*Article* **Patterns of Spatial Overlap between Non-Indigenous and Critically Endangered Freshwater Fishes from a Mediterranean Biodiversity Hotspot †**

**Nicholas Koutsikos 1,2,\*, Leonidas Vardakas 1,3, Olga-Ioanna Kalantzi <sup>2</sup> and Stamatis Zogaris <sup>1</sup>**


**Abstract:** Non-indigenous fish species (NIFS) can cause severe ecological impacts on the invaded ecosystems and are considered as one of the leading factors of freshwater biodiversity loss. Unraveling the spatial overlap between NIFS and critically endangered (CR) fish species can contribute to targeted conservation actions to minimize the potential negative effects. In this study, we applied geostatistical analyses to investigate the spatial overlap of NIFS against fish species that are designated under the CR status according to the IUCN and the Hellenic Zoological Society (HZS) Red Lists. Distributional data (presence–absence) from 800 records of 52 NIFS were compiled for both lentic and lotic ecosystems of Greece. Our results indicate that freshwater ecosystems under high NIFS richness were located mainly in lowland areas and often near large cities and ecosystems with well-developed commercial and recreational fisheries. On the contrary, low NIFS richness was observed in mountainous regions and in relatively small river basins. Overlapping areas of CR species with moderate to high NIFS richness (1.5–4.3 NIFS per 1 km2) were relatively high (~50%). A quarter of the overlapping areas (24.8%) fall within NATURA 2000 network, where legal management bodies could implement specialized programs to minimize the negative impacts. However, the majority of CR fish species' distribution remains in unprotected areas indicating that protected areas should be re-designed to include areas containing freshwater species under the highest threatened category. Our findings demonstrate that whole assemblages of fishes are rapidly changing as NIFS spread into Greece and many freshwater ecosystems of outstanding biodiversity conservation value are under significant invasion pressure.

**Keywords:** invasive species; alien; translocated; critically endangered; freshwater fishes; freshwater ecosystems; conservation; biodiversity; red list
