Taxonomic revision of the Chinese Limnonectes (Anura, Dicroglossidae) with the description of a new species from China and Myanmar

Abstract

Phylogenetic reconstructions derived from DNA sequence data play a central role in documenting the number of species in a complex. Such analyses are pointing to the existence of many cryptic species, especially in poorly understood groups such as the genus Limnonectes, and the L. kuhlii species complex in particular. To understand the Limnonectes frogs of China, we reconstruct the major matrilineal genealogy of Limnonectes from China and Southeast Asia based on 12S rRNA, tRNAVal and 16S rRNA gene sequences. Based on new data we recognize five species of Limnonectes in China including L. bannaensis, L. fujianensis, L. fragilis, L. taylori (new record), and a new species from southern China and Myanmar. Phylogenetically, the new species is more closely related to the clade comprising L. taylori, L. megastomias, L. isanensis, L. nguyenorum, and L. jarujini from Thailand than to other Chinese species. This study supports previous findings of sympatric members of a species complex that are not each other's closest relatives.

Full text

Accepted by J. Rowley: 15 Feb. 2016; published: 22 Mar. 2016
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Taxonomic revision of the Chinese Limnonectes (Anura, Dicroglossidae)
with the description of a new species from China and Myanmar
CHATMONGKON SUWANNAPOOM
1,2, &
, ZHI-YONG YUAN
1,3, &
, JIN-MIN CHEN
4,1
, MIAN HOU
5
,
HAI-PENG ZHAO
6
, LI-JUN WANG
7
, TRUONG SON NGUYEN
8
, TRUONG Q. NGUYEN
8
,
ROBERT W. MURPHY
1,9
, JAQUELINE SULLIVAN
10
, DAVID S. MCLEOD
11
& JING CHE
1,3,12
1
State Key Laboratory of Genetic Resources and Evolution State, Kunming Institute of Zoology, Chinese Academy of Sciences, Kun-
ming 650223, Yunnan, China
2
Division of Fishery, School of Agriculture and Natural Resources, University of Phayao, Phayao, 56000, Thailand
3
Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw 05282, Myanmar
4
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming 650091, China
5
Institute of Continuing Education, Sichuan Normal University, Chengdu 610068, Sichuan, China
6
School of Life Science, Henan University, Kaifeng 475001, Henan, China
7
College of life Sciences, Hainan Normal University, Haikou 570100, Hainan, China
8
Institute of Ecology and Biological Resources Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietna m
9
Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen’s Park, Toronto M5S 2C6, Canada
10
University of Kansas Biodiversity Institute, 1345 Jayhawk Boulevard, Lawrence, Kansas 66045-7561, USA
11
Biology Department, James Madison University, 951 Carrier Dr., Harrisonburg, VA 22807 USA
12
Corresponding author. E-mail: chej@mail.kiz.ac.cn
&
equal first author
Abstract
Phylogenetic reconstructions derived from DNA sequence data play a central role in documenting the number of species
in a complex. Such analyses are pointing to the existence of many cryptic species, especially in poorly understood groups
such as the genus Limnonectes, and the L. kuhlii species complex in particular. To understand the Limnonectes frogs of
China, we reconstruct the major matrilineal genealogy of Limnonectes from China and Southeast Asia based on 12S rR-
NA, tRNA
Val
and 16S rRNA gene sequences. Based on new data we recognize five species of Limnonectes in China
including L. bannaensis, L. fujianensis, L. fragilis, L. taylori (new record), and a new species from southern China and
Myanmar. Phylogenetically, the new species is more closely related to the clade comprising L. taylori, L. megastomias, L.
isanensis, L. nguyenorum, and L. jarujini from Thailand than to other Chinese species. This study supports previous find-
ings of sympatric members of a species complex that are not each other’s closest relatives.
Key words: Indochina, Limnonectes longchuanensis sp. nov., Phylogenetics, Sympatric distribution, Yunnan
Introduction
Limnonectes Fitzinger presently contains 68 species found throughout east and southeast Asia (Frost 2015). These
anurans typically have fang-like odontoid processes on the lower jaw, male-biased size dimorphism, and great
phenotypic similarity. Limnonectes likely contains numerous undescribed cryptic species (Emerson 1996; Emerson
et al. 2000; Evans et al. 2003; McLeod 2010), especially in widespread complexes such as the L. kuhlii complex.
For example, McLeod (2010) suggested that 244 individuals he evaluated in the L. kuhlii complex likely
represented more than 22 new species. Subsequently, seven species (L. taylori, L. jarujini, L. isanensis, L. utara, L.
selatan, L. sisikdagu, and L. nguyenorum) have been described from within the L. kuhlii complex (Matsui et al.
2010b, 2014; McLeod et al. 2011, 2012, 2015). Their discoveries highlight the need for continued exploration,
especially in poorly sampled regions.

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The systematics of Limnonectes in China has been explored previously and three species are currently
recognized within the country (Ye et al. 2007; Fei et al. 2012). Limnonectes fragilis (Liu & Hu 1973) only occurs
in Hainan. The other two species (L. bannaensis Ye, Fei & Jiang and L. fujianensis Ye & Fei), both previously
placed under the nominal species L. kuhlii Tschudi, were recently elevated to full species on the basis of
morphology alone (Ye & Fei 1994; Ye et al. 2007). Limnonectes fujianensis occurs mainly in southeastern China
including Fujian, Hunan, Jiangxi, Zhejiang, and Taiwan provinces (Fei et al. 2012), while Limnonectes bannaensis
has been documented in Yunnan, southern Guangxi, and southern Guangdong provinces (Fei et al. 2012). Based on
molecular evidence, L. bannaensis also occurs in adjacent Vietnam and Laos (McLeod 2010). Recently, Jang-Liaw
& Chou (2011) identified two highly divergent and non-monophyletic mitochondrial DNA (mtDNA) lineages
within “L. bannaensis” from Yunnan province. Their discovery highlighted the likelihood of cryptic species in
China.
Herein we investigate new series of specimens collected from most of the known distribution of Limnonectes
in China. Using mitochondrial DNA, we place these new specimens into the framework of a phylogenetic tree with
congeners from Thailand, Vietnam, Lao, Myanmar, Cambodia, Malaysia, Philippines, and Indonesia. By
combining morphological and molecular data, we seek to clarify the species boundaries between the Chinese
Limnonectes and describe a new species of the Limnonectes kuhlii complex.
Materials and methods
Taxon sampling. A total of 40 specimens of Limnonectes were collected from 23 localities in China and Vietnam
(Fig. 1 and Table 1). Muscle and liver samples were preserved in 95% ethanol. Voucher specimens were fixed in
10% buffered formalin and later transferred to 70% ethanol for preservation. All these new specimens were
deposited the herpetological collection of the Museum of the Kunming Institute of Zoology (KIZ), the Chinese
Academy of Sciences.
TABLE 1 . List of specimens used in the study, their localities, voucher numbers, and GenBank accession numbers for
the 12S rRNA, tRNA Val and 16S rRNA sequences. Museum codes correspond to those of Evans et al. (2003); Matsui et
al. (2010a); Matsui et al. (2010b); McLeod (2010); McLeod et al. (2012) with the addition of KIZ for Kunming Institute
of Zoology and Kunming Institute of Zoology, HN for Hainan Normal University.
Species Locality Catalog number GenBank Source
Ingroup
L. fujianensis
1 China, Fujian, Pu Chang Xian KIZ J28 KU599836 This study
2 China, Fujian, Pu Chang Xian KIZ J27 KU599837 This study
3 China, Fujian, Wuyishan, Sangang KIZ H28 KU599838 This study
4 China, Fujian, Wuyishan, Sangang KIZ H27 KU599839 This study
5 China, Jiangxi, Ji'an, Anfu KIZ012480 KU599840 This study
6 China, Anhui, Qianxian, Hongxing KIZ021935 KU599841 This study
7 China, Anhui, Qianxian, Hongxing KIZ021934 KU599842 This study
8 China, Anhui, Xiuning, Lingnan KIZ07364 KU599843 This study
9 China, Hunan, Guidong KIZYPX8479 KU599844 This study
10 China, Guangdong, Jiguantian KIZ04771 KU599845 This study
11 China, Guangdong, Tianjing Shan KIZ04726 KU599846 This study
12 Taiwan ROC, Taoyuan Co. NMNST 16602 HM067231 McLeod 2010
13 Taiwan ROC, Taoyuan Co. NMNST 16606 HM067235 McLeod 2010
L. bannaensis
14 China, Yunnan, Xishuangbanna, Mengla, Yiwu KIZ024971 KU599847 This study
15 China, Yunnan, Xishuangbanna, Mengla, Yiwu KIZ024970 KU599848 This study
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TABLE 1. (Continued)
Species Locality Catalog number GenBank Source
16 China, Yunnan, Xishuangbanna, Mengla, Bubang KIZ011793 KU599849 This study
17 China, Yunnan, Xishuangbanna, Mengyang,
Long Chang
KIZ011726 KU599850 This study
18 China, Yunnan, Xishuangbanna, Mengyang,
Long Chang
KIZ011727 KU599851 This study
19 China, Yunnan, Xishuangbanna, Mengyang,
Long Chang
KIZ011728 KU599852 This study
20 China, Yunnan, Xishuangbanna, Mengyang,
Caiyanghe
KIZ011735 KU599853 This study
21 China, Yunnan, Xishuangbanna, Mengyang,
Caiyanghe
KIZ011740 KU599854 This study
22 China, Yunnan, Xishuangbanna, Mengyang,
Caiyanghe
KIZ011741 KU599855 This study
23 China, Guangxi, Hulong, Pinglongshan KIZ022207 KU599856 This study
24 China, Guangxi, Shiwandashan1 KU 311786 HM067225 McLeod 2010
25 China, Guangxi, Shiwandashan1 KU 311790 HM067219 McLeod 2010
26 China, Guangxi, Shiwandashan2 KU 311791 HM067220 McLeod 2010
27 Laos, Phongsaly, Phongsaly Dist. FMNH 258519 HM067158 McLeod 2010
28 Laos, Phongsaly, Phongsaly Dist. FMNH 258520 HM067159 McLeod 2010
29 Laos, Huaphahn, Vieng Tong Dist. FMNH 255140 HM067133 McLeod 2010
30 Laos, Huaphahn, Vieng Tong Dist. FMNH 255145 HM067138 McLeod 2010
31 Vietnam, Thanh Hoa, Quan Hoa Dist. KIZ011608 KU599857 This study
32 Vietnam, Vinh Phuc, Tam Dao AMNH 106430 HM067272 McLeod 2010
33 Vietnam, Ha Giang, Yen Minh Dist. AMNH 106558 HM067275 McLeod 2010
34 Vietnam, Ha Giang, Bac Me Dist. IEBR A.2014.347 HM067263 McLeod 2010
35 Vietnam, Ha Giang, Vi Xuyen Dist. IEBR A.2014.3440 HM067266 McLeod 2010
36 Vietnam, Ha Giang, Bac Quang Dist. IEBR A.2014.34 HM067260 McLeod 2010
37 Vietnam, Ha Giang, Bac Me Dist. IEBR A.2014.35 HM067261 McLeod 2010
38 Vietnam, Lao Cai, Van Ban Dist. AMNH 141069 HM067276 McLeod 2010
39 Vietnam, Ha Giang, Vi Xuyen Dist. VNMN A.2015.41 HM067246 McLeod 2010
40 Vietnam, Ha Giang, Vi Xuyen Dist. AMNH 141069 HM067273 McLeod 2010
41 Vietnam, Ha Giang, Vi Xuyen Dist. IEBR A.2014.36 HM067262 McLeod 2010
42 Vietnam, Ha Giang, Vi Xuyen Dist. IEBR A.2014.32 HM067257 McLeod 2010
43 Vietnam, Phu Tho, Xuan Son NP. KIZ011522 KU599858 This study
44 Vietnam, Phu Tho, Xuan Son NP. KIZ011521 KU599859 This study
45 Vietnam, Quang Binh, Phong Nha-Ke Bang KIZ YPX21563 KU599860 This study
46 Vietnam, Quang Binh, Minh Hoa Dist. AMNH 106382 HM067269 McLeod 2010
47 Vietnam, Ha Tinh, Huong Son Dist. AMNH 106384 HM067271 McLeod 2010
48 Vietnam, Ha Tinh, Ke Go AMNH 106381 HM067268 McLeod 2010
49 Vietnam, Quang Tri, Bac Huong Hoa KIZYPX18365 KU599861 This study
L. taylori
50 Thailand, Chiang Mai, Doi Inthanon KUHE19101 AB558929 Matsui et al.
2010b
51 China, Yunnan, Xishuangbanna, Mengyang Dist.,
Caiyanghe
KIZ 011745 KU599862 This study
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TABLE 1. (Continued)
Species Locality Catalog number GenBank Source
52 China, Yunnan, Xishuangbanna,
Mengyang Dist., Caiyanghe
KIZ 011710 KU599863 This study
53 China, Yunnan, Xishuangbanna,
Mengyang Dist.
KIZ022407 KU599864 This study
54 China, Yunnan, Xishuangbanna,
Mengla Dist., Yiwu
KIZ022417 KU599865 This study
55 China, Yunnan, Xishuangbanna,
Mengla Dist., Bubang
KIZ022399 KU599866 This study
56 Laos, Phongsaly, Phongsaly Dist. FMNH 258517 HM067156 McLeod 2010
57 Laos, Phongsaly, Phongsaly Dist. FMNH 258521 HM067160 McLeod 2010
58 Myanmar, Shan State, Kalaw township CAS 221714 HM067306 McLeod 2010
59 Myanmar, Shan State, Taunggyi Dist. CAS 230947 HM067315 McLeod 2010
L. megastomias
60 Thailand, Sa Kaeo, Pang Si Da NP. FMNH 266220 HM067183 McLeod 2010
61 Thailand, Nakhon Ratchasima, Sakaerat KU 307760 HM067201 McLeod 2010
L. isanensis
62 Thailand, Loei, Phu Rua Dist. FMNH 266212 HM067175 McLeod et al.
2012
63 Thailand, Loei, Phu Rua Dist. FMNH 266219 HM067182 McLeod et al.
2012
L. nguyenorum
64 Vietnam, Ha Giang, Vi Xuyen Dist. AMNH 106355 HM067267 McLeod 2010
L. jarujini
65 Thailand, Phetchaburi, Kaeng Krachan Dist. KUHE20127 AB558944 Matsui et al.
2010b
L. longchuanensis sp. nov.
66 China, Yunnan, Dehong, Longchuan KIZ048424 KU599867 This study
67 China, Yunnan, Yingjiang, Tongbiguan KIZYPX33448 KU599868 This study
68 China, Yunnan, Yingjiang, Tongbiguan KIZ048527 KU599869 This study
69 China, Yunnan, Dehong, Longchuan, Husa, KIZ048565 KU599870 This study
70 Myanmar, Kachin State, Putao Dist. CAS 224555 HM067309 McLeod 2010
71 Myanmar, Kachin State, Mohnyin Township CAS 233014 HM067323 McLeod 2010
72 Myanmar, Sagaing Div., Alaungdaw Kathapa CAS 205260 HM067285 McLeod 2010
73 Myanmar, Sagging Div., Mon Ywa Dist. CAS 221717 HM067307 McLeod 2010
74 Myanmar, Sagaing Div., Alaungdaw Kathapa CAS 205280 HM067289 McLeod 2010
75 Myanmar, Sagaing Div., Alaungdaw Kathapa CAS 210186 HM067292 McLeod 2010
76 Myanmar, Sagaing Div., Alaungdaw Kathapa CAS 208057 HM067290 McLeod 2010
77 Myanmar, Sagaing Div., Alaungdaw Kathapa CAS 210183 HM067291 McLeod 2010
78 Myanmar, Chin State, Min Dat Dist. CAS 219994 HM067304 McLeod 2010
79 Myanmar, Chin State, Min Dat Dist. CAS 234727 HM067324 McLeod 2010
80 Myanmar, Chin State, Min Dat Dist. CAS 235239 HM067347 McLeod 2010
81 Myanmar, Chin State, Min Dat Dist. CAS 235006 HM067327 McLeod 2010
82 Myanmar, Chin State, Min Dat Dist. CAS 235100 HM067333 McLeod 2010
83 Myanmar, Chin State, Min Dat Dist. CAS 235132 HM067338 McLeod 2010
84 Myanmar, Sagaing Div., Htamanthi. CAS 232197 HM067318 McLeod 2010
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TABLE 1. (Continued)
Species Locality Catalog number GenBank Source
85 Myanmar, Sagaing Div., Htamanthi. CAS 232281 HM067320 McLeod 2010
L. seletan
86 Malaysia, Pahang, Batang Kali. FRIM 1141 HM067200 McLeod 2010
L. utara
87 Malaysia, Pahang, Sungai Lembing. LSUHC5008 HM067229 McLeod 2010
L. “kuhlii” 1
88 Malaysia, Borneo Island, Sabah State FMNH 257155 HM067144 McLeod 2010
89 Malaysia, Borneo Island, Sabah State FMNH 238471 HM067123 McLeod 2010
90 Malaysia, Borneo Island, Sabah State FMNH 238511 HM067124 McLeod 2010
91 Malaysia, Borneo Island, Sabah State FMNH 234394 HM067118 McLeod 2010
92 Indonesia, Serasan Island BJE 170 HM067283 McLeod 2010
93 Indonesia, Kalimantan AMNH167141 AY313686 Evans et al. 2003
L. “kuhlii” 2
94 Vietnam, Thua Thien Hue,
Phong Dien
KIZ010081 KU599871 This study
95 Laos, Xe Kong, Kaleum Dist.1 FMNH 258506 HM067147 McLeod 2010
96 Laos, Xe Kong, Kaleum Dist.2 FMNH 258512 HM067153 McLeod 2010
97 Cambodia, Ratanakiri, Ta Veng Dist. FMNH 262722 HM067166 McLeod 2010
98 Cambodia, Stung Treng , Siem Pang Dist. FMNH 262727 HM067171 McLeod 2010
L. fragilis
99 China, Hainan, Wuzhi Mountain HN0806062 KU599872 This study
100 China, Hainan, Wuzhi Mountain HN0806009 KU599873 This study
101 China, Hainan, Yingeling Mountain HN0806059 KU599874 This study
102 China, Hainan, Yingeling Mountain HN0806038 KU599875 This study
L. namiyei
103 Japan, Okinawa, Okinawajima Island KUHE L0809191 AB526309 Matsui et al.
2010a
104 Japan, Okinawa, Okinawajima Island KUHE L0809192 AB526310 Matsui et al.
2010a
L. “kuhlii” 3
105 Vietnam, Nghe An, Khe Moi ROM 19384 AF206464,
AF206083
McLeod 2008
L. kadasani Indonesia, Lombok Island LSUMZ 81722 AY313693 Evans et al. 2003
L. microdiscus Indonesia, Java Island, Sukabumi LSUMZ 81739 AY313688 Evans et al. 2003
L. kuhlii Indonesia, Java Island, Sukabumi MZB amph. 6501 AY313687 Evans et al. 2003
L.
microtympanum
Indonesia, Sulawesi Island, Barruo AMNH 16176 AY313743 Evans et al. 2003
L. parvas Philippines, Mindanao Island,
Barangay Malagos
PNM 7447 AY313694 Evans et al. 2003
Outgroup
Fejervarya
limnocharis
Vietnam, Nghe An, Con Cuong AMNH A-161230 AY843588 Evans et al. 2003
Nanorana
yunnanensis
China, Yunnan KIZ047053 KU599876 This study

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FIGURE 1. Sampling localities of the specimens of Limnonectes used in this study. New samples indicated by color: red stars
in China = L. longchuanensis sp. nov., blue squares = L. fujianensis, pink diamonds = L. bannaensis, orange circles = L. taylori,
and green triangles = L. fragilis. Samples downloaded from GenBank were indicated by white symbols. Locality numbers refer
to Table 1 and Fig. 2.
Considering all the recognized Chinese Limnonectes were attributed to the Limnonectes kuhlii complex (Fei et
al. 2012), our analyses focused to this complex below. To evaluate the new samples within the context of the
Limnonectes kuhlii complex, we combined them with 72 previously published sequences from GenBank (Evans et
al. 2003; Matsui et al. 2010b; McLeod 2010; McLeod et al. 2012). The combined sample represents all lineages
currently associated with the Limnonectes kuhlii complex (Table 1; L. kuhlii [Java], L. fujianensis, L. namiyei, L.
bannaensis, L. taylori, L. megastomias, L. isanensis, L. nguynorum, L. jarujini, L. fragilis, undescribed lineages
from Malaysia, Indonesia, Cambodia, Laos and Vietnam) and four congeners outside of the complex (L. kardasani,
L. microdiscus, L. microtympanum, and L. parvus). Nanorana yunnanensis and Fejervarya limnocharis were
chosen as outgroup taxa based on the study of Evans et al. (2003).
Morphology. To assess morphometric differences among the samples, we took the following 18 body
measurements to the nearest 0.1 mm with dial calipers: snout-vent length (SVL); horizontal eye diameter (ED); eye
nostril distance (END); rostrum length distance (RLD); thigh (femur) length (FEL); foot length (FOL); head length
(HL); head width (HW); internarial distance (IN); interorbital width (IO); lower arm length (LAL); mandible-
nostril distance (MN); palm length (PAL); relative finger length (RFL); relative toe length (RTL); shank (tibia)
length (TBL); tympanum diameter (TD); and upper arm length (UAW). Abbreviations and definitions of
morphological features follow Matsui (1984) and McLeod (2008). Except where noted, digital-webbing formulae
are those of Savage (1975). Digits of the hand are numbered II–V based on presumed homology to the digits of
non-anuran tetrapods (Alberch & Gale 1985). Sex and life stage were determined by examination of gonads and by

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inspection of prominent secondary sexual characters (e.g., nuptial pads). Morphological comparisons were based
on specimens at the KIZ, and California Academy of Sciences (CAS) (Tables 2, 3).
TABLE 2 . Mensural and selected meristic data (mm) for the type series of Limnonectes longchuanensis sp. nov. (males).
KIZ = Kunming Institute of Zoology; CAS = California Academy of Sciences.
Extraction, amplification, and sequencing. Genomic DNA was extracted by the standard phenol-chloroform
protocol (Sambrook et al. 1989). A fragment of mtDNA spanning from 12S ribosomal DNA (rDNA) through
transfer RNA for Valine (tRNA
Va l
) to 16S ribosomal DNA (12–16s) was chosen for analysis. Primers used for DNA
amplification by standard polymerase chain reaction (PCR) were 12Sa and 16Sbr of Kocher et al. (1989). Five
internal primers were also used (Table 4) (Kocher et al. 1989; Moriarty & Cannatella 2004). Amplification was
performed in a 25 µL volume reaction with the following procedures: initial denaturation at 95 °C for 5 min, 35
cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min and a final
extension at 72 °C for 10 min. PCR products were purified with a Gel Extraction Mini Kit (Watson
Biotechnologies, Shanghai, China). We conducted all sequencing on ABI PRISM 3730 automated sequencers
KIZ048424 CAS235239 CAS235132 CAS210183 CAS219994
(Holotype) (Paratype) (Paratype) (Paratype) (Paratype)
Sex Male Male Male Male Male
SVL 75.5 57.8 54.9 67.2 77.9
ED 7.8 5.8 6.0 6.5 7.5
END 5.9 4.3 4.4 5.8 5.4
RLD 11.9 8.9 8.5 11.1 11.2
FEL 36.5 27.9 26.5 32.0 36.9
FOL 47.4 37.3 36.7 41.8 48.8
HL 40.3 26.9 24.5 29.6 33.6
HW 36.0 24.7 23.1 27.3 33.0
IN 5.9 5 4.0 4.3 5.1
IO 7.4 4.5 4.3 5.1 6.0
LAL 16.0 11.3 11.0 13.6 14.1
MN 34.5 23.6 21.5 25.3 30.0
PAL 18.6 13.7 13.1 16.0 19.7
RFL IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II
RTL IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I
TBL 33.3 26.2 25.6 29.5 33.0
TD Na Na Na Na Na
UEW 5.1 3.9 3.4 4.5 4.4
ED/HL 0.19 0.21 0.25 0.22 0.22
HW/HL 0.89 1.09 1.06 1.09 1.02
HL/SVL 0.53 0.46 0.45 0.44 0.43
IN/IO 0.79 1.12 0.92 0.84 0.85
IN/SVL 0.07 0.09 0.07 0.06 0.07
END/IN 1 0.85 1.1 1.4 1.07
UEW/IO 0.68 0.86 0.79 0.88 0.74
END/RLD 0.49 0.48 0.51 0.53 0.48
TBL/SVL 0.44 0.45 0.47 0.44 0.42
FEL/SVL 0.48 0.48 0.48 0.48 0.47
LAL/SVL 0.21 0.19 0.2 0.2 0.18

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TABLE 3. Mensural and selected meristic data (mm) for the type series of Limnonectes longchuanensis sp. nov. (females). KIZ = Kunming Institute of Zoology; CAS = California Academy of Sciences.
KIZ048425 KIZ048426 KIZ048427 KIZ048428 KIZ048429 CAS232281 CAS233014 CAS235133 CAS210197 CAS221717
(Paratopotype) (Paratopotype) (Paratype) (Paratopotype) (Paratopotype) (Paratype) (Paratype) (Paratype) (Paratype) (Paratype)
Sex Female Female Female Female Female Female Female Female Female Female
SVL 43.8 48.6 40.6 44.4 43.7 63.0 61.0 48.3 48.9 59.1
ED 6.0 6.2 5.5 5.8 5.9 7.4 6.1 5.2 4.8 4.8
END 3.6 3.8 3.4 3.2 3.4 5.0 5.1 3.6 4.8 5.3
RLD 6.5 7.2 5.8 7.0 8.2 8.7 8.8 6.7 8.0 8.3
FEL 19.9 22.2 19.5 20.1 22.1 28.9 28.7 22.3 23.9 29.2
FOL 29.7 28.8 25.4 26.5 28.6 39.9 37.9 32.9 33.6 37.7
HL 19.4 19.9 16.6 20.3 21.0 24.3 24.5 19.3 20.2 22.9
HW 18.5 19.4 16.6 17.5 19.3 23.5 22.6 18.8 18.2 21.2
IN 3.5 3.8 2.5 3.8 3.7 3.7 5.0 4.0 3.4 4.1
IO 3.7 4.1 3.3 3.6 3.5 3.8 4.0 3.4 2.7 3.6
LAL 9.6 9.4 9.5 9.3 10.4 12.5 10.9 9.9 9.8 10.7
MN 16.0 17.4 14.5 17.3 18.7 21.3 21.4 17.0 17.5 19.9
PAL 11.0 12.3 10.0 10.2 12.0 13.7 13.8 11.7 11.6 13.3
RFL IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II IV-V-III-II
RTL IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I IV-III-V-II-I
TBL 21.0 22.2 18.7 20.0 20.7 26.7 26.2 21.4 22.1 25.9
TD Na Na Na Na Na Na Na Na Na Na
UEW 3.4 3.6 3.0 2.7 3.1 4.6 3.9 3.6 3.3 3.1
ED/HL 0.31 0.31 0.33 0.28 0.28 0.3 0.25 0.27 0.24 0.21
HW/HL 0.95 0.97 1 0.86 0.92 1.03 1.08 1.03 1.11 1.08
HL/SVL 0.44 0.4 0.4 0.45 0.47 0.39 0.4 0.4 0.41 0.39
IN/IO 0.94 0.92 0.76 1.04 1.06 0.97 1.27 1.15 1.26 1.13
IN/SVL 0.08 0.07 0.06 0.08 0.08 0.06 0.08 0.08 0.07 0.07
END/IN 1 0.99 1.35 0.84 0.91 1.35 1.01 0.92 1.4 1.3
UEW/IO 0.91 0.86 0.92 0.73 0.87 1.21 1 1.04 1.23 0.86
END/RLD 0.54 0.53 0.57 0.45 0.41 0.57 0.58 0.54 0.6 0.63
TBL/SVL 0.47 0.45 0.46 0.45 0.47 0.42 0.43 0.44 0.45 0.44
FEL/SVL 0.45 0.45 0.47 0.45 0.5 0.46 0.47 0.46 0.49 0.49
LAL/SVL 0.21 0.19 0.23 0.2 0.23 0.2 0.18 0.2 0.2 0.18

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(Applied Biosystems). All individuals were sequenced in both directions. All sequences were examined for signal
quality and confirmed for complementarities using DNASTAR 5.0 (DNASTAR Inc., Madison, WI, USA).
Maternal genealogy reconstruction. Sequences were aligned with gaps after trimming the ends using
MUSCLE (Edgar 2004) with default settings. The sequence data were submitted to a BLAST search in GenBank to
confirm identity. Uncorrected pair-wise genetic distances (p-distances) were calculated using MEGA 5.0 (Tamura
et al. 2011).
Phylogenetic analyses of the data were conducted using Bayesian inference (BI) and maximum likelihood
(ML). Considering 12–16s are noncoding genes, we did not partition them in the following analyses. We selected
the best-fit nucleotide substitution models for the 12–16s using Akaike Information Criterion (AIC) in
JMODELTEST 2.1.6 (Guindon & Gascue, 2003; Darriba, 2012). BI analyses were performed with MrBayes v3.1.2
(Ronquist & Huelsenbeck 2003). Trees were run for 5 million generations while sampling trees every 1000 trees
generation, and four metropolis-coupled Markov chain Monte Carlo (MCMC) chains (temp = 0.2) runs starting
with a random tree. Convergence was assessed in Tracer v1.5 (Rambaut & Drummond 2007) to confirm that all
parameters had reached stationarity and had sufficient effective sample sizes (> 200). The first 25% were discarded
as burn-in. The frequency of nodal resolution was termed a Bayesian posterior probability (BPP). We conducted
ML analyses in RAxML v7.0.4 (Stamatakis et al. 2008) with 100 bootstrap replicates. The same model as BI
analyses was implemented. Nodal reliabilities were assessed based on 1000 replicates of bootstrap analysis.
TABLE 4. Primers used for amplification and sequencing of the mtDNA genes for samples of the genus Limnonectes.
Results
Sequence variation, matrilineal genealogy. New sequences were deposited in GenBank under accession numbers
KU599836–599876 (Table 1). For all 112 individuals, 1997 base pairs (bp) of 12–16s sequence data (448 bp of 12S
rRNA, 68 bp of tRNA
Val
and 1481 bp of 16S rRNA). The concatenated data set consisted of a matrix of 964 variable
sites, of which 764 were parsimony informative. The best-fit model for 12–16s was GTR+I+G. The BI and ML
analyses produced similar trees. Topologies from the three tree-building methods differed only in their resolution
of poorly supported nodes with BPP < 95% and BS < 70%.
Three major groups (I, II and III) and 10 lineages (lineages A–J) were identified within Limnonectes (Fig. 2).
All Chinese samples were distributed in lineages A, B, C, D, and G with high support. Lineages A was the sister to
B. Lineages C and D were sister groups, and they formed the sister group of A+B. Lineages G was far removed
evolutionarily from A, B, C, and D.
Limnonectes fujianensis and L. namiyei constituted lineage A. Limnonectes fujianensis occurs in southeastern
China and Taiwan, and samples in our study represented localities in Fujian (sites 1–4), Jiangxi (site 5), Anhui
(sites 6–8), Hunan (site 9), Guangdong (sites 10 and 11) and Taiwan (sites 12–13) provinces. Limnonectes namiyei
is a Japanese endemic from Okinawa (sites 103–104).
Lineage B comprises L. bannaensis and L. “kuhlii”3. Limnonectes bannaensis seems to have large geographic
distribution and our samples represent localities in the Chinese provinces of Yunnan (sites 14–22) and Guangxi
(sites 23–26), Phongsaly (sites 27–28) and Huaphahn provinces (sites 29–30) in Laos, and Thanh Hoa, Vinh Phu,
Ha Giang, Lao Cai, Phu Tho, Quang Binh, Ha Tinh, and Quang Tri provinces in Vietnam (sites 31–49). The second
sublineage, L. “kuhlii”3, is represented by a single sample from Khe Moi, Nghe An province, Vietnam (site 105).
Primer Name Primer Sequence Source
12Sa
16Sh
16Sar
16Sbr
HERP177 (forward)
HERP179 (reverse)
HERP180 (reverse)
5'-AAACTGGGATTAGATACCCCACTAT-3'
5'-GCTAGACCATKATGCAAAAGGTA-3'
5'-CGCCTGTTTAYCAAAAACAT-3'
5'-CCGGTYTGAACTCAGATCAYGT-3'
5'-GCCTARAAGCAGCCACC-3'
5'-TCACTGGGCAGGCTRGA-3'
5'-GGTGGCTGCTTYTAGGC-3'
Kocher et al. 1989
Moriarty & Cannatella, 2004
Kocher et al. 1989
Kocher et al. 1989
This study
This study
This study

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FIGURE 2. Bayesian tree of Chinese Limnonectes inferred from mtDNA 12S rRNA, tRNA
Val
and 16S rRNA genes. Colors of
lineages, species names, and symbols correspond to Fig. 1. The numbers above branches represent Bayesian posterior
probabilities (BPP), and bootstrap support (BS) for maximum likelihood (ML); “-” denotes low support (BPP < 95% or BS <
70%) in one analysis; no values on branches represent low support in both analyses. The scale bar represents 0.05 nucleotide
substitutions per site.
Lineage C (Fig. 2) comprises five Indochinese lineages: L. taylori, L. megastomias, L. isanensis, L.
nguyenorum, and L. jarujini. Limnonectes taylori is represented by samples from northern Thailand (site 50, type
locality), northern Laos (sites 56–57), and Myanmar (sites 58–59) as well as some samples from Yunnan province,
China (sites 51–55, Fig. 1). Limnonectes megastomias, L. isanensis, and L. jarujini are endemic to Thailand (sites
60–63, and 65, respectively), and L. nguyenorum is a Vietnamese endemic known only from the type locality in Ha
Giang province (site 64).
Lineage D included samples from Yunnan province, China (Sites 66–69, Fig. 1), from Kachin state, Chin state,
and Sagaing division, Myanmar (sites 70–85). Lineage D represents the new species described below.
Lineage E included two samples from Pahang, Malaysia (sites 86–87), both referred to as “Lineage 6” by
McLeod (2010). One of these (FRIM 1141) is tentatively assigned to Limnonectes seletan and the other (LSUHC
5008) is referred to L. utara based on 99% convergence with previously published GenBank samples (Matsui et al.
2014). Lineage F comprises multiple undescribed lineages from Borneo (sites 88–93).

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Limnonectes fragilis from Hainan Island fell within lineage G. It was distantly related to other Chinese
Limnonectes. It formed the sister group to L. “kuhlii”2 from Vietnam, Laos and Cambodia (sites 94–98).
Lineages H, I, and J formed a clade with weak support at the base and with respect to the inclusion of matriline
I representing Limnonectes kuhlii (from the type locality of Java, Indonesia). Lineages H and J include L. kardasani
and L. microdiscus, and L. microtympanum and L. parvas, respectively.
The p-distances for 12–16s between the 20 sequenced species including the new species ranged from 3.3% (L.
bannaensis and L. “kuhlii”3) to 17.8% (L. microtympanum and L. microdiscus) (Table 5). Distances between the
five species of Chinese Limnonectes ranged from 8.8% (lineage D and L. taylori) to 14.3% (L. fujianensis and L.
fragilis). The p-distance between lineage D and the other congener’s ranged from 7.4% (lineage D and L.
nguyenorum) to 16.0% (lineage D and L. microtympanum). Pair-wise distances between lineage D and L. kuhlii
from Java (Clade I) averaged 15.2%. Within lineage D, p-distances were 1.9%.
Systematic account
Limnonectes longchuanensis sp. nov. Suwannapoom, Yuan, Chen, Sullivan and McLeod
Holotype: Adult male (KIZ048424; Fig. 3 in life and Fig. 4 in preservative), from Longchuan, Dehong, Yunnan,
China (24° 27' 32.40" N, 97° 45' 10.80" E, 1255 m a.s.l.), collected by Jin-Min Chen and Mian Hou on 14 August
2013.
Paratypes: Five paratopotypes and 9 paratypes from China and Myanmar: Four paratopotypes, KIZ048425(f),
KIZ048426(f), KIZ048428(f), KIZ048429(f) from the type locality as above; one paratype KIZ048427(f) from
Yingjiang, Yunnan, China (24° 32' 45.60" N, 97° 44' 32.40" E, 809 m a.s.l.); one paratype, CAS233014 (f) from
Myitkyina District, Kachin State, Myanmar (25° 5' 38.2" N, 96° 22' 49" E); two paratypes, CAS232197(m),
232281(f) from Khandi District, Sagaing Division, Myanmar (25° 19' 20.6" N, 95° 31' 49.8" E, 744–847 m a.s.l.);
one paratype, CAS221717 (f) from Mon Ywa District, Sagaing Division, Myanmar (22° 18' 50.1" N, 94° 24' 25.7"
E); one paratype, CAS210183(m) from Alaungdaw Kathapa National Park, Sagaing Division, Myanmar (22° 18'
32.7" N, 94° 24' 25.8" E); four paratypes, CAS219994(m), 235006(m), 235132(m), 235133(f) from Min Dat
District, Chin State, Myanmar (21° 22' 20.1" N, 93° 58' 34.6" E). Specimens numbered with KIZ (KIZ048425–29)
and CAS (CAS233014, 232197, 232281, 221717, 210183, 219994, 235006, 235132, 235133) were deposited in
KIZ and CAS, respectively.
Diagnosis. Limnonectes longchuanensis sp. nov. is assigned to Limnonectes based on the presence of fanglike
odontoid processes on the lower jaw and male-biased size dimorphism (adult males characterized by hypertrophy
of the head) (Emerson et al. 2000). Adults of the new species are diagnosed from congeners by a combination of
the following characters: (1) a relatively large species of the L. kuhlii species complex; (male SVL = 54.9–77.87
mm, and female SVL = 40.6–63.0 mm); (2) tympanum obscured by thickened skin; (3) tubercles are widely
present on dorsum, lateral surface of body, the dorsum of arms, thighs and tibia; (4) males with nuptial pads on
fingers I only or both fingers I & II; (5) no temporal stripe or dark markings on dorsal sides of body and limbs; (6)
males have longer and wider heads than females (male HL = 24.5–40.3 mm, HW = 23.1–36.0 mm; female HL =
16.6–24.5 mm, HW= 16.6–23.5 mm); (7) vocal sac and vocal slits absent in males; (8) throat and venter
moderately pigmented; (9) relative finger length (longest to shortest) when adpressed: IV–V–III–II; (10) toe
webbing is deeply excised between toes, webbing formula on foot = I0–0
+
II0
+
–0
+
III0
+
–0
+
V0
+
–0V.
Description of holotype. Adult male (Figs 3–4). Head hypertrophied and broader than body, HL 53.4% of
SVL; head longer than wide (HW 89.5% HL); rostrum rounded in dorsal view, projecting beyond lower jaw;
acutely rounded and anteroventerally sloped in profile; nostril dorsolaterally oriented and in line with anterior
mandibular margin, closer to tip of snout than to eye; internarial distance greater than interorbital distance; canthus
rounded; lores sloping, shallowly concave with weak groove; distinct infraorbital groove from anterior margin of
eye to posterior margin of eye continuing indistinctly, not reaching rictus; upper lip moderately straight and flared,
reaching post-rictal tubercle; eye diameter 19.4% of head length; upper eyelid width 68.7% of interorbital distance;
supratypanic fold moderate, extending from eye to angle of jaw (anterior insertion of arm); tympanic anulus not
visible through skin. Vomerine teeth on oblique ridges, separated from each other by much less than width of one
ridge. Choanne oval, perpendicular to longitudinal axis of body. Odontoid processes robust with blunt tips, length

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more than twice depth of mandible at base of process. Symphisial knob at mandibular symphysis. Tongue oval,
wide U-shaped posterior notch. Vocal sac and vocal slits absent. IN 79.0% of IO; IN 7.8% of SVL; END 100.5% of
IN; UEW 68.7% of IO; END 49.9% of RL.
Fingers: finger tips blunt, not expanded, without disk; relative length = IV>V>III>II, no webbing between
fingers; palmar subarticular tubercle formula (digits indicated by Roman numeral): IV(2), V(2), III(1), II(1);
proximal subarticular tubercles prominent, rounded, elevated on all fingers; distal subarticular tubercles low, flat
and indistinct; thenar metacarpal tubercle large, oval, elevated; inner metacarpal tubercle oval, flat, equal in size to
thenar tubercle, not contacting thenar tubercle; outer metacarpal tubercle smaller than inner tubercles, oval, elevate,
contacting inner tubercle; prominent nuptial pad composed of minute spines covering entire upper surface of Digit
II, extending on palmar surface to base of metacarpal tubercle. Tips of toes expanded into discs, toe pads elevated;
decreasing lengths of toes: IV–III–V–II–I; toes webbed from base of toe pad, webbing deeply excised (webbing
formula = I0
+
–0
+
II0
+
–0
+
III0
+
–0
+
IV0
+
–0
+
V); distinct, movable flap of skin on postaxial side of Toe V from base of
toe pad to proximal end of metatarsus; distinct, movable flap of skin on preaxial side of Toe I from base of toe pad
to level of inner metatarsal tubercle, continuing as raised ridge on distal two thirds of tarsus; subarticular tubercles
prominent, elevated, oval (Fig. 4D–E); digits indicated by roman numeral (palmar subarticular tubercle count in
parentheses): V (2), IV (3) III (2), II (1), I (1); inner metatarsal tubercle oval, elongate with elevated post axial
border; TBL 44.1% of SVL; FEL 48.4% of SVL; LAL 21.3% of SVL.
Skin on top of head, throat, dorsal surfaces of forelimbs, and dorsum feebly crenulate; skin on sides rough with
moderately dense, roundish, non-pearl tipped tubercles; skin around vent, “knee”, shank and foot distinctly
tuberculate, covered with moderately dense, small, low tubercles with translucent spinules; ventral skin and
dorsum of thigh smooth; pair of faint, broken dorsolateral folds extending from posterior of eye to vent.
FIGURE 3. (A) Dorsal view, (B) ventral views, (C) palmar view hand, and (D) ventral views of foot in male holotype of
Limnonectes longchuanensis sp. nov. (KIZ048424) in life.

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FIGURE 4. (A) Dorsal view, (B) ventral views, (C) lateral view of head, (D) ventral views of foot and (E) palmar view of hand
in male holotype of Limnonectes longchuanensis sp. nov. (KIZ048424) after preservation.
Coloration: In life brown dorsally and laterally, some tubercles on sides of body appear to have white tops; one
yellowish-brown bar on the center of dorsum extending through the center of upper lip to cloaca; rust colored patch
near angle of jaw; lower lip with distinct dark brown bars separated by irregular white blotches; supratympanic fold
dark brown, dark line extending through eye to nares; eye greyish with dark horizontal bar passing through pupil
and faint vertical bar passing through; skin of arms and legs light brown; throat moderately pigmented,
pigmentation extending onto chest at the level of pectoral girdle, abdomen white with faint brown flecking, ventral
surface of thighs lightly mottled; webbing between toes dark brown with light mottling.
In preservative, brown dorsally and laterally; indistinct dark brown bars on upper lip, lower lip dark with light
mottling; supratympanic fold dark brown; indistinct; throat heavily pigmented; venter immaculate, ventral portions
of limbs mottled around margins; palmar and plantar surfaces dark brown, webbing between toes mottled with
white margin on preaxial sides of digits II, III, IV, V and postaxial side of digit IV.
Va ri at io n . Variation in meristic and mensural characters among the type series shown in Tables 2 and 3. Males
larger than females (SVL). Three of the male specimens in our series (KIZ048424, CAS 235006 and 235132) had
nuptial pads only on the first finger whereas all other males had nuptial pads on the first and second fingers. Males
with larger, more robust odontoid processes than females. One yellowish-brown bar either present or absent on the
center of dorsum extending through the center of upper lip to cloaca.

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FIGURE 5. Dorsal views and tuberculation of leg of (A, B) Limnonectes bannaensis [KIZ022207; male, from Yunnan, China],
(C, D) L. taylori [KIZ022399; male, from Yunnan, China], and (E, F) L. longchuanensis scale bars = 10 mm.
Etymology. The specific epithet longchuanensis is derived from the name of the type locality, Longchuan,
Yunnan, China.
Suggested common names: English: Longchuan big-headed frog.
Chinese name: Longchuan datou wa; ( 陇川大头蛙 ).
Comparisons. Limnonectes longchuanensis sp. nov. resembles other species in the L. kuhlii complex, but is
genetically and morphologically distinct in comparison to other members of this complex (Table 6). Limnonectes
longchuanensis sp. nov. can be distinguished from L. bannaensis by having large, raised tubercles on the dorsum,
lateral surfaces of body, the dorsum of arms, thighs, and tibias (vs. lacking tubercles or having low tubercles on the
dorsum, lateral surfaces of body, the dorsal surface of arms, thighs, and tibias in L. bannaensis giving this species
the appearance of more smooth skin) (Fig. 5).
Limnonectes longchuanensis sp. nov. differs from L. fragilis by having smaller subarticular tubercles and the
possession of nuptial pads in males (vs. no tubercles and nuptial pads in males of L. fragilis). In addition, the back
of L. longchuanensis sp. nov. usually is without distinct ridges whereas L. fragilis possesses them. Limnonectes
fragilis is endemic to Hainan Island (vs. L. longchuanensis sp. nov. does not occur in Hainan).
Limnonectes longchuanensis sp. nov. differs from L. fujianensis in that it normally lacks distinct ridges on the
dorsum (vs. many ridges on the dorsum in L. fujianensis). A dark brown W-shaped marking is visible on the dorsal
view of the head of adult L. fujianensis (vs. W-shaped marking is not on L. longchuanensis sp. nov.). In addition,
the tubercles of L. longchuanensis sp. nov. are larger and more densely arranged than in L. fujianensis.
Limnonectes longchuanensis sp. nov. can be distinguished from L. taylori, L. megastomias, L. isanensis, L.
nguyenorum and L. jarujini by the presence of tubercles on the on lateral surface of body, the dorsum of arms and
thighs (vs. lack of tubercles on the on lateral surface of body, the dorsal surfaces of arms and thighs in all four
species). The first finger is longer than the second finger in L. longchuanensis sp. nov., which distinguishes the
new species from L. taylori and L. jarujini (usually subequal first and second fingers in both species). Limnonectes
longchuanensis sp. nov. is easily distinguished from L. namiyei by the lack of vocal slits (vs. presence of vocal slits
in L. namiyei).

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TABLE 5. The pairwise uncorrected p distance (%) of mtDNA sequence between species of Limnonectes for 1928 bp of mitochondrial 12S rRNA, tRNA
Val
, and 16S rRNA genes.
Species 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 19
1
L. fujianensis
-
2
L. bannaensis
9.0 -
3
L. taylori
11.0 9.3 -
4
L. megastomias
11.1 9.3 6.4 -
5
L. isanensis
11.2 9.8 6.5 6.6 -
6
L. nguyenorum
10.9 9.5 7.1 7.1 7.3 -
7
L. jarujini
11.1 9.7 7.3 7.8 7.9 7.3 -
8
L. longchuanensis sp. nov.
10.5 9.5 8.8 8.3 8.1 7.4 8.0 -
9
L. seletan
12.6 12.1 13.4 13.1 12.4 13.3 13.0 11.7 -
10
L. utara
13.2 11.9 12.9 12.9 13.1 13.1 13.1 12.4 7.4 -
11
L. “kuhlii” 1
13.6 12.6 13.3 13.5 13.3 14.1 13.5 13.4 13.3 14.2 -
12
L. “kuhlii” 2
14.6 13.5 13.7 13.4 13.7 14.0 14.2 14.9 15.7 16.0 14.0 -
13
L. fragilis
14.3 13.3 13.8 12.9 13.3 14.0 14.7 14.0 14.5 14.6 13.4 11.7 -
14
L. namiyei
7.8 8.7 10.8 11.4 11.6 11.0 10.4 10.3 12.5 12.6 13.9 14.7 14.6 -
15
L. “kuhlii” 3
10.1 3.3 10.4 10.0 10.0 10.5 10.8 10.2 12.1 12.4 13.3 14.6 13.4 10.1 -
16
L. kadasani
14.8 14.1 14.9 14.4 14.0 14.6 14.8 14.1 15.1 15.4 15.5 13.3 14.0 14.8 14.3 -
17
L. microdiscus
16.9 15.7 16.8 16.2 16.5 16.7 16.7 15.7 17.0 16.9 17.0 16.3 16.2 16.7 16.2 13.2 -
18
L. kuhlii
16.5 15.9 16.1 14.9 15.0 15.5 16.1 15.2 17.0 16.7 16.1 14.5 15.2 16.9 16.0 16.0 17.7 -
19
L. microtympanum
16.7 15.0 15.2 15.5 14.6 15.6 16.0 16.0 16.9 17.5 16.0 14.1 14.8 16.1 15.8 15.2 17.8 13.7 -
20
L. parvas
14.9 13.0 14.8 14.3 14.1 15.0 14.6 14.3 15.5 14.5 14.1 13.8 13.6 14.0 13.9 13.4 14.6 14.5 14.9

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TABLE 6. Morphological characters for comparison between the new species and its closest relatives (lineage A-D, figure 2); Chinese species are in bold. When nuptial pads are present in a species,
males always possess them on the first finger (F1). In some species, individuals vary with respect to the nuptial pads on second and even third fingers (variability indicated in parentheses)
Limnonectes
longchuanensis
sp. nov.
Limnonectes
bannaensis
Limnonectes
fragilis
Limnonectes
fujianensis
Limnonectes
taylori
Limnonectes
megastomias
Limnonectes
isanensis
Limnonectes
nguyenorum
Limnonectes
jarujini
Limnonectes
namiyei
vocal slits absent absent absent absent absent absent absent absent absent present
nuptial pads F1 (2) F1 (2) absent F1 (2) F1 F1 (2&3) F1 (2) F1&2 F1 F1
dorsal ridges absent absent present present absent absent absent absent absent absent
W on head absent absent absent present absent absent absent absent absent absent
dorsum tubercles present absent present present present absent absent absent absent present
lateral tubercles present absent present present present absent absent absent absent present
arm tubercles present absent absent absent absent absent absent absent absent absent
femoral tubercles present absent absent absent absent absent absent absent absent absent
tibial tubercles present absent present present present absent present present present present
temporal dorsum stripes absent absent present present present absent absent present absent absent
throat pigmented present present present present or
absent present present present present absent present or
absent
venter pigmented absent absent absent present or
absent
present or
absent absent present or
absent absent absent present or
absent

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Distribution. Limnonectes longchuanensis sp. nov. is presently known from China and Myanmar. In China,
the species has been documented in Longchuan (type locality), Husa (24° 25' 22.80" N, 97° 52' 15.60" E, 1576 m
a.s.l.), and Yingjiang (24° 32' 45.60" N, 97° 44' 32.40" E, 809 m a.s.l.), Yunnan, China. In Myanmar, it has been
documented in Kachin State, Chin State and Sagaing Division (Fig. 1). All specimens were collected in evergreen
forests along hillside streams and small tributaries varying in width from 2 m to 5 m (Fig. 6).
FIGURE 6. (A) Longchuan, Yunnan, China, the type locality of Limnonectes longchuanensis sp. nov.; red arrow indicates
Lonchuan forests where the new species was collected. (B) The relatively small size of habitat of the holotype in evergreen
forests; red arrow indicates the closed canopy stream where the holotype of L. longchuanensis sp. nov. was collected.
Discussion
This study examined novel samples of Limnonectes species allied to the L. kuhlii complex from previously
unsampled localities in China and Vietnam. Our results largely corroborate previously published phylogenetic
hypotheses of Matsui et al. (2010), McLeod (2010); McLeod et al. (2012, 2013, 2015). McLeod (2010) recognized
22 distinct lineages allied to L. kuhlii (many undescribed) and restricted the identity of L. kuhlii to the type locality
of Java. Subsequently, 8 species (L. taylori, L. jarujini, L. isanensis, L. utara, L. selatan, L. sisikdagu, L.
nguyenorum, and L. longchuanensis sp. nov.) have been described from within the L. kuhlii complex, bringing the
total number of recognized species allied to L. kuhlii to 13 (Matsui et al. 2010b, 2014; McLeod et al. 2011, 2012,
2015).
From a regional and national perspective, our phylogeny indicates that at least five species of Limnonectes
previously identified as L. kuhlii (= Rana kuhlii Tschudi) occur in China. On the basis of morphological data alone,
Liu et al. (1973), Ye & Fei (1994); Ye et al. (2007) described three of the Chinese Limnonectes species as L.
fragilis, L. fujianensis, and L. bannaensis, respectively. Together with previously sampling efforts (Matsui et al.
2010a; McLeod 2010), the results of our continued survey efforts in China and Indochina demonstrate that L.
taylori (new record for China) and L. longchuanensis sp. nov. also occur in China.
Based on current knowledge, only L. fujianensis and L. fragilis appear to be endemic to China. Limnonectes
bannaensis occurs across Yunnan, Guangdong, and Guangxi province in southern China (Fei et al. 2006) and our
new samples corroborate McLeod (2010), who suggested that L. bannaensis occurs southward to central Vietnam
and eastern Laos. By original description, L. taylori was known only from northwestern Thailand (Matsui et al.
2010b). We extend its distribution into Yunnan province of China, Laos, and Myanmar and refer Lineage 12
(Indochina) of McLeod (2010) to L. taylori based on molecular and morphological data. Low pairwise p-distance
values (1.9%) among samples of L. longchuanensis sp. nov. support the conspecific status of populations in
Yunnan province of China and Myanmar (Fig. 2: locality no. 70–85). We therefore refer Lineage 9 (Myanmar) of
McLeod (2010) to L. longchuanensis sp. nov.
Three species of Limnonectes including L. bannaensis, L. taylori, and L. longchuanensis sp. nov. occur in
Yunnan province, China, and yet these species are not sister taxa (Fig. 2). Since its description (Ye et al. 2007), L.
bannaensis was thought to be the only species of Limnonectes in Yunnan. Matsui et al. (2010b) thought that the
occurrence of more than one species of fanged frogs in Yunnan was unlikely and that all samples should be
considered as L. bannaensis, although they also noted that further study was necessary. Jang-Liaw & Chou (2011)

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identified two highly divergent lineages within L. bannaensis, one of which came from Longchuan. Unfortunately,
we cannot compare their sequence data with ours because different genes were sequenced, and thus cannot
conclusively refer one of their lineages to L. longchuanensis sp. nov.
Limnonectes taylori and L. bannaensis occur sympatrically at Mengla and Mengyang, Xishuangbanna, Yunnan
(Fig. 1). Consistent with other studies of anuran species complexes (e.g., Stuart et al. 2006), our results suggest that
species living in sympatry are not each other’s closest relatives (viz. sister taxa). Previous studies have failed to
discover this pattern in Yunnan. The two sympatric species in Xishuangbanna live in the same habitat and they are
difficult to identify. According to Fei et al. (2012), and our field observations, the breeding season of L. bannaensis
occurs in early April and we found tadpoles during April and May. Matsui et al. (2010b) reported that the breeding
season of L. taylori in Thailand was early August. If this reproductive pattern holds true in the Yunnan populations
it suggests reproductive isolation of the two fanged frogs, a case not unlike that of the Rana longicrus species
group (Yan et al. 2011). These findings highlight the importance of incorporating natural history data in taxonomy.
Our study and others like it highlight the importance of using DNA sequence data in combination with
traditional morphological characters couched within the context of a phylogenetic hypothesis to document species
diversity. This is especially for important for species complexes and congeners that occur sympatrically. Fanged
frogs of the L. kuhlii complex are notoriously difficult to identify based on morphology because of the overall
similarity among species in the group (Emerson et al. 2000). Using molecular analyses to guide morphological re-
examinations can elucidate fine-scale differences in characters that are in fact species-specific variation.
Tuberculation of the leg and thigh has proven to be a particularly effective diagnostic character among the
members of the L. kuhlii complex, though usage of this feature has been restricted to qualitative descriptions.
Further attention should be given to tadpoles, juveniles, and adult females that remain a challenge to identify
correctly in the field.
Acknowledgments
Many thanks are owed to help from Jun-xiao Yang, Zi-jie Zhang, Wei-wei Zhou, Wang-sheng Ji, Shi-yang Dong,
Vo Dina Ba, Nikolai Orlov, Ngoc Sang Nguyen, and Tran Ngoc Ninh in the field. We thank the local conservation
departments for their help in field work of Thailand and Vietnam. The Institute of Animal for Scientific Purposes
Development (IAD) issued permission (No.U1-01205-2558) in Thailand. The Ministry of Agriculture issued
permission (No. 982/TCLN-BTTN) in Vietnam. We thank Liang Fei, Jodi J.L. Rowley and two anonymous
reviewers for the invaluable comments on the manuscript. This work was supported by the program of Chinese
Academy of Sciences (2015CASEABRI002), the National Natural Science Foundation of China (31090250), the
Ministry of Science and Technology of China (MOST 2011FY120200), and the Animal Branch of the Germplasm
Bank of Wild Species of Chinese Academy of Sciences (the Large Research Infrastructure Funding). Chatmongkon
Suwannapoom was sponsored by the Chinese Academy of Sciences Visiting Fellowship for Researchers (Postdoc)
from Developing Countries (2013FFS130015).
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Accepted by J. Rowley: 26 Apr. 2016; Published: 13 Jul. 2016
Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0
599
ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN
1175-5334
(online edition)
Erratum
Copyright © 2016 Magnolia Press
Zootaxa 4137 (4): 599–600
http://www.mapress.com/j/zt/
http://doi.org/10.11646/zootaxa.4137.4.13
http://zoobank.org/urn:lsid:zoobank.org:pub:60B9B6EF-69C3-481F-885A-071710D1F6F9
CHATMONGKON SUWANNAPOOM, ZHI-YONG YUAN, JIN-MIN CHEN, MIAN HOU, HAI-
PENG ZHAO, LI-JUN WANG, TRUONG SON NGUYEN, TRUONG Q. NGUYEN, ROBERT W.
MURPHY, JAQUELINE SULLIVAN, DAVID S. MCLEOD & JING CHE (2016) Taxonomic revision
of the Chinese Limnonectes (Anura, Dicroglossidae) with the description of a new species from China
and Myanmar. Zootaxa, 4093: 181–200.
Correct versions of Figures 1 and 2:
FIGURE 1. Sampling localities of the specimens of Limnonectes used in this study. New samples indicated by color: red stars
in China = L. longchuanensis sp. nov., blue squares = L. fujianensis, pink diamonds = L. bannaensis, orange circles = L. taylori,
and green triangles = L. fragilis. Samples downloaded from GenBank were indicated by white symbols. Locality numbers refer
to Table 1 and Fig. 2.

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FIGURE 2. Bayesian tree of Chinese Limnonectes inferred from mtDNA 12S rRNA, tRNAVal and 16S rRNA genes. Colors
of lineages, species names, and symbols correspond to Fig. 1. The numbers above branches represent Bayesian posterior prob-
abilities (BPP), and bootstrap support (BS) for maximum likelihood (ML); “-” denotes low support (BPP < 95% or BS < 70%)
in one analysis; no values on branches represent low support in both analyses. The scale bar represents 0.05 nucleotide substi-
tutions per site.