ZooKeys 1054: 85-93 (202 1) A peer-reviewed open-access journal doi: 10.3897/zookeys.1054.69075 RESEARCH ARTICLE #ZooKey S https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research New species of the genus Inversidens Haas, 191 (Unionoida, Unionidae, Gonideinae) from Jiangxi Province, China Ruiwen Wu', Xiongjun Liu’, Takaki Kondo?, Shan Ouyang’, Xiaoping Wu* I School of Life Science, Shanxi Normal University, Linfen 041000, China 2 School of Life Science, Jiaying University, Meizhou 514015, China 3 Division of Natural Science, Osaka Kyoiku University, Osaka 582- 8582, Japan 4 School of Life Sciences, Nanchang University, Nanchang 330031, China Corresponding authors: Xiongjun Liu (609449126@qq.com); Xiaoping Wu (xpwu@ncu.edu.cn) Academic editor: Graham Oliver | Received 24 May 2021 | Accepted 9 July 2021 | Published 3 August 2021 http://zoobank.org/BA743F4E-2452-432A-9796-E1DEBCID4CAD Citation: Wu R, Liu X, Kondo T, Ouyang S, Wu X (2021) New species of the genus Jnversidens Haas, 1911 (Unionoida, Unionidae, Gonideinae) from Jiangxi Province, China. ZooKeys 1054: 85-93. https://doi.org/10.3897/ zookeys.1054.69075 Abstract We diagnose and describe a new freshwater mussel species of the genus /nversidens, I. rentianensis sp. nov. from Jiangxi Province, China based on morphological characters and molecular data. This paper includes a morphological description and photograph of the holotype, and partial sequences of mitochondrial COI as DNA barcode data. Keywords COL, freshwater mussel, genetic distances, morphology, taxonomy Introduction The genus /nversidens Haas, 1911 belongs to the subfamily Gonideinae in the family Unionidae. The genus was first depicted by Haas (1911) as a subgenus of Nodularia with two species, i.e., Unio brandtii Kobelt, 1879 and Nodularia parcedentata Haas, 1911, both restricted to Japan. Later, Haas (1969) further classified Unio reinianus Kobelt, 1879, Unio haconensis lhering, 1893, Unio japanensis Lea, 1859, Unio pantoensis Neumayr, 1899 within Inversidens. All species were restricted to Japan, except for Copyright Ruiwen Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 86 Ruiwen Wu et al. / ZooKeys 1054: 85-93 (2021) U. pantoensis, which was distributed in China. By comparing the conchological characters, Kondo (1982) believed that U. brandtii was not morphologically distinct from JN. parcedentata, and regarded N. parcedentata as a variety of U. brandtii. Based on the morphology of the glochidium, Habe (1991) removed U. reinianus from Inversidens, and established a new genus J/nversiunio. Based on morphological characteristics of the shell, Kondo (1998) moved Unio haconensis, which was regarded as a synonym of Unio jokohamensis (Ihering, 1893), into Inversiunio. Furthermore, Starobogatov (1970) used Unio japanensis as the type species for his newly established genus Pronodularia. Currently, only two species are recognized within /nversidens, the Japanese endemic L. brandtii and I. pantoensis in China (Fig. 1A, B; Kondo 2008; He and Zhuang 2013; Lopes-Lima et al. 2020; Graf and Cummings 2021a, b; MolluscaBase eds. 2021). In this study, we diagnose and describe a new /nversidens species from Jiangxi Prov- ince, China. In addition, we provide estimations of the intraspecific and interspecific genetic distances within /nversidens based on the mitochondrial COI barcode to exam- ine species validity. Materials and methods Specimen collection and identification In March 2018, four samples were collected from the Mianshui River, Rentian Town, Ganzhou City, Jiangxi Province, China (25.989557°N, 116.131333°E). All type and voucher specimens are deposited at the Biological Museum of Nanchang University, China (NCFM180325-NCFM180328). DNA extraction and COI amplification Of the four individuals, only two samples had tissues. Total genomic DNA was extract- ed from dissected somatic tissue using TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Mitochondrial cytochrome oxidase subunit I (COI) gene sequences have been widely used for species delimitation of freshwater mussels based on genetic distance and the criteria of monophyly (Elderkin et al. 2016; Lopes-Lima et al. 2019; Smith et al. 2019). Polymerase chain reaction (PCR) amplification of the COI gene with a 680-base pair fragment was performed using a primer pair consisting of LCO1490 and HCO2198 (Folmer et al. 1994). Thermal cycling conditions were 98 °C for 10 s, fol- lowed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1—2 min, and a final extension of 72 °C for 7 min, following the TaKaRa Ex manufacturer's protocol. 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. New species of the genus /nversidens from China 87 DNA barcode dataset construction We constructed a mitochondrial COI dataset with the newly obtained sequences from this study and the available /nversidens brandtii sequences from GenBank. Previously published sequences were downloaded from GenBank and added to the dataset, i.e., 17 species of the subfamily Gonideinae and four species of the subfamily Unioninae for the ingroup, and one species of the subfamily Parreysiinae as the outgroup. As a result, a total of 29 COI sequences were used for this study. Sequence details and GenBank accession numbers are shown in Table 1. All COI nucleotide sequences were translated to amino acid sequences using MEGA 5.0 (Tamura et al. 2011) and aligned based on the amino acid sequences us- ing the program MUSCLE (Edgar 2004) with default settings. We calculated and compared inter-and intraspecific distances with MEGA 5.0 using the uncorrected p- distance. Standard error was assessed using 1000 bootstrap replicates. Table |. List of sequences used in this study. (*) Sequenced from this study. Taxa GenBank accession number UNIONIDAE Rafinesque, 1820 Parreysiinae Henderson, 1935 Indonaia andersoniana (Nevill, 1877) KX865835 Unioninae Rafinesque, 1820 Acuticosta chinensis (Lea, 1868) MG462919 Inversiunio jokohamensis (Ihering, 1893) LC518985 Inversiunio reinianus (Kobelt, 1879) LC518976 Nodularia douglasiae (Griffith & Pidgeon, 1833) NC_026111 Gonideinae Ortmann, 1916 Pseudodon bogani Bolotov, Kondakov & Konopleva in Bolotov et al. 2017 MF352216 Pseudodon manueli Konopleva, Kondakov & Vikrev in Bolotov et al. 2017 MF352228 Monodontina cambodjensis (Petit de la Saussaye, 1865) KP795028 Pilsbryoconcha exilis (Lea, 1838) KP795024 Chamberlainia hainesiana (Lea, 1856) KX822635 Sinohyriopsis cumingii (Lea, 1852) NC_011763 Sinohyriopsis schlegelii (Martens, 1861) NC_015110 Lamprotula caveata (Heude, 1877) KX822646 Lamprotula leaii (Griffith & Pidgeon, 1833) NC_023346 Potomida littoralis (Cuvier, 1798) JN243905 Pronodularia japanensis (Lea, 1859) KX822659 Gonidea angulata (Lea, 1838) DQ272371 Leguminaia wheatleyi (Lea, 1862) KX822651 Microcondylaea bonellii (Férussac, 1827) KX822652 Sinosolenaia carinata (Heude, 1877) KX822669 Ptychorhynchus pfisteri (Heude, 1874) KY067440 Parvasolenaia rivularis (Heude, 1877) KX966393 Inversidens brandtii (Kobelt, 1879) AB040827 Inversidens brandtii (Kobelt, 1879) MT020598 Inversidens brandtii (Kobelt, 1879) MT020597 Inversidens brandtii (Kobelt, 1879) LC519005 Inversidens brandtii (Kobelt, 1879) LC519004 Inversidens rentianensis sp. nov. 1* MZ073336 Inversidens rentianensis sp. nov. 2* MZ073337 88 Ruiwen Wu etal. / ZooKeys 1054: 85-93 (2021) Phylogenetic analysis Bayesian inference (BI) analyses were inferred in MrBayes Version 2.01 (Ronquist et al. 2012), using GTRGAMMAI model of nucleotide substitution. Four chains were run simultaneously for 10 million generations and trees were sampled every 1000 gen- erations. The first 25% of these trees were discarded as burn-in when computing the consensus tree (50% Majority Rule). Sufficient mixing of the chains was considered to have been reached when the average standard deviation of split frequencies was below 0.01. Additionally, IQ- TREE was run for Maximum Likelihood (ML) tree reconstruc- tion, using partition models with 1000 ultrafast bootstraps (Minh et al. 2013). Taxonomy Inversidens rentianensis Wu & Wau, sp. nov. http://zoobank.org/624247 17-95 14-4C7D-9COE-240F1D95F03E Fig. 1C Type specimens. Holotype. Cuina * Jiangxi Province, Ganzhou City, Rentian Town (-EH14), Mianshui River (25.989557°N, 116.131333°E), 13 March 2018, coll. Xiongjun Liu (NCFM180325). Paratypes. Same data as holotype (NCFM180326- NCFM 180328). Diagnosis. /nversidens rentianensis sp. nov. is morphologically distinct from the oth- er two recognized species within the genus by shell shape, beak position and nacre col- our (Table 2). Diagnostic characteristics: shell irregularly subtriangular; curvature of the ventral margin slight, nearly straight; umbo situated 1/2 of shell length; nacre reddish. Description. Shell irregularly subtriangular, medium thickness, and quite inflated. Anterior margin regularly rounded; ventral margin nearly straight; posterior margin obliquely arc-shaped. Umbo prominent and slightly eroded. Umbo sculptured with feebly wavy wrinkles. Posterior slope formed by the ventral margin and posterior mar- gin low, triangular. Epidermis shining black or with brownish-yellow hue. Only one cardinal tooth in each valve, shape triangular. Laterals thick, a little curved, 2 in each valve. Nacre reddish-bronze in colour. Length 43-52 mm, height 29-36 mm. Etymology. The specific epithet is derived from the type locality, Rentian Town. Distribution. The species is known only from Mianshui River, Rentian Town, Ganzhou City, Jiangxi Province, China (present study) (Fig. 2). GenBank accession number. Holotype, NCFM180325: MZ073336; paratypes, NCFM 180326: MZ073337. Molecular analyses. Pairwise COI sequence divergences from Inversidens brandtii and Inversidens rentianensis sp. nov. were conducted using MEGA 5.0. Based on the uncorrected p-distance model, the intraspecific divergences of [. brandtii and I. rentianensis sp. nov. were both 0.00%. The interspecific divergence of J. New species of the genus /nversidens from China 89 Miao Vibe Agy ge AREY we” we Figure |. Photographs of Inversidens taxa A I. brandtii B I. pantoensis C I. rentianensis sp. nov. Photos: [A, B] from the MUSSEL Project, [C] from this study, NCFM180325 (holotype), scale is 2 cm. brandtii and I. rentianensis sp. nov. was 10.1%. Both BI and ML trees obtained a completely consistent topology. Consistent topology relationships are shown in Figure 3. In the phylogenetic trees, /. rentianensis sp. nov. formed a well-supported 90 Ruiwen Wu etal. / ZooKeys 1054: 85-93 (2021) Table 2. Conchological characters of Inversidens rentianensis sp. nov., Inversidens brandtii, Inversidens pantoensis. Characteristic descriptions of 1. brandtii and I. pantoensis are referenced from Kondo (1982, 2008) and He and Zhuang (2013). I. rentianensis sp. nov. I. brandtii I. pantoensis Shell shape Irregularly subtriangular Ovate Inequilateral, quadrate Umbo position 1/2 of shell length 1/4 of shell length 1/3 of shell length Umbo sculpture Feebly wavy wrinkles Rippled Angularly wrinkled Surface sculpture Concentric ridges Concentric ridges Irregular growth lines Nacre colour Reddish Milk-white Bluish Posterior slope Sharp Blunt Blunt Ventral margin Nearly straight Arc-shaped Long and straight mm Figure 2. Photograph of sampling site of Inversidens rentianensis sp. nov. in China. sister-group relationship with /nversidens brandtii (PP = 1.00, BS = 100; Fig. 3). The genera Pronodularia and Inversiunio belong to different clades well-separated from Inversidens (Fig. 3). Remarks. Species delineation can be problematic in the presence of morphologi- cal 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 con- vergence (Pieri et al. 2018; Wu et al. 2018). Lnversidens rentianensis sp. nov. can be New species of the genus /nversidens from China 1/100 0. 81/76 0. 89/97 1/96 0. 82/45 0. 98/88 0. 99/55 0. 87/90 91 Inversidens brandtii AB040827 Inversidens brandtii MT020597 Inversidens brandtii LC519005 Inversidens brandtii LC519004 Inversidens brandtii MT020598 Inversidens rentianensis sp. nov. 1 Inversidens rentianensis sp. nov. 2 Microcondylaea bonellii Leguminaia wheatleyi Gonidea angulata Ptychorhynchus pfisteri Parvasolenaia_ rivularis Lamprotula caveata 1/99 Lamprotula leaii Potomida littoralis Pronodularia japanensis Sinosolenaia carinata Pseudodon bogani Pseudodon manueli Monodontina cambodjensis Pilsbryoconcha exilis Sinohyriopsis cumingii Sinohyriopsis schlegelii 0. 8/89 0. 42/39 0. 82/93 1/99 0. 8/66 1/95 a 0. 59/59 0. 98/98 1/98 0. 59/45 Chamberlainia hainesiana Inversiunio jokohamensis Inversiunio reinianus Nodularia douglasiae Acuticosta chinensis Indonaia andersoniana 1/99 0.99/97 7 0. 98/86 0.09 Figure 3. Phylogenetic tree of freshwater mussels inferred from Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of COI barcode. Support values above the branches are posterior probabilities (PP)/bootstrap support (BS). Red font indicates the new species from this study. distinguished from congeneric species based on diagnostic characteristics of the shell. In this study, we also analyzed the interspecific divergence between Inversidens brandtii and Inversidens rentianensis sp. nov. based on the COI barcode. The results showed that the average interspecific divergence between the two species was 10.1%, which was much higher than intraspecific divergences. Genetic analysis conducted in this study supports J. rentianensis sp. nov. as a valid species, which can be easily distinguished by the COI barcode. 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