Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Rhodopsin gene polymorphism associated with divergent light environments in Atlantic cod.

The spectral sensitivity of visual pigments in vertebrate eyes is optimized for specific light conditions. One of such pigments, rhodopsin (RH1), mediates dim-light vision. Amino acid replacements at tuning sites may alter spectral sensitivity, providing a mechanism to adapt to ambient light conditions and depth of habitat in fish. Here we present a first investigation of RH1 gene polymorphism among two ecotypes of Atlantic cod in Icelandic waters, which experience divergent light environments throughout the year due to alternative foraging behaviour. We identified one synonymous single nucleotide polymorphism (SNP) in the RH1 protein coding region and one in the 3' untranslated region (3'-UTR) that are strongly divergent between these two ecotypes. Moreover, these polymorphisms coincided with the well-known panthophysin (Pan I) polymorphism that differentiates coastal and frontal (migratory) populations of Atlantic cod. While the RH1 SNPs do not provide direct inference for a specific molecular mechanism, their association with this dim-sensitive pigment indicates the involvement of the visual system in local adaptation of Atlantic cod.

Outlier SNP markers reveal fine-scale genetic structuring across European hake populations (Merluccius merluccius).

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.

A genomic island linked to ecotype divergence in Atlantic cod.

The genomic architecture underlying ecological divergence and ecological speciation with gene flow is still largely unknown for most organisms. One central question is whether divergence is genome-wide or localized in 'genomic mosaics' during early stages when gene flow is still pronounced. Empirical work has so far been limited, and the relative impacts of gene flow and natural selection on genomic patterns have not been fully explored. Here, we use ecotypes of Atlantic cod to investigate genomic patterns of diversity and population differentiation in a natural system characterized by high gene flow and large effective population sizes, properties which theoretically could restrict divergence in local genomic regions. We identify a genomic region of strong population differentiation, extending over approximately 20 cM, between pairs of migratory and stationary ecotypes examined at two different localities. Furthermore, the region is characterized by markedly reduced levels of genetic diversity in migratory ecotype samples. The results highlight the genomic region, or 'genomic island', as potentially associated with ecological divergence and suggest the involvement of a selective sweep. Finally, we also confirm earlier findings of localized genomic differentiation in three other linkage groups associated with divergence among eastern Atlantic populations. Thus, although the underlying mechanisms are still unknown, the results suggest that 'genomic mosaics' of differentiation may even be found under high levels of gene flow and that marine fishes may provide insightful model systems for studying and identifying initial targets of selection during ecological divergence.

Variations in the Physical Properties and Microbial Community of Dairy Cow Manure-Implications for Testing and Efficacy of Footbathing Products.

Footbaths containing disinfectants are used on dairy farms to reduce the spread of digital dermatitis; however, they commonly become contaminated with manure. This trial investigated the physical properties and microbial composition of dairy cow manure from two production systems and examined whether the source of manure impacted the efficacy of footbathing disinfectants. Manure was collected from eighteen dairy cows, nine housed and fed grass silage (HOUSED) and nine at pasture (PASTURE). The pH and dry matter content was determined, total DNA was extracted and the region v3-v4 of the 16s rRNA gene sequenced. The efficacy of formalin and two trial products (TP1: peracetic acid and hydrogen peroxide; TP2: chlorocresol and triamine) was evaluated when mixed with manure from the two production systems. Production system differences were found in manure dry matter content, bacterial microbiome and the efficacy of both trial footbathing products but not formalin. The properties of manure affected the results of laboratory testing and therefore have the potential to influence footbathing disinfectant efficacy when footbaths are contaminated with manure. Further research into the impact of organic contaminants on the efficacy of disinfectants could facilitate the development of improved testing programmes and disinfectant products.

Towards vibrant fish populations and sustainable fisheries that benefit all: learning from the last 30 years to inform the next 30 years.

A common goal among fisheries science professionals, stakeholders, and rights holders is to ensure the persistence and resilience of vibrant fish populations and sustainable, equitable fisheries in diverse aquatic ecosystems, from small headwater streams to offshore pelagic waters. Achieving this goal requires a complex intersection of science and management, and a recognition of the interconnections among people, place, and fish that govern these tightly coupled socioecological and sociotechnical systems. The World Fisheries Congress (WFC) convenes every four years and provides a unique global forum to debate and discuss threats, issues, and opportunities facing fish populations and fisheries. The 2021 WFC meeting, hosted remotely in Adelaide, Australia, marked the 30th year since the first meeting was held in Athens, Greece, and provided an opportunity to reflect on progress made in the past 30 years and provide guidance for the future. We assembled a diverse team of individuals involved with the Adelaide WFC and reflected on the major challenges that faced fish and fisheries over the past 30 years, discussed progress toward overcoming those challenges, and then used themes that emerged during the Congress to identify issues and opportunities to improve sustainability in the world's fisheries for the next 30 years. Key future needs and opportunities identified include: rethinking fisheries management systems and modelling approaches, modernizing and integrating assessment and information systems, being responsive and flexible in addressing persistent and emerging threats to fish and fisheries, mainstreaming the human dimension of fisheries, rethinking governance, policy and compliance, and achieving equity and inclusion in fisheries. We also identified a number of cross-cutting themes including better understanding the role of fish as nutrition in a hungry world, adapting to climate change, embracing transdisciplinarity, respecting Indigenous knowledge systems, thinking ahead with foresight science, and working together across scales. By reflecting on the past and thinking about the future, we aim to provide guidance for achieving our mutual goal of sustaining vibrant fish populations and sustainable fisheries that benefit all. We hope that this prospective thinking can serve as a guide to (i) assess progress towards achieving this lofty goal and (ii) refine our path with input from new and emerging voices and approaches in fisheries science, management, and stewardship.

Environmental selection on transcriptome-derived SNPs in a high gene flow marine fish, the Atlantic herring (Clupea harengus).

High gene flow is considered the norm for most marine organisms and is expected to limit their ability to adapt to local environments. Few studies have directly compared the patterns of differentiation at neutral and selected gene loci in marine organisms. We analysed a transcriptome-derived panel of 281 SNPs in Atlantic herring (Clupea harengus), a highly migratory small pelagic fish, for elucidating neutral and selected genetic variation among populations and to identify candidate genes for environmental adaptation. We analysed 607 individuals from 18 spawning locations in the northeast Atlantic, including two temperature clines (5-12 °C) and two salinity clines (5-35‰). By combining genome scan and landscape genetic analyses, four genetically distinct groups of herring were identified: Baltic Sea, Baltic-North Sea transition area, North Sea/British Isles and North Atlantic; notably, samples exhibited divergent clustering patterns for neutral and selected loci. We found statistically strong evidence for divergent selection at 16 outlier loci on a global scale, and significant correlations with temperature and salinity at nine loci. On regional scales, we identified two outlier loci with parallel patterns across temperature clines and five loci associated with temperature in the North Sea/North Atlantic. Likewise, we found seven replicated outliers, of which five were significantly associated with low salinity across both salinity clines. Our results reveal a complex pattern of varying spatial genetic variation among outlier loci, likely reflecting adaptations to local environments. In addition to disclosing the fine scale of local adaptation in a highly vagile species, our data emphasize the need to preserve functionally important biodiversity.

Combining spawn egg counts, individual photo-ID and genetic fingerprinting to estimate the population size and sex ratio of an endangered amphibian.

According to the International Union for Conservation of Nature Red List, 41% of the world's amphibian species are threatened with extinction, making them more threatened than any other vertebrate group nowadays. Given the global amphibian crisis, comprehensive understanding of demographics and population trends of declining and threatened species is essential for effective management and conservation strategies. Counting egg spawns is widely used to assess population abundance in pond breeding anurans. However, it is unknown how such counts translate into robust population size estimations. We monitored the breeding activity of the Natterjack toad (Epidalea calamita), combining egg string counts and individual photo-identification with Capture-Mark-Recapture population size and operational sex ratio estimation. Male Natterjack toads were identified by the pattern of natural markings with repeated ID of the same individual confirmed for 10% of the samples using genetic fingerprinting. We identified 647 unique individuals within a closed study population at Caherdaniel, Co Kerry. Population estimates derived from egg string counts estimated a breeding population of 368 females (95% CI 353-384) and Capture-Mark-Recapture estimated a breeding population of 1698 males (95% CI 1000-2397). The female:male sex ratio was conservatively estimated at 1:5 (95% CI 1:3-1:6) where 62% ± 6% of females were assumed to spawn. These substantially departed from any priori assumption of 1:1 which could have underestimated the breeding population by up to 72%. Where amphibian absolute population size estimation is necessary, methods should include empirical survey data on operational sex ratios and not rely on assumptions or those derived from the literature which may be highly population and/or context-dependent.

Validation of FASTFISH-ID: A new commercial platform for rapid fish species authentication via universal closed-tube barcoding.

Seafood represents up to 20% of animal protein consumption in global food consumption and is a critical dietary and income resource for the world's population. Currently, over 30% of marine fish stocks are harvested at unsustainable levels, and the industry faces challenges related to Illegal, Unregulated and Unreported (IUU) fishing. Accurate species identification is one critical component of successful stock management and helps combat fraud. Existing DNA-based technologies permit identification of seafood even when morphological features are removed, but are either too time-consuming, too expensive, or too specific for widespread use throughout the seafood supply chain. FASTFISH-ID is an innovative commercial platform for fish species authentication, employing closed-tube barcoding in a portable device. This method begins with asymmetric PCR amplification of the full length DNA barcode sequence and subsequently interrogates the resulting single-stranded DNA with a universal set of Positive/Negative probes labeled in two fluorescent colors. Each closed-tube reaction generates two species-specific fluorescent signatures that are then compared to a cloud-based library of previously validated fluorescent signatures. This novel approach results in rapid, automated species authentication without the need for complex, time consuming, identification by DNA sequencing, or repeated analysis with a panel of species-specific tests. Performance of the FASTFISH-ID platform was assessed in a blinded study carried out in three laboratories located in the UK and North America. The method exhibited a 98% success rate among the participating laboratories when compared to species identification via conventional DNA barcoding by sequencing. Thus, FASTFISH-ID is a promising new platform for combating seafood fraud across the global seafood supply chain.

Author Correction: Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Range-wide genomic data synthesis reveals transatlantic vicariance and secondary contact in Atlantic cod.

Recent advances in genetic and genomic analysis have greatly improved our understanding of spatial population structure in marine species. However, studies addressing phylogeographic patterns at oceanic spatial scales remain rare. In Atlantic cod (Gadus morhua), existing range-wide examinations suggest significant transatlantic divergence, although the fine-scale contemporary distribution of populations and potential for secondary contact are largely unresolved. Here, we explore transatlantic phylogeography in Atlantic cod using a data-synthesis approach, integrating multiple genome-wide single-nucleotide polymorphism (SNP) datasets representative of different regions to create a single range-wide dataset containing 1,494 individuals from 54 locations and genotyped at 796 common loci. Our analysis highlights significant transatlantic divergence and supports the hypothesis of westward post-glacial colonization of Greenland from the East Atlantic. Accordingly, our analysis suggests the presence of transatlantic secondary contact off eastern North America and supports existing perspectives on the phylogeographic history of Atlantic cod with an unprecedented combination of genetic and geographic resolution. Moreover, we demonstrate the utility of integrating distinct SNP databases of high comparability.

Natural history and molecular evolution of demersal Mediterranean sharks and skates inferred by comparative phylogeographic and demographic analyses.

BACKGROUND: The unique and complex paleoclimatic and paleogeographic events which affected the Mediterranean Sea since late Miocene deeply influenced the distribution and evolution of marine organisms and shaped their genetic structure. Following the Messinian salinity crisis and the sea-level fluctuations during the Pleistocene, several Mediterranean marine species developed deep genetic differentiation, and some underwent rapid radiation. Here, we consider two of the most prioritized groups for conservation in the light of their evolutionary history: sharks and rays (elasmobranchs). This paper deals with a comparative multispecies analysis of phylogeographic structure and historical demography in two pairs of sympatric, phylogenetically- and ecologically-related elasmobranchs, two scyliorhinid catsharks (Galeus melastomus, Scyliorhinus canicula) and two rajid skates (Raja clavata, Raja miraletus). Sampling and experimental analyses were designed to primarily test if the Sicilian Channel can be considered as effective eco-physiological barrier for Mediterranean demersal sympatric elasmobranchs. METHODS: The phylogeography and the historical demography of target species were inferred by analysing the nucleotide variation of three mitochondrial DNA markers (i.e., partial sequence of COI, NADH2 and CR) obtained from a total of 248 individuals sampled in the Western and Eastern Mediterranean Sea as well as in the adjacent northeastern Atlantic Ocean. Phylogeographic analysis was performed by haplotype networking and testing spatial genetic differentiation of samples (i.e., analysis of molecular variance and of principal components). Demographic history of Mediterranean populations was reconstructed using mismatch distribution and Bayesian Skyline Plot analyses. RESULTS: No spatial genetic differentiation was identified in either catshark species, while phylogeographic structure of lineages was identified in both skates, with R. miraletus more structured than R. clavata. However, such structuring of skate lineages was not consistent with the separation between Western and Eastern Mediterranean. Sudden demographic expansions occurred synchronously during the upper Pleistocene (40,000-60,000 years ago) in both skates and G. melastomus, likely related to optimal environmental conditions. In contrast, S. canicula experienced a slow and constant increase in population size over the last 350,000 years. DISCUSSION: The comparative analysis of phylogeographic and historical demographic patterns for the Mediterranean populations of these elasmobranchs reveals that historical phylogeographic breaks have not had a large impact on their microevolution. We hypothesize that interactions between environmental and ecological/physiological traits may have been the driving force in the microevolution of these demersal elasmobranch species in the Mediterranean rather than oceanographic barriers.

Ecological differences and coexistence in a guild of microparasites: Bartonella in wild rodents.

The study of ecological differences among coexisting microparasites has been largely neglected, but it addresses important and unusual issues because there is no clear distinction in such cases between conventional (resource) and apparent competition. Here patterns in the population dynamics are examined for four species of Bartonella (bacterial parasites) coexisting in two wild rodent hosts, bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus). Using generalized linear modeling and mixed effects models, we examine, for these four species, seasonal patterns and dependencies on host density (both direct and delayed) and, having accounted for these, any differences in prevalence between the two hosts. Whereas previous studies had failed to uncover species differences, here all four were different. Two, B. doshiae and B. taylorii, were more prevalent in wood mice, and one, B. birtlesii, was more prevalent in bank voles. B. birtlesii, B. grahamii, and B. taylorii peaked in prevalence in the fall, whereas B. doshiae peaked in spring. For B. birtlesii in bank voles, density dependence was direct, but for B. taylorii in wood mice density dependence was delayed. B. birtlesii prevalence in wood mice was related to bank vole density. The implications of these differences for species coexistence are discussed.

The dynamics of murid gammaherpesvirus 4 within wild, sympatric populations of bank voles and wood mice.

Murid gammaherpesvirus 4 (MuHV-4) is widely used as a small animal model for understanding gammaherpesvirus infections in man. However, there have been no epidemiological studies of the virus in wild populations of small mammals. As MuHV-4 both infects cells associated with the respiratory and immune systems and attempts to evade immune control via various molecular mechanisms, infection may reduce immunocompetence with potentially serious fitness consequences for individuals. Here we report a longitudinal study of antibody to MuHV-4 in a mixed assemblage of bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus) in the UK. The study was conducted between April 2001 and March 2004. Seroprevalence was higher in wood mice than bank voles, supporting earlier work that suggested wood mice were the major host even though the virus was originally isolated from a bank vole. Analyses of both the probability of having antibodies and the probability of initial seroconversion indicated no clear seasonal pattern or relationship with host density. Instead, infection risk was most closely associated with individual characteristics, with heavier males having the highest risk. This may reflect individual variation in susceptibility, potentially related to variability in the ability to mount an effective immune response.

Outlier Loci Detect Intraspecific Biodiversity amongst Spring and Autumn Spawning Herring across Local Scales.

Herring, Clupea harengus, is one of the ecologically and commercially most important species in European northern seas, where two distinct ecotypes have been described based on spawning time; spring and autumn. To date, it is unknown if these spring and autumn spawning herring constitute genetically distinct units. We assessed levels of genetic divergence between spring and autumn spawning herring in the Baltic Sea using two types of DNA markers, microsatellites and Single Nucleotide Polymorphisms, and compared the results with data for autumn spawning North Sea herring. Temporally replicated analyses reveal clear genetic differences between ecotypes and hence support reproductive isolation. Loci showing non-neutral behaviour, so-called outlier loci, show convergence between autumn spawning herring from demographically disjoint populations, potentially reflecting selective processes associated with autumn spawning ecotypes. The abundance and exploitation of the two ecotypes have varied strongly over space and time in the Baltic Sea, where autumn spawners have faced strong depression for decades. The results therefore have practical implications by highlighting the need for specific management of these co-occurring ecotypes to meet requirements for sustainable exploitation and ensure optimal livelihood for coastal communities.

Inbreeding uncovers fundamental differences in the genetic load affecting male and female fertility in a butterfly.

Inbreeding depression is most pronounced for traits closely associated with fitness. The traditional explanation is that natural selection eliminates deleterious mutations with additive or dominant effects more effectively than recessive mutations, leading to directional dominance for traits subject to strong directional selection. Here we report the unexpected finding that, in the butterfly Bicyclus anynana, male sterility contributes disproportionately to inbreeding depression for fitness (complete sterility in about half the sons from brother-sister matings), while female fertility is insensitive to inbreeding. The contrast between the sexes for functionally equivalent traits is inconsistent with standard selection arguments, and suggests that trait-specific developmental properties and cryptic selection play crucial roles in shaping genetic architecture. There is evidence that spermatogenesis is less developmentally stable than oogenesis, though the unusually high male fertility load in B. anynana additionally suggests the operation of complex selection maintaining male sterility recessives. Analysis of the precise causes of inbreeding depression will be needed to generate a model that reliably explains variation in directional dominance and reconciles the gap between observed and expected genetic loads carried by populations. This challenging evolutionary puzzle should stimulate work on the occurrence and causes of sex differences in fertility load.

Fish product mislabelling: failings of traceability in the production chain and implications for illegal, unreported and unregulated (IUU) fishing.

Increasing consumer demand for seafood, combined with concern over the health of our oceans, has led to many initiatives aimed at tackling destructive fishing practices and promoting the sustainability of fisheries. An important global threat to sustainable fisheries is Illegal, Unreported and Unregulated (IUU) fishing, and there is now an increased emphasis on the use of trade measures to prevent IUU-sourced fish and fish products from entering the international market. Initiatives encompass new legislation in the European Union requiring the inclusion of species names on catch labels throughout the distribution chain. Such certification measures do not, however, guarantee accuracy of species designation. Using two DNA-based methods to compare species descriptions with molecular ID, we examined 386 samples of white fish, or products labelled as primarily containing white fish, from major UK supermarket chains. Species specific real-time PCR probes were used for cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to provide a highly sensitive and species-specific test for the major species of white fish sold in the UK. Additionally, fish-specific primers were used to sequence the forensically validated barcoding gene, mitochondrial cytochrome oxidase I (COI). Overall levels of congruence between product label and genetic species identification were high, with 94.34% of samples correctly labelled, though a significant proportion in terms of potential volume, were mislabelled. Substitution was usually for a cheaper alternative and, in one case, extended to a tropical species. To our knowledge, this is the first published study encompassing a large-scale assessment of UK retailers, and if representative, indicates a potentially significant incidence of incorrect product designation.

SNP discovery using Next Generation Transcriptomic Sequencing in Atlantic herring (Clupea harengus).

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

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 gene-associated 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.

Novel tools for conservation genomics: comparing two high-throughput approaches for SNP discovery in the transcriptome of the European hake.

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).