Specimens were collected by hand. Liver tissues were stored in analytical pure ethanol and toads were preserved in 75% ethanol. All specimens (Fig. 1) were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Figure 1.  

Specimens of Bufo rubroventromaculatus from China in preservative. A Dorsal view; B ventral view.

Measurements were taken with a digital caliper to the nearest 0.1 mm. The methodology of measurements followed Orlov et al. (2024). The following morphological characteristics were noted: snout-vent length (SVL); head length (HL), from the back of the mandible to the tip of the snout; head width (HW), across the angles of the jaws; snout length (SNL), from the front of the eye to the tip of the snout; distance from the back of the mandible to the nostril (MN); distance from the back of the mandible to the front of the eye (MFE); distance from the back of the mandible to the back of the eye (MBE); horizontal eye diameter (ED); internasal distance (IN); distance between the front of the eyes (DAE); distance between the back of the eyes (DPE); nostril–snout distance (NS); eye–nostril distance (EN); distance from the axilla to the elbow (FLL); distance from the elbow to the tip of Finger III (HAL); inner palmar tubercle length (IPT); outer palmar tubercle length (OPT); finger I length (F1); finger II length (F2); finger III length (F3); and finger IV length (F4).

A fragment of the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) was amplified via the polymerase chain reaction (PCR) using the primers L-int: 5′-AGC ATC CTA CCC ACG ATT TCG-3′ (Fu et al. 2005) and H4980: 5′-ACT TTT CGG ATT TGA GTT TGG TT-3′ (Macey et al. 1998). The experimental protocols for amplification and sequencing followed Orlov et al. (2024). Sequences were stitched using SeqMan in Lasergene 7.1 (Burland 2000). All new sequences have been deposited in GenBank under the accessions PQ404040–PQ404048.

Sequences were aligned using ClustalW (Thompson et al. 2002) with default parameters in MEGA 11 (Tamura et al. 2021). Uncorrected pairwise distances were calculated in MEGA 11. The best substitution model was selected using the corrected Bayesian Information Criterion (BIC) in ModelFinder (Kalyaanamoorthy et al. 2017). Bayesian Inference was performed in MrBayes v.3.2.7 (Ronquist et al. 2012) under the selected substitution model (GTR+F+I+G4). The Markov chains were run for 5,000,000 generations and sampled every 100 generations. Maximum Likelihood analysis was conducted in raxmlGUI v.2.0 (Edler et al. 2021) under the selected substitution model (TN+F+I+G4) with 1,000 ultrafast bootstrap replicates.

Morphological measurements of the specimens from China are presented in Table 1. The head width is greater than the head length in all the specimens from China (HW/HL 1.17–1.35), while the head width is smaller than the head length in some individuals of the type series of Bufo rubroventromaculatus (HW/HL 0.88–1.15). Apart from this, there is no significant morphological difference between the specimens from China and the type specimens of B. rubroventromaculatus from Vietnam (Table 2).

Maximum Likelihood analysis and Bayesian Inference result in similar topologies. The specimens from China clustered with Bufo rubroventromaculatus from Vietnam (including the holotype) (Fig. 4). The genetic distances (uncorrected p-distance) between the specimens from China and B. rubroventromaculatus from Vietnam (including the holotype) ranged from 0 to 1.5% (Table 3).

Figure 4.  

Maximum Likelihood phylogenetic tree, based on ND2 sequences. Numbers after and behind the “/” are Maximum Likelihood ultrafast bootstrap values and Bayesian posterior probabilities, respectively. -/- represents less than 90/0.90.

In Orlov et al. (2024), the description of Bufo rubroventromaculatus is very brief, so it is necessary to make a supplementary description of the species. In this study, we confirmed the distribution of this species in China and provided a more detailed description of this species, based on the specimens collected from China.

According to the original description of Bufo rubroventromaculatus, the head width is smaller than the head length in the female holotype and the male paratype, while the head width is greater than the head length in the female paratype (Orlov et al. 2024). However, the head width is greater than the head length in all the specimens we collected from China. Orlov et al. (2024) provided photos of the holotype with a ruler. According to the photos of the holotype and the ruler, it can be seen that the value of the head length is clearly incorrect and the head width is obviously greater than the head length. Therefore, we consider that the head width should be greater than the head length in females of this species. As for males, we consider that it should be the same. Since Orlov et al. (2024) did not provide any photos for the male paratype, it needs to be verified by re-measurement of the specimen.

In the phylogenetic analysis of Orlov et al. (2024), the sequence (GenBank accession AY936852) of a specimen from Jiangkou County, Guizhou Province, China, clustered with the sequences of Bufo rubroventromaculatus. Our analysis confirmed that this specimen should indeed be assigned to B. rubroventromaculatus. However, Jiangkou is far away from the confirmed distribution of B. rubroventromaculatus and there is no other record of this species from the area between Jiangkou and the confirmed distribution of this species. Therefore, we are only certain that this species is distributed in Yunnan and Guangxi in China and whether this species is distributed in Guizhou remains to be verified.