RT PCR outcomes
A total of 1710 qPCR reactions were run over the 73 samples covering the 7 sample categories (Table 1). MarVer1 amplification efficiency reached 92.6%, LOQ corresponded to Ct=34.75; MarVer2 amplification efficiency reached 101%, LOQ corresponded to Ct=35.8; MarVer3 amplification efficiency reached 92.2%, LOQ corresponded to Ct=34; R2≥0.99 for each assay. Melting temperatures were 75.4 +/-0.3, 71.5 +/-0.3, 78.5 +/-0.2 °C, for MarVer1, MarVer2, and MarVer3 respectively. According to the LOQ calculated for each locus, qPCR DNA detection outcomes were divided in three classes: 1) no signal, 2) monk-seal DNA detectable but not quantifiable (DBNQ), 3) positive quantifiable detection (PQD). Over the 73 samples, 26 were PQD positives for at least one of the three markers, of which 19 (90.5%) and 7 (14.6%) fell into the positive control group (n=21) and the trial eDNA samples (n=48), respectively (Figure 1 and Table S1). We found a total of 19 DBNQ positives (for at least one of the three loci), of which only 1 (4.7%) in the positive control group, and the remaining 18 (37.5%) in the two Mediterranean trial eDNA samples (2 in the Strait of Sicily and 16 along the ferry track in the Northern Tyrrhenian, see below). Twenty-eight (28) samples gave no signal of monk seal DNA: they included one positive control (eDNA), sample MmoM+02, and all four negative controls (tissue-extracted DNA and eDNA). The remaining 23 samples were Mediterranean eDNA samples, namely 19 (52.7%) and 4 (33.3%) in the Tyrrhenian and in the Strait of Sicily samples respectively (see below).
Samples being positive for monk seal DNA for all three loci (and sample/experimental replicas) were only found within the positive control group (Categories 1, 2 and 3). Most environmental DNA samples (including 5 of the 8 positive-control eDNA samples from Madeira) show positiveness to monk seal DNA above the LOD with only one or two of the three loci (Figure 1, Table S1). The locus recovering monk seal DNA in more samples (n=22) was MarVer1 locus, followed by MarVer2 (n=20). The 16S primer set, MarVer3, was the one recovering the lower number of positive quantifiable detection (n=12), all of which found in positive-control samples, and only one of these regarding an eDNA sample (MmoM+01). The complete list of Log2 DNA-copies recovered in the complete data set, for all three sample and experimental replicas and markers, is shown in Figure S2.
Positive-control samples (categories 1 to 3). Category 1. Monk seal tissue-extracted DNA (MmoT1), with a concentration of 284 ng/µl (NanoDrop 2000, ThermoFisher Scientific), was used as refence sample. For this sample all three primer sets produced amplicons of the expected size in all 3 reaction replicas, allowing the detection of up to 5.7*10-8 mg/L. Category 2. All 12 DNA extracts from biological residues (MmoR samples in Tables 1 and Table S1) produced at least one PQD in the triplicate test with each one of the assays. The number of samples scoring PQD in all three replicas were 11, 10 and 8 for MarVer1, MarVer2, MarVer3 respectively. Thus, within category 2 samples, MarVer1 produced overall more PQDs (34 out or 36) than the other two loci (31/36 and 27/36 for MarVer2, MarVer3 respectively), although MarVer2 produced the highest number of DNA-copies. Category 3. The results obtained on the positive control eDNA samples collected off the Madeira archipelago and the field observations reported by the rangers at the time of water samples’ collection are shown in Figure 2. Sample MmoM+01, collected at the entrance of the Tabaqueiro cave (attended by approximately 8 seals), was the control eDNA sample producing the strongest signal, in all three sample replicas, all the experimental triplicates and for all three markers (Table S1, Figures 2 and S2).
Mediterranean eDNA sample sets (categories 4 and 5) . Positive quantifiable (PQD, n=7) and unquantifiable (DBNQ, n=18) monk seal DNA detections were found in both Mediterranean eDNA sample sets (Figure 3). Monk seal eDNA was recovered in 47.2% of the ferry samples (Tyrrhenian, n=36) and 66.7% the coastal sample (Pelagie archipelago, Strait of Sicily, n=12). The proportion between PQR and DBNQ detection was much higher in the Pelagian samples (6 PQD and 2 DBNQ detections) than in the ferry sample set (1 PQD and 16 DBNQ detection).
Excluding the two samples collected in the days following sightings (MmoMc11 and MmoMc11), Pelagian monk seal DNA recoveries (n=6) were more frequent in waters far from the main island of Lampedusa (83.3%, n=5, 4 of which in proximity of Lampione) than in the waters surrounding Lampedusa itself (n=1).
Negative-control samples (categories 6 and 7). Both tissue and eDNA negative controls produced no signals in all qPCR runs.
A significant difference was found between the three loci in terms of drop of intensity of the molecular signal (ΔLog2DNA-copies) from the tissue (MmoT01) to the eDNA (MmoM+01) control samples (Kruskal-Wallis test: Χ2= 13.18, df = 2, p-value = 0.001374). The major difference was imputable to locus MarVer3, which showed a significantly higher drop of signal compared with the two 12S-rDNA markers, which instead had a similar decrease of signal (Figure S3).