Molecular Phylogenetics and Evolution 77 (2014) 54–64

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Molecular phylogeny of the Brazilian endemic genus Orthophytum(Bromelioideae, Bromeliaceae) and its implications on morphologicalcharacter evolution

http://dx.doi.org/10.1016/j.ympev.2014.03.0071055-7903/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author.E-mail address: rafael.louzada@ufpe.br (R.B. Louzada).

Rafael B. Louzada a,⇑, Katharina Schulte b,c, Maria das Graças L. Wanderley d, Daniele Silvestro e,Georg Zizka e, Michael H.J. Barfuss f, Clarisse Palma-Silva b

a Universidade Federal de Pernambuco, Departamento de Botânica, Recife 50670-901, Pernambuco, Brazilb Australian Tropical Herbarium, James Cook University, PO Box 6811, Cairns, QLD 4878, Australiac Centre for Tropical Biodiversity and Climate Change, James Cook University, Discovery Drive, Townsville, QLD 4814, Australiad Instituto de Botânica, Secretaria do Meio Ambiente, Avenida Miguel Stéfano 3687, São Paulo 01061-970, São Paulo, Brazile Department of Botany and Molecular Evolution, Research Institute Senckenberg and J.W. Goethe University, Frankfurt am Main D-60325, Germanyf Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Rennweg 14, 1030 Vienna, Austria

a r t i c l e i n f o

Article history:Received 26 January 2013Revised 27 February 2014Accepted 10 March 2014Available online 21 March 2014

Keywords:BromelioideaeEspinhaço RangePhytochrome CPHYCtrnH-psbA spacertrnL-trnF spacer

a b s t r a c t

The saxicolous genus Orthophytum (�60 species, Bromeliaceae) is endemic to eastern Brazil and diversi-fied in xeric habitats of the Atlantic Rainforest, Caatinga and campos rupestres. Within the genus, two maingroups are discerned based on the presence or absence of a pedunculate inflorescence, which are furthersubdivided into several morphological subgroups. However, these systematic hypotheses have not yetbeen tested in a molecular phylogenetic framework. Here we present the first phylogenetic analysis ofOrthophytum using nuclear and plastid markers (phytochrome C, and trnH-psbA and trnL-trnF spacers).Forty species representing the two main groups and all subgroups of Orthophytum, and the related generaCryptanthus (8 spp.) and Lapanthus (2 spp.) were analyzed. The phylogenetic reconstruction revealed awell-supported clade termed Eu-Orthophytum, containing species with pedunculate inflorescences only.The Orthophytum species with sessile inflorescence formed two clades: (1) the amoenum group and (2)the vagans group plus O. foliosum, the only pedunculate Orthophytum species found outside Eu-Orthophy-tum. The vagans clade is in sister group position to Eu-Orthophytum. Within the latter, the subgroupmello-barretoi was sister to the most diversified clade, termed Core Orthophytum. Morphological char-acter state reconstructions of floral characters used in previous taxonomic treatments as key diagnosticcharacters (penduncle presence, corolla form, and petal appendage form) showed different levels ofhomoplasy.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Bromeliaceae (ca. 3400 spp.; Butcher and Gouda, cont. updated)is an almost exclusively Neotropical family, with only one species(Pitcairnia feliciana (A. Chev.) Harms & Mildbraed) occurring inWest Africa. The family has traditionally been divided in three sub-families: Pitcairnioideae, Bromelioideae and Tillandsioideae (Smithand Downs, 1974, 1977, 1979; Smith and Till, 1998). The mono-phyly of Pitcairnioideae has been questioned in several molecularphylogenetic studies (e.g. Barfuss et al., 2005; Crayn et al., 2004;Givnish et al., 2007; Horres et al., 2000; Terry et al., 1997), andrecently a new classification for Bromeliaceae based on molecular

phylogenetic evidence from the gene ndhF was proposed byGivnish et al. (2007, 2011) dividing Bromeliaceae into eight sub-families: Brocchinioideae, Lindmanioideae, Tillandsioideae,Hechtioideae, Navioideae, Pitcairnioideae, Puyoideae andBromelioideae.

Subfamily Bromelioideae comprises 33 genera and approxi-mately 950 species distributed in tropical and subtropical Americawith a center of diversity in southeastern Brazil (Butcher andGouda, cont. updated; Smith and Downs, 1979). The monophylyof the subfamily is supported by both morphological and molecularevidence, with Puya as sister group (Barfuss et al., 2005; Craynet al., 2004; Givnish et al., 2004, 2007, 2011; Horres et al., 2000,2007; Schulte et al., 2005, 2009; Schulte and Zizka, 2008; Terryet al., 1997). Nevertheless the inter- and infrageneric relationshipswithin the subfamily are poorly understood (Brown and Leme,

R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64 55

2000; Schulte et al., 2009). Recent molecular studies based on plas-tid and nuclear data identified several basal lineages within thesubfamily (Greigia Regel, Ochagavia Phil., Fascicularia Mez, Dein-acanthon Mez, Bromelia Juss.) (Schulte et al., 2005, 2009; Schulteand Zizka, 2008). Fernseea Baker was reported as sister to a cladecomprising the remainder of the subfamily, termed Eu-Bromelioi-deae (Schulte et al., 2009; Schulte and Zizka, 2008). Among the lat-ter, the genera Orthophytum Beer, Cryptanthus Otto & A. Dietr.,Ananas Mill., Neoglaziovia Mez, and Acanthostachys Klotzsch wereidentified as early divergent lineages (basal eu-bromelioids)whereas the more advanced bromelioids, characterized by the tankhabit (a central water collecting tank formed by the leaf sheaths),formed a moderately-supported clade, termed the core bromeli-oids (Givnish et al., 2011; Schulte et al., 2009; Schulte and Zizka,2008; Sass and Specht, 2010). Whereas core bromelioids comprisethe majority of species and epiphytes, the more basal lineages lacka central external water reservoir and are mainly terrestrial orlithophytes.

Orthophytum is a saxicolous (rarely terrestrial) genus endemicto eastern Brazil where it underwent considerable diversification(Fig. 1). The species generally inhabit the top of granitic-gneissinselbergs in the regions of the Atlantic Rainforest and the Caatinga,and quartzitic-sandstone outcrops in the Brazilian campos rupestres(‘rocky fields’) along the Espinhaço Range. Two centers of diversitycan be recognized, one in the Espinhaço Range and the other in theAtlantic Rain Forest area in the Brazilian states of Minas Gerais andEspírito Santo (Louzada and Wanderley, 2010).

The genus was described by Beer (1854) based on one unnamedcollection of a pedunculate species known today as Orthophytumglabrum (Mez) Mez (Louzada and Wanderley, 2010). Ule (1908)described two new genera from Brazil (Sincoraea Ule and Cryp-tanthopsis Ule), both with sessile inflorescences, which were subse-quently regarded as synonyms of Orthophytum (Smith, 1955; Smithand Downs, 1979). In the taxonomic treatment for Bromeliaceae inFlora Neotropica (Smith and Downs, 1979), 17 species of Orthophy-tum were recognized. Today the genus comprises about 60 species(Louzada and Wanderley, 2011), the majority described in the lasttwo decades, and a taxonomic revision of the group is urgentlyneeded to assess the conservation status of the species.

Within Orthophytum two main morphological groups are tradi-tionally recognized based on the presence or absence of a peduncle(or stalk, sometimes in bromeliads also called a scape; Leme, 2004;Louzada and Wanderley, 2010; Wanderley, 1990; Wanderley andConceição, 2006). These groups of species were termed ‘‘com-plexes’’ in Leme (2004) and each one was subdivided into sub-groups also called ‘‘subcomplexes’’. However in this study weadopted the terms groups and subgroups instead of complexesand subcomplexes because we understand these morphologicalgroups of species are not species complexes according to its idea.The first group comprises the majority of species and is termedthe ‘‘group with scapose inflorescence’’ which is divided into threesubgroups: disjunctum, leprosum, and mello-barretoi (Leme,2004). The other is the ‘‘group with sessile inflorescence’’ whichcomprises three subgroups: amoenum, vagans, and supthutii(Leme, 2004). Recently, a new genus was established (LapanthusLouzada & Versieux) to better accommodate the species of the sup-thutii subgroup (Louzada and Versieux, 2010). Nevertheless, thevalidity of these taxonomic hypotheses has not yet been testedin a molecular phylogenetic framework.

In previous phylogenetic studies on Bromelioideae, Orthophy-tum has usually been represented by only a few taxa (Ramírez-Morillo, 1996; Schulte et al., 2005, 2009; Schulte and Zizka,2008) and the genus was found to be the sister group of Cryptan-thus. However, due to the low taxon sampling the hypotheses out-lined above and the monophyly of the genus could not be properlytested yet. Therefore, a more comprehensive phylogenetic study is

needed to clarify inter- and intrageneric relationships ofOrthophytum.

Here we present a molecular phylogeny of Orthophytum andrelated genera based on the plastid intergenic spacer regionstrnL-trnF and trnH-psbA, and the low-copy nuclear gene phyto-chrome C (PHYC). The objectives were (1) to assess the phyloge-netic relationships between Orthophytum, Cryptanthus, andLapanthus, and the monophyly of the genera, (2) to elucidate intra-generic relationships in Orthophytum, (3) to investigate the evolu-tion and taxonomic significance of morphological characterspreviously used in the taxonomy of Orthophytum.

2. Materials and methods

2.1. Taxon sampling

In the present study a molecular data set of 54 species from sixgenera (Table 1) was analyzed. In Orthophytum, 40 of the about 60recognized species (i.e. 67% of known diversity) were included toinvestigate all of the morphological groups and subgroupsdescribed by Leme (2004) including the two species of the supth-utii subgroup today recognized as the genus Lapanthus (Louzadaand Versieux, 2010). In addition, eight species of the genus Cryp-tanthus comprising representatives from the two subgenera andsix of eight sections described by Ramírez-Morillo (1996) wereincluded in the data set. Outgroup species were included fromthe early diverging Bromelioideae: Bromelia (2), Ochagavia (1)and from the mono-generic subfamily Puyoideae (1) based onGivnish et al. (2011) and Schulte et al. (2009).

2.2. DNA extraction, amplification and sequencing

Total genomic DNA was extracted from leaf material using acetyltrimethylammonium bromide (CTAB) procedure (Doyle andDoyle, 1987) modified by Horres et al. (2000). The phytochromeC (PHYC) gene was amplified using primers phyc515f-br AAG CCCTTY TAC GCT ATC CTG CAC CG and phyc1699r-br ATW GCA TCCATT TCA ACA TCT TCC CA. Internal primers were used for sequenc-ing (phyc974f-br GCT CCT CAC GGC TGC CAC GCT CA andphyc1145r-mo CCT GMA RCA RGA ACT CAC AAG CAT ATC). ThetrnL-trnF and trnH-psbA regions were amplified using universalprimers described in Shaw et al. (2005) and Sang et al. (1997),respectively. The two plastid regions and the nuclear gene werechosen because they have proven to be most informative markersfor Bromeliaceae (e.g., Barfuss et al., 2005: trnL-trnF; Givnish et al.,2011: trnL-trnF, trnH-psbA; Jabaily and Sytsma, 2010: PHYC).Amplifications were carried out in a Veriti Thermal Cycler (AppliedBiosystem Corp., Foster City, California). Plastid regions wereamplified with 10 lL reactions following Palma-Silva et al.(2009). The nuclear region PHYC was amplified with 10 lL as fol-lows: 1x Taq buffer (Fermentas), 1.5 mM MgCl2 (Fermentas),l00 lmol deoxynucleotide triphosphate, 10 pmol of each primer,1 U Taq DNA polymerase (Fermentas) and 10–20 ng of DNA tem-plate, using a standard cycling program: 2 min denaturation at95 �C followed by 35 cycles of 95 �C denaturation for 30 s, 30 sannealing at 59 �C, and 2 min extension at 70 �C and a final elonga-tion step at 70 �C for 7 min. The PCR products were cleaned usingExoSAP-IT (USB Corp., Cleveland, Ohio) following the manufac-turer’s protocol. Cycle sequencing was carried out with the BigDye Terminator kit v.3.1 (Applied Biosystem Corp., Foster City, Cal-ifornia) with an initial 60 s denaturation at 95 �C, followed by 30cycles at 96 �C denaturation for 10 s, 10 s annealing at 50 �C, and2 min extension at 60 �C. The sequences were generated on anABI 3730 DNA Analyzer sequencer.

Fig. 1. A–U. Diversity of Orthophytum. A. O. argentum. B. O. leprosum. C. O. boudetianum. D. O. braunii. E. O. maracasense. F. O. albopictum. G. O. conquistense. H. O. humile. I. O.disjunctum. J. O. eddie-estevesii. K. O. foliosum. L. O. magalhaesii. M. O. glabrum. N. O. hatschbachii. O. O. ophiuroides. P. O. ulei. Q. O. graomogolense. R. O. grossiorum. S. O. horridum.T. O. sucrei. U. O. lanuginosum (Fotos R.B. Louzada).

56 R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64

2.3. Alignment of sequences and data congruence

The sequences were assembled and edited with the softwareGeneious 5.1.7 (Drummond et al., 2011) and initially aligned withMAFFT (Kazutaka et al., 2002) followed by manual adjustments inGeneious. Congruence among data partitions of the two plastidand one nuclear marker was assessed (a) by visual inspection

of the tree topologies based on the plastid versus the nuclear dataset and (b) with the incongruence length difference (ILD) test(Farris et al., 1994) implemented in PAUP⁄4.0b10 (Swofford,2002) employing 100 replicates (heuristic search, 10 randomaddition replicates, tree-bisection-reconnection (TBR) branchswapping), saving a maximum of 1000 most parsimonious treesper replicate.

Table 1Studied material.

Taxa Locality Voucher GenBank Accession Nos.

trnH-psbA trnL-trnF PHYC

Bromelia pinguin L. ex cult. Schulte 300508-10 (FR) KJ676896 JX649511 JX649422Bromelia serra Griseb. ex cult. Horres 029 (FR) KJ676897 DQ084622 JX649423Cryptanthus bahianus L.B. Sm. ex cult. Gartenherbar 11060a (B) KJ676898 KJ676905 DQ084634Cryptanthus colnagoi Rauh & Leme ex cult. HBV 7103 (WU) KJ676899 KJ676906 JX649432Cryptanthus diamantinense Leme ex cult. Leme 3813 (HB) KJ676900 KJ676907 JX649433Cryptanthus microglaziovii I. Ramírez Brazil, ES, Santa Leopoldina Louzada et al. 12 (SP) KJ666538 KJ676908 KJ716287Cryptanthus odoratissimus Leme ex cult. Kautsky et al. s.n. (HB) KJ676901 KJ676909 JX649435Cryptanthus tiradentensis Leme Brazil, MG, Tiradentes Louzada et al. 158 (SP) KJ666539 KJ676910 KJ716331Cryptanthus warren-loosei Leme ex cult. 0013741 (WU) KJ676902 KJ676911 JX649436Cryptanthus zonatus (Vis.) Beer Brazil, PE, Recife IBT living collection KJ676903 KJ676912 KJ716288Lapanthus duartei (L.B. Sm.) Louzada & Versieux Brazil, MG, Conceição do Mato Dentro Louzada et al. 28 (SP) KJ666540 KJ676913 KJ716289Lapanthus itambensis (Versieux & Leme) Louzada & Versieux Brazil, MG, Santo Antonio do Itambé Louzada et al. 30 (SP) KJ666541 KJ676914 KJ716290Ochagavia litoralis (Phil.) Zizka, Trumpler & Zoellner ex cult. Horres 015a (FR) KJ676904 KJ676955 KJ716329Orthophytum alvimii W. Weber Brazil, MG, Teófilo Otoni Louzada et al. 90 (SP) KJ666542 KJ676915 KJ716291Orthophytum argentum Louzada & Wand. Brazil, BA, Rio de Contas Louzada et al. 110 (SP) KJ666544 KJ676917 KJ716293Orthophytum boudetianum Leme & L. Kollmann Brazil, ES, Afonso Cláudio Louzada 135 (SP) KJ666546 KJ676918 KJ716295Orthophytum braunii Leme Brazil, BA, Seabra Machado 50 (SP) KJ666547 KJ676919 KJ716296Orthophytum burle-marxii L.B. Sm. & Read Brazil, BA, Lençóis Louzada & Moreira 45 (SP) KJ666548 KJ676920 KJ716297Orthophytum conquistense Leme & M. Machado Brazil, BA, Vitória da Conquista Machado 277 (SP) KJ666549 KJ676921 KJ716298Orthophytum diamantinense Leme Brazil, BA, Diamantina Louzada & Ribeiro 146 (SP) KJ666575 KJ676922 KJ716322Orthophytum estevesii (Rauh) Leme Brazil, ES, Santa Teresa Fontana et al. 2959 (MBML) KJ666550 KJ676923 KJ716299Orthophytum falconii Leme Brazil, BA, Candido Sales Reis & Falcon s.n. (HB 89876) KJ666551 KJ676924 KJ716300Orthophytum foliosum L.B. Sm. Brazil, ES, Santa Teresa Louzada et al. 13 (SP) KJ666552 KJ676925 KJ716301Orthophytum fosterianum L.B. Sm. Brazil, ES, Colatina Louzada et al. 17 (SP) KJ666553 KJ676926 KJ716302Orthophytum glabrum (Mez) Mez Brazil, MG, Itaobim Louzada & Medeiros 139 (SP) KJ666554 KJ676927 KJ716303Orthophytum graomogolense Leme & C.C. Paula Brazil, MG, Grão Mogol Louzada & Moreira 42 (SP) KJ666555 KJ676928 KJ716304Orthophytum grossiorum Leme & C.C. Paula Brazil, MG, Carlos Chagas Leme et al. 5584 (HB) KJ666556 KJ676929 KJ716305Orthophytum gurkenii Hutchison Brazil, ES, Baixo Guandu Louzada 133 (SP) KJ666557 KJ676930 KJ716306Orthophytum harleyi Leme & M. Machado Brazil, BA, Érico Cardoso Louzada et al. 108 (SP) KJ666558 KJ676931 KJ716307Orthophytum hatschbachii Leme Brazil, BA, Rio de Contas Louzada et al. 104 (SP) KJ666559 KJ676932 KJ716308Orthophytum heleniceae Leme Brazil, BA, Andaraí Wanderley et al. 2544 (SP) KJ666560 KJ676933 KJ716309Orthophytum horridum Leme Brazil, MG, Pedra Azul Louzada & Medeiros 138 (SP) KJ666561 KJ676934 KJ716310Orthophytum humile L.B. Sm. Brazil, MG, Grão Mogol Louzada & Moreira 41 (SP) KJ666562 KJ676935 KJ716311Orthophytum lanuginosum Leme & C.C. Paula Brazil, MG, Teófilo Otoni Louzada & Medeiros 143 (SP) KJ666563 KJ676936 KJ716312Orthophytum lemei E. Pereira & I.A. Penna Brazil, BA, Morro do Chapéu Louzada et al. 186 KJ666564 KJ676937 KJ716313Orthophytum leprosum (Mez) Mez Brazil, MG, Jacinto Louzada & Medeiros 141 (SP) KJ666545 KJ676938 KJ716294Orthophytum lucidum Leme & H. Luther Brazil, BA, Jequitinhonha Louzada & Medeiros 142 (SP) KJ666565 KJ676939 KJ716314Orthophytum macroflorum Leme & M. Machado Brazil, BA, Licínio de Almeida Machado s.n. (SP 441733) KJ666566 KJ676940 KJ716315Orthophytum magalhaesii L.B. Sm. Brazil, ES, Vila Pavão Louzada 131 (SP) KJ666567 KJ676941 KJ716316Orthophytum mello-barretoi L.B. Sm. Brazil, MG, Santana do Riacho Louzada & Medeiros 84 (SP) KJ666568 KJ676942 KJ716317Orthophytum mucugense Wand. & Conc. Brazil, BA, Mucugê Louzada & Moreira 58 (SP) KJ666569 KJ676943 KJ716318Orthophytum ophiuroides Louzada & Wand. Brazil, BA, Lençóis Louzada & Wanderley 88 (SP) KJ666570 KJ676944 KJ722611Orthophytum pseudostoloniferum Leme & L. Kollmann Brazil, ES, Santa Teresa Leme et al. 6915 (MBML) KJ666571 KJ676945 KJ722610Orthophytum pseudovagans Leme & L. Kollmann Brazil, ES, Águia Branca Demuner et al. 3464 (MBML) KJ666572 KJ676946 KJ716319Orthophytum riocontense Leme Brasil, BA, Abaíra Machado 1206 (SP) KJ666573 KJ676947 KJ716320Orthophytum saxicola (Ule) L.B. Sm. Brazil, BA, Itaberaba Louzada et al. 122 (SP) KJ666574 KJ676948 KJ716321Orthophytum schulzianum Leme & M. Machado Brazil, MG, Diamantina Machado 1218 (SP) KJ666576 KJ676949 KJ716323Orthophytum sucrei H. Luther Brazil, ES, Afonso Cláudio Louzada 136 (SP) KJ666577 KJ676950 KJ716324Orthophytum toscanoi Leme Brazil, BA, Machado 1213 (SP) KJ666578 KJ676951 KJ716325Orthophytum ulei Louzada & Wand. Brazil, BA, Mucugê Louzada & Wanderley 91 (SP) KJ666579 KJ676952 KJ716326Orthophytum vagans M.B. Foster ex cult. Louzada s.n. (SP 442925) KJ666580 KJ676953 KJ716327Orthophytum zanonii Leme Brazil, ES, Pancas Louzada et al. 18 (SP) KJ666581 KJ676954 KJ716328Orthophytum sp. Brazil, BA, Jacaraci Machado 1207 (SP) KJ666543 KJ676916 KJ716292Puya chilensis Molina ex cult. Chase 23824 (K) KJ666582 KJ676956 KJ716330

Abbreviations: B, Herbarium of Botanical Garden of Berlin; FR, Herbarium Senckenbergianum; HB, Herbarium Bradeanum; IBt, Instituto de Botânica, São Paulo; K, Herbariumof Royal Botanical Gardens, Kew; MBML, Herbarium of Museu de Biologia Melo Leitão; SP, Herbarium of Instituto de Botânica, São Paulo; WU, Herbarium of Vienna University.

R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64 57

2.4. Phylogenetic analysis

A maximum parsimony (MP) analysis was performed inPAUP⁄4.0b10. Heuristic searches were conducted with 10,000 ran-dom taxon addition replicates and TBR branch swapping. The sta-tistical support was estimated by bootstrap analysis with 1000pseudoreplicates, each with 10 random taxon addition replicatesand TBR branch swapping. The degree of homoplasy was estimatedusing consistency (CI) and retention (RI) indices.

Bayesian inference analyses (BI) were run in MrBayes 3.2(Ronquist et al., 2012). The best-fit model (GTR + I + G) for thecombined dataset was determined using the Akaike InformationCriterion (Akaike, 1973) as implemented in MrModeltest 2.2

(Nylander, 2004). Four simultaneous Markov chains Monte Carlo(MCMC) were run for 10,000,000 generations sampling every1000 generations. After examining the MCMC convergence usingTracer (Rambaut and Drummond, 2007), the initial 2,000,000generations from each run were discarded from the analysis asburn-in while the remaining trees were used to construct a con-sensus tree with posterior probabilities (PP) assessing the statis-tical nodal support. Two partitioning schemes were tested, oneunlinking the model parameters between nuclear and plastidregions, the other unlinking all markers (i.e. three partitions).The best-fit model was chosen by Bayes factor test based onthe harmonic mean of the respective log likelihoods (Kass andRaftery, 1995).

58 R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64

2.5. State character reconstruction

To examine the evolution of key morphological characters usedin previous taxonomic treatments (Smith and Downs, 1979; Leme,2004) and to assess their taxonomic value, ancestral characterstate reconstructions were performed with maximum parsimonyusing Mesquite 2.75 (Maddison and Maddison, 2011) and a Bayes-ian framework in RASP (Yu et al., 2013). The analyses were run onthe Bayesian consensus tree based on the combined plastid andnuclear data set, exluding the outgroup. Three characters wereexamined: Peduncle presence: [0] absent, [1] present; corollaform: [1] clavate, [2] tubular; and petal appendages form: [1] fim-briate, [2] sacciform lacerate, [3] acute, [4] cuppuliform lacerate,[5] absent). Character states were scored from fresh material, her-barium sample and literature (Louzada and Wanderley, 2010;Smith and Downs, 1979).

3. Results

3.1. Phylogenetic relationship

Sequences for the two plastid and one nuclear loci were gener-ated for 54 accessions of Bromelia, Cryptanthus, Lapanthus, Ochag-avia, Orthophytum and Puya (Table 1). The final alignmentcomprised 605 positions for trnH-psbA, 851 for trnL-trnF intergenicspacer regions, and 1124 for the nuclear gene PHYC. The combineddataset yielded an alignment of 2580 characters in length with 273variable characters. The number of parsimony informative charac-ters was 105 (4%) for the ingroup (Orthophytum, Cryptanthus, andLapanthus), and 78 (3%) for Orthophytum. PHYC alignment pre-sented few double peaks, inferred as allelic variation, which weretreated as ambiguous data. The partition homogeneity test indi-cated that the different data partitions of the combined matrix(PHYC vs. two plastid regions) are not significantly incongruent(P-value = 0.067). The phylogenetic consensus trees based on theplastid versus the nuclear data set did not yield any statisticallysupported incongruent topologies. Thus, in the following we dis-cuss the phylogenetic relationships among Orthophytum andrelated genera based on the combined data set. A comparison ofthe two partitioning schemes in the Bayesian analyses showed thatthe model with three unlinked partitions outperforms the modelwith two partitions (log Bayes factor: 22.7, harmonic means ofthe log-likelihood: �6430.35 and �6441.70 respectively). Theresults of the Bayesian inference (BI) based on the unlinked parti-tion scheme are therefore presented below unless noted otherwise.

In the MP analysis of the combined data matrix 130,093 mostparsimonious trees of 392 steps in length were found (CI = 0.75;RI = 0.89). The MP (not shown) and the BI consensus trees of thecombined data set show a moderate to highly-supported cladecontaining the genera Orthophytum, Cryptanthus and Lapanthus(BS 71, PP 1). This group comprises four main clades (1–4) in theBI (Fig. 2).

The first main clade receives moderate to high statistical sup-port (BS 66, PP 1) and unifies the species of the amoenum subgroupsensu Leme (2004). The clade comprises seven of the ten investi-gated Orthophytum species with sessile inflorescences and shortcaulescent habit (Figs. 2 and 3). Within the first main clade, O. bur-le-marxii, O. heleniceae, O. ophiuroides and O. ulei form a moderatelyto highly-supported clade (BS 69, PP 1) in which the sister grouprelationship between O. burle-marxii and O. ophiuroides receives amoderately to high statistical support (BS 66, PP 0.99).

In the second main clade, the two species of Lapanthus form astrongly supported clade (BS 97, PP 1), which is found as the sistergroup to a clade including Cryptanthus tiradentensis in the firstdiverging lineage plus the highly-supported group (five species)

of Cryptanthus subg. Cryptanthus sensu Ramírez-Morillo (1996)(BS 100, PP 1). Nevertheless, the sister group relationship receivesno statistical support in the BI analysis and the node collapses inthe strict consensus of the MP analysis. Within the Cryptanthussubg. Cryptanthus clade, the first divergent lineage is C. bahianus(sect. Bahianae), sister group to a moderately to well-supportedclade (BS 84, PP 0.99) with C. colnagoi (sect. Cryptanthus), C. dia-mantinesis (sect. Bahianae), C. warren-loosei, (sect. Bahianae), andC. zonatus (sect. Zonatae).

The third main clade shows a highly supported lineage with twolong caulescent species with sessile inflorescences of Cryptanthussubg. Hoplocryptanthus (C. odoratissimus, C. microglaziovii; Fig. 2(BS 95, PP 1) as sister group to a large, highly-supported clade com-prising the remaining species of Orthophytum (BS 96, PP 1). Never-theless, the sister group relationship between the latter and theCryptanthus subg. Hoplocryptanthus clade does not receive statisti-cal support.

The first diverging clade (A) within the large Orthophytum cladeis well supported (BS 91, PP 1) and consists of three species withsessile inflorescences and long caulescent habit, which constitutethe vagans subgroup (O. zanonii, O. vagans, O. pseudovagans) sensuLeme (2004), plus a pedunculate species (O. foliosum) nestedwithin the vagans subgroup. Thus, the clade includes members ofthe two main morphological groups (sessile and pedunculate inflo-rescences; Fig. 2). Within the vagans clade, O. zanonii is sister to awell-supported clade with O. vagans, O. pseudovagans and O. folio-sum (BS 86, PP 0.98).

Next diverging is a well-supported clade (BS 91, PP 1), termedEu-Orthophytum clade in the following, which comprises all spe-cies with pedunculate inflorescences. Its sister group relationshipto the vagans clade is well supported (BS 96, PP 1). The Eu-Ortho-phytum clade splits into two highly supported clades (B, C) andrelationships between the two clades receive high statistical sup-port (Fig. 2). Clade B consists of the species of the mello-barretoisubgroup (BS 96, PP 1), represented in our sampling with fourout of six species and covering almost the complete geographicdistribution of the subgroup. The BI tree shows Orthophytummello-barretoi as sister species of O. schulzianum (BS 75, PP 0.96),both forming a sister group to O. diamantinense and O. graomogo-lense. In the MP strict consensus tree the phylogenetic relation-ships within the mello-barretoi subcomplex remain unresolved.

Clade C, in the following termed the Core Orthophytum clade, isstrongly supported (BS 100, PP 1) and comprises the majority ofOrthophytum species, all possessing pedunculate inflorescences inlax spikes of spikes or spikes densely arranged and petal apicesobtuse to subacute. Several subclades receive moderate to highsupport but relationships between these subclades remain largelyunclear due to a lack of resolution or statistical support. Notewor-thy groups of the Core Orthophytum clade are the glabrum clade(PP 0.97), the fosterianum clade, the saxicola clade (BS 93, PP 1)and the sucrei clade (Fig. 2).

3.2. State character reconstruction

Both Bayesian and maximum parsimony analyzes recon-structed sessile inflorescences as ancestral character state for thegroup, with two independent shifts to pedunculate inflorescences:(1) in Orthophytum foliosum and (2) in the Eu-Orthophytum clade(Figs. 2 and 3). The character state reconstructions of the corollaform indicates that a tubular corolla represents the ancestral char-acter state in the Cryptanthus-Orthophytum clade with at leasttwo shifts to a clavate corolla in the vagans clade (excluding O.foliosum) and the mello-barretoi clade (Fig. 4A). The ancestral char-acter state reconstructions of petal appendages form indicate thatsacciform lacerate petal appendages represent the ancestral statecharacter in the group and that it can be considered a

Fig. 2. Phylogram of Bayesian analysis of the combined nuclear and plastid data (PHYC, trnL-trnF and trnH-psbA). Numbers above the branches represent posteriorprobabilities (PP) and below these branches are bootstrap values (BS). Numbers after terminal names indicate the chromosome counts.

R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64 59

Fig. 3. Cladogram of Bayesian analysis of the combined nuclear and plastid data set (PHYC, trnL-trnF and trnH-psbA) mapping the presence and absence of a peduncle.

60 R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64

synapomorphy of the amoenum clade. The acute petal appendagesare present in Lapanthus, and the absence of petal appendages isshared by at least two clades including Cryptanthus species(Fig. 4B). In the next diverging lineage (vagans clade) cupuliformlacerate petal appendages were reconstructed as ancestral statewith one shift for sacciform lacerate appendages in Orthophytumfoliosum. Fimbriate appendages appear to be a synapomorphy ofthe Core Orthophytum clade.

4. Discussion

In Bromelioideae, the reconstruction of infrageneric relation-ships based on DNA sequence data has proven difficult due tolow sequence divergence of molecular markers used so far, yield-ing phylogenetic reconstructions with generally low resolutionand support values (Sass and Specht, 2010; Schulte et al., 2009;Sousa et al., 2007; Sousa, 2011). In Bromelioideae, those studieswere conducted within the more advanced Eu-Bromelioideae, thecore bromelioids, which are characterized by the presence of a cen-tral water collecting tank and which apparently underwent a rapidradiation starting around 5.5 Ma mainly in a largely continuoushabitat, the Brazilian Atlantic rainforest (Givnish et al., 2011;Schulte et al., 2005, 2009). In contrast, Orthophytum represents

an early diverging lineage within the Eu-Bromelioideae that diver-sified extensively in xeric vegetation types in Southeast and North-east Brazil, the campos rupestres and Caatinga, with their vastdiversity of naturally isolated microhabitats (the inselbergs), whichpromoted genetic isolation and thus fostered the evolution ofnumerous microendemics in Orthophytum (Smith and Downs,1979). Here we present and discuss the first molecular phylogenyof the genus based on a comprehensive sampling of its knowndiversity. This allowed us to identify several highly supported lin-eages within the genus, to elucidate infrageneric phylogenetic rela-tionships, and to highlight critical issues for future research.

4.1. Phylogenetic relationship within Orthophytum and related genera

The phylogenetic reconstructions depict a well-supported cladeunifying the Orthophytum species with pedunculate inflorescenceplus the vagans clade, which comprises three species with sessileinflorescences and short stem, and one species with a pedunculateinflorescence (O. foliosum) (Figs. 2 and 3).

The species belonging to the amoenum subgroup sensu Leme(2004) formed a well-supported clade (Fig. 2), thus supportingLeme’s taxonomic concept. The subgroup is characterized by ses-sile inflorescences (Fig. 3), short and inconspicuous stems, inner

Fig. 4. Ancestral character reconstructions for floral characters based on the Bayesian tree inference of the combined nuclear and plastid data set (PHYC, trnL-trnF and trnH-psbA). (A) Corolla form. (B) Petal appendages form. Branch colors indicate the ancestral reconstruction under maximum parsimony. Pie diagrams at nodes indicate ancestralcharacter reconstructions under Bayesian framework.

R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64 61

leaves and primary bracts contrasting with the outer leaves incolor, and white petals with a pair of sacciform lacerate append-ages inserted laterally to the antepetalous stamens (Leme, 2004;Louzada and Wanderley, 2010). Leme (2007) also noticed a mor-phological affinity between Cryptanthus subgen. Hoplocryptanthussect. Schwackeanae and Orthophytum, stating that species of thissection are morphologically more similar to the species of Ortho-phytum than to any other Cryptanthus species. This morphologicalsimilarity is especially pronounced among species of the amoenumsubgroup and Cryptanthus subgen. Hoplocryptanthus sect.Schwackeanae (here represented by C. tiradentenis), which mainlydiffer in the presence or absence of petal appendages.

Lapanthus, a genus recently erected to accommodate two aber-rant species (L. itambensis and L. duartei) formerly placed withinthe sessile inflorescence group of Orthophytum (Louzada andVersieux, 2010), forms a well-supported monophyletic clade in sis-ter group position to Cryptanthus tiradentensis (subg. Hoplocryptan-thus), and a highly supported clade uniting the species ofCryptanthus subgen. Cryptanthus, however, this sister group rela-tionship is not supported. The species of Cryptanthus subg. Cryptan-thus are andromonoecious, usually short caulescent with sessileinflorescences, never pseudopedunculate, and usually withoutpetal appendages or calli (Ramírez-Morillo, 1996). In Bromeliaceae,andromonoecy is only reported for Cryptanthus subg. Cryptanthus(Ramírez-Morillo, 1996), therefore this feature represents a poten-tial synapomorphy for the clade. Lapanthus is morphologicallymore similar to Cryptanthus subg. Hoplocryptanthus sect. Schwacke-anae because both groups possess scented and hermaphroditicflowers (Louzada and Versieux, 2010; Ramírez-Morillo, 1996).Moreover, the species of Lapanthus and Cryptanthus subg. Hoplo-cryptanthus sect. Schwackeanae share a similar habitat in the south-

ern portion of the Espinhaço Range, inhabiting quartzite-sandstoneand iron rocky outcrops.

In clade 3, the first divergent lineage and sister group of theremaining Orthophytum species comprises Cryptanthus odoratissi-mus and C. microglaziovii, which belong to the subg. Hoplocryptan-thus, sections Mesophyticae and Hoplocryptanthus, respectively.This subgenus is characterized by a usually long caulescent habit(sometimes short caulescent), sessile inflorescences, and hermaph-roditic flowers.

The next diverging clade unifies the three species of the vaganssubgroup sensu Leme (2004) plus Orthophytum foliosum (Fig. 2).The species of the vagans subgroup possess an interesting morpho-logical affinity to Cryptanthus odoratissimus and C. microglaziovii.They have a long caulescent habit with a sessile inflorescence(Fig. 3), which is rare in Orthophytum but not in Cryptanthus subg.Hoplocryptanthus sect. Hoplocryptanthus, including C. microglaziovii.The molecular phylogeny indicates that the vagans subgroup in itsoriginal circumscription may constitute a paraphyletic lineage, andthat Orthophytum foliosum may need to be included. The latter spe-cies has a pedunculate inflorescence and was placed in the disjunc-tum subgroup by Leme (2004). The position of the three speciesunder the subgroup vagans (Leme, 2004; sessile inflorescenceand long caulescent habit), which is closely related to the peduncu-late species of Orthophytum, raises a few questions. Are the plantsreally long caulescent? Is the structure found in Orthophytum folio-sum really homologous to the peduncle of the other Orthophytumspecies? Developmental and anatomical studies of the pedunclesin Orthophytum are required to address these questions.

The outlined morphological affinities between the well-sup-ported clades outside the Eu-Orthophytum clade revealed in themolecular phylogeny lead us to the conclusion that the generic

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boundaries of Orthophytum, Cryptanthus, and Lapanthus need to becarefully revised as in their current circumscription they may notconstitute monophyletic lineages. To this aim, a broader samplingof the genus Cryptanthus appears vital as well as the inclusion offurther molecular markers. Traditionally, the genera Orthophytumand Cryptanthus have been separated based on the presence orabsence of petal appendages (Orthophytum: present, Cryptanthus:absent). The taxonomic utility of this character for the delimitationof genera in Bromeliaceae has been questioned repeatedly (Brownand Terry, 1992; Zizka et al., 1999), and a recent molecular phylo-genetic study demonstrated that this character is homoplastic insubfamily Bromelioideae (Schulte and Zizka, 2008).

Although the chromosome number 2n = 50 prevails in Bromeli-aceae (Brown et al., 1997; Brown and Gilmartin, 1989; Cotias-de-Oliveira et al., 2004; Gitai et al., 2005; Louzada et al., 2010;Palma-Silva et al., 2004; Ramírez-Morillo and Brown, 2001, amongother), Cryptanthus species studied so far exhibited lower chromo-some counts 2n = 34, 36 or 54 (e.g. Ceita et al., 2008; Ramírez-Morillo, 1996; Ramírez-Morillo and Brown, 2001) whereas in Orth-ophytum a polyploidy series of 2n = 50, 100, and 150 has beenreported (Cotias-de-Oliveira et al., 2000; Louzada et al., 2010). InLapanthus the only report is for L. duartei (=O. supthutii) with2n = 50 (Louzada et al., 2010). Therefore, a potentially useful char-acter to delimit Cryptanthus from Orthophytum/Lapanthus may betheir chromosome numbers if the reduction of chromosome num-bers was the initial isolating event that preceded the evolution ofCryptanthus. Nevertheless, this hypothesis remains to be tested ina phylogenetic framework. So far, chromosome counts for Cryptan-thus are available only for a handful of species (9 spp.; Ramírez-Morillo and Brown, 2001).

4.2. Phylogenetic relationships in Eu-Orthophytum

The first diverging lineage within the Eu-Orthophytum claderepresents the mello-barretoi subgroup sensu Leme (2004, 2008),thus supporting the monophyly of the subcomplex. These speciesare characterized by pedunculate inflorescences with basal pri-mary bracts narrowly triangular and elongate, the usually redinflorescences in congested spikes of glomerules, and green petalswith obtuse-cucullate apices. It remains to be evaluated if the sub-group should be recognized with a formal status within the genus(Leme and Paula, 2008) but this should await the inclusion of O.eddie-estevesii, the northernmost species of the group, in the phy-logenetic analysis.

Core Orthophytum (Fig. 2) arises as sister group to the subgroupmello-barretoi and comprises the majority of Orthophytum species,morphologically characterized by lax inflorescences in spikes ofspikes or spikes, greenish-white petals with white lobes, andobtuse to subacute apices. Within Core Orthophytum four lineageswere found with species that, according to their morphological fea-tures, may be included in the leprosum and the disjunctum sub-group sensu Leme (2004). The leprosum subgroup is mainlycharacterized by leaves that do not form a distinct rosette beforeand at anthesis, and the disjunctum subgroup by possessing a dis-tinct rosette before and after anthesis.

The fosterianum clade comprises six pedunculate species (O.alvimii, O. fosterianum, O. grossiorum, O. gurkenii, O. lanuginosum,O. magalhaesii) with inflorescences in lax spikes of spikes, andleaves and primary bracts with lepidote-lanate indumentum, thelatter representing the morphological synapomorphy of the group(Hutchinson, 1983; Leme and Paula, 2003, 2005). The second line-age in Core Orthophytum, named sucrei clade, comprises O. boude-tianum sister to O. sucrei (BS 84, PP 1), and O. estevesii sister to O.pseudostoloniferum (BS 82, PP 1). These are small sized species withinflorescences that are spikes, inhabiting granitic-gneiss rocky out-crops in the central region of the Brazilian state of Espírito Santo.

A highly-supported clade unifies two other groups termed gla-brum and saxicola clades. The glabrum clade shelters four speciesincluding Orthophytum glabrum, the generic type (Smith, 1955).Orthophytum leprosum occurs as sister to a clade with O. glabrum,O. horridum and O. lucidum, and according Leme (2004) the firsttwo species are placed in the leprosum subgroup, and the remain-ing two other species are placed in the disjunctum subgroup. Basedon the present analysis we conclude that the leprosum subgroup isnot monophyletic, firstly due to O. glabrum being more closelyrelated to O. lucidum and O. horridum than to O. leprosum, and sec-ondly by the placement of O. falconii (leprosum subcomplex) in thesaxicola clade. The combination of features such as stiff coriaceousleaves being glabrous or lepidote with adpressed scales, can char-acterize the species included in the glabrum clade. However, moremorphological studies are necessary in order to better define thegroup.

The final diverging clade is a highly supported group termed thesaxicola clade which includes the terrestrial species Orthophytumfalconii in sister group position to the remaining species of the saxi-cola clade. Orthophytum falconii is known only from the type collec-tion and resembles morphologically O. benzingii (=O. leprosum) duethe absence of a rosette at anthesis (Leme, 2003). For that reason, itwas placed in the leprosum subgroup (Leme, 2004).

The saxicola clade certainly represents the most diversifiedgroup within Core Orthophytum with the dwarf O. saxicola andthe robust O. riocontense. As with the glabrum clade, to defineone or more morphological features, which characterize the saxi-cola clade is a challenging task. In contrast to the other subcladeswithin Core Orthophytum, in the saxicola clade both inflorescencestypes (spikes or lax spikes of spikes) can be found in different spe-cies. For example spikes are observed in O. saxicola and O. harleyiwhereas lax spikes of spikes are present in O. argentum and O. tos-canoi or even both inflorescence types can be found in the samespecies (e.g. O. conquistense).

4.3. Character state reconstructions of key morphological characters

In the past, the genus Orthophytum was informally subdividedinto two major groups according to the inflorescence type, the ses-sile and the pendunculate inflorescence group (Leme, 2004; Smithand Downs, 1979; Wanderley, 1990). Mapping this character ontothe obtained phylogeny revealed that pedunculate inflorescencesevolved twice within Orthophytum, once within the vagans clade(O. foliosum) and in a clade that entirely consists of representativeswith pedunculate inflorescences, termed Eu-Orthophytum cladeand comprising the majority of species. In contrast, the Orthophy-tum species with sessile inflorescences were found in two clades:the amoenum and the vagans clade, which both received moderateto high support values (Fig. 2). Relationships between these twoclades remain unclear due to a lack of resolution in the deepernodes of the phylogeny. Besides these two clades, the genera Cryp-tanthus and Lapanthus both consist of species with sessile inflores-cences. Mapping the inflorescence type onto the phylogenyshowed that sessile inflorescences could be regarded as the plesio-morphic condition in Orthophytum, and pedunculate inflorescencesas the derived one (Fig. 3). Although pedunculate inflorescencesapparently arose twice within the genus, the character state pos-sesses a valuable phylogenetic signal as it characterizes a majorlineage, the Eu-Orthophytum clade.

The vagans clade and the Cryptanthus subg. Hoplocryptanthusclade possess an interesting long caulescent habit due to the elon-gation of the vegetative stem bearing the sessile inflorescence,characterizing probably an intermediate stage in the evolution ofOrthophytum. Although three of four species included in the vagansclade have sessile inflorescences and one pedunculate, all are com-posed of congested spikes of glomerules. The same inflorescence

R.B. Louzada et al. / Molecular Phylogenetics and Evolution 77 (2014) 54–64 63

branching type is found in the next diverging mello-barretoi clade.Moreover, O. pseudovagans, O. vagans and O. zanoni have petalswith obtuse-cucullate apices, only present in these two clades,reinforcing the hypothesis of a clade with intermediate features.

Flower characters such as petal appendages and corolla formhave been widely used for classification in Bromeliaceae (Smithand Downs, 1974, 1977, 1979) and are considered valuable forinter- and intraspecific classification in Cryptanthus, Lapanthusand Orthophytum (Leme, 2004; Louzada and Wanderley, 2010;Louzada and Versieux, 2010). Our ancestral state reconstructionsof the form of the corolla and the petal appendages indicated dif-ferent levels of homoplasy (Fig. 4), which implies a limited taxo-nomic value of these characters in the group.

Corolla form mapped in our phylogeny (Fig. 4A) demonstratesthat clavate and tubular corolla arose at least twice in the evolutionof the three genera. The type of corolla can be associated with spe-cific pollinators, however reproductive biology studies in thesegenera are still incipient.

As mentioned above, petal appendages have been used as adiagnostic character at genus level. This feature has been also high-lighted by Schulte and Zizka (2008) where taxonomic significanceof the presence or absence of this character in higher taxonomiclevels was evaluated in a phylogenetic framework. Furthermore,the authors also emphasized that this character is inappropriatefor generic delimitation in Bromelioideae. Taking into accountthe presence or absence of petal appendages, our data confirmthe results published by Schulte and Zizka (2008) (Fig. 4B), how-ever the form of these appendages have not been evaluated atthe infra-generic level before.

The presence of petal appendages seems to be an ancestralcharacter in the group with the appearance of sacciform lacerateappendages in the amoenum clade (Figs. 2 and 4B). In the nexttwo diverging lineages, which comprise two clades of Cryptanthusgenus, the petal appendages are absent and a second independentorigin of petal appendages is inferred in the clade including Eu-Orthophytum and vagans clades.

Our character state reconstruction showed that the sacciformlacerate petal appendages arose two times independently andtherefore are homoplastic. Interestingly, the species of the amoe-num and mello-barretoi clades (Figs. 2 and 4B) that present sacci-form lacerate appendages are distributed in two extremes of thesame mountain chain (Espinhaço Range), indicating the conver-gent evolution of petal appendages form in the same environment.

Fimbriate, acute and cuppuliform lacerate petal appendages areexclusive characters of the Core Orthophytum, Lapanthus andvagans clades respectively, and may be putative synapomorphiesof these clades.

5. Conclusions

The study presents the first phylogeny of the Brazilian endemicgenus Orthophytum. The presented results show the first molecularphylogenetic evidence that Orthophytum as well as its two tradi-tional morphological groups (sessile inflorescence and pedunculategroups) may not be monophyletic. However, with the weak inter-nal node support in trees from the different phylogenetic analyzes,we cannot confirm the non-monophyletic status of Orthophytum.Additionally, Cryptanthus species appear at least in the two lin-eages. However, the two traditional groups amoenum and mello-barretoi were confirmed as natural groups with high statisticalsupport.

Mapping the inflorescence type onto the phylogeny showed thepedunculate condition as derived in the evolution of Orthophytumand related. Bayesian and parsimony ancestral state characterreconstructions indicated varying level homoplasy in the form of

the corolla and the petal appendages, two characters which are fre-quently used as diagnostic in Cryptanthus, Lapanthus andOrthophytum.

Finally, for future studies in Orthophytum we suggest the inclu-sion of additional variable molecular markers to assess the mono-phyly of the genus as well as to include a wider sampling forCryptanthus. Furthermore, phylogeographic studies in closelyrelated Orthophytum species would be desirable to increase ourunderstanding of interspecific relationships.

Acknowledgments

The authors thank São Paulo Research Foundation/FAPESP(graduate research fellowship RBL 08/52912-5 and CP-S 2009/52725-3) and CNPq for financial support; IBAMA, IEF for collectionpermits; Lisa Campbell for language editing; Maria Cláudia Medei-ros, Gisele Silva, Marlon Machado, Geyner Alves, Diego Pinangé,Ana Paula Prata, Daniel Melo, Rodrigo Oliveira for assistance duringfield work; Ana Maria Benko-Iseppon, Fábio Pinheiro, Diego Pin-angé, Geyner Alves, Rodrigo Pegorin and Cesar Redivo for lab assis-tance; Leonardo M. Versieux, Rafaela C. Forzza, Tânia Wendt,Tarciso Filgueiras and two anonymous reviewers and editor fortheir much valuable comments on the paper.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2014.03.007.

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