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Animal conservation relies on assessing the distribution and habitat use of species, but for endangered/elusive animals this can prove difficult. The Monk Seal,  Monachus monachus , is one of the world's most endangered species of pinniped, and the only one endemic to the Mediterranean Sea. During recent decades, direct observations have been few and scattered, making it difficult to determine its distribution away from the Aegean Sea (core distribution area of the post-decline relict population). This study relies on environmental DNA (eDNA) analysis to detect the presence of the Monk Seal in 135 samples collected in 120 locations of the central/western Mediterranean Sea, spanning about 1500 km longitudinally and 1000 km latitudinally. A recently described species-specific qPCR assay was used on marine-water samples, mostly collected during 2021 by a Citizen Science (CS) project. Positive detections occurred throughout the longitudinal range, including the westernmost surveyed area (Balearic archipelago). The distribution of the positive detections indicated six “hotspots”, mostly overlapping with historical Monk Seal sites, suggesting that habitat-specific characteristics play a fundamental role. We applied single-season occupancy models to correct for detection probability and to assess the importance of site-specific characteristics. The distance from small islets and protected (or access-restricted) areas was correlated negatively with the detection probability. This novel molecular approach, applied here for the first time in an extensive CS study, proved its potential as a tool for monitoring the distribution of this endangered/elusive species.

The monk seal is the most endangered pinniped in the world and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. Data on its status are scattered due to both its rarity and evasiveness and records are biased towards occasional, mostly coastal encounters. Nowadays, molecular techniques allow us to detect and quantify minute amounts of DNA traces released into the environment (eDNA) by any organism. A species-specific molecular assay is now available for detecting the recent presence of the monk seal in the water column through the analysis of sea-water samples collected from the sea surface. The project “Spot the Monk” uses this non-invasive detection tool to monitor monk seal occurrence in Mediterranean waters by means of eDNA analysis. The simplicity in the acquisition of samples together with the need to collect samples in multiple points simultaneously made the project well suited to the involvement of the general public. Up to today, about 350 samples have been collected and analysed in the central-western Mediterranean by researchers and a multifarious range of citizen scientists - from recreational sailing organisations, both amateur and competitive sportsmen, to fishermen. This work announces the launch of an open-source Observatory (https://www.spot-the-monk-observatory.com/) where the project outcomes are publicly accessible as soon as they are produced. Embracing the principles of Open Science, we believe that such an approach can contribute to filling the knowledge gap about the distribution of this charismatic species in our seas, providing, at the same time, a proof of concept on how data collected by a variety of actors can be returned to the scientific and non-scientific communities in an innovative format for immediate consultation.

Metabarcoding studies using environmental DNA (eDNA) and high‐throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals, have received less attention. Similarly, there have been few attempts to identify potentially “universal” barcode loci which may be informative across multiple marine vertebrate orders. Here we describe the design and validation of two new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles, and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. “In silico” validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals, have received less attention. Similarly, there have been few attempts to identify potentially “universal” barcode loci which may be informative across multiple marine vertebrate orders. Here we describe the design and validation of two new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles, and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

Collecting fine‐scale occurrence data for marine species across large spatial scales is logistically challenging but is important to determine species distributions and for conservation planning. Inaccurate descriptions of species ranges could result in designating protected areas with inappropriate locations or boundaries. Optimizing sampling strategies therefore is a priority for scaling up survey approaches using tools such as environmental DNA (eDNA) to capture species distributions. In a marine context, commercial vessels, such as ferries, could provide sampling platforms allowing access to undersampled areas and repeatable sampling over time to track community changes. However, sample collection from commercial vessels could be biased and may not represent biological and environmental variability. Here, we evaluate whether sampling along Mediterranean ferry routes can yield unbiased biodiversity survey outcomes, based on perfect knowledge from a stacked species distribution model (SSDM) of marine megafauna derived from online data repositories. Simulations to allocate sampling point locations were carried out representing different sampling strategies (random vs regular), frames (ferry routes vs unconstrained), and number of sampling points. SSDMs were remade from different sampling simulations and compared with the “perfect knowledge” SSDM to quantify the bias associated with different sampling strategies. Ferry routes detected more species and were able to recover known patterns in species richness at smaller sample sizes better than unconstrained sampling points. However, to minimize potential bias, ferry routes should be chosen to cover the variability in species composition and its environmental predictors in the SSDMs. The workflow presented here can be used to design effective sampling strategies using commercial vessel routes globally for eDNA and other biodiversity survey techniques. This approach has potential to provide a cost‐effective method to access remote oceanic areas on a regular basis and can recover meaningful data on spatiotemporal biodiversity patterns. Commercial vessels as sampling platforms can accurately capture species distribution patterns, despite inherent biases associated with constrained spatial coverage. Our workflow can be applied across the global shipping network to upscale sampling using techniques such as environmental DNA to reduce gaps in marine biodiversity knowledge.

Background Citizen Science (CS) offers a promising approach to enhance data collection and engage communities in conservation efforts. This study evaluates the use of CS in environmental DNA (eDNA) monitoring for Mediterranean monk seal conservation. We validated CS by assessing the effectiveness of a newly developed CS-friendly filtration system called “WET” (Water eDNA Trap) in eDNA detection, addressing technical challenges, and analysing volunteer faults. The WET is a 4-litre, manual pump-based filtering system using positive pressure to force water through the filter. We also assessed the use of a retrospective questionnaire as a tool to measure CS’s social impact on participants’ perceived knowledge, attitudes, and conservation behaviours. Results Results suggest the WET performs comparably to traditional methods, with minor technical issues. Despite some faults such as not folding or forgetting to change the filter, volunteers were generally reliable in sample processing. Moreover, CS involvement increased participants’ perceived knowledge, affective attitudes, and conservation behaviours towards seal conservation. Volunteers reported a greater understanding of eDNA monitoring, increased interest in monk seal conservation, and more frequent conservation behaviours, including spreading awareness within their community. While these findings are exploratory due to the small sample size (19 participants) and potential ceiling effects in attitude assessment, they provide an initial validation of the questionnaire as a tool for measuring CS’s social outcomes. Future studies with larger sample sizes are needed to confirm these results and investigate their applicability across broader stakeholder groups. Continuous improvement in volunteer training and equipment design is also recommended. Conclusions This study highlights CS’s potential to improve public engagement and knowledge in conservation. By involving diverse participants, CS can play a critical role in long-term conservation efforts and promote sustainable coexistence between humans and monk seals. Furthermore, the validation of the questionnaire offers a valuable framework for evaluating the social impact of CS initiatives in conservation contexts.

Marine environmental DNA (eDNA) is an important tool for biodiversity research and monitoring but challenges remain in scaling surveys over large spatial areas, and increasing the frequency of sampling in remote locations at reasonable cost. Here we demonstrate the feasibility of sampling from commercial vessels (Mediterranean ferries) while underway, as a strategy to facilitate replicable, systematic marine eDNA surveys in locations that would normally be challenging and expensive for researchers to access. Sixteen eDNA samples were collected from four fixed sampling stations, and in response to four cetacean sightings, across three cruises undertaken along the 300 km ferry route between Livorno (Tuscany) and Golfo Aranci (Sardinia) in the Ligurian/Tyrrhenian Seas, June-July 2018. Using 12SrDNA and 16SrDNA metabarcoding markers, we recovered diverse marine vertebrate Molecular Operational Taxonomic Units (MOTUs) from teleost fish, elasmobranchs, and cetaceans. We detected sample heterogeneity consistent with previously known variation in species occurrences, including putative species spawning peaks associated with specific sea surface temperature ranges, and increased night time abundance of bathypelagic species known to undertake diel migrations through the water column. We suggest commercial vessel based marine eDNA sampling using the global shipping network has potential to facilitate broad-scale biodiversity monitoring in the world’s oceans.

Marine mammal foraging grounds are popular focal points for marine protected area (MPA) implementation, despite being temporally dynamic, requiring continuous monitoring to infer prey availability and abundance. Marine mammal distributions are assumed to be driven by their prey in foraging areas, but limited understanding of prey distributions often prevents us from exploring how shifting prey availability impacts both seasonal and long‐term marine mammal distributions. Environmental DNA (eDNA) metabarcoding could enhance the understanding of marine mammal habitat use in relation to their prey through simultaneous monitoring of both. However, eDNA applications focused on marine mammals or predator–prey dynamics have been limited to date. In this study, we assess spatiotemporal changes in the distribution and abundance of cetaceans, minke whales (Balaenoptera acutorostrata), bottlenose dolphins (Tursiops truncatus) and harbor porpoises (Phocoena phocoena) in relation to key prey species in a newly established MPA, employing eDNA metabarcoding. We recovered 105 molecular operational taxonomic units (OTUs) from marine vertebrates using two primer sets targeting 12S and 16S genes, along with 112 OTUs from a broader eukaryotic primer set targeting 18S rRNA. Overall, key forage fish prey species, sandeels and clupeids, were the most abundant teleost fishes detected, although their availability varied temporally and with distance from shore. We also found clear spatial partitioning between coastal bottlenose dolphins and the more pelagic minke whales and harbor porpoises, paralleling availability of their main prey species. Other species of conservation interest were also detected including the critically endangered European eel (Anguilla anguilla), Atlantic bluefin tuna (Thunnus thynnus), and the invasive pink salmon (Oncorhynchus gorbuscha). This study demonstrates the application of eDNA to detect spatiotemporal trends in the occurrence and abundance of cetacean predators and their prey, furthering our understanding of fine‐scale habitat use within MPAs. Future long‐term monitoring of predator–prey dynamics with eDNA could improve our ability to predict climate‐induced shifts in foraging grounds and enhance rapid responses with appropriate management actions. Prey are an important driver of fine‐scale habitat use by marine mammals, but their distributions are often poorly understood. Here, we demonstrate that eDNA metabarcoding can reveal fine‐scale spatiotemporal differences in cetacean distributions and their key prey species, including spatial partitioning between sympatric cetacean species with different dietary preferences and changing seasonal availability of key prey species.

The monk seal is the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. The limited understanding of the population demographics and conservation status of this species are due to both its rarity and evasiveness, with records biased towards occasional, mostly coastal, encounters. Current molecular techniques allow us to detect DNA traces released into the environment (eDNA) by any organism. We have developed three species-specific qPCR-assays targeting the 12S/16S rRNA mitogenome regions of the monk seal. The assays have been tested extensively on a comprehensive and diverse set of samples (n = 73), including positive controls from a breeding population in Madeira collected during their peak abundance, and two opportunistic collections of Mediterranean eDNA samples (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% of the samples collected from a ferry platform in the Tyrrhenian (2018–2019) and in 66.7% of those obtained in the Pelagie archipelago in the Strait of Sicily (2020). These findings anticipated subsequent visual observations in the proximity of these sampling areas by up to 2 years. At the Tyrrhenian site, monk seal detection increased between 2018 and 2019. The demonstrated approach provides a non-invasive and highly sensitive tool for defining the monk seal actual distribution and home range -enabling monitoring also in nocturnal hours-, for assessing its recovery rate and pinpointing coastal/offshore sites where prioritizing conservation, research, citizen-science, and educational opportunities.