#ZooKeys ZooKeys 1166: 261-270 (2023) DOI: 10.3897/zookeys.1166.104150 Research Article New species of the genus Pseudocuneopsis Huang, Dai, Chen & Wu, 2022 (Bivalvia, Unionidae) from Guangxi Province, China Ruiwen Wu", Lili Liu’, Liping Zhang’, Junli Jia’, Dandong Jin”, Xiaoping Wu®, Xiongjun Liu’ eB wo NY — School of Life Science, Shanxi Normal University, Taiyuan 030031, China Datian High School, Linhai 317004, China School of Life Sciences, Nanchang University, Nanchang 330031, China School of Life Science, Jiaying University, Meizhou 514015, China Corresponding authors: Ruiwen Wu (wurw@sxnu.edu.cn, rwwu@qq.com); Xiongjun Liu (609449126@qq.com) OPEN Qaccess Academic editor: Graham Oliver Received: 28 March 2023 Accepted: 22 May 2023 Published: 12 June 2023 ZooBank: https://zoobank. org/100BA53D-1E5F-490C-B2A9- 7437E80D2DB8 Citation: Wu R, Liu L, Zhang L, Jia J, Jin D, Wu X, Liu X (2023) New species of the genus Pseudocuneopsis Huang, Dai, Chen & Wu, 2022 (Bivalvia, Unionidae) from Guangxi Province, China. ZooKeys 1166: 261-270. https://doi.org/10.3897/ zookeys.1166.104150 Copyright: © Ruiwen Wu et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract A new species of freshwater mussel belonging to the genus Pseudocuneopsis, namely Pseudocuneopsis yangshuoensis sp. nov., is diagnosed and described from Guangxi Province, China. This paper provides a detailed morphological description, photograph of the type specimen, and anatomical characteristics along with partial sequences of mitochondrial COI as DNA barcode data for this novel species. The new species can be distinguished from its congeners (Pseudocuneopsis sichuanensis and Pseudocune- opsis capitata) by shell shape, beak position and surface sculpture. The interspecies genetic distance based on the COI barcode between P yangshuoensis sp. nov. and P. sichuanensis is 8%, while it reaches 9% with P. capitata. Therefore, we provide robust morphological and molecular evidence to support the validity of this new species. Key words: Bivalves, COI, freshwater mussel, morphology, taxonomy Introduction Unionidae Gray, 1840 is a family of bivalves (Mollusca: Bivalvia: Unionida) com- monly referred to as freshwater mussels (Lopes-Lima et al. 2014; Graf and Cummings 2021). These bivalves are essential members of freshwater ecosys- tems, playing a variety of ecosystem services, such as nutrient cycling, increas- ing water purification, bioturbation and habitat provisioning (Vaughn 2018). China is considered to be one of the major biodiversity hotspots for freshwa- ter mussels, with an abundance of rivers and lakes that harbor a wealth of en- demic species (Zieritz et al. 2018; Liu et al. 2022). However, field investigation and research on unionids are concentrated in the middle and lower reaches of the Yangtze River (Wu et al. 2018; Huang et al. 2019; Liu et al. 2020, 2022), with less sampling in the other river basins in Southwest China, for example, Li River in Guangxi Province. These under-investigated areas severely limit the ability to discover new species, which hinders a comprehensive understanding of the phylogeny and evolution within this group. The genus Pseudocuneopsis Huang, Dai, Chen & Wu, 2022 was recently es- tablished by Wu et al. (2022a). Based on mitochondrial phylogenomic analyses, 261 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China Wu et al. (2022a) confirmed that the original genus Cuneopsis sensu lato Simpson, 1900 was polyphyletic, and proposed two new genera: Arcuneopsis and Pseudocuneopsis. Later, the genus name Arcuneopsis was replaced by Tchangsinaia Starobogatov, 1970 because Unio piscicula Heude, 1874 as the type species had previously been classified by Starobogatov (1970) under the genus name Tchangsinaia. Currently, the comprehensive molecular sys- tematics have stabilized the taxonomic status of Pseudocuneopsis, which is under the subfamily Unioninae in Unionidae (Huang et al. 2019; Wu et al. 2019; Wu et al. 2022a). The genus has two recognized species, i.e., Pseudo- cuneopsis sichuanensis Huang, Dai, Chen & Wu, 2022 and Pseudocuneopsis capitata (Heude, 1874); both are endemic to China (Graf and Cummings 2023; MolluscaBase eds 2023). Pseudocuneopsis sichuanensis has a narrow distri- bution reported only in the Sichuan Province, and P. capitata is widely distribut- ed in the Yangtze River basin (Liu et al. 1979; Liu et al. 2022; Wu et al. 2022a). In this study, we diagnose and describe a new Pseudocuneopsis species from Guangxi Province, China. In addition, we provide estimations of the intra- specific and interspecific genetic distances within Pseudocuneopsis based on the mitochondrial COI barcode to examine the species validity. Material and methods Specimen collection, identification and anatomical observations In December 2022, five samples with tissues were collected from the Li Riv- er, Yangshuo County, Guangxi Province, China (24.90099°N, 110.52585°E). All specimens were deposited as vouchers at the Museum of Zoology, Shanxi Normal University (SXNU), China (SXNU22121104-SXNU22121108). We per- formed dissections on all individuals in order to observe soft body characteris- tics through visual examination and a stereoscopic microscope. DNA extraction and COI amplification Total genomic DNA was extracted from dissected somatic tissues using TI- ANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Polymerase chain reaction (PCR) amplification of the COI gene with a 680-base pair fragment was performed using a primer pair consisting of (LCO22me2 + HCO700dy2) (Walker et al. 2007). Thermal cycling conditions were 98 °C for 10 s, followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1-2 min, anda final extension of 72 °C for 7 min, following the TaKaRa Ex manufacturer's proto- col. The amplified PCR products were purified and sequenced by Sangon Biotech (Shanghai). The PCR product size for the COI amplicon was 680 bp. The sequences obtained in this study have been uploaded to GenBank (0Q696218-0Q696222). DNA barcode dataset construction We constructed a mitochondrial COI dataset with the newly obtained sequences from this study and the available Pseudocuneopsis sichuanensis and Pseudocu- neopsis capitata sequences from GenBank. In addition, twenty-five species of the ZooKeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 262 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China subfamily Unioninae for the ingroups, and two species of the subfamily Gonidein- ae as the outgroups were downloaded from GenBank and added to the dataset. As a result, a total of thirty-two COI sequences were used for this study. Se- quence details and GenBank accession numbers are shown in Table 1. COI nucleotide sequences were aligned under the invertebrate mitochondrial code mode in MACSE (Ranwez et al. 2021) with default settings. We calculated and compared inter-and intra-specific distances with MEGA 7.0 (Kumar et al. 2016) using the uncorrected p-distance. Standard error was assessed using 1000 bootstrap replicates. Phylogenetic analysis Bayesian inference (BI) analyses were inferred in MrBayes (Ronquist et al. 2012), using the GTR+I+G model of nucleotide substitution. Four chains were run simultaneously for 10 million generations and trees were sampled every 1000 generations. The first 25% of these trees were discarded as burn-in when Table 1. List of sequences used in this study (*) Sequenced from this study. Taxa GenBank accession number UNIONINAE Rafinesque, 1820 ZooKeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 Lasmigona compressa (Lea, 1829) AF156503 Pyganodon grandis (Say, 1829) AF231734 Strophitus undulatus (Say, 1817) AF156505 Pseudanodonta complanata (Rossmassler, 1835) KX822661 Unio tumidus (Philipsson in Retzius, 1788) KX822672 Nodularia douglasiae (Griffith & Pidgeon, 1833) NC_026111 Aculamprotula scripta (Heude, 1875) MF991456 Aculamprotula tientsinensis (Crosse & Debeaux, 1863) NC_029210 Acuticosta chinensis (Lea, 1868) MG462919 Cuneopsis heudei (Heude, 1874) MG462974 Cuneopsis rufescens (Heude, 1874) MG462982 Inversiunio yanagawensis (Kondo, 1982) LC518988 Pseudocuneopsis capitata (Heude, 1874) MZ540968 Pseudocuneopsis capitata (Heude, 1874) MZ540969 Pseudocuneopsis sichuanensis Huang, Dai, Chen & Wu, 2022 MZ540966 Pseudocuneopsis sichuanensis Huang, Dai, Chen & Wu, 2022 MZ540967 Pseudocuneopsis yangshuoensis sp. nov. 1* 0Q696218 Pseudocuneopsis yangshuoensis sp. nov. 2* 0Q696219 Pseudocuneopsis yangshuoensis sp. nov. 3* 0Q696220 Pseudocuneopsis yangshuoensis sp. nov. 4* 0Q696221 Pseudocuneopsis yangshuoensis sp. nov. 5* 0Q696222 Tchangsinaia piscicula (Heude, 1874) KJ434496 Tchangsinaia piscicula (Heude, 1874) KJ434497 Tchangsinaia piscicula (Heude, 1874) KJ434498 Tchangsinaia piscicula (Heude, 1874) KJ434499 Schistodesmus lampreyanus (Baird & Adams, 1867) MG463038 Schistodesmus spinosus (Simpson, 1900) MG463045 Lanceolaria gladiola (Heude, 1877) KY067441 Lanceolaria grayii (Griffith & Pidgeon, 1833) NC_026686 Lanceolaria lanceolata (Lea, 1856) NC_023955 GONIDEINAE Ortmann, 1916 Lamprotula leaii (Gray in Griffith & Pidgeon, 1833) NC_023346 Sinosolenaia oleivora (Heude, 1877) KX822670 263 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China computing the consensus tree (50% majority rule). Sufficient mixing of the chains was considered to have been reached when the average standard de- viation of split frequencies was below 0.01. Additionally, 1Q- TREE was run for maximum likelihood (ML) tree reconstruction with 1000 ultrafast bootstraps (Minh et al. 2013). The ML and BI trees generated are depicted in Suppl. mate- rial 1 and Suppl. material 2, respectively. Taxonomy Pseudocuneopsis yangshuoensis Wu & Liu, sp. nov. https://zoobank.org/8CF6C1AB-184A-40A4-A05B-4B022A3F7EC3 Fig. 1 Type specimens. Holotype: CHINA » Guangxi Province, Yangshuo County (HHL), Li River (24.90099°N, 110.52585°E), 11 December 2022, coll. Dan- dong Jin (SXNU22121104). Paratypes: same data as holotype (SKNU22121105 - SXNU22121108). Figure 1. Photographs of Pseudocuneopsis yangshuoensis sp. nov. A holotype SXNU22121104 B-E paratype SXNU22121105-SXNU22121108. Sacle bar: 1 cm. ZooKeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 264 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China Diagnosis. Pseudocuneopsis yangshuoensis sp. nov. is morphologically dis- tinct from the other two recognized species within the genus by shell shape, beak position and surface sculpture (Table 2). Diagnostic characteristics: shell wedge-shaped; the ventral margin nearly straight or slightly concave; the umbo situated 1/3 of shell length, and obviously lower than the dorsal margin; nacre orange. Description. Shell wedge-shaped, medium thickness; anterior margin regu- larly rounded and inflated; ventral margin nearly straight or slightly concave; umbo located at 1/3 of shell length and obviously lower than the dorsal margin; umbo sculptured unknown due to severe erosion; posterior slope formed by the ventral margin and dorsal margin low, blunt, approximately 60°; epidermis brownish-black covered with concentric ridges; anterior adductor muscle scars elliptical, deep and unrough; posterior adductor muscle scars long elliptical, shallow and smooth; anterior and posterior retractor muscle scars obvious, with the anterior and posterior irregularly elliptical; mantle muscle scars obvious; left valve with two separate pseudocardinal teeth and two later teeth; the outer and inner pseudocardinal teeth are the same length and projected outward at the same level; right valve with one pseudocardinal tooth and one lateral tooth; lat- eral and pseudocardinal teeth usually curved; nacre orange in colour. Length 41.39-50.51 mm, height 15.34-19.40 mm. Etymology. The specific epithet is derived from the type locality Yangshuo County. For the common name, we recommend “Yangshuo Wedged Mussel” (English) and “Yang Shuo Wei Xie Bang” (BAH (A#ZE) (Chinese). Distribution. Li River, Guangxi, China (Fig. 2). Anatomical characteristics. The soft tissue morphology reveals elongated papillae arranged in two to three rows within the incurrent aperture, with stocky papillae forming the outer row; notable pigmentation and small sarcomas are present along the outer margin of the excurrent aperture; and the size of inner gills exceeds that of outer gills (Fig. 3). Table 2. Conchological characters of Pseudocuneopsis yangshuoensis sp. nov., Pseudocuneopsis capitata, Pseudocune- opsis sichuanensis. Characteristic descriptions of P capitata and P. sichuanensis are referenced from Wu et al. (2022a). Length Width Height Shell shape Umbo position Surface sculpture Nacre colour Dorsal margin Posterior slope Ventral margin P. yangshuoensis sp. nov. P. sichuanensis P capitata 41.39-50.51 (mm) 49.16-62.97 (mm) 101.68-121.32 (mm) 27.25-28.99 (mm) 15.01-22.42 (mm) 37.07-42.72 (mm) 15.34-19.40 (mm) 27.16-36.02 (mm) 49.23-61.02 (mm) Wedge-shaped Oval wedge Elongate wedge 1/3 of shell length; umbo obviously 1/4-1/5 of shell length; umbo 1/6 of shell length; umbo obviously lower than the dorsal margin slightly higher than the dorsal margin higher than the dorsal margin Epidermis brownish-black covered | Epidermis dark brown with growth | Epidermis brownish with low rides, with concentric ridges annulus with 1 or 2 sulci near the which follow the growth lines posterior dorsal margin Orange White Milk-white Anterior margin oval, and inflated, | Anterior margin oval, and inflated, | Anterior margin oval, highly inflated, with the dorsal margin curved with the dorsal margin curved dorsal margin sloped downwards downwards downwards Blunt Blunt Sharp Nearly straight or slightly concave | Slightly concave inward at middle Rounded anteriorly, behind the posterior anterior inflation there is a sinus ZooKeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 265 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China — _ - — —" . ac _ ——— en — IE ene Figure 2. Photograph of the sampling site of Pseudocuneopsis yangshuoensis sp. nov. at the Li River, Guangxi in China. anterior adductor muscle; pam, posterior adductor muscle; exa, excurrent aperture; ia, incurrent aperture; f, foot; ig, inner gill; og, outer gill; Ip, labial palps; m, mantle; p ia, papillae in incurrent aperture; pg exa, pigmentation of excurrent aperture. Zookeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 266 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China Molecular analyses. Pairwise COI sequence divergences from Pseudocune- opsis yangshuoensis sp. nov., Pseudocuneopsis capitata, and Pseudocuneopsis sichuanensis were conducted in MEGA 7.0 with the uncorrected p-distance mod- el. The intraspecific divergence of Pseudocuneopsis yangshuoensis sp. nov. was 0%. The interspecific divergence between Pseudocuneopsis yangshuoensis sp. nov. and P sichuanensis was 8%, and 9% with P. capitata. Both BI and ML trees obtained a consistent topology (Fig. 4). In the phylogenetic trees, Pseudocuneop- sis yangshuoensis sp. nov. formed a well-supported sister-group relationship with P. sichuanensis (BS = 100; PP = 1.00, Fig. 4). Cuneopsis and Tchangsinaia were each separated from Pseudocuneopsis belonging to three different clades (Fig. 4). Remarks. Species delineation can be problematic in the presence of morpho- logical ambiguities due to phenotypic plasticity and convergence (e.g., cryptic species), especially in mollusks (Zieritz et al. 2010; Inoue et al. 2013). The use of molecular genetics can aid species delineation in the case of phenotypic plasticity and/or convergence (Pieri et al. 2018; Wu et al. 2022b). Pseudocu- neopsis yangshuoensis sp. nov. can be distinguished from congeneric species based on the diagnostic characteristics of shells. We also analyze the interspe- cific divergence among P. sichuanensis, P. capitata and P yangshuoensis sp. nov. based on the COI barcode. The results show that the average interspecific Pseudocuneopsis yangshuoensis sp. nov. 1 Pseudocuneopsis yangshuoensis sp. nov. 2 Pseudocuneopsis yangshuoensis_ sp. nov. 3 Pseudocuneopsis yangshuoensis sp. nov. 4 100/1.00 Pseudocuneopsis yangshuoensis sp. nov. 5 98/1.00 Pseudocuneopsis sichuanensis Pseudocuneopsis sichuanensis 60/0.70 Pseudocuneosis capitata Pseudocuneosis capitata 64/0.72 Inversiunio yanagawensis Tchangsinaia piscicula Tchangsinaia piscicula 45/0.92 Tchangsinaia piscicula Tchangsinaia piscicula 100/1.00 Schistodesmus lampreyanus 75/0.90 Schistodesmus spinosus Nodularia douglasiae 70/1.00 Cuneopsis heudei 63/0.90 Cuneopsis rufescens 100/1.00 Aculamprotula scripta Aculamprotula tientsinensis 80/0.94 100/1.00 80/0.80 Unio tumidus 94/0.75 Strophitus undulatus 86/100 Pyganodon grandis gy Lasmigona compressa Pseudanodonta complanata 400/1.00 2/1 .0 Lanceolaria lanceolata Lanceolaria grayii Lanceolaria gladiola Acuticosta chinensis Sinosolenaia oleivora 100/1.00 Lamprotula leaii 96/1.00 65/1.00 0.04 Figure 4. Phylogenetic tree of freshwater mussels inferred from maximum likelihood (ML) and Bayesian inference (BI) analyses of COI barcode. Support values above the branches are bootstrap support (BS)/ posterior probabilities (PP). The red font indicates the species from this study. ZooKeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 267 Ruiwen Wu et al.: New species of the genus Pseudocuneopsis from China divergence between the two species was 8%-9%, which is much higher than intraspecific divergences. Genetic analysis conducted in this study supports P yangshuoensis sp. nov. as a valid species, which can be easily distinguished by COI barcode. Additional information Conflict of interest No conflict of interest was declared. Ethical statement No ethical statement was reported. Funding This work was funded by the National Natural Science Foundation of China (No. 32200370), the Basic Research Program of Shanxi Province, China (No. 20210302124253). Author contributions Ruiwen Wu (RW-W) designed the study; RW-W, Lili Liu (LL-L), Liping Zhang (LP-Z), Xiongjun Liu (XJ-L), Dandong Jin (DD-J) undertook field research and sampling, Xia- oping Wu (XP-W), LL-L, XJ-L and LP-Z conducted the data analysis, RW-W and XJ-L wrote and edited the manuscript. All authors read and approved the final version of the manuscript. Author ORCIDs Ruiwen Wu © https://orcid.org/0000-0002-8936-6054 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Graf DL, Cummings KS (2021) A ‘big data’ approach to global freshwater mussel diversi- ty (Bivalvia: Unionoida), with an updated checklist of genera and species. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1166.104150.suppl1 Supplementary material 2 Phylogenetic tree of freshwater mussels inferred from Bayesian inference (BI) analyses based on COI barcode Authors: Ruiwen Wu, Lili Liu, Liping Zhang, Junli Jia, Dandong Jin, Xiaoping Wu, Xiongjun Liu Data type: Phylogenetic tree Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1166.104150.suppl2 ZooKeys 1166: 261-270 (2023), DOI: 10.3897/zookeys.1166.104150 270