Bioacoustics of Fejervarya zhoushanensis

Geographic distribution and natural history This species is currently known from Zhoushan Archipelago, Zhejiang , China ( Fig. 1 ) and its adjacent coastal Zhejiang . Zhoushan Archipelago located in the East China Sea at the entrance to Hangzhou Bay. It is composed of 1390 islands with varied area, belong to the largest sea archipelago in China . All islands were geologically separated from Chinese mainland at about 7000~9000 years ago. Most individuals of this new species were found in paddy field ( Fig. 9 ), while the remaining were in vegetable field and wasteland. Males were found vocalizing with the head upwards on the margin or adjacent areas of ponds. The reproductive habitats of new species were same as F. kawamurai .

Discussion The unified species concept treats a separately evolving lineage as the only necessary property of species ( de Queiroz 2007 ; Conix 2018 ; Zachos 2018 ). Within the F. limnocharis complex, phylogenetic differences have been crucial in distinguishing the four known species ( Veith et al. 2001 ; Matsui et al. 2007 ; Djong et al. 2011 ). Within the described distribution region (central and eastern China to Japan ) of F. kawamurai ( Djong et al. , 2011 ) , we previously demonstrated that populations from the Zhoushan Archipelago and adjacent Chinese mainland formed two distinct single lineages within this species complex based on mtDNA ( Yang et al. 2022 ). Here, using genomic SNP data, we further proved it through genetic clusters ( Fig. 2 ), phylogenetic ( Fig. 3A ) and species delimitation analyses ( Fig. 3B, C ). Therefore, our genetic and phylogenetic results, based on genomic and previous mtDNA data, provided robust evidences that the Zhoushan Archipelago with coastal Zhejiang and Chinese inland populations belong to two independent species. Interestingly, individuals from Zhejiang populations (Beilun, Pinghu, and Pujiang) clustered with Zhoushan Archipelago individuals on the same branch estimated from GBS data ( Fig. 3 ), while some samples from these localities belong to mtDNA lineage from central China rather than Zhoushan island ( Yang et al. 2022 ), implying the discordance between mtDNA and nDNA due to potential introgression. Moreover, the Admixture analysis indicated by our GBS SNPs data showed that the areas in coastal Zhejiang (Beilun, Pinghu, and Pujiang) and Jiangxi (Jinxian) might serve as a contact zone with historical gene flow between Chinese inland and Zhoushan Archipelago groups ( Fig. 2B ) though it seems that the gene flow did not significantly dilute the phylogenetic structure. Given that the early described lineage of F. kawamurai was frequently referred to central and eastern China in recent publications ( Djong et al. , 2011 ), we described the Zhoushan Archipelago lineage as an independent species based on multiple evidences. Although genetic analysis of a short Cytb fragment revealed small differentiation between the Zhoushan Archipelago populations and a specimen from the Hiroshima ( type locality described for F. kawamurai ) ( Yang et al. 2022 ), we currently refrain from concluding that both Zhoushan and Japanese populations belong to the F. kawamura for the following reasons: (1) a single sample cannot adequately represent the genetic diversity of the entire Japanese population, especially given the partial amplification of ancestral genes; (2) the observed low divergence in this small fragment may result from Cytb pseudogene amplification ( Hazkani-Covo et al. , 2010 ), the potential mtDNA and nDNA discordance as we discovered in Zhejiang coastal region in eastern China , the variable gene histories associated with the potential sampling error of loci; (3) the significant geographical separation and marine barriers between Zhoushan and Japan likely contribute to the distance isolated divergence. Therefore, based on current evidence, it is an effective species for Zhoushan Archipelago, especially through the GBS analysis in this study. Regarding to the samples from Hiroshima or other parts of the Japan , it would be done in the future through international collaboration. Morphological shape characteristics have previously been used for the diagnosis of F. sakishimensis ( Matsui et al. 2007 ) and F. kawamurai ( Djong et al. 2011 ) , serving as secondary species criteria and providing additional lines of evidence relevant to assessing lineage separation ( de Queiroz, 2007 ). In this study, we obtained nearly all the shape characteristics reported in these researches and tested the variations between the F. zhoushanensis sp. nov. and its sister linage, F. kawamurai . The F. zhoushanensis sp. nov. can be quite well diagnosed from F. kawamurai based on overall shape through DFA ( Fig. 4 ). Among the examined traits, several shape characteristics of F. zhoushanensis sp. nov. exhibited significant variations compared to F. kawamurai , including SVL , ED, AED , FLL HL, and FL ( Table 2 ), similar to the diagnosis for F. sakishimensis ( Matsui et al. 2007 ) and F. kawamurai ( Djong et al. 2011 ) . Therefore, these characteristics can also serve as vital morphological diagnostic features for F. zhoushanensis sp. nov. . To data, only shape characteristics have been reported for the diagnosis of species within the F. limnocharis complex. Further investigations of additional morphological features, such as advertisement calls ( Silva et al. 2022 ; Marinho et al. 2024 ), is warranted to enhance our understanding of species differentiation within this group. In summary, we proposed a new species, F. zhoushanensis sp. nov. , which followed the unified species concept, supported by both phylogenetic and morphological evidences. It is worthy to noting that two independent species respective the central China , and the Zhoushan Archipelago, should be recognized. Our results also indicated that the Zhoushan Archipelago may harbor the cryptic diversity of amphibian. Meanwhile, the island ecosystem is fragile and closed ( Quesada-Ruiz & Peña-Alonso 2023 ), which will hinder population expansion of F. zhoushanensis sp. nov. , especially on small islands (e.g., the Shengsi Islands). Therefore, greater attention should be paid to the conservation of this new species in the Zhoushan Archipelago.