New species of the genus Inversidens Haas, 1911 (Unionoida, Unionidae, Gonideinae) from Jiangxi Province, China

Abstract We diagnose and describe a new freshwater mussel species of the genus Inversidens, I.rentianensissp. 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.

In this study, we diagnose and describe a new Inversidens species from Jiangxi Province, China. In addition, we provide estimations of the intraspecific and interspecific genetic distances within Inversidens based on the mitochondrial COI barcode to examine species validity.

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 extracted 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, followed 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.

DNA barcode dataset construction
We constructed a mitochondrial COI dataset with the newly obtained sequences from this study and the available Inversidens 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 using the program MUSCLE (Edgar 2004) with default settings. We calculated and compared inter-and intraspecific distances with MEGA 5.0 using the uncorrected pdistance. Standard error was assessed using 1000 bootstrap replicates.

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 generations. 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 reconstruction, using partition models with 1000 ultrafast bootstraps (Minh et al. 2013). Diagnosis. Inversidens rentianensis sp. nov. is morphologically distinct from the other two recognized species within the genus by shell shape, beak position and nacre colour (Table 2). Diagnostic characteristics: shell irregularly subtriangular; curvature of the ventral margin slight, nearly straight; umbo situated 1/2 of shell length; nacre reddish.

Inversidens rentianensis
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 margin 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.
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 I. brandtii and I. rentianensis sp. nov. were both 0.00%. The interspecific divergence of I. 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, I. rentianensis sp. nov. formed a well-supported  Kondo (1982Kondo ( , 2008 and He and Zhuang (2013  sister-group relationship with Inversidens 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 morphological 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. 2018). Inversidens rentianensis sp. nov. can be 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 I. rentianensis sp. nov. as a valid species, which can be easily distinguished by the COI barcode.