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Molecular phylogenetics of the species-rich genus Habenaria (Orchidaceae)in the New World based on nuclear and plastid DNA sequences

João A.N. Batista a,⇑, Karina S. Borges a, Marina W.F. de Faria a, Karina Proite a, Aline J. Ramalho a,Gerardo A. Salazar b, Cássio van den Berg c

a Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Pampulha, C.P. 486, Belo Horizonte,Minas Gerais 31270-910, Brazilb Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de Mexico, Apartado Postal 70-367, 04510 Mexico, DF, Mexicoc Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina s/n, Feira de Santana, Bahia 44036-900, Brazil

a r t i c l e i n f o

Article history:Received 17 September 2012Revised 2 January 2013Accepted 8 January 2013Available online 19 January 2013

Keywords:OrchidaceaeHabenariaNeotropicalPhylogenyITSmatK

a b s t r a c t

Habenaria is a large genus of terrestrial orchids distributed throughout the tropical and subtropicalregions of the world. The integrity and monophyly of this genus have been under discussion for manyyears, and at one time or another, several genera have been either included in a broadly defined Habena-ria or segregated from it. In this study, the phylogenetic relationships of the Neotropical members of thegenus and selected groups of African Habenaria were investigated using DNA sequences from the nuclearinternal transcribed spacer (ITS) region and the plastid matK gene sampled from 151 taxa of Habenariafrom the Neotropics (ca. 51% of the total) as well as 20 species of Habenaria and Bonatea from the OldWorld. Bayesian and parsimony trees were congruent with each other, and in all analyses, the Neotrop-ical species formed a highly supported group. African species of Habenaria in sections Dolichostachyae,Podandria, Diphyllae, Ceratopetalae and Bilabrellae, and the Neotropical clade formed a highly supported‘‘core Habenaria clade’’, which includes the type species of the genus from the New World. The topologyof the trees indicates an African origin for the Neotropical clade and the low sequence divergence amongthe Neotropical species suggests a recent radiation of the genus in the New World. Species of Bonatea andHabenaria sections Chlorinae and Multipartitae formed a well-supported clade that was sister to the ‘‘coreHabenaria clade’’. The Neotropical clade consists of at least 21 well-supported subgroups, but all Neotrop-ical sections of the current sectional classification are paraphyletic or polyphyletic and will need exten-sive revision and recircumscription. Most of the Neotropical subgroups formed morphologically uniformassemblage of species, but some cases of morphological divergence within subgroups and convergencebetween subgroups indicated that morphology alone can be misleading for inferring relationships withinthe genus. The genera Bertauxia, Kusibabella and Habenella, segregated from New World Habenaria, arenot monophyletic and a revision of the sectional classification rather than a generic division seems mostappropriate. Our results do not support an extensive generic fragmentation of Habenaria as previouslysuggested and will provide a framework for revising the infrageneric classification and investigatingthe patterns of morphological evolution and geographical distribution of the genus in the New World.

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1. Introduction

Habenaria Willd. (Orchidinae, Orchidoideae, Orchidaceae) is alarge genus of approximately 876 (Govaerts et al., 2011) terrestrialspecies distributed throughout the tropical and subtropical regionsof the Old and New World (Pridgeon et al., 2001a) with centers ofdiversity in Brazil, southern and central Africa and East Asia(Kurzweil and Weber, 1992). Most species are perennial, deciduous

geophytes with a growth cycle associated with a wet season fol-lowed by a dormant period in the form of an underground root tu-ber during the dry season. In the New World the genus accounts fora large proportion of the Orchidaceae of tropical and subtropicalgrasslands (Barros, 1987; Toscano de Brito, 1995; Mendonçaet al., 1998; Batista and Bianchetti, 2003; Batista et al., 2004; Zappiet al., 2003), including the savannas of the Brazilian Amazon (Batis-ta et al., 2008a,b), and is poorly represented in tropical forests. Bra-zil and Mexico are the countries with the highest numbers ofspecies in the New World with 167 and 72 species, respectively.In Brazil, the center of diversity of the genus is the cerrado, a spe-cies-rich savanna that covers approximately two million square

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⇑ Corresponding author. Fax: +55 31 3409 2671.E-mail address: [email protected] (J.A.N. Batista).

Molecular Phylogenetics and Evolution 67 (2013) 95–109

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kilometers of central Brazil (Ratter et al., 1997), whereas in Mexico,the greatest diversity is found in tropical and subtropical oak-coniferous forests (Batista et al., 2011a).

Traditionally, Habenaria has been placed in subtribe Habenarii-nae, which, together with Orchidinae, forms tribe Orchideae(Dressler, 1993). The separation of the two subtribes is based pri-marily on stigma morphology: Orchidinae have concave, unstalkedstigmas, often with confluent lobes, whereas Habenariinae havestalked, convex stigma lobes that are usually distinct, but the sep-aration is questionable (Kurzweil and Weber, 1992; Pridgeon et al.,2001a). The tribe Orchideae, with approximately 62 genera and1800 species, is particularly well represented in the Afro-Madaga-scan region (Dressler, 1993; Pridgeon et al., 2001a). Habenaria is byfar the largest genus in Orchideae, comprising approximately 45%of the species assigned to the tribe, followed by Platanthera Rich.(200 spp.) and Cynorkis Thouars (125 spp). Only Habenaria is foundthroughout the American tropics, although a few species of thepredominantly north-temperate genus Platanthera extend southto Mexico and Guatemala.

The integrity of Habenaria as a genus has been under discussionfor many years. Species currently placed in distinct genera such asPlatanthera and Coeloglossum Hartm. were formerly placed in Hab-enaria. Excluding the consistently stalked and strongly convex stig-ma lobes, which are also found in other Habenariinae, thecharacters used for defining Habenaria show extensive variation.Habenaria is currently distinguished from other closely relatedgenera by often bifid petals that are not fused to other parts ofthe flower, a lip that is usually deeply divided and lacking a callusand entire stigma lobes, which are usually free and not adnate tothe petals or lip (Pridgeon et al., 2001a). Some African species for-merly included in Habenaria were segregated to genera such asBonatea Willd., Centrostigma Schltr., Platycoryne Rchb.f. and Roep-erocharis Rchb.f. However, according to Kurzweil and Weber(1992), they are similar in most characters to Habenaria speciesand are better treated as specialized forms of the genus at the sec-tional rank.

The only worldwide revisions of Habenaria were those ofKränzlin (1892, 1901) in which 32 sections were recognized. Char-acterization of the sections was based primarily on the degree ofdissection of the petals and lip and on gynostemium structure,particularly the length of the stigmas. After Kränzlin, few authorshave treated the infrageneric classification of Habenaria. Schlech-ter (1915) renamed some of the sections and Summerhayes(1942, 1960, 1962, 1966) and Hunt (1968) proposed new sections,but these works exclusively addressed African species. For theNew World, Cogniaux (1893) generally followed Kränzlin’s sec-tional characterization in his treatment of Habenaria in Flora Bra-siliensis, whereas Hoehne (1940) used a different approach in therevision of the genus for Flora Brasilica, dividing the Brazilian spe-cies into nine informal groups starting with the vegetative partsand then advancing toward the details of the flowers. In the lastmajor survey of Brazilian orchids, Pabst and Dungs (1975) basi-cally followed the divisions established by Hoehne, using somenew characters to distinguish several groups that they calledalliances.

González-Tamayo (1993) divided the Mexican species into 12tentative natural groups but did not make any reference to pre-vious sectional treatments of the genus. More recently, Szlachet-ko recognized three genera within New World Habenaria:Bertauxia Szlach., Kusibabella Szlach. (Szlachetko, 2004a,b) andHabenella Small (Szlachetko and Kras, 2006). However, hiswork was undertaken on a piecemeal basis based on floral mor-phological characters, and his genera have not been widelyaccepted.

The floral morphology of Southern African Habenariinae wascharacterized in detail by Kurzweil and Weber (1992). A similar

study is not available for the Neotropical Habenaria, neither is acomparative analysis between the New World and Old World spe-cies of the genus. However, an analysis of the literature reveals thatthe floral morphology of the Old World species is much more di-verse than those of the New World. This observation reflectsKränzlin’s sectional classification in which, out of 32 sections,only 12 were from the New World. In fact, compared to the Africanand Asian groups of the genus, the floral morphology of the NewWorld species is more homogenous, particularly for thegynostemium.

Despite being the largest genus in the tribe Orchideae and hav-ing a worldwide distribution, Habenaria is underrepresented inmolecular systematic studies, especially compared with other gen-era in the tribe such as Orchis Tourn. ex L., Ophrys L. and Platanther-a, which have been extensively investigated (Pridgeon et al., 1997;Bateman et al., 1997, 2003). This is most likely because Habenariaoccurs mostly in the Southern Hemisphere and few species are cul-tivated, rendering access to genetic material difficult. Thus far, theonly study addressing the phylogeny of Habenaria using a cladisticapproach and DNA sequence data was the phylogenetic analysis ofOrchidinae and selected Habenariinae of Bateman et al. (2003).Aside from this work, a few species of Habenaria have been se-quenced in the context of general phylogenetic analyses of Orchid-aceae or their infrafamilial ranks (Cameron et al., 1999; Douzeryet al., 1999; Kores et al., 2001; Bellstedt et al., 2001; Ponsie et al.,2007a) and the DNA barcoding of land plants (Lahaye et al.,2008). The study of Bateman et al. (2003) using ITS sequence dataindicated that Habenaria was highly polyphyletic. However, onlyeight species of Habenaria were sampled (�1% of the genus),including only one from the New World. Contrary to the resultsof Bateman et al. (2003), the relatively homogeneous floral mor-phology of the New World species, particularly in regard to thegynostemium, suggests that they could be a monophyletic group.The similarities in floral morphology between New World speciesand some Old World species also suggests a close relationship be-tween some African and American groups of Habenaria, but thecurrently available taxon sampling for molecular studies is insuffi-cient to address these phylogenetic questions. A phylogenetichypothesis for the New World species of the genus should also helpclarify the infrageneric classification of the genus and be useful forredefining the sections as a step toward a generic revision.

In recent years, the use of DNA sequence data has proven usefulfor inferring phylogenetic relationships in Orchidaceae at severaltaxonomic levels (Cameron et al., 1999; van den Berg et al., 2000,2005; Pridgeon et al., 2001b; Williams et al., 2001; Salazar et al.,2003; Freudenstein et al., 2004; review in Cameron, 2007), includ-ing the infrageneric relationships in some large genera of the fam-ily (Bytebier et al., 2007; Fischer et al., 2007; Russell et al., 2009;Whitten et al., 2007). The internal transcribed spacers (ITSs) ofthe nuclear ribosomal multigene family and, to a lesser extent,the maturase K (matK) gene of the plastid genome, have providedgood resolution in phylogenetic analyses of several groups withinOrchidaceae, including the tribe Orchideae (Pridgeon et al., 1997;Bateman et al., 1997, 2003; Ponsie et al., 2007a). Here, we takeadvantage of the large number of Habenaria species occurring inBrazil and Mexico and the availability of some sequence data fromAfrican species in the databanks to investigate the phylogeny of thegenus in the New World, using DNA sequences from the nuclearITS region and the plastid matK gene, focusing on Brazilian andMexican species, with the following objectives: (1) to establishwhether Neotropical species of Habenaria form a monophyleticgroup; (2) to investigate the relationships between Neotropicaland selected groups of African Habenaria and other genera of Habe-nariinae; and (3) to evaluate the current sectional classificationof New World species in light of an explicit phylogenetichypothesis.

96 J.A.N. Batista et al. / Molecular Phylogenetics and Evolution 67 (2013) 95–109


2. Materials and methods

2.1. Taxon sampling

A total of 217 terminals were used consisting of 180 species, ofwhich 152 were Neotropical Habenaria species, corresponding to51% of the total number of species known from the Neotropics (Ba-tista et al., 2011a,b). Additionally, the total included ten AfricanHabenaria species, ten species of Bonatea and eight species ofCynorkis, Gennaria Parl., Satyrium L., Stenoglottis Lindl., Platanthera,Orchis and Disa P.J. Bergius from the Old World (Table 1). The lattergenus (tribe Diseae) was used as a functional outgroup. The sam-pling of the New World Habenaria species was concentrated in Bra-zil and Mexico, included species from all currently recognizedsections and informal groups except the monospecific sectionPycnostachyae Cogn. (Table 2), and covered most of the morpholog-ical variability and geographic distribution of the genus in the Neo-tropics. Most of the plant material was collected in the field anddried in silica gel, but in a few instances, herbarium specimenswere used. Taxa from Brazil were identified by J.A.N. Batista andtaxa from Mexico by G.A. Salazar. Several species with broad geo-graphic distributions or significant morphological variability weresampled more than once. In some cases, additional samples werealso sequenced to confirm the position of a species in the trees.Voucher information, geographic origin and GenBank accessionnumbers are listed in Table 1.

2.2. Molecular markers

Nucleotide sequences from one nuclear (ITS) and one plastid(matK) genome region were used in the analyses. The ITS regionconsisted of the 30 and 50 ends of the 18S and 26S ribosomal RNAgenes, respectively, the internal transcribed spacers (ITS1 andITS2) and the intervening gene 5.8S of the nuclear ribosomal multi-gene family. Amplifications were performed using the primers17SE and 26SE (Sun et al., 1994). For the matK gene, we amplifiedan internal fragment of approximately 630 bp using the primersmatK-F2 (50-CTAATACCCCATCCCATCCAT-30) and matK-R2 (50-CCCAATACAGTACAAAATTGAGC-30). This fragment correspondsapproximately to the same region used in the phylogeny of Bonatea(Ponsie et al., 2007a) and for the barcoding of land plants (Chaseet al., 2007), and it corresponds to the most variable region ofthe gene in several orchid groups (e.g., Whitten et al., 2000). Selec-tion of the markers was based on the ease of amplification, avail-ability of sequences from other genera of Old World Orchideae inpublic data banks and the general use of the markers in phyloge-netic studies of Orchidaceae.

2.3. DNA extraction, amplification and sequencing

Total DNA was extracted from individual plants using a modi-fied version of the 2 � CTAB protocol of Doyle and Doyle (1987).PCR amplifications were performed in a MJ96 Thermocycler. Thegeneral PCR system consisted of 20–50 ng of genomic DNA, 1XPCR Buffer, 2 mM MgCl2, 200 lM dNTPs, 0.4 lM of each primer,2 U of Taq DNA Polymerase (Phoneutria Biotec., Belo Horizonte,Brazil) and water to reach a total volume of 25 ll. Cycling condi-tions were an initial denaturation at 94 �C for 3 min, 35 cycles of94 �C for 45 s, 58 �C for 45 s and 72 �C for 1 min, and a final exten-sion for 3 min at 72 �C. For reactions with low yield or unspecificamplification products, the conditions above in the PCR systemand cycle parameters were individually adjusted. In contrast withother reports (van den Berg et al., 2005), the use of denaturing re-agents such as betaine and DMSO in the ITS amplification did notincrease yield or specificity and were therefore not used. PCR prod-

ucts with single bands were purified using polyethylene glycol pre-cipitation and sequenced in a MegaBACE 1000 (AmershamBiosciences) automatic sequencer following the manufacturer’sprotocol. Some sequences were produced by Macrogen Inc., Korea.All regions were sequenced bi-directionally. Doubtful base callswere, in most cases, verified with a third sequencing reaction.

2.4. Sequence analysis, alignment and pairwise distance calculation

DNA sequence electropherograms were edited with the STADENpackage software (Bonfield et al., 1995) or Sequencher version 4.8(GeneCodes Corp., Ann Arbor, Michigan, USA). The edited se-quences were aligned with MUSCLE (Edgar, 2004), and the result-ing alignment was manually adjusted using MEGA4 software(Tamura et al., 2007). No data were excluded from the analysesdue to ambiguous alignment. Individual gap positions were treatedas missing data. Pairwise distances between the sequences werecalculated using the Maximum Composite Likelihood method inMEGA4 (Tamura et al., 2004, 2007). In the pairwise analyses, thepositions containing gaps and missing data were eliminated fromthe data set (complete deletion option).

2.5. Phylogenetic analyses

The data were analyzed using both parsimony and Bayesianinference. Phylogenetic analyses using parsimony were performedin PAUP� version 4 (Swofford, 1998) with Fitch parsimony (equalweights, unordered characters; Fitch, 1971) as the optimality crite-rion. GenBank sequences from representative species of the OldWorld tribe Diseae (Disa) were used as outgroups. Searches wereinitially performed separately on each data set, and because nocases of strongly supported incongruence were detected (i.e., noconflicting groups were observed between the two data setsobtaining high internal support), a third search was performedwith a combined matrix. Each search consisted of 1000 replicatesof random taxon addition with branch swapping using the TBR(tree-bisection and reconnection) algorithm, retaining only up toten trees per replicate to avoid extensive swapping on suboptimalislands. Internal support was evaluated by character bootstrapping(Felsenstein, 1985) using 1000 replicates, simple addition and TBRbranch swapping, retaining up to ten trees per replicate. For boot-strap support levels, we considered bootstrap percentages (BPs) of50–70% as weak, 71–85% as moderate and >85% as strong (Kresset al., 2002).

A model-based phylogenetic analysis using Markov chainMonte Carlo-based Bayesian inference was performed using MrBa-yes v3.1.2 (Ronquist et al., 2005), treating each DNA region (ITS andmatK) as a separate partition. An evolutionary model for each DNAregion was selected with MrModeltest 2 (Nylander, 2004). For theboth data sets the GTR + I + G model was selected according to theAkaike Information Criterion (AIC) or Hierarchical Likelihood RatioTests (hLRTs). Each analysis consisted of two independent runswith four chains for 3,000,000 generations, sampling one treeevery 100 generations. In the combined analysis, to improve theswapping of chains, the temperature parameter for heating thechains was lowered to 0.05. After discarding the first 25% of thetrees as the burn-in period, the remaining trees were used to assesstopology and posterior probabilities (PP) in a majority-rule consen-sus. Because PP in Bayesian analysis are not equivalent to BP butare generally much higher (Erixon et al., 2003), we used criteriasimilar to a standard statistical test, considering groups withPP > 95% as strongly supported, PP 90–95% as moderately sup-ported and PP < 90% as weakly supported.

J.A.N. Batista et al. / Molecular Phylogenetics and Evolution 67 (2013) 95–109 97


Table 1Voucher information and GenBank accession numbers for the sequences analyzed in this study.

Taxon Voucher Origina ITS matK

DiseaeDisa ochrostachya Rchb.f. GenBank Africa DQ414966 DQ415109Disa uniflora P.J.Bergius GenBank Africa DQ414864 DQ415007Satyrium bicorne (L.) Thunb. GenBank Africa AY704978 EF612539

OrchideaeBonatea antennifera Rolfe GenBank Africa DQ522049 DQ522082Bonatea boltonii (Harv.) Bolus GenBank Africa DQ522054 DQ522083Bonatea bracteata G.McDonald & McMurtry GenBank Africa DQ522057 DQ522084Bonatea cassidea Sond. GenBank Africa DQ522059 DQ522085Bonatea lamprophylla J. Stewart GenBank Africa DQ522060 DQ522086Bonatea polypodantha (Rchb.f.) L. Bolus GenBank Africa DQ522062 DQ522087Bonatea porrecta (Bolus) Summerh. GenBank Africa DQ522064 DQ522088Bonatea pulchella Summerh. GenBank Africa DQ522066 DQ522089Bonatea saundersioides (Kraenzl. & Schltr.) Cortesi GenBank Africa DQ522067 DQ522090Bonatea speciosa (L.f.) Willd. GenBank Africa DQ522069 DQ522091Cynorkis grandiflora Ridl. GenBank Africa EF079186 EF065584Gennaria diphylla (Link) Parl. GenBank Africa AY351380 AY368383Habenaria achalensis Kraenzl. Batista 2506 (BHCB) Brazil, RS HM777526 HM777794Habenaria alata Hook. Nava 1784 (MEXU) Mexico HF560562 HF560586Habenaria alpestris Cogn. Batista 1576 (BHCB) Brazil, DF HM777655 HM777952Habenaria anisitsii Kraenzl. Pereira-Silva 4794 (CEN) Brazil, GO HM777668 HM777920Habenaria araneiflora Barb.Rodr. Batista 2521 (BHCB) Brazil, PR HM777527 HM777795Habenaria aranifera Lindl. Batista 2472 (BHCB) Brazil, RS HM777626 HM777819Habenaria arenaria Lindl. Salazar 6407 (K) Africa HF560563 HF560587Habenaria arenaria Lindl. GenBank Africa DQ522073 DQ522092Habenaria armata Rchb.f. Batista 1297 (CEN) Brazil, DF HM777677 HM777931Habenaria australis J.A.N. Bat., A.A. Vale and Menini Batista 2496 (BHCB) Brazil, RS HM777724 HM777988Habenaria ayangannensis Renz Batista 1919 (BHCB) Brazil, MG HM777706 HM777934Habenaria bahiensis Schltr. Batista 2867 (BHCB) Brazil, MG HM777562 HM777808Habenaria balansae Cogn. Batista 2382 (BHCB) Brazil, GO HM777682 HM777884Habenaria balansae Cogn. Batista 2336 (BHCB) Brazil, MG HM777683 HM777883Habenaria batesii la Croix Pollard 731 (YA) Cameroon HF560564 HF560588Habenaria brachyphyton Schltr. Batista 2515 (BHCB) Brazil, RS HM777557 HM777797Habenaria bractescens Lindl. Batista 2529 (BHCB) Brazil, PR HM777615 HM777839Habenaria brevidens Lindl. Batista 2616 (BHCB) Brazil, MG HM777535 HM777903Habenaria brevidens Lindl. Batista 2617 (BHCB) Brazil, MG KC257473 HM777902Habenaria brevilabiata A. Rich. and Galeotti Nava 1116 (MEXU) Mexico HF560565 HF560589Habenaria caldensis Kraenzl. Batista 1798 (BHCB) Brazil, MG HM777646 HM777882Habenaria caldensis Kraenzl. Batista 250 (CEN) Brazil, GO HM777645 HM777881Habenaria calicis R. González Salazar 8184 (MEXU) Mexico HF560566 HF560590Habenaria canastrensis J.A.N. Bat. and B.M.Carvalho Batista 1806 (BHCB) Brazil, MG HM777726 HM777941Habenaria aff. canastrensis J.A.N. Bat. and B.M. Carvalho Batista 1825 (BHCB) Brazil, MG HM777708 HM777940Habenaria cardiostigmatica J.A.N. Bat. and Bianch. Batista 2939 (BHCB) Brazil, DF HM777575 HM778018Habenaria ciliatisepala J.A.N. Bat. and Bianch. Batista 1610 (BHCB) Brazil, MG HM777567 HM777997Habenaria ciliatisepala J.A.N. Bat. and Bianch. Batista 1558 (CEN) Brazil, GO HM777565 HM777998Habenaria clavata (Lindl.) Rchb.f. GenBank Africa DQ522074 DQ522093Habenaria clypeata Lindl. Jacob 461 (MEXU) Mexico HF560567 HF560591Habenaria coxipoensis Hoehne Batista 372 (CEN) Brazil, DF HM777536 HM777905Habenaria aff. coxipoensis Hoehne Batista 2523 (BHCB) Brazil, PR HM777543 HM777907Habenaria crassicornis Lindl. Salazar 7330 (MEXU) Mexico HF560568 HF560592Habenaria crucifera Rchb.f. & Warm. Batista 1826 (BHCB) Brazil, MG HM777574 HM778014Habenaria crucifera var. brevidactyla J.A.N. Bat. and Bianch. Batista 3062 (BHCB) Brazil, DF KC257471 KC257476Habenaria cryptophila Barb.Rodr. Batista 1488 (CEN) Brazil, GO HM777582 HM777870Habenaria culicina Rchb.f. and Warm. Batista 1345 (CEN) Brazil, GO HM777571 HM778012Habenaria cultellifolia Barb.Rodr. Batista 1487 (CEN) Brazil, GO HM777675 HM777923Habenaria curti-bradei Hoehne Batista 2372 (BHCB) Brazil, DF HM777667 HM777918Habenaria curvilabria Barb.Rodr. Batista 1573 (BHCB) Brazil, GO HM777598 HM777860Habenaria curvilabria Barb.Rodr. van den Berg 1267 (HUEFS) Brazil, GO HM777599 HM777859Habenaria depressifolia Hoehne Batista 2369 (BHCB) Brazil, DF HM777601 HM777868Habenaria distans Griseb. Amaral 18 (CEN) Brazil, DF HM777630 HM777871Habenaria distans Griseb. Jiménez 2662 (AMO) Mexico HF560569 HF560593Habenaria dives Rchb.f. GenBank Africa DQ522075 DQ522095Habenaria edwallii Cogn. Batista 1717 (BHCB) Brazil, MG HM777564 HM777803Habenaria aff. edwallii Cogn. Batista 2395 (BHCB) Brazil, MG HM777553 HM777807Habenaria aff. edwallii Cogn. Batista 247 (CEN) Brazil, GO HM777554 HM777806Habenaria egleriana J.A.N. Bat. and Bianch. Batista 535 (CEN) Brazil, GO HM777694 HM777963Habenaria egleriana J.A.N. Bat. and Bianch. van den Berg 1224 (HUEFS) Brazil, GO HM777692 HM777964Habenaria egleriana J.A.N. Bat. and Bianch. Batista 2378 (BHCB) Brazil, GO HM777695 HM777962Habenaria eustachya Rchb.f. Salazar 6239 (PMA) Panama HF560570 HF560594Habenaria exaltata Barb.Rodr. Batista 2771 (BHCB) Brazil, MG HM777621 HM777829Habenaria aff. fillifera S. Watson Salazar 7324 (MEXU) Mexico HF560571 HF560595Habenaria aff. flexuosa Lindl. Zárate 401 (MEXU) Mexico HF560572 HF560596Habenaria fluminensis Hoehne Mota 3571 (BHCB) Brazil, MG HM777659 HM777945Habenaria glaucophylla Barb.Rodr. var. glaucophylla Batista 761 (CEN) Brazil, DF HM777631 HM777875



Table 1 (continued)


Habenaria glaucophylla var. brevifolia Cogn. Batista 2428 (BHCB) Brazil, MG HM777632 HM777876Habenaria glazioviana Kraenzl. ex Cogn. Pansarin s.n. (BHCB) Brazil, MG HM777545 HM777849Habenaria gonzalez-tamayoi García-Cruz, R. Jiménez and L. Sánchez Salazar 6461 (MEXU) Mexico HF560573 HF560597Habenaria gourlieana Gill. ex Lindl. Batista 344 (CEN) Brazil, DF HM777612 HM777844Habenaria guadalajarana S. Watson Jiménez 2691 (AMO) Mexico HF560574 HF560598Habenaria aff. guadalajarana S. Watson Salas 6025 (MEXU) Mexico HF560575 HF560599Habenaria guilleminii Rchb.f. Batista 1795 (BHCB) Brazil, MG HM777539 HM777900Habenaria guilleminii Rchb.f. Batista 2414 (BHCB) Brazil, MG HM777542 HM777898Habenaria aff. guilleminii Rchb.f. Batista 2592 (BHCB) Brazil, MG HM777541 HM777899Habenaria gustavo-edwallii Hoehne Batista 2537 (BHCB) Brazil, MG HM777529 HM777793Habenaria hamata Hoehne Batista 1519 (CEN) Brazil, DF HM777586 HM777865Habenaria henscheniana Barb.Rodr. Mota 1584 (BHCB) Brazil, SC HM777622 HM777827Habenaria henscheniana Barb.Rodr. Batista 2802 (BHCB) Brazil, MG HM777623 HM777828Habenaria heptadactyla Rchb.f. Batista 674 (CEN) Brazil, DF HM777653 HM777956Habenaria heringeri Pabst Batista 1789 (BHCB) Brazil, DF HM777602 HM777915Habenaria hexaptera Lindl. Batista 59 (CEN) Brazil, DF HM777537 HM777908Habenaria hexaptera Lindl. Batista 2399 (BHCB) Brazil, MG HM777538 HM777909Habenaria hieronymi Kraenzl. Batista 2497 (BHCB) Brazil, RS HM777524 HM777924Habenaria humilis Cogn. Batista 1901 (BHCB) Brazil, MG HM777581 HM777879Habenaria ibarrae R. González Nava s.n. (AMO) Mexico HF560576 HF560600Habenaria imbricata Lindl. Batista 1123 (CEN) Brazil, DF HM777648 HM777926Habenaria imbricata Lindl. Batista 2513 (BHCB) Brazil, RS HM777650 HM777927Habenaria aff. imbricata Lindl. Batista 2950 (BHCB) Brazil, GO HM777678 HM777928Habenaria itacolumia Garay Batista 1380 (CEN) Brazil, MG HM777723 HM777975Habenaria itatiayae Schltr. Mota 3566 (BHCB) Brazil, MG HM777663 HM777894Habenaria jaguariahyvae Kraenzl. Batista 1827 (BHCB) Brazil, MG HM777669 HM777919Habenaria johannensis Barb.Rodr. Mota 2777 (BHCB) Brazil, MG HM777609 HM777841Habenaria josephensis Barb.Rodr. Batista 2452 (BHCB) Brazil, MG HM777596 HM777854Habenaria juruenensis Hoehne Batista 1548 (CEN) Brazil, DF HM777531 HM777897Habenaria kleinii Menini and J.A.N. Bat. Klein 13 (UPCB) Brazil, RS KC257469 KC257477Habenaria laevigata Lindl. GenBank Africa DQ522076 DQ522096Habenaria lavrensis Hoehne Batista 1497 (CEN) Brazil, DF HM777710 HM777938Habenaria lavrensis Hoehne Batista 673 (CEN) Brazil, DF HM777711 HM777937Habenaria leprieurii Rchb.f. Batista 1595 (BHCB) Venezuela HM777660 HM777951Habenaria leprieurii Rchb.f. Batista 1624 (BHCB) Brazil, MG HM777661 HM777950Habenaria aff. leprieurii Rchb.f. Batista 2954 (BHCB) Brazil, GO HM777547 HM777968Habenaria leptoceras Hook. Batista 2658 (BHCB) Brazil, RJ HM777597 HM777855Habenaria leucosantha Barb.Rodr. Batista 1604 (BHCB) Brazil, DF HM777568 HM777790Habenaria lithophila Schltr. GenBank Africa DQ522077 DQ522098Habenaria longicauda Hook. Batista 1590 (BHCB) Brazil, PA HM777608 HM777843Habenaria ludibundiciliata J.A.N. Bat. and Bianch. Jardim 4529 (HUEFS) Brazil, PA HM777639 HM778007Habenaria ludibundiciliata J.A.N. Bat. and Bianch. Batista 1372 (CEN) Brazil, MA HM777638 HM778006Habenaria macilenta (Lindl.) Rchb.f. Batista 2393 (BHCB) Brazil, MG HM777606 HM777811Habenaria macilenta (Lindl.) Rchb.f. Batista 2378a (BHCB) Brazil, GO HM777607 HM777814Habenaria macilenta (Lindl.) Rchb.f. Batista 2354 (BHCB) Brazil, DF KC257472 HM777813Habenaria macroceratitis Willd. Chávez s.n. (MEXU) Mexico HF560577 HF560601Habenaria macronectar (Vell.) Hoehne Batista 2519 (BHCB) Brazil, PR HM777614 HM777833Habenaria macvaughiana R. González Nava s.n. (AMO) Mexico HF560578 HF560602Habenaria magdalenensis Hoehne Batista 2026 (BHCB) Brazil, MG HM777595 HM777858Habenaria magniscutata Catling Batista 1227 (CEN) Brazil, GO HM777641 HM777880Habenaria mannii Hook.f. Salazar 6314 (YA) Cameroon HF560579 HF560603Habenaria aff. meeana Toscano Batista 2028 (BHCB) Brazil, MG HM777713 HM777965Habenaria melanopoda Hoehne and Schltr. Batista 1832 (BHCB) Brazil, MG HM777689 HM777890Habenaria melanopoda Hoehne and Schltr. Batista 1810 (BHCB) Brazil, MG HM777686 HM777887Habenaria melanopoda Hoehne and Schltr. Batista 2471 (BHCB) Brazil, RS HM777687 HM777889Habenaria melanopoda Hoehne and Schltr. Batista 2539 (BHCB) Brazil, MG HM777688 HM777888Habenaria aff. melanopoda Hoehne and Schltr. Batista 2438 (BHCB) Brazil, MG HM777681 HM777892Habenaria mello-barretoi Brade and Pabst Batista 2666 (BHCB) Brazil, MG HM777685 HM777886Habenaria monorrhiza (Sw.) Rchb.f. Salazar 7638A (MEXU) Ecuador HF560580 HF560604Habenaria montevidensis Spreng. Batista 2479 (BHCB) Brazil, RS HM777619 HM777826Habenaria montiswilhelminae Renz Batista 1555 (CEN) Brazil, DF HM777714 HM777957Habenaria montiswilhelminae Renz Batista 2493 (BHCB) Brazil, RS HM777719 HM777960Habenaria montiswilhelminae Renz Batista 1811 (BHCB) Brazil, MG HM777693 HM777961Habenaria aff. rodriguesii Cogn. Batista 970 (CEN) Brazil, DF HM777652 HM777986Habenaria mystacina Lindl. Batista 1812 (BHCB) Brazil, MG HM777728 HM777970Habenaria nabucoi Ruschi Pivari 549 (BHCB) Brazil, MG HM777611 HM777840Habenaria nasuta Rchb.f. and Warm. Batista 1572 (BHCB) Brazil, GO HM777716 HM777959Habenaria nemorosa Barb.Rodr. Batista 2567 (BHCB) Brazil, MG HM777634 HM777872Habenaria nuda Lindl. Batista 2869 (BHCB) Brazil, MG HM777718 HM777981Habenaria aff. nuda Lindl. Batista 1490 (CEN) Brazil, GO HM777664 HM777922Habenaria aff. nuda Lindl. van den Berg 1238 (HUEFS) Brazil, GO HM777665 HM777921Habenaria aff. nuda Lindl. Batista 1368 (CEN) Brazil, MA HM777715 HM777985Habenaria aff. nuda Lindl. Batista 2527 (BHCB) Brazil, PR HM777720 HM777958Habenaria aff. nuda Lindl. Batista 2091 (BHCB) Brazil, MG HM777722 HM777982Habenaria nuda var. pygmaea Hoehne Batista 939 (CEN) Brazil, DF HM777651 HM777984

(continued on next page)



Table 1 (continued)


Habenaria obtusa Lindl. Batista 291 (CEN) Brazil, DF HM777587 HM777862Habenaria cf. odontopetala Rchb.f. Batista 2037 (BHCB) Brazil, MG HM777591 HM777867Habenaria orchiocalcar Hoehne Batista 1570a (BHCB) Brazil, GO HM777662 HM777914Habenaria pabstii J.A.N. Bat. and Bianch. Batista 2360 (BHCB) Brazil, DF HM777666 HM777912Habenaria paranaensis Barb.Rodr. Batista 2436 (BHCB) Brazil, MG HM777528 HM777796Habenaria parviflora Lindl. Batista 1813 (BHCB) Brazil, MG HM777560 HM777800Habenaria parviflora Lindl. Batista 2477 (BHCB) Brazil, RS KC257475 KC257478Habenaria paulensis Porsch Batista 2481 (BHCB) Brazil, RS HM777556 HM777798Habenaria paulistana J.A.N. Bat. and Bianch. Pansarin 726 (UEC) Brazil, SP HM777610 HM777838Habenaria petalodes Lindl. van den Berg 1014 (HUEFS) Brazil, BA HM777583 –Habenaria petalodes Lindl. van den Berg 1481 (HUEFS) Brazil, BA – HM777861Habenaria cf. piraquarensis Hoehne Batista 1050 (CEN) Brazil, DF HM777633 HM777877Habenaria pleiophylla Hoehne and Schltr. Batista 2514 (BHCB) Brazil, RS HM777594 HM777857Habenaria praestans Rendle GenBank Africa DQ522079 DQ522100Habenaria pratensis (Salzm. ex Lindl.) Rchb.f. Batista 2686 (BHCB) Brazil, BA HM777546 HM777847Habenaria psammophila J.A.N. Bat., Bianch. and B.M. Carvalho Batista 1794 (BHCB) Brazil, MG HM777550 HM778000Habenaria pseudoculicina J.A.N. Bat. and Bianch. Batista 1808 (BHCB) Brazil, MG HM777707 HM777943Habenaria pseudoglaucophylla J.A.N. Bat., R.C. Mota and N. Abreu Mota 2818 (BHCB) Brazil, MG HM777590 HM777852Habenaria pseudohamata Toscano Batista 2035 (BHCB) Brazil, MG HM777593 HM777856Habenaria pubidactyla J.A.N. Bat. and Bianch. van den Berg 1360 (HUEFS) Brazil, MG HM777702 HM777972Habenaria pubidactyla spp. brasiliensis J.A.N. Bat. and Bianch. Batista 1785 (BHCB) Brazil, DF HM777690 HM777974Habenaria pubidactyla var. apiculatipetala J.A.N. Bat. and Bianch. Batista 1615 (BHCB) Brazil, MG HM777729 HM777973Habenaria pungens Cogn. Batista 2095 (BHCB) Brazil, GO HM777570 HM778011Habenaria quinqueseta (Michx.) A. Eaton Sánchez s.n. (SERO) Mexico HF560581 HF560605Habenaria regnellii Cogn. Batista 2801 (BHCB) Brazil, MG HM777603 HM777830Habenaria regnellii Cogn. Barfknecht s.n. (BHCB) Brazil, PR HM777604 HM777831Habenaria repens Nutt. Batista 2522 (BHCB) Brazil, PR HM777627 HM777816Habenaria repens Nutt. van den Berg 929 (HUEFS) Brazil, BA HM777628 HM777817Habenaria aff. repens Nutt. Batista 2100 (BHCB) Brazil, MG HM777624 HM777818Habenaria rodeiensis Barb.Rodr. Mota 2824 (BHCB) Brazil, MG HM777577 HM777995Habenaria aff. rodeiensis Barb.Rodr. Batista 2379 (BHCB) Brazil, GO HM777578 HM777992Habenaria aff. rodeiensis Barb.Rodr. Batista 1738 (BHCB) Brazil, MG HM777579 HM777991Habenaria rolfeana Schltr. Mota 3563 (BHCB) Brazil, MG HM777727 HM777977Habenaria rolfeana Schltr. Batista 2467 (BHCB) Brazil, MG HM777730 HM777978Habenaria roraimensis Rolfe Mota 1247 (BHCB) Brazil, RR HM777676 HM777925Habenaria rotundiloba Pabst Batista 2684 (BHCB) Brazil, BA HM777717 HM778017Habenaria rupicola Barb.Rodr. van den Berg 1279 (HUEFS) Brazil, MG HM777534 HM777910Habenaria cf. rupicola Barb.Rodr. Batista 2568 (BHCB) Brazil, MG HM777533 HM777911Habenaria rzedoswkiana R. González Jacob 234 (MEXU) Mexico HF560582 HF560606Habenaria schenckii Cogn. Batista 2882 (BHCB) Brazil, BA HM777580 HM777869Habenaria schwackei Barb.Rodr. Batista 1524 (CEN) Brazil, GO HM777656 HM777954Habenaria schwackei Barb.Rodr. Batista 2524 (BHCB) Brazil, PR HM777657 HM777953Habenaria secunda Lindl. Batista 2640 (BHCB) Brazil, RJ HM777525 HM777791Habenaria secundiflora Barb.Rodr. Batista 2392 (BHCB) Brazil, MG HM777637 HM778004Habenaria secundiflora Barb.Rodr. Batista 2526 (BHCB) Brazil, PR HM777636 HM778005Habenaria setacea Lindl. Mota 3019 (BHCB) Brazil, MG HM777731 HM777980Habenaria setacea Lindl. Batista 1417 (CEN) Brazil, MG KC257474 HM777979Habenaria seticauda Lindl. Batista 1596 (BHCB) Venezuela HM777584 HM777864Habenaria sobraliana J.A.N. Bat., A.A. Vale and Menini Batista 2499 (BHCB) Brazil, RS HM777704 HM777990Habenaria spanophytica J.A.N. Bat. and Bianch. Batista 2408 (BHCB) Brazil, MG HM777576 HM778016Habenaria spathulifera Cogn. Without voucher Brazil, RR HM777544 HM777850Habenaria sprucei Cogn. Batista 3086 (BHCB) Brazil, GO KC257470 KC257479Habenaria strictissima Rchb.f. Leutzi s.n. (MEXU) Mexico HF560583 HF560607Habenaria subauriculata Robinson and Greenm. García-Mendoza 7988 (MEXU) Mexico HF560584 HF560608Habenaria subfiliformis Cogn. Batista 1597 (BHCB) Venezuela HM777572 HM778009Habenaria subfiliformis Cogn. Batista 2022 (BHCB) Brazil, MG HM777573 HM778008Habenaria aff. subfiliformis Cogn. Batista 1788 (BHCB) Brazil, DF HM777705 HM777936Habenaria aff. subfiliformis Cogn. Batista 2808 (BHCB) Brazil, MG HM777709 HM778010Habenaria subviridis Hoehne and Schltr. Batista 1814 (BHCB) Brazil, MG HM777679 HM777929Habenaria subviridis Hoehne and Schltr. Batista 2605 (BHCB) Brazil, MG HM777680 HM777930Habenaria tamanduensis Schltr. Batista 1784 (BHCB) Brazil, DF HM777600 HM777993Habenaria tridens Lindl. GenBank Africa DQ522080 DQ522101Habenaria trifida Kunth Batista 1571a (BHCB) Brazil, GO HM777671 HM777916Habenaria trifida Kunth Batista 1783 (BHCB) Brazil, DF HM777672 HM777917Habenaria cf. uliginosa Rchb.f. Batista 1620 (BHCB) Brazil, MG HM777625 HM777824Habenaria umbraticola Barb.Rodr. Mota 3569 (BHCB) Brazil, MG HM777605 HM777874Habenaria urbaniana Cogn. Batista 911 (CEN) Brazil, MG HM777658 HM777944Habenaria warmingii Rchb.f. and Warm. Batista 2409 (BHCB) Brazil, MG HM777616 HM777821Habenaria warmingii Rchb.f. and Warm. Batista 2584 (BHCB) Brazil, MG HM777617 HM777820Habenaria weileriana Schltr. Salazar 6310 (YA) Cameroon HF560585 HF560609Orchis quadripunctata Cirillo ex Ten. GenBank Europe Z94105/Z94106 AY368385Platanthera chlorantha (Custer) Rchb. GenBank Europe AY704975 DQ522103Stenoglottis longifolia Hook.f. GenBank Africa AF348065 AY368387

a Abbreviations for Brazilian states are: BA = Bahia; DF = Distrito Federal; MA = Maranhão; MG = Minas Gerais; GO = Goiás; MT = Mato Grosso; PA = Pará; PR = Paraná;RJ = Rio de Janeiro; RR = Roraima; RS = Rio Grande do Sul; SC = Santa Catarina; SP = São Paulo.



3. Results

3.1. Sequence divergence

Sequence divergence among the Neotropical taxa of Habenariawas low. The mean pairwise distance for the Neotropical ITS se-quences (152 taxa) was only 0.018, whereas the mean pairwisedistance for the analyzed African sequences of Habenaria (10 spe-cies) was 0.142 (Table 3). To analyze whether these results wererelated to the difference in the number of taxa sampled betweenthe two groups, pairwise distances were calculated for selectedtaxa within the Neotropical Habenaria; the results were similarlylow independent of the number or taxa sampled (data not shown).This divergence among African Habenaria was surprisingly high be-cause it was similar to or greater than that obtained for othergroups represented by different genera: 0.083 for Habenariinae(Cynorkis and Stenoglottis) and 0.134 for Orchidinae (Orchis and Pla-thantera). Among the groups included in the analysis, only the Bon-atea species had mean pairwise distances similar to theNeotropical Habenaria species (0.015). The monospecific genusGennaria showed the greatest divergence from other groups dueto its high number of autapomorphic characters. Divergenceamong matK sequences was similar to that of the ITS region, albeitless. The mean pairwise distance was 0.009 for the NeotropicalHabenaria sequences and 0.029 for the African species (data notshown).

As a result of low divergence, alignments were straightforwardand unambiguous for the Neotropical ITS and all of the matK se-quences. In contrast, the Old World ITS sequences were more diffi-cult to align, and some ambiguous positions were present in thealignment. We attempted to include the basal genus CodonorchisLindl. (Kores et al., 2001) as an outgroup in the phylogenetic anal-yses, but aligning its ITS sequence proved much more difficult andambiguous, for which reason it was not used. Accordingly,

Chemisquy and Morrone (2012) suggested that the published ITSsequence of Codonorchis is most likely a pseudogene, as it lacksconserved motifs and was highly divergent in comparison to otheranalyzed sequences.

3.2. Parsimony analyses

Initially, we performed separate analyses for the ITS and matKdata sets. The ITS matrix consisted of 762 characters, of which365 (48%) were parsimony-informative. The parsimony analysisfound 4340 shortest trees with a length of 1454 steps, consistencyindex (CI) of 0.53 and retention index (RI) of 0.81. The matK matrixdid not have a single indel due to its low sequence divergence. Thelatter matrix had 627 characters, of which 122 (19%) were parsi-mony-informative. The parsimony analysis found 8830 shortesttrees with a length of 403 steps, CI of 0.60, and RI of 0.85 (Table 4).Overall, the resolution of the strict consensus and bootstrap treesof the matK matrix was lower than that obtained with the ITS dataset. Because the consensus and bootstrap trees of the matK matrixwere largely unresolved and no supported conflict was found be-tween the trees of the individual analyses, only the results of thecombined analyses will be presented and described. The generalfeatures of the datasets including taxon sampling and parsimonystatistics are presented in Table 4.

The combined matrix consisted of 1389 characters, of which487 (35%) were parsimony-informative. A total of 5110 shortesttrees were found, with a length of 1897 steps, CI of 0.53 and RIof 0.81 (Table 4). Because the parsimony trees are largely congru-ent with the Bayesian trees but are less resolved and with weakeroverall support, a strict consensus tree of the combined datasets ispresented as Supplementary Material (Fig. S1 and S2). The Neo-tropical taxa of Habenaria formed a highly supported group (99%BS). Habenaria tridens Lindl. from Africa was sister to the Neotrop-ical clade (100% BS), whereas H. batesii, also from Africa, was sisterto the Neotropical Habenaria–H. tridens (82% BS). Three other Afri-can species of Habenaria (H. dives Rchb.f., H. clavata (Lindl.) Rchb.f.and H. lithophila Schltr.) formed a strongly supported clade (100%BS) that was sister to the Neotropical Habenaria–H. tridens–H.batesii clade (100% BS). All species of Bonatea along with Habenaria

Table 2Sections of Neotropical Habenaria following Kränzlin (1892, 1901) and Cogniaux(1893), with the number of taxa in each section sampled for this study, excludingsynonyms.

Section Abbreviation No. of taxa/no. of taxasampled

% of taxasampled

Clypeatae CLY 10/5 50Macroceratitae MAC 14/10 71Maculosae MCU 3/2 67Micranthae MTH 14/13 93Microdactylae MDA 6/4 67Microstylinae MST 5/3 60Nudae NUD 12/11 92Odontopetalae ODO 4/2 50Pentadactylae PEN 32/18 56Pratenses PRA 6/5 83Pycnostachyae PYC 1/0 0Quadratae QUA 9/6 67Seticaudae SET 5/3 60Spathaceae SPA 12/9 75

Table 3Mean pairwise distances within and between the ITS sequences of the major groupsused in this study.

1 2 3 4 5 6

1. Neotropical Habenaria 0.0182. African Habenaria 0.132 0.1423. Habenariinae 0.127 0.133 0.0834. Bonatea 0.117 0.116 0.102 0.0155. Gennaria 0.217 0.221 0.185 0.199 n/c6. Orchidinae 0.199 0.196 0.150 0.168 0.246 0.134

Table 4Taxon sampling, matrix values and parsimony statistics of the ITS, matK andcombined data sets.

ITS matK ITS + matK

No. taxa 180 180 180No. Neotropical Habenaria 152

(51%)152(51%)

152 (51%)

No. old world orchidiinae and disinae 28 28 28No. sequences 217 217 217No. Neotropical Habenaria sequences 188 188 188No. old world orchidiinae and disinae

sequences29 29 29

Aligned length 762 627 1389Variable parsimony-uninformative

characters97(12.7%)

67(10.7%)

164(11.8%)

Parsimony-informative characters 365(48%)

122(19%)

487 (35%)

Best trees length 1454 403 1897No. of trees 4340 8830 5110Consistency index (CI) 0.53 0.60 0.53Retention index (RI) 0.81 0.85 0.81No. of internal nodes MP/BIa 109/124 62/73 131/151No. nodes with bootstrap >85% 46 (42%) 14

(22.6%)65 (49.6%)

No. nodes with posterior probabilities>0.95

86 (69%) 60 (82%) 106 (70%)

a Abbreviations: MP = Maximum parsimony; BI = Bayesian inference.



Fig. 1. Bayesian tree from the combined ITS and matK datasets. Bootstrap percentages from the parsimony analysis and posterior probabilities are shown next to nodes.Neotropical subgroups are boxed and numbered. The generic name for all Neotropical species is abbreviated. The three letter abbreviation to the right of the species nameindicates its sectional classification (Table 2). The sectional classifications of African Habenaria are also shown. Alternative recent generic circumscriptions are indicated inbrackets. Available somatic chromosome numbers are shown in the terminal branches for a few species. The type species of Habenaria, H. macroceratitis, is highlighted in bold.For species sampled more than once, the two or three letter abbreviation after the species name indicates the geographic origin of the sample. Abbreviations areMEX = Mexico; VEN = Venezuela; and for Brazilian states: BA = Bahia; DF = Distrito Federal; MA = Maranhão; MG = Minas Gerais; GO = Goiás; PA = Pará; PR = Paraná; RS = RioGrande do Sul; and SC = Santa Catarina.



laevigata Lindl. formed a strongly supported clade (100% BS). Allspecies of Habenaria along with Bonatea and Gennaria formed amoderately supported clade (78% BS). The Habenaria–Bonatea–

Gennaria clade was sister successively to Cynorkis (87% BS), Steno-glottis (66% BS), an Orchis–Platanthera clade (86% BS), and Satyrium(100% BS).

Fig. 2. Continuation of the tree of Fig. 1. A single tree with proportional branch lengths is shown in the upper left-hand corner to show the low levels of divergence within theNeotropical clade. For abbreviations, see Fig. 1.



3.3. Bayesian analyses

Similarly to the parsimony analyses, the ITS and matK data setswere initially analyzed separately. Because no significant incon-gruences were detected between the plastid and nuclear data, a fi-nal search was performed with the combined matrices. Theresulting Bayesian majority-rule consensus tree was fully congru-ent with the strict consensus tree of the combined parsimony anal-yses but was more resolved and with stronger overall support.Because the combined ITS/matK data set included a broad taxo-nomic sample and is, overall, better resolved and supported thaneither of the other analyses, it is the tree that best represents ourphylogenetic hypothesis and was chosen for presentation and dis-cussion (Figs. 1 and 2).

The Neotropical Habenaria formed a monophyletic group withstrong support (1.00 PP), with Habenaria tridens (section Dolicho-stachyae) sister to it (1.00 PP). Habenaria batesii (section Podandria)was sister to the Neotropical Habenaria–H. tridens clade (0.91 PP),whereas Habenaria dives (section Bilabrellae), H. clavata (sectionCeratopetalae) and H. lithophila (section Diphyllae) formed astrongly supported clade (1.00 PP) that was sister to the Neotrop-ical–H. tridens–H. batesii clade (1.00 PP). All sampled species ofBonatea along with H. laevigata and H. arenaria Lindl. (section Chlo-rinae) also formed a strongly supported clade (1.00 PP). The othersampled African Habenaria (H. mannii, H. praestans and H. weileri-ana) formed with the Bonatea–H. laevigata–H. arenaria clade astrongly supported clade (0.97 PP), which was sister to the Neo-tropical–H. tridens–H. batesii–H. dives–H. clavata–H. lithophilaclade, but with low support (0.66 PP). The Habenaria–Bonatea cladewas sister successively to Gennaria (1.00 PP), Cynorkis (1.00 PP),Stenoglottis (0.86 PP), an Orchis–Platanthera clade (0.92 PP), andSatyrium (1.00 PP). The Neotropical species formed several well-supported subgroups, but many species were either unresolvedor weakly supported as sisters to the subgroups. Relationshipsamong the Neotropical subgroups were poorly resolved, as supportfor the internal nodes of the tree was low overall.

4. Discussion

4.1. Generic limits of Habenaria

Irrespective of the method of inference, in all of our analyses ofthe combined ITS–matK dataset, the Neotropical Habenaria formeda strongly supported monophyletic group (99% BS; 1.00 PP) (Fig. 1,clade A). Furthermore, the African species Habenaria dives, H. clav-ata and H. lithophila were strongly supported as sister (100% BS;1.00 PP) to the clade formed successively by the African speciesH. batesii and H. tridens plus the whole Neotropical clade. These re-sults show that Neotropical Habenaria and African representativesof the genus in the sections Dolichostachyae, Podandria, Bilabrellae,Ceratopetalae, and Diphyllae form a strongly supported ‘‘core Habe-naria clade’’ (Fig. 1, clade B) that includes the type species of thegenus, H. macroceratitis from the New World.

Another group strongly supported by the trees was the ‘‘Bonateaclade’’ formed by the species of Bonatea (formerly a section of Hab-enaria) plus Habenaria laevigata (section Chlorinae) (100% BS; 1.00PP). In the Bayesian analysis, this clade also included Habenariaarenaria (section Chlorinae) (1.00 PP), H. mannii and H. praestans(section Multipartitae) plus H. weileriana (section Chlorinae) (0.97PP) (Fig. 1, clade C), but this result was not supported in the parsi-mony analysis. Relationships between the ‘‘core Habenaria clade’’and the ‘‘Bonatea clade’’ were not resolved. In the Bayesian analy-sis, the ‘‘Bonatea clade’’ was sister to the ‘‘core Habenaria clade’’,rendering all sampled taxa of Habenaria plus Bonatea as monophy-letic, but support was low (0.66 PP) (Fig. 1). The monospecific

genus Gennaria, formerly included in Habenaria, was highly sup-ported (1.00 PP) as sister to the Habenaria–Bonatea clade. However,the Gennaria sequences were full of autapomorphies, and the posi-tion of this species varied in other analyses according to the datasets, species sampled and method of analysis. A division of the Afri-can Habenaria into two major clades was first envisioned in thephylogenetic analysis of Bellstedt et al. (2001), albeit with a muchlower sampling that used the trnL intron and the trnL-trnF spacerregion to investigate phylogenetic relationships in Disa. In thatwork, H. pseudociliosa Schelpe ex J.C. Manning (section Chlorinae),H. malacophylla Rchb.f. (section Ceratopetalae), and H. laevigataformed one strongly supported clade and H. tysonii Bolus (sectionDiphyllae) and H. dives formed another.

Beyond the division of the Neotropical and African Habenariainto two major clades, in the study of Bateman et al. (2003), Asianspecies of Habenaria (H. sagittifera Rchb.f. [section Cruciatae], H.tibetica Schltr., H. delavayi Finet) were more closely related toPecteilis Raf. (Asian) and Herminium L. (Euro–Asian), whereas Afri-can (H. arenaria, H. procera (Afzel. ex Sw.) Lindl., H. tridactylitesLindl. [section Tridactylae]) and a single Neotropical species (H.odontopetala Rchb.f.) were grouped with Bonatea (African) andGennaria (Canary Islands, west and central Mediterranean). A sim-ilar result was found in a molecular phylogenetic analysis of Diseaethat also used the ITS region (Douzery et al., 1999), where H. sagit-tifolia (a misspelling of H. sagittifera, from China and Japan) formeda strongly supported group (95% BS) with Herminium, and Bonateaspeciosa (L.f.) Willd., Habenaria arenaria and H. procera (sectionChlorinae), all from tropical Africa, formed a clade with moderatebootstrap support (77% BS). An exception was the highly supportedclade (100% BS) formed by H. repens (Neotropical) and Holothrix, amember of Orchidinae s.s. from tropical and southern Africa. How-ever, this last result is doubtful because the two sequences areidentical and according to Bateman et al. (2003), H. repens is eithermisnamed or misidentified in this work. Although Asian species ofHabenaria were not included in our analyses, these results are con-sistent with ours and indicate a strong relationship between geo-graphical and phylogenetic structure with a separation betweenAfrican-Neotropical and some Asian groups of Habenaria. Furtherinferences on the generic limits of Habenaria are limited by theavailability of molecular data. Only a few Habenaria from the OldWorld and eight of the 23 genera of Habenariinae (sensu auct.)listed in Genera Orchidacearum (Pridgeon et al., 2001a) are cur-rently available for molecular analyses.

4.2. Comparison of the New World sectional classification with thephylogenetic analysis

The current infrageneric classification of Habenaria is basedmostly on Kränzlin’s (1892, 1901) sectional treatments. In his sys-tem, the Neotropical species were divided into 12 sections. To facil-itate the comparison of this sectional treatment with the results ofour cladistic analyses, the sectional assignment of the taxa accord-ing to Kränzlin (1892, 1901) and Cogniaux (1893) is indicated afterthe species’ name in Figs. 1 and 2. Abbreviations for the Neotropi-cal sections, number of species, and species sampled in each sec-tion are shown in Table 2. All sections of the current sectionalclassification are paraphyletic or polyphyletic with regard to thesubgroups recovered in the molecular tree. Species from the sec-tion Macroceratitae Kraenzl. (MAC) are concentrated in subgroup4, but other species assigned to this section are dispersed acrosssubgroups 2, 7, 8, 13, 16 and 17. Similarly, many species from sec-tion Pratensis Kraenzl. (PRA) are concentrated in subgroup 1, butother species assigned to this section are in subgroup 5 or are unre-solved in different positions of the tree. The species in section Cly-peatae Kraenzl. (CLY) are dispersed across subgroups 2, 6, 8 and 9of the tree. Section Pentadactylae Kraenzl. (PEN), the largest among



the Neotropical sections, and section Micranthae Kraenzl. (MTH)are also polyphyletic, and the species assigned to these sectionsare either placed in several different subgroups or are unresolved.The species in sections Quadratae Kraenzl. (QUA) and SeticaudaeKraenzl. (SET) are concentrated in subgroup 10, but this subgroupalso includes species assigned to section Micranthae. Even the spe-cies belonging to the smaller sections Microstylinae Kraenzl. (MST)and Microdactylae Kraenzl. (MDA) are dispersed across the tree. Forthe Old World sections of the genus, the current taxon sampling isnot sufficient for an evaluation of the infrageneric groups, and theywill not be discussed.

4.3. Phylogenetic relationships within New World Habenaria

Several terminal nodes of the Neotropical clade formed mono-phyletic subgroups (Figs. 1 and 2), and approximately 21 such sub-groups were recovered that comply with at least one of thefollowing criteria: (1) the group is moderately to strongly sup-ported in the phylogenetic analyses; and (2) the species are mor-phologically similar. However, several species were unresolvedrelative to these subgroups, and the relationships between the sub-groups are not clear because most of the internal nodes of the treeswere poorly resolved or weakly supported. Although the relation-ships among the subgroups are mostly unresolved, a general char-acterization and discussion of the subgroups is presented as anattempt to correlate the results of the phylogenetic analyses withthe most salient taxonomic, morphological, biogeographical andevolutionary aspects of each group/subgroup of the Neotropicalspecies.

4.3.1. Basal clades: subgroups 1–6In the Bayesian analysis of the ITS–matK dataset, the Neotropi-

cal group formed a polytomy at the base of the clade that includedH. monorrhiza, subgroup 1 and the clade formed by all other Neo-tropical species. Habenaria monorrhiza is a weedy species com-monly found by the side of roads along most of Mexico, CentralAmerica and northern South America. Subgroup 1 consists of asmall group of four species, of which three were sampled here, thatdiffers from all other Neotropical Habenaria in its yellow to orangeperianth, laterally expanded segments of the petals and labellum,and diurnal fragrance (Hoehne, 1940). Based on these features,Singer and Cocucci (1997) suggested butterfly pollination for thisgroup, in contrast with most other Habenaria species, which arefragrant at night and have flower syndromes associated with mothpollination.

Subgroups 2–6 and some unplaced species form a clade.Although support is low, (0.81 PP) all species in these subgroupshave patent, spreading leaves and are concentrated mostly insouthern South America, but they vary widely in flower size andmorphology. Subgroups 2 and 3 are strongly supported in all anal-yses and form a well-supported clade including H. leucosantha(0.96 PP). Based on morphological similarities, subgroup 2 is com-posed of approximately 11 species (five sampled) and subgroup 3eight species (five sampled). Species in subgroup 2 were assignedto sections Macroceratitae, Micranthae, Pentadactylae and Clypeatae,whereas the species in subgroup 3 were placed in sections Micrant-hae and Microstylinae in the sectional treatments of Kränzlin (1892,1901) and Cogniaux (1893). The length of the lateral segments ofthe petals and labellum, one of the characters used by Kränzlinfor the characterization of the sections, is highly variable in sub-group 2, which explains the placement of species in this subgroupin different sections, but this subgroup is otherwise homogeneousin vegetative and floral characters. The center of diversity of bothsubgroups is southeastern and southern Brazil.

The monophyly of subgroups 4–6 was strongly supported in allanalyses and in the Bayesian analysis of the combined data sets;

they formed a strongly supported clade with H. macilenta sisterto the group (Fig. 1) (1.00 PP). However, there are no evident floralmorphological similarities between the species, and the inclusionof H. macilenta in this clade was unexpected because this specieshas a dissimilar morphology and geographic distribution (centraland northern Brazil and the Guianas) and on the basis of flowermorphology was associated by previous authors (Hoehne, 1940;Pabst and Dungs, 1975) with the species recovered here in sub-group 12. Subgroup 4 consists of a group of nine taxa (seven sam-pled) with several morphological similarities. A distinctivecharacter of this subgroup is the long (8–14 mm), involute stigmalobes, a character not found in any other Neotropical group of thegenus. Also distinct from other Neotropical Habenaria is the short,erect, tooth-like process, originated from the lip, in front of the en-trance to the spur found in some species of this subgroup. This fea-ture is one of the characters used to separate Bonatea fromHabenaria, but our results show that it is homoplasious. The spe-cies in subgroup 4 were previously assigned to section Macrocerat-itae (Kränzlin, 1901; Cogniaux, 1893; Batista et al., 2006), but H.macroceratitis, the type species of this section, is morphologicallydistinct and distantly related in the sequence data trees (Fig. 1).Species in subgroup 4 have the longest spurs among NeotropicalHabenaria, reaching up to 20 cm in H. longicauda (Batista et al.,2006).

Subgroup 5 is composed of approximately seven species (threesampled), found mostly in southern Brazil, Uruguay and Argentina.Subgroup 6 is composed of approximately 12 species (three sam-pled) including H. repens, the most widespread species of the genusin the Neotropics, extending from the southern USA to northernArgentina. Habenaria repens is typically aquatic, whereas the otherspecies in the subgroup usually grow in water-logged places. Hab-enaria warmingii has never been formally associated with H. repensand was placed in section Pentadactylae by Kranzlin (1892, 1901)and Cogniaux (1893). However, the two species agree well in theirvegetative and floral morphology.

4.3.2. Forest clades: subgroups 7–10In the Bayesian analysis of the combined ITS and matK datasets,

subgroups 7–21 formed a clade (0.96 PP) with subgroups 7–10placed in a basal polytomy along with a few other unresolved spe-cies. Although the relationships among subgroups 7–10 were notresolved, the species in these subgroups are mostly forest-dwell-ing, in contrast with most other New World Habenaria, which oc-cur in open savanna or grasslands. Forest species are commonamong African and Asian Habenaria but in the New World, are re-stricted to these subgroups, suggesting a relationship betweenthem. The species in subgroups 7–10 always have well-developedelliptical, oblong or lanceolate leaves, but flower morphology isvariable. Subgroup 7 consists of approximately nine species (sixsampled) and in the New World, is the only subgroup formedexclusively by species from tropical forest. The support for thissubgroup varied between the analyses (53% BS; 1.00 PP), but thesimilarities in habitat, habit and flower morphology support a closerelationship between the species in this subgroup. Subgroup 8 in-cludes H. macroceratitis, the type species of the genus. This sub-group consists of a small group of approximately five species(two sampled) that are primarily Mexican but also extend overthe southern USA, the Caribbean and northern South America.The species in subgroup 9 are all Mexican. This subgroup mostlikely consists of approximately 56 taxa, mostly from Mexico (10sampled), with a few species extending to Guatemala and otherMesoamerican countries. The low resolution and limited taxonsampling currently available for this subgroup did not allow a com-parison with the 12 informal groups proposed by González-Tamayo (1993) for Mexican Habenaria. Considering the highnumber of species and the variation in flower size and morphology



displayed by the species of this subgroup, which range from min-ute flowers a few millimeters in diameter to large green (H.rzedowkiana) or white (H. clypeata) flowers, it is likely that a largersampling and better resolution might reveal subdivisions. Contraryto most Neotropical species, which inhabit open, grassland habitatsand, less frequently, tropical forests, the species in this subgroupprimarily inhabit subtropical to warm-temperate conifer-oakforests along the mountains of much of Mexico. In all analyses,subgroup 9 is sister to subgroup 8, and the two form a stronglysupported clade (97% BS; 1.00 PP).

Subgroup 10 was strongly supported in all combined and indi-vidual analyses (98% BS; 1.00 PP). It is composed of a large group ofapproximately 34 species (16 sampled) distributed throughout theNeotropics. These species primarily inhabit tropical forests, but thegroup formed by H. seticauda, H. obtusa and H. hamata prefers drygrasslands, indicating a probable reversal. Subgroup 10 is the onlyone among the Neotropical Habenaria that includes species withentire petals and labellum. Entire flower segments were used tocharacterize the genera Habenella (Small, 1903; Szlachetko andKras, 2006) and Platantheroides Szlach. (Szlachetko, 2004b), bothsegregated from Habenaria. However, our results do not supportthis division because subgroup 10 also includes two groups of spe-cies with short lateral segments on the labellum and petals (H.josephensis–H. leptoceras and H. pleiophylla–H. pseudohamata), indi-cating that entire segments evolved independently in two or threespecies groups within subgroup 10 (e.g., H. strictissima and H. brev-ilabiata; H. curvilabria and H. magdalenensis; and H. odontopetalathrough H. seticauda). On the basis of chromosome numbers andstructure, Felix and Guerra (1998) were the first to suggest a closerelationship between these two groups of species, e.g., those withentire segments and with short lateral segments. In accordancewith this relationship, the morphology of the gynostemium ishomogeneous across all of subgroup 10. Our results indicate thatthe recently described H. pseudoglaucophylla J.A.N. Bat., R.C. Motaand N. Abreu (Batista et al., 2008b), similar in gynostemium mor-phology but with long lateral segments of the labellum, is sisterto subgroup 10, which suggests a progressive reduction in the sizeof the lateral segments within the subgroup. This subgroup has aunique triplet insertion mutation in positions 293–295 of ITS1.However, this insertion is absent in H. pseudoglaucophylla. All spe-cies sampled in subgroup 9 and some species of Bonatea have dif-ferent insertions at the same position.

Two species, Habenaria schenckii and H. depressifolia, are of par-ticular interest among the species in the forest clades because theyare vegetatively identical to each other and differ from all otherNeotropical species in having 1–2 basal, orbiculate, fleshy leavesthat lay adpressed to the ground. These vegetative characters arecommon among Old World species of Habenaria and characterizesect. Diphyllae, but in the New World are found only found in thesetwo species. A close relationship between the two species was notconfirmed in our analyses, but the unresolved positions in the treesindicate that the available molecular data has not been sufficient toresolve the relationship between them.

4.3.3. Subgroups 11–15Similarly to subgroups 1–10, most species in subgroups 11–15

have elliptical to lanceolate spreading leaves, whereas flower mor-phology is variable. Some species in these subgroups are wide-spread, but most occur in central and southeastern Brazil.Subgroup 11 consists of approximately eight species (five sam-pled), usually occurring in high-altitude grasslands or in temperateareas at lower altitudes. Subgroup 12 consists of six species, ofwhich all were sampled here. Habenaria trifida, distributed fromMexico to northern Argentina, has one of the broadest geographicranges among the Neotropical species in the genus, but other spe-cies of this group are concentrated in the cerrado vegetation of cen-

tral Brazil. They typically occur in grasslands and are characterizedby a few-flowered inflorescence, medium to large flowers, a whitecorolla and a long pedicel. Subgroup 13 is composed of approxi-mately 11 species (eight sampled), most of them occurring in cen-tral and southeastern Brazil. The inclusion of H. rupicola and H.coxipoensis in this subgroup was unexpected, as these two specieshave flower morphologies remarkably similar to H. repens (sub-group 6) and H. subviridis (subgroup 11), most likely indicating pol-linator-driven homoplasy. Subgroup 14 consists of twomorphologically dissimilar species with similar geographical dis-tributions, concentrated on the rocky fields of the Espinhaço rangein Minas Gerais and the highlands of Central Brazil. Subgroup 15was not strongly supported, but the four species in the subgroupcomprise a morphologically uniform assemblage.

4.3.4. Cerrado clades: subgroups 16–21The species in subgroups 16–21 formed a well-supported clade

(Clade D, Fig. 2) in the Bayesian analysis of the combined data sets(1.00 PP). In contrast with subgroups 1–15, most species in sub-groups 16–21 have linear, grass-like leaves, which are commonlyadpressed to the stem. Based on these characters, these speciesbest correspond to sect. Nudae Cogn. They are primarily Brazilianand are mostly concentrated in the cerrado and campos rupestres(rocky fields) of the central and southeastern regions of the coun-try. Regarding their vegetative parts, subgroup 16 is the onlyexception in clade D, as most species in this subgroup have lance-olate, spreading leaves. Subgroup 18 consists of a group of approx-imately eight taxa (five sampled). Some species in this subgroupwere recently revised by Batista and Bianchetti (2010), but theircircumscription of the H. crucifera group conflicts with the resultsof the molecular trees, indicating that the morphological charac-ters they used to define the group are homoplasic. Habenaria pun-gens Cogn. from the cerrado of central Brazil and Bolivia isunequivocally placed in subgroup 18. This is one of the most inter-esting and distinct Habenaria species in the Neotropics because itsflowers form a dense, umbel-like inflorescence and are bright-yel-low and non-resupinate, resembling in their general aspect theAfrican genus Platycoryne Rchb.f., formerly segregated from Habe-naria. Unfortunately, no Platycoryne material was available formolecular study and it was therefore not possible to test whethersuch a floral similarity is indicative of a close relationship betweenH. pungens and Platycoryne or resulted from convergence. Never-theless, the position of H. pungens deep in the Neotropical cladeand the distant position of all African taxa sampled in the molecu-lar trees suggest that a direct relationship between the two taxa isunlikely.

Subgroups 19–21 along with some unresolved species formed awell-supported clade in the Bayesian analysis of the combined datasets (0.99 PP), but the relationships within this clade were unclear.Subgroup 20 is composed of a small group of four species (all sam-pled) concentrated in the cerrado biome of central Brazil. Speciesin this subgroup belong to four sections in the sectional classifica-tion of Kränzlin (1892, 1901) and Cogniaux (1893), but are mor-phologically cohesive and well-supported in the molecular tree(1.00 PP). Support for subgroup 21 was low, and relationshipswithin the subgroup were poorly resolved. This subgroup currentlyconsists of approximately 23 taxa (13 sampled) distributed fromnortheastern to southern Brazil but concentrated in the cerradoand rocky fields of central and southeastern Brazil.

4.4. Taxonomic implications

The results of Bateman et al. (2003) indicated that Habenaria ishighly polyphyletic, and those authors envisioned an extensive dis-mantling of Habenaria into smaller monophyletic genera. However,only eight species of Habenaria (approximately 1% of the genus)



were included in that analysis. At about the same time, Szlachetko(2003a,b, 2004a,b; Szlachetko and Kras, 2006) begin a worldwidedivision of the genus, recognizing three genera segregated fromNew World Habenaria: Bertauxia Szlach., Kusibabella Szlach. andPlatantheroides Szlach. (a synonym of Habenella Small). In our phy-logenetic analysis, Bertauxia is polyphyletic, with the three speciesassigned to that genus dispersed among clades 4 (H. vaupelliiRchb.f. and Warm. = H. johanennsis Barb.Rodr.), 16 (H. rodeiensisBarb.Rodr.) and 21 (H. nasuta Rchb.f. and Warm.) (Figs. 1 and 2).Even on morphological grounds, this genus has no support (Batistaet al., 2006). In turn, Kusibabella and Habenella are paraphyletic.Kusibabella includes most species of subgroup 4 but with H. crypto-phila Barb.Rodr. embedded in it and H. johannensis not included,whereas Habenella (formerly described as superfluous Platanthero-ides Szlach.) includes some of the species in subgroup 10 alongwith other African and Asian species.

Although more narrowly circumscribed genera such as Kusiba-bella and Habenella could become monophyletic with some adjust-ments, we do not favor a generic fragmentation of the New WorldHabenaria on the basis of the following arguments: (1) the Neo-tropical Habenaria are monophyletic; (2) many lineages are com-posed of one or few taxa and consequently many genera withone or a few species would have to be created; (3) phylogeneticrelationships between the Neotropical clade and many Africanand Asian clades of the genus are unresolved, and a subdivisionof the Neotropical clade will require a corresponding extensivegeneric fragmentation of the African and Asian groups, for whichthere is limited molecular data available; and (4) the creation ofnew genera will not provide any additional information whencompared to a sectional subdivision and will require extensivenomenclatural changes, whereas a sectional classification wouldnot require major nomenclatural changes, just realignments ofspecies as required to comply with monophyly and a morphologi-cal recircumscription.

Rather than a generic fragmentation, we favor a revision of thecurrent sectional classification of the Neotropical species togetherwith a morphological, cytogenetic and biogeographic characteriza-tion of the subgroups, work that is already underway. Regardingthe genus as a whole, it is clear that, as currently circumscribed,Habenaria is polyphyletic relative to Asian species in sections Cru-ciatae and Peristyloideae. However, a massive segregation of newgenera is unlikely. Our results indicate that the Neotropical Habe-naria form a strongly supported clade with African species in sec-tions Diphyllae, Dolichostachyae, Ceratopetalae, Podandria andBilabrellae, and they are likely to be kept together. However, itshould be noted that the sectional assignment of the African spe-cies sampled in the molecular analyses was based primarily onKränzlin’s sectional classification, which is highly artificial, at leastfor the Neotropical species. Nevertheless, despite the correct sec-tional classification of the African species sampled, our results indi-cate that some of them are more closely related to the Neotropicalspecies than to other African species. On the other hand, the rela-tionships of the Neotropical clade with other African sections of thegenus such as Chlorinae and Multipartitae and other former sectionsnow treated at the generic rank (Bonatea) are unresolved or weaklysupported and will require a more comprehensive sampling ofthese groups.

With the sampling currently available, the recognition of Bona-tea at the generic level renders Habenaria paraphyletic and requireseither the recognition of other African groups of Habenaria in sects.Chlorinae and Multipartitae as independent genera or the inclusionof Bonatea in a broadly circumscribed Habenaria. In this context,the recent transfer of Bonatea bracteata and B. tentaculifera to Hab-enaria (Ponsie et al., 2007b) is provisional, and a decision on thetaxonomic status of Bonatea and other genera formerly placed inHabenaria such as Centrostigma, Platycoryne and Roeperocharis will

have to wait for a more comprehensive sampling, as our under-standing of these groups is currently limited by the availabilityof molecular data.

4.5. Evolutionary trends in Habenaria

Compared to African species, sequence polymorphism in the ITSand matK regions among New World Habenaria was much less fre-quent. The morphological variability of the Neotropical species iscorrespondingly smaller than that found in African and Asiangroups of the genus. Furthermore, the generic diversity of tribeOrchideae in the Neotropics is much lower than tropical Africaand Asia. These results, together with the paraphyletic positionof African species of Habenaria in relation to the Neotropical clade,indicate an African origin for the Neotropical clade and suggest arecent dispersal and radiation of the genus in the New World. Atime calibration of our reconstructed phylogeny will provide in-sight into this hypothesis.

The only chromosome counts for Neotropical Habenaria indi-cated a diploid number of 42 with other values such as 44, 50,80 and 84 (Daviña et al., 2009; Felix and Guerra, 1998). Whenthe available counts are plotted in our trees, the chromosomenumber increases in the derived lineages, suggesting that polyplo-idization and other forms of genome evolution may be related tothe evolution and speciation of the genus in the New World. How-ever, chromosome counts are still few, and only in one case (sub-group 10) was more than one species in the same group counted,making it difficult to extrapolate the number to each clade. If chro-mosome numbers are constant within each clade, chromosomenumber could be a useful character for the characterization ofsome clades, but additional data are necessary to confirm thispossibility.

Similarly to other large genera, the extent to which molecularphylogenies will be translated into systematic classifications is un-clear. The relationships between the groups recovered in themolecular trees and morphological traits were not always clear.Many of the groups in the trees formed uniform assemblages ofspecies, but in several instances, morphologically dissimilar spe-cies where grouped, whereas in other cases, species with similarmorphological traits were dispersed among the subgroups, indicat-ing homoplasy of these characters. These results indicate that mor-phological resemblance alone can be misleading for inferringrelationships within the genus.

4.6. Conclusions and perspectives

Our results establish unambiguously that Neotropical Habenariaare monophyletic and closely related to some African species of thegenus. Furthermore, the topology of the trees indicates an Africanorigin and the low divergence among the Neotropical sequencessuggest a recent radiation of the genus in the Neotropics. Furtherwork with a molecular dating approach will provide more insightinto the timing of the radiation and other aspects of this issue.The precise relationships between Neotropical and African Habena-ria and other groups of African Habenariinae could not be confi-dently resolved because sampling of African taxa is still limited.Currently, only a few African Habenaria and eight of the 23 generaof Habenariinae listed in Genera Orchidacearum (Pridgeon et al.,2001a) are available in GenBank. The inclusion of more Africanspecies and sections of Habenaria as well as the closely related gen-era Centrostigma, Platycoryne and Roeperocharis will be necessary todefine generic limits.

Among the Neotropical species, the identification of severalwell-supported subgroups will provide the basis for a revision ofthe current sectional classification. Taxon sampling of the Neotrop-ical Habenaria has increased greatly, but additional fieldwork is re-



quired in some groups. Relative to the number of species, the Mex-ican clade (subgroup 9) is still poorly sampled, and the addition ofmore taxa is necessary to resolve the relationships in this largegroup. Taxon sampling of some Andean species also needs to beimproved, particularly for a few morphologically distinct speciesrestricted to this region. On the other hand, analyses to resolverelationships between and within subgroups 16–21, which arenow well-sampled, will require the addition of more DNA regionsin the analyses, preferably nuclear genes with high variation.

The few chromosome counts available suggest that karyotypeevolution is related to the evolution and diversification of thegenus in the New World, but more studies with a higher numberof species are necessary. The inclusion of other features such aschromosome morphology may also provide insight into this ques-tion. Finally, for the first time, we have an explicit phylogenetichypothesis for the Neotropical species of Habenaria that will pro-vide a basis for investigating the patterns of morphological evolu-tion, diversification and distribution of the genus in the NewWorld.

Acknowledgments

The authors thank Rubens C. Mota, Nara F.O. Mota, E. Pansarin,Marco O.D. Pivari, Geraldo Barfknecht, João B.A. Bringel, Eric Smidt,Jacques Klein and Rolando J. Machorro for providing samples, Ben-ny Bytebier, Graham Grieve and Rolando J. Machorro for some ofthe photographs used in graphical abstract and Fig. S1, IBAMAand IEF Minas Gerais for providing scientific collection permitsand two anonymous reviewers who provided useful commentsand corrections. G.A.S. thanks Laura Vázquez Valedelamar for herassistance with DNA sequencing. This research was financially sup-ported by the Fundação de Amparo a Pesquisa do Estado de MinasGerais – FAPEMIG, Conselho Nacional de Desenvolvimento Cientí-fico e Tecnológico – CNPq, and Pró-Reitoria de Pesquisa da Univer-sidade Federal de Minas Gerais – UFMG. CvdB and JANB alsoacknowledge scholarships received from CNPq (Pq-1D and Pq-2).

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.2013.01.008.

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