genealogy and palaeodrainage basins in yunnan province: …labs.eeb.utoronto.ca/murphy/pdfs of...

Molecular Ecology (2010) 19, 3406–3420 doi: 10.1111/j.1365-294X.2010.04747.x

Genealogy and palaeodrainage basins in YunnanProvince: phylogeography of the Yunnan spiny frog,Nanorana yunnanensis (Dicroglossidae)

DONG-RU ZHANG,* 1 MING-YONG CHEN,*†‡1 ROBERT W. MURPHY,*§ J ING CHE,* JUN-FENG

PANG,* J IAN-SHENG HU,† J ING LUO,†– SHAN-J IN WU,*** HUI YE† and YA-PING ZHANG*–

*State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences,

Kunming 650223, China, †College of Life Sciences, Yunnan University, Kunming 650091, China, ‡Institute of National Nature

Reserves, the Dai Nationality Autonomous Prefecture, Xishuangbanna 666100, China, §Centre for Biodiversity and Conservation

Biology, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ont., M5S 2C6, Canada, –Laboratory for Conservation and

Utilization of Bio-resources, Yunnan University, Kunming 650091, China, **Sichuan Key Laboratory of Conservation Biology on

Endangered Wildlife, College of Life Science, Sichuan University, Chengdu 610064, China

Corresponde

E-mail: zhan

5033724; E-m1These autho

Abstract

Historical drainage patterns adjacent to the Qinghai-Tibetan Plateau differed markedly

from those of today. We examined the relationship between drainage history and

geographic patterns of genetic variation in the Yunnan spiny frog, Nanorana yunnanensis,

using approximately 981 base pairs of mitochondrial DNA partial sequences from

protein-coding genes ND1 and ND2, and intervening areas including complete tRNAIle,

tRNAGln and tRNAMet. Two null hypotheses were tested: (i) that genetic patterns do not

correspond to the development of drainage systems and (ii) that populations had been

stable and not experienced population expansion, bottlenecking and selection. Genea-

logical analyses identified three, major, well-supported maternal lineages, each of which

had two sublineages. These divergent lineages were completely concordant with six

geographical regions. Genetic structure and divergence were strongly congruent with

historical rather than contemporary drainage patterns. Most lineages and sublineages

were formed via population fragmentation during the rearrangement of paleodrainage

basins in the Early Pliocene and Early Pleistocene. Sympatric lineages occurred only in

localities at the boundaries of major drainages, likely reflecting secondary contact of

previously allopatric populations. Extensive population expansion probably occurred

early in the Middle Pleistocene accompanying dramatic climatic oscillations.

Keywords: China, drainage history, mtDNA, Nanorana bourreti, Yunnan Plateau

Received 14 December 2009; revision received 9 May 2010; accepted 12 May 2010

Introduction

Genealogical analyses, including phylogeography, can

discover mechanisms that shape genetic structure in

natural populations. These mechanisms might promote

future diversification and preserve the evolutionary

potential of extant species (Moritz 2002). Mitochondrial

nce: Prof. Ya-Ping Zhang, Fax: 86-871-5195430;

[email protected] and Prof. Hui Ye, Fax: 86-871-

ail: [email protected].

rs contributed equally.

DNA (mtDNA) is often chosen as a marker in genealog-

ical studies because it is inherited clonally through

maternal lineages as a single linked locus. Gene flow

and genetic recombination do not obscure its genealogi-

cal history. However, mtDNA has a smaller effective

population size and it can provide only part of a spe-

cies’ history (Zhang & Hewitt 2003; Ballard & Whitlock

2004). Thus, inferences on the history of species ⁄ popula-

tions should be taken with caution (Ballard & Whitlock

2004).

The Qinghai-Tibetan Plateau (QTP) and its adjacent

areas span three biodiversity hotspots: Himalaya,

� 2010 Blackwell Publishing Ltd

DRAIN AGE HISTORY AND YUNNAN SPI NY FROG PHYLOGEOGRAPHY 3407

Indo-Burma and the mountains of southwestern China

(Myers et al. 2000; Biodiversity-hotspots 2005). The

QTP’s dramatic variation in climate and topography

supports a wide array of habitats (Biodiversity-hotspots

2005) and forms a model ecosystem for investigating

speciation. The Late Cenozoic uplifting of the QTP

seems to be a main driving force for shaping the recent

genetic structure and biodiversity of organisms in the

region (e.g. Liu et al. 2006).

Being adjacent to the QTP, the Yunnan Plateau is

extremely complex in topography and climate and of

great interest to biologists and geologists alike. It

increasingly plays an important role in revealing biolog-

ical consequences of the Late Cenozoic orogenic events

(Cheng et al. 2001). Different regions on the Yunnan

Plateau responded uniquely to each period of uplifting,

as did the evolution of paleo-drainage systems (Cheng

et al. 2001).

Historically, drainage of the major continental East

Asian rivers on the southeastern margin of the Tibet

plateau differed markedly from their current directions

(Ruber et al. 2004) (Fig. S1). On the Yunnan Plateau,

river capture and reversal events occurred with the Late

Cenozoic uplifting of QTP (Cheng et al. 2001; Clark

et al. 2004).

Previous investigations into freshwater fishes in

China suggested that historical drainage rearrange-

ments (e.g. basin connection, river reversal and river

capture) generated genetic structure and biodiversity

(e.g. Perdices et al. 2004, 2005; Yang et al. 2009). How-

ever, these studies mainly involved drainage basins out-

side of Yunnan province. Here, we investigate the

association between drainage histories in areas adjacent

to the QTP and mtDNA genetic structure in a species of

dicroglossid frog, Nanorana yunnanensis.

Nanorana yunnanensis occurs in southwestern China

(Guizhou, Yunnan and Sichuan provinces), northern

Vietnam and Myanmar and presumably in intervening

Laos (Frost 2008). It has a low dispersal capability and

shows a preference for living near moss-covered rocks

in cold montane streams at higher elevations (from

about 1500–2500 m; personal observations). These eco-

logical features make N. yunnanensis an ideal species

for investigating the genetic consequences of the geolog-

ical history of drainage basins.

Our study provides the first test of the vicariance

hypotheses for this region using a semi-aquatic anuran.

Our null hypothesis states that geographic and genea-

logical histories correspond poorly to one another, i.e.

that genetic patterns do not correspond to the develop-

ment of drainage systems. If true, we expect to observe

broadly mixed haplotype groups among nearby rivers,

while allowing for isolation-by-distance. The null

hypothesis is rejected upon discovering that lineage his-


tory corresponds to the historical development of drain-

age systems. If this null hypothesis is rejected, then a

new hypothesis can be tested further. The new hypothe-

sis states that genetic patterns correspond to geological

history. Rejection of this hypothesis requires one of two

explanations: either the geological history is inaccurate,

or historically the frogs have dispersed. These two pos-

sibilities can be tested using several genetic predictions.

An inaccurate geological history can be supported by

discovering multiple taxa that have the same pattern of

genealogical history, and dispersal can be identified by

discovering genealogical relationships in the form of a

Hennigean comb where relationships correspond with

geographic distance (e.g. Murphy & Aguirre-Leon

2002).

Materials and methods

Sampling

A total of 1193 individuals of N. yunnanensis were col-

lected from 71 localities throughout the Chinese range

plus one sample site from Vietnam (Table 1; Fig. 1).

Sampling emphasized different river systems and

mountain ranges, rather than from a same area. Sample

sizes mostly range from 10 to 30 individuals per loca-

tion depending on population density. Following

approved Animal Use Protocols, five voucher speci-

mens were collected from each locality with sample size

‡5. Toe clips taken for the remaining samples were pre-

served in 95% ethanol, and transferred and frozen at

)80 �C in the laboratory; live frogs were then released.

The sister taxa to N. yunnanensis, Nanorana quadranus

and Nanorana unculuanus (Che et al. 2009) were used as

outgroup taxa for genealogical reconstruction. Voucher

specimens were deposited in the Kunming Institute of

Zoology, Chinese Academy of Sciences; Yunnan Univer-

sity and Sichuan University Museum (Table S1).

Laboratory procedures

Genomic DNA was extracted with proteinase K fol-

lowed by the standard 3-step phenol-chloroform

method (Sambrook et al. 1989). Partial segments of ND1

and ND2 (subunits one and two of NADH) genes and

the three complete intervening tRNA genes were

obtained for all individuals. PCR amplification used the

primer combinations L3878 (Macey et al. 1998) and

H4980 (Macey et al. 1997). Amplified fragments were

sequenced with PCR primer combinations and an inter-

nal primer L4160 (Kumazawa & Nishida 1993). Amplifi-

cation was conducted in a 25 lL volume reaction

initiated at 95 �C for 5 min followed by 35 cycles of

94 �C for 1 min, 46 �C for 45 s, 72 �C for 1 min and a

Table 1 Summary of sample site details for Nanorana yunnanensis. For each population sampled, geographic origin, identified sublin-

eage (W1a, W1b, W2a, W2b, C1, C2a, C2b, E1 and E2), sample size (N) and coordinates (latitude ⁄ longitude) are given. Haplotype diver-

sity (h) and nucleotide diversity (p) for each population with sample size ‡10 are presented

Sample

site Locality Lineage N Latitude Longitude h p

1 Husa, Longchuan Co.,YN W1b 7 24�27¢N 97�53¢E 0.7143 ± 0.1809 0.001553 ± 0.001207

2 Chenzi, Longchuan Co., YN W1b 19 24�21¢N 97�57¢E 0.6959 ± 0.0619 0.002897 ± 0.001784

3 Jietou, Tengchong Co., YN W1b 19 25�30¢N 98�40¢E 0.7544 ± 0.0904 0.002229 ± 0.001442

4 Gaoligong mountain, YN W1b 4 25�07¢N 98�42¢E5 Caizidi, Longlin Co., YN W1a 1 24�35¢N 98�41¢E6 Xiyi, Longyang Borough, YN W1a, W2b 24 24�53¢N 99�17¢E 0.5616 ± 0.0468 0.025691 ± 0.013027

7 Wumulong, Yongde Co., YN W2b 9 24�22¢N 99�39¢E8 Boshang, Linxiang Borough, YN W2b 24 23�43¢N 100�03¢E 0.8514 ± 0.0491 0.005274 ± 0.002950

9 Mengku, Shuangjiang Co., YN W2b 18 23�37¢N 99�52¢E 0.9020 ± 0.0374 0.004990 ± 0.002852

10 Wangjiao, Cangyuan Co., YN W2b 3 23�09¢N 99�15¢E11 Mengsuo, Ximeng Co., YN W2a 2 22�45¢N 99�28¢E12 Nanding, Lancang Co., YN W2b 21 22�54¢N 100�10¢E 0.7619 ± 0.0688 0.002961 ± 0.001807

13 Mengma, Menglian Co., YN W2a 18 22�15¢N 99�24¢E 0.8105 ± 0.0703 0.013139 ± 0.006945

14 Bada, Menghai Co., YN W2a 19 21�50¢N 100�25¢E 0.2924 ± 0.1274 0.005782 ± 0.003241

15 Yiwu, Mengla Co., YN W2b 22 21�58¢N 101�27¢E 0.5065 ± 0.0496 0.003098 ± 0.001871

16 Tongxin, Ninger Co., YN W2b 7 22�57¢N 101�04¢E17 Daying, Binchuan Co., YN W2b 6 25�46¢N 100�27¢E18 Fenghuangshan, Nanjian Co., YN W2b 30 24�58¢N 100�20¢E 0 0

19 Huangcaoling, Jingdong Co., YN W2b 28 24�26¢N 100�50¢E 0.4841 ± 0.1094 0.003913 ± 0.002257

20 Shuitang, Xinping Co., YN W2b 22 24�07¢N 101�29¢E 0.7100 ± 0.0601 0.004157 ± 0.002404

21 Xin’an, Mojiang Co., YN W2b 30 23�34¢N 101�35¢E 0.8437 ± 0.0378 0.007253 ± 0.003895

22 Lianzhu, Mojiang Co., YN W2b 9 23�26¢N 101�41¢E23 Huanglianshan,Lvchun Co., YN W2b 11 22�51¢N 102�16¢E 0.7636 ± 0.1066 0.001001 ± 0.000825

24 Fengshuiling, Jinping Co., YN W2b 7 22�47¢N 103�04¢E25 Sa Pa vicinity, Lao Cai, Vietnam W2b 2 22�18¢N 103�46¢E26 Yilong, Shiping Co., YN C2a 23 23�43¢N 102�28¢E 0.6877 ± 0.0545 0.004118 ± 0.002379

27 Baoxiu, Shiping Co., YN C2a 11 23�46¢N 102�23¢E 0.7818 ± 0.1073 0.004856 ± 0.002900

28 Dutian, Shuangbai Co., YN C2a 4 24�33¢N 101�28¢E29 Fangtun, Yimen Co., YN C2a 3 24�40¢N 102�10¢E30 Tongchang, Yimen Co., YN C2a 17 24�42¢N 102�03¢E 0.4044 ± 0.1304 0.001649 ± 0.001148

31 Pubei, Yimen Co., YN C2a 3 24�36¢N 102�10¢E32 Zhongcun, Lufeng Co., YN C2a, C2b, E1 34 25�15¢N 102�04¢E 0.8752 ± 0.0332 0.027285 ± 0.013629

33 Yuanmou Co., YN C2b 13 25�42¢N 101�52¢E 0.9487 ± 0.0423 0.007031 ± 0.003977

34 Wanma, Yongren Co., YN C2b 31 26�15¢N 101�26¢E 0.6882 ± 0.0624 0.005134 ± 0.002851

35 Menghu, Yongren Co., YN C2b 7 26�05¢N 101�34¢E36 Shalong, Xiangyun Co., YN C2b 20 25�28¢N 100�32¢E 0.8211 ± 0.0598 0.007259 ± 0.003970

37 Zhoucheng, Binchuan Co., YN C2b 20 25�44¢N 100�34¢E 0.6316 ± 0.0875 0.001620 ± 0.001122

38 Fengyu, Eryuan Co., YN C2b 4 25�59¢N 99�55¢E39 Dengchuan, Eryuan Co., YN C2b 12 25�58¢N 100�04¢E 0.6818 ± 0.0786 0.002008 ± 0.001373

40 Xintun, Heqing Co., YN C2b 19 26�36¢N 100�13¢E 0.6491 ± 0.1085 0.004972 ± 0.002833

41 Yangping, Yongsheng Co., YN C2b 21 26�44¢N 100�48¢E 0.9000 ± 0.0390 0.005650 ± 0.003157

42 Yongxing, Huaping Co., YN C2b 19 26�50¢N 101�13¢E 0.8421 ± 0.0473 0.006724 ± 0.003714

43 Mingyin, Yulong Co., YN C2b 22 21�17¢N 100�21¢E 0.0909 ± 0.0809 0.001112 ± 0.000847

44 Lugu Lake, Ninglang Co., YN C2b 7 27�47¢N 100�46¢E45 Yanyuan Co., SC C2b 18 27�25¢N 101�30¢E 0.6993 ± 0.0669 0.004004 ± 0.002353

46 Qiaowa, Muli Co., SC C2b 19 27�56¢N 101�16¢E 0 0

47 Yandai, Jiulong Co., SC C2b 2 28�28¢N 101�44¢E48 Chengxiang, Mianning Co., SC C2b 26 28�33¢N 102�10¢E 0.5077 ± 0.0403 0.000518 ± 0.000504

49 Xichang, SC C2b 27 27�53¢N 102�16¢E 0.6040 ± 0.0850 0.003851 ± 0.002230

50 Leibo Co., SC C2b 3 28�15¢N 103�34¢E51 Rehe, Dechang Co., SC C1, C2b 12 27�19¢N 101�57¢E 0.8636 ± 0.0716 0.008495 ± 0.004767

52 Cida, Dechang Co., SC C1 30 27�11¢N 102�04¢E 0.8299 ± 0.0329 0.001676 ± 0.001130

53 Tong’an, Huili Co., SC C1, C2b 22 26�21¢N 102�18¢E 0.8225 ± 0.0561 0.006297 ± 0.003472

54 Daqiao, Huidong Co., SC C1, C2b 19 26�40¢N 102�49¢E 0.7602 ± 0.0899 0.005925 ± 0.003313

3408 D. -R . ZH ANG ET AL.


Table 1 (Continued)

Sample

site Locality Lineage N Latitude Longitude h p

55 Qiaojia Co., YN C1, C2b 12 26�55¢N 102�55¢E 0.8636 ± 0.0550 0.014379 ± 0.007818

56 Sayingpan, Luquan Co., YN C1, C2a, C2b, E1, E2 54 26�00¢N 102�31¢E 0.8358 ± 0.0374 0.045260 ± 0.022109

57 Near Caohai, Weining Co., GZ E2 30 26�51¢N 104�14¢E 0.7747 ± 0.0520 0.005739 ± 0.003152

58 Zhehai, Huize Co., YN E2 21 26�33¢N 103�37¢E 0.7952 ± 0.0594 0.003155 ± 0.001905

59 Xintian, Dongchuan Co., YN E2 15 25�58¢N 103�06¢E 0.6000 ± 0.1129 0.001728 ± 0.001200

60 Xuanwei Co., YN E2 18 26�12¢N 104�06¢E 0.8627 ± 0.0470 0.003258 ± 0.001974

61 Qixing, Xundian Co., YN E2 20 25�31¢N 103�19¢E 0.1895 ± 0.1081 0.000193 ± 0.000286

62 Baisha, Zhanyi Co., YN E2 10 25�36¢N 103�48¢E 0.8000 ± 0.1001 0.001359 ± 0.001039

63 Jiuxian, Malong Co., YN E2 16 25�22¢N 103�22¢E 0.9000 ± 0.0461 0.004808 ± 0.002782

64 Sanchahe, Luliang Co., YN E2 21 25�02¢N 103�47¢E 0 0

65 Yangqiao, Songming Co., YN E2 21 25�20¢N 103�02¢E 0.7333 ± 0.0609 0.003311 ± 0.001983

66 Shuanglong, Panlong Borough, YN E2 33 25�06¢N 102�48¢E 0.5833 ± 0.0944 0.002170 ± 0.001378

67 Bajie, Anning, YN E2 20 24�40¢N 102�21¢E 0.8579 ± 0.0599 0.004743 ± 0.002710

68 Banqiao, Shiling Co., YN E2 7 24�40¢N 103�15¢E69 Longjie, Chengjiang Co., YN E2, E1 17 24�40¢N 102�55¢E 0.8162 ± 0.0607 0.027913 ± 0.014400

70 Chading, Wuding Co., YN E1, C2a, C2b 30 25�39¢N 102�17¢E 0.8000 ± 0.0561 0.036184 ± 0.018032

71 Hongta, Yuxi, YN E1 18 24�20¢N 102�30¢E 0.6993 ± 0.0840 0.001646 ± 0.001142

YN, Yunnan Province; SC, Sichuan Province; GZ, Guizhou Province.

Fig. 1 Map showing the geographic distribution of populations of Nanorana yunnanensis included in this study. Populations are

numbered as in Table 1 and presented as pie-diagrams, with slice size proportional to the frequency of the lineages ⁄ sublineages

occurring in the site. Inset in upper right corner shows an enlarged view of the population from Luquan. The rivers indicated in

lower right corner are as follows: I, Irrawaddy; S, Salween; M, Mekong; R, Red River; P, Pearl; and Y, Yangtze.




single final extension at 72 �C for 10 min. Negative con-

trols were run for all amplifications. PCR products were

purified with Gel Extraction Mini kit (Watson BioTech-

nologies, Shanghai, China). The purified product was

used as the template DNA for cycle sequencing reac-

tions performed using BigDye Terminator Cycle

Sequencing kit (version 2.0, Applied Biosystems), and

sequencing was conducted on an ABI PRISM 3730

(Applied Biosystems) automatic DNA sequencer with

both forward and reverse primers.

DNA sequence alignment

DNA sequences were edited using DNASTAR 5.0

(DNASTAR Inc.). Nucleotide sequences were aligned

using Clustal X 1.81 (Thompson et al. 1997) with default

parameters and then revised by eye in MEGA 3.1 (Kumar

et al. 2004). Protein-coding nucleotide sequences were

translated to amino acid using MEGA 3.1 (Kumar et al.

2004) to confirm alignment. Alignments of tRNA

sequences were constructed manually based on second-

ary structural models (Kumazawa & Nishida 1993) to

ensure proper alignment (Macey & Verma 1997). Posi-

tions of ambiguous alignment and those from variable

length intergenic regions were excluded from the analy-

ses. No gaps existed in the final database of aligned

sequences. Identical haplotypes were collapsed using

DnaSP 4.10.4 (Rozas et al. 2003).

Genealogical reconstruction

Historical relationships among haplotypes were recon-

structed using Bayesian inference (BI) as implemented

in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003), based

on the HKY+I+G (Hasegawa et al. 1985) model of

sequence evolution, as determined using ModelTest 3.7

(Posada & Crandall 1998). Two independent runs start-

ing from different random trees were performed with

four Markov chains. The analysis was run for 3 · 106

generations, and the chain was sampled every 100 gen-

erations. The first 8000 trees were discarded as burn-in,

after the log-likelihood scores stabilized and average

standard deviation of split frequencies approached zero.

Convergence onto the stationary distribution between

two independent runs was confirmed. A 50% majority

rule consensus of the sampled trees was constructed to

obtain Bayesian posterior probabilities (BPP) of the tree

nodes.

Molecular diversity and genetic structure

ARLEQUIN 3.1 (Excoffier et al. 2005) was used in the anal-

ysis as follows. Molecular diversity indices were esti-

mated for each sampled population with sample size

‡10 and for each lineage ⁄ sublineage. Population struc-

ture was measured by an analysis of molecular vari-

ance (AMOVA) (Excoffier et al. 1992) with significance

assessed by 10 000 permutations. Populations were

grouped according to either the mtDNA lineages iden-

tified by genealogical reconstruction, or major drainage

basins. The most plausible geographical subdivision

was assumed to be the one where among-group varia-

tion had the greatest statistical significance. Genetic dif-

ferentiation among populations was evaluated by

pairwise values of FST, and statistical significance was

assessed by 10 000 permutations. Both AMOVA and FST

used Tamura & Nei (1993) genetic distance with

gamma correction (C = 0.9030) for heterogeneity of

mutation rates.

Population demography

Historical population dynamics of the major lin-

eages ⁄ sublineages was examined with mismatch distri-

bution analysis (MDA) (Rogers & Harpending 1992)

implemented in ARLEQUIN 3.1. This analysis compared

the observed frequencies of pairwise differences in

haplotypes with those expected under a single sudden

expansion model (Rogers & Harpending 1992). An

expected distribution under a model of sudden demo-

graphic expansion was generated with a total of 5000

permutations. Under the null hypothesis of sudden

expansion, the raggedness index quantifying the

smoothness of the observed mismatch distribution

(Harpending 1994) and the sum of squares deviations

(SSD) between the observed and expected mismatch

distributions were assessed, and statistical significance

was tested by PRag and PSSD, respectively. While

demographic stability produces multimodal distribu-

tions, unimodal patterns occur during sudden popula-

tion expansion (Slatkin & Hudson 1991). The

raggedness index was expected to have a higher value

in relatively stable populations. If the sudden expan-

sion model was not rejected, a time of expansion (t)

was evaluated using the equation s = 2 ut (Rogers &

Harpending 1992), where s was the mode of mismatch

distribution, expressed in units of evolutionary time,

and u was the mutation rate for the entire sequence

under study. The value of u was calculated using the

formula u = lk, where l was the mutation rate per

nucleotide and k was the number of nucleotides

assayed.

Tajima’s D (Tajima 1989) and Fu’s FS statistics (Fu

1997), calculated using ARLEQUIN 3.1, were used to seek

evidence of demographic expansions within individual

lineages and sublineages. The statistical significance

was tested with 5000 permutations. The null hypothesis

was that of a stable population.



Dating

In the absence of fossil or geological data, the molecular

clock provides a valuable means of inferring the ages of

lineage (Bromham & Penny 2003) yet such should be

taken with caution. Increasing evidence suggests that

the DNA of closely related species can evolve at differ-

ent rates (Welch & Bromham 2005). The molecular clock

is usually calibrated using the fossil record or estab-

lished biogeographic events (Bromham & Penny 2003)

yet such may be a source of considerable error (Arbo-

gast et al. 2002).

The rate of divergence in ND1–ND2 was approxi-

mately constant across lineages of amphibians and rep-

tiles (e.g. Crawford 2003; Crawford & Smith 2005).

Therefore, we adopted a rate of 1.91% divergence per

million years obtained by Crawford (2003) by applying

a model-based correction to the ND2 data of Macey

et al. (1998). To test for rate heterogeneity among lin-

eages, we employed relative-rate tests using PHYLTEST

(Kumar 1996).

Time to the most recent common ancestor (TMRCA)

was estimated using the strict-clock Bayesian Markov

chain Monte Carlo method as implemented in BEAST

v1.5.4 (Drummond & Rambaut 2007). Analyses were

performed for 50 million generations using an HKY

model of nucleotide substitution with gamma distrib-

uted rate variation among sites, assuming a Yule specia-

tion process and a constant global molecular clock

0.957% ⁄ Myr. The effective sample size for parameter

estimates and convergence was checked using TRACER

(Rambaut & Drummond 2007).

Results

Authenticity of mitochondrial DNA

The fragment examined included six segments: partial

ND1 and ND2, tRNAIle, one unknown sequence frag-

ment and two copies of tRNAMet. In the ingroup sam-

ples and one outgroup taxon, N. quadranus, the

unknown sequence fragment between tRNAIle and

upstream tRNAMet exhibited considerable length varia-

tion. Sequences of less than about 50 bp could not be

folded into secondary structure typical of tRNAGln.

Thus, they were excluded from further analyses. A

complete tRNAGln occurred in the other outgroup

taxon, N. unculuanus, and it was typical of most meta-

zoans.

No premature stop codons were observed in the pro-

tein-coding genes, and the tRNAs had stable secondary

structures, indicating that the sequences were obtained

from functional genes. Light strand sequences showed a

strong bias against guanine (G = 11.3%, A = 33.5%,


T = 26.9%, and C = 28.3%), as characteristic of mtDNA.

Thus, the DNA sequences were not nuclear-integrated

copies of mitochondrial genes (Zhang & Hewitt 1996).

Sequence characteristics

Of the 981 bp of aligned ingroup nucleotides, 309 poly-

morphic sites had a transition ⁄ transversion ratio of

8.2033, and 230 sites were potentially phylogenetically

informative. The gamma-shape parameter and propor-

tion of invariable sites were estimated at 0.9030 and

0.4452, respectively. Among the 1193 specimens of

N. yunnanensis and two outgroup taxa, 251 haplotypes

were detected (GenBank Accession no. HM054164–

HM054414. DRYAD identifier for Data file: http://

hdl.handle.net/10255/dryad.1581).

Tamura-Nei-corrected sequence divergence between

haplotypes ranged from 0.1% to 11.4%. Uncorrected

p-divergence ranged from 0.1% to 9.38%.

Genealogy reconstruction

The lineages of N. yunnanensis exhibited strong geo-

graphical structure, and they clustered together and

with robust statistical support (Fig. 2). Three major

putatively independent evolutionary lineages were

identified by three phylogenetically and geographically

distinct haplogroups as follows: W (western), C (cen-

tral) and E (eastern) (Figs 1 and 2). Lineage W, distrib-

uted in Hengduan Mountains region (populations 1–

25), contained haplotypes from tributaries of Irrawaddy,

Salween, Mekong and Red rivers. Lineage C, located in

the Chuxiong Basin and the southwestern Sichuan Pla-

teau (populations 26–56), mainly contained haplotypes

from most of the tributaries of Jinsha River (upper and

part of the middle Yangtze River) plus a few from the

Red River. Lineage E, located on the central and eastern

Yunnan Plateau (populations 57–71), contained haplo-

types from tributaries of the Jinsha and Pearl rivers.

Co-occurring lineages occurred only in locations where

different drainages conjoined historically. Lineages C

and E co-occurred in Luquan, Lufeng and Wuding

(populations 56, 32 and 70, respectively) on the central

Yunnan Plateau (Fig. 1; Table 1). Lineages W and C

were syntopic in Binchuan (17 and 37; Fig. 1; Table 1).

Lineage W contained two sublineages, W1 and W2

(Fig. 2), separated by Yongde Snow Mountain. Sublin-

eage W1 (Fig. 1, populations 1–6) was restricted geo-

graphically to northern Yongde Snow Mountain and

W1b (populations 1–4) was separated from W1a (popula-

tions 5–6) by Gaoligong Mountain. Sublineage W2

(Fig. 1, populations 7–25) from southern Yongde Snow

Mountain contained sublineage W2a, comprised of pop-

ulations from MengHai (population 14), Menglian (13)

Fig. 2 Bayesian inference tree for Nanorana yunnanensis based on the 251 haplotypes from ND1–ND2 (including two outgroups) and

assuming the evolutionary model HKY+I+G. Numbers above the nodes are Bayesian posterior probability values. Vertical bars show

the lineage ⁄ sublineage assignment.


and Ximeng (11), and sublineage W2b, including the

remaining populations. Baoshan (Fig. 1, population 6)

had haplotypes from both W1 and W2.

Lineage C was comprised of sublineage C1, contain-

ing populations 51–56, and sublineage C2, mainly con-

sisting of populations 26–50. Sublineages C1 and C2

occurred syntopically in populations 51–56.

Sublineages E1 and E2 were syntopic only in popula-

tion 69. Sublineage E1 was geographically restricted to

Luquan, Wuding, Yuxi and Chengjiang (populations 56,

70, 71 and 69, respectively), most of which occurred on

the central Yunnan Plateau. Sublineage E2 occurred in

populations 57–69 on eastern Yunnan Plateau.

Net average genetic distances (Tamura-Nei with

gamma correction) between lineages were as follows:

W ⁄ C, 7.20% (±0.86%); W ⁄ E, 6.81% (±0.86%); C ⁄ E,

7.27% (±0.90%). Divergences between sublineages were

as follows: W1 ⁄ W2, 2.95% (±0.52%); C1 ⁄ C2, 2.12%

(±0.45%); E1 ⁄ E2, 5.43% (±0.84%).

Population genetic diversity

Overall haplotype (h) and nucleotide diversity (p) of

N. yunnanensis were 0.9897 ± 0.0006 and 0.056854 ±

0.027182, respectively. Variation in h among populations

was considerable, ranging from 0 to 0.9487 ± 0.0423



(Table 1). Similarly, p ranged from 0 to

0.045260 ± 0.022109. These values were higher in Luquan

(population 56), Wuding (population 70), Chengjiang

(population 69), Lufeng (population 32) and Baoshan

(population 6) (Table 1). Luquan had the highest p val-

ues, a reflection of the sympatric occurrence of haplo-

types from four sublineages: E1, E2, C1 and C2 (Fig. 1).

Lufeng and Wuding, which harboured particularly

higher p values, contained haplotypes from sublineages

E1 and C2 (Fig. 1). Chengjiang was the only site having

haplotypes from sublineages E1 and E2 (Fig. 1). Baoshan

contained haplotypes from sublineages W1 and W2.

Population genetic structure

Examination of the geographical structure with AMOVA

involved five grouping options (Table 2). First, all indi-

viduals were grouped based on lineages E, W and C.

Second, all sampled populations were grouped accord-

ing to three current drainage basins and without regard

to which lineages occurred in each zone: the eastward

flowing Jinsha River basin; southward drainage basins

including the Irrawaddy, Salween, Mekong and Red

rivers; and the eastward flowing Pearl River drainage

spanning most of the range of N. yunnanensis in the

eastern Yunnan Plateau. In the third through fifth

groupings, each of the three major lineages was consid-

ered independently. Within lineage W, populations

were subdivided into three geographical regions recov-

ered by the phylogeny, and lineages C and E had two

geographical divisions each. Thus, some groupings con-

sidered population structure within the context of their

current distributions.

AMOVA showed significant genetic structure at all hier-

archical levels examined (P << 0.01; Table 2). Of the

first two grouping options, the mtDNA lineages

explained greater amount of variation (FCT = 0.77411).

In contrast, partitioning the genetic variation by con-

temporary drainage basins explained much less varia-

tion (FCT = 0.37521). Analysis confined to each of the

three lineages while grouping them according to geog-

Table 2 Summary of results of the hierarchical analysis of molecul

five grouping options. All P << 0.01

Grouping option

% Among

groups

% Among

populations

within groups

Three lineages 77.41 16.66

Three drainage basins 37.52 51.42

Within west lineage 62.15 22.37

Within east lineage 84.03 7.91

Within central lineage 52.96 26.57


raphy detected virtually identical patterns of genetic

structure. The greatest portions of the overall variation

(62.15%, 84.03% and 52.96%) were accounted for by

among-subgroup diversity, and the within-population

component (15.48%, 8.06% and 20.47%) also contrib-

uted to overall variation.

Pairwise FST estimates among populations showed

the widest possible variation, ranging from 0.00 to 1.00

(Table S2). Three geographical zones and six subdivi-

sions coincided to a great extent with three lineages

and six sublineages, respectively. The highest and most

significant values of FST occurred among distinct geo-

graphical zones, averaging more than 0.906 (Table 3

and Table S2). An almost identical pattern occurred

among distinct subdivisions within each geographical

zone (Table 3 and Table S2). Lower, though mostly sig-

nificant, FST values were observed within individual

geographical zones and within each subdivision. A

strong, mostly significant pattern of differentiation was

observed in FST estimates in subdivisions within W1

(Table 3) as suggested by the phylogeny (Fig. 2). There-

fore, significant geographical structure occurred in the

mtDNA of N. yunnanensis.

Population historical demography

Historical demographics for lineages W, C and E pro-

vided important insights into the history of N. yunnan-

ensis (Fig. S2; Table 4). The lineages had substantially

different MDAs and genetic diversities. Although all

three lineages had multimodal distributions with steep

curves and a high frequency of diverged haplotypes,

the values of both PRag and PSSD were not significant,

indicating that mismatch distributions did not deviate

from expectations of expansion. The raggedness index

for each lineage was low. Within lineages W and E, FS

values were not significant. However, a significant, neg-

ative FS value ()23.2704, P = 0.0126) and lower nucleo-

tide diversity (p = 0.0162 ± 0.0080) were observed

within lineage C, strongly indicating historical demo-

graphic growth.

ar variance (AMOVA) for Nanorana yunnanensis conducted using

% Within

populations FSC FST FCT

5.93 0.73763 0.94073 0.77411

11.06 0.82293 0.88937 0.37521

15.48 0.59093 0.84517 0.62151

8.06 0.49506 0.91937 0.84031

20.47 0.56484 0.79532 0.52964

Table 3 Summary of average pairwise FST values for Nanorana

yunnanensis within and among geographical zones and subdivi-

sions

FST FST

Within W lineage 0.707 Between W1 and W2 0.829

Within C lineage 0.654 Within W1 0.722

Within E lineage 0.550 Within W1a 0.743

Between W and C 0.928 Within W1b 0.055

Between W and E 0.926 Between W1a and W1b 0.789

Between C and E 0.906 Within W2 0.624

Between C1 and C2 0.687 Between E1 and E2 0.771

Within C1 0.239 Within E1 0.280

Within C2 0.659 Within E2 0.481


Population expansion was indicated within sublineag-

es W2, C2, C1 and E2 (Fig. S2; Table 4). Smoother MDA

curves fit well with the expected unimodal model of

sudden expansion and, statistically, population growth

was strongly supported by both SSD and raggedness

index. Significant population growth was also inferred

by FS and lower nucleotide diversity. Estimates of

expansion time ranged from approximately 160 000–

620 000 years before present (BP), mostly from 490 000

to 620 000 years BP. In contrast, neither MDA nor neu-

trality tests supported population expansion for sublin-

eages W1 and E1.

Tajima’s D values were not significant. However, they

were negative and P-values were lower in lineages in

which MDA and Fu’s FS test indicated population

expansion.

Divergence time estimates

Relative-rate tests suggested that a molecular clock was

compatible with our data. The estimated TMRCA of the

ingroup was 5.25 Mya (Early Pliocene) with a 95%

HPD of (4.36, 6.20). The TMRCA of lineages W and C

Table 4 Statistics of genetic diversity, neutrality test and results of th

lineages of Nanorana yunnanensis

Lineage ⁄Sublineage Gene diversity

Nucleotide

diversity

Tajima’s D

(P value)

W 0.9746 ± 0.0026 0.0211 ± 0.0103 )0.4922 (0.3774

C 0.9763 ± 0.0020 0.0162 ± 0.0080 )0.6813 (0.2806

E 0.9485 ± 0.0059 0.0194 ± 0.0095 0.3413 (0.7240

W2 0.9666 ± 0.0036 0.0125 ± 0.0063 )0.9921 (0.1544

W1 0.9127 ± 0.0156 0.0190 ± 0.0095 1.2438 (0.9176

C2 0.9722 ± 0.0026 0.0117 ± 0.0059 )1.0867 (0.1272

C1 0.8498 ± 0.0269 0.0024 ± 0.0015 )1.2634 (0.0786

E2 0.9318 ± 0.0080 0.0069 ± 0.0036 )1.0923 (0.1240

E1 0.8702 ± 0.0283 0.0046 ± 0.0026 0.6144 (0.7700

was 4.78 Mya (Early Pliocene) with a 95% HPD of

(3.95, 5.66). Comparisons of TMRCA estimates of sub-

lineages W1 ⁄ W2 (TMRCA 2.56 Mya, 95% HPD: 2.01–

3.11), C1 ⁄ C2 (TMRCA 1.62 Mya, 95% HPD: 1.21–2.06)

and E1 ⁄ E2 (TMRCA 3.26 Mya, 95% HPD: 2.48–4.09)

indicated Late Pliocene and Early Pleistocene diver-

gences.

Discussion

The mtDNA

The tandem duplication of tRNAMet was the third dis-

covery in an amphibian mitochondrial genome, being

previously reported in Fejervarya cancrivora (Ren et al.

2009) and Fejervarya limnocharis (Liu et al. 2005). The

common ancestor of the sister genera Fejervarya and

Nanorana (Chen et al. 2005; Che et al. 2009) may have

had the tandem duplication. Both N. yunnanensis and

N. quadranus have the apomorphic, short sequence. The

reduced sequence can serve to unite N. yunnanensis and

N. quadranus as sister taxa. This is an alternative set of

relationships for N. yunnanensis, N. quadranus and

N. unculuanus from those resolved by Che et al. (2009),

where N. unculuanus and N. yunnanensis were weakly

resolved as being sister taxa.

Population genetic diversity

Compared with other amphibians (e.g. 0.00967

± 0.000001, Crinia georgiana, Edwards et al. 2007), higher

levels of diversity (0.056854 ± 0.027182) were found

within N. yunnanensis, which might be attributed to a

long evolutionary history, a broad distribution and a

geologically complex history. A long evolutionary his-

tory may account for high level of diversity in ND1–

ND2. Their most recent common ancestor appeared

about 5.25 Mya. The long evolutionary history allowed

e mismatch distribution analysis for three lineages and six sub-

FS (P-value) SSD (PSSD)

Raggedness index

(PRag)

) )10.8666 (0.1096) 0.0135 (0.1962) 0.0036 (0.1748)

) )23.2704 (0.0126) 0.0061 (0.3142) 0.0042 (0.2416)

) )0.4315 (0.5400) 0.0154 (0.3202) 0.0054 (0.9336)

) )15.6260 (0.0208) 0.0065 (0.1278) 0.0051 (0.5276)

) 5.4116 (0.9312) 0.0447 (0.0580) 0.0353 (0.0140)

) )23.4382 (0.0048) 0.0029 (0.3646) 0.0055 (0.2152)

) )8.1049 (0.0070) 0.0043 (0.5816) 0.0245 (0.7496)

) )10.6819 (0.0294) 0.0027 (0.9350) 0.0057 (0.9540)

) 0.1129 (0.5608) 0.0776 (0.0248) 0.1207 (0.0240)



the species to accumulate mutations. Notwithstanding,

N. yunnanensis is broadly distributed and geologically

this region experienced a complex evolution. The most

significant geological changes in the Yunnan Plateau

occurred from the Pliocene to the Early Pleistocene

(Cheng et al. 2001), when a third uplifting on Qinghai-

Tibet Plateau occurred more strongly and frequently,

starting about 3.4 Mya (Yu et al. 2000). The drainage

system experienced a complicated evolution involving

erosion and river capture (e.g. Cheng et al. 2001; Clark

et al. 2004) that must have lead to repeated isolation

and fragmentation events in N. yunnanensis. The pro-

cess of diversification is somewhat similar to that

hypothesized for plants on the QTP and its adjacent

areas, and for freshwater fishes outside of Yunnan

province. The great species diversity and higher popu-

lation differentiation in the areas likely originated par-

tially through repeated shifts of species’ ranges and

their genetic isolation in diverse habitats (Wu 1980;

Axelrod et al. 1996).

Genetic structure and drainage history

Studies indicate strong associations between the evolu-

tion of palaeodrainage systems and current genetic pat-

terns in drainage-associated organisms (Perdices et al.

2005; Kozak et al. 2006; Zemlak et al. 2008). Geomor-

phological evidence suggests that paleodrainage and

contemporary hydrological systems differed signifi-

cantly in Yunnan province (Clark et al. 2004) (Fig. S1).

The distribution of N. yunnanensis is largely confined to

drainage systems. Consequently, its genetic structure

might correspond to the region’s paleogeography. To

evaluate this possibility, we formed an H0 stating that

contemporary gene flow was unrestricted within con-

temporary lineages. This H0 would be rejected by find-

ing that overall variation was attributable to historical

among-lineage associations. The AMOVA analysis

(Table 2) showed that the greatest amount of overall

genetic variation occurred when group partitioning was

based on genealogy alone. When grouping was based

on the current drainage system, the level of differentia-

tion among populations within a single drainage was

almost twice as large as among different drainages

(51.42% and 37.52%, respectively; Table 2). Thus, we

could not reject the H0. This pattern indicated that con-

temporary drainage basins are composites of histori-

cally isolated drainage systems.

Lineages W and C clustered together with strong sup-

port (Fig. 2), which supported the hypothesis that the

modern upper and middle Yangtze River drainage and

most of drainages distributed in Hengduan Mountains

Region were originally major tributaries to the paleo-

Red River system (Clark et al. 2004). The estimated


divergence times between lineage W and C

(�4.78 Mya) approximately corresponded to an early

Pliocene transition from being exterior to interior in the

drainage system in central Yunnan (Cheng et al. 2001)

and the differential uplift of the Ailao Shan-Diancang

Shan Range (Wang et al. 2006).

Within the middle Yangtze River basin, a distinctive

mtDNA discordance was observed between haplotypes

from some tributaries (including populations 57–59, 61,

63, 65–67 and 70, belonging to lineage E; Figs 1 and 2)

and those from others (populations 33–56, lineage C).

This discovery supported the hypothesis that the mod-

ern middle Yangtze River drainage was formed by con-

nection of historically independent palaeodrainage

basins. A freshwater fish phylogeographic study is con-

cordant with this hypothesis (Yang et al. 2009). Most

tributaries of the middle Yangtze River historically flo-

wed southward into the South China Sea through the

Paleo-Red River before their capture and reversal (Clark

et al. 2004). The estimated diversification times among

the lineages of N. yunnanensis (�5.25 Mya) approxi-

mately corresponded to an Early Pliocene watershed

formation of the central Yunnan Plateau (Cheng et al.

2001). This was a deformation response to the extrusion

and uplift of the Central Yunnan block in the early Plio-

cene (Zhang et al. 2003), and it divided the ancient Red,

Pearl and Jinsha Rivers (Wang & Wang 2005).

The relatively low levels of genetic divergence (1.0%)

between lineages C2a and C2b demonstrate the impor-

tant roles that historical drainage patterns play in shap-

ing the modern genetic structure of N. yunnanensis.

Lineage C2a (mostly from populations 26–31), composed

of haplotypes from tributaries of Red River basin, lar-

gely corresponds to historical drainages (Clark et al.

2004). Lineage C2b (mostly from populations 32–56) pri-

marily includes haplotypes from tributaries of the Jin-

sha River basin. These two lineages are associated with

different drainage basins yet they are sister groups with

shallower divergence than those among C2a and C1

(2.6%), C2b and C1 (2.2%); here the BPP is moderate

(0.90; Fig. 2). This pattern supports the hypothesis that

the tributaries of the Jinsha River basin historically flo-

wed southward and connected with the paleo-Red

River basin (Clark et al. 2004).

Cladogenesis within lineage W is also congruent with

the history of drainage patterns. Lineage W1b contains

haplotypes from tributaries of the Irrawaddy River

basin, W1a from those of Salween River basin and W2

from both the Mekong River and Red river basins

(Fig. 1). These drainage basins in western Yunnan have

experienced little change since the early Pliocene

(Cheng et al. 2001) with the exception of their upper

portions in southeastern Tibet (Clark et al. 2004). The

AMOVA analysis supports their antiquity. The greatest


amount of genetic variation occurs among groups

(62.15%, Table 2) when the data are partitioned as con-

temporary drainage basins.

Two geographical subdivisions of lineage E explain

the greatest amount of variation (84.03%, Table 2). Sub-

lineage E2 is distributed across the eastern Yunnan Pla-

teau, and E1 is on the central Yunnan Plateau at

Luquan, Wuding, Lufeng and Yuxi (Figs 1 and 2).

These lineages have an average divergence of 5.43%.

Sublineage E1 is strongly correlated with the Late Plio-

cene–Early Pleistocene catchment basin and its many

lakes, both large and small (Wang et al. 1995).

Sublineages C1 and C2 likely originated through allo-

patric fragmentation of ancient populations during the

early Pleistocene when the southward flowing

Dadu ⁄ Anning River was captured. One part became the

eastward flowing lower Dadu River (Clark et al. 2004).

New, steep gradients in the uppermost Anning River

were formed with the uplift of adjacent Gonga Shan

(Clark et al. 2004).

In some cases, the genealogical history of N. yunnan-

ensis substantially corresponds with historical, rather

than contemporary drainage patterns. Thus, acceptance

of our H0 varies among the clades. Recent biogeograph-

ic studies of Southeast Asian fishes also demonstrate

that the evolution of drainage basins played a key role

in shaping current geographic patterns of genetic and

species diversity (e.g. Ruber et al. 2004).

Many of the vicariance patterns recovered herein are

novel. Vicariance theory assumes that common distribu-

tional patterns originated via shared vicariance events.

Thus, further study involving a diversity of species

endemic to adjacent areas of the QTP is required to test

the ubiquity of our vicariance hypotheses.

Restricted gene flow among populations of N. yun-

nanensis is strongly implied (Table S2). Long-term

genetic isolation among lineages is corroborated by FST

values >0.94 (Table 2). Low dispersal ability and this

species preference for sitting on moss-covered rocks

near cold montane streams at higher elevations may be

responsible for the restricted gene flow. This type of

habitat is never continuous.

Population expansion and secondary contact duringthe Pleistocene

Sympatric maternal lineages occur in a few populations.

This pattern could result from either incomplete lineage

sorting, or secondary contact between previously allo-

patric lineages (Barber 1999). Lineages W and C co-

occur in Binchuan (populations 17, 37; Fig. 1). The

higher nucleotide diversity (0.031086 ± 0.015631), geog-

raphy and the absence of intermediate divergent haplo-

types strongly indicate secondary contact. Secondary

contact is also indicated in Luquan, Lufeng and Wud-

ing, where lineages C and E co-occur.

If secondary contact occurred, then population expan-

sion is expected. Thus, the H0 states that populations

are stable in their occurrence, and the H1 is one of the

range expansions. MDA and some neutrality tests

(Fig. S2; Table 4) reject the H0 and suggest population

expansion in sublineages W2, C1, C2 and E2. Secondary

contact among lineages is also strongly supported by

the higher average nucleotide diversity within these

populations (Table 1). Secondary admixture of previ-

ously allopatric populations likely produced substantial

levels of sympatric genetic diversity (e.g. Fu et al. 2005).

Presumably, gene flow is unabated in these areas, but

this remains to be documented. Secondary contact

between lineages occurs in areas adjacent to major

drainage divides (Fig. 1), and this strongly indicates

that drainage basins profoundly influenced contempo-

rary genetic structure. For example, sympatry occurs

between lineages C and E along the central Yunnan Pla-

teau, which is divided by the Jinsha, Red and Pear riv-

ers (Wang & Wang 2005).

Although MDA analyses suggest demographic expan-

sion, neutrality tests reveal no demographic growth,

except in lineage C (Table 4). This incongruence could

be attributed to the occurrence of strong subdivisions

within each lineage (Rogers & Harpending 1992). Line-

age C has two subgroups, but the extent of differentia-

tion between them is far lower than that within

lineages W and E (Table 3). MDA is robust and hardly

affected by population structure (Harpending 1994) and

should approximately hold true even when populations

are completely isolated (Rogers 1995), yet population

subdivision lowers the power of neutrality tests (Ray

et al. 2003).

The estimated expansion time suggests extensive

demographic growth from the onset of the Middle

Pleistocene (about 0.6 Mya) triggered by climatic oscil-

lations. Indeed, 0.8 Mya marks an important climate

transition period (Jiang et al. 2002; Ming 2007). Higher

levels of haplotype diversity and lower levels of nucle-

otide diversity occur in sublineages undergoing histor-

ical demographic expansion as opposed to sublineages

exhibiting demographic stability (Table 4). Populations

likely rapidly colonized or recolonized some areas

during climatic oscillations. This latter possibility

would reduce allelic diversity within lineages (Hewitt

2000).

Implications for conservation

The total genetic diversity is a key to evaluating the

persistence and conservation of a species (Rauch & Bar-

Yam 2005). The maintenance of genetic diversity is



required for long-term survival of species (Frankel &

Soule 1981) and for the long-term persistence of ecosys-

tem functions (Frankham et al. 2002; Frankham 2005).

The loss of variation may largely limit the adaptability

of populations to changing environments (Lacy 1997).

Two morphologically very similar species, Nanorana

liui (=Rana muta, Su & Li 1986) and Nanorana (Paa) bour-

reti (Dubois 1987) (as Rana bourreti), have been

described from this region. Che et al. (2009) discovered

that N. yunnanensis (then Paa yunnanensis) was not

monophyletic with respect to N. liui and N. bourreti.

They considered the latter two species to be junior syn-

onyms of N. yunnanensis. However, Huang et al. (2009)

reported that the population from Jingdong was a new

record of N. bourreti rather than N. yunnanensis, based

on both molecular and morphological evidence. Huang

et al. (2009) provide evidence that N. yunnanensis may

be a species complex, and this is important because bad

taxonomy kills (Daugherty et al. 1990; May 1990). Our

study reveals pronounced mtDNA intraspecific genetic

divergence. The mean among-lineage divergence

(7.09%) is higher than intraspecific divergences reported

for many other species of anurans (e.g. 1.42%, Edwards

et al. 2007; 1.5%, Kozak et al. 2006; 3%, Hoffman &

Blouin 2004), suggesting that N. yunnanensis may be a

species complex. Widespread, high-density species are

less likely to succumb to extinction than small isolated

species (Macarthur & Wilson 1963, 1967). Cryptic species

can be lost or further endangered through bad taxon-

omy, and at the expense of conserving more widespread

taxa. If the recognition of N. bourreti (lineage W) is cor-

rect, then our genealogy implies that at least three (W, C

and E) and as many as five species (W1, W2, C, E1 and

E2) occur in the complex, each having a far-more-

restricted geographic distribution. However, the sympat-

ric occurrences of several lineages also suggest that rec-

ognition of N. bourreti might not be justified. Nuclear

gene data and ⁄ or morphometric examination is required

to test the possibility of a species complex. Such would

imply that the occurrence of sympatric lineages owes to

the introgressive capture of mitochondrial genomes via

interspecific hybridization.

Nanorana yunnanensis is listed as a vulnerable species

by Zhao (1998). Over past few decades, the species has

suffered rapid population declines mainly owing to

over-harvesting for human consumption, but also

because of habitat destruction associated with agricul-

tural expansion, deforestation, community development

and water pollution. Conservation requires a reduction

in human disturbance, possibly through establishing

several protected areas encompassing all of the lin-

eages.

Two major Chinese government policies promote con-

servation in southwestern China. Nowadays, logging is


completely banned within most of China’s biodiversity

hotspots, a response to the catastrophic floods of 1998.

In addition, the Land Conversion Program (= the Grain

to Green Policy) facilitates conservation by banning

agriculture on steep slopes, prohibiting the clearing of

forests for shifting agriculture, and enacting laws for

protecting species. Farmers can receive subsidies for

replanting barren slopes, albeit initially with cultures of

pine and fruit trees. Diverse, native vegetation would

promote biodiversity throughout the region (Biodiver-

sity-hotspots 2005).

Acknowledgements

Jiatang Li, Jiawei Wu, Chunling Zhu, Wanhe Zhang, Jianping

Jiang and Amy Lathrop assisted in the field or kindly provided

samples. Tiao Ning, Weiwei Zhou, Liang Xie and Dingqi Rao

provided valuable comments on early drafts of the manuscript.

Lianming Gao assisted in locating botanical literature. Techni-

cal support from the laboratory of Ya-ping Zhang is gratefully

acknowledged. Kai He helped with rate tests. The Protected

Areas in Yunnan and Sichuan graciously provided collecting

permits. This work was supported by grants from the National

Basic Research Program of China (973 Program,

2007CB411600), the National Natural Science Foundation of

China, the Natural Science Foundation of Yunnan Province,

Bureau of Science and Technology of Yunnan Province, and

the State Key Laboratory of Genetic Resources and Evolution.

Manuscript preparation was supported by the Natural Sciences

and Engineering Research Council of Canada Discovery Grant

A3148.

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graphy and conservation genetics of Asian amphibians and

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Supporting information

Additional supporting information may be found in the online

version of this article.

Table S1 Voucher specimen numbers for each population.

Acronyms are YU for College of Life Sciences, Yunnan Univer-

sity; SKLGRE for State Key Laboratory of Genetic Resources

and Evolution, Kunming Institute of Zoology, Chinese Acad-

emy of Sciences; SCUM for Sichuan University Museum.

Table S2 Pairwise FST values among the 71 populations of the

Nanorana yunnanensis. Populations are numbered as in Table 1.

Significant level set to P < 0.05 after 10 000 permutations. FST

values are below diagonal, and P values are above diagonal,

where ‘+’ indicates significance and ‘)’ P > 0.05.

Fig. S1 Reconstruction of drainage basin history owing to river

capture ⁄ reversals of major rivers in eastern Tibet (from Clark

et al. 2004). Colours represent individual drainage basins drawn

on top of grayscale topography. (a) Interpreted pattern prior to

the major captures, where the upper Yangtze, middle Yangtze,

upper Mekong, upper Salween and the Tsangpo rivers drained

together to the South China Sea through the paleo-Red River

(blue). (b) Capture ⁄ reversal of the middle Yangtze River redi-

rects drainage away from Red River and into the East China

Sea through the lower Yangtze River (green). (c) Capture of the

upper Yangtze River to the east into the lower Yangtze River,

and the upper Mekong and upper Salween rivers obtain their

modern drainage positions (green, yellow and orange, respec-

tively). Capture of the Tsangpo River to the south through the

Irrawaddy River (red). (d) Capture of the Tsangpo River

through the Brahmaputra River into its modern course (pink).

This final configuration is the modern drainage basin pattern.

Fig. S2 Mismatch distributions for each major haplotype line-

age and some sublineages of Nanorana yunnanensis. The

abscissa shows the number of pairwise differences between

compared haplotypes. The ordinate shows the frequency for

each value. The histograms represent the observed frequencies

of pairwise divergences among haplotypes and the line refers

to the expectation under the model of population expansion.

(a–i): Mismatch distributions for the lineages and sublineages

W, W1,W2, C, C1, C2, E, E1 and E2, respectively.

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directed to the corresponding author for the article.


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