Freshwater mussels (Bivalvia: Unionidae) from the rising sun (Far East Asia): phylogeny, systematics, and distribution.

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.

Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia: Unionoidea) macroevolution.

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.

The complete mitochondrial genome of the hermaphroditic freshwater mussel Anodonta cygnea (Bivalvia: Unionidae): in silico analyses of sex-specific ORFs across order Unionoida.

BACKGROUND: Doubly uniparental inheritance (DUI) of mitochondrial DNA in bivalves is a fascinating exception to strictly maternal inheritance as practiced by all other animals. Recent work on DUI suggests that there may be unique regions of the mitochondrial genomes that play a role in sex determination and/or sexual development in freshwater mussels (order Unionoida). In this study, one complete mitochondrial genome of the hermaphroditic swan mussel, Anodonta cygnea, is sequenced and compared to the complete mitochondrial genome of the gonochoric duck mussel, Anodonta anatina. An in silico assessment of novel proteins found within freshwater bivalve species (known as F-, H-, and M-open reading frames or ORFs) is conducted, with special attention to putative transmembrane domains (TMs), signal peptides (SPs), signal cleavage sites (SCS), subcellular localization, and potential control regions. Characteristics of TMs are also examined across freshwater mussel lineages. RESULTS: In silico analyses suggests the presence of SPs and SCSs and provides some insight into possible function(s) of these novel ORFs. The assessed confidence in these structures and functions was highly variable, possibly due to the novelty of these proteins. The number and topology of putative TMs appear to be maintained among both F- and H-ORFs, however, this is not the case for M-ORFs. There does not appear to be a typical control region in H-type mitochondrial DNA, especially given the loss of tandem repeats in unassigned regions when compared to F-type mtDNA. CONCLUSION: In silico analyses provides a useful tool to discover patterns in DUI and to navigate further in situ analyses related to DUI in freshwater mussels. In situ analysis will be necessary to further explore the intracellular localizations and possible role of these open reading frames in the process of sex determination in freshwater mussel.

Evaluation of the Applicability of the Comparatory Method for Species Diagnosis of Unionidae (Bivalvia) by Genetic Analysis.

Five genetically distant groups of mussels possessing high intragroup homogeneity were identified among 65 specimens of 14 East European Unionidae "comparatory species" by genetic analysis of nuclear and mitochondrial markers. By shell morphology other than the shape of the convex contour of the shell, the identified groups correspond to five "taxonomic species" according to Zhadin's classification. The use of the comparatory method for Unionidae species identification is unjustified.

Identification and characterization of two LBP/BPI genes involved in innate immunity from Hyriopsis cumingii.

Lipopolysaccharide-binding protein and bactericidal permeability-increasing protein (LBP/BPI) play crucial role in modulating cellular signals in response to Gram-negative bacteria infection. In the present study, two isoforms of LBP/BPI genes, designated as HcLBP/BPI1 and HcLBP/BPI2, respectively, were cloned from the mussel Hyriopsis cumingii by RACE approach. The full-length cDNA sequences of HcLBP/BPI1 and HcLBP/BPI2 were 1887 and 2227 bp and encoded two secreted proteins of 501 and 518 amino acid residues, respectively. The deduced amino acid of HcLBP/BPI1 and HcLBP/BPI2 contained several conserved domains, such as signal peptide, two BPI/LBP and one central domain. Phylogentic analysis further supported that HcLBP/BPI1 and HcLBP/BPI2 belonged to new members of invertebrate LBP/BPI family. The mRNA transcripts of HcLBP/BPI1 and HcLBP/BPI2 were ubiquitously expressed in all examined tissues, and the expression level of HcLBP/BPI1 was higher than that of HcLBP/BPI2. The mRNA expression of HcLBP/BPI1 in hepatopancreas and hemocytes was significantly up-regulate after Aeromonas hydrophila and LPS challenge, and HcLBP/BPI2 in hepatopancreas was only up-regulated at 6 and 12 h after LPS challenge and at 12 h after A. hydrophila challenge. In addition, the recombinant HcLBP/BPIs displayed antibacterial activity against Gram-negative bacteria, and the antibacterial index of HcLBP/BPI1 was higher than that of HcLBP/BPI2. These results indicated that HcLBP/BPI1 and HcLBP/BPI2 probably played distinct roles in bacterial mediating immune response in Mollusca.

Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels.

Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species' presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.

A galectin from Hyriopsis cumingii involved in the innate immune response against to pathogenic microorganism and its expression profiling during pearl sac formation.

Hyriopsis cumingii is the most important freshwater pearl mussel cultured in China. The operation for implantation is one necessary technical step for pearl culture. However, implantation-induced trauma results in a series of immune responses and can enable the invasion of pathogenic microbes. Lectin proteins are found widely in nature and play important roles in innate immunity. Galectins are members of the lectin superfamily and are characterized by one or several carbohydrate recognition domains (CRDs) that produce multiple sugar binding sites on the protein. Here we cloned and characterized the H. cumingii galectin gene HcGal1, which encodes a 312 amino acid galectin protein. The HcGal1 transcript was detected in all tested H. cumingii tissues and showed higher expression specifically in immune tissues. The significant upregulation of HcGal1 expression was observed after challenging the mussel with lipopolysaccharide or Gram-negative and Gram-positive bacteria. After implantation, significant downregulation of the HcGal1 transcript was noted in the mantle, hemocytes, and pearl sac in the acute-stress stage (0-24 h) and the stage of wound healing and pearl-sac formation (24 h-7 d). In addition, significant upregulation of HcGal1 expression was observed in the liver in the stage of wound healing and pearl-sac formation. In the pearl-secretion stage (7-35 d), the HcGal1 transcript levels returned to normal in all tested tissues. We also show that recombinantly expressed and purified HcGal1 can agglutinate some Gram-negative and Gram-positive bacteria. In addition, in vivo experiments showed that the recombinant protein HcGal1 could promote phagocytosis by hemocytes. Our data suggest that HcGal1 plays a role in innate immune responses involved in pathogen recognition and wound healing.

Cloning and analysis of gene expression of interleukin-17 homolog in triangle-shell pearl mussel, Hyriopsis cumingii, during pearl sac formation.

Successful allograft of mantle tissues in certain bivalve mollusks can form pearl sacs secreting nacre for pearl production. Little was known, however, about the immune consequences in response to the tissue transplantation. In the present study, interleukin (IL)-17, one of the key regulatory genes of alloimmunity, was cloned from the triangle-shell pearl mussel (HcIL-17) Hyriopsis cumingii by high-throughput sequencing of the mantle transcriptome. The sequence of HcIL-17 contains an open reading frame of 567 bp encoding a putative protein of 188 amino acid residues. Analysis of sequence characteristics, multiple sequence alignment and phylogenetic analysis indicated HcIL-17 was a novel member in the mollusk IL-17 family. Expression of the HcIL-17 gene in donor mantle tissues and in hemocytes of recipient mussel was up-regulated dramatically within 7 days in response to the mantle tissue allograft for pearl aquaculture, suggesting remarkable proinflammatory responses during pearl sac formation in triangle-shell pearl mussels. Analysis of the time-course expression of HcIL-17 gene revealed the induction of HcIL-17 was time-dependent, reflecting the different periods of alloimmune events in triangle-shell mussels. The results of this study provide essential background information for further investigation of mollusk alloimmunity.

[The Microsculpture of Glochidia of Some Anodontine Bivalves (Unionidae)].

Glochidia of three freshwater anodontine bivalves: Kunashiria Starobogatov in Zatrawkin, 1983, Sinanodonta Modell, 1944, from the Far East, and Anodonta Lamarck, 1799, were investigated by scanning electron microscopy. Data on the microsculpture of the outer surface of glochidial valves are given. Among the three genera discussed, the glochidia of Anodonta are the largest, with a loose-looped outer microsculpture and numerous granules. The glochidia of Kunashiria and Sinanodonta differ by the valve height--length proportions and some details of the outer microsculpture: glochidia of Kunashiria have a tight-looped outer sculpture while the glochidia of Sinanodonta have a loose-net outer sculpture.

Taxonomic revision of two species in the genus Ptychorhynchus Simpson, 1900 (Bivalvia: Unionidae: Gonideinae), with description of a new species.

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.

Comparison of population genetic patterns in two widespread freshwater mussels with contrasting life histories in western North America.

We investigate population genetic structuring in Margaritifera falcata, a freshwater mussel native to western North America, across the majority of its geographical range. We find shallow rangewide genetic structure, strong population-level structuring and very low population diversity in this species, using both mitochondrial sequence and nuclear microsatellite data. We contrast these patterns with previous findings in another freshwater mussel species group (Anodonta californiensis/A. nuttalliana) occupying the same continental region and many of the same watersheds. We conclude that differences are likely caused by contrasting life history attributes between genera, particularly host fish requirements and hermaphroditism. Further, we demonstrate the occurrence of a 'hotspot' for genetic diversity in both groups of mussels, occurring in the vicinity of the lower Columbia River drainage. We suggest that stream hierarchy may be responsible for this pattern and may produce similar patterns in other widespread freshwater species.

Novel protein genes in animal mtDNA: a new sex determination system in freshwater mussels (Bivalvia: Unionoida)?

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.

Pyganodon (Bivalvia: Unionoida: Unionidae) phylogenetics: a male- and female-transmitted mitochondrial DNA perspective.

Species boundaries, evolutionary relationships and geographic distributions of many unionoid bivalve species, like those in the genus Pyganodon, remain unresolved in Eastern North America. Because unionoid bivalves are one of the most imperiled groups of animals in the world, understanding the genetic variation within and among populations as well as among species is crucial for effective conservation planning. Conservation of unionoid species is indispensable from a freshwater habitat perspective but also because they possess a unique mitochondrial inheritance system where distinct gender-associated mitochondrial DNA lineages coexist: a female-transmitted (F) mt genome and a male-transmitted (M) mt genome that are involved in the maintenance of separate sexes (=dioecy). In this study, 42 populations of Pyganodon sp. were sampled across a large geographical range and fragments of two mitochondrial genes (cox1 and cox2) were sequenced from both the M- and F-transmitted mtDNA genomes. Our results support the recency of the divergence between P. cataracta and P. fragilis. We also found two relatively divergent F and M lineages within P. grandis. Surprisingly, the relationships among the P. grandis specimens in the F and M sequence trees are not congruent. We found that a single haplotype in P. lacustris has recently swept throughout the M genotype space leading to an unexpectedly low diversity in the M lineage in that species. Our survey put forward some challenging results that force us to rethink hybridization and species boundaries in the genus Pyganodon. As the M and F genomes do not always display the same phylogeographic story in each species, we also discuss the importance of being careful in the interpretation of molecular data based solely on maternal transmitted mtDNA genomes. The involvement of F and M genomes in unionoid bivalve sex determination likely played a role in the genesis of the unorthodox phylogeographic patterns reported herein.

Molecular phylogenetic analysis of tropical freshwater mussels (Mollusca: Bivalvia: Unionoida) resolves the position of Coelatura and supports a monophyletic Unionidae.

In previous molecular phylogenetic analyses of the freshwater mussel family Unionidae (Bivalvia: Unionoida), the Afrotropical genus Coelatura had been recovered in various positions, generally indicating a paraphyletic Unionidae. However that result was typically poorly supported and in conflict with morphology-based analyses. We set out to test the phylogenetic position of Coelatura by sampling tropical lineages omitted from previous studies. Forty-one partial 28S nuclear rDNA and partial COI mtDNA sequences (1130 total aligned nucleotides) were analyzed separately and in combination under both maximum parsimony and likelihood, as well as Bayesian inference. There was significant phylogenetic incongruence between the character sets (partition homogeneity test, p<0.01), but a novel heuristic for comparing bootstrap values among character sets analyzed separately and in combination illustrated that the observed conflict was due to homoplasy rather than separate gene histories. Phylogenetic analyses robustly supported a monophyletic Unionidae, with Coelatura recovered as part of a well-supported Africa-India clade (=Parreysiinae). The implications of this result are discussed in the context of Afrotropical freshwater mussel evolution and the classification of the family Unionidae.

New freshwater mussels from two Southeast Asian genera Bineurus and Thaiconcha (Pseudodontini, Gonideinae, Unionidae).

The Mekong and Chao Phraya rivers harbor a species-rich freshwater mussel assemblage containing a large radiation of the Pseudodontini species. Members of the genera Bineurus Simpson 1900 and Thaiconcha Bolotov et al., 2020 primarily inhabit small and medium-sized tributaries of these rivers. Here, we present an integrative taxonomic review of these genus-level clades. We show that Bineurus contains four species: B. mouhotii (Lea, 1863), B. exilis (Morelet, 1866) stat. rev., B. anodontinum (Rochebrune, 1882) stat. rev., and B. loeiensis sp. nov. In its turn, Thaiconcha comprises three species: T. callifera (Martens, 1860), T. munelliptica sp. nov., and T. thaiensis sp. nov. Two species, Pseudodon ovalis Morlet, 1889 and P. thomsoni Morlet, 1884, are considered here as questionable taxa. These findings further highlight that Southeast Asia represents a significant evolutionary hotspot of freshwater mussels, which requires further international collaborative research and conservation efforts.

Reproductive aspects of freshwater unionid mussel sperm: Seasonal dynamics, male-to male variability, and cell quantification.

Freshwater unionid mussel diversity is decreasing because of species extirpation or extinction. While little can be done to recover lost species, there is an opportunity to develop techniques to save other species. This can be facilitated through gene banking and assisted reproduction. Unfortunately, limited information is available on mussel reproduction, especially relating to sperm quality. Objectives, therefore, were to quantify seasonal changes in sperm concentration and morphology for two unionid mussels, Ligumia subrostrata and Lampsilisstraminea, measure intraspecific heterogeneity for sperm morphometry, and develop an efficient method to quantify sperm concentration using a microspectrophotometer. There were no differences in sperm concentration when cells were extracted from the center or at a half centimeter on either side of the visceral mass, during the spawning season. There was a seasonal change in sperm concentration, such that concentration for L. subrostrata ranged from 1.1 × 109 to 19.60 × 109 cells/mL with there being the largest counts between 26 September to 7 November. L. straminea sperm concentration was greatest (20.0 × 109 cells/mL) on 13 September and subsequently decreased. Sperm were uniflagellated and SEM results for L. subrostrata and L. straminea showed mean head length and width (mid-spawning) were 3.38 ± 0.04 µm and 1.61 ± 0.01 µm and 3.37 ± 0.04 µm and 1.61 ± 0.01 µm, respectively. There were close (R2 ≥ 0.85) quadratic associations between hemocytometer counts and absorbance (300, 600, 700 nm). These results provide baseline information to further investigate sperm quality, fertilizing capacity, and cryopreservation for freshwater mussels.

Genome-wide SNPs redefines species boundaries and conservation units in the freshwater mussel genus Cyprogenia of North America.

Detailed information on species delineation and population genetic structure is a prerequisite for designing effective restoration and conservation strategies for imperiled organisms. Phylogenomic and population genomic analyses based on genome-wide double digest restriction-site associated DNA sequencing (ddRAD-Seq) data has identified three allopatric lineages in the North American freshwater mussel genus Cyprogenia. Cyprogenia stegaria is restricted to the Eastern Highlands and displays little genetic structuring within this region. However, two allopatric lineages of C. aberti in the Ozark and Ouachita highlands exhibit substantial levels (mean uncorrected FST = 0.368) of genetic differentiation and each warrants recognition as a distinct evolutionary lineage. Lineages of Cyprogenia in the Ouachita and Ozark highlands are further subdivided reflecting structuring at the level of river systems. Species tree inference and species delimitation in a Bayesian framework using single nucleotide polymorphisms (SNP) data supported results from phylogenetic analyses, and supports three species of Cyprogenia over the currently recognized two species. A comparison of SNPs generated from both destructively and non-destructively collected samples revealed no significant difference in the SNP error rate, quality and amount of ddRAD sequence reads, indicating that nondestructive or trace samples can be effectively utilized to generate SNP data for organisms for which destructive sampling is not permitted.

[Analysis of complete mitochondrial genome of Cristaria plicata].

The complete mitochondrial genome of Cristaria plicata was obtained using long amplification polymerase chain reaction (LA-PCR). Analysis showed that the total length of sequence was 15 712 bp, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 26 non-coding regions ranged from 2 bp to 328 bp in size. The nucleotide composition of A, T, C, G were 36.54% 27.22% 23.22% and 13.02%, respectively. Most genes were encoded on the L strand, While ND3-ND5, ND4L, COI-COIII, ATP6, ATP8, tRNAAsp, and tRNAHis were encoded on the H strand. The arrangement of genes in C. plicata was identical to that of Lampsilis ornata, but was different from that of Hyriopsis cum-ingii between COII and 12S rRNA in the same family. Thirteen protein genes contained 3 initiation codons, i.e., I (AUU, AUC), V (GUG), and M (AUA, AUG), all of which had complete stop codons (UAA or UAG), except for the stop codon in ND2 that had an incomplete T. Fifteen tRNAs had typical cloverleaf structure, except for tRNAThr, tRNALys, tRNASer(UCN), tRNAAsp, tRNAArg, tRNATyr, and tRNAMet. Like other freshwater bivalvia, C. plicata contained ATP8 gene, which might be related to the balance of osmotic pressure in the cytoplasm.

Identification of complete F-type mitochondrial genome in Lamprotula scripta and Lamprotula caveata and analysis on DUI.

Mitochondrial DNA is typically passed to offspring through maternal inheritance. However, in mussels, two kinds of mitochondrial DNA exist: F and M type, which are referred to as doubly uniparental inheritance (DUI). Studies have shown that DUI may be related to gender determination. In this study, we obtained the first complete F-type mitochondrial genome of Lamprotula scripta and Lamprotula caveata which were 16,250 bp and 16,641 bp in length, respectively, and had 13 protein coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs and 27 non-coding (NC) regions. The largest NC region of L. scripta was 639 bp and located between ND5 and tRNAGln. The largest NC of L. caveata was 1046 bp and also located between ND5 and tRNAGln. The overall AT content of L. scripta and L. caveata was 58.95% and 58.66%, respectively, which were lower than Lamprotula leai, Lamprotula gottschei and Lamprotula tortuosa. We next compared F and M mitochondrial genomic data on freshwater mussels and established a phylogenetic tree based on amino acid sequences of 13 PCGs and COII gene. Our results showed that F- and M-type mitochondria were significantly separated into two branches, and the basic structure of phylogenetic trees were divided into four distinct groups: Unioninae, Anodontini, Gonideinae and Ambleminae. Relatives of Gonideinae and Ambleminae were more closely related than Unioninae and Anodontini, indicating significant differences in mtDNA between the two mitogenome types. Moreover, we revealed that L. scripta and L. caveata are closely relatives, suggesting that they are both subordinates of the Gonideinae subfamily. Consequently, we speculate that the formation of DUI hinders their disappearance, which provides a basis for further studies into the mechanisms and genetic diversities of DUI formation.

Ecological correlates and phylogenetic signal of host use in North American unionid mussels.

Mussels in the order Unionoida comprise ∼75% of the world's freshwater bivalve species and are free-living apart from a brief larval stage that parasitizes fish. We investigated the relationships among species of North American unionid mussels and their known host fishes from a macroevolutionary perspective to test whether and how ecological and evolutionary factors correlate with patterns of host use. A subset of 69 mussel species was chosen based on data availability regarding their fish host repertoires, phylogenetic relationships, and ecology. Despite the brevity of their parasitic life stages, the mussels conformed to the right-skewed distribution of host specificity typical of parasitic taxa, in which most species are specialists and a few are generalists. Phylogenetic least squares regression models identified affinity for low-gradient and riffle habitats, and colonization of post-glacial watersheds as the best predictors for the number of fish host species per mussel. However, the second-best model identified citation number as a predictor of the number of hosts, implying that many mussel-host interactions still remain to be identified. A Multiple Regression Mantel test was performed to identify factors associated with the proportion of hosts shared between pairs of mussel species. Range overlap, citations, genetic distance, and similarity in host infection strategy were significantly correlated with the proportion of hosts shared, yet total variation as explained by the best model was low (R2 = 0.14). There was evidence of a topological association between mussels and their hosts (P = 0.001) and a significant phylogenetic signal of host specificity (λ = 0.81, P = 0.003), indicating closely related mussels that overlap in range are more likely to be competing for hosts. Our results provide an initial macroevolutionary framework for studying the evolution of host infection strategies in these mussels but also highlights gaps still remaining in our fundamental ecological knowledge of this endangered clade.