research article phylogeographic structure of a...

Research ArticlePhylogeographic Structure of a Tethyan Relict Capparis spinosa(Capparaceae) Traces Pleistocene Geologic and Climatic Changesin the Western Himalayas Tianshan Mountains and AdjacentDesert Regions

Qian Wang12 Ming-Li Zhang13 and Lin-Ke Yin1

1Key Laboratory of Biogeography and Bioresource in Arid Land Xinjiang Institute of Ecology and GeographyChinese Academy of Sciences Urumqi Xinjiang 830011 China2University of Chinese Academy of Sciences Beijing 100049 China3Institute of Botany Chinese Academy of Sciences Beijing 100093 China

Correspondence should be addressed to Ming-Li Zhang zhangmlibcasaccn and Lin-Ke Yin yinlkmsxjbaccn

Received 27 February 2016 Accepted 27 April 2016

Academic Editor William H Piel

Copyright copy 2016 Qian Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Complex geological movements more or less affected or changed floristic structures while the alternation of glacials andinterglacials is presumed to have further shaped the present discontinuous genetic pattern of temperate plants Here we considerCapparis spinosa a xeromorphic Tethyan relict to discuss its divergence pattern and explore how it responded in a stepwise fashionto Pleistocene geologic and climatic changes 267 individuals from 31 populations were sampled and 24 haplotypes were identifiedbased on three cpDNA fragments (trnL-trnF rps12-rpl20 and ndhF) SAMOVA clustered the 31 populations into 5 major cladesAMOVA suggests that gene flow between themmight be restricted by vicariance Molecular clock dating indicates that intraspecificdivergence began in early Pleistocene consistent with a time of intense uplift of the Himalaya and Tianshan Mountains andintensified in mid-Pleistocene Species distribution modeling suggests range reduction in the high mountains during the LastGlacial Maximum (LGM) as a result of cold climates when glacier advanced while gorges at midelevations in Tianshan appear tohave served as refugia Populations of low-altitude desert regions on the other hand probably experienced only marginal impactsfrom glaciation according to the high levels of genetic diversity

1 Introduction

An essential purpose of biogeography is to comprehend themechanisms resulting in the current distribution patterns oforganisms during their evolutionary histories [1 2] Eventssince the late Paleogene especially the Quaternary geologicand climatic changes likely had a far-reaching influenceon the spatial geographic structure and genetic diversifica-tion of temperate species [3 4] After the Oligocene theinland Tethys or Paratethys Sea retreated accompanied byan increasingly droughty climate resulting in elaboration ofthe xerophytic Tethyan Flora [5 6] From the Neogene toQuaternary with the increasing aridification and uplift of theHimalayas Tibet Plateau and Tianshan Mountains [7 8]

the originally continuous arid Tethyan Flora graduallyevolved into the North Temperate Mediterranean WestAsian-Central Asian Central Asian andHimalayan elementsof today [9 10] Complex geological movements more or lessaffected or changed floristic structures while rapid climaticshifts likewise impacted plant life worldwide during thistime Significantly the alternation of glacials and interglacialsduring the Pleistocene is presumed to have played a vital rolein further shaping the present discontinuous genetic patternfor a majority of temperate plant species in the NorthernHemisphere [6 11 12]

Phylogeography a discipline which began more than 20years ago has mostly considered the response of geographicdistribution and genetic structure of current species to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 5792708 13 pageshttpdxdoiorg10115520165792708

2 BioMed Research International

historical events and the cycles of glaciation [13ndash16] Chloro-plastDNA (cpDNA) suitable for reconstructing phylogeneticpatterns of plant species is effective in exploring and estimat-ing phylogeographical history of temperate plants caused byclimatic change [17ndash20] Recent scholars have concentratedon the refuge during the Last Glacial Maximum (LGM) andrange expansion during inter- or postglacial periods [21ndash24] With the most focus on mountain plants in EuropeNorth America and East Asia [25ndash27] phylogeographicpatterns and demographical history of eremophytes in aridNorthwestern China are also receiving increasing attention[28ndash30]

We use Capparis spinosa a typical xerophyte to verifythe hypothesis of Pleistocene historical influence throughphylogeographic analysis and discuss how its spatial geneticstructure responded to paleogeologic and paleoclimaticchanges The perennial creeping subshrub is a type memberof a relatively large genus of the Capparaceae with a naturaldistribution in the Mediterranean West and Central Asia[31 32] In arid Western China there is only one singlegenus (Capparis Tourn ex L) with one single species (Cspinosa L) belonging exclusively to this family [33] Cspinosa ranges over the Western Himalayas Eastern PamirPlateau Tianshan Mountains Turpan-Hami Basin and theHexi Corridor Previous studies on Capparis have paid moreattention to autecology phenology quantitative morphologyand reproductive ecology [34ndash37] Molecular studies relatedto the genetic variation of the species are little explored[38 39] and have not considered genetic divergence from aphylogeographic point of view

Here we employed three maternally inherited cpDNAspacers trnL-trnF rps12-rpl20 and ndhF to trace historicaldivergence patterns of C spinosa We design to addressthe following concrete issues (1) According to phylogeneticrelationships yielded by cpDNA sequence variation whatis the genetic pattern of the species (2) When and howhave allopatric divergence and genetic differentiation ofthis xerophyte been influenced by glacial-interglacial cyclesassociated with geologic and climatic changes

2 Materials and Methods

21 Population Sampling A total of 267 individuals of Cspinosa from 31 natural populations were investigated inthe present study (Table 1) throughout almost the entiregeographic distribution of the species in EasternCentral Asiaincluding Xinjiang western Gansu and western Tibet Foreach population leaf samples were collected from 5 to 12mature and large perennial individuals separated by at least100m in order to avoid inbreeding and clones The latitudelongitude and altitude at each collection center were mea-sured using a global positioning system (GPS) receiver About10 g of fresh leaf materials was gathered per individual driedin silica gel in the field and stored at minus20∘C in the laboratoryCapparis bodinieri Levl was chosen as an outgroup for thephylogenic study based on previous taxonomic studies onCapparis [31] Voucher specimens for all individuals weredeposited in the Herbarium of the Xinjiang Institute of Ecol-ogy and Geography Chinese Academy of Sciences (XJBI)

22 DNA Extraction Amplification and Sequencing Silica-gel-dried leaf tissues were ground into powder in liquidnitrogen with a Fastprep-24 device (Sample Preparation Sys-tem MP Biomedicals USA) Total genomic DNA wasextracted from about 50mg of powdered tissues using amodified 2x CTAB method [40] Through preliminaryscreening from eighteen pairs of primers (see Table S1 inSupplementary Material available online at httpdxdoiorg10115520165792708) three cpDNA regions trnL-trnFrps12-rpl20 and ndhF (329F 927R) were found becausethey have more sequence variation in individuals amongpopulations Polymerase chain reaction (PCR) amplificationand DNA sequencing were performed using primer pairsfor these three regions PCRs were carried out in 30120583Lreaction volumes including 15 120583L of 10x buffer 2 120583L of25mmolLMgCl

2 22 120583L of 50 ng120583L each primer (Sangon

Shanghai China) 3120583L of 25mmolL dNTP 03 120583L of 5U120583LTaq DNA polymerase and 10 120583L of 5 ng120583L genomic DNAPCR amplification was run on a Gene-Amp PCR system9700 DNA Thermal Cycler (Applied Biosystems FosterCity CA USA) with parameters set as follows an initialstart at 94∘C with 4min followed by 30 cycles of 94∘C with30 s then 59∘C 52∘C and 52∘C respectively for trnL-trnFrps12-rpl20 and ndhF with 30 s and 72∘C with 90 s plusa subsequent additional extension at 72∘C with 10minSizes of PCR products were visualized on 1 TAE-agarosegel electrophoresis All PCR products were purified withPCR product purification kit (Shanghai SBS Biotech LtdChina) and then sequenced in both directions for trnL-trnFrps12-rpl20 and ndhF using an ABI Prism 3730 xl automatedsequencer in Sangon Biological Engineering Technology ampService Co Ltd Shanghai China

23 Data Analysis The chloroplast DNA sequences wereedited by SeqMan (Lasergene DNASTAR Inc MadisonWisconsin USA) All sequences were aligned with ClustalX version 181 [41] then refined and adjusted All resultingcpDNA sequences identified by nucleotide variations signi-fying different haplotypes were deposited in GenBank Thecorresponding accession numbers are KU940216ndashKU940218for trnL-trnF KU866560ndashKU866562 and KU940220 forrps12-rpl20 and KU866548ndashKU866558 and KU940221ndashKU940223 for ndhF (329F 927R) and accessions numbersof C bodinieri as the outgroup are KU940219 for trnL-trnFKU866563 for rps12-rpl20 and KU866559 for ndhF (329F927R)Different cpDNAhaplotypeswere identified byDnaSP50 [42] Geographic distribution of detected haplotypeswas mapped using ArcMap 93 (ESRI Redlands CA USA)Phylogenetic relationships among cpDNA haplotypes wereassociated via amedian-joining network (MJN)method [43]which was constructed using Network 4610 (available athttpwwwfluxus-engineeringcomsharenet rnhtm)

To maximize the differences of haplotype compositionbetween geographical locations spatial analysis of molecularvariance algorithm was conducted using SAMOVA 10 pro-cedure This method divided the sampled populations intoinferred partition (119870 groups) based on genetic differentiationand geographical homogeneity [44] We initially set the 119870values ranged from 2 to 12 by repeated simulated annealing

BioMed Research International 3

Table1Detailsof

geograph

icallocatio

nssam

ples

izescpD

NAhaplotypesand

diversity

indicesfor

31po

pulations

ofC

spinosa

Cod

ePo

pulationlocality

Latitud

elong

itude

Altitude

(m)

No

Haplotype

119867119889

(plusmnSD

)120587(plusmnSD

)Overall

267

08925plusmn00075

00037plusmn00019

(I)Th

eWestern

Him

alayas

grou

p(1)Z

DZa

ndaTibet

31∘

301015840

N79∘381015840

E3598

9H22H

23H

2404167plusmn01907

00021plusmn00013

(2)Q

SQusum

Tibet

31∘

291015840

N79∘481015840

E364

55

H22

00

(II)Th

eEastern

Pamirgrou

p(3)Y

GS

Yeng

isarXinjiang

38∘

431015840

N76∘141015840

E2010

9H6

00

(4)T

XGTaxkorgan

Xinjiang

37∘

471015840

N75∘131015840

E3694

5H6

00

(5)A

KTAktoXinjiang

38∘

591015840

N75∘321015840

E244

19

H6

00

(6)W

QWuq

iaX

injiang

39∘

441015840

N75∘401015840

E19

4510

H6H21

046

67plusmn01318

000

06plusmn000

05(7)A

TXArtux

Xinjiang

39∘

521015840

N76∘471015840

E13

0310

H6H7

046

67plusmn01318

000

06plusmn000

05(8)JS

JiashiXinjiang

39∘

561015840

N78∘011015840

E1162

9H6

00

(III)Th

eIliVa

lleygrou

p(9)G

LGon

gliuX

injiang

43∘

371015840

N81∘ 4

91015840E

710

9H7

00

(10)

YNYining

Xinjiang

43∘

371015840

N82∘081015840

E750

9H7

00

(11)BL

Bole

Xinjiang

44∘

421015840

N82∘051015840

E470

9H7

00

(12)

WS

WusuXinjiang

44∘

191015840

N84∘191015840

E685

9H7H12

05000plusmn01283

00013plusmn000

09(IV)Th

enorth

sideo

fthe

Tianshan

Mou

ntains

grou

p(13)

KRMY

KaramayX

injiang

44∘

561015840

N84∘461015840

E296

5H12

00

(14)T

LTo

liXinjiang

45∘

551015840

N83∘361015840

E684

5H17

00

(15)

SHZ

ShiheziXinjiang

44∘

161015840

N85∘571015840

E356

5H11

00

(16)

SWShaw

anX

injiang

44∘

191015840

N85∘351015840

E584

9H5

00

(17)

MNS

ManasX

injiang

44∘

111015840

N86∘081015840

E64

28

H5H10

04286plusmn01687

000

02plusmn000

02(18)

URM

QUrumqiX

injiang

43∘

441015840

N86∘571015840

E10

608

H10H

1104286plusmn01687

00010plusmn000

07(19)

JMS

JimsarXinjiang

43∘

551015840

N89∘071015840

E945

8H5

00

(V)Th

esou

thsid

eofthe

Tianshan

Mou

ntains

grou

p(20)

KPKa

lpinX

injiang

40∘

351015840

N79∘361015840

E1131

10H1H5

03556plusmn01591

000

08plusmn000

06(21)WNS

WensuX

injiang

41∘

291015840

N79∘541015840

E12

969

H1H8H20

07500plusmn00786

00016plusmn00010

(22)

BCBa

icheng

Xinjiang

41∘

591015840

N83∘031015840

E13

698

H1H8

05714plusmn00945

000

03plusmn000

03(23)

LTLu

ntaiX

injiang

41∘

141015840

N84∘121015840

E921

10H8H14

03556plusmn01591

00011plusmn000

07(24)

KRL

Korla

Xinjiang

41∘

501015840

N86∘031015840

E10

728

H1H8H13

06071plusmn01640

000

04plusmn000

04(25)

HJ

HejingXinjiang

42∘

311015840

N86∘161015840

E14

0310

H5H8

046

67plusmn01318

00012plusmn000

08(26)

TKS

Toksun

Xinjiang

42∘

491015840

N88∘381015840

E95

12H1H5

05455plusmn00615

00012plusmn000

08(27)

TRP

TurpanX

injiang

42∘

521015840

N89∘111015840

Eminus84

12H1H5H11H

18H

1908485plusmn00586

00017plusmn00010

(28)

SSShanshanX

injiang

43∘

111015840

N90∘091015840

E10

439

H10H

15H

1607222plusmn00967

00015plusmn000

09(29)

HM

Ham

iXinjiang

42∘

311015840

N94∘091015840

E773

9H9H10H

1107500plusmn00786

00013plusmn000

09(30)

DH

Dun

huangGansu

40∘

251015840

N94∘441015840

E10

5110

H1H2H3

03778plusmn01813

000

05plusmn000

04(31)GZ

Guazhou

Gansu

40∘

221015840

N95∘341015840

E1133

10H1H2H4H5

07333plusmn01005

00011plusmn000

07Nonum

bero

fsam

pleind

ividuals119867119889haplotype

diversity

120587nucleotided

iversity


process until largest 119865CT index was obtained However when119870 ge 6 at least one of the groups contained only one popula-tion implying that information on the group structure wouldhave been disappearing [45ndash48] To maintain the groupingpattern and exclude configurations with a single populationin any of the groups finally valid range of 119870 was set as 2ndash5

Parameters of Neirsquos diversity containing haplotype diver-sity (119867

119889) and nucleotide diversity (120587) were computed in

Arlequin 31 program [49 50] To evaluate genetic varia-tion within populations among populations within groupsand among groups AMOVA (analysis of molecular vari-ance) was performed in the program Arlequin version 31[50] and significance tests were conducted using 1000 per-mutations The statistics of average gene diversity and pop-ulation differentiation that is the average gene diversitywithin populations (119867

119878) total gene diversity across all popu-

lations (119867119879) the number of substitution types (119873ST) and

genetic differentiation over populations (119866ST) were calcu-lated using Permut version 10 [51] (available at httpwwwpierrotoninrafrgeneticslaboSoftwarePermutCpSSRindexhtml) To test for the existence of phylogeographical struc-ture the comparison of 119866ST versus119873ST was conducted usingPermut based on 1000 random permutations of haplotypesacross populations If 119873ST is significantly higher than 119866STthen genealogically closely related haplotypes tend to occurtogether within populations indicating the presence ofphylogeographical structure [51] Significance of 119875 valueswas examined by the 119880 Test using the Haplonst procedure[51]

To estimate the divergence times of cpDNA haplotypesthe BEAST version 161 [52] was carried out for lineage anal-ysis following a Bayesian relaxedmolecular clock ABayesianMarkov Chain Monte Carlo (MCMC) procedure was run for20000000 generations with a coalescent-based tree priorityrule and a HKY substitution model Given the uncertainty ofthe cpDNA mutation rate the normal distribution prioritywith a mean value of 2 times 10minus9 s sminus1 yrminus1 (substitutions per siteper year) and a SD of 608 times 10minus9 for most angiosperm specieswere adopted as a criterion for the analytical operation [2453] to cover the rate variationwithin the 95 range of the dis-tribution for the present calculation of molecular divergencetimes of genotypesThe combined parameters were validatedin Tracer version 15 [52] to check whether the parametervalue was greater than 1 and the effective sample size valuewas larger than 200 as an indication that these sequence datawere suitable for a relaxed molecular clock model Trees werefinally compiled in FigTree 131 The statistical support ofclades depended on Bayesian posterior probability

To investigate spatial genetic patterns of geographicaldistance among populations for each defined regional groupgenetic landscape shape analysis was performed using AllelesIn Space [54] Primarily a connective Delaunay triangu-lation network was constructed based on the geographiccoordinates of all sampled points according to the Watson(1992) and Brouns (2003) methods [55 56] Secondly theaverage genetic distances resulting from the cpDNA sequencedata were calculated among populations in the networkAfterwards the genetic distancematrix was interpolated ontospatial grids of the connective network forming a landscape

shape A final three-dimensional surface plot was generatedwhere the abscissa and ordinate signified geographical coor-dinates and the vertical coordinate were equivalent to geneticdistance

Possible demographic historical expansions for definedgroups were inferred bymismatch distribution analysis usingArlequin with 1000 parametric bootstrap replicates [57]Populations that have experienced recent expansion areexpected to have a unimodal curve in the pairwise mismatchdistribution whereas stable populations should have a bi-or multimodal shape [58 59] Neutrality tests (Tajimarsquos 119863and Fursquos 119865

119904statistics) [60 61] were used to detect further

evidence for probable historical processes [62 63] A positiveor negative 119863 value (not 0) may imply that a populationunderwent expansion or bottlenecks [64] while a negative 119865

119904

value probably indicates a recent population expansion [61]To explore the potential distribution changes of the spe-

cies between the present-day and the Last Glacial Maximum(LGM approximately 18ndash21 ka) the ecological niche model-ing was performed in MAXENT version 331 [65] followingthemaximum entropy algorithmWe input an available coor-dinate set of occurrence points from the speciesrsquo entire geo-graphical distribution in Eastern Central Asia based on theChinese Virtual Herbarium (httpwwwcvhorgcncms)and our field survey locations (Table 1) The current climaticenvelopes were predicted based on WorldClim dataset [66]and the LGM layers in accordance with the Model forInterdisciplinary Research on Climate (MIROC) [67] andthe Community Climate System Model (CCSM) [68] Weacquired 19 BIOCLIM variables to model ecological niche at25 arcmin resolution from the WorldClim database (avail-able at httpwwwworldclimorgdownload) To avoid thepotential problems of overfitting [69] we removed the highlycorrelated bioclimatic variables retaining nine least corre-lated ones Model performance was identified by the areaunder the receiver operating characteristic curve (AUC)Thedefault parameters to form ecological niche modeling werechosen as follows regularization multiplier = 10 percentageof the data set for train the model = 75 percentage fortesting = 25 and the threshold for available presence data= 10 percentiles In addition a Jackknife test was used tomeasure the significant contributions of the BIOCLIM vari-ables on the occurrence prediction for each model Finallypotential distribution ranges for the present-day and LGMwere projected in ArcMap 93 (ESRI Redlands CA USA)

3 Results

31 Chloroplast Variation and Haplotype Geographical Dis-tribution The aligned sequence length of the three cpDNAregions was 2254 bp (923 bp for trnL-trnF 728 bp for rps12-rpl20 and 603 bp for ndhF (329F 927R)) Based on the totalsequences thirty-six polymorphic sites (thirty nucleotidesubstitutions and six indels) were obtained (Table S2) A totalof 24 haplotypes (H1ndashH24) were identified from 267 samplesamong 31 populations across the entire geographic range ofC spinosa (Figure 1(a) Table 1)

According to the results of SAMOVA the 31 studiedpopulations were divided into five defined phylogeographical


GZDH

HM

SSTRPTKSHJ

Ili valley

KRLLTBC

WNSKP

YGSTXGEastern PamirAKT

WQ

ATX

JSGL

YNBL

WSKRMYTL SHZ SW

MNSURMQ

JMS

ZDQS

Tian Shan Tian Shan

Tarim Basin

Western Himalayas

Kun

Plateau of Tibet

Hexi Corridor

Turpan-Hami

H1H7H13H19

H2H8H14H20

H3H9H15H21

H4H10H16H22

H5H11H17H23

H6H12H18H24

35∘N

40∘N

45∘N

70∘E 80∘E 90∘E

Lun Mountains

(a)

H1H5H11

H8H10H18

H7

H6

H24 H23

H21

H22H12

H17

H19

H4 H9

H16

H20

H14 H2

H13

H15H3

(b)

(c) (d)

Figure 1 Geographical distribution and phylogenetic network of haplotypes in sampled locations of Capparis spinosa (a) Distribution ofthe cpDNA haplotypes The abbreviated letters of population localities correspond to those shown in Table 1 Pie charts show the differenthaplotypes and their proportions in each population different colors are consistent withmatching haplotypes in the figure legend (b)Median-joining network reflecting the cpDNAhaplotype relationships Small open red circles represent potentially intermediate genotypes with othermutational steps between real haplotypes circle sizes are proportional to haplotype frequencies (c d) Two different kinds of habitats of thespecies (c) eroding mountain slopes and (d) gravel deserts


groups (I) the Western Himalayas Group (II) the EasternPamir Group (III) the Ili Valley Group (IV) the northside of the Tianshan Mountains Group and (V) the southside of the Tianshan Mountains Group (Table 1) The maingenealogical relationships of the network connection dia-gram (Figure 1(b)) as well as the BEAST tree (Figure 2)suggested the similar divergence trend

Distribution of the haplotypes showed high geographicstructure Haplotypes H22 H6 H7 H1 H8 H5 and H10 hadthe most frequent and widespread distributions H22 onlywas distributed in the populations of Group I H6 occurredin all populations of Group II H7 only occurred in the popu-lations of Group III H1 and H8 were dominantly distributedin the populations of GroupV while H5 andH10 weremainlypresent in the populations of Group IV (Figure 1(a))

32 Genetic Diversity and Population Genetic StructureGenetic diversity analysis revealed that total genetic diversity(119867119879= 0916 se = 00172) was much higher than average

gene diversity (119867119878= 0316 se = 00533) which indicated

that there was considerable population differentiation acrossthe distributional range The 119880 Test showed that total 119873ST(0843 se = 0033) was significantly higher than 119866ST (0655se = 0057) (119880 = 691 119875 lt 001) indicating cpDNA varia-tion with signal phylogeographic structure for the species

AMOVA revealed that a larger proportion of the vari-ation was interpopulational than intrapopulational (8520versus 1480) Differences among geographical groups weresignificant and AMOVA results showed that 7183 (119865CT =071831 119875 lt 0001) of total variation existed between thefive groups (Table 2) For the two genetic diversity indexeshaplotype diversities (119867

119889) within the 31 populations ranged

from 0 to 08485 and nucleotide diversities (120587) ranged from0 to 00021 Considering the whole species high degrees ofhaplotype diversity (119867

119889= 08925) and nucleotide diversity

(120587 = 00037) were found At the level of population sixpopulations (TRP HM WNS GZ SS and KRL) in GroupV showed considerably high degrees of genetic diversity(Table 1)

33 Molecular Divergence Time Estimates and DemographicHistory Analyses The phylogenetic tree yielded by BEASTanalysis was strongly coupled with geography demonstratingthat the coalescent sequences were suitable to be employedin phylogenetic and phylogeographic analysis The resultsindicate that the identified cpDNA haplotypes are clusteredinto six main clades Divergence times estimated fromBEAST analysis were Pleistocene and the main divergencebetween the geographical groups began at 1179Ma in earlyPleistocene (Figure 2)

The three-dimensional surface plot produced by geneticlandscape shape analyses showed that spatial genetic dis-tances gradually increased from the western populations toeastern populations (Figure 3) For mismatch analysis theobservedmismatch curve was not unimodal but strongly dis-cordant with a model of sudden range expansion (Figure 4)indicating the stable populations Additionally no supportingevidence was provided by Tajimarsquos 119863 and Fursquos 119865

119904values for

recent range expansion events

34 Potential Distribution Modeling Under the selectedmodel the test AUC value for the ENM of C spinosa inthe present-day was 0995 and under the MIROC climatescenario the value was 0997 in the LGM projection SDMdid not yield suitable or credible distribution in LGM basedon the CCSM model The Jackknife analysis of regularizedtraining gain indicated that there were five environmen-tal variables contributing more highly to the distributionmodel in the present-day precipitation of the wettest month(2264) mean temperature of the coldest quarter (2297)precipitation of the coldest quarter (1548) mean tem-perature of the driest quarter (825) and precipitationseasonality (637) The five most contributive bioclimaticvariables of the MIROC model in the LGM were annualmean temperature (2432) precipitation of the coldestquarter (2018) mean temperature of the coldest quarter(1387) mean temperature of the driest quarter (1218)and precipitation of the wettest month (1189) The rangedifferences under the distinct climatic conditions of the twomodels revealed a diminished amount of suitable habitatfor the species during the LGM period In comparisonwith the present potential distribution the MIROC scenarioindicated that the high-altitude ranges of the species in theWestern Himalayas eastern Pamir and western TianshanMountains were diminished (Figure 5) according to theLGM modeling distribution However some localities in themiddle Tianshan such as WNS and KRL were not affectedby this in the LGM model (Figure 5) and thus midelevationgorges environments might have provided suitable survivalhabitats for the species in the glacial epoch

4 Discussion

41 Genetic Variation At the species level a high total genediversity (119867

119879= 0916) was detected Compared with other

species where three intergenic spacers have been sequencedgene diversity inC spinosawas higher for instance Juniperussabina (119867

119879= 0577) [70] and Gymnocarpos przewalskii

(119867119879= 0903) [29] The results of genetic diversity analysis

also give indication of a high level of total haplotype diversity(119867119889= 08925) (Table 1) High values of the diversity indices

might have been promoted by the accumulation of variationin C spinosa which to a great extent may have beenassociated with differences in geological and topographicalconditions complex habitat patterns and gradually intensivearidification occurring in Eastern Central Asia during theQuaternary period

Dramatic genetic variation among the total populationswas shown accounting for a far greater proportion thanthat within populations (Table 2) The genetic differencesamong populations are believed to be closely related tothe degree of gene exchange [71] which depends on seedtransfer for cpDNA in most angiosperms [72] The seeddispersal modes of C spinosa mainly include anemochorymyrmecochory and endozoochory by rodent vector [73]In the current survey regions the mountains and sprawlingdesert zones isolated habitat into disjunctive geographicalunits In this situation gene flow between populationswas potentially restricted in short distance Especially in


H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13

Miocene Pliocene PleistocenePresent

5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3

Holocene2 Ma4 Ma6 Ma

Capparis bodinieri

Figure 2 Phylogenetic chronogram of 24 haplotypes in C spinosa generated from BEAST analysis Divergence time (million years ago) ofnodes are shown blue horizontal bars indicating the 95 ranges of highest posterior density

Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S

Figure 3 Spatial genetic landscape shapes constructed by interpolation analysis for C spinosa The abscissae and ordinates correspond togeographical coordinates covering the entire distributional populations and the vertical axes represent genetic distances


Table 2 Results of AMOVA analysis of molecular variance for populations and geographical groups of C spinosa

Source of variation df SS VC PV () Fixation indexAmong groups 4 738961 3723 7183 119865CT 071831

lowastlowast

Among populations within groups 26 175341 0693 1337 119865SC 047458lowastlowast

Within populations 236 181039 0767 1480 119865ST 085199lowastlowast

Total 266 1095341 5183df degrees of freedom SS sum of squares VC variance components PV percentage of variation 119865SC correlation of chlorotypes within populations relativeto groups 119865ST correlation within populations relative to the total 119865CT correlation within groups relative to the total lowastlowast119875 lt 0001 1000 permutations

ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y

3 5 7 9 11 13 15 17 191Pairwise differences

Figure 4 Pairwise mismatch distributions of cpDNA sequences ofC spinosa

small sized populations individuals might be susceptible toinbreeding Therefore gene flow was limited attributing tothe incapacity of seed dispersal across impassable physicalbarriers by anemochory or zoochory resulting in populationdifferentiation

42 Influence of Pleistocene Geological and Climatic Changeson Phylogeographical Structure The timescales of intraspe-cific divergences are usually related to historical geologic andclimatic events [3 12] The estimated genetic divergence timebetween the five geographical groups started at approximately1179 million years ago based on molecular dating resultsof the BEAST analysis (Figure 2) which falls into earlyPleistocene the phase of intense uplift of Himalayas [74]From early to middle Pleistocene most large-scale piedmontglaciers developed on the northern slope of the Himalayas[75] which may have been the impetus causing haplotypesH23H24 andH22 belonging to theWesternHimalayan pop-ulations to branch off the phylogenetic tree (0808ndash0621Ma)(Figure 2) At the intersection of the Himalaya Kunlunand Tianshan Mountains the Pamir uplift was structuredfrom at least the end of the Paleogene In the Quaternarystage owing to at least three large-scale glaciations piedmontmoraines intermittently formed in this region [76] In con-nection with these fluctuations the Western Himalayan andEastern Pamirian populations from high-altitude localitiesexperienced large-scale differentiation accompanied withadaptation to colder climates resulting in different degreesof genetic diversity and conspicuous population differencesWe found that haplotypes H22 andH6were respectively dis-tributed over every sampled location in Western Himalayan

and Eastern Pamir region while the rare haplotypes H23H24 and H21 were randomly distributed in single pop-ulations Although BEAST analysis and network diagramdid not clearly show that haplotype H22 clustered into thesame clade with H23 and H24 it occupies a key transitionalposition (Figures 1(b) and 2) Genetically H22 contained ahaplotype approximating those in the Tianshan region whilegeographically it is distributed in the Western Himalayanregion indicating a possible common Tethyan origin andthen allopatric divergence and local evolution

In comparison with the formation of the Himalayas andPamirs neotectonic movement of Tianshan Mountains wassomewhat lagging occurring in Pliocene and intensivelyuplifting in early Pleistocene [7 8] Since Quaternary dias-trophism and climatic changes together with more thanthree glacial-interglacial cycles [77] not only changed thetopography but also resulted in temperature and precipitationdifferences between western and eastern ranges north andsouth slopes and high and low altitudes [74] rapid upliftof the Tianshan range as a barrier would of necessity havechanged the local atmospheric circulation and screened thesources of moist airflow intensifying the arid climate inadjacent regions Later since the mid-Pleistocene enhanceddrought and cold occurred in the Tianshan and the surround-ings at 06ndash02Ma [78] as evidenced by loess sediments onnorthern slope of the Tianshan Mountains [74] During thisperiod a weakened southwest monsoon and strengthenedplateau winter monsoon resulted in increasing aridificationand desert expansion [79] Compared with the geographicpatterns under present-day climatic conditions distributionranges of C spinosa during the LGM period were con-tracted (Figure 5) Although distribution areas of the specieschanged the main distribution in the low-altitude easterndesert region was stable during glacial periods and onlymarginally influencedThe great spatial and genetic distancesamong locations in the eastern Tianshan and Hexi Corridorcan be perceived in the 3D surface plots (Figure 3) Thischaracteristic of the spatial genetic landscape analysis alsodemonstrates that the species has had relatively stable habitatsin the low-altitude desert region during an extensive evolu-tionary history At the stage of the mid-Pleistocene periodthe continuously expanding Taklimakan [80] and intensivearidification accelerated the worsening drought and heat forthe lower elevations of the eastern Tianshan Turpan-HamiBasin and Hexi Corridor populations which must haveinfluenced recent divergence among these areas In generalgenetic divergence among geographic groups in Pleistoceneappears to have been driven mainly by intense uplift of the


TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

Figure 5 Species distributionmodeling ofC spinosa during present-day and LGMperiods in arid EasternCentral Asia Potential distributionin the LGM epoch is based on scenarios of the MIROC model Yellow localities populations sensitive to glaciation in the high mountainsBlue localities populations at middle elevations Green localities gorges served as refugia in the mid-Tianshan Red localities populations oflow-altitude desert region

Himalayas and Tianshan Mountains The cold-dry to warm-humid climatic cycles during the late Quaternary are inferredto have promoted genetic divergence within groups

43 Potential Glacial Refugia and Demographic Dispersal inPast Scenarios According to previous glaciological researchmaximum glaciation was likely to have occurred in theearly phase of the mid-Pleistocene (08ndash06Ma) in EasternCentral Asia [74 77] During this period and the next twoglacial stages owing to large-scale glacial advances glaciers

in Tianshan at one time extended as far down as the pied-mont belts [74] In comparison with the present distributionrange the LGM localities of C spinosa were reduced in thehigh-altitude ranges of the Western Himalayas Pamirs andwestern Tianshan Mountains (Figure 5) as a result of frigidclimate in the wake of glacial advances The increasinglysevere climate in the glacial epoch would have damagedthe ecological habitats of some xeric plant species [81]however it seems likely that C spinosa could have survivedin some stable gorges of Tianshan Mountains These suitable


refugia were less influenced by climatic oscillations andtherefore could maintain richer degrees of genetic diversityamong populations [25 82] Refugial zones are generallyrecognizable because of high levels of genetic diversity andunique genotypes in species populations [12 22] and areusually located in the middle altitude of mountains whichcould supply warmer habitats for species to resist cold glacialclimates [83] A number of glacial refugia have been revealedworldwide on the basis of molecular approaches [84ndash86]However refugia localities in the Tianshan and adjacent aridregions have remained rather cryptic as there seems to be alack of applicable studies [23] As a representative xerophyteC spinosa selected relatively warm gorges (WNS and KRL) inthe middle Tianshan Mountains as potential refugia duringglacial epochs in accordance with the high levels of geneticdiversity within these populations

Unlike the traditional northward expansions duringwarm interglacial periods [18 87] in the current study thedispersal of C spinosa was profoundly affected not only bytemperature variation but also by aridification in EasternCentral Asia thus xeric species may well prefer relativelyarid areas during the warm and moist interglacial stagesConsidering the network diagram genotypes H1 and H8were found with high rates of incidence on the south side ofTianshan group indicating that these two were predominanthaplotypes in Group V (Figure 1)These characteristics implythat the species should have undergone large-scale inter-glacial expansions from glacial refugia in this region How-ever multimodalmismatch distribution shape (Figure 4) andpositive Fursquos119865

119904value failed to provide any significant evidence

for recent range expansion According to Printzen et al(2003) extensive intraspecific differentiation is probably con-nected with range fragmentation or long-distance dispersal[88] Evidenced from our field observations C spinosa is axeromorphic species that prefers to distribute itself in erodingrocky mountains and arid piedmont proluvial or alluvialgravelly fans (Figures 1(c) and 1(d)) In the end of early Pleis-tocene and the earlymid-Pleistocene intensified aridificationand the formation of Taklimakan desert resulted in dry andrainless conditions in piedmont zones of Tianshan [74 89]However in contrast with the cold climate in glacial phasein the interglacial and postglacial period the climate becamewarm and humid At this time glacier-melt water increasedthe volume of river runoff on the edge of the TarimBasin [74]Thus the movement of the wind and river runoff providedappropriate conditions for survival and seed dispersal of CspinosaThese could harbor relatively stable habitats preserv-ing the speciesrsquo existing genetic variation and genetic dif-ferences among populations There is considerable evidencesuch as increasing 12057518119874 values palynological componentsmultiple alluvial sequences and lake sediments that revealsthe alternation of glacials and interglacials occurring in theTianshan Mountains during the Pleistocene [74 90 91] Thiscircularly fluctuant climate propelled demographic dispersalin the interglacial intervals Interestingly unlike most otherspecies with decreased levels of genetic diversity related todesert expansion during interglacial and postglacial coloniza-tion [29 30] as xeric Tethyan relic C spinosa showed suc-cessively increased levels of genetic diversity and population

sizes especially when it contacted themore droughty and hotgravelly deserts in Turpan-Hami Basin and Hexi CorridorOne reasonable explanation for this phenomenon is the prob-ability that the suitable warm temperate continental climatein eastern gravel deserts is similar to the ancient hot and drysubtropical summer climate of the Tethyan Flora preservingthe characteristics of the original climate and environmentThe other corollary is that the desert region could play therole of a shelter influenced only marginally by glaciation

5 Conclusions

The current study focuses on how complicated Pleistocenegeological and cyclical climatological events influenced thephylogeographic structure and genetic divergence of Cspinosa in arid Eastern Central Asia Strongly spatial phylo-geographic patterns were documented in the species Intenseuplift of the Himalaya and Tianshan Mountains around theearly Pleistocene coupled with a cold-dry climate and conse-quent aridification during the stage of Quaternary glaciationin this survey region played important roles both in trig-gering and in shaping the current phylogeographic structureof the species group There were at least three glacial-interglacial cycles in the Himalayas as well as the Pamir andTianshan Mountains during the Pleistocene During glacialepochs potential refugia were inferred for the gorges of themiddle Tianshan Mountains while in interglacials and thepostglacial period the species experienced dispersal Thephylogeography ofC spinosa in the present research providesthe basis for future studies on how xerophilous plant speciesacross both arid high-altitude rocky mountains (Figure 1(c))and low-altitude gravel deserts (Figure 1(d)) responded in astepwise fashion to Quaternary geologic and climatic events

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Dr Stewart C Sanderson inthe Shrub Sciences Laboratory Rocky Mountain ResearchStation US Department of Agriculture Utah USA for hishelpful suggestions and English improvement of the paperThis study was financially supported by China National KeyBasic Research Program (2014CB954201) CAS Key Labo-ratory of Biogeography and Bioresource in Arid Landand Xinjiang Institute of Ecology and Geography ChineseAcademy of Sciences

References

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[2] R IMilne andR J Abbott ldquoTheorigin and evolution of tertiaryrelict florasrdquoAdvances in Botanical Research vol 38 pp 281ndash3142002

[3] G M Hewitt ldquoThe genetic legacy of the quaternary ice agesrdquoNature vol 405 no 6789 pp 907ndash913 2000


[4] G M Hewitt ldquoGenetic consequences of climatic oscillations inthe Quaternaryrdquo Philosophical Transactions of the Royal SocietyB Biological Sciences vol 359 no 1442 pp 183ndash195 2004

[5] E Palamarev ldquoPaleobotanical evidences of the Tertiary historyand origin of the Mediterranean sclerophyll dendroflorardquo PlantSystematics and Evolution vol 162 no 1 pp 93ndash107 1989

[6] H Sun ldquoTethys retreat and Himalayas-Hengduanshan Moun-tains uplift and their significance on the origin and developmentof the Sino-Himalayan elements and Alpine florardquo Acta Botan-ica Yunnanica vol 24 no 3 pp 273ndash288 2002 (Chinese)

[7] J-M Sun R-X Zhu and J Bowler ldquoTiming of the TianshanMountains uplift constrained by magnetostratigraphic analysisof molasse depositsrdquo Earth and Planetary Science Letters vol219 no 3-4 pp 239ndash253 2004

[8] L-NWang J-Q Ji D-X Sun et al ldquoTheuplift history of south-western Tianshan-Implications from AFT analysis of detritalsamplesrdquo Chinese Journal of Geophysics vol 53 no 4 pp 931ndash945 2010 (Chinese)

[9] A Takhtajan T J Crovello and A Cronquist Floristic Regionsof the World University of California Press Berkeley CalifUSA 1986

[10] Z-Y Wu H Sun Z-K Zhou D-Z Li and H Peng Floristicsof Seed Plants from China Science Press Beijing China 2010(Chinese)

[11] L L Anderson F S Hu D M Nelson R J Petit and K NPaige ldquoIce-age endurance DNA evidence of a white sprucerefugium in Alaskardquo Proceedings of the National Academy ofSciences of the United States of America vol 103 no 33 pp12447ndash12450 2006

[12] R J Petit I Aguinagalde J-L de Beaulieu et al ldquoGlacialrefugia hotspots but not melting pots of genetic diversityrdquo Sci-ence vol 300 no 5625 pp 1563ndash1565 2003

[13] J C Avise ldquoThe history and purview of phylogeography apersonal reflectionrdquoMolecular Ecology vol 7 no 4 pp 371ndash3791998

[14] A Terrab P Schonswetter S Talavera E Vela and T F StuessyldquoRange-wide phylogeography of Juniperus thurifera L a pre-sumptive keystone species of western Mediterranean vegeta-tion during cold stages of the Pleistocenerdquo Molecular Phyloge-netics and Evolution vol 48 no 1 pp 94ndash102 2008

[15] M J Hickerson B C Carstens J Cavender-Bares et al ldquoPhylo-geographyrsquos past present and future 10 years after Avise 2000rdquoMolecular Phylogenetics and Evolution vol 54 no 1 pp 291ndash3012010

[16] J-Q Liu Y-S Sun X-J Ge L-M Gao and Y-X QiuldquoPhylogeographic studies of plants in China advances in thepast and directions in the futurerdquo Journal of Systematics andEvolution vol 50 no 4 pp 267ndash275 2012

[17] A C Newton T R Allnutt A C M Gillies A J Lowe andRA Ennos ldquoMolecular phylogeography intraspecific variationand the conservation of tree speciesrdquoTrends in Ecology and Evo-lution vol 14 no 4 pp 140ndash145 1999

[18] I V Bartish J W Kadereit and H P Comes ldquoLate Quaternaryhistory of Hippophae rhamnoides L (Elaeagnaceae) inferredfrom chalcone synthase intron (Chsi) sequences and chloroplastDNA variationrdquo Molecular Ecology vol 15 no 13 pp 4065ndash4083 2006

[19] X-J Ge C-C Hwang Z-H Liu et al ldquoConservation genet-ics and phylogeography of endangered and endemic shrub Tet-raena mongolica (Zygophyllaceae) in Inner Mongolia ChinardquoBMC Genetics vol 12 article 1 2011

[20] Y-Y Liao A W Gichira Q-F Wang and J-M Chen ldquoMole-cular phylogeography of four endemic Sagittaria species (Alis-mataceae) in the Sino-Japanese Floristic Region of East AsiardquoBotanical Journal of the Linnean Society vol 180 no 1 pp 6ndash20 2016

[21] P Schonswetter I Stehlik R Holderegger and A TribschldquoMolecular evidence for glacial refugia of mountain plants inthe European AlpsrdquoMolecular Ecology vol 14 no 11 pp 3547ndash3555 2005

[22] J R Stewart A M Lister I Barnes and L Dalen ldquoRefugiarevisited individualistic responses of species in space and timerdquoProceedings of the Royal Society B Biological Sciences vol 277no 1682 pp 661ndash671 2010

[23] Y-X Qiu C-X Fu and H P Comes ldquoPlant molecular phylo-geography in China and adjacent regions tracing the geneticimprints of Quaternary climate and environmental change inthe worldrsquos most diverse temperate florardquoMolecular Phylogenet-ics and Evolution vol 59 no 1 pp 225ndash244 2011

[24] D-R Jia R-J Abbott T-L Liu K-S Mao I V Bartish andJ-Q Liu ldquoOut of the Qinghai-Tibet Plateau evidence for theorigin and dispersal of Eurasian temperate plants from a phy-logeographic study of Hippophae rhamnoides (Elaeagnaceae)rdquoNew Phytologist vol 194 no 4 pp 1123ndash1133 2012

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[26] O Bettin C Cornejo P J Edwards andRHolderegger ldquoPhylo-geography of the high alpine plant Senecio halleri (Asteraceae)in the European Alps in situ glacial survival with postglacialstepwise dispersal into peripheral areasrdquoMolecular Ecology vol16 no 12 pp 2517ndash2524 2007

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[28] Z Su M Zhang and S C Sanderson ldquoChloroplast phylogeo-graphy of Helianthemum songaricum (Cistaceae) from north-western China implications for preservation of genetic diver-sityrdquo Conservation Genetics vol 12 no 6 pp 1525ndash1537 2011

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[30] H-H Meng and M-L Zhang ldquoDiversification of plant speciesin arid Northwest China species-level phylogeographical his-tory of Lagochilus Bunge ex Bentham (Lamiaceae)rdquo MolecularPhylogenetics and Evolution vol 68 no 3 pp 398ndash409 2013

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[32] M-L Zhang and G C Tucker ldquoCapparaceaerdquo in Flora ofChina Z-Y Wu and P H Raven Eds pp 433ndash450 SciencePressMissouri Botanical Garden Pres Beijing China 2008

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[36] T Zhang and D-Y Tan ldquoAn examination of the function ofmale flowers in an andromonoecious shrub Capparis spinosardquoJournal of Integrative Plant Biology vol 51 no 3 pp 316ndash3242009

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[38] H Nosrati M A H Feizi M Mazinani and A R HaghighildquoEffect of population size on genetic variation levels inCapparisspinosa (Capparaceae) detected by RAPDsrdquo EurAsian Journal ofBioSciences vol 6 pp 70ndash75 2012

[39] O Ozbek andAKara ldquoGenetic variation in natural populationsof Capparis from Turkey as revealed by RAPD analysisrdquo PlantSystematics and Evolution vol 299 no 10 pp 1911ndash1933 2013

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[51] O Pons and R J Petit ldquoMeasuring and testing genetic differ-entiation with ordered versus unordered allelesrdquo Genetics vol144 no 3 pp 1237ndash1245 1996

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[58] M Slatkin and R R Hudson ldquoPairwise comparisons of mito-chondrial DNA sequences in stable and exponentially growingpopulationsrdquo Genetics vol 129 no 2 pp 555ndash562 1991

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[62] L A Hurtado T Erez S Castrezana and T A Markow ldquoCon-trasting population genetic patterns and evolutionary histo-ries among sympatric Sonoran Desert cactophilic DrosophilardquoMolecular Ecology vol 13 no 6 pp 1365ndash1375 2004

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[70] Y-P Guo R Zhang C-Y Chen D-W Zhou and J-Q LiuldquoAllopatric divergence and regional range expansion of junipe-rus sabina in Chinardquo Journal of Systematics and Evolution vol48 no 3 pp 153ndash160 2010

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[72] D E McCauley ldquoThe use of chloroplast DNA polymorphism instudies of gene flow in plantsrdquo Trends in Ecology amp Evolutionvol 10 no 5 pp 198ndash202 1995

[73] T ZhangTheReproductive Ecology onCapparis spinosa L (Cap-paraceae) Xinjiang Agricultural University Urumqi China2008 (Chinese)

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[75] S C Porter ldquoQuaternary glacial record in Swat Kohistan WestPakistanrdquo Geological Society of America Bulletin vol 81 no 5pp 1421ndash1446 1970

[76] Xinjiang Integrated Survey Group of Chinese Academy of Sci-ences Xinjiang Geomorphology Science Press Beijing China1978 (Chinese)

[77] X Xu A Kleidon L Miller S Wang L Wang and G DongldquoLate Quaternary glaciation in the Tianshan and implicationsfor palaeoclimatic change a reviewrdquo Boreas vol 39 no 2 pp215ndash232 2010

[78] J-J Li ldquoThe patterns of environmental changes since late Pleis-tocene in Northwestern Chinardquo Quaternary Science Reviewsvol 3 pp 197ndash204 1990

[79] X Wu F Wang and Z-S An ldquoThe phase and height of tibetanplateau rise in late cenozoicrdquo in Loess Quaternary GeologyGlobal Changes D-S Liu and Z-S An Eds pp 1ndash13 ScientificPublishing House Beijing China 1992

[80] ZWu ldquoApproach to the genesis of the Taklamakan desertrdquoActaGeographica Sinica vol 36 no 3 pp 280ndash291 1981 (Chinese)

[81] Y-X Liu ldquoA study on origin and formation of the Chinesedesert florasrdquoActa Phytotaxonmica Sinica vol 33 no 2 pp 131ndash143 1995 (Chinese)

[82] AWidmer and C Lexer ldquoGlacial refugia sanctuaries for allelicrichness but not for gene diversityrdquo Trends in Ecology andEvolution vol 16 no 6 pp 267ndash269 2001

[83] Y-J Zhang M Stock P Zhang X-L Wang H Zhou and L-H Qu ldquoPhylogeography of a widespread terrestrial vertebratein a barely-studied Palearctic region green toads (Bufo viridissubgroup) indicate glacial refugia in Eastern Central AsiardquoGenetica vol 134 no 3 pp 353ndash365 2008

[84] R J Abbott L C Smith R I Milne R M M Crawford KWolff and J Balfour ldquoMolecular analysis of plantmigration andrefugia in the Arcticrdquo Science vol 289 no 5483 pp 1343ndash13462000

[85] T Lacourse RWMathewes andDW Fedje ldquoLate-glacial veg-etation dynamics of the Queen Charlotte Islands and adjacentcontinental shelf British Columbia Canadardquo PalaeogeographyPalaeoclimatology Palaeoecology vol 226 no 1-2 pp 36ndash572005

[86] L-Y Wang R J Abbott W Zheng P Chen Y Wang and JLiu ldquoHistory and evolution of alpine plants endemic to theQinghai-Tibetan Plateau Aconitum gymnandrum (Ranuncu-laceae)rdquoMolecular Ecology vol 18 no 4 pp 709ndash721 2009

[87] G M Hewitt ldquoQuaternary phylogeography the roots of hybridzonesrdquo Genetica vol 139 no 5 pp 617ndash638 2011

[88] C Printzen S Ekman and T Toslashnsberg ldquoPhylogeography ofCavernularia hultenii evidence of slow genetic drift in a widelydisjunct lichenrdquoMolecular Ecology vol 12 no 6 pp 1473ndash14862003

[89] H-B Zheng C M Powell K Butcher and J Cao ldquoLate Neo-gene loess deposition in southern Tarim Basin tectonic andpalaeoenvironmental implicationsrdquoTectonophysics vol 375 no1ndash4 pp 49ndash59 2003

[90] QWen andY Shi ldquoThequaternary climo-environment changesin Chaiwopu basin of Xinjiang regionrdquo Chinese GeographicalScience vol 3 no 2 pp 147ndash158 1993

[91] H Lu D W Burbank and Y Li ldquoAlluvial sequence in thenorth piedmont of the Chinese Tian Shan over the past 550 kyrand its relationship to climate changerdquo Palaeogeography Palaeo-climatology Palaeoecology vol 285 no 3-4 pp 343ndash353 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


historical events and the cycles of glaciation [13ndash16] Chloro-plastDNA (cpDNA) suitable for reconstructing phylogeneticpatterns of plant species is effective in exploring and estimat-ing phylogeographical history of temperate plants caused byclimatic change [17ndash20] Recent scholars have concentratedon the refuge during the Last Glacial Maximum (LGM) andrange expansion during inter- or postglacial periods [21ndash24] With the most focus on mountain plants in EuropeNorth America and East Asia [25ndash27] phylogeographicpatterns and demographical history of eremophytes in aridNorthwestern China are also receiving increasing attention[28ndash30]

We use Capparis spinosa a typical xerophyte to verifythe hypothesis of Pleistocene historical influence throughphylogeographic analysis and discuss how its spatial geneticstructure responded to paleogeologic and paleoclimaticchanges The perennial creeping subshrub is a type memberof a relatively large genus of the Capparaceae with a naturaldistribution in the Mediterranean West and Central Asia[31 32] In arid Western China there is only one singlegenus (Capparis Tourn ex L) with one single species (Cspinosa L) belonging exclusively to this family [33] Cspinosa ranges over the Western Himalayas Eastern PamirPlateau Tianshan Mountains Turpan-Hami Basin and theHexi Corridor Previous studies on Capparis have paid moreattention to autecology phenology quantitative morphologyand reproductive ecology [34ndash37] Molecular studies relatedto the genetic variation of the species are little explored[38 39] and have not considered genetic divergence from aphylogeographic point of view

Here we employed three maternally inherited cpDNAspacers trnL-trnF rps12-rpl20 and ndhF to trace historicaldivergence patterns of C spinosa We design to addressthe following concrete issues (1) According to phylogeneticrelationships yielded by cpDNA sequence variation whatis the genetic pattern of the species (2) When and howhave allopatric divergence and genetic differentiation ofthis xerophyte been influenced by glacial-interglacial cyclesassociated with geologic and climatic changes

2 Materials and Methods

21 Population Sampling A total of 267 individuals of Cspinosa from 31 natural populations were investigated inthe present study (Table 1) throughout almost the entiregeographic distribution of the species in EasternCentral Asiaincluding Xinjiang western Gansu and western Tibet Foreach population leaf samples were collected from 5 to 12mature and large perennial individuals separated by at least100m in order to avoid inbreeding and clones The latitudelongitude and altitude at each collection center were mea-sured using a global positioning system (GPS) receiver About10 g of fresh leaf materials was gathered per individual driedin silica gel in the field and stored at minus20∘C in the laboratoryCapparis bodinieri Levl was chosen as an outgroup for thephylogenic study based on previous taxonomic studies onCapparis [31] Voucher specimens for all individuals weredeposited in the Herbarium of the Xinjiang Institute of Ecol-ogy and Geography Chinese Academy of Sciences (XJBI)

22 DNA Extraction Amplification and Sequencing Silica-gel-dried leaf tissues were ground into powder in liquidnitrogen with a Fastprep-24 device (Sample Preparation Sys-tem MP Biomedicals USA) Total genomic DNA wasextracted from about 50mg of powdered tissues using amodified 2x CTAB method [40] Through preliminaryscreening from eighteen pairs of primers (see Table S1 inSupplementary Material available online at httpdxdoiorg10115520165792708) three cpDNA regions trnL-trnFrps12-rpl20 and ndhF (329F 927R) were found becausethey have more sequence variation in individuals amongpopulations Polymerase chain reaction (PCR) amplificationand DNA sequencing were performed using primer pairsfor these three regions PCRs were carried out in 30120583Lreaction volumes including 15 120583L of 10x buffer 2 120583L of25mmolLMgCl

2 22 120583L of 50 ng120583L each primer (Sangon

Shanghai China) 3120583L of 25mmolL dNTP 03 120583L of 5U120583LTaq DNA polymerase and 10 120583L of 5 ng120583L genomic DNAPCR amplification was run on a Gene-Amp PCR system9700 DNA Thermal Cycler (Applied Biosystems FosterCity CA USA) with parameters set as follows an initialstart at 94∘C with 4min followed by 30 cycles of 94∘C with30 s then 59∘C 52∘C and 52∘C respectively for trnL-trnFrps12-rpl20 and ndhF with 30 s and 72∘C with 90 s plusa subsequent additional extension at 72∘C with 10minSizes of PCR products were visualized on 1 TAE-agarosegel electrophoresis All PCR products were purified withPCR product purification kit (Shanghai SBS Biotech LtdChina) and then sequenced in both directions for trnL-trnFrps12-rpl20 and ndhF using an ABI Prism 3730 xl automatedsequencer in Sangon Biological Engineering Technology ampService Co Ltd Shanghai China

23 Data Analysis The chloroplast DNA sequences wereedited by SeqMan (Lasergene DNASTAR Inc MadisonWisconsin USA) All sequences were aligned with ClustalX version 181 [41] then refined and adjusted All resultingcpDNA sequences identified by nucleotide variations signi-fying different haplotypes were deposited in GenBank Thecorresponding accession numbers are KU940216ndashKU940218for trnL-trnF KU866560ndashKU866562 and KU940220 forrps12-rpl20 and KU866548ndashKU866558 and KU940221ndashKU940223 for ndhF (329F 927R) and accessions numbersof C bodinieri as the outgroup are KU940219 for trnL-trnFKU866563 for rps12-rpl20 and KU866559 for ndhF (329F927R)Different cpDNAhaplotypeswere identified byDnaSP50 [42] Geographic distribution of detected haplotypeswas mapped using ArcMap 93 (ESRI Redlands CA USA)Phylogenetic relationships among cpDNA haplotypes wereassociated via amedian-joining network (MJN)method [43]which was constructed using Network 4610 (available athttpwwwfluxus-engineeringcomsharenet rnhtm)

To maximize the differences of haplotype compositionbetween geographical locations spatial analysis of molecularvariance algorithm was conducted using SAMOVA 10 pro-cedure This method divided the sampled populations intoinferred partition (119870 groups) based on genetic differentiationand geographical homogeneity [44] We initially set the 119870values ranged from 2 to 12 by repeated simulated annealing


Table1Detailsof

geograph

icallocatio

nssam

ples

izescpD

NAhaplotypesand

diversity

indicesfor

31po

pulations

ofC

spinosa

Cod

ePo

pulationlocality

Latitud

elong

itude

Altitude

(m)

No

Haplotype

119867119889

(plusmnSD

)120587(plusmnSD

)Overall

267

08925plusmn00075

00037plusmn00019

(I)Th

eWestern

Him

alayas

grou

p(1)Z

DZa

ndaTibet

31∘

301015840

N79∘381015840

E3598

9H22H

23H

2404167plusmn01907

00021plusmn00013

(2)Q

SQusum

Tibet

31∘

291015840

N79∘481015840

E364

55

H22

00

(II)Th

eEastern

Pamirgrou

p(3)Y

GS

Yeng

isarXinjiang

38∘

431015840

N76∘141015840

E2010

9H6

00

(4)T

XGTaxkorgan

Xinjiang

37∘

471015840

N75∘131015840

E3694

5H6

00

(5)A

KTAktoXinjiang

38∘

591015840

N75∘321015840

E244

19

H6

00

(6)W

QWuq

iaX

injiang

39∘

441015840

N75∘401015840

E19

4510

H6H21

046

67plusmn01318

000

06plusmn000

05(7)A

TXArtux

Xinjiang

39∘

521015840

N76∘471015840

E13

0310

H6H7

046

67plusmn01318

000

06plusmn000

05(8)JS

JiashiXinjiang

39∘

561015840

N78∘011015840

E1162

9H6

00

(III)Th

eIliVa

lleygrou

p(9)G

LGon

gliuX

injiang

43∘

371015840

N81∘ 4

91015840E

710

9H7

00

(10)

YNYining

Xinjiang

43∘

371015840

N82∘081015840

E750

9H7

00

(11)BL

Bole

Xinjiang

44∘

421015840

N82∘051015840

E470

9H7

00

(12)

WS

WusuXinjiang

44∘

191015840

N84∘191015840

E685

9H7H12

05000plusmn01283

00013plusmn000

09(IV)Th

enorth

sideo

fthe

Tianshan

Mou

ntains

grou

p(13)

KRMY

KaramayX

injiang

44∘

561015840

N84∘461015840

E296

5H12

00

(14)T

LTo

liXinjiang

45∘

551015840

N83∘361015840

E684

5H17

00

(15)

SHZ

ShiheziXinjiang

44∘

161015840

N85∘571015840

E356

5H11

00

(16)

SWShaw

anX

injiang

44∘

191015840

N85∘351015840

E584

9H5

00

(17)

MNS

ManasX

injiang

44∘

111015840

N86∘081015840

E64

28

H5H10

04286plusmn01687

000

02plusmn000

02(18)

URM

QUrumqiX

injiang

43∘

441015840

N86∘571015840

E10

608

H10H

1104286plusmn01687

00010plusmn000

07(19)

JMS

JimsarXinjiang

43∘

551015840

N89∘071015840

E945

8H5

00

(V)Th

esou

thsid

eofthe

Tianshan

Mou

ntains

grou

p(20)

KPKa

lpinX

injiang

40∘

351015840

N79∘361015840

E1131

10H1H5

03556plusmn01591

000

08plusmn000

06(21)WNS

WensuX

injiang

41∘

291015840

N79∘541015840

E12

969

H1H8H20

07500plusmn00786

00016plusmn00010

(22)

BCBa

icheng

Xinjiang

41∘

591015840

N83∘031015840

E13

698

H1H8

05714plusmn00945

000

03plusmn000

03(23)

LTLu

ntaiX

injiang

41∘

141015840

N84∘121015840

E921

10H8H14

03556plusmn01591

00011plusmn000

07(24)

KRL

Korla

Xinjiang

41∘

501015840

N86∘031015840

E10

728

H1H8H13

06071plusmn01640

000

04plusmn000

04(25)

HJ

HejingXinjiang

42∘

311015840

N86∘161015840

E14

0310

H5H8

046

67plusmn01318

00012plusmn000

08(26)

TKS

Toksun

Xinjiang

42∘

491015840

N88∘381015840

E95

12H1H5

05455plusmn00615

00012plusmn000

08(27)

TRP

TurpanX

injiang

42∘

521015840

N89∘111015840

Eminus84

12H1H5H11H

18H

1908485plusmn00586

00017plusmn00010

(28)

SSShanshanX

injiang

43∘

111015840

N90∘091015840

E10

439

H10H

15H

1607222plusmn00967

00015plusmn000

09(29)

HM

Ham

iXinjiang

42∘

311015840

N94∘091015840

E773

9H9H10H

1107500plusmn00786

00013plusmn000

09(30)

DH

Dun

huangGansu

40∘

251015840

N94∘441015840

E10

5110

H1H2H3

03778plusmn01813

000

05plusmn000

04(31)GZ

Guazhou

Gansu

40∘

221015840

N95∘341015840

E1133

10H1H2H4H5

07333plusmn01005

00011plusmn000

07Nonum

bero

fsam

pleind


diversity

120587nucleotided

iversity















119904



3 Results




GZDH

HM

SSTRPTKSHJ

Ili valley

KRLLTBC

WNSKP


WQ

ATX

JSGL

YNBL

WSKRMYTL SHZ SW

MNSURMQ

JMS

ZDQS

Tian Shan Tian Shan

Tarim Basin

Western Himalayas

Kun

Plateau of Tibet

Hexi Corridor

Turpan-Hami

H1H7H13H19

H2H8H14H20

H3H9H15H21

H4H10H16H22

H5H11H17H23

H6H12H18H24

35∘N

40∘N

45∘N

70∘E 80∘E 90∘E

Lun Mountains

(a)

H1H5H11

H8H10H18

H7

H6

H24 H23

H21

H22H12

H17

H19

H4 H9

H16

H20

H14 H2

H13

H15H3

(b)

(c) (d)















119904values for



4 Discussion










H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13


5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3


Capparis bodinieri


Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S





lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Detailsof

geograph

icallocatio

nssam

ples

izescpD

NAhaplotypesand

diversity

indicesfor

31po

pulations

ofC

spinosa

Cod

ePo

pulationlocality

Latitud

elong

itude

Altitude

(m)

No

Haplotype

119867119889

(plusmnSD

)120587(plusmnSD

)Overall

267

08925plusmn00075

00037plusmn00019

(I)Th

eWestern

Him

alayas

grou

p(1)Z

DZa

ndaTibet

31∘

301015840

N79∘381015840

E3598

9H22H

23H

2404167plusmn01907

00021plusmn00013

(2)Q

SQusum

Tibet

31∘

291015840

N79∘481015840

E364

55

H22

00

(II)Th

eEastern

Pamirgrou

p(3)Y

GS

Yeng

isarXinjiang

38∘

431015840

N76∘141015840

E2010

9H6

00

(4)T

XGTaxkorgan

Xinjiang

37∘

471015840

N75∘131015840

E3694

5H6

00

(5)A

KTAktoXinjiang

38∘

591015840

N75∘321015840

E244

19

H6

00

(6)W

QWuq

iaX

injiang

39∘

441015840

N75∘401015840

E19

4510

H6H21

046

67plusmn01318

000

06plusmn000

05(7)A

TXArtux

Xinjiang

39∘

521015840

N76∘471015840

E13

0310

H6H7

046

67plusmn01318

000

06plusmn000

05(8)JS

JiashiXinjiang

39∘

561015840

N78∘011015840

E1162

9H6

00

(III)Th

eIliVa

lleygrou

p(9)G

LGon

gliuX

injiang

43∘

371015840

N81∘ 4

91015840E

710

9H7

00

(10)

YNYining

Xinjiang

43∘

371015840

N82∘081015840

E750

9H7

00

(11)BL

Bole

Xinjiang

44∘

421015840

N82∘051015840

E470

9H7

00

(12)

WS

WusuXinjiang

44∘

191015840

N84∘191015840

E685

9H7H12

05000plusmn01283

00013plusmn000

09(IV)Th

enorth

sideo

fthe

Tianshan

Mou

ntains

grou

p(13)

KRMY

KaramayX

injiang

44∘

561015840

N84∘461015840

E296

5H12

00

(14)T

LTo

liXinjiang

45∘

551015840

N83∘361015840

E684

5H17

00

(15)

SHZ

ShiheziXinjiang

44∘

161015840

N85∘571015840

E356

5H11

00

(16)

SWShaw

anX

injiang

44∘

191015840

N85∘351015840

E584

9H5

00

(17)

MNS

ManasX

injiang

44∘

111015840

N86∘081015840

E64

28

H5H10

04286plusmn01687

000

02plusmn000

02(18)

URM

QUrumqiX

injiang

43∘

441015840

N86∘571015840

E10

608

H10H

1104286plusmn01687

00010plusmn000

07(19)

JMS

JimsarXinjiang

43∘

551015840

N89∘071015840

E945

8H5

00

(V)Th

esou

thsid

eofthe

Tianshan

Mou

ntains

grou

p(20)

KPKa

lpinX

injiang

40∘

351015840

N79∘361015840

E1131

10H1H5

03556plusmn01591

000

08plusmn000

06(21)WNS

WensuX

injiang

41∘

291015840

N79∘541015840

E12

969

H1H8H20

07500plusmn00786

00016plusmn00010

(22)

BCBa

icheng

Xinjiang

41∘

591015840

N83∘031015840

E13

698

H1H8

05714plusmn00945

000

03plusmn000

03(23)

LTLu

ntaiX

injiang

41∘

141015840

N84∘121015840

E921

10H8H14

03556plusmn01591

00011plusmn000

07(24)

KRL

Korla

Xinjiang

41∘

501015840

N86∘031015840

E10

728

H1H8H13

06071plusmn01640

000

04plusmn000

04(25)

HJ

HejingXinjiang

42∘

311015840

N86∘161015840

E14

0310

H5H8

046

67plusmn01318

00012plusmn000

08(26)

TKS

Toksun

Xinjiang

42∘

491015840

N88∘381015840

E95

12H1H5

05455plusmn00615

00012plusmn000

08(27)

TRP

TurpanX

injiang

42∘

521015840

N89∘111015840

Eminus84

12H1H5H11H

18H

1908485plusmn00586

00017plusmn00010

(28)

SSShanshanX

injiang

43∘

111015840

N90∘091015840

E10

439

H10H

15H

1607222plusmn00967

00015plusmn000

09(29)

HM

Ham

iXinjiang

42∘

311015840

N94∘091015840

E773

9H9H10H

1107500plusmn00786

00013plusmn000

09(30)

DH

Dun

huangGansu

40∘

251015840

N94∘441015840

E10

5110

H1H2H3

03778plusmn01813

000

05plusmn000

04(31)GZ

Guazhou

Gansu

40∘

221015840

N95∘341015840

E1133

10H1H2H4H5

07333plusmn01005

00011plusmn000

07Nonum

bero

fsam

pleind


diversity

120587nucleotided

iversity















119904



3 Results




GZDH

HM

SSTRPTKSHJ

Ili valley

KRLLTBC

WNSKP


WQ

ATX

JSGL

YNBL

WSKRMYTL SHZ SW

MNSURMQ

JMS

ZDQS

Tian Shan Tian Shan

Tarim Basin

Western Himalayas

Kun

Plateau of Tibet

Hexi Corridor

Turpan-Hami

H1H7H13H19

H2H8H14H20

H3H9H15H21

H4H10H16H22

H5H11H17H23

H6H12H18H24

35∘N

40∘N

45∘N

70∘E 80∘E 90∘E

Lun Mountains

(a)

H1H5H11

H8H10H18

H7

H6

H24 H23

H21

H22H12

H17

H19

H4 H9

H16

H20

H14 H2

H13

H15H3

(b)

(c) (d)















119904values for



4 Discussion










H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13


5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3


Capparis bodinieri


Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S





lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















119904



3 Results




GZDH

HM

SSTRPTKSHJ

Ili valley

KRLLTBC

WNSKP


WQ

ATX

JSGL

YNBL

WSKRMYTL SHZ SW

MNSURMQ

JMS

ZDQS

Tian Shan Tian Shan

Tarim Basin

Western Himalayas

Kun

Plateau of Tibet

Hexi Corridor

Turpan-Hami

H1H7H13H19

H2H8H14H20

H3H9H15H21

H4H10H16H22

H5H11H17H23

H6H12H18H24

35∘N

40∘N

45∘N

70∘E 80∘E 90∘E

Lun Mountains

(a)

H1H5H11

H8H10H18

H7

H6

H24 H23

H21

H22H12

H17

H19

H4 H9

H16

H20

H14 H2

H13

H15H3

(b)

(c) (d)















119904values for



4 Discussion










H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13


5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3


Capparis bodinieri


Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S





lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


GZDH

HM

SSTRPTKSHJ

Ili valley

KRLLTBC

WNSKP


WQ

ATX

JSGL

YNBL

WSKRMYTL SHZ SW

MNSURMQ

JMS

ZDQS

Tian Shan Tian Shan

Tarim Basin

Western Himalayas

Kun

Plateau of Tibet

Hexi Corridor

Turpan-Hami

H1H7H13H19

H2H8H14H20

H3H9H15H21

H4H10H16H22

H5H11H17H23

H6H12H18H24

35∘N

40∘N

45∘N

70∘E 80∘E 90∘E

Lun Mountains

(a)

H1H5H11

H8H10H18

H7

H6

H24 H23

H21

H22H12

H17

H19

H4 H9

H16

H20

H14 H2

H13

H15H3

(b)

(c) (d)















119904values for



4 Discussion










H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13


5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3


Capparis bodinieri


Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S





lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














119904values for



4 Discussion










H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13


5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3


Capparis bodinieri


Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S





lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


H23

H24

H7H6

H21

H22

H19

H17

H12

H9

H10H16

H5H14

H20

H4

H11H18

H15H3

H8H1H2

H13


5560

1179

0808

0097

02700143

06210213

0067

0998

0748

03050142

03770169

0082

0051

Clade 1

Clade 2

Clade 4

Clade 5

Clade 6

Clade 3


Capparis bodinieri


Gen

etic

dist

ance

752∘E

952∘E 315∘N

456∘N

NEW

S





lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




lowastlowast




ObservedSimulated

0500

100015002000250030003500400045005000

Freq

uenc

y








TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

TianShan

Western

Himalayas

CorridorTurpan-Hami

Eastern

Basin

Pamir

Tian Shan

Hexi

Present-day

LGM (MIROC)

45∘N

40∘N

35∘N

45∘N

40∘N

35∘N

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E

75∘E 80

∘E 85∘E 90

∘E 95∘E 100

∘E











5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusions


Competing Interests


Acknowledgments


References






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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