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).

Figure 1.  

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 (CCGGTYTGAACTCAGATCAYGT) (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. 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).

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).

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.

Figure 2.  

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.

Table 1

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), 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 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.