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The quality of spatial analyses of biodiversity is improved by (i) utilizing study areas with well defined physiogeographical boundaries, (ii) limiting the impact of widespread species, and (iii) using taxa with heterogeneous distributions. These conditions are typically met by ecosystems such as oceanic islands or ancient lakes and their biota. While research on ancient lakes has contributed significantly to our understanding of evolutionary processes, statistically sound studies of spatial variation of extant biodiversity have been hampered by the frequently vast size of ancient lakes, their limited accessibility, and the lack of scientific infrastructure. The European ancient Lake Ohrid provides a rare opportunity for such a reliable spatial study. The comprehensive horizontal and vertical sampling of a species-rich taxon, the Gastropoda, presented here, revealed interesting patterns of biodiversity, which, in part, have not been shown before for other ancient lakes. In a total of 284 samples from 224 different locations throughout the Ohrid Basin, 68 gastropod species, with 50 of them (= 73.5%) being endemic, could be reported. The spatial distribution of these species shows the following characteristics: (i) within Lake Ohrid, the most frequent species are endemic taxa with a wide depth range, (ii) widespread species (i.e. those occurring throughout the Balkans or beyond) are rare and mainly occur in the upper layer of the lake, (iii) while the total number of species decreases with water depth, the proportion of endemics increases, and (iv) the deeper layers of Lake Ohrid appear to have a higher spatial homogeneity of biodiversity. Moreover, gastropod communities of Lake Ohrid and its feeder springs are both distinct from each other and from the surrounding waters. The analysis also shows that community similarity of Lake Ohrid is mainly driven by niche processes (e.g. environmental factors), but also by neutral processes (e.g. dispersal limitation and evolutionary histories of species). For niche-based mechanisms it is shown that large scale effects such as type of water body or water depth are mainly responsible for the similarity of gastropod communities, whereas small scale effects like environmental gradients affect gastropod compositions only marginally. In fact, neutral processes appear to be more important than the small scale environmental factors, thus emphasizing the importance of dispersal capacities and evolutionary histories of species.