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A New Species of Phytotelm Breeding Frog (Anura: Rhacophoridae) from the Central Highlands of Vietnam

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A New Species of Phytotelm Breeding Frog (Anura: Rhacophoridae) from the Central Highlands of Vietnam Zootaxa 4779 (3): 341–354 ISSN 1175-5326 (print edition) https://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2020 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4779.3.3 http://zoobank.org/urn:lsid:zoobank.org:pub:74D8F480-0D91-4060-9B77-F366442C0D38 A new species of phytotelm breeding frog (Anura: Rhacophoridae) from the Central Highlands of Vietnam JODI J. L. ROWLEY1,2,*, DUONG THI THUY LE3,4,5, HUY DUC HOANG3,4,6, TRUNG TIEN CAO7 & VINH QUANG DAU8 1Australian Museum Research Institute, Australian Museum, 1 William Street, Sydney, New South Wales 2010, Australia. jodi.row- [email protected]; https://orcid.org/0000-0002-2011-9143; Phone: +61 2 9320 6014 2Centre for Ecosystem Science, School of Biological, Earth and Environmental Sciences,University of New South Wales, Sydney, New South Wales 2052, Australia. 3University of Science, Faculty of Biology and Biotechnology, 227 Nguyen Van Cu, 5 District, Ho Chi Minh City, Vietnam. 4Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City. 5 [email protected]; https://orcid.org/0000-0002-3832-7944 6 [email protected]; https://orcid.org/0000-0001-6528-193X 7Institute of Biology, Chemistry and Environment, Vinh University, 182 Le Duan St, Vinh City, Vietnam. [email protected]; https://orcid.org/0000-0002-4256-8951 8Hong Duc University, 565 Quang Trung Street-Dong Ve Ward-Thanh Hoa City, Vietnam. [email protected]; https://orcid.org/0000-0002-8976-1644 *Corresponding author Abstract We describe a new species of phytotelm-breeding rhacophorid frog from central Vietnam. 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. The species occurs in syntopy with G. lumarius. At present, Gracixalus trieng sp. nov. is known only from montane bamboo and evergreen forest (>1700 m) on Mount Ngoc Linh and adjacent peaks; and it is likely to be restricted to high-elevation forest with an estimated geographical distribution of <1000 km2. Key words: Anura, Gracixalus, Southeast Asia, Vietnam Introduction The family Rhacophoridae contains over 400 species of primarily arboreal frogs distributed throughout sub-saharan Africa, China, Southeast Asia, Japan, Taiwan, the Philippines, and the Greater Sunda Islands (AmphibiaWeb 2020; Frost 2020). The genus Gracixalus Delorme, Dubois, Grosjean & Ohler 2005 is known from southern and south- western China, Vietnam, Laos, Thailand, and Myanmar and presently contains 16 species (Frost 2020). Although evolutionary relationships in the group are not well-resolved, species in the genus generally fall within two pheno- typic and phylogenetic groups; small-bodied frogs with a greenish dorsum and often a pointed snout (‘Clade I’ of Rowley et al. 2011), and medium-bodied frogs with a brownish dorsum and rounded snout (‘Clade II’ of Rowley et al. 2011). The recognized species diversity in the group has increased dramatically in recent years, with over half of the known species of Gracixalus described in the last 10 years (AmphibiaWeb 2020; Frost 2020). During field work at high-elevations in central Vietnam in 2009 and 2010, we discovered two sympatric, medi- um-sized phytotelm-breeding rhacophorid resembling species in the Gracixalus ‘Clade II’ of Rowley et al. (2011). Accepted by E. Quah: 21 Apr. 2020; published: 20 May 2020 341 Licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ We were unable to assign either to known species, and the most morphologically distinct—a striking taxon with a distinctive tuberculate dorsal texture and yellow and pink nocturnal colouration—was later described as the Thorny Tree Frog (Gracixalus lumarius; Rowley et al. 2014). Here we describe the less morphologically distinct of the two forms as a new species. Materials and methods Specimens were deposited at the Australian Museum (AMS), the University of Science Ho Chi Minh City (UNS), and the North Carolina Museum of Natural Sciences (NCSM). We recorded morphological data from specimens fixed in 10% formalin and then stored in 70% ethanol. Morphometric data were taken (to the nearest 0.1 mm) with digital callipers. Measurements include snout-vent length (SVL); head length from tip of snout to rear of jaws (HDL); head width at the commissure of the jaws (HDW); snout length from tip of snout to the anterior corner of eye (SNT); diameter of the exposed portion of the eyeball (EYE); interorbital distance (IOD); horizontal diameter of tympanum (TMP); distance from anterior edge of tympanum to posterior corner of the eye (TEY); internarial space (IN); distance from front of eye to nostril (EN); tibia length with the hindlimb flexed (TIB); manus length from tip of third digit to base of tubercle on prepollex (ML); and pes length from tip of fourth toe to base of the inner metatarsal tubercle (PL). We use a traditional formula for finger numbering rather than one based on homology (e.g., Alberch & Gale 1985). Sex was determined by the presence of nuptial pads, vocal sacs and/or gonadal inspection. Mass was recorded in life (to the nearest 0.1 g), using Pesola scales. Radiographs of the holotype were prepared to examine osteological features including the presence of intercalary cartilage between terminal and penultimate phalanges of digits, and the shape of the distal end of the terminal phalanges. Webbing formula follows that of Myers and Du- ellman (1982) modified by Savage and Heyer (1997). We obtained comparative morphological data from specimens (Appendix I), photographs of these specimens in life and from the literature (original descriptions supplemented by information from Bossuyt & Dubois 2001; Rowley et al. 2011, 2014; Fei et al. 2009, 2010; Mo et al. 2013; Matsui et al. 2015, 2017; Zeng et al. 2017; Chen et al. 2018; Wang et al. 2018; Yu et al. 2019). Photographs of G. carinen- sis type material were also examined (lectotype BMNH 1947.2.6.24 and 1947.2.6.27, and MSNG 29852.A [three specimens]). In order to support the generic placement of the new species, we obtained tissue samples from the new spe- cies, sequenced one fragments of mitochondrial DNA (see below) and compared these sequences to species cur- rently assigned to the genus Gracixalus (Frost 2020) and for which GenBank data (trimmed to match the length of the fragment obtained here) were available (Table 1). Specimens of Kurixalus effingeri (type species of the genus Kurixalus), Philautus aurifasciatus (type species of the genus Philautus) and Rhacophorus reinwardtii were used as outgroups (sensu Rowley et al. 2011). Total genomic DNA was extracted from tissues using DNeasy tissue extraction kits (Qiagen). We used the prim- ers 16SAR and 16SBR of Palumbi et al. (1991) to amplify ~550 base pairs of the 16S rRNA mitochondrial gene. PCR amplification was carried out in 25μl reactions with 100 ng of genomic DNA, 1 x Reaction Buffer (Bioline My Taq Red Reagent Buffer), 2 pmol corresponding primers and Bioline My Taq Red DNA polymerase (0.5 unit). Negative controls were included in each PCR. Thermocycling was performed on an Eppendorf Mastercycler EpS (Eppendorf, Hamburg, Germany) under the following conditions: 16S, initial denaturation 94ºC (2 mins), 2 cycles of 94ºC (20s) denaturation, 52ºC (40s) annealing and 72ºC (60s) extension, followed by 33 cycles of 94ºC (20s) denaturation, 50ºC (40s) annealing and 72ºC (50s) extension, followed by a final extension step at 72ºC for 5 mins. PCR products were purified using ExoSap-IT (USB Corporation, OH, USA). Purified templates were sequenced directly by Macrogen (Seoul, Korea). Sequences were edited using Sequencher 4.10 (Gene Codes, Ann Arbor, MI) and aligned using MAFFT (Katoh et al. 2002) on the CIPRES Science Gateway (Miller et al. 2010). We used Akaike Information Criterion as implemented in jModelTest 2.1.6 (Darriba et al. 2012) and installed on the CIPRES Gateway to select the best-fit model of nucleotide substitution, which was then used in model-based phylogenetic inference. Maximum likelihood (ML) analyses were performed on the data matrix using RAxML, also installed on the CIPRES Gateway, using default settings, a GTRCAT model and 100 bootstrap replicates (Stamatakis et al. 2007). We consider branches receiving ≥70% bootstrap support to be well-supported following Hillis and Bull (1993). Uncorrected pairwise sequence divergence (with pairwise deletion of gaps and missing data) was calculated using MEGA 5 (Tamura et al. 2011). 342 · Zootaxa 4779 (3) © 2020 Magnolia Press ROWLEY ET AL. TABLE 1. Samples and sequences used in molecular analyses. Locality Voucher no. GenBank no. Gracixalus cf. ananjevae Nghe An Province, Vietnam VNMN 03012 JN862546 Gracixalus gracilipes Yunnan Province, China KIZ060821196
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