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Home , Euthynnus, Little tunny

SCRS/2019/113 Collect. Vol. Sci. Pap. ICCAT, 76(7): 149-155 (2019)

TWO SPECIES WITHIN THE ( alletteratus)

Judith Ollé1, Laura Vilà1, Jordi Viñas1*

SUMMARY

With the aim to determine whether Euthynnus alletteratus present population structure in its area of fishery. We genetically analyzed up to 175 individuals captured in the localities of Portugal, Tunisia, Senegal and Côte d’Ivoire. The results of the analyses of three unlinked molecular markers (mitochondrial DNA, rhodopsin and calmodulin) showed strong population structure separating in two clades the individuals of Portugal and Tunisia from the other two localities: Senegal and Côte d’Ivoire. The high levels of genetic differentiation are equivalent with the levels of differentiation observed between Euthynnus species. These findings suggest that E. alletteratus fishery is actually composed by two different species distributed in separated areas within the .

RÉSUMÉ

Afin de déterminer si Euthynnus alletteratus présente une structure de population dans la zone où cette espèce est pêchée, une analyse génétique de 175 spécimens capturés dans des localités de l’UE- Portugal, de la Tunisie, du Sénégal et de la Côte d’Ivoire a été réalisée. Les résultats des analyses de trois marqueurs moléculaires non liés (ADN mitochondrial, rhodopsine et calmoduline) ont montré une forte structure de population divisant en deux clades les spécimens du Portugal et de la Tunisie de deux autres localités : Sénégal et Côte d’Ivoire. Les niveaux élevés de différenciation génétique sont équivalents aux niveaux de différenciation observés entre les espèces d'Euthynnus. Ces résultats suggèrent que la pêcherie d'E. Alletteratus est en réalité composée de deux espèces différentes réparties dans des zones séparées de l'océan Atlantique. RESUMEN Con el objetivo de determinar si Euthynnus alletteratus presenta una estructura de población en su área de pesca, se analizaron genéticamente hasta 175 ejemplares capturados en lugares de Portugal, Túnez, Senegal y Côte d’Ivoire. Los resultados de los análisis de tres marcadores moleculares no vinculados (ADN mitocondrial, rodopsina y calmodulina) mostraron una fuerte estructura de población que dividió a la población en dos clados; separando los ejemplares de Portugal y Túnez de los de Senegal y Cote d’Ivoire. Los altos niveles de diferenciación genética son equivalentes a los niveles de diferenciación observados entre las especies de Euthynnus. Estos hallazgos sugieren que la pesquería de E. alletteratus está compuesta en realidad por dos especies diferentes distribuidas en áreas separadas dentro del océano Atlántico.

KEYWORDS

Small , Stock identification, Little tunny (LTA) (Euthynnus alletteratus), Phylogenetics.

1Laboratori Ictiologia Genètica, Departament de Biologia, Universitat de Girona. 17071, Girona, Spain. *Corresponding author:[email protected]. 149 Introduction

Euthynnus alletteratus (little tunny) is a member of the Small Tuna species, as other members of this group it has relevant economic impact due to their exploitation by traditional along the Atlantic and Mediterranean coasts. Nowadays the available knowledge referring to the life-history traits of the E. alletteratus is still scarce. To address this problem, the recollection of biological data it is a key factor. With this aim, Small Tuna Year Program seek to improve the knowledge on three biological aspects: age and growth, reproduction and stock structure.

Captures on E. alletteratus can highly fluctuate among years, but due to their exploitation by traditional fisheries and as ICCAT state in the last report, decreasing trends can be mask by dominant single species landings, thus secondary species are discarded and not reported (ICCAT, 2018). For any commercial species, overexploitation can reduce the genetic diversity of the species, narrowing their adaptive capacity and compromising its future viability.

Moreover, as members of the family, the biological traits of this species, should avoid the presence of population structure. Because their pelagic habitat, the large population size and their migratory behavior, should promote gene flow between locations and keep high levels of genetic diversity (Waples, 1998). However, many studies have shown how indeed some scombrid species can have structured populations (Alvarado Bremer et al., 1998; Vinãs, Alvarado Bremer and Pla, 2004a; Viñas, Alvarado Bremer and Pla, 2004b; Zardoya et al., 2004).

In the present work we assess the stock structure of this species using population genetic methodologies in the E. alletteratus fishery from the Atlantic and .

Material and Methods

For this study, we did analyze 175 individuals of E. alletteratus from 4 different locations distributed in two areas: North Atlantic-Mediterranean which includes Portugal (32) and Tunisia (46); Tropical Atlantic with Senegal (50) and Côte d’Ivoire (47). Additionally, for the analysis we included 20 individuals of from Vietnam. See Table 1 and Figure 1 for a detailed description of the sampling.

DNA was extracted using a commercial kit (Real pure genomic DNA Extraction Kit, Durviz, Valencia, Spain) and amplified for the mitochondrial DNA control region marker (mtDNA CR) (Viñas, Alvarado Bremer and Pla, 2004a). A batch of individuals were also amplified for two nuclear markers, rhodopsin Rhod2F (5’- GGTCCCGTTACATCCCTGA-3’) Rhod1R (5’-CATTGGGTTGTAGATGGAGGA-3’) and calmodulin (Chow and Takeyama, 2000). All PCR products were cleansed and sequenced one way with the corresponding forward primer.

Sequences were edited and aligned using Geneious 7.1.9 (Kearse et al., 2012) (MUSCLE alignment), for each individual the species assignation was corroborated comparing its sequence with previously annotated sequences available in the GenBank public databases (BLAST, NCBI) (Altschul et al., 1990). In order to improve the phylogeny reconstruction sequences for rhodopsin and calmodulin markers were concatenated and analyzed as a single alignment. For the mtDNA CR, haplotypes were collapsed using DnaSP6 (Rozas et al., 2017). ɸst, AMOVA, nucleotide diversity (π) and haplotype diversity (h) were estimated with Arlequin 3.5.2. (Excoffier and Lischer, 2010). Genetic distances (DA) and phylogeny reconstruction were conducted with MEGA 7 (Kumar et al., 2016).

Results

When analyzing E. alletteratus mtDNA CR marker, comparison of the sequences to GenBank (NCBI) database using BLAST, results from Portugal and Tunisia locations showed expected levels of sequence identity (about 99.7%; e-value 0) to the ones of E. alletteratus. In contrast, BLAST results for all sequences of Senegal and Côte d’Ivoire locations showed less identity than the expected to E. alletteratus sequences from GenBank (about 90%; e-value 1.33e-136).

Population genetic analysis resulted in no genetic heterogeneity between Portugal and Tunisia (ɸst = 0.007; p = 0.198) and between Senegal and Côte d’Ivoire (ɸst = 0.004; p = 0.162). However, an extremely high differentiation was found when comparing the Tropical Atlantic individuals (Côte d’Ivoire and Senegal) to the ones from the North Atlantic-Mediterranean locations (Portugal and Tunisia) (ɸst = 0.931; p = 0.000), that is, 93% of the genetic variation in those individuals is due to differences among populations (Table 2). Similar levels of genetic

150 differentiation were found in the comparisons within Euthynnus sp., E. alletteratus from North Atlantic- Mediterranean vs. E. affinis from Vietnam (ɸst = 0.969; p = 0.000) and E. alletteratus from Tropical Atlantic vs. E. affinis from Vietnam (ɸst = 0.923; p = 0.000). Furthermore, the genetic variability in these two locations is distributed differently. Where in North Atlantic-Mediterranean population the 78 sequences resulted in only 16 different haplotypes, with a maximum frequency of 46 individuals in a single haplotype. In the Tropical Atlantic locations, a total amount of 97 sequences resulted in a collection of 59 unique haplotypes, being the maximum frequency of a single haplotype in 11 individuals. None of the haplotypes was shared between these two locations (Figure 2). Consequently, haplotype diversity for North Atlantic-Mediterranean and Tropical Atlantic population was estimated in 0.641 and 0.976, respectively. Plus, nucleotide diversity was also lower for North Atlantic- Mediterranean (0.0037) than for Tropical Atlantic (0.0095) (see Table 1).

For rhodopsin and calmodulin nuclear makers we found three specific single nucleotide polymorphisms (SNP) that differentiate the North Atlantic-Mediterranean population from the Tropical Atlantic and E. affinis. Further, genetic distance between the two clades of E. alletteratus was higher than the distance between E. alletteratus and E. affinis (DA = 0.005; SD = 0.003) (Table 3) (Figure 3).

Discussion

Small tuna species as members of Scombridae family are expected to not present genetically structured populations. Large populations sizes, pelagic habitat and migratory behavior should act as a force increasing the genetic flow between locations and avoiding the loss of genetic diversity. Thus, when genetic structure for these pelagic species is observed, it is usually low (Waples, 1998; Waples and Gaggiotti, 2006). It should be noted, however, in case genetic structure is not detected, does not imply absence of genetic heterogeneity, since multiple factors such as improper sampling and lack of resolution of genetic markers could affect in detecting genetic differentiation. Thus, usually low but significant ɸst values are the common scenario for these pelagic species (Waples, 1998; Waples and Gaggiotti, 2006). However, several studies are demonstrating that some of these species show population differentiation, even in a relatively small geographic scale. For instance, Scomber scombrus, revealed genetic differentiation between western Atlantic, eastern Atlantic and Mediterranean locations (Zardoya et al., 2004; Rodríguez-Ezpeleta et al., 2016). Or in the case for Sarda sarda, where two distinct mtDNA clades are heterogeneously distributed in the Mediterranean Sea (Viñas, Alvarado Bremer and Pla, 2004b). In addition, at least in two tuna species, obesus and Thunnus alalunga, genetic heterogeneity has also been described (Alvarado Bremer et al., 1998; Vinãs, Alvarado Bremer and Pla, 2004a). In this study, genetic analyses did not show significant genetic differences between Atlantic Ocean and Mediterranean Sea, when comparing de locations of Portugal and Tunisia, as well no heterogeneity between Senegal and Côte d’Ivoire, but, as commented above the use of other markers could unravel a hidden genetic structure. However, genetic and phylogenetic analyses revealed the presence of two clearly differentiated clades between North Atlantic-Mediterranean and Tropical Atlantic populations. The value of population differentiation (ɸst = 0.931), close to the maximum one, indicates a lack of genetic flow between these populations and thus, reproductive isolation between them. Surprisingly, these highly significant levels of genetic differentiation where similar to the values when two Euthynnus species are compared. Moreover, the patterns of genetic diversity for the mtDNA are clearly different between North Atlantic-Mediterranean and Tropical Atlantic populations, validating the idea of no gene flow between these areas. In agreement with mtDNA markers, the results for the nuclear markers, were specific SNPs were found for each group and genetic distances where even higher within E. alletteratus populations than the distances between E. alletteratus and E. affinis.

In conclusion, the extremely high level of population structure found for all markers sets the scenario of an event of possible speciation within the E. alletteratus distribution. These findings urgently demand for a reevaluation of the policies for the fishery management and, to assure the species survival must be considered as independent stocks.

References

Altschul, S. F. et al. (1990) ‘Basic local alignment search tool’, Journal of Molecular Biology. Academic Press, 215(3), pp. 403–410. doi: 10.1016/S0022-2836(05)80360-2. Alvarado Bremer, J. R. et al. (1998) ‘Genetic evidence for inter-oceanic subdivision of (Thunnus obesus) populations’, Marine Biology, 132, pp. 547–557.

151 Chow, S. and Takeyama, H. (2000) ‘Nuclear and mitochondrial DNA analyses reveal four genetically separated breeding units of the ’, Journal of Fish Biology. John Wiley & Sons, Ltd (10.1111), 56(5), pp. 1087–1098. doi: 10.1111/j.1095-8649.2000.tb02125.x. Excoffier, L. and Lischer, H. E. L. (2010) ‘Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows’, Molecular Ecology Resources. Blackwell Publishing Ltd, 10(3), pp. 564–567. doi: 10.1111/j.1755-0998.2010.02847.x. ICCAT (2018) International Commission for the Conservation of Atlantic : for biennial period , 2018-19 Part I (2018)- Vol. 2. Available at: www.iccat.int (Accessed: 13 June 2019). Kearse, M. et al. (2012) ‘Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data’, Bioinformatics, 19(12), pp. 34–17. doi: 10.1093/bioinformatics/bts199. Kumar, S. et al. (2016) ‘MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets Downloaded from’, Mol. Biol. Evol, 33(7), pp. 1870–1874. doi: 10.1093/molbev/msw054. Rodríguez-Ezpeleta, N. et al. (2016) ‘Population structure of Atlantic inferred from RAD-seq-derived SNP markers: effects of sequence clustering parameters and hierarchical SNP selection’, Molecular Ecology Resources, 16, pp. 991–1001. doi: 10.1111/1755-0998.12518. Rozas, J. et al. (2017) ‘DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets’, Molecular Biology and Evolution, 34(12), pp. 3299–3302. doi: 10.1093/molbev/msx248. Vinãs, J., Alvarado Bremer, A. J. R. and Pla, A. C. (2004a) ‘Inter-oceanic genetic differentiation among (Thunnus alalunga) populations’, Marine Biology, 145, pp. 225–232. doi: 10.1007/s00227-004-1319-5. Viñas, J., Alvarado Bremer, J. and Pla, C. (2004b) ‘Phylogeography of the (Sarda sarda) in the northern Mediterranean: the combined effects of historical vicariance, population expansion, secondary invasion, and isolation by distance’, Molecular Phylogenetics and Evolution. Academic Press, 33(1), pp. 32–42. doi: 10.1016/J.YMPEV.2004.04.009. Waples, R. S. (1998) ‘Separating the Wheat From the Chaff: Patterns of Genetic Differentiation in High Gene Flow Species’, The American Genetic Association, 89, pp. 438–450. Waples, R. S. and Gaggiotti, O. (2006) ‘What is a population? An empirical evaluation of some genetic methods for identifying the number of gene pools and their degree of connectivity’, Molecular Ecology, 15(6), pp. 1419–1439. doi: 10.1111/j.1365-294X.2006.02890.x. Zardoya, R. et al. (2004) ‘Differential population structuring of two closely related fish species, the mackerel (Scomber scombrus) and the chub mackerel (Scomber japonicus), in the Mediterranean Sea’, Molecular Ecology. John Wiley & Sons, Ltd (10.1111), 13(7), pp. 1785–1798. doi: 10.1111/j.1365- 294X.2004.02198.x.

152 Table 1. Sampling description, locations code, number of individuals (n), number of haplotypes (M), haplotype diversity (h), nucleotide diversity (π) and standard deviation (SD), based on data from mtDNA CR.

Location Code n M h ± SD π ± SD Euthynnus alletteratus North Atlantic-Mediterranean 78 16 0.641 ± 0.061 0.0037 ± 0.0025 Portugal PRT 32 9 0.643 ± 0.094 0.0027 ± 0.0020 Tunisia TUN 46 12 0.644 ± 0.077 0.0044 ± 0.0029

Tropical Atlantic 97 59 0.976 ± 0.007 0.0095 ± 0.0054 Senegal SEN 50 31 0.958 ± 0.018 0.0090 ± 0.0051 Côte d’Ivoire CIV 47 38 0.988 ± 0.008 0.0100 ± 0.0057

Euthynnus affinis Vietnam VNM 20 12 0.879 ± 0.065 0.0053 ± 0.0034

Table 2. Pairwise genetic differentiation between North Atlantic-Mediterranean (North Atl.-Med) and Tropical Atlantic locations and between species E. alletteratus (North Atl.-Med and Tropical Atlantic) and E. affinis (Vietnam), below diagonal ɸst values, above diagonal p-values, based on data from mtDNA CR.

North Atl.-Med. Tropical Atlantic Vietnam North Atl.-Med. - 0.000 ± 0.000 0.000 ± 0.000 Tropical Atlantic 0.931 - 0.000 ± 0.000 Vietnam 0.969 0.923 -

Table 3. Below diagonal genetic distances (DA) between populations or species pairs, above diagonal Std. Err., based on data from rhodopsin and calmodulin markers.

Tropical North Atl.-Med. Vietnam Atlantic North Atl.-Med. - 0,004 0,005 Tropical Atlantic 0,010 - 0,003 Vietnam 0,010 0,005 -

153

Figure 1. Locations were individuals were captured, for E. alletteratus Portugal (PRT) and Tunisia (TUN) belong to North Atlantic-Mediterranean population and Senegal (SEN) and Côte d’Ivoire (CIV) form the Tropical Atlantic population. All individuals for E. affinis belong to the same location of Vietnam (VNM).

Figure 2. Phylogenetic reconstruction of the mtDNA CR marker. Neighbor-joining tree and Tamura 3-parameter distance. Circles for E. alletteratus from Tropical Atlantic, triangles for E. alletteratus from North Atlantic- Mediterranean, squares for E. affinis, stars for A. rochei as outgroup. Bootstrap values were depicted on each branch.

154

Figure 3. Unrooted Neighbor-joining tree with Jukes-Canto distance model for calmodulin and rhodopsin nuclear genes. Circles for E. alletteratus from Tropical Atlantic, triangles for E. alletteratus from North Atlantic- Mediterranean, squares for E. affinis.

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