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.

Low impact of different SNP panels from two building-loci pipelines on RAD-Seq population genomic metrics: case study on five diverse aquatic species.

BACKGROUND: The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer's 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control. RESULTS: Despite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons. CONCLUSIONS: Tested building-loci pipelines for selection of SNP panels seem to have low influence on population genetics inference across the diverse case-study scenarios here studied. However, preliminary trials with different bioinformatic pipelines are suggested to evaluate their influence on population parameters according with the specific goals of each study.

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.

Effects of water pollution and river fragmentation on population genetic structure of invasive mosquitofish.

We analyzed variation at the GPI-2 locus and eleven microsatellite loci of eastern mosquitofish Gambusia holbrooki populations introduced to the Ebro River (Spain), sampling above and below a dam (Flix Reservoir) where severe chronic pollution has been well documented. Allele frequency changes at the GPI-2 locus in the sites nearest to the polluted sediments agree with previous results from studies in mercury-exposed populations of this highly invasive fish. Genetic distinction of the mosquitofish collected close to the polluted sediments was detected at the GPI locus but also at the presumptive neutral microsatellite loci. Recent migration rates estimated from microsatellites indicated that around 30% of fish collected in a specific location were immigrants from upstream and downstream sources. Such high migration rates probably contribute to the mosquitofish's invasive success and suggest that the consequences on the mosquitofish regional genetic structured of high levels of water toxicants could be mediated by immigration from other sites, but the effect of pollutants on local diversity might be higher than observed here.

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.

Genomic survey provides insights into the evolutionary changes that occurred during European expansion of the invasive mosquitofish (Gambusia holbrooki).

Biological invasions rank among the main global threats for biodiversity. The Eastern mosquitofish (Gambusia holbrooki) is considered one of the 100 world worst invasive species due to its high adaptation capability to new environments. Using the restriction-site-associated DNA tags (RADtags), introduced European locations were compared against native US mosquitofish populations to analyse genomic changes that occurred during invasive process of European locations. After filtering, 7724 RADtags containing only one SNP were retained for population studies. Comparative genomics indicated that 186 of these RADtags matched sequences in the transcriptome of Xyphophorus maculatus, the most closely related genome available. Genomic analyses showed that invasive populations show high reductions in diversity. Further, analyses of population structuring based on these data are concordant with previous analyses based on microsatellites. It is concluded that during the invasion process genetic drift was the main evolutionary force affecting patterns of diversity and population structure. While recognizing that positive selection could be masked by the strong drift during founder events, adaptive processes were evidenced in a reduced number of RADtags (<2%), with only one of these in a putative coding region. Surprisingly, balancing selection was detected in several coding RADtags, suggesting that the preservation of polymorphism in specific genes could be more important than the average population diversity for the population maintenance at any location, particularly for the survival of introduced populations.

Gene flow and maintenance of genetic diversity in invasive mosquitofish (Gambusia holbrooki).

Genetic analyses contribute to studies of biological invasions by mapping the origin and dispersal patterns of invasive species occupying new territories. Using microsatellite loci, we assessed the genetic diversity and spatial population structure of mosquitofish (Gambusia holbrooki) that had invaded Spanish watersheds, along with the American locations close to the suspected potential source populations. Mosquitofish populations from the Spanish streams that were studied had similar levels of genetic diversity to the American samples; therefore, these populations did not appear to have undergone substantial losses of genetic diversity during the invasion process. Population structure analyses indicated that the Spanish populations fell into four main clusters, which were primarily associated with hydrography. Dispersal patterns indicated that local populations were highly connected upstream and downstream through active dispersal, with an average of 21.5% fish from other locations in each population. After initially introducing fish to one location in a given basin, such dispersal potential might contribute to the spread and colonization of suitable habitats throughout the entire river basin. The two-dimension isolation-by-distance pattern here obtained, indicated that the human-mediated translocation of mosquitofish among the three study basins is a regular occurrence. Overall, both phenomena, high natural dispersal and human translocation, favor gene flow among river basins and the retention of high genetic diversity, which might help retain the invasive potential of mosquitofish populations.

Temporal and spatial diversification of the African disjunct genus Androcymbium (Colchicaceae).

The genus Androcymbium (Colchicaceae) includes 57 species that are distributed in the extreme northern and southern portions of Africa, mainly in regions with a Mediterranean climate. We present the first phylogeographic analysis of the genus with species from all five of its distribution areas (North Africa, Horn of Africa, Namibia, western South Africa, and eastern South Africa). We used sequence data from six chloroplast regions and one nuclear region. Phylogeographic reconstructions were conducted using both parsimony and Bayesian inference methods. Molecular dating estimates using a Bayesian approach suggest a middle Miocene (13.4+/-1.5 mya) origin of the genus; this approach also provides support for a late Miocene (9.6+/-1.7 mya) diversification in the winter-rainfall area of western South Africa-south of Namibia and strongly influenced by the Benguela current. Three northward dispersion events have been reported in Androcymbium. The first dated to the end of the Miocene (7.0+/-2.0 mya) and gave rise to the genus Colchicum. The second and the third dispersion events took place in the mid Pliocene, rising one from eastern South Africa and originating the only species found in the Horn of Africa (3.0 mya), and the other from Namibia to the Mediterranean basin (4.0 mya). The formation of a late Miocene-Pliocene arid track in the east of Africa is of a great importance to explain these northward dispersions in Androcymbium.

Role of genetic refuges in the restoration of native gene pools of brown trout.

Captive-bred animals derived from native, alien, or hybrid stocks are often released in large numbers in natural settings with the intention of augmenting harvests. In brown trout (Salmo trutta), stocking with hatchery-reared non-native fish has been the main management strategy used to maintain or improve depleted wild brown trout populations in Iberian and other Mediterranean regions. This measure has become a serious threat to the conservation of native genetic diversity, mainly due to introgressive hybridization. Aware of this risk, the agency responsible for management of brown trout in the eastern Pyrenees (Spain) created "brown trout genetic refuges" to preserve the integrity of brown trout gene pools in this region. Within refuge areas, the prerefuge status with respect to fishing activities has been maintained, but hatchery releases have been banned completely. We evaluated this management strategy through a comparison of the stocking impact on native populations that accounted for stocking histories before and after refuge designations and fishing activities. In particular we examined the relevant scientific, cultural, and political challenges encountered. Despite agency willingness to change fishery policies to balance exploitation and conservation, acceptance of these new policies by anglers and genetic monitoring of refuge populations should also be considered. To improve management supported by genetic refuges, we suggest focusing on areas where the public is more receptive, considering the situation of local native diversity, and monitoring of adjacent introgressed populations. We recommend the use of directional supportive breeding only when a population really needs to be enhanced. In any case, management strategies should be developed to allow for protection within the context of human use.

Polygenic analysis of ammonia-oxidizing bacteria using 16S rDNA, amoA, and amoB genes.

Finding a unique molecular marker capable of quickly providing rigorous and useful phylogenetic information would facilitate assessing the diversity of ammonia-oxidizing bacteria in environmental samples. Since only one of several available markers can be used at a time in these kinds of studies, the 16S rDNA, amoA and amoB genes were evaluated individually and then compared in order to identify the one that best fits the information provided by the composite dataset. Distance-based neighbor-joining and maximum parsimony trees generated using the sequences of the three mentioned genes were analyzed with respect to the combined polygenic trees. Maximum parsimony trees were found to be more accurate than distance-based ones, and the polygenic topology was shown to best fit the information contained in the sequences. However, the taxonomic and phylogenetic information provided by the three markers separately was also valid. Therefore, either of the functional markers (amoA or amoB) can be used to trace ammonia oxidizers in environmental studies in which only one gene can be targeted.

Polygenic analysis of ammonia-oxidizing bacteria using 16S rDNA, amoA, and amoB genes / Análisis poligénico de cepas de bacterias oxidadoras de amoníaco por medio de los genes 16S rDNA, amoA y amoB

Finding a unique molecular marker capable of quickly providing rigorous and useful phylogenetic information would facilitate assessing the diversity of ammonia-oxidizing bacteria in environmental samples. Since only one of several available markers can be used at a time in these kinds of studies, the 16S rDNA, amoA and amoB genes were evaluated individually and then compared in order to identify the one that best fits the information provided by the composite dataset. Distance-based neighbor-joining and maximum parsimony trees generated using the sequences of the three mentioned genes were analyzed with respect to the combined polygenic trees. Maximum parsimony trees were found to be more accurate than distance-based ones, and the polygenic topology was shown to best fit the information contained in the sequences. However, the taxonomic and phylogenetic information provided by the three markers separately was also valid. Therefore, either of the functional markers (amoA or amoB) can be used to trace ammonia oxidizers in environmental studies in which only one gene can be targeted (AU)

Encontrar un marcador molecular único capaz de proporcionar rápidamente información filogenética rigurosa y útil facilitaría evaluación de la diversidad de las bacterias oxidadoras de amoníaco en muestras ambientales. En esta clase de estudios no se puede utilizar simultáneamente más que uno de los marcadores disponibles. Los genes 16S rDNA, amoA y amoB se evaluaron individualmente para identificar el que se ajusta mejor a la información proporcionada por el conjunto de datos de los tres genes. Se compararon los árboles de Neighbor-Joining, basados en las distancias, y los árboles de máxima parsimonia basados en las secuencias conocidas de los tres genes mencionados, y se analizaron en relación con los árboles poligénicos construidos con la información combinada proporcionada por los tres genes. Los árboles de máxima parsimonia resultaron más fieles que los basados en las distancias, y la topología poligénica era la que mejor se ajustaba a la información contenida en las secuencias. Sin embargo, la información taxonómica y filogenética proporcionada por los tres marcadores por separado también resultó válida. Por tanto, cualquiera de los dos marcadores funcionales (amoA o amoB) se puede utilizar para detectar los oxidantes del amoníaco en estudios ambientales en los que solamente puede usarse un gen (AU)

Historical biogeography of Mediterranean trout.

Complete sequencing of the mitochondrial control region was used to describe phylogenetic relationships of brown trout populations (Salmo trutta) in the Mediterranean river basins of Iberia and to review the historical biogeography of trout from the Mediterranean regions. Phylogenetic relationships among trout lineages suggested that the Danubian one is the most ancestral, in accordance with the eastern origin of most of the European freshwater fish species. Nested-clade and mismatch analyses suggested that the present distribution of haplotypes of the Adriatic and Mediterranean lineages resulted from population expansions originated, respectively, from central and western Europe, which favoured extensive secondary contacts between lineages. Reduced diversity detected within 50% of the analysed populations and large intrabasin differentiation indicated restricted gene flow in post-glacial periods.