Environmental DNA as a tool for monitoring species of EU interest in Italian freshwaters. Management and conservation implications

Freshwater habitats occupy less than the 1% of the Earth but host almost 10% of the known biodiversity and act as hotspots of biological diversity. Nevertheless, freshwaters stand out as perhaps the most endangered ecosystem globally. Unaltered aquatic communities are fragile and often affected by some complications that can hinder their management and conservation. Moreover, native species related to water environments can be cryptic and difficult to monitor. This is especially concerning about those species in danger of extinction. Invasive Alien Species (IAS) are among the most impacting threats to biodiversity globally, but are particularly harmful for freshwater ecosystems. Biological invasions alter ecosystem composition and structure, leading to the impoverishment of local native communities, in which endemic species are particularly vulnerable. Native species are directly threatened by IAS due to the negative interactions of competition, predation, and disease transmission. Italian freshwaters host at least 100,000 species, and the highest number of endemism and diversity on fish in Europe. The European Union adopted several regulations to halt the loss of biodiversity at the global scale. The Habitats Directive 92/43/EEC preserve natural habitats and wild fauna and flora in a favourable conservation status. The Water Framework Directive 2000/60/EC promotes protection of entire river catchments to ensure adequate water quality and support local biodiversity. The Regulation on IAS 1143/2014 prevent new introductions and manage the spreading of invasive alien species in the Union territory. These three regulations rely both on monitoring obligations and on spatial planning to establish protected areas (the firsts) and manage eradications (the last). Environmental DNA (eDNA) using quantitative PCR (qPCR) is a sensitive tool to detect rare and elusive species, such as endangered or invasive alien species (IAS) during the initial stages of colonization. With this thesis we evaluate the potential applications of a qPCR eDNA-based approach for large-scale routine monitoring of freshwater species of European Union interest and their implication in conservation planning in accordance to the willingness declared by EU to expand the coverage of protected areas in its territory. First, we selected 22 target species belonging to Fungi, Mollusca, Arthropoda and Chordata. Fourteen of them are endangered species listed in the Habitats Directive 92/43/EEC, whereas the remaining eight are IAS listed under the EU Regulation 1143/2014. We designed designed and tested an eDNA protocol for the simultaneous detection of 22 freshwater species of conservation concern, to be tested in two temporal replicates at 53 sampling sites located on standing and running water of Central Italy. This first study also aimed at identifying the environmental factors influencing species detection, and assessing the efficiency and cost-effectiveness of an eDNA-based multi-species detection versus a taxon-specific traditional survey method. We designed for the first time eDNA assays for 13 species, nine of them resulting accurate and specific. PCA and biplots were used to explore the relationships between species detection and 25 fine-and large-scale environmental variables measured at each sampling site. Cost for eDNA included assay design, validation and purchase, laboratory equipment and consumables, personnel and cost for sampling, filtration, extraction, and amplification stages. Costs for traditional surveys were based on taxon and habitat specific sampling techniques according to published guidelines. We detected 18 out of the 22 target species at 45 sites. Highest detection rates were observed for fish (0.59 ± 0.2), amphibians (0.43 ± 0.3), molluscs (0.30 ± 0.12) and mammals (0.27 ± 0.29). Species were mostly detected during spring. PCA highlighted a relationship between a subset of environmental factors for four species. Compared to traditional techniques, a qPCR-based eDNA approach demonstrated as a faster (<46% of time), accurate (+49 new occurrences), and affordable (<88% of costs) approach for multiple species detection, offering great potential for large-scale monitoring of freshwater wildlife, especially for routine monitoring under EU regulations. We also analyzed eDNA occurrences to update the occurrence data and identify priority areas of conservation concern pursuant to European regulations. We developed indexes and prioritization scores for the 53 sampling sites according to the occurrence and prevalence of native species and IAS. As to species listed in Appendix II of HD occurring in running water sites, we interpolated the species presences 10 km-upstream the river network and identified gaps in the Natura 2000 Network, consisting in 808 km of unprotected river stretches. To prioritize areas of conservation concern in accomplishment of regulation obligations (such as Habitats Directive, IAS regulation, and Water Framework Directive), for each site we computed a Prioritizing Protection Score (PPS), a Prioritizing Eradication Score (PES), and a Freshwater Conservation Score (FCS). Results from qPCR eDNA survey revealed new occurrences of species listed in Annexes II, IV and V, and IAS, highlighting 53 new grid cells that will contribute to the next reporting for both HD and IAS Regulation. PPS, PES, and FCS identified 39 priority sites claiming for Natura 2000 designation or extension, 22 sites for IAS eradication and management, and 33 sites in need of freshwater protection. This innovative approach enhances monitoring efficiency, fills data gaps, and can strengthen freshwater conservation and restoration across Europe. Moreover, we focused on a flagship and umbrella species which conservation action could benefit the whole aquatic ecosystem, the Eurasian otter (Lutra lutra). We compared the efficiency of eDNA and traditional field survey techniques in detecting otters at the border of its current expansion range in Central Italy. We contemporary run a systematic standard field survey (n=192) and a qPCR-based eDNA sampling (n=17) in six river basins of the Latium region. We compared results of these methods at the scale of i) sampling site, ii) 5 km and iii) 10 km radius upstream. Field survey allowed to detect otter signs at 23 sites in one river catchment in the southern part of the study area. Both eDNA and standard survey allowed to detect otters at seven sites in southern catchments and one located northward in a tributary of the river Tiber. Both techniques confirmed otter absence in northern Latium. eDNA showed a higher detection rate (0.41) compared to the standard field survey, both at sampling site (0.06), 5 km (0.23) or 10 km radius (0.35) scale. By excluding sites negative to both eDNA and standard survey, eDNA detection rate was significantly different at sampling site scale only (p=0.01). These results suggest eDNA as a more fast and sensitive method in revealing otter presence. Finally, we present some guidelines explaining how to plan and implement a monitoring project on freshwater species based on qPCR eDNA in Central Italy. Guidelines focused on sampling design, field sample collection, and DNA filtering operations. The guidelines do not go into detail of further phases of an eDNA project, such as the amplification of eDNA, or the design of genetic assays, as these are largely available in the literature. The guidelines include an introduction to qPCR eDNA, the ecology of eDNA, practical issues related to sampling strategies and techniques, and advices on species and sampling sites selection. We then discussed about pitfalls and limitations to be accounted when planning eDNA-based monitoring.