Non-invasive monitoring of freshwater species in Central and Southern Italian basins. From standard survey to environmental DNA

Freshwater ecosystems, including rivers, lakes, wetlands, and riparian zones, are vital for the Earth's biosphere but have been significantly altered by human activities. Despite occupying less than 1% of the Earth's surface, freshwater ecosystems support significant biodiversity, including approximately one-third of vertebrate species and 10% of all species. These habitats host diverse taxa such as freshwater mammals, dragonflies, turtles, birds, fishes, and crabs, many of which are endemic to specific regions. Freshwater ecosystems provide essential services such as fisheries, water provisioning, transportation, and cultural benefits. These ecosystems are highly sensitive to environmental changes, making them particularly vulnerable to human impacts such as water resource development, land cover modifications, habitat destruction, fragmentation, eutrophication, chemical pollution, invasive species, overexploitation, and climate change, all of which contribute to the rapid loss of freshwater biodiversity and the degradation of ecosystem services. Human activities and environmental changes continue to pose significant threats to the integrity and biodiversity of freshwater ecosystems. Invasive species intensify these challenges, making urgent conservation efforts necessary. Italian freshwater habitats are home to at least 100,000 species, highlighting their ecological richness. However, invasive species, pollution, and habitat degradation are among the major threats to Italian freshwater ecosystems. The introduction of non-native species such as crayfish and fish has disrupted ecological balances, leading to habitat destruction and competition with native species. Invasive Alien Species (IAS) are non-native organisms introduced to natural environments where they cause significant harm to ecosystems, biodiversity, and human health. In Europe, IAS are a primary driver of biodiversity loss. The EU has implemented regulatory measures to prevent, minimize, and mitigate the negative impacts of IAS, including the EU Biodiversity Strategy for 2030, which aims to manage established invasive species and reduce species threatened on the Red List by 50% by 2030. Regulation (EU) 1143/2014 outlines specific measures to limit the introduction, sale, reproduction, and spread of IAS. Biodiversity monitoring is crucial for detecting deviations from desired states, assessing the effectiveness of management actions, and identifying impacts from disturbances. Traditional techniques, though useful, present various challenges, including standardization of protocols and the invasiveness of methodologies. The EU Habitats Directive aims to conserve biodiversity through the designation of protected areas and the creation of the Natura 2000 network. Monitoring techniques must be sufficiently sensitive to predict the future evolution of ecosystems. Environmental DNA (eDNA) monitoring offers numerous advantages, making it a valuable tool for biodiversity assessment and conservation. eDNA sampling is highly standardized across different habitats, providing consistent data collection compared to traditional methods, which often yield variable results. This non-invasive technique does not harm species or habitats, unlike traditional surveys that may disturb organisms and ecosystems. eDNA's sensitivity is notable, particularly for detecting cryptic species or juvenile stages that are challenging to identify with traditional methods. Cost-effectiveness is another significant advantage, with eDNA often having shorter handling times and lower costs compared to traditional techniques. In aquatic ecosystems, eDNA is especially powerful, improving the efficiency and accuracy of sampling for organisms like freshwater mussels. Despite these advantages, challenges such as PCR inhibition and contamination must be acknowledged and mitigated. eDNA monitoring also presents several significant challenges. Contamination can lead to false positives at various stages, from field sampling to laboratory analysis. Inhibition by humic acids in samples can interfere with PCR reactions, resulting in false negatives and underestimating species occurrence. Errors in DNA sequences, whether from PCR or sequencing, can bias results and obscure biodiversity signals. The reliance on reference DNA-sequence databases for identifying environmental DNA sequences poses additional challenges, with gaps in geographical and taxonomic coverage hindering eDNA effectiveness. Addressing these challenges is crucial for ensuring the reliability and effectiveness of eDNA monitoring in conservation efforts. The main objectives of this PhD were to: I) Investigate and contribute to the development of new techniques for monitoring species of conservation interest; II) Evaluate the strengths of standard monitoring techniques and the potential of new non-invasive techniques to improve the current state of the art; III) Assess the weaknesses of these techniques, especially innovative ones, to propose new directions for study that fill current knowledge gaps as soon as possible. Evaluating the strengths and weaknesses between traditional and innovative techniques could lead to the contextualized application of one or both methods, depending on their complementary usefulness. The focus was on animal species in freshwater ecosystems listed in annexes II, IV, and V of the Habitats Directive 92/43/CEE, which are threatened and require rapid monitoring by Member States every six years, as well as invasive alien species that pose threats to these species or their habitats. Over the three-year period, the target species varied. As part of a PSR project funded by the Lazio Region, the initial list was provided by the Region, focusing on developing a protocol for rapidly monitoring rare and elusive freshwater species using eDNA. During the preparatory stages, species-specific primers were developed using DNA sequences from genetic databases. Limitations included insufficient genetic sequences for some species and genetic similarity among invertebrates, making differentiation difficult. After assessing suitability, the final target list consisted of 22 species across 10 taxa. This PhD thesis comprises three main chapters focusing on two primary monitoring techniques: Chapter I: Environmental DNA. This chapter explores eDNA as an effective method for species detection, especially for elusive species. In the highly biodiverse Latium region of Central Italy, eDNA assays were developed for 22 native endangered and invasive alien freshwater species. The study aimed to design accurate and sensitive eDNA assays for robust molecular analyses and effective conservation management. Chapter II: Environmental DNA and Bombina pachypus. Focusing on Bombina pachypus, an endangered species endemic to the Italian Apennines, this chapter highlights the severe decline and fragmentation of its habitat. Despite conservation efforts, B. pachypus continues to face population declines. The project aimed to develop specific primers for eDNA detection of B. pachypus, detect its presence and that of the pathogenic fungus B. dendrobatidis in 20 freshwater bodies in Central Italy, and evaluate factors affecting eDNA detection. The goal was to provide valuable insights into the distribution and conservation status of B. pachypus and its habitat. Chapter III: Standard Survey Techniques. This chapter reports on the Eurasian otter's (Lutra lutra) trophic niche, emphasizing the relevance of standard survey monitoring for this formerly endangered top predator of riverine ecosystems. Despite recovery efforts, the otter's range in Italy remains limited and isolated. Standard survey techniques are essential for assessing population trends, habitat use, and ecological requirements. The project aimed to analyze the trophic niche of otters along three neighboring rivers in Southern Italy by examining prey remains in otter scats. Understanding the otter's resource exploitation patterns is crucial for effective conservation and management strategies. This PhD project highlights the potential and challenges of integrating traditional and innovative monitoring techniques, emphasizing the importance of developing robust, non- invasive methods like eDNA to improve biodiversity conservation efforts. The findings underscore the need for continuous advancement in monitoring technologies to address current gaps and enhance the effectiveness of conservation strategies for threatened and invasive species in freshwater.