Benthic meiofauna as indicator of ecological changes in estuarine ecosystems: The use of nematodes in ecological quality assessment

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ContentslistsavailableatSciVerseScienceDirect

Ecological

Indicators

j ou rna l h o m e pa ge : w w w . e l s e v i e r . c o m / l o c a t e / e c o l i n d

Benthic

meiofauna

as

indicator

of

ecological

changes

in

estuarine

ecosystems:

The

use

of

nematodes

in

ecological

quality

assessment

A.S.

Alves

a,d,∗

_,

_H.

_Adão

b,d

_,

_T.J.

_Ferrero

c

_,

_J.C.

_Marques

a

_,

_M.J.

_Costa

d

_,

_J.

_Patrício

a

a_IMAR_–_Institute_of_Marine_Research,_c/o_Department_of_Life_Sciences,_Faculty_of_Sciences_and_Technology,_University_of_Coimbra,_3004-517_Coimbra,_Portugal b_University_of_Évora,_Biology_Department,_Apartado_94,_7002-554_Évora,_Portugal

c_The_Natural_History_Museum,_Department_of_Zoology,_Cromwell_Road,_London_SW7_5BD,_UK

d_Centre_of_{Oceanography,}_Faculty_of_Sciences,_University_of_Lisbon,_Campo_Grande,_1749-016_Lisbon,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received25April2012

Receivedinrevisedform12July2012 Accepted21July2012 Keywords: Meiobenthos Free-livingnematodes Indicators Biodiversity Estuaries

a

b

s

t

r

a

c

t

Estuarinemeiofaunacommunitieshavebeenonlyrecentlyconsideredtobegoodindicatorsof ecolog-icalquality,exhibitingseveraladvantagesovermacrofauna,suchastheirsmallsize,highabundance, rapidgenerationtimesandabsenceofaplanktonicphase.Inestuarieswemustaccountnotonlyfora greatnaturalvariabilityalongtheestuarinegradient(e.g.sedimenttypeanddynamics,oxygen availabil-ity,temperatureandflowspeed)butalsofortheexistenceofanthropogenicpressures(e.g.highlocal populationdensity,presenceofharborsanddredgingactivities).

Spatialandtemporalbiodiversitypatternsofmeiofaunaandfree-livingmarinenematodeswere stud-iedintheMondegoestuary(Portugal).Bothtaxonomicandfunctional approacheswereappliedto nematodecommunitiesinordertodescribethecommunitystructureandtorelateitwiththe envi-ronmentalparametersalongtheestuary.Atallsamplingevents,nematodeassemblagesreflectedthe estuarinegradient,andsalinityandgrainsizecompositionwereconfirmedtobethemainabioticfactors controllingthedistributionoftheassemblages.

Moreover,thelowtemporalvariabilitymayindicatethatnaturalvariabilityissuperimposedbythe anthropogenicpressurespresentinsomeareasoftheestuary.Thecharacterizationofbothmeiofaunaand nematodeassemblageshighlightedtheusefulnessoftheintegrationofbothtaxonomicandfunctional attributes,whichmustbetakenintoconsiderationwhenassessingtheecologicalstatusofestuaries.

1. Introduction

Meiofaunafeaturesareagoodindicatorofenvironmental

con-ditions and changes in their density, diversity, structure and

functioning may indicate alterations in the system. Although

notbeingincludedinthebiologicalcompartment thatneedsto

be monitored in the scopeof the Water Framework Directive

(WFD,Directive2000/60/EC),meiofaunagivesvaluable

informa-tionregardingecosystemshealth.AccordingtoSheppard(2006),

marinescientistsneedtoincreaseawarenessofandemphasizethe

importanceofthemanyspeciesthathavenoappeal,whicharenot

attractiveand,forthemostpart,arenotseen,likemeiofauna.

Despitethesedifficulties,meiofaunacommunitiesare

reason-ablywellcharacterizedaround theworld,withstudiesranging

fromthedeepseafloortoalpinelakes,aswellasfromtropical

∗ Correspondingauthorat:IMAR–InstituteofMarineResearch,c/oDepartmentof LifeSciences,FacultyofSciencesandTechnology,UniversityofCoimbra,3004-517 Coimbra,Portugal.

E-mailaddress:asalves@uc.pt(A.S.Alves).

reefstopolarseaice(Giere,2009).InEurope,studieson

meioben-thiccommunitiesmostlyencompassthemorenortherlyestuarine

ecosystems(e.g.WarwickandGee,1984;LiandVincx,1993;Smol

etal.,1994;Soetaertetal.,1995;Ferreroetal.,2008).In

south-ernEuropethereisaseriousgapinknowledge.Particularlyinthe

IberianPeninsula,thereisalackofinformationonbothspatialand

temporaldistributionofmeiofaunaandfreelivingnematodesin

estuarineenvironments,beingessentialtodescribethose

biodi-versitypatterns.

Meiobenthiccommunitiesprovideinformationofgreatinterest

notonlyduetotheirimportantroleinmarinebenthicfoodchains

(Heipetal.,1985;Moensetal.,2005)butalsoduetotheir

ecolog-icalcharacteristics(smallsize,highabundance,rapidgeneration

timesandabsenceofaplanktonicphase),givingmeiofaunaseveral

advantagesoverthecommonlyusedmacrofaunacommunitiesas

monitoringorganisms(KennedyandJacoby,1999;Schratzberger

etal.,2000;AustenandWiddicombe,2006).In fact,nematodes

have beenpointedout aspotential indicatorsof anthropogenic

disturbanceinaquaticecosystems(e.g.CoullandChandler,1992;

Schratzbergeret al., 2004; Steyaert et al., 2007;Moreno et al.,

2008).Theinclusionofinformationregardingtheirfunctionaltraits

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(e.g.trophicstructure,lifestrategy)canprovidecriticalinformation

onthefunctioningofecosystems(Norlingetal.,2007;Danovaro

etal.,2008).

Estuariesarenaturallystressedsystemswithahighdegreeof

variabilityintheirphysical–chemicalcharacteristics.Thenatural

gradient of salinity, linked withother gradients(e.g. bed

sedi-ment type and dynamics,oxygen availability, temperature and

currentspeed),arewelldocumentedasimportantfactorsin

deter-miningtemporalandspatialvariationsofmeiofaunacommunities

(Bouwman,1983;Heipetal.,1985;AustenandWarwick,1989;

Soetaertetal.,1995;Lietal.,1997;Forster,1998;MoensandVincx,

2000;Steyaertetal.,2003;Deryckeetal.,2007;Alvesetal.,2009;

Adãoetal.,2009)butstudiesencompassingtheentiresalinityrange

frommarinetofreshwaterconditionsarefew(e.g.Portugal:Alves

etal.,2009;Adãoetal.,2009;Patrícioetal.,2012;United

King-dom:Ferreroetal.,2008;TheNetherlands:Soetaertetal.,1994,

Australia:Hourstonetal.,2011).Moreover,moststudiescovera

smalltemporalrange,providingonlylimitedinformationonthe

behaviorofassemblagesoverlongertimescales.

Thepresentstudycomparesthecharacteristicsofmeiofauna

andfreelivingnematodesassemblagesinthesubtidalsediments

of differentlocationsfromEuhaline toOligohalineareas ofthe

Mondegoestuary.Furthermore,thetemporal(seasonal)variability

betweentheassemblagesofdifferentlocationsisassessedandthe

useofnematodesasbiologicalindicatorsofenvironmentalquality

isconsidered.

Thisstudyaimedtoinvestigatechangesinpatternsofmeiofauna

andnematodeassemblagecompositionandnematodediversity,

trophiccompositionandlifestrategiesbetweendifferentestuarine

locationsandsamplingoccasions

Thefollowingnullhypothesesweretested:(a)therewouldbe

nodifferencesinmeiofaunataxonandnematodeassemblage

com-positionandtrophiccompositionalongtheestuary;and(b)there

wouldbenodifferencesinthemeiofaunaltaxonand nematode

assemblagecompositionandtrophiccompositionatdifferent

sea-sonalsamplingevents.

2. Materialsandmethods

2.1. Studyarea

TheMondegoestuary(Fig.1),locatedontheAtlanticcoastof

Portugal(40◦₀₈′_N,₈◦₅₀′_W),_is_a_polyhaline_system_influenced_by_a

warm-temperateclimate.Theestuaryis21kmlong(basedonthe

extentoftidalinfluence)withanareaofabout8.6km2_and,_in_its

terminalpart(atadistanceof7kmfromthesea)itdividesinto

twoarms,northernandsouthern,separatedbyanalluvialisland

(Murraceiraisland),whichrejoinneartheestuary’smouth.Thetwo

armshaveverydifferenthydrologicalcharacteristics.The

north-ernarmisdeeper(5–10mduringhightide),receivesmostofthe

system’sfreshwaterinput,beinginfluencedbyseasonalfluctuation

inwaterflow(Flindtetal.,1997),andformsthemainnavigation

channelonwhichtheFigueiradaFozharborislocated.The

south-ernarmisshallower(2–4mduringhightide),haslargeareasof

intertidalmudflats(almost75%ofthearea)exposedduringlow

tideand,untiltheSpringof2006,wasalmostsiltedupintheupper

zones.InMay2006,thecommunicationbetweenbotharmswas

re-establishedinordertoimprovethewaterqualityintheterminal

partoftheestuarybyreducingtheresidencetimeinthesouthern

arm(Netoetal.,2010).

TheMondegoestuarysupportsnotonlytheFigueiradaFoz

har-bor(regulardredgingiscarriedouttoensureshippingconditions)

butalsonumerousindustriesandreceivesagriculturalrun-offfrom

riceandcornfieldsintheLowerRivervalley(Marquesetal.,2003).

2.2. Samplingstrategy

Thesubtidalsoft-bottommeiobenthicassemblageswere

sam-pled along thesalinitygradient ofthe Mondegoestuaryonsix

samplingoccasions:August2006(summer,Su06),November2006

(autumn,Au06),March2007(winter,Wi07),June2007(spring,

Sp07), September 2009 (summer, Su09) and December 2009

(autumn,Au09).

Elevensamplingstationswereselectedfollowingthedivisionof

theestuaryproposedbyTeixeiraetal.(2008)(Fig.1).Theestuary

wasthusdividedinfivedifferentareas:Euhaline(station4;salinity

30–34);PolyhalineoftheSouthArm(st6,7and9;salinity18–30),

PolyhalineoftheNorthArm(st12and13;salinity18–30),

Meso-haline(18and19;salinity5–18)andOligohaline(st21,23and25;

salinity0.5–5).

2.2.1. Environmentaldata

Ateachsamplingstation,bottomwaterparameterswere

mea-suredinsituwithaYSIData SondeSurvey4:salinity (Practical

SalinityScale)(inAutumn2009–nosalinitydatawasrecorded),

temperature(◦_C),_pH,_and_dissolved_oxygen_(DO)_(mg_L−1_)._Water

sampleswerecollectedfordeterminationofnutrientsand

chloro-phyll a (mgm−3₎ _in _laboratory: _nitrate _(NO₃−_-N) _and _nitrite

(NO2−-N)concentrations(mmolL−1)wereanalyzedaccordingto

standard methods described in Strickland and Parsons (1972)

and ammonium (NH4+-N) and phosphate (PO43−-P)

concentra-tions(mmolL−1₎_were_analyzed_following_the_Limnologisk_Metodik

(1992). Chlorophyll a (Chl a) determinations were performed

accordingtoParsonsetal.(1985).Sedimentsamplesweretaken

ateachstationtodeterminetheorganicmattercontentandgrain

size.Sedimentorganicmatter(OM)contentwasdefinedasthe

dif-ferencebetweentheweightofeachsampleafteroven-dryingat

60◦_C_for₇₂_h_followed_by_combustion_at₄₅₀◦_C_for₈_h,_and_was

expressedasthepercentageofthetotalweight.Grainsizewas

ana-lyzedbydrymechanicalseparationthroughacolumnofsievesof

Fig.1. Mondegoestuary(Portugal):stationlocation(blackcircles).Areas:Euhaline(station4),PolyhalineoftheSouthArm(stations6,7and9),PolyhalineoftheNorthArm (stations12and13),Mesohaline(stations18and19)andOligohaline(stations21,23and25).

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differentmeshsizes,correspondingtothefiveclassesdescribed

byBrownandMcLachlan (1990): (a)gravel(>2mm),(b)coarse

sand(0.500–2.000mm),(c)meansand(0.250–0.500mm),(d)fine

sand(0.063–0.250mm),and(e)siltandclay(<0.063mm).The

rel-ativecontentofthedifferentgrain-sizefractionswasexpressedas

apercentageofthetotalsampleweight.

2.2.2. Biologicaldata

Threereplicatesamplesofsubtidalmeiobenthoswerecollected,

ateachsamplingstation,byforcingaKajaksedimentcorer(inner

diameter:4.6cm)3cmintothesediment.Allsampleswere

pre-servedin4%bufferedformaldehydeandweresievedthrough1mm

and 38mm mesh size sieves(material retained on thesmaller

meshwascollected).Meiofaunawasextractedfromthesediment

fractionusingLudoxHS-40colloidalsilicaataspecificgravityof

1.18gcm−3₍_Vincx,₁₉₉₆_)._All_meiobenthic_organisms_were

iden-tifiedtomajortaxalevelunderastereomicroscopeusingHiggins

andThiel(1988)andGiere(2009)andthedensity(individualsper

10cm2₎_of_each_taxon_was_quantified.

Fromeachreplicate,arandomsetof120nematodes,orthetotal

numberofindividualsinsampleswithlessthan120nematodes,

werepicked,clearedinglycerol–ethanolsolution,transferredto

anhydrousglycerolbyevaporationandmountedonslidesfor

iden-tification(Vincx,1996).Allnematodeswereidentifiedtogenus

levelusingamicroscopefittedwitha100×oilimmersion

objec-tiveandbasedonthepictorialkeysofPlattandWarwick(1983,

1988),Warwicketal.(1998),theonlineinformationsystemNeMys

(Steyaertetal.,2005)andonAbebeetal.(2006).

2.3. Dataanalysis

Univariate and multivariate analyses to detect spatial and

temporal changes in thecommunity structure wereperformed

accordingtotheproceduresdescribedbyClarke(1993),usingthe

PRIMERv6softwarepackage(ClarkeandWarwick,2001)withthe

PERMANOVAadd-onpackage(Andersonetal.,2008).

2.3.1. Environmentalvariables

A PrincipalComponentAnalysis (PCA)of theenvironmental

variables was performed tofind patterns in multi-dimensional

databyreducing thenumberof dimensions,withminimalloss

of information. Prior to the calculation of the environmental

parameterresemblancematrixbasedonEuclideandistance,the

environmentalvariables(temperature,salinity,dissolvedoxygen,

pH,ammonium,nitrate,nitrite,phosphate,silicates,organic

mat-terandeachofthefivegranulometricclasses)weresquare-root

transformed(exceptdissolvedoxygenandpHdata)andfollowed

normalization.

2.3.2. Meiofaunaassemblages

Totalmeiofaunadensityanddensityofindividualmajor

maio-faunataxa(individualsper10cm2₎_were_calculated,_for_each_area

andsamplingoccasion.

In order to test the hypothesis that the composition of

meiofauna changes spatially and seasonally, a two-way

PER-MANOVA analysis was carried out with the following crossed

factor design: “area” and “sampling occasion” as fixed factors,

with five (Euhaline, Polyhaline North Arm, Polyhaline South

Arm, Mesohaline and Oligohaline) and six levels (Su06, Au06,

Wi07,Sp07,Su09 andAu09),respectively.Meiofaunataxa

den-sitydatawere square roottransformedin order toscaledown

densities of highly abundant taxa and therefore increase the

importanceoftheless abundanttaxa intheanalyses. The

PER-MANOVA test was conducted on Bray–Curtis similarity matrix

andtheresidualswerepermutatedunderareducedmodel,with

9999permutations.Thenull hypothesiswasrejected whenthe

significance level p was <0.05 (if the number of permutation

waslowerthan150,theMonteCarlopermutationpwasused).If

significantdifferencesweredetected,thesewereexaminedusinga

posterioripair-wisecomparisons,using9999permutationsunder

areduced model.Afterwards,thesimilaritybetweenmeiofauna

assemblagesalongtheestuary,inthedifferentsamplingoccasions,

wasplotted usingnon-metricmultidimensionalscaling(nMDS),

withBray–Curtisassimilaritymeasure(ClarkeandGreen,1988).

2.3.3. Nematodesassemblages

AstheNematodawasalwaysthedominantmeiofaunalgroup,

wedecidedtostudythisgroupinparticulardepth.Therefore,total

density, generadiversity, trophic composition and several

eco-logical indicators, either basedon diversity (Margalef Index, d;

Shannon-Wienerdiversity,H′₎_or_on_ecological_strategies_(Index_of

TrophicDiversity,ITD;MaturityIndex,MI),werecalculatedusing

thenematodesdataset,foreachareaandsamplingoccasion.

Inordertoinvestigatethetrophiccompositionofthe

assem-blages,marinenematodesgenerawereassignedtooneofthefour

functionalfeedinggroups,designatedbyWieser(1953),basedon

buccalcavitymorphology:selective(1A)andnon-selective(1B)

depositfeeders,epigrowthfeeders(2A)andomnivores/predators

(2B).Thetrophicclassificationofthefreshwaternematodeswas

basedondietandbuccalcavitystructureinformation(Yeatesetal.,

1993;Traunspurger,1997).

TheIndexofTrophicDiversity(Heipetal.,1985)wascalculated

as:ITD=

P

2_,_where_is_the_density_contribution_of_each_trophic

grouptototalnematodedensity,rangingfrom0.25(highesttrophic

diversity,i.e.,eachofthefourtrophicguildsaccountfor25%ofthe

nematodedensity),to1.0(lowesttrophicdiversity,i.e.,onetrophic

guildaccountsfor100%ofthenematodedensity).TheMaturity

Index(Bongers,1990;Bongersetal.,1991)wasusedtoanalyze

nematodeslifestrategy.Nematodegenerawereassignedavalueon

ascale(c–pscore)accordinglytheirabilityforcolonizingor

persist-inginacertainhabitat,from“colonizers”(c;organismswithahigh

tolerancetodisturbanceevents)to“persisters”(p;lowtolerance).

Thus,theindexisexpressedasac–pvalue,rangingfrom1(extreme

colonizers)to5(extremepersisters)representinglife-history

char-acteristicsassociatedwithr-andK-selection,respectively(Bongers

andBongers,1998;BongersandFerris,1999)andvariesfrom1,

underdisturbedconditions,to3or4,underundisturbedconditions.

Theindexwascalculatedastheweightedaverageoftheindividual

colonizer–persister(c–p)valuesasMI=

P

_i=1n

v

(i)_·f(i),where

v

(i)

isthec–pvalueofthetaxoniandf(i)isthefrequencyofthattaxon.

Two-waypermutational analysesof variance (PERMANOVA)

wereappliedtotestthenullhypothesesthatnosignificant

spa-tial(betweenareas)andtemporal(betweensamplingoccasions)

differencesexisted,inthenematodeassemblagedescriptors(total

density,generadiversity,trophiccomposition,d,H′_,_ITD_and_MI).

PERMANOVA was used as an alternative to ANOVA since its

assumptionswerenotmet,evenafterdatatransformation.

Two-wayPERMANOVAanalyseswerecarriedoutwiththesamedesign

describedformeiofaunaanalysis.AllPERMANOVAtestswere

con-ductedonEuclidean-distancesimilaritymatricesandtheresiduals

werepermutatedunderareducedmodel,with9999permutations.

Thenullhypothesiswasrejectedwhenthesignificancelevelpwas

<0.05(ifthenumberofpermutationwaslowerthan150,theMonte

Carlopermutationpwasused).Wheneversignificantdifferences

weredetected,thesewereexaminedusingaposterioripair-wise

comparisons,using9999permutationsunderareducedmodel.

In orderto test fortemporal and spatial differences

regard-ingnematodesassemblages’composition,atwo-wayPERMANOVA

analysis was carried out with the previously described design

(“area”:5levels;“samplingoccasion”:6levels),usingBray–Curtis

as similarity measure. The null hypothesis was rejected when

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waslowerthan150,theMonteCarlopermutationpwasused).

If significant differences were detected, these were examined

usingaposterioripair-wisecomparisons,using9999permutations

underareducedmodel.Nematodegeneradensitydatawerefirst

squareroottransformedinordertoscaledowndensitiesofhighly

abundant generaand therefore increase the importanceof the

lessabundantgeneraintheanalyses,andthesimilaritybetween

communities along theestuary, inthedifferent sampling

occa-sions,wasplottedbynon-metricmultidimensionalscaling(nMDS),

usingtheBray–Curtissimilaritymeasure(ClarkeandGreen,1988).

Afterwards,therelative contributionof eachgenustothe

aver-agedissimilarities betweenareas and samplingoccasionswere

calculatedusingtwo-waycrossedsimilaritypercentageanalysis

procedure(SIMPER,cut-offpercentage:90%).

2.3.4. Nematodesassemblagesvs.environmentalvariables

The relationship between environmental variables and the

structure of thenematodescommunity wasexploredby

carry-ingouttheBIOENVprocedure(ClarkeandAinsworth,1993),using

Spearman’scorrelation.

3. Results

3.1. Environmentalvariables

Alongtheestuary,salinityandnutrientconcentrationsshowed

opposite trends,withhigher salinity values andlower nutrient

concentrationsdownstreamandlowersalinityvaluesandhigher

nutrientconcentrationsupstream.Adecreaseingrainsizewasalso

observedfromOligohalineareatowardthemouthoftheestuary.

ThePCAordinationoftheenvironmentalfactorsshowedthat

thefirsttwocomponents(PC1,29.0%andPC2,23.8%)accounted

forabout53%ofthevariabilityofthedata(Fig.2).TheOligohaline

andMesohalinesampleswerecharacterizedbyhighnutrients

con-centration,atallsamplingoccasions,whileinAutumn2006,Winter

2007andSpring2007,thesamplesfromthesetwoupstreamareas

wereclearly separated fromtheremainingonesmainly dueto

higherpercentageofcoarsersediments.

Ingeneral,independentlyfromthesamplingoccasion,higher

salinity, finer sediments and lower nutrient concentrations

characterizedthesamplesfromthePolyhalineNA,PolyhalineSA

and Euhaline areas.Witha few exceptions (mainly in Summer

2009),thetwoPolyhalineareaspresenteddifferent

environmen-talattributes:thePolyhalineNAsampleshavingcoarsersediments

andthePolyhalineSAsamplesbeingcharacterizedbyfiner

sedi-mentsandhigherOMcontent.

3.2. Meiofaunaassemblages

Fourteenmajortaxawereidentifiedalongtheestuaryduringthe

samplingperiodwithNematodathedominanttaxon(92.4%),

fol-lowedbyPolychaeta(4.7%)andHarpacticoidcopepods(1.5%).All

othertaxaattainedlessthan1%[e.g.Bivalvia(0.4%),Oligochaeta

(0.4%), Ostracoda (0.2%), Tardigrada (0.1%), Gastropoda (0.1%),

Amphipoda(0.1%),Nauplii(0.1%)]andsometaxapresentedvery

low density (lessthan 0.03%),such asCiliophora, Halacaroidea,

TurbellariaandCladocera.

Total meiofauna density (±sd) ranged from 25.4±25.9ind

10cm−2_{(Oligohaline,}_Sp07)_to_1383.5_±_687.9_ind₁₀_cm−2

(Euha-line, Su06) and the number of taxa present varied from three

(Mesohaline,Sp07;Euhaline,Au06andAu09)toeleven(Polyhaline

SAandEuhalineinSu06),withnoclearincreasefromOligohaline

toEuhalineareas(Table1).

PERMANOVA analysisof meiofaunaassemblage composition

datashoweda significantinteractionbetween“area”and

“sam-plingoccasion”(Table2A).TheOligohalineareawasdifferentfrom

allothersonallsamplingoccasions,withminorexceptionsinAu06

(OligohalinesimilartoEuhaline,t=1.35,p=0.143),inWi07

(Oligo-halineonlydifferentfromthePolyhalineSA,t=2.94,p=0.002)and

inSp07(OligohalinesimilartoMesohaline,t=1.57,p=0.104).This

patternisdistinctlyvisibleinthenMDSordination(Fig.3),with

aclearseparation ofOligohalineandMesohalineareasfromthe

remainingones.

3.3. Nematodesassemblages

3.3.1. Structureandtrophiccomposition

The density (N) of nematodes ranged from 21.4±23.5ind

10cm−2 _in _the _Oligohaline _area _(Sp07) _to _1323.1_±_674.7_ind

10cm−2_in_the_Euhaline_area_(Su06)._Over_the_whole_estuary,_mean

Fig.2. PrincipalComponentAnalysis(PCA)plotbasedontheenvironmentalvariablesmeasuredineach“area”(Oligohaline,Mesohaline,PolyhalineNorthArm,Polyhaline SouthArmandEuhaline)and“samplingoccasion”(Summer06,Autumn06,Winter07,Spring07,Summer09andAutumn09).PC1=29.0%,PC2=23.8%.

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466 Table 1 Mean density ± standard deviation (number of individuals per 10 cm 2) of meiofaunal taxa in each area (Oligohaline, Mesohaline, Polyhaline North Arm, Polyhaline South Arm and Euhaline) and sampling occasion (Summer 2006, Su06; Autumn 2006, Au06; Winter 2007, Wi07; Spring 2007, Sp07; Summer 2009, Su09 and Autumn 2009, Au09). Area Sampling occasion Nematoda Polychaeta Copepoda Bivalvia Oligochaeta Ostracoda Gastropoda Nauplii Tardigrada Amphipoda Ciliophora Halacaroidea Turbellaria Cladocera Total Euhaline Su06 1323.1 ± 674.7 4.8 ± 2.2 30.9 ± 14.0 6.4 ± 0.7 4.0 ± 1.5 4.0 ± 3.1 3.2 ± 3.1 5.2 ± 4.1 0.8 ± 0.3 0.6 ± 0.6 0.4 ± 0.7 1383.5 ± 687.9 Au06 52.6 ± 19.9 0.6 ± 1.0 0.2 ± 0.3 53.4 ± 20.5 Wi07 332.7 ± 134.2 5.0 ± 1.5 33.5 ± 34.4 0.2 ± 0.3 0.2 ± 0.3 1.2 ± 1.2 1.6 ± 0.3 374.5 ± 160.2 Sp07 139.3 ± 9.9 8.8 ± 4.6 3.6 ± 0.6 1.0 ± 1.7 152.7 ± 10.5 Su09 157.5 ± 63.4 0.6 ± 1.0 2.8 ± 2.4 1.0 ± 1.3 0.2 ± 0.3 1.2 ± 1.2 0.2 ± 0.3 163.6 ± 65.7 Au09 103.6 ± 22.9 1.2 ± 0.6 4.2 ± 4.2 109.0 ± 26.2 Polyhaline SA Su06 617.0 ± 468.7 42.3 ± 22.8 14.2 ± 18.3 0.6 ± 0.5 0.4 ± 0.5 5.9 ± 8.4 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.2 1.1 ± 1.0 0.1 ± 0.1 681.9 ± 500.5 Au06 172.0 ± 150.7 15.7 ± 14.9 1.1 ± 1.8 0.1 ± 0.2 189.0 ± 161.8 Wi07 526.1 ± 506.0 8.7 ± 7.7 7.8 ± 7.7 0.4 ± 0.5 0.9 ± 1.1 0.1 ± 0.2 0.1 ± 0.1 544.0 ± 521.8 Sp07 196.9 ± 134.9 9.9 ± 9.4 2.9 ± 3.4 0.1 ± 0.1 0.7 ± 0.5 0.1 ± 0.1 210.6 ± 145.0 Su09 201.2 ± 81.0 7.8 ± 1.5 2.4 ± 2.3 0.3 ± 0.3 0.1 ± 0.1 1.1 ± 0.9 0.1 ± 0.1 212.9 ± 77.5 Au09 182.6 ± 70.7 9.5 ± 4.5 1.5 ± 1.3 0.1 ± 0.2 0.2 ± 0.2 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 194.3 ± 69.2 Polyhaline NA Su06 238.4 ± 13.6 16.8 ± 10.4 4.0 ± 4.0 1.2 ± 0.6 3.2 ± 2.8 0.1 ± 0.1 1.0 ± 1.4 0.4 ± 0.3 1.8 ± 3.6 0.1 ± 0.1 267.0 ± 1.3 Au06 259.9 ± 14.8 2.3 ± 1.3 0.1 ± 0.1 0.1 ± 0.1 262.4 ± 16.0 Wi07 72.8 ± 103.0 1.7 ± 2.4 0.3 ± 0.3 0.4 ± 0.6 0.1 ± 0.1 0.1 ± 0.1 75.5 ± 106.7 Sp07 173.5 ± 98.3 10.1 ± 9.5 0.2 ± 0.3 0.1 ± 0.1 0.1 ± 0.1 184.0 ± 108.1 Su09 303.7 ± 115.9 2.4 ± 0.0 1.1 ± 1.3 0.1 ± 0.1 5.7 ± 7.5 0.1 ± 0.1 313.2 ± 121.9 Au09 247.0 ± 100.2 2.4 ± 2.8 0.7 ± 1.0 0.2 ± 0.3 1.9 ± 1.8 252.3 ± 97.9 Mesohaline Su06 183.8 ± 1.7 63.8 ± 24.4 2.2 ± 2.6 0.5 ± 0.4 0.5 ± 0.7 1.2 ± 0.3 0.2 ± 0.0 0.3 ± 0.4 0.1 ± 0.1 0.2 ± 0.3 252.9 ± 28.7 Au06 260.5 ± 16.5 2.0 ± 0.6 0.1 ± 0.1 0.1 ± 0.1 0.2 ± 0.3 0.1 ± 0.1 263.0 ± 15.2 Wi07 209.8 ± 106.3 5.4 ± 0.3 0.2 ± 0.3 0.2 ± 0.3 0.1 ± 0.1 0.5 ± 0.7 216.2 ± 105.7 Sp07 68.0 ± 74.6 2.4 ± 1.1 0.2 ± 0.3 70.6 ± 75.5 Su09 55.6 ± 43.7 6.0 ± 1.7 0.5 ± 0.1 0.3 ± 0.4 1.6 ± 2.3 64.0 ± 44.6 Au09 55.1 ± 17.5 1.0 ± 0.0 0.6 ± 0.9 0.5 ± 0.4 1.1 ± 1.0 58.3 ± 17.2 Oligohaline Su06 85.8 ± 41.4 29.2 ± 11.7 1.5 ± 1.3 12.3 ± 18.8 0.5 ± 0.8 0.1 ± 0.1 0.2 ± 0.2 0.2 ± 0.2 129.7 ± 43.5 Au06 23.9 ± 6.0 0.7 ± 0.6 0.1 ± 0.1 1.9 ± 3.2 0.1 ± 0.1 0.1 ± 0.1 26.8 ± 7.5 Wi07 67.4 ± 82.0 4.5 ± 6.9 0.3 ± 0.4 0.3 ± 0.6 0.1 ± 0.1 0.1 ± 0.1 5.4 ± 9.4 78.2 ± 98.5 Sp07 21.4 ± 23.5 1.9 ± 2.3 1.6 ± 0.9 0.2 ± 0.3 0.2 ± 0.2 0.1 ± 0.1 25.4 ± 25.9 Su09 29.7 ± 22.8 1.5 ± 1.0 3.0 ± 4.5 2.2 ± 3.8 0.1 ± 0.2 36.6 ± 19.5 Au09 32.6 ± 17.3 1.2 ± 0.2 0.7 ± 0.5 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.2 0.1 ± 0.1 2.5 ± 3.6 0.2 ± 0.2 37.5 ± 21.2

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467 Table2

Detailsofthetwo-factorPERMANOVAtest(“area”with5levels,and“samplingoccasion”with6levels,asfixedfactors)forallvariablesanalyzed.Boldvaluesstandforthe significantdifferences(p<0.05).A–meiofaunacomposition;B–nematodesdescriptors.

Sourceofvariation Degreesoffreedom Sumofsquares Meansquares Pseudo-F P(perm) A.Meiofauna

Composition Area 4 39,752 9937.9 16.28 0.0001

Samplingoccasion 5 23,716 4743.3 7.77 0.0001

Area×samplingoccasion 19 24,391 1283.7 2.10 0.0001

Residual 139 84,871 610.58

Total 167 175,020

B.Nematodes

Totaldensity Area 4 2,423,900 605,970 24.31 0.0001

Samplingoccasion 5 2,012,300 404,860 16.24 0.0001

Area×samplingoccasion 19 4,162,200 219,060 8.79 0.0001

Residual 139 3,464,500 24,925

Total 167 10,996,000

Numberofgenera Area 4 471.19 117.8 10.37 0.0001

Samplingoccasion 5 318.13 63.626 5.60 0.0001

Area× samplingoccasion 19 373.84 19.676 1.73 0.0401

Residual 139 1578.6 11.357

Total 167 2823.6

Trophiccomposition Area 4 19,645 4911.3 8.10 0.0001

Residual 139 84,261 606.2

Total 167 150,940

Composition Area 4 98,388 24,597 16.37 0.0001

Residual 139 208,840 1502.4

Total 167 420,420

MargalefIndex Area 4 48.505 12.126 21.99 0.0001

Area×samplingoccasion 19 19.238 1.0125 1.84 0.025

Residual 139 76.665 0.55154

Total 167 155.88

Shannon-WienerIndex Area 4 13.633 3.4082 8.22 0.0001

Residual 139 57.633 0.41462

Total 167 87.925

IndexofTrophicDiversity Area 4 0.31339 0.078347 3.05 0.0203

Residual 139 3.5658 0.025653

Total 167 4.5852

MaturityIndex Area 4 4.1698 1.0425 9.86 0.0001

Area× samplingoccasion 19 3.5231 0.18543 1.75 0.0438

Residual 139 14.701 0.10576

Total 167 24.568

Fig.3.nMDSordinationbasedonmeiobenthosineachofthesamplingstationsineach“area”(Oligohaline,Mesohaline,PolyhalineNorthArm,PolyhalineSouthArmand Euhaline)and“samplingoccasion”(Summer06,Autumn06,Winter07,Spring07,Summer09andAutumn09).

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468

Fig.4.Nematodecommunityineach“area”(Oligohaline,Mesohaline,PolyhalineNorthArm,PolyhalineSouthArmandEuhaline)duringthestudyperiod(Su06,Summer 2006;Au06,Autumn2006;Wi07,Winter2007;Sp07,Spring2007;Su09,Summer2009;Au09,Autumn2009).(A)Averagedensity(ind10cm−2_);_(B)_number_of_genera_(S).

density(±sd)washighestinWi07(363.40±343.16ind10cm−2_),

andlowestduringAu09(123.04±154.79ind10cm−2_)._Generally,

thehighestdensitieswerereached intheEuhalineand

Polyha-lineareas(Fig.4A).PERMANOVAanalysisofdensitydatashowed

asignificantinteractionbetween“area”and“samplingoccasion”

(Table2B).Individualpair-wisecomparisonsoninteractionfactor

(“area”×“samplingoccasion”)showedthattheOligohalinearea,in

general,showedsignificantlylowerdensityvaluesthantheother

areas,regardlessofthesamplingoccasion.Moreover,the

Polyha-lineNAdidnotshowsignificantdifferencesthroughtimewhile

allotherareasshowedsignificantdifferencesindensitybetween

oneormoresamplingoccasions(seeSupplementarymaterial–

Annex).

Nematodesaccountedforbetween88%(Su06)and95%(Au06)

ofthetotalmeiofaunaldensityandatotalof106nematodegenera,

belongingto40families,wereidentifiedalongtheestuaryduring

thestudyperiod.ThemostabundantorderswereChromadorida

(46.3%),Monhysterida(36.7%)andEnoplida(11.7%)andthemost

abundantfamilieswereComesomatidae(25.3%),Xyalidae(16.7%),

Linhomoeidae(11.8%),Chromadoridae(10.3%)and

Sphaerolaimi-dae(8.6%).

Thenumberofgenera(S)rangedbetween8inthePolyhaline

NAarea(Su09)and19intheEuhalinearea(Su06)(Fig.4B).

PER-MANOVArevealedasignificantinteractionoffactors“area”and

“samplingoccasion”forthenumberofgenera(Table2B).The

pair-wisetestsperformedontheinteractiontermshowedthatinAu06,

Sp07andAu09there werenosignificantdifferencesin number

ofgenerabetweenareas,whileintheremainingsampling

occa-sionstheEuhaline areashowedhigherdiversity thantheother

areas.Allareasshowedsignificantvariationinthenumberof

gen-erabetweenatleasttwosamplingoccasions(seeSupplementary

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469 Table3

Averagedensity(¯x;numberindividualsper10cm2_),_percentage_of_contribution_(%)_and_rank_by_density_(Rk)_of_nematode_genera_in_each_area_of_the_Mondego_estuary_derived

frompooleddatafromallsamplingoccasions.Tableonlyliststhegenerathatcontributed>0.5%tothetotaldensityandthefivemostabundantgeneraineachareaare bolded.

Genera Totalaverage

density

% Euhaline PolyhalineSA PolyhalineNA Mesohaline Oligohaline

¯x % Rk ¯x % Rk ¯x % Rk ¯x % Rk ¯x % Rk Sabatieria 249.9 23.5 38.5 10.9 2 87.5 31.9 1 121.7 51.9 1 1.6 1.0 12 0.5 1.1 16 Daptonema 163.1 15.3 36.4 10.3 3 45.9 16.7 3 22.2 9.5 3 47.6 29.8 1 11.1 25.8 1 Terschellingia 86.7 8.2 7.3 2.1 13 49.3 18.0 2 7.8 3.3 7 21.6 13.5 3 0.8 1.8 12 Metachromadora 86.0 8.1 76.7 21.8 1 5.6 2.0 9 3.6 1.5 10 0.1 0.0 45 0.1 0.1 47 Sphaerolaimus 84.6 8.0 18.9 5.4 6 34.9 12.7 4 22.2 9.5 2 8.5 5.3 6 0.1 0.3 30 Anoplostoma 75.5 7.1 25.3 7.2 5 9.5 3.5 6 10.2 4.3 5 28.2 17.7 2 2.2 5.2 4 Dichromadora 47.1 4.4 5.0 1.4 18 5.5 2.0 10 20.9 8.9 4 13.6 8.5 4 2.2 5.1 5 Viscosia 37.0 3.5 14.4 4.1 7 8.9 3.3 7 9.1 3.9 6 4.3 2.7 8 0.3 0.7 19 Ptycholaimellus 35.4 3.3 8.4 2.4 11 10.5 3.8 5 4.8 2.1 8 10.4 6.5 5 1.3 3.1 7 Microlaimus 30.1 2.8 29.7 8.4 4 0.4 0.2 16 0.0 0.1 56 Linhomoeus 18.5 1.7 8.8 2.5 10 7.7 2.8 8 1.8 0.8 11 0.1 0.0 38 0.1 0.3 28 Axonolaimus 14.4 1.4 11.1 3.2 8 0.1 0.0 26 0.5 0.2 14 1.6 1.0 11 1.0 2.4 9 Paracyatholaimus 13.4 1.3 2.4 0.7 22 0.1 0.0 28 0.3 0.1 17 7.4 4.6 7 3.3 7.6 3 Mesodorylaimus 12.5 1.2 0.4 0.1 35 2.8 1.7 10 9.4 21.8 2 Prochromadorella 11.0 1.0 10.7 3.0 9 0.1 0.0 27 0.1 0.1 23 Leptolaimus 8.4 0.8 1.5 0.4 27 0.6 0.2 16 4.5 1.9 9 1.4 0.9 13 0.4 0.9 18 Molgolaimus 8.0 0.8 7.7 2.2 12 0.2 0.1 25 0.1 0.0 28 0.1 0.0 40 Calyptronema 7.0 0.7 6.4 1.8 14 0.7 0.2 15 Chromadora 6.4 0.6 5.2 1.5 16 0.6 0.2 17 0.5 0.2 13 Spilophorella 5.6 0.5 0.2 0.1 23 1.7 0.7 12 3.5 2.2 9 0.1 0.3 29 Aegialoalaimus 5.5 0.5 5.5 1.6 15 Halalaimus 5.4 0.5 3.9 1.1 20 0.9 0.3 13 0.3 0.1 18 0.2 0.1 26 0.2 0.4 26 Paralinhomoeus 5.2 0.5 5.1 1.4 17 0.1 0.0 41 Oncholaimellus 5.0 0.5 4.7 1.3 19 0.2 0.1 29 0.1 0.1 40 Othergenera 41.3 3.9 17.8 5.1 5.3 1.9 1.9 0.8 6.5 4.1 9.8 22.8 Meandensity 351.7 274.0 234.8 159.6 43.0

Totalgeneranumber 53 33 35 58 84

Throughout the study period, fifteen genera dominated

the nematodeassemblages (90.8%): Sabatieria, Daptonema,

Ter-schellingia,Metachromadora,Sphaerolaimus,Anoplostoma,

Dichro-madora,Viscosia,Ptycholaimellus,Microlaimus,Linhomoeus,

Axono-laimus, Paracyatholaimus, Mesodorylaimus and Prochromadorella

(Table3).Theremaininggeneraallrepresentedabundanceslower

than 1%.The mostspatially widespread genuswas Daptonema

(present along the whole length of the estuary through the

entiresamplingperiod),followedbySabatieriaandDichromadora

(Table3).Freshwaternematodescomprised3.5%ofthetotal

nema-todesdensity(1%inSp07to4.4%inWi07).

The five dominant genera showed clear variation over the

studyperiod, asshownin Fig.5,and a distinctpattern of

gen-era turnover along theestuary is visible.Non-selective deposit

feeders(1B)likeSabatieriaand Daptonema,showedanopposite

densitycontributiontrendinthePolyhalineareas,withthe

contri-butionofSabatieiraincreasingfromWi07toAu09,andDaptonema

decreasing in thesame period.Sabatieria wasalmost absentin

theMesohalineandOligohalineareas,whereDaptonemashowed

ahighcontribution.Terschellingia,aselectivedepositfeeder(1A),

showedhighcontributionsinWi07,especially inthePolyhaline

SA and Mesohaline areas. Predators (2B), like Metachromadora

andSphaerolaimus,peakedondifferentsamplingoccasions,with

ahighcontributionofMetachromadoraintheEuhalinearea,while

SphaerolaimuswasmostlyobservedinthePolyhalineNA(Au06)

andMesohaline(Wi07)areas.

Throughout the estuary, the nematodes community was

characterized by a dominance of non-selective deposit feeders

(52.0±12.1%)duringtheentirestudyperiod,followedby

omni-vores/predators(23.2±8.1%),epigrowthfeeders(15.9±3.3%)and

selectivedepositfeeders(8.9±4.8%).Non-selectivedeposit

feed-ers werethe mostabundant trophic group,in almost allareas

andsamplingoccasions,rangingfrom22.5%(Euhalinearea,Au06)

to 81.6% (Polyhaline NA area, Au09). In the Mesohaline and

Oligohalineareastherewasalowercontributionofpredatorsonall

samplingoccasions(rangingfrom1.7%inAu06to16.6%inWi07,

bothintheMesohalinearea)comparedwiththeremainingareas

(rangingfrom7.3%inAu09,PolyhalineNAareato56.7%inAu06,

Euhalinearea)(Fig.6).PERMANOVAanalysisoftrophicstructure

datashoweda significantinteractionbetweenfactor“area”and

“samplingoccasion”(Table2B).Individualpair-wisecomparisons

performedontheinteractionfactorshowedsignificantdifferences

introphiccompositionbetweenareasonallsamplingoccasions

andalsosignificantdifferencesateachareathroughoutthestudy

period(seeSupplementarymaterial–Annex).

Regardingtheoverallcomposition,multivariatePERMANOVA

analysis showedthat the estuarineassemblages weredifferent

between areas and samplingoccasions (Table 2B). In concrete,

depending on the chosen area, there were significant

differ-encesbetweendistinctpairofsamplingoccasions.Theresultsare

supportedby a visualassessment ofthe patternsin the nMDS

ordinationofsquare-roottransformeddata,usingBray–Curtis,as

showninFig.7.

Two-waySIMPERanalysis showedhowthe nematodes

gen-eracontributedtosimilarityvaluesoftheaprioridefinedgroups.

MaximumdissimilaritieswereobtainedbetweentheOligohaline

areaandboththePolyhalineareas(80.15%withPolyhalineSAand

79.57%withPolyhalineNA)andEuhalinearea(79.78%).Maximum

dissimilaritieswerealsoobservedbetweenSummer06andthe

fol-lowingthreesamplingoccasions,Autumn06(71.57%),Winter07

(68.59%)andSpring07(68.58%).Thegenerathatcontributedmost

tothesimilaritywithinbothsamplingoccasionsandareaswere

Daptonema,Sabatieria,SphaerolaimusandDichromadora.

3.3.2. Indicesestimation

Margalef Index (d) and Shannon-Wiener index values (H′₎

(Fig. 8A), followed the trend shown by the number of genera

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470

Fig.5. Percentageofcontributionofthefivemostabundantnematodegenera(Sabatieria,Daptonema,Terschellingia,MetachromadoraandSphaerolaimus)ineach“area” (Oligohaline,Mesohaline,PolyhalineNorthArm,PolyhalineSouthArmandEuhaline)and“samplingoccasion”(Summer06,Autumn06,Winter07,Spring07,Summer09 andAutumn09).

MargalefIndexshowedasignificantinteractionbetween“area”

and“samplingoccasion”(Table2B).TheMesohalineandEuhaline

areas didnot showsignificant differences in richness

through-outthestudyperiod,whiletheOligohalineareashowedseveral

pairsof samplingoccasionswithsignificantly differentrichness

values, higher in Wi07and Au09. Moreover,nosignificant

dif-ferences where found between areas in Su06 and Sp07 (see

Supplementary material – Annex). The Shannon-Wiener index

showedsignificantdifferencesbetweenallpairsofareas(Table2B)

except between Oligohaline–Mesohaline (t=1.27, p=0.21) and

Mesohaline–PolyhalineSA(t=1.24;p=0.22).Ingeneral,both

indi-catorsshowedalowerdiversityinthePolyhalineareas(Fig.8A).

The Index of Trophic Diversity ranged from 0.31 (Euhaline,

Su06)to0.62(PolyhalineNA,Sp07).Significantdifferenceswere

observedbetweenareas(Table2B),withhighervaluesinthe

Oligo-halineand Mesohalineareas,indicating lowertrophic diversity,

Fig.6.Percentageofcontributionofthedifferenttrophicgroups,ineach“area”(Oligohaline,Mesohaline,PolyhalineNorthArm,PolyhalineSouthArmandEuhaline)and “samplingoccasion”(Summer06,Autumn06,Winter07,Spring07,Summer09andAutumn09).1A–selectivedepositfeeders;1B–non-selectivedepositfeeders;2A– epigrowthfeeders;2B– omnivores/predators.

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471

Fig.7.nMDSordinationbasedonnematodesdatasetineach“area”(Oligohaline,Mesohaline,PolyhalineNorthArm,PolyhalineSouthArmandEuhaline)and“sampling occasion”(Summer06,Autumn06,Winter07,Spring07,Summer09andAutumn09).

Table4

BIOENVresultscarriedoutfornematodesassemblagesandenvironmentaldata,in eachsamplingoccasion.

Samplingoccasion Spearman’srank correlation

Variables

Summer2006 0.938 Salinity,NO3−,meansand,

coarsesand,Chla

Autumn2006 0.245 pH,finesand,coarsesand

Winter2007 0.636 Salinity,pH,meansand

Spring2007 0.839 Salinity,NO3−

Summer2009 0.862 Salinity,NO3−

Autumn2009 0.642 NO3−,silicates,%OM,mean

sand

andlowervaluesinthePolyhalineandEuhalineareas(Polyhaline

NA>PolyhalineSA,PolyhalineNA>Euhaline),indicativeofahigher

trophicdiversity(Fig.8B).

TheMaturityIndex(MI)rangedbetween2.1(PolyhalineNAin

Wi07,Sp07,Su09andAu09; MesohalineinSu06andSp07)and

3.0(Oligohaline,Su06)(Fig.8B)andmostnematodesshowedac–p

valueof2(average=70%),followedbyc–pvaluesof3(26%).The

MIshowedasignificantinteractionbetweenthefactors“area”and

“samplingoccasion”(Table2B).Individualpair-wisecomparisons

performedontheinteractionrevealednoseasonaldifferencesin

thePolyhalineSAarea.TheMIvaluesoftheMesohalinearea

exhib-itedthehighesttemporalvariations.Interestingly,inAu06(flood

period),nosignificantdifferencesinMIwererecordedalongthe

estuary.

3.4. Environmentalvariablesvs.nematodeassemblages

SeparateBIOENVanalysiswereperformedforeach sampling

occasioninordertoanalyzethemainfactorsresponsibleforthe

distributionofnematodesalongtheestuaryineachsampling

occa-sion,withsalinity,grainsizevariablesandnutrientsalwaysbeing

correlatedwiththenematodeassemblagecomposition(Table4).

4. Discussion

Thecombination ofthetemporaland spatialinformation on

meiofaunaandnematodesoftheMondegoestuaryallowedafull

descriptionofthemeiobenthiccommunitiesalongtheestuarine

gradienttomemade.Theinformationwasthenanalyzedinthe

contextoftheecologicalassessmentoftransitionalwatersusing

thesecommunities,makingavailableinformationonthe

ecologi-calconditionsofthesystemandinitiatingabaselineforlong-term

monitoringstudies.Previous studieshave onlybeenfocusedon

oneseason,lackingtemporalreplication(Alvesetal.,2009;Adão

etal.,2009;Patrícioetal.,2012),andthepresentstudy,aswell

asintegratingthecompleteestuarinegradient,wasrepeatedon

sixsamplingoccasions,allowingamoreextensivedatabasetobe

analyzedandrelatedtotheenvironmentalgradient.

The environmentalcharacterization of theMondego estuary

wasbasedonabioticmeasurementscollected ateach sampling

event.Thecharacterizationofasystembasedonchemical

param-eters only provides information about quality at the time of

measurement,lackingthesensitivitytodeterminetheimpactof

previouseventsontheecologyofthesystem(SpellmanandDrinan,

2001).However,bioindicatorsprovideindicationsaboutpast

con-ditionsandaccuratelyassessecologicalconditionsitisnecessary

touseasetofindicatorswhichrepresentthestructure,function

andcompositionofthesystem.Inthisstudy,meiobenthic

commu-nitieswerestudiedindetail,withspecialemphasisonnematodes

assemblages.

A clearestuarinegradient, fromtheoligohalineareatoward

theeuhalinezonewasobservedduringthesurveyperiod,mainly

causedbyvariationsinsalinity,nutrientconcentrationsand

sed-imentgrainsize.TheidentificationofbotharmsoftheMondego

estuaryas two differentsubsystems was confirmed,

represent-ingdistincthydrologicalregimes.Salinityincreasedfromupstream

towardthemouthoftheestuaryonallsamplingoccasionsexceptin

Autumn2006.Duringthisseason,aperiodofheavyrainand

flood-ingoccurred(INAGsource),loweringsalinityvaluesandconfirming

theimportanceofextremeeventsinchangingtheenvironmental

characteristicsofestuaries.Thenematodecommunitywasaffected

atthistimesincetheseparationofsalinityzonesalongtheestuary

wasnotsodistinct.Theseverefloodmayhavecausedsediment

displacementanderosionaswellaschangingtheinterstitialwater

salinity(Santosetal.,1996),andorganismsmayhavebeenwashed

away,leadingtothelowdensityvaluesobservedduringthisseason.

Bothsalinityandsediment structure aremajorfactors

influ-encingmeiobenthiccommunitystructure(Heipetal.,1985)and

results fromthe BIOENVanalysis showed that thedistribution

patternofnematodeswasmainlystructuredbydistinct

environ-mentalfactorslikesalinity,sedimentgrainsizeandwaternutrients,

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472

Fig.8.Ecologicalindicatorsvaluesineach“area”(Oligohaline,Mesohaline,PolyhalineNorthArm,PolyhalineSouthArmandEuhaline)and“samplingoccasion”(Summer06, Autumn06,Winter07,Spring07,Summer09andAutumn09).(A)MargalefIndex(d±standarddeviation)andShannon-Wienerindex(H′_±_standard_deviation)_(bits_ind−1_);

(B)IndexofTrophicDiversity(ITD±standarddeviation)andMaturityIndex(MI±standarddeviation).

nematodecommunitypatterns(AustenandWarwick,1989;Vincx

etal.,1990;Coull,1999;Ferreroetal.,2008;Schratzbergeretal.,

2008; Adão et al., 2009). However, despite the other

environ-mentaldifferencesbetweenthepolyhalineareas,themeiofauna

andnematodecommunitiesweresimilar,emphasizingtheprime

importanceofsalinity indefining andlimiting species

distribu-tionin transitionalwater systems (Austenand Warwick,1989;

Vincxetal.,1990;Soetaertetal.,1995;Attrill,2002;Ferreroetal.,

2008),itseffectsoverridingthatofsedimentgrainsizecomposition

(AustenandWarwick,1989;Adãoetal.,2009).

Meiofaunadensityanddiversityweresimilartoother

meio-fauna communities, with densities falling within the range

observedinotherEuropeanestuaries(Smoletal.,1994;Soetaert

etal., 1994,1995).Thedominanceof nematodesover allother

taxaiswelldocumented,withNematodatypicallybeingthemost

abundanttaxon(usually60–90%)(Coull,1999).Polychaetaranked

second,contrarytothecommonobservationthatcopepods are

usuallymoreabundant(Coull,1999).Harpacticoidcopepodsare

sensitivetoenvironmentalperturbation(Hicksand Coull,1983;

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473

indicateanthropogenicdisturbancesintheMondegoestuary.Low

densityofharpacticoidcopepodswasalsoobservedinthe

West-erschelde(VanDammeetal.,1984;Soetaertetal.,1995)andwas

ascribedtopollutioneffects.

Theincreaseintaxonomicresolution (frommeiofaunamajor

taxatonematodegenuslevel)enhancedourknowledgeofthe

sys-tem,suggestingthat highertaxonomicresolution maybemore

informativefor measurementof changesin meiofauna

commu-nitystructure.However,somestudiesofmeiofaunacommunities

asindicatorsofstatusinmarineenvironments(Schratzbergeretal.,

2000)and as indicators of pollution in harbors (Morenoet al.,

2008),forinstance,haveshownthatmeiofaunataxonassemblages

couldprovideasensitiveandclearmeasureofenvironmentalstatus

whencomparinginshoreandoffshorelocationsandthatindicators

basedonmeiofaunataxademonstrateda significantcorrelation

withtheconcentrationofcontaminants.

Nematodescommunitiescomprisedahighnumberofgenera

butwithfewdominantones,asobservedinotherestuaries(Austen

et al.,1989; Liand Vincx,1993; Soetaert etal., 1995;

Rzeznik-Orignac et al.,2003;Steyaert et al.,2003; Ferreroet al.,2008).

ThedominantgeneraweresimilartothosefoundintheBrouage

mudflat(France)(Rzeznik-Orignacetal.,2003)andintheThames

estuary(United Kingdom)(Ferrero at al.,2008), indicating that

speciesthatareabletotoleratethehighlyvariablesalinityin

estu-ariestendtobeabundant,takingadvantageoftheplentifulfood

resourcesofestuaries(Hourstonetal.,2011).Also,thewide

dis-tributionrangeof Daptonema,Sabatieriaand Dichromadora,also

observedbyFerreroetal.(2008),reflectsthewidesalinityrange

tolerated bythesegenera (Heip etal., 1985;Moens and Vincx,

2000;Ferreroetal.,2008).Moreover,Sabatieria,Daptonemaand

Terschellingia,thethreemostabundantgenerainthepresentstudy,

areknowntobetoleranttopollution(Soetaertetal.,1995;Austen

andSomerfield,1997;Schratzbergeretal.,2006;Steyaertetal.,

2007;Gambietal.,2009;Armenterosetal.,2009),andtheirhigh

densitiesalongtheMondegoestuarymaybeindicativeofthe

pres-suresfromwhichthisestuarysuffers.Infact,Morenoetal.(2011),

inanevaluationoftheuseofnematodesasbiologicalindicators

ofenvironmentalqualityinsedimentsoftheMediterraneanSea

statedthatthepresenceofsomegeneraprovidedaccurate

informa-tionontheecologyandadaptationoforganismstoenvironmental

conditions.Inthisstudy,disturbedplaceswerecharacterizedby

ahighdensityofTerschellingia,ParacomesomaandSabatieira,and

sitesclassifiedasinmoderateorpoorecologicalqualitystatuswere

alsodominatedbyDaptonema,indicatingthatsuchinhospitable

habitatconditionscanonlybetoleratedbygeneraabletothrivein

extremeconditions(Morenoetal.,2008).

GeneradiversitybroadlyfollowedtheRemane’sdiagram(1934)

for the effect of the salinity gradient on benthic invertebrates

speciesrichness(postulatedfortheBalticSea),withhigh

diver-sityinthemorestablemarineandfreshwaterwaters.Accordingto

Attrill(2002),salinityvariationovertimemaybemoreimportant

thanaveragesalinityforthedistributionofnematodesalongthe

estuary(alsoconfirmedbyFerreroetal.,2008).

Thepremisethatenvironmentalvariablesinfluence

meioben-thiccommunitiesiswelldescribed,butthequestionofhowfar

backweshouldconsidertheenvironmentalhistoryofasystemin

ordertoexplainthedistributionofthecommunitiesdependson

thelife-historycharacteristicsofthespeciesand,coupledwiththe

characterizationoftheenvironment,extremeeventsshouldalso

betakeninconsideration(Soetaertetal.,1995).

Spatialvariability,withthetransitionbetweenareasbeing

char-acterizedbydifferentassemblagesandwithstrongvariationsin

generadominance, wasdetected.Theshift fromanoligohaline

nematodecommunity,characterizedbylowdensity,high

nema-tode diversity and high abundance of Daptonema, to a typical

estuarinecommunity, characterized byhigh nematodedensity,

wasobserved,asintheThamesestuary(Ferreroetal.,2008).The

remainingareaswerealsodiscrete,eachonecharacterizedbya

dif-ferentcommunity,withtheexceptionofthePolyhalineareas(see

above).

Inthepresentstudy,besidestheclearspatialpattern,some

tem-poralvariationswerealsoobserved.Similarresultswereobserved

intheSwanRiverestuary,Australia(Hourstonetal.,2009),with

nematode species being markedly influenced by both site and

season,withsitebeingthemostimportantfactor. Intemperate

regions,nematodedensitiesusuallypeakinthewarmestmonths

(HicksandCoull,1983;Smoletal.,1994)andinthisstudy,although

thehighestdensitywasobservedinSummer2006,thepatternwas

notrepeatedintheotherwarmseasons.

The multivariate analysis allowed a representation of both

environmentaland biological (meiofauna and nematodes)data,

showingthattheestuarineabioticgradientwasmostlyreflected

inthebiologicalcommunities.

Spatialandtemporalvariationsofnematodeassemblageshas

been studied in several systems (e.g. Yodnarasri et al., 2008;

Armenterosetal.,2009;Hourstonetal.,2009,2011;Semprucci

etal.,2010)and,in ordertousethatinformationforecological

assessment,theapplicationofecologicalindicestothenematodes

assemblages enhanced our knowledge onthe benthic

environ-ment.Coupledwiththetaxonomicdiversity,functionaldiversity

isimportantforinterpretingdistributionpatternsofthe

commu-nities(Schratzbergeretal.,2008).Inwhatreferstomeiobenthic

communities,andbesidesthecommondiversitymeasures,

spe-cificindicatorsrelyonnematodesinformation,suchastheMaturity

Index andtheIndex ofTrophicDiversity.Thesetwo indices do

notdependonthesystem,notsufferingfromlackofgenerality

andtheuseofindicatorsbasedondifferentecologicalprinciples

is,accordingtoDaueretal.(1993)highlyrecommendedin

deter-miningtheenvironmentalqualitystatusofanecosystem(Marques

etal.,2009).

KnowingthattheMondegoestuarysuffersfromanthropogenic

pressures,especiallyinthePolyhalineareas(Northernarm–

dredg-ingactivities,harbor;Southernarm–inputsfromthePrantoRiver

andagriculturalrunoffs),wecanevaluatetheperformanceofthe

indicesindifferentiatinghomogeneoussectorsofimpactalongthe

estuary.Theresultsverifiedthattheindicesbehaveddifferently.

Forexample,theIndexofTrophicDiversity,generallyusedto

cor-relatetrophic diversitywithpollutionlevels(Heip etal.,1985),

appearedonly todifferentiate“extreme” conditionssuchasthe

relativelygoodecologicalconditionsinthemouthoftheestuary

(reflectedinhightrophicdiversityindexvalues)andtheupstream

partoftheestuaryhavinglowerecologicalstatus.Intheupstream

zone,theincorporationoffeedinginformationonthefreshwater

genera,mostlypredators,mayhavecontributedtotheobserved

pattern.However,ifthisdominanceisanaturalfeaturein

estu-aries,theparametersofthisindexshouldbereadjustedsothat

thepredominanceoffreshwaternematodesdoesnotexclusively

implyaclassificationofbadecologicalconditions.Asimilarresult

wasobservedbyMorenoetal.(2011),withtheITDnotseparating

siteswithdifferentecologicalclassificationsandevenindicatinga

goodEcologicalQualityStatusindisturbedsites.

Furthermore,theclassificationoffeedingcomplexity,asfirst

described by Wieser (1953), hasthe disadvantageof confining

nematodespecies toa single trophic status (Heip etal., 1985),

whichmaynotrepresenttherealcomplexityoffeedinghabitatsof

nematodes(MoensandVincx,1997),withtrophicplasticitybeing

describedformostfeedingtypes(Moensetal.,2005;Schratzberger

etal.,2008).

Ontheotherhand,thelowMaturityIndexvaluesobservedin

boththepolyhalineandeuhalineareassuggestedahighstresslevel,

sinceopportunisticgeneraincreaseinabundanceinadverse

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474

Anoppositetrendwasobservedintheoligohalinearea,wherethe

MIreachedmaximumvalues,indicatingabetterecologicalstatus,

withtheMIalsocapturingthecompositionvariationsthatoccurred

intheupstreamareaovertime(higherdispersionofoligohaline

samplesinthenMDS).Theseobservationsmayberelatedtothe

originoftheindexwhich,contrarytotheIndexofTrophic

Diver-sity,wasdevelopedforsoiland freshwaternematodes(Bongers

andBongers,1998)andlatelyextendedtoassessingthecondition

ofmarineandbrackishsediments,beinglessfrequentlyappliedto

marinenematodes(Bongersetal.,1991),partlyduetoalackof

empiricalsupportfortheclassificationofsomemarinegeneraand

theabsenceorrarityofextremecolonizersandpersistersinmost

marinehabitats(Schratzbergeretal.,2006).AccordingtoMoreno

etal.(2011),theanalysisofthepercentagecompositionofthe

dif-ferentc–pclassesineachsiteallowedabetterclassificationofthe

studiedsitesthantheapplicationoftheMI.

This study emphasized the need for the development of

a nematode-based multimetric index (Patrício et al., 2012),

takingin consideration density,composition, and genera

sensi-tivity/tolerance tostress, as proposedby Morenoet al. (2011).

Moreover,thismultimetricindexshouldincludeinformationwith

parametersmoreaccuratelybasedonmarine/estuarinenematodes

includingmaturityandtrophicvaluesspecificallycalculatedforthe

genera.Thereisalsotheneedforre-evaluationoftheboundaries

oftheindicesused,asanindexcanprovideagood

characteriza-tionofthesystembutmaybelimitedtoaspecificspatialarea.The

correctapplication ofnematodeinformation anditsintegration

intoa multimetricindex,withasuitablecombinationofseveral

indicators,would provideclearerinformation regarding

ecosys-temstatus,sinceitwouldovercomethelimitationsofindividual

analyses.Itisalsoimportanttobearinmindthattheevaluation

ofreferenceconditionsinordertoprovidecomparisonswith

dis-turbedenvironmentsisusuallyrequired.Sincemeiobenthicstudies

arequiterecentinPortugueseestuaries,itmaybeinterestingto

determineiftheanalysisofmeiobenthiccommunitiesinan

estu-arywherehumanperturbationsarealmostabsent(Miraestuary–

Alvesetal.,2009;Adãoetal.,2009)maybeusedinthe

establish-mentofreferenceconditions.

Acknowledgements

Thepresentstudywascarriedoutusingfundsprovidedbythe

researchprojectsRECONNECT(PTDC/MAR/64627/2006)and

3M-RECITAL(LTER/BIA-BEC/0019/2009).Additionally,itbenefitedfrom

onegrant,givenbyFCT–(SFRH/BD/62000/2009).Thestudywas

alsosubsidizedbytheEuropeanSocialFundandMCTESnational

funds,throughtheQRENandPOPH:HumanPotentialOperational

Programme–NSRF:NationalStrategicReferenceFramework–4.4.

Aspecialthankstoallwhoassistedusduringfieldandlaboratory

work.Theseniorauthor(J.Patrício)providedamajorcontribution

asproject’sleader.

AppendixA. Supplementarydata

Supplementary data associated with this article can

be found, in the online version, at http://dx.doi.org/

10.1016/j.ecolind.2012.07.013.

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