Next-generation phylogeography of the cockle Cerastoderma glaucum: Highly heterogeneous genetic differentiation in a lagoon species.

AIM: Coastal lagoons form an intriguing example of fragmented marine habitats. Restricted gene flow among isolated populations of lagoon species may promote their genetic divergence and may thus provide a first step toward speciation. In the present study, the population genetic structure of the lagoon cockle Cerastoderma glaucum has been investigated to clarify the complex phylogeographic pattern found in previous studies, to localize major genetic breaks, and to discuss their origin and maintenance. LOCATION: The Atlantic and Mediterranean coasts, including the Baltic, North Sea, and Black Sea. METHODS: A total of 204 C. glaucum individuals from 14 populations were genotyped using restriction site-associated DNA sequencing (RADseq). The genetic diversity, divergence, and structure were analyzed using genome-wide single nucleotide polymorphisms (SNPs). Phylogenetic relationships were inferred under a coalescent model using svdquartets. RESULTS: The RADseq approach allowed inferring phylogeographic relationships with an unprecedented resolution. Three deeply divergent lineages were identified within C. glaucum that are separated by many genetic barriers: one lineage in the Aegean-Black Sea region, one in the Ionian Sea, and the last one widely distributed from the Western Mediterranean to the Baltic Sea. The nested branching pattern displayed on the species tree largely agrees with the likely scenario of C. glaucum postglacial expansion from the Mediterranean to the Baltic Sea. MAIN CONCLUSION: The genetic differentiations between geographically separated lagoons proved to be strong, highlighting the evolutionary influence of these naturally fragmented habitats. The postglacial expansion created complex patterns of spatial segregation of genetic diversity with allele frequency gradients in many outlier loci, but also discrepancies between the nuclear and mitochondrial genetic markers that probably arose from genetic surfing of mitochondrial variation.

Abnormalities in bivalve larvae from the Puck Bay (Gulf of Gdansk, southern Baltic Sea) as an indicator of environmental pollution.

This study described the occurrence of abnormalities in bivalve larvae from the Puck Bay. Analyses of plankton samples collected in 2012-2013 showed that larval Mytilus trossulus, Mya arenaria, and Cerastoderma glaucum exhibited abnormalities that could indicate adverse environmental impacts. The deformities were mainly in shells, but missing soft tissue fragments and protruding vela were also noted. In addition to larval studies, we analyzed benthic postlarvae of Mytilus trossulus. Interestingly, grooves and notches at different locations of the prodissoconch, dissoconch, and shell margin were observed. Some of these deformations were reminiscent of the indentations found on the shell edge of larvae. Comparing the proportion of abnormal postlarvae to larvae with shell abnormalities suggested that the survival of larvae with shell abnormalities was low. Overall, our results suggested that the ratio of abnormal bivalve larvae could be used as an indicator of the biological effects of hazardous substances in the pelagic environment.

Sex-related differences in steroid concentrations in the blue mussel (Mytilus edulis trossulus) from the southern Baltic Sea.

This paper reports on sex-related differences in free steroid hormone concentrations including the concentrations of three naturally occurring estrogens (17ß-estradiol E2, estrone E1, and estriol E3) and one androgen (testosterone T) in the tissues (gills and gonads) of the blue mussel Mytilus edulis trossulus sampled from the Gulf of Gdansk (Baltic Sea, Poland). The dissimilarity in steroid concentrations between tissues was particularly evident in the T concentration with a level in gills almost three times higher compared to gonads (on average, 15.38 ng/g w.w. and 5.31 ng/g w.w., respectively, p=0.00008), suggesting its exogenous origin. In general, a tendency towards a skewed steroid profile related to sex, with E2 more abundant for males and T for females, was observed. Female gonads were characterized by a higher level of T than testis (4.61 ng/g w.w. for females and 0.70 ng/g w.w. for males, p=0.0121). At the same time, the level of E2 found in the testis was higher than in the ovary (4.81 ng/g w.w. and 3.86 ng/g w.w., respectively); however, the difference was not statistically significant. As for gills, similar trend with T and E2 being more abundant in males was observed. At the same time, no disturbances in the sex ratio and gametogenesis process were observed which suggests i) efficient deactivation of free forms of steroids, and/or ii) their little or no physiological role.

Comparison of trace metal bioavailabilities in European coastal waters using mussels from Mytilus edulis complex as biomonitors.

Mussels from Mytilus edulis complex were used as biomonitors of the trace metals Fe, Mn, Pb, Zn, and Cu at 17 sampling sites to assess the relative bioavailability of metals in coastal waters around the European continent. Because accumulated metal concentrations in a given area can differ temporally, data were corrected for the effect of season before large-scale spatial comparisons were made. The highest concentration of Fe was noted in the North Sea and of Mn in the Baltic. Increased tissue concentrations of Pb were recorded in the mussels from the Bay of Biscay and the Baltic Sea. Low concentrations of metals were determined in the mussels from the Mediterranean Sea and the Northern Baltic. Relatively low geographic variations of Cu and Zn indicate that mussels are able to partially regulate accumulated body concentrations, which means Cu and Zn are, to some extent, independent of environmental concentrations.

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.

Geographic and seasonal patterns and limits on the adaptive response to temperature of European Mytilus spp. and Macoma balthica populations.

Seasonal variations in seawater temperature require extensive metabolic acclimatization in cold-blooded organisms inhabiting the coastal waters of Europe. Given the energetic costs of acclimatization, differences in adaptive capacity to climatic conditions are to be expected among distinct populations of species that are distributed over a wide geographic range. We studied seasonal variations in the metabolic adjustments of two very common bivalve taxa at European scale. To this end we sampled 16 populations of Mytilus spp. and 10 Macoma balthica populations distributed from 39 degrees to 69 degrees N. The results from this large-scale comprehensive comparison demonstrated seasonal cycles in metabolic rates which were maximized during winter and springtime, and often reduced in the summer and autumn. Studying the sensitivity of metabolic rates to thermal variations, we found that a broad range of Q (10) values occurred under relatively cold conditions. As habitat temperatures increased the range of Q (10) narrowed, reaching a bottleneck in southern marginal populations during summer. For Mytilus spp., genetic-group-specific clines and limits on Q (10) values were observed at temperatures corresponding to the maximum climatic conditions these geographic populations presently experience. Such specific limitations indicate differential thermal adaptation among these divergent groups. They may explain currently observed migrations in mussel distributions and invasions. Our results provide a practical framework for the thermal ecophysiology of bivalves, the assessment of environmental changes due to climate change and its impact on (and consequences for) aquaculture.

Metal sources to the Baltic clam Macoma balthica (Mollusca: Bivalvia) in the southern Baltic Sea (the Gulf of Gdansk).

Metal concentrations of Cu, Fe, Mn, Ni, Pb and Zn in an infaunal facultative deposit-feeding bivalve, the Baltic clam Macoma balthica, in the Gulf of Gdansk (southern Baltic Sea) were assessed and compared to selected concentrations of metals in the environment. Between October 1996 and September 1997, dissolved and easy extractable (by 1M HCl) metal fractions of total suspended particulate matter (TPM) in the overlying water and of surficial sediments (<63 microm) were measured monthly at five sublittoral sites in the Gulf of Gdansk, and accumulated tissue metal concentrations in M. balthica were determined simultaneously. The study highlights the importance of sediment geochemistry as a factor modifying ambient trace metal bioavailabilities. Surficial sediments appeared to contribute most to the accumulation of Cu and Pb in M. balthica, reflecting the high metal availability in the Gulf. Assimilation of Cu from sediments is controlled by Mn components possibly through an inhibitory effect of Mn oxyhydroxides, while Pb accumulation from sediments depends on the organic content of the sediment. A dual metal uptake pathway, with a suspended particulate-bound fraction and surficial sediments, was apparent for Mn and Zn. Partitioning of Mn in sediments was related to the concentration of labile Fe, with increased levels of Fe tending to inhibit the accumulation of Mn by the clam. Tissue accumulated Zn might have been altered by the clam's internal regulation, making Zn tissue concentrations, to some degree, independent of its environmental level. The principal source of Ni accumulated by the clams exists in the soluble phase.

First record of the occurrence of "gill disease" in Mytilus edulis trossulus from the Gulf of Gdansk (Baltic Sea, Poland).

We are presenting the first report of the occurrence of "gill disease" in Mytilus edulis trossulus from the southern Baltic Sea, Gulf of Gdansk (Poland). The disease preliminary diagnosis was based on the presence of white and yellow "spots" causing deep indentations in the gills with degeneration, destruction and necrosis of gill filaments. Average prevalence of gill erosion in the blue mussels population was 15.8%. According to other authors, gill erosion may affect over 80% of the bivalve population resulting in mortality rates of up to 40%. The origin of the gill erosion remains unknown, but viruses are most likely involved in the etiology of these pathological conditions. The disease as such may indicate a decrease in the immunological resistance of organisms to infections and inflammations directly or indirectly caused by harmful factors in the ambient environment. Furthermore, the occurrence of numerous pathologies in bivalves is a particular problem in the Gulf of Gdansk being a low biodiversity ecosystem. Pathologies reduce bilvalves' reproduction ability, worsen their physiological condition and increase their mortality rate. Those factors may also pose a significant ecological danger and lead to negative alterations of the ecosystem.

Ultrastructural study of neoplastic cells in Macoma balthica (Bivalvia) from the Gulf of Gdansk (Poland).

In the Baltic clam Macoma balthica from the Gulf of Gdansk, neoplasia is considered as a serious epizootic linked to bad environmental conditions and high levels of pollutants. Previous research was focused on the diagnosis, prevalence, seasonality and histopathological characteristic of the cancer. This study is focused on electron microscopy analyses describing cell ultrastructure abnormalities related to neoplasia. Examinations using the electron microscopy highlighted changes confined to anatomic ultrastructures, shapes and functions of neoplastic cells. The lobulated appearance of the nucleus, changes in cellular matrix and the occurrence of large granular cells with hyperchromatic nuclei, atypical Golgi structures and deterioration of rough endoplasmic reticulum manifested the disease. The presence of atypical mitochondria, free ribosomes and hypertrophic nuclei suggests the adaptation of neoplastic cells to increased mitotic activity, while the observed modification of cellular membranes may reflect functional changes connected to increased pinocytotic activity or intercellular transport. The cancer cells were found to appear in two types, abnormal round-shaped cells and spindle-shaped cells, both with increased frequencies of cell division. Round-shaped cells typical for disseminated neoplasia were observed in all affected bivalves, in a few cases co-occurring with abnormal spherical cells. Spindle-shaped cells containing some intracytoplasmic filaments, and with a tendency of the nuclei to be orientated as in a palisade were interpreted as myofibroblasts-like cells and were observed in five out of eleven clams diagnosed as neoplastic. This finding represents the first demonstration of phenotypic differences in the cell types co-occurring in animals diagnosed as neoplastic and by that suggests coexistence of two types of bivalve cancer, disseminated neoplasia and probable fibrosarcoma.

Abnormal features of Macoma balthica (Bivalvia) in the Baltic Sea: alerting symptoms of environmental adversity?

Recent studies of the Baltic clam Macoma balthica (L.) from the southern Baltic (the Gulf of Gdansk) have revealed striking morphological, histological and cytogenetic features. Strong deformation of the shell, including elongation of the posterior end and the appearance of an easily visible flexure in this part, has been recorded. The population contribution of the deformed blunt shelled ("irregular") clams ranged from 0% to 65% and tended to increase with depth. The morphologically "irregular" clams had higher accumulated tissue concentrations of trace metals (As, Ag, Cd, Pb, Cu and Zn), indicating a different metal handling ability. Adverse conditions in deeper water regions of the Gulf (e.g. hypoxia, hydrogen sulphide, elevated bioavailability of contaminants) have been suggested as inducers of the phenotypical changes (morphological deformation) in part of the population and, in parallel, of the specific physiological adaptations that result in higher metal accumulation in the "irregular" clams. Cytogenetic and histological analyses showed the presence of tumours in gill cells and digestive system of the affected clams, the prevalence of disseminated neoplasia ranging from 0% to 94% depending on the site. The disease was manifested by a modified karyotype (i.e. an abnormal number and morphology of chromosomes), a higher activity of nucleolar organizer regions (AgNORs), and tissue lesions (enlarged cells, actively proliferative with pleomorphic nuclei). Bottom sediments showed acute toxicity and have been proposed as a source of an initialising carcinogenic factor. However, none of the ecotoxicological studies provided was successful in the clear demonstration of a single (or multifactorial) agent that can account for the disseminated neoplasia.

Free amino acids in the clam Macoma balthica L. (Bivalvia, Mollusca) from brackish waters of the southern Baltic Sea.

Fourteen acidic and neutral free amino acids (FAA) were investigated in soft tissue of Macoma balthica from different depth zones of the Gulf of Gdansk (Baltic Sea) over a full seasonal cycle. The dry weight of the bivalves and physico-chemical parameters of overlying bottom water and surface sediments were measured simultaneously at each site. In the brackish waters of the Baltic, the main pool of FAA is composed of Ala, Gln, Arg, Gly and Orn which represent approximately 80% of the total. Compared to the full saline environments, the composition of FAA in the clams from the Baltic differs substantially. The differences can be attributed to the lower salinity of the Baltic. In the Baltic, Gly appears to play a most important role in regulating intracellular osmolarity in the clams, a function performed primarily by Tau in Atlantic and North Sea populations. Spatio-temporal variations of the FAA are affected by biotic and environmental parameters; their respective influence differs with the amino acids. The concentration of Arg depends on its uptake from the external medium. However, its level might be temporarily modified by stress-induced metabolic transformation (e.g. hydrolysis to Orn) caused by changes in the ambient environment. The concentration of Ala increases with depth, probably because of physiological adaptations of the animal to diminishing oxygen concentration through anaerobic glucose catabolism. Biosynthesis of Ala, similarly to Gln, in the shallower zone is generally related to the physiological state of an organism. The concentration of Gly is most likely regulated by internal mechanisms driven by gonadal development and reproduction.

The relationship between metal concentrations and phenotypes in the Baltic clam Macoma balthica (L.) from the Gulf of Gdansk, southern Baltic.

Soft tissue accumulated concentrations of nine metals (V, As, Se, Ag, Cd, Pb, Mn, Cu and Zn) were studied in two main phenotypes (1) according to external shell colour (white and pink), and (2) according to shell shape (shell with a rounded posterior end--"regular" and with an elongate posterior end and a notable flexure--"irregular") of the Baltic clam Macoma balthica from southern Baltic Sea off Poland. No differences in metal concentrations were observed between colour-based phenotypes. By contrast, "irregular" clams exhibited generally higher concentrations of all elements in their tissues than "regular" bivalves. This finding provides the first reference on a potential linkage of shell deformation with tissue metal concentrations within one entire population of clams living in the same habitat. Different ability of metal handling in the shape-based phenotypes is presumably related to different physiological capacity of the bivalves induced by the selection effect of specific environmental conditions. It is suggested that unfavourable conditions in deep waters of the Gulf of Gdansk (e.g. hypoxia/anoxia, hydrogen sulphide, elevated bioavailability of metals) induces, in a certain part of the population, morphological deformation of shell (thereby leading to irregular shape) and in parallel physiological adaptations which result in greater sensitivity to trace metals of "irregular" clams. This hypothesis however, requires further investigation with special focus on genetic divergences between phenotypes because till now we cannot exclude the co-occurrence of two types (semi-species) of clams in the Gulf: an Atlantic type and a Baltic type. Genetic analysis with a use of DALP technique revealed strong intrapopulational polymorphism but no fingerprints or intraspecific polymorphism characterising any of the phenotypes considered (both colour- and shape-based). Since eight polymorphic loci were clearly identified further studies of population genetic structure hold optimistic promise.