Molecular and morphological evidence reveals a hidden new taxon in the endemic genus Pseudocuneopsis (Bivalvia, Unionidae) from China Author Liu, Lili https://orcid.org/0009-0009-1164-7735 School of Life Science, Shanxi Normal University, Taiyuan 030031, China Author Zhang, Liping School of Life Science, Shanxi Normal University, Taiyuan 030031, China Author Jin, Dandong Datian High School, Linhai 317004, China Author Wang, Haotian China Development Bank Guangxi Zhuang Autonomous Region Branch, Nanning 530025, China Author Liu, Xiongjun https://orcid.org/0000-0002-5333-6670 School of Life Science, Jiaying University, Meizhou 514015, China Author Wu, Ruiwen https://orcid.org/0000-0002-8936-6054 School of Life Science, Shanxi Normal University, Taiyuan 030031, China 494212953@qq.com text ZooKeys 2023 2023-09-11 1179 219 229 http://dx.doi.org/10.3897/zookeys.1179.109817 journal article http://dx.doi.org/10.3897/zookeys.1179.109817 1313-2970-1179-219 76563AB89F814B91BB6EC4743C97E9E7 942544F72F6758AB9893FBB379095F6E Pseudocuneopsis wuana Liu & Wu sp. nov. Fig. 1 Type specimens. Holotype : China • Guangxi Province, Nanning City (南宁市), Qingshui River ( 23.4075°N , 108.7557°E ), 22 June 2023, Ruiwen Wu leg. (SXNU23062201). Paratypes : same data as holotype (voucher numbers SXNU23062202-SXNU23062206). Figure 1. Photographs of Pseudocuneopsis wuana sp. nov. A holotype (SXNU23062201) B-F paratypes (SXNU23062202-SXNU23062206). All specimens shown at the same scale. Photographs by Lili Liu and Liping Zhang. Diagnosis. Pseudocuneopsis wuana sp. nov. is morphologically distinct from the other three recognized species within the genus by shell shape, beak position and sculpture, and surface sculpture (Table 2 ). Diagnostic characteristics: shell ovaliform; ventral margin somewhat prominent at middle; umbo situated 1/3-1/4 of shell length and higher than dorsal margin; epidermis tawny to dark brown, shell surface sculptured with concentric ridges; nacre silvery-white, umbo pocket light yellow. Table 2. Conchological characters of Pseudocuneopsis wuana sp. nov., P. yangshuoensis , P. capitata , and P. sichuanensis . Characteristic descriptions of P. capitata , P. sichuanensis , and P. yangshuoensis are referenced from published works ( Wu et al. 2022 ; Wu et al. 2023 ).

-	P. yangshuoensis	P. sichuanensis	P. capitata	P. wuana sp. nov.
Length	41.39-50.51 (mm)	49.16-62.97 (mm)	101.68-121.32 (mm)	24.97-35.91 (mm)
Width	15.34-19.40 (mm)	15.01-22.42 (mm)	37.07-42.72 (mm)	10.72-15.74 (mm)
Height	27.25-28.99 (mm)	27.16-36.02 (mm)	49.23-61.02 (mm)	15.49-21.95 (mm)
Shell shape	Wedge-shaped	Oval	Elongate	ovaliform
Umbo position	1/3 of shell length; umbo obviously lower than the dorsal margin	1/4-1/5 of shell length; umbo slightly higher than the dorsal margin	1/6 of shell length; umbo obviously higher than the dorsal margin	1/3-1/4 of shell length; umbo higher than the dorsal margin
Surface sculpture	Epidermis brownish-black covered with concentric ridges	Epidermis dark brown with growth annuli with 1 or 2 sulci near posterior dorsal margin	Epidermis brownish with low rides, which follow growth lines	Epidermis tawny to dark brown covered with concentric ridges
Nacre colou r	Orange	White	Milk-white	Silvery-white, umbo pocket light yellow
Dorsal margin	Anterior margin oval, and inflated, with the dorsal margin curved downwards	Anterior margin oval, and inflated, with dorsal margin curved downwards	Anterior margin oval, highly inflated, dorsal margin sloped downwards	Anterior margin round, and inflated, with dorsal margin curved downwards
Posterior slope	Blunt	Blunt	Sharp	Blunt
Ventral margin	Nearly straight or slightly concave	Slightly concave inward at middle posterior	Rounded anteriorly, with sinus behind anterior inflation	Somewhat prominent at middle

Description. Shell ovaliform, medium-thick; anterior margin rounded and inflated; ventral margin somewhat prominent in the middle; umbo located at 1/3-1/4 of shell length and higher than dorsal margin; umbo sculptured with nodes or nodulose wrinkles, or severely eroded; posterior slope formed by ventral margin and dorsal margin low, blunt, located at almost 1/3 of shell height; epidermis tawny to dark brown covered with concentric ridges; anterior adductor muscle scars elliptical, deep, and smooth; posterior adductor muscle scars round to elliptical, shallow and smooth; anterior and posterior retractor muscle scars noticeable, with anterior and posterior irregularly oval; mantle muscle scars obvious; left valve with two separate pseudocardinal teeth and two lateral teeth; outer and inner pseudocardinal teeth of different lengths and projecting outward at different levels, outer and inner pseudocardinal teeth roughly the same size; right valve with one well-developed pseudocardinal tooth and one lateral tooth; nacre silvery-white, umbo pocket light yellow. Length 24.97-35.91 mm, height 15.49-21.95 mm. Etymology. This species' name is dedicated to Dr Ruiwen Wu, who collected these specimens. For the common name, we recommend "Wu ovaliform Mussel" (English) and "Wu Shi Wei Xie Bang" (武氏伪楔蚌) (Chinese). Distribution. Qingshui River, Guangxi, China (Fig. 2 ). Figure 2. The type locality of Pseudocuneopsis wuana sp. nov. in Qingshui River, Nanning City, China. Anatomical characteristics. Within the incurrent aperture, there are elongated papillae arranged in three or four rows; these have a slight swelling at their base; papillae of the excurrent aperture well developed, stubby, and arranged in two rows. The inner gills are larger than the outer gills. Labial palps are medium-thick and elongated (Fig. 3 ). Figure 3. Anatomical features of Pseudocuneopsis wuana sp. nov. with right valve removed. Abbreviations: aam, anterior adductor muscle; pam, posterior adductor muscle; exa, excurrent aperture; ia, incurrent aperture; f, foot; ig, inner gill; og, outer gill; lp, labial palps; m, mantle; p ia, papillae in incurrent aperture; p exa, papillae in excurrent aperture. Molecular analyses. Pairwise COI sequence divergences from Pseudocuneopsis wuana sp. nov., P. yangshuoensis , P. capitata , and P. sichuanensis were calculated in MEGA 7.0 with the uncorrected p -distance model. The intraspecific divergence of the newly discovered species, P. wuana sp. nov., ranged from 0% to 0.5%. The genetic divergence between P. wuana and P. yangshuoensis was 5.1%, while that between P. wuana and the other two species, namely P. sichuanensis and P. capitata , was 8.2% and 10.2%, respectively. The BI and ML trees based on the mitochondrial COI gene yielded incongruent topologies (Figs 4 , 5 ). However, both phylogenetic trees consistently supported the sister-group relationship between Pseudocuneopsis wuana sp. nov. and P. yangshuoensis (BS = 100%; PP = 1.00, Figs 4 , 5 ). The genus Pseudocuneopsis exhibited monophyly with robust bootstrap support (BS = 98%) and full Bayesian posterior probability (PP = 1.00) (Figs 4 , 5 ). Figure 4. Phylogenetic tree of freshwater mussels inferred by maximum-likelihood (ML) analysis of the COI barcode fragment. Bootstrap-support (BS) values are shown at the nodes. The new species is indicated in red. Figure 5. Phylogenetic tree of freshwater mussels inferred from Bayesian-inference (BI) analysis of the COI barcode fragment. Posterior probabilities (PP) are shown at the nodes. The new species is indicated in red. Remarks. The placement of the new species in Pseudocuneopsis is supported by both morphological characteristics and phylogenetic analyses. Pseudocuneopsis wuana sp. nov. can readily be distinguished from congeneric species by its distinctive ovaliform shell, tawny to dark-brown epidermis covered with concentric ridges, and somewhat prominent at the middle of ventral margin. We conducted an analysis of interspecific divergence among P. sichuanensis , P. capitata , P. yangshuoensis , and the newly described species P. wuana using the COI gene. The results indicate that the genetic distances between P. wuana and its congeneric species, namely P. yangshuoensis , P. sichuanensis , and P. capitata , were 5.1%, 8.2%, and 10.2%, respectively, which are distinctly higher than intraspecific divergences. We conclude that the genetic analyses support the recognition of P. wuana sp. nov. as a valid new species, which can easily be distinguished by its unique COI barcode sequences. In recent years, through more extensive investigations and field surveys, several new species of freshwater mussels, such as Postolata guangxiensis and Pseudocuneopsis yangshuoensis , have been discovered in Guangxi Province, China ( Dai et al. 2023 ; Wu et al. 2023 ). As a major ecological barrier in southwest China, Guangxi is one of the significant watershed areas of the Pearl river basin. The province is noted for its distinctive karst landscape which harbors a rich diversity of species. However, due to the insufficient attention paid to freshwater mussels in the region, the mussel diversity in China is poorly understood. The lack of data regarding species' distributions, population trends, threats, and accurate taxonomic information has severely impeded conservation efforts for the unionids in this area. Our findings suggest that there is still much to be discovered regarding the diversity of freshwater mussels in Guangxi Province. Additional extensive exploration may reveal other species that have yet to be documented.