Integrative taxonomic analyses reveal first country records of Occidozygashiwandashanensis Chen, Peng, Liu, Huang, Liao & Mo, 2022 and Hylaranalatouchii (Boulenger, 1899) (Anura, Dicroglossidae, Ranidae) from Vietnam

Abstract Background Occidozygashiwandashaensis was recently discovered from Guangxi Province of China. Hylaranalatouchii is a widespread species in southern China, including Hong Kong and Taiwan. Both species are expected to be found in the border areas between Vietnam and China; however, no records of these frogs have been documented from Vietnam so far. New information We record two species of amphibians for the first time from Vietnam, namely Occidozygashiwandashaensis from Bac Giang Province and Hylaranalatouchii from Hai Phong City and Quang Ninh Province in northern Vietnam. Morphologically, the Vietnamese representatives of O.shiwandashanensis resemble the type series from China. The specimens of H.latouchii from Vietnam slightly differ from the type series from China by having a larger size (SVL 48.6–51.7 mm in males, SVL 58.4 mm in the females vs. 36.0–40.0 mm in males, 42.0–53.0 mm in females). Genetic distances between the Vietnamese records and the type specimens of O.shiwandashanensis from China varied from 0 to 1.5% (16S gene). Genetic divergences between the Vietnamese records and H.latouchii from the type locality were 2.0–2.6% (16S gene). In addition, morphological data and natural history notes of the aforementioned species are provided, based on the new records from Vietnam.


Sampling
Field surveys were conducted in Tay Yen Tu Nature Reserve, Bac Giang Province in June 2007 and in May 2015; in Bai Tu Long National Park, Quang Ninh Province in May 2011, in June 2017 and in June 2023; and in Cat Ba National Park, Hai Phong City in July 2020 (Fig. 1).The coordinates (WGS 84) and elevations were determined by using the GPS Garmin 60CX.Amphibians were collected between 19:00 h and 23:00 h.After taking photographs of the individuals in life, frogs were anaesthetised and euthanised in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002), fixed in 80% ethanol for four hours, then later transferred to 70% ethanol for permanent storage.For molecular analysis, tissue samples of muscle and liver were preserved separately in 95% ethanol.Preserved specimens were deposited in the collection of the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.

Molecular analysis
One sample of Occidozyga and two samples of Hylarana were amplified fo r ~ 560 base pairs length fragment of the 16S rRNA mitochondrial gene (Suppl.material 1).Tissue samples were extracted using PureLink™ RNA Micro Scale Kit (Thermo Fisher Scientific company), following the manufacturers' instructions.Total DNA was amplified using PCR Applied Biosystems; the PCR volume consisted of 25 μl, including 12 μl of Mastermix, 6 μl of water, 1 μl of each primer at concentration of 10 pmol/μl and 5 μl of DNA.Primers used in PCR and sequencing were as follows: LR-N-13398 (5'-CGCCTGTTTACCAAAAACAT -3'; forward) and LR-J 12887 (5'-CCGGTCTGAACTCAGATCACGT -3'; reverse) (Simon et al. 1994).PCR conditions: 94°C for 5 minutes of initial denaturation; with 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 45 s; and the final extension at 72°C for 7 minutes.PCR products were sent to Apical Scientific company for sequencing (https://apicalscientific.com).The obtained sequences were deposited in GenBank under the accession numbers R656682 of Occidozyga sample and OR656680-OR656681 of Hylarana samples.
In addition to the sequence of the newly-collected sample of Occidozyga from Vietnam, we used 14 available sequences of 16S rRNA of 11 species of Occidozyga from GenBank (Chen et al. 2022) for phylogenetic analyses.Sequences of Limnonectes jarujini and Ingerana tenasserimensis were included in the analysis as outgroups.Locality information and accession numbers for all sequences included in the analysis can be found in Suppl.material 1.
In addition to the two sequences of the newly-collected samples of Hylarana from Vietnam, we used 29 available sequences of 16S rRNA of seven species of Hylarana from GenBank for phylogenetic analyses.Sequences of Babina holsti were included in the analysis as an outgroup.Locality information and accession numbers for all sequences included in the analysis can be found in Suppl.material 1.
Chromas Pro software (Technelysium Pty Ltd., Tewantin, Australia) was used to edit the sequences, which were aligned using the ClustalW (Thompson et al. 1997) option in MEGA 7.0 (Kumar et al. 2016) with default parameters and subsequently optimised manually in BioEdit 7.0.5.2 (Hall 1999).Locality information and GenBank accession numbers for all new sequences in this study can be found in Suppl.material 1. Pairwise comparisons of uncorrected sequence divergences (p-distance) were calculated with MEGA 7.0 (Kumar et al. 2016) where the outgroup was excluded.Variance was estimated using the bootstrap method with 1000 replicates using nucleotide substitution, while gap/ missing data were treated via pairwise deletion.Phylogenetic trees were constructed using Maximum Likelihood (ML) and Bayesian Inference (BI).Prior to ML and Bayesian phylogenetic analyses, we chose the optimum substitution models for entire sequences using Kakusan 4 (Tanabe 2011), based on the Akaike Information Criterion (AIC).The BI was performed in MrBayes 3.2 (Ronquist et al. 2012).The BI summarised two independent runs of four Markov Chains for 10,000,000 generations.A tree was sampled every 100 generations and a consensus topology was calculated for 70,000 trees after discarding the first 30,001 trees (burn-in = 3,000,000).We checked parameter estimations and convergence using Tracer version 1.5 (Rambaut and Drummond 2009).The strength of nodal support in the ML tree was analysed using nonparametric bootstrapping with 1000 replicates.We regarded tree nodes in the ML tree with bootstrap values of 75% or greater as sufficiently resolved (Hillis andBull 1993, Huelsenbeck andHillis 1993) and nodes with a BPP of 95% or greater as significant in the BI analysis (Leaché and Reeder 2002).

Morphological examination
Measurements were taken on preserved specimens with a digital caliper to the nearest 0.1 mm.The following abbreviations were used: SVL = snout-vent length, HL = head length (measured as a parallel line with the vertebral column from posterior margin of mandible to tip of snout), HW = maximum head width (across angles of jaws), RL = rostral length (from anterior corner of orbit to tip of snout), NS = distance from nostril to the tip of snout, EN = distance from anterior corner of orbit to the nostril, IND = internarial distance, IOD = interorbital distance, ED = eye diameter, UEW = maximum width of upper eyelid, MN = posterior margin of mandible to nostril, MFE = posterior margin of mandible to anterior corner of orbit, MBE = posterior margin of mandible to posterior corner of orbit; DAE = distance between anterior corners of orbits, DPE = distance between posterior corners of orbits, TD = tympanum diameter, TYE = distance from anterior margin of tympanum to posterior corner of orbit, FLL = forearm length, from elbow to base of outer palmar tubercle, HAL = hand length, from base of outer palmar tubercle to tip of third finger, FL1-4 = Finger length I-IV, NPL = nuptial pad length, FeL = femur length (from vent to knee), TbL= tibia length (from knee to tarsus), TbW = maximum tibia width, FoL = foot length (from tarsus to the tip of fourth toe), TL1-5 = toe length I-V.For webbing formula, we followed Glaw and Vences (2007).Sex was determined by the presence of nuptial pads and based on gonadal inspection.

Data resources
The aligned 16S dataset contained a total of 560 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 Occidozyga was strongly supported and in agreement with results of Chen et al. ( 2022).The specimen collected from Bac Giang Province of Vietnam, clustered with the specimens (including type specimens) of O. shiwandashanensis from China (Fig. 2).Genetic divergence between the specimen from Vietnam and the type specimens of O. shiwandashanensis is approximately 1.5% (Suppl.material 2).It is comparable to the interspecific genetic divergence (uncorrected p-distance) between the type samples of O. shiwandashanensis which is up to 1.5% (Suppl.material 2).Morphologically, the specimen from Bac Giang Province shows a similar appearance compared with the original description of O. shiwandashanensis.Therefore, we considered the population from Bac Giang, Vietnam to be conspecific with O. shiwandashanensis.
The aligned 16S dataset contained a total of 564 nucleotide base pairs (bp) in length, with 100 variable positions and 84 parsimony informative sites (including outgroups).The BI and ML analyses showed consistent topology (Fig. 3).The results indicated that the monophyly of Hylarana was strongly supported and two samples from Hai Phong City and Quang Ninh Province were closest to a sample which was collected from Jiulongshan National Nature Reserve, Zhejiang, China by Sun et al. (2021) (voucher specimen LSU20200422001ZL, GenBank accession number MT702387), a sample that was collected from Jinggangshan, Jiangxi Province in China by Xiao et al. (2019) (GenBank accession number MN241431) and a sample that was collected from Taiwan by Sumida et al. (2003) (GenBank accession number AB058880).The specimens collected from Hai Phong City and Quang Ninh Province in Vietnam clustered with those of H. latouchii from China (Fig. 3).Genetic divergences between the specimens from Vietnam and the type series of H. latouchii were 2.0-2.6%(Suppl.material 3).It is comparable to interspecific genetic divergence (uncorrected p-distance) between the samples of H. latouchii from China which varied from 0-2% and the samples of H. nigrovittata which varied from 0.2-2.8%(Suppl.material 3).Morphologically, the specimens from Quang Ninh Province show a similar appearance compared to the original description of H. latouchii.Therefore, we considered the frog population from Hai Phong and Quang Ninh, Vietnam to be conspecific with H. latouchii.Skin: Dorsal surface shagreened with small, raised tubercles, more prominent and dense on tibia; distinctly raised supratympanic fold stretching from corner of eye to shoulder; dorsolateral fold absent; ventral surface of throat, chest, abdomen and thighs scattered with small glands.

Taxon treatments
Colouration in life: Dorsum pale brown with irregular pale dark spots with a light yellow vertebral stripe; dorsal surface of hind limbs pale brown with dark crossbars; ventral surface creamy-white with brown spots on lateral margin and throat; ventral surface of limbs yellow-white with dense brown spots; ventral surfaces of palm and feet brown; pupil reddish-brown; iris pale brown (Fig. 4) (determination after Chen et al. ( 2022)).

Distribution
The species was previously known only from the Shiwandashan Mountain, Fangcheng, Guangxi, China (Chen et al. 2022).The new record of this species in Bac Giang Province of Vietnam is approximately 180 km distant from the type locality in China.Integrative taxonomic analyses reveal first country records of Occidozyga ...

Ecology
The specimens were found between 19:00 h and 23:00 h on the ground, in small ponds and in small streams.The surrounding habitat was mixed secondary evergreen forest consisting of larger and medium hardwoods, shrubs and arrowroot.The females contained yellowish-cream eggs with melanic poles.The specimens from Bac Giang Province were found at elevations of 300-400 m a.s.l., lower than the known altitude range in Guangxi, China (550-650 m a.s.l.) (Chen et al. 2022).

Notes
The specimen from Vietnam slightly differs from the type series from China by having the snout slightly shorter than eye diameter (vs.eye diameter less than snout length) and the presence of a light yellow vertebral stripe on the dorsum.Colouration in life: Iris black, surrounded by red-golden network; dorsum light yellow or grey yellow; flanks yellowish-white or with dark spots; dorsal surface of fore-and hindlimbs brown with dark brown cross bands; upper lip white; throat, chest, belly and ventral surface of thigh cream with dark brown mottling (Fig. 5) (determination after Fei et al. (2009) and Fei et al. (2012)).

Distribution
The species was previously known only from southern China (Zhejiang, Fujian, Guangxi, Hong Kong, Guangdong, Hunan, Jiangxi, Jiangxu and Anhui), including Taiwan (Frost 2023).The new record of this species in Hai Phong City and Quang Ninh Province, Vietnam is approximately 1,300 km distant from the type locality in Fuzhou, Fujian Province, China.

Ecology
The specimens were found between 19:00 h and 23:00 h on the ground, in small ponds and in small rocky streams.The surrounding habitat was mixed secondary karst forest and evergreen forest of medium hardwoods, shrubs and arrowroot.

Notes
The specimens from Vietnam slightly differ from the type series from China by having a slightly larger size (SVL 48.6-51.7 mm in males, SVL 58.4 mm in females vs. 36.0-40.0mm in males, 42.0-53.0 in females).
The new country record of Hylarana latouchii from Vietnam, which was already mentioned by Gawor et al. (2016), however, without specific identification "the taxonomic status of the Hylarana from Bai Tu Long needs further clarification", brings the total number of Hylarana to 15 in Vietnam.
Our research also showed that the genus Hylarana contains several species complexes.Interspecific genetic divergences of the species complexes is relatively high, for example, between population of H. latouchii, these were up to 2.6%, but still lower than those of H. maosonensis (up to 4.44%), H. annamitica (3.55%) and H. nigrovittata (2.8%).
These new discoveries highlight that the knowledge on the herpetofauna of Vietnam, particularly in the border region between China and Vietnam, is still incomplete and that additional field research is warranted.

Figure 2 .
Figure 2. The Bayesian Inference (BI) tree of Occidozyga, based on the partial 16S rRNA mitochondrial gene.Values at nodes correspond to BI/ML support values, respectively.

Hylarana latouchii (Boulenger, 1899) Materials
Fei et al. (2012)metrics of the specimens are provided in Suppl.material 5. Morphological characters of the specimens from Hai Phong City and Quang Ninh Province agreed well with the descriptions ofFei et al. (2009)andFei et al. (2012).