRJPOR 8234 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This studyUFRJPOR 8235 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This study
UFRJPOR 8237 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This studyUFRJPOR 8238 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This studySaba (n=3)UFRJPOR 8040 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This studyUFRJPOR 8041 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This studyUFRJPOR 8042 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This studyAntigua and Barbuda (n=12)UFRJPOR 7936 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7938 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7939 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7940 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7942 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7943 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7949 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7950 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7952 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 8093 Five Islands 17°04.990'N, 61° 54.840'W 4 This studyUFRJPOR 8185 Diamond_Bank, Saint Paul 17°12.000'N, 61°52.800'W 7 - 8 This studyUFRJPOR 8186 Diamond Bank, Saint Paul 17°12.000'N, 61°52.800'W 7 - 8 This studyGuadeloupe (n=9)UFRJPOR 7655 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This studyUFRJPOR 7659 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This studyUFRJPOR 7648 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This studyUFRJPOR 7827 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8087 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8558 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8091 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8092 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8340 Cave Cathédrale, ? 16°27.740'N, 61°31.837'W 13.7 This studyMartinique (n=23)UFRJPOR 7403 Grottes des couleurs, Pointe Burgos, Grande
Anse, Les Anses d’Arlet14°29.787'N, 61°05.351'W <10 m This study
UFRJPOR 7404 Pointe Burgos, Grande Anse, Anses d'Arlet <10 m This studyUFRJPOR 7405 Grottes des couleurs, Pointe Burgos, Grande
Anse, Les Anses d’Arlet14°29.787'N, 61°05.351'W <10 m This study
UFRJPOR 7406 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m This study
UFRJPOR 7407 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m This study
UFRJPOR 7408 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m This study
UFRJPOR 7409 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m This study
UFRJPOR 7410 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m KX355573
UFRJPOR 7411 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m KX355574
UFRJPOR 7412 Les Anses d’Arlet <10 m KX355575UFRJPOR 7423 Grottes des couleurs, Pointe Burgos, Grande
Anse, Les Anses d’Arlet14°29.787'N, 61°05.351'W <10 m This study
UFRPOR 7424 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet
14°29.787'N, 61°05.351'W <10 m This study
SP2 Les Anses d’Arlet <10 m This studyUFRJPOR 7777 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7778 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7845 Pointe Burgos, Grande Anse, Les Anses
d’Arlet14°29.787'N, 61°05.351'W? This study
UFRJPOR 7859 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 3? This studyUFRJPOR 7861 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 3 This studyUFRJPOR 7862 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 5 This studyUFRJPOR 7863 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7865 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7871 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7877 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studySouthwestern Caribbean ecoregion (n=33)Panamá (n=19)UFRJPOR 6389 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? This studyPC BT 12 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? EU781989PC BT 22 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? EU781990PC BT 23 Bocas del Toro 09º20.914'N, 82º9.394'W <20? EU781991UFRJPOR 6948 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394'W <20? This studyUFRJPOR 6951 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394'W <20? This studyUFRJPOR 6953 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394' W <20? This studyUFRJPOR 6954 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This studyUFRJPOR 6956 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This study
UFRJPOR 6959 Bocas del Toro <20? This studyUFRJPOR 6960 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This studyUFRJPOR 6961 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This studyUFRJPOR 6975 Bocas del Toro <20? This studyUFRJPOR 6976 Bocas del Toro 17 This studyUFRJPOR 6977 =MNRJ 15748B
Bocas del Toro <20? This study
UFRJPOR 6978 =MNRJ 15754B
Bocas del Toro <20? This study
UFRJPOR 6988 Bocas del Toro <20? This studyUFRJPOR 6989 Cayo Zapatilla, Bocas del Toro 09º14.881'N, 82º01.952'W <20? This studyUFRJPOR 6989A Cayo Zapatilla, Bocas del Toro 09º14.881'N, 82º01.952'W <20? This studyColombia (n=14)SM01 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 21 This studySM02 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 19 This studySM03 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 20 This studySM04 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 11 This studySM06 Punta Venado, Ensenada de Taganga, Santa
Marta11°16'25.00"N, 74°12'24.00"W 18 This study
SM07 Punta Venado, Ensenada de Taganga, Santa Marta
11°16'25.00"N, 74°12'24.00"W 18.6 This study
SM12 Punta Gaira, Gaira Bay, Santa Marta 11°13'08.00"N, 74°14'30.00"W 14 This study UFRJPOR 5357 Bajo Agua Viva, Urabá 07°53''N, 76°38'W? 15 EU781970 UFRJPOR 5359 Bajo Agua Viva, Urabá 07°53''N, 76°38'W? 15 EU781969UFRJPOR 5360 “Bajo Agua Viva”, Urabá 07°53''N, 76°38'W? 15 EU781968UFRJPOR 5363 “West View”, Leward-reef, San Andrés 12°33'N, 81°43'W? 5 EU781971UFRJPOR 5364 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781972UFRJPOR 5366 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781973UFRJPOR 5367 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781974Southern Caribbean ecoregion: Curaçao (n=2)UFRJPOR 6726 Water Factory, Willemstadt 12°06'30.88"N, 68°57'13.53"W 17.8 Curaçao paperUFRJPOR 6765 Hook’s Hut, Willemstadt 12°07'18.94"N, 68°58'11.46"W 13.3 Curaçao paperTropical Southwestern AtlanticNortheastern Brazil ecoregion: Brazil (n=9)BPOTPOR 200 Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 This studyBPOTPOR 202 Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 EU781985BPOTPOR 610 Risca das Bicudas, RGN 04º57'00.9"S, 36º07'49.7"W 8-10? EU781978BPOTPOR 634 Urca do Tubarao, RGN 04º50'52.7”S, 36º27'02.1"W 8 This study
MNRJ 8440 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781983MNRJ 8445 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781984MNRJ 8465 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781982MNRJ 8481 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 This studyMNRJ 8488 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781980Fernando de Noronha and Atoll das Rocas ecoregion: Brazil (n=5)MNRJ 7725 Barretinha, Rocas Atoll, RGN 03°51'36''S, 33°49'04''W 12 EU781975MNRJ 8609 Sela Gineta Island, Fernando de Noronha
Archipelago, PE03°48'49''S, 32°23'29''W 7 EU781976
MNRJ 8602 Ressurreta, Fernando de Noronha Archipelago, PE
03°48'49''S, 32°23'29''W 4 EU781977
UFRJPOR 6480 Ressurreta, Fernando de Noronha Archipelago, PE
03°48'49''S, 32°23'29''W 7.3 This study
UFRJPOR 6481 Ressurreta, Fernando de Noronha Archipelago, PE
03°48'49''S, 32°23'29''W 7.3 This study
Eastern Brazil ecoregion (n=1): NE BrazilUFRJPOR_4703 Parcel das Paredes, Abrolhos Archipelago,
BA17°58'S, 38°40'W 8 EU781979
211
Table S2. ST-Groups and its corresponding STs as suggested by the network topology of Leucetta floridana. (Number of sequences within each STG and ST)
STGA (110) STGB (1) STGC (4)
STGD (24) STGE (5) STGF (9) STGG (6) STGH (3)
ST1(98) ST21 (1) ST2 (3) ST12 (3) ST7 (1) ST4 (6) ST6 (5) ST17 (1)
ST3 (2) ST8 (1) ST14 (14) ST10 (4) ST5 (1) ST9 (1) ST18 (2)
ST13 (2) ST15 (4) ST11 (2)
ST19 (4) ST16 (3)
ST20 (1)
ST22 (1)
ST23 (1)
ST24 (1)
Table S3. AMOVA. Hypotheses of ST-groups (STG-Tests) for Leucetta floridana.with the obtained F.statistics: CT=variation among hypotheticalɸSTGs, SC= variation within STs among hypothetical groups and ST= variation within STs. All values were significant (10 000 permutations). Theɸ ɸchosen hypothese is indicated in bold. (Reference to principal text: STG1=STGA+STGB+STGC+STGD, STG2=STGE, STG3=STGF, STG4=STGG andSTG5=STGH).
STGS within Clade 1 Clade2 Clade3 ɸCT ɸSC ɸST
STG-Test1 STGA STGB STGC STGD STGE STGF STGG STGH 0.679 0.943 0.982STG-Test2 STGA STGB STGC STGD STGE STGF STGG STGH 0.759 0.931 0.983STG-Test3 STGA STGB STGC STGD STGE STGF STGG STGH 0.767 0.929 0.983STG-Test4 STGA STGB STGC STGD STGE STGF STGG STGH 0.757 0.922 0.981STG-Test5 STGA STGB STGC STGD STGE STGF STGG STGH 0.768 0.919 0.981STG-Test6 STGA STGB STGC STGD STGE STGF STGG STGH 0.667 0.939 0.980STG-Test7 STGA STGB STGC STGD STGE STGF STGG STGH 0.689 0.910 0.972STG-Test8 STGA STGB STGC STGD STGE STGF STGG STGH 0.752 0.882 0.971STG-Test9 STGA STGB STGC STGD STGE STGF STGG STGH 0.729 0.922 0.979STG-Test10 STGA STGB STGC STGD STGE STGF STGG STGH 0.724 0.894 0.971STG-Test11 STGA STGB STGC STGD STGE STGF STGG STGH 0.749 0.883 0.970STG-Test12 STGA STGB STGC STGD STGE STGF STGG STGH 0.692 0.903 0.970STG-Test13 STGA STGB STGC STGD STGE STGF STGG STGH 0.704 0.926 0.978STG-Test14 STGA STGB STGC STGD STGE STGF STGG STGH 0.719 0.895 0.970STG-Test15 STGA STGB STGC STGD STGE STGF STGG STGH 0.757 0.886 0.972
Table S4. AMOVA. Hypotheses of population structure (H) and the obtained F-statistics: ɸCT=variation among hypothetical groups, ɸSC= variation withinlocalities among hypothetical groups and ɸST= variation within localities. Significant values are indicated in bold (10 000 permutations). Ecoregions: SC=Southern Caribbean, NE Brazil=Northeastern Brazil, FN and RA=Fernando de Noronha and Atoll das Rocas and EB=Eastern Brazil. Localities:PR=Puerto Rico, AG=Anguilla, SN=Saint Martin, SB=Saba, AN=Antigua, SNT=Les Saintes, GU=Guadeloupe, MT=Martinique, SAN=San Andrés,URA=Uraba, SM=Santa Marta, CUR=Curaçao, RN=Rio Grande do Norte, CE=Ceará, AT= Rocas Atoll, FN=Fernando de Noronha Archipelago,ABR=Abrolhos Archipelago.
Tropical Northern Atlantic Tropical Southern Atlantic F- statisticsGreater Antilles
Eastern Caribbean SouthwesternCaribbean
SC NE Brazil FN andRA
EB ɸCT ɸSC ɸST
H1 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR -0.054 0.433 0.402H2 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR -0.061 0.433 0.398H3 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.251 0.318 0.489H4 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.170 0.347 0.458H5 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.319 0.271 0.503H6 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.317 0.271 0.502H7 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.270 0.305 0.492H8 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.262 0.308 0.490H9 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.377 0.223 0.516H10 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.399 0.201 0.520H11 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.388 0.209 0.516H12 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.378 0.218 0.514H13 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.5488 -0.1148 0.4970H14 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.421 0.081 0.468H15 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.436 0.061 0.471H16 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.532 -0.121 0.475H17 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.538 -0.148 0.470H18 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.532 -0.170 0.452H19 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.5494 -0.1192 0.496H20 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.541 -0.157 0.469H21 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.429 0.096 0.483H22 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.423 0.102 0.482H23 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.376 0.130 0.457H24 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.341 0.204 0.476H25 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.362 0.177 0.476H26 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.354 0.184 0.474
214
Table S5. Mismatch distribution values. SSD=sum of squared deviation, r=Harpending's raggedness index.
SSD SSD P value r r p value
Groups
Antigua 0.0280 0.18 0.1717 0.5357
Les Saintes 0.2791 0.071 0.3125 0.4099
Les Saintes + Guadaloupe 0.3067 0.0889 0.3580 0.2832
Saint Martin 0.0059 0.4314 0.2760 0.5558
Panama 0.1336 0.0666 0.2870 0.0246
San Andrés 0.1243 0.3188 0.2778 0.7281
Santa Marta 0.4535 0 0.7279 0.9399
Uraba 0.0898 0.6033 0.5555 0.8964
Puerto Rico 0.0229 0.2384 0.4722 0.4358
Fernando de Noronha 0.02194 0.6456 0.25 0.9243
Ceará 0.0542 0.5037 0.4 0.3706
Eastern Caribbean 0.4050 0.0003 0.1161 0.9996
Southwestern Caribbean 0.0515 0.0201 0.0829 0.054
Northeast Brazil 0.0062 0.452 0.2006 0.4564
FN and Rocas Atoll 0.0542 0.5042 0.4 0.3863
Tropical Southwestern Atlantic 0.0116 0.3149 0.1619 0.4790
Tropical Northwestern Atlantic 0.4707 0.0001 0.0677 1.0
Clade 1 0.3638 0.0 0.0931 1.0
Clade 2 = STG3 0.0275 0.2272 0.2037 0.3274
Clade 3 0.0330 0.487 0.1088 0.6829
STG1 0.3337 0 0.1130 0.9996
STG2 0.0072 0.7642 0.2 0.9411
STG4 0.003 0.745 0.2222 0.9268
STG5 0.0898 0.5988 0.5556 0.8925
All 0.4539 0.0001 0.0629 1.0
Chapter 4
GENERAL DISCUSSION
216
This study contributed to the knowledge of the biodiversity of calcareous sponges from the
Western Tropical Atlantic (WTA). Nineteen species, including 15 new species and four new
records, were added to the 67 previously known species from this area (Van Soest et al., 2016;
Van Soest, 2017; Azevedo et al., submitted), summing up a total of 86 calcareous sponges in the
WTA (Table 1).
Compared to regions with coastline extensions similar to the study area (ca. 29 000 km) such
as Australia and Japan, whose coastlines cover 25,670 and 29,751 km, respectively (CIA, 2013),
the richness of Calcarea is, unexpectedly, higher in the WTA. Sixty-seven species have been
reported from Australia (Leocorny et al., 2016; Van Soest et al., 2016) and 79 from Japan (Van
Soest et al., 2016). This is very surprising as the calcareous sponges from those Indo-Pacific
regions have been thoroughly studied (e.g. Japan: Hôzawa, 1916, 1918, 1929, 1933, 1940;
Hôzawa & Tanita, 1941; Tanita, 1942, 1943 and Australia: Lendenfeld, 1885; Carter, 1886;
Dendy, 1892, 1893; Dendy & Row, 1913; Dendy & Frederick, 1924; Row & Hôzawa, 1931;
Wörheide & Hooper, 1999, 2003).
Considering the biogeographical provinces within the study area, the major contribution was
to the TNA (Tropical Northwestern Atlantic), as the previous number of reported species from
this province was 23 and it was raised to 45. Twelve new species (Amphoriscus micropilosus sp.
nov., Arthuria vansoesti sp. nov., Ascandra torquata sp. nov., Clathrina aspera sp. nov., C.
curaçaoensis sp. nov., Ernstia sp. nov. , Leucandra caribea sp. nov., Leucandrilla
pseudosagittata sp. nov., Leucilla antillana sp. nov., Grantessa tumida sp. nov., Sycon
conulosum sp. nov., Sycon magniapicalis sp. nov.) and nine new records (Arthuria hirsuta,
Borojevia tenuispinata, Clathrina aurea, C. blanca, C. cylindractina, C. insularis, C. lutea, C.
mutabilis, and Nicola tetela) were reported herein.
The new 21 records from the TNA have certainly changed the distribution of the species
richness within the ecoregions of this province. Before this study, the Greater Antilles presented
217
the highest number (8) of species (Haeckel, 1870, 1872; Lehnert & Van Soest, 1998) and it was
followed by Bermuda, with six species (Poléjaeff, 1883; de Laubenfels, 1950). Now, the richest
ecoregion is the Southern Caribbean, with 23 species (Arndt, 1927; Haeckel, 1872; this study),
followed by the Eastern Caribbean, with 13 species (Duchassaing & Michelotti, 1864;
Schuffner, 1877; this study) and the Southwestern Caribbean, with nine species (Rozemeijer &
Dulfer, 1987; Valderrama et al., 2009; Klautau et al., 2013; this study). Additionally, all these
new records are from localities that once had very few or no valid records of Calcarea.
Previously, three species were recorded from Colombia (Rozemeijer & Dulfer, 1987;
Valderrama et al., 2009), two from Panama (Valderrama et al., 2009; Klautau et al., 2013), one
from Curaçao (Arndt, 1927) and none from Martinique. Nowadays, those numbers have
increased and there are five, seven, eight and 20 calcareous sponges from Colombia, Panama,
Curaçao and Martinique, respectively.
In the Tropical Southwestern Atlantic (TSA), the richness of calcareous sponges has
increased from 48 (Muricy et al., 2011; Cavalcanti et al., 2014, 2015; Azevedo et al., submitted)
to 54 species. Although known species were found again in the Abrolhos Archipelago (e.g. C.
lutea, C. aurea, L. roseus), three new species (Amphoriscus hirsutus sp. nov., Leucascus
luteoatlanticus sp. nov. and Grantia grandisapicalis sp. nov.) and two new records (C.
luteoculcitella and Ernstia vansoesti) were described from there. This evidenced that sampling
in unexplored sites within “known” localities can still reveal novelties in species richness and
distribution. Furthermore, the six records raised the number of valid species from the Eastern
Brazil ecoregion from 25 to 30, and now it is the richest ecoregion of the TSA (Borojevic, 1971;
Borojevic & Peixinho, 1976; Cavalcanti et al., 2014, 2015; Azevedo et al., submitted; this
study). Nonetheless, in some areas or localities within this ecoregion, such as the littoral of
Espírito Santo State, no Calcarea has ever been recorded (Muricy et al., 2011).
218
In the North Brazilian Shelf (NBS), only 11 species have been reported so far (Borojevic &
Peixinho, 1976; Cavalcanti et al., 2013; Van Soest, 2017). This apparent poor diversity of
Calcarea may be an artefact of the lack of investigation on sponge diversity in this province.
Future samplings in this region may provide important insights for understanding former
disjunct species distribution in the Caribbean Sea and NE Brazilian coast.
Comparative information on Porifera2 richness within the WTA is available across some
ecoregions. Considering the Southern Caribbean (SC) and the Eastern Brazil (EB),
Demospongiae is accounted as the richest class with 201 (88.2%, Van Soest et al., 2012; 2014;
Alvizu et al., 2013) and 121 (79.6%, Van Soest et al., 2016) species, respectively. Within
Calcarea, 23 species were recorded from the SC (10.1%) and 30 from the EB (19.7%, this
study). Homoscleromorpha has four species from the SC (1.7%) and one single record from the
EB (0.7%, Domingos et al., 2016). Interestingly, in these “well-sampled” ecoregions, the
observed percentages are close to the ones obtained for global diversity of Porifera:
Demospongiae, 83%; Calcarea, 8% and Homoscleromorpha: 1% (Van Soest et al., 2012).
Despite the limited knowledge on calcareous sponges within certain localities in the WTA, it
is possible to elucidate some general distribution patterns. Likewise Demospongiae (Van Soest
& Hajdu, 1997) and Homoscleromorpha species (Domingos et al., 2016), most of the calcareous
sponges (65.1%=56 species) presented herein are endemic from the WTA. Among the non-WTA
endemic species, 13 species (15.3%) are amphi-Atlantic3, eight (9.4%) are restricted to the
2 Hexactinellida and certain Demospongiae recorded at more than 200 m (published in VanSoest et al., 2014; Hestetum et al., 2016 for the SC) were not considered as the depthboundary for MEOW is 200 m deep (Spalding et al., 2007).
3 Five from the West African Transition (Tropical Atlantic realm; Borojevic & Peixinho,1872; Thacker, 1908), three from the Temperate Southern Africa realm (Klautau &Valentine, Urban 1908; Brøndsted, 1931) and five from the Temperate Northern Atlanticrealm (Topsent, 1892, Miklucho-Maclay, 1868; Borojevic & Boury-Esnault, 1987, Sarà &Gaino, 1971; Klautau et al., 2016).
219
Western Atlantic4, four are Atlanto-Pacific5 and the other four are cosmopolitan6. Although the
amphi-Atlantic distribution of certain calcareous sponges proved to be the result of
overconservative systematics (Solé-Cava et al., 1991; Klautau et al., 1994); the presence of
Arthuria hirsuta, originally described from South Africa (Klautau & Valentine, 2003), was
confirmed in the Caribbean (La Martinique, Pérez et al., submitted, Appendix: Table 1). In this
study, the occurrence of the Australian Clathrina luteoculcitella in NE Brazil was corroborated
by molecular methods and whether or not this correspond to a natural distribution should be
investigated. Unpublished results from the Pacotilles campaign revealed the presence of this
species also in Bequia (Eastern Caribbean), which would give some cues for the understanding
these unexpected distribution patterns. The records from the WTA of the cosmopolitan species
should be revised, except for those of Vosmaeropsis sericata as its type locality is included in
the WTA (Cavalcanti et al., 2015).
A higher number of Calcaroneans was recorded from the WTA (46 spp.) compared to
Calcineans (39 spp.); however, the proportion of trans-Amazonian species is higher in Calcinea
(33.3%=13 spp.7) than in Calcaronea (15.2%=7 spp.8). It is possible that there is some kind of
bias in this result, but the possibility that the calciblastula larva (from Calcinea) can disperse
better than the amphiblastula (from Calcaronea) underneath the Amazon plume should not be
discarded. However, this hypothesis requires further investigation.
4 Seven that can also be found in SE or S Brazil (Warm Temperate Southwestern Atlantic;Muricy et al., 2011; Azevedo et al., submitted) and one also recorded in the Northern Gulfof Mexico (WTNA; Haeckel, 1872).
5 Three with records in the Indo-Pacific (Wörheide & Hooper, 1999; Van Soestet al., 2015;Poléjaeff, 1883; Kelly et al., 2009) and one in the SE Pacific (Azevedo et al., 2015).
6 As the type locality of Vosmaeropsis levis is unclear, its distribution was not consideredherein.
7 Arthuria vansoesti, Ascandra torquata, Borojevia tenuispinata, Clathrina aspera, C. aurea,C. cylindractina, C. insularis, C. lutea, C. luteoculcitella (considering material fromPacotilles), C. mutabilis, Leucaltis clathria, Leucetta floridana and Nicola tetela.
8 Grantia kempfi, Leucandra barbata, L. rudifera, L. serrata, Leucilla uter, Sycettusa flamma,and Vosmaeropsis complanatispinifera.
220
A biogeographic province is a large geographic area characterised by the presence of
endemic species and delimited by particular geomorphological, hydrogeographic and
geochemical factors (Spalding et al., 2007). The >10% endemism criterion proposed by Briggs
(1974) has been widely employed for defining biogeographic provinces within the Tropical
Atlantic (e.g. Floeter & Soares-Gomez, 1999; Floeter & Gasparini, 2000; Briggs & Bowen,
2012). In this study, the percentage of endemic species in both the TNA and TSA surpassed the
10%, being 46.7% (21 out of 45 species) in the former and 40.7% (22 out of 54 species) in the
latter. Therefore, it is possible to say that the existence of the TNA and TSA provinces is
supported by the observed high endemism of calcareous sponges within these areas.
Nonetheless, there are several species that occur in more than one province. Among the 86
species from the WTA, 16 (18.6%) species are shared between the TNA, NBS and/or TSA.
Leucaltis clathria and Leucetta floridana have been recorded from the three provinces
(Borojevic & Peixinho, 1976; Haeckel, 1872; Klautau et al., 2013; Valderrama et al., 2009) and
they are considered species with a wide distribution. Grantia kempfi, Sycettusa flamma and
Vosmaeropsis complanatispinifera are shared between the NBS and the TSA (Borojevic &
Peixinho, 1976; Cavalcanti et al., 2015; Van Soest, 2017). Eleven species, Arthuria vansoesti,
Borojevia tenuispinata, Clathrina aspera, C. aurea, C. insularis, C. lutea, C. mutabilis,
Leucandra barbata, L. rudifera, Leucilla uter and Nicola tetela were recorded from the TNA
and TSA (Azevedo et al., submitted; Cóndor-Luján & Klautau, 2016, Muricy et al., 2011; this
study) but not in the NBS.
The occurrence of 13 species in the TNA and TSA provinces suggests a Caribbean-Brazilian
affinity within the class Calcarea. Although this affinity constitutes a novelty for Calcarea, it
was already observed in Demospongiae some decades ago (Hechtel, 1976; Colette & Rützler,
1977) and indicated in more recent studies (Moraes, 2011; Muricy et al., 2011; Moura et al.,
2016; Soares et al., 2016).
221
Hechtel (1976) reported numerous Demospongiae from both the West Indies and Brazil and
suggested that larvae of those species would cross the Orinoco and Amazon barriers,
considering the assumption of hard substrate availability close to the river's discharge which
would permit sponge settlement. One year later, Colette & Rützler (1977) not only reported
“Caribbean” sponges underneath the mouth of the Amazon River but also mentioned that there
was no apparent effect of freshwater and that there was abundant hard substrata on the bottom
of the Amazon mouth. Recently, Moura et al. (2016) reported the existence of a calcium
carbonate system underneath the Amazon river plume, which would act as a connectivity
corridor for wide depth–ranging reef-associated species, such as sponges.
The presence of calcareous species in both provinces also suggests a possible connectivity
between the TNA and TSA populations, which could be explained by a stepping-stone model of
population structure in the North Brazil Shelf Province (NBS) (in the Venezuelan Southeastern
Coast, Guyanas or in the Northern Brazilian Coast) facilitating the gene flow between these two
provinces. Therefore, it is quite probable to find several of the 13 shared species in the NBS (up
to date, only Leucetta floridana and Leucaltis clathria had been reported from the three
provinces).
In this study, L. floridana, one of the most widespread species in the WTA, was used as a
model to assess connectivity between TNA and TSA populations. The results evidenced five
structured populations: one widespread population maintaining gene flow between the TNA and
TSA provinces and four other isolated populations within the Caribbean Sea. The finding of a
panmitic widespread population in the WTA was really unexpected as it rejects the hypothesis
that the discharge of the Amazon River is an effective barrier to the maintenance of gene flow
among trans-Amazonian populations of calcareous sponges. Interestingly, a similar genetic
222
pattern was observed in the Demospongiae Chondrilla aff. nucula collected in the Caribbean
and in the Brazilian coast (Boury-Esnault et al., 20169).
Cryptic species are not unusual in Calcarea because of the plasticity of some characters in
certain genera and species. Studies assessing the genetic identity of former Brazilian
populations of Clathrina clathrus, C. primordialis and Borojevia cerebrum revealed that they
were cryptic species and a posterior discovery of diagnostic morphological characters justified
the erection of new species: C. aurea, C. cylindractina, C. conifera, and B. brasiliensis (Klautau
et al., 1994; Solé-Cava et al., 1991). In L. floridana, the observed genetic variability (0-1.8%)
is within the expected intraspecific range estimated for other leucettas (Valderrama et al., 2009)
and other Calcinean species (Rossi et al., 2011). Moreover, the high morphological variability of
L. floridana did not match the structured populations found in this study. Individuals with
different morphotypes (based on the shape of the oscula) and/or colour-morphs (light blue, pink
and white) were found in the same population or ST-group. Likewise, several individuals
collected in different habitats (completely protected from light, such as underneath boulders or
inside crevices, and semi-exposed to light, such as vertical slopes), depths (- 2 and - 40 m) or
localities (adjacent or distant) grouped in the same population. Therefore, there is no evidence to
support an ongoing cryptic speciation in L. floridana.
The high intraspecific variation within Leucetta floridana is comparable to that of other two
species with similar distribution in the WTA: C. lutea with 0-1.6% and C. mutabilis with 0-2%
(values taken from Chapter 3.4). An exploratory ITS sequence-type network of these two
Clathrina species (Appendix: Figures 2 and 3) showed that each species presented an ITS
sequence-type shared by Caribbean and Brazilian individuals (Brazil-Saint Martin and Brazil-
Curaçao-La Martinique-Panama, respectively). Furthermore, genetic analyses10 with C. lutea
sequences evidenced a similar phylogeographic and population pattern as those of L. floridana.
9 Results recently presented by N. Boury-Esnault in the II Workshopt ABC/CNRS (LIA MARRIO).
10 Results I recently presented in the II Workshop ABC/CNRS (LIA MARRIO).
223
According to Avise (2000), phylogeographic breaks shared among taxa with varying life
histories provide a rational basis for determining where the effective gene flow is restricted or
totally absent and thus, facilitate the detection of physical features that may act as important
barriers. Consequently, further studies should be addressed to verify the congruence of the
phylogeographic and population patterns of C. lutea and L. floridana as they might reveal
important traits for understanding connectivity and species evolutionary history in the WTA.
Leucetta floridana and L. potiguar are morphologically very similar species but present
strong diagnostic morphological characters (Lanna et al., 2009). Different from L. floridana, L.
potiguar is a NE Brazilian endemic species. In all known phylogenetic trees (Valderrama et al.,
2009; Klautau et al., 2013; Azevedo et al., submitted; this study), they appear as sister species.
Given their current geographic distribution and their phylogenetic position (Figure 2 of Chapter
3.4 of this study), it is possible to infer that the ancestral species of these pair of eucettas had a
widespread distribution in the WTA (including the Caribbean Sea and the NE Brazilian coast).
In this study, the genetic divergence estimated by the uncorrected p distance between L.
floridana and L. potiguar varied from 2.5 to 3.4%. If considered the mutation rate of 1% per
MY calculated for Demospongiae (Wörheide et al., 2004), it is possible to infer that the
cladogenetic event occurred ~4 MY BP, during the Pliocene (~5.3 – 2.6 MY BP). Interestingly,
the Amazon River became fully established at ~7 MY BP (Figuereido et al., 2009) and during
the early Pliocene, it started to drain water and sediments on a large scale to the Atlantic Ocean
(Latrubesse et al., 2010). As the discharge of freshwater and sediments from the Amazon River
has been recognised as responsible for the formation of pairs of sister species of reef fishes in
the WTA (Rocha, 2003 and references therein), this can also be suggested for this pair of sisters
species of Leucetta.
Table 1. List of recorded species from the Western Tropical Atlantic. Abbreviations: TL=type locality. Brazilian States - AL: Alagoas, BA: Bahia, CE:Ceará, ES: Espírito Santo, MA: Maranhão, PB: Paraíba, PA: Pará, PE: Pernambuco, RN: Rio Grande do Norte, RJ: Rio de Janeiro, SC: Santa Catarina,SP: São Paulo. FN: Fernando de Noronha. Provinces in the Tropical Atlantic (TA) – NBS: North Brazil Shelf, TNA: Tropical Northwestern Atlantic,TSA: Tropical Southwestern Atlantic, WAT: West African Transition. Other provinces - WTNA: Warm Temperate Northwest Atlantic, WTSA: WarmTemperate Southwestern Atlantic, WTSEP: Warm Temperate Southeastern Pacific. Realms - CIP: Central Indo-Pacific, TAA: Temperate Australasia,TNA: Temperate Northern Atlantic, TNP: Temperate Northern Pacific, TSAfrica: Temperate Southern Africa, TSAmerica: Temperate South America. E:Eastern, N: Northern, NE: Northeastern, S: Southern, SE: Southeastern, SW: Southwestern, W:Western. USA: United States of America. ** Indicates new species and * new records obtained in this study.
Species Distribution Ecoregion, Province, Realm
References
Subclass Calcaronea
1 Amphoriscus ancora Van Soest, 2017 Guyana Shelf (TL) Guianan, NBS Van Soest, 2017
2 Amphoriscus hirsutus sp. nov.** Abrolhos Archipelago (BA), NE Brazil(TL)
Eastern Brazil, TSA This study
3 Amphoriscus micropilosus sp. nov.** Curaçao (TL) S Caribbean, TNA This study
4 Amphoriscus synapta (Schmidt in Haeckel, 1872)
BA, NE Brazil (TL) NE or E Brazil, TSA Haeckel, 1872
5 Amphoriscus testiparus (Haeckel, 1872) Cuba (TL) Greater Antilles, TNA Haeckel, 1872
6 Amphoriscus urna Haeckel, 1872 Venezuela (TL) S Caribbean, TNA Haeckel, 1872
7 Grantessa anisactina Borojevic & Peixinho, 1976
Paraíba, NE Brazil (TL) E Brazil, TSA Borojevic & Peixinho, 1976
8 Grantessa tumida sp. nov. ** Curaçao (TL) S Caribbean, TNA This study
9 Grantia atlantica Ridley, 1881 Vitória Bank (ES), SE Brazil E Brazil, TSA Ridley, 1881
10 Grantia grandisapicalis sp. nov. ** Abrolhos Archipelago (BA), NE Brazil(TL)
E Brazil, TSA This study
11 Grantia kempfi Borojevic & Peixinho, 1976 Guyana Shelf Guianan, NBS Van Soest, 2017
AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
PE (TL), NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Off Abrolhos, BA, NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976
12 Leucandra amorpha Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883
13 Leucandra armata (Urban, 1908) Off Marajó Island, PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
CE, PE, AL, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Off Abrolhos, BA, NE Brazil and ES, SE Brazil Brazil
E Brazil, TSA Borojevic & Peixinho, 1976
Francis Bay (TL), South Africa Natal, Agulhas, TSAfrica (realm)
Brøndsted, 1931; Urban, 1908, 1909
14 Leucandra barbata (Duchassaing & Michelotti, 1864)
Dry Tortugas Floridian, TNA de Laubenfels, 1936
Virgin Islands (TL) E Caribbean, TNA Duchassaing & Michelotti, 1864
Colombia SW Caribbean, TNA Rozemeijer & Dulfer, 1987
PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Off Abrolhos, BA, NE Brazil and ES, SE Brazil
E Brazil, TSA Borojevic & Peixinho, 1976
15 Leucandra caribea sp. nov.** Curaçao (TL) S Caribbean, TNA This study
16 Leucandra crassior Ridley, 1881 PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Vitória Bank (TL), ES, SE Brazil E Brazil, TSA Ridley, 1881
17 Leucandra crosslandi Thacker, 1908 PB, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Boa Vista Island, Cape Verde (TL) Cape Verde, WAT Thacker, 1908
18 Leucandra crustacea (Haeckel, 1872) Bermuda Bermuda, TNA De Laubenfels, 1950
Venezuela (TL) S Caribbean, TNA Haeckel, 1872
19 Leucandra curva (Schuffner, 1877) Barbados E Caribbean, TNA Schuffner, 1877
20 Leucandra hentschelii Brøndsted, 1931 NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Simonstown, South Africa (TL) Agulhas Bank, Agulhas,TSAfrica (realm)
Brøndsted, 1931
21 Leucandra multiformis Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883
22 Leucandra rudifera Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883
Trindade Island Trindade and Martin Vaz Islands, TSA
Moraes et al.., 2006
Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA, TSAmerica
Muricy et al., 1991, Muricy & Silva, 1999.
Cape Verde Cape Verde, WAT Thacker, 1908
23 Leucandra serrata Azevedo & Klautau, 2007 RN, NE Brazil NE Brazil, TSA Lanna et al., 2009
RJ, SE Brazil (TL) SE Brazil, WTSA, TSAmerica
Azevedo & Klautau, 2007
24 Leucandra typica (Poléjaeff, 1883) Bemuda (TL) Bermuda, TNA Poléjaeff, 1883
25 Leucandrilla pseudosagittata sp. nov.** Curaçao (TL) S Caribbean, TNA This study
26 Leucilla amphora Haeckel, 1872 Puerto Rico (TL) Greater Antilles, TNA Haeckel, 1872
Curaçao S Caribbean Arndt, 1927
Senegal, West Africa Sahelian Upwelling, WAT
Borojevic & Boury-Esnault, 1987
27 Leucilla antillana sp. nov.** Curaçao (TL) S Caribbean, TNA This study
La Martinique* E Caribbean, TNA Pérez et al., submitted: Leucilla sp. nov.
28 Leucilla sacculata (Carter, 1890) FN (TL), NE Brazil FN and Atoll das Rocas, TSA
Carter, 1890
29 Leucilla uter Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883
Curaçao S Caribbean, TNA Arnd, 1927
CE, RN, PB, PE, AL, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Abrolhos Archipelago*, Off Abrolhos and Off Belmonte, BA, NE Brazil and ES, SE Brazil
E Brazil, TSA Borojevic & Peixinho, 1976, this study
Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA, TSAmerica
Muricy et al., 1991; Muricy & Silva, 1999; Borojevic & Peixinho, 1976.
Philippines E Philippines, Western Coral Triangle, CIP
Poléjaeff, 1883
30 Leucosolenia horrida (Schmidt in Haeckel, 1872)
Florida. USA (TL) Floridian, TNA Haeckel, 1872
31 Leucosolenia salpinix Van Soest, 2017 Guyana Shelf (TL) Guianan, NBS Van Soest, 2017
32 Paraleucilla incomposita Cavalcanti et al., 2014
Arraial d'Ajuda (TL), BA, NE Brazil E Brazil, TSA Cavalcanti, et al., 2014
33 Paraleucilla oca Cavalcanti et al., 2014 Itaparica Island (TL), BA, NE Brazil E Brazil, TSA Cavalcanti, et al., 2014
34 Paraleucilla solangeae Cavalcanti et al., 2014
Guarajuba, BA, NE Brazil (TL) NE Brazil, TSA Cavalcanti, et al., 2014
35 Paraleucilla sphaerica Lanna et al., 2009 Potiguar Basin, NE Brazil (TL) NE Brazil, TSA Lanna et al., 2009
36 Sycettusa flamma (Poléjaeff, 1883) AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
Off Bahia (TL), NE Brazil NE or E Brazil, TNA Poléjaeff, 1883
37 Sycon ampulla (Haeckel, 1872) Venezuela (TL?) S Caribbean, TNA Haeckel, 1972
RJ, SE Brazil and Florianópolis, SC, S Brazil (TL?)
SE Brazil, WTSA, TSAmerica
Haeckel, 1972; Mello-Leitão et al., 1961; Muricy & Silva, 1999.
Azores, Portugal Azores Canaries Madeira, Lusitanian, TNA (realm)
Topsent, 1892
38 Sycon barbadense (Schuffner, 1877) Barbados (TL) E Caribbean, TNA Schuffner, 1877
39 Sycon brasiliense Borojevic, 1971 Cabo São Tomé, RJ, SE Brazil (TL) E Brazil, TSA Borojevic, 1971
40 Sycon conulosum sp. nov.** Curaçao (TL) S Caribbean, TNA This study
41 Sycon formosum (Haeckel, 1870) Cuba (TL) Greater Antilles, TNA Haeckel, 1870
42 Sycon frustulosum Borojevic & Peixinho, 1976
PE (TL), NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
43 Sycon magniapicalis sp. nov.** Curaçao (TL) S Caribbean, TNA This study
44 Sycon vigilans Sarà & Gaino, 1971 Punta Manara (TL), Italy Mediterranean Sea, TNA (realm)
Sarà & Gaino, 1971
PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Urca, RJ (SE) and SC (S), Brazil SE Brazil, WTSA Borojevic &Peixinho, 1976; Mothes & Lerner, 1994.
45 Vosmaeropsis complanatispinifera Cavalcantiet al., 2015
AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
RN (TL), NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976; Cavalcanti et al., 2015
46 Vosmaeropsis levis Hozawa, 1940 Mexico (TL) Atlantic or Pacific Atlantic?
TNA? Hôzawa, 1940
Cabo de São Tomé, RJ E Brazil, TSA Borojevic, 1971
47 Vosmaeropsis sericata Ridley, 1881Cosmpolitan
Vitória Bank, SE Brazil (TL) E Brazil, TSA Ridley 1881
Cape Verde Cape Verde, WAT Thacker, 1908
Chile WTSEP, TSAmerica Breitfuss, 1898
SUBCLASS CALCINEA
48 Arthuria hirsuta Klautau & Valentine, 2003* Stil Bay (TL), Cape Town, South Africa
Agulhas Bank, Agulhas,TSAfrica (realm)
Klautau & Valentine, 2003
La Martinique* E Caribbean, TNA Pérez et al., submitted
49 Arthuria trindadensis Azevedo et al., submitted
Trindade Island Trindade and Martin Vaz Islands, TSA
Azevedo et al., submitted
50 Arthuria vansoesti sp. nov.**
Curaçao (TL) S Caribbean, TNA This study
La Martinique* E Caribbean, TNA Pérez et al., submitted: Arthuria sp. nov.
Abrolhos Archipelago, BA, NE Brazil*
E Brazil, TSA This study
51 Ascaltis agassizii Haeckel 1872 Florida (TL), United States of America Floridian, TNA Haeckel, 1872
Gulf of Mexico TNA? or TA? (realms) Haeckel, 1872
52 Ascalits panis (Haeckel, 1870) Florida, United States of America Floridian, WTNA Haeckel, 1870
Carrier Bow Cay, Belize W Caribbean, TNA Rutzler et al., 2014
Cape Verde, West Africa Cape Verde, WAT Thacker, 1908
53 Ascaltis poterium (Haeckel, 1872) PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Cosmopolitan Australia (TL) CIP? or TAA? (realms) Haeckel, 1872
Arctic Artcic (realm) Breitfuss, 1932
Chile TSA (realm) Ridley, 1881; Breitfuss, 1898
54 Ascaltis reticulum (Schmidt, 1862) PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
Cosmopolitan PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
ES, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976
Banyuls-sur-Mer (TL) and Adriatic Sea, Mediterranean Sea
Mediterranean Sea, TNA (realm)
Schmidt, 1862; Klautau et al., 2016 (more references in it)
Arctic Artcic (realm) Breitfuss, 1932
Japan TNP (realm) Hôzawa, 1940; Tanita, 1943
55 Ascandra ascandroides (Borojevic, 1971) PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
Cabo São Tomé (TL), RJ, SE Brazil E Brazil, TSA Borojevic, 1971
Arraial do Cabo, RJ (SE) and SC (S) SE Brazil, WTSA Klautau & Borojevic, 2001; Mothes & Lerner, 1994
Bay of Biscay S European Atlantic Shelf, Lusitanian
Borojevic & Boury-Esnault, 1987
56 Ascandra atlantica (Thacker, 1908) NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976
Vitoria, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976
Boa Vista (TL), Cape Verde Cape Verde, WAT Thacker, 1908Klautau & Valentine
57 Ascandra torquata sp. nov.** Bocas del Toro, Panama* SW Caribbean, TNA Personal observation
Santa Marta, Colombia* SW Caribbean, TNA Personal observation
Curaçao S Caribbean, TNA This study
Arvoredo Archipelago (TL), SC, S Brazil
S Brazil, WTSA, TSAmerica
This study
58 Borojevia brasiliensis (Solé-Cava et al., 1991)
CE, NE Brazil* NE Brazil, TSA This study
Cabo de São Tomé, RJ, SE Brazil E Brazil, TSA Borojevic, 1971
Arraial do Cabo, RJ, SE Brazil (TL) SE Brazil, WTSA, TSAmerica
Solé-Cava et al., 1991Muricy et al., 2011 (and references in it)
59 Borojevia tenuispinata Azevedo et al, submitted
Curaçao* S Caribbean, TNA This study*
São Pedro e São Paulo Archipelago (TL), NE Brazil
São Pedro e São Paulo Islands, TSA
Azevedo et al, submitted
60 Borojevia trispinata Azevedo et al, submitted São Pedro e São Paulo Archipelago (TL), NE Brazil
São Pedro e São Paulo Islands, TSA
Azevedo et al, submitted
Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA Azevedo et al, submitted
61 Clathrina aspera sp. nov.** Curaçao (TL) S Caribbean, TNA This study
RN, NE Brazil NE Brazil, TSA This study
RJ, SE Brazil SE Brazil, WTSA This study*
62 Clathrina aurea (Solé-Cava et al., 1991) Bocas del Toro, Panama* SW Caribbean, TNA Personal observation
Martinique* Eastern Caribbean, TNA
Pérez et al., submitted*
FN, NE Brazil FN Archipelago and Atoll das Rocas, TSA
Muricy et al., 2001 (more references in it)
Potiguar Basin, NE Brazil NE Brazil, TSA Lanna et al.., 2009
Abrolhos Archipelago*, BA, NE Brazil E Brazil, TSA Azevedo et al., in press; this study
RJ (TL) and SP, SE Brazil SE Brazil, WTSA Solé-Cava et al., 1999; Muricy et al., 2011 (references in it)
S Peru WTSEP, TSAmerica Azevedo et al., 2015
63 Clathrina blanca Miklucho-Maclay, 1868*Cosmopolitan
Curaçao* S Caribbean, TNA This study
SP, SE Brazil and SC, S Brazil SE Brazil, WTSA Borojevic, 1971
Canary Islands (TL) and Norway Lusitanian and N European Sea, TNA (realm)
Miklucho-Maclay, 1868; Rapp,2006 (references in it)
Antarctic Southern Ocean (realm) Brøndsted, 1931
Japan TNP (realm) Hôzawa, 1929; Tanita, 1943
64 Clathrina conifera (Klautau & Borojevic, 2001)
NE Brazil NE Brazil, TSA Borojevic & Pexinho, 1976; Klautau & Valentine, 2003
Arraial do Cabo, RJ (TL), SE brazil SE Brazil, WTSA, TSAmerica
Borojevic & Pexinho, 1976; Klautau & Valentine, 2003
Adriatic Sea Mediterranean Sea, TNA (realm)
Klautau et al., 2006
65 Clathrina curaçaoensis sp. nov.** Curaçao (TL) S Caribbean, TNA This study
66 Bocas del Toro, Panama* SW Caribbean, TNA Personal observation
Clathrina cylindractina (Klautau et al., 1994)*
Arraial do Cabo, RJ, SE Brazil (TP) SE Brazil, WTSA Klautau et al., 1994
67 Clathrina hondurensis Klautau & Valentine, 2003
Belize (TP) W Caribbean, TNA Klautau & Valentine, 2003
Carrie Bow Cay, Belize W Caribbean, TNA Rützler et al., 2014
Bocas del Toro, Panama* SW Caribbean, TNA Personal observation
Curaçao* S Caribbean, TNA This study
68 Clathrina insularis Azevedo et al., submitted Curaçao* S Caribbean, TNA This study
Martinique* E Caribbean, TNA Pérez et al., submitted: Clathrina sp. nov. 1
FN (TL), NE Brazil FN and Atoll das Rocas, TSA
Azevedo et al., submitted
69 Clathrina luteoculcitella Wörheide & Hooper, 1999*
Abrolhos, NE Brazil* Eastern Brazil, TSA This study
Great Barrier Reef (TL), Australia NE Australian Shelf, CIP
Wörheide & Hooper, 1999
70 Clathrina lutea Azevedo et al., submitted Florida, USA Floridian, TNA Klautau et al., 2013; Zea et al.,2014
Little San Salvador, Bahamas Bahamian, TNA Zea et al., 2014
Jamaica* Greater Antilles, TNA Lehner & Van Soest, 1998 (C. primordialis ), this study
Curaçao* S Caribbean, TNA This study
Virgin Islands E Caribbean, TNA Klautau et al., 2013
Abrolhos Archipelago (TL) E Brazil, TSA Azevedo et al.., submitted; thisstudy*
71 Clathrina mutabilis Azevedo et al., submitted Panamá and Colombia* SW, TNA Personal observation*
Curaçao* S Caribbean, TNA This study*
Martinique* E Caribbean, TNA Pérez et al., submitted: Clathrina sp. nov. 2*
FN Archipelago, NE Brazil (TL) FN and Atoll das Rocas, TSA
Azevedo et al., submitted
RN, NE Brazil* NE Brazil, TSA This study
72 Ernstia citrea Azevedo et al., submitted Rocas Atoll, NE Brazil FN and Atoll das Rocas, TSA
Azevedo et al., submitted
Abrolhos Archipelago, NE Brazil* Eastern Brazil, TSA This study
73 Ernstia multispiculata Azevedo et al., submitted
Rocas Atoll, NE Brazil FN and Atoll das Rocas, TSA
Azevedo et al., submitted
74 Ernstia santipauli Azevedo et al., submitted São Pedro e São Paulo Archipelago (TL), NE Brazil
São Pedro and São Paulo Islands
Azevedo et al., submitted
75 Ernstia sp. nov. ** La Martinique (TL) E Caribbean, TNA Pérez et al., submitted; Fontana et al., in prep.
76 Ernstia rocasensis Azevedo et al., submitted Rocas Atoll (TL) and FN Archipelago, NE Brazil
FN and Atoll das Rocas, TSA
Azevedo et al., submitted; this study
Abrolhos Archipelago, BA, NE Brazil* E Brazil, TSA Azevedo et al., submitted; this study
77 Ernstia sulphurea Miklucho-Maclay, 1868 FN Archipelago, NE Brazil FN and Atoll das Rocas, TSA
Azevedo et al., submitted
Trindade Island, SE Brazil Trindade and Martin Vaz Islands, TSA
Azevedo et al., submitted
Lanzarote Beach, Canary Islands (TL) Azores Canaries Madeira, Lusitanian
Miklucho-Maclay, 1868
78 Leucaltis clathria Haeckel, 1872 Florida, USA (TL) Floridian, TNA Haeckel, 1872
Bocas del Toro, Panama SW Caribbean, TNA Klautau et al., 2013;Zea et al., 2014; personal observation*
Guyana Shelf Guianian, NBS Van Soest, 2017
PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976;
CE, RG, AL, SE, NE BraziL NE Brazil, TSA Borojevic & Peixinho, 1976; Lanna et al., 2009
ES, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976.
79 Leucascus luteoatlanticus sp. nov.** Abrolhos Archipelago (TL), BA, NE BraziL
E Brazil, TSA This study
80 Leucascus roseus Lanna et al., 2007 Potiguar Basin, RN, NE Brazil NE Brazil, TSA Lanna et al., 2009
Abrolhos Archipelago, BA, NE Brazil Eastern Brazil, TSA Cavalcanti et al., 2013; this study*
RJ and SP (TL), SE Brazil Southeastern Brazil, WTSA
Cavalcanti et al., 2013; Lanna et al., 2007;
81 Leucascus aff. simplex sensu Cavalcanti et al., 2013
PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976; Cavalcanti et al., 2013
82 Leucetta floridana (Haeckel, 1872) Bermuda Bermuda, TNA de Laubenfels, 1950
Bahamas Bahamian, TNA Zea et al., 2014
Florida (TL) Floridian, TNA Haeckel, 1872
Jamaica and Cayman Islands Greater Antilles, TNA Lehner & van Soest, 1998; Milaslovich et al., 2011
Panama and Colombia (Santa Marta*) SW Caribbean, TNA Klautau et al., 2013; this study (Santa Marta)*; Valderrama et al., 2009; Zea et al., 2014
Curaçao* S Caribbean, TNA This study
Lesser Antilles* including St. Eustatius E Caribbean, TNA García-Hernández et al., 2016; Pérez et al., submitted; this study;
MA and PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976
MA, CE, RN, PA, PE and AL, NE Brazil
NE Brazil, TSA Borojevic & Peixinho, 1976; Lanna et al, 2009; Valderrama et al., 2009
FN Archipelago and Rocas Atoll FN and Atoll das Rocas, TSA
Borojevic & Peixinho, 1976; Moraes et al., 2006; Valderrama et al., 2009
Abrolhos, BA, NE Brazil and ES, SE Brazil.
E Brazil, TSA Borojevic & Peixinho, 1976; Valderrama et al., 2009
83 Leucetta imberbis (Duchassaing & Michelotti, 1864)
Virgin Islands (TP) E Caribbean, TNA Duchassaing & Michelotti, 1864
Santa Marta, Colombia SW Caribbean, TNA Rozemeijer & Dulfer, 1987
Jamaica Greater Antilles, TNA Lehner & Van Soest, 1998
84 Leucetta potiguar Lanna et al., 2009 Potiguar Basin (TL), RN and CE, NE Brazil
NE Brazil, TSA Lanna et al., 2009; Valderramaet al., 2009
85 Leucettusa corticata (Haeckel, 1872) Cuba Greater Antilles, TNA Haeckel, 1872
New Zealand S New Zealand?, TAA Kelly et al., 2009
86 Nicola tetela (Borojevic & Peixinho, 1976) Curaçao* S Caribbean, TNA This study
Sint Eustatius E Caribbean, TNA García-Hernández et al., 2016
Abrolhos (TL), BA, NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976
Chapter 5
CONCLUSIONS AND PERSPECTIVES
234
This study certainly contributed to our comprehension of the marine biodiversity of the Western
Tropical Atlantic, filling in the gap of knowledge on calcareous sponges from poorly studied areas
such as the Caribbean Sea. The 86 species now reported from the WTA represents approximately
12% of all known Calcarea (ca. 730 species; Van Soest et al., 2012; Rapp, 2013; Cavalcanti et al.,
2013, 2014, 2015 ; Azevedo et al., 2015, submitted; Van Soest & Voogd, 2015; Klautau et al., 2016;
Leocorny et al., 2016; Van Soest, 2017; Azevedo et al., submitted). However, the richness of this
region is still underestimated as new species are still being discovered from new localities in the
Caribbean Sea (Lesser Antilles, Pacotilles campaign, pers. obs.) and in the NE Brazil (Bahia, F.
Cavalcanti, pers. comm.). Besides, new species of the subclass Calcaronea from Colombia and
Panama (not included in this study) are being described.
As suggested for other marine taxa (Ludt & Rocha 2005), the current distribution of calcareous
sponges may be related to the major changes occurred during the Pleistocene in the WTA. It would
be interesting to compare phylogeographic patterns across different genera in Calcarea (e.g.
Clathrina and Leucetta) to see if they corroborate this hypothesis.
As mentioned before, the systematics of calcareous sponges is considered particularly difficult
because of the plasticity of some of the characters (Van Soest et al., 2012). In this study, molecular
approaches played an important role in providing useful information for the identification of species
whose morphological characters were very plastic (e.g., diactines in Ascandra torquata) and for the
allocation of certain species in its correct genus (e.g. Nicola tetela). In the latter case, I should point
out the importance of revising the type material and not to limit to the examination of the original
description.
The Amazon River outflow may constitute a semi-permeable barrier to calcareous sponges. It
would allow the dispersal of some species such as Leucetta floridana, Clathrina aurea, C. insularis,
C. lutea, and C. mutabilis (whose occurrences in the TNA and TSA were corroborated by
morphological and molecular methods) but also, restrict the dispersal of other species (the TNA and
235
TSA endemic species). At least, for one calcareous species, L. floridana, the discharge of freshwater
and sediments from the rivers of the TWA (Orinoco and Amazonas) does not constitute an effective
barrier to gene flow. Studies using other molecular markers are recommended to give more support
to this finding.
In order to fully understand marine connectivity, it is also very important to perform studies on
the reproduction of the target species (e.g. larval behaviour) as it provides quite useful information
to explain the observed genetic patterns. Non-physical barriers such as chemical defenses and adult
ecology should be considered as well. As most of the mentioned information is scarce or even
inexistent in Calcarea, it is crucial to begin investigating these aspects if a more precise
understanding of calcareous sponges connectivity in the Western Tropical Atlantic is desired.
Chapter 6
REFERENCES
237
ANTCLIFFE, J. B., CALLOW, R. H. T. & BRASIER, M. D. (2014) Giving the early fossil
record of sponges a squeeze. Biological Reviews, 89: 972–1004.
ANTHONY, E.J., GARDEL, A., GRATIOT, N., PROISY, C., ALLISON, M.A., DOLIQUE, F.,
FROMARD, F. 2010.The Amazon-influenced muddy coast of South America: A review of mud-
bank-shoreline interactions. Earth-Science Reviews, 103(3–4): 99–121.
AZEVEDO, F. Sistemática e Biogeografia de esponjas calcareas (Porifera, Calcarea) da
América do Sul. Tese (Doutorado) - UFRJ/MN/Programa de Pós-graduação em Ciências
Biológicas (Zoologia).
AZEVEDO, F.; CÓNDOR-LUJÁN, B.; WILLENZ, P.; HAJDU, E.; HOOKER, Y. &
KLAUTAU, M. (2015) Integrative taxonomy of calcareous sponges (subclass Calcinea) from
the Peruvian coast: morphology, molecules and biogeography. Zoological Journal of the
Linnean Society, 173: 787–817.
AZEVEDO, F., PADUA, A., MORAES, F., ROSSI, A., MURICY, G. & KLAUTAU, M.
(submitted). Taxonomy and phylogeny of calcareous sponges (Porifera: Calcarea: Calcinea)
from Brazilian mid-shelf and oceanic islands. Zootaxa.
ALMANY, G., BERUMEN, M.L., THORROLD, S.R., PLANES, S. & JONES, G.P. (2007)
Local Replenishment of Coral Reef Fish Populations in a Marine Reserve. Science, 316: 742-
744.
ALVIZU, A.; DÍAZ, M.C.; BASTIDAS, C.; RÜTZLER, K.; THACKER, R.W.; MARQUEZ,
L.M. (2013) A skeleton-less sponge of Caribbean mangroves: invasive or undescribed?
Invertebrate Biology 132(2): 81-94.
AMADO-FILHO, G.M.; MOURA, R.L.; BASTOS, A.C.; FRANCINI-FILHO, R.B.,
PEREIRA-FILHO, G.H.; BAHIA, R.G.; MORAES, F.C. & MOTTA, F.S. (2016) Mesophotic
ecosystems of the unique South Atlantic atoll are composed by rhodolith beds and scattered
consolidated reefs. Marine Biodiversity; 933-936.
238
ARNDT, W. (1927) Kalk- und Kieselschwämme von Curaçao. Bijdragen tot de Dierkunde 25:
133-158.
BIDDER, G.P. (1898) The Skeleton and Classification of Calcareous Sponges. Proceedings of
the Royal Society 64: 61-76.
BOROJEVIC, R. (1971) Éponges calcaires de la côte sudest du Brésil, épibiontes sur Laminaria
brasiliensis et Sargassum cymosum. Revista Brasileira de Biologia, 31; 525–530.
BOROJEVIC, R. & BOURY-ESNAULT, N. (1987) Calcareous sponges collected by N.O.
Thalassa on the continental margin of the Bay Biscaye: I – Calcinea. In: VACELET, J. &
BOURY-ESNAULT, N. (eds) Taxonomy of Porifera from NE Atlantic and Mediterranean Sea.
Berlin, Alemania: Editorial Sprinter, 1-27 pp.
BOROJEVIC, R., BOURY-ESNAULT, N. & VACELET, J. (1990) A revision of the
supraspecific classification of the subclass Calcinea (Porifera, class Calcarea). Bulletin du
Muséum National d’Histoire Naturelle, Paris, Zoologie, 2:243–246.
BOROJEVIC, R., BOURY-ESNAULT, N. & VACELET, J. (2000) A revision of the
supraspecific classification of the subclass Calcaronea (Porifera, class Calcarea). Zoosystema,
22: 203–263.
BOROJEVIC, R. & PEIXINHO, S. (1976) Éponges calcaires des côtes nord et nord-est du
Brésil. Bulletin du Muséum d’histoire Naturelle de Paris, 402: 987–1036.
BOSCHI, E. (2000) Species of decapod crustaceans and their distribution in the American
marine zoogeographic provinces. Revista de Investigación y Desarrollo Pesquero 13: 7–136.
BOURY-ESNAULT, N., ERESKOVSKY, A., LAZOSKI, C., LAPORT, M., HARDOIM, C.,
SOLÉ-CAVA, A.M. & ZILBERBERG, C. Biodiversity and phylogeography of Chondrilla along
a N-S gradient in the Tropical Western Atlantic: an integrative approach. Program and Book of
Abstracts. II Workshop ABC/CNRS (LIA-MARRIO).
239
BOWEN, B.W., BASS, A.L., MUSS, A.J., CARLIN, J. & ROBERTSON, D.R. (2006)
Phylogeography of two Atlantic squirrelfishes (family Holocentridae): exploring pelagic larval
duration and population connectivity. Marine Biology, 149, 899– 913.
BOWERBANK, J.S. (1862) On the Anatomy and Physiology of the Spongiadae. Part II.
Philosophical Transactions of the Royal Society, 152(2): 747-829
BRADBURY, I. R. & BENTZEN, P. (2007) Non-linear genetic isolation by distance:
implications for dispersal estimation in anadromous and marine fish populations. Marine
Ecology Progress Series 340: 245–257.
BREITFUSS, L. (1932) Die Kalkschwammfauna des arktischen Gebietes. Fauna Arctica, 6,
235–252.
BREITFUSS, L. (1898c) Die Kalkschwämme der Sammlung Plate. (Fauna Chilensis, I).
Zoologische Jahrbücher Suppl., 4, 455–470.
BRIGGS., J.C. (1974) Marine zoogeography. New York: McGraw-Hill.
BRIGGS, J.C. (1995) Global biogeography. Amsterdam: Elsevier.
BRIGGS, J.C. & BOWEN, B.W. (2012) A realignment of marine biogeographic provinces with
particular reference to fish distributions. Journal of Biogeography 39: 12–30.
BURKE, K., FOX, P.J. & $ENGÖR, A.M.C. (1978) Buoyant Ocean Floor and the Evolution of
the Caribbean . Journal of Geophysical Research, 83(138): 3949-3954.
CAVALCANTI, F.F.; BASTOS, N. & LANNA, E. (2015) Two new species of the genus
Vosmaeropsis Dendy, 1892 (Porifera, Calcarea), with comments on the distribution of V. sericata
(Ridley, 1881) along the Southwestern Atlantic Ocean. Zootaxa 3956 (4): 476–490.
CAVALCANTI, F. & KLAUTAU, M. (2011) Solenoid: a new acquiferous system to Porifera.
Zoomorphology, 130(4): 255-260
240
CAVALCANTI, F.F.; MENEGOLA, C. & LANNA, E. (2014) Three new species of the genus
Paraleucilla Dendy, 1892 (Porifera, Calcarea) from the coast of Bahia State, Northeastern
Brazil. Zootaxa 3764(5):537–554.
CAVALCANTI, F.F., RAPP, H.T. & KLAUTAU, M. (2013) Taxonomic revision of Leucascus
Dendy, 1892 (Porifera: Calcarea) with revalidation of Ascoleucetta Dendy & Frederick, 1924
and description of three new species. Zootaxa, 3619 (3), 275–314.
CARTER, H.J. (1886). Descriptions of Sponges from the Neighbourhood of Port Phillip Heads,
South Australia, continued. Annals and Magazine of Natural History 17 : 34- 149. Descriptions
of sponges from the neighbourhood of Port Phillip Heads. Annals and Magazine of Natural
History, 18
CARTER, H.J. (1890) Porifera. Notes on the Zoology of Fernando de Noronha. Journal of the
Linnean Society, 20, 564–569.
CHAVES-FONNEGRA, A.; FELDHEIM, K.A.; SECORD, J. & LOPEZ, J. V. (2015).
Population structure and dispersal of the coral excavating sponge Cliona delitrix. Molecular
Ecology, 24: 1447-1466.
CHÉRUBIN, L.M. & RICHARDSON, P.L. (2007) Caribbean current variability and the
influence of the Amazon and Orinoco freshwater plumes . Deep Sea Research Part I:
Oceanographic Research Papers 54(9): 1451-1473.
COELHO-SOUZA, S.A., LÓPEZ, M.S., DAVEE GUIMARÃES, J.R., COUTINHO,R.,
CANDELLA, R.N. (2012) Biophysical interactions in the Cabo Frio upwelling system,
Southeastern Brazil. Brazilian Journal of Oceanography, 60(3): 353-365.
COLL, M., PIRODDI, C., STEENBEEK, J., KASCHNER, K., LASRAM, F.B.R., AGUZZI, J.,
BALLESTEROS, E., BIANCHI, C.N., CORBERA, J., DAILIANIS, T., DANOVARO, R.,
ESTRADA, M., FROGLIA, C., GALIL, B.S., GASOL, J.M., GERTWAGEN, R., GIL, J.,
GUILHAUMON, F., KESNER-REYES, K., KITSOS, M.-S., KOUKOURAS, A.,
241
LAMPADARIOU, N., LAXAMANA, E., LÓPEZ-FÉ DE LA CUADRA, C.M., LOTZE, H.K.,
MARTIN, D., MOUILLOT, D., ORO, D., RAICEVICH, S., RIUS-BARILE, J., SAIZ-
SALINAS, J.I., SAN VICENTE, C., SOMOT, SAMUEL, TEMPLADO, J., TURON, X.,
VAFIDIS, D., VILLANUEVA, R., VOULTSIADOU, E. The Biodiversity of the Mediterranean
Sea: Estimates, Patterns, and Threats . PLoS ONE 5(8): e11842.
COLLETTE, B.B. & RÜTZLER, K. (1977) Reef fishes over sponge bottoms off the mouth of
the Amazon River. Third International Coral Reef Symposium: 305–310.
CÓNDOR-LUJÁN, B. & KLAUTAU, M. (2016) Nicola gen. nov. with redescription of Nicola
tetela (Borojevic & Peixinho, 1976) (Porifera: Calcarea: Calcinea: Clathrinida). Zootaxa, 4103,
230–238.
COWEN, R.K., LWIZA, K.M.M, SPONAUGLE, S., PARIS, C.B. & OLSON, D.B. (2000)
Connectivity of marine populations: open or closed? Science 287:857–59.
COWEN, R.K., PARIS, C.B. & SRINIVASAN, A. (2006) Scaling of connectivity in marine
populations. Science 311:522–27.
COWEN, R. K. & SPONAUGLE, S. (2009) Larval Dispersal and Marine Population
Connectivity. The Annual Review of Marine Science, 1:443–66
DE BAKKER, D.M., MEESTERS. E.H.W.G, VAN BLEIJSWIJK, J.D.L, LUTTIKHUIZEN,
P.C., BREEUWER, H.J.A.J., BECKING, L.E. (2016) Population genetic structure, abundance,
and health status of two dominant benthic species in the Saba Bank National Park, Caribbean
Netherlands: Montastraea cavernosa and Xestospongia muta. PLoS ONE, 11(5): e0155969.
DEBIASSE, M. B.; RICHARDS, V. P.; SHIVJI, M. S. (2010) Genetic assessment of
connectivity in the common reef sponge, Callyspongia vaginalis (Demospongiae:
Haplosclerida) reveals high population structure along the Florida reef tract. Coral Reefs, 29:
47–55.
242
DEBIASSE, M. B.; RICHARDS, V. P.; SHIVJI, M. S. & HELLBERG, M. E. (2016) Shared
phylogeographical breaks in a Caribbean coral reef sponge and its invertebrate commensals.
Journal of Biogeography.43: 2136–2146.
JASPER M. DE GOEIJ, J.M., OEVELEN, D.,VERMEIJ, M.J.A, OSINGA, R.,
MIDDELBURG, J.J., DE GOEIJ, A.F.P.M., ADMIRAAL, W. (2013) Surviving in a Marine
Desert: The Sponge Loop Retains Resources Within Coral Reefs. Science, 342 (6154): 108-110.
DE LAUBENFELS, M.W. (1936) A Discussion of the Sponge Fauna of the Dry Tortugas in
Particular and the West Indies in General, with Material for a Revision of the Families and
Orders of the Porifera. Carnegie Institute of Washington Publication, 467, 1–225.
DE LAUBENFELS, M.W. (1950) The Porifera of the Bermuda Archipelago. Transactions of
the Zoological Society of London, 27 (1), 1–154.
DENDY, A. (1892) Synopsis of the Australian Calcarea Heterocoela, with a proposed
classification of the group and descriptions of some new genera and species. Proceedings of the
Royal Society of Victoria, 5, 69–116.
DENDY, A. (1893). On a New Species of Leucosolenia from the neighbourhoodof Port Phillip
Heads. Proceedings of the Royal Society of Victoria(New Series) 5: 178-180.
DENDY, A. & FREDERICK, L. M. (1924). On a Collection of Sponges from the Abrolhos
Islands, Western Australia. Journal of the Linnean Society. Zoology. 35: 477-519, pls 25-26.
DENDY, A. & ROW, R.W.H. (1913) The classification and phylogeny of the calcareous
sponges, with a reference list of all the described species, systematically arranged. Proceedings
of the Zoological Society of London, 3, 704– 813.
DOMINGUEZ, J.M.L., MARTIN, L. & BITTENCOURT, A.C.S.P. (1983) Sea-level history and
Quaternary evolution of river mouth-associated beach-ridge plains along the east-southeast
Brazilian coast: a summary. The Society of Economic Paleontologists and Mineralogists
243
DOMINGOS, C., LAGE, A. & MURICY, G. (2016) Overview of the biodiversity and
distribution of the Class Homoscleromorpha in the Tropical Western Atlantic . Marine
Biological Association of the United Kingdom, 96(2), 379 –389.
DUCHASSAING DE FONBRESSIN, P. & MICHELOTTI, G. (1864). Spongiaires de la mer
Caraibe. Natuurkundige verhandelingen van de Hollandsche maatschappij der wetenschappen
te Haarlem. 21(2): 1-124.
DRAPER, G., JACKSON, T.A. & DONOVAN, S.K. (1994) Geologic Provinces of the
Caribbean Region . In: Donovan & Jackson (eds). Caribbean Geology. An Introduction. The
University of the West Indies Publishers' Association (UWIPA), Jamaica, 3 - 12 pp.
ERESKOVSKY, A.V. (2010) The Comparative Embryology of Sponges. Springer-Verlag,
Dordrecht Heidelberg London.
ERESKOVSKY, A.V. & WILLENZ, P. (2008) Larval development in Guancha arnesenae
(Porifera, Calcispongiae, Calcinea). Zoomorphology, 127: 175-187.
EKMAN, S. 1953. Zoogeography of the sea. Londres, Sidwick & Jackson (ed.)
FLOETER, S.R., ROCHA, L. A., ROBERTSON, D. R., JOYEUX, J. C., SMITH-VANIZ, W. F.,
WIRTZ, P., EDWARDS, A. J., BARREIROS, J. P., FERREIRA, C. E. L., GASPARINI, J. L.,
BRITO, A., FALCÓN, J.M. BOWEN, B. W. & BERNARDI, G. (2008) Atlantic reef fish
biogeography and evolution. Journal of Biogeography, 35:22–47
J. FIGUEIREDO, C. HOORN, P. VAN DER VEN, E. SOARES. (2009) Late Miocene onset of
the Amazon River and the Amazon deep-sea fan: Evidence from the Foz do Amazonas Basin.
Geology 37, 619 -622.
FLOETER, S.R. & GASPARINI, J.L. (2000) The southwestern Atlantic reef fish fauna:
composition and zoogeographic patterns . Journal of Fish Biology 56, 1099–1114.
FLOETER, S.R. & SOARES-GOMEZ, A. (1999) Biogeographic and Species Richness Patterns
of Gastropoda on the Southwestern Atlantic . Revista Brasiliera de Biologia, 59(4): 567-575.
244
FROMONT, J., HUGGETT, M.J., LENGGER, S.K., GRICE, K. & SCHÖNBERG, C.H.L.
(2016). Characterization of Leucetta prolifera, a calcarean cyanosponge from south-western
Australia, and its symbionts, Journal of the Marine Biological Association of the United
Kingdom, 96(2), pp. 541–552.
FRY, W.G. (1971). The biology of larvae of Ophlitaspongia seriata from two Nord Wales
populations. In: Crisp, D.J. (Eds.). Proceedings of 4th European Marine Biology Symposium,
Bangor, North Wales, UK, Cambridge University Press, Cambridge. pp. 155–178.
GAGNAIRE, P.-A., BROQUET, T, AURELLE, D., VIARD, F., SOUISSI, A., BONHOMME,
F., ARNAUD-HAOND, S. & BIERNE, N. 2015. Using neutral, selected, and hitchhiker loci to
assess connectivity of marine populations in the genomic era . Evolutionary Applications, 8(8):
769-786.
GARCÍA-HERNÁNDEZ, J.E., DE VOOGD, N.J., VAN SOEST, R.W.M. (2015) Sponges
(Porifera) of St. Eustatius . In: Marine biodiversity survey of St. Eustatius, Dutch Caribbean.
Hoeksema, B.W. (eds). Naturalis Biodiversity Center, Leiden, and ANEMOON Foundation.
HAECKEL, E. (1870) Prodromus eines Systems der Kalkschwämme. Jenaische Zeitschrift für
Medicin und Naturwissenschaft, 5, 176–191.
HAECKEL, E. (1872) Die Kalkschwämme. Eine Monographie in zwei Bänden Text und einem
Atlas mit 60 Tafeln Abbildungen. G. Reimer, Berlin, 1: 484pp, 2: 418 pp, 3: 60 pp.
HELFMAN, G.S., COLLETTE, B.B. & FACEY, D.E. (1997) The diversity of fishes, p. 528.
Blackwell Science, Inc., Malden, MA.
HELLBERG, M.E. (2009) Gene Flow and Isolation among Populations of Marine Animals .
Annual Review of Ecology, Evolution, and Systematics, 40:291–310.
HECHTEL, G.J. (1976) Zoogeography of Brazilian marine Demospongiae. In: Harrison, F.W. &
Cowden, R.R. (Eds.), Aspects of sponge biology. Academic Press, New York, pp. 237–259.
245
HOOPER, J. & VAN SOEST, R.W.M. 2002. Systema Porifera: A Guide to the Classification of
Sponges. Kluwer Academic/Plenum Publishers, New York.
HÔZAWA, S. (1916) On some Japanese calcareous sponges belonging to the family
Heteropiidae. Journal of the College of Sciences, Imperial University of Tokyo, 38, 1–41.
HÔZAWA, S. (1918) Reports on the calcareous sponges collected during 1906 by the United
States Fisheries Steamer Albatross in the Northwestern Pacific. Proceedings of the United
States National Museum 54: 525-556.
HÔZAWA, S. (1929) Studies on the calcareous sponges of Japan. Journal of the Faculty of
Sciences, Imperial University of Tokyo, 1, 277–389.
HÔZAWA, S. (1933) Report on the calcareous sponges obtained by the survey of the
Continental Shelf bordering on Japan. Science Reports of the Tohoku Imperial University 8: 1-
20.
HÔZAWA, S. (1940) Report on the calcareous sponges obtained by the Zoological Institute and
Museum
of Hamburg, Part I. Science Reports of the Tôhoku Imperial University, Series IV, 15(2), 131–
163.
HÔZAWA, S. & Tanita (1941) The fauna of Akkeshi Bay: XII Calcarea. Journal of the Faculty
Science Hokkaido Imperial University (V1, Zoology) 7 (4): 421-429.
IMESEK, M., PLESE, B., PFANNKUCHEN, M., GODRIJAN, J., PFANNKUCHEN, D.M.,
KLAUTAU, M. & CETKOVIC, H. (2014) Integrative taxonomy of four Clathrina species of
the Adriatic Sea, with the first formal description of Clathrina rubra Sarà, 1958. Organisms
Diveristy & Evolution, 14, 21–29.
JANUSSEN, D. & RAPP, H.T. (2011) Redescription of Jenkina articulata Broendsted from the
deep Eckström Shelf, E-Weddell Sea, Antarctica and a comment on the possible mass
occurrence of this species. Deep-Sea Research II, 58: 2022-2026.
246
JOHNS, W.E., TOWNSEND, T.L., FRATANTONI, D.M. & WILSON, W.D. (2002). On the
Atlantic inflow to the Caribbean Sea. Deep-Sea Research I, 49: 211–243.
JONES, G.P., PLANES, S. & THORROLD, S.R. (2005) Coral Reef Fish Larvae Settle Close to
Home. Current Biology 15: 1314–1318.
KELLY, M.; EDWARDS, A.R.; WILKINSON, M.R.; ALVAREZ, B.; COOK, S. DE C.;
BERGQUIST, P.R.; BUCKERIDGE, ST J.; CAMPBELL, H.J.; REISWIG, H.M.;
VALENTINE, C. & VACELET, J. (2009). Phylum Porifera: sponges. in: Gordon, D.P. (Ed.)
(2009). New Zealand inventory of biodiversity: 1. Kingdom Animalia: Radiata,
Lophotrochozoa, Deuterostomia. pp. 23-46.
KINDER, T.H., HEBURN, G.W. & GREEN, A.W. (1985) Some aspects of the Caribbean
circulation. Marine Geology 68, 25–52.
KLAUTAU, M. (2016) Capítulo 7 Porifera. In: Zoologia dos Invertebrados. Adilson Fransozo e
Maria Lucia Negreiros-Fransozo (eds). Editora Guanabara Koogan, Rio de Janeiro.
KLAUTAU, M., SOLÉ-CAVA, A. & BOROJEVIC, R. (1994) Biochemical systematics of
sibling sympatric species of Clathrina (Porifera, Calcarea). Biochemical Systematics and
Ecology, 22, 367–375.
KLAUTAU, M. & BOROJEVIC, R. (2001) Sponges of the genus Clathrina Gray, 1867 from
Arraial do Cabo, Brazil. Zoosystema, 23 (3), 395–410.
KLAUTAU, M. & VALENTINE, C. (2003) Revision of the Genus Clathrina (Porifera,
Calcarea). Zoological Journal of the Linnean Society, 139:1-62.
KLAUTAU, M. ;AZEVEDO, F.; CÓNDOR-LUJÁN, B.; RAPP, H.T.; COLLINS, A. & RUSSO,
C.A.M. (2013). A molecular phylogeny for the Order Clathrinida rekindles and refines
Haeckel’s taxonomic proposal for calcareous sponges. Integrative and Comparative Biology
53(3):447-461.
247
KLAUTAU, M., IMESEK, M., AZEVEDO, F.C., PLESE, B., NIKOLIC, V. & CÉTKOVIC, H.
(2016) Adriatic calcarean sponges (Porifera, Calcarea) with description of six new species and
richness analysis. European Journal of Taxonomy, 178, 1–52.
Koechlin, N. (1977) Installation d’une épifaune á Spirographis spallanzani Viviani, Sycon
ciliatum Fabricius et Ciona intestinalis (L.) dans le port de plaisance de Lézardrieux (Cotes du
Nord). Cahiers De Biologie Marine,18: 325–337.
LANNA, E.; CAVALCANTI, F.; CARDOSO, L.; MURICY, G. & KLAUTAU, M. (2009).
Taxonomy of calcareous sponges (Porifera, Calcarea) from Potiguar Basin, NE Brazil. Zootaxa
1973: 1–27
LANNA, E., ROSSI, A.L., CAVALCANTI, F.F., HAJDU, E. & KLAUTAU, M. (2007)
Calcareous sponges from São Paulo state, Brazil (Porifera: Calcarea: Calcinea) with the
description of two new species. Journal of the Marine Biological Association of the United
Kingdom, 87, 1553–1561.
LAZOSKI, C.; SOLÉ-CAVA, A.M.; BOURY-ESNAULT, N.; KLAUTAU, M. & RUSSO, C.
2001. Cryptic speciation in hight gene flow scenario in the oviparous marine sponge Chondrosia
reniformis. Marine Biology, 139: 421-429.
LATRUBESSE, E., COZZUOL, M., DA SILVA-CAMINHA, S.A.F., RIGSBY, C.A., ABSY,
M.L. & JARAMILLO, C. (2010) The Late Miocene paleogeography of the Amazon Basin and
the evolution of the Amazon River system. Earth-Science Reviews 99(3–4): 99–124.
LEHNERT, H. & VAN SOEST, R.W.M. (1998) Shallow water sponges of Jamaica. Beaufortia,
48 (5), 71–103.
LENDENFELD, R. von (1885) A monograph of the Australian sponges. Part III. The
Calcispongiae. Proceedings of the Linnean Society of New South Wales, 4, 1083–1150.
LEYS, S. & RIESGO, A. (2012). Epithelia, an Evolutionary Novelty of Metazoans. Journal of
Experimental Zoology Part B Molecular and Developmental Evolution, 318(6):438-47.
248
López-Legentil, S. & Pawlik, J.R. 2009. Genetic structure of the Caribbean giant barrel
sponge Xestospongia muta using the I3-M11 partition of COI. Coral Reefs, 28:157–165.
LOWE, W. & ALLENDORF, A. (2010) What can genetics tell us about population
connectivity? Molecular Ecology, 19: 3038–3051.
MAGALHAES, G.M., AMADO-FILHO, G.M., ROSA, M.R., MOURA, R.L., BRASILEIRO,
P., MORAES, F.C., FRANCINI-FILHO , R.B., PEREIRA-FILHO, G.H. (2015) Changes in
benthic communities along a 0–60 m depth gradient in the remote St. Peter and St. Paul
Archipelago (Mid-Atlantic Ridge, Brazil). Bulletin of Marine Science, 91(3):377–396.
MALDONADO, M. (2006) The ecology of the sponge larva. Canadian Journal of Zoology, 84:
175–194.
MALDONADO, M. & RIESGO, A. (2008). Reproduction in the phylum Porifera: A synoptic
overview. Treballs de la Societat Catalana de Biologia 59: 29-49.
MANUEL, M.; BOROJEVIC, R.; BOURY-ESNAULT, N. & VACELET, J. (2002) Class
Calcarea
Bowerbank, 1864. In: HOOPER, John and VAN SOEST, Rob (eds.) Systema Porifera. A guide
to
the classification of sponges. Nueva York, Estados Unidos, Klumer Academic / Publishers.
2002, p.
1103 -1110. ISBN: 0306472600.
MATTHEWS, J.E. & HOLCOMBE, T.L. (1985) Venezuela Basin of the Caribbean Sea -
stratigraphy and sediment distribution. Marine Geology 68, 1–23.
MELLO-LEITÃO, A., PÊGO, A.F. & LOPES, W.M. (1961) Poríferos assinalados no Brasil.
Avulsos do Centro de Estudos Zoológicos, 10, 1–29.
MESCHEDE, M. & FRISCH, W. (1998) A plate-tectonic model for the Mesozoic and Early
Cenozoic history of the Caribbean plate. Tectonophysics 296: 269–291
249
MIKLUCHO-MACLAY, N. (1868) Beiträgezur Kenntniss der Spongien I. Jenaische Zeitschrift
für Medicin und Naturwissenschaft, 4, 221–240.
MILOSLAVICH P., DÍAZ J.M., KLEIN E., ALVARADO J.J., DÍAZ C., GOBIN J., ESCOBAR-
BRIONES E., CRUZ-MOTTA J.J., WEIL E., CORTÉS J., BASTIDAS A.C., ROBERTSON R.,
ZAPATA F., MARTÍN A., CASTILLO J., KAZANDJIAN, A. AND ORTIZ M. (2010) Marine
Biodiversity in the Caribbean: Regional Estimates and Distribution Patterns. PLoS ONE 5(8),
e11916.
MOLINARI, R. L., SPILLANE, I. BROOKS, D., ATWOOD, D. & DUCKETT, C. (1981)
Surface currents in the Caribbean Sea as deduced from Lagrangian observations. Journal of
Geophysical Research., 86, 6537– 6542.
MORAES, F.C. (2011) Esponjas das Ilhas Oceânicas Brasileiras. Museu Nacional, Série Livros
44, Rio de Janeiro, 252 pp.
MORAES, F.C., VENTURA, M., KLAUTAU, M., HAJDU, E.C.M. & MURICY, G. (2006)
Biodiversidade de Esponjas das Ilhas Oceânicas Brasileiras. In: Alves, R.J.V. & Castro, J.W.A.
(Eds.). Ilhas Oceânicas Brasileiras - da pesquisa ao manejo. Ministério do Meio Ambiente,
Brasília, pp. 1–298.
MOURA, R.L., SECCHIN, N.A., AMADO-FILHO, G.M., FRANCINI-FILHO, R.B.,
FREITAS, M.O., MINTE-VERA, C.V., TEIXEIRA, J.B., THOMPSON, F.L., DUTRA, G.F.,
SUMIDA, P.Y.G., GUTH, A.Z., LOPES, R.M. & BASTOS, A.C. (2013) Spatial patterns of
benthic mega habitats and conservation planning in the Abrolhos Bank. Continental Shelf
Research, 70, 109–117.
MOURA, R.L, AMADO-FILHO, G.M., MORAES, F.C., BRASILEIRO, P.S., SALOMON,
P.S., MAHIQUES, M.M., BASTOS, A.C., MARCELO, G. ALMEIDA, SILVA, J.M., ARAUJO,
B.F., BRITO, F.P., RANGEL, T.P., OLIVEIRA, B.C.V., BAHIA, R.G., PARANHOS, R.P.,
DIAS, R.J.S., SIEGLE, E., FIGUEIREDO, A.G., PEREIRA, R.C., LEAL, C.V., HAJDU, E.,
250
ASP, N.E., GREGORACCI, G.B., NEUMANN-LEITÃO, S., YAGER, P.L., FRANCINI-
FILHO, R.B., FRÓES, A., CAMPEÃO, M., SILVA, B.S.S., MOREIRA, A.P.B., OLIVEIRA, L.,
SOARES, A.C., ARAUJO, L., OLIVEIRA, N.A., TEIXEIRA, J.B., VALLE, R.A.B.,
THOMPSON, C.C., REZENDE, C.E., THOMPSON, F.L. (2016) An extensive reef system at
the Amazon River mouth. Science Advances, 2 (4): e1501252.
MURICY, G.; LOPES, D.A.; HAJDU, E.; CARVALHO, M.S.; MORAES, F.C.; KLAUTAU,
M.; MENEGOLA, C. & PINHEIRO, U. (2011) Catalogue of Brazilian Porifera. Série Livros 46,
Museu Nacional, Rio de Janeiro, 300 pp.
MURICY, G., HAJDU, E., CUSTÓDIO, M., KLAUTAU, M., RUSSO, C.A.M. & PEIXINHO,
S. (1991) Sponge distribution at Arraial do Cabo, SE Brazil. Proceedings of the VII Symposium
on Coastal and Ocean Management. Long Beach: ASCE Publications, 2, 1183–1196.
MURICY, M. & SILVA, O.C (1999) Esponjas marinhas do Estado do Rio de Janeiro: um
recurso renovável inexplorado. In: Silva, S.H.G., Lavrado, H.P. (Eds.), Ecologia dos Ambientes
Costeiros do Estado do Rio de Janeiro. Série Oecologia Brasiliensis 7, pp.155–178.
O’DEA, A., LESSIOS, H.A., COATES, A.G., EYTAN, R.I., RESTREPO-MORENO, S.A.,
CIONE, A.L., COLLINS, L.S., DE QUEIROZ, A., FARRIS, D.W., NORRIS, R.D.,
STALLARD, R.F., WOODBURNE, M.O., AGUILERA, O., AUBRY, M.-P., BERGGREN,
W.A., BUDD, A.F., COZZUOL, M.A., COPPARD, S.E., DUQUE-CARO, H., FINNEGAN, S.,
GASPARINI, G.M., GROSSMAN, E.L., JOHNSON, K.G., KEIGWIN, L.DL., KNOWLTON,
N., LEIGH, E.G., LEONARD-PINGEL, J.S., MARKO, P.B., PYENSON, N.D., RACHELLO-
DOLMEN, P.G., SOIBELZON, E., SOIBELZON, L., TODD, J.A., VERMEIJ, G.J.,
JACKSON, J.B.C. (2016) Formation of the Isthmus of Panama. Science Advances, 2(8):
e1600883
O´DEA, A., RODRÍGUEZ, F., DE GRACIA, C., COATES, A.G. 2007. Patrimonio
paleontológico, La paleontología marina en el Istmo de Panamá. Canto Rodado 2: 149 – 179.
251
PADUA, A. Q. (2012). Estudo da conectividade de populações de Clathrina aurea (Porifera:
Calcarea) na costa do Brasil. Dissertação (Mestrado) – UFRJ/MN/Programa de Pós-graduação
em Ciências Biológicas (Zoologia).
PAUDA, A., LANNA, E. & KLAUTUA, M. (2013) Macrofauna inhabiting the sponge
Paraleucila magna (Porifera: Calcarea) in Rio de Janeiro, Brazil. Journal of the Marine
Biological Association of the United Kingdom, 93(4), 889–898.
PALACIO, F. J. (1982) Revisión zoogeográfica marina del sur del Brasil. Boletim do Instituto
Oceanográfico 31(1):69-92.
PARIS, CB, CHÉRUBIN, LM, COWEN, RK (2007) Surfing, spinning, or diving from reef to
reef: effects on population connectivity. Marine Ecology Progress Series 347:285-300.
PERDICARIS S, VLACHOGIANNI T, VALAVANIDIS A (2013) Bioactive Natural Substances
from Marine Sponges: New Developments and Prospects for Future Pharmaceuticals. Natural
Products Chemistry Research 1:114.
PINDELL, J.L. (1994) Evolution of the Gulf of Mexico and the Caribbean. In: Donovan, S.K.,
Jackson, T.A. (Eds.), Caribbean Geology: An Introduction, U.W.I. Publ. Ass. Kingston, pp. 13–
39
POLÉJAEFF, N. (1883) Report on the Calcarea dredged by H.M.S.‘Challenger’, during the
years 1873-1876. Report on the Scientific Results of the Voyage of H.M.S. ‘Challenger’, 1873-
1876. Zoology, 8 (2), 1–76.
Rapp, H.T. (2006) Calcareous sponges of the genera Clathrina and Guancha (Calcinea,
Calcarea, Porifera) of Norway (north-east Atlantic) with the description of five new species.
Zoological Journal of the Linnean Society 147 (3): 331-365.
RAPP, H.T.; GÖCKE, C.; TENDAL, O.S.; JANUSSEN, D. (2013) Two new species of
calcareous sponges (Porifera: Calcarea) from the deep Antarctic Eckström Shelf and a revised
list of species found in Antarctic waters. Zootaxa 3692 (1): 149-159.
252
RAPP, H.T.; JANUSSEN, D. & TENDAL, O. (2011) Calcareous sponges from abyssal and
bathyal depths in the Weddell Sea, Antarctica. Deep-Sea Research II, 58: 58-67.
RICHARDS, V.P.; BERNARD, A.; FELDHEIM, K & SHIVJI, M.S. (2016) Patterns of popu -
lation structure and dispersal in the long-lived “redwood” of the coral reef, the giant barrel
sponge (Xestospongia muta). Coral Reefs, 35(3):1097–1107.
RIDLEY, S.O. (1881) Spongida collected during the Expedition of H.M.S Alert in the Straits of
Magellan and on the coast of Patagonia. Proc. zool. Soc. Lond.: 107-139.
ROCHA, L.A. (2003) Patterns of distribution and processes of speciation in Brazilian reef
fishes. Journal of Biogeography, 30: 1161–1171.
ROCHA, L.A.; BASS, A.L., ROBERTSON, R. & BOWEN, B.W. (2002) Adult habitat
preferences, larval dispersal, and the comparative phylogeography of three Atlantic
surgeonfishes (Teleostei: Acanthuridae). Molecular Ecology, 11:243-252.
ROSSI, A.L., RUSSO, C.A.M., SOLÉ-CAVA, A.M., RAPP, H.T. & KLAUTAU, M. (2011)
Phylogenetic signal in the evolution of body colour and spicule skeleton in calcareous sponges.
Zoological Journal of the Linnean Society, 163 (4), 1026–1034.
ROW, R.W.H. & HÔZAWA, S. (1931) Report on the Calcarea obtained by the Hamburg South-
West Australian Expedition of 1905. Science Reports of the Tôhoku University, 6(1), 727–809.
ROZEMEIJER, M; DULFER, W. (1987) A quantitative analysis of the cryptofauna of the Santa
Marta area (Colombia). Report of the University of amsterdam. 100 pp.
SARÀ, M. & GAINO, E. (1971) Sycon vigilans, nuova specie di Calcispongiae dal littorale
ligure (Porifera). Bolletino dei Musei e degli Istituti Biologici della Universitá di Genova.
9(268): 21-28.
SCHIZAS, N. (2012) Misconceptions regarding nuclear mitochondrial pseudogenes (Numts)
may obscure detection of mitochondrial evolutionary novelties. Aquatic Biology 17: 91–96
253
SCHUFFNER, O. (1877) Beschreibung einiger neuer Kalkschwämme. Jenaische Zeitschrift für
Naturwissenschaft 11: 403-433.
SELKOE, K.A., WATSON, J.R., WHITE, C., HORIN, T.B., IACCHEI, M., MITARAI, S.,
SIEGEL, D.A., STEVEN, G.D., & TOONEN, R.J. (2010) Taking the chaos out of genetic
patchiness: seascape genetics reveals ecological and oceanographic drivers of genetic patterns in
three temperate reef species. Molecular Ecology, 19:3708–3726.
SETON, M., MÜLLER, R.D., ZAHIROVIC, S., GAINA, C., TORSVIK,T., SHEPHARD, G.,
TALSMA, A., GURNIS, M. TURNER, M., MAUS, S., CHANDLER, M. (2012) Global
continental and ocean basin reconstructions since 200 Ma M. Earth-Science Reviews 113: 212–
270.
SILVEIRA, I.C.A, MIRANDA, L.B. & BROWN W.S. On the origins of the North Brazil
Current. (1994) Journal of Geophysical Research, 99(11): 22501–12.
SOARES, M., DA CRUZ LOTUFO, T.M., VIEIRA, L.M., SALANI, S., HAJDU, E.,
MATTHEWS- CASCON, H., LEÃO, Z.M.A.N. & KIKUCHI, R.K.P. (2016) Brazilian Marine
Animal Forests: A New World to Discover in the Southwestern Atlantic . Springer International
Publishing. S. Rossi (ed.)
SOLÉ-CAVA, A.M., KLAUTAU, M., BOURY-ESNAULT, N., BOROJEVIC, R. & THORPE, J.
(1991) Genetic evidence for cryptic speciation in allopatric populations of two cosmopolitan
species of the calcareous sponge Clathrina. Marine Biology, 111, 381–386.
SPALDING, M.D., FOX, H.E., ALLEN, G.R., DAVIDSON, N., FERDAÑA, Z.A.,
FINLAYSON, M., HALPERN, B.S., JORGE, MA, LOMBANA, A., LOURIE, S.A., MARTIN
K.D., MCMANUS, E., MOLNAR, J., RECCHIA, C.A. & ROBERTSON, J. (2007) Marine
ecoregions of the world: a bioregionalization of coastal and shelf areas. Bioscience 57, 573–583.
STRAMMA, L. 1991. Geostrophic transport of the South Equatorial Current in the Atlantic.
Journal of Marine Research, 49(2): 281–94.
254
SWEARER, S.E., THORROLD, S.R., SHIMA, J.S., HELLBERG, M.E., JONES, G.P.,
ROBERTSON, D.R., SELKOE, K.A., RUIZ, G.M., MORGAN, S.G., & WARNER, R.R.
(2002). Evidence of self-recruitment in demersal marine populations. Bulletin of Marine
Science 70, 251–272.
TANITA, S. (1942) Report on the calcareous sponges obtained by the Zoological Institute and
Museum of Hamburg. Part II. Science Reports of the Tôhoku University, (4) 17 (2), 105–135.
TANITA, S. (1943) Studies on the Calcarea of Japan. Science Reports of the Tohoku Imperial
University, 17, 353– 490.
THACKER, A. G. (1908) On collections of the Cape Verde Islands fauna made by Cyril
Crossland, M.A. The Calcareous sponges. Proceedings of the Zoological Society of London, 49,
757–782.
THORROLD, S., ZACHERL, D., & LEVIN, L.A. (2007) Population Connectivity and Larval
Dispersal. Using Geochemical signatures in Calcified Structures. Oceanography, 20(3): 80-89.
TOPSENT, E. (1892). Contribution à l’étude des Spongiaires de l’Atlantique Nord (Golfe de
Gascogne, Terre-Neuve, Açores). Résultats des campagnes scientifiques accomplies par le
Prince Albert I. Monaco. 2: 1-165, pls I-XI.
VALDERRAMA, D., ROSSI, A.L., SOLÉ-CAVA, A.M., RAPP, H.T. & KLAUTAU, M. (2009)
Revalidation of Leucetta floridana (Haeckel, 1872) (Porifera, Calcarea): a widespread species in
the tropical western Atlantic. Zoological Journal of the Linnean Society, 157 (1), 1–16.
VAN SOEST, R. W. M. (2017). Sponges of the Guyana Shelf. Zootaxa. 4217: 1-225.
VAN SOEST, R.W.M, BOURY-ESNAULT, N., HOOPER, J.N.A., RÜTZLER, K., DE VOOGD,
N.J., ALVAREZ DE GLASBY, B., HAJDU, E., PISERA, A.B., MANCONI, R.,
SCHOENBERG, C., JANUSSEN, D., TABACHNICK, K.R., KLAUTAU, M., PICTON, B.,
KELLY, M., VACELET, J., DOHRMANN, M., DÍAZ, M.C. & CÁRDENAS, P. (2016) World
255
Porifera database. Available from: http://www.marinespecies.org/porifera (accessed on
November 2016).
VAN SOEST, R.W.M.; BOURY-ESNAULT, N.; VACELET, J.; DOHRMANN, M.;
ERPENBECK, D.; DE VOOGD, N.J.; SANTODOMINGO, N.; VANHOORNE, B.; KELLY, M.
& HOOPER, J.N.A. 2012. Global Diversity of Sponge (Porifera). PLoSONE 7(4): e35105.
VAN SOEST, R.W.M; DE VOOGD, N. (2015) Calcareous sponges of Indonesia. Zootaxa
3951(1):1-105.
VAN SOEST, R.W.M. & HAJDU. E. (1997) Marine area relationships from twenty sponge
phylogenies. A comparison of methods and coding strategies. Cladistics. 13:1–20.
VAN SOEST, R.W.M.; MEESTERS, E.H & BECKING, L.E. (2014). Deep-water sponges
(Porifera) from Bonaire and Klein Curaçao, Southern Caribbean. Zootaxa. 3878(5): 401-443.
VOIGT, O. & WÖRHEIDE, G. (2016) A short LSU rRNA fragment as a standard marker for
integrative taxonomy in calcareous sponges (Porifera: Calcarea). Organisms Diversity &
Evolution, 16 (1), 53–64.
VOIGT, O., WÜLFING, E., & WÖRHEIDE, G. (2012). Molecular phylogenetic evaluation of
classification and scenarios of character evolution in calcareous sponges (Porifera, Class
Calcarea).PLoS ONE, 7(3), e33417.
WHITE, C. (2010). Measuring Connections in the Sea: Pushing the Boundaries of Seascape
Genetics at Channel Islands National Park. The George Wright Forum 27 (3): 280–291 © The
George Wright Society.
WHITE, C., SELKOE, K.A., WATSON, J., SIEGEL, D.A., ZACHER, D.C., & TOONEN, R.J.
(2010) Ocean currents help explain population genetic structure . Proceedings of the Royal
Society B, 7;277(1688):1685-94.
256
WILLIAMS, S.T. & KNOWLTON, N. (2001) Mitochondrial pseudogenes are pervasive and
often insidious in the snapping shrimp genus Alpheus. Molecular Biology and Evolution, 18:
1484−1493.
WÖRHEIDE, G. & HOOPER, J.N.A. (1999) Calcarea from the Great Barrier Reef. 1: Cryptic
Calcinea from Heron Island and Wistari Reef (Capricorn-Bunker group). Memoirs of the
Queensland Museum 43:859-891.
WÖRHEIDE, G., NICHOLS, S. & GOLDBERG, J. (2004). Intragenomic variation of the rDNA
internal transcribed spacers in sponges (Phylum Porifera): implications for phylogenetic studies.
Molecular Phylogenetics and Evolution, 33: 816-830.
ZILBERBERG, C. (2006) Reprodução clonal e sistemática molecular de esponjas (Porifera:
Demospongiae). Tese (doutorado) - UFRJ/Departamento de Genética.
APPENDIX 1
Calcareous sponge species recorded in Martinique . C= Cave (dark, semi-dark caves, tunnels, overhangs), M= Mangrove, R= Reef (hard bottoms ingeneral, including coral reefs). Species marked by an * indicate new records for the Eastern Caribbean. Modified from Pérez et al., submitted.
Order Family Species External traits Genbank Accesion Numbers
Habitat
Clathrinida Clathrinidae
Arthuria hirsuta (Klautau & Valentine, 2003) *
White, massive to semi-spherical clathroid body. Hispid.
KX355564 C
Arthuria sp. nov. Yellow, clathrate with water-collecting tube. Soft.
C
Clathrina aurea Solé-Cava, Klautau, Boury-Esnault, Borojevic & Thorpe, 1991*
Light yellow, clathrate with several oscula.
KX355565 -KX355567
C
Clathrina sp. nov. 1* Light yellow, clathrate with water-collecting tube. Soft.
KX355568 -KX355571
C
Clathrina sp. nov. 2* Light yellow, clathrate and thin encrusting. Soft.
KX355572 C
Clathrina sp. White clathrate. CErnstia sp. nov. Lemon yellow clathrate. Soft. C
Leucosolenida Amphoriscidae Leucilla sp. nov. Bright white, tubular with apical osculum. Delicate.
C
Leucilla sp. 1 CLeucilla sp. 2 CAmphoriscus sp. C
Grantiidae Leucandra sp. CLeucettidae Leucetta floridana (Haeckel,
1872)*White, massive with apical osculum. Rough.
KX355573 -KX355575
C, R
Sycettidae Sycon sp. Beige, globular with apical osculum surrounded by crown.
M
APPENDIX 2
Median-Joinig network of Clathrina lutea. ST2 is shared between Caribbean (Saint Martin)and Brazilian specimens of C. lutea. Taken from presentation at II Workshop MARRIO.
APPENDIX 3
Preliminary Median-Joining network of Clathrina mutabilis. ST1 is shared betweenCaribbean (Curaçao, Martinique and Panama) and Brazilian specimens of C. mutabilis.