Background: The bullet tuna (Auxis rochei) is an epipelagic fish with a worldwide distribution that is highly targeted by fisheries. Genetic diversity and population genetics are good indicators of population structure and thus, essential tools for fisheries management. Knowing which factors (biotic and abiotic) might be shaping such structure is crucial for management plans. In the present study, we assessed the population structure of the bullet tuna in the western and central Mediterranean Sea. Methods: We used two types of genetic data: the mitochondrial DNA control region and seven microsatellite loci. The analysis of 431 sequences with a length of 386 bp from the mtDNA CR and the results from 276 individuals were genotyped by seven microsatellite loci. Results: Both types of markers coincided in showing significant genetic differences between an Iberian Peninsula–Strait of Gibraltar stock in comparison with a North African stock. Conclusions: We argue that this differentiation pattern is likely caused by reproductive strategies such as coastal spawning, larval retention, and natal homing behavior. These results should endorse the implementation of management plans for a resource that currently is not being managed. Thus, to ensure sustainability, these new policies should consider the presence of at least two genetically identified stocks.

Conservation genetics has become a valuable resource for a proper stock management in wild fish populations. The analysis of the genetic variability permits a better identification of the species but it also allows assessing the status of the populations. Small tuna is a well characterised group of pelagic and migratory fish species usually exploited by traditional fisheries. In spite of that, morphological similarity between species of same genus can lead to incorrect species identification. Thus, the presence of multiple species within a single fishery can complicate its management hampering the viability of the populations. In addition, due to their life history traits -pelagic habitat, migratory behaviour, large population size, larval stage- and the apparent lack of natural barriers in their habitat, it has been postulated that these species are not prone to present population heterogeneity. However, several studies have shown population differentiation in some species. The aim of the present work, is to genetically validate the species of two sets of individuals from both the fishery of Euthynnus alletteratus and Auxis rochei, and later determine a putative population structure by using classic genetic markers. We analysed 175 individuals of E. alletteratus from four locations (Portugal, Tunisia, Ivory Coast and Senegal) and 438 individuals of A. rochei from eight populations (Portugal, Ceuta, Malaga, Cartagena, Valencia, Tarragona, Algeria and Tunisia). All specimens were sequenced for the mitochondrial DNA control region marker (mtDNA CR). Additionally, for E. alletteratus a subset of individuals was also tested for two unlinked nuclear genes, Rhodopsin and Calmodulin. For A. rochei a selection of individuals was sequenced for the Tmo4c4 nuclear gene. Sequences for all markers were analysed and aligned using Geneious 7.1.9 software. For both mtDNA CR alignments a list of unique haplotypes was created with DNAsp 6.12.03 and pairwise differences and FST were estimated in Arlequin 3.5.2.2. When analysing E. alletteratus, the BLAST results for Ivory Coast and Senegal showed less identity than the expected in same species for mtDNA CR sequences in GenBank (about 90%; e-value 1.33e-136), while Portugal and Tunisia showed the expected levels of sequence identity (about 99,7%; e-value 0). The analysis of mtDNA CR showed an extremely high differentiation comparing the southern individuals (Ivory Coast and Senegal) to the ones from the two northern locations (Portugal and Tunisia) (φst = 0.931; p = 0.000), that is, 93% of the genetic variation in those individuals is due to differences among populations. This value, close to the maximum, indicates a lack of genetic flow between those populations and a degree of differentiation at species level. In accordance with these results, in each group of individuals (Northern vs. Southern) and for both nuclear markers we did detect specific SNPs (single nucleotide polymorphism) that differentiate each putative species. Furthermore, we found low genetic heterogeneity between Portugal and Tunisia individuals (φst = 0.007; p = 0.198), as well low heterogeneity between Ivory Coast and Senegal locations (φst = 0.004; p = 0.162), which, based on previous reports, are in agreement with the expected population differentiation for these species. In the case of A. rochei, the mtDNA CR BLAST analysis revealed the presence of 7 Auxis thazard individuals within A. rochei fishery in the Ceuta population (identity about 98%; e-value 6e-173). These individuals were also tested for the Tmo4c4 nuclear marker, their sequences were identical with previous annotated A. thazard Tmo4c4 sequences, further, the comparison of the region here analysed exhibit one specific SNP between both species. The analysis of the mtDNA CR for this species resulted in a lack of genetic heterogeneity among locations on the Iberian Peninsula - Portugal, Ceuta, Malaga, Cartagena, Valencia and Tarragona- (φst = 0,000; p= 0,472), but significant differences between those locations and the two North-African locations, Algeria and Tunisia (φst = 0.034; p= 0.000). In conclusion, the extremely high level of population structure within E. alletteratus that displays the mtDNA CR marker and the specific SNPs found for each group, sets the scenario in which no genetic flow occurs between those populations and thus, a possible event of speciation may or might have started to occur. Regarding A. thazard, this is the first evidence of this species within the Mediterranean waters but also, this warns us of a putative mixed fishery with A. rochei. Still, more research is needed in order to clarify why these individuals were caught in the Mediterranean Sea. If they are no fruit of a stochastic event but there is a displacement on this species distribution, will be necessary to upgrade the present management directives for this fishery

Atlantic bonito (Sarda sarda) is an epipelagic migratory species of the family Scombridae and it is usually classified under the common name of small tuna. This species is heavily targeted by artisanal and commercial fisheries in the northeast Atlantic and Mediterranean. However, no management directive has been proposed for this species. In 2019, ICCAT (International Commission for the Conservation of Atlantic tuna) identified the Atlantic bonito as a critical species to be studied, due to the lack of knowledge in several key biological aspects such as growth, reproduction, and stock structure. In a preliminary genetic analysis of more than 600 individuals from the Northeast Atlantic and Mediterranean, using a single molecular marker (mitochondrial DNA control region), we detected a significant genetic differentiation between two groups of locations. One group with two locations within the Mediterranean (Spain and Tunisia) and one location in the Atlantic near to the strait of Gibraltar (Portugal); and another group comprising locations along the African coast (Morocco, Mauritania, Senegal, and Côte d’Ivoire). A new analysis, using a genomic approach (ddRadseq analysis) confirm the same populations structure. Population genetic analysis of 95 individuals from seven locations recovered more than 8500 SNPs with a coverage greater than 20X (mean = 27.9; SD = 16.8) and confirmed that the genetic discontinuity is not placed in the Strait of Gibraltar. The locations from north and south Portugal were grouped together with the two locations within the Mediterranean Sea (Spain and Tunisia) and clearly differentiated from the rest of locations in the African coast (Morocco, Senegal, and Côte d’Ivoire). These results updated the genetic population structure of Atlantic bonito, and they can be used as starting point to infer a proper management regulation for this relevant commercial species.