Genetic and morphological divergence between populations of the flatfish Platichthys flesus (L.) (Pleuronectidae)

Populations of the flounder, Platichthys flesus , were screened for electrophoretically detectable protein variation at up to 37 loci. Atlantic and North Sea populations (subspecies flesus ) were genetically very similar to one another ( I >0.99) but different from Adriatic (subspecies italicus ) and Black Sea (subspecies luscus ) populations. The values for genetic identity between subspecies were around 0.9. Diagnostic loci enabled specimens of flesus, italicus and luscus to be differentiated from one another. Samples of flesus showed two to three times the heterozygosity levels of italicus or luscus , consistent with the greater population size of flesus . Morphological comparisons enabled populations to be categorized to subspecies. Platichthys flesus italicus and luscus are valid subspecies: the terms are not synonyms. The genetic distance data provide an estimate of divergence time of the three subspecies at about 2 My ago, an estimate that seems reasonable in the light of present knowledge concerning the recent geological histories of the Mediterranean and Black Sea basins.     

Alimentary tract helminths of four pleuronectid flatfish in relation to host phylogeny and ecology

Eighteen species of helminths were analysed in relation to host–parasite specificity and the effect of host ecological preferences on the establishment of the parasite fauna in the alimentary tract of four pleuronectid flatfish, flounder Pleuronectes flesus , witch flounder Glyptocephalus cynoglossus , American plaice Hippoglossoides platessoides , and Atlantic halibut Hippoglossus hippoglossus in northern Norway. Thirteen species were generalists and the diversity of the parasite faunas decreased with increasing depths inhabited by the host. Similarities were greatest between the parasite faunas of flounder and American plaice and least between flounder and witch flounder. Host ecology, rather than phylogenetic relationships of these hosts, mainly influences the composition and diversity of the parasite communities of flatfishes in northern Norway.     

The GENIPOL European flounder Platichthys flesus L. toxicogenomics microarray: application for investigation of the response to furunculosis vaccination

The GENIPOL cDNA microarray, comprising some 14 000 elements representing 3336 unique expressed sequence tag clusters, was developed for studies on pollutant chemical effects on hepatic gene expression in the sentinel flatfish species Platichthys flesus , the European flounder. This array derived from probes obtained by targeted cloning of xenobiotic metabolising and responsive genes, subtractive suppressive hybridization of pollutant-treated fish and subtracted normalized cDNA libraries of treated fish has been successfully utilized for diagnosing effects of prototypical pollutants, endocrine disruptors and analysis of environmental effects. In the latter context, it has been recommended by the International Council for Exploration of the Sea that the array is tested in European monitoring programmes for assessment of the biological effects of contaminants. Sequence homology is sufficiently great between P. flesus and other pleuronectid flatfish to enable satisfactory use with other species, including American winter flounder and Japanese flounder, plaice, Atlantic halibut and sole. Elucidation of the effects of pollutants requires knowledge of other affectors of gene expression, and in this context the observed effects of a bacterial infection have been studied. The 'infection' was experimentally applied by challenge with a vaccine containing Aeromonas salmonicida , the bacterial agent causing furunculosis in flounder and salmonids. These results show that, despite its currently limited gene coverage, application of this array is not confined to toxicology.     

Novel microsatellites from the European plaice (Pleuronectes platessa)––identification by data mining and cross-species amplification in other flatfishes

The European plaice (Pleuronectes platessa; Pleuronectidae) is considered the most important flatfish for fisheries in Europe. Overall catches have been reduced drastically throughout the past decades, indicating a severe decline in the natural populations. However, a limited set of genetic tools are available to improve stock assessment and management for the species. Sequences of European plaice and its congener, the winter flounder (Pleuronectes americanus) deposited in the public database were searched for short tandem repeats. Out of 22 loci derived from database sequences, eight were amplified successfully and genotyping of 23 wild-caught individuals showed expected heterozygosities ranging from 0.18 to 0.84. The average allele number was 4.75 per locus (range 2–11). Successful cross-species amplification of the markers was demonstrated from two related flatfishes. All eight markers were successfully amplified from the European flounder (Platichthys flesus), a phylogenetically closely related species, whereas three of them worked in Achiroides melanorhynchus, an endemic species of Southeast Asia for which no microsatellites have been described to date.     

Evolutionary mechanisms shaping the genetic population structure of marine fishes; lessons from the European flounder (Platichthys flesus L.)

A number of evolutionary mechanisms have been suggested for generating low but significant genetic structuring among marine fish populations. We used nine microsatellite loci and recently developed methods in landscape genetics and coalescence-based estimation of historical gene flow and effective population sizes to assess temporal and spatial dynamics of the population structure in European flounder (Platichthys flesus L.). We collected 1062 flounders from 13 localities in the northeast Atlantic and Baltic Seas and found temporally stable and highly significant genetic differentiation among samples covering a large part of the species' range (global F(ST) = 0.024, P < 0.0001). In addition to historical processes, a number of contemporary acting evolutionary mechanisms were associated with genetic structuring. Physical forces, such as oceanographic and bathymetric barriers, were most likely related with the extreme isolation of the island population at the Faroe Islands. A sharp genetic break was associated with a change in life history from pelagic to benthic spawners in the Baltic Sea. Partial Mantel tests showed that geographical distance per se was not related with genetic structuring among Atlantic and western Baltic Sea samples. Alternative factors, such as dispersal potential and/or environmental gradients, could be important for generating genetic divergence in this region. The results show that the magnitude and scale of structuring generated by a specific mechanism depend critically on its interplay with other evolutionary mechanisms, highlighting the importance of investigating species with wide geographical and ecological distributions to increase our understanding of evolution in the marine environment.     

Prevalence of viral haemorrhagic septicaemia virus in Danish marine fishes and its occurrence in new host species

In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine waters around Denmark, staff from the Danish Institute for Food and Veterinary Research participated in 5 research cruises during 1998 to 2002 as a follow-up to 4 research cruises performed in 1996 to 1997. In total, 16,655 fish were examined virologically as 3569 samples. Forty fish species and 3 invertebrate species were represented. VHSV was isolated from 133 samples representing 8 species: herring Clupea harengus, sprat Sprattus sprattus, dab Limanda limanda, flounder Platichthys flesus, plaice Pleuronectes platessa, cod Gadus morhua, sand eel Ammodytes sp. and sand goby Pomatochistus minutus. Calculations showed that VHSV was more prevalent in the Baltic Sea in an area between Zealand and the island of Bornholm and the waters surrounding Bornholm than in the Kattegat, Skagerrak and along the North Sea coast of Denmark. This is the first report on the isolation of VHSV from dab, flounder and plaice and the first publication on VHSV from sand eel from Europe and sand goby.     

Adaptive divergence in a high gene flow environment: Hsc70 variation in the European flounder (Platichthys flesus L.)

Little is known about local adaptations in marine fishes since population genetic surveys in these species have typically not applied genetic markers subject to selection. In this study, we used a candidate gene approach to investigate adaptive population divergence in the European flounder (Platichthys flesus L.) throughout the northeastern Atlantic. We contrasted patterns of genetic variation in a presumably neutral microsatellite baseline to patterns from a heat-shock cognate protein gene, Hsc70. Using two different neutrality tests we found that the microsatellite data set most likely represented a neutral baseline. In contrast, Hsc70 strongly deviated from neutral expectations. Importantly, when estimating standardized levels of population divergence (F(ST)'), we also found a large discrepancy in the patterns of structuring in the two data sets. Thus, samples grouped according to geographical or historical proximity with regards to microsatellites, but according to environmental similarities with regards to Hsc70. The differences between the data sets were particularly pronounced in pairwise comparisons involving populations in the western and central Baltic Sea. For instance, the genetic differentiation between geographically close Baltic Sea and North Sea populations was found to be 0.02 and 0.45 for microsatellites and Hsc70 respectively. Our results strongly suggest adaptive population divergence and indicate local adaptations at the DNA level in a background of high levels of gene flow, typically found in many marine fish species. Furthermore, this study highlights the usefulness of the candidate gene approach for demonstrating local selection in non-model organisms such as most marine fishes.     

Infection of estuarine fish populations by Cryptocotyle lingua (Creplin)

Fish populations in the Medway Estuary become infected with Cryptocotyle lingua (Creplin) shortly after their inshore migrations. The present study examines infection levels between 1973 and 1975 in four fish species; whiting, Merlangius merlangus (L.), pouting, Trisopterus luscus (L.), plaice, Pleuronectes platessa L. and flounder, Platichthys flesus (L.). Frequency of infection was highest in flounder, followed by pouting, plaice and whiting. Unusually, parasite density was lowest in plaice even though gastropod molluscs (the parasite's first intermediate host) were most common in their diet. Seasonal levels of infection are discussed and prove useful indicators to the migratory movements of individual fish populations. In whiting and pouting, incidence of infection increased with length of host while no relationship between infection and age of host was observed in either flatfish. Metacercariae were not distributed randomly over the body surface; in gadoids they were concentrated above the lateral line while in pleuronectids they were most intense in the fins and gills.     

Diel variability in catch rate of juvenile flatfish on two small nursery grounds (Port Erin Bay, Isle of Man and Porto Pirn Bay, Faial, Azores)

Variation in beach seine catches was examined in two juvenile flatfish populations (Port Erin, Isle of Man and Porto Pirn, Azores) to determine the importance of diel cycles while controlling for tidal rhythmicity. At Port Erin sampling was undertaken at each low water on consecutive days over a 2-week period in May/June and September 1991. There was no periodicity in the catches of plaice ( Pleuronectes platessa ) in May/June, but in September catches were higher at night. Other flatfish species ( Limanda limanda, Pleuronectes flesus, Scophthalmus maximus and S. rhombus ) sampled at Port Erin were nocturnal. At Porto Pirn, Azores, fish were sampled over 24 h each month from July 1989 to June 1990 and at each low water over a 2-week period in July 1991. The only flatfish species present was Bothuspodas , which had a diurnal pattern in catches over a 1-year period. Sampling at low water in July gave high catches at both midnight and midday.     

Strong population genetic structure and contrasting demographic histories for the small-spotted catshark (Scyliorhinus canicula) in the Mediterranean Sea

Coastal and demersal chondrichthyans, such as the small-spotted catshark, are expected to exhibit genetic differentiation in areas of complex geomorphology like the Mediterranean Basin because of their limited dispersal ability. To test this hypothesis, we used a fragment of the mitochondrial cytochrome c oxidase subunit I gene and 12 nuclear microsatellite loci in order to investigate the genetic structure and historical demography of this species, and to identify potential barriers to gene flow. Samples were collected from the Balearic Islands, the Algerian Basin, the Ionian Sea, the Corinthian Gulf and various locations across the Aegean Sea. Additional sequences from the Atlantic and the Levantine Basin retrieved from GenBank were included in the mitochondrial DNA analysis. Both mitochondrial and nuclear microsatellite DNA data revealed a strong genetic subdivision, mainly between the western and eastern Mediterranean, whereas the Levantine Basin shared haplotypes with both areas. The geographic isolation of the Mediterranean basins seems to enforce the population genetic differentiation of the species, with the deep sea acting as a strong barrier to its dispersal. Contrasting historical demographic patterns were also observed in different parts of the species'' distribution, most notably a population growth trend in the western Mediterranean/Atlantic area and a slight decreasing one in the Aegean Sea. The different effects of the Pleistocene glacial periods on the habitat availability may explain the contrasting demographic patterns observed. The current findings suggest that the small-spotted catshark exhibits several genetic stocks in the Mediterranean, although further study is needed.     

Comparative Analysis of a Mitochondrial DNA Control Region Fragment Amplified from Three Adriatic Flatfish Species and Molecular Phylogenesis of Pleuronectiformes

The 5′-end of the mitochondrial control region of three Pleuronectiformes from the Adriatic Sea, Platichthys flesus italicus (Adriatic flounder), Solea vulgaris (common sole), and Solea kleini (Klein's sole), was sequenced and compared with that of six other flatfish species from the families Pleuronectidae and Bothidae. The sequence structures of all flatfishes appear very similar and consist of alternate short segments with low, medium, and high rates of nucleotide substitution. Four conserved 19-bp repeats occur at the beginning of the European and Adriatic flounder sequences. The common occurrence of tandem arrays in fish control regions could be related to a stable secondary structure. Molecular phylogenetic relationships among Pleuronectiformes agree well with previous morphologic data at all taxonomic levels. Molecular analyses could therefore contribute to resolving phylogenetic and taxonomic debates within the Pleuronectiformes. Received December 1, 1997; accepted June 30, 1998.     

Polymorphic microsatellite loci for the cardinal fish (<Emphasis Type="Italic">Apogon imberbis</Emphasis>)

Eight polymorphic microsatellite loci were isolated and characterized for the Cardinal fish (Apogon imberbis), a coastal-reef fish endemic to the Mediterranean Sea. Characterization of 30 Cardinal fish individuals form the western Mediterranean showed moderate to high allelic diversity ranging from 6 to 19 alleles per locus. Two loci showed significant departures from Hardy-Weinberg equilibrium presumably due to null alleles. No evidence of linkage disequilibrium was found for any locus pairwise comparasions. This microsatellite set could be useful for any basic population genetic studies of this species.     

Molecular and morphologic approaches to discrimination of variability patterns in chub mackerel, Scomber japonicus

The systematic status and the evolutionary biology of chub mackerel (Scomber japonicus) in the South West Atlantic Ocean is confusing with an unknown degree of genetic differentiation and reproductive isolation between units. Simultaneous genetic and morphologic analyses were made on 227 fish collected from two areas of the South West Atlantic Ocean and one from the Mediterranean Sea. The genetic analysis was based on 36 protein-coding loci, 16 of which were variable. The morphologic analyses include six morphometric length measurements and a meristic character. Correspondence between genetic and morphologic variability patterns indicates isolated Mediterranean and Southwest Atlantic subgroups of S. japonicus and, less clearly, possible additional divergence in two regional stocks within the latter group. The most conservative approach to management is to manage the stocks independently of one another.     

New microsatellites primers for plaice,Pleuronectes platessa L. (Teleostei: Pleuronectidae)

Seven microsatellite loci were developed for Pleuronectes platessa, a commercially important flatfish found throughout northern European waters. The number of alleles per locus ranged from 11 to 33 and heterozygosities from 0.74 to 0.96. Cross‐reactivity was tested against six other flatfish species. Significant amplification was found in all species from one to all seven of the loci.     

Mitochondrial DNA reveals a mosaic pattern of phylogeographical structure in Atlantic and Mediterranean populations of anchovy (Engraulis encrasicolus)

This study extends the geographic coverage of a previous study of mitochondrial DNA restriction fragment length polymorphism in European anchovy. Both studies together include 24 samples representing 17 localities extending from the Black Sea, through the Mediterranean Sea to the eastern Atlantic as far south as Dakar, Senegal. Eighty-eight haplotypes define two clades (A and B) separated by 3.2% sequence divergence. Clade A has a star-like genealogy indicative of a recent population expansion. Clade B has a more complex genealogy, consisting of several haplotypes at intermediate frequencies. The distributions of these clades consist of a mosaic with abrupt changes between some areas and gradients between other areas. Clade A predominates the Black and Aegean seas, but is present throughout the Mediterranean. Unexpectedly, new data show that clade A is also at a high frequency in the Atlantic, from Portugal to at least Senegal. Overall, the level of genetic differentiation among populations is high (F(ST)=0.148, p<0.0001), with the greatest differences between basins. AMOVA reveals four main geographical groups: Atlantic, central Mediterranean, Aegean Sea, and Black Sea. Mismatch distribution clearly indicates historical bottleneck and population expansion for clade A, while for clade B such evidence is equivocal. This difference may reflect a range expansion for both clades, but with higher gene flow (Nm values) between demes for clade A. Both contemporary and historical processes are important in shaping the complex genetic population structure of European anchovy.     

Fine Spatial Structure of Atlantic Hake (<Emphasis Type="Italic">Merluccius merluccius</Emphasis>) Stocks Revealed by Variation at Microsatellite Loci

Genetic variation at 5 microsatellite loci was analyzed for European hake Merluccius merluccius sampled from 9 different regions in the Atlantic Ocean and the Mediterranean Sea. Significant genetic differentiation was found between samples, suggesting a fine subdivision of Atlantic and Mediterranean hake stocks. These results are discussed in the context of the decline of demersal fish species, probably due to overfishing.     

Exploitation of a turbot (Scophthalmus maximus L.) immune-related expressed sequence tag (EST) database for microsatellite screening and validation

In this study, we identified and characterized 160 microsatellite loci from an expressed sequence tag (EST) database generated from immune-related organs of turbot (Scophthalmus maximus). A final set of 83 new polymorphic microsatellites were validated after the analysis of 40 individuals of Atlantic origin including both wild and farmed individuals. The allele number and the expected heterozygosity ranged from 2 to 18 and from 0.021 to 0.951, respectively. Evidences of null alleles at moderate-high frequencies were detected at six loci using population data. None of the analysed loci showed deviations from Mendelian segregation after the analysis of five full-sib families including approximately 92 individuals/family. The markers are used to consolidate the turbot genetic map, and because they are mostly EST-derived, they will be very useful for comparative genomic studies within flatfishes and with model fish species. Using an in silico approach, we detected significant homologies of microsatellite sequences with the EST databases of the flatfish species with highest genomic resources (Senegalese sole, Atlantic halibut, bastard halibut) in 31% of these turbot markers. The conservation of these microsatellites within Pleuronectiformes will pave the way for anchoring genetic maps of different species and identifying genomic regions related to productive traits.     

Isolation and characterization of 28 new microsatellite markers for European flounder (Platichthys flesus L.)

The European flounder (Platichthys flesus L.) is used in ecotoxicological studies to provide detailed information on the effects of pollution on individual fish. Data on population and evolutionary level effects are, however, limited. Here, the isolation and characterization of 28 novel species specific microsatellite loci are presented. The number of alleles ranged from 8 to 38, and observed heterozygosity from 0.542 to 1.     

Parasites as biological markers: evolutionary relationships in the heterospecific combination of helminths (monogeneans) and teleosts (Gadidae)

Analysis of the genetic structure (11 loci) of helminth populations (Monogenea, Diclidophora ), parasites of two teleost species (Gadidae, Trisopterus luscus and T. minutus minulus ) which are sympatric in the Atlantic, revealed the existence of two species which were specific to each of the two hosts. A strong genetic similitude was found between one of these species ( Diclidophora luscae ), a parasite of T. luscus and a monogenean population parasitizing a third Gadidae ( Trisopterus minutus capelanus ) in the Mediterranean. These results led to a re-examinination of the evolutionary systematics of the three host taxa of the genus Trisopterus. Genetic analysis showed differences between the two subspecies T. minutus minutus (Atlantic) and T. minutus capelanus (Mediterranean) at eight of the 16 loci studied. However, there was no significant genetic divergence between T. luscus and T.m. capelanus. A new phyletic structure in the genus Trisopterus is proposed and the evolutionary trends within the Gadidae/Monogenea system are discussed.     

Genetic diversity of the imperilled bath sponge Spongia officinalis Linnaeus, 1759 across the Mediterranean Sea: patterns of population differentiation and implications for taxonomy and conservation

The Mediterranean bath sponge Spongia officinalis is an iconic species with high socio‐economic value and precarious future owing to unregulated harvesting, mortality incidents and lack of established knowledge regarding its ecology. This study aims to assess genetic diversity and population structure of the species at different geographical scales throughout its distribution. For this purpose, 11 locations in the Eastern Mediterranean (Aegean Sea), Western Mediterranean (Provence coast) and the Strait of Gibraltar were sampled; specimens were analysed using partial mitochondrial cytochrome oxidase subunit I (COI) sequences, along with a set of eight microsatellite loci. According to our results (i) no genetic differentiation exists among the acknowledged Mediterranean morphotypes, and hence, S. officinalis can be viewed as a single, morphologically variable species; (ii) a notable divergence was recorded in the Gibraltar region, indicating the possible existence of a cryptic species; (iii) restriction to gene flow was evidenced between the Aegean Sea and Provence giving two well‐defined regional clusters, thus suggesting the existence of a phylogeographic break between the two systems; (iv) low levels of genetic structure, not correlated to geographical distance, were observed inside geographical sectors, implying mechanisms (natural or anthropogenic) that enhance dispersal and gene flow have promoted population connectivity; (v) the genetic diversity of S. officinalis is maintained high in most studied locations despite pressure from harvesting and the influence of devastating epidemics. These findings provide a basis towards the effective conservation and management of the species.