Evidence of backcross inviability and mitochondrial DNA paternal leakage in sea turtle hybrids.

Hybridization is known to be part of many species' evolutionary history. Sea turtles have a fascinating hybridization system in which species separated by as much as 43 million years are still capable of hybridizing. Indeed, the largest nesting populations in Brazil of loggerheads (Caretta caretta) and hawksbills (Eretmochelys imbricata) have a high incidence of hybrids between these two species. A third species, olive ridleys (Lepidochelys olivacea), is also known to hybridize although at a smaller scale. Here, we used restriction site-associated DNA sequencing (RAD-Seq) markers, mitogenomes, and satellite-telemetry to investigate the patterns of hybridization and introgression in the Brazilian sea turtle population and their relationship with the migratory behaviours between feeding and nesting aggregations. We also explicitly test if the mixing of two divergent genomes in sea turtle hybrids causes mitochondrial paternal leakage. We developed a new species-specific PCR-assay capable of detecting mitochondrial DNA (mtDNA) inheritance from both parental species and performed ultra-deep sequencing to estimate the abundance of each mtDNA type. Our results show that all adult hybrids are first generation (F1) and most display a loggerhead migratory behaviour. We detected paternal leakage in F1 hybrids and different proportions of mitochondria from maternal and paternal species. Although previous studies showed no significant fitness decrease in hatchlings, our results support genetically-related hybrid breakdown possibly caused by cytonuclear incompatibility. Further research on hybrids from other populations in addition to Brazil and between different species will show if backcross inviability and mitochondrial paternal leakage is observed across sea turtle species.

Heterogeneous microgeographic genetic structure of the common cockle (Cerastoderma edule) in the Northeast Atlantic Ocean: biogeographic barriers and environmental factors.

Knowledge of genetic structure at the finest level is essential for the conservation of genetic resources. Despite no visible barriers limiting gene flow, significant genetic structure has been shown in marine species. The common cockle (Cerastoderma edule) is a bivalve of great commercial and ecological value inhabiting the Northeast Atlantic Ocean. Previous population genomics studies demonstrated significant structure both across the Northeast Atlantic, but also within small geographic areas, highlighting the need to investigate fine-scale structuring. Here, we analysed two geographic areas that could represent opposite models of structure for the species: (1) the SW British Isles region, highly fragmented due to biogeographic barriers, and (2) Galicia (NW Spain), a putative homogeneous region. A total of 9250 SNPs genotyped by 2b-RAD on 599 individuals from 22 natural beds were used for the analysis. The entire SNP dataset mostly confirmed previous observations related to genetic diversity and differentiation; however, neutral and divergent SNP outlier datasets enabled disentangling physical barriers from abiotic environmental factors structuring both regions. While Galicia showed a homogeneous structure, the SW British Isles region was split into four reliable genetic regions related to oceanographic features and abiotic factors, such as sea surface salinity and temperature. The information gathered supports specific management policies of cockle resources in SW British and Galician regions also considering their particular socio-economic characteristics; further, these new data will be added to those recently reported in the Northeast Atlantic to define sustainable management actions across the whole distribution range of the species.

Fitness consequences and ancestry loss in the Apennine brown bear after a simulated genetic rescue intervention.

Reduction in population size, with its predicted effects on population fitness, is the most alarming anthropogenic impact on endangered species. By introducing compatible individuals, genetic rescue (GR) is a promising but debated approach for reducing the genetic load unmasked by inbreeding and for restoring the fitness of declining populations. Although GR can improve genetic diversity and fitness, it can also produce loss of ancestry, hampering local adaptation, or replace with introduced variants the unique genetic pools evolved in endemic groups. We used forward genetic simulations based on empirical genomic data to assess fitness benefits and loss of ancestry risks of GR in the Apennine brown bear (Ursus arctos marsicanus). There are approximately 50 individuals of this isolated subspecies, and they have lower genetic diversity and higher inbreeding than other European brown bears, and GR has been suggested to reduce extinction risks. We compared 10 GR scenarios in which the number and genetic characteristics of migrants varied with a non-GR scenario of simple demographic increase due to nongenetic factors. The introduction of 5 individuals of higher fitness or lower levels of deleterious mutations than the target Apennine brown bear from a larger European brown bear population produced a rapid 10-20% increase in fitness in the subspecies and up to 22.4% loss of ancestry over 30 generations. Without a contemporary demographic increase, fitness started to decline again after a few generations. Doubling the population size without GR gradually increased fitness to a comparable level, but without losing ancestry, thus resulting in the best strategy for the Apennine brown bear conservation. Our results highlight the importance for management of endangered species of realistic forward simulations grounded in empirical whole-genome data.

Consecuencias en la aptitud y pérdida de ascendencia del oso pardo de los Apeninos después de un rescate genético simulado Resumen La reducción del tamaño poblacional, con los previsibles efectos sobre su aptitud, es el impacto antropogénico más alarmante sobre las especies amenazadas. Mediante la introducción de individuos compatibles, el rescate genético (RG) es una estrategia prometedora para reducir la carga genética revelada por la endogamia y restaurar la aptitud de las poblaciones en declive, aunque todavía se debate la eficiencia de esta. Aunque el RG puede mejorar la diversidad genética y la aptitud, también puede producir pérdida de ascendencia, lo que puede dificultar la adaptación local, o sustituir con variantes introducidas por los migrantes los acervos genéticos únicos que han evolucionado en grupos endémicos. En este trabajo realizamos simulaciones genéticas a futuro basadas en datos genómicos empíricos para evaluar los beneficios del RG en términos de aptitud y los riesgos de la pérdida de ascendencia en el oso pardo de los Apeninos (Ursus arctos marsicanus). Quedan aproximadamente 50 individuos de esta subespecie aislada que cuentan con una menor diversidad genética y un mayor nivel de endogamia comparado con otros osos pardos europeos y se ha sugerido que el RG podria reducir el riesgo de extinción de esta población. Comparamos 10 escenarios de RG en los que variaban el número y las características genéticas de los osos migrantes con un escenario sin RG con aumento demográfico causado por factores no genéticos. La introducción de 5 individuos procedentes de una población europea de oso pardo con mayor aptitud o niveles menores de mutaciones deletéreas que el oso pardo de los Apeninos produjo un rápido aumento de la aptitud del 10-20% en la subespecie y hasta un 22.4% de pérdida de ascendencia durante 30 generaciones. En las simulaciones sin un aumento demográfico, la aptitud empezó a disminuir de nuevo después de unas pocas generaciones. La duplicación del tamaño de la población sin RG aumentó gradualmente la aptitud hasta un nivel comparable al de algunos escenarios de RG, pero sin pérdida de ascendencia, por lo que parece ser la mejor estrategia para la conservación del oso pardo de los Apeninos. Nuestros resultados resaltan la importancia que tienen las simulaciones realistas a futuro basadas en datos empíricos del genoma completo para la gestión de especies amenazadas.

A genome-wide association study, supported by a new chromosome-level genome assembly, suggests sox2 as a main driver of the undifferentiatiated ZZ/ZW sex determination of turbot (Scophthalmus maximus).

BACKGROUND: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. RESULTS: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. CONCLUSIONS: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families.

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.

Genomic survey of edible cockle (Cerastoderma edule) in the Northeast Atlantic: A baseline for sustainable management of its wild resources.

Knowledge on correlations between environmental factors and genome divergence between populations of marine species is crucial for sustainable management of fisheries and wild populations. The edible cockle (Cerastoderma edule) is a marine bivalve distributed along the Northeast Atlantic coast of Europe and is an important resource from both commercial and ecological perspectives. We performed a population genomics screening using 2b-RAD genotyping on 9309 SNPs localized in the cockle's genome on a sample of 536 specimens pertaining to 14 beds in the Northeast Atlantic Ocean to analyse the genetic structure with regard to environmental variables. Larval dispersal modelling considering species behaviour and interannual/interseasonal variation in ocean conditions was carried out as an essential background to which compare genetic information. Cockle populations in the Northeast Atlantic displayed low but significant geographical differentiation between populations (F ST = 0.0240; p < 0.001), albeit not across generations. We identified 742 and 36 outlier SNPs related to divergent and balancing selection in all the geographical scenarios inspected, and sea temperature and salinity were the main environmental correlates suggested. Highly significant linkage disequilibrium was detected at specific genomic regions against the very low values observed across the whole genome. Two main genetic groups were identified, northwards and southwards of French Brittany. Larval dispersal modelling suggested a barrier for larval dispersal linked to the Ushant front that could explain these two genetic clusters. Further genetic subdivision was observed using outlier loci and considering larval advection. The northern group was divided into the Irish/Celtic Seas and the English Channel/North Sea, while the southern group was divided into three subgroups. This information represents the baseline for the management of cockles, designing conservation strategies, founding broodstock for depleted beds and producing suitable seed for aquaculture production.

Genome-wide analysis clarifies the population genetic structure of wild gilthead sea bream (Sparus aurata).

Gilthead sea bream is an important target for both recreational and commercial fishing in Europe, where it is also one of the most important cultured fish. Its distribution ranges from the Mediterranean to the African and European coasts of the North-East Atlantic. Until now, the population genetic structure of this species in the wild has largely been studied using microsatellite DNA markers, with minimal genetic differentiation being detected. In this geographically widespread study, 958 wild gilthead sea bream from 23 locations within the Mediterranean Sea and Atlantic Ocean were genotyped at 1159 genome-wide SNP markers by RAD sequencing. Outlier analyses identified 18 loci potentially under selection. Neutral marker analyses identified weak subdivision into three genetic clusters: Atlantic, West, and East Mediterranean. The latter group could be further subdivided into an Ionian/Adriatic and an Aegean group using the outlier markers alone. Seascape analysis suggested that this differentiation was mainly due to difference in salinity, this being also supported by preliminary genomic functional analysis. These results are of fundamental importance for the development of proper management of this species in the wild and are a first step toward the study of the potential genetic impact of the sea bream aquaculture industry.

A Useful SNP Panel to Distinguish Two Cockle Species, Cerastoderma edule and C. glaucum, Co-Occurring in Some European Beds, and Their Putative Hybrids.

Cockles are highly appreciated mollusks and provide important services in coastal areas. The two European species, edible (Cerastodermaedule) and lagoon (Cerastodermaglaucum) cockles, are not easily distinguishable, especially when young. Interestingly, the species show different resistance to Marteilia cochillia, the parasite responsible for marteiliosis outbreaks, which is devastating cockle production in some areas. C.edule is severely affected by the parasite, while C. glaucum seems to be resistant, although underlying reasons are still unknown. Hybrids between both species might be interesting to introgress allelic variants responsible for tolerance, either naturally or through artificial selection, from lagoon into edible cockle. Here, we used 2b restriction site-associated DNA sequencing (2b-RAD) to identify single nucleotide polymorphisms (SNP) diagnostic for cockle discrimination (fixed for alternative allelic variants). Among the nine diagnostic SNPs selected, seven were validated using a SNaPshot assay in samples covering most of the distribution range of both species. The validated SNPs were used to check cockles that were suggested to be hybrids by a claimed diagnostic tool based on the internal transcribed spacers of the ribosomal RNA. Although these were shown to be false positives, we cannot rule out the fact that hybrids can occur and be viable. The SNP tool here developed will be valuable for their identification and management.

Tracing seafood at high spatial resolution using NGS-generated data and machine learning: Comparing microbiome versus SNPs.

Developing reliable tools to trace food origin represents a major goal for producers and control authorities. Here, we test the hypothesis whether NGS-generated data could provide a reliable tool to ensure seafood traceability. As a test case, we used the Manila clam Ruditapes philippinarum, a bivalve mollusk of high commercial interest with worldwide distribution, collected in the Venice lagoon sites subjected to prohibition of clam harvesting because of chemical contamination as well as in authorized clam harvesting areas. The results obtained demonstrated that the geographic origin of Manila clam may be more accurately determined basing on microbiome data than single nucleotide polymorphisms. In particular, combining microbiome data with machine-learning techniques, we provide the experimental evidence that it is possible to trace the clam place of origin at high spatial resolution. Considering its low cost and portability, NGS-analysis of microbiome data might represent a cost-effective, high-resolution tool for reliable food traceability.

Disentangling Genetic Variation for Resistance and Endurance to Scuticociliatosis in Turbot Using Pedigree and Genomic Information.

Selective breeding for improving host responses to infectious pathogens is a promising option for disease control. In fact, disease resilience, the ability of a host to survive or cope with infectious challenge, has become a highly desirable breeding goal. However, resilience is a complex trait composed of two different host defence mechanisms, namely resistance (the ability of a host to avoid becoming infected or diseased) and endurance (the ability of an infected host to survive the infection). While both could be targeted for genetic improvement, it is currently unknown how they contribute to survival, as reliable estimates of genetic parameters for both traits obtained simultaneously are scarce. A difficulty lies in obtaining endurance phenotypes for genetic analyses. In this study, we present the results from an innovative challenge test carried out in turbot whose design allowed disentangling the genetic basis of resistance and endurance to Philasterides dicentrarchi, a parasite causing scuticociliatosis that leads to substantial economic losses in the aquaculture industry. A noticeable characteristic of the parasite is that it causes visual signs that can be used for disentangling resistance and endurance. Our results showed the existence of genetic variation for both traits (heritability = 0.26 and 0.12 for resistance and endurance, respectively) and for the composite trait resilience (heritability = 0.15). The genetic correlation between resistance and resilience was very high (0.90) indicating that both are at a large extent the same trait, but no significant genetic correlation was found between resistance and endurance. A total of 18,125 SNPs obtained from 2b-RAD sequencing enabled genome-wide association analyses for detecting QTLs controlling the three traits. A candidate QTL region on linkage group 19 that explains 33% of the additive genetic variance was identified for resilience. The region contains relevant genes related to immune response and defence mechanisms. Although no significant associations were found for resistance, the pattern of association was the same as for resilience. For endurance, one significant association was found on linkage group 2. The accuracy of genomic breeding values was also explored for resilience, showing that it increased by 12% when compared with the accuracy of pedigree-based breeding values. To our knowledge, this is the first study in turbot disentangling the genetic basis of resistance and endurance to scuticociliatosis.

Parallel evolution and adaptation to environmental factors in a marine flatfish: Implications for fisheries and aquaculture management of the turbot (Scophthalmus maximus).

Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot (Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range (672 individuals; 20 populations) by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double-digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming.

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish.

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.

RAD SNP markers as a tool for conservation of dolphinfish Coryphaena hippurus in the Mediterranean Sea: Identification of subtle genetic structure and assessment of populations sex-ratios.

Dolphinfish is an important fish species for both commercial and sport fishing, but so far limited information is available on genetic variability and pattern of differentiation of dolphinfish populations in the Mediterranean basin. Recently developed techniques allow genome-wide identification of genetic markers for better understanding of population structure in species with limited genome information. Using restriction-site associated DNA analysis we successfully genotyped 140 individuals of dolphinfish from eight locations in the Mediterranean Sea at 3324 SNP loci. We identified 311 sex-related loci that were used to assess sex-ratio in dolphinfish populations. In addition, we identified a weak signature of genetic differentiation of the population closer to Gibraltar Strait in comparison to other Mediterranean populations, which might be related to introgression of individuals from Atlantic. No further genetic differentiation could be detected in the other populations sampled, as expected considering the known highly mobility of the species. The results obtained improve our knowledge of the species and can help managing dolphinfish stock in the future.