Assays overview
Specificity and sensitivity . The high degree of molecular
differentiation of the three presented probes compared to the homologous
regions in the sister species -Hawaiian monk seal- genome suggested high
specificity of the proposed assays, assumption that was confirmed in all
positive and negative control samples sets. High specificity to target
species is a fundamental prerequisite for the dissemination of an
innovative method given the conservation relevance of the study species.
Our experimental design and trial suggest that there is little room for
false positives (except those attributable to lab contamination, for
which maximum caution should be posed, as in any experiment that is
based on eDNA analysis), while incidence of false negative should be
more of a concern, as PCR inhibitors present in environmental matrices
is recognized as one the main limitations of eDNA-based detection
approaches (Loeza-Quintana et al ., 2020).
Reliability . We reckon that also the samples being positive in
only 1 or 2 of the three amplification replicas represent genuine monk
seal detection, considering the following points:
- The high homology of the probe to -and only- monk seal DNA (see
above), supported by the specific melting temperatures recorded,
suggests that any positive detection has to denote monk seal DNA
presence.
- Failing amplification in replicas of a positive sample may be
ascribable to the extremely low concentration of the target molecule.
Marine vertebrate DNA counts for only about 0.004% of the eDNA
retrieved from marine samples (Stat et al ., 2017). In the DNA
metabarcoding analysis carried out on the ferry sample subset (2018) and
same loci, over 90% of the detected reads was attributable to bony fish
community (decreasing of one more order of magnitude the incidence of
rare vertebrates, such as marine mammals, on the overall marine
vertebrate fauna), and no read was attributable to monk seal (Valsecchiet al ., in prep.). Replications (from sampling to DNA
amplification) reduces the rate of false negatives; however, the optimal
level of replication is difficult to be estimated, since it strongly
depends on the detection probability of the target taxon.
- With the exception of MmoM+1, collected in proximity of the entrance
of the cave most densely populated by monk seals (n=8 ca) and that
returned positiveness for all three loci, all remaining Madeira
control-eDNA samples (n=7, 87.5%) resulted positive to monk-seal DNA
either only in some sample/reaction replicas and/or for only some of the
loci (typically the two 12S-rDNA ones), although seals were definitely
around in those waters.
In some cases, a positive detection was found in only one of the sample
replicates (each originating from one of the three filters used to
process a single sample). This effect can be more easily explained,
considering the specific characteristics of the animal and the eDNA
state released (Lacoursière‐Roussel & Deiner, 2019; Turner et
al ., 2014). Large animals, such as marine mammals, release in the
surrounding water biological traces often consisting of large clusters
of cells rather than of single or a few cells (Valsecchi et al .,
1998). In this way, when the sample is divided into two or more
subsamples, it is likely that the cells, being clustered, are not
distributed homogeneously among the various subsamples.
Imperfect detection is an unavoidable feature of most data on species
presence/absence even during traditional ecological field studies, when
individuals and species that are present at one site are not always all
detected, and failure in accounting for imperfect detection may result
in biased inference. eDNA assays are clearly imperfect, relying on small
amounts of degraded DNA, and still laboratory and sequencing costs limit
replication (Ficetola et al ., 2015). However, the high
sensitivity and specificity of well-developed eDNA-based approaches can
counteract these limitations.
Readiness . Quantitative PCR assays allow to obtain quick outcomes
as opposed to DNA metabarcoding approach (requiring library preparation
and high-throughput DNA sequencing).