Impacts of late Quaternary environmental change on the long-tailed ground squirrel (Urocitellus undulatus) in Mongolia

2018·0.52 MB·

by  Mclean, Bryan S

ZOOLOGICAL RESEARCH
Impacts of late Quaternary environmental change on
the long-tailed ground squirrel (Urocitellus undulatus)
in Mongolia
BryanS.McLean1,*,BatsaikhanNyamsuren2,AndreyTchabovsky3,JosephA.Cook4
1UniversityofFlorida,FloridaMuseumofNaturalHistory,Gainesville,FL32611,USA
2DepartmentofBiology,SchoolofArtsandSciences,NationalUniversityofMongolia,UlaanBaatar 11000,Mongolia
3LaboratoryofPopulationEcology,A.N.SevertsovInstituteofEcologyandEvolution,Moscow119071,Russia
4UniversityofNewMexico,DepartmentofBiologyandMuseumofSouthwesternBiology,Albuquerque,NM87131,USA
ABSTRACT of range shifts into these lowland areas in response
to Pleistocene glaciation and environmental change,
Impacts of Quaternary environmental changes on followed by upslope movements and mitochondrial
mammal faunas of central Asia remain poorly lineage sorting with Holocene aridification. Our study
understood due to a lack of comprehensive illuminatespossiblehistoricalmechanismsresponsible
phylogeographic sampling for most species. To for U. undulatus genetic structure and contributes to
help address this knowledge gap, we conducted a framework for ongoing exploration of mammalian
the most extensive molecular analysis to date of responsetopastandpresentclimatechangeincentral
thelong-tailedgroundsquirrel(Urocitellusundulatus Asia.
Pallas 1778) in Mongolia, a country that comprises
Keywords: Central Asia; Gobi Desert; Great Lakes
thesoutherncoreofthisspecies’range. Drawingon
Depression;Mongolia;Phylogeography
material from recent collaborative field expeditions,
we genotyped 128 individuals at two mitochondrial INTRODUCTION
genes (cytochrome b and cytochrome oxidase I; Urocitellus undulatus Pallas 1778 is a charismatic,
1 797 bp total). Phylogenetic inference supports the medium-bodied ground-dwelling sciurid distributed across
existenceoftwodeeplydivergentinfraspecificlineages central Asia, including portions of Siberia, Mongolia,
northwestern China, and easternmost Kazakhstan and
(corresponding to subspecies U. u. undulatus and
Kyrgyzstan (Helgen et al., 2009; Kryštufek & Vohralík, 2013;
U.u.eversmanni),aresultinagreementwithprevious
Ognev, 1947; Wilson & Reeder, 2005). Although the genus
molecular investigations but discordant with patterns
of range-wide craniometric and external phenotypic
variation. In the widespread western eversmanni
Received: 06September2017; Accepted: 02January2018; Online:
lineage,werecoveredgeographically-associatedclades
08March2018
from the: (a) Khangai, (b) Mongolian Altai, and Foundationitems:Expeditionswerefundedprimarilybygrantsfromthe
(c) Govi Altai mountain ranges. Phylogeographic NationalScienceFoundation(USA;DBI-9411976supplement(1999),
structure in U. u. eversmanni is consistent with an DEB-0717214(2009-2012),DEB-1258010(2015-2016)). B.S.M.was
isolation-by-distancemodel;however,geneticdistances partiallysupportedbyaPeterBuckPredoctoralFellowshipduringthe
are significantly lower than among subspecies, and 2015Mongolianexpedition. Datagenerationandanalysishereinwas
intra-clade relationships are largely unresolved. The supportedbytheNationalScienceFoundation(DEB-1258010)andthe
latterpatterns,aswellastherelativelyhighernucleotide AmericanSocietyofMammalogists(ASMFellowshiptoB.S.M.)
polymorphism of populations from the Great Lakes *Correspondingauthor,E-mail:[email protected]
DepressionofnorthwesternMongolia,suggestahistory DOI:10.24272/j.issn.2095-8137.2018.042
364 SciencePress ZoologicalResearch39(5):364–372,2018
Urocitellus(comprisedof12speciesformerlysubsumedwithin Mongolian range of U. undulatus at two mitochondrial genes
Spermophilus;Helgenetal.,2009)isdistributedacrossmuch (cytochrome b (cyt b) and cytochrome oxidase I (COI), 1
of the Holarctic region, U. undulatus is the only exclusively 797 bp total). We document population genetic variation and
Palearctic species in this clade (Wilson & Reeder, 2005; structure, and use those data to explore the potential effects
McLean et al., 2016b). To date, various single- and multilocus of known late Pleistocene environmental changes on genetic
investigations (Tsvirka et al., 2008; Ermakov et al., 2015; patterns. Ourworkprovidesnewinformationontheevolutionary
McLeanetal.,2016b;Simonovetal.,2017)haverevealedthat and biogeographic history of U. undulatus in western Mongolia
U. undulatus is comprised of two deeply divergent lineages and lays a foundation for further analyses in this and similarly
recognizable as well-defined subspecies (Kryštufek & Vohralík, distributedcentralAsianmammals.
2013)orsemi-species(Pavlinov&Lissovsky,2012). Theseare
MATERIALSANDMETHODS
an eastern lineage (undulatus) in western and central Siberia,
northernMongolia,andtheAmurregionofsoutheasternSiberia, Samplesandsequencing
and a western lineage (eversmanni) in western Mongolia, Specimens used in this study are housed at the University
northern China, and Kazakhstan. However, our understanding of New Mexico Museum of Southwestern Biology (MSB).
ofthefulldiversityandevolutionaryandbiogeographichistoryof Mongolian samples were collected during joint MSB-National
thisspeciesacrossitsvastrangeremainsincomplete. University of Mongolia expeditions in 1999 (Tinnin et al.,
Although U. undulatus has been the subject of persistent 2002), 2009–2012 (with University of Kansas and University
morphologicalandmolecularfocusoverthepastthreedecades of Nebraska), and 2015–2016 (with Northern Michigan
(Ermakov et al., 2015; Linetskaya & Linetskii, 1989; McLean University). Cumulative efforts of these expeditions include
et al., 2016b; Simonov et al., 2017; Tsvirka et al., 2008; >6 500 cataloged mammal specimens from across major
Vorontsov et al., 1980), more expansive genetic datasets Mongolianvegetativeandfaunalprovinces,manyofwhichare
are necessary to test existing taxonomic hypotheses and associatedwithecto-andendoparasitespecimensarchivedat
illuminate the historical demography and biogeography of MSB Division of Parasites or University of Nebraska Manter
this species. Unfortunately, however, a lack of spatially Lab of Parasitology. All field methods followed guidelines
comprehensive sampling and associated genetic data exists of institutional animal care and use committees as well
for this and many other central Asian vertebrate taxa. This as the American Society of Mammalogists Guide for Use
data gap precludes identification of the broader abiotic and of Wild Mammals in Research (Sikes et al., 2011), and
bioticprocessesactingtoshapephylogeographicpatternsand were focused on collection of “holistic” mammal specimens
vertebrate community structure across this expansive region. (e.g.,Dunnum&Cook,2012;McLeanetal.,2016a;Yatesetal.,
Fortaxawithrelativelyhighmorphologicalconservatism(such 1996).Cumulatively,thesematerialsrepresentanunparalleled
asUrocitellus), suchdatasetsareparticularlycrucialtorefine resourceforestablishingMongolianfaunalbaselinesinanera
our understanding of the true genomic and biogeographic ofongoingglobalclimateandenvironmentalchange.
historiesoflineages. Weselected128specimensofU.undulatusforsequencing
Themostsignificantlackofphylogeographicsamplingfrom and analysis (Figure 1; Supplementary Table S1; GenBank
U. undulatus is in Mongolia, a country that nevertheless accessionNos.MG883400–MG883654).Thedatasetincluded
comprises the southern core of this species range. Several 119 individuals from 11 different Mongolian aimags (political
pressingevolutionaryandbiogeographicalquestionshingeon landdesignationsanalogoustoprovincesorstates)aswellas
improved genetic sampling of Mongolian populations. First, putative representatives of both subspecies (as delineated by
although each of the subspecific lineages of U. undulatus Kryštufek & Vohralík, 2013). The dataset also included nine
(undulatusandeversmanni)isdocumentedwithinthecountry, individualsofU.u.undulatusfromSakhaRepublicinnorthern
what are their exact geographic distributions? Second, do Siberia. We selected sequences of the Columbian ground
anypopulationsinMongoliadisplaypatternsofmixedmtDNA squirrel (U. columbianus) from GenBank as the outgroup
ancestry and, if so, where are these populations located? for phylogenetic analysis. Frozen tissue samples of all
Third,howhaveknownlateQuaternaryenvironmentalchanges U. undulatus individuals (liver, muscle, or dried muscle) were
shaped phylogeographic structure within the widespread subjected to lysis in a solution of 600 µL tissue lysis buffer
western lineage (eversmanni)? Specifically, this lineage and 12–15 µL reconstituted proteinase K (Omega E.Z.N.A.
occupies an environmentally and climatically heterogeneous kit; Omega Bio-tek, Inc., USA) for up to 24 hours. Genomic
range in Mongolia, including multiple mountain systems DNA was isolated using a standard salt/ethanol extraction
(Khangai, Mongolian Altai, Govi Altai) that were subject to procedure. ToreducethepotentialforPCRinhibition,alldried
latePleistoceneglaciation,downwardexpansionofpermafrost, muscle samples were processed prior to lysis by removing
andotherenvironmentalchanges. debris, cutting into sub-centimeter sized pieces, and washing
in 100% ethanol for 15 min at room temperature, vortexing
Inthispaper,wepresentthemostcomprehensivemolecular
several times; these were then washed in STE buffer under
phylogeographic analysis of U. undulatus in Mongolia to
refrigeration for 12–16 h. Final extractions were quantified
date. Drawing on material collected during expeditions of
flourometrically using a Qubit Broad Range assay kit (Life
the Museum of Southwestern Biology (New Mexico, USA)
TechnologiesCorp.,USA).
from1999–2016,wegenotypedsamplesfromacrosstheentire
ZoologicalResearch39(5):364–372,2018 365
G R E A T
M
O L A K E S KHENTII
N
G +
O
L K H A N G A I Ulaanbaatar
I
A A
LTN
A
I GOVI ALTGAI O B I D E S E R T
Figure1MapofMongoliashowingmajorlandscapefeaturesandsamplinglocalitiesofU.undulatus
Higherelevationsareshownindarkercolorsandmajormountainrangesandlandscapefeaturesareindicatedwithtext.Collectionlocalitiesofsamplesusedin
thisstudyindicatedbyredcircles(U.u.eversmanni)andsquares(U.u.undulatus).
We used the primer pair MVZ05/MVZ14 (5(cid:48)-CGAAGC estimateposteriordistributionsoftreetopologies,runningboth
TTGATATGAAAAACCATCGTTG/CTTGATATGAAAAACCATCG analyses for 10 000 000 generations, sampling every 1 000
TTG-3(cid:48); Smith & Patton, 1993) to amplify all 1 140 bp of the generations,anddiscardingthefirst25%ofsamplesasburn-in.
mitochondrialcytbgene. WeusedtheprimerpairHCO2198/ ConvergenceofallparameterswasassessedinTracerv1.6.0
LCO1490 (5(cid:48)-TAAACTTCAGGGTGACCAAAAAATCA/GGTC (Rambautetal.,2014)byvisualizingtraceplotsandensuring
AACAAATCATAAAGATATTGG-3(cid:48); Folmer et al., 1994) to effectivesamplesizes>200.
amplify 657 bp of the mitochondrial COI gene. Amplification To characterize population structure in U. undulatus, we
of both mtDNA regions took place in 25 µL reactions, with performed an analysis of molecular variance (AMOVA) of all
annealing temperatures of 52 °C and 48 °C, respectively. samples in the R package poppr (Kamvar et al., 2014). For
Purified PCR products were sequenced using Big Dye each gene, we included subspecies (undulatus, eversmanni)
Terminator 3.1 technology (Applied Biosystems, USA) on an and aimag of origin (12 total in our dataset) as factors. We
ABI3130automatedDNAsequencerintheMolecularBiology alsotestedsignificanceoftheobservedvariancepatternswith
Facility in the Department of Biology at University of New 4999randomizations. Becausepoliticalboundariesmayonly
Mexico. Sequences were manually edited in Sequencher weakly capture landscape-scale genetic structure, we next
v5.3 (Gene Codes Corp., Michigan, USA) and aligned with tested consistency of our data with an isolation-by-distance
MUSCLE v3.7 (Edgar, 2004) using default settings on the (IBD) model, focusing only on U. u. eversmanni. For that
CIPRESsciencegateway(www.phylo.org;Milleretal.,2010). test,weusedfunctionsintheRpackagesape(Paradisetal.,
WeusedtheRpackagespegas(Paradisetal.,2017b)and 2017a) and raster (Hijmans et al., 2017) to compute pairwise
popGenome(Pfeiferetal.,2017)tocalculatestandardpopulation genetic (P-values) and geographic (in meters) distances,
geneticstatistics,andtotestforsignalsofpopulationexpansion respectively. Wecalculatedcorrelationsbetweenthesematrices
basedontheTajima’sD statistic. Partialdeletionofpositions using the mantel function in vegan (Oksanen et al., 2017) and
with missing data was performed when calculating pairwise assessed statistical significance using 4 999 permutations of
nucleotide-based metrics (nucleotide diversity and pairwise the geographic distance matrix. Finally, we visualized spatial
number of nucleotide differences). We inferred phylogeny patternsofmtDNAdiversitybycomputingaminimum-spanning
for all samples in a Bayesian framework. First, we used haplotypenetwork(Bandeltetal.,1999)inPopART(http://popart.
PartitionFinder v2.1 (Lanfear et al., 2017) to simultaneously otago.ac.nz),usingjustthesignificantlymorevariablecytbgene.
inferthebest-fitpartitioningschemeandmodelsofsequence
RESULTS
evolution for the concatenated mtDNA matrix, as evaluated
using the AICc metric. We conducted Bayesian phylogenetic The best-fit partitioning scheme for the concatenated
inference in MrBayes v3.2.3 (Ronquist & Huelsenbeck, 2003) mtDNA matrix included a different partition for each codon
on CIPRES, using the optimal partitioning scheme inferred position within the cyt b and COI genes (although codon
above. Two independent MCMC analyses composed of 4 position 2 for both genes shared the same partition;
Metropolis-coupled chains each (the default) were used to Table 1). The Tamura-Nei (TrN) substitution model or
366 www.zoores.ac.cn
2_Urocitellus_columbianus
1_Urocitellus_columbianus
NK139016_Sakha
NK139006_Sakha
NK139009_Sakha
NK139021_Sakha
NK139034_Sakha
NK100786_Tov
NK100788_Tov
NK100796_Tov
NK289433_Khentii
NK272178_Khovsgol
NK272177_Khovsgol
NK272179_Khovsgol
NK272176_Khovsgol
NK270901_Uvs
NK270904_Uvs
NK270900_Uvs
NK270902_Uvs
NK272034_Uvs
NK224672_Bayankhongor
NK224682_Bayankhongor
NK289154_Bulgan
NK289155_Bulgan
NK100607_Ovorkhangai
NK270021_Arkhangai
NK270023_Arkhangai
NK100650_Ovorkhangai
NK100591_Ovorkhangai
NK100601_Ovorkhangai
NK100604_Ovorkhangai
NK100605_Ovorkhangai
NK100606_Ovorkhangai
NK100649_Ovorkhangai
NK272326_Bulgan
NK272327_Bulgan
NK272328_Bulgan
NK272329_Bulgan
NK270002_Arkhangai
NK270019_Arkhangai
NK224681_Bayankhongor
NK224683_Bayankhongor
NK224702_Bayankhongor
NK270007_Arkhangai
NK289182_Bulgan
NK289183_Bulgan
NK224669_Bayankhongor
NK224670_Bayankhongor
NK224717_Bayankhongor
NK270020_Arkhangai
NK270022_Arkhangai
NK270621_Uvs
one of its extensions (TrN with equal base frequencNieK2s7,0683_Uvms aximum inter-clade distance for COI (3.16%) is concordant
NK270800_Uvs
NK194291_Bayan-Olgii
gamma-distributed heterogeneity, and/or invariant sites) waNsK192374_Gwoivtih-AltaEirmakov et al.’s (2015) estimate of 3.5% using this
NK192410_Govi-Altai
preferred for all partitions (Table 1). Phylogenetic inferenNNcKKe119933550098__GGosovvaii--mAAlltteaaii markerbutdifferentindividuals. Forreference, average
NK192392_Govi-Altai
in MrBayes recovered two major clades (U. u. undulatus anNdK192400_Guonvic-Aoltrariected distances between all samples of U. undulatus
NK192406_Govi-Altai
U.u.eversmannisensuKryštufek&Vohralík,2013)withstronNNgKK119944348307__BBaaanyyaadnn--OOthllggiieii two U. columbianus outgroups are 8.21%±0.14 and
NK194441_Bayan-Olgii
support (PP=1; Figure 2). The average uncorrected genetiNNcKK119944239533__4BB.aa9yyaa9nn%--OOll±ggii0ii.16forcytbandCOI,respectively. However,wenote
NK194407_Bayan-Olgii
distance(mean±SD)betweenthesecladesis5.84%±0.19foNrK194436_thBaaytanU-O.lguiindulatus and U. columbianus may not share a most
NK194463_Bayan-Olgii
cyt b, but a more modest 2.67%±0.20 for COI. Notably, thNNeKK27207108188_9B_rBaeyacaynae-nOn-Oltgligciiiommonancestor(McLeanetal.,2016b).
NK270489_Bayan-Olgii
NK270577_Bayan-Olgii
NK270581_Bayan-Olgii
NK270553_Bayan-Olgii
NK270572_Bayan-Olgii
NK270573_Bayan-Olgii
NK223726_Khovd
NK223741_Khovd
2_Urocitellus_coluNmKb2ia2n3u7s42_Khovd
1_Urocitellus_coluNmKb2i2a3n8u7s3_Khovd
NK270576_Bayan-OlgiNiK139016_Sakha
NK270588_Bayan-Olgii NK139006_Sakha
NK193544_Govi-Altai NK139009_Sakha
U. u. undulaNtKu1s94292_Bayan-Olgii NK139021_Sakha
NK192362_Govi-Altai NK139034_Sakha
NNNNNKKNKKKNN11K121KK9997922222202277333033007773758831556855_____356GGGAG___ooGrUUookvvvvovvhii--iivss-a-AAiAAn-llAttllNgttaaaalNNaKiitiiaiKK2i722277122711NNN877NKKK_79112KK__008KK1h0090ohh7740voo8837svv863g9ss___ogg6TTKloo_oollThvveonvtii Khovsgol
NK223514_Govi-AlNtaKi272176_Khovsgol
NK223527_Govi-AltaNiK270901_Uvs *
NK223528_Govi-AltNaKi270904_Uvs
NNKK227700990002__UUvvss Uvs*
NK272034_Uvs
NK224672_Bayankhongor
NK224682_Bayankhongor
0.0060 NK289154_Bulgan
NK289155_Bulgan
U. u. eversmanni NK100607_Ovorkhangai
NK270021_Arkhangai
NK270023_Arkhangai
NK100650_Ovorkhangai
NK100591_Ovorkhangai
NK100601_Ovorkhangai
NK100604_Ovorkhangai
NK100605_Ovorkhangai
NK100606_Ovorkhangai
NNKK217020362469__BOuvlgoarknhangai Khangai
NK272327_Bulgan
NK272328_Bulgan
NK272329_Bulgan
NK270002_Arkhangai
NK270019_Arkhangai
NK224681_Bayankhongor
0.52 NK224683_Bayankhongor
NK224702_Bayankhongor
NK270007_Arkhangai
NK289182_Bulgan
NK289183_Bulgan
NK224669_Bayankhongor
NK224670_Bayankhongor
NK224717_Bayankhongor
NK270020_Arkhangai
NK270022_Arkhangai
NK270621_Uvs
NK270683_Uvs *
NK270800_Uvs
NK194291_Bayan-Olgii
NK192374_Govi-Altai
NK192410_Govi-Altai
NK193508_Govi-Altai
NK193509_Govi-Altai
NK192392_Govi-Altai
NK192400_Govi-Altai
0.64 NK192406_Govi-Altai
NK194380_Bayan-Olgii
NK194437_Bayan-Olgii
NK194441_Bayan-Olgii
NK194293_Bayan-Olgii
NNKK119944345037__BBaayyaann--OOlglgiiii Western
NK194436_Bayan-Olgii
NK194463_Bayan-Olgii
NK270188_Bayan-Olgii
0.63 NK270189_Bayan-Olgii
NK270489_Bayan-Olgii
NK270577_Bayan-Olgii
NK270581_Bayan-Olgii
NK270553_Bayan-Olgii
NK270572_Bayan-Olgii
NK270573_Bayan-Olgii
NK223726_Khovd
NK223741_Khovd
NK223742_Khovd
NK223873_Khovd
NK270576_Bayan-Olgii
NK270588_Bayan-Olgii
NK193544_Govi-Altai
NK194292_Bayan-Olgii
NK192362_Govi-Altai
NK192371_Govi-Altai
NK192373_Govi-Altai
NK192375_Govi-Altai
NK192376_Govi-Altai
NK270035_Arkhangai
NNKK227700885556__UUvvss * Govi
NK223583_Govi-Altai
NK223514_Govi-Altai
NK223527_Govi-Altai
NK223528_Govi-Altai
0.84
Figure2Majority-ruleconsensusphylogramofUrocitellusundulatusbasedonBayesianinferenceinMrBayes
0.0060
Subspeciesandmajorclades(withinU.u.eversmannionly)areindicatedbytext.ThetreeisrootedonthesplitfromtheNearcticU.columbianus.Brancheswith
posteriorprobabilitieslessthan0.9(i.e.,between0.5and0.9)areindicatedbyarrows.Identicalcytbhaplotypesarerepresentedbyamaximumof3individuals
inthetree;additionalduplicatehaplotypesweretrimmedforclarity(n=32).AsterisksdenoteindividualsfromUvsAimag.
ZoologicalResearch39(5):364–372,2018 367
Table1Best-fitmodelsofevolutionaccordingtotheAICc Aimags(Figure2),althoughwenotethatsomecladesexhibit
metricforpartitionsoftheconcatenatedmtDNAmatrix incompletelineagesorting. Forexample,individualsfromUvs
Model No.sites Aimag are associated with four distinct genetic clusters or
subclades in the Bayesian phylogeny (asterisks in Figure 2).
cytbposition1 TrNEF+G 380
Finer-scaleassociationswithlandscapefeaturessuchasrivers
cytbposition2+COIposition2 Trn+I 599
werenotevidentinourdataset.
cytbposition3 TrN+G 380
Despite the incomplete geographic sorting of mtDNA
COIposition1 TrN 219
haplotypesandgenerallackoffine-scalepopulationpatterning,
COIposition3 TrN 219
thereisdetectablephylogeographicstructurewithinMongolian
U.u.eversmanni. AMOVAssupportaroleforbroadprovincial
Both the genotyping results (Table 2) and phylogenetic
classifications in mtDNA variation, with 21.7% and 33.9% of
inference confirmed hypotheses that the U. u. eversmanni
molecularvarianceincytb andCOI,respectively, attributable
lineageiswidespreadacrosswesternMongolia,occurringinfar
to aimag of origin after accounting for subspecific variation
northern (Khövsgöl), southern (Govi Altai), and westernmost
(Table 3). Both values are greater than expected by chance
(Bayan-Ölgii)aimags.Conversely,thenominaleasternlineage
(Table 3). A statistically significant correlation was also found
(U. u. undulatus) occupies a more restricted range in the
between matrices describing raw genetic distances and raw
country;individualstakenfromasfareastasKhövsgölAimag,
geographicdistanceswithinU.u.eversmanni incytb(r=0.58,
Bulgan Aimag and the southeastern Khangai Mountains P<0.01) and COI (r2=0.38, P<0.01), thereby supporting an
maintainevolutionaryaffinitywiththewesternU.u.eversmanni
isolation-by-distancehypothesisinthissubspecies.
lineage(Figure2).WefoundnoevidenceformtDNAadmixture
(i.e., presenceofhaplotypesfrommultiplesubspecies)inany
populationsurveyed,includingthoseproximatetotheapparent Table2PopulationgeneticsummarystatisticsforUrocitellus
phylogeographic break between undulatus and eversmanni, undulatuseversmanni,partitionedbygene
suggesting a persistent lack of gene flow between these two n H Hd S k π
subspecificlineages.
cytb(1140bp)
Within the widespread U. u. eversmanni lineage, three
110 47 0.91 67 9.60 8.71×10-3
geographically-associated subclades are strongly supported
COI(657bp)
in the MrBayes topology, from (1) the Khangai Mountains
andsurroundingregions(“Khangai”clade); (2)theMongolian 111 22 0.88 23 2.16 3.29×10-3
and Govi Altai and surrounding highland regions (“Western”
n: Numberofsamples,H:Numberofhaplotypes,Hd: Haplotype
clade);and(3)additionalrangesoftheGoviAltai(“Govi”clade).
diversity,S:Numberofpolymorphicsites,k: Averagenumberof
Additional,morenarrowlydistributedgeneticclusterswerealso
nucleotidedifferences,π:Nucleotidediversity.
recovered and include populations from Khövsgöl and Uvs
Table3Resultsofanalysisofmolecularvariance(AMOVA)forbothgenesandallsamplesofUrocitellusundulatus
Degrees Sumof Variancerelative
Variance P
offreedom squareddeviations toexpected
cytb
Betweensubspecies 1 2.84 0.05(8.9) Greater 0.01
Amongaimags 10 15.66 0.12(21.7) Greater 0.01
Withinaimags 115 42.88 0.37(69.3) Less 0.01
Total 126 61.38 0.54(100)
COI
Betweensubspecies 1 4.25 0.08(14.9) Greater 0.01
Amongaimags 10 21.33 0.18(33.9) Greater <0.01
Withinaimags 116 31.65 0.27(51.2) Less <0.01
Total 127 57.23 0.53(100)
Nevertheless, we emphasize that divergences among and U. u. undulatus (Ermakov et al., 2015; this study).
U. undulatus subclades are very low. This can be visualized TheyarealsolowerthanthosefoundwithinU.u.eversmanni
in the minimum-spanning haplotype network (Figure 3), and populationsfromtheadjacentAltairegionofsouthernRussia
is borne out in uncorrected pairwise genetic distances (Simonov et al., 2017), although the latter study used
computed within the clade of (mean±SD) 0.63%±0.39 for cyt the noncoding and more variable mtDNA control region.
b and 0.33%±0.22 for COI. Those distances are roughly an In addition, although there was more variation within than
order of magnitude lower than between U. u. eversmanni among aimags, AMOVAs suggest that there is a significantly
368 www.zoores.ac.cn
lower amount of molecular variance within aimags than U.u.eversmanni (cytb, D=–2.09; COI,D=–1.47)andacross
expected by chance, highlighting the shallow differentiation all samples of U. undulatus (cyt b, D=–1.52; COI, D=–0.82),
that exists in broad geographic regions. Finally, we althoughtheresultwasonlysignificantattheP<0.01levelfor
recovered negative values of Tajima’s D for both genes in U.u.eversmanni cytb(P>0.10forallothers).
Khov.
Uvs
B.-O. Bul.
Ark.
Khovd
Ovor.
Govi Bayan.
Figure3Minimum-spanninghaplotypenetworkofallUrocitellusundulatuseversmannisamplesusedinthisstudy
Individuals in haplotype network (top) are colored by aimag of origin, with colors corresponding to the map (below). Notches indicate single nucleotide
substitutions. Note the map does not include the distribution of U. u. undulatus (B.-O.=Bayan-Olgii, Govi=Govi-Altai, Khov.=Khovsgol, Bul.=Bulgan,
Ark.=Arkhangai,Ovor.=Ovorkhangai,Bayan.=Bayankhongor).
DISCUSSION Meriones gerbils and Allactaga, Dipus jerboas, Liao et al.,
2016) in central Asia to date hint at significant impacts of
late Pleistocene environmental change on population genetic
Relativelylittleisknownaboutrange-widepatternsofgenetic
diversityandgeographicstructureinthisregion. Ouranalysis
structure and endemicity in many central Asian mammal
of U. undulatus allows us to establish preliminary hypotheses
species. This information gap precludes tests of existing
of mammalian spatiotemporal response to late Quaternary
taxonomic hypotheses and limits deeper knowledge of how
change within Mongolia that can be further tested in this and
past environmental changes across this vast region have
othersympatricspecies.
influencedmammaliandiversification. Thisisparticularlytrue
forecomorphologicallyconservativetaxa,suchasU.undulatus, On a range-wide scale, our results support the existing
where molecules and morphology might be expected to give systematic hypothesis (Kryštufek & Vohralík, 2013; Pavlinov
cryptic or conflicting historical signals. Indeed, the few & Lissovsky, 2012) that U. undulatus is comprised of two
phylogeographic investigations of non-volant vertebrate taxa deeply divergent lineages (undulatus and eversmanni). The
(e.g.,Phrynocephaluslizards,Wang&Fu,2004;Rhombomys strong phylogeographic break between these lineages, which
gerbils, Ning et al., 2007; Bufo toads, Zhang et al., 2008; stretchesfromsouthernLakeBaikal(Russia)througheastern
ZoologicalResearch39(5):364–372,2018 369
Selenge and Töv Aimags (Mongolia), contrasts, however, our data, including low population structure and negative
with previously published patterns of cranial shape variation. values for Tajima’s D in U. u. eversmanni, suggest recent
Specifically,morphologicalstudies(Kryštufek&Vohralík,2013; upslope range expansions from lowland refugia. Therefore,
Linetskaya & Linetskii, 1989; Vorontsov et al., 1980) found fromanelevationalperspective,thesestudiessupportopposite
populations from Yakutia and the Amur region of Russia to historical scenarios that likely reflect differences in the need
be highly divergent in cranial shape, while populations from of amphibians to track water availability versus that of steppe
theremainderoftherangeextendingacrosssouthernSiberia mammals. Gür (2013) and Liao et al. (2016) describe
and northern Mongolia exhibited a broad longitudinal cline a third pattern in Anatolian ground squirrels (Spermophilus
in cranial shape. Because body size varies significantly in xanthoprymnus) in Turkey and gerbils and jerboas (Meriones
U. undulatus, and cranial morphology is highly allometric in meridianus and Dipus sagitta) in northern China, suggesting
Urocitellusgroundsquirrels(e.g.,Robinson&Hoffmann,1975), that these species expanded their areal, but not elevational,
itseemslikelythatcranialshapedata(especiallythosebased distributions during the late Pleistocene in conjunction with
on linear measurements) largely reflect body size differences, expansionofcoldsteppehabitatsanddeserts.
which may or may not be useful for elucidating evolutionary If our hypothesis of lowland Pleistocene range shifts is
structure in this species. More robust tests of species limits correct, the most extensive corridors for gene flow among
and phylogeographic hypotheses in U. undulatus, as well as ancient populations of U. u. eversmanni may have been
of our assertion of a lack of gene flow between undulatus in the “Great Lakes Depression” and “Valley of the Govi
and eversmanni, await data from additional and independent Lakes”. Those lowland regions, located between major
regionsofthenucleargenome. Mongolian mountain ranges, are mostly contiguous with a
Considering the eversmanni lineage which makes up broad longitudinal band of mesic steppe that transverses
the bulk of our sampling, our results provide new insights Mongolia and forms a corridor for more arid-adapted taxa
into effects of late Quaternary climate change on historical suchasMongoliangazelle(Procapragutturosa)andTolaihare
biogeographyofthistaxonacrossMongolia.Therecordoflate (Lepus tolai; Batsaikhan et al., 2014). However, Pleistocene
PleistoceneandHoloceneenvironmentalchangeinthisregion environmentsinthisregionwerelikelymoremesicthantoday
includes extensive plateau and mountain valley glaciation, andmayhaveincludedamixtureofsteppeandforestelements
specifically in the Khangai, Mongolian Altai, and Govi Altai (Grunert et al., 2000; Böhner & Lehmkuhl, 2005). Downward
ranges; extensive downward expansion of permafrost; and expansion of mesic floral and faunal elements into the Great
intermittentformationanddrainingoflakesatbothhigherand Lakes Depression during the late Pleistocene would have
lower elevations (Böhner & Lehmkuhl, 2005; Grunert et al., provided suitable habitat for an increasingly mesic-adapted
2000; Lehmkuhl, 1998; Lehmkuhl & Lang, 2001). Montane suiteofvertebratespeciessuchasU.undulatus.
glaciationandtheexpansionofpermafrostshouldhavedriven
As a post hoc investigation of this scenario, and to more
downsloperangeshiftsinU.undulatus,asthisspeciesprefers
thoroughly parse AMOVA results, we calculated population
mesic steppe habitats and requires deeper permafrost levels
genetic statistics (haplotype and nucleotide diversity) for all
for construction of hibernacula. These range shifts, in turn,
aimagswithatleast15sampledindividuals(Uvs,Bayan-Ölgii,
shouldhavepromotedincreasedmixingofpopulationsacross
and Govi Altai). While Uvs Aimag contains lower haplotype
lowlandsofwesternMongoliabetween30–12kya.
diversity (0.71) than either Bayan-Ölgii or Govi Altai aimags
Consistentwiththatscenario,wefoundshallowdivergence (0.91and0.80,respectively),ithashighernucleotidediversity
between major mtDNA clusters within U. u. eversmanni. (6.69x10-3 vs. 2.37x10-3 and 4.92x10-3, respectively). This
However, because many of these mitochondrial lineages are increased nucleotide polymorphism could have resulted from
restricted to mid- and high-elevation steppe habitats (e.g., confinement of multiple U. u. eversmanni lineages within
in the Govi Altai) and unlikely to experience high levels of a Pleistocene refugium spread across the Great Lakes
gene flow, low divergences are likely to be signatures of past Depression and surrounding basins, followed by rapid range
(i.e., latest Quaternary) population mixing across lowlands of expansion into favorable montane habitats that became
western Mongolia. The shallow but significant geographic increasingly disjunct with Holocene climate changes, yielding
structurethatdoesexistamonggeographicallyandecologically the phylogeographic and demographic signals we detected.
disparate populations of U. u. eversmanni could, in turn, Simonovetal. (2017)demonstratedelevatedmtDNAdiversity
havebeengeneratedbypartiallineagesortingandhaplotypic in U. u. eversmanni from the southern Altai Mountains in
divergencefollowingexpansionintomorefavorableareasand Russia, and suggested that those populations may also have
cessationofpopulationconnectivity. been isolated in lowland glacial refugia. Our data strongly
Zhang et al. (2008) found a similar pattern of reduced support their hypothesis that one of these refugia was in the
haplotypeandnucleotidediversityingreentoads(Bufoviridis) Great Lakes Depression. However, we cannot completely
inhabitingeasternCentralAsia.Theirdatasupportahistoryof rule out refugia elsewhere in northern Mongolia, such as
refugial isolation in montane regions of northwest China and in Khövsgöl Aimag, a region proximate to the Great Lakes
eastern Kazakhstan followed by rapid postglacial expansion Depression, but from which we were only able to sample six
intosurroundingbasins. Liaoetal. (2016)describedasimilar individualsfromarelativelysmallarea.
pattern in the jerboa Allactaga sibirica in China. Conversely, Currently, Pleistocene paleoenvironments of western
370 www.zoores.ac.cn
Mongolia are somewhat poorly constrained, preventing more withoutwhoseeffortsthisstudywouldnothavebeenpossible.
precise and detailed links between small mammal historical
biogeography and past environmental change. Grunert et al. REFERENCES
(2000) proposed a lowstand (i.e., lowered lake levels due to
localorregionalenvironmentalchange)forbothUvsNuurand
BandeltHJ,ForsterP,RöhlA.1999.Median-joiningnetworksforinferring
Bayan Nuur during the Last Glacial Maximum (LGM), which
intraspecificphylogenies.MolecularBiologyandEvolution,16(1):37–48.
would have led to exposure of even more extensive areas in
BatsaikhanN,SamiyaR,SharS,LkhagvasurenD,KingSRB.2014.AField
the Great Lakes Depression than are available today. While
these lake lowstands are somewhat counterintuitive given Guide to the Mammals of Mongolia. 2nd ed. London: Zoological Society
the relatively mesic conditions inferred for other mid-latitude ofLondon.
regionsduringtheLGM,asimilarpatternofglaciallowstands BöhnerJ,LehmkuhlF.2005.EnvironmentalchangemodellingforCentral
hasbeendescribedfromlakesinnearbynorthwesternChina and High Asia: pleistocene, present and future scenarios. Boreas, 34(2):
(Fang, 1991). Conversely, the southern Altai Mountains in 220–231.
Russia experienced formation of large glacial lakes during DunnumJL,CookJA.2012.Gerritsmithmiller:hisinfluenceontheenduring
the late Pleistocene (e.g., Rudoy, 2002). Understanding the
legacyofnaturalhistorycollections.Mammalia,76(4):365–373.
extenttowhichtheseidiosyncraticlandscape-levelresponses
EdgarRC.2004.MUSCLE:multiplesequencealignmentwithhighaccuracy
interacted with regional-scale environmental variability to
andhighthroughput.NucleicAcidsResearch,32(5):1792–1797.
impact the distribution and demography of U. undulatus will
Ermakov OA, Simonov E, Surin VL, Titov SV, Brandler OV, Ivanova NV,
require linking all currently available sequence datasets with
BorisenkoAV.2015.Implicationsofhybridization,NUMTs,andoverlooked
newgeneticdatainarange-widephylogeographicframework.
diversityforDNAbarcodingofEurasiangroundsquirrels.PLoSOne,10(1):
CONCLUSION e0117201.
Fang,J.1991.Lakeevolutionduringthepast30,000yearsinChina,andits
We analyzed the phylogeography of the long tailed ground
squirrel (Urocitellus undulatus) across the southern core implicationsforenvironmentalchange.QuaternaryResearch,36:37–60.
of its large central Asian range. Phylogenetic and FolmerO,BlackM,HoehW,LutzR,VrijenhoekR.1994.DNAprimersfor
population genetic inferences based on mtDNA strongly amplificationofmitochondrialcytochromecoxidasesubunitIfromdiverse
support the presence of two major lineages in Mongolia metazoaninvertebrates.MolecularMarineBiologyandBiotechnology,3(5):
(U. u. undulatus and U. u. eversmanni). Within the 294–299.
more widespread U. u. eversmanni, we identified statistically Grunert J, Lehmkuhl F, Walther M. 2000. Paleoclimatic evolution of the
significant but extremely shallow phylogeographic structure, Uvs Nuur basin and adjacent areas (Western Mongolia). Quaternary
with modern genetic clusters associated with Mongolian
International,65–66:171–192.
mountain systems (Khangai Mountains, Mongolian and Govi
GürH.2013.TheeffectsoftheLateQuaternaryglacial–interglacialcycles
Altai). Together, our analyses support a late Pleistocene
onAnatoliangroundsquirrels: rangeexpansionduringtheglacialperiods?
historyofextensivepopulationadmixtureinU.u.eversmanni,
BiologicalJournaloftheLinneanSociety,109(1):19–32.
possibly across the Great Lakes Depression and contiguous
HelgenKM,ColeFR,HelgenLE,WilsonDE.2009.Genericrevisioninthe
lowlands of north-west Mongolia, followed by geologically
recent diversification in postglacial isolation. In addition to HolarcticgroundsquirrelgenusSpermophilus.JournalofMammalogy,90(2):
providingnewgeographiccontextforU.undulatussystematics 270–305.
and phylogeography, our study establishes hypotheses of Hijmans RJ, van Etten J, Cheng J, Mattiuzzi M, Sumner M, Greenberg
distributionalanddemographicresponsetopastenvironmental JA, Lamigueiro OP, Bevan A, Racine EB, Shortridge A, Ghosh A.
changeinmesic-adaptedcentralAsianmammalspecieswhich 2017(2017-11-13).Raster: geographicdataanalysisandmodelingVersion
maybetestedusingrobust,genomic-scaledatasets. 2.6-7.https://cran.r-project.org/web/packages/raster/.
Kamvar, ZN, Tabima JF, Grünwald NJ. 2014. Poppr: an R package for
COMPETINGINTERESTS
genetic analysis of populations with clonal, partially clonal, and/or sexual
Theauthorsdeclarethattheyhavenocompetinginterests. reproduction.PeerJ,2:e281.
KryštufekB,VohralíkV.2013.TaxonomicrevisionofthePalaearcticrodents
AUTHORS’CONTRIBUTIONS
(Rodentia).Part2.Sciuridae:Urocitellus,MarmotaandSciurotamias.Lynx,
B.S.M. designed the study, extracted genomic DNA, sequenced 44:27–138.
mitochondrial genes of interest, and performed all statistical analyses. Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. 2017.
B.S.M.,B.N.,A.T.,andJ.A.C.wroteandrevisedthemanuscript.Allauthors PartitionFinder2:newmethodsforselectingpartitionedmodelsofevolution
readandapprovedthefinalmanuscript. formolecularandmorphologicalphylogeneticanalyses.MolecularBiology
andEvolution,34(3):772–773.
ACKNOWLEDGEMENTS
LehmkuhlF.1998.QuaternaryglaciationsincentralandwesternMongolia.
Wethankthemanyresearchers, students, andassistantsinvolvedinour QuaternaryProceedings,6:153–167.
logistically challenging Mongolian fieldwork over the past two decades, Lehmkuhl F, Lang A. 2001. Geomorphological investigations and
ZoologicalResearch39(5):364–372,2018 371
luminescencedatinginthesouthernpartoftheKhangayandtheValleyof Version2.2.4.https://cran.r-project.org/web/packages/PopGenome/.
theGobiLakes(CentralMongolia).JournalofQuaternaryScience, 16(1): RambautA,SuchardMA,XieW,DrummondAJ.2014.Tracer.Version1.6.0.
69–87. http://beast.community/tracer.
Liao J, Jing D, Luo G, Wang Y, Zhao L, and Liu N. 2016. Comparative RobinsonJW,HoffmannRS.1975.Geographicalandinterspecificcranial
phylogeography of Meriones meridianus, Dipus sagitta, and Allactaga variationinbig-earedgroundsquirrels(Spermophilus): amultivariatestudy.
sibirica: PotentialindicatorsoftheimpactoftheQinghai-TibetanPlateau SystematicZoology,24(1):79–88.
uplift.MammalianBiology,81:31–39. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic
LinetskayaON,LinetskiiAI.1989.Speciesdifferencesinthecraniometric inferenceundermixedmodels.Bioinformatics,19(12):1572–1574.
variation among ground squirrels of the Eastern Palearctic (subgenus Rudoy AN. 2002. Glacier-dammed lakes and geological work of glacial
Urocitellus). In: Sovremennye Podkhody K Izucheniyu Izmenchivosti superfloods in the late Pleistocene, southern Siberia, Altai mountains.
(Modern Approaches to the Study of Variability). Vladivostok: QuaternaryInternational,87(1):119–140.
Dal’nevostochnoeOtdelenie,AkademiyaNaukSSSR,99–105. Sikes RS, Gannon WL, the Animal Care and Use Committee of the
McLeanBS,BellKC,DunnumJL,AbrahamsonB,ColellaJP,DeardorffER, American Society of Mammalogists. 2011. Guidelines of the American
WeberJA,JonesAK,Salazar-MirallesF,CookJA.2016a.Naturalhistory SocietyofMammalogistsfortheuseofwildmammalsinresearch.Journal
collections-basedresearch: progress,promise,andbestpractices.Journal ofMammalogy,92(1):235–253.
ofMammalogy,97(1):287–297. SimonovEP,DvilisAE,LopatinaNV,LitvinovYN,MoskvitinaNS,Ermakov
McLeanBS,JacksonDJ,CookJA.2016b.Rapiddivergenceandgeneflow OA. 2017. The influence of late Pleistocene mountain glaciations on the
athighlatitudesshapethehistoryofHolarcticgroundsquirrels(Urocitellus). geneticdifferentiationoflong-tailedgroundsquirrel(Urocitellusundulatus).
MolecularPhylogeneticsandEvolution,102:174–188. RussianJournalofGenetics,53(5):614–622.
Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Smith MF, Patton JL. 1993. The diversification of South American murid
Gatewayforinferenceoflargephylogenetictrees.In: Proceedingsofthe rodents:evidencefrommitochondrialDNAsequencedatafortheakodontine
2010GatewayComputingEnvironmentsWorkshop(GCE).NewOrleans,LA, tribe.BiologicalJournaloftheLinneanSociety,50(3):149–177.
USA:IEEE,1–8. TinninDS,DunnumJL,Salazar-BravoJ,BatsaikhanN,BurtMS,Gardner
NingSL,ZhouLZ,ZhangBW,ZhaoTB,ZouGY.2007.Phylogeographic SL, Yates TL. 2002. Contributions to the Mammalogy of Mongolia, with
patterns of the great gerbil Rhombomys opimus in China based on achecklistofspeciesforthecountry.Albuquerque, NM:TheMuseumof
mitochondrial cytochrome b gene sequence variations. Acta Zoologica SouthwesternBiology.
Sinica,53(4):630–640.(inChinese) Tsvirka MV, Spiridonova LN, Korablev VP. 2008. Molecular genetic
Ognev SI. 1947. Zveri SSSR I Prilezhashchikh Stran. Gryzuny relationships among East Palaerctic ground squirrels of the genus
(Prodolzhenie). (Zveri Vostochnoi Evropy I Severnoi Azii) (Mammals of Spermophilus (Sciuridae, Rodentia). Russian Journal of Genetics, 44(8):
the USSR and Adjacent Countries: Rodents (continued). (Mammals of 966–974.
Eastern Europe and Northern Azii)). 5th ed. Moscow: Akademiya Nauk Vorontsov NN, Frisman LV, Lyapunova EA, Mezhova ON, Serdyuk VA,
SSSR. Fomicheva II. 1980. The effect of isolation on the morphological and
Oksanen J, Blanchet FG, Friendly, M, Kindt R, Legendre P, McGlinn D, geneticaldivergenceofpopulations.Genetica,52(1):339–359.
Minchin PR, O’Hara RB, Simpson GL, Solymos P, Henry M, Stevens Wang YZ, Fu JZ. 2004. Cladogenesis and vicariance patterns in the
H, Szoecs E, Wagner H. 2017(2017-12-01). Vegan: community ecology toad-headed lizard Phrynocephalus versicolor species complex. Copeia,
packageVersion2.4-5.https://cran.r-project.org/web/packages/vegan/ 2004(2):199–206.
Paradis E, Bolker B, Claude J, Cuong HS, Desper R, Durand B, Dutheil Wilson DE, Reeder DM. 2005. Mammal Species of the World. 3rd ed.
J,GascuelO,HeiblC,LawsonD,LefortV,LegendreP,LemonJ,NoelY, Baltimore,Maryland:JohnsHopkinsUniversityPress.
NylanderJ,Opgen-RheinR,PopescuAA,SchliepK,StrimmerK,deVienne Yates TL, Jones C, Cook JA. 1996. Preservation of voucher specimens.
D.2017a.Package“ape.”Version4.0.http://ape-package.ird.fr/. In: Wilson DE, Cole FR, Nichols JD, Rudran R, Foster MS. Measuring
Paradis E, Jombart T, Schliep K, Potts A, Winter D. 2017b(2017-05-03). and Monitoring Biological Diversity: Standard Methods for Mammals.
Pegas: populationandevolutionarygeneticsanalysissystemVersion0.10. Washington,DC:SmithsonianInstitutionPress,265–274.
http://ape-package.ird.fr/pegas.html Zhang YJ, Stöck M, Zhang P, Wang XL, Zhou H, Qu LH. 2008.
PavlinovIY,LissovskyAA.2012.TheMammalsofRussia:ATaxonomicand Phylogeography of a widespread terrestrial vertebrate in a barely-studied
GeographicReference.Moscow:KMKScientificPress. Palearcticregion:greentoads(Bufoviridissubgroup)indicateglacialrefugia
Pfeifer B, Wittelsbuerger U, Li H, Handsaker B. 2017(2017-07-04). inEasternCentralAsia.Genetica,134(3):353–365.
PopGenome:anefficientSwissarmyknifeforpopulationgenomicanalyses
372 www.zoores.ac.cn