Bioacoustics of Gracixalus trieng

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. TABLE­2 . Measurements (mm) of Gracixalus trieng sp.­nov. Abbreviations defined in text. *holotype. UNS 00342/ AMS R176204 UNS 00343/ AMS R176205 AMS­ R176206* AMS R176207 NCSM 79748 UNS 00351/ AMS R176208 AMS R176209 Sex adult male adult male adult male adult male adult male juvenile juvenile SVL 40.9 37.8 37.2 38.2 41.4 24.3 19.9 HDL 12.5 12.2 13 13.4 13.8 9.4 7.5 HDW 14.9 14.2 14.3 14.5 15.1 9.4 7.5 SNT 6.1 5.3 5.9 5.6 6.0 3.5 2.9 EYE 4.4 4.0 4.4 4.7 4.8 3.0 2.9 IOD 4.6 3.9 4.1 4.0 3.9 2.9 2.7 TMP 1.8 1.7 2.2 2.1 2.0 1.3 1.1 TEY 1.1 1.5 1.2 1.6 1.7 0.8 0.7 IN 4.4 4.2 4.1 4.2 4.5 2.9 2.3 NS 2.5 2.3 2.6 2.7 2.3 1.5 0.9 EN 3.2 2.8 2.9 3.2 3.7 2.2 1.9 TIB 19.2 19.3 18.3 18.7 20.2 12.3 9.3 ML 14.0 13.3 12.7 14.0 14.0 7.6 6.4 PL 18.6 17.8 18.1 18.7 19.7 10.8 7.1 IMT 2.1 1.9 2.0 2.2 2.2 0.9 - HW/HL 1.19 1.16 1.10 1.08 1.09 1.00 1.00 HL/HW 0.84 0.86 0.91 0.92 0.91 1.00 1.00 TIB/SVL 0.47 0.51 0.49 0.49 0.49 0.51 0.47 HDL/SVL 0.31 0.32 0.35 0.35 0.33 0.39 0.38 TMP/EYE 0.41 0.43 0.50 0.45 0.42 0.43 0.38 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).