ChapterPDF AvailableLiterature Review

The Gulf of Ambracia's Common Bottlenose Dolphins, Tursiops truncatus: A Highly Dense and yet Threatened Population

Authors:
  • Tethys Research Institute

Abstract and Figures

The common bottlenose dolphin (Tursiops truncatus) is the only cetacean present in the semiclosed waters of the Gulf of Ambracia, Western Greece. This increasingly degraded coastal ecosystem hosts one of the highest observed densities in the Mediterranean Sea for this species. Photo-identification data and tissue samples collected through skin-swabbing and remote biopsy sampling techniques during boat-based surveys conducted between 2006 and 2015 in the Gulf, were used to examine bottlenose dolphin abundance, population trends, site fidelity, genetic differentiation and toxicological status. Bottlenose dolphins showed high levels of year-round site fidelity throughout the 10-year study period. Dolphin population estimates mostly fell between 130 and 170 with CVs averaging about 10%; a trend in population size over the 10 years was a decline of 1.6% per year (but this was not significant). Genetic differentiation between the bottlenose dolphins of the Gulf and their conspecifics from neighbouring populations was detected, and low genetic diversity was found among individuals sampled. In addition, pesticides where identified as factors posing a real toxicological problem for local bottlenose dolphins. Therefore, in the Gulf of Ambracia, high dolphin density does not seem to be indicative of favourable conservation status or pristine habitat.
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1!
Bottled(nose)+dolphins+Tursiops(truncatus+in+the+Gulf+of+Ambracia,+western+Greece+
Joan%Gonzalvo1,%Giancarlo%Lauriano2,%Philip%S.%Hammond3,%Karine%A.%Viaud-Martinez4,%Maria%Cristina%Fossi5,%Ada%
Natoli6,%Letizia%Marsili5+
1Tethys%Research%Institute,%Viale%G.%B.%Gadio%2,%20121%Milan,%Italy%
2Institute%for%Environmental%Protection%and%Research%(ISPRA),%Via%V.%Brancati%60,%00144%Roma,%Italy%
3%Sea%Mammal%Research%Unit,%Gatty%Marine%Laboratory,%University%of%St%Andrews,%St%Andrews,%Fife%KY16%8LB,%Scotland,%
UK%
4illumina,%Inc.,%5200%illumina%Way,%San%Diego,%CA%92122,%USA%
5Department%of%Environmental,%Earth%and%Physical%Sciences,%University%of%Siena,%Via%Mattioli%4,%53100%Siena,%Italy.%%
6UAE%Dolphin%Project,%PO%Box%211973,%Dubai,%United%Arab%Emirates.%
Abstract+%
The! bottlenose! dolphin! is! the! only! cetacean! present! in! the! semi-closed! waters! of! the! Gulf! of! Ambracia,! western!
Greece.! This! increasingly! degraded! coastal! ecosystem! hosts! one! of! the! highest! observed! densities! in! the!
Mediterranean! for! this! species.! Photo-identification! data! and! tissue! samples! collected! through! skin-swabbing! and!
remote!biopsy! sampling!techniques!during!boat-based!surveys!conducted!between!2006-2015!in!the!Gulf,!were!used!
to! examine! bottlenose! dolphin! abundance,! population! trends,! site! fidelity,! genetic! differentiation! and! toxicological!
status.! Bottlenose! dolphins! showed! high! levels! of! year-round! site! fidelity! throughout! the! 10-year! study! period.!
Dolphin!population!estimates!mostly!fell!between! 130!and!170!with! CVs!averaging!about! 10%;!a!trend! in!population!
size! over! the! 10! years! was! a! decline! of! 1.6%! per! year! but! this! was! not! significant.!A! clear! genetic! differentiation!
between!the!bottlenose!dolphins! of! the! Gulf! and! their! conspecifics! from! neighboring!populations!was!detected!and!
low!genetic! diversity! was! also! found.! In! addition,! pesticides! where! identified! as! factors!posing!a! real! toxicological!
problem!for! local! bottlenose! dolphins.!Therefore,!in! the!Gulf!of!Ambracia,! high! dolphin!density!does!not!seem!to!be!
indicative! of! favorable! conservation! status! or! pristine! habitat.! On! the! contrary,! Ambracian! dolphins! are! probably!
facing!a!high!risk!of!extinction.!
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Key+ words:!Bottlenose! dolphin,! Tursiops% truncatus,! photo-identification,! site-fidelity,! abundance! estimate,! mark-
recapture,!pollution,!genetics,!Gulf!of!Ambracia,!Amvrakikos.!
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Introduction+
The!common!bottlenose! dolphin! Tursiops%truncatus,!hereafter!bottlenose! dolphin,!is!the!most!common! cetacean!on!
the!continental! shelf!of! the! Mediterranean! Sea,! where! its! distribution!appears!to! be! scattered!and! fragmented! into!
small!units!(Notarbartolo!di!Sciara,!2002).!Despite!a!worldwide!distribution!and!their!capability!of!moving!across!long!
distances,!bottlenose!dolphins!show!a!marked!population!genetic!structure!throughout!their!range!(Natoli!et!al.,!2004;!
Tezanos-Pinto! et! al.,! 2009).! Coastal! bottlenose! dolphin! populations! in! particular! are! often! resident! and! prone! to!
population!fragmentation!sometimes!exhibiting!strong!population!differentiation!even!across!a!small!geographic!scale!
(Natoli!et!al.,!2005;!Richards!et!al.,! 2013;!Mirimin!et!al.,!2011).!!Identifying!those!population!units!and!assessing!their!
boundaries!is! crucial!to!be!able!to! implement! effective!conservation!measures! to! protect!small!resident!populations!
and!ensure!the!survival!of!this!species!across!its!range.!!
In! 2006,! the! International! Union! for! Conservation! of! Nature! (IUCN)! Red! List! Authority! and! the! Agreement! on! the!
Conservation!of!Cetaceans!in! the!Black!Sea,!Mediterranean!Sea!and!contiguous!Atlantic!area!(ACCOBAMS)!agreed! to!
classify! the! Mediterranean! sub-population! of! bottlenose! dolphins! as! ‘Vulnerable’! according! to! the! IUCN! Red! List!
criteria!and!based!on! a!suspected!population!decline!of!at!least!30%! over!the!last!60!years! (Reeves!and!Notarbartolo!
di!Sciara,!2006).!Intentional!kills!and!extermination!campaigns! conducted!until!at!least!the! early!1960s!in! portions!of!
the!basin!and!recent!and!on-going!incidental! mortality!in!fishing!gear!(i.e.,by-catch)! are!considered!to!be!among!the!
main! factors! contributing! to! such! decline,! including! also! other! ongoing! threats! derived! from! overfishing! and! from!
generalized!habitat!degradation!caused! by!contamination! and!disturbance!from!marine!traffic!(Bearzi!et!al.,!2008b).!
The!bottlenose!dolphin!is!listed!in!the!Appendix!II!of!the!Convention!on!the!Conservation!of!Migratory!Species!of!Wild!
Animals! (CMS),! in! the! Appendix! II! (Strictly! Protected! Fauna! Species)! of! the! Convention! on! the! Conservation! of!
European!Wildlife!and! Natural!Habitats!(Bern!Convention),!and! in! the! Annexes!II!and!IV!of!the! EU! Habitats! Directive!
(Council!Directive!92/43/EEC).!
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Greek!waters! still! harbor! a!remarkable! richness!of! cetacean! fauna!compared! to! the!rest! of! the! Mediterranean.! And!
yet,! such! richness! is! progressively! eroding! due! to! rapidly! growing! degradation! of! the! marine! environment!
(Notarbartolo!di!Sciara!and!Bearzi,!2010).!The!bottlenose!dolphin!is!the!cetacean!species!that!is!most!commonly!found!
in! Greek! coastal! waters! and! the! second! most! abundant! species! after! the! striped! dolphins!Stenella% coeruleoalba!
(Frantzis,!2009).!Cetaceans!living!in!coastal!areas,!and!particularly!in!semi-closed!inshore!habitats,!such! as!the!Gulf!of!
Ambracia!in!western!Greece,!are!exposed!to!risks!deriving!from!a!variety!of!anthropogenic!impacts!and!are!especially!
vulnerable! because! they! often! have! restricted! geographic! ranges,! disjoined!distributions! and! limited! movements!
(Reeves!et!al.,!2003).!In!this!increasingly!degraded!Gulf,!where!the!bottlenose!dolphin!is!the!only!cetacean!present!at!
an!average! density! of! 0.37! animals!km2,! one! of! the! highest!observed! densities! in!the!Mediterranean! for! this!species!
(Bearzi!et!al.,! 2008a),! dolphins!may!be!suffering!significant!physiological!stress! by!anthropogenic!activities!(Gonzalvo!
et!al.,! 2015).! This! study! focuses! in! the!conservation!status!of!bottlenose!dolphins! in! the! Gulf! of!Ambracia!(Fig.!1)!by!
examining!their!abundance,!population!trends,!site!fidelity,!toxicological!status!and!genetic!differentiation.!
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Materials+and+Methods+
Study+Area+
The!Gulf!of! Ambracia,! also! referred!to!as! Amvrakikos! Gulf,! is! a!shallow,!semi-closed! embayment! of!405! km2!whose!
only!communication!with!the!open! Ionian! Sea! is! through! the! Preveza!Channel,!a!narrow!(minimum!width! of! 370m)!
and! shallow! (212! m! deep)! 3! km-long! corridor!(Fig.! 2).! On! average,! the! depth! of! the! Gulf! is! approximately! 30! m!
(maximum!60!m!deep),!and!its!bottom!mostly!consists!of!mud!or!sand!(Ferentinos!et!al.,!2010).!It!is!characterized!by!
its!abundant!wildlife!and,!in!addition!to!providing!key!habitat!for!bottlenose!dolphins!(Bearzi!et!al.,!2008a;!Gonzalvo!et!
al.,!2015),!it!is!an!important!foraging!place!for!the!endangered!loggerhead!sea!turtle!Caretta%caretta!(Rees!et!al.,!2013)!
and!a! breeding!site!for!the!also!critically!endangered!Dalmatian!pelican!Pelecanus% crispus!(Catsadorakis! et!al.,!2015).!
Its!northern!side,!a!complex!ecosystem,!is!composed!of!a!double!delta!from!the!rivers!Arachthos!and!Louros!and!their!
associated! marshes! and! lagoons! are! of! particular! importance! for! bird! diversity.! The! Gulf! of! Ambracia! is! partially!
included! in! Natura! 2000! an d! Ramsar! sites! and! is! protected! by! national,! Europ ean! and!international! regulations.! In!
2008!it!was!designated!as!a!‘National!Park’!in!accordance!with!the!Greek!national!legislation!(11989/08!KYA).!!
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Due!to!its!isolated!character!the!water!quality!is!strongly!influenced!by!man-made!processes;!input!of!organic!matter!
and!pollutants!comes!from!various!sources!(Friligos! et!al.,!1997;!Karras!et! al.,! 2007;! Kountoura!and!Zacharias,! 2013;!
Tsangaris!et!al.,!2010).!Rivers!Louros!and!Arachthos!are!the!main!pathways!bringing!agricultural!runoff!(Tsabaris!et!al.,!
2015;!Tsangaris!et!al.,!2010).!The!use!of! Dichlorodiphenyltrichloroethane!group!(DDTs),!an!organochlorine!known!for!
its! insecticidal! properties,! as! well! as! of!other! xenobiotic! compounds!such! as! polychlorinated! biphenyls! (PCBs)! and!
hexachlorobenzene!(HCB),!was!banned!by!the!Stockholm!Convention!in!2001!because!of!their!environmental!impacts.!
Nevertheless,! in! the! Gulf! of! Ambracia,! high! levels! of! these! compounds! have! been! detected!in! eggs! of! Dalmatian!
pelicans! as! well! as! in! eels! Anguila% anguila!(Albanis! et! al.,! 1995),! mussels! Mytilus% galloprovincialis!(Tsangaris! et! al.,!
2010)!and!fishes!(Hatzianestis!et!al.,!2001).!Although!the!use!of!these!chemicals!reached!its!peak!between!the!decades!
of!the! 40s! and! 50s,!and!continued!until!the! late! 70s,!nowadays!they!can!still!be! considered! priority!contaminants! in!
toxicological! studies.! Hence,! they! are! often! referred! as! legacy! contaminants,! which! persist! in! the! environment,!
bioaccumulate!through!the!food!web,!and!pose!a!risk!to!human!health!and!the!environment.!!
Fish! farms,! agriculture,! livestock! and! discharges! of! domestic! sewage! from! coastal! towns! and! villages! further!
contribute!to!the!nutrient!enrichment!of!the!Ambracian!waters!(Ferentinos!et!al.,!2010;!Gonzalvo!et!al.,!2014),!which!
are! rather! murky! and! highly! eutrophic,! with! Secchi! disk! readings! often! as! small! as! 2! m! (Bearzi! et! al.,! 2008a).! The!
western!part! of! the!Gulf!is!seasonally!hypoxic!while!the!eastern!part! is! seasonally!anoxic!(Kountoura!and!Zacharias,!
2013).!The!local! active! fishing!fleet! totals! about!360! boats! and! is! composed! exclusively! of! small-scale! fishing! boats!
working!primarily!with! set!nets!(i.e.!trammel!and! gill!nets),!targeting!mainly!small!pelagic/epipelagic!fish!and!shrimp!
Penaeus%kerathurus!(Gonzalvo!et!al.,!2014).!
Boat-based+survey+data+collection+
Boat! surveys! were! conducted!between! 2006! and! 2015!from! a! 5.80! m! long! inflatable! boat! with! fiberglass! keel!
(Novamarine!RH-580)!powered!by!a!100!HP! four-stroke!outboard!engine,!based!on!predefined!routes!(i.e.,!transects)!
designed!to!guarantee!a!uniform!effort!coverage!of!the!whole!Gulf!of!Ambracia!on!a!monthly!basis.!Survey!conditions!
were! considered! as! “positive”! under! daylight! and! good! visibility,! sea! state! ≤3! Beaufort! (large! wavelets,! crests!
beginning!to!break! and! scattered!whitecaps)!and! with,! at! least,!two! observers! scanning! the! sea!surface! looking! for!
dolphins.!When! spotted,! dolphin! groups! were!approached! at! low! speed,!progressively!converging!with! their!routes!
and! avoiding! sudden! changes! of! speed! and! directionality! to! minimize! potential! disturbance.! As! proposed!by! Mann!
(2000),!when!observing!a!dolphin! group!both!dolphin!activity!and!distance!among!individuals!was!considered;!hence,!
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5!
a!group! was! defined! as! ‘that! composed! of! dolphins! within! approximately! 100! m! radius! of! each! other!(Irvine! et! al.,!
1981)!that!were!moreover!observed!in!apparent!association,!moving!in!the!same!direction!and!often,!but!not!always,!
engaged! in! the! same! activity! (Shane,! 1990)’.! Group! size! estimates! and! composition! where! recorded.! The! latter!
included!four!age!categories!(i.e.,!new-born,!calf,!juvenile!and!adult)!based!on!visual!assessment!of!sizes!in!relation!to!
average!adult!size!(Bearzi!et!al.,!1997).!
Photo-identification++
At!each!dolphin!sighting,!photo-identification!effort!was!dedicated!to!obtain!as!many!good!images!as!possible!of!every!
individual! present! in! our! focal! g roup! throughout! the! duration! of! the! observation,! using! digital! Canon! SLR! cameras!
equipped!with!Canon! EF! 70200!mm! f/2.8L! USM! zoom! lens,!avoiding!bias! toward! any! particular!individuals.!Photo-
identification!was!consistently!based!on!long-term!natural!marks!such!as!notches!and!nicks!in!the!dolphins’!dorsal!fins!
(Würsig!and!Würsig,!1979;!Würsig!and!Jefferson,!1990;!Wilson!et!al.,!1997),!as!well!as!on!any!additional!mark!in!other!
parts!of!the!body.!!
Photos! were! then! cropped! around! the! dorsal! fin! and! visible! part! of! the! body.! Selection! of! photographs! followed!
recommendations! provided!by! Read! et! al.! (2003);!therefore,! it! was! exclusively! based! on! photographic! quality! using!
consistent!criteria! (i.e.!entire!dorsal!fin! visible,!fin!perpendicular!to! camera,!high!sharpness!and!resolution,! no!water!
spray!or!other!interference!masking!fin!profile).!The!best!images!of!every!dolphin!during! each!sighting!were! selected!
and!compared!with!a!catalogue!of!identified!individuals,!where!dolphins!were!classified,!depending!on!their!degree!of!
distinctiveness,!into!one! of!the!following!four!categories:!D1!-!highly! marked!(i.e.!very!distinctive);!D2!-!marked;!D3!-!
moderately!marked;!and!D4!-! slightly! marked!(poorly! distinguishable).! When! a!match! was! not! found,!the! individual!
was!given!a!unique!identification!code!and! added!to!the!catalogue.!The!matching!procedure!was! done!twice!by!two!
different! experienced! researchers,! working! independently! and! using! exactly! the! same! protocol! to! minimize! the!
number!of!matching!errors.!Identifications!and!details!relating!to!dolphin!group/sighting!membership!were!recorded!
on!a! database! to! construct! individual!sighting!histories.!In!most! cases,! calves! were! recognized! in!the! field! primarily!
based!on!their!regular!association!with!an!identifiable!adult!dolphin!(i.e.!mother);!therefore,!only!calves!and!juveniles!
with!distinctive!marks!were!included!in!the!analysis.!
Estimation+of+population+size+!
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Population!size!was!estimated!within!each!year!separately!using!the!multi-sample!closed!population!models!available!
in!CAPTURE,!run!within!software!MARK!(Otis,! et!al.,!1978;!http://www.phidot.org/software/mark/).!The!assumptions!
of! closed! models! are! well! known!and! discussed! in,! for! example,! Hammond! (2009).! ! All! closed! population! models!
assume!that!no!births,!death,!permanent!immigration!or!emigration!occur!within!the!study!period.!!In!addition,!there!
are!three!fundamental! assumptions! related! to!the! data:! marks! are!unique;! marks!cannot! be! lost;! and! all! marks! are!
correctly! recorded! and! reported.! Other! assumptions! that! relate! to! variation! in! capture! probability! depend! on! the!
particular!mark-recapture!model!used.! Failure!to!meet!these!assumptions!can!lead!to!bias!in!estimates!of!population!
size!(Hammond,!2009,!2010).!In!this!case,!the!assumptions!were!mostly!addressed!as!described!by!Bearzi!et!al.!(2008),!
who!produced!the!first!population!estimate! for! bottlenose! dolphins!living!in!the!Gulf! of! Ambracia! based! on! photo-
identification!effort!conducted!between!2002!and!2005.!
Capture!histories!for! each!individual!bottlenose!dolphin!identified!in!the!Gulf!of!Ambracia!were!constructed!for!each!
year!using!a!calendar!month!as!a!sampling!occasion.!A!time!frame!of!one!month!was!considered!a!good!compromise!
between!three!factors:!having!a!reasonable!number!of!sampling!occasions!per! year;!maximising!capture!probability!
within! each! capture! occasion;! and! ensuring! that! the! population! mixes! between! sampling! occasions! (Genov! et! al.,!
2008;!Hammond,!2010).!For!the!years!2006-2014,!five!sampling!occasions!(i.e.,!May-September)!were!chosen!for!each!
year.!In!2015,!the!season!was!shorter!resulting!in!only!four!sampling!occasions!(i.e.,!June-September).!
The! models! available! in! program! CAPTURE! are:! M0,! no! variation! in! capture! probability;! Mt,! variation! in! capture!
probability! among! sampling! occasions! (Darroch,! 1958);! Mh,! variation! in! capture! probability! among! individuals!
(Burnham!and!Overton,!1978);!and!Mb,!variation!in!capture!probability!as!a!behavioral!response! to!first!capture! (Otis!
et!al.,! 1978).! Mod els! Mth!(Chao! et! al.! 1992),! Mtb!(Pollock,! 1975)! and! Mbh!(Norris! and! P ollock,! 1996)! combine! these!
effects.! ! In!analysis,! program! CAPTURE! was! first! used! to! determine! the! most! appropriate!o f!these! models! for! each!
data!subset! D1+D2!and!D1+D2+D3!in!each! year!according!to!the! model! section!criteria!based! on! the!goodness!of!fit!
tests!conducted.!Behavioral!response!models!are!not!generally!considered!appropriate!in!a!photo-identification!study!
because! there! is! no! reason! why! dolphins! should! react! differently! following! first! capture.! Nevertheless,! they! were!
included!here!to!investigate!whether!they!were!selected!as!the!most!appropriate!in!any!year.!In!addition,!a!consistent!
set! of! models! was! run! on! all! years! (Mt,! Mh,! Mth)! with! the! aim! o f! constructing! consistent! time! series! to! investigate!
trends!in!population!size!over!time.!!
!
7!
Markrecapture! estimates! refer! to! the! population! of! animals! that! have! appropriate! marks,! as! defined! above.! To!
derive! an! estimate! of! the!total! population,! the! estimated! number! of! marked! individuals! must! be! divided! by! the!
proportion!of!marked!animals!in!the!population!(e.g.,!Wilson!et!al.,!1999).!!
The!proportion!of!marked!individuals!(θ)!was!estimated!for!each!year!as!the!mean!of!the!proportion!of!“marked”!and!
“unmarked”!individuals!in!each!encounter!for!the!D1+D2!and!D1+D2+D3!data!subsets.!The!variance!of!estimated!total!
population!size!was!estimated!as:!
!"# !!"!#$ = !!"!#$
!!"#(!)
!!+
!"#(!)
!!!
where!N!is!the!number!of!marked!individuals!in!the!population!and!Ntotal!is!total!population!size!(Urian!et!al.,!2015)!.!!
In! analysis,! two! data! subsets ! were! considered.! The! first! was! restricted! to! th e! most! distinctively! marked! individuals!
(D1+D2).!The!second!also!included!moderately!marked!individuals!(D1+D2+D3).!Including!moderately!distinct! animals!
maximizes!sample!size! but!may! cause! positive! bias! because! of! missed! matches!between! individuals.! Using!only! the!
most!distinct!animals!minimizes!the!possibility!of!bias!resulting!from!missed!matches!but!the!reduced!sample!size!will!
result!in! poorer! precision.!Slightly!marked!individuals!(D4)!were!not!considered!at! all!in!analysis.!The!estimate!of! the!
number!of! marked! animals! in! the!D1+D2! data! subset! is! expected!to! be! smaller! than! in!the!D1+D2+D3! data! subset.!
However,! the! proportion! of! marked! animals! in! the! population! is! will! also! be! smaller! for! D1+D2! subset! than! the!
D1+D2+D3! subsets! and,! theoretically,! estimates! of! the! total! population! from! both! analyses! should! be! the! same.!
Consideration!of!both!sets!of!estimates!allowed!the!trade-off!between!bias!and!precision!to!be!investigated.!
Tissue+sampling+(skin+swabbing+for+genetics+and+biopsy+sampling+for+toxicology)+
Remote! biopsy! sampling! of! free-ranging! cetaceans! is! an! inexpensive! tool! used! to! collect! valuable! data! for!
conservation! and! management.! Analyses! of! skin! and! blubber! samples! have! provided! information! on! population!
genetics,! hormone! levels,! and! contaminant! loads! (Gorgone! et! al.,!2008).! Between! 2001! and! 2007,! 20! skin! samp les!
were!collected! from! bow-riding! dolphins! with! the! use! of! a!sterilized! nylon!scrub! pad!affixed! to!the! round!end! of! a!
wooden!pole!through!“skin!swabbing”;!a!method,!adapted!from!Harlin!et!al.!(1999),!that!provides!samples!suitable!for!
amplification! and! sequencing! of! mitochondrial! DNA.! Other! skin! samples! were!also! opportunistically! obtained! from!
four!dead!individuals!found!stranded!or!floating!adrift.!!
!
8!
In!July!2013!a!1-week!biopsy!sampling!campaign!was!conducted!using!standard!procedures!and!equipment!(Gorgone!
et!al.,!2008;!Kiszka!et!al.,!2010).!Skin!and!blubber!tissue!samples!were!collected!using!a!crossbow!(Barnett!Panzer!V,!68!
kg!draw!weight)!and!custom!made!bolts!and!tips!(Ceta!Dart,!Copenhagen,!Denmark).!Tips!were!25!mm!in!length!and!7!
mm!in! internal!diameter.!A! high-pressure!molded!stopper!prevented!the!bolt!from!penetrating! more! than!about!20!
mm!forcing!it!to!bounce!back.!The!dorso-lateral!area!below!the!dorsal!fin!was!the!body!region!aimed!at.!Sampling!was!
only!attempted!on!adult!dolphins.!No!individuals!accompanied!by!offspring!(or!in!groups!in!which!offspring!is!present)!
were!sampled.! In! case! of! doubt! a! precautionary! approach! was!applied! (i.e.,! no! sampling! attempted).! Continuous!
photo-identification!effort!allowed!identifying!15!of!the!16!sampled!animals.!!
Genetic+analysis+
A!total!of!74!bottlenose!dolphin!samples!from!5!different!populations!were!included!in!this!study!(Fig.!2).!From!the!24!
skin!samples!of! bottlenose! dolphins! from! the! Gulf!of! Ambracia! (GA),! only!19! were!viable!(Table! 1).!Those! nineteen!
dolphins!were! sequenced! for! the! first! time,! and! compared! with! previously! analyzed! samples! from! the! Western!
Mediterranean!Sea!(WM,!n!=! 13),!Adriatic!Sea!(ADR,!n=20),!Inner! Ionian! Sea!Archipelago!(IISA,!n=9)! and! the! Aegean!
Sea!(AEG,! n=13)!(Natoli!et!al.,! 2005;! Viaud-Martinez!et!al.,!2008).! The!methodology!followed!for! tissue!preservation,!
DNA!isolation!and! the! sequencing! of! the! first!442!base!pairs!(bp)!of!the!mitochondrial!DNA!(mtDNA)!control!region!
was!followed!as!described!in!Viaud-Martinez!et!al.!2008.!
Sequence! alignment! was! performed! using! 650! bp! of! mtDNA! obtained! from! previously! published! studies! collected!
from!dolphins!in!the!ADR!(n!=16),!IISA!(n!=!9)!(Natoli!et!al.!2005),!and!442!bp!of!mtDNA!from!dolphins!in!the!ADR!(n!=!
4)! and! AEG! (n! =! 13)! (Viaud-Martinez! et! al.! 2008).! ! These! sequences! were! aligned! with! new! dolphin! sequences!
obtained!from!the!Gulf!of!Ambracia!(n!=!19).!!Sequence!alignment!was!performed!using!Sequencher!3.1!(Gene!Codes!
Corporation).!!Final!haplotypes!were!reported!for!the!442!bp!shared!between!these!populations.!!
Genetic! diversity! within!dolphin! populations! was! measured! using!haplotype! diversity! (He),! which! is! equivalent! to!
expected! heterozygosity! after! adjustment! to! population! size,!and! nucleotide! diversity! (π;! Nei,! 1987)! using! Arlequin!
v.3.5.1.2!(Excoffier!and!Lischer,!2010).!Analysis!of! molecular! variance! (AMOVA)!was!used! to! estimate! the!degree!of!
subdivision!among!the!5!dolphin!populations.!The!null!hypothesis!of!no!genetic!divergence!between!the!5!populations!
was!tested!using!Contingency!or!Exact!Test!in! Arlequin!v.3.5.1.2!(Excoffier!and!Lischer,!2010);!this!test!has!previously!
been!demonstrated!to!be!a!robust!measurement!for!identifying!population!boundaries!(Waples!and!Gaggiotti,!2006).!
!
9!
Genetic!differentiation!among! pairwise! population! was! inferred! as! FST!(Weir! and! Cockerham,!1984),! Tajima’s!D! and!
Fu’s!Fs!estimates!for!each!population!were!calculated!with!Arlequin!v.3.5.1.2!(Excoffier!and!Lischer,!2010).!Haplotype!
genealogy! was! reconstructed! using! Haplotype! Viewer! (Ewing,! 2010)! using!maximum! parsimony! (Phylip! v.3.69;!
Felsenstein,!1989),!where!the!size!of!each!node!(circle)!represents!the!frequency!of!a!haplotype!and!the!length!of!the!
links!(or!number!of!black!nodes)!represents!the!genetic!divergence!among!haplotypes.!
Toxicology+analysis+
HCB,! DDTs! and! PCBs! were! measured! in! subcutaneous! blubber! of! 14! bottlenose! dolphins! according! to! the! EPA!
(Environmental! Protection! Agency)! meth od! 8081/8082! with! modifications! (Marsili! and! Focardi,! 1997 ).! The! samples!
(about! 0.1! g)! were! lyophilized! in! an! Edwards! freeze! drier! for! 2! days! and! extracted! with! n-hexane! for! gas!
chromatography! in! a! Soxhlet! apparatus!(Marsili! et! al.,! 2014).! Capillary! gas -chromatography! revealed! HCB,! op’-! and!
pp’-!isomers!of!DDT!and!its!derivatives!DDD!and!DDE,!and!30!PCB!congeners.!Total!PCBs!were!quantified!as!the!sum!of!
individual!congeners!(IUPAC!no.!95,!101,!99,151,!144,!135,!149,!118,!146,!153,!141,!138,!178,!187,!183,!128,!174,!177,!
156,! 171,! 202,! 172,! 180,! 199,! 170,! 196,! 201,! 195,! 194,! 206;! Ballschmiter! and! Zell,! 1980),! while! total! DDTs! were!
calculated!as! the! sum! of! pp’DDT,! op’DDT,! pp’DDE,! op’DDE,! pp’DDD! and! op’DDD.! The! detection! limit! was!0.1! ng/kg!
(ppt)!for!all!the!OC! analysed.!The!extracted!organic!matter!(EOM%)!from!freeze-dried!samples!has!been! evaluated!in!
all!samples.!!
Data!were!processed!using!Statistica!5.0!(Statsoft).!Precise!information!on!the!distribution!of!values!was!obtained!by!
the!ShapiroWilks!W!(significance!level:!p<0.05),!a!normality!test.!Most!of!the!variables!were!not!normally!distributed;!
so! non-parametric! tests! should! be! used.! However,!do! to! the! small! number! of! samples! no!subsequent! statistical!
elaboration!was!possible!to!evaluate!differences!between!groups.!!
!
Results+
Boat-based+survey+
Across!10!years!of!research!in!the!Gulf!of!Ambracia,!with!74!months!spent!in!the!field!and!a!total!of!770!daily!surveys,!
more!than!13000!km!of! survey! effort!were!conducted!under!positive!conditions,! resulting! in!631!bottlenose!dolphin!
!
10!
sightings!and!almost! a! thousand! hours! spent! with! dolphins!(table! 2).!In! years! 2006!and!2007! monthly!surveys! were!
also!conducted!in!the!Ionian! Sea!open!waters!to! look!for!possible! sightings!of!bottlenose! dolphins!in!the!vicinities!of!
the!mouth!of!the!Gulf,!where!a!total!of!667!km!of!survey!effort!under!favorable!conditions!produced!only!one!sighting!
in!which!15!bottlenose!dolphins!were!photo-identified;!none!of!them!had!(or!has!been)!ever!found!inside!the!Gulf.!
Photo-identification++
A!total!of!185!dolphins!were!identified.!The!rate!at!which! new! individuals! were! photo-identified!during! the! 10-year!
study!period!is! shown! in! Figure!3.!This! discovery!curve! rose! sharply!in! 2006,! coinciding!with! the! start!of! the! photo-
identification! work,! and! then! increased! more! slowly! followed! by! an! asymptotic! pattern! from! 2007! onwards.! The!
progressive!flattening!of!the!discovery!curve!and!the!high!site-fidelity!shown!by!the!dolphins!(Fig.!4),!indicate!that!the!
population! was! effectively! geographically!closed! (i.e.! confined! to! the! Gulf! of! Ambracia )! during! breeding! (sampling)!
seasons!during!the!period!of!the!study.!
Population+size+and+trend+
The! data! subsets! D1+D2! and! D1+D2+D3! included! 108! and! 148! individuals,! respectively.! The! population! estimates!
produced!for!both! subsets!are!shown! in! Table! 3.!The!model! selected!as!most! appropriate!varied! from! year! to!year.!
Model!Mtb!was!selected!in!2009!for!both!data!subsets!but!the!estimates!were!considerably!larger!than!any!other!and!
were!very! imprecise;!they! were!therefore!considered!unreliable!and! excluded!from!further!consideration.!Estimates!
for!the! D1+D2+D3!data! subset! were!larger!and!slightly!more!precise! than!for!the!D1+D2!data!subset!in! each!year,!as!
expected!because!they!include!additional!less!distinct!animals!and!therefore!larger!sample!sizes.!The!D1+D2+D3!data!
subset!estimates!were,!however,!more!variable!among!years.!
Estimates!for!each!year!did! not!vary!greatly!among! models.!Model!M0!was!selected!as!the!most!appropriate!in!three!
of!the!10! years! for! each! data! subset;! in! each!case!the! estimates! were! the! same! as!for!model! Mt.! Models! including!
heterogeneity!of!capture!probabilities,!Mh!and! Mth,!were!selected!as!the!most!appropriate!in!four!of!the!10!years!for!
each!data!subset.!Estimates!from!these!models!were!almost!always!larger!than!from!model!Mt,!which!is!indicative!of!
heterogeneity!of!capture!probabilities!in!the!data!causing!a!negative!bias!if!not!accounted!for.!Estimates!from!model!
Mth!were!usually!the!least!precise!but!CVs!were!generally!less!than!0.1.!
!
11!
All!things!considered,!the!series!of!estimates!from!model!Mth!are!identified!as!the!most!robust.!These!estimates!from!
the!same!model!are!much!more!consistent!over!the!10!year!study!period!than!estimates!chosen!as!most!appropriate!
by!CAPTURE.! !They!are!also!likely!to!be!the!least! biased!estimates!because! they!allow!capture!probability!to!vary!by!
both!time!and!individual.!
Table! 4! shows! these! estimates! from! model! Mth!corrected! for! the! estimated! proportion! of! marked! animals! in! the!
population! for! each! year! and! each! data! subset.! The! estimated! proportion! of! marked! animals! in! the! population! is!
larger!in!each!year!for!data!subset!D1+D2+D3!than!for!data!subset!D1+D2,!as!expected.!The!estimated!proportions!are!
quite!consistent!for!data!subset!D1+D2!(0.432-0.645)!and! highly!consistent!for!data! subset!D1+D2+D3!(0.636-0.772).!
Estimates!of! total!population!size! for!each!data!subsets!are! very!consistent!in!most!years,!differing!by!less!than!10%!
except! in! 2007! (D1+D2! higher)! and! 2013! (D1+D2+D3! higher).! Thus,! there! is! no! evidence! of! any! bias! caused! by!
including! moderately! distinct ive! animals! in! analysis.! In! addition,! estimates! of! total! population ! size! for! data! subset!
D1+D2+D3! are! consistently! more! precise! than! those! from! the! D1+D2! data! subset! (mean! CV! of! 0.096! for! D1+D2,!
compared!to!0.118!for!D1+D2+D3).!
The! best! series! of! population! estimates! is! therefore! considered!to! be! the! one! using! the! Mth!model! applied! to! the!
D1+D2+D3! data! subset!(Fig.! 5).! A! regression! of! log! total! population! size! generated! an! annual! decline! of! 1.64%!
(SE=1.54%;!95%!CI!=!-5.04%!-!1.89%)!but!this!was!not!significant!(p=0.31).!!
Genetics+
The!analysis!of!74!sequences!for!442!bp!identified!16!unique!haplotypes!characterized!by!22!polymorphic!sites!(Table!
5;!Genbank!accession! in! progress).! ! The! Gulf! of! Ambracia!population! (GA)! was! characterized!by!only!two!haplotypes!
and!one!of!those!was!observed!in!18/19!individuals.!Both!haplotypes!were!unique!to!the!GA!population.!GA!dolphins!
did! not! share! any! haplotype! with! the! other!4! populations.!The! haplotype!network! identifies! a! highly! represented!
haplotype!among!all!the!populations!except!for!the!GA!and!IISA!populations!(Fig.! 6).!The!two!unique!haplotypes!that!
characterize!the! GA! population! were! one! and! five!mutation! steps! away!from! a! haplotype!observed! in! the! ADR!and!
AEG!populations,!respectively.!
Both!haplotype!and!nucleotide!diversities!were!lower!for!the!GA!population!(He!=!0.11,!π!=!0.003)!when!compared!to!
all! the! other! populations! (0.74! ≤! He!≤! 0.92,! and! 0.009! ≤! π! ≤! 0.0016).! ! Tajima’s! D! were! not! significant! for! all! the!
!
12!
populations!except!for!GA!(Tajima’s!D!=!-2.273,!p!value!=!0.001),!whereas!Fu’s!values!were!all!not!significant!including!
for!the!GA!population.!!
The!analysis!of! population! differentiation!resulted!highly!significant!(AMOVA! FST!=! 0.375,! df! =! 73,! overall! p! <! 0.001).!!
Exact! Tests! of! haplotype! distributions! supported! significant! differences! between! the! 5! populations.! ! The! null!
hypothesis!of!no!genetic!divergence!was!rejected!(p!<!0.001).!!Genetic!differentiation!among!pairwise!populations!is!
reported!in! Table!6.!Significant!genetic!differentiation!was!observed!among! all!populations,!except!between!the!ADR!
and!AEG!population.!The!GA!population!showed!the!highest!FST!values!when!compared!with!all!the!other!populations!
analyzed.!!
Toxicology+
The!OCs!levels!found!in!the!subcutaneous!blubber!of!the!14!sampled!bottlenose!dolphins,!reported!as!dry!weight!and!
in!lipid!weight!values!(d.w.!and!l.w.,!respectively),!together!with!the!lipid!percent!(EOM%),!water!percent!(H2O%)!and!
information! on! their! sex! and! sexual! maturity,! are! shown! in! Table! 7.! The! sex! of! the! individuals! was! established!
genetically.!Males!of!at!least!10!years!of!age!and!5-10!years!old!females!were!considered!sexually!mature!(Wells!and!
Scott,!1999).!The!age!of!the!dolphins!was!estimated!based!on!their!photo-identification!stories.!For!instance,!a!dolphin!
recorded!as! a!new-born!in!September!2006,!when!first!identified,!was!considered!to!be!7!years!old!when!sampled!in!
July!2013.!The!individual!AMV12!was!genetically!recognized!as!a!male!but,!since!it!was!not!photo-identified!during!the!
sampling,!his!age!could!not!be!determined.!!
To!make!comparisons!between!the!different!xenobiotic!levels!detected!in!each!single!individual!and!within!the!various!
interest!groups!(gender! and/or! sexual! maturity),!the!results! were! considered!on!the!basis!of! lipid!content.!From!the!
analyzed! OCs,! HCB! was! the! compound! with! the! lowest! levels! and! with! relatively! similar! values! amon g! individuals!
(Table!7).!Large!differences!were!detected!in!the!levels!of!DDTs!and!PCBs! between!individual!blubber!samples,!which!
ranged!from!7.6!µg/g!l.w.!to!399.7!µg/g!l.w.!and!from!3.0!µg/g!l.w.!to!108.7!µg/g!l.w.,!respectively.!DDTs!values!were!
higher!for!all!the!analyzed!animals,!except!for!dolphin!AMV04,!who!had!almost!the!same!levels!of!the!two!xenobiotics,!
and!for!AMV11,!who!had!three!times!more!PCBs!than!the!DDTs!(Fig.!7).!
The!ratio!between!DDTs!and!PCBs! (ΣDDT/ΣPCB)! has!been!used!for!characterizing!the!magnitude!of!the!contributions!
from!agricultural!and!industrial!sources!to!marine!mammal!contamination!(Aguilar!et!al.,!1999),!because!generally!it!is!
!
13!
higher!in!water!masses!closer!to!agricultural!areas!and!lower!in!waters!closer!to!industrialized!areas.!In!the!Ambracian!
dolphins,!the!ΣDDT/ΣPCB!ratio!varied!between!0.352!and!7.463,!with!a!mean!value!of!2.43.!!A!typical!technical!DDT!is!
composed! of! pp’DDT! (77.1%),! opʹDDT! (14.9%),! ppʹDDD! (0.3%),! opʹDDD! (0.1%),! ppʹDDE! (4.0%),! opʹDDE! (0.1%)! and!
unidentified! compounds! (3.5%)! (WHO,! 1979)! and! presents! a! pp’DDE/pp’DDT! ratio! of! 0.05.! Therefore,!if! the! ratio!
pp'DDE/pp'DDT! has!high! values,! it! can! be! deduced! that! the! majority! of! the! active! substance! (pp’DDT)! has! been!
degraded! to! pp’DDE,! which! means! that! no!recent! entries! o f! insecticide! occurred!(Aguilar,! 1984).! In! all! bottlenose!
dolphins,! pp'DDE/pp’DDT! ratio! was! always!higher! than! 10,! reaching! a! maximum!value! of! 147! in! blubber! of! AMV6.!
pp'DDE/DDTs!ratio,!as!well!as!having!a!similar!meaning!to!the!pp’DDE/pp’DDT!ratio,!can!also!indicate!the!efficiency!of!
the! metabolic! processes! of! a! population! (Borrell! and! Aguilar,! 1987).! In! fact,! the! ppDDE/DDTs! ratio! indicates! the!
relative!abundance!of!metabolized!forms!of!DDT;!with!values!from!0.81!to!0.96.!!
Sexually!mature!males!(i.e.,!adults)!had!the!higher!levels!of!all!xenobiotics,!than!immature!dolphins!of!the!same!sex!
(Table!8).!This! difference! was!particularly!high!in!the!case!of!DDTs!and!was!observed!independently! of! the! group! in!
which! male! dolphin! AMV12,! whose! sexual! maturity! was! considered! uncertain,! was! included.! Higher! levels! of!
xenobiotics!where! also! detected!in!females,!when!sexually!mature.!Mean!value! of! DDTs!was!higher!in! males!than!in!
the!females.!Contrarily,!females!had!higher!HCB!and!PCBs!mean!values.!!
!
Discussion+
Site-fidelity+
Bottlenose! dolphins! in! the! Gulf! of! Ambracia! showed! high!levels! of! year-round! site! fidelity! throughout!the! 10-year!
study! period,! which! is! consistent! with! previous! findings! (Bearzi! et! al.,! 2008).! ! Dolphin! groups! encountered! in! the!
southwest! portion! of! the! Gulf,! in! areas! closer! to! the! Preveza! channel,! were! followed! for! periods! of! up! to! several!
hours,!but! they! never! entered! the! narrow! and! shallow! corridor!leading! to! open! seawaters.!Three! individuals!firstly!
photo-identified!in!the!Gulf!of!Ambracia!and!regularly!observed!between!2003-2008,!were!subsequently!found!in!the!
Inner!Ionian! Sea! archipelago!and!in!the! Gulf!of!Corinth!(Bearzi!et!al.,!2010),!not! to! be! seen!ever!again!in!Ambracian!
waters!(Gonzalvo,!unpublished!data;!Fig.!4).!Based!on!photographs!of!their!genital!area,!all!three!animals!were!males.!
This!is!consistent!with!the!hypothesis!that!males!are!more!wide-ranging!than!females,!and!they!may!therefore!be!the!
!
14!
primary! vectors!of! genetic! exchange! (Wells!et! al.! 1987;! Bearzi! et!al.! 1997).!Such! observations!indicate!some! small!
degree!of!emigration,!but!no!immigration!into!the!Gulf!has!been!recorded!so!far.!The!observed!extent!of!occurrence!
of! the! population,!defined! as!the! area! contained! within! the! shortest! continuous! imaginary! boundary! which! can! be!
drawn!to!encompass!all!the! known,! inferred! or!projected! sites! of! present! occurrence!of!a! taxon,! excluding! cases!of!
vagrancy,!was!about!three!quarters! of! the!total!area!of!the!Gulf!(i.e.,! approx.!300!km2,! excluding!enclosed! marshes!
and!lagoons),!falling!within!the!range!of!values!required!(<! 5000!km2)!for!classification!as!Endangered!under!criterion!
B1!of!the!IUCN!Red!List!(IUCN,!2012).!!!
Population+size+and+trend+
The!approach!taken!to!analyze!the!photo-identification!data!to!estimate!population!size!was!to!use!closed!population!
models!applied!to!monthly!(during!summer)!sampling!occasions!within!years!rather!than!open!population!models!
applied!to!annual!sampling!occasions.!Open!models!are!best!suited!for!estimation!of!survival!rate!and!tend!to!be!less!
robust!than!closed!models!for!estimating!population!size;!in!particular,!they!are!unable!to!take!account!of!
heterogeneity!of!capture!probabilities!(Hammond!2009;!2010),!which!was!shown!to!be!influential!in!our!case.!An!
alternative!could!be!to!use!the!robust!design!analytical!framework,!which!would!combine!the!use!of!closed!models!
based!on!monthly!sampling!occasions!to!estimate!population!size!within!years!with!open!models!based!on!annual!
data!to!estimate!survival!rate.!Temporary!emigration/immigration!rates!between!years!can!also!be!estimated!and!this!
could!be!interesting!to!investigate!here.!However,!robust!design!models!are!data-hungry!and!the!small!number!of!
animals!in!this!population!may!limit!the!utility!of!this!approach.!
Estimates!of!the!number!of!marked!individuals!from!the!closed!population!models!were!variable!from!year!to!year!if!
the!model!selected!as!the!most!appropriate!was!used!in!each!year.!There!was!much!more!consistency!when!a!single!
model!was!used!for!each!year.!Models!accounting!for!heterogeneity!of!capture!probabilities!among!individuals!
consistently!gave!larger!annual!estimates!than!models!without!heterogeneity,!indicating!that!even!when!these!models!
were!not!chosen!there!was!still!likely!some!heterogeneity!in!the!data.!Thus,!we!feel!confident!that!choosing!the!time!
series!of!estimates!from!a!single!model!with!heterogeneity!is!justified!as!the!best!approach!in!this!study.!A!similar!
approach!was!taken!in!a!study!of!northern!Norwegian!killer!whales!(Kuningas!et!al.!2014).
Analysing!data!subsets!with!and!without!moderately!distinct!individuals!to!explore!the!trade-off!between!bias!and!
precision!in!estimates!showed!that!including!moderately!distinct!individuals!increased!precision,!as!expected!because!
!
15!
of!larger!sample!size,!but!found!no!evidence!that!including!moderately!distinct!individuals!caused!bias.!Thus,!our!
estimates!based!on!the!D1+D2+D3!data!subset!were!the!best!estimates.!!
These!population! estimates! over!the!10-year!study!mostly!fell!between!130!and! 170! with! CVs! averaging! about!10%.!
Confidence! that! the! true! size! of! the! population! lies! within! this! range! is! therefore! high.! The! estimated! trend! in!
population!size!over!the!10!years!was!a!decline!of!1.6%!per!year!but!this!was!not!significant.!A!simple!power!analysis!
(Gerrodette,!1987)!indicates!that!the!power!of! the! data! to! detect! a!rate!of!decline!of!this!magnitude! is! only! around!
30%.!!For!the!power!to!increase!to!the!typically!accepted!desirable!level!of!80%,!there!would!need!to!be!an!additional!
five!years!of!survey!with!estimates!at!the!same!precision!to!detect!a!decline!of!1.6%!per!year.!
A! population! size! estimated! to! number! fewer! than! 250! mature! individuals! can! be! classified! as! Endangered! under!
criterion! D! (IUCN,! 2012).! ! Our! most! robust! estimates! of! the! total! number! of! bottlenose! dolphins! in! the! Gulf! of!
Ambracia!never!exceeded!170!individuals!and!even!the!widest!95%!confidence!interval!did!not!exceed!250.!
Genetics+
Despite!our!relatively!small!sample!sizes,!our!results!indicate!that!the!dolphins!of!the!Gulf!of!Ambracia!are!genetically!
differentiated!from!the!surrounding!Mediterranean! populations.!Furthermore,!the!genetic!diversity!observed!for!the!
GA!population!was!the! lowest!when!compared!with!the!rest! of!populations!analyzed!in!this!study!and!with!previous!
studies!found!in!the!literature!(Curry,!1997;!Natoli!et!al.,!2005;!Viaud-Martinez!et!al.,!2008)!which!suggests!that!the!GA!
population!is!highly!isolated.!!
Samples!used! in! this! study! were! obtained! by! different! agencies! through! a! variety! of! means!and! over! the!course! of!
several!years.!In!the!case! of!GA!samples!(Table!1),!whenever!a!sample!was!taken,! the!research!boat!abandoned!that!
particular!dolphin!group! and! headed! to! a! different! side! of! the! Gulf! in! search!for! other! specimens,! to! minimize!the!
possibility! of! samples! taken! the! same! day! representing!closely! related! individuals.! Bottlenose!dolphins! from! the!
neighboring! IISA! area,! which! belong! to! an! even! smaller! resident! population,! despite! their! smaller! sample! size,!
exhibited!a!higher!genetic!diversity.!Nevertheless,!relatedness!among!GA!samples!cannot!be!ruled!out.!
The!genetic!uniqueness!of!the!dolphins!of!the!Gulf!of!Ambracia,!as!shown!by!low!mtDNA!diversity!and!the!presence!of!
unique! haplotypes,! demonstrates! low! connectivity! with!neighboring! Adriatic! Sea! and! Ionian! Sea! and! could!be!
explained! by! historical! events,! and/or! low! gene! flow! among! populations.!Additionally,! the!fact! that! the! Tajima’s! D!
!
16!
estimate! was! negative! and! significant! for! the! GA! population! indicates! that! the! population! may! have! undergone! a!
recent!reduction,!however!Fu’s!Fs! was! not! in!support!of!this!scenario.!To! estimate!both!historical!and!contemporary!
connectivity! between! the! GA! and! the! neighboring! populations,! as! well! as! understanding! the! ancestry! of! the! GA!
population,!the!analysis!of!a!larger!sample!set!and!the!use!of!nuclear!marker!would!be!required.!
Toxicology+
Free-ranging!bottlenose!dolphins!sampled! in! the! Gulf! of! Ambracia,! were! considered! to! be! in! good!nutritional!status!
(no!signs! of! emaciation)! and! were!frequently! engaged! in! surface-feeding.! Nevertheless,!lipid! content! in! their!
subcutaneous!blubber!(represented!by!EOM%)!resulted!in!28.6%.!!This!value!is!much!lower!than!what!was!reported!in!
bottlenose!dolphins!of!the!Sea!of! Cortez!(n!=!11;!43.5%),!the!Gibraltar!Strait!(N!=!3;!48.9%),!the!Strait!of!Sicily!(n!=!4;!
60.8%),!and! a! single!specimen!from! the!Ionian! Sea!with! 74.0%, ! but!it! was!similar!to! EOM%! from! specimens! of! the!
Croatian!Adriatic!Sea!(n! =! 14;! 24.5%)! (Marsili,! unp ublished! data;! Marsili! et! al.,! 1996).! ! Variables!such! as! nutritional!
status,!reproductive! status,! ontogeny,! environmental!temperature! changes!and! pathologies! have! been! observed! to!
affect!blubber! composition!(Wells!and!Scott,! 1999;!Struntz!et!al.,!2004;! Durkin!et!al.,!2005).! Although!some!of!those!
variables! may! have! contributed! to! the! variation! in! lipid! content! (range!14.0-54.7),! a! mean! value! as! low! as! that!
detected!in!the!present!study!is!indication!of!a!generalized!poor!health!condition.!!
With! respect! to! the! dolphin’s! toxicological! status,! it! must! be! also! considered!that! when! lipids! mobilize! from! the!
blubber,!lipophylic!contaminants!redistribute!leading!to!higher!tissue!concentrations.!Consequently,!specimens!with!a!
low!EOM%,!may!have!a!higher!risk!of!suffering!exposure-related!adverse!health!effects!(Yordy!et!al.,!2010).!Generally!
in!waters! near! industrialized! areas! ΣDDT/ΣPCB! ratio!is!<1! and! in!waters! close!to! agricultural! areas! is! >1.! Hence,! our!
results! indicate! that!contamination! in! the! Gulf! has!predominantly! an!agricultural! origin.! This! is! in! accordance! with!
other! studies,! reporting! a! predominance! of! DDTs! rather! than! PCBs,! conducted! in! this! part! of! the! Mediterranean!
(Marsili!and!Focardi,!1996;!Marsili,!2000;!Fossi!et!al.,!2004;!Marsili!et!al.,!2014).!!Values!of!pp’DDE/pp’DDT!ratio!>0.05!
for!all! sampled! animals!showed! that!DDT! contamination!was!not!recent.!Moreover,!based!on!the!values!obtained!of!
the!pp'DDE/DDTs!ratio,!varying!from!0.81!to!0.96,!bottlenose!dolphins!of!the!Gulf!of!Ambracia!manifested!a!very!high!
metabolism!of!this!pesticide.!
Gender!and!sexual!maturity!are!important!variables!when!assessing!dolphins’!toxicological!status.!Females!lose!up!to!
90%!of!their!total!body!burden!of!OCs!during!pregnancy!and!lactation!(Borrell!and!Aguilar,!2005;!Tanabe!et!al.,!1982).!
!
17!
In! addition,! the! milk! of! these! marine! mammals! contains! very! high! levels! of! fats,! mostly! triglycerides;! the! milk! of!
striped!dolphin,! for! instance,!contains!258!mg/g!of!triglycerides! in!280!mg/g!total!fats!(Kawai!and!Fukushima,! 1981).!
Consequently,!males! accumulate!persistent!organic! pollutants!for!their!entire! life,!increasing!in! contamination!levels!
with!age,! while! females! do! not.! The! mean! value!of!DDTs! found! in!bottlenose! dolphins!of! the! Gulf!of! Ambracia! was!
higher!in!males!than!in!females,!but!contrarily!to!what!we!expected,!the!exact!opposite!was!found!for!HCB!and!PCBs.!
Three!sexually! mature! females!had! levels! of! average! HCB,! DDTs! and! PCBs! higher! than!younger! im mature! females.!
However,!when!looking! at! their!personal! history! (i.e.,! health,!nutritional!status,! pregnancies/lactation),! only! female!
AMV15!(firstly! identified!in!July!2003!as! an!adult)!had!been!observed!with! offspring;!one!in!2005!and!another! one!in!
2008!(both!currently!alive! according! to! our!photo-identification! records).!In!fact!this! female!had! very! low! OC! levels,!
contrarily! to! the! other!two,! which!were! never! observed! in! association! with! offspring!and! are! unlikely! to! have!
reproduced.!
Anthropogenic! pollution! may! have! important! consequences! in! dolphin! population! dynamics! (Garcia-Alvarez! et! al.,!
2014).! Most! studies! on! the! toxicological! status! from! OCs! in! bottlenose! dolphins! in! the! Mediterranean! Sea! were!
exclusively!based!on!stranded! animals! (Marsili!and! Focardi,! 1997;!Romanic! et! al.,! 2014;! Shoham-Frider! et! al.,! 2009;!
Storelli!et!al.,!2007;!Storelli!and!Marcotrigiano,!2003;!Wafo!et!al.,!2005).!A!recent!review!paper!by!Jepson!et!al.!(2016),!
using!samples!from!both!stranded!and!free-living!biopsied!animals,!reported!high!mean!∑PCB!concentrations!(>!100.0!
mg/kg!l.w.)!in!bottlenose!dolphins!from!western!Mediterranean!and!northern!Adriatic!Sea.!The!present!study!is!one!of!
the!few! works! conducted!in!the!Mediterranean!Sea! based!uniquely!in!free-ranging!bottlenose!dolphins!and!provides!
unprecedented!information!on!this! matter!for!the! Gulf.!OCs!levels! found! in! bottlenose! dolphins!resulted!very! similar!
to!those!reported!by!Fossi!et!al.!(2003)!for!the! same!species!in!the!neighboring!waters!of!the!Ionian!Sea!for!HCB!and!
PCBs!while! for! DDTs!were!4! times!higher,! which! indicates!that!the! latter!legacy! pesticide!poses! a! real! toxicological!
problem!for!the!dolphins!of!the!Gulf!of!Ambracia.!
!
Conclusions+
Our!findings!indicate!that!bottlenose!dolphins!in!the!Gulf!Ambracia!constitute!a!geographically!and!otherwise!distinct!
group! with! little! demographic! exchange! that! are! exposed! to! high! levels! of! pollution,! mostly! derived! from! local!
agriculture!(i.e.,!pesticides).!!Based!on!photographic!mark-recapture!estimates,!134!animals!(CV!=!0.11)!resided!in!the!
!
18!
Gulf!in!2015.!!By!applying!standard!criteria!provided!by!the!IUCN!Red!List!of!Threatened!Species!we!conclude!that!this!
‘subpopulation’! would! qualify! as! Endangered!according!to!Red!List!criteria.!!While!local!density!of!dolphins!is!among!
the! highest! recorded! anywhere! in! the! Mediterrane an! Sea,! this! is! not! indicative! of! favorable! conservation! status! or!
pristine!habitat.!!On!the!contrary,!these!dolphins!face!a!high!risk!of!extinction!due!to!their!likely!reproductive!isolation,!
small!population!size!and!small!extent!of!occurrence,!as!well!as!to!acute!and!growing! anthropogenic!impacts!in!their!
semi-closed!shallow!habitat.! ! Management! of! human! pressures! is!an!obvious!way!of!reducing! such! a! risk,!consistent!
with!national!and!regional!commitments!to!protect!this!coastal!area!and!cetaceans!generally.!!
+
Acknowledgements+
This!work!was!supported!in!part!by!OceanCare!and!UNEP's!Regional!Activity!Centre!for!Specially!Protected!Areas!(RAC-
SPA).!Special!thanks!to!Stefano!Agazzi,!Giovanni!Bearzi,!Silvia!Bonizzoni,!Marina!Costa,!Tilen!Genov!and!Ioannis!Giovos.!
We!are! also!grateful!for!the! work!carried!out!by!participants!in! our! citizen! science!program! handling! and! cataloging!
hundreds!of!digital!images.!The!Milan!Civic!Aquarium!and!Hydrobiological!Station,!Lefkas!Marina!and!Jimmy!Pandazis!
provided!logistical!support.!!! !
!
19!
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24!
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Environ.!Toxicol.!Chem.!29,!12631273.!
!!
!
! !
!
26!
Table!1.!Detail!on!skin!samples!of!bottlenose!dolphins!taken!in!the!Gulf!of!Ambracia!used!for!genetic!analysis;!20!
obtained!by!skin!swabbing!from!bow-riding!specimens!and!4!from!dead!animals.!
!
Sample
code!
Date!
D-M-YYYY!
Time!
hh:mm!
Sampling!Method!
Haplotype!
1!
4-8-2004!
11:26!
skin!swabbing!!
IV!
2!
4-8-2004!
11:40!
skin!swabbing!!
IV!
3!
4-8-2004!
11:50!
skin!swabbing!!
Failed!sequencing!
4!
6-8-2004!
10:42!
skin!swabbing!!
Failed!sequencing!
5!
6-8-2004!
11:21!
skin!swabbing!!
XXXXII!
6!
6-8-2004!
13:09!
skin!swabbing!!
Failed!sequencing!
7!
9-8-2004!
11:32!
skin!swabbing!!
Failed!sequencing!
8!
15-8-2004!
9:40!
skin!swabbing!!
Failed!sequencing!
9!
14-8-2006!
9:40!
skin!swabbing!!
IV!
10!
14-8-2006!
11:50!
skin!swabbing!!
IV!
11!
8-9-2006!
10:33!
skin!swabbing!!
IV!
12!
8-9-2006!
11:11!
skin!swabbing!!
IV!
13!
8-9-2006!
11:22!
skin!swabbing!!
IV!
14!
15-9-2006!
10:40!
skin!swabbing!!
IV!
15!
28-9-2006!
19:05!
dead!stranded!!
IV!
16!
20-6-2007!
19:15!
dead!stranded!!
IV!
17!
26-6-2007!
8:52!
skin!swabbing!!
IV!
18!
26-6-2007!
9:04!
skin!swabbing!!
IV!
19!
27-6-2007!
11:38!
skin!swabbing!!
IV!
20!
4-7-2007!
11:47!
dead!floating!adrift!
IV!
21!
19-8-2007!
11:01!
skin!swabbing!!
IV!
22!
19-8-2007!
11:19!
skin!swabbing!!
IV!
23!
27-8-2007!
10:45!
skin!swabbing!!
IV!
24!
8-9-2009!
14:40!
dead!stranded!!
IV!
!
! !
!
27!
Table!2:!Summary!of!research!effort!conducted!in!the!Gulf!of!Ambracia!(2006-2015).!Framed!dataset!refers!exclusively!
to!the!number!of!sightings!and!photo-Id!effort!used!for!the!population!estimates.!
Year!
Research!
calendar!!
(Months!at!sea)!
Days!
at!sea!
Km!
survey!
"positive"!
Total!!
Sightings!
Time!with!
dolphins!
Dataset+used+for+Mark-Recapture+Population+estimates+
(Sightings!on!transect!between!May-September)+
Total+Sightings++
Photos+taken+
Photos+selected++
2006!
Apr-Dec!
(9)%
107!
1630!
70!
99!h!32!min!
38!
5872!
2770!
2007!
Year-round!
(12)%
136!
2776!
101!
139!h!45!min!
25!
4674!
2656!
2008!
Year-round!
(12)%
105!
1648!
77!
110!h!52!min!
25!
2466!
1561!
2009!
May-Oct!
(6)%
82!
1310!
76!
105!h!50!min!
25!
5478!
2241!
2010!
Apr-Oct!
(7)%
88!
1234!
80!
97!h!36!min!
29!
5478!
2241!
2011!
Apr-Sep!
(6)%
43!
785!
35!
46!h!56!min!
23!
4765!
2045!
2012!
Apr-Sep!
(6)%
55!
656!
50!
75!h!19!min!
37!
8873!
3296!
2013!
Apr-Sep!
(6)%
60!
667!
55!
85!h!17!min!
37!
8216!
2992!
2014!
Apr-Sep!
(6)%
56!
707!
50!
77!h!30!min!
33!
9695!
3287!
2015!
Jun-Sep!
(4)%
38!
412!
37!
54!h!10min!
30!
5462!
2674!
Total!
74!months!
770!
13303!
631!
992!h!47!min!
302+
60979+
25763+
! !
!
28!
Table!3:!Mark-recapture!population!estimates!of!the!number!of!marked!individuals!generated!by!different!models!for!
data! subsets! D1+D2! (top)! and! D1+D2+D3! (bottom).! ! Estimates! from! models! identified! by! CAPTURE! as! the! most!
appropriate! are! shown! in! bold.! The! model! identified! as! most! appropriate! in! 2008! for! data! subset! D1+D2+D3! was!
undefined.!Shaded!columns!correspond!to!the!results!from!Mt,!Mh,!and!Mth!models!run!for!each!year.!
!
D1-D2+
+
Year+
M(tb)+
SE!
CV%
M(0)+
SE!
CV%
M(t)+
SE!
CV%
M(h)+
SE!
CV%
M(th)+
SE!
CV%
!
2006+
!
!
%
!
!
%
71!
2,38!
0,033%
82!
5,68!
0,069%
78+
6,28!
0,081%
!
2007+
!
!
%
!
!
%
82+
3,58!
0,044%
93!
6,33!
0,068%
85!
7,07!
0,083%
!
2008+
!
!
%
74+
3,52!
0,048%
74!
3,24!
0,044%
86!
6,86!
0,080%
83!
8,14!
0,098%
!
2009+
126+
244,63!
1,942%
!
!
%
78!
5,25!
0,067%
101!
11,48!
0,114%
98!
15,12!
0,154%
!
2010+
!
!
%
66+
3,40!
0,051%
66!
3,14!
0,048%
76!
6,13!
0,081%
73!
7,27!
0,100%
!
2011+
66+
4,05!
0,061%
!
!
%
66!
2,54!
0,038%
75!
5,40!
0,072%
73!
6,24!
0,085%
!
2012+
!
!
%
!
!
%
72!
1,34!
0,019%
78+
4,50!
0,058%
75!
3,62!
0,048%
!
2013+
!
!
%
!
!
%
73!
2,20!
0,030%
80!
9,80!
0,123%
72+
2,91!
0,040%
!
2014+
!
!
%
!
!
%
63!
2,07!
0,033%
69+
5,03!
0,073%
63!
3,79!
0,060%
!
2015+
!!
!!
%%
66+
3,23!
0,050%
66!
3,03!
0,050%
77!
6,66!
0,090%
80!
10,33!
0,130%
!
D1-D2-D3+
+
Year+
M(tb)+
SE!
CV%
M(0)+
SE!
CV%
M(t)+
SE!
CV%
M(h)+
SE!
CV%
M(th)+
SE!
CV%
!
2006+
!
!
%
!
!
%
93!
2,80!
0,030%
105!
6,31!
0,060%
99+
6,62!
0,067%
!
2007+
!
!
%
!
!
%
104!
4,21!
0,040%
119!
7,67!
0,064%
111+
8,93!
0,080%
2008+
!
!
%
!
!
%
86!
3,22!
0,037%
99!
6,53!
0,066%
94!
7,67!
0,082%
!
2009+
174+
391,10!
2,248%
!
!
%
94!
6,06!
0,064%
114!
11,57!
0,101%
107!
14,31!
0,134%
!
2010+
!
!
%
84+
3,92!
0,047%
84!
4,83!
0,058%
94!
6,33!
0,067%
86!
6,90!
0,080%
!
2011+
100+
15,27!
0,153%
!
!
%
94!
3,64!
0,039%
112!
9,33!
0,083%
111!
9,99!
0,090%
!
2012+
!
!
%
!
!
%
104!
3,47!
0,033%
116!
5,81!
0,050%
111+
5,18!
0,047%
!
2013+
!
!
%
74+
3,03!
0,041%
73!
2,81!
0,038%
82!
5,53!
0,067%
76!
5,45!
0,072%
!
2014+
!
!
%
!
!
%
93!
2,443!
0,026%
103+
5,97!
0,058%
95!
4,97!
0,052%
!
2015+
!!
!!
%%
90+
3,96!
0,044%
90!
3,75!
0,040%
100!
6,23!
0,060%
95!
8,81!
0,090%
!
! !
!
29!
!
Table!4:! Estimates! of!the!number!of! marked!individuals!from! model! Mth,! of!the!proportion! of! marked!animals!in!the!
population,!and!of!total!population!size!for!data!subsets!D1+D2!and!D1+D2+D3.!
Year+
Subset!
CAPTURE+estimate+
HM!θ!
Corrected+estimate+
CV!
SE!
95%CI!
2006+
D1+D2!
78%
0.552!
141+
0.094!
13.30!
117-170!
+
D1+D2+D3!
99!
0.702!
141+
0.086!
12.25!
119-167!
2007+
D1+D2!
85!
0.582!
145+
0.121!
17.72!
115-185!
+
D1+D2+D3!
111!
0.679!
163+
0.107!
17.575!
132-201!
2008+
D1+D2!
83!
0.645!
128+
0.118!
15.20!
102-161!
+
D1+D2+D3!
94!
0.734!
127+
0.094!
12.099!
106-153!
2009+
D1+D2!
98!
0.560!
174+
0.167!
29.34!
126-242!
+
D1+D2+D3!
107!
0.636!
168+
0.145!
24.432!
126-223!
2010+
D1+D2!
73!
0.535!
136+
0.117!
15.95!
108-171!
+
D1+D2+D3!
86!
0.652!
131+
0.102!
13.451!
107-160!
2011+
D1+D2!
73!
0.469!
155+
0.133!
20.73!
119-201!
+
D1+D2+D3!
111!
0.661!
167+
0.110!
18.470!
135-207!
2012+
D1+D2!
75!
0.516!
145+
0.077!
11.29!
124-169!
+
D1+D2+D3!
111!
0.772!
143+
0.054!
7.862!
129-159!
2013+
D1+D2!
72!
0.541!
133+
0.077!
10.26!
114-154!
+
D1+D2+D3!
76!
0.708!
107+
0.080!
8.636!
91-125!
2014+
D1+D2!
63!
0.432!
145+
0.119!
17.42!
115-184!
+
D1+D2+D3!
95!
0.685!
138+
0.067!
9.394!
121-158!
2015+
D1+D2!
80!
0.582!
137+
0.153!
21.12!
101-185!
+
D1+D2+D3!
95!
0.707!
134!
0.110!
14.84!
110-170!
+
!
!
30!
Table!5:!Haplotype!affiliations!across!the!5!putative!populations.!Shared!haplotypes!are!shaded.!!
!
%
%
Populations%
%
%
%
#%
Haplotype%
WM%
ADR%
IISA%
AEG%
GA%
1!
!
!
I!
2!
11!
!
6!
!
2!
IV!
!
!
!
!
18!
3!
XIV!
!
3!
!
!
!
4!
XV!
!
2!
!
3!
!
5!
XVI!
6!
!
1!
!
!
6!
XVII!
1!
!
!
!
!
7!
XVIII!
2!
!
!
!
!
8!
XIX!
!
2!
3!
1!
!
9!
XXII!
!
!
3!
2!
!
10!
XXIII!
1!
!
!
!
!
11!
XXXVIII!
!
!
!
1!
!
12!
XXXXI!
1!
1!
!
!
!
13!
XXXXII!
!
!
!
!
1!
14!
XXXXIII!
!
1!
!
!
!
15!
XXXXIV!
!
!
1!
!
!
16!
XXXXV!
!
!
1!
!
!
%
n%=16%
n%=%13%
n%=%20%
n%=%9%
n%=%13%
n%=%19%
!
!
!
!
Table!6:+Genetic!differentiation!estimated!with!Fst!based!on!mtDNA!data.!Statistical!significance:!*!Fst!>!0!(p!<!0.05),!
**!Fst!>!0!(p!≤!0.001).!!
! !
n%
Populations%
WM%
ADR%
IISA%
GA%
AEG%
13%
WM%
!
!
!
!
!
20%
ADR%
0.199**!
!
!
!
!
9%
IISA%
0.150**!
0.226**!
!
!
!
19%
GA%
0.600**!
0.600**!
0.627**!
!
!
13%
AEG%
0.172**!
-0.005!
0.141*!
0.612**!
!
!
31!
Table!7:! Sex!and! sexual! maturity,!lipid!percent! (EOM%),! water!percent! (H2O%)! and! OCs!levels!(i.e.,!HCB,!DDT,! PCB)!
reported!as!d.w.!(and!l.w.)!for!all!14!sampled!dolphins.!
Dolphin+
code+
Sex+
Sexual+
maturity+
Photo-Id+info!(to!estimate!minimum!age!and!
sexual!maturity)+
EOM
%+
H2O+
%+
HCB++ng/g!
d.w.!(l.w.)+
DDTs+ng/g!
d.w.!(l.w.)+
PCBs+ng/g!
d.w.!(l.w.)+
AMV01!
M!
No!
Firstly!identified!in!September!2009!as!a!calf!
30.3!
55.0!
6.45!
(21.28)!
25182.4!
(83110.2)!
7344.5!
(24239.1)!
AMV03!
M!
Yes!
Firstly!identified!in!September!2004!as!an!adult!
29.7!
59.3!
6.63!
(22.32)!
2830.0!
(9528.7)!
1312.6!
(4419.6)!
AMV04!
M!
Yes!
Firstly!identified!in!June!2010!as!an!adult!
14.0!
55.2!
5.80!
(41.43)!
1133.2!
(8094.2)!
1236.9!
(8834.6)!
AMV05!
F!
No!
Firstly!identified!in!June!2012!as!a!juvenile!
54.7!
51.0!
10.02!
(18.33)!
14249.5!
(26050.3)!
5628.2!
(10289.2)!
AMV06!
M!
Yes!
Firstly!identified!in!September!2002!as!an!adult!
28.7!
58.9!
6.11!
(21.28)!
14586.3!
(50823.4)!
5647.4!
(19677.4)!
AMV07!
M!
No!
Firstly!identified!in!April!2011!as!a!calf!
21.1!
56.4!
6.21!
(29.41)!
1593.3!
(7551.2)!
626.1!
(2967.3)!
AMV08!
F!
No!
Firstly!identified!in!May!2011!as!a!juvenile!
17.3!
54.8!
7.54!
(43.56)!
3072.1!
(17757.7)!
1348.3!
(7793.7)!
AMV09!
M!
Yes!
Firstly!identified!in!January!2007!as!an!adult!
37.8!
52.5!
11.79!
(31.20)!
11941.1!
(31590.1)!
7346.5!
(19435.2)!
AMV10!
M!
Yes!
Firstly!identified!in!July!2003!as!an!adult!
21.2!
53.6!
16.75!
(79.00)!
13025.2!
(61439.7)!
10302.3!
(48595.5)!
AMV11!
F!
Yes!
Firstly!identified!in!July!2003!as!an!adult!
18.6!
58.6!
15.12!
(81.29)!
7123.5!
(38298.1)!
20218.9!
(108704.0)!
AMV12!
M!
?%
Unknown;!no!photo-identified!
34.4!
55.8!
13.97!
(40.62)!
18066.6!
(52519.1)!
9859.1!
(28660.1)!
AMV13!
F!
Yes!
Firstly!identified!in!June!2004!as!an!adult!
22.5!
56.6!
12.81!
(56.92)!
16097.3!
(71543.4)!
6603.3!
(29347.9)!
AMV14!
M!
Yes!
Firstly!identified!in!July!2001!as!an!adult!
40.3!
57.3!
20.61!
(51.14)!
161091.2!
(399730.0)!
21584.2!
(53558.9)!
AMV15!
F!
Yes!
Firstly!identified!in!July!2003!as!an!adult.!Two!
offspring!recorded!(in!2005!and!2008)!
30.0!
55.2!
12.72!
(42.39)!
6539.6!
(21798.8)!
2463.9!
(8212.9)!
!
! !
!
32!
Table!8:!Arithmetic!mean!levels!and!Standard!Deviation!(in!brackets)!of!HCB,!DDTs!and!PCBs!in!the!bottlenose!
dolphins!of!the!Gulf!of!Ambracia,!divided!by!gender!(males!and!females)!and!sexual!maturity.!
!
+
n+
HCB+ng/g+l.w.+
DDTs+µg/g+l.w.+
PCBs+µg/g+l.w.+
MALES!&!FEMALES!
14!
41.44!(20.19)!
62.85!(99.92)!
26.77!(28.27)!
MALES!
9!
37.52!(18.70)!
78.27!(123.4)!
23.38!(18.00)!
FEMALES!
5!
48.50!(23.01)!
35.09!(21.78)!
32.87!(43.33)!
Sexually!mature!males!
6!
41.06!(21.80)!
93.53!(151.5)!
25.75!(20.56)!
No-sexually!mature!males!
3!
30.44!(9.71)!
47.73!(38.01)!
18.62!(13.74)!
No-sexually!mature!males!
(AMV12%not%included)!
2!
25.35!(5.75)!
45.33!(53.43)!
13.60!(15.04)!
Sexually!mature!females!
3!
60.20!(19.66)!
43.88!(25.34)!
48.75!(52.98)!
No-sexually!mature!females!
2!
30.94!(17.84)!
21.90!(5.86)!
9.04!(17.64)!
!
! !
!
33!
!Figure!1:!Common!bottlenose!dolphins!photographed!in!the!Gulf!of!Ambracia!showing!the!characteristic!morphology!
of!the!species.!Bottom-right!image!shows!an!adult!bottlenose!severely!affected!by!a!skin!condition!firstly!reported!by!
Gonzalvo!et!al.!(2015).!Photos!by!J.Gonzalvo/Tethys!Research!Institute.!
!
Figure!2:!(Top)!Map!of!the!Gulf!of!Ambracia.!(Bottom)!Geographic!origin!of!samples!analysed!for!mtDNA;!WM-western!
Mediterranean,!ADR-Adriatic!Sea,!IISA-Inner!Ionian!Sea!Archipelago,!GA-Gulf!of!Ambracia!and!AEG-Aegean!Sea!
(numbers!indicate!the!sample!size).!
!
Figure!3:!Discovery!curves,!taking!into!account!the!degree!of!distinctiveness!(from!D1-highly!marked!to!D4-poorly!
marked),!for!individually!identified!bottlenose!dolphins!across!2006-2015!in!the!Gulf!of!Ambracia.!
!
Figure! 4:! Residency!pattern! of! marked! individuals! (D1-D2)! in! the! Gulf!of! Ambracia.! Grey!cells! indicate! presence!
documented!through!digital!photo-identification.!Months!included!in!the!mark-recapture!model!are!shown!with!white!
letters!on!black!background.!Three!individuals!with!“¢”!besides!their!ID-Code!were!last!seen!in!our!study!area!in!2008;!
they!were! identified! in!June! 2010! in! the!Inner!Ionian!Sea! Archipelago!and! two! of! them!were!spotted! in! the! Gulf!of!
Corinth!in!August!that!same!year!(T1,!T2!and!T3!in!Bearzi!et!al.,!2010).!
!
Figure!5:!Estimates!of!the!number!of!identifiable!individuals!D1+D2+D3!(grey!squares)!and!total!population!size!(black!
circles)!within!years!using!the!closed!model!Mth!Chao.!The!fitted!regression!line!shows!an!exponential!decline!at!a!
rate!of!1.6%!(SE=1.5%)!per!year.!
!
Figure!6:!Maximum!parsimony!network!illustrating!the!relationships!among!the!74!bottlenose!dolphin!samples!from!
WM-western!Mediterranean,!ADR-Adriatic!Sea,!IISA-Inner!Ionian!Sea!Archipelago,!GA-Gulf!of!Ambracia!and!AEG-
Aegean.!Each!one!of!the!16!haplotypes!is!represented!by!a!circle!with!a!corresponding!Roman!numeral!and!number!of!
individuals!represented!per!population.!
!
Figure!7:!Plot!of!DDT!and!PCB!levels!(ng/g!lipid!weight)!in!the!subcutaneous!blubber!of!the!single!specimens.!
!
!
... Its waters are rich in nutrients and characterized by abundant wildlife. The dolphins of the Gulf of Ambracia are genetically differentiated from the surrounding Mediterranean metapopulation [54]. The area is recognized as IMMA, where the bottlenose dolphin is the only qualifying species [55]. ...
... Due to the peculiar physiography of this semi-closed gulf and the resident behaviour of the dolphins, this result should be considered quite reliable. In fact, the bottlenose dolphins living in the Gulf of Ambracia are considered a subpopulation, spending their entire life in the waters of the gulf [54]. ...
... Behavioural plasticity is a key feature of the bottlenose dolphin and is probably transmitted from one generation to the next one as a local cultural tradition, allowing the dolphins to improve their ability to exploit the residency area and to adapt to changes. As part of this plastic behaviour, bottlenose dolphins can learn to obtain food from trawlers, gillnets and aquaculture fish cages, and these opportunistic strategies can become an integral part of their feeding habits [30,54,77,[94][95][96][97][98][99][100][101][102][103][104][105][106]. ...
Article
The common bottlenose dolphin (Tursiops truncatus) is a cosmopolitan delphinid, regularly present in the Mediterranean Sea. According to previous studies, this dolphin tends to form resident geographical units scattered on the continental shelf. We investigated how the physiographic characteristics of the area of residence, with special reference to the size and shape of the continental shelf, affect the home range and the group size of the local units. We analysed and compared data collected between 2004–2016 by 15 research groups operating in different study areas of the Mediterranean Sea: the Alboran Sea, in the South-Western Mediterranean, the Gulf of Lion and the Pelagos Sanctuary for the marine mammals, in the North-Western Mediterranean, and the Gulf of Ambracia, in the North-Central Mediterranean Sea. We have found that in areas characterised by a wide continental platform, dolphins have wider home ranges and aggregate into larger groups. In areas characterized by a narrow continental platform, dolphins show much smaller home ranges and aggregate into smaller groups. The results obtained from this collective research effort highlight the importance of data sharing to improve our scientific knowledge in the field of cetaceans and beyond.
... In Greek waters, the bottlenose dolphin is found in coastal areas, including both rugged coastlines with no continental shelf (Bearzi et al., 2005;Bearzi, Agazzi, Bonizzoni, Costa, & Azzellino, 2008;Frantzis et al., 2003) and semienclosed gulfs of eutrophic waters, such as the Gulf of Ambracia (Bearzi et al., 2008;Gonzalvo et al., 2016). ...
... No precise information is available on the stock status of any of the fish species in the Gulf of Ambracia (European Commission, 2009). Nevertheless, fish farms, agriculture, livestock, and discharges of domestic sewage from coastal towns and villages contribute to the nutrient enrichment of the Ambracian waters, which are highly eutrophic (Ferentinos et al., 2010;Gonzalvo et al., 2015Gonzalvo et al., , 2016. The western part of the Gulf is seasonally hypoxic, while the eastern part is seasonally anoxic (Kountoura & Zacharias, 2013). ...
Article
The Gulf of Ambracia, in northwestern Greece, hosts a highly threatened community of about 150 common bottlenose dolphins (Tursiops truncatus ). Until now, information on their feeding habits was derived exclusively from fish scale samples collected during surface‐feeding events targeting small schooling epipelagic fish. The aim of this study was to determine the diet of bottlenose dolphins living in the Gulf of Ambracia through the application of Bayesian isotopic mixing models. Skin biopsy samples of 16 dolphins were analyzed and no difference related to sex or age‐class was found in δ13C and δ15N values. Results suggested that the dolphin diet was mainly based on Trachurus trachurus , species belonging to the family Sparidae: Diplodus annularis , Lithognathus mormyrus , and Sepia officinalis , which represented together about 42% ± 15% of the biomass ingested, followed by species belonging to the order Clupeiformes (Engraulis encrasicolus, Sardinella aurita, and Sardina pilchardus ) and the genus Gobius (37% ± 17%). A better understanding of the feeding habits of these dolphins sheds light on the feeding ecology of this highly threatened population by, for instance, evidencing interactions with artisanal fisheries sharing the same target species, and is key for identifying adequate management measures consistent with an ecosystem‐based approach.
... Στον Αμβρακικό κόλπο στην δυτική Ελλάδα τα ρινοδέλφινα εμφανίζουν αρκετά μεγάλη πυκνότητα. Σε 10ετή έρευνα (2006-2015) οπτικής παρακολούθησης με πλοίο και λήψη φωτογραφικών δεδομένων σε 13.000 km προσπάθειας καταγράφηκαν 631 παρατηρήσεις(Gonzalvo et al., 2016). Στην ίδια περιοχή οι(Bearzi et al., 2008) κατέγραψαν την υψηλή πυκνότητα των 0,37 ατόμων ανά km 2 . ...
Thesis
Full-text available
Η παρούσα εργασία καταγράφει και περιγράφει τις τεχνικές παρακολούθησης που εφαρμόζονται στην σύγχρονη επιστημονική έρευνα για τα κητώδη, με ειδίκευση τη συλλογή δεδομένων για την εκτίμηση των πληθυσμών και της κατανομής των ειδών. Βασική μεθοδολογία για τη συγγραφή της αποτέλεσε η διεξοδική μελέτη της πρόσφατης βιβλιογραφίας στο πεδίο της επιστήμης των θαλάσσιων θηλαστικών. Η εργασία χωρίζεται σε τέσσερα κεφάλαια. Στο πρώτο κεφάλαιο αποτυπώνονται τα γενικά στοιχεία γύρω από τα κητώδη. Περιγράφονται βασικά βιολογικά και οικολογικά χαρακτηριστικά των κητωδών και γίνεται λόγος για την συμβολή τους στα οικοσυστήματα και την αξία τους γενικότερα. Το κεφάλαιο κλείνει με μια αναδρομή στη σχέση του ανθρώπου με τα κητώδη από την εποχή της φαλαινοθηρίας στις σημερινές προσπάθειες προστασίας. Το δεύτερο κεφάλαιο περιγράφει τις σημαντικότερες τεχνικές παρακολούθησης της αφθονίας και της κατανομής των κητωδών. Με βάση τα τεχνικά μέσα και τον τρόπο που χρησιμοποιούνται για τη δειγματοληψία των δεδομένων, διακρίνονται πέντε τεχνικές και επεξηγούνται τα βασικά χαρακτηριστικά, τα πλεονεκτήματα, τα μειονεκτήματα και η μελλοντική τους βελτίωση. Στο τρίτο κεφάλαιο παρουσιάζεται η σημερινή γνώση της αφθονίας και της κατανομής των ειδών στη Μεσόγειο και τη Μαύρη θάλασσα, με βάση τα αποτελέσματα των επιστημονικών ερευνών τις τελευταίες δεκαετίες. Αναφέρονται παραδείγματα από τις έρευνες για κάθε είδος ξεχωριστά και οι συγκεκριμένες τεχνικές που εφαρμόστηκαν. Τέλος, στο τέταρτο κεφάλαιο παρατίθενται τα συμπεράσματα που προκύπτουν από το σύνολο της εργασίας και η συζήτηση για την αποτελεσματική προστασία και διατήρηση των κητωδών που παραμένει ανοικτή.
... The Strait of Gibraltar killer whale (Esteban and Foote, 2019) and the Gulf of Corinth common dolphin subpopulations (Bearzi, Bonizzoni and Santostasi, 2020) have recently both been added to the Red List with a status of CR. The Red List assessments of the Gulf of Ambracia common bottlenose dolphins (Gonzalvo et al., 2016), the Gulf of Corinth striped dolphins (Bearzi et al., 2016) and the Mediterranean long-finned pilot whale subpopulations (Globicephala melas) (Verborgh et al., 2016) are currently being reviewed. The Mediterranean Sea rough-toothed dolphins (Steno bredanensis) (Kerem et al., 2016) and the Iberian/North West African subpopulation (Fontaine, 2016) of harbour porpoises are also being assessed. ...
... For the vast majority of these species, only presence data are available in this sea. Conversely, other contiguous areas (e.g., the Black Sea, Gulf of Corinth, Hellenic Trench, Ambracian Gulf, and the Inner Ionian Sea) are well studied for their cetofauna (e.g., [21][22][23][24][25][26]). Due to the availability of data for these seven species from areas other than the Aegean Sea, this chapter will not be an in-depth review of their ecology. ...
Chapter
The underwater habitat complexity of the Aegean Sea renders it home to a diversity of cetacean species. From shallow-diving piscivores to teuthophagous deep divers, this sea hosts seven regular cetacean species (Delphinidae, Phocoenidae, Physeteridae, and Ziphiidae), while five more species have been reported as visitors. Despite this high biodiversity, little is known about this taxon, with most available information referring only to presence data. Due to an ever-increasing human development in the area, seven threats can be identified so far: interactions with fisheries and plastic debris, noise and chemical pollution, shipping, climate change, and epizootics. The concurrent absence of abundance data for these apex predators and the lack of protection areas and law enforcement undermine the survival of the cetaceans in the Aegean Sea. Based on these deficiencies and the scarce governmental funding, we propose that research effort should focus on three main topics: abundance, fine-scale distribution, and ecology. Only with this information available, the monitoring of anthropogenic threats will allow the assessment of the effectiveness of potential conservation measures.
... Our results, despite being acquired from a small number of animals, suggest that this approach could represent a first step in overcoming the difficulties, already reported in other studies, associated with a drone-based sampling technique for wild small cetaceans [15]. A major limitation with using UAVs for blow sampling is the impossibility to match the samples with the individuals because bottlenose dolphin groups are relatively large, densely concentrated and highly dynamic, with frequent changes in group size and composition of individuals [26]. The contemporary use of a sampling tool with a camera mounted on the UAV, as already tested by Raudino and colleagues (2019) [15], was not possible in our study; this technical limitation should be addressed in future studies by using different and more proficient UAVs. ...
Article
Full-text available
Recent studies describe the use of UAVs in collecting blow samples from large whales to analyze the microbial and viral community in exhaled air. Unfortunately, attempts to collect blow from small cetaceans have not been successful due to their swimming and diving behavior. In order to overcome these limitations, in this study we investigated the application of a specific sampling tool attached to a UAV to analyze the blow from small cetaceans and their respiratory microbiome. Preliminary trials to set up the sampling tool were conducted on a group of 6 bottlenose dolphins (Tursiops truncatus) under human care, housed at Acquario di Genova, with approximately 1 meter distance between the blowing animal and the tool to obtain suitable samples. The same sampling kit, suspended via a 2 meter rope assembled on a waterproof UAV, flying 3 meters above the animals, was used to sample the blows of 5 wild bottlenose dolphins in the Gulf of Ambracia (Greece) and a sperm whale (Physeter macrocephalus) in the southern Tyrrhenian Sea (Italy), to investigate whether this experimental assembly also works for large whale sampling. In order to distinguish between blow-associated microbes and seawater microbes, we pooled 5 seawater samples from the same area where blow samples’ collection were carried out. The the respiratory microbiota was assessed by using the V3-V4 region of the 16S rRNA gene via Illumina Amplicon Sequencing. The pooled water samples contained more bacterial taxa than the blow samples of both wild animals and the sequenced dolphin maintained under human care. The composition of the bacterial community differed between the water samples and between the blow samples of wild cetaceans and that under human care, but these differences may have been mediated by different microbial communities between seawater and aquarium water. The sperm whale’s respiratory microbiome was more similar to the results obtained from wild bottlenose dolphins. Although the number of samples used in this study was limited and sampling and analyses were impaired by several limitations, the results are rather encouraging, as shown by the evident microbial differences between seawater and blow samples, confirmed also by the meta-analysis carried out comparing our results with those obtained in previous studies. Collecting exhaled air from small cetaceans using drones is a challenging process, both logistically and technically. The success in obtaining samples from small cetacean blow in this study in comparison to previous studies is likely due to the distance the sampling kit is suspended from the drone, which reduced the likelihood that the turbulence of the drone propeller interfered with successfully sampling blow, suggested as a factor leading to poor success in previous studies.
... sardine, Sardina pilchardus: Somarakis, Tsianis, Machias, & Stergiou, 2006) and large (swordfish, Xiphias gladius and Thunnus thynnus: ICCAT, 2003) pelagic fish species, as well as for marine megafauna, actively foraging on the continental shelf and interacting with local fisheries (i.e. monk seals: Karamanlidis et al., 2008;marine turtles: Margaritoulis et al., 2003;dolphins: Bearzi et al., 2010;Gonzalvo et al., 2016). ...
Article
Full-text available
The present study deals with the monitoring for a real implementation of management policies in marine en-vironments and the potential conflicts between professional and recreationalfishery in the coastal areas of theMediterranean Sea. A comprehensive database offishing effort andfisheries infringements from professionalsmall-scale and recreationalfisheries was screened to identify hotspots areas offishing pressure in the coastalzones of Ionian Sea. Mapping points showed that the number of the recorded infringements conducted both byprofessional and recreationalfishers are too low (1 and 6 recorded infringements per 104km2of vessel days peryear) and thatfishing effort, and subsequently the recorded infringements, are not evenly distributed but con-centrated on specificfishing grounds. These revealed high-risk areas prone to illegalfishing activities and areimplying problems in the implementation of thefisheries regulations rather than a low delinquency of thefishersto comply with the rules. Findings represents a step forward in applying tracking technology to the surveillanceof small-scalefishery and are crucial towards the specification of the critical zones for setting an efficient controlsystem.
Article
Although banned for years, organochlorine pesticides and PCBs continue to affect aquatic life, dolphins being particularly exposed. The concentrations of 31 PCB congeners, and 15 banned pesticides or metabolites were measured in 5 tissues of 68 striped dolphins stranded in the Northwestern Mediterranean coast in 2010–16. The results were compared to historical data (1988–2009) and, even though there is a slow decreasing trend, the levels in the 2010–2016 samples were still elevated based on common cetacean toxicological thresholds. A transition period in 2007–08, probably caused by a morbillivirus epizootic amplified the stranding, espacially of highly contaminated specimens. From 2010, higher proportions in parent compounds towards metabolites were observed yet again. These changing patterns were likely reflect the exposure of dolphins to the remobilization of pollutants from contaminated soils and sediments, with a prominent role of rivers. This should lead to an even slower decline of these contaminants that could last for decades, requiring new efforts to reduce their dispersal to aquatic ecosystems.
Article
• Marine aquaculture, and its fast‐growing development, has the potential for wider environmental, ecosystem, and biodiversity impacts. This study assesses the impact of fish farming on a bottlenose dolphin (Tursiops truncatus) population within the Gulf of Ambracia (western Greece) between 2007 and 2018. Two different study areas were defined in order to investigate differences on occurrence, abundance, behaviour, and seasonal fluctuations between them: a ‘control area’, in the north‐western side of the Gulf; and a ‘fish farm area’, in the south west, where the highest density of fish farm cages is found. • A total of 169 daily surveys were dedicated to the control area and 74 days were dedicated to the fish farm area, yielding 104 and 37 sightings, respectively. Both the probability of detecting dolphins (U = 6,763.000, P = 0.002) and the group sizes (U = 578.000, P < 0.001) were smaller around fish farms. • Seasonality analyses were restricted to 2007–2008, as this was the period with year‐round effort. Results revealed that dolphins were more frequently seen around fish farms in the winter (Kruskal–Wallis test, P = 0.036). • From the 40 identified individuals that were re‐sighted at least 10 times during the study period, 21 used the fish farm area less than expected according to sampling effort, and 10 of them were never observed in that area (binomial test, P < 0.05). • The results revealed an impact of fish farms on the distribution of bottlenose dolphins in the Gulf of Ambracia. This information should be taken into consideration when defining ecosystem‐based management measures within the management plan, which is currently in preparation for this Natura 2000 site.
Article
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