Population genetics and morphometric variation of blue mussels in the western Baltic Sea

The investigation of hybridizing taxa can provide intriguing insights into the process of speciation and the adaptive potential of populations to environmental change. Due to its gradual nature in terms of ecology and genetics, the European blue mussel hybrid zone between North Sea Mytilus edulis and Baltic M. trossulus is an ideal study system to analyse the significance of inter-specific hybridization in evolution. Although much research has already focussed on the genetic and phenotypic structures in the Baltic Proper, less is known about the situation in the western transition area. By means of a multi-locus genotypic assessment and multivariate morphometric analyses I have examined the genetic and morphological constitutions of several Baltic Mytilus populations. Paying special attention to the poorly resolved outer parts, I find that most blue mussels in the western Baltic Sea and Skagerrak are higher generation backcross hybrids (99 % in the Kiel Fjord; 81 % in Tjärnö). While my results suggest that gene variants of M. edulis still prevail over that of M. trossulus in these mytilids, they also demonstrate that a minority of individuals in the Kiel Fjord are introgressed by M. galloprovincialis alleles. Although the origin of these alien polymorphisms cannot be told from my data, I postulate that their movement into the Baltic gene pool is most likely human-mediated (e.g. ship traffic). Considering the increasing records of anthropogenic introductions of Mytilus species to non-native habitats and the limited power of the applied molecular markers to discriminate M. galloprovincialis, it is to expect that more cryptic invasions are detected in future, technically advanced investigations. My analysis of M. edulis allele frequency changes from the North Sea to the Baltic Proper extends and supports an earlier work by Stuckas et al. (2009), providing evidence for discordant patterns of gene flow across the hybrid zone. These discrepancies probably arise from the concerted action of direct selection, genetic hitchhiking, stochastic evolutionary forces and shifts in the geographical position of the secondary contact area. Different to this previous study and in line with the observations by Kijewski et al. (2006), I find that introgression into the central Baltic is restricted at least for some M. edulis maternal haplotypes (D-loop), indicative of cytonuclear incompatibilities between inner and outer Baltic mussels. While differential environmental pressures might contribute to the maintenance of semi-permeable genetic barriers between M. edulis-like and M. trossulus-like hybrid populations, they can also account for the observed dissimilarities in shell morphology and phenotypic diversity. In contrast to individuals of the Baltic Proper, which express a Abstract | 7 population-specific, M. trossulus-like morphotype, mussels of the transition zone build a morphological continuum between parental forms. Following the reasoning by Gardner (1996) I propose that the strong environmental variability in the western Baltic selects for multiple phenotypes, while the constant and extreme conditions (e.g. low salinities, absence of predators) in the eastern Baltic favour only a single shell morphotype. It remains to be shown whether these opposing patterns are consequences of environmentally-induced discrepancies in genetic variability at fixed causal loci (level of heterozygosity, selection for multiple alleles) and/or differences in phenotypic plasticity

Population structure and connectivity in Indo-Pacific deep-sea mussels of the Bathymodiolus septemdierum complex

Current pressures to mine polymetallic sulfide deposits pose threats to the animal communities found at deep-sea hydrothermal vents. Management plans aimed at preserving these unusual communities require knowledge of historical and contemporary forces that shaped the distributions and connectivity of associated species. As most vent research has focused on the eastern Pacific and mid-Atlantic ridge systems less is known about Indo-Pacific vents, where mineral extraction activities are imminent. Deep-sea mussels (Bivalvia: Mytilidae) of the genus Bathymodiolus include the morphotypic species B. septemdierum, B. brevior, B. marisindicus, and B. elongatus which are among the dominant vent taxa in western Pacific back-arc basins and the Central Indian Ridge. To assess their interpopulational relationships, we examined multilocus genotypes based on DNA sequences from four nuclear and four mitochondrial genes, and allozyme variation encoded by eleven genes. Bayesian assignment methods grouped mussels from seven widespread western Pacific localities into a single cluster, whereas the Indian Ocean mussels were clearly divergent. Thus, we designate two regional metapopulations. Notably, contemporary migration rates among all sites appeared to be low despite limited population differentiation, which highlights the necessity of obtaining realistic data on recovery times and fine-scale population structure to develop and manage conservation units effectively. Future studies using population genomic methods to address these issues in a range of species will help to inform management plans aimed at mitigating potential impacts of deep-sea mining in the Indo-Pacific region

Intra-host symbiont diversity in eastern Pacific cold seep tubeworms identified by the 16S-V6 region, but undetected by the 16S-V4 region

Vestimentiferan tubeworms are key taxa in deep-sea chemosynthetic habitats worldwide. As adults they obtain their nutrition through their sulfide-oxidizing bacterial endosymbionts, which are acquired from the environment. Although horizontal transmission should favor infections by various symbiotic microbes, the current paradigm holds that every tubeworm harbors only one endosymbiotic 16S rRNA phylotype. Although previous studies based on traditional Sanger sequencing have questioned these findings, population level high-throughput analyses of the symbiont 16S diversity are still missing. To get further insights into the symbiont genetic variation and uncover hitherto hidden diversity we applied state-of-the-art 16S-V4 amplicon sequencing to populations of the co-occurring tubeworm species Lamellibrachia barhami and Escarpia spicata that were collected during E/V Nautilus and R/V Western Flyer cruises to cold seeps in the eastern Pacific Ocean. In agreement with earlier work our sequence data indicated that L. barhami and E. spicata share one monomorphic symbiont phylotype. However, complementary CARD-FISH analyses targeting the 16S-V6 region implied the existence of an additional phylotype in L. barhami. Our results suggest that the V4 region might not be sufficiently variable to investigate diversity in the intra-host symbiont population at least in the analyzed sample set. This is an important finding given that this region has become the standard molecular marker for high-throughput microbiome analyses. Further metagenomic research will be necessary to solve these issues and to uncover symbiont diversity that is hidden below the 16S rRNA level

Population connectivity and speciation of vent mussels from the Mid-Atlantic Ridge: An interdisciplinary approach

Knowledge about the genetic connectivity of populations and the mechanisms underlying speciation is an important requirement for biodiversity conservation. However, despite the fact that such information is generally sparse for deep sea biota, the demands for mining of seafloor mineral resources are increasing. Using mussels of the genus Bathymodiolus as a model system and integrating expertise from various research disciplines, the present study aims to elucidate patterns of geneflow between hydrothermal vent populations of the Mid-Atlantic Ridge. To characterize the level of genetic exchange on ecological time scales, we will genotype mussels with species- diagnostic SNP markers designed from high-throughput transcriptomic data. By performing hybridization experiments, we will study patterns of reproductive isolation between B. azoricus and B. childressi from shallower sites. Moreover, we will measure larval survival and behaviour of B. azoricus under a variety of environmental conditions to identify factors that influence the spreading of propagules between vents. The results from the ecophysiological experiments will be incorporated into larval dispersal studies performed with Lagrangian simulations utilizing a high-resolution model of the global ocean under realistic forcing. By comparing the outcomes of the genetic, biophysical and ecological approaches, we expect that this work will make fundamental contributions to a better understanding of population connectivity and diversification in the deep sea as well as help to manage anthropogenic threats to hydrothermal vent ecosystems

Deep-sea mussels from a hybrid zone on the Mid-Atlantic Ridge host genetically indistinguishable symbionts

The composition and diversity of animal microbiomes is shaped by a variety of factors, many of them interacting, such as host traits, the environment, and biogeography. Hybrid zones, in which the ranges of two host species meet and hybrids are found, provide natural experiments for determining the drivers of microbiome communities, but have not been well studied in marine environments. Here, we analysed the composition of the symbiont community in two deep-sea, Bathymodiolus mussel species along their known distribution range at hydrothermal vents on the Mid-Atlantic Ridge, with a focus on the hybrid zone where they interbreed. In-depth metagenomic analyses of the sulphur-oxidising symbionts of 30 mussels from the hybrid zone, at a resolution of single nucleotide polymorphism analyses of ~2500 orthologous genes, revealed that parental and hybrid mussels (F2–F4 generation) have genetically indistinguishable symbionts. While host genetics does not appear to affect symbiont composition in these mussels, redundancy analyses showed that geographic location of the mussels on the Mid-Atlantic Ridge explained most of the symbiont genetic variability compared to the other factors. We hypothesise that geographic structuring of the free-living symbiont population plays a major role in driving the composition of the microbiome in these deep-sea mussels

Allopatric and Sympatric Drivers of Speciation in Alviniconcha Hydrothermal Vent Snails

Despite significant advances in our understanding of speciation in the marine environment, the mechanisms underlying evolutionary diversification in deep-sea habitats remain poorly investigated. Here, we used multigene molecular clocks and population genetic inferences to examine processes that led to the emergence of the six extant lineages of Alviniconcha snails, a key taxon inhabiting deep-sea hydrothermal vents in the Indo-Pacific Ocean. We show that both allopatric divergence through historical vicariance and ecological isolation due to niche segregation contributed to speciation in this genus. The split between the two major Alviniconcha clades (separating A. boucheti and A. marisindica from A. kojimai, A. hessleri, and A. strummeri) probably resulted from tectonic processes leading to geographic separation, whereas the splits between co-occurring species might have been influenced by ecological factors, such as the availability of specific chemosynthetic symbionts. Phylogenetic origin of the sixth species, Alviniconcha adamantis, remains uncertain, although its sister position to other extant Alviniconcha lineages indicates a possible ancestral relationship. This study lays a foundation for future genomic studies aimed at deciphering the roles of local adaptation, reproductive biology, and host–symbiont compatibility in speciation of these vent-restricted snails

Combined genotyping, microbial diversity and metabolite profiling studies on farmed Mytilus spp. from Kiel Fjord

The blue mussel Mytilus is a popular food source with high economical value. Species of the M. edulis complex (M. edulis, M. galloprovincialis and M. trossulus) hybridise whenever their geographic ranges overlap posing difficulties to species discrimination, which is important for blue mussel aquaculture. The aim of this study was to determine the genetic structure of farmed blue mussels in Kiel Fjord. Microbial and metabolic profile patterns were studied to investigate a possible dependency on the genotype of the bivalves. Genotyping confirmed the complex genetic structure of the Baltic Sea hybrid zone and revealed an unexpected dominance of M. trossulus alleles being in contrast to the predominance of M. edulis alleles described for wild Baltic blue mussels. Culture-dependent and -independent microbial community analyses indicated the presence of a diverse Mytilus-associated microbiota, while an LC-MS/MS-based metabolome study identified 76 major compounds dominated by pigments, alkaloids and polyketides in the whole tissue extracts. Analysis of mussel microbiota and metabolome did not indicate genotypic dependence, but demonstrated high intraspecific variability of farmed mussel individuals. We hypothesise that individual differences in microbial and metabolite patterns may be caused by high individual plasticity and might be enhanced by e.g. nutritional condition, age and gender

Host hybridization as a potential mechanism of lateral symbiont transfer in deep‐sea vesicomyid clams

Deep‐sea vesicomyid clams live in mutualistic symbiosis with chemosynthetic bacteria that are inherited through the maternal germ line. On evolutionary timescales, strictly vertical transmission should lead to cospeciation of host mitochondrial and symbiont lineages; nonetheless, examples of incongruent phylogenies have been reported, suggesting that symbionts are occasionally horizontally transmitted between host species. The current paradigm for vesicomyid clams holds that direct transfers cause host shifts or mixtures of symbionts. An alternative hypothesis suggests that hybridization between host species might explain symbiont transfers. Two clam species, Archivesica gigas and Phreagena soyoae, frequently co‐occur at deep‐sea hydrocarbon seeps in the eastern Pacific Ocean. Although the two species typically host gammaproteobacterial symbiont lineages marked by divergent 16S rRNA phylotypes, we identified a number of clams with the A. gigas mitotype that hosted symbionts with the P. soyoae phylotype. Demographic inference models based on genome‐wide SNP data and three Sanger sequenced gene markers provided evidence that A. gigas and P. soyoae hybridized in the past, supporting the hypothesis that hybridization might be a viable mechanism of interspecific symbiont transfer. These findings provide new perspectives on the evolution of vertically transmitted symbionts and their hosts in deep‐sea chemosynthetic environments

Differential patterns of connectivity in Western Pacific hydrothermal vent metapopulations: A comparison of biophysical and genetic models

Hydrothermal ecosystems face threats from planned deep-seabed mining activities, despite the fact that patterns of realized connectivity among vent-associated populations and communities are still poorly understood. Since populations of vent endemic species depend on larval dispersal to maintain connectivity and resilience to habitat changes, effective conservation strategies for hydrothermal ecosystems should include assessments of metapopulation dynamics. In this study, we combined population genetic methods with biophysical models to assess strength and direction of gene flow within four species of the genus Alviniconcha (A. boucheti, A. kojimai, A. strummeri and A. hessleri) that are ecologically dominant taxa at Western Pacific hydrothermal vents. In contrast to predictions from dispersal models, among-basin migration in A. boucheti occurred predominantly in an eastward direction, while populations within the North Fiji Basin were clearly structured despite the absence of oceanographic barriers. Dispersal models and genetic data were largely in agreement for the other Alviniconcha species, suggesting limited between-basin migration for A. kojimai, lack of genetic structure in A. strummeri within the Lau Basin and restricted gene flow between northern and southern A. hessleri populations in the Mariana back-arc as a result of oceanic current conditions. Our findings show that gene flow patterns in ecologically similar congeneric species can be remarkably different and surprisingly limited depending on environmental and evolutionary contexts. These results are relevant to regional conservation planning and to considerations of similar integrated analyses for any vent metapopulations under threat from seabed mining

Divergent paths in the evolutionary history of maternally transmitted clam symbionts

Vertical transmission of bacterial endosymbionts is accompanied by virtually irreversible gene loss that results in a progressive reduction in genome size. While the evolutionary processes of genome reduction have been well described in some terrestrial symbioses, they are less understood in marine systems where vertical transmission is rarely observed. The association between deep-sea vesicomyid clams and chemosynthetic Gammaproteobacteria is one example of maternally inherited symbioses in the ocean. Here, we assessed the contributions of drift, recombination and selection to genome evolution in two extant vesicomyid symbiont clades by comparing 15 representative symbiont genomes (1.017–1.586 Mb) to those of closely related bacteria and the hosts' mitochondria. Our analyses suggest that drift is a significant force driving genome evolution in vesicomyid symbionts, though selection and interspecific recombination appear to be critical for maintaining symbiont functional integrity and creating divergent patterns of gene conservation. Notably, the two symbiont clades possess putative functional differences in sulfide physiology, anaerobic respiration and dependency on environmental vitamin B12, which probably reflect adaptations to different ecological habitats available to each symbiont group. Overall, these results contribute to our understanding of the eco-evolutionary processes shaping reductive genome evolution in vertically transmitted symbioses