Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Diversity and host specificity of monogenean gill parasites (Platyhelminthes) of cichlid fishes in the Bangweulu-Mweru ecoregion.

This study represents the first exploration of the parasite fauna of cichlid fishes in the Mweru-Luapula subregion (Central Africa). Twelve species of cichlids and 14 species of Monogenea from three genera (Cichlidogyrus, Gyrodactylus and Scutogyrus) were collected. We present a first record of the gill parasite fauna of eight host species, Oreochromis mweruensis, Orthochromis sp. 'Mambilima', Sargochromis mellandi, Serranochromis angusticeps, S. stappersi, S. thumbergi and Tylochromis mylodon. The host range of ten parasite species was expanded. The study further includes the description of Cichlidogyrus consobrini sp. n. from S. mellandi and Orthochromis sp. 'Mambilima'. A new morphotype of C. halli is characterized, and three species - C. papernastrema, C. quaestio and C. zambezensis - are redescribed. Furthermore, the biodiversity and host specificity of these parasites is compared with that of cichlid parasites from Lake Kariba and Cameroon. Two species, including C. consobrini sp. n. and a new morphotype of C. halli, are putative endemics. The parasite fauna in Bangweulu-Mweru is highly similar in species composition to Lake Kariba, but in Bangweulu-Mweru the same parasite species are more host-specific, probably because of hydrogeographical differences between the two regions.

Parasite escape through trophic specialization in a species flock.

Adaptive radiation occurs when species diversify rapidly to occupy an array of ecological niches. As opportunities for parasite infection and transmission may greatly vary among these niches, adaptive radiation is expected to be associated with a turnover of the parasite community. As major agents of natural and sexual selection, parasites may play a central role in host diversification. The study of parasite turnover may thus be of general relevance and could significantly improve our understanding of adaptive radiation. In this study, we examined the parasite faunas of eleven species belonging to the tribe Tropheini, one of several adaptive radiations of cichlid fishes in Lake Tanganyika. The most parsimonious ancestral foraging strategy among the Tropheini is relatively unselective substrate browsing of aufwuchs. Several lineages evolved more specialized foraging strategies, such as selective combing of microscopic diatoms or picking of macro-invertebrates. We found that representatives of these specialized lineages bear reduced infection with food-web-transmitted acanthocephalan helminths, but not with parasites with a direct life cycle. Possibly, the evolution of selective foraging strategies entailed reduced ingestion of intermediate invertebrate hosts of acanthocephalans. We conclude that some species belonging to the Tropheini virtually escape acanthocephalan infection as a by-product of trophic specialization.

Weak link between dispersal and parasite community differentiation or immunogenetic divergence in two sympatric cichlid fishes.

Geographical isolation, habitat variation and trophic specialization have contributed to a large extent to the astonishing diversity of cichlid fishes in the Great East African lakes. Because parasite communities often vary across space and environments, parasites can accompany and potentially enhance cichlid species diversification. However, host dispersal may reduce opportunities for parasite-driven evolution by homogenizing parasite communities and allele frequencies of immunity genes. To test for the relationships between parasite community variation, host dispersal and parasite-induced host evolution, we studied two sympatric cichlid species with contrasting dispersal capacities along the shores of southern Lake Tanganyika. Whereas the philopatric Tropheus moorii evolved into several genetically differentiated colour morphs, Simochromis diagramma is phenotypically rather uniform across its distribution range and shows only weak population structure. Populations of both species were infected with divergent parasite communities and harbour differentiated variant pools of an important set of immune genes, the major histocompatibility complex (MHC). The overall extent of geographical variation of parasites and MHC genes was similar between host species. This indicates that immunogenetic divergence among populations of Lake Tanganyika cichlids can occur even in species that are strongly dispersing. However, because this also includes species that are phenotypically uniform, parasite-induced evolution may not represent a key factor underlying species diversification in this system.

Intermediate number of major histocompatibility complex class IIB length variants relates to enlarged perivisceral fat deposits in the blunt-head cichlid Tropheus moorii.

Studying the genetic basis of host-parasite interactions represents an outstanding opportunity to observe eco-evolutionary processes. Established candidates for such studies in vertebrates are immunogenes of the major histocompatibility complex (MHC). The MHC has been reported to reach high intra- and interindividual diversity, and a diverse MHC might be advantageous when facing infections from multiple parasites. However, other studies indicated that individuals with an intermediate number of MHC alleles are less infected with parasites or have other fitness advantages. In this study, we assessed the optimal number of MHC alleles in the blunt-head cichlid Tropheus moorii from Lake Tanganyika. We investigated the influence of the interindividual variation in number of MHC length variants on parasite infection and body condition, measured by the amount of perivisceral fat reserves. Surprisingly, there was no correlation between parasite infection and number of MHC length variants or perivisceral fat deposits. However, the individual number of MHC length variants significantly correlated with the amount of perivisceral fat deposits in males, suggesting that male individuals with an intermediate number of alleles might be able to use their fat reserves more efficiently.

Inbreeding within human Schistosoma mansoni: do host-specific factors shape the genetic composition of parasite populations?

The size, structure and distribution of host populations are key determinants of the genetic composition of parasite populations. Despite the evolutionary and epidemiological merits, there has been little consideration of how host heterogeneities affect the evolutionary trajectories of parasite populations. We assessed the genetic composition of natural populations of the parasite Schistosoma mansoni in northern Senegal. A total of 1346 parasites were collected from 14 snail and 57 human hosts within three villages and individually genotyped using nine microsatellite markers. Human host demographic parameters (age, gender and village of residence) and co-infection with Schistosoma haematobium were documented, and S. mansoni infection intensities were quantified. F-statistics and clustering analyses revealed a random distribution (panmixia) of parasite genetic variation among villages and hosts, confirming the concept of human hosts as 'genetic mixing bowls' for schistosomes. Host gender and village of residence did not show any association with parasite genetics. Host age, however, was significantly correlated with parasite inbreeding and heterozygosity, with children being more infected by related parasites than adults. The patterns may be explained by (1) genotype-dependent 'concomitant immunity' that leads to selective recruitment of genetically unrelated worms with host age, and/or (2) the 'genetic mixing bowl' hypothesis, where older hosts have been exposed to a wider variety of parasite strains than children. The present study suggests that host-specific factors may shape the genetic composition of schistosome populations, revealing important insights into host-parasite interactions within a natural system.

Regional environmental pressure influences population differentiation in turbot (Scophthalmus maximus).

Unravelling the factors shaping the genetic structure of mobile marine species is challenging due to the high potential for gene flow. However, genetic inference can be greatly enhanced by increasing the genomic, geographical or environmental resolution of population genetic studies. Here, we investigated the population structure of turbot (Scophthalmus maximus) by screening 17 random and gene-linked markers in 999 individuals at 290 geographical locations throughout the northeast Atlantic Ocean. A seascape genetics approach with the inclusion of high-resolution oceanographical data was used to quantify the association of genetic variation with spatial, temporal and environmental parameters. Neutral loci identified three subgroups: an Atlantic group, a Baltic Sea group and one on the Irish Shelf. The inclusion of loci putatively under selection suggested an additional break in the North Sea, subdividing southern from northern Atlantic individuals. Environmental and spatial seascape variables correlated marginally with neutral genetic variation, but explained significant proportions (respectively, 8.7% and 10.3%) of adaptive genetic variation. Environmental variables associated with outlier allele frequencies included salinity, temperature, bottom shear stress, dissolved oxygen concentration and depth of the pycnocline. Furthermore, levels of explained adaptive genetic variation differed markedly between basins (3% vs. 12% in the North and Baltic Sea, respectively). We suggest that stable environmental selection pressure contributes to relatively strong local adaptation in the Baltic Sea. Our seascape genetic approach using a large number of sampling locations and associated oceanographical data proved useful for the identification of population units as the basis of management decisions.

Hybridisation between the two major African schistosome species of humans.

It is generally accepted that Schistosoma mansoni and Schistosoma haematobium, causing intestinal and urinary schistosomiasis, respectively, are not able to hybridise, due to the high phylogenetic distance between them. Cloning of nuclear internal transcribed spacer rDNA and partial mitochondrial cytochrome c oxidase 1 fragments revealed two internal transcribed spacer rDNA genotypes within single eggs and miracidia, one identical to S. mansoni and the other identical to S. haematobium, suggesting hybrid ancestry. The cytochrome c oxidase 1 clones always belonged to only one of the parental species. This demonstrates that offspring of heterologous pairing between these two species is not (always) parthenogenetic.

Regular treatments of praziquantel do not impact on the genetic make-up of Schistosoma mansoni in Northern Senegal.

The Senegal River Basin (SRB) experienced a major epidemic of intestinal schistosomiasis in the early nineties, after the construction of a dam for irrigation purposes. Exceptionally low cure rates following praziquantel (PZQ) treatment at the onset of the epidemic raised concerns about PZQ resistant strains of Schistosoma mansoni, although they could also be attributed to the intense transmission at that time. A field study in the same region more than 15 years later found cure rates for S. mansoni still to be low, whereas Schistosomahaematobium responded well to treatment. We collected S. mansoni miracidia from children at base-line prior to treatment, six months after two PZQ treatments and two years after the start of the study when they had received a total of five PZQ treatments. In total, 434 miracidia from 12 children were successfully genotyped with at least six out of nine DNA microsatellite loci. We found no significant differences in the genetic diversity of, and genetic differentiation between parasite populations before and after repeated treatment, suggesting that PZQ treatment does not have an impact on the neutral evolution of the parasite. This is in stark contrast with a similar study in Tanzania where a significant decrease in genetic diversity was observed in S. mansoni miracidia after a single round of PZQ treatment. We argue that PZQ resistance might play a role in our study area, although rapid re-infection cannot be excluded. It is important to monitor this situation carefully and conduct larger field studies with short-term follow-up after treatment. Since PZQ is the only general schistosomicide available, the possibility of PZQ resistance is of great concern both for disease control and for curative use in clinical practice.

Gene transcription reflects poor health status of resident European eel chronically exposed to environmental pollutants.

Understanding the effects of chronic exposure to pollutants on the genome and transcriptome of diadromous fish populations is crucial for their resilience under combined anthropogenic and environmental selective pressures. The catadromous European eel (Anguilla anguilla L.) has suffered a dramatic decline in recruitment for three decades, necessitating a thorough assessment of the transcriptional effects of environmental pollutants on resident and migrating eels in natural systems. We investigated the relationship between muscular bioaccumulation levels of metals (Hg, Cd, Pb, Cu, Zn, Ni, Cr, As and Se), PCBs and organochlorine pesticides (DDTs), the health status (condition factor and lipid reserves) and the associated transcriptional response in liver and gill tissues for genes involved in metal detoxification (metallothionein, MT) and oxidative metabolism (cytochrome P4501A, CYP1A) of xenobiotic compounds. In total 84 resident eels originating from three Belgian river basins (Scheldt, Meuse and Yzer) were analyzed along with five unpolluted aquaculture samples as control group. There was a large spatial variation in individual contaminant intensity and profile, while tissue pollution levels were strongly and negatively associated with condition indices, suggesting an important impact of pollution on the health of sub-adult resident eels. Gene transcription patterns revealed a complex response mechanism to a cocktail of pollutants, with a high variation at low pollution levels, but strongly down-regulated hepatic and gill gene transcription in highly polluted eels. Resident eels clearly experience a high pollution burden and seem to show a dysfunctional gene transcription regulation of detoxification genes at higher pollutant levels, correlated with low energy reserves and condition. To fully understand the evolutionary implications of pollutants on eel reproductive fitness, analyses of mature migrating eels and the characterization of their transcriptome-wide gene transcription response would be appropriate to unveil the complex responses associated with multiple interacting stressors and the long-term consequences at the entire species level. In the meanwhile, jointly monitoring environmental and tissue pollution levels at a European scale should be initiated, while preserving high quality habitats to increase the recovery chance of European eel in the future.

Temporal genetic stability and high effective population size despite fisheries-induced life-history trait evolution in the North Sea sole.

Heavy fishing and other anthropogenic influences can have profound impact on a species' resilience to harvesting. Besides the decrease in the census and effective population size, strong declines in mature adults and recruiting individuals may lead to almost irreversible genetic changes in life-history traits. Here, we investigated the evolution of genetic diversity and effective population size in the heavily exploited sole (Solea solea), through the analysis of historical DNA from a collection of 1379 sole otoliths dating back from 1957. Despite documented shifts in life-history traits, neutral genetic diversity inferred from 11 microsatellite markers showed a remarkable stability over a period of 50 years of heavy fishing. Using simulations and corrections for fisheries induced demographic variation, both single-sample estimates and temporal estimates of effective population size (N(e) ) were always higher than 1000, suggesting that despite the severe census size decrease over a 50-year period of harvesting, genetic drift is probably not strong enough to significantly decrease the neutral diversity of this species in the North Sea. However, the inferred ratio of effective population size to the census size (N(e) /N(c) ) appears very small (10(-5) ), suggesting that overall only a low proportion of adults contribute to the next generation. The high N(e) level together with the low N(e) /N(c) ratio is probably caused by a combination of an equalized reproductive output of younger cohorts, a decrease in generation time and a large variance in reproductive success typical for marine species. Because strong evolutionary changes in age and size at first maturation have been observed for sole, changes in adaptive genetic variation should be further monitored to detect the evolutionary consequences of human-induced selection.

Optimal sample storage and extraction procotols for reliable multilocus genotyping of the human parasite Schistosoma mansoni.

Genotyping individual larval stages and eggs of natural parasite populations is complicated by the difficulty of obtaining reliable genotypes from low quantity DNA template. A suitable storage and extraction protocol, together with a thorough quantification of genotyping errors are therefore crucial for molecular epidemiological studies. Here we test the robustness, handling time, ease of use, cost effectiveness and success rate of various fixation (Whatman FTA(®) Classic and Elute Cards, 70% EtOH and RNAlater(®)) and subsequent DNA extraction methods (commercial kits and proteinase K protocol). None of these methods require a cooling chain and are therefore suitable for field collection. Based on a multiplex microsatellite PCR with nine loci the success and reliability of each technique is evaluated by the proportion of samples with at least eight scored loci and the proportion of genotyping errors. If only the former is taken into account, FTA(®) Elute is recommended (83% success; 44% genotyping error; 0.2 €/sample; 1h 20 m handling time). However, when also considering the genotyping errors, handling time and ease of use, we opt for 70% EtOH with the 96-well plate technology followed by a simple proteinase K extraction (73% success; 0% genotyping error; 0.2 €/sample; 15m handling time). For eggs we suggest (1) to pool all eggs per person in 1.5 ml tubes filled with 70% EtOH for transport and (2) to identify each egg to species level prior to genotyping. To this end we extended the Rapid diagnostic PCR developed by Webster et al. (2010) with a S. mansoni-specific primer to discriminate between S. mansoni, S. haematobium and S. bovis in a single PCR reaction. The success rate of genotyping eggs was 75% (0% genotyping error). This is the first study to incorporate genotyping errors through re-amplification for the evaluation of schistosome sampling protocols and the identification of error-prone loci.

Signature of selection on the rhodopsin gene in the marine radiation of American seven-spined gobies (Gobiidae, Gobiosomatini).

In comparison with terrestrial and freshwater ecosystems, information about speciation modes and the role of selection in marine environments is scarce. Recent studies have indicated that spectral adaptation could play an important role in the diversification of marine species flocks. Natural selection influences specific amino acids (AAs) that are involved in the spectral tuning mechanism of visual pigment genes. To study the wider occurrence and the characteristics of spectral adaptation in marine radiations, a reinterpretation of the rhodopsin (RH1) data of American seven-spined gobies (genus Elacatinus; Gobiidae; Teleostei) was carried out. Reanalysis revealed that some AAs, which are well known in the literature as spectral tuning sites, are variable in Elacatinus. Those crucial AA substitutions originated polyphyletically, indicating convergent evolution within the genus Elacatinus. Moreover, statistical tests based on the d(N)/d(S) ratio detected selection in several phylogenetic lineages and at specific AAs. Many of these AAs were previously shown to be under selection in other marine radiations. Therefore, the current phylogenetic approach provided an extended list of AAs that are probably involved in spectral tuning, and which should be validated by mutagenic experiments.

Mito-nuclear discordance in the degree of population differentiation in a marine goby.

An increasing number of phylogeographic studies on marine species shows discordant patterns in the degree of population differentiation between nuclear and mitochondrial markers. To understand better which factors have the potential to cause these patterns of discordance in marine organisms, a population genetic study was realized on the sand goby Pomatoschistus minutus (Pallas 1770; Gobiidae, Teleostei). Sand gobies from eight European locations were genotyped at eight microsatellite markers. Microsatellites confirmed the global phylogeographical pattern of P. minutus observed with mitochondrial DNA (mtDNA) markers and nuclear allozyme markers. Three groups consistent with the mitochondrial lineages were defined (the Mediterranean, Iberian and North Atlantic groups) and indications of a recent founder event in the northern Baltic Sea were found. Nevertheless, differences in the degree of population differentiation between the nuclear and mitochondrial markers were large (global F(ST)-values for microsatellites=0.0121; for allozymes=0.00831; for mtDNA=0.4293). Selection, sex-biased dispersal, homoplasy and a high effective population size are generally accepted as explanations for this mitonuclear discrepancy in the degree of population differentiation. In this study, selection on mtDNA and microsatellites, male-biased dispersal and homoplasy on microsatellite markers are unlikely to be a main cause for this discrepancy. The most likely reason for the discordant pattern is a recent demographical expansion of the sand goby, resulting in high effective population sizes slowing down the differentiation of nuclear DNA.

QTL for body weight, morphometric traits and stress response in European sea bass Dicentrarchus labrax.

Natural mating and mass spawning in the European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) complicate genetic studies and the implementation of selective breeding schemes. We utilized a two-step experimental design for detecting QTL in mass-spawning species: 2122 offspring from natural mating between 57 parents (22 males, 34 females and one missing) phenotyped for body weight, eight morphometric traits and cortisol levels, had been previously assigned to parents based on genotypes of 31 DNA microsatellite markers. Five large full-sib families (five sires and two dams) were selected from the offspring (570 animals), which were genotyped with 67 additional markers. A new genetic map was compiled, specific to our population, but based on the previously published map. QTL mapping was performed with two methods: half-sib regression analysis (paternal and maternal) and variance component analysis accounting for all family relationships. Two significant QTL were found for body weight on linkage group 4 and 6, six significant QTL for morphometric traits on linkage groups 1B, 4, 6, 7, 15 and 23 and three suggestive QTL for stress response on linkage groups 3, 14 and 23. The QTL explained between 8% and 38% of phenotypic variance. The results are the first step towards identifying genes involved in economically important traits like body weight and stress response in European sea bass.

Evolutionary history shapes the association between developmental instability and population-level genetic variation in three-spined sticklebacks.

Developmental instability (DI) is the sensitivity of a developing trait to random noise and can be measured by degrees of directionally random asymmetry [fluctuating asymmetry (FA)]. FA has been shown to increase with loss of genetic variation and inbreeding as measures of genetic stress, but associations vary among studies. Directional selection and evolutionary change of traits have been hypothesized to increase the average levels of FA of these traits and to increase the association strength between FA and population-level genetic variation. We test these two hypotheses in three-spined stickleback (Gasterosteus aculeatus L.) populations that recently colonized the freshwater habitat. Some traits, like lateral bone plates, length of the pelvic spine, frontal gill rakers and eye size, evolved in response to selection regimes during colonization. Other traits, like distal gill rakers and number of pelvic fin rays, did not show such phenotypic shifts. Contrary to a priori predictions, average FA did not systematically increase in traits that were under presumed directional selection, and the increases observed in a few traits were likely to be attributable to other factors. However, traits under directional selection did show a weak but significantly stronger negative association between FA and selectively neutral genetic variation at the population level compared with the traits that did not show an evolutionary change during colonization. These results support our second prediction, providing evidence that selection history can shape associations between DI and population-level genetic variation at neutral markers, which potentially reflect genetic stress. We argue that this might explain at least some of the observed heterogeneities in the patterns of asymmetry.

Conservation of the introgressed European water frog complex using molecular tools.

In Belgium, the Pelophylax esculentus complex has recently been subjected to multiple introductions of non-native water frogs, increasing the occurrence of hybridisation events. In the present study, we tested the reliability of morphometric and recently developed microsatellite tools to identify introgression and to determine the origin of exotic Belgian water frogs. By analysing 150 individuals of each taxon of the P. esculentus complex and an additional 60 specimens of the introduced P. cf. bedriagae, we show that neither of the currently available tools appears to have sufficient power to reliably distinguish all Belgian water frog species. We therefore aimed at increasing the discriminatory power of a microsatellite identification tool by developing a new marker panel with additional microsatellite loci. By adding only two new microsatellite loci (RlCA5 and RlCA1b20), all taxa of the P. esculentus complex could be distinguished from each other with high confidence. Three more loci (Res3, Res5 and Res17) provided a powerful discrimination of the exotic species.

Influence of DNA isolation from historical otoliths on nuclear-mitochondrial marker amplification and age determination in an overexploited fish, the common sole (Solea solea L.).

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole (Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material.

Morphological and genetic seasonal dynamics of European eel Anguilla anguilla recruitment in southern France.

The fine scale morphological and genetic dynamics of successive waves of Anguilla anguilla glass eel recruitment was studied over a 2 year period at a southern European Mediterranean location (Camargue, France) with continuous recruitment. Using morphometric [total length (L(T)), mass (M), condition (K) and pigmentation stage] as well as genetic (allozyme) markers, the aim was to test for the existence of temporally separated spawning groups and explore the relation between genetic variability and morphological heterogeneity of recruits. The results showed that L(T), M and K varied over time, being highest from the end of summer to winter (peaking in December) and lowest in spring (lowest in April). The pigmentation stages within monthly samples were highly diverse with a heterogeneous seasonal pattern. Allozyme data showed high genetic variability values within samples, but low genetic differentiation among samples (F(ST) = 0.003, P < 0.05). Pairwise comparisons between samples indicated a positive correlation between genetic differentiation and difference in recruitment time (days), with a marked increase in genetic differentiation around 250 days between monthly recruitment samples. Furthermore, genetic diversity increased with the number of pigmentation stages per sample and was negatively correlated with the North Atlantic Oscillation (NAO) index during the putative year of trans-oceanic migration. No correlation, however, was found between the level of multilocus heterozygosity (MLH) and growth variables. A situation of genetic patchiness with fluctuating parental contribution can thus best explain the patterns observed, although the existence of two separate spawning periods cannot be excluded. More discriminatory and sensitive genetic markers, such as (neutral and adaptive) microsatellites, could probably provide additional insights into the most probable hypothesis explaining the population structure and recruitment heterogeneity of A. anguilla.

Matrilinear phylogeography and demographical patterns of Rutilus rutilus: implications for taxonomy and conservation.

A phylogeographic analysis of mitochondrial DNA sequence variation was carried out to infer the geographical distribution of the genealogical lineages and the historical demography of roach Rutilus rutilus (L.). A total of 265 individuals from 52 sites covering most of the Eurasian distribution range were sequenced for a 475 bp fragment of the mitochondrial cytochrome b gene. The monophyletic roach contained two deep clades that dated back to the Pliocene. The Ponto-Caspian clade comprised populations from Greece to Siberia with a likely palaeorefugium at the west coast of the Caspian Sea. This clade largely corresponds to individuals with morphological features described as Rutilus heckelii. The west European clade included individuals from central and western Europe with the Danube and Dniester basins as possible palaeorefugia. This clade largely corresponds to individuals with morphological features described as R. rutilus. A suture-zone of the two main lineages was observed along the coastal region of the Black Sea. The neutrality tests and the mismatch distributions indicated a demographic expansion during the Middle-Pleistocene for both clades.