Rediscovering the Endangered Mediterranean Killifish Aphanius iberus : Environmental DNA Reveals Updated Distribution and Conservation Implications in C

ABSTRACT

Aquatic ecosystems are among the most biodiverse and ecologically important habitats on Earth. Despite this, they are increasingly threatened by habitat degradation and biological invasions. In Europe, intensive agriculture and urbanization along the Mediterranean coasts have disrupted hydrological connectivity and altered natural habitats of aquatic fauna. One of the most affected species is the endangered Spanish toothcarp ( Aphanius iberus ), a small euryhaline killifish endemic to Spain's Mediterranean coast. Its populations are confined to fragmented habitats facing environmental pressures, particularly competition with invasive Eastern mosquitofish ( Gambusia holbrooki ). The assessment of the Spanish toothcarp's distribution is critical for its effective conservation management; however, traditional methods are labor‐intensive, invasive, and often fail to detect low abundance species. Therefore, we applied an environmental DNA (eDNA) metabarcoding approach targeting the mitochondrial 12 S rRNA gene to reassess its distribution. From 2022 to 2024, 135 water samples were collected across 16 sites encompassing current, reintroduced, and historical locations of the species. Sampling was conducted in autumn and spring along the Mediterranean coast and included freshwater, brackish and saline habitats. Our results identified population strongholds for the Spanish toothcarp and confirmed the success of recent reintroduction efforts, particularly the inland Villena population. The accompanying fish assemblages remained consistent throughout the monitoring period, and invasive Eastern mosquitofish was present at all locations. Detections of Spanish toothcarp at historical sites, notably Guardamar del Segura, highlight the sensitivity of eDNA for identifying remnant populations undetectable by conventional methods. However, the low sequence read counts and sporadic occurrence reaffirm the species' endangered status, emphasizing the need for frequent monitoring and the integration of habitat restoration and invasive species control. Overall, our findings underscore eDNA metabarcoding as an efficient, non‐invasive method for monitoring Spanish toothcarp, with the ability to advance conservation efforts for threatened aquatic fauna in transitional habitats facing analogous environmental challenges.