Next-generation sequencing techniques for conservation: development of barcode libraries for animal species and their application for biodiversity monitoring, protection and management

The ongoing sixth mass extinction is marked by a rapid acceleration of biodiversity loss in recent decades. In response, the scientific community has adopted a range of strategies to help mitigate this crisis, with molecular methods playing a valuable role in conservation efforts. Effective approaches to counter biodiversity loss prioritise species identification and description, monitoring their global distribution and abundance, and preserving both species and ecosystems. This thesis introduces novel methods, strategies, and applications of genetic approaches for biodiversity conservation and management. A molecular pipeline employing high-throughput sequencing technologies is presented to develop reference barcode libraries for large-scale biodiversity monitoring, along with a practical case study. This pipeline allows the simultaneous barcoding of approximately 1,000 samples, proving remarkable efficiency for projects aimed at establishing a global biodiversity surveillance system. The thesis also explores the application of genetic methods for biodiversity monitoring through environmental DNA (eDNA) analysis. Two research studies in freshwater systems of northwestern Italy are presented: one monitoring target species, including the Eurasian otter and the white-clawed crayfish, and the other characterizing freshwater-associated communities. Additionally, eDNA metabarcoding is employed to assess the effectiveness of terrestrial Natura 2000 protected areas in protecting animal communities in Northern Apennines. A multi-marker metabarcoding approach is applied to analyse riverine eDNA samples and describe invertebrate and vertebrate assemblages living in and associated with watercourses in protected and unprotected areas. Biodiversity monitoring is integrated with the assessment of chemical and geomorphological parameters of the rivers. The protocols, methodologies, and findings presented in this work advance the field of conservation genetics, providing innovative tools and strategies for biodiversity protection and management. By enhancing the application of genetic barcoding and eDNA metabarcoding, this thesis contributes to more effective monitoring, management, and preservation of biodiversity across diverse ecosystems and conservation contexts.