Yunming MO, Weicai CHEN*, Huaying WU, Wei ZHANGand Shichu ZHOU

1Natural History Museum of Guangxi, Nanning 530012, Guangxi, China2School of Life Sciences, East China Normal University, Shanghai 200062, China

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

Yunming MO1, Weicai CHEN1*, Huaying WU1, Wei ZHANG2and Shichu ZHOU1

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

A new species of the genus 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 fi rst fi nger expanded with circummarginal grooves; smooth, gr ass-green dorsum with irregular brown mottling; pineal body invisible; throat to upper abdomen with gray mottling; do rsal 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 fi nger 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 rR NA 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.

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

1. Introduction

The genus Odorrana Fei, Ye and Huang (Fei et al., 1990) was originally erected for species of Chinese odorous frogs, then Fei et al. (1990) pl aced 12 species of Chinese frogs of the genus Hylarana into a new genus, Odorrana. The genus is diagnosed primarily by external morphology and sternal elements, with mesosternum long and slender and proximally much thicker; a very small and cartilaginous omosternum, with the omosternal bone not forked; and xiphisternal cartilage much larger than omosternum and deeply notched posteriorly (Fei et al., 1990). Currently, th e genus Odorrana contains approximately 55 species occurring in high-gradient streams of Japan, southern China and Indochina west to northeastern India, Myanmar and Thailand and south to Sumatra and Borneo (Fei et al., 2009, 2012; Kuramoto et al., 2011; Frost, 2014). Recent molecular phylogenetic analyses suggested Od orrana formed a well-supported monophyletic group (Chen et al., 2013). All are known to be associated with mountain streams except O. wuchuanensis, which occurs in dark caves.

During 2013–2014, eight Odorrana specimens were collected inside a completely dark karst cave in Lipu County, Guangxi Province, China. Morphologically, these specimens most closely resemble O. yizhangensis and O. schmackeri (Fei et al., 2009, 2012), but differ from O. yizhangensis, O. schmackeri and all other Odorrana from China and adjoining countries. They occur in a similar environment to O. wuchuanensis, inhabiting a dark cave, but are morphologically very different from O. wuchuanensis. Herein, we describe this population as a new species of Odorrana.

2. Materials and Methods

2.1 Sampling During 2013 and 2014, fieldwork was carried out in northeastern Guangxi, China (Figure 1A). Specimens were collected by hand, euthanized and fi xed in 10% formalin and subsequently transferred to 75% ethanol for storage. Muscle tissues from three individuals were sampled and preserved in 100% ethanol for DNA extraction prior to fi xing in formalin. All specimens andtissue samples were deposited in the Natural History Museum of Guangxi (NHMG), China.

2.2 Mor phological analysis Morphological data are obtained from preserved specimens, using dial calipers to the nearest 0.1 mm. The following abbreviations were used: SV L, snout-vent length (from tip of snout to vent); HL , head length (distance between angle of jaws and snout-tip); HW, head width (across angle of jaws); SL, snout length (from the front of eye to the tip of snout); IND, internarial distance; IOD, interorbital distance; UEW, maximum width of upper eyelid; ED, eye diameter; TD, tympanum diameter; TED, tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye); LAHL, length of lower arm and hand; HAL, hand len gth (from elbow to the tip of third fi nger); LAN, width of lower arm; FD3, diameter of third finger disc; HLL, length of hind leg (from vent to tip of the longest toe); TL, t ib ia length; TW, tibia width, FOL, foot length (from the base of inner metatarsal tubercle to the tip of fourth toe) (Table 1). Webbing formula followed Savage and Heyer (1967), and that modified by Myers and Duellman (1982), and Savage and Heyer (1997).

2.3 Genetic analysis Genomic DNA was isolated from three individuals (Voucher Nos.: NHMG1303018; NHMG1303019; NHMG1306002) fol lowing the QIAGEN DNeasy Blood and Tissue Kit according to the manufacturer’s protocol. We used the primers 16SAR and 16SBR of Palumbi et al. (1991) to amplify ~550 base pairs of the 16S rRNA gene. The PCR cycles included an initial denaturation step of 4 min at 95°C and 35 cycles of denaturation for 30s at 94°C, primer annealing for 30s at 52°C, and extension for 1 min at 72°C. The PCR products were electrophoresed on a 0.8% agarose gel and sequenced directly using an ABI 3730IPV automatic sequencer. New DNA sequences obtained in this study were aligned automatically using Clustal X (version 1.83; Thompson et al., 1997) and deposited in GenBank (Accession Nos.: KM388699-KM388701). Homologous fragments of 16S for Odorrana species were downloaded from GenBank (Appendix I). These sequences were trimmed to match the length of the f ragments obtained in this study. Uncorrected pairwise sequence divergence was calculated using MEGA version 5 (Tamura et al., 2011).

3. Results

3.1 Species description

Odorrana lipuensis sp. nov.

Holotype: NH MG1306001, adult male, from a co mpletely dark karst cave of Lipu County, Guangxi, China (110°26′ E, 24°38′ N; altitude: 182 m a.s.l.), collected on June 15, 2013 by Yunming MO and Shichu ZHOU (Figu re 2 A, B). The holotype was collected about 80 m from the entrance (Figure 1 B), which is in perpetual darkness.

Table 1 Measurements (in mm) of O. lipuensis sp. nov. (Abbreviations see Material and Methods). Percent means ratio (R) of each morphometric character to SVL.

Para types: Seven individuals collected at the same locality as holotype. NHMG1303018, NHMG1303019, adult females collected by Weicai CHEN, Yunm ing MO and Shichu ZHOU on March 16, 2013; NHMG1306002, adult male, NHMG1306003, adult female collected by Yunming MO and Shichu ZHOU on June 15, 2013; NHMG20140702, NHMG20140703, adult males, NHMG20140701, adult female collected by Weicai CHEN, Yunming MO and Shichu ZHOU on June 26, 2014.

Diagnosis: Od orrana lipuensis sp. nov. differs from its congeners by the following combination of characters: medium body size (SVL: 40.7–47.7 mm in four adult males, 51.1–55.4 mm in four adult females) (Table 1); dorsolateral fold absent; all except fi rst fi nger expandedwith circummarginal grooves; smooth, grass-green dorsum with irregular brown mottling; pineal body invisible; throat to upper abdomen with gray mottling; ventral part of forelimbs and hindlimbs light pink with mottling; dorsal surfaces of limbs with brown bands; tiny spinules on lateral parts of body, temporal region, and anterior and posterior edge of tympanum; inner metacarpal tubercle oval, elongate; outer metacarpal tubercle oval; white nuptial pad present on fi nger I; males lacking vocal sacs and linea musculina; females having creamy yellow eggs without black poles (Figure 2 A, B, C, E, F).

Descr iption of holotype: Adult male; SVL 47.7 mm; habitus slender; head length slight ly greater than head width (HL = 15.6 mm; HW = 14.6 mm); snout obtusely rounded in dorsal view, projecting beyond lower jaw, round in profile, not depressed; nostril lateral, closer to snout tip than eye; canthu s distinct; pupil horizontally oval; loreal region slightly concave and oblique; snout length (SL = 6.5 mm) greater than eye diameter (ED = 5.5 mm); interorbital distance smaller than width of upper eyelid and horizontal diameter of tympanum; pineal body invisible; tympanum distinct, rounded, 75% eye diameter, not depressed relative to skin of temporal region, tympanic rim slightly elevated relative to tympanum; vomeri ne teeth moderately developed, on two oblique ridges; tongue cordiform, deeply notche d posteriorly; odor absent.

Tips of all except first finger expanded with circummarginal groove s, horizontal grooves present; width of finger III disc about 46% the diameter of tympanum; relative finger lengths, III > IV > II = I ; webbing absent; subarticular tubercles prominent: 1, 1, 2, 2; inner metacarpal tubercle oval, elongate; outer metacarpal tubercle oval; white nuptial pads present (Figur e 2 E).

Ti ps of toes expanded into disc with circummarginal grooves; relative length of toes, I < II < I II < V < IV; webbing formula I 2–– 2 II 1+– 2 III 1+– 3–IV 3–– 2–V; subarticular tubercles present 1, 1, 2, 3, 2; elongate, oval inner metatarsal tubercle, larger than toe I; outer metataral tubercle absent (Figure 2 F).

Skin smooth on upper surfaces; venter smooth; tiny spinules on lateral body, upper edge of lid, temporal region, and anterior and posterior edge of tympanum; weak supratympanic fold from posterior edge of eye to posterior edge of tympanum; dorsolateral fold absent.

Color of holotype in life: Dorsal parts of head and dorsum, fl ank, forelimb, thigh, tibia and foot grass-green w ith irregular brown mottling ; throat, chest, and abdomen with gray mottling; dorsal surfaces of limbs with brown bands; iris black, surrounded by green gold network (Figure 2 A, B).

Color of holotype in preservative: Dorsal parts of head and dorsum, and upper part of flanks brown with gray spots; throat, chest and upper abdomen creamy-white with brown mottling; ventral surface of forelimbs and hindlimbs creamy-white with brown mottling.

Variation: Tip of first finger with expanded disc in females; venter surface creamy-white with gray pigments and throat, chest and upper abdomen with brown mottling in NHMG1306006 and NHMG1303019; vomerine teeth strongly developed in NHMG1303018 and NHMG1303019, the others moderately developed; brown dorsum without gray spots in NHMG1303018 and NHMG1303019; interorbital distance as wide as tympanum diameter in NHMG1303018; skin smooth on upper surfaces with small fl at granules in NHMG1303018.

Measurements of holotype (in mm): SVL = 47.7, HL = 15.6, HW = 14.6, SL = 6.5, IND = 4.4, IOD = 3.7, UEW = 4.0, ED = 5.5, TD = 4.1, TED = 0.8, LAHL = 22.4, HAL = 12.8, LAN = 4.7 , FD3 = 1.9, HLL = 74.6, TL = 24.1, TW = 5.6, LFT = 33.8, FOL = 23.0

Genetic Analyses: Uncorrected sequence divergences between O. lipuensis sp. nov. sequences and all homologous sequences of Odorrana avai lable on GenBank is equal to or greater than 4.9%. Odorrana lipuensis sp. nov. and O. geminata display the smallest distance of genetic differentiation, at 4.9%. The genetic distance between O. lipuensis sp. nov. and O. yizhangensis is 7.9%, O. schmackeri is 10.7%, and O. wuchuanensis is 7.2%. Details are provided in Appendix II.

Etymology: This species is named after the locality where it was collected. The suggested English name is the Lipu Odorous Frog.

Habitat and d istribution: Odorrana lip ue nsis sp. nov. is known only from the type locality. A total of 22 individuals, including adults and sub-adults, were found in a small approximate 2 m2pool (Figure 1 C), about 80 m from the entrance of the cave. The collecting site was in complete darkness. The depth of water was about 60 cm, the water temperature was 20–21°C, and the air temperature was 18–19°C in the cave, tested in March, May, June, July, August, October and December. During the rainy season (generally from May to August), we found that spring water overfl ows from the pool, and some individuals were up to 15 m from the pool (Figure 1 D). In June, we found metamorphs and amplexus behavior (Figure 2 G, H). Two adult females (NHMG1306003 and NHMG20140701, collected in June) contained 59 and 32 creamy yellow eggs respectively, with 3.2–4.2 mm in diameter (Figure 2 C). Thus, it is assumed that the breeding season begins in May or June, and lasts until July. In the same cave, some Diestrammena species and Cryplothelea variegata are found. Outside the cave, no adults and tadpoles of the new species were found during a herpetological survey in the vicinity. However, other species of anurans, including Hylarana guenther i, Microhyla butleri , Microhyla fissipes, Duttaphrynus melanostictus, Fejervarya multistriata, and Rhacophorus mutus, were documented in the vicinity of the cave. Because karst landscapes are well-developed in this area, the new species may range more widely in the region.

3.2 Comparisons Odorrana lipuensi s sp. nov. is morphologically similar to O. yizhangensis and O. schmackeri. However, O. yizhangensis differs from O. lipuensis sp. nov. in having tiny spinules on the chest, visible pineal body and vocal sac (Fei et al., 2009, 2012); Odorrana schmackeri differs from O. lipuensis sp. nov. in having deep brownish markings on dorsum, dark spots on its fl ank, throat to abdomen whitish, males with vocal sacs, and females with eggs with pigmented pole (Fei et al., 2009, 2012). Ecologically, O. lipuensis sp. nov. and O. wuchuanensis occur in similar environments, but O. wuchuanensis differs from O. lipuensis sp. nov. in having a larger body size (SVL, 71–77 mm male versus 40.7–47.7 mm male in O. lipuensis sp. nov.), dark gray venter with whitish texture, and visible pineal body (Figure 2 D).

4. Discussion

Most species of Odorrana occur in cascading streams or slow-moving streams. Prior to this report, we have known only one specie s of Odorrana, O. wuchuanensis, to o ccur inside dark caves. Here we reported a second odorous frog, O. lipuensis sp. nov., which is ecologically similar to O. wuchuanensis, but differs greatly from it in morphology and mitochondrial DNA (Figure 2 B, D). Although O. lipuensis sp. nov. appears similar to O. yizhangensis and O. schmackeri, O. lipuensis sp. nov. is very different from them in ecology and morphology (Fei et al., 2009, 2012). Odorrana yizhangensis and O. schmackeri are cascade frogs, but only O. lipuensis sp. nov. is known to inhabit dark caves, similar to O. wuchuanensis. Genetic distances of 16S rRNA between them is greater than 7%, which is greater than that usually representing differentiation at the species level in frogs (> 3%) (Vences et al., 2005).

Odorrana lipuensis sp. nov. is currently known from the type locality. Considering well-developed karst landscapes in this area, we believe that this new species may range more widely in the region. More fieldwork need to be done to know its actual distribution and population size, and to learn how the new species has adapted to subterranean environments.

Acknowledgements We thank Jianping JIANG, Jiatang LI and Yuchi ZHENG from Chengdu Institute of Biology, Chinese Academy of Sciences, who allowed us to check Chinese odoriferous species, and two anonymous reviewers improved this manuscript. This work was supported by the Natural Science Foundation of Guangxi Province, China (Grant Nos: 2012GXNSFAA053057, 0832238) and the ‘Xibuzhiguang’ Project of Chinese Academy of Sciences.

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Appendix

Material examined

Amolops ricketti (NHMG1305073, NHMG1305074) Mengshan County, Guangxi, China; O. versabilis (NHMG0804069, NHMG1407003) Jinxiu County, Guangxi, China; O. schmackeri (NHMG200805097, NHMG200805098) Jinxiu County, Guangxi, China; O. hejiangensis (CIB93489, CIB93488) Hejiang County, Sichuan, China; O. jingdongensis (CIB98026, CIB98025) Jingdong County, Yunnan, China; O. junlianensis (CIB98027, CIB98028) Puyi County, Guizhou, China; O. kuangwuensis (CIB40990, CIB40991) Nanjiang County, Sichuan, China; O. livida (NHMG1305067, NHMG1305068) Jinxiu County, Guangxi, China; O. tiannanensis (CIB41794) Hekou County, Yunnan, China; O. wuchuanensis (CIB90555, CIB90550) Wuchuan County, Guizhou, China.

NHMG: Natural History Museum of Guangxi

CIB: Chengdu Institute of Biology, Chinese Academy of Sciences

Appendix I Comparative mtDNA sequences examined

Appendix II Uncorrected p-distance distances among some Odorrana species based on partial mitochondrial 16S rRNA sequences.

Dr. Weicai CHEN, from the Natural History Museum of Guangxi, Nanning, China, with his research focusing on taxonomy, population genetics and molecular ecology of amphibian. E-mail: chenweicai2003@126.com

7 September 2014 Accepted: 1 February 2015