Molecular Phylogenetics and Evolution 57 (2010) 176–188
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Molecular Phylogenetics and Evolution
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Strong phylogeographic pattern of cpDNA variation reveals multiple glacial refugia for Saruma henryi Oliv. (Aristolochiaceae), an endangered herb endemic to China
Tian-Hua Zhou a,b, Shan Li c, Zeng-Qiang Qian d, Hai-Lun Su a, Zhao-Hui Huang a, Zhi-Gang Guo a, Pan-Feng Dai a, Zhan-Lin Liu a, Gui-Fang Zhao a,* a Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education), College of Life Sciences, Northwest University, Xi’an 710069, PR China b College of Life Sciences and Engineering, Shaanxi University of Technology, Hanzhong 723001, PR China c College of Life Sciences and Technology, Tongji University, Shanghai 200092, PR China d School of Marine and Tropical Biology, and Comparative Genomics Centre, James Cook University, Townsville, QLD 4811, Australia article info abstract
Article history: Saruma henryi Oliv. (Aristolochiaceae) is an endangered herb endemic to China. In this study, chloroplast Received 13 November 2009 microsatellites (cpSSRs) and sequences of the atpB–rbcL intergenic spacers were employed to reveal its Revised 23 June 2010 genetic diversity and phylogeographic patterns. We detected high within-species genetic diversity Accepted 2 July 2010 (H = 0.939 for cpSSR; H = 0.862 for atpB–rbcL) and pronounced among-population genetic differentia- Available online 14 July 2010 T T tion (HS = 0.182, GST = 0.811, RST = 0.9, FST = 0.93 for cpSSR; HS = 0.238, GST = 0.724, NST = 0.758, FST =
0.79 for atpB–rbcL) with a strong phylogeographic pattern (RST > GST, P < 0.01 for cpSSR; NST > GST, Keywords: U = 0.25 for atpB–rbcL). Eleven haplotypes were distinguished by cpSSRs and atpB–rbcL intergenic spac- Saruma henryi Oliv. ers, respectively. The molecular phylogenetic data, together with the geographic distribution of the hap- Endemic species Phylogeography lotypes, suggested the existence of multiple localized glacial refugia in Mts. Qinling, eastern Mts. Bashan cpSSR and the southeastern edge of Yunnan-Guizhou Plateau. Nested clade analysis (NCA) and population atpB–rbcL genetic analyses supported the limited gene flow (caused by low dispersal capacity and complex topog- Population genetics raphy of its habitats) as the major factor responsible for the strong population differentiation and phylog- eographic pattern. Past fragmentation and allopatric fragmentation were inferred as important processes responsible for the modern phylogeograhpic pattern. In addition, contiguous range expansions occurred in western Mts. Qinling and eastern Mts. Bashan. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Union for Conservation of Nature (IUCN) (China Plant Specialist Group, 2004), and needs urgent protection and restoration. Saruma Oliv. is an isolated relic genus within the family Aristolo- To date, previous studies of S. henryi have been mainly focused chiaceae, and comprises a single species, Saruma henryi Oliv. (Hsu, upon its morphology and anatomy (Dickison, 1996; González, 2005). As a typical Central China distributed endemic species (Wang 1999; González et al., 2001; González and Stevenson, 2002; Kelly and Zhang, 1994), this plant is mainly dispersed in Gansu, Guizhou, and González, 2003), cytology (Li et al., 1994b; Sugawara, 1987), sys- Hubei, Jiangxi, Henan, Shaanxi and Chongqing Provinces (see Fig. 1), tematics (González, 1999; Gregory, 1956; Ma, 1990; Neinhuis et al., occurring primarily in shady and moist locations with an elevation 2005; Jaramillo and Kramer, 2007; Wanke et al., 2006), reproductive of 600–1600 m (Cheng et al., 1988; Ying and Zhang, 1994). It pos- biology (Zhao et al., 2006, 2005), pharmacognosy (Liu et al., 1994; sesses high phylogenetic, ecological and medicinal significances Peng et al., 2005) and chemical contents (Dong et al., 2009; Iwashina (Gregory, 1956; Ma, 1990; Igarashi and Fukuda, 1997; Cheng et al., et al., 2002). Using dominant inter-simple sequence repeat (ISSR) 1988; reviewed by Zhou et al. (2010)). However, due to habitat dete- markers, Zhou et al. (2010) reported low within-population genetic rioration and artificial over-exploitation, S. henryi has been listed as diversity and high among-population genetic differentiation in this an endangered species by both local and central governments in species. However, such information may be incomplete, due to the China (Di and Yu, 1989; Fu et al., 1993) and by the International limitations of the used molecular marker system. Additional studies based on other marker systems may help better reveal the ‘real’ pat- tern of genetic diversity in this species. China has the most diverse flora in the Northern Temperate * Corresponding author. Address: College of Life Sciences, Northwest University, Zone. Despite its being approximately the same size as Europe or 229, Northern Taibai Road, Xi’an, Shaanxi Province 710069, PR China. Fax: +86 29 88303572. continental United States, the number of vascular plant species E-mail address: [email protected] (G.-F. Zhao). in China is twice that of North America and three times that of
1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.07.001 T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188 177
Fig. 1. Geographic distribution of 16 S. henryi populations and the detected cpSSR (H1–H11) and atpB–rbcL haplotypes (A–K) in China. The triangles denote the localities of the sampled populations. The haplotypes for each population are marked out together above a black line. The circles and sequels represent cpSSR and atpB–rbcL haplotypes, respectively.
Europe. Hengduan range, Central China (the region marked in red przewalskii, a tree endemic to the Qinghai-Tibet Plateau region oval in Fig. 1, usually refers to the region along the middle and (Zhang et al., 2005); Alsophila spinulosa, a relictual tree fern distrib- downstream of Yangtze River, and mainly includes Hunan, Hubei, uted in southern China (Su et al., 2005a,b; Zhang et al., 2005); and Jiangxi, Anhui and Chongqing Provinces) and Lingnan region are Dunnia sinensis, an endangered, endemic shrub restricted to the regarded as the three regions with the highest levels of plant diver- southern part of Guangdong Province (Ge et al., 2002). Although sity and endemism in China (Ying and Zhang, 1994). Among these these studies have enriched our knowledge about the effects of his- regions, Central China is characterized by significant variation in torical events on the current geographic distributions of plant spe- topography, climatic and ecological conditions, and was spared cies in China, due to large gaps in taxonomic and habitat sampling, from direct effects of the repeated Pleistocene continental glacia- for example, there are few published data examining phylogeo- tions (Hu, 1980; Liu and Basinger, 2000). Up to 2001, about 6390 graphic patterns across a wide geographic range (Ran et al., species had been identified in this region, with 63.11% of them 2006) and relating to herbaceous plants’ phylogeography (Huang being endemic to China; a total of 92 endemic plant genera had et al., 2005). Our understanding of the historical biogeographic been recorded there, approximately accounting for 37.19% of the events in China still remains incomplete, and the formation mech- total of China (Ying, 2001). Thus, this region is also considered to anism of high diversity and endemism in Central China has yet to harbor the most typical and centralized flora of China. be further investigated. And, with short lifecycles and sensitivity to Although no massive ice sheet was developed in most areas of environmental changes, S. henryi may be an ideal candidate plant China during glacial periods, the tremendous global climatic for molecular phylogeographic studies to reveal the evolutionary changes, together with the local climatic changes caused by the history of plants in Central China since the late Tertiary and Qinghai-Tibet Plateau uplift particularly during Quaternary glacia- throughout the Quaternary. tions, have affected the distribution and evolution of many plant Molecular techniques, using organelle markers, have provided species in this area (Wang and Ge, 2006; Zhang et al., 2005). Recent powerful tools for studying the phylogeography or migratory phylogeographic studies of flora in China have mainly focused on footprints of species (Avise, 2000; Gao et al., 2007). In plants, the endangered or endemic species with narrow distribution chloroplast DNA (cpDNA) is thought to evolve slowly, with low ranges. And most phylogeographic studies of plant migration in mutation (Li and Fu, 1997; Wolfe et al., 1987) and recombination China have dealt with woody, long-lived tree or shrub species, rates (Clegg and Zurawski, 1992). Moreover, chloroplast genomes e.g. Cathaya argyrophylla, an endangered conifer restricted to sub- are particularly sensitive to the effects of fragmentation due to tropical mountains of China (Wang and Ge, 2006); Juniperus their smaller effective population sizes than nuclear genomes as 178 T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188 well as their restricted seed-mediated gene dispersal compared (10 mM pH 8.0 Tris–HCl; 1 mM pH 8.0 EDTA) for the subsequent with pollen-mediated gene flow. Thus, chloroplast-specific mark- use. ers should theoretically provide good indicators of historical bot- tlenecks, founder effects and genetic drift (Petit et al., 1997). In 2.3. CpSSR-PCR amplification the past decades, chloroplast simple sequence repeats (cpSSRs, a.k.a chloroplast microsatellites) have successfully established In this study, the universal cpSSR primers were described by their applications in studies of plant population genetics, phyloge- Weising and Gardner (1999) and Chung and Staub (2003), and syn- netics, germplasm identification and resource conservation thesized by Shanghai Sangon Biological Engineering Technology & (Parducci et al., 2001; Provan et al., 2001). Service Co., Ltd. (China). Fifteen primer pairs were initially Here we report our recent application of cpSSRs and sequencing screened, and 12 of them, which yielded bright and discernible of chloroplast DNA to the populations of S. henryi. Our specific bands, were used for the analyses of all 290 samples (Table 2). goals were to: (i) further reveal the genetic diversity and structure PCRs were performed in a 10-lL reaction volume containing: of this species; (ii) further understand the phylogeography of this 1.0 lL10 PCR buffer, 2 mM Mg2+, 0.2 mM each of dNTPs, 0.5 U species; and (iii) propose possible explanations for the high diver- Taq DNA polymerase, 0.4 lM primer and 50 ng DNA template. sity and endemism in Central China. The amplifications were performed in the thermal cycler PTC-200 (MJ Research) with the following program: initial denaturation at 2. Materials and methods 94 °C for 5 min; 35 cycles of 94 °C for 30 s, appropriate annealing temperature (see Table 2 for details) for 45 s, 72 °C for 55 s; and 2.1. Plant materials last synthesis at 72 °C for 7 min. A negative control with no DNA added was included in each PCR run. In all, 16 populations of S. henryi were sampled across seven prov- The amplification products (2 lL) were mixed with 0.5 lL load- inces in China, including Gansu, Shaanxi, Henan, Hubei, Jiangxi, ing buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol Chongqing and Guizhou Provinces (Table 1; Figs. 1 and 2). Among blue), denatured at 94 °C for 3 min and separated in 12% denatur- these populations, eight were sampled from Mts. Qinling region, ing polyacrylamide gels (7 M urea) in 1 TBE buffer at 180–220 V four from Mts. Bashan region, and four from the region south of Yan- for 6–8 h, along with pBR322DNA/BsuRI (HaeIII) size marker (MBI). gtze River. This sampling strategy covered most of its presently The bands were visualized by silver staining (Xu et al., 2002), and known populations (Ying and Zhang, 1994). Twenty individuals photographed using Bio-Rad Gel Documentation System (Bio-Rad were sampled from most of the studied populations, except three Laboratories, UK). of them (FX, ZN and XR) from which only 10 were collected due to their small population sizes. In addition, a population of Asarum pul- 2.4. Amplification and sequencing of atpB–rbcL intergenic spacers chellum Hemsl (Aristolochiaceae) was included as the outgroup (see Table 1). Fresh leaves were collected, dried in a ziplock bag with sil- Using universal primers, an initial screen for DNA sequence var- ica gel, transported back to the laboratory and kept in 80 °C freezer. iability of various chloroplast markers was conducted against 32 The neighboring individuals were at least 2 m apart, so as to avoid samples, with two from each population. The intergenic spacers resampling from the same individual. Corresponding to each popu- (IGSs) of the following pairs of genes were amplified using previ- lation, parameters such as longitude, latitude and altitude were re- ously published primers: trnL–trnF(Taberlet et al., 1991), trnT–trnF corded for further analysis. Voucher specimens were deposited in (Taberlet et al., 1991), trnV–trnM(Cheng et al., 2005), atpB–rbcL the Laboratory of Molecular Ecological Genetics, College of Life Sci- (Chiang et al., 1998), rpl20–rps12 and psbB–psbF(Hamilton, ences, Northwest University (see Table 1 for details). 1999). The sequences of trnT–trnF and trnV–trnM IGSs failed to be obtained due to weak sequencing signals, while no variation 2.2. DNA extraction was detected in the IGSs of trnL–trnF, rpl20–rps12 and psbB–psbF. Only atpB–rbcL revealed sequence variation in the screened indi- Total genomic DNA was extracted using a modified CTAB meth- viduals, and were used thereafter for the large-scale survey of hap- od (Doyle and Doyle, 1987), and then dissolved in 0.1 TE buffer lotype variation within S. henryi.
Table 1 Sampling details and genetic diversity of S. henryi populations.
Populations Locations Longitude (°E)/latitude (°N) Voucher number cpSSR atpB–rbcL
nNHe Hd nNPi Hd DC Dongcha, Tianshui, Gansu 34°190/106°410 SH06GS1 20 2 0.3947 0.10 5 1 0 0.20 MN Moonxia, Huixian, Gansu 33°390/106°170 SH06GS2 20 2 0.3947 0.10 5 2 0.00072 0.40 LB Miaotaizi, Liuba, Shaanxi 33°410/106°500 SH06SX1 20 2 0.4421 0.10 5 2 0.0024 0.40 TB Taibaishan, Meixian, Shaanxi 34°060/107°500 SH07SX2 20 2 0.2684 0.10 5 1 0 0.20 TP Taipingyu, Huxian, Shaanxi 33°540/108°390 SH07SX3 20 1 0.0000 0.05 5 1 0 0.20 LS Xiaogouhe, Lushi, Henan 33°490/112°120 SH06HN1 20 1 0.0000 0.05 5 2 0.0012 0.40 LC Danangou, Luanchuan, Henan 33°440/111°360 SH06HN2 20 1 0.0000 0.05 5 2 0.00576 0.40 YX Huaishu, Yunxi, Hubei 33°120/109°500 SH06HB5 20 1 0.0000 0.05 5 1 0 0.20 WD Wudangshan, Shiyan, Hubei 32°250/110°010 SH06HB4 20 2 0.2684 0.10 5 2 0.00144 0.40 FX Yangchashan, Fangxian, Hubei 31°540/110°300 SH06HB3 10 1 0.0000 0.10 5 1 0 0.20 SB Songbai, Shennongjia, Hubei 31°450/110°420 SH06HB2 20 1 0.0000 0.05 5 3 0.0012 0.60 HH Longmenhe,Xingshan, Hubei 31°240/110°300 SH06HB1 20 2 0.5053 0.10 5 1 0 0.20 WG Wugongshan, Luxi, Jiangxi 27°340/114°150 SH07JX1 20 1 0.0000 0.05 5 1 0 0.20 ZN Tanshan, Zhenning, Guizhou 25°460/105°510 SH07GZ1 10 1 0.0000 0.10 5 1 0 0.20 XR Huaishuping, Xingren,Guizhou 25°220/105°080 SH07GZ2 10 2 0.5333 0.20 4 2 0.0006 0.50 JF Jinfoshan, Nanchuan, Chongqing 29°040/107°170 2007CQ1 20 1 0.0000 0.05 4 1 0 0.25 Total – – – 290 11 0.8478 0.038 78 11 0.0024 0.128
Notes: n, sample size; N, number of haplotypes; He, gene diversity; Pi, nucleotide diversity; Hd, haplotype diversity. T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188 179
Fig. 2. Most parsimonious tree (A) and the 95% plausible network (B) of 11 cpSSR haplotypes of S. henryi (H1–H11). Bootstrap values (based on 1000 permutations) higher than 60% are indicated above branches of MP tree. On the network, the size of circles corresponds to the frequency of each haplotype. Each solid line represents one mutational step that interconnects two haplotypes for which parsimony is supported at the 95% level. The small solid circles in network indicate inferred intermediate haplotypes not detected in this investigation. In both MP tree and NCA network, the 11cpSSR haplotypes are formed into two grades. Grade I of MP tree corresponds to Clade 2–3, and Grade II to Clade 2–1.
Table 2 The length of amplified cpDNA fragments (in bp) for the analyzed loci and definition of 11 cpSSR haplotypes.
Primers 6# 7# 9# 13# 15# 16# 17# 18# 19# 20# 22# 23# Genes Rpob PsbbC–TrnS Ycf3 Rps3–Rpl22 Rpl2–Rpl23 TrnL Rrn5–TrnR Ycf5 Ycf5 NdhD–PsaC TrnL–16SrRNA Rpl12–TrnH
TA 60 62 59 60 62 60 60 59 60 60 59 60 H1 299 342 244 264 264 360 236 264 365 343 180 331 H2 299 342 238 264 268 360 236 264 365 343 180 331 H3 299 342 238 264 264 360 236 264 365 339 175 331 H4 299 342 238 264 264 360 236 264 365 343 175 331 H5 299 342 238 264 264 360 236 264 365 343 180 331 H6 299 342 244 239 264 360 236 264 365 343 180 331 H7 299 342 238 264 264 360 236 264 365 350 180 331 H8 299 346 238 260 264 360 236 264 365 350 180 331 H9 299 346 238 260 264 360 228 264 365 350 180 331 H10 299 346 238 260 264 360 228 260 365 350 180 331 H11 299 342 238 260 264 360 228 275 365 350 180 331 Asarum pulchellum 299 342 238 260 280 366 233 270 365 346 285 335
Notes: TA, annealing temperature (°C).
Four or five samples were randomly chosen from each popula- (Chiang et al., 1998). The amplifications were conducted in a 50- tion, and used for sequencing (Table 1). PCRs and DNA sequencing lL reaction volume containing: 5.0 lL10 PCR buffer, 2 mM were performed with universal primers: 50-CRGGTTGAGGAG Mg2+, 0.25 mM each of dNTPs, 2 U Taq DNA polymerase, 10 lM TTACTCG-30, and 50-GACCRGAAGTAGTAGGATT-30 for atpB–rbcL of each primer and 50 ng DNA template. PCRs were performed 180 T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188
Table 3 Chloroplast DNA sequence polymorphisms detected in the atpB–rbcL intergenic spacer (IGS) of S. henryi defining 11 haplotypes (A–K). The GenBank accession number of haplotype was given in the right column. ‘–’ indicates alignment gap.
Haplotype 33 79 101 148 152 156 167 204 301 462 463 465 673 676 GenBank Accession Nos. A G G T A C T G G G – G A T C GU190134 B G G T A C T A A G – G A T C GU190135 C G G T A C T A G G – G A T C GU190136 D AGTACTAGG–––TCGU190137 E G C T A C T A G G – G A T C GU190138 F G G T A C G A A G A G A T C GU190139 G G G T A A G A G G – G A C C GU190140 H G G T A C G A G C – G A C G GU190141 I G G T A C G A A C – G A C G GU190142 J AGC–CTAGG–––TCGU190144 K G G T A C T G G G A G A T C GU190143
in the thermal cycler PTC-200 (MJ Research) with the following (Swofford, 2002) using A. pulchellum as outgroup. Branch support program: initial denaturation at 94 °C for 4 min; followed by 35 cy- was assessed by bootstrap analysis with 1000 replicates of full heu- cles of 94 °C for 1 min, 50 °C for 1 min and 2 min extension at ristic searches. In addition, the phylogeographic history of this spe- 72 °C; and last synthesis at 72 °C for 7 min. cie was evaluated by nested clade analysis (NCA, Templeton et al., The sizes of PCR products were determined by agarose electro- 1995), which takes into account the geographic processes that cause phoresis. All PCR products were purified from agarose gels using genetic differences in natural populations. NCA is intended to dis- PCR Product Purification Kit (Shanghai Sangon Biological Engineer- criminate among a wide array of geographic processes and events ing Technology & Service Co., Ltd.), and sequenced in both direc- that shape species history, such as allopatric fragmentation, contig- tions by standard methods on an ABI 377 automated sequencer uous range expansion, and restricted gene flow caused by isolation in Shanghai Sangon Biological Engineering Technology & Service by distance (Templeton et al., 1995). Despite some criticisms over Co., Ltd. this technique (Knowles and Maddison, 2002; Petit, 2008), it is still the only method with the potential for disentangling multiple and overlying effects of historical and recurrent events within a given 2.5. Data analysis data set (Templeton, 2004). We reconstructed the network and NCA with the program AeNCA (Panchal, 2007). Firstly, haplotype 2.5.1. cpSSR data network was reconstructed with TCS, a subprogram of AeNCA; then, Variation within populations was estimated by dividing the GeoDis (another subprogram of AeNCA) was implemented based on number of haplotypes present by the number of individuals as- the network. Clade distances (Dc), a measure of the geographic range sayed (Hd, haplotype diversity) and by calculating the gene diver- of a clade, and nested clade distances (Dn), a measure of the distribu- sity (He) (equivalent to the expected heterozygosity for diploid tion range of a particular clade relative to its closest sister clades, data) (Weir, 1996) for each population based on haplotype fre- were defined based on geographic locations of samples in the nest- quencies. Estimates of average gene diversity within populations ing cladogram. Differences between interior (ancestral) and tip (re- (HS) and total gene diversity (HT) were calculated using the pro- cent) clades, D and D distances, were calculated, where I and T were gram PERMUT (RJ Petit, available at http://www.pierroton.inra.fr/ c n interior and tip clades, respectively. The null hypothesis of no geo- genetics/labo/software/Permut/)(Pons and Petit, 1996). To com- graphic associations for tip clades and interior clades was tested pare the amount of total genetic variability partitioned within by considering that the dispersion distances of clades were not and among populations or regions, the hierarchical analysis of greater or less than that expected by chance, and by comparing ob- molecular variance (AMOVA) was performed using Arlequin v3.0 served D and D values with a distribution of such values, calculated (Excoffier et al., 2005). The significance of covariance components c n for each 10,000 random permutations of clades against sampling was tested using permutation tests (1000 permutations) at differ- locations at the 0.05 significance level (Templeton et al., 1995). Per- ent levels (haplotypes among populations among regions, haplo- mutation tests were conducted separately for each level of the types among populations within regions and populations among nested cladogram. Following the determination of significance lev- regions). Only P-values lower than 0.05 were considered signifi- els for Dc and Dn, inferences about the historical processes that were cant. With the same program, FST measures between all pairwise likely to be responsible for observed patterns of clade structure were populations were obtained. Gene flow (Nm) was estimated using given by the program. the expression FST = 1/(1 + 2Nm), where N is the female effective population size and m is the female migration rate. In addition, geographic distances were calculated using GenAlEx v6.2 (Peakall 2.5.2. Chloroplast sequence data and Smouse, 2006). Mantel test was conducted using the program Sequences were edited and assembled with DNAStar (Gene TFPGA v1.3 (Miller, 1997) to evaluate the relationship between Codes Corporation). Variable sites in the data matrix were dou- pairwise FST and geographic distances. ble-checked against the original electrophorogram. Multiple align- To establish the extent to which mutational differences between ments of the cpDNA sequences were manually performed with the haplotypes contributed towards population differentiation, the pro- assistance of ClustalX v1.83 (Thompson et al., 1997) and subse- gram PERMUT was used to estimate RST and GST. RST (or NST) takes quently adjusted in BioEdit v7.0.4.1 (Hall, 1999). Insertions/dele- into account mutational differences between the haplotypes while tions (indels) were generally placed so as to increase the number the latter only makes use of haplotype frequencies. A higher RST than of matching nucleotides in a sequence position. Nucleotide diver- GST usually indicates the presence of phylogeographic structure sity (Pi)(Nei, 1987), haplotype diversity (Hd)(Nei and Tajima, (Pons and Petit, 1996) with closely related haplotypes found more 1983) and gene flow (Nm) among-population were calculated using often in the same area than relatively distantly related haplotypes. the program DNA Sequence Polymorphism (DnaSP) (Rozas et al., Phylogenetic relationships between cpSSR haplotypes of S. henryi 2003). Neutrality tests of Tajima’s D, Fu and Li’s D* and F* were also were assessed under maximum parsimony (MP) in PAUP 4.0b10 conducted using this program. T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188 181
We also calculated within-population diversity (HS), total diver- between 0 and 0.533 at the population level, but was relatively sity (HT) and level of population differentiation (GST) at both spe- higher at species level (He = 0.848). This result was also revealed cies and regional levels. To incorporate the relationships between by PERMUT (HS = 0.182, HT = 0.939). haplotypes, an estimate of population subdivision for phylogenet- The AMOVA analysis revealed extremely high genetic differen- ically ordered alleles (NST) was obtained, and the test statistic U, tiation, with 93.17% of the total variability partitioned among pop- comparing the values of NST and GST, was calculated. A higher NST ulations. It was also shown that 50.90% of total variability occurred than GST usually indicates the presence of phylogeographic struc- among the three geographic units (See Table 5 for details). Gene ture (Pons and Petit, 1996). All aforementioned parameters were flow (Nm, in this study, refers to seed flow) was limited with the va- calculated using the program HAPLONST (Pons and Petit, 1996). lue of 0.018, in line with the pronounced among-population In order to describe genetic structure and variability among differentiation. populations, AMOVA analysis was performed using squared Euclid- The test for phylogeographic structure in haplotype variation ean distances (Excoffier et al., 1992). Variance was partitioned to revealed that population differentiation estimating substitutions the following components: among individuals within populations, (RST = 0.913) was significantly higher than that over all populations among populations within regions and among regions. Genetic based on haplotype frequencies alone (GST = 0.806) (P < 0.01), analyses were performed using Arlequin v3.0 (Excoffier et al., implying the presence of phylogeographic structure. 2005). In addition, Mantel test revealed a significant correlation be- Relationships between all chloroplast haplotypes were esti- tween genetic and geographic distances (r = 0.371, P = 0.001), indi- mated by PAUP 4.0b10 and AeNCA with the same method as de- cating the role of geographic isolation in shaping the present scribed in cpSSR data analysis. The network was reconstructed population genetic structure of S. henryi. using the nucleotide characters (10 substitutions and four indels). Finally, the correlation between geographic distances and pairwise 3.1.2. Haplotype distribution and relationship F among all populations was tested using the program TFPGA ST The geographic distribution and the respective frequencies of v1.3 (Miller, 1997). cpSSR haplotypes within populations were shown in Fig. 1. The dis- tribution of the 11 haplotypes was not at random, but showed 2.5.3. Comparison among the cpSSR-, atpB–rbcL- and ISSR-based strong geographic patterns. Eight haplotypes (H1, H2, H3, H4, H5, results H6, H9 and H10) were distributed in multiple populations, while With the aim to further reveal the genetic structure of S. henryi, the other three (H7, H8 and H11) were unique to a specific popu- we compared results based on cpSSRs and atpB–rbcL sequences lation, respectively. Among the 16 populations sampled, seven and the previously published ISSR-based results (Zhou et al., populations harbored more than one haplotypes, while only a sin- 2010). Furthermore, the MP trees and NCA networks based on gle haplotype was detected in all the other populations (Tables 1 cpSSR and atpB–rbcL data were also compared in order to under- and 4). Although most of the haplotypes were shared by two or stand the phylogeography of the structure. more adjacent populations, there existed no widespread haplo- type. For example, haplotype H2 was shared by populations DC, 3. Results MN, LB and TB, all of which were located in western Mts. Qinling, within 100 km distances from one another. 3.1. cpSSR data With A. pulchellum used as outgroup, maximum parsimony analysis of the 11 cpSSR haplotypes of S. henryi generated three 3.1.1. Genetic diversity and differentiation equally parsimonious trees with a consistency index (CI) of 0.823 Twelve cpSSR primers were used to amplify all 290 DNA sam- and a retention index (RI) of 0.764. As shown in the MP tree ples, with eight of them proving polymorphic. In all, 23 bright (Fig. 2), the 11 cpSSR haplotypes were clustered into two major and discernible DNA bands were scored (Table 2), and 11 haplo- grades: haplotypes H1, H2, H3, H4 and H11 formed Grade I and types were distinguished (H1–H11). Despite being significantly af- Glade II included the other six haplotypes. This was confirmed by fected by sample sizes, the haplotype diversities (Hd) were the NCA network, in which the haplotypes located in Mts. Qinling relatively low, ranging from 0.05 to 0.2. Gene diversity (He) varied (H1/H2/H3/H4) and population JF (H11) formed Clade 2–3, and
Table 4 The distribution of each haplotype in the populations of S. henryi.
Pop cpSSR haplotypes atpB–rbcL haplotypes H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 A B C D E F G H I J K DC 5 15 5 MN 13 7 3 2 LB 16 4 41 TB 5 15 5 TP 20 5 LS 20 14 LC 20 3 2 YX 20 5 WD 8 12 2 3 FX 10 5 SB 20 1 1 3 HH 3 17 5 WG 20 5 ZN 10 5 XR 6 4 1 3 JF 20 4 Total 21 37 42 40 31 22 6 20 37 14 20 27 14 9 4 3 1 5 1 2 5 7 Freq. 0.072 0.128 0.145 0.138 0.107 0.076 0.02 0.069 0.127 0.048 0.069 0.346 0.179 0.115 0.051 0.038 0.013 0.064 0.013 0.026 0.064 0.090
Notes: Freq., the frequency of each haplotype. 182 T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188
Table 5 Analysis of molecular variance (AMOVA) of S. henryi based on cpSSR and atpB–rbcL data.
Source of variation df SS VC Variation (%) Fixation index cpSSR Among groups 1 295.330 1.856 50.91 Among populations within groups 14 392.189 1541 42.26 Within populations 274 68.250 0.249 6.83
Total 289 755.769 3.645 FST = 0.932 atpB–rbcL Among groups 2 27.146 0.385 23.82 Among populations within groups 13 56.347 0.799 49.48 Within populations 62 19.400 0.43145 26.70
Total 77 101.766 1.54781 FST = 0.733
those in Mts. Bashan and in the region south of Yangtze River (H5/ 26.70% and 23.82%, respectively (Table 5). In addition, the estimated H6/H7/H8/H9/H10) formed Clade 2–1 (Fig. 2). gene flow (here refers to seed flow only) was also low, with the value
As inferred by NCA, Clade 1–1 (including haplotypes H7 and H10) Nm = 0.09. could be explained by past fragmentation, while Clade 1–2 (includ- Maximum parsimony analyses based on the 11 atpB–rbcL hap- ing haplotypes H8 and H9) by restricted gene flow with isolation by lotypes of S. henryi generated 16 equally parsimonious trees with a distance. Contiguous range expansion was inferred for nested Clade consistency index (CI) of 0.776 and a retention index (RI) of 0.612. 1–3, which comprised two haplotypes detected from Mts. Bashan, In the MP tree, 11 haplotypes were clustered into two major grades H5 and H6. Past fragmentation was inferred for the both two-step (Fig. 3). Grade I comprised haplotypes D and J. Haplotypes A, C, K clades, 2–1 and 2–3. The three-step clade 3–1 included all haplo- and E clustered together formed a group, while haplotypes B, F types revealed by cpSSR markers, and allopatric fragmentation and G, H, I formed another; then the two groups clustered together was proposed to explain its distribution (see Table 7). and formed Grade II. Nested diagram was also reconstructed from cpDNA haplotypes (Fig. 3), and two groups (Clade 3–2 and Clade 3.2. atpB–rbcL sequence data 2–4, see Fig. 3) were detected among the 11 haplotypes in the network. Haplotypes A, K, C, E, B and F formed Clade 2–2, while 3.2.1. Sequence characteristics and haplotype distribution haplotypes G, H and I formed Clade 2–3; then these two clades Sequences of the cpDNA atpB–rbcL intergenic spacers ranged clustered together and formed a group (Clade 3–2). The remaining from 831 to 834 bp in length. The length after sequence alignment haplotypes (D and J) formed another group (Clade 2–4). Clade 3–2 was 835 bp. There were 10 nucleotide substitutions and four in- in the network roughly corresponded to the Grade II in the MP tree, dels, from which a total of 11 haplotypes (A–J) were identified except the positions of haplotypes B and F. Another group of the (Table 3). The sequences of these haplotypes have been deposited network, Clade 2–4, was exactly consistent with Grade I in the in the GenBank (Accession Nos. from GU190134 to GU190144). MP tree. No alternative connection between haplotypes (‘loops’) Haplotype frequencies in each population and geographic distribu- was observed. Clade 1–4 included haplotypes A and K, which were tion are presented in Table 3 and Fig. 1. The most widespread hap- mainly distributed in Mts. Qinling and Mts. Bashan, and could be lotypes were haplotype A (in seven of the 16 populations), B (in explained by contiguous range expansion as inferred by NCA. For four populations) and C (in four populations). All the other eight the two-step clade 2–2, which comprised most of the haplotypes haplotypes were unique to a specific population, respectively (Ta- from most populations, restricted gene flow with isolation by dis- ble 4 and Fig. 1). tance was uncovered by distance analyses. Clade 2–3 included the haplotypes G, H and I, all of which had lower haplotypic frequen- 3.2.2. Genetic diversity and genetic structure cies, and the inference from the NCA was restricted gene flow with Neutrality tests were performed to determine whether atpB–rbcL isolation by distance. Allopatric fragmentation was inferred for locus is subject to selection or has evolved neutrally. Neither Taj- nested clade 2–4, which included haplotypes found in populations ima’s D (D = 0.327, P>0.1) nor Fu and Li’s D* and F* (D* = 1.43, WG and JF. As for the three-step clade 3–2, which harbored most of P> 0.05; F* = 0.975, P > 0.10) rejected the null hypothesis of neutral atpB–rbcL haplotypes, restricted gene flow with isolation by dis- evolution. tance was inferred by NCA. However, NCA gave no inference for
Haplotype diversity (Hd) varied significantly among popula- the total cladogram (Table 6). tions, ranging from 0.2 (DC/TB/TP/YX/FX/HH/WG/ZN) to 0.6 (SB). Mantel test revealed a significant correlation between genetic
Nucleotide diversity (Pi) showed a similar pattern (Table 1). How- and geographic distance matrices (r = 0.436, P = 0.004), which con- ever, the haplotype diversity at species level was relatively high firmed the role of geographic isolation in shaping the present pop-
(0.761), as confirmed by HAPLONST (HS = 0.238, HT = 0.862). ulation genetic structure of S. henryi. Population subdivision of all 16 populations was very high
(GST = 0.724). When taking into account the relationships among 3.2.3. Comparison among cpSSR-, atpB–rbcL- and ISSR-based results haplotypes, genetic differentiation was further increased (NST = A comparative analysis was performed among the present 0.758, HS = 0.209, HT = 0.863, where HS and HT represent within- cpSSR-/atpB–rbcL-based and the previously published ISSR-based population and total genetic diversity of ordered alleles, respec- results (Zhou et al., 2010) on the genetic variation and differentia- tively). The U-test showed that NST was larger than GST (U= 0.25, tion. Generally, three different markers revealed more or less the P< 0.01). AMOVA analysis indicated strong genetic differentiation same profile that S. henryi possessed high species level genetic among the sampled populations or among the populations within diversity, strong among-population genetic differentiation, very the regions (P < 0.001). It was also shown that most variation oc- limited among-population gene flow and obvious correlation be- curred among populations within groups (49.48%), and that the tween genetic and geographic distances (see Table 8). However, amounts of variation within populations and among groups were there were also differences; for example, the estimates of gene T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188 183
Fig. 3. Most parsimonious tree (A) and 95% plausible network (B) of the 11 atpB–rbcL haplotypes for S. henryi. Bootstrap values (based on 1000 permutations) higher than 60% are indicated above branches. The atpB–rbcL haplotypes were identified by letters A–J. On the network, the size of circles corresponds to the frequency of each haplotype. Each solid line represents one mutational step that interconnects two haplotypes for which parsimony is supported at the 95% level. The small solid circles indicate inferred intermediate haplotypes not detected in this investigation. In both MP tree and NCA network, the 11 haplotypes are divided into two grades; Grade I in MP tree corresponds to Clade 2–4 of network, and Grade II to Clade 3–2.
flow from cpSSR and atpB–rbcL data were even much weaker than process was revealed in populations from eastern Mts. Bashan as that from ISSR data. well as Mts. Qinling (Clade 1–4 and 2–3). Furthermore, past frag- In addition, we also compared the NCA results based on cpSSR mentation was uncovered as the inference for the two-step clades and atpB–rbcL sequences. Both cpSSR and atpB–rbcL data yielded 2–1 and 2–3 as inferred from cpSSR data, but was not found for any 11 haplotypes, and showed that allopatric fragmentation, contigu- clade based on atpB–rbcL data. ous range expansion and restricted gene flow with isolation by dis- tance were the reasons for phylogeographic pattern for S. henryi. However, there occurred some differences. Allopatric fragmenta- 4. Discussion tion was suggested as the inference responsible for the phylogeo- graphic pattern of Clade 3–1, which included all cpSSR 4.1. Genetic diversity and population differentiation haplotypes, while in the NCA result based on atpB–rbcL data, re- stricted gene flow with isolated by distance was inferred for Clade In this study, the total genetic diversity (HT)ofS. henryi uncov- 3–2 which comprised most haplotypes. In addition, cpSSR data re- ered by cpSSR and atpB–rbcL noncoding region was relatively high, vealed allopatric expansion in the population (Clade 1–3) located compared with the mean genetic diversity value estimated from in eastern Mts. Bashan; by contrast, based on atpB–rbcL data, this cpDNA-based studies of 170 plant species (HT = 0.67) (Petit et al., 184 T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188
Table 6
Nested clade and geographical distance analyses for S. henryi based on the nested design of Fig. 2. For each clade and interior-tip clade comparison (I–T), the clade distance (Dc), and nested clade distance (Dn) are reported. Significantly small (*S) or large (*L) values are indicated for each clade and I–T status. Tip (T) or interior (I) clades (haplotypes) are also indicated.
Nested clades and haplotypes of cpSSR Type of geographical distance Nested clades and haplotypes of atpB–rbcL Type of geographical distance
Within Clade (Dc) Nested Clade (Dn) Within Clade (Dc) Nested Clade (Dn) Clade 1–1 Clade 1–3 H10 (T) 40.1755*s 49.215*s F (T) 0.0 233.6306 H7(I) 0.0*s 49.2645*l B (I) 87.9523 90.4879 I–T 40.1755*s 0.0495*l I–T 87.9523 143.1427 Clade 1–2 Clade 1–4 H8 (T) 0.0*s 363.1531*l K (T) 132.0924 301.3918*l H9 (I) 19.2007*s 200.6166*s A (I) 131.5354*s 156.2836*s I–T 19.2007 162.5365*s I–T 0.557 145.1082*s Clade 1–3 Clade 1–5 5 (T) 82.8676*l 83.5247*l E (T) 0.0 170.1916 6 (I) 33.8351*s 41.9527*s C (I) 276.7492 237.9869 I–T 49.0325*s 41.572*s I–T 276.7492 67.7952 Clade 1–6 Clade 1–7 4 (T) 28.5107*s 186.3516 I (T) 0.0 146.914 3 (I) 64.6541*s 177.9235 H (I) 0.0 293.9219 I–T 36.1434*l 8.4281 I–T 0.0 147.0079 Clade 2–1 Clade 2–2 1–2 (T) 258.6654*s 317.6585*s 1–3 (T) 100.0308*s 255.6947*s 1–3 (T) 64.788*s 271.8281*s 1–4 (T) 186.1589*s 333.8522*s 1–1(I) 49.2299*s 646.5873*l 1–5 (I) 218.6168 633.866*l I–T 115.1649 351.2134*l I–T 58.8237 323.9396*l Clade 2–3 Clade 2–3 1–6 (T) 182.0348*s 213.0544*s 1–7 (T) 195.9166*l 250.1572*l 1–5 (I) 26.2165*s 215.6313*s 1–2 (I) 0.0*s 47.6053*s 1–7 (I) 0.0*s 477.4048*l 1–T 195.9166*s 202.5518*s I–T 168.6068*s 130.2713*l Clade 2–4 Clade 3–1 1–10 (I) 0.0*s 394.0827*l 2–1 (T) 376.9591 349.0991 1–6 (I) 0.0*s 317.3399*s 2–3 (I) 256.4782*s 347.9713 Clade 3–2 I–T 120.4809*s 1.1278 2–3 (T) 94.3481*s 228.5811*s Clade 3–2 2–2 (I) 407.1083*l 398.4455*l 2–2 (T) 0.0*s 331.0415*l I–T 312.7602*l 169.8644*l 2–4 (I) 51.9567*s 101.342*s Clade 4–2 I–T 51.9567 229.6995*s 3–2 (T) 371.8402 379.0827 Total cladogram 3–3 (I) 351.4478 489.1871 3–1 (T) 348.6208 344.7907 I–T 20.3924 110.1044 3–2 (T) 150.2142*s 325.1393 Total cladogram 4–1 (T) 0.0 298.0096 4–2 (T) 389.8538 388.9279
Table 7 Nested contingency analysis of geographic associations and phylogeographic inferences obtained from a nested haplotype analysis of S. henryi. Numbers in parentheses indicate choice made in the dichotomous key given in Templeton (2004).
Clade Permutational Chi-squared statistic Clade key Inferences cpSSR Clade 1–1 8.5714* 1-2-11-12-13-14 NO Past fragmentation Clade 1–2 56.0* 1-19 NO Allopatric fragmentation Clade 1–3 34.6944* 1-2-11-12 NO Contiguous range expansion Clade 1–4 56.0000 1-2 NO Inconclusive outcome Clade 1–5 34.6944 1-2 NO Inconclusive outcome Clade 1–6 82.0* 1-19-20-2-3-4 NO Restricted gene flow with isolation by distance Clade 2–1 246.0315* 1-2-11-12-13-14 NO Past fragmentation Clade 2–3 246.0* 1-2-11-12-13-4 NO Past fragmentation Clade 3–1 1-19 NO Allopatric fragmentation Clade 3–2 1-19-20-2-11-12-13-14 NO Past fragmentation Total Cladogram 289.0000 1-2 NO Inconclusive outcome atpB–rbcL Clade 1–3 15.0 Null hypothesis cannot be rejected Clade 1–4 34.0* 1-19-20-2-11-12 NO Contiguous range expansion Clade 1–5 8.0* Null hypothesis cannot be rejected Clade 1–7 3.0* Null hypothesis cannot be rejected Clade 2–2 92.5525* 1-2-3-4 NO Restricted gene flow with isolation by distance Clade 2–3 8.0* 1-19-20-2-11-12 NO Contiguous range expansion Clade 2–4 9.0* 1-2-3-4 NO Allopatric fragmentation Clade 3–2 49.4877* 1-2-3-4 NO Restricted gene flow with isolation by distance Clade 4–2 78.0* Null hypothesis cannot be rejected Total Cladogram 79.0* 1-2-3-4 NO Null hypothesis cannot be rejected T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188 185
Table 8 gether with local climatic changes caused by the Qinghai-Tibet The genetic diversity indices based on cpSSR, rbcL–atpB and ISSR data. Plateau uplift particularly during Quaternary glaciations, have af-
HT HS GST FST NM Mantel Test fected the distribution and evolution of many plant species in this cpSSR 0.94 0.18 0.81 0.93 0.02 r = 0.371, P = 0.001 area (Wang and Ge, 2006; Zhang et al., 2005). Previous studies atpB–rbcL 0.86 0.24 0.73 0.72 0.09 r = 0.436, P = 0.004 have suggested that the lowest temperature during the glacial ISSR 0.26 0.08 0.63 0.67 0.29 r = 0.779, P = 0.001 age was 9–13 °C lower than that in the present period, and that the highest temperature during the interglacial age was 2 °C higher than that in the present period in Central China (Shi et al., 1993), 2005). However, this was confirmed by the previously published where the majority of S. henryi populations are located. In general, ISSR-based result (Zhou et al., 2010). The high cpDNA diversity of with the decrease of environmental temperature, plants tend to S. henryi may be attributed to its long evolutionary history and migrate towards lower altitudes or latitudes, but migrate towards its diversified living conditions, which have allowed it to accumu- higher altitudes or altitudes as the temperature increases. Central late considerable genetic variability. China, with altitudes ranging from 100 to 3000 m, is characterized However, the within-population diversity of this species by its complex topography. There are lots of high mountains and revealed by both cpDNA markers was pretty low, coupled with basins, as well as Yangtze River, Yellow River, Pearl River and their strong population differentiation (HS = 0.182, GST = 0.91, RST = 0.81, branches running between mountains, in the valleys and through FST = 0.93 for cpSSR; HS = 0.238, GST = 0.724, NST = 0.758, FST = 0.79 plains. The present habitats of the study species are distributed be- for atpB–rbcL). The haplotype distribution, AMOVA analysis and tween 800 and 1500 m asl. Furthermore, the temperate-deciduous phylogeographic inferences also showed that cpDNA variation was forest in Central China displays a discontinuous geographic distri- highly structured, in line with the previously published ISSR result bution pattern due to the invasion of coniferous forest during the (Zhou et al., 2010). Two factors may roughly explain the high glacial age and the invasion of evergreen forest during the intergla- cpDNA-based population subdivision within S. henryi. On one hand, cial age in the late Tertiary and Quaternary (Harrison et al., 2001; it can be partly attributed to the lack of an efficient seed dispersal Qian and Ricklefs, 2000; Qiu et al., 2009). S. henryi usually grows mechanism in this species. In plants, the modes of pollen and seed in the temperate-deciduous forests. We assume that S. henryi pop- dispersal determine the gene flow among populations (Li and Chen, ulations survived climatic changes in situ, perhaps by moving 2004). The cpDNA data only reflect the gene flow via seed dispersal upwards and downwards in their mountain ranges and tracking (Petit et al., 2003), and thus the genetic differentiation from cpDNA favorable humidity conditions as imposed by the East Asian analyses should be negatively related to the dispersal ability of monsoon, rather than migrating southwards long distances and seeds. In S. henryi, ripe fruits are normally dispersed by gravity, backwards in the interglacial age. Accompanied with several inter- and have a restricted dispersal radius. One the other hand, changes of glacial and interglacial ages, such elevational moves geographic isolation may also partly account for the pronounced precedently occurred multiple times. Consequently, the migratory genetic differentiation. In this study, the smallest, largest and mean mode of S. henryi in face of dramatic climatic oscillations during among-population geographic distances were 25.4 km (SB vs. FX), the Quaternary was significantly different from those examples 1104.6 km (LS vs. XR) and 430.6 km, respectively. Neighboring pop- of European and North American plants, which migrated long dis- ulations were usually separated by geographic barriers (e.g. undu- tances to the south and took refugia there, and then returned dur- lating mountains, rivers), which largely hindered gene flow via ing the interglacial age (Hewitt, 1999). seed and pollen dispersal among populations. In general, refugia should be places with stable climatic condi- The result of this study was roughly consistent with the previ- tions so that a species can be conserved during the glacial age ously published ISSR-based result on the genetic diversity and (Tzedakis et al., 2002). The genetic diversity of a species differs be- structure of S. henryi (Zhou et al., 2010). Both studies supported tween the populations in refugia and those migratory ones. The for- that weak dispersal ability of seeds, geographic isolation and hab- mer usually possess more genetic variability than the latter, since itat fragmentation should be responsible for the low within-popu- the latter usually suffered from founder or bottleneck effects during lation genetic diversity and strong among-population genetic the migratory process, which may decrease its genetic diversity differentiation. Based on this conclusion, the protection of its nat- (Avise, 2000; Hewitt, 1996, 2000). In addition, the populations in ural habitats and in situ conservation are also proposed as the con- refugia usually harbor not only relatively higher genetic diversity servation policy. but also haplotypic uniqueness (Petit et al., 2003). In our study, pop- However, there were minor differences. For example, the gene ulations MN, LB, WD and XR also harbored relatively higher genetic flow from ISSR data (Nm = 0.29) was much higher than those from and haplotype diversities (Table 1 and Fig. 1) as revealed by both cpSSR and atpB–rbcL data. This can be roughly attributed to the dif- cpSSR and atpB–rbcL. From the haplotype abundance and unique- ferent modes of inheritance of the three marker systems. The ISSR- ness, in line with the geological and climatic history in Central China, based gene flow reflects both seed and pollen flows while the we can deduce the existence of several Pleistocene refugia of S. hen- atpB–rbcL and cpSSR-based ones only reflect seed flow. ryi, including Mts. Qinling (population MN, LB), eastern Mts. Bashan (population WD) and the southeastern edge of Yunnan-Guizhou Pla- 4.2. Glacial refuge areas teau (population XR). In Mts. Qinling and Mts. Bashan, there are many valleys and basins with altitudes ranging from 200 to Climatic oscillations during the Pleistocene resulted in several 3000 m, especially in the upper reaches of Han River and Jialin River glacial–interglacial cycles which caused expansion and contrac- (both being branches of Yangtze River). Desirable climatic condition tions of habitats (Abbott and Brochmann, 2003; Hewitt, 2004). and mature vegetation render this region a good shelter for S. henryi. Geographic areas exhibiting increased levels of genetic diversity In the upper reach of Pearl River in the southern edge of Yunnan- are ideal candidates in search of past glacial refugia. These regions Guizhou Plateau, there are also lots of valleys with altitudes ranging should be characterized by comparably stable ecological condi- from 200 to 2000 m, which can also provide good shelters for this tions during environmental fluctuations, fostering the accumula- species. All these three regions have long been regarded as the main tion of genetic diversity as has been demonstrated in many cases centers where endemic plants are distributed in China (Ying and (Tzedakis et al., 2002). Zhang, 1994). Some well-known plants which found glacial refugia Although no massive ice sheet was developed in Central China there include Cathaya argyrophlla (Wang and Ge, 2006) and Ginkgo during glacial periods, the tremendous global climatic changes, to- biloba (Gong et al., 2008). 186 T.-H. Zhou et al. / Molecular Phylogenetics and Evolution 57 (2010) 176–188
4.3. Geographic distribution of haplotypes and phylogeographic Thirdly, although no significant range expansion was detected implications from the refuge areas, contiguous expansion in restricted regions (Mts. Qinling and eastern Mts. Bashan) was revealed by NCA based The strong geographic structures among cpSSR haplotypes on both cpSSR and atpB–rbcL data. In western Mts. Qinling, cpSSR
(RST > GST, P < 0.01) and among atpB–rbcL haplotypes (NST > GST, haplotype H2 was shared by four contiguous populations (DC, U = 0.25) both reflected that different haplotype compositions MN, LB and TP), probably due to range expansion. In addition, were found in different geographic regions. Combining all these atpB–rbcL haplotype A was found in most populations in Mts. Qin- findings, several factors were identified to explain the present ling (MN, DC, LB, TB, TP and LC) and adjacent population WD (be- cpDNA-based phylogeographic pattern of S. henryi. long to Mts. Bashan), which are geographically nearby; haplotype B Firstly, restricted gene flow as a result of low seed dispersal was shared by populations from Mts. Bashan (WD, SB and HH) and ability and complex topography, may be the main factor responsi- YX, and these populations are also geographically close to one an- ble for the high genetic and geographic differentiation as well as other. This also indicated the occurrence of contiguous range for the formation of notable phylogeographic pattern. Restricted expansion in both Mts. Qinling and in eastern Mts. Bashan. Also, gene flow with isolation by distance was also revealed as the his- haplotypes F, K and I, all of which were at the tips of nested clades, torical process for clade 3–2, which included most of the atpB– were found in eastern Mts. Qinling. These three haplotypes have a rbcL haplotypes. The gene flows reported in this study was 0.018 relatively recent origination, and have evolved from haplotypes B, for cpSSRs and 0.09 for arpB–rbcL intergenic spacers, respectively. A and H, respectively.
In population genetics, a value of gene flow (Nm) <1.0 (less than In summary, the following historical scenario of Saruma can be one migrant per generation into a population) is generally re- drawn. The species found glacial refugia in situ during the glacial garded as the threshold quantity beyond which significant popula- age and experienced limited range expansion during the intergla- tion differentiation occurs (Slatkin, 1987). It has been reported that cial age. Habitat fragmentation induced by climatic oscillations S. henryi adopts a predominantly selfing breeding system (Zhao during the Quaternary and the complex topography rendered S. et al., 2005); in addition, the seeds of S. henryi are generally grav- henryi isolated in several geographic units, which resulted in pro- ity-dispersed, and are characterized by complex and tough coats nounced genetic differentiation and phylogeographic pattern. We and low germination rate (Zhao et al., 2006). Thus, the gene flow may also conclude that high plant diversity and endemism in Cen- via seed dispersal was very limited. Mantel test revealed a signifi- tral China have mainly resulted from allopatric speciation due to cant correlation between genetic and geographic distances, and complex topography in this area and allopatric fragmentation dur- thus supported isolation-by-distance (IBD) model (Wright, 1943). ing the late Tertiary and Quaternary. Central China, where most S. henryi populations are located, is characterized by undulating mountains, e.g. Mts. Qinling and Mts. Bashan. Furthermore, there also lie valleys, basins, plains Acknowledgments and plateaus among them. The complex topography of the area is a natural barrier that renders it difficult for the seeds to disperse. This study was financially co-supported by the National Natural Thus, the divergent gene pools emerged; the species experienced Science Foundation of China (Grant No. 30800087), the Program for only limited gene flow, and remained genetically isolated. Changjiang Scholars and Innovative Research Team in University Secondly, allopatric fragmentation may explain the phylogeo- (PCSIRT) and the Postgraduate Innovation & Education Program graphic structure of S. henryi populations. As revealed by NCA, it of Northwest University (NWU) (Grant No. 08YZZ37) The authors might occur in most distribution areas of S. henryi. For example, it thank two anonymous reviewers for their critical comments on was obvious in populations WG and JF (both from the region south the earlier drafts of this manuscript. of Yangtze River), which was confirmed by the distribution of both cpSSRs and atpB–rbcL haplotypes. Many populations possess their References own distinct haplotypes, or several populations share one haplo- type. Fragmented populations are predicted to experience stochas- Abbott, R.J., Brochmann, C., 2003. History and evolution of the arctic flora: in the tic losses of rare alleles, since only a small portion of the original footsteps of Eric Hultén. Mol. Ecol. 12, 299–313. gene pool remains after fragmentation (Barrett and Kohn, 1991; Ell- Avise, J.C., 2000. Phylogeography: the History and Formation of Species. Harvard University Press, Cambridge, MA, USA. strand and Elam, 1993). Subsequently, random genetic drift will re- Barrett, S.C., Kohn, J.R., 1991. Genetic and Evolutionary Consequences of Small duce allelic richness and increase among-population genetic Population Size in Plants: Implications for Conservation. Oxford University divergence, although it takes several generations for genetic drift Press, New York, NY, USA. Cheng, C.-Y., Yang, C.-S., Hwang, S.-M., 1988. Aristolochiaceae. In: Kiu, H.-S., Ling, Y.- to have significant impacts. Due to the invasion of coniferous forest R. (Eds.), Flora Reipublicae Popularis Sinicae, vol. 24. Science Press, Beijing, during that glacial age and of evergreen forest during the interglacial China, pp. 159–245. age in the late Tertiary and Quaternary, the temperate-deciduous Cheng, Y.P., Hwang, S.Y., Lin, T.P., 2005. Potential refugia in Taiwan revealed by the phylogeographical study of Castanopsis carlesii Hayata (Fagaceae). Mol. Ecol. 14, forest in Central China displays a fragmented distribution pattern 2075–2085. (Harrison et al., 2001; Qian and Ricklefs, 2000; Qiu et al., 2009). S. Chiang, T.Y., Schaal, B.A., Peng, T., 1998. Universal primers for amplification and henryi usually occurs under the canopy of temperate-deciduous for- sequencing a noncoding spacer between atpB and rbcL genes of chloroplast DNA. Bot. Bull. Acad. Sin. 39, 245–250. est, and accordingly its geographic distribution was fragmented; China Plant Specialist Group, 2004. Saruma henryi. In: IUCN 2008 (Ed.), 2008 IUCN allopatric fragmentation occurred inevitably. Allopatric fragmenta- Red List of Threatened Species.
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