A species-specific qPCR assay provides novel insight into range expansion of the Mediterranean monk seal (Monachus monachus) by means of eDNA analysis

Abstract

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.

Playing “hide and seek” with the Mediterranean monk seal: a citizen science dataset reveals its distribution from molecular traces (eDNA)

“…The screening of the 135 samples analysed in this study involved the filtering of a total of 1431 L of sea water, filtered on a total of 370 membranes, the extracted DNA from which was run in triplicate for a total screening of 1100 (370 × 3) qPCR reactions (Supplementary Table 1 S). Since the purpose of the study was to have an overview on the presence of the Monk Seal on a wide geographical area, we used a single locus, locus MarVer2, which was the best performing of the 3 loci described in Valsecchi et al 32 . The locus choice was motivated for 3 reasons: (1) the high detectability rate; (2) the small size of the targeted amplicon (71 bp), particularly suitable for highly degraded DNA, such as eDNA; (3) the presence of a long string of unique bases (n = 8) at the 3' end of the Monk-Seal specific primer, significantly increasing its specificity.…”

“…entering the food web) and abiotic (e.g. UV, currents) factors [26][27][28][29][30][31] , it can still provide presence-absence data witnessing the passage of elusive species, such as the Monk Seal 32 .…”

Towards a strategy for the recovering of the Mediterranean monk seal in the Adriatic-Ionian Basin

“…An essential first step for MMS conservation is, thus, to determine habitat availability/suitability in areas not surveyed yet (Calabria, Sicily, parts of Apulia, Croatia and of the Greek Ionian Sea). The e-DNA analysis of samples from areas throughout the Basin might yield useful results (Valsecchi et al, 2022) as a non-invasive, low-cost method on where to concentrate conservation activities, compared to time-consuming, expensive questionnaire/field surveys with often questionable results. Questionnaire surveys focusing on fishers in areas already pre-selected via e-DNA analysis, along with broad public awareness actions encouraging the reporting of sightings, may further help to prioritize core areas with recurring sightings.…”

“…Further research into various aspects such as the species' genetics, movements, dietary preferences, etc., is required for improving our knowledge and, thus, support effective management options. Novel, non-invasive techniques are available such as e-DNA analysis (Valsecchi et al, 2022) or microplastics analysis (Hernandez-Milian et al, 2021), and may greatly facilitate the filling of gaps in the species' biology and ecology.…”

Environmental DNA (eDNA) for monitoring marine mammals: Challenges and opportunities

“…In recent years, eDNA studies in seawater have been used to monitor the diversity of marine vertebrate communities, primarily focusing on teleost fish and elasmobranchs (Thomsen et al, 2012;Miya et al, 2015;Andruszkiewicz et al, 2017;Truelove et al, 2019). However, eDNA studies on marine mammals are scarce and, to date, have mainly focused on the detection and identification of single species (Baker et al, 2018;Valsecchi et al, 2022), especially elusive or rare species (Foote et al, 2012;Ma et al, 2016), rather than the monitoring of marine mammals diversity. This is due, in part, to the lack of efficient taxon-specific metabarcoding primers.…”