Molecular phylogeny, divergence time estimates and historical biogeography within one of the world's largest monocot genera
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Molecular phylogeny, divergence time estimates and historical biogeography within one of the world's largest monocot genera
Qin-Qin Li 0 1
Song-Dong Zhou 1
De-Qing Huang 1
Xing-Jin He 1
Xian-Qin Wei 1
Associate Editor: Chelsea D. Specht
0 College of Life Science and Technology, Inner Mongolia Normal University , Hohhot 010022 , China Inner Mongolia
1 Key Laboratory of Bio-Resources and Eco-Environment, MOE, College of Life Sciences, Sichuan University , Chengdu 610064 , China Sichuan
A primary aim of historical biogeography is to identify the causal factors or processes that have shaped the composition and distribution of biotas over time. Another is to infer the evolution of geographic ranges of species and clades in a phylogenetic context. To this end, historical biogeography addresses important questions such as: Where were ancestors distributed? Where did lineages originate? Which processes cause geographic ranges to evolve through time? Allium subgenus Anguinum comprises approximately twelve taxa with a disjunct distribution in the high mountains from south-western Europe to eastern Asia and in northeastern North America. Although both the systematic position and the geographical limits of Anguinum have been identified, to date no molecular systematic study has been performed utilizing a comprehensive sampling of these species. With an emphasis on the Anguinum eastern Asian geographical group, the goals of the present study were: (i) to infer species-level phylogenetic relationships within Anguinum, (ii) to assess molecular divergence and estimated the times of the major splits in Anguinum and (iii) to trace the biogeographic history of the subgenus. Four DNA sequences (ITS, matK, trnH-psbA, rps16) were used to reconstruct the phylogeny of Allium subgen. Anguinum. RbcL sequences were used to estimate divergences time for Allium, and sequences of ITS were used to estimate the divergence times for Anguinum and its main lineages and to provide implications for the evolutionary history of the subgenus. Phylogenetic analyses for all Allium corroborate that Anguinum is monophyletic and indicate that Anguinum is composed of two sister groups: one with a Eurasian-American distribution, and the other restricted to eastern Asia. In the eastern Asian geographical group, incongruence between gene trees and morphology-based taxonomies was recovered as was incongruence between data from plastid and nuclear sequences. This incongruence is likely due to the combined effects of a recent radiation, incomplete lineage sorting, and hybridization/introgression. Divergence time estimates suggest that the crown group of Anguinum originated during the late Miocene (ca. 7.16 Mya) and then diverged and dispersed. Biogeographic analyses using statistical dispersal-vicariance analysis (S-DIVA) and a likelihood method support an eastern Asia origin of Anguinum. It is inferred that in the late Pliocene/Early Pleistocene, with cooling climates and the uplift of the Himalayas and Hengduan Mountains, the ancestor of the eastern Asian alliance clade underwent a very recent radiation.
A primary aim of historical biogeography is to identify
the causal factors or processes that have shaped the
composition and distribution of biotas over time
). Another major focus is to infer the
evolution of geographic ranges of species and clades in a
(Ree and Smith 2008)
. To this end,
many questions about the historical distributions of
species may be of interest, such as: Where were ancestors
distributed? Where did lineages originate? Which
processes (e.g., vicariance or dispersal) cause geographic
ranges to evolve through time?
(Ree and Smith 2008; Xie
et al. 2009)
. Many studies have addressed these
biogeographic questions using phylogenetic analyses,
molecular dating, and reconstruction of ancestral geographic
(e.g., Sytsma et al. 2004; Nie et al. 2006; Bell
2007; Sanmartın et al. 2008; Xie et al. 2009)
A brief introduction of the paleontological history of
the Northern Hemisphere since the late Cretaceous helps
to understand the biogeographic history of plant biota.
Overall, the Earth’s climate became cooler through the
(Zachos et al. 2001)
, and the climate cooled
gently from 50 to 35 million years (Myr) ago, then fluctuated
until 15 Myr ago, after which the climate cooled
progressively, culminating in the Quaternary (2–0 Myr ago)
(Milne and Abbott 2002)
. Cooling climates in the
latter part of the Tertiary forced the boreotropical flora
retreat southwards to large refugial regions that
preserved the warm wet climate that they needed. These
refugia include eastern Asia, south-eastern Europe,
eastern and western North America, and western Asia, and
the floras concerned are termed Tertiary relict floras
(Tiffney 1985a,b; Wen 1999; Milne and Abbott 2002)
Over the 2 million years of the Quaternary, these data
indicate considerable and continuous climatic variation
(Mitchell 1976; Smiley et al. 1991)
. Up to 24 glacial
events of about 50–100 000 years each have occurred
(van Donk 1976). The climatic oscillations of the
Quaternary resulted in repeated drastic environmental
changes that profoundly shaped the current distributions
and genetic structures of many plant species in
temperate zones of the Northern Hemisphere
. When the Tertiary period began, North
America and Eurasia were each separated into western
and eastern portions by epicontinental seaways
1985a; Tiffney and Manchester 2001)
. At the same time
western North America was connected to East Asia
across the Bering land bridge (BLB), while eastern North
America was connected via Greenland to north-eastern
Europe—a connection known as the North Atlantic land
(NALB; Tiffney 1985b)
. Different perspectives exist
for the timing of the break up of the NALB
a, b; Tiffney 2000; Wen 2000; Tiffney and Manchester
2001; Milne and Abbott 2002; Milne 2006)
, but it is
thought that the NALB was available for plant exchanges
from the early Tertiary to possibly as late as 15 Myr ago
(Milne and Abbott 2002). It is known that the BLB
connected eastern Asia and western North America at one
time or another throughout the late Cretaceous and the
Tertiary and was available for floristic exchanges until
ca. 3.5 millions of years
(Ma; Tiffney 1985 a, b; Wen 1999;
Gladenkov et al. 2002; Milne 2006)
The genus Allium comprises about 920 species
(Seregin et al. 2015)
, making it one of the largest
monocotyledonous genera. Allium is a member of order
Asparagales, family Amaryllidaceae, subfamily Allioideae
(Fay and Chase 1996; APG III 2009; Chase et al. 2009)
Fay and Chase (1996)
Friesen et al. (2000)
Chase et al. (2009)
, Allium (including Caloscordum Herb.,
Milula Prain and Nectaroscordum Lindl.) is the only genus
in tribe Allieae. Previous molecular data suggested that
Allium evolution proceeded in three separate
evolutionary lines; subgenus Anguinum is a member of the second
(Fritsch 2001; Fritsch and Friesen 2002;
Friesen et al. 2006; Li et al. 2010; Choi et al. 2012)
Anguinum contains approximately twelve taxa (nine
species and three varieties) with a disjunct distribution in the
high mountains from south-western Europe to eastern
Asia and in northeastern North America
. It is characterized by specific root
, leaf and bulb structure
(Pastor and Valdes 1985), hypogeal seed germination
and A. victorialis-type seedlings
, narrow, branched and
lengthwise-twisted septal nectarines
a short vegetative period with the adaptation of the light
regime under deciduous forest conditions
Li et al. 2010)
. Unlike other Allium lineages, the seed
testa sculpturing is very simple among species of
(Kruse 1984, 1988)
. Species of Anguinum also
share similar metaphase chromosomes and the basic
chromosome number x ¼ 8 and all reported karyotypes
are 2A type (Jing et al. 1999). Based on the consistency
of its morphological, anatomical and cytological
characteristics, it is a rather distinct and specialized group
et al. 2010)
. Previous molecular studies indicated that
Anguinum is monophyletic and shares a more recent
common ancestor with Vvedenskya, Porphyroprason and
Melanocrommyum and is the sister group to
(Friesen et al. 2006; Li et al. 2010)
According to Friesen et al. (2006) and
Li et al. (2010)
sister groups comprise this subgenus: one with a
Eurasian–American distribution, and the other restricted
to the Hengduan Mountains and adjacent areas.
Although both the systematic position and the
geographical limits of Anguinum have been identified, to
date no molecular systematic study has been performed
utilizing a comprehensive sampling of these species.
Thus, in order to better understand the phylogeny and
historical biogeography of Anguinum, an extended
population sampling of Anguinum species endemic to eastern
Asia was incorporated in this study. With an emphasis on
the Anguinum eastern Asian geographical group, the
goals of the present study were: (i) to infer species-level
phylogenetic relationships within Anguinum using four
molecular markers ITS, matK, trnH-psbA, and rps16, (ii)
to assess molecular divergence and estimated the times
of the major splits in Anguinum and related these
divergence times with external factors that might have
contributed to the diversification of the subgenus and (iii) to
trace the biogeographic history of the subgenus by
provided a time frame to the biogeographic studies.
Our sampling strategy was designed to cover those
taxonomic and geographic Anguinum groups that were
underrepresented in previous analyses, especially from
eastern Asia, and to build on previous studies
et al. 2006; Li et al. 2010)
. Fifty samples (ITS sequences
for four samples downloaded in Genbank) representing
twelve taxa of Anguinum with a focus on the eastern
Asian geographical group, were sampled as the ingroup
for phylogenetic reconstructions. Two species from
subgenus Caloscordum (A. neriniflorum, A. tubiflorum) were
designated as outgroups according to previous studies
(Fritsch and Friesen 2002; Friesen et al. 2006; Nguyen
et al. 2008; Li et al. 2010)
. GenBank accession numbers
and voucher details referred to the above-mentioned
taxa are given in [see Supporting Information,
There are no known fossils of Anguinum or even
Allium, so we first intend to estimate the divergence time
of Allium, based on a relatively broad analysis of rbcL
sequences of Allium (i.e., representatives almost in every
subgenus of Allium) together with other samples from
the order Asparagales and other monocots. According to
previous systematic studies
(e.g. Fay and Chase 1996;
Vinnersten and Bremer 2001; Davis et al. 2004; Tamura
et al. 2004; Leebens-Mack et al. 2005; M u¨ller et al. 2006;
Meng et al. 2008; Kim et al. 2010)
and our preliminary
phylogenetic analysis for 255 sequences of the groups of
monocots, 152 sequences were chosen covering all
orders and as many families as possible referred to the
present study, in which 32 sequences (26 sequences
were generated in this study) representing Allium spp.
[see Supporting Information, Appendix S2]. To infer
divergence times within Anguinum, 144 ITS sequences (34
sequences were generated in this study) that
represented Anguinum and other subgenera and sections in
Allium, plus two sequences of Nothoscordum gracile
(family Amaryllidaceae, subfamily Allioideae, tribe
Gilliesieae) and Tulbaghia violacea (family
Amaryllidaceae, subfamily Allioideae, tribe Tulbaghieae)
were used, and the selection of Anguinum taxa is based
on haplotype analysis, and sequences belong to the same
haplotype form the same taxa are removed (except A.
nanodes) [see Supporting Information, Appendix S3].
DNA extraction, amplification and sequencing
Genomic DNA was extracted from silica gel-dried or fresh
leaves using the method of
Doyle and Doyle (1987)
Primers ITS4 and ITS5
(White et al. 1990)
were used to
amplify the ITS region followed the protocol of
Li et al.
. The rps16 intron was amplified with primers rpsF
(Oxelman et al. 1997)
in accordance with the
Marazzi et al. (2006)
. The intergenic spacer
trnH-psbA was amplified using the primers trnH (GUG) F
. The PCR programme was as
follows: 94 C for 4 min; 30 cycles of 94 C for 30 s, 52 C
for 30 s, 72 C for 1 min; and 72 C for 7 min. The rbcL was
amplified with primers rbcL N’ and DBRBAS2
et al. 1987)
for 26 sequences. The matK was amplified
with primers 3F_KIM and IR_KIM (Kim, unpublished).
Their PCR parameters were same as follows: 94 C for
4 min; 30 cycles of 94 C for 1 min, 52 C for 1 min, 72 C
for 1 min 30 s; and 72 C for 10 min. PCR products were
separated using 1.5 % (w/v) agarose TAE gel and purified
using Wizard PCR preps DNA Purification System
(Promega, Madison, WI, USA) following the
manufacturer’s instructions. The purified PCR products were
sequenced in an ABI 310 Genetic Analyzer (Applied
Biosystems Inc.) using the PCR primers.
Sequence comparisons and phylogenetic analyses
Forward and reverse sequences were assembled and
edited with SeqMan (DNAstar package; DNAStar Inc.,
Madison, WI, USA). DNA sequences were initially aligned
using the default pairwise and multiple alignment
parameters in Clustal X
(Jeanmougin et al. 1998)
rechecked and adjusted manually as necessary using
(Tamura et al. 2007)
. Gaps were positioned to
minimize nucleotide mismatches and treated as missing
data in phylogenetic analyses. Phylogenetic analyses
were conducted by employing maximum-parsimony
(MP) criteria and Bayesian inference (BI), using the
programs PAUP* version 4.0b10
MrBayes version 3.1.2
(Ronquist and Huelsenbeck 2003)
respectively. For MP, heuristic searches were carried with
1000 random addition sequence replicates. One tree was
saved at each step during stepwise addition, and
treebisection-reconnection (TBR) was used to swap
branches. All characters were unordered and equally
weighted. Gaps were treated as missing data. Bootstrap
values were calculated from 6000 000 replicate analyses
using ‘fast’ stepwise-addition of taxa and only those
values compatible with the majority-rule consensus tree
were recorded. Prior to a Bayesian analysis, MrModeltest
was used to select a best-fit
model of nucleotide substitution under the Akaike
infomation criterion. The Bayesian Markov chain Monte Carlo
(MCMC) settings consisted of four independent runs with
four chains each (one cold chain and three incrementally
heated chains) for 6 million generations starting from
random trees and sampling one of every 100
generations, by using default priors and estimating all
parameters during the analysis. The first 25 % of the trees were
discarded as burn-in. A 50 % majority-rule consensus
tree of the remaining trees was produced.
The incongruence length difference (ILD) test of ITS vs.
the combined chloroplast sequences (rps16, matK, and
trnH-psbA) was carried out in PAUP*
(Farris et al. 1994)
assess potential conflicts between the phylogenetic
signal from different genomes. This test was implemented
with 100 partition-homogeneity test replicates, using a
heuristic search option with the simple addition of taxa,
TBR branch swapping and MaxTrees set to 1000.
Divergence time estimation
Since the strict molecular clock model was rejected for
rbcL with P < 0.05 in Likelihood ratio tests
, divergences time of Allium was
estimated using a Bayesian approach with an uncorrelated
lognormal relaxed molecular clock model
et al. 2006)
, as implemented in the program BEAST
(Drummond and Rambaut 2007)
. An advantage of
BEAST is its ability to estimate the topology and
divergence times simultaneously. The BEAST analysis was run
for 5 107 generations with parameters sampled every
1000 generations and used the GTR þ IþG substitution
model selected by MrModeltest and randomly generated
Starting Tree and the Yule tree prior. The fossil record of
monocots is comparatively poor primarily due to
problems of preservation
(Herendeen and Crane 1995;
Crepet, Nixon and Gandolfo 2004)
. A few fossils of the
Asparagales have been reported from the late Eocene
(Couper 1960; Muller 1981; Herendeen and Crane 1995)
and they all may be too young to calibrate the crown
clade of the order
(Wikstro¨m et al. 2001; Janssen and
. Based on five molecular markers,
Magallon and Castillo (2009)
estimated the age of the
crown Asparagales as 124.95 million years ago (Mya) for
relaxed datings. We therefore used this age as the
calibration point for the crown group Asparagales node,
with a normally distributed standard deviation of 0.35.
Based on eight reference fossils,
estimated the split between Acorus and all other monocots
to be more than 134 Myr old. Using a calibration point
outside the monocots (within the eudicot order Fagales),
Wikstro¨m et al. (2001) produced an age for extant
(crown group node) monocots of 127–141 Mya. These
two estimates fall into the range of ages estimated by
Sanderson and Doyle (2001)
and are in fairly close
Good-Avila et al. (2006)
who estimated the
crown age of monocots to be 132 Myr old. As
wrote, we can draw some generalities about the
ages of monocots from these studies. Thus, following
Janssen and Bremer (2004)
Meng et al. (2008)
calibrated the crown age of monocots as 134 Mya, with
a normally distributed standard deviation of 4.25. We
also set the crown age of Amaryllidaceae at 87 Mya
Janssen and Bremer (2004)
, with a normally
distributed standard deviation of 0.01. The results were
evaluated by the Tracer program 1.4
and the first 10 % of the generations
were discarded as burn-in. Samples from the posterior
were summarized on the maximum clade credibility tree
using TreeAnnotator version 1.5.2.
and the tree was visualized using FigTree
. The divergence times are
given as the mean node heights and the 95 % highest
posterior density (HPD) intervals in millions of years (Ma).
Magallon and Castillo (2009)
, we rooted the
tree with four species (Ascarina swamyana, Chloranthus
nervosus, Hedyosmum arborescens and Sarcandra
chloranthoides) from order Chloranthales.
Because the likelihood ratio tests rejected the
molecular clock for the data (P < 0.05), divergence times within
Anguinum were estimated using the same methods
stated above and the GTR þ IþG substitution model was
selected by MrModeltest. The crown age of Allium was
set to 34.26 Mya based on previous analysis, with a
normally distributed standard deviation of 0.1. According to
previous phylogenetic analyses
(Fay and Chase 1996;
Mes et al. 1997; Fay et al. 2000; Friesen et al. 2000, 2006;
Nguyen et al. 2008; Li et al. 2010)
, Nothoscordum gracile
and Tulbaghia violacea were used to root the tree.
To perform our analysis, we used the maximum clade
credibility (MCC) phylogeny from the ITS data set by
BEAST. Distribution areas of Anguinum and its close allies
(Caloscordum, Vvedenskya, Porphyroprason, and
Melanocrommyum) were defined according to the World
Checklist of Selected Plant Families maintained by the
Royal Botanic Gardens, Kew, UK (http://apps.kew.org/
wcsp/home.do) and taxonomic and geographical studies
of these Allium species
(e.g. Xu and Kamelin 2000; Dale
et al. 2002; Choi and Oh 2011)
. Potential biogeographical
scenarios of Anguinum were investigated using
statistical dispersal–vicariance analysis
(S-DIVA; Yu et al.
and a maximum likelihood-based method
(LAGRANGE; Ree et al. 2005; Ree and Smith 2008)
implemented in the computer software Reconstruct Ancestral
States in Phylogenies (RASP; Yu et al. 2015). In the
SDIVA analysis, allowing reconstruction, the number of
ancestral areas was restricted to two. The rationale for
such a constraint is that vicariance is a proximate
consequence of dispersal. Moreover, extant taxa used in the
analyses rarely occur in more than two individual areas.
The ML inferences of geographic range evolution using
LAGRANGE were conducted for the same distribution
matrix under the constraints of maximum areas of two.
The connectivity between areas was not constrained.
The ILD test conducted on the combined data matrix of
common ITS and chloroplast sequences (matK þ
trnH-psbA þ rps16) accessions was significant (ILD
probability value ¼ 0.01), indicating that the two datasets are
. Finally, four
different datasets were generated: dataset 1 for Anguinum ITS
phylogenetic analyses, dataset 2 for Anguinum
combined chloroplast phylogenetic analyses, dataset 3 for
Allium divergence time estimation, and dataset 4 for
Anguinum divergence time estimation. After introducing
the necessary gaps, the ITS alignment for dataset 1 was
649 bp in length and resulted in 498 constant characters
and 150 variable characters, 110 of which were
potentially parsimony-informative; the mean G þ C content
was 47.5 %. For dataset 2, the combined chloroplast
sequences (matK þ trnH-psbA þ rps16) produced a matrix
2296 bp in length, and for which 2193 characters were
constant, 39 autapomorphic and 56 potentially
parsimony-informative; the mean G þ C content was
31.3 %. The statistics of chloroplast sequences and
nuclear ITS are shown in Table 1. For dataset 3, the aligned
rbcL sequences produced a matrix 1328 bp in length, and
for which 741 characters were constant, and 418
potentially parsimony-informative; the mean G þ C content
was 43.6 %. For dataset 4, the aligned ITS sequences
produced a matrix 754 bp in length, and for which 99
characters were constant, and 547 potentially
parsimonyinformative; the mean G þ C content was 46.6 %.
ITS phylogeny. For dataset 1, trees inferred from BI and
MP showed no significant difference in their topologies,
and therefore only the Bayesian tree with posterior
probabilities (PP) and bootstrap support values (BS) was
shown in Fig. 1. In all analyses, the subgen. Anguinum
proved to be monophyletic and robustly separated from
the outgroup species (PP ¼ 1.00, BS ¼ 100 %). The
Information ITS matK trnH-psbA rps16 Combined cpDNA data
Range of length (bp) 642–646 840–855 548–584 813–838 –
Aligned length (bp)
Number of variable characters
Number of parsimony- informative characters
Consistency index (CI)
Retention index (RI)
Homoplasy index (HI)
Anguinum contains two sister groups: one with a
Eurasian–American distribution (including A. victorialis,
A. listera, A. microdictyon, A. ochotense, A. tricoccum var.
tricoccum, A. tricoccum var. burdickii), and the other
restricted to eastern Asia (i.e., the Hengduan Mountains
and adjacent areas, including A. ovalifolium var.
ovalifolium, A. ovalifolium var. cordifolium, A. ovalifolium var.
leuconeurum, A. funckiifolium, A. nanodes, A. prattii).
Within the Eurasian–American distribution clade,
A. ochotense, A. tricoccum, and three species from
Eurasia (A. listera, A. microdictyon, and A. victorialis) form
a trichotomy (PP ¼ 0.85, BS ¼ 80 %); three accessions of
A. listera join together (PP ¼ 0.99, BS ¼ 63 %). Within the
eastern Asia distribution clade, species from eastern Asia
(A. ovalifolium var. ovalifolium, A. ovalifolium var.
cordifolium, A. ovalifolium var. leuconeurum, A. nanodes,
A. funckiifolium, A. prattii) form a large basal-most
polytomy (PP ¼ 1.00, BS ¼ 100 %), and inside some small
polytomies; two accessions of A. nanodes grouped together
(PP ¼ 0.95, BS ¼ 63 %). In all cases, although there are
small clades of alleles from the same taxa, A. ovalifolium
var. ovalifolium, A. ovalifolium var. leuconeurum and
A. prattii are non-monophyletic. Considering only one
accession involved in the present study, the monophyly/
non-monophyly of A. ovalifolium var. cordifolium and
A. funckiifolium requires further investigation. Overall,
ITS phylogeny is populated by both short internal and
terminal branches; therefore, it is not surprising that
there are unresolved polytomies.
Chloroplast sequence phylogeny
In the combined cpDNA analyses, the topology of the
Bayesian tree was similar to that of the MP consensus
tree. The 50 % majority-rule consensus tree from BI is
presented in Fig. 2, with PP and BS support values. The
monophyly of subgen. Anguinum was also recovered
(PP ¼ 1.00, BS ¼ 100 %). Within Anguinum, two sister
groups are evident. Within the Eurasian–American
distribution clade (PP ¼ 1.00, BS ¼ 100 %), accessions of A.
listera and A. victorialis form a polytomy; within the
eastern Asia distribution clade (PP ¼ 0.89, BS ¼ 95 %),
accessions of A. ovalifolium var. ovalifolium, A. ovalifolium
var. cordifolium, A. ovalifolium var. leuconeurum, A.
nanodes, A. funckiifolium, and A. prattii form a large
basalmost polytomy, and also some small polytomies inside
the polytomy. The topological patterns of the combined
cpDNA phylogeny are more complex than that of ITS.
Accessions from following four taxa did not form
monophyletic groups: A. ovalifolium var. ovalifolium, A.
ovalifolium var. leuconeurum, A. nanodes, A. prattii, although
part of the sequences for the same taxa grouped
together. Overall, the cpDNA phylogenetic hypothesis is
also rich in polytomies.
Estimation of divergence time
The crown group of genus Allium originated during the
late Eocene (ca. 34.26 Mya) and then diverged into three
clades (Fig. 3). The chronogram and results of
divergence-time estimation of Anguinum are shown in
Fig. 4. The crown group of Anguinum originated during
the late Miocene (ca. 7.16 Mya). Within Anguinum, the
divergence of the Eurasian-American alliance clade dates
to the Mid-Pliocene (ca. 3.64 Mya), and the origin of the
eastern Asian alliance clade to the early Pleistocene (ca.
Eight areas were considered for biogeographical analysis
of Anguinum and its allies: eastern Asia (a), eastern
North America (b), western North America (c), Europe
and adjacent areas (d), Siberia (e), West Asia and
adjacent areas (f), Central Asia (g) and the Mediterranea (h).
The inferred historical biogeographic scenarios from
analyses using S-DIVA and LAGRANGE are shown in Fig. 4.
Both the analyses strongly supported East Asia as the
ancestral area of Anguinum.
Sequence divergence and polytomies
For ITS sequences, the average pairwise K2P
two-parameter; Kimura 1980)
distance between ingroup
Anguinum and outgroup Caloscordum ranged from
15.73 to 19.58 %. The average pairwise K2P distance
within Anguinum was very low (range 0.00– 6.37 %), and
the average distance was 1.60 %. In the
EurasianAmerican alliance clade, the average distance was
1.40 % (range 0.00–3.69 %), and in the eastern Asia
alliance clade, the average distance was 0.27 % (range
0.00–1.09 %). For chloroplast sequences, the average
pairwise K2P distance between ingroup Anguinum and
outgroup Caloscordum ranged from 1.40 to 2.02 %. The
average pairwise K2P distance within Anguinum was
very low (range 0.00–1.17 %), and the average distance
was 0.32 %. In the sister group formed by A. victorialis
and A. listera, the average distance was 0.15 % (range
0.00–0.27 %), and in the eastern Asia alliance clade, the
average distance was 0.23 % (range 0.00–0.74 %).
Overall, ITS data show remarkably low levels of
variability within Anguinum, and the variation is low in
comparison to subgenus Melanocrommyum in the same
(Gurushidze et al. 2008)
sequence data also show remarkably low levels of
variability within Anguinum. Another conspicuous result is the
different level of differentiation at the ITS and the
chloroplast sequences within and among species of subgen.
Anguinum. In several species intraspecies diversity is
quite high, and several species show almost no
interspecies diversity. Low sequence divergence results in an
unresolved ITS and chloroplast phylogeny of Anguinum,
with both short internal and terminal branches; thus, it is
not surprising that there are rich in polytomies. Further
studies (utilizing low-copy nuclear gene phylogenies
and/or employing marker technology that has broad
genomic coverage) are needed to obtain more
information on species-level relationships of Anguinum.
Phylogenetic implications, incomplete lineage sorting, hybridization/introgression
The results presented here support the earlier finding
that subgen. Anguinum is monophyletic
(Friesen et al.
2006; Li et al. 2010)
. In agreement with Friesen et al.
Li et al. (2010)
, two sister groups comprise
this subgenus: one with a Eurasian–American distribution
and the other restricted to eastern Asia. In the Eurasian–
American geographical group, A. listera, A. microdictyon,
and A. victorialis are closely related species and form a
polytomy, which may indicate that this group diverged
rapidly. In the ITS tree, accessions of three A. listera
populations are clustered together, implying that A.
listera is monophyletic. In the eastern Asian geographical
group, for both the ITS and the cpDNA tree, accessions of
A. ovalifolium var. ovalifolium, A. ovalifolium var.
cordifolium, A. ovalifolium var. leuconeurum, A. nanodes,
A. funckiifolium, and A. prattii form a large basal-most
polytomy, and also some small polytomies inside the
polytomy. Accessions of two A. nanodes populations
form a monophyletic group in the ITS tree, and are not
clustered together in chloroplast tree. Both ITS tree and
chloroplast tree sufficiently indicate that A. ovalifolium
var. ovalifolium, A. ovalifolium var. leuconeurum and
A. prattii are non-monophyletic, although there are small
clades of alleles from the same taxa. In total, in the
eastern Asian geographical group, incongruence between
gene trees and morphology-based taxonomies, and
incongruence between trees from plastid sequences and
nuclear sequences are recovered. This leaves the
phylogenetic relationships among the species ambiguous.
Incongruence between molecular data and
(e. g. Gurushidze et al.
2008, 2010; Aduse-Poku et al. 2009; Waters et al. 2010)
and incongruence between nuclear and plastid data
g. Fehrer et al. 2007; Kim and Donoghue 2008; Pelser
et al. 2010)
have been repeatedly reported. Experimental
and theoretical studies
(e. g. reviewed in Wendel and
Doyle 1998; Whitfield and Lockhart 2007; Waterman
et al. 2009; Gurushidze et al. 2010)
have shown that
some factors can lead to gene trees incongruence and
gene tree—species tree incongruence, including
stochastic error, systematic error, convergence,
evolutionary rate heterogeneity, lineage sorting, and reticulation,
namely hybridization, introgression, homoploid and
polyploid. When incongruence is recovered among gene
trees, the first consideration is whether it is due to
inadequate sampling. The second consideration is stochastic
error and systematic error. Once these factors are ruled
out, biological factors in the evolutionary process may be
considered, including convergence and rapid radiation,
horizontal gene transfer, hybridization/introgression, and
incomplete lineage sorting. Our sampling scheme was
designed to cover all taxa of the Anguinum eastern Asia
geographical group; with the exception of A. ovalifolium
var. cordifolium and A. funckiifolium, all remaining taxa
were represented by accessions from multiple
populations. ITS sequences and three chloroplast sequences
(matK, trnH-psbA, rps16) were selected as molecular
markers in order to have markers representing different
genomic compartments. Phylogenetic analyses of the
ITS sequences and the combined chloroplast sequences
were performed separately, and in BI, a best-fit model of
nucleotide substitution was selected for each chloroplast
sequence. Consistency index
(CI; Kluge and Farris 1969)
and the retention index
(RI; Farris 1989)
the parsimony analysis are as follows: CI and RI obtained
from the ITS sequences were separately 0.94 and 0.98,
and CI and RI obtained from the combined chloroplast
sequences were separately 0.89 and 0.94, which
illustrate that the levels of homoplasy found in our data set
are similar to those of other angiosperm groups
and Wendel 2003)
. Given these tests, it is likely that
inadequate sampling as well as stochastic error and
systematic error can be ruled out, indicating a biological factor
for the observed incongruence.
As a single genetic change in a regulatory region can
cause dramatic morphological transformations, which
typically are unaccompanied by similar levels of
molecular divergence, and the processes that drive molecular
divergence can lag behind the phenotypic divergence, the
result will be incongruence between molecular
phylogenies and morphology
(reviewed in Wendel and Doyle
. While, data from ITS sequences and three
chloroplast sequences provide neither resolved topologies nor
congruent hypotheses about species-level relationships
in the Anguinum eastern Asia geographical group, it is
possible that other loci or genomic elements that are
more rapidly evolving may provide phylogenetic
resolution for this group.
Given the nature of the data, however, it is most
probable that incomplete lineage sorting and hybridization/
introgression are the main contributing factors to the
conflicts found among sequences for the Anguinum
eastern Asia geographical group. The incongruence
occurs between gene trees and morphology-based
taxonomies, and between trees from plastid sequences
and nuclear ribosomal sequences. These findings are
consistent with other studies in Allium subgen.
(Gurushidze et al. 2010)
(e.g., Dobes et al. 2004; Jakob and Blattner
2006; Vargas et al. 2009)
indicating that the combined
effects of incomplete lineage sorting and hybridization/
introgression can obscure organismal-level relationships
in a phylogenetic framework. Incomplete lineage sorting
(ILS) is the persistence of ancestral polymorphisms
through speciation events; with time and subsequent
extinction of gene lineages, descendant populations will
have randomly sorted separate nuclear and organelle
(Wendel and Doyle 1998; Avise 2000)
. ILS is
especially likely when species rapidly radiate and
population sizes are large
(Maddison 1997; Sang 2002; Funk
and Omland 2003)
. A consequence of incomplete
lineage sorting is that species will appear to be
nonmonophyletic. The ITS tree indicates that, A. nanodes is
monophyletic, while A. ovalifolium var. ovalifolium,
A. ovalifolium var. leuconeurum, and A. prattii are
resolved as non-monophyletic. Evidence suggests that
nuclear genes move across hybrid boundaries less freely
than organellar genes
(Rieseberg and Wendel 1993;
Soltis and Soltis 1995; Hardig et al. 2000)
, so we conclude
that incomplete lineage sorting could most likely
account for the difference in the plastid tree vs. the nuclear
ITS tree for this group. It cannot be ruled out from these
data, however, that ancient reticulation has occurred in
the past. During glacial periods in the Quaternary,
previously isolated populations of the Anguinum eastern
Asian geographical group may have come into contact,
allowing hybridisation and introgression, resulting in
species non-monophyly as evidenced by the DNA data
presented here. The combined plastid DNA tree indicated
that A. nanodes, A. ovalifolium var. ovalifolium, A.
ovalifolium var. leuconeurum, and A. prattii are
nonmonophyletic, while accessions of partial populations of
A. ovalifolium var. ovalifolium, A. ovalifolium var.
cordifolium, and A. prattii are clustered together (Fig. 2, node c),
and accessions of partial populations of A. ovalifolium
var. ovalifolium, A. ovalifolium var. leuconeurum, and
A. prattii are clustered together (Fig. 2, node d). Above
evidence suggests that, the lineage sorting process is
either not finished, or that some hybridization is still going
on. The coalescence of organelle DNA is four times faster
than nuclear genes
, and therefore it is
unlikely that the lineage sorting for nuclear genes had been
completed if lineage sorting for chloroplast genes is not
yet complete. Overall, we propose that the process of
lineage sorting is ongoing for both nuclear ribosomal and
chloroplast genes in the Anguinum eastern Asian
geographical group, and any lineage sorting for A. nanodes
may have been completed, while this process for the
other taxa is not yet complete.
Incongruence between phylogenies inferred from
nuclear and chloroplast regions, is partially interpreted as
being a result of incomplete lineage sorting. However,
the incomplete lineage sorting is not the only process
that could produce such incongruence. Incongruence
between nuclear and chloroplast data, and the sharing
of chloroplast haplotypes between species, are also
usually interpreted as being a result of
hybridization/introgression which results in the chloroplast genome of one
species being replaced by that of another species, and
such reticulation is easily reflected in the form of cpDNA/
nuclear gene tree incongruencies
(Anderson 1949; Soltis
and Soltis 1995; Rautenberg et al. 2010)
. By means of
molecular markers, ‘footprint’ of
hybridization/introgression in the Anguinum eastern Asian geographical group
can be found. A prominent example is phenomenon in
A. nanodes. Lineage sorting for ITS in this species has
been completed (Fig.1), but in chloroplast tree (Fig. 2), A.
nanodes 1 and A. nanodes 2 are not clustered together.
A. nanodes 1 is clustered together with multiple
populations of A. prattii (Fig. 2, node a) and is the sister group
with A. prattii 11 from close localities (Fig. 2, node b),
which fully demonstrate that after complete lineage
sorting, subsequent hybridization or introgression
occurred, and the choloplast genome of A. nanodes 1
was replaced by that of A. prattii from close localities. It
is thus possible that currently delimited morphospecies
had insufficient time to develop reproductive barriers,
thus promoting hybridisation/introgression. Previous
(Jing et al. 1999; Xu and Kamelin 2000)
suggested that polyploids existed in Allium ovalifolium var.
ovalifolium (2n ¼ 16, 24) and A. prattii (2n ¼ 16, 32),
which perhaps also imply hybridization/introgression
exists in the Anguinum eastern Asian geographical
group. While it may be possible to detect hybridization/
introgression by means of multiple base calls in ITS
(Noyes 2006; Carine et al.
, the homogenous nature of the ITS data we obtain
means that detecting hybridization/introgression using
this approach is not possible. Young species groups in
particular should be affected by incomplete lineage
sorting while its influence should decrease with increasing
time due to the gradual loss of polymorphisms and
fixation of lineage-specific alleles
(Maddison 1997; Wendel
and Doyle 1998)
. As hybridization/introgression also
occurs mostly between young and reproductively not
completely isolated species
(Comes and Abbott 2001;
Ramdhani et al. 2009)
, incomplete lineage sorting and
hybridization/introgression may not be mutually
exclusive. It is much more difficult to distinguish between
incomplete lineage sorting and hybridization/introgression
because these processes can produce almost identical
outputs at the level of tree discordances
Doyle 1998; Ramdhani et al. 2009; Lu et al. 2010)
the existing information it is impossible to distinguish the
relative impact of incomplete lineage sorting vs.
hybridization/introgression in the Anguinum eastern Asian
A hypothesis of the evolutionary history of
Our analyses revealed that the crown group of
Anguinum originated during the late Miocene (ca. 7.16
Mya), and eastern Asia was the ancestral area for
Anguinum (node a), where it underwent duplication, and
gave rise to two different lineages. during the
MidPliocene (ca. 3.64 Mya), one of them, the ancestor of the
Eurasian-American alliance clade began to diverge
(node b), one descendant probably dispersed to the
northeastern North America by the BLB or by the long
distance dispersal and diverged into two varieties (A.
tricoccum var. tricoccum and A. tricoccum var. burdickii);
the other descendant stayed in eastern Asia (node c)
and began to diverge at ca. 3.09 Mya, in which A.
ochotense is the earliest-branching species and gradually
dispersed to western North America (Attu Island) and
Siberia—while ancestor of the remaining species (A.
listera, A. microdictyon, and A. victorialis) originated at the
Mid-Pleistocene (ca. 0.98 Mya), in which A. victorialis is
the early-branching species and gradually dispersed to
Europe and A. microdictyon gradually dispersed to
Siberia and Central Asia. Compared to the origin of the
Eurasian-American alliance clade, the origin of the
eastern Asian alliance (eastern Himalaya and areas south of
the Qinlin Mountains of China) clade (node d) is a
relatively recent event, which began to diverge at the early
Pleistocene (ca. 1.56 Mya), and A. nanodes originated at
the late Pleistocene (ca. 0.14 Mya). Geological estimates
date the start of the uplift of the Himalayas and
Hengduan Mountains unequivocally within the Late
(ca. 15–10 Ma; Royden et al. 2008)
and episodes of uplift probably continued throughout
the Late Pliocene (ca. 3 Ma) and well into the Quaternary
(Li et al. 1979; Zhou et al. 2006)
. Severe climatic
oscillations had dramatic effects on the evolution and
distribution of plants in this region
(Sun 2002; Qiu et al. 2011)
is inferred that during the late Pliocene/Early Pleistocene,
with cooling climates and the uplift of the Himalayas
and Hengduan Mountains, the ancestor of the Anguinum
eastern Asian lineage underwent a very recent radiation,
and gave rise to several closely related species
constituting A. ovalifolium var. ovalifolium, A. ovalifolium var.
cordifolium, A. ovalifolium var. leuconeurum, A.
funckiifolium, A. nanodes, and A. prattii. Recent rapid
radiations could result in morphospecies appearing
nonmonophyletic in DNA-based studies
Seehausen 2004; Ramdhani et al. 2009, Lu et al. 2010)
as we have found in the Anguinum eastern Asian lineage.
These taxa may have had insufficient time to develop
reproductive barriers following recent radiation, thus
(Ramdhani et al. 2009)
, and also
insufficient time to finish the lineage sorting process.
Due to rapid radiation, phylogenetically inferred
internodes on gene trees may be short and difficult to resolve
with confidence. This short interior branch phenomenon
may be also a common cause of phylogenetic
(Wendel and Doyle 1998)
. Hybridization appears to
facilitate the ability of some plant species to adapt to or
evolve in response to changes in climate
. It is inferred that,
hybridization/introgression in the Anguinum eastern Asian lineage could be
promoted by the ecological heterogeneity and rapidly
changing environment resulting from the intense uplift
of the Himalaya, resulting in rampant species
nonmonophyly, and also partially explaining the
inconguence between ITS tree and cpDNA tree.
Sources of Funding
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 31270241, 31470009,
31570198, 31460051), and the National Specimen
Information Infrastructure, Educational Specimen
SubPlatform (Web, http://mnh.scu.edu.cn/).
Contributions by the Authors
Qin-Qin Li conceived the idea and performed the
experiment, and then wrote the manuscript. Xian-Qin Wei
conducted 1 years of fieldwork. De-Qing Huang made
valuable comments on the manuscript. Xing-Jin He and
Song-Dong Zhou made valuable comments on the
manuscript and provided technical support in the experiments.
Conflicts of Interest Statement
We thank Jie-Mei Xu for identification of some
specimens. We also thank Dr. Yun-Dong Gao for providing
some samples analysed in this study. We are grateful for
the helpful comments of two anonymous reviewers and
from Chief Editor J. Hall Cushman and Associate Editor
Chelsea D. Specht.
The following additional information is available in the
online version of this article —
Appendix S1. Anguinum material of 50 samples plus
two species from Caloscordum (A. neriniflorum, A.
tubiflorum), with corresponding locality, voucher information,
GenBank reference numbers.
Appendix S2. Taxa, and GenBank accession numbers
for all rbcL sequences used in the present study.
Appendix S3. Taxa, references, and GenBank accession
numbers for all ITS sequences used in the Anguinum
divergence time estimation.
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