Life in sympatry: coexistence of native Eurytemora affinis and invasive Eurytemora carolleeae in the Gulf of Finland (Baltic Sea)

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Finland (Baltic Sea)

Natalia Sukhikh, Anissa Souissi, Sami Souissi, Anne-Catherine Holl, Nikolaos

Schizas, Victor Alekseev

To cite this version:

Natalia Sukhikh, Anissa Souissi, Sami Souissi, Anne-Catherine Holl, Nikolaos Schizas, et al.. Life in

sympatry: coexistence of native Eurytemora affinis and invasive Eurytemora carolleeae in the Gulf of

Finland (Baltic Sea). Oceanologia, Polish Academy of Sciences, 2018, �10.1016/j.oceano.2018.11.002�.

�hal-01977016�

ORIGINAL

RESEARCH

ARTICLE

Life

in

sympatry:

coexistence

of

native

Eurytemora

affinis

and

invasive

Eurytemora

carolleeae

in

the

Gulf

of

Finland

(Baltic

Sea)

Natalia

Sukhikh

*

,

Anissa

Souissi

,

Sami

Souissi

,

Anne-Catherine

Holl

,

Nikolaos

V.

Schizas

,

Victor

Alekseev

a

ZoologicalInstitute,RussianAcademyofScience,SaintPetersburg,Russia

b_{LilleUniversity,CNRS,ULCO,LOG,Wimereux,France} c

LilleUniversityofScienceandTechnology,CNRS,Villeneuved'AscqCedex,France

d_{DepartmentofMarineSciences,UniversityofPuertoRico,Mayagüez,PuertoRico}

Received18June2018;accepted2November2018

KEYWORDS

Eurytemoraspecies; Copepoda;

Zooplankton; Invasiveandnative species;

GulfofFinland

Summary Theinvasionofexoticspeciesintonativeecosystemsisbecomingacrucialissuein globalbiology.Overthelasttenyears,atleast45invasionsofaquaticspecieshavebeenreportedin theeasternpartoftheGulfofFinland;themajorityofthemwereintroducedthroughballastwater. Recently,invasionoftheestuarinecalanoidcopepodEurytemoracarolleeae(Temoridae), origi-natingfromNorthAmerica,hasbeenreportedinseveralEuropeanestuariesandparticularlyinthe GulfofFinland.ThisspeciesismorphologicallyverysimilartothenativeEurytemoraaffinis,butitis easilydiscriminatedbymolecularmarkers.Inthisstudy,wemonitoredthedistributionareaofthe invasivecopepodspeciesinEuropeanwaters,aswellasthepopulationstructureof(native)E. af_finisand(invasive)E.carolleeae,from2006to2018intheGulfofFinland.Thepopulationdensity ofE.affiniswassignificantlyhigher,comparedtoE.carolleeae,duringmostofthestudyperiod.The onlyexceptionwasNevaBayin2010,whereintheinvasivespeciesdominatedpossiblyduetohigh temperaturesanddifferencesinthelevelsoffishpredation.ThereproductiveperformanceofE. carolleeaewasalsohigherthanthatofE.affinis.Theseresultsshowdifferentpopulationdynamics betweenthetwospecies.ItwasrevealedthatinvasiveE.carolleeaedevelopsinsomeofthevery

PeerreviewundertheresponsibilityofInstituteofOceanologyofthePolishAcademyofSciences.

* Correspondingauthor.Universitetskayaemb.,1,St.-Petersburg,199034,Russia.Tel.:+78123281311;fax:+78123280221. E-mailaddresses:Susikh1@mail.ru(N.Sukhikh),anissa.ben-radhia@univ-lille1.fr(A.Souissi),Sami.Souissi@univ-lille1.fr(S.Souissi),

Anne-Catherine.Holl@univ-lille1.fr(A.-C.Holl),nschizas@gmail.com(N.V.Schizas),alekseev@zin.ru(V.Alekseev).

Availableonlineatwww.sciencedirect.com

ScienceDirect

jo u rn al ho m e p age : w w w. jo ur na ls .e l se v i er.c o m / o ce an o lo g i a/

https://doi.org/10.1016/j.oceano.2018.11.002

0078-3234/©2018InstituteofOceanologyofthePolishAcademyofSciences.ProductionandhostingbyElsevierSp.zo.o.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.

Introduction

Anestimated140—171aquaticinvasionshavebeenreported in the Baltic Sea during the last two centuries (www. stateofthebalticsea.helcom.fi;www.corpi.ku.lt).The geolo-gically young ecosystem ofthe Baltic Sea,in combination withsalinitygradients,hasresultedinmanynewecological niches.Thesefactorshavebeenhypothesizedtoprovidethe key necessary conditions for the spread of new invasive species and their naturalization in the area (Leppäkoski etal., 2002a,b). Previous and ongoing intensive maritime traffic,however,resultsinthedisplacementofmillionoftons of ballast water from site to site (www.helcom.fi/Lists/ Publications).ThesetransfersareimpactingtheBaltic'sflora andfauna,andthey maybeamajorfactorinthemultiple invasions recorded in the region during the last century (OjaveerandKotta,2015).

TheGulfofFinlandisoneofthemostdensemaritimetraffic areasintheBalticSea;itincludesseveralactiveinternational shipping routes and large ports (Pollumaea and Valjataga, 2004).Consequently,morethan 40alienspecieshavebeen foundduringthelasttenyearsinonlytheeasternpartofthe GulfofFinland,mostofwhichwereinvertebrates(Lehtiniemi etal.,2016).Mostofthesespecieswereintroducedthrough ballastwater(Berezinaetal.,2011; Katajisto etal., 2013; Lehtiniemietal.,2016;Panovetal.,2003;www.helcom.fi/ Lists/Publications; www.stateofthebalticsea.helcom.fi), including:Cercopagis pengoi(Ostroumov, 1891) (Crustacea: Cladocera), Mytilopsis leucophaeata (Conrad, 1831), (Mol-lusca:Bivalvia),Palaemonserratus(Pennant,1777) (Crusta-cea: Decapoda), Eriocheir sinensis (Milne-Edwards, 1853) (Crustacea: Decapoda), Palaemon elegans (Martin Rathke, 1837)(Crustacea:Decapoda),Neogobiusmelanostomus (Pal-las,1814)(Fish).

Theinvasivespecieslistincludesseveralcopepodspecies, amongwhichthereisareportofasubtleinvasionin2007of the estuarine North American copepod Eurytemora carol-leeaeAlekseevandSouissi,2011intheeasternpartofthe GulfofFinland(Alekseevetal.,2009;Sukhikhetal.,2013). Later,thisspecieswasalsodetectedintheGulfofRigaandin theAmsterdamchannels(Sukhikhetal.,2013),aswellasin additional locations (Wasmund et al., 2013), namely: Kiel Bight,MecklenburgBight,ArkonaSea,BornholmSea,andin EasternGotlandSea.

It isinterestingthat,accordingtopictures and descrip-tionsofEurytemoraspeciesinEnglishwaters(Gurney,1931), E. carolleeae already inhabited this area of water at the beginningof20thcentury.Possibly,itwasaninvasionthrough shipballastwater,similartothecaseofEurytemora amer-icana Williams, 1906, which was originally discovered in 1933inthesamearea(Sukhikhetal.,2016a).Recentgenetic studies of Eurytemora populations have not revealed the

presence of E. carolleeae in English waters (Lee, 2000; Sukhikhetal.,2016b;Winkleretal.,2011).However,genetic studiestargetedfew crustaceanspecimens, anditis likely thattheymissedE.carolleeae.Inaddition,early morpholo-gicalstudiesmayhavemisidentifiedthisspeciesas Euryte-moraaffinis(Poppe,1880).

The E. affinis species complex is a group of species inhabitingtheHolarctic(Sukhikhetal.,2013).Thespecies complexiscurrentlyrepresentedbythreespecies:E.affinis withPalearctic distribution;NorthAmericanE.carolleeae; andAsianEurytemoracaspicaSukhikhandAlekseev,2013.All ofthesespeciesinhabitestuariesandfreshwaterreservoirs wheretheyarethedominantpelagicspeciesandconstitute themainfoodsourceforanimalsathighertrophiclevels(e.g. Devrekeretal.,2008,2010;Duretal.,2009;Lee,2000).

The E. affinis species complex has been well studied (Devreker et al., 2008, 2010; Dur et al., 2009; Hirche, 1992;Knatz, 1978;Lajus etal.,2015; Lloydetal.,2013). Experimental studies comparing the reproductive traits (developmenttime, clutchsizeandlongevity) ofE.affinis (fromtheSeineestuary,France)andE.carolleeae(fromSt. Lawrencesaltmarshes, Canada;andChesapeakeBay,USA) have confirmed the higher fitness of the North American population (Beyrend-Dur et al., 2009; Devreker et al., 2012) compared to the European one (Devreker et al., 2009,2012).Inaddition,fieldmeasurementshavesuggested that,in both populations,egg productiondecreasedwhen temperatures roseabove 188C (Lloydetal.,2013; Pierson et al., 2016). This corroborates results from laboratory experiments(Devrekeretal.,2012).

Inthispaper,weinvestigatedthecoexistenceofthesetwo EurytemoraspeciesintheGulfofFinland.Thepresenceof both species in the Baltic Sea is the result of secondary contact. Historically, only E. affinis inhabited the studied region,whereasthenativehabitatofE.carolleeaewasthe North AmericanAtlanticcoast.E.affinisandE.carolleeae divergedapproximately5.1millionyearsago,datingtothe time of theMiocene/Pliocene boundary (Lee,2000). They have a mean sequence divergence of 15% in part of the mitochondrialcytochromecoxidaseI(COI)gene.

ThedetectionoftheserelatedspeciesinBalticwatersis likelytheresultofrecentinvasionbyE.carolleeaeviathe ballastwaterofships(Alekseevetal.,2009;Sukhikhetal., 2013).ThemostlikelysourceofthisinvasionistheAtlantic coast of theUnitedStates (Alekseevet al.,2009;Sukhikh etal.,2013).

E.carolleeaeandE.affinisareverysimilar morphologi-callyanditappearsasthoughtheyoccupy,moreorless,the sameecologicalniches.Likeotherinvasivespecies,however, displacementscanbedetrimentaltoecosystemstability.At thebeginningoftheinvasion,siblingspeciescause uniden-tifiablechangesinbiologicaldiversity,followedby

rearran-samehabitatsasnativeE.affinis,therebypotentiallybecomingasignificantcomponentofthe zooplanktoninthestudiedarea.Moreover,invaderhasthepotentialtodisplacenativeE.affinis. ©2018 Instituteof OceanologyofthePolishAcademy ofSciences. Productionand hostingby ElsevierSp.zo.o.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(

gementoftheaquaticcommunities(Gelembiuketal.,2006). Infact,suchspeciescanexhibitdistincthabitatpreferences definedbydepth,salinity,orexposure.Successional differ-encesbetweensiblingspecies,reflectingtemporal partition-ing of resources in response to seasonal change or disturbance, have also been documented (Knowlton, 1993). This may be the result of different physiologies. Moreover,hybridization experiments,betweentheseNorth American and Europeanspecies, have shown reproductive incompatibility among them (S. Souissi, unpublished). For example,six Tubifex tubifex(oligochaetes)lineages living sympatricallydifferedintheirtolerancetocadmium( Sturm-bauer etal.,1999) andin their resistance to infection by Myxoboluscerebralis(Beauchampetal.,2001).

Previous dataon theregion'szooplanktoncommunity is ratherlimitedandhasbeenpublishedmainlyinRussian.The zooplanktoncommunity ofthe easternpartof theGulf of Finland is represented mainly by freshwater species. The bulkofzooplankton,bymass,usuallyconsistsofCladocera (PollumaeandKotta,2007;Uittoetal.,1999),while cope-podsdominatenumerically(OgorodnikovaandVolkhonskaya, 2006;Ostov,1971;RyabovaandPogrebov,1991).Ingeneral, zooplanktonin the Russian Gulf ofFinland are distributed irregularly,andtheareasofhighestzooplanktonabundance arelocatedinthesouthernandeasternregions(Ostov,1971). Dependingontheyearofthestudy,zooplanktonbiomasses have varied from 140 to 1000mgm
3 (Antsulevich et al., 1995;Basova,1983;LavrentievaandFinogenova,1999).Asa result,LugaBayandNevaBay(bothsituatedinthe south-eastern Gulf of Finland) serve as the main areas for fish feedingandbreeding(Golubkov,2009).Themainconsumers ofzooplanktonintheGulfofFinlandareBalticherring.Since the mid-1990s, however, Sprat (Sprattus sprattus (Lin-naeus)),whichisrecoveringfrom overfishinginthe1970s, has also begun to play a significantrole as a zooplankton predator(Alimovetal.,2004).

Zooplanktonaggregationsarerepresentedbyboth brack-ishandfreshwaterspeciesinLugaBay.Studies(Lavrentieva and Finogenova, 1999; Ogorodnikova and Volkhonskaya, 2006; Ryabova and Pogrebov, 1991; Sergeev et al., 1971) haveshownthatdifferentspecieshavedominated aggrega-tionsindifferentyears: Keratellaquadrata (Muller); Kera-tella cochlearis (Gosse); Synchaeta baltica Ehrenberg; Synchaeta oblonga Ehrenberg; Bosmina obtusirostris Sars; Acartia clausi Giesbrecht; Daphnia cristata Sars; Daphnia cucullata Sars; and Eurytemora spp. Generally speaking, thesedominantspeciesoccurinotherspartsoftheGulfof Finlandaswell(PollumaeandKotta,2007;Uittoetal.,1999). Eurytemoraspp.areinvariablypresentinthesespecieslists (Uittoetal.,1999).Itisoneofthedominantmembersinthe Gulf of Finland (https://www.st.nmfs.noaa.gov/copepod/ time-series/fi-30103/), and they reach up to 50% of all zooplanktonbiomassinthestudyarea(Sukhikh,unpublished data).Eurytemoraspp.consistupto45%ofallstomachsof cyprinidspeciesandareabundantlyfoundinthestomachsof sticklebacks(Demchuketal.,2017).

LittleisknownaboutlocalEurytemoraspp.populations andeven lessisknown about thenew invasivespecies, E. carolleeae, inthe BalticSea. Thisis thefirst studyof the populationstructureandreproductivetraitsoftworelated specieslivingtogetherintheBalticSea:nativeE.affinisand invasiveE.carolleeae(ofwesternAtlanticorigin).Wehave

usedgeneticmarkerstoexaminethepotentialfor hybridiza-tionbetweenthesetwocloselyrelatedspecies(E.affinisand E.carolleeae)whicharelivinginsympatry.

AstheinvasionofE.carolleeaeseemstobearecentand rapidprocess,wehypothesizeherethatithasthepotential todisplacenativeE.affinisintheGulfofFinlandecosystem andpossibly in the entire Baltic Sea.Such an outcome is especiallypossibleunder certain conditions,such as force majeureeventsthatcauseprofoundenvironmentalchanges. Weseektoclarifyspatialandtemporaldifferencesintheir distributionsthatarerelatedto,ordependenton, environ-mentalparametersinordertogainabetterunderstandingof thepotentialfornativeE.affinistobedisplacedbyinvasive E.carolleeae.

2.

Material

and

methods

2.1. Sampling

InordertorevealthedistributionofinvasiveE.carolleeaein Europeanwaters,copepodswerecollectedfrom11European sitesbetween2004and2017:channelsin Amsterdam;the Elbe,Seine,Schelde,Loire,andGirondeestuaries;theLake of the Bois de Boulogne (Paris); Umeå Seaport (Sweden); VistulaLagoon;theGulfsofRigaandFinland(theBalticSea); andtheNorthern DvinaRiver(Fig. 1,Table1).Threesites wereanalyzedintheGulfofFinland:theGulfofVyborg,Neva BayandLugaBay.

Monitoringofinvasivespecieshasbeen carriedout per-iodicallysince2004inNevaBayandsince2006inLugaBay.To estimate the relative percentage of the two Eurytemora speciesin Neva andLugaBays, samplingswereperformed onceperyear, usuallyduringAugustwhenhigh population densitiesare observed;Luga Bay sampling in2010 was an exceptionandoccurredinSeptember.Thetotalnumberof studiedspecimensrangedfrom15to181persite(thenumber of specimens obtained in three replicates, three nets in each).

Seasonal monitoring of adult population densities (E. affinisandE.carolleeae)inLugaBay(GulfofFinland)was carried out from 10.06 to 27.08 in 2006, from 19.04 to 17.09 in 2008 and from 16.06 to 27.09 in 2015. Sampling wasperformedatthemouthofLugaRiver,everytendaysin 2006 and in 2008, and every twenty days in 2015. Water salinityandtemperatureatthemouthoftheLugaRiverwere measuredusing a COM-100 waterproof combinationmeter (HMDigital,USA).

Samples were collected with 100mm or 230mm mesh planktonnetsbyverticaltowsfromdepthtosurfaceinthree replicatesand preserved in 96%ethanol or in 4% formalin solution(samplinginformationisgiveninTable1).

2.2. Speciesidentification

IdentificationofadultE.affinisandE.carolleeaecopepods wasaccomplishedbyfollowingpublishedtaxonomicalkeys (Alekseev and Souissi, 2011; Sukhikh and Alekseev, 2013). Morphological analysis of adult copepods was performed under an SZX2 dissection microscope (Olympus) with a 5mmresolutionocularmicrometer.E.carolleeaetype mate-rialfrom theRussianAcademyofSciencesZoological

Insti-tute collection was used for reference in this study (type collection#55052-55054).Identificationofspecimens from theScheldeRiver,Seineestuary,GulfofRiga,GulfofFinland, VistulaLagoon,Loireestuary,LakeintheBoisdeBoulogne, andNorthernDvinaRiverwasalsosupportedbyDNA sequen-cingof aportionof themitochondrialcytochrome oxidase subunit1gene(COI,seebelow).Instudyingandestimating population densities in Luga Bay, only adult stages of E. affinisandE.carolleeaewereanalyzedastherearenoclear morphological features distinguishing the juvenile stages (naupliiand copepodites)ofthese closelyrelated species. Moreover, an additional Eurytemora species, Eurytemora lacustris (Poppe, 1887), was present in the zooplankton communityofthe sampledarea. The juvenilestages ofE. lacustrisare also indistinguishable fromthose ofE. affinis andE. carolleeae. As aresult, it was impossible for usto separately distinguish or estimate nauplii andcopepodites densitiesforthesethreeEurytemoraspecies.

2.3. Morphological andreproductivetraits measurements

Formeasurementof reproductiveparameters,20E. carol-leeae females and 23 E. affinis females were randomly selected from the same sample collected in July 2015 in LugaBay(watertemperature17.38C).The numberofeggs perclutchand theeggdiametersof 5—10eggsfrom each

clutchwerecalculatedforeachfemale ofbothspecies.In addition,thelengthsandwidthsoftheprosomeandtheegg sacweremeasuredunderadissectionmicroscope(asabove).

2.4. Statisticalanalysis

Differencesbetween thespecies,interms ofreproductive parametersaswellasinthelengthsandwidthsofprosomes andeggsacs,werequantifiedusingthenonparametric Krus-kal—WallistestasimplementedintheStatistica7software package.Therelationshipsbetweenfemaleprosomelength andclutchsize,inbothstudiedspecies,wereshownbylinear regressionanalysis(Statistica7).Thesignificancelimitwas setatp<0.05.

2.5. Materialusedforgeneticanalysis

Thenuclearribosomal18Sgene,ITSregions(including5.8S), and one mitochondrial (COI) gene were analyzed in the present study. Specimens used for genetic analysis were obtainedfrom: NevaBay (Russia),July2014 (E.affinis, E. carolleeae);theLoireandSeineRivers(France),April2011 (E.affinis);theSaint-Lawrenceestuary(France),September 2014(E.carolleeae);andalaboratorycollection(E. carol-leeae),originallyfromChesapeakeBay(U.S.A.).Atotalof18 E.affinisindividualsand23E.carolleeaeindividualswere analyzedwithgenetictools.

Figure1 Samplinglocations analyzedby theauthors (rhombi)and literature data onthedistribution of invasive Eurytemora carolleeaeinEurope(circles).Black_figuresrepresentthepresenceofinvasiveEurytemoracarolleeaeinstudiedarea.1_—Gironde Estuary;2—LoireEstuary;3—ChelsonMeadow,Plymouth,Britishwaters(Gurney,1931);4—SeineEstuary;5—LakeinBoisde Boulogne(Paris);6_—ScheldtEstuary;7_—Amsterdamchannels(Sukhikhetal.,2013);8_—ElbeEstuary;9_—13:9_—KielBight,10_— MecklenburgBight,11—ArkonaSea,12—BornholmSeaand13—EasternGotlandSea(Wasmundetal.,2013);14—GulfofBothnia, Umeå;15_—Stockholm(Gorokhovaetal.,2013);16_—Vistulalagoon;17_—GulfofRiga;18_—GulfofFinland;19_—theWhiteSea

(Sukhikhetal.,2016a,bandpers.comm.ofPolyakovaN.V.);20—22—theCaspianSeaandthedrainagebasinofVolgaRiver(Lazareva

2.6. DNAextraction,amplification,and sequencing

Genomic DNA was extracted from single adult copepods preservedin96%ethanolusingastandardmethoddescribed byAljanabiandMartinez(1997)orusing acelllysisbuffer withProteinase-KprotocolmodifiedfromHoelzelandGreen (1992)andLee(2000).Polymerasechainreaction(PCR),in ordertoachievecytochromeoxidasesubunit1(COI) ampli-fication, utilized both universal (COIH, COIL) and specific (EuF1, EuR2)primers.Their sequencesare: COIH2198 (50 -TAAACTTCAGGGTGACCAAAAAATCA-30); COIL 1490 (50 -GGTCAACAAATCATAAAGATATTGG-30; Folmer et al., 1994); EuF1 (50-CGTATGGAGTTGGGACAAGC-30); and EuR2 (50 -CAAAATAAGTGTTGGTATAAAATTGGA-30; Winkler et al., 2011). Two thermocycling programs, modified from Lee (2000),wereusedforPCRamplification.Thefirstwas5cycles of908C(30s),458C(60s),728C(90s);followedby27cycles of908C(30s),558C(45s),728C(60s);andendingwith5min at728C.Thesecondprogramfeaturedaninitialdenaturation at 958C for 30s; followedby 5cycles of 908C(30s), 558C (60s),728C(90s);followedby27cyclesof908C(30s),558C

(45s), 728C (60s); and ending with 5min at 728C. These conditions and methods were used in our previous work (Sukhikh etal.,2016a,b).

Complete18SrDNAswereamplifiedusingtheprimerpair 18A1 mod (50-CTGGTTGATCCTGCCAGTCATATGC-30) and 1800 mod(50-GATCCTTCCGCAGGTTCACCTACG-30) (Raupach etal., 2009). The ITS-4 and ITS-5 universal nITS (nuclear ribosomalDNAinternaltranscribed spacer)primers(White et al., 1990) were used for amplification of the ITS1-5.8SrRNA-ITS2region.PCRconditionsforbothsetsofprimers (18SrRNAand nITS) were: initial denaturation at 958C for 30s;followedby38cyclesof958C(30s),annealing(508Cfor nITSor 558Cfor 18SrRNA)for 30s, 728C(70s);andafinal extensionat728Cfor7min.

Amplified products were purified with a QIAquick PCR purificationkit(Qiagen,Valencia,CA,USA)andsequenced using an ABI 3100 or 3130 automated sequencer (Applied BiosystemsInc.,FosterCity,CA,USA).BothDNAstrandswere sequencedtoconfirmtheaccuracyofeachsamplesequence. Sequenceswerealigned using theCLUSTALWalgorithm (Thompsonetal.,1994)implementedinBIOEDITv.7.2(Hall, 1999)withmanualeditingofambiguoussites.Thenumberof

Table1 SamplinglocationsofpopulationsofEurytemoraaffinisandEurytemoracarolleeae. Samplinglocations Samplingdate Samplesize

forgenetic analysisa Samplesize for morphological analysisb Latitude Longitude

Elbeestuary March2006 50 53853024N 09808044E

ScheldtRiver Antwerp Duaene April2011 April2011 7 1 15 15 51813042N 518N 04823086E 048E Seineestuary April2011

May2008 July2008 37 10 8 9 49828033N 49₈₂₈033N 498N 00827054W 00₈₂₇054W 008W GulfofRiga CityPort Aug.2008 14 29 57804044N 23804044E GulfofFinland: GulfofVyborg NevaR.estuary LugaR.estuary Sep.2007 Aug.2009 Aug.2010 Aug.2004,2007,2010_—12,2014_—15 Aug.2006—09,2011,2015 Sep.2010 35 30 30 10 110 227 60823039N 59832036N 59824013N 28826074E 29828017E 28811006E

VistulaLagoon Oct.2007 Jun.2015

5 30

30

54865002N 20823037E NorthernDvinaRiver Aug.2015 5 10 64833000N 40832000E GulfofBothnia,Umeå May2010 10 6384903000N 2081505000E Loireestuary St.1 St.2 April2011 July2009 52 10 9 47₈₁₇023N 02₈₀₁052W Girondeestuary St.1 St.3 May2005 July2009 10 4 45804010N 00838030W

LakeintheBoisdeBoulogne(Paris) July2010 3 48851048N 2815007E Saint-LawrenceEstuary Sep.2014 4 488101N 6982008W

ChesapeakeBay Feb.2013 3 38836015N 7684054W

a_{Numberofindividualssequencedperlocation.}

polymorphicsiteswasestimatedusingDNASPv6(Libradoand Rozas,2009).The levelofnucleotide differencesbetween thespecieswascalculated using theTamura-Nei93model withtheMEGA6.06softwarepackage(Tamuraetal.,2013).

3.

Results

3.1. Distributionofinvasivespecies inEuropean waters

Apart from the Gulf of Finland, the presence of invasive AmericanEurytemoraspecieswasmonitoredat11sampling locations (Table 1) over the last 12 years. As a result, E. carolleeae was detected in Riga Bay and in Amsterdam channels.ThedensityofAmericanEurytemora inRigaBay didnotexceed2%oftotaldensity(bothEurytemoraspecies). Incontrast,E.carolleeaewasmoreprevalentinAmsterdam channelswithatotalofabout30%ofthecombined Euryte-moradensity.E.carolleeaewasabsentfromallsamplesfrom theSchelde,Seine, Loire,andGironde estuaries,andalso absentfromtheBoisdeBoulogne(Paris),VistulaLagoon,the GulfofBothnia(theBalticSea),andNorthernDvinaRiver.

3.2. Coexistenceofnativeandinvasive Eurytemoraspecies intheGulfofFinland

Duringtheentirestudyperiod,E.affinisnumerically domi-nated the Eurytemora species assemblage in the Gulf of Finland(Fig. 3a, b).Eurytemora carolleeaeaccounted for 2—30%inLugaBayandfrom0%to100%inNevaBay.During the whole study period, E. carolleeae occurred in fewer numbers than E. affinis in Neva and Luga Bay regions in the Gulf of Finland. The maximum E. carolleeae density percentageswereobservedin 2010and2015 (Fig. 3a).At thesametime,thedensitiesofE.carolleeaeadultfemales duringtheunusualtemperatureconditionsin2010and2015, weresimilartothoseseenduringthethermallynormalyear 2011,inwhichE.affiniswasprevalent(631259indm
3). Indeed, thedensity of E. carolleeae adultfemales in mid September2010 in Neva Bay was 2411indm
3. In July 2011,thedensityofE.carolleeaeadultfemaleswasabout 1610indm
3.InmidAugust2015,thedensityofE. car-olleeae adultfemales was 248indm
3. E. affinis adult females densities in 2010 and 2015 were low: 108 51indm
3and11indm
3,respectively.

3.3. SalinityandtemperatureconditionsinLuga Bay,GulfofFinland

WatersalinityinthestudiedareaatthemouthoftheLuga Riverchangedfrom0.67to2.31psuduringthemonitoring period. Temperatures during the summers of 2006, 2008, 2015,and2017rangedfrom aminimumof12.88C(inJune 2015)toamaximumof23.28C(inJuly2006)(Fig.2).Inthis regionoftheGulfofFinland,meanwatertemperatureinJuly isusuallybetween18and208C(http://weatherarchive.ru). During2010,2015,and2017,however,watertemperatures wereunexpectedlydifferent.In2010,forexample,23.58C wasrecorded.Yet,thesummers of2015and2017,by con-trast, wererather cooland meanJuly temperatureswere 17.18Cand15.88C,respectively(http://weatherarchive.ru).

3.4. Densitychanges inadultE.affinisand E. carolleeae populations

The average density of the zooplankton community (repre-sentedmainlybyRotifera,Cladocera,andCopepoda)wasabout 105indm
3inallstudiedperiods.TheOrderCopepoda domi-natedthesummerzooplanktoncommunity(50,000indm
3). Thepredominantzooplankton speciesweretherotifer Kera-tellaquadrata,thecalanoidcopepodE.affinis,andthe cla-doceranBosminalongispina.E.carolleeaewaspresentinallof theGulfofFinlandstudylocations.SeasonalmonitoringofE. carolleeaeandE.affinisinLugaBayshowedthatbothspecies exhibited two summer population density peaks (in years 2015 and in 2008) and one strongpeak in 2006 (Fig. 4). In 2006,themajorpeakforbothspecieswasobservedinthe end-of-Junetobeginning-of-Julytimeframe,yetwithanalmostfive foldhigherdensityforE.affinisthanforE.carolleeae.Aminor peakwasnotedforE.affinisatthebeginningofAugust,aswell asaslightincreaseattheendofthemonth(Fig.4a).

In2008,thehighestdensitywasobservedbetween mid-JuneandthebeginningofJulyforE.affinis,andasecond peakwasrecordedattheendofAugust.Atthesametimes, twopeaksofdensitywerealsoobservedforE.carolleeaebut ofsmallermagnitude(Fig.4b).

In 2015, the first density peak, for both species, was recorded during mid-June and the beginning of July, and thesecondonewasobservedatthebeginningofSeptember (Fig.4c).NevaBaysamplinginsummer2017(24.07.17)did notdetectanyEurytemoraspecimens.

Figure2 MeanchangesinLugaBay,GulfofFinland,surfacewatertemperature(8C)duringspring,summer,andearlyautumninthe years:2006(fullline);2008(dottedline);and2015(dashedline).

Overall, E. affinis population densities were generally severaltimeshigherthanthoseofE.carolleeae.The max-imaldensitieswereobservedforbothspeciesin2006,namely 1295indm
3forE.affinis,and201indm
3forE.carolleeae. Theminimalpopulationdensitieswereobservedduringthe summer of 2015 in which no more than 117indm
3 were observedforE.affinisand24indm
3forE.carolleeae.

InFig.5,thedetaileddensitiesofmalesandfemales,of bothspecies,areshown.During2006and2008,therewere moremalesthanfemalesinbothE.affinis(Fig.5a,b)andE. carolleeae populations (Fig. 5d, e). However, during 2015 (Fig. 5c, f), the oppositeoccurred, andthe sex ratio was generallyinfavoroffemalesexceptforonedate(beginning ofJuly,E.affinis)(Fig.5c).

3.5. ReproductiveparametersofEurytemora females

Thetwo EurytemoraspeciesstudiedintheGulfofFinland weresignificantlydifferent(p<0.05)intheirmorphological (prosomelengthandwidth,eggsacwidth)andreproductive

(clutchsize)characteristics (Table 2).The respective pro-some lengths and widths were 830.27.0mm and 310.1 6.4mminE. carolleeaeand744.015.5mm and247.9 5.3mminE.affinis.

ClutchsizewasalmosttwotimeslargerinE.carolleeae than in E. affinis: 61.72.4 and 34.01.4, respectively Analysisofeggsizeandeggsaclengthdidnotreveal sub-stantialdifferencesbetweenthetwospecies.Thedifference ineggsacwidthbetweenthetwospecies(252.311.5mm inE.carolleeaeversus226.95.1mminE.affinis)reflects differencesintheshapeofthesac,whichismoreroundedin E.carolleeaeandmoreovalinE.affinis.Atthesametime, prosomelengthsandclutchsizesinfemalesofbothspecies hadalinear relationship(E.affinis, r2_{=0.59, p<0.05;E.}

carolleeae,r2=0.35,p<0.05).

Figure5 Populationdensitychangesinmales(dottedlines)andfemales(solidlines)ofEurytemoraaffinis(a,b,c)andEurytemora carolleeae(d,e,f)duringthe2006(a,d),2008(b,e),and2015(c,f)summerseasons.

Figure 3 Percent ratio of invasive Eurytemora carolleeae (blacksector)andnativeEurytemoraaffinis(graysector),during thelast10years,inNeva(a)andLugaBays(b),GulfofFinland.

Figure 4 Population density changes in adult Eurytemora affinis (dotted line) and Eurytemora carolleeae (solid line) duringthe2006(a),2008(b),and2015(c)summerseasons.

3.6. DNApolymorphismdataand hybridization betweenthespecies

Morphological observation revealed clear differences betweenthe two species, andspecimens exhibiting inter-mediatecharacterswerenottypicallyseenduringthestudy period.Very rare specimens (about 1%)with intermediate featureswereobservedandtheyweretentativelypresumed tobehybrids.

These intermediate phenotypes usually featured inter-mediatenumbersofeggsinthe eggsac,intermediateegg sizes,bodysizes,orcaudalramishapes.Somehad segment-likedivisionsinsetaeandgenitalsomitewithoutgrowth,as inE.carolleeae,yettheyalwaysdifferedfromthe morphol-ogyofE.carolleeaetypespecimensbyawing-likeoutgrowth inthedistalpartofbody,adiagnosticcharacterofE.affinis. Genetic analyseswereperformedwithacompletedata setof86sequences(75originaland11previouslypublished; Sukhikhetal.,2016b).Theobtainedsequenceswere com-paredwithexistingsequencesofEurytemoraanddeposited in GenBank (accession numbers 18SrRNA KX400968— KX400986; COIKX400987—KX401004, KX401042—KX401328; nITSKX401005—KX401041). The ITSand18S nuclear genes wereanalyzedtogetherwiththeCOIgeneinorderto deter-minewhetherhybridizationoccursand,ifso,whetheronly F1individualsareobservedoraretheresubsequent genera-tionsofintrogression.

SixteenE.carolleeaeCOIsequencesandthirteenE.affinis COIsequenceswereanalyzed.Samplessourceswere:eightE. carolleeaeandeightE.affinissampledfromNevaBay,fourE. affinisandfourE.carolleeaesampledfromLugaBayand3E. carolleeaefromChesapeakeBay.Inbothspecies,a544b.p. COI product was amplified. Overall, E. carolleeae (COI) sequencescontained38polymorphicsitesand13haplotypes; E.affinissequencescontained4polymorphicsitesand4 hap-lotypes.The level of pairwise divergence in theCOI gene betweenthetwospecieswas15%,whichisindicativeofhigh divergencebetweenthese2species.

In terms of the 18SrRNA gene (length of 1690bp), 15sequences weresuccessfully obtainedfor E. carolleeae and 9 for E. affinis. There were no observed nucleotide differencesbetween the speciesand no polymorphic sites wereobserved.Thissuggeststhatthe18SrRNAgeneismore usefulinwidephylogeneticanalysis ofCopepoda,andless usefulinworkwithcloselyrelatedspecies.

ITSgenesequenceswereobtainedandanalyzed(E. car-olleeaen=17;E.affinisn=12)from samplescollected as follows:14E.carolleeaeand12E.affinissampledfromNeva Bay;andthree E.carolleeaefromChesapeakeBay. Dueto polymorphism,ITSampliconswere791bpfromE.carolleeae and 783bp from E. affinis. Overall, E. carolleeae ITS

sequences(794bpinlength,includingsiteswithalignment gaps)containedonepolymorphicsite,whereasE.affinisITS sequences(795bpinlength,includingsiteswithalignment gaps)hadnopolymorphicsites.Thelevelofpairwise diver-gence, in the ITS1-5.8SrRNA-ITS2 region betweenthe two species,was 4.9%.E. affinissequencesfrom theLoireand SeineRiverswerenotavailable.

4.

Discussion

4.1. DistributionofinvasiveE.carolleeaein Europe

The presenceof theinvasiveE. carolleeaespecies in Eur-opean watershasonlybeen reportedinspecificlocations, namely: theGulf of Finland,theGulf of Riga,Amsterdam channels (Sukhikh et al., 2013), Kiel Bight; Mecklenburg Bight,theArkonaSea,theBornholmSea,theEasternGotland Sea (Wasmundet al., 2013)and perhaps in British waters (Gurney, 1931) (Fig. 1). The presence of E. carolleeae in these areas is a noteworthy result since there are many previous reports, from a wide variety of European fresh andmarinewaters,showingnoevidenceofE.carolleeae.

Accurate identification ofdifferent speciesis necessary due to the fact that they feature evident differences in physiology,andthosedifferencesmaycauseharmfulchanges inecosystemfunctionorproductivity.Populationshiftsmay eventually have important consequences for biodiversity, biogeography, conservation, or fisheries management (Gelembiuketal.,2006;Knowlton,1993;Lee,2000).Such invasions might have important implications for disease transmissionas well. Eurytemoraare major hostsof many pathogens, including Vibriocholerae,V. vulnificus, and V. parahaemolyticus(Colwell,2004;Leeetal.,2007;Piasecki etal.,2004).Theyare alsoprobablehostsandvectors for plerocercoidsthatcaninfectsomefishspecies(Arnoldand Yue,1997).

E.carolleeaewasnotfoundintheElbe,Schelde,Seine, LoireorGironde estuaries,norwasitdetectedinthelake nearParis,the VistulaLagoon,or theGulfofBothnia (the Baltic Sea) in 2006—2011 (Table 1). In addition, we have previouslyshownthatitisnotpresentinsamplesfromWhite Searockpools (Sukhikh etal.,2016a,b),inthe WhiteSea itself (pers. comm.of Polyakova N.V.),or in the Northern DvinaRiver.Inaddition,specieslistsfromthePechora Estu-ary(Cherevichko,2017;Fefilova,2015),theCaspianSea,and theVolgaRiverdrainagebasin(Lazarevaetal.,2018;Sukhikh etal.,2018)didnotincludeE.carolleeae.Finally,previous geneticstudiesoftheE.affinisspeciescomplexinanumber of locations (theSwedish coast — Gorokhovaet al.,2013;

Table2 MeanvaluesofreproductiveparametersinfemalesofEurytemoracarolleeaeandEurytemoraaffinisfromtheGulfof Finland.Meanstandarddeviation.

Species/measuring Eggnumber Eggsize ProsomeL (_mm) ProsomeW (_mm) ClutchL (_mm) ClutchW (_mm) Numberof studied individuals E.carolleeae 61.72.4 76.70.8 830.27.0 310.16.4 476.614.7 252.311.5 20 E.affinis 34.01.4 77.60.57 744.015.5 247.95.3 466.811.1 226.95.1 23

Winkler et al.,2011; theElbe, Schelde, Seine,Loire, and Girondeestuaries—Winkleretal.,2011)havenotdetected E.affinis.

4.2. PopulationdynamicsofE.carolleeaeandE. affinisintheGulfofFinland

Seasonal study of E.carolleeae andE. affinis in Luga Bay revealed no substantial differences in their population dynamics.Thehighestdensitieswereobservedduringearly summer of2006for bothspecies(Fig. 4a).Thesemaximal densitiesmaybetheresultofdredgingactivityintheLuga Bay study area that occurred during the summer of 2006 (Spiridonovetal.,2011).Thiseventcausedresuspensionof nutrientsinthewatercolumn which,inresult,inducedan increaseofphytoplankton(themainfoodsourcefor Euryte-mora)density(Spiridonovetal.,2011).

The lowest population densities (both species) were observedduringthe summerof 2015(Fig. 4c). Theperiod wascharacterizedbyunusuallylowtemperatures,including aminimumof12.88CinJune.Theconditionslikelyreduced phytoplanktondensities,andtheeffectisapossiblereason forthedecreasedpopulationdensitiesrecordedforbothE. affinis andE.carolleeae.Nevertheless, nooverall correla-tionwasfoundbetweenpopulationdensityandwater tem-peratureduringthesummer.

TheabsenceofEurytemoraspeciesinthe2017samples waspossiblyduetoashiftoftheresidentmarinezooplankton communitytoariverineone,sincesummer2017wasrather rainyandriverflowhadincreased.Duringthesamesampling period, Eurytemoraspecieswere observedinmoreor less usualdensitiesinthecentralpartofNevaBayoftheGulfof Finland(pers.comm.ofLitvinchukL.),anareaunaffectedby river outflow-associatedsalinity decreases.In thesummer 2018,E.carolleeaeinLugaBaywas alsoobservedinusual density.

Throughoutthestudyperiod,thepopulationdensityofE. affiniswasseveraltimeshigherthanthatofE.carolleeaein LugaBay(Fig.4).However,inSeptember2010andinAugust 2015,Neva Bay samples contained onlyE. carolleeae;this suggestsamajorshiftinzooplanktonpopulations,featuringa replacementofE.affinisbyinvasiveE.carolleeae(Fig.3). However,theshiftinzooplanktonwastemporarysince sam-plesdevoidofE.affiniswererecordedonlythosetwotimes. Interestingly,bothsummers2010and2015featuredunusual temperatures: hot2010 andcold 2015.Recordheat levels were observed in summer 2010, resulting in the warmest summer of the last 100 years in the region (https://en. wikipedia.org; https://en.wikipedia.org/wiki/ 2010_Northern_Hemisphere_summer_heat_waves). Conse-quently, duringthat summer,the warmestwater tempera-tureswerealsorecorded.Watertemperaturesabove15—208C areknowntobeunfavorableforE.affinis(Devrekeretal., 2008,2010;Duretal.,2009;Hirche,1992;Knatz,1978).

Theseuncommontemperatureconditionsprobably nega-tively affected native E. affinis populations, yet without reducing population densities of invasive E. carolleeae.Thetemperaturetoleranceoftheinvasive cope-pod speciesis possiblywider as watertemperatures inits nativeChesapeakeBayrangebetween5and258C(Kimmel etal.,2006).E.carolleeaeisalsocharacterizedbyhighegg

productivity(Piersonetal.,2016),whichcouldfavoritsrapid spreadinthearea.IntheeasternpartoftheGulfofFinland, yearlymeanwatertemperaturevariedbetween0(winter) and 18—208C (summer) (http://weatherarchive.ru/Sea/ Ust-luga/July). In such an environment, invasive species maybemore successfulthan native ones infast changing environmentalandtemperatureconditions.Furthermore,E. carolleeaedensitieswerenotobservedtodependonsummer temperaturesindifferentyears.

In2010and2015, analysisof LugaBay samplesdidnot revealreplacementofE.affinisbyE.carolleeae.This indi-catesthatsite-specificfactorslikelyplayasignificantrolein thepopulationdynamicsofthespecies.Infact,the popula-tiondensity trends are similar to the other years studied (Fig. 3)eventhoughtheproportions ofE.carolleeaewere slightlyhigherduringthesetwoyears(30%in2010and14%in 2015).Therelativelylower2010densitiesofE.carolleeaein LugaBay, incomparison toNeva Bay,could bedueto the sample collection timing. Plankton samples were not col-lectedduringAugust,asinotheryears,butlater,attheend ofSeptember,whenwatertemperaturewas188C.However, during September of 2008 and 2015, water temperatures werenothigherthan158C,andneitherEurytemoraspecies wasfoundthere.Theseobservationsreinforcethepossibility thattemperaturefluctuationsmayaffectthedevelopment ofbothspeciesintheGulfofFinland.

LugaBayisknowntobeoneofthemostimportantregions intheGulfofFinlandforfishfeeding,breeding,andspawning (Golubkov,2009).Therefore,itispossiblethatfishpredation oncopepodswashigherinLugaBay.Prosomesize(lengthand width)waslargerinE.carolleeaethaninE.affinis(Table2); thismakesthemmoresusceptibletovisualpredators.Ithas beendemonstratedthatfisheatlargerzooplanktonfirstand smallonesafterwards(BrooksandDodson,1965).Inaddition, thisinvasivespecieshasalargereggsac(Table2),anditwas shownbyMahjoubetal.thatfishprefertofeedonovigerous females.Therefore,withtheirbiggerprosomesandeggsacs, E.carolleeaemaybemorevisibletofishpredatorsandmore susceptibletosubsequentpredation.Therefore,inaddition totemperature,fish predationpressure maybeoneofthe limitingfactorsinpopulationgrowthofE.carolleeaeinLuga Bay.Ideally,laboratoryexperimentswouldtestthese hypoth-eses.

4.3. Reproductivecharacteristicsofthestudied species

StudyofthereproductiveparametersofthetwoEurytemora specieslivinginsympatryrevealedasignificantdifferencein clutchsize,butnotineggsize.E.carolleeae,fromasummer 2015 sample, was characterized by higher reproductive potential.TheinvasiveE.carolleeaeproducedalmostdouble theclutchsize(62eggsfemale
1)thanthatofthenativeE. affinis (34 eggsfemale
1).In Chesapeake Bay (the native habitat of E. carolleeae), the species is characterized by salinitytolerance,temperaturetolerance,andhigh fecund-ity(Piersonetal.,2016).Beyrend-Duretal.(2009)compared twoformerlytransatlanticEurytemorapopulationscollected fromtheSeineestuary(France)andfromtheSaintLawrence saltmarshes(Canada)andshowedthatAmericanEurytemora had higher fecundity, higher salinity tolerance, shorter

developmenttime,andalongerlifespan(Beyrend-Duretal., 2009).Thesereproductiveandphysiologicaldifferencesmay enhancetheabilityofE.carolleeaetoinvadeandspreadinto newareas.Thisabilitymayfurtherbeenhancedinregions whereconditionshavebecomemorefavorable, overtime, duetoclimatechange.Ageneraltrendofdecreasingsalinity intheBalticSeaisonesuchexample(https://www.st.nmfs. noaa).

4.4. ComparisonbetweeninvasiveandnativeE. carolleeaepopulations

IncomparisonsbetweentheinvasiveE.carolleeaefoundin theGulfofFinland(thisstudy)andthenativeE.carolleeae fromChesapeakeBay(Lloydetal.,2013),nativeE. carol-leeaehadalowerclutchsize(around50eggsfemale
1)anda smallerprosomelength(about780mm)at thesamewater temperatures.ChesapeakeBayisapossiblesourceof inva-sive copepods (Sukhikh et al., 2013), and it is likely that invasiveE.carolleeaeencounteredmorefavorable environ-mentalconditionsin theGulf ofFinlandthan inits native area.ThisinterpretationissupportedbyLajusetal.(2015), who compared levels of fluctuating asymmetry (FA) for populationsofE.carolleeaefromChesapeakeBayandfrom theGulfofFinland.Fluctuatingasymmetryrepresents ran-domdeviationsfrom perfect symmetry,andis aproxy for developmentalinstability(Zakharov,1989).FAisoftenused tomonitorstressofdifferentorigins(Beasleyetal.,2013; Grahametal.,2010).

FAwaslargerfornativeE.carolleeae(ChesapeakeBay), comparedtoinvasiveE.carolleeae(GulfofFinland). Inter-estingly,E.affinisfrom theGulfofFinlandhasalmostthe sameFAastheinvasiveE.carolleeaespecies.Thismaybethe resultofgenerallylessstressfulenvironmentalconditionsin theGulfof Finlandin comparisonto ChesapeakeBay. The Gulf features different temperature conditions and fewer salinitychangesduetotheabsenceoftides.Infact,theE. affinispopulationfromtheSeineestuary,withitshightides, hadthehighestFA ofallof thestudiedpopulations(Lajus etal.,2015).Thosefindingsfitswithourdatashowinghigher FAfornativeE.carolleeae(fromChesapeakeBay)thanfor invasiveE.carolleeae(fromtheBaltic).

4.5. Interaction betweensympatricspecies

Long-termmonitoringofthepopulationdensitiesofthetwo Eurytemoraspecieslivinginsympatry,aswellasanalysisof theirmorphologicalandreproductiveparameters,revealed that invasive E. carolleeae and native E. affinis have remained reproductively isolated from one another. How-ever,rareindividualswithintermediatemorphological fea-tureswereobserved.Similarcases areknown,andhybrids within zooplanktonspecies/lineagesare notunheard ofin studies of planktonic dispersers, and in particular within Copepoda(Makino and Tanabe, 2009; Parentet al.,2012; Petrusek et al., 2008; Pritchard et al., 2012; Taylor and Hebert,1993).

Analysis of nuclear ITS genes confirmed that the gene pools of the two studied species have remained largely geneticallyisolated.Morevariable(andthusmorepowerful) molecularmarkersshouldbedevelopedtotestforthe

pre-sence of subtle introgression between these two closely relatedandsympatricspecies.

5.

Conclusion

WehavedemonstratedthattwoEurytemoraspecies(native E.affinisandinvasiveE.carolleeae)co-existinthesamearea intheGulfofFinland.Althoughpreviouslypublishedworkhas establishedthepresenceofthesespeciesintheGulfofRiga and in Amsterdam channels, Wasmund et al. (2013) have demonstrated theirexpandedco-distributionin KielBight, MecklenburgBight,ArkonaSea,BornholmSea,andinEastern GotlandSea.

Thepopulationdynamicsofbothspeciesarelargely par-allel.InvasiveE.carolleeaeisusuallysecondtoE.affinisin termsof density.Inaddition, thelargerbodysizeand dif-ferentreproductivetraitsofE.carolleeaeconferapotential forittodisplacenativeE.affinisspecies.Futureworkwhich aimstoassesstheprospectsforfurthergeographicexpansion of E. carolleeae should take into consideration not only interspecificcompetitionbetweenthesetwocloselyrelated Eurytemoraspecies,butalsospeciespresentathigherand lowertrophiclevelsthatinteractwithEurytemoracopepods.

Acknowledgments

WeareverygratefultoG.Winkler,J.Pierson,O.Glippa,M. Tackx, L. Samchishina, J. Polunina, E. Naumenko, S. Mal-javin,andS.Korochinfortheirhelpinsampling.Wethankthe staffoftheGEPVlaboratory,LilleUniversity,andespecially VincentCastricforprovidingthegeneticworkandvaluable commentsonthispaper.WethankthestaffoftheZoological Institute Laboratory of Molecular-Genetic Systematics, wherethegeneticanalysiswasperformed.Wearethankful to L.F. Litvinchuk, V.I. Lazoreva, and N.V. Polyakova for informationonthedistributionofE.carolleeaeinEuropean waters. We thankanonymous reviewers for valuable com-mentsthatsubstantiallyimprovedthemanuscript.Forthis work,theFederalCollectionN96-03-16,ZoologicalInstitute oftheRussianAcademyofSciences(St.Petersburg,Russia) wasused.Thisworkwasconductedinaccordancewiththe national initiative !!!!-!17-117041910019-2 and sup-portedby:theBIODISEINE;ZOOGLOBALSeineAvalproject; and the Mechnikov postdoctoral program in France; the “Biodiversity”grantfromthePresidiumoftheRussian Acad-emyofSciences;andRussianFoundationforBasicResearch (CKK3 17-04-00027 !). We are also grateful to Edward RamsayforEnglishediting.

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