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Molecular Phylogenetics and Evolution 44 (2007) 942–952

Molecular phylogeography of endangered sharp-snouted pitviper(Deinagkistrodon acutus; Reptilia, Viperidae) in Mainland China

Song Huang a,b,c, Shunping He b,*, Zuogang Peng b, Kai Zhao b, Ermi Zhao a,*

a School of Life Sciences, Sichuan University, Chengdu 610064, Chinab Laboratory of Fish Phylogenetics and Biogeography, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

c Department of Biology, Huangshan University, Huangshan 245021, China

Received 6 August 2006; revised 3 April 2007; accepted 23 May 2007Available online 9 June 2007

Abstract

Using phylogenetic and population genetic approaches, the present study reports the phylogeographic structure of the sharp-snoutedpitviper (Deinagkistrodon acutus), a threatened snake species with commercial and medicinal importance in China. The entire mitochon-drial ND2 gene (NADH dehydrogenase subunit 2) sequences of 86 individuals of D. acutus from 14 localities across its range in Chinawere determined. Based on the results of phylogenetic analyses, distribution of diagnostic sites, haplotype network, and AMOVA hier-archical analysis, an east–west division of the whole D. acutus population could be observed. Geographically, a line formed by a lake,river, and mountain chain (the Poyang Lake, Gan River to the southern end of the Wuyi Mountains), results in vicariance and approx-imately vertically splits the range into two and the whole population into two main lineages (western and eastern). The bifurcating treesuggested generally west to east dispersal trend. The data fit the isolation by distance (IBD) model well. Star-like clusters in haplotypenetwork, significantly negative values of Fs statistics, and unimodal mismatch distributions all suggest recent demographic expansions infour areas. The results show that isolation, dispersal, bottleneck, and expansion jointly constitute the history of D. acutus. In a haplotypenetwork, the excessive predominance of central haplotypes, few medium-frequency haplotypes, predominance (73.1%) of the singletonsamong the derived haplotypes, most of which are connected to the central haplotype by only one mutational step, unsymmetrical cam-panulate unimodal curve of mismatch distributions and leftwards shift of the peaks, all suggest that the whole D. acutus population is ayoung population with low genetic diversity. Based on the data, the first priority for conservation action should be given to the Huang-shan unit.� 2007 Elsevier Inc. All rights reserved.

Keywords: Phylogeography; Deinagkistrodon acutus; Reptilia; ND2; Isolation; Dispersal; Expansion; Conservation

1. Introduction

Studies on molecular phylogeography and conservationgenetics have increased rapidly during the last decade. Theinformation derived from molecular data of these studies,undetectable through traditional biogeography anddemography, is essential to examining population dynam-ics and understanding evolutionary processes (Avise,

1055-7903/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2007.05.019

* Corresponding authors. Fax: +86 27 68780071 (S. He), +86 2885222753 (E. Zhao).

E-mail addresses: clad@ihb.ac.cn (S. He), zhaoermi@cib.ac.cn(E. Zhao).

1994; Prior et al., 1997), and could provide the foundationfor formulating and implementing any conservation andmanagement plan (e.g., Frankham et al., 2002; Hrbeket al., 2005; Ma et al., 2006). In Mainland China, few suchstudies have been reported, and most of them involveendotherm species, fishes, and amphibians (e.g., Maceyet al., 1998b; Murphy et al., 2000; Su et al., 2001; Panget al., 2003; Perdices et al., 2004, 2005; Fu et al., 2005;Li et al., 2005; Zhao et al., 2005). Few such studies involveterrestrial poikilotherms with narrow habitat requirementsand limited dispersal potentials such as venomous snakes;this is partly also due to their secretive habits, difficultyin obtaining representative samples (described by Ursenb-

S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952 943

acher et al., 2006), and their toxicity to humans. Despitethis, the above-mentioned ecological traits of snakes arelikely to make them good models for phylogeographicalstudies. Creer et al. (2001) presented a clear phylogeo-graphical pattern of the bamboo viper within Taiwan,which indicated the presence of two geographically over-lapping clades, and supports the existence of at least onecolonization event from the Mainland in the Pliocene.However, the present study may be the first report regard-ing phylogeography of reptiles in Mainland China.

The sharp-snouted pitviper, Deinagkistrodon acutus

(Gunther, 1888), belonging to the monotypic genus Dei-

nagkistrodon, occurs in 10 provinces of southern and wes-tern China from Zhejiang and Fujian in the west toGuizhou and Chongqing in the east, as well as in Taiwan(Zhao and Adler, 1993), and southwestwards to Laos andnorthern Vietnam (Gumprecht et al., 2004). More than90% of the populations of this species occur in China(http://www.chinabiodiversity.com/redlist). In spite of itscommercial utilization, more precise information regardingits natural distribution in the area north of the Chang Jiang(Yangtze River) is lacking. The sharp-snouted pitviper isone of the main snake species in China with commercialand medicinal importance; it can grow up to 2 m in lengthand can weigh over 5 kg. However, it is difficult to findindividuals over 2.5 kg in weight in the field at the presenttime. It is an oviparous, terrestrial venomous snake, mov-ing slowly, inhabiting lower montane forests (under1200 m), and breeding once a year from the age of fouryears (Huang and Huang, 2003).

Many wildlife species have experienced populationdeclines as a result of commercial over-exploitation and hab-itat destruction (e.g., Pang et al., 2003; Hrbek et al., 2005),and D. acutus likewise. In 2004, D. acutus was listed in ChinaSpecies Red List as a vulnerable endangered species (VUA2a) because there has been not less than 30% populationreduction during the last decade, and the reduction is stillcontinuing (http://www.chinabiodiversity.com). Chineseophiologists recognized the severe threatened plight of thisspecies, and launched artificial breeding efforts in the1980s, and have successfully reproduced a second generationin captive conditions (Huang and Huang, 2003).

The present study involves a complete ND2 genesequence-based phylogeographic study across the wholerange of D. acutus in Mainland China. Our aims are toexamine the population genetic structure of D. actus, pres-ent a hypothesis of population dynamics explaining thecurrent distribution, and further develop effective conserva-tion recommendations for this species. The reason that weselected the ND2 gene was based on the finding reported byAshton and de Queiroz (2001) that the ND2 gene is prefer-able for examining intraspecific relationships in snakes.

2. Materials and methods

A dense and relevant geographical sampling wasdesigned to include individuals from most of the distribu-

tion range of the sharp-snouted pitviper, in order to com-prehensively represent the inter- and intra-populationvariation of the species (Verheyen et al., 2003). A total of86 specimens of D. acutus were collected from 14 samplinglocalities in 9 provinces of Mainland China (Fig. 1 andTable 1). Samples were obtained either by ourselves fromwild-caught snakes (40 D. acutus and 1 Gloydius brevicau-

dus, including all samples from the Huangshan unit, seebelow), or by purchasing them from native mountaineersat country fairs, who could supply information about thelocation of the snakes. However, since time constraints pre-cluded each site being visited by the authors to record posi-tional and habitat information, we used the commonlatitude and longitude of the county town for all specimensused from that county. The diameter of each locality there-fore spans approximately 100 km. Seventy-two specimensof D. acutus (specimen numbers: SCUM045401-045472)and 1 G. brevicaudus (SCUM045473) were deposited inthe Sichuan University Museum (SCUM). Fourteen speci-mens of D. acutus were deposited in Huangshan institute ofsnakes (specimen numbers: HSIOS04101-04114). For easeof description and discussion, sampling localities were arti-ficially grouped into five geographic units according to thegeographic distances and geographic similarity (see Fig. 1and Table 1): Westmost unit (WMU, including samplingsites located at eastern edge of Yunnan-Guizhou Plateau);Central unit (CU, including most area of Hunan andJiangxi Pro.); Wuyishan unit (WYU, located in WuyiMts.); Zhejiang unit (ZJU, Zhejiang Pro.); and Huangshanunit (HSU, including Qimen, Dongzhi, Taipin Co., AnhuiPro.).

The fresh liver tissues of all D. acutus individuals and aG. brevicaudus (as one of two outgroups) were removedand immediately preserved in 95% ethanol for sequencingtheir mitochondrial complete ND2 gene. The completesequence of ND2 gene of Crotalus enyo (1032 bp) retrievedfrom GenBank (AY016246, Ashton and de Queiroz, 2001)was selected as another outgroup. Total genomic DNA wasextracted from livers according to the phenol/chloroformextraction procedure (Sambrook et al., 1989). The ND2gene region was amplified from the total DNA extractsusing the polymerase chain reaction (PCR) with one primerpair. The forward primer was L4437c (50-CAG CTA AATAAG CTA TCG GGC CCA TAC C-30) according toKumazawa et al. (1996) with slight modification, and thereverse primer was tRNA-trpR’ (50-GGC TTT GAAGGC TYC TAG TTT-30) according to Ashton and deQueiroz (2001) with slight modification. The PCR con-tained approximately 100 ng of template DNA, 1 lL ofeach primer, 5 lL of 10· reaction buffer, 2 lL dNTPs (each2.5 mM), and 2.0 U of Taq DNA polymerase in total 50 lLvolume. The reactions were cycled at the following temper-atures: an initial 94 �C denaturation for 3 min, 30 cycles of94 �C denaturation for 1 min, 58–64 �C annealing for1 min, 72 �C extension for 1 min, and a final 72 �C exten-sion for 5 min. Amplified DNA was fractionated by elec-trophoresis through 0.8% low-melting agarose gels,

Fig. 1. Map of the sampling localities for this study, showing the five geographic units grouped according to the geographic distances and geographicsimilarity. The abbreviation and coordinates of localities are listed in Table 1. The bold bar with interspersed dots roughly represents the division line fromthe Poyang Lake, Gan River to the southern end of the Wuyi Mts.

944 S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952

recovered from the gels, and purified using BioStar Glass-milk DNA purification Kit according to manufacturer’sinstructions. Purified DNA was sequenced with the Per-kin-Elmer BigDye DNA Sequencing Kit according to themanufacturer’s protocol with the primers used in PCR.

Eighty-six entire newly determined ND2 sequences ofD. acutus (homologous protein-coding genes) with twosequences from G. brevicaudus (newly determined) andC. enyo (retrieved from GenBank) were aligned using Clus-tal X (Thompson et al., 1997), and translated into aminoacid sequences to check for the presence of stop codonswithin the sequences which might indicate the amplificationof pseudogenes.

Phylogenetic and population genetic methods wereapplied to investigate the patterns of phylogeographicalvariation of D. acutus. Phylogenetic analysis of all ND2sequences including two outgroups was performed toreconstruct bifurcating trees using neighbor-joining (NJ)and maximum-parsimony (MP) approaches using PAUP*

v4b10 (Swofford, 2002). An unrooted tree of haplotypeswas reconstructed using Bayesian inference (BI) usingMrBayes v3.0 (Huelsenbeck and Ronquist, 2001). Model-test 3.7 (Posada and Crandall, 1998) was used to find themodel of nucleotide evolution with AIC criterion for NJand BIC criterion for BI. To assess statistical support for

hypothesized clades, bootstrap analysis was performedwith 1000 replicates for the NJ (under the TrN + G modelof evolution, �lnL = 3003.8127) and MP analyses. In theBayesian analysis, posterior distributions were obtainedby Markov chain Monte Carlo (MCMC) analysis withone cold chain and three heated chains. The maximum like-lihood model employed six substitution types (Nst = 6)with a proportion of sites being invariant (rates = pro-pinv). Samples of trees and parameters were drawn every100 steps from a total of 1,000,000 MCMC generations.The first 100,000 generations (1000 trees) were discardedas burn in (chain had not become stationary) and onlythe results from the last 900,000 generations (9000 trees)were used to compute a consensus tree. We ran three addi-tional analyses starting with random trees. Consensus of allof the post-burn in generations (3,600,000 generationsresulting in 36,000 trees) was computed from all four runs.

The haplotype network (statistical parsimony) was gen-erated with the program TCS version 1.21 (Clement et al.,2000) in order to further investigate the more specificgenetic variation in various populations. In the event thatthe network revealed a star-like phylogeny indicative of apopulation bottleneck followed by expansion (Slatkinand Hudson, 1991; Rogers and Harpending, 1992; Vinaset al., 2004), mismatch distributions and Fu’s Fs test were

Tab

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S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952 945

subsequently carried out using Arlequin ver. 2000 (Excof-fier et al., 2000) on the ‘‘star’’ with adequate sample sizeto evaluate their historical demography. Unimodal mis-match distributions and negative values of Fs are expected,and would be interpreted as recent range expansions fol-lowing recovery from a bottleneck (Slatkin and Hudson,1991; Rogers and Harpending, 1992; Fu, 1997).

Mantel test (1000 permutations) was performed usingArlequin ver. 2000 to test for the presence of isolation bydistance (IBD; Wright, 1943) by calculating the correlationcoefficient between two pairwise matrices of geographicaldistances and genetic differentiation. Geographic distances(great circle distance) between sampling sites were deter-mined from longitudinal and latitudinal coordinates.

To assess the most probable population configurationand geographical subdivision, the hierarchical analysis ofAMOVA (analysis of molecular variance) was performedusing Arlequin ver. 2000. According to different geograph-ical hierarchies, all local populations were, respectively,grouped into 2, 3, 4, and 5 groups. The arrangement withthe highest value of among group variation (hCT), respec-tively, in each grouping scheme was inferred as being themost probable geographical subdivision.

Any expansion events might be affiliated with some pastgeological events and climatological changes. Dating suchexpansions has always attracted phylogeographers, eventhough molecular clocks vary in rate to some extent(Thorpe et al., 2005). The likelihood ratio test (LRT, Huel-senbeck and Crandall, 1997) was performed by comparingthe likelihood ratio test statistic (LR = 2[lnL1 � lnL2]) todetermine whether more complex models provided a signif-icantly better fit to the data than more simple models. Fol-lowing a test of v2 distribution with n � 2 degrees offreedom (where n is the number of sequences), this testswhether the sequences of the present study fit the molecularclock hypothesis (i.e., equal rates across all sequences). Theequation s = 2ut (Nei and Tajima, 1981; Rogers and Har-pending, 1992) was used to estimate the approximateexpansion time in generations (t) for D. acutus populations,where s is the date of the growth or decline measured inunits of mutational time and u is the mutation rate persequence and per generation. The value of u was calculatedfrom u = 2lk, where l is the mutation rate per nucleotideand k is the number of nucleotides of the analyzed frag-ment. Finally, the approximate time of expansion in yearscould be calculated by multiplying t by the generation time(4 years; Huang and Huang, 2003) of D. acutus. No fossilrecords or biogeographical events isolating distinct popula-tions of D. acutus are available to calibrate the substitutionrate of this species. The substitution rate of mtDNAsequences, mainly including the ND2 gene, had been cali-brated in studies of lizards (Macey et al., 1998a) and othervertebrates (Macey et al., 1998b), as approximately 0.65%per million years. This rate was used in the present study.

Information about the genetic diversity of threatenedspecies is a prerequisite to designing suitable strategiesfor genetic conservation and establishing effective and effi-

946 S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952

cient conservation practices (Falk and Holsinger, 1991). Inthe present study, the nucleotide diversity (p), the averagenumber of nucleotide differences per site (Nei, 1987) wascalculated in Arlequin ver. 2000 to explore the geneticdiversity within different geographic units of D. acutus, inorder to assess the implications for future conservationand management programs.

3. Results

The entire ND2 gene sequences, 1032 bp (i.e., 344codons) in length, from 86 ingroup individuals ofD. acutus, and 1026 bp (i.e., 342 codons) from one out-group individual of G. brevicaudus (GenBank AccessionNo. DQ839399) were obtained, both including the stopcodon TAG located at the 30 terminal end. No stop codonwas found in the inside of all ND2 sequences, and no indelwas found in ingroup sequences. The alignment includingin- and out-group performed in Clustal X revealed thatcodon deletions occurring in G. brevicaudus ND2 sequencewere found at nucleotide positions 664–666 and 676–678,corresponding to codon positions 222 and 226. Nucleotidecompositions of complete ND2 gene of D. acutus, G. brev-icaudus, and C. enyo were, respectively: C = 34.2%, 32.8%,and 34.4%; T = 21.9%, 23.2%, 21.9.0%; A = 34.3%, 33.7%,and 33.8%; and G = 9.6%, 10.3%, and 9.9%. Within totalND2 data set from D. acutus, 51 sites were variable(4.9%) including 38 (3.7%) that were parsimony informa-tive, Ti/Tv ratio = 6.7.

A total of 32 haplotypes (from haplotype 1 to 32 corre-spond to GenBank Accession Nos. from DQ836185 toDQ836216) defined from all ingroup individuals were listedin Table 1 to show their distribution in subpopulations.Only 3 haplotypes (9.4%), i.e., haplotype 6, 14, and 24,were shared by individuals from two adjacent units, theothers were all restricted either to the same sampling local-ity (75%) or to the same unit (15.6%). This genetic rangerestriction was further revealed in the result of the Manteltest. A Mantel test of association of geographical distancesand genetic differentiation showed that D. acutus fits theisolation by distance (IBD) model well (r = 0.57,P < 0.01). Both results indicate that local populations ofD. acutus have evolved relatively independently.

The NJ and MP analyses produced an identical topol-ogy which suggested the monophyly of the whole popula-tion of D. acutus with 100% support, and symmetricallyassigned all the ingroup sequences into two main phyloge-netic clades (i.e., main lineages) (Fig. 2a). Identical topol-ogy was also revealed in the pattern of unrooted treefrom Baysian inference on haplotypes (Fig. 2b), as wellas in the map of diagnostic sites (Fig. 3), haplotype net-work (Fig. 4), and the result of AMOVA hierarchical anal-ysis. From these results, an east–west division of D. acutus

population with strong geographic configuration could beobserved. Geographically, the two main clades are dividedby a division line, i.e., a chain consisting of lake, river andmountains (the Poyang Lake, Gan River, and the Wuyi

Mts., see Fig. 1). Main clade W (western lineage) was com-posed of all individuals from western area of the line, aswell as including 8 individuals from eastern area of the line.For the main clade E (eastern lineage), in spite of one indi-vidual (YZ3, from western area of the line) forming basalclade with very low support (just more than 50), all the restwere confined to eastern area of the line (including all indi-viduals from Wuyi Mts., see Fig. 1). The main clade W wassplit into two groups: subclade W1 and W2, the formercomprised all individuals from Westmost unit and twofrom the Central unit at the base; the latter grouped 9 indi-viduals from the Central unit and 8 individuals fromHuangshan unit (Fig. 2a). Within the main clade E, subc-lade E1 comprised individuals from Huangshan, Zhejiang,and Wuyishan unit; however, subclade E2 consisted ofindividuals from Wuyishan unit only. The tight topologicalstructure of the bifurcating tree (Fig. 2a) suggested rela-tively low differentiation among sequences within subc-lades. However, it is noteworthy that two sequences (YZ3and ZX7, i.e., haplotype 25 and 20) fall outside the tightclusters of sequences (especially see Fig. 2b) and are locatedat a basal position within main clade E (Fig. 2a). Thesetherefore seem comparatively ancient. These two oldsequences also were singletons (only found in one individ-ual, see Fig. 2b and Table 1).

The east–west division of D. acutus population was alsorevealed in the AMOVA hierarchical analysis. In each ofthe grouping schemes dividing the whole D. acutus popula-tion into 2, 3, 4, or 5 groups, among all possible arrange-ments, the arrangement that produced the highest valueof hCT (among group variation) is shown in Table 2. Ofall rearrangements, the grouping scheme of two groupsdivided by the above-mentioned division line (PoyangLake, Gan River, to the southern end of the Wuyi Mts.)maximized the value of hCT (0.682). This suggested thatthe most probable (i.e., natural) geographic differentiationof the whole D. acutus population was tightly correlatedwith this division line, which is consistent with the resultssuch as the phylogenetic analysis in the present study.Furthermore, even the rearrangements with 3, 4, and 5groups all revealed the highest value of hCT when splitalong same geographical division. Any additional attemptsat grouping sequences from both sides of the line togetherall resulted in the sharp falls in the values of hCT (resultsnot shown). In the 5-groups scheme, the arrangement pro-ducing the highest values of hCT was identical with theaforementioned five geographic units grouped artificiallyaccording to geographic distance and similarity (see Table2 and Fig. 1), suggesting the rationality of this artificialcompartmentalization.

The map of variable sites (Fig. 3) showed a clear clusterrelationship of haplotypes, consistent with the topology ofthe bifurcating tree (Fig. 2). Within site 714–963 in ND2gene, seven variable sites were found to be diagnostic,i.e., they could definitely distinguish sequences belongingto western or eastern lineages. If we try to group thesequence of haplotype 25 (i.e., YZ3, forming a basal

Fig. 2. (a) Phylogenetic relationships of all individuals based on the entire ND2 gene sequences derived from NJ (TrN + G model) and MP analyses. Thetopology of NJ tree illustrated here is identical to that of MP tree. Numbers above and below branches are NJ and MP bootstraps values, respectively,only showing those higher than 50%. (b) Unrooted 50% majority-rule BI consensus tree of haplotypes. Numbers of haplotypes (1–32) correspond to Table1. Posterior probability values are shown at the corresponding nodes.

S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952 947

subclade in main clade E with very low support) into mainclade W we could also find six diagnostic sites which werelocated at the middle of the sequence of ND2 from site 484to 687. This suggested that this old sequence was an inter-mediate type, presumably occurring at the early period ofdifferentiation of eastern and western lineages. In addition,we also found five diagnostic sites which can locate asequence in a subclade or a localized area. The subclade

W1 could be distinguished from the others by site 600.The site 29, 193, and 453 could be used to distinguish thesubclade E1 from the others. The ‘‘A’’ at site 567 onlyoccurred in the haplotypes from Wuyi Mts.

The haplotype network showed a fairly star-like cluster-ing pattern (Fig. 4). Within the four ‘‘stars’’, there werefewer unobserved haplotypes than observed haplotypes,demonstrating a good degree of geographic coverage in

Fig. 3. Map of variable sites in ND2 gene of D. acutus, show the diagnostic sites. Numbers of haplotypes correspond to Table 1. Long grey bars show thediagnostic sites distinguishing two main clades. Short grey bars show those can distinguish the sequences from subclades or local range from the others.The sites pointed by arrows below the figure would be diagnostic sites, if haplotype 25 is grouped in main clade W.

948 S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952

the sampling in the present study. The four ‘‘stars’’ corre-sponded to the four phylogenetic mtDNA subclades. Theircentral haplotypes predominated, while the majority of theother haplotypes were connected by, and therefore derivedfrom, them. For the ‘‘star’’ cluster W1, W2, and E2, thecentral haplotypes excessively predominated. First, thenumbers of individuals sharing the central haplotypes wereeither considerably more than (W1: 23 > 8) or almost equalto (W2 and E2) the numbers of individuals sharing theirderived haplotypes. Second, few medium-frequency haplo-types could be found in their star-like phylogeny. Third,most of the derived haplotypes (73.1%) were singletonsconnected to the central haplotye by only one mutationalstep. Those observations should suggest a populationbottleneck followed by sudden expansion. Having anadvantage over the bifurcating tree in detail at the intraspe-cific level, the haplotype network could intuitively reflectthe genetically greater distances between the two oldsingletons and the central haplotypes (8 mutational steps,see Fig. 4).

Unimodal mismatch distributions were observed in allof the four star-like clusters (Fig. 5). The higher frequenciescorresponding with the smaller pairwise differences resultedin leftwards shifts of the peaks, especially in W1. Signifi-cant negative values for Fu’s Fs were tested in all of them(Fig. 5). Both showed a significant signal of populationexpansion for all of the four ‘‘stars’’, i.e., sublineages.The molecular clock hypothesis was not rejected by theLRT test (LR = 93.32, df = 86, P = 0.28). Using the evolu-tionary rate of approximately 0.65% per million years here,the four expansion times were estimated as about 166,000–252,000 years before the present (yr. B.P.), which are listedin Fig. 5.

4. Discussion

4.1. Dispersal and origin

Phylogenetic branching patterns can suggest dispersaldirection and the location of origin of a population. These

Fig. 4. Statistical parsimony network showing genetic relationships and distance among 32 haplotypes of D. acutus in this study. Numbers of haplotypescorrespond to Table 1. Hypothetically intermediate haplotypes unobserved are shown as black dots. Each mutation step is shown as a either very short orlonger line connecting neighboring haplotypes (including observed and unobserved one). The sizes of circles roughly represent the numbers ofindividual(s), the scale is given below the figure.

Table 2Hierarchical analysis of AMOVA (analysis of molecular variance) of D. acutus

Groups hST hSC hCT % Amonggroups

% Withinpopulations

P

All populations 0.815 — — 81.49 18.51 <0.0012 Groups: <YH,TP,QM,DZ,GZ,ZX>, <JA,AF,YZ,GL,LS,LF,JK,PS> 0.872 0.597 0.682 68.18 12.81 <0.0013 Groups: <YH,TP,QM,DZ,GZ,ZX>, <JA,AF,YZ>, <GL,LS,LF,JK,PS> 0.857 0.575 0.662 66.20 14.35 <0.0014 Groups: <TP,QM,DZ>, <YH,GZ,ZX>, <JA,AF,YZ>, <GL,LS,LF,JK,PS> 0.840 0.509 0.674 67.35 16.03 <0.0015 Groups: <YH>, <TP,QM,DZ>, <GZ,ZX>, <JA,AF,YZ>, <GL,LS,LF,JK,PS> 0.836 0.514 0.662 66.19 16.44 <0.001

Fig. 5. Unimodal mismatch distributions for the four sublineages of D. acutus with a star-like phylogeny in the haplotype network (Fig. 4). Open circles,simulated data; full circles, observed data. The values of Fu’s Fs with P values, Tau, and expansion time are listed below each graphic.

S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952 949

have been demonstrated successfully by some studies onamphibians and reptiles with limited dispersal capabilities(e.g., Carranza et al., 2000; Fu et al., 2005). In the presentstudy, a generally west to east dispersal trend of D. acutus

populations could be suggested by our phylogenetic tree.The topology reveals that all individuals grouped intotwo main clades, one of which (main clade E) dominatelyconsists of individuals from the eastern area of the D. acu-

tus distribution range (only one old singleton forming abasal clade with very low support was from the west),and the other (main clade W) consists of the individuals

primarily from the western area, as well as a few fromthe east (8 individuals, 16.3%). Those few individuals clus-tered in western clade could occur in the eastern area, pre-sumably as a result of west to east dispersal events. Thishypothesis of west to east dispersal trend of D. acutus,derived from the existing data, is consistent with that ofthe Bufo gargarizans species group (Fu et al., 2005) whosedistribution range overlaps with that of D. acutus.

Four star-like phylogenies of haplotypes (Fig. 4) mightsuggest the possibly geographical origins of regional popu-lations and the short-distance migrations. For star W1 the

950 S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952

possible origin is in WMU, where the central haplotype 28(distributed over whole WMU and shared by 23 individu-als all from WMU) gave origin to five singletons fromWMU, and also gave origin to haplotypes 22 and 26 bothfrom CU. Clade W2 presumably originated in CU from itscentral haplotype 24 which is shared by 7 individuals (78%)from CU and 2 individuals (22%) from HSU. During theexpansion, haplotype 24 became a widespread haplotypein CU and migrated to HSU to give origin to four single-tons (5, 9, 10, and 11). Star E2 have a possible origin inWYU from haplotype 15, where it gave origin to five differ-ent singletons all limited to this unit. Star E1 have a plau-sible origin in ZJU with haplotype 6 (shared by 2individuals, one from ZJU and the other from HSU),where it gave origin to three derivate singletons (1, 2, and3). Presumably, some individuals carrying the haplotype6 migrated from ZJU to HSU and gave origin to haplo-types 4 and 14. Haplotype 14 gave origin to haplotypes 7and 8 in HSU, and migrated to WYU where it expandedand gave origin to haplotye 12. In further studies, if morehaplotypes could be found, the dispersal routes and origi-nal locality of D. acutus might be more clearly revealed.

4.2. Bottleneck and range expansions

Within a total of 86 specimens sampled randomly, onlytwo old singletons are found which are genetically apartfrom the other tightly clustered sequences (Fig. 2a andb). Furthermore, in the haplotype network (Fig. 4), thereare many unobserved intermediate haplotypes missingalong the long branches between two old singletons andcentral haplotypes, whereas almost all possible haplotypesare represented within each ‘‘stars’’ cluster. Both phenom-ena could result from a bottleneck event causing massiveextinction of populations before subsequent recent expan-sions (the earliest recent expansion is roughly estimatedto have occurred at about 252,000 years B.P.; see Fig. 5).Poikilotherms are very sensitive to the change of tempera-ture; therefore, a plausible explanation for this massiveextinction of populations may be Pleistocene climatic fluc-tuation, although no distinct Quaternary glacier landformor deposits have been found within the distribution rangeof D. acutus so far (Li et al., 2004).

Four star-like phylogenies with geographically localizeddistribution (Fig. 4), coupled with significantly negativevalues of Fu’s Fs tests (Fig. 5), and unimodal mismatch dis-tributions of the frequencies of pairwise differences in localpopulations (Fig. 5), all indicated that D. acutus popula-tions had passed through recent demographic expansions,respectively, at four locations after the bottleneck. Themost recent expansion occurred at the Westmost unitabout 166,000 years B.P. (Fig. 5). The maximum frequen-cies corresponded to the minimum pairwise differencesbetween individuals in westmost population, resulted inan extreme shift to the left of the peak, therefore givingan appearance of a non-campanulate unimodal curve.Three other range expansions occurred at about 169,000–

252,000 years B.P. During this period, the expansion ofsublineage W2 crossed the present-day Poyang Lake withone side now being part of the central and the other beingpart of the Huangshan unit (see Fig. 4, W2 and Fig. 1).After Late Pleistocene, the water-system of the ancientGan River began to develop. At around 2.3 ka B.P., underthe joint action of Yangtze River and ancient Gan River,the Meijiazhou, a natural levee, was formed at the southshore of Yangtze River. Its resistance to the water fromancient Gan River, which used to flow into Yangtze River,resulted in the formation of Poyang Lake and its enlarge-ment southward (Wu, 2001). According to the dating, theexpansion of sublineage W2 occurred before the develop-ment and formation of ancient Gan River and PoyangLake.

4.3. Isolations and west–east division

Taking no account of anthropogenic influence, isolationby distance or/and geography might play important role(s)in the evolutionary history of amphibians and reptiles withlimited vagility (e.g., Kuchta and Tan, 2005). In the presentstudy, isolation-by-distance appears to be a factor involvedin the population genetic structuring of D. acutus. Addi-tionally, the range-restriction of main clades and subclades,the localized distribution of haplotypes and diagnosticsites, also distinctly corroborate the existence of D. acutus

population isolations at various geographical scales. Themost noteworthy isolation within the whole D. acutus pop-ulation on the Mainland is attributed to natural barriers,i.e., a division line formed by Poyang Lake, Gan River,and the Wuyi Mts. This hypothesis of vicariance is consis-tent with the geological history and topography of the dis-tribution range of D. acutus. Subclade W2 clustersindividuals from both sides of Poyang Lake (numbersapproximately equal). At c. 169,000 years B.P., rangeexpansion and broad gene flow of D. acutus populationhad presumably occurred at this area. There was no vicar-iance at that time. After Late Pleistocene, along with thedevelopment of the ancient Gan River and the formationof an interlaced river network, the water system of ancientGan River gradually acted as a barrier to dispersal andgene flow of D. acutus. Vicariance came into being gradu-ally until the formations of Poyang Lake and Gan RiverDrainage. The Wuyishan Mts., combined with PoyangLake and Gan River Drainage, approximately verticallydivided the range into two areas (west and east) and thewhole population into two (western and eastern) mainlineages.

Thus isolation, dispersal, bottlenecks, and expansionjointly constitute the history of D. acutus. This differs par-tially from the conclusion of Fu et al.’s (2005) study onthe B. gargarizans species group whose distribution rangeoverlaps with that of D. acutus and is larger than latter.Their phylogenetic analysis suggested that ‘‘dispersal,instead of vicariance, dominated the history of the speciesgroup’’. Although vicariance was not observed in Fu

S. Huang et al. / Molecular Phylogenetics and Evolution 44 (2007) 942–952 951

et al.’s data, they also predicted that: ‘‘If a new phyloge-netic analysis reveals a symmetric tree with two distinctclades, which are dominantly composed of western oreastern populations, respectively, a vicariant explanationwould be preferred’’. Our data completely fit thisprediction.

4.4. Implications for conservation

The genetic structure revealed by the analyses in thepresent study suggests that D. acutus is a young populationwith low genetic diversity, likely still in the process of pop-ulation development and expansion. However, consideringthe status of its population declined rapidly in recent years,conservation measures should be taken into considerationright away. The concepts of Evolutionarily Significant Unit(ESU) and Management Unit (MU) are useful for identify-ing and prioritizing conservation units within a species.Moritz (1994) advanced the operational definitions: ESUsshould be reciprocally monophyletic for mtDNA allelesand show significant divergence of allele frequencies atnuclear loci, while MUs are recognized as populations withsignificant divergence of allele frequencies at mitochondrialor nuclear loci, regardless of the phylogenetic distinctive-ness of the alleles. According to the definitions above,our data could not identify ESUs within D. acutus, butappear to suggest at least two MUs—one in WMU andthe other in WYU—each of which deserves conservationattention. Based on our phylogenetic tree there also mightbe some minor candidate MUs in local populations withinHSU, CU, and ZJU, respectively, which require furtherconfirmation by more samples from local populationsand higher-resolution phylogenetic analyses.

At present, consensus has not been reached on how tobest delineate units for conservation (Crandall et al.,2000; Moritz, 2002; Gompert et al., 2006). Based on ourgenetic data, the highest priority for conservation seemsto be given to the HSU, although no ESU or explicitMUs could be identified for this unit. This conclusion issupported by two facts. First, among the five geographicalunits, only the Huangshan population is derived from bothmain lineages, therefore is diphyletic, and houses bothgenetic types. Second, the most cogent evidence is thatthe highest nucleotide diversity (1.37%) among all unitswas found in Huangshan unit, more than other units by3.5–29.5 times (Table 1), and make a dominant contribu-tion to the overall nucleotide diversity of whole population(1.41%). Protection of the Huangshan population will pro-tect the majority of overall nucleotide diversity, and willproduce an optimal input–output ratio in conservationeconomics. Thus our recommend action is that: (1), crea-tion of a nature reserve and establishment of a special fundfor protection of wild D. acutus in the Huangshan unit; (2),establishment of a well designed and large-scale captive-breeding program in the Huangshan unit, to maintain ahealthy and genetically diverse captive population for pre-serving the genetic diversity of this area, and to reproduce

large numbers of captive-bred individuals for the market toalleviate the pressure on the wild population of D. acutus.

Acknowledgments

This research was funded by grants from National Nat-ural Science Foundation of China (30225008 and30530120). We thank Drs. Shun-qing Lu (Huangshan Uni-versity), Peng Guo (Yibin University), and Jun-feng Pang(Kunming Institute of Zoology, CAS) for their suggestionsat the early stage of the present study. We are indebted toProf. Jie-tang Huang (Huangshan Institute of Snake) whocollected most of the specimens. We also thank Mr. Hong-hu Cheng (Yinkesong.com) for his help with the artwork.We would like to extend our thanks to two anonymousreviewers and the associate editor for their valuable com-ments on this manuscript. We particularly thank one ofthe reviewers who did extensive corrections on the manu-script, and Dr. Anita Malhotra (University of Wales, Ban-gor) for her valuable comments and extensive revisions onthis manuscript.

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