Mussel Diversity Research Articles

Lateral connectivity maintains higher freshwater mussel biodiversity

Abstract The loss of lateral hydrological connectivity (LLHC) in freshwater ecosystems is increasingly threatening biodiversity; however, studies reporting this situation are limited, especially those assessing multi‐measure biodiversity features at large spatial scales. Here, and using one the most threatened taxonomic groups in freshwater ecosystems, we examined the possible influence of LLHC on the species diversity, density, and biomass of mussel assemblages and the genetic diversity and structure of Nodularia douglasiae (a common and widespread native species) in the Yangtze River floodplain. Results showed that the species diversity of freshwater mussels (α and β diversity) significantly differed between the connected and the disconnected areas, with higher α diversity and lower β diversity in the connected areas. At the same time, relative abundance, density, biomass, and genetic diversity of freshwater mussels were higher in the connected areas. High genetic differentiation and low gene flow were found among the 18 analysed N. douglasiae populations, indicating that LLHC is affecting genetic structure of this particular freshwater mussel species. Overall, results indicated that river–lake connectivity is affecting freshwater mussel biodiversity. In addition, this study highlights that multi‐measure biodiversity features provide distinct information about biodiversity dynamics, and are essential to evaluate the effects of floodplain disconnection. We advocate that an integrative approach to floodplain management embracing species and genetic diversity is needed for effective biodiversity conservation in large river ecosystems.

A revision of Rhombuniopsis Haas, 1920 (Unionida, Unionidae) endemic to the ancient lakes of Yunnan, China, with descriptions of two new species

Freshwater mussels are an important component of the macrobenthic fauna but due to modern environmental pollution and destruction, the abundance of freshwater mussels has declined rapidly, and this fauna is one of the most threatened animal groups in the world. China is the country with the second highest diversity of freshwater mussels in the world. Rhombuniopsis is a genus of freshwater mussels endemic to the ancient lakes and surrounding area in Yunnan Province, China. Species in this genus are threatened with extinction and have not been recorded alive for many years. We present a revision of Rhombuniopsis based on the morphological study of the museum materials and newly collected specimens, and propose two species new to science: Rhombuniopsis linan sp. nov. and Rhombuniopsis songmeng sp. nov. The two new species are considered extinct because the lakes they inhabited were completely dried up due to human activity. We compare our recent data with the historical studies to trace the population change of the Rhombuniopsis species in different lakes. We revise the distribution and assess the conservation status for each species. The present work confirms the importance of conservation of molluscs in the ancient lakes of Yunnan and suggest that several species are already extinct.

High endemic freshwater mussel (Bivalvia: Unionida) diversity in western Borneo, with description of three new species

Abstract The freshwater mussels (Bivalvia: Unionida) of the biodiversity hotspot Sundaland are experiencing severe anthropogenic threats, whilst their diversity and distribution remain poorly understood. Here, we present the first modern-day data on Unionida diversity and distribution across western Borneo. Mussels were surveyed and collected in the upper Kapuas and Pawan river basins in West Kalimantan, Indonesia, and the Sambas Besar, Sarawak and Batang Sadong river basins in Sarawak, Malaysia. DNA sequencing (COI + 16S + ND1 + 18S + 28S) and morphological analyses were conducted to delineate and identify species, and reconstruct phylogenetic relationships and population genetics. Specimens belonged to six native genera and nine species, of which seven are endemic to Borneo and three are new to science, i.e. Ctenodesma mawonae Zieritz et al., sp. nov., Sarawak basin, and Ctenodesma bersinara Zieritz et al., sp. nov., and Rectidens lauris Zieritz et al., sp. nov., both Pawan basin. The monotypic genera Caudiculatus and Discomya were phylogenetically highly divergent from other known Gonideinae taxa, potentially indicating a separate tribe (for Caudiculatus) and subfamily (for Discomya). In addition, we report new records of the non-native Sinanodonta pacifica Heude, 1878 in the Batang Sadong and Kapuas river basins.

Population genomics of the endangered freshwater mussel, Arcidens wheeleri (Unionoidea: Unionidae: Anodontini), in the Little River, Arkansas, USA

ABSTRACT North America is a hotspot of freshwater mussel diversity. However, over the last century, many anthropogenic stressors have led to substantial declines in freshwater mussels of the families Unionidae and Margaritiferidae in North America. Conserving the remaining mussel fauna is a priority, as they play an integral role in freshwater ecosystems. The Little River in Arkansas is home to one of the last remaining populations of the federally endangered freshwater mussel, Arcidens wheeleri (Unionidae: Anodontini). Population dynamics information is lacking for A. wheeleri, and no population genetics study has been conducted on this species. A greater understanding of the genetic diversity within a population can serve as a benchmark for developing an effective management plan. We sampled A. wheeleri from three locations in the Little River. Genomic data were generated with a single-enzyme restriction-site-associated DNA sequencing approach to assess genetic diversity and structure of A. wheeleri in the Little River. Genetic structure analyses indicated one genetic population among the three locations, with limited, fine-scale subpopulation structure. Observed heterozygosity values were considerably lower than expected heterozygosity values, with Ho = 0.14 and He = 0.22, likely indicating a genetic bottleneck. Demographic analysis of the Little River population of A. wheeleri also suggests a historical bottleneck. Furthermore, a high inbreeding coefficient (FIS = 0.33) indicates A. wheeleri in the Little River is losing genetic diversity. Data generated indicate considerable risk of extirpation for A. wheeleri from the Little River and should serve as a baseline for future monitoring. Given its high risk of extinction, we recommend increased study of A. wheeleri across its range and on-the-ground conservation actions that include habitat protection and restoration, which are the only options until a successful host fish and protocols are identified for propagation.

Addition to the known diversity of Chinese freshwater mussels: integrative description of a new species of Postolata Dai et al., 2023 (Bivalvia, Unionidae, Gonideinae)

In this study, we present a new species of freshwater mussel in the genus PostolataDai 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 (i.e., Postolata guangxiensis) by its shell shape, beak position, surface sculpture, nacre color, and hinge structure. Molecular 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.

Diversity, morphology, and phylogeny of freshwater mussels of the genus Nodularia (Bivalvia: Unionidae) from China, with descriptions of four new species

AbstractFreshwater bivalves (Bivalvia, Unionida) are one of the most threatened groups of animals in the world. Defining species boundaries and understanding the phylogeny and genetic diversity of these species is key to guiding their conservation and management. However, the presence of significant phenotypic plasticity and convergence within this group complicates species delimitation. This includes the freshwater mussel genus Nodularia, endemic to East Asia, for which a comprehensive understanding of species diversity and phylogenetic relationships remains elusive due to inadequate sampling in previous studies, particularly in China, a widely recognized biodiversity hotspot for freshwater mussels. Here, we conduct comprehensive taxonomic and phylogenetic analyses of Nodularia species based on extensive sampling across 23 provinces in China and multiple data sources, including shell morphology, soft body anatomy, six‐gene (COI + ND1 + 16S + 18S + 28S + histone H3) and mitogenome datasets. The integrative systematics approach used here reveals 10 distinct species in this genus, four of which are new to science, i.e. Nodularia hanensis sp. nov., Nodularia huana sp. nov., Nodularia fusiformans sp. nov., Nodularia dualobtusus sp. nov. and two of which are new records for China, i.e. Nodularia dorri (Wattebled [Journal de Conchyliologie, 34, 1886, 54]) and Nodularia micheloti (Morlet [Journal de Conchyliologie, 34, 1886, 75]). We also propose that the nominal species Nodularia jourdyi (Morlet [Journal de Conchyliologie, 34, 1886, 75]) syn. nov. is a new synonym for Nodularia douglasiae (Griffith & Pidgeon, 1833) based on molecular data. BI, ML, and BEAST analyses based on the six‐gene dataset and mitochondrial phylogenomics consistently support the following phylogenetic relationships: (N. dorri + (N. hanensis sp. nov. + N. micheloti)) + (N. breviconcha + (N. huana sp. nov. + (N. fusiformans sp. nov. + ((N. nuxpersicae + N. nipponensis) + (N. dualobtusus sp. nov. + N. douglasiae))))). The molecular clock with fossil calibration indicates that Nodularia originated in the Late Cretaceous period (ca. 73.78 Mya). It then diverged into two independent clades during the Middle Paleogene (ca. 45.01 Mya), followed by a rapid burst of extant speciation during the Neogene (mean age 28.28 to 4.79 Mya). Nodularia breviconcha is the earliest differentiated taxon among the 10 Nodularia taxa, appearing during the Paleogene‐Neogene transition (28.28 Mya; 95% HPD = 14.35–48.44 Mya). Taken together, we provide a robust systematic framework for Nodularia species, addressing phylogenetic relationships, taxonomy, and evolutionary history of this group.

Microsatellite Analysis of the Genetic Diversity of Mussels from the North-Western Region of the Black Sea

Using four microsatellite DNA markers (<i>Mch 5, Mch 8, MT 203, MT 282</i>), the genetic diversity and population structure of three groups of mussels from the north-western section of the Black Sea were analyzed, and mussels from the Baltic and North Seas were studied as outgroups. By the studied microsatellite (MS) loci, 59 alleles were determined in five mussels' groups (<i>n</i> = 118). The observed heterozygosity (<i>H<sub>O</sub></i>) ranged from 0.083 to 0.538 and was lower than expected (<i>H<sub>E</sub></i> = 0.344-0.858). All mussel groups showed heterozygote deficiency, which suggests the possibility of inbreeding. Weak (<i>F<sub>ST</sub></i> = 0.036-0.048) and moderate (<i>F<sub>ST</sub></i> = 0.054-0.127) genetic differentiation between the studied groups of mussels was revealed. Genetic distances between the mussels' groups from different locations were calculated, which ranged from 0.211 to 1.130. The obtained data were confirmed by cluster and coordinate analyzes carried out using genetic distances between the groups by distribution of alleles frequencies.

A trait dataset for freshwater mussels of the United States of America

The United States of America has a diverse collection of freshwater mussels comprising 301 species distributed among 59 genera and two families (Margaritiferidae and Unionidae), each having a unique suite of traits. Mussels are among the most imperilled animals and are critical components of their ecosystems, and successful management, conservation and research requires a cohesive and widely accessible data source. Although trait-based analysis for mussels has increased, only a small proportion of traits reflecting mussel diversity in this region has been collated. Decentralized and non-standardized trait information impedes large-scale analysis. Assembling trait data in a synthetic dataset enables comparison across species and lineages and identification of data gaps. We collated data from the primary literature, books, state and federal reports, theses and dissertations, and museum collections into a centralized dataset covering information on taxonomy, morphology, reproductive ecology and life history, fish hosts, habitats, thermal tolerance, geographic distribution, available genetic information, and conservation status. By collating these traits, we aid researchers in assessing variation in mussel traits and modelling ecosystem change.

Sixty years of species diversity and population density decline of freshwater mussels in a global biodiversity hotspot

Sixty years of species diversity and population density decline of freshwater mussels in a global biodiversity hotspot

Unveiling the identity of Diaurora Cockerell, 1903 (Bivalvia, Unionidae): morphology, molecular phylogenetics, and the description of a new species.

The monotypic freshwater mussel genus Diaurora Cockerell, 1903 has long been enigmatic due to its rarity and morphological confusion with Acuticosta. In this study, we comprehensively redescribed Diauroraaurorea (Heude, 1883) through a detailed analysis of shell morphology and molecular phylogenetics of recently collected specimens. Moreover, a new species, Diauroralaevesp. nov., was identified from the Fuyishui River, a tributary of the Zishui River in Shaoyang County, Shaoyang City, Hunan Province, China. Molecular phylogenetic analyses showed that D.aurorea and D.laevesp. nov. were reciprocally monophyletic and formed a clade as sister to Schistodesmus. Our study underscores the necessity of further exploring the diversity of freshwater mussels in understudied small tributaries throughout China.

Limitations of beta diversity in conservation site selection

Preservation of diversity is a key issue in many conservation efforts. It has recently been suggested that beta diversity should be considered in conservation planning and that sites with high local contribution to beta diversity (LCBD) may be valuable for conservation. Yet, such an approach has not been applied to freshwater mussels, a highly imperiled group of organisms. We used a dataset on freshwater mussel from the five largest rivers of southwestern Ontario, Canada to examine whether LCBD would be an efficient approach for assessing the conservation value of sites and compared it to more traditional conservation approaches (biomonitoring, high richness, high abundance). Sites with high LCBD value were associated with low mussel richness and were often characterized by mussels that are regionally widespread and usually abundant in lentic systems or smaller streams, limiting site selection based on beta diversity. Species at risk (including IUCN categories of Critically Endangered through Near Threatened) were more likely to occur in communities with high richness and abundance at sites chosen by the more traditional approaches. Thus, protecting sites with higher alpha diversity of mussels would also support higher gamma diversity of mussels. The provision of important ecosystem services by higher abundances of mussels which was also correlated with local richness should be considered as well. Our study suggests that there likely is an important link between high richness and abundance of mussels and the presence of species at risk which we argue is driven by facilitation (i.e., positive species interaction), an important, yet understudied, process that deserves more attention in conservation.

Assessment of the genetic diversity of Chinese freshwater mussels and refuge areas in the Yangtze River floodplain

Abstract Freshwater mussels are one of the most diverse and endangered faunas worldwide. Owing to the continuing global biodiversity loss, it is essential to identify and protect populations that are not severely affected by human disturbances and ensure that natural genetic diversity is not inevitably lost. The generation of baseline genetic diversity data will help guide species conservation and restoration plans in the Yangtze River Basin where freshwater mussels are facing multiple threats and where strong declines have been observed. The genetic diversity of freshwater mussels was assessed using 818 mtDNA cytochrome c oxidase subunit 1 (COI), 565 28S rRNA and 509 COI + 28S datasets, and refuge areas in the Yangtze River floodplain were predicted using a Maxent habitat suitability model. The results showed that the genetic diversity of Aculamprotula spp., Sinosolenaia carinata, Cuneopsis rufescens, Lamprotula cornuumlunae, Lanceolaria lanceolata, Ptychorhynchus pfisteri, Schistodesmus spp. and Sinohyriopsis cumingii was lower than that of other freshwater mussels, which indicated that the conservation status of these species should be prioritized. The habitat suitability modelling showed that Poyang Lake and Dongting Lake basins (lakes and tributary rivers connected to the Yangtze River mainstem) present the most suitable habitats for freshwater mussels, indicating that lateral hydrological connectivity may be the most important factor in maintaining the genetic diversity of Chinese freshwater mussels. Consequently, Poyang Lake and Dongting Lake basins should be established as protected areas. This study provides valuable information for the conservation and management of freshwater mussel biodiversity in the Yangtze River basin, which will not only help conserve this vulnerable group but will also provide wider benefits to freshwater ecosystems.

Effect of complex hydraulic variables and physicochemical factors on freshwater mussel density in the largest floodplain lake, China

BackgroundHabitat degradation and flow regime alterations are two of the most prominent and common impact factors to freshwater mussel populations. Knowledge of the correlation between freshwater mussel distribution, density and habitat characteristics is important for maintaining and restoring their biodiversity and ecological functions. Information on predicting habitat suitability of freshwater mussels is lacking in China. Here, we aimed to analyze the correlation between freshwater mussel density and complex hydraulic and physicochemical variables to predict habitat suitability.ResultsThe results showed that four complex hydraulic variables (boundary Reynolds number, critical shear stress, bed roughness and mean sediment particle size) and four physicochemical variables (water temperature, chlorophyll-a, transparency and pH) were key factors for predicting habitat suitability of freshwater mussels. Freshwater mussel density was significantly correlated with Froude number, water temperature and chlorophyll-a.ConclusionsOur results confirmed that higher freshwater mussel density would be associated with areas that are stable in complex hydraulic and physicochemical variables. These results provide an important insight into the conservation of freshwater mussel diversity and their habitat restoration in China and globally.

Multilocus and mitogenomic phylogenetic analyses reveal a new genus and species of freshwater mussel (Bivalvia: Unionidae) from Guangxi, China

Freshwater mussels are essential for the integrity of freshwater ecosystems but numbers of these organisms are declining rapidly at regional and global scales. The phylogenetic and biogeographic aspects of the rich unionoid fauna of the Indo-Burma region are becoming increasingly well understood. Guangxi is part of the Chinese portion of the Indo-Burma biodiversity hotspot but regional studies of the freshwater mussel diversity are scarce. In this study, we report a new genus and species of freshwater mussel from Guangxi, China. Genetic datasets including three genes (COI, 16S rRNA and 28S rRNA) and complete maternal mitogenomes were compiled to infer the phylogenetic history of the group. Molecular phylogenetic analyses showed that the new species formed a monophyletic group and was closely related to Obovalis and Ptychorhynchus in the tribe Gonideini of the subfamily Gonideinae. Morphological and molecular evidence supported that these specimens represent an undescribed genus and species that we describe as Postolata guangxiensis gen. nov., sp. nov. The discovery of this new taxon adds to the known level of endemism of freshwater mussels in Guangxi and a detailed survey of uncharted areas should reveal new diversity in the future. We also suggest that complete mitogenomes or even genome-scale nuclear data should be used for phylogenetic reconstructions when proposing major taxonomic changes. ZooBank: urn:lsid:zoobank.org:pub:76FC5A1D-7507-4F26-A12C-EC08AB333274

Status and Distribution of Freshwater Mussels in the Louisiana Section of Bayou Bartholomew

As a major tributary to the species-rich Ouachita River and one of the few remaining non-channelized rivers in the Alluvial Plains physiographic division, Bayou Bartholomew is a key ecosystem for the freshwater mussel diversity of southeast Arkansas and northeast Louisiana. Bayou Bartholomew is known to contain 16 mussel Species of Greatest Conservation Need (SGCN) but the mussel assemblage of the Louisiana section of Bayou Bartholomew has not been assessed since the early 2000s. In 2021, we sampled over 100 river km on the main channel of Bayou Bartholomew and four tributaries from just downstream of the Louisiana/Arkansas border to the confluence with the Ouachita River. We used a novel two-phase timed-search protocol to characterize the mussel assemblage and measured shell length (long axis) to determine the size distribution of each species encountered. A suite of relevant habitat variables and site attributes were also quantified or qualitatively noted. One site was selected for sampling using a grid survey to determine quantitative estimates of mussel density. Our survey confirmed the presence of 35 species including 12 SGCN in the Louisiana section of the Bayou Bartholomew drainage. In total, 3,292 live mussels representing 33 species were collected in the main channel, 234 live mussels representing 18 species were found in a quantitative grid, and four species were found in tributaries. Two additional species were only found in tributaries as dead shells. Length frequencies of all common and abundant species indicated ongoing annual recruitment. Patterns of ubiquity in the main channel of Bayou Bartholomew varied by species, and mussels were rare in the tributaries. Generalized linear modelling and non-metric multidimensional scaling analyses identified relationships between mussel assemblage composition and the distance from the confluence with the Ouachita River and habitat variables including substrates, depth, and mesohabitat types. A comparison with previous studies did not provide any evidence of major changes in overall mussel species distributions or occurrence in Bayou Bartholomew in Louisiana. We attribute the apparent stability in the mussel assemblage to the fact that major anthropogenic alterations such as channelization, impoundments, and watershed urbanization have been minimal since the earlier studies, and we conclude that Bayou Bartholomew will likely continue to be a hotspot of regional mussel diversity and a haven for several of Louisiana's SGCN if large-scale anthropogenic alterations to the system do not occur and management actions are taken to protect and allow for the persistence of heterogenous habitats.

Erratum

[This corrects the article DOI: 10.1002/ece3.9717.].

Two for the price of one: eDNA metabarcoding reveals temporal and spatial variability of mussel and fish co‐distributions in Michigan riverine systems

AbstractFreshwater mussels (family Unionidae) are among the world's most endangered taxa, with almost 75% of North American taxa classified as a species of concern, threatened, or endangered. Despite the critical importance of comprehensive distributional data for the conservation of unionids and fishes, these data are often lacking because of the labor and resources associated with traditional survey methods. During their larval stage, unionid mussels use various fish species as obligate hosts, making native fish species vital to unionid persistence and an understanding of host distribution similarly important. Here, we utilized an eDNA metabarcoding approach to evaluate patterns of co‐distribution of unionid mussels and fishes along ~362 km of the densely sampled Grand River network as well as the outlets of 19 tributaries along the eastern shore of Lake Michigan, USA. We detected a total of 21 mussel and 40 fish taxa, with distinctive composition of both mussel and fish assemblages across tributaries and differences in fish taxa between sampling periods. Notably, we detected more mussel taxa within the Grand River watershed than at the outlets of all 20 rivers combined. Within the Grand River network, two fish taxa (Pylodictus olivaris and Cyprinella) were found more frequently in areas of high mussel diversity, and three fish taxa more frequently in areas of low mussel diversity (Umbra, Leuciscidae, and Etheostoma). There was little difference between eDNA detections of mussels from samples collected in June versus August, but we detected significantly more fish taxa in August compared to June. Taken together, our findings demonstrate the value of eDNA metabarcoding for evaluating co‐distribution of ecologically connected taxa. The use of eDNA as a tool for determining distributions of mussels and their obligate hosts may facilitate conservation efforts for these imperiled taxa.

Phylogenetic assessment of endangered and look-alike Pigtoe species in a freshwater mussel diversity hotspot.

The Green River in Kentucky in the eastern United States is a freshwater mussel biodiversity hotspot, with 71 known species. Among them, the endangered Pleurobema plenum coexists with other morphologically similar species in the genera Fusconaia and Pleurobema, known colloquially as "pigtoes." Identification of species in these genera is challenging even for mussel experts familiar with them. In our study, the correct identification of these species by experts ranged from 57% to 83%. We delineated taxonomic boundaries among seven species and tested for cryptic biodiversity among these look-alike mussels utilizing mitochondrial and nuclear DNA sequence variation. Phylogenetic analysis of combined (1215 bp) mitochondrial DNA cytochrome oxidase I (COI) and NADH dehydrogenase 1 (ND1) genes showed five well-diverged groups that included F.flava, F.subrotunda, P.cordatum, and P.plenum as distinct clades, with P.sintoxia and P.rubrum grouped into a single clade. While our mitochondrial DNA analyses did not distinguish P.sintoxia and P.rubrum as phylogenetically distinct species, the typical shell forms of these two nominal taxa are very distinct. Further phylogenetic analysis using nuclear ribosomal transcribed spacer region subunit I (ITS1) DNA sequences also showed that P.sintoxia and P.rubrum were not distinct lineages. No cryptic species were detected in the Fusconaia and Pleurobema samples analyzed from the Green River. The highest haplotype diversity (h), average number of nucleotide differences (k), and nucleotide diversity (π) were observed for F.subrotunda at both the COI (h=0.896, k=3.805, π=0.00808) and ND1 (h=0.984, k=6.595, π =0.00886) markers, with similarly high genetic diversity in the other taxa. Our results give managers confidence that cryptic taxa do not occur within or among these morphologically similar species in the Green River, and populations appear genetically diverse, indicative of large and healthy populations.

Microhabitat and Landscape Drivers of Richness and Abundance of Freshwater Mussels (Unionida: Unionidae) in a Coastal Plain River

Although rivers support significant unionid mussel (Unionida: Unionidae) diversity, Gulf of Mexico tributary rivers have been subject to changes in water quality and habitat due to altered watershed land use. We quantified mussel species richness and relative abundance and environmental factors in small tributary streams of the Pearl River, Mississippi-Louisiana. Freshwater mussel and habitat surveys were conducted at 27 stream sampling sites over two summers (9 sites revisited), and coverage of seven land use categories and seven geological categories above each reach were calculated. Mussels were patchily distributed (53% of sites sampled yielded mussels) and typically not abundant (only 26% of sites yielded >10 mussels). Surveys revealed nine species, with total abundance ranging from 0–66 mussels and richness ranging from 0–5 species per site. Assemblages were driven by an upper to lower watershed gradient of decreasing CPUE and richness, with microhabitat and water quality, land cover, and geology locally modifying this gradient. Environmental variables did not seem of sufficient magnitude to account for the patchy distributions and low abundances of mussels at most study sites, and we hypothesize that high discharge events related to tropical storm passage may have exerted an overriding influence on mussel assemblages in these streams through direct mortality and/or altered availability of suitable glochidial hosts.

Resolving species-level diversity of Beringiana and Sinanodonta mussels (Bivalvia: Unionidae) in the Japanese archipelago using genome-wide data

Accurate species identification is of primary importance in ecology and evolutionary biology. For a long time, the unionid mussels Beringiana and Sinanodonta have puzzled researchers trying to unravel their diversity because of their poorly discernible morphologies. A recent study conducted species delineation of unionid mussels based on mitochondrial DNA variation, opening up a new avenue to grasp species diversity of the mussels. However, mtDNA-based classification may not align with species boundaries because mtDNA is prone to introgression and incomplete lineage sorting that cause discordance between species affiliation and gene phylogeny. In this study, we evaluated the validity of the mtDNA-based classification of unionid mussels Beringiana and Sinanodonta in Japan using mitochondrial sequence data, double digest restriction site-associated DNA library (ddRAD) sequencing, and morphological data. We found significant inconsistencies in the mitochondrial and nuclear DNA phylogenies, casting doubt on the reliability of the mtDNA-based classification in this group. In addition, nuclear DNA phylogeny revealed that there are at least two unionid lineages hidden in the mtDNA phylogeny. Although molecular dating technique indicates that Beringiana and Sinanodonta diverged >35 million years ago, their shell morphologies are often indistinguishable. Specifically, morphological analyses exhibited the parallel appearance of nearly identical ball-like shell forms in the two genera in Lake Biwa, which further complicates species identification and the morphological evolution of unionid mussels. Our study adds to a growing body of literature that accurate species identification of unionid mussels is difficult when using morphological characters alone. Although mtDNA-based classification is a simple and convenient way to classify unionid mussels, considerable caution is warranted for its application in ecological and evolutionary studies.