Genome-wide nucleotide diversity of hatchery-reared Atlantic and Mediterranean strains of brown trout Salmo trutta compared to wild Mediterranean populations.

A genome-wide assessment of diversity is provided for wild Mediterranean brown trout Salmo trutta populations from headwater tributaries of the Orb River and from Atlantic and Mediterranean hatchery-reared strains that have been used for stocking. Double-digest restriction-site-associated DNA sequencing (dd-RADseq) was performed and the efficiency of de novo and reference-mapping approaches to obtain individual genotypes was compared. Large numbers of single nucleotide polymorphism (SNP) markers with similar genome-wide distributions were discovered using both approaches (196 639 v. 121 016 SNPs, respectively), with c. 80% of the loci detected de novo being also found with reference mapping, using the Atlantic salmon Salmo salar genome as a reference. Lower mapping density but larger nucleotide diversity (π) was generally observed near extremities of linkage groups, consistent with regions of residual tetrasomic inheritance observed in salmonids. Genome-wide diversity estimates revealed reduced polymorphism in hatchery strains (π = 0·0040 and π = 0·0029 in Atlantic and Mediterranean strains, respectively) compared to wild populations (π = 0·0049), a pattern that was congruent with allelic richness estimated from microsatellite markers. Finally, pronounced heterozygote deficiency was found in hatchery strains (Atlantic FIS = 0·18; Mediterranean FIS = 0·42), indicating that stocking practices may affect the genetic diversity in wild populations. These new genomic resources will provide important tools to define better conservation strategies in S. trutta.

Shape plasticity in response to water velocity in the freshwater blenny Salaria fluviatilis.

A non-random association between an environmental factor and a given trait could be explained by directional selection (genetic determinism) and by phenotypic plasticity (environmental determinism). A previous study showed a significant relationship between morphology and water velocity in Salaria fluviatilis that conformed to functional expectations. The objective of this study was to test whether this relationship could be explained by phenotypic plasticity. Salaria fluviatilis from a Corsican stream were placed in four experimental channels with different water velocities (0, 10, 20 and 30 cm s(-1)) to test whether there was a morphological response associated with this environmental factor. After 28 days, fish shape changed in response to water velocity without any significant growth. Fish in higher water velocities exhibited a more slender body shape and longer anal and caudal fins. These results indicate a high degree of morphological plasticity in riverine populations of S. fluviatilis and suggest that the previous relationship between morphology and water velocity observed in the field may largely be due to an environmental determinism.

Three brown trout Salmo trutta lineages in Corsica described through allozyme variation.

The brown trout Salmo trutta is represented by three lineages in Corsica: (1) an ancestral Corsican lineage, (2) a Mediterranean lineage and (3) a recently stocked domestic Atlantic S. trutta lineage (all are interfertile); the main focus of this study was the ancestral Corsican S. trutta, but the other lineages were also considered. A total of 38 samples captured between 1993 and 1998 were analysed, with nearly 1000 individuals considered overall. The Corsican ancestral lineage (Adriatic lineage according to the mitochondrial DNA control region nomenclature, AD) mostly inhabits streams in the southern half of the island; the Mediterranean lineage (ME) is present more in the north, especially in Golu River, but most populations are an admixture of these lineages and the domestic Atlantic S. trutta (AT). Locations where the Corsican ancestral S. trutta is dominant are now protected against stocking and sometimes fishing is also forbidden. The presence of the Corsican S. trutta is unique in France.

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes.

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

Morphological variation in the freshwater blenny Salaria fluviatilis from Corsican rivers: adaptive divergence, phenotypic plasticity or both?

The first goal of this study was to determine whether morphological variation in the freshwater blenny Salaria fluviatilis results in spatially structured populations distributed around Corsica, France, which would suggest genetically differentiated populations through reproductive isolation by distance. The second goal was to determine whether some morphological traits are related to water velocity, one of the most contrasting habitat characteristics in these rivers, which would suggest an adaptation to local conditions. The results showed that the morphology of S. fluviatilis differed among the three main geographic areas studied in Corsica and that geographically distant populations of S. fluviatilis were less similar morphologically and genetically than close ones. The results also indicated that the morphological differences among populations conformed to functional expectations. Overall, the results suggest that the morphological variation of S. fluviatilis from Corsican rivers is an adaptive response to water velocity and that these populations are in a process of reproductive isolation by distance.

Permanent genetic resources added to molecular ecology resources database 1 April 2013-31 May 2013.

This article documents the addition of 234 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acipenser sinensis, Aleochara bilineata, Aleochara bipustulata, Barbus meridionalis, Colossoma macropomum, Delia radicum, Drosophila nigrosparsa, Fontainea picrosperma, Helianthemum cinereum, Liomys pictus, Megabalanus azoricus, Pelteobagrus vachelli, Pleuragramma antarcticum, Podarcis hispanica type 1A, Sardinella brasiliensis and Sclerotinia homoeocarpa. These loci were cross-tested on the following species: Acipenser dabryanus, Barbus balcanicus, Barbus barbus, Barbus cyclolepis, Drosophila hydei, Drosophila melanogaster, Drosophila obscura, Drosophila subobscura, Fontainea australis, Fontainea fugax, Fontainea oraria, Fontainea rostrata, Fontainea venosa, Podarcis bocagei, Podarcis carbonelli, Podarcis liolepis, Podarcis muralis and Podarcis vaucheri.

Species boundaries in the Himantura uarnak species complex (Myliobatiformes: Dasyatidae).

Samples of the 'Himantura uarnak' species complex (H. leoparda, H. uarnak, H. undulata under their current definitions), mostly from the Coral Triangle, were analyzed using nuclear markers and mitochondrial DNA sequences. Genotypes at five intron loci showed four reproductively isolated clusters of individuals. The COI sequences showed four major mitochondrial lineages, each diagnostic of a cluster as defined by nuclear markers. No mitochondrial introgression was detected. The average Kimura-2 parameter nucleotide distance separating clades was 0.061-0.120 (net: 0.055-0.114), while the distance separating individuals within a clade was 0.002-0.008. Additional, partial cytochrome-b gene sequences were used to link these samples with previously published sequences of reference specimens of the three nominal species. One of the clusters was identified as H. undulata and another one, as H. uarnak, while two cryptic species were uncovered within the recently-described H. leoparda, challenging the current morphology-based taxonomy of species within the H. uarnak species complex.

Distinguishing species of European sturgeons Acipenser spp. using microsatellite allele sequences.

Five microsatellite markers were analysed and their alleles were sequenced for the three sturgeon species that lived in western Europe: the European sturgeon Acipenser sturio, the Atlantic sturgeon Acipenser oxyrinchus and the Adriatic sturgeon Acipenser naccarii. A total of 94 different allele sequences were obtained. Fixed mutations in the flanking regions or in the core repeat of microsatellites provided a clear distinction between the different species. Comparison of allele sequences also provided some insights into microsatellites and the evolution of Acipenser species. These nuclear markers can be used to solve species determination problems, and combined with mitochondrial markers, will be useful to identify introgression and hybridization among the three species. Moreover, because they are short and with a limited allele size range, they are particularly suited for analysis of museum specimens or archaeological remains.

Population genetic structure of Cichla pleiozona (Perciformes: Cichlidae) in the Upper Madera basin (Bolivian Amazon): sex-biased dispersal?

This study investigates the population structure of the Tucunaré (Cichla pleiozona) in the Bolivian Amazon (Upper Madera) by using nuclear (EPIC-PCR, 67 individuals) and mitochondrial (Control Region, 41 published and 76 new sequences) DNA analyses, in relation with ecological (water quality: muddy, clear and mix) and geographic factors. Our analyses of both markers showed the highest diversity in clear waters (Yata, Middle and Upper Iténez), and the existence of two populations in muddy waters (Sécure and Ichilo) and one in mix waters (Manuripi). On the other hand, mitochondrial analyses identified three populations in clear waters where nuclear analyses identified a panmictic population. The highest diversity observed in the Yata-Iténez system suggests that an aquatic refuge occurred during the past in this area. The possible explanations for the observed discrepancy between nuclear and mitochondrial markers are discussed, and a sex-biased dispersal seems to be the most plausible hypothesis in the light of the available information and field observations.

Polymorphic microsatellite loci in the widespread amphidromous goby Sicyopterus lagocephalus and cross-genus amplification among Sicydiinae.

Microsatellite DNA markers were isolated in an amphidromous goby (Sicyopterus lagocephalus) from a partial genomic library enriched for AC repeats. Eight microsatellites were highly polymorphic with six to 33 alleles per locus and expected heterozygosities ranging from 0.53 to 0.97. Cross-species amplifications were performed within the sub-family Sicydiinae by genotyping individuals from two species of the genus Cotylopus. Some of these loci were successfully amplified and showed polymorphism in the second genus.

Rare and asymmetrical hybridization of the endemic Barbus carpathicus with its widespread congener Barbus barbus.

In endemic species that co-occur with widespread congeners, hybridization can lead to an influx of novel and beneficial genetic variation, but high rates of introgression may cause genetic swamping of the endemic species and have detrimental effects on its survival potential. This study examines hybridization between sympatric populations of the Carpathian barbel Barbus carpathicus, a recently discovered cryptic species with a restricted range, and the widespread common barbel Barbus barbus. Based on six diagnostic allozyme loci, a microsatellite locus and mtDNA, hybrids were found to be present at multiple localities within the Vistula River drainage (Baltic Sea) as well as in the Tisza River system of the Danube River drainage (Black Sea). However, the numbers of hybrids were very low; four individuals of 230 fish sampled from the Vistula drainage. Bayesian assessment of their nuclear genotypes suggested that two hybrids in the Vistula drainage and nine in the Tisza system were F1 generation, and one in the Vistula drainage and one in the Tisza system were backcrosses (BC) to B. barbus, while no F2 or BC to B. carpathicus were detected. No hybrid carried B. carpathicus mtDNA and cytonuclear linkage disequilibria showed significant positive associations between hybrid genotypes and B. barbus mtDNA, suggesting unidirectionality in the interspecific mating with a disproportionate contribution of B. barbus mothers. Despite geographically broad occurrence of hybrids, these data provide evidence of strong constraints on hybridization in the native breeding habitats and the lack of introgression towards B. carpathicus.

Development of a key using morphological characters to distinguish south-western Indian Ocean anguillid glass eels.

Two morphological characters were examined in 4099 anguillid glass eels sampled in four south-western Indian Ocean islands, and resulting identifications were tested using genetic analysis. Distance between the origin of the dorsal and anal fins as related to total length and tail and caudal fin pigmentation enabled formulation of a useful field identification key, which was able to discriminate 4036 glass eels of the regional species Anguilla bicolor bicolor, Anguilla marmorata and Anguilla mossambica, and excluded the occurrence of Anguilla nebulosa labiata in the sample.

Ubiquity of Sicyopterus lagocephalus (Teleostei: Gobioidei) and phylogeography of the genus Sicyopterus in the Indo-Pacific area inferred from mitochondrial cytochrome b gene.

Sicyopterus lagocephalus is a Gobiidae Sicydiinae (Teleostei) thought to inhabit Indo-Pacific island rivers from Comoros Islands in the Indian Ocean to Australs Islands (French Polynesia) in the Pacific Ocean. Its biological cycle comprises a marine planctonic larval phase of several months allowing it to migrate from island to island, but the other species of the genus, with such a larval stage, have generally a more restricted range and are often endemic. To understand the organisation of a species with such a wide distribution, mtDNA cytochrome b sequences were amplified for 55 specimens of this genus covering most of its distribution range together with six close endemic species and other gobiids used as outgroups. The main result is the confirmation of the ubiquity of S. lagocephalus that occurs over a range of 18,000 km in the Indian and Pacific Oceans. Two clades were identified within this species, one clustering most of French Polynesian haplotypes and the other clustering most of Mascarene (including Comoros) haplotypes. The overall pattern of distribution and phylogenetic relationship suggests that the lineages leading to endemic species originated earlier than S. lagocephalus. This latter seems to be a secondary migrant species, having colonised both Indian and Pacific Oceans with a few exceptions, situated at the border of the range (Madagascar, Marquesas, Rapa). According to the results, the phylogeny of the Sicyopterus group, the age of the different lineages and the past history of the colonisation of the Indo-Pacific islands are discussed.

Intron polymorphism (EPIC-PCR) reveals phylogeographic structure of Zacco platypus in China: a possible target for aquaculture development.

The present study aims at a phylogeographic description of Zacco platypus from southeast China, in order to detect subdivisions within the nominal species. Two main basins were sampled: the Chang Jiang (Yangstze River) in central and east China (Hunan and Sichuan provinces) and the Xi Jiang, the more southern main tributary of the Zhu Jiang (Pearl River, Guangxi province). A total of 27 intron systems were tested, five of them were informative and gave 12 interpretable and polymorphic loci. Within the diversity of Z. platypus, four genetic groups were identified by multidimensional (FCA) analyses, corresponding to distinct genetic pools. The geographical distribution of the genetic groups corresponds neither with the drainage structure, nor the geographic distances between samples. It follows that isolation by distance and limited migration are insufficient to explain this geographic structure. The history of the river network therefore appears to have played an important role.

Autosomal differences between males and females in hybrid zones: a first report from Barbus barbus and Barbus meridionalis (Cyprinidae).

Narrow hybrid zones are generally subjected to the action of two forces: dispersal, which tends to homogenise the hybridising taxa, and selection against hybrids, which, in contrast, produces steep clines of introgression for diagnostic markers. Although differences between sexes in dispersal abilities or in susceptibility to hybrid counterselection are common in hybrid zones, autosomal genetic differences between males and females have never been reported to our knowledge. Barbus barbus and Barbus meridionalis (Cyprinidae) form a hybrid zone along the Lergue river. By carrying out a genetic analysis of males and females in six samples from two central stations of the hybrid zone using codominant markers (six allozymes and four microsatellite loci), we revealed significant multilocus and monolocus differences between the sexes. This could reflect a genetic difference among sexes within a same cohort, caused either by a survival (or fertility) differential among sexes or by a sex-specific pattern of dispersal. Alternatively, this may be due to genetic differentiation between cohorts, since male and female barbel exhibit different maturation, growth and survival patterns leading to different age distributions among sexes, and particularly among reproducers.

Genetic introgression between wild and stocked salmonids and the prospects for using molecular markers in population rehabilitation: the case of the Adriatic grayling (Thymallus thymallus L. 1785).

In the north Adriatic basin, a morphologically and genetically distinct lineage of grayling is found, designated as the Adriatic grayling. In Slovenia, the Adriatic grayling is restricted to the Soca river system, where it is critically endangered. The most pertinent threat is stocking with non-native, highly divergent Sava (Danubian) drainage stock, and this activity has been going on for more than four decades. The present study was designed to characterise the genetic structure of the Adriatic grayling in Slovenia, with particular emphasis on estimating the degree of introgression with non-indigenous stocked grayling. We analysed polymorphism at 154 microsatellite loci in samples representing grayling from the Adriatic and Danubian drainage stock. A relatively high number (12) of alleles, diagnostic for the Adriatic grayling, were identified. However, a correspondence analysis based on individual multilocus genotypes also revealed that there is no distinctive Adriatic group but rather a dispersed multitude of individuals that cannot be unambiguously distinguished from the more homogenous Danubian population. A Bayesian analysis of individual admixture coefficients confirmed this pattern and revealed extensive introgression between the Adriatic grayling and stocked grayling of Danubian origin. Average individual admixture coefficients showed that only between 50 and 60% of the original gene pools remained, and only few non-introgressed indigenous individuals could be identified. Microsatellite-based individual admixture analysis appear to be an important tool for identifying remaining non-introgressed indigenous individuals that could be used for restoring the original populations.

Extreme genetic differentiation among the remnant populations of marble trout (Salmo marmoratus) in Slovenia.

Populations of the marble trout (Salmo marmoratus) have declined critically due to introgression by brown trout (Salmo trutta) strains. In order to define strategies for long-term conservation, we examined the genetic structure of the 8 known pure populations using 15 microsatellite loci. The analyses reveal extraordinarily strong genetic differentiation among populations separated by < 15 km, and extremely low levels of intrapopulation genetic variability. As natural recolonization seems highly unlikely, appropriate management and conservation strategies should comprise the reintroduction of pure populations from mixed stocks (translocation) to avoid further loss of genetic diversity.

Natural and artificial secondary contact in brown trout (Salmo trutta, L.) in the French western Pyrenees assessed by allozymes and microsatellites.

Analysis of allozyme polymorphism in brown trout (Salmo trutta) populations from south-western France shows that two genetically differentiated wild forms (characterised by the LDH-C1*100 and 90 alleles) introgress in this area. As allozymes could not evaluate the impact of stocking in the Atlantic basin, microsatellites have been necessary to detect the influence of hatchery fish and to confirm that the observed structure was natural. Microsatellites confirm the distinctness of the two wild forms based on allozyme loci. This situation provides a new example of secondary contact for this species in the Atlantic basin, with various levels of mixing being seen between the two population groups. The origin of these forms is discussed in the light of previous studies concerning modern and ancestral Atlantic trout (Hamilton et al, 1989) and lineages stemming from different glacial refuges (Garcia Marin et al, 1999; Weiss et al, 2000). This local analysis provides new insights in defining the evolutionary history of this species and confirms the important role of glaciation events in this history.

Multiple origins of polyploidy in the phylogeny of southern African barbs (Cyprinidae) as inferred from mtDNA markers.

The cyprinid genus Barbus, with more than 800 nominal species, is an apparently polyphyletic assemblage to which a number of unrelated species, groups and/or assemblages have been assigned. It includes species that exhibit three different ploidy levels: diploid, tetraploid and hexaploid. Several lineages of the family Cyprinidae constitute a major component of the African freshwater ichthyofauna, having about 500 species, and fishes assigned to the genus 'Barbus' have the most species on the continent. We used complete sequences of the mitochondrial cytochrome b gene in order to infer phylogenetic relationships between diploid, tetraploid and hexaploid species of 'Barbus' occurring in southern Africa, the only region where representatives of all of the three ploidy levels occur. The results indicate that most of the lineages are incorrectly classified in the genus 'Barbus'. The southern African tetraploids probably originated from southern African diploids. They constitute a monophyletic group distinct from tetraploids occurring in the Euro-Mediterranean region (Barbus sensu stricto). The 'small' African diploid species seem to be paraphyletic, while the 'large' African hexaploid barbs species are of a single, recent origin and form a monophyletic group. The evidence of multiple, independent origins of polyploidy occurring in the African cyprinine cyprinids thus provides a significant contribution to the knowledge on the systematic diversity of these fishes, and warrants a thorough taxonomic reorganization of the genus.