Annals of Botany 116: 35–48, 2015
doi:10.1093/aob/mcv062, available online at www.aob.oxfordjournals.org
Species delimitation in plants using the Qinghai–Tibet Plateau endemic Orinus
(Poaceae: Tridentinae) as an example
Xu Su1,2, Guili Wu1, Lili Li1 and Jianquan Liu1,*
1
State Key Laboratory of Grassland Agro-Ecosystem, School of Life Science, Lanzhou University, Lanzhou 730000,
PR China and 2Key Laboratory of Education Ministry of Environments and Resources in the Qinghai–Tibet Plateau,
School of Geography and Life Science, Qinghai Normal University, Xining 810008, PR China
* For correspondence. E-mail [email protected]
Received: 26 July 2014 Returned for revision: 27 November 2014 Accepted: 31 March 2015 Published electronically: 18 May 2015
Background and Aims Accurate identification of species is essential for the majority of biological studies.
However, defining species objectively and consistently remains a challenge, especially for plants distributed in remote regions where there is often a lack of sufficient previous specimens. In this study, multiple approaches and
lines of evidence were used to determine species boundaries for plants occurring in the Qinghai–Tibet Plateau, using the genus Orinus (Poaceae) as a model system for an integrative approach to delimiting species.
Methods A total of 786 individuals from 102 populations of six previously recognized species were collected for
niche, morphological and genetic analyses. Three plastid DNA regions (matK, rbcL and trnH-psbA) and one nuclear
DNA region [internal transcribed space (ITS)] were sequenced.
Key Results Whereas six species had been previously recognized, statistical analyses based on character
variation, molecular data and niche differentiation identified only two well-delimited clusters, together with a third
possibly originating from relatively recent hybridization between, or historical introgression from, the other two.
Conclusions Based on a principle of integrative species delimitation to reconcile different sources of data, the
results provide compelling evidence that the six previously recognized species of the genus Orinus that were
examined should be reduced to two, with new circumscriptions, and a third, identified in this study, should be
described as a new species. This empirical study highlights the value of applying genetic differentiation, morphometric statistics and ecological niche modelling in an integrative approach to re-circumscribing species boundaries.
The results produce relatively objective, operational and unbiased taxonomic classifications of plants occurring in
remote regions.
Key words: Orinus, Poaceae, species delimitation, genetic gaps, principal co-ordinate analysis, PCoA, niche
differentiation, Qinghai–Tibet Plateau.
INTRODUCTION
Species are fundamental in almost all biological disciplines for
numerous studies (Mayr, 1982), and they are especially important in the understanding of biodiversity. Any error in determining species units can lead to more serious errors in other
scientific analyses that use species as the basic units of analysis,
and such errors may result in an unpredictable waste of effort
or increase the costs of species conservation (Wiens, 2007).
However, what defines a species remains a matter of debate,
and several different species criteria have been proposed. The
combination of these two facts in our taxonomic treatments of
both plants and animals make it necessary to delimit species
more effectively. An emerging consensus is that species should
be delimited as evolutionarily distinct lineages, reconciling different sources of data, including morphological distinction, genetic distance from other species due to reproductive isolation
and termination of gene flow, niche differentiation and other
lines of evidence (de Queiroz, 1998, 2007; Stockman and
Bond, 2007; Bond and Stockman, 2008; Fujita et al., 2012;
Hendrixson et al., 2013; Mckay et al., 2013). Such approaches
have proved useful in addressing taxonomic debates about species delimitation in animals (Carstens and Dewey, 2010;
Harrington and Near, 2012; Salter et al., 2013). In addition, it
has been suggested that species delimitation in this way is testable and relatively objective, rather than subjective and biased
as in the past (Salter et al., 2013). Species-level difficulties in
assigning taxonomy are more serious in plants than in animals
(Levin, 1979; Briggs and Walters, 1997). There has even been
doubt as to whether the concept of a species can truly be applied to plants, because the large intralineage phenotypic variation and frequent interlineage introgressions that they display
make it difficult to separate them into discrete clusters
(Bachmann, 1998). It is therefore necessary to assess the discreteness of plant species, or the objectivity with which these
species have been delimited, using genetic and morphological
distinctions and other available data under an integrative species concept that reconciles different sources of data and meets
multiple criteria of the diverse species concepts. This is especially important for groups occurring in remote regions where
most taxonomic species have been established on the basis of
C The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.
V
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36
Su et al. — Determining species delimitation within the genus Orinus
only a few specimens, or even a single type specimen, and there
have been no statistical analyses of morphological variation
within and between species.
Genetic differentiation between distinct lineages can be determined quickly and cheaply by sequencing the commonly
used DNA fragments (e.g. DNA barcodes) for multiple individuals (Mallet, 1995). These fragments were selected from among
numerous candidate DNAs as being particularly effective in
discriminating between different taxonomic species (Besansky
et al., 2003; Kress and Erickson, 2008; Li et al., 2011).
Therefore, the species delimited based on genetic differentiation with these DNA fragments cannot only be compared across
different genera and family, but can also be used for further molecular identifications in the future. Based on genetic differentiation, numerous cryptic species lacking morphological
differentiation have been revealed in both animals and plants
(Yassin et al., 2008; Ragupathy et al., 2009). In plants, three
plastid DNA fragments, rbcL, matK and trnH-psbA, were initially selected as the core DNA barcodes (Knoop, 2004; Kress
and Erickson, 2007; Fazekas et al., 2008; Hollingsworth et al.,
2009). Later, through analysis of a large comparative data set,
the nuclear rDNA internal transcribed spacer (ITS) region was
identified as a novel core barcode (Chen et al., 2010; Li et al.,
2011; Wang et al., 2011). It should be noted that plastid DNA
is usually inherited maternally in angiosperms, whereas the ITS
region is derived from both parents (McCauley et al., 2007).
Inconsistent interspecific relationships inferred from ITS data
on the one hand and plastid DNA data sets on the other may
suggest a hybrid origin, or the effects of introgression, for the
sampled species (Besnard et al., 2007). Additive sites with mutations making them divergent from those of the parents also
occur in hybrid species or populations with a hybrid introgression history if the number of generations has been insufficient
to homogenize all ITS copies within an individual genome by
concerted evolution (Alvarez and Wendel, 2003). Due to the
random nature of concerted evolution, the hybrid descendants
sometimes comprise a paraphyletic lineage (Alvarez and
Wendel, 2003; Harpke and Peterson, 2006; Grimm and Denk,
2008). Genetic data based on sequencing of plastid DNA and
ITS can therefore not only discriminate between closely related
species that have undergone dichotomous divergence, but also
identify species or populations that putatively originated as hybrids between independent lineages (Harding et al., 2000).
However, few empirical studies in plants are involved in these
lines of genetic and other evidence necessary for delimitating
species. In this study, we present such a case study using
Orinus (Poaceae) occurring in the Qinghai–Tibet Plateau
(QTP) as a model system.
As the largest and highest plateau, with an average elevation
of >4000 m, the QTP has received a great deal of attention
from botanists in many countries. Around 9000 plant species
have been recorded as occurring there, and >18 % of the species are endemic (Wu, 2008). However, only approx. 20 genera
are believed to be endemic (Wu et al., 1995). Because it is difficult to access the QTP, the numbers of specimens collected for
most endemic genera and species are much smaller than those
available for species occurring in other regions. Some endemic
species have been established based on a few specimens, or
even just only the type specimen. In addition, the species-rich
genera in the QTP have diversified extensively within a short
time scale, and ongoing hybridization events have therefore
been frequent due to incomplete reproductive isolation (Liu
et al., 2006). It is likely that some endemic ‘species’ have been
described based on plants from the hybrid zones between two
closely related species (Zha et al., 2010). If this is the case,
these hybrids have not evolved into distinct lineages that lack
distinct niche differentiation from their parental species through
occupying new habitats. In contrast, the new habitats created by
the extensive QTP uplifts and the Quaternary climatic oscillations favoured the development of homoploid hybrid species as
distinct lineages evolving independently from their parental
species (Liu et al., 2014; Sun et al., 2014). Despite the rarity of
homoploid speciation in plants (Rieseberg et al., 1997), three
diploid hybrid species have been found in the QTP, and they all
occupy different niches from those of their parental species
(Ma et al., 2006; Liu et al., 2014; Sun et al., 2014).
In the genus Orinus Hitchc., three widely distributed species
in the QTP were described in the 1960s: O. thoroldii (Stapf ex
Hemsl.) Bor, O. kokonorica (K.S.Hao) Tzvelev and O. anomala
Keng f. (Tzvelev, 1968). The latter two species have largely
overlapping distributions in the north-eastern QTP. However,
three more species, occurring within the distributional regions
of these species, have since been described. For example, Zhao
and Li (1994) reported on O. tibeticus N.X.Zhao, based on only
type specimens, from a small area in Tibet, and O. alticulmus
L.B.Cai & T. L.Zhang and O. longiglumis L.B.Cai & X.Su
from several small locations of Qinghai and Xizang (Zhao and
Li, 1994; Chen and Phillips, 2006; Zhang and Cai, 2008; Su
and Cai, 2009). All of the six Orinus species referred to above
are distributed in alpine regions at elevations between 2230 and
5200 m. The wide distribution of the genus and the relatively
small number of species makes Orinus eminently suitable as an
exemplar for examining species delimitation in the remote
QTP. We explored the sites where the holotype of each species
was obtained and collected all possible representative populations of Orinus across its distribution range in the QTP. We first
examined species delimitation based on the genetic differentiations revealed by the maternally inherited plastid DNA fragments and the biparentally inherited nuclear ITS. We then
established morphological clusters based on statistical analyses
of variable traits at the population level. Finally, we tested
whether the clusters obtained based on genetic and morphological distinctions show distinct niche differentiation. Our aim was
to use an integrative approach to reconcile multiple species criteria to delimit operational species boundaries in the genus
Orinus.
MATERIALS AND METHODS
Field exploration and sample collections
We explored all localities in which type specimens for six species of Orinus Hitchc. had been collected previously, and successfully collected material in these locations. We further
extended our field expedition to cover all possible distribution
localities of each species according to the records for all specimens held in the herbaria. In total, we collected 786 individuals
from 102 populations of the six species across all distributions
of the genus over the QTP (Fig. 1; Supplementary Data Table
S1). We used a hand-held eTrex GIS (Garmin) to measure the
Su et al. — Determining species delimitation within the genus Orinus
37
O.kokonorica
O.anomala
O.thoraldii
O.tibeticus
glacier
5000 m a. s. I.
4500 m
4000 m
O.longiglumis
3000 m
2000 m
1000 m
0m
0
50
100
150 200 km
O.alticulmus
FIG. 1. Current geographical distribution and sampling sites for six previously established species (shown in different colours) in the genus Orinus on the
Qinghai–Tibet Plateau. Three morphological clusters are outlined with dotted lines in different colours.
latitude, longitude and elevation of each sampling site. We
identified each population as one of the six previously recognized species on the basis of species morphological characteristics described in the past (Zhao and Li, 1994; Chen and
Phillips, 2006; Zhang and Cai, 2008; Su and Cai, 2009) and locality records for these species in the herbaria. Voucher specimens of representatives of all populations were stored at
Lanzhou University. All the populations examined in the field
were used for niche modelling and comparisons. At least five
individuals from each population, separated by at least 20 m,
and having an inflorescence and/or fruits, were dried as specimens for use in further statistical analyses of morphological
variation. At the same time, fresh leaves from each individual
were immediately dried with silica gel and the dried materials
were subsequently used for extracting total DNA.
DNA sequences and phylogenetic analyses
For each sample, total genomic DNA was extracted from silica-dried leaves using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). We amplified
and sequenced three plastid DNA regions (matK, rbcL and
trnH-psbA) and one nuclear region (ITS). The forward and reverse primers used for the polymerase chain reaction (PCR)
were as follows: ‘390F’ and ‘1326R’ for matK (Cuénoud et al.,
2002); ‘1F’ and ‘724R’ (or ‘627F’ and ‘1504R’ as an
alternative reverse primer) for rbcL (Lledó et al., 1998); ‘trnH’
and ‘psbA’ (Sang et al., 1997) for trnH-psbA; and ‘ITS1’ and
‘ITS4’, respectively, for ITS; this primer pair amplifies both internal spacers and the 58S gene (White et al., 1990). The PCR
amplifications were performed in 25 mL reaction mixtures containing 25 mL of 10 PCR buffer, 025 mL of Taq DNA polymerase (5 U mL–1; TaKaRa Biotech, Dalian, China),
025 mmol L–1 dNTPs, 02 mmol L–1 MgCl2, 20 mmol L–1 each
primer and 10 mL of template DNA. The cycling parameters
used for matK, trnH-psbA, rbcL and ITS were 94 C for 5 min,
followed by 36 cycles of 94 C for 50 s, 49–58 C for 50 s and
72 C for 1 min or 1 min and 40 s, with a final extension for
10 min at 72 C. The PCR products were purified using a
TIAN-quick Midi Purification Kit (Tiangen Biotech, Beijing,
China) according to the manufacturer’s instructions and sequenced in both directions with PCR primers using an ABI
3130 l automated sequencer (Applied Biosystems, Foster
City, CA, USA). All sequences reported in this study have been
deposited
in GenBank
under accession
numbers
KP302391–KP303274.
All plastid DNA fragments were linked together as a single
data set. Alignments of plastid DNA and ITS sequences were
performed using MUSCLE (Edgar, 2004) and refined manually
in MEGA 5 (Tamura et al., 2011). We tested congruence between the ITS and plastid DNA data sets by applying the incongruence length difference (ILD) test, with 1000 replicates, to
potentially parsimony-informative characters using the tree
38
Su et al. — Determining species delimitation within the genus Orinus
bisection–reconnection (TBR) branch swapping algorithm and
the number of trees retained for each replicate limited to 1000
(Farris et al., 1995). Both plastid DNA and ITS data sets individually, and a combination of both data sets, were subjected to
maximum parsimony (MP) and maximum likelihood (ML)
analysis using PAUP* 40b10 (Swofford, 2002) and Bayesian
inference (BI) using MrBayes 3.1 (Ronquist and Huelsenbeck,
2003). To evaluate clade support, bootstrap values were calculated from 1000 replicates (Felsenstein, 1985). Analysis of the
data sets for ML and BI was performed using a general timereversible model with gamma distribution split into four categories (GTR þ G4), as determined by the Akaike information
criterion in PAUP/ModelTest 3.7 (Posada and Crandall, 1998;
Swofford, 2002). Other parameters were set as the default. BI
consisted of two parallel runs each of four incrementally heated
chains, and 3 million generations sampled every 1000 generations. The output was diagnosed for convergence using Tracer
v.1.3 (Rambaut and Drummond, 2007), and summary statistics
and trees were generated using the last 2 million generations,
well beyond the point at which convergence of parameter estimates had taken place. For the ML trees, the reliability of each
internal branch was evaluated based on 1000 bootstrap replicates. In MP analyses, heuristic searches were carried with
1000 random addition sequence replicates. Ten trees were
saved at each step during stepwise addition, and TBR was used
to swap branches. Characters were unordered and equally
weighted. Gaps were treated as missing data, and multistate
data were interpreted as uncertainty because there were no distinct differences when they were coded as additional characters
(not shown here). To evaluate clade support, bootstrap values
were calculated from 1 000 000 replicates. Ten trees were held
at each step during stepwise addition for bootstrap. Leptochloa
viscida (Scribn.) Beal was used as the outgroup. We further estimated divergence time based on plastid DNA and ITS variation between the three major clades. We used BEAST to
estimate divergence between these clades (Drummond and
Rambaut, 2007) based on plastid DNA and ITS mutation rates
[03 10–9–10 10–9 per site year–1 for plastid DNA
(Romaschenko et al., 2014); 58 10–9–81 10–9 per site
year–1 for ITS (Wolfe et al., 1987)] previously reported for
other perennial genera in the same family.
Species delimitation
In addition to the phylogenetic analyses described above, we
constructed genetic clusters based on the following two
approaches. First, we used TCS 1.21 (Clement et al., 2000) to
construct statistical parsimony networks and we calculated
maximum connection steps at 95 % boundaries. We examined
discontinuities in sequence variation by using a statistical parsimony analysis. Secondly, we used the GMYC model (Pons
et al., 2006) to identify the possible species units within each
data set. This approach identifies the point in the phylogeny
where coalescent branching events within species transition to
those corresponding to species-level divergence. Gene tree uncertainty was estimated by Bayesian implementation (bGMYC;
Reid and Carstens, 2012) through sampling over the posterior
distribution of sampled gene trees. All ultrametric gene trees
were constructed by BEAST v1.6.1 (Drummond and Rambaut,
2007) under a strict clock model using a MCMC (Markov chain
Monte Carlo) run of 5 107 generations with sampling every
5 103 generations for the bGMYC runs. Following 40 %
burn-in, we trimmed the posterior distributions to 100 trees
(evenly sampled throughout the posterior), and input them for
bGMYC analyses. We used default settings, and the parameter
py2 was set to 1.2 and the starting number of species was set to
half the total number of tips. For all analyses, we ran bGMYC
for 20 000 generations, with a burn-in of 10 000 generations,
and sampled every 200th generation.
We used the genealogical sorting index (gsi; Cummings
et al., 2008) to assess the taxonomic distinctiveness of the species delimited by phylogenetic analyses, TCS and bGMYC.
The putative species delimited by these approaches were tested
against a null hypothesis of no divergence. We then calculated
an ensemble gsi (egsi) and gsi for each delimited species data
set using the Genealogical Sorting Index web server (http://
www.genealogicalsorting.org). We used the 50 % majority rule
consensus gene trees as input trees. We estimated a P-value by
using 10 000 permutations through the null hypothesis that the
degree of exclusive ancestry is observed by chance alone (i.e.
no divergence). The significance was inferred at P < 001.
Finally, we compared genetic divergence within and between
‘species’ (as defined previously), and within and between
‘clades’ (as identified here) using uncorrected sequence divergence p-distances.
Clustering analyses of morphological variations
We first looked for morphological variations within populations and compared these variations against the identifying descriptions available for each species. In order to test for
consistent differences between species, we observed and measured six characters for the vegetative organs (plant height,
presence/absence of rhizome scales, leaf length, leaf width, and
presence/absence of hairs covering leaf blades and sheaths; if
hairs are present, a sparse or dense state was scored), and eight
characters for the reproductive organs (panicle length, spikelet
length, floret number per spikelet of the almost whole inflorescence, upper glume length and presence/absence of hairs covering upper glumes, the lowest lemma length and presence/
absence of lemma hairs, and spikelet colour). A total of 420 individuals from 64 populations were selected for morphological
analysis (Supplementary Data Table S1; n ¼ 5–10 individuals
per population). To produce a graphical representation of variation among species, a principal co-ordinate analysis (PCoA)
was performed (Bitner-Mathé and Klaczko, 1999) using the
Multivariate Statistics Package (MVSP 3.1; http://www.kovcomp.com/, accessed March 2008) and the PCoA component
scores were used to group all individuals evaluated.
Ecological niche modelling
In order to examine niche divergence between the targeted
species, we used the maximum entropy model and machinelearning algorithm implemented in MAXENT version 3.3.3k
(Phillips et al., 2006; Phillips and Dudı́k, 2008) to predict niche
models based on the collection sites used in our field study and
on herbarium records. At the same time, we compiled an
Su et al. — Determining species delimitation within the genus Orinus
environmental data set comprising 19 ecological variables together with elevation information from the WORLDCLIM
database at a resolution of 2.5 in (Hijmans et al., 2005; www.
worldclim.org). We tested pairwise correlations between the 20
variables within the distribution of each species and also across
different species. We selected seven environmental variables
(i.e. elevation, mean diurnal range, minimum temperature in
the coldest month, annual temperature range, annual precipitation, precipitation in the driest month and seasonality of precipitation) and used high pairwise Pearson correlation coefficients
of r 06 to perform the tests below, in order to minimize biased fitting of niche models. Default parameters for MAXENT
were adopted, and 80 % of the species records were used for
training and 20 % for testing the model. Graphics were drawn
using DIVA-GIS 75 (http://www.worldclim.org/).
In order to measure the niche similarity between species, we
used ENMTools 1.3 (Warren et al., 2008, 2010) to calculate
Schoener’s D (Schoener, 1968) and standardized Hellinger distance (calculated as I). We performed an identity test and background test using ENMTools 1.3. The null distribution of niche
models in the identity test was obtained based on 200 pseudoreplicates generated by random sampling from the data points
pooled for each pair of species. We used 200 replications to calculate the null distribution for each pair of species in both directions in the background test. We determined measures of niche
similarity (D and I) between species with null distributions. We
tested significance and drew histograms using R 213 (http://
www.r-project.org/).
RESULTS
Phylogenetic analyses of plastid DNA and ITS sequences
After sequencing matK, rbcL and trnH-psbA for all the individuals examined from the six species, 48 different sequences
(haplotypes) were obtained from the combination of all variations identified across the three plastid DNA fragments. The
aligned data set consisted of 2898 bp, of which 19 were potentially parsimony informative. Parsimony analysis identified 152
trees with 76 steps, a consistency index (CI) of 097 and a retention index (RI) of 099. The strict MP consensus tree (Fig. 2A)
was generally congruent with the ML tree (–lnL ¼ 43980004
for the best model, GTR þ G þ I) (Fig. 2A). All analyses identified only two clades (A and B): one consisted of O. anomala,
O. kokonorica and O. alticulmus, and the other comprised
O. thoroldii, O. tibeticus and O. longiglumis.
We also recovered 48 different ITS sequences (ribotypes)
from these sampled individuals. The aligned ITS data set was
624 bp in length with 43 variable sites, of which 37 were potentially parsimony informative. Parsimony analysis identified 222
equally parsimonious trees with 146 steps, CI 093 and RI 097.
The strict MP consensus tree (Fig. 2B) and ML tree
(–lnL ¼ 15190023, the best-fit model being GTR þ G þ I)
(Fig. 2B) were mostly congruent in topology with the 50 % majority rule consensus tree derived from Bayesian analysis (under
the GTR þ G þ I model) (Fig. 2B). Four clades (A, B, C1 and
C2) were recovered with a high level of statistical support:
clade A was made up of individuals from O. anomala, O. kokonorica and O. alticulmus, whereas clade B consisted of O. thoroldii, O. tibeticus and O. longiglumis. The sequences in clade
39
C1 were those recovered from O. anomala and O. thoroldii
populations distributed in western Sichuan (populations 26–39,
Fig. 1). Clade C2 was composed of two O. thoroldii populations from the south-eastern QTP, but near to Tibet. It should
be noted that all populations in clades C1 and C2 shared plastid
DNA haplotypes with clade A. We also found that ITS sequences in clades C1 and C2 showed distinctly additive sites,
with double peaks of two different nucleotides that distinguish
the two major clades A and B, although such a scenario also occurred in some ribotypes of clades A and B (Fig. 3) in the populations collected from those sites adjacent to those of clades C1
and C2 (Fig. 1: populations 18–22, 40 and 50–68 of clade A,
and populations 70–78 of clade B). In addition, the other nucleotides that are differentiated between the two major clades are
the same as those of either clade A or clade B in these two
small clades. Seven mutations shared nucleotides between
C1–C2 and A (Fig. 3). However, we found that only two mutations (Fig. 3: variable positions 198 and 487) were shared between C1–C2 and B, although five additional mutations (Fig. 3:
variable position 63, 109, 207, 526 and 581) were shared between C2 and B. Seven mutations were found to be unique to
clade C2, and these were totally different from those identified
in clades A and B (Fig. 3: variable position 45, 66, 118, 197,
464, 525 and 566). Because of these endemic mutations, and
since there is less sharing of mutations between A and C2 than
between clades A and C1, C1 and C2 should be regarded as
monophyletic, rather than paraphyletic.
Partition homogeneity analyses showed weak incongruence
between the plastid DNA data set and the nuclear ITS data set
(P ¼ 0658). This incongruence might derive from hybridization and back introgression, as indicated by the presence of
some additive ITS sites. Analyses of the combined data set produced clades similar to that based on the ITS data set because
the latter furnished a larger number of informative sites (results
not shown here). We further estimated divergence among the
three major clades (A, B and C) based on published mutation
rates for Poaceae. The time of divergence between clades A
and B based on the plastid DNA or ITS data set was estimated
to be 203 (95 % HPD: 116–301) million years ago (Ma) or
258 [95 % higheest posterior density (HPD): 178–351] Ma,
respectively. Additionally, the divergence time between clade
C1 or C2 and A based on the ITS genetic distances was estimated to be 101 (95 % HPD: 060–147) or 181 (95 % HPD:
118–250) Ma.
Species delimitation and tests based on genetic evidence
Both plastid DNA and ITS data sets were subjected to TCS
network analyses based on statistical parsimony in order to detect the presence of species-level groups by identifying independent clusters (Templeton et al., 1992). The network
analyses recovered either two or four clusters (Fig. 4), as did
phylogenetic analyses. We also constructed logarithmic lineage
through time plots based on fitting of the position of the speciation to coalescence transition from the GMYC model. Again,
two or four clusters with >95 % of the posterior distribution
were identified based on the plastid DNA or the ITS data set
(results not shown here). We then tested the genealogical distinctness of these putative ‘species’ clusters identified by
Su et al. — Determining species delimitation within the genus Orinus
40
A
B
A
A
100/100/100
100/100/100
C1
100/100/100
C
C2
100/100/100
100/100/100
100/100/100
100/100/100
B
L. viscida
B
100/100/100
L. viscida
FIG. 2. Strict MP consensus tree based on (A) plastid DNA and (B) nuclear ITS sequences. Each tree is topologically congruent with the ML tree and Bayesian consensus tree based on the same data set. Bootstrap support values from Bayesian and ML analyses and the corresponding posterior probabilities from MP analyses are
given above the branches (>50 % values). The colours represent different species as in Fig. 1. If a sequence was identified in two or more different species, only the
species with the largest number of individuals is shown.
phylogenetic, TCS and GMYC analyses based on the estimated
gsi values (Table 1). All clusters identified by these analyses received high support from gsi tests (Table 1).
Finally, we compared genetic divergence between and within
the ‘species’ described previously and the ‘clades’ identified
here (using uncorrected sequence divergence p-distances)
(Fig. 5). Based on the plastid DNA data set, most inter-‘species’
divergence values were smaller than intra-‘species’ differentiation (Fig. 5). However, the interclade divergences were greater
than the intraclade divergences. Similarly, based on the ITS data
set, we found that intra-‘species’ divergence values were greater
than inter-‘specific’ values. However, the reverse pattern was
found if comparisons were made between and within the clades.
We further compared genetic divergence values between
C1 þ C2 and clade A or B. Again we found that the divergences
between them were clearly larger than those within them.
Morphological clustering based on statistical analyses
of variable traits
We observed and measured 14 morphological traits comprising plant height, leaf length, leaf width, presence/absence of
hairs covering the leaf blades and sheaths, panicle length, spikelet length, floret number per spikelet of the almost whole inflorescence, upper glume length and presence/absence of hairs
covering upper glumes, the length of the lowest lemma and
presence/absence of lemma hairs, spikelet colour and presence/
absence of rhizome scales (see Fig. 7A, B). Some of these traits
were previously used to delimit the six QTP Orinus species discussed herein. The data measurements obtained for the above
14 characters were subjected to PCoA. Based on the magnitudes of eigenvalues, the variance values obtained for the first
three principal components were 40953, 22164 and 12946 %,
respectively (cumulative value: 76063 %). Of these, the first
two principal components accounted for 63117 % of the variation in 14 characters, and they thus provided the main information used for further clustering. We made a two-dimensional
scatter plot using the first two principal components. PCoA
analysis identified three distinct clusters (Fig. 6). The first cluster (A) consisted of 126 individuals sampled from 22 populations of O. anomala, O. kokonorica and O. alticulmus. This
cluster corresponded closely to the clade A which had been
identified from plastid DNA and ITS sequence variations. It differed from the second cluster (B) in that plants in cluster A had
Su et al. — Determining species delimitation within the genus Orinus
41
Clade
3
2
4
5
6
3
6
6
9
0
9
4
1
0
7
1
0
9
1
1
8
1
5
1
1
7
6
1
9
1
1
9
2
1
9
3
1
9
4
Variable positions
1 1 2 2 4
9 9 0 1 1
7 8 7 9 2
4
2
8
4
4
5
4
6
1
4
6
4
4
8
7
5
2
5
5
2
6
5
2
8
5
5
1
5
5
4
5
6
6
5
8
1
5
9
4
6
0
4
A-1 (45 individuals)
C
T
A
T
T
G G
T
G
C
T
A
C
A
C
C
T
A
C
A
A
T
G G
K
C
T
A
T
G G
T
C
G
A-2 (38 individuals)
C
T
A
T
T
G G
T
G
C
T
A
C
A
C
C
Y
A
C
A
A
T
G G
K
C
T
A
T
G G
T
C
G
A-3 (30 individuals)
C
T
A
T
T
G G
T
G
C
T
A
C
A
C
C
T
A
C
A
A
T
G G
T
C
T
A
T
G G
Y
C
G
A-4 (24 individuals)
C
T
R
T
T
G G
T
G
C
T
A
C
A
C
C
T
A
C
A
A
T
G G
T
C
T
A
T
R
G
T
C
G
C1-1 (13 individuals)
C
T
A
T
C
G G
T
G
M
T
A
C
A
C
C
C
A
C
A
A
T
G G
G
C
T
A
T
A
G
C
Y
G
C1-2 (10 individuals)
C
T
A
T
C
G G
T
G
A
T
A
C
A
C
C
C
A
C
A
M
T
G G
G
C
T
A
T
A
G
C
C
G
C1-3 (12 individuals)
C
T
A
T
C
G G
T
G
C
T
A
C
A
C
C
C
A
C
A
A
T
G G
G
C
T
A
T
A
G
C
C
G
C2-1 (7 individuals)
C
C
G
C
T
G G
C
T
C
W
A
C
R
C
T
C
G
C
A
A
Y
C
T
G
T
G W
T
A
T
T
C
R
C2-2 (5 individuals)
C
C
G
C
T
G G
C
T
C
T
A
C
A
C
T
C
G
C
W
R
C
C
T
G
T
G
A
T
R
T
T
C
G
C2-3 (8 individuals)
C
C
G
C
T
G G
C
T
C
T
A
C
R
C
T
C
G
C
A
A
Y
C
T
G
T
G
A
T
A
T
T
Y
G
C2-4 (5 individuals)
C
Y
R
C
T
G G
C
T
C
T
A
C
A
C
T
C
G
C
A
R
T
C
T
G
T
G
A
T
A
T
T
C
G
B-1 (62 individuals)
T
T
G
T
T
G
C
G
C
A
C
T
G
Y
C
C
G
T
T
C
T
G G
G
C
G
T
C
A
G
T
T
A
B-2 (36 individuals)
T
T
G
T
T
A
A
C
G
C
A
C
T
G
Y
C
C
G
T
T
C
T
G G
G
C
G
T
Y
A
G
T
T
A
B-3 (45 individuals)
T
T
G
T
Y
A
A
C
G
C
A
C
T
G
T
C
C
G
T
T
C
T
G G
G
C
G
T
C
A
G
T
T
A
B-4 (49 individuals)
T
T
G
T
T
R
A
C
G
C
A
C
T
G
T
C
C
G
T
T
C
T
G G
G
C
G
T
C
A
G
T
T
A
A
FIG. 3. Nucleotide sites showing variation between the major ITS sequences in the clades A, B, C1 and C2 identified in the present study.
A
B
Clade A
Clade B
Clade C
FIG. 4. Cluster networks obtained by TCS analyses based on (A) plastid DNA and (B) ITS sequences
TABLE 1. Genealogical sorting index (gsi) and P-values of species
clusters identified by phylogenetic, TCS and GMYC analyses
Gene
Plastid DNA
ITS
Clade A
Clade C (1–2)
Clade B
0.9192 (<00001)
10000 (<00001)
–
06923 (<00001)
10000 (<00001)
10000 (<00001)
P-values are based on 10 000 permutations and are given in parentheses.
short and scaly rhizomes, 3–10 cm panicles, yellow spikelets,
rough and hairless or occasionally villous leaf blades, sheaths
and glumes, and lemmas that are densely villous on ridges and
margins. The second cluster (B) comprised 199 individuals
sampled from 26 populations of three previously identified species (O. thoroldii, O. tibeticus and O. longiglumis) from the
western QTP. It corresponded well to clade B which had
been identified on the basis of plastid DNA and ITS
42
Su et al. — Determining species delimitation within the genus Orinus
A
Maximum intra-cluster P-distance
Minimum inter-cluster P-distance
0·0025
0·0020
0·0015
0·0010
0·0005
B
B
O
.l
O
O
C
la
de
on
gi
gl
um
is
us
.t
.t
ib
et
ic
ho
ro
ld
ii
A
O
.a
lti
C
la
de
cu
lm
us
a
ic
ok
on
or
.k
O
O
.a
no
m
al
a
0
Maximum intra-cluster P-distance
Minimum inter-cluster P-distance
0·040
0·035
0·030
0·025
0·020
0·015
0·010
0·005
C
B
O
.l
on
C
la
de
de
la
gl
gi
ib
.t
O
C
um
is
us
et
ro
O
.t
ho
la
C
ic
ld
ii
A
de
us
lti
.a
O
O
.k
ok
on
cu
or
m
no
.a
O
lm
ic
al
a
a
0
FIG. 5. Genetic distances between and within the six previously described species and between and within the ‘clades’ identified in this study, based on plastid DNA
(A) and nuclear ITS (B) sequences.
sequence variation. This cluster was distinguished by long and
scaly rhizomes, 95–128 cm panicles, purple-brown spikelets
and densely villous leaf blades, sheaths, glumes and lemmas.
The third group (C1–2) comprised the remaining individuals
from 16 populations of O. anomala and O. thoroldii distributed
in the south-eastern QTP. Most of the morphological traits of
plants in this cluster are intermediate between those of the other
two clusters: rough or hairless leaf blades, sheaths and glumes,
and yellow or purple-brown spikelets. It should be noted that
this cluster is distinguished from the other two clusters by having scaleless rhizomes (Fig. 7C). The eastern populations in
this group are more similar to O. anomala than to O. thoroldii,
whereas the western populations are more similar to O. thoroldii than to O. anomala with respect to morphological variation.
Niche differentiation between clusters and the species previously
described
We first examined niche differentiation between the three
morphological clusters. Ecological niche models were
constructed using information from 66 cluster A sites in the
eastern QTP, 71 cluster B sites from the western QTP and 20
sites of the third cluster (C1–2) (Supplementary Data Table S2)
to predict the geographical distributions of each cluster based
on our field studies and specimen records from herbaria
(Fig. 8A). Areas under the receiver operating characteristic
(ROC) curve had values of 0978, 0986 and 0988 for clusters
A, B and C (1–2), respectively, indicating that differences varied greatly from random expectation. Tests of identity between
the three clusters showed that there was distinct niche differentiation (P < 001) (Fig. 8B). Background tests between them
also showed that the ecological niches of the three clusters
were well differentiated (P < 001), no matter in which direction comparisons were made (Supplementary Data Fig. S1).
The niche of the third cluster differs from the other two mainly
in that it is characterized by high precipitation and temperature.
We further compared niche differentiation between the six previously acknowledged species, based on specimen records and
our field collections (Fig. S2). We did not find any distinct
niche differentiation between O. anomala, O. kokonorica and
O. alticulmus, or between O. thoroldii, O. tibeticus and
Su et al. — Determining species delimitation within the genus Orinus
43
3·0
2·5
2·0
Cluster B
1·5
Axis 2
1·0
0·5
0
–0·5
–1·0
–1·5
–2·0
Cluster A
Cluster C
–3·0 –2·5 –2·0 –1·5 –1·0 –0·5
0
0·5
1·0
1·5
2·0
2·5
3·0
Axis 1
FIG. 6. Scatterplots based on principal co-ordinate analysis scores for each individual (shown as a dot) evaluated. The dot colours represent different species as indicated in Fig. 1.
A
C
B
FIG. 7. Rhizomes of typical plants from the morphological clusters A, B and C (C includes clades C1 and C2 according to ITS sequence analysis). The rhizomes of
all individuals in cluster C were found to be smooth and lacking in scales.
O. longiglumis, although when any species from one of the two
groups was compared with any species from the other group,
ecological differentiation was distinct (P < 001).
DISCUSSION
In this study, we used an integrative approach based on multiple
evidence from population genetic data, statistical analyses of
morphological variation and niche differentiation to delimit
plant species boundaries, using the QTP endemic genus Orinus
(Poaceae) as a model. Our results are most unexpected, because
all six previously published species need to be taxonomically
revised. On the basis of morphological variation, all populations
of the genus examined could be grouped into two discrete clusters distributed in the west and in the east, and one cluster, intermediate between them, occurring in the south-east. These
three clusters showed clear ecological preferences and niche
Su et al. — Determining species delimitation within the genus Orinus
44
A
B
Cluster A
D
I
0-0·3
0·3-0·5
0·5-0·7
0·7-0·9
0·9-1·0
No data
60
70
50
60
Frequency
Frequency
Cluster A vs. Cluster C
40
30
20
40
30
20
0·7481
10
50
0·4858
10
0
0·2
0·4
0·6
0·8
0
0·2
1·0
0·4
I
0·6
0·8
1·0
0·8
1·0
0·8
1·0
D
Cluster A vs. Cluster B
25
30
20
25
Frequency
Frequency
Cluster C
15
10
5
0-0·3
0·3-0·5
0·5-0·7
0·7-0·9
0·9-1·0
No data
0
0·2
20
15
10
5
0·7155
0·4176
0
0·4
0·6
0·8
1·0
0·2
0·4
I
0·6
D
Cluster B vs. Cluster C
Cluster B
60
50
40
Frequency
Frequency
50
30
20
0-0·3
0·3-0·5
0·5-0·7
0·7-0·9
0·9-1·0
No data
0
0·2
30
20
0·4329
0·7303
10
40
10
0
0·4
0·6
I
0·8
1·0
0·2
0·4
0·6
D
FIG. 8. (A) Predicted distributions of cluster A, cluster B and cluster C (1–2) based on ecological niche modelling using Maxent. (b) Results of identity tests. Bars indicate the null distributions of D and I. Both are generated from 100 randomizations. The x-axes indicate values of I and D, and the y-axes indicate the number of
randomizations. Arrows indicate values obtained in actual Maxent runs.
differentiation. East–west delimitation was further supported by
plastid DNA and ITS data sets. However, using plastid DNA
data, the morphologically intermediate cluster was found to be
nested within the eastern clade, whereas ITS sequences for this
cluster, which had a distinct signature containing additive sites
suggestive of hybridization in the past, indicated that it comprised two paraphyletic sub-clades. The results of these analyses suggested that all populations of this genus should be
assigned to two discrete species occurring in the eastern and the
western QTP, respectively, and a third one, with intermediate
morphology possibly indicating hybrid origin, distributed in the
south-eastern QTP.
West–east delimitation
In the past, different species concepts have been proposed
and debated. However, ‘good species’ are more likely to
comprise evolutionarily independent lineages with genetic gaps
(de Queiroz, 2005, 2007). However, a major disadvantage of
this lineage species concept is that it does not necessarily tie
species delimitation to the identification of any one particular
visible trait. Although such genetic differentiation is desirable,
it should not be considered the only criterion for taxonomic
classification for operational practices. However, if welldifferentiated lineages are supported by more lines of evidence
and congruent with more than one species concept, the delimited
species seem to be more testable and usable. Nowadays, it is
common to combine evidence from modern, DNA-based techniques with traditional morphological taxonomy (Ackermann
and Bishop, 2010). Undoubtedly, such an integrative delimitation
of species is more operational than the others based on one single
criterion (Fujita et al., 2012; Mckay et al., 2013).
Our extensive exploration of Orinus identified two distinct
clusters in the west and east of the QTP, and delimitation between them fulfils at least three of the criteria required to define
Su et al. — Determining species delimitation within the genus Orinus
species. First, multiple morphological traits are distinctly different in the two groups. For example, spikelets are yellow in the
eastern cluster, but purple to brown in the western cluster. Leaf
blades, sheaths and glumes are hairless in the eastern cluster,
whereas they are villous in the western cluster. These two clusters also differ from each other in other morphological traits.
Secondly, there is clear genetic differentiation on the basis of
both plastid DNA and ITS data (Fig. 2). All analyses of these
two data sets were able to distinguish the eastern and western
clusters clearly. Finally, the west–east delimitation was supported by niche differentiation (Fig. 8). Within the western
cluster, three species, i.e. O. thoroldii, O. tibeticus, and O. longiglumis, had previously been identified, based on hairs covering
the leaf sheath, panicle and spikelet length, floret number per
spikelet and the relative length of a glume and the adjacent floret (Zhao and Li, 1994; Chen and Phillips, 2006; Zhang and
Cai, 2008; Su and Cai, 2009). Our morphological analyses suggested that none of these species could be distinguished from
the other two on the basis of the above morphological characters. These characters showed considerable variation between
individuals even within the same population. Similarly, the
characters used to define the other three species, those of the
eastern cluster, i.e. O. anomala, O. kokonorica and O. alticulmus, for example leaf spinule, panicle shape, spikelet length,
floret number per spikelet, lemma hairs and the relative lengths
of palea and lemma (Zhao and Li, 1994; Chen and Phillips,
2006; Zhang and Cai, 2008; Su and Cai, 2009), also varied
greatly within populations, and showed no distinct clustering in
our statistical analyses.
Within either the eastern or the western cluster, further
within-cluster differentiation could not be achieved using the
plastid DNA or the ITS data set (Fig. 2; Table 1). In addition,
we found that the niches preferred by the three species within
the eastern or the western group showed no distinct differentiation. All these findings suggest that the west–east delimitation
within the genus may represent two real ‘species’ units and that
no further taxonomic division within each group is warranted.
This delimitation may mirror the history of speciation of
Orinus in this region. According to our dating, based on plastid
DNA or ITS sequence variation between the two clades, the divergence occurred between 20 and 25 Ma. Geological evidence indicates that the QTP underwent extensive uplift
between 36 and 18 Ma (Li et al., 1995; Shi et al., 1998). Most
other alpine genera that occur mainly in the QTP have also
been reported as having diversified within this period (e.g. Liu
et al., 2002, 2006; Wang et al., 2004; Zhang et al., 2009). It is
likely that the extensive plateau uplifts and accompanying
habitat changes (Tang and Shen, 1996) that took place during
this period promoted the east–west divergence of Orinus in
the QTP.
An intermediate cluster in the south-east
In addition to the two well-delimited clusters described
above, we identified a third. This cluster differs from the other
two in lacking scales on the rhizomes (Fig. 7). However, the
other traits examined were found to be intermediate between
those of the eastern and the western cluster. For example, in
this third cluster, the leaf blades, sheaths and glumes are rough
45
and hairless, or villous. Spikelets are yellow or purple-brown.
Paleas are sharply acute or obtuse. Phylogenetic analysis of
plastid DNA sequence variation suggested that this cluster was
nested entirely within the eastern clade, whereas analyses of
ITS sequences revealed two clades without sister relationships
(Fig. 2). We found that ITS sequences in these two clades, particularly clade C2, showed a random distribution of divergent
sites corresponding to those of the eastern and of the western
clade. In addition, some sampled individuals show additive
double peaks from these divergent sites. These genetic signatures all suggest the occurrence of recent hybridization and introgression events (Arnold, 1992; Mallet, 2008; Soltis and
Soltis, 2009; Wang et al., 2009; Abbott et al., 2010, 2013;
Abbott and Rieseberg, 2012). We treated this cluster as an independently evolved lineage (species) originating from hybridization or introgression, rather than as a hybrid, for the following
three reasons. First, hybrids generally occur in the regions intermediate between two parental species, whereas hybrid species
differ from the two parental species in having distinctly differentiated niches. Two confirmed diploid hybrid species in the
QTP were found to occur in different distributions from those
of their respective parental species (Ma et al., 2006; Sun et al.,
2014). The results of the series of tests that we applied to the
three clusters suggested that this cluster, which occurs in
the south-eastern QTP, occupies different niches from those of
the other two clusters, which are distributed in the east and
west. Secondly, in the process of occupying niches, hybrid species usually develop new morphological or physiological traits.
This may explain the presence of scaleless rhizomes in this
south-eastern cluster. In contrast, hybrids usually lack such innovative traits other than those which are intermediate between
those of the two parental species. Finally, we found that C2 had
accumulated seven mutations that were distinctly different from
those found in either of the eastern and western (A and B, Fig.
3) clades, according to the ITS sequence variation matrix.
These endemic mutations suggested that C2 has evolved as an
independent lineage since the putative hybridization event.
The random fixture, with or without additional nucleotides, of
mutations derived from either clade A or clade B almost certainly reflects either the origin of C2 as a hybrid between A and
B, or subsequent introgression from one or the other of these
clades. Why did these endemic mutations not appear in clade
C1? It is likely that C1 experienced more back introgression
with clade A, leading to the homogenization of these mutations
back into the clade A forms. Because additional nucleotides in
the differentiated mutations were also found in clade A and
clade B, it is likely that A and B diverged early on, and subsequently met again to produce ancestral populations like clade
C2. This clade then developed into a new lineage and some of
its populations back-crossed with clade A to produce clade C1,
although morphological traits in C1 are still the same as those
in clade C2 with scaleless rhizomes. Early hybridization events
between clades A and B or later back introgression with C1 or
C2 resulted in the additional nucleotides of the differentiated
mutations in some ITS sequences being recovered from clade
A or B. Up to now, we failed to find morphological differentiation between these two clades and it remains unknown whether
reproductive isolation has evolved between them. Overall,
based on the integrative principles of species delimitations,
these findings suggest that the third morphological cluster
46
Su et al. — Determining species delimitation within the genus Orinus
(including both ITS clades C1 þ C2), which occurs in the
south-eastern QTP, may represent a new ‘species’ of the genus
originating through hybridization or introgression, although it
remains to be determined whether hybridization contributed to
the reproductive isolation of this lineage from the others.
Our cytogenetic examination of the three clusters (results not
shown) revealed that they all are diploid. The hybridization or
introgression events therefore probably occurred at the diploid
level. Hybrid species are usually found to occupy new niches,
different from those of the two parental species, in order to
avoid competition (Arnold, 1997). Three probable homoploid
hybrid species have been reported from the QTP (Ma et al.,
2006; Liu et al., 2014; Sun et al., 2014). It has been suggested
that the latter two of these potential hybrid species originated
from the Quaternary climatic oscillations, which led to a second
period of contact and subsequent hybridization between two parental species (Liu et al., 2014; Sun et al., 2014). The new habitats created by the QTP uplifts and the Quaternary climate
changes also favoured the survival and expansions of these new
lineages which originated from hybridization or introgression.
Because the divergence between clades A and B occurred earlier (dated as 258–203 Ma according to our results) than the
Quaternary, the climatic oscillations during the latter period
might have driven clades A and B to retreat to the same area,
where they hybridized with each other to produce a hybrid lineage. However, at the end of the climatic oscillations, both
clades recolonized their original areas of distribution, although
repeated back introgression between them and their hybrid offspring still occurred. The hybrid or introgressed populations
gradually developed into a new lineage. Confirmation of these
hypotheses will require further evidence particularly that based
on modelling allele changes at multiple nuclear loci as has been
done for other diploid hybrid species (Liu et al., 2014; Sun
et al., 2014).
Conclusions
In recent years, a major advance in species delimitation has
resulted from a move from studies using single lines of evidence or a single criterion towards increasing the number of different lines of evidence used, or addressing multiple species
concepts with an integrative approach. This trend has been
driven partly by a reduction in the cost of obtaining different
types of evidence and using different methods, but also by an
increasing recognition that a ‘good species’ is one delimited using multiple criteria under an integrative species concept.
According to our statistical analyses of morphological variation, most traits previously used as diagnostic for the six species
established within Orinus are highly variable both within and
between populations, suggesting that none of these previously
circumscribed ‘species’ is independently evolving lineages.
Both genetic and niche analyses further confirmed this inference. Instead, our integrative analyses identified two welldifferentiated clusters in the east and the west, respectively,
which meet three species delimitation criteria due to their long
divergence history. However, the third one in the south-east is
an independently evolving lineage probably at the early speciation stage because only two of three examined criteria are fulfilled. These three clusters may together represent relatively
objective, operable and unbiased species units, although further
evidence is needed before a definitive taxonomic revision of
the genus can be made.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Figure S1: results of
background tests, indicating the null distributions of D and I.
Figure S2: niche differentiation between six previously
described species based on specimen records and our field collections, indicating predicted distributions, results of identity
tests and results of background tests. Table S1: provenance of
samples used in multilocus species delimitation analyses. Table
S2: data used in niche modelling.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Key
Project for Basic Research (2014CB954100), the National
Natural Science Foundation of China (grant no. 31260052),
and the Foreign Collaboration program ‘111’ and the
Collaboration Program of the Ministry of Science and
Technology of China (2010DFA34610).
LITERATURE CITED
Abbott RJ, Albach D, Ansell S, et al. 2013. Hybridization and speciation.
Journal of Evolutionary Biology 26: 229–246.
Abbott RJ, Hegarty MJ, Hiscock SJ, Brennan AC. 2010. Homoploid hybrid
speciation in action. Taxon 59: 1375–1386.
Abbott RJ, Rieseberg LH. 2012. Hybrid speciation. In: eLS. Chichester: John
Wiley and Sons, Ltd, Doi: 10.1002/9780470015902.a0001753.pub2.
Ackermann RR, Bishop JM. 2010. Morphological and molecular evidence reveals recent hybridization between gorilla taxa. Evolution 64: 271–290.
Alvarez I, Wendel JF. 2003. Ribosomal ITS sequences and plant phylogenetic
inference. Molecular Phylogenetics and Evolution 29: 417–434.
Arnold ML. 1992. Natural hybridization as an evolutionary process. Annual
Review of Ecology and Systematics 23: 237–261.
Arnold ML. 1997. Natural hybridization and evolution. New York: Oxford
University Press.
Bachmann K. 1998. Species as units of diversity: an outdated concept. Theory
in Biosciences 117: 213–230.
Besansky NJ, Krzywinski J, Lehmann T, et al. 2003. Semipermeable species
boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation. Proceedings of the
National Academy of Sciences, USA 100: 10818–10823.
Besnard G, de Casas RR, Vargas P. 2007. Plastid and nuclear DNA polymorphism reveals historical processes of isolation and reticulation in the olive
tree complex (Oleaeuropaea). Journal of Biogeography 34: 736–752.
Bitner-Mathé BC, Klaczko LB. 1999. Plasticity of Drosophila melanogaster
wing morphology: effects of sex, temperature and density. Genetica 105:
203–210.
Bond JE, Stockman AK. 2008. An integrative method for delimiting cohesion
species: finding the population–species interface in a group of Californian
trapdoor spiders with extreme genetic divergence and geographic structuring. Systematic Biology 57: 628–646.
Briggs D, Walters SM. 1997. Plant variation and evolution. Cambridge:
Cambridge University Press.
Carstens BC, Dewey T. 2010. Species delimitation using a combined coalescent
and information-theoretic approach: an example from North American
Myotis bats. Systematic Biology 59: 400–414.
Chen SL, Phillips SM. 2006. Orinus (Poaceae). In: ZY Wu, PH Raven, eds.
Flora of China, Vol. 22. Beijing: Science Press; St. Louis, MO: Missouri
Botanical Garden Press, 464–465.
Chen SL, Yao H, Han JP, et al. 2010. Validation of the ITS2 region as a novel
DNA barcode for identifying medicinal plant species. PLoS One 5: e8613.
Su et al. — Determining species delimitation within the genus Orinus
Clement M, Posada D, Crandall K. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657–1660.
Cuénoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase WW.
2002. Molecular phylogenetics of the Caryophyllales based on 18S
rDNA, rbcL, atpB and matK sequences. American Journal of Botany 89:
132–144.
Cummings MP, Neel MC, Shaw KL. 2008. A genealogical approach to quantifying lineage divergence. Evolution 62: 2411–2422.
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities
of fresh leaf material. Phytochemical Bulletin 19: 11–15.
Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by
sampling trees. BMC Evolutionary Biology 7: 214.
Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy
and high throughput. Nucleic Acids Research 32: 1792–1797.
Farris JS, Kallersjo M, Kluge AG, Bult C. 1995. Testing significance of incongruence. Cladistics 10: 315–319.
Fazekas AJ, Burgess KS, Kesanakurti PR, et al. 2008. Multiple multilocus
DNA barcodes from the plastid genome discriminate plant species equally
well. PLoS One 3: e2802.
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the
bootstrap. Evolution 39: 783–791.
Fujita MK, Leaché AD, Burbrink FT, McGuire JA, Moritz C. 2012.
Coalescent-based species delimitation in an integrative taxonomy. Trends in
Ecology and Evolution 27: 480–488.
Grimm GW, Denk T. 2008. ITS evolution in Platanus (Platanaceae): homoeologues, pseudogenes and ancient hybridization. Annals of Botany 101:
403–419.
Harding H, Zhang Y, Bertolotti A, Zeng H, Ron D. 2000. Perk is essential for
translational regulation and cell survival during the unfolded protein
response. Molecular Cell 5: 897–904.
Harpke D, Peterson A. 2006. Non-concerted ITS evolution in Mammillaria
(Cactaceae). Molecular Phylogenetics and Evolution 41: 579–593.
Harrington RC, Near TJ. 2012. Phylogenetic and coalescent strategies of species delimitation in Snubnose Darters (Percidae: Etheostoma). Systematic
Biology 61: 63–79.
Hendrixson BE, DeRussy BM, Hamilton CA, Bond JE. 2013. An exploration
of species boundaries in turret-building tarantulas of the Mojave Desert
(Araneae, Mygalomorphae, Theraphosidae, Aphonopelma). Molecular
Phylogenetics and Evolution 66: 327–340.
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. 2005. Very high
resolution interpolated climate surfaces for global land areas. Internal
Journal of Climatology 25: 1965–1978.
Hollingsworth ML, Clark AA, Forrest LL, et al. 2009. Selecting barcoding
loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants. Molecular Ecology Resources
9: 439–457.
Knoop V. 2004. The mitochondrial DNA of land plants: peculiarities in phylogenetic perspective. Current Genetics 46: 123–139.
Kress WJ, Erickson DL. 2007. A two-locus global DNA barcode for land
plants: the coding rbcL gene complements the non-coding trnH-psbA
spacer region. PLoS One 2: e508.
Kress WJ, Erickson DL. 2008. DNA barcoding – a windfall for tropical biology? Biotropica 40: 405–408.
Lledó DM, Crespo MB, Cameron KM, Fay MF, Chase MW. 1998.
Systematics of Plumbaginaceae based upon cladistic analysis of rbcL sequence data. Systematic Biology 23: 21–29.
Levin DA. 1979. The nature of plant species. Science 204: 381–384.
Li DZ, Gao LM, Li HT, et al., 2011. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the
core barcode for seed plants. Proceedings of the National Academy of
Sciences, USA 108: 19641–19646.
Li JJ, Shi YF, Li BY. 1995. Uplift of the Qinghai–Xizang (Tibet) Plateau and
global change. Lanzhou: Lanzhou.University Press.
Liu BB, Abbott RJ, Lu ZQ, Tian B, Liu JQ. 2014. Diploid hybrid origin of
Ostryopsis intermedia (Betulaceae) in the Qinghai–Tibet Plateau triggered
by Quaternary climate change. Molecular Ecology 23: 3013–3027.
Liu JQ, Gao TG, Chen ZD, Lu AM. 2002. Molecular phylogeny and biogeography of the Qinghai–Tibet Plateau endemic Nannoglottis (Asteraceae).
Molecular Phylogenetics and Evolution 23: 307–325.
Liu J, Wang Y, Wang A, Hideaki O, Abbott RJ. 2006. Radiation and diversification within the Ligularia–Cremanthodium–Parasenecio complex
(Asteraceae) triggered by uplift of the Qinghai–Tibetan Plateau. Molecular
Phylogenetics and Evolution 38: 31–49.
47
Ma XF, Szmidt AE, Wang XR. 2006. Genetic structure and evolutionary
history of a diploid hybrid pine Pinus densata inferred from the nucleotide variation at seven gene loci. Molecular Biology and Evolution 23:
807–816.
Mallet J. 1995. A species definition for the modern synthesis. Trends in Ecology
and Evolution 10: 294–299.
Mallet J. 2008. Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation. Philosophical Transactions of the
Royal Society B: Biological Sciences 363: 2971–2986.
Mayr E. 1982. The growth of biological thought: diversity, evolution and inheritance. Cambridge, MA: Belknap Press of Harvard University Press.
McCauley DE, Sundby AK, Bailey MF, Welch ME. 2007. Inheritance of chloroplast DNA is not strictly maternal in Silene vulgaris (Caryophyllaceae):
evidence from experimental crosses and natural populations. American
Journal of Botany 94: 1333–1337.
McKay BD, Mays HL Jr, Wu Y, et al. 2013. An empirical comparison of character-based and coalescent-based approaches to species delimitation in a
young avian complex. Molecular Ecology 22: 4943–4957.
Pang XH, Song JY, Zhu YJ, Xu HX, Huang LF, Chen SL. 2011. Applying
plant DNA barcodes for Rosaceae species identification. Cladistics 27:
165–170.
Phillips SJ, Anderson RP, Schapire RE. 2006. Maximum entropy modeling of
species geographic distributions. Ecological Modelling 190: 231–259.
Phillips SJ, Dudı́k M. 2008. Modeling of species distributions with Maxent:
new extensions and a comprehensive evaluation. Echography 31: 161–175.
Pons J, Barraclough TG, Gomez-Zurita J, et al. 2006. Sequence-based species
delimitation for the DNA taxonomy of undescribed insects. Systematic
Biology 55: 595–609.
Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818.
de Queiroz K. 1998. The general lineage concept of species, species criteria, and
the process of speciation: a conceptual unification and terminological recommendations. In: DJ Howard, SH Berlocher, eds. Endless forms: species
and speciation. Oxford: Oxford University Press, 57–75.
de Queiroz K. 2005. Different species problems and their resolution. BioEssays
27: 1263–1269.
de Queiroz K. 2007. Species concepts and species delimitation. Systematic
Biology 56: 879–886.
Ragupathy S, Newmaster SG, Murugesan M, Balasubramaniam V. 2009.
DNA barcoding discriminates a new cryptic grass species revealed in an ethnobotany study by the hill tribes of the Western Ghats in southern India.
Molecular Ecology Resources 9: 164–171.
Rambaut A, Drummond AJ. 2007. Tracer. Available from: http://beast.bio.ed.
ac.uk/tracer.
Reid NM, Carstens BC. 2012. Phylogenetic estimation error can decrease the
accuracy of species delimitation: a Bayesian implementation of the general
mixed Yule–coalescent model. BMC Evolutionary Biology 12: 196.
Rieseberg LH. 1997. Hybrid origins of plant species. Annual Review of Ecology
and Systematics 28: 359–389.
Romaschenko K, Garcia-Jacas N, Peterson PM, Soreng RJ, Vilatersana R,
Susanna A. 2014. Miocene–Pliocene speciation, introgression, and migration of Patis and Ptilagrostis (Poaceae: Stipeae). Molecular Phylogenetics
and Evolution 70: 244–259.
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
Sang T, Crawford DJ, Stuessy TF. 1997. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). American
Journal of Botany 84: 1120–1136.
Satler JD, Carstens BC, Hedin M. 2013. Multilocus species delimitation in a
complex of morphologically conserved trapdoor spiders (Mygalomorphae,
Antrodiaetidae, Aliatypus). Systematic Biology 62: 805–823.
Schoener TW. 1968. The Anolis lizards of Bimini: resource partitioning in a
complex fauna. Ecology 49: 704–726.
Shi YF, Li JJ, Li BY. 1998. Uplift and environmental changes of
Qinghai–Tibetan Plateau in the Late Cenozoic. Guangzhou: Guangdong
Science and Technology Press.
Soltis PS, Soltis DE. 2009. The role of hybridization in plant speciation. Annual
Review of Plant Biology 60: 561–588.
Stockman AK, Bond JE. 2007. Delimiting cohesion species: extreme population structuring and the role of ecological interchangeability. Molecular
Ecology 16: 3374–3392.
Su X, Cai LB. 2009. Orinus longiglumis (Poaceae: chloridoideae), a new species
from Xizang (Tibet), China. Annales Botanici Fennici 46: 143–147.
48
Su et al. — Determining species delimitation within the genus Orinus
Sun YS, Abbott RJ, Li LL, Li L, Zou JB, Liu JQ. 2014. Evolutionary history
of purple cone spruce (Picea purpurea) in the Qinghai–Tibet Plateau: homoploid hybrid origin and Pleistocene expansion. Molecular Ecology 23:
343–359.
Swofford DL. 2002. PAUP*: phylogenetic analysis using parsimony (*and other
methods), version 4. Sunderland, MA: Sinauer Associates.
Tamura K, Peterson D, Peterson N, et al. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Molecular Biology and Evolution 28:
2731–2739.
Tang LY, Shen CM. 1996. Late Cenozoic vegetational history and climatic
characteristics of Qinghai–Xizang Plateau. Acta Micropalaeontologica
Sinica 13: 321–337.
Templeton AR, Crandall KA, Sing CF. 1992. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease
mapping and sequencing data. III. Cladogram estimation. Genetics 132:
619–633.
Tzvelev NN. 1968. Plantae Asiae Centralis, Vol. 4. Leningrad: Nauka
Publishers.
Wang LY, Abbott RJ, Zheng W, Chen P, Wang YJ, Liu JQ. 2009. History
and evolution of alpine plants endemic to the Qinghai–Tibetan Plateau:
Aconitum gymnandrum (Ranunculaceae). Molecular Ecology 18: 709–721.
Wang Q, Yu QS, Liu JQ. 2011. Are nuclear loci ideal for barcoding plants? A
case study of genetic delimitation of two sister species using multiple loci
and multiple intraspecific individuals. Journal of Systematics and Evolution
49: 182–188.
Wang YJ, Li XJ, Hao G, Liu JQ. 2004. Molecular phylogeny and biogeography of Androsace (Primulaceae) and the convergent evolution of cushion
morphology. Acta Phytotaxonomica Sinica 42: 481–499.
Warren DL, Glor RE, Turelli M. 2008. Environmental niche equivalency versus conservatism: quantitative approaches to niche evolution. Evolution 62:
2868–2883.
Warren DL, Glor RE, Turelli M. 2010. ENMTools: a toolbox for
comparative studies of environmental niche models. Echography 33:
607–611.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing
of fungal ribosomal RNA genes for phylogenetics. In: M Innis, D Gelfand, J
Sninsky, T White, eds. PCR protocols. San Diego: Academic Press,
315–322.
Wiens JJ. 2007. Species delimitation: new approaches for discovering diversity.
Systematic Biology 56: 875–878.
Wolfe KH, Li WH, Sharp PM. 1987. Rates of nucleotide substitution
vary greatly among plant mitochondrial, chloroplast, and nuclear
DNAs. Proceedings of the National Academy of Sciences, USA 84:
9054–9058.
Wu SG, Yang YP, Fei Y. 1995. On the flora of the alpine region in the
Qinghai–Xizang (Tibet) plateau. Acta Botanica Yunnanica 17: 233–250.
Wu YH. 2008. The vascular plants and their eco-geographical distribution of
the Qinghai–Tibetan Plateau. Beijing: Science Press.
Yassin A, Capy P, Madi-Ravazzi L, Ogereau D, David JR. 2008. DNA barcode discovers two cryptic species and two geographical radiations in the invasive drosophilid Zaprionus indianus. Molecular Ecology Resources 8:
491–501.
Zha HG, Milne RI, Sun H. 2010. Asymmetric hybridization in Rhododendron
agastum: a hybrid taxon comprising mainly F1s in Yunnan, China. Annals
of Botany 105: 89–100.
Zhang TL, Cai LB. 2008. A new species of Orinus (Poaceae) from Qinghai,
China. Novon 18: 273–278.
Zhang XL, Wang YJ, Ge XJ, Yuan YM, Yang HL, Liu JQ. 2009.
Molecular phylogeny and biogeography of Gentiana sect. Cruciata
(Gentianaceae) based on four chloroplast DNA datasets. Taxon 58:
862–870.
Zhao NX, Li MF. 1994. New taxa and new recording species of Gramineae
from Tibet. Acta Botanica Yunnanica 16: 228–230.