Chun-Hua Zhou

The Complete Maternally and Paternally Inherited Mitochondrial Genomes of the Endangered Freshwater Mussel Solenaia carinatus (Bivalvia: Unionidae) and Implications for Unionidae Taxonomy

Doubly uniparental inheritance (DUI) is an exception to the typical maternal inheritance of mitochondrial (mt) DNA in Metazoa, and found only in some bivalves. In species with DUI, there are two highly divergent gender-associated mt genomes: maternal (F) and paternal (M), which transmit independently and show different tissue localization. Solenaia carinatus is an endangered freshwater mussel species exclusive to Poyang Lake basin, China. Anthropogenic events in the watershed greatly threaten the survival of this species. Nevertheless, the taxonomy of S. carinatus based on shell morphology is confusing, and the subfamilial placement of the genus Solenaia remains unclear. In order to clarify the taxonomic status and discuss the phylogenetic implications of family Unionidae, the entire F and M mt genomes of S. carinatus were sequenced and compared with the mt genomes of diverse freshwater mussel species. The complete F and M mt genomes of S. carinatus are 16716 bp and 17102 bp in size, respectively. The F and M mt genomes of S. carinatus diverge by about 40% in nucleotide sequence and 48% in amino acid sequence. Compared to F counterparts, the M genome shows a more compact structure. Different gene arrangements are found in these two gender-associated mt genomes. Among these, the F genome cox2-rrnS gene order is considered to be a genome-level synapomorphy for female lineage of the subfamily Gonideinae. From maternal and paternal mtDNA perspectives, the phylogenetic analyses of Unionoida indicate that S. carinatus belongs to Gonideinae. The F and M clades in freshwater mussels are reciprocal monophyly. The phylogenetic trees advocate the classification of sampled Unionidae species into four subfamilies: Gonideinae, Ambleminae, Anodontinae, and Unioninae, which is supported by the morphological characteristics of glochidia.

Complete maternal mitochondrial genome of freshwater mussel Anodonta lucida (Bivalvia: Unionidae: Anodontinae)

Abstract The taxonomy of genus Anodonta is rather ambiguous, as it has great variation on the shell shape. Anodonta lucida is an endemic species of freshwater mussel in China, characterized by shining epidermis. The complete maternal mitochondrial genome of freshwater mussel A. lucida was first determined (GenBank accession no. KF667529). The genome is 16,285 bp long with an AT content of 64.02%. All the 37 typical animal mitochondrial genes are found, including 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The genome also contains 24 unassigned regions, ranking from 1 to 830 bp in length, the largest of which is the putative control region (CR). The base composition of the genome is A (36.32%), G (13.01%), T (27.70%) and C (22.98%). Gene order is identical to other species of Unionidae except Gonideinae.

Complete maternal mitochondrial genome of freshwater mussel Aculamprotula tientsinensis (Bivalvia: Unionidae: Unioninae)

Abstract Aculamprotula tientsinensis is a rare and endemic species of freshwater mussel in China. This study firstly determined the complete F-type mitochondrial genome of A. tientsinensis. The circle genome (15 695 bp) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, 1 FORF gene. Except for cob, nad5 and nad6, the remaining protein-coding genes initiate with the orthodox start codon (ATG, ATA, ATT). There are 26 non-coding regions in the mitogenome of A. tientsinensis, ranging in size from 1 to 229 bp. The base composition of the genome is A (37.83%), G (12.69%), T (25.43%) and C (24.06%). Gene order is identical to other female species of Unionidae but for Gonideinae. The phylogenetic analyses of Unionidae indicate that A. tientsinensis is closely related to A. tortuosa and A. coreana, which belong to Unioninae. The complete mitogenome can deepen comparative and evolutionary genomics of Unionidae and be more comprehensive to parse the genetic relationship between the species and the ownership beyond species.

The complete F-type mitochondrial genome of threatened Chinese freshwater mussel Solenaia oleivora (Bivalvia: Unionidae: Gonideinae).

Abstract The freshwater mussel Solenaia oleivora is considered threatened due to anthropogenic habitat modification and pollution. The complete F-type mitochondrial genome (mitogenome) of S. oleivora was first determined (GenBank accession no. KF296320). The length of this mitogenome is 16,392 bp with an AT content of 59.9%. There are 37 typical animal mitochondrial genes and a putative control region. Within the control region, 5 tandem repeats of a 106 bp element were identified. The gene order rearrangement of cox2-rrnS in F-type mitogenome compared to that of other freshwater mussels, is not unique for S. oleivora, but is shared with all other available freshwater mussels in the subfamily Gonideinae. The complete mitogenome can provide a basic data for the further studies on molecular taxonomy, phylogenetic analyses and conservation genetics.

The complete maternal mitochondrial genome of rare Chinese freshwater mussel Lepidodesma languilati (Bivalvia: Unionidae: Unioninae).

Abstract Lepidodesma languilati is considered threatened because of the influence of human activities in China. The complete F-type mitochondrial genome of L. languilati was determined in this study (GenBank accession no. KT381195). It is a 15 754-bp-long circular molecule that consists of 37 genes that are typically found in other invertebrates. The overall base composition of the entire sequence is as follows: A (39.1%), T (25.7%), C (23.4%), and G (11.8%). Except for cox1 (TTG), cob (ATT), nad1 (ATT), nad6 (ATA), nad4 (TTG), and atp8 (GTG), 7 of the 13 protein-coding genes initiate with orthodox ATG start codon. All the 13 protein-coding genes have complete termination codon TAA or TAG. Phylogenetic tree indicates that L. languilati belongs to Unioninae. The newly sequenced complete mitogenome can provide basic data for comparative studies on mitochondrial genomes of Unionidae. It could also lay the important theoretical foundation for phylogenetics, population genetics, germplasm resources protection, sustainable, and reasonable utilization.

The complete F-type mitochondrial genome of Chinese Anodonta arcaeformis (Bivalvia: Unionidae: Anodontinae)

Abstract Anodonta arcaeformis is a Chinese common species. The complete F-type mitochondrial genome was first determined. The complete genome is 15,672 bp in length, with AT content 64.59%. All the 37 typical animal mitochondrial genes were identified, including 13 protein coding genes, 22 tRNA genes and 2 rRNA genes. And a novel FORF (277 bp, 92aa) was found between tRNAGlu and ND2, which was considered to be involved in sex determination. The putative control region (270 bp) is located between ND5 and tRNAGln, with an A + T content of 70.07%. The gene order is identical to other species of Unionidae female mitochondrial except Gonideinae.

Genetic structure and diversity of Nodularia douglasiae (Bivalvia: Unionida) from the middle and lower Yangtze River drainage

The Yangtze River drainage in China is among the most species rich rivers for freshwater mussels (order Unionida) on Earth with at least 68 species known. The freshwater mussels of the Yangtze River face a variety of threats with indications that species are declining in abundance and area of occupancy. This study represents the first analyses of the genetic structure and diversity for the common and widespread freshwater mussel Nodularia douglasiae based on microsatellite DNA genotypes and mitochondrial DNA sequences. Phylogenetic analysis a fragment of the COI mitochondrial gene indicated that N. douglasiae collected from across the middle and lower Yangtze River drainage are monophyletic with N. douglasiae from Japan, Russia, and South Korea. The results of the analysis of both the mtDNA and microsatellite datasets indicated that the seven collection locations of N. douglasiae in the middle and lower Yangtze River drainage showed high genetic diversity, significant genetic differentiation and genetic structure, and stable population dynamics over time. Moreover, we found that the connections among tributaries rivers and lakes in the Yangtze River drainage were important in maintaining gene flow among locations that N. douglasiae inhabits. An understanding of the genetic structure and diversity of a widespread species like N. douglasiae could be used as a surrogate to better understand the populations of other freshwater mussel species that are more rare in the Yangtze River drainage. At the same time, these results could provide a basis for the protection of genetic diversity and management of unionid mussels diversity and other aquatic organisms in the system.

Genetic diversity analysis of mitochondrial DNA control region in artificially propagated Chinese sucker Myxocyprinus asiaticus.

Abstract The genetic diversity of the three major artificially propagated populations of Chinese sucker, an endangered freshwater fish species, was investigated using the sequences of mitochondrial DNA (mtDNA) control regions. Among the 89 individuals tested, 66 variable sites (7.26%) and 10 haplotypes were detected (Haplotype diversity Hd = 0.805, Nucleotide diversity π = 0.0287). In general, genetic diversity was lower in artificially propagated populations than in wild populations. This reduction in genetic diversity may be due to population bottlenecks, genetic drift and human selection. A stepping-stone pattern of gene flow was detected in the populations studied, showing much higher gene flow between neighbouring populations. To increase the genetic diversity, wild lineages should be introduced, and more lineages should be shared among artificially propagated populations.

Reclassification of Lamprotula rochechouartii as Margaritifera rochechouartii comb. nov. (Bivalvia: Margaritiferidae) revealed by time-calibrated multi-locus phylogenetic analyses and mitochondrial phylogenomics of Unionoida

The family Margaritiferidae encompasses 12 valid species, which are distributed widely but disjunctively in the Northern Hemisphere. A lack of a well resolved and temporally calibrated phylogenetic framework of Margaritiferidae has made it difficult to discuss the evolutionary pattern and process. Phylogenetic relationships between five major clades, which were revealed in earlier studies, remain elusive and unresolved. Lamprotula rochechouartii has long been classified within the family Unionidae based on shell morphology, but our preliminary molecular study on this species made us hypothesize that it has an affinity with margaritiferids. Hence, five loci (COI, 16S, 18S, 28S and histone H3) were used to investigate the phylogenetic position of L. rochechouartii and intra-familial relationships within Margaritiferidae using various partitioning strategies. Moreover, two mitochondrial genomes were newly obtained to further resolve and validate the five-clade relationships within Margaritiferidae in a broad view of Unionoida evolution. Both five-gene and mitogenome datasets strongly advocated treating Lamprotula rochechouartii as Margaritifera rochechouartiicomb. nov. Maximum likelihood and Bayesian inference analyses using partitioned five-gene dataset resulted in various topologies, but five well-supported clades were obtained. The most probable cladistic relationships generated by five-gene dataset analyses were identical to subsequent whole mitogenome analyses except the position of M. monodonta. M. rochechouartii and M. laosensis had a well-supported sister relationship and formed a basal clade splitting from the rest of the family. Based on six reliable fossils, crown age of the extant Margaritiferidae was estimated during the Late Cretaceous at 88.3 Ma (95% HPD = 66.2-117.4). But we hypothesized a much earlier origin of this family due to the Permian stem age (mean = 257 Ma, 95% HPD = 230.0-296.0) and a high extinction rate in the whole order. Biogeographic scenarios supported a Laurasian origin of extant Margaritiferidae during the Late Cretaceous, and suggested that Asian margaritiferids may have had two origins, having either Asia (M. rochechouartii, M. laosensis) or North America (M. dahurica, M. laevis, and M. middendorffi) as ancestral. The newly added Margaritiferidae species M. rochechouartii expands our recognized distribution range of modern margaritiferids. Our results indicate that whole mitogenome sequences can be used to reconstruct robust phylogenetic relationships for freshwater mussels, especially with the help of adding M-type mitogenomes.

Towards a global phylogeny of freshwater mussels (Bivalvia: Unionida): Species delimitation of Chinese taxa, mitochondrial phylogenomics, and diversification patterns

The Yangtze River Basin in China is one of the global hotspots of freshwater mussel (order Unionida) diversity with 68 nominal species. Few studies have tested the validity of these nominal species. Some taxa from the Yangtze unionid fauna have not been adequately examined using molecular data and well-positioned phylogenetically with respect to the global Unionida. We evaluated species boundaries of Chinese freshwater mussels, and disentangled their phylogenetic relationships within the context of the global freshwater mussels based on the multi-locus data and complete mitochondrial genomes. Moreover, we produced the time-calibrated phylogeny of Unionida and explored patterns of diversification. COI barcode data suggested the existence of 41 phylogenetic distinct species from our sampled 40 nominal taxa inhabiting the middle and lower reaches of the Yangtze River. Maximum likelihood and Bayesian inference analyses on three loci (COI, 16S, and 28S) and complete mitochondrial genomes showed that the subfamily Unioninae sensu stricto was paraphyletic, and the subfamily Anodontinae should be subsumed under Unioninae. In addition, we described two new tribes (Aculamprotulini tribe nov. and Lepidodesmini tribe nov.) in the subfamily Unioninae and one new genus (Parvasolenaiagen. nov.) in the subfamily Gonideinae. Molecular dating analysis suggested freshwater mussels diversified at 346.1 Mya (HPD = 286.6-409.9). The global diversification rate for Unionida was estimated to be 0.025 species/Myr. Our study found only a single well-supported rate shift in Unionida diversification, occurring at the base of the subfamily Ambleminae. The evolution of active host-attraction may have triggered the burst of speciation in Ambleminae, and the environment and geography of the Mississippi River Basin likely sustained this radiation.