First national record of Microhylahmongorum Hoang, Nguyen, Phan, Pham, Ninh, Wang, Jiang, Ziegler and Nguyen, 2022 (Anura, Microhylidae, Microhyla) in China

Abstract Background To date, 10 species of the genus Microhyla have been recorded in China, of which six were distributed in Yunnan Province. Microhylahmongorum 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. New information We report the first country record of Microhylahmongorum, based on specimens collected from Yunnan border region. Morphologically, the specimen was consistent with the original descriptions of M.hmongorum. Phylogenetically, the sequences of the specimens from China clustered with the sequence of type specimens of M.hmongorum from Vietnam, with uncorrected pairwise distances of 0.9% at the 16S gene fragment analysed. Therefore, we report M.hmongorum as a new record species in China.

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.

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).
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 μl 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.First national record of Microhyla hmongorum Hoang, Nguyen, Phan, Pham, ... 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 M. heymonsi complex and outgroup ( M. 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).

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 BI 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 M. 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, ...
Dorsal skin surface relatively smooth with small tubercles; ventral surfaces of body and limbs smooth; flanks of body relatively smooth (Fig. 3).
In preservation.Dorsal surfaces of body and limbs greyish or brown, with usually a yellow hair-fine median line from snout to anus and two very small black spots on back, forming " ()" -shape; ventral surfaces of body whitish obscured by many brown marblings; flanks and lateral side of head dark with a dark lateral stripe; ventral side of throat of adult male black; dorsal parts of limbs, fingers and toes with brown crossbars (Fig. 3).

Distribution
Microhyla hmongorum was previously known in Lai Chau Province, northern Vietnam, Phongsali and Luang Prabang Provinces of Laos and Kachin of Myanmar (Hoang et al. 2022).Our study further extends the species' distribution range northwards to Mengla, Xishuangbanna and Yuanyang, Honghe, Yunnan Province, China.

Ecology
The species is often found in areas that are highly disturbed by human activity.The habitat of the species mainly includes paddy fields, still ponds and rain puddles.Breeding season of the species is mainly during April to September.This species is in sympatric distribution with M. butleri, M. mukhlesuri and F. multistriata.

Notes
Morphological characters of the specimen from China agreed well with the original description of Hoang et al. (2022).Based on the type locality in northern Vietnam, we suggest "Yuè Běi Jī Wā (越北姬蛙)" as its Chinese common name.First national record of Microhyla hmongorum Hoang, Nguyen, Phan, Pham, ...

Discussion
Yunnan Province, renowned for its diverse range of species, is located in southwest China and lies at a biological crossroads of three biodiversity hotspots; the Himalaya, mountains of southwest China and Indo-Burma (Mittermeier et al. 2004).China tops the list of amphibian diversity amongst these biodiversity hotspots (Jiang et al. 2016).In addition, 36 new amphibian species from Yunnan have been discovered and identified over the past three years (AmphibiaChina 2023).These results indicate that the region's rich amphibian diversity is still underestimated.Our study confirms that specimens from Mengla, Xishuangbanna and Yuanyang, Honghe, Yunnan Province, China belong to M. hmongorum, representing the first record of this species in China.The discovery of M. hmongorum in this study raises the overall number of known amphibian species in China from 634 (AmphibiaChina 2023) to 635, along with the known number of Microhyla species from 10 to 11 (Frost 2023).Notably, this brings the number of Microhyla species recorded in Yunnan, China, to seven, namely M. hmongorum, M. berdmorei, M. butleri, M. fissipes, M. heymonsi, M. mukhlesuri and M. pulchra.Our findings further validate the underestimation of amphibian diversity in Yunnan.Furthermore, this species is currently known from two isolated areas in Yunnan: Xishuangbannan and Honghe which are separated by a straight-line distance of approximately 200 km.However, the species is likely to occur in other parts of Yunnan, China.Therefore, more samples from China should be included to conduct detailed studies on M. heymonsi complex to clarify the distribution range of species, especially to further confirm the distribution of M. heymonsi in Yunnan in the future.
Our findings further support the need to prioritise future attention to the diversity and taxonomy of amphibians in the southwest border region.Although various studies have clarified the taxonomy and geographic ranges of some species, such as Amolops viridimaculatus (Zhang et al. 2021, Mahony et al. 2022) inornata (Liu et al. 2021) continue to be controversial.Moreover, one of the newly-recorded species we reported was found along the China-Laos border.Given its location in the same zoogeographic region, we presume that this species might also be present in northern Laos.As a result, we recommend international collaboration to strengthen fieldwork along the south-western borders paired with the integration of molecular and acoustic data in order to uncover more new species and new record species and, therefore, clarify taxonomy questions.

Figure 1 .
Figure 1.Map showing the new record in China (red circle) and the type locality of M. hmongorum (blue triangle) in Vietnam.

Figure 3 .
Figure 3. Male of M. hmongorum (KIZ 027488) in preservative.A Dorsal view; B. Ventral view; C Ventral view of figure; D Ventral view of foot.

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.

,
Limnonectes limborgi (Huang et al. 2022) and published a series of new recorded species, new recorded genera and new species have been discovered in border region in recent years (e.g.Yuan et al. (2019), Wu et al. (2021), Zhang et al. (2022)), Amphibian diversity still remains underrated while the taxonomy of several species, such as A. mengyangensis (Wu et al. 2020) and Micryletta