Genome-wide insights into introgression and its consequences for genome-wide heterozygosity in the Mytilus species complex across Europe.

The three mussel species comprising the Mytilus complex are widespread across Europe and readily hybridize when they occur in sympatry, resulting in a mosaic of populations with varying genomic backgrounds. Two of these species, M. edulis and M. galloprovincialis, are extensively cultivated across Europe, with annual production exceeding 230,000 tonnes. The third species, M. trossulus, is considered commercially damaging as hybridization with this species results in weaker shells and poor meat quality. We therefore used restriction site associated DNA sequencing to generate high-resolution insights into the structure of the Mytilus complex across Europe and to resolve patterns of introgression. Inferred species distributions were concordant with the results of previous studies based on smaller numbers of genetic markers, with M. edulis and M. galloprovincialis predominating in northern and southern Europe respectively, while introgression between these species was most pronounced in northern France and the Shetland Islands. We also detected traces of M. trossulus ancestry in several northern European populations, especially around the Baltic and in northern Scotland. Finally, genome-wide heterozygosity, whether quantified at the population or individual level, was lowest in M. edulis, intermediate in M. galloprovincialis, and highest in M. trossulus, while introgression was positively associated with heterozygosity in M. edulis but negatively associated with heterozygosity in M. galloprovincialis. Our study will help to inform mussel aquaculture by providing baseline information on the genomic backgrounds of different Mytilus populations across Europe and by elucidating the effects of introgression on genome-wide heterozygosity, which is known to influence commercially important traits such as growth, viability, and fecundity in mussels.

Deciphering mollusc shell production: the roles of genetic mechanisms through to ecology, aquaculture and biomimetics.

Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing (RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are still debated, with CaCO3 precipitation estimates ranging from 1-2 J/mg to 17-55 J/mg depending on experimental and environmental conditions. However, organic components are more expensive (~29 J/mg) and recent data indicate transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi) and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1 gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs. Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.

Population Genetic Structure is Unrelated to Shell Shape, Thickness and Organic Content in European Populations of the Soft-Shell Clam Mya Arenaria.

The soft-shell clam Mya arenaria is one of the most ancient invaders of European coasts and is present in many coastal ecosystems, yet little is known about its genetic structure in Europe. We collected 266 samples spanning a latitudinal cline from the Mediterranean to the North Sea and genotyped them at 12 microsatellite loci. In parallel, geometric morphometric analysis of shell outlines was used to test for associations between shell shape, latitude and genotype, and for a selection of shells we measured the thickness and organic content of the granular prismatic (PR), the crossed-lamellar (CL) and the complex crossed-lamellar (CCL) layers. Strong population structure was detected, with Bayesian cluster analysis identifying four groups located in the Mediterranean, Celtic Sea, along the continental coast of the North Sea and in Scotland. Multivariate analysis of shell shape uncovered a significant effect of collection site but no associations with any other variables. Shell thickness did not vary significantly with either latitude or genotype, although PR thickness and calcification were positively associated with latitude, while CCL thickness showed a negative association. Our study provides new insights into the population structure of this species and sheds light on factors influencing shell shape, thickness and microstructure.

RAD sequencing sheds new light on the genetic structure and local adaptation of European scallops and resolves their demographic histories.

Recent developments in genomics are advancing our understanding of the processes shaping population structure in wild organisms. In particular, reduced representation sequencing has facilitated the generation of dense genetic marker datasets that provide greater power for resolving population structure, investigating the role of selection and reconstructing demographic histories. We therefore used RAD sequencing to study the great scallop Pecten maximus and its sister species P. jacobeus along a latitudinal cline in Europe. Analysis of 219 samples genotyped at 82,439 single nucleotide polymorphisms clearly resolved an Atlantic and a Norwegian group within P. maximus as well as P. jacobeus, in support of previous studies. Fine-scale structure was also detected, including pronounced differences involving Mulroy Bay in Ireland, where scallops are commercially cultured. Furthermore, we identified a suite of 279 environmentally associated loci that resolved a contrasting phylogenetic pattern to the remaining neutral loci, consistent with ecologically mediated divergence. Finally, demographic inference provided support for the two P. maximus groups having diverged during the last glacial maximum and subsequently expanded, whereas P. jacobeus diverged around 95,000 generations ago and experienced less pronounced expansion. Our results provide an integrative perspective on the factors shaping genome-wide differentiation in a commercially important marine invertebrate.

2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador.

BACKGROUND: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. METHODOLOGY/PRINCIPAL FINDINGS: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment.