New records of amphibians from Bac Kan Province, Vietnam

Abstract Background Since the establishment of the Nam Xuan Lac Habitat and Species Conservation Area in 2003 in Bac Kan Province, northern Vietnam, only two herpetological studies have been conducted: One recorded 14 species of amphibians from Ban Thi-Xuan Lac area in 2004 and a recent study reported 32 species of amphibians from this protected area in 2019. New information As a result of our field surveys in 2020 and 2021, a total of 23 species of amphibians was recorded from the Nam Xuan Lac Habitat and Species Conservation Area. Eight of them are recorded for the first time from Bac Kan Province, comprising one species of Microhylidae, two species of Megophryidae, one species of Dicroglossidae, two species of Ranidae and two species of Rhacophoridae. Besides morphological descriptions, we provide ecological notes of newly-recorded species of amphibians from Bac Kan Province.


Introduction
The Nam Xuan Lac Habitat and Species Conservation Area (HSCA) was established in 2003 by the People's Committee of Bac Kan. This HSCA encompasses a core zone of 1,788 ha and a buffer zone of 7,508 ha limestone karst forest (Forest Protection Department of Bac Kan Province 2013). In terms of amphibian diversity, Bac Kan Province is one of the most poorly-studied Provinces in northern Vietnam. In their herpetofaunal book of Vietnam,  recorded 27 species of amphibians from Bac Kan Province and most of them were reported from Ba Be National Park. Le et al. (2004) recorded 14 species of amphibians from Ban Thi-Xuan Lac Commune. Recently, Tran (2019) reported 32 species of amphibians from Nam Xuan Lac Habitat and Species Conservation Area.
As a result of our recent field surveys in the Nam Xuan Lac Habitat and Species Conservation Area, Bac Kan Province, we herein report eight new records from this Province.

Materials and methods
Sampling Field surveys were conducted by Anh Mai Luong, Cuong The Pham, Dung Trung Le, Quyen Hanh Do, Tien Quang Phan and Truong Quang Nguyen (hereafter Luong et al.) from 24 to 29 August 2020 and from 22 to 28 April 2021 in Nam Xuan Lac HSCA, Bac Kan Province (Figs 1, 2, 3). The coordinates (WGS 84) and elevations were determined by using the GPS Garmin 60CX.
Specimens were collected between 19:00 and 24:00 h. After taking photographs in life, specimens were euthanised in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002), fixed in 80% ethanol for five hours and then later transferred to 70% ethanol for permanent storage. Tissue samples were preserved separately in 70% ethanol prior to fixation. Specimens referred in this paper are deposited in the collection of the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.

Morphological examination
Measurements were taken on preserved specimens with a set of digital calipers to the nearest 0.1 mm. The following abbreviations are used: SVL = snout-vent length, HL = head length (measured as a parallel line to the vertebral column from posterior margin of mandible to tip of snout), HW = maximum head width (across angles of jaws), RL = rostral length (from anterior corner of orbit to tip of snout), NS = distance from nostril to the tip of snout, EN = distance from anterior corner of orbit to the nostril, IND = internarial distance, IOD = interorbital distance, ED = eye diameter, UEW = maximum width of upper eyelid, DAE = distance between anterior corners of orbits, MN = posterior margin of mandible to nostril, MFE = posterior margin of mandible to anterior corner of orbit, MBE = posterior margin of mandible to posterior corner of orbit; DPE = distance between posterior corners of orbits, TYD = tympanum diameter, TYE = distance from anterior margin of tympanum to posterior corner of orbit, FLL = forearm length, from elbow to base of outer palmar tubercle, HAL = hand length, from base of outer palmar tubercle to tip of third finger, FL1-4 = Finger length I-IV, OPT = outer palmar tubercle length, IPT = inner palmar tubercle length, NPL = nuptial pad length, FeL = femur length (from vent to knee), TbL= tibia length (from knee to tarsus), TbW = maximum tibia width, FoL = foot length (from tarsus to the tip of fourth toe), TL1-5 = toe length I-V and IMT = inner metatarsal tubercle length. For the webbing formula, we followed Glaw and Vences (2007). Sex was determined by the presence of nuptial pads and based on gonadal inspection.  New records of amphibians from Bac Kan Province, Vietnam absent; tongue notched posteriorly. Forelimbs: Forearm slender (FLL 4.2-5.4 mm), hand length (HAL 8.7-10.4 mm); relative finger lengths I < II < IV < III, tips of fingers pointed; fingers free of webbing. Hind-limbs: Thigh slender (FeL 9.1-12.8 mm); tibia five times longer than wide (TbL 10.5-14.6 mm, TbW 1.9-3.1 mm); relative toe lengths I < II < V < III < IV; webbing formula I1-1½II1-2III2-3IV3-2V; tibio-tarsal articulation reaching to the eye when leg adpressed along body. Skin: Dorsum smooth, but with some rather large smooth flattened pustules on front part of dorsum; supratympanic fold indistinct; throat, chest, belly and underside of limbs smooth; cloacal region granular.

Microhyla butleri
Colouration in life: Dorsal surface of head and body grey with brownish and reddish marking, in X-shape; a whitish stripe from eye to anterior shoulder; flank grey with black spots, dorsal surface of limbs grey with dark transverse bars; belly cream; throat and chest mottled with dark brown (Fig. 4) (determination after Bourret (1942), Taylor (1962)).

Distribution
This is a common species in Vietnam , Frost 2021. Elsewhere, this species has been recorded from China, Myanmar, Laos, Thailand, Cambodia, Malaysia and Singapore , Frost 2021.

Ecology
Specimens were found between 19:30 and 20:30 h on the ground. The surrounding habitat was mixed secondary forest of small hardwoods and shrubs.
Colouration in life: Dorsal surface of head and body brown grey with triangular marking between eyes, some grey spots in middle of back; dorsal surface of fore-and hind- limbs brown grey with dark bars; throat and chest transparent grey, border of throat grey with white spots; belly white ( Fig. 5) (determination after Taylor (1962), ).

Distribution
In Vietnam, L. minima was known from Son La, Dien Bien, Hoa Binh, Quang Ninh and Thanh Hoa Provinces (Frost 2021). Elsewhere, this species has been recorded from Thailand and Laos (Frost 2021).

Ecology
Specimens were found on the trees or on the ground along the stream between 19:30 and 21:30 h. The surrounding habitat was mixed secondary forest of hardwoods and shrubs.  Lathrop et al. (1998)).

Distribution
In Vietnam, L. nahangensis was previously known only from Tuyen Quang Province , Frost 2021. The new record of the species from Bac Kan found approximately 19 km from the type locality in Tuyen Quang Province.

Ecology
Specimens were found on the trees along the stream, between 20:00 and 22:00 h. The surrounding habitat was mixed secondary forest of small hardwoods and shrubs.   (1950)).

Distribution
In Vietnam, Q. boulengeri was known from Cao Bang, Son La, Tuyen Quang and Nghe An Provinces , Frost 2021. Elsewhere, this species has been recorded from China (Frost 2021).

Ecology
Specimens were found in the stream, at 20:30 h. The surrounding habitat was mixed secondary forest of medium hardwoods and shrubs.

Description
Size medium (SVL 45.2 mm in male; SVL 53.7 mm in female); head longer than wide (HL 17.1 mm, HW 14.8 mm in male; HL 21.4 mm, HW 18.6 mm in female); snout obtusely round in dorsal view, longer than eye diameter (RL 6.6 mm, ED 5.1 mm in male; RL 8.0 mm, ED 6.6 mm in female); nostrils round, closer to the tip of snout than to eye (NS 2.6 mm, EN 4.1 mm in male; NS 3.4 mm, EN 5.3 mm in female); canthus rostralis distinct, loreal region slightly concave and oblique; tympanum distinct round; vomerine teeth present; tongue deeply notched posteriorly. Forelimbs: Forearm slender (FLL 10.3 mm in male; FLL 11.8 mm in female), hand length (HAL 24.0 mm in male; HAL 30.1 mm in female); relative finger lengths I < II < IV < III, tips of fingers enlarged into discs; fingers free of webbing. Hind-limbs: Thigh slender (FeL 23.7 mm in male; FeL 29.7 mm in female); tibia five times longer than wide in the male (TbL 26.3 mm, TbW 4.6 mm), six times longer than wide in the female (TbL 32.8 mm, TbW 5.0 mm); relative toe lengths I < II < III < V < IV; webbing formula I½-½II0-1III0-1½IV1-0V; tibiotarsal articulation reaching to the nostril when leg adpressed along body. Skin: Dorsal surface of head and body smooth; tiny spinules on flanks, upper edge of eyelid; anterior and posterior edge of tympanum; supratympanic fold indistinct, dorsolateral fold absent; throat, chest, belly and ventral surface of thigh smooth.
Colouration in life: Dorsum and upper part of flanks moss green with brown mottles, dorsal surface of limbs moss green with dark brown cross bars; upper lip with dark brown bars; throat, chest and belly cream with dark brown mottles (Fig. 8) (determination after Mo et al. (2015), ).

Distribution
In Vietnam, O. lipuensis was known from Cao Bang and Tuyen Quang Provinces , Frost 2021. Elsewhere, this species has been recorded from China (Frost 2021).

Ecology
Specimens were found on trees, between 20:00 and 20:30 h, near the waterfall in a rocky stream. The surrounding habitat was mixed secondary forest of small hardwoods, shrubs and vines.
Colouration in life: Dorsal surface light brown; flanks whitish-brown; dorsal surface of limbs with dark brown transverse bars; a small black stripe from nostril to eye; sides of limbs with dark pattern; ventral surface yellowish-white; gular region marbled with grey; ventral surface of hind-limbs yellow ( Fig. 9) (determination after Bourret (1942), Inger et al. (1999)).

Distribution
In Vietnam, R. johnsi was known from Lao Cai and Ha Giang Provinces in the North to Lam Dong and Dong Nai Provinces , Frost 2021. Elsewhere, this species has been recorded from China, Taiwan, Thailand and Cambodia , Frost 2021.

Ecology
Specimens were found on the ground, between 19:00 and 20:30 h. The surrounding habitat was mixed secondary forest of small hardwoods and vines.
Colouration in life: Dorsal surface of head and body, flank green olive, a dark green irregular patch running from between eyes to shoulder; upper and lower lip green olive with some creamy white spots; dorsal part of limbs green olive with transverse dark green bands; throat and margin of throat, chest and belly marbled with white; ventral part of limbs greyish-white ( Fig. 10) (determination after , ).

Ecology
Specimens were found on trees along the trail, between 20:00 and 21:00 h, 2-3 m from the ground. The surrounding habitat consisted of mixed secondary forest composed of small hardwoods and vines.

Discussion
Our new findings bring the total number of amphibian species to 43 in Bac Kan Province ). Le et al. (2004) provided a list of the herpetofauna of Ba Be National Park with 16 recorded species of amphibian.  report six new species of amphibians from Kim Hy Nature Reserve. These authors also recorded Rhacophorus viridimaculatus, a species originally described by  from Ha Giang Province, for the first time from Bac Kan Province. However, the specimens from Nam Xuan Lac HSCA differ from those of R. viridimaculatus by having a smaller interorbital distance in males (IOD 5.1-5.6 mm vs. 8.76 mm in R. viridimaculatus) and the absence vocal sacs in males (vs. present in R. viridimaculatus) ). Further phylogenetic studies will help to elucidate the taxonomic position of these morphologicallysimilar species. The amphibian fauna of Nam Xuan Lac HSCA, Bac Kan Province contains two species of conservation concern: one species endemic to Vietnam (Leptobrachella nahangensis) and one species (Quasipaa boulengeri) listed as Endangered by IUCN (2021). Additional surveys are required to obtain further data on the actual herpetofaunal diversity of Nam Xuan Lac Habitat and Species Conservation Area, as well as of Bac Kan Province. Figure 11.