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Article

Marine Mammals’ Fauna Detection via eDNA Methodology in Pagasitikos Gulf (Greece)

by
Elena Akritopoulou
1,2,
Athanasios Exadactylos
1,
Anastasia Komnenou
2,3,
Joanne Sarantopoulou
1,
Christos Domenikiotis
1 and
Georgios A. Gkafas
1,*
1
Department of Ichthyology and Aquatic Environment, University of Thessaly, Fytokou Str., 38446 Volos, Greece
2
ARION—Cetacean Rescue and Rehabilitation Research Center, M. Botsari 110, 54453 Thessaloniki, Greece
3
School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(10), 692; https://doi.org/10.3390/d17100692
Submission received: 18 June 2025 / Revised: 13 September 2025 / Accepted: 26 September 2025 / Published: 3 October 2025
(This article belongs to the Special Issue Applications of Environmental DNA in Biomonitoring Across Multiple Trophic Levels)
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Abstract

Marine mammals are important ecological bio-indicators of marine ecosystems impacted by a plethora of anthropogenic and environmental threats. Genomics detects genetic variation, adaptation to environmental shifts, and susceptibility to diseases in marine mammal species. In this study, eDNA was utilized for the first time in the Pagasitikos Gulf over three consecutive years (2022–2024) in order to detect marine mammal species. Additionally, visual monitoring and eDNA results were compared to reveal the pros and cons of the two methodologies. The gulf was zoned into five different areas with respect to oceanographic features for sampling. DNA extraction was assessed by using a standard protocol of phenol–chloroform followed by PCR amplification using the 16S rRNA gene. A total of 5,209,613 highly filtered sequence reads were attributed to 108 species. Among these, Monachus monachus, Tursiops truncatus, and Ziphius cavirostris species were detected. This novel detection of Z. cavirostris in the relatively shallow waters of the Gulf of Pagasitikos raised the question of whether it was a random event or a new ecological trend. Z. cavirostris and M. monachus appeared to share the same marine areas within the gulf. In the era of the climate crisis, eDNA provides essential information on marine mammals’ ecological status, yields novel detections, and predicts behavioral changes essential to deep-diving species.

1. Introduction

Marine mammal species are characterized as important ecological bio-indicators of marine ecosystems [1]. They are apex predators holding a high trophic position in the food web [2]. They play a critical role in maintaining ecosystem stability and function by facilitating the vertical transport of metabolic by-products, such as in fecal plumes [3,4,5], and by depositing carcasses, which support deep-sea and benthic ecosystems over long periods [6,7,8]. Recent bioenergetic modeling has shown that nutrient availability is substantially influenced by cetacean communities, including deep divers and small cetaceans. This influence depends on species assemblage and geography [9]. On the other hand, the “EU Biodiversity Strategy” requires the European conservation network to incorporate 30% of territorial seas and land by 2030 [10].
The strategy highlights and is focused on the essence of pinpointing and securing key marine habitats for marine megafauna within the framework of wide conservation efforts [11]. Nonetheless, in order to form effective conservation strategies and policy for marine mammals, the monitoring and assessment of both marine mammals’ populations and the threats they face is required [1,12,13]. A plethora of laws and directives dictate the implementation of monitoring programs (via different methodologies) in order to efficiently assess marine mammal populations and mitigate the threats impacting them [14].
Traditional monitoring methodologies to estimate the abundance, distribution, and density of marine mammals include visual, acoustic, and aerial surveys [15,16,17,18]. However, the majority of these methods are time-, cost-, and resource-consuming and regularly sunlight- and weather-dependent [19,20,21].
Genomic monitoring provides detailed information regarding the population structure, connectivity, and demographic history of marine mammals. Genome-wide analyses have revealed fine-scale structuring in Tursiops spp. that is associated with habitat and environmental shifts [22,23,24]. Via these analyses, bottlenecks and post-bottleneck recovery in killer whales and northern elephant seals have been identified [23,25]. Also, phylogeographic patterns across basins have been resolved [26].
Genomics can also be used to detect previously unrecorded species or cryptic lineages [27,28,29,30], evaluate health and immune function [31], and inform life history and migration dynamics [32,33,34,35]. This provides direct support for conservation and management planning.
In recent years, the methodology of environmental DNA (eDNA) has been utilized in marine species detection, including mobile marine fauna such as marine mammal species. So far, eDNA has been used as a complementary methodology alongside traditional marine mammal monitoring methods, such as visual surveys and passive acoustic monitoring (PAM), rather than as a replacement. Although visual and acoustic methods are still the main methods used for estimating abundance, distribution, and behavior [36,37], eDNA has the advantage of being able to detect elusive, deep-diving, or low-density species [38,39,40]. eDNA is a widely used methodology in marine biodiversity research, but it is still developing in marine mammal species research, with promising results in marine mammal genetic species detection, monitoring, and abundance estimation [38,41].
Marine mammals represent a polyphyletic group [42] that have been independently adapted to marine life from different evolutionary lineages [43,44]. This group includes members of different mammalian orders, such as Cetacea (whales, dolphins, and porpoises); Sirenia (dugongs and manatees); and Carnivora, which consists of the suborder the Pinnipedia (seals, sea lions, and walruses) and the Marine Fissipedia (sea otters and polar bears).
Given the latter, the phylogenetic diversity of this grouping can affect eDNA assay design, as primer binding sites may differ in sequence conservation across these orders, which can impact amplification efficiency and detection sensitivity [34,38]. Moreover, the application of environmental DNA (eDNA) metabarcoding for marine mammals is subject to significant limitations. These include primer bias due to low sequence variation in mitochondrial markers and low genomic representation in public databases, which may result in potential false positive and/or false negative results, especially within closely related taxa such as delphinids [34,38,45,46]. The false positives may arise from contamination, sequencing artefacts, or taxonomic misassignments, while false negatives can result from DNA degradation, PCR inhibition, or insufficient template material [47].
To this extent, eDNA can identify down to species-specific level and confirm “elusive”/dubious sightings of the past, such as the harbour porpoise (Phocoena phocoena) in Danish waters and the Mediterranean monk seal (Monachus monachus) in surface water samples that visual monitoring failed to yield [30,48,49]. Therefore, eDNA expands the capacity for species detection, especially in challenging conditions or for cryptic species [40,48,49,50]. Studies indicated a positive correlation between DNA concentration and estimated population density rooted in traditional monitoring methodologies [48,51].
Nonetheless, there are challenges that eDNA methodology faces regarding marine mammal research. Although marine mammal metabarcoding primers already exist [37,38,49,52,53,54], many eDNA monitoring studies still rely on species-specific assays, particularly for rare or elusive taxa. It has been addressed that universal primer sets can vary in efficiency across species and taxonomic groups [34]. This has limited the routine application of metabarcoding for comprehensive marine mammal diversity assessments in practice.
In this study, we use the term “marine mammals” to refer to a group of animals that are classified together ecologically rather than taxonomically. This group includes members of different mammalian orders, such as Cetacea, Sirenia, and Carnivora. The phylogenetic diversity of this grouping can affect eDNA assay design, as primer binding sites may differ in sequence conservation across these orders, which can impact amplification efficiency and detection sensitivity [34,38].
Molecular identification of certain marine mammal species can be challenging due to low interspecific divergence in mitochondrial loci or limited availability of reference sequences. For instance, Tursiops truncatus and Tursiops aduncus [38] and Delphinus delphis and Delphinus capensis [55] exhibit minimal genetic differences in standard barcoding regions, which complicates species-level resolution.
Furthermore, rare or elusive taxa, such as Ziphius cavirostris, may be underrepresented in public sequence databases, which further limits the confidence in taxonomic assignment [38,51,56,57]. These constraints were considered in primer selection and the interpretation of eDNA metabarcoding results.
Also, the abiotic and biotic parameters of the marine environment may interfere with the persistence of eDNA in the marine environment. Hence, the probability of detection may fluctuate according to these parameters [21,39,57].
In this study, eDNA was applied as a marine biodiversity species detection and monitoring methodology focused on marine mammal species in the Pagasitikos Gulf (Greece, Eastern Mediterranean). Due to the fact that the gulf is characterized by high ecological value and abundant marine mammal fauna [58,59,60,61] but, simultaneously, by significant high fishing activity [62,63,64], the main hypotheses were to explore the potential correlations and associations between the different marine mammal species and between marine mammal species and total abundance within the gulf. To mitigate false positives/negatives risks, we implemented stringent quality filtering, chimera removal, and technical replication and relied on curated reference databases for taxonomic assignment.
Finally, challenges and limitations associated with eDNA methodology, as well as benefits and potential future prospects, are discussed.

2. Materials and Methods

2.1. Water Sampling

The Pagasitikos Gulf is a semi-closed gulf with bathymetry characterized by an average depth of 75 m and a deepest point of 108 m [64,65,66] (Figure 1). Water samples were derived from five areas (A, B, C, D, and E) in the Pagasitikos Gulf, covering the wide breadth of the gulf over three consecutive years, 2022 to 2024, as shown in Figure 1.
In this study, from each sampling location, 2 samples of 5 L of marine water were obtained, following [39], on each fieldwork day. Hence, overall, 20 samples per year were obtained, apart from the year 2023. Due to the two consecutive catastrophic storms “Daniel” and “Elias” in the area, only 10 samples overall were possible to be collected. Also, an additional replicate sample was taken in 2022 from areas E and B due to a high rate of dolphin encounters during the sampling process. Similarly, an additional replicate sample was taken from area A in 2024 for the same reason.
The repeat-sampling-based design increased the probability of amplifying target DNA in subsequent PCRs. Water collection was achieved via the eDNA Citizen Scientist Sampler [67], where self-preserving filter housing is provided by the manufacturer. The self-preserving filter housing provides the opportunity for the filter to be handled safely back in the laboratory. Additionally, in order to avoid “false positives” and/or “false negatives”, a strict protocol was used regarding the sampling design and implementation. This involves the amount of water processed, repetitive sampling in each station, and the process of potential PCR negative controls.
Also, filtration occurred on-site, minimizing the time of the process and potential contamination. The choice of this eDNA sample was based on the manufacturer’s specification of preloaded and individually sterilized packaged filters, leading to self-preserving filter housing and safe sample handling in the laboratory. Hence, the filter is never handled in situ, and the chance of sample contamination is low.
In order to minimize the potential DNA degradation due to environmental factors of the marine water, sampling took place as soon as marine mammals were encountered during visual surveys. In days when no marine mammal was encountered, sampling was achieved following the transect line.
The filtration of each sample involved 10 L of marine water passing through a PES filter (Smith Root, Vancouver, WA, USA, 0.45 µm pore size). After completion of fieldwork, filter samples were transferred to the laboratory, where each filter was removed from the filter housing, folded, and stored at −20 °C.

2.2. DNA Extraction and PCR Amplification

After filtration, the standard protocol of phenol-chloroform [68] was implemented. The filters were soaked in lysis buffer (Tris-Hcl 10 mM, NaCl 100 mM, EDTA 1 mM) with proteinase K+ (20 mg/mL) overnight at 60 °C. They were centrifuged twice using 25:24:1 phenol-chloroform-isoamyl alcohol, and for the DNA precipitation, cold pure ethanol and 3M Sodium Acetate were added for ~2 h at −20 °C. A final washing step was applied, and the pellet was diluted in TE (10 mM Tris-Hcl, 1 mM EDTA) and stored at −20 °C. All samples were calculated for DNA concentration in nanodrop to secure a 20 ng/μL of DNA in each PCR sample, while 260/280 threshold peaks were within the range of 1.8–2.2.
The 16S-rRNA gene (MarVer3) [45] was used for a final amplicon product of 220 bp. The PCR annealing temperature was at 60 °C for 30 cycles, using the following reagent concentrations: 5X Green GoTaq Buffer (Promega, Madison, WI, USA) (6 μL of the Buffer), 0.25 μΜ of each primer, and ddH2O to reach a final volume of 20 μL. GoTaq Green Master Mix is a premixed, ready-to-use solution containing bacterially derived Taq DNA polymerase, dNTPs, MgCl2, and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR.
Negative samples (ddH20) were also used to evaluate potential contaminations. Lastly, in order to avoid contamination, PCRs of the negative and the tested samples were run independently in separate rooms following specific laboratory precautions for eDNA analysis. Illumina barcode-tagged primers were generated and loaded in an Ion-Torren sequencer.
This study employed primers targeting the mitochondrial 16S rRNA gene, selected for their demonstrated utility in marine mammal eDNA studies [45]. On the other hand, these primers potentially allow surveys of complete marine vertebrate communities in single, high-throughput sequencing (HTS) metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators
Also, primer binding in this study’s target species (Tursiops truncatus, Monachus monachus, and Ziphius cavirostris) has been confirmed using in silico alignment against publicly available mitogenomes to ensure sufficient coverage and minimal mismatches across taxa.

2.3. Bioinformatics

Fastq reads were demultiplexed, checked for quality profiles, trimmed, and filtered using the dada2 program [68] through the R platform (version 4.3). Briefly, the frequency of each quality score at each base position of the reads was drawn and validated, chimeras were removed, and then the reads were truncated at position 210. The Dada2 denoising algorithm was used to infer the exact biological sequences present in the sample.
Also, the Learn Error Rates method was utilized to model specific error profiles, and through the dereplication method, identical reads were combined. The denoising algorithm compares sequences and, using its error model, decides whether a difference between two sequences is due to a sequencing error (and should be corrected) or true biological variation (and should be kept). It “infers” the true sequences (Amplicon Sequence Variants—ASVs) that were originally in the sample.
After the creation of the ASVs, the taxonomic origin of each unique sequence was attributed by blasting against the GenBank NT database using the local blastn+ engine [69] (the NT database was downloaded via FTP from https://ftp.ncbi.nlm.nih.gov/blast/db/—accessed on 1 March 2025). The level of homology of the hits with an e-value less than 5 × 10−5 and at least 98% identity was set. Species-level assignments were accepted only when the ASV matched with ≥98–100% sequence identity to a single species without equivalent matches to other taxa. When multiple taxa shared identical or nearly identical sequences in the database, the ASV was conservatively assigned to the lowest unambiguous rank (e.g., genus or family). This conservative approach minimized potential false positives due to incomplete reference databases or low marker resolution.

2.4. Biotic and Abiotic Parameters Assessment

Chlorophyll a and sea surface temperature SST values were retrieved from Copernicus Marine Services [70] over the sampling periods in order to assess whether biotic and abiotic parameters may acquire a role in marine mammal presence and abundance via the marine biodiversity distribution within the gulf. All biotic and abiotic factors are instrumental in marine mammal species’ abundance, habitat synthesis, and their prey items [71,72,73,74,75]. However, due to time limitations, this study is focused solely on Chl-a and SST.

2.5. Statistical Analysis

The statistical analysis of the marine mammals’ sequence reads was conducted in Jamovi (version 2.5) [76] and R [77] platforms in order to explore potential correlations and significance, while a linear regression took place. An ANOVA was used in order to test the overall significance of the linear regression. The basic hypotheses of the regression, like normality, were tested through the Shapiro–Wilk test; Q-Q Plots were produced, while autocorrelation in the residuals was tested via the Durbin–Watson test. A correlation matrix was produced in the R platform using the Pearson correlation coefficient (r). A linear regression in Jamovi was applied in order to explore potential patterns and correlations with marine mammal species.

3. Results

3.1. Overview and Taxa Detected

Overall, 53 samples were obtained from five areas over the period 2022–24. After filtering and chimera removal, the final DNA reads yield 5,209,613 reads of abundance (Bio project accession number: PRJNA1327453). After omitting the unassigned taxa, the assigned taxa resulted in 108 species (Supplementary Materials Tables S1 and S2) and 5,202,885 reads. Six different kingdoms were detected, out of which Animalia (99.1%) was the most dominant kingdom, followed by Bacteria (0.80%). Also, 18 phyla were identified, with Chordata being the most abundant one (98.97%), followed by Pseudomonadota (0.34%) and Chlorophyta (0.11%). A total of 27 classes and 54 orders were assigned, with the majority of sequences belonging to Mammalia (57.4%) and, at the order level, to Primates (54.6%).
Overall, three species of marine mammals were detected via eDNA belonging to Mammalia and in the orders of Cetardiodactyla (11,142 reads) and Carnivora (125,059 reads) (Figure 2a,b).
The species of Ziphius cavirostris and Tursiops truncatus were assigned in Cetardiodactyla, resulting in 8.35% and 91.65%, respectively (Figure 2a), while in Carnivora, the species of Monachus monachus, the sole pinniped species existing in Greek waters, made up 91.2% of the order (Figure 2b).

3.2. Data Categorisation and Statistics

The sequence reads were indexed by the taxonomic orders of the species and analyzed. In order to focus solely on marine mammal species, only the sequence reads that referred to marine mammal species detected were kept in this study. The sequence reads of the Mediterranean monk seal (Monachus monachus) were separated from the sequence reads of the other identified carnivora species and were categorized as “READSMon”. Likewise, in the order of Cetardiodactyla, the sequence reads of Cuvier’s beaked whale (Ziphius cavirostris) and bottlenose dolphin (Tursiops truncatus) were separated and classified as “READSZiph” and “READSTur”, respectively. Finally, the overall abundance (DNA reads of all species detected in the survey) was labeled as “READST”.
A correlation matrix was produced in R platform to explore correlations and statistical significance (Table 1) for each species of marine mammals, total abundance, year, and area, as well as between area and year. For the statistical investigation of the data, Pearson correlation coefficients (r) between the variables YEAR, AREA, READSMon, READSTur, READSZiph, and READST were calculated. The values of the correlation matrix were extracted, and then the corresponding p-values were calculated based on the t-statistic.
The factors of “AREA” and “YEAR” displayed almost null correlation with each species, whilst a strong positive correlation between the species of Cuvier’s beaked whale (READSZiph) and the Mediterranean monk seal (READSmon) occurred. Also, a strong positive correlation was found between the Mediterranean monk seal and the overall abundance within the gulf. Likewise, a low degree of positive correlation was displayed between the species of bottlenose dolphin (READSTur) and total abundance. On the contrary, a low degree of negative correlation between bottlenose dolphins and the factor of “YEAR” was observed.
The p values of the correlation matrix (Table 1) produced in R confirm the results of the correlation matrix, pointing out statistical significances between Cuvier’s beaked whale and the Mediterranean monk seal, and additionally reveal an effect of the overall marine biodiversity abundance (READST) on the Mediterranean monk seal.
The species of Cuvier’s beaked whale was detected in 2022 and 2024 in two marine areas (central and western), while the bottlenose dolphin was recorded in three locations (central, western, and south-central) over the period 2022–2024. Similarly, the Mediterranean monk seal was reported in all areas of the gulf, with main presence in central, western, and south-central areas over the period 2022–2024. However, no statistical significance was found regarding area or year.

3.3. Biotic and Abiotic Factors

Finally, Chlorophyll a (Chl-a) and SST values revealed little information in this study. Chlorophyll a displayed a maximum value of 0.519 in September 2024 and a minimum value of 0.218 in September 2022, whilst SST exhibited a max 28.4 °C in September 2024 and a min 26.3 °C in September 2022.
Overall, SST displayed small temperature variability (approx. 2 °C) throughout the years (Figure 3) whilst, similarly, Chl-a followed the same pattern, apart from August 2023 values (Figure 4).
Finally, due to the low level of variability displayed by the data, the linear regression yields no results between marine mammal species abundance, Chl-a, and SST in this study.

4. Discussion

Overall, a plethora of multilevel taxonomy richness was detected via eDNA analysis in this study, comprising 108 species, 6 kingdoms, 18 phyla, 27 classes, and 54 orders. Compared to the limited number of sampling days in the field, this indicates that the Pagasitikos Gulf is ecologically abundant and resonates with other studies using different abundance measurement methodologies conducted within the gulf [78,79,80].
Overall, so far, five species of marine mammals have been identified via visual monitoring to use the marine areas of the Pagasitikos Gulf. These species are the Mediterranean monk seal (Monachus monachus), bottlenose dolphin (Tursiops truncatus), common dolphin (Delphinus delphis), striped dolphin (Stenella coeruleoalba), and, occasionally, Risso’s dolphin (Grampus griseus) (Marine Mammal Monitoring Unit of University of Thessaly, unpublished data).
The molecular identification of three species of marine mammals in the Pagasitikos Gulf lines up with the existing scientific information regarding the marine mammal fauna detected in the past via other monitoring methodologies (i.e., visual) in the gulf (Marine Mammal Monitoring Unit, University of Thessaly, unpublished data) and resonates with the marine mammal fauna of Greece [81,82,83,84,85,86].
Additionally, a strong positive correlation was found between the Mediterranean monk seal and the overall marine biodiversity abundance within the gulf. Hence, the richer the resources in an area within the gulf, the higher the possibility of the Mediterranean monk seal using that habitat.

4.1. Marine Mammals’ Species Identification and Distribution

(i)
Distribution of Ziphius cavirostris
In this study, the detections of Cuvier’s beaked whale were unexpected, as no sightings or strandings had been recorded in the area of Pagasitikos Gulf before. That makes it the first-ever detection in the area of the Pagasitikos Gulf.
Although recent long-term scale research recorded the movements of Cuvier’s beaked whale in the Mediterranean Sea [85,86], the scientific information still remains limited. In Greece, Z. cavirostris has been detected in the North Ionian Sea; the Hellenic Trench; Southwestern Crete; and specific areas in the Aegean Sea, including North Icaria, Rhodes, and Cyclades [81,87,88].
Cuvier’s beaked whale is an oceanic species found in steep slopes, submarine canyons, and escarpments, with required habitat depths of at least 1000 m [81,88,89,90,91]. Hence, recording the species over the years 2022 and 2024 in different areas within the Pagasitikos Gulf raises the question whether these detections in the “abnormal” habitats for species were random or a future new ecological trend may be displayed within the gulf.
Furthermore, a strong positive correlation between Cuvier’s beaked whale and the Mediterranean monk seal occurred, with the former species found to have an effect on the latter. Consequently, it appears that it is a great possibility that the two species share the same habitats within the gulf, and especially marine areas with rich marine biodiversity.
The marine oceanography, biodiversity, substrate type, and biodiversity gene flow in the gulf differentiate within the gulf, leading to almost distinctive areas [64,78,80,92,93,94]. The central and eastern areas in the Pagasitikos Gulf are considered richer in biodiversity and biomass than the western areas based on various ecological indices [78,80]. In this study, the central and west marine areas are the main areas where all three species were detected, and combined with the correlation results on the Mediterranean monk seal, Cuvier’s beaked whale, and biodiversity abundance, it may indicate these areas as marine mammal habitat preference.
Although Cuvier’s beaked whale habitat preferences in general are totally different from those detected in this study, there are a few cases where the species has been recorded close to coastal waters. Boldrocchi et al. [95] argue that the detection of the species via eDNA in close proximity to coastal waters may involve possible migration of its cephalopod prey over fall or a potential prey-preference shift.
However, in this study, detections of the species were solely over the springtime, but at least five species of cephalopods are known to be Pagasitikos Gulf benthic fauna, with large Illex coindetii congregations mainly found in the eastern area of the gulf [78]. The main prey item of Cuvier’s beaked whale in the Mediterranean is a variety of nine families of cephalopods, including the family of Ommastrephidae, where Illex coindetii belongs taxonomically [96]. Therefore, the presence of Cuvier’s beaked whale in the Pagasitikos Gulf may be attributed to Illex coindetii groups inhabiting the muddy waters of the eastern and western areas of the gulf.
Nonetheless, other scenarios could explain the presence of the species, including seabed morphology, ballast waters, boat activity within the gulf, etc. However, scientific literature suggests that genetics and genomics research to provide robust information regarding the marine mammals’ species site-fidelity [97,98]. Thus, the presence of high-filtered reads of Cuvier’s beaked whale on an annual basis, as seen in this study, may yield similar information regarding the species’ site fidelity patterns in the Pagasitikos Gulf.
Consequently, in the era of climate crisis, eDNA can provide crucial information on marine mammals’ ecological status; identify new areas that marine mammal species have never been reported; and predict behavioral and feeding changes, especially regarding deep-diving species, such as Cuvier’s beaked whale, in specific, relatively small, and geographically separate marine areas, as seen in this study.
(ii)
Distribution of Tursiops truncantus
On the other hand, it appears that the documented marine biodiversity abundance plays a key role, to a certain extent, in the populations of bottlenose dolphin (T. truncatus) within the gulf based on the low degree of positive correlation displayed. The species has been identified as one of the main marine mammals in the area of the Pagasitikos Gulf since 2022 via visual data (Marine Mammal Monitoring Unit, University of Thessaly, unpublished data).
However, the limited data provided by this study may explain the low degree of correlations or total absence of statistical significance or correlation between these factors and marine mammals in the area. Further sampling could possibly change this result in the future, especially since our model appears to be stable (Supplementary Materials Tables S4–S6 and Figure S1). Hence, the low degree of negative correlation between bottlenose dolphins and the factor of “YEAR” found could be justified, rather than the species over the course of time, to exhibit a decline in abundance.
The distribution of bottlenose dolphins usually depends on habitat type and prey availability [71,99]. Simultaneously, the habitats of the Pagasitikos Gulf are considered rich in marine biodiversity abundance and biomass, including prey items of Tursiops truncatus such as European hake (Merluccius merluccius), pandora (Pagellus erythrinus), annular seabream (Diplodus annularis), European conger (Conger conger), and the cephalopod European squid (Loligo vulgaris) [79,100,101,102,103,104].
Additionally, the site fidelity of the species itself should be taken into account, as it may play an important role in species habitat usage [105]. Previous studies point out that the species of bottlenose dolphin expresses a preference for shallow waters of less than 100 m deep in the Mediterranean Sea and exhibits a residential pattern of behaviour [86,106].
(iii)
Correlations between marine mammals’ distribution
Overall, the species of Cuvier’s beaked whale was detected in 2022 and 2024 in central and western marine areas of the gulf, while the species of bottlenose dolphin was detected in all years of the study (2022–2024), mainly in the central and western areas, followed by relatively fewer detections in the south-central areas. Finally, the Mediterranean monk seal (Monachus monachus) was also identified in all three years of the study, covering the entire area of Pagasitikos Gulf, with the main significant areas being the central, western, and south-central areas of the gulf.
The total absence of striped dolphin (Stenella coeruleoalba) in this eDNA study is also considered a “pitfall” since the species, along with T. truncatus, is the main cetal species in the Pagasitikos Gulf and is regularly reported [107]. Other eDNA studies had similar issues where the eDNA data compared to visual data displayed identification gaps attributed to the primers used, resulting in possible influence of species detection [45,108,109]. In this study, the same set of primers was used (MarVer3), and consequently, the possibility for biased results in species identification requires further investigation.

4.2. Biotic and Abiotic Factors’ Spatial Distribution

In 2023, the area of Thessaly, where the Pagastikos Gulf is located, was severely impacted by the consecutive storms of Daniel and Elias, causing extensive floods resulting in an enormous amount of transported both terrestrial and riverine materials to enter the system of the gulf via runoff waters [110]. In six hours, the cumulative rain heights were 159.6 mm (“Daniel”) and 201.6 mm (“Elias”), respectively, over each storm [111]. Finally, these circumstances brought the overall fieldwork of that year to a standstill since no additional days at sea took place after the storms.
The Pagasitikos Gulf is a semi-enclosed basin mainly characterized by shallow depths (maximum of ~108 m), complex inflows of freshwater (rivers and torrents), and an extended summer thermocline. Hence, these characteristics reinforce the seasonal variability in salinity and frequent algal blooms [64,112,113,114]. Consequently, these oceanographic conditions affect primary productivity, with chlorophyll-a (Chl-a) serving as an indicator of phytoplankton biomass, and sea surface temperature (SST) being a key physical factor in ecosystem dynamics.
However, the present analysis detected no statistically significant effect of either Chl-a or SST on the presence of marine mammals or overall biodiversity. This may be due to the relatively narrow range of SST variation in the gulf, which is enclosed, and the decoupling of surface Chl-a from trophic availability at higher levels of the food web due to the spatial and temporal patchiness of blooms.
However, these two factors play a crucial role in the composition of biocommunities and the abundance of marine species, including marine mammal species and their prey items [59,65,71,72,73,74,75]. Hence, a further systematic comparative investigation is suggested regarding the effect of abiotic and biotic factors on the marine mammal diversity of the gulf, involving data from before, during, and after the storms.

4.3. Limitations of the Study

Although fieldwork was conducted over the same time periods each year for this study, due to a lack of funding, the number of sampling days at sea was limited over the years 2022–2024.
Moreover, the fact that only two main environmental factors were explored in this study, and considering the results provided by the analysis, indicates the need for additional abiotic factors to be included in the future.
Also, the existing scientific literature to date indicates that the detection of “loose” DNA in the marine environment is challenging to achieve due to its limited lifespan in this environment (approx. 15–20 days), depending on the oceanographic properties of each marine area [21,58,109].
Other challenging points in this study could involve the primer set used. For example, in Valsecchi et al.’s study [109], MarVer 3 (the primer set producing the largest amplicon) expressed a relative difficulty in amplification and/or in the production of DNA. Alongside the signal variability detection, which is dependent on the DNA concentration and quality from the sample collected, the absence of other marine mammal species known to use the Pagasitikos Gulf, such as the striped dolphin (Stenella coeruleoalba), could be justified.
Additionally, Monachus monachus, when it comes to eDNA sequencing, is represented by a comparatively small number of genetic sequences in public repositories, with mainly 63 nucleotide sequences and 107 proteins in GenBank. This possibly reflects its scarcity and relatively small population size [38,51,115]. This limited dataset reduces taxonomic assignment confidence in eDNA analyses and should be considered when interpreting metabarcoding results.
Regarding the two cetacean species detected in this study, the accurate molecular identification of certain marine mammal species can be hindered by low interspecific divergence in commonly used mitochondrial loci, as well as by limited taxon representation in reference databases.
For example, ecotypes of the bottlenose dolphin (Tursiops truncatus and Tursiops aduncus) and species of the common dolphin (Delphinus delphis and Delphinus capensis) exhibit minimal sequence variation in standard barcoding markers, complicating species-level identification. Furthermore, rare or elusive species, such as the Cuvier’s beaked whale (Ziphius cavirostris), are often underrepresented in public sequence repositories, which further reduces the reliability of taxonomic assignments [38,51,56,57]. These limitations were explicitly considered during primer selection and when interpreting the eDNA metabarcoding results presented here.
Also, 2023 was substantially distressful for the marine ecosystems and civil communities of the Pagasitikos Gulf due to the two consecutive devastating storms, “Daniel” and “Elias”, in the area. The storms impacted the balance, biodiversity synthesis, and water quality of the gulf and held local communities at a standstill for months. Unfortunately, only a single fieldwork project was achieved that year. Overall, the low number of fieldwork days combined with the events of 2023 most likely had repercussions regarding the actual numbers indicating species abundance.
However, new scientific information was produced regarding the marine mammal fauna of the Pagasitikos Gulf via eDNA. The methodology of eDNA, regardless of the challenges of this study, proved to be a robust way to identify species of marine mammals in a non-invasive way and overcome limitations other monitoring methodologies disclose (visual or acoustics) [51], revealing important species new to the area, such as Cuvier’s beaked whale, and new areas for marine mammal species.
The marine mammal fauna of the Pagasitikos Gulf is rich and has been studied since 2021 via visual and molecular methodologies. This study validates eDNA as an identification and monitoring methodology for marine mammal species, pointing out possible areas in the Pagasitikos Gulf as important areas for marine mammals. Systematic research is needed in order to unveil the role of the gulf in supporting deep-diving cetal species such as Cuvier’s beaked whale and define the habitat usage by the marine mammal species in the gulf (i.e., nursery or feeding ground) utilizing eDNA.
Also, in due course, the beneficial attribute of eDNA methodology regarding the actual population sizes of each marine mammal using the waters of the gulf is yet to be revealed [116]. Despite these precautions, eDNA studies remain subject to false positives and false negatives, and results should therefore be interpreted with caution and within the context of known methodological limitations [47].
Finally, to the best of our knowledge, this is the first study conducted in Greece investigating marine mammal fauna via the eDNA methodology.

5. Conclusions

The Pagasitikos Gulf is considered to be an ecologically rich marine area, justified not only by the existing scientific literature [79,80,81] but also by the relevant small number of fieldwork days in this study and the great variety of species that were detected and distributed in 6 different kingdoms, 18 phyla, 27 classes, and 54 orders via eDNA.
In the Pagasitikos Gulf, there was no scientific literature regarding the marine mammals’ fauna till the Marine Mammal Monitoring Unit of the University of Thessaly initiated visual surveys in 2021. Since then, five species of marine mammals have been identified via visual monitoring within the gulf, including the Mediterranean monk seal (Monachus monachus), bottlenose dolphin (Tursiops truncatus), common dolphin (Delphinus delphis), striped dolphin (Stenella coeruleoalba), and, occasionally, Risso’s dolphin (Grampus griseus) (Marine Mammal Monitoring Unit of the University of Thessaly, unpublished data).
Out of these species, two (2) main species of the gulf, such as the Mediterranean monk seal and bottlenose dolphin, and one (1) species, such as Cuvier’s beaked whale, which has never been observed via traditional monitoring methods nor by strandings, were detected via eDNA in this study. Hence, these molecular detections of the latter species are the first ever in the area of the Pagasitikos Gulf.
The detections of Cuvier’s beaked whale over 2022 and 2024, along with the existing scientific literature that indicates the species moves closer to coastal waters in the Mediterranean basin due to prey movements or to a potential diet shift [96], point out the efficiency of eDNA as a monitoring and detection method and reveal a few of its benefits. These benefits include the identification of “new” to science areas for marine mammals and the prediction of ecological changes, especially regarding deep-diving species in proportionally small and geographically isolated marine areas.
Although the bottlenose dolphin (T. truncatus) is one of the main marine mammal species recorded by visual monitoring within the gulf [108], in this study, the species yielded a low number of DNA reads over the years, adding more to the challenges of the study. However, the Mediterranean monk seal (Monachus monachus) appeared in all designated study areas within the gulf over time with a strong genetic signal.
The limited reference sequenced data available for M. monachus, along with the low genetic divergence observed among certain cetacean species (e.g., Tursiops spp. and Delphinus spp.), and the underrepresentation of rare taxa such as Z. cavirostris, all lower the taxonomic confidence that can be placed in eDNA analyses [38,51,55,56,57,116]. These factors were considered during primer selection and when interpreting the results.
All marine mammal species detected in this study are found in the highest level of the trophic chain feeding on a variety of prey items, including European hake (Merluccius merluccius), pandora (Pagellus erythrinus), annular seabream (Diplodus annularis), European conger (Conger conger), and the cephalopod European squid (Loligo vulgaris). These species of prey items are found in great abundance in the Pagasitikos Gulf, making the gulf rich in biodiversity and a good feeding ground for marine mammals.
The abiotic and biotic factors, such as Chl-a and SST, were found to be statistically insignificant on marine mammal species. However, more research is needed, and more environmental factors should be investigated since biocommunity synthesis and marine mammals’ prey item abundance are dependent on them [52,57,64,65,66,67,117].
In order to tackle the primer limitations, in this study, primer sets with demonstrated amplification efficiency for the detected species (M. monachus, T. truncatus, and Z. cavirostris) were chosen based on available sequence data and in consideration of database coverage.
This study faced challenges, such as the aftermath of the two storms that halted the overall fieldwork in 2023, a lack of funding that limited the seasonality of the sampling, potential primer “pitfalls”, and other factors, including the “delicate” lifespan of “loose” DNA in the marine environment. Nonetheless, this is the first eDNA study in Greece involving marine mammals and specifically in the marine area of the Pagasitikos Gulf, as marine mammal scientific information was only initiated in 2021. The amount of novel scientific information produced in this study establishes eDNA as a non-invasive, essential molecular methodology for marine mammals in Greece.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d17100692/s1, Figure S1: Q-Q plot of standardized residuals. Figure S2: Scatter plot of areas. Figure S3: Scatter plot of reads. Table S1: Species detected via eDNA in this study. Table S2: Phyla detected via eDNA in this study. Table S3: Shapiro–Wilk results. Table S4: Model Fit results. Table S5: Durbin–Watson test. Table S6: Variance Inflation Factor displaying the stability of the model in this study.

Author Contributions

Conceptualization, G.A.G. and E.A.; methodology, G.A.G., J.S. and E.A.; software, G.A.G., E.A. and C.D.; validation, G.A.G.; formal analysis, G.A.G. and E.A.; resources, A.E.; data curation, G.A.G., A.E., J.S. and A.K.; writing—original draft preparation, E.A.; writing—review and editing, G.A.G., A.E. and A.K.; supervision, G.A.G.; funding acquisition, G.A.G., A.E. and A.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Filtered fastq files were submitted to the NCBI database under the PRJNA1327453 BioProject.

Acknowledgments

We thank local fishermen and the ARION Cetacean Rescue and Rehabilitation Research Center for the sampling surveys.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Map of the location of Pagasitikos Gulf (PG) and the area zone divisions (A, B, C, D, and E) within PG in this study. The areas are divided according to the oceanographic features of the region. Red dots correspond to the sampling sites.
Figure 1. Map of the location of Pagasitikos Gulf (PG) and the area zone divisions (A, B, C, D, and E) within PG in this study. The areas are divided according to the oceanographic features of the region. Red dots correspond to the sampling sites.
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Figure 2. (a) Marine mammal classification by phylum, order, and species focused on cetacean species (Cetardiodactyla order (b) and on pinniped species detected in Pagasitikos Gulf via eDNA (Carnivora order).
Figure 2. (a) Marine mammal classification by phylum, order, and species focused on cetacean species (Cetardiodactyla order (b) and on pinniped species detected in Pagasitikos Gulf via eDNA (Carnivora order).
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Figure 3. Sampling stations (A, B, C, D and E) of this study and spatial distribution of sea surface temperature in Pagasitikos Gulf in August 2023.
Figure 3. Sampling stations (A, B, C, D and E) of this study and spatial distribution of sea surface temperature in Pagasitikos Gulf in August 2023.
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Figure 4. Sampling stations (A, B, C, D and E) of this study and spatial distribution of Chl-a in Pagasitikos Gulf in August 2023.
Figure 4. Sampling stations (A, B, C, D and E) of this study and spatial distribution of Chl-a in Pagasitikos Gulf in August 2023.
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Table 1. Summarized tables displaying statistical significance * between different marine mammal species (READSMon, READSTur, and READSZiph), overall abundance (READST) with area, and year in Pagasitikos Gulf (p < 0.05).
Table 1. Summarized tables displaying statistical significance * between different marine mammal species (READSMon, READSTur, and READSZiph), overall abundance (READST) with area, and year in Pagasitikos Gulf (p < 0.05).
YEARAREAREADSMonREADSTurREADSZiphREADST
YEAR1.000.8690.6670.9540.3630.601
AREA0.8691.000.5080.1510.3030.304
READSMon0.6670.5081.000.4150.022 *0.039 *
READSTur0.9540.1510.4151.000.3140.568
READSZiph0.3630.3030.022 *0.3141.000.083
READST0.6010.3040.039 *0.5680.0831.00
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MDPI and ACS Style

Akritopoulou, E.; Exadactylos, A.; Komnenou, A.; Sarantopoulou, J.; Domenikiotis, C.; Gkafas, G.A. Marine Mammals’ Fauna Detection via eDNA Methodology in Pagasitikos Gulf (Greece). Diversity 2025, 17, 692. https://doi.org/10.3390/d17100692

AMA Style

Akritopoulou E, Exadactylos A, Komnenou A, Sarantopoulou J, Domenikiotis C, Gkafas GA. Marine Mammals’ Fauna Detection via eDNA Methodology in Pagasitikos Gulf (Greece). Diversity. 2025; 17(10):692. https://doi.org/10.3390/d17100692

Chicago/Turabian Style

Akritopoulou, Elena, Athanasios Exadactylos, Anastasia Komnenou, Joanne Sarantopoulou, Christos Domenikiotis, and Georgios A. Gkafas. 2025. "Marine Mammals’ Fauna Detection via eDNA Methodology in Pagasitikos Gulf (Greece)" Diversity 17, no. 10: 692. https://doi.org/10.3390/d17100692

APA Style

Akritopoulou, E., Exadactylos, A., Komnenou, A., Sarantopoulou, J., Domenikiotis, C., & Gkafas, G. A. (2025). Marine Mammals’ Fauna Detection via eDNA Methodology in Pagasitikos Gulf (Greece). Diversity, 17(10), 692. https://doi.org/10.3390/d17100692

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