Abstract Subtribe Spiranthinae is the most species-rich lineage of terrestrial Neotropical orchids, encompassing > 500 species and 40 genera. We conducted maximum parsimony and maximum likelihood phylogenetic analyses of DNA sequence data of plastid matK-trnK and trnL-trnF and nuclear ribosomal ITS sequences for 36 genera and 182 species of Spiranthinae plus appropriate outgroups. The results strongly support monophyly of Spiranthinae (minus Discyphus, Discyphinae and Galeottiella, Galeottiellinae) and five major lineages, namely monospecific Cotylolabium (sister to the remaining Spiranthinae) and the Eurystyles, Pelexia, Spiranthes and Stenorrhynchos clades. Eighteen of the 27 genera of Spiranthinae for which more than one species was included in our analyses are monophyletic. Paraphyly of large genera, such as Cyclopogon and Sarcoglottis, resulted from segregation of particular species or groups of species exhibiting minor modifications of structures directly involved in pollination (e.g. nectary, rostellum and viscidium). Conversely, polyphyly has resulted from convergent evolution of floral attributes in distantly related species (e.g. Mesadenus). Some of the morphological characters used traditionally for generic delimitation and in non-molecular cladistic analyses of Spiranthinae are discussed against the evolutionary framework set by our molecular trees, emphasizing putative synapomorphies and problems derived from inappropriate character coding or incorrect homology assessments. Our ancestral area analysis indicates that Spiranthinae originated in eastern South America, with subsequent migrations and secondary radiations in Mesoamerica and North America, plus a derived migration from the latter region to the Old World (Spiranthes). ‘Modern orchidologists tend to take the view that any modifications in the reproductive organs of orchids, no matter how obscure, are probably of evolutionary importance because of the close correlation in the whole family between flower structure and pollinators. It is tempting to think that any character of evolutionary importance is ipso facto a character that will define a distinct genus, but obviously this is not true.’ Rogers McVaugh, Orchidaceae, In Flora Novo-Galiciana Vol. 16 (1985). INTRODUCTION Subtribe Spiranthinae is the most species-rich clade of terrestrial orchids in the Neotropics, where most of the c. 40 genera and 520 species are found (Garay, 1982; Salazar, 2003b; Chase et al., 2015). Spiranthinae were first recognized formally as ‘division’ Spiranthidae of tribe Neottieae in the early orchid classification of Lindley (1840), but 20th century systematists largely followed the circumscription of Spiranthinae outlined in the posthumously published synoptic classification of Orchidaceae by Schlechter (1926). In that work, Spiranthinae were distinguished by their more or less erect anther, fasciculate roots, basal leaves (Fig. 1) and margins of the labellum adherent (‘adnate’) to the sides of the column. Schlechter (1920) also carried out the first modern revision of the generic classification of Spiranthinae, recognizing 24 genera, including many proposed there for the first time, based on floral features such as presence of a spur, relative length and thickness of the column, presence of a column foot, lobulation of the stigma, and details of the rostellum and viscidium (Fig. 2). Using characters of the rostellum and viscidium, Schlechter grouped the genera into four unnamed alliances or Gattungsreihen, to which he gave informal names in a later work (Schlechter, 1926). Figure 1. View largeDownload slide Habit and vegetative features of selected Spiranthinae. A. Sacoila hassleri growing in a sandy savanna (Brazil, Batista et al. 3137). B. Aulosepalum hemichreum shortly before shedding the leaves growing on limestone (Mexico, Salazar 6044). C. Cyclopogon calophyllus in leaf litter (Brazil, Salazar et al. 7793). D. Coccineorchis cernua in deep leaf mould in an Andean cloud forest (Peru, Edquen s.n.). E. Dichromanthus cinnabarinus in a periodically mowed lawn on a traffic island (Mexico, Salazar & Cabrera 6879). F. Epiphytic Lankesterella ceracifolia (above, Argentina, Salazar 7535) and Eurystyles auriculata (below, El Salvador, Salazar & Linares 7646). G. Sarcoglottis sceptrodes, plant removed from soil to show the dense fascicle of fleshy roots (Mexico, Salazar et al. 6584). H. Greenwoodiella wercklei, roots produced at intervals on the rhizome (Dominican Republic, Fragoso et al. 518). Photographers: João A. N. Batista (A), Gerardo A. Salazar (B, C, E–H), José D. Edquen (D). Figure 1. View largeDownload slide Habit and vegetative features of selected Spiranthinae. A. Sacoila hassleri growing in a sandy savanna (Brazil, Batista et al. 3137). B. Aulosepalum hemichreum shortly before shedding the leaves growing on limestone (Mexico, Salazar 6044). C. Cyclopogon calophyllus in leaf litter (Brazil, Salazar et al. 7793). D. Coccineorchis cernua in deep leaf mould in an Andean cloud forest (Peru, Edquen s.n.). E. Dichromanthus cinnabarinus in a periodically mowed lawn on a traffic island (Mexico, Salazar & Cabrera 6879). F. Epiphytic Lankesterella ceracifolia (above, Argentina, Salazar 7535) and Eurystyles auriculata (below, El Salvador, Salazar & Linares 7646). G. Sarcoglottis sceptrodes, plant removed from soil to show the dense fascicle of fleshy roots (Mexico, Salazar et al. 6584). H. Greenwoodiella wercklei, roots produced at intervals on the rhizome (Dominican Republic, Fragoso et al. 518). Photographers: João A. N. Batista (A), Gerardo A. Salazar (B, C, E–H), José D. Edquen (D). Figure 2. View largeDownload slide Floral features of selected Spiranthinae. A–D. Funkiella hyemalis (A, Mexico, Salazar 7633; B–D, Mexico, Salazar 6904). A. Inflorescence. B. Proximal half of labellum showing the basal nectary (above) and thickened orange–red areas. C. Ventral view of column apex prior to removal of the pollinarium. D. Ventral view of column apex after removal of the pollinarium. E–H. Funkiella parasitica (Mexico, Soto & Soto 10902). E. Inflorescence. F. Labellum showing thickened orange–red areas. G. Ventral view of column apex prior to removal of the pollinarium. H. Ventral view of column apex after removal of the pollinarium. I–L. Funkiella minutiflora (Mexico, Salazar et al. 9918). I. Flower. J. Flower with the sepals and petals excised showing the labellum partially enfolding the column. K. Labellum showing thickened orange–red areas. L. Ventral view of column apex prior to removal of the pollinarium. M–O. Sarcoglottis scintillans (Mexico, Salazar et al. 7436). M. Flower. N. Longitudinal section of ovary and nectary. O. Ventral view of column prior to the removal of the pollinarium. P–T. Sarcoglottis sceptrodes (P: Mexico, Figueroa 85; Q–T, Mexico, Martínez s.n.). P. Longitudinal section of ovary and nectary. Q. Dorsal view of column apex showing the viscidium among the divergent pollinium apices. R. Ventral view of column apex showing the viscidium. S. Ventral view of pollinarium. T. Ventral view of column apex after removal of the pollinarium. Photographer: Gerardo A. Salazar. Scale bars: 10 mm (A, B, M, N, P); 5 mm (E); 3 mm (C, D, F, I, O); 1 mm (J, K, R, S, T); 0.5 mm (G, H, L, K). Figure 2. View largeDownload slide Floral features of selected Spiranthinae. A–D. Funkiella hyemalis (A, Mexico, Salazar 7633; B–D, Mexico, Salazar 6904). A. Inflorescence. B. Proximal half of labellum showing the basal nectary (above) and thickened orange–red areas. C. Ventral view of column apex prior to removal of the pollinarium. D. Ventral view of column apex after removal of the pollinarium. E–H. Funkiella parasitica (Mexico, Soto & Soto 10902). E. Inflorescence. F. Labellum showing thickened orange–red areas. G. Ventral view of column apex prior to removal of the pollinarium. H. Ventral view of column apex after removal of the pollinarium. I–L. Funkiella minutiflora (Mexico, Salazar et al. 9918). I. Flower. J. Flower with the sepals and petals excised showing the labellum partially enfolding the column. K. Labellum showing thickened orange–red areas. L. Ventral view of column apex prior to removal of the pollinarium. M–O. Sarcoglottis scintillans (Mexico, Salazar et al. 7436). M. Flower. N. Longitudinal section of ovary and nectary. O. Ventral view of column prior to the removal of the pollinarium. P–T. Sarcoglottis sceptrodes (P: Mexico, Figueroa 85; Q–T, Mexico, Martínez s.n.). P. Longitudinal section of ovary and nectary. Q. Dorsal view of column apex showing the viscidium among the divergent pollinium apices. R. Ventral view of column apex showing the viscidium. S. Ventral view of pollinarium. T. Ventral view of column apex after removal of the pollinarium. Photographer: Gerardo A. Salazar. Scale bars: 10 mm (A, B, M, N, P); 5 mm (E); 3 mm (C, D, F, I, O); 1 mm (J, K, R, S, T); 0.5 mm (G, H, L, K). The generic classification of Spiranthinae proposed by Schlechter (1920) was criticized by the influential Harvard orchidologist Ames (e.g. Ames, 1922) for relying on ‘recondite’ column characters, and such disagreement among leading orchid specialists resulted in a long-standing lack of consensus in the approach to the generic classification of Spiranthinae. For instance, Harvard botanists who prepared orchid floras of various New World countries, strongly influenced by Ames’ views, placed nearly all species of Spiranthinae in an exceedingly broad genus Spiranthes Rich. s.l. (e.g. Correll, 1950; Williams, 1951; Ames & Correll, 1952; Schweinfurth, 1958), whereas other botanists followed Schlechter (Hoehne, 1945; Correa, 1955; Brieger, 1974–75; Garay, 1978). Two further generic revisions of Spiranthinae were published, nearly simultaneously, in the early 1980s (Balogh, 1982; Garay, 1982). The most salient feature of these two treatments was their disagreement in the number of genera recognized and in the species composition of the genera (McVaugh, 1985: 295–296). Garay (1982), whose publication gained priority by 2 months, admitted that he had previously supported the ‘lumping’ approach of his earlier Harvard colleagues but radically changed his views when he had a chance to study the whole complex on his own, increasing the number of genera recognized to 44. Garay (1982) distinguished the genera based on the structure of the rostellum, but he also considered important the degree of fusion of the lateral sepals (forming a floral tube) and position of the stigma, i.e. ‘terminal’ vs. ‘anterior’. In contrast, Balogh (1982) recognized only 16 genera based on characteristics of the rostellum, pollinarium and viscidium, position of the entrance of the stylar channel and position of the lateral sepals, grouping most of the genera in four alliances similar in composition to the Gattungsreihen of Schlechter (1920, 1926). Balogh pioneered the application of cladistic methods to orchid classification, applying Hennigian argumentation (after Hennig, 1966) and manual optimization (i.e. without using specific algorithms) of the characters on a cladogram to assess relationships in the Pelexia Poit. ex Lindl. alliance (Burns-Balogh & Robinson, 1983) and her version of Deiregyne Schltr. (sensuBurns-Balogh, 1988). However, she did not attempt to carry out a phylogenetic assessment of the whole subtribe. Dressler (1993) reviewed the classification of the orchid family, stressing the different generic treatments of Spiranthinae of Balogh (1982; also as Burns-Balogh, 1986b) and Garay (1982) and the scant discussion supporting either. Dressler (1993) adopted the generic scheme of Garay (1982), but stated that, at that point, one could not evaluate either of those classifications without redoing much of the work. Soon after, Szlachetko (1995a) proposed a new classification of Orchidaceae in which he divided Spiranthinae into three less-inclusive subtribes, namely Spiranthinae, Cyclopogoninae and Stenorrhynchidinae, based on differences in the structure of the rostellum and viscidium. He referred to these three groups as ‘subclades’ but did not provide any clear indication of which synapomorphies diagnose them, leaving aside the contradiction arising from his explicit rejection of cladistic methods in favour of the search for polythetic, ‘homogeneous’ groups in an evolutionary taxonomic context (Szlachetko, 1995a: 6). Szlachetko and co-workers subsequently proposed several new genera of Spiranthinae (e.g. Szlachetko, 1991a, b, 1993a, 1994a, b; González & Szlachetko, 1995; Szlachetko & González, 1996a, b, c; Szlachetko, González & Rutkowski, 2000, 2001), often splitting genera that they considered ‘highly heterogeneous and difficult to define’ on morphological grounds (e.g. Szlachetko et al., 2001: 3). Salazar et al. (2003) carried out the first molecular phylogenetic analysis of Spiranthinae, conducting maximum parsimony (MP) and Bayesian inference analyses of DNA sequences and insertion/deletion (indel) data from four plastid (rbcL, matK−trnK, trnL intron and trnL−trnF spacer) and one nuclear region (the ITS region of nuclear ribosomal DNA, hereafter nrITS) for 50 taxa, of which 24 species/21 genera were previously included in Spiranthinae. Their results showed that, with the exclusion of Galeottiella Schltr. (removed to a monogeneric subtribe by Salazar, Chase & Soto, 2002), Spiranthinae are monophyletic and strongly supported, whereas Schlechter’s (1920) Gattungsreihen, Balogh’s (1982) generic alliances and the narrowly defined subtribes of Szlachetko (1995a) are not monophyletic. Salazar et al. (2003) suggested that conflicts between strongly supported clades recovered in their analysis and the limits of taxa based solely on floral structures directly involved in pollination, such as the rostellum and viscidium, might reflect homoplasy in floral characters resulting from pressures from similar pollinators in distantly related groups. However, because they only analysed a fraction of the known diversity of Spiranthinae, the ensuing synoptical treatments of the genera of Galeottiellinae and Spiranthinae by Salazar (2003a, b, respectively) followed the generic concepts of Garay (1982) to minimize arbitrary changes lacking phylogenetic support, except for some changes resulting from the phylogenetic analysis of Salazar et al. (2003). Szlachetko, Rutkowski & Mytnik (2005) criticized the analysis of Salazar et al. (2003) for having included only 24 (c. 6%) of the species of Spiranthinae. However, Szlachetko and co-workers approached the issue by conducting a molecular phylogenetic analysis of only 19 species of Spiranthinae s.l. and a single genomic region (nrITS), without offering a rationale for excluding taxa for which sequences of this and other DNA regions were already available (Górniak et al., 2006). In any event, the analysis of Górniak et al. (2006) corroborated the results of Salazar et al. (2003) regarding both the non-monophyly of Szlachetko’s narrowly delimited subtribes and the discovery of some ‘unexpected’ groupings in the molecular tree relative to morphological classifications. Rutkowski, Szlachetko & Górniak (2008) published a book on the phylogeny and taxonomy of Spiranthinae, ‘Stenorrhynchidinae’ and ‘Cyclopogoninae’ in Central and South America (but also including Mexico, located in North America), in which they conducted phenetic and cladistic analyses of vegetative and floral morphological characters and molecular characters (DNA sequences of plastid matK and nrITS regions). All such analyses used the genera as terminal taxa, and therefore those authors did not attempt to evaluate generic monophyly. Moreover, their analyses were hampered by methodological inconsistencies, such as arbitrarily excluding Spiranthes from the morphological analyses, confounding the results of distance analyses with cladograms, using different sets of ingroup and outgroup taxa in their separate and combined DNA analyses without any justification, and failing to explain clearly how they conducted the analyses. Rutkowski et al. (2008) did not provide an articulate discussion summarizing the results of their various morphological analyses, which produced different groupings depending on whether floral, vegetative or both classes of characters were included. They conceded that their DNA analyses ‘do not confirm the monophyletic character of Spiranthinae and Stenorrhynchidinae sensu Szlachetko’ (Rutkowski et al., 2008: 95), but failed to cite their own previous molecular analysis (Górniak et al., 2006), in which they arrived at similar conclusions. Most surprisingly, the taxonomic synopsis of Rutkowski et al. (2008) ignored their own molecular phylogenetic results and continued using artificial, non-monophyletic ‘subtribes’. Several recent molecular phylogenetic studies, although not focused specifically on Spiranthinae, have contributed to clarification of subtribal limits and relationships in Cranichideae. Figueroa et al. (2008) analysed DNA sequence data and anatomical root characters to assess relationships and morphological evolution among representatives of various genera of Cranichidinae, Prescottiinae and Spiranthinae. In turn, Álvarez-Molina & Cameron (2009) and Salazar et al. (2009) independently assessed the limits and relationships of Cranichidinae and Prescottiinae, but also included several representatives of Spiranthinae. Those studies supported the results and conclusions of Salazar et al. (2003) regarding subtribal relationships and the limits and monophyly of Spiranthinae to the exclusion of Galeottiella (removed to Galeottiellinae). Likewise, Salazar (2009) and Salazar et al. (2009, 2011b) clarified the systematic position of Galeoglossum A.Rich. & Galeotti, including its synonym, Pseudocranichis Garay, showing that it belongs in Cranichidinae and not in Spiranthinae as believed by Garay (1982) in the case of G. thysanochilum (Rob. & Greenm.) Salazar. Recently, several papers have focused on molecular phylogenetics and floral evolution of particular species and clades of Spiranthinae, elucidating the systematic position of some taxa of uncertain affinity (Salazar & Ballesteros-Barrera, 2010; Batista et al., 2011; Salazar & Dressler, 2011; Salazar & Jost, 2012; Borba et al., 2014; Salazar, Cabrera & Figueroa, 2011a; Salazar, van den Berg & Popovkin, 2014; Salazar et al., 2016). For instance, Batista et al. (2011) showed that Nothostele Garay is a member of Spiranthinae, not of Cranichidinae as suggested by Dressler (1993), and the analysis of Salazar et al. (2014) revealed that Discyphus Schltr., a morphologically distinctive monospecific genus previously included in Spiranthinae, does not fit there. They proposed a new subtribe for its single species, Discyphinae. The contribution by Borba et al. (2014) was significant in that it identified the monospecific, south-eastern Brazilian endemic Cotylolabium Garay as sister to all other Spiranthinae, which has important implications for inferring morphological character evolution. Two recurring conclusions have arisen from the above-mentioned molecular phylogenetic and morphological studies. The first is that floral evolution in Spiranthinae is much more complex than cursory comparisons suggest. For instance, Salazar et al. (2011a) showed that the hummingbird-pollination syndrome, which includes odourless flowers, tubular, showily coloured perianth and bracts in tones of red, pink, orange or yellow, and a long, narrow rostellum with stiff, bristle-like rostellum remnant, evolved independently in distantly related clades such as Dichromanthus Garay, Stenorrhynchos Rich. ex Spreng. and other genera belonging to different major clades of Spiranthinae. Taxonomists focused only on floral characters have traditionally grouped these distantly related taxa into polyphyletic genera such as the different versions of Stenorrhynchos s.l. held, for instance, by Schlechter (1920), Balogh (1982), Garay (1982), Szlachetko (1995a) and Szlachetko et al. (2005). The second recurring conclusion is that molecular studies have consistently recovered five major clades in Spiranthinae, namely: (1) the monospecific, eastern Brazilian endemic Cotylolabium as the strongly supported sister of the remainder of the subtribe; (2) the Stenorrhynchos clade; (3) the Pelexia clade; (4) the Eurystyles Wawra clade; and (5) the Spiranthes clade (clade names, for example, after Salazar et al., 2003, 2011a, 2014, 2016; Batista et al., 2011; Borba et al., 2014). However, as pointed out by Borba et al. (2014), not all genera of Spiranthinae have been included in molecular phylogenetic analyses and several groups distributed in relatively inaccessible areas, mainly in South America, have not yet been analysed. Moreover, previous molecular phylogenetic studies of Spiranthinae have focused mostly on subtribal and generic relationships or on particular species or groups, and monophyly has not been assessed for most genera. In this work, we build upon our previous studies to investigate phylogenetic relationships in Spiranthinae, analysing a nearly complete generic sample (sensuChase et al., 2015; Salazar et al., 2016) and over one-third of the species currently accepted in the subtribe. We include DNA sequence data from one nuclear and three plastid DNA regions. The nuclear region consists of the internal transcribed spacers 1 and 2 and the intervening gene 5.8S of the nuclear ribosomal multigene family (nrITS; Baldwin et al., 1995). The plastid regions include the matK gene plus partial 3′ trnK intron downstream matK (Hilu & Liang, 1997; Barthet et al., 2015), the trnL group I intron and the trnL−trnF spacer (subsequently together referred to as the trnL-F region; Taberlet et al., 1991). All these regions have been used previously, individually or in various combinations, for phylogenetic inference in Spiranthinae (Salazar et al., 2003, 2011a, 2014, 2016; Górniak et al., 2006; Rutkowski et al., 2008; Salazar & Ballesteros-Barrera, 2010; Batista et al., 2011; Salazar & Dressler, 2011; Salazar & Jost, 2012; Borba et al., 2014) and other Cranichideae (Figueroa et al., 2008; Álvarez-Molina & Cameron, 2009; Salazar et al., 2009, 2011b; Cisternas et al., 2012). We made an effort to achieve the best representation, as availability of material permitted, of the structural and ecological diversity of the subtribe over its geographical distribution worldwide, which required years of coordinated collecting effort by several collaborators. Our main aim is to assess generic monophyly and relationships as a foundation for subsequent systematic and evolutionary studies. We also discuss the value as phylogenetic and taxonomic markers of some floral morphological features in the light of our results and conduct an exploratory analysis of the ancestral distribution areas of the subtribe, major clades and genera. MATERIAL AND METHODS Taxon sampling In total, 230 terminals were included in the phylogenetic analyses. These represent 182 species and 36 genera of Spiranthinae and 21 species/18 genera belonging to other subtribes of Cranichideae, namely Cranichidinae, Discyphinae, Galeottiellinae, Goodyerinae and Manniellinae (Salazar et al., 2003; Álvarez-Molina & Cameron, 2009; Salazar et al. 2009; Batista et al., 2011; Chase et al., 2015; Supporting Information, Table S1). We are missing only monospecific Aracamunia Carnevali & I.Ramírez, monospecific Cybebus Garay, monospecific Degranvillea Determann and Helonoma Garay, which includes four species. Molecular methods Genomic DNA was extracted from fresh- or silica gel-dried plant tissue or from small leaf fragments, flower buds or pollinia taken from herbarium specimens. Extraction, amplification (PCR) and Sanger sequencing of DNA were carried out using standard protocols and the primers of Salazar et al. (2003). Sequence editing and alignment The bidirectional sequence reads were assembled and edited with Sequencher version 4 or 5 (GeneCodes Corp., Ann Arbor, MI, USA). Each DNA region (matK−trnK, trnL−trnF and ITS) was aligned separately using the L-INS-i algorithm implemented in the online interface of the software package MAFFT version 7 (Katoh & Standley, 2013; http://mafft.cbrc.jp/alignment/server/), with minor manual adjustment with Mesquite version 3.11 (Maddison & Maddison, 2016). In several instances, only partial sequences of one or more of the regions analysed were obtained, or one or two of the regions could not be sequenced for particular samples. The unavailable sequences or sequence portions were scored as missing data. The aligned matrix in Nexus format was deposited in the Dryad repository (doi:10.5061/dryad.9b9c1). Phylogenetic analyses We conducted MP and maximum likelihood (ML) analyses with the aim of comparing the patterns of relationship and clade support generated by a method that does not require explicit models of nucleotide substitution (MP) with another that does (ML). Separate and combined MP analyses of plastid (matK−trnK/trnL−trnF regions) and nuclear DNA data (nrITS) were conducted with the software PAUP* version 4.0a150 for 32-bit Microsoft Windows (Swofford, 2016). Each analysis consisted of a heuristic search with 1000 replicates of random taxon order for the starting trees and TBR branch-swapping, saving in memory up to 20 most-parsimonious trees (MPTs) from each replicate to limit the time spent swapping in large islands of trees (Maddison, 1991). All characters were treated as unordered and equally weighted, and the individual positions of indel events postulated to account for length differences among sequences were treated as missing data. Clade support was evaluated with 1000 bootstrap replicates (Felsenstein, 1985), each consisting of 20 heuristic searches with random taxon order for the starting trees and TBR branch-swapping, saving up to 20 shortest trees per search. ML analyses were conducted for separate and combined plastid and nuclear data with the program RAxML-HPC2 on XSEDE version 8.2.9 (Stamatakis, 2014) implemented in the Cyberinfrastructure for Phylogenetic Research (CIPRES) Portal 2.0 (Miller, Pfeiffer & Schwartz, 2010). One thousand rapid bootstrap replicates (Stamatakis, Hoover & Rougemont, 2008) were followed by a thorough ML search with the default value of 25 rate categories and the GTRGAMMA model for nucleotides, allowing separate estimation of all free model parameters for the matK gene, trnK intron excluding matK, trnL intron, trnL−trnF intergenic spacer and the nrITS region. The phylogenetic trees were edited with FigTree version 1.4.0 (Rambaut, 2012) and Photoshop CC (Adobe Systems Inc., San Jose, CA, USA). Bootstrap percentages of 51–74, 75–89 and 90–100 were arbitrarily considered as weak, moderate and strong support, respectively. Ancestral area reconstruction We conducted an ancestral area analysis using the Bayesian binary Markov chain Monte Carlo method (BMM) for ancestral states implemented in the RASP software package (reconstruct ancestral state in phylogenies; Yu et al., 2015). The tree obtained in our ML analysis of combined plastid and nuclear sequences was loaded into RASP and ten Markov chains were run for 5000000 generations, sampling every 1000 generations and discarding 20% of the trees sampled as burn-in. State frequencies were estimated using the F81 model and the among-site rate variation model was set to gamma. The maximum number of reconstructed ancestral areas for a clade was set to five. Eleven distributions were considered that represent major areas of endemism for the species included in the phylogenetic analyses: (A) temperate North America; (B) Mesoamerica (including Mexico and Central America south to the Panama/Colombia border); (C) Andean South America; (D) eastern South America; (E) Amazonia; (F) tropical Asia; (G) west tropical Africa; (H) New Caledonia; (I) Malagasy region; (J) Caribbean; and (K) temperate Eurasia and northern Africa. Distribution data were recorded from specimens housed in the herbaria studied (AMES, AMO, ANDES, ARIZ, ASU, BHCB, BM, CAS, CHAPA, COL, CORU, ENCB, F, FCME, GH, IBUG, IEB, JBSD, K, LL, MEXU, MG, MHES, MO, NY, PMA, QCA, QCNE, R, RB, SEL, SERO, TEX, UAMIZ, US, USJ, UVAL, VEN, W and XAL; acronyms according to Thiers, 2017), complemented with records from public databases. The latter include Portal de datos abiertos UNAM (https://datosabiertos.unam.mx/), SEINET (http://swbiodiversity.org/seinet/), Tropicos (http://www.tropicos.org/Home.aspx), REFLORA (http://floradobrasil.jbrj.gov.br/reflora/PrincipalUC/PrincipalUC.do) and the World Checklist of Selected Plant Families (http://apps.kew.org/wcsp/prepareChecklist.do;jsessionid=DC970EDFBCF052B4F2BFA4BCE576E9A2?checklist=selected_families%40%40002020120150657121). RESULTS Phylogenetic analyses The plastid dataset consisted of 224 terminals and 4171 characters, of which 1237 (30%) were potentially parsimony-informative. The plastid MP analysis found 4160 MPTs with a length of 5397 steps, consistency index (CI) excluding uninformative characters of 0.40 and retention index (RI) of 0.80. The strict consensus included 4160 trees with clade support from the bootstrap analysis (Supporting Information, Fig. S1). The ML tree of the plastid dataset was topologically similar to that from the MP analysis, but support was usually slightly higher in the ML analysis (Supporting Information, Fig. S2). The major difference between the MP and ML plastid trees was the position of Coccineorchis Schltr., placed by MP as sister to the Eurystyles clade and by ML as sister to the Pelexia clade, but neither position obtained a bootstrap percentage (BP) > 50. The nrITS dataset included 221 terminals and 760 characters, of which 382 (50%) were potentially parsimony-informative. The nrITS MP analysis found 11500 MPTs with a length of 2691 steps, CI of 0.30 and RI of 0.79. The consensus tree (Supporting Information, Fig. S3) is topologically similar to the tree obtained in the ML nrITS analysis, but, as in the plastid dataset, resolution and clade support were overall higher in the ML analysis (Supporting Information, Fig. S4). The major groups recovered by the MP and ML nrITS analyses are for the most part the same as those found in the plastid trees, except for the Eurystyles clade being nested in the Spiranthes clade in the nrITS analyses. The sister-group relationship between the Eurystyles clade and the clade that includes species of Hapalorchis Schltr. [and in the nrITS analyses Pseudoeurystyles lorenzii (Cogn.) Hoehne] was weakly (BP 73) and strongly supported (BP 90) by the MP and ML nrITS analyses, respectively (Supporting Information, Figs S3, S4). The combined matrix consisted of 230 terminals and 4931 characters, of which 1619 (33%) were potentially parsimony-informative. The combined MP analysis resulted in 6400 MPTs with a length of 8158 steps, CI of 0.36 and RI of 0.79 (Supporting Information, Fig. S5). As in the separate analyses, resolution increased and more clades obtained strong support (BP ≥ 90) in the ML analysis (Figs 3–6) relative to the MP analysis, with some exceptions. These exceptions include the Stenorrhynchos clade, which in the MP combined analysis was strongly supported (BP 100), whereas in the ML analysis received weak support (BP 68). Likewise, a subclade of the Pelexia clade containing, among others, the species of Cyclopogon C.Presl s.l. was strongly supported by MP (BP 100), but weakly so by ML (BP 68). Given the similarity in the groups recovered by all analyses, and the greater resolution and overall bootstrap support of the tree resulting from the ML analysis of combined plastid and nuclear data, in the following we will use the latter for describing the phylogenetic results. For ease of visualization, Figures 3–6 show only the portion of the tree corresponding to Spiranthinae, divided into their major clades. The full combined ML tree is displayed in Supporting Information, Figure S6. Cotylolabium lutzii (Pabst) Garay is sister of the remaining Spiranthinae, which consist of four major clades corresponding to the Eurystyles, Spiranthes, Stenorrhynchos and Pelexia clades identified in previous molecular phylogenetic analyses. The Eurystyles clade includes monospecific Quechua Salazar & Jost as the sister (BP 87) of a strongly supported clade (BP 100) including monophyletic Lankesterella Ames and Eurystyles. The Eurystyles clade is the strongly supported sister (BP 93) of the Spiranthes clade (BP 100), and the latter includes four main subclades, marked with numbered circles 1–4 in Figure 3. The first of these subclades (BP 100) includes Pseudoeurystyles lorenzii and Hapalorchis. The second subclade consists of Funkiella Schltr. as sister of a group encompassing Sotoa Salazar plus Svenkoeltzia Burns-Bal. in turn sister to Beloglottis Schltr. plus Aulosepalum Garay; all genera in this subclade for which more than one species was analysed are monophyletic and strongly supported. The third main subclade is Spiranthes (BP 100), and the fourth one encompasses a group (BP < 50) consisting of weakly supported Physogyne Garay plus Pseudogoodyera Schltr. (BP 77) and Mesadenus Schltr. plus Greenwoodiella Salazar, Hern.-López & J.Sharma (BP 79), and another clade that includes Kionophyton Garay, Schiedeella Schltr., Dichromanthus Garay and Deiregyne Schltr. (sensuGaray, 1982; Salazar, 2003b), all with BP 100. Figure 3. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences. Numbers above branches indicate bootstrap percentages > 50. For simplicity, outgroups were excluded. Numbered black circles mark clades discussed in the text. Figure 3. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences. Numbers above branches indicate bootstrap percentages > 50. For simplicity, outgroups were excluded. Numbered black circles mark clades discussed in the text. The Stenorrhynchos clade (Fig. 4; BP 68) includes Mesadenus glaziovii (Cogn.) Schltr. (which renders Mesadenus polyphyletic), monophyletic Stenorrhynchos (clade 5) and monospecific Thelyschista Garay plus Buchtienia ecuadorensis Garay as successive sisters of a major group consisting of two subclades. The first subclade (6) is strongly supported (BP 99) and encompasses Nothostele acianthiformis (Rchb.f. & Warm.) Garay sister to Eltroplectris Raf. (BP 100). The second subclade (7) consists, on the one hand, of a weakly supported group (BP 63) with various species of Pteroglossa Schltr. and Mesadenella Pabst & Garay and another group (BP 65) that in turn consists of two clades: Lyroglossa grisebachii (Cogn.) Schltr. sister to Pteroglossa macrantha (Rchb.f.) Schltr. plus Sacoila hassleri (Cogn.) Garay (BP 66) and Skeptrostachys Garay (BP 95) sister to Sacoila lanceolata (Aubl.) Garay (BP 67). Hence, neither Pteroglossa nor Sacoila Raf. is monophyletic. Figure 4. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences (continuation of Fig. 3). Numbers above branches indicate bootstrap percentages > 50. Numbered black circles mark clades discussed in the text. Figure 4. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences (continuation of Fig. 3). Numbers above branches indicate bootstrap percentages > 50. Numbered black circles mark clades discussed in the text. In the Pelexia clade, Coccineorchis is weakly associated with the rest (Fig. 5; BP 61) and Sauroglossum elatum Lindl. diverges next (BP 68). Sarcoglottis C.Presl (clade 8) is strongly supported (BP 100) and includes a group of Mexican/Central American species, S. corymbosa Garay to S. cerina (Lindl.) P.N.Don nested among mostly South American species. The sister of Sarcoglottis is a strongly supported clade (Fig. 5, clade 9; BP 100) encompassing polyphyletic Pelexia and some members of Odontorrhynchus M.N.Correa, Brachystele Schltr. and Andean Sauroglossum corymbosum (Lindl.) Garay. Pelexia weberbaueriana (Kraenzl. ex Schltr.) Schltr., Sauroglossum corymbosum and Odontorrhynchus chlorops (Rchb.f.) Garay form a strongly supported group (clade 10; BP 100) that is sister to a ‘core’ Pelexia clade, which includes three strongly supported groups: Pelexia section Pachygenium Schltr. (clade 11), Brachystele (with Odontorrhynchus variabilis Garay nested; clade 12) and Pelexia section Pelexia (clade 13). Relationships among these are not clearly resolved (e.g. the sister-group relationship between clades 12 and 13 attained a BP < 50). The remaining members of the Pelexia clade (Fig. 6) consist mostly of species of Cyclopogon s.l., with Veyretia Szlach. and Brachystele guayanensis (Lindl.) Schltr. embedded in a derived position; hence, both Brachystele and Cyclopogon are polyphyletic. Figure 5. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences (continuation of Fig. 4). Numbers above branches indicate bootstrap percentages > 50. Numbered black circles mark clades discussed in the text. Figure 5. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences (continuation of Fig. 4). Numbers above branches indicate bootstrap percentages > 50. Numbered black circles mark clades discussed in the text. Figure 6. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences (continuation of Fig. 5). Numbers above branches indicate bootstrap percentages > 50. Numbered black circles mark clades discussed in the text. Figure 6. View largeDownload slide Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences (continuation of Fig. 5). Numbers above branches indicate bootstrap percentages > 50. Numbered black circles mark clades discussed in the text. Ancestral area analysis The analysis identified eastern South America as the ancestral area for Spiranthinae, with a major dispersal to Mesoamerica in the last common ancestor (LCA) of the Spiranthes clade (Fig. 10; Supporting Information, Fig. S8). Two separate dispersals from Mesoamerica to North America [one for the LCA of the clade consisting of S. lucida (H.H.Eaton) Ames and S. romanzoffiana Cham. and the other for the LCA of the remainder of North American Spiranthes] and one to the Old World are inferred from our data. Additional dispersals to Mesoamerica include one of the two main subclades of Sarcoglottis, Pelexia section Pelexia (or Pelexia s.s.; see Discussion) and particular species or groups in Eurystyles, Stenorrhynchos, Mesadenella and Cyclopogon. DISCUSSION Overall phylogenetic relationships in Spiranthinae The data sets analysed, separate and combined, irrespective of the method of analysis (MP or ML) recovered the same five main clades of Spiranthinae, with some topological differences among analyses that did not obtain strong bootstrap support. Such major lineages, namely Cotylolabium and the Stenorrhynchos, Eurystyles, Spiranthes and Pelexia clades (Figs 3–6) fully agree with those groups found in previous analyses of the same DNA regions but which included a much smaller taxonomic sample (e.g. Salazar et al., 2003, 2011a, 2016; Batista et al., 2011; Borba et al., 2014). As in those works, the major clades received varying degrees of support, but the relationships among them are not clearly resolved with the exception, in the present analysis, of a strongly supported sister-group relationship between the Eurystyles and Spiranthes clades (BP 91 and 98 in the combined MP and ML analyses, respectively). Lack of supported resolution for the relationships among the Pelexia and Stenorrhynchos clades with respect to one another and to the group formed by the Eurystyles/Spiranthes clades might be a reflection of the short branches subtending those portions of the tree (Supporting Information, Fig. S7). The small number of molecular changes contrasts with the noticeable structural, ecological and distributional differences among the major clades of Spiranthinae (see later; Salazar et al., 2003), a combination suggestive of a succession of cladogenetic events in a geologically short time interval during which little genetic change accumulated. Generic monophyly and relationships Of the 27 genera of Spiranthinae for which more than one species was included in our analyses, 18 were recovered as monophyletic. In the following, the genera are commented upon, in the context of the major clade to which they belong, in ascending branching order according to the tree depicted in Figures 3–6. Cotylolabium The single species in this genus was described originally as a species of Stenorrhynchos, but our results, like those from previous analysis that have included it, clearly place it as the sister of the rest of Spiranthinae (Borba et al., 2014; Salazar et al., 2016). Borba et al. (2014) provided detailed illustrations and discussion of the distinctive vegetative and floral attributes of C. lutzii, hypothesizing that the scented, bright yellow flowers with partially spreading lateral sepals and distally broadened labellum (Fig. 7A) correspond to a mellitophilous pollination syndrome. Figure 7. View largeDownload slide Inflorescences of selected members of Spiranthinae. A. Cotylolabium lutzii (Brazil, Martins da Costa 326). B. Quechua glabrescens (Ecuador, Jost 7916). C. Lankesterella gnoma (Brazil, Batista s.n.). D. Eurystyles actinosophila (Brazil, Batista s.n.). E. Hapalorchis aff. lineatus (Guatemala, Salazar et al. 7699). F. Funkiella hyemalis (Mexico, Salazar et al. 9177). G. Sotoa confusa (Mexico, Hernández-López & Treviño-Carreón 85). H. Svenkoeltzia congestiflora (Mexico, Salazar 9507). I. Beloglottis mexicana (Mexico, Salazar et al. 9349). J. Aulosepalum tenuiflorum (Mexico, Salazar et al. 7427). K. Spiranthes nebulorum (Mexico, Beutelspacher s.n.). L. Physogyne gonzalesii (Mexico, Jiménez-Machorrro s.n.). Photographers: Eduardo L. Borba (A), Lou Jost (B), João A. N. Batista (C, D), Gerardo A. Salazar (E, F, H–J), Tania Hernández-López (G), Carlos R. Beutelspacher (K), Rolando Jiménez-Machorro (L). Figure 7. View largeDownload slide Inflorescences of selected members of Spiranthinae. A. Cotylolabium lutzii (Brazil, Martins da Costa 326). B. Quechua glabrescens (Ecuador, Jost 7916). C. Lankesterella gnoma (Brazil, Batista s.n.). D. Eurystyles actinosophila (Brazil, Batista s.n.). E. Hapalorchis aff. lineatus (Guatemala, Salazar et al. 7699). F. Funkiella hyemalis (Mexico, Salazar et al. 9177). G. Sotoa confusa (Mexico, Hernández-López & Treviño-Carreón 85). H. Svenkoeltzia congestiflora (Mexico, Salazar 9507). I. Beloglottis mexicana (Mexico, Salazar et al. 9349). J. Aulosepalum tenuiflorum (Mexico, Salazar et al. 7427). K. Spiranthes nebulorum (Mexico, Beutelspacher s.n.). L. Physogyne gonzalesii (Mexico, Jiménez-Machorrro s.n.). Photographers: Eduardo L. Borba (A), Lou Jost (B), João A. N. Batista (C, D), Gerardo A. Salazar (E, F, H–J), Tania Hernández-López (G), Carlos R. Beutelspacher (K), Rolando Jiménez-Machorro (L). Eurystyles clade The monospecific, central Andean Quechua (Fig. 7B) was proposed as a distinct genus only recently based on MP and Bayesian inference analyses of the same DNA regions used here and detailed morphological comparisons (Salazar & Jost, 2012). Previously, Quechua glabrescens (T.Hashim.) Salazar & Jost had been included in Spiranthes or Cyclopogon. Overall, floral structure of Q. glabrescens is reminiscent of that of some species of Hapalorchis (Fig. 7E), and the early-diverging position of both Quechua and Hapalorchis in their respective major clades suggests that such floral structure might represent plesiomorphic (shared ancestral) traits in the group formed by the Eurystyles and Spiranthes clades. Vegetatively, Q. glabrescens sharply differs from Hapalorchis in having a rosette or sessile, linear-oblanceolate, fleshy leaves, in contrast to the petiolate, ovate, membranaceous leaves of Hapalorchis. The leaves of Quechua appear to persist over more than 1 year, a feature shared with its closest relatives (Lankesterella and Eurystyles). Eurystyles and Lankesterella (Fig. 7C, D) form a strongly supported group, corroborating the results of previous molecular phylogenetic studies (e.g. Górniak et al., 2006; Salazar & Dressler, 2011). Their unusual (in Spiranthinae) epiphytic habit and small rosettes of persistent, usually ciliate leaves (Fig. 1F) led Dressler (1981), Soto (1993) and Salazar (2005b) to argue for a close relationship between these two genera, despite differences in inflorescence and flower morphology. Burns-Balogh, Robinson & Foster (1985) stressed the unique features of the leaves, inflorescence and column of Eurystyles, Synanthes Burns-Bal., H.Rob. & M.S.Foster (here considered a synonym of Eurystyles; see later) and Pseudoeurystyles Hoehne and treated them as a distinct alliance in Spiranthinae. However, they did not compare them with Lankesterella, which Balogh (1982) had previously sunk as a section of Stenorrhynchos. Concerning the relationships of the Eurystyles alliance to other Spiranthinae, Burns-Balogh et al. (1985) considered the presence in some of its members of a supposedly plesiomorphic type of rostellum (i.e. one with an excised rostellar remnant similar to that in Spiranthes) as evidence of its early divergence from the ‘basal stock’ of the subtribe. Such a claim, however, is inconsistent with the derived position of Spiranthes in a different major clade (see below). Szlachetko (1992) noticed similarities in labellum morphology between some species of Eurystyles and Lankesterella, but in his classification (Szlachetko, 1995a; Szlachetko et al., 2005; Rutkowski et al., 2008), which emphasized characters of the column, placed the former in his version of Spiranthinae and the latter in ‘Stenorrhynchidinae’. Such segregation is untenable on phylogenetic grounds given the sister-group relationship between these genera, strongly supported by vegetative and genetic evidence. Other than the recurrence of autogamy, nothing is known of natural pollination of Eurystyles and Lankesterella; Salazar & Dressler (2011) proposed that the differences in reproductive structure between these sister genera could be a reflection of different pollination mechanisms. Mesoamerican Eurystyles borealis A.H.Heller has been associated, on morphological grounds, with Paraguayan E. bertonii (Burns-Bal., H.Rob. & M.S.Foster) Szlach. in Synanthes Burns-Bal., H.Rob. & M.S.Foster (Burns-Balogh et al., 1985). However, these two species, each distributed at one extreme of the Neotropics, are auto-pollinating and the character that distinguishes them from other Eurystyles spp. (absence of a rostellum) probably evolved convergently. Previous studies have shown that absence of a rostellum is recurrent in auto-pollinating variants of various species of Spiranthinae (e.g. Catling, 1990; Szlachetko, 1992; Salazar et al., 2016). Eurystyles cornu-bovis Szlach. is unique in the genus in having entire (i.e. not ciliate) leaf margins and pink (vs. greenish or whitish) flowers, among other minor details of its floral morphology (Szlachetko, 1992), but these features probably represent derived autapomorphies. Spiranthes clade This strongly supported group includes four main clades (1–4 in Fig. 3). In the first of these, our accession of Pseudoeurystyles lorenzii is sister, with strong support (BP 100), to Hapalorchis. Vegetative and floral similarities of P. lorenzii to the Eurystyles clade, and especially to Lankesterella (Szlachetko, 1992), might represent symplesiomorphies inherited from the most recent common ancestor shared by these clades, as suggested earlier for Quechua. However, because we were not able to include sequences of the plastid regions, this result should be considered with caution and will be explored further in another contribution (A. A. Bernal & E. C. Smidt, unpubl. data). In the second major subclade of the Spiranthes clade (Fig. 3, clade 2), Funkiella s.l. (including Microthelys Garay, Ecuadoria Dodson & Dressler and, probably, Stalkya Garay; Salazar, 2003b; Solano-Gómez, Salazar & Jiménez-Machorro, 2011) is sister to a clade with Sotoa plus Svenkoeltzia and Beloglottis as the successive sisters of Aulosepalum. The monophyly of Funkiella, Beloglottis and Aulosepalum is strongly supported, as are the relationships among all these genera. Funkiella includes high-elevation species distributed on most major cordilleras from southern North America, Central America and the Greater Antilles south to Ecuador, which display a noticeable variation in flower size and rostellum remnant/pollinarium morphology (Fig. 2A–L). For instance, flowers of F. minutiflora (A.Rich. & Galeotti) Salazar & Soto Arenas are c. 3 mm long or less (Fig. 2I–K), have a shortly apiculate rostellum remnant and the viscidium is located centrally on the ventral surface of the pollinarium (Fig. 2L). This last feature, a centrally placed viscidium, is shared also by species of the polyphyletic and distantly related Mesadenus Schltr. (see later), and, outside Spiranthinae, by Galeottiella (Salazar et al., 2002, 2003; Salazar, 2003a). In contrast, the sepals of F. hyemalis (A.Rich. & Galeotti) Schltr. (Figs 2A, 7F), the largest-flowered species of the genus, can exceed 25 mm in length, and the rostellum remnant is distinctly elongate and basally tridentate, with the viscidium attached to the distal part of the pollinarium (Fig. 2C, D). However, all Funkiella spp. share a putative morphological synapomorphy, i.e. possession of a red or orange thickened area on the labellum (Fig. 2B, F, K), and differences in flower size and column/pollinarium morphology probably reflect different pollination syndromes. Monotypic Sotoa (Fig. 7G) is a geophyte restricted to the outskirts of the Chihuahuan Desert and other semi-arid regions of southern North America (southern USA south to the Mexican state of Oaxaca; Salazar & Ballesteros-Barrera, 2010). Its flowers are fragrant during daytime hours, predominantly white, sometimes with rosy suffusion, and the sepals and petals bear contrasting green or brownish veining, all of which suggests mellitophily. Svenkoeltzia encompasses four tenuously defined species, plants grow epiphytically or lithophytically in moist oak–coniferous forests in southern Mexico, and, in contrast to Sotoa, they have a more or less one-sided raceme with bright yellow flowers probably pollinated by hummingbirds (Fig. 7H). Sotoa confusa (Garay) Salazar and Svenkoeltzia congestiflora (L.O.Williams) Burns-Bal. have been placed by taxonomists in various versions of Funkiella (e.g. Garay, 1982; Szlachetko, 1993b; Szlachetko et al., 2005), but their phylogenetic position precludes their inclusion in Funkiella (Fig. 3). An as-yet undescribed species recently discovered in the Chihuahuan Desert (north-eastern Mexico) ‘blurs’ the morphological and genetic distinction between Sotoa and Svenkoeltzia, and ongoing phylogenetic studies might result in the merging of these two small genera (G. A. Salazar & T. J. Hernández-López, unpubl. data). Beloglottis (Fig. 7I) is widespread in the mainland Neotropics (Mexico to Bolivia) and occurs in moist to wet tropical forests and cloud forests, usually living as a lithophyte or epiphyte. As in previous molecular analyses (e.g. Salazar et al., 2003, 2011a, 2016), in the present study Beloglottis is strongly supported as the sister of Aulosepalum (Fig. 7J). Szlachetko (1996) considered the distinctive Guiana Highland/Andean genus Helonoma as a synonym of Beloglottis. No material of Helonoma suitable for DNA analysis has been available to us, but such an approach is unsustained on morphological and ecological grounds. Helonoma spp. occur in the highly specialized, wet, oligotrophic environments on top of Guiana Highland tepuis and Andean tepui habitats of Colombia, Ecuador and Peru in the case of H. peruviana (Szlach.) Salazar, Dueñas & Fern.-Alonso (formerly Wallnoeferia peruviana Szlach.; Dueñas & Fernández-Alonso, 2009). Indeed, Helonoma is similar to the Guiana Highland endemic, monospecific Aracamunia in its rhizomatous habit, roots covered by silvery pubescence, few-flowered raceme, flowers provided with a long mentum (and correspondingly long column foot), partially fused sepals and spatulate petals partially adnate to the sepals. Aracamunia liesneri Carnevali & I.Ramírez is distinctive, however, in the clavate, glandular processes arising from the leaf axils, which have been suggested to be compatible with carnivory (Carnevali & Ramírez in Steyermark & Holst, 1989; Salazar, 2003b). Aracamunia has not been available for molecular study. Aulosepalum (Fig. 7J) occurs from Mexico to Costa Rica, and its species inhabit predominantly tropical deciduous and semi-deciduous forests, oak–coniferous forests and xerophilous scrub (Salazar 2003b, 2005a). Aulosepalum has been the subject of taxonomic contention (see discussion under Aulosepalum and, especially, Deiregyne in Salazar, 2003b). Garay’s (1982) concept of Aulosepalum required only a few adjustments, such as inclusion of A. pyramidale (Lindl.) M.A.Dix & M.W.Dix (placed by Garay in his Kionophyton) and an additional species, A. riodelayense (Burns-Bal.) Salazar, to match the strongly supported monophyletic group identified in this study. González & Szlachetko (1995) segregated A. pyramidale and A. riodelayense in their new genus, Gracielanthus R.González & Szlach., which is polyphyletic (Fig. 3). Other than their shorter floral tubes and correspondingly shorter labellum bases, these two species fit well with the rest of Aulosepalum. To further complicate nomenclature, Rutkowski, Mytnik & Szlachetko (2004) treated Aulosepalum as a subgenus of their concept of Deiregyne (which corresponds to Garay’s Aulosepalum, as argued by Catling, 1989; Salazar, 2003b), but soon after they changed their minds and considered Aulosepalum as a monospecific, distinct genus (Szlachetko et al., 2005). The details of this nomenclatural fiasco are beyond the focus of the present paper and will be dealt with in another contribution (G. A. Salazar, R. Chalqueño & S. A. Adachi, unpubl. data). Spiranthes (Fig. 3, clade 3) is strongly supported in our analyses. Dueck, Aygoren & Cameron (2014) thoroughly assessed phylogenetic relationships in this genus based on a nearly complete sample of the c. 36 currently accepted species using several plastid and nuclear DNA regions. Our results, based on a limited sample of taxa (14 species), agree in most details with theirs, placing S. romanzoffiana plus S. lucida as the sister of the rest, matching the ‘mainly western North American clade’ of Dueck et al. (2014) Our analysis also recovers an Old World group, including S. aestivalis (Poir.) Rich., S. sinensis (Pers.) Ames and S. spiralis (L.) Chevall., that is sister to their ‘midwestern and eastern North American clade’, and the relationships in the latter are congruent with their results, too. Of particular interest was the inclusion, in our analysis, of Mesoamerican S. graminea Lindl. and S. nebulorum Catling & V.R.Catling (Fig. 7K), which were unavailable to Dueck et al. (2014). Based on cytogenetic and morphological similarities, e.g. to S. praecox (Walter) S.Watson, Dueck et al. (2014) suggested that S. graminea could belong in the primarily western North American clade, but our results place S. graminea plus S. nebulorum as sister to the rest minus the western clade. Both our molecular results and our ancestral area reconstruction agree with the hypothesis posed by Dueck et al. (2014) that Spiranthes is derived from Mesoamerican ancestors (Figs 3, 10; Supporting Information, Fig. S8). Catling (1983) reviewed the pollination mechanisms of several northern North American Spiranthes spp. Except for auto-pollinating and apomictic races that occur in various species, flowers of Spiranthes are pollinated by several kinds of bees, mainly of Apidae (bumblebees, Bombus spp., and honey bees, Apis mellifera) but also members of Halictidae, Megachilidae and Andrenidae. In species pollinated by Bombus and megachilids, e.g. S. lacera (Raf.) Raf. and S. romanzoffiana, nectar accumulates at the bottom of the floral tube. The bees land on the lowermost open flowers and crawl upward on the raceme, probing the flowers for nectar. The viscidium in these species is comparatively long and rigid, adhering to the dorsal surface of the bee’s galea. However, in S. lucida, pollinated by halictid bees, the bees visit many flowers that they reach by flight. In this species, nectar accumulates on the ventral surface of the column and the oval viscidium is attached to the clypeus (see Catling, 1983; Salazar, 2003b). Likewise, S. spiralis, distributed in Ireland, southern Britain, central Europe and the Mediterranean, is pollinated by Bombus and Apis (e.g. Darwin, 1877; reviewed by Jacquemyn & Hutchings, 2010). The last main group in the Spiranthes clade (Fig. 3, clade 4) includes an assortment of genera centred in Mexico/northern Central America and the Caribbean. Physogyne (Fig. 7L) and Pseudogoodyera (Fig. 8A) were only recently included in a molecular phylogenetic analysis (Salazar et al., 2016). Physogyne includes two or three species restricted to steep slopes and rocky outcrops in tropical deciduous forest and its ecotones with warm pine–oak forest on the Pacific slope of Mexico. Pseudogoodyera consists of two species, one of them, P. pseudogoodyeroides (L.O.Williams) R.González & Szlach., widespread on the Atlantic slope of Mexico south to Belize and the other, P. wrightii (Rchb.f.) Schltr., endemic to Cuba. Both species live in small soil pockets on karstic outcrops in areas of moist, semi-evergreen tropical forests. Pseudogoodyera pseudogoodyeroides and Physogyne gonzalezii (L.O.Williams) Garay were both placed in Pseudogoodyera by Burns-Balogh (1986b), but she oddly included Physogyne sparsiflora (C.Schweinf.) Garay in Schiedeella. Both these genera are only rarely collected and little is known of any aspect of their biology; further study is required to determine whether they should be merged in a single genus. Our results show that, as currently delimited, Mesadenus is polyphyletic: ‘core’ Mesadenus, i.e. the clade that includes M. polyanthus (Rchb.f.) Schltr. (the type species; Fig. 8B), is weakly supported as sister to Greenwoodiella in a strongly supported subgroup of the Spiranthes clade (Fig. 3, clade 4). However, Brazilian M. glaziovii does not group with Mexican, Caribbean and Central American representatives of the genus, being weakly (BP 68 in our combined ML analysis; Fig. 4) to strongly supported (BP 100 in our combined MP analysis; Supporting Information, Fig. S5) as belonging in the Stenorrhynchos clade. Mesadenus is distinctive in its tiny flowers with all perianth segments similar in shape and colour (the labellum at most slightly wider than the other perianth parts) and the viscidium positioned on the centre of the ventral surface of the pollinarium (e.g. Salazar, 2003b: fig. 191.1). However, small flowers and a central viscidium have evolved in distantly related groups of Spiranthinae, such as Funkiella minutiflora (see earlier; Fig. 2L). The central position of the viscidium could be correlated with the shortening of the rostellum to match the reduction of the flower as a whole, but this hypothesis will be explored further elsewhere (G. A. Salazar & J. A. N. Batista, unpubl. data). On the other hand, most Greenwoodiella spp. were placed formerly in Schiedeella, but our results, and the previous study of Salazar et al. (2016), confirm that Schiedeella as interpreted by most previous taxonomists is polyphyletic. Mesadenus and Greenwoodiella differ in vegetative and floral attributes that have been discussed elsewhere (Salazar et al., 2016). Figure 8. View largeDownload slide Inflorescences of selected members of Spiranthinae (continuation). A. Pseudogoodyera pseudogoodyeroides (Mexico, Francke s.n.). B. Mesadenus polyanthus (Mexico, Salazar 7370). C. Greenwoodiella micrantha var. garayana (Mexico, Salazar et al. 7420). D. Kionophyton sawyeri (Mexico, Salazar 7252). E. Schiedeella transversalis (Mexico, Salazar 6873). F. Dichromanthus yucundaa (Mexico, García-Mendoza & Franco 8744). G. Deiregyne densiflora (Mexico, Reyes s.n.). H. Stenorrhynchos glicensteinii (El Salvador, Salazar & Linares 7532). I. Thelyschista ghillanyi (Brazil, van den Berg 1435). J. Buchtienia ecuadorensis (Peru, Simpson s.n.). K. Nothostele acianthiformis (Brazil, Viana 767). L. Eltroplectris triloba (Brazil, Batista 3293). Photographers: Gerardo A. Salazar (A–F, H), Jerónimo Reyes (G), Cássio van den Berg (I), Phillip Simpson (J), João A. N. Batista (K, L). Figure 8. View largeDownload slide Inflorescences of selected members of Spiranthinae (continuation). A. Pseudogoodyera pseudogoodyeroides (Mexico, Francke s.n.). B. Mesadenus polyanthus (Mexico, Salazar 7370). C. Greenwoodiella micrantha var. garayana (Mexico, Salazar et al. 7420). D. Kionophyton sawyeri (Mexico, Salazar 7252). E. Schiedeella transversalis (Mexico, Salazar 6873). F. Dichromanthus yucundaa (Mexico, García-Mendoza & Franco 8744). G. Deiregyne densiflora (Mexico, Reyes s.n.). H. Stenorrhynchos glicensteinii (El Salvador, Salazar & Linares 7532). I. Thelyschista ghillanyi (Brazil, van den Berg 1435). J. Buchtienia ecuadorensis (Peru, Simpson s.n.). K. Nothostele acianthiformis (Brazil, Viana 767). L. Eltroplectris triloba (Brazil, Batista 3293). Photographers: Gerardo A. Salazar (A–F, H), Jerónimo Reyes (G), Cássio van den Berg (I), Phillip Simpson (J), João A. N. Batista (K, L). Kionophyton, the clade that includes the type species of Schiedeella, S. transversalis (A.Rich. & Galeotti) Schltr. (see discussion in Salazar et al., 2016), Dichromanthus and Deiregyne are all strongly supported genera. Burns-Balogh (1986a) placed Kionophyton sawyeri (Standl. & L.O.Williams) Garay (Fig. 8D) in monotypic Greenwoodia Burns-Bal. but K. seminuda (Schltr.) Garay in Stenorrhynchos section Mesadenella (Pabst & Garay) Burns-Bal.; our results confirm that these two morphologically similar species belong together. Our sample of Schiedeella s.s. includes S. transversalis (Fig. 8E), S. crenulata (L.O.Williams) Espejo & López-Ferrari, S. affinis (C.Schweinf.) Salazar and S. durangensis (Ames & C.Schweinf.) Garay. Schiedeella affinis has been treated as a member of Mesadenus (Garay, 1982) and Brachystele (e.g. Burns-Balogh, 1986b; Szlachetko et al., 2005) based on its minute flowers, which are among the smallest in Spiranthinae, but similarity is probably due to simplification resulting from extreme size reduction. Vegetatively and eco-geographically S. affinis fits well with other Schiedeella. Dichromanthus (Fig. 8F) sensuSalazar et al. (2002, 2011a) and Salazar & García-Mendoza (2009) and Deiregyne sensuGaray (1982) form a strongly supported sister-pair with similar habitat preferences, vegetative morphology and overall floral structure. The main differences are flower colour, relative length of the floral tube and the structure of the rostellum and viscidium, all these related to differences in pollination syndrome (see Salazar et al., 2011a). In Dichromanthus cinnabarinus (Lex.) Garay, the rostellar remnant is soft, pliable and notched/hollow as a result of the apical portion of the rostellum being removed together with the viscidium, whereas in D. aurantiacus (Lex.) Salazar & Soto Arenas, D. michuacanus (Lex.) Salazar & Soto Arenas and D. yucundaa Salazar & García-Mend. (Fig. 8F) the viscidium is sheath-like and its removal leaves a relatively hard (but flexible), pointed rostellar remnant. However, in all four species the flowers are tubular, and the labellum has a narrow, conduplicate basal channel with submarginal nectar glands (see Salazar et al., 2011a: figs 4, 5). Dichromanthus spp. display an ornithophilous pollination syndrome, except for D. michuacanus, which is pollinated by bumblebees (Salazar et al., 2011a; Figueroa et al., 2012). Deiregyne spp. display a mellitophilous pollination syndrome, and there are some occasional field observations of bumblebees pollinating their flowers, as in D. densiflora (C.Schweinf.) Salazar & Soto Arenas (Fig. 8G). Deiregyne has had an unnecessarily complex taxonomic story caused by undue concern over interpretation of imprecise generic definitions, such as the original formulation of Deiregyne by Schlechter (1920), which encompassed a heterogeneous assortment of species currently placed in Deiregyne (sensuGaray, 1982), Schiedeella (sensuSalazar et al., 2016) and Aulosepalum (sensuGaray, 1982; Salazar, 2003b). As delimited by Garay (1982), who was the first to lectotypify the genus, Deiregyne only required the transfer of a few species described since, usually as members of Oestlundorchis Szlach. (a synonym of Deiregyne; e.g. Soto et al., 2007) and a species segregated by Garay (1982) in monotypic Dithyridanthus Garay to achieve monophyly (Salazar & Ballesteros-Barrera, 2010). Deiregyne spp. are relatively homogeneous in habit and floral structure, and they share diaphanous floral bracts with dark veins that permit recognition of the genus at a glance even from herbarium specimens (Fig. 8G; see Hágsater et al., 2005: figs 445–450). Balogh (1981, 1982) and Burns-Balogh (1986b) placed most Deiregyne spp. (as interpreted here) in Schiedeella, whereas her concept of Deiregyne (Burns-Balogh, 1986b, 1988) is equivalent to Garay’s (1982) circumscription of Aulosepalum (see above). Recently, Szlachetko & Kolanowska (2013) contributed further to this nomenclatural mayhem, proposing conservation of Deiregyne sensu Burns-Balogh based on recycling of Szlachetko’s (1995b) old argument on interpretation of the imprecise original diagnosis of the genus by Schlechter (1920). Stenorrhynchos clade This clade was recovered consistently by our analyses with varying degrees of support (Fig. 4; Supporting Information, Figs S1–S6) and most previous molecular phylogenetic studies of Spiranthinae (e.g. Salazar et al., 2003, 2011a, 2016; Batista et al., 2011; Borba et al., 2014). Like the Spiranthes clade (see earlier), the Stenorrhynchos clade displays several pollination syndromes, including halictid bee pollination (Mesadenella: Singer, 2002), hummingbird pollination (Stenorrhynchos: Siegel, 2011; Sacoila: Singer & Sazima, 2000) and butterfly pollination (Pteroglossa spp.: Pansarin & Ferreira, 2015). The taxonomy of this natural group has been confounded by floral homoplasy, as taxonomists focused only on floral characters are easily misled by convergent features due to similar pollination syndromes such as adaptation to hummingbird pollination (van der Pijl & Dodson, 1966; Salazar et al., 2003, 2011a; Batista et al., 2011). Many species in this major clade have a narrowly pointed, stiff rostellum remnant, with some noticeable exceptions, such as Buchtienia Schltr. and Thelyschista (see below). As noted earlier, ‘Mesadenus’ glaziovii is sister to the rest of the Stenorrhynchos clade, which includes three main clades (Fig. 4). Stenorrhynchos s.s. (clade 5; see Salazar et al., 2011a) is strongly supported as monophyletic and includes about seven species that florally are nearly indistinguishable from one another but exhibit distinctive vegetative attributes, ecological preferences and distributions (Salazar, 2003b; Christenson, 2005; Salazar et al., 2011a). Next, two morphological ‘oddballs’, Thelyschista and Buchtienia, form a grade with a group that includes the remainder of this major clade. Monospecific Thelyschista has green sepals and white labellum and petals, the latter forming a narrow, somewhat recurved floral tube with the dorsal sepal and the labellum (Fig. 8I); its column is distinctive in the tridentate rostellum with the massive viscidium wedged between the three teeth (Salazar, 2003b: fig. 207.1). On the other hand, Buchtienia (Fig. 8J) includes four species with greenish to pinkish or brownish flowers, vegetatively and florally reminiscent of Eltroplectris, except for lack of a spur and their peculiar column structure. The column in Buchtienia is abruptly expanded laterally from a narrow base and somewhat sigmoid when seen from one side, with a shortly oblong, pliable rostellum that, after removal of the pollinarium, ends in a membranaceous, emarginate lamina (Salazar, 2003b; de Fraga, Meneguzzo & Saddi, 2015). This contrasts with the straight, clavate column ending in a bristle-like, hard rostellum/rostellum remnant that is common in the Stenorrhynchos clade. The other taxa of the Stenorrhynchos clade form a weakly supported group consisting of two subclades. The first of these (Fig. 4, clade 6) is strongly supported (BP 99) and includes Nothostele acianthiformis (Fig. 8K) sister to Eltroplectris (Fig. 8L), in agreement with the analysis of Batista et al. (2011), who discussed in detail similarities and differences between these two genera and chose to maintain them separate. The second subclade is weakly supported (BP 67; Fig. 4, clade 7) and in turn encompasses two weakly supported groups. One of these (BP 63) includes the species of Mesadenella (Fig. 9A) and some Pteroglossa (Fig. 9B). Of these, Szlachetko (in Rutkowski et al., 2008) segregated P. roseoalba (Rchb.f.) Salazar & M.W.Chase to Callistanthos Szlach. and both P. euphlebia (Oliv. ex Rchb. f.) Garay and P. glazioviana (Cogn.) Garay to Cogniauxiocharis (Schltr.) Szlach. The other one (BP 65) consists of two major groups: the first with Lyroglossa grisebachii (Cogn.) Schltr. (Fig. 9D) sister to a species pair of Pteroglossa macrantha (Rchb.f.) Schltr. (type species of Pteroglossa; Fig. 9C) and Sacoila hassleri (Cogn.) Garay (BP 95), and a second including Skeptrostachys (Fig. 9E) plus Sacoila lanceolata (Fig. 9F; BP 67). Thus, Mesadenella, Sacoila and Pteroglossa are non-monophyletic. Szlachetko (in Rutkowski et al., 2008) created an additional monospecific genus, Lyrochilus, for Pteroglossa hilariana (not sampled by us), which according to him is similar in habit to Lyroglossa and in floral structure to Pteroglossa. Salazar (2003b) proposed that distinguishing Eltroplectris from Pteroglossa, each including about ten species, has been problematic because of use of inconsistent characters, such as the degree of adnation of the spur to the ovary (e.g. Szlachetko, 1995c). This is corroborated by the recent discovery of a new Peruvian species closely related to P. macrantha that completely lacks a spur (Damián & Salazar, 2017). Szlachetko & González (1996b) transferred E. triloba (Lindl.) Pabst and several other species to their new genus, Ochyrella Szlach. & R.González, but our study indicates that E. triloba is closely related to E. calcarata (Sw.) Garay & H.R.Sweet, the type species of Eltroplectris, and that neither the generic limits of Szlachetko and co-workers nor those accepted by Salazar (2003b) for Eltroplectris and Pteroglossa represent natural groups. Salazar (2003b) argued that the short, ventrally channelled column, narrowly triangular rostellum, concave anther and marginal, completely adnate nectar glands of Lyroglossa are reminiscent of those of Pteroglossa, and such morphological similarity is consistent with their close relationship to one another revealed by this study. Salazar (2003b) also stressed the morphological similarity of Sacoila to Skeptrostachys, which agrees with their close relationship revealed by our analysis. Nevertheless, considerable work remains to be done on a critical reassessment of generic limits in this major clade, which will probably result in a reduction of the number of genera. Pelexia clade Our combined analyses weakly support the association of Coccineorchis with the rest of the Pelexia clade (ML BP 61, Fig. 5; MP BP 69; Supporting Information, Fig. S5). Coccineorchis is not closely related to Stenorrhynchos and its close kin, despite similar overall flower shape and the presence in both genera of a hard, bristle-like rostellum sheathed by the viscidium. These features, together with the somewhat nodding, dense raceme of bright yellow to red tubular flowers are suggestive of hummingbird pollination in Coccineorchis, which has not been corroborated in the field (Fig. 9G; Salazar et al., 2011a). Figure 9. View largeDownload slide Inflorescences of selected members of Spiranthinae (continuation). A. Mesadenella petenensis (Mexico, Jiménez-Machorro 3002). B. Pteroglossa euphlebia (Brazil, Guimarães 191). C. Lyroglossa grisebachii (Brazil, Batista 1821). D. Pteroglossa macrantha (Argentina, Singer s.n.). E. Skeptrostachys gigantea (Brazil, Batista Bianchetti 3352). F. Sacoila lanceolata (Mexico, Amith 1922). G. Coccineorchis cernua (Peru, Edquen s.n.). H. Sauroglossum elatum (Brazil, Smidt 1007). I. Sarcoglottis cerina (El Salvador, Batlle s.n.). J. Odontorrhynchus chlorops (Argentina, Rodríguez s.n.). K. Pelexia funckiana (Mexico, Figueroa 6). L. Pelexia hirta (Ecuador, Tobar 15). M. Brachystele cyclochila (Brazil, Batista et al. 2225). N. Cyclopogon epiphyticum (Ecuador, Salazar et al. 9764). O. Cyclopogon ovalifolius (Peru, Morón s.n.). P. Veyretia rupicola (Brazil, van den Berg 1477). Photographers: Gerardo A. Salazar (A, I, K, N), Leonardo R. S. Guimarães (B), João A. N. Batista (C, E, M), Rodrigo B. Singer (D), Jonathan Amith (F), José D. Edquen (G), Eric C. Smidt (H), Juan J. Rodríguez (J), Francisco Tobar (L), Érica Morón (O), Cássio van den Berg (P). Figure 9. View largeDownload slide Inflorescences of selected members of Spiranthinae (continuation). A. Mesadenella petenensis (Mexico, Jiménez-Machorro 3002). B. Pteroglossa euphlebia (Brazil, Guimarães 191). C. Lyroglossa grisebachii (Brazil, Batista 1821). D. Pteroglossa macrantha (Argentina, Singer s.n.). E. Skeptrostachys gigantea (Brazil, Batista Bianchetti 3352). F. Sacoila lanceolata (Mexico, Amith 1922). G. Coccineorchis cernua (Peru, Edquen s.n.). H. Sauroglossum elatum (Brazil, Smidt 1007). I. Sarcoglottis cerina (El Salvador, Batlle s.n.). J. Odontorrhynchus chlorops (Argentina, Rodríguez s.n.). K. Pelexia funckiana (Mexico, Figueroa 6). L. Pelexia hirta (Ecuador, Tobar 15). M. Brachystele cyclochila (Brazil, Batista et al. 2225). N. Cyclopogon epiphyticum (Ecuador, Salazar et al. 9764). O. Cyclopogon ovalifolius (Peru, Morón s.n.). P. Veyretia rupicola (Brazil, van den Berg 1477). Photographers: Gerardo A. Salazar (A, I, K, N), Leonardo R. S. Guimarães (B), João A. N. Batista (C, E, M), Rodrigo B. Singer (D), Jonathan Amith (F), José D. Edquen (G), Eric C. Smidt (H), Juan J. Rodríguez (J), Francisco Tobar (L), Érica Morón (O), Cássio van den Berg (P). Sauroglossum elatum (Fig. 9H) diverges next (BP 68), and its position agrees with previous molecular phylogenetic analyses that have included this species and a few other representatives of the Pelexia clade (Borba et al., 2014; Salazar et al., 2016). Sauroglossum is polyphyletic, as Andean S. corymbosum does not group with south-eastern Brazilian/Argentinian S. elatum, the type species of that genus. Flowers of S. elatum have a short rostellum with a ventrally adhesive viscidium that, upon removal of the pollinarium, leaves a notch at the apex of the column (Singer, 2002; Salazar, 2003b). Singer (2002) studied the reproductive biology of S. elatum, demonstrating protandry and pollination by noctuid moths. The remainder of the Pelexia clade forms three major groups, the first of which is Sarcoglottis (Fig. 2M–T; Fig. 5, clade 8). This mostly South American genus includes a derived, strongly supported Mesoamerican subclade (S. corymbosa to S. cerina; BP 92; Fig. 9I). Sarcoglottis sceptrodes Schltr. also occurs in Mesoamerica, but it is closer to Caribbean/northern South American S. acaulis (Sm.) Schltr. and Andean S. speciosa C.Presl (the latter the type species of Sarcoglottis). The recent segregates Zhukowskia (Schltr.) Szlach., R.González & Rutk. and Potosia (Schltr.) R.González & Szlach., typified by Sarcoglottis smithii (Rchb.f.) Schltr. and S. schaffneri (Rchb.f.) Ames, respectively, are nested in the Mesoamerican subclade and are thus phylogenetically untenable and taxonomically superfluous. Traditionally, Sarcoglottis was distinguished from Pelexia by characters of the nectary, which in the former supposedly is completely fused with the ovary, with neither a prominent spur nor a clearly visible line of adnation (e.g. Garay, 1982), whereas in the latter the nectary is prominent and chin-like, saccate or spurred. However, there is substantial variation in this feature in Sarcoglottis (Fig. 2M–P), and reliance on this single character has led some taxonomists to create new genera, such as Zhukowskia, to accommodate the ‘intermediate’ species (see Szlachetko et al., 2000). Potosia, on the other hand, was first created as a section of Pelexia by Schlechter (1920) and recently raised to generic level, without any meaningful discussion supporting such a decision, in a minimal paper published in a journal of invertebrate zoology (Mytnik, 2003). Subsequently, Mytnik-Ejsmont & Rutkowski (2006) attempted [sic] ‘to verify a legitimacy of distinguishing particular genera within the subtribe Cyclopogoninae’, including among others Pelexia, Sarcoglottis, Potosia and Zhukowskia. For this, they conducted phenetic analyses of (mostly floral) morphological characters, but because they used the genera as terminals, their analyses provided no evidence on generic limits and composition; at most, their phenograms depict overall morphological similarities among genera that were arbitrarily delimited beforehand. Sarcoglottis is the strongly supported sister of a clade that includes non-monophyletic Pelexia that has nested in it several species assigned to other genera (Fig. 5, clade 9). A first group (clade 10) includes Andean Pelexia weberbaueriana (Kraenzl.) Schltr., Sauroglossum corymbosum and Odontorrhynchus chlorops (Rchb.f.) Garay (Fig. 9J); the oldest available generic name for such a group is Synassa Lindl., typified by Synassa corymbosa Lindl. (=Sauroglossum corymbosum). Rutkowski et al. (2008) revived Synassa but only to include the type species and the morphologically similar Sauroglossum aurantiacum (C.Schweinf.) Garay, but our results show that this group is more diverse than previously thought. It is noteworthy that Schweinfurth (1951) originally described Sauroglossum aurantiacum as a variety of Pelexia weberbaueriana, which is consistent with the close genetic relationship found here between the latter and S. corymbosum. Excluding the aforementioned P. weberbaueriana, the remaining Pelexia spp. are found in a strongly supported clade in which most Brachystele spp. and Odontorrhynchus variabilis are nested (Fig. 5, clades 11–13). Two clades of Pelexia were consistently recovered, largely corresponding to Schlechter’s (1920) sections [‘Eu-’]Pelexia (Fig. 9K) and Pachygenium (Fig. 9L). The latter is distinguished by its oblanceolate-spathulate leaves that attenuate basally and comparatively fleshy flowers with a usually saccate spur, versus the distinctly petiolate, obliquely ovate leaves and somewhat membranaceous flowers with cylindrical, retrorse spur of the former. Szlachetko et al. (2001) raised section Pachygenium to generic rank. Our results are consistent with recognition of those two clades as distinct genera, although some features that Szlachetko et al. (2001) cited to differentiate Pachygenium from Pelexia s.s. (e.g. the attributes of the rostellum, viscidium and stigma) do not hold true as distinguishing characters. However, these clades diverge significantly in ecological preferences and overall floral morphology. Species of section Pachygenium usually inhabit open grasslands, rocky fields and forest savannas; their flowers (Fig. 9L) have a broadly channelled labellum and diurnal perfume and are pollinated by bumblebees, at least P. eckmanii (Kraenzl.) Schltr. (Dressler 1981, 1993) and P. oestrifera (Rchb.f. & Warm.) Schltr. (Singer & Sazima, 1999). In contrast, species of section Pelexia (or Pelexia s.s.) inhabit forests and its narrow flowers (Fig. 9K) are apparently odourless, but no information is available on their natural pollination. Like Pelexia, Brachystele (Fig. 9M) is polyphyletic. Brachystele guayanensis is deeply embedded among Cyclopogon spp. as sister to Veyretia in a different main subclade of the Pelexia clade (Fig. 6, clade 17). Conversely, Chilean Odontorrhynchus variabilis is nested in ‘core’ Brachystele (Fig. 5, clade 12) and morphologically is barely distinguishable from B. unilateralis (Poir.) Schltr., the type species of Brachystele. Rutkowski et al. (2008) placed Brachystele and Sauroglossum in their polyphyletic version of Spiranthinae and Odontorrhynchus in polyphyletic ‘Stenorrhynchidinae.’ Core Brachystele shows geographical structure: western South American species B. unilateralis and O. variabilis form a strongly supported group and south-eastern South American B. subfiliformis (Cogn.) Schltr. to B. cyclochila (Kraenzl.) Schltr. form another clade (although with BP < 50 in our combined ML analysis). Our analyses are not decisive about whether Brachystele is closer to Pelexia s.s. or to Pachygenium, although Brachystele is similar in habitat preference and overall distribution to Pachygenium, both centred in open habitats in south-eastern South America (Supporting Information, Fig. S8). Moreover, pollination by native and introduced bumblebees has been reported for Brachystele unilateralis (Sanguinetti & Singer, 2014). All this suggests a close relationship between Pachygenium and Brachystele. Cyclopogon (Fig. 6) is perhaps the taxonomically most challenging genus of Spiranthinae. Taxonomists have recognized several genera, most of them segregated from Cyclopogon s.l. (except Veyretia; see below), based on single floral attributes, such as whether the lateral sepals are partially connate to form ‘a distinct sepaline tube’, on the basis of which Garay (1978, 1982) treated Cyclopogon as monospecific and moved all other species to Beadlea Small. Similarly, Garay (1982) created Stigmatosema Garay to include two former Cyclopogon spp. in which the apex of the rostellum remnant is ‘sulcate’ (i.e. it has the lateral margins upturned), and about a dozen additional species have been subsequently transferred to, or described as, Stigmatosema. Szlachetko (1994b) segregated Cocleorchis Szlach., with deflexed (‘revolute’) rostellum margins, and Warscaea Szlach., with a broad and short rostellum that upon removal of the viscidium is deeply notched. However, Cyclopogon (Cocleorchis) dressleri Szlach. (of which C. sarcoglottidis Szlach., the type species of Cocleorchis, is considered here as a synonym) is strongly supported by our analyses as the sister of Cyclopogon ovalifolius C.Presl, the type species of Cyclopogon, demonstrating the meaninglessness of segregating genera based on minor floral attributes in this florally labile clade. The sister group of the rest in Cyclopogon s.l. is a strongly supported, small subclade including C. variegatus Barb.Rodr. to C. olivaceus (Rolfe) Schltr. (Fig. 6, clade 14), which is distinctive in its dark brownish- to purplish-green leaves, often dotted with white or pink, and a relatively simple labellum (i.e. not abruptly expanded into an apical lobe or epichile; Fig. 9N). Should Cyclopogon be divided into sections, the name available for this clade is section Beadlea (Small) Burns-Bal. Next, there is a large group that includes many species with homogeneous floral morphology (Fig. 6, clade 15), in which C. (‘Warscaea’) apricus (Lindl.) Schltr. is sister to the rest. The third main clade of Cyclopogon (Fig. 6, clades 16 and 17) includes an assortment of species that have been attributed to several genera, namely Cyclopogon [C. micranthus (Barb.Rodr.) Schltr., C. ovalifolius, C. elatus (Sw.) Schltr., C. luteo-albus (A.Rich. & Galeotti) Schltr., C. obliquus (J.J.Sm.) Szlach., C. saccatus (A.Rich. & Galeotti) Schltr. and C. truncatus (Lindl.) Schltr.], Stigmatosema [C. inaequilaterus (Poepp. & Endl.) Schltr., Cocleorchis (C. dressleri), ‘Brachystele’ guayanensis and Veyretia. Veyretia spp. were formerly included in Sarcoglottis section Aphylla Burns-Bal. (Burns-Balogh, 1983). Szlachetko (1995a) segregated Veyretia from Sarcoglottis mainly based on the presumed absence of leaves at flowering time (although leaves can be present or absent at flowering time; instead of flat and broad as in Sarcoglottis, they are grass-like and convolute; Hoehne, 1945; Salazar, 2003b) and the bifurcate nectar chamber. The strongly supported embedded position of Veyretia in Cyclopogon s.l. in our trees is unexpected, but the vegetative and floral peculiarities of the species of Veyretia probably represent derived modifications of the otherwise conservative, symplesiomorphic vegetative and floral morphology of the Cyclopogon clade. The association of ‘Brachystele’ guayanensis with Veyretia is also surprising at first glance, given the obvious difference in the appearance of the minute flowers of the former. However, ‘B.’ guayanensis shares with Veyretia a preference for open grassland habitats and, upon close examination, it is evident that its flowers have the two-chambered nectary of Veyretia (although more shallowly so in B. guayanensis, in proportion to its noticeable reduction in flower size). As in the Stenorrhynchos clade, much work remains to be done to sort out generic limits in this group. Such work ideally should include detailed morphological and developmental comparative studies, coupled with observations on natural pollination, to achieve a better understanding of structural homology and functionally driven homoplasy. Floral morphological characters as phylogenetic and taxonomic markers As indicated above, Burns-Balogh & Robinson (1983) carried out a cladistic analysis of floral morphological characters for the ‘Pelexia alliance’, including Cyclopogon, Pelexia and Sarcoglottis (Veyretia spp. were then included in Sarcoglottis section Aphylla). Some characters used by Burns-Balogh & Robinson (1983) exhibit continuous variation (e.g. flowers erect or horizontal, pollinarium oblong vs. wishbone-shaped, position of the sepals relative to the labellum), and their coding in discrete states is questionable. Hence, discussion here is restricted to discrete characters. Burns-Balogh & Robinson (1983) identified several putative synapomorphies for the Pelexia alliance, including apiculate anther, oblong, truncate or shallowly notched rostellum remnant (Fig. 2O, R, T) and apical viscidium held between the apices of the pollinia and located on the dorsal side of the rostellum (Fig. 2Q, S), thus corresponding to the ‘wedge-type’ viscidium of Greenwood (1982). However, an apiculate anther is not exclusive to Cyclopogon, Pelexia and Sarcoglottis but is also present in Brachystele, Odontorrhynchus and Sauroglossum, and hence is a putative synapomorphy of the Pelexia clade except Coccineorchis (Salazar, 2003b). On the other hand, the truncate rostellum remnant and a wedge-type viscidium are structurally and functionally linked because the viscidium corresponds to the distal portion of the rostellum and when it is detached it leaves a straight or somewhat concave zone of rupture that produces the ‘truncate or shallowly notched rostellum remnant’ (Fig. 2T). Both attributes are absent in the species of Sauroglossum, Odontorrhynchus and Brachystele but present in Sarcoglottis, Pelexia s.s., Pachygenium, Cyclopogon s.l. and Veyretia. In all these genera the pollination mechanism involves release of the viscid matter by the viscidium when its dorsal surface is pressed by the underside of the labrum of their pollinators (several types of bees) when they extend their mouthparts to probe the flower for nectar (Singer & Coccuci, 1999; Singer & Sazima, 1999; field observations not available for Veyretia). Another unique trait of the wedge-type viscidium is that it is located between the divergent apices of the pollinia, which is linked to the aforementioned pollination mechanism, since the labrum could not contact the dorsal surface of the viscidium if the apices of the pollinia were parallel and connivent over the dorsal surface of the viscidium, as in other Spiranthinae (Fig. 2Q, S; cf. Greenwood, 1982). The fact that those three features of the rostellum and viscidium are always present together strongly suggests that they are linked functionally, and their use as independent characters in a cladistic analysis is unadvisable because they ‘overweigh’ as three characters what is actually one. The same reasoning is applicable to the suite of co-occurring characters that characterize the hummingbird pollination syndrome evolved convergently in the Pelexia, Stenorrhynchos and Spiranthes clades (Salazar et al., 2011a). Burns-Balogh & Robinson (1983) identified two synapomorphies supporting a clade formed by Pelexia and Sarcoglottis (the latter including Veyretia as section Aphylla), i.e. subulate (slender, long and pointed) basal nectar glands in the labellum (Fig. 2N, P) and a non-basal position of the entrance of the stylar channel in the stigma. The relationships recovered by our analyses suggest that possession of subulate nectar glands is a putative synapomorphy of the clade that includes Sarcoglottis plus Pelexia s.l. and most species of Brachystele and Odontorrhynchus, with subsequent reversals (secondary losses) in clades 10 and 12 (Fig. 5). The second putative synapomorphy of Pelexia and Sarcoglottis according to Burns-Balogh & Robinson (1983), a non-basal position of the entrance of the stylar channel, is based on an incorrect interpretation of the homology of the structures concerned. According to Burns-Balogh & Robinson (1983), in Pelexia and Sarcoglottis the entrance to the stylar channel is located ‘above the stigmatic area [. . .] at the base of the sterile rostellum’, whereas in Cyclopogon, Spiranthes and other Spiranthinae it is located ‘in an area between the two [fertile, or receptive] stigmatic lobes, sometimes very near the base of the lobes’. Obviously, they considered that the often bilobed or bipartite receptive stigmatic surface of Spiranthinae represents the lateral lobes of the stigma, with the ‘sterile rostellum’ representing the median lobe. However, micromorphological and developmental studies conducted by Rasmussen (1982), Kurzweil (1988), Figueroa et al. (2012) and Figueroa (2014) clearly showed that, in Spiranthinae and other Cranichideae, the median carpel develops before the lateral carpels, enlarges considerably and gives rise to both the receptive stigmatic area(s) and a non-receptive portion that bears the viscidium. The last, non-receptive portion conforms to the original definition of rostellum by Richard (1817) and upheld by, among others, Vermeulen (1959), Dressler (1993), Kurzweil (1988, 1998) and Salazar et al. (2011a), which is the one followed here (see Rasmussen, 1982, for a different interpretation). Those developmental studies also showed that the lateral carpel apices (corresponding to the lateral stigma lobes of most plants, but not Cranichideae) arise fused as a transverse ridge adjacent to the base of the receptive area of the median stigma lobe and appear to contribute little to the receptive surface itself. Therefore, the supposed lateral stigma lobes of Burns-Balogh & Robinson (1983) are part of the receptive portion of the median lobe. Moreover, in all flowers of Cyclopogon, Pelexia, Sarcoglottis and Veyretia that we have examined the entrance to the stylar channel is located near the base of the stigmatic area. Therefore, the entrance of the stylar channel in the Pelexia clade is always located at the confluence of the three carpel apices and interpretation of the alternative condition as a synapomorphy for a Sarcoglottis–Pelexia clade to the exclusion of other genera is unsustained. The cladistic analysis of ‘Deiregyne’ sensuBurns-Balogh (1988; =Aulosepalum Garay as interpreted here), based on 25 floral attributes and one vegetative attribute, will be discussed elsewhere against the framework of a detailed study of the phylogenetic relationships of Aulosepalum based on a multilocus molecular analysis and comparative morphological observations (G. A. Salazar, R. Chalqueño & S. A. Adachi, unpubl. data). On the other hand, the six vegetative and 43 floral features used in the phenetic and cladistic analyses of genera for which monophyly was not assessed (because the genera were used as terminals) by Rutkowski et al. (2008), briefly described in their appendix 1, deserve more careful discussion than the focus of the present work permits. However, a perusal of their definition of the characters and their states reveals many potential problems, including, among others: (1) character redundancy in, for instance, characters 5 (leaf petiole narrow = no/yes) and 6 (leaf petiole gradually transforming into the blade = no/yes), which are clearly a single attribute that was scored twice; likewise, characters describing the apex of the rostellum, e.g. 39 (rostellum bilobed = no/yes), 44 (rostellum furculate = no/yes), 45 (rostellum subulate = no/yes), 46 (rostellum tridentate = no/yes), represent an ‘inflation’ of the weight assigned to one and the same structure; (2) gradual attributes arbitrarily made discrete, e.g. character 13 (most of the spur united to the ovary, but with a free top = no/yes) and 14 (spur united with ovary basally only, mostly free = no/yes); and (3) autapomorphic attributes, which are uninformative about relationships among the genera and irrelevant in this context for generic delimitation, because the genera were delimited a priori, e.g. characters 2, 4, 9, 10, 18, 34 and 36. Overall, there is a lack of discussion backed by detailed comparative and developmental evidence from studies conducted by those authors or referred to the literature, a requisite for any solid interpretation of structural homology and evolution. These issues will have to be addressed in monographs of the major clades and monophyletic genera, as applicable. There is little point in trying to make sense of the complex evolution of floral morphology in reference to artificial major groups (‘subtribes’) and arbitrarily delimited genera such as those recognized by Rutkowski et al. (2008). Biogeographical considerations Our ancestral area analysis indicates a Neotropical origin for Spiranthinae, with eastern South America attaining the highest probability as the area of origin of the subtribe. Migrations associated with secondary diversification in Mesoamerica and subsequently North America/Eurasia are indicated for the Spiranthes clade, and again in Mesoamerica for several subclades of the Pelexia and Eurystyles clades (Fig. 10; Supporting Information, Fig. S8). Previous hypothetical scenarios about historical biogeography of Spiranthinae such as those in Rutkowski et al. (2008) are hardly comparable because they are based on groupings that, according to our results, do not represent clades. However, as noted earlier, both our phylogenetic and biogeographical results agree with the proposal of Dueck et al. (2014) that Spiranthes, a predominantly temperate North American/Eurasian clade, is derived from Mesoamerican ancestors. Figure 10. View large Download slide Ancestral area reconstruction under the BMM method with RASP. The most probable ancestral areas of clades are indicated as coloured circles. For simplicity, outgroups and areas not directly relevant to Spiranthinae were trimmed (see the full set of reconstructed probabilities in Supporting Information, Fig. S8). Figure 10. View large Download slide Ancestral area reconstruction under the BMM method with RASP. The most probable ancestral areas of clades are indicated as coloured circles. For simplicity, outgroups and areas not directly relevant to Spiranthinae were trimmed (see the full set of reconstructed probabilities in Supporting Information, Fig. S8). Cyclopogon obliquus has a puzzling distribution and a tortuous taxonomic history, having been described originally from a plant found at the Buitenzorg (Bogor) Botanic Garden in Java, Indonesia, and assigned then to the ‘catch-all’ genus Spiranthes s.l. Later it was re-described twice, once as Manniella hongkongensis S.Y.Hu & Barretto (Hu & Barretto, 1976) from a plant found, as the name implies, on the island of Hong Kong, and the other as Pelexia hameri Garay on the basis of a specimen from El Salvador, Central America (Garay, 1978). The species has been found since in other locations in both hemispheres, including Samoa, Sri Lanka, the West Indies (Guadeloupe and Cuba), Nicaragua, Costa Rica (see Blanco, 2002, and references therein) and Mexico (Soto et al., 2007). Nevertheless, as for all its close relatives, this species is most likely of Neotropical origin and its first discovery in an Asian botanical garden, associated with a cultivated plant of the Neotropical family Cyclanthaceae, suggests an accidental introduction into south-eastern Asia, where it appears to be spreading (cf. Comber, 1990; Cribb & Ormerod, 1999). FINAL CONSIDERATIONS AND FUTURE WORK This study represents the most thorough phylogenetic analysis of Spiranthinae conducted to date and is based on an intensive effort to sample the structural, ecological and geographical diversity displayed by this subtribe worldwide. Our generic sample is nearly complete according to the genera recognized in the phylogenetic classification of the orchid family by Chase et al. (2015). Genera that have not been available for molecular analysis include mostly monospecific taxa, such as Cybebus, restricted to Andean Ecuador and Colombia, and Degranvillea, a mycoheterotrophic taxon endemic to lowland, seasonally dry tropical forests in French Guiana (Determann, 1985). Cybebus shares some vegetative characters with the Pelexia clade, including rhizomatous habit and long-petiolate leaves with several sunken longitudinal veins (reminiscent of plants of Coccineorchis and some Pelexia s.s.), but the small, stiff rostellum remnant suggests a possible relationship to the Stenorrhynchos clade. Overall appearance of its flowers is reminiscent of members of Eltroplectris and Pteroglossa, but no spur is present (Salazar, 2003b). On the other hand, Degranvillea shows some floral features in common with the Pelexia clade, including the narrowly conical sepaline spur, subulate nectar glands of the labellum and truncate rostellum with terminal viscidium (as in some species of Sauroglossum and Pelexia s.s.), but its highly modified vegetative organs do not offer any obvious clue about its phylogenetic affinities. Lastly, Aracamunia and Helonoma are two distinctive genera highly specialized to the wet, oligotrophic environments of tepuis of the Guayana Highlands and the Andean tepuis. As mentioned earlier, Helonoma was sunk in Beloglottis by Szlachetko (1996), but these two groups are too distinctive morphologically and ecologically to accept such an idea. Resolution of this issue will have to wait until samples suitable for DNA analyses become available. The phylogenetic framework generated by this study, which includes many clades not only supported by the DNA sequences but also by structural, ecological and distributional data (e.g. Funkiella s.l.; see earlier and Fig. 2A–L), provides an objective basis for subsequent macroevolutionary analyses, including detailed morphological and developmental studies and systematic monographs of natural taxa. Those parts of the phylogenetic tree that still lack clear resolution or support, such as the relationships among some of the major clades of Spiranthinae and interspecific relationships in species-rich genera (i.e. Cyclopogon, Pelexia, Sarcoglottis) will benefit from further work aimed at increasing the sample of both taxa and characters. Affordable access to genome-scale data offers a promising possibility, although difficulty of accessing restricted, rare taxa is a problem likely to be solved only through involvement of local researchers and students in the regions where the diversity of this subtribe is concentrated. The results presented here offer a framework for designing future collecting efforts and focusing monographic work. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Taxa studied, voucher information and GenBank accession numbers. Figure S1. Strict consensus from the MP analysis of plastid DNA sequences. Numbers above branches indicate bootstrap percentages > 50. Figure S2. Maximum likelihood tree from the ML analysis of plastid DNA sequences. Numbers above branches indicate bootstrap percentages > 50. Figure S3. Strict consensus from the MP analysis of nuclear DNA sequences. Numbers above branches indicate bootstrap percentages > 50. Figure S4. Maximum likelihood tree from the ML analysis of nuclear DNA sequences. Numbers above branches indicate bootstrap percentages > 50. Figure S5. Strict consensus from the MP analysis of combined plastid and nuclear DNA sequences. Numbers above branches indicate bootstrap percentages > 50. Figure S6. Maximum likelihood tree from the ML analysis of combined plastid and nuclear DNA sequences. Numbers above branches indicate bootstrap percentages > 50. Figure S7. Maximum likelihood phylogram from the ML analysis of combined plastid and nuclear DNA sequences. Branch lengths are drawn proportional to the amount of character change. Figure S8. Ancestral area reconstruction under the BMM method with RASP. Ancestral areas with different probabilities are indicated as portions of coloured rings. The most probable ancestral areas are indicated by letters at the centre of each ring. ACKNOWLEDGEMENTS This work was supported by a scholarship for graduate study from Dirección General del Personal Académico, Universidad Nacional Autónoma de México, a graduate fellowship from the American Orchid Society, a grant (Apoyo Complementario a Proyectos de Investigación Científica para Investigadores en Proceso de Consolidación) from the Mexican Consejo Nacional de Ciencia y Tecnología and institutional research funds provided by the Instituto de Biología, Universidad Nacional Autónoma de México (all to G.A.S.). C.R.B., R.B.S. and C.v.d.B. acknowledge the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, Brazil (process 470353/2011–2) for financial support. The authors thank James D. Ackerman, Carlos R. Beutelspacher, Eduardo L. Borba, Haydée Cabassi, Mark Clements, José D. Edquen, Coyolxauhqui Figueroa, Eduardo Flachsland, Abisaí García-Mendoza, Giovanni Giraldo, Douglas Goldman, Héctor M. Hernández, Tania J. Hernández-López, Wesley E. Higgins, Alexander Hirtz, Carlos René Huerta Alvízar, Thorsten Krömer, Carlos Lehnebach, Alex Portilla, José Portilla, Franco Pupulin, Marco A. López Rosas, Érika Morón, Daniel Nickrent, Ricardo Penz, Rosiela Pérez-Bravo, Jerónimo Reyes, Juan José Rodríguez, Erik Rothacker, Octavio Suárez, Jorge Warner and the late Andrés Maduro and Miguel Ángel Soto for plant material, photographs and information. 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