In the last few years microsatellites have become one of the most popular molecular markers used with applications in many different fields. High polymorphism and the relative ease of scoring represent the two major features that make microsatellites of large interest for many genetic studies. The major drawback of microsatellites is that they need to be isolated de novo from species that are being examined for the first time. The aim of the present paper is to review the various methods of microsatellite isolation described in the literature with the purpose of providing useful guidelines in making appropriate choices among the large number of currently available options. In addition, we propose a fast and easy protocol which is a combination of different published methods.

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The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.Notothenioidei | Icefish | Percomorpha | molecular clock | buoyancy

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Illegal, unreported and unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using geneassociated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome-wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large- and fine-scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F(CT) = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F(CT) range 0.275-0.705) and fine-scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.

The Strait of Gibraltar has been proposed to be the divide between two marine biogeographical regions, the Mediterranean Sea and the Northeast Atlantic. Intraspecific studies have shown, for several of the examined species, a reduction of gene flow between the two basins. The present study examines genetic variation at nuclear and mitochondrial loci in five marine teleost species belonging to the family Sparidae. Four samples for each species were analysed spanning the Northeast Atlantic and the Mediterranean. For all individuals 17 allozyme loci were scored and a combined single strand conformation polymorphism‐sequencing approach was used to survey approximately 190 bp of the mitochondrial DNA (mtDNA) D‐loop region. All five species share similar biological features. For three species, namely Lithognathus mormyrus, Spondyliosoma cantharus, and Dentex dentex, large mtDNA divergence was observed between Atlantic and Mediterranean samples. Little or no mtDNA differentiation was found in the other two species, Pagrus pagrus and Pagellus bogaraveo. Allozyme data revealed strong differentiation when comparing Atlantic and Mediterranean samples of L. mormyrus and D. dentex, moderate for P. pagrus, and no differentiation for P. bogaraveo and S. cantharus. These results provide evidence for a sharp phylogeographical break (sensu Avise) between the Atlantic and the Mediterranean for two (or possibly three) sparid species of the five investigated. At the same time, the obtained results for the other two species raise the question on which ecological/historical factors might have caused the observed discrepancy in the geographical distribution of genetic variation among otherwise biologically similar species.

Antarctic waters represent a unique marine environment delimited by an oceanographic barrier, the Polar Front Zone, and characterized by constant subzero temperatures and presence of sea ice. A group of teleost fish, the Notothenioidei, have adapted to these challenging environmental conditions, undergoing a remarkable diversification. In the present study a total of 798 base pairs, generated from partial sequencing of 16S and 12S mitochondrial ribosomal RNA genes, were examined in 33 notothenioid species representative of all families included in the suborder Notothenioidei. Phylogenetic trees, reconstructed on the basis of sequence data by different methods, indicate that traditional hypotheses on notothenioid systematics and biogeography might be in need of reexamination. Molecular evidence suggests that vicariant speciation could be invoked to explain the early divergence of Eleginops maclovinus, a species previously included in the family Nototheniidae, which is now proposed as the closest sister group to all the rest of notothenioids apart from bovichtids. On the other hand, repeated, independent dispersal through the Polar Front is proposed for the divergence of other subantarctic notothenioid species. Likewise, multiple, independent transitions from benthic to pelagic habit are inferred from molecular data, at variance with the more conservative hypothesis based on cladograms reconstructed from morphological data.