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Article

Genetic Structuring and Connectivity of European Squid Populations in the Mediterranean Sea Based on Mitochondrial COI Data

by
Vasiliki Pertesi
,
Joanne Sarantopoulou
,
Athanasios Exadactylos
,
Dimitrios Vafidis
and
Georgios A. Gkafas
*
Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, 38446 Volos, Greece
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(8), 394; https://doi.org/10.3390/fishes10080394
Submission received: 9 June 2025 / Revised: 25 July 2025 / Accepted: 1 August 2025 / Published: 7 August 2025
(This article belongs to the Section Genetics and Biotechnology)
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Abstract

Understanding population connectivity and genetic structure is crucial for the effective management of exploited marine species. This study investigates the population genetics of the common European squid (Loligo vulgaris) across the Mediterranean Sea, focusing on samples from the Aegean Sea and comparative sequences from Western Mediterranean, Eastern Mediterranean, and Atlantic coasts. A total of 67 COI mitochondrial sequences were analyzed, identifying 12 haplotypes and 27 polymorphic sites. Population-level genetic diversity, pairwise FST values, and haplotype network analyses revealed pronounced genetic differentiation in the eastern Mediterranean contrasting with the genetic homogeneity observed among Western populations. The low haplotype diversity observed in the Greek populations of L. vulgaris may be influenced by a combination of ecological and historical factors, as the Aegean region is recognized as a hotspot of endemism and historical population fragmentation. The results indicate the existence of at least two genetically differentiated clusters within the Mediterranean basin. This study advances current knowledge of the genetic structure of Loligo vulgaris by providing novel genetic data on populations from the eastern Mediterranean, offering valuable insights for future conservation and management strategies for the species.
Keywords:
European squid; COI gene; genetic structure; population genetics; Mediterranean Sea
Key Contribution: This study provides one of the first evidence of genetic structuring in Mediterranean populations of the European squid (Loligo vulgaris Lamarck, 1978) using mitochondrial COI sequences. It identifies an east–west phylogeographic break within the basin, with significant differentiation in the Aegean and Turkish regions compared to the genetically homogeneous western populations. This genetic data may be evidence for the further study of the species with respect to the documented high fishing pressure of the species in question in the area. Recognizing multiple management units within the species’ range along with understanding the biogeography of the species with respect to potential dynamics-rate shifts is vital for species genetic conservation. The work also lays the groundwork for future genomic investigations into adaptive variation, population connectivity, gene flow, and the associations with environmental alterations and the impact of overfishing.

1. Introduction

European squid (Loligo vulgaris Lamarck, 1978) is a neritic cephalopod species classified within the Loliginidae family. The distribution of L. vulgaris [1,2] spans the Eastern Atlantic Ocean as follows: from approximately 55° N, around the British Isles, the North Sea (including the Skagerrak, the Kattegat and the western Baltic Sea), to 20° S, off the southwestern coast of Africa, including Madeiran waters and also the Mediterranean Sea: from the western to the eastern basins, including the Adriatic Sea.
With respect to climate change, rising sea temperatures (~1.5 °C since 1980) are driving distributional shifts northward and into deeper waters while increasing larval mortality during marine heatwaves [3,4]. Optimal spawning temperatures (14–20 °C) are being disrupted, with a 30% reduction in egg survival observed during extreme warming events [5,6]. These thermal stressors may alter population genetic structure by selecting heat-tolerant genotypes or reducing gene flow due to fragmented habitats.
On the other hand, ocean acidification-driven declining pH (<7.8) impairs statolith formation in paralarvae, reducing swimming ability and survival rates by 20% under high CO2 conditions [7,8]. Such developmental disruptions could lead to genetic bottlenecks if only acidification-resistant individuals contribute to future generations. Thus, expanding hypoxic zones force squid into shallower, more vulnerable habitats, increasing predation risk [9].
As for fishing efforts, demersal trawling destroys benthic egg masses, reducing recruitment, though artificial reefs may mitigate this [10,11], driving habitat loss, and synergistic climate-fishing pressures may cause local extirpations, with models predicting 40% habitat loss by 2050 in southern regions [12,13]. These disruptions could fragment populations, reducing genetic diversity. Moreover, heavy metals (Hg, Cd) that are bioaccumulated in tissues potentially affect reproductive fitness and introducing selection pressures on detoxification mechanisms [14]. Also, microplastics may cause intestinal blockages and sublethal physiological stress [15], possibly altering population health and adaptive potential. To mitigate these threats and maintain genetic diversity, measures such as seasonal no-trawl zones, pollution monitoring, and dynamic quota systems are urgently needed [16,17]. Protecting spawning grounds and reducing cumulative stressors may enhance evolutionary adaptability in the face of rapid environmental change
L. vulgaris is harvested all year round across its entire distribution area, primarily as bycatch from multi-species bottom and pelagic trawl fisheries [1]. This myopsid species holds considerable commercial value, particularly in regions including the French and Iberian coasts, the Sahara Bank, and the Mediterranean Sea [18]. Over the past decade, capture fisheries’ production of loliginid squids has increased [19], highlighting the growing commercial importance of the species. Due to the growing fishing pressure on squid populations in recent years, it is essential to understand their population structure and connectivity in order to implement informed, data-driven approaches to fishery resource management [20,21].
Fishery stock delineation has often relied on geographical or political boundaries rather than biological data, risking the depletion of vulnerable subpopulations, even when quotas are met [21]. Despite assumptions of genetic homogeneity in marine species due to a lack of obvious barriers, many populations show subtle but ecologically significant structuring [22,23]. While marine genetic differentiation is typically lower than in freshwater or terrestrial species, even minor divergence can reflect adaptation to environmental variation, highlighting the need to incorporate genetic and ecological insights into management strategies [21].
The Mediterranean Sea, although appearing open and continuous, is shaped by intricate oceanographic fronts, depth gradients, and semi-enclosed basins that act as natural barriers to gene flow for many marine species—including loliginid squids. Several studies have revealed a clear genetic structure across the region, reflecting both ancient vicariant events and contemporary ecological boundaries. Comparable barriers, such as the Almeria-Oran front [24], the Strait of Sicily [25,26], and the Korinthiakos Gulf [27], have similarly been implicated in limiting gene flow in fishes [28,29], bivalves [30], and marine mammals [31,32].
Loliginid squids, which rely on localized spawning and exhibit limited larval dispersal potential [33] are particularly vulnerable to such barriers. Pleistocene glaciations, which dropped sea levels by up to 120 m [34], fragmented marine habitats, generating isolated refugia and promoting genetic divergence [35,36]. Post-glacial recolonization likely proceeded asymmetrically, further shaping population structure. Ecological specialization and site fidelity, similar to those observed also in other species [37,38], may reinforce these patterns.
This combination of historical and contemporary factors reveals how invisible seascape boundaries mold biodiversity. Understanding these processes in loliginid squids not only deepens our biological insight but is essential for effective conservation planning in a rapidly changing marine environment.
Research on the population genetic structure of L. vulgaris remains limited, with few studies available and a scarcity of comprehensive genetic data for the species. Only a few studies have previously explored the connectivity of the L. vulgaris population. Garoia et al. (2004) [39] analyzed six microsatellite loci from four populations across the Adriatic Sea, revealing a genetically homogeneous single population, indicative of panmixia. Likewise, García-Mayoral et al. (2020) [40] examined the genetic variability of paralarvae sampled from locations approximately 300 km apart, north and south of Galicia, in northwestern Spain, using mitochondrial COI markers, but they detected no evidence of genetic differentiation between the groups.
Olmos-Pérez et al. (2018) [41] used DNA barcoding of the mitochondrial COI gene to identify loliginid squid paralarvae and assess their genetic diversity along the northwestern Atlantic coast of Spain. Their study included Loligo vulgaris, Alloteuthis media, and A. subulata. For L. vulgaris, the analysis revealed high genetic diversity compared to the other two species. Despite sampling from two regions, they found no genetic differentiation among the individuals, indicating a panmictic population structure. The results support the idea that L. vulgaris in this region forms a genetically homogeneous population, at least at the mitochondrial level, and emphasize the role of larval dispersal in limiting genetic structuring in marine cephalopods.
More recent studies have employed advanced genomic techniques to uncover subtle population structures. García-Mayoral et al. (2024) [21] investigated the population of European squid along the western Iberian Peninsula, revealing both high genetic diversity and genetic homogeneity among local populations, as indicated by neutral genetic markers. Concurrently, the analysis of adaptive SNPs demonstrated moderate genetic differentiation with a geographic trend, suggesting local adaptation within an otherwise panmictic population.
Based on the previous studies, European squid is generally considered to lack significant genetic structuring across its distribution [39, 40, 41]. This pattern is not unexpected, considering that L. vulgaris is capable of long-distance swimming [42] and extended planktonic larval duration (PLD) [43,44], allowing larvae to disperse widely, depending on prevailing oceanographic features and environmental conditions such as currents, temperature gradients, and salinity regimes. Additionally, recent research has reported high levels of genetic diversity within this species, particularly in populations along the western Iberian Peninsula [21]. In line with these findings, this study is guided by the following two hypotheses: (1) that no significant genetic differentiation exists among L. vulgaris populations within the Mediterranean Sea, and (2) that these populations exhibit high genetic diversity. To test these hypotheses, we investigated the genetic variation in the species across the Mediterranean using mitochondrial cytochrome c oxidase subunit I (COI) sequences. The mitochondrial cytochrome c oxidase subunit I (COI) gene was selected as the genetic marker for this study due to its proven efficacy in species identification and population genetic analyses across a broad range of animal taxa. COI is among the most commonly used mitochondrial markers, as it combines several advantages as follows: a relatively high mutation rate, conserved primer binding regions, and a high copy number per cell. This facilitates amplification even from degraded or low-concentration DNA samples [45]. Furthermore, the use of COI as a standardized DNA barcode for animals has been demonstrated to enable highly effective species-level identifications. It provides sufficient interspecific divergence to distinguish closely related taxa in most animal phyla, delivering an identification resolution that captures nearly the full extent of animal biodiversity [46]. These characteristics render COI particularly suitable for the initial investigation of population structure in L. vulgaris, especially in contexts where genomic resources are limited or incomplete.
The aim of this study is to examine the population structure of European squid in the Mediterranean Sea and to expand the current understanding of the species in this region, as available data remain limited. Such insights are crucial for the development of more effective and sustainable management strategies.

2. Materials and Methods

2.1. Sampling

In the present study, 20 L. vulgaris specimens were collected from local fishermen in the marine waters surrounding Andros Island in the Aegean Sea. To evaluate genetic structure within the Mediterranean Sea, supplementary sequences were retrieved from the NCBI GenBank database (France: 11; Italy: 12; Iberian Peninsula (Portugal: 4—Spain: 9): 13; Turkey: 11;) (last date of access 7 March 2025). The dataset included samples collected from natural populations as well as from cephalopod products obtained through seafood authenticity testing conducted in retail settings, such as markets and restaurants (French and Italian data). Samples were initially stored on ice until DNA extraction was performed.

2.2. Molecular Procedures

Genomic DNA was extracted using the Invitrogen DNA Extraction Kit, following the manufacturer’s protocol. The quality and quantity of the extracted DNA were subsequently assessed by agarose gel screening (0.8% w/v).
Amplification of COI gene fragments was carried out using our newly designed primers (F: 5′-CAGATATAGCTTTCCCACG-3′; R: 5′-TGCTCCAGCTAAAACAGG-3′). Primer design was based on the sequences obtained from the NCBI GenBank database of the cytochrome oxidase subunit I gene. These sequences were aligned using AliView software [47] and conserved regions were identified as target sites for primer development. Different flanking regions of 18–22 bp were chosen with a C/G content between 50 and 60%. The final set of primer set had no significant hairpin (ΔG > −3 kcal/mol) with no secondary structures. We further evaluate the primer set through BLASTN (NCBI) to verify primer uniqueness.
PCR amplification was conducted using the following thermal cycling conditions: 95 °C for 15 min, followed by 30 cycles of 95 °C for 1 min, 58 °C for 25 s and 72 °C for 45 s, with a final hold at 72 °C for 10 min. Amplification reaction mixtures contained 1 μL of the extracted genomic DNA, 1 μL of each primer (10 μM), 6 μL of GoTaq Green Master Mix (Promega) and PCR water was added for a final volume of 20 μL. PCR products were visualized on a 1.2% agarose gel and sequenced in forward direction using standard Sanger sequencing protocols in a ABI3700 Genetic Analyser.

2.3. Statistical Analysis

All datasets were aligned using AliView software [47], using the MUSCLE [48] algorithm as implemented in the software, as it offers a higher alignment accuracy compared with currently available programs. Haplotype frequencies and their distribution across populations were analyzed with FABOX v.1.61 [49]. FABOX is a collection of simple and intuitive web services that enable us to quickly perform typical tasks with sequence data such as collapsing a set of sequences into haplotypes and automated formatting of input files for a number of population genetics programs, such as Arlequin. Genetic diversity indices (nucleotide diversity, mean number of pairwise differences), pairwise FST values, and ΦST pairwise differences were calculated using Arlequin v.3.5 [50]. A median-joining haplotype network was constructed with Hapsolutely software [51], following the method described by Bandelt et al. [52]. The median-joining network (MJN) approach is particularly suitable for intraspecific datasets as it combines the principles of minimum-spanning trees and parsimony to reconstruct all shortest trees in a single network [52]. Unlike bifurcating phylogenetic trees, MJNs are better suited to represent ambiguous or non-hierarchical relationships often found in population-level mitochondrial data, where recombination is absent and the divergence among haplotypes is low. This method has been successfully applied in studies of cephalopod population genetics [53], making it a robust and appropriate choice for our dataset.
Aligned fasta was converted to Structure format through Fasta2Structure software [54]. Then, cluster analysis was performed using LEA software [55], as implemented in R-platform [55]. We performed runs for 7 values of K and chose the value of K for which the cross-entropy [56,57] curve exhibits a plateau. Significance was corrected for multiple tests using Bonferroni correction.

3. Results

3.1. Haplotype Diversity

A 213 bp fragment of the COI gene was analyzed using 67 sequences collected from six different geographic regions (Figure 1). Sequence analysis identified 12 haplotypes and revealed 27 polymorphic sites. The Greek population exhibited only two haplotypes (h1 and h2), with h1 being dominant. While the highest haplotype diversity detected in the French population, with nine distinct haplotypes (h1, h2, h3, h4, h5, h6, h8, h9, and h10), four of which (h3–h6) were region-specific. In the Italian population, five haplotypes were identified (h1, h2, h8, h9, and h10), partially overlapping with both the French and Greek populations. In Iberian samples, five haplotypes (h1, h2, h7, h8, and h10) were identified. In Turkey, two distinct haplotypes (h11 and h12) were observed, both of which were unique to this region and not found in any other area. Region-specific haplotypes included h3, h4, h5, and h6 for France; h7 for Iberia; and h11 and h12 for Turkey.

3.2. Nucleotide Diversity Analysis

The nucleotide diversity (Table 1) varied between 0.0009 (Greece) and 0.0126 (France). Italy reported almost the same nucleotide diversity as France (0.011). The Iberian Peninsula exhibited a moderate nucleotide diversity (0.0078), followed by Turkey with 0.0024. The Greek and Turkish populations each exhibited only one polymorphic site, while the highest number of polymorphic sites was observed in the French population (9), followed by Italy (6) and the Iberian Peninsula (6).

3.3. Genetic Differentiation (ΦST and FST Values)

ΦST and FST pairwise values (Table 2) indicate a highly significant genetic differentiation for the Greek population compared to all other regions. The highest values were found in the comparisons between Greece and Turkey (FST = 0.947*) and the Iberian Peninsula (FST = 0.748*), all of which were statistically significant (after Bonferroni correction—p-value < 0.001). Similarly, notable differentiation was recorded between Greece and Italy (FST = 0.579*), as well as between Greece and France (FST = 0.651*). The Turkish population also exhibited strong and significant differentiation from Iberian populations (FST = 0.752*), Italy (FST = 0.707*), and France (FST = 0.667*) (Table 2), suggesting a marked genetic distinctiveness of this population. Similarly, ΦST values were significantly higher between Greece and Turkey, with the lowest and non-significant variation among Atlantic populations. Also, significant differentiation was observed between Turkey and the rest.
In contrast, FST values among the Western European populations were generally low and non-significant, with negative or near-zero values observed in comparison. These results suggest a relative genetic homogeneity within the Western Mediterranean and Atlantic populations, in contrast to the high differentiation seen in the easternmost regions.
The overall pattern of genetic differentiation indicates variable levels of population structure across the five geographical areas, with marked divergence detected between certain population pairs and near-zero differentiation among others.

3.4. Median-Joining Network

The median-joining network constructed from COI gene sequences (Figure 2) revealed the presence of several distinct haplotype clusters among the five populations examined (Figure 2). The largest cluster was primarily composed of individuals from Greece, with minor contributions from Italy, the Iberian Peninsula, and France, indicating a degree of haplotype sharing among these regions. A second major cluster was predominantly represented by individuals from Turkey, suggesting relative genetic distinctiveness. Additional smaller clusters consisted mainly of haplotypes from France and Italy. Iberian individuals were broadly distributed among various clusters, reflecting a pattern of admixture. Overall, the network structure suggests extensive gene flow among Mediterranean populations, although specific haplotypes associated with Turkey and Greece exhibited a degree of separation, indicating localized genetic differentiation.

3.5. Cluster Analysis

For the analysis in LEA, the cross-entropy curve reached a plateau after K = 3 (Figure 3A) suggesting a clear cut differentiation between Levantine Sea (Turkey population) and the rest of the Mediterranean Sea and Atlantic Ocean. Furthermore, Greece exhibits a single genetic cluster with shared frequencies with further western populations. On the other hand, a third cluster was identified in Italy and the Atlantic Ocean, showing the genetic homogeneity of the species in these areas (Figure 3B).

4. Discussion

This research is, to the best of our knowledge, the first thorough investigation into the genetic variation and population structure of the European squid (Loligo vulgaris Lamarck, 1798) in the eastern Mediterranean, with a particular focus on samples collected from the Aegean Sea. By analyzing the mitochondrial cytochrome c oxidase subunit I (COI) gene, we identified two distinct haplotypes within Greek waters and a total of 12 haplotypes across the entire sample set.
The pairwise FST analysis revealed strong genetic structuring among L. vulgaris populations across the different regions, particularly highlighting significant genetic differentiation in the eastern Mediterranean. The Greek population exhibited the highest and most significant FST values. Similarly, the Turkish population also displayed strong genetic divergence from all the other regions suggesting a high degree of genetic isolation.
In contrast, populations from the western Mediterranean and Atlantic (Spain, Portugal, Italy, France) showed low or even negative FST values among themselves, indicating a lack of significant genetic differentiation and suggesting a relatively panmictic or well-connected population structure in those regions. The low FST values suggest that there is a strong genetic homogeneity among the populations in these areas. Recent research has also highlighted the genetic uniformity of Loliginid squid populations found in the northeastern Atlantic waters of Europe [41,58].
Overall, genetic homogeneity was observed in these areas, which is not surprising given that adult L. vulgaris are capable of swimming long distances [42], and their planktonic paralarvae exhibit a high dispersal potential. These findings support the hypothesis of widespread genetic homogeneity among L. vulgaris populations in the northeastern Atlantic.
The marked genetic differentiation of both Greek and Turkish populations from their western Mediterranean and Atlantic counterparts suggests localized isolation potentially driven by reduced gene flow and region-specific oceanographic conditions. These results support the hypothesis of population fragmentation and restricted gene flow in the eastern Mediterranean, likely due to the oceanographic complexity of the area [59] or ecological barriers. Four major phylogeographic barriers have been recognized within the Mediterranean Sea. Located at the: (a) Dardanelles Strait (separating the Black Sea from the Mediterranean Sea), (b) the Levantine Basin (separating the north-eastern from the south-eastern Mediterranean), (c) the Adriatic Sea (differentiating the eastern from the southern Mediterranean), and (d) the Italian Peninsula (acting as a barrier between the eastern and western Mediterranean) [60,61,62,63] (Figure 1). Such physical discontinuities are likely to contribute to the observed genetic structuring in marine populations across these regions [64,65]. The low haplotype diversity observed in the Greek populations of L. vulgaris may be influenced by a combination of ecological and historical factors, as the Aegean region is recognized as a hotspot of endemism and historical population fragmentation. The Aegean Archipelago, situated at the intersection of three continents (Europe, Asia, and Africa), has undergone repeated episodes of tectonism, volcanism, and sea-level fluctuations, leading to the isolation of numerous landmasses and the formation of distinct biogeographic zones [66]. Such processes have likely influenced the demographic history and genetic structure of marine and coastal species, including L. vulgaris. Moreover, environmental factors such as elevated salinity and warmer water temperatures [67], which are characteristic of the eastern Mediterranean, may act as limiting forces for gene flow and population connectivity. These conditions could contribute to the observed genetic heterogeneity in the region by imposing physiological or ecological constraints on dispersal and successful colonization.
These findings support the existence of at least two genetically distinct clusters in the Mediterranean, outlined as follows: one in the eastern basin and another broadly encompassing western populations (Figure 3). This genetic divergence in Greek and Turkish populations may be shaped by the region’s unique hydrographic features (e.g., complex water mass structures [68], thermohaline circulation and water mass formation [69], and mesoscale circulation patterns [70]).
Fishes 10 00394 g003
Figure 3. Cluster analysis for the five populations examined. (A) Cross-entropy curve for best K. (B) The three genetic clusters of the five populations.
Figure 3. Cluster analysis for the five populations examined. (A) Cross-entropy curve for best K. (B) The three genetic clusters of the five populations.
Fishes 10 00394 g003
The observed genetic differentiation between eastern and western Mediterranean populations is a common pattern across many marine species, and several interconnected factors help explain why this occurs. This pattern is primarily shaped by longstanding geographical and oceanographic barriers that limit gene flow, often in subtle but persistent ways. One of the most influential of these is the Strait of Sicily, often acting as a transition zone between two semi-independent basins with differing environmental dynamics [25,26].
The eastern Mediterranean is more enclosed, with complex gyres, sharper salinity gradients, and lower productivity compared to the west [59]. These factors can restrict the dispersal of larvae and constrain adult movement, particularly for species that rely on nearshore habitats and have limited migratory capacity—such as many cephalopods. In contrast, the western basin is more open and connected to the Atlantic through the Strait of Gibraltar, allowing more consistent gene flow [28,30].
Specifically, hydrodynamic modeling studies have confirmed that oceanographic features in the Mediterranean can strongly influence larval dispersal patterns and connectivity [71]. In particular, the complex hydrodynamics and erratic larval transport in the Aegean, Ionian, and Levantine basins are likely to hinder effective gene flow between local populations [71]. Planktonic larval duration (PLD) is a key factor influencing the genetic composition and spatial distribution patterns of marine organisms [72]. In species with extended PLDs (>2 weeks) such as L. vulgaris, whose paralarval phase lasts approximately 2 to 3 months depending on sea temperature [43,44], larval dispersal is strongly influenced by oceanographic features, including prevailing currents [73,74], mesoscale eddies [75], and oceanographic fronts [76]. The complex circulation regimes—dominated by gyres, eddies, and island wakes—create fragmented hydrodynamic units that act as partial barriers to larval exchange [71]. According to Shaw et al. [58], physical barriers such as deep-sea basins play a pivotal role in shaping the migratory patterns of Loligo forbesii.
The genetic differentiation observed between the two Mediterranean basins may be partly explained by historical biogeographic processes, particularly those occurring during the Late Pleistocene [77], as documented in other marine species [32,78]. During the Pleistocene epoch, alterations in sea levels, occurrences of upwelling, temperature changes, fluctuations in food resources, isolation of marine populations in refugia, and growing divergence that persists to this day [35,36] may have affected the population patterns of the European squid. This notion is also supported by Göpel et al. [78] for the species L. forbesii. Together, these historical and ecological forces have left a lasting imprint on the genetic landscapes of Mediterranean marine life.
Several studies have previously reported similar findings regarding the genetic differences between eastern and western Mediterranean populations of cephalopod species. Pérez-Losada et al. [79] observed that populations of Sepia officinalis from the Aegean and Ionian Seas are genetically distinct from the rest of the Mediterranean basin. Likewise, Göpel et al. (2022) [78] identified gene flow among populations of L. forbesii across the Strait of Gibraltar, while also detecting a slight genetic differentiation, which suggested a reduction in gene flow between eastern and western populations.
On the other hand, the European common squid is a key species in Mediterranean fisheries, supporting both artisanal and industrial fleets [4,79], with recent studies indicating declining L. vulgaris populations in several Mediterranean regions, attributed to overexploitation and environmental changes. Landings data from the Adriatic and Tyrrhenian Seas show significant reductions over the past two decades [16]. In the eastern Mediterranean, biomass estimates have decreased by ~30% since the early 2000s [79]. To this extent, climate change exacerbates these declines, as rising sea temperatures affect spawning success and larval survival [80]. Additionally, habitat degradation (e.g., trawling damage to spawning grounds) further threatens populations [81].
The notably low haplotype diversity in the Greek samples may reflect a combination of ecological and historical factors. The eastern Mediterranean is known to have lower primary productivity and more oligotrophic conditions compared to the western basin [8], which could possibly influence population size and genetic variability through reduced recruitment success and population bottlenecks. Additionally, historical events such as Pleistocene sea-level fluctuations and associated habitat contractions [64] may have led to population isolation and genetic drift, reducing genetic diversity in these regions. Also, the eastern Mediterranean experiences significantly higher evaporation rates compared to the western basin, leading to increased salinity and a net decrease in surface water levels from west to east [8]. Combined with warmer water temperatures in the eastern region [67], these environmental gradients can act as physiological or ecological barriers to gene flow, potentially limiting larval dispersal and further maintaining population differentiation and reduced genetic variation in eastern populations. These factors together could explain the reduced diversity observed in Greek samples and highlight the need for region-specific conservation and management strategies.
It is important to note that due to the limited number of sequences used, as well as the uncertain origin of some data—since they were derived from quality control and adulteration testing (e.g., sequences from France and Italy)—we maintain a level of caution in interpreting the results. This highlights the urgent need for further enrichment of genetic data related to the population structure of the European squid. Current information about the Mediterranean populations remains extremely limited, and a comprehensive understanding of population dynamics is essential for the effective management and conservation of the species.
Although this study did not detect significant signs of demographic expansion or selection, the presence of unique and geographically restricted haplotypes underscores the potential for local adaptation. Future work using higher-resolution genomic tools (e.g., RAD-seq and whole-genome sequencing) is warranted to further elucidate the fine-scale population structure and adaptive variation in L. vulgaris. The results of this study emphasize the need to integrate genetic data into fisheries’ management, especially in areas like the eastern Mediterranean, where defining stocks based on genetic evidence could be vital for the sustainable use and conservation of this economically significant species.

5. Conclusions

This study offers one of the first assessments of the population structure of Loligo vulgaris in the Mediterranean Sea, based on mitochondrial COI data. The findings reveal notable genetic divergence between eastern and western populations, with Greek and Turkish samples showing clear distinctions from those in the Western Mediterranean and Atlantic regions. These patterns reflect reduced gene flow, likely influenced by oceanographic discontinuities, larval dispersal barriers, and historical events. The presence of unique, region-specific haplotypes supports the hypothesis of localized adaptation and population fragmentation in the eastern basin. In contrast, western populations exhibited low and often non-significant FST values, suggesting a broadly panmictic structure and high gene flow among France, Spain, Portugal, and Italy.
The differentiation observed in the east is likely maintained by the complex hydrography of the Aegean and Levantine Seas, which includes gyres, island wakes, and mesoscale eddies that act as partial barriers to dispersal. Furthermore, historical factors such as Pleistocene sea-level changes may have contributed to the fragmentation of eastern populations. The restricted gene flow and the presence of geographically constrained haplotypes in the eastern Mediterranean underscore the necessity of recognizing multiple, region-specific management units for L. vulgaris.
Given the species’ commercial value and ongoing fishing pressure, these results highlight the importance of integrating molecular data into fishery management frameworks. Unlike finfish, cephalopod fisheries in the Mediterranean are often unregulated or managed under broad categories [79]. The absence of Minimum Landing Sizes (MLS) for L. vulgaris allows the harvest of juveniles, worsening overfishing [4]. A more nuanced understanding of L. vulgaris connectivity and population dynamics is vital for ensuring the long-term sustainability and resilience of this ecologically and economically important cephalopod.

Author Contributions

Conceptualization, G.A.G.; methodology, V.P., J.S. and G.A.G.; software, V.P. and G.A.G.; validation, G.A.G., D.V. and A.E.; formal analysis, V.P. and G.A.G.; investigation, V.P. and G.A.G.; resources, D.V., A.E. and G.A.G.; data curation, G.A.G.; writing—original draft preparation, V.P. and G.A.G.; writing—review and editing, J.S., D.V., A.E. and G.A.G.; supervision, G.A.G.; project administration, G.A.G.; funding acquisition, A.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The test samples were dead fish, and no ethical approval was required.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fishes 10 00394 g001
Figure 1. Map showing the genetic structure of the five Loligo vulgaris populations for the 12 COI haplotypes. 1—France (11); 2—Iberian Peninsula (Spain and Portugal) (3); 4—Italy (12); 4—Greece (20); 5—Turkey (11). Numbers in parentheses are sample sizes. Different colors corresponded to the 12 haplotypes. Also, phylogeographic barriers are presented (a: Dardanelles strait; b: Levantine Basin; c: Adriatc Sea; d: Italian Peninsula; e: Almeria-Oran front).
Figure 1. Map showing the genetic structure of the five Loligo vulgaris populations for the 12 COI haplotypes. 1—France (11); 2—Iberian Peninsula (Spain and Portugal) (3); 4—Italy (12); 4—Greece (20); 5—Turkey (11). Numbers in parentheses are sample sizes. Different colors corresponded to the 12 haplotypes. Also, phylogeographic barriers are presented (a: Dardanelles strait; b: Levantine Basin; c: Adriatc Sea; d: Italian Peninsula; e: Almeria-Oran front).
Fishes 10 00394 g001
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Figure 2. Median-joining haplotype network for European squid constructed from COI gene sequences. Each node (circle) corresponds to a unique haplotype (h1–h12), with accompanying numbers indicating the count of individuals possessing that haplotype. Colors represent the geographical source of each sample. Black dots represent unsampled intermediate haplotypes.
Figure 2. Median-joining haplotype network for European squid constructed from COI gene sequences. Each node (circle) corresponds to a unique haplotype (h1–h12), with accompanying numbers indicating the count of individuals possessing that haplotype. Colors represent the geographical source of each sample. Black dots represent unsampled intermediate haplotypes.
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Table 1. Polymorphic sites, and nucleotide diversity across the five Loligo vulgaris populations.
Table 1. Polymorphic sites, and nucleotide diversity across the five Loligo vulgaris populations.
PopulationSample SizePolymorphic Sites (S)Nucleotide Diversity
France1190.0126 +/− 0.008
Greece2010.0009 +/− 0.001
Italy1260.0110 +/− 0.007
Iberian13 0.0078 +/− 0.006
Turkey1110.0024 +/− 0.002
Table 2. Pairwise FST comparisons (below diagonal) and corrected ΦST pairwise values (above diagonal) between the six Loligo vulgaris populations. Statistically significant values (p < 0.01, after Bonferroni correction) denoted with asterisk.
Table 2. Pairwise FST comparisons (below diagonal) and corrected ΦST pairwise values (above diagonal) between the six Loligo vulgaris populations. Statistically significant values (p < 0.01, after Bonferroni correction) denoted with asterisk.
ItalyFranceIberian PeninsulaTurkeyGreece
1.305 *1.914 *2.233 *5.387 *-Greece
3.548 *3.218 *3.472 *-0.947 *Turkey
−0.026−0.116-0.752 *0.748 *Iberian Peninsula
−0.103-−0.0540.668 *0.652 *France
-−0.042−0.0120.707 *0.579 *Italy
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Pertesi, V.; Sarantopoulou, J.; Exadactylos, A.; Vafidis, D.; Gkafas, G.A. Genetic Structuring and Connectivity of European Squid Populations in the Mediterranean Sea Based on Mitochondrial COI Data. Fishes 2025, 10, 394. https://doi.org/10.3390/fishes10080394

AMA Style

Pertesi V, Sarantopoulou J, Exadactylos A, Vafidis D, Gkafas GA. Genetic Structuring and Connectivity of European Squid Populations in the Mediterranean Sea Based on Mitochondrial COI Data. Fishes. 2025; 10(8):394. https://doi.org/10.3390/fishes10080394

Chicago/Turabian Style

Pertesi, Vasiliki, Joanne Sarantopoulou, Athanasios Exadactylos, Dimitrios Vafidis, and Georgios A. Gkafas. 2025. "Genetic Structuring and Connectivity of European Squid Populations in the Mediterranean Sea Based on Mitochondrial COI Data" Fishes 10, no. 8: 394. https://doi.org/10.3390/fishes10080394

APA Style

Pertesi, V., Sarantopoulou, J., Exadactylos, A., Vafidis, D., & Gkafas, G. A. (2025). Genetic Structuring and Connectivity of European Squid Populations in the Mediterranean Sea Based on Mitochondrial COI Data. Fishes, 10(8), 394. https://doi.org/10.3390/fishes10080394

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