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1. Habitat diversity plays a significant role in shaping the genetic structure of cetacean populations. However, the processes involved in defining the genetic differentiation of these highly mobile marine mammals are still largely unknown. 2. Levels of genetic differentiation and dispersal patterns of common bottlenose dolphins (Tursiops truncatus) were assessed in the north-eastern Mediterranean Sea, with a focus on the Adriatic Sea. This is a region characterized by diverse marine ecosystems and high levels of human-induced habitat degradation. 3. Although this species seems almost uniformly distributed throughout the Adriatic Basin, genetic evidence rejected the hypothesis of a single stock. Pairwise estimates of genetic differentiation at 12 microsatellite loci, and mitochondrial DNA (entire control region, 920bp), revealed diverse levels of genetic differentiation among five putative populations from the Tyrrhenian Sea to the Aegean Sea. 4. A fine-scale genetic structure was recorded within the Adriatic Sea, where females appear to be the principal gene flow mediators. The assessment of recent migration rates indicates a relatively high level of gene flow from the North Adriatic towards adjacent areas. 5. Indication of a fine-scale population structure across the Adriatic Sea is a factor to be carefully considered in the emerging marine management scenario set by the implementation of the EU Marine Strategy Framework Directive (2008/56/CE), particularly when it comes to assessing and managing direct mortality caused by human activities (e.g. fisheries or maritime traffic). A good knowledge of population structure at the basin level is also fundamental for the identification of potential Adriatic Special Areas of Conservation for the bottlenose dolphin under the Habitats Directive (Council Directive 92/43/EEC).
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Population genetic structure of common bottlenose dolphins
(Tursiops truncatus) in the Adriatic Sea and contiguous regions:
implications for international conservation
STEFANIA GASPARI
a,
*, DRAŠKO HOLCER
b,c
, PETER MACKELWORTH
c
, CATERINA FORTUNA
d
,
ALEXANDROS FRANTZIS
e
, TILEN GENOV
f
, MORGANA VIGHI
a
, CHIARA NATALI
a
, NIKOLINA RAKO
c
,
ELISA BANCHI
a
, GUIDO CHELAZZI
a
and CLAUDIO CIOFI
a
a
Department of Biology, University of Florence, Florence, Italy
b
Department of Zoology, Croatian Natural History Museum, Zagreb, Croatia
c
Blue World Institute of Marine Research and Conservation, Veli Lošinj, Croatia
d
Italian National Institute for Environmental Protection and Research, Rome, Italy
e
Pelagos Cetacean Research Institute, Vouliagmeni, Greece
f
Morigenos - Slovenian Marine Mammal Society, Piran, Slovenia
ABSTRACT
1. Habitat diversity plays a signicant role in shaping the genetic structure of cetacean populations. However, the
processes involved in dening the genetic differentiation of these highly mobile marine mammals are still largely
unknown.
2. Levels of genetic differentiation and dispersal patterns of common bottlenose dolphins (Tursiops truncatus)
were assessed in the north-eastern Mediterranean Sea, with a focus on the Adriatic Sea. This is a region
characterized by diverse marine ecosystems and high levels of human-induced habitat degradation.
3. Although this species seems almost uniformly distributed throughout the Adriatic Basin, genetic evidence
rejected the hypothesis of a single stock. Pairwise estimates of genetic differentiation at 12 microsatellite loci,
and mitochondrial DNA (entire control region, 920bp), revealed diverse levels of genetic differentiation among
ve putative populations from the Tyrrhenian Sea to the Aegean Sea.
4. A ne-scale genetic structure was recorded within the Adriatic Sea, where females appear to be the principal
gene ow mediators. The assessment of recent migration rates indicates a relatively high level of gene ow from the
North Adriatic towards adjacent areas.
5. Indication of a ne-scale population structure across the Adriatic Sea is a factor to be carefully considered in
the emerging marine management scenario set by the implementation of the EU Marine Strategy Framework
Directive (2008/56/CE), particularly when it comes to assessing and managing direct mortality caused by human
activities (e.g. sheries or maritime trafc). A good knowledge of population structure at the basin level is also
fundamental for the identication of potential Adriatic Special Areas of Conservation for the bottlenose dolphin
under the Habitats Directive (Council Directive 92/43/EEC).
Copyright #2013 John Wiley & Sons, Ltd.
Received 22 April 2013; Revised 29 August 2013; Accepted 20 September 2013
*Correspondence to: S. Gaspari, Department of Biology, University of Florence, Via Madonna del Piano 6, 50019 Sesto Fiorentino (FI), Italy. Email:
stefania.gaspari@uni.it, stefaniagaspari@gmail.com
Copyright #2013 John Wiley & Sons, Ltd.
AQUATIC CONSERVATION: MARINE AND FRESHWATER ECOSYSTEMS
Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
Published online in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/aqc.2415
KEY WORDS: cetacean conservation; Tursiops truncatus; Mediterranean Sea; Adriatic Sea; population structure; gene ow
INTRODUCTION
Several cetacean species have greater population
structure than would be expected over relatively
small geographic scales. This is predominantly due
to their demographic history, habitat association
and foraging behaviour (Hoelzel et al., 2002).
Furthermore, dispersal may be limited by
oceanographic processes such as salinity and
temperature gradients (Fullard et al., 2000;
Jørgensen et al., 2005; Natoli et al., 2005). The
common bottlenose dolphin (Tursiops truncatus)
shows strong genetic structure among populations
across its worldwide range (Hoelzel et al., 1998;
Natoli et al., 2004). This pattern is not always
associated to geographical distance and appears to
be highly dependent on the type of environment
that the constituent individuals inhabit. The
identication of genetic discontinuities is critical
when evaluating processes affecting the distribution
of genetic variation both within and among
populations. This is particularly important for
marine species, such as Tursiops truncatus,thatare
often discretely distributed, but can nevertheless be
genetically structured.
One previous population genetic study on
common bottlenose dolphins was conducted on a
broad geographic scale from the eastern North
Atlantic to the Black Sea. It reported a general
pattern of genetic divergence between the east and
west Mediterranean basins (Natoli et al., 2005).
Common bottlenose dolphins have been studied in
only relatively limited parts of the Mediterranean,
therefore wide areas remain largely unexplored,
particularly in the eastern basin (see Bearzi et al.,
2009 for a review). Moreover, the distinction
between inshore and offshore populations has yet
to be dened, especially in the Adriatic Sea, which
is characterized by very diverse marine coastal
habitats within a relatively small geographic area
(Cushman-Roisin et al., 2010).
The common bottlenose dolphin, living mainly in
coastal areas, faces numerous human-induced
threats and has therefore attracted the attention of
both national and international conservation
authorities (Bearzi et al., 2009). The European
Habitats Directive (Council Directive 92/43/EEC)
lists Tursiops truncatus in Annex II and requires
European Union (EU) member states to establish
Special Areas of Conservation where populations
are resident. This species is also listed in Annex
IV, which demands national authorities to
monitor the status of extant populations and
human-induced mortality. Moreover, the Marine
Strategy Framework Directive (MSFD, Directive
2008/56/EC) requires all EU member states to
assess and monitor the status of the marine
environment in relation to the main pressures
caused by human activities in order to achieve a
Good Environmental Statusby 2020. Core
aspects of this framework are the development of
monitoring and management programmes at the
regional level. This includes the assessment of the
status of cetacean species and analysis of
population genetic structure (MSFD indicator
1.3.2) that may help clarify migratory routes and
identify distinct units for conservation (European
Commission, 2011). This is of particular importance
in the Adriatic Sea, a semi-enclosed basin that
represents one of the four Mediterranean MSFD
sub-regions and is affected, particularly on the
north-west coast, by human encroachment and
water pollution.
This study evaluated whether common bottlenose
dolphins living in the Adriatic Sea represent a
genetically distinct unit and/or show patterns of
population genetic structure. Dispersal routes were
also assessed across the Adriatic, Ionian and
Aegean Seas, and the non-contiguous area of the
Tyrrhenian Sea.
MATERIALS AND METHODS
Study area
This study was conducted in the Adriatic, Ionian,
Aegean and Tyrrhenian Seas (Figure 1). The Adriatic
Sea shows clear differences in coastal and submarine
topography along its longitudinal and transverse
axes, and it is divided into three sub-basins
(Artegiani et al., 1997; Cushman-Roisin et al., 2010).
S. GASPARI ET AL.
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
The northern section is shallow (average depth of
35 m) and extends south-eastwards to the 100 m
bathymetric contour. The central Adriatic is a
transition sub-basin with an average depth of 140 m
and extends as far as the 170 m deep Pelagosa sill.
The southern sub-basin is characterized by a wide
depression, more than 1200 m deep and is divided
from the Ionian Sea by the Otranto sill. The west
coast of the Adriatic Sea is fairly linear, sandy
with gentle slopes, while the east coast is irregular,
has many islands and a rocky, steeply sloping
bathymetry. Despite the north-westsouth-east
topographic division, Artegiani et al. (1997) and
Cushman-Roisin et al. (2010) considered the North
Adriatic sub-basin dynamically independent from
the Central and South Adriatic sections. Therefore,
samples were collected from the central and
southern sections of the basin and pooled as the
Central-South Adriatic sampling area.
Sample collection and genetic analysis
Tissue samples from 89 adult common bottlenose
dolphins were collected between 1992 and 2009
from stranded animals (N= 69) and free-ranging
specimens (N= 20) in the Adriatic Sea (43 stranded
and 20 free-ranging), and in the Ionian (N=6
stranded), Aegean (N= 6 stranded) and Tyrrhenian
(N= 14 stranded) Seas. Biopsy samples from
free-ranging animals were collected from an average
of one individual per social group encountered
during surveys. Samples from stranded dolphins
were collected opportunistically from isolated
individuals at different times of the year.
DNA was extracted with phenol/chloroform and
ethanol precipitation from tissue samples preserved
in salt-saturated 20% DMSO. Samples were
genotyped at 12 microsatellite loci, including
EV37Mn and EV14Pm (Valsecchi and Amos,
1996), TtruGT6 (Caldwell et al., 2002), D08
(Shinohara et al., 1997) and Ttr04, Ttr11, Ttr19,
Ttr34, Ttr58, Ttr63, TtrRH1 and TtrRC12 (Rosel
et al., 2005). To ensure accuracy in genotyping and
to standardize allele sizing for each locus, about
30% of the samples were re-amplied as controls.
Microsatellite genotypes were screened for duplicate
sampling using the Excel Microsatellite Toolkit
3.1.1 (Park, 2001) and tested for scoring errors due
to allelic dropout, null alleles and stuttering with
Micro-Checker 2.2.3 (Van Oosterhout et al., 2004).
Aegean
Ionian
Tyrrhenian
Adriatic
NORTH AFRICA
EUROPE
1234567891011121314151617181920212223242526272829
1212 8
11121 1 3 1
2
12 3 2 1
5321 111
12 111
11111
North Adriatic
Tyrrhenian
Ionian
Aegean
Central-South Adriatic
Haplotype
Figure 1. Map of the study area. Samples were obtained from stranded and free-ranging bottlenose dolphins in the Tyrrhenian, Adriatic, Ionian and
Aegean Seas (dashed circles). Dotted line in the Adriatic Sea shows approximate limit between north and central-south basins. Number of haplotypes
is reported for each sampling area.
POPULATION STRUCTURE OF TURSIOPS TRUNCATUS IN THE EAST MEDITERRANEAN
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
The mitochondrial DNA (mtDNA) entire
control region was amplied and sequenced
using the light-strand primer TURCRL5483
(5- GGTCTTGTAAACCGGAAAAGG - 3)
and the heavy-strand primer TURCRH6379
(5- GCAGACTTACACATGCAAGCA - 3)
designed specically on the threonine tRNA and
12S rRNA genes, respectively. Primer numbers refer
to the 3base of the published T.truncatus
mitochondrial genome sequence (Xiong et al., 2009).
Microsatellite alleles and cycle sequencing
reactions were resolved on an Applied Biosystems
3100 genetic analyser. Raw sequence chromatographs
from both strands were edited and aligned using
CodonCode Aligner 3.0.1 (CodonCode Corporation).
The consensus sequence consisted of 920 nucleotides
corresponding to the entire common bottlenose
dolphin mtDNA control region sequence.
Analysis of genetic variation and population structure
Allele diversity, observed heterozygosity, and
unbiased gene diversity were assessed using
GenAlEx 6.5 (Peakall and Smouse, 2012), and
tested for departure from HardyWeinberg
equilibrium (HWE) using the Markov chain
randomization implemented in Genepop 4.1
(Rousset, 2008). Allelic richness was calculated to
account for variation in sample size using the
rarefaction method implemented in Fstat 2.9.3.2
(Goudet, 1995). Haplotype and nucleotide
diversity for mtDNA sequences and population
comparisons based on F-statistics for mtDNA
and microsatellite loci were calculated using
Arlequin 3.5 (Excofer and Lischer, 2010).
Genetic differentiation among populations was also
evaluated using principal component analysis
(PCA) on multilocus genotypes implemented in
GenAlEx 6.5. This analysis transforms a number
of correlated variables (the alleles) into a smaller
number of uncorrelated variables (the principal
components) to best represent the original
relationships among sampling sites and alleles. A
Mantel test for matrix correspondence and a spatial
autocorrelation analysis were also performed using
GenAlEx 6.5 to compare patterns of genetic
variability as a function of geographic distances in
the Adriatic Sea. Pairwise genetic distance matrix
was calculated following Peakall et al. (1995) and
Smouse and Peakall (1999). Pairwise geographic
distances were calculated based on geographic
coordinates of individual sampling locations. The
spatial autocorrelation coefcient of genetic
distance (r) was calculated for 10 geographic
distance classes of 20 km and 95% condence
intervals were generated based on 1000 bootstrap
replicates. Signicance of Mantel test was based on
1000 permutations. The Bayesian clustering method
of Structure 2.3.3 (Pritchard et al., 2000; Falush
et al., 2003) was implemented to estimate patterns
of genetic structure. Markov Chain Monte Carlo
(MCMC) runs were conducted without prior
population information to assess the most
appropriate number (K) of populations needed for
interpreting the observed genotypes. The analysis
was conducted for Kvalues ranging from one to
seven (number of sampling areas plus two) using a
burn-in period of 100 000 iterations followed by
runs of 10
6
steps. Given the contiguous geographic
range and the probability of gene ow, the
admixture and the correlated frequency models
were chosen. The LOCPRIOR option (Hubisz
et al., 2009) was used to include sampling locations
information and assist clustering at lower levels of
divergence or with fewer data, as was the case with
the Ionian and Aegean sample set. The mean
likelihood L(K) was calculated over 10 runs for each
K. The mean difference was assessed between
successive likelihood values of K,L(K) and the
absolute value of the difference between successive
values of L(K), |L′′(K)|. The modal value of ΔK
(the mean of |L′′(K)| averaged over 10 runs
divided by the standard deviation of L(K)) was
estimated using Structure Harvester (Earl and
vonHoldt, 2012) and it was taken as the most likely
number of populations Kas described in Evanno
et al. (2005). The Kvalue was then used as prior
information to estimate the probability that an
individual belongs to a given population. The
program BayesAss (Wilson and Rannala, 2003) was
used to estimate bi-directional recent migration
rates among populations. The program implements
a Bayesian procedure using MCMC procedure
allowing for deviations from HardyWeinberg
equilibrium. MCMC runs of 10 × 10
6
iterations
were conducted and sampled every 1000 iterations
S. GASPARI ET AL.
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
to infer posterior probability distribution using
default Delta values. The Bayesian coalescent
approach implemented in Migrate 3.4.2 was also
applied to estimate average mutation-scaled
effective migration rates (M) between sampling
areas assuming migration-drift equilibrium (Beerli,
2006). An innite allele microsatellite mutation
model (IAM) with constant mutation rate, an
F
ST
based measure as starting parameter and an
uniform prior distribution were used. The Bayesian
run consisted of one long chain with 5 × 10
6
parameter values sampled every 100 iterations and a
burn-in of 10 000 genealogies discarded at the
beginning of each chain.
RESULTS
Measures of genetic diversity
Common bottlenose dolphins showed a relatively
high degree of genetic diversity. Mean number
of alleles per sampling area varied from 4.9 to 9
and allelic richness was similar among areas,
ranging from 4.1 to 4.3. Expected and observed
heterozygosities were 0.764 ± 0.005SE and
0.692 ± 0.023SE, respectively (Table 1). There was
no evidence of scoring errors due to stuttering,
large allele dropout or null alleles. However,
signicant departure from HardyWeinberg
equilibrium was recorded in the North Adriatic
and Tyrrhenian seas after applying sequential
Bonferroni correction. Deviation from equilibrium
was detected at four loci for the Adriatic and
Tyrrhenian populations, suggesting possible
population substructure. Population comparison
analyses were run removing those loci that
showed heterozygosity deciency for one or more
populations (Supplementary material, Table S1).
However, patterns of population divergence did
not change signicantly and therefore the original
set of 12 loci was maintained. Sequencing of
the mitochondrial DNA control region revealed a
total of 29 haplotypes with ve to 12 haplotypes
per sample area (Table 1). Mean number of
pairwise differences was 9.8 ± 0.9SE with the
lowest value (6.7 ± 1.5SE) recorded in the
Tyrrhenian Sea and the highest (11.8 ± 2.8SE)
reported for the Aegean Sea. Haplotypic diversity
was generally high, ranging from 0.67 in the
Tyrrhenian to 1 in the Aegean Sea. Twenty-four
unique haplotypes were recorded. The Aegean Sea
was represented by ve samples and each revealed
a unique haplotype. The Ionian and Tyrrhenian
Sea had 60% and 80% unique haplotypes,
respectively. The North and Central-South Adriatic
shared three haplotypes, and the most common
sequence was shared by 25 dolphins in the
Tyrrhenian and Adriatic Sea (Figure 1).
Population structure
Genetic divergence among common bottlenose
dolphins from the ve sampling areas of the
Mediterranean Sea at microsatellite loci and
mtDNA is reported in Table 2. Microsatellite data
detected signicant differentiation among all
putative populations except for the Aegean Sea,
which showed a low level of genetic divergence
from the Ionian and Adriatic Sea. A low but
signicant F
ST
wasalsoestimatedbetweentheNorth
and Sentral-South Adriatic areas. This pattern of
genetic structuring was inpartcorroboratedby
Table 1. Microsatellite loci and mitochondrial DNA diversity measures for bottlenose dolphins from ve sampling regions across the Mediterranean
basin. N, sample size; A, allele diversity; A
R
, allelic richness; H
E
, mean expected heterozygosity; H
O
, mean observed heterozygosity; k, number of
haplotypes; S, number of segregating sites; h, haplotype diversity; π, nucleotide diversity. Standard error values in parenthesis
Microsatellites Mitochondrial DNA
Sampling site NA A
R
H
E
H
O
NkShπ 10
-2
)
North Adriatic 39 9.0 (1.0) 4.3 (0.2) 0.77 (0.02) 0.71 (0.03)** 29 12 43 0.82 (0.02) 1.07 (0.09)
Central-South Adriatic 24 8.1 (0.9) 4.4 (0.2) 0.78 (0.02) 0.77 (0.03)* 16 8 23 0.87 (0.02) 0.95 (0.15)
Ionian 6 4.9 (0.4) 4.2 (0.3) 0.76 (0.03) 0.68 (0.05) 6 5 26 0.93 (0.05) 1.26 (0.32)
Aegean 6 4.9 (0.5) 4.3 (0.3) 0.76 (0.04) 0.63 (0.04) 5 5 24 1.00 (0.06) 1.28 (0.37)
Tyrrhenian 14 6.1 (0.5) 4.1 (0.2) 0.75 (0.03) 0.67 (0.05)** 14 5 20 0.67 (0.06) 0.73 (0.11)
*and **denote signicant heterozigosity deciency (P<0.05 and P<0.01, respectively) before Bonferroni correction.
POPULATION STRUCTURE OF TURSIOPS TRUNCATUS IN THE EAST MEDITERRANEAN
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
principal component analysis (PCA) (Figure 2). The
rst three components of PCA explained 88% of the
total inertia, with components 1 and 2 explaining
44% and 25% of the variation, respectively. The
Mantel test found a weak correlation between
geographic and genetic distance in the Adriatic Sea
(Rxy = 0.009, P= 0.048). Spatial autocorrelation
analysis revealed no signicant correlation. Analysis
of mtDNA sequences partially supported these
results. Signicant differentiation was, in fact,
recorded between the North Adriatic and the
Aegean sampling locations and no signicant
divergence was recorded between the Tyrrhenian
and the Adriatic Sea. Genetic comparison
between West and East Adriatic animals was
highly signicant at nuclear DNA loci (P<0.001)
but not signicant for mitochondrial DNA
sequence comparison.
Mean values of the log likelihood of the data
estimated using the Bayesian clustering approach
did not provide a Kvalue with a signicantly high
posterior probability. Conversely, using the statistic
ΔKbased on the rate of change in the log
probability of the data between successive Kvalues,
a modal value of ΔK= 21.1 was found for K=5.
This value was used as prior population
information for calculating the posterior probability
of individual assignment. However, different
proportions of dolphin multilocus genotypes were
assigned to different clusters and no strong pattern
of population structure could be detected. High
rates of gene ow were observed from the North
Adriatic to the other regions (Table 3). Migration
rates among the Central-South Adriatic, Ionian,
Aegean and Tyrrhenian seas appeared to be
negligible. Comparison between the West and East
Adriatic coastal areas revealed a pattern of gene
ow from west to east. For the West Adriatic, the
proportions of resident individuals and migrants
from the East Adriatic were 0.970 and 0.029,
respectively. For the East Adriatic, the proportions
of residents and migrants from the West Adriatic
were 0.680 and 0.320, respectively. The coalescent
approach recovered relatively high migration rates
from the Adriatic to the Ionian Sea and between the
Ionian and the Aegean Sea (range of posterior
distribution values of M: 93.1133.6). Minor rates
of gene ow were instead recorded among other
possible migration routes (Mvalues from 2.9 to 55.1).
DISCUSSION
Results from this study revealed different degrees of
genetic differentiation across the study area. An
overall pattern of population structuring was
detected by principal component analysis and, to a
minor extent, by a Bayesian clustering approach.
Pairwise comparisons based on F-statistics showed
Table 2. Genetic differentiation based on F-statistics for the bottlenose dolphin. F
ST
values are reported on the left triangulation matrix for
microsatellite loci (below diagonal) and mtDNA sequences (above diagonal)
Sampling site North Adriatic Central-South Adriatic Ionian Aegean Tyrrhenian
North Adriatic ―–0.003 0.131(*) 0.112(*) 0.021
Central-South Adriatic 0.009(*) 0.080(*) 0.073 0.057
Ionian 0.026(*) 0.025(*) 0.034 0.221(**)
Aegean 0.010 0.011 0.015 0.200(*)
Tyrrhenian 0.051(**) 0.033(**) 0.042(*) 0.043(*)
Signicance of xation indices at 5% (*) and 1% (**) levels was tested by 10 000 permutations.
Aegean
Ionian
North Adriatic Central-South Adriatic
Tyrrhenian
PC1
PC2
Figure 2. Principal component analysis of common bottlenose dolphin
multilocus genotypes from the ve study areas (North Adriatic,
Central-South Adriatic, Ionian, Aegean, and Tyrrhenian Seas).
S. GASPARI ET AL.
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
signicant differences in nuclear DNA between all
areas except between the Aegean Sea and the other
east Mediterranean sampling sites. Mitochondrial
DNA data revealed a strong divergence between the
geographically distant sampling sites of the North
Tyrrhenian Sea and Aegean Sea, but did not show a
clear separation between the Tyrrhenian Sea and
the Adriatic Sea. These results are in general
agreement with the previous study conducted by
Natoli et al. (2005) in which an overall pattern of
genetic differentiation was found between the west
and east Mediterranean basins, and mtDNA
sequence divergence was weaker than nuclear DNA
data. Lack of genetic differentiation among the
Aegean, Ionian and Central-South Adriatic in both
mitochondrial and nuclear markers mirrored the
general south-eastwardly pattern of gene ow
detected by the Bayesian inference methods.
Further subdivision was found within the
Adriatic Sea, where common bottlenose dolphins
revealed a ne-scale genetic structure at nuclear
DNA markers, showing a differentiation between
north and central-south sub-basins, as well as
between the west and east coasts. This subdivision
seems to reect the physiographic differences found
along both latitudinal and longitudinal axes of the
Basin. The Adriatic Sea presents very diverse
oceanographic features between the sandy and
fairly linear west coast and the karst rocky and
rugged east coast. Important environmental
differences are also present between the North and
Central-South Adriatic sub-basins, which are
characterized by different depth gradients and
water mass circulations (Artegiani et al., 1997).
Differences in marine habitats and resources
could be among the mechanisms by which this
differentiation has evolved and is maintained.
Different degrees of resource specialization, with
respect to prey and specic habitats, have been
documented in bottlenose dolphins by Barros and
Wells (1998), Connor (2000), and Sargeant et al.
(2005). Combined results between genetics,
photo-identication and stable isotope analyses
suggest, as a preliminary hypothesis, that factors
such as local site delity and/or physiographic
features, rather than prey specialization, are the
mechanisms maintaining the observed population
structure. In fact, stable isotopes analyses conducted
in the central Adriatic shows that common
bottlenose dolphins readily shift prey, probably
dependent on which prey is available (Holcer, 2012).
The genetic structure of bottlenose dolphin
populations at a relatively small geographic
scale was also suggested by Krützen et al.(2004)at
Shark Bay, Australia, by Sellas et al. (2005) in the
western North Atlantic, and recently by Ansmann
et al. (2012) for inshore bottlenose dolphins
(Tursiops aduncus) in Moreton Bay, Australia.
These studies and our data imply a broader,
worldwide pattern of small-scale differentiation for
this genus. Photo-identication data have suggested
that common bottlenose dolphins of the Adriatic
Sea are structured in putative local populations
(Fortuna, 2006; Genov et al., 2008, 2009; Holcer,
2012; Pleslićet al., in press), corroborating the results
of this study. For instance, in the North Adriatic, no
matches were found between individuals from the
Gulf of Trieste (Italy, Slovenia and Croatia) and the
Table 3. Means and 95% condence intervals of the posterior probabilities for migration rates between bottlenose dolphin populations. Sampling sites
listed in the rows represent populations of origin of migrants. Recipient populations are listed in the columns
Recipient population
Population of origin North Adriatic Central-South Adriatic Ionian Aegean Tyrrhenian
North Adriatic 0.981 0.298 0.219 0.189 0.273
(0.951, 0.997) (0.249, 0.327) (0.103, 0.307) (0.073, 0.298) (0.200, 0.324)
Central-South Adriatic 0.004 0.679 0.023 0.026 0.015
(0.000, 0.025) (0.667, 0.711) (0.000, 0.097) (0.000, 0.107) (0.000, 0.065)
Ionian 0.005 0.007 0.711 0.026 0.012
(0.000, 0.022) (0.000, 0.034) (0.667, 0.810) (0.000, 0.110) (0.000, 0.052)
Aegean 0.004 0.006 0.023 0.728 0.012
(0.000, 0.022) (0.000, 0.032) (0.000, 0.101) (0.669, 0.858) (0.000, 0.053)
Tyrrhenian 0.004 0.007 0.023 0.029 0.688
(0.000, 0.020) (0.000, 0.037) (0.000, 0.102) (0.000, 0.113) (0.667, 0.735)
POPULATION STRUCTURE OF TURSIOPS TRUNCATUS IN THE EAST MEDITERRANEAN
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
Kvarnerićarchipelago, Croatia, only 120150 km
apart (Genov et al., 2008, 2009). Similarly, long-term
studies based on photo-identication show an
even ner-scale population structure in this
region, reporting very little exchange among local
populations along the Croatian coast (Holcer,
2012). Conversely, some individuals were found in
both catalogues of the Kvarnerićand Kornati
archipelagos (North Adriatic), approximately
80100 km apart (Fortuna, 2006; Kammigan
et al., 2008). Data from the Vis and Lastovo
archipelagos (Central-South Adriatic), 80150 km
south of Kornati archipelago, (Holcer, 2012) did
not show any match with other sites in the region.
The use of stranded samples may lead to an
underestimate of genetic differentiation, particularly
when population structure is assessed over a
relatively small geographic scale (Bilgmann et al.,
2011). This may render difcult the identication of
management units for conservation. Although the
majority of tissue samples collected for this study
were obtained from stranded specimens, the results
did show evidence of population structure in the
Adriatic Sea, which was also strengthened by a
strong match with photo-identication and stable
isotopes data.
When considering the discrepancies between
nuclear and mitochondrial DNA the data may be
inuenced by sex-biased gene ow. Differential
dispersal of males and females can have a major
inuence on the distribution of maternally and
bi-parentally inherited genes in bottlenose dolphin
populations (Hoelzel et al., 1998). Diverse dispersal
behaviours may be adopted in different regions. In
some populations, male bottlenose dolphins disperse
more often and further than females (Krützen et al.,
2004; Möller and Beheregaray, 2004), while in other
populations there are no signicant differences in
dispersal between the sexes (Natoli et al., 2005).
Unlike the nuclear DNA data, the mitochondrial
DNA sequences comparisons recovered no
differences among sampling sites in the Adriatic
Sea, suggesting that female bottlenose dolphins may
have an important role in mediating gene ow
across the basin.
The absence of a clear assignment of individual
multilocus genotypes to distinct genetic clusters,
despite the Bayesian clustering approach
distinguishing ve partitions, may be due to
limitations of the software in detecting genetic
differentiation when F
ST
values are low (Latch et al.,
2006). The existence of both resident and visiting
individuals, as indicated by photo-identication
studies (Genov et al., 2008; Pleslićet al., in press)
may be an additional or concomitant cause of why
consistent assignment to distinct clusters could not
be detected. In particular, a signicant proportion
of either migrants or individuals of mixed ancestry
may result in a degree of gene ow that potentially
obscures population structure.
Analysis of recent migration rates recorded a
signicant rate of gene ow from the North
Adriatic towards the other study areas, and
negligible movements of individual dolphins
between all other sampled areas. This study
supports the ndings that there are differences in
movement patterns of resident and visiting animals
according to the data derived from different
projects in the North Adriatic (Fortuna, 2006;
Genov et al., 2008, 2009; Kammigan et al., 2008;
Holcer, 2012; Pleslićet al., in press). The
Kvarnerićbottlenose dolphin population showed
up to 30% rate of non-random temporary
emigration (Fortuna, 2006), while in the Vis and
Lastovo archipelagos Holcer (2012) described
areas with locally dened groups mixing with
transient individuals. Similarly, 48% of about 100
identied individuals in the Gulf of Trieste were
recorded only once and therefore considered
transient or visiting dolphins (Genov et al., 2008).
Conservation implications
The Adriatic Sea common bottlenose dolphins
inhabit an environment greatly affected by human
activities, including intensive shery, gas and oil
exploitation, maritime trafc, tourism and chemical
pollutants. These pressures, particularly shery
bycatch, may have a strong, adverse impact on
population viability and need to be carefully
assessed and managed at scales that are consistent
with the population structure of bottlenose dolphins
(Fortuna et al., 2010). Indeed, scientic assessment
of population structure, distribution and status,
identication of threats affecting a speciesviability
and implementation of conservation and
S. GASPARI ET AL.
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
management measures should not prescind from
an international and coordinated plan of actions.
The Mediterranean subpopulation(sensu IUCN)
of Tursiops truncatus is listed as Vulnerable (A2cde)
in the IUCN Red List (http://www.iucnredlist.
org/details/16369383/0). The IUCN assessment
noted that the listing of Mediterranean common
bottlenose dolphins as a single subpopulation should
not be interpreted to mean there is no further
subpopulation structure within the region.This
study provides evidence of ne-scale population
structure and dispersal patterns in the Adriatic Sea
and the eastern Mediterranean basin and supports
the necessity of identifying separate areas for
conservation. Genetic divergence between the North
and Central-South Adriatic dolphin populations
strengthens the case for conservation actions
targeting different sites. Population management
actions should also consider how the impact of
human activities differs across geographically distinct
areas. The results, describing possible south-eastward
migration routes from the North Adriatic,
advocate the development of an international
network of marine protected areas and connecting
corridors (Bearzi et al., 2011). The Convention for
the Protection of the Marine Environment and the
Coastal Region of the Mediterranean (Barcelona
1995) considers the development of protected
areas for cetacean species in the Adriatic basin.
Croatia joined the EU in 2013, and although there
is no control over marine regions currently
under the jurisdiction of Bosnia-Herzegovina,
Montenegro and Albania, the application of these
countries to become EU members would require a
certain level of cooperation. In this instance, the
development of the EU Natura 2000 network of
the Habitats Directive (Directive 92/43/EEC) and
the extension of the Pan European Ecological
Network of the Convention on the conservation
of European wildlife and natural habitats (Bern,
1979) to non-EU States, provide the instruments
to coordinate international conservation strategies
within the Adriatic Sea (see Mackelworth et al.,
2011, 2013 for a review; Genov et al., 2012). In
addition, the implementation of the Marine
Strategy Framework Directive (MSFD) (Directive
2008/56/CE) and its monitoring activities (Article
11) calls for the cooperation between EU and
third party States in regions of shared marine
waters (Article 13). This is of particular importance
as the Adriatic Sea is one of only four dened
sub-regions of the Mediterranean basin and it is
considered to be the future pilot area for the
development of marine spatial planning which
includes the denition of MPAs. While there are
clear arguments for the development of spatial
conservation measures for the Adriatic Sea, the
paucity of data available requires that further
genetic research is undertaken to help dene these
regionsinthenearfuture,especiallytohelpinthe
effective evaluation of shery-induced mortality at
the level of units-to-conserve(Taylor et al.,
2010). The MSFD requires member states to work
together to establish programmes for monitoring
the Good Environmental Statusof shared waters
by July 2014. This would be an excellent
timeframe for the development of further genetic
research in the Adriatic region.
ACKNOWLEDGEMENTS
Thanks to The Marine Mammal Tissue Bank,
University of Padua, Carola Vallini, ARCHE,
Marco Affronte, Fondazione Cetacea, Paola Pino
dAstore; Petros Lyberakis, Natural History
Museum of Crete, Elisabeth Dimitra, University
of the Aegean, Joan Gonzalvo, Tethys Research
Institute, Christos Delistathis, Spyros Eleftheriou
and Letizia Marisili. We are also grateful to Scott
Baker and two anonymous referees for useful
comments on an early version of the manuscript.
This study was funded by the Italian Ministry of
Agriculture Food and Forestry Policies (MPAAF).
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SUPPORTING INFORMATION
Additional supporting information may be found in
the online version of this article at the publishers
web site:
Table S1. Observed and expected heterozygosity per
locus per putative populations (four loci were
excluded).
POPULATION STRUCTURE OF TURSIOPS TRUNCATUS IN THE EAST MEDITERRANEAN
Copyright #2013 John Wiley & Sons, Ltd. Aquatic Conserv: Mar. Freshw. Ecosyst. (2013)
... Previous studies suggested the occurrence of genetic structure within the Adriatic Sea (Gaspari et al. 2015a), although no fine-scale estimates of recent gene flow currently exist for this region. A recent mtDNA study on bottlenose dolphins from the Gulf of Ambracia suggests a high degree of differentiation relative to other Mediterranean locations (Gonzalvo et al. 2016). ...
... All genotyped produced are novel, using a new genotyping protocol developed specifically for this study (see details below). Samples were divided into potential resident communities based on their geographic origin, and the pattern of population structure identified by Gaspari et al. (2015a). We considered 5 locations within the Adriatic Sea, corresponding to: (1) the Gulf of Trieste and adjacent waters (shortened as GoT elsewhere in the manuscript; Genov et al. 2008Genov et al. , 2019a (2) the complex island archipelago in Northern Croatia, referred to as CroatiaN (Bearzi et al. 1997;Pleslić et al. 2015;2021); ...
... Previous studies suggested the presence of fine-scale population structure of bottlenose dolphins in the Adriatic Sea (namely, some degree of differentiation between North-South and East-West; Gaspari et al. 2015a). However, details on connectivity patterns and gene flow dynamics were incomplete, due to limited sampling and lower resolution (Gaspari et al. 2015b). ...
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Accurate description of population structure and genetic connectivity is essential for efficient conservation efforts. Along the European coastline, Tursiops truncatus typically shows high site fidelity to relatively small areas, often semi-enclosed waters, but patterns of genetic connectivity among such areas are often poorly understood. In this study, we investigate the patterns of genetic structure and connectivity of Tursiops truncatus in the Adriatic Sea and contiguous Mediterranean, using multilocus microsatellite genotypes. We focus particularly on areas where photo-ID studies suggest the occurrence of local ‘resident communities’. Patterns of geographic structure were identified using multivariate methods, Bayesian assignment methods, and analyses of relatedness. Our results are consistent with the occurrence of communities with high site fidelity to the Gulf of Ambracia, Croatian island archipelagos, and the Gulf of Trieste. Dolphins in these regions do not fit a model of complete panmixia, but neither do they exhibit multiple discrete population units. Even for the community in the Gulf of Ambracia, which is well separated by several population genetic estimates, we can unambiguously identify individual dispersal to the most distant area in the Northern Adriatic Sea. We suggest that the population structure patterns in these animals might be best described as a stable metapopulation and discuss the implications of such a model for regional conservation efforts. The critically endangered Ambracian sub-population is particularly well differentiated, and is therefore at high risk of local extinction due to relatively small size, high degree of isolation and exposure to several anthropogenic pressures. The exact geographic boundaries of individual sub-populations cannot always be determined due to lack of sampling and low resolution of the methods used. Nevertheless, our results have important implications for effective conservation of local communities showing strong site fidelity.
... Conservation measures that could effectively reduce such threats should be primarily focused on regulating all human activities, raising awareness of the local community, and increasing surveillance (Figure 3 and Tables S2, S3). In addition, the expansion of the size of the N2K site and protected area network beyond Cres-Losǐnj and Visǩi akvatorij would guarantee the preservation of a larger portion of the home range of this highly mobile species (Pleslićet al., 2015;Fortuna et al., 2018), enhance genetic exchange between populations (Gaspari et al., 2015), and allow a better management of the source of threats that may be located outside the current borders of the N2K sites (Fortuna et al., 2018). The implementation of these measures would favor the resilience of the species to the effects of climate change (Wild et al., 2019;van Weelden et al., 2021). ...
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... These variables can be used to derive performance indicators such as the proportion of injured individuals, the interaction rate with fishing activities, and change in population demography (Figure 3 and Tables S2, S3). In addition, BWI recommended biopsy sampling for genetic and contamination analyses to better understand processes affecting the well-being of the local dolphin population (Gaspari et al., 2015;Zanuttini et al., 2019). ...
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Since the adoption of the Habitats and Birds Directives by EU governments, marine Natura 2000 (N2K) sites have been established in the European Mediterranean Sea, creating one of the largest international networks of protected areas. Nevertheless, to date, marine N2K sites are generally scarcely implemented, studied and monitored, and thus their management effectiveness is weak, and their environmental status is often unknown. The Interreg Italy-Croatia ECOSS project aimed at establishing the ECOlogical observing system of the Adriatic Sea (ECOAdS), to integrate the existing research and monitoring activities in the area, and to promote data sharing at international level, for enhancing monitoring and conservation in Adriatic N2K network. In the framework of ECOSS, a conceptual model was developed and applied to selected N2K sites, to review the existing knowledge, assess site effectiveness, and suggest possible improvements in their monitoring and management based on the contribution that ECOAdS can provide to their implementation. Information on social, ecological, and oceanographic elements related to the conservation and management of these case studies was gathered by consulting the project partners involved in the management and monitoring of the sites and through a literature review. The results of this study revealed a discouraging condition with no management plan in most of the sites, while regulatory measures are generally in place but without surveillance. Monitoring activities are performed occasionally, and information on presence and status of protected species is often lacking or outdated. Although the N2K network provides a unique opportunity to advance marine conservation and achieve the 30% conservation target by 2030, the biggest challenge ahead is the proper management and monitoring of N2K sites. The proposed conceptual model may be taken as a framework to properly set up ecological observing systems in the N2K network and help overcome current limitations, integrating scientific research within the N2K conservation strategies.
... Previous studies suggested the occurrence of genetic structure within the Adriatic Sea (Gaspari et al. 2015a), although no nescale estimates of recent gene ow currently exist for this region. A recent mtDNA study on animals from the Gulf of Ambracia suggest a high degree of differentiation relative to other Mediterranean locations (Gonzalvo et al. 2016). ...
... Previous studies suggested the presence of ne-scale population structure of bottlenose dolphins in the Adriatic Sea (namely, some degree of differentiation between North-South and East-West;Gaspari et al., 2015a). However, details on connectivity patterns and gene ow dynamics were lacking, due to low sampling and low resolution(Gaspari et al. 2015b). ...
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Accurate description of population structure and genetic connectivity is essential for efficient conservation efforts. Along the European coastline, Tursiops truncatus typically shows high site fidelity to relatively small areas, often semi-enclosed waters, but patterns of genetic connectivity among such areas are often poorly understood. In this study, we investigate the patterns of genetic structure and connectivity of Tursiops truncatus in the Adriatic Sea and contiguous Mediterranean, where photo-ID studies suggest the occurrence of local ‘resident communities’, and a complex pattern of geographic population structure has previously been suggested. Our results are consistent with the occurrence of communities with high site fidelity to the Gulf of Ambracia, Croatian island systems and the Gulf of Trieste. Dolphins in this region do not fit a model of complete panmixia, but neither do they exhibit multiple discrete population units. Even for the community in the Gulf of Ambracia, which is well separated by several population genetic estimates, we can unambiguously identify individual dispersal to the most distant area in the Northern Adriatic Sea. We suggest that the population structure patterns in these animals might be best described as a stable metapopulation and discuss the implications of such a model for regional conservation efforts. The critically endangered Ambracian sub-population is particularly well differentiated and is therefore at high risk of local extinction due to relatively small size, high degree of isolation and exposure to several anthropogenic pressures. The exact geographic boundaries of individual sub-populations cannot always be determined due to lack of sampling and low resolution of the methods used. Nevertheless, our results have important implications for effective conservation of local communities showing strong site fidelity.
... The fact that Lino has been determined to be a male is consistent with the general notion that males tend to be the dispersing sex, both in this genus (Krützen et al. 2004;Möller and Beheregaray 2004;Bearzi et al. 2011) and in mammals generally (Greenwood 1980;Dobson 1982;Lawson Handley and Perrin 2007), although genetic evidence from the Mediterranean Sea and the North Atlantic Ocean has suggested that female bottlenose dolphins may be equally important (Natoli et al. 2005;Rosel et al. 2009) or potentially even more important (Gaspari et al. 2015a) in promoting gene flow. Kin cooperation and familiarity with the natal area, particularly in relation to potential foraging specialisation, may oppose sex-biased dispersal and promote natal philopatry (Lawson Handley and Perrin 2007;Rosel et al. 2009). ...
... The existing evidence from photo-ID, genetics and ecological markers such as stable isotopes largely suggests that most local populations or subpopulations of bottlenose dolphins in the Mediterranean Sea are largely discrete or at least somewhat well-defined units (Natoli et al. 2005;Genov et al. 2009;Gnone et al. 2011;Gaspari et al. 2015aGaspari et al. , 2015bGonzalvo et al. 2016;Giménez et al. 2018;Pleslić et al. 2019). However, bottlenose dolphins in the region have also been documented undertaking movements over substantial distances (Bearzi et al. 2011;Gnone et al. 2011;Genov et al. 2016). ...
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Information on movements and connectivity among populations of animals is important for the delineation of units to conserve, so that demographic parameters, such as abundance, fecundity and mortality, can be placed in an appropriate population and conservation context. Common bottlenose dolphins (Tursiops truncatus) are often considered relatively ‘resident’ and demonstrating strong site fidelity to specific areas. However, this perception may partly be an artefact of the distribution and ‘habitat use’ of cetacean researchers, rather than animals themselves, and bottlenose dolphins have been shown to be capable of substantial movements, often in relatively short periods of time. Here, we report on two long-distance movements of a common bottlenose dolphin within the Mediterranean Sea, across the Tyrrhenian, Ionian and Adriatic Seas, and subsequently back across all three seas to Ligurian Sea, making these the two longest recorded movement for this species in the Mediterranean Sea to date and some of the longest in the world. We also review published records of long-distance movements in this species worldwide. This study highlights the utility of photo-identification and the importance of regional data sharing. We argue that photo-identification comparisons are always worthwhile and the results are informative regardless of the presence or absence of matches, especially with the ongoing advances in automated matching software.
... While an early study did not detect evidence of population struc- Table 3. Abundance estimates of bottlenose dolphins in the Adriatic Sea, including information on observation platform, years of sampling, Adriatic Sea sector (N = northern, C = central, S = south-ern, E = eastern, W = western) and geographic area, surface area, model used for abundance estimates, number of groups, estimated abundance, standard error (SE), coefficient of variation (CV), 95% confidence interval (CI), and literature source. ture among Adriatic bottlenose dolphins (Galov et al., 2011), such finding could be due to small sample size and geographic limitations; a subsequent study suggested a general north-south and east-west split based on nuclear DNA, but not mitochondrial DNA (Gaspari et al., 2015a). Additional work was consistent with the hypothesis of a north/central/south division, with samples from the Gulf of Trieste separating clearly from all other Adriatic samples, and instead clustering with samples from the Aegean Sea (Gaspari et al., 2015b). ...
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The Adriatic Sea is one of the Mediterranean areas most exposed to cumulative human stressors. This semi-enclosed and largely shallow basin has been subjected to intensive exploitation and destructive fishing practices for decades, resulting in biodiversity decline and poor ecosystem health. Diversity of cetaceans is lower than in other parts of the Mediterranean, and the extant dolphins and whales have been facing threats that include the combined effects of habitat loss and degradation, prey depletion, incidental mortality and injury caused by fisheries, anthropogenic noise, chemical contamination and climate change. Here, we report information for the nine cetacean species known to occur in the basin (classified as either regular, visitor or vagrant), plus three species characterized by a single record. For these species, we review evidence from field research and other studies-with a bias towards the common bottlenose dolphin *Tursiops truncatus* (by far the most intensively-studied cetacean in this area). We also describe and characterize the main threats to cetaceans in the Adriatic, relying on recent literature as well as historical information that helps frame the present status of cetaceans in the context of past human impacts (particularly the extensive killing campaigns conducted until the 1960s). Finally, we provide management recommendations to inform and guide the action that must be taken in compliance with extant legislation, marine conservation directives and international commitments to protect marine biodiversity.
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Book
Because of its centrallocation in the Old World, the Adriatic Sea has long been explored and studied. Modern methods of investigation, however, have accelerated the pace of study during the last decade. These are the ADCP currentmeter, satellite imagery, drifter technology, and, last but not least, the computer with its arsenal of tools for data analysis and model simulations. As a result of this renaissance, the Adriatic Sea and its sub-basins are currently the object of intensified scrutiny by a number of scientific teams, in Europe and be­ yond. Questions concerning the mesoscale variability that dominates regional motions, the seasonal circulation of the sea, and its long-term climatic role in the broader Mediterranean, have become topics of lively discussions. The time was ripe then when an international workshop dedicated to the physical oceanography of the Adriatic Sea was convened in Trieste on 21-25 September 1998. Its objectives were to assess the current knowledge of the oceanography of the Adriatic Sea, to review the newly acquired observations, to create syn­ ergy between model simulations and observations, and to identify directions for future Adriatic oceanography. This book, however,is not the mere proceedings of the workshop. It was written as a monograph synthetizing the current knowledge of the physical oceanography of the Adriatic Sea, with the hope that it will serve as a reference to anyone interested in the Adriatic. The book also identifies topics in need of additional inquiry and proposes research directions for the next decade.
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