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Accurate identification and precise classification of freshwater mussel species that are among the most threatened freshwater taxa in the world, play a crucial role in informing conservation and management efforts for these organisms. However, due to the variability in shell morphology, relying solely on shell characteristics for species taxonomy poses significant challenges, thereby impeding effective conservation planning and management. The freshwater mussel genus Ptychorhynchus Simpson, 1900 is one such group in need of study. We integrate molecular phylogeny, shell morphology and soft-body anatomy to examine the classification of Ptychorhynchus denserugata (Haas, 1910) and Ptychorhynchus resupinatus (von Martens, 1902). The COI barcoding data support the clustering of P. denserugata and Nodularia douglasiae within a single clade, and P. denserugata shares the diagnostic feature of the genus Nodularia , i.e. knobs or bumps on the inner mantle surface in the excurrent aperture. Therefore, by integrating molecular data and anatomical characteristics, we confirm that the nominal species P. denserugata syn. nov. is a new synonym for N. douglasiae . The multi-locus (COI + ND1 + 16S rRNA + 18S rRNA + 28S rRNA ) phylogeny and mitochondrial phylogenomics support the transfer of P. resupinatus from Ptychorhynchus to the newly elevated genus Cosmopseudodon stat. rev., as Cosmopseudodon resupinatus stat. rev. that is still considered the designated type species. We also describe a new species based on integrative taxonomy, i.e. Cosmopseudodon wenshanensis sp. nov. The comprehensive understanding of the taxonomy and diversity of the revised Cosmopseudodon species, and shell heteromorphism of N. douglasiae (=P. denserugata syn. nov.), will serve as a crucial foundation for further scientific assessment and conservation strategies pertaining to these taxa. ZooBank: urn:lsid:zoobank.org:pub:E48968B1-DF0F-42AD-8F31-B8C95F23CE57.

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Located at the junction between Europe, Africa, and Asia, with distinct evolutionary origins and varied ecological and geographical settings, together with a marked history of changes in orogeny and configuration of the main river basins, turned the Eastern Mediterranean into a region of high diversity and endemism of freshwater taxa. Freshwater mussels (Bivalvia, Unionidae) from the Western Palearctic have been widely studied in their European range, but little attention has been dedicated to these taxa in the Eastern Mediterranean region and their diversity and phylogeography are still poorly understood. The present study aims to resolve the diversity, biogeography, and evolutionary relationships of the Eastern Mediterranean freshwater mussels. To that end, we performed multiple field surveys, phylogenetic analyses, and a thorough taxonomic revaluation. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens across Turkey, Israel, and Iran, combining COI + 16S + 28S and COI phylogenies with molecular species delineation methods. Phylogeographical patterns were characterized based on published molecular data, newly sequenced specimens, and species distribution data, as well as ancestral range estimations. We reveal that Unionidae species richness in the Eastern Mediterranean is over 70% higher than previously assumed, counting 19 species within two subfamilies, the Unioninae (14) and Gonideinae (5). We propose two new species, Anodonta seddonisp. nov. and Leguminaia anatolicasp. nov. Six additional taxa, Unio delicatusstat. rev., Unio eucirrusstat. rev., Unio huetistat. rev., Unio sesirmensisstat. rev., Unio terminalisstat. rev. removed from the synonymy of Unio tigridis, as well as Unio damascensisstat. rev. removed from the synonymy of Unio crassus, are re-described. The nominal taxa Unio rothi var. komarowi O. Boettger, 1880 and Unio armeniacus Kobelt, 1911 are proposed as new synonyms of Unio bruguierianus, and Anodonta cyrea Drouët, 1881 and Anodonta cilicica Kobelt & Rolle, 1895 as new synonyms of Anodonta anatina. Also, the presence of Unio tumidus in the Maritza River is confirmed. The phylogeographic patterns described here are interpreted concerning major past geological events. Conservation needs and implications are presented, together with populations and species conservation priorities., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Data on the historical change of the Transbaikalian malacofauna in the Neopleistocene and Holocene is presented. Aquatic mollusc shells from archaeological excavations of the ancient settlements dating from the Neolithic period to Medieval and also from a drill hole of the Neopleistocene alluvial deposits were collected. In total eight species of bivalve molluscs from the families Margaritiferidae, Unionidae, Lymnocardiidae, Glycymerididae [marine], and two gastropod species from families Viviparidae and Planorbidae were identified. These species were aged using radiocarbon dating. It was found that the species ranged in age from more than 50.000 to 2.080-1.210 years BP. Five species inhabited the Transbaikal region which are locally extirpated today. Their disjunctive ranges in the past included southern Europe and Western and Eastern Siberia to Transbaikalia and in the east to Far East and Primorye Territory of Russia. A remarkable finding is that of the bivalve genus Monodacna, which was found very far from its native range, the Ponto-Caspian region. The time of existence and extirpation of the thermophilic species of genera Monodacna, Planorbis, Lanceolaria and Amuropaludina corresponds to cycles of the warming and cooling in Pleistocene and Holocene according to regional climate chronological scales. These species can be used as palaeoclimate indicators. Change of the regional malacofaunal species composition is connected with the natural climatochron cycles in the Pleistocene and Holocene resulting in evidence for succession. In the course of this succession, these stenothermal species became extirpated on a regional level, decreasing their global ranges., Competing Interests: The authors have declared that no competing interests exist

The Viviparidae, commonly known as River Snails, is a dominant group of freshwater snails with a nearly worldwide distribution that reaches its highest taxonomic and morphological diversity in Southeast Asia. The rich fossil record is indicative of a probable Middle Jurassic origin on the Laurasian supercontinent where the group started to diversify during the Cretaceous. However, it remains uncertain when and how the biodiversity hotspot in Southeast Asia was formed. Here, we used a comprehensive genetic data set containing both mitochondrial and nuclear markers and comprising species representing 24 out of 28 genera from throughout the range of the family. To reconstruct the spatiotemporal evolution of viviparids on a global scale, we reconstructed a fossil-calibrated phylogeny. We further assessed the roles of cladogenetic and anagenetic events in range evolution. Finally, we reconstructed the evolution of shell features by estimating ancestral character states to assess whether the appearance of sculptured shell morphologies was driven by major habitat shifts. The molecular phylogeny supports the monophyly of the three subfamilies, the Bellamyinae, Lioplacinae, and Viviparinae, but challenges the currently accepted genus-level classification in several cases. The almost global distribution of River Snails has been influenced both by comparatively ancient vicariance and more recent founder events. In Southeast Asia, Miocene dispersal was a main factor in shaping the modern species distributions. A recurrent theme across different viviparid taxa is that many species living in lentic waters exhibit sculptured shells, whereas only one strongly sculptured species is known from lotic environments. We show that such shell sculpture is habitat-dependent and indeed evolved several times independently in lentic River Snails. Considerably high transition rates between shell types in lentic habitats probably caused the co-occurrence of morphologically distinct shell types in several lakes. In contrast, directional evolution toward smooth shells in lotic habitats, as identified in the present analyses, explains why sculptured shells are rarely found in these habitats. However, the specific factors that promoted changes in shell morphology require further work. [biogeographical analyses; fossil-calibrated phylogeny; fossil-constrained analyses; Southeast Asia; stochastic character mapping.]., (© The Author(s) 2020. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Freshwater mussels (Bivalvia: Unionidae) is a diverse family with around 700 species being widespread in the Northern Hemisphere and Africa. These animals fulfill key ecological functions and provide important services to humans. Unfortunately, populations have declined dramatically over the last century, rendering Unionidae one of the world's most imperiled taxonomic groups. In Far East Asia (comprising Japan, Korea, and Eastern Russia), conservation actions have been hindered by a lack of basic information on the number, identity, distribution and phylogenetic relationships of species. Available knowledge is restricted to studies on national and sub-national levels. The present study aims to resolve the diversity, biogeography and evolutionary relationships of the Far East Asian Unionidae in a globally comprehensive phylogenetic and systematic context. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens from across Japan, South Korea, and Russia, based on molecular (including molecular species delineation and a COI + 28S phylogeny) and comparative morphological analyses. Biogeographical patterns were then assessed based on available species distribution data from the authors and previous reference works. We revealed that Unionidae species richness in Far East Asia is 30% higher than previously assumed, counting 43 species (41 native + 2 alien) within two Unionidae subfamilies, the Unioninae (32 + 1) and Gonideinae (9 + 1). Four of these species are new to science, i.e. Beringiana gosannensissp. nov., Beringiana fukuharaisp. nov., Buldowskia kamiyaisp. nov., and Koreosolenaia sitgyensisgen. & sp. nov. We also propose a replacement name for Nodularia sinulata, i.e. Nodularia breviconchanom. nov. and describe a new tribe (Middendorffinaiini tribe nov.) within the Unioninae subfamily. Biogeographical patterns indicate that this fauna is related to that from China south to Vietnam until the Mekong River basin. The Japanese islands of Honshu, Shikoku, Kyushu, Hokkaido, and the Korean Peninsula were identified as areas of particularly high conservation value, owing to high rates of endemism, diversity and habitat loss. The genetically unique species within the genera Amuranodonta, Obovalis, Koreosolenaiagen. nov., and Middendorffinaia are of high conservation concern., (Copyright © 2020 Elsevier Inc. All rights reserved.)

While a growing body of modern phylogenetic research reveals that the Western Indochina represents a separate biogeographic subregion having a largely endemic freshwater fauna, the boundaries of this subregion are still unclear. We use freshwater mussels (Unionidae) as a model to reconstruct spatial patterns of freshwater biogeographic divides throughout Asia. Here, we present an updated freshwater biogeographic division of mainland Southeast Asia and describe 12 species and 4 genera of freshwater mussels new to science. We show that the Isthmus of Kra represents a significant southern biogeographic barrier between freshwater mussel faunas of the Western Indochina and Sundaland subregions, while the Indian and Western Indochina subregions are separated by the Naga Hills, Chin Hills, and Rakhine Yoma mountain ranges. Our findings highlight that the freshwater bivalve fauna of Southeast Asia primarily originated within three evolutionary hotspots (Western Indochina, Sundaland, and East Asian) supplemented by ancient immigrants from the Indian Subcontinent.

Freshwater mussels are ecosystem engineers and keystone species in aquatic environments. Unfortunately, due to dramatic declines this fauna is among the most threatened globally. Here, we clarify the taxonomy and biogeography of Russian Unionidae species based on the most comprehensive multi-locus dataset sampled to date. We revise the distribution and assess the conservation status for each species. This fauna comprises 16 native species from 11 genera and 4 tribes: Anodonta, Pseudanodonta (Anodontini); Amuranodonta, Beringiana, Buldowskia, Cristaria, Sinanodonta (Cristariini); Middendorffinaia, Nodularia, Unio (Unionini); and Lanceolaria (Lanceolariini). No country-level endemic species are known in Russia, except for Buldowskia suifunica that may also occur in China. Sinanodonta woodiana, a non-native species, was introduced from China. Russia comprises the northern parts of Western and Eastern Palearctic subregions. The first subregion with six species encompasses a huge area from the western boundary of Russia to the Lena Basin in Siberia. The second subregion with 10 species covers the Amur Basin, rivers east of the Lena Basin, coastal basins of the Japan Sea, and the North Pacific Islands. The fauna of Russia primarily includes widespread generalist species that are here considered Least Concern (LC). However, Buldowskia suifunica and Sinanodonta lauta have restricted distributions and are assessed here as Vulnerable (VU) and Endangered (EN), respectively.

Using a new fossil-calibrated mitogenome-based approach, we identified macroevolutionary shifts in mitochondrial gene order among the freshwater mussels (Unionoidea). We show that the early Mesozoic divergence of the two Unionoidea clades, Margaritiferidae and Unionidae, was accompanied by a synchronous split in the gene arrangement in the female mitogenome (i.e., gene orders MF1 and UF1). Our results suggest that this macroevolutionary jump was completed within a relatively short time interval (95% HPD 201-226 Ma) that coincided with the Triassic-Jurassic mass extinction. Both gene orders have persisted within these clades for ~200 Ma. The monophyly of the so-called "problematic" Gonideinae taxa was supported by all the inferred phylogenies in this study using, for the first time, the M- and F-type mitogenomes either singly or combined. Within Gonideinae, two additional splits in the gene order (UF1 to UF2, UF2 to UF3) occurred in the Mesozoic and have persisted for ~150 and ~100 Ma, respectively. Finally, the mitogenomic results suggest ancient connections between freshwater basins of East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a continuous paleo-river system or along the Tethys coastal line, which are well supported by at least three independent but almost synchronous divergence events.

Background: Supernumerary ORFan genes (i.e., open reading frames without obvious homology to other genes) are present in the mitochondrial genomes of gonochoric freshwater mussels (Bivalvia: Unionida) showing doubly uniparental inheritance (DUI) of mitochondria. DUI is a system in which distinct female-transmitted and male-transmitted mitotypes coexist in a single species. In families Unionidae and Margaritiferidae, the transition from dioecy to hermaphroditism and the loss of DUI appear to be linked, and this event seems to affect the integrity of the ORFan genes. These observations led to the hypothesis that the ORFans have a role in DUI and/or sex determination. Complete mitochondrial genome sequences are however scarce for most families of freshwater mussels, therefore hindering a clear localization of DUI in the various lineages and a comprehensive understanding of the influence of the ORFans on DUI and sexual systems. Therefore, we sequenced and characterized eleven new mitogenomes from poorly sampled freshwater mussel families to gather information on the evolution and variability of the ORFan genes and their protein products., Results: We obtained ten complete plus one almost complete mitogenome sequence from ten representative species (gonochoric and hermaphroditic) of families Margaritiferidae, Hyriidae, Mulleriidae, and Iridinidae. ORFan genes are present only in DUI species from Margaritiferidae and Hyriidae, while non-DUI species from Hyriidae, Iridinidae, and Mulleriidae lack them completely, independently of their sexual system. Comparisons among the proteins translated from the newly characterized ORFans and already known ones provide evidence of conserved structures, as well as family-specific features., Conclusions: The ORFan proteins show a comparable organization of secondary structures among different families of freshwater mussels, which supports a conserved physiological role, but also have distinctive family-specific features. Given this latter observation and the fact that the ORFans can be either highly mutated or completely absent in species that secondarily lost DUI depending on their respective family, we hypothesize that some aspects of the connection among ORFans, sexual systems, and DUI may differ in the various lineages of unionids.

Freshwater mussels (Unionida) are one of the most imperiled animal groups worldwide, revealing the fastest rates of extinction. Habitat degradation, river pollution and climate change are the primary causes of global decline. However, biological threats for freshwater mussels are still poorly known. Here, we describe a diverse ecological group of leeches (Hirudinea: Glossiphoniidae) inhabiting the mantle cavity of freshwater mussels. So far, examples of mussel-associated leech species are recorded from East Asia, Southeast Asia, India and Nepal, Africa, and North America. This group comprises a dozen glossiphoniid species with a hidden life style inside the mantle cavity of their hosts largely overlooked by researchers. We show that the association with freshwater mussels evolved independently in three leech clades, i.e. Batracobdelloides, Hemiclepsis, and Placobdella, at least since the Miocene. Seven mussel-associated leech species and two additional free-living taxa are described here as new to science.

Freshwater mussels are sensitive to habitat and water quality, revealing the fastest rates of human-mediated global extinction among aquatic animals. These animals are especially diverse in tropical Asia, the faunas of which are characterized by high levels of endemism. Here we describe four new species and four new subspecies of freshwater mussels from Myanmar. Leoparreysia whitteni sp. nov., the smallest representative of this genus, was discovered from the Ayeyarwady and Chindwin rivers. Radiatula myitthanensis sp. nov. and R. chindwinensis sp. nov. were recorded from the Chindwin Basin, and R. mouhoti haungthayawensis ssp. nov. has been discovered from the Haungthayaw River. Indochinella pugio has been revised with a description of three subspecies: I. pugio viridissima ssp. nov. from the Sittaung, Bilin and Bago rivers, I. pugio daweiensis ssp. nov. from the Dawei River, and I. pugio paradoxa ssp. nov. from the Haungthayaw River. Yaukthwa elongatula sp. nov., a peculiar species, conchologically resembling representatives of the genus Solenaia (Gonideinae) with ultra-elongated shell was found in the Chindwin Basin. Our records highlight that tropical Asia harbors numerous, but still overlooked local endemic lineages of freshwater bivalves, which may be on the brink of extinction due to the high anthropogenic and climate change impacts.

Uniorectangularis Tapparone-Canefri, 1889 is a little-known nominal species of freshwater mussels described from a tributary of the Ayeyarwady River in Myanmar. This taxon was considered a synonym of Gibbosulalaosensis (Lea, 1863), a margaritiferid species. However, the range of Gibbosulalaosensis does not encompass the Ayeyarwady River watershed. Here we re-examine the holotype of Uniorectangularis and provide a conchological re-description of this species. Based on conchological features such as the shell shape, elevated umbo, and the structure of lateral and pseudocardinal teeth, we transfer this taxon to the genus Indonaia Prashad, 1918 and propose I.rectangularis (Tapparone-Canefri, 1889), comb. nov. It appears to be a rare freshwater mussel species with a restricted range, because it has not been found since the original description. Two additional species in this genus are known from Myanmar, i.e. Indonaiaandersoniana (Nevill, 1877) and I.subclathrata (Martens, 1899).

Two Unionida (freshwater mussel) families are present in the Northern Hemisphere; the Margaritiferidae, representing the most threatened of unionid families, and the Unionidae, which include several genera of unresolved taxonomic placement. The recent reassignment of the poorly studied Lamprotula rochechouartii from the Unionidae to the Margaritiferidae motivated a new search for other potential species of margaritiferids from members of Gibbosula and Lamprotula. Based on molecular and morphological analyses conducted on newly collected specimens from Vietnam, we here assign Gibbosula crassa to the Margaritiferidae. Additionally, we reanalyzed all diagnostic characteristics of the Margaritiferidae and examined museum specimens of Lamprotula and Gibbosula. As a result, two additional species are also moved to the Margaritiferidae, i.e. Gibbosula confragosa and Gibbosula polysticta. We performed a robust five marker phylogeny with all available margaritiferid species and discuss the taxonomy within the family. The present phylogeny reveals the division of Margaritiferidae into four ancient clades with distinct morphological, biogeographical and ecological characteristics that justify the division of the Margaritiferidae into two subfamilies (Gibbosulinae and Margaritiferinae) and four genera (Gibbosula, Cumberlandia, Margaritifera, and Pseudunio). The systematics of the Margaritiferidae family is re-defined as well as their distribution, potential origin and main biogeographic patterns., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Deforestation, climate change and invasive species constitute three global threats to biodiversity that act synergistically. However, drivers and rates of loss of freshwater biodiversity now and in the future are poorly understood. Here we focus on the potential impacts of global change on freshwater mussels (Order Unionida) in Sundaland (SE Asia), a vulnerable group facing global declines and recognized indicators of overall freshwater biodiversity. We used an ensemble of distribution models to identify habitats potentially suitable for freshwater mussels and their change under a range of climate, deforestation and invasion scenarios. Our data and models revealed that, at present, Sundaland features 47 and 32 Mha of habitat that can be considered environmentally suitable for native and invasive freshwater mussels, respectively. We anticipate that by 2050, the area suitable for palm oil cultivation may expand between 8 and 44 Mha, representing an annual increase of 2-11%. This is expected to result in a 20% decrease in suitable habitat for native mussels, a drop that reaches 30% by 2050 when considering concomitant climate change. In contrast, the habitat potentially suitable for invasive mussels may increase by 44-56% under 2050 future scenarios. Consequently, native mussels may compete for habitat, food resources and fish hosts with invasive mussels across approximately 60% of their suitable range. Our projections can be used to guide future expeditions to monitor the conservation status of freshwater biodiversity, and potentially reveal populations of endemic species on the brink of extinction. Future conservation measures-most importantly the designation of nature reserves-should take into account trends in freshwater biodiversity generally, and particularly species such as freshwater mussels, vital to safeguard fundamental ecosystem services., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Freshwater mussels (Bivalvia: Unionida) serve an important role as aquatic ecosystem engineers but are one of the most critically imperilled groups of animals. Here, we used a combination of sequencing strategies to assemble and annotate a draft genome of Venustaconcha ellipsiformis, which will serve as a valuable genomic resource given the ecological value and unique "doubly uniparental inheritance" mode of mitochondrial DNA transmission of freshwater mussels. The genome described here was obtained by combining high-coverage short reads (65× genome coverage of Illumina paired-end and 11× genome coverage of mate-pairs sequences) with low-coverage Pacific Biosciences long reads (0.3× genome coverage). Briefly, the final scaffold assembly accounted for a total size of 1.54 Gb (366,926 scaffolds, N50 = 6.5 kb, with 2.3% of "N" nucleotides), representing 86% of the predicted genome size of 1.80 Gb, while over one third of the genome (37.5%) consisted of repeated elements and >85% of the core eukaryotic genes were recovered. Given the repeated genetic bottlenecks of V. ellipsiformis populations as a result of glaciations events, heterozygosity was also found to be remarkably low (0.6%), in contrast to most other sequenced bivalve species. Finally, we reassembled the full mitochondrial genome and found six polymorphic sites with respect to the previously published reference. This resource opens the way to comparative genomics studies to identify genes related to the unique adaptations of freshwater mussels and their distinctive mitochondrial inheritance mechanism.

The diversity and taxonomy of anodontine species in Lake Baikal and Transbaikalia region has been contentious since it is based on a typological species concept, the so called "Comparatory Method". Using this method, six Comparatory anodontine species have been described for the study area as belonging to the genus Colletopterum. This genus was separated from Anodonta based on shell characteristics and further split into two subgenera, i.e. Colletopterum sensu stricto and Colletopterum (Piscinaliana). However, many authors do not recognize this separation maintaining all Colletopterum forms within Anodonta. The current study clarifies the taxonomy and systematics of Anodontinae in this region, using a combination of molecular, morphological and anatomical data. All previously recognized Comparatory forms are here recognized as a single species, i.e. Anodonta anatina.

Doubly uniparental inheritance (DUI) describes a mode of mtDNA transmission widespread in gonochoric freshwater mussels (Bivalvia: Palaeoheterodonta: Unionida). In this system, both female- and male-transmitted mtDNAs, named F and M respectively, coexist in the same species. In unionids, DUI is strictly correlated to gonochorism and to the presence of the atypical open reading frames (ORFans) F-orf and M-orf, respectively inside F and M mtDNAs, which are hypothesized to participate in sex determination. However, DUI is not found in all three Unionida superfamilies (confirmed in Hyrioidea and Unionoidea but not in Etherioidea), raising the question of its origin in these bivalves. To reconstruct the co-evolution of DUI and of ORFans, we sequenced the mtDNAs of four unionids (two gonochoric with DUI, one gonochoric and one hermaphroditic without DUI) and of the related gonochoric species Neotrigonia margaritacea (Palaeoheterodonta: Trigoniida). Our analyses suggest that rearranged mtDNAs appeared early during unionid radiation, and that a duplicated and diverged atp8 gene evolved into the M-orf associated with the paternal transmission route in Hyrioidea and Unionoidea, but not in Etherioidea. We propose that novel mtDNA-encoded genes can deeply influence bivalve sex determining systems and the evolution of the mitogenomes in which they occur.

Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Freshwater mussels (Bivalvia, Unionida) fulfil important ecosystem functions and are one of the most threatened freshwater taxa globally. Knowledge of freshwater mussel diversity, distribution and ecology in Peninsular Malaysia is extremely poor, and the conservation status of half of the species presumed to occur in the region has yet to be assessed. We conducted the first comprehensive assessment of Peninsular Malaysia's freshwater mussels based on species presence/absence and environmental data collected from 155 sites spanning all major river catchments and diverse habitat types. Through an integrative morphological-molecular approach we recognised nine native and one widespread non-native species, i.e. Sinanodonta woodiana. Two species, i.e. Pilsbryoconcha compressa and Pseudodon cambodjensis, had not been previously recorded from Malaysia, which is likely a result of morphological misidentifications of historical records. Due to their restriction to single river catchments and declining distributions, Hyriopsis bialata, possibly endemic to Peninsular Malaysia, Ensidens ingallsianus, possibly already extinct in the peninsula, and Rectidens sumatrensis, particularly require conservation attention. Equally, the Pahang, the Perak and the north-western river catchments are of particular conservation value due to the presence of a globally unique freshwater mussel fauna. Statistical relationships of 15 water quality parameters and mussel presence/absence identified acidification and nutrient pollution (eutrophication) as the most important anthropogenic factors threatening freshwater mussel diversity in Peninsular Malaysia. These factors can be linked to atmospheric pollution, deforestation, oil-palm plantations and a lack of functioning waste water treatment, and could be mitigated by establishing riparian buffers and improving waste water treatment for rivers running through agricultural and residential land., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The number of anodontine bivalve species placed in the genus Cristaria (Bivalvia, Unionidae) from the Russian Far East is still not stable among authors. Some recognize only one valid species Cristaria plicata (Leach, 1815) while others accept two additional species, Cristaria tuberculata Schumacher, 1817 and Cristaria herculea (Middendorff, 1847). In the present study, these taxonomic doubts are addressed using analyses of mitochondrial DNA sequences and shell morphometry. No significant differences have been revealed by the COI DNA sequences or the main statistical morphometric indices from the three Cristaria forms. In the specimens analysed, changes in shell morphometry with age suggest that original descriptions of the different forms may be attributed solely to differences in age and sex. We consider that Cristaria plicata, Cristaria tuberculata and Cristaria herculea from the Russian Far East should be considered as a single species, namely Cristaria plicata (Leach, 1815), with Cristaria tuberculata and Cristaria herculea as junior synonyms. The geographic range of Cristaria plicata and its conservation status are also presented here.

The number of species in the freshwater mussel genus Cristaria Schumacher, 1817 recognized from Far East Russia has varied over the last several decades. While some authors consider the occurrence of only one species, Cristaria plicata (Leach, 1815), widespread in East Asia, others, recognize two separate species Cristaria herculea (Middendorff, 1847) and Cristaria tuberculata Schumacher, 1817 from Far East Russia, distinct from C. plicata. For the present study, freshwater mussels, identified as C. herculea, were collected in the Upper Amur basin (Transbaikalia, Russia). The shell morphology and the whole soft body anatomy were analysed in detail and compared with previously published information on other Cristaria spp.. Additionally, a cytochrome oxidase subunit 1 (CO1) gene fragment was sequenced from foot tissue samples of selected animals, collected from the same region, and compared with published data. Based upon morphological similarities of glochidia and adult morphology and anatomy as well as the mitochondrial DNA sequence analysis, we consider C. herculea as a synonym of C. plicata. Further analysis of Far East Russia C. herculea and C. tuberculata specimens using both molecular and morphological characters should be carried in the future to enhance our knowledge about the taxonomy within the Cristaria genus. Moreover, a comprehensive revision of the genus Cristaria is needed, restricting the type locality and comparing topotypic specimens for both C. plicata and C. tuberculata, and including all recognized Cristaria species.

Mitochondrial (mt) function depends critically on optimal interactions between components encoded by mt and nuclear DNAs. mitochondrial DNA (mtDNA) inheritance (SMI) is thought to have evolved in animal species to maintain mito-nuclear complementarity by preventing the spread of selfish mt elements thus typically rendering mtDNA heteroplasmy evolutionarily ephemeral. Here, we show that mtDNA intraorganismal heteroplasmy can have deterministic underpinnings and persist for hundreds of millions of years. We demonstrate that the only exception to SMI in the animal kingdom, that is, the doubly uniparental mtDNA inheritance system in bivalves, with its three-way interactions among egg mt-, sperm mt- and nucleus-encoded gene products, is tightly associated with the maintenance of separate male and female sexes (dioecy) in freshwater mussels. Specifically, this mother-through-daughter and father-through-son mtDNA inheritance system, containing highly differentiated mt genomes, is found in all dioecious freshwater mussel species. Conversely, all hermaphroditic species lack the paternally transmitted mtDNA (=possess SMI) and have heterogeneous macromutations in the recently discovered, novel protein-coding gene (F-orf) in their maternally transmitted mt genomes. Using immunoelectron microscopy, we have localized the F-open reading frame (ORF) protein, likely involved in specifying separate sexes, in mitochondria and in the nucleus. Our results support the hypothesis that proteins coded by the highly divergent maternally and paternally transmitted mt genomes could be directly involved in sex determination in freshwater mussels. Concomitantly, our study demonstrates novel features for animal mt genomes: the existence of additional, lineage-specific, mtDNA-encoded proteins with functional significance and the involvement of mtDNA-encoded proteins in extra-mt functions. Our results open new avenues for the identification, characterization, and functional analyses of ORFs in the intergenic regions, previously defined as "noncoding," found in a large proportion of animal mt genomes.

Background: Doubly uniparental inheritance (DUI) is an atypical system of animal mtDNA inheritance found only in some bivalves. Under DUI, maternally (F genome) and paternally (M genome) transmitted mtDNAs yield two distinct gender-associated mtDNA lineages. The oldest distinct M and F genomes are found in freshwater mussels (order Unionoida). Comparative analyses of unionoid mitochondrial genomes and a robust phylogenetic framework are necessary to elucidate the origin, function and molecular evolutionary consequences of DUI. Herein, F and M genomes from three unionoid species, Venustaconcha ellipsiformis, Pyganodon grandis and Quadrula quadrula have been sequenced. Comparative genomic analyses were carried out on these six genomes along with two F and one M unionoid genomes from GenBank (F and M genomes of Inversidens japanensis and F genome of Lampsilis ornata)., Results: Compared to their unionoid F counterparts, the M genomes contain some unique features including a novel localization of the trnH gene, an inversion of the atp8-trnD genes and a unique 3'coding extension of the cytochrome c oxidase subunit II gene. One or more of these unique M genome features could be causally associated with paternal transmission. Unionoid bivalves are characterized by extreme intraspecific sequence divergences between gender-associated mtDNAs with an average of 50% for V. ellipsiformis, 50% for I. japanensis, 51% for P. grandis and 52% for Q. quadrula (uncorrected amino acid p-distances). Phylogenetic analyses of 12 protein-coding genes from 29 bivalve and five outgroup mt genomes robustly indicate bivalve monophyly and the following branching order within the autolamellibranch bivalves: ((Pteriomorphia, Veneroida) Unionoida)., Conclusion: The basal nature of the Unionoida within the autolamellibranch bivalves and the previously hypothesized single origin of DUI suggest that (1) DUI arose in the ancestral autolamellibranch bivalve lineage and was subsequently lost in multiple descendant lineages and (2) the mitochondrial genome characteristics observed in unionoid bivalves could more closely resemble the DUI ancestral condition. Descriptions and comparisons presented in this paper are fundamental to a more complete understanding regarding the origins and consequences of DUI.

Doubly uniparental inheritance (DUI) of mitochondrial DNA in marine mussels (Mytiloida), freshwater mussels (Unionoida), and marine clams (Veneroida) is the only known exception to the general rule of strict maternal transmission of mtDNA in animals. DUI is characterized by the presence of gender-associated mitochondrial DNA lineages that are inherited through males (male-transmitted or M types) or females (female-transmitted or F types), respectively. This unusual system constitutes an excellent model for studying basic aspects of mitochondrial DNA inheritance and the evolution of mtDNA genomes in general. Here we compare published mitochondrial genomes of unionoid bivalve species with DUI, with an emphasis on characterizing unassigned regions, to identify regions of the F and M mtDNA genomes that could (i) play a role in replication or transcription of the mtDNA molecule and/or (ii) determine whether a genome will be transmitted via the female or the male gamete. Our results reveal the presence of one F-specific and one M-specific open reading frames (ORFs), and we hypothesize that they play a role in the transmission and/or gender-specific adaptive functions of the M and F mtDNA genomes in unionoid bivalves. Three major unassigned regions shared among all F and M unionoid genomes have also been identified, and our results indicate that (i) two of them are potential heavy-strand control regions (O(H)) for regulating replication and/or transcription and that (ii) multiple and potentially bidirectional light-strand origins of replication (O(L)) are present in unionoid F and M mitochondrial genomes. We propose that unassigned regions are the most promising candidate sequences in which to find regulatory and/or gender-specific sequences that could determine whether a mitochondrial genome will be maternally or paternally transmitted.

Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.