Genomic and Phylogenetic Insights into the Hybridogenetic Origin of the Probably Extinct Iberian Endemic Squalius palaciosi ^†

Introduction: Squalius palaciosi (Doadrio, 1980; Leuciscidae) is a highly threatened freshwater fish species with an extremely restricted distribution, currently confined to a few tributaries on the right bank of the Guadalquivir River basin. During the 1980s, its populations were abundant and constituted a dominant component of local fish communities. However, multiple threats led to a drastic population decline, bringing the species to the brink of extinction. From an evolutionary perspective, S. palaciosi is particularly remarkable due to its polyploid condition and its potential involvement in hybridogenetic complexes, a rare phenomenon in the Iberian Peninsula. Hybridogenetic systems are well documented in its congeners Squalius alburnoides, widely distributed across Iberian river basins, and Squalius sp., restricted to the Guadiana basin. In these systems, the maternal lineage is shared (Squalius pyrenaicus), whereas the paternal lineage varies and remains unknown in S. palaciosi. Objective: This study aims to generate the first genomic data for S. palaciosi and to elucidate its evolutionary origin, as well as its mitochondrial and nuclear phylogenetic relationships within hybridogenetic complexes. Methodology: Genomic DNA was extracted from skeletal remains of preserved specimens housed in the fish collection of the National Museum of Natural Sciences (MNCN-CSIC) and subjected to Illumina short-read sequencing. After quality filtering, potential contaminant reads were removed. The complete mitochondrial genome and several nuclear gene fragments were assembled. Mitochondrial phylogenetic analyses were conducted using publicly available whole-genome sequencing data from Iberian freshwater fish species. Nuclear gene fragments were taxonomically assigned using BLAST analyses. Results: Phylogenetic analyses revealed that S. palaciosi is closely related at the mitochondrial level to S. alburnoides and S. tartessicus, with strong statistical support. BLAST-based taxonomic assignments of nuclear markers suggest the involvement of multiple Iberian freshwater fish species in the hybridogenetic origin of S. palaciosi. Conclusions: Our results provide novel insights into the evolutionary history of S. palaciosi and support a complex hybridogenetic origin involving multiple parental lineages. This study contributes to a better understanding of hybridogenetic speciation in freshwater fishes, a rare but evolutionarily significant process.