Botanical Journal of the Linnean Society, 2018, 186, 273–303. With 10 figures.
Phylogenetic systematics of subtribe Spiranthinae
(Orchidaceae: Orchidoideae: Cranichideae) based on
nuclear and plastid DNA sequences of a nearly complete
generic sample
Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado
Postal 70-367, 04510 Mexico City, Mexico
2
Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av.
Antônio Carlos 6627, Pampulha, C.P. 486, 31270-910 Belo Horizonte, Minas Gerais, Brazil
3
Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina
s.n., 44036-900 Feira de Santana, Bahia, Brazil
4
Florida Museum of Natural History, Dickinson Hall, 1659 Museum Road, Gainesville, FL 32611-7800, USA
5
Departamento de Botânica, sala 446 Setor de Ciências Biológicas, Programa de Pós-graduação em
Ciências Ambientais," after Ciências Biológicas, Centro Politécnico, Universidade Federal do Paraná,
Caixa Postal 19031, 81531-990 Curitiba, Paraná, Brazil
6
Universidade de Passo Fundo, Instituto de Ciências Biológicas, Campus I, Bairro São José, BR 285,
99052-900, Passo Fundo, Rio Grande do Sul, Brazil
7
Universidade Federal do Rio Grande do Sul, Departamento de Botânica, Av. Bento Gonçalves, 9500,
Bloco IV, Prédio 43433, Campus do Vale, Agronomia, 91501-970 Porto Alegre, Rio Grande do Sul, Brazil
8
Botanischer Garten München-Nymphenburg, Menzinger Str. 61, D-80638 Munich, Germany
9
Herbario AMO, Montañas Calizas 490, Lomas de Chapultepec, 11000 Mexico City, Mexico
10
Dirección de Biodiversidad, Ministerio de Ecología y Recursos Naturales Renovables, Calle San
Lorenzo 1538, Código Postal 3300, Posadas, Misiones, Argentina
11
Misiones 4091, 3300 Posadas, Misiones, Argentina
12
Instituto de Botânica, Av. Miguel Stéfano, 3687, Água Funda, São Paulo 04301-902, Brazil
13
Herbario Nacional del Ecuador, Instituto Nacional de Biovidersidad, Casilla Postal 17-07-8976
Avenida Río Coca E6-115 e Isla Fernandina, Sector Jipijapa, Quito, Ecuador
14
Centro Universitario Regional del Litoral Atlántico, Universidad Autónoma de Honduras, La Ceiba,
Honduras
15
Jardín Botánico de Pinar del Río, Km 1½ Carretera al Hoyo Guamá, Pinar del Río 20100, Cuba
16
Jardín Botánico Lankester, Universidad de Costa Rica, PO Box 1031-7050, Cartago, Costa Rica
17
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
18
School of Biological Sciences, University of Western Australia, Crawley, WA 6009, Australia
1
Received 9 July 2017; revised 13 November 2017; accepted for publication 4 December 2017
*Corresponding author. E-mail: gasc@ib.unam.mx
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
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GERARDO A. SALAZAR1*, JOÃO A. N. BATISTA2, LIDIA I. CABRERA1,
CÁSSIO VAN DEN BERG3, W. MARK WHITTEN4, ERIC C. SMIDT5,
CRISTIANO ROBERTO BUZATTO6, RODRIGO B. SINGER7, GÜNTER GERLACH8,
ROLANDO JIMÉNEZ-MACHORRO9, JOSÉ A. RADINS10, IRMA S. INSAURRALDE11,
LEONARDO R. S. GUIMARÃES12, FÁBIO DE BARROS12, FRANCISCO TOBAR13,
JOSÉ L. LINARES14, ERNESTO MÚJICA15, ROBERT L. DRESSLER16, MARIO A. BLANCO16,
ERIC HÁGSATER9 and MARK W. CHASE17,18
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G. A. SALAZAR ET AL.
ADDITIONAL KEYWORDS: Ancestral area – floral morphology – homology – homoplasy – molecular
phylogenetics – pollination syndrome – taxonomy.
‘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).
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
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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 nonmolecular 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).
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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,
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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.
(sensu Burns-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,
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MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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, southeastern 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 (sensu
Figure 1. 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).
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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.
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MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
MATERIAL AND METHODS
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 geldried 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).
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,
PHYLOGENETIC ANALYSES
We conducted MP and maximum likelihood (ML)
analyses with the aim of comparing the patterns
Figure 2. 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).
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Chase et al., 2015; Salazar et al., 2016) and over onethird 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.
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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 5 000 000 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 amongsite 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=DC970ED
FBCF052B4F2BFA4BCE576E9A2?checklist=selec
ted_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
11 500 MPTs with a length of 2691 steps, CI of 0.30 and
RI of 0.79. The consensus tree (Supporting Information,
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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 branchswapping, 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.
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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. (sensu Garay, 1982; Salazar, 2003b), all
with BP 100.
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.
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
p o l y p h y l e t i c Pe l e x i a a n d s o m e m e m b e r s o f
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.
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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 sistergroup 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 BurnsBal. in turn sister to Beloglottis Schltr. plus Aulosepalum
Garay; all genera in this subclade for which more than
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Figure 3. 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.
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
283
ANCESTRAL AREA ANALYSIS
DISCUSSION
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.
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
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Figure 4. 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.
284
G. A. SALAZAR ET AL.
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Figure 5. 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.
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
285
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Figure 6. 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.
them are not clearly resolved with the exception, in
the present analysis, of a strongly supported sistergroup 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
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286
G. A. SALAZAR ET AL.
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.
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, linearoblanceolate, 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, BurnsBalogh 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.
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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.
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
287
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Figure 7. 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).
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
288
G. A. SALAZAR ET AL.
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
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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
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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
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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
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Figure 8. 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).
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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 (sensu Garay, 1982), Schiedeella (sensu
Salazar et al., 2016) and Aulosepalum (sensu Garay,
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 BurnsBalogh based on recycling of Szlachetko’s (1995b) old
argument on interpretation of the imprecise original
diagnosis of the genus by Schlechter (1920).
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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).
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) sensu Salazar et al.
(2002, 2011a) and Salazar & García-Mendoza
(2009) and Deiregyne sensu Garay (1982) form a
strongly supported sister-pair with similar habitat
preferences, vegetative morphology and overall floral
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MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
Stenorrhynchos clade
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 bristlelike, 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
Figure 9. 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).
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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
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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).
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
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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.
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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)
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(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 oblanceolatespathulate 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
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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 BurnsBalogh & 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
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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.
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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.
Cyclopogon obliquus has a puzzling distribution and
a tortuous taxonomic history, having been described
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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 BurnsBalogh & 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’ sensu BurnsBalogh (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
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Figure 10. 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).
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
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
MOLECULAR PHYLOGENETICS OF SPIRANTHINAE
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.
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. The Curators of 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
graciously assisted us during study of the collections
in their charge. G.A.S. thanks Martin Ingrouille for
supervision, friendship, field companion in Mexico
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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).
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and useful suggestions to a very early version of this
work. The staff of the Molecular Systematics Section,
Jodrell Laboratory, Royal Botanic Gardens, Kew, and
Laura Márquez Valdelamar, Instituto de Biología,
Universidad Nacional Autónoma de México, helped
with laboratory work.
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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.
© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 186, 273–303
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