Asian Herpetological Research 2015, 6(1): 11–17 ORIGINAL ARTICLE DOI: 10.16373/j.cnki.ahr.140054

A New of Inhabiting Complete Darkness in a Karst Cave in Guangxi,

Yunming MO1, Weicai CHEN1*, Huaying WU1, Wei ZHANG2 and Shichu ZHOU1

1 Natural History Museum of Guangxi, Nanning 530012, Guangxi, China 2 School of Life Sciences, East China Normal University, Shanghai 200062, China

Abstract A new species of the Odorrana is described from a completely dark karst cave of northeastern Guangxi, southern China. The new species, Odorrana lipuensis sp. nov., can be distinguished from its congeners by a combination of the following characters: medium size (SVL: 40.7–47.7 mm in males, 51.1–55.4 mm in females); tips of all but first finger expanded with circummarginal grooves; smooth, grass-green dorsum with irregular brown mottling; pineal body invisible; throat to upper abdomen with gray mottling; dorsal surfaces of limbs with brown bands; dorsolateral fold absent; tiny spinules on lateral body, temporal region, and anterior and posterior edge of tympanum; white nuptial pad present on finger I; males lacking vocal sacs; females having creamy yellow eggs, without black poles. Uncorrected sequence divergences between O. lipuensis sp. nov. and all homologous 16S rRNA sequences of Odorrana available on GenBank is equal to or greater than 4.9%. Currently, the new species is only known from the type locality.

Keywords Odorrana lipuensis sp. nov., karst cave, Guangxi, southern China

1. Introduction monophyletic group (Chen et al., 2013). All are known to be associated with mountain streams except O. The genus Odorrana Fei, Ye and Huang (Fei et al., wuchuanensis, which occurs in dark caves. 1990) was originally erected for species of Chinese During 2013–2014, eight Odorrana specimens were odorous , then Fei et al. (1990) placed 12 species of collected inside a completely dark karst cave in Lipu Chinese frogs of the genus Hylarana into a new genus, County, Guangxi Province, China. Morphologically, these Odorrana. The genus is diagnosed primarily by external specimens most closely resemble O. yizhangensis and morphology and sternal elements, with mesosternum O. schmackeri (Fei et al., 2009, 2012), but differ from long and slender and proximally much thicker; a very O. yizhangensis, O. schmackeri and all other Odorrana small and cartilaginous omosternum, with the omosternal from China and adjoining countries. They occur in a bone not forked; and xiphisternal cartilage much larger similar environment to O. wuchuanensis, inhabiting a than omosternum and deeply notched posteriorly (Fei dark cave, but are morphologically very different from O. et al., 1990). Currently, the genus Odorrana contains wuchuanensis. Herein, we describe this population as a approximately 55 species occurring in high-gradient new species of Odorrana. streams of Japan, southern China and Indochina west to northeastern India, Myanmar and Thailand and south to 2. Materials and Methods Sumatra and Borneo (Fei et al., 2009, 2012; Kuramoto et al., 2011; Frost, 2014). Recent molecular phylogenetic 2.1 Sampling During 2013 and 2014, fieldwork was analyses suggested Odorrana formed a well-supported carried out in northeastern Guangxi, China (Figure 1A). Specimens were collected by hand, euthanized and fixed

* Corresponding author: Dr. Weicai CHEN, from the Natural History in 10% formalin and subsequently transferred to 75% Museum of Guangxi, Nanning, China, with his research focusing on ethanol for storage. Muscle tissues from three individuals , population genetics and molecular ecology of . E-mail: [email protected] were sampled and preserved in 100% ethanol for DNA Received: 7 September 2014 Accepted: 1 February 2015 extraction prior to fixing in formalin. All specimens and 12 Asian Herpetological Research Vol. 6 tissue samples were deposited in the Natural History Table 1 Measurements (in mm) of O. lipuensis sp. nov. Museum of Guangxi (NHMG), China. (Abbreviations see Material and Methods). Percent means ratio (R) of each morphometric character to SVL. 2.2 Morphological analysis Morphological data are obtained from preserved specimens, using dial calipers Males (n = 4) Females (n = 4) Character to the nearest 0.1 mm. The following abbreviations were Range Mean R Range Mean R used: SVL, snout-vent length (from tip of snout to vent); SVL 40.7–47.7 43.4 51.1–55.4 54.1 HL, head length (distance between angle of jaws and HL 14.8–15.6 15.2 35.0% 18.2–20.7 19.1 35.3% snout-tip); HW, head width (across angle of jaws); SL, HW 13.6–14.6 13.9 32.0% 18.2–20.7 19.1 35.3% snout length (from the front of eye to the tip of snout); IND, internarial distance; IOD, interorbital distance; SL 6.5–6.6 6.6 15.2% 7.5–8.0 7.7 14.2% UEW, maximum width of upper eyelid; ED, eye diameter; IND 4.2–4.4 4.3 9.9% 4.3–5.4 5.1 9.4% TD, tympanum diameter; TED, tympanum-eye distance IOD 3.2–3.8 3.6 8.3% 4.1–4.9 4.5 8.3% (from anterior edge of tympanum to posterior corner of UEW 3.7–4.2 4.0 9.2% 4.4–5.4 4.8 8.9% the eye); LAHL, length of lower arm and hand; HAL, ED 4.3–5.5 5.1 11.8% 5.5–6.4 5.9 10.9% hand length (from elbow to the tip of third finger); LAN, TD 3.4–4.1 3.7 8.5% 3.9–4.3 4.1 7.6% width of lower arm; FD3, diameter of third finger disc; HLL, length of hind leg (from vent to tip of the longest TED 0.7–0.8 0.8 1.8% 1.1–1.4 1.3 2.4% toe); TL, tibia length; TW, tibia width, FOL, foot length LAHL 20.1–22.4 21.3 49.1% 26.9–29.1 27.5 50.8% (from the base of inner metatarsal tubercle to the tip of HAL 11.6–12.8 12.3 28.3% 15.6–16.9 16.2 29.9% fourth toe) (Table 1). Webbing formula followed Savage LAN 4.7–5.0 4.9 11.3% 3.3–4.7 4.1 7.6% and Heyer (1967), and that modified by Myers and FD3 1.7–2.0 1.9 4.4% 2.4–3.3 2.9 5.4% Duellman (1982), and Savage and Heyer (1997). HLL 68.2–74.6 73.3 167.3% 87.7–91.8 89.7 165.8% 2.3 Genetic analysis Genomic DNA was isolated TL 24.1–24.5 24.3 56.0% 28.3–30.2 29.6 54.7% from three individuals (Voucher Nos.: NHMG1303018; TW 5.0–5.6 5.3 12.2% 4.7–6.7 5.7 10.5% NHMG1303019; NHMG1306002) following the LFT 30.9–33.8 32.5 74.9% 38.5–41.2 39.9 73.8% QIAGEN DNeasy Blood and Tissue Kit according to the manufacturer’s protocol. We used the primers 16SAR FOL 20.9–23.0 21.7 50.0% 25.9–28.4 27.6 51.0% and 16SBR of Palumbi et al. (1991) to amplify ~550 base pairs of the 16S rRNA gene. The PCR cycles included an completely dark karst cave of Lipu County, Guangxi, initial denaturation step of 4 min at 95°C and 35 cycles China (110°26´ E, 24°38´ N; altitude: 182 m a.s.l.), of denaturation for 30s at 94°C, primer annealing for collected on June 15, 2013 by Yunming MO and Shichu 30s at 52°C, and extension for 1 min at 72°C. The PCR ZHOU (Figure 2 A, B). The holotype was collected about products were electrophoresed on a 0.8% agarose gel 80 m from the entrance (Figure 1 B), which is in perpetual and sequenced directly using an ABI 3730IPV automatic darkness. sequencer. New DNA sequences obtained in this study Paratypes: Seven individuals collected at the same were aligned automatically using Clustal X (version locality as holotype. NHMG1303018, NHMG1303019, 1.83; Thompson et al., 1997) and deposited in GenBank adult females collected by Weicai CHEN, Yunming MO (Accession Nos.: KM388699-KM388701). Homologous and Shichu ZHOU on March 16, 2013; NHMG1306002, fragments of 16S for Odorrana species were downloaded adult male, NHMG1306003, adult female collected by from GenBank (Appendix I). These sequences were Yunming MO and Shichu ZHOU on June 15, 2013; trimmed to match the length of the fragments obtained in NHMG20140702, NHMG20140703, adult males, this study. Uncorrected pairwise sequence divergence was NHMG20140701, adult female collected by Weicai calculated using MEGA version 5 (Tamura et al., 2011). CHEN, Yunming MO and Shichu ZHOU on June 26, 2014. 3. Results Diagnosis: Odorrana lipuensis sp. nov. differs from its congeners by the following combination of characters: 3.1 Species description medium body size (SVL: 40.7–47.7 mm in four adult Odorrana lipuensis sp. nov. males, 51.1–55.4 mm in four adult females) (Table 1); Holotype: NHMG1306001, adult male, from a dorsolateral fold absent; all except first finger expanded No. 1 Yunming MO et al. A New Species of Odorrana 13

Figure 1 (A) Distribution of O. lipuensis sp. nov. and O. wuchuanensis. Red circle represents O. lipuensis sp. nov.; blue circle is O. wuchuanensis; (B) The entrance of karst cave at the type locality of O. lipuensis sp. nov.; (C) Type locality of O. lipuensis sp. nov. in March, 2013; (D) Type locality of O. lipuensis sp. nov. in June, 2013 (rainy season). with circummarginal grooves; smooth, grass-green eyelid and horizontal diameter of tympanum; pineal body dorsum with irregular brown mottling; pineal body invisible; tympanum distinct, rounded, 75% eye diameter, invisible; throat to upper abdomen with gray mottling; not depressed relative to skin of temporal region, ventral part of forelimbs and hindlimbs light pink with tympanic rim slightly elevated relative to tympanum; mottling; dorsal surfaces of limbs with brown bands; vomerine teeth moderately developed, on two oblique tiny spinules on lateral parts of body, temporal region, ridges; tongue cordiform, deeply notched posteriorly; and anterior and posterior edge of tympanum; inner odor absent. metacarpal tubercle oval, elongate; outer metacarpal Tips of all except first finger expanded with tubercle oval; white nuptial pad present on finger I; males circummarginal grooves, horizontal grooves present; lacking vocal sacs and linea musculina; females having width of finger III disc about 46% the diameter of creamy yellow eggs without black poles (Figure 2 A, B, tympanum; relative finger lengths, III > IV > II = I; C, E, F). webbing absent; subarticular tubercles prominent: 1, Description of holotype: Adult male; SVL 47.7 mm; 1, 2, 2; inner metacarpal tubercle oval, elongate; outer habitus slender; head length slightly greater than head metacarpal tubercle oval; white nuptial pads present width (HL = 15.6 mm; HW = 14.6 mm); snout obtusely (Figure 2 E). rounded in dorsal view, projecting beyond lower jaw, Tips of toes expanded into disc with circummarginal round in profile, not depressed; nostril lateral, closer to grooves; relative length of toes, I < II < III < V < IV; snout tip than eye; canthus distinct; pupil horizontally webbing formula I 2– – 2 II 1+ – 2 III 1+ – 3– IV 3– – 2– V; oval; loreal region slightly concave and oblique; snout subarticular tubercles present 1, 1, 2, 3, 2; elongate, oval length (SL = 6.5 mm) greater than eye diameter (ED = inner metatarsal tubercle, larger than toe I; outer metataral 5.5 mm); interorbital distance smaller than width of upper tubercle absent (Figure 2 F). 14 Asian Herpetological Research Vol. 6

Figure 2 (A) Dorsolateral view of the holotype (NHMG1306001) of O. lipuensis sp. nov. in life. Photo: Yunming MO. (B) Ventral view of the holotype (NHMG1306001) of O. lipuensis sp. nov. in life. Photo: Yunming MO. (C) Female with creamy yellow eggs without pigmented poles (NHMG1306003). Photo: Yunming MO. (D) Ventral view of O. wuchuanensis in life. Photo: Jian XU. (E) Ventral view of right hand (NHMG1306001). (F) Ventral view of right foot (NHMG1306001). (G) Tadpole of O. lipuensis sp. nov.. (H) Amplexus behavior of O. lipuensis sp. nov. No. 1 Yunming MO et al. A New Species of Odorrana 15

Skin smooth on upper surfaces; venter smooth; tiny temperature was 18–19°C in the cave, tested in March, spinules on lateral body, upper edge of lid, temporal May, June, July, August, October and December. During region, and anterior and posterior edge of tympanum; the rainy season (generally from May to August), we weak supratympanic fold from posterior edge of eye to found that spring water overflows from the pool, and some posterior edge of tympanum; dorsolateral fold absent. individuals were up to 15 m from the pool (Figure 1 D). Color of holotype in life: Dorsal parts of head and In June, we found metamorphs and amplexus behavior dorsum, flank, forelimb, thigh, tibia and foot grass-green (Figure 2 G, H). Two adult females (NHMG1306003 with irregular brown mottling; throat, chest, and abdomen and NHMG20140701, collected in June) contained 59 with gray mottling; dorsal surfaces of limbs with brown and 32 creamy yellow eggs respectively, with 3.2–4.2 bands; iris black, surrounded by green gold network mm in diameter (Figure 2 C). Thus, it is assumed that (Figure 2 A, B). the breeding season begins in May or June, and lasts Color of holotype in preservative: Dorsal parts of head until July. In the same cave, some Diestrammena species and dorsum, and upper part of flanks brown with gray and Cryplothelea variegata are found. Outside the cave, spots; throat, chest and upper abdomen creamy-white no adults and tadpoles of the new species were found with brown mottling; ventral surface of forelimbs and during a herpetological survey in the vicinity. However, hindlimbs creamy-white with brown mottling. other species of anurans, including Hylarana guentheri, Variation: Tip of first finger with expanded disc Microhyla butleri, Microhyla fissipes, Duttaphrynus in females; venter surface creamy-white with gray melanostictus, Fejervarya multistriata, and Rhacophorus pigments and throat, chest and upper abdomen with mutus, were documented in the vicinity of the cave. brown mottling in NHMG1306006 and NHMG1303019; Because karst landscapes are well-developed in this area, vomerine teeth strongly developed in NHMG1303018 the new species may range more widely in the region. and NHMG1303019, the others moderately developed; 3.2 Comparisons Odorrana lipuensis sp. nov. is brown dorsum without gray spots in NHMG1303018 morphologically similar to O. yizhangensis and O. and NHMG1303019; interorbital distance as wide as schmackeri. However, O. yizhangensis differs from O. tympanum diameter in NHMG1303018; skin smooth on lipuensis sp. nov. in having tiny spinules on the chest, upper surfaces with small flat granules in NHMG1303018. visible pineal body and vocal sac (Fei et al., 2009, 2012); Measurements of holotype (in mm): SVL = 47.7, HL = differs from O. lipuensis sp. nov. 15.6, HW = 14.6, SL = 6.5, IND = 4.4, IOD = 3.7, UEW in having deep brownish markings on dorsum, dark spots = 4.0, ED = 5.5, TD = 4.1, TED = 0.8, LAHL = 22.4, on its flank, throat to abdomen whitish, males with vocal HAL = 12.8, LAN = 4.7 , FD3 = 1.9, HLL = 74.6, TL = sacs, and females with eggs with pigmented pole (Fei et 24.1, TW = 5.6, LFT = 33.8, FOL = 23.0 al., 2009, 2012). Ecologically, O. lipuensis sp. nov. and Genetic Analyses: Uncorrected sequence divergences O. wuchuanensis occur in similar environments, but O. between O. lipuensis sp. nov. sequences and all wuchuanensis differs from O. lipuensis sp. nov. in having homologous sequences of Odorrana available on a larger body size (SVL, 71–77 mm male versus 40.7– GenBank is equal to or greater than 4.9%. Odorrana 47.7 mm male in O. lipuensis sp. nov.), dark gray venter lipuensis sp. nov. and O. geminata display the smallest with whitish texture, and visible pineal body (Figure 2 D). distance of genetic differentiation, at 4.9%. The Odorrana lipuensis sp. nov. differs from O. genetic distance between O. lipuensis sp. nov. and O. amamiensis, O. andersonii, O. aureola, O. bacboensis, yizhangensis is 7.9%, O. schmackeri is 10.7%, and O. O. chapaensis, O. geminata, O. grahami, O. hainanensis, wuchuanensis is 7.2%. Details are provided in Appendix II. O. indeprensa, O. ishikawae, O. jingdongensis, Etymology: This species is named after the locality where O. junlianensis, O. kuangwuensis, O. livida, O. it was collected. The suggested English name is the Lipu lungshengensis, O. macrotympana, O. margaretae, Odorous . O. nasuta, O. orba, O. splendida, O. supranarina, O. and distribution: Odorrana lipuensis sp. tiannanensis, O. versabilis, and O. wuchuanensis in nov. is known only from the type locality. A total of 22 having a smaller body size (SVL > 60 mm male versus individuals, including adults and sub-adults, were found 40.7–47.7 mm male in O. lipuensis sp. nov., and SVL in a small approximate 2 m2 pool (Figure 1 C), about > 70 mm female versus 51.1–55.4 mm female in O. 80 m from the entrance of the cave. The collecting site lipuensis sp. nov.) (Matsui, 1994; Bain et al., 2003; Bain was in complete darkness. The depth of water was about and Stuart, 2005; Stuart and Bain, 2005; Stuart et al., 60 cm, the water temperature was 20–21°C, and the air 2006; Orlov et al., 2006; Li et al., 2008; Bain et al., 2009; 16 Asian Herpetological Research Vol. 6

Fei et al., 2009, 2012). Odorrana lipuensis sp. nov. differs on tympanum (Bain et al., 2003). Odorrana swinhoana from O. andersonii, O. anlungensis, O. cangyuanensis, O. differs from O. lipuensis sp. nov. in having vocal sac exiliversabilis, O. grahami, O. graminea, O. hainanensis, and rough dorsum (Fei et al., 2009, 2012). Odorrana O. hejiangensis, O. huanggangensis, O. lungshengensis, trankieni differs from O. lipuensis sp. nov. in its relative O. macrotympana, O. nanjiangensis,O. nasuta, O. length of finger, III = I > II > IV(versus III > IV > II = I in schmackeri, O. swinhoana, O. tianmuii, O. tiannanensis, O. lipuensis sp. nov.) (Orlov et al., 2003). Odorrana zhaoi O. tormota, O.versabilis, O. yizhangensis, and O. zhaoi differs from O. lipuensis sp. nov. in having tiny spinules in having males without vocal sacs (Fei et al., 2009, on the chest and visible pineal body. 2012). Odorrana lipuensis sp. nov. differs from O. andersonii, O. exiliversabilis, O. grahami, O. graminea, 4. Discussion O. hejiangensis, O. huanggangensis, O. junlianensis, O. kuangwuensis, O. lungshengensis, O. macrotympana, O. Most species of Odorrana occur in cascading streams margaretae, O. nanjiangensis,O. nasuta, O. schmackeri, or slow-moving streams. Prior to this report, we have O. swinhoana, O. tianmuii, and O. versabilis in having known only one species of Odorrana, O. wuchuanensis, females with creamy yellow eggs without pigmented to occur inside dark caves. Here we reported a second poles (Fei et al., 2009, 2012). Odorrana lipuensis sp. nov. odorous frog, O. lipuensis sp. nov., which is ecologically differs from O. absita, O. banaorum, O. exiliversabilis, similar to O. wuchuanensis, but differs greatly from it O. gigatympana, O. hosii, O. monjerai, O. narina, in morphology and mitochondrial DNA (Figure 2 B, O. nasica, O. tormota, O. utsunomiyaorum, and O. D). Although O. lipuensis sp. nov. appears similar to O. yentuensis in lacking dorsolateral folds (Maeda and yizhangensis and O. schmackeri, O. lipuensis sp. nov. Matsui, 1990; Matsui, 1994; Bain et al., 2003; Stuart and is very different from them in ecology and morphology Chan-ard, 2005; Orlov et al., 2006; Matsui and Jaafar, (Fei et al., 2009, 2012). Odorrana yizhangensis and 2006; Tran et al., 2008; Fei et al., 2009, 2012). Odorrana O. schmackeri are cascade frogs, but only O. lipuensis anlungensis, O. chloronota, O. graminea, O. khalam and sp. nov. is known to inhabit dark caves, similar to O. O. rotodora differ from O. lipuensis sp. nov. in having a wuchuanensis. Genetic distances of 16S rRNA between uniform green dorsum with or without dark spots (lacking them is greater than 7%, which is greater than that usually a uniform green dorsum in O. lipuensis sp. nov.) (Bain representing differentiation at the species level in frogs (> et al., 2003; Stuart et al., 2005; Fei et al., 2009, 2012). 3%) (Vences et al., 2005). Odorrana bolavensis differs from O. lipuensis sp. nov. Odorrana lipuensis sp. nov. is currently known from in having spinules on dorsum and venter (versus smooth the type locality. Considering well-developed karst dorsum and venter in the latter) and having black spots on landscapes in this area, we believe that this new species upper and low lip (absent in O. lipuensis sp. nov.) (Stuart may range more widely in the region. More fieldwork and Bain, 2005). Odorrana hejiangensis differs from O. need to be done to know its actual distribution and lipuensis sp. nov. in having prominent tubercles on flank, population size, and to learn how the new species has visible pineal body and vocal sacs (Fei et al., 2009, 2012). adapted to subterranean environments. Odorrana huanggangensis differs from O. lipuensis sp. Acknowledgements We thank Jianping JIANG, Jiatang nov. in having whitish spinules on throat and abdomen LI and Yuchi ZHENG from Chengdu Institute of Biology, and having green dorsum with circular brown spots (Chen Chinese Academy of Sciences, who allowed us to et al., 2010). Odorrana leporipes differs from O. lipuensis check Chinese odoriferous species, and two anonymous sp. nov. in having white supratympanic fold, weak reviewers improved this manuscript. This work was dorsolateral fold, and absence of transverse bands on the supported by the Natural Science Foundation of Guangxi arms and legs (Bain et al., 2003). Odorrana morafkai Province, China (Grant Nos: 2012GXNSFAA053057, differs from O. lipuensis sp. nov. in having yellow-white 0832238) and the ‘Xibuzhiguang’ Project of Chinese lip-stripe extending across upper lip, terminating in Academy of Sciences. glandule above insertion of arm (absent in O. lipuensis sp. nov.) (Bain et al., 2003). Odorrana nanjiangensis References differs from O. lipuensis sp. nov. in its visible pineal body and vocal sacs (Fei et al., 2009, 2012). Bain R. H., Lathrop A., Murphy R. W., Orlov N. L., Cuc H. T. differs from O. lipuensis sp. nov. in having large, round 2003. Cryptic species of a cascade frog from Southeast Asia: black spots on its flanks and covering a layer of skin Taxonomic revisions and descriptions of six new species. Am No. 1 Yunming MO et al. A New Species of Odorrana 17

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