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NEW RECORDS OF CASCADE FROGS OF THE GENUS Odorrana (Amphibia: Anura: Ranidae) FROM VIETNAM

Odorrana fengkaiensis was recorded for the first time from Vietnam based on a new amphibian collection from Cao Bang, Bac Kan, Bac Giang, Quang Ninh provinces. Sequences of the specimens from Vietnam were identical to those of the paratypes of O. fengkaiensis from China. In addition, O. trankieni was newly recorded from Hoa Binh and Bac Giang provinces, Northern Vietnam, including the first description of female individuals. Genetic divergence between O. trankieni from Vietnam and its sister species, O. nasuta from China, ranged from 1.42 to 2.66%. The intra-specific genetic distance within O. trankieni ranged from 0.68 to 1.88% between the northeastern and northwestern populations.


INTRODUCTION
Cascade frogs of the genus Odorrana have a wide distribution in Asia, from Northeastern India and Southern China eastwards to Japan, throughout Indochina and southwards to Sumatra and Borneo (Frost, 2020). Odorrana belongs to one of the most diverse groups of amphibians with 59 recognized species and more than twenty new species have been described in the last ten years (Frost, 2020). Due to morphologically similarity in the genus, species that were formally thought to be widespread are now being recognized as complexes of species with much narrower distributions (Wang et al., 2015). In Vietnam, Pham et al. (2016a, b) recently discovered a new species (Odorrana mutschmanni Pham, Nguyen, Le, Bonkowski & Ziegler, 2016) and a new country record (O. lipuensis Mo, Chen, Wu, Zhang & Zhou, 2015).
Based on newly collected specimens of amphibians from northern Vietnam, we herein record Odorrana fengkaiensis for the first time from Vietnam. In addition, we report new provincial records of O. trankieni from Hoa Binh and Bac Giang provinces, including the first description of female individuals.

MATRIAL AND METHODS
Field surveys were conducted in Tay (Fig. 1). Specimens were collected between 19:00 and 24:00 h. After photographing in life, specimens were euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons, 2002), fixed in 80% ethanol for five hours, and then transferred to 70% ethanol for permanent storage. Tissue samples were preserved separately in 70% ethanol prior to fixation. Specimens referred to in this paper are deposited in the collections of the Institute of Ecology and Biological Resources (IEBR) and the Vietnam National Museum of Nature (VNMN), Ha Noi, Vietnam. Morphological characters. Measurements were taken with a digital caliper to the nearest 0.1 mm. Abbreviations are as follows: SVL: snout-vent length, HL: head length (from the back of mandible to tip of snout), HW: maximum head width (across angle of jaws), RL: rostral length (from anterior corner of orbit to tip of snout), ED: eye diameter, UEW: maximum width of upper eyelid, IND: internarial distance, IOD: interorbital distance, DAE: distance between anterior corners of eyes, DPE: distance between posterior corners of eyes, NS: distance from nostril to the tip of snout , EN: distance from anterior corner of eye to the nostril, TD: tympanum diameter, TYE: distance from anterior margin of tympanum to posterior corner of the eye, UAL: upper arm length (from axilla to elbow), FAL: fore arm length (from elbow to tip of third finger), TFL: third finger length, fd3: width of disc of finger III, IPT: inner palmar tubercle length, OPT: outer palmar tubercle length, NPL: nuptial pad lengthfinger I, FeL: femur length (from vent to knee), TbL: tibia length (from knee to tarsus), TbW: tibia width, FoL: foot length (from tarsus to the tip of fourth toe), FTL: fourth toe length, IMT: inner metatarsal tubercle length, td4: width of disc of toe IV. For webbing formula we followed Glaw & Vences (2007).
Molecular analysis. The tissue samples of Odorrana were extracted using Dneasy Blood and Tissue kits (Qiagen, Germany) following protocols by the manufacturer. Extracted DNA was then amplified by HotStar Taq Mastermix (Qiagen, Germany). The standard PCR conditions were 95 o C for 15 min to active Taq, 35 cycles at 95 o C for 30 s, 50 o C for 45 s, 72 o C for 1 min, a final elongation at 72 o C for 10 min. The PCR components were 2 µl of each primer at 10 µmol/µl, 5 µl water, 10 µl of Mastermix and 2 µl DNA template. The primers used to amplify a fragment of the mitochondrial DNA 16S gene using a pair of primers AR (5'-CGCCTGTTTATCAAAAA CAT-3') and BR (5'-CCGGTCTGAACTC AGATCACGT-3') (Palumbi et al., 1991). PCR products were visualized using electrophoresis through a 1% agarose gel, marker 1kb, 1X TBE and stained with ethidium bromide and photographed under UV light. Successful amplifications were purified using GeneJet PCR Purification Kit (ThermoFisher Scientific, Lithuania). Cleaned PCR products were sent to 1 st Base (Malaysia) for sequencing in both directions. Sequences were validated with Sequencher v4.10 (Gene Codes, Ann Arbor, MI) with default setting and analyzed using BLAST Tool as implemented in the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov/). The sequences were uploaded on Genbank under accession numbers KX893887-KX893890, KX893900, KX893909.  Description. Morphological characters of specimens from Bac Giang, Cao Bang, Bac Kan and Quang Ninh provinces agreed well with the description of Wang et al. (2015). Size lage (SVL 96.5-105.5 mm, TB ± SD 100.9 ± 2,8 mm, n = 8 in females); head longer than wide (HL/HW 1.07-1.14); snout rounded in dorsal view, projecting beyond lower jaw; nostril lateral, closer to tip of snout than to eye (NS 6.2-7.6 mm, EN 7.8-8.8 mm); canthus distinct; pupil horizontally oval; loreal region slightly concave and oblique; snout length greater than eye diameter (RL/ED 1.14-1.53); internarial distance wider than interorbital distance and upper eyelid (IND 9.7-11.9 mm, IOD 8.1-11.3 mm, UEW 6.4-7.7 mm); tympanum distinct, rounded smaller than eye diameter (TD/ED 0.43-0.54); vomerine teeth present, in two oblique ridges; tongue cordiform, deeply notched posteriorly; females contained creamy yellow eggs with black poles.
Hindlimbs robust, long, thigh shorter than tibia (FeL 47.0-53.4 mm, TbL 56.6-63.9 mm); tibia approximately four to five times longer than wide (TbL/TbW 3.9-4.9); tips of toes expanded with circummarginal grooves; width of toe IV disc smaller than two times of the width of phalange, smaller than width of finger III disc; relative toe lengths I < II < III < V < IV; webbing well developed, to posterior of terminal phalanx; subarticular tubercles present, formula 1, 1, 2, 3, 2; inner metatarsal tubercle elongate; outer metatarsal tubercle absent; tibio-tarsal articulation reaching beyond the tip of snout.
Skin. Dorsal surface of head and body smooth; flanks with tubercles; tiny spinules on upper edge of eyelid, anterior and posterior edge of tympanum; supratympanic fold weakly developed; dorsolateral fold absent; dorsal surface of limbs smooth; throat, chest, belly and ventral surface of thigh smooth.
Coloration in life. Iris black, surrounded by golden network; head and anterior of dorsum green with brown mottling; posterior of dorsum and flanks brown, with some large black spots; dorsal surface of fore and hindlimbs brown with black cross bands; upper lip with black bars; throat, chest cream with dark brown mottling; belly immaculate white.
Ecological notes. The specimens were found between 19:00 and 23:00 on the rocks or on the ground in rocky streams. The surrounding habitat was a mixed secondary evergreen forest or karst forest of medium and small hardwoods and shrubs.
Skin. Dorsal surface of head and body smooth; tiny spinules on flanks, upper edge of eyelid, anterior and posterior edge of tympanum; supratympanic fold present; dorsolateral fold well developed; throat, chest, belly and ventral surface of thigh smooth.
Coloration in life. Iris black, surrounded by red golden network; dorsum olive brown or dark green with some red brown spots; flanks brown with or without dark spots; dorsal surface of fore and hindlimbs brown with dark brown cross bands; upper lip brown or white; throat, chest cream with dark brown mottling; belly immaculate white or cream with dark brown mottling.
Sexual dimorphism. Males are smaller than females (SVL 75.2-84.1 mm, n = 7 vs. SVL 86.2-95.8 mm mm, n = 5, respectively). Males have strong arms, finger I with white nuptial pad, and a pair of gular pouches below jaw articulations. Gravid females containing creamy yellow eggs without black poles.
Ecological notes. The specimens were found between 19:00 and 22:00 on the rocks, in the rocky streams. The surrounding habitat was mixed secondary evergreen forest of medium and small hardwoods with bamboo, shrubs and arrowroot.
Distribution: This species is currently known only from the type locality in Son La Province, northern Vietnam (Frost, 2020). This is the first record of O. trankieni from Hoa Binh and Bac Giang provinces.
Revised diagnosis.
Genetically, Odorrana fengkaiensis samples from Vietnam are identical to those from China, suggesting that the species is widely distributed across southeastern Asia. The species is placed in the same clade as O. hainanensis and O. bacboensis and recovered as a sister species to the former species (Subgroup 4 of Group D) in Ngo et al. (2016). Pairwise genetic divergence between species of the group is between 1.37-3.48%. On the other hand, O. trankieni and O. nasuta are shown to be sister taxa in the Subgroup 3 of Group D (Ngo et al., 2016). The two species have pairwise genetic distance of 1.42-2.66%. The observed molecular variation within O. trankieni ranging from 0.68% to 1.88% is quite substantial, considering the distance between the two closely related species, and warrants further investigation of the different geographic populations in Vietnam.