Postglacial colonization of the Tibetan plateau inferred from the matrilineal genetic structure of the endemic red-necked snow finch,
Pyrgilauda ruficollis
YAN HUA QU,
*
PER G. P . ERICSON,
†
FU MIN LEI
*
and SHOU HSIEN LI
‡
*
Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuanxi Road, Haidian District, Beijing 100080, People’s Republic of China,
†
Department of Vertebrate Zoology, Swedish Museum of Natural History, PO Box 50007, SE-10405 Stockholm, Sweden,
‡
Department of Life Sciences, National Taiwan Normal University, Taiwan
Abstract
Most phylogeographical studies of postglacial colonization focus on high latitude locationsin the Northern Hemisphere. Here, we studied the phylogeographical structure of thered-necked snow finch
Pyrgilauda ruficollis
, an endemic species of the Tibetan plateau. Weanalysed 879 base pairs (bp) of the mitochondrial cytochrome
b
gene and 529 bp of thecontrol region in 41 birds from four regional groups separated by mountain ranges. Wedetected 34 haplotypes, 31 of which occurred in a single individual and only three of whichwere shared among sampling sites within regional groups or among regional groups.Haplotype diversity was high (
h
= 0.94); nucleotide diversity was low (
d
= 0.00415) and geneticdifferentiation was virtually non-existent. Analyses of mismatch distributions and geographi-cally nested clades yielded results consistent with contiguous range expansion, and theexpansion times were estimated as 0.07–0.19 million years ago (Ma). Our results suggest that
P. ruficollis
colonized the Tibetan plateau after the extensive glacial period (0.5–0.175 Ma),expanding from the eastern margin towards the inner plateau. Thus, in contrast to many ofthe postglacial phylogeographical structures known at high latitudes, this colonizationoccurred without matrilineal population structuring. This might be due to the short glacialcycles typical of the Tibetan plateau, adaptation of
P. ruficollis
to cold conditions, or refugiaand colonized habitat being semicontinuous and thus promoting population mixing.
Received 5 September 2004; revision received 19 November 2004; accepted 7 February 2005
Introduction
Postglacial colonization has created a variety of phylogeo-graphical structures in species from different latitudes(Rising & Avise 1993; Hewitt 1996; Merila
et al
. 1997). Previ-ously glaciated areas in the Arctic and the sub-Arctic regionscontain species with low levels of clade divergence, indicat-ing recent colonization followed by population expansion.In Europe and North America, such areas contain specieswith intermediate clade divergences, indicating their sur-vival during several ice ages. In the tropics, this area containsspecies with deeply diverged clades, often within small geo-graphical areas, indicating their survival there since thePliocene (Hewitt 2000, 2004). Whereas many comparisons
of phylogeographical structures are available for regions atdifferent latitudes, studies that focus on previously glaciatedmontane areas are rare. Herein we present a study of a speciesendemic to the Tibetan plateau.
The Tibetan plateau occupies an area of 2.5 million km
2
, orapproximately one-quarter of China, and has an averagealtitude of 4500 m above sea level (a.s.l.). It is the youngestplateau on Earth; the most recent uplift event occurringbetween 3.6 and 1.7 million years ago (Ma) (Li & Zhou 1998).The uplift caused great climatic changes: grasslands replacedforests while the climate gradually became drier, colder andwindier, and glaciers and deserts developed (Wu
et al
. 2001).The unique geomorphological configuration, the complex landconditions, the diversified climate, and the unique geologicalevolution combine to make the Tibetan plateau an area of world-wide importance for the evolution of endemic, specializedmontane species (Cheng 1981; Tang 1996; Macey
et al
. 1998).
Correspondence: Yan Hua Qu, Fax: 0086 10 62565689; E-mail:[email protected]
Glacial cycles in alpine regions have generated variedphylogeographical structures that reflect different routesof postglacial colonization (Mardulyn 2001; Despres
et al
.2002; Kropf
et al
. 2003). The topographical diversity of theTibetan plateau might have created both networks of refugiaduring glaciation and complex barriers to subsequent expan-sion (Hewitt 2004). In comparison to species that colonizedtheir present-day ranges from lower latitudes, montanespecies undergoing postglacial colonization would haveneeded to undertake altitudinal shifts, and might have beenable to spread more widely across tundra and steppe plains.In this study, we assessed these possibilities by conductinga phylogeographical study of an alpine bird endemic tothe Tibetan plateau, the red-necked snow finch
Pyrgilaudaruficollis
.
Pyrgilauda ruficollis
is one of the four species of the genus
Pyrgilauda
(Eck 1996), three of which (
Pyrgilauda ruficollis
,
Pyrgilauda blanfordi
and
Pyrgilauda davidiana
) have similarranges in the Tibetan plateau (Qu
et al
. 2002). This speciesis a year-round resident across the mountain steppe zoneat altitudes of 3500–5300 m a.s.l., or higher (Fig. 1a), whereit occurs in alpine meadows and breeds inside pika(
Ochotona
spp.) burrows.
Pyrgilauda ruficollis
makes irregularaltitudinal movements, descending to lower altitudes inlarge flocks during autumn and winter when driven byextreme weather conditions (Cramp & Perrins 1994). Thehighest known records of
P. ruficollis
are from 5300 m a.s.l.in the Tanggula Mountains. Several mountain ranges,some with peaks over 6500 m a.s.l., occur within the distribu-tion of
P. ruficollis
, and these might create barriers to geneflow because they are believed to be major zoogeographi-cal barriers associated with evolutionary divergence(Mayr 1963; Macey
Here, we assume that the present-day distribution of
P. ruficollis s
tems directly from postglacial colonization. Wehypothesize that isolation in different refugia surroundingthe Tibetan plateau led to phylogeographical divergence inthis species. We also hypothesize that the mountain rangeswithin the present distribution range constitute barriers togene flow that have led to population differentiation. Thegoal of the study is to test these hypotheses by describing thephylogeographical and population structures of
Pyrgilaudaruficollis
in the Tibetan plateau and using these structuresto infer evidence for population bottlenecks and expansion,and genetic divergence.
Materials and methods
Study area and sample collections
The birds were collected using mist nets from 10 sites coveringmajor parts of the range of
Pyrgilauda ruficollis
. Each birdwithin a site was taken from a different part of the colony
to avoid sampling relatives (Hansen
et al
. 1997). Blood ortissue samples were obtained from 43 birds. Groups withadequate sample sizes were created by pooling birds intofour regional groups: QR (Qinghai region, average altitude4000 m a.s.l.), TR (Tanggulashan region, average altitude5500 m a.s.l.), WTR (west Tibet region, average altitude 4800 ma.s.l.), and ETR (east Tibet region, average altitude 4500 ma.s.l.) (Fig. 1b, c and Table 1).
DNA extraction, polymerase chain reaction and sequencing
Genomic DNA was extracted from blood or tissue samplesusing the QIAamp DNA Mini Kit (QIAGEN) following manu-facturer’s instructions. Initially, 879 bp of the cytochrome
b
gene was amplified as a single fragment with the primer pair
L14841
(5
′
-CCATCCAACATCTCAGCATGATGAAA-3
′
)(Kocher
et al
. 1989) and
H15915
(5
′
-AACTGCAGTCATCT-CCGGTTTACAAGAC-3
′
) (Edwards & Wilson 1990). Thethermocycling program consisted of an initial denaturationat 94
°
C for 5 min, followed by 40 cycles of 94
°
C for 40 s, 49
°
Cfor 40 s, and 72
°
C for 5 min. For the sequencing reactions,the following primers were used:
L14841
,
H15915
,
P5L
(5
′
-CCTTCCTCCACGAAACAGGCTCAAACAACCC-3
′
) and
H658
(5
′
-TCTTTGATGGAGTAGTAGGGGTGGAATGG-3
′
)(Irestedt
et al
. 2002), with
P5L
and
H658
as internal primerson the light and heavy strands, respectively.
A 529-bp fragment of the control region was amplifiedusing the primer pair,
F304
(5
′
-CTTGACACTGATGCAC-TTG-3
′
) and
H1261
(5
′
-AGGTACCATCTTGGCATCTTC-3
′
) (Marshall & Baker 1997). The thermocycling programconsisted of an initial denaturation at 94
°
C for 5 min,followed by 40 cycles of 94
°
C for 40 s, 56
°
C for 40 s, and72
°
C for 5 min. The same primers were used for the sequen-cing reactions.
The polymerase chain reaction (PCR) products werepurified using QIAquickTM PCR purification Kit (QIAGEN),and then sequenced on a Perkin-Elmer 377 semiautomatedDNA sequencer (Applied BioSystems), using Perkin-ElmerPrism terminator cycle sequencing kits (Applied BioSystems)with Ampli
Taq
FS polymerase with BigDye terminators.Both strands of each PCR product were sequenced. Thesequencing program consisted of 25 cycles of denaturationat 96
°
C for 30 s, annealing at 50
°
C for 15 s, and extensionat 60
°
C for 4 min.Multiple sequence fragments were obtained by sequen-
cing with different primers for each gene and individual.While a pair of internal primers (
P5L
and
H658
) of the cyto-chrome
b
was used to sequence approximately half of thegene (about 400 bp and 500 bp, respectively), other pair ofprimers (
L14841
and
H15915
) obtained whole sequence.No length variation in the control region was found, mak-ing alignment straightforward. Complete sequences wereassembled using
seqman
II (DNASTAR). Sequences were
P O S T G L A C I A L C O L O N I Z A T I O N O F R E D - N E C K E D S N O W F I N C H
Fig. 1 The study area and locations of thefour regional groups separated by mountainranges. (a) The distribution records andrange of Pyrgilauda ruficollis. (b) The samplingsites and regional groups. (c) The mountainranges in the study area. Notes: BayanHar mountains at 5300 m a.s.l. on averageand A’nyemaqen mountains at 5500 m a.s.l.on average separate QR from other regionalgroups. Tanggula mountains with an averagealtitude of 6000 m a.s.l. separate TR from otherregional groups. Gangdise-Nyaingentanglhamountains at an average altitude of 6000 ma.s.l. separate WTR from ETR.
compared visually to the original chromatograms to avoidreading errors. Complete sequences were aligned by eye.All sequences are accessible at GenBank (Accession nosAY825286–AY825329, AY961957–AY961980).
Sequence variation and genetic diversity
Numbers of haplotypes (
HT
), and values of haplotypediversity (
h
) (Nei 1987), nucleotide diversity (
3
; Nei &Tajima 1981) and nucleotide divergence (
d
; Nei & Tajima1981) were computed using the package,
dnasp
(version4.0; Rozas
et al
. 2003).
Hierarchical analysis of molecular variance
A hierarchical analysis of molecular variance (
amova
;Excoffier
et al
. 1992) was implemented using the
arlequin
version 2.0 package (Schneider
et al
. 1997). The
F
statistics werecomputed using haplotype frequencies alone, and the signi-ficance of departures from zero of
F
statistics and geneticvariance components was tested using 10 000 permutations.
Mismatch distribution analysis
Mismatch distributions were calculated using
arlequin
2.0, and their fit to Poisson distributions was assessedby Monte Carlo simulations of 1000 random samples. Thesum of squared deviations (SSD) and raggedness indexes
(
r
) between observed and expected mismatch distributionswere used as a test statistic and their
P
value representedthe probability of obtaining a simulated sum of squareddeviation larger or equal to the one observed.
Values of Tajima’s
D
(Tajima 1989) were calculated fromthe total number of segregating sites and used to assessevidence for population expansion, under which negativevalues are expected (Aris-Brosou & Excoffier 1996). Esti-mation and testing were done by bootstrap resampling(10 000 replicates) using
arlequin
2.0.The relationship
τ
= 2
ut
(Rogers & Harpending 1992)was used to estimate a time of expansion (
t
), where
τ
is themode of the mismatch distribution, expressed in units ofevolutionary time, and
u
is the mutation rate for the wholesequence. The value
u
was calculated using the formula
u
=
µ
k
, where
µ
is the mutation rate per nucleotide and
k
isthe number of nucleotides assayed. A mutation rate of 2%per million years (Myr) (
µ
= 2.0
× 10−8) was used, as a standardevolutionary rate of mitochondrial DNA used in most studiesof avian species. A generation time of 1.5 years was used inall calculations (Summers-Smith 1988).
Coalescent-based estimation of gene flow
We used the program migrate version 1.7.6 (Beerli 1997) toestimate maximum-likelihood migration rates among fourregional groups. This approach, based on coalescence usingMarkov chain Monte Carlo (MCMC) searches, takes account
Table 1 Details of sampled and sampling sites, and values of nucleotide divergence, diversity and haplotype diversity in regional groups
Regional groups
Sampling sites
UTM coordinates
Haplotypes present N
Nucleotide divergence (d)
Nucleotide diversity (3)
Haplotype diversity (h)
Qr (Qinghai region) Huashixia –1 194 125.22 4 041 084.6
Three haplotypes (in bold) occurred in more than one bird from different regional groups (haplotype 8) and different sampling sites (haplotypes 6 and 23) (numbers of occurrence in italics).
P O S T G L A C I A L C O L O N I Z A T I O N O F R E D - N E C K E D S N O W F I N C H 1771
of unequally effective population sizes and asymmetricalgene flow (Beerli & Felsenstein 1999). Effective populationsizes and gene flow rates were estimated from FST valuesand were set as initial values. We performed 10 short chains(500 trees used out of 10 000 sampled) and three long chains(5000 trees used out of 100 000 sampled). Adaptive heatingwith four chains of different temperature (1, 3, 5, 8) was used.These runs were repeated using the same condition untilconsistent results were obtained.
Nested design and nested cladistic analysis of geographical distances
The networking algorithm developed by Templeton et al.(1992) was used to construct the intraspecific maximumparsimony phylogenetic relationship among haplotypesusing the program tcs version 1.18 (Clement et al. 2000).Clade distances (Dc) and nested clade distances (Dn) weredefined based on the geographical locations of samples inthe nesting cladogram, and were estimated as described inTempleton et al. (1995). The differences between interior(ancestral) and tip (recent) clade Dc and Dn distances werecalculated to yield DcI − DcT and DnI − DnT values, whereI and T were interior and tip clades, respectively.
The null hypothesis of no geographical associations of tipclades and interior clades was tested by considering thatthe dispersion distance of clades was not greater or lessthan expected by chance, and comparing observed Dc andDn values with a distribution of such values, calculated foreach 10 000 random permutations of clades against sam-pling locations (Templeton & Sing 1993; Templeton 1995).Permutation tests were conducted separately for each levelof the nested cladogram using geodis version 2.2 (Posadaet al. 2000). As soon as significance levels for Dc and Dnwere determined, inferences about the processes that werelikely to be responsible for observed patterns of cladestructure were made using the latest inference keys pro-vided at http://darwin.uvigo.es (updated July 2004).
Results
Sequence variation and genetic diversity
Full-length DNA sequences of the cytochrome b gene andthe control region (CR) were obtained for 41 of 43 birds.
The combined length of these sequences (1405 bp) contained49 polymorphic sites, 27 of which were parsimony informative.The cytochrome b gene sequences contained 32 polymorphicsites, 20 of which were parsimony informative, and 19 ofwhich were characterized by T↔C transitions, nine by A↔Gtransitions, two by both a transition and a transversion(C↔T↔G; A↔G↔C), one by T↔G transversion and oneby G↔C transversion. Seven variable amino acid positionswere detected in cytochrome b. The CR sequences contained17 polymorphic sites, seven of which were parsimony infor-mative, and 11 of which were characterized by T↔C transi-tions, two by A↔G transitions, two by T↔G transversion,one by A↔C transversion and one by C↔G transversion.
These polymorphic sites defined 34 unique haplotypes,31 of which were observed in a single bird each and onlythree of which were shared among different regional groupsor different sampling sites from same regional groups.Haplotype 8 was shared between the groups QR and WTR,and the sampling sites within each group. Two individualsin ETR shared haplotype 6, and haplotype 23 was sharedby four individuals in QR. Within regional groups, haplotypediversity values were nearly maximal; nucleotide diversitywas the highest in QR, whereas the other three groups hadthe similar values; pairwise nucleotide divergence valueswere highest in QR and lowest in ETP (Table 1). The valueof Tajima’s D test was −1.3254 in the group of all haplo-types combined (P > 0.1), suggesting that the observednucleotide polymorphism is selectively neutral.
Hierarchical analysis of molecular variance
Virtually all of the total genetic variance was located atthe smallest geographical scale: among individuals withinsampling sites. The only remaining component that wassignificantly greater than zero was located among siteswithin regional groups. The component located among theregional groups was not significantly different from zero(Table 2).
Mismatch distribution analysis
The mismatch distributions for the three largest regionalgroups and for the group containing all haplotypes com-bined consisted of distinct unimodal curves (Fig. 2). The TRgroup was excluded from this analysis because of its low
Table 2 Hierarchical analysis of molecular variance for Pyrgilauda ruficollis
Source of variation Variance component Φ-statistics Variance explained (%) P Fixation indices
Among regions 0.0008 0.17 0.393 FCT = 0.0017Among samples/within regions 0.0292 7.93 0.002 FSC = 0.0794Within samples 0.454 91.9 0.0004 FST = 0.0810
sample size. Both the variance (SSD) and raggedness index(r) tests suggested that the curves did not significantly differfrom the distributions expected from a model of populationexpansion. Tajima’s D statistics showed negative valuesfor all three regional groups (Table 3). These resultsare consistent with the groups having undergone recentpopulation expansion. The estimates of expansion timeranged from approximately 72 000–190 000 years.
Coalescent-based estimate of gene flow
Significant levels of historical gene flow were detected for4 of the 12 possible source-recipient relationships betweenpairs of regional groups (Fig. 3 and Table 4). The gene flowestimates for the remaining eight possible relationshipswere close to zero. The estimates of female effective popu-lation sizes scaled by mutation rate were larger for QRand ETR, than TR and WTR (Table 4).
Nested cladistic analysis of mitochondrial DNA haplotypes
The nested clade network of haplotypes of Pyrgilaudaruficollis was centred on haplotype 8, which occurred ineach of four sites in two regional groups. Many of the otherhaplotypes were derivable from it by one or two muta-tions, and the network contained several extinct or unsampledhaplotypes (Fig. 4). The most diverged pair of haplotypesdiffered by six substitutions. The parsimony network ofhaplotypes was resolved, except for the presence of one
loop of ambiguity (loop 1 in Fig. 4). Three major haplotypegroups (haplogroups) were identified: 4.1, 4.2 and 4.3, allof which contained haplotypes from all four regional groups.
Some phylogeographical structures within clades weredetected (Table 5). Three associations between clade andgeographical location were significant (1-5, 1-18 and 2-12),all at low nesting levels (one-step to two-step haplogroups).These patterns are consistent with contiguous range expan-sion. For three-step (3-2) and four-step (4-1 and 4-2)haplogroups, results were ambiguous because of inadequategeographical sampling. Geographical associations were eitherdue to long-distance colonization and past fragmenta-tion in the scenario where P. ruficollis was absent in theintermediate areas, or due to contiguous range expansionin the scenario where P. ruficollis was present in these areas.To discriminate the type of movement leading to thispattern, we performed mismatch distribution analysis andcalculated SSD and raggedness index for haplogroups 3-2,4-1 and 4-2. A model of demographic expansion wasstatistically supported for these clades: P(SSDobs) valueswere 0.38, 0.86 and 0.33; raggedness indexes were 0.04, 0.05and 0.02; and P(Ragobs) values were 0.77, 0.55 and 0.63,respectively. A contingency test detected a significantgeographical association of haplotypes contained withinhaplogroups 3-5, 4-2 and the total cladogram.
Discussion
The combination of low nucleotide diversity and high haplo-type diversity, and the shape of the mismatch distributions,both suggest that Pyrgilauda ruficollis underwent a rapid rangeexpansion following a population bottleneck. Time estimatesderived from the mismatch distributions suggest that thispostglacial colonization occurred at 0.07–0.19 Ma, which isconsistent with expansion occurring after the extensiveglacial period (0.5–0.175 Ma). The nested cladistic analysissuggests that the range expansion was contiguous withgradual movement, with no strong geographical differ-entiation for any clade. amova and migrate indicate that
The parameters of the model of sudden expansion (Rogers & Harpending 1992) are presented as well as goodness-of-fit test to the model; SSD, sum of squared deviations; r, raggedness indexes. Tajima’s (1989) D test values and their statistical significance are also given (S, number of polymorphic sites, θ0, pre-expansion and θ1, postexpansion population size, τ, time in number of generations, elapsed since the sudden expansion episode).
Table 4 Estimates of gene flow (Nem) and theta between regionalgroups of Pyrgilauda ruficollis
Table 5 The nested cladistic analysis of geographical distances for the mitochondrial DNA haplotypes of Pyrgilauda ruficollis. Thehaplotype designations are given at the top and are boxed together to reflect the one-step nested design given in Fig. 4
Tests determine whether the within-clade (Dc) or nested clade (Dn) geographical distances are significantly large (L) or significantly small (S) at the 0.05 (*), 0.01 (**) or 0.001 (***) levels. Where interior and tip clades are present, significance is also tested for the average difference between these two types of clades (I-T). Interior clades are in bold. Note that the number of steps indicated refers to the clades within the nested clades.
no significant genetic divergence exists among regionalgroups separated by mountain ranges.
Inference of recent population expansion in Pyrgilauda ruficollis
The levels of haplotype diversity in Pyrgilauda ruficollispopulations are higher than in most other avian speciesfrom previously glaciated areas, whereas the values ofnucleotide diversity are lower (Table 6). This relationshipbetween haplotype and nucleotide diversity is also evidentfrom the unimodal mismatch distributions. Such a patternis frequently attributed to population expansion, whichenhances the retention of novel mutations (Watterson 1984;Avise & Walker 1998) and creates an excess of haplotypesdiffering by one or a few mutations (Slatkin & Hudson 1991;Rogers & Harpending 1992).
The analyses of matrilineal gene flow and of nestedclades provide evidence for range expansion at the scale of
the Tibetan plateau. Three of the low-level clades providethe clearest evidence that range expansion was contiguouswith gradual movement. The inadequate geographicalsampling prevents resolution between long-distance colo-nization, combined with population fragmentation andgradual movement as the mechanism to explain rangeexpansion based on high-level clades. For clarifying thisquestion, further work based on microsatellites will becontinued.
Postglacial colonization of P. ruficollis populations
Analyses of the cytochrome b sequences from four speciesof the genus Pyrgilauda suggest that the speciation ofP. ruficollis occurred at 1.0–1.5 Ma (Qu 2003), which is approxi-mately contemporaneous with the uplift of the Tibetanplateau. The plateau has since undergone four or fiveglaciations (Shi 2002; Zheng et al. 2002). The largest glacierof the Tibetan plateau occurred in middle Pleistocene (0.5
Fig. 2 Mismatch distributions for the three largest regional groups and for the entire sample. The histograms represent the observedfrequencies of pairwise differences among haplotypes and the line shows the curve expected for a population that has expanded. (a)Mismatch distribution in the QR group. (b) Mismatch distribution in the ETR group. (c) Mismatch distribution in the WTR group. (d)Mismatch distribution in the entire sample.
Table 6 Comparison of nucleotide diversity, haplotype diversity, phylogeographical pattern and expansion time estimates of Pyrgilauda ruficollis in the Tibetan plateau with values frombirds from other glaciated areas. See References for details
Species Study areas MarkerNucleotide diversity (3)
Haplotype diversity (h)
Phylogeographicalstructure
Expansion time Glaciations Reference
Sooty tern Sterna fuscata
Atlantic, Pacific, and Indian oceans
mtDNA, CR 0.0007–0.0012 0.53 Three clades: Atlantic, Indo-Pacific and southwest Pacific
10 kyr Final Pleistocene glacial cycle (12.5–17.5 kyr)
Peck & Congdon 2004
Sooty tern Sterna fuscata
Atlantic, Pacific, andIndian oceans
mtDNA, CR 0.029 0.82–0.90 Two clades: Atlantic and Indo-Pacific
7.5–187 kyr Final Pleistocene glacial cycle (12.5–17.5 kyr)
Avise et al. 2000
Bearded vulture Gypaetus barbatus
Temperate and tropical regions
mtDNA, CR 0.0292 0.932 Two clades: Western, and Eastern and tropic
12–14 kyr Pleistocene glacial maxima
Godoy et al. 2004
Razobill Alca torda
Atlantic Ocean mtDNA, CR 0.0093–0.0198 0.81–0.97 Two clades Pleistocene glaciations
Moum & Arnason 2001
Common guillemot Uria aalge
0.0042–0.0066 0.68–0.89 Lack of geographical structure
Pied flycatch Ficedula hypoleuca
Sweden mtDNA RFLP Too small sample size to determine subdivision
100 kyr Last Pleistocene glaciation
Tegelström et al. 1990
Eider duck Somateria mollissima
Northern Europe mtDNA, CR 0.004–0.033 0.6–1 Isolation by distance 10 kyr Weichsel glaciation (20 kyr)
Tiedemann et al. 2004
Greenfinch Carduelis chloris
Europe mtDNA, CR 0.00134 0.612 Two clades: northern European and southern European
5–8 kyr Post-Pleistocene glaciations
Merila et al. 1997
Great bustard Otis tarda
Europe mtDNA CR, cyt b, tRNA’s
0.0032 0.17 Two clades: Iberian Peninsula and European mainland
200 kyr Last glacial period/or several Quaternary cold periods
Pitra et al. 2000
Bluethroat Luscinia svecica
Eurasia mtDNA CR, cyt b
0.00023–0.003 0.86 Two clades: southern and northern groups
15 kyr Post-Pleistocene glaciations
Zink et al. 2003
Yellow wagtail Motacilla flava
Eurasia mtDNA, cyt b, ND3
0.0025–0.0047 0.76 Three clades: Europe, southwestern Asia, northeastern Asia and southeastern Asia
Citrine wagtail Motacilla citreola
Eurasia 0.0014–0.0022 0.86 Two clades: Eastern and Western
Last Pleistocene glaciation
Pavlova et al. 2003
Rock Patridge Alectoris graeca
Mid-latitude temperate zone
mtDNA, CR 0.008 0.76 Two clades: Sicily and other regions
Myr). The extensive glacial advance continued until 0.17Ma, after the penultimate glaciation (0.3–0.13 Ma) (Zhuoet al. 1998; Zhang et al. 2000; Shi 2002; Zheng et al. 2002).Our estimates of time since population expansion are thusconsistent with range expansion that occurred after theextensive glacial period.
The different estimates of time since expansion suggestthat these populations are still in genetic disequilibria. Thisinference is consistent with our analysis of matrilinealgene flow, which identified QR and ETR as the source ofgene flow to other regions and identified these groups aspotential refugia during glacial advance. The larger esti-mates of female effective population size for these groupsalso suggested that they were important historical sourcesof migrants.
Geological evidence suggests that during the maximumglacial advance, an ice sheet covered an area five to seventimes larger than it does today (Shi et al. 1990; Wu et al.2001; Zheng et al. 2002). At that time, ice cover would havebeen permanent in the highest altitude and central regionsof the Tibetan plateau (Shi et al. 1990; Shi 1996), and thefrequency of glaciers in the east was less than that in thewest (Zhang et al. 2000). QR is located in the northeastern
margin of the Tibetan plateau, which was free of ice cover,whereas ETR is located on the southeastern margin, whichwas covered by ice (Fig. 3; Li 1986; Shi et al. 1990; Shi 1996).Some ice-free areas might have existed around QR andETR and these could have provided suitable refugia(Cheng 1981; Tang 1996). Pikas (Ochotona spp.) migrated toETR during the late Pleistocene (Li 1986). Abandoned bur-rows of pikas provide the only nest site used by P. ruficollis(Dementiev & Gladkov 1954; Cheng 1981; Fu 1998). It istherefore plausible that P. ruficollis existed in refugiaaround the eastern margin of the Tibetan plateau duringthe glacial advance. From there, populations could haveexpanded towards the inner plateau after the retreat ofthe glaciers.
Population structure in P. ruficollis
We found that genetic differentiation among sampling sitesand regional groups was extremely low and that most ofthe variance occurred within sampling sites. Consequently,we found no evidence that mountain ranges created barriersto gene flow. This conclusion was supported by the nestedcladistic analysis, in which most of the haplotypes and
Fig. 3 Gene flow connections among regional groups. The shadow area represented the range of ice cover during the maximum Pleistoceneglaciation (data from 1: 4 000 000 digital elevation data developed by Institute of Geography, Chinese Academy of Sciences).
haplogroups showed no strong geographical divergence.Extensive gene flow after postglacial colonization canexplain this pattern of low divergence. At equilibrium,gene flow of the order of a few individuals per generationis theoretically sufficient to prevent the genetic divergence(Hartl & Clark 1989). Nonetheless, our results suggest thatP. ruficollis has undergone recent range expansion and is stillin genetic disequilibrium. We therefore favour extensivegene flow occurred during a range expansion and an insuffi-cient time for genetic differentiation as the best workinghypothesis to explain the virtual lack of matrilineal geneticstructure in P. ruficollis.
Comparison of the phylogeographical structure of P. ruficollis with that of avian species from other glaciated areas
We compared the phylogeographical structure of P. ruficolliswith that of avian species from other glaciated areas(Table 6). This comparison is preliminary and needs to beconfirmed using larger data sets. The phylogeographicalstructure of P. ruficollis is most similar to that of Arcticbirds, with some clear differences, and is very differentfrom the marked phylogeographical structure typical ofEuropean, North American and tropical birds.
Fig. 4 Nested clades of Pyrgilauda ruficollis haplotypes. The zeros refer to unobserved haplotypes intermediate between observedhaplotypes. One-step clades are indicated by ‘1-#’; two-step clades by ‘2-#’, three-step clades by ‘3-#’ and four-step clades by ‘4-#’, where #is the number assigned to the clades within each level.
P O S T G L A C I A L C O L O N I Z A T I O N O F R E D - N E C K E D S N O W F I N C H 1779
Whereas Arctic birds have shallow clades with significantgenetic divergence, P. ruficollis has weak phylogeographicalstructure and no trace of genetic divergence. The possiblereason might be the sampling at different geographicalscale. While our sampling in the Tibetan plateau was atsmaller geographical scale, most studies on Arctic avianspecies were done on the circum-Arctic range, which widergeographical scale allows population subdivision andsubspecies speciation (Wenink et al. 1993; Holder et al. 1999,2000).
The influence of the ice ages on climate was long termand continuous in temperate region (Hewitt 1996, 2004).Most temperate species experienced the postglacial expan-sion after the retreat of the Last Glacial Maximum (23–18bp). Phylogeographical studies reveal the survival of deeplineages, often in several glacial refugia, indicating survivalof populations in these southern refugia over many ice ages.With repeated range changes, survival populations maypass through many such adaptations and reorganizations,which allowed their lineages to diverge and accumulategenetic differences (Hewitt 1996, 2000, 2004).
In contrast, the Tibetan plateau has been less affected byice sheets than its highly glaciated neighbouring regionsduring last two glacial cycles (Sharma & Ower 1996; Zhenget al. 2002). When the plateau uplifted to 4500 m a.s.l., thecold conditions restricted glacier growth. The largest glacierdevelopment in the Tibetan plateau occurred during themiddle Pleistocene (0.5 Myr). Glacial retreat has occurredsince 0.17 Ma (Zhang et al. 2000; Shi 2002; Zheng et al. 2002).Our results suggest that P. ruficollis populations expandedfollowing this retreat, and that the glacial period might havebeen too short for genetic divergence among refugia to arise.
Moreover, P. ruficollis is an endemic species of theTibetan plateau and is well adapted to this extreme en-vironment. Its high elevation meadow and steppe habitatswere probably less fragmented during the glacial advancethan at present. Comprehensive pollen analyses indicatethat both alpine meadow and steppe extended in the eastpart of the Tibetan plateau during the ice ages (Kong & Du1980; Ke & Sun 1992; Liu et al. 2002). It is plausible that thepopulations of this cold-tolerant bird were widespread inthese habitats, and that its distribution range was displacedeastward during glaciation. If so, haplotype compositioncould have remained homogeneous during range shiftsand demographic fluctuations. By contrast, the considerablelevels of haplotype divergence in many birds in previouslyglaciated regions of Europe and North America are generatedby isolation in different refugia during several glacial cycles(Hewitt 1996, 2000, 2004).
Conclusion
The goal of this study was to test hypotheses about theeffects of postglacial colonization on the phylogeographical
structure of an endemic bird in the Tibetan plateau. Ourresults suggest that this bird experienced rapid populationexpansion after the retreat of extensive glaciers. However,unlike the strong phylogeographical structures that arewell known in temperate birds, postglacial colonizationhas led to very weak phylogeographical structure and nodetectable genetic divergence. Potential explanations forthis result are short glacial cycles, adaptation to cold con-ditions, and semicontinuous habitats and refugia. Overall,this study provided new evidence for the role of post-glacial colonization in shaping the phylogeographicalstructure of endemic species of the Tibetan plateau.
Acknowledgements
Our sincere thanks to the following people for their help in obtain-ing samples for this study: Zuo Hua Yin, Gang Wang, Hong FengZhao, Qi Sen Yang and Jian li Lu. We thank Martin Irestedt, PiaEldenäs and Elisabeth Köster for help in laboratory work. Authorsthank Yohannes Elizabeth for the useful comments. The editors ofMolecular Ecology and three anonymous referees provided valu-able comments on the manuscripts. The research was supportedby NSFC 30170126, 30270182, as well as by the Swedish ResearchCouncil (grant no. 621- 2001-2773 to P.E.).
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