Fejervarya zhoushanensis, Tong & Wang & Xu & Guo & Guo & Shi & Jin, 2025

Tong, Haojie, Wang, Kaixin, Xu, Jun, Guo, Junru, Guo, Yaxin, Shi, Shuangyue & Jin, Yuanting, 2025, A new species in the Fejervarya limnocharis species complex (Amphibia: Dicroglossidae), Zootaxa 5660 (4), pp. 529-546 : 538-543

Fejervarya zhoushanensis sp. nov.

( Figs. 5–8 )

Diagnosis

A species that has independent systematic taxonomic status in F. limnocharis species complex. It is genetically closest to F. kawamurai , both sister lineages diverge last in the complex. The adult SVL is 35.0– 46.3 mm in males, 33.7–50.1 mm in females ( Table 2 ). This new species is differentiated from F. kawamurai by larger snout-to-vent length (SVL), longer head length (HL), larger eye diameter (ED) and aeddistance between anterior corners of eyes (AED), longer forearm length (FLL) and foot length (FL) for both sexes; in larger tympanum diameter (TD), snout length (SL) and inner metatarsal tubercle length (IMTL) for males; in longer hindlimb length (HLL) for females.

Holotype

CJLU ZSBD01 M ( Fig. 5 ). An adult female collected from paddy field in the main island of Zhoushan Archipelago, Zhejiang, China (122.2125° E, 30.0749° N, alt. 10.2 m a.s.l) on July, 2024 by Haojie Tong and Yuanting Jin.

Paratypes

Four paratypes were collected from Zhoushan Archipelago in July, 2024 by Haojie Tong and Yuanting Jin. CJLU ZSBD02 F (adult female, Fig. 6A, E ) and CJLU ZSBD03M (adult male, Fig. 6B, F ) were collected at the same locality as the holotype. CJLU ZSDS04 F (adult female, Fig. 6C, G ) and CJLU ZSDS05M (adult male, Fig. 6D, H ) were collected from Daishan island (122.1515° E, 30.2621° N, alt. 14.2 m a.s.l) .

Description of holotype

Adult female, SVL 43.12 mm ( Fig. 5 ). Head triangular, longer than wide, HW/HL 88%; HL/SVL 33%; HW/SVL 29%. Snout slightly pointed in dorsal and lateral views. Nostrils subcircular with low dermal flaps, below canthus, slightly closer to tip of snout (2.75 mm) than to eye (2.93 mm); canthus rostralis indistinct and slightly convex; loreal region slightly concave; ED/HW 36% and ED/HL 32%; IOD/IND 89% and IOD/UEW 81%; distance between posterior tips of eyes (8.76 mm) is wider than distance between anterior tips of eyes (5.11 mm). Tympanum distinct, round, small, TD/ED 60%; distance between eye and tympanum (0.92 mm) is about 1/3 of TD. Tympanic annulus rounded, with upper portion hidden by the supratympanic fold. Supratympanic fold evident from the upper part of the tympanum to close to the insertion of the arm. Tongue deeply notched, without papillae. Vomerine teeth in short with two barely separated transverse series. Vocal sac subgular, median, externally expanded, like a bilobate shape when deflated and inflated, on each side of mouth.

Forelimbs moderately hypertrophied; upper arm thinner than forearm. Fingers thin, short, unwebbed, fringed; finger tips bluntly rounded and not enlarged to disks; relative finger length III>I>IV>II ( Fig. 7A ). Subarticular tubercles single, rounded, prominent; prepollex oval, prominent; two oval, distinct palmar tubercles; supernumerary tubercles absent.

Hindlimbs relatively long, about 1.2 times length of SVL, TL/HLL 33%, FL/HLL 37%. Toes long and thin, toe tips blunt, slightly rounded, not enlarged to disks, relative toe length IV>III>V>II>I ( Fig. 7B ). Subarticular tubercles single, prominent and oval-shaped. Inner metatarsal tubercle single and elliptical, IMTL/1TL 67%; outer metatarsal tubercle single and round. Toe webs feeble, webbing formula: I1–2II1– 21/2 III2–3IV3–1V ( Fig. 7B ). Cloacal opening directed posteriorly at upper level of thighs.

Skin texture on dorsal and lateral surface of head, body and body flanks shagreened. Distinct, round glandular warts on whole parts of dorsum, continuing on dorsal surfaces of legs and arms. Small and scattered tubercles present on ventral surface of thighs, sub-cloacal region. Skin smooth on abdomen, ventral surfaces of forelimbs, tibiae, and tarsi. Dorsal skin showing small and longitudinal dermal ridges arranged in series.

Measurements of holotype (in millimeters)

SVL 43.12; HL 14.20; HW 12.47; ED 4.48; TD 2.71; BL 20.47; FLL 19.67; HLL 52.54; SL 6.38; IND 2.74; IOD 2.45; UEW 3.04; AED 5.64; TL 17.51; IMTL 2.62; 1TL 3.90; FL 19.69.

Color of the holotype

In life, male dorsal ground color grayish brown with large dark brown blotches; no vertebral line medially. Dark brown wide and incomplete crossbars on dorsal surface of limbs. Upper lip and superior parts of lower lip with consecutive dark bars. Upper half of tympanum coved by dark brown band. White on abdomen, ventral surfaces of thigh and tibia; black M-shaped bands across throat ( Fig. 5 ). In preservative, the coloration pattern has slightly faded, but no obvious changes in color patterns ( Fig. 8 ).

Variation in the type series

Morphological measurements are presented in Table 2 . Toe webbing formula varies as follows: I (1– 11/2) – (2– 21/2) II (1– 11/2) – (21/2) III 2 – (3– 31/2) IV (3– 31/2) – 1 V. In life ( Figs. 5–6 ), dorsal ground color grayish brown, some individual slightly light green (e.g., CJLU ZSDS 05M), some with paint red in intermediate region of forelimbs (e.g., CJLU ZSDS 04F). Red on cloacal region and ventral surfaces of thigh (e.g., CJLU ZSBD 01M, CJLU ZSBD 02F, CJLU ZSDS 04F and CJLU ZSDS 05M). In preservative ( Fig. 8 ), coloration patterns are similar, but some specimens became lighter ( CJLU ZSDS 04F, CJLU ZSDS 05M) after fixation in 10% formalin.

Comparisons

Fejervarya zhoushanensis differs from its congeners as follows: From F. kawamurai by larger SVL (35.0– 46.3 mm in males, 33.7–50.1 mm in females of F. zhoushanensis vs 26.2–39.4 mm in males, 26.0– 41.4 mm in females of F. kawamurai ), larger ED (4.0– 6.7 mm in males, 4.3–7.1 mm in females of F. zhoushanensis vs 2.7–4.4 mm in males, 2.9–5.0 mm in females of F. kawamurai ) and AED (4.8–6.8 mm in males, 5.2–7.5 mm in females of F. zhoushanensis vs 4.6–6.3 mm in males, 5.0– 6.9 mm in females of F. kawamurai ), longer FLL (13.9–23.5 mm in males, 11.5–27.4 mm in females of F. zhoushanensis vs 14.2–23.0 mm in males, 13.9–21.6 mm in females of F. kawamurai ) and FL (15.2–21.3 mm in males, 18.2–25.7 mm in females of F. zhoushanensis vs 15.3–19.9 mm in males, 16.2–22.2 mm in females of F. kawamurai ); larger TD (2.7–4.7 mm of F. zhoushanensis vs 2.1–3.1 mm of F. kawamurai ), SL (5.3–7.2 mm of F. zhoushanensis vs 5.4–6.5 mm of F. kawamurai ) and IMTL (1.3–2.2 mm of F. zhoushanensis vs 1.2–2.0 mm of F. kawamurai ) for males; and longer HLL (50.3–73.7 mm of F. zhoushanensis vs 38.1–60.6 mm of F. kawamurai ) for females; from F. sakishimensis and F. multistriata by having smaller size ( SVL 35.0– 46.3 mm in males, 33.7–50.1 mm in females of F. zhoushanensis vs 45.2–55.5 mm in males, 48.5–69.2 mm in females of F. sakishimensis , 40.4–44.4 mm in males, 44.6–52.4 mm in females of F. multistriata ); it further differs from F. sakishimensis by larger TD (ratio TD/ SVL 0.06–0.13, mean 0.09, in F. zhoushanensis vs 0.06–1.00, mean 0.07, in F. sakishimensis ); from F. cancrivora (Gravenhorst, 1829) and F. moodiei (Taylor, 1920) by having an outer metatarsal tubercle (absent in F. cancrivora and F. moodiei ), and by its smaller size ( SVL 35.0– 46.3 mm in males, 33.7–50.1 mm in females of F. zhoushanensis vs 58.6–77.2 mm in males, 76.4–99.1 mm in females of F. cancrivora , 44.5–74.9 mm in males, 46.1–88.1 mm in females of F. moodiei ); from F. kupitzi Köhler, Mogk, Khaing, and Than, 2019 by having smaller size ( SVL 35.0– 46.3 mm in males, 33.7–50.1 mm in females of F. zhoushanensis vs 44.2–49.2 mm in males, 51.2–63.6 mm in females of F. kupitzi ), and by having a head width less than head length (vs head that is about as long as wide in F. kupitzi ); from F. iskandari Veith, Kosuch, Ohler, and Dubois, 2001 by its larger size ( SVL 35.0– 50.1 mm in F. zhoushanensis versus 40.4–42.7 mm in F. iskandari ), and by larger TD (ratio TD/ SVL 0.06–0.13, mean 0.09, in F. zhoushanensis vs 0.06–0.08, mean 0.07, in F. iskandari ); from F. limnocharis and F. orissaensis (Dutta, 1997) by larger TD (vs ratio TD/ SVL 0.07–0.08, mean 0.08, in F. limnocharis , 0.05–0.09, mean 0.7, in F. orissaensis ); it further differs from F. orissaensis by having slighter smaller size ( SVL 35.0– 46.3 mm in males, 33.7–50.1 mm in females of F. zhoushanensis vs 37.8–51.4 mm in males, 41.1–60.0 mm in females of F. orissaensis ); from F. triora Stuart, Chuaynkern, Chanard, and Inger, 2006 by smaller size ( SVL 35.0– 50.1 mm in F. zhoushanensis vs 54.9–60.2 of F. triora ); from F. vittigera (Wiegmann, 1834) by having a free fap of skin along outer edge of fifth toe and metatarsal (vs absent in F. vittigera ).

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 .

Etymology

The specific epithet zhoushanensis is derived from the Zhoushan city, Zhejiang, China.

Suggested common names

We suggest the following common name: Zhoushan Rice Frog (English), ḂƜ¨ǟ(Chinese).

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.

Figures

FIGURE 1. Map showing collection locations sampled for GBS analysis in China. Alphanumeric codes refer to collection location details in Table 1. Orange symbols are populations records from Chinese mainland, purple symbols—Zhoushan Archipelago populations, black symbols—Hainan population. Map B refers to enlarged information in box area of map A.

FIGURE 2. Genetic analysis for Zhoushan Archipelago populations and Chinese mainland populations based on SNP data. (A) PCA analysis. (B) ADMIXTURE barplot.

FIGURE 3. The phylogenetic tree and species tree estimated from GBS data of F. kawamurai, F. zhoushanensis sp. nov. and F. multistriata. (A) The topologies obtained from RAxML phylogenetic tree estimated from concatenated SNPs. Node labels represent RAxML bootstrap values. (B) The SNAPPER species tree with posterior probabilities showed on each node. (C) The densitree species tree plot from the posterior distribution of species tree.

FIGURE 4. Plot of individual scores on the first two discriminant functions (DF1 and DF2) from the DF analysis of morphological characteristics.

FIGURE 5. Fejervarya zhoushanensis sp. nov., holotype in life (CJLU ZSBD01M). Body in (A) dorsal, (B) ventral, (C) dorsolateral, and (D) anterior views. Scale bar (while lines), 10 mm.

FIGURE 6. Four paratypes of F. zhoushanensis sp. nov. in life. Dorsal views of (A) CJLU ZSBD02F (female), (B) CJLU ZSBD03M (male), (C) CJLU ZSDS04F (female) and (D) CJLU ZSDS05M (male). Ventral views of (E) CJLU ZSBD02F, (F) CJLU ZSBD03M, (G) CJLU ZSDS04F and (H) CJLU ZSDS05M. Scale bar (while lines), 10 mm.

FIGURE 7. Ventral views of right hand (A) and foot (B) of the male holotype of F. zhoushanensis sp. nov.. Scale bar (while lines), 10 mm.

FIGURE 8. The holotype and four paratypes of F. zhoushanensis sp. nov. in preservation with 10% formalin. Dorsal views of (A) CJLU ZSBD01M, (B) CJLU ZSBD02F, (C) CJLU ZSBD03M, (D) CJLU ZSDS04F and (E) CJLU ZSDS05M. Ventral views of (F) CJLU ZSBD01M, (G) CJLU ZSBD02F, (H) CJLU ZSBD03M, (I) CJLU ZSDS04F and (J) CJLU ZSDS05M. Scale bar (while lines), 10 mm.

FIGURE 9. Habitat at the type locality of F. zhoushanensis sp. nov., main island of Zhoushan Archipelago, Zhejiang Province, China. (122.11° E, 30.371° N, alt. 34.1 m a.s.l).