Potential factors driving the distribution of subterranean invertebrates in karst groundwaters of the Rotolo Cave (southern Italy).

The study of subterranean environments is hindered by the lack of information that does not allow to fully explain the subsurface abiotic and biotic dynamics, especially when considering the 3D interactions in the deep karst. The fragmented knowledge of mechanisms and factors regulating the subsurface environment and its biological features makes it difficult to assess the connections between the various components, and the changes related to anthropogenic impacts. Challenges for data collection in karst are mainly referred to explorations and surveying in caves, because they are difficult to access. Despite the limitation in accessing the subsurface, karst groundwaters offer privileged sites to study ecosystems of high intrinsic value due to their specialized fauna (stygofauna), often comprising rare and endemic species. The aquatic cave-dwelling organisms live in fissures of the rock mass and in larger conduits and channels, resulting in consequential relations with the aquifer hydrogeological dynamics. The extreme vulnerability of karst and the occurrence of low resilient communities therein, requires urgent monitoring and conservation tools to protect the underground ecosystems in their entirety. This work offers the basis for setting practical conservation actions by means of a systematic and long-term scientific program in the Apulian karst. The carbonates of Apulia host significant groundwater resources and over 2000 caves in need of protection. Many efforts have to be made to assess the abiotic dynamics, together with the status of the stygodiversity. A first attempt was carried out at Grave Rotolo, the deepest cave in Apulia, where cavers have reached the groundwater bodies. A preliminary screening of both saturated and unsaturated karst (May-October 2021) allowed us to assess the minimum theoretical number of species living in the system through asymptotic species richness estimators (Chao1, Chao2, Abundance Coverage Estimator, Incidence Coverage Estimator). This karst system harbors a diverse and composite array of species across groundwater habitat types. Indeed, the faunal sampling underestimated the theoretical true baseline richness level of the cave (range 16.77-76.05%). In addition, the sites where the groundwater fauna live in the cave could be related to complex subsurface flow pathways or different recharge areas, as suggested by the differences observed in species compositions and species abundances among different cave water bodies. Therefore, the combined biological and hydrogeological approach could shed light on their mutual information, where the biological component may work as hydrogeological tracer of the karst system. These tools are necessary to select proper monitoring sites, and define the most appropriate sampling and analytic techniques for achieving a better understanding of the groundwater dynamics and species partitioning in deep karst environments.