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Biological Journal of the Linnean Society, 2014, 111, 777–788. With 2 figures
Phylogeography of the genus Dasiphora (Rosaceae)
in the Qinghai-Tibetan Plateau: divergence blurred
by expansion
YA-ZHEN MA1, ZHONG-HU LI1,2, XI WANG1, BAO-LONG SHANG1, GUI-LI WU1 and
YU-JIN WANG1,3*
1
State Key Laboratory of Grassland Agro-Ecosystem, School of Life Science, Lanzhou University,
Lanzhou 730000, China
2
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education,
College of Life Sciences, Northwest University, Xi’an 710069, China
3
State Key Laboratory of Systematics and Evolution Botany, Beijing 100093, China
Received 23 September 2013; revised 2 December 2013; accepted for publication 2 December 2013
Plateau uprisings and climatic oscillations are considered to have caused extensive allopatric divergences that
account for the rich species diversity of the Qinghai-Tibetan Plateau (QTP). However, secondary contact during
range shifts in the Quaternary glacial cycles or inter-uplift stages may have restored the gene flow between species
and so counteracted these divergences, particularly in rapidly-adapting dominant elements. We tested this
hypothesis by determining the phylogeographical history of Dasiphora (Rosaceae), a genus of two species that are
widely distributed on the QTP and co-exist in numerous localities. We sequenced two chloroplast DNA fragments
(rbcL, trnT-L) for 559 individuals from 87 populations. Bayesian methods were used to identify phylogenetic
relationships and to estimate divergence times. Demographic histories were inferred using neutrality tests,
mismatch distribution analysis, and coalescent simulation. A total of 112 haplotypes that clustered into three major
groups were identified. The formation of these groups and their subgroups was dated to between the Pliocene and
the late Pleistocene. In addition, we found that some groups underwent multiple extensive expansions. Speciesspecific haplotypes were identified for each species, although these haplotypes phylogenetically intermixed. These
results suggest that recent plateau uplifts and climatic oscillations might have caused the deep divergences observed
within this genus. However, later range expansions probably blurred these divergences and possible species
boundaries. Our results shed new light on the complex evolutionary history of the QTP alpine plants. © 2014 The
Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788.
ADDITIONAL KEYWORDS: climatic oscillations – demographic history – dominant element – gene flow –
range shifts – Quaternary.
INTRODUCTION
The Qinghai-Tibetan Plateau (QTP) is the largest and
highest plateau in the world, with a mean altitude of
more than 4000 m a.s.l. It and its adjacent regions are
a major global biodiversity hotspot, housing numerous endemic species (Wu, 1988; Mittermeier et al.,
2005). It has been suggested that the high species
diversity in this region is a result of allopatric
*Corresponding author. E-mail: [email protected]
speciation following the extensive Miocene-Pliocene
plateau uplifts, which broke large contiguous areas of
land up into smaller fragments, and the Quaternary
climatic oscillations (Liu et al., 2002, 2006; Liu,
2004; Wang et al., 2009a; Mao et al., 2010; Xu et al.,
2010; Tian et al., 2011). However, range shifts introduced by the geological changes, as well as the
glacial–interglacial cycles, may have counteracted
these allopatric divergences by enabling mutual
introgressions. Evidence for such events has been
found in species complexes with recent divergences
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
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(Wang et al., 2009b; Du et al., 2011). Homogenization
of this type may have prevented the formation of new
species and blurred the boundaries between species
that were once well differentiated genetically. This
may be particularly common among dominant components because they would have colonized unoccupied habitats more rapidly than their competitors
(Avise, 2000). It is therefore likely that their range
shifts during the intergeological and/or interglacial stages may have caused more introgressions
and homogenizations between the diverging lineages.
However, the genetic signatures of these ancient
divergences may still be present within current populations, particularly within the cytoplasmic DNA that
does not undergo extensive recombination.
In the present study, we tested this hypothesis
by performing a phylogeographical investigation of
the shrub genus Dasiphora (Rosaceae). This genus
comprises two morphologically different species: the
yellow-petaled Dasiphora fruticosa and the whitepetaled Dasiphora glabra (see Supporting information, Fig. S1). Both species were previously placed
within the genus Potentilla, although they are clearly
distinct from the herbaceous species of this genus
both morphologically and genetically, and were therefore separated as an independent genus, Dasiphora
(Klackenberg, 1983; Dobeš & Paule, 2010). These two
species are widely distributed in the QTP where they
occupy diverse of habitats with a range of altitudes
from 500 m to 5000 m (Li, Ikeda & Ohba, 2003) and
appear to be unaffected by frosts (Elkington &
Woodell, 1963). Because of their widespread distribution and a myriad of individuals, this genus provided
an ideal proxy to explore the impact of geographical
or climate changes of the QTP on genetic pattern and
demographic history of alpine plants, and inspired a
number of phylogeographical studies. These investigations provided clear evidence of range expansions,
especially in the highlands, and deeply diverged lineages or clades within both species (Li et al., 2009;
Wang et al., 2009c; Shimono et al., 2010; Sun et al.,
2010).
Nevertheless, all of these studies included only one
member of the genus, either D. fruticosa or D. glabra;
thus, no measure has been made to exclude the
impact of hybridization or introgression on the demographic inference, which is highly possible considering their co-existence in numbers of localities.
To obtain a sound understanding of its evolutionary history, it is therefore rewarding to study both
members applying a uniform procedure. In the
present study, we conducted an extensive sampling of
both species, covering more than two-thirds of the
total area of the QTP along with a few representative
populations from outside this area. The chloroplast
(cp)DNA rbcL fragment, being demonstrated to be
useful for species delimitation (Li et al., 2011),
together with the cpDNA trnT-L fragment, which
is very sutitable for inferring the demographic history of the genus (Wang et al., 2009b; Sun et al.,
2010), were sequenced and analyzed, applying a
phylogeographical approach. Our specific objectives
were to: (1) detect and date deep divergences and
range expansions within the genus; (2) determine
whether the identified genetic divergences corresponded to the currently recognized interspecific
delimitation, which is based on morphological differences; and (3) test the hypothesis that later range
shifts may have blurred previous deep divergences.
MATERIAL AND METHODS
PLANT SAMPLING
Leaf samples were collected from 559 individuals
belonging to 87 Dasiphora populations (Fig. 1; see
also Supporting information, Table S1). Eighty-two of
these populations were found in the QTP and the
adjacent Himalayan region, whereas the other five
populations were sampled from the north (P68), the
east (P66, P67, P71), and the north-east (P69) to the
QTP. Fifty-one of the sampled localities consisted
exclusively of D. fruticosa, four consisted exclusively
of D. glabra, and the remainder contained individuals of both species. Each sampled individual were
assigned to one of the two species according to petal
colour.
Fresh leaves were collected from six to eight individuals from each population, except two populations
(P2 and P57), where two or three individuals were
sampled, respectively, and dried over silica gel. The
sampled individuals were separated by at least 50 m.
All samples and voucher specimens were stored at
Lanzhou University.
DNA EXTRACTION, POLYMERASE CHAIN REACTION
(PCR) AMPLIFICATION, AND SEQUENCING
Total DNA was extracted from approximately 20 mg
of dried leaf tissue using the DNeasy Tissue Kit
(Qiagen) in accordance with the manufacturer’s
instructions and stored in a final volume of 200 μL
of AE Buffer (i.e. the elution buffer provided with the
kit). The targeted cpDNA sequences (rbcL and trnT-L)
were amplified for all collected individuals and a few
outgroups (Potentilla anserina, Potentilla caliginosa,
Potentilla griffithii, and Potentilla potaninii). PCR
was performed using previously reported primers
(see Supporting information, Table S2; Taberlet et al.,
1991; Little & Barrington, 2003) in a 25-μL reaction
volume containing 10–40 ng of plant DNA, 0.5 mM
dNTPs, 50 mM Tris-HCl, 1.5 mM MgCl2, 2 μM of each
primer and 0.75 units of rTaq DNA polymerase
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
Figure 1. Geographical distribution of chloroplast (cp)DNA genetic variation within Dasiphora. The figure on the upper left (A) shows the overview of the studied
area and two populations situated far away from the Qinghai-Tibetan Plateau (QTP) which is outlined with a dotted line. For each population, the solid colour
blocks within the circles denote the detected haplotypes and their relative abundance, the small annotated circle with white background indicates the number
of private haplotypes, and the red or green outer ring marks Dasiphora fruticosa or Dasiphora glabra, respectively.
PHYLOGEOGRAPHY OF THE GENUS DASIPHORA
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
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(Takara). Amplification was achieved using a thermal
cycle comprising: an initial 5-min denaturation period
at 94 °C, followed by 37 cycles of denaturation at
94 °C for 40 s, annealing at 55 °C (rbcL) or 53.5 °C
(trnT-L) for 40 s, and extension at 72 °C for 1 min 20 s
(rbcL) or 1 min 10 s (trnT-L), with a final extension
period at 72 °C for 7 min. PCR products were purified
using a TIANquick Midi Purification Kit (Tiangen) in
accordance with the manufacturer’s instructions. The
purified PCR products were sequenced with the PCR
primers described above and an original designed
internal primer for the rbcL fragment (primer ‘rbcLIR’; see Supporting information, Table S2) using
the ABI Prism Bigdye Terminator Cycle Sequencing
Kit, version 3.1 (Applied Biosystems). The purified
DNA fragments were then separated and called
using an ABI 3130XL DNA Analyzer (Perkin Elmer
Applied Biosystems). All newly obtained Dasiphora
sequences have been submitted to GenBank under
the accession numbers KC544517–KC544604 and
KC544637–KC544690.
POPULATION
GENETIC ANALYSIS
The haplotype diversity (HE) of the cpDNA for each
population was calculated using DNASP, version 5.0
(Librado & Rozas, 2009). The mean genetic diversity
within populations (HS), total genetic diversity (HT),
and the coefficient of differentiation (GST, NST) for each
species, separately and combined, were also estimated based on the observed cpDNA variation
using PERMUT (Pons & Petit, 1996). The GST value
reflects haplotype frequencies alone, whereas NST
also accounts for the genetic similarity between
haplotypes. The phylogeographical structure of the
data was evaluated by determining whether NST was
significantly larger than GST over 1000 random permutations (Pons & Chaouche, 1995).
Analysis of molecular variance (AMOVA) (Excoffier,
Smouse & Quattro, 1992) was applied to the cpDNA
datasets to evaluate the hierarchical partitioning
of genetic variation among species, populations, and
individuals. This approach considers both differences
in sequence frequency and the number of variable site
differences among observed sequences. The significance of the results was tested using a nonparametric
permutation procedure with 1000 permutations.
A Mantel test was performed to assess the significance of isolation by distance between populations
with 1000 random permutations on matrices of
pairwise population differentiation values (FST) and
the natural logarithm of geographical distances
(Rousset, 1997). All of these analyses were performed
using ARLEQUIN, version 3.5 (Excoffier & Lischer,
2010).
PHYLOGENETIC
ANALYSIS
The obtained sequences were aligned using
CLUSTALW as implemented in MEGA, version 5.0
(Kumar et al., 2008) and revised manually. A total of
22 indels within the cpDNA sequences were coded
as binary states (0 or 1) using GAPCODER (Young &
Healy, 2003); mononucleotide microsatellite repeats
were removed as a result of the high mutation
rate and associated significant risk of homoplasy.
Haplotypes were identified and distinguished using
the DNASP, version 5.0 (Librado & Rozas, 2009).
The two cpDNA fragments, without inconsistent
signal detected by incongruence length difference test
(Farris et al., 1995), were combined to conduct all the
analyses.
To confirm that all the detected haplotypes of
Dasiphora comprised a monophyly and no hybridization or introgression had occurred between this genus
and other genera, four Potentilla species (GenBank
accession numbers: KF855326–KF855333) and one
Fragaria species (Fragaria vesca, downloaded from
GenBank with accession number: JF345175), which
was previously recovered to be closely related to
Dasiphora (Eriksson et al., 2003; Dobeš & Paule,
2010), were included in the phylogenetic analysis
and selected as outgroups in succession. The phylogenetic relationship was inferred using MrBayes,
version 3.1.2 (Huelsenbeck et al., 2001; Ronquist &
Huelsenbeck, 2003), with the best-fitting GTR+I+G
model selected by MRMODELTEST, version 2.0
(Nylander, 2004). Four chains (one cold and three
heated with temperature 0.20), each with a different
starting seed, were run for 3.5 × 107 generations, and
each analysis was repeated twice. Trees were sampled
every 500 generations. Stationary states were typically attained within 8.75 × 106 generations, and so
the first 17 500 trees were discarded as ‘burn-ins’ and
excluded from all inferential analyses. We then used
Bayesian posterior probabilities to assess the branch
support in each case. To detect the genealogical relationships among haplotypes with shallow genetic
divergences, a haplotype network was constructed
using NETWORK, version 4.5.0.0 (Bandelt, Forster &
Röhl, 1999).
We also used a Bayesian approach implemented
in BEAST, version 1.7.2 (Drummond & Rambaut,
2007) to estimate the diversification times of the
cpDNA haplotypes. Using the GTR+I+G substitution
model, a constant population size coalescent tree
prior and a random starting tree, we performed
three independent runs of 2 × 108 generations and
sampled all parameters once every 2000 generations,
with a burn-in of 5 × 107 generations. TRACER,
version 1.4 (Rambaut & Drummond, 2007) was used
to check the convergence of chains to the stationary
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
PHYLOGEOGRAPHY OF THE GENUS DASIPHORA
distribution and the results were summarized using
TREEANNOTATOR, version 1.7.2 (part of the
package BEAST, version 1.7.2). Because there are no
known fossilized examples of Dasiphora or any of its
close relatives that could be used to calibrate the
nucleotide substitution rate, we used a normally
distributed prior with a mean of 2 × 10−9 and a SD
of 6.080 × 10−10 substitutions per site per year
(s s−1 year−1) to cover a majority of the possible cpDNA
substitution rates for angiosperm species: the 95%
confidence interval surrounding the mean rate for
angiosperms ranges from 1 × 10−9 to 3 × 10−9 s s−1
year−1 (Wolfe, Li & Sharp, 1987).
DEMOGRAPHIC
781
(g) suggests population expansion, whereas a negative value indicates population contraction.
For datasets that were well supported by the
results of the expansion tests, we used the mode of
the mismatch distribution τ, expressed in units of
evolutionary time, as well as the formulas τ = 2ut
(Rogers & Harpending, 1992) and u = μkg to calculate
the expansion times. In these expressions, u is the
mutation rate for the whole sequence per generation,
μ is the mutation rate per nucleotide site per year (s
s−1 year−1), k is the mean sequence length and g is the
generation time in years, which we took to be 5 years.
As before, cpDNA rates ranging from 1 × 10−9 to
3 × 10−9 s s−1 year−1 (Wolfe et al., 1987) were used to
estimate the expansion timescales.
TESTS BASED ON CHLOROPLAST
DNA
SEQUENCES
To detect demographic expansions in the genus’
history, mismatch distribution analyses were performed for total populations and for samples from
each cpDNA group identified by the phylogenetic
analyses. Demographic equilibrium is expected to
yield multimodal or random and rough distributions
of pairwise differences, whereas unimodal and smooth
distributions usually suggest a sudden demographic
expansion (Slatkin & Hudson, 1991; Rogers &
Harpending, 1992). We also tested the validity of the
expansion model using the sum of squared deviations
between the observed and expected mismatch distributions. In addition, the smoothness of the distribution curves was evaluated using Harpending’s
raggedness index and calculated P values. A low raggedness is usually considered to indicate that the
population recently experienced a nonstationary
history. We also performed Fu’s FS test (Fu, 1997) and
calculated Tajima’s D (Tajima, 1989) for the population samples to investigate their recent demographic
expansions. All of these analyses were performed
with ARLEQUIN, version 3.5, using 10 000 parametric bootstrap replicates.
Based on the population datasets, the identified
potential demographic expansions were further tested
using LAMARC, version 2.1.8 (Kuhner, 2006) with a
coalescent simulation approach that considered the
genealogical relationships among the haplotypes. The
analysis was based on a Bayesian approach using
the Metropolis-coupled Markov chain Monte Carlo
(MCMC) method with replication of chains and adaptive heating to achieve optimal sampling of the
parameter space. The MCMC runs were performed
three times with random seeds; each run used 10
initial chains with 5000 samples and two long final
chains with 50 000 samples. All initial chains and
final chains were simulated using a sampling interval
of 30 and a burn-in of 10 000 samples. Within this
analysis, a large and positive exponential growth rate
RESULTS
CHLOROPLAST DNA
SEQUENCE VARIATION
The rbcL fragment was 1166 bp in length and had 24
variable sites, 17 of which were parsimonyinformative. The total alignment length of the trnT-L
sequence was 963 bp. We detected 65 nucleotide
substitutions and 46 indels, 76 of which were
parsimony-informative. After concatenation of the two
fragments, the sequences were aligned, giving a consensus length of 2129 bp. Based on the identified
polymorphic sites, a total of 112 haplotypes were
identified, of which 86 were private to a single population (Fig. 1; see also Supporting infromation,
Table S1). Nine haplotypes were shared between
species, 73 haplotypes were found only in D. fruticosa,
and 30 were specific to D. glabra (Fig. 2). Three
widely shared haplotypes, H30, H40, and H47, were
discovered in 22.6%, 10.9%, and 14.2% of all samples,
respectively. The most common haplotype, H30, was
widely distributed across the northern and eastern
regions of the plateau, and occurred in 42 populations. H40 and H47 were widely distributed in the
eastern QTP (which includes the upper reaches of
the Yellow River). However, H40 was also detected
in the south-eastern plateau, whereas H47 was found
in the north-east (Fig. 1; see also Supporting information, Table S1).
Haplotype diversity (HE) was calculated based on
the cpDNA haplotype frequencies for each population
(see Supporting information, Table S1). Based on the
observed cpDNA variation, the mean genetic diversity
within populations (HS) was high for both D. fruticosa
and D. glabra (0.525 and 0.652), and the total genetic
diversity HT (0.918) across the whole genus was
greater than HS (0.558) (Table 1). Permutation tests
between NST and GST indicated the existence of a
well-defined phylogeographical structure that extends
across the sampling range because NST was always
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
Figure 2. Phylogenetic tree and media-joining network constructed for chloroplast (cp)DNA haplotypes recovered for all sampled Dasiphora individuals. The
numbers above the branches of the phylogenetic tree (left) with the format ‘xx/yy’ indicate BEAST posterior probabilities (xx) and Bayesian posterior probabilities
from MrBayes (yy). Nodes representing major divergences are indicated by black circles containing different annotated numbers, whereas the stem age of the genus
is highlighted with a red circle [95% highest posterior density (HPD)]. The haplotype number and detected frequency are indicated at the end of each branch. The
sizes of the circles in the median-joining network (right) reflect the frequency of each haplotype. Small black dots denote the number of steps separating adjacent
haplotypes. The two species are indicated by the different colours of the outer rings. The three major haplogroups are labelled Clade A, Grade B and Grade C and
indicated by different colours. The three subgroups of clade A are labelled A1, A2, and A3.
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© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
PHYLOGEOGRAPHY OF THE GENUS DASIPHORA
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Table 1. Result of genetic diversity analysis and Mantel test
Groups
HS
HT
GST
NST
Mantel test r (P)
Dasiphora fruticosa
Dasiphora glabra
Total
0.525
0.652
0.558
0.909
0.952
0.918
0.423
0.315
0.392
0.641**
0.494*
0.596**
0.472 (0.000)
0.316 (0.002)
0.385 (0.000)
HS, mean genetic diversity within populations; HT, total genetic diversity; GST, interpopulation differentiation; NST, the
number of substitution types; *P < 0.05, **P < 0.01 (both indicating that NST is significantly larger than GST); r, correlation
coefficient obtained from the appropriate matrix.
Table 2. Result of analysis of molecular variance within Dasiphora
Groups
Source of variation
d.f.
SS
VC
V%
F-statistic
All samples
Among
Among
Within
Total
Among
Within
Total
Among
Within
Total
1
85
471
557
66
382
448
19
89
108
59.763
1096.911
651.327
1808.002
762.655
392.583
1155.238
334.256
258.744
593.000
0.265
1.798
1.383
3.447
1.573
1.028
2.600
2.719
2.907
5.627
7.71
52.18
40.11
FCT = 0.077*
FSC = 0.565*
FST = 0.599*
60.48
39.52
FST = 0.605*
48.33
51.67
FST = 0.483*
Dasiphora fruticosa
Dasiphora glabra
groups
populations within groups
populations
populations
populations
populations
populations
VC, variance component; V%, percent variation; FST, differentiation among populations; FSC, differentiation among
populations within species; FCT, differentiation among species; *P < 0.001, 1000 permutation.
significantly larger than GST, especially in D. fruticosa
(Table 1). Mantel tests for isolation-by-distance
revealed a significant correlation between the genetic
differentiation of populations and the geographical distance (Table 1). Hierarchical AMOVA results
showed that there was relatively little interspecific
differentiation (FCT = 0.077) but the interpopulation
differentiation within each species was much more
pronounced (Table 2).
Based on the phylogenetic analyses, we divided the
112 haplotypes into three groups (clade A, grade B,
and grade C), which were also recovered from the
network relationships (Fig. 2). A similar topology was
also obtained when considering only one fragment,
either rbcL or trnT-L sequences (results not shown).
The 24 basal haplotypes within grade C had a disjunctive distribution pattern, occurring mainly in the
southern and south-eastern regions of the plateau
(Fig. 1). Four of the haplotypes within this group were
specific to D. fruticosa; one was found in both species;
and the others were exclusive to D. glabra (Fig. 2).
The haplotypes of grade B occurred primarily in the
western and southern parts of the plateau (Fig. 1).
Most of its haplotypes were specific to D. fruticosa
and derived from the central haplotype, H7 (Fig. 2).
The derived haplotypes from this group were also
detected in populations from outside the QTP (Fig. 1).
The third group (clade A) encompassed 35 haplotypes,
including three that were widely shared between
the two species (H30, H40, and H47) (Fig. 2). Of the
minor haplotypes within this group, only one
haplotype was specific to D. glabra and the others
were specific to D. fruticosa. Haplotypes of this group
were mainly distributed across the eastern and northern regions of the plateau, although they were also
detected in four Himalayan populations (Fig. 1). The
network analysis revealed clear radiative patterns
from the three most common haplotypes and also
highlighted two tentative subclades (Fig. 2).
DATING
HAPLOTYPE DIVERSIFICATION AND
DEMOGRAPHIC EXPANSIONS
Although the phylogenetic relationships between the
covered cpDNA haplotype groups were poorly supported at some nodes (Fig. 2), we used BEAST to date
the crown times of the 12 most interesting nodes
(nodes 1–12; Fig. 2) for the recovered haplotypes of
the total genus which represent the major divergence
events within Dasiphora (Fig. 2, Table 3). The stem
age of Dasiphora was estimated to be 13.32 Mya [95%
highest posterior density (HPD): 4.99–23.51 Mya]
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
τ, time in number of generations elapsed since the sudden expansion episode; θ0 and θ1, pre-expansion and post-expansion population sizes; SSD, sum of squared deviations; RAG,
Harpending’s raggedness index; tmin, absolute expansion time estimated with a high substitution rate, 3 × 10−9 s s−1 year−1; tmax, absolute expansion time estimated with a low
substitution rate, 1 × 10−9 s s−1 year−1; tave, mean expansion time; kya, thousand years ago; CI, confidence interval; NC, not calculated.
−2.098 (0.001)
−1.992 (0.001)
−2.080 (0.001)
−1.869 (0.006)
−1.655 (0.014)
−0.680 (0.276)
−1.945 (0.001)
−7.811 (0.000)
−4.960 (0.001)
−22.531 (0.000)
−27.589 (0.000)
−25.218 (0.000)
−2.928 (0.176)
−24.295 (0.002)
76.923
76.923
4.512
48.615
152.250
NC
211.230
51.282–153.846
51.282–153.846
3.008–9.025
32.410–97.230
101.470–304.410
NC
140.820–422.461
0.431 (0.632)
0.638 (0.730)
0.076 (0.837)
0.025 (0.894)
0.020 (0.142)
0.017 (0.045)
0.006 (0.979)
0.00002 (0.533)
0.00064 (0.246)
0.003 (0.690)
0.001 (0.824)
0.008 (0.068)
0.009 (0.138)
0.007 (0.750)
0.247
0.122
29.617
6.770
36.600
386.25
8.955
Growth rate (95% CI)
Tajima’s
D (P value)
Fu’s FS
(P value)
tave
(kya)
tmin – tmax (kya)
RAG
(P value)
3.000 (0.395–3.500)
3.000 (0.486–3.500)
0.176 (0.000–2.836)
1.896 (0.326–3.586)
5.936 (3.219–7.766)
9.971 (5.523–25.088)
8.238 (1.783–13.617)
The present study reports a large-scale phylogeographical study on the shrub genus Dasiphora (Rosaceae).
We sampled 559 individuals from 87 populations and
Clade A1
Clade A2
Grade A3
Clade A
Grade B
Grade C
Total
DISCUSSION
SSD (P value)
(Fig. 2). However, the crown age of the total current
haplotypes was approximately 4.64 Mya (node 1) and
those of the other groups ranged from 3.25 Mya to
0.32 Mya (nodes 2 to 12), corresponding to a timescale
ranging from the mid-Pliocene to the late Pleistocene
(Fig. 2, Table 3).
The distributions of pairwise differences for the
samples from the identified haplotype groups were
uni- or bi-modal in all cases, except for those of total
samples and grade C, for which the observed curves
appeared to be multimodal (see Supporting information, Fig. S2). Further analyses using the nonsignificant variance and raggedness index tests suggested
that most of the observed distributions did not differ
significantly from those expected under a sudden
expansion model (Table 4). These results are consistent with sudden demographic expansions among most
groups or subgroups, which is further supported
by the star-like patterns of the haplotype network
(Fig. 2). The significant negative calculated values
for Fu’s FS and Tajima’s D, as well as the large ‘g’
value (which indicates the exponential population
growth rate), were also consistent with repeated
range expansions (Table 4). Given substitution rates
of 1 × 10−9 to 3 × 10−9 s s−1 year−1 (Wolfe et al., 1987),
the mean expansion times were calculated to be 152
and 49 kya for grade B and clade A (Table 4), respectively. The expansions of three subgroups (A1, A2, and
A3) were dated to around 77 kya (A1, A2) and 4.5 kya
(A3) (Table 4).
θ1
HPD, highest posterior density.
θ0
1.81–8.26
1.30–5.71
0.98–4.45
0.76–3.54
0.50–3.69
0.54–2.87
0.61–2.80
0.44–2.50
0.38–1.76
0.28–1.37
0.05–0.57
0.08–0.61
τ (95% CI)
4.64
3.25
2.54
2.02
1.97
1.59
1.58
1.39
0.95
0.75
0.29
0.32
Groups
1
2
3
4
5
6
7
8
9
10
11
12
Neutrality tests
95% HPD
Mismatch distribution
Time (Mya)
Table 4. Results of mismatch distribution analysis, neutrality test (Tajima’s D, Fu’s FS tests), and LAMARC analysis
Nodes
LAMARC
Table 3. Estimates of divergence times within Dasiphora
for the major nodes by BEAST analysis
930.558 (−444.149–976.158)
440.544 (−462.489–964.075)
934.012 (−295.897–996.471)
964.825 (52.310–996.674)
925.027 (208.045–990.022)
430.644 (92.540–854.333)
385.386 (131.574–706.017)
Y.-Z. MA ET AL.
0.000
0.000
0.751
0.018
0.000
6.729
0.000
784
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
PHYLOGEOGRAPHY OF THE GENUS DASIPHORA
sequenced two cpDNA regions (rbcL, trnT-L). The
cpDNA sequence datasets revealed high levels of
genetic variation among the samples. Phylogenetic
constructions based on cpDNA haplotypes showed that
all of the sampled Dasiphora individuals comprised a
monophyletic lineage (Fig. 2), indicating that no introgression or hybridization had occurred between this
genus and other genera. Our dating of the stem age of
Dasiphora suggests that it diverged from its sister
genus Fragaria around 13.32 Mya, with a 95% HPD
interval ranging from 4.99 Mya to 23.51 Mya (Fig. 2).
This is reasonably consistent with the recent dating
of the divergence between these two genera, which is
estimated to have occurred between 12 Mya and 27
Mya (Dobeš & Paule, 2010). By contrast to the early
origin of Dasiphora, the intrageneric divergences
appear to have occurred relatively recently, around
4.64 Mya (95% HPD: 1.81–8.26 Mya) (based on the
crown age for all identified haplotypes; see node 1 in
Fig. 2, Table 3). This relatively recent diversification
atop an unbranched stem lineage was considered
to reflect ancient extinctions (Harvey, May & Nee,
1994). Deep divergences appear to have occurred on an
ongoing basis within this genus since the mid-Pliocene
(Fig. 2, Table 3). Although results from such calibrations must be interpreted with caution, the estimated
timescales for the deep divergences (major nodes) are
in good agreement with the timings of the recent QTP
uplifts (which occurred between 5 Mya and 1.8 Mya)
and the extensive glaciations that occurred during the
Quaternary period (Li, Shi & Li, 1995; Shi, Li & Li,
1998; Zheng, Xu & Shen, 2002). A number of species
diversifications and deep intraspecific divergences
appear to have occurred in multiple genera and species
during this period (Liu et al., 2002; Wang et al., 2009a,
b; Jia et al., 2011). Therefore, it is very likely that the
deep divergences identified within Dasiphora in the
present study were caused by allopatric differentiation
and reductions in gene flow following the plateau
uplifts and climate oscillations.
The sampled individuals were assigned to
D. fruticosa or D. glabra according to the colours of
their petals, although a number of haplotypes were
detected in both species. Moreover, phylogenetic
analyses suggested that the cpDNA haplotypes specific to D. fruticosa and D. glabra did not cluster into
completely separate lineages or clades (Fig. 2). Similarly, AMOVA indicated that the genetic differentiation between the two species was quite low, whereas
the population differentiation within each species was
fairly high (Table 2). These results implied that the
interspecific delimitation based on petal colour is
not exactly correlated to the cpDNA differentiation.
However, it is interesting that most basal haplotypes
in grade C of the phylogenetic tree primarily fixed
for D. glabra individuals (i.e. Dasiphora plants with
785
white petals) were located in the southern and southeastern regions of the plateau, and those from grade
B mainly fixed for D. fruticosa individuals (i.e. plants
with yellow petals) were mostly found in the more
western regions. By contrast, haplotypes from the
derived clade (clade A), especially H30, H40, and H47,
which were found to be shared between the two
species, inhabited almost exclusively in the northern
region (Figs 1, 2). Although the alternative possibility
(i.e. the retention of ancient polymorphism) could
not be excluded, such a genetic pattern is suggestive
of earlier allopatric divergences and later hybridizations (Avise, 2000), which might be facilitated by
range shifts that enabled the mixing of the different
lineages (Wang et al., 2009c; Sun et al., 2010).
This proposal is further supported by the fact that
the majority of haplotypes from the two basal
haplogroups (grade B and C) are specific to
D. fruticosa and D. glabra, respectively. Our findings
additionally indicate that the use of choloroplast loci
as the only fragments of plant molecular barcoding
may not be sufficient and that information from
the nuclear genome is indispensable (Li et al., 2011).
It should be noted that hybridizations and
introgressions between closely related species but
with morphological distinctness are very common in
plants as a result of incomplete reproductive isolations (Li et al., 2012). Molecular barcoding of these
species should rely on more nuclear markers and such
analyses can determine the nuclear compositions of
these hybrids contributed respectively by each parental species (Avise, 2000).
In line with the previously published studies (Li
et al., 2009; Wang et al., 2009c; Shimono et al., 2010;
Sun et al., 2010), our results indicate that both
species of this genus have experienced repeated
demographic expansions, as revealed by the formation of two cpDNA haplotype groups: grade B and
clade A (Table 4; see also Supporting information,
Fig. S2). The earlier expansion of grade B might have
occurred across the whole QTP, whereas the recent
one may have been localized to the northern or
eastern regions of the plateau (Figs 1, 2). These two
major expansions were estimated to have occurred
before the Last Glacial Maximum (LGM: 0.018–
0.024 Mya) (Shi et al., 1998) (Table 4), likely during
the inter-uplift and/or interglacial stages when the
climate was still relatively cold, because Dasiphora,
as a dominant, cold-preferring and pioneering shrub,
would have been able to occupy newly-formed niches
more rapidly than other plants (Avise, 2000). The
frequent occurrence of polyploids in both species
(Elkington & Woodell, 1963; Klackenberg, 1983)
might have accelerated these expansions because
polyploids tend to occupy newly available niches as
a result of the geological and/or climatic changes
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788
786
Y.-Z. MA ET AL.
(Ramsey, 2011). In addition, population-private haplotypes derived from these historical expansions were
widely detected across the plateau (Fig. 1; see also
Supporting information, Table S1), reflecting that
most studied populations survived the LGM in situ
or nearby localities (Liu et al., 2012). We further
inferred that the southern and south-eastern plateau
have served as refugia for the genus throughout
the Quaternary and thus provided resources for
the later diversification and expansion because: (1)
some haplotypes in the basal group of the cpDNA
phylogenetic tree were disjunctively detected in populations of the southern and south-eastern plateau
(Fig. 1); (2) Dasiphora has a high tolerance for
extreme conditions (Elkington & Woodell, 1963); and
(3) the existence of multiple high-altitude refugia
that would have facilitated survival during the LGM
has previously been proposed for other alpine plants
occurring in the QTP (Wang et al., 2009b; Opgenoorth
et al., 2010; Jia et al., 2011; Gao et al., 2012; Liu et al.,
2012).
Overall, deep divergences and range expansions
within the genus were estimated to have occurred
between the Pliocene and the late Pleistocene.
However, these genetic divergences do not correlate
with the morphological delimitations of the two
current species based on their petal colours. Some
haplotypes or genetic lineages were shared by the two
species. Nevertheless, the distributions of some recovered lineages were geographically correlated. In addition, these cpDNA lineages contained clear evidence
of demographic expansions. Our results support
the hypothesis that range shifts taking place in the
late Pleistocene (32–304 kya) and middle-late Holocene (3–9 kya) might have blurred early divergences
created by the uplifts of the plateau in the Pliocene
(3.25–4.64 Mya) and the climatic oscillations during
the Pleistocene (0.32–2.54 Mya). These findings
clearly illustrate the complex evolutionary histories of
alpine plants.
ACKNOWLEDGEMENTS
We are highly indebted to Professor Jian-Quan Liu
for his direction on this work and for revising the
manuscript. We appreciate the help received from
Hiroshi Ikeda, Richard Milne, Kang-Shan Mao, Bin
Tian, Teng-Liang Liu, Li-Li Wu, Rui-Rui Liu, Jun
Zeng, Yi-Xuan Kou, Ping Chen, Ru Zhang, and two
anonymous reviewers with respect to field work,
experimental help, data analysis or constructive comments on the draft of the manuscript. This study was
supported by the Natural Science Foundation of
China (81274024, 30800064), the Open Funds of the
State Key Laboratory of Systematic and Evolutionary
Botany, Institute of Botany, Chinese Academy of Sci-
ences (lzusm-wyj), and the Program for New Century
Excellent Talents in University (NCET-08-0261).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Figure S1. Photographs of Dasiphora fruticosa (A, LiuJQ-08KLS-011) and Dasiphora glabra (B, LiuJQ-GN2011-114).
Figure S2. Mismatch distribution analyses for total populations and the samples from the main haplotype
groups/subgroups. The red line represents the observed distribution of pairwise differences among the samples,
whereas the blue line indicates the distribution expected under the sudden expansion model.
Table S1. Geographical information, haplotype frequency, haplotype diversity (HE), and number of private
haplotypes (Nph) of the sampled population.
Table S2. Information of five primers used for polymerase chain reaction amplification and sequencing.
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 777–788