ORIGINAL PAPER

Chloroplast diversity and population differentiationof Castanopsis fargesii (Fagaceae): a dominant tree speciesin evergreen broad-leaved forest of subtropical China

Ye Sun & Huaqiang Hu & Hongwen Huang &

Carlos Fabián Vargas-Mendoza

Received: 6 February 2013 /Revised: 29 May 2014 /Accepted: 4 July 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Subtropical forests in China constitute the majorexpanse of evergreen broad-leaved forest in East Asia. Thesignificant genetic divergence of the keystone tree speciesshould be expected due to the huge geomorphological andenvironmental changes fromwest to east in subtropical China.In this study, a total of 652 individuals from 27 populations ofCastanopsis fargesii throughout its natural range in mainlandChina were genotyped with eight chloroplast microsatellitemarkers to investigate genetic diversity, population differenti-ation, and demographic history ofC. fargesii. Phylogeograph-ic structure among populations of C. fargesii was evidencedby the permutation test, revealing that NST was significantlyhigher than GST. The strong genetic differentiation foundamong populations was well in accordance with isolation-by-distance model. In addition, significant isolation by eleva-tion was detected among populations. Significant geneticdifferentiations were revealed among the west, center, andeast regions by approximate Bayesian computations (ABC).The genetic divergence might reflect the regional responses tothe fast and dramatic uplift of Yunnan-Guizhou Plateau andWuyi mountain range in the Pleistocene. In the present study,

contraction-expansion process was detected in the west, cen-ter, and east regions, indicating that geomorphological remod-eling together with climatic changes in the Pleistocene hadstrong impact on genetic structure of C. fargesii.

Keywords Castanopsis fargesii . Chloroplast microsatellite .

Evergreenbroad-leaved forest .Genetic structure .SubtropicalChina

Introduction

Subtropical evergreen broad-leaved forest (EBLF) is one ofthe most important vegetation types in the world. This type offorest is widely distributed in East Asia, the North AmericanAtlantic coast, the Mediterranean coast of Europe, and anumber of localities in Oceania, South America and Africa(Song et al. 2005; Chen and Li 2008). Subtropical EBLFoccurs in China from 24 to 32° N latitude and from 99 to123° E longitude, and covers approximately 25 % of the areaof the country (Wang et al. 2007).

Evergreen broad-leaved forest has sustained extraordinarychanges beginning in the late Tertiary period (Song et al.2005). Based on pollen data, broad-leaved evergreen/warmmixed forests are believed to be forced to retreat southward inthe lowlands as far as 24° N at the last glacial maximum inQuaternary (Yu et al. 2000; Harrison et al. 2001; Ni et al.2010). However, phylogeographic studies of plant speciesfrom subtropical China do not reveal clear patterns of post-glacial re-colonization from the south, and suggested a patternof range fragmentation and only localized range expansion(Gao et al. 2007; Qiu et al. 2011; Lei et al. 2012). Thus, moreancient events before the last glacial maximum (LGM) shouldbe considered in phylogeographic studies of subtropical or-ganisms in China (Zhang et al. 2013), particular for old-growth forest taxa (Cannon and Manos 2003).

Communicated by A. Kremer

Electronic supplementary material The online version of this article(doi:10.1007/s11295-014-0776-3) contains supplementary material,which is available to authorized users.

Y. Sun (*) :H. Hu :H. HuangKey Laboratory of Plant Resources Conservation and SustainableUtilisation, South China Botanical Garden, Chinese Academy ofSciences, Guangzhou 510650, People’s Republic of Chinae-mail: sun-ye@scbg.ac.cn

H. HuUniversity of Chinese Academy of Sciences, Beijing 100049, China

C. F. Vargas-MendozaEscuela Nacional de Ciencias Biológicas-I.P.N., Prolongación deCarpio y Plan de Ayala s/n, 11340 Mexico, D.F., Mexico

Tree Genetics & GenomesDOI 10.1007/s11295-014-0776-3

Geographical processes such as tectonic events have servedas major driving force for regional climate shifts (Ravelo et al.2004) and population diversification of organisms (Yan et al.2013). Uplift of the Qinghai-Tibetan Plateau, particularlyduring the Quaternary period, remodeled the geomorphologyof China and changed the terrain to a hypsographic ladder inthree wide steps from high in the west to low in the east(Zhang et al. 2000). Three gradient terrains have far-reaching effects on climatic environments (Yang et al. 1989),as a result, climatic difference between western and easternChina became evident, bringing about a longitudinal differen-tiation of the flora in this region (Jin et al. 2003). Forestfragmentation and population isolation should be expectedduring the formation of three gradient terrains. Recent studieshave revealed west–east lineage split in the southwesternregion of subtropical China and highlighted the impact ofgeological and ecological factors on phylogeographic diver-gence (Yan et al. 2012, 2013; Fan et al. 2013; Liu et al. 2013).However, the impacts of the gradient terrains on geneticstructuring of plant species in central and eastern China arestill little known. A large-scale genetic study of the keystonespecies with highest diagnostic value for EBLFs in subtropicalChina would be beneficial to improve our understanding ofthe impacts of gradient terrains on phylogeographic pattern ofthis region (Qiu et al. 2011), especially for population diver-sification from west to east.

The species composition of EBLF in China is extremelydiverse and complex. Natural subtropical EBLFs in this regionare dominated by genera such as Castanopsis, Lithocarpus,Cyclobalanopsis (Fagaceae), Machilus (Lauraceae), Schima(Theaceae), Distylium (Hamamelidaceae), Magnolia, andMichelia (Magnoliaceae) (Tang and Ohsawa 2009). The ge-nus Castanopsis is one of the most important taxa in tropicaland subtropical regions of China, with about 58 species dis-tributed in China (Huang and Bruce 1999), primarily in flo-ristic regions of the Dian-Qian-Gui biogeographical region(bordering areas of the Provinces of Yunnan, Guizhou, andGuangxi), the Yunnan Plateau, and South China (Liu andZhou 2006).

Castanopsis fargesii Franch. is a major dominant treespecies in EBLFs of subtropical China. The trees of thisspecies are 10–30 m tall and capable of exploiting lightenvironments of different forest gaps. C. fargesii is mainlydistributed in the south of the Yangtze River and displayssubstantial variation on leaves and cupules (Huang andBruce 1999). The significant genetic divergence should beexpected due to the huge geomorphological and environmen-tal changes from west to east in subtropical China. Investigat-ing the geographic pattern of genetic diversity of C. fargesiiwill further facilitate our understanding of the role of geologyand climate in shaping the evolutionary history of the organ-ism in subtropical EBLF of China. In this study, populationgenetic analysis of C. fargesii was performed with extensive

sampling using chloroplast microsatellite markers in order to:(1) get insight into the genetic diversity and population struc-ture of this tree species and (2) investigate the possible geneticdivergence among the east, center, and west regions, and testdifferent scenarios of population divergence with approximateBayesian computations (ABC).

Materials and methods

Population sampling

A total of 27 populations comprising 652 individuals ofC. fargesii (Table 1 and Fig. 1a) were sampled throughoutits distribution range in China. Ten to 44 individuals from eachpopulation were sampled at intervals of at least 20 m to avoidcollecting close relatives. Fresh leaves were collected anddried in silica gel. Voucher specimens were made for eachpopulation and have been stored in the Herbarium of theSouth China Botanical Garden (formerly the South ChinaInstitute of Botany, IBSC), Chinese Academy of Sciences.

Chloroplast microsatellite genotyping

Total DNAwas extracted using the CTAB procedure of Doyleand Doyle (1987). In a pilot experiment, 11 polymorphicchloroplast microsatellites were screened from those de-scribed in Weising and Gardner (1999), Deguilloux et al.(2003), and Sebastiani et al. (2004). Polymerase chain reac-tion (PCR) was performed in 10 μL of a reaction mixtureconsisting of 1×PCR buffer, 0.2 mM dNTPs, 1.5 mMMgCl2,0.25 μM of each primer, 40 ng of DNA, and 1 U of Taq DNApolymerase (TaKaRa, Dalian, China). Amplification was per-formed in a PTC-200 thermocycler (Bio-Rad Laboratory,Hercules, California, USA) as follows: initial denaturationfor 10 min at 95 °C, followed by 30 cycles of 1 min at95 °C, 1.5 min at annealing temperature (see Table S1),1.5 min at 72 °C, and final extension for 10 min at 72 °C. PCRproducts were separated on denaturing 6 % polyacrylamidegels and visualized by staining with silver nitrate. Alleles weresized by comparison with relative fragment positions in a 25-bp DNA ladder (Promega, Madison, WI, USA). Each ampli-fied PCR product at all loci was sampled and sequenced toinvestigate allele-size variation across populations (GenBankaccession numbers: JX839991-JX840066). Finally, eightchloroplast microsatellites (see Table S1) were used in geneticanalysis.

Genetic analysis

A unique combination of size variants at eight microsatelliteswas defined as a different haplotype (Pardo et al. 2008).Effective number of haplotypes (Ne), unbiased haplotype

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diversity (He), and haplotypic richness (Hr) were estimated ineach population, using the program CONTRIB version 1.02(Petit et al. 1998). Genetic diversity and genetic differentiationparameters including mean within-population gene diversity(HS), total gene diversity (HT), the coefficient of genetic

differentiation across all populations (GST), as well as thecorresponding parameter NST obtained by taking into accountthe evolutionary distance between haplotypes, were calculatedwith PERMUTCPSSR v2.0 (Pons and Petit 1996). Existenceof phylogeographic structure was inferred by testing for

Table 1 Geographic characteristics, sample size, and haplotype diversity in 27 populations of Castanopsis fargesii

Province Population (code) Samplesize

° Nlatitude

° Elongitude

Elevation(m)

Haplotypes Haplotypediversity(He)

Haplotypicrichness(Hr)

Tajima’sD

SSD value(mismatchanalysis)

West region 14.000 0.848 1.372 0.110*

Sichuan Yingjing (YJ) 10 29°46′ 102°52′ 1,015 H12 (10) 0.000 0.000 0.000 0.000

Chongqing Jinyunshan (JYS) 28 29°50′ 106°23′ 732 H13 (28) 0.000 0.000 0.000 0.000

Yunnan Daweishan (DWS) 24 22°55′ 103°42′ 2,039 H14 (24) 0.000 0.000 0.000 0.000

Malipo (MLP) 24 23°10′ 104°47′ 1,975 H11 (7) H15 (1) H12(15) H6 (1)

1.823 0.543 1.915 0.449

Funing (FN) 21 23°28′ 105°36′ 1,436 H1 (3) H2 (1) H3 (1)H4 (4) H5 (1) H7(1) H8 (8) H9 (2)

4.463 0.819 2.184 0.116

Guizhou Xishui (XS) 24 28°32′ 106°23′ 848 H10 (22) H14 (2) 0.670 0.159 −0.890 0.034

Central region 21.388 0.932 2.126 0.096*

Guizhou Fanjingshan (FJS) 27 27°53′ 108°43′ 930 H25 (27) 0.000 0.000 0.000 0.000

Libo (LB) 44 25°29′ 107°53′ 728 H22 (8) H29 (10)H30 (24) H46 (2)

2.252 0.630 0.415 0.169

Guangxi Damingshan (DMS) 24 23°29′ 108°26′ 1,207 H21 (4) H28 (1)H45 (19)

1.322 0.359 0.503 0.121

Dayaoshan (DYS) 24 24°09′ 110°07′ 927 H28 (5) H48 (19) 0.953 0.344 0.780 0.163

Huaping (HP) 24 25°38′ 109°54′ 700 H23 (1) H24 (1) H31(7) H49 (15)

1.823 0.543 2.229 0.217

Hunan Longshan (LSH) 24 29°26′ 109°43′ 630 H35 (6) H36 (1) H22(9) H37 (3) H38(1) H39 (1) H43 (3)

3.866 0.793 2.217 0.166

Huitong (HT) 24 26°56′ 109°55′ 499 H23 (21) H28 (3) 0.820 0.228 −0.446 0.075

Yuelushan (YLS) 24 28°11′ 112°56′ 160 H20 (24) 0.000 0.000 0.000 0.000

Guangdong Tianjingshan (TJS) 20 24°41′ 112°59′ 568 H17 (1) H20 (16) H22(2) H27 (1)

1.763 0.363 −1.616* 0.086

Chebaling (CBL) 24 24°43′ 114°15′ 402 H33 (1) H34 (12) H18(1) H21 (9) H42 (1)

2.248 0.630 0.851 0.217

Nankunshan (NKS) 24 23°38′ 113°53′ 351 H40 (21) H19 (1) H21(2)

1.087 0.236 −0.706 0.053

Jiangxi Lushan (LS) 20 29°31′ 115°54′ 239 H44 (20) 0.000 0.000 0.000 0.000

Dagangshan (DGS) 24 27°35′ 114°33′ 345 H22 (1) H30 (1) H47(22)

0.833 0.163 −1.495 0.024

Jinggangshan (JGS) 22 26°32′ 114°11′ 528 H47 (22) 0.000 0.000 0.000 0.000

Jiulianshan (JLS) 24 24°31′ 114°27′ 646 H22 (23) H47 (1) 0.417 0.083 −1.884* 0.010

East region 10.206 0.770 2.272 0.170*

Zhejiang Ningbo (NB) 26 29°48′ 121°47′ 314 H23 (26) 0.000 0.000 0.000 0.000

Gutianshan (GTS) 24 29°15′ 118°07′ 462 H29 (14) H30 (7)H44 (1) H46 (2)

2.077 0.591 −0.228 0.164

Fujian Wuyishan (WYS) 24 27°37′ 117°48′ 384 H34 (23) H41 (1) 0.417 0.083 −2.210* 0.010

Jianning (JN) 24 26°45′ 116°48′ 430 H34 (24) 0.000 0.000 0.000 0.000

Dehua (DH) 24 25°38′ 118°22′ 390 H34 (24) 0.000 0.000 0.000 0.000

Anxi (AX) 26 25°16′ 117°38′ 355 H16 (2) H26 (11)H28 (1) H30 (8)H32 (4)

2.885 0.723 0.963 0.310

*P<0.05

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significant differences between GST and NST usingPERMUTCPSSR v2.0 with 1,000 permutations. A reducedmedian-joining network was constructed with NETWORKv4.5.1.6 (Bandelt et al. 1999) to infer haplotype relationships.

Correlations between the genetic distance matrix and thegeographic distance matrix or difference in elevation wereevaluated by Mantel test as implemented in IBDWS v3.23(Jensen et al. 2005) with 1,000 randomizations. Geographicaldistance between two pairs of coordinates was obtained fromhttp://www.gpsvisualizer.com/calculators. Analysis ofmolecular variance (AMOVA) was conducted by usingARLEQUIN v3.1 (Excoffier et al. 2005) to partition geneticvariation among different hierarchical levels under two hy-potheses: the first considered that there was no geographicseparation among populations. The second hypothesis com-pared three different groups of populations in the west(Yingjing, Jinyunshan, Daweishan, Malipo, Funing, andXishui), central (Fanjingshan, Libo, Damingshan, Dayaoshan,Huaping, Longshan, Huitong, Yuelushan, Tianjingshan,Chebaling, Nankunshan, Lushan, Dagangshan, Jinggangshan,and Jiulianshan), and east (Ningbo, Gutianshan, Wuyishan,Jianning, Dehua, and Aanxi) regions of the study area, takinginto account hypsographic terrains of the topography in sub-tropical China and the fact that the Wuyi mountain range is amajor dividing line between the center and the east in China.

Distribution of pairwise size differences between haplo-types was examined within populations using the nucleotide

mismatch distribution test (Rogers and Harpending 1992) toinfer demographic history. Frequency distribution was pre-dicted to be unimodal and with a Poisson-shaped distributionin lineages that had undergone recent population expansions,and multimodal in those whose populations were in equilibri-um. The sum of squared deviations (SSD) for observed andexpected mismatches was compared in order to test the hy-pothesis of pure demographic expansion, and theHarpending’s raggedness index of the observed distributionwas used as a criterion of spatial expansion (Schneider andExcoffier 1999), a significant P value rejected the fit of thedata to the expansion model. All tests were performed withARLEQUIN v3.1. Demographic history (population spatialexpansion) was also tested by calculating the significance ofTajima’s D (Tajima 1989) and Fu’s FS (Fu 1997) withARLEQUIN v3.1.

The DIYABC v. 1.0.4.39 (Cornuet et al. 2008) was used toinvestigate the possible genetic divergence among the east,center, and west regions, and test different scenarios of popu-lation divergence (Fig. S1). We pooled all populations in eachregion into three big populations accordingwithAMOVAsecondhypothesis, and adopted a stepwise procedure to performABC analysis. We firstly defined four competing scenarios(scenarios 1–4, Fig. 2) regarding the pattern of genetic diver-gence among the three regions. In these cases, each regionremained at a constant effective population size until presenttime. Once the divergence pattern among the three regions

Fig. 1 a Sampling localities andchloroplast haplotypes of the 27populations of Castanopsisfargesii. Black and red dashedlines show geographical divisionof west, center, and east regions ofthe study area. The inset mapindicates the geographicalfeatures of China and thedistribution range of C. fargesii inChina. b Haplotype relationshipsshown on median-joiningnetwork. Open circles representunsampled or extinct haplotypes;dashed line represents ambiguousconnections; size of each circle isproportional to the haplotypefrequency

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was given, several hypotheses (scenarios 1.1–1.4, Fig. 2) ofold contraction-expansion processes were tested. After the oldcontraction-expansion process has been determined, recentpopulation expansion processes (scenarios 1.3.1–1.3.4,Fig. 2) were checked. Summary statistics for each populationincluded mean number of alleles, mean gene diversity, meanallele-size variance, and FST between two samples (Weir andCockerham 1984). One million simulations were run for eachscenario. Models were compared by estimating their posteriorprobabilities using the direct estimation and logistic regressionmethod (Cornuet et al. 2008).

Results

Genetic diversity and differentiation

A total of 27 size variants at the eight chloroplastmicrosatellites and 49 haplotypes were identified among the27 populations of C. fargesii (see Table S1, S2). Size variantcombinations and the sequences of each haplotype wereshown in Table S2. Six loci (cmcs5, ucd4, udt1, udt3, ukk3,ccmp3) were confirmed to be variable as microsatellite, butsize variants at locus cmcs14 were due to two insertions (1-bpand 7-bp), and locus ccmp10 had two insertions (1-bp and 12-bp).

The most polymorphic populations were Funing (FN) inYunnan province and Longshan (LSH) in Hunan provincewith 8 and 7 haplotypes, respectively. The most commonhaplotype was H34, found mainly at Jianning (JN), Dehua(DH), and Wuyishan (WYS) in Fujian province, but alsooccurring at Chebalin (CBL) in Guangdong province. In thewest region populations (Yingjing, Jinyunshan, Daweishan,Malipo, Funing, and Xishui), the haplotype H13 obtained thehighest frequency. No haplotype was shared between the westregion populations and the rest of the populations sampled.Ten populations were fixed by a single haplotype. Total ge-netic diversity (HT=0.973) across all populations was muchhigher than within-population gene diversity (HS=0.270), andpopulation differentiation values were high (GST=0.722,RST=0.847).

Population genetic structure

A permutation test showed that NST (0.831) was significantlyhigher than GST (0.722), suggesting a phylogeographic struc-ture occurred among populations of C. fargesii. Relationshipsamong haplotypes were shown in Fig. 1b. Haplotypes 1 to 15were detected only in the west region populations of Funing(FN), Malipo (MLP), Daweishan (DWS), Yingjing (YJ),Jinyunshan (JYS), and Xishui (XS). Haplotypes 1–15 consti-tuted a haplogroup together with H34, H35, H37, H38, and

H40, and closely connected with H17, H18, H23, H24, andH49, but divergent from other haplotypes.

Significant correlations between genetic distance and geo-graphic distance were determined by Mantel test (r=0.4305,P<0.001) (Fig. 3a), and significant correlations between ge-netic distance and difference in elevation were also detected(r=0.2919, P<0.001) among the 27 populations ofC. fargesii(Fig. 3b). The AMOVA analysis showed 82.64 % of molec-ular variation partitioned among populations and 17.36 %within populations in the one-group hypothesis (Table 2).Testing of the three-group hypothesis showed strong separa-tion among populations in the east, center, and west regions(FCT=0.349, P=0.000).

Historical demography

The value obtained for Tajima’s D was positive but did notdiffer significantly from zero in the overall data set (D=2.920,P=0.983). Estimates at the population level were negative ineight populations, but significantly different only in theJiulianshan (JLS, −1.884, P=0.01), Wuyishan (WYS,−2.210, P=0.001), and Tianjingshan (TJS, −1.616, P=0.037) populations. The value obtained for Fu’s FS was neg-ative but not significantly different in the overall data set (D=−4.173, P=0.358). At the population level, negative estimateswere obtained only in the Dagangshan (DGS) population(−0.142,P=0.355). For overall data set, mismatch distributionanalysis rejected the hypothesis of historical expansion(Harpending’s raggedness index=0.289, P=0.000). At thepopulation level, mismatch distribution analysis showed thatthe DGS, JLS, WYS, XS, DMS, and TJS populations did notreject the hypothesis of historical population expansion(Harpending’s raggedness index=0.727, P=0.637; 0.854,P=0.867; 0.854, P=0.836; 0.757, P=0.625; 0.577, P=0.409; and 0.499, P=0.448, respectively). On the other hand,the distribution of pairwise mutation differences in the GTS,LB, CBL, FN, AX, and LSH populations was significantlydifferent from that expected in a Poisson distribution(Harpending’s raggedness index=0.620, P=0.035; 0.631,P=0.002; 0.495, P=0.003; 0.342, P=0.004; 0.924, P=0.000; and 0.484, P=0.000, respectively) and did not fit amodel of spatial expansion. Demographic equilibrium wasalso assumed to occur in the HT, DYS, HP, MLP, and NKSpopulations since these rejected the hypothesis of pure demo-graphic expansion (the sum of squared deviations (SSD)=0.075, P=0.018; 0.163, P=0.034; 0.217, P=0.050; 0.449,P=0.000; and 0.053, P=0.036, respectively). Remaining pop-ulations could not be analyzed due to the low geneticvariation.

The ABC analysis gave the greatest support for the scenar-io 1 regarding to the pattern of divergence among the threeregions. The posterior probabilities of this scenario were 0.872(95 % CI=0.579–1.000) and 0.930 (95 % CI=0.923–0.936)

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for direct estimation and logistic regression, respectively. Thishypothesis established that the west was the first region to beseparated. Then the east region was diversified, and finally thecenter population detached from the east region. In the secondstep of ABC analyses, scenario 1.3 that the west region branchexperienced an old bottleneck followed by a population

expansion was highly supportive, the posterior probability oflogistic regression was 0.335 (95 % CI=0.305–0.366). Thedirect estimate of probability for this scenario was not highest,but without significant difference to other scenarios. In thethird step of ABC analyses, scenario 1.3.4 that the center andeast regions have experienced a recent contraction-expansion

Fig. 2 Different evolutionary scenarios tested on three pooled big pop-ulations in the west (BW), center (BC), and east (BE) regions withapproximate Bayesian computations (ABC) step by step. Scenarios 1–4regarding the pattern of genetic divergence among the three regions, eachregion remained at a constant effective population size in these cases.Once the divergence pattern among the three regions was given, scenarios

1.1–1.4 hypothesized old contraction-expansion. Scenarios 1.3.1–1.3.4checked recent population expansion processes. An ancestral population(NT) is in grey dotted line; effective population size in an old event ofbottleneck (blue dotted line) and expansion (red solid line). Green dottedline is a recent event of bottleneck, and the dark red solid line is a recentexpansion event

Fig. 3 Isolation by distance (a) and isolation by elevation (b) among the 27 populations of Castanopsis fargesii. (Distance is expressed logarithmicallyand elevation difference is expressed as meters)

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process obtained highest posterior probabilities. The posteriorprobabilities of this scenario were 0.434 (95 % CI=0.000–0.868) and 0.700 (95 % CI=0.672–0.727) for direct estima-tion and logistic regression, respectively. The divergence ofthe west region was the oldest and happened about 33,300generations ago, while the split between the center and the eastoccurred 2,730 generations ago (Table S3). An old bottleneck-expansion happened about 32,794 generations ago in the westregion, while a recent expansion occurred in both the centerand east region 1,910 generations ago.

Discussion

Genetic diversity was high in C. fargesii (HT=0.973, 49haplotypes based on 8 cpSSR loci) compared with otherspecies in the family Fagaceae, such as Castanopsis hystrix(HT=0.686, 14 haplotypes based on 7 cpSSR loci) in southChina (Li et al. 2007), Lithocarpus densiflorus (HT=0.40, 6haplotypes based on 5 cpSSR loci) in California (Nettel et al.2009), Quercus semiserrata (HT=0.93, 16 haplotypes basedon 9 cpSSR loci) in northern Thailand (Pakkad et al. 2008),and Quercus lobata (HT=0.979, 39 haplotypes based on 6cpSSR loci) in California (Grivet et al. 2006). Although directcomparison of genetic diversity among these species was notpossible due to differences in sampling and cpSSR loci, likelyexplanation for the difference observed might be thatC. fargesii populations had persisted in subtropical China fora long time as stone oaks (Cannon and Manos 2003) and notundergone a severe demographic change like tanoak andEuropean oaks (Grivet et al. 2006; Nettel et al. 2009). Main-taining high level of genetic diversity within a species wascrucial for its survival in face of climate change. Ten popula-tions (Yingjing (YJ), Jinyunshan (JYS), Daweishan (DWS),Fanjingshan (FJS), Yuelushan (YLS), Lushan (LS),Jinggangshan (JGS), Ningbo (NB), Jianning (JN), and Dehua(DH)) were fixed by single haplotype. Most of these

populations were geographically located on the periphery ofthe distribution range of C. fargesii. These geographicallyperipheral populations should exhibit lower genetic diversityand higher genetic differentiation as a result of smaller effec-tive population size and high geographic isolation relative togeographically central populations (Eckert et al. 2008). Ge-netic differentiation among populations was high (GST=0.722, RST=0.847) and followed an isolation-by-distancemodel, this was consistent with the observation that the dis-persal distance of seeds of C. fargesii by rodents was quiteshort (Zhang et al. 2006) and partially explained significantdifferentiation among populations within the region. The geo-graphical topologies were very complex due to mosaic distri-bution of mountains, which might further increase populationisolation within regions.

Rapid and dramatic uplifting of plateau and mountainranges would cause habitat fragmentation and influence thegenetic discontinuities of organisms (Yan et al. 2013). In thisstudy, significant genetic differentiations were revealedamong the west, center, and east region populations (Table 2,Fig. 2). The genetic divergence might reflect the regionalresponses to the fast and dramatic uplift of the Yunnan-Guizhou Plateau and Wuyi mountain range. In the west re-gion, Daweishan (DWS), Malipo (MLP), Funing (FN),Yingjing (YJ), Jingyunshan (JYS), and Xishui (XS) popula-tions bordered the Sichuan Basin and the Yunnan-GuizhouPlateau, which is geographically located on the second step ofthe hypsographic ladder of China. These six populations in thewest were genetically distinguished from the rest of the pop-ulations sampled in the third level of ladder of the Chineseterrain. There were not shared haplotypes between the westregion and center or east region, suggesting the divergencewas ancient. The ABC analysis also indicated that the westregion population separated from the other two regions longago. By accounting for the generation time of Castanopsisspecies which was about 25 years (Liu et al. 2008), thisdivergence time of the west was about 0.833 million years(Ma) before the present, and the differentiation between the

Table 2 Results of AMOVA on27 populations of Castanopsisfargesii

***P<0.000

Source of variation Degree offreedom

Sum ofsquares

Variancecomponents

Percent variation

Single group

Among populations 26 1111.61 1.75810 82.64 FST=0.826***

Within populations 625 230.789 0.36926 17.36

Total 651 1342.402 2.12736

Three groups

Among the east, central,and west regions

2 774.886 1.72295 34.9 FCT=0.349***

Among populationswithin groups

24 1448.340 2.47527 50.144 FSC=0.770***

Within populations 625 461.578 0.73852 14.96 FST=0.850***

Total 651 2684.804 4.77728

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center and the east along the Wuyi mountain range was about0.068 Ma ago. These two periods were broadly consistentwith the rapid and dramatic uplifting of the Yunnan-GuizhouPlateau and Wuyi mountain range. Neotectonic movementduring the Pliocene and Pleistocene led to the uplift of westernChina, with more vigorous speed of uplifting since the mid-Pleistocene (around 0.9 Ma) and subsequent phased upliftscontinuing up to the present (Ming 1987; Yang et al. 1989).The uplift of the Yunnan-Guizhou Plateau began since 2.8 Maand had a fast and dramatic uplift around 0.8 Ma (Tang et al.1994), which was very close to the divergence time of the westregion population in the present study. The Wuyi mountainrange gradually uplifted following the Himalayan movement,with concussive uplifting in the late Pleistocene (Chen andZhou 1993; Luo et al. 2010), which was also consistent withthe divergence time of the center and east region populations.Thus, a significant pattern of isolation by elevation (Fig. 3b)should be expected in this study and indicated the role ofgeography in shaping evolutionary history.

Castanopsis species had a wide distribution in Asia duringthe Eocene and Miocene, and underwent range reductiontoward the south triggered by global cooling in the Plioceneand aridification in central China (Liu and Zhou 2006), butpersisted in subtropical China as suggested by a recent paly-nological study that clearly showed the forest taxa, includingCastanopsis, existed in the Dajiuhu (31°29′ N, 110°00′ E) ofCentral China throughout the past 40,000 years, with conifersand evergreen tree dominating the glacial period (Li et al.2012). Morphotectonic events in the Pliocene and Pleistocenecaused fragmentation of evergreen broad-leaved forest insubtropical China, and genetic differentiation built up amongfragmented forests. In the middle and late Pleistocene, theclimate in subtropical China was characterized by repeatedalternation by cold and warm (Cao 1990), and mountainvegetation in south China moved up and down (Jin et al.2003). Contraction-expansion process detected in each regionby ABC analyses highlighted the climatic oscillation andforest vegetation change during this period. The strongestsignals of demographic expansion at population levels weredetected in the east populations (Jiulianshan (JLS),Tianjingshan (TJS), and Wuyishan (WYS)) by the analysisof mismatch distribution and Tajima’s D might relate to thefact that climatic shifts were more obvious in eastern andcentral China during the middle Plistocene (Yang et al. 1989).

The haplotypes from the west and east regions had fairlyradical differences in their structure—the east very compactand frequent with less missing haplotypes and the west quitesparse with many missing haplotypes would suggest intro-gression of types from other species. It is quite possible thatthe western populations were so unique from the easternpopulations because of secondary contact with another spe-cies. However, it is impossible to make a definitive statementwithout sampling other sympatric Castanopsis species. This

should be tested with a specific fieldwork and with moremarkers.

Acknowledgments We thank three anonymous reviewers for theircritical comments. This study was financially supported by the NationalNatural Science Foundation of China (31170512, 30871959, and31370668), and by the Knowledge Innovation Program of the ChineseAcademy of Sciences (KSCX2-EW-J-28).

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