Đang tải... (xem toàn văn)
DSpace at VNU: First Record of the Genus Oreolalax (Anura: Megophryidae) from Vietnam with Description of a New Species...
First Record of the Genus Oreolalax (Anura: Megophryidae) from Vietnam with Description of a New Species Author(s): Truong Q Nguyen , Trung M Phung , Minh D Le , Thomas Ziegler , and Wolfgang Böhme Source: Copeia, 2013(2):213-222 2013 Published By: The American Society of Ichthyologists and Herpetologists DOI: http://dx.doi.org/10.1643/CH-12-021 URL: http://www.bioone.org/doi/full/10.1643/CH-12-021 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use Usage of BioOne content is strictly limited to personal, educational, and non-commercial use Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research Copeia 2013, No 2, 213–222 First Record of the Genus Oreolalax (Anura: Megophryidae) from Vietnam with Description of a New Species Truong Q Nguyen1, Trung M Phung2, Minh D Le3,4,5, Thomas Ziegler6, and Wolfgang Bo ă hme7 The genus Oreolalax is reported from Vietnam for the first time and a new species is described based on morphological differences, molecular divergence, and phylogenetic placement Morphologically, the new species is distinguishable from its congeners on the basis of a combination of the following diagnostic characters: size small; tympanum hidden; toes with webbing at base; dorsum with distinct, round, spiny warts; flanks with white, spiny spots; belly and lower surface of limbs smooth, with dark marbling; interorbital region without dark triangular pattern; upper surface of thigh with dark bars; male with black spines present on margin of lower lip, spinal patches on chest small with fine spines, nuptial spines on fingers small, and without vocal sacs In phylogenetic analyses, the new species is unambiguously nested within the genus Oreolalax T HE genus Oreolalax was created by Myers and Leviton (1962) for four species of Scutiger described by Liu (1943, 1947) from western China Dubois (1980, 1987) treated Oreolalax as a subgenus of Scutiger Ohler and Dubois (1992) also assigned Megalophrys weigoldi Vogt, 1924 to the genus Scutiger (subgenus Aelurolalax) However, the placement of this species in the genus Oreolalax was mentioned by Fei (1999) and subsequently followed by Delorme et al (2006), Wei et al (2007, 2009), and Fei et al (2009, 2010) On the other hand, Oreolalax was recently considered as a distinct genus based on both morphological and phylogenetic data (e.g., Wu et al., 1993; Zhao and Adler, 1993; Fu and Murphy, 1997; Zheng et al., 2004; Delorme et al., 2006; Fu et al., 2007; Wei et al., 2007) Members of the genus Oreolalax are characterized by having maxillary teeth short; pupils vertically elliptical; tongue deeply notched posteriorly; eustachian tubes with large, rounded openings; tympana hidden beneath skin; fingers free of webbing; toes with some webbing; outer metatarsals united; dorsum glandular with numerous warts; males with large, flat, but prominent pectoral and axillary glands; and palmar tubercles large, flat, and not extending onto first metatarsal (Myers and Leviton, 1962) The genus Oreolalax currently consists of 17 species and so far is known only from southwestern China (Frost, 2011) Frost (2011) assumed that it might occur in Laos and Vietnam; however, no voucher specimen has been reported previously During recent field work in the Hoang Lien Mountain Range, we discovered a species of frog which resembled Oreolalax, but it was not assignable to any of the named species so far Herein, we describe this species as new, assign it to Oreolalax based on morphological characters and phylogenetic analysis of nucleotide sequence data, and report the genus from Vietnam for the first time MATERIALS AND METHODS Sampling.—A field survey was conducted in April 2010 in the Hoang Lien Mountain Range, Sa Pa District, Lao Cai Province, northern Vietnam Specimens were collected by T M Phung Samples of muscle tissue were preserved separately in 95% ethanol and voucher specimens were fixed in approximately 80% ethanol, and then later transferred to 70% ethanol for permanent storage DNA sequence data from our new samples were combined with a previously collected mitochondrial sequence dataset for species of Oreolalax and relevant out-groups, including Leptobrachium boringiae, L xanthospilum, Megophrys omeimontis, and Scutiger chintingensis (Zheng et al., 2004; Fu et al., 2007; Table 1) Sequences of Oreolalax granulosus, O puxiongensis, O weigoldi, and O xiangchengensis are unobtainable from GenBank; therefore, these species were excluded from our phylogenetic analysis in this study Specimens referred to in this paper are deposited in the collections of the Chengdu Institute of Biology (CIB), Chinese Academy of Sciences, Sichuan, China; the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam; the Institute of Zoology (IOZ), Chinese Academy of Sciences, Beijing, China; the Royal Ontario Museum (ROM), Toronto, Canada; the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences, Yunnan, China; and the Zoologisches Forschungsmuseum Alexander Koenig (ZFMK), Bonn, Germany Nucleotide sequencing and phylogenetic analyses.—We used Le et al.’s (2006) protocols for extraction, amplification, and DNA sequencing Two fragments of the mitochondrial genes 12S and 16S were amplified using two pairs of primers, L1991 + H1478 for 12S (Kocher et al., 1989) and 16Sar + 16Sbr for 16S (Palumbi et al., 1991) After sequences were Department of Vertebrate Zoology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam; Present address: Department of Terrestrial Ecology, Cologne Biocenter, University of Cologne, Zu ă lpicher Strasse 47b, D-50674 Cologne, Germany; E-mail: nqt2@yahoo.com Send reprint requests to this address Dong Khoi 9A, Tam Hiep, Bien Hoa, Dong Nai Province, Vietnam; E-mail: pmytrung@yahoo.com Centre for Natural Resources and Environmental Studies, Hanoi National University, 19 Le Thanh Tong, Hanoi, Vietnam; E-mail: minh.le.cres@gmail.com Faculty of Environmental Sciences, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai Road, Hanoi, Vietnam Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, New York 10024-5192 AG Zoologischer Garten Ko ă ln, Riehler Strasse 173, D-50735 Cologne, Germany; E-mail: ziegler@koelnerzoo.de Herpetology Section, Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, D-53113, Bonn, Germany; E-mail: w.boehme@zfmk.de Submitted: 15 February 2012 Accepted: 15 November 2012 Associate Editor: B Stuart DOI: 10.1643/CH-12-021 F 2013 by the American Society of Ichthyologists and Herpetologists 214 Copeia 2013, No Table Samples Used in Molecular Analyses Species information Oreolalax chuanbeiensis Oreolalax jingdongensis Oreolalax liangbeiensis Oreolalax lichuanensis Oreolalax major Oreolalax multipunctatus Oreolalax nanjiangensis Oreolalax omeimontis Oreolalax pingii Oreolalax popei Oreolalax rhodostigmatus Oreolalax rugosus Oreolalax schmidti Oreolalax sp (China) Oreolalax sterlingae, new species (Vietnam) Oreolalax sterlingae, new species (Vietnam) Oreolalax xiangchengensis Leptobrachium boringiae Leptobrachium xanthospilum Megophrys omeimontis Scutiger chintingensis GenBank no (12S) GenBank no (16S) Locality Voucher EF397266 EF544236 EF397253 EF544237 EF397252 EF397268 EF397265 EF397264 EF397259 — EF397248 EF397254 EF397258 EF397256 KC569979 EF397266 EF544236 EF397253 EF544237 EF397252 EF397268 EF397265 EF397264 EF397259 EF397267 EF397248 EF397254 EF397258 EF397256 KC569981 Mao Xian, Sichuan, China — Puxiong, Sichuan, China — Hongya, Sichuan, China Omei, Sichuan, China Nan Jiang, Sichuan, China Omei, Sichuan, China Xi Chang, Sichuan, China Penxian, Sichuan, China Da Fang, Guizhou, China Shi Mian, Sichuan, China Da Yi, Sichuan, China Pengxian, Sichuan, China Sa Pa, Lao Cai, Vietnam CIB-XM 074 IOZCAS 2691–2692 IOZCAS 3796 IOZCAS 2675 ROM 40452 ROM 40463 CIB-XM 804 CIB-XM 439 CIB-XM 980 CIB-XM 107 CIB-ZYCA 746 CIB-XM 340 CIB-XM 417 CIB-XM 092 IEBR A.2012.1 KC569980 KC569982 Sa Pa, Lao Cai, Vietnam ZFMK 92830 EF397250 EF397247 EF397245 EF397242 EF397269 EF397250 EF397247 EF397245 EF397242 AY526209 Li Jiang, Yunnan, China Leishan, Guizhou, China Gia Lai, Vietnam Omei, Sichuan, China Hongya, Sichuan, China CIB-3LW 008 CIB-XM 594 ROM 32184 ROM 40462 ROM 39065 (12S) /XM 1045 (16S) aligned by Clustal X v2 (Thompson et al., 1997), data were analyzed using maximum parsimony (MP) and Bayesian analysis (BA), as implemented in PAUP*4.0b10 (Swofford, 2001) and MrBayes v3.2 (Huelsenbeck and Ronquist, 2001), respectively Settings for these analyses followed Le et al (2006), except that the number of generations in the Bayesian analysis was increased to 13107 The optimal model for nucleotide evolution was set to GTR+I+C as selected by Modeltest v3.7 (Posada and Crandall, 1998) Nodal support was evaluated using Bootstrap replication (BP) as calculated in PAUP and posterior probability (PP) in MrBayes v3.2 Uncorrected pairwise divergences were calculated in PAUP*4.0b10 Morphological analysis.—We compared the new species with other members of the genus (see Material Examined) and data obtained from the literature (Liu and Hu, 1960; Liu et al., 1979; Tian, 1983; Fei and Huang, 1983; Yang et al., 1983; Fei et al., 1992, 1999, 2009, 2010; Ohler and Dubois, 1992; Fu and Murphy, 1997; Wei et al., 2009) Measurements were taken with digital callipers to the nearest 0.1 mm The following abbreviations were used: SVL: snout–vent length, HL: head length (from posterior margin of lower jaw to the tip of snout), HW: maximum head width (across angle of jaws), SNL: snout length (from anterior corner of eye to the tip of snout), NS: distance from nostril to the tip of snout, EN: distance from anterior corner of eye to the nostril, IN: internarial distance, IOD: interorbital distance, ED: eye diameter, UEW: maximum width of upper eyelid, DAE: distance between anterior corners of eyes, DPE: distance between posterior corners of eyes, MAE: distance between posterior margin of lower jaw and anterior corner of eye, MPE: distance between posterior margin of lower jaw and posterior corner of eye, MN: distance from posterior margin of lower jaw to the nostril, FLL: forelimb length (from axilla to elbow), HAL: hand length (from elbow to the tip of third finger), FFL: first finger length, TFL: third finger length, IPT: inner palmar tubercle length, OPT: outer palmar tubercle length, FeL: femur length (from vent to knee), TbL: tibia length (from knee to tarsus), FoL: foot length (from tarsus to the tip of fourth toe), FTL: fourth toe length, IMT: inner metatarsal tubercle length, OMT: outer metatarsal tubercle length, ParL: parotoid length (from posterior corner of eye to the end of parotoid gland), SPW: maximum width of spinal patch on chest, CW: chest width (distance between axillae) We followed Wei et al (2009) to define the size of spinal patches on chest: small if the ratio of SPW/CW ,0.3 and large if this ratio 0.4 Photos of the preserved specimen were taken with a digital microscope Keyence VHX-500F RESULTS Phylogenetic analyses.—The combined matrix contains 867 aligned characters, 375 of 12S and 492 of 16S MP analysis of the dataset recovered nine most parsimonious trees with 609 steps (CI 0.69, RI 0.63) One of the nine trees is shown in Figure Fifty percent of the in-group nodes in our preferred tree received strong support (Bootstrap value $ 70%; Hillis and Bull, 1993), and the species from Sa Pa is unambigously nested in the genus Oreolalax (Bootstrap value 97) In the Bayesian analysis, –lnL scores reached stationarity after 11,000 generations in both runs The Bayesian topology is generally in agreement with the MP topology, but we note a few significant discrepancies In the Bayesian topology, the species of Oreolalax from Sa Pa along with other clades, including O multipunctatus and other species, O jingdongensis and others, and O lichuanensis and others, are unresolved In addition, the positions of O Nguyen et al.—New Oreolalax from Vietnam 215 Fig The maximum parsimony tree based on partial 12S and 16S genes This is one of nine most parsimonious trees (TL 609, CI 0.69, RI 0.63) The dataset includes 867 aligned characters of which 156 are potentially parsimony informative Numbers above and below branches are bootstrap (.50%) values and Bayesian posterior probabilities (.95%), respectively chuanbeiensis and O nanjiangensis are interchanged, and O liangbeiensis and O major become sister with O rugosus replacing the position of O liangbeiensis as shown in Figure Both MP and BA analyses strongly support the monophyly of the genus Oreolalax including the new species (BP 97, PP 98), and demonstrate that the Vietnamese species is not closely related to any other members of the genus (Fig 1) This species is largely divergent from others within Oreolalax in terms of genetic distance, with the minimum pairwise divergence of approximately 5% in 16S, the fastest evolving marker in this study (Table 2) Morphological comparisons.—The new Vietnamese species of Oreolalax differs from previously named species as follows: Table Uncorrected (‘‘p’’) Distance Matrix Showing Pairwise Genetic Divergence between Vietnamese Oreolalax and Other Species in the Genus (except O granulosus, O puxiongensis, O weigoldi, and O xiangchengensis) Oreolalax sterlingae, new species Species name Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax chuanbeiensis jingdongensis liangbeiensis lichuanensis major multipunctatus nanjiangensis omeimontis pingii popei rhodostigmatus rugosus schmidti sp (China) xiangchengensis (16S) 0.073 0.058 0.054 0.073 0.054 0.068 0.068 0.066 0.064 0.073 0.073 0.048 0.066 0.056 0.056 Oreolalax sterlingae, new species (12S) 0.046 0.022 0.022 0.035 0.022 0.051 0.046 0.040 0.022 — 0.051 0.022 0.024 0.022 0.022 Oreolalax sterlingae, new species (total) 0.061 0.042 0.040 0.056 0.040 0.061 0.059 0.055 0.046 0.072 0.063 0.036 0.048 0.041 0.041 216 from O chuanbeiensis by having a smaller size (SVL 36.8 mm [n 1] vs 47.5–56.3 mm [n 20] in males and 44.6 mm [n 1] vs 55.5–59.0 mm [n 3] in females), white spots on flanks present (vs absent in O chuanbeiensis), and fringes along the side of toes absent (vs present in O chuanbeiensis); from O granulosus by having a smaller size (SVL 36.8 mm [n 1] vs 48.6–61.5 mm [n 16] in males and 44.6 mm [n 1] vs 57.4–60.0 mm [n 3] in females), spinal patches on chest smaller (vs very large in O granulosus), toes with webbing at base only (vs webbing well developed in O granulosus), and white spots on flanks present (vs absent in O granulosus); from O jingdongensis by having a smaller size (SVL 36.8 mm [n 1] vs 48.1–60.3 mm [n 16] in males), spines on chest small (vs large in O jingdongensis), nuptial spines on fingers small (vs large in O jingdongensis), webbing on toes basal (vs 1/3 webbed in O jingdongensis), and triangular pattern between the eyes absent (vs present in O jingdongensis); from O liangbeiensis by having a smaller size (SVL 36.8 mm [n 1] vs 45.1–56.3 mm [n 20] in males and 44.6 mm [n 1] vs 56.0–65.7 mm [n 8] in females), spinal patches on chest small (vs very large in O liangbeiensis), and belly with dark marbling (vs immaculate in O liangbeiensis); from O lichuanensis by having a smaller size (SVL 36.8 mm [n 1] vs 50.0–64.8 mm [n 2] in males and 44.6 mm [n 20] vs 57.3–62.2 mm [n 4] in females), spines on chest small (vs large in O lichuanensis), nuptial spines on fingers small (vs large in O lichuanensis), and white spots on flanks present (vs absent in O lichuanensis); from O major by having a smaller size (SVL 36.8 mm [n 1] vs 59.2–68.7 mm [n 6] in males and 44.6 mm [n 1] vs 65.0–70.0 mm [n 2] in females), spinal patches on chest small (vs very large in O major), webbing on toes less developed (at base vs 1/3–2/3 webbed), fringes along the side of toes absent (vs present in O major), and white spots on flanks present (vs absent in O major); from O multipunctatus by having a smaller size (SVL 36.8 mm [n 1] vs 47.4–49.8 mm [n 4] in males), spines on chest small (vs large in O multipunctatus), nuptial spines on fingers small (vs large in O multipunctatus), white spots on flanks present (vs absent in O multipunctatus), and round black spots on dorsum absent (vs present in O multipunctatus); from O nanjiangensis by having a smaller size (SVL 36.8 mm [n 1] vs 52.6–60.0 mm [n 10] in males and 44.6 mm [n 1] vs 53.3–58.2 mm [n 8] in females), spines on margin of lower jaw present (vs absent in O nanjiangensis), spines on chest small (vs large in O nanjiangensis), nuptial spines on fingers small (vs large in O nanjiangensis), belly with dark marbling (vs immaculate in O nanjiangensis), and white spots on flanks present (vs absent in O nanjiangensis); from O omeimontis by having a smaller size (SVL 36.8 mm [n 1] vs 49.5–58.4 mm [n 15] in males and 44.6 mm [n 1] vs 51.2–56.1 mm [n 3] in females), spines on margin of lower jaw present (vs absent in O omeimontis), vocal sac absent in males (vs present in O omeimontis), triangular pattern between eyes absent (vs present in O omeimontis), and white spots on flanks present (vs absent in O omeimontis); from O pingii by having a dorsum with large round warts (vs small warts in O pingii), belly with dark marbling (vs immaculate in O pingii), and dark bars on upper side of limbs present (vs absent in O pingii); from O popei by having a smaller size (SVL 36.8 mm [n 1] vs 60.0–69.0 mm [n 20] in males and 44.6 mm [n 1] vs 51.5–67.0 mm [n 10] in females), spines on chest small (vs large in O popei), nuptial spines on fingers small (vs large in O popei), Copeia 2013, No dark spots with light centers on dorsum absent (vs present in O popei), and white spots on flanks present (vs absent in O popei); from O puxiongensis by having a dorsum with round warts (vs back with strongly developed spiny ridges in O puxiongensis), belly with marbling (vs immaculate in O puxiongensis), nuptial spines on fingers small (vs large in O puxiongensis), and triangular pattern between eyes absent (vs present in O puxiongensis); from O rhodostigmatus by having a smaller size (SVL 36.8 mm [n 1] vs 57.5–73.5 mm [n 7] in males and 44.6 mm [n 1] vs 62.4–70.6 mm [n 2] in females), tympanum hidden (vs distinct in O rhodostigmatus), and large orange round warts on axilla and at the back of thigh absent (vs present in O rhodostigmatus); from O rugosus by having toes with less developed webbing (at base vs 1/4 webbed in O rugosus), toes with lateral fringes absent (vs present in O rugosus), and light spots on flanks present (vs absent in O rugosus); from O schmidti by having a belly with marbling (vs immaculate in O schmidti), nuptial spines on fingers small (vs large in O schmidti), and dark triangular pattern between the eyes absent (vs present in O schmidti); from O weigoldi by having a smaller size (SVL 36.8 mm [n 1] vs 58.2 mm [n 1] in males), two clearly separated spinal patches on chest (vs one large spinalpatch in O weigoldi), toes with webbing at base (vs well developed in O weigoldi), and belly smooth (vs granular in O weigoldi); from O xiangchengensis by having a smaller size (SVL 36.8 mm [n 1] vs 45.4–50.6 mm [n 10] in males and 44.6 mm [n 1] vs 54.3–61.4 mm [n 10] in females), spinal patches on chest small (vs very large in O xiangchengensis), and toes with webbing at base (vs almost completely webbed in O xiangchengensis; Table 3) Based on the results of the molecular analyses and morphological comparisons, we argue that the Vietnamese specimens represent a new species, which is assigned to the genus Oreolalax and is described below Oreolalax sterlingae, new species Holotype.—IEBR A.2012.1, adult male, northern Vietnam, Lao Cai Province, Sa Pa District, Hoang Lien Mountain, 2900 m, Phung My Trung, 30 April 2010 Paratype.—ZFMK 92830, adult female, same collection data as the holotype Diagnosis.—The new species differs from its congeners by a combination of the following morphological characters: size small (SVL 36.8 mm in male, 44.6 mm in female); tympanum hidden; toes with webbing at base; dorsum with distinct round spiny warts; flanks with white spiny spots; belly and lower surface of limbs smooth, with dark marbling; interorbital region without dark triangular pattern; upper surface of thigh with three wide dark bars; male with black spines present on margin of lower lip, spinal patches on chest small with fine spines, nuptial spines on fingers small, and without vocal sacs Description of holotype.—Size small (SVL 36.8 mm) Head wider than long (HL 13.3 mm, HW 13.7 mm); snout protruding, rounded anteriorly, snout length (SNL 6.1 mm) greater than horizontal diameter of eye (ED 5.1 mm); interorbital region flat, wider (IOD 4.3 mm) than upper eyelid (UEW 3.3 mm); canthus rostralis distinct, loreal region concave; nostril oval, on lateral side, lying midway SVL (mm) Males Females Tympanum (hidden 0, distinct 1) Wart (round 0, elongated ridge 1) Belly (smooth 0, granular 1) Belly (immaculate 0, with marbling 1) Triangular pattern between eyes (absent 0, present 1) Toe webbing Vocal sac (absent 0, present 1) Spines on lip margin (absent 0, present 1) Spinal patches on chest (small 0, large 1) Spines on chest (small 0, large 1) Nuptial spines on fingers (small 0, large 1) Dark bars on limbs n (from literature) n (examined) SVL (mm) Males Females Tympanum (hidden 0, distinct 1) Wart (round 0, elongated ridge 1) Belly (smooth 0, granular 1) Belly(immaculate50, withmarbling51) Triangular pattern between eyes (absent 0, present 1) Toe webbing Vocal sac (absent 0, present 1) Male with spines on lip margin (absent 0, present 1) Spinal patches on chest (small 0, large 1) Spines on chest (small 0, large 1) Nuptial spines on fingers (small 0, large 1) Dark bars on limbs n (from literature) n (examined) — 0 — 20M, 3M — at base 0 distinct — M, 1F 49.5–58.4 51.2–56.1 0–1 1 at base 0 distinct 15M, 3F — 36.8 44.6 0 at base 0 distinct — M, 1F Oreolalax sterlingae, new species (Vietnam) omeimontis 47.5–56.3 55.5–59.0 — 0 36.8 44.6 0 Oreolalax sterlingae, new species (Vietnam) chuanbeiensis popei — 20M, 8F 1M 1/4 at base 0 1 0 1 absent distinct 20M, 20F 20M, 10F — — 0 — 45.1–56.3 56.0–65.7 0–1 0 liangbeiensis — 20M, 4F — 1 — 50.0–64.8 57.3–62.2 — lichuanensis at base 0 — 20M, 10F 1M 1/3 0 0 — 7M, 2F — 57.5–73.5 62.4–70.6 — 1/4 0 — 10M, 10F 2F at base 0 indistinct 30M, 3F — 44.3–52.6 40.0–47.0 45.0–55.3 48.0–54.0 0 — — 0 0 schmidti — 6M, 2F — 0 1/3–2/3 distinct 10M, 8F — 1 — — 52.6–60.0 53.3–58.2 0 0 — — — — — — distinct 1M — 58.2 — — 1 — — 1 0 absent 10M, 10F — 45.4–50.6 54.3–61.4 0 weigoldi xiangchengensis — 4M — 1 1/3 47.4–49.8 — — 0–1 indistinct major multipunctatus nanjiangensis 59.2–68.7 65.0–70.0 0 puxiongensis rhodostigmatus rugosus — 16M, 4F 3M, 3F 1 1/3 48.1–60.3 48.7–56.5 0 1 jingdongensis 43.4–51.0 60.0–69.0 41.3–45.3 46.8–54.4 51.5–67.0 43.0–50.0 0 very small 0 1 0 pingii — 16M, 3F — 0 1/2–2/3 48.6–61.5 57.4–60.0 — granulosus Table Morphological Comparison of Species of Oreolalax (M = male, F = female, — = data unavailable) Nguyen et al.—New Oreolalax from Vietnam 217 218 Copeia 2013, No Fig Holotype (A: IEBR A.2012.1, male) and paratype (B: ZFMK 92830, female) of Oreolalax sterlingae, new species, in life Nguyen et al.—New Oreolalax from Vietnam 219 Fig Ventral side of hand (A) and foot (B) of the holotype (IEBR A.2012.1) of Oreolalax sterlingae, new species Hindlimbs: Femur slightly longer than tibia (FeL 17.8 mm, TbL 17.3 mm, TbL/SVL 47%); heels overlapping when held at right angles to the body; foot long (FoL 28.3 mm); toes long and thin, toe IV (FTL 14.5 mm) shorter than femur; relative length of toes: I,II,V#III,IV; tip of toes rounded, not enlarged, without discs, webbing at base only; dermal fringe along toe V absent; subarticular tubercles indistinct; inner metatarsal tubercle present but not developed, rounded (IMT 2.8 mm); outer metatarsal tubercle absent; tarsal fold absent Skin: Snout and dorsal head with flattened warts; dorsum and upper flanks with distinct large warts, bearing small black spines; dorsolateral folds absent; upper surface of forelimbs, tarsus, and legs with flattened warts, bearing small black spines; parotoid glands well developed with small black spines; black spines also present in loreal region, upper and lower jaws, tympanic region, and more distinct on margin of lower lip; flank with white spiny spots; chest with two distinctly separated, oval patches of small black spines (CW 14.2 mm, SPW 4.1 mm, ratio SPW/CW 0.29, narrowest distance between patches 3.3 mm); throat, venter, and lower surface of limbs smooth Fig Ventral side (A) and chest region (B) of the holotype (IEBR A.2012.1) of Oreolalax sterlingae, new species between tip of snout and eye (NS 3.2 mm, EN 3.2 mm); pupil vertical; tympanum hidden; parotoid gland elongate (ParL 8.3/8.5 mm), about three times longer than wide; canthal, preorbital, supraorbital, postorbital, supratympanic, and parietal ridges absent; maxillary teeth present; vomerine teeth absent; tongue longer than wide, free at the back, notched posteriorly Forelimbs: Arm relatively short (FLL 7.4 mm), shorter than half of hand (HAL 18.4 mm), distinctly enlarged; relative length of fingers: I,II,IV,III, tip of finger rounded, not enlarged, without groove; fingers without dermal fringe and webbing; subarticular tubercles absent on fingers I and II, indistinct on fingers III and IV; palmar tubercles flat, rounded; inner and upper sides of fingers I and II with small black nuptial spines Coloration in preservative.—Upper head grayish brown; dorsum and upper flanks grayish brown with small black spines; upper lip grayish brown with some indistinct whitish spots; upper surface of arm and thigh with whitish spots or bars; chest with two patches of black spines; lower zone of flanks grayish brown with whitish spots; chin, throat, and belly cream with brown mottlings; lower surface of limbs grayish brown with whitish spots Coloration in life.—Dorsum and upper flanks brown with small black spines; upper lip brown with some indistinct whitish spots; upper surface of arm and thigh with whitish spots or bars; lower zone of flanks brown with yellowish white spots; chin, throat, and belly cream with brown mottling; lower surface of limbs dark brown with whitish spots (Figs 2, 3, 4) Sexual dimorphism.—Male is smaller than the female (Table 4) and has lower lip with distinct black spines, dorsal 220 Copeia 2013, No Table Measurements (in mm) of the Holotype and Paratype of Oreolalax sterlingae, New Species Abbreviations defined in the text SVL HL HW SNL NS EN IN IOD ED UEW DAE DPE MAE MPE MN FLL HAL FFL TFL IPT OPT TbL FoL FeL FTL IMT OMT ParL (left/right) IEBR A.2012.1 Holotype (Male) ZFMK 92830 Paratype (Female) 36.8 13.3 13.7 6.1 3.2 3.2 4.3 5.1 3.3 6.9 11.7 9.2 5.2 11.4 7.4 18.4 4.3 8.4 1.9 2.3 17.3 28.3 17.8 14.5 2.8 absent 8.3/8.5 44.6 15.3 15.8 6.9 3.5 3.4 4.3 5.4 5.6 3.7 8.1 13.1 10.1 6.9 12.9 9.7 23.2 5.7 10.8 2.0 2.1 19.7 32.4 20.3 16.8 2.9 absent 5.5/5.5 warts more prominent, chest with two patches of small spines, inner and upper sides of fingers I and II with black nuptial spines Vocal sacs are absent in both sexes Natural history.—Specimens of Oreolalax sterlingae were found at night in a rocky rivulet in bamboo forest at an elevation of 2900 m (Fig 5) Other species of Oreolalax occur at 700–3550 m (Wei et al., 2007; Fei et al., 2010; Frost, 2011) The female (ZFMK 92830) contained yellow eggs (3.6 mm in diameter) Distribution.—Oreolalax sterlingae is known only from Hoang Lien Mountain Range, Lao Cai Province, northern Vietnam (Fig 6) Etymology.—The specific epithet is dedicated to Dr Eleanor J Sterling from the Center for Biodiversity and Conservation, American Museum of Natural History in New York, to acknowledge her outstanding contribution to biodiversity research and conservation in Vietnam As common names we suggest Sterling’s Toothed Toad (English), Co´c nu´i s-tecling (Vietnamese) DISCUSSION Although our morphological and molecular results clearly show that the new species belongs to the genus Oreolalax, its position within the genus is still largely unresolved The sister relationship between O sterlingae and the clade Fig Habitat of Oreolalax sterlingae, new species, on Hoang Lien Mountain Range, Lao Cai Province, Vietnam containing O multipunctatus and other species (see Fig 1) is only weakly supported in the MP analysis (BP , 50%), and the two groups become unresolved in the Bayesian analysis Moreover, many nodes in the genus receive low support values in both analyses Additional data from both mitochondrial and nuclear genes might help provide a more robust phylogenetic hypothesis of the genus, as shown in previous study (e.g., Fu et al., 2007) The first discovery of a member of the genus Oreolalax outside China once again confirms the close affinity between northern Vietnam and southern China as shown in other taxonomic groups (see Sterling et al., 2006) The new record also highlights the unusual amphibian diversity of the Hoang Lien Mountain Range, which is known as one of the most famous regions in terms of herpetofaunal diversity in Vietnam, and situated in one of the highest phylogenetic diversity zones of amphibians in the world (Fritz and Rahbek, 2012) Ohler et al (2000) reported a total of 42 species of amphibians from Hoang Lien Range with descriptions of two new species, namely Leptolalax pluvialis and Rhacophorus duboisi Orlov et al (2001) listed 20 species of rhacophorids and discovered another new species of tree frog, viz Rhacophorus hoanglienensis In addition, a number of new species of amphibians have been discovered from this region during the last decade: Leptobrachium echinatum (Dubois and Ohler, 1998), Amolops cucae (Bain et al., 2006), Amolops minutus and A splendissimus (Orlov and Ho, 2007), and Theloderma lateriticum (Bain et al., 2009) It is important to note the rarity of this species in Vietnam Although numerous surveys have been conducted in Lai Chau, Lao Cai, and Yen Bai provinces since 1998, this is the first record of Oreolalax from the Hoang Lien Mountain Range Because the actual distribution of the new species is unknown, we suggest the species should be considered Data Deficient following IUCN’s Red List categories (IUCN, 2001) Among 17 species of Oreolalax known from China, 13 are listed in the IUCN Red List (2012) as near threatened to crictically endangered due to anthropogenic threats and small distribution ranges MATERIAL EXAMINED O jingdongensis: (6) China: KIZ 91006–91007, 91011, 91155– 91157, Yunnan, Jingdong Huashan Nguyen et al.—New Oreolalax from Vietnam 221 LITERATURE CITED Fig Map showing the type locality (circle) of Oreolalax sterlingae, new species, from Hoang Lien Mountain Range, Sa Pa District, Lao Cai Province, Vietnam O liangbeiensis: (1) China: KIZ 65II0684, Sichuan, Puxiong, Liangbei O puxiongensis: (1) China: KIZ 19810703001, Sichuan, Puxiong O rugosus: (2) China: KIZ 820857, 821129, Yunnan, Yangbi ACKNOWLEDGMENTS We thank the directorate of Hoang Lien National Park for issuing relevant permits For the loan of specimens, we are grateful to C Le (Hanoi), T Nguyen (Hanoi), J Che and J Wang (Kunming) Special thanks to D Kizirian for commenting on the manuscript, E Sterling and K Koy for providing the map T Phung thanks T Nguyen, H Vu, and L Do for assistance in the field T Nguyen would like to thank M Bonkowski for support of work in Germany and J Jiang for providing literature M Le was supported by the National Foundation for Science and Technology Development of Vietnam (NAFOSTED: Grant No 106.15-2010.30) Research of T Nguyen in Germany is funded by the Alexander von Humboldt Foundation (VIE 1143441) Bain, R H., T Q Nguyen, and K V Doan 2009 A new species of the genus Theloderma Tschudi, 1838 (Anura: Rhacophoridae) from northwestern Vietnam Zootaxa 2191:58–68 Bain, R H., B L Stuart, and N L Orlov 2006 Three new Indochinese species of cascade frogs (Amphibia: Ranidae) allied to Rana archotapnus Copeia 2006:43–59 Delorme, M., A Dubois, S Grosjean, and A Ohler 2006 Une nouvelle ergotaxinomie des Megophryidae (Amphibia, Anura) Alytes 24:6–21 Dubois, A 1980 Notes sur la syste´matique et la re´partition des amphibiens anoures de Chine et des regions avoisinantes IV Classification ge´ne´rique et subge´ne´rique des Pelobatidae Megophryinae Bulletin Mensuel de la Socie´te´ Linne´enne de Lyon 49:469–482 Dubois, A 1987 ‘‘1986’’ Miscellanea taxinomica batrachologica (I) Alytes 5:7–95 Dubois, A., and A Ohler 1998 A new species of Leptobrachium (Vibrissaphora) from northern Vietnam, with a review of the taxonomy of the genus Leptobrachium (Pelobatidae, Megophryinae) Dumerilia 4:1–32 Fei, L 1999 Atlas of Amphibians of China Henan Press of Science and Technology Fei, L., S.-Q Hu, C.-Y Ye, and Y.-A Huang 2009 Fauna Sinica Amphibia Volume Anura Science Press, Chinese Academy of Science, Beijing Fei, L., and Y.-Z Huang 1983 A new species of pelobatid toad from western Sichuan Acta Herpetologica Sinica 2:71–75 Fei, L., C.-Y Ye, and H.-J Chen 1992 A new pelobatid toad of genus Oreolalax (Amphibia: Anura) from YunnanGuizhou Plateau of China Acta Biologica Plateau Sinica 6:39–44 Fei, L., C.-Y Ye, and J.-P Jiang 2010 Colored Atlas of Chinese Amphibians Sichuan Publishing House of Science and Technology Fei, L., C.-Y Ye, and C Li 1999 Description of a new pelobatid toad of genus Oreolalax–Oreolalax nanjiangensis from Daba Mountain Region of China Acta Zootaxonomica Sinica 24:107–113 Fritz, S A., and C Rahbek 2012 Global patterns of amphibian phylogenetic diversity Journal of Biogeography 39:1373–1382 Frost, D R 2011 Amphibian Species of the World: an Online Reference Version 5.5 (31 January 2011) Electronic Database accessible at http://research.amnh.org/ herpetology/amphibia/index.html American Museum of Natural History, New York Fu, J., and R W Murphy 1997 Phylogeny of Chinese Oreolalax and the use of functional outgroups to select among multiple equally parsimonious trees Asiatic Herpetological Research 7:38–43 Fu, J., C J Weadick, and K Bi 2007 A phylogeny of the high-elevation Tibetan megophryid frogs and evidence for the multiple origins of reversed sexual size dimorphism Journal of Zoology 273:315–325 Hillis, D M., and J J Bull 1993 An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis Systematic Biology 42:182–192 Huelsenbeck, J P., and F Ronquist 2001 MRBAYES: Bayesian inference of phylogeny Bioinformatics 17: 754–755 222 IUCN 2001 IUCN Red List Categories and Criteria: Version 3.1 IUCN Species Survival Commission IUCN, Gland, Switzerland and Cambridge, U.K IUCN 2012 IUCN Red List of Threatened Species Version 2012.1 Downloaded on 02 August 2012 www.iucnredlist org Kocher, T D., W K Thomas, A Meyer, S V Edwards, S Pa ăa ă bo, F X Villablanca, and A C Wilson 1989 Dynamics of mitochondrial DNA evolution in mammals: amplification and sequencing with conserved primers Proceedings of the National Academy of Sciences of the United States of America 86:6196–6200 Le, M., C J Raxworthy, W P McCord, and L Mertz 2006 A molecular phylogeny of tortoises (Testudines: Testudinidae) based on mitochondrial and nuclear genes Molecular Phylogenetic and Evolution 40:517–531 Liu, C.-C 1943 Two new Scutiger from Chao-Chiao-Hsien, Sikang Journal of the West China Border Research Society 14(B):35–39 Liu, C.-C 1947 Two new frogs of the genus Scutiger from West China Copeia 1947:123–126 Liu, C.-C., and S.-Q Hu 1960 New Scutiger from China with a discussion about the genus Scientia Sinica 9:760–779 Liu, C.-C., S.-Q Hu, and L Fei 1979 Five new pelobatid toads from China Acta Zootaxonomica Sinica Beijing 4:83–92 Myers, G S., and A E Leviton 1962 Generic classification of the high-altitude pelobatid toads of Asia (Scutiger, Aelurophryne, and Oreolalax) Copeia 1962:287–291 Ohler, A., and A Dubois 1992 The holotype of Megalophrys weigoldi Vogt, 1924 (Amphibia: Anura: Pelobatidae) Journal of Herpetology 26:245–249 Ohler, A., O Marguis, S R Swan, and S Grosjean 2000 Amphibian biodiversity of the Hoang Lien Nature Reserve (Lao Cai Province, northern Vietnam) with description of two new species Herpetozoa 13:71–87 Orlov, N L., and C T Ho 2007 Two new species of cascade ranids of Amolops genus (Anura: Ranidae) from Lai Chau Province (Northwest Vietnam) Russian Journal of Herpetology 14:211–228 Orlov, N L., A Lathrop, R W Murphy, and C T Ho 2001 Frogs of the family Rhacophoridae (Anura: Amphibia) in the northern Hoang Lien Mountains (Mount Fan Si Copeia 2013, No Pan, Sa Pa District, Lao Cai Province), Vietnam Russian Journal of Herpetology 8:17–44 Palumbi, S R., A P Martin, S Romano, W O McMillan, L Stice, and G Grabowski 1991 The Simple Fool’s Guide to PCR Version 2.0 Privately published Posada, D., and K A Crandall 1998 MODELTEST: testing the model of DNA substitution Bioinformatics 14:817– 818 Sterling, E J., M H Hurley, and M D Le 2006 Vietnam: A Natural History Yale University Press, New Haven, Connecticut Swofford, D L 2001 PAUP*: phylogenetic analysis using parsimony (*and other methods), version Sinauer Associates, Sunderland, Massachusetts Thompson, J D., T J Gibson, F Plewniak, F Jeanmougin, and D G Higgins 1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Research 25:4876–4882 Tian, W.-S 1983 A new species of Oreolalax—O chuanbeiensis Acta Herpetologica Sinica 2:59–62 Vogt, T 1924 Reptilien und Amphibien aus Szetschwan, Osttibet und Tschili Zoologischer Anzeiger 60:337–344 Wei, G., Z.-Z Li, N Xu, and J.-P Jiang 2007 Review on the systematics of the genus Oreolalax Chinese Journal of Zoology 42:169–173 Wei, G., B Wang, N Xu, Z.-Z Li, and J.-P Jiang 2009 Morphological evolution from aquatic to terrestrial in the genus Oreolalax (Amphibia, Anura, Megophryidae) Progress in Natural Science 19:1043–1048 Wu, G.-F., E.-M Zhao, R F Inger, and H B Shaffer 1993 A new frog of the genus Oreolalax (Pelobatidae) from Sichuan, China Journal of Herpetology 27:410–413 Yang, D.-T., D.-S Ma, H.-J Chen, and F.-L Li 1983 Descriptions of two new pelobatid toads from Yunnan Acta Zootaxonomica Sinica 8:323–327 Zhao, E.-M., and K Adler 1993 Herpetology of China Society for the Study of Amphibians and Reptiles, Oxford, Ohio Zheng, Y.-C., B.-H Mo, Z.-J Liu, and X.-M Zeng 2004 Phylogenetic relationships of megophryid genera (Anura: Megophryidae) based on partial sequences of mitochondrial 16S rRNA gene Zoological Research 25:205–213 ... Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax Oreolalax chuanbeiensis jingdongensis liangbeiensis lichuanensis major... nanjiangensis Oreolalax omeimontis Oreolalax pingii Oreolalax popei Oreolalax rhodostigmatus Oreolalax rugosus Oreolalax schmidti Oreolalax sp (China) Oreolalax sterlingae, new species (Vietnam) Oreolalax. .. Molecular Analyses Species information Oreolalax chuanbeiensis Oreolalax jingdongensis Oreolalax liangbeiensis Oreolalax lichuanensis Oreolalax major Oreolalax multipunctatus Oreolalax nanjiangensis