IntroductionTunas (and tuna-like species) are among the most important fish species commercialized worldwide. Despite the slight decrease recorded in 2019 due to the COVID-19 restrictions, which impacted the export and sashimi markets (FAO 2022b; FAO 2024), tunas reached a record level of annual landings of 8.3 million tonnes in 2022. From a global trade perspective, tunas and tuna-like species (including bonitos and billfishes) worthed €16.2 billion in exports revenue in 2022, which represented roughly 9% of the total value for aquatic animal products exports (FAO 2024).There are seven different tuna species with major commercial importance at a global scale (ISSF 2024). The most globally caught species is skipjack tuna (Katsuwonus pelamis , Linnaeus, 1758, SKJ), corresponding to 58% of the total catch, followed by yellowfin tuna (Thunnus albacares , Bonnaterre, 1788, YFT) with 30% of the global catch, bigeye tuna (Thunnus obesus , Lowe, 1839, BET) with 7%, albacore tuna (Thunnus alalunga , Bonnaterre, 1788, ALB) with 4%, and Atlantic Bluefin tuna (Thunnus thynnus , Linnaeus, 1758, BFT) with only 1% (ISSF 2024). At the global level, all mentioned species are considered Least Concern by the IUCN (Collette et al. 2015a; Collette et al. 2021a; Collette et al. 2021b; Collette et al. 2021d), with the exception of BET which has maintained its Vulnerable conservation status throughout the years (Collette et al. 2021c). However, certain tuna populations may hold a regional conservation status that differs from the global assessment (Collette et al. 2015b).In 2022, 65% of the total tuna catches came from stocks that were not experiencing overfishing, mostly due to SKJ healthy stocks, but 13% still came from overfished YFT, BET, ALB and BFT stocks (ISSF 2024).Tuna can be sold fresh, frozen or cured, and its trade is divided into two main groups of commodities: the first comprised of processed and preserved tuna, mainly canned, and the second comprised of fresh and frozen tuna, mostly for sushi and sashimi markets (Klapper et al. 2023; Silva et al. 2024). The canned tuna market accounts for more than half of global tuna exports, primarily using SKJ and/or YFT, and with the European Union and the United States serving as key markets (Guillotreau et al. 2016). Canned tuna products experienced a decline in 2021 (FAO 2024), but made a robust rebound in 2022, increasing both in value and volume, and this increase is expected to continue over the next decades (Kawamoto 2022). In contrast, the fresh tuna market, which is lower in volume but commands a much higher trade value, has shown increased growth since 2021 (FAO 2024). A major share of this trade corresponds to the tuna sashimi market, which uses mostly BFT and BET (FAO 2022a; Klapper et al. 2023; Servusova and Piskata 2021) and is almost exclusively directed to Japan, which retains 60 to 80% of all global demand (Guillotreau et al. 2016). The market value of tuna species varies considerably depending on factors such as demand, quality, but also the species, origin and conservation status, influencing the allocation of specific species to different markets (Carreiro et al. 2023; Chapela et al. 2007; Kappel et al. 2017; Pecoraro et al. 2020).Over the last years, the importance and need for seafood to be traced back to its origins (traceability) has become evident (Cawthorn and Mariani 2017), with different mechanisms emerging to guarantee transparency and promote sector sustainability (Thorpe et al. 2022). Generally, food authenticity and traceability refers to a product’s taxonomical identification and geographical origins, respectively. The European Union’s regulations (2013) require seafood products in general, to have an indication of the scientific names and commercial designations of the species used in them. However, canned and processed products are excluded from this obligation, and according to the Council Regulation ECC 1536/1992 (1992), only the commercial designation is mandatory. Nevertheless, this regulation not only states that preserved tuna and bonito products must be prepared exclusively from designated species, namely those in the Thunnus genus or SKJ , but also explicitly prohibits the mixing of different species within the same container (1992). Beyond these regulations, companies can voluntarily identify the species used in their products, but some EU countries, such as Spain, impose additional rules, restricting the species that can be canned under each designation and mandating the use of precise labelling on packaging (Klapper et al. 2023).Different studies (Mariani et al. 2015) have suggested that seafood mislabelling is decreasing in European countries, but a study by Sotelo et al. (2018) revealed that 7.84% of canned tuna products, sampled from at least six European countries, incur in mislabelling practices. Incorrect identification and consequent mislabelling of tuna species, either intentionally or unintentionally, results in more than just economic deception of consumers. These errors can have far-reaching implications from inaccurate or erroneous landing reports, stock quota enforcement measures, fish species value, conservation assessments, population’s health parameters, loss of genetic diversity (Pardo et al. 2016), up to impaired sustainability evaluations of a specific fishing industry (Carreiro et al. 2023). Therefore, ensuring accurate species identification is essential for preserving both the economic and the ecological integrity of global tuna fisheries. However, species identification in the tuna canning industry, is particularly challenging since the extensive processing steps often hinder the identification of tuna species through morphological traits (Liu et al. 2016), and identification relies heavily on DNA-based methods (Klapper et al. 2023; Pardo et al. 2016).In Portugal, the canning industry is present in the mainland territory, which represents 80% of the national market, and in the Azores archipelago and, in 2022, the tuna canning industry alone accounted for 32.8% of all fishing and aquaculture processing industry’s production in Portugal (INE 2024). Portugal follows the standard European Union legislation for canned tuna products, and in some canning industries, such as the Azores canning industry, species scientific names are voluntarily indicated in cans, together with the capture origin and fishing methods used. However, the same does not happen for most of the other companies operating in the mainland, and little information is provided in the cans. In light of this, our study aimed to: 1) identify species used by the Portuguese tuna canning industry and determine if any of these species are classified under any conservation status, 2) test whether the canning industry shows any seasonal trends in the tuna species used or if these are randomly sourced throughout the year, 3) assess compliance of this industry with the current legislation, specifically with regards to the mixing of species (1992) within processed products.

AbstractThe development of management strategies for the promotion of sustainable fisheries relies on a deep knowledge of ecological and evolutionary processes driving the diversification and genetic variation of marine organisms. Sustainability strategies are especially relevant for marine species such as the European sardine (Sardina pilchardus), a small pelagic fish with high ecological and socioeconomic importance, especially in Southern Europe, whose stock has declined since 2006, possibly due to environmental factors. Here, we generated sequences for 139 mitochondrial genomes from individuals from 19 different geographical locations across most of the species distribution range, which was used to assess genetic diversity, diversification history and genomic signatures of selection. Our data supported an extensive gene flow in European sardine. However, phylogenetic analyses of mitogenomes revealed diversification patterns related to climate shifts in the late Miocene and Pliocene that may indicate past divergence related to rapid demographic expansion. Tests of selection showed a significant signature of purifying selection, but positive selection was also detected in different sites and specific mitochondrial lineages. Our results showed that European sardine diversification has been strongly driven by climate shifts, and rapid changes in marine environmental conditions are likely to strongly affect the distribution and stock size of this species. IntroductionUnderstanding the ecological and evolutionary mechanisms driving the diversification and dispersal of marine organisms is essential for elucidating their genetic variation patterns. This knowledge underpins the development of conservation and management strategies for species of economic and ecological importance 1,2. Advances in marine genomics provide new insights into the evolutionary history and population structure of marine organisms, as well as into the evolutionary consequences of selective harvest, local adaptation, and response to climate change 2-5. Genomic data have also become increasingly relevant for the assessment and promotion of sustainable fisheries 6,7, for example, by enabling demographic analyses for stock identification and management and the assessment of connectivity among geographically delimited stocks.Population structure in marine fishes has been presumed to be minimal, since marine environments have fewer barriers to gene flow compared to terrestrial ecosystems, resulting in high levels of connectivity among populations and large population sizes in marine species1,8. Consequently, it was posited that adaptive divergence would be limited or non-existent in marine fishes due to the overwhelming effect of genetic drift and gene flow. However, large population sizes may increase the probability of retention of advantageous alleles, a phenomenon facilitated by local selective pressures 9, and adaptive processes have been shown to shape genetic patterns in oceanic fish populations8,10-16.Local adaptation is the driving force behind divergent selection17, when different alleles are selected in different subpopulations, in contrast with global adaptation, when the same allele is selected across all species populations. Local adaptation results from two antagonistic forces, natural selection, which promotes intraspecific differentiation, and gene flow, which promotes homogenization. Identifying genomic signatures of natural selection is pivotal for unravelling the molecular mechanisms underlying adaptation18. Genomic signatures can result from different types of natural selection, manifesting in two main forms: positive and negative (or purifying) selection. Positive selection, which promotes the proliferation of beneficial mutations within a population, can be divided into balancing selection, preserving genetic polymorphisms, and directional selection, driving advantageous alleles to fixation, in contrast to purifying selection that works to eliminate deleterious mutations within a population 19.The European sardine, Sardina pilchardus (Walbaum, 1792), is a small pelagic fish from the Alosidae family, inhabiting the Northeast Atlantic Ocean, from the North Sea to Mauritania and Senegal and with populations in the Azores, Madeira and Canaries, and the MeditFerranean Sea 20. The European sardine is a migratory and schooling species that forms schools potentially comprising millions of individuals and is known to prefer colder water for living and spawning21-24. The European sardine plays an important role in marine ecosystems, as both a consumer of plankton and a prey for larger predators 25,26. Moreover, it is one of the most important marine fish resources in Southern Europe and Morocco27, especially in the Iberian Peninsula28, where its landings represent ~40% of the total capture 29. It constitutes the main target species for the purse-seine fleets operating in Portugal and Spain, thereby serving as a critical revenue stream for the respective local economies 30. The biomass of the Ibero-Atlantic stock has been declining since 2006, as its recruitment is strongly related to environmental conditions 31. This decline has led sardine abundance to fall to its historical minimums32, triggering profound socio-economic impacts on fishing communities. Consequently, this species has been subject to numerous studies, namely on its biology and ecology21,22,33-36, phenotypic variation37-39, population genetics 40-42, besides a complete genome sequencing 43,44.The mitochondrial genome (mitogenome), a maternally inherited, circular DNA molecule, has been a focal point in the study of evolutionary biology and population genetics due to its relatively high mutation rate, lack of recombination, and haploid nature. The positive and negative features of mitochondrial DNA (mtDNA) for population genetics, phylogeographic and phylogenetic studies have been extensively discussed45-47. Nevertheless, mitogenomes can provide valuable information at a relatively low cost as a byproduct of whole genome resequencing. Positive selection in mtDNA can be detected due to direct selection on the mitogenome or indirect selection in the nuclear genes that compose the mito-nuclear complex. The mitogenome contains 13 protein-coding genes that contribute to four Electron Transport System (ETS) complexes, whose function requires over 500 proteins encoded in the nuclear genome 48. This interaction between the mitogenome and the nuclear genome could generate diverse response patterns with compensatory mechanisms and coevolution between the two genomic compartments. A study on 70 mitogenomes of Clupeoid fishes49, including the European sardine, concluded for the prevalence of purifying selection, but also for the observed shift in codon preference patterns between marine and euryhaline/freshwater Clupeoids, indicating possible selection for improved translational efficiency while adapting to low-salinity habitats. This mitogenomic plasticity and enhanced efficiency of the metabolic machinery may have contributed to the evolutionary success and abundance of Clupeoid fish49. Mitogenomes can also harbour rare mutations that provide a selective advantage through the interaction with environmental factors such as temperature 45, raising the hypothesis that the wide temperature range of the European sardine increases its potential for local adaptation due to divergent selection.Previous studies on European sardine 50 have largely focused on mtDNA fragments to infer population structure, historical demography, and signatures of molecular adaptation. However, the resolution provided by partial mitochondrial data is limited, often obscuring finer-scale evolutionary processes and the detection of adaptive genetic variation. In this study, complete mitochondrial genomes from individuals collected across the entire distribution range of the European sardine were sequenced and analysed with three main objectives: 1) to assess the population genetic structure inside the species; 2) to reconstruct the phylogenetic relationships among lineages and explore the timing of diversification events within the species; and 3) to evaluate the role of natural selection (both positive and negative) in shaping mitochondrial genome variation, and its potential for local adaptation or association with intraspecific lineage.