Phylogeny of the Asian spiny frog tribe Paini

Molecular Phylogenetics and Evolution 50 (2009) 59–73
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Phylogeny of the Asian spiny frog tribe Paini (Family Dicroglossidae) sensu Dubois
Jing Che a,1, Jian-sheng Hu b,a,1, Wei-wei Zhou a, Robert W. Murphy c,a, Theodore J. Papenfuss d,
Ming-yong Chen a,b, Ding-qi Rao e, Pi-peng Li f, Ya-ping Zhang a,g,*
a
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming 650223, PR China
College of Life Sciences, Yunnan University, Kunming 650091, PR China
c
Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ont., Canada M5S 2C6
d
University of California, Berkeley, Department of Integrative Biology, Museum of Vertebrate Zoology, 3101 Valley of Life Sciences Building #3160, Berkeley, CA 94720-3160, USA
e
Division of Amphibians and Reptiles, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming 650223, PR China
f
Liaoning Key Laboratory of Biodiversity and Evolution, Shenyang Normal University, Shenyang 110034, PR China
g
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming 650091, PR China
b
a r t i c l e
i n f o
Article history:
Received 11 May 2008
Revised 3 September 2008
Accepted 6 October 2008
Available online 21 October 2008
Keywords:
Tribe Paini
Dicroglossidae
Mitochondrial DNA
Nuclear DNA
Phylogeny
Geography
China
Southeast Asia
a b s t r a c t
The anuran tribe Paini, family Dicroglossidae, is known in this group only from Asia. The phylogenetic
relationships and often the taxonomic recognition of species are controversial. In order to stabilize the
classification, we used approximately 2100 bp of nuclear (rhodopsin, tyrosinase) and mitochondrial
(12S, 16S rRNA) DNA sequence data to infer the phylogenetic relationships of these frogs. Phylogenetic
trees reconstructed using Bayesian inference and maximum parsimony methods supported a monophyletic tribe Paini. Two distinct groups (I, II) were recovered with the mtDNA alone and the total concatenated data (mtDNA + nuDNA). The recognition of two genera, Quasipaa and Nanorana, was supported.
Group I, Quasipaa, is widespread east of the Hengduan Mountain Ranges and consists of taxa from relatively low elevations in southern China, Vietnam and Laos. Group II, Nanorana, contains a mix of species
occurring from high to low elevation predominantly in the Qinghai-Tibetan Plateau and Hengduan
Mountain Ranges. The occurrence of frogs at high elevations appears to be a derived ecological condition.
The composition of some major species groups based on morphological characteristics strongly conflicts
with the molecular analysis. Some possible cryptic species are indicated by the molecular analyses. The
incorporation of genetic data from type localities helped to resolve some of the taxonomic problems,
although further combined analyses of morphological data from type specimens are required. The two
nuDNA gene segments proved to be very informative for resolving higher phylogenetic relationships
and more nuclear data should be explored to be more confident in the relationships.
Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
Genus Paa, Dubois, 1975, and its allies including Chaparana
Bourret, 1939 and Nanorana Günther, 1896, are a rather poorly
investigated group of dicroglossid frogs (Frost, 2008). Dubois
(1992) established the tribe Paini to include the genera Paa and
Chaparana. The tribe now also includes the genus Nanorana (Ohler
and Dubois, 2006) (Table 1). These aquatic and semi-aquatic species live mostly in swift, boulder-strewn rivers and streams in
the mountains (400–4700 m) of South and Southeast Asia. Most
male frogs are characterized by keratinized spines on the chest,
belly, or on other parts of their bodies (Fig. 1). In addition,
most males also possess the secondary sex characteristic of
* Corresponding author. Address: The Chinese Academy of Sciences, State Key
Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology,
Kunming 650223, PR China. Fax: +86 871 5032804.
E-mail address: zhangyp1@263.net.cn (Y. Zhang).
1
These two authors contributed equally to this work.
1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2008.10.007
hypertrophied forearms during the breeding season. These characters are thought to be adaptations to males grasping females during amplexus (Ohler and Dubois, 2006). Because they are closely
tied to water, the species are probably poor overland dispersers.
If true, then it is likely those populations in different drainages
have high levels of genetic divergence and that more species exist
than are currently recognized.
Taxonomic controversy has reigned supreme in this group of
frogs, and since Boulenger (1920), the taxonomy of this group
has been revised numerous times (Dubois, 1975, 1987 (1986),
1992, 2005; Liu and Hu, 1961; Fei et al., 1990, 2005; Fei, 1999;
Frost et al., 2006; Jiang et al., 2005; Ohler and Dubois, 2006; Roelants et al., 2004). Dubois (1975) established the subgenus Paa
(genus Rana) for those species with distinct male secondary sexual
characters and large eggs. Subsequently, Paa was given generic status, and together with the genus Chaparana, most of which lack
spines, were united to form the tribe Paini within the subfamily
Raninae (Dubois, 1992) (Table 1). The tribe Paini has been resolved
to nest within the subfamily Dicroglossinae based on molecular
60
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Table 1
Different classifications of dicroglossid frogs in the tribe Paini.
Specific
epithet
Dubois 1987 (1986)
Fei et al. (1990)
Dubois (1992)
Fei (1999)
Fei et al. (2005)
Frost et al.
(2006)
Ohler and Dubois
(2006)
Ranidae
Raninae
Ranidae
Raninae
Paini
Paa
Gynandropaa
Gynandropaa
Gynandropaa
Ranidae
Raninae
Ranidae
Dicroglossinae
Dicroglossidae
bourreti
yunnanensis
liui
Ranidae
Raninae
Ranini
Rana
Paa (yunnanensis group)
Paa (yunnanensis group)
—
Paa
—
Paa (yunnanensis group)
Paa (yunnanensis group)
Paa
—
Paa (yunnanensis group)
Paa (yunnanensis group)
Nanorana
Nanorana
Nanorana
conaensis
liebigii
arnoldi
chayuensis
medogensis
maculosa
boulengeri
robertingeri
spinosa
shini
exilispinosa
jiulongensis
verrucospin osa
Paa
Paa
Paa
—
—
Paa
Paa
—
Paa
Paa
Paa
—
—
Paa
Paa
—
Paa
—
Paa
Paa
—
Paa
Paa
Paa
Paa
—
Paa
Paa
—
Paa
Paa
Paa
Paa
—
Paa
Paa
Paa
Paa
—
Paa
Paa
—
Paa
Paa
Paa
Paa
Paa
Paa
Paa
Paa
Paa
—
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
unculuana (us)
aenea
taihangnica
Rana (lateralis group)
Paa (liebigii group)
—
Unculuana
—
—
Paa (liebigii group)
Paa (liebigii group)
Paa (maculosa group)
Paa (maculosa group)
—
Paa (maculosa group)
Quasipaa
—
Quasipaa
Quasipaa
Quasipaa
Quasipaa
—
Chaparana
Chaparana
Chaparana
—
Unculuana
—
—
Unculuana
—
Feiana
Nanorana
Nanorana
quadrana (us)
yei
Paa (delacouri group)
—
Quadrana
—
Feirana
—
Feirana
—
Feiana
Feiana
Nanorana
Quasipaa
delacouri
Paa (delacouri group)
—
—
—
Nanorana
pleskei
ventripunctata
parkeri
Nanorana
—
Altirana
Nanorana
Nanorana
Altirana
Annandia
Ranini
Nanorana
Nanorana
Nanorana
Altirana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Ranidae
Dicroglossinae
Paini
Gynandropaa
Gynandropaa
Gynandropaa
Gynandropaa
Chaparana
Paa
Paa
Paa
Paa
Paa
Paa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Chaparana
Chaparana
Chaparana
Paa
Gynandropaa
Feirana
Quasipaa
Limnonectini
Annandia
Paini
Nanorana
Nanorana
Nanorana
Altirana
(liebigii group)
(liebigii group)
(maculosa group)
(maculosa group)
(spinosa group)
(spinosa group)
(spinosa group)
(spinosa group)
Paa
Paa
Paa
Paa
Fig. 1. Male Paa yunnanensis collected from Luquan, Yunnan, China exhibiting
nuptial spines and hypertrophied forearms.
data (e.g., Roelants et al., 2004). Recent studies also indicated that
Paa sensu Dubois (1992) is not monophyletic with respect to Chaparana and Nanorana (Che et al., 2007; Chen et al., 2005; Jiang
et al., 2005; Roelants et al., 2004). Species related to Nanorana were
originally treated as a subgenus of Rana by Boulenger (1920), and
assigned to Altirana and Nanorana by Liu and Hu (1961). Subsequently, these species were included in the tribe Ranini in a single
genus, Nanorana, by Dubois (1992) (Table 1). Ohler and Dubois
(2006) proposed six genera including Allopaa, Chaparana, Chrysopaa, Gynandropaa, Nanorana, and Quasipaa within the tribe Paini
based on 31 morphological characters. Curiously, their new generic
assignments within the Paini were at odds with their own molecular assessment (Jiang et al., 2005) (see Fig. 2); a total evidence approach was not pursued.
(liebigii group)
(liebigii group)
(liebigii group)
(liebigii group)
(liebigii group)
(boulengeri group)
(spinosa group)
(boulengeri group)
(spinosa group)
(spinosa group)
(liebigii group)
(liebigii group)
(maculosa group)
(maculosa group)
(maculosa group)
(boulengeri group)
(boulengeri group)
(spinosa group)
(boulengeri group)
(spinosa group)
(spinosa group)
Many studies have resolved the tribe Paini as a monophyletic
group (Che et al., 2007; Chen et al., 2005; Roelants et al., 2004).
Jiang et al. (2005) depicted two groups (I, II) in the Paini when evaluating mtDNA data (12S, 16S rRNA) using phenetic neighbor-joining clustering. However, the monophyly of these groups became
ambiguous when the analysis was based on a phylogenetic approach. Using the data of Jiang et al. (2005) and their own data,
Frost et al. (2006) proposed two generic divisions for the tribe Paini: Nanorana and Quasipaa. The latter genus was earlier suggested
by Jiang et al. (2005) (Table 1). Although much progress has been
made in understanding the relationships of the tribe Paini, the relationships and taxonomic assignments within the group have remained obscure. In part, the various taxonomic arrangements
arose from different approaches to systematics. The taxonomy of
Dubois (1992) was based mostly on degrees of overall similarity,
and not phylogeny (Inger, 1996).
Presently, the tribe Paini includes 42 species (Ohler and Dubois,
2006), the majority of which (27) occur in China though the validation of some species deserve further study. Fei (1999) and Fei et al.
(1990) placed the Chinese species into a single genus Paa, including
three subgenera: P. (Paa), P. (Unculuanus), and P. (Feirana). The subgenera Unculuanus and Feirana corresponded to the genus Chaparana (Dubois, 1992), and were subsequently elevated to distinct
generic status by Fei et al. (2005). Dubois 1987 (1986) proposed
division of species groups within Paa. Subsequently, Dubois
(1992) further delimited subgenera and species groups (Table 1).
Mostly based on the suggestion of Dubois (1987 ‘‘1986”, 1992),
Fei (1999) further divided the Chinese species of Paa into four species groups. Most recently, Fei et al. (2005) divided Paa into six
species groups: P. spinosa, P. boulengeri, P. liebigii, P. yunnanensis,
P. blanfordii, and P. maculosa (Table 1).
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
61
Fig. 2. Previous phylogenetic hypotheses: (a) phenetic relationships of 30 species of the tribe Paini inferred from 31 morphological characters (Ohler and Dubois, 2006), (b)
cladistic relationships of 19 species of the tribe Paini estimated from molecular data (Jiang et al., 2005). Drawings are not to scale. The vertical and horizontal lines with
different colors show the taxonomic rearrangements suggested by Ohler and Dubois (2006). The horizontal solid and dotted lines with the same color represent different
assignments of subgenera.
Many of these species show great phenotypic similarity to the
extent that recognizing species is often a challenge. The species
have been historically defined largely on the presence and distribution of body spinules in males during the breeding season. However, females, juveniles and males outside of the breeding season
can be difficult to identify to species, much less to associate with
a particular group. Fortunately, molecular genetic tools are very
effective at identifying species, and especially at revealing morphologically cryptic species (e.g., Bain et al., 2003; Stuart et al., 2006).
Phylogenetic reconstruction using molecular data now typically
involves a diverse array of genes. Because mtDNA has limitations
when used alone to form phylogenetic hypotheses (Moore, 1995),
investigations have exploited other, independent sources of phylogenetic characters, e.g., nuDNA. Roelants et al. (2004) provided a
molecular analysis of the ranoid relationships while incorporating
sequence data from rhodopsin and tyrosinase. Their analysis only
included five species of the tribe Paini. Frost et al. (2006) used
additional nuclear genes in analyzing the phylogenetic relationships of extant amphibians. However, for the three species in tribe
Paini included in their analysis, nuDNA data were obtained for two
species only. Jiang et al. (2005) proposed a molecular phylogeny of
the tribe Paini using approximately 900 bp of mtDNA sequence
data. Herein, we extend pervious investigations and employ nuclear DNA, including exon 1 sequences from rhodopsin and tyrosinase
in a phylogenetic study of the nominal tribe Paini. We have also expanded taxon sampling, and included portions of two mtDNA
genes (12S, 16S rRNA). Our objectives were as follows: (1) test
the monophyly of the tribe Paini sensu Dubois; (2) investigate
the presence of cryptic species in the tribe; (3) investigate the
validity of the current species groupings of Fei et al. (2005) based
on the presence and distribution of body spines; and (4) provide
a molecular phylogenetic framework for a phylogeny-based
taxonomy.
2. Materials and methods
2.1. Taxon sampling
Species in the genera Paa, Nanorana, and Chaparana formed our
ingroup, and species in the genera Hoplobatrachus, Limnonectes,
and Fejervarya were used as outgroup taxa (Table 2). Ninety-nine
specimens in total were used, among which 84 individuals representing 25 species formed the ingroup. For the Paini, the taxonomic
arrangements by Dubois (1992) were followed for the purpose of
discussion. Regarding P. taihangnica and P. yei, we used the original
generic assignments (Chen and Jiang, 2002; Chen et al., 2002).
Fieldwork in China coincided with the breeding season so that
females and males in breeding condition would be included in
the sampling. When possible, male specimens in breeding condition were used to confirm identity of the species and specimens
were obtained from or nearby type localities to assure accurate genetic assessments of species. Two species, P. polunini and P. blanfordii, documented to occur in China (Fei et al., 2005) were not
included due to difficulty in sampling. In addition, P. feae has remained unknown since its discovery and could not be included.
For those species occuring in Laos and Vietnam, we included tissues from the FMNH (Field Museum of Natural History, Chicago,
USA), the MVZ (Museum of Vertebrate Zoology, Berkeley, USA),
62
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Table 2
Sampling information for dicroglossid frogs in the tribe Paini including voucher specimens, localities and GenBank accession numbers for species used in this study. Asterisks
mark 26 sequences retrieved from Genbank. KIZ, Kunming Institute of Zoology, the Chinese Academy of Sciences; SCUM, Sichuan University Museum; YNU, Yunnan University;
SYNU, Shenyang Normal University; CIB, Chengdu Institute of Biology, the Chinese Academy of Sciences.
Species
Locality
Altitude (m)
Reference
specimens No.
GenBank Accession Nos. mtDNA
Nuclear DNA
12S-1
12S-2
16S
Rhodopsin
Tyrosinase
Ingroup
genus Paa
P. arnoldi 1
P. arnoldi 2
P. arnoldi 3
P. arnoldi 4
P. boulengeri 1
P. boulengeri 2
P. boulengeri 3
P. boulengeri 4
P. boulengeri 5
P. cf. boulengeri 6
P. boulengeri 7
P. boulengeri 8
P. boulengeri 9
P. boulengeri 10
P. chayuensis 1
P. chayuensis 2
P. chayuensis 3
P. conaensis 1
P. exilispinosa 1
P. exilispinosa 2
P. exilispinosa 3
P. jiulongensis 1
P. jiulongensis 2
P. liebigii 1
P. liebigii 2
P. liui 1
P. liui 2
P. maculosa 1
P. maculosa 2
P. medogensis 1
P. medogensis 2
P. robertingeri 1
P. robertingeri 2
P. shini 1
P. shini 2
P. shini 3
P. spinosa 1
P. spinosa 2
P. spinosa 3
P. spinosa 4
P. spinosa 5
P. spinosa 6
P. spinosa 7
P. cf. spinosa 8
P. sp.
P. taihangnica 1
P. taihangnica 2
P. verrucospinosa 1
P. verrucospinosa 2
P. verrucospinosa 3
P. verrucospinosa 4
P. yei 1
P. yunnanensis 1
P. yunnanensis 2
P. yunnanensis 3
P. yunnanensis 4
P. yunnanensis 5
P. yunnanensis 6
P. yunnanensis 7
P. yunnanensis 8
P. yunnanensis 9
P. bourreti
P. sp.
P. sp.
China: Pian ma, Lushui Co., Yunnan (1)
China: Pian ma, Lushui Co., Yunnan (1)
China: Shangpa, Fugong Co., Yunnan (2)
China: Shangpa, Fugong Co., Yunnan (2)
China: Mt. Emei, Sichuan (3)
China: Hunan (4)
China: Maolan nature reserve, Guizhou (5)
China: Longqing, Shizong Co., Yunnan (6)
China: Longqing, Shizong Co., Yunnan (6)
Vietnam: Tam Dao, Vinh Phu Prov. (7)
China: Yihuang, Jiangxi (8)
China: Yichang, Hubei (9)
China: Yichang, Hubei (9)
China: Lichuan, Hubei (10)
China: Cibagou nature reserve, Xizang (11)
China: Shama Village, Zayü Co., Xizang (12)
China: Shama Village, Zayü Co., Xizang (12)
China: Mama, Cona Co., Xizang (13)
China: Sangang, Wuyi, Fujian (14)
China: Sangang, Wuyi, Fujian (14)
China: Hong Kong (15)
China: Sangang, Wuyi, Fujian (16)
China: Sangang, Wuyi, Fujian (16)
China: Yakong Co., Xizang (17)
China: Yakong Co., Xizang (17)
China: Lugu Lake, Ninglang Co., Yunnan (18)
China: Lugu Lake, Ninglang Co., Yunnan (18)
China: Xinmin, Jingdong Co., Yunnan (19)
China: Xinmin, Jingdong Co., Yunnan (19)
China: 62 k, Medôg Co., Xizang (20)
China: Medôg Co., Xizang (21)
China: Zihuai, Hejiang Co., Sichuan (22)
China: Zihuai, Hejiang Co., Sichuan (22)
China: Huaping, Longsheng Co., Guangxi (23)
China: Huaping, Longsheng Co., Guangxi (23)
China: Dayao shan, Guangxi (24)
China: Mt.Dawei, Pingbian Co., Yunnan (25)
China: Mt.Dawei, Pingbian Co., Yunnan (25)
China: Damenglong, Jinghong, Yunnan (26)
China: Damenglong, Jinghong, Yunnan (26)
China: Lu shan, Jiujiang, Jiangxi (27)
China: Lu shan, jiujiang, Jiangxi (27)
China: Huang shan, Anhui (28)
Vietnam: Con Cuong vicinity, Nghe An (29)
Vietnam: Ngoc Linh vicinity, Kon Tum (30)
China: Taihangshan, Jiyuan, Henan (31)
China: Taihangshan, Jiyuan, Henan (31)
China: Mengsong, Jinghong, Yunnan (32)
China: Mengsong, Jinghong, Yunnan (32)
Vietnam: Tam Dao, Vinh Phu Prov. (33)
Vietnam: Tam Dao, Vinh Phu Prov. (33)
China: Shangcheng Co., Henan (34)
China: Jiefanggou, Xichang, Sichuan (35)
China: Mazong, Huili, Sichuan (36)
China: Dongping, Qiaojia Co., Yunnan (37)
China: Shiping Co., Yunnan (38)
China: Pubei, Yimen Co., Yunnan (39)
China: Leqiu, Nanjian Co., Yunnan (40)
China: Jinping Co., Yunnan (41)
China: Wulongmu, Yongde Co., Yunnan (42)
Vietnam: Sa Pa vicinity, Lao Cai (43)
Vietnam: Sa Pa vicinity, Lao Cai (44)
Lao PDR: Phongsaly Dist, Phongsaly Prov (45)
Lao PDR: Kaleum Dist, Xe Kong Prov (46)
2000
2000
1250
1250
1300
1050
900
1100
1100
950
800
<800
<800
1500
1593
1413
1413
3100
900
900
260
900
900
2700
2700
2600
2600
1900
1900
2126
2790
760
800
1000
1000
1000
1450
1450
1400
1400
500-1260
500-1260
500-600
308
1070
800
800
1600
1600
900
900-1100
400
2560
2300
1500
1400
1560
1600
1560
1700
1900
1900
600-800
1100-1280
SCUM050410CHX
SCUM05152123WD
YNU-HU200109012
YNU-HU200109006
SCUM37989
YNU-HUHU01
YNU-HU2003061301
YNU-HU20024060
YNU-HU20024061
MVZ226340
KIZ-JX246
KIZ-HUB292
KIZ-HUB293
KIZ-HUB274
SYNU-XZ54
SYNU-XZ67
SYNU-XZ64
KIZ-YP152
YNU-HU20026023
YNU-HU20026022
MVZ230391
YNU-HU200206036
YNU-HU200206037
KIZ-RDXZL1
KIZ-RDXZL2
YNU-HU200107010
YNU-HU200107016
YNU-HU2002322
YNU-HU2002308
SYNU-XZ75
SYNU-XZ35
YNU-HU20025105
YNU-HU20025106
YNU-HU20025002
YNU-HU20025001
SCUM060702L
YNU-HU20024040
YNU-HU20024042
YNU-HU20051005
YNU-HU20051004
KIZ-JX0709001
KIZ-JX0709002
KIZ-C21
ROM35181
ROM37390
KIZ-HN0709001
KIZ-HN0709002
YNU-HU20030724006
YNU-HU20030724005
MVZ223858
MVZ223934
YNU-HU200205151
SCUM045183CJ
SCUM20030091GP
YNU-HU200208005
YNU-HU2004009
YNU-HU20024012
YNU-HU20010401
YNU-HU20010505
YNU-HU20011102
ROM27866
ROM41507
FMNH258389
FMNH258383
EU979714
EU979711
EU979712
EU979713
EU979674
EU979680
EU979679
EU979678
EU979677
No data
EU979675
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EU979646
EU979645
EU979650
EU979665
EU979657
EU979724
EU979725
EU979658
EU979660
EU979669
No data
No data
EU979683
EU979685
EU979684
EU979687
EU979686
EU979690
EU979692
EU979691
EU979688
EU979689
EU979663
EU979656
EU979778
EU979776
DQ118467
EU979777
DQ118433
EU979761
EU979760
EU979759
DQ118435
EU979791
EU979757
EU979755
EU979758
EU979756
DQ116484
DQ118466
DQ118465
EU979774
DQ118440
DQ118439
EU979739
DQ118441
EU979741
DQ118455
DQ118456
DQ118449
EU979762
DQ118468
EU979775
DQ118463
DQ118462
EU979754
DQ118434
DQ118442
DQ118443
EU979742
DQ118436
DQ118437
EU979747
EU979746
EU979738
EU979737
EU979740
EU979751
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EU979783
EU979745
DQ118438
EU979753
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DQ118444
DQ118448
EU979764
EU979763
DQ118450
EU979765
EU979768
DQ118451
EU979769
EU979766
EU979767
EU979749
EU979743
EU979838
EU979836
DQ118511
EU979837
DQ118477*
EU979821
EU979820
EU979819
DQ118479
EU979851
EU979817
EU979815
EU979818
EU979816
DQ118508
DQ118510
DQ118509
EU979834
DQ118484
DQ118483
EU979799
DQ118485
EU979801
DQ118499
DQ118500
DQ118493
EU979822
DQ118512
EU979835
DQ118507
DQ118506
EU979814
DQ118478
DQ118486
DQ118487
EU979802
DQ118480
DQ118481
EU979807
EU979806
EU979798
EU979797
EU979800
EU979811
EU979804
EU979842
EU979843
EU979805
DQ118482
EU979813
EU979850
DQ118488
DQ118492*
EU979824
EU979823
DQ118494
EU979825
EU979828
DQ118495
EU979829
EU979826
EU979827
EU979809
EU979803
EU979854
EU979856
EU979857
EU979858
DQ458264*
EU979916
EU979917
EU979919
EU979918
EU979911
EU979939
EU979940
EU979942
EU979941
EU979855
EU979852
EU979853
EU979874
EU979925
EU979924
EU979922
EU979926
EU979927
EU979864
EU979863
EU979876
EU979875
EU979860
EU979859
EU979861
EU979862
EU979915
EU979914
EU979906
EU979907
EU979908
EU979912
EU979913
EU979899
EU979898
EU979892
EU979891
EU979923
EU979901
EU979921
EU979893
EU979894
EU979896
EU979897
EU979909
EU979910
EU979905
DQ458263*
EU979880
EU979879
EU979878
EU979877
EU979883
EU979885
EU979884
EU979881
EU979882
EU979903
EU979920
EU979945
EU979947
EU979948
EU979949
DQ458279*
EU980007
EU980008
EU980010
EU980009
EU980002
EU980030
EU980031
EU980033
EU980032
EU979946
EU979943
EU979944
EU979965
EU980016
EU980015
EU980013
EU980017
EU980018
EU979955
EU979954
EU979967
EU979966
EU979951
EU979950
EU979952
EU979953
EU980006
EU980005
EU979997
EU979998
EU979999
EU980003
EU980004
EU979990
EU979989
EU979983
EU979982
EU980014
EU979992
EU980012
EU979984
EU979985
EU979987
EU979988
EU980000
EU980001
EU979996
DQ458278*
EU979971
EU979970
EU979969
EU979968
EU979974
EU979976
EU979975
EU979972
EU979973
EU979994
EU980011
genus Chaparana
C. cf. delacouri 1
C. cf. delacouri 2
C. cf. delacouri 3
Lao PDR: Kaleum Dist, Xe Kong Prov (47)
Vietnam: Con Cuong Dist, Nghe An (48)
Lao PDR: Phongsaly Dist, Phongsaly Prov (45)
920-1000
700
600-800
FMNH258619
FMNH255623
FMNH258628
EU979666
EU979664
EU979662
EU979752
EU979750
EU979748
EU979812
EU979810
EU979808
EU979904
EU979900
EU979902
EU979995
EU979991
EU979993
63
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Table 2 (continued)
Species
C.
C.
C.
C.
C.
C.
C.
C.
quadranus 1
quadranus 2
quadranus 3
quadranus 4
quadranus 5
unculuanus 1
unculuanus 2
aenea
Locality
Altitude (m)
Reference
specimens No.
GenBank Accession Nos. mtDNA
Nuclear DNA
12S-1
12S-2
16S
Rhodopsin
Tyrosinase
China: Sangzhi, Hunan (49)
China: Maowen Co., Sichuan (50)
China: An Co., Sichuan (51)
China: Guanyang,Wushan Co., Chongqing (52)
China: Guanyang,Wushan Co., Chongqing (52)
China: Jingdong Co., Yunnan (53)
China: Jingdong Co., Yunnan (53)
Vietnam: Sa Pa vicinity, Lao Cai (54)
no data
1370
1200
1750
1750
2150
2150
1400
KIZ-JJ7
SCUM20045195CJ
SCUM20030031GP
YNU-HU20025111
YNU-HU20025113
YNU-HU2002502601
YNU-HU2002502702
ROM37984
EU979696
EU979695
EU979694
EU979697
EU979698
EU979699
EU979700
EU979693
EU979772
DQ118470
EU979771
DQ118471
EU979773
DQ118446
DQ118447
EU979770
EU979832
DQ118514
EU979831
DQ118515
EU979833
DQ118490*
DQ118491
EU979830
EU979888
EU979887
EU979886
EU979890
EU979889
DQ458262*
EU979865
EU979895
EU979979
EU979978
EU979977
EU979981
EU979980
DQ458277*
EU979956
EU979986
China:
China:
China:
China:
China:
China:
China:
China:
China:
Nyingchi, Xizang (55)
Damxung Co., Xizang (56)
Damxung Co., Xizang (56)
Zöiga, Sichuan (57)
Zöiga, Sichuan (57)
Zöiga, Sichuan (57)
Zhongdian Co., Yunnan (58)
Zhongdian Co., Yunnan (58)
Zhongdian Co., Yunnan (58)
3436
4200
4200
3446
3446
3500
3284
3280
3280
CIB-XM1096
KIZ-RD005
KIZ-RD015
SCUM045853WD
SCUM045860WD
SCUM045856WD
SCUM045886WD
SCUM045881WD
SCUM045887WD
EU979722
EU979723
EU979721
EU979718
EU979719
EU979720
EU979716
EU979715
EU979717
DQ118454
DQ118452
DQ118453
DQ118460
EU979780
EU979781
DQ118459
EU979779
DQ118457
DQ118498*
DQ118496
DQ118497
DQ118504
EU979840
EU979841
DQ118503
EU979839
DQ118501
DQ458261*
EU979873
EU979872
EU979869
EU979870
EU979871
EU979867
EU979866
EU979868
DQ458276*
EU979964
EU979963
EU979960
EU979961
EU979962
EU979958
EU979957
EU979959
Outgroup
genus Hoplobatrachus
H. rugulosus 1
China: Xishuangbanna, Yunnan
H. rugulosus 2
Vietnam: Tam Dao, Vinh Phu Prov.
H. occipitalis 1
Mauritania: Nouakchott District
H. occipitalis 2
Tanzania: Mwanza Region
500
900
280
1145
SCUM0437941
MVZ224079
MVZ235754
MVZ234146
EU979726
EU979727
EU979728
EU979729
DQ458237*
EU979784
EU979785
EU979786
DQ458251*
EU979844
EU979845
EU979846
DQ458258*
EU979933
EU979934
EU979935
DQ458273*
EU980024
EU980025
EU980026
genus Limnonectes
L. fragilis 1
L. fragilis 2
L. fujianensis
L. kuhlii 1
L. kuhlii 2
L. shompenorum
L. grunniens
L. modestus
China: Mt.Limu, Hainan
China: Wuzhi Shan, Hainan
China: Sangang,Wuyi, Fujian
China: Jinping Co., Yunnan
Vietnam: Tam Dao, Vinh Phu Prov.
Indonesia: Bengkulu Prov.
Indonesia: Sulawesi Tenggara Prov.
Indonesia: Sulawesi Tenggara Prov.
600
no data
920
900
900
30
94
76
SCUM-H008
MVZ236675
YNU-HU20026017
KIZ-RD05DT1
MVZ223972
MVZ239426
MVZ239149
MVZ239413
EU979640
EU979641
EU979639
EU979637
EU979638
EU979642
EU979643
EU979644
DQ458235*
EU979733
DQ118473
DQ118475
EU979732
EU979734
EU979735
EU979736
DQ458249*
EU979793
DQ118517*
DQ118519*
EU979792
EU979794
EU979795
EU979796
DQ458270*
EU979929
DQ458260*
DQ458269*
EU979928
EU979931
EU979932
EU979930
DQ458285*
EU980020
DQ458275*
DQ458284*
EU980019
EU980022
EU980023
EU980021
genus Fejervarya
F. limnocharis 2
F. limnocharis 3
F. cancrivora
Vietnam: Tam Dao, Vinh Phu Prov.
China: Sanya, Hainan
Indonesia: Sulawesi Selatan Prov.
950
10
315
MVZ226347
SCUM-H003CJ
MVZ239403
EU979730
EU979731
No data
EU979787
EU979788
EU979789
EU979847
EU979848
EU979849
EU979936
EU979937
EU979938
EU980027
EU980028
EU980029
genus Nanorana
N. parkeri 1
N. parkeri 2
N. parkeri 3
N. pleskei 1
N. pleskei 2
N. pleskei 3
N. ventripunctata 1
N. ventripunctata 2
N. ventripunctata 3
and the ROM (Royal Ontario Museum, Toronto, Canada). Most of
the identifications for these specimens took place in the field and
are largely based on either female and/or juveniles and are considered to be very tentative (Table 2).
A total of 15 specimens from 10 species were designated as outgroup taxa based on the results of Frost et al. (2006) and Roelants
et al. (2004). All sampled species and locations are given in Table 2
and Fig. 3.
2.2. Extraction, amplification, and sequencing
Muscle or liver tissue samples were stored in 95% or 100%
ethanol, or frozen at 80 °C. DNA was extracted using the standard 3-step phenol/chloroform extractions (Sambrook et al.,
1989). Amplification was performed in a 25–50 ll volume reaction with the following procedures for both the rhodopsin and
tyrosinase genes: initial denaturation step with 4 min at 94 °C;
35 cycles of denaturation 1 min at 94 °C, annealing for 1 min
at 51 °C (rhodopsin) and 49 °C (tyrosinase), extension for 1 min
at 72 °C; final extension for 10 min at 72 °C. The same PCR protocols were used for the mtDNA genes except for annealing at
55 °C. Primer sequences are given in Table 3. Purified PCR products were directly sequenced with an ABI automated DNA sequencer in both directions for each species. They were
submitted for a BLAST search in GenBank to ensure the target
sequences had been amplified.
2.3. Data analysis
Nucleotide sequences were aligned for each gene using Clustal
X 1.81 (Thompson et al., 1997) with default parameters, and then
verified by eye using MEGA 3.1 (Kumar et al., 2004). We plotted
the transition to transversion ratios as determined by DAMBE
(Xia, 2000) against the sequence divergence using F84 to test our
data sets for saturation. The five sequence data sets showed no saturation effects, and thus all substitutions in these genes were used
for further analyses. These plots are available from the authors
upon request.
Because mtDNA genes do not freely segregate and recombine,
and are virtually inherited as one linkage group, the three mtDNA
gene segments (12S rRNA-1, -2, 16S rRNA) were concatenated into
a single partition and analyzed simultaneously. Two hypervariable
regions, with a total of 110 bp, were excluded from further analysis
due to ambiguity of alignment. Such exclusion increases the reliability of the phylogenetic analysis (Swofford et al., 1996).
Phylogenetic history was hypothesized using Bayesian inference (BI) as implemented in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) incorporating both nonpartitioned and partitioned
strategies. Each data set followed its own best-fit model in the
analysis of the combined data set. The data were partitioned by
gene and by codon position. The best-fitting nucleotide substitution models for each of the seven partitions and the unpartitioned
data set were selected by using the Akaike Information Criterion as
64
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Fig. 3. Sampling sites of all ingroup taxa used in this study. Site names and coordinates are listed in Table 2. Two groups (I, II) were mapped, including Clade A (green circle), B
(blue circle), C (red circle), D (yellow circle), and E (purple circle). Map is from Google Earth, Copyright 2008. All rights reserved. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this paper.)
Table 3
Primers used in PCR and sequencing of dicroglossid frogs in the tribe Paini.
Locus
Primer
Primer sequence
Size (bp)
Cited source
12S rRNA-1
FS01
R16
L02510
H03063
L1091
H1478
Rhod1A
Rhod1D
Tyr1G
Tyr1B
50 -AAC GCT AAG ATG AAC CCT AAA AAG TTC T-30
50 -ATA GTG GGG TAT CTA ATC CCA GTT TGT TTT-30
50 -CGC CTG TTT ATC AAA AAC AT-30
50 -CTC CGG TTT GAA CTC AGA TC-30
50 -AAA AAG CTT CAA ACT GGG ATT AGA TAC CCC ACT AT-30
50 -TGA CTG CAG AGG GTG ACG GGC GGT GTG T-30
50 -ACC ATG AAC GGA ACA GAA GGY CC-30
50 -GTA GCG AAG AAR CCT TCA AMG TA-30
50 -TGC TGG GCR TCT CTC CAR TCC CA-30
50 -AGG TCC TCY TRA GGA AGG AAT G-30
411
Sumida and Ogata (1998)
Sumida et al. (2000a,b)
kocher et al. (1989)
12S rRNA-2
16S rRNA
Exon 1 of rhodopsin gene
Exon 1 of tyrosinase gene
implemented in Modeltest 3.7 (Posada and Crandall, 1998). The
model GTR + I + G was selected for the mtDNA sequences. The following models were selected for analysis of the nuDNA data:
TrN + I + G for the first codon position in the rhodopsin partition;
F81 + I for the second codon position in the rhodopsin partition;
HKY + G for the third codon position in the rhodopsin partition;
TVM + I + G for the first codon position in the tyrosinase partition;
TVM + G for the second codon position in the tyrosinase partition;
and GTR + G for the third codon position in the tyrosinase partition.
BI involved 300 million generations with sampling trees every
100th generation. The first 8000 trees were discarded as burn-in,
i.e., before the log-likelihood scores stabilized. Two independent
runs with four Markov chains were performed. A 50% majority rule
consensus of the sampled trees was constructed to calculate the
Bayesian posterior probabilities (BPP) of the tree nodes.
Maximum parsimony (MP) analyses were implemented using
PAUP 4.0b10a (Swofford, 2003). For the final combined analyses,
heuristic MP searches were executed for 1000 replicates with all
characters treated as unordered and equally weighted. Treesearching used tree bisection reconnection (TBR) branch swapping.
A heuristic MP search proved to be impossible for the separated
data sets, i.e., mtDNA and nuDNA alone, owing to computational
400
550
316
Bossuyt and Milinkovitch (2000)
532
limitations. To assess nodal reliabilities using bootstrap analysis
(BBP), a ‘‘fast” stepwise-addition bootstrap analysis (BBP) was conducted using 1000 replicates. The BBPs were subsequently mapped
on the 50% majority rule BI tree.
For the nodes in the total evidence dataset, we also conducted a
partitioned Bremer support analysis (PBS; Bremer, 1988, 1994)
using TreeRot.v2 (Sorenson, 1999). This measured the relative contribution of each gene class (mtDNA and nuDNA) towards the total
Bremer support (Fig. 6b).
3. Results
3.1. Sequence characteristics
All 99 specimens were sequenced for the 12S rRNA-2 and 16S
rRNA fragments. Sequences of the 12S rRNA-1 fragment were
incomplete for four specimens (see Table 2) although 422 bp of
nucleotide positions were obtained for most specimens. For 12S
rRNA-2, one region with 28 bp was excluded from analysis due
to ambiguous alignment; the remaining 352 bp were used for analyses. The alignment of 16S gene sequences produced fragments
601 bp in length, of which one region with 82 bp was excluded
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
from further analysis due to ambiguous alignment. The final matrix
of all aligned mtDNA data comprised 1292 nucleotide positions of
which 528 were variable and 469 were potentially phylogenetically informative.
For the two nuclear fragments, all 99 specimens were successfully sequenced (Table 2). All sequences were translated into amino acids without stop codons and the alignment did not contain
indels. A total of 521 bp of tyrosinase and 315 bp of rhodopsin
were resolved. Among these combined sequences, 243 sites were
variable and 204 sites were potentially phylogenetically informative. All sequences were deposited in GenBank (Table 2).
3.2. Phylogenetic analysis of the nuDNA
We analyzed the nuclear gene data using a total evidence approach. All nuDNA sequence data were combined unconditionally.
The 50% majority rule consensus tree (partitioned strategies) inferred from BI resolved five Clades (A–E) (Fig. 4). No significant differences occurred between the topology of the nonpartitioned and
partitioned strategies. All analyses differed only in the relationships
among Clades A, B, and D, which were weakly supported (BPP < 90,
BBP < 50). In total, 47 nodes received strong support (BPP > 90),
among which 31 received good BBPs (>80) and 15 had poor BBPs
(<50).
The monophyly of the tribe Paini, comprising those taxa from
genera Paa, Nanorana, and Chaparana, was strongly supported
(99% BPP and 96% BBP). Clade A included Paa yei (34), P. exilispinosa
(14, 15), P. spinosa (25, 27, and 28), P. jiulongensis (16), P. shini
(23, 24), P. verrucospinosa (33), P. robertingeri (22), P. boulengeri
(3–10), and two unidentified species of Paa (30, 46). Within Clade
A, five subclades (A1–A5) were recovered but with unresolved relationships. Clade B contained two subclades (B1, B2). Subclade B1
only included one population of C. delacouri (47). Another two populations of C. delacouri (45, 48), one population of P. verrucospinosa
(32), two population of P. spinosa (26, 29), and one unidentified
species of Paa (45) constituted subclade B2. Clade C included four
subclades (C1–C4). Paa yunnanensis (35–43), P. liui (18), and P.
bourreti (44) constituted subclade C1. Subclade C2 contained C. aenea (54) and C. unculuanus (53). Subclades C3 and C4 only included
C. quadranus (49–52), and P. taihangnica (31), respectively. Clade D
consisted of two specimens of P. liebigii (17) only. Clade E contained three species of Nanorana sensu stricto, including N. ventripunctata (58), N. pleskei (57), and N. parkeri (55, 56) as subclade E2,
and other five species of Paa, including P. conaensis (13), P. maculosa (19), P. medogensis (20, 21), P. arnoldi (1, 2), and P. chayuensis
(11, 12) as subclade E1.
3.3. Phylogenetic analysis of the mtDNA
Fig. 5 shows the 50% majority rule consensus tree from the BI
analysis. In total, 72 nodes received strong support (BPP P 90),
among which, 46 received strong BBPs (P90), 13 with moderate
support (90 > BBP P 70) and 13 with poor BBPs (<50).
Congruent with the results of the nuDNA data, monophyly of the
tribe Paini was supported with a high BPP (100) and moderate BBP
(86). Whereas analysis of the nuDNA data resolved five Clades, the
mtDNA data yielded a tree with two distinct groups (I, II). Group I,
with a high BPP (100) and moderate BBP (79), included those taxa resolved in Clades A and B with the nuDNA data. The monophyly of
Clade A recovered with the nuDNA data was not recovered by the
mtDNA analyses. The relationships of subclades A1–A5 and Clade
B remained ambiguous. Group II included those taxa related to
Clades C–E based on the nuDNA sequences. This group received high
nodal support (BPP = 100, BBP = 99). The monophyly of Clade E
recovered with nuDNA was not supported by mtDNA (Fig. 5), but
mtDNA still recovered two subclades (Fig. 5: E1 and E2).
65
3.4. Phylogenetic analysis of the combined mitochondrial and nuclear
genes (mtDNA + nuDNA)
When mapped geographically, the clades contained neighboring localities (Fig. 3). Names were applied to these clades (Table
4) with fair certainty when the samples included the type localities
of species, which was true for most taxa.
Monophyly of the tribe Paini was supported by the combined
data with strong nodal support (Fig. 6). The total evidence analysis
resulted in a tree having a topology similar to that of our mtDNA
data alone and major groups I and II were obtained with greater
support (I: BPP = 99, BBP = 95; II: BPP = 99, BBP = 100). The five major Clades recovered with the nuDNA data were also supported by
the combined data (Fig. 6). Clades A and B constituted Group I.
However, the branching sequence for taxa within Clade A still remained enigmatic. Within Group II, Clades C and E were supported,
but the relationships of Clade D (P. liebigii) remained unclear.
The partitioned Bremer support analysis indicated that, of the
83 resolved nodes on the concatenated MP tree (Fig. 6), 50 received
significant support (PBS > 4) from the mtDNA data alone. In particular, substantial support occurred in the terminal taxa (Fig. 6). Substantial Bremer support for 30 nodes was attributable to the
nuDNA data, including four nodes with conflicting PBS values compared to the mtDNA data. Overall, the nuDNA data tended to resolve more basal nodes within all groups.
4. Discussion
4.1. Trees
Our results presently provide the most comprehensive phylogeny for the endemic Asian tribe Paini. The study encompasses a
substantial portion of the range of the majority of recognized species (25 species; Table 2) and it includes intraspecific samplings for
several species. Analyses of these data recover major clades in the
tribe Paini, and many terminal relationships are resolved.
Analysis of the mtDNA (Fig. 5) recovered two major groups (I
and II) within the tribe Paini as also attained by Jiang et al.
(2005: Fig. 2b). Compared with Jiang et al. (2005), we added eight
species to the phylogeny and, importantly, used a large number of
samples for those species that are suspected to include cryptic
species.
Herein, we used both mtDNA and two nuclear genes to infer the
phylogenetic relationships of the Asian spiny frogs. When evaluated separately, the rhodopsin and tyrosinase gene trees (not
shown) resolved fewer branches than the combined nuDNA data.
This result owed to limited sequence divergence. Nonetheless,
our results indicated that these two nuclear genes were informative at the base of the tree, especially in recovering the five major
clades (A–E) in the tribe Paini (Fig. 4).
Analysis of the final concatenated data (mtDNA + nuDNA;
Fig. 6) did not remarkably improve resolution and confidence, as
might be expected, but rather resulted in loss of resolution and/
or support. This was particularly apparent in basal branching relationships. The total evidence data produced almost identical
results to the mtDNA alone, particularly in recovering two groups.
The mtDNA genes examined herein evolved at a much faster rate
than the nuDNA and consequently the former data provided a
greater number of informative characters. The difference in number of characters could have resulted in the mtDNA overpowering
the signal from the nuDNA when the two data sets were combined.
For example, nuDNA strongly supported the monophyly of Clade A
(BPP = 100, BBP = 96) with five subclades (Fig. 4), but the monophyly was not recovered by the mtDNA alone. The partitioned Bremer support analyses (Fig. 6b) indicated that the mtDNA data had
conflicting PBS values compared to the nuDNA data for Clade A
66
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Fig. 4. Phylogenetic hypothesis derived from a Bayesian inference analysis of nuclear rhodopsin and tyrosinase DNA sequence data. Numbers near branches are Bayesian
posterior probabilities (P90) and bootstrap proportions from a maximum parsimony analysis (P70). Vertical bars indicate clade designation. Bold letters and numbers at the
nodes refer to the clades recovered in the nuDNA analysis. Species names and localities are shown on the tips of the tree.
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
67
Fig. 5. Phylogenetic hypothesis derived from a Bayesian inference analysis of mtDNA sequence data. Numbers near branches are Bayesian posterior probabilities (P90) and
bootstrap proportions from a maximum parsimony analysis (P70). Vertical bars indicate clade designation. Bold letters and numbers at the nodes refer to the clades
recovered in the nuDNA analysis. Species names and localities are shown on the tips of the tree.
(1/11). Clearly, Clade A based on the final concatenated data
(Fig. 6a) had a lower support index of BPP = 99 and BBP = 87 compared to the nuclear data alone. Conflicting PBS values between
mtDNA and nuDNA were also present in the basal nodes of Clade
C. The clade is supported as being a monophyletic group in all analyses with high BPP support only. The PBS analysis indicated that
most of the support may be attributed to the nuDNA gene sequence data, though still with low resolution (Fig. 6b: 1/3).
68
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Fig. 6. The phylogenetic hypothesis derived from the combined data including mtDNA and nuclear sequences. (a) The 50% majority rule consensus from a Bayesian inference
analysis. Numbers near branches are Bayesian posterior probabilities (P90) and bootstrap proportions from a maximum parsimony analysis (P70). (b) The strict consensus
tree from the maximum parsimony analysis. Numbers above the lines or beside the nodes are partitioned Bremer support (mtDNA/nuDNA). Vertical bars indicate species and
clade designation. Species names and localities are shown on the tips of the tree.
The trees obtained from the analyses of mtDNA and nuDNA sequence data were usually congruent. In areas where the two genomes disagreed, the conflicting nodes either were not resolved or
they received low bootstrap support, reflecting a dearth of data.
For example, the status of Clade D within the tribe Paini remained
unresolved. Additional nuDNA sequences should be added to better test these hypotheses.
The hypothesis based on the total evidence analysis (Fig. 6) forms
the best current estimate of the phylogenetic relationships within
the tribe Paini. This tree will be used for the discussions that follow.
4.2. Monophyly of the tribe Paini
All analyses (Figs. 4–6) supported the monophyly of the nominal
tribe Paini with strong nodal support. Partitioned Bremer support
analyses indicated that the nuDNA data contributed 81% of the support and the mtDNA data offered 19% toward the concatenated parsimony tree (Fig. 6b: 13/3). These findings were concordant with the
results of Che et al. (2007), Chen et al. (2005), and Roelants et al.
(2004), but conflicted with the tree of Frost et al. (2006). The most
likely explanation for the difference is taxon sampling. Frost et al.
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
Table 4
Indicated nomendatorial adjustments for dicroglossid frogs in the tribe Paini
evaluated in this study.
Major
clade
Subclade
Subclade
Node
Genus
Species
I
A
A1
A1a
Quasipaa
A2
A1b
A2a
Quasipaa
Quasipaa
A2b
A3
A4
A5
Bl
Quasipaa
Quasipaa
Quasipaa
Quasipaa
Quasipaa
B2
Quasipaa
C1
Nanorana
spinosa?
exilispinosa?
jiulongensis
verrucospinosa?
sp. nov.?
boulengeri
yei
shini
sp. nov.
delacouri? sp.
nov. ?
verrucospinosa?
sp. nov.?
yunnanensis
B
II
C
C2
C2a
C2b
El
E1a
E1b
E1c
E1d
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
Nanorana
E2
E2a
E2b
E2c
Nanorana
Nanorana
Nanorana
C3
C4
D
E
Synonyms
robertingeri
liui,
bourreti
aenea
unculuanus
quadranus
taihangnica
liebigii
conaensis
medogensis
maculosa
chayuensis?
arnoldi?
ventripunctata
pleskei
parkeri
(2006) evaluated all living groups of amphibians and most relationships were well resolved, particularly at higher taxonomic levels.
However, their study included only three species in the tribe Paini.
The large sampling of outgroup taxa may have caused a bias, possibly
involving either long-branch attraction (Felsenstein, 1978) or longbranch repulsion (Siddall and Whiting, 1999). Furthermore, the difference between our trees and those of Frost et al. (2006) might owe
to their use of incomplete sequence data for several taxa. The absence of nuDNA data for N. pleskei in Frost et al. (2006) precluded
our re-evaluating their paraphyly of the tribe Paini.
Using partial sequences of 12S and 16S rRNA, Jiang et al. (2005)
obtained a united tribe Paini in their phenetic, neighbor-joining
analysis, but this arrangement received no nodal support. Consistent with Frost et al. (2006, 138), our revaluation of Jiang et al.’s
(2005) data confirmed that the monophyly of the group was
ambiguous in their MP (Fig. 2b) and ML analyses. Our enlarged
mtDNA data set supported the monophyly of the tribe (Fig. 5),
and stronger support was obtained from the nuDNA and combined
data (Figs. 4 and 6). Jiang et al. (2005) excluded all indel, missing
and ambiguous sites, but in doing so likely deleted some useful
characters (Giribet and Wheeler, 1999). The choice and number
of outgroup taxa may also be contributing to the discrepancies between the results of Jiang et al. (2005) and this study because outgroup choice is critical (Cameron et al., 2004; Milinkovitch and
Lyons-Weiler, 1998; Tarrio et al., 2000; Ware et al., 2008).
4.3. Geographic correspondence of clades
4.3.1. Alternative hypotheses
In the following discussion, the primary subclades are denoted
by a letter. The alphabetical sequence is derived from the total evidence tree (Fig. 6). In general, there is a high correspondence between DNA variation and location of samples (Figs. 3 and 6). On
one hand, this is not surprising. These frogs occur in montane
streams and most of the sample sites occur on isolated mountains.
Thus, it is anticipated that most populations can be identified
69
genetically. On the other hand, because the montane streams are
often part of the same drainage system, we could expect possible
gene flow. The general absence of gene flow suggests that each isolated mountain region serves as a sky island for these frogs. If true,
then the total number of species could be grossly underestimated.
The labeled nodes on the cladogram of Fig. 6 correspond to
grouped localities depicted in Fig. 3. Group I largely consists of
species that occur east of Hengduan Mountain Ranges, including
southern China, Vietnam, and Laos, and from relatively low elevations. Group II containes samples distributed in low to high elevations predominantly in Qinghai-Tibetan Plateau and Hengduan
Mountain Ranges. The sister groups to the Paini (genera Hoplobatrachus, Limnonectes, and Fejervarya) occur mostly at low elevations (Table 2), suggesting that high elevation appears to be the
derived ecological state for Paini. For the purpose of this discussion, we have defined low elevations as those occurring below
about 1500 m, moderate elevations as 1500–2500 m, and high elevations as above 2500 m. These are generalized ranges of elevations for species and there are exceptions to distributional
patterns, especially among the deep river valleys, particularly in
the Hengduan Mountains of western China where opportunities
for short distance vertical migration are possible (Yang, 1993).
For example, P. arnoldi could be found from 1000 to 2000 m in
western Yunnan (Table 2 and Appendix A).
4.3.2. Major Clade I
Group I contains two major lineages, Clade A and Clade B
(Fig. 6), which were also recovered by analyses of the nuDNA data
only (Fig. 4). Clade A consists of low elevation frogs (mostly
<1500 m; Table 2 and Appendix A) occurring throughout China
southward from Sichuan province in the west to Henan Province
in the east extending into south-central Vietnam and southern
Laos (Fig. 3). Among the five major Clades, Clade A has the widest
distribution. Subclade A1 (Fig. 6) includes samplings from five
localities and could be divided into two populations (A1a, A1b).
With strong nodal support, the phylogenetic analysis clearly unites
these five localities. Localities 27 and 14 form a sister group relationship, and yet locality 14 is geographically near locality 16.
Subclade A2a contains two populations, one from southern
Yunnan province, China (25) near the border of Vietnam and the
other from isolated Tam Dao Mountain in northern Vietnam
(7, 33). These two populations are genetically well differentiated
in their mtDNA only (Fig. 5). An assessment of gene flow within
this assemblage using highly variable nuDNA markers is essential
for recognizing subdivisions deserving of species recognition.
North and east of these two sample sites, there is a widespread
assemblage of slightly genetically differentiated populations, Subclade A2b (3–10, and 22).
Subclade A3, along with A4 and A5, are sister group lineages of the
monophyletic subclades A1 and A2. Resolution of relationships
among subclades A3–A5 differs among the analyses of mtDNA, nuDNA and the total evidence data (Figs. 4–6). The nodes involved in the
various associations are not supported and the relationships among
these three clades could be considered to be unresolved. All three lineages (A3–A5) are very distinct genetically and those that include
two sampling locations (A4, A5) show little differentiation within
them.
Clade B forms the sister group of Clade A. Subclade B1 (47) is a
population in southern Laos. It occurs near another specimen from
locality 46 in subclade A5. The two lineages exhibit significant
mtDNA and nuDNA divergence, and undoubtedly constitute two
different species. Subclade B2 contains a suite of localities ranging
from southern Yunnan province, China southward into extreme
northern Laos and northern Vietnam (Fig. 3). There is some mixing
of haplotypes between Laos and Vietnam suggesting the occurrence
of gene flow.
70
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
4.3.3. Major Clade II
This group contains three major Clades: C–E (Fig. 6). The phylogenetic associations of these Clades vary depending on the data
partition.
Most species of Clade C, a mix of taxa occurring in low to high
elevations, occur in the Qinghai-Tibetan Plateau and Hengduan
Mountain Ranges. There are four major subclades: C1–C4. The
monophyly of Clade C only received substantial BPP support (94,
95, and 99) in all analyses (Figs. 4–6). Subclade C1, a moderatehigh elevation group (Table 2 and Appendix A), is distributed from
southern Sichuan province southward through Yunnan province
into northern Vietnam. Given the topographic complexities in this
region, it is possible that more detailed collecting might reveal substantial substructuring. Subclade C2 contains two highly differentiated populations that occur at moderate elevations (Table 2;
Appendix A). One population (53) occurs in southern Yunnan province, China and the other (54) in Sa Pa, extreme northern Vietnam.
The high level of divergence in both nuDNA and mtDNA supports
the division of the two species. Interestingly, unlike the Vietnamese specimens (Dubois and Ohler, 2005), those from southern
Yunnan lack nuptial spines in males (Liu and Hu, 1961; Yang,
1991).
Monophyletic subclade C3 occurs in central China at low to
moderate elevations. The clade has a wide distribution mostly
occurring along the Qinling, Taihang, and Daba mountains. The
group exhibits little genetic differentiation among the sample sites.
Finally, subclade C4, located at a low elevation in east-central
China, consists of a single sampling site. These frogs (C3, C4), which
are also distinctly differentiated genetically, also lack nuptial
spines.
Clade D, as previously noted, has ambiguous relationships with
respect to clades C and E. Further study will be needed to clarify
the relationships of these groups. The isolated population (17)
from the southern Himalayas occurs in Yakong County, Xizang
province at a high elevation and it is very distinctive genetically.
Finally, Clade E, a moderate to high altitude group, was recovered by an analysis of nuDNA only (Fig. 4). The mtDNA data
(Fig. 5) did not resolve this association. All of the samples in this
Clade occur on the Qinghai-Xizang (Tibetan) Plateau and in the
Hengduan Mountains. Within this group, two major subclades occur. Subclade E1 mostly occurs from the slopes of the southern
Himalayas eastward to the Hengduan Mountains of western Yunnan province, China. Subclade E2 is distributed on the hinterland
of Xizang region with the highest elevation (mostly above
3000 m) among all species in the tribe Paini. This group is most often referred to the genus Nanorana.
4.4. Names and geography: taxonomic implications
4.4.1. Genera allocation
Undoubtedly, the tribe Paini is monophyletic. The species
among the nominal tribe Paini sensu Dubois have been suggested
to comprise two to six genera (Liu and Hu, 1961; Fei, 1999; Fei
et al., 2005; Frost et al., 2006; Dubois, 1987 ‘‘1986”, 1992; Dubois,
2005; Ohler and Dubois, 2006). Presently, a consensus for the taxonomy among members of this tribe Paini is wanting.
Ohler and Dubois (2006) proposed six generic divisions including
Allopaa, Chaparana, Chrysopaa, Gynandropaa, Nanorana, and Quasipaa (Fig. 2a, Table 1), among which Allopaa and Chrysopaa were
monotypic genera. This taxonomic arrangement strongly conflicts
with our analysis. The designation of genera in the absence of a phylogeny, whether based on morphology, molecular genetics or both,
can result in taxonomic instability, if not chaos. Our phylogenetic
hypothesis does not support the validity of two genera, Chaparana,
and Gynandropaa. The taxa allied to Gynandropaa, including P. liui,
P. yunnanensis, P. bourreti, and C. quadranus, did not constitute a
monophyletic group based on any partition of our data or the combined data (Figs. 4–6). Furthermore, those species of Chaparana
including C. aenea, C. unculuanus, P. taihangnica, P. arnoldi, P. conaensis, P. medogensis, P. maculosa, P. chayuensis, P. arnoldi, and P. liebigii,
were not resolved as a monophyletic group.
Nanorana (E2) sensu stricto is the only clearly supported monophyletic group of Ohler and Dubois (2006). The three species of Nanorana
represent a classic example of adaptive evolution in high elevations
(see Table 2 and Appendix A). For example, these species have secondarily reduced vocal sacs and columella (Hu et al., 1985; Zhao and Yang,
1997). Our molecular analyses supported the opinion of Boulenger
(1920) that the high elevation species of Nanorana represented dwarfed, degraded forms derived from lower elevation ‘‘Paa”.
Taxonomy should reflect evolutionary history and only monophyletic groups should be recognized as taxonomic ranks. A taxonomic arrangement based on phylogeny could unite all species in
Group I into a single genus. The oldest available name for Group
I is Quasipaa, as noted by Jiang et al. (2005). Similarly, all species
in Group II should be referred to the genus Nanorana as suggested
by Chen et al. (2005) and Frost et al. (2006) (Table 4).
4.4.2. Species designation
Given the taxonomic confusion that often accompanies the
identification of species in this tribe; we mapped the resolved
clades before assigning names (Figs. 3 and 6). This served two
goals: first, it allowed for the detection of any widespread species
and second it allowed for the visualization of the geographic limits
of each clade. Some, but not all samples were collected from type
localities. Where type localities were sampled, it is possible to
comment on the validity of some species.
Paa spinosa is likely a species complex, rather than a single species. Both mitochondrial and nuclear data suggested that the populations currently under the name P. spinosa belong to at least
three independent evolutionary lineages (subclades A1a, A2a, and
B2). Unfortunately, we did not have samples from the type locality
of P. spinosa. Further study should be conducted to clarify the relationship between P. spinosa and P. exilispinosa in A1a (Fig. 6). Furthermore, we identified the samples from Fujian province, China
(A1b, 16) as P. jiulongensis. Clearly, present molecular data supported the morphological recognion that A1b is highly differentiated genetically from the remaining populations (A1a).
Recently, Hu et al. (2005) reported P. verrucospinosa from Mengsong, Yunnan, China. We used the same specimens as Hu et al.
(2005) and these specimens (32) unambiguously nested within
subclade B2. They did not cluster with samples of P. verrucospinosa
from Tam Dao, Vietnam (33) (A2a). Because it is possible that P.
verrucospinosa from Sa Pa, Vietnam, the supposed type locality (given as Chapa), is not the same species as the populations from Tam
Dao, Vietnam (designated as part of the original type series), specimens of P. verrucospinosa from Sa Pa are required to resolve this
taxonomic conundrum.
Subclade B2, including ‘‘P. spinosa”, P. verrucospinosa, P. sp., and
C. cf. delacouri from locations 26, 29, 32, 45, and 48, received strong
support. Samples from 47 were also originally identified as C. delacouri, which was highly differentiated genetically from the other
two C. delacouri in B1. Unfortunately, the three C. delacouri we sequenced were not from the type locality, Bac-Kan, Vietnam.
In the interest of nomenclatorial stability, any assignment of
species names within subclades A1a, A2a, B1, and B2 is premature
until the type localities of P. spinosa, P. exilispinosa, P. verrusospinosa, and C. delacouri can be sampled and analyzed genetically.
Moreover, combined analyses of morphological data from type
specimens are urgent to clarify the present taxonomic chaos (Table
4). Our molecular data clearly supported the subclade A5 as a new
undescribed species, which coincided with our morphological
observation (unpublished data).
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
71
Paa robertingeri nested deeply within P. boulengeri and does not
appear to be a valid species. We therefore suggest that P.
robertingeri should be synonymized into P. boulengeri. The specimens of P. boulengeri (9) and P. robertingeri (22) were collected
from their respective type localities. Both P. liui (P. muta Su and
Li, 1986) (18) and P. bourreti (43) from their respective type localities nested within P. yunnanensis (subclade C1), and they appear to
be junior synonyms of the latter (Table 4).
Given the plethora of available names in the tribe Paini, our
analyses emphasize the necessity to morphologically evaluate type
material and to collect molecular samples from type localities before proposing taxonomic changes. Informed sampling is essential
for taxonomic accuracy and stability. Our analyses indicate that
molecular tools, an important complement of morphology, are very
effective at detecting cryptic species.
ensis, was supported. This result is not surprising given that these
species were once considered to be either conspecific with, or subspecies of P. maculosa. Due to the absence of specimens from the
type locality of P. arnoldi (Pangnamdim, Nam Tamai Valley, Triangle, northern Myanmar), we could not evaluate the validity of this
species in China (Yang, 1991), which is currently recognized as a
junior synonym P. chayuensis (Fei et al., 2005).
In summary, because the presence and distribution of nuptial
spines are highly homoplastic, classifications based on these attributes do not reflect evolutionary history. This is particularly true in
the formation of species groups. Regardless, spines can serve as
informative characters for recognizing species. Further phylogenetic investigations should shed light on the evolution of spines.
4.5. The validity of utility of spinules
Karyotype variation appears to correspond to the phylogeny of
these frogs. As Figs. 4–6 reveal, P. taihangnica, C. quadranus, C.
unculuanus, P. liui, and P. yunnanensis are members of Clade C.
Within Clade C, P. yunnanensis, P. liui, and C. unculuanus, for which
karyotypic data are known, have an increased number of chromosomes (Wu and Zhao, 1984; Liu and Jiu, 1984; Tan and Wu, 1987;
Li and Hu, 1994, 1996). The karyotype of P. yunnanensis and P. liui,
consists of 64T, and that of C. unculuanus is 2n = 40. These states
represent chromosomal deviations from the standard ranid karyological formula of 2n = 26 biarmed chromosomes (Li and Hu, 1999).
Fig. 6 shows that those species with 2n = 26 including P. boulengeri,
P. chayuensis, P. exlispinosa, P. maculosa, P. jiulongensis, P. robertingeri, P. spinosa, P. taihangnica, C. quadranus, P. verrucospinosa, and
N. parkeri (Hu, 2004; Pang et al., 2002) did not constitute a monophyletic group. They exhibit the plesiomorphic karotype. In contrast, the derived state of 64T is a clear synapomorphy for P. liui
and P. yunnanensis. And it is possible that the 40 biarmed karyotype of C. unculuanus constitutes either a synapomorphic transitional state to the 64T karyotype or an autapomorphy.
Body spinules, a secondary sexual characteristic of adult males,
play an important role in the life history of these frogs and their allies. The lack of congruence between the molecular and morphological data sets indicates that some species have independently,
secondarily, lost the nuptial spines. The parallel losses create havoc
in the present classification.
Clade C is composed of C. quadranus, C. unculuanus, P. taihangnica,
P. yunnanensis, P. bourreti, and P. liui. Within this clade, C. quadranus,
C. unculuanus, and P. taihangnica lack spines, and C. aenea, P. yunnanensis, P. bourreti, and P. liui are characterized by spines forming two
patches on the chest (Dubois and Ohler, 2005; Fei et al., 2005). Our
tree strongly conflicts with morphological assessments suggested
by Ohler and Dubois (2006). For example, in their analyses, P. yunnanensis and P. bourreti were depicted as being distantly related to
C. quadranus and C. unculuanus (Fig. 2a).
The taxonomic validity of the two Chinese species, C. quadranus
and C. unculuanus, has always been controversial. Dubois (1992)
assigned these two species to genus Chaparana. Initially they were
classified as Rana quadranus and R. unculuanus by Liu and Hu
(1961), who suggested that R. quadranus is related to group Spinosae (now species of Paa) in ecological habits and body features. Dubois (1987 ‘‘1986”) once placed C. quadranus into Rana (Paa), and C.
unculuanus in Rana (Rana). However, Zhao et al. (2000) proposed
the two species should be retained in Rana due to their ambiguous
status. Although these two species were placed in Paa by Fei et al.
(1990) and Fei (1999), they were subsequently assigned to the genera Feirana and Unculuanus, respectively (Fei et al., 2005). The taxonomic disagreements reflect the fact that neither species
possesses hypertrophied forearms and keratinized spines on chest
of males, i.e., the secondary sex characters characteristic of male
Paa. Paa yei (in Group I) and P. taihangnica (in Group II) are distinct
species separated from C. quadranus (Chen and Jiang, 2002; Chen
et al., 2002). However, these species were incorrectly transferred
to Feirana by Fei et al. (2005), mainly due to the absence of hypertrophied forearms and keratinized spines.
Our phylogeny also conflicts with other species groups assignments. Paa shini (A4), along with P. boulengeri and P. robertingeri
(A2b), were placed in the P. boulengeri species group by Fei
(1999) and Fei et al. (2005) due to similar distributions of spines
(Table 1). However, our molecular phylogeny did not support this
arrangement. Similarly, monophyly was not supported for the
morphologically based P. liebigii species group, including P. liebigii
and P. conaensis (Table 1).
Unfortunately, we cannot evaluate the validity of the P. yunnanensis species group of Fei et al. (2005) because P. feae has remained
unknown since its discovery (Sclater, 1892) in Yunnan, China (Yang
and Wu, personal communication). Presently, only the P. maculosa
species group, including P. chayuensis, P. maculosa, and P. medong-
4.6. Karyotypic evolution
4.7. Implications for conservation
Most species of frogs in the Paini and their tadpoles live in swift,
rocky streams in forested areas. The specific habitat requirements
suggest that populations are likely to have high genetic divergences because the species are probably poor overland dispersers.
Given the extensive genetic differentiation among sampling sites,
this prediction appears to be reasonable. The recognition of subclades A1a, A2a, A5, B1, and B2 as separate evolutionary lineages
should be further studied. It is important to analyze type material
morphologically and give exact species designations for the five
subclades. Individual cryptic species have smaller ranges than
the entire group. Consequently, individual species have greater
vulnerability to extinction. Biodiversity inventories of frogs based
on morphology alone will not be sufficient to capture the extent
of variability that conservation requires.
Acknowledgments
We would like to acknowledge G.-f. Wu, H. Zhao, S.-q. Lu,
S. Huang, D. Wang, P. Guo, X.-m. Wen, H.-x. Cai, J.-t. Li, X.-m. Zeng,
and J.-p. Jiang for assistance in fieldwork or providing tissues in
their care. We also would like to extend our sincere gratitude to
the following people and institutions for the help during loan of
material: H. Voris and A. Resetar (Field Museum of Natural History,
Chicago, USA), A. Lathrop and R. MacCulloch (Royal Ontario Museum, Canada) and C. Cicero (Museum of Vertebrate Zoology,
Berkeley, USA). A. Lathrop kindly helped to prepare Figs. 1 and 3
and provided other essential assistance and feedback. R. MacCulloch assisted with accurate French translations. Technical support
72
J. Che et al. / Molecular Phylogenetics and Evolution 50 (2009) 59–73
from workers in the lab of Y.-p. Zhang is gratefully acknowledged.
We thank Dr. K. Adler, C. Blair, A. Lathrop, R. MacCulloch,
E.-m. Zhao, and four anonymous reviewers for insightful comments on an earlier version of the manuscript. This work was supported by grants from the National Basic Research Program of
China (973 Program, 2007CB411600), the National Natural Science
Foundation of China (30621092), the Chinese Academy of Sciences,
the Natural Science Foundation of Yunnan Province, the Applied
Fundmental Research Foundation of Yunnan Province, and the Bureau of Science and Technology of Yunnan Province.
Appendix A
The elevation information of some known species in the tribe
Paini for reference in this study (Chen and Jiang, 2002; Chen
et al., 2002; Fei, 1999; Fei et al., 1990; Hu, 2004; Hu et al., 2005;
Yang, 1991).
P. boulengeri:
P. exlispinosa:
P. jiulongensis:
P. robertingeri:
P. shini:
P. spinosa:
P. verrucospinosa:
P. yei:
P. chayuensis:
P. conaensis:
P. liebigii:
P. liui:
P. maculosa:
P. medogensis:
P. taihangnica:
P. arnoldi:
P. yunnanensis:
C. quadranus:
C. unculuanus:
N. parkeri:
N. pleskei:
N. ventripunctata:
700–1900 m
500–1400 m
>800 m
650–1500 m
1000 m
500–1500 m
1400, 1600 m
424 m
1000–1540 m
2900–3400 m
2000–3500 m
2100–2650 m
1800–2600 m
1000-2100 m
728–1230 m
1000–1540 m
1500–2400 m
500–1830 m
1650–2200 m
2850–4700 m
3300–4500 m
3120–4100 m
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