Genetic relations between the bivalves Cardium (Cerastoderma) edule,Cardium lamarcki and Cardium glaucum,studied by means of crossed immunoelectrophoresis

Rabbit antisera were raised against Cardium edule (L.) from the Waddensea locality, Skallingen, against Cardium lamarcki (Reeve) from the Danish locality, Dybsø Fjord, and against Cardium glaucum (Bruguière) from the French Mediterranean coast at Ile des Embiez. Allopatric and sympatric populations of C. lamarcki and C. edule from Swedish and Danish localities, and allopatric populations of C. glaucum from the Mediterranean Sea, Red Sea and Black Sea were studied by means of the described method and by starch gel electrophoresis of malate dehydrogenase (MDH). By means of crossed immuno-electrophoresis, using intermediary gels containing antibodies against one of the Cardium types and receiver gels containing antibodies against one of the others, type-specific and type-non-specific reactions were recorded. The results are compared with a similar investigation of Mytilus edulis, M. galloprovincialis and M. californianus. Based on immunological properties, it is shown that C. edule is a valid species different from the C. lamarcki/C. glaucum complex. It is further suggested that C. lamarcki and C. glaucum should still be referred to by these two names as significant genetic differences between them is revealed by this immunological study.     

Habitat selection of two congeneric bivalves,Cardium edule and C. glaucum in sympatric and allopatric populations

Habitat selection of two bivalve species, Cardium edule (L.) and C. glaucum (Bruguière) is described for sympatric and allopatric populations of both sibling species. It is demonstrated that C. edule grows faster than C. glaucum and is absent from localities with periodic low food-availability. The ability to burrow of C. glaucum is low compared with that of C. edule; the former species does not occur in very loose, well-sorted sand near the coast. Earlier studies which suggest that tidal range determine the species distribution could not be confirmed.     

Biological studies on Cardium glaucum,based on some Baltic and Mediterranean populations

The recent separation of Cardium glaucum Bruguière from C. edule L. and the scarcity of reports of populations containing both species has necessitated further ecological study to demonstrate differences in habitat preferences. Some mixed and unispecific populations of the cockles Cardium edule and C. glaucum, situated in fjords around the Kattegat and Baltic coasts of Denmark were studied; it was demonstrated that some degree of tidal influece was essential for the survival of C. edule, and that C. glaucum was being restricted by the exposure of the site. A study of the cockle populations of some étangs on the Mediterranean coast of France demonstrated that, despite earlier reports of its presence, C. edule was not present in that area. A brief survey of the Bassin d'Arcachon (France) showed that it contained a mixed cockle population. It was concluded that C. edule was unable to occupy localities having a tidal amplitude of less than 0.2 m, and that high temperatures also restricted occupation of semitidal habitats; similarly, the exposure of the site restricted colonisation by C. glaucum.     

Studies on the reproductive cycles of Cardium edule and C. glaucum

Two allopatric populations of Cardium edule and two of C. glaucum were sampled over a period of 16 months and their gonad maturity cycles compared by histological methods. The onset of both spermatogenesis and oogenesis in C. edule took place between September and October; in C. glaucum it was slightly later, between October and November. Development of the gonads continued slowly throughout the winter, C. glaucum developing marginally faster than C. edule. Maturation accelerated in April and May, with the gonads of both species becoming fully ripe in late May and early June. Spawning of the two species approximately coincided at all the localities investigated. Spawning in C. edule was better coordinated than in C. glaucum. Transplanting cockles of one species to the typical habitat of the other did not appreciably alter the spawning period. The separation of spawning periods in sympatric associations of the two cockles is discussed.     

Upper temperature tolerances of some European molluscs

The upper temperature tolerances of three European species of Cardium [C. (Cerastoderma) glaucum Bruguière, C. tuberculatum L., C. (Cerastoderma) edule L.] are compared by means of median lethal temperature (LT₅₀) and median burial temperature (BT₅₀) determinations for periods of exposure up to 96 h. The influence of previous acclimation temperatures on these determinations is also considered. C. glaucum from shallow water (0.5 to 1 m), lagoonal, habitats in the Mediterranean Sea shows a greater thermal tolerance than C. tuberculatum from deeper water (4 to 5 m) in the Mediterranean. The thermal tolerance of C. edule from the intertidal zone, but at higher latitudes, is intermediate. Young C. glaucum show a greater resistance to short exposures to high temperatures than adults, but for longer exposures their tolerance is the same. All 3 species exhibit a significant effect of previous acclimation temperature on the burrowing response, and C. glaucum and C. tuberculatum a significant but lesser effect on LT₅₀. LT₅₀ determinations with C. edule showed secondary mortality effects which may have obscured any effect of acclimation temperature.     

Enzymatic and morphological criteria for distinguishing between Cardium edule and C. glaucum of the Portuguese coast

Cardium edule L. and C. glaucum Bruguière are two sibling species of cockles commonly found in littoral systems along the Portuguese coast, where they sometimes occur as mixed populations. The problem of morphological separation of these two cockle species has not been completely solved. Indeed, the distinctive morphological criterion proposed by Brock after identifying the specimens by means of electrophoretic patterns of hepatopancreatic malate dehydrogenase was not effective when applied to Portuguese Cardium spp. The object of the present study was to establish morphological discrimination criteria for distinguishing Portuguese C. edule and C. glaucum on the basis of initial identification of individuals through the enzymatic criterion established by Brock. Cockles were collected from 1988 to 1991 at 16 estuarine and lagoonal systems along the Portuguese coast. Hepatopancreatic extracts were submitted to horizontal starch gel electrophoresis following Brock's procedure. The malate dehydrogenase patterns obtained were identical to those found by Brock. Clear interspecific differences in the leucine aminopeptidase and esterase isozyme systems were also detected. The morphological study of the shells involved 7 morphometric and 9 qualitative and semiquantitative characters. Canonical variate analysis of the morphometric characters showed that these could not be used for identification purposes. However, a clear separation between the two species was obtained by a principal-component analysis of qualitative and semiquantitative characters. The characters mainly useful for distinguishing between the two species were valve profile, shape of ventral valve junction, type of calcareous scales and shell colour.     

Does displacement of spawning time occur in the sibling species Cerastoderma edule and C. lamarcki?

The spawning cycles of the sibling species Cerastoderma edule (L.) and C. lamarcki (Reeve) were studied in allopatric and sympatric Danish populations from 1978–1980. The results do not support earlier findings of reproductive character displacement in sympatric populations of the two species. New records of hybrid formation and intermediate forms are presented and discussed.     

An electron-microscope study of periostracum formation in some marine bivalves. I. The origin of the periostracum

Electron microscopy has revealed the presence of specialised basal cells at the base of the periostracal groove in the bivalves Mytilus edulis (L), Nucula sulcata Bronn., Cardium edule (L) and Macoma balthica (L). A common basal cell type is observed which gives rise to a pellicle in all cases except N. sulcata. The pellicle is the first part of the periostracum to be elaborated, and it is suggestd that it may be an indication of specialisation in bivalves. In N. sulcata and C. edule, an additional cell type is present. In N. sulcata, this second basal cell type appears to be directly involved in periostracum formation, whereas in C. edule an indirect role is proposed.     

Morphological and DNA sequence variation in the kelp Costaria costata (Phaeophyta)

Kelp species show substantial intraspecific morphological variation. The annual kelp, Costaria costata (C. Agardh) Saunders, is found in the lower intertidal and subtidal regions of shores which vary in exposure from strongly wave-exposed to sheltered. Phenotypic variation was studied in this monotypic genus. Individuals of C. costata were collected from two morphologically-distinct populations (one wave-exposed and the other wave-sheltered) at Cape Beale and Stanley Park, Vancouver, Canada, in May 1985. Analysis of morphological variation, using multivariate statistical techniques, indicated significant differentiation between plants from the two sites. Blade widths and thicknesses were the major discriminating variables. Restriction fragment length polymorphism (RFLP) analysis of nuclear DNA with 42 anonymous probes demonstrated one polymorphism. Most of the probes are hypothesized to encode highly repeated, dispersed sequences. These repeat sequence probes comprised 80.9% of all cloned fragments. Primary sequence analysis of 1595 base pairs of small-subunit ribosomal DNA and 204 base pairs of upstream sequence (probe pCc 18) failed to show any divergence between plants from the two sites.     

Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.     

Phylogeography of Cerastoderma glaucum (Bivalvia: Cardiidae) across Europe: a major break in the Eastern Mediterranean

Molecular variation of the lagoon cockle Cerastoderma glaucum (Poiret, 1789) was examined across the species range along European coasts, from the northern Baltic to the Black Sea and the Caspian Sea. A major phylogeographic break in mitochondrial COI gene sequences (divergence 6.2%) separated a group of Ponto-Caspian and Aegean Sea haplotypes from those to the west of the Peloponnese peninsula in the Mediterranean and in the Atlantic-Baltic sector. A similar subdivision, yet not entirely geographically coincident, was found at 16 allozyme loci (Nei's distance D=0.15). The results imply a long-term isolation of populations in parts of the Eastern Mediterranean or Black Sea basins through the Pleistocene. The subdivision does not concur with previous views on the systematics of C. glaucum complex, but the pattern is notably similar to that described earlier from some fish species. Marked phylogeographic structuring was also found at lower level within the major Mediterranean-Atlantic phylogroup of C. glaucum, which was divided into six regional or local haplotype subgroups. Divergence of these groups may date back one or several major Pleistocene climatic cycles. Local mtDNA diversity was particularly high in a sample from the Ionian Sea, whose mitochondrial identity was of the Mediterranean-Atlantic type, while its nuclear characters were more strongly associated with the Ponto-Caspian type. Patterns of shallower, star-phylogeny type diversity within the Ponto-Caspian phylogroup and in the Baltic Sea area may represent more recent, post-glacial generation of variation.     

The cockle <Emphasis Type="Italic">Cerastoderma edule</Emphasis> at Northeast Atlantic shores: genetic signatures of glacial refugia

The cockle Cerastoderma (Cardium) edule (L.) ranges from NW Africa to northern Scandinavia. Abundance in shallow coastal sediment is often high, and it attracts commercial harvest. In this study, a complex genetic pattern has been revealed by mitochondrial DNA in 383 individuals from 19 sampling sites. Parsimony network analysis of 79 haplotypes identified two dominant central haplotypes separated by low divergence. One is characteristic for a homogenous southwestern group of populations from Africa to the British Isles. The other is characteristic for a heterogeneous northern group with a deviant Arctic population. At the entrance of the Baltic Sea, a mixture zone of the dominant haplotypes was found. The estimated population expansion time for the northern haplotype group predates that of the southwestern one, suggesting northern glacial refugia and a subsequent southern expansion of C. edule populations.     

An electron-microscope study of periostracum formation in some marine bivalves. II. The cells lining the periostracal groove

The histology and ultrastructure of the epithelial cells of the outer and middle mantle folds of the bivalves Mytilus edulis (L), Cardium edule (L), Macoma balthica (L) and Nucula sulcata Bronn are described. The cells lining the inner face of the outer fold exhibit a prominent granular endoplasmic reticulum, and Golgi complexes which are concerned with the elaboration of granules and vesicles eventually incorporated into the periostracum. A gradual reduction in the protein synthetic apparatus occurs towards the tip of the fold. Within the cells, it is proposed that the ovoid inclusion bodies are lysosomes and that they control the rate of secretion. The cells of the middle fold are cuboidal in appearance. Those of M. edulis and N. sulcata exhibit prominent granules, whereas those of C. edule and M. balthica possess vesicles. The cells of M. edulis differ from the others in possessing stout bundles of filaments, which occupy large areas of the cell and constitute a cell web. The cells of the epithelium in all cases do not appear to be implicated in periostracum formation.     

Evolutionary relationships of deep-sea hydrothermal vent and cold-water seep clams (Bivalvia: Vesicomyidae): results from the mitochondrial cytochrome oxidase subunit I

Phylogenetic relationships among vesicomyid clams (Bivalvia: Vesicomyidae) and their placement within the order Heterodonta were examined using mitochondrial encoded cytochrome oxidase subunit I (COI) DNA sequences. The presently analyzed vesicomyids represent a recent monophyletic radiation that probably occurred within the Cenozoic. Nucleotide phylogenetic analyses resolved discrete clades that were consistent with currently recognized species: Calyptogena magnifica, C. ponderosa, Ectenagena extenta, C. phaseoliformis, Vesicomya cordata, Calyptogena n. sp. (Gulf of Mexico), C. kaikoi, C. nautilei, C. solidissima and C. soyoae (Type-A). However, specimens variously identified as: V. gigas, C. kilmeri, C. pacifica, and V. lepta comprised two “species complexes”, each composed of multiple evolutionary lineages. Most taxa are limited to hydrothermal-vent or cold-seep habitats, but the “vent” versus “seep” clams do not constitute separate monophyletic groups. Current applications of the generic names Calyptogena, Ectenagena, and Vesicomya are not consistent with phylogenetic inferences. Received: 24 July 1997 / Accepted: 22 August 1997     

Genetic divergence of Maldivian and Micronesian demes of the damselfishes Stegastes nigricans,Chrysiptera biocellata,C. glauca and C. leucopoma (Pomacentridae)

Samples representing the damselfishes (Perciformes: Pomacentridae) Stegastes nigricans, Chrysiptera biocellata, C. glauca, and C. leucopoma from North Male Atoll, Republic of Maldives, and Guam, Southern Marianas, were surveyed electrophoretically. A sample representing C. leucopoma from Ngemelis Island, Republic of Palau, was included in the analysis to provide a within-Micronesia comparison. On average, products of 23 presumptive protein-coding loci were examined in each sample. In comparisons of Maldivian and Guamanian samples, absolute or virtually fixed allelic differences were detected at: (1) ADA ^* and PEPS ^* for Stegastes nigricans; (2) ADA ^*, AAT-1 ^*, AAT-2 ^*, and mMDH ^* for C. biocellata, and; (3) ADA ^*, AAT-1 ^*, and AAT-2 ^* for C. glauca. At three polymorphic loci, ADA ^*, sMDH-1 ^*, and sMDH-2 ^*, similar allele frequencies were found in Palauan and Guamanian samples representing C. leucopoma, suggesting that these samples share a gene pool. Significantly different allele frequencies at ADA ^* and sMDH-1 ^* and a virtually fixed allelic difference at sMDH-2 ^* were detected between the Maldivian samples and each of the Micronesian samples representing C. leucopoma. The observed patterns of allele frequency differentiation suggest that Maldivian and Micronesian samples of each of the four study species represent separate demes.     

Human trampling as short-term disturbance on intertidal mudflats: effects on macrofauna biodiversity and population dynamics of bivalves

The effect of physical disturbance in the form of trampling on the benthic environment of an intertidal mudflat was investigated. Intense trampling was created as unintended side-effect by benthic ecologists during field experiments in spring and summer 2005, when a mid-shore area of 25 × 25 m was visited twice per month by on average five researchers for a period of 8 months. At the putatively-impacted location (I) (25 × 25 m) and two nearby control locations (Cs) (25 × 25 m each), three sites (4 × 4 m) were randomly selected and at each site, three plots (50 × 50 cm) were sampled after 18 and 40 days from the end of the disturbance. Multivariate and univariate asymmetrical analyses tested for changes in the macrofaunal assemblage, biomass of microphytobenthos and various sediment properties (grain-size, water content, NH₄ and NO₃ concentrations in the pore water) between the two control locations (Cs) and the putatively-impacted location (I). There were no detectable changes in the sediment properties and microphytobenthos biomass, but variability at small scale was observed. Microphytobenthos and NH₄ were correlated at I to the number of footprints, as estimated by the percentage cover of physical depressions. This indicated that trampling could have an impact at small scales, but more investigation is needed. Trampling, instead, clearly modified the abundance and population dynamics of the clam Macoma balthica (L.) and the cockle Cerastoderma edule (L.). There was a negative impact on adults of both species, probably because footsteps directly killed or buried the animals, provoking asphyxia. Conversely, trampling indirectly enhanced recruitment rate of M. balthica, while small-sized C. edule did not react to the trampling. It was likely that small animals could recover more quickly because trampling occurred during the growing season and there was a continuous supply of larvae and juveniles. In addition, trampling might have weakened negative adult-juvenile interactions between adult cockles and juvenile M. balthica, thus facilitating the recruitment. Our findings indicated that human trampling is a relevant source of disturbance for the conservation and management of mudflats. During the growing season recovery can be fast, but in the long-term it might lead towards the dominance of M. balthica to the cost of C. edule, thereby affecting ecosystem functioning.     

Effect of the presence of the shore crab, <Emphasis Type="Italic">Carcinus maenas</Emphasis>, on burrowing behaviour and clearance rate of the common cockle, <Emphasis Type="Italic">Cerastoderma edule</Emphasis>

Bivalves demonstrate various morphological and behavioural adaptations to reduce the risk of being attacked by predators. This paper examines how the presence of the crab Carcinus maenas (L.), a natural predator of the cockle Cerastoderma edule (L.), affects its burrowing depth and clearance or feeding rate. Cockles were placed in experimental tanks and treated with three levels of predatory disturbance: (1) unfed crab loose inside the tank, (2) unfed crab inside a cage suspended in the water column and (3) no crab present. Cockles’ burrowing depth was measured in two sediment types: mud and sand. Cockles burrowed more deeply in treatments with no crabs. Burrowing depth in sand was significantly greater than in mud. Two factors may contribute to the reduction in burial depth of C. edule in the presence of C. maenas: the change in the vertical orientation of the cockle and the ‘cough response’. No significant difference was found in the cockles’ clearance rate among the different levels of predator threat.     

Minimal predation upon meiofauna by endobenthic macrofauna in the Exe Estuary,south west England

Field manipulation experiments were performed in the Exe Estuary, south west England, in October 1988, to investigate the importance of the meiofaunamacroinfauna trophic link in benthic trophodynamics. Four hypothetically meiofauna-predacious endobenthic macrofauna species were selected for manipulation using the criteria of high abundance and different modes of feeding: Cerastoderma edule (filter-feeder), Nereis diversicolor (omnivorous scavenger), Ophelia bicornis (sand-ingester), Scrobicularia plana (deposit-feeder). Enclosures constructed from plastic tubing, 63 m nylon monofilament mesh and galvanized steel were deployed, containing adult members of these taxa at densities raised to approximately four times that of the surrounding sediment. The experiments ran for 12 tidal cycles. Differences in phyletic meiofaunal abundance between treatment and control enclosures at the termination of the experiment were assessed using both uni- and multivariate dataanalysis techniques. Only two univariate significant differences (p<0.05) existed for the N. diversicolor treatment and two for the S. plana treatment. Annelida, Turbellaria and copepod nauplii were the only meiofauna taxa affected. No univariate significant differences were recorded for either the C. edule or O. bicornis treatments. Multidimensional scaling ordination of the data revealed no consistent changes in community composition between treatments and controls. It is concluded that the experiments provide evidence of minimal predation by macroinfauna upon meiofauna.     

Cetacean DNA fingerprinting using the oligonucleotide (GGAT)4 as a probe

Since the discovery in 1985 of hypervariable minisatellite regions in human DNA, DNA fingerprinting has been widely used in animals and plants. Although many different probes have been developed recently, most investigators still use 33.15 and 33.6 human polycore minisatellites for hybridization studies of cetaceans. This animal order shows considerably less variability than humans, and thus some limitations have to be taken into account when using these probes. The present study demonstrates that the short repetitive tandem oligonucleotide (GGAT)₄ hybridizes with cetacean (Globicephala macrorhynchus andGrampus griseus) DNA, yielding individual-specific patterns of bands or DNA fingerprints. Freedom of design and shorter hybridization times of oligonucleotides allow an increase in the number of usable probes used, helping to overcome some of the problems found when only human polycore minisatellites are used.     

Highly repetitive DNA sequences provide evidence for a lack of gene flow between two morphological forms of Herdmania momus (Ascidiacea: Stolidobranchia)

The genomic structure of two morphological forms of the pyurid ascidian Herdmania momus — H. momus forma curvata and H. momus forma grandis—are compared using a rapidly evolving highly repetitive element isolated from the genome of H. momus forma curvata. A 663 bp (base pair) Cla I satellite sequence is present only in the genome of H. momus forma curvata, and hence can be used as a form-specific marker. Low-stringency Southern blot analyses, using the Cla I satellite as a probe, revealed that the genomes of H. momus forma grandis and another pyurid ascidian, Pyura stolonifera, do not contain similar sequences to the H. momus forma curvata repetitive element. In addition to this genomic dissimilarity, there are a number of significant reproductive and developmental differences between the two H. momus forms, and interform fertilisation rates are significantly lower than intraform rates. The molecular, reproductive and developmental differences between H. momus forma curvata and H. momus forma grandis indicate the presence of strong barriers to gene flow.