Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study

ÂngelaBritoa,b_,VitorRamosa,b,c_,RuiSeabrab,c_{,ArleteSantos}a,b_{,CatarinaL.Santos}a_,

MiguelLopoa_{,SérgioFerreira}a_{,AntónioMartins}c_,RitaMotaa,b_{,BárbaraFrazão}b,c_,

RosárioMartinsc,d_{,VitorVasconcelos}b,c_{,PaulaTamagnini}a,b,∗

a_{IBMC–InstitutodeBiologiaMoleculareCelular,UniversidadedoPorto,RuadoCampoAlegre823,4150-180Porto,Portugal} b_{FaculdadedeCiências,DepartamentodeBiologia,UniversidadedoPorto,RuadoCampoAlegre,EdifícioFC4,4169-007Porto,Portugal}

c_{InterdisciplinaryCentreofMarineandEnvironmentalResearch(CIIMAR/CIMAR),UniversityofPorto,RuadosBragas289,4050-123Porto,Portugal}

d_{CISA–CentrodeInvestigac¸ãoemSaúdeeAmbiente,EscolaSuperiordeTecnologiadaSaúde,InstitutoPolitécnicodoPorto,RuaValentePerfeito322,4400-330VilaNovadeGaia,}

Portugal

a r t i c l e i n f o

Articlehistory: Received15April2011

Receivedinrevisedform21July2011 Accepted28July2011 Keywords: Cyanobacteria Marine 16SrRNAgene Nitrogenfixation Phylogeny Toxins a b s t r a c t

Cyanobacteriaareimportantprimaryproducers,andmanyareabletofixatmosphericnitrogenplayinga keyroleinthemarineenvironment.However,notmuchisknownaboutthediversityofcyanobacteriain Portuguesemarinewaters.Thispaperdescribesthediversityof60strainsisolatedfrombenthichabitats in9sites(intertidalzones)onthePortugueseSouthandWestcoasts.Thestrainswerecharacterizedby amorphologicalstudy(lightandelectronmicroscopy)andbyamolecularcharacterization(partial16S rRNA,nifH,nifK,mcyA,mcyE/ndaF,sxtIgenes).Themorphologicalanalysesrevealed35morphotypes(15 generaand16species)belongingto4cyanobacterialOrders/Subsections.Thedominantgroupsamong theisolatesweretheOscillatoriales.Thereisabroadcongruencebetweenmorphologicalandmolecular assignments.The16SrRNAgenesequencesof9strainshavelessthan97%similaritycomparedtothe sequencesinthedatabases,revealingnovelcyanobacterialdiversity.Phylogeneticanalysis,basedon partial16SrRNAgenesequencesshowedatleast12clusters.One-thirdoftheisolatesarepotentialN2-

fixers,astheyexhibitheterocystsorthepresenceofnifgeneswasdemonstratedbyPCR.Additionally, noconventionalfreshwatertoxinsgenesweredetectedbyPCRscreening.

© 2012 Elsevier GmbH. All rights reserved.

Introduction

Continental Portugal has an extensive coastline, of about 940km,facingtheNorthAtlanticOcean.Itisoneofthewarmest Europeancountries,anditsclimateisclassifiedasMediterranean typeCsainthesouth(C–warmtemperate;s–summerdry;a– hotsummer)andCsbinthenorth(C–warmtemperate;s–sum- merdry;b–warmsummer),accordingtotheKöppen’sscheme

[23].Thenear-shorewaveenergyhasastrongspatialandseasonal variability[30].Westernandsoutherncoastsareevidentlyunder differentwaveregimes,withtheunshelteredwestcoastsitesexpe- riencinghigherwaveheightandpowerthansouthernones,anda moderatedecreasinggradientfromnorthtosouthcanbeobserved. Waveheightandpowerinthewinterarealsomuchhigherthanin thesummer[30],andalongthecoastitispossibletoencounter

∗ Correspondingauthorat:IBMC–InstitutodeBiologiaMoleculareCelular, UniversidadedoPorto,RuadoCampoAlegre823,4150-180Porto,Portugal. Tel.:+351226074900;fax:+351226099157.

E-mailaddress:[email protected](P.Tamagnini).

differenttopographiesandbeachmorphologies–rockybeaches, beacheswithsandandrocksand sandybeacheswithdispersed rocks,resultingindiverselevelsofexposuretotheprevailingwave regimen.

Cyanobacteriaarephotosyntheticprokaryoteswithawidegeo- graphical distribution that are present in a broad spectrum of environmentalconditions [49]. Taxonomy of cyanobacteriais a controversialsubject,withtwoprevailingapproaches,the“botan- ical”and“bacterial”.Thereorganizedtaxonomicrevisionbasedon thebotanicalcodeuses morphological,biochemical and molec- ularcharacters[19–21].Aftertherecognitionoftheprokaryotic featuresofcyanobacteria,Rippkaetal.[36]publishedabacterio- logicaltaxonomybasedonmorphological,biochemicalandgenetic charactersofaxeniccultures,whileBergey’sManualofSystematic Bacteriology[5]usesaphylogeneticapproachprimarilybasedon 16SrRNAsequencecomparisons.Insummary,cyanobacteriacan beclassifiedintofiveSubsections[5,36]thatbroadlycoincidewith Ordersofthebotanicalapproach[19–21].Cyanobacteriabelonging tosubsectionsI(Chroococcales)andII(Pleurocapsales)areunicel- lular,butwhiletheorganismsinsubsectionIdivideexclusively bybinary fission,theonesfromsubsection IIcanalso undergo

0723-2020/$–seefrontmatter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2011.07.003

Samplingsitelatitude/longitude Waveexposure Beachtype 1.MoledodoMinho High Rocky N41◦₅₀_58.68_/W8◦₅₂_0.18 _(Rivermouth)

2.S.BartolomeudoMar High Sandandrocks N41◦₃₄_25.59_/W8◦₄₇_54.81

3.Lavadores High Sandandrocks N41◦₀₇_45.07_/W8◦₄₀_6.88

4.Aguda High Sandandrocks

N41◦0258.35/W8◦3919.22

5.FozdoArelho High Sandy N39◦₂₅_59.79_/W9◦₁₃_48.99 _(Rivermouth)

6.Martinhal Intermediate Sandy N37◦₀₁_07.30_/W8◦₅₅_36.17

7.Burgau Intermediate-low Sandandrocks N37◦0415.84/W8◦4634.97

8.Luz Intermediate-low Rocky N37◦₀₅_10.38_/W8◦₄₃_31.35

9.Olhosd’Água

N37◦₀₅_23.01_/W8◦₁₁_27.66 _{Low Sandy}

multiplefissionproducingsmalleasilydispersiblecellscalledbaeo- cytes.ThesubsectionIII(Oscillatoriales)includesthefilamentous strainswithoutcelldifferentiation.SubsectionsIV(Nostocales)and V(Stigonematales)comprisethefilamentousstrainsthatareable todifferentiateheterocystsandakinetes.Inaddition,filamentsof cyanobacteriafrom subsectionV are able todividein multiple planesdisplayingtruebranching.

Benthiccyanobacteriagrowalongtheshoreondifferentsub- strata,mainlyintheintertidalzones,aspartofcomplexmulti-taxa communities,oftenformingcohesivematsandbiofilms.Inthese habitats, theyare exposed toa range of daily stresses suchas nutrientlimitation,highUV-radiationanddesiccation[1,6].The successfuladaptationtotheseharshenvironmentsislargelydue totheirmorphologicalandfunctionalversatility[31].Cyanobac- teria playa major role in theglobalcarbon cycleas important primaryproducersperformingoxygenicphotosynthesis,andthe diazotrophictaxaarefundamental tothenitrogencycle,partic- ularlyinoceans[18].Inadditiontotheirecologicalimportance, cyanobacteriaarealsorecognizedasbeingaprolificsourceofbio- logicallyactivenaturalproducts; someof thesecompoundsare toxictoawidearrayoforganisms[50].Nevertheless,littleisknown aboutthediversityoftheseorganismsalongthePortuguesecoast withonlyafewreportspublished[e.g.2,9].Araújoetal.[2]provided anupdatedchecklistofthebenthicmarinealgaeofthenorthern Portuguesecoast,including26speciesofcyanobacteria.However, theseauthorsbasedtheirworkonnewrecords,literaturerefer- encesandherbariumdatabutdidnotisolateormaintaincultures oftheobservedspecimens.

Theaimofthisworkwastoidentifythecyanobacteriapresent intheintertidalzonesofthePortuguesecoastusingapolyphasic approach.TheisolatedspecimensarekeptatLEGECultureCollec- tion,andavailableforfurtherstudies.Inadditiontotheassessment of cyanobacterial diversity, a PCR-based screen for putative diazotrophsand toxin-producerswasperformedtounveiltheir role(s)intheecosystem.

Materialsandmethods

Samplingsites

The sites (Table1)were selected in order to represent the diversity of the Portuguese coast. Along the coast it is possi- bletodiscriminatebetweenrockybeaches(samplingsites1and 8),beacheswithsandand rocks(sampling sites2,3, 4,7) and sandybeacheswithdispersed rocks(sampling sites5,6 and9) (Table1).Severalrelevantvariables:wavepower[30],seasurface

temperature[SST[25]],riverrunoffandotherimportantclimatic variablessuchasairtemperatureandprecipitation(Institutode Meteorologia,IP,Portugal)werealsotakenintoaccount.Inbrief, Westcoastsamplingsitesexperiencegenerallyhigherenergetic waveregimes,loweroverall meanSSTsandmeanair tempera- tures[40],andhigherfreshwaterinputs,bothfromriverrunoff andprecipitation,thantheirSouthcoastcounterparts.

Cyanobacteriasampling,isolation,andculturing

Biologicalsampleswerecollectedinbothsummerandautumn of2006,andspringof2007.Samplecollectionwasperformedby scrapingthesurfaceofawiderangeofsubstrates(e.g.seaweeds, rocksurfaces,seashells,Sabellariaalveolatareefs)presentonbare rocksorshallowpuddlestidalpools.FortheisolateLEGE06009see also[9].Inaddition,seawatersamplesfromthesurfzonewerealso collected.

Rawbiologicalsampleswerescreenedforcyanobacterialspec- imensusingalightmicroscope(Leica DMLB),andsubsequently subjectedtoliquid cultureenrichment,agarplates streakingor micromanipulation.Wheneverpossiblesinglecells/filamentswere isolatedand transferredtoliquid orsolid mediumusinga Pas- teurpipette[34].Whenmicromanipulationwasfoundunsuitable, aliquotsoftheenrichedliquidculturesweretransferredtoliquid mediumorstreakedonagarplates.Seawatersampleswerefiltered withsterileglassfiberfilters(GF/C–Ø47mm,Whatman),andsub- sequentlyplacedonliquidmediumuntilavisiblecolonyappeared. Single colonies were picked and streaked on agarplates. New coloniesweretransferredintoliquidmedium.Culturesweremain- tainedusingthefollowingmedia:MN[34],BG110,BG11[44],and Z8[22]supplementedwith25gL−1NaCl,furthernamedZ825‰. ThemediaweresupplementedwithB12vitamin,andwhennec- essarywithcycloheximideoramphothericinB[34].Thecultures werekeptunder14hlight (10–30␮molphotonsm−2s−1)/10h darkcyclesat25◦C.CyanobacterialisolatesaredepositedatLEGE Culture Collection (Laboratório de Ecotoxicologia, Genómica e Evoluc¸ão;CIIMAR,Porto,Portugal).Additionally,theisolateLEGE 06123isalsodepositedatCultureCollectionofAlgaeandProtozoa (CCAP1425/1),fordetailsonthisorganismsee[33].

Lightandtransmissionelectronmicroscopy(TEM)and morphologicalidentification

Cells were observed using a Leica DMLB microscope (Leica MicrosystemsGmbH),imageswerecapturedwithaLeicaICCAAna- logueCameraSystem,and thecells weremeasuredusingQwin

parameterwasmeasured20timesindifferentindividuals. ForTEMstudies,cellswerecollected,centrifugedandprocessed asdescribedbySeabraetal.[39].Sectionswereexaminedusinga ZeissEM902.

Themorphologicalidentificationwascarriedoutfollowingthe criteria of Komárek and Anagnostidis [19–21], Waterbury and Stanier[47]andbyusingBergey’sManualofSystematicBacteriol- ogy[4],i.e.whenevertheidentificationdiffersinthetwosystems (“botanical”and“bacteriological”),thecorrespondingform-genus in Bergey’s classification is also mentioned. For Pleurocapsales WaterburyandStanier[47]classificationwasfollowed.

DNAextraction,purification,PCRsandsequencing

CellswereharvestedbycentrifugationandDNAwasextracted using the Maxwell® _{16 System, and the Cell DNA Purifi-} cation Kit for Gram-negative bacteria (Promega Corporation) according to the instructions of the manufacturer. DNA frag- ments within the following genes were amplified using the oligonucleotide primers listed on Table 2: 16S rRNA gene (smallsubunit ribosomal gene), nifK (dinitrogenase_{␤ subunit),} nifH (dinitrogenase reductase), mcyA and mcyE (microcystin synthetase), ndaF (nodularin synthetase), and sxtI (saxitoxin biosynthesis). The 16S rRNA gene was amplified using two primerpairs, eachincludingoneuniversalprimer andone spe- cific for cyanobacteria [8-27F(universal)/CYA781R(specific), and CYA361F(specific)/1492R(universal)],thisallowedustoamplifya longerfragment,andtoavoidunspecificamplificationssincethe culturesarenot axenic.FornifK,thefirstgroupofprimer pairs (seeTable2)wasdesignedforfilamentouscyanobacteria,whereas thesecondgroupwasdesignedforunicellularcyanobacteria.PCR reactionswereperformedusingathermalcyclerMyCyclerTM(Bio- RadlaboratoriesInc.,Hercules,CA,USA)following[45].ThePCR profiles,aftera2–5minat94◦Cofdenaturationstep,werethefol- lowing:16SrRNAgene–35cyclesof94◦C1min,50◦C1min,and 72◦C1min30s;nifK–30cyclesof94◦C1min,50◦C1min,and 72◦Cfor1min15s;mcyA,mcyE,andndaF–30cyclesof95◦C1min 30s,52◦C30s,and72◦C50s;sxtI–30cyclesof94◦C10s,52◦C 20s,and72◦C1min.Inallcasesafinalextensionof7minat72◦C wasperformed.AmplificationofthenifHfragmentwasperformed accordingtothemethodsdescribedpreviously[29].ThePCRprod- uctswereseparatedbyagarosegelelectrophoresisusingstandard protocols[38].DNAfragmentswereisolatedfromgelsusingthe NZYGelpureKit(NZYtech,Lda.INOVISA,Lisbon,Portugal),accord- ingtothemanufacturer’sinstructions.PurifiedPCRproductswere clonedintopGEM®_{-TEasyvector(Promega,Madison,WI,USA),and} transformedintoEscherichiacoliDH5␣competentcellsfollowing themanufacturer’sinstructions,andthemethodologydescribedin

[33].SomepurifiedPCRproductsweredirectlysequencedatSTAB Vida(Lisbon,Portugal).

Nucleotidesequenceaccessionnumbers

Novel sequences associated with this study are avail- able in GenBank under the accession numbers FJ589716, HQ832895–HQ832951,JF708120andJF708121.

Phylogeneticanalysis

InordertointegratethecyanobacterialdiversityalongthePor- tuguesecoastintoabroaderphylogeneticcontext,the16SrRNA genesequenceswereanalyzed,comparedwiththecurrentlyavail- able databases and used to construct phylogenetic trees. Each sequencewasindependentlyusedasthequeryinaBLASTsearch against thenon-redundant nucleotide databaseof theNational

toincludefull-lengthsequencesandtoobtainareliableoverallpic- tureofthecyanobacterialdiversity,anumberofreferencestrains from each subsections/ordersrepresented in oursamples were retrievedfromGenBankandwereincludedinthefollowingphylo- geneticanalyses.Thereferencestrainswereselectedaccordingto theBergey’sManualofSystematicBacteriology(2001),andfrom thosetheonescloselyrelatedtooursampleswereused.Amultiple alignmentencompassing16SrRNAgenesequencesfromtheiso- lates,thereferencestrainsandChloroflexusaurantiacusJ-10-flas theoutgroupwasperformedusingtheClustalW2algorithm[24], withallthedefaultparameters.Duetothesizedifferencesinthe amplificationproductsofeachisolate,andtoavoidtheconsequent biaseffectonthetreeconstruction, thisalignment waspruned toaninternalcoreof655nucleotidespresentinallsequences– correspondingtonucleotides 519to1172 inE.coli.The phylo- genetictreewascomputedusingtheMaximum-likelihood(ML) methodology[8].Thealignment wasimported toGeneiousPro

[7], and the tree wasbuild withthe PhyML [12] plugin,using theHKY85asthesubstitutionmodeland1000pseudo-replicates forthebootstrapanalysis,andallowingthesoftwaretoestimate thetransition/transversionratio,theproportionofinvariablesites and thegammadistributionparameterwith4substitution rate categories.

Resultsanddiscussion

Thisworkpresentsapolyphasicstudyofcyanobacterialiso- latesfromselected intertidalzones alongthePortuguese coast, combiningtheisolationofstrains, andtheircharacterizationby microscopicobservationsandmolecularanalysis.

Morphologicalcharacterization

Toevaluatethediversityofcyanobacteria,ninebeachesthat reflect the heterogeneity of habitats present along the conti- nental Portuguesecoastwereselected(Table1).Approximately 100cyanobacterialisolateswereretrieved, fromwhich60 were characterizedandshowntobelongto35differentmorphotypes. Fifteen genera and 16 species belonging tofour cyanobacterial orders/subsectionswereidentifiedbasedonmorphologicalchar- acters(TableS1,supplementarymaterial).Intermsoftheisolate’s spatial distribution noclear pattern was observed, i.e. the dif- ferent cyanobacterialgroupsare distributedby allthedifferent beachestypes.However,onemusttakeintoaccountthatthenum- berofisolatesofcertaingeneraismuchhigherthanothers,and that thedifferentnumberof isolatesobtainfor eachgenuscan be due to their ubiquity or to the fact that they are easier to isolate.Theisolationmediawereselecteddue totheirdifferent compositioninordertoexposethehighestpossiblediversity.MN mediumrevealedthehighestdiversity,Z825‰wasparticularly successfulfor coccoidsand Leptolyngbya spp.,while BG110 and BG11didnotcontributetoahigher diversitythantheZ825‰, withtheexceptionofNostocsp.LEGE06158.Themostrepresen- tativegroupamongtheisolatescomprisesthenonheterocystous forms,notablythefilamentousOscillatorialesandtheunicellular Chroococcales.RepresentativesfromtheunicellularPleurocapsales werealsofound,aswellasheterocystoustaxa:Nostocales(Fig.1). Notrue-branchingfilamentous,Stigonematales,werefound.The nonheterocystous cyanobacteria are, indeed, reported as the predominantformsinmarineenvironments[3,6,43,46].Thisseems tobe alsothecase for Portugal, theChecklistof benthic marine algaeandcyanobacteriaofnorthernPortugal(“Checklist”)reported 26 species ofcyanobacteria,15 of which areOscillatoriales[2]. Strains belonging to the genera Hyella, Myxosarcina, Lyngbya,

Targetgenes Primerpaira _Sequence5_→3 _{PCRproductexpectedsize(bp) Reference}

16SrRNA 8-27F AGAGTTTGATCCTGGCTCAG [48]

CYA781R(A)b _{GACTACTGGGGTATCTAATCCCATT 773 [28]}

CYA781R(B) GACTACAGGGGTATCTAATCCCTTT [28]

CYA361F GGAATTTTCCGCAATGGG 1131 [27]

1492R GGTTACCTTGTTACGACTT [48]

nifK nifK0F(A) CAAGGTTCTCAAGGTTGCGTT 1139 [33]

nifK1F(B) CAAGGTTCTCAAGGTTGTGTG [33]

nifK4R TGTAAGTGGTGGCGATCAAAGA [33]

nifK0F(A)/nifK1F(B) Seeabove 407 [33]

nifK3_{R GGGATGAAGTTGATTTTGCCGTT Thiswork}

nifk146F CCTGGGAATATCGGGAA 319 Thiswork

nifk465R GCCATACAAGTTGTACAGACA Thiswork

nifk310F CGTCACTTCAAAGAGCCTT 1084 Thiswork

nifk1394R GGCGATCAAAGATAGGATA Thiswork

nifH4 TTYTAYGGNAARGGNGG [29]

nifH nifH3 ATRTTRTTNGCNGCRTA 361 [29]

nifH2 ADNGCCATCATYTCNCC [29]

nifH1 TGYGAYCCNAARGCNGA [29]

mcyA mcyA-Cd1F AAAAGTGTTTTATTAGCGGCTCAT 297 [14]

mcyA-Cd1R AAAATTAAAAGCCGTATCAAA [14]

mcyE/ndaF HEPF TTTGGGGTTAACTTTTTTGGGCATAGTC 472 [16]

HEPR AATTCTTGAGGCTGTAAATCGGTTT [16]

sxtI sxtI-F2 GGATCTCAAAGAAGATGGCA 991 Thiswork

sxtI-R GGTTCGCCGCGGACATTAAA [17]

a_{F(forward)andR(reverse).}

b _{(A)and(B)–primersusedtogetherinamixturewithequimolarconcentration.}

Pseudophormidium and Calothrix were found in the same area (northofPortugal)asreportedintheChecklist.Cyanobium,Lep- tolyngbyaandPseudanabaenaarethemostnumerousamongour isolates(TableS1andTable3).Somegeneradescribedby[2]were notfoundinourstudy(Chamaecalyx,Dermocarpella,Entophysalis, Hydrococcus,Trichocoleus,Porphyrosiphon,Sirocoleum,and Spiro- coleus),whereas Xenococcus,Microcoleus,Spirulina morphotypes werepresent inourfieldsamplesbutitsisolationwasnotsuc- cessful.Ontheotherhand,generathatarenotdescribedbythese authorswere present in oursamples (Aphanothece, Cyanobium, Synechocystis,Chroococcidiopsis,Chroococcopsis,Leptolyngbya,Pseu- danabaena, Romeria, Schizothrix, Nostoc, and Scytonema), some of them also confirmedby a recent studies onthe Portuguese coast:Synechocystis,Cyanobium,andLeptolyngbya[9,26].However, Araújoetal.[2]basedtheirworknotonlyonnewrecordsbutalso onliteraturereferencesandherbariumdata,anddidnotisolatethe observedspecimens.RepresentativesoftheorderStigonematales werenotfoundinthisstudynorreportedbyAraújoetal.[2].This wasexpectedsinceitisknownthattheseorganismsarepoorlyrep- resentedinmarineenvironments[15],andmainlyencounteredin theflowingwatersofhotspringsandsoils[11,36,52].

Fig.1. Distributionoftheisolatespertaxonomicgroup.

The diversity of our cyanobacterial isolates is depicted in

Figs.2and3(aswellasinFigs.S1andS2), whereitispossible toobservegenerabelongingtothefourorders.TheChroococcales Synechocystisand Cyanobium dividingin oneplane(Fig.2a and b,Fig.3aandb),thePleurocapsalesChroococcopsis,Myxosarcina andHyelladividinginmorethanoneplane(Fig.2c–g,Fig.3d)and producingbaeocytes(Fig.2f),theOscillatorialesRomeria,Phormid- ium, Leptolyngbya, Pseudophormidium, Lyngbya and Plectonema (Fig. 2h–q, Fig.3e–f), withLeptolyngbya cf. halophila exhibiting nodule-likestructures(Fig.2m,Fig.3f)andPlectonemacf.radio- sumgeminatefalsebranches(Fig.2q),andtheNostocalesgenera Nostoc,CalothrixandScytonema(Fig.2r–u,Fig.3g),withintercalar (Fig.2t)orterminal(Fig.2s)heterocysts.InCalothrixsp.LEGE06100 hormogoniawerefound(Fig.2u).Additionally,theultrastructural imagesprovidedinformationonthesizeandshapeofthesheath (Fig.3e,fandi),andthedifferentarrangementofthethylakoids (Fig.3a–c,e,gandh).Itwasalsopossibletoobservethedisrup- tionofthemothersheathleadingtodisintegrationofthecolonies aftercelldivision(Fig.3c),andanoduledetailofLeptolyngbyacf. halophilaLEGE06102(Fig.3f).Abriefdescriptionofeachoftheiso- latesandthemorphologicalcharacteristicsusedtotaxonomically affiliatethemisdepictedinTableS1,aswellastheirdistribution andhabitatdescription.

DNAsequenceandphylogeneticanalysis

Partial16SrRNAgenesequenceswereobtainedforalltheiso- lates.Thesesequenceswerecomparedwiththeonesavailablein theNCBIdatabase(March2011)usingBLASTn,andtheresultsare showninTable3.Therewasaquitegoodcorrelationbetweenthe phenotypicandgenotypicbasedidentifications,formorethanone- thirdoftheisolates(Table3,highlightedingrey).Fortheremaining isolatesdiscrepancieswereobservedbetweenthetwoidentifica- tions,possiblyresultingfromlimitationsofthedatabasesand/or taxonomicalconstraints–itiswellrecognizedthatcyanobacterial taxonomyfacesseveralproblems,andiscurrentlyunderrevision

[46].Oneshouldpointout that9ofourisolateshave lessthan 97%similaritytothe16SrRNAgenesequencesinthedatabase,

Fig.2. LightmicrographsillustratingthediversityofcyanobacterialmorphotypesisolatedfromtheintertidalzonesofninebeachesonthePortuguesecoast.(a)Synechocystis salinaLEGE06099,(b)Cyanobiumsp.LEGE06184,(c)Chroococcopsissp.LEGE07168,(d)Chroococcopsissp.LEGE07187,(e)Myxosarcinasp.LEGE06146,(f)Chroococcopsis sp.LEGE07161,(g)Hyellasp.LEGE07179,(h)Romeriasp.LEGE06013,(i)Phormidiumsp.1LEGE06111,(j)PhormidiumlaetevirensLEGE06103,(k)Leptolyngbyamycoidea LEGE06126,(l)LeptolyngbyamycoideaLEGE06118,(m)Leptolyngbyacf.halophilaLEGE06102,(n)Leptolyngbyasp.LEGE06188,(o)Pseudophormidiumsp.LEGE06125,(p) Lyngbyacf.aestuariiLEGE07165,(q)Plectonemacf.radiosumLEGE06105,(r)Nostocsp.1LEGE06106,(s)Calothrixsp.LEGE06122,(t)Scytonemasp.LEGE07189,(u)Calothrix sp.LEGE06100.b–baeocytes;fb–falsebranching;hg–hormogonia;h–heterocysts;n–nodule.Scalebars–10␮m.

Fig.3.Transmissionelectronmicrographsofselectedisolatesfromthesamplingsites.(a)SynechocystissalinaLEGE06099,(b)Cyanobiumsp.LEGE06097,(c)Gloeocapsopsis cf.crepidinumLEGE06123,(d)Chroococcopsissp.LEGE07187,(e)LeptolyngbyamycoideaLEGE06118,(f)Leptolyngbyacf.halophilaLEGE06102,insert–noduledetail(g) Nostocsp.2LEGE06158,(h)Calothrixsp.2LEGE06122,(i)Rivulariasp.LEGE07159.het–heterocyst;sh–sheath;sp–septum;th–thylakoid;veg–vegetativecell.arrow head–disruptionofthemothersheath.Scalebars–1␮m.

emphasizingthepresenceofnovelcyanobacterialdiversityinPor- tugueseshorewaters(Table3,dashedboxes).

Phylogeneticanalyseswereperformed toassess therelative positioning of the cyanobacteria isolated in this study. An ML algorithm was applied to a multiple alignment of partial 16S