Zoosyst. Evol. 100 (3) 2024, 769-778 | DOI 10.3897/zse.100.126069 > PENSUFT. yee BERLIN Addition to the known diversity of Chinese freshwater mussels: integrative description of a new species of Postolata Dai et al., 2023 (Bivalvia, Unionidae, Gonideinae) Lili Liu’, Liping Zhang**, Kaiyu Hou!, Liyang Ning!, Ruiwen Wu! 1 School of Life Science, Shanxi Normal University, Taiyuan 030031, China https://zoobank. org/354401E7-E9A 4-45 F'D-83 DA-8 76 BB4A09650 Corresponding author: Ruiwen Wu (wurw@sxnu.edu.cn) Academic editor: Matthias Glaubrecht # Received 24 April 2024 # Accepted 30 May 2024 Published 13 June 2024 Abstract In this study, we present a new species of freshwater mussel in the genus Postolata Dai et al., 2023, from Guangxi Province, China, by integrating morphological, anatomical, and molecular data. Postolata longjiangensis Liu & Wu, sp. nov. is distinguished from its congener (1.e., Postolata guangxiensis) by its shell shape, beak position, surface sculpture, nacre color, and hinge structure. Mo- lecular species delimitation results based on the mitochondrial COI gene support the separation of Postolata longjiangensis Liu & Wu, sp. nov. from its congener. The multi-locus (COI + 16S rRNA + 28S rRNA) phylogeny reveals that this species forms the sister lineage to Postolata guangxiensis in the tribe Gonideini. Key Words China, cryptic species, freshwater mussels, integrative taxonomy, multi-locus phylogeny, Postolata Introduction Freshwater mussels (order Unionida) are renowned for their distinctive life cycle, characterized by a parasitic phase primarily reliant on fish hosts and an uncommon doubly mitochondrial inheritance (Barnhart et al. 2008; Modesto et al. 2018; Guerra et al. 2019). They are glob- ally distributed in freshwater habitats, with the highest levels of diversity observed in East Asia and North Amer- ica (Zieritz et al. 2018; Graf and Cummings 2023). This group plays crucial ecological roles, with certain mussel Species simultaneously fulfilling the criteria of indica- tor, umbrella, and flagship species (Howard and Cuffey 2006; Vaughn et al. 2008; Vaughn 2018). Unfortunate- ly, freshwater mussels have become one of the most 1m- periled faunas worldwide, with an increasing number of species at risk of extinction (Lydeard et al. 2004; Haag and Williams 2014; Ferreira-Rodriguez et al. 2019). * These authors contributed equally to this work. Consequently, the urgency of describing their diversity and systematics is highlighted by the growing research efforts and conservation attention dedicated to this group (Lopes-Lima et al. 2017a, 2017b; Do et al. 2018; Huang et al. 2019; Liu et al. 2022). The Guangxi Zhuang Autonomous Region (hereinaf- ter referred to as Guangxi), located in southern China and sharing a border with Vietnam, plays a significant role within the Indo-Burma biodiversity hotspot situated in the Chinese region (Tordoff et al. 2012). In recent years, new species of freshwater mussels such as Postolata guangxiensis Dai et al., 2023, Pseudocuneopsis yang- Shuoensis Wu & Liu, 2023 and Pseudocuneopsis wuana Liu & Wu, 2023 have been discovered in Guangxi (Dai et al. 2023; Wu et al. 2023b; Liu et al. 2023). It suggests that the unique habitats and geographical flora of this region may harbor previously undescribed species, while still underestimating the level of species diversity. Copyright Liu, L. 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, distri- bution, and reproduction in any medium, provided the original author and source are credited. 770 The freshwater mussel genus Postolata Dai et al., 2023 belongs to the tribe Gonideini in the subfamily Go- nideinae (Dai et al. 2023; Wu et al. 2024). It was recently established as a monotypic genus comprising only one species, namely Postolata guangxiensis Dai et al., 2023, which is endemic to Guangxi province in China. In this study, another new species of Postolata, also from Guangxi, is diagnosed and described. We employ an integrative taxonomic approach that incorporates mor- phological, anatomical, and molecular phylogeny to iden- tify and differentiate this species. Materials and methods Specimen collection, identification, and anatomical observations InApril 2024, six freshwater mussel specimens were collected from a rural streamlet at an altitude of approximately 150.18 m in Hechi City, Guangxi Province, China (24.530716°N, 108.5762°E; Fig. 1). Meanwhile, two specimens of Postola- ta guangxiensis were collected from the type locality (Luo- qing River, Guangxi Province; Fig. 1). All specimens were deposited as vouchers at the Museum of Zoology, Shanxi Normal University (SXNU), China (voucher numbers SXNU_24040701-SXNU_ 24040706 for Postolata longji- angensis sp. nov.,; voucher numbers SXNU_PG_ 22102301 and SXNU_PG_ 22102303 for Postolata guangxiensis). 105° E 106°E 107°E 24° Altitude/m 2124 21? —_ 1048 -29 108° E ee ————— Km Sampling site 0 35 70 140 Liu, L. et al.: Integrative description of a new species of Posto/ata The conchological and anatomical features of all indi- viduals were visually examined with the naked eye and under a stereoscopic microscope (CX31-12C03, Olym- pus Corporation, Japan), including shell shape, umbo position and sculpture, shell surface sculpture, hinge structure, muscle attachment, and papillae in the incur- rent and excurrent apertures (Figs 2, 3). The anatomical features of the soft body were described according to Williams et al. (2008). DNA extraction, amplification, and sequencing According to the manufacturer’s instructions, a small piece of foot tissue was excised for DNA extraction us- ing the TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China). Three gene fragments, 1.e., the mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA), and the nucle- ar gene of 28S ribosomal RNA (28S rRNA), were se- quenced based on our previous studies (Wu et al. 2024). PCR amplification was implemented in a 25-uL volume using the following thermal cycling conditions: 3.5 min at 94 °C, followed by 35 cycles of 94 °C for 30 sec, 50 °C for 30 sec, 72 °C for 1 min, and a final extension of 72 °C for 5 min. The amplified PCR products were purified and sequenced by Sangon Biotech (Shanghai). All newly obtained sequences in this study have been submitted to GenBank. 109°E 110° E WE. 12" E e Postolata guangxiensis @ Postolata longjiangensis sp. nov. Figure 1. Map (Guangxi Province) of sampling localities of Postolata species and habitat of Postolata longjiangensis sp. nov. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 769-778 Alignments, partitioning strategies, and model selection In this study, we constructed two datasets. First, a DNA barcoding (COI) dataset for molecular species delimita- tion was compiled (Table 1). We downloaded the COI se- quences of eight species in the tribe Gonideini, as well as all published COI sequences of Postolata guangxiensis, along with COI sequences from the new species described in this study. Two species from the tribe Lamprotulin1, i.e., Lamprotula caveata (Heude, 1877) and Lamprotula leaii (Gray, 1833), were selected as outgroups. Second, a three-gene dataset for phylogenetic analysis was prepared (Table 2). This dataset contains species from all eight rec- ognized tribes in the subfamily Gonideinae. In addition, Margaritifera dahurica (Middendorff, 1850) and M. mar- garitifera (Linnaeus, 1758) from the family Margaritife- ridae were selected as outgroups. The molecular data analyses and phylogenetic recon- struction were consistent with the methods used in our previous studies (Wu et al. 2023a; Wu et al. 2024). Pro- tein-coding genes (COI) were aligned by built-in MACSE with invertebrate mitochondrial codon modes imple- mented in PhyloSuite v1.2.3 (Zhang et al. 2020). Ribo- somal genes (16S rRNA and 28S rRNA) were aligned using MAFFT v7.2 (Katoh and Standley 2013) with the L-INS-i algorithm. Ambiguous alignment areas were trimmed by Gblocks (Castresana 2000), the parameter ri- bosomal gene block with a minimum length was set to 2 base pairs (bp), and the allowed gap position was selected with half; the minimum length of the protein-coding gene block was set to 3 bp, and the allowed gap position was also selected with half. For the barcoding dataset, the COI sequence fragment length was 510 bp after alignment and trimming. For the three-gene dataset, COI, 16S, and 28S sequences were aligned and trimmed to lengths of 624 bp, 471 bp, and 751 bp, respectively. Sequences of the multi-gene dataset were concatenated using Phylosuite v1.2.3. The three-gene dataset was analyzed with partition schemes based on the genes and codons. PartitionFind- er (Lanfear et al. 2017) was used to select the mod- els for Bayesian inference (BI) analyses. ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the maximum likelihood (ML) analysis models in IQ-TREE (Minh et al. 2020). The selection of best-fit models was based on the corrected Akaike Information Criteri- on (AICc). Substitution models assigned to each parti- tion by PartitionFinder and ModelFinder are listed in Suppl. material 1. Neighbor-joining clustering and phylogenetic analyses We used an integrative approach that combined molecu- lar and morphological analyses for species delimitation and diagnosis. Based on the COI dataset, the NJ tree was constructed using the uncorrected p-distance model in TAK Table 1. List of COI sequences used in this study. Species GenBank accession number Postolata guangxiensis Dai et al., 2023 OP009379 Postolata guangxiensis Dai et al., 2023 OP009380 Postolata guangxiensis Dai et al., 2023 OP009381 Postolata guangxiensis Dai et al., 2023 OP009382 Postolata guangxiensis Dai et al., 2023 OP009383 Postolata guangxiensis Dai et al., 2023 OP009384 Postolata guangxiensis Dai et al., 2023 OP009385 Postolata longjiangensis Liu & Wu, sp. nov.1* = PP713224 Postolata longjiangensis Liu & Wu, sp. nov. 2* = PP713225 Postolata longjiangensis Liu & Wu, sp. nov. 3* = PP713226 Postolata longjiangensis Liu & Wu, sp. nov. 4* = PP713227 Postolata longjiangensis Liu & Wu, sp. nov.5* =PP713228 Postolata longjiangensis Liu & Wu, sp. nov. 6* = PP713229 Obovalis omiensis (Heimburg, 1884) LC518997 Sinosolenaia carinata (Heude, 1877) MG742248 Sinosolenaia oleivora (Heude, 1877) MG742249 Ptychorhynchus pfisteri (Heude, 1874) MG742247 Gonidea angulata (Lea, 1838) DQ272372 Leguminaia anatolica Girlek et al., 2021 MZ511008 Leguminaia saulcyi (Bourguignat, 1852) MZ510997 Inversidens rentianensis Wu & Wu, 2021 OR826138 Lamprotula caveata (Heude, 1877) KJ434503 Lamprotula leaii (Gray, 1833) MFO72503 *Sequences from this study. MEGA 7.0 (Kumar et al. 2016) with 1000 bootstrap rep- licates. Intraspecific and interspecific genetic distances were calculated based on the COI barcoding dataset using the uncorrected p-distance model in MEGA 7.0. For the multi-locus dataset, the IQ-TREE web serv- er (http://igtree.cibiv.univie.ac.at/) performed maximum likelihood (ML) phylogenetic analysis using the ultrafast bootstrapping algorithm with 1000 repetitions. Bayesian inference (BI) phylogenetic analyses were carried out in MrBayes v2.01 (Ronquist et al. 2012) with generated mod- els in PartitionFinder (Lanfear et al. 2017). Four indepen- dent Markov Chain Monte Carlo (MCMC) models were run simultaneously for ten million generations, and sam- pling was conducted every 1000 generations with a burn- in of 25%. The process terminated when the average stan- dard deviation of the splitting frequency fell below 0.01. Ultimately, the constructed phylogenetic trees were imple- mented in the online 1TOL (https://itol.embl.de/itol.cgi) to realize editing and visualization (Letunic and Bork 2007). Results Systematics Family Unionidae Rafinesque, 1820 Subfamily Gonideinae Ortmann, 1916 Tribe Gonideini Ortmann, 1916 Genus Postolata Dai, Huang, Guo & Wu, 2023 Type species. Postolata guangxiensis Dai et al., 2023. zse.pensoft.net 772 Liu, L. et al.: Integrative description of a new species of Postolata Table 2. Sequences from the three-gene dataset used for molecular analyses and corresponding GenBank numbers. Family Subfamily Tribe Taxa COl 16S 28S Unionidae Gonideinae Gonideini Ptychorhynchus pfisteri (Heude, 1874) MG463034 KY067440 MG595562 Obovalis omiensis (Heimburg, 1884) LC518995 LC223994 LC519064 Postolata longjiangensis Liu & Wu, sp. nov. 1* =PP713224 PP717959 PP717965 Postolata longjiangensis Liu & Wu, sp. nov. 2* =PP713225 PP717960 PP717966 Postolata longjiangensis Liu & Wu, sp. nov. 3* = PP713226 PP/17961 PP/17967 Postolata longjiangensis Liu & Wu, sp. nov. 4* PP713227 PP717962 PP7/17968 Postolata longjiangensis Liu & Wu, sp. nov. 5* =PP713228 PP/17963 PP717969 Postolata longjiangensis Liu & Wu, sp. nov. 6* =PP713229 PP/717964 PP7/17970 Postolata guangxiensis Dai et al., 2023 1 OP009379 OPO020466 OP020470 Postolata guangxiensis Dai et al., 2023 2 OP009380 OP020467 OP020470 Postolata guangxiensis Dai et al., 2023 3 OP009381 OP020468 OP020470 Postolata guangxiensis Dai et al., 2023 4 OP009382 OP020469 OP020471 Postolata guangxiensis Dai et al., 2023 5 OP009383 OP020467 OP020472 Postolata guangxiensis Dai et al., 2023 6 OP009384 OP020468 OP020470 Postolata guangxiensis Dai et al., 2023 7 OP009385 OP020469 OP020471 Parvasolenaia rivularis (Heude, 1877) KX966393 KX966393 MG595632 Koreosolenaia sitgyensis Lee et al., 2020 GQ451872 GQ451859 MT020817 Sinosolenaia carinata (Heude, 1877) KX822669 MK683025 KX822626 Gonidea angulata (Lea, 1838) DQ272371 KFO11258 AF400691 Microcondylaea bonellii (Férussac, 1827) KX822652 KP218021 KX822609 Pseudodontini Bineurus loeiensis Konopleva et al., 2021 KX865879 KX865650 KX865750 Bineurus anodontinum (Rochebrune, 1882) MW603662 MZ684076 MZ684018 Thaiconcha callifera (Martens, 1860) KX865862 KX865633 KX865734 Pseudodon mekongi (Bolotov et al., 2020) KX865861 KX865632 KX865733 Pseudodon vondembuschianus (Lea, 1840) KP795029 KP795052 MZ684028 Pilsbryoconcha exilis (Lea, 1838) KX051291 KX865646 KX822613 Indopseudodon kayinensis (Bolotov et al., 2020) MZ678754 MZ684081 MZ684033 Indopseudodon bogani (Bolotov et al., 2017) MF352218 MF352292 MF352350 Schepmaniini Schepmania sp. 5973 MZ678755 MZ684082 MZ684035 Lamprotulin' §Lamprotula caveata (Heude, 1877) KX822646 NC_030336 KX822603 Lamprotula leaii (Gray, 1833) NC_023346 NC_023346 MG595524 Potomida littoralis (Cuvier, 1798) JN243905 NC_030073 JN243883 Contradentini Yaukthwa inlenensis Konopleva et al., 2019 KX865927 KX865681 KX865798 Yaukthwa paiensis Konopleva et al., 2019 MH345972 MH346012 MH345992 Yaukthwa elongatula Bolotov et al., 2019 MK372408 MK372456 MK372486 Lens contradens (Lea, 1838) MG581991 MT993693 MT993745 Lens eximius (Lea, 1856) KX865941 KX865689 KX865812 Physunio superbus (Lea, 1843) MG582020 MT993689 MT993741 Trapezoideus foliaceus (Gould, 1843) MH345985 MH346025 MH346005 Rectidentini = Hyriopsis bialata Simpson, 1900 KX051274 MT993644 MT993697 Hyriopsis desowitzi Brandt, 1974 KX822644 MT993679 KX822601 Rectidens sumatrensis (Dunker, 1852) KX051314 MW242818 KX822620 Ensidens sagittarius (Lea, 1856) KX865950 KX865696 KX865821 Ctenodesmini Khairuloconcha lunbawangorum Zieritz et al., MN900790 MZ684078 MN902294 2021 Khairuloconcha sahanae Zieritz et al., 2021 MZ678752 MZ684079 MZ684024 Chamberlainiini Chamberlainia somsakpanhai Kongim et al., 2023 KX822635 MK994770 KX822592 Margaritiferidae Margaritifera dahurica (Middendorff, 1850) KJ161516 KJ943526 KT343747 Margaritifera margaritifera (Linnaeus, 1758) KX550089 KX550091 KX550093 *Sequences from this study. Postolata longjiangensis Liu & Wu, sp. nov. https://zoobank.org/615D6E27-0F F5-4A62-8287-817768787DD0 Fig. 2 Type materials. Holotype (Fig. 2E): SXNU_24040702 (length 53.06 mm, height 27.11 mm, width 17.28 mm); Long River, Hechi City, Guangxi Province, China. Paratypes (Fig. 2A—D, F): five specimens, SXNU_ 24040703, SXNU_ 24040705, SXNU_ 24040706, SXNU_ 24040704, and SXNU_ 24040701. Same collec- tion location as the holotype. zse.pensoft.net Morphological diagnosis. Postolata longjiangensis sp. nov. can be distinguished from Postolata guangxien- sis by the shell shape, beak position, surface sculpture, nacre color, and hinge structure (Table 3). Diagnostic characteristics: shell elongated, irregularly rectangular; the umbo situated at 1/4 of the shell length; epidermis brown with greenish tinge; nacre blue-white; and hinge tooth weaker than that of Postolata guangxiensis. Molecular diagnosis. Postolata longjiangensis sp. nov. and Postolata guangxiensis formed a closely relat- ed group within the tribe Gonideini. The sequences of Zoosyst. Evol. 100 (3) 2024, 769-778 773 Figure 2. Shells of Postolata longjiangensis sp. nov. A. Paratype: SXNU_24040703; B. paratype: SXNU_24040705; C. Paratype: SXNU_ 24040706; D. Paratype: SXKNU_24040704; E. Holotype: SXNU_24040702; and F. Paratype: SXNU_24040701. Postolata longjiangensis sp. nov. revealed a well-sup- ported lineage that is distinct from its congener (Fig. 4). The genetic distance between the new species and Posto- lata guangxiensis is 8.13% based on the COI barcoding sequences. Description. Shell elongated, irregularly rectangular, slightly thickened, moderately inflated; anterior margin rounded and short; ventral margin nearly straight; poste- rior margin wide and long; posterior slope significantly prominent; dorsal margin nearly straight, with an up- ward tilt angle; umbo located at 1/4 of the shell length and sculptured with wavy ridges; epidermis brown with greenish tinge; shell surface sculptured with fine con- centric growth lines (Fig. 2; Table 3). Anterior adductor muscle attachment oblong, little deep, and smooth; pos- terior adductor muscle attachment round to oval, shal- low, and smooth; anterior retractor muscle attachment completely integrated with anterior adductor muscle at- tachment; posterior retractor muscle attachment irregu- larly round and fused with the posterior adductor muscle attachment; mantle muscle attachment obvious. Hinge weakly developed; anterior tooth extremely small, pos- terior tooth small, thin, and pyramidal in the left valve; anterior tooth upright pyramidal; posterior tooth degen- erate and merge into the lateral teeth in the right valve; there is one short lateral tooth of both shells; nacre-bule- white (Fig. 2; Table 3). Papillae in the incurrent aperture short and cylindrical, arranged in two rows; papillae in the excurrent aperture weakly developed, sparsely ar- ranged in one row; and the pigmentation of the incurrent and excurrent aperture significant; the size of inner gills exceeds that of outer gills; labial palps medium-thick, flat elliptical (Fig. 3; Table 3). Etymology. This species’ name is dedicated to its col- lection location, the Long River in Hechi City, Guangxi Province, China. For the common name, we recommend “Longjiang Rear-wide Mussel” (English) and “Long Ji- ang Hou Ju Bang” (Jé 11 Ja HEE) (Chinese). Distribution. Long River at Hechi City, Guangxi Province, China. zse.pensoft.net 774 Liu, L. et al.: Integrative description of a new species of Postolata Figure 3. Anatomical features of Postolata longjiangensis sp. nov. and Postolata guangxiensis, A1—3. Postolata longjiangensis sp. nov.; B1I-3. Postolata guangxiensis. Abbreviations: aam, anterior adductor muscle; pam, posterior adductor muscle; exa, excur- rent aperture; 1a, incurrent aperture; f, foot; ig, inner gill; og, outer gill; lp, labial palps; m, mantle; p ia, papillae in incurrent aperture; p exa, papillae of excurrent aperture. Table 3. Conchological and soft-body characteristics of Postolata longjiangensis sp. nov. and Postolata guangxiensis. Features Postolata longjiangensis sp. nov. Postolata guangxiensis Length (mm) 41.14-49.93 49.22-57.76 Width (mm) 13.82-17.28 19.95-21.42 Height (mm) 22.89-27.63 33.47-39.34 Shell shape Elongated, irregularly rectangular lrregularly rectangular Shell thickness Slightly thick Moderately thick Umbo position and sculpture 1/4 of shell length; umbo sculptured with wavy ridges 1/3 of shell length; umbo often eroded Surface sculpture Epidermis is brown with a bit green; shell surface — Epidermis is black-brown; shell surface sculptured with sculptured with fine concentric growth lines fine concentric growth lines; there is one sulcus near the posterior dorsal margin Nacre colour Blue-white Milky-white Posterior slope Significantly prominent Insignificant Dorsal margin Nearly straight, with an upward tilt angle Slightly curved downwards Hinge Weakly developed Well developed Pseudocardinal teeth of the Anterior tooth extremely small, posterior tooth small, Anterior tooth small, posterior tooth thick and left valve thin, and pyramidal pyramidal Pseudocardinal teeth of the Anterior tooth upright pyramidal, posterior tooth — Anterior tooth well-developed, posterior tooth reduced right valve degenerate and merge into the lateral teeth Lateral teeth One tooth on both valves, nearly straight One tooth on both valves, small and short Incurrent aperture Papillae is short cylindrical, arranged in two rows; and Papillae is distinctly short cylindrical, arranged in one pigmentation is significant to two rows Excurrent aperture Papillae is weakly developed, sparsely arranged in Papillae is short and dense; pigmentation unnoticeable one row; and pigmentation is significant Labial palps Medium-thick, flat elliptical Medium-thick, elliptical zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 769-778 0.97 0.4 0.22 0.39 0.47 0.33 0.72 0.95 0.35 0.95 7715 Postolata guangxiensis 6 Postolata guangxiensis 4 Postolata guangxiensis 7 Postolata guangxiensis 3 Postolata guangxiensis 1 Postolata guangxiensis 5 Postolata guangxiensis 2 Postolata longjiangensis sp. nov. 4 Postolata longjiangensis sp. nov. 5 Postolata longjiangensis sp. nov. 2 Postolata longjiangensis sp. nov. 1 Postolata longjiangensis sp. nov. 6 Postolata longjiangensis sp. nov. 3 Obovalis omiensis Ptychorhynchus pfisteri Sinosolenaia oleivora Leguminaia anatolica Leguminaia saulcyi Gonidea angulata Inversidens rentianensis Sinosolenaia carinata Lamprotula caveata Lamprotula leaii Tree scale: 0.01 H—-— Figure 4. Neighbor-joining tree generated from 23 COI sequences (12 taxa) based on the uncorrected p-distance model. The num- bers at the nodes indicate bootstrap supports (BS). The red fonts represent the species defined in this study. Phylogenetic analyses Multilocus phylogenies that were reconstructed using Bayesian inference (BI) and maximum likelihood (ML) analyses produced consistent topologies (Fig. 5). Both BI and ML analyses indicated that Postolata longjiangensis sp. nov. formed the sister lineage to Postolata guangxien- sis in the tribe Gonideini with high support values (BS/PP = 96/0.96) (Fig. 5). All eight recognized tribes in the subfamily Gonidein- ae formed monophyletic groups with the following rela- tionships: ((Gonideini + (((Contradentini + Rectidentin1) + Ctenodesmini) + (Lamprotulini + Chamberlainiini)) + (Pseudodontini + Schepmaniini)) (Fig. 5). Discussion We integrated comprehensive molecular evidence, shell morphology, and soft-body anatomy into the identifica- tion and classification of the new species from Guangx1, namely Postolata longjiangensis sp. nov. The topology of our phylogenetic tree (Fig. 5) basically corresponds to those generated in previous studies, except for some nodes (Wu et al. 2024). In our phylogenetic tree, the six individuals of P. /ongjiangensis occupy a single branch that shares a sister-group relationship with Postolata guangxiensis in the tribe Gonideini (Fig. 5). The long branch of P. longjiangensis unequivocally indicates its distinct (species-level) divergence from congeneric spe- cies (uncorrected p-distance = 8.13%; Fig. 4). In addition to the molecular phylogenetic evidence, Postolata longjiangensis and Postolata guangxiensis also display significant disparities in both shell morphol- ogy and soft-body anatomy (Table 3). The shell thick- ness of P. longjiangensis is relatively lower compared to that of P. guangxiensis, and the hinge is weakly de- veloped, with only one prominent pseudocardinal tooth. The morphological characteristics of the apertures and labial palps also differ significantly (Fig. 3). The con- vergence of shell and anatomical features in freshwater mussels is an important factor contributing to the diffi- culty in species definition (Inoue et al. 2013; Lopes-Lima et al. 2024). However, both species of Postolata possess inter-specific diagnostic features in terms of both shell morphology and anatomy. zse.pensoft.net 776 Bl by fr f ( Tribe \ == Gonideini — Contradentini ~~ Rectidentini Ctenodesmini Lamprotulini == Chamberlainiini 0.98 == Pseudodontini \ == Schepmaniini \ / eee _ 0.96 0.89 Liu, L. et al Postolata guangxiensis Postolata longjiangensis sp. nov. Obovalis omiensis Ptychorhynchus pfisteri Koreosolenaia sitgyensis Parvasolenaia rivularis Gonidea angulata Microcondylaea bonellii Sinosolenaia carinata Yaukthwa inlenensis Yaukthwa paiensis Yaukthwa elongatula Lens contradens Lens eximius Physunio superbus Trapezoideus foliaceus Hyriopsis desowitzi Rectidens sumatrensis Hyriopsis bialata Ensidens sagittarius Khairuloconcha lunbawangorum Khairuloconcha sahanae Lamprotula caveata Lamprotula leaii Potomida littoralis Chamberlainia somsakpanhai Bineurus anodontinum Bineurus loeiensis Thaiconcha callifera 96 99 100 100 100 98 100 44 100 100 55 100 86 63 .. Integrative description of a new species of Postolata ML 65 67 64 100 Pseudodon mekongi 100 || 96 Pseudodon vondembuschianus Pilsbryoconcha exilis 1 Indopseudodon bogani 908 0.81 Indopseudodon kayinensis 64 63 Schepmania sp. 5973 1 Margaritifera dahurica 100 Margaritifera margaritifera Tree scale: 1. _-———————————— Tree scale:0.1 -——H Figure 5. Bayesian inference (BI) and maximum likelihood (ML) trees reconstructed from the three-gene dataset (COI + 16S rRNA + 28S rRNA). Numbers at the nodes indicate the statistical support values for posterior probability (PP) and bootstrap support (BS). Color-coded clades and shadows represent eight tribes in the subfamily Gonideinae. The southern region of Guangxi, situated in the In- do-Burma hotspot area, has garnered significant attention and conservation efforts for its rich biodiversity (Tordoff et al. 2012). However, there remains a dearth of research on freshwater mussels in this area, including accurate species distribution, precise classification, and popula- tion dynamics (Zieritz et al. 2018; Liu et al. 2022). This knowledge gap severely impedes the progress of mussel conservation in this region. The construction and plan- ning of hydraulic projects for large rivers in recent years have led to the emergence of small rivers and tributaries as vital habitats for aquatic life (Jiang et al. 2011; Sabo et al. 2012; Xie 2017). The type locality of Postolata guangxiensis 1s a small tributary of the Luoqging River, characterized by good water quality and a silt bottom, with numerous residential structures in close proximity (Dai et al. 2023). It is interesting that Postolata longji- angensis was discovered 100 km away in the Long River and shares a similar habitat type (Fig. 1). The river habi- tats housing endemic mussel species are highly vulnera- ble and require immediate attention and protection due to the impacts of urbanization and human activities. There- fore, we advocate for the implementation of in situ con- servation measures for select endemic and endangered freshwater mussels through the establishment of nature reserves. Additionally, comprehensive research on arti- ficial breeding techniques and their practical application zse.pensoft.net is imperative to facilitate the recovery of this critically endangered species. Acknowledgements This work was funded by the National Natural Sci- ence Foundation of China (No. 32200370), the Ba- sic Research Program of Shanxi Province, China (No. 20210302124253), the Research Project Supported by the Shanxi Scholarship Council of China (2024-088), and the Innovation and Entrepreneurship Training Program for college students in Shanxi Province (2023DXCM-31). References Barnhart MC, Haag WR, Roston WN (2008) Adaptations to host in- fection and larval parasitism in Unionoida. Journal of the North American Benthological Society 27(2): 370-394. https://doi. org/10.1899/07-093.1 Castresana J (2000) Selection of conserved blocks from multiple align- ments for their use in phylogenetic analysis. 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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 au- thor(s) are credited. Link: https://doi.org/10.3897/zse.100.126069.suppl1