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

Abstract

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.

Full text

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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.!
!
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.!
!
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! (2–12! 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,!

!
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! 70–200!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.!!

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7!
Mark–recapture! 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!Shapiro–Wilks!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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Norris,!J.L.,!Pollock,!K.H.,!1996.!Nonparametric!MLE!under!two!closed! capture-recapture! models! with! heterogeneity.!
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Environ.!Toxicol.!Chem.!29,!1263–1273.!
!!
!
! !

!
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!
Notes!
Haplotype!
1!
4-8-2004!
11:26!
skin!swabbing!!
No!Identified!
IV!
2!
4-8-2004!
11:40!
skin!swabbing!!
Identified!as!3022!
IV!
3!
4-8-2004!
11:50!
skin!swabbing!!
No!Identified!
Failed!sequencing!
4!
6-8-2004!
10:42!
skin!swabbing!!
No!Identified!
Failed!sequencing!
5!
6-8-2004!
11:21!
skin!swabbing!!
No!Identified!
XXXXII!
6!
6-8-2004!
13:09!
skin!swabbing!!
No!Identified!
Failed!sequencing!
7!
9-8-2004!
11:32!
skin!swabbing!!
Identified!as!3036!
Failed!sequencing!
8!
15-8-2004!
9:40!
skin!swabbing!!
No!Identified!
Failed!sequencing!
9!
14-8-2006!
9:40!
skin!swabbing!!
No!Identified!
IV!
10!
14-8-2006!
11:50!
skin!swabbing!!
No!Identified!
IV!
11!
8-9-2006!
10:33!
skin!swabbing!!
No!Identified!
IV!
12!
8-9-2006!
11:11!
skin!swabbing!!
No!Identified!
IV!
13!
8-9-2006!
11:22!
skin!swabbing!!
No!Identified!
IV!
14!
15-9-2006!
10:40!
skin!swabbing!!
No!Identified!
IV!
15!
28-9-2006!
19:05!
dead!stranded!!
No!Identified!!
(Juvenile,!male,!length=210!cm)!
IV!
16!
20-6-2007!
19:15!
dead!stranded!!
No!Identified!!
(Adult,!male,!length=257!cm)!
IV!
17!
26-6-2007!
8:52!
skin!swabbing!!
No!Identified!
IV!
18!
26-6-2007!
9:04!
skin!swabbing!!
No!Identified!
IV!
19!
27-6-2007!
11:38!
skin!swabbing!!
No!Identified!
IV!
20!
4-7-2007!
11:47!
dead!floating!adrift!
Male!new!born;!length!about!100!cm!!
(mother!identified!as!03046)!
IV!
21!
19-8-2007!
11:01!
skin!swabbing!!
No!Identified!
IV!
22!
19-8-2007!
11:19!
skin!swabbing!!
No!Identified!
IV!
23!
27-8-2007!
10:45!
skin!swabbing!!
No!Identified!
IV!
24!
8-9-2009!
14:40!
dead!stranded!!
No!Identified!!
(Adult,!male,!length=270!cm)!
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.!
!
!