Biodiversity Data Journal 11: e103580 OO) doi: 10.3897/BDJ.11.e103580 open access Taxonomy & Inventories First national record of Microhyla hmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler and Nguyen, 2022 (Anura, Microhylidae, Microhyla) in China Yun-He Wu#§, Zhong-Bin Yu*$, Chen-Qi Lu*!, Kasyoka Kilunda Felista*!, Shao-bing HouF, Jie-Qiong Jin*§, Jin-Min Chen*, Dong-Ru Zhang}, Zhi-Yong Yuan", Jing Che? + State Key Laboratory of Genetic Resources and Evolution & Yunnan key laboratory of biodiversity and ecological conservation of Gaoligong Mountain, Kunming Institute of Zoology, Chinese Academy of Sciences, 650223, Kunming, Yunnan, China § Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, 05282, Yezin, Nay Pyi Taw, Myanmar | Kunming College of Life Science, University of the Chinese Academy of Sciences, 650204, Kunming, Yunnan, China | Key Laboratory of Conserving Wildlife with Small Populations in Yunnan, Southwest Forestry University, 650224, Kunming, Yunnan, China Corresponding author: Jing Che (chej@mail.kiz.ac.cn) Academic editor: Truong Nguyen Received: 16 Mar 2023 | Accepted: 01 Apr 2023 | Published: 10 Apr 2023 Citation: Wu Y-H, Yu Z-B, Lu C-Q, Felista KK, Hou S-b, Jin J-Q, Chen J-M, Zhang D-R, Yuan Z-Y, Che J (2023) First national record of Microhyla hmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler and Nguyen, 2022 (Anura, Microhylidae, Microhyla) in China. Biodiversity Data Journal 11: e103580. https://doi.org/10.3897/BDJ.11.€103580 Abstract Background To date, 10 species of the genus Microhyla have been recorded in China, of which six were distributed in Yunnan Province. Microhyla hmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler, and Nguyen, 2022 was also speculated to be distributed in Xishuangbana, Yunnan Province, China. However, there is no evidence of documentation of M. hmongorum. © Wu Y et al. 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. 2 Wu Y et al New information We report the first country record of Microhyla hmongorum, based on specimens collected from Yunnan border region. Morphologically, the specimen was consistent with the original descriptions of iM. hmongorum. Phylogenetically, the sequences of the specimens from China clustered with the sequence of type specimens of IV. hmongorum from Vietnam, with uncorrected pairwise distances of 0.9% at the 16S gene fragment analysed. Therefore, we report VV. hmongorum as a new record species in China. Keywords Amphibia, Microhyla hmongorum, new record, China Introduction Microhyla Tschudi, 1838, a genus of the family Microhylidae, is widely distributed throughout India and Sri Lanka eastwards to the Ryukyu Archipelago of Japan and southwards to Indonesia and presently includes 51 recognised species (Frost 2023). To date, 10 species have been recorded in China, namely M. beilunensis Zhang, Fei, Ye, Wang, Wang and Jiang, 2018, MV. berdmorei (Blyth 1856), VM. butleri Boulenger, 1900, MW. dabieshanensis Zhang, Chen and Zhang, 2022, M. fanjingshanensis Li, Zhang, Xu, Lv and Jiang, 2019, M. fissipes Boulenger, 1884, M. heymonsi Vogt, 1911, M. mixture Liu and Hu, 1966, M. mukhlesuri Hasan, Islam, Kuramoto, Kurabayashi and Sumida, 2014 and WM. pulchra (Hallowell, 1861) (AmphibiaChina 2023). Of these 51 species, six occur in Yunnan, M. berdmorei, M. butleri, M. fissipes, M. heymonsi, M. mukhlesuri and M. pulchra (Yuan et al. 2022). Hoang et al. (2022) studied the distribution pattern of the VM. heymonsi group with descriptions of two new species (IV. hmongorum and M. xodangorum) from Vietnam, which postulated the distribution of M. hmongorum in Xishuangbana, Yunnan Province, China. However, there is no evidence of documentation of M. hmongorum in China. To date, this species was known only from the type locality in Vietnam. Yunnan Province borders Vietnam, Laos and Myanmar. Recently, several cryptic and novel amphibians’ species have been described in the border region (e.g. Yang and Huang (2019), Yuan et al. (2019), Chen et al. (2020), Liu et al. (2021), Wu et al. (2021), Zhang et al. (2022)). These findings imply that the amphibian diversity in the border region may still be diverse and largely underestimated. During our fieldworks in Yunnan, China, we collected some specimens of rice frogs, which can be assigned to the genus M. heymonsi complex. Detailed morphological comparisons and molecular analysis indicated that these specimens should be categorised as M. hmongorum; thus, we herein describe the new records in details. First national record of Microhyla hmongorum Hoang, Nguyen, Phan, Pham, ... 3 Materials and methods Field surveys were conducted in Xishuangbanna and Yuanyang, Yunnan Province, China in April 2016 and September 2017 (Fig. 1). These specimens were euthanised and fixed in 10% formalin and then transferred to 70% ethanol for permanent storage. Liver tissue samples were preserved in absolute ethyl alcohol for molecular analysis. The tissue sample was deposited in the Herpetological Museum of Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). 100°E 102°E 104°E 106°E 24°N 22°N 22°N 20°N 20°N 100°E 102°E 104°E 106°E Figure 1. EES] Map showing the new record in China (red circle) and the type locality of M. hmongorum (blue triangle) in Vietnam. Total genomic DNA was extracted from liver tissues using the standard phenol-chloroform extraction protocol (Sambrook et al. 1989). The mitochondrial gene 16S ribosomal RNA gene (16S rRNA) was amplified and sequenced from six specimens using the primer pairs (5-3’) 16S rRNA-F (CGCCTGTTTAYCAAAAACAT) and 16S rRNA-R (CCGGTYT GAACTCAGATCAYGT) (Kocher et al. 1989). PCR amplifications were performed in a 25 ul reaction volume with the following procedure: initial denaturing step at 95°C for 4 min, 35 cycles of denaturing at 94°C for 40 s, annealing at 55°C for 1 min and extending at 72°C for 1 min and a final extension at 72°C for 10 min. The amplified PCR product was purified using Qiagen PCR purification kit and then sequences in both directions were obtained from an ABI 3100 automated sequencer. New sequences were deposited in GenBank under accession numbers (the GenBank accession numbers are available in Suppl. 4 Wu Y etal material 1). Newly-obtained sequences were first assembled and edited using AutoSeqMan (Sun 2018). Phylogenetic relationships amongst M. heymonsi complex were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI) to reconstruct phylogenetic relationships. Homologous sequences of VM. heymonsi complex and outgroup (//. marmorata) were obtained from GenBank (Suppl. material 1). New sequences incorporated with homologous data retrieved from GenBank were aligned using MUSCLE 3.8 (Edgar 2004) and then inspected by eye for accuracy and trimmed to minimise missing characters in MEGA6 ( Tamura et al. 2013). Both BI and ML analyses were executed in the CIPRES web server ( Miller et al. 2010). The GTR+I+G model was selected as the best substitution model by jModelTest 2.1.4 (Darriba et al. 2012). The BI analyses used Metropolis Coupled Markov Chain Monte Carlo (MCMC) with three heated chains and one cold chain for 10 million generations and sampled every 1,000 generations, with the first 25% of samples discarded as burn-in. Maximum Likelihood analyses were performed using RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014). The analyses used the proportion of invariable sites estimated from the data and 1,000 bootstrap pseudoreplicates under the GTR+G model. Pairwise divergences (uncorrected p-distance) between species on 16S dataset were calculated using MEGA6 (Tamura et al. 2013). Measurements were recorded to the nearest 0.1 mm with digital calipers by Zhong-Bin Yu following Fei et al. (2009). Measurements included: SVL (Snout-vent length); HL (Head length); HW (Head width); SL (Snout length); INS (Internasal space); IOS (Interorbital space); NED (nasal to eye distance); UEW (Upper eyelid width); ED (Eye diameter); TD (tympanum diameter); LAL (lower arm length); LAHL (Length of lower arm and hand); HAL (Hand length); LAD (Diameter of lower arm); FEM (Femoral length); TL (Tibia length); FTL (Foot length). Data resources The aligned 16S dataset contained a total of 1153 nucleotide base pairs (bp) in length, with 269 variable positions and 176 parsimony informative sites (including outgroups). The Bl and ML analyses showed consistent topology (Fig. 2). The results indicated that the monophyly of the Microhyla heymonsi group was strongly supported and in agreement with results of Hoang et al. (2022). These specimens collected from Mengla and Yuanyang, Yunnan, China, clustered with the specimens (including the type specimens) of /. hmongorum from Vietnam (Fig. 2). Genetic divergence between the specimens from China and the type specimens of M. hmongorum was only 0.9% (Table 1). It is comparable to interspecific genetic divergence (uncorrected p-distance) between the new sample obtained from Yunnan, China and the other species of Microhyla heymonsi group varied from 4.0% (versus M. cf. heymonsi) to 11.1% (versus M. neglecta) (Table 1). Morphologically, the specimen from Yunnan Province shows a similar appearance to the Original description of M. hmongorum. Therefore, we considered the Yunnan, China population to be conspecific with M. hmongorum. First national record of Microhyla hmongorum Hoang, Nguyen, Phan, Pham, ... Table 1. Uncorrected p-distances amongst the Microhyla heymonsi group (below the diagonal) and standard error estimates (above the diagonal). The ingroup mean uncorrected p-distances are shown on the diagonal. Species 1 2 3 4 5 6 7 8 9 M. ninhthuanensis 1.4 1.0 0.9 1.0 1.3 1.5 1.2 0.8 1.5 M. daklakensis 5.4 0.2 0.9 1.0 1.3 455 ste) 1.0 1.6 M. cf. heymonsi 4.5 4.8 1.7 0.7 1.2 1.4 1.1 0.8 1.5 M. heymonsi 4.8 5.3 3.3 — 1.3 Ps) axe) 0.9 15 M. pineticola 9.1 8.4 7.7 8.4 — 1.1 1.3 1.3 1.6 M. neglecta 10.8 10.2 9.7 9.9 5.2 — tes 1:6 1.6 M. xodangorum 7.8 7.7 6.6 7.9 8.6 11.4 — 1.2 1.6 M. hmongorum 4.1 5.6 4.0 4.5 9.3 11.1 6.6 0.9 1.5 M. marmorata 12.4 12.1 11.6 12.5 13.0 13.2 13.2 11.3 — Microhyla cf. heymonsi IEBR A.5057 1/100} Microhyla cf. heymonsi IEBR A.5058 Microhyla cf. heymonsi IEBR A.5059 0.99/-1 EMicrohyla cf. heymonsi IEBR A.5060 1/-,Microhyla cf. heymonsi IEBR A.5062 Microhyla cf. heymonsi IEBR A.5063 ‘Microhyla cf. heymonsi 1EBR A.5061 1/98 Microhyla cf. heymonsi VNMN 04450 Microhyla cf. heymonsi IEBR A.5056 1/96 pMicrohyla cf. heymonsi IEBR AS075 90) °Microhyla cf. heymonsi VNMN 04244 0.977) UMicrohyla cf. heymonsi VNMN 04245 0.98/95 Microhyla cf. heymonsi TEBR A.5054 ‘Microhyla cf. heymonsi 1EBR AS074 Microhyla cf. heymonsi IEBR A.5055 Microhyla cf. heymonsi YEBR A5072 0.99/82" Microhyla cf, heymonsi: IEBR AS073 ».98/100¢ Microhyla cf. heymonsi IEBR A.5064 Microhyla cf. heymonsi IEBR A.5065 1/100] _jpMicrohyla cf. heymonsi EBR A.5066 Microhyla cf. heymonsi IEBR A.5067 Microhyla cf. heymonsi AMNH A163850 Microhyla cf. heymonsi 0.97/74 i ‘Microhyla cf. heymonsi IEBR A5071 0.98/98 ag Microhyla cf, heymonsi ND.17.16 e -Microhyla cf. heymonsi ND.17.17 \ Microhyla cf. heymonsi 1EBR A.5068 Microhyla cf. heymonsi 1EBR A.5069 Microhyla cf. heymonsi EBR A.S070 Microhyla heymonsi KUHE 50505 * Microhyla hmongorum lEBR A.4905* Microhyla hmongorum IEBR A.4906* Microhyla hmongorum 1EBR A.4907* Microhyla hmongorum 1EBR A.4910* Microhyla hmongorum 1EBR A.4908* , Microhyla hmongorum IEBR A.4909* 0.97/79 Microhyla hmongorum IEBR A.4911* Microhyla hmongorum 2004.0414 Microhyla hmongorum KIZ 032775 Microhyla hmongorum K1IZ 032776 193 | ~83] -Microhyla hmongorum KIZ 032771 Microhyla hmongorum KIZ 032772 Microhyla hmongorum Tissue YPX46569 Microhyla hmongorum KIZ 027488 ‘Microhyla ninhthuanensis IEBR A.S052 Microhyla heymonsi group 0.97/81] “Microhyla_ninhthuanensis 1EBR A.5053 Microhyla ninhthuanensis VNMN 2021.05 Microhyla ninhthuanensis CIB (HAO185)* Microhyla ninhthuanensis KUHE 23856 Microhyla ninhthuanensis ZMMU NAP-03780 1/99 Microhyla pineticola VNMNO7719 Microhyla neglecta VNMN07344 Microhyla xodangorum \EBR A.4913 1/98 ‘Microhyla daklakensis ZISP 14249* Microhyla daklakensis CIB (VNMN 06858)* ee -Microhyla marmorata IEBR A5076 0.02 Figure 2. EES Phylogenetic tree of M@. heymonsi complex, based on Bayesian Inference of a fragment of the mitochondrial 16S gene. Nodal support values with Bayesian posterior probability (BPP) > 0.95 / ML inferences (BS) > 70 are performed near the respective nodes. A “-” Bayesian posterior probability < 0.95 and bootstrap support < 70. Bayesian posterior probability (BPP) < 0.95 / ML inferences (BS) < 70 are not shown. 1/100 0.98/94 1/100 1/97 6 Wu Y et al Taxon treatment Microhyla hmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler and Nguyen, 2022 Material a. acceptedNameUsage: Microhyla hmongorum; class: Amphibia; order: Anura; family: Microhylidae; genus: Microhyla; specificEpithet: hmongorum; country: China; countryCode: CHN; stateProvince: Yunnan; county: Yuanyang; locality: Panzhihua; verbatimElevation: 1375 m; verbatimLatitude: 23°3'14.89"; verbatimLongitude: 102°44'58.09"; individualCount: 1; sex: male; lifeStage: adult; catalogNumber: KIZ 027488; basisOfRecord: preserved specimen Description Morphmetrics of the speciemen are provided in see Suppl. material 2. Small size frog, body triangle, adult male with SVL 19.9 mm; head length (HL 6.2 mm, 32.1% of SVL) slightly longer than width (HW 5.8 mm, 29.1% of SVL); snout rounded in profile, projecting beyond the lower jaw, its length (SL 2.9 mm, 14.6% of SVL) longer than horizontal diameter of eye (ED 1.9 mm, 9.5% of SVL); canthus rostralis round, loreal region vertical and slightly concave; interorbital space flat, larger (IOS 2.1 mm, 10.6% of SVL) than width of upper eyelid (UEW 1.4 mm, 7.0% of SVL) and internarial distance (INS 1.9 mm, 9.5% of SVL); snout longer than eye diameter (SL/ED 152.6%); tympanum hidden; vomerine teeth absent; tongue posteriorly oval and not notched behind; supratympanic fold weak, extending from posterior corner of eye to arm insertion; male with internal single subgular vocal sac; nuptial pad absent (Fig. 3). Forelimbs slender; lower arm length (LAL 3.4 mm, 17.1% of SVL) shorter than hand length (HAL 4.8 mm, 24.1% of SVL); relative finger lengths: I