María Inés Roldán

Population genetic structure of European hake, Merluccius merluccius

Allozyme analysis of tissue samples of 910 European hake Merluccius merluccius from six North Atlantic Ocean and four Mediterranean Sea locations revealed polymorphism at 21 out of 34 protein-coding loci examined. A major subdivision between Atlantic and Mediterranean collections (mean Neis genetic distance within regions 0.005; between regions 0.014) was particularly evident from allelic differences at the GAPDH-1* and GR-2* loci. Further population subdivision was indicated within both regions, and gene flow from adjacent Atlantic to proximal Mediterranean populations was suggested by allele frequencies and relevant oceanographic and geological information. The data support present management based on distinct Atlantic and Mediterranean populations and indicate that the population structure within the Atlantic is more complex than the discrete northern and southern stocks proposed by ICES.

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.

A genetic assessment of the population structure of swordfish (Xiphias gladius) in the Mediterranean Sea

Nine samples of swordfish from different Mediterranean areas plus a sample obtained in Atlantic waters adjacent to the Strait of Gibraltar were examined for temporal and spatial heterogeneity using allozyme analysis. Only two out of 38 loci (IDHP-2* and LDH-3*) were polymorphic based on a frequency of less than 0.95 in one or more samples. Both polymorphism and heterozygosity revealed a low genetic variability for swordfish in comparison with other pelagic species. No heterogeneity due to differences between age groups was observed for those samples including juvenile and adult specimens. The lack of temporal or spatial heterogeneity found in this study is consistent with a single population of swordfish in the Mediterranean Sea and adjacent waters of the Atlantic Ocean.

Phylogenetic inference in Odontesthes and Atherina (Teleostei: Atheriniformes) with insights into ecological adaptation

This contribution provides an insight into Atheriniformes systematics based on four mitochondrial regions: 12S rRNA, cytb, COI and control region (2794bp in total). In the Atherinopsoidei (New World silversides), comparisons among five species of Odontesthes, O. argentinensis, O. bonariensis, O. smitti, O. hatcheri and O. incisa revealed a putative marine-freshwater pairing pattern of Odontesthes species, possibly driven by sea level fluctuations of South American waters. This study represents the first data on molecular phylogeny of Odontesthes species that can be of usefulness to biodiversity conservation policies. In the Atherinoidei (Old World silversides), Atherina boyeri was corroborated as a species complex constituted by a marine form, a marine with dark spots form and a brackish form. Concretely, Odontesthes and Atherina may represent geographically replicated models to study genetic adaptation and speciation of marine species to brackish and freshwater habitats. In addition, phylogenetic analyses supported Odontesthes and Atherina as monophyletic taxa and their separation into two differentiated suborders Atherinopsoidei and Atherinoidei, respectively.

Multilocus comparative phylogeography of two aristeid shrimps of high commercial interest (Aristeus antennatus and Aristaeomorpha foliacea) reveals different responses to past environmental changes.

Phylogeographical studies can reveal hidden patterns in the evolutionary history of species. Comparative analyses of closely related species can further help disentangle the relative contributions of processes responsible for such patterns. In this work, the phylogeography of two aristeid species, Aristeus antennatus and Aristaeomorpha foliacea, was compared through multiple genetic markers. These marine shrimp species are of high commercial importance, and are exploited in the Mediterranean Sea (MED) and in Mozambique Channel (MOZ) where they occur in partial sympatry. Aristeus antennatus (N = 50) from Western and Eastern Mediterranean (WM and EM, respectively), Atlantic Ocean (AO) and MOZ, and Aristaeomorpha foliacea (N = 40) from WM, EM, MOZ North-Western Australia (AUS) were analyzed with two nuclear genes (PEPCK and NaK) and one mitochondrial (COI) gene. Within the study area differences were found between the two species in their phylogeographical patterns, suggesting distinct responses to environmental changes. Monophyly of Aristeus antennatus was found across its distributional range. This pattern contrasted by a deep evolutionary split within Aristaeomorpha foliacea where genetic diversity followed geography distinguishing MED-MOZ and AUS. We propose that the AUS lineage of A. foliacea warrants consideration as a distinct species, with consequent implications in systematics and resource management.

Clarifying the taxonomic status of Merluccius spp. in the northeastern Pacific: a combined morphological and molecular approach

The taxonomic status of hake (Merluccius spp.) in the northeastern Pacific is unclear. Hakes in this region are Merluccius productus, M. angustimanus, M. hernandezi, and a morphotype known as dwarf hake. Of these, only the first two species are currently valid. Descriptions in previous studies have been limited by overlapping morphological characteristic, lack of biological material, and limited numbers of sampling localities. To clarify their taxonomy, 461 hake were obtained from eight localities along the North American coast for morphological and mitochondrial DNA sequence analyses (cytochrome b, cytochrome c oxidase subunit I, and 16S ribosomal rDNA). Morphological and molecular analyses suggest that hake in this region represent a continuum of a single species with some levels of morphological and genetic intra-specific variation. In light of these results, we propose that M. productus is the only species of hake present along the North American and northern Central American coast.

Development and characterization of novel microsatellite markers by Next Generation Sequencing for the blue and red shrimp Aristeus antennatus.

The blue and red shrimp, Aristeus antennatus, is a commercially important crustacean, in the Mediterranean Sea, which has been listed as a priority species for fishery management. Hypervariable microsatellite markers could be a useful tool to identify genetic stocks among geographically close fishing grounds. Potential microsatellite markers (97) identified from next-generation sequencing of an individual shrimp using a 454 GS Junior Pyrosequencer were tested on a preliminary panel of 15 individuals representing the four worldwide genetic stocks of the species from which 35 polymorphic loci were identified and used to characterize an additional 20 individuals from the Western Mediterranean Sea. In the Western Mediterranean sample, 32 out of 35 were polymorphic loci and the number of alleles per locus ranged from 2 to 14 and expected heterozygosity ranged from 0.050 to 0.968. No linkage disequilibrium was detected, indicating the independence of the loci. These novel microsatellites provide additional tools to address questions relating to genetic diversity, parentage studies and connectivity patterns of A. antennatus populations and help develop effective strategies to ensure long-term sustainability of this resource.

Shaken not stirred: A molecular contribution to the systematics of genus Mugil (Teleostei, Mugilidae).

With this work we addressed some molecular systematic issues within the Mugil cephalus species complex. Particular attention was paid to the debated situations of: (i) Mugil liza, occurring in partial sympatry with Mugil cephalus in the northwestern Atlantic, and (ii) Mugil platanus, considered by some authors a synonymy of the former species and distributed in the southwestern Atlantic. We sequenced 79 individuals of a 465-bp portion of the mitochondrial control region (CR) from 8 western Atlantic and 2 Mediterranean localities. In addition, all CR sequences available from GenBank for the studied taxa were added to our dataset, for a total of 323 individuals. Overall, 229 haplotypes corresponding to 8 divergent monophyletic lineages were detected. Results of phylogenetic analyses were consistent with the occurrence of past speciation events producing the observed lineages. Of these lineages, 7 correspond to cryptic species and one is constituted by M. liza and M. platanus. As a matter of fact, these 2 taxa constitute a single lineage within the M. cephalus species complex. However, individuals of M. liza/M. platanus lineage analyzed by means of the 18 mitochondrial markers available in GenBank exhibited a degree of genetic diversity consistent with highly divergent populations. Of the 8 lineages detected, the Mediterraean one (type locality) corresponds to M. cephalus; the lineage M. liza/M. platanus should be named M. liza, under the priority principle, and the left 6 lineages need formal description.

An optimized high quality male DNA extraction from spermatophores in open thelycum shrimp species.

The crucial step of most of the current genetic studies is the extraction of DNA of sufficient quantity and quality. Several genomic DNA isolation methods have been described to successfully obtain male DNA from shrimp species. However, all current protocols require invasive handling methods with males for DNA isolation. Using Aristeus antennatus as a model we tested a reliable non-invasive differential DNA extraction method to male DNA isolation from spermatophores attached to female thelycum. The present protocol provides high quality and quantity DNA for polymerase chain reaction amplification and male genotyping. This new approach could be useful to experimental shrimp culture to select sires with relevant genetic patterns for selective breeding programs. More importantly, it can be applied to identify the mating pairs and male structure in wild populations of species as A. antennatus, where males are often difficult to capture. Our method could be also valuable for biological studies on other spermatophore-using species, such as myriapods, arachnids and insects.