Are River Springs Devoid of Fish?—The Case of the Maciço Calcário Estremenho ^†

The evolution of strictly freshwater fishes in the Iberian Peninsula is correlated with the paleogeomorphological evolution of Iberian basins and mountain ranges. Located in Portugal’s central region, the Maciço Calcário Estremenho (MCE) is a unique limestone geological formation with river springs that separates two different biogeographic regions for freshwater ichthyofauna: at its western slope, small coastal streams with ancient connections to the Mondego Riverland, and at its eastern slope, sub-basins of the Tagus River. Springs from these rivers are located only a few kilometers apart and are typically seen as devoid of fish due to their intermittent regime, shallow water column, high slope, and/or existence of insurmountable barriers that virtually prevent upstream fish migration. Although the species richness is known for the region, little is known about which species have colonized the headwaters. The objectives of this study were to (1) assess which fish species are present at the springs and (2) characterize the genetic diversity at the inter- and intrapopulation levels of two target species (Achondrostoma oligolepis and Squalius pyrenaicus) existing on opposite slopes of the MCE, using one mitochondrial and one nuclear gene. Genetic analyses showed differences between populations from distinct rivers but also at the intrapopulation level, with unique haplotypes being found in some locations within a river basin. This study demonstrated that springs are not devoid of fish in the MCE and may instead be important conservation areas for native species, benefiting from the existing Natural Park established in the region. Despite their geographical proximity, the isolation of populations of the same species in distinct rivers resulted in significant interpopulation genetic differences. Moreover, intrapopulation genetic differences were also found, highlighting two important topics: the sampling scheme can influence the obtained genetic data and the genetic diversity may differ between upstream and downstream stretches of the same river. Sampling several locations within the same river leads, therefore, to more reliable results, and future landscape genetic studies using fast-paced markers are necessary to assess genetic connectivity and to depict how the genetic diversity of strictly freshwater fish species is distributed along a river course.