Molecular Ecology (2013) 22, 1120–1133 doi: 10.1111/mec.12153
Geological events play a larger role than Pleistocene climatic fluctuations in driving the genetic structure of Quasipaa boulengeri (Anura: Dicroglossidae)
FANG YAN,*† WEIWEI ZHOU,* HAITAO ZHAO,‡ ZHIYONG YUAN,*† YUNYU WANG,* KE JIANG,* JIEQIONG JIN,* ROBERT W. MURPHY,*§ JING CHE* and YAPING ZHANG*¶ *State Key Laboratory of Genetic Resources and Evolution, and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China, †Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, ‡College of Life Sciences, Bijie University, Bijie 551700, China, §Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario, Canada M5S 2C6, ¶Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming 650091, China
Abstract Paleoclimatic and paleogeological events have been identified as being the two main drivers of genetic structuring in extant organisms. We used a montane stream-dwelling frog, Quasipaa boulengeri, to explore the relative roles played by these drivers on species in southern China, a region needing thorough studies. We detected four major matrilines, and no broadly distributed haplotypes occurred. The complex orogenesis of south-western China drove matrilineal divergence in Q. boulengeri into highly struc- tured geographical units. These matrilines subsequently persisted in situ with stable populations rather than undergoing expansions during glacial cycling. The unification of the upper and middle Yangtze River in the Three Gorges mountain region mediated downstream colonization of this frog. Analyses identified geological events as playing a larger role than climatic fluctuations in driving the population history of Q. boulen- geri. Nuclear allele analyses indicated gene flow; this maintained genetic cohesion of the species. South-eastern Sichuan Basin was identified as the area of secondary contact for several matrilines, and this area deserves further study and special protection.
Keywords: phylogeography, Pleistocene climatic fluctuations, secondary contact, southern China, Yangtze River Received 12 July 2012; revision received 23 October 2012; accepted 25 October 2012
tain systems, resulted in habitat fragmentation which Introduction formed barriers to gene flow, leading to genetic diversi- Climate, geography and/or a combination of these fication and even speciation (e.g. Hughes & Eastwood factors usually drive the patterns of genetic diversity seen 2006; Liu et al. 2006; Antonelli et al. 2009; Che et al. today. On the one hand, climatic fluctuations during the 2010; Chaves et al. 2011; Li et al. 2012). Sometimes the Pleistocene are responsible for the isolation of popula- two drivers work together hand-in-hand within a tions in multiple refugia. Isolation into small refugia species (e.g. Brunhoff et al. 2003; Lemmon et al. 2007; resulted in lower genetic diversity and population diver- Macqueen et al. 2011; Zhou et al. 2012). The relative roles gence via bottlenecking and genetic drift (reviewed by climate and geography play in driving genetic patterns Hewitt 2000, 2004). On the other hand, geological has important implications for speciation and diversifi- changes, such as the uplifting of Andes and Tibet moun- cation, conservation, biogeography and geology, among other phenomena (e.g. Hewitt 1996; Shih et al. 2006; Correspondence: Yaping Zhang, Fax: 86-871-5195430, E-mail: Fouquet et al. 2010). Southern China, an area spanning [email protected] and Jing Che, Fax: 86-871-5133185, from the eastern Tibetan Plateau to the Pacific Ocean, E-mail: [email protected] harbours high levels of species diversity (Zhang 1999;
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Myers et al. 2000; Qian & Ricklefs 2000), which provides (Paini) exemplify the important role physiographic an excellent study system for detecting the relative con- change plays in shaping the evolutionary history of tributions of various processes. species (Che et al. 2010). Most species of spiny frogs are Orogenesis of the Qinghai-Tibetan Plateau (QTP) and aquatic and occur in remote, swift and rocky montane associated climatic changes appear to be the most streams. No evidence suggests that the species dis- important drivers of speciation and population diversi- perses long distances across terrestrial landscapes. Such fication in western China (He et al. 2001; Liu et al. 2006; species are likely to have very high genetic divergences Che et al. 2010; Li et al. 2012). For example, because of between populations, as occurs in the largely aquatic orogenesis, western China has a highly complex moun- frog, Nanorana yunnanensis (Zhang et al. 2010b). The tain system including the QTP with an average eleva- restrictions to remote montane aquatic habitats make tion of about 4500 m above sea level (asl.) (Shi et al. spiny frogs an interesting species for studying the rela- 1998), the Himalayas and the Hengduan Mountains. tive roles played by geologic events and climatic shifts The eastern boundary of the montane system involves in shaping population structures. Among the species of the Three Gorges mountain region (TGMR), which aver- spiny frogs, Quasipaa boulengeri has keratinized nuptial ages only about 1000 m asl. These mountain ranges excrescences that cover the venter of males during the remain geographically and ecologically dynamic, and breeding season. This species mainly occurs in montane the perpetuation of the geomorphic evolution in this streams of southern China (Fig. 1), primarily in the region is responsible for the isolation and differentia- upper and middle reaches of the Yangtze River. This tion of many populations including plants and animals distribution makes it as an ideal model organism for (e.g. Yuan et al. 2008; Zhang et al. 2010a; Li et al. 2012). testing hypotheses regarding the role played by histori- Uplifting of the eastern QTP rearranged drainage sys- cal drainage rearrangement on species vicariance and tems and this may have influenced the distribution and dispersion. population structure of species (He & Chen 2006; Zhang Using DNA sequence data and a phylogenetic et al. 2010b; Zhang & Sun 2011). South-western China approach, we test hypotheses regarding the driving experienced significant changes in paleodrainage forces that generated the extraordinary high level of patterns (Clark et al. 2004). For example, during the biodiversity in the western mountains of China, in Pliocene the upper reaches of the Yangtze River formed particular the relative roles played by Pleistocene a tributary of the paleo-Red River, which drained south- climatic cycling and topological shifts related to oro- wards into the South China Sea (Clark et al. 2004); it was genesis. If geologic deformations are the most important isolated from the middle drainage system of the Yangtze driving force, then we expect to observe high levels of River at the TGMR. This historical break is recorded in genetic differences between isolated populations of the level of genetic divergence between populations of Q. boulengeri due to older, complex landscape features; fishes (Xiao et al. 2001; Perdices et al. 2004; Yang et al. current populations are predicted to have persisted in 2009). The connection of the watercourses now facilitates situ throughout the Pleistocene climatic oscillations. species dispersal from west to east. This hypothesis predicts much local genetic differentia- Cyclical cooling–warming events during the Quater- tion and population structuring with no signals of nary Period are responsible for periodic habitat contrac- extirpation and expansion. Alternatively, the detection tions and expansions. These events shaped the of low levels of genetic divergence between collecting distributions of species and their genetic attributes, localities will suggest that Pleistocene climatic cycling especially in Europe and North America (e.g. Taberlet played a greater role in the evolution of this frog than et al. 1998; Hewitt 2004; Soltis et al. 2006). In contrast, did physiography. East Asia did not experience major glaciations (Shi 1986; Liu 1988) and southern China had a relatively Materials and methods mild Pleistocene climate (Weaver et al. 1998; Pinot et al. 1999; Ju et al. 2007). The great topographical diversity Sample collection involving a mosaic of mountains also potentially pro- vided a variety of relatively stable habitats (Qian & A total of 340 samples were collected from 45 localities Ricklefs 2000; Lopez-Pujol et al. 2011). Thus, climatic throughout the range of Quasipaa boulengeri between cycling might not have led to localized extirpations and 2008 and 2010 (Fig. 1; Table S1, Supporting informa- population shrinkages or expansions in southern China tion). Sample sizes mostly reached 10 individuals per (e.g. Wang & Ge 2006; Gong et al. 2008; Gao et al. 2011). location. Quasipaa robertingeri from the type locality Amphibians not only serve as environmental indica- (Tiantangba subcounty, Hejiang Co., Sichuan, China, tors, they are also ideal model organisms for discerning 900 m) was included in the ingroup (Table S1, Support- historical processes (Zeisset & Beebee 2008). Spiny frogs ing information) because the species was suggested to
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Fig. 1 Map showing the distribution of Quasipaa boulengeri. Localities are detailed in Table S1 (Supporting information) and populations are presented as pie-dia- grams with slice-size proportional to the frequency of the major matrilines. Inset in upper right corner shows the simpli- fied maternal genealogy with major matrilines A, B, C and D. Colours of pie-diagrams and tree correspond to the matrilines in Figure 2.
be a junior synonym of Q. boulengeri (Che et al. 2009). BigDye Terminator Cycle Sequencing Kit on an ABI Tissue samples, including liver, muscle, toe clips, and PRISM 3730 DNA analyser (Applied Biosystems). tadpoles, were preserved in 95% ethanol for subsequent Sequences of each segment were proofread and assem- DNA sequencing. Voucher specimens were deposited bled with DNASTAR 5.0 (DNASTAR Inc., Madison, WI, in the Kunming Institute of Zoology, Chinese Academy USA), initially aligned using CLUSTAL W with default of Sciences (KIZ). Five samples of three species includ- parameters (Thompson et al. 1994), and then adjusted ing Quasipaa spinosa, Quasipaa shini, Quasipaa jiulongensis by eyes if required using MEGA 4 (Tamura et al. 2007). were chosen as the outgroup taxa based on the study of Che et al. (2009) (Table S1, Supporting information). Maternal genealogy and nuclear allele tree Phylogenetic relationship among mitochondrial haplo- Sequencing and alignment types were reconstructed using Bayesian inference (BI) Total DNA was extracted with the standard three-step and maximum parsimony (MP) methods using MRBAYES phenol–chloroform method (Sambrook et al. 1989), then 3.1.2 (Ronquist & Huelsenbeck 2003) and PAUP* 4.0b10a frozen at À20 °C. A partial fragment of the mitochon- (Swofford 2003), respectively. We selected the best-fit drial cytochrome b gene (Cytb) (Table S2, Supporting nucleotide substitution models for the three codon posi- information) was amplified for all samples. Partial tions using MODELTEST 3.7 (Posada & Crandall 1998). For sequences from an intron of proglucagon (GCG) BI, four independent runs were performed of the Cytb (Table S2, Supporting information) were obtained from data with each partition model for 10 million genera- a subset of samples consisting of 53 individuals repre- tions. Trees were sampled every 100th generation senting each mtDNA lineage (hereafter termed a matri- resulting 100 000 trees and the first 25% were discarded line). PCR was carried in a 25-lL reaction volume as burn-in. MP analyses used full heuristic tree searches l 9 l containing 2.5 Lof10 buffer with 2 mM MgCl2,1 L with 1000 replications, random addition of sequences, of dNTP (0.125 mM), 1 lL of each primer (3 pM), 1U and tree-bisection-reconnection (TBR) branch swapping. Taq DNA polymerase, 30 ng total DNA for Cytb and Bootstrap support for the MP tree involved 1000 repli- 50 ng for GCG. Sterile water was added to complete the cates of full heuristic searches. final volume. The reactions were performed using the For the nuclear allelic tree, the best-fit model was esti- following procedure: initial denaturation at 94 °C for mated using MODELTEST. Allele sequences of heterozy- 5 min, 35 cycles of denaturation at 94 °C for 45 s, gous individuals were inferred using PHASE 2.1 (Stephens annealing at 45 °C for Cytb and 56 °C for GCG per 45 s, et al. 2001; Stephens & Donnelly 2003), for which the extension at 72 °C for 45 s, and a final extension at 72 °C input files were prepared using SEQPHASE (Flot 2010). for 7 min. PCR products were purified with a Gel We constructed the BI tree using GCG alleles under Extraction Mini Kit (Watson Biotechnologies, Shanghai, the same settings as for Cytb, except for model of China), and then sequenced in both directions with substitution. Finally, the longest nonrecombined region
© 2012 Blackwell Publishing Ltd OROGENESIS OUTWEIGHS THE PLEISTOCENE IN A FROG 1123 of nuDNA sequences was inferred by IMGC (Woerner skyline plots (Drummond et al. 2005) were performed et al. 2007). An allele network was generated from the using BEAST 1.6.1 (Drummond & Rambaut 2007) to longest nonrecombined region using the median-joining describe demographic history by assessing the time var- (MJ) method (Bandelt et al. 1999) implemented in the iation of effective population size. This analysis was software NETWORK 4.6.0.0 (available at http://www. performed using the same settings as above for the fluxus-engineering.com/sharenet.htm). divergence time estimation, except the coalescent tree prior was specified as the Bayesian skyline with four groups. Molecular diversity and genetic structure Molecular diversity for Cytb was estimated both overall Results and each population using the number of haplotypes (n), haplotype diversity (h), and nucleotide diversity Sequence data (p). To investigate the level of genetic variation between populations, we performed a two-level hierarchical For 340 ingroup individuals, 810 base pairs (bp) of Cytb analysis of molecular variance (AMOVA; Excoffier et al. data were aligned after trimming the ends. Sequences 1992). Pairwise population differentiation was estimated from the five outgroup individuals unambiguously by computing pairwise Φst for the Cytb data between aligned with the ingroup at all 810 bp. All nucleotide populations having more than five samples in ARLEQUIN sequences successfully translated to amino acids 3.5 (Excoffier et al. 2005). Divergence between the matri- without premature stop codons and heterozygotes. lines was estimated by Kimura’s (1980) two-parameter The expected bias against guanine was observed (K2P) model as implemented in MEGA 4. (G = 14.5%, A = 24.9%, T = 29.2%, C = 31.4%). These data indicated that the mitochondrial gene was sequenced, and not a nuclear copy. The best-fit models Spatio-temporal reconstruction for the first, second and third codon positions of Cytb To estimate the divergence time and ancestral distribu- sequence were K80, K81uf and TIM+G, respectively. tion of the extant matrilines, we used a coalescent time Ingroup sequences generated 114 haplotypes that con- estimation method in BEAST and a statistical dispersal- tained 196 potentially parsimony informative sites. The vicariance analysis implemented in S-DIVA (Nylander haplotype distributions were restricted geographically: et al. 2008; Yu et al. 2010). For molecular time estima- 85.22% of the haplotypes occurred in one location only. tion, the molecular clock was assessed by relative-rate No broadly distributed haplotypes occurred. tests using PHYLTST (Kumar 1996). Lacking of fossil For GCG, we sequenced a subset of all samples (Table evidence, we assumed a range of substitution rate of S1, Supporting information). In total, we analysed 53 0.65–1.00% per Myr for Cytb based on evolutionary individuals of Q. boulengeri representing matrilines A, rates commonly proposed for anurans (Macey et al. B, C1, C2, and D1–D4. The 926 bp fragment involved 45 1998, 2001; Monsen & Blouin 2003; Prohl€ et al. 2010) polymorphic sites and 13 indels. Thirty-two individuals and generally for mtDNA (Brown et al. 1979). Analyses were heterozygous. In all cases, both alleles within an were performed for 30 million generations assuming individual were the same length. The fragments con- a Yule speciation process with different models for sisted of 84 alleles. The longest nonrecombined region three codon positions. Ancestral distribution analyses finally generated 30 alleles consisting of 572 bp. The used 30 000 trees from Cytb and the final tree was best model of nucleotide substitution for the intron of retained. GCG was GTR+I+G.
Historical demography Matrilineal genealogy and nuDNA allele tree Three methods were used with the Cytb data to infer The BI and MP trees based on the mtDNA data the demographic history of matrilines A, B, C1, C2, D1, resolved extremely similar matrilines and only the BI D2, D3 and D4. First, Tajima’s D (Tajima 1989) and Fu’s tree was shown in Figure 2. The ingroup contained four Fs (Fu 1997) neutrality tests were examined using highly supported matrilines. Matriline A, the sister ARLEQUIN. In the neutrality test, population growth was group of all other matrilines, consisted of specimens indicated by significantly negative values. Second, from a small area in south-eastern Sichuan Basin mismatch distributions (Slatkin & Hudson 1991) calcu- (Fig. 1). Matriline B, sister group of matrilines C and D, lated with ARLEQUIN were used to detect past population occurred in southern Sichuan Basin (Fig. 1). Major matr- expansions. Significant sum of square deviations (SSD) iline C contained two minor matrilines, one in southern rejected the rapid expansion model. Third, Bayesian and northern Sichuan Basin (C1), and the other in
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Fig. 2 Maternal genealogy from a Bayesian 1.00/-- inference analysis for Quasipaa boulengeri. D4 Numbers near branches are Bayesian pos- terior probabilities (90) and bootstrap proportions from a maximum parsimony analysis (70), which are only shown for D major matrilines and minor matrilines. 1.00/81 D3 Four major matrilines are identified by different colour as follows: blue, matriline 0.99/-- A; cyan, matriline B; red, matriline C; and 1.00/93 green, matriline D. D2 0.97/-- 1.00/84 Q. boulengeri 1.00/72 D1 1.00/100 C2 1.00/100 C1 C 1.00/98 1.00/100 1.00/100 B 1.00/100 1.00/100 1.00/100 A
0.03 southern Sichuan Basin and northern Yunnan (C2) although some patterns existed (Fig. 3). The allele (Fig. 1). These three matrilines were associated with the tree clustered Q. robertingeri within Q. boulengeri upper Yangtze River. Finally, matriline D was broadly (Fig. 3a). Two well-supported groups of alleles, AI and distributed mainly in the midstream basin of the AII, occurred in the tree (Fig. 3a) and the MJ network Yangtze River, with some individuals occurring in the displayed this scenario (Fig. 3d). AI contained all indi- Pearl River. It consisted of four well-supported minor viduals from matriline A (Fig. 2) (Fig. 1: locality 9, 10, matrilines whose relationships were unresolved. The 11), as well as alleles of some individuals from other four major matrilines (A–D) were geographically matrilines, including matriline B from Changning, restricted; only five of the 45 localities (9–11, 13, 14; Xuyong, and Mt. Jinfoshan (Fig. 2) (Fig. 1: locality 8, 9, Fig. 1) contained individuals from more than one 12, respectively), matriline C from Kangxian, Xuyong, matriline. and Hejiang (Fig. 2) (Fig. 1: locality 1, 9, 10, respec- Compared with analyses of the mtDNA sequences, tively), and matriline D from Yuanhou, Shizhu, and those for GCG did not show strong historical patterning, Jiannan (Fig. 2) (Fig. 1: locality 11, 13, 14, respectively).
(a) Fig. 3 Allele tree and MJ network for (d) Quasipaa boulengeri constructed from the AII nuclear gene GCG. Colours correspond to matrilines in Figure 2 where blue is for matriline A, cyan for matriline B, red for matriline C and green for matriline (b) 1 9 D. (a) A Bayesian tree based on alleles of AI 0.96 8,9,11,13,14 GCG. Numbers above the nodes are 11 Q. boulengeri 0.97 Bayesian posterior probabilities. (b, c) 13 1.00 10 Magnified topology of allelic groups AI 10 and AII from Fig. 3a. Numbers on termi- 0.96 AI 12 1.00 nals refer to localities (Figure 1). Identical 0.91 1 symbols, including the triangle, circle 10 and square, indicate the same, heterozy- gous individual. (d) MJ network. Two 1.00 1.00 AII clusters were identified that correspond (c) 14 to allelic groups AI and AII in (a). 1.00 12 1.00 2,13,14 1.00 0.99 2 9 0.98 0.97 2 outgroup 2 0.1
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Excluding locality 1 from northern Sichuan Basin, all matrilines D1–D4 were distributed in two or three areas frogs in AI were from south-eastern Sichuan Basin (UM 50%, UMP 50%; Fig. 6). (localities 8–14). AII contained the remaining individu- als of matriline B (Fig. 2) (Fig. 1: locality 2, 3, 13, 14). Historical demography The relationships of other individuals in matrilines C and D (Fig. 2) were unresolved. Three heterozygous Demographic expansion of Q. boulengeri was not individuals had alleles from both AI and AII: supported by the three methods we employed. The KIZYPX17780, KIZ07923 and KIZ03143 from Xuyong expansion hypothesis was significantly rejected by mis- (locality 9), Mt. Jinfoshan (locality 12), and Shizhu match analyses in matrilines A, C1, C2 and D4. Values (locality 13), respectively (Fig. 3b, c). of SSD and raggedness index did not reject population expansions for matrilines B, D1, D2, and D3 (Table 3), however, the mismatch distributions of D1–D3 were not Molecular diversity and genetic structure unimodal (Fig. 4). In neutrality tests, only matriline B Overall h and p for Cytb were 0.9821 Æ 0.0022 and showed significant negative value for Fu’s Fs and 0.0406 Æ 0.0196, respectively. The number of haplotypes demographic expansion was not supported in all other (n), h, and p varied among populations (Table 1). Popu- matrilines (Table 3). Bayesian skyline plots suggested lations from Xuyong, Hejiang, Yuanhou and Mt. Lei- that the effective population size was relatively stable gongshan (localities 9–11, 35; Fig. 1) had remarkably or slowly decreasing in all tested matrilines (Fig. 5). higher values as expected given the co-occurrences of matrilines. The population from Xuyong had the high- Discussion est p value, which corresponded to the co-occurrence of haplotypes from matrilines A, B and C. The population Deep pre-Pleistocene split from Mt. Leigongshan, which harboured the highest h value, contained haplotypes from matrilines D2 and D3. Populations from Sichuan Basin (matrilines A, B and C) Populations from Hejiang and Yuanhou contained two have a 4.77% to 12.77% mtDNA difference (Fig. 1). The matriline each, A and C, and A and D, respectively, high levels of splits likely reflect long-term isolation, and also had high values of p and h. Results from the which is likely associated with the complex geological two-level hierarchical AMOVA suggested Q. boulengeri history of Southwest China. The divergence of matriline was highly structured geographically: 72.45% of the A dates to about 8.72 Ma and matrilines B, C, and D from genetic variation was attributed to differentiation 3.18 to 3.46 Ma (Fig. 6), suggesting that these matrilines among populations, and this was highly significant separated during the late Miocene and again in the mid- (P < 0.001). Except for seven pairs of populations, pair- dle Pliocene. These two periods are broadly consistent wise Φst values were statistically significant ranging with the rapid and drastic uplifting of the QTP and adja- from 0.0616 to 1.000 (P < 0.001 to P = 0.027). Genetic cent Southwest China, the oldest starting about 8 Ma divergence ranged from 4.12% to 12.77% between matri- (Harrison et al. 1992) followed by mid-Pliocene events lines A–D, and from 2.66% to 4.30% between minor (Cui et al. 1996; Shi et al. 1999; Li et al. 2001a). Each phase matrilines C1–C2 and D1–D4 (Table 2). of geological movement is responsible for habitat frag- mentation, which could hinder or stop dispersion and gene flow between populations. Similar effects are Spatio-temporal reconstruction reported for several species of plants (Liu et al. 2006; The null hypothesis of clock-like evolution was not Yuan et al. 2008) and animals (He et al. 2001; Zhang et al. rejected at the 5% level. Therefore, we used a strict 2010a,b; Li et al. 2012). Thus, the complex geological molecular clock. Time since the most recent common history appears responsible for driving the high level of ancestor of the entire ingroup was estimated to be species diversity in the western mountains of China. 16.04 Ma (95% HPD, 12.43–21.01 Ma). Matriline A Mitochondrial DNA analyses suggest that Quasipaa diverged at 8.72 Ma (95% HPD, 6.42–11.73 Ma) and ma- boulengeri is geographically restricted to local montane trilines B, C and D diverged between 3.18 to 3.46 Ma areas. About 85% of the haplotypes occur in one location (95% HPD, 2.39–4.25 to 2.59–4.65 Ma). Matrilines C1–C2 only. The AMOVA and Φst analysis obtain a high level of diverged at 2.39 Ma (95% HPD, 1.69–3.29 Ma), and ma- geographic structuring. In addition to complex topologi- trilines D1–D4 diverged from 1.93 to 2.36 Ma (95% cal changes, this pattern may result from the isolation of HPD, 1.34–2.66 to 1.75–3.16 Ma). Ancestral area recon- populations due to specific habitat requirements. Quasi- structions for matrilines A–D, corresponding to ances- paa boulengeri mainly occurs in rocky montane streams. tral nodes 1, 2 and 3, were from the upper Yangtze This specific habitat requirement may limit gene flow River (U, 100%; Fig. 6). Ancestral populations of and result in a pattern structured by genetic drift.
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Table 1 Grouping and summary of p genetic diversity of each population in Locality Grouping Hap n/N h Quasipaa boulengeri 1 U 1,39,96 3/9 0.6389 Æ 0.1258 0.0040 Æ 0.0026 2 U 2,4,10,14,16 5/12 0.5758 Æ 0.1634 0.0008 Æ 0.0008 3 U 10,60 2/10 0.3556 Æ 0.1591 0.0009 Æ 0.0008 4 U 86,93,95,97 4/12 0.7576 Æ 0.0806 0.0091 Æ 0.0052 5 U 3 1/10 —— 6 U 3 1/10 —— 7 U 100,101,102,3 4/9 0.7778 Æ 0.1100 0.0025 Æ 0.0018 8 U 98,99 2/11 0.1818 Æ 0.1436 0.0002 Æ 0.0004 9 U 99,103–109 8/13 0.9231 Æ 0.0500 0.0677 Æ 0.0352 10 U 110–114 5/14 0.6593 Æ 0.1227 0.0509 Æ 0.0264 11 U 26–29 4/10 0.7111 Æ 0.1175 0.0525 Æ 0.0282 12 U 9 1/1 —— 13 U 7–9,11 4/10 0.6444 Æ 0.1518 0.0242 Æ 0.0132 14 U 8,9,64 3/8 0.4643 Æ 0.2000 0.0194 Æ 0.0110 15 U 12,13 2/10 0.4667 Æ 0.1318 0.0029 Æ 0.0019 16 U 12,13,18,19 4/10 0.7333 Æ 0.1005 0.0079 Æ 0.0046 17 U 12,15,17 3/10 0.6444 Æ 0.1012 0.0009 Æ 0.0008 18 U 30–33 4/10 0.5333 Æ 0.1801 0.0044 Æ 0.0028 19 M 67,33 2/2 —— 20 U 20 1/1 —— 21 U 24,25 2/3 —— 22 U 21–23 3/9 0.7222 Æ 0.0967 0.0030 Æ 0.0020 23 U 38,40,41 3/10 0.6444 Æ 0.1012 0.0032 Æ 0.0021 24 P 5,6 2/2 —— 25 P 42,43 2/10 0.3556 Æ 0.1591 0.0044 Æ 0.0028 26 M 34–37 4/11 0.7091 Æ 0.0990 0.0029 Æ 0.0019 27 P 54–57 5/10 0.6593 Æ 0.1227 0.0026 Æ 0.0017 28 M 64 1/10 —— 29 M 66,64 2/2 —— 30 M 62 1/1 —— 31 M 68–72,17,12 7/10 0.9111 Æ 0.0773 0.0071 Æ 0.0042 32 M 69,73–75 4/10 0.6444 Æ 0.1518 0.0024 Æ 0.0017 33 M 80 1/1 —— 34 M 67,69,76–79 6/11 0.8909 Æ 0.0633 0.0097 Æ 0.0055 35 P 44–50,37 8/10 0.9556 Æ 0.0594 0.0170 Æ 0.0095 36 P 51–53 3/10 0.7333 Æ 0.1199 0.0041 Æ 0.0026 37 M 81 1/1 —— 38 M 58,59,61 3/6 0.733 Æ 0.1552 0.0035 Æ 0.0024 39 P 62 1/1 —— 40 M 84,85,87 3/6 0.8000 Æ 0.1217 0.0013 Æ 0.0012 41 M 82 1/3 —— 42 M 83 1/2 —— 43 M 65 1/2 —— 44 M 88–92 5/7 0.9048 Æ 0.1033 0.0063 Æ 0.0040 45 M 77,79,67,94 4/10 0.7778 Æ 0.0907 0.0111 Æ 0.0063
Hap, Cytb haplotype; n, the number of Cytb haplotypes; N, the number of individuals; h, haplotype diversity; p, nucleotide diversity; U, upstream Yangtze River; M, mid- stream Yangtze River; P, Pearl River.
River. Dispersal from the UTYR best explains the occur- Drainage-mediated dispersal from upper to middle rence in MTYR, both from the matrilineal pattern and Yangtze River the spatio-temporal reconstruction analyses (Fig. 6). The Data analyses imply one possible historical dispersal maternal ancestor of the species appears to have event for Q. boulengeri. Matrilines A, B and C occur in occupied the UTYR from 8.72 to 3.18 Ma. About upper tributaries of the Yangtze River (UTYR) and 2.36 Ma, matriline D appears to have dispersed to the matriline D mainly occupies midstream tributaries of MTYR (Fig. 6: UM) or the MTYR and Pearl River the Yangtze River (MTYR), with some in the Pearl (Fig. 6: UMP). The exact origin (UTYR or MTYR) of
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Table 2 Genetic distance for Cytb between major and minor matrilines esti- A B C C1 C2 D D1 D2 D3 mated in Kimura’s two-parameter model A B 0.1251 C 0.1277 0.0477 C1 0.1231 0.0477 — C2 0.1285 0.0480 — 0.0430 D 0.1243 0.0412 0.0506 0.0530 0.0492 D1 0.1264 0.0432 0.0534 0.0536 0.0532 — D2 0.1208 0.0401 0.0482 0.0531 0.0451 — 0.0348 D3 0.1174 0.0391 0.0514 0.0522 0.0510 — 0.0334 0.0297 D4 0.1266 0.0416 0.0505 0.0510 0.0501 — 0.0361 0.0305 0.0266
Table 3 Statistics of neutrality tests and results of the mismatch distribution anal- Neutrality tests Mismatch distribution yses for matrilines of Quasipaa boulengeri Tajima’s D Fu’s Fs SSD Raggedness Matrilines (P-value) (P-value) (P-value) index (P-value)
Matriline A 0.9578 (0.8810) 2.3970 (0.8530) 0.8408 (0.0000) 0.0808 (1.0000) Matriline B À1.3036 (0.0750) À6.2030 (0.0010) 0.0061 (0.1800) 0.0697 (0.2500) Matriline C1 0.5635 (0.7660) 1.5609 (0.7790) 0.0681 (0.0250) 0.1117 (0.0820) Matriline C2 À0.6859 (0.2490) 0.4954 (0.6130) 0.5708 (0.0000) 0.1033 (1.0000) Matriline D1 0.2210 (0.6910) À0.3370 (0.5290) 0.0121 (0.3210) 0.0113 (0.4470) Matriline D2 À0.6138 (0.3140) À4.7921 (0.1300) 0.0112 (0.2740) 0.0168 (0.1750) Matriline D3 À0.8564 (0.2010) À1.9077 (0.2160) 0.0051 (0.8240) 0.0161 (0.8400) Matriline D4 2.1285 (0.9950) 4.2255 (0.9650) 0.1066 (0.0010) 0.1061 (0.2800)
SSD, Sum of squares deviations. populations in the Pearl River remains unknown. The population expansion during interglacial times seems to unresolved relationships of matrilines D1–D4 suggest have contributed to the current patterns of genetic diver- the rapid diversification into newly available habitat. sity. However, this scenario is not the case for Q. boulen- Drainage evolution can drive geographic patterns of geri. The species seems to have survived environmental genetic diversity for stream-associated amphibians perturbations intact and, consequently, its genetic varia- (Jones et al. 2006; Kozak et al. 2006; Zhang et al. 2010b; tion is highly structured geographically as shown by the Zhou et al. 2012). Historically, the UTYR flowed south- AMOVA and Φst analysis. All divergences predate Pleisto- wards into the Red River (Clark et al. 2004; Yang 2006) cene climatic changes and none of our analyses detect and the MTYR and lower Yangtze River flowed into the distinct expansions of populations, which are typical East China Sea (Yang 2006). Although the exact time for responses to climatic oscillations. The mismatch analyses the connection at the TGMR is controversial, this significantly reject the expansion hypothesis in matriline appears to have occurred sometime between the late Pli- A, C1, C2, and D4 (Table 3). The neutrality tests and ocene and early Pleistocene, when the Yangtze River Bayesian skyline plots do not consistently detect rapid shifted its drainage network (Li et al. 2001b). The esti- population expansion. For example, though population mated time of dispersal for Q. boulengeri (3.18–2.36 Ma; expansion for matriline D1 to D3 are not rejected by Fig. 6) corresponds exactly to this period. mismatch distributions, and multimodal shapes for D1 to D3 (Fig. 4) do not support this hypothesis. Long-term stability of populations also occurs in Cathaya argyrophy- Persistence during Pleistocene climatic oscillations lla (Wang & Ge 2006), Ginkgo biloba (Gong et al. 2008) Population demography of species is often associated and Spizixos semitorques (Gao et al. 2011). Thus, Pleisto- with cyclical Pleistocene glaciations in Europe and cene climatic cycling does not appear to be an important North America (Hewitt 2000, 2004). In southern China, driver for some species in southern China. this scenario also exists for some plants (Qiu et al. Two major factors may explain the absence of expan- 2009; Huang et al. 2010), and animals (Li et al. 2009; sion. First, West China is a complex mosaic of several Zhan & Fu 2011; Gao et al. 2012). In these cases, rapid mountains that experienced a relatively stable climate
© 2012 Blackwell Publishing Ltd 1128 F. YAN ET AL.
(a) 140 (b) 400 Lineage A Lineage B 120 350 100 300 250 80 200 60 150 Frecuency Frecuency 40 100 20 50 0 0 0 5 10 15 20 25 0123456 Pairwise differences Pairwise differences
(c) 50 (d)1200 45 Lineage C1 Lineage C2 40 1000 35 800 30 25 600 20 Frecuency 15 Frecuency 400 10 200 5 0 0 0246810 0481216 Pairwise differences Pairwise differences
(e) 350 Lineage D1 (f) 700 Lineage D2 300 600 250 500 200 400 150 300 Frecuency 100 Frecuency 200 50 100 0 0 1 6 11 16 21 26 1 6 11 16 21 Pairwise differences Pairwise differences
(g) 50 (h) 16 Lineage D4 45 Lineage D3 14 40 12 35 30 10 25 8 20 6 Frecuency 15 Frecuency 4 10 5 2 0 0 147101316 14710131619 Pairwise differences Pairwise differences
Fig. 4 Mismatch distributions for each matriline of Quasipaa boulengeri. The abscissa indicates the number of pairwise differences between compared sequences. The ordinate is the frequency for each value. Histograms are the observed frequencies of pairwise divergences among sequences and the line refers to the expectation under the model of population expansion. (a–h) Mismatch distri- butions for the matrilines A, B, C1, C2, D1, D2, D3 and D4, respectively. throughout the Pleistocene (Weaver et al. 1998; Pinot Secondary contact et al. 1999; Ju et al. 2007). Second, Q. boulengeri dwells in cold, montane streams. This aquatic habitat provides South-eastern Sichuan Basin is an area of secondary relatively stable conditions today and probably during contact for several matrilines. Although nuclear data Pleistocene climatic cycling. The restriction to an aquatic from GCG do not well-resolve relationships among habitat predicts that populations of Q. boulengeri would Q. boulengeri due to implied gene flow or a recent ori- have been isolated in the fragmented mountain habitats. gin, especially for matrilines C and D, they show some Hilly mountains and river valleys may facilitate short interesting features. The allele tree (Fig. 3a) and the MJ distance vertical migrations (Zhang et al. 2010a). network (Fig. 3d) identify two groups, AI and AII.
© 2012 Blackwell Publishing Ltd OROGENESIS OUTWEIGHS THE PLEISTOCENE IN A FROG 1129
(a) (b) 1.0E1 1.0E1 Lineage A Lineage B
1.0E0 1.0E0
1.0E-1 1.0E-1
Effective population size (Neτ)
Effective population size (Neτ) 1.0E-2 1.0E-2 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.0 0.01 0.02 0.03 0.04 Time (Millons of years before present) Time (Millons of years before present)
(c) (d) 1.0E1 1.0E1 Lineage C1 Lineage C2
1.0E0 1.0E0
1.0E-1 1.0E-1
Effective population size (Neτ)
Effective population size (Neτ) 1.0E-2 1.0E-2 0.0 0.05 0.1 0.15 0.2 0.0 0.1 0.2 0.3 0.4 Time (Millons of years before present) Time (Millons of years before present)
(e) (f) 1.0E1 1.0E1 Lineage D1 Lineage D2
1.0E0 1.0E0
Effective population size (Neτ)
Effective population size (Neτ) 1.0E-1 1.0E-1 0.0 0.25 0.5 0.75 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Time (Millons of years before present) Time (Millons of years before present)
(g) (h) 1.0E1 1.0E1 Lineage D3 Lineage D4
1.0E0
1.0E0
1.0E-1
Effective population size (Neτ)
Effective population size (Neτ) 1.0E-1 1.0E-2 0.0 0.05 0.1 0.15 0.2 0.25 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Time (Millons of years before present) Time (Millons of years before present)
Fig. 5 Bayesian skyline plot estimated by BEAST for each matriline of Quasipaa boulengeri: x-axis, time in millions of years; y-axis, effective population size (units = Neτ, the product of effective population size and generation length in millions of years). The mean estimate and both 95% HPD limits are indicated. (a–h) Bayesian skyline plots for matrilines A, B, C1, C2, D1, D2, D3 and D4, respectively.
Whereas AII has a loose association with matriline B, indicate that contemporary gene flow mainly occurs almost all alleles in AI are from matriline A along with between matriline A and other matrilines. Alleles in AI some alleles from matrilines B–D. These patterns occur mostly in individuals from south-eastern Sichuan
© 2012 Blackwell Publishing Ltd 1130 F. YAN ET AL.
Fig. 6 Time tree for Quasipaa boulengeri. 22 20 18 1614 12 10 8 6 4 2 0 Ma Branch lengths are proportional to diver- gence times. Tree topology is derived D4 from BEAST. Bars on the nodes are 95% confidence intervals. Matrilines A, B, C1, C2 and D1–D4 correspond to those in UM 50% Figure 2. Labels above nodes 1, 2, 3 and UMP 50% 4 4 indicate the results from S-DIVA.U, upstream Yangtze River; M, midstream D3 Yangtze River; P, Pearl River.
U 100% 3 D2
Q. boulengeri D1 U 100% 2 C2
U 100% C1 1 B
A
2.0
Basin (localities 8–14; Fig. 1). For example, populations et al. 2011). This species is geographically restricted to from Xuyong, Hejiang and Yuanhou (localities 9–11, several local montane areas only. Because isolated mon- respectively) have extremely high levels of lineage tane populations may easily lose variation via genetic diversity (Fig. 2: matrilines ABC, AC and AD, respec- drift and inbreeding (Frankham 1996; Young et al. 1996; tively), yet all individuals cluster into AI (Fig. 3a, d). Keller & Waller 2002), continued monitoring and assess- Moreover, three individuals in matriline B are clearly ment is necessary to assure perpetuation of the evolution- heterozygous for GCG and have alleles from both AI ary potential of the species. Owing to limited resources, it and AII (Fig. 3b, c). These observations provide strong may be necessary to prioritize the protection of remain- evidence for gene flow between matrilines A and B. ing populations. For example, because locality 9 has the Our data support the hypothesis that despite the high highest level of mtDNA diversity and involves most level of mtDNA divergence between matriline A and matrilines, its protection may be a priority. South-eastern matrilines B–D, unabated gene flow maintains genetic Sichuan Basin is a major genetic reservoir for Q. boulenge- cohesion. Alleles in AI occur mostly in individuals from ri. This region holds four major matrilines and the region south-eastern Sichuan Basin (localities 8–14; Fig. 1), and requires continual monitoring and special protection. this may be an area of secondary contact. South-eastern Sichuan Basin is the contact zone of Conclusions four matrilines. Panmixia may occur here. Further study of populations from this area using multiple hypervari- Orogenesis of the Qinghai-Tibetan Plateau in West able nuDNA loci, such as short tandem repeats (micro- China built a complex montane system that involved satellites), may facilitate an understanding of the changing paleodrainages. Reconstructed population drivers of differentiation. histories suggest that these developing montane system and paleodrainages drove the formation of high levels Implications for conservation of genetic divergence in Q. boulengeri. Pleistocene climatic oscillations do not appear to be of consequence Our study may serve as a guide for monitoring and for this species. Gene flow maintains genetic cohesion developing a conservation strategy for endangered among deeply diverged matrilines. Habitat protection is Q. boulengeri (Hilton-Taylor 2000; Lau et al. 2004). As a necessary and the careful management of human montane, stream-dwelling frog, three factors may be consumption is required to conserve this species. major threats: (i) habitat destruction and (ii) degradation mainly due to the deforestation, and (iii) water pollution due to economic and agricultural development. Over- Acknowledgements exploitation for human consumption in southern China We thank Peng Guo, Yongbiao Xu, Shaoneng Wang, Yunke is another problem (Lau et al. 2004; Xie et al. 2007; Dai Wu and Li Ding for providing tissues and helping during
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