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Phylogeography of the Fanged Dicroglossine Frog, Limnonectes fujianensis(Anura, Ranidae), in TaiwanAuthor(s): Nian-Hong Jang-Liaw and Wen-Hao ChouSource: Zoological Science, 28(4):254-263. 2011.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.28.254URL: http://www.bioone.org/doi/full/10.2108/zsj.28.254

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2011 Zoological Society of JapanZOOLOGICAL SCIENCE 28: 254–263 (2011)

Phylogeography of the Fanged Dicroglossine Frog, Limnonectes fujianensis (Anura, Ranidae), in Taiwan

Nian-Hong Jang-Liaw1 and Wen-Hao Chou1,2,3*

1Department of Zoology, National Museum of Natural Science, 1st Kuang-Chien Rd.,

Taichung 404, Taiwan2Department of Life Sciences, National Chung Hsing University, 250 Kuo-Kuang Rd.,

Taichung 402, Taiwan3Graduate School of Museum Studies, Taipei National University of the Arts,

1st Hsueh-Yuan Rd., Peitou, Taipei 112, Taiwan

A phylogenetic analysis of Taiwanese fanged dicroglossine frog, Limnonectes fujianensis (Anura,

Ranidae), was conducted to examine its genetic diversification using sequence data from a portion

of the mitochondrial DNA (mtDNA) cytochrome b sequences. We collected genetic data from 200

individuals at 23 localities in Taiwan and three localities in China. A neighbor-joining tree of 39 hap-

lotypes revealed two clades in Taiwan and a clade in China, each showing restricted geographical

distribution. The pattern of geographical divergence suggests a single invasion into Taiwan. Diver-

gence times between clades were inferred using molecular clock tests. The population relationship

of L. fujianensis between Taiwan and mainland China, and the phylogenetic relationships with its

congeners, e.g., L. bannaensis, L. fragilis and L. kuhlii, were obtained and discussed.

Key words: Limnonectes fujianensis, Taiwan, phylogeography, mtDNA, cytochrome b

INTRODUCTION

Phylogeographical studies that analyze lineages with

disjunctive distribution have recently attracted attention,

largely because they provided data critical to our under-

standing of evolutionary processes associated with lineage

divergence and geohistorical changes. Due to limited disper-

sal abilities across topological barriers and their interactions

with biotic or abiotic conditions of their microhabitats, we

consider amphibians to be ideal materials for such research.

In general, amphibians’ unique characters, such as perme-

able skins and aquatic larval stage, crucially enable these

animals to respond to microhabitat changes. They, there-

fore, are sensitive to the driving forces of adaptation, migra-

tion, and even extinction. Their susceptibility to conditions

such as vegetation, light, temperature, and precipitation in

micro-environments makes them good indicators of environ-

mental changes (Lips, 1998; Pounds et al., 1999).

The fanged dicroglossine frogs of genus Limnonectes

comprise a group of primarily forest-dwelling frogs, mainly

distributed in South Asia, Southeast Asia, the Philippines,

and Wallacea (Inger, 1999). Fanged frogs are morphologi-

cally distinctive from other anurans in that sexual size dimor-

phism is strongly male-biased, and the males have greatly

enlarged odontoid processes (Inger, 1966; Emerson and

Berrigan, 1993; Tsuji, 2004). The Chinese Fujian large-

headed fanged L. fujianensis, previously known as Rana

(Limnonectes) kuhlii (Boulenger, 1920; Pope, 1931; Pope

and Boring, 1940; Liu and Hu, 1961; Lue and Chen, 1986),

was only regarded as distinct by Ye and Fei (1994), and

occurs in Fujian, Hunan, Jangxi, and Zhejiang. Recently,

Zhang et al. (2005) further recognized additional distribution

range of L. fujianensis to include Anhui of China and Tai-

wan, based on partial sequences of the mitochondrial 12S

rRNA and 16S rRNA genes. Zhang et al. (2005) also sug-

gested that both fanged frogs from Hainan and Yunnan

were taxonomically distinct, i.e., L. fragilis and L. kuhlii,

respectively. In addition, other fanged frog occur in China,

e.g., L. bannaensis from western-southern China, mainly in

Yunnan Province (Ye et al., 2007). Matsui et al. (2010),

however, recognized three species, L. fujianensis, L.

bannaensis, L. fragilis, present in China, Taiwan and

Hainan, and stressed that L. “kuhlii” from northern Laos and

central Vietnam and L. bannaensis are conspecific. The

Chinese and Taiwanese fanged frogs are all morphologically

similar, suggesting that their evolutionary picture may have

been blurred. Molecular biogeographic information will be

helpful to reveal their natural history with respect to geogra-

phy, geology, and paleoclimatology.

In Taiwan, this frog prefers shallow, slowly flowing

waters of streams and ditches with overgrowing grass or

fallen leaves in mountain regions, and can be found

throughout most of the year (Chou and Lin, 1997; Lue et al.,

1999). Though widely distributed across a vast area of five

provinces in China, our intensive investigation suggests that

L. fujianensis in Taiwan is limited in western and northern

part of this island. A specimen collected from Lai-yi, Pintung

(NTU-A0012-1690; deposited at National Taiwan University)

suggests its distribution might have covered the southwest-

ern area (Fig. 1); unfortunately, we could not find any spec-

* Corresponding author. Phone: +886-4-23226940;

Fax : +886-4-23232146;

E-mail: whchou@mail.nmns.edu.tw

doi:10.2108/zsj.28.254

Phylogenetic of Taiwan Limnonectes fujianensis 255

imen for DNA analysis during our collecting period in this

study. This sexually dimorphic anuran has advertisement

calls and nuptial pads, and the large male exerts mating

advantage through the size-dependent spatial movement

(Tsuji, 2004; Yang, 2006). Some congeners from Borneo

exhibit parental care by carrying tadpoles on males’ backs

(Inger and Voris, 1988), but the Taiwanese L. fujianensis

lacks such parental care behaviors (Tsuji, 2004). The long

breeding season and high fidelity of habitats disclosed the

possibility of limited dispersal ability in this species.

Taiwan is a subtropical to tropical island located off the

coast of southeastern China with high biodiversity (Shao,

2006; Kier et al., 2009). Uplift of the mountains caused by

collision of the Eurasian and Philippine plates resulted in this

island’s steep topography and craggy mountain ranges

about 2.5–1.0 Ma (Lin, 1966; Huang et al., 1997). The

ancient fauna and flora of Taiwan are thought to originate in

continental China via landbridges across the Taiwan Strait

(currently 200 km wide and 50 m deep on average) initially

during the Pliocene (Yu and Lu, 1995) and potentially at

least twice during the Pleistocence (Emery et al., 1971;

Zhao, 1982; Yang, 1991). The Central Mountain Range of

Taiwan approaches an elevation of 4000 m rising steeply

from the eastern coast and giving way to a broad western

plain. The large-scale Central Mountain Range and the

Taiwan Strait may form vicariant forces causing allopatric

barriers to gene flow. Within such a large island of

36,000 km2 in size, the flora and fauna of Taiwan are

reputed to show significant divergence, owing to its complex

topography, microclimates, and ecological complexity. The

high biodiversity makes Taiwan a suitable locale for phylo-

geographic research. Several phylogenetic and phylogeo-

graphic investigations using molecular approaches have

been conducted on small vertebrates, in particular freshwa-

ter fishes (Wang et al., 1999; Wang et al., 2000; Wang et

al., 2004; Cheng et al., 2005; Ma et al., 2006; Watanabe et

al., 2007) and amphibians (Yang et al., 1994; Jang-Liaw et

al., 2008; Jang-Liaw and Lee, 2009). We thus applied

molecular markers to elucidate the evolutionary relation-

ships within Taiwanese L. fujianensis, particularly focused

on (1) describing the population genetic diversity and phy-

logeny, (2) discussing relationships between phylogenetic

structures and geographic characters, (3) demonstrating the

possible migration history into/within Taiwan, and (4) com-

paring phylogeographic characters with other vertebrates in

Taiwan. In addition, the phylogenetic relationships to its rel-

atives, including L. bannaensis, L. fragilis from China and L.

kuhlii from Java, are discussed.

MATERIALS AND METHODS

Sample collection

A total of 200 specimens of adult Limnonectes fujianensis were

analyzed in this study. One hundred

and ninety-three individuals were from

23 collection sites in Taiwan, others

were collected from China, including

five individuals from two sites in Fujian

Provinces, and two individuals from

Guangdung Provinces (Table 1). Col-

lecting sites are shown in Fig. 1. Mus-

cular tissue samples were preserved in

99.5% ethanol for laboratory analyses.

Most L. fujianensis specimens used in

this study have been deposited in the

National Museum of Natural Science

(NMNS), Taichung, Taiwan and in the

National Chung Hsing University

(NCHU), Taichung, Taiwan (Table 1).

We also analyzed three related fanged

frogs, of which the voucher numbers

are listed in Table 1. A sequence of

Rana zhenhaiensis (GenBank acces-

sion number FJ349554) was applied as

an outgroup in this study.

DNA amplification and sequencing

Genomic DNA was isolated from a

piece of muscle tissue (about 5 mg)

using the Tissue and Cell Genomic

DNA Purification Kit (Hopegen Biotech-

nology Development Enterprises). The

extraction of crude DNA was performed

according to the manufacturer’s instruc-

tions with repeat membrane binding,

salt washing, and centrifugation. A 868-

bp region of the cytochrome b gene

was selected for amplification with the

polymerase chain reaction (PCR) using

primers L14850 (5′-TCTCATCCTGAT-

GAAACTTTGGCTC-3′) and Ptacek 2H

Fig. 1. Locations of sampling sites for Limnonectes fujianensis, L. bannaensis, and L. fragilis in

this study. Site numbers and locality codes are listed in Table 1. In the map of Taiwan (right),

dashed line represents postulated boundary of two biogeographic districts based on the results

of phylogenetic analyses: midwestern-to-northeastern district (clade I) and midwestern-to-

southern district (clade II). Moreover, the dotted line indicates the boundary of subclades within

clade I. The solid arrow points out a possible geographic isolating feature, the Chousui River,

bordering the clades I and II. Solid star ( ) indicates a population site inferred from a specimen

record (NTU-A0012-1690). In the large scale map (upper left), the gray-shaded zone (mainland

China district; clade IV) denotes the distributional range of L. fujianensis (modified from Fei,

1999). The collection sites of L. bannaensis ( ) and L. fragilis ( ) specimens are indicated, but

the L. kuhlii specimen from Java is not shown here.

N.-H. Jang-Liaw and W.-H. Chou256

(5′-TCTTCTACTGGTTGTCCTCCGATTCA-3′) designed by

Tanaka-Ueno et al. (1998) and Ptacek et al. (1994), respectively.

PCR conditions consisted of 35 cycles of denaturation (95°C, 50 s),

annealing (46°C, 1 min), and extension (72°C, 1 min 20 s) (modified

from Saiki, 1990) on a RoboCycler Gradient 96 temperature cycler

(Stratagene Inc.) with PCR Master Mix Kit (Hopegen Biotechnology

Development Enterprises). PCR products were purified with the

HiYield Gel/PCR DNA Fragments Extraction Kit (RBC Bioscience)

and used for sequencing. Sequences were obtained by the multiple

fluorescent dyes method using an ABI PRISM 3130xl Genetic

Analyzer, and were aligned with the aid of MegAlign ver. 4.0 (DNA

Star Inc.) and by eye, using the complementary strand for verifica-

tion. Sequences of 200 L. fujianensis samples, four L. bannaensis,

four L. fragilis and one L. kuhlii were deposited in the GenBank

database (accession numbers FJ349345–FJ349553).

Data analysis

Preliminary phylogenetic and molecular evolutionary analyses

were conducted using MEGA version 4.0 (Tamura et al., 2007) and

DNA SP version 4.00.2 (Rozas et al., 2003). We constructed phy-

logenetic trees of L. fujianensis and other three fanged frogs using

neighbor-joining (NJ), maximum parsimony (MP), and maximum

likelihood (ML) analyses. All these analyses were performed on

unique haplotypes by PAUP* version 4 beta (Swofford, 2001). Like-

lihood settings from the best-fit model HKY + I + G (gamma shape =

0.8965) with base frequencies of A = 0.2684, C = 0.3415, G =

0.1216, T = 0.2685 and a transition/transversion ratio of 6.3924,

selected by hLRTs (hierarchical likelihood ratio tests) were obtained

from MODELTEST 3.7 (Posada and Crandall, 1998). Replicate hap-

lotypes were excluded from the analysis to reduce computational

time. NJ tree construction was based on the probability model iden-

tified above, with ties broken randomly. MP and ML analyses were

conducted using a random addition heuristic search with tree-

bisection-reconnection (TBR) branch swapping. Bootstrapping

[1,000 replicates for NJ (NJ option), MP (fast-heuristic search), and

ML (full-heuristic search, TBR branch swapping)] was performed to

obtain a relative measure of node support for the resulting tree

(Felsenstein, 1985). The selective neutrality of all sequences was

assessed by Tajima’s D (Tajima, 1989) statistic and Fu and Li’s D

and F statistics (Fu and Li, 1993) within each clade and local pop-

ulation.

A statistical parsimony network was constructed by linking hap-

lotypes in a hierarchical manner based on the variations between

sequences. For the purpose of analyzing the possibilities of genetic

relationships between populations, multifurcation and/or reticulation

network analysis is useful and expected. In this study, we used TCS

Table 1. Sampling localities, sample sizes (Ns), haplotypes and specimen numbers analyzed in this study. Sampling locality

numbers correspond to those in Fig 1. ENS = Eric N. Smith field catalogue; KIZ = Kunming Institute of Zoology, the Chinese

Academy of Sciences, Kunming,Yunnan, China; NCHU = National Chung Hsing University, Taichung, Taiwan; NMNS =

National Museum of Natural Science, Taichung; YNU = Yunnan University, Kunming, Yunnan, China.

Species/Sampling locality Ns Haplotypes (no. of individuals) Specimen

Limnonectes fujianensis 200

1. Yangmingshan (YMS) 10 h01(4), h02(5), h03(1) NMNS17373–17382

2. Sindian (SD) 10 h01(7), h04(2), h05(1) NMNS17092–17101

3. Fuhsing (FH) 10 h06(1), h07(3), h08(6) NMNS16602–16611

4. Wufeng (WE) 10 h06(9), h09(1) NMNS16631–16640

5. Nanjuang (NJ) 4 h07(3), h10(1) NCHU14957, NCHU14965–14966, NCHU14979

6. Taian (TA) 10 h07(1), h11(1), h12(6), h13(2) NMNS17332–17343

7. Sanyi (SY) 10 h12(3), h14(5), h15(2) NMNS16671–16680

8. Dongshih (DS) 7 h16(5), h17(2) NMNS16821–16822, 16915–16919

9. Taiping (TP) 10 h18(9), h19(1) NMNS16762–16771

10. Yuchih (YC) 10 h20(9), h21(1) NMNS16650–16659

11. Lugu (LG) 10 h20(5), h22(5) NMNS17308–17312, 17320–17324

12. Gukeng (GK) 10 h23(8), h24(2) NMNS16698–16704, 16735–16737

13. Fanlu (FL) 10 h24(6), h25(4) NMNS16723–16730, 17034–17035

14. Jhongpu (JP) 10 h24(5), h26(5) NMNS16749–16758

15. Sanmin (SM) 10 h24(7), h27(3) NMNS16713–16722

16. Nan-oa (NA) 6 h28(6) NMNS16792, 17430–17434

17. Datong (DT) 5 h01(5) NCHU608–610, NCHU623–624

18. Chilan (CL) 3 h01(3) NMNS16591, 17441–17442

19. Yuanshan (YS) 10 h01(8), h29(2) NMNS17105–17112, 17436–17437

20. Toucheng (TC) 10 h01(9), h30(1) NMNS17405–17414

21. Shuangxi (SX) 10 h01(10) NMNS17385–17394

22. Nankang (NK) 5 h01(3), h31(1), h32(1) NMNS17113–17114, 17369–17371

23. Keelung (KL) 3 h01(3) NMNS15942, 17041, 17384

24. Changting (FJ1) 3 h33(1), h34(1), h35(1) NMNS17039–17040

25. Wuyishan (FJ2) 2 h36(1), h37(1) YNU-HU20026040, 20026017

26. Nankunshan (GD) 2 h38(1), h39(1) NMNS17007–17008

L. bannaensis 4

27. Monla (YN1) 2 b01(2) KIZ-YN0705144–0705145

28. Longchuan (YN2) 2 b02(1), b03(1) KIZ-YN070552–070553

L. fragilis 4

29. Yinggeling (HN) 4 f01(3), f02(1) KIZ-HN0806055–0806058

L. kuhlii 1

30. Java 1 k01(1) ENS7395

Phylogenetic of Taiwan Limnonectes fujianensis 257

1.21 (Clement et al., 2000) to reconstruct the parsimonious network

for all haplotypes of L. fujianensis populations. We set the connec-

tion limit in the TCS program to 25 steps. No specific setting on

gaps was involved as no gaps occurred in our data set.

For estimating divergence times between clades, we con-

ducted the two-cluster test of constancy of evolutionary rates based

on the K2P-distance model for all haplotypes using LINTREE

(Takezaki et al., 1995). The test employed the NJ method to estab-

lish tree topology, and Rana zhenhaiensis (GenBank accession

number FJ349554) was used as an outgroup. We calculated the

height of the branch point of two clades, defined as one-half the

average of the mean nucleotide differences between the two

clades. Divergence time between clades was estimated by the ratio

of the height to the divergence rate. In this study, we applied 1.41%

per lineage per million years as the divergence rate followed Jang-

Liaw et al. (2008) inferred from the molecular evident on population

diversity of Taiwanese Sylvirana latouchii.

RESULTS

Phylogenetic analysis

We amplified cytochrome b partial genes sequence from

193 Taiwanese Limnonectes fujianensis specimens and

seven individuals from China for reconstructing the popula-

tion genetic structure of this fanged frog within Taiwan.

Three relatives of L. fujianensis, including four L. bannaensis

specimens from Yunnan Province (China), four L. fragilis

from Hainan Is. (China) and one L. kuhlii from Java

(Indonesia) were also sequenced. The examined sequences

are 868 bp in length. No insertions, deletions or stop codons

were found in these sequences. In L. fujianensis, 39 haplo-

types were detected from the specimens. Totally 78 poly-

morphic sites were identified among them, and all these

sites were parsimony informative, i.e. valuable mutation

sites for phylogeny analyses.

We detected a total of 45 haplotypes from all Limnonectes

specimens studied. The sequence of Rana zhenhaiensis

was treated as outgroup. The neighbor-joining haplotype

tree with NJ, MP and ML bootstrap values (Fig. 2) topolog-

ically showed three major clades among all L. fujianensis

samples: 1) clade I, defined by 26 haplotypes from 148

specimens representing the midwestern-to-northeastern

populations in Taiwan; 2) clade II, defined by six haplotypes

from 45 specimens representing the midwestern-to-southern

populations in Taiwan; and 3) clade III, defined by seven

specimens from mainland China. Two subclades (Ia and Ib)

Fig. 2. Neighbor-joining tree of 868-bp-long haplotypes inferred

from partial cytochrome b sequences. Branch lengths are propor-tional to the scale given in nucleotide substitutions per site. Num-bers at internal nodes are bootstrap probabilities (> 50%) for the values of major clades only in order for NJ/ MP/ ML based on 1000 replications for each tree.

Table 2. Distances inferred from haplotypes of clades, populations of Limnonectes fujianensis, and all species in this study. Above diagonals indicate numbers of differences, and below diagonals indicate estimated pairwise distant values by K2P model. The column to the right of the matrix shows within-lineage distance values inferred from the haplotypes with standard errors inferred from 1000 replicates bootstraps. In parentheses: all = all tested L. fujianensis samples in this study; I, II, III, Ia and Ib refer to clades I, II, III, and subclades Ia, Ib, respectively; TW = Taiwan; FJ = Fujian; GD = Guangdung; YN = Yunnan (1 and 2 correspond to localities YN1 and YN2, respectively in Fig. 1).

1 1a 1b 1c 1d 1e 1f 1g 1h 2 2a 2b 3 4 d × 102 within clade

1. L. fujianensis (all) – – – – – – – – 167.56 154.23 174.22 180.97 196.82 1.7520 ± 0.2536

1a. L. fujianensis (Ia) – 6.6 – 15.91 – 30.74 29.33 30.34 166.56 155.33 173.17 181.78 194.83 0.3575 ± 0.0883

1b. L. fujianensis (Ib) – 0.0077 – 16.21 – 30.82 29.38 30.41 167.54 156.38 173.13 179.50 196.50 0.2020 ± 0.0725

1c. L. fujianensis (I) – – – 16.00 – 30.77 29.35 30.36 166.86 154.27 173.15 181.80 195.35 0.5247 ± 0.1201

1d. L. fujianensis (II) – 0.0186 0.0190 0.0188 – 28.47 28.17 28.38 168.44 154.67 175.33 184.00 199.50 0.6661 ± 0.1732

1e. L. fujianensis (TW) – – – – – 30.34 29.13 29.99 167.16 154.34 173.56 181.63 196.13 0.9537 ± 0.1670

1f. L. fujianensis (FJ) – 0.0366 0.0367 0.0366 0.0338 0.0361 16.30 – 169.00 153.60 176.70 177.80 200.60 0.6268 ± 0.1932

1g. L. fujianensis (GD) – 0.0349 0.0350 0.0349 0.0334 0.0346 0.0191 – 170.33 154.00 178.50 178.50 198.50 0.3468 ± 0.1801

1h. L. fujianensis (III) – 0.0361 0.0362 0.0361 0.0337 0.0357 – – 169.38 153.71 177.21 178.00 200.00 1.2263 ± 0.2562

2. L. bannaensis 0.2297 0.2279 0.2296 0.2284 0.2310 0.2289 0.2326 0.2305 0.2333 – – 172.33 175.67 14.8590 ± 1.2489

2a. L. bannaensis (YN1) 0.2080 0.2066 0.2114 0.2081 0.2084 0.2081 0.2073 0.2078 0.2074 – 162.50 183.00 170.00 n/c

2b. L. bannaensis (YN2) 0.2405 0.2386 0.2387 0.2386 0.2423 0.2393 0.2453 0.2486 0.2463 – 0.2223 167.00 178.50 0.1153 ± 0.1077

3. L. fragilis 0.2529 0.2542 0.2504 0.2530 0.2582 0.2540 0.2473 0.2486 0.2476 0.2357 0.2541 0.2265 168.00 0.6961 ± 0.2743

4. L. kuhlii 0.2801 0.2766 0.2794 0.2775 0.2848 0.2789 0.2871 0.2832 0.2859 0.2413 0.2331 0.2454 0.2301 n/c

N.-H. Jang-Liaw and W.-H. Chou258

were detected within clade I, in which the subclade Ia is

defined by 18 haplotypes from 82 specimens representing

the midwestern-to-northern populations in Taiwan, and the

subclade Ib is defined by eight haplotypes from 66 speci-

mens representing the northern and northeastern popula-

tions in Taiwan. Fig. 1 shows the biogeographic districts of

these clades. Samples from Lugu (LG) contain haplotypes

from clades I and II. Both strict consensus trees from MP

and ML analyses (not shown) exhibited minor discrepancies

with the NJ tree in topology within major clades.

Within L. fujianensis, the average sequence difference

among all haplotypes was 1.75 ± 0.25% (mean ± SD; see

Table 2) and range from 0.12−4.07% (data matrix for the

number of base substitutions per site from analysis between

sequences is not shown). There were 32 haplotypes in

Taiwanese L. fujianensis specimens. Among Taiwanese

specimens the average sequence difference was 0.95 ±0.17%, ranging from 0.12−2.59%. Among L. fujianensis

specimens from China the average sequence difference

was 1.23 ± 0.26% (range 0.12−2.24%). The Fst values were

from 0.72 to 0.85 between major clades inferred from all

sequences (data not shown). The distances between

clades/subclades inferred from haplotypes ranged from

0.77−3.61%, clades/subclades from 0.20−1.23% (Table 2).

The genetic variation analysis within L. fujianensis

population showed nucleotide diversity (π) among 26 sam-

pling populations ranging from 0.00−0.83%; the mean of all

L. fujianensis individuals was 1.10% (SD = 0.08%). The hap-

lotype diversity (Hd) within each locality ranged variably

from 0.00−1.00; the nucleotide diversity (π) among clades/

subclades ranged from 0.05−1.21%. The haplotype diversity

(Hd) within each clade/subclades ranged from 0.37−0.92

(except for the Chinese clade, 1.00) (Table 3).

Table 2 shows the distances between/within clades and

populations of L. fujianensis and other three Limnonectes

Table 3. Genetic variation of sampling localities of Limnonectes

fujianensis in this study. See Table 1 for locality codes and Fig. 2 for

grouping of clades. Ns, number of specimens; h, number of haplo-

types observed; s, number of segregating sites; Hd, estimates of

haplotype diversity; π, nucleotide diversity.

Clades/Locality Ns h

Numbers of individuals

of each clades s Hd π × 102

Ia Ib II III

Clade I 148 26 31 0.851 0.434

Subclade Ia 82 18 20 0.917 0.298

1. YMS 10 3 5 5 – – 6 0.644 0.343

2. SD 10 3 1 9 – – 7 0.511 0.179

3. FH 10 3 10 – – – 4 0.600 0.161

4. WE 10 2 10 – – – 3 0.200 0.069

5. NJ 4 2 4 – – – 1 0.500 0.058

6. TA 10 4 10 – – – 8 0.644 0.392

7. SY 10 3 10 – – – 4 0.689 0.177

8. DS 7 2 7 – – – 2 0.476 0.110

9. TP 10 2 10 – – – 1 0.200 0.023

10. YC 10 2 10 – – – 1 0.200 0.023

Subclade Ib 66 8 7 0.374 0.046

16. NA 6 1 – 6 – – 0 0.000 0.000

17. DT 5 1 – 5 – – 0 0.000 0.000

18. CL 3 1 – 3 – – 0 0.000 0.000

19. YS 10 2 – 10 – – 1 0.356 0.041

20. TC 10 2 – 10 – – 1 0.200 0.023

21. SX 10 1 – 10 – – 0 0.000 0.000

22. NK 5 3 – 5 – – 2 0.700 0.092

23. KL 3 1 – 3 – – 0 0.000 0.000

Clade II 45 6 14 0.751 0.478

11. LG 10 2 5 – 5 – 13 0.556 0.832

12. GK 10 2 – – 10 – 9 0.356 0.369

13. FL 10 2 – – 10 – 6 0.533 0.369

14. JP 10 2 – – 10 – 1 0.556 0.064

15. SM 10 2 – – 10 – 2 0.467 0.108

Clade III 7 7 24 1.000 1.207

24. FJ1 3 – – – – 3 3 1.000 0.230

25. FJ2 2 – – – – 2 1 1.000 0.115

26. GD 2 – – – – 2 3 1.000 0.341

All specimens 200 39 82 66 45 7 78 0.906 1.097

Table 4. Neutrality test statistics of Limnonectes fujianensis within

each population (clades/subclades and localities; Figs. 1 and 2)

using the total number of mutations of partial cytochrome b

sequence for calculations. n: numbers of sequences. *: statistically

significant at the 5% level. **: statistically significant at the 2% level.

n/c: not calculated because of too small number of sequences (less

than four sequences). –: not calculated due to lack of polymor-

phisms in data.

Clade/Locality n Tajima’s D Fu and Li’s D Fu and Li’s F

Clade I 148 –0.9465 –1.5737 –1.5839

Subclade Ia 82 –1.0344 –0.8206 –1.0724

1. YMS 5Ia + 5Ib 1.6714 0.7749 1.1163

2. SD 1Ia + 9Ib –1.5729 –1.6342 –1.8179

3. FH 10 –0.0379 –0.3383 –0.2967

4. WE 10 –1.5622 –1.7844 –1.9338

5. NJ 4 –0.6124 –0.6124 –0.4787

6. TA 10 0.8729 0.9621 1.0562

7. SY 10 0.3242 1.2391 1.1372

8. DS 7 0.6873 1.1781 1.1451

9. TP 10 –1.1117 –1.2434 –1.3467

10. YC 10 –1.1117 –1.2434 –1.3467

Subclade Ib 66 –1.8146* –2.0530 –2.3260

16. NA 6 – – –

17. DT 5 – – –

18. CL 3 n/c n/c n/c

19. YS 10 0.0150 0.8042 0.6840

20. TC 10 –1.1117 –1.2434 –1.3469

21. SX 10 – – –

22. NK 5 –0.9726 –0.9726 –0.9544

23. KL 3 n/c n/c n/c

Clade II 45 0.9693 1.5392* 1.5938

11. LG 5Ia + 5II 2.6013** 1.4995** 1.9955**

12. GK 10 0.0256 1.4351* 1.2221

13. FL 10 2.1048** 1.3461 1.7192**

14. JP 10 1.4636 0.8042 1.0688

15. SM 10 1.0330 1.0262 1.1460

Clade III 7 0.3930 0.7781 0.7633

24. FJ1 3 n/c n/c n/c

25. FJ2 2 n/c n/c n/c

26. GD 2 n/c n/c n/c

All specimens 200 –0.8664 0.0671 –0.4314

Phylogenetic of Taiwan Limnonectes fujianensis 259

species. The distances between L. fujinanensis and other

fanged frogs were from 23.0–28.0%. The pairwise distances

of Taiwanese L. fujinanensis clades/subclades were from

0.77–1.90%, and 3.34–3.67% between Taiwanese and

Chinese populations.

Neutrality tests

In this study, the values of neutrality test statistics are

shown in Table 4. Neutral theory has become the primary

null hypothesis used to test for the effects of natural selec-

tion. If a molecular marker that is assumed to be evolving

neutrally is found to be subject to selection, conclusions

based on patterns of dissimilarity at the marker could be

misleading (Ford, 2002). Tajima’s D values did not signifi-

cantly deviate from zero in most localities, except for the

Lugu and Fanlu populations (sites 11 and 13). Fu and Li’s

D and F values did not deviate significantly from zero in

most populations, except for Lugu, Gukeng (sites 11, 12)

and Lugu, Fanlu (sites 11, 13) populations, respectively. Fu

and Li’s D value of clade II showed significant deviation from

zero, so did the Tajima’s D value of subclade Ib. Overall,

neutrality test values among all L. fujianensis individuals did

not significantly deviate from zero. These results indicate

that the cytochrome b gene of L. fujianensis evolves neu-

trally, and could be considered as a neutral marker.

Parsimonious network analysis

and phylogeographic information

A statistical parsimony network

was constructed by linking the L.

fujianensis haplotypes in a hierarchi-

cal manner, based on the variations

between sequences by TCS 1.21

(Fig. 3). In this network, three major

clades were separated clearly and

largely agreed with the neighbor-

joining tree of PAUP. Some haplo-

types were located at the interior

nodes between clades.

Clade I included two distinct

subclades. In subclade Ia, haplo-

types showed close relationships

with each other, however, the valid-

ity of this group was less strongly

supported by phylogenetic analysis

results. Two major haplotypes (h20

and h18, represented by 14 and 9

individuals, respectively) was

located interiorly among haplotypes

of this subclade. At two localities

(sites 1 and 2) individuals from both

subclades Ia and Ib occurred sym-

patrically (see Table 3). A major

haplotype, h01 represented by 52

individuals, was located the center

among haplotypes of subclade Ib.

The haplotype h01 was the most

abundant and widely distributed

one, found in nine collecting sites. In

clade II, which was closer to clade I

than to III, most haplotypes were

found only from a single locality, except that h24 was

extended in four sites represented by 20 individuals. Collec-

tively, most L. fujianensis haplotypes were distributed at sin-

gular location; only six haplotypes were sampled from two or

more sites.

Mostly, each sampling site contained haplotypes from

one particular clade, except for sites 1 (Yangmingshan) and

2 (Sindian) which contained haplotypes from subclades Ia

and Ib, and site 11 (Lugu) which contained haplotypes of

subclade Ia and clade II (Table 3). However, samples from

sites 1 and 2 did not exhibit high π values (0.34% and

0.18%, respectively), given the small numbers of segregat-

ing sites (s = 6 and 7, respectively). Instead, site 11 had a

higher π value (0.83%) and more segregating sites (s = 13),

and was considered as a boundary locality between clades

I and II.

Divergence times among major clades within Limnonectes fujianensis

We conducted the two-cluster test by including all 39

haplotypes of L. fujianensis used for the phylogenetic anal-

ysis, and a cytochrome b sequence of L. kuhlii (k01 of this

study) was used as the outgroup. The results of the test are

summarized in Table 5. CP value is the confidence proba-

bility (1 – P-value) computed in the two-cluster test. Differ-

entiation events between clades showed no significant rate

Fig. 3. Parsimony network for each clade constructed by TCS 1.21. Numbers inside circles are

haplotypes. The number in parentheses denotes the number of localities the corresponding hap-

lotype was sampled. Haplotypes without number in parentheses represent that the haplotype

was found from a single location. Circle size reflects the number of individuals having the corre-

sponding haplotype. Small solid dots signify possible missing haplotypes. A line connecting two

haplotypes represents one nucleotide substitution.

Table 5. Results of two-cluster test and estimated divergence times of Limnonectes fujianensis

inferred from haplotype clades (Fig. 2). Divergence time was calculated from its height. b1 and b2

represent the lengths of two clades devided by the same branching point. Z = delta/s.e.; delta =

| b1–b2 |; height = average (b1, b2); divergence time = height/divergence rate (= 1.41% per lin-

eage per million years followed Jang-Liaw et. al., 2008); and CP value = 1–p value.

Sister groups

(1 vs 2)CP (%) Z delta s.e. b1 b2 height (s.e.)

Divergence time

(mean ± SE; mya)

Ia vs Ib 15.86% 0.2060 0.0004 0.0019 0.0040 0.0036 0.0038 (0.0012) 0.27 ± 0.09

I vs II 27.36% 0.3555 0.0012 0.0035 0.0087 0.0099 0.0093 (0.0020) 0.66 ± 0.14

(I+II) vs III 66.80% 0.9745 0.0064 0.0066 0.0209 0.0145 0.0177 (0.0028) 1.25 ± 0.20

FJ vs GD 29.60% 0.3839 0.0015 0.0040 0.0103 0.0087 0.0095 (0.0021) 0.67 ± 0.15

N.-H. Jang-Liaw and W.-H. Chou260

differences. The divergence time between clades/subclades

within Taiwan was estimated from 0.27 ± 0.09 (mean ± SE)

to 0.66 ± 0.14 million years ago (MYA). The divergence time

between the Taiwanese and Chinese populations (clades I

and II vs. clade III) was estimated as 1.25 ± 0.20 MYA.

Within the Chinese clade, the estimated divergence time

between Fujian and Guangdung samples is 0.67 ± 0.15

MYA.

DISCUSSION

Divergence and biogeography

In our L. fujianensis samples, the resultant tree topolo-

gies for all phylogenetic analyses were basically identical

and congruent with the network analysis. This study sug-

gests the presence of three genetically differentiated clades

in L. fujianensis, of which the Chinese clade III is distinctly

separated from the Taiwanese clades with high bootstrap

replications in PAUP analysis (Fig. 2). Taiwan Strait, 200 km

wide and 50 m deep on average, is regarded as an effective

isolation mechanism for fauna on its either sides (Tzeng,

1986; Cheng and Fang, 1999; Jang-Liaw et al., 2008). How-

ever, this also provides opportunities for animals to migrate

between mainland China and Taiwan via landbridge forma-

tion more than once, due to sea level changes during the

Pleistocene Ice Ages (Emery et al., 1971; Zhao, 1982;

Yang, 1991). Examples of multiple invasions have been

studied in certain animals, e.g., the Bamboo Viper

Trimeresurus stejnegeri (Creer et al., 2001), the La Touche’s

Frog Sylvaticus latouchii (Jang-Liaw et al., 2008), Sauter’s

Frog Rana sauteri (Jang-Liaw and Lee, 2009), and some

rodents (Yu, 1995). A single invasion of L. fujianensis from

China to Taiwan was derived from the phylogenetic analy-

ses in this study.

The two clades in Taiwan are almost allopatrically dis-

tributed. The K2P genetic distance between clades I and II

reaches 0.019 (Table 2), and the estimated divergence time

0.66 Mya for clades I vs. II accounts for mid-Pleistocene

diversification on either side of the Chousui River (Fig. 1).

Chousui River has long been considered an effective geo-

graphic barrier for gene flows or a boundary of differentiation

in some vertebrates, including fishes (Wang et al., 2004;

Watanabe et al., 2007), and frogs (Jang-Liaw et al., 2008).

However, we note that the sample from Lugu (site 11),

upstream of Chousui River, possesses haplotypes of both

clades I and II. The sampled 10 individuals at Lugu contain

five h20s (clade I) and five h22s (clade II) (Table 1). Both

h20 and h22 are interior haplotypes and remotely differenti-

ated with 13 nucleotide substitutions, making this site bear-

ing the highest nucleotide diversity value (π = 0.83%) among

all the collecting sites. The composition of the Lugu sample

may feature a location of secondary contact, likely reflecting

post-glacial colonization dynamics.

Subclades Ia and Ib showed much closer genetic dis-

tance (0.0077) than they did to others, implying their more

recent differentiation. As described above, both subclades

seem to be not to be clearly isolated, owing to low bootstrap

values from the MP and ML analyses. The MP analysis

even treated subclades Ia and Ib as a single lineage (tree

not shown). Subclade Ib has particularly low haplotype

diversity (Hd = 0.374) and π value (0.046%) (Table 3), which

suggests a “recent population bottleneck” or “founder event

by single or few mtDNA lineages” had occurred during the

history of subclade Ib (see Grant and Bowen, 1998). It is

possibly due to a bottleneck effect exhibited by the data that

haplotype h01 can be found from nine out of ten sampling

sites in subclade Ib (Table 1, Fig. 2).

Along with the estimated divergence times inferred from

two-cluster test, the order of phylogenetic isolation events

for L. fujianensis in Taiwan began with 1) Taiwanese popu-

lation (clades I and II) becoming separated from Chinese

populations through isolation of the Taiwan Strait (1.25 ±0.20 mya) during the early Pleistocene; 2) followed by clade

I separating from clade II caused by the isolation of Chousui

River (0.66 ± 0.14 mya) during the middle Pleistocene; and

3) subclade Ib separating from subclade Ia in northern

Taiwan (0.27 ± 0.09 mya) during the middle Pleistocene.

These data indicate that the ancestors of subclade Ia

can be considered the first colonies into Taiwan Island,

which then distributed southward and northward, forming

distinct clades/subclades sequentially. Nevertheless, sub-

clades Ia and Ib are bounded by an hypothetical line con-

necting sites 1 and 2 located on northern and southern sides

of Taipei Basin, respectively. No obvious topographical bar-

rier can be found there. It is not clear why Taipei Basin has

become the boundary of subclades Ia and Ib and caused

this bottleneck effect.

The distributional range of L. fujianenesis in mainland

China is projected to be from Zhejiang to Guangdong

Provinces (Fei, 1999). In this study, the Taiwanese popula-

tions are monophyletic with respect to the limited number of

Chinese populations sampled. This may suggest that the

observed phylogeographical patterns be the result of the

historical lineage sorting of the original L. fujianenesis

isolated when Taiwan first became isolated from China.

Parsimonious network analysis (Fig. 3) indicates that the

haplotype h35 of the Changting, Fujian (site 24) was closest

to the Taiwanese clades of the seven specimens from main-

land China used in this study. This implies that Taiwanese

L. fujianenesis may have arrived from Fujian when the land-

bridge across Taiwan Strait appeared during the Ice Ages.

Biogeographic characters comparing to other animals in

Taiwan

The multiple-invasion hypothesis regarding populations

of the Taiwanese fauna has been tested and supported by

using various vertebrates (Yu, 1995; Yu et al., 1996; Wang

et al., 1999; Hsu et al., 2000; Creer et al., 2001; Creer et al.,

2004; Oshida et al., 2006; Jang-Liaw et al., 2008; Jang-Liaw

and Lee, 2009). To date, only Sylvirana latouchii and

Limnonectes fujianensis of the Taiwanese frogs have been

studied to obtain the divergence scenarios with their China

ancestral populations. S. latouchii, a common ranid frog in

the lowlands of Taiwan (Jang-Liaw et al., 2008), is widely

distributed all over this island from the low hills up to eleva-

tions ca. 1,500 m above sea level (Lue et al., 1999). S.

latouchii breeds all the year (Huang et al., 2004) and its tad-

poles are commonly found in various aquatic environments,

such as slow-moving water in ditches, small streams, and

static rain pools (Chou and Lin, 1997; Wu and Kam, 2005).

According to our investigation, L. fujianensis is also com-

monly found in the south (but not the lowest south), mid-

west, and north-to-northeast of Taiwan. Its tadpoles share

Phylogenetic of Taiwan Limnonectes fujianensis 261

several microhabitats with S. latouchii (Chou and Lin, 1997).

However, L. fujianensis seems to show higher fidelity to

habitats than S. latouchii (Yang, 2006), providing a potential

reason for its limited distribution area and single-invasion

history in Taiwan. In comparison with the inferred migration

history of R. sauteri and S. latouchii in Taiwan (Jang-Liaw

et al., 2008; Jang-Liaw and Lee, 2009), L. fujianensis is a

more recent invader, and probably entered Taiwan simulta-

neously with the second invasion of S. latouchii in middle

Pleistocene. It is not known why L. fujianensis reached

Taiwan at the central-western Taiwan while S. latouchii

arrived via northern Taiwan.

Like other vertebrates, the Taiwan L. fujianensis seems

to be impeded by some topological barriers. The Central

Mountain Range composed of a series of high mountains

over 3000 m of elevation apparently obstruct L. fujianensis

from moving eastward. Isolation by the Central Mountain

Range is also noticeable in freshwater fishes (Tzeng, 1986;

Cheng and Fang, 1999) and in other terrestrial vertebrates

(Chang and Liu, 1997; Oshida et al., 2006; Jang-Liaw et al.,

2008; Jang-Liaw and Lee, 2009). Chousui River is also a dif-

ficult barrier for faunal exchange (Wang et al., 2004;

Watanabe et al., 2007; Jang-Liaw et al., 2008). Jang-Liaw et

al. (2008) discussed that the genetic divergence of S.

latouchii on both sides of Chousui River was evident, with a

lately developed haplotype distributed across the river. A

similar situation is present in L. fujianensis. Haplotype 20 of

subclade Ia occurring in Lugu (site 11), located south bank

of Chousui River, indicates migration across the river,

though few, is possible.

Another important topology, Miaoli Plateau, that seems

to be effective to the genetic differentiation of S. latouchii

(Jang-Liaw et al., 2008), did not serve as an isolation factor

in L. fujianensis. The Miaoli Plateau is located near sites 4,

5 and 6, and a boundary of distribution for some freshwater

fishes (Tzeng, 1986; Wang et al., 1999; Wang et al., 2004;

Watanabe et al., 2007). Haplotypes of clade I of L. fujianensis

distributed over Miaoli Plateau suggests different natural his-

tories for L. fujianensis and S. latouchii. Nevertheless, north-

ern Taiwan often exhibits unique freshwater fish fauna

(Oshima, 1923; Tzeng, 1986; Cheng and Fang, 1999) and

distinct genetic structures of some animals, such as bagrid

catfishes (Watanabe et al., 2007), cyprinids (Wang et al.,

1999; Wang et al., 2000; Wang et al., 2004; Wu et al.,

2007), frogs (Jang-Liaw et al., 2008; Jang-Liaw and Lee,

2009), snakes (Creer et al., 2001) and squirrels (Oshida et

al., 2006). In L. fujianensis of this study, no such well

defined northern Taiwan district is recognized, but a hypo-

thetical line connecting sites 1 and 2 (Fig. 1) near the Taipei

Basin divided subclades Ia and Ib. Supposedly, the trans-

gressions of Taipei Basin performed an isolation mechanism

in this frog that was not found in other animals mentioned

above. After all, the low genetic divergence between sub-

clades Ia and Ib of L. fujianensis could merely be an out-

come of a bottleneck effect in northern Taiwan.

Taxonomic implication of Limnonectes fujianensisTaxonomical studies on the Limnonectes species in

China and the adjacent areas have been relatively less

intensive until late 1900s. Several previously recognized L.

kuhlii are presently regarded distinct, for example, L. fragilis

from Hainan Island of China (Liu et al., 1973); L. bannaensis

from southern Yunnan of China (Ye et al., 2007); L.

fujianensis from Hunan, Jiangxi, Anhui, Zhejiang, and Fujian

of China and Taiwan (Ye and Fei, 1994; Zhang et al., 2005),

L. megastomias from eastern Thailand (McLeod, 2008). The

taxonomy of Limnonectes frogs has never been easy, due

to the similar morphology among the congeners. When

described (Ye and Fei, 1994) the Fujian large-headed

fanged L. fujianensis was thought to occur in south-eastern

China and Taiwan. On the contrary, Ye et al. (2007) pro-

posed that the Taiwan fanged frog be a cryptic species. Our

genetic data of distance comparison, however, do not sup-

port this proposal. The genetic distances between Taiwan-

ese and Chinese L. fujianensis samples (3.3–3.7%) were far

less than the distances between Taiwanese samples and

other Limnonectes species (23.0–28.0%; Table 2). Interest-

ingly, there might be a cryptic species within currently rec-

ognized L. bannaensis sampled from Yunnan based on the

results of our phylogenetic analyses. Three Yunnan haplo-

types (b01–03) exhibited non-monophyletic relationships

(Fig. 2).

Phylogenetic studies frequently result in useful informa-

tion for taxonomic considerations. Emerson et al. (2000) and

Evans et al. (2003) tested the molecular phylogenies of

Limnonectes from Southeast Asia and suggested four major

groups present within this genus. Although their samples

included a Taiwanese L. kuhlii (currently known as L.

fujianensis) sample, they did not discuss the phylogenetic

positions of the Taiwanese and Chinese species. Zhang et

al. (2005), by inference from partial sequences of mitochon-

drial 12S rRNA and 16S rRNA genes, concluded that three

Limnonectes species from Taiwan and China, i.e., L. fragilis,

L. fujianensis and L. bannaensis, composed a monophyletic

group distinct from the four major groups in Southeast Asia.

A recent study of molecular systematic relationships

between fanged frogs from China and adjacent areas con-

firmed the close relationships among L. bannaensis, L.

fujianensis, and L. namiyaei (Matsui et al., 2010). Both

Zhang et al. (2005) and Matsui et al. (2010) agreed that L.

fragilis exhibited remote genetic distances with L. fujianensis

and L. bannaensis, respectively, as shown in our results

(Fig. 2). Matsui et al. (2010) indicated that the Hainan L.

fragilis exhibited a closer relationship with the true L. kuhlii

from Java than to other L. “kuhlii” from various localities in

China and Southeast Asia, and revealed the presence of

much more species diversity in fanged frogs than we now

understand.

Despite the previous efforts on the taxonomy of

Limnonectes species in China, the issue of the

Nyctibatrachus sinensis, a synonym of Rana kuhlii (Mell,

1922; currently L. kuhlii), remains a mystery. This species

was first described by W. Peters (1882) from Loufushan,

Guangdung (Mons Lofau, Provincia Canton), China. Mell

(1922) examined the types and determined it to be a syn-

onym of Rana kuhlii, which was followed by Pope (1931)

and Liu (1950). Without any explanation, Liu and Hu (1961)

changed this previous view and listed N. sinensis as a syn-

onym of Rana spinosa (currently Quasipaa spinosa; see

Frost et al., 2006; Frost, 2009). Ye (2009), however, stated

that Pope and Boring (1940) dissected the type specimen

and found the base of omossternum not to be forked, which

N.-H. Jang-Liaw and W.-H. Chou262

is not a character of fanged frogs. It is worthy of attention

that our samples with haplotypes h38 and h39 collected

from site 26, i.e., Nankunshan, 45 km north of Loufushan,

Guangdung, China, were included in clade IV of L. fujianensis

(Fig. 2). It is quite evident that, if N. sinensis Peters 1882

and L. fujianensis Ye and Fei 1994 are synonymous, the L.

sinensis shall have priority. As a matter of fact, the genera

Limnonectes and Quasipaa are morphologically distinct,

and easily distinguishable from each other. An inspection to

the types of N. sinensis is essential to the nomenclature of

L. fujianensis.

ACKNOWLEDGMENTS

We would like to express our sincerest appreciation to Sheng-

Hai Wu, Jing Che, Eric N. Smith, Hsiao-Yin Su, and Chin-Ming

Tseng for their generous donation of tissue samples; Ping Wei and

Hua-Gu Ye of the South China Botanical Garden for providing vehi-

cles and field guidance; Shui-Hua Chen, Tsang-Sung Chen, and

Chun-Mo Tsai of the Zhejiang Museum of Natural History for sup-

porting the field work; Jia-Lin Zhu of the Xiamen Daily, and Ruei-

Yun Lin of the Hwa-An Science Association for their enthusiasm in

field work planning and participation. Special thanks also to three

anonymous reviewers for their helpful comments on an earlier ver-

sion of this manuscript.

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(Received December 15, 2009 / Accepted September 12, 2010)