Gracixalus trieng

Rowley, Jodi J. L., Le, Duong Thi Thuy, Hoang, Huy Duc, Cao, Trung Tien & Dau, Vinh Quang, 2020, - A-new-species-of-phytotelm-breeding-frog- (Anura: - Rhacophoridae) - from-the-Central-Highlands-of-Vietnam, Zootaxa 4779 (3), pp. 341-354 : 343-351

Gracixalus trieng ­sp.­nov.

Holotype: AMS R176206 , adult male, collected from tree hole (approx. 10 cm diameter entrance, 45 cm off ground) with eggs ( AMS R176211 ), in montane evergreen forest in Ngoc Linh Nature Reserve , Dak Glei District, Kon Tum Province, Vietnam (15.063º N, 107.865º E, 2045 m; Fig. 1). Collected at 19:45 h on 3 April 2010 by J. J. L. Rowley, Le T. T. D., Dau Q. V. and Hoang D. H.

Paratypes: UNS 00342 / AMS R176204 , adult male, on dead fallen tree, 1 m from ground, in montane evergreen forest in Ngoc Linh Nature Reserve , Dak Glei District, Kon Tum Province, Vietnam (15.034º N, 107.827º E, 2055 m), collected at 19:30 h on 29 March 2010 by J. J. L. Rowley, Le T. T. D., Dau Q . V. and Hoang D. H . UNS 00343 / AMS R176205 , adult male, in tree hole filled with water (35 cm long, 5 cm deep, 1.5 m above ground), in montane evergreen forest in Ngoc Linh Nature Reserve , Dak Glei District, Kon Tum Province, Vietnam (15.063º N, 107.864º E, 2014 m), collected at 19:55 h on 3 April 2010 by J. J. L. Rowley, Le T. T. D., Dau Q . V. and Hoang D. H . AMS R176207 and NCSM 79748 , adult males, in tree hole filled with water (approximately 1 m above ground), in montane evergreen forest in Ngoc Linh Nature Reserve , Dak Glei District, Kon Tum Province, Vietnam (approximately 15.063º N, 107.860º E, 2000 m), collected at approximately 11:00 h on 3 April 2010 by local Gie-Trieng guides (A Phuoc, A Tru, A Nap, A Doi) . UNS 00351 / AMS R176208 , juvenile, on epiphyte leaf, 1 m above ground in montane evergreen/bamboo forest in Ngoc Linh Nature Reserve , Dak Glei District, Kon Tum Province, Vietnam (15.063º N, 107.863º E, 1960 m), collected at 20:10 h on 3 April 2010 by J. J. L. Rowley, Le T. T. D., Dau Q . V. and Hoang D. H . AMS R176209 , juvenile, on plant lead in stream bed in montane evergreen/bamboo forest 3 m from 3 m wide, rocky stream in Ngoc Linh Nature Reserve , Dak Glei District, Kon Tum Province, Vietnam (15.063º N, 107.859º E, 1716 m), collected at night on 4 April 2010 by J. J. L. Rowley, Le T. T. D., Dau Q . V. and Hoang D. H .

Referred­Specimen:­ AMS R176211 , eggs, collected at same time and location as holotype .

FIGURE­1. Collection sites of Gracixalus trieng sp.­nov.­ Collection locality of holotype is marked with a yellow star. Note that the high elevation habitat of the new species is geographically isolated and of all known species in ‘Clade II’ ( Rowley et al. 2011) of the genus, only Gracixalus lumarius occurs in the mapped area (in syntopy with Gracixalus trieng sp.­nov.).

Etymology: The specific name is in reference to the Giẻ Triêng people, most of whom live in Kon Tum Prov- ince in the Central Highlands of Vietnam, and who assisted us greatly during our surveys. The species epithet is used as a noun in apposition.

Suggested­Common­Name: Trieng tree frog (English). Ếch cây giẻ triêng (Vietnamese) .

Diagnosis: The new species is assigned to the family Rhacophoridae by the presence of intercalary cartilage between the terminal and penultimate phalanges of digits, tips of digits expanded into large discs bearing circummarginal grooves, vomerine teeth absent, pupil horizontal, large, conical tubercles/spines present on the upper eye- lid ( Liem 1970; Duellman & Trueb 1986; Brown & Alcala 1994; Delorme et al. 2005), and to the genus Gracixalus by the presence of a dark X, inverted V or inverted Y-shape marking present on the dorsal surface of the trunk ( Fei et al. 2009). Gracixalus trieng sp.­nov.­ is distinguished from all congeners by a combination of 1) body size medium (37.2–41.4 mm in five adult males), (2) snout rounded in dorsal and lateral views, (3) dorsal surface brown or yellowish with a darker brown interorbital crossbar and inverse-Y shape on the back, (4) throat and chest yellow or yellowish brown with pinkish mottling and belly and ventral surfaces of limbs including hands and feet pinkish, (5) tympanum and supratympanic fold distinct, (6) iris pale gold with darker gold radiating out from anterior and posterior edges of pupil, (7) majority of dorsal body and limb surfaces smooth in adults, with some individuals having sparsely distributed low, irregular tubercles, (8) nuptial pads on fingers I and II in adult males, and (9) eggs deposited as a tightly spaced array of non-pendent eggs on the wall of a phytotelmon.

FIGURE­2. Diurnal dorsolateral view (A), nocturnal dorsolateral view (B), dorsal view (C) and ventral view (D) of the holotype of Gracixalus trieng sp.­nov. (AMS R176206) in life.

Description­of­holotype: A medium-sized rhacophorid (37.2 mm), body robust (Figs. 2,3), head length 91% of head width, snout broadly rounded in dorsal view, rounded in profile, canthus rostralis rounded; loreal region slightly sloping, interorbital region convex, nostrils rounded, slightly protuberant, without flap of skin laterally, closer to tip of snout than eye; pupil oval, horizontal, tympanic annulus distinct, approximately one-third of eye diameter, pineal ocellus absent, skin not co-ossified to skull, vomerine teeth absent, choanae small, round, separated by about six times that of choanae diameter; tongue attached anteriorly, deeply notched posteriorly, tooth-like projections on lower jaw absent, supratympanic fold distinct, extending from eye to axilla. Vocal sac present, connected to buccal cavity by pair of distinct, oval openings at bases of jaws, only slightly baggy gular region. Forelimbs relatively robust, fingers narrow; relative length of fingers I <II <IV <III. Tips of all fingers with well-developed discs with circummarginal grooves; discs relatively wide compared to finger width (third finger disc 239% of third finger width at penultimate phalange), third finger disc width greater (131%) than tympanum diameter; slight dermal fringes on fingers, basal webbing at base of fingers II–IV. Subarticular tubercles prominent, rounded, formula 1, 1, 2, 2. Accessory palmar tubercles present; supernumerary tubercles absent; palmar tubercle divided, irregular, flat; thenar tubercle indistinct; prepollex elongate, with oval tubercle; distinct (Fig. 4A), nuptial pads present on inner surface of the prepollex and dorsal and inner surfaces of fingers I and II (Fig. 4B). Relative length of toes I <II <V <III <IV, tips of toes with well-developed discs with distinct circummarginal grooves, wide when compared to toe length. Webbing present, formula I 2 - – 2 + II 1 + – 2 + III 2 - – 3 IV 2 + – 1 V, with slight dermal fringes, subarticular tubercles prominent, rounded, formula 1, 1, 2, 3, 2, inner metatarsal tubercle distinct, outer metatarsal tubercle and supernumerary tubercles absent (Fig. 4C). Dorsal surface smooth, with only sparsely distributed low, irregular, smooth tubercles. Ventral surface of thighs and abdomen coarsely granular, throat finely granular. No dermal fringes on limbs, no tibiotarsal projection, no distinct supracloacal glands.

FIGURE­3. Dorsal (A) ventral (B) view of preserved male holotype of Gracixalus trieng sp.­nov. (AMS R176206). Scale bar = 5 mm.

Colour­of­holotype­in­life: Dorsal surface brown (diurnally) or yellowish-tan (nocturnally) with a darker interorbital crossbar and inverted Y-shaped marking on the back starting between the eye and covering most of head; limbs with dark brown barring (Fig. 2). Patterning more distinct diurnally. Fingers brown or yellowish-tan, webbing pinkish brown. Diffuse darker brown line under canthus rostralis from eye to nostril, no obvious tympanic markings. Flanks mottled brown and dark pink (diurnally) or yellow and pink (nocturnally). Throat and chest mostly yellowish brown, with dark pinkish brown mottling (diurnally) or yellow with pink mottling (nocturnally); belly and ventral surfaces of limbs including hands and feet pinkish brown (diurnally) or pink (nocturnally). Iris pale gold with a sparse network of fine dark gold reticulations darker gold radiating out of anterior and posterior edges of pupil; iris periphery black; eye periphery pale blue posteriorly.

Colour­of­holotype­in­preservative: Dorsal surface as in life, but paler with less distinct markings (Fig. 3). Ventral surface paler, throat pale brown with pale creamy yellow mottling concentrated on chest. Belly and ventral surface of limbs pinkish grey. Dorsal and ventral surfaces of discs on fingers and toes grey.

Measurements: Holotype: SVL 37.2, HDL 13.0, HDW 14.3, SNT 5.9, EYE 4.4, IOD 4.1, TMP 2.2, TEY 1.2, IN 4.1, NS 2.6, EN 2.9, TIB 18.3. ML 12.7, PL 18.1, IMT 2.0.

Variation: Measurements of the type series are shown in Table 2. Paratypes vary in slightly in their colouration and pattern (Fig. 5). Skin texture also varies from almost completely smooth to sparsely distributed with low, irregular tubercles. Tiny, conical tubercles are present on the eyelids, under the eye and on the tympanum in some individuals ( UNS 00342/ AMS R176204 , AMS R176207 and to a lesser extent, NCSM 79748 ). The tympanic annulus is less distinct in NCSM 79748 , UNS 00343 / AMS R176205 and AMS R176206 . Considerable variation in colouration occurs in individuals over time in life, with several specimens changing from mostly pale yellow dorsally and pale pink ventrally without distinct dorsal markings (typical nocturnal coloration), to dark brown dorsally and dark pinkish brown ventrally, with distinct dark brown dorsal markings (typical diurnal coloration) over the course of 24 h (Fig. 2B; AMS R176206 ; UNS 00342/ AMS R176204 , AMS R176207 ). In life, juveniles of the new species ( AMS R176207 , UNS 00351/ AMS R176208 ) have a pale tan dorsum with an indistinct underlying pattern, yellowish flanks, and anterior and posterior surfaces of thighs, with dark brown blotches, and transparent creamy yellow venters ( UNS 00351/ AMS R176208 ) or transparent pale pink venters with white blotches on chest and throat ( AMS R176209 ). In preservative, all but AMS R176207 have a distinct cross-mark on the back. In preservative, all adult paratypes are pinkish-grey ventrally, with variable amounts of indistinct, darker greyish-brown blotches concentrated anteriorly. UNS 00342 / AMS R176204 and the holotype AMS R176206 have darker brown throats and juveniles of the new species ( AMS R176207 , UNS 00351/ AMS R176208 ) have pale grey venters with darker blotches. All paratypes have distinct supratympanic folds and dark brown canthal and supratympanic lines. A single adult male paratype ( AMS R176207 ) had fine, small tubercles on the dorsum in life, and the juvenile paratype UNS 00351 / AMS R176208 (Fig. 5D) was slightly shagreened. All male paratypes have nuptial pads on fingers I and II.

FIGURE­4. (A) Plantar surface of left hand showing nuptial pad on prepollex (marked with red arrow), (B) dorsal surface of left hand showing nuptial pads of Fingers I and II (marked with red arrows), and (C) plantar surface of left foot of the male holotype of Gracixalus trieng sp.­nov. (AMS R176206). Scale bars = 2 mm.

Ecology:­ All specimens were found in ­ montane evergreen forest (Fig. 6A). The holotype was found in a tree hole, with 130 eggs deposited on the trunk of the tree above the water (Fig. 6B). All other adult males in the type series were found in tree holes, and both juveniles were found on arboreal vegetation. All but juvenile AMS R176209

were found away from any streams or ponds. The species occurs in syntopy with G. lumarius . The females, tadpoles and advertisement call of the new species are unknown.

Distribution­and­conservation­status: The new species is known only from between 1716–2055 m asl within Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam. Mount Ngoc Linh and adjacent high-elevation peaks form an isolated area of high elevation and many faunal elements at this location are thought to be endemic ( Abramov et al. 2006, Jenkins et al. 2007). The species was not located at lower elevations, despite surveys over 19 nights during March/ April 2009 and July 2010 at elevations from ~ 930 m and above (Rowley et al. unpub. data). The range of the new species is not expected to extend outside of Mount Ngoc Linh and closely surrounding peaks but may occur in adjacent Quang Nam Province. Thus, we assume that the new species is restricted geographically, likely having an extent of occurrence (EOO) of <1000 km 2. The new species also most likely occurs in one threat-defined location, which has been characterized as an area with continuing decline in the quality of its habitat due to deforestation ( Meyfroidt et al. 2013). Gracixalus trieng ­sp.­nov. therefore likely qualifies as Endangered in accordance with the IUCN Red List of Threatened Species categories and criteria ( IUCN 2012) B1ab(iii).

Comparisons: Within the genus Gracixalus , Gracixalus trieng sp.­nov. is distinguished from Clade I of Rowley et al. (2011); G. gracilipes (Bourret) G. quangi Rowley, Dau, Nguyen , Cao & Nguyen, G. quyeti (Nguyen, Hendrix, Böhme, Vu & Ziegler) and G. supercornutus (Orlov, Ho, & Nguyen) by having a medium body size (adult males 37.2–41.4 mm SVL in five adult males) versus small body size (males <25 mm SVL), brown or yellowish dorsum and a pink or pinkish brown venter (versus greenish dorsum and white or yellow venter), and eggs deposited on wall of a phytotelmon (versus on vegetation above pools for G. gracilipes , G. quangi , and G. supercornutus , unknown in G. quyeti ). The round snout (versus pointed snout) further differentiates Gracixalus trieng sp.­nov. from G. gracilipes , G. quangi and G. supercornutus .

FIGURE­5. Gracixalus trieng sp.­nov.­ (A) ­ male paratype UNS 00342/AMS R176204, nocturnal coloration, (B) ­ male paratype UNS 00343/AMS R176205, diurnal coloration, (C) male holotype ­ AMS R176206, diurnal coloration, (D) juvenile paratype UNS 00351/AMS R176208, diurnal coloration.

FIGURE­6. (A) Montane evergreen and bamboo forest at ~ 2000 m elevation, habitat of Gracixalus trieng sp.­nov. and (B) tree hole with Gracixalus trieng sp.­nov.­ eggs (AMS R176211) deposited, and where the male holotype of G. trieng (AMS R176206) was found.

The new species differs from Gracixalus ananjevae (Matsui & Orlov) by having a medium body size (adult males 37.2–41.4 mm SVL in five adult males), compared to 32.4 mm in one adult male; a yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus greyish white venter [likely in preservative] with no mottling), and adult males with nuptial pads on fingers I and II (versus finger I only).

Gracixalus trieng sp.­nov. is distinguished from Gracixalus carinensis (Boulenger) by having a yellow or yellowish-brown throat and chest with pinkish mottling and pinkish belly (versus white venter with no mottling), adult males with nuptial pads on fingers I and II (versus finger I only; Bossuyt & Dubois 2001). In addition, Gracixalus trieng sp.­nov.­ has less developed webbing (Fig. 4C) compared to Gracixalus carinensis (see Fig. 4B in Matsui et al. 2017).

From Gracixalus guangdongensis (Wang, Zeng, Liu & Wang) , Gracixalus trieng sp.­nov.­ differs by having a greater body size (adult males 37.2–41.4 mm SVL in five adult males, compared to SVL 26.1–34.7 mm), yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus ventral surface of throat, chest and forelimbs dirty white with small dark specks; belly white anteriorly with large dark blotches).

From Gracixalus jinggangensis (Zeng, Zhao, Chen, Chen, Zhang & Wang) , the new species differs by having a larger body size (adult males 37.2–41.4 mm SVL in five adult males, compared to SVL 27.9–33.8 mm), and yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus ventral surface of throat, chest and forelimbs dirty white with small dark specks; belly white anteriorly with large dark blotches).

From Gracixalus jinxiuensis (Hu) , the new species is distinguished by having a larger body size (37.2–41.4 mm SVL in five adult males compared to 23.5 mm in adult male holotype), yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus pale grey-brown with dark marbling), and adult males with nuptial pads on fingers I and II (versus finger I only).

From Gracixalus lumarius Rowley, Le, Dau, Hoang , & Cao, the new species differs by having large, white conical tubercles on the dorsum absent (versus at most sparsely distributed low, irregular tubercles present), iris pale gold with a sparse network of fine dark gold reticulations darker gold radiating out of anterior and posterior edges of pupil (versus a dark gold iris with a dense, relatively uniformly distributed network of black reticulations), yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus uniformly pinkish venter), and adult males with nuptial pads on fingers I and II (versus finger I only).

From Gracixalus medogensis (Ye & Hu) , the new species is distinguished by having a larger body size (37.2– 41.4 mm SVL in five adult males compared to 26.5 mm in one adult male), yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus light grey or whitish venter), adult males with nuptial pads on fingers I and II (versus finger I only), and linea masculina absent (versus present).

From Gracixalus nonggangensis Mo, Zhang, Luo, Zhou & Chen and Gracixalus waza Nguyen, Le, Pham, Nguyen, Bonkowski & Ziegler , the new species differs by having a larger body size (37.2–41.4 mm SVL in five adult males versus <36 mm in adult males), and a yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus white venter with brown spots), adult males with nuptial pads on fingers I and II (versus absent or finger I only).

From Gracixalus sapaensis Matsui, Ohler, Eto & Nguyen , the new species differs by having a yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus ventral surface of throat, chest, belly, and forelimb light yellow), adult males with nuptial pads on fingers I and II (versus finger I only).

From Gracixalus seesom Matsui, Khonsue, Panha & Eto , the new species differs by having a larger body size (37.2–41.4 mm SVL in five adult males versus 21.6–23.0 mm), round (versus pointed) snout, yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus opaque white venter), and adult males with nuptial pads on fingers I and II (versus nuptial pads absent).

From Gracixalus tianlinensis Chen, Bei, Liao, Zhou & Mo , the new species differs by having a larger body size (37.2–41.4 mm SVL in five adult males versus 30.3–35.9 mm), yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus ventral surface of the throat, chest grey with small dark specks and belly creamy white).

From Gracixalus yunnanensis Yu, Li, Wang, Rao, Wu & Yang , Gracixalus trieng sp.­nov.­ differs by having a larger body size (37.2–41.4 mm SVL in five adult males versus 26.0– 34.2 mm), yellow or yellowish brown throat and chest with pinkish mottling and pinkish belly (versus semi-transparent orangish with yellow spots), linea masculina absent (versus present), and adult males with nuptial pads on fingers I and II (versus finger I only).

Mitochondrial­sequence­divergence: The phylogenetic placement of the new species was not well-resolved (Fig. 7). Uncorrected sequence divergences between Gracixalus trieng sp.­nov.­ and all other Gracixalus species for which comparable molecular data are available is> 3.6% at the 16S rRNA gene fragment examined, with the least divergence between the new species and G. guangdongensis (3.6%) and G. sapaensis (3.9%). This degree of pairwise divergence in the 16S rRNA gene in frogs has been interpreted previously as indicative of differentiation at the species level ( Vences et al. 2005).

FIGURE­7. Maximum likelihood tree on ~550 bp section of 16S (mtDNA) gene for Gracixalus trieng sp.­nov.­ and ­ all Gracixalus species for which comparative sequences are available and for outgroups ( Kurixalus effingeri , Philautus aurifasciatus and Rhacophorus reinwardtii ). Node support is indicated on the branches as Maximum Likelihood bootstrap support (<70% = grey. 100% = black).