epidendrum flammeus complete integrative
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Pessoa & al. Different tools to delimitEpidendrumspeTAXON61 (4) August 2012: 721734
INTRODUCTION
Species delimitation is an old issue with an extensie theo-
retical and methodological body of literature (de Queiro, 1998;Sites & Marshall, 2004 and references therein). This subject
has recently attracted renewed attention, which is emphasied
by the wide interest in barcoding approaches (Lahaye & al.,
2008; Hollingsworth & al.,2009; Pires & Marinoni, 2010). Be-
cause species are the main units of ecological and eolutionary
studies, the identification of boundaries among closely related
species, and therefore the inference of the number of extant
species, is an essential target of current systematic studies (Du-
minil & al.,2006; Petit & Excoffier, 2009). To achiee this goal,
the cross-alidation of species identification using different
approaches is desirable (but see Padial & al., 2010), and dif-
ferent tools are aailable to depict complex morphological and
genetic patterns of ariation, such as morphometry (Pinheiro
called integratie taxonomy (Dayrat, 2005; Padial & al., 20
Schlick-Steiner & al.,2010).
The perspectie that species can be delimited based
multiple lines of eidence has been boosted by the increing eidence that reproductie barriers are permeable to g
flow (porous genomes, Wu, 2001), that species lineages m
dierge despite hybridiation and introgression and that a sin
species can originate polyphyletically by parallel eolut
(reiewed in Hausdorf, 2011). Indeed, the general lineage c
cept of species (de Queiro, 1998) can be used as a pro
conceptual framework related to the use of different type
data to identify species. According to this concept, differ
patterns of diergence for different types of data and deli
tation criteria may arise at different times in the proces
speciation (de Queiro, 1998). For example, different spe
criteria such as intrinsic reproductie isolation (Mayr, 194
reciprocal monophyly (de Queiro & Donoghue, 1988)
Integrating different tools to disentangle species complexes:A case study in Epidendrum(Orchidaceae)
Edlley Max Pessoa,Marccus Alves,Anderson Alves-Arajo,Clarisse Palma-Silva& Fbio Pinheiro
1 Universidade Federal de Pernambuco, Centro de Cincias Biolgicas, Departamento de Botnica, 50670-420, Recife, PE, Brazil
2 Instituto de Botnica, 04301-902, So Paulo, SP, Brazil
Author for correspondence:Fbio Pinheiro, biopinheiro@yahoo.com.br
Abstract Species delimitation remains a central problem in systematic, taxonomic and evolutionary studies. However, the
precise delimitation of species depends on the criteria used to identify lineages and the specific species concept that is applied.
Recently, multidisciplinary studies using different data sources have significantly improved the delimitation of species within
complex taxonomic groups, leading to an integrative taxonomy. To investigate the species delimitation within the Atlantic
clade ofEpidendrum(subg.Amphyglottium), four different species criteria were examined (phenetic differentiation, mono-
phyly, diagnosability, absence of genetic intermediates). Morphometrics, plastid DNA sequences and nuclear microsatellite
markers were used to explore the agreement between patterns recovered and species criteria tested. The conflicts amongspecies criteria are discussed in light of pollination ecology, patterns of gene f low, reproductive isolation mechanisms and
selective pressures currently acting in deceptive orchid species. Four currently recognized species from the Atlantic clade
could be delimitated, including one newly described species,Epidendrum f lammeus. Three out of f ive species satisfied the
monophyly criterion, and few diagnostic flower characters were found among species. In contrast, nuclear microsatellite
data correctly assigned individuals to their respective species, even in the presence of weak reproductive isolation and ex-
tensive hybridization events reported in the literature. One important implication of this finding is that phylogenetic studies
inEpidendrumspp. should make use of single- or low-copy nuclear loci instead of plastid markers, which may be true for
other plant groups. The results also indicate that the generalized pollination syndrome found among species of the Atlantic
clade, the different levels of gene flow observed between nuclear and plastid markers, and hybridization events are com-
monly observed as the main evolutionary forces within this orchid group. Finally, we discuss evolutionary processes and
taxonomic limits among these species, and we highlight the need to increase the inter-disciplinary approach to investigate
species limits in complex plant groups.
Keywords hybridization; orchid evolution; reproductive barriers; speciation; species complex; species delimitation
Supplementary Material Tables S1S2 and Figures S1S3 (in the Electronic Supplement) and the alignment are available in the
Supplementary Data section of the online version of this article(http://www.ingentaconnect.com/content/iapt/tax).
http://www.ingentaconnect.com/content/iapt/taxhttp://www.ingentaconnect.com/content/iapt/tax -
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TAXON61 (4) August 2012: 721Pessoa & al. Different tools to delimitEpidendrumspecies
scenarios has been inestigated, leading to the conclusion that
the application of seeral different criteria should be ealu-
ated considering the releant forces driing speciation process
(reiewed by Padial & al., 2010).
Whereas qualitatie traits are relatiely easy to measure,
the ast majority of phenotypic differences between popula-
tions, species, or higher-order taxa are quantitatie(Lynch
& Walsh, 1998) and will thus require accurate and objectie
measurement and analysis. The use of multiariate methods
to analye quantitatie morphometric data proides the op-
portunity to summarie large numbers of characters in few
discriminant components, allowing the identification of cryptic
borders between closely related species (Borba & al.,2002) or
the mosaic nature of recombinant hybrids (Lexer & al.,2009).
Furthermore, traditional characters used to differentiate spe-
cies, such as morphology, are increasingly supplemented by
approaches based on molecular genetic data. Distinguish-
ing species that hae low leels of genetic diergence, eitherbecause speciation is recent or because species continue to
exchange genes remains a challenging task in eolutionary
biology and molecular taxonomy (Duminil & al., 2006; Lexer
& al., 2009). The inestigation of species limits is greatly fac
tated by the aailability of molecular markers informati
the species leel, from different genomic compartments, m
typically nuclear and cytoplasmic genomes (Duminil &
2006; Moccia & al.,2007; Palma-Sila & al.,2011). Accord
to Petit & Excoffier (2009) and Hausdorf & Hennig (2010),
use of multiple unlinked nuclear markers experiencing h
intraspecific gene flow leels coupled with the use of mod
based assignment methods proides the best results to deli
species. Nuclear microsatellites hae been pointed out as hig
informatie markers for interspecific comparisons (Dum
& al.,2006; Hausdorf & Hennig, 2010; Palma-Sila & al.,20
High cross-transferability among species has been obsered
nuclear microsatellites (Barbar & al., 2007; Pinheiro &
2009b), and the occurrence of homoplasy is oercome by
use of multiple unlinked loci (Duminil & Di Michele, 200
Our study group, Epidendrum L. (Orchidaceae), is
largest (1500 species) and most widespread (Southern UnStates to North Argentina) Neotropical orchid genus (Hgs
& Soto-Arenas, 2005).Epidendrumis renowned for uncerta
ties regarding delimitation within species complexes, beca
Fig. .Geographical distribution
ofEpidendrumspecies of the
Atlantic clade. A,Latin Ameri-
can map; B,detail of Brailian
Atlantic coast.
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many species show impressie morphological ariation. Studies
within the genus are primarily limited to the description of new
species based on qualitatie morphological data, and comple-
mentary taxonomic and eolutionary studies are comparatiely
rare (see reiew by Hgsater & Soto-Arenas, 2005). Blurred
morphological boundaries between many closely related spe-
cies are common within the genus, and detailed data describing
patterns of intraspecific morphological ariation are restricted
to few species (Pinheiro & Barros, 2007a, b). Recently, a highly
informatie set of nuclear (Pinheiro & al., 2008a, b) and plastid
markers (Pinheiro & al.,2009c) was deeloped for Epiden-
drum, which contributed substantially to the inestigation of
eolutionary processes at low taxonomic leels within the genus
(Pinheiro & al., 2010, 2011).
Species of Epidendrum subg. Amphyglottium (Salisb.)Brieger were first recognied as a monophyletic group by Hg-
sater & Soto-Arenas (2005). Recently, Pinheiro & al. (2009a)
expanded the preious phylogenetic study of Hgsater & Soto-Arenas (2005) by analying 14 species. Strongly supported
clades representing distinct biogeographical regions were re-
coered, such as the Andean clade, with species mainly dis-
tributed along the Andean and Guianan mountain ranges, and
the Atlantic clade, which includes species distributed along
the Brailian coast, in the Cerrado and Caatinga egetation
domains (Fig. 1). The Atlantic clade comprises four species
(Epidendrum cinnabarinumSalm. ex Lindl., E. denticula-
tumBarb. Rodr., E. fulgensBrongn. and E. puniceoluteum
Pinheiro & Barros) (Fig. S1 in the Electronic Supplement).
Extensie ariation of morphological traits, allopatric s. sym-
patric/parapatric occurrence and wide geographic distribution
offer interesting possibilities for inestigating species limits,
reproductie isolation and phylogeography within this group.
Hybridiation and late generation introgression betweenE. ful-
gensandE. puniceoluteumwere identified using nuclear and
plastid microsatellites (Pinheiro & al.,2010), which suggests
an important role of interspecif ic gene flow in generating mor-
phological and chromosome number diersification. Moreoer,
pure parental indiiduals of both species and hybrid indiiduals
were identified with high confidence using Bayesian assign-
ment analysis, in agreement with preious obserations based
on morphological characters (Pinheiro & Barros, 2006). Eenwithin the geographical range of E. fulgens it was possible
to identify important phylogeographic breaks and historical
demographic eents (gene flow, bottleneck) that shaped the
current genetic patterns obsered in natural populations (Pin-
heiro & al.,2011).
To explore species limits and speciation hypotheses among
closely related species, the general lineage concept of species
was used as the conceptual framework to test the utility of dif-
ferent species criteria, in the context of integratie taxonomy.
The following four criteria for species delimitation, based on
morphological and DNA polymorphism, were tested: (1) phe-
netic differentiation (Sokal & Croello, 1970); (2) reciprocal
monophyly (de Queiro & Donoghue, 1988); (3) diagnosability,
of a new species was also tested in our study, described be
asE. flammeus. Qualitatie morphological characters, morp
metric data and multiple nuclear and plastid markers cle
indicate species limits within the Atlantic clade, clarify
eolutionary and speciation processes within this group.
cross-alidation of species identification using different to
following the general lineage species concept and an integ
tie taxonomic approach, proed to be useful for depict
complex eolutionary relationships that are commonly fo
among many plant groups.
MATERIALS AND METHODS
Group of interest. All species of the Atlantic clade w
analyed in this study.Epidendrum flammeusoccurs within
geographical domain of the Atlantic clade, and shares m
morphological characters withE. fulgensandE. denticulatAll of the specimens ofE.flammeuswere collected in one
nitic rock outcrop within the Borborema mountain range
northeastern Brail. Samples ofE. flammeuswere found du
recent fieldwork conducted to make floristic inentories of r
outcrops in the Serra da Borborema massif, within Caatin
Preliminary qualitatie morphological comparisons with o
specimens of subg.Amphyglottiumindicated that these col
tions might represent a new species. Additional herbarium m
rial was also found from close geographic localities, identi
asE. fulgens(see commentaries in species description). For
reason, samples ofE. flammeuswere included in this study
its status as a new species was inestigated. Population or
and sample sies are described in Table 1.Morphometric analysis. To quantify continuous
terns of morphological ariation among species of the Atla
clade (E. cinnabarinum,E. denticulatum,E. fulgens,E. pu
ceoluteum) and the new species described, ariation inflower traits (TableS1 in the Electronic Supplement) was m
ured in 420 indiiduals, using a digital ruler and follow
the procedure described in Pinheiro & Barros (2007a). Dis
minant function analysis (DA) was used to assess multi
ate morphological differentiation among populations. Wi
Lambda, jackknifed classification, which assigns unclassispecimens to groups, and F-to remoe statistics, which pro
an indication of the relatie importance of each ariable,
also reported. Mean and standard deiation alues for e
character and each species were calculated, and are depic
in Box-plot graphics. Data were analysed and tested for m
tiariate normality using SYSTAT .11.0. (SYSTAT Softw
Richmond, California, U.S.A.).
Molecular phylogenetic study. A recent molec
phylogenetic study of subg. Amphyglottium(Pinheiro &
2009a) was used as the basic framework to test the phylogen
position ofE. flammeus. The precise phylogenetic relationsh
of the new species were then determined by sequencing th
plastid regions, trnT-trnL, trnL-trnFand rpl32-trnLfor all s
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described E. flammeus. Epidendrum campestreLindl. and
E. nocturnumJacq. were used as outgroups. DNA extraction,
amplification reactions and plastid DNA sequencing were car-
ried out following Pinheiro & al. (2009c). All of the sequences
used in this study were submitted to GenBank (Appendix). A
total of 20 terminal operational taxonomic units (OTUs) were
selected for this study, and fie of them were represented by
more than one accession each (Epidendrum cinnabarinum,
E. fulgens,E. puniceoluteum,E. denticulatum,E. f lammeus).
Each sequence accession of the trnL-trnF, rpl32-trnLand
trnL-trnTregions was aligned with Clustal W option in the
BioEdit Sequence Alignment Editor (Hall, 1999). The resulting
automated alignment was manually edited in BioEdit .5.0.6
and then was exported as a Nexus file for maximum pa
mony (MP) and maximum likelihood (ML) analyses. The
analysis was performed following Pinheiro & al. (2009a), us
the criterion of Fitch (1971), excluding uninformatie char
ters, and with ACCTRAN optimiation. Ten thousand ad
tion sequence replicates were performed by stepwise addit
and holding 10 trees per replicate, and TBR branch swapp
on best trees. The MP analysis was performed with PAU
.4.0b10 (Swofford, 2002). The ML analysis was conduc
using RAxML .7.0.4 (Stamatakis, 2006) and RAxML-G
.1.1 (Silestro & Michalak, 2011). The GTR+ substitut
model, which allows rate ariation among sites, was de
mined using jModeltest .0.1.1 (Posada, 2008) under the Aka
Table .Epidendrumspecies and populations analyed in this study. Sample sies for the morphometric analysis, nuclear microsat-ellite markers (SSR) assays for assignment tests and plastid intergenic spacer sequences (CP) are indicated.
Species Population
Sample Sie
Morphometry SSR CP
Epi
dendrums
ubg.
Amphyglottium
Atlanticclade
E. cinnabarinumSalm. ex Lindl. Pirambu 25 1
Lagoa do Abaet 13 1
E. denticulatum Barb. Rodr. Oliena 9 20
Alcobaa 11
Poos de Caldas 5
Marambaia 10 20 1
Botucatu 13
So Paulo 22
Itapea 9 1
E. flammeusE. Pessoa & M. Ales Pedra do Cachorro 51 53 4
E. fulgens Brongn. Parati 22 1
Bertioga 26 20
Canania 30
Itaja 26
Florianpolis 33
Imbituba 32 20 1
Porto Alegre 17
E. puniceoluteum Pinheiro & Barros Imbituba 13 20 1
Canania 26
Pontal do Sul 27 20 1
E. calanthumRchb. f. & Wars. Serra Pacaraima 1
E. macrocarpum Rich. Recife 1
E. myrmecophorum Barb. Rodr. Araruama 1
E. radicansPa. ex Lindl. Oaxaca 1
E. secundum Jacq. Serra do Rio do Rastro 1
E. xanthinumLindl. Santa Brbara 1
group
E. campestreLindl. Santana do Riacho 1
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information criterion (AIC). To find the optimal likelihood tree,
we ran 100 independent tree searches on the combined matrix.
The support for indiidual branches was ealuated using non-
parametric bootstrapping (Felsenstein, 1985) with 1000 thor-
ough bootstrap replicates. The categories of bootstrap support
(BS) considered were unsupported ( 0.01 for all comparisons). The ordination diagram of
DA indicates a clear separation among the species, consider
the first three canonical axes (Fig. 2, Wilks Lambda = 0.00
P< 0.0001) which represent 97.5% of the total sample ariat
The fie most discriminant ariables in the model are len
of the callus of the lip, column length, length and width of
central lobe of the lip, and lip length. The jackknifed clafication matrix produced by DA shows an aerage of 98%
correct classification of indiiduals into the preiously assig
species, ranging from 100% correct classification inE. fulg
andE. cinnabarinumto 94% inE. f lammeus(Table S2 in
Electronic Supplement). The pattern of ariation of the ab
characters is represented by box-plots (Fig. S2 in the Electro
Supplement). High intraspecific ariability is obsered for
most all of the characters, which oerlap among most spec
OnlyE. cinnabarinumshows clear discontinuities, mainl
the length of the dorsal and lateral sepals, the petal and
callus of the lip (Fig. S2).
The trnL-trnFgene sequences were 1040 bp long, rp
trnL832 bp, and trnL-trnT683 bp, excluding flanking regio
insertions and ambiguously aligned fragments. The combi
molecular matrix consisted of 2557 aligned bp from the
terminal OTUs with nucleotide frequencies of: A = 0.3855,
0.1258, G = 0.1462, and T = 0.3425, with a transition/trans
sion ratio = 2 (kappa = 4.9217825).
The topologies of MP and ML trees recoered in this st
were identical, and for this reason only results from the
analysis are shown (Fig. 3). The main difference obsered
comparison to the study of Pinheiro & al. (2009a), was
position ofE. radicansnested within the Andean clade (FigSubgenusAmphyglottiumis supported as a monophyletic gr
(BS 100; Fig. 3). Strong support (BS 85) was obsered
both the Atlantic clade and subsect. Tuberculata. In the And
cladeE. radicansis sister to the other species,E. calanthum
E. macrocarpum. Within the Atlantic clade,E. cinnabarinu
sister to the other species with BS = 98. BothE. denticula
and E. flammeusare monophyletic, with bootstrap alue
90% and 92%, respectiely. In contrast to the results obtai
by Pinheiro & al. (2009a) using AFLP,E. fulgensandE. p
ceoluteumwere not monophyletic (Fig. 3).
Bayesian assignment results obtained by STRUCTU
and STRUCTURAMAcorrectly assigned all indiidual
preiously recognied species. The statisticL(K) proposed
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Canonical Axis 1 (59.0%)
lacinonaC
)%1.8(3si
xA
lacinonaC
)%4.03(2si
xA
A
B
Fig. .Discriminant scatter
plots showing morphological
similarities amongE. cinnabar-
inum(),E. denticulatum(),
E. f lammeus(),E. fulgens()
andE. puniceoluteum().
A,first and second axes repre-sent 89.4% of the ariation of
the dataset; B,first and third
axes represent 67.1% of the total
ariation among samples.
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probabilities calculated by STRUCTURAMA also showed
K=4 as the most probable number of genetic clusters (Table 2).
Assignment probabilities were higher than 0.9 for almost all
indiiduals, indicating a clear clustering and no indication of
admixture for almost all specimens (Fig. 4). Only indiiduals
ofE. puniceoluteumshowed signs of admixture withE. fulgens.
The eleen nuclear microsatellite loci analyed reealed
a total of 109 alleles among species of the Atlantic clade (ex-
cluding E. cinnabarinum). Of those, PAA identified 49 pri-
ate alleles distributed amongE. denticulatum,E. f lammeus,
E. fulgensand E. puniceoluteum. The number of diagnostic
alleles ranged from 16 inE. denticulatumandE. fulgensto 7 in
E. f lammeus(Table 3). Diagnostic characters were also foundfor both quantitatie and qualitatie morphological traits. Four
exclusie morphological characters were found inE. cinnabari-
num, two inE. denticulatumand one inE. fulgens. Diagnostic
morphological characters were not found inE. f lammeusand
E. puniceoluteum(Table 3).
DISCUSSION
When combining different data types to test hypotheses
about lineage diersification, authors go beyond the naming of
species by reconstructing the eolutionary processes inoled
in their origin (Schlick-Steiner & al., 2010). The characteria-
E. secundum
E. xanthinum
E. cinnabarinum
E. fulgens
E. puniceoluteum
E. denticulatum
E. calanthum
E. radicans
E. myrmecophorum
E. campestre
E. nocturnum
E. macrocarpum
E. cinnabarinum
E. fulgens
E. puniceoluteum
E. denticulatum
E. flammeus
E. flammeus
E. flammeus
E. flammeus
outgroup
100
100
64
92
100
98
100
90
0.0030 substitution/site
I II
III
IV
100
Fig. .Maximum-likelihood
phylogram ofEpidendrum
subg.Amphyglottium, includ
E. campestre andE. nocturn
as outgroups, based on com-
bined trnT-trnL, trnL-trnFa
rpl32-trnL data. The phylogshown is based on the best-
scoring maximum-likelihoo
tree. Clades recognied in th
study are indicated as follow
(I)E. subg.Amphyglottium,
(II)E. subsect. Tuberculata,
(III) Andean clade,(Iv) Atl
tic clade. Maximum likeliho
bootstrap support alues abo
50% are indicated on branch
Table .Estimates of cluster number (K) from STRUCTURE andSTRUCTURAMA analyses using nine microsatellite loci for threedatasets, includingE. flammeusandE. denticulatum,E. flammeus
E. fulgens, andE. flammeusandE. puniceoluteum. STRUCTURE hoc statisticsL(K) and Kand STRUCTURAMA posterior probabdistributionsE(K) are gien.
No. of
popula-tions (K)
STRUCTURE
ad hoc statistics
STRUCTURAposterior probab
distributions
L(K) K E(K)
1 4620.73 0.00
2 3781.25 8.40 0.00
3 3423.44 0.77 0.01
4 3103.32 32.72 0.43
5 3041.53 8.24 0.37
6 3067.23 0.41 0.14
7 3068.70 0.25 0.03
8 3091.25 1.06 0.01
9 3230.99 0.74 0.00
10 3125.18
TheL(K) alue in bold points at the beginning of similar estimatesobtained by log Pr(X|K) and indicates the correct number of group
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cryptic species hae been described, and the number of species
has decreased through the demonstration of conspecificity of
nominal taxa (Schlick-Steiner & al., 2010). Through its inter-
disciplinary approach, in which phylogeography, systemat-
ics, population genetics, ecology and morphology proide the
necessary framework for comparatie analyses of biodiersity,
integratie taxonomy should hae a deep impact across all
leels of biological organiation (Dayrat, 2005; Hendry & al.,
2010). Gien that the Neotropics harbour an impressie number
of species, and a still unknown number of species remain to
be described, the integration of seeral disciplines should be
especially helpful in this region (Dini-Filho & al., 2008; Pires
& Marinoni, 2010; Antonelli & Sanmartn, 2011).
Integratie taxonomy has included new concepts and
methods in order to delimit species, but a lack of consensus
about how data from different sources should be integrated is
still eident (Padial & al., 2010). Congruence among differ-
ent sources of data strengthens species delimitation in mostcases (Saolainen & al., 2006; Moccia & al., 2007; Reees
& Richards, 2011). This approach, named integration by con-
gruence (Padial & al., 2010), tends to promote taxonomic stabil-
ity, since most taxonomists will share similar opinions on the
alidity of a species supported by seeral datasets. Howeer,
recent radiations may often be oerlooked under this po
of iew (Padial & al., 2010; Barrett & Freudenstein, 20
On the other hand, the heterogeneous nature of eolution
forces often precludes full character congruence in specie
complete reproductie isolation, and such conflicts could in
cate ongoing speciation (Borba & al., 2002; Pillon & al., 20
Palma-Sila & al., 2011; Duminil & Di Michele, 2009). Beca
different species concepts often refer to different stages dur
speciation, full congruence among criteria will be achie
only in a late stage of differentiation (de Queiro, 1998)
such situations, species recognition could be based on a sin
dataset, following the framework of integration by cumulat
(Padial & al., 2010). A major adantage of this approach is
species which originated from recent radiations could be ea
delimited (Padial & al., 2010). Howeer, an oerestimation
species number is expected when only a single line of eide
is considered (Padial & al., 2010).
The contrasting approaches of integration by congrueand integration by cumulation need to be coordinated tak
into account the eolutionary forces associated leading
speciation in the taxonomic group of interest (Hendry &
2010; Padial & al., 2010). Since the concordance among d
types is not strictly necessary for justifying the existence
0%
10%
20%
30%
40%
50%
60%70%
80%
90%
100%
E. flammeus E. fulgens E. denticulatum E. puniceoluteum
Bertioga Imbituba OlivenaImbituba Marambaia Pontal do Sul Imbituba
Fig. .Maximum posterior assignment probabilities for 173 specimens ofE. f lammeus(Pedra do Cachorro population),E. fulgens(Bertioga
Imbituba populations),E. denticulatum(Oliena and Marambaia populations) andE. puniceoluteum(Imbituba and Pontal do Sul populatio
analyed with STRUCTURE withK= 4. Each ertical bar represents an indiidual. The proportion of color in each bar represents an indiiduassignment probability to different species. vertical dotted lines delimit different populations within species. See Table 1 for population det
Table .Number of fixed (priate) alleles and presence (1) and absence (0) of diagnostic morphological characters amongE. cinnabarinum,E. denticulatum,E. flammeus,E. fulgensandE. puniceoluteumdetected by PAA.
Character E. cinnabarinum E. denticulatum E. flammeus E. fulgens E. puniceoluteu
Fixed microsatellite alleles 16 7 16 10
Lip with brown dots 0 0 0 1 0
Flowers pink 0 1 0 0 0
Lip callus white 0 1 0 0 0
Dorsal sepal >18 mm long 1 0 0 0 0
Mid-lobe of lip entire 1 0 0 0 0
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distinct species (de Queiro, 1998; Padial & al., 2010; Barrett
& Freudenstein, 2011), we adopted the cumulatie integration
of multiple lines of eidence as a criterion to combine the dif-
ferent data sources inestigated in this study. Furthermore, spe-
cies delimitation will be discussed in the light of eolutionary
trends (Padial & al., 2010), based mainly on specif ic ecological
and genetic characteristics obsered in subg.Amphyglottium.
The phylogeny recoered in this study shows a topology
similar to the tree published by Pinheiro & al. (2009a). Within
the Atlantic clade, support for the monophyly criterion (de
Queiro & Donoghue, 1988) is obsered only for E. cinna-
barinum,E. denticulatumandE. flammeus(Table 4).Epiden-
drum fulgensandE. puniceoluteumare not monophyletic in this
clade. Thus, although monophyly is a proper criterion for spe-
cies delimitation under the general lineage concept, monophy-
letic groups might not be detected in the face of gene flow and
incomplete lineage sorting, factors that are common at lower
taxonomic leels (Knowles & Carstens, 2007).Epidendrum ful-gensoccurs in sympatry with three other species (E. denticula-
tum,E. puniceoluteum,E. secundum), and hybridiation eents
are known to occur among them (Pinheiro & al., in prep.). In
fact, hybridiation and introgression between E. fulgensand
E. puniceoluteumin six sympatric populations were detected
by nuclear and plastid microsatellites (Pinheiro & al., 2010).
Indeed, reproductie isolation mechanisms are not complete
among the species of the Atlantic clade (Pansarin & Amaral,
2008; Pinheiro & al., 2010; Pinheiro, unpub.), and support for
the reproductie isolation criterion (Mayr, 1942) was obsered
only forE. cinnabarinum. Moreoer, shared haplotypes were
detected by Pinheiro & al. (2010) in some indiiduals, suggest-
ing late generation backcrossing, which can mislead results
from phylogenetic trees. Howeer, the monophyly obsered for
E. cinnabarinum,E. denticulatum, andE. f lammeussuggests
the absence of haplotype sharing caused by late-generation
introgression, indicating the existence of strong postygotic
barriers, as F1 hybrids can be easily produced in crossing ex-
periments (Pinheiro, unpub.). Further studies inestigating the
genetic structure of other hybrid ones in the range of species of
the Atlantic clade will help to clarify the strength of reproduc-
tie barriers and species cohesion within this group.
Although plastid markers did not support a pattern ofmonophyly for all species, nuclear microsatellite markers cor-
rectly assigned all indiiduals to the known species (Fig. S3;
Fig. 4), and genetic intermediates were rarely found (E. fulgens
E. puniceoluteum,Fig. 4), strongly supporting the genetic
intermediates criteria (Table 4) (Mallet, 1995). The different
leels of resolution recoered by plastid and nuclear markers
could result from differential rates of gene flow between the
two genomes. According to Petit & Excoffier (2009), genomic
compartments with high leels of intraspecific gene flow are
predicted to experience less introgression (interspecif ic gene
flow), because genetic drift reduces the probability of an in-
trogressed allele to increase in frequency by chance. In plants
pollen moement is often the predominant form of gene f low
Recent empirical data show that seeds hae poorer disp
ibility than pollen, which could explain the higher leel
introgression obsered for maternally inherited plastid D
(Du & al., 2009; Palma-Sila & al., 2011). In fact, Pinheiro &
(2010, 2011) found that gene flow ia pollen inEpidendrum
more than ten-fold higher than that ia seeds. Consequeneen in the presence of hybridiation and introgression (P
heiro & al., 2010), nuclear markers are less introgressed beca
they experience high leels of gene flow. These results stron
agree with patterns that hae been obsered in many ot
angiosperms (reiewed in Petit & al., 2005; Duminil &
2007), and suggest that nuclear markers coupled with mult
cus assignment methods (e.g., Duminil & al., 2006; Hausd
& Hennig, 2010) represent the best option for species delim
tion. Howeer, additional analyses including additional pop
tions may be required to demonstrate that species show d
genetic structuring among populations, which might resu
an oerestimation of the number of true species (Pritch
& al., 2010). One important implication of this finding is
phylogenetic studies in Epidendrumspp. should make us
single- or low-copy nuclear loci instead of plastid mark
which might also be true for other plant groups.
Few morphological diagnostic characters supporting b
the phenetic (Sokal & Croello, 1970) and diagnosability (C
craft, 1983) criteria were found among species of the Atla
clade (Table 3). Four of seen morphological characters w
restricted to E. cinnabarinum(Table 3; Fig. S2), which h
larger flowers and are isited by hummingbirds (Pinhe
unpub.), in contrast to the other species, which hae smaflowers and are mainly pollinated by butterflies (Alme
& Figueiredo, 2003; Pansarin & Amaral, 2008; Fuhro &
2010).Epidendrum denticulatumshowed two diagnostic m
phological characters,E. fulgensone, and no exclusie flo
trait could be found for eitherE. flammeusorE. puniceolute
The morphological delimitation ofE. puniceoluteum(Pinh
& Barros, 2006) andE. flammeus(this study) was possible o
when considering the combination of multiple morphome
characters (Fig. 2; Table S1). Usually,Epidendrumspecie
subsect.Amphyglottiumlack strong premating barriers beca
many species share an extensie number of pollinator spec
(Byerychudek, 1981; Almeida & Figueiredo, 2003; Pans
& Amaral, 2008; Hgsater & Soto-Arenas, 2005). The oe
Table .Conformance ofEpidendrumspecies of the Atlantic cladewith four criteria for species delimitation.
Species Phenetic
Mono-
phyly
Diagnos-
ability
Genoty
clust
E. cinnabarinum Yes Yes Yes Yes
E. denticulatum Yes Yes Yes YesE. flammeus Yes Yes Yes Yes
E. fulgens Yes No Yes Yes
E. puniceoluteum Yes No Yes Yes
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TAXON61 (4) August 2012: 721Pessoa & al. Different tools to delimitEpidendrumspecies
the Atlantic clade. In general, plants with generalist pollination
systems might be less likely to experience strong directional
selection on floral traits (Waser, 1998), and diagnostic flower
characters could be less eident in such species (Pellegrino
& al., 2005).
The relaxed selection related to pollinator behaior could
be responsible for high leels of intraspecific morphological
ariation (Juillet & Scopece, 2010). In food-deceptie or-
chids, outcrossing preails because pollinators aoid plants
in the same patch, promoting gene flow by pollen oer long
distances and reducing the chances of geitonogamous pollina-
tion (Coolino & Widmer, 2005). This behaior is likely to
result in low stabiliing selection for floral traits in deceptie
species, and thus floral polymorphisms arising from muta-
tions can accumulate and increase intraspecific ariation in
natural populations (Juillet & Scopece, 2010). According to
Pinheiro & al. (2010), habitat selection contributes to species
cohesion as a potential post-mating, postygotic barrier thatlimits gene f low. Future studies should include long-term field
experiments, including reciprocal transplants and reproductie
manipulations, to proide direct tests of this hypothesis.
According to the results obtained in this study, species of
the Atlantic clade satisfy most of the species criteria examined
(Table 4), and thus merit recognition as distinct species. All
of the species satisfy at least three of the four species criteria
inestigated: (1) phenetic differentiation based on morphologi-
cal discontinuities obsered on morphometric results (Sokal
& Croello, 1970); (2) monophyly (de Queiro & Donoghue,
1988); (3) diagnosability (Cracraft, 1983), which was found
in both molecular and morphological traits; and (4) geno -
typic clusters recognied by a deficit of genetic intermediates
(Mallet, 1995), as shown by nuclear microsatellite data. The
multidisciplinary approach adopted here to inestigate spe-
cies limits in this orchid group allowed for the delimitation of
closely related species of the Atlantic clade and the recognition
of a new species,E. f lammeus. The new species is described
and illustrated below. An identification key for species from
the Atlantic clade is also proided. Species of subg.Amphy-
glottiumwere used as models to understand diersification
patterns inEpidendrum. The huge number of species in this
genus is an intrinsic impediment for multidisciplinary stud-ies, but different studies focusing on specific and smallerEpi-
dendrumgroups should be a suitable approach to understand
evolutionary patterns within this genus. Moreover, future
studies should consider the use of multiple lines of eidence to
inestigate species limits in different plant groups, in order to
depict eolutionary trends and speciation processes according
to an integratie taxonomic approach.
DESCRIPTION OF THE NEW SPECIES
Epidendrum flammeusE. Pessoa & M. Ales, sp. nov. Type:
Brail. Pernambuco, Municpio de So Caetano, RPPN
Epidendrum flammeus is morphologically related
E. denticulatum, butdiffersby haing yellow, orange or
flowers, these with a cured lip.
Description. Rupicolous, caespitose herb. Stems 10
73.0 cm long, 0.51.0 cm wide, simple, cane-like, terete. Lea
3.09.0 cm long, 0.83.0 cm wide, distichous, distribu
throughout the stems, coriaceous, lanceolate to elliptic, a
rounded, slightly emarginate, margin entire. Inflorescence
minal, racemose; peduncule 14.077.0 cm long, coered by ac
amplexcaulous sheaths; rachis of inflorescence 3.04.0 cm lo
Floral bracts 0.30.9 cm long, 0.10.2 cm wide, much sho
than oary, triangular, lanceolate, apex acute. Flowers 5
simultaneous, nonresupinate, yellow, orange or red. O
pedicellate, 1.22.9 cm long, 0.200.25 cm wide; dorsal se
0.71.2 cm long, 0.320.55 cm wide, oblong-oboate, apex ac
margin entire; lateral sepals 0.71.4 cm long, 0.390.60 cm w
oboate, falcate, apex acute, margin entire; petals 0.81.3
long, 0.300.59 cm wide, oboate to elliptic, apex acute, marentire thoughout or apical half erose. Lip 0.300.67 cm lo
0.91.6 cm wide, united with column, 3-lobed, base of the d
with a pair of ooid callus, and a longitudinal keel 0.250.48
long, 0.140.30 cm wide; lateral lobes 0.350.70 cm long, 0.
0.75 cm wide, semiorbicular, margin serrate to fimbriate, m
lobe 0.220.38 cm long, 0.300.68 cm wide, flabellate, a
bilobed, margin serrate to fimbriate. Column 0.51.1 cm lo
0.300.35 cm wide, apex of entral face with a pair of proj
tions on both sides, directed to lip base. Anther apical, o
Pollinia 4, ooid, laterally compressed. Cuniculus 0.70.9
long, penetrating about half the oary. Capsule 5.05.2 cm lo
1.31.9 cm wide, globose to ooid. Figure 5.
Paratypes. BRAzIL. Alagoas, Quebrangulo, REBIO
dra Talhada, 091517 S 362536 W, 25 Out 2011,B.Amo
1149(UFP); Paraba, So Joo do Tigre, APA das Onas, 6
2005,Dantas & al. 1442(JPB); Pernambuco, Bonito, Rese
municipal, 20 Jul 1995, M. Alves & al. 34545 (UFP!); i
Brejo da Madre de Deus, Faenda Bituri, 19 Apr 1959,D.
drade-Lima 59-3354 (IPA); ibid. So Caetano, RPPN Pedra
Cachorro, 081406 S 361125.5 W, 14 Jul 2007, P. Gom
684 (UFP!, MO!); ibid. So Caetano, RPPN Pedra do Cacho
0814 21.8 S 361120.3 W, 29 Aug 2010,D. Cavalcanti &
289(UFP!, PEUFR!).Etymology. The new species is named on behalf of
color of its flowers, which are mainly red, orange and yell
similar to a flame.
Distribution and ecologyEpidendrum flammeusis kno
from the states of Pernambuco, Paraba and Alagoas in the no
ern part of northeastern Brail (Fig. 1). The species occur
granitic rock outcrops (up to 1000 m high) on the Borbore
plateau, within the Caatinga biome. It grows in association w
other rupicolous plants such as bromeliads and orchid in isla
of egetation. Flowers hae been obsered during the entire y
with a clear flowering peak from December to March.
Many inentories from northeastern Brail (Pereira, 19
Gomes-Ferreira, 1990; Felix & Caralho, 2002) list specim
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Almeida, A.M. & Figueiredo, R.A. 2003. Ants isit nectaries of
dendrum denticulatum (Orchidaceae) in a Brailian rainforEffects on herbiory and pollination.Brazil. J. Biol. 63: 551
Antonelli, A. & Sanmartn, I. 2011. Why are there so many plant cies in the Neotropics? Taxon60: 403414.
Barbar, T., Palma-Silva, C., Paggi, G.M., Bered, F., Fay, M.FLexer, C. 2007. Cross-species transfer of nuclear microsate
markers: Potential and limitations.Molec. Ecol. 16: 3759376Barrett, C.F. & Freudenstein, J.V. 2011. An integratie approac
delimiting species in a rare but widespread mycoheterotrop
orchid.Molec. Ecol. 20: 27712786.Barros, F. de, Vinhos, F., Rodrigues, V.T., Fraga, C.N. & Pes
E.M. 2011. Orchidaceae. In: Lista de Espcies da Flora do BrJardim Botnico do Rio de Janeiro. http://floradobrasil.jbrj.
.br/2011/FB000179, accessed 20 Apr. 2012).Bierzychudek, P. 1981.Asclepias, Lantana andEpidendrum: A fl
mimicry complex?Biotropica 13: 5458.
Borba, E.L., Shepherd, G.J., Van den Berg, C. & Semir, J. 2Floral and egetatie morphometrics of fiePleurothallis (Ordaceae) species: Correlations with taxonomy, phylogeny, genariability, and pollination systems.Ann. Bot. 90: 219230
Buzzato, C.R., Davies, K.L., Singer, R.B., Santos, R.P. & V
den Berg, C. 2012. A comparatie surey of floral characin Capanemia (Orchidaceae: Oncidiinae).Ann. Bot.109: 1
144.Cozzolino, S. & Widmer, A. 2005. Orchid diersity: An eolution
consequence of deception? Trends Ecol. Evol. 20: 487494.
Cracraft, J. 1983. Species concept and speciation analysis. C
Ornithol. 1: 159187.Davis, J.I. & Nixon, K.C. 1992. Populations, genetic ariation,
the delimitation of phylogenetic species. Syst. Biol. 41: 4214Dayrat, B. 2005.Towards integratie taxonomy.Biol. J. Linn. S
85:407415.De Queiroz, K. 1998. The general lineage concept of species, spe
criteria, and the process of speciation. Pp. 5775 in: Howard,
& Berlocher, S.H. (eds.),Endless forms: Species and speciatOxford: Oxford Uniersity Press.
De Queiroz, K. & Donoghue, M. 1988. Phylogenetic systematics
the species problem. Cladistics4: 317338.Diniz-Filho, J.A.F., Telles, M.P.C., Bonatto, S.L., Eizirik, E., Frei
T.R.O., Marco, P., Santos, F.R., Sole-Cava, A. & Soares, T2008. Mapping the eolutionary twilight one: Molecular mark
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flow: Chloroplast ersus mitochondrial DNA in the Picea aspe
Epidendrum fulgensandE. puniceoluteumgrow in south-
ern and southeastern Brail, in coastal sand areas locally called
restingas (Barros & al., 2011).Epidendrum cinnabarinumis
known from restingas and the granitic rock outcrops found in
the Caatinga ecosystem in northeastern Brail, andE. denticu-
latumoccurs in southern, southeastern, and southern parts of
northeastern Brail, also growing on sandy soils along the coast
and in saanna egetation (Cerrado) (Barros & al., 2011, Fig. 1).
Morphological aff inities. The new species described
here has preiously been wrongly identified as E. fulgens,
because of similarities in flower color which ranges from red
to orange or yellow in the latter species (Fig. S1). The mor-
phology of the sepals, petals and lip are also similar in these
two species, as well as in all species ofEpidendrumplaced in
the Atlantic clade.
Epidendrum fulgensandE. cinnabarinumshare a similar
column morphology, but both species lack the pair of lateral
projections on the entral face of the apex directed to the lipsbase seen in other species of the Atlantic clade (E. denticula-
tum,E. flammeus,E. punniceoluteum). AlthoughE. flammeus
andE. cinnabarinum are sympatric (Fig. 1), their flowers differ
in sie and morphology. InE. puniceoluteum, the flowers are
red-purple and larger than inE. denticulatumandE. flammeus,
which hae flowers of similar sie.Epidendrum denticulatum
has pink flowers, whereas in E. flammeus they are yellow,
orange or red. In addition, E. flammeusshows a cured lip,
which is erect inE. denticulatum.
Two morphotypes can be recognied in the populations
ofE. flammeus(Fig. 5). In one of them, plants hae yellow
and slightly larger flowers, and the lip is more incured. The
second morphotype has red and smaller flowers and the lip is
only slightly incured. Specimens with intermediate charac-
teristics such as orange flowers, and with ariation in flower
sie and lip curature are common within populations. Clear
discontinuities between morphotypes were not obsered in
the morphometric analysis (Fig. 2). Fine-scale genetic studies
within populations could clarify the role of microhabitat pref-
erences between morphotypes, and a pollination surey could
clarify the role of color selection and the maintenance of the
flower ariability in this species.
Key to Epidendrum flammeusand related species
of the Atlantic clade of subg.Amphyglottium
1. Dorsal sepal >4 cm long; lateral lobes of the lip fimbriate
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. cinnabarinum
1. Dorsal sepal
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Appendix.Specimens used in morphological and molecular analyses with oucher information, countr y, locality, and GenBank accession numbers forrpl32-trnL, trnT-trnLand trnL-trnF, respectiely. Newly generated sequences are indicated by an asterisk.
Epidendrum cinnabarinumSalm. ex Lindl., Brail, Sergipe, Pirambu,F. Pinheiro 609(SP), JQ645971*, JQ645991*, JQ646011*;Epidendrum cinnabarinBrail, Bahia, Lagoa do Abaet,F. Pinheiro 608(SP), JQ645970*, JQ645990*, JQ646010*;Epidendrum dent iculatumBarb. Rodr., Brail, Bahia, OlieF. Pinheiro 626(SP);Epidendrum denticulatum, Brail, Bahia, Alcobaa, F. Pinheiro 627(SP);Epidendrum denticulatum, Brail, Minas Gerais, Pde Caldas,F. Pinheiro 628(SP);Epidendrum denticulatum, Brail, Rio de Janeiro, Marambaia,F. Pinheiro 610(SP), JQ645972*, JQ645992*, JQ6460Epidendrum denticulatum, Brail, So Paulo, Botucatu, F. Pinheiro 629(SP); Epidendrum denticulatum, Brail, So Paulo, So Paulo, F. Pinheiro(SP);Epidendrum denticulatum, Brail, So Paulo, Itapea, F. Pinheiro 611(SP), JQ645973*, JQ645993*, JQ646013*;Epidendrum f lammeusE. PessoM. Ales, Brail, Pernambuco, Pedra do Cachorro,F. Pinheiro 612, JQ645974*, JQ645994*, JQ646014*;Epidendrum flammeus, Brail, Pernambuco, PedrCachorro,F. Pinheiro 613, JQ645975*, JQ645995*, JQ646015*;Epidendrum flammeus, Brail, Pernambuco, Pedra do Cachorro,F. Pinheiro 614, JQ6459
JQ645996*, JQ646016*;Epidendrum flammeus, Brail, Pernambuco, Pedra do Cachorro,F. Pinheiro 615, JQ645977*, JQ645997*, JQ646017*;EpidendfulgensBrongn., Brail, Rio de Janeiro, Parati,F. Pinheiro 631(SP), JQ645978*, JQ645998*, JQ646018*;Epidendrum fulgens,Brail, So Paulo, BertiF. Pinheiro 616(SP);Epidendrum fulgens,Brail, So Paulo, Canania,F. Pinheiro 632(SP);Epidendrum fulgens,Brail, Santa Catarina , Itaja,F. Pinh633(SP);Epidendrum fulgens,Brail, Santa Catarina, Florianpolis,F. Pinheiro 634(SP);Epidendrum fulgens,Brail, Santa Catarina, Imbituba,F. Pinh617(SP), JQ645979*, JQ645999*, JQ646019*;Epidendrum fulgens,Brail, Rio Grande do Sul, Porto Alegre,F. Pinheiro 635(SP);Epidendrum puniceolutPinheiro & Barros, Brail, So Paulo, Ilha Comprida, F. Pinheiro 622(SP), JQ645984*, JQ646004*, JQ646024*;Epidendrum puniceoluteum,Brail,Paulo, Canania,F. Pinheiro 636(SP);Epidendrum puniceoluteum,Brail, Paran, Pontal do Sul,F. Pinheiro 621(SP), JQ645983*, JQ646003*, JQ6460Epidendrum calanthumRchb.f. & Wars., Brail, Roraima, Serra Pacaraima,F. Pinheiro 606(SP), JQ645968*, JQ645988*, JQ646008*;Epidendrum mrocarpumRich., Brail, Pernambuco, Recife,F. Pinheiro 618(SP), JQ645980*, JQ646000*, JQ646020*;Epidendrum myrmecophorumBarb. Rodr., BrRio de Janeiro,Araruama,F. Pinheiro 619(SP), JQ645981*, JQ646001*, JQ646021*;Epidendrum radicansPa. ex Lindl., Mexico, Oaxaca,F. Pinheiro(SP), JQ645985*, JQ646005*, JQ646025*;Epidendrum secundum Jacq., Brail, Santa Catarina, Serra do Rio do Rastro, F. Pinheiro 624 (SP), JQ6459JQ646006*, JQ646026*; Epidendrum xanthinumLindl., Brail, Minas Gerais, Santa Brbara, F. Pinheiro 625 (SP), JQ645987*, JQ646007*, JQ6460OUTGROUP:Epidendrum campestre Lindl., Brail, Minas Gerais, Santana do Riacho,F. Pinheiro 607(SP), JQ645969*, JQ645989*, JQ646009*;Epidrum nocturnumJacq., Brail, So Paulo, Canania,F. Pinheiro 620(SP), JQ645982*, JQ646002*, JQ646022*.
http://pritch.bsd.uchicago.edu/structure.htmlhttp://pritch.bsd.uchicago.edu/structure.htmlhttp://dx.doi.org/10.1007/s13127-http://dx.doi.org/10.1007/s13127-http://dx.doi.org/10.1007/s13127-http://dx.doi.org/10.1007/s13127-http://pritch.bsd.uchicago.edu/structure.htmlhttp://pritch.bsd.uchicago.edu/structure.html -
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Electronic Supplement (1 pp.) to: Pessoa & al. Different tools to delimitEpidendrumspeciesTAXON61 (4) August 2012Vol. 61 August 2012
International Journal of Taxonomy, Phylogeny and Evolution
Electronic Supplement to
Integrating different tools to disentangle speciescomplexes: A case study in Epidendrum (Orchidaceae)
Edlley Max Pessoa, Marccus Alves, Anderson Alves-Arajo, Clarisse Palma-Silva
& Fbio Pinheiro
Taxon:
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Table S.Morphological characters used in morphometric analyses, means and standard deviations ofEpidendrum cinnabarinum(Ec),E. denticu-
latum(Ed),E. flammeus(Efl),E. fulgens(Ef) andE. puniceoluteum (Ep) and results of discriminant analysis, including F-to-remove and canonicaldiscriminant values for the first three axes (CA 1, CA 2 and CA 3).
Characters Ec Ed Efl Ef Ep
F-to-
remove CA 1 CA 2 CA 3
Pedicel length 31.56 4.80 21.75 4.24 19.27 3.60 19.56 2.92 25.50 3.34 15.12 0.070 0.099 0.052
Dorsal sepal length 20.15 1.45 10.54 1.08 10.18 1.36 13.39 0.98 14.48 1.25 0.74 0.015 0.107 0.183Dorsal sepal width 5.94 0.91 3.85 0.44 4.26 0.52 5.34 0.50 5.72 0.53 3.06 0.062 0.283 0.443
Lateral sepal length 21.56 2.28 11.11 1.07 10.53 1.43 14.06 1.04 15.13 1.29 2.73 0.004 0.199 0.067
Lateral sepal width 5.72 0.62 4.13 0.52 4.70 0.57 5.80 0.46 6.24 0.65 15.21 0.459 0.897 1.032
Petal length 21.24 2.43 10.95 1.02 10.29 1.29 13.31 0.91 14.82 1.28 7.07 0.194 0.403 0.269
Petal width 5.23 1.02 3.63 0.64 4.27 0.72 5.44 0.81 5.82 0.75 4.19 0.082 0.13 0.325
Lip length 8.29 1.16 6.59 1.00 5.25 0.77 6.05 0.59 6.58 0.65 20.96 0.315 0.208 0.617
Lip width 15.41 1.79 12.14 1.62 12.09 1.58 14.56 1.38 17.09 1.51 18.72 0.226 0.013 0.443
Column length 14.56 1.11 6.82 0.91 7.98 1.36 12.21 0.89 10.38 0.87 58.59 0.493 1.000 0.013
Lateral lobe of lip length 7.06 0.36 6.31 0.88 5.15 0.83 6.25 0.67 7.33 0.67 19.26 0.838 0.423 0.279
Lateral lobe of lip width 7.42 0.65 6.05 0.98 5.77 0.86 8.27 0.98 7.80 1.16 20.09 0.241 0.276 0.359
Central lobe of lip length 5.54 0.50 4.28 0.86 3.11 0.39 3.38 0.49 5.15 0.61 21.6 0.201 0.679 0.629Central lobe of lip width 3.70 0.34 6.46 1.00 4.85 0.92 4.69 0.78 7.20 1.22 23.82 0.482 0.093 0.300
Callus of lip length 7.81 0.35 2.71 0.41 3.51 0.54 4.43 0.51 4.04 0.58 77.87 1.404 0.441 0.453
Callus of lip width 1.58 0.19 2.00 0.40 2.35 0.44 2.48 0.39 3.14 0.56 15.46 0.545 0.596 1.001
Table S.Results of jackknifed classification matrix from discriminant analysis performed on 420 individuals from Epidendrum cinnabarinum(38),E. denticulatum(79),E. f lammeus(51),E. fulgens(186) andE. puniceoluteum(66), indicating the percentage of correct classification of individualsin each species. Wilks Lambda = 0.011,P= 0.0000.
Species E. cinnabarinum E. denticulatum E. f lammeus E. fulgens E. puniceoluteum % correct
E. cinnabarinum 38 0 0 0 0 100
E. denticulatum 0 76 3 0 0 96
E. f lammeus 0 0 48 3 0 94
E. fulgens 0 0 0 186 0 100
E. puniceoluteum 0 1 0 0 65 98
Total 38 77 51 189 65 98
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Fig. S.Morphological and color variation within and amongEpidendrumspecies from the Atlantic clade.
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Fig. S.Box-plots for the 16 quantitative characters measured onEpidendrum fulgens(Ef),E. puniceoluteum(Ep),E. f lammeus(Efl),E. den-
ticulatum(Ed) andE. cinnabarinum(Ec.). Rectangles define 25 and 75 percentiles; horizontal lines show median; whiskers are from 10 to 90
percent ile; asterisks indicate extreme values.
Ef Ep Efl Ed Ec Ef E p E fl Ed Ec Ef Ep Efl Ed Ec
Ef E p Efl Ed Ec Ef Ep Efl Ed Ec Ef Ep Efl Ed Ec Ef E p Efl Ed Ec
Ef Ep Efl Ed Ec Ef Ep Efl Ed Ec Ef Ep Efl Ed Ec Ef E p E fl Ed Ec
Ef Ep Efl Ed EcEf Ep Efl Ed EcEf Ep Efl Ed Ec Ef Ep Efl Ed Ec
Ef Ep Efl Ed Ec10
20
30
40
50
PedicelLength[mm]
0
10
20
30
Dorsa
lSepalLength[mm]
2
3
4
5
6
7
8
Dors
alSepalWidth[mm]
0
10
20
30
Latera
lSepalLength[mm]
2
3
4
5
6
7
8
LateralSepalWidth[mm]
0
10
20
30
PetalLength[mm]
2
3
4
5
6
7
8
9
PetalWidth[mm]
3
4
5
6
7
8
9
10
11
LipLength[mm]
0
5
10
15
20
25
LipWidth[mm]
5
10
15
20
ColumnLength[mm]
2
3
4
5
6
7
8
9
10
LateralLobeofLipLength[mm]
2
3
4
5
6
7
8
9
10
11
LateralLobeofLipWidth[mm]
2
3
4
5
6
7
CentralLobeofLipLength[mm
]
2
3
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6
7
8
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CentralLobeofLipWidth[mm]
1
2
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5
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9
CallusofLipLength[mm]
1
2
3
4
5
CallusofLipWidt[mm]h
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Fig. S.Graphical methods allowing detection of the true number of groupsK, based on ad hoc statistics provided by STRUCTURE. A, Mean log
probabil ity of dataL(K) (SD) as a function ofK, where the values plateaus atK= 4; B, the magnitude of Kas a function ofK, where the modal
value of this distribution indicates the trueKor the uppermost level of structure, here four clusters.
K
L(K)
K
K
A
B
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