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The rst internal molecular phylogeny of the animal phylumEntoprocta (Kamptozoa)

Judith Fuchs a, * , Tohru Iseto b , Mamiko Hirose c, Per Sundberg a , Matthias Obst aa Department of Zoology, University of Gothenburg, Box 463, 40530 Gteborg, Swedenb Seto Marine Biological Laboratory, Field Science Education and Research Center, Kyoto University, 459 Shirahama, Nishimuro, Wakayama 649-2211, Japanc Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan

a r t i c l e i n f o

Article history:Received 16 November 2009Revised 5 April 2010Accepted 7 April 2010Available online 14 April 2010

Keywords:SpiraliaLophotrochozoaLoxosomaTrochophoraCycliophoraBayesianCytochrome c oxidase IRibosomal

a b s t r a c t

This article provides the rst molecular phylogenetic study of the enigmatic invertebrate phylum Ento-procta and was designed to resolve the internal phylogenetic relationships of the taxon. The study isbased on partial and combined analyses of the mitochondrial gene cytochrome c oxidase subunit I(COI), as well as the nuclear ribosomal genes 28S rDNA and 18S rDNA. A short morphological charactermatrix was constructed to tracecharacter evolution along the combined molecular phylogenetic tree. Thecombined analyses of all three genes strongly support the monophyly of the phylum Entoprocta and asister group relationship of Entoprocta and Cycliophora, a result which is consistent with a number of previous morphological and molecular assessments. We nd evidence for two separate lineages withinthe Entoprocta, one lineage leading to all recent colonial taxa, Coloniales, another representing the cladeof solitary entoprocts, Solitaria. Our study suggests that Loxosomella is a paraphyletic assembly withregard to the genera Loxomitra , Loxosoma , and Loxocorone . The results imply that the ancestral entoproctwas a solitary, marine organism with an epizoic life style. The groundplan of the entoproct adult stageprobably included a bilobed centralized nervous system, and the larva was assumedly planktonic, witha gut and a ciliated creeping sole.

2010 Elsevier Inc. All rights reserved.

1. Introduction

The enigmatic phylum Entoprocta (Kamptozoa) includesapproximately 180 species to date, of whichmost are marine ( Isetoet al., 2008 ). Few species of the genus Loxosomatoides live in brack-ish waters, and only two species, Urnatella gracilis and Loxosomato-ides sirindhornae , have yet been described from freshwater ( Wood,2005 ). The current classication recognizes two orders withinEntoprocta, the Solitaria and the Coloniales ( Emschermann,1972 ). Within Solitaria, one family and commonly ve genera aredened, while colonial entoprocts comprise three families andsix genera ( Table 1 ). The main characters for distinguishing be-tween entoproct genera are the arrangement of the body muscula-ture, the form of the attachment structure, and the buddingpatterns ( Emschermann, 1985; Iseto, 2002 ). For an overview of entoproct diversity see Fig. 1 .

A large number of solitary entoprocts are commensals of bot-tom dwelling marine animals and are often found inside theirhosts burrows or tubes. Common hosts for entoprocts are inverte-brates, which produce ventilating currents, such as sponges, poly-chaetes, or bryozoans. Almost all members of the genus Loxosoma

live associated with polychaetes ( Nielsen, 1996 ). The ecology of such associations is little investigated, but the hosts seem to pro-vide both water current and protection for their minute symbionts(Iseto, 2005; Nielsen, 1964 ). In contrast to this, some solitary ento-procts are found on non-living substrata and most colonial speciesnaturally occur on various living and non-living, submerged sub-strates ( Iseto, 2003; Wasson, 2002 ).

Entoprocts have two modes of reproduction, sexual reproduc-tion with a larval stage and asexual budding. Buds are releasedfrom the parental calyx (i.e., the body including the tentacles) inthe solitary Loxosomatidae, while the colonial species bud fromthe base of the stalk or from growing tips of stolons ( Nielsen,2001 ). Some solitary species even perform budding from the larvalstage (see below). The entoproct larva is either a creeping-type lar-va that bears a ciliated foot, or a swimming-type larva, muchresembling a trochophore of mollusks or annelids (when deninga trochophore as a larva with a prototroch sensu Rouse, 1999 ). Bothlarval types are found among Solitaria and Coloniales, but a singlespecies typically produces either one or the other type, with onlyfew exceptions ( Nielsen, 1971 ). Most larvae in the solitary genusLoxosomella and in all colonial species settle upon liberation fromthe parent organism and metamorphose into the adult forms ( Niel-sen, 2002 ). Exceptions to this general pattern are found in somesolitary species, which produce larvae that do not metamorphose,

1055-7903/$ - see front matter 2010 Elsevier Inc. All rights reserved.doi: 10.1016/j.ympev.2010.04.009

* Corresponding author.E-mail address: [email protected] (J. Fuchs).

Molecular Phylogenetics and Evolution 56 (2010) 370379

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

j ou r na l hom e pa ge : www.e l s e v i e r. c om / l oc a t e / ym pe v
http://dx.doi.org/10.1016/j.ympev.2010.04.009mailto:[email protected]://www.sciencedirect.com/science/journal/10557903http://www.elsevier.com/locate/ympevhttp://www.elsevier.com/locate/ympevhttp://www.sciencedirect.com/science/journal/10557903mailto:[email protected]://dx.doi.org/10.1016/j.ympev.2010.04.009

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but produce buds instead. However, larval budding is rather theexception in entoprocts and is likely to be the derived mode (seealso Nielsen, 2002 ).

Concerning entoproct phylogenetic relationships, the taxon isstill one of the most enigmatic in the metazoan tree of life. Upontheir rst discoveries, entoprocts were described as special polypsor rotifers and later treated as Bryozoa, together with Ectoprocta(van Beneden, 1845; Ellis, 1755; Pallas, 1774; Sars, 1835 ; and oth-ers). However, ever since their discovery, anatomical differencesbetween entoprocts and all other animal groups were realizedand Nitsche (1870) proposed to separate entoprocts from Bryozoa,and Entoprocta were raised to phylum state in 1888 (Hatschek).The main anatomical and developmental differences betweenentoprocts and ectoprocts are the position of the anus (inside vs.outside the tentacle crown), the cleavage pattern (spiral vs. radial),and the body cavities (acoelomate vs. coelomate). However, theoccurrence of similar features in both phyla, especially in the larvalmorphologies and the life cycles, has nourished the discussionabout a close relationship of entoprocts and ectoprocts until today(Nielsen, 2001 ). More support for an entoproctectoproct relation-ship was added by the description of the enigmatic Cycliophora(Funch and Kristensen, 1995 ), a new animal phylum that was orig-inally described to share features with both entoprocts and ecto-procts. In contrast to this, some more recent microscopicexaminations describe a number of remarkable morphologicalsimilarities of larval entoprocts and basal molluscs, leading theauthors to suggest an entoproct-mollusk clade ( Haszprunar andWanninger, 2008; Wanninger et al., 2007 ).

The inclusion of entoprocts in phylogenetic analyses has so far

lead to various results. Cladistic studies of anatomical features sup-port relationships to molluscs ( Haszprunar, 1996 ), or cycliopho-rans ( Obst, 2003; Srensen et al., 2000; Zravy et al., 1998 ).Phylogenies based on ribosomal genes consolidate the position of entoprocts within Lophotrochozoa ( Giribet et al., 2000; Mackeyet al., 1996; Peterson and Eernisse, 2001; Zravy et al., 1998 ) andsupport a relationship to Cycliophora ( Passamaneck and Halanych,2006 ), and two recent studies recover a group consisting of (Entoprocta + Cycliophora) + Ectoprocta, with high support valuesfor the former clade only ( Mallatt et al., 2010; Paps et al., 2009b ).In accordance with this, recent phylogenomic analyses of Metazoaplace entoprocts within Lophotrochozoa ( Dunn et al., 2008; Papset al., 2009a ). Mitochondrial gene organization of two entoproctsshowed highest similarity to that of mollusks, and phylogeny

reconstruction based on mitochondrial protein coding genesshoweda close relationshipof Entoprocta andPhoronida ( Yokobori

et al., 2008 ). An EST based study suggested a sistergroup relation-ship of entoprocts + ectoprocts ( Hausdorf et al., 2007 ), while one of the most comprehensive analysis to date supports a clade consist-ing of (Entoprocta + Cycliophora) + Ectoprocta, with high supportvalues for the former clade only ( Hejnol et al., 2009 ).

The rather conicting results outlined above can most probablybe resolved by adding more entoproct taxa and/or higher gene

sampling to the datasets of future analyses. However, understand-ing the evolution of entoproct diversity requiresnot only a rm po-sition of the phylum among other protostome phyla, but also asound assessment of the phylogenetic relationships within thephylum. To date, no such analysis exists, and entoproct taxonomyas well as the few notions about entoproct internal evolution, aresolely based on morphological characters, mainly of the adult stageonly.

Here, we present the rst phylogenetic study of the internalrelationships of the phylumEntoprocta, based on partial sequencesof the mitochondrial gene cytochrome c oxidase subunit I, and thenuclear genes 28S rDNA and 18S rDNA. We included most of therepresentative genera in the phylum as well as a number of crucialoutgroups in order to reconstruct the evolution of important ento-proct and lophotrochozoan characters, especially with regard tothe larval anatomy, the patterns of asexual reproduction, and thediverse commensal associations.

2. Materials and methods

2.1. Sampling

Animals were collected in Belize, Sweden, Thailand, and Japanbetween 2004 and 2009, and were subsequently determined bythe authors. The sampling included 18 entoproct species repre-senting three out of four entoproct families and seven out of 11entoproct genera ( Tables 1 and 2 ). The sampling was especially in-tense in the most diverse entoproct family, the Loxosomatidae.Most entoproct species were newly sequenced and some se-quences were retrieved from GenBank. We furthermore addedten species from potential outgroup phyla to the analysis (all taxaare listed in Table2 ). Voucher specimens (specimens that were col-lected together with the sequenced material) were deposited at theGothenburg Museum of Natural History, Sweden ( Table 2 ).

2.2. DNA extraction, amplication, and sequencing

Specimens were preserved and stored in 7096% ethanol untilextraction of genomic DNA using the DNeasy Tissue Kit followingthe manufacturers protocol (Quiagen, Valencia, CA, USA). Polymer-ase chain reaction (PCR) amplications of partial mitochondrialcytochrome c oxidase subunit I (COI hereafter), as well as partialnuclear 28S rDNA (28S hereafter), and partial nuclear 18S rDNA

(18S hereafter) were accomplished with primers COI (LCO1490-HCO2198), 28S (28SC1-28SC2), 18S (1F-4R or 1F-5R; 3F-18SBI;18SA29R) and are described in Fuchs et al. (2009) . PCRs contained1 l l of each primer, 2 l l DNA template, ready-to-go PCR beads(Amershal Biosciences), and distilled water to a nal volume of 25 l l. Amplications were carried out in thermo cyclers 2720 (Ap-plied Biosystems). The temperature proles were as follows: forCOI: 95 C/5 min, 35 cycles 95 C/40 s, 45 C/45 s, 72 C/1 min,and nal extension at 72 C/8 min; for 28S: 95 C/5 min, 35 cycles95 C/40s, 52 C/40s, 72 C/1 min, and nal extension 72 C/8 min; for 18S: 94 C/2 min, 35 cycles 94 C/45s, 49 C/45 s,72 C/1 min and nal extension at 72 C/6 min. Sequencing wasperformed on an ABI 3730XL DNA Analyser (Applied Biosystems)by the Macrogen Sequencing System, Korea. Some PCR amplica-

tions of the partial COI gene were performed using Ex Taq DNAPolymerase (Takara) under the temperature condition: 94 C/

Table 1

Current systematics of Entoprocta based on morphological characters ( Iseto, 2002 ;ITIS (Integrated taxonomic information system) webservices; Nielsen, 1996; Wasson,2002; Wood, 2005 ). In brackets, approximate species numbers are indicated,however, only the species number of Loxosomatidae has recently been revised(dHondt and Gordon, 1999; Nielsen, 2010; Wasson, 1997 ; etc.). Asterisks indicate thegenera with species included in this study.

Family Genus

SolitariaLoxosomatidae (>140) Loxosoma *

Loxosomella *

Loxomitra *

Loxocorone *

Loxomespilon

ColonialesPedicellinidae (20) Pedicellina *

Loxosomatoides *

Myosoma

Barentsiidae (20) Barentsia *

Urnatella

Loxokalypodidae (2) Loxokalypus

J. Fuchs et al. / Molecular Phylogenetics and Evolution 56 (2010) 370379 371

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Table 2

Taxa included in phylogenetic analyses. GB: from GenBank. (a) Yokobori et al. (2008) , (b) Giribet et al. (2004) , (c) Giribet et al. (2006) , (d) Fuchs et al. (2009) , (e) Baker and Giribet(2007) , (f) Okusu et al. (2003) , (g) Rousset et al. (2007) , (h) Passamaneck et al. (2004) . Voucher deposited at GNM, Gothenburg Museum of Natural History, Sweden.

Species Substrate Collection site Collectionyear

COIAccessionNo.

28SAccessionNo.

18SAccessionNo.

Voucher

IngroupOrder Solitaria Loxosomella vivipara Tedania ignis ,

Porifera

Belize 2004 GU125760 GU125730 GU125745 GNM Entoprocta 1

Loxosomella parguerensis Tedania ignis ,Porifera

Belize 2004 GU125761 GU125731 GU125746 GNM Entoprocta 2

Loxosomella varians Nephtys sp.,Polychaeta

Tjrn, Sweden 2008 GU125762 GU125732 GU125747 GNM Entoprocta 3

Loxosoma pectinaricola Pectinaria belgica ,Polychaeta

Tjrn, Sweden 2008 GU125763 GU125733 GU125748

Loxosomella harmeri Gattyana cirrosa ,Polychaeta

Sweden, lpnr.318 2008 GU125764 GU125734 GU125749 GNM Entoprocta 4

Loxosomella sp. Golngia sp.,Sipuncula

Friday Harbor.USA

2006 GU125765 GU125735 GU125750 GNM Entoprocta 5

Loxosomella sp. Palmiskeneaskenei , Bryozoa

Sweden, lpnr252 2007 GU125766 GU125736 GU125751 GNM Entoprocta 6

Loxosomella plakorticola Plakortis sp.,Porifera

Manza, Okinawa, Japan


Loxosomella stomatophora Non-living(glass slides)

Mizugama,Okinawa, Japan


Loxomitra mizugamaensis Non-living(glass slides) Mizugama,Okinawa, Japan 2004 GU125769 GU125739 GU125754 GNM Entoprocta 9

Loxomitra tetraorganon Non-living(glass slides)

Mizugama,Okinawa, Japan

2008 GU125770 GU125740 GU125755

Loxosomella aloxiata GB AB264800 aLoxocorone allax GB AB264799 aLoxosomella murmanica GB AY218083 b DQ279950 c AY218100 b

OrderColoniales

Loxosomatoides sirindhornae Non-living(nylon rope)

Mae Klong River,Thailand


Barentsia discreta Non-living(stones)

Shimoda,Shizuoka, Japan


Barentsia gracilis GB FJ196079 d FJ196138 d FJ196109 dPedicellina cernua GB FJ196081 d FJ196111 d

OutgroupsPhoronida Phoronis ovalis Sweden, lpnr.362 2008 GU125773 GU125743 GU125758 GNM Phoronida 9

Phoronis vancouverensis GB FJ196088 d FJ196145 d FJ196118 d

Brachiopoda Terebratulina retusa Sweden, lpnr.263 2007 GU125774 GU125744 GU125759Macandrevia cranium Tjrn, Sweden 2007 GU125775 GNM Brachiopoda 193Terebratalia transversa GB FJ196085 d FJ196143 d FJ196115 d

Cycliophora Symbion americanus GB EF140787 e EF142102 e EF142081 e

Bryozoa Pectinatella magnica GB FJ196095 d FJ196151 d FJ196124 d Asajirella gelatinosa GB FJ196096 d FJ196153 d FJ196126 d

Mollusca Chaetoderma nitidulum GB AY377726 f AY340387 g AY340425 gHelicoradomenia sp. GB AY377725 f AY145409 h AY145377 h

Table 3

Support values for the various hypotheses tested during analyses of the different datasets (COI, 28S, 18S, nuclear, molecular). Bootstrap support values for ML (maximumlikelihood), and posterior probabilities for BI (Bayesian Inference).

Entoprocta Coloniales Solitaria Ento + Cycliophora

ML BI ML BI ML BI ML BI

COI 0 0.00 82 1.00 0 0.00 0 0.0028S 99 1.00 78 1.00 97 0.87 55 0.6818S 100 1.00 99 1.00 100 1.00 80 0.00NUC 100 1.00 100 1.00 100 1.00 89 0.93MOL 100 1.00 100 1.00 100 1.00 84 0.99

Loxosomella Loxomitra Barentsia

ML BI ML BI ML BI

COI 0 0.00 76 1.00 0 0.0028S 0 0.00 100 1.00 65 0.8718S 0 0.00 100 1.00 69 0.00NUC 0 0.00 100 1.00 0 0.00MOL 0 0.00 100 1.00 0 0.00


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via the Cipres Portal v1.15 and Bayesian algorithm using MrBayes3.1.2 ( Ronquist and Huelsenbeck, 2003 ). Models used for ML were specied as GTRGAMMA (GTR model with mixed rate het-erogeneity, searching under the CAT model and returning toGAMMA scores). Bootstrapping was performed with 10,000 repli-cates in RAxML 7.0.4, using a switch of the model GAMMA toCAT for rapid bootstrapping and a nal ML search under theGAMMA + P-Invar Model, i.e., all free model parameters esti-mated by RAxML ( Stamatakis et al., 2008 ). The models for Bayes-ian analyses were selected among 24 models of evolution usingMrModeltest 2.2 ( Nylander, 2004 ). Best models for all datasets(including the three codon positions of COI) were determinedas GTR + I + G under the Akaike Information Criterion (AIC). Fortesting convergence, each Bayesian analysis was run three timesfor each dataset, each run with two chains and for 1,000,000 gen-erations, whereby the rst 2500 trees were discarded as burn-in.Tree reconstructions were visualized herein using TreeViewX andAdobe Illustrator CS3.

2.4. Coding of morphological characters

In order to trace character evolution on the molecular trees, wedened a matrix including 13 relevant morphological, ecological,and life history characters for the species included in this study(Table 4 and Supplementary material ). We imported the Bayesiantree of the combined analysis (MOL) as well as the matrix intoMacClade 4 ( Maddison and Maddison, 2005 ) and traced characterevolution on the provided tree. Characters were dened as eitherbinary or multistate, and unordered.

3. Results

The COI partition from 27 terminals (18 entoprocts and 9 out-groups) had a length alignment of 601 bp with 62.2% variable

and 52% parsimony informative positions. The 28S fragment from24 species (incl. 15 entoprocts) had a length of 335 bp, containing44% variable and 37.6% parsimony informative sites. The 18S par-tition of 25 species (incl. 16 entoprocts) had a size of 1668 bp with33.6% variable and 23.4% parsimony informative sites. The datasetswere combined in a nuclear alignment NUC (28S, 18S) and amolecular alignment MOL (COI, 28S, 18S). The NUC dataset con-sisted of 2003 bp from 25 species (incl. 16 entoprocts). The MOL dataset consisted of 2604 bp from 25 species (incl. 16 entoprocts)with 41.6% variable and 31.8% were parsimony informative sites.

3.1. Internal relationships

The datasets were analyzed separately for every gene and com-bined for all nuclear and molecular data ( Figs. 2 and 3 ). Exceptfrom analysis of COI, all analyses conrmed the monophyly of Entoprocta with high support, i.e., BS (bootstrap support) >99%and BPP (Bayesian posterior probability) = 1. Likewise, Coloniales

are recovered in all analyses with maximum nodal support values(MOL BS/BPP = 100/1.00). The support for Solitaria was very strong(MOL BS/BPP = 100/1.00) in all but the COI analysis. In this case,Loxosomella murmanica groups with the cycliophoran Symbionamericanus outside the Entoprocta ( Fig. 2 a). Among the tentativeoutgroups, the analyses consistently recovered a well-supportedrelationship between Cycliophora and Entoprocta (MOL BS/BPP = 84/0.99, NUC BS/BPP = 89/0.93), the only exception beingCOI (Table 3 ).

Within Coloniales, most analyses suggest that the freshwaterentoproct Loxosomatoides sirindhornae is the sistergroup to a cladeincluding the marine Barentsia gracilis , Barentsia discreta , and Pedi-cellina cernua (Figs. 2 and 3 ). The Barentsiidae are paraphyletic inmost analyses with regard to Pedicellina cernua , and some analysesrendered a polytomy including Barentsia and Pedicellina (Figs. 2, 3and Table3 ). The analysesalso suggestthat Loxosomella , within Sol-itaria,is aparaphyletic groupwithregardto severalothergenera,i.e.,Loxosomitra , Loxosoma , and Loxocorone (Figs. 2 and 3 ). The last genuswas only represented in the COI analysis and remains unsettledwithin Solitaria. The two species of the genus Loxomitra includedin this study showed a well-supported sister group relationship inallanalyses, and a weakafnity toa cladecomposed of L. murmanicaand L. varians . The only representative of the genus Loxosoma in ourstudy, consistently nested within Loxosomella without support forany particular sister group relationship ( Figs. 2 and 3 ).

3.2. Character evolution

Following character evolution along the MOL tree ( Fig.4 , Table4and Supplementary material ) showed little homoplasy for most of the morphological, ecological, and life history characters (overallCI = 0.78). Solitarity is plesiomorphic for Entoprocta, while colo-niality is apomorphic for Coloniales. Living in a marine habitat isancestral, while limnic habitats are derived for L. sirindhornae .Association with benthic invertebrates is ancestral for Entoprocta,while a free-living life style has evolved several times within thephylum and is apomorph for Loxomitra , L. stomatophora , and equiv-ocal for Loxosomatoides sirindhornae and Barentsia gracilis , sinceboth species live on living as well as non-living substrates . Contin-uous muscles that reach from the foot into the feeding apparatusare an apomorphy for Solitaria. A star cell complex is apomorphfor the Pedicellina /Barentsia clade. A stolon as attachment structureis an apomorphy for the colonial entoprocts investigated herein,

while the ancestral condition for the attachment structure inEntoprocta remains unresolved. Adult budding from the stolon isapomorphic for Coloniales, while budding from the calyx is con-ned to Solitaria. The ancestral pattern, however, remains unsuresince the outgroup Cycliophora shows internal budding in allstages. A bilobed central nervous system is probably the ancestralfeature of adult Entoprocta, while the ancestral larval condition inEntoprocta includes the presence of a gut and a ciliated creepingfoot as well as the absence of larval budding and larval eyes.

4. Discussion

4.1. Evolution of entoproct anatomy and life history

The monophylyof the phylum Entoprocta is supported in all ouranalyses, except for the analysis based solely on COI, thus corrob-

Table 4

Morphological character matrix, which was used to reconstruct character evolution in

Entoprocta ( Fig. 4 and Supplementary material ). Key references: Iseto, 2002; Fuchsand Wanninger, 2008; Fuchs et al., 2006; Funch, 1996; Nielsen, 1964, 1966, 1967,1971, 1989, 1996; Wanninger, 2005; Wanninger et al., 2007; Wood, 2005 .

Character 1. Coloniality: solitary; colonial with stolonsCharacter 2. Habitat: marine; limnicCharacter 3. Commensalism: absent; associated with a benthic animal hostCharacter 4. Continuous muscles between stalk and calyx: absent; presentCharacter 5. Star cell complex: absent; presentCharacter 6. Muscle packets alternating with non-muscular regions in the

stalk: absent; presentCharacter 7. Attachment structure of zooid: (a) stolon; (b) foot with muscle

tissue; (c) disc with muscle tissue; (d) disc without muscle tissueCharacter 8. Adult budding: (a) from stolon; (b) from calyx; (c) internalCharacter 9. Peanut shaped CNS: absent; presentCharacter 10. Larval budding: absent; presentCharacter 11. Larval ciliated foot: absent; presentCharacter 12. Larval gut: absent; presentCharacter 13. Larval eyes: absent; present

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orating long standing hypotheses based on morphological data(Emschermann, 1972; Hatschek, 1888; Nitsche, 1870 ). The COIanalysis places the entoproct L. murmanica as sister taxon to thecycliophoran Symbion americanus , and in a clade with two ecto-proct species. However, we interpret this result as a long branch ef-fect ( Felsenstein, 1978 ), caused by high sequence divergence in theCOI fragment of these two species.

Our analyses recover the two earlier recognized entoproct or-ders, Solitaria and Coloniales. In addition, our results suggest thatsolitarity may have been in the ground pattern of Entoprocta.How-ever, these issues cannot entirely be resolved, since our study does

not include a representative of Loxokalypodidae. This family in-cludes colonial entoprocts without a stolon ( Emschermann,1972 ) and thus forms a kind of intermediate stage between solitaryand colonial forms. To date, only two species of Loxokalypodidaehave been described ( Emschermann, 1972; dHondt and Gordon,1999 ) and observations of both species are so rare that it wasimpossible to obtain samples for this study.

Solitaria comprises the single family Loxosomatidae and thesystematics within this taxon has in the past been subject to sev-eral changes, and might still be modied in the future due tonew species discoveries and new data becoming available. Based

Fig. 3. Tree based on Bayesian analysis (BI) of combined data set (MOL). Node support is indicated above (posterior probability values) and below (maximum likelihoodbootstrap values) each branch.


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on morphology, however, the taxon Loxosomatidae is currentlysubdivided into Loxosomella , Loxosoma (including the formermonotypic Loxostemma ), Loxomitra , Loxocorone , and Loxomespilon(Nielsen, 1996, 2010; Iseto, 2002 ; Table 1 ). Loxosomella is a para-phyletic grouping in our analyses, which suggests that typical ana-tomical characters of the genus may belong to the solitarianground pattern, e.g., the continuous musculature between theattachment structure, the stalk, and the calyx, as well as buddingfrom the calyx. Furthermore, the attachment structure in theancestral solitarian might have been a muscular foot. The distribu-

tion of commensal species in our trees suggests that commensal-ism was present in the Solitarian ground pattern and that

Loxomitra acquired a free-living life style independently from colo-nial taxa. Within Coloniales, the unresolved clade containingBarentsia and Pedicellina shows a ground pattern with a muscularstar cell complex at the junction between the stalk and the calyx.

The character evolution in entoprocts described above reectsthe general focus of entoproct studies on the adult body bauplan.However, the entoproct larval stage certainly is an important lifecycle stage, as it is the main vector for a species dispersal, andwe consider the entoproct larval stage at least as informative forphylogenetic inferences as the adult stage. However, coding ento-

proct larval characters is still in its infancy, since entoproct larvaeare generally poorly known and only few detailed, comparable

Fig. 4. Combined MOL tree based on the maximum likelihood and the Bayesian analyses; branches with support values

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descriptions exist to date ( Fuchs andWanninger, 2008; Haszprunarand Wanninger, 2008; Nielsen, 1971; Wanninger et al., 2007 ).However, the distribution of available larval characters in our phy-logenetic reconstruction supports an ancestral entoproct with aplanktotrophic larva, which had a ciliated creeping foot. The creep-ing-type larva probably evolved into the swimming-type formswithin the Coloniales and Solitaria. Further, our analyses supportthe absence of larval budding in the entoproct ground pattern,implying that cycliophorans and Loxosomella vivipara have evolvedthis feature independently.

4.2. Entoproct relations to Cycliophora and other lophotrochozoans

In accordance with several previous comparative studies, wend strong support for a sister group relationship between Entopr-octaand Cycliophora. The growing condence in an entoproctcyc-liophoran clade is supported by morphological as well as moleculardata ( Funch and Kristensen, 1995, 1997; Hejnol et al., 2009; Obst,2003; Paps et al., 2009b; Passamaneck and Halanych, 2006; Sren-sen et al., 2000; Zravy et al., 1998 ), and suggests homology of sev-eral similar characters in these two phyla, which allows forreconstructing their evolutionary history beyond the phylum level.A basic morphological apomorphycombining Entoprocta with Cyc-liophora are mushroom-shaped extensions, which extend from thebasal lamina into the epidermis ( Funch and Kristensen, 1997; Niel-sen and Jespersen, 1997 ). Studies of the nervous systems of bothtaxa further indicate structural similarity between adult ento-procts ( Fuchs et al., 2006 ) and the cycliophoran free-living stages(Funch, 1996; Funch and Kristensen, 1997; Neves et al., 2010a,b;Wanninger, 2005 ), which points towards the derived nature of allfree-swimming stages in the cycliophoran life cycle. However,the complicated life cycle of Cycliophora yet prevents a clearhomology assignment of all life cycle stages of Cycliophora andEntoprocta. Nevertheless, according to our analyses, the ancestorof Entoprocta and Cycliophora canprobably be interpreted as a sol-itary, marine organism, which lived associated with a benthic ani-mal host and possessed a well-pronounced ability for asexualreproduction.

It should be mentioned here once more, that a monophyleticclade Tetraneuralia was proposed for Entoprocta + Mollusca, so-lely based on morphological similarities between an entoproctcreeping-type larva and basal mollusks ( Wanninger, 2009 ; Wann-inger et al., 2007 ). Clear structural similarities between the taxa ex-ist, e.g., in the nervous system (complex apical organ andtetraneury) and the ciliated creeping foot (with mucous cells, alarge pedal gland, and unique muscle setup). However, improvedcladistic and phylogenetic analyses will be prerequisite for resolv-ing the evolutionary history of Lophotrochozoa and reconstructtheir last common ancestor.

In conclusion, this study suggests an entoproct ancestor with a

marine, probably solitary, epizoic adult stage with a bilobed gan-glion, and a planktotrophic larva with a ciliated creeping foot. Inorder to fully understand character evolution in Entoprocta, futurestudies are needed that describe the neuromuscular anatomy of yet unstudied entoproct larvae as well as the cycliophoran feedingstage with modern microscopic methods. Moreover, enigmaticentoproct key taxa such as Loxokalypus , are important to includein future phylogenetic analyses. Finally, the spiralian nature of the entoproct cleavage needs to be veried with modernapproaches.

Acknowledgments

The authors thank Timothy Wood for providing the freshwater

entoproct sample. The authors are grateful to the marine team of the Swedish Taxonomy Initiative, as well as the crews of Oscar

von Sydow, Arne Tiselius, and Nereus at the Sven Lovn Centerfor Marine Sciences Kristineberg and Tjrn for technical supportduring collections. The authors also thank twoanonymous refereeswho, with their suggestions, helped to improve the manuscript.This work was supported by grants from the Swedish TaxonomyInitiative to P.S., J.F., and M.O.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, inthe online version, at doi:10.1016/j.ympev.2010.04.009 .

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