Why three barcode assays
The first motivation for looking at multiple loci was driven by the possibility of picking the best possible candidate, among 3 loci still poorly explored for species detection purposes. Secondarily, we also made sure to draw probes targeting at fragments of different lengths in order to: a) verify whether amplicon size matters when dealing with degenerate templates such as eDNA; b) testing the possibility of multiplexing all or a combination of the 3 barcodes (see below).
Regarding the first point, we do find that the primer set producing the largest amplicon (MarVer3, 216bp) is in fact the one that amplifies with more difficulty and/or producing fewer DNA copies than the other two assays (MarVer1 and MarVer2, 146bp and 71bp respectively). This is true also when employing high-molecular-weight DNA as template. The rate of signal drop between a high-concentration/quality DNA sample and environmental DNA is significantly higher than that observed in the two markers targeting smaller fragments (Figure S3). If differential drops in recorded DNA signal is correlated to DNA degradation and thus to temporal variables (time since DNA release into the environment) could be explored in future studies.
The fact that the performance of the three markers does not show a consistent ranking across all samples, but that simultaneously it is not randomly distributed, as patterns seem perceptible within eDNA samples’ categories (e.g. MarVer1 performed better in category-3 samples, MarVer2 in category-4 samples, and MarVer3 was the only marker detecting -weak- signals in some category-5 samples, see Figures 3 and S2, and Table S1), suggests further investigations should be encouraged. In Figure S4 we advance a provisional hypothesis, of the temporal driven “environmental marker-selection”, which attempts to explain the observed phenomena.
At the moment, our study suggests that the most informative and efficient markers are the two 12S-rDNA loci (MarVer1 and MarVer2). MarVer3 (16S-rDNA) could be employed in the markers panel and, in case of positive detection, it could indeed -but cautionary- provide a proof of signal freshness (see below in the Mediterranean sample set discussion).