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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

Lindl., and L. rubescens Lindl. have been recognized, but the
Phylogenetic relationships within Laelia Lindl. have re- former has been considered morphologically intermediate be-
mained only partially resolved, due in part to the limited taxon tween the Mexican Laelia and Schomburgkia (Van den Berg
sampling of the genus in its new broader circumscription. As & al., 2000, 2009; Van den Berg & Chase, 2004a).
a consequence, the group is recognized as the Laelia alliance Reticulation events are often difficult to identify, and mor-
even when Schomburgkia Lindl. is included within Laelia (Van phology (the main source of data used to hypothesize hybrid-
den Berg & al., 2000, 2009; Van den Berg & Chase, 2004a; ization) can be sometimes misleading when the descendants
Chase & al., 2015). Published phylogenetic analyses have in- are closer to one parent and not intermediate (Rieseberg, 1995;
cluded at most 12 species, mainly from the Mexican Laelia. The Wissemann, 2007); furthermore, the assumption of bifurcating
most thorough published work on the phylogeny of this group patterns of evolution complicates the discovery of reticulation
of taxa was based only on the Mexican species of Laelia and events (Legendre, 2000; McBreen & Lockhart, 2006; Huson &
was focused on the analysis of only morphological characters al., 2010). The incongruence between gene trees of uniparen-
(Halbinger & Soto, 1997). The molecular phylogenies published tally (mitochondrial or plastid) or biparentally (nuclear) inher-
so far had a wider scope but lower taxon sampling; the topolo- ited markers has been used as evidence of reticulation events
gies based on molecular data differ from those of morphology in a wide variety of phylogenetic analyses (e.g., Pelser & al.,
in both internal relationships and the placement of Schomburg- 2010; Yu & al., 2013).
kia (Halbinger & Soto, 1997; Van den Berg & al., 2000, 2009). Hybridization has been reported in several groups of
The monophyly of the alliance has been corroborated by several orchids using molecular markers (Gravendeel & al., 2004;
molecular analyses (Van den Berg & al., 2009; Freudenstein Azevedo & al., 2006; Russell & al., 2010). In Laeliinae,
& Chase, 2015). The relationships between Laelia superbiens 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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Brassavola R.Br., and Laelia, where many hybrids, natural were transferred to Sophronitis Lindl. (Van den Berg & Chase,
or artificial, have been described to date (Halbinger & Soto, 2000, 2004b). These Brazilian species were later transferred to
1997; Salazar & al., 2014; Van den Berg, 2014a). However, Cattleya based on further molecular evidence (Van den Berg,
there are few studies assessing hybridization patterns in these 2008). At this point, Laelia was limited to the predominantly
genera with molecular methods, and it has only been inferred Mexican species and those previously included in Schomburg-
in Cattleya from conflicting topologies of nuclear and plastid kia (most species previously combined in Laelia by Williams,
trees (Van den Berg & al., 2009; Santos, 2011). Recently, Van 1941). Historically, many botanists and orchid growers have
den Berg (2014a, b) suggested that reticulation events are an regarded Schomburgkia as separate from Laelia for most of the
evolutionary driving force in Cattleya, based on the abundance period prior to molecular data. Molecular phylogenetic evidence
of putative natural hybrids described. It will not be surprising demonstrated that the genus was composed of two clades, and
if similar patterns of evolution have also influenced the evolu- L. anceps and L. rubescens were closer to species assigned to
tion of Laelia, especially because of its similar generalized Schomburgkia. Based on these results, many authors preferred
type of bee-pollination indicated by morphology. The impor- the expanded circumscription of Laelia, including Schomburg-
tance of reticulate evolution within the genus is unclear, even kia (Williams, 1941), to yield a workable and monophyletic con-
when molecular evidence from both maternal (plastid) and cept of the genus (Van den Berg & al., 2000, 2009; Van den Berg
bi-parental (nuclear) inheritance is available for a subset of & Chase, 2004a; Soto & al., 2006; Chase & al., 2015; Govaerts
the genus; nevertheless, previous phylogenies did not find any & al., 2015). However, some have kept the two genera distinct in
incongruence between both types of evidence, probably as a their phylogenetic analyses (e.g., Freudenstein & Chase, 2015).
consequence of limited sampling of taxa and poor resolution Recently, a different point of view was established by Archila
when using plastid or nuclear regions individually (Van den & Szlachetko (2014) when they regarded L. rubescens, L. aurea
Berg & al., 2000, 2009). A.V.Navarro, and a new species they described as a different
Insertion-deletions (indels) are evolutionary events occur- genus, Encabarcenia Archila & Szlach. They stated, based on
ring in coding and, more frequently, noncoding regions of DNA, the morphological phylogenetic analyses of Halbinger & Soto
detectable when comparing the sequences of different organ- (1997), that these species were indeed a distinct genus, stress-
isms; their presence will vary depending on the region of DNA ing its floral similarity to Broughtonia R.Br. However, this is in
analyzed and organisms studied. Indels are common events in contrast to all previous molecular evidence of the phylogenetic
evolution as obvious from the comparison of many orchid plastid placement of these species according to the principle of mono-
genomes and other groups of plants (e.g., Barrett & al., 2014, phyly currently used in phylogenetic systematics (Van den Berg
2016; Luo & al., 2014; Carbonell-Caballero & al., 2015). How- & al., 2000, 2009; Soto & al., 2006).
ever, indels are not always considered as evidence in phyloge- Disagreements on the limits of genera are common among
netic analysis even though coding schemes have been developed taxonomists, dubbed either lumpers or splitters depending on
to incorporate them in the analyses (González, 1996; Simmons & their wide or narrow delimitation viewpoints. Examples of such
Ochoterena, 2000; Müller, 2005). There is increasing evidence disagreements are frequent among orchid specialists, and a clear
that indels can improve the resolution and branch support of example is the Trichocentrum clade within Oncidiinae. Some
phylogenetic trees in some cases (Simmons & Ochoterena, 2000; taxonomists treat it as a single genus, Trichocentrum Poepp. &
Simmons & al., 2001; Ogden & Rosenberg, 2007). Endl. s.l. (Chase & al., 2009) while others consider the group
Laelia in its current circumscription (Soto & al., 2006) is a as composed of four genera: Lophiaris Rafinesque (“mule ear”
genus of about 24 species distributed from northern Mexico to species), Cohniella Pfitzer (“rat-tail” species), Lophiarella
southeastern Brazil, including the Antilles (Cuba and Jamaica), Szlach. (Oncidium microchilum group), and Trichocentrum s.str.
in a wide variety of environments, but most of its species are (Carnevali & al., 2013). The debate on lumping or splitting is far
associated with seasonally dry forests. It was first described from over with no scientific consensus in sight.
with two Mexican species, L. grandiflora (Llave & Lex.) Lindl. The previous molecular phylogenetic analyses recovered
(= L. speciosa (Kunth) Schltr., the type) and L. autumnalis (Llave two groups of species within Laelia s.l.; the first included the
& Lex.) Lindl., but at least 157 specific names were associated long known Mexican mid- to high-altitude species and the sec-
with Laelia later in various circumscriptions of the genus. When ond included L. rubescens, L. anceps, L. superbiens, and all spe-
we account for infraspecific taxa that number would increase cies of Schomburgkia sampled (Sch. crispa Lindl., Sch. lyonsi
to more than 200 names. Describing all taxonomic changes in Lindl., Sch. splendida Schltr., Sch. undulata Lindl.). However,
the genus would be outside the scope of this paper. However, support values for these two groups were relatively low and
we here summarize some of the most relevant changes. The interspecific relationships were not resolved. For that reason,
phylogenetic analysis by Van den Berg & al. (2000) based on we focused our research on the evolutionary relationships within
nuclear ITS resulted in a clearer understanding of the evolu- Laelia s.l. and, mainly, its relation to those species previously
tionary relationships that would eventually be used, along with included within Schomburgkia. Our main objectives were to
additional evidence, to establish new taxonomic circumscrip- analyze the evolutionary history of this group, evaluate the re-
tions in the Laeliinae. Laelia then included mainly Mexican sults of different phylogenetic methods, and evaluate how indel
and Brazilian species as well as the taxa traditionally referred coding affects them. Some authors have suggested that hybrid-
to Schomburgkia, but suffered a great change in its circum- ization could be an important evolutionary force in some groups
scription when 50 species and 5 hybrids of Brazilian Laelia of Laeliinae but we do not know to what extent it influences the

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evolution of Laelia (Van den Berg & al., 2009; Santos, 2011; Van DNA sampling. — We amplified and sequenced seven re-
den Berg, 2014a, b); thus we included molecular evidence from gions of plastid DNA and the ITS region (5′ partial 18S, ITS1,
both uniparentally (plastid) and biparentally (nuclear) inher- 5.8S, ITS2, 3′ partial 26S) after testing for variation, ease of am-
ited markers. For achieving these objectives we sequenced one plification, and sequencing. The plastid regions amplified and
nuclear and seven plastid regions of DNA and analyzed them sequenced were previously reported to contain high variation
with maximum parsimony, maximum likelihood, and Bayesian in plants and, specifically, in close relatives of the study group
inference, separately. We also coded indels in order to explore (Shaw & al. 2005, 2007; Neubig & al., 2009; Santos, 2011).
their effect on the topology of the three phylogenetic methods. 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
MATERIALS AND METHODS amplification are detailed in Table 1. Primers for rps16-trnK
and trnQ-rps16 were designed for this study due to problems
Plant material. — We sampled as many species and mor- of amplification and sequencing with the primers reported by
phological variants as were available to us from the geographic Shaw & al. (2007).
range of Laelia s.l. Our ingroup consisted of 21 species of the DNA isolation and sequencing. — DNA was isolated from
genus, and some infraspecific taxa or morphologically distinct silica gel dried leaves using a rescaled protocol of the 2× CTAB
geographic samples for L. anceps, L. autumnalis, L. (Schom- method (Doyle & Doyle, 1987). Isolated DNA was stored at
burgkia) gloriosa, L. speciosa, L. (Sch.) superbiens, and L. (Sch.) −80°C at the UEFS DNA bank. All primers and protocols used
undulata; a recently described hybrid, Laelia ×oaxacana Sala- to amplify and sequence each region are listed in Table 1. The
zar & R.Jiménez, was also included within our ingroup. In amplified fragments were purified by precipitation using PEG-
total, we analyzed 27 different accessions of Laelia (Appen- 8000 11% and 70% ethanol (Paithankar & Prasad, 1991). The
dix 1). Our outgroup included three distant species of Laeliinae, purified fragments were then sequenced using the Big Dye 3.1
Broughtonia sanguinea Sw. (R.Br.), Dinema polybulbon Lindl., kit (Applied Biosystems, Foster City, California, U.S.A.) and
and Homalopetalum pachyphyllum (L.O.Williams) Dressler; an automatic ABI3130XL capillary sequencer at Universidade
thus, our analysis included 30 taxa (Appendix 1). 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
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- by jModelTest are summarized in Table 2). These models serve
ited and assembled using Geneious v.5.3.6 (Drummond & al., as input parameters to ML and BI analyses; the ML analyses
2010). Edited sequences were aligned using MUSCLE v.3.8.31 were run with RAxML v.8.1.21 (Stamatakis, 2014) through the
(Edgar, 2004) through the Geneious platform. Alignments raxmlGUI v.1.5b1 (Silvestro & Michalak, 2012) platform; we
were manually inspected, and modified if needed, to avoid used a single model of evolution, the MULTIGAMMA, as most
problems of automated alignment (e.g., misplacement of nucle- aligned regions had a GTR + gamma model of evolution (Table
otides in gap regions). Sequences were deposited at GenBank 2). We analyzed support through a bootstrap analysis with
(Appendix 1). The nuclear and plastid matrices were analyzed 10,000 replicates. The strategy used for parsimony bootstrap
independently. The gaps resulting in the alignment were ana- was adopted for the ML bootstrap analyses. BI analyses were
lyzed as missing data, as most likelihood algorithms do, based done through MrBayes v.3.2 (Ronquist & al., 2012). Analy-
on studies that report no effects on the results (Drummond ses were run for 50 million generations with two independent
& Bouckaert, 2015; Zheng & Wiens, 2015). Gaps were also runs with four chains each and setting the Temp and Nswaps
coded and added to matrices for analyses (details of coding and parameters to 0.05 and 2, respectively. We considered that
analysis below). Alignments of both matrices were deposited runs converged when the standard deviation among split fre-
in TreeBASE (S19331). We used maximum parsimony (MP), quencies achieved a value of less than 0.01, and the estimated
maximum likelihood (ML) and Bayesian inference (BI) to sample size (ESS) was over 200; these values were considered
analyze the phylogenetic relationships among the species of after 25% of the generations were discarded as burn-in. This
Laelia. Preliminary tests of the ITS and plastid alignments was achieved either by manually inspecting MrBayes log files
provided conflicting topologies and accordingly were analyzed or by analyzing the trace files through Tracer v.1.6 (Rambaut
separately; they represent two different kinds of inheritance & al., 2014). We considered posterior probabilities (PP) poor
and trees obtained represent two different evolutionary his- (< 0.90), weak (0.90 to < 0.95), moderate (0.95 to < 0.98), and
tories. MP analyses were run with TNT v.1.5 (Goloboff & strong (≥ 0.98) (Erixon & al., 2003). All nodes below 50% BS
Catalano, 2016). They consisted of traditional searches with (MP and ML, they are reported always in this order) and PP
10,000 random taxon-addition replicates to identify multiple were collapsed in the trees shown. We searched for deviations
islands of optimal trees (Maddison, 1991); saved trees were of GC content in the nuclear ITS as a way to determine if se-
input into the swapping process to increase our probabilities quences were subject to more extensive contraction/expansion
to find the optimal tree. We set a maximum of 10 trees held at phenomena as can occur in regions with lower GC content
each replicate, TBR swapping for every rearrangement tried, (Carels & Bernardi, 2000). We also searched for recombina-
and a maximum of 1000 trees held for the whole search; all tion events using the Maximum Chisquare test of Smith (1992)
other parameters were set to default. A bootstrapping analy- implemented in RecombiTEST (Piganeau & al., 2004), a web-
sis consisting of 10,000 replicates using the same search pa- based platform (http://www.lifesci.sussex.ac.uk/CSE/test/).
rameters was used to evaluate support (Felsenstein, 1985). We displayed the extent of phylogenetic incongruence be-
We considered that bootstrap support (BS) values were poor tween phylogenetic trees with a galled network constructed
(< 50%), weak (50%–70%), moderate (> 70%–85%), or strong with Dendroscope v.3 (Huson & Scornavacca, 2012) using the
(> 85%) (Kress & al., 2002). We identified the model of evo- Bayesian majority consensus trees from the plastid and nrITS
lution of each region with jModelTest v.2.1.7 (Darriba & al., analyses. The galled network was used to illustrate the differ-
2012), using the Akaike information criterion (AIC) as selec- ent phylogenetic positions of taxa in the two trees and to help
tion criterion and default parameters of search (models selected 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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Indel coding and analysis. — A large amount of variation in ITS1, and from 63.6% to 67.8% in ITS2 (common values as
in the matrices was composed of indels (insertion-deletion compared to other species of Laeliinae). The GC content of
events) for various regions (see Table 2). We coded the indels plastid regions varied from 22.9% to 36.6% (Table 2), similar
using the simple coding scheme of Simmons & Ochoterena values when compared to the same DNA fragments from the
(2000), automatically coded and added to matrix data through complete plastid genome of Cattleya crispata (Thunb.) Van den
the SeqState v.1.4.1 platform (Müller, 2005). Indels were ana- Berg (Da Rocha Perini & al., 2015). The evolutionary models
lyzed as standard data in MP, ML, and BI (using the default selected by jModelTest for nuclear ITS1 and ITS2 were K80 + G
models and settings for this kind of data). All these analyses and HKY + G, respectively. The models for the plastid regions
were completed using the same settings as described above for were GTR + G for rpl32-trnL, rps16-trnK, trnQ-rps16, and
each method. Preliminary tests for the phylogenetic relation- trnS-trnfM, GTR + I for the 3′ portion of ycf1, GTR + I + G for
ships of Laelia with and without coding indels showed a con- trnD-trnT, and the HKY + I + G model for trnH-psbA (Table 2).
flict in the position of L. halbingeriana Salazar & Soto Arenas. Phylogenetic analyses. — The trees of the MP, ML and
Thus, we analyzed the matrices with and without this species BI analyses were congruent at most internal clades with col-
to evaluate how this exclusion affects support of other clades. lapsed nodes in the MP and ML topologies. The BI trees for
Hypothesis testing. — We tested different topologies using both the nuclear and plastid regions were best resolved, and
both a Bayesian and a likelihood approach. The former con- BS (MP and ML) and PP values are shown on these BI trees.
sisted of constrained and unconstrained BI analyses with the The main differences between topologies were the recognition
same data and search parameters as used in the previously run of both samples of L. (Sch.) gloriosa (Rchb.f.) L.O.Williams as
BI analyses; we also estimated marginal likelihoods with a BI sister to each other with ML (76% BS), whereas MP and BI
stepping stone analysis, a supposedly better likelihood estimate recovered them as an unresolved clade with both samples of
for hypothesis testing under Bayesian analyses (Drummond L. (Sch.) undulata (Lindl.) L.O.Williams. Laelia (Sch.) moyo-
& Bouckaert, 2015); the stepping stone analysis consisted of bambae (Schltr.) C.Schweinf. and L. (Sch.) marginata (Lindl.)
51 runs of one million generations each discarding the first L.O.Williams were also recovered as sister lineages in the ML
25% generations as burn-in. The likelihoods obtained were analysis (88% BS), but this group was not present in the MP
then used to compute a Bayes factor. The likelihood approach and BI analyses. The MP analyses of the nuclear ITS and plas-
consisted of three different approaches of hypothesis test- tid DNA regions produced 16 and 2 most parsimonious trees,
ing, Kishino-Hasegawa (KH; Kishino & Hasegawa, 1989), respectively (Table 3).
Shimodaira-​Hasegawa (SH; Shimodaira & Hasegawa, 1999), The topologies of all analyses agreed in the presence
and Approximately Unbiased (AU; Shimodaira, 2002) tests; of two clades. The first contains the long recognized group
we obtained the site individual likelihood values for each indi- of highland Mexican Laelia (clade A: L. albida Bateman ex
vidual topology through PAUP v.4.0b10 (Swofford, 2003) and Lindl., L. autumnalis, L. crawshayana Rchb.f., L. eyermani-
imported them into CONSEL v.1.20 (Shimodaira & Hasegawa, ana Rchb.f., L. furfuracea Lindl., L. speciosa) (Electr. Suppl.:
2001) for all three analyses using default parameters. 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-
RESULTS tion of L. (Sch.) superbiens was different in the nuclear DNA
analyses; it was recovered as sister to L. halbingeriana in a
Matrix description. —The nuclear ITS dataset was 489 bp clade with L. aurea and L. rubescens (BS < 50%, < 50%, PP
long (we excluded the sequenced fragments of 18S [partial], 0.87) and not among those Laelia species traditionally classi-
26S [partial], and 5.8S to avoid uncertainties of sequencing for fied as Schomburgkia as occurred in the plastid DNA (Electr.
initial and final fragments of the sequences [18S and 26S], and Suppl.: Fig. S1). The hybrid species included in the analyses,
to exclude a conserved and invariant region [5.8S] among se- L. ×oaxacana, was recovered within the L. anceps clade using
quences) and the aligned plastid (rpl32-trnL, rps16-trnK, trnD- both nuclear and plastid DNA (Electr. Suppl.: Fig. S1). Laelia
trnT, trnH-psbA, trnQ-rps16, trnS-trnfM intergenic spacers, gouldiana Rchb.f. was recovered in either clade A (nuclear
and the 3′ portion of the ycf1 gene) dataset was 7497 bp long. DNA) in an unresolved clade with L. autumnalis f. xanthotropis
The total number of variable characters from nrITS and plastid (Rchb.f.) Halbinger & Soto Arenas, L. crawshayana, and L. ey-
matrices was 641 (233 of them parsimony informative). Some ermaniana or in clade B (plastid DNA) as sister of L. anceps
sequences had large unique indels; the most striking was found subsp. anceps. Laelia ×oaxacana, the supposed hybrid between
in L. speciosa in the trnQ-rps16 region with one single indel of L. halbingeriana and L. anceps subsp. anceps was always re-
527 bp in length. The most important features of the aligned covered as sister of the latter, with plastid and nuclear DNA.
sequences are described in Table 2. All analyzed sequences Plastid DNA analysis recovered a weakly (BS 59%, 57%)
of nrITS and plastid were generated for this study, except for or poorly (PP 0.80) supported Laelia s.l. Clade A was strongly
those indicated in Appendix 1. No recombination event was de- supported by BS (98%, 100%) and PP (1) values. Clade B was
tected in the nrITS sequences, and all species analyzed showed moderately to weakly supported by BS (74%, 69%) but strongly
no evidence of nrITS pseudogenes or polymorphism within supported by PP (0.99). Three main internal clades were recov-
them (amplification and sequencing were straightforward). ered within clade B; the clade of L. aurea + L. rubescens was
The nrITS GC content (Table 2) ranged from 54.6% to 60.5% strongly supported by BS (99%, 99%) and PP (1); the clade of

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Table 3. Summary of parsimony data and tree descriptions for the matrices analyzed.
Matrix Taxa V.C. C.Is. PIC PIC I Tree length Trees rec.
Plastid, no indels 30 349 – 195 – 699 2
ITS, no indels 28 59 – 38 – 129 16
Plastid, indels 30 467 206 281 86 964 1
ITS, indels 28 72 26 51 13 171 15
Plastid, L. halbingeriana excluded, no indels 29 338 – 193 – 632 2
ITS, L. halbingeriana excluded, no indels 27 56 – 38 – 126 16
Plastid,, L. halbingeriana excluded, indels 29 450 197 278 85 906 1
ITS, L. halbingeriana excluded, indels 27 71 26 49 11 166 42
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 of L. (Sch.) gloriosa and L. (Sch.) undulata. The ML analysis
by both BS (99%, 100%) and PP values (1); the clade of Lae- recovered this species in a position identical to that when indels
lia (Sch.) gloriosa + L. (Sch.) undulata ​+ L. halbingeriana was were not coded (trees not shown).
poorly or strongly supported by BS (<50%, 91%) and strongly The analyses of the nuclear data with indels coded were
supported by PP (1) (Electr. Suppl.: Fig. S1). almost identical to the analyses without indels coded but sup-
Nuclear DNA analysis recovered a weakly (BS 53%, 65%, port values were slightly different (Electr. Suppl.: Figs. S1, S2).
PP 0.56) supported Laelia s.l. clade; clade A was poorly or mod- Furthermore, Laelia aurea and L. rubescens were recovered
erately supported by BS (< 50%, 78%) and strongly supported as sister species with weak and moderate BS (64%, 71%) and
by PP (1); clade B was moderately to strongly supported by BS weak PP (0.88) support. Laelia halbingeriana was recovered as
(74%, 89%) and strongly supported by PP (1); internal relation- sister to L. (Sch.) superbiens with strong BS (93%, 98%) and PP
ships within clades A and B were in general less resolved than (1) support. Laelia (Sch.) splendida (Schltr.) L.O.Williams was
in the plastid tree (Electr. Suppl.: Fig. S1). Laelia autumnalis f. recovered as sister to L. (Sch.) gloriosa, L. (Sch.) elata (Schltr.)
xanthotropis is more closely related to other species of Laelia J.M.H.Shaw, L. (Sch.) lueddemannii (Prill.) L.O.Williams, and
from clade A (BS 63%, 65%, PP 0.94) than to L. autumnalis L. (Sch.) undulata with strong BS (85%, 98%) and PP (1) sup-
f. atrorubens (Gower) Halbinger. Laelia (Sch.) superbiens is port. The same group excluding L. (Sch.) splendida was re-
sister to L. halbingeriana within the clade of L. aurea and covered with weak and moderate BS (56%, 74%), and poor PP
L. rubescens (BS < 50%, < 50%, PP 0.87). The positions of (0.54) support.
L. autumnalis f. xanthotropis and L. (Sch.) superbiens described Phylogenetic analyses without Laelia halbingeriana. — The
above were not recovered in the analysis of the plastid regions. MP, ML, and BI analyses of plastid and nuclear DNA without
Indel analysis. — The coding of indel characters with the coding indels and excluding L. halbingeriana produced the same
simple coding scheme yielded 206 variable characters for the general clade topologies as those including this species. How-
plastid regions, 86 of them parsimony informative, and 26 ever, there was a little improvement of the BS and PP values of
variable characters for the ITS regions, 13 of them parsimony most clades (Electr. Suppl.: Fig. S3). Laelia s.l. (clades A and B)
informative (Table 3; Electr. Suppl.: Tables S1, S2). The results and the internal clades within clade B had better BS values than
for the MP, ML, and BI analyses including indels were similar in the analysis without coding indels and including L. halbin-
to the analysis without coding the indels but some clades had geriana (Electr. Suppl.: Figs. S1, S3). In the analysis of plastid
higher PP support values in the indel coded analyses (Electr. regions some clades, such as the Laelia albida + ​L. furfuracea
Suppl.: Fig. S2). A clade of L. albida + ​L. furfuracea was re- clade with weak BS (58%, 83%) and weak PP (0.91) support
covered in the MP analysis but collapsed in the ML analysis were recovered; Laelia s.l. had weak BS (59%, 59%) and PP
(Electr. Suppl.: Fig. S2). The BI analysis without coded indels (0.90) support; clade A had strong BS (99%, 100%) and PP (1)
recovered the same clade with poor PP (0.86) but with weak support; clade B had moderate BS (75%, 70%) and strong PP
BS (58%, 54%) support. The BI analysis with coded indels pro- (0.99) support; the clade of Laelia (Sch.) had strong BS (89%,
duced a different topology with L. albida sister to L. autumnalis 91%) and PP (1) support (Electr. Suppl.: Fig. S3). In the nrITS
f. xanthotropis with high PP (0.97) but poor BS (< 50%). That analyses there was a clade consisting of L. autumnalis f. xan-
clade was sister to L. crawshayana with moderate PP (0.95); thotropis together with L. crawshayana, L. eyermaniana, and
this greater clade was sister to L. autumnalis f. atrorubens with L. gouldiana with lower support in the MP and ML analyses
moderate PP (0.97). Laelia furfuracea was sister to all of these (BS 61%, 66%) but with unchanged and weak PP (0.94) support.
species with high PP (0.99) and poor to weak BS (< 50%, 69%). Indel analyses without Laelia halbingeriana. — This set
Notably, L. halbingeriana was sister to all Laelia species that of analyses produced the same general topologies as those in-
had been classified as Schomburgkia (BS < 50%, < 50%, PP cluding L. halbingeriana. The BS and PP values for various
0.78) and it was not recovered as a clade with the accessions clades were higher when compared to the same set of analyses

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

Plastid ITS

Homalopetalum pachyphyllum Homalopetalum pachyphyllum

Dinema polybulbon Broughtonia sanguinea */*

Broughtonia sanguinea Dinema polybulbon 0.64

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

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

Laelia furfuracea
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
1 Laelia gouldiana Laelia anceps subsp. dawsonii

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

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

Laelia (Sch.) undulata Laelia (Sch.) gloriosa
Laelia (Sch.) moyobambae Laelia (Sch.) elata */76
1 Laelia (Sch.) marginata Laelia (Sch.) lueddemannii 0.57
1 Laelia (Sch.) elata Laelia (Sch.) undulata 85/98
0.94 Laelia (Sch.) lueddemannii Laelia (Sch.) undulata 2 53/73
1 Laelia (Sch.) lyonsii Laelia (Sch.) splendida 0.98

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

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

of plastid and nrITS sequences.


Light gray branches lead to taxa


Laelia ey


which are incongruently placed in ia





the analyses of the two datasets.

La tu


eli na



s f sha


Laelia speciosa ur lis f

. a ya

fur . x



ac an
tro n


ea th

ws )
rub a

ot n a a


ro ca . d (W

Laelia pi
xa bsp ns

specio s
oa su rbie iens

sa ‘Pag

et’ × s e b
ia ep up er

el anc .) s sup

L lia ch .)
e (S ch
La elia a (S
Homalopetalum pachyphyllum i
La ael
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

Laelia (Sch.) marginata

or os la at

a io a ta a

Broughtonia sanguinea sa (E 2


La S)






1.0E-4 Clade B

Laelia (Sch.)


lia h

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

Table 5. Summary of characters of Laelia and Schomburgkia.

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

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to keep both clades in an expanded circumscription of Laelia this case more DNA regions only confirmed previous results,
including Schomburgkia, instead of recognizing L. anceps and which are fully compatible with a broadly defined Laelia, and
L. rubescens as members of Schomburgkia. If we chose to the need for resurrecting Schomburkgia is based in taxonomic
keep an expanded Laelia, more than 60% of its species would opinion rather than new results.
be different from the typical Laelia morphology. Furthermore, Recently, Archila & Szlachetko (2014) proposed that
we would be neglecting the phylogenetic information and the L. rubescens and L. aurea were sufficiently different from the
molecular evidence explained in detail below. rest of Laelia to merit generic status, for which they described
We will not elaborate on the delimitation of Laelia (defined Encabarcenia Archila & Szlach. We argue that they simply
as the high-elevation Mexican Laelia species) as a separate stressed some morphological peculiarities present in these two
genus since this is a group that is morphologically, geographi- species, but that this group does not deserve generic status.
cally (Halbinger & Soto, 1997), and phylogenetically distinct Additionally, the recognition of this genus would make the
(Van den Berg & al., 2000, 2009; Soto & al., 2006; this study) concept of Schomburgkia here proposed (or even a broader
from Schomburgkia. The delimitation of the latter would be circumscription of Laelia) non-monophyletic, or else would
controversial for some due to the placement of L. anceps (and require the creation or recognition of further genera, making
allies) and L. rubescens (and allies) within it. However, this the nomenclature of the Laelia alliance unnecessarily compli-
circumscription results in a monophyletic, coherent taxon cated. Encabarcenia, albeit morphologically distinctive, shares
characterized by morphological and biogeographic features with Schomburgkia, as here circumscribed, the compressed
(Table 5). Laelia anceps and allies have overall morphological pseudobulbs, and the long-peduncled and sub-umbellate in-
similarities with core Schomburgkia, as stated above; Lae- florescences bearing short-lived flowers. It is also biogeo-
lia rubescens and allies are apparently intermediate between graphically congruent with Schomburgkia in that it inhabits
Laelia and Schomburgkia by having a somewhat elongated low-elevation environments and is absent from the Mexican
rhizome as in the latter but sharing floral similarities with highlands (Table 5).
Laelia. In addition, L. rubescens and allies feature strongly
compressed pseudobulbs and evanescent flowers arranged in New combinations
long-peduncled, sub-umbellate inflorescences; furthermore,
the flowers have a labellum with a pubescent throat (except. × Schombolaelia gouldiana (Rchb.f.) Peraza & Carnevali,
for L. rubescens var. peduncularis (Lindl.) Halb.) and a small comb. nov. ≡ Laelia gouldiana Rchb.f. in Gard. Chron.,
column (Table 5). The phylogenetic analysis of molecular data ser. 3, 3: 41. 1888 – Holotype: a cultivated plant, Dec 1887,
provides evidence that this group should be kept within Schom- Sander s.n. (W).
burgkia. The phylogenetic topologies recovered from nuclear Parentage: Schomburgkia anceps × Laelia autumnalis
evidence (ITS) show a possible relationship of Sch. superbiens
with L. rubescens, albeit with weak BS and PP support; this Schomburgkia anceps (Lindl.) Peraza & Carnevali, comb. nov.
may be related to past or present introgression of DNA between ≡ Laelia anceps Lindl. in Edwards’s Bot. Reg. 21: t. 1751.
them. Phylogenetic analysis of plastid DNA grouped L. rube- 1835 (“1836”) ≡ Amalias anceps (Lindl.) Hoffmanns. in
scens and L. aurea as sister to L. anceps (and allies) and core Linnaea 16: 228. 1842 ≡ Cattleya anceps (Lindl.) Beer in
Schomburgkia. Molecular sequence changes seem to character- Prakt. Stud. Orchid.: 208. 1854 ≡ Bletia anceps (Lindl.)
ize an expanded Schomburgkia as a unit; all species have a 9 bp Rchb.f., Xenia Orchid. 2: 47. 1862 – Holotype: [illustration
insertion (TGATACCGC) in the 3′ partial portion of the ycf1 in] Edwards’s Bot. Reg. 21: t. 1751. 1835.
gene; a 14 bp fragment (TAAAGTATATGTAT), a 8 bp deletion
(TAATAAAT), and a 6 bp deletion (TAGTAC) in the trnD- Schomburgkia aurea (A.V.Navarro) Peraza & Carnevali,
trnT ; and, a 9 bp deletion (CAGGTTCAT) in trnH-psbA; the comb. nov. ≡ Laelia aurea A.V.Navarro in Orquídea
latter seems to have undergone a duplication in all other Laelia (México City), n.s., 12(1): 42. 1990 ≡ Laelia rubescens var.
species except for L. eyermaniana and L. speciosa. Additional aurea (A.V.Navarro) M.Wolff & O.Gruss, Orchid. Atlas:
single-nucleotide synapomorphies support the separation of 183. 2007 ≡ Encabarcenia aurea (A.V.Navarro) Archila &
Laelia from Schomburgkia. These shared molecular characters Szlach. in Revista Guatemalensis 17(2): 27. 2014 – Holo-
provide evidence that L. rubescens (and allies) should be kept type: Mexico, Nayarit, Las Coloradas, E. Hágsater 450
within Schomburgkia and not as a separate genus. Furthermore, (AMO; isotype: K).
our analyses of hypothesis testing provided very strong sup-
port or strong support for this conclusion from both Bayesian Schomburgkia dawsonii (J.Anderson) Peraza & Carnevali,
analyses and the AU test; the KH and SH tests yielded less comb. nov. ≡ Laelia anceps var. dawsonii J.Anderson in
strong support but still had high values of support for the same Gard. Chron. 1868: 27. 1868 ≡ Laelia dawsonii (J.Anderson)
hypothesis. De B.Crawshay in Gard. Chron., ser. 3, 32: 414. 1902 ≡
On the other hand, CvdB considers that no generic taxo- Laelia anceps subsp. dawsonii (J.Anderson) Rolfe in Orch.
nomic change from a broadly defined Laelia would be needed Rev. 30: 10. 1922 – Lectotype (designated by Soto Arenas
since the topologies found for Laelia anceps and L. rubescens in Orquídea (Mexico City) 13: 133. 1993): [illustration]
are virtually the same as found in his previous analyses based “Laelia anceps Dawsoni” in Warner, Select Orchid. Pl.
on ITS, matK and trnL-F (Van den Berg & al., 2000, 2009). In 2: t. 34. 1865.

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Schomburgkia halbingeriana (Salazar & Soto Arenas) Peraza regarded as Sch. anceps, the populations from the mountains
& Carnevali, comb. nov. ≡ Laelia halbingeriana Salazar in the Pacific slope of Mexico should be considered Sch. daw-
& Soto Arenas in Phytotaxa 178(3): 162. 2014 – Holotype: sonii, and the populations from Chiapas (Mexico), Guatemala,
Mexico, Oaxaca, Portillo de Coyula, unos 2 km antes del and Honduras should be recognized as Sch. mottae. We were,
poblado de Coyula subiendo desde Quiotepec, donde la unfortunately, unable to sample Sch. mottae but we consider,
línea eléctrica cruza el camino, 1160 m elevation, 11 Jul based on morphology, phenology and geographic distribu-
2004, pressed in cultivation 17 Oct 2006, Salazar & al. tion, that it should be expanded to include those populations
6695 (MEXU). from Chiapas previously included as part of Sch. anceps (as
L. anceps subsp. anceps) by Halbinger & Soto (1997). Thus, we
Schomburgkia mottae (Archila, Chiron, Szlach. & Pérez- propose to treat them as different species and we transfer them
García) Peraza & Carnevali, comb. nov. ≡ Laelia mottae to Schomburgkia following the logic stated above. Schomburg-
Archila, Chiron, Szlach. & Pérez-García in Bot. Sci. 92 (3): kia dawsonii has a 16 bp deletion (TATTAGTATTAGTATA)
345. 2014 – Holotype: Guatemala, Chiquimula, Esquipu- in the trnH-psbA intergenic spacer, a 5 bp deletion (TATAT)
las, alt. ca. 400 m, bosque tropical caducifolio, Sep 2010, in the rpl32-trnL intergenic spacer, a 19 bp insertion (TAGT​
F. Archila s.n. (BIGU). TACTAATTTAGTACA) in the trnD-trnT intergenic spacer,
a 9 bp insertion (TTTTTTACA) in the trnS-trnfM intergenic
Schomburgkia ×oaxacana (Salazar & R.Jiménez) Peraza & spacer (compared to Schomburgkia anceps); these are the most
Carnevali, comb. nov. ≡ Laelia ×oaxacana Salazar & obvious sequence differences between them.
R.Jiménez. in Phytotaxa 178(3): 167. 2014 – Holotype: Hybridization seems to have played an important role in
Mexico, Oaxaca, Barranca del Río Santo Domingo, muy the evolution of both genera. There are several hypothesized
al E de Tecomavaca, cerca de Buenos Aires (entre Coyula hybrids, including ×Schombolaelia gouldiana, a natural hybrid
y el río Santo Domingo), ca. 1600–1700 m elev., Jun 1986, between Laelia and Schomburgkia (Fig. 2); the age of this hy-
fl. in cult. 4 Nov 1991, Lau sub Hágsater 9539 (AMO; bridization event is unknown, but probably not recent because
isotypes: MEXU, SERO). the nrITS regions display no polymorphisms characteristic of
F1 or recent hybrids (Baldwin, 1992; Baldwin & al., 1995). A
Schomburgkia perezgarciae (Archila & Szlach.) Peraza & similar pattern in nrITS is found in Sch. ×oaxacana whose
Carnevali, comb. nov. ≡ Encabarcenia perezgarciae plastid and nuclear DNA are closely related to Sch. anceps;
Archila & Szlach. in Revista Guatemalensis 17(2): 27. however, it has the morphological characters of Sch. halbin-
2014 – Holotype: Guatemala, Suchitepequez, Mazat- geriana; the fixation of one single copy of nuclear ITS is a
enango, sobre árboles de Tabebuia rosea (Bert.) DC, Jun common result of concerted evolution (Baldwin, 1992; Baldwin
1999, F. Archila s.n. (BIGU). & al., 1995). However, we cannot discard another cause for the
patterns of relationships among these species; lineage sorting
Schomburgkia rubescens (Lindl.) Peraza & Carnevali, comb. is difficult to distinguish from hybridization and cannot be
nov. ≡ Laelia rubescens Lindl. in Edwards’s Bot. Reg. 26: fully discarded here due to the absence of more exhaustive
t. 41. 1840 ≡ Cattleya rubescens Beer, Prakt. Stud. Orchid.: tests. The position of Sch. halbingeriana is uncertain; when
208. 1854 ≡ Bletia rubescens Rchb.f. in Walpers, Ann. Bot. we treated indels as “missing” data it was recovered as sister
Syst. 6: 425. 1862 – Holotype: Probably from Mexico, Mr. to Sch. undulata and Sch. crispa, but when we coded the indels
Barker, imported by Mr. J. Knight (K). it was recovered as sister to all other species of Schomburgkia
(in the traditional sense). It was also recovered with different
Schomburgkia rubescens f. peduncularis (Lindl.) Peraza & relationships when plastid or nrITS sequences were analyzed.
Carnevali, comb. nov. ≡ Laelia peduncularis Lindl. in We cannot conclude if this behavior is the result of hybridiza-
Edwards’s Bot. Reg. 28: t. 9. 1842 ≡ Cattleya peduncu- tion or incomplete lineage sorting. When excluded from the
laris (Lindl.) Beer in Prakt. Stud. Orchid.: 213. 1854, nom. analyses, several nodes had higher support values in the plastid
inval. ≡ Bletia peduncularis (Lindl.) Rchb.f. in Walpers, analyses. nrITS data did not result in good resolution and prob-
Ann. Bot. Syst. 6: 426. 1862 ≡ Laelia rubescens f. pedun- ably other nuclear data would contribute to solving this open
cularis (Lindl.) Halb. in Orquidea (Mexico City) 13: 294. question. The clade containing L. halbingeriana or the sister
1993 ≡ Encabarcenia rubescens f. peduncularis (Lindl.) clade had weak BS and PP values when using plastid DNA;
Archila & Szlach. in Revista Guatemalensis 17(2): 26. 2014 when we used the nuclear regions we noticed that it was always
– Holotype: A mexican plant flowered in Birmingham, recovered as sister taxon of Sch. superbiens in a clade distinct
Geo. Barker s.n. (K). from the remaining Schomburgkia (in the old sense); which
seem to conserve a nuclear relationship with Sch. rubescens
The evidence of this molecular analysis, morphological and and Sch. aurea or show evidence of introgression from one of
geographic data detailed in Halbinger & Soto (1997), Archila & these species; this is difficult to assess at this time. When we
al. (2014), and Salazar & al. (2014) provides evidence that the removed Sch. halbingeriana from the analyses, the support
previously known subspecies of L. anceps are following differ- values for related clades improved significantly in the plastid
ent evolutionary trajectories. We propose that the populations ML and BI analyses.
from the mountains of the Gulf of Mexico slopes should be

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ACKNOWLEDGEMENTS genomes elucidates the relationships between wild and domestic

species within the genus Citrus. Molec. Biol. Evol. 32: 2015–2035.
The first author is grateful to Juliana Freitas, Juliana Santos,
Carels, N. & Bernardi, G. 2000. Two classes of genes in plants. Genetics
Michella Teixeira, Ricardo Vilas-Boas, and Evandro Santos for train- 154: 1819–1825. http://www.genetics.org/content/154/4/1819.long
ing in the laboratory. The authors thank Gerardo A. Salazar, Eric Carnevali, G., Cetzal-Ix, W., Balam, R., Leopardi, C. & Romero-
Hágsater, Luis M. Sánchez, and Rolando Jiménez for plant material González, G.A. 2013. A combined evidence phylogenetic re-​
and/or helpful discussions on orchid taxonomy, variation, and evolu- circumscription and a taxonomic revision of Lophiarella (Orchid­
tion. They also kindly hosted the first author at MEXU and AMO aceae: Oncidiinae). Syst. Bot. 38: 46–63.
herbaria when analyzing their orchid collections. We are grateful to Chase, M.W., Veitch, N. & Grayer, R. 2009. Subtribe Oncidiinae. Pp.
Dr. Lynn Clark, Dr. Michael Pirie, and four anonymous reviewers for 211–220 in: Pridgeon, A.M., Cribb, P.J., Chase, M.W. & Rasmussen,
comments that greatly improved the manuscript. Authors are grateful F.N. (eds.), Genera orchidacearum, vol. 5, Epidendroideae (part 2).
to FAPESB and CNPq for financial support through a research grant Oxford: Oxford University Press.
(PRONEX grant PNX0014/2009). Finally, LNPF thanks Consejo Na- Chase, M.W., Cameron, K.M., Freudenstein, J.V., Pridgeon, A.M.,
Salazar, G., Van den Berg, C. & Schuiteman, A. 2015. An up-
cional de Ciencia y Tecnología (CONACyT) for two grants (204216
dated classification of Orchidaceae. Bot. J. Linn. Soc. 177: 151–174.
and 239018) to fulfill a postdoctoral fellowship during two years at http://dx.doi.org/10.1111/boj.12234
Universidade Estadual de Feira de Santana, and CvdB thanks CNPq Cummings, M.P., Handley, S.A, Myers, D.S., Reed, D.L., Rokas, A.
for a productivity scholarship (PQ-1B). & Winka, K. 2003. Comparing bootstrap and posterior probability
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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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