Scaphopoda is the sister taxon to Bivalvia: Evidence of ancient incomplete lineage sorting.

The almost simultaneous emergence of major animal phyla during the early Cambrian shaped modern animal biodiversity. Reconstructing evolutionary relationships among such closely spaced branches in the animal tree of life has proven to be a major challenge, hindering understanding of early animal evolution and the fossil record. This is particularly true in the species-rich and highly varied Mollusca where dramatic inconsistency among paleontological, morphological, and molecular evidence has led to a long-standing debate about the group's phylogeny and the nature of dozens of enigmatic fossil taxa. A critical step needed to overcome this issue is to supplement available genomic data, which is plentiful for well-studied lineages, with genomes from rare but key lineages, such as Scaphopoda. Here, by presenting chromosome-level genomes from both extant scaphopod orders and leveraging complete genomes spanning Mollusca, we provide strong support for Scaphopoda as the sister taxon of Bivalvia, revitalizing the morphology-based Diasoma hypothesis originally proposed 50 years ago. Our molecular clock analysis confidently dates the split between Bivalvia and Scaphopoda at ~520 Ma, prompting a reinterpretation of controversial laterally compressed Early Cambrian fossils, including Anabarella, Watsonella, and Mellopegma, as stem diasomes. Moreover, we show that incongruence in the phylogenetic placement of Scaphopoda in previous phylogenomic studies was due to ancient incomplete lineage sorting (ILS) that occurred during the rapid radiation of Conchifera. Our findings highlight the need to consider ILS as a potential source of error in deep phylogeny reconstruction, especially in the context of the unique nature of the Cambrian Explosion.

Phylogenomics of Bivalvia Using Ultraconserved Elements (UCEs) Reveal New Topologies for Pteriomorphia and Imparidentia.

Despite significant advances in phylogenetics over the past decades, the deep relationships within Bivalvia (phylum Mollusca) remain inconclusive. Previous efforts based on morphology or several genes have failed to resolve many key nodes in the phylogeny of Bivalvia. Advances have been made recently using transcriptome data, but the phylogenetic relationships within Bivalvia historically lacked consensus, especially within Pteriomorphia and Imparidentia. Here, we inferred the relationships of key lineages within Bivalvia using matrices generated from specifically designed ultraconserved elements (UCEs) with 16 available genomic resources and 85 newly sequenced specimens from 55 families. Our new probes (Bivalve UCE 2k v.1) for target sequencing captured an average of 849 UCEs with 1085-bp in mean length from in vitro experiments. Our results introduced novel schemes from six major clades (Protobranchina, Pteriomorphia, Palaeoheterodonta, Archiheterodonta, Anomalodesmata and Imparidentia), though some inner nodes were poorly resolved, such as paraphyletic Heterodonta in some topologies potentially due to insufficient taxon sampling. The resolution increased when analyzing specific matrices for Pteriomorphia and Imparidentia. We recovered three Pteriomorphia topologies different from previously published trees, with the strongest support for ((Ostreida + (Arcida + Mytilida)) + (Pectinida + (Limida + Pectinida))). Limida were nested within Pectinida, warranting further studies. For Imparidentia, our results strongly supported the new hypothesis of (Galeommatida + (Adapedonta + Cardiida)), while the possible non-monophyly of Lucinida was inferred but poorly supported. Overall, our results provide important insights into the phylogeny of Bivalvia and show that target enrichment sequencing of UCEs can be broadly applied to study both deep and shallow phylogenetic relationships.

Comparative mitochondrial genome analysis provides new insights into the classification of Modiolinae.

BACKGROUND: Mitochondrial genomes have become a powerful tool for studying molecular genetics and phylogeny of mollusks. Currently, the position of Modiolinae within Mytilidae and the taxonomic and phylogenetic relationships within Modiolinae were still controversial. This study focuses on the complete mitochondrial genomes of two species: Modiolus modulaides (Röding, 1798) and Modiolus auriculatus Krauss, 1848, which have not been sequenced before. METHODS AND RESULTS: We assembled and characterized the mitochondrial genomes of M. modulaides and M. auriculatus and then analyzed the phylogenetic relationships. The mitochondrial genomes of M. modulaides and M. auriculatus were 15,422 bp and 16,027 bp, respectively. Both of them were composed of 36 functional genes, including 12 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. All protein-coding genes showed A + T bias, positive GC skews, and negative AT skews in nucleotide composition. Phylogenetic analysis based on the mitochondrial genomes showed that Modiolinae and Bathymodiolinae clustered together to form a sister relationship. Seven Modiolinae species were divided into two clades: L1 (M. modulaides, M. auriculatus and Modiolus philippinarum Hanley, 1843) and L2 [Modiolus modiolus (Linnaeus, 1758), Modiolus kurilensis Bernard, 1983, Modiolus nipponicus (Oyama, 1950), and Modiolus comptus (Sowerby III, 1915)]. The divergence time of the two clades was approximately 105.75 Ma. Furthermore, the transfer RNA gene rearrangement, longer genetic distance, and greater genetic differentiation were confirmed between the L1 and L2 clades, as well as differences in the external characteristics of the shells of the two clades. CONCLUSIONS: Based on the molecular data, it was speculated that species from the L1 clade might belong to other genera or new genera. This study provides molecular information for further taxonomic and phylogenetic studies of Mytilidae.

Construction and characterization of microsatellite markers for the Neptune whelk, Neptunea cumingii.

BACKGROUND: The Neptune whelk, Neptunea cumingii, is an economic gastropod endemic to the East Asia Warm Temperate Biotic Subregion. The study of the development in microsatellite markers maybe beneficial for assessing the genetic diversity and conservation of resources on this dwindling species. METHODS AND RESULTS: The microsatellite markers were constructed and characterized through Illumina high-throughput sequencing and capillary electrophoresis techniques. Eleven polymorphic microsatellite loci were screened and validated. The observed heterozygosity and expected heterozygosity of each locus showed a range from 0.0600 to 0.6508 and from 0.7380 to 0.9375, respectively. The average Shannon's information index and polymorphism information content were 2.0828 and 0.8325, respectively. Deviation from all loci was generated from Hardy-Weinberg equilibrium. CONCLUSIONS: The 11 microsatellite markers developed in this study show polymorphic status. These markers are expected to be highly informative for further analysis of genetic diversity in N. cumingii.

Complete mitochondrial genome of Cultellus attenuatus and its phylogenetic implications.

BACKGROUND: The mitochondrial genomes of three species in Solenoidea of Heterodonta have been reported, but the mitochondrial genes and phylogenetic relationships of Cultellus attenuatus, which also belongs to this superfamily and has high economic value, are unknown. METHODS AND RESULTS: The complete mitochondrial genome of C. attenuatus was sequenced and compared with mitogenomes of seven species of Heterodonta bivalve mollusks in GenBank. The mitochondrial genome of C. attenuatus has a length of 16,888 bp and contains 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs. In comparison with C. attenuates, the mitochondrial genes of Sinonovacula constricta from the same family were not rearranged, but those of six other species from different families were rearranged to different degrees. The location, size, and composition of the largest noncoding regions in eight species suggested a closer relationship between C. attenuatus and S. constricta. The phylogenetic analysis showed that C. attenuatus and S. constricta belonging to Cultellidae cluster into one branch and that two species of Solenidae (Solen grandis and Solen strictus) clustered as their sister taxa. CONCLUSIONS: Overall, we used mitochondrial genome data to demonstrate that C. attenuatus and S. constricta exhibit the closest relationship in Heterodonta. These data and analyses provide new insights into the phylogenetic relationships in Heterodonta.

Reconstruction of the evolutionary biogeography reveal the origins and diversification of oysters (Bivalvia: Ostreidae).

Oysters (Bivalvia: Ostreidae Rafinesque, 1815) live in the intertidal and shallow subtidal areas worldwide. Despite their long evolutionary histories, abundant fossil records, global distribution, and ecological significance, a systematic time-dependent biogeographical analysis of this family is still lacking. Using combined mitochondrial (COI and 16S rRNA) and nuclear (18S rRNA, 28S rRNA, H3 and ITS2) gene makers for 80% (70/88) of the recognized extant Ostreidae, we reconstructed the global phylogenetic and biogeographical relationships throughout the evolutionary history of oysters. The result provided a holistic view of the origin, migration and dispersal patterns of Ostreidae. The phylogenetic results and fossil evidence indicated that Ostreidae originated from the circum-Arctic region in the Early Jurassic. The widening of the Atlantic Ocean and changes in the Tethys Ocean further facilitated their subsequent diversification during the Cretaceous and the Palaeogene periods. In particular, Crassostrea and Saccostrea exhibited relatively low dispersal abilities and their major diversifications were consistent with the tectonic events. Environmental adaptations and reproductive patterns, therefore, should play key roles in the formation of oyster distribution patterners, rather than the dispersal ability of their planktonic larvae. The diversity dynamics inferred by standard phylogenetic are consistent with the fossil record, however, further systematic classification, especially for fossil genus Ostrea, would enhance our understanding on extant and fossil oysters. The present study of the historical biogeography of oysters provides new insights into the evolution and speciation of oysters. Our findings also provide a foundation for the assessment of evolutionary patterns and ecological processes in intertidal and inshore life.

Integrated Metagenomic and Metabolomic Analysis on Two Competing Mussels, Mytella strigata and Perna viridis, in China.

Biological invasion is a primary direct driver of biodiversity loss. Recently, owing to exploitation competition with an invasive mussel, Mytella strigata (Hanley, 1843), there has been a drastic decrease in the population of native Perna viridis (Linnaeus, 1758) in several western Pacific regions. In the present study, intestinal microbiota, metabolome, and key digestive enzyme activities were compared between the two competing mussels, M. strigata and P. viridis, to elucidate the differences in intestinal microbiota and metabolic points. We observed that Proteobacteria, Firmicutes, and Bacteroidota were the three predominant bacterial phyla in the two species. The relative abundance of Bacteroidota related to carbohydrate-degrading ability was significantly higher in M. strigata than in P. viridis. Compared to P. viridis, different metabolites including maltose and trehalose were enriched in M. strigata. Lastly, higher carbohydrases activities of alpha-amylase, cellulase, and xylanase were observed in M. strigata than in P. viridis. These differences might play an important role in the adaptation process of M. strigata to the new environment. This study provides important basic knowledge for investigating the competition between M. strigata and P. viridis in terms of food resources utilization.

Sequencing of the Complete Mitochondrial Genome of the Big Brown Mactra Clam, Mactra grandis (Venerida: Mactridae).

Mitochondrial genomes are playing an increasingly important role in molluscan taxonomy, germplasm, and evolution studies. The first complete mitochondrial genome of the commercial big brown mactra clam, Mactra grandis, was characterized using Illumina next-generation sequencing in this study. The 17,289 bp circular genome has a typical gene organization of 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs, with an obvious (A + T)-bias of 64.54%. All PCGs exhibited a homogeneous bias in nucleotide composition with a (A + T)-bias, a positive GC skew, and a negative AT skew. Results of phylogenetic analysis showed that Mactra grandis was most closely related to Mactra cygnus. The functional gene arrangement of the two species was identical but different from other Mactra species. The congeneric relationships among Mactra species were demonstrated by genetic distance analysis. Additionally, the selective pressure analysis suggested that cox1 was highly efficient for discriminating closely related species in genus Mactra, while nad2 was the most appropriate marker for population genetic analysis.

Network Pharmacology Analysis on the Mechanism of Xihuangwan in Treating Rectal Cancer and Radiation Enteritis.

BACKGROUND: Recent studies have shown that XihuangWan (XHW) is a kind of Chinese medicine with significant anti-tumor and anti-inflammatory activities. However, its mechanism for preventing and treating radiation proctitis in rectal cancer patients during radiotherapy remains unclear. METHODS: This study employed the network pharmacology to establish a "drug-active ingredient-target genedisease" network via using TCMSP, SymMap, GeneCard, and OMIM databases. The PPI network was conducted by the String tool. The core targets of XHW in the treatment of rectal cancer and radiation enteritis were identified by topological analysis, and the functional annotation analysis and pathway enrichment analysis were performed. RESULTS: A total of 61 active ingredients of XHW ingredients, 4607 rectal cancer-related genes, 5803 radiation enteritis-related genes, and 68 common targets of XHW in the treatment of rectal cancer and radiation enteritis were obtained. PTGS1 and NR3C2, as identified potential targets, were significantly associated with OS of colorectal cancer patients. GO and KEGG enrichment analysis showed that bioinformatics annotation of these common genes was mainly involved in DNA-binding transcription factor, PI3K/Akt, TNF, HIF-1 signaling pathway, and colorectal cancer pathway. CONCLUSION: The active ingredients of XHW, mainly including Quercetin, Ellagic acid, and Stigmasterol, might act on common targets of rectal cancer and radiation enteritis, such as PTGS1, NR3C2, IL-6, EGFR, HIF-1A, CASP3, BCL2, ESR1, MYC, and PPARG, and regulate multiple signaling pathways like PI3K-Akt, TNF, and HIF-1 to inhibit tumor proliferation, tumor angiogenesis, inflammatory responses, and oxidative stress, thereby achieving prevention and treatment of radiation enteritis in rectal cancer patients during radiotherapy. It provided an important reference for further elucidating the anti-inflammation and anti-tumor mechanism and clinical application of XHW.

The complete mitochondrial genome of a fouling mussel, Xenostrobus atratus (Mollusca: Mytilidae), and its phylogenetic implication.

In this study, we report the female-lineage mitochondrial genome of Xenostrobus atratus for the first time. The circular mitochondrial genome is 14,806 bp in length and contains 12 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. All genes are encoded on the heavy strand. The genome composition is A + T biased (66.6%), with 25.2% A, 41.4% T, 21.7% G and 11.7% C. A Bayesian inference (BI) phylogenetic tree was constructed based on the mitochondrial genomes of X. atratus and 46 other Mytilidae species. Our results demonstrate that X. atratus and Limnoperna fortunei have distinct lineages, opposing synonymizing Xenostrobus within Limnoperna. According to this study, the validity of the subfamily Limnoperninae and genus Xenostrobus is strongly supported. However, there is still an urgent need for more mitochondrial data to decide to which subfamily X. atratus belongs.

Genetic Analysis Based on Mitochondrial nad2 Gene Reveals a Recent Population Expansion of the Invasive Mussel, Mytella strigata, in China.

Mytella strigata is a highly adaptable invasive alien species that has been established in coastal China since 2014. Mitochondrial DNA (mtDNA) is an important tool for studying the evolution and population genetics of invasive species. In this study, the mitochondrial genome of M. strigata from China was sequenced by Illumina high-throughput sequencing and characterized with 13 protein-coding genes (PCGs). By assessing the selective pressure of 13 PCGs, the nad2 gene had the fastest evolutionary rate and was finally selected for population genetic analysis. A total of 285 nad2 sequences from seven M. strigata populations in China were analyzed and showed obviously T-rich and C-rich characteristics. According to population genetic diversity analysis, all the seven populations had haplotype (gene) diversity (Hd) ≥ 0.5 and nucleotide diversity (Pi) < 0.005. Haplotype networks showed a "star" distribution. Population historical dynamic analyses showed that Fu's Fs and Tajima's D values of all populations were negative except the Qukou (QK) and Beihai (BH) populations. The Zhangzhou (ZJ) and Xiamen (XM) populations were unimodal while the other populations were multimodal. These results suggested that the population of M. strigata in China may have passed the bottleneck period and is currently in a state of population expansion.

Comparative analysis of the mitochondrial genomes of the family Mactridae (Mollusca: Venerida) and their phylogenetic implications.

Taxonomy and phylogenetic relationships within the family Mactridae have remained debatable because of the plasticity of morphological characteristics and the lack of accurate molecular data, thereby resulting in abundant synonyms and taxa rearrangements. Mitochondrial genomes (mitogenomes) have been widely used as powerful tools to reconstruct phylogenies of various groups of mollusks; however, they have not been used for studying the phylogeny of mactrids specifically. In the present study, mitogenomes of seven Mactridae species, namely Mactra chinensis, Mactra cygnus, Mactra quadrangularis, Mactra cumingii, Mactrinula dolabrata, Raeta pulchella, and Raeta sp., were sequenced by Illumina high-throughput sequencing, and a comparative mitochondrial genomic analysis was conducted. The newly sequenced mitogenomes were double-stranded circular molecules, with all functional genes encoded on the heavy strand. All the new mactrid mitogenomes had two rRNA genes (12S and 16S), 13 protein-coding genes (PCGs) (atp6, cox1, cox2, cox3, cytb, nad1, nad2, nad3, nad4, nad4l, nad5, nad6, and atp8), and 22 tRNAs. The mitogenomes showed considerable variation in AT content, GC skew, and AT skew. The results of the phylogenetic analysis confirmed monophyly of the family Mactridae and suggested that genera Mactrinula, Spisula, Rangia, and Mulinia should not be placed under subfamily Mactrinae. Our results supported that potential cryptic species existed in Mactra antiquata. We also proposed subfamily Kymatoxinae should belong to the family Mactridae rather than Anatinellidae and Mactra alta in China should be Mactra cygnus. Additionally, conservation in functional gene arrangement was found in genera Mactra, Raeta, and Lutraria. But gene orders in S. sachalinensis and S. solida were quite different, questioning their congeneric relationship. Our results further suggested that the taxonomy within the family Mactridae requires an integrative revision.

The Complete Mitochondrial Genome of Entemnotrochus rumphii, a Living Fossil for Vetigastropoda (Mollusca: Gastropoda).

Pleurotomarioidea represents a truly isolated and basally diverging lineage in Vetigastropoda (Mollusca: Gastropoda) whose fossil record can date back to the late Cambrian, thus providing rare insights into the evolutionary history of molluscs. Here, we sequenced and assembled the complete mitochondrial genome of one representative species from Pleurotomarioidea-Entemnotrochus rumphii (Schepman, 1879)-of which the mitogenome is 15,795 bp in length, including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The nucleotide composition was biased toward AT, and A + T content reached 65.2%. E. rumphii was recovered as sister to all other living vetigastropods according to mitogenome-based phylogenetic analysis. The mitochondrial gene order was consistent with major vetigastropods and the hypothetical ancestral gastropoda, suggesting the deep conservation of mitogenome arrangement in Vetigastropoda.

The complete mitochondrial genome of a marine mussel, Modiolus comptus (Mollusca: Mytilidae), and its phylogenetic implication.

In this study, the complete mitochondrial genome of Modiolus comptus was determined and annotated for the first time. The 15,591 bp circular genome has a base composition of 24.3% A, 38.6% T, 12.5% C, and 24.5% G, demonstrating a bias of higher AT content (63.0%) than GC content (27.0%). The mitochondrial genome contains 12 protein-coding genes (PCGs), 20 transfer RNA genes (tRNA), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and one control region. All genes of M. comptus were encoded on the heavy strand, except trnT(ugu) gene. The whole mitochondrial genome of M. comptus and 21 mitogenomes of other Mytilidae species were used for phylogenetic analysis. The result indicated the newly sequenced species had the closest relationship with Modiolus nipponicus (MK721547) and was clustered within the clade of genus Modiolus.

Transcriptomic analysis of Crassostrea sikamea × Crassostrea angulata hybrids in response to low salinity stress.

Hybrid oysters often show heterosis in growth rate, weight, survival and adaptability to extremes of salinity. Oysters have also been used as model organisms to study the evolution of host-defense system. To gain comprehensive knowledge about various physiological processes in hybrid oysters under low salinity stress, we performed transcriptomic analysis of gill tissue of Crassostrea sikamea ♀ × Crassostrea angulata♂ hybrid using the deep-sequencing platform Illumina HiSeq. We exploited the high-throughput technique to delineate differentially expressed genes (DEGs) in oysters maintained in hypotonic conditions. A total of 199,391 high quality unigenes, with average length of 644 bp, were generated. Of these 35 and 31 genes showed up- and down-regulation, respectively. Functional categorization and pathway analysis of these DEGs revealed enrichment for immune mechanism, apoptosis, energy metabolism and osmoregulation under low salinity stress. The expression patterns of 41 DEGs in hybrids and their parental species were further analyzed by quantitative real-time PCR (qRT-PCR). This study will serve as a platform for subsequent gene expression analysis regarding environmental stress. Our findings will also provide valuable information about gene expression to better understand the immune mechanism, apoptosis, energy metabolism and osmoregulation in hybrid oysters under low salinity stress.

[Effects of ggpS over-expression on glycosylglycerol and glycerol biosynthesis of Synechocystis sp. PCC 6803].

To study the roles of glucosylglycerol phosphate synthase (Ggps) in glucosylglycerol (GG) and glycerol biosynthesis, we over-expressed Ggps from either Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 in a Synechocystis strain with a high GG titer, and determined the GG and glycerol accumulation in the resultant mutants grown under different NaCl-stress conditions. Ion chromatography results revealed that GG yield was not improved, but glycerol production was significantly enhanced by over-expression of Ggps from Synechocystis sp. PCC 6803 (6803ggpS). In addition, increasing the NaCl concentration of medium from 600 to 900 mmol/L led to a further 75% increase of glycerol accumulation in the mutant strain with 6803ggpS over-expression. These findings show the role of ggpS in driving the carbon flux to the glycerol biosynthesis pathway, and will be helpful for further improvement of GG and glycerol production in Synechocystis.