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.     

Evolution of Cardium (Cerastoderma) edule,C. lamarcki and C. glaucum: studies of DNA-variation

Genetic relations between populations of Cardium (Cerastoderma) edule (L.) and C. lamarcki (Reeve) from Danish waters and C. glaucum (Bruguiére) from the Mediterranean Sea were studied using DNA analysis. Repetitive sequences of nuclear DNA were compared by Southern blotting of enzyme digested DNA fragments, separated on agarose gel by electrophoresis. Fragments were visualized by autoradiography using ³²P labelled probes. These were nick translated, randomly sheared fragments of the whole chromosomal genomes of two species of distant taxa — Mytilus edulis and Mya arenaria, respectively. The number of detected DNA bands was 18 for C. edule, 32 for C. lamarcki, and 24 for C. glaucum. Measurements of genetic distances, based on comparisons of these repetitive sequences using a simple dissimilarity coefficients, show the greatest distance, i.e., 0.84 between C. edule and C. lamarcki, 0.69 between C. edule and C. glaucum, and the smallest, 0.52 between C. lamarcki and C. glaucum. Comparisons of the three Cardium types, using gene spliced and cloned C. edule nuclear DNA (nDNA) fragments as probes, accorded with these findings. Also, analysis of mitochondrial DNA (mtDNA) from eggs and hepatopancreas, although not in itself conclusive, accorded with the results of the analyses of nDNA. MtDNA was measured by electron micrograph to 15 000 base pairs (bp), and sizes of restriction enzyme digested fragments were compared by gel electrophoresis. The results of the comparisons based on repetitive sequences supported by the two other studies suggest that C. edule resembles the ancestral form more than the two others, and that C. lamarcki is the most different and recently evolved form. This hypothesis is discussed in relation to the geographical distribution of the species complex.     

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.     

<Emphasis Type="Italic">Patella aspera</Emphasis> and<Emphasis Type="Italic"> P. ulyssiponensis</Emphasis>: genetic evidence of speciation in the North-east Atlantic

The group of subspecies of Patella ulyssiponensis, described by Christiaens, was widely known as Patella aspera until recently. The group extends throughout the Mediterranean, on all Macaronesian islands, along the North African coast, and in Europe, as far north as southern Norway. Throughout its range it displays great variation in shell sculpture and colour. The aim of this work was to re-examine the various subspecies proposed by Christiaens and, genetically, to test the hypothesis that European continental populations belong to a different species than the one composed by Macaronesian (north-west African) populations. In the present work, this group was studied by allozyme electrophoresis of 21 loci and by six morphological variables. The monomorphic locus of malate dehydrogenase (Mdh-1) was found to be diagnostic for distinguishing European continental populations from those of north-west African archipelagos, confirming the specific status of both groups. The allele observed at this locus in the Macaronesian populations was novel, while European continental populations showed a plesiomorphic one, shared with all other north-west Atlantic patellids with the exception of Patina pellucida, suggesting a more recent origin of the Macaronesian species from the continental forms. Both species showed a genetic identity of 0.730±0.061, which allowed rough estimations of 6.5–3 Mya since speciation. The subspecies Patella ulyssiponensis deserta described by Christiaens was not confirmed by our genetic data. Other loci (glyceraldehyde-3-phosphate dehydrogenase and the second locus of lactate dehydrogenase) were partially diagnostic, in which both species showed different most common alleles. Morphologically, both species are easily recognised by shell characters and the results agree with previous findings, that continental populations are more homogeneous in shell morphology and radula characters than populations from the Macaronesia. This work supports retention of the earliest valid name, Patella ulyssiponensis Gmelin, with Lisbon, Portugal as type locality, for the European continental species, and Patella aspera Röding for the Macaronesian populations. Population subdivision within species was measured by theta, the estimator of F_st, showing in both P. aspera and P. ulyssiponensis a high degree of genetic structuring (=0.226 and 0.182, respectively) mostly explained by the large distances separating the populations within species.Communicated by J.P.Thorpe, Port Erin     

Allozymes and morphometric characters of three species ofMytilus in the Northern and Southern Hemispheres

Many authors have considered the common mussels in temperate waters of the Northern and Southern Hemispheres to be a single cosmopolitan species,Mytilus edulis Linnaeus, 1758. Others have divided these mussels into several subspecies or species. Samples of mussels were collected from 36 locations in the Northern Hemisphere and nine locations in the Southern Hemisphere. Electrophoretic evidence from eight loci indicates that the Northern Hemisphere samples consist of three electrophoretically distinguishable species:M. edulis from eastern North America and western Europe;M. galloprovincialis Lamarck, 1819 from the Mediterranean Sea, western Europe, California, and eastern Asia; andM. trossulus Gould, 1850 from the Baltic Sea, eastern Canada, western North America and the Pacific coast of Siberia. Mussels from Chile, Argentina, the Falkland Islands and the Kerguelen Islands contain alleles characteristic of all three Northern Hemisphere species, but because they are most similar toM. edulis from the Northern Hemisphere, we suggest that they tentatively be included inM. edulis. These South American samples are morphologically intermediate between Northern HemisphereM. edulis andM. trossulus. Mussels from Australia and New Zealand are similar in allele frequency and morphometric characters toM. galloprovincialis from the Northern Hemisphere. FossilMytilus sp. are present in Australia, New Zealand and South America, which suggests that the Southern Hemisphere populations may be native, rather than introduced by humans. Morphometric characters were measured on samples which the allozyme data indicated contained a single species. Canonical variates analysis of the morphometric characters yields functions which distinguish among our samples of the species in the Northern Hemisphere.     

Evaluation of combined morphological and molecular techniques for marine nematode (<Emphasis Type="Italic">Terschellingia</Emphasis> spp.) identification

Marine nematodes, which play an important role in many ecosystems, include a number of apparently cosmopolitan taxa that exhibit broad biogeographic ranges even though there is no obvious dispersal phase in their lifecycle. In this study, standard taxonomic approaches to marine nematode identification in conjunction with multivariate statistical analysis of morphometric data were compared with molecular techniques. Specimens of the marine nematode Terschellingia longicaudata that had been identified by their morphological features were investigated from a range of localities (East and West Atlantic, Bahrain, Malaysia) and habitats (estuarine, intertidal, subtidal) using molecular approaches based on the amplification and sequencing of the small subunit ribosomal RNA (18S rRNA). The study revealed that the majority of the morphologically defined T. longicaudata specimens share a single 18S rRNA sequence and apparently belong to a single taxon distributed from the British Isles to Malaysia. In addition, 18S rRNA analysis also revealed two additional sequences. One of these sequences was found in both the British Isles and Mexico, the other was recorded only from British waters. Individuals collected in Bahrain and identified from their morphology as T. longicaudata had two highly divergent 18S rRNA sequences. Separate morphological and morphometric approaches to identification of specimens from the same sites that had been formalin-preserved did not support evidence of multiple genotypes revealed previously by molecular analysis. Current taxonomy based on morphological characters detected using light-microscopy may be unable to discriminate possible species complexes. Biodiversity of marine nematodes may often be underestimated due to the presence of morphologically cryptic species complexes. High-throughput techniques such as DNA barcoding would aid in species identification but may require thorough analysis of multiple nuclear and mitochondrial molecular markers.     

<Emphasis Type="Italic">Euplotes uncinatus</Emphasis> (Ciliophora,Hypotrichia), a new species with zooxanthellae

During the observations of a field collection of Maristentor dinoferus with fluorescence microscopy, we discovered a smaller ciliate with zooxanthellae. On the basis of morphological, morphometric and ultrastructural characters, this ciliate has been recognized as a new species of the genus Euplotes. To emphasize the peculiar shape and extension of the right buccal margin, the species has been named Euplotes uncinatus sp. n. The most distinctive traits of E. uncinatus are the presence of mucocyst-like vesicles, a kind of extrusome not previously found in Euplotes, and the presence of zooxanthellae. Evidence from ciliate behavior and zooxanthellae ultrastructure make it very likely that the zooxanthellae are symbiotic, even though some are evidently consumed by the host.     

Morphological and molecular data suggest a cosmopolitan distribution of the polychaete <Emphasis Type="Italic">Proscoloplos cygnochaetus</Emphasis> Day, 1954 (Annelida,Orbiniidae)

The discovery of a possibly invasive Proscoloplos species on the French Atlantic coast led to a detailed morphological and molecular investigation. Proscoloplos consists of three nominal species, but molecular analyses of the variable ITS1 and ITS2 region revealed no clade support for samples from South Africa, France and Australia, representing at least two of these nominal species. We found no unambiguous diagnostic characters for the three different species even with scanning electron microscopy. Two main characters—first appearance of branchiae and hooks—that were used for species delineation show considerable intrapopulational variation and do not withstand critical evaluation. The obtained data and the observed regenerative capabilities point to a cautious use of the position of branchiae and hooked chaetae as taxonomic marker in Proscoloplos. After experimental bisection of animals, the recovery led to a phenotype without recognizable signs of regeneration, but neither branchiae nor hooked chaetae reoccurred at the same initial segmental position. Summarising the molecular and morphological data we suggest the synonymy of the known Proscoloplos species under the senior synonym Proscoloplos cygnochaetus Day, 1967. The enormous geographic range of this species comprises the temperate waters of the whole southern hemisphere and includes the French population. We discuss a dispersal enabled through the effective anchorage on vessels using mucous glands and chaetae possibly combined with architomic reproduction.     

Using a combined approach to explain the morphological and ecological diversity in <Emphasis Type="Italic">Phanogenia gracilis</Emphasis> Hartlaub, 1893 (Echinodermata: Crinoidea) <Emphasis Type="Italic">sensu lato</Emphasis>: two species or intraspecific variation?

Phanogenia gracilis sensu lato is a shallow-water crinoid distributed throughout the Indo-western Pacific. The taxonomy of P. gracilis s.l. is clouded by the presence of two distinct morphotypes, each differing in morphology and ecology. The goal was to determine the taxonomic status of P. gracilis s.l. using partial gene sequences of two mitochondrial DNA genes, cytochrome oxidase c subunit I and NADH dehydrogenase subunit II, in conjunction with morphological and ecological data. The molecular phylogenies revealed three lineages separated by 5.0–6.6% corrected genetic distance, which is consistent with the genetic distances among other echinoderm species. Neither morphotype was monophyletic, nor was any examined morphological character exclusive to any one lineage. Discriminant function analysis (DFA) of the morphological and ecological data yielded significant results when grouping P. gracilis by morphotype and by clades recovered in the phylogenetic analyses, but grouping by sample locality was rejected. Although DFA results of grouping by clade were significant, jackknife support was weak, while only correctly grouping specimens by their respective clades 65% of the time. The results suggest the possibility of cryptic species, but additional molecular and morphological data are needed to confirm this. This study demonstrates the need to reevaluate the taxonomy of crinoid species and their respective diagnostic characters.     

Isozymes and general protein patterns as taxonomic markers in the taxon Nephtyidae (Annelida Polychaeta)

Seven Nephtys species and one species of the genus Aglaophamus (collected from different European tidal and subtidal locations between 1989 and 1991) were compared with respect to six isozyme systems (-amylase, esterase, hexokinase, malate dehydrogenase, malic enzyme, phosphoglucoseisomerase) as well as with respect to general protein patterns with non-specific staining. The proteins were obtained from the tissue (proboscis, individual segments) of single individuals, the species of which had previously been accurately determined, and were separated by isoelectric focusing (IEF) in polyacrylamide gels. The enzymes were identified by their specific catalytic activities, and the general proteins were visualized by silver staining. All the isozymes proved to be monomorphic within each of the various polychaete populations. With a single exception, it was impossible to distinguish geographically separated populations of the same species, because the band patterns were completely consistent within the species. The individual enzymes varied in their suitability for species differentiation. Only with respect to esterase and -amylase could all species be distinguished; the other enzymes studied were identical in morphologically similar species. In contrast, each species could be identified by its general protein pattern, although some species differed from others in the position of a few bands only. Individuals of N. hombergii, N. caeca and N. cirrosa with abnormally shaped parapodia, as well as juvenile nephtyids, could be unequivocally assigned to their respective species by IEF. Only in the case of N. longosetosa were two morphological variants found to differ in their electrophoretic characteristics; one of them is interpreted as a new species, not previously described.     

Biodiversity in the ecosystem of the Portuguese continental shelf: distributional ecology and the role of benthic amphipods

The biodiversity and distributional ecology of amphipod crustaceans were analysed from benthic surveys in 1985 and 1986 along the coast of Portugal, from the Tagus canyon to the coast of Minho, in order to determine their role in the ecosystem of the Portuguese continental self. In samples obtained from different types of substrate between 18 and 545 m, we identified 113 species belonging to 51 genera of 24 families. Seventeen of these species were new records for the Portuguese coast. The relative frequencies and number of individuals of all species were analysed. The genus Ampelisca clearly dominated the group of the most frequent and abundant species, which also exhibited a large range of vertical distribution. Most of the 113 species were found only down to 150 m depth, corresponding roughly to the lower limit of the continental shelf. Fifteen species exhibited clear bathyal traits, being consistently found deeper than 100 m, especially in the Tagus canyon and the upper zone of the continental slope. The most frequent and abundant species were found predominatly or in large numbers in medium to fine sand bottoms. This type of substrate also contained the highest number of species. The granulometric structure of the habitat could constitute the most important factor controlling both biodiversity and development of large amphipod populations, since oxygen level, water exchange, number of available shelters, and probably the quantity and quality of nutritional resources all vary as a function of substrate texture. Because of their dominance in both frequency and abundance, several Ampelisca species may constitute an important food source for many secondary consumers of the shelf sediments, especially for demersal fishes.     

Polymorphism in the brown alga<Emphasis Type="Italic"> Dictyota dichotoma</Emphasis> (Dictyotales,Phaeophyceae) from Korea

Two morphotypes of Dictyota dichotoma of uncertain taxonomic status have been identified in Korea. The broad thallus type is common on the south and east coasts of Korea, and is similar to European D. dichotoma in gross morphology. The slender thallus type is only found on the west coast. Morphological differences between the two plant types are evident in their thallus width, their terminal bifurcations, the shapes of apices, and their thallus thickness. The differences in the size and shape of the tetrasporangia and tetraspores are also notable. Plants from the east coast exhibit a dominant asexual annual life-cycle involving sporophytes but not gametophytes, and maintain their population by the production of in-situ germlings. In the slender plants from the west coast, a sexual life-cycle dominates. The rbcL, rbcS, psaA and psbA gene sequence differences between the two morphotypes were congruent with morphological, anatomical and phenological characteristics. In spite of these differences, there is no known inbreeding barrier between these two morphotypes. The L_m/L_c values of the second internodes from two types are also within the range of the species criterion specified by Hörnig et al. These results suggest that the two types of D. dichotoma from Korea should be classified as a single species and that they may be in the process of speciation due to geographical isolation (allopatric speciation).Communicated by O. Kinne, Oldendorf/Luhe     

Genetic evidence for cryptic speciation in the marine bryozoan Alcyonidium gelatinosum

The common subtidal marine bryozoan Alcyonidium gelatinosum (L.) is one of the most confusing species of a taxonomically very difficult genus. It shows massive variation for almost all morphological characters. A number of sympatric colonies collected off the coast of Lundy (Bristol Channel) could be clearly divided on morphological grounds into two quite distinct types. Using starch gel electrophoresis the products of a range of genetic enzyme loci were compared between the two morphotypes and also between these and other Bristol Channel populations of A. gelatinosum. Minimal differences were observed between one Lundy morphotype and the other allopatric populations, which it was consequently concluded were conspecific. The second morphotype showed considerable genetic differentiation [genetic identity (Nei, 1972) =0.417] from the other Lundy population, with no common alleles at some loci. The probability of obtaining the observed results by chance in a single population is significantly low (P<10^–14). It is therefore concluded that the two morphotypes of A. gelatinosum from Lundy are not conspecific.     

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.     

Genetic evidence for the occurrence of a cryptic species with the littoral nemerteans Lineus ruber and L. viridis (Nemertea: Anopla)

Lineus ruber and L. viridis are common intertidal heteronemerteans found on the coasts of northern Europe and the east coast of North America. The two species resemble each other morphologically and were synonymised for many years prior to being separated on the basis of larval development. In this study, specimens of L. ruber and L. viridis were collected from sites along the west and southwest coast of Britain, northern France and the east coast of North America. The external morphology and allele frequencies of isozymes of the two species were compared from all sites. The external morphology of L. ruber and L. viridis was similar but they could generally be separated by colour. Allele frequencies for up to 13 enzyme loci between sympatric populations of L. ruber and L. viridis indicated that these two species are genetically very different (Nei's genetic identity=0.090 to 0.083). Allele-frequency data also indicated the presence of a third genetic type occurring sympatrically with populations of both L. ruber and L. viridis on the coasts of Britain and France but not on the coast of North America. Fixed differences in allele frequencies between populations of the third genetic type and sympatric populations of L. ruber and L. viridis were observed across multiple loci. Genetic identity between the third genetic type and sympatric populations of L. ruber and L. viridis were extremely low (Nei's genetic identity =<0.078). Such large genetic differences between populations indicate a barrier to gene flow and reproductive isolation. The aberrant type, which exhibits sufficient morphological variation to prevent individuals being distinguished from those of both L. ruber and L. viridis, therefore represents a separate species. The low genetic identities found in intrageneric comparisons of species found in this study have been found in other studies on nemerteans. They may indicate systematic problems within these groups or other phenomena such as morphological stasis.     

Chemotaxonomy ofGyrocotyle (Platyhelminthes: Cercomeria) species,parasites of chimaerid fish (Holocephali), by chemometry of their fatty acids

The validity of some morphological gyrocotylid species, exclusive gut parasites of ratfishes (Holocephali), is contested. Non-morphological characters, such as fatty acid profiles, may be used to resolve taxonomic problems. FiveGyrocotyle species were studied: three inChimaera monstrosa collected between 1985 and 1987 from the north-east Atlantic (on the Norwegian coast); and two inHydrolagus colliei, collected in 1987 from the north-east Pacific (in Nanaimo, British Columbia, Canada). Their fatty acids were obtained by methanolytic extraction of worm tissue samples, followed by gas chromatography of the fatty acid methyl esters, and finally multivariate data treatment (principal component analysis) of gas chromatographic results. Complete separation of the five species was obtained; classification agrees well with that arrived at by morpholog and enzyme electrophoresis.