sequencingwallbase

SPECIALISSUEAscomycetefungiondimensionstoneofthe''BurgGleichen'',ThuringiaChristineHallmannDianaFritzlarLorenaStannekMichaelHoppertReceived:7January2011/Accepted:22April2011/Publishedonline:13May2011TheAuthor(s)2011.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractInthepresentstudy,thediversityofascomycetefungiwasinvestigatedontwowallareasofthe''BurgGleichen'',Thuringia(Germany),madeofvarioustypesofsandstones,travertineandGrenzdolomit.
FromaW-exposed,shadedwallarea,free-livingascomycetes(mainly''blackfungi'')andgreenalgaecouldberetrievedfromsandstonelithologies.
SandstonefromanESE-exposedareawasmainlycolonizedbylichenascomycetesandthelichenalgaTrebouxia.
Bothareasshareasmallnumberofgeneralistspecies,relatedtotheascomyceteblackfungiSarcinomycespetricola,PhaeococcomyceschersonesosandStichococcusmirabilis.
Free-livingblackfungiwereisolatedandcharacterizedwithrespecttocellwallmorphologyandmelanincontent.
Aremarkablyrigidmelaninlayer,incorporatedinthecellwallofaCladosporiumisolateispresentedindetail.
KeywordsDimensionstoneBiolmAscomycetefungiGreenalgaeCellwallMelaninIntroductionAscomycetefungalorganismsaremostsuccessfulcolo-nizersofallterrestrialhabitats,suchasrockandsoil(e.
g.
GorbushinaandKrumbein2000;AndersonandCairney2004).
Theyinteractindifferentwayswithotherorgan-isms,assymbionts(inmycorrhizaorlichensymbiosis),aspathogensorasimportantdestruentsofmostorganiccompounds,especiallyofplantlitter(Hattenschwileretal.
2005).
Althoughtheyareheterotrophic,i.
e.
theydependonorganicsubstratesproducedbyotherorganisms,manyofthemareadaptedtolowavailabilityofnutrients(Wainrightetal.
1993).
Moreover,fungalsporesareresistanttodes-iccationandradiation.
Owingtotheirsmallsize,thesporesaredispersedbyavarietyofvectors,inparticularbywind,butalsobyanimalsandbyplantdiaspores.
Fungalhyphaepenetratesurfacesandgrowinsidesoil,butalsoinvarioustypesofclasticrocks(e.
g.
sandstone)andhomogeneousmaterial,e.
g.
dolomiticlimestone(Gorbushinaetal.
1993,Hoppertetal.
2004,Gorbushina2007).
Thefungalhyphaisamongthefastestgrowingcellinnature:hyphaegrowwithavelocitybetween10and40lm/min(TrinciandSaunders1977).
Anactivegrowthzoneof10–15lmextendsbehindtheapex(Steinberg2007).
Theactivegrowthzoneisalsoimportantforsurfaceadhesion,theexcretionoflyticenzymes(e.
g.
forpenetrationofwood)andtheexertionofmechanicalforcesonthesurface.
Forendo-lithicgrowth,activedissolutionofcalciticmatricesbyorganicacids(e.
g.
Ascasoetal.
1998)isoneimportantmechanismforactivepenetration.
Fungalhyphaendapathinsedimentarymaterial(cf.
Fig.
1)bygrowingaroundsmallparticles.
Thisfeatureisbroughtaboutbyfastchangesinthegrowthdirectionofthehyphaltipandcouldalsobeobservedinorganismsofotherphyla,suchasplantroothairsorstreptomycetes(GeitmanandEmons2000;Fla¨rdh2003).
C.
HallmannAbt.
ExperimentellePhykologieundSammlungfu¨rAlgenkulturen,Albrecht-von-Haller-Institutfu¨rPanzenwissenschaften,Georg-August-Universita¨tGo¨ttingen,UntereKarspu¨le2,37073Go¨ttingen,GermanyD.
FritzlarL.
StannekM.
Hoppert(&)Institutfu¨rMikrobiologieundGenetik,Georg-August-Universita¨tGo¨ttingen,Grisebachstrae8,37077Go¨ttingen,Germanye-mail:mhopper@gwdg.
deM.
HoppertCourantResearchCentreGeobiology,Georg-August-Universita¨tGo¨ttingen,37077Go¨ttingen,Germany123EnvironEarthSci(2011)63:1713–1722DOI10.
1007/s12665-011-1076-yAscomycetefungionbuildingstonearewellknownandhavebeenfrequentlydescribed,mostlyincontexttobio-deterioration(e.
g.
Gorbushinaetal.
1993;Diakumakuetal.
1995,Steringeretal.
1997;GorbushinaandKrumbein2000;Simonovicovaetal.
2004).
Oneimportantgroupofcolonizers,theDematiaceae,producemelanininvegetativehyphae(Cooke1961;Wollenzienetal.
1995).
Thoughallfungiproducevarioustypesofpigments,melaninispre-dominantincellwallsofreproductivestructures,suchasconidiaorfruitingbodies,includingalltypesofspores(BellandWheeler1986).
ThemelanisationofvegetativecellsinDematiaceaeleadstoaheavyblackpigmentationofallcolonizedsurfaces(Saiz-Jimenez1995).
ThefrequentlydescribedmeristematicgrowthformofDematiaceaewithsphericalinsteadoflamentouscellsmaybeanadditionaladaptationtoadversegrowthconditionsinrockandstonehabitats,e.
g.
desiccationstress(Wollenzienetal.
1995).
ThepresentedstudyispartofacomparativeanalysisofmicrobialcommunitiesfromtwonaturalstonewallsfromtheBurgGleichen(cf.
Stu¨cketal.
2011[thisissue]).
Certaindifferencesintheabundanceofcyanobacteriaonwallsectionshavealreadybeendescribed(Hoppertetal.
2010).
Here,weshowtheoccurrenceofascomycetefungiontwowallsec-tions.
Typically,thefungalisolatesproducemelanininvege-tativecells.
Thesemelanindepositsformdistinctlayers,extractablefromcellwallsofoneCladosporiumisolate.
MaterialsandmethodsSamplingThesamplingsiteswereseveralwallsectionsoftheBurgGleichen,nearGotha,Thuringia(50°5204900N,10°5002000E).
Samples(approx.
100lldryvolumepersample)werecollectedinMay2009fromESE(wallareaA)andW-exposed(wallareaB)wallswithdifferentlithologies(Fig.
2).
Thesampleswerescrapedoffwithasterilescalpelandcollectedinsterile2-mlreactiontubes.
Samplesforclonelibrarieswererandomlytakenfromsites,wheresufcientmaterialcouldberemovedwithascalpel.
AllsamplesinareaAweretakenfromthewallbase(Fig.
2)madeofeitherGleichenbergerRa¨tsandsteinorSem-ionotussandstein.
Walljointsweremostlyclosed,i.
e.
lledwithmortar.
AreaBexhibitedavarietyoflithologies(travertine,Grenzdolomit,Ra¨tsandstein).
Mostwalljointswereopen.
SamplesfromRa¨tsandsteinfromthewallbaseweretakenintoconsiderationforthisstudy.
EachsamplewaslabeledwitheitherAorB(accordingtothewallsection)andwitharespectiverunningcodenumber.
Thesubsetofsamplesfromsandstonelithologieswasprocessedforthedetectionoffungalclones.
CultivationandisolationFortheenrichmentandisolationoflamentousfungi,smallamountsofthebiolmsampleswereusedtoinoculateculturemediapreparedaccordingtoStaley1968,withmodications(peptone/yeastextract/glucosemedium,PYG):Bacto-peptone0.
25g/l,yeastextract0.
25g/l,glucose0.
25g/l,basalsaltsolution19ml/l,traceelementsolution1ml/l(basalsaltsolutionin500ml:nitrilotriaceticacid3.
0g,MgSO497H2O7.
2g,CaCl292H2O1.
7g(NH4)6Mo7O2494H2O3.
0mg,FeSO497H2O49.
5mg.
Traceelementsolutionin100ml:EDTA250.
0mg,ZnSO497H2O1.
1mg,MnSO491H2O154.
0mg,FeSO497H2O500.
0mg,CuSO495H2O39.
2mg,CoSO397H2O19.
6mg,Na2B4O79.
34mg).
Forsolidmedia,1.
5%(w/v)Bacto-Agarwasadded.
Isolationofrepresentativestrainswasperformedbyrepeatedstreakingeithersinglecoloniesorconidiaonagarplatesuntilapparentmacro-scopicandmicroscopichomogeneityofcoloniesandconidia.
Isolateswereincubatedabout2weeksatroomtemperatureunderambientlight.
Liquidcultureswereincubatedinashakingwaterbathinthedark.
Forsomegrowthexperiments,apieceofSeebergerSandsteinwasgroundedinamortarandsterilizedbyautoclaving.
EqualvolumesofisolatedstrainsweremixedwithPYGagarmedium(keptliquidat95°C).
Themixturewasappliedonasterilemicroscopicslideandinoculatedaftersolidication.
Thesampleswereinspectedbylightmicroscopy(Axioscope40,includingAxiocamMRm,CarlZeissMicro-imaging,Go¨ttingen).
DNAextraction,PCR,cloningandsequencingForisolationofenvironmentalDNAaswellasDNAfromfungalisolates,cloningandsequencingstepswereper-formedasalreadydescribed(Hallmannetal.
2010[thisissue])withfollowingmodications:Fortwosamples(A1-4andB18)celldisruptionfor30sand50sofbeatFig.
1GrowthexperimentwithaCladosporiumisolate.
aGrowthinagarmediumwithoutsolidparticles.
bGrowthinagarmediummixedwithofsandstoneclasts.
Hyphaearetraced(whitelines).
Notetherapidchangesofgrowthdirectionsascomparedwitha1714EnvironEarthSci(2011)63:1713–1722123beatingwasapplied(ifnotespeciallymentioned,resultsof30sbeatbeatingwereshown).
Foridenticationofascomycetes,theprimerpairITS1(50-TCCGTAGGTGAACCTGCGG)andITS4(50-TCCTCCGCTTATTGATATGC)fortheITS(inter-naltranscribedspacer)wereused(AndersonandCairney2004).
Accessionnumbersofsequencesrelatedtothisstudyaredepositedonline(GenBankTM:http://www.
ncbi.
nlm.
nih.
gov/genbank)withthispublicationasareference.
ElectronmicroscopyForelectronmicroscopy,vegetativelamentswereeithertakenfromsolidorliquidcultures,concentratedbylteringovera0.
45-lmsizeporelterandembeddedinresinaccordingtoSpurr(1969).
Agarblockswerechemicallyxedovernightin2.
5%(v/v)glutaraldehyde(gradeIforelectronmicroscopy,Sigma–Aldrich,St.
Louis,MO,USA)andfor2hinosmiumtetroxide(1%,w/v,aqueoussolution,ScienceServices,Munich).
SamplesweredehydratedinagradedethanolseriesandinltratedwithSpurrresinover48hbeforepolymerizationat70°Cfor12h.
Ultrathinsectionsof80-nmthicknesswerecutwithglassknives.
Post-stainingofsectionswasperformedwith1.
5%(w/v)phosphotungsticacidfor5min(Hoppert2003).
ElectronmicroscopywasperformedwithaZeissEM902transmissionelectronmicroscope,equippedwitha1Kdigitalcamera(CarlZeissNTS,Oberkochen).
Fig.
2WallareasoftheBurgGleichenselectedforsampling.
aWallareaA(RomanesqueHall).
bWallareaB(sectionoftheringwall).
Samplingsitesaremarkedbyredsquares.
Theschematicdrawing(originalgure:WanjaWedekind)showsthelocationofthewallsinthecastlecomplexEnvironEarthSci(2011)63:1713–17221715123ForimmunogoldlabelingwiththelectinconcanavalinA(ConA),theprotocolaccordingtoHallmannetal.
2010(thisissue)wasapplied.
Goldmarkersinimagesweredigitallyenhancedasdescribed(HoppertandHolzenburg1998).
Melaninpreparationforelectronmicroscopywasper-formedaccordingtoRosasetal.
2000,withmodications.
AmelanisedCladosporiumisolatewasculturedonPYG-mediumandthencentrifugedfor30minat3,0009g.
Thecellswerewashedin100mMpotassium-phosphatebuffer,supplementedwith0.
9%(w/v)sodiumchloride(phos-phate-bufferedsaline,PBS).
Then,cellswereresuspendedin1.
0Msorbitol/0.
1Msodiumcitratesolution(pH5.
5),containing10mg/mllysisenzymefromTrichodermaharzianum(Sigma–Aldrich)andincubatedatroomtem-peratureovernight.
Cellswereagaincentrifuged,resus-pendedinPBSandincubatedin4Mguanidiniumthiocyanatesolutionfor12hatroomtemperature.
AfterwashingthecellsinPBSbycentrifugationandresuspen-sioninreactionbuffer(10mMTris,1mMCaCl2,0.
5%,w/vSDS),1mg/mlnalconcentrationproteinaseKwasadded.
Thesuspensionwasincubatedfor4hat65°C.
CellswereagainwashedinPBSandboiledfor6hin6MaqueousHClsolution.
Theremainingmelaninpreparationwasdialyzedfor2daysagainstdistilledwater(Viskingdialysistubing,molecularweightcutoff12,000–14,000,Serva,Heidelberg).
Thepreparedmelaninwasthensub-jectedtoembeddinginSpurrresinasdescribedabove.
ResultsBothareassampled(Fig.
2)exhibitedobviouscolonizationbygreenalgae(inaddition,cyanobacteriaonareaA)andepilithicorendolithiclichens(cf.
Fig.
3).
Approximately,one-thirdofthecollectedsampleswasusefulforcloningoffungalgenera.
Forgenerationoffungalclonelibraries,sequencingwithITSprimersturnedouttobemostappropriate(AndersonandCairney2004).
FromareasAandB,65(48)and30(75)fungal(algal)cloneswereretrieved,respectively.
IthastobekeptinmindthattheorganismswereidentiedbytheirsequencesofclosestknownrelativebyBLASTnanalysis(NationalCenterforBiotechnologyInformation,Bethesda,MD,USA).
ThegenusandspeciesnamesgiveninTables1and2representtheclosestknownrelativeaccordingtothesesequencesimilarities.
Table1liststhefungiidentiedfromtheclonelibraries,alongwiththealgalclones.
Mostly,theapparentcoloni-zationcorrelateswithclonebankdata.
AllsitesfromareaAwereapparentlycolonizedbyendolithiclichens.
Accordingly,amajorpartofalgaeandfungiretrievedfromtheclonebankswere,infact,lichenassociated.
SamplesB13,B14andB18weretakenfromsitesapparentlycolo-nizedbygreenalgae.
The(non-lichenassociated)algaeStichococcusandPseudochlorellacouldalsoberetrievedfromtheclonebanks.
Inaddition,numerousSarcinomycesandPhaeococcomyces-relatedfungalcloneswerepresent.
Becausethecelldisruptionmethodmayhavedetri-mentaleffectsonthequalityofclonelibraries(i.
e.
withrespecttospeciesrichness),differentintensitiesofbeadbeatingwereappliedontwosamples.
ThedatainTable1(sampleB1830sand50sbeatbeatingtime;sampleA1-430sand50s)documentaminorvariationintheretrievedclones,butdonotshowtheoccurrenceofessentiallydif-ferentspeciesgroups.
Thus,bothpairsofsamplescouldbeassignedtoeitherwallareaAorwallareaB-typicalgroupsofclones.
Althoughtheretrievedclonebanksalsodifferfromsampletosample,twogroupsofspeciestypicalforeitherofthewallareascanbeclearlydistinguished.
ItisobviousthatthefungaldiversityonareaAishigherwhencom-paredwithareaB,wheremorealgalthanfungalspeciescouldbeidentied.
Justasmallnumberofidenticalspecies(representedbytherespectiveclones)werepresentinbothFig.
3Macroscopicappearanceof(microbially)colonizedsandstonesurfaces.
aEndolithiclichensinconjunctionwithscalingandbackweatheringofthesurface.
bLayerofgreenalgae1716EnvironEarthSci(2011)63:1713–1722123areas:representativesoftwoascomycetefungi,Sarcino-mycespetricola-andPhaeococcomyceschersonesos-aswellasStichococcusmirabilis-clones(greenalgae)wereretrievedwithhighclonenumbers.
Onelichenascomycete(fromCaloplacadecipiens)andaTrebouxiasp.
lichenalgawerefoundinsamplesfrombothwalls,butjustinlownumbers.
Inparticular,thefungalclonesinbothgroupsofsamplesaredifferent(Fig.
4).
OnareaA,mostascomy-cetesarerelatedtogeneraknownfromlichensymbiosis(cf.
Wirth1995).
Alsothepresenceofseverallichenalgaclones(Trebouxia)reectsthisfeature.
Thisisalsoinaccordancewiththeobservationthatmainlyatthewallbase,thesandstonewasintensivelycolonizedbyendolithiclichens.
AlthoughalsoareaBexhibitedcolonizationbyendo-lithiclichensatthewallbase,mainlynon-lichenizedorganismscouldberetrieved:Here,justonecloneofthetypicallichenalgaandtwolichenfungiclonescouldbefound.
Amongthefungalorganisms,especiallySarcino-mycesandPhaeococcomycescloneswereretrieved,Table1DistributionoffungalandalgalclonesonwallsectionsClosestrelativespeciesA2-1A1-4A1-4(50s)A1-8B13B14B17B18B18(50s)B22%sequencesimilaritytoclosestrelativespeciesSarcinomycespetricola*111652597-98Phaeococcomyceschersonesos*223141196-97UnculturedCladosporiumclone*1199-100Caloplacadecipiens1299Capnobotryellasp.
.
*6399Cladosporiumcladosporioides*1199Phaeobotrysphaeriacitrigena190UnculturedDothideomycetes31082-83Anisomeridiumpolypori16186-88Dolichousnealongissima196Phaeophysciaciliata889-90Pseudocyphellariafimbriatoides183Umbilicariaarctica185Erysiphealphitoides199Pleosporaherbarum1100Stichococcusmirabilis1741675158789-96Trebouxiasp.
1198-99UnculturedTrebouxiaphotobiont411193-100Trebouxiaarboricola197Chlorellasp.
192Stichococcusrelated12185179-82Pseudochlorellasp.
112286-89Blackfungiaremarkedbyan(*),lichenalgaeandlichenfungiaremarkedinredBoxedareasindicatespeciespresentinbothwallareas(green),exclusivelypresentinareaA(blue)orinareaB(beige)ThepredominanceofSarcinomycespetricolainareaAisindicatedbyadarkgreencolorTable2BLASTsearchresultsoffungalisolatesSampleno.
Closestrelativespecies,accessionno.
PercentagesequencesimilaritytoclosestrelativespeciesB1-2Beauveriabassiana,GQ302680100A1-2,B1-3,B12,B2-5,B10CladosporiumcladosporioidesstrainF12,HQ38076699–100B1-8,B14Phaeococcomyceschersonesos,AJ50732396EnvironEarthSci(2011)63:1713–17221717123i.
e.
representativesoffree-living,non-lichenizedblackfungi.
Theoveralldiversitywasconsiderablylower(10differentblastresponses)thanonareaA(19differentblastresponses).
Severalascomycetousblackfungicouldbeisolatedfromthesamples,especiallyfromareaB,aslistedinTable2.
AlthoughthediversityofascomycetecloneswashigheronareaA,justonefungalisolatecouldbeobtainedfromthisarea.
SincetheabundantlichenascomycetesfromareaAareratherdifculttocultureinstandardmedia,theycouldnotberetrievedaspurecultures.
SeveralClado-sporiumandPhaeococcomyces-relatedisolatescouldbeobtainedfromareaB.
ThoughjustoneCladosporium-relatedclonecouldberetrievedfromclonebanks,severalCladosporiumstrainscouldbeisolated,accountingforahighabundanceofdiaspores,butalownumberofactivelygrowingorganismsatthetimeofsampling.
Allisolatesbelongtotheblack(melanised)fungiandservedasmodelorganismsforfurtherstudieslikeobser-vationofmelaninproductionandgrowthexperiments.
The0%10%20%30%40%50%60%70%80%90%100%wallareaAwallareaBascomyceteslichenfungilichenalgaegreenalgaeFig.
4RelativeamountsofclonenumbersoflargephylogeneticgroupsinwallareasAandBFig.
5MelaninproductioninCladosporiumandPhaeococcomycesstrains.
Cladosporium(a)andPhaeococcomyces(b)onsolidmedium.
cCladosporiumstraininliquidculture;vegetativehyphaeformglobulesduetoculturingconditionsandshowadarkstain.
dGrowthofCladosporiumwithoutformationofmelaninafterseveralculturepassages1718EnvironEarthSci(2011)63:1713–1722123growthexperimentinFig.
1illustratesthatCladosporium,likeallotherisolates,isabletogrowinvasiveinagarmediacontainingasuspendedfractionofcrushedsand-stone.
Oneascomycete(Beauveriabassiana)couldbeobtainedinpureculture,butnotfromtheclonebanks.
TheformationofmelanininsolidandliquidculturesisshowninFig.
5a–c.
Themelanisedisolatesexhibitthick,multi-layeredcellwalls(Fig.
6).
InPhaeococcomyces,twochemicallydistinctcellwalllayerscouldbeobserved:theinnerlayerislabeledbythelectinmarkerconcanavalinA(cf.
Hallmannetal.
2010[thisissue]),i.
e.
theConA-goldparticlesbindtothisfeature.
Anoutermostlayerconsistsoflessdenselypackedextracellularpolymers(exopolymers;Fig.
6a,b).
InCladosporiumisolates,thicklayersofexopolymersbetweenadjacentcellscouldbeobserved(Fig.
6c).
Thewallsareapproximately400nminthick-ness(Fig.
6d).
AliquidcultureshowninFig.
5dillustratesthatmelaninproductionmayalsogetlostafterseveralpassagesofculturinginthelaboratory.
Thereisastrikingdifferenceincellwallthicknessbetweenmelanisedandnon-melanisedCladosporiumisolates(Fig.
7).
Thenon-melanizedcellwallappearsbyafactorof10thinner,withoutanysignofexopolymerformation(Fig.
7b).
Althoughthemelanisedcellwallappearsthick,themelaninitselfrepresentsjustasmall,butveryrigidpro-portionofthecell.
WhileitwasnotpossibletoextractmelaninfromtheCladosporiumisolates,distinctmelanincellwallfragments(''melaninghosts'')fromPhaeococc-omycescouldberetrieved.
Here,themelaninrepresentsonedistinctlayerofthecellwallthatcouldbeisolatedafterharshtreatmentwithproteolyticandglycolyticenzymesaswellasboilinginhydrochloricacid.
Evenafterthistreatment,theoriginalshapeofthecellwallwasstillpreserved(Fig.
8).
Fig.
6PhaeococcomycesandCladosporiumcellwalls;transmissionelectronmicroscopyofultrathinsections.
a,bPhaeococcomycesshowingamultilayeredcellwall(arrows)withanoutermostexopolymerlayer.
bDetailwithaConA-goldlabeledwall(arrows;goldlabelencircled)andanunlabeledoutermostexopolymerlayer(asterisk).
Cladosporiumstrain:crosssectionsofthreecells(c),embeddedinanextracellularmatrix(asterisks).
Multilayeredcellwallofasinglecell(d)EnvironEarthSci(2011)63:1713–17221719123DiscussionThisstudyisfocusedonascomycetefungi,buttheclonelibrariesrevealedalsogreenalgalclones.
TheselectedprimerpairexhibitsabroadspecicityforITSsequencesoffungal(aswellasalgal)organismsandhasalreadybeensuccessfullyappliedforasimilarstudy(BerdoulayandSalvado2009).
However,aphylogeneticanalysiscom-prisingallphylotypeswithinfungi(andothereukaryoticmicroorganisms)wouldrequireawholesetofprimers(AndersonandCairney2004andreferencestherein).
Thus,thoughwecouldstatecleardifferencesbetweenthewallareas,thewholediversityofpresentphylotypes,ase.
g.
analyzedinastudyofrock-inhabitingfungirelatedtoDothideomycetes,couldnotberetrieved(Ruibaletal.
2009).
Itshouldbenotedthatalsothelackofsequenceinformationfromwell-characterizedisolatesinthepublicdatabaseslimitsthe''taxonomicresolution''inourapproach(cf.
AndersonandCairney2004).
Hence,some-timesjustlowsequencesimilaritiesofthecloneswithalreadyknownrelativespeciescouldberetrieved(cf.
Table1).
Especially,thedataonlichenascomycetegeneramustbeinterpretedcarefullyandshallbetakenasanindicatorforthepresenceofascomycetesinvolvedinlichensymbiosis,butnotnecessarilyfortheoccurrenceofadenedlichenspecies.
Generally,thesignicantdifferencesbetweenascomy-cetefungiondifferentwallsectionsofthe''BurgGlei-chen''areobvious.
Thoughallsamplesinthisstudyweretakenfromsandstones,someimpactfromsurroundinglimestonelithologies(inareaB),nesoilinwalljointsandmortarsmayinuencedirectlyorindirectlythesituation(pH,availablenutrientsorions)forcolonizingmicroor-ganisms.
Particularly,gypsum-containingmortarinwallareaA(cf.
Hoppertetal.
2010)mayhaveinuencedspeciesdiversityonthissite.
Moreover,openwalljointsinareaBprovidenumeroussmallnichesfordepositionsofsoil,moisture,birddroppingsandothernutrientsourcesthatmayinuencemicrobialgrowth.
Theseeffectscannotbecompletelyexcludedinmost''eld''situations,andaredifculttoquantify.
Thereis,however,nodirectevidencefortheinuenceofthelimestoneinareaB.
Otherwise,morelichenfungalclonesderivingfromcalcicolousandornitrophilouslichensshouldbeexpected(cf.
Arinoetal.
1997),whichis,infact,notthecase.
Generally,wallareaAisdirectlyexposedtosunlight,andisthereforemoresubjectedtodesiccationstressthantheW-exposedwallareaB,directlylocatedintheshadowofanadjacenttower(cf.
Fig.
2).
Clearly,lichenfungiandlichenalgaerepresentamajorpartofthemicrobialoraonthesun(ESE)-exposedwallsurface.
AttheW-exposedwallsurface(areaB),thenon-lichenizedblackfungiSarcinomycespetricolaandPhae-ococcomyceschersonesosaswellasStichococcusmirabilisandotherStichococcus-relatedalgalclonescouldberetrievedinhighabundance,butclonesofnon-lichenizedascomycetesaswellaslichen-associatedgenerawererare.
SarcinomycesandPhaeococcomyceswerealsopresentinareaAaccountingforbroadecologicalamplitudesoftheseorganisms(cf.
Wollenzienetal.
1997;BogomolovaandMinter2003;Michailyuk2008;Hallmannetal.
2010[thisissue]).
Sarcinomyces,however,couldonlyberetrievedinlowabundancefromareaA.
IthastobediscriminatedbetweentheStichococcusmirabilis-relatedclones(abun-dantinbothareas),andclones,moredistantlyrelatedtothegenusStichococcus(''Stichococcus-related''inTable1).
ThelattergroupcouldbeclearlyassignedexclusivelytowallareaB(cf.
Table1).
Fig.
7Cellwallswithandwithoutmelaninincomparison;trans-missionelectronmicroscopyofultrathinsections.
Cladosporiumstrain,liquidculture.
aMelanin-producingcellincrosssectionwithamultilayeredcellwall(arrows)ofseveral100nminthickness(cf.
Fig.
5c).
bThincellwall(betweenbotharrows)ofsometensofnm(non-melanisedvariant,cf.
Fig.
5d)1720EnvironEarthSci(2011)63:1713–1722123Thesedataimplythatlichenizedorganismsaresuc-cessfulcompetitorsonareaA,comparedwithsomenon-lichenizedgreenalgae,butalsocomparedwiththeblackfungusSarcinomyces.
TheequaldistributionofPhaeo-coccomyceschersonesos-andStichococcusmirabilis-rela-tedclonesinbothareasindicatethatsomespeciesremainobviouslycompletelyunaffected.
InwallareaB,althoughlichencolonizationwasobservable,non-lichenizedfungiandalgaedominated.
Itmaybepossiblethattherathermoistandnutrient-richconditionsaremorefavor-ablefornon-lichenizedgeneralists(cf.
HoppertandKo¨nig2006).
Mostoftheascomycetes,eitheridentiedbytheirsequencesorisolatedfromthestonesurfaceareknownasmelanisedstrains.
Althoughmelanisationisalsoknownfromlichenascomycetes,thenon-lichenizedgenerahavebeenintensivelystudiedwithrespecttotheirpigmentation(e.
g.
Diakumakuetal.
1995).
Besidesendolithicgrowth(inparticular,theformationofmicropits),pigmentationisanobvioushazardonstonesurfacescausedbytheseorgan-ismsonstonesurfaces.
Thiseffectisratherimportantfortheappearanceofsmoothsculpturedsurfaces(especiallymarble)thanforthenaturalbuildingstoneaspresentedhere.
However,ithastobekeptinmindthatanysurfacecolorofabuildingstoneisdarkenedbythemelanisedorganisms.
Incontrasttothegreencolorofalgae(andsomebrightcolorsofcrustoselichens),thisdarkeningisnotperceivedasa''biogenic''stain,butratherasanaturalcolorofthestoneorasasuccessivedarkeningbyotherfactors,e.
g.
sootdeposits.
Melanizationisanessentialfeatureforprotectionagainsthighlightintensities,ultravioletandevenionizingradiation(BellandWheeler1986;DadachovaandCasa-devall2008.
However,melaninisnotessentialforgrowthinthedarkandmaybenotexpressedinisolatesgrownunderlaboratoryconditions(Fig.
5).
Accordingly,inlaboratoryculturesofCladosporiumisolates,justverythincellwallscouldbeobserved.
TheisolationprocedureofmelaninsfromcellwallsofPhaeococcomycesillustratestherigidityofthemelan-isedcellwall.
Evenboilingindilutedhydrochloridesolutionoverseveralhoursdidneitherdestroythemel-anisedcellwalllayernorthemoleculeitself.
Allotherfeaturesofthecell,includingallstructuresofthecellenvelopeweredestroyed(Fig.
8).
Onbuildingstone,aftercelldeathanddecayofallotherorganiccom-pounds,themelanisedcellwallfragmentsremainonthesiteforrelativelylongtimes.
Sincethelamentousfungiareendolithic,theseparticlesarenotjustattachedtothebuildingsurface,butaredepositedindeeperlayersofthematerial.
ConclusionAlthoughcolonizationofbuildingsurfacesbymicroor-ganismsandtheircontributiontobiogenicweatheringisawellknownfact,differencesinthespeciescompositionorinspeciesdiversityhasbeenrarelyaddressed.
Thepre-sentedstudyshowsacleardistinctionbetweencertainspecialists,justpresentineitherofthebothwallareasunderinvestigationandalownumberofgeneralists.
Organismsfrombothgroupsmayaffectthebuildingmaterial.
Somelichensmaytakepartintheformationoflargescales(cf.
Fig.
3a)onsandstonesubstrata.
Algaeandparticularlyblackfungicontributesurfacestains.
Itislikelythatexpositionandmoistureregimestronglyinu-encethedominanceofeitherofthesegroups,butdoesnotnecessarilyreduceorevenexcludealgalorfungalgrowth.
Thus,alsointerventioninmoistureregimesonbuildingsurfacesmaychange,butnotnecessarilyreducemicrobialgrowthonbuildingmaterial.
Fig.
8''Melaninghosts''ofPhaeococcomyces,transmissionelectronmicroscopyofultrathinsections.
aCrosssection.
bSagittalsection,depictingtheareaofaformerseptum(asterisk)EnvironEarthSci(2011)63:1713–17221721123AcknowledgmentsFundingofthisprojectbytheDBU(DeutscheBundesstiftungUmwelt)isgratefullyacknowledged.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionNoncommercialLicensewhichper-mitsanynoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthor(s)andsourcearecredited.
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