CONFERENCE PAPERS / SYSTEMATICS / HÁGSATER ET AL.

PHYLOGENOMICS OF EPIDENDRUM:
UNTANGLING A NEOTROPICAL
MEGA-DIVERSIFICATION

Eric Hágsater*1, Carolina Granados Mendoza², Gerardo A. Salazar², Sebastián

Quiroga-González², Susana Magallón², Cassio van den Berg3, Emily Moriarty
Lemmon4, and Alan R. Lemmon5
1
Herbario AMO, Montañas Calizas 490, Miguel Hidalgo, Mexico City, 11000,
Mexico; ²Departamento de Botánica, Instituto de Biología, Universidad Nacio-
nal Autónoma de México, Apartado Postal 70-367, 04510 Mexico City, Mexi-
co; 3Departamento de Ciências Biológicas, Universidade Estadual de Feira de
Santana, Av. Transnordestina s.n., 44036-900 Feira de Santana, Bahia, Brazil;
4
Department of Biological Science, Florida State University, 319 Stadium Drive,
P.O. Box 3064295, Tallahassee, FL 32306-4295, USA; 5Department of Scientif-
ic Computing, Florida State University, Dirac Science Library, Tallahassee, FL
32306-4102, USA
*Corresponding author. E-mail: erichag_1@msn.com

Two of the major challenges in plant evolutionary studies
are the development of orthologous molecular markers
variable enough to resolve interspecific relationships and
generating such data for a large number of species in ‘me-
ga-diverse’ lineages. One such lineage is the Neotropical
orchid genus Epidendrum, as our recent estimates indicate
that it might include about 2400 extant species. As part of
our long-term systematic and evolutionary studies of this
genus, we are employing a novel phylogenomic approach,
anchored hybrid enrichment, for selectively sampling up to
517 single- or low-copy nuclear loci potentially informative
at a range of phylogenetic depths (including interspecific
relationships). Our previous phylogenetic studies, based on
a combination of Sanger sequencing of nuclear ITS and
plastid matK-trnK and trnL-trnF regions, recovered several
major clades but neither the ‘backbone’ relationships of
Epidendrum nor the relationships among closely related spe-
cies were reliably resolved or supported. Our preliminary
tests showed that over 335 single-copy nuclear loci could
be reliably sequenced, and maximum likelihood analyses
of the concatenated matrix resulted in a fully resolved tree
with strong bootstrap support for many clades. Our cur-
rent work is focused on attaining an appropriate coverage
of the diversity of this huge genus, emphasizing a full repre-
sentation of the main clades and a complete species’ level
sample of selected, focal clades as a basis for a densely sam-
pled, well-resolved phylogeny for future macroevolutionary
studies.
Epidendrum L. is perhaps the largest orchid ge-
nus in the New World, with an estimated 1500
accepted described species (Hágsater and Soto
2005) out of about 3000 published names, many
having been transferred to other genera or ac-
cepted as synonyms, and several-hundred addi-
tional ones pending formal description, and for
which we have some kind of material (herbari-
um specimen, digital image, illustration, etc.);
our current list of probable species totals some
2400 (Hágsater et al. 2016). Thirty-eight new spe-
cies have been recently published (Hágsater and
Santiago 2018). Most species live as epiphytes or
lithophytes in moist to wet forests, but particular
species thrive in every environment suitable for
plant life, from coastal sand dunes to Andean
páramos and from pristine forests to roadside em-
bankments. This group exhibits an enormous di-
versity of growth forms and architectures, which
enables them to thrive in the highly competitive
epiphytic biotope. Such growth forms include
caespitose plants with slender, cane-like stems or,
less commonly, thickened pseudobulbs; densely

256

or loosely branching erect or pendent shrubs;
monopodial, monocaulescent herbs or more of-
ten forming a branched crown. Likewise, there
is a vast array of inflorescence and floral mor-
phologies, including apical, lateral or rarely basal
inflorescences that vary from single-flowered to
subcorymbose, racemose or paniculate; flowers
may be produced only once or new racemes arise
from old inflorescences over several years. Both
the unparalleled taxic diversity and the notice-
able structural and ecological disparity displayed
by Epidendrum make an ideal model to investigate
intrinsic and extrinsic factors underlying evolu-
tionary diversification of species-rich lineages. To
do this, the first requisite is a robust, well-resolved
phylogenetic framework as a basis for inferences
on the evolutionary dynamics of the group.
Early molecular phylogenetic studies of Laeliinae
have included some species of Epidendrum, but
they were focused mostly on deeper-level relation-
ships, including subtribal and generic limits (van
den Berg et al. 2000, 2009), providing limited in-
formation on its intrageneric relationships. The
analysis of van den Berg et al. (2000) was based
on a single marker, the nuclear ribosomal inter-
nal transcribed spacer (ITS) region, which provid-
ed limited phylogenetic support for relationships
among the species of Epidendrum analyzed. Subse-
quently, van den Berg et al. (2009) increased their
sample of characters to include, besides ITS,
three plastid markers, namely the trnL intron,
trnL-trnF intergenic spacer, and the matK-trnK
region, obtaining similar results in terms of sup-
port for the phylogenetic relationships recovered
among Epidendrum representatives. Hágsater and
Soto-Arenas (2005) analyzed a much more inclu-
sive sample of species of Epidendrum (ca. 80 spp.)
and DNA sequences from the nuclear ITS region
and plastid matK-trnK region. In their analysis,
phylogenetic support was mainly restricted to
some intermediate and relatively shallow (recent)
divergences, whereas the ‘backbone’ and most
relationships among shallow clades were only
weakly supported.
CONFERENCE PAPERS / SYSTEMATICS / HÁGSATER ET AL.
A few recent studies have focused on inferring
phylogenetic relationship among species of Epi-
dendrum. Pinheiro et al. (2009) assessed mono-
phyly and internal relationships of Epidendrum
subgenus Amphiglottium Lindl., analyzing DNA
sequences of the plastid trnL-trnF region and
AFLP data. Pessoa et al. (2012) explored species
limits in a clade from eastern Brazil belonging to
E. subgenus Amphiglottium using DNA sequences
of non-coding plastid DNA (intergenic spacers
trnT-trnL, trnL-trnF and rpl32-trnL), nuclear mi-
crosatellites, and morphometric data. Marques et
al. (2014) assessed the genetic structure and the
outcomes of hybridization among three species
of Epidendrum in Ecuador based on AFLPs and
sequences of both coding and non-coding plas-
tid DNA (trnL-trnF, rps16, rpoC1, psbK-psbI, matK,
and rbcL). However, all these studies were focused
on small groups of closely related species or on
particular suspected hybrid swarms, and the over-
all relationships in Epidendrum remain largely un-
explored.
Recently, Quiroga-González (2017) investigat-
ed the phylogenetic relationships of a Mexican
group of species represented by E. anisatum Lex.
Such a group was originally recognized by Hág-
sater (1985) among the 40 groups of his mor-
phology-based informal classification, and sub-
sequently the E. anisatum group was redefined
by García-Cruz (1995) on the basis of a phenet-
ic analysis of morphological data. The species
belonging to this group inhabit moist pine-oak
forests and cloud forests of the Mexican Pacific
slope and, according to García-Cruz (1995), in-
clude E. anisatum, E. costatum A.Rich. & Galeot-
ti., E. cusii Hágsater, E. dorsocarinatum Hágsater,
E. gasteriferum Scheeren, E. guerrerense Hágsater
& García-Cruz, E. gomezii Schltr., E. hueycante-
nangense Hágsater & García-Cruz, E. juerguensenii
Rchb.f., E. lowilliamsii García-Cruz, E. matudae
L.O.Williams, E. mixtecanum Hágsater & García-
Cruz, E. neogaliciense Hágsater & R.González, E.
oaxacanum Rolfe, E. pastranae Hágsater, E. rosilloi
Hágsater, and E. vandifolium Lindl. In his study,
Quiroga-González (2017) included 13 of the 17
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CONFERENCE PAPERS / SYSTEMATICS / HÁGSATER ET AL.
Fig. 1. Majority-rule (50%) consensus tree from a Bayesian analysis of six plastid and one nuclear DNA regions (see text); num-
bers below branches are posterior probabilities. Modified from Quiroga-González (2017).
species recognized by García-Cruz (1995) in the
group and analyzed DNA sequences of six plas-
tid markers, namely matK-trnK, trnH-psbA, and
trnL-trnF regions, trnS-trnfM and atpI-atpH inter-
genic spacers, and the intron of gene rps16, plus
the nuclear ITS region. The phylogenetic signal
provided by this set of uni- and biparentally in-
herited markers confirmed monophyly of the E.
anisatum group and provided support for some of
its internal relationships (Fig. 1). However, sever-
al clades subtended by short branches lacked sup-
port. Moreover, some topological incongruences
were detected between the patterns resulting from
the plastid and nuclear data, which may result
from limited phylogenetic signal. All the above
point toward the need to use more variable and
informative molecular markers, preferably from
different genome compartments. Shallow-level
relationships in Epidendrum recovered in previ-
ous phylogenetic studies are in general character-
ized by short or nearly null subtending branch
258
lengths (e.g. Pessoa et al. 2012; Quiroga-González
2017), likely due to the sparse informative charac-
ters marking clades for recent evolutionary time
frames. Plastid markers in general are known to
be less variable than nuclear markers (Clegg et al.
1994; Sang 2002), resulting in less phylogenetic
utility, especially for resolving recent divergences.
Additionally, it has been proposed that plastid
markers in Epidendrum might be subjected to
higher levels of introgression compared to nu-
clear markers due to the lower dispersal capacity
of seeds relative to pollinia (Pessoa et al. 2012).
From about a dozen molecular markers used in
previous shallow-level phylogenetic studies in Epi-
dendrum, 11 are plastid markers, ITS being the
only nuclear marker (Hágsater and Soto 2005;
Pessoa et al. 2012; Quiroga-González 2017; van
den Berg et al. 2000, 2009). Therefore, most
data used in phylogenetic analyses conducted to
date for Epidendrum are prone to be strongly in-
fluenced by hybridization or introgression events

or to provide limited phylogenetic utility due to
overall low sequence variation. Although the nu-
clear ITS region is more variable than the plastid
markers used in phylogenetic inference for Epi-
dendrum (e.g. Quiroga-González 2017), this region
occurs as multiple copies in the genome, making
orthology assessments uncertain if incomplete
concerted evolution has occurred (Chase et al.
2007; Zhang et al. 2012).
Hence, to overcome the current lack of resolu-
tion and support among closely related species of
Epidendrum it will be necessary to use additional
independent, highly variable, bi- and uniparen-
tally inherited markers. Nuclear single- or low-co-
py genes are a promising source of phylogenetic
information. When present as a single copy, or-
thology of nuclear genes is straightforward, and
their variability is usually higher compared to
many plastid markers (e.g. Granados Mendoza et
al. 2015). Evolutionary biologists now have access
to a diverse array of high-throughput techniques
for producing massive DNA sequence data at
a comparatively low cost (e.g. versus Sanger se-
quencing) and in a reasonable amount of time.
Plant phylogenomics is being increasingly ap-
proached in evolutionary studies through a vari-
ety of highly efficient genome-partitioning strate-
gies (GPS), which are a set of methods focused on
sequencing specific regions of the genomes that
are expected to be highly informative for partic-
ular phylogenetic questions (Lemmon and Lem-
mon 2013). Hybrid enrichment strategies (HES)
are a subset of genome-partitioning methods
aimed at capturing specific genomic regions pri-
or to high-throughput sequencing (Mamanova et
al. 2010; Summerer 2009). All HES require an
initial phase of probe design in which the genom-
ic regions of interest are delimited from existing
genomic resources, such as complete or partially
sequenced genomes and transcriptomes. Partial-
ly overlapping short nucleotide sequences of 60
to 120 base pairs (bp) long are designed based
on the target regions. Capture probes are then
synthesized, usually as RNA oligonucleotides.
As in most methods implying high-throughput
CONFERENCE PAPERS / SYSTEMATICS / HÁGSATER ET AL.
sequencing, a library preparation step is required
in which the genomic DNA from the species of
interest is fragmented, indexed, and linked to
special adapters appropriate for the specific se-
quencing platform being employed.
The next step is called enrichment, in which a
target genomic DNA library is mixed with the
RNA capture probes. During enrichment, the
genomic regions complementary to the capture
probes hybridize with them, forming RNA-DNA
hybrids that are subsequently separated from
the remaining, non-target genomic DNA using
streptavidin magnetic beads. After high-through-
put sequencing, raw reads are de-multiplexed
using the indexes and assembled against the
reference sequences used during the probe de-
sign step.We are currently starting to apply the
anchored hybrid enrichment method (AHE;
Lemmon et al. 2012), a highly efficient HES first
developed for vertebrates and recently modified
for its application in plants (Buddenhagen et al.
2016; Fragoso-Martínez et al. 2017) to investigate
phylogenetic relationships in Epidendrum (Grana-
dos Mendoza et al. 2016; Granados Mendoza,
E. Hágsater et al., unpubl. data). As designed by
Buddenhagen and collaborators, angiosperm
AHE probes are expected to perform well in
most flowering plant lineages because its de-
sign was based on a variety of species distribut-
ed across the angiosperm phylogenetic tree. The
angiosperm AHE capture probe set targets 517
low-copy nuclear loci expected to show different
degrees of variability, thus being potentially infor-
mative at a wide range of phylogenetic depths.
Our AHE study initially aimed at evaluating the
effectiveness of gene capture and sequencing in
Epidendrum and assessing the performance of the
loci recovered in resolving and supporting phylo-
genetic relationships at a range of Taxonomical
depths. For this we used a representative sample
of taxa including 18 species of Epidendrum and
one species each of other genera of Laeliinae,
including Arpophyllum Lex., Barkeria Knowles &
Westc., Broughtonia R.Br., and Caularthron Raf.,
as well as one species of Pleurothallis R.Br., of sub-
259
CONFERENCE PAPERS / SYSTEMATICS / HÁGSATER ET AL.
Fig. 2. Maximum likelihood tree from concatenate analysis of 335 single-copy nuclear loci generated by Anchored Hybrid En-
richment. A. Tree drawn with branch lengths proportional to number of changes. B. Tree with bootstrap percentages indicated
under branches.
tribe Pleurothallidinae, to be used as functional
outgroup. Our preliminary results indicate that
the AHE technique was able to recover 335 loci,
with an average copy number ranging from 0.66
to 0.89 and average length of 581 bp (range: 163
to 1495 bp). A maximum likelihood analysis of
all the loci concatenated retrieved a fully resolved
phylogenetic tree with all nodes obtaining strong
support (bootstrap percentages ≥97; Fig. 2). This
demonstrates the feasibility of applying AHE to
Epidendrum and, because the loci are informative
at a range of phylogenetic depths, including rela-
tionships of pairs of closely related species, this
method is promising for untangling phyloge-
netic relationships both within Epidendrum and
between this genus and its closest relatives. The
latter will likely facilitate the identification of its
sister genus, which has important implications
for comparative studies such as reconstructing
the evolution of morphological attributes.
As noted earlier, inclusion of data from different
genomic compartments is highly desirable for as-
sessing phylogenetic relationships among species
of Epidendrum. An ongoing study (C. Granados
260
Mendoza, E. Hágsater et al., in prep.) is using
out-of-target raw sequences from AHE data for
assembling partial chloroplast genomes of Epi-
dendrum species. Since the AHE probes are not
100% efficient in capturing the targeted nuclear
regions, some non-nuclear regions are also cap-
tured during enrichment and sequenced along
with the nuclear loci. This approach will increase
our character sampling to include plastid data as
well at no extra cost. Application of these meth-
ods to a large sample of species representing most
major clades identified by prior morphological
and molecular studies will greatly improve our
understanding of the phylogenetic relationships
of this spectacular Neotropical radiation as a ba-
sis for understanding the factors underlying it.
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