Molecular Phylogenetics, Floral Convergence and Systematics Of

Botanical Journal of the Linnean Society, 2011, 167, 1–18. With 5 ﬁgures

Molecular phylogenetics, ﬂoral convergence and systematics of Dichromanthus and Stenorrhynchos (Orchidaceae: Spiranthinae)

GERARDO A. SALAZAR*, LIDIA I. CABRERA and COYOLXAUHQUI FIGUEROA

Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-367, 04510 Mexico City, Distrito Federal, Mexico

Received 11 January 2011; revised 16 April 2011; accepted for publication 18 May 2011

Contrasting generic concepts of Dichromanthus and Stenorrhynchos held recently by taxonomists were assessed by means of parsimony and Bayesian cladistics analyses of 40 species/22 genera of Spiranthinae and over 2400 base pairs of non-coding nuclear and plastid DNA. Floral structure of relevant taxa was compared using fresh, pickled and herbarium specimens. Our results indicate that a broad concept of Dichromanthus corresponds to a strongly supported monophyletic group sharing several ecogeographical attributes and at least one putative floral synapo- morphy (nectary formed by a narrow channel at the base of the labellum). Dichromanthus s.l. occupies a derived position within the Spiranthes clade, with Deiregyne being strongly supported as its sister genus. Stenorrhynchos, as delimited by most previous taxonomists, is shown to be polyphyletic; monophyly requires a narrower delimitation and this narrow concept is strongly supported as belonging in the Stenorrhynchos clade, which also includes members of the genera Eltroplectris, Mesadenella, Pteroglossa and Sacoila. The scant published information on natural pollination and inferences made from flower colouration and morphology suggest convergence in floral characteristics between species of Dichromanthus, Stenorrhynchos and probably other distantly related genera as a result of independent adaptation to pollination by hummingbirds. Bee-pollinated D. michuacanus is recovered in a derived position relative to hummingbird-pollinated Dichromanthus spp., suggesting a secondary reversal in this trait. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 1–18.

ADDITIONAL KEYWORDS: convergent evolution – ﬂoral morphology – internal transcribed spacer (ITS) – phylogenetics – pollination – trnL/trnF.

‘A point which has been generally overlooked in taxonomy in median carpel apex, a portion of which develops into the orchids is that the characters which result from adapta- the viscidium; Richard, 1817; Vermeulen, 1959; tions to bird-pollination are often striking. These characters Dressler, 1993; Kurzweil, 1998), have been considered are commonly employed by taxonomists in separating genera, of prime importance for generic delimitation in sub- with the result that closely related species may be placed in tribe Spiranthinae Lindl. (Lindley, 1840; Schlechter, distinct genera.’ 1920; Balogh, 1982; Garay, 1982; Greenwood, 1982; L. van der Pijl & C. H Dodson (1966) in Orchid flowers: their Burns-Balogh, 1986; Szlachetko, 1995; Szlachetko, pollination and evolution. Rutkowski & Mytnik, 2005; Rutkowski, Szlachetko & Górniak, 2008) and in other orchids (e.g. Micheneau, Johnson & Fay, 2009). However, reliance on such a INTRODUCTION limited source of character information for classifying > Floral characters, and especially attributes of the a large group such as Spiranthinae ( 400 species; rostellum (the modified, non-receptive part of the Salazar, 2003) by intuitively weighting character similarities and differences, has resulted in perplex- ing discrepancies among the classifications that have *Corresponding author. E-mail: been proposed for Spiranthinae (e.g. see discussion in [email protected] McVaugh, 1985: 295–296).

One of the genera for which delimitation has been genus were its type species, S. speciosum (Jacq.) Rich. inconsistent among the different classifications is ex Spreng. and three additional species morphologi- Stenorrhynchos Rich. ex Sprengl. On the one hand, cally and ecologically similar to it. Recently, Christen- John Lindley (1840) distinguished Stenorrhynchos son (2005) described several additional species fitting from the other five genera he included in his ‘division in this restricted concept of Stenorrhynchos. Through- Spiranthidae’ (= Spiranthinae), namely Pelexia Poit. out this paper, we will refer to Stenorrhynchos in this ex Rich., Sauroglossum Lindl., Spiranthes Rich., narrow sense as Stenorrhynchos sensu stricto (s.s.), in Synnasa Lindl. and Cnemidia Lindl. (now a synonym contrast with the more inclusive delimitation of Szla- of Tropidia Lindl., a member of Epidendroideae chetko and co-workers (e.g. Szlachetko et al., 2005), known not to be closely related to Spiranthinae) by referred to here as Stenorhynchos sensu lato (s.l.). the large showy flowers, large coloured bracts, The genus Dichromanthus Garay was proposed absence of ‘calli’ (swollen nectar glands) at the base of originally to include a single species, D. cinnabarinus the labellum and possession of a hard, sharply (Lex.) Garay (Garay, 1982). This species had been pointed rostellum remnant (i.e. what remains of the placed by both Lindley (1840) and Schlechter (1920) rostellum after the removal of the pollinarium). On in Stenorrhynchos, in spite of its ‘soft, pliable, linear the other hand, in the first modern generic revision of oblong, blunt rostellum’ [remnant] (Garay, 1982). Spiranthinae, Schlechter (1920) raised the number of Balogh (1982), Balogh & Greenwood (1982) and genera to 24, seven of which showed the rostellum Greenwood (1982) also noticed the distinctive rostel- characteristics of Stenorrhynchos sensu Lindley lum structure of ‘Stenorrhynchos’ cinnabarinus, (1840). These seven genera were grouped into one of describing its rostellum remnant as ‘tubular-tipped’ the four greges or generic alliances (Gattungsreichen) and the viscidium as having a ‘plug-like’ extension proposed by Schlechter (1920, 1926), namely grex that fits in the tube prior to its removal by the Raphiorrhyncha. Over half a century later, Garay pollinator. Balogh & Greenwood (1982) proposed the (1982) revised the generic classification of Spiranthi- new genus, Cutsis Burns-Bal., E.W.Greenw. & nae and recognized 44 genera, which he did not group R.González, to accommodate this atypical species, but into alliances. For the species with ‘stenorrhynchoid’ Dichromanthus has nomenclatural priority. Lately, floral attributes, Garay retained most of the generic two contrasting views concerning the circumscription concepts of Schlechter but transferred some species of of Dichromanthus have been held by taxonomists. On Stenorrhynchos sensu Schlechter (1920) to additional the one hand, Salazar and co-workers (Salazar, Chase genera, including Cotylolabium Garay, Dichroman- & Soto, 2002; Salazar, 2003, 2009; Salazar et al., thus Garay, Sacoila Raf. and Skeptrostachys Garay. 2003, 2006; Hágsater et al., 2005; Soto et al., 2007; Almost simultaneously, Balogh (1982) published a Figueroa et al., 2008; Salazar & García-Mendoza, competing classification in which she accepted only 16 2009; Salazar & Ballesteros-Barrera, 2010) embraced genera of Spiranthinae, recognized four alliances a broader concept of Dichromanthus to include three similar to the greges of Schlechter (1920, 1926) and additional species, namely D. aurantiacus (Lex.) embraced generic concepts substantially broader than Salazar & Soto Arenas, D. michuacanus (Lex.) those of both Schlechter and Garay. For instance, she Salazar & Soto Arenas and the recently discovered treated nearly all the previously recognized genera D. yucundaa Salazar & García-Mend. (Salazar & with a stiff, sharply pointed rostellum remnant as García-Mendoza, 2009), based on their vegetative, sections of a broadly defined Stenorrhynchos. reproductive and genetic similarities to D. cinnabari- More recently, Szlachetko (1995) split Spiranthinae nus. This broader generic circumscription will be into three less inclusive subtribes, namely Spiranthi- hereafter referred to as Dichromanthus s.l. On the nae, Cyclopogoninae Szlach. and Stenorrhynchidinae other hand, Szlachetko et al. (2005) considered Szlach. The circumscription of the stenorrhynchoid Dichromanthus as a monotypic genus and included genera in his system largely followed Garay (1982), D. aurantiacus and D. michuacanus (plus a few seg- although subsequently he and his co-workers created regates from these species here considered as their additional genera (see Szlachetko et al., 2005; Rut- synonyms) in the genus Stenorrhynchos,asdid kowski et al., 2008). In contrast, in a synopsis of the Lindley (1840), Schlechter (1920) and Garay (1982). genera of Spiranthinae, Salazar (2003) adopted the Burns-Balogh (1986) also treated Dichromanthus as generic concepts of Garay (1982) to minimize arbi- monotypic but placed D. aurantiacus in Stenorrhyn- trary changes not supported by explicit phylogenetic chos and D. michuacanus in Schiedeella. hypotheses, making minor adjustments to match the Salazar et al. (2003) assessed the phylogenetic rela- results of a molecular phylogenetic study (Salazar tionships of tribe Cranichideae Endl., with a special et al., 2003). The concept of Stenorrhynchos upheld by focus on Spiranthinae, analysing sequence and Salazar, however, was narrower than that of any insertion/deletion (indel) data from five regions of previous treatment. The only species retained in this plastid and nuclear DNA. They considered 24 species

© 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 1–18 DNA PHYLOGENETICS AND FLORAL CONVERGENCE 3 that had been assigned previously to Spiranthinae, less flowers, a tubular, showily coloured perianth in finding that, with the removal of Galeottiella (as tones of red, pink, orange or yellow and a long, proposed by Salazar et al., 2002), Spiranthinae were narrow rostellum with stiff, bristle-like rostellum strongly supported as monophyletic and consisted of remnant, seem to conform to a syndrome of pollina- three major clades, identified by Salazar et al. (2003) tion by hummingbirds (van der Pijl & Dodson, 1966; as the Stenorrhynchos, Pelexia and Spiranthes clades. Dressler, 1981; Catling, 1987; Galetto, Bernardello & However, such clades did not correspond entirely Rivera, 1997; Singer & Sazima, 2000). It has been either with the limits of previously proposed generic suggested that the similarity in overall floral struc- alliances (Schlechter, 1920; Balogh, 1982) or with the ture between the otherwise distinctive species of narrowly defined subtribes of Szlachetko (1995). Some Dichromanthus s.l., Coccineorchis and Stenorrhyn- of the relationships recovered by that analysis were chos s.s. might have resulted from convergent adap- unexpected, such as the association (with low boot- tation to hummingbird pollination (Salazar et al., strap support) of Coccineorchis Schltr. with members 2002, 2003; Salazar, 2003, 2005; Salazar & García- of the so-called Pelexia alliance (after Balogh, 1982; Mendoza, 2009). However, previous phylogenetic Burns-Balogh & Robinson, 1983) instead of the Sten- studies of Spiranthinae have only sparsely sampled orrhynchos alliance, as may be expected from similari- the species of these genera. A more thorough sam- ties in gross floral morphology to the latter. The genus pling of the species involved would allow us to attain Stenorrhynchos, as delimited by most previous a clearer picture of their relationships, setting a firm workers (e.g. Lindley, 1840; Schlechter, 1920; Balogh, ground for carrying out inferences about the evolution 1982; Garay, 1982; Szlachetko, 1994), was shown to be of floral morphology and pollination syndromes. polyphyletic, as ‘Stenorrhynchos’ aurantiacus was Here, we assessed the phylogenetic relationships of strongly supported as the sister of Dichromanthus Dichromathus and Stenorrhynchos using a more com- cinnabarinus but only distantly related to the strongly prehensive set of representatives of subtribe Spiran- supported Stenorrhynchos s.s. clade. The latter thinae than previous analyses, including exemplars of included S. glicensteinii Christenson, a recently all four species of Dichromanthus s.l. and over half of described species hardly distinguishable from S. spe- the species of Stenorrhynchos s.s. recognized in recent ciosum, the type species of the genus Stenorrhynchos works (Salazar et al., 2002, 2003; Salazar, 2003; (Salazar et al., 2003; Christenson, 2005). Christenson, 2005; Salazar & García-Mendoza, 2009). Górniak et al. (2006) explored the phylogenetic Our sampling included the type species of Dichro- relationships of 19 species of Spiranthinae using manthus and Stenorrhynchos. We performed cladistic nuclear ribosomal internal transcribed spacer analyses of sequence and indel data from two highly (nrITS) DNA sequences, largely corroborating previ- variable, non-coding DNA regions: nuclear ribosomal ous findings by Salazar et al. (2003). Their study did ITS region (including the internal transcribed not include Dichromanthus cinnabarinus, but the spacers, ITS1 and ITS2, and the intervening 5.8S accession of D. aurantiacus analysed by them gene; Baldwin et al., 1995) and plastid trnL/trnF grouped with Deiregyne (as its synonym Burns- region (encompassing the intron of trnL, the inter- baloghia Szlach.) and Schiedeella within a clade genic spacer between trnL and trnF and partial exon similar in composition to the Spiranthes clade of portions; Taberlet et al., 1991). Both these regions Salazar et al. (2003), which was only distantly have succeeded, alone or in combination with one related to Stenorrhynchos speciosum. another or with other DNA regions, in resolving intra- Recently, Figueroa et al. (2008) carried out an and intergeneric phylogenetic relationships in Spiran- assessment of the phylogenetic relationships of 26 thinae (Salazar et al., 2003; Górniak et al., 2006; species of Cranichideae based on nrITS and plastid Figueroa et al., 2008; Salazar & Ballesteros-Barrera, matK–trnK DNA sequences, plus three structural 2010; Batista et al., 2011; Salazar & Dressler, in characters, with the aim of examining possible evo- press) and other orchidoid lineages (e.g. Bellstedt, lutionary paths and the systematic value of several Linder & Harley, 2001; Kores et al., 2001; Clements anatomical characters of the root. They included two et al., 2002; Bateman et al., 2003; van der Niet et al., accessions of Dichromanthus cinnabarinus and one of 2005; Álvarez-Molina & Cameron, 2009; Salazar D. michuacanus. In their analysis, D. michuacanus et al., 2009). We are particularly interested in: testing was strongly supported as the sister of D. cinnabari- the monophyly of Dichromanthus s.l. and Stenorrhyn- nus but only distantly related to Sacoila lanceolata chos s.s.; clarifying their relationships to one another; (Aubl.) Garay (a member of the Stenorrhynchos exploring the implications of the inferred relation- clade). ships for interpreting the evolution of hummingbird Notably, the floral characteristics shared by most pollination in Spiranthinae; and discussing the role of species of Spiranthinae that at one time or another the rostellum and other floral characters in pollina- have been included in Stenorrhynchos, namely odour- tion and assessing their value as taxonomic markers.

MATERIAL AND METHODS SEQUENCE EDITION, ALIGNMENT AND GAP CODING STUDIED SPECIES Bidirectional sequence reads were obtained for both Fifty-three exemplars of 40 species and 22 genera of DNA regions and the chromatograms were edited Spiranthinae, approximately 10% of the species and and assembled with Sequencher version 4.8 > 50% of the genera of the subtribe (sensu Salazar, (GeneCodes Corp., Ann Arbor, MI, USA). Alignment 2003), were analysed. All four species of Dichroman- was carried out by visual inspection and trying to thus s.l. were included. Three accessions from differ- maximize sequence similarity (Simmons, 2004). ent localities of Dichromanthus aurantiacus, and four Non-autapomorphic indels were coded as binary each of D. cinnabarinus and D. michuacanus, were (presence/absence) characters, with the exception of analysed to check for species exclusivity (Velasco, those located in mononucleotide repeat (microsatel- 2009). Four of the seven species of Stenorrhynchos s.s. lite) regions, which were excluded because of uncer- recognized by Christenson (2005) were included in the tainty of their homology (Kelchner, 2000). The analyses (one accession each). These represent much individual gap positions were treated as missing of the morphological variation and geographical range data and the coded indels were appended to the of the genus. Representative species of subtribes sequence matrices. Cranichidinae, Galeottiellinae, Goodyerinae and Manniellinae were used as outgroups, following previous phylogenetic studies (Kores et al., 1997, 2001; PHYLOGENETIC ANALYSES Salazar et al., 2003, 2009; Figueroa et al., 2008; Parsimony analyses of three data sets (nrITS, trnL/ Álvarez-Molina & Cameron, 2009). A list of the taxa trnF and both regions combined) were conducted studied, including voucher information and GenBank with the computer programme PAUP* version 4.02b accessions, is provided in Table 1. The aligned matrix for Macintosh (Swofford, 2002). Initially, we analy- is available on request from G.A.S. sed separately plastid and nuclear DNA to check for potential incongruence, comparing the trees resulting from each data set in search of strongly sup- DNA EXTRACTION, AMPLIFICATION AND SEQUENCING ported, conflicting groups (see below). Each analysis Genomic DNA was extracted from fresh or silica gel- consisted of a heuristic search with 1000 replicates dried plant tissue using a 2 ¥ cetyltrimethylammo- of random addition of sequences for the starting nium bromide (CTAB) protocol based on Doyle & trees; branch-swapping using the tree bisection– Doyle (1987), which was modified by the addition of reconnection (TBR) algorithm with the ‘MULTREES’ 2% polyvinyl pyrrolidone (PVP) to the extraction option was activated to allow for storage of multiple buffer. Amplification (PCR) was carried out using a trees in memory, saving all most-parsimonious trees commercial kit (Taq PCR Core Kit; Qiagen, Hilden, (MPTs) found. The optimality criterion was Fitch Germany) following the manufacturer’s protocols, but parsimony (Fitch, 1971), i.e. all characters were adding to the reaction mix 0.5 mL of a 0.4% aqueous treated as unordered and equally weighted. Internal solution of bovine serum albumin (BSA) to neutralize support for clades was assessed by 300 non- potential inhibitors (Kreader, 1996) and, in the case parametric bootstrap replicates (Felsenstein, 1985), of nrITS, 0.5 mL of dimethylsulphoxide (DMSO) to with heuristic searches consisting of 20 random reduce problems associated with DNA secondary sequence additions and TBR branch-swapping, structure. The nrITS region was amplified and saving up to 20 MPTs per heuristic replicate. We sequenced with primers ITS5 and ITS4 (White et al., arbitrarily considered bootstrap percentages (BP) of 1990), whereas, for the trnL/trnF region primers, c 51–70 as weak support, 71–84 as moderate support and f were used for amplification and sequencing was and 85–100 as strong support. carried out with primers c, d, e and f (all from Additionally, a model-based phylogenetic analysis Taberlet et al., 1991). PCR profiles were as in Salazar of the combined data set was conducted using et al. (2003). The PCR products were cleaned with Markov chain Monte Carlo Bayesian inference as QIAquick silica columns (Qiagen) and used in cycle implemented in MrBayes version 3.1.2 (Ronquist, sequencing reactions with the ABI Prism Big Dye® Huelsenbeck & Van Der Mark, 2005). The model of Terminator Cycle Sequencing Ready Reaction kit with molecular evolution chosen for both the nrITS and AmpliTaq® DNA polymerase version 3.1 (Applied Bio- the trnL/trnF sequence data sets according to the systems Inc., Foster City, CA, USA). Cycle sequencing Akaike information criterion (Akaike, 1974) with the products were purified with Centri-Sep sephadex program Modeltest 3.7 (Posada & Crandall, 1998) columns (Princeton Separations Inc., Adelphia, NJ, was GTR + I +G. The ‘standard discrete model’ for USA) and sequenced in a 3100 Genetic Analyzer morphological data implemented in MrBayes (based (Applied Biosystems). on Lewis, 2001) was set for the indel data. All

Table 1. Voucher information and GenBank accessions for DNA sequences

Taxon Voucher specimen nrITS trnL-trnF

Subtribe Cranichidinae Ponthieva racemosa (Walt.) Mohr Mexico, Salazar 6049 (MEXU) AJ539508 AJ544490 Prescottia plantaginea Lindl. Brazil, Salazar 6350 (K) AJ539511 AJ544493 Subtribe Galeottiellinae Galeottiella sarcoglossa (A.Rich. & Mexico, Jiménez 2334 (AMO) AJ539518 AJ544500 Galeotti) Schltr. Subtribe Goodyerinae Goodyera pubescens (Willd.) R.Br. USA, Chase 212 (NCU) AJ539519 AM419815 Subtribe Manniellinae Manniella cypripedioides Salazar, Cameroon, Salazar 6323 (YA) AJ539516 AJ544498 T.Franke, Zapfack & Benkeen Subtribe Spiranthinae Aulosepalum tenuiﬂorum (Greenm.) Mexico, Salazar 6150 (MEXU) AJ539591 AJ544474 Garay Beloglottis costaricensis (Rchb.f.) Mexico, Soto 8129 (MEXU) AJ539492 AJ544475 Schltr. Coccineorchis cernua (Lindl.) Garay Panama, Salazar 6249 (MEXU) AJ539502 AJ544485 Coccineorchis standleyi (Ames) Garay Panama, Salazar 6248 (MEXU) FN996949 FN996961 Cyclopogon epiphyticum (Dodson) Ecuador, Salazar 6345 (K) AJ539499 AJ544482 Dodson Deiregyne albovaginata (C.Schweinf.) Mexico, Jiménez 2164 (AMO) FN641870 FN641882 Garay Deiregyne densiﬂora (C.Schweinf.) Mexico, Salazar 6125 (MEXU) FN641874 FN641886 Salazar & Soto Arenas Deiregyne diaphana (Lindl.) Garay Mexico, Salazar 6172 (MEXU) AJ539484 AJ544467 Deiregyne eriophora (B.L.Rob. & Mexico, Salazar 6104 (MEXU) FN641873 FN641885 Greenm.) Garay Deiregyne falcata (L.O.Williams) Mexico, Salazar 6212 (MEXU) FN641871 FN641883 Garay Deiregyne pseudopyramidalis Mexico, Salazar 6126A (MEXU) FN641872 FN641884 (L.O.Williams) Garay Deiregyne rhombilabia Garay Mexico, Salazar 6138 (MEXU) FN641869 FN641881 Dichromanthus aurantiacus (Lex.) (1) Mexico, Francke s.n. (MEXU) FN996956 FN996970 Salazar & Soto Arenas (2) Mexico, Salazar 6717 (MEXU) FN996957 FN996971 (3) Mexico, Salazar 6351 (K) AJ539485 AJ544468 Dichromanthus cinnabarinus (Lex.) (1) Mexico, Linares 4469 (MEXU) AJ539486 AJ544469 Garay (2) Mexico, Salazar 6493bis (MEXU) FN996951 FN996963 (3) Mexico, Figueroa s.n. (MEXU) FN996952 FN996964 (4) Mexico, Salazar 6895 (MEXU) AM778176 FN996965 Dichromanthus michuacanus (Lex.) (1) Mexico, Salazar 6047 (MEXU) AM778177 FN996966 Salazar & Soto Arenas (2) Mexico, Salazar 7403 (MEXU) FN996953 FN996967 (3) Mexico, Salazar 7034 (MEXU) FN996954 FN996968 (4) Mexico, Salazar 7456A (MEXU) FN996955 FN996969 Dichromanthus yucundaa Salazar & Mexico, García-Mendoza & Franco 8774 (MEXU) FN996950 FN996962 García-Mendoza Eltroplectris calcarata (Sw.) Garay & Brazil, Soares s.n. (MEXU, photograph) AJ519448 AJ519452 H.R.Sweet Eltroplectris triloba (Lindl.) Pabst Argentina, Munich Bot. Gard. 96/4474 (M) FN641864 FN641875 Eurystyles borealis A.H.Heller Mexico, Soto 9149 (AMO) AJ539497 AJ544480 Funkiella hyemalis (A.Rich. & Mexico, Salazar 6128 (MEXU) AJ539495 AJ544478 Galeotti) Schltr. Lankesterella gnoma (Kraenzl.) Brazil, Warren s.n. (K) FN556163 FN556168 Hoehne

Table 1. Continued

Taxon Voucher specimen nrITS trnL-trnF

Mesadenella petenensis (L.O.Williams) Mexico, Salazar 6069 (MEXU) AJ539503 AJ544486 Garay Mesadenus lucayanus (Britt.) Schltr. Mexico, Salazar 6043 (MEXU) AJ539488 AJ544471 Microthelys minutiﬂora (A.Rich. & Mexico, Salazar 6129 (MEXU) AJ539494 AJ544477 Galeotti) Garay Odontorrhynchus variablis Garay Chile, Wallace 130/85 (CANB) AJ539498 AJ544481 Pelexia adnata (Sw.) Poit. ex Spreng. Mexico, Salazar 6012 (MEXU) AJ539501 AJ544484 Pteroglossa roseoalba (Rchb.f.) Salazar El Salvador, Salazar 6023 (MEXU) FN868839 FN868837 & M.W.Chase Sacoila lanceolata (Aubl.) Garay Guatemala, Förther 2545 (M) AJ539504 – Brazil, da Silva 874 (MG) – AJ544529 Sarcoglottis acaulis (J.E.Sm.) Schltr. Trinidad, Salazar 6346 (K) AJ544483 AJ539500 Schiedeella crenulata (L.O.Williams) Mexico, Goldman 902 (BH) FN641868 FN641880 Espejo & López-Ferrari Schiedeella durangensis (Ames & Mexico, Soto 10673 (AMO) FN641867 FN641879 C.Schweinf.) Burns-Bal. Schiedeella faucisanguinea (Dod) Mexico, Jiménez s.n. (AMO) AJ539496 AJ544479 Burns-Bal. Schiedeella llaveana (Lindl.) Schltr. Mexico, Salazar 6105 (MEXU) AJ539487 – Mexico, Salazar 6073 (MEXU) – AJ544470 Spiranthes cernua (L.) Rich. USA, Nickrent 4188 (MEXU) AJ539489 AJ544472 Spiranthes spiralis (L.) Cheval. UK, Rudall & Bateman s.n. (K) AJ539490 AJ544473 Stenorrhynchos albidomaculatum Costa Rica, Salazar 7662 (MEXU) FN996948 FN996960 E.A.Christ. Stenorrhynchos glicensteinii Mexico, Salazar 6090 (MEXU) AJ539505 AJ544487 E.A.Christ. Stenorrhynchos millei Schltr. Ecuador, Portilla s.n. (MEXU) FN996946 FN996958 Stenorrhynchos speciosum (Jacq.) Virgin Islands, Salazar 7661 (MEXU) FN996947 FN996959 Rich. ex Spreng. Svenkoeltzia congestiﬂora Mexico, Salazar 6143 (MEXU) AJ539493 AJ544476 (L.O.Williams) Burns-Bal.

model parameters were unlinked among three MORPHOLOGICAL OBSERVATIONS character partitions (nrITS sequences, trnL/trnF sequences and indels) to allow each group of char- Fresh or pickled flowers of the species of Spiranthinae acters to have its own set of parameters, as sug- included in the cladistic analyses were dissected and gested by Ronquist et al. (2005). Two simultaneous examined under a stereomicroscope (Stemi SV 6; Carl analyses were run for 1 000 000 generations under Zeiss Mikroskopie, Jena, Germany) to determine the the default conditions for the Markov chains, sam- structure of the rostellum and other floral parts. The pling trees every 100 generations. Convergence was exemplars listed in Table 1 for DNA sequences also determined by examining the average standard vouch for the morphological observations, and we deviation of split frequencies among the two runs studied boiled flowers from additional specimens kept and by plotting the log–likelihood scores of the trees at the herbaria AMES, AMO, COL, K, ENCB, IEB, visited by the cold chain. The first 200 000 genera- JBSD, MEXU, NY, SEL, US and VEN. Floral parts of tions of each run were discarded as the burn-in and plants representing all four species of Dichromanthus the remaining trees from both runs were pooled to s.l., as well as Deireyne obtecta (C.Schweinf.) Garay, build a majority-rule consensus tree, on which infer- Stenorrhynchos glicensteinii and Svenkoeltzia conges- ences about relationships and posterior probabilities tiflora (L.O.Williams) Burns-Bal. were photographed of clades were based. Posterior probabilities (PP) of in vivo with a digital camera (Coolpix 5200; Nikon, 0.95–1.00 were considered as strong support, 0.90– Tokyo, Japan) to illustrate their floral characteristics, 0.94 as moderate support and < 0.90 as weak especially the structure of the gynostemium and the support. rostellum/rostellum remnant.

POLLINATION DATA Eurystyles (c) and Spiranthes (d) clades are strongly Information on the pollination of species of Spiran- supported (BP 100, 100 and 92, respectively), but < thinae was gathered from the literature, unless oth- Coccineorchis grouped (with BS 50) to the Eury- erwise indicated. styles clade instead of other members of the Pelexia clade (b). The relationships among these major clades lack bootstrap support > 50. The position of Dichro- RESULTS manthus s.l. and Stenorrhynchos s.s. in the trnL/trnF PARSIMONY ANALYSES analysis mirrors the nrITS analysis (Fig. 1A), with The nrITS matrix consisted of 782 characters, of which Dichromanthus embedded in the Spiranthes clade 253 (32%) were potentially informative to parsimony. and with Deiregyne as its closest relative (BP 89). The analysis found 216 MPTs with a length of 1018 Within Dichromanthus s.l., D. cinnabarinus diverged steps, consistency index (CI) excluding uninformative first, but relationships among the other three species characters = 0.45 and retention index (RI) = 0.70. The were unresolved. In turn, Stenorrhynchos belongs in strict consensus of the 216 MPTs is depicted in the Stenorrhynchos clade as sister to a monophyletic Figure 1A. Spiranthinae s.l. received strong support group encompassing Eltroplectris, Mesadenella, Ptero- (BP 100) and, within them, four major clades (a–d in glossa and Sacoila (BP 92). Fig. 1A) were recovered. These clades correspond to The relationships recovered by the separate analy- moderately supported Stenorrhynchos (a; BP 72), ses of nrITS and the trnL/trnF region were similar, Pelexia (b; BP 64) and Spiranthes clades (d; BP 74) of and none of the few discordant groups between the Salazar et al. (2003), plus a strongly supported group two analyses received strong support from the boot- consisting of Eurystyles borealis A.H.Heller and strap in either (Fig. 1). Therefore, the topological dif- Lankesterella gnoma (Kraenzl.) Hoehne, which is here- ferences are interpreted as resulting from insufficient after referred to as the Eurystyles clade (c; BP 95). phylogenetic signal in the data sets and not as real The latter was strongly supported as the sister of the conflict (Wiens, 1998). The combined matrix of the Spiranthes clade (BP 92), whereas support for the nrITS and trnL/trnF regions consisted of 2441 Pelexia clade as sister to [Eurystyles clade/Spiranthes characters, of which 503 (21%) were potentially clade] was weak (BP 63). Overall, generic relation- parsimony-informative. The heuristic search found 14 ships mirror the results of Salazar et al. (2003), MPTs with a length of 1862 steps, CI = 0.49 and Salazar & Ballesteros-Barrera (2010), Batista et al. RI = 0.74. The 14 MPTs were nearly completely (2011) and Salazar & Dressler (in press). The four resolved, except for the intraspecific relationships species of Stenorrhynchos s.s. analysed here form a among accessions of Dichromanthus cinnabarinus moderately supported monophyletic group (BP 75) and D. michuacanus. Spiranthinae sensu Salazar within the Stenorrhynchos clade; the relationships (2003) were strongly supported as monophyletic among Stenorrhynchos s.s., Pteroglossa roseoalba (BP 100); within them, the same four major clades (Rchb.f.) Salazar & M.W.Chase and the clade that recovered by the nrITS analysis were recovered but includes Mesadenella petenensis (L.O.Williams) Garay, obtaining stronger bootstrap support (Fig. 2A). The Sacoila lanceolata and Eltroplectris triloba (Lindl.) Stenorrhynchos clade (a; BP 96) was the first to Pabst/E. calcarata (Sw.) Garay & H.R.Sweet are unre- diverge and consisted of Stenorrhynchos s.s. (BP 100) solved in the strict consensus (Fig. 1A). Dichroman- as the sister of a clade (BP 87), within which thus s.l. is strongly supported as monophyletic (BP 99) (Eltroplectris calcarata/E. triloba) (BP 100) is in turn and it occupies a derived position within the Spiran- sister to weakly supported (Mesadenella petenen- thes clade, being moderately supported (BP 74) as the sis(Pteroglossa roseoalba/Sacoila lanceolata)). The sister of Deiregyne. The accessions of both Dichroman- Pelexia clade (b; BP 77) diverges next but is only thus michuacanus and D. cinnabarinus form strongly weakly supported (BP 55) as sister to (Eurystyles supported exclusive groups, as do two of the three clade/Spiranthes clade) The position of Coccineorchis samples of D. aurantiacus, but the position of the third as the sister of the other constituents of the Pelexia accession of the last species is unresolved. The single clade received moderate support (BP 77), whereas accession of D. yucundaa was weakly associated with (Cyclopogon epiphyticum(Sarcoglottis acaulis(Odont- D. cinnabarinus (BP 69). orrhynchus variabilis/Pelexia adnata))) is strongly The trnL/trnF matrix included 1659 characters, supported (BP 100). The Eurystyles clade (c; BP 100) 250 (15%) of which were potentially parsimony- obtained strong support as the sister of the Spiran- informative. The analysis found four MPTs with a thes clade (d; BP 96). The latter is in turn strongly length of 834 steps, CI = 0.54 and RI = 0.78. The strict supported (BP 100) and, within it, the first group to consensus of the four MPTs is depicted in Figure 1B. diverge encompasses (Svenkoeltzia congestiflora(Au- As in the nrITS analysis, Spiranthinae s.l. are losepalum tenuiflorum/Beloglottis costaricensis)) as strongly supported (BP 98). The Stenorrhynchos (a), the sister of (Schiedeella faucisanguinea(Microthelys

© 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 1–18 8 SALAZAR TAL ET . 01TeLnenSceyo London, of Society Linnean The 2011 © oaia ora fteLnenSociety Linnean the of Journal Botanical 2011, ,

Figure 1. Strict consensus trees from the separate analyses of (A) the nuclear ribosomal internal transcribed spacer (nrITS) region and (B) the trnL/trnF region.

167 Numbers above branches are bootstrap percentages > 50%. Type species of genera are indicated in boldface. Bars a–d mark the four major clades or groups of

1–18 , Spiranthinae referred in the text. 01TeLnenSceyo London, of Society Linnean The 2011 © oaia ora fteLnenSociety Linnean the of Journal Botanical N HLGNTC N LRLCONVERGENCE FLORAL AND PHYLOGENETICS DNA 2011, , 167 1–18 ,

Figure 2. A, strict consensus tree from the parsimony analysis of combined nuclear ribosomal internal transcribed spacer (nrITS) and trnL/trnF regions; numbers above branches are bootstrap percentages > 50. B, Bayesian summary tree of combined nrITS and trnL/trnF regions; numbers above branches are posterior probabilities > 0.50. Type species of genera are indicated in boldface. Circles a–d mark the four major clades or groups of Spiranthinae referred in the text. Shaded boxes indicate species for which pollination by hummingbirds has been confirmed; clear boxes indicate species for which hummingbird pollination has been inferred from floral morphology and colouration. 9 10 SALAZAR ET AL. minutiflora/Funkiella hyemalis)). All these groups SYSTEMATIC POSITION AND ECOGEOGRAPHICAL are strongly supported. Spiranthes s.s. (BP 100) CHARACTERISTICS OF DICHROMANTHUS diverges next, being sister, with BP < 50, to the AND STENORRHYNCHOS remaining of the Spiranthes clade; the latter includes Mesadenus lucayanus as sister (BP 98) to a clade On the one hand, our data strongly support mono- that encompasses two strongly supported sub- phyly of Dichromanthus s.l., in agreement with the clades: Schiedeella s.s. (S. durangensis(S. llaveana/S. vegetative, reproductive and ecogeographical features crenulata)) (BP 98) and (Deiregyne/Dichromanthus shared by the four species that we include in this s.l.). Dichromanthus s.l. is strongly supported as genus (Salazar et al., 2002, 2003, 2006; Salazar, 2003, monophyletic (BP 100) and its three species for which 2009; Hágsater et al., 2005; Soto et al., 2007; Figueroa more than one accession were analysed turned out to et al., 2008; Salazar & García-Mendoza, 2009). Like- be exclusive. Dichromanthus cinnabarinus was recov- wise, the derived position of Dichromanthus s.l. ered as the sister of a weakly supported clade (BP 61) within the Spiranthes clade (Fig. 2, clade d) as sister in which D. aurantiacus is sister to |(D. yucundaa/ to Deiregyne (sensu Salazar, 2003) is strongly sup- D. michuacanus). This last association, however, ported (see also Salazar & Ballesteros-Barrera, 2010). obtained a BP < 50. On the other hand, monophyly of Stenorrhynchos s.s. received moderate to strong support in our analyses, but this genus belongs in the strongly supported Stenorrhynchos clade (Fig. 2, clade a). Therefore, BAYESIAN ANALYSIS inclusion of Dichromanthus aurantiacus, D. michua- The Bayesian summary tree was topologically similar canus, Deiregyne albovaginata and D. densiflora to the strict consensus of the combined parsimony in Stenorrhynchos, as in Szlachetko et al. (2005), analysis, differing only in the relative positions of two renders Stenorrhynchos polyphyletic and results in an outgroup species (Prescottia plantaginea Lindl. and unnatural assemblage of species with disparate eco- Ponthieva racemosa (Walt.) Mohr) and in recovering logical preferences and overall morphology (Salazar, the Stenorrhynchos and Pelexia clades as forming a 2003; see below). weakly supported group (PP 0.65; Fig. 2B). Spiranthi- Dichromanthus yucundaa is known only from a nae and its four major clades were supported by high small area in the Mexican state of Oaxaca (Salazar & posterior probabilities (PP 1.00). Overall, patterns of García-Mendoza, 2009), but the other three species of supported resolution were similar in the combined Dichromanthus s.l. are widespread and relatively parsimony and Bayesian analyses, and both the phy- common throughout most mountain ranges and pla- logenetic position and the interspecific relationships teaux from the southern USA (Arizona and Texas) of Dichromanthus s.l. and Stenorrhynchos s.s. were through Mexico to northern Central America (Salazar, identical in both instances (Fig. 2A, B). 2003). All four species thrive in forest clearings and other open, disturbed or marginal habitats, such as basaltic or limestone rocky fields, natural and human- induced grasslands and roadside banks, in areas DISCUSSION dominated by seasonally dry coniferous–oak forest, The present parsimony and Bayesian analyses tropical deciduous forest and xerophilous scrub (Luer, strongly support the monophyly of Spiranthinae 1975; Soto, 2002; Salazar, 2003, 2009; Coleman, 2005; sensu Salazar (2003; Salazar et al., 2003) and confirm Hágsater et al., 2005; Salazar et al., 2006; Salazar & the non-monophyly of the narrowly defined subtribes García-Mendoza, 2009). Members of Deiregyne, the into which Szlachetko (1995) divided Spiranthinae, as sister of Dichromanthus, are also characteristic ele- previously noted by Salazar et al. (2003) and Górniak ments of seasonally dry/cool montane habitats in et al. (2006). The relationships among the four major Mexico and Guatemala and the distribution ranges of clades of Spiranthinae (a–d in Fig. 2) are only weakly Deiregyne and Dichromanthus largely overlap supported, but each of these clades received moderate (Salazar, 2003). Most species of Deiregyne grow in leaf to strong support. The generic relationships within litter under shade provided by the forest canopy or at this subtribe are beyond the focus of this paper and the forest edge, although D. pseudopyramidalis will be discussed elsewhere (G. A. Salazar, unpubl. (L.O.Williams) Garay and D. rhombilabia Garay data). In the following, discussion will be restricted to favour the same exposed conditions preferred by the implications of our results for the classification of Dichromanthus and in some areas they have been Dichromanthus s.l. and Stenorrhynchos s.s., struc- found living side by side with species of Dichroman- tural aspects of some floral characteristics related to thus (G. A. Salazar, pers. observ.). In contrast, the pollination and the utility of floral characters as taxo- seven or so species currently recognized in Stenor- nomic markers. rhynchos s.s. (Salazar, 2003; Christenson, 2005) dwell

© 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 1–18 DNA PHYLOGENETICS AND FLORAL CONVERGENCE 11 in moist to wet cloud forests and Andean páramos, Wilson, 2008). However, there are documented ranging from southern Mexico through Central instances of secondary adaptation to pollination by America and the Antilles to Andean Colombia, hymenopterans in hummingbird-pollinated clades, as Venezuela, Ecuador and Peru (Salazar, 2003; in Euphorbia [Pedilanthus] diazlunana (J.A.Lomelí & Christenson, 2005; Hágsater et al., 2005). Sahagún) V.W.Steinm. (Euphorbiaceae; Sahagún- Godínez & Lomelí-Sención, 1997; Cacho et al., 2010). Further, the most salient floral difference between FLORAL STRUCTURE AND POLLINATION SYNDROMES D. michuacanus and its congeners is flower colour. Dichromanthus s.l. is unusual in the Spiranthes clade Experimental studies have indicated that pollinator in that most of its species (except D. michuacanus) shifts might be initiated by a single major mutation; display a hummingbird pollination syndrome, which for instance, by substitution of an allele by another at includes odourless, brightly coloured flowers and floral a locus that controls the expression of a floral pigment bracts in tones of red, orange or a combination of (Bradshaw & Schemske, 2003). However, interspecific these, and a tubular perianth producing nectar at the relationships in Dichromanthus received only weak bottom of the floral tube (Fig. 3A–C). In contrast, and moderate support in our parsimony and Bayesian pollination by nectar-foraging bees such as bumble- analyses, respectively, and in our view additional bees (Bombus spp.) apparently predominates and phylogenetic work incorporating other plastid and probably represents the plesiomorphic condition in the nuclear regions is required to clarify the relationships Spiranthes clade (Luer, 1975; Catling, 1983; Salazar, among the species of Dichromanthus. The issue of a 2003; Salazar & Ballesteros-Barrera, 2010). putative backward shift from bird to bee pollination Pollination of D. aurantiacus (Fig. 3A) and D. cinna- in D. michuacanus will then have to be reconsidered. barinus (Fig. 3B) by the hummingbirds Amazilia Singer & Sazima (2000) documented the pollination berylina and Hylocharis leucotis has been recorded in of Sacoila lanceolata (Fig. 3G), a member of the Sten- central Mexico (Sarmiento & Romero, 2000; Hágsater orrhynchos clade, by three species of hummingbird in et al., 2005). The pollinator of D. yucundaa (Fig. 3C) is southern Brazil and Siegel (2011) published a photo- not known, but on account of its floral characteristics graph of the hummingbird Calliphlox bryantae visiting Salazar & García-Mendoza (2009) suggested that this flowers of Stenorrhynchos glicensteinii (as S. specio- species might also be hummingbird-pollinated. The sum) in Costa Rica. The bird bears a pollinarium of the flowers of D. michuacanus, however, differ from the orchid stuck on its beak, which suggest that it is a previous three species in being whitish, yellow or green genuine pollinator of this species. Moreover, humming- with dark green to brown veins on sepals and petals, bird pollination has been inferred, on the basis of and in producing an intense diurnal odour (Fig. 3D). flower morphology and colouration, for Stenorrhynchos These floral features are compatible with bee pollina- s.s. (Fig. 3H) and other members of the Stenorrhynchos tion (Salazar, 2003) and, recently, the pollination of clade (van der Pijl & Dodson, 1966; Singer & Sazima, D. michuacanus by the bumblebee Bombus diligens 2000; Salazar, 2003) and for Svenkoeltzia (Spiranthes was observed for the first time in central Mexico (M. A. clade; Fig. 3I) (Salazar, 2003). In the Pelexia clade bee López Rosas, pers. comm.). Natural pollination of pollination predominates, with species of Cyclopogon Deiregyne, the sister genus of Dichromanthus, has not C.Presl being pollinated by various halictid bees been observed, but flowers of the species of Deiregyne (Singer & Cocucci, 1999; Singer & Sazima, 1999; (Fig. 3E, F) display the stereotyped nectar–bee syn- Benítez et al., 2006), Pelexia by Bombus spp. (Dressler, drome characteristic of the Spiranthes clade (Salazar, 1993; Singer & Sazima, 1999) and Sarcoglottis Schltr. 2003; Salazar & Ballesteros-Barrera, 2010). The by Euglossa spp. (Singer & Sazima, 1999). Neverthe- flowers of Schiedeella, the sister of the clade formed by less, noctuid moth pollination has been documented for Deiregyne and Dichromanthus, are similar to those of Sauroglossum elatum Lindl. (Singer, 2002), a close Deiregyne. Luer (1975) recorded the pollination of relative of Pelexia (see Álvarez-Molina & Cameron, S. durangensis (Ames & C.Schweinf.) Burns-Bal. 2009). There are no observations of the natural polli- in central Mexico by the bumblebee Bombus nation of Coccineorchis, but in this member of the steindachneri. Pelexia clade flowers are brightly coloured, tubular, Hummingbird pollination probably represents a scentless and provided with a narrow, stiff rostellum specialization of Dichromanthus and the derived posi- remnant, together suggesting hummingbird pollination of bee-pollinated D. michuacanus within Dichro- tion (Salazar, 2003, 2005). manthus in the trees of Figure 2 is suggestive of a Occurrence of hummingbird pollination in indi- reversal to bumblebee pollination. This result is unex- vidual species or groups of species belonging to pected, given the generalized directionality of shifts distantly related clades, i.e. some species of from bee to hummingbird pollination (Beardsley, Yen Dichromanthus and (probably) Svenkoeltzia in the & Olmstead, 2003; Wilson et al., 2007; Thomson & Spiranthes clade; Sacoila and Stenorrhynchos in the

Figure 3. Inflorescences representative of studied species of Spiranthinae. A, Dichromanthus aurantiacus (from Salazar 7724). B, D. cinnabarinus (from Salazar & Cabrera 6728). C, D. yucundaa (from García-Mendoza and Franco 8744). D, D. michuacanus (from Salazar 7250). E, Deiregyne rhombilabia (from Salazar et al. 7400). F, D. eriophora (from Salazar et al. 8183). G, Sacoila lanceolata (from a cultivated specimen of unspecified origin). H, Stenorrhynchos glicensteinii (from Salazar & Linares 7532). I, Svenkoeltzia congestiflora (from Andreano s.n.). ᭣

Stenorrhynchos clade; probably Coccineorchis in manthus s.l.InD. aurantiacus, D. michuacanus and the Pelexia clade, each of them being closely related to D. yucundaa, the viscidium is sheathing and, upon groups exhibiting other pollination syndromes, sug- removal of the pollinarium (by hand or by a pollina- gests that hummingbird pollination has evolved tor), the rostellum remnant is a narrow, hard bristle several times independently within subtribe Spiran- (Fig. 4A–F). In D. aurantiacus (Fig. 4A, B) the thinae (Fig. 2). Parallel adaptation to hummingbird rostellum/rostellum remnant is proportionately pollination leading to floral resemblance to ornitho- longer than in D. yucundaa and D. michuacanus philous members of Spiranthinae also occurred in the (Fig. 4C–F), but in all three species the viscidium distantly related Neotropical orchid, Corymborkis covers most of the length of the rostellum, whereas flava (Sw.) Kuntze (Epidendroideae, Tropidieae; the viscidia of Stenorrhynchos and Svenkoeltzia Abreu & Vieira, 2004; Vieira et al., 2007). Moreover, only cover the distal half or so of the rostellum multiple origins of hummingbird pollination have also (Fig. 4M–P). been documented in explicit phylogenetic contexts As mentioned earlier, Dichromanthus cinnabarinus in various other lineages of predominantly insect- is distinctive in that the broadly notched rostellum pollinated plants, including, for instance, the monocot remnant is provided with an apical pouch or cavity genus Costus L. (Costaceae; Kay et al., 2005) and in which a retrorse extension of the viscidium is eudicots such as Mimulus L. (Phrymaceae; Beardsley ‘plugged’ before its removal (Fig. 4G–J). Nevertheless, et al., 2003) and Penstemon Schmidel (Plantagi- such an evident structural difference between D. cin- naceae; Wilson et al., 2007). nabarinus and the other species here included in Dichromanthus, which has been considered worthy of generic distinction by previous taxonomists, does not FLORAL CHARACTERS AS TAXONOMIC MARKERS appear to imply a significant functional difference as Salazar et al. (2003) noted several instances of incon- D. cinnabarinus is pollinated in a similar fashion to, gruence between clades recovered by their molecular and by the same species of hummingbirds as, D. analyses and previous taxonomic groupings based on aurantiacus (Sarmiento & Romero, 2000; Hágsater floral morphology. They proposed that some of the et al., 2005). floral characters used to define the genera are directly Incongruence between groups delimited on the involved in pollination and thus prone to homoplasy basis of particular floral characters involved in polli- resulting from selective pressures from pollinators. nation and clades recovered in the molecular trees Hence, similarity in such characters between dis- does not preclude the possibility that other structural tantly related groups (as evidenced by the DNA trees) features might reliably mark such clades. For might reflect convergent adaptation to pollinators instance, despite the above-mentioned differences in rather than phylogenetic proximity. Our results lend flower coloration, odour and rostellum/viscidium mor- support to that hypothesis with regard to Dichroman- phology among the species of Dichromanthus s.l., all thus and Stenorrhynchos, in which similar overall of them share a putative morphological synapomor- flower shape and colouration probably evolved inde- phy, i.e. a nectary formed by a narrow channel at the pendently as a result of convergent adaptation to base of the labellum with an irregularly thickened hummingbird pollination. It is not clear, however, intramarginal nectar gland on each side (Fig. 5A–D). whether the hard, sharply pointed rostellum remnant In contrast, in Stenorrhynchos s.s. (Fig. 5E) and Sven- shared by most hummingbird-pollinated members of koeltzia (Fig. 5F) the nectary is a broad, round cavity Spiranthinae is also convergent or if it represents the lacking obvious nectar glands. A channelled lip base is plesiomorphic (ancestral) condition in the subtribe. also present in Deiregyne diaphana (Lindl.) Garay, Ongoing comparative (including developmental) but the channel is proportionately longer and shal- studies of floral morphology in Spiranthinae (C. lower than in Dichromanthus and the nectar glands Figueroa & G. A. Salazar, unpubl. data) will shed occupy a distal position instead of being located near light on the homology and evolutionary patterns of the channel base. No other genus of Spiranthinae has this and other floral structures. a nectary like that of Dichromanthus s.s., and there- It is worthy to note the variation in rostellum and fore the genus can be unambiguously diagnosed by pollinarium structure among the species of Dichro- this feature (cf. Salazar, 2003).

Figure 4. Gynostemium and rostellum/rostellum remnant structure of selected species of Spiranthinae. The apices of structures point upwards. A–B, Dichromanthus aurantiacus (from Salazar 7724). C–D, D. yucundaa (from García- Mendoza & Franco 8744). E–F, D. michuacanus (from Salazar 7250). G–J, D. cinnabarinus (from Salazar & Cabrera 6728). K–L, Deiregyne obtecta (from Salazar et al. 7704). M–N, Stenorrhynchos glicensteinii (from Salazar & Linares 7532). O–P, Svenkoeltzia congestiﬂora (from Salazar 8103). Scale bars, 2 mm (A–G, K–O); 1 mm (H–J, P). Abbreviations: co, column; pl, viscidium plug; po, pollinium; ro, rostellum; rp, rostellum pouch; rr, rostellum remnant; st, stigma; vi, viscidium. ᭣

Figure 5. Nectary structure in selected species of Spiranthinae. The labellum apex points upwards. A, Dichromanthus aurantiacus (from Salazar 7724). B, D. yucundaa (from García-Mendoza & Franco 8744). C, D. cinnabarinus (from Salazar & Cabrera 6728). D, D. michuacanus (from Salazar 7250). E, Stenorrhynchos glicensteinii (from Salazar & Linares 7532). F, Svenkoeltzia congestiﬂora (from Salazar 8103). Scale bars, 3 mm.

From all the above, it is now clear that the broad Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski concept of Stenorrhynchos held by Szlachetko et al. MF, Campbell CS, Donohue MJ. 1995. The ITS region of (2005; Rutkowski et al., 2008), and most previous nuclear ribosomal DNA: a valuable source of evidence on taxonomists, represents an artificial assemblage angiosperm phylogeny. Annals of the Missouri Botanical based on floral characteristics that have probably Garden 82: 247–277. evolved convergently as a result of adaptation to the Balogh P. 1982. Generic redefinition in subtribe Spiranthi- same pollinator group (i.e. hummingbirds). The nae (Orchidaceae). American Journal of Botany 69: 1119– observed variation in flower colour and in the struc- 1132. Balogh P, Greenwood EW. 1982. Cutsis Balogh, Greenwood ture of the rostellum and other floral organs associ- and Gonzales [sic], a new genus from México. Phytologia 51: ated with pollination among closely related species, as 297–298. within Dichromanthus s.l., indicates that floral mor- Bateman RM, Hollingsworth PM, Preston J, Yi-bo L, phology is evolutionarily labile (see also Salazar & Pridgeon AM, Chase MW. 2003. Molecular phylogenetics Dressler, in press) and the reliability of such charac- and evolution of Orchidinae and selected Habenarinae. ters to delimit natural (i.e. monophyletic) genera and Botanical Journal of the Linnean Society 142: 1–40. higher-level taxa should not be assumed a priori. This Batista JAN, Meneguzzo TEC, Salazar GA, Bianchetti L work highlights the need for detailed, comparative de B, Ramalho AJ. 2011. Phylogenetic placement, taxo- structural and pollination studies which, coupled with nomic revision and a new species of Nothostele (Orchi- formal phylogenetic analyses of both molecular and daceae), an enigmatic genus endemic to the cerrado of structural characters, will allow us to achieve a sound central Brazil. Botanical Journal of the Linnean Society understanding of the role of floral organs in pollina- 165: 348–363. tion and their usefulness for delimiting historically Beardsley PM, Yen A, Olmstead RG. 2003. AFLP phylog- meaningful taxonomic groups in Spiranthinae. eny of Mimulus section Erythranthe and the evolution of hummingbird pollination. Evolution 457: 1397–1410. Bellstedt DU, Linder HP, Harley E. 2001. 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