Research Article |
Corresponding author: Ruichang Quan ( quanrc@xtbg.ac.cn ) Corresponding author: Song Li ( lis@mail.kiz.ac.cn ) Academic editor: Günter Gollmann
© 2022 Shuo Liu, Dingqi Rao, Dongru Zhang, Ye Htet Lwin, Mingzhong Mo, Changsheng Zuo, Fawang Yin, Ruichang Quan, Song Li.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Liu S, Rao D, Zhang D, Lwin YH, Mo M, Zuo C, Yin F, Quan R, Li S (2022) Phylogenetic position of Odorrana macrotympana (Yang, 2008) (Anura, Ranidae) and extension of its geographical distribution. Herpetozoa 35: 9-19. https://doi.org/10.3897/herpetozoa.35.e77147
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Based on a 16S rRNA gene fragment, a molecular phylogeny for the genus Odorrana Fei, Ye & Huang, 1990 was reconstructed, the validity of the poorly-known ranid species O. macrotympana (Yang, 2008) was confirmed and its phylogenetic position was evaluated. In addition, we report the first country record of O. macrotympana from Myanmar, based on our new records from Htamanthi Wildlife Sanctuary, Sagaing Division and present a supplementary description of this species. This report also constitutes the first record of O. macrotympana from outside of China.
16S rRNA, China, Htamanthi Wildlife Sanctuary, morphology, Myanmar, new record, western Yunnan
Odorrana macrotympana is a poorly-known ranid species described, based on only two specimens from Xueli Village, Taiping Town, Yingjiang County, Dehong Prefecture, Yunnan Province, China, close to the border with Myanmar (
Odorrana macrotympana most closely resembles O. tiannanensis (Yang & Li, 1980), but it is characterised by the following characters: lips uniform white without any spots or streaks, dorsal skin smooth and intermittent dorsolateral folds present, tympanum large and external vocal sac present in males (
As no molecular data were available for Odorrana macrotympana so far, the phylogenetic position of O. macrotympana has never been inferred and, thus, its systematic affinities remain unresolved. During our field surveys in Yunnan Province, China and in northern Myanmar from 2009 to 2021, we collected one topotype of O. macrotympana from its type locality, Yingjiang County, Dehong Prefecture, Yunnan Province, China; five specimens of Odorrana cf. macrotympana from Htamanthi Wildlife Sanctuary, northern Myanmar; and four topotypes of O. tiannanensis from its type locality, Hekou County, Honghe Prefecture, Yunnan Province, China, respectively. We collected DNA sequence data of these specimens in order to reconstruct a molecular phylogeny and evaluate the phylogenetic position of O. macrotympana.
In addition, our phylogenetic analysis of molecular data demonstrated that the specimens from northern Myanmar should be assigned to Odorrana macrotympana. Thus, this report constitutes the first record for the species outside China and from the country of Myanmar.
Field survey in Yingjiang County, Yunnan Province, China, was conducted under permits from Tongbiguan Provincial Natural Reserve Management and Protection Bureau; field survey in Hekou County, Yunnan Province, China, was conducted under permits from Honghe Prefecture Forestry and Grassland Bureau of Yunnan Province; and the field survey in northern Myanmar was undertaken at the invitation of the Republic of the Union of Myanmar, Ministry of Natural Resources and Environmental Conservation, Forest Department, Forest Research Institute. After live photographs were taken, specimens were collected, euthanised with ethyl acetate and fixed in 75% ethanol for storage. Liver tissue samples were preserved in 99% ethanol for molecular analysis. The specimens from China were deposited in Kunming Institute of Zoology, Chinese Academy of Sciences (abbreviation:
Measurements were taken with a digital caliper to the nearest 0.1 mm by Shuo Liu. We measured: snout-vent length (SVL, from tip of snout to vent); head length (HL, from tip of snout to rear of jaw); head width (HW, width of head at widest point); snout length (SL, from tip of snout to anterior border of eye); eye diameter (ED, diameter of exposed portion of eyeball); tympanum diameter (TD, maximum diameter of tympanum); forearm and hand length (FHL, from elbow to tip of third finger); hand length (HAL, from the base of the outer palmar tubercle to the tip of third finger); total leg length (LEG, from vent to tip of fourth toe); tibia length (TL, distance from knee to heel); and tarsus and foot length (TFL, from heel to the tip of fourth toe). Morphological data of the type specimens of Odorrana macrotympana were taken from
Total genomic DNA was extracted from liver tissues using the DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). A fragment encoding mitochondrial 16S rRNA gene was amplified and sequenced. All new sequences have been deposited in GenBank, other sequences being downloaded from GenBank (Table
Samples used for phylogenetic analyses of molecular sequence data. * = type locality.
Species | Locality | Voucher No. | GenBank No. |
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Odorrana absita | Xe Sap, Xe Kong, Laos* | FMNH258109 | EU861542 |
Odorrana amamiensis | Tokunoshima, Ryukyu, Japan | KUHE24635 | AB200947 |
Odorrana anlungensis | Anlong, Guizhou, China* | HNNU1008I109 | KF185049 |
Odorrana aureola | Phu Luang, Loei, Thailand* | ZMKU AM 01137 | KT002162 |
Odorrana bacboensis | Khe Moi, Nghe An, Vietnam* | FMNH255611 | DQ650569 |
Odorrana banaorum | Tram Lap, Gia Lai, Vietnam | ROM7472 | AF206487 |
Odorrana chapaensis | Lai Chau, Vietnam | AMNH A161439 | DQ283372 |
Odorrana chloronota | Darjeeling, West Bengal, India* | BMNH 1947.2.28.6 | DQ650594 |
Odorrana daorum | Sa Pa, Lao Cai, Vietnam | ROM19053 | AF206482 |
Odorrana dulongensis | Dulongjiang, Yunnan, China* | KIZ035027 | MW128102 |
Odorrana exiliversabilis | Wuyishan, Fujian, China* | HNNU0607032 | KF185056 |
Odorrana fengkaiensis | Shiwanshan, Guangxi, China | HNNU295 7k | KF185033 |
Odorrana geminata | Cao Bo, Ha Giang, Vietnam | AMNH 163782 | EU861546 |
Odorrana grahami | Kunming, Yunnan, China* | HNNU1008II016 | KF185051 |
Odorrana graminea | Wuzhishan, Hainan, China* | HNNU0606123 | KF185038 |
Odorrana hainanensis | Wuzhishan, Hainan, China* | HNNU0606105 | KF185032 |
Odorrana hejiangensis | Hejiang, Sichuan, China* | HNNU1007I202 | KF185052 |
Odorrana hosii | Kuala Lumpur, Malaysia | IABHU 21004 | AB511284 |
Odorrana huanggangensis | Wuyishan, Fujian, China* | HNNU0607001 | KF185059 |
Odorrana ishikawae | Amami Island, Ryukyu, Japan | No voucher | AB511282 |
Odorrana jingdongensis | Jingdong, Yunan, China* | 20070711017 | KF185050 |
Odorrana junlianensis | Junlian, Sichuan, China* | HNNU002 | KF185058 |
Odorrana kuangwuensis | Nanjiang, Sichuan, China* | HNNU0908II185 | KF185034 |
Odorrana kweichowensis | Shilian, Meitan, Guizhou, China | CIBGYU20130917004 | MH193548 |
Odorrana leporipes | Shaoguan, Guangdong, China* | HNNU1008I099 | KF185036 |
Odorrana lipuensis | Lipu, Guilin, Guangxi, China* | NHMG1306002 | KM388699 |
Odorrana livida | Thagata Juwa, Myanmar* | BMNH 1889.2.25.48 | DQ650615 |
Odorrana lungshengensis | Longsheng, Guangxi, China* | HNNU70028 | KF185054 |
Odorrana macrotympana | Yingjiang, Yunnan, China* |
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OL831010 |
Odorrana macrotympana | Htamanthi, Sagaing, Myanmar | SEABRI 2019120040 | OL831011 |
Odorrana macrotympana | Htamanthi, Sagaing, Myanmar | SEABRI 2019120041 | OL831012 |
Odorrana macrotympana | Htamanthi, Sagaing, Myanmar | SEABRI 2019120072 | OL831013 |
Odorrana macrotympana | Htamanthi, Sagaing, Myanmar | SEABRI 2019120073 | OL831014 |
Odorrana margaretae | Emei, Sichuan, China | HNNU20050032 | KF185035 |
Odorrana morafkai | Tram Lap, Gia Lai, Vietnam | ROM7446 | AF206484 |
Odorrana nanjiangensis | Nanjiang, Sichuan, China* | HNNU1007I291 | KF185042 |
Odorrana narina | Okinawa Island, Ryukyu, Japan | No voucher | AB511287 |
Odorrana nasica | Ha Tinh, Vietnam | AMNH A161169 | DQ283345 |
Odorrana nasuta | Wuzhishan, Hainan, China* | HNNU051119 | KF185053 |
Odorrana sangzhiensis | Sangzhi, Hunan, China* | CSUFT 4305220051 | MW464865 |
Odorrana schmackeri | Yichang, Hubei, China* | HNNU0908II349 | KF185047 |
Odorrana supranarina | Iriomotejima, Ryukyu, Japan | KUHE2898 | AB200950 |
Odorrana swinhoana | Nantou, Taiwan, China | HNNUTW9 | KF185046 |
Odorrana tianmuii | Tianmushan, Zhejiang, China* | NHMG1303018 | KT315390 |
Odorrana tiannanensis | Hekou, Yunnan, China* | KIZ20193272 | OL831009 |
Odorrana tiannanensis | Hekou, Yunnan, China* | KIZ20193273 | OL831008 |
Odorrana tiannanensis | Hekou, Yunnan, China* | KIZ20193274 | OL831007 |
Odorrana tiannanensis | Hekou, Yunnan, China* | KIZ20215191 | OL831006 |
Odorrana tiannanensis | Hekou, Yunnan, China* | SCUM50510CHX | EF453751 |
Odorrana tormota | Huangshan, Anhui, China* | No voucher | DQ835616 |
Odorrana trankieni | Ta Xua, Son La, Vietnam* | VNMN04035 | KX893900 |
Odorrana utsunomiyaorum | Iriomotejima, Ryukyu, Japan | KUHE12896 | AB200952 |
Odorrana versabilis | Leishan, Guizhou, China* | HNNU003 LS | KF185055 |
Odorrana wuchuanensis | Wuchuan, Guizhou, China* | HNNU019 L | KF185043 |
Odorrana yentuensis | Tay Yen Tu, Bac Giang, Vietnam* | IEBRA.2015.38 | KX893891 |
Odorrana yizhangensis | Yizhang, Hunan, China* | HNNU1008I075 | KF185048 |
Odorrana yunnanensis | Longchuan, Yunnan, China* | HNNU001YN | KF185057 |
Pelophylax nigromaculatus | Aichi, Japan | No voucher | LC389208 |
Rana chensinensis | Ningshan, Shanxi, China | HNNU20060359 | KF185061 |
Sequences were aligned using MAFFT 7 (
The sequence alignment is 1095 bp in length. Topologies resulting from BI and ML analyses were consistent. The sequences of the specimens from Myanmar clustered with the sequence of our single topotype of Odorrana macrotympana; together, they represent a distinct lineage in the genus Odorrana, which is sister to O. tiannanensis with strong support (Fig.
Genetic divergence (uncorrected p-distance) between the specimens from Myanmar and the topotype of Odorrana macrotympana was only 0.43% (Suppl. material
Xueli Village, Taiping Town, Yingjiang County, Dehong Prefecture, Yunnan Province, China.
Type specimens of Odorrana macrotympana in preservative: A. Dorsal and B. Ventral, view of the holotype (
Measurements and the comparison with the type specimens are represented in Table
Measurements (mm) of Odorrana macrotympana. See text for character definitions. Data of the type specimens were obtained from the original description (
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SEABRI 2019120040 | SEABRI 2019120041 | SEABRI 2019120072 | SEABRI 2019120073 | SEABRI 2019120074 | |
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Holotype | Paratype | ||||||
Female | Male | Male | Female | Male | Female | Male | |
SVL | 95 | 50 | 56.8 | 111.2 | 59.1 | 91.6 | 56.7 |
HL | 35.5 | 20 | 22.3 | 40.6 | 24.8 | 33.4 | 23.8 |
HW | 32 | 17 | 21.2 | 37.2 | 22.0 | 32.5 | 22.2 |
SL | 15 | 8 | 10.1 | 18.2 | 10.3 | 15.6 | 10.8 |
ED | 13 | 7.5 | 7.9 | 11.7 | 8.2 | 9.2 | 8.0 |
TD | 6 | 5.5 | 5.6 | 6.6 | 5.8 | 5.7 | 6.2 |
FHL | 41.5 | 24 | 28.5 | 50.6 | 29.7 | 45.2 | 27.2 |
HAL | 16 (26) | 15.5 | 16.4 | 27.7 | 18.6 | 26.5 | 17.9 |
LEG | 185 | 100 | 117.0 | 219.0 | 121.0 | 195.5 | 117.0 |
TL | 63.5 | 32.5 | 36.2 | 68.7 | 38.5 | 60.2 | 35.9 |
TFL | 75.5 | 42.5 | 46.2 | 84.5 | 49.7 | 76.5 | 46.3 |
Forelimbs robust in males and relatively weak in females; relative lengths of fingers III > IV > I > II; all finger tips slightly expanded; lateral fringes and webbing on fingers absent; subarticular tubercle round and prominent; inner metacarpal tubercle and outer metacarpal tubercle distinct; greyish-yellow glandular nuptial pad on finger I in males.
Hind limbs long, tibia slightly longer than thigh; toes long and thin, relative lengths IV > V > III > II > I; all toe tips slightly expanded; entire webbing; subarticular tubercles prominent and longitudinally ovoid; inner metatarsal tubercle prominent and oval; outer metatarsal absent.
Dorsal skin smooth, lateral skin granular, ventral skin smooth. Dorsolateral folds distinct.
Dorsum greyish-brown, brownish-green or solid green with a few or many tiny black spots and several distinct or indistinct large black or brown spots. Upper and lower lips greyish-white, yellow, light green or brown with a few or many brown or black spots. Lateral side of head and body greyish-brown with many large or tiny black spots. Dorsal surfaces of limbs greyish-brown with some brown or black bands. Ventral surface of throat, chest and belly uniform white or some stains on ventral surface of throat, chest and anterior part of belly. Ventral surface of limbs white or pink. Tympanum brown, iris greyish-brown, pupil black.
In preservative, dorsum fades to greyish-black. Ventral surface of throat, chest, belly and limbs pale white. Iris black, pupil white.
Body size much smaller in adult males than in adult females, adult males have relatively stouter forelimbs than females and greyish-yellow glandular nuptial pad on finger I, external vocal sacs present in adult males.
In Myanmar, all specimens of Odorrana macrotympana were found on the banks of large rivers. During December, we heard the courtship calls from males on the river banks at night, the sound of the calls being similar to those of other Odorrana species. We also observed males and females in amplexus on the banks of the rivers. No eggs or tadpoles were found.
According to
Previously, Odorrana macrotympana was known only from its type locality (
Morphologically, the characters of the specimens from northern Myanmar mostly agree with the original description of Odorrana macrotympana apart from a few differences. According to the original description of O. macrotympana, the dorsum is greyish-brown and upper and lower lips are uniform white without any spots or streaks (
The type specimens of Odorrana macrotympana were collected in December 1994 and females have been observed to deposit eggs in February of the following year (
Our field survey in Myanmar was undertaken at the invitation of the Republic of the Union of Myanmar, Ministry of Natural Resources and Environmental Conservation, Forest Department, Forest Research Institute. We thank the Ministry of Myanmar for its invitation and its staff and the local guides for their help in the field. Thank Bin Yang for helping us collect specimens. Thanks to Xiaofeng Ma and Hong Hui for providing photos and Xinwang Yang for providing information about specimen collection. We also thank the editors and reviewers for their working on the manuscript.
Table S1
Data type: Table (MS EXCEL file)
Explanation note: Genetic divergence (uncorrected p-distance) (%) based on the mitochondrial 16S rRNA gene sequences.