Regional environmental and climatic concerns on preserving native gene pools of a least concern species: Brown trout lineages in Mediterranean streams.

The European brown trout, Salmo trutta, is a cold-adapted fish reported as a Least Concern species in the IUCN Red List. This species colonized new territories from southern refuges during the last glacial melting, but during the 20th century suffered from anthropic impacts on its habitats. The long-time survival of the species relies on the genetic diversity within and among populations. Brown trout is among the genetically most diverse vertebrate species; however, native populations in Mediterranean rivers have dramatically suffered of introgressive hybridization from extensive releases of evolutionary distant non-native Atlantic stocks. In addition, in Mediterranean rivers climate change will result in unsuitable conditions for the species during the 21st century. Using brown trout populations at the headstreams of a Pyrenean river as a model, this paper revised how hatchery releases have affected the native gene pools and how environmental and climatic variables controlled the amount of local introgression at intra-basin level. Introgressive hybridization was detected in all studied sites. Ten times larger divergence was observed among populations at tributaries than among populations along the main stem. A highly impacted population distributed in a long transect in the main stem suggested that hatchery fish move towards the main stem wherever released. From already highly impacted populations and despite the cessation of hatchery releases, warmer temperatures and lower precipitation expected from climate change will extend the introgressive hybridization along the basin, contributing to the extinction of the native gene pools. Based on available morphological distinction of native, hatchery and hybrid brown trout, we advocate the involvement of regional social groups (e.g. riverside dwellers, anglers, conservationists, hikers) in citizen science programs to detect the spread of non-native phenotypes along the rivers. These are cheap and fast methods to collaborate with fishery managers in the preservation and recovery of the regional native populations.

Genetic Demography of the Blue and Red Shrimp, Aristeus antennatus: A Female-Based Case Study Integrating Multilocus Genotyping and Morphometric Data.

In this study, we quantified the three key biological processes, growth, recruitment, and dispersal pattern, which are necessary for a better understanding of the population dynamics of the blue and red shrimp Aristeus antennatus. This marine exploited crustacean shows sex-related distribution along the water column, being females predominate in the middle slope. The present study attempts to fill the existing gap in the females' genetic demography, as scarce knowledge is available despite being the most abundant sex in catches. We analyzed morphometric data and genotyped 12 microsatellite loci in 665 A. antennatus females collected in two consecutive seasons, winter and summer 2016, at the main Mediterranean fishing ground as a model. Almost every female in summer was inseminated. Five modal groups were observed in both seasons, from 0+ to 4+ in winter and from 1+ to 5+ in summer. Commercial-sized sorting based on fishermen's experience resulted in a moderate-to-high assertive method concerning cohort determination. Genetic data pointed out females' horizontal movement between neighboring fishing grounds, explaining the low genetic divergence detected among western Mediterranean grounds. Our results could represent critical information for the future implementation of management measures to ensure long-time conservation of the A. antennatus populations.

Genomic Hatchery Introgression in Brown Trout (Salmo trutta L.): Development of a Diagnostic SNP Panel for Monitoring the Impacted Mediterranean Rivers.

Brown trout (Salmo trutta L.) populations have been restocked during recent decades to satisfy angling demand and counterbalance the decline of wild populations. Millions of fertile brown trout individuals were released into Mediterranean and Atlantic rivers from hatcheries with homogeneous central European stocks. Consequently, many native gene pools have become endangered by introgressive hybridization with those hatchery stocks. Different genetic tools have been used to identify and evaluate the degree of introgression starting from pure native and restocking reference populations (e.g., LDH-C* locus, microsatellites). However, due to the high genetic structuring of brown trout, the definition of the "native pool" is hard to achieve. Additionally, although the LDH-C* locus is useful for determining the introgression degree at the population level, its consistency at individual level is far from being accurate, especially after several generations were since releases. Accordingly, the development of a more powerful and cost-effective tool is essential for an appropriate monitoring to recover brown-trout-native gene pools. Here, we used the 2b restriction site-associated DNA sequencing (2b-RADseq) and Stacks 2 with a reference genome to identify single-nucleotide polymorphisms (SNPs) diagnostic for hatchery-native fish discrimination in the Atlantic and Mediterranean drainages of the Iberian Peninsula. A final set of 20 SNPs was validated in a MassARRAY® System genotyping by contrasting data with the whole SNP dataset using samples with different degree of introgression from those previously recorded. Heterogeneous introgression impact was confirmed among and within river basins, and was the highest in the Mediterranean Slope. The SNP tool reported here should be assessed in a broader sample scenario in Southern Europe considering its potential for monitoring recovery plans.

Male Deep-Sea Shrimps Aristeus antennatus at Fishing Grounds: Growth and First Evaluation of Recruitment by Multilocus Genotyping.

The population biology of the deep-sea shrimp Aristeus antennatus, as with other exploited demersal species, is usually studied using data from fishery statistics. Such statistical analyses have shown female-biased sex ratios during the spawning season in this species. Because the abundance of males increases at greater depths that are not exploited by fisheries (virgin grounds), knowledge on their recruitment is limited. Here, the growth and recruitment of A. antennatus males at fishing grounds was evaluated. This was achieved by integrating information on previously identified breeding behaviours and by tracing the young-of-year cohort through genotyping at 10 microsatellite loci. Using a codend and a codend cover with distinct meshed windows, four groups of males were collected in winter and in a subsequent spawning summer season. Summer collections were mostly composed of pre-adult males, reaching sizes that are to be expected from the growth of winter juveniles; however, many specimens also originated from nearby grounds. This result indicates the horizontal dispersal of male juveniles via intermediate and deep oceanographic currents. Such dispersal complements passive larval dispersal in surface waters, and contributes to the weak genetic divergence among regional fishing grounds. These features could be shared by other deep-sea crustacean and fish species, and should be considered for the sustainable exploitation of demersal fisheries.

Genetic analyses reveal temporal stability and connectivity pattern in blue and red shrimp Aristeus antennatus populations.

Temporal variability of the genetic structure and connectivity patterns of the blue and red shrimp Aristeus antennatus in the seven most important fishing grounds of the Western Mediterranean Sea, were assessed using twelve microsatellite loci during 2 consecutive years (2016 and 2017), in a total of 1403 adult individuals. A high level of geographical connectivity among groups was observed in the two studied years. In fact, no significant geographical differentiation was found in 2016 (FST = 0.0018, p > 0.05), whereas it was indicated in 2017 (FST = 0.0025, p < 0.05). This small divergence in 2017 was not attributed to the distance among locations nor to the effect of the Ibiza Channel. Significant allele frequency changes were found at local level between the 2 years (FCT = 0.0006, p < 0.05), mainly due to Blanes' fishing ground. Larval dispersal from the North to the South through the main superficial current supports the high level of connectivity pattern found. The temporal genetic instability detected in the Blanes' fishing ground could be explained by oceanographic temporary features. Our findings evidence only one biological unit in the study region and establish the baseline for an inter-federal management plan of A. antennatus.

Genetic structure and population connectivity of the blue and red shrimp Aristeus antennatus.

The blue and red shrimp Aristeus antennatus is a demersal marine species harvested by bottom trawling in the Mediterranean Sea, the adjacent Atlantic Ocean (AO) waters, and the Mozambique Channel in the Indian Ocean (IO). As it is considered to be a priority species for sustainable fishing, identification of its genetic stocks and the connectivity between them is essential. Using 12 microsatellite loci we detected at least four genetic stocks distributed in the Western Mediterranean (WM), Eastern Mediterranean (EM), AO, and IO and signals for a possible fifth stock in the Alborán Sea. We detected no additional population structuring within the WM. Thus, although the Almería-Orán Front exerts some isolating effect, high genetic homogeneity and gene flow are present within the WM Basin. The IO stock is genetically closer to the AO stock than to the others; thus, the species dispersion route is more likely via the Atlantic Ocean than via the Red Sea. Large effective population sizes suggest population sustainability, but moderate genetic diversity values indicate to proceed with caution. Our genetic results serve as a basis for species conservation to ensure long-term sustainability of this marine resource.

Mating structure of the blue and red shrimp, Aristeus antennatus (Risso, 1816) characterized by relatedness analysis.

Understanding life history variation and strategies is crucial for stock assessment and fisheries management due to the direct effects on population dynamics, effective population size, sex-ratios, levels of inbreeding, and relatedness among individuals. Aristeus antennatus (En ─ Blue and red shrimp; Fr ─ Crevette rouge; Sp ─ Gamba rosada) is one of the most exploited demersal resources in the Western Mediterranean Sea. However, information regarding the mating system and mate choice preferences remains largely unknown. Advances in molecular genetic markers and methods of inferring biological relationships among individuals have facilitated new insights into the reproductive dynamics of the species in the wild. Here, we used microsatellite markers to examine the A. antennatus mating system and putative mate choice preferences. Our results provided clear evidence of polyandry and polygyny. Relatedness analyses, together with FST and DAPC values showed females exhibited a mating bias towards unrelated males. Mating males were inferred from spermatophores and suggested males were sympatric with females and were also from other spawning grounds. Our findings provided the first description of the reproductive behavior of blue and red shrimp.

Morphological identification and molecular confirmation of the deep-sea blue and red shrimp Aristeus antennatus larvae.

The early life stages of the blue and red shrimp Aristeus antennatus (Decapoda: Dendrobranchiata: Penaeoidea: Aristeidae) were described by Heldt in 1955 based on plankton samples, larval rearing and assumptions of species habitat. Even with adequate keys, identification of its first larval stages remained a difficult task due to the lack of specific morphological characters which would differentiate them from other Penaeoidea species. Larvae of Aristeus antennatus were collected in the continental slope off the Spanish Mediterranean coast in August 2016 with a neuston net and preserved in ethanol 96%. DNA from the larvae was extracted and the molecular markers Cytochrome Oxidase I and 16S rDNA were sequenced and compared to that of adults with the objective of confirming the previous morphological description. Then, we present additional information to the morphological description of Aristeus antennatus larval stages through scanning electron microscopy and molecular analysis. This represents the first documented occurrence of Aristeus antennatus larvae off the Catalan coast and sets the grounds for further work on larval ecology and population connectivity of the species, which is an important contribution to a more sustainable fishery.

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.

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.

Genetic analyses of two spawning stocks of the short-finned squid (Illex argentinus) using nuclear and mitochondrial data.

Short-finned squid Illex argentinus (Omastrephidae) is an economically important species which supports a long-term and intensive commercial fishery in the South-West Atlantic Ocean. In this study, the genetic variability of allozymes (40 loci) and mitochondrial regions, COI (556 bp) and 16S rDNA (439 bp), were addressed in the two most important species' spawning stocks, the Summer Spawning Stock (SSS) and the South Patagonic Stock (SPS). Five out of 20 polymorphic allozyme loci were polymorphic at 95% criterion and heterozygosity levels were low. The concatenated analysis of mitochondrial molecular markers revealed high to low values of haplotype and nucleotide diversity, respectively. Nuclear and mitochondrial data revealed no significant genetic differences between these two spawning stocks. Our results and the biological characteristics of I. argentinus deal with a no simple genetic structure of populations described as spatial and temporal chaotic patchiness. SSS and SPS spawning stocks of I. argentinus could not be elevated to the status of genetic stocks.

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.

Analysis of genetic variability in Aristaeomorpha foliacea (crustacea, aristeidae) using DNA-ISSR (Inter Simple Sequence Repeats) markers.

This work reports the first genetic data of Aristaeomorpha foliacea, a marine decapod of high commercial value, from six Mediterranean localities and one new fishing ground in the Mozambique Channel. The use of five Inter Simple Sequence Repeat (ISSR) primers provided 150 polymorphic loci. Average estimates of genetic diversity did not significantly differ among sampled localities, with a mean value of heterozygosity H=0.105±0.015. Analysis of molecular variance (AMOVA) allocated>98% of genetic variability to the within-sample component, displaying values higher than those previously reported in ISSR studies on marine invertebrates. Cluster analyses did not detect geographically or genetically distinct groups. The observed lack of large-scale genetic differentiation is discussed in relation to the high potential for larval dispersal of the species and to features of the marker employed.

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.