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\begin{document}

\title{Newly developed \emph{ad hoc} molecular assays shows how eDNA can
witness and anticipate the monk seal recolonization of central
Mediterranean}



\author[1]{Elena Valsecchi}%
\author[2]{Emanuele Coppola}%
\author[3]{Rosa Pires}%
\author[1]{Andrea Parmegiani}%
\author[1]{Maurizio Casiraghi}%
\author[4]{Paolo Galli}%
\author[1]{Antonia Bruno}%
\affil[1]{University of Milan–Bicocca}%
\affil[2]{Gruppo Foca Monaca APS}%
\affil[3]{Instituto das Florestas e Conservação da Natureza}%
\affil[4]{University of Milano Bicocca}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
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.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

The Mediterranean monk seal (\emph{Monachus monachus} ) is the only
pinniped historically and permanently present in the Mediterranean
basin. Originally Mediterranean monk seals were found regularly
throughout the Mediterranean, Marmara and Black Seas, along the West
African coast to as far south as Cap Blanc, and in Cape Verde, Canary,
Madeira and Azores Islands. In the 20\textsuperscript{th}~century, the
species was drastically reduced, primarily by fishermen, over most of
its range, leading to severe bottleneck and inbreeding depression
signatures in the refugial populations in eastern Mediterranean
(Stoffel\emph{et al.} , 2018; Karamanlidis \emph{et al.} , 2021).
However, in the last decade, occasional sightings on Italian coastal
waters have been recorded (Figure S1 in Supporting Information).

The Mediterranean monk seal current distribution is only partially known
with most available data retrieved from sites where adult females are
known to return every year to reproduce. Thanks to the predictability of
these events, some of the delivery caves are monitored year-round using
camera-trap (e.g. Martinez-Jauregui \emph{et al} ., 2012), allowing us
to learn much about mother/pup relation. Yet, the home ranges of these
marine mammals are wide, as adult individuals are known to be able to
cover several tens of kilometers per day (Adamantopoulou \emph{et al} .,
2011), and little is known about milling and foraging areas and which is
the actual distributional range during the non-breeding season. This
kind of information is difficult to retrieve as monk seals are often
elusive. The recently become available possibility to molecularly
monitor the presence of any target species through the analysis of
traces of its DNA released in the environment (eDNA) provides a precious
and absolutely non-invasive means by which unveiling and oversee the
Mediterranean monk seal population.

In this letter we desire to make available a triple set of
species-specific molecular assays able to detect the presence of
residual Mediterranean monk seal DNA both from soil and water
environmental matrices. The developed assays were validated both in
silico and in the wet lab on a wide range of DNA templates, including
tissue (n=3) and residual (n=12) extracts and marine eDNA samples
(n=58). The overall sample (n=73) was split into 7 categories: 3
positive and 2 negative controls and 2 sets (offshore/coastal) of marine
eDNA samples. Our results show that 1) the assays are successful in
identifying the smallest amount of monk seal eDNA (up to
5.7*10\textsuperscript{-8} mg/L); 2) the screening on both Mediterranean
marine eDNA sample sets (from previous and ongoing studies) detected the
presence of monk seal in about 50\% of the samples of either set, and
showed that eDNA analysis can anticipate the detection of the monk seal
presence in the study areas before human eye; 3) finally, the proposed
approach can potentially unveil still unknown aspects of the biology of
this charismatic marine mammal species, by allowing its identification
in inaccessible contexts (i.e. offshore waters and during night-time).

\textbf{MATERIALS AND METHODS}

\textbf{Species specific primer sets design}

Candidate regions for designing monk seal specific primers were
identified within the mtDNA regions targeted by MarVer primers
(Valsecchi \emph{et al} ., 2020), as the amplicons produced by the three
primer sets -MarVer1, MarVer2 and MarVer3 - were all highly polymorphic
also between the two sister species of the Mediterranean (\emph{Monachus
monachus} ) and the Hawaiian (\emph{Neomonachus schauinslandi} ) monk
seals: 12 (out of 199bp), 14 (out of 87bp) and 24 (out of 237bp)
variable sites were found in the fragments amplified by MarVer1, MarVer2
and MarVer3 respectively. Internally to each amplicon, one monk-seal
specific primer was designed from the stretch of sequence showing the
largest number of diagnostic sites. Each monk-seal specific primer was
paired for amplification to the corresponding MarVer universal primer
(Valsecchi \emph{et al} ., 2020) on the opposite strand. In those
instances where the universal primer presented one degenerate base, the
mammalian variant was used (Valsecchi \emph{et al} ., 2020). For the
reasons exposed in the Discussion, another prerequisite we included in
the search for adequate priming sites was that of aiming at producing
amplicons sufficiently different in size between the three loci.
Hereafter we use the terms ``MarVer1'', ``MarVer2'' and ``MarVer3'' to
designate the three loci, rather than the primer sets.

\textbf{Sample set}

The three assays were tested on seven typologies of samples, including
both tissue/biological residues DNA (n=3) and marine eDNA (n=4)
templates, encompassing three kinds of positive controls, two negative
controls and two trials on Mediterranean marine eDNA ``opportunistic''
samples already available from ongoing projects from EV's research group
(Table 1). The seven categories are described in details in Box 1.

The DNA from the tissue sample (category 1) was extracted using the
Qiagen DNeasy Blood and Tissue Kit, while all remaining DNA samples
(categories 2-6) were extracted using the Qiagen DNeasy PowerSoil Kit
according to the manufacturer protocol. When biological residual samples
(MmoR, category 2) were in liquid form (e.g. feces dissolved in
ethanol), an aliquot was filtered through a membrane that was
subsequently rinsed with distilled water prior DNA extraction with the
same extraction-protocol used for eDNA samples. Table S1 shows a
detailed description of all 73 samples.

The four sets of eDNA samples (categories 3 to 5 and 7), coming from
different projects and having been collected in different contexts and
by different operators, were not always homogeneous in terms of number
of liters of marine water being processed and porosity of filters used.
However, they were all collected from the sea surface, with the
exception of ferry samples collected from sea water intake placed 4.5m
below sea level (Valsecchi \emph{et al} ., in prep.). Water volumes were
collected and stored in the Bag-in-Box Sampling System (BiBSS,
Valsecchi\emph{et al} . in prep.).

The two samples collected directly from shore (MmoMc11 and MnoMc12,
category 4) underwent through a 10-fold template dilution prior
amplification, a procedure used to minimize the interference of PCR
inhibitors (Gasparini \emph{et al} . 2020), known to be particular
abundant in proximity of sediments (Lance \& Guan 2019).

\textbf{qPCR experiment design}

Quantitative Real Time PCR (qPCR) assays were performed with AB 7500
(Applied Biosystem) to test the three primer pairs.~First, for each
primer pair, \emph{M. monachus} tissue samples were used to set
amplification efficiency (E) and limit of detection (LOD) and limit of
quantification (LOQ), according to Bustin \emph{et al} ., 2009 and
Klymus\emph{et al} ., 2019. Ten-fold serial dilutions were used to
generate the standard curve.

All 73 samples plus no-template negative controls were run in
triplicate, using the following qPCR conditions: an initial denaturation
at 95 \selectlanguage{ngerman}°C for 10 min, followed by 40 cycles of denaturation at 95 °C for
15 s and annealing-elongation at 56 °C for 1 min. A final dissociation
stage was performed. Amplification reaction consisted of 5.0 \selectlanguage{greek}μ\selectlanguage{english}l SsoFast
EvaGreen Supermix with Low ROX (Bio-Rad), 0.1 \selectlanguage{greek}μ\selectlanguage{english}l each {[}10 \selectlanguage{greek}μ\selectlanguage{english}M{]} primer
solution, 2 \selectlanguage{greek}μ\selectlanguage{english}l DNA sample, and 2.8 \selectlanguage{greek}μ\selectlanguage{english}l of Milli-Q water.

Ct (Threshold Cycles) values were converted into counts (DNA copies)
according to Bruno \emph{et al} ., 2017. When qPCR copy number outputs
were below the limit of quantification (LOQ), but above the theoretical
limit of qPCR (three copies per reaction according to Bustin \emph{et
al} . (2009)), they were addressed as `detectable but not quantifiable'
(DBNQ). For all the reactions, primer specificity was verified by the
calculated TM.

\textbf{Testing for correlation between marker's amplicon size and DNA
degradation}

Since the primer set producing the larger amplicon (MarVer3, 216bp) was
found to perform less efficiently than the other two (see Results), we
tested whether the drop of signal between a high-quality DNA sample and
a degraded DNA sample (as eDNA) was the same for all three loci (whose
sizes are multiple of ca 70bp) regardless the dimension of the fragment
they amplify. This was possible thanks to the availability of two
exceptional samples in our data set: 1) a high molecular weight DNA
sample, such as MmoT01, freshly extracted from a tissue collected in the
immediate \emph{post-mortem} and 2) the best possible eDNA sample, such
as MmoM+01, collected in a site at high monk seal eDNA concentration.
This sample was collected in a site characterized by two peculiar
conditions: a) proximity to a cave contextually inhabited by many seals
and b) limited mixing with surrounding waters (thus little signal
dispersal) due to a counter-current flux pushing the water against the
opening of the cave (see Discussion). All three loci produced a strong
signal in both samples and all replicas thus allowing to test for
correlation between amplicon size and DNA quality. We applied a
Kruskal-Wallis test, as not requiring data being normally distributed
and having similar levels of variance.

\textbf{RESULTS}

\textbf{Primer sets}

Within each of the three MarVer loci (Valsecchi \emph{et al} ., 2020)
one\emph{Monachus monachus} specific priming site, fulfilling the
desirable criteria of high specificity for the target species, was
identified (Table 2). The degree of differentiation of the three
species-specific priming sites between the Mediterranean monk seal
(GenBankAN: GU174602) and its closest relative, the Hawaiian monk seal
(GenBankAN: MscNC\_008421), was as follows: 6bp (24\%), 9bp (32.1\%) and
8bp (22.2\%) for MarVer1, MarVer2 and MarVer3 respectively. Primers'
sequences have been deposited on the BOLD System repository
(Ratnasingham \& Hebert 2007).

\textbf{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
\selectlanguage{ngerman}°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 Log\textsubscript{2} DNA-copies
recovered in the complete data set, for all three sample and
experimental replicas and markers, is shown in Figure S2.

\textbf{Positive-control samples (categories 1 to 3). Category 1.} Monk
seal tissue-extracted DNA (MmoT1), with a concentration of 284 ng/\selectlanguage{greek}µ\selectlanguage{english}l
(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\textsuperscript{-8} mg/L. \textbf{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. \textbf{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).

\textbf{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).

\textbf{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
(\selectlanguage{greek}Δ\selectlanguage{english}Log\textsubscript{2}DNA-copies) from the tissue (MmoT01) to the eDNA
(MmoM+01) control samples (Kruskal-Wallis test: \selectlanguage{greek}Χ\selectlanguage{english}\textsuperscript{2}=
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).

\textbf{DISCUSSION}

\textbf{Assays overview}

\emph{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 \emph{et al} ., 2020).

\emph{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 \emph{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
(Valsecchi\emph{et 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 (Lacoursi\selectlanguage{ngerman}ère\selectlanguage{english}-Roussel \& Deiner, 2019; Turner \emph{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 \emph{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 \emph{et al} ., 2015). However, the high
sensitivity and specificity of well-developed eDNA-based approaches can
counteract these limitations.

\emph{Readiness} . Quantitative PCR assays allow to obtain quick
outcomes as opposed to DNA metabarcoding approach (requiring library
preparation and high-throughput DNA sequencing).

\textbf{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).

\textbf{Traditional PCR and assays' multiplexing~}

The possibility to visualize the assays' outcomes using the traditional
PCR, rather than qPCR, is a desirable feature, as it allows the test to
be carried out in any structure equipped with essential molecular
biology laboratory apparatus, besides being cheaper. Although the
diagnostic sensitivity decreases in the case of traditional PCR, our
study indicates that when the signal is not too weak (see below), even
basic PCR allows the identification of traces of monk seal DNA. The
possibility of using the traditional PCR, in turn, entails further
advantages, such as the possibility of multiplex run, particularly
advantageous when multiple assays are available. This procedure reserves
more plusses. Firstly, it allows saving on the amount of eDNA template
(precious as typically limited) to be used. Another advantage is the
possibility of amplifying the template molecule with different probes at
once, a strategy that may reveal useful when dealing with highly-diluted
eDNA of the desirable target species and especially for large body size
species which are likely to shed ``clustered'' eDNA (e.g. only one of
the three replica-filters, may contain the target species DNA, although
they all come from the same water sample). In these instances,
multiplexing could minimize the possibility of false negatives while
possibly gathering clues about the signal age (see above), although the
latter aspect needs further deepening. A preliminary multiplex test
indicates that all combinations of the three proposed assays are
compatible for multiplexing and that the approach is efficient also with
eDNA samples, providing a relatively high (say \textgreater{}10
log\textsubscript{2}DNA copies/liter) monk seal DNA content such as in
Madeira control sample MmoM+01 (Figure S5).

\textbf{Currents matter}

This study provides indirect evidence of how marine eDNA suffers
susceptibility to currents disturbance: Madeira samples' stations 1
(MmoM+01) and 2 (MmoM+02) are only 112 meters apart from each other, and
yet, in the first one (at the entrance of the cave frequented by
\textgreater{}8 seals) the molecular signal was the loudest of our eDNA
sample set, while in the latter no signal at all was found over 27
reactions (3 filters, 3 replicas, 3 markers). So, apparently along those
112 meters there is (or there was at the time of sampling) an invisible
barrier that prevents the two water masses from merging (Figure S6).
This also implicitly means that around station 1 there is a sort of
pocket or relatively unstirred water, where dated and recent monk seal
biological traces may accumulate overtime. This fortuitous circumstance
makes station 1 the ideal scenario where testing the differential
ability of three markers to perceive the temporal scale of eDNA
traces.~The discrepancy in monk seal eDNA content between the two
closely adjacent sampling stations if, on one hand, highlights relevance
of currents in swiping away eDNA signals, on the other hand, it adds a
reassuring element on the fact that when a signal is indeed intercepted,
this probably reflects the presence of the animal in a limited
spatio/temporal range (as signals that disperse with currents become
shortly extremely diluted and therefore remain undetected).

\textbf{Detection of monk seal in Mediterranean eDNA data sets and
anticipation on visual data~}

Positive detections were found in both the Tyrrhenian and the Strait of
Sicily trial sample sets, both collected in marine districts where the
monk seal is occasionally encountered. DNA recovery was more prevalent
in the Pelagian (especially around Lampione Island) than in the ferry
sample. Monk seal sightings in waters surrounding the Italian peninsula
have been occurring rarely, but constantly in the last decades (Figure
S1). According to available published data (some sightings may remain
unrevealed for the species safeguard), no sighting was recorded in 2019,
while at least 9 events were recorded in 2020, suggesting an increased
monk seal occurrence in Italian waters. The same trend, but anticipated
by a year, was found in our two-year spanning Tyrrhenian sample: the 17
ferry eDNA samples in which monk seal DNA was detected were more
abundant in 2019 (n=13, 65\% of yearly total) than in 2018 (n=4, 25\% of
yearly total).

The strongest monk seal DNA recovery within the ferry-route eDNA
collection was found in sample MmoMo17 (sample 19-1LiGA1), collected on
the 20\textsuperscript{th} of June 2019 at about 11km from Elba Island
and 26km from Capraia Island, where the monk seal has been sighted at
least twice during summer 2020 (June/July). In the same site, exactly
one year before (18\textsuperscript{th} of June 2018), a detectable but
non-quantifiable signal was identified, witnessing the presence of the
monk seal in the Tuscany archipelagos up to two years before the
recent\emph{de-visu} identifications in the waters surrounding Capraia
island (June-July 2020).

In the Pelagian data, both samples (MmoMc09, and MmoMc11) scoring the
strongest detection signals were somehow related to sightings. Sample
MmoM+09 was collected in the northern shore of Lampedusa close to where
two consecutive sightings would have occurred two months later (Figure
3). Sample MmoMc11 was instead purposely collected two days after a
sighting occurred nearby. In this case, at least for MarVer2, the signal
was the strongest within the Pelagian data
(5.36*10\textsuperscript{3}DNA copies per liter of marine water),
comparable in value to the signal obtained in the control eDNA of
Madeira sample MmoM+01 (6.38*10\textsuperscript{3}). We generally found
agreement with data reporting a signal persistence of about 48 hours
from release (Collins\emph{et al} ., 2018), although other factors
should be kept in consideration, such as the possible unsubstantiated
repeated presence of the animal in the two days between sighting and
sampling (which could have increased the signal intensity). Also, we are
unaware if the animal/s has/have engaged in behaviors that facilitate
the release of biological material (e.g. landing or rubbing on the
shoreline), and finally it should be bear in mind the differential eDNA
persistence between coastal and deep pelagic waters (Collins \emph{et
al} ., 2018).

\textbf{Relevance of nocturnal samples}

This study provides the first example not only of molecular detection of
the monk seal presence based on marine water analysis, but it also
represents the first case where monk seals are looked for in open waters
and, moreover, at any time of day. Despite diurnal (n=17, 47.2\%) and
nocturnal (n=19, 52.8\%) ferry samples were roughly equally represented,
positive monk seal DNA detections were more commonly found at night
(64.7\%, n=11) than in samples collected during light hours (35.3\%,
n=6). Although we have denominated the ferry samples as ``offshore
eDNA'' (Category 5), the surveyed Livorno-Golfo Aranci route crosses
inevitably twice the continental shelf. Thus, 22 (61.1\%) of our 36
ferry-based samples were collected off the continental shelf
(depth\textgreater{}200mt). These included all the 7 samples collected
in fix stations 3 and 4 (n=14), plus all the sighting samples (n=8), all
occurring along the study route between fix stations 3 and 4 (Figure 3).
About half (n=12, 54.5\%) of these 22 deep water samples were collected
at night. Interestingly, despite the sample size is not appropriate to
establish statistical significance, the instances (n=9) in which monk
seal traces were found in offshore waters originated prevalently from
nocturnal samples (n=6). These DNA recoveries were however all weak
(DBNQ), denoting either diluted (relatively far from the point of
shedding) or faded (not freshly released in the environment) signals.
Interestingly however, in most of the 22 truly pelagic samples (off the
continental shelf) positivity was given by locus MarVer3 (the largest in
amplicon size), thus suggesting the presence of not too degraded
(recently shed?) eDNA. If confirmed by future offshore screening, the
finding would indicate that monk seals are likely to frequent deep
waters -probably foraging- at night, while they have more coastal habits
during day time. Interestingly, the metabarcoding analysis on the 2018
ferry-sample subset showed significantly higher vertebrate read counts
(\textgreater{}95\% attributable to bony fish) in nocturnal-vs-diurnal
samples (Valsecchi \emph{et al} ., in prep.). The sample recording the
highest monk seal signal (MmoMo17) was diurnal, but collected on the
continental shelf, at about ten kilometers from two islands. This might
explain why this species is more commonly seen by the human eye (thus
during day-light hours) in coastal water rather than in offshore waters,
which are probably more frequently attended by monk seals at night.

\textbf{Value of offshore molecular surveillance over monk seal
occurrence}

Although recognized as the most endangered pinniped species worldwide,
the status of the Mediterranean monk seal population is currently
considered ``data-deficient'' as, so far, its study has been focused on
resting/reproductive coastal areas, where paradoxically the research is
necessarily limited by the concern of disturbing such a vulnerable
species, thus the adoption of camera traps. Yet, beside the
above-mentioned constraints, limiting the study of the monk seal to its
coastal occurrence would inevitably limit and bias our knowledge of the
species, as it brings insights on limited aspects of its life cycle. The
possibility to monitor the species in offshore waters opens the prospect
to fill the knowledge gaps about still uninvestigated facets of the
biology of this threatened species, such as feeding habits, movements
during non-reproductive season, species boundaries etc.

\textbf{Conclusions}

We present three molecular assays tailored for the detection of monk
seal DNA, validated on three sets of positive control samples, including
eDNA samples collected during the 2020 monk seal breeding season off
Madeira (Desertas Islands). The eDNA-based assays have proven to be able
to successfully detect the presence of this threatened species in both
tested sets of coastal and offshore Mediterranean eDNA, witnessing its
presence long before being recorded by human eyes. The outcomes also
suggest that this emblematic species is progressively re-appropriating
its original range. Not only could this approach represent an innovative
and absolutely non-invasive way to monitor the status of this unique
pinniped, but it also has the potential to highlight still
uninvestigated aspects of its behavior, such as its nocturnal hunting
nature. Coupled with traditional ecological surveys, the proposed
approach appears an ideal molecular surveillance tool valuable in many
applications relevant to the monk seal conservation/study (reviewed in
Appendix S1).

\textbf{Figure legends}

\textbf{Figure 1} Log\textsubscript{2} DNA-copies distribution across
positive quantifiable and detectable (PQD) samples. Values (expressed
per liter of marine water in the case of eDNA samples) for all three
markers across the entire set of positive control samples are shown in
comparison with the same values recorded in the 7 Mediterranean eDNA
samples (last ones on the right) reporting unambiguous signals of the
monk seal presence (i.e. above the detectable threshold).

\textbf{Figure 2} Madeira eDNA positive-control (sample category 3)
sampling sites and results. In the map the eight sampled sites are
indicated with color-coded dots according to which marker detected monk
seal DNA in that specific site. The result table reports on the
left-hand side the field notes of each sampling site as reported from
the rangers at the moment of sample collection and release to the
molecular team after disclosure of results. On the right-hand side are
shown the molecular results for each of the three assays expressed in
ranges of logarithmic scale of number of DNA copies per liter of sampled
marine water.

\textbf{Figure 3} Calendar of sample collection and sightings along with
molecular results in the two tested Mediterranean areas for which eDNA
samples were available from on-going projects: offshore Tyrrhenian
(above) and coastal Strait of Sicily, Pelagie archipelago (bottom
section). In the tables black squares indicate positive and quantifiable
(PQD) monk seal DNA detection by one or more of the three loci (red
symbols in the map). The grey squares show samples reporting weak -below
the detection threshold- monk-seal signals (yellow symbols in the map),
defined in the text as DBNQ (detectable but not quantifiable). The
number of squares reflects the number of the sample replicas with
positive detection. Coloured cells indicate sampling events, with darker
shading showing nocturnal samples. Positions and dates of recent monk
seal sightings in the two study areas are reported (in red in the
calendar tables).

\textbf{Box 1} Details of the seven sample categories. ``+C'' and ``-C
`` denote positive and negative control sample sets respectively.

\textbf{ACKNOWLEDGEMENTS}

We thank Giuseppe Sciancalepore of the Dept. of Comparative Biomedicine
and Food Science of the University of Padova for providing the monk seal
archival sample from the Mediterranean marine mammal tissue bank
(\emph{www.marinemammals.eu}). We are grateful to Corsica and Sardinia
Ferries, in the person of Cristina Pizzutti, for welcoming our proposal
of attempting eDNA sampling from their fleet, thanks to the brokerage of
Dr Antonella Arcangeli of ISPRA who set up the FLT network within which
our protocol was firstly proposed. Thanks to rangers from the IFCN for
the samples collection off Desertas Islands, to Moby Diving for giving
support in sample collection off Lampedusa, and Franco D'Urso of the
KRCA for promptly supplying BiBSS to Madeira's team. Thanks to Gruppo
Foca Monaca APS - Rome- and Grupa Sredozemna Medvjedica - Zagabira- for
providing the biological residues samples from their archives and the
data on occasional observations of the last decades.Thank also to
University of Bicocca Marine Science past and current master students
Roberto Lombardi, Sofia Frappi, Simone Innocente and Francesca Gagliardi
for lending aliquots of the samples collected for their theses used to
test the assays proposed in this study. Finally, we would like to
publicly thank all the supporters of our ``MeD for Med'' (``Marine eDNA
for the Mediterranean'') crowdfunding project, who enthusiastically
believed in our initiative despite the fundraising campaign was launched
during COVID pandemic, restricting our sampling design.

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\textbf{Summary of Supporting Information}

\textbf{Figure S1} Map of monk seal sightings

\textbf{Table S1} Samples' and results' description

\textbf{Figure S2} Log DNA copies graphs across all 73 samples

\textbf{Figure S3} Signal drop tissue vs eDNA statistics

\textbf{Figure S4} Hypothesis of ``Environmental Marker Selection''

\textbf{Figure S5} Multiplex-test gel

\textbf{Figure S6} Map of MmoM+01 and MmoM+02 sampling sites

\textbf{Appendix S1} Possible applications of monk-seal molecular
monitoring

\textbf{Table 1} The seven categories of screened samples (n=73). In the
``Expected outcome'' column, green, yellow and red dots indicate sample
categories where the monk seal presence is expected (positive controls),
unknown but possible (Mediterranean trial samples) and impossible
(negative controls), respectively. The number of dots reflects strength
of expected outcome.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/471141/articles/562811-newly-developed-ad-hoc-molecular-assays-shows-how-edna-can-witness-and-anticipate-the-monk-seal-recolonization-of-central-mediterranean}

\textbf{Table 2} Primer sets' sequences for the three monk-seal specific
molecular assays. Oligos named ``Mmo\ldots{}'' are the species-specific
primers (in bold), while those labelled ``MarVer\ldots{}'' are the
universal marine vertebrate primers described in Valsecchi et al.
(2020). The suffixes ``nd'' followed by a letter (e.g. ``ndC'') indicate
the non-degenerate form of a specific primer where the letter indicates
the mammalian specific nucleotide used in place of the degenerate base
(see Appendix S2 in Valsecchi et al. 2020).

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/471141/articles/562811-newly-developed-ad-hoc-molecular-assays-shows-how-edna-can-witness-and-anticipate-the-monk-seal-recolonization-of-central-mediterranean}

\textbf{Box 1} Sample categories.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/471141/articles/562811-newly-developed-ad-hoc-molecular-assays-shows-how-edna-can-witness-and-anticipate-the-monk-seal-recolonization-of-central-mediterranean}

\textbf{Figure 1} Distribution of Log\textsubscript{2} DNA-copies in
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\textbf{Figure 2} Madeira map, samples and results\selectlanguage{english}
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