Saving the locals: a conservation genomics approach to the Endangered Spanish Toothcarp, Aphanius iberus (Valenciennes, 1846)

Saving the locals: a conservation genomics approach to the Endangered Spanish Toothcarp, Aphanius iberus (Valenciennes, 1846) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Saving the locals: a conservation genomics approach to the Endangered Spanish Toothcarp, Aphanius iberus (Valenciennes, 1846) Maria Estarellas, Alfonso López-Solano, Gabriel Mochales-Riaño, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7494783/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Understanding the genetic structure and evolutionary history of endangered species is crucial for effective conservation planning. The Spanish toothcarp, Aphanius iberus (Valenciennes, 1846), an endemic and euryhaline fish of the Mediterranean coast of the Iberian Peninsula, is currently threatened by habitat destruction, climate change, and anthropogenic translocations. Here, we employed genome-wide SNP data from medium- to low-coverage whole genomes to investigate the population structure, genetic diversity, and demographic history of A. iberus , especially focussing on its northern distribution, which has remained poorly studied. Our analyses revealed a well-structured genetic pattern across the species’ range, with four main genetic lineages: Northern Catalonia, Southern Catalonia, Levantine, and Murcian. Genomic indicators, including heterozygosity, ROHs, and migration analyses, suggest higher inbreeding and genetic erosion in the northernmost populations, likely due to long-term isolation, whereas southern populations maintain healthier genetic diversity. We also identified several admixed and potentially introduced populations. These findings underscore the importance of accurately determining the origin of populations before any translocation or reintroduction, as misguided management may compromise the genetic integrity of natural lineages. This work provides essential genomic insights to guide conservation strategies and emphasizes the need for lineage-aware management of endemic species like A. iberus . Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Evolution Biological sciences/Genetics Genomics translocations population structure genetic diversity Aphanius iberus Figures Figure 1 Figure 2 Figure 3 Introduction Conservation initiatives involving fauna and flora are frequently enhanced nowadays by a comprehensive understanding of genomics [ 1 , 2 ]. Further insight in this area has the potential to optimize the allocation of economic resources and facilitate the implementation of more effective conservation strategies [ 3 ]. Concerning these decision-making processes, population genomics emerges as a powerful tool, as it focuses on the study of genomic variation within and among populations, as well as the evolutionary factors shaping this variation [ 4 , 5 ]. The results may be used to characterize the genomic profile of populations, elucidating their evolutionary history and, for example, enabling their classification into different Operational Conservation Units (similar to Designatable Units (or DUs)) for conservation management purposes [6, 7; 8]. Genomic data also provide information on connectivity, genetic diversity, inbreeding, and signatures of local adaptation. Combined approaches such as structure inference, phylogenomic reconstructions, migration modelling, and demographic history analyses offer a comprehensive view of population dynamics, evolutionary trajectories, and vulnerabilities, thereby supporting evidence-based conservation strategies [ 9 , 10 , 11 ]. The application of these techniques is especially valuable for continental aquatic fauna, where dispersal and colonization are limited by geographical barriers [ 12 ]. While research has often focused on model species, domestic animals, or charismatic taxa [ 13 ], many species with unique evolutionary traits remain overlooked, even when they can adapt to highly variable habitats. One such trait is euryhalinity, an ancestral adaptation that allows fish to thrive across contrasting salinity regimes but is retained by only ~ 3–5% of fish species [ 14 , 15 , 16 , 17 ]. This limited occurrence suggests that the physiological plasticity associated with euryhalinity entails significant costs, yet these species have attracted considerable scientific interest and serve as key models in ecological and physiological research [18, 19, 20; 21, 22, 23]. The Spanish toothcarp, Aphanius iberus (Valenciennes, 1846), is an endemic species to the Mediterranean coast of the Iberian Peninsula, currently found in a wide range of habitats, including groundwater springs (locally known as ullals ), coastal lagoons, river mouths, salt marshes, and even salt evaporation ponds where salinity can reach twice or more than that of seawater [ 6 , 24 , 25 ]. Listed as Endangered in the Spanish National Catalogue of Threatened Species (RD 139/2011[ 26 ]), its populations face isolation and fragmentation due to habitat destruction, introductions of invasive species translocations, and climate change among other threats [ 27 , 28 ]. The species’ limited natural dispersal is mainly triggered by extreme weather events, such as intense storms and floods, often linked to Isolated High-level Atmospheric Depressions (locally known as DANAs ), and, less frequently, by strong coastal waves [ 28 , 29 ]. These events, which are becoming more frequent under current climate change, can mobilize large volumes of water, transporting individuals across fragmented habitats and influencing the species’ biology and ecology. At the same time, global warming is not only leading to progressively warmer and saltier conditions, particularly in southern Spain, but also causing severe desiccation in many habitats [ 30 , 31 ], making reintroduction and translocation strategies especially relevant in the species’ conservation management [ 32 , 33 ]. The geographic distribution of the Spanish toothcarp has been previously studied, revealing a well-structured genetic pattern composed of several distinct lineages that reflect the spatial segregation of the species across its natural range (Fig. 1 a) [ 6 , 11 , 25 , 28 , 34 , 35 ]. These lineages include: (1) the Northern Catalonia lineage, comprising two populations from Girona province, near the French border; (2) the Southern Catalonia lineage, which extends southwards to the Ebro River Delta; (3) the Levantine lineage (corresponding to Iberian Levante), represented by a single genetic population covering most of the Valencian region; and (4) the Murcian lineage (already referred to as such in Doadrio et al., 1996), stretching from Cape of Nao (southern Alicante province) through the province of Murcia to Rambla Moreras, the southernmost limit of the species’ range (Fig. 1 a) [ 6 , 25 , 34 ]. Subsequent studies have refined this geographical and genetic gradient, identifying admixed populations located between these genetically distinct lineages, which show signs of historical or ongoing gene flow and may have important implications for the genetic diversity and evolutionary history of the species [ 7 , 11 , 28 , 35 , 36 , 37 ]. Translocations and reintroductions have been carried out across the distribution of A. iberus , sometimes without complete knowledge of the genetic origin of the source populations [ 11 , 25 ]. A clear example is the Adra population (locality 28), at the southernmost limit of the species’ range (Fig. 1 a), which recent evidence suggests originated from translocations from the Albuixech population (locality 23 [ 11 ]). Notably, this population of Albuixech is the only genetic population representing the entire Levantine region and has served as the source for several reintroductions within this lineage’s range. Similar cases of uncertain origin have been reported elsewhere, such as the Prat de Llobregat population (localities 7 and 8, Fig. 1 a), declared extinct in the 1960s and later reintroduced in the mid-1990s with individuals provided by local aquarists [ 38 , 39 ]. In contrast, the A. iberus populations of the Ebro River Delta (localities 18–21, Fig. 1 a) are original and have remained stable over time. Only two small-scale experimental releases were conducted under the LIFE project (1996–1999) with captive-bred fish in newly created habitats. While such kind of interventions can help restore extirpated populations, they may also compromise the integrity of natural lineages if performed without an adequate genetic and ecological basis [ 32 , 40 ]. The translocation of non-native individuals can potentially disrupt the local adaptations that are critical for the survival of A. iberus in its varied habitats, leading to a decline in fitness and resilience. This is particularly concerning for populations surviving exclusively in artificial habitats, such as irrigation ponds in Sax and Adra (localities 24 and 28), which face compounded threats to their long-term survival. A primary objective of this study was to reconstruct and understand the evolutionary history and population structure of Aphanius iberus along its distribution range, with particular emphasis on the Catalonia region (Northern and Southern Catalonia lineages), located in the northward region of the species’ distribution range and which has not been extensively studied. We hypothesize that the present distribution of genetic lineages reflects a combination of historical isolation and recent anthropogenic influences, including translocations and habitat fragmentation. Given the well-documented phenomenon of mito-nuclear discordance, where mitochondrial and nuclear markers can reveal different phylogenetic relationships [ 3 , 41 , 42 , 43 ], we applied next-generation sequencing (NGS) to generate genome-wide SNP data, enabling us to assess whether genomic-based population relationships align with those previously inferred from mitochondrial data, while providing a more comprehensive view of genomic diversity and lineage origins. This study included the most representative conservation units from all lineages of A. iberus , as updated by Nester et al. [ 11 ], together with nearly all currently documented populations from the species’ northern distribution range. This framework allowed us to identify populations of allochthonous origin and to confirm the natural status of others whose provenance has been debated. By applying a conservation genomics approach to this non-model, endangered species, we aim not only to inform region-specific management actions but also to contribute to the broader understanding of how genomic tools can enhance conservation strategies. This approach, centered on preserving the genetic identity and evolutionary legacy of local populations, underscores the value of “saving the locals” as a guiding principle for biodiversity conservation in fragmented and rapidly changing ecosystems. Results Sample selection and dataset construction Sequencing of the Cytochrome b gene resulted in an alignment of 985 base pairs. The haplotype network revealed 75 unique haplotypes for A. iberus and an additional five haplotypes for its closest relative, A. baeticus (Table S2 , Fig. S1 ). Within A. iberus , haplotypes from the Murcian lineage formed two main clusters: one comprising coastal localities 25–27 and 30–41 (Mar Menor, Santa Pola, Vinalopó, Rambla Moreras, and Chícamo), and another corresponding to inland water localities 24 and 29 (Sax and Villena, respectively), in agreement with previous genetic studies [ 11 , 28 ]. The Levantine lineage, including the Albuixech population (locality 23) and the allochthonous population from Adra (locality 28), also formed a distinct cluster. In contrast, haplotypes from the northern Mediterranean coast (Northern and Southern Catalonia lineages) grouped into three clusters, encompassing 21 unique haplotypes. The Southern Catalonia lineage (localities 9, 11, 15–21) clustered closely with localities 6–8 from the Llobregat River Delta. While a second and third cluster grouped localities 12 and 17 (Tributaris–Sèquia Major and Torrent del Pi) and another one the Northern Catalonia lineage (localities 1, 2, 10, 13 and 14) (Table S2 , Fig. S1 ). A total of 46 A. iberus individuals were selected for WGS, ensuring representation of all mitochondrial haplotypes from localities 1–21 (Catalonia region), complemented by seven individuals from the remaining distribution (localities 22–28) and two outgroups ( A. baeticus and A. anatoliae ). DNA concentrations averaged 10 ng/µL (range: 6.5–20 ng/µL). Sequencing generated between 17,293,672 and 90,793,054 raw reads per individual (mean: 65,273,642), corresponding to 2.594–13.619 GB of data (mean: 9.791 GB) per sample. After duplicate removal, quality filtering, and exclusion of repetitive regions, Dataset 1 (all 46 individuals) comprised 7,248,998 SNPs, including 1,160,109 unlinked SNPs (uSNPs). Dataset 2 (restricted to the 44 A. iberus individuals) contained 8,952,228 SNPs, of which 1,339,194 were uSNPs. Population structure, phylogenomic reconstructions, and migratory events Principal Component Analysis: The PCA performed revealed clear geographic structuring in A. iberus populations (Fig. 1 b). PC1 (14.6% of the variance) separated the Levantine lineage (locality 23) and the Murcian lineage (localities 24–27) from the Catalonia region (Northern and Southern Catalonia lineages, localities 1–21). PC2 reflected a north–south gradient among Catalan populations. Therefore, based on the PCA, six genetic clusters were identified: three corresponding to previously described lineages (Levantine, Murcian, and Northern Catalonia) and three subclusters within the Southern Catalonia lineage: Ebro River Delta (localities 9, 11, 18–21), Tarragona (localities 12, 15–17), and Prat de Llobregat (localities 6–8). Several geographically discordant populations were also detected, such as Adra (locality 28) and Mollet del Vallès (locality 3) clustering with Levantine. Localities 10, 13, 14 group with Northern Catalonia despite locating geographically to the Southern Catalonia lineage. Some populations, such as Cabanes (locality 22) and Sant Cugat del Vallès (locality 5), did not group with any cluster and occupied intermediate positions between clusters. Notably, several populations from the Southern Catalonia lineage (localities 10, 13, and 14) clustered genetically with the Northern Catalonia lineage. Population structure with Structf4: The optimal K from the Structf4 analysis was determined to be 5 (Fig. S2 ), corresponding to the four main lineages (Northern Catalonia, Southern Catalonia, Levantine and Murcian) plus the outgroups (Fig. 2 b, c). The Murcian lineage (localities 24–27) showed nearly homogeneous ancestry, except locality 24, which shared some ancestry with the outgroup. The Levantine lineage (locality 23) displayed an ancestry profile almost identical to Adra (locality 28) and Mollet del Vallès (locality 3). In Catalonia, two main ancestry components matched the two described lineages, with additional admixed groups. The Northern Catalonia lineage shares ancestry with southern localities 10, 13, 14. Southern Catalonia lineage shares ancestry with Rubí (locality 4), which is geographically distant. Two further admixed groups within the Southern Catalonia region corresponded to the Tarragona cluster (localities 12, 15–17) and the Prat de Llobregat cluster (localities 6–8), the latter showing slightly more Northern Catalonia ancestry along a geographic gradient. Tarragona itself was split into a northern group (locality 12) and a southern group (localities 15–17). Sant Cugat del Vallès (locality 5) exhibited a complex profile combining Levantine, Northern, and Southern Catalonia ancestry. Cabanes (locality 22) showed admixture between both Catalonia lineages and Levantine lineage, consistent with its geographic location (Fig. 1 a). Maximum Likelihood tree with IQTree: The ML phylogeny, rooted with A. anatoliae (branch omitted in Fig. 2 a), recovered A. baeticus as sister to all A. iberus samples, which formed four well-supported and geographically structured clades. The Murcian lineage was the first to diverge, followed by the Levantine lineage, both separated by Cape of Nao (Fig. 2 c). Adra (locality 28) clustered with Levantine lineage rather than Murcian lineage, matching previous results [ 11 ]. Northern and Southern Catalonia lineages were reciprocally monophyletic, each forming subgroups consistent with the PCA and Structf4 results. The Northern Catalonia clade included the northernmost populations plus localities 10, 13, 14 and Sant Cugat del Vallès (locality 5). Some Southern Catalonia populations (e.g. Bassa Nova in Gaià, Madrigueres, and Rubí (localities 9, 11 and 4 respectively)) showed shared ancestry with more distant populations. Relationships among Tarragona and nearby populations (localities 12, 15–17) were less resolved, with shorter branches and lower support, suggesting lower divergence. The Prat de Llobregat population (localities 6–8) was phylogenetically closer to Northern Catalonia, consistent with introgression prior to its extirpation during the construction of the Barcelona Airport and later reintroduction. Migration patterns: Migration analysis with divMigrate revealed directional gene flow from the Northern Catalonia lineage (localities 1, 2, 10, 13, 14) towards Tarragona (localities 12, 15, 17) within the Southern Catalonia region, with no evidence of reverse flow. This indicates that the northern lineage acts as a genetic source rather than a recipient. In contrast, Ebro River Delta (localities 9, 11, 18–21), Tarragona (localities 12, 15–17), and Prat de Llobregat (localities 6–8) appeared genetically interconnected, suggesting recent or ongoing exchange among geographically close populations (Fig. 3 d). Gst yielded the same pattern as Nm, and Jost’s D results are shown in Fig. S3 . Genome-wide diversity A genome-wide assessment of heterozygosity was conducted for all individuals (Fig. 3 a). The values varied substantially, both between and within populations, with most falling within the range of 1,000 to 3,000 heterozygous SNPs per megabase pair (SNPs/Mbp). The lowest heterozygosity was recorded in the Murcian lineage population of Sax (locality 24), with approximately 500 SNPs/Mbp. In contrast, the highest value was observed in the nearby population of Santa Pola (locality 25), with approximately 3,800 SNPs/Mbp. In Catalan lineages, the lowest heterozygosity levels were observed in the northern one. Conversely, the admixed Sant Cugat population (locality 5) showed the highest values in the region (3,000 SNPs/Mbp). Populations from Southern Catalonia exhibited very diverse levels of heterozygosity (ranging between 1,150 and 3,000 SNPs/Mbp). The analysis of ROHs showed consistent patterns across populations (Fig. 3 b). As expected from its low heterozygosity, the Sax population (locality 24) exhibited approximately 80% of its callable genome within ROHs, with nearly 35% in long ROHs. In contrast, Santa Pola (locality 25) showed the lowest ROH percentage and virtually no long ROHs (both from the Murcian lineage). Among Catalan populations, those from the Southern Catalonia lineage: Ebro River Delta (localities 9, 11 and 18–21) and Tarragona (localities 12 and 15–17) had lower ROH proportions, primarily composed of short segments. In contrast, populations from the Northern Catalonia lineage displayed a higher percentage of the genome within ROHs, including a greater presence of long segments. The admixed populations: Tarragona (localities 12 and 15–17) and Prat de Llobregat (localities 6–8) did not exhibit particularly low ROH values. Localities 9 and 11 exhibited both a higher percentage and greater length of ROHs compared to other Ebro River Delta localities (18–21), likely reflecting their captive-breeding origin, despite their close geographic and genetic proximity. Individuals from localities 10, 13 and 14 had ROH profiles similar to those from the Northern Catalonia lineage (localities 1 and 2), from which they likely originated. Prat de Llobregat populations (Localities 6–8) in Barcelona exhibited around 40% of their genome in ROHs, but these occurred in longer stretches compared to most other populations. Demographic history To infer the demographic history, we used a high-coverage dataset comprising 1,198,608,839 base pairs obtained from three individuals: two Aphanius iberus individuals, one from the Southern Catalonia lineage (locality 9) and one from the Northern Catalonia lineage (locality 2), and one individual of Aphanius baeticus as an outgroup. All three populations showed signs of demographic decline during the Pleistocene. In the Northern Catalonia lineage, this decline appears to have been continuous and uninterrupted. In contrast, the Southern Catalonia lineage experienced a partial demographic recovery after the initial decline, although recent trends indicate that the populations may be declining once again. A similar pattern was observed in A. baeticus , which also exhibited a phase of recovery followed by a renewed decline, paralleling the demographic trajectory of the Southern Catalonia lineage of A. iberus . Discussion Aphanius iberus exhibits a distinctive geographic genetic distribution pattern, characterized by distinct genetic lineages and the intermingling of admixed populations [ 6 , 11 , 28 ]. This genetic structure reflects the extensive evolutionary history shaped by local adaptations to diverse environmental pressures yet remains vulnerable to anthropogenic disturbances such as non-native translocations. The reconstruction of the history of its populations was one of the primary objectives of this study. To this end, analyses of population structure, phylogenetic inference, genome-wide heterozygosity, analysis of ROHs, migration, and demographic history were conducted, revealing a robust population structure indicative of long-established populations [ 6 , 39 ]. Additionally, the unique adaptability of A. iberus to varying salinity levels is believed to have evolved in response to historical climatic events, such as the Messinian salinity crisis [ 36 ]. During this period, drastic changes in the Mediterranean basin led to the formation of highly saline environments. The ability of the species to thrive in these fluctuating conditions reflects a costly yet effective evolutionary strategy, enabling them to become euryhaline and eurythermal. This adaptability contrasts with the more stable habitat preference of other Mediterranean closely related cyprinodontiform, like Valencia hispanica , which occupy consistent freshwater environments and follow a less physiologically demanding, but potentially less versatile strategy [ 36 , 44 , 45 ]. However, despite its broad salinity tolerance, A. iberus exhibits very limited natural dispersal capacity across open sea, a constraint that has likely reinforced the historical isolation among lineages. These historical adaptations underscore the resilience of the species and highlight the evolutionary pressures that have shaped its current distribution and genetic diversity. The three major genetic lineages originally described by Doadrio et al. [ 6 ], Catalonia, Levantine, and Murcian, have since been refined, with the Levantine clade subdivided into the Southern Catalonia lineage (Ebro River Delta populations, localities 18–21) and the Levantine lineage sensu stricto (represented by the Albuixech population, locality 23) [ 11 , 28 ]. Our mitochondrial haplotype network, phylogenomic and population structure analyses confirmed the presence of these four main genetic lineages: Northern Catalonia, Southern Catalonia, Levantine, and Murcian; and revealed genetically intermediate and admixed populations between the first two. Using the mentioned genomic approaches, we distinguished populations of natural origin from those likely resulting from translocations. For example, some populations within the Southern Catalonia lineage (localities 10, 13, and 14) show introgression from the Northern Catalonia lineage, whereas the Prat de Llobregat (localities 7 and 8) and Salou (Tributaris–Sèquia Major, locality 12) populations likely have a natural origin (Figs. 1 , S1). In contrast, other populations deviated from the expected geographic genetic gradient, clustering instead with distant populations and sharing their ancestry, migration patterns, and heterozygosity levels; this was the case for most localities 9–17, excluding aforementioned locality 12. These results, integrated with historical and institutional records, provide a detailed framework, expanded in the Supplementary Results & Discussion, for clarifying the origin of each population and guiding lineage-specific conservation actions. The results for the populations located outside the Catalonia region are consistent with previous studies. Both phylogenetic relationships and PCA clustering (and the haplotype network) aligned with the species’ known geographic gradient (Figs. 1 b, 2 , S1) [ 6 , 7 , 11 , 28 , 36 ]. The StructF4 results identified the Cabanes population (locality 22) as admixed between the Levantine, Northern Catalonia, and Southern Catalonia lineages. While previous studies classified this population as admixed between the Levantine and Southern Catalonia lineages, the current result may reflect the limited sampling in the dataset (only one individual was included and the absence of intermediate populations). Still, the phylogenetic tree places the Cabanes population (locality 22) as a sister branch to the Catalan lineages (Northern and Southern Catalonia) and the Levantine lineage, supporting previous findings. Similarly, the Murcian lineage populations clustered together, despite being previously recognized as distinct conservation units [ 11 , 28 ]. The Adra population (locality 28), which used to represent the southernmost population of the species’ distribution, exhibits genetic similarity to the Albuixech population in Valencia (Levantine lineage, locality 23). These distinctive characteristics, recently elucidated by Nester et al. [ 11 ] and confirmed in this study, support the hypothesis of a translocation in the 1980s from Albuixech, a population that has since undergone extensive captive breeding, effectively becoming a genetic refuge in captivity. Similarly, the Mollet del Vallès population (locality 3) in the suburbs of Barcelona appears to have originated from the same source (Levantine lineage). Migration analyses, restricted to natural populations to avoid translocation bias (Fig. 3 d), revealed directional gene flow from the Northern Catalonia lineage to the south, with no evidence of incoming migration. These patterns likely reflect historical (multi-generational) gene flow, rather than current, ongoing migration. The absence of significant recent inflow toward the Northern Catalonia lineage suggests long-term isolation, which may have been reinforced by oceanographic and geographic barriers. Along the Levantine coast, sediment accumulation from the Ebro River has created shallow waters that could facilitate dispersal, particularly under the climatic conditions described, whereas the Northern Catalonia coast is characterized by much deeper waters, potentially acting as a barrier to dispersal. Furthermore, prevailing Mediterranean currents in this Levantine region generally flow southward, possibly enhancing past dispersal in that direction, although seasonal northward currents may occur locally during summer. The current absence of populations between the Barcelona area (Prat de Llobregat, localities 7–8) and the Girona populations of Northern Catalonia (localities 1–2), a gap of roughly 70 km, may indicate historical local extinctions that further reduced connectivity. In contrast, high levels of interpopulation exchange were observed between the Ebro River Delta (localities 18–21) and Tarragona (localities 12, 15, 16 and 17), as well as with the Prat de Llobregat (localities 7–8), suggesting that historical connectivity existed among central-to-southern Catalan populations despite geographic separation. The overall levels of genomic diversity appear relatively high for an Endangered species (ranging between 1,000 and 3,000 SNPs/Mbp). By comparison, the Pyrenean desman ( Galemys pyrenaicus ), Tasmanian devil ( Sarcophilus harrisii ), and Sumatran orangutan ( Pongo abelii ) have reported heterozygosity values as low as 12, 320, and 1,200 SNPs/Mbp, respectively [ 3 ]. The Spanish toothcarp has long been recognized for its high genetic variability [ 6 , 11 , 25 , 28 ]. In our study, populations from the Northern Catalonia lineage (localities 1–2) displayed the lowest levels of heterozygosity, consistent with a likely southern origin of the species, as peripheral populations at the northern distribution limit often harbor reduced genetic diversity, and the highest ROH proportions, suggesting a decline in genetic diversity due to repeated bottlenecks and increased susceptibility to genetic drift. Conversely, introduced populations in the Barcelona province (such as localities 3, 4, and 5) showed high heterozygosity and low ROH levels. While these metrics may suggest good genetic health, they are likely artifacts of recent admixture, particularly in Rubí and Sant Cugat (localities 4 and 5, respectively), which show genetic signatures from the Southern Catalonia lineage. Other populations showing higher percentages of ROHs are localities 7 and 8, which were reintroduced from an ex-situ breeding program (currently locality 6). Besides presenting a larger amount of ROHs, it is notable that around 15% of the genome is in long stretches of homozygosity, an indicator of recent inbreeding loops likely caused by the captive breeding program [ 46 ]. The demographic history analysis revealed a persistent decline Ne size through the Pleistocene, particularly in the Northern Catalonia lineage. In contrast, the Southern Catalonia lineage also experienced demographic contractions, but these were not continuous, with a period of recovery before a more recent decline. Although PSMC results cannot directly infer dispersal, the sustained low Ne in the north is consistent with long-term isolation, as discussed in the migration section, where no evidence of recent incoming gene flow was detected. This isolation likely reduced opportunities for replenishment of genetic diversity, increasing the susceptibility of Northern Catalonia populations to genetic drift and inbreeding, as reflected in their higher frequencies and longer runs of homozygosity. Despite these demographic constraints, the species exhibits traits that may enhance its resilience, such as high reproductive output, rapid sexual maturation, and fast growth rates [ 7 , 39 ]. Supporting this, ROH patterns in southernmost populations (excluding locality 24) and, to a lesser extent, in Southern Catalonia indicate relatively good genomic health. ROHs in these groups are predominantly short, covering 20–25% of the genome, which is consistent with ancient, rather than recent, bottlenecks. In contrast, long ROHs are mainly observed in Northern Catalonia, consistent with smaller population sizes and mating between closely related individuals. The role of anthropogenic translocations (whether intentional or accidental) is of particular concern, as they can disrupt natural evolutionary trajectories and erode locally adapted population structures. This is especially problematic for Aphanius iberus , a species with well-defined genetic lineages and likely local adaptations critical for survival. The translocation of non-native individuals may dilute these adaptations, thereby reducing fitness and resilience. Habitat transformation driven by agricultural expansion and coastal development further threatens population integrity. In highly anthropized environments, such as the irrigation ponds inhabited by the Sax population (locality 24), selective pressures differ markedly from those in natural habitats, potentially driving divergent evolutionary trajectories. Peripheral populations, such as those in Northern Catalonia (localities 1–2), face additional challenges due to their position at the range margin, where vulnerability is heightened. Conversely, our results suggest that some populations from the Levantine and Murcian regions may be better equipped to cope with rising temperatures and climatic variability. Nonetheless, a comprehensive conservation strategy must consider all populations, especially those in the north, to safeguard the species’ long-term resilience and genetic diversity. In light of our findings, the integration of high-resolution genomic tools emerges as essential to detect hidden admixture, resolve the origins of uncertain populations, and design targeted, locally adapted management strategies. Such approaches provide a robust framework for addressing historical uncertainties and ensuring the preservation of A. iberus in the face of ongoing environmental change. Materials and methods Sample Collection, DNA Extraction, and Mitochondrial Data Screening Tissue samples were obtained by collecting caudal fin clips, following standard protocols and in coordination with the relevant authorities. Adult individuals of A. iberus (average standard length 2–4 cm; body weight 0.5–1 g) were euthanized by immersion in an overdose of buffered tricaine methanesulfonate (MS-222, 0.1%), in accordance with internationally approved protocols for small cyprinodontiform fishes. All procedures were performed by qualified personnel under the responsibility of the competent authorities (Consorci del Delta del Llobregat, Parc Natural del Delta de l’Ebre, and Departament de Recerca i Universitats de la Generalitat de Catalunya), with collection and handling authorized within the framework of the research project SGR-00420. The study protocol and methods were approved by the Animal Experimentation Ethics Committee of the National Museum of Natural Sciences (CEEA-MNCN; Spanish Animal Experimentation Research Centre no. ES280790000189), in strict accordance with current Spanish law (RD53/2013), transposed from European Union Directive 2010/63/EU (art. 2, 5f). All experimental protocols were conducted in accordance with relevant guidelines and regulations and are reported in compliance with the ARRIVE guidelines ( https://arriveguidelines.org ). Humane endpoints consisted of monitoring for the immediate loss of opercular movement and reflexes after immersion in the anesthetic solution, thereby confirming the effectiveness of euthanasia and safeguarding animal welfare. Fin clips were stored in 95% ethanol and deposited in a − 80°C freezer until processing. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer’s specifications. DNA quantification was carried out through fluorometry using Qubit Broad Range (Thermofisher). Suitable DNA samples were then selected for sequencing. Detailed information about samples and locations can be found in Table S1 . In order to account for the full genetic diversity present in the Catalonia region (Northern and Southern Catalonia lineages), located along the northeastern coast of the Iberian Peninsula, we initially sequenced the cytochrome b gene for all 174 collected individuals using the primers GluF and ThR [ 47 ]. The primer sequences and PCR conditions are detailed in Table S3 . Newly generated sequences were supplemented with an additional 192 sequences from Doadrio research group (obtained from NCBI GenBank corresponding to 2001 and 2015) obtained from GenBank (Sample information and GenBank accession numbers can be found in Table S2 ). These sequences were aligned using MAFFT implemented in Geneious Prime v.2023.0.4. The resulting alignments were used to construct a haplotype network using the TCS Network Builder [ 48 ], with the "gap" parameter set to missing. The resulting graph file was visualized using TCSbu [ 49 ]. The study area was expanded to include seven additional samples of A. iberus from across the species' entire distribution range, ensuring representation of all known four genetic lineages previously identified, namely, Levantine, and Murcian lineages. Additionally, a sample of sister species Aphanius baeticus (Doadrio, Carmona & Fernández-Delgado, 2002) was included as a closest related outgroup, while a sample of Anatolichthys anatoliae (Leidenfrost, 1912) was incorporated as a more distantly outgroup (Fig. 1 a). Data generation and processing: High quality DNA samples were then sent for Illumina library preparation and sequencing on a NovaSeq X Plus (PE150) targeting at least 10X coverage for 42 individuals and 15X for four samples (Table S1 ). Raw reads were filtered, and adapters were removed using fastp [ 50 ] with a minimum base quality score of 30, activating automatic adapter detection for paired-end sequencing and trimming polyG/X tails. To assure the quality of the data and correct trimming, sequences were checked with FastQC v.0.20.0 [ 51 ]. Filtered reads were mapped against the reference genome of Aphanius iberus [ 52 ] with bwa-mem v0.7.17 [ 53 ] and sorted using Samtools v1.9 [ 54 ]. Duplicate reads were removed using Picard [ 55 ] and reads with a mapping quality below 30 were discarded using Samtools view. SNPcalling was done using Haplotypecaller from GATK [ 56 ] with BP_resolution. Output files were combined using CombineGVCFs, and genotyping was carried out with GenotypeGVCFs from GATK [ 56 ]. At this stage, we generated two different datasets: Dataset 1, containing all 46 individuals of Aphanius iberus along with the two outgroups, Aphanius baeticus and Anatolichthys anatoliae ; and Dataset 2, containing exclusively the 44 individuals of Aphanius iberus. Datasets are detailed in Table S1 . Coverage was calculated and is detailed in Table S1 . The four samples sequenced at higher coverage were downsampled to a similar coverage to the other samples using Picard [ 55 ] for inclusion in Datasets 1 and 2. The reference genome was scanned for repetitive regions, which were subsequently removed. Datasets 1 and 2 were filtered to exclude variants matching at least one of the following criteria: Quality by Depth (QD) < 10.0, Mapping Quality (MQ) 50.0, StrandOddsRatio (SOR) > 5.0, MQRankSum 5.0, and ReadPosRankSum 5.0. Further filtering of genotypes was performed using VCFtools, selecting variants with a quality score above 30, retaining only biallelic SNPs, removing indels, a minor allele frequency (MAF) of 0.001, and no missing data. To account for linkage disequilibrium, a secondary dataset was generated for each of the Datasets 1 and 2 retaining only unlinked SNPs (uSNPS) using bcftools with r2 ≤ to 0.5. Population structure and phylogenomic analyses To gain a preliminary insight into the variability among the samples, a Principal Component Analysis (PCA) was conducted using Plink v1.9 [ 57 ] on A. iberus individuals (Dataset 2, unlinked SNPs). Additionally, we conducted a population structure analysis using Structf4 [ 58 ], considering 2–7 ancestral components and 50,000,000 iterations for both the first and second MCMC chains. Results were visualized with R v.4.3.2 [ 59 ]. These analyses were performed on Dataset 1 and 2 (unlinked SNPs), which included the two outgroups to determine if some lineages were external. To investigate phylogenomic relationships among populations, we constructed a maximum-likelihood (ML) phylogeny. For this, we filtered Dataset 1 to exclude missing data and randomly selected one allele per genomic site (i.e., each SNP position in the alignment), resulting in a total of 5,808,219 SNPs. The dataset was then divided into non-overlapping 100 kbp windows, and each window was analyzed independently using IQ-TREE2 [ 60 ] with the GTR + ASC model and 1,000 bootstrap replicates. The best-scoring trees from each window were subsequently combined using ASTRAL v5.7.8 [61]. Genome-wide diversity We estimated genome-wide heterozygosity for each individual in Dataset 1 (including both genera Aphanius and Anatolichthys ; unfiltered). To do so, we divided the reference genome into non-overlapping 100 kb windows. Within each window, heterozygous sites were counted using vcfhetcount from vcflib [ 62 ]. A site was considered callable if it was not an indel and had a mapping quality score above 30. Results were plot in R v.4.3.2 [ 59 ]. Runs of Homozygosity (ROHs) were calculated for each individual of A. iberus based on the density of heterozygous sites in the genome using the implemented Hidden Markov Model (HMM) in the bcftools roh function with the default parameter –AF-dflt 0.4 [ 63 ], on the filtered Dataset 2. A. baeticus and A. anatoliae were excluded from this analysis to prevent them from influencing the results by causing noise, as the patterns of homozygosity can vary considerably between species. We kept ROHs with a Phred Score of at least 70 and with a minimum length of 100 Kbp. ROHs are considered long when above 1Mbp, medium between 500 Kbps and 1 Mbp and short below 100 Kbps. Visualization for all analyses was carried out in Rv.4.3.2 [ 59 ] using ggplot2.61 [ 64 ]. Migration We estimated gene flow among different population groups within the Northern and Southern Catalonia lineages. Populations were grouped according to the genetic clusters retrieved in the PCA and, in some cases, further subdivided based on their geographic location. Because a minimum number of individuals per group is required to run the analysis, it was conducted only within Catalonian populations (localities 1–21). This approach also provides valuable insight into the status and potential origin of these populations. To do this, we generated a genepop file from Dataset 2 using the vcf2genepop function in the R package gwscaR [ 66 ]. Gene flow was then estimated with the function divMigrate in the R package diveRsity [ 66 ]. We calculated the number of effective migrants per generation (Nm), Jost’s D (D), and Nei’s GST (GST). The resulting matrix was plotted with the R package qgraph [ 67 ], disregarding edges below 0.22. Demographic history We inferred the demographic history of the species using Pairwise Sequential Markovian Coalescent (PSMC) [68]. This analysis was run on two individuals of A. iberus (one from the Northern and one from the Southern Catalonia lineages) and one A. baeticus , all three sequenced aiming at a 15X coverage. Heterozygous positions were obtained from bam files with Samtools v1.9 [ 54 ] and the data was filtered to assure a mapping and base quality > 30. A generation time (θ) of 0.5 and a mutation rate of 3.5 x 10 − 9 per bp per generation were assumed [ 69 ]. Ten bootstrap replicates were performed on each sample. Declarations Acknowledgements and funding We would like to thank the Spanish Ministry of Science and Innovation and the State Agency for Research for their financial support through Project Aphanius (PID2019-103936GB-C22) and Project Saltfish (PID2023-146173NB-C22), both awarded to ID. We are grateful to Miriam Casal, Pilar Risueño, Equipo de Ectotermos from the Zoo of Barcelona and Juan Miguel Galindo Galindo from Junta de Andalucía for their effort in sample collection, and to Pablo Librado (Institute of Evolutionary Biology, CSIC-UPF) for his assistance with the Structf4 analysis. We also wish to thank Lourdes Alcaraz for her laboratory support. Finally, we acknowledge CESGA, the Supercomputing Center of Galicia, for providing computational resources, and the Universidad Complutense de Madrid (UCM) for hosting AL-S and TLN within the Doctorado en Biología program. This project was supported by the Fundació Barcelona Zoo and the Departament de Medi Ambient i Sostenibilitat , through grant SGR-00420 from the Departament de Recerca i Universitats de la Generalitat de Catalunya to SC. ME is funded by an FPI fellowship from the Ministerio de Ciencia, Innovación y Universidades, Spain (PRE2022-101473). AL-S is funded by an FPI fellowship from the same governmental institution (PRE2020-092988). GM-R was also funded by an FPI fellowship (PRE2019-088729). BB-C and TLN are supported by FPU fellowships (FPU2018/04742 and FPU2020/04500) both from the Ministerio de Ciencia, Innovación y Universidades, Spain. AT is supported by the “la Caixa” doctoral fellowship programme (LCF/BQ/DR20/11790007). ST is funded by an AGAUR-FI Joan Oró fellowship from the Departament de Recerca i Universitats de la Generalitat de Catalunya and the European Social Fund Plus (2024_FI-1_01035). Author contributions All authors contributed to conceive and design the study. A.L.-S., S.P., T.L.N., N.F., J.X., J.R.-O., E.d.R. and I.D. collected the field samples. M.E., A.L.-S., S.P. and T.L.N. carried out laboratory work. M.E., A.L.-S., G.M-R., S.P., A.T., B.B.-C. and S.T. carried out data analysis and all authors performed the interpretation. M.E., A.L.-S., G.M.-R., S.P., A.T. and B.B.-C. produced the workflow of all scripts. M.E. and A.L.-S. wrote the first draft of the manuscript, based on ideas conceived also with G.M-R., S.P., A.T., B.B.-C., T.L.N., S.T., I.D. and S.C. All authors discussed ideas and contributed with valuable scientific interpretations and carefully reviewed and approved the final manuscript. Data availability statement The datasets generated and/or analyzed during the current study are available in NCBI/GenBank repository under BioProject PRJNA1320828, with accession numbers provided in Supplementary Table S1. https://dataview.ncbi.nlm.nih.gov/object/PRJNA1320828?reviewer=mi56tduse3pe847n42t926s3e3 Additional Information The authors declare no competing interests. References Khan, S. et al. Overview on the role of advanced genomics in conservation biology of endangered species. International Journal of Genomics , 3460416. (2016). https://doi.org/10.1155/2016/3460416 (2016). Supple, M. A. & Shapiro, B. 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10:09:34","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186619,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/54e39c1d46291ba74cf90860.html"},{"id":92249004,"identity":"c091c1a9-701e-4426-b3ee-38a03eff7792","added_by":"auto","created_at":"2025-09-26 10:09:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120990,"visible":true,"origin":"","legend":"\u003cp\u003ea) Distribution of the populations of A. iberus included in the study. Genetic lineages are highlighted, and localities are colored according to the results of the PCA. b) PCA analysis of A. iberus. c) Representation of male and female individuals of A. iberus.\u003c/p\u003e","description":"","filename":"FIG1NEWCOLORSMORENAMES.png","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/b76cb251a2ed701bc0ce3d72.png"},{"id":92249005,"identity":"902df37a-f7f4-48e6-9a10-2df82ab8fcdc","added_by":"auto","created_at":"2025-09-26 10:09:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102459,"visible":true,"origin":"","legend":"\u003cp\u003ea) Phylogenetic Tree inferred using a ML approach based on genomic data. Bootstraps \u0026gt; 95 are marked with a black circle and \u0026gt; 75 with a white one. The tree was rooted with A. anatoliae, but the branch is not shown for visualization purposes. b) and c)Structf4 K=5 analysis as bar charts or on the map as pie charts.\u003c/p\u003e","description":"","filename":"FIG2mapstructtreeV2.png","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/be85e7bcd00eeb86444b0737.png"},{"id":92249206,"identity":"3f04853e-f471-4f59-864c-e0c574389377","added_by":"auto","created_at":"2025-09-26 10:17:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116948,"visible":true,"origin":"","legend":"\u003cp\u003ea) Genome-wide heterozygosity levels of all individuals of A. iberus included in the study. b) Percentage of the genome in Runs of Homozygosity (ROHs). c) Demographic history of two populations of A. iberus and one A. baeticus during the Pleistocene. d) Migration network (Effective Migrants per generation Nm) amongst the genetic groups identified in the population structure analyses.\u003c/p\u003e","description":"","filename":"FIG3new.png","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/563b95d6cc218e320a719677.png"},{"id":98243517,"identity":"9bfff36d-100f-49ec-a379-6309d3a3f85e","added_by":"auto","created_at":"2025-12-15 16:08:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1239286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/07b4c4e2-c4d6-4ac1-8623-751a87ca5e42.pdf"},{"id":92249008,"identity":"1d22db90-3b46-4d22-9da9-c05e06003012","added_by":"auto","created_at":"2025-09-26 10:09:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12772,"visible":true,"origin":"","legend":"","description":"","filename":"DEFTableS1wgssampels.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/ea962b368fac520feee9d30c.xlsx"},{"id":92249207,"identity":"561bf662-7c3c-4dcd-baf8-149d3ba18292","added_by":"auto","created_at":"2025-09-26 10:17:33","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":42509,"visible":true,"origin":"","legend":"","description":"","filename":"DEFTableS2allsamples.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/a752874428b0f72056fe3362.xlsx"},{"id":92249011,"identity":"70933255-d953-4072-9c7e-e6d4e4b12659","added_by":"auto","created_at":"2025-09-26 10:09:33","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5372,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3PCRconditions.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/cd74905394af3b1c4476e0be.xlsx"},{"id":92250157,"identity":"ad334a4e-cf37-4253-b358-ef7e186d4312","added_by":"auto","created_at":"2025-09-26 10:25:33","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1218982,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryresultsdiscussionandfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7494783/v1/8c67f6dbf7c906366c35852a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Saving the locals: a conservation genomics approach to the Endangered Spanish Toothcarp, Aphanius iberus (Valenciennes, 1846)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eConservation initiatives involving fauna and flora are frequently enhanced nowadays by a comprehensive understanding of genomics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Further insight in this area has the potential to optimize the allocation of economic resources and facilitate the implementation of more effective conservation strategies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Concerning these decision-making processes, population genomics emerges as a powerful tool, as it focuses on the study of genomic variation within and among populations, as well as the evolutionary factors shaping this variation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The results may be used to characterize the genomic profile of populations, elucidating their evolutionary history and, for example, enabling their classification into different Operational Conservation Units (similar to Designatable Units (or DUs)) for conservation management purposes [6, 7; 8]. Genomic data also provide information on connectivity, genetic diversity, inbreeding, and signatures of local adaptation. Combined approaches such as structure inference, phylogenomic reconstructions, migration modelling, and demographic history analyses offer a comprehensive view of population dynamics, evolutionary trajectories, and vulnerabilities, thereby supporting evidence-based conservation strategies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe application of these techniques is especially valuable for continental aquatic fauna, where dispersal and colonization are limited by geographical barriers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While research has often focused on model species, domestic animals, or charismatic taxa [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], many species with unique evolutionary traits remain overlooked, even when they can adapt to highly variable habitats. One such trait is euryhalinity, an ancestral adaptation that allows fish to thrive across contrasting salinity regimes but is retained by only\u0026thinsp;~\u0026thinsp;3\u0026ndash;5% of fish species [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This limited occurrence suggests that the physiological plasticity associated with euryhalinity entails significant costs, yet these species have attracted considerable scientific interest and serve as key models in ecological and physiological research [18, 19, 20; 21, 22, 23].\u003c/p\u003e\u003cp\u003eThe Spanish toothcarp, \u003cem\u003eAphanius iberus\u003c/em\u003e (Valenciennes, 1846), is an endemic species to the Mediterranean coast of the Iberian Peninsula, currently found in a wide range of habitats, including groundwater springs (locally known as \u003cem\u003eullals\u003c/em\u003e), coastal lagoons, river mouths, salt marshes, and even salt evaporation ponds where salinity can reach twice or more than that of seawater [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Listed as Endangered in the Spanish National Catalogue of Threatened Species (RD 139/2011[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]), its populations face isolation and fragmentation due to habitat destruction, introductions of invasive species translocations, and climate change among other threats [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The species\u0026rsquo; limited natural dispersal is mainly triggered by extreme weather events, such as intense storms and floods, often linked to Isolated High-level Atmospheric Depressions (locally known as \u003cem\u003eDANAs\u003c/em\u003e), and, less frequently, by strong coastal waves [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These events, which are becoming more frequent under current climate change, can mobilize large volumes of water, transporting individuals across fragmented habitats and influencing the species\u0026rsquo; biology and ecology. At the same time, global warming is not only leading to progressively warmer and saltier conditions, particularly in southern Spain, but also causing severe desiccation in many habitats [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], making reintroduction and translocation strategies especially relevant in the species\u0026rsquo; conservation management [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe geographic distribution of the Spanish toothcarp has been previously studied, revealing a well-structured genetic pattern composed of several distinct lineages that reflect the spatial segregation of the species across its natural range (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These lineages include: (1) the Northern Catalonia lineage, comprising two populations from Girona province, near the French border; (2) the Southern Catalonia lineage, which extends southwards to the Ebro River Delta; (3) the Levantine lineage (corresponding to Iberian Levante), represented by a single genetic population covering most of the Valencian region; and (4) the Murcian lineage (already referred to as such in Doadrio et al., 1996), stretching from Cape of Nao (southern Alicante province) through the province of Murcia to Rambla Moreras, the southernmost limit of the species\u0026rsquo; range (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Subsequent studies have refined this geographical and genetic gradient, identifying admixed populations located between these genetically distinct lineages, which show signs of historical or ongoing gene flow and may have important implications for the genetic diversity and evolutionary history of the species [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTranslocations and reintroductions have been carried out across the distribution of \u003cem\u003eA. iberus\u003c/em\u003e, sometimes without complete knowledge of the genetic origin of the source populations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A clear example is the Adra population (locality 28), at the southernmost limit of the species\u0026rsquo; range (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), which recent evidence suggests originated from translocations from the Albuixech population (locality 23 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]). Notably, this population of Albuixech is the only genetic population representing the entire Levantine region and has served as the source for several reintroductions within this lineage\u0026rsquo;s range. Similar cases of uncertain origin have been reported elsewhere, such as the Prat de Llobregat population (localities 7 and 8, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), declared extinct in the 1960s and later reintroduced in the mid-1990s with individuals provided by local aquarists [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In contrast, the \u003cem\u003eA. iberus\u003c/em\u003e populations of the Ebro River Delta (localities 18\u0026ndash;21, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) are original and have remained stable over time. Only two small-scale experimental releases were conducted under the LIFE project (1996\u0026ndash;1999) with captive-bred fish in newly created habitats. While such kind of interventions can help restore extirpated populations, they may also compromise the integrity of natural lineages if performed without an adequate genetic and ecological basis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The translocation of non-native individuals can potentially disrupt the local adaptations that are critical for the survival of \u003cem\u003eA. iberus\u003c/em\u003e in its varied habitats, leading to a decline in fitness and resilience. This is particularly concerning for populations surviving exclusively in artificial habitats, such as irrigation ponds in Sax and Adra (localities 24 and 28), which face compounded threats to their long-term survival.\u003c/p\u003e\u003cp\u003eA primary objective of this study was to reconstruct and understand the evolutionary history and population structure of \u003cem\u003eAphanius iberus\u003c/em\u003e along its distribution range, with particular emphasis on the Catalonia region (Northern and Southern Catalonia lineages), located in the northward region of the species\u0026rsquo; distribution range and which has not been extensively studied. We hypothesize that the present distribution of genetic lineages reflects a combination of historical isolation and recent anthropogenic influences, including translocations and habitat fragmentation. Given the well-documented phenomenon of mito-nuclear discordance, where mitochondrial and nuclear markers can reveal different phylogenetic relationships [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], we applied next-generation sequencing (NGS) to generate genome-wide SNP data, enabling us to assess whether genomic-based population relationships align with those previously inferred from mitochondrial data, while providing a more comprehensive view of genomic diversity and lineage origins. This study included the most representative conservation units from all lineages of \u003cem\u003eA. iberus\u003c/em\u003e, as updated by Nester et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], together with nearly all currently documented populations from the species\u0026rsquo; northern distribution range. This framework allowed us to identify populations of allochthonous origin and to confirm the natural status of others whose provenance has been debated. By applying a conservation genomics approach to this non-model, endangered species, we aim not only to inform region-specific management actions but also to contribute to the broader understanding of how genomic tools can enhance conservation strategies. This approach, centered on preserving the genetic identity and evolutionary legacy of local populations, underscores the value of \u0026ldquo;saving the locals\u0026rdquo; as a guiding principle for biodiversity conservation in fragmented and rapidly changing ecosystems.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample selection and dataset construction\u003c/h2\u003e\u003cp\u003eSequencing of the Cytochrome b gene resulted in an alignment of 985 base pairs. The haplotype network revealed 75 unique haplotypes for \u003cem\u003eA. iberus\u003c/em\u003e and an additional five haplotypes for its closest relative, \u003cem\u003eA. baeticus\u003c/em\u003e (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Within \u003cem\u003eA. iberus\u003c/em\u003e, haplotypes from the Murcian lineage formed two main clusters: one comprising coastal localities 25\u0026ndash;27 and 30\u0026ndash;41 (Mar Menor, Santa Pola, Vinalop\u0026oacute;, Rambla Moreras, and Ch\u0026iacute;camo), and another corresponding to inland water localities 24 and 29 (Sax and Villena, respectively), in agreement with previous genetic studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The Levantine lineage, including the Albuixech population (locality 23) and the allochthonous population from Adra (locality 28), also formed a distinct cluster. In contrast, haplotypes from the northern Mediterranean coast (Northern and Southern Catalonia lineages) grouped into three clusters, encompassing 21 unique haplotypes. The Southern Catalonia lineage (localities 9, 11, 15\u0026ndash;21) clustered closely with localities 6\u0026ndash;8 from the Llobregat River Delta. While a second and third cluster grouped localities 12 and 17 (Tributaris\u0026ndash;S\u0026egrave;quia Major and Torrent del Pi) and another one the Northern Catalonia lineage (localities 1, 2, 10, 13 and 14) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA total of 46 \u003cem\u003eA. iberus\u003c/em\u003e individuals were selected for WGS, ensuring representation of all mitochondrial haplotypes from localities 1\u0026ndash;21 (Catalonia region), complemented by seven individuals from the remaining distribution (localities 22\u0026ndash;28) and two outgroups (\u003cem\u003eA. baeticus\u003c/em\u003e and \u003cem\u003eA. anatoliae\u003c/em\u003e). DNA concentrations averaged 10 ng/\u0026micro;L (range: 6.5\u0026ndash;20 ng/\u0026micro;L). Sequencing generated between 17,293,672 and 90,793,054 raw reads per individual (mean: 65,273,642), corresponding to 2.594\u0026ndash;13.619 GB of data (mean: 9.791 GB) per sample. After duplicate removal, quality filtering, and exclusion of repetitive regions, Dataset 1 (all 46 individuals) comprised 7,248,998 SNPs, including 1,160,109 unlinked SNPs (uSNPs). Dataset 2 (restricted to the 44 \u003cem\u003eA. iberus\u003c/em\u003e individuals) contained 8,952,228 SNPs, of which 1,339,194 were uSNPs.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePopulation structure, phylogenomic reconstructions, and migratory events\u003c/h3\u003e\n\u003cp\u003ePrincipal Component Analysis:\u003c/p\u003e\u003cp\u003eThe PCA performed revealed clear geographic structuring in \u003cem\u003eA. iberus\u003c/em\u003e populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). PC1 (14.6% of the variance) separated the Levantine lineage (locality 23) and the Murcian lineage (localities 24\u0026ndash;27) from the Catalonia region (Northern and Southern Catalonia lineages, localities 1\u0026ndash;21). PC2 reflected a north\u0026ndash;south gradient among Catalan populations. Therefore, based on the PCA, six genetic clusters were identified: three corresponding to previously described lineages (Levantine, Murcian, and Northern Catalonia) and three subclusters within the Southern Catalonia lineage: Ebro River Delta (localities 9, 11, 18\u0026ndash;21), Tarragona (localities 12, 15\u0026ndash;17), and Prat de Llobregat (localities 6\u0026ndash;8). Several geographically discordant populations were also detected, such as Adra (locality 28) and Mollet del Vall\u0026egrave;s (locality 3) clustering with Levantine. Localities 10, 13, 14 group with Northern Catalonia despite locating geographically to the Southern Catalonia lineage. Some populations, such as Cabanes (locality 22) and Sant Cugat del Vall\u0026egrave;s (locality 5), did not group with any cluster and occupied intermediate positions between clusters. Notably, several populations from the Southern Catalonia lineage (localities 10, 13, and 14) clustered genetically with the Northern Catalonia lineage.\u003c/p\u003e\u003cp\u003ePopulation structure with Structf4:\u003c/p\u003e\u003cp\u003eThe optimal \u003cem\u003eK\u003c/em\u003e from the Structf4 analysis was determined to be 5 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), corresponding to the four main lineages (Northern Catalonia, Southern Catalonia, Levantine and Murcian) plus the outgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). The Murcian lineage (localities 24\u0026ndash;27) showed nearly homogeneous ancestry, except locality 24, which shared some ancestry with the outgroup. The Levantine lineage (locality 23) displayed an ancestry profile almost identical to Adra (locality 28) and Mollet del Vall\u0026egrave;s (locality 3). In Catalonia, two main ancestry components matched the two described lineages, with additional admixed groups. The Northern Catalonia lineage shares ancestry with southern localities 10, 13, 14. Southern Catalonia lineage shares ancestry with Rub\u0026iacute; (locality 4), which is geographically distant. Two further admixed groups within the Southern Catalonia region corresponded to the Tarragona cluster (localities 12, 15\u0026ndash;17) and the Prat de Llobregat cluster (localities 6\u0026ndash;8), the latter showing slightly more Northern Catalonia ancestry along a geographic gradient. Tarragona itself was split into a northern group (locality 12) and a southern group (localities 15\u0026ndash;17). Sant Cugat del Vall\u0026egrave;s (locality 5) exhibited a complex profile combining Levantine, Northern, and Southern Catalonia ancestry. Cabanes (locality 22) showed admixture between both Catalonia lineages and Levantine lineage, consistent with its geographic location (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMaximum Likelihood tree with IQTree:\u003c/p\u003e\u003cp\u003eThe ML phylogeny, rooted with \u003cem\u003eA. anatoliae\u003c/em\u003e (branch omitted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), recovered \u003cem\u003eA. baeticus\u003c/em\u003e as sister to all \u003cem\u003eA. iberus\u003c/em\u003e samples, which formed four well-supported and geographically structured clades. The Murcian lineage was the first to diverge, followed by the Levantine lineage, both separated by Cape of Nao (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Adra (locality 28) clustered with Levantine lineage rather than Murcian lineage, matching previous results [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Northern and Southern Catalonia lineages were reciprocally monophyletic, each forming subgroups consistent with the PCA and Structf4 results. The Northern Catalonia clade included the northernmost populations plus localities 10, 13, 14 and Sant Cugat del Vall\u0026egrave;s (locality 5). Some Southern Catalonia populations (e.g. Bassa Nova in Gai\u0026agrave;, Madrigueres, and Rub\u0026iacute; (localities 9, 11 and 4 respectively)) showed shared ancestry with more distant populations. Relationships among Tarragona and nearby populations (localities 12, 15\u0026ndash;17) were less resolved, with shorter branches and lower support, suggesting lower divergence. The Prat de Llobregat population (localities 6\u0026ndash;8) was phylogenetically closer to Northern Catalonia, consistent with introgression prior to its extirpation during the construction of the Barcelona Airport and later reintroduction.\u003c/p\u003e\u003cp\u003eMigration patterns:\u003c/p\u003e\u003cp\u003eMigration analysis with divMigrate revealed directional gene flow from the Northern Catalonia lineage (localities 1, 2, 10, 13, 14) towards Tarragona (localities 12, 15, 17) within the Southern Catalonia region, with no evidence of reverse flow. This indicates that the northern lineage acts as a genetic source rather than a recipient. In contrast, Ebro River Delta (localities 9, 11, 18\u0026ndash;21), Tarragona (localities 12, 15\u0026ndash;17), and Prat de Llobregat (localities 6\u0026ndash;8) appeared genetically interconnected, suggesting recent or ongoing exchange among geographically close populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Gst yielded the same pattern as Nm, and Jost\u0026rsquo;s D results are shown in Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eGenome-wide diversity\u003c/h3\u003e\n\u003cp\u003eA genome-wide assessment of heterozygosity was conducted for all individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The values varied substantially, both between and within populations, with most falling within the range of 1,000 to 3,000 heterozygous SNPs per megabase pair (SNPs/Mbp). The lowest heterozygosity was recorded in the Murcian lineage population of Sax (locality 24), with approximately 500 SNPs/Mbp. In contrast, the highest value was observed in the nearby population of Santa Pola (locality 25), with approximately 3,800 SNPs/Mbp. In Catalan lineages, the lowest heterozygosity levels were observed in the northern one. Conversely, the admixed Sant Cugat population (locality 5) showed the highest values in the region (3,000 SNPs/Mbp). Populations from Southern Catalonia exhibited very diverse levels of heterozygosity (ranging between 1,150 and 3,000 SNPs/Mbp).\u003c/p\u003e\u003cp\u003eThe analysis of ROHs showed consistent patterns across populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). As expected from its low heterozygosity, the Sax population (locality 24) exhibited approximately 80% of its callable genome within ROHs, with nearly 35% in long ROHs. In contrast, Santa Pola (locality 25) showed the lowest ROH percentage and virtually no long ROHs (both from the Murcian lineage). Among Catalan populations, those from the Southern Catalonia lineage: Ebro River Delta (localities 9, 11 and 18\u0026ndash;21) and Tarragona (localities 12 and 15\u0026ndash;17) had lower ROH proportions, primarily composed of short segments. In contrast, populations from the Northern Catalonia lineage displayed a higher percentage of the genome within ROHs, including a greater presence of long segments. The admixed populations: Tarragona (localities 12 and 15\u0026ndash;17) and Prat de Llobregat (localities 6\u0026ndash;8) did not exhibit particularly low ROH values. Localities 9 and 11 exhibited both a higher percentage and greater length of ROHs compared to other Ebro River Delta localities (18\u0026ndash;21), likely reflecting their captive-breeding origin, despite their close geographic and genetic proximity. Individuals from localities 10, 13 and 14 had ROH profiles similar to those from the Northern Catalonia lineage (localities 1 and 2), from which they likely originated. Prat de Llobregat populations (Localities 6\u0026ndash;8) in Barcelona exhibited around 40% of their genome in ROHs, but these occurred in longer stretches compared to most other populations.\u003c/p\u003e\n\u003ch3\u003eDemographic history\u003c/h3\u003e\n\u003cp\u003eTo infer the demographic history, we used a high-coverage dataset comprising 1,198,608,839 base pairs obtained from three individuals: two \u003cem\u003eAphanius iberus\u003c/em\u003e individuals, one from the Southern Catalonia lineage (locality 9) and one from the Northern Catalonia lineage (locality 2), and one individual of \u003cem\u003eAphanius baeticus\u003c/em\u003e as an outgroup. All three populations showed signs of demographic decline during the Pleistocene. In the Northern Catalonia lineage, this decline appears to have been continuous and uninterrupted. In contrast, the Southern Catalonia lineage experienced a partial demographic recovery after the initial decline, although recent trends indicate that the populations may be declining once again. A similar pattern was observed in \u003cem\u003eA. baeticus\u003c/em\u003e, which also exhibited a phase of recovery followed by a renewed decline, paralleling the demographic trajectory of the Southern Catalonia lineage of \u003cem\u003eA. iberus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eAphanius iberus\u003c/em\u003e exhibits a distinctive geographic genetic distribution pattern, characterized by distinct genetic lineages and the intermingling of admixed populations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This genetic structure reflects the extensive evolutionary history shaped by local adaptations to diverse environmental pressures yet remains vulnerable to anthropogenic disturbances such as non-native translocations. The reconstruction of the history of its populations was one of the primary objectives of this study. To this end, analyses of population structure, phylogenetic inference, genome-wide heterozygosity, analysis of ROHs, migration, and demographic history were conducted, revealing a robust population structure indicative of long-established populations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, the unique adaptability of \u003cem\u003eA. iberus\u003c/em\u003e to varying salinity levels is believed to have evolved in response to historical climatic events, such as the Messinian salinity crisis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. During this period, drastic changes in the Mediterranean basin led to the formation of highly saline environments. The ability of the species to thrive in these fluctuating conditions reflects a costly yet effective evolutionary strategy, enabling them to become euryhaline and eurythermal. This adaptability contrasts with the more stable habitat preference of other Mediterranean closely related cyprinodontiform, like \u003cem\u003eValencia hispanica\u003c/em\u003e, which occupy consistent freshwater environments and follow a less physiologically demanding, but potentially less versatile strategy [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, despite its broad salinity tolerance, \u003cem\u003eA. iberus\u003c/em\u003e exhibits very limited natural dispersal capacity across open sea, a constraint that has likely reinforced the historical isolation among lineages. These historical adaptations underscore the resilience of the species and highlight the evolutionary pressures that have shaped its current distribution and genetic diversity.\u003c/p\u003e\u003cp\u003eThe three major genetic lineages originally described by Doadrio et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], Catalonia, Levantine, and Murcian, have since been refined, with the Levantine clade subdivided into the Southern Catalonia lineage (Ebro River Delta populations, localities 18\u0026ndash;21) and the Levantine lineage sensu stricto (represented by the Albuixech population, locality 23) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our mitochondrial haplotype network, phylogenomic and population structure analyses confirmed the presence of these four main genetic lineages: Northern Catalonia, Southern Catalonia, Levantine, and Murcian; and revealed genetically intermediate and admixed populations between the first two. Using the mentioned genomic approaches, we distinguished populations of natural origin from those likely resulting from translocations. For example, some populations within the Southern Catalonia lineage (localities 10, 13, and 14) show introgression from the Northern Catalonia lineage, whereas the Prat de Llobregat (localities 7 and 8) and Salou (Tributaris\u0026ndash;S\u0026egrave;quia Major, locality 12) populations likely have a natural origin (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S1). In contrast, other populations deviated from the expected geographic genetic gradient, clustering instead with distant populations and sharing their ancestry, migration patterns, and heterozygosity levels; this was the case for most localities 9\u0026ndash;17, excluding aforementioned locality 12. These results, integrated with historical and institutional records, provide a detailed framework, expanded in the Supplementary Results \u0026amp; Discussion, for clarifying the origin of each population and guiding lineage-specific conservation actions.\u003c/p\u003e\u003cp\u003eThe results for the populations located outside the Catalonia region are consistent with previous studies. Both phylogenetic relationships and PCA clustering (and the haplotype network) aligned with the species\u0026rsquo; known geographic gradient (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, S1) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The StructF4 results identified the Cabanes population (locality 22) as admixed between the Levantine, Northern Catalonia, and Southern Catalonia lineages. While previous studies classified this population as admixed between the Levantine and Southern Catalonia lineages, the current result may reflect the limited sampling in the dataset (only one individual was included and the absence of intermediate populations). Still, the phylogenetic tree places the Cabanes population (locality 22) as a sister branch to the Catalan lineages (Northern and Southern Catalonia) and the Levantine lineage, supporting previous findings. Similarly, the Murcian lineage populations clustered together, despite being previously recognized as distinct conservation units [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The Adra population (locality 28), which used to represent the southernmost population of the species\u0026rsquo; distribution, exhibits genetic similarity to the Albuixech population in Valencia (Levantine lineage, locality 23). These distinctive characteristics, recently elucidated by Nester et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and confirmed in this study, support the hypothesis of a translocation in the 1980s from Albuixech, a population that has since undergone extensive captive breeding, effectively becoming a genetic refuge in captivity. Similarly, the Mollet del Vall\u0026egrave;s population (locality 3) in the suburbs of Barcelona appears to have originated from the same source (Levantine lineage).\u003c/p\u003e\u003cp\u003eMigration analyses, restricted to natural populations to avoid translocation bias (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), revealed directional gene flow from the Northern Catalonia lineage to the south, with no evidence of incoming migration. These patterns likely reflect historical (multi-generational) gene flow, rather than current, ongoing migration. The absence of significant recent inflow toward the Northern Catalonia lineage suggests long-term isolation, which may have been reinforced by oceanographic and geographic barriers. Along the Levantine coast, sediment accumulation from the Ebro River has created shallow waters that could facilitate dispersal, particularly under the climatic conditions described, whereas the Northern Catalonia coast is characterized by much deeper waters, potentially acting as a barrier to dispersal. Furthermore, prevailing Mediterranean currents in this Levantine region generally flow southward, possibly enhancing past dispersal in that direction, although seasonal northward currents may occur locally during summer. The current absence of populations between the Barcelona area (Prat de Llobregat, localities 7\u0026ndash;8) and the Girona populations of Northern Catalonia (localities 1\u0026ndash;2), a gap of roughly 70 km, may indicate historical local extinctions that further reduced connectivity. In contrast, high levels of interpopulation exchange were observed between the Ebro River Delta (localities 18\u0026ndash;21) and Tarragona (localities 12, 15, 16 and 17), as well as with the Prat de Llobregat (localities 7\u0026ndash;8), suggesting that historical connectivity existed among central-to-southern Catalan populations despite geographic separation.\u003c/p\u003e\u003cp\u003eThe overall levels of genomic diversity appear relatively high for an Endangered species (ranging between 1,000 and 3,000 SNPs/Mbp). By comparison, the Pyrenean desman (\u003cem\u003eGalemys pyrenaicus\u003c/em\u003e), Tasmanian devil (\u003cem\u003eSarcophilus harrisii\u003c/em\u003e), and Sumatran orangutan (\u003cem\u003ePongo abelii\u003c/em\u003e) have reported heterozygosity values as low as 12, 320, and 1,200 SNPs/Mbp, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The Spanish toothcarp has long been recognized for its high genetic variability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, populations from the Northern Catalonia lineage (localities 1\u0026ndash;2) displayed the lowest levels of heterozygosity, consistent with a likely southern origin of the species, as peripheral populations at the northern distribution limit often harbor reduced genetic diversity, and the highest ROH proportions, suggesting a decline in genetic diversity due to repeated bottlenecks and increased susceptibility to genetic drift. Conversely, introduced populations in the Barcelona province (such as localities 3, 4, and 5) showed high heterozygosity and low ROH levels. While these metrics may suggest good genetic health, they are likely artifacts of recent admixture, particularly in Rub\u0026iacute; and Sant Cugat (localities 4 and 5, respectively), which show genetic signatures from the Southern Catalonia lineage. Other populations showing higher percentages of ROHs are localities 7 and 8, which were reintroduced from an ex-situ breeding program (currently locality 6). Besides presenting a larger amount of ROHs, it is notable that around 15% of the genome is in long stretches of homozygosity, an indicator of recent inbreeding loops likely caused by the captive breeding program [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe demographic history analysis revealed a persistent decline Ne size through the Pleistocene, particularly in the Northern Catalonia lineage. In contrast, the Southern Catalonia lineage also experienced demographic contractions, but these were not continuous, with a period of recovery before a more recent decline. Although PSMC results cannot directly infer dispersal, the sustained low Ne in the north is consistent with long-term isolation, as discussed in the migration section, where no evidence of recent incoming gene flow was detected. This isolation likely reduced opportunities for replenishment of genetic diversity, increasing the susceptibility of Northern Catalonia populations to genetic drift and inbreeding, as reflected in their higher frequencies and longer runs of homozygosity. Despite these demographic constraints, the species exhibits traits that may enhance its resilience, such as high reproductive output, rapid sexual maturation, and fast growth rates [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Supporting this, ROH patterns in southernmost populations (excluding locality 24) and, to a lesser extent, in Southern Catalonia indicate relatively good genomic health. ROHs in these groups are predominantly short, covering 20\u0026ndash;25% of the genome, which is consistent with ancient, rather than recent, bottlenecks. In contrast, long ROHs are mainly observed in Northern Catalonia, consistent with smaller population sizes and mating between closely related individuals.\u003c/p\u003e\u003cp\u003eThe role of anthropogenic translocations (whether intentional or accidental) is of particular concern, as they can disrupt natural evolutionary trajectories and erode locally adapted population structures. This is especially problematic for \u003cem\u003eAphanius iberus\u003c/em\u003e, a species with well-defined genetic lineages and likely local adaptations critical for survival. The translocation of non-native individuals may dilute these adaptations, thereby reducing fitness and resilience. Habitat transformation driven by agricultural expansion and coastal development further threatens population integrity. In highly anthropized environments, such as the irrigation ponds inhabited by the Sax population (locality 24), selective pressures differ markedly from those in natural habitats, potentially driving divergent evolutionary trajectories. Peripheral populations, such as those in Northern Catalonia (localities 1\u0026ndash;2), face additional challenges due to their position at the range margin, where vulnerability is heightened. Conversely, our results suggest that some populations from the Levantine and Murcian regions may be better equipped to cope with rising temperatures and climatic variability. Nonetheless, a comprehensive conservation strategy must consider all populations, especially those in the north, to safeguard the species\u0026rsquo; long-term resilience and genetic diversity. In light of our findings, the integration of high-resolution genomic tools emerges as essential to detect hidden admixture, resolve the origins of uncertain populations, and design targeted, locally adapted management strategies. Such approaches provide a robust framework for addressing historical uncertainties and ensuring the preservation of \u003cem\u003eA. iberus\u003c/em\u003e in the face of ongoing environmental change.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eSample Collection, DNA Extraction, and Mitochondrial Data Screening\u003c/h2\u003e\u003cp\u003eTissue samples were obtained by collecting caudal fin clips, following standard protocols and in coordination with the relevant authorities. Adult individuals of \u003cem\u003eA. iberus\u003c/em\u003e (average standard length 2\u0026ndash;4 cm; body weight 0.5\u0026ndash;1 g) were euthanized by immersion in an overdose of buffered tricaine methanesulfonate (MS-222, 0.1%), in accordance with internationally approved protocols for small cyprinodontiform fishes. All procedures were performed by qualified personnel under the responsibility of the competent authorities (Consorci del Delta del Llobregat, Parc Natural del Delta de l\u0026rsquo;Ebre, and Departament de Recerca i Universitats de la Generalitat de Catalunya), with collection and handling authorized within the framework of the research project SGR-00420.\u003c/p\u003e\u003cp\u003eThe study protocol and methods were approved by the Animal Experimentation Ethics Committee of the National Museum of Natural Sciences (CEEA-MNCN; Spanish Animal Experimentation Research Centre no. ES280790000189), in strict accordance with current Spanish law (RD53/2013), transposed from European Union Directive 2010/63/EU (art. 2, 5f). All experimental protocols were conducted in accordance with relevant guidelines and regulations and are reported in compliance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Humane endpoints consisted of monitoring for the immediate loss of opercular movement and reflexes after immersion in the anesthetic solution, thereby confirming the effectiveness of euthanasia and safeguarding animal welfare.\u003c/p\u003e\u003cp\u003eFin clips were stored in 95% ethanol and deposited in a \u0026minus;\u0026thinsp;80\u0026deg;C freezer until processing. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer\u0026rsquo;s specifications. DNA quantification was carried out through fluorometry using Qubit Broad Range (Thermofisher). Suitable DNA samples were then selected for sequencing. Detailed information about samples and locations can be found in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eIn order to account for the full genetic diversity present in the Catalonia region (Northern and Southern Catalonia lineages), located along the northeastern coast of the Iberian Peninsula, we initially sequenced the cytochrome b gene for all 174 collected individuals using the primers GluF and ThR [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The primer sequences and PCR conditions are detailed in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e. Newly generated sequences were supplemented with an additional 192 sequences from Doadrio research group (obtained from NCBI GenBank corresponding to 2001 and 2015) obtained from GenBank (Sample information and GenBank accession numbers can be found in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These sequences were aligned using MAFFT implemented in Geneious Prime v.2023.0.4. The resulting alignments were used to construct a haplotype network using the TCS Network Builder [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], with the \"gap\" parameter set to missing. The resulting graph file was visualized using TCSbu [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe study area was expanded to include seven additional samples of \u003cem\u003eA. iberus\u003c/em\u003e from across the species' entire distribution range, ensuring representation of all known four genetic lineages previously identified, namely, Levantine, and Murcian lineages. Additionally, a sample of sister species \u003cem\u003eAphanius baeticus\u003c/em\u003e (Doadrio, Carmona \u0026amp; Fern\u0026aacute;ndez-Delgado, 2002) was included as a closest related outgroup, while a sample of \u003cem\u003eAnatolichthys anatoliae\u003c/em\u003e (Leidenfrost, 1912) was incorporated as a more distantly outgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eData generation and processing:\u003c/h3\u003e\n\u003cp\u003eHigh quality DNA samples were then sent for Illumina library preparation and sequencing on a NovaSeq X Plus (PE150) targeting at least 10X coverage for 42 individuals and 15X for four samples (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRaw reads were filtered, and adapters were removed using fastp [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] with a minimum base quality score of 30, activating automatic adapter detection for paired-end sequencing and trimming polyG/X tails. To assure the quality of the data and correct trimming, sequences were checked with FastQC v.0.20.0 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Filtered reads were mapped against the reference genome of \u003cem\u003eAphanius iberus\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] with bwa-mem v0.7.17 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and sorted using Samtools v1.9 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Duplicate reads were removed using Picard [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and reads with a mapping quality below 30 were discarded using Samtools view. SNPcalling was done using Haplotypecaller from GATK [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] with BP_resolution. Output files were combined using CombineGVCFs, and genotyping was carried out with GenotypeGVCFs from GATK [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt this stage, we generated two different datasets: Dataset 1, containing all 46 individuals of \u003cem\u003eAphanius iberus\u003c/em\u003e along with the two outgroups, \u003cem\u003eAphanius baeticus\u003c/em\u003e and \u003cem\u003eAnatolichthys anatoliae\u003c/em\u003e; and Dataset 2, containing exclusively the 44 individuals of \u003cem\u003eAphanius iberus.\u003c/em\u003e Datasets are detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Coverage was calculated and is detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The four samples sequenced at higher coverage were downsampled to a similar coverage to the other samples using Picard [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] for inclusion in Datasets 1 and 2.\u003c/p\u003e\u003cp\u003eThe reference genome was scanned for repetitive regions, which were subsequently removed. Datasets 1 and 2 were filtered to exclude variants matching at least one of the following criteria: Quality by Depth (QD)\u0026thinsp;\u0026lt;\u0026thinsp;10.0, Mapping Quality (MQ)\u0026thinsp;\u0026lt;\u0026thinsp;55.0, Fisher Strand test (FS)\u0026thinsp;\u0026gt;\u0026thinsp;50.0, StrandOddsRatio (SOR)\u0026thinsp;\u0026gt;\u0026thinsp;5.0, MQRankSum \u0026lt; -5.0 \u0026amp;\u0026amp; MQRankSum\u0026thinsp;\u0026gt;\u0026thinsp;5.0, and ReadPosRankSum \u0026lt; -5.0 \u0026amp;\u0026amp; ReadPosRankSum\u0026thinsp;\u0026gt;\u0026thinsp;5.0. Further filtering of genotypes was performed using VCFtools, selecting variants with a quality score above 30, retaining only biallelic SNPs, removing indels, a minor allele frequency (MAF) of 0.001, and no missing data. To account for linkage disequilibrium, a secondary dataset was generated for each of the Datasets 1 and 2 retaining only unlinked SNPs (uSNPS) using bcftools with r2\u0026thinsp;\u0026le;\u0026thinsp;to 0.5.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePopulation structure and phylogenomic analyses\u003c/h2\u003e\u003cp\u003eTo gain a preliminary insight into the variability among the samples, a Principal Component Analysis (PCA) was conducted using Plink v1.9 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] on \u003cem\u003eA. iberus\u003c/em\u003e individuals (Dataset 2, unlinked SNPs). Additionally, we conducted a population structure analysis using Structf4 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], considering 2\u0026ndash;7 ancestral components and 50,000,000 iterations for both the first and second MCMC chains. Results were visualized with R v.4.3.2 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. These analyses were performed on Dataset 1 and 2 (unlinked SNPs), which included the two outgroups to determine if some lineages were external.\u003c/p\u003e\u003cp\u003eTo investigate phylogenomic relationships among populations, we constructed a maximum-likelihood (ML) phylogeny. For this, we filtered Dataset 1 to exclude missing data and randomly selected one allele per genomic site (i.e., each SNP position in the alignment), resulting in a total of 5,808,219 SNPs. The dataset was then divided into non-overlapping 100 kbp windows, and each window was analyzed independently using IQ-TREE2 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] with the GTR\u0026thinsp;+\u0026thinsp;ASC model and 1,000 bootstrap replicates. The best-scoring trees from each window were subsequently combined using ASTRAL v5.7.8 [61].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eGenome-wide diversity\u003c/h2\u003e\u003cp\u003eWe estimated genome-wide heterozygosity for each individual in Dataset 1 (including both genera \u003cem\u003eAphanius\u003c/em\u003e and \u003cem\u003eAnatolichthys\u003c/em\u003e; unfiltered). To do so, we divided the reference genome into non-overlapping 100 kb windows. Within each window, heterozygous sites were counted using vcfhetcount from vcflib [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. A site was considered callable if it was not an indel and had a mapping quality score above 30. Results were plot in R v.4.3.2 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRuns of Homozygosity (ROHs) were calculated for each individual of \u003cem\u003eA. iberus\u003c/em\u003e based on the density of heterozygous sites in the genome using the implemented Hidden Markov Model (HMM) in the bcftools roh function with the default parameter \u0026ndash;AF-dflt 0.4 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e63\u003c/span\u003e], on the filtered Dataset 2. \u003cem\u003eA. baeticus\u003c/em\u003e and \u003cem\u003eA. anatoliae\u003c/em\u003e were excluded from this analysis to prevent them from influencing the results by causing noise, as the patterns of homozygosity can vary considerably between species. We kept ROHs with a Phred Score of at least 70 and with a minimum length of 100 Kbp. ROHs are considered long when above 1Mbp, medium between 500 Kbps and 1 Mbp and short below 100 Kbps. Visualization for all analyses was carried out in Rv.4.3.2 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] using ggplot2.61 [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMigration\u003c/h2\u003e\u003cp\u003eWe estimated gene flow among different population groups within the Northern and Southern Catalonia lineages. Populations were grouped according to the genetic clusters retrieved in the PCA and, in some cases, further subdivided based on their geographic location. Because a minimum number of individuals per group is required to run the analysis, it was conducted only within Catalonian populations (localities 1\u0026ndash;21). This approach also provides valuable insight into the status and potential origin of these populations. To do this, we generated a genepop file from Dataset 2 using the vcf2genepop function in the R package gwscaR [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Gene flow was then estimated with the function divMigrate in the R package diveRsity [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. We calculated the number of effective migrants per generation (Nm), Jost\u0026rsquo;s D (D), and Nei\u0026rsquo;s GST (GST). The resulting matrix was plotted with the R package qgraph [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], disregarding edges below 0.22.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eDemographic history\u003c/h2\u003e\u003cp\u003eWe inferred the demographic history of the species using Pairwise Sequential Markovian Coalescent (PSMC) [68]. This analysis was run on two individuals of \u003cem\u003eA. iberus\u003c/em\u003e (one from the Northern and one from the Southern Catalonia lineages) and one \u003cem\u003eA. baeticus\u003c/em\u003e, all three sequenced aiming at a 15X coverage. Heterozygous positions were obtained from bam files with Samtools v1.9 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and the data was filtered to assure a mapping and base quality\u0026thinsp;\u0026gt;\u0026thinsp;30. A generation time (θ) of 0.5 and a mutation rate of 3.5 x 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e per bp per generation were assumed [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Ten bootstrap replicates were performed on each sample.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements and funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Spanish Ministry of Science and Innovation and the State Agency for Research for their financial support through Project Aphanius (PID2019-103936GB-C22) and Project Saltfish (PID2023-146173NB-C22), both awarded to ID. We are grateful to Miriam Casal, Pilar Risue\u0026ntilde;o, \u003cem\u003eEquipo de Ectotermos\u003c/em\u003e from the Zoo of Barcelona and Juan Miguel Galindo Galindo from \u003cem\u003eJunta de Andaluc\u0026iacute;a\u003c/em\u003e for their effort in sample collection, and to Pablo Librado (Institute of Evolutionary Biology, CSIC-UPF) for his assistance with the Structf4 analysis. We also wish to thank Lourdes Alcaraz for her laboratory support. Finally, we acknowledge CESGA, the Supercomputing Center of Galicia, for providing computational resources, and the Universidad Complutense de Madrid (UCM) for hosting AL-S and TLN within the \u003cem\u003eDoctorado en Biolog\u0026iacute;a\u003c/em\u003e program.\u003c/p\u003e\n\u003cp\u003eThis project was supported by the \u003cem\u003eFundaci\u0026oacute; Barcelona Zoo\u003c/em\u003e and the \u003cem\u003eDepartament de Medi Ambient i Sostenibilitat\u003c/em\u003e, through grant SGR-00420 from the \u003cem\u003eDepartament de Recerca i Universitats de la Generalitat de Catalunya\u003c/em\u003e to SC. ME is funded by an FPI fellowship from the \u003cem\u003eMinisterio de Ciencia, Innovaci\u0026oacute;n y Universidades,\u0026nbsp;\u003c/em\u003eSpain (PRE2022-101473). AL-S is funded by an FPI fellowship from the same governmental institution (PRE2020-092988). GM-R was also funded by an FPI fellowship (PRE2019-088729). BB-C and TLN are supported by FPU fellowships (FPU2018/04742 and FPU2020/04500) both from the \u003cem\u003eMinisterio de Ciencia, Innovaci\u0026oacute;n y Universidades,\u0026nbsp;\u003c/em\u003eSpain. AT is supported by the \u0026ldquo;la Caixa\u0026rdquo; doctoral fellowship programme (LCF/BQ/DR20/11790007). ST is funded by an AGAUR-FI Joan Or\u0026oacute; fellowship from the \u003cem\u003eDepartament de Recerca i Universitats de la Generalitat de Catalunya\u003c/em\u003e and the European Social Fund Plus (2024_FI-1_01035).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to conceive and design the study. A.L.-S., S.P., T.L.N., N.F., J.X., J.R.-O., E.d.R. and I.D. collected the field samples. M.E., A.L.-S., S.P. and T.L.N. carried out laboratory work. M.E., A.L.-S., G.M-R., S.P., A.T., B.B.-C. and S.T. carried out data analysis and all authors performed the interpretation. M.E., A.L.-S., G.M.-R., S.P., A.T. and B.B.-C. produced the workflow of all scripts. M.E. and A.L.-S. wrote the first draft of the manuscript, based on ideas conceived also with G.M-R., S.P., A.T., B.B.-C., T.L.N., S.T., I.D. and S.C. All authors discussed ideas and contributed with valuable scientific interpretations and carefully reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in NCBI/GenBank repository under BioProject PRJNA1320828, with accession numbers provided in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003ehttps://dataview.ncbi.nlm.nih.gov/object/PRJNA1320828?reviewer=mi56tduse3pe847n42t926s3e3\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhan, S. et al. 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Ecolog\u0026iacute;a y conservaci\u0026oacute;n del fartet (\u003cem\u003eLebias ibera\u003c/em\u003e) en las marismas del Ampurd\u0026aacute;n (Catalu\u0026ntilde;a). En (ed Planelles-Gomis, M.) Peces ciprinod\u0026oacute;ntidos Ib\u0026eacute;ricos: Fartet y Samaruc, 151\u0026ndash;161. Generalitat Valenciana, Conselleria de Medio Ambiente. (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDoadrio, I., Perea, S., Garz\u0026oacute;n-Heydt, P. \u0026amp; Gonz\u0026aacute;lez, L. \u003cem\u003eIctiofauna continental espa\u0026ntilde;ola. Bases para su seguimiento\u003c/em\u003e (Direcci\u0026oacute;n General de Medio Natural y Pol\u0026iacute;tica Forestal, 2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerea, S., Vukić, J., Šanda, R. \u0026amp; Doadrio, I. 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Recurrent evolution of small body size and loss of the sword ornament in Northern swordtail fish. \u003cem\u003eEvolution\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/evolut/qpae124\u003c/span\u003e\u003cspan address=\"10.1093/evolut/qpae124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Genomics, translocations, population structure, genetic diversity, Aphanius iberus","lastPublishedDoi":"10.21203/rs.3.rs-7494783/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7494783/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the genetic structure and evolutionary history of endangered species is crucial for effective conservation planning. The Spanish toothcarp, \u003cem\u003eAphanius iberus\u003c/em\u003e (Valenciennes, 1846), an endemic and euryhaline fish of the Mediterranean coast of the Iberian Peninsula, is currently threatened by habitat destruction, climate change, and anthropogenic translocations. Here, we employed genome-wide SNP data from medium- to low-coverage whole genomes to investigate the population structure, genetic diversity, and demographic history of \u003cem\u003eA. iberus\u003c/em\u003e, especially focussing on its northern distribution, which has remained poorly studied.\u003c/p\u003e\u003cp\u003eOur analyses revealed a well-structured genetic pattern across the species\u0026rsquo; range, with four main genetic lineages: Northern Catalonia, Southern Catalonia, Levantine, and Murcian. Genomic indicators, including heterozygosity, ROHs, and migration analyses, suggest higher inbreeding and genetic erosion in the northernmost populations, likely due to long-term isolation, whereas southern populations maintain healthier genetic diversity. We also identified several admixed and potentially introduced populations.\u003c/p\u003e\u003cp\u003eThese findings underscore the importance of accurately determining the origin of populations before any translocation or reintroduction, as misguided management may compromise the genetic integrity of natural lineages. This work provides essential genomic insights to guide conservation strategies and emphasizes the need for lineage-aware management of endemic species like \u003cem\u003eA. iberus\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Saving the locals: a conservation genomics approach to the Endangered Spanish Toothcarp, Aphanius iberus (Valenciennes, 1846)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 10:09:28","doi":"10.21203/rs.3.rs-7494783/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-10T10:00:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T14:53:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T13:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196403279278625956314884520182609991723","date":"2025-10-13T08:41:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136027470322226582845401892020607962602","date":"2025-10-13T07:04:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302500240441594569851976964673371542297","date":"2025-10-09T09:13:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T08:24:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-07T08:03:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-23T15:34:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T08:17:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-19T08:13:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6e647f97-8070-4550-b739-b76fa1c2317a","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55291824,"name":"Biological sciences/Ecology"},{"id":55291825,"name":"Earth and environmental sciences/Ecology"},{"id":55291826,"name":"Biological sciences/Evolution"},{"id":55291827,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2025-12-15T16:00:57+00:00","versionOfRecord":{"articleIdentity":"rs-7494783","link":"https://doi.org/10.1038/s41598-025-31909-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-11 15:57:18","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-09-26 10:09:28","video":"","vorDoi":"10.1038/s41598-025-31909-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31909-y","workflowStages":[]},"version":"v1","identity":"rs-7494783","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7494783","identity":"rs-7494783","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}