Salinity is a major ecological driver in aquatic environments and strongly influences the physiology, distribution, and survival of fish species. While many fishes are restricted to narrow salinity ranges, euryhaline species can tolerate large osmotic fluctuations despite the substantial physiological adjustments required. The Spanish toothcarp, Aphanius iberus, an endemic Mediterranean killifish, is one of such exceptional species capable of inhabiting environments ranging from freshwater to hypersaline systems. Despite this remarkable resilience, the molecular mechanisms underlying salinity tolerance and osmoregulatory plasticity in this species remain poorly understood. We used RNA sequencing to analyze gill and gastrointestinal tract transcriptomes from individuals belonging to four wild populations distributed along a natural salinity gradient: freshwater-like low-salinity habitats (0.76 PSU); brackish habitats (4.8 and 7.4 PSU at sampling); and hypersaline lagoon-associated habitats (52 PSU). Differential gene expression analyses revealed strong tissue-specific patterns and salinity-associated differences among population contrasts. A substantially higher number of differentially expressed genes was detected in the gills, where genes associated with ion transport, cytoskeletal remodeling, and energy metabolism varied across contrasting osmotic environments, consistent with the central role of this tissue in osmoregulation. In the gastrointestinal tract, pathways related to lipid and carbohydrate metabolism were particularly represented, especially in brackish population contrasts. In addition, genes previously associated with osmotic stress, cellular remodeling and genome regulation, including ATPases, histones and transposable element-related sequences, showed significant expression differences across populations. These findings offer novel insights into the molecular architecture of salinity tolerance in euryhaline fishes, highlighting coordinated transcriptional responses across tissues involved in ion regulation and metabolic adjustment and improving our understanding of how euryhaline fishes cope with extreme and fluctuating osmotic environments. From a conservation perspective, identifying the molecular basis of this physiological plasticity contributes to the management of this highly threatened endemic species and the dynamic coastal habitats it inhabits.

Freshwater salinization is an emerging threat impacting approximately one-third of the world's freshwater bodies. However, the salinity tolerance of many inland fishes remains understudied, despite being a crucial factor in determining species distributions and fitness. We updated and analysed a comprehensive global database of experimental salinity tolerance, compiled from 128 sources, to investigate geographical and taxonomic biases in research on the salinity tolerance of inland fishes. Additionally, we examined how salinity tolerance relates to fish traits and how experimental data align with field-reported salinity levels. Experimental data on salinity tolerance was available for < 1% of the world's inland fishes, with data severely lacking from diverse tropical regions and species-rich orders such as Characiformes and Siluriformes. Most salinity tolerance studies focused on relatively large and widely distributed species from North America, Europe and Oceania and certain orders such as Acipenseriformes, Centrarchiformes, Cyprinodontiformes or Lepisosteiformes. Our results showed that fish salinity tolerance was strongly associated with fish habitat type, taxonomy, aspects of morphology, migratory behaviour and the experimental approach used (direct vs. gradual). Our results also showed significant, though sometimes weak relationships between field-reported salinity levels and experimental estimates. Improved understanding of fish salinity tolerance will be essential for assessing impacts of freshwater salinization and forecasting the invasion potential of non-native species.

Geological and climatic events frequently represent the primary explanations to describe evolutionary processes among species.Freshwater fishes have been used previously as models to uncover evolutionary and historical biogeographic patterns in central Mexico,hydrologic systems and biotas.Xenotoca variata(Cyprinodontiformes:Goodeidae)is one of the most widely distributed species across central Mexico.The species represents a highly dimorphic and sexually selective species.In this study,the phylogenetic and phylogeographic patterns of populations of X.variata,using one mitochondrial locus(cytb)and three nuclear loci(S7,RHO,and RAG1),were described in order to understand the evolutionary history of the species throughout its distributional range.Two well-defined and highly supported clades were recovered with all analyses and genes studied,with an estimated divergence time of ca.2.42 Mya,corroborating the existence of an independent evolutionary unit in Cuitzeo Lake and its recognition as a putative new species.Also,a phylogeographic structure in two genes was found within the widely distributed clades.The role played by climate change events and geological history as well as the possible influence of reproductive traits in the phylogeographic pattern of the species are discussed.

Invasive non-native species are an important cause of biodiversity loss, particularly in fresh waters. The mosquitofish Gambusia holbrooki are among the world’s worst invasive species: they have caused extirpations of native species and are known to sometimes cause trophic cascades and ecosystem effects. This invasive species is also known to impact threatened fishes such as the Spanish toothcarp (Aphanius iberus), which is endemic to Mediterranean Spain. However, it is unclear if the impact of mosquitofish on many fishes is more through resource competition, agonistic interactions or predation, and how often mosquitofish cause trophic cascades. To clarify these questions, we performed a 48-day mesocosm experiment in eutrophic conditions to test for interspecific effects and clarify the impact mechanism using six treatments: the two fish species alone each at two densities, and the two fish species mixed or separated with a net that prevented direct interactions among them. We observed clear fish treatment effects on several variables. At low initial fish densities, the population growth rate of mosquitofish was orders of magnitude greater than that of the Spanish toothcarp, likely contributing to its invasive success and ecological impact. At high fish densities, turbidity, chlorophyll a concentration and daytime dissolved oxygen percentage increased, whereas total phosphorus decreased; crucially, the trophic cascade caused by mosquitofish was stronger than that by toothcarp. The experiment also demonstrated that the interspecific effects of mosquitofish on toothcarp were more important than those of intraspecific competition. The invasive species produced effects on population growth rate, size structure, and fish condition (mass-length relationship) of toothcarp. Effects on population growth rate of toothcarp seem more caused by resource competition, whereas impacts on size structure and condition seem also caused by more direct interactions. The diversity of effects of mosquitofish underscores the difficulty of predicting the impact of invasive species. Our study further provides an approach to differentiate the effects of resource competition from other more direct ecological interactions and so to clarify the impact mechanism of aquatic invasive species.

BACKGROUND:The sequencing of non-model species has increased exponentially in recent years, largely due to the advent of novel sequencing technologies. In this study, we construct the Reference Genome of the Spanish toothcarp (Aphanius iberus (Valenciennes, 1846)), a renowned euryhaline fish species. This species is native to the marshes along the Mediterranean coast of Spain and has been threatened with extinction as a result of habitat modification caused by urbanization, agriculture, and its popularity among aquarium hobbyists since the mid-twentieth century. It is also one of the first Reference Genome for Euro-Asian species within the globally distributed order Cyprinodontiformes. Additionally, this effort aims to enhance our comprehension of the species' evolutionary ecology and history, particularly its remarkable adaptations that enable it to thrive in diverse and constantly changing inland aquatic environments. RESULTS:A hybrid assembly approach was employed, integrating PacBio long-read sequencing with Illumina short-read data. In addition to the assembly, an extensive functional annotation of the genome is provided by using AUGUSTUS, and two different approaches (InterProScan and Sma3s). The genome size (1.15 Gb) is consistent with that of the most closely related species, and its quality and completeness, as assessed with various methods, exceeded the suggested minimum thresholds, thus confirming the robustness of the assembly. When conducting an orthology analysis, it was observed that nearly all genes were grouped in orthogroups that included genes of genetically similar species. GO Term annotation revealed, among others, categories related with salinity regulation processes (ion transport, transmembrane transport, membrane related terms or calcium ion binding). CONCLUSIONS:The integration of genomic data with predicted genes presents future research opportunities across multiple disciplines, such as physiology, reproduction, disease, and opens up new avenues for future studies in comparative genomic studies. Of particular interest is the investigation of genes potentially associated with salinity adaptation, as identified in this study. Overall, this study contributes to the growing database of Reference Genomes, provides valuable information that enhances the knowledge within the order Cyprinodontiformes, and aids in improving the conservation status of threatened species by facilitating a better understanding of their behavior in nature and optimizing resource allocation towards their preservation.

Genomic data has revealed hybridisation is common in nature. Highly divergent allopatric species are often overlooked in our efforts to characterise the prevalence and consequences of hybridisation in natural systems, presumably because they are viewed as less likely to hybridise. Yet, such species are models to investigate later stages of the speciation continuum. Here, we study such a system-the Iberian chubs (Squalius spp.), a group of primary fish species with allopatric distributions across distinct environments and river catchments. Throughout their evolutionary history, the rivers inhabited by these species suffered quite dramatic changes, potentially allowing for multiple periods of isolation and secondary contact. To investigate if such a history left traces, we generated low-coverage whole-genome resequencing data for 125 individuals from eight Iberian chub species. Our results showed high levels of inter (0.44 ≤ FST ≤ 0.88) and intra-specific (0 ≤ FST ≤ 0.61) genetic differentiation. We uncovered four contrasting cases of potential hybridisation, spanning different geographical and time scales. First, we found evidence of ancient hybridisation on a species now inhabiting a whole river catchment. Second, we uncover recent hybridisation restricted to a localised stream, with introgression of nuclear and mitochondrial DNA. Third, we detected, at a regional scale, a case with nuclear introgression but no detectable mitochondrial DNA introgression. Finally, we uncover a case where two very distinct mitochondrial lineages persist at balanced frequencies in a putative hybrid population, despite no detectable nuclear introgression. Our findings suggest hybridisation and introgression led to assimilation or local exclusion of one or both parental species.

Hybridization - the successful reproductive cross between individuals from different species - is increasingly recognized as a relatively common phenomenon in nature, mostly due to the increasing availability of genomic data for many organisms and the development of sophisticated methods to detect past gene flow. As a result, evidence of past gene flow has been detected in the genomes of many species, allowing us to investigate the evolutionary forces that act upon hybrid genomes. Biological systems where multiple instances of hybridization have been detected at different time scales are especially useful for this, as they allow investigating the effects of selection and recombination at different time scales post-hybridization. The Iberian chubs ( Squalius spp. ) present an ideal study system for this. In this group of primary fish inhabiting the rivers of the Iberian Peninsula, multiple instances of past hybridization at different geographical and time scales were reported between different species. However, before we investigate the effects of selection and recombination, it is crucial that we date the hybridization events and quantify the contribution from each parental species. In this study, we infer the demographic history of two Iberian chub hybrid lineages, one resulting from a proposed ancient hybridization event ( S. pyrenaicus ) and another potentially much more recent (São Martinho). We generated whole genome resequencing data for 52 individuals from the two hybrid lineages and their putative parental populations. Our demographic modelling results confirm the hypothesis of ancient hybridization between S. carolitertii and S. tartessicus around 600,000-800,000 years ago, giving rise to the S. pyrenaicus lineage. We also confirm that S. carolitertii is the major parental, with S. tartessicus only contributing <10% to the S. pyrenaicus genome. Furthermore, we confirm the more recent hybridization between S. caetobrigu s and S. pyrenaicus (around 4500 years ago) in the São Martinho population. We observe similar contributions from both parental species (around 50%) to the genome of the individuals of this population. Overall, our results give us a deeper understanding of how and when these past gene flow events happened and the contributions of each parental species to the genomes of the individuals of the hybrid lineages. ### Competing Interest Statement The authors have declared no competing interest.

A new species, Squalius caetobrigus sp. nov., is described based on morphological and genetic traits. Squalius caetobrigus sp. nov. can be distinguished from other Squalius species through a combination of morphometric, meristic and genetic characteristics: 39-42 ((X) under bar =40.7; (X) over tilde =41) canaliculate scales on the lateral line; 6-7 ((X) under bar =7; (X) over tilde =7) scales above the lateral line; 3-4 ((X) under bar =3.1; (X) over tilde =3) scales below the lateral line; short preorbital distance HL/PrOL is 4.2-6.2 ((X) under bar =5); no contact between the fourth infraorbital bone and the preopercular; short dentary with a very developed coronoid process; the posterior process of the maxilla is long and thin as well as the lower branch of the pharyngeal bone; small pharyngeal plate of the pharyngeal bone and 5 autapomorphies in the mitochondrial cytochrome b gene. Squalius caetobrigus is restricted to the Sado River basin in southern Portugal.

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 both a single genetic marker (cytochrome b) and genome-wide SNP data from medium-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 higher genetic diversity. We also identified several admixed and potentially translocated 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 native 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.

The Neretva River drainage, a unique biogeographic unit within the Balkans, is a typical karstic river system with complex hydrological connections, hosting many endemic fishes, among which also the Neretva bleak Alburnus neretvae (Cypriniformes: Leuciscidae). It has one of the smallest distribution ranges among its congeners. Moreover, its habitat is especially susceptible to ongoing climate change and human induced alterations, thus the species may face a severe reduction and fragmentation of habitat in the near future. We present the first population genetic study of the Neretva bleak, assessing the current state of the species, based on analyses of mitochondrial gene cytochrome b of 89 individuals from 11 localities across the Neretva drainage, representing five populations. Our results show that the species is undergoing recent population expansion since the end of the Pleistocene and exhibits a high haplotype diversity at most localities. Our results further indicate the existence of genetic structure within this species; pronounced genetic differentiation was revealed between all populations apart from the pairs Trebižat–Ričina and Neretva–Trebišnjica. Although, based on our results, the species is currently in good condition, monitoring and conservation measures should be established with an emphasis on maintaining the genetic diversity within the species.

To date, the phylogeny of Alburnus, one of the most speciose western Palearctic freshwater fish genera, remains unresolved and its monophyly questioned. Here, we present a taxon-dense phylogeny of the genus and related genera, based on mitochondrial and nuclear DNA sequence data. A special focus is put on relationships between the Peri-Adriatic species. Our analyses confirmed the paraphyly of Alburnus with the retrieval of Leucaspius delineatus, Anaecypris punica and Anaecypris hispanica as three separate clades within it. This finding challenges the current taxonomic framework of the genus and highlights the need for a re-evaluation of its classification. The remaining three well-supported clades are currently assigned to Alburnus: Clade I from Levant and the Tigris and Euphrates basins, Clade V with shemayas and Clade VI comprising all remaining species: from Europe, Ponto-Caspian and Aral regions and Anatolia. Most relationships within Clade V and VI were not resolved likely due to rapid diversification in the late Pliocene and Pleistocene. Monophyly of the Peri-Adriatic group, suggested in previous works, was not confirmed. Moreover, Alburnus arborella did not form a monophyletic lineage and displayed extraordinary diversity both in its native and introduced range. Finally, new evidence for introductions of species were provided.

Cobitis maroccana, also known as the Moroccan loach, is an endemic freshwater fish found in the rivers of the Loukkos and Sebou basins in Morocco. In order to gain a better understanding of the genetic differentiation within C. maroccana from the Loukkos and Sebou populations, a comprehensive study was conducted, focusing on the mitochondrial cytochrome b gene. A total of 30 sequences of the complete cytochrome b (1140 bp) were obtained from C. maroccana specimens. Through a combination of phylogenetic analysis, phylogeographic assessments, as well as the estimation of the Fst index, the research outcomes revealed significant genetic differentiation between the two juxtaposed basin populations. Notably, each of these basins displayed unique and distinct haplotype groups within the C. maroccana specimens. These intriguing findings strongly suppose that the populations in the Loukkos and Sebou basins have become reproductively isolated from each other over time, indicating limited or no interbreeding between them.

The evolutionary potential of a species directly impacts its ability to survive in fluctuating environments. A fundamental goal in wildlife conservation is enhancing this potential since anthropogenic pressures and rapid climate change are shifting environments at an alarming rate. One way to increase a species’ adaptive potential is through the delineation of management units based on population genomic analyses. Such units consist of evolutionarily significant gene pools requiring immediate conservation action. The delineation of priority units is fundamental for species on the brink of extinction, a predicament shared by numerous killifish species worldwide which face human-driven habitat transformations leading to the destruction of functioning ecosystems. The Mediterranean coast of the Iberian Peninsula has undergone one of the greatest human-driven habitat transformations in Europe since the turn of the 20th century as a result of agricultural exploitation and urbanization, imposing novel environmental pressures on various aquatic organisms including killifish species, such as the endangered and endemic Spanish toothcarp (Aphanius iberus Valenciennes, 1846). In the present study, we performed a SNP-based genetic analysis to delineate management units, or Operational Conservation Units (OCUs) for A. iberus, sampling a total of 176 individuals from 18 sample locations and analyzing their genetic structure, diversity, levels of gene flow, and degrees of genetic differentiation. Overall, the populations were highly structured with low genetic diversity values. Little to no gene flow was detected and FST values were high, indicating a large degree of genetic differentiation between populations, most likely attributable to habitat fragmentation. The results of our genetic analyses suggested the recommendation of nine OCUs for A. iberus, which should be implemented immediately into recovery programs to enhance the conservation management of this species. Using A. iberus as a study model, our research exemplifies how to delineate conservation priorities pertinent to killifish species with limited dispersal opportunity as a result of disruptions in population connectivity.

Three new species, Cobitis almadae sp. nov., Cobitis atlantica sp. nov., and Cobitis mellaria sp. nov. are described on the basis of morphological and genetic traits. Cobitis almadae sp. nov. is restricted to the Sizandro Drainage in Portugal and can be distinguished from other Cobitis species through a combination of morphometric and genetic traits including large and low peduncle depth, lateral ethmoid (suborbital spine) well developed with long narrow mediocaudal, laterocaudal and mediorostral processes, an elongated and narrow frontoparietal fontanel and a wide third Gambetta’s zone sprinkled with numerous black spots. Furthermore, two autapomorphies are found within its mitochondrial cytochrome b gene. Cobitis atlantica sp. nov. inhabits the northern Atlantic rivers of the Iberian Peninsula from the Minho Drainage to the Alcoa Drainage and can be differentiated from other Cobitis species through a set of morphometric and genetic traits including short and high peduncle depth,a well-developed lateral ethmoid (suborbital spine) with short and wide mediocaudal, laterocaudal and mediorostral processes, wide frontoparietal fontanel, and developed ventral pigmentation in adult individuals. In females, the Gambetta’s fourth row has 10-16 blotches reaching the ventral pigmentation in the caudal region and the third Gambetta’s zone is narrow with black spots. Cobitis mellaria sp. nov inhabits the Valle Drainage in southern Spain and is distinguished from other Cobitis species through the following morphometric and genetic traits: low peduncle depth, lamina circularis with convex outer edge, lateral ethmoid (suborbital spine) with short laterocaudal process and large mediorostral process, elongated frontoparietal fontanel, no ventral pigmentation in adults. In females, Gambetta’s fourth row has 10-15 blotches. One autapomorphy is found within the mitochondrial cytochrome b gene of Cobitis mellaria sp. nov.

Biodiversity conservation entails not only the preservation of specific taxa but also genetic diversity. Despite the crucial role of molecular data in freshwater fish conservation management, there is a scarcity of information regarding the genetic diversity of Luciobarbus Heckel, 1843 (Actinopterygii, Cyprinidae) populations in the Aral system. Therefore, the primary aim of this study was to provide genetic information on two native species of the Luciobarbus genus found in the Aral system: L. conocephalus (Kessler, 1872) and L. brachycephalus (Kessler, 1872). These species, like many others in the Aral system, confront the imminent threat of extinction due to system alterations. However, genetic studies on these species at the nuclear level are challenging because Luciobarbus is an allotetraploid genus. Consequently, genetic investigations thus far have focused mainly on sequencing mitochondrial genes due to their haploid nature. This study has successfully developed fifteen new polymorphic microsatellite loci, which can prove to be valuable for population genetics, conservation, and other pertinent research on these species.

Livestock guarding dogs (LGDs) have been used to protect livestock for millennia. While previous works suggested a single origin of modern LGDs, the degree and source of shared ancestry have not been tested. To address this, we generated genome-wide SNP data from 304 LGDs and combined it with public genomic data from 2,183 modern and 22 ancient dogs. Our findings reveal shared ancestry and extensive gene flow among modern LGD breeds which we attribute to historical livestock migrations. Additionally, admixture between LGDs and free-ranging dogs argues against reproductive isolation as a core mechanism for maintaining the specialized skills of LGDs. Finally, we identify two lineages within modern LGDs and uncover multiple ancestries tracing back to distinct Eurasian ancient dogs, concordant with the absence of a single ancestor. Overall, our work explores the complex evolutionary history of LGDs, offering valuable insights into how human and livestock co-migrations shaped this functional group.

Se describen dos nuevas especies, Squalius gaditanus sp. nov. y Squalius tartessicus sp. nov. sobre la base de caracteres morfológicos y genéticos. Squalius gaditanus está restringida a las cuencas de Barbate, Jara y Miel en la provincia de Cádiz (sur de España). Squalius gaditanus sp. nov. se puede distinguir de otras especies del género Squalius, de la Península Ibérica, a través de una combinación de caracteres morfométricos, merísticos y genéticos: 36-‍40 (x̄=38) escamas canaliculadas en la línea lateral; 6-‍7 (x̄=6.7) escamas por encima de la línea lateral; 2-‍3 (x̄=2,8) escamas debajo de la línea lateral; vértebras 37-‍39 (x̄=38); en los ejemplares adultos el segundo infraorbitario es más estrecho que el tercero; maxilar con su proceso anterior poco puntiagudo; proceso posterior del maxilar largo y delgado; la rama inferior del hueso faríngeo es corta y robusta; placa faríngea del basioccipital redondeada y dos autapomorfias en el gen mitocondrial citocromo b. Squalius tartessicus sp. nov. vive en las cuencas de los ríos Almargem, Gilao, Odiel, Guadiana, Guadalquivir, Guadalete, Guadalhorce, Vélez, Guadalfeo y Segura en el sur de la Península Ibérica. Squalius tartessicus sp. nov. se puede distinguir de otras especies del género Squalius, de la Península Ibérica,a través de una combinación de caracteres morfométricos, merísticos y genéticos; 37-‍41 (x̄=38.8), escamas canaliculadas en la línea lateral; 6-‍7 (x̄=7) escamas por encima de la línea lateral; 2-‍3 (x̄=2.9) escamas por debajo de la línea lateral; 37-‍39 (x̄=38) número de vértebras; en los adultos infraorbitarios excepcionalmente anchos; maxilar con la apófisis anterior puntiaguda; apófisis posterior del maxilar larga y delgada; la apófisis inferior del hueso faríngeo es corta y robusta; placa faríngea del basioccipital de forma triangular; lámina posterior de cleitro extendida.

Nemacheilid fishes in the genus Turcinoemacheilus are physically small members of the ichthyofauna communities of high-altitude and mountainous freshwater ecosystems. They are all distributed in Western Asia apart from a single species, described in the Himalayas. They are usually very similar in appearance, which complicates their proper identification and/or description. This is why it is important to use multidisciplinary and integrative taxonomical approaches in order to study their true diversity. In this study, three new species of Turcinoemacheilus are described from Iran, raising the total number of valid species to nine. Turcinoemacheilus ansari new species, is distinguished by the anus being situated behind the midpoint of the pelvic-fin and anal-fin origins and the short anal-fin base length. Turcinoemacheilus christofferi new species, differs by the anus being situated behind the midpoint of the pelvic-fin and anal-fin origins, with a complete lateral line reaching to the anterior part of the caudal fin. Turcinoemacheilus moghbeli new species, is distinguished by the anus being situated at or in front of the midpoint of the pelvic-fin and anal-fin origins, with a great pre-pelvic distance and a caudal peduncle length 1.5–2.3 times its length. In Western Asia, all Turcinoemacheilus species are well separated by molecular characters, showing between 3.6 and 14.1% uncorrected p genetic distances in the COI barcode region. This work shows the importance of studying the hidden diversity of under-sampled and understudied groups of organisms to have a clear image of true biodiversity in order to effectively conserve and protect it.

We analyzed two morpho-species, Astyanax aeneus and Astyanax caballeroi , coexisting in Lake Catemaco, with ecological differences previously associated with different trophic niches. While A. aeneus is a widely distributed species, A. caballeroi is endemic to Lake Catemaco. Due to the contrasting morphology between these two sympatric species, they were originally assigned to different genera, but little is known about their genetic differentiation. We hypothesized that these two species, which present differentiation in morphology and in trophic niches, co-occur in concomitance with assortative mating and form distinct genetic clusters. We tested this prediction by typing a set of 12 microsatellites in 348 individuals of the two morpho-species, comparing their genetic structure patterns with that of an allopatric population of A. aeneus (i.e., the Maquinas population). Genetic structure analysis, assignment analyses, and estimation of gene flow between the sympatric morpho-species were carried out. Contrary to expectations, there is a lack of genetic differentiation between the A. aeneus and A. caballeroi morpho-species. However, there was a genetic differentiation between Lake Catemaco and Río Maquinas populations of A. aeneus . In addition, we found an asymmetric gene flow pattern, with a larger migration rate from A. aeneus to A. caballeroi than in the other direction. Finally, the allopatric population of A. aeneus showed lower levels of genetic diversity than those reported for both morpho-species in the lacustrine system. Consequently, our results support the notion that these morpho-species are either in an early phase of speciation or represent a single polymorphic species. This model provides relevant information to understanding the presence of polymorphisms under gene flow and their potential impact on the speciation process.