Molecular Phylogenetics and Evolution 67 (2013) 129–139

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Molecular Phylogenetics and Evolution

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Quaternary climate and environmental changes have shaped genetic differentiation in a Chinese pheasant endemic to the eastern margin of the Qinghai-Tibetan Plateau ⇑ Langyu Gu a,1,3, Yang Liu b,2,3, Pinjia Que a, Zhengwang Zhang a, a Ministry of Education Key Laboratory for Biodiversity Sciences and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing 100875, China b Computational and Molecular Population Genetics, Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland article info abstract

Article history: The geological complexity generated by the uplift of the Qinghai-Tibetan Plateau and the resulting hab- Received 14 July 2012 itat heterogeneity have functioned together with climatic oscillations in the Quaternary to have a pro- Revised 13 December 2012 found impact on the patterns of genetic diversity and demography of the fauna in this region. To Accepted 15 December 2012 understand the effect of the climatic and environmental shifts of the Quaternary on intraspeciﬁc genetic Available online 29 December 2012 patterns and evolutionary history, we investigated the population genetic structure of the blue eared pheasant (Crossoptilon auritum), an endemic bird inhabiting the easternmost region of the plateau. Our Keywords: phylogeographic analysis of mitochondrial DNA sequences and eight autosomal microsatellites reveals Pheasant that the blue eared pheasant is subdivided into four distinct subpopulations: a central group (Huzhu Qinghai-Tibetan Plateau Quaternary and Taizi Mountains), a southern Zoige group, a southernmost Wanglang group and the northernmost Conservation units Helan Mountain group. These groups are likely to have diverged in the Pleistocene, corresponding to geo- Phylogeography logical changes and the interglacial–glacial climate oscillations that occurred at the eastern margin of the Qinghai-Tibetan Plateau. These subpopulations thus represent major conservation units, especially for the isolated Helan subpopulation. Our ﬁndings provide evidence of population divergence driven by complex Quaternary climate and environmental changes and, once more, highlight the importance of phylogeographic studies for conservation endeavours. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction in the peripheral areas) that generated habitat heterogeneity (Wil- liams et al., 1993). These factors are likely to have created opportu- The Quaternary ice ages played a major role in shaping the pres- nities for the isolation of alpine species populations in the QTP, ent distribution of organisms and the geographical structure of ge- which may have eventually led to inter- and intra-speciﬁc diver- netic diversity in the Northern Hemisphere (Hewitt, 2000, 2004). gence (Zeng et al., 2008; Tu et al., 2010). Assessing the genetic var- Phylogeographic studies of various species living in regions of iation of endemisms in the QTP thus provides a valuable Western Eurasia and North America that were glaciated have opportunity to understand the effect of the environmental changes shown that spatial patterns of genetic diversity are strongly asso- in the Quaternary on the evolution of organisms (Zhang et al., ciated with putative refugial areas during the glacial and intergla- 2005; Chen et al., 2008; Yang et al., 2008; Wang et al., 2009). cial periods (Hewitt, 2000, 2004; Avise, 2009). Although no major The eastern margin of the QTP harbours many endemic species continental ice sheets were present in Eastern Asia during the Qua- and is renowned as a biodiversity hotspot (http://www.biodiversi- ternary (Zhang et al., 2000), the uplift of the Qinghai-Tibetan Pla- tyhotspots.org). The concentration of endemisms and the excep- teau (QTP) generated a structurally complex landscape (e.g., high tional magnitude of biodiversity were hypothesised to correlate mountains and deep valleys) and climatic perturbations (e.g., a with the uplift of the QTP and the environmental changes of the harsh climate in the central area of the QTP and a mild climate Quaternary (Yang et al., 2009; Lei, 2012). However, not all organisms were similarly affected. A recent study on birds, for example, showed that two species distributed at the eastern edge of QTP ⇑ Corresponding author. Fax: +86 1058807721. E-mail address: [email protected] (Z. Zhang). maintained stable demographic levels during the glacial period, 1 Present address: Zoological Institute, University of Basel, Vesalgasse 1, 4051 indicating that suitable habitats might have existed during that Basel, Switzerland. period (Qu et al., 2010). This scenario is also supported by geolog- 2 Present address: State Key Laboratory of Biocontrol and School of Life Sciences, ical evidence indicating that the margins of the QTP might have Sun Yat-sen University, Guangzhou 510275, China. been relatively ice-free compared to the platform during the Ice 3 These authors contributed equally to this work.

Age (Zhang et al., 2000). While several large-scale phylogeographic Mountain (TZ) in Gansu were sampled at the terrain transition studies have investigated the glacial refugium in the QTP using zone between the QTP and the Loess Plateau. The two southern several alpine species (e.g., Zhang et al., 2005; Qu et al., 2010; populations from Zoige (ZO) and Wanglang (WL) in Sichuan inha- Qiu et al., 2011; Zhao et al., 2011a), the general scenario of glacial bit the lower terrain at the eastern margin of QTP (Fig. 1). The pop- refugiums for species inhabiting the margins of the QTP is re- ulation in Helan Mountain (HLS) is isolated from the other stricted to only a few taxa (Zhan et al., 2011). populations by the Tengger Desert (Fig. 1). Fifty-one blood samples Several avian groups distributed along the eastern margin of the were collected from individuals that were rescued in nature re- QTP exhibit different levels of morphological divergence, reflecting serves or confiscated individuals from the local forestry adminis- different evolutionary trajectories (Qu et al., 2005; Martens et al., trations. Forty naturally shed feather samples were collected in 2011; Zhan et al., 2011). Although phylogeographic studies of the field surveys during 2010–2011. Prior to extraction, the blood avian species distributed along the edge of the QTP have received was preserved in 95% ethanol at 80 °C and the feathers were great attention (Qu et al., 2005, 2010), comprehensive analyses stored at 80 °C. Genomic DNA was extracted using DNA extrac- based on a temporal and spatial framework remain sparse, most tion kits (TianGen Biotech, Beijing, China). likely because of sampling difficulties for species living at high alti- tudes and the low resolution of mtDNA markers (Edwards and 2.2. Mitochondrial DNA sequencing Bensch, 2009). Besides, the impact of the Quaternary climate and environmental changes on the diversification and spatial organisa- We amplified a 393 bp fragment of the mitochondrial control tion varies between species with different habitat requirement and region (CR) using the primer pair CRL (Desjardins and Morais, dispersal ability. For example, some alpine species in the QTP could 1990) and CRH2 (this study). We also amplified a 977 bp fragment shift their distribution through altitudinal movement rather than of the NADH dehydrogenase subunit 2 mitochondrial gene (ND2) long-distance migration (Zhou et al., 2010; Zhan et al., 2011). using the L5215 (Hackett, 1996) and H1064 (Drovetski et al., Hence, more studies are required to further our understanding of 2004) primers (Table S1). To handle the issue of fragmented DNA the Quaternary climate and environmental changes responsible found in some samples, additional internal primers (ND2P1F, for driving population divergence in this region. ND2P1R, ND2P2F, ND2P2R, ND2P3F, and ND2P3R; for detailed In this study, we investigated the genetic structure of the blue information, see Table S1) were designed and used to amplify three eared pheasant (BEP), Crossoptilon auritum, using both maternally separate segments of ND2. This process generated a concatenated inherited sequences (mtDNA) and bi-parentally inherited autoso- sequence with the same length (977 bp) as the target region. All mal microsatellites. The BEP is endemic to China and distributed of the fragments were amplified in a reaction volume of 20 lL con- along the northeastern margin of the QTP, being specifically lo- taining 100 ng of template DNA, 10 lL of pre-mix (Takara), and cated at the topographic transition zone from the QTP to the Loess 0.25 lM of each primer. The PCR cycle utilised the following proce- Plateau and the Sichuan Basin (Cheng, 1978; del Hoyo et al., 1996; dure: denaturation at 95 °C for 5 min followed by 35 cycles at 94 °C Lei and Lu, 2006). The BEP is a resident species and has no morpho- for 45 s, denaturation at 50 °C (CR) or 54 °C (ND2) for 45 s and then logically recognised subspecies (Johnsgard, 1999; Clements et al., extension at 72 °C for 70 s, and a final extension at 72 °C for 2012), and the population residing at the northern limit of the spe- 10 min. To check the amplification success of the PCRs, we checked cies’ distribution on Helan Mountain may be isolated from other each PCR product for size on a 1.5% agarose gel and compared the populations. This scenario is supported by the fact that the south- products to a 100 bp ladder (Takara Company). The successful PCR ern side of the mountain was surrounded by large deserts (MacK- products were precipitated with ethanol and sequenced using Ter- innon and Phillipps, 2000; Li et al., 2009). The BEP shows a habitat minator Ready Reaction Mix Big Dye (v3.1, Applied Biosystems) on preference for coniferous and broad-leaf mixed forests, subalpine the ABI 3730 and 3100 Genetic Analyzers at the BGI (Beijing Geno- coniferous forest and scrub meadow at 2000–4000 m.a.s.l. (Lei mic Institute). To avoid co-amplification of nuclear copies (Numts) and Lu, 2006; Wu and Liu, 2010a). There is no systematic popula- of mtDNA, we took several experimental controls as described in tion census of BEP and the overall population size was believed to Liu et al. (2010) in order to obtain authentic mtDNA sequences. Fi- be less than 10,000 mature individuals (Birdlife International, nally, a total of 48 BEPs (34 blood and 14 feather samples) were 2012). Given that its distribution range encompasses several ter- successfully sequenced and thus available for the subsequent anal- rain transition zones and eco-zones with varied environmental ysis (Table 1). The resulting sequences were aligned using Mega conditions and the fact that the BEP has relatively low dispersal 5.03 (Tamura et al., 2011). ability, this organism is a suitable model for studying the possible relationship between genetic divergence and the historic climatic 2.3. Microsatellite genotyping effect on the eastern QTP. We predicted that BEP may be spatially split into distinct genetic groups, subpopulations north and south All of the samples were genotyped at eight microsatellite loci to Yellow River and the isolated Helan Mountain subpopulations that were developed from other pheasants (primer details and because of the major geographic barriers in its range. PCR profiles in Gu et al. (2012)). The fragments were separated To study the evolutionary history of BEP, we combined popula- on an ABI PRISM 3100 Genetic Analyzer (Tsingke Company, Bei- tion genetics and phylogenetic analyses to establish the spatial and jing), and the fragment lengths were determined against an inter- temporal patterns of genetic divergence in the BEP. We also esti- nal size standard (GeneScan™ 500 LIZÒ Size Standard, Applied mated the time of intraspecific divergence of the BEP and investi- Biosystems) with GeneMapper v3.7 (Applied Biosystems). To en- gated the correlation between divergence times and historical sure genotype repeatability, the PCR amplifications and fragment environmental changes. genotypes were repeated independently: three to four times for the DNA extracted from the feathers and twice for the other types 2. Materials and methods of samples.

2.1. Sample collection and DNA extraction 2.4. Statistical analysis

A total of 91 BEP individuals were collected from ﬁve sites with- Congruence tests for the two mitochondrial fragments were in the species’ distributional range in western China. The popula- performed using the Incongruence Length Difference Test (ILD) tions from the Huzhu Mountain (HZ) in Qinghai and the Taizi (homogeneity test) implemented in PAUP⁄4.0b10 (Swofford, L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139 131

Fig. 1. The sampling sites for blue eared pheasant Crossoptilon auritum and the 11 deﬁned mitochondrial haplotypes based on 1370 bp of the concatenated mitochondrial sequences (containing 393 bp of the CR and 977 bp of the ND2) from 48 individuals of blue eared pheasant were indicated.

2002). From these results (P > 0.99), the CR and ND2 sequences For the microsatellite data, we estimated the standard genetic were concatenated into a 1370 bp sequence alignment for further diversity for each site using the following indices: the number of genetic and phylogenetic analyses. We calculated the following alleles at each locus, the observed (HO) and expected (HE) heterozy- indices of genetic diversity for each sampling site with DnaSP gosities using Arlequin v3.11 (Excofﬁer et al., 2005), the mean alle- v5.10.01 (Librado and Rozas, 2009): the number of polymorphic lic richness (AR) using FSTAT v2.9.3.2 (Goudet, 2002) and the sites (S), the number of haplotypes (Na), the haplotype diversity number of private alleles (PA) using HP-Rare v1.1 (Kalinowski (h), the nucleotide diversity (p) and the average number of nucle- et al., 2006). We tested the allelic dropout and false alleles using otide differences (k). MicroChecker v2.2.3 (Oosterhout et al., 2004) and calculated the 132 L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139

Table 1 Estimates of genetic diversity based on mtDNA and microsatellites in blue eared pheasant Crossoptilon auritum. Sample sizes (n) for each population are given; For mtDNA, number of polymorphic sites (S), number of haplotypes (Na), haplotype diversity (h), nucleotide diversity as percentage (p), and the average number of nucleotide differences (k) are given. For microsatellites, the means of observed heterozygosity (HO), expected heterozygosity (HE), allele richness (AR), number private allele (PA), inbreeding coefﬁcient (FIS) are indicated.

Pop Latitude Longitude n (mtDNA) SNah p (%) Kn(STR) HO HE AR PA FIS HZ 36°500N 101°570E 16 6 6 0.68 0.09 1.17 16 0.73 0.75 4.25 1.00 0.02 TZ 35°170N 102°590E 15 4 3 0.59 0.09 1.16 18 0.60 0.63 3.66 0.74 0.03 HLS 38°370N 105°500E 7 1 1 0.00 0.00 0.00 36 0.36 0.49 2.85 0.40 0.19** ZO 33°340N 102°570E 5 3 2 0.40 0.09 1.20 16 0.63 0.69 3.70 0.65 0.05 WL 32°550N 104°090E 5 3 2 0.40 0.09 1.20 5 0.75 0.78 4.33 1.11 0.01

** P < 0.01.

Fig. 2. Bayesian STRUCTURE clustering results based on microsatellite genotypes of 91 individuals of blue eared pheasant Crossoptilon auritum from five populations. (a) full data, indicating three genetic clusters (DK = 3); (b) samples of south of the low-elevation region, indicating two genetic clusters (DK = 2); (c) samples of north of the low- elevation region, indicating one genetic cluster (K = 1). Note that it is impossible to infer DK for K = 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) null allele frequency using FreeNA (Chapuis and Estoup, 2007). best-fitting nucleotide substitution model for the CR and ND2 joint Furthermore, we assessed deviations from Hardy–Weinberg equi- dataset by the Akaike Information Criterion in jModelTest v0.1.1 librium (HWE) and genotypic linkage equilibrium (within popula- (Posada, 2008). For the Bayesian inference analysis, two indepen- tions) using Arlequin v3.11 (Excoffier et al., 2005). We also dent runs with default heating temperatures were performed with calculated the inbreeding index (FIS) for each population and as- MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001). This analysis sessed the significance of this index on the basis of 10,000 permu- was run for 10,000,000 steps and sampled every 1000 steps. The tations in Arlequin software. The sequential Bonferroni correction first 25% of the samples were discarded as burn-in. The conver- (Rice, 1989) was used to adjust the significance levels for multiple gence of the MCMC chains was assessed using Tracer v1.5 (Ram- testing. baut and Drummond, 2009). For ML tree reconstruction, we used Using the concatenated mitochondrial CR and ND2 sequences, a web server (http://www.atgc-montpellier.fr/phyml/) to run the we reconstructed the phylogenetic relationships among the sam- PhyML v.3.0 program (Guindon et al., 2010). We started with the pled individuals using the Bayesian inference and Maximum Like- heuristic searching strategy to identify the best topology from five lihood approaches. The Hasegawa–Kishino-and-Yano model (HKY, random BIONJ trees. Those trees were moved by nearest-neighbour Hasegawa et al., 1985) with invariant sites was identified as the interchange (NNI) and the subtree pruning and regrafting (SPR) L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139 133 approaches. The approximate Likelihood Ratio Test was used to pendent runs for each K-value (K = 1–6) for the entire dataset. We estimate the nodal support for the phylogenetic inferences (Ani- used Structure Harvester v0.6.8 (Earl and von Holdt, 2011) to iden- simova and Gascuel, 2006) by using a Shimodaira-Hasegawa-like tify the most likely number of genetic clusters on the basis of the procedure in PhyML. The phylogenetic trees were rooted using ad hoc statistics described in Evanno et al. (2005). The final results homologous sequences derived from the complete mitochondrial of the bar plot for individual memberships were visualised with genome of the White Eared Pheasant, C. crossoptilon (GenBank DISTRUCT v1.1 (Rosenberg, 2004). accession NC_016679.1) (Wu et al., 2005). We further constructed The between-population migration rates (M) were estimated unrooted haplotype networks using the median-joining algorithm with MDIV software (Nielsen and Wakeleya, 2001). We used the (Bandelt et al., 1999) implemented in Network v4.6.0.0 (http:// HKY model and performed 2,000,000 MCMC sampling runs with www.fluxus-engineering.com). This method allows the visualisa- 500,000 burn-in iterations. The value M in MDIV represents the tion of mtDNA haplotype relationships and frequencies. average number of migrants moving between two populations The Time to the Most Recent Common Ancestry (TMRCA) for the each generation (Nielsen and Wakeleya, 2001). The parameters populations was estimated using a Bayesian Markov Monte Carlo for M were limited from 0 to 10. We performed three independent method implemented in BEAST v1.6.1 (Drummond and Rambaut, runs to confirm convergence. 2007). Because there is no suitable fossil record available to cali- Because the resulting phylogenetic trees were not always bifur- brate the mutation rates for the eared pheasant or other closely re- cating, we used all of the mtDNA data according to network results lated species, we adopted a substitution rate for CR (7.23 ± 1.58% to test for historical demographic changes in the BEP using differ- divergence per million years divided by two) derived from the ent methods. We first calculated Tajima’s D (Tajima, 1989) and Fu’s grouse (Tetraoninae) (Drovetski, 2003), which belongs to the same Fs (Fu, 1997) for the mtDNA in DnaSP. Both of these values are family (Phasianidae) with BEP. The substitution rate for ND2 was based on a sudden expansion in population, which may leave a set to 0.614 ± 0.1% per million years based on a study that used footprint in the DNA sequence and cause a shift in the shape of multiple fossil time records throughout the phylogenetic tree. This the frequency distribution compared to the neutral Wright–Fisher rate for ND2 was calculated especially for the node between the model. Significant negative values indicate a recent population Coturnix and Gallus genera (Pereira and Baker, 2006). Because dat- expansion. We then applied the R2 statistic, which has a greater ing the divergent time is a challenge without calibration of the power to test population expansion when the sample size is small molecular clock from fossil and geological evidence, our tentative (Ramos-Onsins and Rozas, 2002). Based on the difference between results for BEP should be interpreted in caution. We further parti- the number of mutations and nucleotide substitutions, a signifi- tioned the CR and ND2 datasets to consider the different nucleotide cant positive R2 value is evidence of a population expansion (Pil- substitution rates in BEAUti software. Again, we used C. crossopti- kington et al., 2008). Additionally, we tested demographic and lon as the outgroup (Wu et al., 2005) and the Tamura–Nei model spatial population expansion of BEP by conducting mismatch dis- as the unlinked evolutionary model of nucleotide substitution for tribution using Arlequin. The shape of the mismatch distribution both datasets, as suggested by jModelTest v0.1.1. The best clock of a constant size population can be distinguished from an expan- model and tree prior model were determined from the Bayes Fac- sion population. The raggedness statistic r is a measure of this rag- tors calculated in Tracer (Rambaut and Drummond, 2009). The gedness (Harpending et al., 1993). The sum of squared differences BEAST analysis was performed with 100,000,000 generations, with (SSD) was also investigated to test departure from the sudden the first 25% of the generations being discarded as burn-in. The expansion model by comparing the distribution between the ob- mean TMRCA was obtained and checked in Tracer, and the station- served and estimated results with coalescent simulations (Schnei- ary distribution of this measure was ensured when the effective der and Excoffier, 1999; Excoffier, 2004). A significant SSD sample size (ESS) exceeded 200. We generated clade credibility (P < 0.05) indicates that there is no sudden population expansion. with TREEANNOTATOR (http://beast.bio.ed.ac.uk/TreeAnnotator) We also obtained the relative expected and observed trends using and generated a final preparation of the tree using FIGTREE Arlequin software. (http://tree.bio.ed.ac.uk/software/figtree). For each data set, analyses of molecular variance (AMOVA) 3. Results (Excoffier et al., 1992) and population differentiation were conducted in Arlequin to assess the population subdivision among 3.1. Molecular diversity the five sites. We tested all of the grouping options in the five populations with AMOVA and defined the optimal grouping as the We obtained 1370 bp of the concatenated mitochondrial se- largest value of genetic variation among groups (FCT) with the quences (containing 393 bp of the CR and 977 bp of the ND2) from smallest value of genetic variation among populations within 48 BEP individuals. No internal stop codons or indels were found in groups (FSC). We calculated the global UST and pairwise UST for the ND2 sequences, but one base-pair insertion was found in the the mtDNA data set with the Tamura–Nei model (Tamura and CR sequences. Ten and three haplotypes were obtained for CR

Nei, 1993) and the global FST and pairwise FST for the microsatellite and ND2 segment, respectively. And the concatenated CR and data set with the Weir and Cockerham estimator (1984) in Arle- ND2 dataset yielded 11 haplotypes (GenBank accession quin. The signiﬁcance levels of all analyses were assessed on the KC462573-KC462594 provided in Table S2). One haplotype found basis of 10,000 permutations and were adjusted using a sequential in ZO exhibited a mutation from CAT to TAT in the partial ND2 Bonferroni correction for multiple testing. gene, resulting in an amino acidic change from histidine to tyro- The genetic relationship for the microsatellites was visualised sine. The haplotype diversity varied between regions, with the with principal coordinates analyses (PCoA) by using pairwise highest value observed at HZ and the lowest value observed at Euclidian distances between individual genotypes in GENALEX HLS (Table 1). v6.3 (Peakall and Smouse, 2006). We further tested for genetic Ninety-one individuals from the ﬁve populations were geno- structure (assuming no prior imposing spatial information for the typed at eight microsatellite loci (Table 1). No locus showed BEP samples) using a Bayesian clustering method, which was large-allele dropout or false alleles. No linkage disequilibrium implemented in STRUCTURE v2.3 (Pritchard et al., 2000; Falush was found after a sequential Bonferroni correction across all of et al., 2003). We used an admixture model with correlated allele the loci. Two loci (3D2 in HLS and TT06 in ZO) showed higher null frequencies and performed 300,000 Markov chain Monte Carlo allele frequencies (>0.20). Given that FST is very sensitive to the null (MCMC) steps with 200,000 burn-in steps. We conducted 10 inde- allele effect (Chapuis and Estoup, 2007), we obtained two pairwise 134 L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139

genetic differentiation (FST) matrices with and without these two ation. The AMOVA for the mtDNA showed that an arrangement of loci. We did not find any significant influence of these two loci four groups, with HZ and TZ grouped together against HLS, ZO and 2 on the population structuring estimation (Mantel test: R = 0.97, WL individually, resulted in the greatest value (FCT = 0.53, P = 0.10) P = 0.01). To increase the statistical power, we included these loci (Table S1). The pairwise molecular variations among the mtDNA of in further population genetic analyses. the populations (UST) were significantly different from zero, with Genetic diversity based on microsatellites varied between popu- the exception of the comparison between HZ and TZ (Table 2). lations. The HZ and WL populations showed high genetic diversity, The results of the AMOVA for the microsatellite markers showed and the HLS population showed the lowest mean heterozygosity, a similar group-partitioning pattern (FCT = 0.19, P = 0.10). However, allelic richness and rare allele incidence values (Table 1). The FIS val- the genetic variation among the populations within groups, which ues were not significantly different from zero in any of the popula- was generated for HZ and TZ, was significantly larger than zero tions except HLS, which had a significantly positive FIS value (0.19). (FSC = 0.11, P < 0.001) (Table S2). This finding of genetic variation This finding was most likely caused by the fact that five out of the between HZ and TZ was consistent with the results of the pairwise eight microsatellites showed significant deviation from HWE after comparisons of the microsatellites of these two populations

Sequential Bonferroni correction of the HLS population. (FST = 0.10, P < 0.001), though this value was generally lower than the other comparisons (Table 2). Furthermore, our Bayesian clus- 3.2. Phylogeographic analysis tering analysis with STRUCTURE revealed congruent partitioning of three genetic clusters in all 10 replicates (high peak occurred Phylogenetic reconstruction using Bayesian inference and Max- at DK = 3 for K =3)(Evanno et al., 2005), with HZ and TZ grouped imum Likelihood revealed a similar topology with short branch together, WL and ZO forming one cluster, and HLS forming a single length (Figs. S1 and S2). Again, the mtDNA haplotype networks cluster (Fig. 2a). A plot of the principal coordinate analyses based showed a shallow and star-like topology with 1–6 mutational steps on the microsatellite data revealed a similar pattern (Fig. 3). To ex- between deﬁned haplotypes (Fig. 3). All of the individuals from the plore the potential substructures within these three genetic clus- HLS population shared one unique haplotype that was one muta- ters, we completed further analyses for the central cluster (HZ, tional step away from the most common haplotype. TZ) and the southern cluster (ZO, WL) using the same parameter Based on the Bayes Factor results, the uncorrelated exponential setting in STRUCTURE. Our result revealed that a genetic substruc- relaxed clock (Drummond et al., 2006) with constant size model ture existed within the southern cluster (ZO, WL) (Fig. 2b) but was selected as a prior set for running BEAST. The BEAST analyses failed to identify a population subdivision within the central clus- indicated that the TMRCA between the ZO population and the ter (HZ, TZ) (Fig. 2c, considering the highest log likelihood values other populations was approximately 0.14 (0.05–0.28, 95% HPD) for K = 1 and the impossible to infer DK for K = 1). mya, the TMRCA between the WL population and other populations was 0.10 (0.03–0.17, 95% HPD) mya, and the TMRCA between 3.4. Demographic changes the HLS population and other populations was approximately 0.08 (0.02–0.12, 95% HPD) mya (Fig. 4). The HZ and TZ populations The posterior probability distribution of M (as simulated using exhibited polytomy on the tree (Fig. 4). MDIV) indicated very low migration rates between populations, with the rates almost identical to ze (Fig. 5). 3.3. Population genetic analyses We found no evidence for demographic expansion in the mtDNA data. We found non-signiﬁcant results for the R2 statistic

The global test for mtDNA (UST = 0.47, P < 0.001) and microsat- (0.11, P = 0.13), Tajima’s D (1.09, P = 0.15) and Fu’s Fu (2.70, ellites (FST = 0.27, P < 0.001) indicated signiﬁcant genetic differenti- P = 0.08). Besides, the observed trend in the mismatch distribution

Fig. 3. The phylogenetic relationships of blue eared pheasants Crossoptilon auritum based on: (a) Unrooted median joining networks based on 1370 bp of the concatenated mitochondrial sequences (containing 393 bp of the CR and 977 bp of the ND2) from 48 individuals; (b) A principal coordinates analysis based on 91 individuals at eight microsatellite loci. Different colors represent different populations. L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139 135

Fig. 4. Chronogram based on mtDNA tree of blue eared pheasant Crossoptilon auritum obtained using an uncorrelated exponential relaxed clock model assuming a constant population size. Datings of divergence times with ranging the 95% posterior probability interval are indicated above the branches, and posterior probability under the branches. The branch of outgroup was too long so we used short bars to abbreviate it.

likely requires additional conservation efforts. We did not detect Table 2 Pairwise genetic differentiation among blue eared pheasant Crossoptilon auritum strong evidence of a demographic population expansion for the based on mtDNA (below the diagonal: UST) and microsatellites (above the diagonal: BEP. This ﬁnding is in contrast to the ﬁndings for other species FST). from the QTP platform, including plants (Zhang et al., 2005) and

Location HZ TZ HLS ZO WL birds (Qu et al., 2005, 2010), which exhibited a population expansion following the dynamics of the ice age. However, consistent HZ – 0.10** 0.31** 0.17** 0.14** with our results, population size-constancy during the Quaternary TZ 0.04 – 0.37** 0.27** 0.15** HLS 0.54** 0.56** – 0.33** 0.30** has been described in two passerine species, the twite (Carduelis ZO 0.61** 0.62** 0.86** – 0.15** flavirostris) and the black redstart (Phoenicurus ochruros), which WL 0.35** 0.39** 0.71** 0.45* – are both distributed on the eastern margin of the QTP (Qu et al., The significant genetic differentiation after sequential Bonferroni correction (the 2010). It is thus likely that the observed stability in the size of largest P value we used for the correction was 0.05 and 0.01, respectively) was the BEP population was caused by mitigative habitat pressure on indicated as the eastern margin of the QTP (Qu et al., 2010), considering that * P < 0.05. there was little glacier cover in this area during the Quaternary ** P < 0.01. period (Zhang et al., 2000). There was also evidence indicating that the alpine meadow and steppe retreated eastward during the ice deviated significantly from the expected demographic expansion age (Williams et al., 1993), which might have provided habitat model (SSD = 0.06, P = 0.01, Fig. 6a). However, this result was not suitable for the BEP. the case for the expected spatial expansion model (SSD = 0.01, P = 0.29, Fig. 6b), indicating some evidence of a sudden spatial 4.1. Phylogeographic patterns in the BEP expansion in the BEP. The most common haplotype was observed in three popula- 4. Discussion tions, namely ZO, HZ and TZ (Fig. 3a); another haplotype was shared between WL and HZ. Considering the fact that no significant The combination of maternally inherited mtDNA and bi-paren- migration was detected (Fig. 5) and the shared haplotypes were tally inherited microsatellites provides a comprehensive frame- widespread, these haplotypes are most likely the retained ances- work to analyse the genetic structure and demographic history of tral haplotypes (Crandall and Templeton, 1993). The ZO, WL and the BEP, a Chinese endemic pheasant. We found significant genetic HLS populations contained private haplotypes that were only a differentiation within the BEP, which appears to be separated into few mutational steps from the most common haplotype, indicating one central group (HZ + TZ), the northernmost HLS, the southern that these regions might have been historically colonised from the ZO and the southernmost WL. Our results suggest that the genetic central group (HZ and TZ), a finding that is consistent with the spa- divergence in the BEP in the eastern margin of the QTP in China tial expansion we identified (Fig. 6b). correlates with geological changes and the interglacial–glacial cli- Notably, we found that the northernmost HLS is genetically dis- mate oscillations. The geographically isolated HLS population tinct from the other populations. This population exhibited low ge- showed relatively low levels of genetic diversity and thus most netic diversity in both mtDNA and microsatellites and showed 136 L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139

Fig. 5. The posterior probability distribution of migration rates based on mtDNA sequence dataset between populations of blue eared pheasant Crossoptilon auritum estimated by MDIV.

Fig. 6. Mismatch distribution calculated based on mtDNA dataset of the blue eared pheasant Crossoptilon auritum (observed: line; expected: dotted). The raggedness index r and SSD statistics with P value were presented under (a) sudden demographic expansion model; (b) sudden spatial expansion mode.

genetic signs of inbreeding, as evidenced by a low mtDNA haplo- junctive habitat distribution resulting from the Late Pleistocene type diversity and significantly positive FIS value (Table 1). Further- expansion of the adjacent Tengger desert (Williams et al., 1993). more, the genetic distance between the unique HLS haplotype and While the formation of the Tengger desert had already started the most common haplotype was only one mutational step as we in the Middle Pleistocene, this feature reached its maximum range mentioned above (Fig. 3). These findings suggest that the HLS pop- during the Late Pleistocene (Williams et al., 1993). Evidence of ulation was likely colonised from the adjacent populations and sand accumulation in the Qinghai and Lanzhou areas suggests that subsequently became isolated, with enhanced genetic drift or this desert once intruded into the northeast margin of the QTP selection eventually shaping the observed phylogeographic pattern (Dong et al., 1997). The Tengger desert contracted in size under (Mayr, 1942; Yeung et al., 2011). The TMRCA of the HLS population the warm and humid climate that occurred during the interglacial and the other populations was dated to the Late Pleistocene. The period but expanded southward in the subsequent glacial period isolation of the HLS population is most likely a consequence of dis- (Dong et al., 1997; Liu et al., 2000). As an alpine species, the BEP L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139 137 might have been able to stay in the high latitude Helan Mountains ent environmental features of the ZO and WL could also increase instead of migrating southward during the warm interglacial per- genetic differentiation. iod. However, the expansion of the Tengger desert in the subsequent glacial periods caused the gene flow between the HLS 4.3. Implications for conservation population and the other populations to eventually cease, leading to the isolation of the HLS population. Similar to this scenario, Genetic analysis is useful for determining which conservation the Tengger desert was also used to explain the population subdi- measurements enable effective management efforts (Hammer vision in the Sichuan subspecies of blue sheep (Pseudois nayaur et al., 2010; Osborne et al., 2012). This study suggests that the pop- szechuanensis), whose population in the Helan Mountains might ulation from the Helan Mountains should be treated as an Evolu- be designated as a new subspecies, as this population exhibits sig- tionarily Significant Unit (ESU) for its reciprocal monophyly for nificant genetic divergence from the populations in the adjacent mtDNA alleles and significant divergence in the allele frequencies Qinghai, Gansu and Sichuan regions (Zeng et al., 2008). found at microsatellites (Moritz, 1994). However, the reciprocal Although the BEP is ground-dwelling species that excels in run- monophyly requirement of an ESU may in some cases be too strin- ning, this pheasant is not skilled in flying because of the degener- gent, as a reciprocal monophyletic status can be overturned by just ation of the wings. We thus expected genetic differentiation a single anomalous individual in the entire sample (Fraser and Ber- between the HZ and TZ populations because these two populations natchez, 2001). For example, the ZO population in this study is are located on different sides of the Yellow River, which acts as a monophyletic and may form a subgroup to all of the other groups bio-geographical barrier in plants and lizards (Chen et al., 2008; with the exception of one individual, who shares one ancestral Zhao et al., 2011b). In contrast to our expectation, we detected haplotype with other populations. To circumvent the problem of no significant genetic differentiation in the mtDNA and microsatel- less phylogenetic separation than reciprocal monophyly in prac- lites of the HZ and TZ populations (Table 2; Fig. 2). In fact, these tice, Moritz (1994) proposed the management unit (MU), which populations shared the two common haplotypes (Fig. 1). One pos- is appropriate for the conservation status definition for the ZO sible reason for this finding might be the recent formation of the and WL populations in this study because this classification only Yellow River, which has not provided enough time for the popula- requires different allele frequencies (nuclear or mitochondrial) tions to retain genetic differences. However, the Yellow River had without the monophyletic phylogeny requirement. already formed between the regions where HZ and TZ populations The existing population survey data for the BEP is not system- inhabited during the Kunlun–Huanghe Tectonic Movement (1.1– atic and needs to be updated (del Hoyo et al., 1996), with many 0.6 mya) (Li et al., 2007). Compared to the population divergence surveys being limited to the area south of Gansu (Wu and Liu, time within the BEP (Fig. 4), it should have provided sufficient 2010b, 2011). Little recent survey data are available for the duration to accumulate genetic differences. Another possible inbreeding HLS population, despite the fact that the need for these explanation is that the Yellow River did not always act as a barrier data is urgent and important for developing a conservation plan. due to the frequent climatic fluctuations in the Late Pleistocene; as Additionally, considering the fact that the BEP is distributed across the river flow decreased, the riverbed could have been filled with different eco-zones with varied environmental conditions, investi- silt during the dry and cold periods (Zhang et al., 2001), providing gations into local adaptation through the genetic divergence data effective opportunities for the BEP to disperse across the river and in this study (Myers et al., 2012) might be needed to provide addi- resulting in genetic connectivity. Comparably, Zhao et al. (2011b) tional understanding of the evolution and adaptation of the BEP to found that the Yellow River served as a barrier for the less mobile the climate and environmental changes of the Quaternary. lizard species Eremias brenchleyi but not for sister species E. argus, which exhibits a better dispersal ability during the Yellow River zero flow. A study indicating that the Yellow, Yangtze and Mekong Acknowledgments rivers did not present a physical barrier for the blood pheasant (Ith- aginis cruentus) in the QTP (Zhan et al., 2011) also provided strong We thank Jiang Chang, Ying Liu, Jiliang Xu, Xinkang Bao, and support to the hypothesis that some pheasants (like the BEP) can Helanshan National Nature Reserve in Ningxia and Inner Mongolia, cross the Yellow River. Forestry Bureau of Zoige County, Beijing Zoo, Linxia Dongjiao Zoo, Yinchuan Zoo, Xining Zoo, and Wanglang National Nature Reserve for the collection of samples. We are grateful to Walter Salzburger, 4.2. The regional population substructure is influenced by changes in Shou-Hsien Li, Ming Li, Xiangjiang Zhan, Yanhua Qu, Lu Dong for Quaternary climate and environment the valuable comments. We also thank Jiang Chang, Ning Wang and De Chen for data analysis support, Xiuqi Fang and Weili Qiu Significant genetic differentiation was found between the ZO for geographical knowledge support. We also thank Jien Zhang and WL populations (Fig. 2b and Table 2), which are located at for preparing the map. We thank the two anonymous reviewers the topographic transition zone between the QTP and the Sichuan for suggestions as well. Funding for field work and genetic analyses Basin. The regional tectonic movement in the eastern area of the was provided by the National Science and Technology Ministry QTP during the Late Pleistocene increased the relative elevation (2012BAC01B06), the National Natural Science Foundation of Chi- of many mountains and valleys (Zhang et al., 2003), which might na (Nos. 30770314 and 31272296) and the ‘‘985 Project’’ of Beijing have resulted in geographic isolation and prevented effective gene Normal University. Yang Liu was supported by the Career Develop- flow between these two populations. Additionally, the paleocli- ment Bursary award of the British Ornithologists’ Union and the mate conditions during that period were considerably different be- ‘‘Hundred Talent Program (Bai Ren Ji Hua)’’ from Sun Yat-sen Uni- tween the ZO and WL regions. The ZO was characterised as a cold versity, China. climate in Late Pleistocene, as evidenced by the woolly rhinoceros (Coelodonta antiquitatis) fossil record (Zong et al., 1985), while the WL had a warmer and precipitation-rich climate during the same Appendix A. Supplementary material period (Williams et al., 1993). Considering that climate transition or habitat preference can be the impetus for population divergence Supplementary data associated with this article can be found, in (Liu et al., 2010; Yeung et al., 2011) even in the face of considerable the online version, at http://dx.doi.org/10.1016/j.ympev.2012. gene flow (Yeung et al., 2011), the process of adapting to the differ- 12.013. 138 L. Gu et al. / Molecular Phylogenetics and Evolution 67 (2013) 129–139

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