Habitat niches of fish species can exert a strong influence on population structure, even on a small geographical scale. In this scope, Pelasgus thesproticus is a great model species to study connectivity in riverine environments owing to its naturally patchy habitat distribution. Furthermore, it is important to conduct such studies in near-natural systems to avoid the impact of human disturbances on the river, such as fragmentation, morphological changes and habitat degradation. In this sense, the Vjosa in Albania is an excellent study area. A total of 204 individuals were sampled from five locations in the lower Vjosa and two tributaries and genotyped with 33 newly designed microsatellites loci using high throughput sequencing. The application of microsatellite genotyping by sequencing revealed genetic structure and some differentiation, even at a small spatial scale (< 65 river km). A total of 500 alleles were found with an average of 0.93 private alleles among sites with rather low FST values (< 0.04). The extent of admixture observed in some populations indicate that the genetic structure is mainly influenced by upstream populations, either from the main river itself or from tributaries. In addition, the connection between a tributary and the other sites is disrupted by the flow regime, which is reflected in a high degree of divergence from the other populations. Our results indicate that hydrological conditions of the flowing river present strong barriers to gene flow, particularly in the upstream direction, but at the same time act as dispersal corridors in the downstream direction and exhibit source-sink dynamics in which upstream populations contribute disproportionately to downstream populations for this habitat specialist along the river. It is suggested that processes of colonization and reinforcement may play an important role in shaping the genetic structure of patchily distributed fish species in natural river systems. Future studies should increase the knowledge of dispersal factors, habitat heterogeneity, consequence of source-sink dynamics, and gene flow within the system, which will help to understand and maintain important processes related to metapopulation theory and the potential evolutionary consequences of habitat loss and fragmentation.
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