TAXON 65 (6) • December 2016: 1249–1262 Peraza-Flores & al.

• Phylogeny of the Laelia alliance

A molecular phylogeny of the Laelia alliance (Orchidaceae)

and a reassessment of Laelia and Schomburgkia
Lizandro N. Peraza-Flores,1 Germán Carnevali2 & Cássio van den Berg1
1 Universidade Estadual de Feira de Santana, Departamento de Ciências Biológicas, Av. Transnordestina s.n., 44036-900 Feira de
Santana, Bahia, Brazil
2 Herbario CICY, Centro de Investigación Científica de Yucatán, A.C. (CICY), Calle 43 No. 130, Colonia Chuburná de Hidalgo,
C.P. 97200, Mérida, Yucatán, México
Author for correspondence: Lizandro N. Peraza-Flores, lizandropf@gmail.com
ORCID  LNPF, http://orcid.org/0000-0001-9534-0523; GC, http://orcid.org/0000-0002-2659-9352; CvdB, http://orcid.org/0000-0001-5028-0686

DOI  https://doi.org/10.12705/656.3

Abstract  We provide a molecular phylogenetic analysis of the Laelia alliance based on sampling of 20 of an estimated 24 spe-
cies, and seven plastid regions plus nrITS 1 & 2 analyzed using maximum parsimony, maximum likelihood, and Bayesian
inference. Furthermore, we used coded indels to evaluate their effect on the phylogenetic relationships and / or support values
(bootstrap and posterior probabilities). Our results confirm the existence of two clades, the long known Laelia (endemic to
México at middle to high elevations) and Schomburgkia (a more widely distributed taxon). Both clades were strongly sup-
ported by PP values and moderate BS; thus, we preferred to recognize them as separate genera with a reappraised morphology
and taxonomic delimitation. The inclusion of coded indels in the analyses was of great utility; we could identify the probable
hybrid origin of L. gouldiana and L. halbingeriana. We propose new combinations to accommodate the taxonomic changes
resulting from our study. We transfer L. anceps subsp. anceps, L. anceps subsp. dawsonii, L. aurea, L. mottae, and L. rubescens
to Schomburgkia, with the second taxon elevated to species level; the molecular evidence together with previous published
work on the morphology, phenology and geography of the group support these decisions. Lastly, the recently described genus
Encabarcenia is considered as a synonym of Schomburgkia and, as a result, a new combination is proposed.

Keywords  hybridization; indel coding; molecular phylogeny; Orchidaceae; splitting

Supplementary Material  The Electronic Supplement (Tables S1 & S2; Figs. S1–S3) is available in the Supplementary Data
section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax; DNA sequence alignments
are available from TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S19331)

INTRODUCTION Lindl. (and/or other members of Schomburgkia), L. anceps

Phylogenetic relationships within Laelia Lindl. have re-
mained only partially resolved, due in part to the limited taxon
sampling of the genus in its new broader circumscription. As
a consequence, the group is recognized as the Laelia alliance
even when Schomburgkia Lindl. is included within Laelia (Van
den Berg & al., 2000, 2009; Van den Berg & Chase, 2004a;
Chase & al., 2015). Published phylogenetic analyses have in-
cluded at most 12 species, mainly from the Mexican Laelia. The
most thorough published work on the phylogeny of this group
of taxa was based only on the Mexican species of Laelia and
was focused on the analysis of only morphological characters
(Halbinger & Soto, 1997). The molecular phylogenies published
so far had a wider scope but lower taxon sampling; the topolo-
gies based on molecular data differ from those of morphology
in both internal relationships and the placement of Schomburg-
kia (Halbinger & Soto, 1997; Van den Berg & al., 2000, 2009).
The monophyly of the alliance has been corroborated by several
molecular analyses (Van den Berg & al., 2009; Freudenstein
& Chase, 2015). The relationships between Laelia superbiens
Lindl., and L. rubescens Lindl. have been recognized, but the
former has been considered morphologically intermediate be-
tween the Mexican Laelia and Schomburgkia (Van den Berg
& al., 2000, 2009; Van den Berg & Chase, 2004a).
Reticulation events are often difficult to identify, and mor-
phology (the main source of data used to hypothesize hybrid-
ization) can be sometimes misleading when the descendants
are closer to one parent and not intermediate (Rieseberg, 1995;
Wissemann, 2007); furthermore, the assumption of bifurcating
patterns of evolution complicates the discovery of reticulation
events (Legendre, 2000; McBreen & Lockhart, 2006; Huson &
al., 2010). The incongruence between gene trees of uniparen-
tally (mitochondrial or plastid) or biparentally (nuclear) inher-
ited markers has been used as evidence of reticulation events
in a wide variety of phylogenetic analyses (e.g., Pelser & al.,
2010; Yu & al., 2013).
Hybridization has been reported in several groups of
orchids using molecular markers (Gravendeel & al., 2004;
Azevedo & al., 2006; Russell & al., 2010). In Laeliinae,
clear examples are found within and among Cattleya Lindl.,

Received: 3 Feb 2016 | returned for (first) revision: 24 Mar 2016 | (last) revision received: 16 Sep 2016 | accepted: 18 Sep 2016 || publication date(s):
online fast track, n/a; in print and online issues, 22 Dec 2016 || © International Association for Plant Taxonomy (IAPT) 2016

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Peraza-Flores & al. • Phylogeny of the Laelia alliance
Brassavola R.Br., and Laelia, where many hybrids, natural
or artificial, have been described to date (Halbinger & Soto,
1997; Salazar & al., 2014; Van den Berg, 2014a). However,
there are few studies assessing hybridization patterns in these
genera with molecular methods, and it has only been inferred
in Cattleya from conflicting topologies of nuclear and plastid
trees (Van den Berg & al., 2009; Santos, 2011). Recently, Van
den Berg (2014a, b) suggested that reticulation events are an
evolutionary driving force in Cattleya, based on the abundance
of putative natural hybrids described. It will not be surprising
if similar patterns of evolution have also influenced the evolu-
tion of Laelia, especially because of its similar generalized
type of bee-pollination indicated by morphology. The impor-
tance of reticulate evolution within the genus is unclear, even
when molecular evidence from both maternal (plastid) and
bi-parental (nuclear) inheritance is available for a subset of
the genus; nevertheless, previous phylogenies did not find any
incongruence between both types of evidence, probably as a
consequence of limited sampling of taxa and poor resolution
when using plastid or nuclear regions individually (Van den
Berg & al., 2000, 2009).
Insertion-deletions (indels) are evolutionary events occur-
ring in coding and, more frequently, noncoding regions of DNA,
detectable when comparing the sequences of different organ-
isms; their presence will vary depending on the region of DNA
analyzed and organisms studied. Indels are common events in
evolution as obvious from the comparison of many orchid plastid
genomes and other groups of plants (e.g., Barrett & al., 2014,
2016; Luo & al., 2014; Carbonell-Caballero & al., 2015). How-
ever, indels are not always considered as evidence in phyloge-
netic analysis even though coding schemes have been developed
to incorporate them in the analyses (González, 1996; Simmons &
Ochoterena, 2000; Müller, 2005). There is increasing evidence
that indels can improve the resolution and branch support of
phylogenetic trees in some cases (Simmons & Ochoterena, 2000;
Simmons & al., 2001; Ogden & Rosenberg, 2007).
Laelia in its current circumscription (Soto & al., 2006) is a
genus of about 24 species distributed from northern Mexico to
southeastern Brazil, including the Antilles (Cuba and Jamaica),
in a wide variety of environments, but most of its species are
associated with seasonally dry forests. It was first described
with two Mexican species, L. grandiflora (Llave & Lex.) Lindl.
(= L. speciosa (Kunth) Schltr., the type) and L. autumnalis (Llave
& Lex.) Lindl., but at least 157 specific names were associated
with Laelia later in various circumscriptions of the genus. When
we account for infraspecific taxa that number would increase
to more than 200 names. Describing all taxonomic changes in
the genus would be outside the scope of this paper. However,
we here summarize some of the most relevant changes. The
phylogenetic analysis by Van den Berg & al. (2000) based on
nuclear ITS resulted in a clearer understanding of the evolu-
tionary relationships that would eventually be used, along with
additional evidence, to establish new taxonomic circumscrip-
tions in the Laeliinae. Laelia then included mainly Mexican
and Brazilian species as well as the taxa traditionally referred
to Schomburgkia, but suffered a great change in its circum-
scription when 50 species and 5 hybrids of Brazilian Laelia
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TAXON 65 (6) • December 2016: 1249–1262
were transferred to Sophronitis Lindl. (Van den Berg & Chase,
2000, 2004b). These Brazilian species were later transferred to
Cattleya based on further molecular evidence (Van den Berg,
2008). At this point, Laelia was limited to the predominantly
Mexican species and those previously included in Schomburg-
kia (most species previously combined in Laelia by Williams,
1941). Historically, many botanists and orchid growers have
regarded Schomburgkia as separate from Laelia for most of the
period prior to molecular data. Molecular phylogenetic evidence
demonstrated that the genus was composed of two clades, and
L. anceps and L. rubescens were closer to species assigned to
Schomburgkia. Based on these results, many authors preferred
the expanded circumscription of Laelia, including Schomburg-
kia (Williams, 1941), to yield a workable and monophyletic con-
cept of the genus (Van den Berg & al., 2000, 2009; Van den Berg
& Chase, 2004a; Soto & al., 2006; Chase & al., 2015; Govaerts
& al., 2015). However, some have kept the two genera distinct in
their phylogenetic analyses (e.g., Freudenstein & Chase, 2015).
Recently, a different point of view was established by Archila
& Szlachetko (2014) when they regarded L. rubescens, L. aurea
A.V.Navarro, and a new species they described as a different
genus, Encabarcenia Archila & Szlach. They stated, based on
the morphological phylogenetic analyses of Halbinger & Soto
(1997), that these species were indeed a distinct genus, stress-
ing its floral similarity to Broughtonia R.Br. However, this is in
contrast to all previous molecular evidence of the phylogenetic
placement of these species according to the principle of mono-
phyly currently used in phylogenetic systematics (Van den Berg
& al., 2000, 2009; Soto & al., 2006).
Disagreements on the limits of genera are common among
taxonomists, dubbed either lumpers or splitters depending on
their wide or narrow delimitation viewpoints. Examples of such
disagreements are frequent among orchid specialists, and a clear
example is the Trichocentrum clade within Oncidiinae. Some
taxonomists treat it as a single genus, Trichocentrum Poepp. &
Endl. s.l. (Chase & al., 2009) while others consider the group
as composed of four genera: Lophiaris Rafinesque (“mule ear”
species), Cohniella Pfitzer (“rat-tail” species), Lophiarella
Szlach. (Oncidium microchilum group), and Trichocentrum s.str.
(Carnevali & al., 2013). The debate on lumping or splitting is far
from over with no scientific consensus in sight.
The previous molecular phylogenetic analyses recovered
two groups of species within Laelia s.l.; the first included the
long known Mexican mid- to high-altitude species and the sec-
ond included L. rubescens, L. anceps, L. superbiens, and all spe-
cies of Schomburgkia sampled (Sch. crispa Lindl., Sch. lyonsi
Lindl., Sch. splendida Schltr., Sch. undulata Lindl.). However,
support values for these two groups were relatively low and
interspecific relationships were not resolved. For that reason,
we focused our research on the evolutionary relationships within
Laelia s.l. and, mainly, its relation to those species previously
included within Schomburgkia. Our main objectives were to
analyze the evolutionary history of this group, evaluate the re-
sults of different phylogenetic methods, and evaluate how indel
coding affects them. Some authors have suggested that hybrid-
ization could be an important evolutionary force in some groups
of Laeliinae but we do not know to what extent it influences the
TAXON 65 (6) • December 2016: 1249–1262 Peraza-Flores & al. • Phylogeny of the Laelia alliance

evolution of Laelia (Van den Berg & al., 2009; Santos, 2011; Van
den Berg, 2014a, b); thus we included molecular evidence from
both uniparentally (plastid) and biparentally (nuclear) inher-
ited markers. For achieving these objectives we sequenced one
nuclear and seven plastid regions of DNA and analyzed them
with maximum parsimony, maximum likelihood, and Bayesian
inference, separately. We also coded indels in order to explore
their effect on the topology of the three phylogenetic methods.
MATERIALS AND METHODS
Plant material. — We sampled as many species and mor-
phological variants as were available to us from the geographic
range of Laelia s.l. Our ingroup consisted of 21 species of the
genus, and some infraspecific taxa or morphologically distinct
geographic samples for L. anceps, L. autumnalis, L. (Schom-
burgkia) gloriosa, L. speciosa, L. (Sch.) superbiens, and L. (Sch.)
undulata; a recently described hybrid, Laelia ×oaxacana Sala-
zar & R.Jiménez, was also included within our ingroup. In
total, we analyzed 27 different accessions of Laelia (Appen-
dix 1). Our outgroup included three distant species of Laeliinae,
Broughtonia sanguinea Sw. (R.Br.), Dinema polybulbon Lindl.,
and Homalopetalum pachyphyllum (L.O.Williams) Dressler;
thus, our analysis included 30 taxa (Appendix 1).
DNA sampling. — We amplified and sequenced seven re-
gions of plastid DNA and the ITS region (5′ partial 18S, ITS1,
5.8S, ITS2, 3′ partial 26S) after testing for variation, ease of am-
plification, and sequencing. The plastid regions amplified and
sequenced were previously reported to contain high variation
in plants and, specifically, in close relatives of the study group
(Shaw & al. 2005, 2007; Neubig & al., 2009; Santos, 2011).
The plastid regions amplified and sequenced were rpl32-trnL,
rps16-trnK, trnD-trnT, trnH-psbA, trnQ-rps16, trnS-trnfM, and
the 3′ portion of the ycf1 gene. Primers and protocols used for
amplification are detailed in Table 1. Primers for rps16-trnK
and trnQ-rps16 were designed for this study due to problems
of amplification and sequencing with the primers reported by
Shaw & al. (2007).
DNA isolation and sequencing. — DNA was isolated from
silica gel dried leaves using a rescaled protocol of the 2× CTAB
method (Doyle & Doyle, 1987). Isolated DNA was stored at
−80°C at the UEFS DNA bank. All primers and protocols used
to amplify and sequence each region are listed in Table 1. The
amplified fragments were purified by precipitation using PEG-
8000 11% and 70% ethanol (Paithankar & Prasad, 1991). The
purified fragments were then sequenced using the Big Dye 3.1
kit (Applied Biosystems, Foster City, California, U.S.A.) and
an automatic ABI3130XL capillary sequencer at Universidade
Estadual de Feira de Santana (UEFS), Bahia, Brazil.

Table 1. Primers and protocols used to amplify the DNA regions used in the phylogenetic analyses.
Region Primer Protocol Notes
ITS ITS16SE- 5′-ACG AAT TCA TGG TCC GGT GAA GTG TTC G -3′ 94°C for 3 min, 28 × (94°C for ITS16SE and 26SE
ITS26SE- 5′-TAG AAT TCC CCG GTT CGC TCG CCG TTA C -3′ 1 min, 50°C for 1 min, 72°C for (Sun & al., 1994); ITS4
ITS92- 5′-AAG GTT TCC GTA GGT GAA C -3′ 3 min), 72°C for 7 min (White & al., 1990)
ITS4- 5′-TCC TCC GCT TAT TGA TAT GC -3′ ITS92 and ITS4, for
sequencing
rpl32-trnL rpl32-F 5′-CAG TTC CAA AAA AAC GTA CTT C -3′ 80°C for 2 min, 30 × (95°C for Shaw & al. (2007)
trnL(UAG) 5′- CTG CTT CCT AAG AGC AGC GT -3′ 1 min, 50°C for 1 min, 65°C for
2 min), 65°C for 3 min
rps16-trnK rps16-trnK F- 5′-AGG KGY TCA ACC CAC ARR AAC TG -3′ 80°C for 5 min, 35 × (95°C for Designed in this study
rps16-trnK R- 5′-ACC TTT CAG GAT CAG TCG YGG TC -3′ 30 s, 50°C for 20–30 s, 65°C for
30–45 s), 65°C for 5 min
trnD-trnT trnD-T 100F - 5′ - GGA CTT GAA TGA GAA TGG CAT AG -3′ 80°C for 5 min, 30 × (95°C for Designed by C.v.d.B.
trnD-T 1463R -5′ - CGC TGA ACG AAG CAA TGA G -3′ 1 min, 54°C–55°C for 1 min, trnD-T 673F was used
trnD-T 673F - 5′ - GAA TGA GCT ATC CCA TAC TCC -3′ 65°C for 2 min), 65°C for 5 min for sequencing
trnH-psbA F- 5′-CGC GCA TGG TGG ATT CAC AAA T -3′ 80°C for 5 min, 30 × (95°C for trnH (Tate & Simpson,
R- 5′-GTT ATG CAT GAA CGT AAT GCT C -3′ 1 min, 50°C for 1 min, 65°C for 2003); psbA (Sang & al.,
4 min), 65°C for 5 min 1997)
trnQ-rps16 trnQ-rps16 F- 5′- CGT TAY TCG GRG GTT CGA ATC -3′ 80°C for 5 min, 30 × (95°C for Designed in this study
trnQ-rps16 R- 5′- CAT GTC CTT CAA GTC GCR CGT TG -3′ 1 min, 50°C for 1 min, 65°C for
4 min), 65°C for 5 min
trnS-trnfM trnSUGA 5′- GAG AGA GAG GGA TTC GAA CC -3′ 80°C for 3 min, 30 × (94°C for Demesure & al. (1995)
trnfMCAU 5′- CAT AAC CTT GAG GTC ACG GG -3′ 1 min, 55°C for 1 min [ramp
of 0.3°C/s from 55°C to 65°C],
65°C for 2 min), 65°C for 5 min
3′ portion 3720F- 5′- TAC GTA TGT AAT GAA CGA ATG G -3′ 94°C for 3 min, 8 × (94°C for 30 s, Neubig & al. (2009)
of ycf1 5500R- 5′- GCT GTT ATT GGC ATC AAA CCA ATA GCG -3′ 58°C–51°C for 1 min [reducing IntF and IntR were used
IntF- 5′- GAT CTG GAC CAA TGC ACA TAT T -3′ 1°C/cycle], 72°C for 3 min), 30 × for sequencing
IntR- 5′- TTT GAT TGG GAT GAT CCA AGG -3′ (94°C for 30 s, 50°C for 1 min,
72°C for 3 min), 72°C for 3 min

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Phylogenetic analysis. — Electropherograms were ed-
ited and assembled using Geneious v.5.3.6 (Drummond & al.,
2010). Edited sequences were aligned using MUSCLE v.3.8.31
(Edgar, 2004) through the Geneious platform. Alignments
were manually inspected, and modified if needed, to avoid
problems of automated alignment (e.g., misplacement of nucle-
otides in gap regions). Sequences were deposited at GenBank
(Appendix 1). The nuclear and plastid matrices were analyzed
independently. The gaps resulting in the alignment were ana-
lyzed as missing data, as most likelihood algorithms do, based
on studies that report no effects on the results (Drummond
& Bouckaert, 2015; Zheng & Wiens, 2015). Gaps were also
coded and added to matrices for analyses (details of coding and
analysis below). Alignments of both matrices were deposited
in TreeBASE (S19331). We used maximum parsimony (MP),
maximum likelihood (ML) and Bayesian inference (BI) to
analyze the phylogenetic relationships among the species of
Laelia. Preliminary tests of the ITS and plastid alignments
provided conflicting topologies and accordingly were analyzed
separately; they represent two different kinds of inheritance
and trees obtained represent two different evolutionary his-
tories. MP analyses were run with TNT v.1.5 (Goloboff &
Catalano, 2016). They consisted of traditional searches with
10,000 random taxon-addition replicates to identify multiple
islands of optimal trees (Maddison, 1991); saved trees were
input into the swapping process to increase our probabilities
to find the optimal tree. We set a maximum of 10 trees held at
each replicate, TBR swapping for every rearrangement tried,
and a maximum of 1000 trees held for the whole search; all
other parameters were set to default. A bootstrapping analy-
sis consisting of 10,000 replicates using the same search pa-
rameters was used to evaluate support (Felsenstein, 1985).
We considered that bootstrap support (BS) values were poor
(< 50%), weak (50%–70%), moderate (> 70%–85%), or strong
(> 85%) (Kress & al., 2002). We identified the model of evo-
lution of each region with jModelTest v.2.1.7 (Darriba & al.,
2012), using the Akaike information criterion (AIC) as selec-
tion criterion and default parameters of search (models selected
by jModelTest are summarized in Table 2). These models serve
as input parameters to ML and BI analyses; the ML analyses
were run with RAxML v.8.1.21 (Stamatakis, 2014) through the
raxmlGUI v.1.5b1 (Silvestro & Michalak, 2012) platform; we
used a single model of evolution, the MULTIGAMMA, as most
aligned regions had a GTR + gamma model of evolution (Table
2). We analyzed support through a bootstrap analysis with
10,000 replicates. The strategy used for parsimony bootstrap
was adopted for the ML bootstrap analyses. BI analyses were
done through MrBayes v.3.2 (Ronquist & al., 2012). Analy-
ses were run for 50 million generations with two independent
runs with four chains each and setting the Temp and Nswaps
parameters to 0.05 and 2, respectively. We considered that
runs converged when the standard deviation among split fre-
quencies achieved a value of less than 0.01, and the estimated
sample size (ESS) was over 200; these values were considered
after 25% of the generations were discarded as burn-in. This
was achieved either by manually inspecting MrBayes log files
or by analyzing the trace files through Tracer v.1.6 (Rambaut
& al., 2014). We considered posterior probabilities (PP) poor
(< 0.90), weak (0.90 to < 0.95), moderate (0.95 to < 0.98), and
strong (≥ 0.98) (Erixon & al., 2003). All nodes below 50% BS
(MP and ML, they are reported always in this order) and PP
were collapsed in the trees shown. We searched for deviations
of GC content in the nuclear ITS as a way to determine if se-
quences were subject to more extensive contraction/expansion
phenomena as can occur in regions with lower GC content
(Carels & Bernardi, 2000). We also searched for recombina-
tion events using the Maximum Chisquare test of Smith (1992)
implemented in RecombiTEST (Piganeau & al., 2004), a web-
based platform (http://www.lifesci.sussex.ac.uk/CSE/test/).
We displayed the extent of phylogenetic incongruence be-
tween phylogenetic trees with a galled network constructed
with Dendroscope v.3 (Huson & Scornavacca, 2012) using the
Bayesian majority consensus trees from the plastid and nrITS
analyses. The galled network was used to illustrate the differ-
ent phylogenetic positions of taxa in the two trees and to help
identify taxa with different positions.

Table 2. Summary of the alignments, including parsimony information, for the nuclear and plastid regions analyzed.
ITS1 ITS2 rpl32-trnL rps16-trnK trnD-trnT trnH-psbA trnQ-rps16 trnS-trnfM ycf1 SIC Total
Species 28 28 30 30 28 24 30 29 21 30 30
Var. char. (%) 53 (22.1) 44 (17.7) 120 (13.2) 48 (7) 69 (4.5) 24 (2.7) 155 (17.7) 78 (7.4) 50 (3.2) 204 641 (8)
PIC (%) 20 (8.3) 18 (7.2) 46 (4.7) 22 (3.2) 27 (1.8) 6 (0.7) 14 (1.6) 46 (4.3) 34 (1.6) 86 233 (2.9)
Length var. 227–233 242–245 748–902 589–641 1144–1371 580–810 279–846 802–1017 1482–1497 – 6642–7563
Matrix length 240 249 984 690 1518 872 876 1060 1497 – 7986
% gaps 2.9–5.4 1.6–2.4 5–18.8 7.4–14.9 9.7–24.6 7.1–33.5 3.4–68.2 4.1–5.9 0–1 – 5.3–16.5
% missing 7 6.8 0 0 8 21.7 0 3.7 30.1 – 11
GC content 54.6–60.5 63.6–67.8 22.9–25.7 29.8–31.3 28.2–30.4 33.7–36.6 25.7–33.5 33.3–35.3 26.8–28.1 – 30.2–34.8
Models (AIC) K80 + G HKY + G GTR + G GTR + G GTR + I + G HKY + I + G GTR + G GTR + G GTR + I – –
SIC = simple indel coding. Total = total values for aligned sequences without coded indels. Var. char. = variable characters in matrix. PIC = par-
simony informative characters. Length var. = length variation of sequences in matrix. % gaps = minimum and maximum percentage of gap char-
acters of the sequences from matrix. % missing = percentage of missing data in matrix, not counting gaps. GC content = minimum and maximum
percentage of GC content of sequences in matrix. Models (AIC) = models selected with AIC through jModelTest v.2.1.7.

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TAXON 65 (6) • December 2016: 1249–1262
Indel coding and analysis. — A large amount of variation
in the matrices was composed of indels (insertion-deletion
events) for various regions (see Table 2). We coded the indels
using the simple coding scheme of Simmons & Ochoterena
(2000), automatically coded and added to matrix data through
the SeqState v.1.4.1 platform (Müller, 2005). Indels were ana-
lyzed as standard data in MP, ML, and BI (using the default
models and settings for this kind of data). All these analyses
were completed using the same settings as described above for
each method. Preliminary tests for the phylogenetic relation-
ships of Laelia with and without coding indels showed a con-
flict in the position of L. halbingeriana Salazar & Soto Arenas.
Thus, we analyzed the matrices with and without this species
to evaluate how this exclusion affects support of other clades.
Hypothesis testing. — We tested different topologies using
both a Bayesian and a likelihood approach. The former con-
sisted of constrained and unconstrained BI analyses with the
same data and search parameters as used in the previously run
BI analyses; we also estimated marginal likelihoods with a BI
stepping stone analysis, a supposedly better likelihood estimate
for hypothesis testing under Bayesian analyses (Drummond
& Bouckaert, 2015); the stepping stone analysis consisted of
51 runs of one million generations each discarding the first
25% generations as burn-in. The likelihoods obtained were
then used to compute a Bayes factor. The likelihood approach
consisted of three different approaches of hypothesis test-
ing, Kishino-Hasegawa (KH; Kishino & Hasegawa, 1989),
Shimodaira-​Hasegawa (SH; Shimodaira & Hasegawa, 1999),
and Approximately Unbiased (AU; Shimodaira, 2002) tests;
we obtained the site individual likelihood values for each indi-
vidual topology through PAUP v.4.0b10 (Swofford, 2003) and
imported them into CONSEL v.1.20 (Shimodaira & Hasegawa,
2001) for all three analyses using default parameters.
RESULTS
Matrix description. —The nuclear ITS dataset was 489 bp
long (we excluded the sequenced fragments of 18S [partial],
26S [partial], and 5.8S to avoid uncertainties of sequencing for
initial and final fragments of the sequences [18S and 26S], and
to exclude a conserved and invariant region [5.8S] among se-
quences) and the aligned plastid (rpl32-trnL, rps16-trnK, trnD-
trnT, trnH-psbA, trnQ-rps16, trnS-trnfM intergenic spacers,
and the 3′ portion of the ycf1 gene) dataset was 7497 bp long.
The total number of variable characters from nrITS and plastid
matrices was 641 (233 of them parsimony informative). Some
sequences had large unique indels; the most striking was found
in L. speciosa in the trnQ-rps16 region with one single indel of
527 bp in length. The most important features of the aligned
sequences are described in Table 2. All analyzed sequences
of nrITS and plastid were generated for this study, except for
those indicated in Appendix 1. No recombination event was de-
tected in the nrITS sequences, and all species analyzed showed
no evidence of nrITS pseudogenes or polymorphism within
them (amplification and sequencing were straightforward).
The nrITS GC content (Table 2) ranged from 54.6% to 60.5%
Version of Record
Peraza-Flores & al. • Phylogeny of the Laelia alliance
in ITS1, and from 63.6% to 67.8% in ITS2 (common values as
compared to other species of Laeliinae). The GC content of
plastid regions varied from 22.9% to 36.6% (Table 2), similar
values when compared to the same DNA fragments from the
complete plastid genome of Cattleya crispata (Thunb.) Van den
Berg (Da Rocha Perini & al., 2015). The evolutionary models
selected by jModelTest for nuclear ITS1 and ITS2 were K80 + G
and HKY + G, respectively. The models for the plastid regions
were GTR + G for rpl32-trnL, rps16-trnK, trnQ-rps16, and
trnS-trnfM, GTR + I for the 3′ portion of ycf1, GTR + I + G for
trnD-trnT, and the HKY + I + G model for trnH-psbA (Table 2).
Phylogenetic analyses. — The trees of the MP, ML and
BI analyses were congruent at most internal clades with col-
lapsed nodes in the MP and ML topologies. The BI trees for
both the nuclear and plastid regions were best resolved, and
BS (MP and ML) and PP values are shown on these BI trees.
The main differences between topologies were the recognition
of both samples of L. (Sch.) gloriosa (Rchb.f.) L.O.Williams as
sister to each other with ML (76% BS), whereas MP and BI
recovered them as an unresolved clade with both samples of
L. (Sch.) undulata (Lindl.) L.O.Williams. Laelia (Sch.) moyo-
bambae (Schltr.) C.Schweinf. and L. (Sch.) marginata (Lindl.)
L.O.Williams were also recovered as sister lineages in the ML
analysis (88% BS), but this group was not present in the MP
and BI analyses. The MP analyses of the nuclear ITS and plas-
tid DNA regions produced 16 and 2 most parsimonious trees,
respectively (Table 3).
The topologies of all analyses agreed in the presence
of two clades. The first contains the long recognized group
of highland Mexican Laelia (clade A: L. albida Bateman ex
Lindl., L. autumnalis, L. crawshayana Rchb.f., L. eyermani-
ana Rchb.f., L. furfuracea Lindl., L. speciosa) (Electr. Suppl.:
Fig. S1). The other group (clade B) was composed of L. au-
rea, L. rubescens, L. anceps, and all the species historically
known as Schomburgkia (Electr. Suppl.: Fig. S1). The posi-
tion of L. (Sch.) superbiens was different in the nuclear DNA
analyses; it was recovered as sister to L. halbingeriana in a
clade with L. aurea and L. rubescens (BS < 50%, < 50%, PP
0.87) and not among those Laelia species traditionally classi-
fied as Schomburgkia as occurred in the plastid DNA (Electr.
Suppl.: Fig. S1). The hybrid species included in the analyses,
L. ×oaxacana, was recovered within the L. anceps clade using
both nuclear and plastid DNA (Electr. Suppl.: Fig. S1). Laelia
gouldiana Rchb.f. was recovered in either clade A (nuclear
DNA) in an unresolved clade with L. autumnalis f. xanthotropis
(Rchb.f.) Halbinger & Soto Arenas, L. crawshayana, and L. ey-
ermaniana or in clade B (plastid DNA) as sister of L. anceps
subsp. anceps. Laelia ×oaxacana, the supposed hybrid between
L. halbingeriana and L. anceps subsp. anceps was always re-
covered as sister of the latter, with plastid and nuclear DNA.
Plastid DNA analysis recovered a weakly (BS 59%, 57%)
or poorly (PP 0.80) supported Laelia s.l. Clade A was strongly
supported by BS (98%, 100%) and PP (1) values. Clade B was
moderately to weakly supported by BS (74%, 69%) but strongly
supported by PP (0.99). Three main internal clades were recov-
ered within clade B; the clade of L. aurea + L. rubescens was
strongly supported by BS (99%, 99%) and PP (1); the clade of
1253
Peraza-Flores & al. • Phylogeny of the Laelia alliance
Table 3. Summary of parsimony data and tree descriptions for the matrices analyzed.
Matrix Taxa V.C.
Plastid, no indels 30 349
ITS, no indels 28 59
Plastid, indels 30 467
ITS, indels 28 72
Plastid, L. halbingeriana excluded, no indels 29 338
ITS, L. halbingeriana excluded, no indels 27 56
Plastid,, L. halbingeriana excluded, indels 29 450
ITS, L. halbingeriana excluded, indels 27 71
V.C. = variable characters parsimony-uninformative. C.Is. = characters from indels. PIC = parsimony-informative characters. PIC I = parsimony-
informative characters from indels. Trees rec. = number of trees recovered.
L. anceps + L. gouldiana + L. ×oaxacana was strongly supported
by both BS (99%, 100%) and PP values (1); the clade of Lae-
lia (Sch.) gloriosa + L. (Sch.) undulata ​+ L. halbingeriana was
poorly or strongly supported by BS (<50%, 91%) and strongly
supported by PP (1) (Electr. Suppl.: Fig. S1).
Nuclear DNA analysis recovered a weakly (BS 53%, 65%,
PP 0.56) supported Laelia s.l. clade; clade A was poorly or mod-
erately supported by BS (< 50%, 78%) and strongly supported
by PP (1); clade B was moderately to strongly supported by BS
(74%, 89%) and strongly supported by PP (1); internal relation-
ships within clades A and B were in general less resolved than
in the plastid tree (Electr. Suppl.: Fig. S1). Laelia autumnalis f.
xanthotropis is more closely related to other species of Laelia
from clade A (BS 63%, 65%, PP 0.94) than to L. autumnalis
f. atrorubens (Gower) Halbinger. Laelia (Sch.) superbiens is
sister to L. halbingeriana within the clade of L. aurea and
L. rubescens (BS < 50%, < 50%, PP 0.87). The positions of
L. autumnalis f. xanthotropis and L. (Sch.) superbiens described
above were not recovered in the analysis of the plastid regions.
Indel analysis. — The coding of indel characters with the
simple coding scheme yielded 206 variable characters for the
plastid regions, 86 of them parsimony informative, and 26
variable characters for the ITS regions, 13 of them parsimony
informative (Table 3; Electr. Suppl.: Tables S1, S2). The results
for the MP, ML, and BI analyses including indels were similar
to the analysis without coding the indels but some clades had
higher PP support values in the indel coded analyses (Electr.
Suppl.: Fig. S2). A clade of L. albida + ​L. furfuracea was re-
covered in the MP analysis but collapsed in the ML analysis
(Electr. Suppl.: Fig. S2). The BI analysis without coded indels
recovered the same clade with poor PP (0.86) but with weak
BS (58%, 54%) support. The BI analysis with coded indels pro-
duced a different topology with L. albida sister to L. autumnalis
f. xanthotropis with high PP (0.97) but poor BS (< 50%). That
clade was sister to L. crawshayana with moderate PP (0.95);
this greater clade was sister to L. autumnalis f. atrorubens with
moderate PP (0.97). Laelia furfuracea was sister to all of these
species with high PP (0.99) and poor to weak BS (< 50%, 69%).
Notably, L. halbingeriana was sister to all Laelia species that
had been classified as Schomburgkia (BS < 50%, < 50%, PP
0.78) and it was not recovered as a clade with the accessions
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TAXON 65 (6) • December 2016: 1249–1262
C.Is. PIC PIC I Tree length Trees rec.
– 195 – 699 2
– 38 – 129 16
206 281 86 964 1
26 51 13 171 15
– 193 – 632 2
– 38 – 126 16
197 278 85 906 1
26 49 11 166 42
of L. (Sch.) gloriosa and L. (Sch.) undulata. The ML analysis
recovered this species in a position identical to that when indels
were not coded (trees not shown).
The analyses of the nuclear data with indels coded were
almost identical to the analyses without indels coded but sup-
port values were slightly different (Electr. Suppl.: Figs. S1, S2).
Furthermore, Laelia aurea and L. rubescens were recovered
as sister species with weak and moderate BS (64%, 71%) and
weak PP (0.88) support. Laelia halbingeriana was recovered as
sister to L. (Sch.) superbiens with strong BS (93%, 98%) and PP
(1) support. Laelia (Sch.) splendida (Schltr.) L.O.Williams was
recovered as sister to L. (Sch.) gloriosa, L. (Sch.) elata (Schltr.)
J.M.H.Shaw, L. (Sch.) lueddemannii (Prill.) L.O.Williams, and
L. (Sch.) undulata with strong BS (85%, 98%) and PP (1) sup-
port. The same group excluding L. (Sch.) splendida was re-
covered with weak and moderate BS (56%, 74%), and poor PP
(0.54) support.
Phylogenetic analyses without Laelia halbingeriana. — The
MP, ML, and BI analyses of plastid and nuclear DNA without
coding indels and excluding L. halbingeriana produced the same
general clade topologies as those including this species. How-
ever, there was a little improvement of the BS and PP values of
most clades (Electr. Suppl.: Fig. S3). Laelia s.l. (clades A and B)
and the internal clades within clade B had better BS values than
in the analysis without coding indels and including L. halbin-
geriana (Electr. Suppl.: Figs. S1, S3). In the analysis of plastid
regions some clades, such as the Laelia albida + ​L. furfuracea
clade with weak BS (58%, 83%) and weak PP (0.91) support
were recovered; Laelia s.l. had weak BS (59%, 59%) and PP
(0.90) support; clade A had strong BS (99%, 100%) and PP (1)
support; clade B had moderate BS (75%, 70%) and strong PP
(0.99) support; the clade of Laelia (Sch.) had strong BS (89%,
91%) and PP (1) support (Electr. Suppl.: Fig. S3). In the nrITS
analyses there was a clade consisting of L. autumnalis f. xan-
thotropis together with L. crawshayana, L. eyermaniana, and
L. gouldiana with lower support in the MP and ML analyses
(BS 61%, 66%) but with unchanged and weak PP (0.94) support.
Indel analyses without Laelia halbingeriana. — This set
of analyses produced the same general topologies as those in-
cluding L. halbingeriana. The BS and PP values for various
clades were higher when compared to the same set of analyses
TAXON 65 (6) • December 2016: 1249–1262 Peraza-Flores & al. • Phylogeny of the Laelia alliance

including L. halbingeriana (Fig. 1; Electr. Suppl.: Fig. S2).
Other support values for some clades were lower than in the
analyses with the full taxa set, for both nuclear and plastid
DNA (Electr. Suppl.: Figs. S1, S3). In the analysis of plastid re-
gions, Laelia autumnalis f. xanthotropis was sister to L. albida
with poor BS (< 50%, < 50%) but moderate PP (0.95) support;
L. autumnalis f. atrorubens is placed in a clade with L. craw-
shayana, L. autumnalis f. xanthotropis and L. albida with poor
BS (< 50%, < 50%) and moderate PP (0.95) support (Fig. 1). In
the nrITS analyses L. autumnalis f. xanthotropis appeared in
the same position with weak BS (63%, 64%) and moderate PP
(0.95) values as in the analyses without indel coding (Fig. 1).
Parsimony analysis and indel coding. — There were con-
trasting results when indels were coded in the MP analyses; the
support values in plastid analyses were improved while those
of nrITS analyses were lower (Fig. 1; Table 3; Electr. Suppl.:
Figs. S1–S3). We found the most striking differences between
coded and non-coded indels when we excluded L. halbingeri-
ana from the analyses, mainly in internal relationships of the
Laelia (Schomburgkia) clade in the plastid phylogenetic trees
(Fig. 1; Electr. Suppl.: Figs. S1–S3).
Galled network. — The network tree highlighted the dis-
cordant positions of some taxa between the plastid and nrITS
phylogenetic trees; most evident were the relationships between
the taxa of the Laelia (Schomburgkia) clade, the L. anceps
clade, and the L.  rubescens + L. aurea clade. Furthermore,
L. gouldiana is recovered in either clade A or clade B (Fig. 2).
Hypothesis testing. — We tested if the clade of L. rube-
scens + L. aurea is a member of clade A or clade B because
of the weak to moderate support for its position in the MP,
ML, and BI analyses. We constrained the analysis so that this
clade would be considered to be a member of clade A; the
marginal likelihood of the analysis of plastid data (−14036.54)
was 4.48 log units worse than that of the unconstrained analy-
sis (−14032.06). Furthermore, we computed marginal likeli-
hoods from a Bayesian stepping stones (BSS) analysis using
the same data to compare results; the marginal likelihood of
the constrained model was −14268.51, 7.93 log units worse
than that of the unconstrained analysis (−14260.58). The mar-
ginal likelihood from the constrained analysis of nrITS data
(−1467.47) was 20.23 log units worse than the unconstrained
model likelihood (−1447.24). The marginal likelihoods from the
BSS were similar; the constrained model (−1537.36) was 18.75
log units worse than the unconstrained model (−1518.61). Kass
& Raftery (1995) argued that a difference of 3–5 log units is
strong evidence while a difference larger 5 log units is regarded
as very strong evidence favoring the better model.
We also computed the KH, SH, and AU tests to assess if
the L. rubescens + L. aurea clade is a member of clade A or
clade B. We used the trees from the above Bayesian analyses

Plastid ITS
Outgroup

Homalopetalum pachyphyllum Homalopetalum pachyphyllum

Dinema polybulbon Broughtonia sanguinea */*

Broughtonia sanguinea Dinema polybulbon 0.64

*/*
*/*
Laelia albida
0.93
0.95
Laelia albida
*/* Laelia autumnalis f. xanthotropis Laelia autumnalis f. xanthotropis
0.95
Laelia crawshayana Laelia crawshayana
Clade A

* /75
63/64
0.95
Laelia autumnalis f. atrorubens
99/100 Laelia eyermaniana 0.95
*/73
0.85 1 Laelia furfuracea Laelia gouldiana 55/87
100/100 Laelia eyermaniana 1
1 Laelia autumnalis f. atrorubens
Laelia speciosa
Laelia

100/100
Laelia furfuracea
1
Laelia speciosa ‘Paget’ Laelia speciosa 91/99
99/99 Laelia aurea ‘Paget’ Laelia speciosa 1
72/58 1 Laelia rubescens Laelia anceps subsp. anceps
0.95 68/80 63/76
0.98 Laelia anceps subsp. anceps Laelia ×oaxacana 0.99 */80
*/63
0.57
91/96
1 Laelia gouldiana Laelia anceps subsp. dawsonii
0.96

99/100
1 Laelia ×oaxacana Laelia aurea 65/70
62/67
0.99
Laelia anceps subsp. dawsonii Laelia rubescens 0.90
*/ *
Laelia (Sch.) gloriosa (ES) Laelia (Sch.) superbiens (W) 100/100
0.55
Clade B

†3, 130,
144, 205
99/100
1
Laelia (Sch.) gloriosa Laelia (Sch.) superbiens 1 68/86
1
59/* 82/100 Laelia (Sch.) undulata 2 Laelia (Sch.) gloriosa (ES)
Schomburgkia

*/78
0.93
84/85
1
Laelia (Sch.) undulata Laelia (Sch.) gloriosa
1
91/99
1
Laelia (Sch.) moyobambae Laelia (Sch.) elata */76
1 Laelia (Sch.) marginata Laelia (Sch.) lueddemannii 0.57
89/97
1 Laelia (Sch.) elata Laelia (Sch.) undulata 85/98
1
*/*
0.94 Laelia (Sch.) lueddemannii Laelia (Sch.) undulata 2 53/73
99/100
1 Laelia (Sch.) lyonsii Laelia (Sch.) splendida 0.98

82/93 Laelia (Sch.) splendida Laelia (Sch.) lyonsii

1
99/100 Laelia (Sch.) superbiens (W)
1
Laelia (Sch.) superbiens
0.0050 0.0050

Fig. 1. 50% majority-rule trees of the Laelia alliance from Bayesian analysis of plastid and nuclear datasets with indels coded and excluding Laelia
halbingeriana. Maximum parsimony and maximum likelihood support values are indicated above, and Bayesian posterior probability values
below branches. The traditional classification of Laelia and Schomburgkia is highlighted with light gray bars. The circumscription adopted here
is indicated with dark gray bars. Putative hybrids are marked with small light gray arrows. Asterisks indicate support values below 50% BS or 0.5
PP. Long branches were trimmed (a double slash on the branch) to fit trees into a single readable figure. † Indels supporting the clade. Pictures by
GC, line drawings by LP.

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Peraza-Flores & al. • Phylogeny of the Laelia alliance TAXON 65 (6) • December 2016: 1249–1262

(constrained and unconstrained topologies) and the original DISCUSSION

matrices to estimate the site-wise log-likelihoods of trees in
PAUP. The results from the CONSEL analyses are summarized The results of all analyses were, in general, congruent with
in Table 4. The AU test with plastid data supported the hypoth- topologies of previous analyses that included species of the
esis of L. rubescens + L. aurea belonging to clade B (p-value Laelia alliance (Van den Berg & al., 2009). However, most
= 0.971); the other tests had smaller p-values (0.904 for both internal relationships recovered here are new due to the more
KH and SH) for this hypothesis. The tests for the nrITS data comprehensive sampling relative to previous studies (Van den
had higher p-values than those for the plastid data for the same Berg & al., 2000, 2009). Laelia speciosa was recovered as sister
hypothesis (AU: 0.978, KH: 0.953, SH: 0.953). to all other Laelia s.str. (clade A) as previously reported from
molecular data but discordant with the result of a morpho-
logical phylogeny where it was recovered as the most derived
Table 4. CONSEL output for for the position of the Laelia rubescens + ​
Laelia aurea clade. species (Halbinger & Soto, 1997; Van den Berg & al., 2000,
2009). Internal relationships within Laelia are more difficult to
Matrix Rank Tree AU NP BP PP KH SH
compare with other phylogenies as the trees of those investiga-
Plastid 1 2 0.971 0.932 0.930 1.000 0.904 0.904 tions consisted mostly of unresolved clades (Halbinger & Soto,
2 1 0.029* 0.068 0.070 2e−04 0.096 0.096 1997; Van den Berg & al., 2000) or included too few species
ITS 1 2 0.978 0.972 0.971 1.000 0.953 0.953 to unambiguously assess the internal relationships (Van den
Berg & al., 2009). The same holds for clade B; the topologies
2 1 0.022* 0.028* 0.029* 5e−08 0.047* 0.047*
included a grade with L. rubescens as sister to all other species
AU = p-value of the approximately unbiased test; NP = bootstrap in the clade (Van den Berg & al., 2009) or unresolved clades
probability; BP = bootstrap probability from replicates; PP = Bayesian with no clear relationships (Van den Berg & al., 2000). Despite
posterior probability; KH = p-value of the Kishino-Hasegawa test; SH
= p-value of the Shimodaira-Hasegawa test. Tree: 1 = Laelia rubes-
the smaller sampling in those studies, the placement of L. an-
cens + Laelia aurea sister to all other Laelia s.str.; 2 = Laelia rubescens ​ ceps and L. rubescens in the Schomburgkia clade instead of
+ Laelia aurea sister to all other Laelia (Schomburgkia). * significant in Laelia s.str. was recovered unambiguously in Van den Berg
at the 0.05 level. & al. (2000, 2009).

Fig. 2. Galled network from the Clade A

trees of the Bayesian analysis
La

of plastid and nrITS sequences.

eli

Light gray branches lead to taxa

aa

La
Laelia ey

el
Laelia
utu

which are incongruently placed in ia

na

au

s
mn
La

ldia

the analyses of the two datasets.

ep
La tu
el

m
nc
ali

eli na
ia

albida

gou

.a
s f sha

er

af
Laelia speciosa ur lis f
cr

sp
m
aw
. a ya

fur . x
lia

ub

ii
an

ac an
tro n

on
Lae

ea th
ss

ws )
ia
rub a

ot n a a
na

ep

ro ca . d (W
en

Laelia pi
xa bsp ns
nc

specio s
oa su rbie iens
s

sa ‘Pag
aa

et’ × s e b
ia ep up er
eli

el anc .) s sup
La

a
L lia ch .)
e (S ch
La elia a (S
Homalopetalum pachyphyllum i
La ael
L
Laelia (Sch.) lueddemannii
Dinema polybulbon Laelia (Sch.) elata
L L La
La ael aeli elia
el ia a ( (S
ia (S S c
(S ch ch h.
s ch .) .) u ) u
en .) glo nd nd
esc gl ri u ul
Laelia (Sch.) lyonsii

rub
Laelia (Sch.) marginata

or os la at
a
re

a io a ta a
eli
a

Broughtonia sanguinea sa (E 2
au

moyobambae
did

La S)
ia

len
el
La

sp

Outgroup
h.)

a
Sc

rian
a(

1.0E-4 Clade B
e

Laelia (Sch.)
eli

ing
La

alb
lia h
Lae

1256 Version of Record

TAXON 65 (6) • December 2016: 1249–1262 Peraza-Flores & al. • Phylogeny of the Laelia alliance

The support values from the MP and ML analyses differed
substantially from those of the BI analyses. Support values
from MP and ML analyses were in general weak or moderate,
a behavior of clade support already reported by Erixon & al.
(2003); furthermore, it has been suggested that BS values of
70% correspond to a 95% accuracy (“true topology”) of clades
(Hillis & Bull, 1993). BI posterior probabilities can underesti-
mate or, in some cases overestimate, the support of some clades,
but they are thought to yield better results when considering the
relation between accuracy and support than bootstrap-based
methodologies (Alfaro & al., 2003); however, there is evidence
that Bayesian PP could be overestimating the support of clades
(Suzuki & al., 2002; Cummings & al., 2003; Simmons & al.,
2004). Our taxonomic and nomenclatural proposal is based
upon these criteria of clade support, considering only those
clades with high clade support for both BS and PP. However,
the cases when poor PP support was predominant were few
(Fig. 1; Electr. Suppl.: Figs. S1–S3).
The analyses with the coding of indels improved the sup-
port of some clades and the internal resolution in both clade
A and B. This is true for ML and BI, but MP did not recover
fully resolved internal relationships when indels were coded;
the MP analyses were in general less resolved when the indels
were coded (Fig. 1; Electr. Suppl.: Figs. S1–S3).
Clade A, equivalent to Laelia s.str., is characterized by
short rhizomes, fusiform, non-laterally compressed pseudo-
bulbs, a racemose inflorescence bearing bracts shorter than
pedicels and internodes, and flowers with sepals and petals
entire and not undulate (see Halbinger & Soto, 1997); this clade
occurs at middle to high elevations in tropical or semitropi-
cal habitats (Table 5). The placement of L. gouldiana within
clade A with nuclear and within clade B with plastid DNA is ev-
idence of a putative hybrid origin as suggested by Reichenbach
(1888) but not confirmed previously (Halbinger & Soto, 1997).
The putative parental species are L. anceps for the maternal line
and L. autumnalis for the paternal line. However, we cannot
discard the possibility that other events were responsible for
the patterns here observed. Events such as incomplete lineage
sorting are difficult to distinguish from hybridization and we
did not explore this possibility more profoundly with appropri-
ate methodologies.
Species in clade B, Laelia (Sch.) + L. anceps (and allies) + ​
L. rubescens and L. aurea, are characterized by long rhizomes,
elongated pseudobulbs and long flower bracts, at least for the
previously known species of Schomburgkia (e.g., Sch. crispa
and Sch. lyonsii) and to a smaller degree for L. anceps (and close
relatives). Laelia rubescens and L. aurea are morphologically
aberrant within this clade; although they possess somewhat
elongated rhizomes, their flower morphology and the small
flower bracts are similar to those of species of clade A. All
members of this clade have laterally compressed pseudobulbs,
either strongly (as in L. rubescens and allies) or moderately (as
in all other members of clade B). The floral similarities with
other species of Laelia could be explained by the preserva-
tion of plesiomorphic characters; additionally, both species,
as a group, have a unique character, compressed pseudobulbs
that separates them from other Laelia s.str. (Halbinger & Soto,
1997). The placement of these species within clade B will be
discussed below.
Taxonomic rearrangements. — We (LP and GC) argue
that in order to obtain a classification resulting in monophy-
letic, morphologically coherent and easily diagnosable taxa, we
should treat clades A and B as separate genera. We also make
other new combinations in both groups, all of them formally
transferred below. Based on similar molecular results, Van
den Berg & al. (2000, 2009) and Soto & al. (2006) preferred

Table 5. Summary of characters of Laelia and Schomburgkia.

Character Laelia Lindl.
Pseudobulb cross-section Round
Arrangement of flowers on rachis Laxly arranged, individual pedicels shorter than
the rachis
Floral bracts Triangular to triangular elliptic, inconspicuous,
always much shorter than pedicels and internodes
of rachis.
Bracts of the lower half of inflorescence Usually shorter than internodes
Elevation range Mostly high elevation, usually above 2000 m;
most species withstand light frost
Distribution Restricted to the Mexican Plateau west and north
of the Tehuantepec Isthmus
Flowering season During the peak or towards the end of the rainy
season (more rarely in May as in L. speciosa)
Schomburgkia Lindl.
Always somewhat compressed laterally
Crowded to subumbelliform, individual pedicels
longer than the rachis
Linear to linear elliptic, always exceeding half the
pedicel length, longer than internodes of rachis;
inconspicuous and rarely subequal in the Schom-
burgkia rubescens comb. nov. complex
Exceeding the internodes
Mostly low elevation, usually below 1500 m; most
species cannot withstand any frost
South America, Central American Isthmus, and
Megamexico east and south of the Tehuantepec
Isthmus; Schomburgkia anceps comb. nov. also at
intermediate elevations in Mexico along the Sierra
Madre Oriental and the Sierra Madre Occidental
During the peak of the dry season (February–May
in the Northern Hemisphere). Schomburgkia
anceps comb. nov. in autumn to spring

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Peraza-Flores & al. • Phylogeny of the Laelia alliance
to keep both clades in an expanded circumscription of Laelia
including Schomburgkia, instead of recognizing L. anceps and
L. rubescens as members of Schomburgkia. If we chose to
keep an expanded Laelia, more than 60% of its species would
be different from the typical Laelia morphology. Furthermore,
we would be neglecting the phylogenetic information and the
molecular evidence explained in detail below.
We will not elaborate on the delimitation of Laelia (defined
as the high-elevation Mexican Laelia species) as a separate
genus since this is a group that is morphologically, geographi-
cally (Halbinger & Soto, 1997), and phylogenetically distinct
(Van den Berg & al., 2000, 2009; Soto & al., 2006; this study)
from Schomburgkia. The delimitation of the latter would be
controversial for some due to the placement of L. anceps (and
allies) and L. rubescens (and allies) within it. However, this
circumscription results in a monophyletic, coherent taxon
characterized by morphological and biogeographic features
(Table 5). Laelia anceps and allies have overall morphological
similarities with core Schomburgkia, as stated above; Lae-
lia rubescens and allies are apparently intermediate between
Laelia and Schomburgkia by having a somewhat elongated
rhizome as in the latter but sharing floral similarities with
Laelia. In addition, L. rubescens and allies feature strongly
compressed pseudobulbs and evanescent flowers arranged in
long-peduncled, sub-umbellate inflorescences; furthermore,
the flowers have a labellum with a pubescent throat (except.
for L. rubescens var. peduncularis (Lindl.) Halb.) and a small
column (Table 5). The phylogenetic analysis of molecular data
provides evidence that this group should be kept within Schom-
burgkia. The phylogenetic topologies recovered from nuclear
evidence (ITS) show a possible relationship of Sch. superbiens
with L. rubescens, albeit with weak BS and PP support; this
may be related to past or present introgression of DNA between
them. Phylogenetic analysis of plastid DNA grouped L. rube-
scens and L. aurea as sister to L. anceps (and allies) and core
Schomburgkia. Molecular sequence changes seem to character-
ize an expanded Schomburgkia as a unit; all species have a 9 bp
insertion (TGATACCGC) in the 3′ partial portion of the ycf1
gene; a 14 bp fragment (TAAAGTATATGTAT), a 8 bp deletion
(TAATAAAT), and a 6 bp deletion (TAGTAC) in the trnD-
trnT ; and, a 9 bp deletion (CAGGTTCAT) in trnH-psbA; the
latter seems to have undergone a duplication in all other Laelia
species except for L. eyermaniana and L. speciosa. Additional
single-nucleotide synapomorphies support the separation of
Laelia from Schomburgkia. These shared molecular characters
provide evidence that L. rubescens (and allies) should be kept
within Schomburgkia and not as a separate genus. Furthermore,
our analyses of hypothesis testing provided very strong sup-
port or strong support for this conclusion from both Bayesian
analyses and the AU test; the KH and SH tests yielded less
strong support but still had high values of support for the same
hypothesis.
On the other hand, CvdB considers that no generic taxo-
nomic change from a broadly defined Laelia would be needed
since the topologies found for Laelia anceps and L. rubescens
are virtually the same as found in his previous analyses based
on ITS, matK and trnL-F (Van den Berg & al., 2000, 2009). In
1258 Version of Record
TAXON 65 (6) • December 2016: 1249–1262
this case more DNA regions only confirmed previous results,
which are fully compatible with a broadly defined Laelia, and
the need for resurrecting Schomburkgia is based in taxonomic
opinion rather than new results.
Recently, Archila & Szlachetko (2014) proposed that
L. rubescens and L. aurea were sufficiently different from the
rest of Laelia to merit generic status, for which they described
Encabarcenia Archila & Szlach. We argue that they simply
stressed some morphological peculiarities present in these two
species, but that this group does not deserve generic status.
Additionally, the recognition of this genus would make the
concept of Schomburgkia here proposed (or even a broader
circumscription of Laelia) non-monophyletic, or else would
require the creation or recognition of further genera, making
the nomenclature of the Laelia alliance unnecessarily compli-
cated. Encabarcenia, albeit morphologically distinctive, shares
with Schomburgkia, as here circumscribed, the compressed
pseudobulbs, and the long-peduncled and sub-umbellate in-
florescences bearing short-lived flowers. It is also biogeo-
graphically congruent with Schomburgkia in that it inhabits
low-elevation environments and is absent from the Mexican
highlands (Table 5).
New combinations
× Schombolaelia gouldiana (Rchb.f.) Peraza & Carnevali,
comb. nov. ≡ Laelia gouldiana Rchb.f. in Gard. Chron.,
ser. 3, 3: 41. 1888 – Holotype: a cultivated plant, Dec 1887,
Sander s.n. (W).
Parentage: Schomburgkia anceps × Laelia autumnalis
Schomburgkia anceps (Lindl.) Peraza & Carnevali, comb. nov.
≡ Laelia anceps Lindl. in Edwards’s Bot. Reg. 21: t. 1751.
1835 (“1836”) ≡ Amalias anceps (Lindl.) Hoffmanns. in
Linnaea 16: 228. 1842 ≡ Cattleya anceps (Lindl.) Beer in
Prakt. Stud. Orchid.: 208. 1854 ≡ Bletia anceps (Lindl.)
Rchb.f., Xenia Orchid. 2: 47. 1862 – Holotype: [illustration
in] Edwards’s Bot. Reg. 21: t. 1751. 1835.
Schomburgkia aurea (A.V.Navarro) Peraza & Carnevali,
comb. nov. ≡ Laelia aurea A.V.Navarro in Orquídea
(México City), n.s., 12(1): 42. 1990 ≡ Laelia rubescens var.
aurea (A.V.Navarro) M.Wolff & O.Gruss, Orchid. Atlas:
183. 2007 ≡ Encabarcenia aurea (A.V.Navarro) Archila &
Szlach. in Revista Guatemalensis 17(2): 27. 2014 – Holo-
type: Mexico, Nayarit, Las Coloradas, E. Hágsater 450
(AMO; isotype: K).
Schomburgkia dawsonii (J.Anderson) Peraza & Carnevali,
comb. nov. ≡ Laelia anceps var. dawsonii J.Anderson in
Gard. Chron. 1868: 27. 1868 ≡ Laelia dawsonii (J.Anderson)
De B.Crawshay in Gard. Chron., ser. 3, 32: 414. 1902 ≡
Laelia anceps subsp. dawsonii (J.Anderson) Rolfe in Orch.
Rev. 30: 10. 1922 – Lectotype (designated by Soto Arenas
in Orquídea (Mexico City) 13: 133. 1993): [illustration]
“Laelia anceps Dawsoni” in Warner, Select Orchid. Pl.
2: t. 34. 1865.
TAXON 65 (6) • December 2016: 1249–1262
Schomburgkia halbingeriana (Salazar & Soto Arenas) Peraza
& Carnevali, comb. nov. ≡ Laelia halbingeriana Salazar
& Soto Arenas in Phytotaxa 178(3): 162. 2014 – Holotype:
Mexico, Oaxaca, Portillo de Coyula, unos 2 km antes del
poblado de Coyula subiendo desde Quiotepec, donde la
línea eléctrica cruza el camino, 1160 m elevation, 11 Jul
2004, pressed in cultivation 17 Oct 2006, Salazar & al.
6695 (MEXU).
Schomburgkia mottae (Archila, Chiron, Szlach. & Pérez-
García) Peraza & Carnevali, comb. nov. ≡ Laelia mottae
Archila, Chiron, Szlach. & Pérez-García in Bot. Sci. 92 (3):
345. 2014 – Holotype: Guatemala, Chiquimula, Esquipu-
las, alt. ca. 400 m, bosque tropical caducifolio, Sep 2010,
F. Archila s.n. (BIGU).
Schomburgkia ×oaxacana (Salazar & R.Jiménez) Peraza &
Carnevali, comb. nov. ≡ Laelia ×oaxacana Salazar &
R.Jiménez. in Phytotaxa 178(3): 167. 2014 – Holotype:
Mexico, Oaxaca, Barranca del Río Santo Domingo, muy
al E de Tecomavaca, cerca de Buenos Aires (entre Coyula
y el río Santo Domingo), ca. 1600–1700 m elev., Jun 1986,
fl. in cult. 4 Nov 1991, Lau sub Hágsater 9539 (AMO;
isotypes: MEXU, SERO).
Schomburgkia perezgarciae (Archila & Szlach.) Peraza &
Carnevali, comb. nov. ≡ Encabarcenia perezgarciae
Archila & Szlach. in Revista Guatemalensis 17(2): 27.
2014 – Holotype: Guatemala, Suchitepequez, Mazat-
enango, sobre árboles de Tabebuia rosea (Bert.) DC, Jun
1999, F. Archila s.n. (BIGU).
Schomburgkia rubescens (Lindl.) Peraza & Carnevali, comb.
nov. ≡ Laelia rubescens Lindl. in Edwards’s Bot. Reg. 26:
t. 41. 1840 ≡ Cattleya rubescens Beer, Prakt. Stud. Orchid.:
208. 1854 ≡ Bletia rubescens Rchb.f. in Walpers, Ann. Bot.
Syst. 6: 425. 1862 – Holotype: Probably from Mexico, Mr.
Barker, imported by Mr. J. Knight (K).
Schomburgkia rubescens f. peduncularis (Lindl.) Peraza &
Carnevali, comb. nov. ≡ Laelia peduncularis Lindl. in
Edwards’s Bot. Reg. 28: t. 9. 1842 ≡ Cattleya peduncu-
laris (Lindl.) Beer in Prakt. Stud. Orchid.: 213. 1854, nom.
inval. ≡ Bletia peduncularis (Lindl.) Rchb.f. in Walpers,
Ann. Bot. Syst. 6: 426. 1862 ≡ Laelia rubescens f. pedun-
cularis (Lindl.) Halb. in Orquidea (Mexico City) 13: 294.
1993 ≡ Encabarcenia rubescens f. peduncularis (Lindl.)
Archila & Szlach. in Revista Guatemalensis 17(2): 26. 2014
– Holotype: A mexican plant flowered in Birmingham,
Geo. Barker s.n. (K).
The evidence of this molecular analysis, morphological and
geographic data detailed in Halbinger & Soto (1997), Archila &
al. (2014), and Salazar & al. (2014) provides evidence that the
previously known subspecies of L. anceps are following differ-
ent evolutionary trajectories. We propose that the populations
from the mountains of the Gulf of Mexico slopes should be
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Peraza-Flores & al. • Phylogeny of the Laelia alliance
regarded as Sch. anceps, the populations from the mountains
in the Pacific slope of Mexico should be considered Sch. daw-
sonii, and the populations from Chiapas (Mexico), Guatemala,
and Honduras should be recognized as Sch. mottae. We were,
unfortunately, unable to sample Sch. mottae but we consider,
based on morphology, phenology and geographic distribu-
tion, that it should be expanded to include those populations
from Chiapas previously included as part of Sch. anceps (as
L. anceps subsp. anceps) by Halbinger & Soto (1997). Thus, we
propose to treat them as different species and we transfer them
to Schomburgkia following the logic stated above. Schomburg-
kia dawsonii has a 16 bp deletion (TATTAGTATTAGTATA)
in the trnH-psbA intergenic spacer, a 5 bp deletion (TATAT)
in the rpl32-trnL intergenic spacer, a 19 bp insertion (TAGT​
TACTAATTTAGTACA) in the trnD-trnT intergenic spacer,
a 9 bp insertion (TTTTTTACA) in the trnS-trnfM intergenic
spacer (compared to Schomburgkia anceps); these are the most
obvious sequence differences between them.
Hybridization seems to have played an important role in
the evolution of both genera. There are several hypothesized
hybrids, including ×Schombolaelia gouldiana, a natural hybrid
between Laelia and Schomburgkia (Fig. 2); the age of this hy-
bridization event is unknown, but probably not recent because
the nrITS regions display no polymorphisms characteristic of
F1 or recent hybrids (Baldwin, 1992; Baldwin & al., 1995). A
similar pattern in nrITS is found in Sch. ×oaxacana whose
plastid and nuclear DNA are closely related to Sch. anceps;
however, it has the morphological characters of Sch. halbin-
geriana; the fixation of one single copy of nuclear ITS is a
common result of concerted evolution (Baldwin, 1992; Baldwin
& al., 1995). However, we cannot discard another cause for the
patterns of relationships among these species; lineage sorting
is difficult to distinguish from hybridization and cannot be
fully discarded here due to the absence of more exhaustive
tests. The position of Sch. halbingeriana is uncertain; when
we treated indels as “missing” data it was recovered as sister
to Sch. undulata and Sch. crispa, but when we coded the indels
it was recovered as sister to all other species of Schomburgkia
(in the traditional sense). It was also recovered with different
relationships when plastid or nrITS sequences were analyzed.
We cannot conclude if this behavior is the result of hybridiza-
tion or incomplete lineage sorting. When excluded from the
analyses, several nodes had higher support values in the plastid
analyses. nrITS data did not result in good resolution and prob-
ably other nuclear data would contribute to solving this open
question. The clade containing L. halbingeriana or the sister
clade had weak BS and PP values when using plastid DNA;
when we used the nuclear regions we noticed that it was always
recovered as sister taxon of Sch. superbiens in a clade distinct
from the remaining Schomburgkia (in the old sense); which
seem to conserve a nuclear relationship with Sch. rubescens
and Sch. aurea or show evidence of introgression from one of
these species; this is difficult to assess at this time. When we
removed Sch. halbingeriana from the analyses, the support
values for related clades improved significantly in the plastid
ML and BI analyses.
1259
Peraza-Flores & al. • Phylogeny of the Laelia alliance
ACKNOWLEDGEMENTS
The first author is grateful to Juliana Freitas, Juliana Santos,
Michella Teixeira, Ricardo Vilas-Boas, and Evandro Santos for train-
ing in the laboratory. The authors thank Gerardo A. Salazar, Eric
Hágsater, Luis M. Sánchez, and Rolando Jiménez for plant material
and/or helpful discussions on orchid taxonomy, variation, and evolu-
tion. They also kindly hosted the first author at MEXU and AMO
herbaria when analyzing their orchid collections. We are grateful to
Dr. Lynn Clark, Dr. Michael Pirie, and four anonymous reviewers for
comments that greatly improved the manuscript. Authors are grateful
to FAPESB and CNPq for financial support through a research grant
(PRONEX grant PNX0014/2009). Finally, LNPF thanks Consejo Na-
cional de Ciencia y Tecnología (CONACyT) for two grants (204216
and 239018) to fulfill a postdoctoral fellowship during two years at
Universidade Estadual de Feira de Santana, and CvdB thanks CNPq
for a productivity scholarship (PQ-1B).
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Appendix 1. List of taxa included in the phylogenetic analyses with voucher information and GenBank accession numbers (nrITS, rpl32-trnL, rps16-trnK,
trnD-trnT, trnH-psbA, trnQ-rps16, trnS-trnfM, the 3′ portion of ycf1). Sequences not generated in this research are marked with *.
Broughtonia sanguinea R.Br., cult., Brieger Coll. 14440 (ESA), AF260186*, KR908830, KR908956, –, KR908825, KR908990, KR908903, –; Dinema
polybulbon Lindl., México, Brieger Coll. 6052 (ESA), AF260154*, KR908832, KR908957, KR908871, –, KR908991, KR908904, –; Homalopetalum
pachyphyllum (L.O.Williams) Dressler, México, M.A. Soto Arenas 7640 (AMO), AF260155*, KR908834, KR908958, –, KR908808, KR908992, KR908905,
–; Laelia albida Bateman ex Lindl., México, M.W. Chase 6407 (K), KR816305, KR908836, KR908959, KR908872, KR908811, KR908993, KR908906,
KR908935; Laelia anceps subsp. anceps Lindl., México, R. Jimenez 111 (AMO), KR816306, KR908837, KR908960, KR908873, KR908821, KR908994,
KR908907, KR908936; Laelia anceps subsp. dawsonii (J.Anderson) Rolfe, México, M.A. Soto Arenas 7426 (AMO), KR816304, KR908839, KR908963,
KR908876, KR908824, KR908997, KR908910, KR908937; Laelia aurea A.V.Navarro, México, M.A. Soto Arenas 8690 (AMO), KR816307, KR908841,
KR908964, KR908877, KR908827, KR908998, KR908911, KR908938; Laelia autumnalis f. xanthotropis (Rchb.f.) Halb. & Soto Arenas, México,
R. Jimenez 2665 (AMO), KR816308, KR908843, KR908966, KR908878, KR908813, KR908999, KR908912, KR908939; Laelia autumnalis f. atro-
rubens (Backh.) Halb., México, M.W. Chase 6406 (K), KR816309, KR908842, KR908967, KR908879, KR908814, KR909001, KR908913, KR908940;
Laelia crawshayana Rchb.f., México, M.A. Soto Arenas 8065 (AMO), KR816310, KR908845, KR908968, KR908880, KR908816, KR909002, KR908914,
KR908941; Laelia eyermaniana Rchb.f., México, M.A. Soto Arenas 8912 (AMO), KR816311, KR908846, KR908969, KR908881, KR908817, KR909003,
KR908915, KR908942; Laelia furfuracea Lindl., México, M.W. Chase 6410 (K), KR816312, KR908847, KR908970, KR908882, KR908812, KR909004,
KR908916, KR908943; Laelia gouldiana Rchb.f., México, M.W. Chase 6408 (K), KR816313, KR908848, KR908971, KR908883, KR908820, KR909005,
KR908917, KR908944; Laelia halbingeriana Salazar & Soto Arenas, México, G.A. Salazar 6740 (AMO), KR816314, KR908849, KR908972, KR908884,
KR908823, KR909006, KR908918, KR908945; Laelia rubescens Lindl., México, R. Jimenez 2562 (AMO), KR816316, KR908851, KR908973, KR908885,
KR908828, KR909007, KR908919, KR908946; Laelia speciosa Schltr., México, M.W. Chase 6088 (K), KR816317, KR908853, KR908975, KR908887,
KR908815, KR909009, KR908921, KR908947; Laelia speciosa ‘Paget’, México, M.W. Chase 6411 (K), AY008578*, KR908854, KR908976, KR908888,
–, KR909010, KR908922, KR908948; Laelia ×oaxacana Salazar & R.Jiménez, México, E. Hagsater 9539 (AMO), KR816315, KR908850, KR908977,
KR908889, KR908822, KR909011, KR908923, KR908949; Laelia (Sch.) elata (Schltr.) J.M.H.Shaw, Venezuela, G. Carnevali s.n. (CICY), KU232396,
KR908862, KR908980, KR908892, –, KR909014, KR908926, –; Laelia (Sch.) gloriosa (Rchb.f.) L.O.Williams, Brazil, Brieger Coll. 33686 (ESA),
KR816318, KR908861, KR908979, KR908891, –, KR909013, KR908925, KR908951; Laelia (Sch.) gloriosa (Rchb.f.) L.O.Williams (ES), Brazil, Brieger
Coll. 15922 (ESA), KR816331, KR908858, KR908978, KR908890, KR908804, KR909012, KR908924, KR908950; Laelia (Sch.) lueddemannii (Prill)
L.O.Williams, Panamá, Brieger Coll. 277 (ESA), KR816319, KR908863, KR908981, KR908893, KR908798, KR909015, KR908927, KR908952; Laelia
(Sch.) lyonsii (Lindl.) L.O.Williams, cult., Brieger Coll. 16846 (ESA), KR816320, KR908864, KR908982, KR908894, KR908796, KR909016, KR908928,
–; Laelia (Sch.) marginata (Lindl.) L.O.Williams, Venezuela, G. Carnevali s.n. (CICY), –, KR908865, KR908983, KR908895, –, KR909017, KR908929,
–; Laelia (Sch.) moyobambae (Schltr.) C.Schweinf., cult., G. Carnevali s.n. (CICY), –, KR908866, KR908984, KR908896, –, KR909018, KR908930, –;
Laelia (Sch.) splendida (Schltr.) L.O.Williams, cult., C. van den Berg s.n. (UEFS), KR816321, KR908867, KR908985, KR908898, KR908807, KR909019,
–, KR908953; Laelia (Sch.) superbiens Lindl., México, M.W. Chase 5934 (K), KR816323, KR908868, KR908987, KR908900, KR908809, KR909021,
KR908932, KR908955; Laelia (Sch.) superbiens Lindl. (W), México, E. Hagsater 12490 (AMO), KR816322, KR908855, KR908986, KR908899, KR908810,
KR909020, KR908931, KR908954; Laelia (Sch.) undulata (Lindl.) L.O.Williams, Venezuela, G. Carnevali s.n. (CICY), AF260223*, KR908870, KR908989,
KR908902, KR908795, KR909023, KR908934, –; Laelia (Sch.) undulata (Lindl.) L.O.Williams 2, cult., M.W. Chase 1251 (K), KU232397, KR908869,
KR908988, KR908901, KR908794, KR909022, KR908933, –.

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