Genetic diversity in mitochondrial DNA reveals the effect of a Fisheries Protection Zone on exploited marine species in the Menorca Channel (Western Mediterranean)

doi:10.21203/rs.3.rs-7271616/v1

Overexploitation can drive evolutionary changes and erode genetic diversity, reducing species’ adaptive capacity to environmental and anthropogenic pressures. Spatial marine conservation measures, such as Marine Protected Areas and Fisheries Protection Zones (FPZs), aim to mitigate these impacts by preserving biodiversity and promoting sustainable fisheries. Recently, nucleotide diversity of the mitochondrial Cytochrome C Oxidase subunit I (COI) marker has emerged as a promising proxy for assessing species conservation status. To evaluate the effectiveness of an FPZ established in 2016 in the Menorca Channel, COI genetic diversity was assessed in four exploited marine species across three areas: the FPZ and two nearby non-protected zones. All species exhibited consistently higher genetic diversity within the FPZ, despite evidence of high gene flow among areas. Coalescent simulations were used to model expected genetic diversity under neutral scenarios of bottlenecks and expansions, with magnitudes estimated from differences in nucleotide diversities observed between fished and non-fished zones. Simulations supported a scenario of population expansion in the FPZ, contrasting with signs of genetic erosion in fished areas. These patterns align with Vessel Monitoring System (VMS) data, which show a post-protection-establishment shift in fishing effort toward non-protected zones, potentially contributing to population declines outside the FPZ. This study provides genetic evidence of the positive effects of fishing restrictions on fishery resources in the Menorca Channel, supporting the FPZ’s role in preserving genetic diversity and promoting population recovery. Furthermore, it highlights COI nucleotide diversity as a simple, cost-effective tool for monitoring marine species’ conservation status and guiding resource management strategies.

Nucleotide diversity

COI

Coalescent simulations

Fisheries resources

Protected areas

Mediterranean Sea

In the last few decades, the implementation of marine protected areas (MPAs) and fisheries protection zones (FPZs) has increased worldwide, implemented as tools to simultaneously achieve both biodiversity conservation and fisheries management objectives (Hilborn 2004; Gaines et al. 2010). Protected areas play an important role in fisheries management due to two main ecological mechanisms: the dispersal of adults and juveniles, defined as "spill-over" (Rowley 1994), and the larval supply. These areas contribute to the restoration of overfished fish stocks and provide net benefits to nearby areas (Di Lorenzo et al. 2016, 2020; Andrello et al. 2017). In the Mediterranean Sea, a clear increase in biomass and abundance of exploited marine species within protected areas has been observed, with consequent benefits for fishing activities in areas adjacent to the reserves (Guidetti and Sala 2007; Harmelin-Vivien et al. 2008; Follesa et al. 2011; Giakoumi et al. 2017). These benefits have also been observed in individual fish conditions, assessed through physiological indicators (Lloret and Planes 2003; Lloret et al. 2005; Sillero-Ríos et al. 2018). These findings suggest that some fish species show a preference for habitats with better conditions for development, survival, feeding, and reproduction.

Furthermore, it is known that the exploitation of fishery resources can affect the evolutionary dynamics of fish populations in different ways: i) loss of genetic diversity or adaptive potential (Pinsky and Palumbi 2014; Petit-Marty et al. 2022); ii) variations in the power of detection of population structure (Gandra et al. 2020); and iii) evolutionary changes due to fishing pressures (Allendorf et al. 2008). Intense fishing pressure leads to population declines, which will significantly reduce the levels of genetic diversity if declines are strong enough to produce changes in the frequency of alleles within the affected populations (Hauser et al. 2002; Hutchinson et al. 2003; Ruggeri et al. 2016). Since genetic variation is the basis of natural selection, its degradation can reduce the ability of a species to adapt to changing environments produced directly or indirectly by anthropogenic pressures, increasing the risk of local extinction (Spielman et al. 2004; Allendorf et al. 2008; Vásquez et al. 2023).

In this sense, overfishing drives population decline, which in turn causes the loss of genetic diversity in exploited species and, therefore, potentially affects their conservation and adaptive capacity to environmental changes (Petit-Marty et al. 2022; Sadler et al. 2023; Mendoza-Portillo et al. 2023).

In this context, mitochondrial genes can provide information not only on demographic changes and declines in population sizes but can also serve as diagnostic tools for assessing the conservation status of exploited populations (Johnson et al. 2018; Petit-Marty et al. 2020; Righi et al. 2020; Ilham Syahadah Mohd Yusoff et al. 2021; Canteri et al. 2021; Ferragut-Perello et al. 2023; Zlateva et al. 2023). Recently, some studies have demonstrated that estimates of genetic diversity—particularly the nucleotide diversity index—derived from the mitochondrial marker Cytochrome C Oxidase subunit I (COI) can be effective, simple and cost-efficient indicators of the conservation status of commercially exploited (Yorisue et al. 2020; Petit-Marty et al. 2022) and vulnerable (Ham-Dueñas et al. 2020; Petit-Marty et al. 2020; Ferragut-Perello et al. 2023) marine fish species.

The genetic diversity observed in mitochondrial markers, combined with coalescent simulations, also provides insights into the demographic history of marine species over time. Coalescent simulations are a well-established method for generating population samples under different evolutionary scenarios (Ewing and Hermisson 2010) and have been used to estimate expected decreases in genetic diversity—under a neutral model of evolution—resulting from population declines driven by sustained harvesting pressure (Pinsky and Palumbi 2014; Arenas 2019; Petit-Marty et al. 2022; Reid and Pinsky 2022).

The present study was carried out in the Menorca Channel, which is located between the islands of Mallorca and Menorca in the Western Mediterranean and represents nearly 20% of the coastal continental shelf of the Balearic Archipelago (40 to 100 m depth). Due to the hydrodynamic conditions, the area hosts a wide distribution of habitats and species of conservation interest, such as coralligenous outcrops, maërl, and biogenic detrital beds (Barberá et al. 2012; Moranta et al. 2014; Farriols et al. 2025). Red algae beds, composed of both Peyssonnelia and rhodoliths, have a high biodiversity and secondary macrobenthic production, which positively influence the abundance, physiological status, and key vital aspects of demersal resources on the Balearic continental shelf (Ordines et al. 2009, 2015). Maërl and coralligenous beds are classified as sensitive habitats (HSs) and essential fish habitats (EFHs) (Ardizzone 2006). Following the implementation of Council Regulation (EC) No. 1967/2006, which establishes management measures for the sustainable exploitation of fishery resources in the Mediterranean Sea, rhodolith beds and coralligenous bottoms were recognized as protected habitats. Owing to the presence of these essential habitats and their spatial overlap with trawling activities and associated impacts, the Menorca Channel was declared a Site of Community Importance (SCI) in 2014. In 2016, trawling was subsequently banned in certain areas within the SCI, designated as Fisheries Protection Zone (FPZ).

This study aims to assess the effect of a Fisheries Protection Zone (FPZ) on the conservation status of exploited marine species by analysing genetic diversity levels using the mitochondrial COI marker, comparing the FPZ located in the Menorca Channel with the surrounding areas subjected to fishing activity. To do this, three species of teleosts were used as models: Mullus surmuletus (red striped mullet), Serranus cabrilla (comber) and Scorpaena notata (small red scorpionfish), and one cephalopod species: Octopus vulgaris (common octopus). All these species are widely distributed throughout the East Atlantic and the Mediterranean Sea, inhabiting the narrow continental shelf areas characterized by rocky or sandy substrates and seagrass meadows (Ordines et al. 2009; Alós et al. 2011; Fadhlaoui-Zid et al. 2012; Félix-Hackradt et al. 2013; Bos et al. 2020; Pérez et al. 2023). Furthermore, in the Balearic Islands all species are commercially exploited, whether they are target species (M. surmuletus, S. cabrilla, and O. vulgaris) or bycatches (S. notata) (Quetglas et al. 1998; 2016).

Sampling

The samples of the studied species were collected during the MEDITS (International bottom trawl survey in the Mediterranean) and CANAL scientific surveys, which were partially or totally conducted in the Menorca Channel between 2021 and 2023. In these surveys, the experimental bottom trawl GOC-73 was used to sample demersal communities and resources from seafloor areas subject to fishing activity (for specific information see Spedicato et al. 2019). Specifically, the samples were obtained from FPZs located in the Menorca Channel and from surrounding fished area within the SCI and adjacent areas (ADJ), located south of the Menorca Channel. Sampling sites for the studied species are indicated in Fig. 1. For all the studied species, tissue samples were collected from at least 30 specimens per zone (FPZ, SCI, and ADJ), preserved in 96% ethanol and stored at -20ºC. The total number of tissue samples is indicated in Table 1.

Fishing footprint

Data from the Vessel Monitoring by satellite System (VMS) collected between 2009 and 2023, were analysed to assess the distribution of bottom trawl fishing efforts along the Menorca Channel before and after the establishment of the FPZ. Prior to analysis, VMS data were filtered to exclude signals produced during sailing and manoeuvring. To do so, only VMS signals recorded between 05:00 am and 05:00 pm (the period when trawlers are allowed to fish in the Balearic Islands) and with an instantaneous speed from 2 to 3.6 knots (the velocity range during fishing operations in the Balearic Islands) were considered. Finally, the mean annual number of VMS signals per cell in a 0.01º resolution grid was mapped with ArcGIS Desktop 10.8 (ESRI 2020) for the periods 2009-2015 and 2017-2023, corresponding to the sampling periods before and after the FPZ implementation, respectively.

Laboratory procedures

Total genomic DNA (gDNA) was extracted using the DNeasy Blood and Tissue kit (Qiagen, West Sussex, UK). Polymerase chain reaction (PCR) was used to amplify the genetic marker Cytochrome C Oxidase subunit I (COI) using different primer sets: FF2d/FR1d (600 bp for M. surmuletus, 642 bp for S. notata;Ivanova et al. 2007), FishF1/FishR1 (612 bp for S. cabrilla; Ward et al. 2005), and LCO1490/HCO2198 (657 bp for O. vulgaris; Folmer et al. 1994). PCR reactions were carried out in a 25 μL volume containing: 2.5 μL of 10X Buffer (BIOLINE, London, UK), 1.75 μL of 50 mM MgCl₂, 1 μL of 100 mM dNTPs, 0.5 μL of each primer (200 mM), 0.05 μL of DNA polymerase (BIOLINE) (5 U/μL), and 1 μL of gDNA template. PCR program conditions for each species and primer set are detailed in Table S1. The amplification products were purified using the MicroCLEAN kit (Microzone, Haywards Heath, UK) and sent to MACROGEN (Madrid, Spain) for Sanger sequencing.

The sequences were edited and aligned using MEGA X (Kumar et al. 2018) with the ClustalX method. All sequences have been deposited in GenBank under the following accession numbers: M. surmuletus (PQ339824 - PQ339921), S. cabrilla (PQ339926 - PQ340023), S. notata (PQ363178 - PQ363270), and O. vulgaris (PQ340025 - PQ340124).

Genetic diversity and differentiation

For data analysis, samples from each species were grouped into three zones according to the sampling sites: FPZ, SCI, and ADJ. For each zone, genetic diversity statistics were estimated using DnaSP v6 (Rozas et al. 2017) . Non‑parametric Kruskal-Wallis and post hoc Dunn’s multiple comparison tests were performed to test statistical differences between zones for each species using the R package FSA (Ogle et al. 2023). The 95% confidence intervals (CIs) for the mean nucleotide diversity values were obtained by 1,000 bootstrap replicates of COI sequences for zone and species, using the pegas R package. Violin plots were generated with ggplot2 package in R (Wickham 2016) to visualize the distribution of nucleotide diversity values of bootstrapped samples across zones.

For each species, haplotype networks were created using median-joining network analysis implemented in PopArt v1.7 (Leigh and Bryant 2015). Moreover, genetic differentiation and its statistical significance among the three zones for each species were calculated through pairwise estimates (Φ_st) in Arlequin v3.5 (Excoffier and Lischer 2010), with 10,000 permutations.

Demographic history

To investigate the demographic history of the studied species in the Menorca Channel, on the one hand, Tajima’s D, Fu’s Fs, and Ramos-Onsins and Rozas's R₂neutralitystatistics (Tajima 1989; Fu 1997; Ramos-Onsins and Rozas 2002) were calculated with DnaSP v6 (Rozas et al. 2017), with statistical significance assessed through 10,000 permutations. The same software was used to estimate mismatch distributions by comparing the observed distribution of pairwise nucleotide differences with the expected distribution under a model of exponential demographic expansion. To assess how well our experimental data matched the model distribution, the sum of squared deviations (SSD) between the observed and expected mismatch distributions, as well as the raggedness index (rg; Watterson 1975), were used as test statistics. These statistics were performed using 10,000 bootstrapped replicates in Arlequin v3.5 (Excoffier and Lischer 2010). For these analyses, a single population from the Menorca Channel was considered for each species by pooling individuals from the three zones, as long as no clear genetic differentiation was found among them.

On the other hand, coalescent simulation analyses using msms program (Ewing and Hermisson 2010) were performed to assess the demographic effects of fisheries protection in the Menorca Channel.

Based on the Wright-Fisher neutral model (Wright 1931), the simulations modelled two demographic scenarios starting eight years ago, coinciding with the FPZ declaration: a potential demographic bottleneck in non-protected zones (SCI and ADJ considered separately) with respect to the FPZ, and a potential population expansion in the FPZ with respect to non-fishing protected zones. The simulations assumed that genetic diversity before the establishment of the protected FPZ zone was similar to that in non-protected areas. The simulations assumed no recombination, no migration, and no subsequent demographic events following the initial population size change. The composite parameter θ = 4Neu was approximated by the Watterson’s estimator (θ_W; Watterson 1975) calculated from the number of segregating sites (S) in DnaSP v6 as theta (θ) per COI sequence and sampling zone, as follows:

i) For modelling bottlenecks, the observed θ was approximated by the current θ_W in the Fisheries Protection Zone (FPZ): θ_F = 0.00452 for M. surmuletus; θ_F = 0.00805 for S. cabrilla; θ_F = 0.00708 for S. notata; and θ_F = 0.00346 for O. vulgaris. Bottleneck intensity was calculated as the ratio of nucleotide diversity in individuals from SCI or ADJ (π_S and π_A,respectively) to that in individuals from FPZs (π_F).

ii)For modelling expansion, the observed θwas approximated by the average θ_W in the non-protected zones SCI and ADJ: θ_S-A = 0.00330 for M. surmuletus; θ_S-A = 0.00768 for S. cabrilla; θ_S-A = 0.00504 for S. notata; and θ_S-A = 0.00351 for O. vulgaris. Expansion intensity was estimated as the ratio of nucleotide diversity in FPZ individuals (π_F) to the average nucleotide diversity in non-protected areas (π_S-A).

To assess changes in genetic diversity, each simulation included 1,000 random samplings of N individuals per species, where N equals the analysed sample size of each species.

Fishing footprint

The spatial distribution of fishing effort in the studied zones showed no evidence of fishing activity within the Fisheries Protection Zone (FPZ) from 2017 to 2023, in contrast to the period from 2009 to 2015 (Fig. 2a and b), confirming the full enforcement of the FPZ. Vessel Monitoring by satellite System (VMS) data also revealed an increase in fishing effort outside the FPZ, especially in southern SCI (Fig. 2c), where bottom trawling activity is now concentrated. In contrast, in the ADJ zone, the intensity of trawling activity remained nearly constant after the FPZ implementation compared to the 2009–2015 period, although fishing effort remained high.

Genetic diversity and differentiation

Genetic diversity statistics from the COI fragment for each species within each studied zone are presented in Table 1, and the 95% CIs of the mean nucleotide diversity are shown in Table S2.

Considering the overall estimates across all zones, Scorpaena notata exhibited the highest number of segregating sites and haplotypes (35 and 32, respectively), whereas Octopus vulgaris presented the lowest values (14 and 11, respectively). On the other hand, Serranus cabrilla showed the highest values across haplotype diversity (Hd = 0.8502), mean number of nucleotide differences (Kt = 5.5620), and nucleotide diversity (π = 0.0091), while Mullus surmuletus (Hd = 0.6933; Kt = 1.5464; π = 0.0026) showed the lowest values.

In general, the individuals of the studied species collected from the FPZ displayed the highest nucleotide diversity values compared with those from the non-protected zones, the closest SCI and the farthest ADJ (Table 1, Fig. 3). The lowest levels of nucleotide diversity were observed in the SCI in all three teleost fishes, whereas in O. vulgaris, the lowest value was found in the ADJ. According to the results of the non-parametric Kruskal-Wallis and Dunn’s post hoc tests (Table S3), significant differences were observed between the FPZ and the other two zones for all four species, except between FPZ and ADJ in M. surmuletus.

Haplotype networks (Fig. 4) and pairwise estimates (Φ_st; Table S4) for the studied species revealed no population differentiation based on geographic origin within the Menorca Channel, with the main haplotypes shared by individuals from all zones, as expected given the proximity of the areas. Scorpaena notata showed a star-like network, with nearly half of the samples (47%) across all three zones—SCI, FPZ and ADJ—sharing the main haplotype. The other species exhibited two or three main haplotypes. Among them, S. cabrilla presented the most diverse network, with the main haplotype (Hap_2) separated from haplotype Hap_1 by 14 mutational steps. The greatest number of unique haplotypes were found in M. surmuletus and S. notata (both with 72% of their haplotype being unique).

Demographic history

Negative Tajima’s D and Fu’s Fs values, along with low and significant Ramos-Onsins and Rozas's R₂ values, indicate a demographic expansion in M. surmuletus and S. notata in the Menorca Channel population (Table 2). In contrast, non-significant values were found for S. cabrilla and O. vulgaris populations. Although S. cabrilla showed slightly negative values and O. vulgaris slightly positive values, both values were close to zero, suggesting demographic stability in both species. The unimodal mismatch distributions observed for M. surmuletus and S. notata matched the expected distributions under a sudden expansion model, supporting the neutrality test results. Conversely, S. cabrilla and O. vulgaris exhibited bimodal mismatch distributions (Fig. 5), strongly deviating from the expansion model.

From the coalescent simulation tests of bottleneck scenario, the observed nucleotide diversity at the non-protected SCI and ADJ zones for M. surmuletus and S. notata was lower than the expected 95% upper CI limit of nucleotide diversity replicates generated from the simulation (Table 3). This suggests that the proposed model of population bottlenecks caused by fishing in the SCI and ADJ could explain the observed nucleotide diversity estimates. In contrast, the observed nucleotide diversity values for S. cabrilla and O. vulgaris from the SCI and ADJ zones, although lower than in FPZ, did not fall within the 95% upper CI limit of nucleotide diversity estimates expected by the bottleneck model, suggesting no significant effect of the current fishing activities on the genetic diversity levels of these two species.

According to the hypothesis of population expansion after banning trawling activities in the protected zone (FPZ), the observed nucleotide diversity values were significantly higher than the expected values from a population expansion derived from the coalescent simulations for all species (Table 3). This could suggest that protection may have allowed genetic diversity recovery to levels higher than expected by our tested model. It could be because the nucleotide diversity in FPZ before protection was higher than the current diversity observed in the other two zones, or because growing rate is higher than in the model.

In the present study, we evaluated how a Fisheries Protection Zone in the Menorca Channel influences genetic diversity by analysing the mitochondrial COI gene in four exploited marine species, three teleost fishes (Mullus surmuletus, Serranus cabrilla, and Scorpaena notata) and one cephalopod (Octopus vulgaris). Overall, individuals collected from the FPZ displayed the highest nucleotide diversity values compared to those from non-protected zones, the closest SCI and the farthest ADJ. These results suggest a better conservation status for the studied species within the protected area.

Recent studies have reported positive effects from the establishment of the FPZ in the Menorca Channel, demonstrating that the 2016 trawling ban has helped protect 80% of rhodolith beds and 95% of coralligenous bottoms from trawling. This protection has led to increased biomass of rhodolith beds and Laminaria rodriguezii forests, thereby contributing to the recovery of epibenthic communities (Farriols et al. 2021; 2025). The distribution of these habitats has increased by 6% and 54%, respectively, in recent years, highlighting the FPZ as an effective conservation measure for benthic habitats, communities, and species in the Menorca Channel (Farriols et al. 2025). The recovery of these significant marine habitats is enhancing marine biodiversity in the area due to their structural and functional complexity (Barberá et al. 2012; Joher et al. 2012, 2015), which benefits both exploited and unexploited species.

In addition to the implementation of the FPZ, some areas of the Menorca Channel had already undergone previous changes. On the one hand, submarine cables were installed in the 1970s, in a narrow area within what is now the FPZ, connecting Mallorca and Menorca (Cabrito et al. 2024), which resulted in trawling being excluded from this area for decades, potentially providing long-term benefits to marine communities. On the other hand, a general reduction in fishing effort has been recorded across the Balearic Islands in recent decades (Quetglas et al. 2017). Therefore, the recovery trend observed in this study may have originated from these prior changes and was further enhanced by the implementation of the protected area.

Specimens of the four studied species collected from non-protected areas in the Menorca Channel showed high nucleotide diversity values, although lower compared to those from the protected area. This is to be expected given the geographical proximity to the FPZ, suggesting that adjacent areas may benefit from population growth in the protected zone. It is therefore a clear example of how a marine reserve can play a crucial role in sustaining the productivity of fishing zones and contributing to the consequent gene flow between protected and near non-protected areas, which is essential for preserving and restoring ecological processes and for ensuring genetic diversity, which supports species’ ability to adapt to global environmental changes (Gandra et al. 2020). In this sense, the lack of genetic structuring observed in the haplotype networks (Fig. 4) and the low and non-significant Φ_stvalues (Table S4) suggest high levels of gene flow between the FPZ and nearby zones. In the Mediterranean, similar patterns have been observed in fish species such as Diplodus vulgaris, Mullus surmuletus and M. barbatus, where high genetic diversity was found across both protected and adjacent fished areas (Félix-Hackradt et al. 2013; Sahyoun et al. 2016).

However, for the three teleost fish species analysed in this study, the lowest nucleotide diversity values were unexpectedly observed in the SCI, the non-protected zone closest to the protected FPZ. As this zone presents similar habitat characteristics to those of protected area (Farriols et al. 2025), the observed reduction in genetic diversity could be attributable to increased fishing pressure rather than habitat differences. Supporting this, Vessel Monitoring System (VMS) data indicate that, following the establishment of the FPZ, trawling activities in the Menorca Channel shifted mainly toward the southern area of the SCI (Fig. 2).

While all target species in this study exhibited higher genetic diversity values within the protected area, interspecific differences in nucleotide diversity levels were observed. Specifically, S. cabrilla showed the highest nucleotide diversity, followed by O. vulgaris, whereas M. surmuletus and S. notata presented the lowest values. These differences could be related to differences in the life-history traits of the species studied. A key distinction is S. cabrilla’s reproductive strategy asa simultaneous hermaphrodite that does not self-fertilize, which reduces homozygosity and inbreeding risk, thereby enhancing genetic diversity by promoting spawning events and offspring heterozygosity (Avise and Mank 2009; Coscia et al. 2016). Since each individual in the population contributes mitochondrial DNA to the next generation—unlike in non-hermaphroditic species, where only females do—the resulting nucleotide diversity is not directly comparable to that of the other species. Regarding O. vulgaris, this species exhibits a fast growth rate, a short semelparous life cycle that allows rapid generational turnover, and high fecundity (Hanlon and Messenger 1996), traits that confer relative resilience to fishing pressure and environmental perturbations compared with many teleost species (Faure et al. 2000). Lastly, M. surmuletus and S. notata possess typical life-history traits of marine fish: they are oviparous species with external fertilization, their eggs are embedded in a gelatinous matrix, and they have relatively short generation times—3.8 and 2.9 years, respectively (Table 4).

In order to compare the nucleotide diversity estimates of the studied species in the Menorca Channel with those reported for other species across the Balearic Islands, only two studies were found (Petit-Marty et al. 2022; Riera et al. 2025), both focusing on teleost species (Fig. 6). Compared with the estimate reported by Petit-Marty et al. (2022) (π = 0.0047), M. surmuletus exhibited lower nucleotide diversity in this study (π = 0.0026), whereas S. notata presented slightly greater diversity (π = 0.0025) than the estimate for the entire population across the Balearic Archipelago (π = 0.0020; Riera et al. 2025). These intraspecific differences may reflect variations in sampling effort and geographic coverage across the Balearic Islands. Additionally, M. surmuletus displayed lower nucleotide estimates than its sister species, M. barbatus (π = 0.0035) (Fig. 6).

Both M. surmuletus and S. notata from the Menorca Channel exhibited nucleotide diversity values below the lower limit of the 95% confidence interval for Mediterranean fish species as calculated by Petit-Marty et al. (2022) (mean 95% CI = 0.0034–0.0047) (Fig. 6). In this context, both species exhibited genetic diversity levels slightly higher than two overexploited species, M. merluccius (π = 0.0018) and Lophius budegassa (π = 0.0016) (Fig. 6), both characterized by large body size and long generation times (~10 years) (Petit-Marty et al. 2022). Although the comparison should be interpreted with caution, as the mutation rate may vary among genera, these results suggest that both species could be affected by the impact of fishing. In the case of S. notata, this aligns with the findings of Riera et al. (2025), who reported signs of fishing pressure on this species in the Balearic Islands, despite it being a bycatch species.

Finally, S. cabrilla exhibited the highest values even exceeding the upper limit of the 95% confidence interval for Mediterranean fish species (Petit-Marty et al. 2022) (Fig. 6), as expected given its reproduction strategy described previously.

The genetic diversity values estimated in the four target species also varied when compared with those of the same species from other areas across the Mediterranean Sea and Northeast Atlantic. M. surmuletus from the Menorca Channel (π = 0.0026) exhibited similar values to populations in Egypt (π = 0.0030) (Soliman et al. 2024). S. cabrilla showed nucleotide diversity values consistent with those reported in the Eastern Mediterranean (π = 0.0118), although populations from Turkey exhibited lower genetic diversity (π = 0.0035) (Bos et al. 2020). Lastly, O. vulgaris exhibited nucleotide diversity levels (π = 0.0045) consistent with estimates from Central Mediterranean (Sardinia, π = 0.0046) (Melis et al. 2018) and Northeast Atlantic populations (Portugal, π = 0.0040; and Canary Islands, π = 0.0059) (Pérez et al. 2023).

Regarding demographic history and coalescent simulations analyses, the target species displayed distinct patterns. Populations of M. surmuletus and S. notata in the Menorca Channel showed signs of recent expansion, as indicated by significant neutrality statistics and “star” or “complex”-star-shaped haplotype networks (Table 2, Fig. 4). Similar results have been observed in S. notata population across the Balearic Islands (Riera et al. 2025). In contrast, S. cabrilla and O. vulgaris presented non-significant neutrality statistics and bimodal mismatch distributions, suggesting a stable population size. Bimodal distributions may also indicate complex population structures, such as the presence of distinct lineages (Jenkins et al. 2018), although this was not clearly evident in the haplotype networks. For octopus, two different mitochondrial haplotype lineages have been observed along the East Atlantic coast and locally along Sardinia’s coast (Melis et al. 2018; Quinteiro et al. 2020), as well as a complex genetic pattern across the Mediterranean (Fadhlaoui-Zid et al. 2021). Further analyses should be realised to explore the genetic structure and demographic dynamics of octopus and comber species in the Balearic Islands.

Coalescent simulations help assess whether observed results align with expected demographic events such as expansions or bottlenecks in the different zones studied within the Menorca Channel. Coalescent simulations indicated that FPZ populations grew more than expected by a population expansion if the levels of nucleotide diversity in the FPZ populations before protection were equal to those observed in the non-protected zones. The contrary was also true, bottleneck simulations showed that the observed values of nucleotide diversity in the fished zones were significantly lower than expected by a reduction of population starting with the levels observed in the FPZ population. Differences in genetic diversity between ADJ and SCI may stem from historically low diversity levels in the Menorca Channel prior to FPZ implementation. Before protection, high gene flow combined with fishing pressure likely homogenized genetic diversity across zones. However, the establishment of the FPZ enabled population growth within protected areas. In contrast, individuals outside the FPZ faced intense fishing pressure, leading to population decline. The impact was stronger in the closest SCI zone where trawling activities were concentrated (Fig. 2) likely leading to greater genetic erosion than in the farthest ADJ zone, where fishing efforts remained relatively stable following the establishment of the trawling ban. In this scenario, the fishing-driven decline in SCI may have exceeded the demographic benefits of gene flow from the FPZ, whereas the ADJ—although also affected—experienced milder losses.

In contrast, for S. cabrilla and O. vulgaris, coalescent simulations also suggested population expansion within the FPZ, but no decline in non-protected zones under the tested conditions. This pattern may result from a) a bottleneck less severe than modelled, b) a recent demographic decline not yet detectable through genetic signals, or c) no significant effect of current trawling activities on the genetic diversity of these species. Additionally, the relatively resilient life history traits of S. cabrilla and O. vulgaris—such as hermaphroditism in the former and rapid turnover in the latter—may have caused these species to recover more quickly from the effects of fishing. These characteristics likely contributed to the higher genetic diversity observed in the present study, making these two species good indicators of conservation effectiveness in both protected and non-protected zones.

This study provides genetic evidence of the positive impact of a Fisheries Protection Zone (FPZ) on overexploited marine species in the Menorca Channel, employing mitochondrial COI nucleotide diversity as a novel proxy for species conservation status. Although mitochondrial DNA has several limitations due to its characteristics—maternally inhered, haploidy and lack of recombination—it remains a valuable marker for genetic conservation studies. A positive correlation between mitochondrial and nuclear genetic diversity in fish species has been reported (Piganeau and Eyre-Walker 2009), suggesting that a reduction in mitochondrial COI diversity may reflect broader nuclear genomic erosion and, therefore, can provide useful insight into the conservation status of marine species populations.

Overall, our findings indicate a positive trend in the recovery of genetic diversity in exploited species, enhanced by the establishment of the Fisheries Protection Zone, which has contributed to the improved condition of marine habitats within the study area—improvements that are closely linked to the conservation status of marine species. Moreover, this study highlights the value of genetic diversity approaches as effective diagnostic tools to assess protected and non-protected zones, and to support ongoing monitoring of vulnerable and exploited species.

Funding

This study is part of the MARFISH project (PDR2020/69–ITS 2017-006), included in the Projectes de Recerca Científica i Tecnològica of the Direcció General de Política Universitària i Recerca, which are funded by the Comunitat Autònoma de les Illes Balears (CAIB) through the Direcció Conselleria de Fons Europeus, Universitat i Cultura, with resources from the Tourist Stay Tax Law. The CAIB also funded the 2022 grants for the training of research personnel (N. Pasini, research grant nº FPI_026_2022). The MEDITS surveys are cofunded by the European Union through the European Maritime Fisheries and Aquaculture Fund (EMFAF) within the National Program of collection, management, and use of data in the fisheries sector and support for scientific advice regarding the Common Fisheries Policy. The CANAL surveys were part of the SosMed project (Improvement in scientific and technical knowledge for the sustainability of demersal fisheries in the western Mediterranean), funded by the European Union—Next Generation (Recovery, Transformation and Resilience Plan), with an agreement between the Ministry of Agriculture, Fisheries and Food and the Spanish National Research Council, through the Spanish Institute of Oceanography.

Conflicts of interest

The authors declare no conflict of interest.

Author Contributions

Conceptualization, F.O., A.P. and S.R.-A.; methodology, N.P.; data analysis, N.P., S.R.-A., M.B., J.F.F, M.T.F. and N.P.-M.; writing—original draft preparation, N.P.; writing—review and editing, M.B., J.F.F., M.T.F., N.P.-M., F.O., S.R.-A. and A.P.; funding acquisition, F.O. and A.P. All authors have read and agreed to the published version of the manuscript.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. COI mitochondrial sequences were deposited into the GenBank (NCBI) database under accession numbers: PQ339824-PQ339921 (Mullus surmuletus); PQ339926-PQ340023 (Serranus cabrilla); PQ363178-PQ363270 (Scorpaena notata); and PQ340025-PQ340124 (Octopus vulgaris).

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Table 1 Genetic and haplotype diversity indices of COI marker in four species across the three studied zones: the Fishery Protection Zone (FPZ), where trawling is banned; the Site of Community Importance (SCI), the nearest open-to-fishing zone to the FPZ; and the ADJ zone, the farthest open-to-fishing area from the FPZ. Amplified COI fragment lengths: Mullus surmuletus = 600 bp, Serranus cabrilla = 612 bp, Serranus notata = 642 bp, Octopus vulgaris = 657 bp. Symbols are defined in the footnote^a

	ZONE	N	S	h	Hd ± SD	Kt	π ± SD
*Mullus surmuletus*	FPZ	33	11	9	0.6987 ± 0.0740	1.6894	0.0028 ± 0.0004
	SCI	34	7	7	0.6292 ± 0.0630	1.2674	0.0021 ± 0.0002
	ADJ	31	9	8	0.7505 ± 0.0500	1.6430	0.0027 ± 0.0003
	Total	98	20	18	0.6933 ± 0.0360	1.5464	0.0026 ± 0.0002
*Serranus cabrilla*	FPZ	33	19	11	0.8712 ± 0.0290	5.8371	0.0095 ± 0.0006
	SCI	34	17	10	0.7825 ± 0.0630	5.3957	0.0088 ± 0.0009
	ADJ	31	19	12	0.8645 ± 0.0390	5.5828	0.0091 ± 0.0006
	Total	98	28	21	0.8502 ± 0.0200	5.5620	0.0091 ± 0.0004
*Scorpaena notata*	FPZ	30	18	13	0.7954 ± 0.0680	2.2643	0.0035 ± 0.0007
	SCI	31	9	10	0.6237 ± 0.0990	0.8086	0.0013 ± 0.0003
	ADJ	32	17	16	0.8528 ± 0.0570	1.5806	0.0025 ± 0.0004
	Total	93	35	32	0.7632 ± 0.0460	1.5680	0.0024± 0.0003
*Octopus vulgaris*	FPZ	30	9	6	0.7770 ± 0.0380	3.5012	0.0053 ± 0.0004
	SCI	34	10	7	0.7683 ± 0.0450	3.4029	0.0052 ± 0.0003
	ADJ	36	9	6	0.7492 ± 0.0460	3.3397	0.0051 ± 0.0005
	Total	100	14	11	0.7713 ± 0.0180	3.3851	0.0052 ± 0.0002

^aN = number of individuals; S = number of segregating sites; h = number of haplotypes; Hd = haplotype diversity; Kt = mean nucleotide difference; π = nucleotide diversity; SD = standard deviation

Table 2 Tajima’s D, Fu’s FS, Ramos-Onsins and Rozas's R₂ indices, as well as mismatch distribution metrics (including the sum of squared deviations from the sudden expansion model, SSD, and the Harpending raggedness index, rg), are indicated for each species. The corresponding p-values are reported in brackets. Values in bold were significant at the 95% confidence level

Species	D	Fs	R₂	SSD	rg
*Mullus surmuletus*	-1.7452 (0.0145)	-10.6423 (0.0002)	0.0371 (0.0391)	0.0261 (0.2509)	0.0883 (0.1621)
*Serranus cabrilla*	-0.0332 (0.6000)	-1.9033 (0.3163)	0.0958 (0.5727)	0.0307 (0.2609)	0.0333 (0.4172)
*Scorpaena notata*	-2.4020 (0.0000)	-28.0227 (0.0000)	0.0210 (0.0000)	0.0134 (0.2220)	0.0758 (0.2260)
*Octopus vulgaris*	0.6886 (0.8018)	0.8488 (0.6770)	0.1173 (0.7849)	0.0786 (0.0537)	0.1050 (0.1579)

Table 3 Observed (π_ob) and expected (π_ex)nucleotide diversity values are shown for each species in the three zones: the Fishery Protection Zone (FPZ), where trawling is banned; the Site of Community Importance (SCI), the nearest open-to-fishing zone to the FPZ; and the ADJ zone, the farthest open-to-fishing area from the FPZ. Observed values were calculated using DnaSP v6. Expected values were modelled through coalescent simulations assuming equal genetic diversity across zones prior to the FPZ’s establishment. Each simulation used θ (θ = 4Neu) as starting parameter, estimated by the observed Watterson’s estimator (θ_W) in FPZ samples (for bottleneck scenarios) and by the observed average θ_W in the non-protected zones SCI and ADJ specimens (for expansion scenarios), with 1,000 random samplings of N individuals per species (N = observed sample size). Bottleneck intensity was calculated as the ratio of nucleotide diversity in individuals from SCI or ADJ (π_S and π_A, respectively) to those from FPZs (π_F); expansion intensity was calculated as the ratio of π_F to the average nucleotide diversity in non-protected areas (π_S-A). The 95% upper CI limit of the 1,000 nucleotide diversity replicates obtained from the msms program are indicated

*Bottleneck simulations*
	*Mullus surmuletus*				*Serranus cabrilla*
	Bottleneck intensity	π_ob	π_ex	95% upper CI limit	Bottleneck intensity	π_ob	π_ex	95% upper CI limit
SCI	0.74822	0.00211	0.00332	0.00345	0.95597	0.00882	0.00725	0.00750
ADJ	0.97163	0.00274	0.00427	0.00443	0.92453	0.00912	0.00766	0.00794
	*Scorpaena notata*				*Octopus vulgaris*
	Bottleneck intensity	π_ob	π_ex	95% upper CI limit	Bottleneck intensity	π_ob	π_ex	95% upper CI limit
SCI	0.69688	0.00126	0.00270	0.00282	0.95310	0.00518	0.00348	0.00361
ADJ	0.35694	0.00246	0.00482	0.00499	0.97186	0.00508	0.00339	0.00353
*Population expansion simulations*
	*Mullus surmuletus*				*Serranus cabrilla*
	Expansion intensity	π_ob	π_ex	95% upper CI limit	Expansion intensity	π_ob	π_ex	95% upper CI limit
FPZ	1.16289	0.00282	0.00232	0.00240	1.06354	0.00954	0.00535	0.00550
	*Scorpaena notata*				*Octopus vulgaris*
	Expansion intensity	π_ob	π_ex	95% upper CI limit	Expansion intensity	π_ob	π_ex	95% upper CI limit
FPZ	1.89785	0.00353	0.00303	0.00310	1.03899	0.00533	0.00243	0.00250

Table 4 Characteristics of the studied species. Max. size and gen. times define the maximum recorded size in centimetres and the mean generation time in years, respectively. Information on commercial importance, fishing gear, IUCN status, and movement/habitats was obtained from IUCN reports for the evaluated species and their populations in the Mediterranean. Data on maximum size and generation times were retrieved from FishBase. N/A= not available

Species

Commercial importance

Fishing gear

IUCN

Population trends

Threats

Movements/Habitats

Max. size (cm)/gen. times (year)

Mullus surmuletus

High

(Carpenter et al. 2015)

Bottoms trawls, trammel nets

(Carpenter et al. 2015)

LC

(Carpenter et al. 2015)

Decreasing

(Quetglas et al. 2016)

Overexploitation

(FAO 2024)

Oceanodromous/

Marine oceanic - Epipelagic (0-200 m)

(Carpenter et al. 2015)

40.0 cm/3.8 years

Serranus cabrilla

Minor