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 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.

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. 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 in the environment (eDNA) by any organism. We present three qPCR-assays targeting the monk seal mitogenome. The assays were soundly tested on an extensive and diversified sample set (n=73), including positive controls from Madeira breeding population collected during the peak of abundance, and two opportunistic Mediterranean eDNA-sample collections (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% and 66.7% of the samples collected in the Tyrrhenian from a ferry platform (2018-2019) and in the Pelagie archipelago -Strait of Sicily- (2020) respectively, anticipating (up to 2 year) visual observations occurred subsequently in proximity of the sampled areas. In the Tyrrhenian, detection occurrence increased between 2018 and 2019. Monk seal DNA recoveries were commoner in night-time ferry-samples, suggesting nocturnal predatory activity in pelagic waters. The proposed technique provides a non-invasive and yet highly-sensitive tool for defining the monk seal actual distribution and home range, its recovery rate and pinpoint coastal/offshore localities where prioritizing conservation, research, citizen science and education initiatives.

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The monk seal, the most endangered pinniped worldwide and the only one found in the Mediterranean, has suffered a drastic decline in the last few decades. Nowadays molecular techniques allow to detect minute amounts of DNA released in the environment (eDNA) by any organism. We present three qPCR-assays targeting the monk seal mitogenome. The assays were soundly tested on an extensive/diversified sample set (n=73), including positive controls from Madeira breeding population and two opportunistic Mediterranean eDNA-sample collections (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% and 66.7% of the samples collected in the Tyrrhenian from a ferry platform (2018-2019) and in the Pelagie archipelago (2020) respectively, anticipating (up to 2 year) visual observations occurred subsequently in proximity of the sampled areas. In the Tyrrhenian, detection occurrence increased between 2018 and 2019. Monk seal DNA recoveries were commoner in night-time ferry-samples, suggesting nocturnal predatory activity in pelagic waters. The proposed technique provides a non-invasive and yet highly-sensitive tool for defining the monk seal actual range, its recovery rate and pinpoint coastal/offshore localities where prioritizing conservation.