Two Genetically Distinct Lineages in Galician Populations of the Three-Spined Stickleback (Gasterosteus aculeatus)

The three-spined stickleback (Gasterosteus aculeatus) is a widely used model for studying the genetic basis of phenotypic variation. However, genomic studies have focused primarily on a limited number of geographic regions, leaving some regions underrepresented. In this study, we investigated genetic variation in stickleback populations from Galicia in northwestern Spain, a region near the southern limit of the species' European distribution that has been proposed as a glacial refugium but remains underrepresented in genomic analyses. Using double-digest RAD sequencing, we identified two genetically distinct lineages within Galician populations. These lineages showed substantial genetic differentiation (mean F _ST = 0.435) and also exhibited fine-scale population structure within each lineage. We detected morphological variation among Galician populations, although its association with lineage identity and ecology remains unclear. Most individuals carried the low-plated allele at a major armor plate locus (Eda), which is commonly found in freshwater populations throughout the species' range. Using whole-genome sequencing, we showed that both lineages were assigned to the Southern European Clade of the three-spined stickleback. Our results reveal previously unrecognized phylogeographic structure within Galician sticklebacks and highlight the importance of sampling underrepresented regions in model systems. These populations provide a useful system for investigating how geographic structure shapes patterns of genetic and phenotypic variation.