Distinction of Gracixalus carinensis from Vietnam and Myanmar, with description of a new species

RESEARCH ARTICLE 2017 VOLUME 33 ISSUE 1-4 PAGES 25-37 Distinction of Gracixalus carinensis from Vietnam and Myanmar, with description of a new species Masafumi Matsui 1 *, Annemarie Ohler 2, Koshiro Eto 1, Nguyen Thien Tao 3 1. Graduate School of Human and Environmental Studies, Kyoto University, Yoshida, Nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8501 Japan 2. Institut de Systématique, Évolution, Biodiversité, ISYEB UMR 7205 CNRS, MNHN, UPMC, EPHE, Muséum national d Histoire naturelle, Sorbonne Universités, 57 rue Cuvier, 75005, Paris, France 3. Department of Nature Conservation, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam Gracixalus carinensis was originally described from Myanmar, but samples of the species reported in molecular phylogenetic works were all from Vietnam, far apart from the type locality. Moreover, the voucher specimens used for sequencing seem to have never been critically studied. We newly sequenced specimens from Vietnam and also closely examined morphology of vouchers. As a result, we confirmed that samples treated under this name from Vietnam constitute a single species. Although no molecular data are available for topotypic samples from Myanmar, detailed morphological comparisons revealed that samples from Vietnam are constantly separated from the topotypic samples of G. carinensis by much poorly developed toe webbing. We thus consider the Vietnamese samples as an undescribed species and describe them as G. sapaensis sp. nov. INTRODUCTION In describing a new species of small rhacophorid of the genus Gracixalus Delorme, Dubois, Grosjean, & Ohler, 2005, Matsui et al. (2015) examined mitochondrial phylogeny of the genus including a pair of samples [KUHE (Graduate School of Human and Environmental Studies, Kyoto University) 46401 (GenBank accession LC011938) and 46402 (LC011939)] collected from northern Vietnam and tentatively identified as G. carinensis (Boulenger, 1893). This identification was made based on the fact that the two specimens formed a clade with sequences of G. carinensis already reported by that time (e.g., Yu et al., 2009; Li et al., 2009). However, the DNA sequences used as those of G. carinensis were derived from only several voucher specimens from Vietnam, and it is uncertain whether or not the previous authors actually examined the voucher specimens of the sequences. As noted by Matsui et al. (2015), the species identity of G. carinensis in the previous studies (Delorme et al., 2005; Li et al., 2008, 2009) is doubtful, because at least Vietnamese samples used by Matsui et al. (2015) are morphologically different from Ixalus carinensis from Myanmar housed in the Natural History Museum, London (BM, type series: Bossuyt & Dubois, 2001), Museum für Naturkunde, Berlin (Zoologisches Museum Berlin: ZMB), and Naturhistorisches Museum Basel (NMB), all of which were examined long ago by one of us (M. Matsui). We therefore made literature survey to check identification of vouchers of G. carinensis hitherto reported in molecular phylogenetic works and confirmed that samples previously treated under this name from Vietnam are conspecific with some of the vouchers and newly obtained samples. Unfortunately, no molecular data are Received 06 April 2016 Accepted 14 June 2016 Published Online 17 March 2017 *Corresponding author fumi@zoo.zool.kyoto-u.ac.jp Corresponding editor Gonçalo M. Rosa ISSCA and authors 2017

MASAFUMI MATSUI et al. available for topotypic samples from Myanmar, but detailed morphological comparisons revealed that samples of G. carinensis from Vietnam are constantly separated from Myanmar samples and considered as an undescribed species. MATERIALS AND METHODS This published work has been registered in ZooBank. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser. The LSID for this publication is: urn:lsid:zoobank.org:pub:715aeb13-a80a-489d-a699-44ba34cd2bcd The first specimen now called G. carinensis based on mitochondrial DNA sequence is MNHN (Muséum National d Histoire Naturelle, Paris) 1999.5961 from Lai Chau, Vietnam (as subgenus Aquixalus of genus Aquixalus Delorme, Dubois, Grosjean, & Ohler, 2005: Delorme et al., 2005: GenBank accession AY880589 for 12S and AY880503 for 16S rrna; see discussion). Two specimens (MNHN 1999.5964 and 1999.5966) in a series of MNHN collection containing the above voucher (MNHN 1999.5961) were available for obtaining new sequences. Additionally, we examined sequences of newly obtained samples from Lao Cai [Vietnam National Museum of Nature (VNMN) 4211, 4212, 4358, 4359, and 4366]. We followed Kuraishi et al. (2013) for methods of DNA extraction from tissues preserved in 99% ethanol, and amplification and sequencing of the mtdna fragments. We deposited the resultant sequences [ca. 500 base pairs (bp) of partial sequences of mitochondrial 16S rrna gene] in GenBank (Accession numbers listed in tab. 1). Using the sequences thus obtained, and adding published sequences of G. carinensis, we examined phylogenetic relationships from 443 bp of 16S rrna sequences. As shown in tab. 1, GenBank data of all hitherto known members of the genus, i.e., G. gracilipes Bourret, 1937, G. jinxiuensis (Hu, 1978), G. lumarius Rowley, Le, Dau, Hoang, & Cao, 2014, G. nonggangensis Mo, Zhang, Luo, Zhou, & Chen, 2013, G. quangi Rowley, Dau, Nguyen, Cao, & Nguyen, 2011, G. quyeti (Nguyen, Hendrix, Böhme, Vu, & Ziegler, 2008), G. seesom Matsui, Khonsue, Panha, & Eto, 2015, G. supercornutus (Orlov, Ho, & Nguyen, 2004), and G. waza Nguyen, Le, Pham, Nguyen, Bonkowski, & Ziegler, 2013 were utilized for comparisons. We also added sequences of 12 newly obtained samples of G. jinxiuensis from Lao Cai (tab. 1). As outgroup species, we used sequences of Kurixalus odontotarsus (Ye & Fei, 1993), Philautus aurifasciatus (Schlegel, 1837), and Rhacophorus borneensis Matsui, Shimada, & Sudin, 2013. For tree construction and calculation of genetic distances (uncorrected p-distance), we followed Kuraishi et al. (2013). Morphological study was conducted for vouchers with sequence data (MNHN 1999.5961, 1999.5964, and 1999.5966), together with the remaining 14 specimens in the MNHN series (MNHN 1999.5895 5900, 5903, 5905-5908, 5963, 5965, 5975; catalogued as Philautus carinensis), KUHE 46401 and 46402, and VNMN 4211, 4212, 4358, 4359, and 4366. We took the following 20 body measurements to the nearest 0.1 mm with a dial caliper under a binocular microscope, following Matsui (1984, 1994): (1) snout-vent length (SVL); (2) head length (HL); (3) head width (HW); (4) internarial distance (IND); (5) interorbital distance (IOD); (6) upper eyelid width (UEW); (7) nostril-eyelid length (N-EL); (8) snout length (SL); (9) eye length (EL); (10) tympanum diameter (TD); (11) tympanum-eye length (T-EL); (12) forelimb length (FLL); (13) lower arm and hand length (LAL); (14) first finger length (1FL); (15) inner palmar tubercle length (IPTL); (16) hindlimb length (HLL); (17) tibia length (TL); (18) foot length (FL); (19) inner metatarsal tubercle length (IMTL); and (20) first toe length (1TOEL). We also took measurements for finger and toe disks to the nearest 0.01 mm using a binocular dissecting microscope equipped with a micrometer: (21-24) first to fourth finger disk diameter (1-4FDW); and (25-29) first to fifth toe disk diameter (1-5TDW). We followed the system of description of toe-webbing states used by Savage (1997). For comparisons, we used the data taken for a total 12 samples of true C. carinensis from Myanmar (BM 1974.2.6.24-27, four individuals, including a lectotype); ZMB 11576 (four individuals): NMB 1242-1245. In the univariate comparisons, SVL was compared by Tukey-Kramer test, while the ratios (R) of the remaining characters to SVL were compared by Dunn s multiple comparisons test. The significance level was set at 0.95. SYSTEMATICS In both the maximum-likelihood (ML) and Bayesian (BI: fig. 1) trees, samples of Gracixalus formed a well-supported clade (BS = 94%, BPP = 1.00), and were split into two major groups. Of these, one group containing a clade of G. gracilipes and its relatives (BS = 90%, BPP = 0.99), and G. lumarius was not well supported (BS = 76%, BPP = 0.81), but another group formed a clade with substantial support (BS = 95%, BPP = 0.99) and contained G. jinxiuensis, G. waza, G. nonggangensis, and a clade of Vietnamese G. carinensis (including 26

ALYTES 2017 33 Table 1. Sample of Gracixalus and other species used for DNA analysis (16S rrna, 443 bp) in this study together with the information on voucher, collection locality and GenBank accession numbers. Voucher abbreviations: AMS: Australian Museum; BORN: BORNEENSIS, University Malaysia, Sabah; CIB: Chengdu Institute of Biology; IEBR: Institute of Ecology and Biological Resources, Hanoi; KIZ: Kunmin Institute of Zoology; KUHE: Graduate School of Human and Environmental Studies, Kyoto University; MNHN: Muséum national d Histoire naturelle, Paris; MZB: Museum Zoologicum Bogoriense; NHMG: Natural History Museum of Guangxi; VNMN: Vietnam National Museum of Nature; VNUH: Vietnam National University, Hanoi. Species name Voucher Locality Reference Acc. No. 1 Gracixalus carinensis MNHN 1999.5964 Vietnam, Lao Cai This study LC140969 2 Gracixalus carinensis MNHN 1999.5966 Vietnam, Lao Cai This study LC140970 3 Gracixalus carinensis KUHE 46401 Vietnam, Lao Cai Matsui et al. (2015) LC011938 4 Gracixalus carinensis KUHE 46402 Vietnam, Lao Cai Matsui et al. (2015) LC011939 5 Gracixalus carinensis CIB XM-439 Vietnam, Lao Cai Li et al. (2009) GQ285670 6 Gracixalus jinxiuensis IEBR 2351 Vietnam, Lai Chau Li et al. (2009) EU871425 7 Aquixalus odontotasrus MNHN 1999.5961 Vietnam, Lao Cai Delorme (2004) AY880503 8 Gracixalus sp. VNMN 4211 Vietnam, Lao Cai This study LC140971 9 Gracixalus sp. VNMN 4212 Vietnam, Lao Cai This study LC140972 10 Gracixalus sp. VNMN 4358 Vietnam, Lao Cai This study LC140973 11 Gracixalus sp. VNMN 4366 Vietnam, Lao Cai This study LC140974 12 Gracixalus sp. VNMN 4359 Vietnam, Lao Cai This study LC140975 13 Gracixalus cf. jinxiuensis AMS R173454 Vietnam, Nghe An Rowley et al. (2011) JN862547 14 Gracixalus jinxiuensis KUHE 32453 Laos, Houapan Matsui et al. (2015) LC011937 15 Gracixalus jinxiuensis KIZ 060821126 China, Yunnan Li et al. (2009) EF564525 16 Gracixalus jinxiuensis VNMN 4209 Vietnam, Lao Cai This study LC140976 17 Gracixalus jinxiuensis VNMN 4210 Vietnam, Lao Cai This study LC140977 18 Gracixalus jinxiuensis VNMN 4213 Vietnam, Lao Cai This study LC140978 19 Gracixalus jinxiuensis VNMN 4216 Vietnam, Lao Cai This study LC140979 20 Gracixalus jinxiuensis VNMN 4217 Vietnam, Lao Cai This study LC140980 21 Gracixalus jinxiuensis VNMN 4218 Vietnam, Lao Cai This study LC140981 22 Gracixalus jinxiuensis VNMN 4230 Vietnam, Lao Cai This study LC140982 23 Gracixalus jinxiuensis VNMN 4231 Vietnam, Lao Cai This study LC140983 24 Gracixalus jinxiuensis VNMN 4232 Vietnam, Lao Cai This study LC140984 25 Gracixalus jinxiuensis VNMN 4355 Vietnam, Lao Cai This study LC140985 26 Gracixalus jinxiuensis VNMN 4357 Vietnam, Lao Cai This study LC140986 27 Gracixalus jinxiuensis VNMN 4371 Vietnam, Lao Cai This study LC140987 28 Gracixalus jinxiuensis CIB HN 201108200 China, Hunan Matsui et al. (2015) LC011936 29 Gracixalus jinxiuensis KIZ 060821013 China, Yunnan Yu et al. (2008) EF564524 30 Gracixalus sp. CIB 03320Rao China, Yunnan Li et al. (2009) GQ285669 31 Cracixalus nonggarensis NHMG 20091010 China, Guangxi Mo et al. (2013) JX841318 32 Gracixalus waza IEBR A.2012.2 Vietnam, Cao Bang Nguyen et al. (2013) JX896684 33 Gracixalus quyeti VNUH 160706 Vietanm, Quang Binh Li et al. (2009) EU871428 34 Gracixalus quangi AMS R173417 Vietnam, Nghe An Rowley et al. (2011) JN862539 35 Gracixalus supercornutus AMS R173887 Vietnam, Kon Tum Rowley et al. (2011) JN862545 36 Gracixalus gracilipes MNHN1999.592 Vietnam, Lao Cai Yu et al. (2009) AY880504 37 Gracixalus seesom KUHE 35084 Thailand, Kanchanaburi Matsui et al. (2015) LC011932 38 Gracixalus lumarius AMS R 176202 Vietnam, Kon Tum Rowley et al. (2014) KF918412 39 Kurixalus odontotarsus KIZ 201307071 China, Yunnan Nguyen et al. (2014a) AB933303 40 Philautus aurifasciatus MZB 16395 Indonesia, Java Nguyen et al. (2014b) KJ802924 41 Rhacophorus borneensis BORN 22410 Malaysia, Sabah Matsui et al. (2013) AB781693 27

MASAFUMI MATSUI et al. Figure 1. Maximum-likelihood (ML) tree from a 443 bp sequence of mitochondrial 16S rrna gene for putative Gracixalus carinensis from Vietnam, members of the species currently assigned to Gracixalus, and for outgroups. Numbers above or below branches represent bootstrap supports for ML inferences and Bayesian posterior probabilities. Aquixalus). In the well-supported G. carinensis clade (BS = 93%, BPP = 0.99), all samples, except for one (VNMN 4359), were very similar in sequences. One sequence reported as Aquixalus odontotarsus from Lai Chou (AY880503: MNHN 1999.5961) diverged from Kurixalus odontotarsus with a large distance (p-distance = 14.4%: tab. 2), but was in the G. carinensis clade with small divergences between the others in this clade (p-distance = 0.2-2.7%). Of the other sequences in this group, our new sequences for MNHN 1999.5964 and 1999.5966 were identical, and were very similar to those of KUHE 46401 and 46402 (p-distance = 0.2-0.5%). These sequences also differed only slightly (p-distance = 0.2-1.0%) from those of new sequences of Vietnamese samples (Gracixalus sp.: VNMN 4211, 4212, 4358, 4359, and 4366), and of reported G. carinensis (GQ285670: p-distance = 0-0.5%). One sequence of reported G. jinxiuensis (EU871425) was also within this group with the p-distance of 0.2-0.5%. One exceptional sample (VNMN 4359) differed from the others by the p-distance of 2.4-2.9%. Other samples studied differed from samples in the G. carinensis clade with the p-distance of 3.9% or larger. From these results, it is certain that samples of Vietnamese G. carinensis clade [including reported A. odontotarsus and one, probably misidentified, G. jinxiuensis (EU871425)] hitherto reported are conspecific from the genetic point of view. The vouchers, MNHN1999.5961, 1999.5964, and 1999.5966, were nearly identical morphologically, and were also very similar with the other 14 specimens in the MNHN series. Other specimens in the G. carinensis clade also did not differ essentially from the MNHN series. Thus, all the samples treated as G. carinensis from Vietnam in the previous works are regarded as conspecific with our samples. On the other hand, specimens of G. carinensis from Myanmar in the BM, ZMB, and NMB collections are morphologically nearly uniform, and are generally similar to the Vietnamese G. carinensis. However, they are clearly different in the development of toe webbing, and the Myanmar samples show better development than the Vietnamese ones even in the specimen with the least developed webbing. Unfortunately, molecular information on topotypic G. carinensis from Myanmar is not available at present, but from discrete difference in toe web morphology, we conclude G. carinensis from Vietnam as a new species and describe it as follows: Gracixalus sapaensis sp. nov. Synonymy. Philautus carinensis: Ohler, Marquis, Swan & Grosjean, 2000, Herpetozoa, 13: 71-87; Aquixalus (Aquixalus) carinensis: Delorme, Dubois, Grosjean & Ohler, 2005, Bulletin Mensuel de la Société Linnéenne de Lyon, 74: 166; Kurixalus carinensis: Nguyen, Ho & Nguyen, 2009, Herpetofauna of Vietnam: 527; Gracixalus carinensis: Li, Che, Murphy, Zhao, Zhao, Rao & Zhang, 2009, Molecular Phylogenetics and Evolution, 53: 509. 28

ALYTES 2017 33 Table 2. Uncorrected p-distances (in %) for fragment of 16S rrna between samples of putative Gracixalus carinensis from Vietnam, members of the species currently assigned to Gracixalus, and outgroups. 1, 2 3 4 5 6 7 8-11 12 13 14 15 16-27 28 29 30 31 32 33 34 35 36 37 38 39 40 1, 2 Gracixalus carinensis MNHN 1999.5964, 5966 3 Gracixalus carinensis (LC011938) 0.24 4 Gracixalus carinensis (LC011939) 0.24 0.49 5 Gracixalus carinensis (GQ285670) 0.24 0.49 0.00 6 Gracixalus jinxiuensis (EU871425) 0.24 0.49 0.49 0.49 7 Aquixalus odontotasrus (AY880503) 0.49 0.73 0.73 0.73 0.20 8-11 Gracixalus sp. VNMN 4211-4366 0.73 0.98 0.98 0.98 0.49 0.24 12 Gracixalus sp. VNMN 4359 2.69 2.69 2.93 2.93 2.93 2.69 2.44 13 Gracixalus cf. jinxiuensis (JN862547) 4.65 4.65 4.89 4.89 4.40 4.16 3.90 5.10 14 Gracixalus jinxiuensis (LC011937) 4.65 4.65 4.89 4.89 4.40 4.16 3.90 5.10 0.00 15 Gracixalus jinxiuensis (EF564525) 4.65 4.65 4.89 4.89 4.40 4.16 3.90 5.10 0.00 0.00 16-27 Gracixalus jinxiuensis VNMN 4209-4371 4.65 4.65 4.89 4.89 4.40 4.16 3.90 5.10 0.00 0.00 0.00 28 Gracixalus jinxiuensis (LC011936) 4.89 4.89 5.13 5.13 4.60 4.40 4.60 5.40 2.20 2.20 2.20 2.20 29 Gracixalus jinxiuensis (EF564524) 6.36 6.36 6.60 6.60 6.60 6.85 7.10 7.80 6.40 6.40 6.40 6.40 5.62 30 Gracixalus sp. (GQ285669) 6.36 6.60 6.60 6.60 6.11 6.36 6.11 7.09 5.13 5.13 5.13 5.13 5.38 7.58 31 Cracixalus nonggarensis (JX841318) 7.82 7.82 8.07 8.07 7.58 7.82 8.07 9.05 7.33 7.33 7.33 7.33 7.82 8.07 9.29 32 Gracixalus waza (JX896684) 7.09 7.09 7.33 7.33 6.85 6.60 6.85 7.82 5.87 5.87 5.87 5.87 6.85 7.82 9.29 2.69 33 Gracixalus quyeti (EU871428) 9.54 9.54 9.78 9.78 9.78 10.02 10.27 10.51 10.02 10.02 10.02 10.02 9.78 9.78 10.76 11.20 11.70 34 Gracixalus quangi (JN862539) 7.82 7.82 8.07 8.07 8.07 8.31 8.56 9.54 8.07 8.07 8.07 8.07 8.31 9.29 9.78 9.50 10.30 6.60 35 Gracixalus supercornutus (JN862545) 9.54 9.54 9.78 9.78 9.78 10.02 10.27 11.49 9.54 9.54 9.54 9.54 9.29 10.76 10.76 11.00 11.70 6.85 2.69 36 Gracixalus gracilipes (AY880504) 9.78 9.78 10.02 10.02 10.02 10.27 10.51 10.51 10.76 10.76 10.76 10.76 10.51 11.25 12.47 11.70 12.50 8.80 5.38 7.09 37 Gracixalus seesom (LC011932) 7.09 7.09 7.33 7.33 7.33 7.58 7.58 8.07 8.31 8.31 8.31 8.31 7.82 9.05 9.54 10.00 10.00 7.33 5.13 6.36 6.60 38 Gracixalus lumarius (KF918412) 15.89 15.65 16.14 16.14 15.65 15.65 15.89 15.65 14.43 14.43 14.43 14.43 14.18 16.38 15.16 15.65 15.89 14.91 14.43 15.89 14.91 14.67 39 Kurixalus odontotarsus (AB933303) 14.43 14.43 14.67 14.67 14.67 14.43 14.67 14.91 13.94 13.94 13.94 13.94 14.43 13.94 16.63 16.38 15.65 14.91 16.14 15.65 17.85 15.65 19.32 40 Philautus aurifasciatus (KJ802924) 15.40 15.65 15.16 15.16 15.40 15.65 15.65 15.40 15.16 15.16 15.16 15.16 15.89 15.65 14.91 14.91 14.43 16.63 15.40 16.14 16.63 15.16 19.56 15.89 41 Rhacophorus borneensis (AB781693) 7.82 7.82 8.07 8.07 8.07 7.82 8.07 8.07 7.58 7.58 7.58 7.58 7.33 8.80 9.54 10.76 9.78 10.02 10.51 11.98 11.49 9.78 18.09 13.45 15.89 29

MASAFUMI MATSUI et al. Etymology. The specific epithet is derived from Sa Pa, a district in Lao Cai Province, northern Vietnam, where the new species occurs. Holotype. MNHN 1999. 5961 (fig. 2-4), an adult male from Fan Si Pan (Phan Xi Păng) mountains, Hoang Lien Nature Reserve, Sa Pa District, Lao Cai Province, Vietnam (1250 m a.s.l.), collected on 11 July 1998 by A. Ohler and S. Grosjean. Paratypes. MNHN 1999.5896 and 5906, from Sapa (22 18 14 N, 103 49 02 E, 1650-2000 m a.s.l.) on 4 and 17 February 1998 by S. Swan; MNHN 1999.5895, 5897-5900, 5903, 5905, 5907, and 5908 from Sapa (22 16 26 N, 103 53 36 E, 1620-1660 m a.s.l.) on 19 and 20 February 1998 by S. Swan; MNHN 1999.5963-5966 from Sapa on 12 August 1998 by S. Grosjean; MNHN 1999.5975 from Sapa by S. Swan. KUHE 46401, 46402, two adult males from Tramton Pass, entrance to Mt. Fan Si Pan (1600 m a.s.l.), collected on 30 June 2012 by Kunio Araya. Referred specimens. VNMN 4211, 4212, 4358, 4359, and 4366 (four males and one female) from Y Ty mountain (22 36 38 N, 103 38 37 E, 2200-2340 m a.s.l.), Bat Xat District, Lao Cai Province, collected on 12 October 2014 by Nguyen Thien Tao. Diagnosis. The new species is assigned to the genus Gracixalus from its inclusion in the clade with the type species, G. gracilipes, in the mitochondrial phylogeny. Although it is not synapomorphy of the genus Gracixalus, the diagnosis used for describing the other congeners [presence of intercalary cartilage between Figure 2. Dorsal (A, C) and ventral (B, D) views: A-B. male holotype (MNHN 1999. 5961) of Gracixalus sapaensis sp. nov.; C-D. male lectotype of Ixalus carinensis (BM 1974.2.6.24) after preservation. Scale bar = 10 mm. 30

terminal and penultimate phalanges of digits, tips of digits expanded into large disks bearing circummarginal grooves, vomerine teeth absent, horizontal pupil, and small to medium size (males <40 mm SVL)] applies to the new species. Gracixalus sapaensis sp. nov. is distinguishable from its congeners by a combination of (1) size small to medium (males 21-37 mm, females 27-40 mm SVL), (2) snout rounded, (3) dorsal skin nearly smooth, (4) upper eyelid and dorsum lacking spines, (5) tibiotarsal projection absent, (6) hindlimb moderately long with tibiotarsal articulation usually reaching to center of eye when fully stretched leg adpressed to body, (7) web poorly developed with phalanges free of broad web on fourth toe usually 3 on inner side, and 2 3/4 on outer side, (8) dorsal surface grayish to reddish brown, with darker brown marking on upper eyelid and across back, forming X marking (fig. 3.A), and (9) flank and ventral surface lacking clear dark marking (fig. 3.B). Description of holotype (measurements in mm). Adult male, SVL 35.4 mm; body moderately elongate; head slightly shorter (HL 12.5, 35.4%SVL) than wide (HW 12.6, 35.6% SVL), wider than body; snout rounded in dorsal and lateral views, length (SL 5.6, 15.8% SVL) longer than eye length (EL 5.1, 14.4% SVL), slightly protruding beyond mouth; canthus rostralis rounded; loreal region vertical and slightly concave; nostril oval without flap of skin laterally, slightly closer to tip of snout (S-NL 2.1, 5.9% SVL) than to eye (N-EL 2.4, 6.8% SVL); internarial distance ALYTES 2017 33 Figure 3. Dorsal (A) and dorsolateral (B) views of male holotype (MNHN 1999. 5961) of Gracixalus sapaensis sp. nov. in life. (IND 3.4, 9.7% SVL) slightly wider than interorbital distance (IOD 3.3, 9.4% SVL), which in turn slightly wider than upper eyelid (UEW 3.2, 9.0% SVL); eye large, pupil horizontal; tympanum rather indistinct, subcircular, diameter (TD 2.6, 7.4% SVL) one-half of eye length and separated from eye by one-third of tympanum diameter (T-EL 0.9, 2.5% SVL); pineal ocellus absent; vomerine teeth absent; tongue notched posteriorly, without lingual papilla. Forelimb long (FLL 27.2, 76.8% SVL) and slender; hand and forearm long (LAL 19.7, 55.7% SVL); finger length formula: I < II < IV < III; expanded disks each with circummarginal groove and transverse ventral groove; disks on third and fourth fingers (3FDW 2.3, 6.4% SVL; 4FDW 2.2, 6.2% SVL) only slightly wider than those on second (2FDW 2.2, 6.1% SVL) but much wider than first (1FDW 1.3, 3.6% SVL), all smaller than tympanum; remnant of webbing between inner three fingers; subarticular tubercles prominent, rounded, single, formula 1, 1, 2, 2; supernumerary tubercles present on outer three fingers; inner palmar tubercle flat (IPTL 1.8, 4.9% SVL); outer palmar tubercle divided into two. Hindlimb long (HLL 59.9, 169.2% SVL); tibiotarsal articulation reaching to anterior border of eye when fully stretched leg adpressed to body; heels overlapping each other when thigh (THIGH 16.6, 46.9% SVL) and tibia (TL 18.1, 51.1% SVL) placed at right angle to body; foot (FL 17.3, 48.9% SVL) shorter than tibia; toe length formula I < II < III < V < IV (fig. 4.A); toes bearing expanded disks each with circummarginal groove and transverse ventral groove; widths of outer toe disks (4TDW 2.2, 6.2% SVL; 5TDW 2.0, 5.7% SVL) narrower than those of outer finger disks; webbing formula I 2-2 2/3 II 1 1/2-3 III 2-3 1/3 IV 2 2/3-1 1/2 V (fig. 4.A); subarticular tubercles distinct, rounded, formula 1, 1, 2, 3, 2; supernumerary tubercles present; inner metatarsal tubercle (IMTL 1.2, 3.5% SVL) oval and flat, about one-fourth length of first toe (1TOEL 4.7, 13.4% SVL); no outer metatarsal tubercle (fig. 4.A). Dorsal surface nearly smooth, scattered with minute tubercles with a blunt tip on shout and interorbital 31

32 MASAFUMI MATSUI et al. Figure 4. Ventral views of right foot after preservation of: A. male holotype (MNHN 1999. 5961) of Gracixalus sapaensis sp. nov.; B. male lectotype of Ixalus carinensis (BM 1974.2.6.24) after preservation. Scale bar = 5 mm. space, and sparsely scattered with minute, blunt tubercles between shoulder and sacral regions; skin of head not coossified to forehead; supratympanic fold distinct, continuing as glandular fold to above insertion of forelimb; skin of lower jaw posterolaterally lacking short fold at anterior base of upper arm; side of body coarsely scattered with low, large round tubercles, continuing to flat granules on belly; tarsal fold absent; no tubercles at tibiotarsal articulation; prepollex oval, distinct; dermal fringe on inside of all fingers; dermal fringe along fifth toe, from tip of toe to base of metatarsal; nuptial spines absent on first finger; a pair of long vocal slits on both sides of mouth floor well anterior to jaw commissure. Color. In life, dorsal surface golden ochre with a darker brownish marking on upper eyelid and across back, forming X marking (fig. 3); an oval light yellow transparent spot laterally in front of groin; dorsal side of thigh and tibia golden ochre barred with faint dark brown; rear of thigh light grey with yellow spots; ventral surface of throat, chest, belly, and forelimb light yellow, hindlimb darker yellow; webbing yellowish; iris golden with greenish reflection, with a broad horizontal band and a narrow fine band in lower part. In alcohol, color and pattern generally faded. Dorsal surface grayish brown, with darker brown marking on upper eyelid and across back, forming X marking (fig. 2.A); an oval white spot laterally in front of groin; dorsal side of thigh and tibia barred with faint dark brown (fig. 2.A); ventral surface, including throat, chest, belly, forelimb, and hindlimb whitish (fig. 2.B). Variation. As shown in tab 3, females (mean SVL ± 1 SD: 34.8 ± 3.6 mm) are significantly larger than males (29.9 ± 5.0 mm) (p < 0.05). In body proportions (R), males have significantly smaller R1TOEL (median: 10.2% SVL) than females (12.8% SVL), but otherwise there was no statistically significant sexual dimorphism. Tibiotarsal articulation, when fully stretched leg adpressed to body, usually reaches to posterior border of eye (22.2%), posterior border to center of eye (11.1%), center of eye (55.6%), between eye and nostril (5.6%), and anterior border of eye (5.6%). Degree of toe webbing slightly varies and phalanges free of broad web in adults are 2 3 4 (23.1%), 3 (38.5%) and 3 1 3 (38.5%) on inner side, and 2 1/3 (15.4%), 2 1/2 (7.7%), 2 3/4 (30.8%), 3 (23.1%), and 3 1/3 (23.1%) on outer side on the fourth toe. The color in the paratypes is generally less faded than in the holotype: dorsal part of head, dorsum, and limbs grayish brown or brown, and sometimes reddish brown; flanks lighter than dorsum; interocular crossbar and a cross-mark on back starting between eyes usually dark brown, and sometimes marginally dotted by dark brown; loreal and tympanic regions sometimes dark brown, and tympanum also dark brown in some individuals; posterior part of thigh sometimes darker; two darker bands on forearm and one to three darker bands on tibia; throat whitish but light brown in some; chest and belly usually whitish, but with scattered minute brown spots in some specimens; ventral part of thigh and tibia whitish, but sometimes scattered with minute brown spots or a few large dark blotches; webbing whitish scattered dotted with dark brown. Range. Known only from northwestern Vietnam (fig. 5): Lai Chau (Phong Tho, Tam Duong), Lao Cai (Sa Pa, Bat Xat), and Dak Lak (Chu Yang Sin: Nguyen et al., 2009, as Aquixalus). The known localities vary from 1250-2340 m in altitude. Natural history. Ohler et al. (2000) reported the new species to occur at all the vegetation types they classified (agriculture, scrub, submontane forest, and montane forest) from 1260-2020 m a.s.l. on Fan Si Pan mountains. Specimens were usually found sitting on leaves, rarely branches, up to 2 m from the ground, in the vicinity of mountain streams (median distance observed 3 m). They also reported the new species (as Philautus carinensis) to breed mainly in October-November unlike Ph. odontotarsus (now Kurixalus), and Ph. jinxiuensis and Ph. gracilipes (both now Gracixalus) that breed in July. Comparisons. From phylogenetic analyses using mtdna sequences, Matsui et al. (2015) recognized two genetic clades among species of Gracixalus. Together with G. sapaensis sp. nov. (as G. carinensis), G. jinxiuensis from southeastern China, northern Vietnam, and Laos, G. nonggangensis from Vietnamese border of China, and G. waza from northern Vietnam formed a clade. Gracixalus sapaensis sp. nov. resembles these species in having brownish dorsum and rounded snout. Of these, G. sapaensis sp. nov. most resembles G. carinensis with

ALYTES 2017 33 Table 3. Measurements in types of Gracixalus sapaensis sp. nov. and G. carinensis. SVL (mean ± 1SD, in mm) and medians of ratios (R) of other characters to SVL, followed by ranges in parenthesis. See text for character abbreviations. Gracixalus sapaensis Gracixalus carinensis 7 males 10 females 10 males 2 females SVL 29.2 ± 6.1 (20.8-36.9) 35.1 ± 3.7 (27.2-39.5) 35.3 ± 2.5 (30.2-38.1) 41.1 (40.3-41.8) RHL 36.8 (34.1-39.0) 35.8 (34.0-39.0) 34.3 - RHW 36.9 (34.1-38.6) 37.2 (36.5-40.0) 36.2 (34.6-38.1) 37.0 (36.0-38.0) RIND 9.7 (9.0-12.1) 9.8 (8.8-11.3) 9.5 (9.1-10.1) - RIOD 10.1 (9.4-13.0) 11.3 (9.7-12.6) 8.8 (7.4-10.7) - RUEW 9.0 (7.0-11.0) 9.5 (8.8-11.7) 11.3 (9.6-11.7) - RSL 15.0 (13.7-15.8) 14.4 (13.2-16.2) 12.4 - RN-EL 7.0 (6.7-8.6) 6.7 (5.8-9.3) 7.6 - REL 14.8 (14.2-18.2) 13.2 (13.0-16.2) 13.7 (13.5-13.9) 14.1 (13.4-14.8) RTD 6.4 (4.9-7.4) 6.5 (4.9-7.5) 4.8 (13.5-13.9) 4.9 (4.7-5.0) RE-TL 2.5 (1.6-3.6) 2.0 (1.5-2.8) 2.5 - RLAL 53.7 (48.8-55.6) 54.7 (52.4-56.7) 54.2 - RFLL 67.3 (62.3-76.8) 69.5 (66.3-74.1) - - RIPL 3.8 (3.3-5.2) 4.1 (2.9-5.9) - - R1FL 8.8 (6.7-10.5) 10.8 (8.9-11.9) - - RTL 49.6 (44.7-51.1) 49.3 (48.4-53.4) 48.1 (43.4-50.3) 46.8 (46.7-46.9) RFL 46.0 (42.0-48.9) 47.2 (45.6-49.8) 45.5 (44.3-45.8) 45.7 (45.0-46.4) RHLL 152.0 (144.2-169.2) 156.8 (150.7-169.8) - - RIMTL 3.8 (2.4-5.8) 4.5 (3.3-5.3) 4.5 - R1TOEL 10.2 (7.8-13.4) 12.7 (10.8-14.2) 12.6 - R1FD 3.4 (3.1-3.6) 3.1 (2.0-4.4) 3.4 - R2FD 5.0 (4.0-6.1) 5.2 (4.9-6.0) 5.3 - R3FD 6.1 (4.2-6.5) 5.9 (5.2-7.2) 5.7 (5.3-6.2) 5.7 (5.2-6.2) R4FD 5.3 (4.0-6.2) 5.7 (4.8-6.4) 5.9 - R1TD 3.3 (2.7-4.0) 3.3 (2.0-4.1) 2.8 - R2TD 4.0 (3.5-4.5) 4.2 (3.8-4.4) 3.4 - R3TD 4.4 (3.5-5.0) 4.4 (4.0-5.0) 4.8 - R4TD 4.6 (3.5-6.2) 4.8 (4.2-5.4) 4.5 (4.2-5.3) 4.7 (4.7-4.8) R5TD 4.7 (4.3-5.6) 4.7 (4.4-5.3) 4.8 - which it has long been confused, but differs from it by having less developed toe webbing (tab. 4). On the fourth toe, phalanges free of broad web are 3 1/3 (2 3/4-3 1/3 ) on inner side, and 2 3/4 (2 3/4-3 1/3 ) on outer side (vs. 2 1/2 and 2, respectively, free in G. carinensis) in males. More distinctly, webbing between toes is much more concave than in G. carinensis (fig. 4). Furthermore, in males, eye length relative to SVL is larger [14.8 (14.2-18.2)%] than in G. carinensis [13.7 (13.5-13.9)%]. The new species also looks like G. jinxiuensis but differs from it by larger body size (mean male SVL 29 mm) and smooth dorsum (vs. SVL 24 mm in topotypic male and dorsum scattered with tubercles in G. jinxiuensis). Similarly, G. sapaensis sp. nov. differs from G. medogensis from Xizang, China just as does G. jinxiuensis (SVL 26.5 mm and dorsum scattered with tubercles in G. medogensis). From G. nonggangensis and G. waza, G. sapaensis sp. nov. differs by having nearly smooth dorsum, and venter with dark marking (vs. dorsum with small tubercles, and venter white with dark marbling in G. nonggangensis and G. waza). In having rounded snout G. sapaensis sp. nov. differs from G. gracilipes from southern China and northern Vietnam, G. supercornutus from central Vietnam, G. quangi from north central Vietnam, and G. seesom from Thailand, all with pointed snout. In addition to the snout shape, the new species differs from G. gracilipes in 33

MASAFUMI MATSUI et al. Figure 5. Map of Southeast Asia showing the type localities of Gracixalus sapaensis sp. nov. in Vietnam (1) and of G. carinensis in Myanmar (2), and the locality where putative G. carinensis is recoded in Thailand (3). lacking light temporal marking, and tibiotarsal projection (vs. with white patch under eye to tympanum, and having tibiotarsal projection in G. gracilipes). From G. supercornutus, G. sapaensis sp. nov. differs in lacking spines on dorsum and light marking around eye and tympanum (vs. dorsum covered with large spines, and white patch present under eye to tympanum in G. supercornutus). Gracixalus sapaensis sp. nov. differs from G. quangi in having nearly smooth dorsum and lacking tibiotarsal projection (vs. dorsum scattered with small asperities, and tibiotarsal projection present in G. quangi). The new species differs from G. seesom in in larger body size (SVL 21-37 mm in males and 27-40 mm in females) and having dark brown markings on dorsum (vs. males 21-24 mm, females 23-25 mm SVL, with monotonous tan dorsum in G. seesom). Gracixalus sapaensis sp. nov. resembles G. quyeti from central Vietnam in having a round snout, but differs from it in dorsal skin texture and coloration (dorsum smooth and gray brown with dark marking vs. dorsum with strong tubercles and yellowish-olive to moss green in G. quyeti). The new species differs from G. lumarius from central Vietnam in smaller body size and (SVL 21-37 mm in males and 27-40 mm in females) and lack of conical asperities on dorsum (vs. SVL 39-42 mm, dorsum with distinctive, white conical asperities in G. lumarius). DISCUSSION The accuracy of phylogenetic hypotheses largely depends upon the correct identification of the taxa studied. In the case of Vietnamese G. carinesis, here described as G. sapaensis, the name was first used for the specimen MNHN 1999.5961 from Lai Chau. Until that time, the species (G. carinesis) was not recorded from Vietnam (e.g., Bourret, 1941). According to GenBank records, mitochondrial DNA sequence of MNHN 1999.5961 was first studied and registered as AY880589 for 12S rrna and AY880503 for 16S rrna by Delorme in her unpublished doctoral thesis of Museum National d Histoire Naturelle (Delorme, 2004), and published in Delorme et al. (2005), where the specimen was placed in a new genus/subgenus and called Aquixalus (Aquixalus) carinensis. Details of the original identification of the voucher specimen (as Philautus: Ohler et al., 2000) are unknown. However, Aquixalus sensu stricto was later synonymized with the genus Kurixalus Ye, Fei, & Dubois in Fei, 1999, because the type species, Philautus odontotarsus Ye & Fei, 1993 was later transferred to Kurixalus, while Vietnamese A. (A.) carinensis proved to be close to A. (Gracixalus) gracilipes, which subgenus was then elevated to a genus rank (Li et al., 2008). Through this process, the voucher specimen of AY880503 (MNHN 1999.5961) from Vietnam became Gracixalus carinensis (Li et al., 2009). Additional sequences reported as G. carinensis are few [e.g., GQ285670 for ROM 39660 (CIB XM-439), Li et al. (2009)]. The DNA sequences previously reported as those of G. carinensis were derived from only several voucher specimens, whose identification is uncertain regarding the authors actual examination of the vouchers. In the collection of MNHN, the series of G. carinensis from Lai Chau is reported to contain 26 specimens other than 1999.5961 (Ohler et al., 2000: 1999.5895-5915, 1999.5963-5966, 1999.5975). In addition to sequence MNHN 1999.5964 and 1999.5966, we morphologically examined 14 of them (MNHNP 1999.5895-5900, 5903, 5905-5908, 5963, 5965, 5975), as well as MNHN 1999.5961, because the series actually includes several heterospecific specimens. Our mtdna trees suggest that the sequence of a Vietnamese G. jinxiuensis [EU871425, voucher IEBR 2351 (Truong et al., 2008; Li et al., 2009)], forms a strongly supported clade with the Vietnamese G. carinensis, with the genetic distance (uncorrected p-distance) from G. carinensis of only 0.4%. We also confirmed close 34

ALYTES 2017 33 Table 4. Development of toe webbing in two species of Gracixalus as shown by the number of phalanges free of broad web. Medians marked by gray. 1 st outer 2 nd inner 2 nd outer 3 rd inner 3 rd outer 4 th inner 4 th outer 5 th inner 1 1/3 1 1/2 1 3/4 2 2 2 1/3 2 1/2 2 3/4 1 1/3 1 1/2 1 3/4 2 2 2 1/3 2 1/2 2 3/4 3 1 1/3 1 1/2 1 3/4 2 2 1/3 2 2 1/3 2 1/2 2 3/4 3 3 1/3 1 1/4 2 2 1/3 2 1/2 2 3/4 3 3 1/3 1 1 1/3 1 1/2 1 3/4 2 males 5 2 1 2 4 1 1 2 2 1 4 1 1 3 3 1 1 2 2 1 G. sapaensis females 2 2 3 2 3 3 2 5 1 1 4 1 1 2 2 3 1 2 4 2 2 1 1 2 2 1 6 1 young 2 1 1 2 3 2 2 1 2 1 1 1 1 1 1 1 2 1 1 2 total 2 4 9 3 5 4 4 2 12 1 1 1 5 5 5 3 5 8 1 1 4 6 6 1 2 1 5 5 3 4 9 4 males 1 1 1 3 2 1 1 1 1 1 1 1 3 3 2 1 females 1 1 1 1 1 1 1 1 G. carinensis total 1 1 2 4 3 1 1 1 2 1 2 1 1 3 3 1 2 2 morphological uniformity of the MNHN series, suggesting their conspecific relationships (i.e., G. sapaensis sp. nov.). Therefore, we surmise that the Vietnamese frogs so far called G. carinensis, including at least part of the Vietnamese sample of K. odontotarsus in Delorme et al. (2005) and Grosjean et al. (2008), and G. jinxiuensis in Nguyen et al. (2008) and Li et al. (2009), are regarded as a single species, G. sapaensis sp. nov. This conclusion also confirms Yu et al. (2009), who reported that the sample of A. odontotarsus from Vietnam is not conspecific with K. odontotarsus from the type locality in China. Compared with type series of G. carinensis, the new species is morphologically distinguishable with distinctly more poorly developed and distinctly concave toe webbing, although they are otherwise very similar. Gracixalus carinensis was collected from Karin Bia-po, Karin Hills (2900-3500 ft), and Thao, all in Burma (now Myanmar: Boulenger, 1893; Bossuyt & Dubois, 2001) (fig. 5). Otherwise, records of the species are available from Thailand (Chan-ard, 2003; Khonsue & Thirakhupt, 2001), although these records are incomplete and not traceable with regard to the species identification. These records seem to be based on Chan-ard et al. (1999:18), where Philautus carinensis is listed as unpublished data of Chan-ard. Van Dijk & Swan (2004) noted that the species (as Kurixalus) was recorded twenty years ago in Thailand from 1300 m a.s.l. in Doi Inthanon National Park in Chiang Mai Province, but no reference is provided. Doi Inthanon is situated much nearer to the type locality Karin Bia-po than to northern Vietnam (fig. 5), and although the possibility of morphocline from Myanmar through Thailand to Vietnam cannot be precluded, the Thai population, if indeed the records are correct, seems to be assignable to G. carinensis and not to G. sapaensis. Molecular information on topotypic G. carinensis is not available, and recent papers of Myanmar amphibians (e.g., Wilkinson et al., 2003; Wogan et al., 2003; Wogan, 2012; Zug, 2015) do not refer to the species. It is thus inevitable in the future to obtain detailed information of G. carinensis from Myanmar so as to further clarify specific distinctness from G. sapaensis and clarify its phylogenetic relationships within the genus Gracixalus. ACKNOWLEDGEMENTS We thank the directorate of the Forest Protection Department of Lao Cai Provinces for issuing relevant permit and support of his fieldwork. We also thank K. Araya for providing specimens, and N. Kuraishi and K. Nishikawa for laboratory assistance and facilities. Matsui thanks C. McCarthy and B. T. Clarke (BM), R. Gu nther and U. Manthey (ZMB), and A. Hänggi (NMB) for allowing him to examine specimens under their care, S. Panha and W. Khonsue for providing important information, and L. Pierre and the staff of the Reptiles and Amphibian collections, Direction des collections (MNHNP) for the loan of specimens. Examination of museum specimens by Matsui was made possible by grants from the Monbusho International Scientific Research Programs (Field Research Nos. 01041051, 02041051). Fieldwork of Swan and Ohler was conducted in the framework of biodiversity studies of Frontiers Vietnam and collaboration of IEBR (Hanoi, Vietnam). 35

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