LAMP-based molecular sexing in a gonochoric marine bivalve (Macoma balthica rubra) with divergent sex-specific mitochondrial genomes.

Taking advantage of the unique system of doubly uniparental inheritance (DUI) of mitochondria, we developed a reliable molecular method to sex individuals of the marine bivalve Macoma balthica rubra. In species with DUI (~100 known bivalves), both sexes transmit their mitochondria: males bear both a male- and female-type mitogenome, while females bear only the female type. Male and female mitotypes are sufficiently divergent to reliably PCR-amplify them specifically. Loop-mediated isothermal amplification (LAMP) is a precise, economical and portable alternative to PCR for molecular sexing and we demonstrate its application in this context. We used 154 individuals sampled along the Atlantic coast of France and sexed microscopically by gonad examination to test for the congruence among gamete type, PCR sexing and LAMP sexing. We show an exact match among the sexing results from these three methods using the male and female mt-cox1 genes. DUI can be disrupted in inter-specific hybrids, causing unexpected distribution of mitogenomes, such as homoplasmic males or heteroplasmic females. To our knowledge, DUI disruption at the intra-specific scale has never been tested. We applied our sexing protocol to control for unexpected heteroplasmy caused by hybridization between divergent genetic lineages and found no evidence of disruption in the mode of mitochondrial inheritance in M. balthica rubra. We propose LAMP as a useful tool to accelerate eco-evolutionary studies of DUI. It offers the opportunity to investigate the potential role of, previously unaccounted-for, sex-specific patterns such as sexual selection or sex-specific dispersal bias in the evolution of free-spawning benthic species.

Mitochondrial genomes of the Baltic clam Macoma balthica (Bivalvia: Tellinidae): setting the stage for studying mito-nuclear incompatibilities.

BACKGROUND: Allopatric divergence across lineages can lead to post-zygotic reproductive isolation upon secondary contact and disrupt coevolution between mitochondrial and nuclear genomes, promoting emergence of genetic incompatibilities. A previous F ST scan on the transcriptome of the Baltic clam Macoma balthica highlighted several genes potentially involved in mito-nuclear incompatibilities (MNIs). As proteins involved in the mitochondrial oxidative phosphorylation (OXPHO) chain are prone to MNIs and can contribute to the maintenance of genetic barriers, the mitochondrial genomes of six Ma. balthica individuals spanning two secondary contact zones were sequenced using the Illumina MiSeq plateform. RESULTS: The mitogenome has an approximate length of 16,806 bp and encodes 13 protein-coding genes, 2 rRNAs and 22 tRNAs, all located on the same strand. atp8, a gene long reported as rare in bivalves, was detected. It encodes 42 amino acids and is putatively expressed and functional. A large unassigned region was identified between rrnS and tRNA (Met) and could likely correspond to the Control Region. Replacement and synonymous mutations were mapped on the inferred secondary structure of all protein-coding genes of the OXPHO chain. The atp6 and atp8 genes were characterized by background levels of replacement mutations, relative to synonymous mutations. However, most nad genes (notably nad2 and nad5) were characterized by an elevated proportion of replacement mutations. CONCLUSIONS: Six nearly complete mitochondrial genomes were successfully assembled and annotated, providing the necessary roadmap to study MNIs at OXPHO loci. Few replacement mutations were mapped on mitochondrial-encoded ATP synthase subunits, which is in contrast with previous data on nuclear-encoded subunits. Conversely, the high population divergence and the prevalence of non-synonymous mutations at nad genes are congruent with previous observations from the nuclear transcriptome. This further suggest that MNIs between subunits of Complex I of the OXPHO chain, coding for NADH dehydrogenase, may play a role in maintaining barriers to gene flow in Ma. balthica.

SNP detection from de novo transcriptome sequencing in the bivalve Macoma balthica: marker development for evolutionary studies.

Hybrid zones are noteworthy systems for the study of environmental adaptation to fast-changing environments, as they constitute reservoirs of polymorphism and are key to the maintenance of biodiversity. They can move in relation to climate fluctuations, as temperature can affect both selection and migration, or remain trapped by environmental and physical barriers. There is therefore a very strong incentive to study the dynamics of hybrid zones subjected to climate variations. The infaunal bivalve Macoma balthica emerges as a noteworthy model species, as divergent lineages hybridize, and its native NE Atlantic range is currently contracting to the North. To investigate the dynamics and functioning of hybrid zones in M. balthica, we developed new molecular markers by sequencing the collective transcriptome of 30 individuals. Ten individuals were pooled for each of the three populations sampled at the margins of two hybrid zones. A single 454 run generated 277 Mb from which 17K SNPs were detected. SNP density averaged 1 polymorphic site every 14 to 19 bases, for mitochondrial and nuclear loci, respectively. An [Formula: see text] scan detected high genetic divergence among several hundred SNPs, some of them involved in energetic metabolism, cellular respiration and physiological stress. The high population differentiation, recorded for nuclear-encoded ATP synthase and NADH dehydrogenase as well as most mitochondrial loci, suggests cytonuclear genetic incompatibilities. Results from this study will help pave the way to a high-resolution study of hybrid zone dynamics in M. balthica, and the relative importance of endogenous and exogenous barriers to gene flow in this system.

Shell deformations in the Baltic clam Macoma balthica from southern Baltic Sea (the Gulf of Gdansk): hypotheses on environmental effects.

High-resolution digital photography and graphical image analyses systems have been used to define external morphometric characters of shell deformations in four populations of the Baltic clam Macoma balthica from the Gulf of Gdansk (southern Baltic Sea). The proposed shell deformation indices (SDI), which were based on the relationship of selected dimensions in the posterior and the anterior part of the shell, showed at least three morphological features that provide a distinctive diagnosis of "regular" and "deformed" clams: the presence of flexure on the posterior side (SDI1), elongated posterior region (SDI2), and shell growth (SDI3). The degree and prevalence of deformed clams varied locally over space. Increase in percentage contribution of aberrated shells with depth, corresponding to oxygen depletion profile in the Gulf, suggests low oxygen concentrations as the main agent exerting a deforming influence. The observed morphological aberrations developed with age (size) of a bivalve, suggesting a long-term effect of causal factors, and were accompanied by lightening shell weight, possibly due to decalcification of previously deposited calcareous material during anaerobic metabolism. It is hypothesized that hypoxic/anoxic conditions and a subsequent presence of hydrogen sulfide on a deep organic-rich sea bottom induce shell form alterations that enable the pumping of oxygenated water from above the anoxic layer. Such a morphological modification highlights the functional significance of shell deformations in protective response to the ambient low-oxygen concentrations. Sediment organotin concentrations fall within moderate to high contamination range and, therefore, may also have an adverse impact on the shell form. DNA analyses of the fragment of mitochondrial gene cytochrome oxidase I (COI, 393 base pairs) showed homogenous genetic structure of regular and deformed clams, indicating that shell deformations in M. balthica are primarily driven by acclimatization to the ambient environmental conditions.