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.