Journal of cave and karst studies

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Journal of cave and karst studies

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Title:
Journal of cave and karst studies
Series Title:
Journal of Cave & Karst Studies
Alternate Title:
Continues NSS bulletin (OCLC: 2087737)
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National Speleological Society
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National Speleological Society
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English

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Geology ( local )
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serial ( sobekcm )
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United States

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Open Access - Permission by Publisher
Original Version:
Vol. 76, no. 2 (2014)

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University of South Florida Library
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University of South Florida
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K26-02067 ( USFLDC DOI )
k26.2067 ( USFLDC Handle )
20949 ( karstportal - original NodeID )
0146-9517 ( ISSN )

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Journal of Cave and Karst Studies Volume 76 Number 2 August 2014Article 69Karst Evolution of the Garraf Massif (Barcelona, Spain): Doline Formation, Chronology and Archaeopalaeontological Archives John Daura, Montserrat Sanz, Joan Josep Forns, Antoni Asensio, and Ramon JuliArticle 88Biogenicity and Characterization of Moonmilk in the Grotta Nera (Majella National Park, Abruzzi, Central Italy) Paola Cacchio, Gianluca Ferrini, Claudia Ercole, Maddalena Del Gallo, and Aldo LepidiArticle 104Adaptations of Indigenous Bacteria to Fuel Contamination in Karst Aquifers in South-Central Kentucky Tom D. Byl, David W. Metge, Daniel T. Agymang, Mike Bradley, Gregg Hileman, and Ron W. HarveyArticle 114Aerosolized Microbes from Organic Rich Materials: Case Study of Bat Guano From Caves in Romania Daniela R. Borda, Ruxandra M. Na stase-Bucur, Marina Spnu, Raluca Uricariu, and Janez MulecArticle 127Glacial Lake Schoharie: An Investigative Study of Glaciolacustrine Lithofacies in Caves, Helderberg Plateau, Central New York Jeremy M. Weremeichik and John E. MylroieArticle 139Diet Analysis of Leopoldamys Neilli, A Cave-Dwelling Rodent In Southeast Asia, Using Next-Generation Sequencing From Feces Alice Latinne, Maxime Galan, Surachit Waengsothorn, Prateep Rojanadilok, Krairat Eiamampai, Kriangsak Sribuarod, and Johan R. MichauxArticle 146Microclimate Effects on Number and Distribution of Fungi in the Wodarz Underground Complex in the Owl Mountains (Gry Sowie), Poland Rafa Ogrek, Wojciech Pusz, Agnieszka Lejman, and Cecylia Uklan ska-PuszBook Review 154Sources et Sites des Eaux KarstiquesJournal of Cave and Karst StudiesVolume 76 Number 2 August 2014 Journal of Cave and Karst Studies Distribution Changes During the November 9, 2013, Board of Governors meeting, the BOG voted to change the Journal to electronic distribution for all levels of membership beginning with the April 2014 issue. Upon publication, electronic les (as PDFs) for each issue will be available for immediate viewing and download through the Member Portal on www.caves.org. For those individuals that wish to continue to receive the Journal in a printed format, it will be available by subscription for an additional fee. Online subscription and payment options will be made available through the website in the near future. Until then, you can arrange to receive a print subscription of the Journal by contacting the NSS ofce at (256) 852-1300. August 2014 Volume 76, Number 2 ISSN 1090-6924 A Publication of the National Speleological Society JOURNAL OF CAVE AND KARST STUDIES rf

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GUIDE TO AUTHORS The Journal of Cave and Karst Studies is a multidisciplinary journal devoted to cave and karst research. The Journal is seeking original, unpublished manuscripts concerning the scientic study of caves or other karst features. Authors do not need to be members of the National Speleological Society, but preference is given to manuscripts of importance to North American speleology. LANGUAGES: The Journal of Cave and Karst Studies uses American-style English as its standard language and spelling style, with the exception of allowing a second abstract in another language when room allows. In the case of proper names, the Journal tries to accommodate other spellings and punctuation styles. In cases where the Editor-in-Chief nds it appropriate to use nonEnglish words outside of proper names (generally where no equivalent English word exists), the Journal italicizes them. However, the common abbreviations i.e., e.g., et al., and etc. should appear in roman text. Authors are encouraged to write for our combined professional and amateur readerships. CONTENT: Each paper will contain a title with the authors names and addresses, an abstract, and the text of the paper, including a summary or conclusions section. Acknowledgments and references follow the text. ABSTRACTS: An abstract stating the essential points and results must accompany all articles. An abstract is a summary, not a promise of what topics are covered in the paper. STYLE: The Journal consults The Chicago Manual of Style on most general style issues. REFERENCES: In the text, references to previously published work should be followed by the relevant authors name and date (and page number, when appropriate) in parentheses. All cited references are alphabetical at the end of the manuscript with senior authors last name rst, followed by date of publication, title, publisher, volume, and page numbers. Geological Society of America for mat should be used (see http://www.geosociety.org/pubs/geoguid5. htm). Please do not abbreviate periodical titles. Web references are acceptable when deemed appropriate. The references should follow the style of: Author (or publisher), year, Webpage title: Publisher (if a specic author is available), full URL (e.g., http://www. usgs.gov/citguide.html) and date when the web site was accessed in brackets; for example [accessed July 16, 2002]. If there are specic authors given, use their name and list the responsible organization as publisher. Because of the ephemeral nature of websites, please provide the specic date. Citations within the text should read: (Author, Year). SUBMISSION: Effective February 2011, all manuscripts are to be submitted via Peertrack, a web-based system for online submission. The web address is http://www.edmgr.com/jcks. Instructions are provided at that address. At your rst visit, you will be prompted to establish a login and password, after which you will enter information about your manuscript (e.g., authors and addresses, manuscript title, abstract, etc.). You will then enter your manuscript, tables, and gure les separately or all together as part of the manuscript. Manuscript les can be uploaded as DOC, WPD, RTF, TXT, or LaTeX. A DOC template with additional manuscript specications may be downloaded. (Note: LaTeX les should not use any unusual style les; a LaTeX template and BiBTeX le for the Journal may be downloaded or obtained from the Editor-inChief.) Table les can be uploaded as DOC, WPD, RTF, TXT, or LaTeX les, and gure les can be uploaded as TIFF, EPS, AI, or CDR les. Alternatively, authors may submit manuscripts as PDF or HTML les, but if the manuscript is accepted for publication, the manuscript will need to be submitted as one of the accepted le types listed above. Manuscripts must be typed, double spaced, and single-sided. Manuscripts should be no longer than 6,000 words plus tables and gures, but exceptions are permitted on a case-bycase basis. Authors of accepted papers exceeding this limit may have to pay a current page charge for the extra pages unless decided otherwise by the Editor-in-Chief. Extensive supporting data will be placed on the Journals website with a paper copy placed in the NSS archives and library. The data that are used within a paper must be made available. Authors may be required to provide supporting data in a fundamental format, such as ASCII for text data or comma-delimited ASCII for tabular data. DISCUSSIONS: Critical discussions of papers previously published in the Journal are welcome. Authors will be given an opportunity to reply. Discussions and replies must be limited to a maximum of 1000 words and discussions will be subject to review before publication. Discussions must be within 6 months after the original article appears. MEASUREMENTS: All measurements will be in Systeme Internationale (metric) except when quoting historical references. Other units will be allowed where necessary if placed in parentheses and following the SI units. FIGURES: Figures and lettering must be neat and legible. Figure captions should be on a separate sheet of paper and not within the gure. Figures should be numbered in sequence and referred to in the text by inserting (Fig. x). Most gures will be reduced, hence the lettering should be large. Photographs must be sharp and high contrast. Color will generally only be printed at authors expense. TABLES: See http://www.caves.org/pub/journal/PDF/Tables. pdf to get guidelines for table layout. COPYRIGHT AND AUTHORS RESPONSIBILITIES: It is the authors responsibility to clear any copyright or acknowledgement matters concerning text, tables, or gures used. Authors should also ensure adequate attention to sensitive or legal issues such as land owner and land manager concerns or policies. PROCESS: All submitted manuscripts are sent out to at least two experts in the eld. Reviewed manuscripts are then returned to the author for consideration of the referees remarks and revision, where appropriate. Revised manuscripts are returned to the appropriate Associate Editor who then recommends acceptance or rejection. The Editor-in-Chief makes nal decisions regarding publication. Upon acceptance, the senior author will be sent one set of PDF proofs for review. Examine the current issue for more information about the format used. ELECTRONIC FILES: The Journal is printed at high resolution. Illustrations must be a minimum of 300 dpi for acceptance.The Journal of Cave and Karst Studies (ISSN 1090-6924, CPM Number #40065056) is a multi-disciplinary, refereed journal published three times a year by the National Speleological Society, 2813 Cave Avenue, Huntsville, Alabama 35810-4431 USA; Phone (256) 852-1300; Fax (256) 851-9241, email: nss@caves.org; World Wide Web: http://www.caves.org/pub/journal/. Check the Journal website for subscripion rates. Back issues and cumulative indices are available from the NSS ofce. POSTMASTER: send address changes to the Journal of Cave and Karst Studies, 2813 Cave Avenue, Huntsville, Alabama 35810-4431 USA. The Journal of Cave and Karst Studies is covered by the following ISI Thomson Services Science Citation Index Expanded, ISI Alerting Services, and Current Contents/Physical, Chemical, and Earth Sciences. Copyright 2014 by the National Speleological Society, Inc. Front cover: Moonmilk stalatites in Grotta Nera, Italy. See Cacchio et al., in this issue.Published By The National Speleological SocietyEditor-in-Chief Malcolm S. FieldNational Center of Environmental Assessment (8623P) Ofce of Research and Development U.S. Environmental Protection Agency 1200 Pennsylvania Avenue NW Washington, DC 20460-0001 703-347-8601 Voice 703-347-8692 Fax eld.malcolm@epa.govProduction EditorScott A. EngelCH2M HILL 2095 Lakeside Centre Way, Suite 200 Knoxville, TN 37922 865-560-2954 scott.engel@ch2m.comJournal Copy EditorBill MixonJOURNAL ADVISORY BOARD Penelope Boston Gareth Davies Luis Espinasa Derek Ford Louise Hose Leslie Melim Wil Orndorf Bill Shear Dorothy Vesper BOARD OF EDITORS AnthropologyGeorge Crothers University of Kentucky211 Lafferty Hall george.crothers@uky.eduConservation-Life SciencesJulian J. Lewis & Salisa L. LewisLewis & Associates, LLC. lewisbioconsult@aol.comEarth SciencesBenjamin SchwartzDepartment of Biology Texas State University bs37@txstate.eduRobert BrinkmanDepartment of Geology, Environment, and Sustainability Hofstra University robert.brinkmann@hofstra.eduMario PariseNational Research Council, Italy m.parise@ba.irpi.cnr.itExplorationPaul BurgerCave Resources Ofce National Park Service Carlsbad, NM paul_burger@nps.govMicrobiologyKathleen H. LavoieDepartment of Biology State University of New York, Plattsburgh, lavoiekh@plattsburgh.eduPaleontologyGreg McDonaldPark Museum Management Program National Park Service, Fort Collins, CO greg_mcdonald@nps.govSocial SciencesJoseph C. DouglasHistory Department Volunteer State Community College joe.douglas@volstate.eduBook ReviewsArthur N. Palmer & Margaret V. PalmerDepartment of Earth Sciences State University of New York, Oneonta palmeran@oneonta.edu

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KARSTEVOLUTIONOFTHEGARRAFMASSIF (BARCELONA,SPAIN):DOLINEFORMATION, CHRONOLOGYANDARCHAEOPALAEONTOLOGICALARCHIVESJOANDAURA1,MONTSERRATSANZ1,JOANJOSEPFORNO S2,ANTONIASENSIO3,ANDRAMONJULIA `4Abstract: Karstlandscapeevolutionhasbeenwidelystudiedinrecentyearsonkarst plateaus,buttheuseofdatingmethodshasnotusuallybeenpossibleowingtoalackof data.TheintenselykarstifiedGarrafMassif,however,presentslargesolutiondolinesand severalshaftsthatcontainarchaeologicalandpalaentologicalremainsthatcanbeused fordeterminingthechronologicalframeworkofthekarstificationprocesses.Thesesites havebeendatedusingvarioustechniques,andtheresultingdatacombinedwithevidence frompreviousgeomorphologicalstudies.Theresultsallowustodefinethepatternand timingoftheevolutionofkarstmorphologiesinthenortheastoftheIberianPeninsula. Weproposeasimplefive-stagegeomorphologicalmodeloftheevolutionoftheGarraf dolineslocatedontheplateau,betweentheMiddletoUpperMioceneandtheHolocene. Thestudyalsoprovidesimportantinformationforanalyzinglandscapehistoryinhigh plateausofkarstregions.INTRODUCTIONVariousmodelsofthegeomorphologicalformationand evolutionofdolineshavebeenproposed,basedonthestudy ofsedimentaryfills(Sauroetal.,2009),host-rockdissolution processes(ZamboandFord,1997),anddepositmorphology andarchitecture(FordandWilliams,2007;Walthametal., 2005).Solutiondolinesarethemostcommonlystudiedofthe karstlandforms,havingbeendescribedindetailsincetheend ofthenineteenthcentury(Cvijic,1893),whilecollapse sinkholesareperhapsthemostspectacular,withthe tiankengs inthekarstareasofChinaprovidingthebestexamples(Zhu andWaltham,2005).Recently,manystudiesconductedin differentkarstregionshaveproposednewmodelsforthefull evolutionarysequenceofdolinedevelopment(Gibbardetal., 1986;Bruxellesetal.,2008,2012;Luzo netal.,2008;Siart etal.,2010). Chronologicalapproachestothedatingofdolineand karstevolutionarelimitedandinvolveassociatingthese depressionswiththearchaeologicalorpalaeontological record(Ufrecht,2008).Sediment-filleddepressions,mainly dolines,havebeenusedaspromisingrecordsforgeoarchaeologicalresearch(VanAndel,1998;Bruxellesetal., 2006),especiallyaspartofamultidisciplinaryapproach (Siartetal.,2010),andhaveledtoabetterunderstanding ofthechronologicalframework.Suchstudiesprovide absoluteagesofthesedimentsandthematerialspreserved inthedolines,whichmakesitpossibletodatethedoline formation.Thishasbeenthecase,forexample,ofthe DolinedOrgnac3(Moncel,2003;Monceletal.,2005),the dolineofCantalouette(Bourguignon,2004),thedolinesin theCaussesregion(Quile`setal.,2002),andthedolinein Visogliano(Falgue`resetal.,2008),amongothers. Morecommonistheuseofthesesediment-filled depressionsforarchaeologicalandpalaeoenvironmental reconstruction(Gibbardetal.,1986,Bruxellesetal.,2008; Siartetal.,2010;Bruxellesetal.,2012).Mostofthese reconstructionshavebeenconductedinancientMediterraneanlandscapes(MarrinerandMorhange,2007; Fouacheetal.,2008),wheretheabundanceofkarst regionswithoutrunningsurfacewaterandanyassociated sedimentaccumulationshashinderedreconstructionowing totheabsenceofcorrespondingnon-karstarchives.These studiesemphasizetheimportanceofkarstdepressionsas highlyvaluable,uniquesourcesofinformation.These depressionsactastrapsforerodedsoilsthatmay accumulateandbepreservedfromfurthererosion,thereby facilitatingtheanalysisoflandscapeevolution,chronology, andassociatedpalaeoenvironmentalphases(Hempel, 1991). Morethanthreehundredshaftsaredocumentedinthe centralGarrafMassif,andstudiesofthiskarstregionhave alonggeologicalandspeleologicaltraditiondatingbackto theendofthenineteenthcentury(Font,1897).Work carriedoutduringthe1940sand1950sofferedaninitial modeloftheGarrafkarstsoriginanddevelopment, providinganinitialchronologicalapproachtounderstandingdolineformation(Llopis,1943,1947;Montoriol,1950, 1G.R.Q.GrupdeRecercadelQuaternari.SERP.Dept.Prehisto`ria,H.Antigai Arqueologia.FacultatdeGeografiaiHisto`ria.UniversitatdeBarcelona.C/ Montalegre,6.08001,Barcelona,Spain.grq@ub.edu2DepartamentdeCie`nciesdelaTerra,UniversitatdelesIllesBalears.Ctra. Valldemossakm7,5.07122IllesBalears,Spain3S.E.O.delC.E.V.SeccioEspeleolo`gicadelOrdaldelCentreExcursionistade Vallirana.C/Major,402.08759.Vallirana,Barcelona,Spain4InstitutCie`nciesdelaTerraJaumeAlmera.CSIC.C/Llu sSoleSabar ss/n. 08028,Barcelona,SpainJ.Daura,M.Sanz,J.J.Fornos,A.Asensio,andR.Julia`KarstevolutionoftheGarrafMassif(Barcelona,Spain):dolineformation, chronologyandarchaeo-palaeontologicalarchives. JournalofCaveandKarstStudies, v.76,no.2,p.69.DOI:10.4311/2011ES0254JournalofCaveandKarstStudies, August2014 N 69

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1954;MontoriolandMuntan,1959,1961),whilemore recentstudieshavefocusedtheirattentiononotherkarst features(Freixes,1989;Custodioetal.,1993).However, thesomewhatsimplisticandschematicstudiesundertaken duringthe1950sdonotprovideanydirectdatingofthe regionsdolineformation.Morerecently,theGrupde RecercadelQuaternarioftheUniversityofBarcelona (GRQ-UB)hasinitiatedsedimentologicalandarchaeologicalworkaimedatobtainingmoreprecisedatesforthe PleistocenesedimentaryfillsintheGarrafkarst(Daura, 2008). Thefindingsfromtheseearlierstudies,combinedwith morerecentgeomorphologicalandchronologicaldata, allowustoinitiatedevelopmentofachronologicalmodel thatcanbeextrapolatedtootherkarstmassifsinthe IberianPeninsulaandtheWesternMediterraneanandto establishthechronologyofdolinesedimentationstages. Furthermore,wepresentthekarstcavitiesoftheGarraf Massifasaspecificcasestudythatcanbeusedtoexplain theapparenthiatusinPlioceneandPleistocenesedimentaryfillonkarstplateaus.GEOLOGICALSETTINGANDSTUDYAREAGARRAFMASSIFOccupyinganareaofaround500km2(Salas,1987), theGarrafMassifisahorstlocatedinthenortheastof theIberianPeninsula,30kmsouthwestofthecityof Barcelona.Themassif,composedmainlyofJurassicand Cretaceouslimestoneanddolostone(Figs.1and2),isa low-reliefmountainrangethatrisestoaheightoflessthan 600masaresultofgentletilting.Ontheeasternsideofthe massif,Palaeozoic(Benet,1990)andTriassicmaterialscan befound(Marzo,1979). Cretaceouscarbonaterocks(Andreuetal.,1987;Salas, 1987;Albichetal.,2006;Moreno,2007)dominatethe centralpartofthemassif,wheremostofthekarstlandforms havedeveloped(Borra`s,1974;Lloret,1979;Rubinat,1981; Asensio,1993;Rubinat,2004).TheCretaceousformations consistoflimestonewithdolomiticintercalationsfromthe ValanginianandBarremianstages.OutcropsofCretaceous marlsandmarlylimestone(Salas,1987)ofAptianage (Moreno,2007)arealsofoundinafewareas. Figure1.Locationofthestudyareaandoftheprincipaldolinesandtheirassociateddeposits(Source:X.EsteveandICC, InstitutCartogra`ficdeCatalunya).KARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES70 N JournalofCaveandKarstStudies, August2014

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Figure2.A:GeologicmapofthecentralpartoftheGarrafMassifwiththelocationofitsmaindolinesandtheirassociated deposits.B:DigitalelevationmodeloftheGarrafplateaushowingthelocationofmaindolinesandshafts.J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 71

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Garrafkarstificationisconcentratedinthemassifs fracturedcarbonaterocks,anditsmainkarstfeatures includedolines,shafts,andcavescontainingPleistocene sedimematryfill(Dauraetal.,2005,2010a,b).Structurally, thelimestonemassifisanantiform(Bartrinaetal.,1992), resultingfromanortheasttosouthwestcompressionofthe alpineorogeny,andaseriesoffracturestrendingpredominantlynortheasttosouthwestrunacrosstheentire complex(Guimera`,1988).Fracturescontrolalmostall thedevelopmentofshaftsinthemassif,includingthose beneaththedolineperimeters.GARRAFDOLINESANDPLATEAUGenerally,theGarrafdolinesarefoundinCretaceous rocks.TheJurassicstratainthehighplainspresentless significantkarstdevelopmentandfewerdolines.The Triassiccarbonatescomprisestratathatonlycropoutin thenortheastoftheGarrafMassifrim.OntheGarraf plateau,severalareas(Llopis,1941)ofdolinedensitycan bediscerned(Fig.1).Themostsignificantaccumulation liesbetweenCampgra`s,PuigdelOlla,andSotdelInfern, withatotalofthirteendolines,followedbyColldelOrdalLledoner,withtwelvedepressions,andPladArdenyawith fourpreserveddolines(Fig.2). AccordingtothemodelsproposedbyWalthametal. (2005,p.26andsubseq.)andFordandWilliams(2007, p.339andsubseq.),thesedolinescanbeclassifiedintotwo maintypes.Thefirstincludesdolineswithhigherconcavity indices,withnoorveryscarcesedimentarydeposits,and withwell-developedshaftsinthecenter.Thesedolines,for example,theDolinadelSotarroorthedolinesinsoutheastCampgra`s,havepreviouslybeendescribedbyMontoriol(1950,1954)andMontoriolandMuntan(1958, 1959)andwillnotbedealtwithhere. Thesecondtypeincludesdolineswithlowconcavity indicesanddepressionsthatareassociatedwithundevelopedshafts.Thesedolinesusuallypresentlargesedimentaryterra-rossatypedepositsproducedbychemical weatheringwithintheirperimeters.Mostdolinesinthe GarrafMassifareofthistype,andexamplesincludethe dolinesinlesAlzines,EsquerdadelesAlzines,Campgra `s, PladeQuerol,BassotdelArbre,PuigdelOlla,andSerra delMasamongothers(Fig.1). Thedolinesoflowconcavityandtheirassociatedshafts intheGarrafplateaupresentthebestmorphologiesfor studyingkarst-landscapeevolutionthankstotheirsedimentaryfill.Thisplateau(Fig.2-B)isanerosionalsurface that,followinganepisodeofupliftduringthelate Miocene,nowliesbetween450and550mamsl,as determinedbyLlopis(1947),andcorrespondstoaperiod duringtheMiocenewhenthesealevel(baselevelhere)was probablystableforalongperiod.Correlationbetweenthe erosionalsurfaceofthelimestonemassifandsealevels havebeenbasedonmarinedeposits(Llopis,1947)located onthenorthwestsideofthemassif.Thesedeposits correspondtotheMiddletoUpperMiocene(Machpherson,1994;dePortaandCivis,1996).Duringthattimemost ofthelittoralrangewouldhavebeensubmerged(Gallart, 1980),andwhiletheemergingpartoftheGarrafMassif wasdevelopinganerosionalsurface,thesunkenpartofthe depressionpreserveditspre-floodingreliefintact,duetoits ownfossilization.Someofthehighestpeaksinthemassif todayareevidenceoftheaforementionedprocessof erosionandlevelling.ExamplessuchasMorellaand Montau(Fig.1)havebeenconsideredmonadnocks,i.e., rockymassesthatsurvivedtheerosionandstandisolated abovethegeneralleveloftheplateau.AfterLlopis(1947), subsequentauthorshaveconsideredthiskarstplaintobe apeneplain(Montoriol,1954;MontoriolandMuntan, 1961). Todaythekarstplainisnolongercontinuous,as faultinghasseparateditintodifferentblocks.Themain faultsintheGarrafblockarearrangedintwoorthogonal systems,onealignedwiththeGarrafMassif(NE-SW)and theotherperpendiculartotherange(NW-SE)(Fig.2). Someminorfaultsalsofollowthispattern.Thisgroupof faultsisresponsibleforthesinkingofsomeblocksandthe elevationofothers.ThemostsignificantcaseistheBegues plain,whereapoljesubsequentlyevolvedanddividesthe twomainareaswithahighconcentrationofdolines. Shaftscontainingsedimentaryfillandlocatedunderthe Garrafplateauareespeciallyrelevantherebecausethey preserverecordsthatcanbeusedtounderstandthe evolutionandchronologyofthekarstplain.Theseshafts havedevelopedalongverticaltectonicfractures,predominantlyatthebaseoftheepikarstzone.Theseshafts,coneorbell-shapedandblind,reachupwardstonearthe surface,butmanyofthemhavenonaturalopeningtothe surface,andthosethatdo,oftenowetheirdevelopment tosurfaceactivity(tree-rootaction,speleologicalworks, subsoilcorrosion,etc.).Noneofthemhasaknown accessibleconnectiontoanactivehorizontalcavesystem. Thismorphology,whichiscommonintheGarraf Massif,suggestsanupwardshaftdevelopmentformedby ascendingerosion(Maucci,1960;Jennings,1985;Klimchouk,2000;Baron ,2002;Jonesetal.,2004);however, neitherhypogeneticprocessesnorup-wellingdeepwaters havebeenrecordedintheGarrafplateau.SITEDESCRIPTIONThisstudyfocusesonfoursitesintheGarrafplateau containingarchaeologicalandpalaeontologicalremains (Fig.2).Thefirsttwosites,site1,EsquerdadelesAlzines andsite2,Alzines,werediscoveredin2004bytwoofthe authors(JDandMS).Theyaresolutiondolines( dolina ) excavatedbetween2004and2009bytheGrupdeRecerca delQuaternari,andbothcontainshafts( avenc )belowthe depression.Besidesthesedolines,twoadditionalkarst formationsontheGarrafkarstplain,site3,AvencMarcel andsite4,theCave( Cova )Bonica,werediscoveredduring speleologicalworkinthetwentiethcenturyandareoftheKARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES72 N JournalofCaveandKarstStudies, August2014

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utmostimportancewhenestablishingachronological frameworkforthefirstQuaternarysedimentdeposition atthislevelofthegeneralerosionsurface.SITE1:ESQUERDADELESALZINESSite1istheDolinadeEsquerdadelesAlzines(DEA) anditssubterraneannetworkofshafts(Fig.3).Itisa solutiondolinelocatedattheconvergenceoftwofaults(d1 andd2)extendingoverapproximately2km.Thedolineis locatedatbetween530and523.6mamslandisellipticalin shape.Thedepressionis40-mlongby30-mwide,witha maximumdepthof11m.Itscentralsectionhasbeen significantlymodifiedasaconsequenceofspeleological work(GIRES,1995)carriedoutduringthe1990s. Thesite,delimitedbythedolineperimeter,includes fourshafts(AvencdelEsquerdadelesAlzines,Avenc GrandelEsquerdadelesAlzines,AvencdelsArqueo`legs andAvencPetitdelEsquerdadelesAlzines)thattogether formasubterraneannetworkthathasdevelopedalong faultsd1andd2. ArchaeologicalworkundertakeninLayerCandinthe mixedsedimentsoftheDEAbytheGRQ-UBrecovered1,067 artifacts(Fig.4).Theyarech aracterizedbyapredominanceof flakesfromthereductionofco resandartifactsofvarious morphologies.However,thec hronologicalfr ameworkand technologicaltraits identifieddonotallowthesitetobeplaced withinaparticularoneoftheU pperorMiddlePalaeolithic toolgroups,althoughtherecover yofsomeartifactscommonly foundinUpperPalaeolithicass emblagessuggestsaconnection tothisperiod(Dauraetal.,2011).SITE2:ALZINESSite2,orAlzines,correspondstoasolutiondolineand itssubterraneannetworkofshafts.Thedoline,knownas DolinaIIdelesAlzines(DA)(Fig.3),islocated290mto thenortheastofSite1.Locatedonthesameplateau, between528.2and523.6mamsl,itismoreellipticalin shapethanDEAandhasalsodevelopedonfaultd1.Its presentdaysurfacehasbeenmodifiedbyspeleological workcarriedoutinthe1960sbytheGrupExploracions Subterra`nies(Montoriol,1964)andbysubsequentwork bytheGrupdInvestigacions,RecerquesiEspeleologia SesroviresandtheSeccioEspeleolo`gicaOrdaldelCentre ExcursionistadeVallirana. Site2,delimitedbytheperimeterofDA,containsa significantsubterraneannetwork,comprisingtwomain shafts.Thefirstofthese,AvencdelesDesil ? lusions,also namedAvencNoudelesAlzines(Valdepenas,2012),was discoveredduringGRQ-UBexcavations(DauraandSanz, 2010)anddoesnotcontainanyPleistoceneremains.The second,AvencdelesAlzines,containsarchaeologicaland palaeontologicalremainsthatappeartohaveenteredthe shaftfromParetsdelsOssos(Table1). Largemammalboneshavebeenrecoveredatthebase oftheDAinamassofcementedredbreccia.Asthesites faunalremainsarestillbeingrestored,palaeontological analysesarenotyetavailable.ThereforeTable1only providesapreliminaryassignmentofbearremainsas Ursus sp.CollapsedsedimentsthroughoutParetsdelsOssos providerhinocerosremains( Stephanorhinusetruscus ) correspondingtoposteriorextremities,isolatedhyenateeth andcarpals,andafewstonetools(Daura2008).SITE3:AVENCMARCELLocatedintheTriassic(UpperMuschelkalk)dolostone andlimestone,AvencMarcel(Fig.5)isacomplexsystem of80mdeep,spindle-shapedshafts,developedasaresult ofrockfracturingandcliffregression.Itwasdiscoveredin 1982duringtheerectionofatransmissiontowerand exploredfullyforthefirsttimein1989byoneofthe authors(AA).Palaeontologicalremainswererecoveredat theoriginalcollapsedentranceduringthefirstexploration (Asensio,1993)(Table1).In2002,apalaeontological excavationbyB.Mart nez-Navarro,J.Agust ,andM. LlenasfromtheInstitutCatala`dePaleontologiaMiquel Crusafontwascarriedoutintheshaft,butnoarchaeologicalorpalaeontologicalremainswerefound(Mart nezNavarroetal.,2002).SITE4:COVABONICACovaBonicaisasmallcavechambercontainingavertical shaftnearasignificantfracture,whichisalsoincontactwith theTriassicdolostoneandlimestone.Sedimentshavebeen displacedthroughtheverticalshaftandfissuresfromanarea oftheplateauwithahighdensityofdolines.Thecavewas firstexploredduringthenineteenthcentury(Font,1899)and atvarioustimesinthetwentieth(Montoriol,1954;Borra `s, 1974)byspeleologistsandgeologicalgroups.Studieshave beenconductedonthepalaeontologicalremains(Table1), focusingonsmallmammals(Agust ,1988;BlainandBailo n, 2006;Blain,2009)andprimates(Delson,1971,1973,1974; DelsonandNicolaescu-Plopsor,1975;Crusafont-Pairo and Golpe-Posse,1984).METHODSTodeterminethechronologyofdolineformationand evolution,aseriesoffieldsurveyswereconductedatsites1, thedolinadeEsquerdadelesAlzines,and2,theDolinaIIde lesAlzines.Severaltestpitsweredugwithapowershovel, reachingamaximumdepthof6mbelowthecurrentfloor leveltowheretheMesozoichostrockcroppedout.Sections fromeachtestpitwerestratigraphicallydescribed,and sedimentsamplesweretakentostudythefill. Thesamplesandcoreswerebagged,numbered,and takentotheEarthSciencesDepartmentattheUniversity ofIllesBalears,wheretheywereopened,sectioned lengthwise,photographed,andsampledatdifferentintervalsinstratigraphicalorder,accordingtothedifferent layersobserved.Thepresenceofsedimentarystructures, suchaslaminations,andothergeneralobservationswere noted.J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 73

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Figure3.Cross-section(A)andplan(B)ofsite1,EsquerdadelesAlzines,andCross-section(C)andplan(D)ofsite2, DolinaIIdelasAlzines.KARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES74 N JournalofCaveandKarstStudies, August2014

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Archaeo-palaeontologicalremainsweresubsequently discoveredatsites1and2.AtDEA,theexcavation focusedonatwenty-square-meterareausingstandard archaeologicalmethods,includingthree-dimensionalplottingoffindsandtheirfeatures.Archaeologicalremains weremappedinsitupriortoremoval,andsedimentswere dry-sievedusingsuperimposed5and0.5mmmesh screens. Figure4.Site1,EsquerdadelesAlzines,rawmaterials(A)andstructuralcategories(B)ofstoneartifactsbasedonDaura etal.(2011). Table1.Faunalremainsrecoveredfromthebasesofdolinesandinsideofshafts,aswellasfromothercavesthathavean openingtotheGarrafplateau. PlateauSites Site2,Alzines Site3,CovaBonica Site4,AvencMarcelCollSostrell Perissodactyla Primates Carnivora UnidentifiedaStephanorhinusetruscusaMacaca cf.s ylvanusbFelidaeindet.eArtiodactyla DolichopithecusarvernensisbMegantereon sp.eCervidaesp.IaLagomorpha Lynx sp.aCaprinaeindetaProlagusmichauxidPanthera sp.aCarnivora Insectivora Homotherium sp.eUrsus sp. BeremendiafissidensdCanis sp.eLynx sp. Rodentia Vulpes sp.eEliomysintermediusdMartes sp.eCastillomyscrusafontidPerissodactyla ApodemusmystacinusdEquus sp.eApodemusjeantetidStephanorhinusetruscuseAllophaiomys sp.?dArtiodactyla TrilophomysvandeweerdidCervidaesp.IeStephanomysdonnezanidCervidaesp.IIeMimomysmedasensisdOviboviniindetaff. Soergelia sp.eSciurus sp.dCaprinaeindetaPliopetauristapliocaenicadProboscidea Blackia sp.dElephas sp.?ePteromys sp.dTestudines Testudo sp.aRodentia MimomysmedasensiseMimomys aff. tornensiseApodemus aff. mystacinuseaDaura(2008).bDelson(1971;1973;1974),CrusafontandGolpe-Posse(1974)andDelsonandPlopsor(1975).cAgust (1988).dDescribedhereforthefirsttime,followingtheidentificationcurrentlylistedinthecollectionsoftheMuseudeGeologiadeBarcelona(MGB).eAsensio(1993).J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 75

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AtDA,levelspresentingpalaeontologicalremains compriseverycompact,hardcalcareousbrecciasthatare cementedatthedolinebase.AccordingtothemethodologicalproposalsmadebyRamosetal.(2008)forsitesof thistype,excavationshouldinvolvetheremovalof sedimentindifferentblocks.Usinghammersandchisels, eachrecoveredblockwastakentothearchaeological laboratoryoftheGrupdeRecercadelQuaternariatthe SeminaridEstudisiRecerquesPrehisto`riques(laGuixera) operatedbytheCastelldefelsTownCouncil,treated,and excavatedwithapneumaticmicrohammer. Atalaterdate,aspeleologicalexpeditionwas undertakenbytheGRQandtheSeccioEspeleo`logicade lOrdaldelCentreExcursionistadeValliranatodetectany newcavities(AvencdelesDesil ? lusionsatsite2andAvenc delsArqueo`legsatsite1;Fig.3)andtoexplorecaves (CovaBonicaandAvencMarcel;Fig.2)andshaftsunder theGarrafplateau.Atopographicalandgeologicalstudy wasalsocarriedouttodeterminetheevolutionofthe dolineandkarstplainandtherelationshipbetweenthe endokarstandexokarstmorphologies. Sedimentdateswereanaly zedbyopticallystimulated luminescenceandthermoluminescenceattheRadiochemistryandDatingLaboratoryoftheUniversidad Auto nomadeMadridusingtheadditivedosemethod andstandardprocedures(F leming,1970;Zimmerman, 1971;Aitken,1985;NambiandAitken,1986;Arribaset al.,1990)andby14CattheBetaAnalyticLaboratory, USA.Todeterminetheageo fmaterialsbeyondthe radiocarbonandtheOSLdat inglimits,thepalaeontoFigure5.Cross-section(A)andplan(B)ofsite3,AvencMarcel.KARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES76 N JournalofCaveandKarstStudies, August2014

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logicalrecordsfromAvencMarcel,DEA,CollSostrell, andCovaBonicawerealsoincludedinthestudy.The latterarebasedonprevious studies(Delson,1971,1973, 1974;DelsonandNicolaescu -Plopsor,1975;CrusafontPairo andGolpe-Posse,1984;Agust ,1988;Asensio, 1993;Daura,2008).RESULTSSTRATIGRAPHICSETTINGSedimentaryfillfromsite1,theDolinadelEsquerda delesAlzines,isthemostsignificantforstratigraphic reconstruction.Currentfieldworkfocusedontheexcavationoffivetestpits(Fig.3),twoinsectorsI,IIandIII,and oneintheAvencdelsArqueo`legsarea(Fig.6).Theresults formasingle,unifiedsequence4.5minlength,butthe correlationsbetweenthepitsaremerelytentative,giventhe discontinuitiesbetweenthem. Thelowestunitcorrespondstohighlycemented brecciaandconglomerates(layersE,G1andI),witha reddish,siltymatrixformedbydecalcification.The coarsestfractionishighlyweatheredandroundedby dissolutionandtypicallycontainsspariticcalcitefragments,consistingoflargedogtoothsparcrystalsof unusuallyhighpurityandcrystallinity.LayerH,above, iscomposedofnon-cemented,compactlutites,separated fromtheupperpartofthesequencebysub-angular boulders. Atthetopofthesequence,layersC,C2,D2,B1,D1,B, andD,infillingsaretypicallycharacterizedbydepositsup to2-mthick(Table2),correspondingtolutitesthatare occasionallyburned,withalmostnon-existenthostrock components,indicativeofthehighdegreeofsolution activityinthedolineformationprocess.Infact,silt-size grainsofquartzarepredominantinthesamplesanalysed (84.2to90.6%)followedbyfeldspars(4.9to10.6%),while hostrockfragmentsareveryscarce(0to5.2%).Illiteis presentinminoramounts(0to4.5%). Theuppermostlayer,layer1,contains10-to20-cm thicksuperficial,roundedandsub-angularlimestonegravel withoutlutitesandmayrepresentthecurrentsoillayer. ThestratigraphyissummarizedinTable2andillustrated inFigure6. Site2wasstudiedviathirteentestpits(Fig.3).The idealizedschematicprofilepresentedinFigure6shows similaritiestoDEA.Moreover,threebroadepisodesof sedimentationcanbeidentified.TheunderlyinglayerEis composedofcementedbrecciaandconglomerateswith spariticcalcitefragmentsadheredtothebedrock,representingtheearliestdepositionfoundintestpitS-11,which collapsedalongPoudelaRevoluciointothesubterranean network.Thisunitcontainsbonesoflargemammalsfrom theLowerPleistocenethatapparentlyfellfromtheParet delsOssos.Thesecondpackage,layerD,iscomposed mainlyofcementedredclaywithironoxidesandwithouta coarsefraction.Ontop,subsequentlayersCandBare dominatedbyalutiticmatrixwithoutgravelsandcontaina fewsmallpiecesofrubifiedsedimentandsparseHolocene faunalremains.Theuppermost10cmcomprisethecurrent soillayerandcontainsub-angularlimestonegravels. Othersites,becausetheydonotpresentacomplete stratigraphicsequence,arelesssignificanttoourunderstandingofthesedimentaryfillontheGarrafplateau. However,theseunitsconstitutetheoldeststratigraphiclevels exposed.Sedimentaryfillsthatcontainfossilremainshave beenidentifiedinthesubaerialexposureofcementedbreccia inCollSostrell(layerK);inveryhighlycementedbreccia adheredtoexternalcavewalls(layerK)andinfiltratedfrom theGarrafkarstplain(layerJ)atsite4,CovaBonica;andin cementedredbreccia(layerK)andcompactedlutiteswith ironoxides(layerJ)atSite3,theAvencMarcel. Sedimentcollapsesintotheunderlyingcavitiesare commonintheGarrafsystem.Stratigraphiccorrelations betweentheexternalandinternalsedimentsaretentative, giventhemajordiscontinuities,includingtheBigGalleryin site1andtheParetsdelOssosinsite2(Fig.3).DATINGOurdatingofthedolineandshaftfillundertheGarraf plateauindicatesachronologyrangingbetweenthe PlioceneandHolocene.Sevenradiometricdateswere obtainedfromtestpits,two14Cdatesoncharcoaland boneandfiveopticallystimulatedluminescencedateson sediment.These,alongwithonethermoluminescesence date,arepresentedinTable3anddiscussedbelow.The datingofthedolinedepositsishinderedbytaphonomic andgeologicalprocesses,theabsenceorscarcityoforganic material,andwildfiredisturbanceofsediments. Todeterminetheageofthefirstsedimentaccumulation intheplateau,thechronologicalrangeofthemammalian speciespresentinCovaBonica(site4)andAvencMarcel (site3)isclearlydiagnosticofPlioceneandLowerPleistocenecontexts(Fig.7).AvencMarcel(Fig.5)developed onthisancientsurface,anditssedimentaryfilloriginated fromtheancientplateaubase,whichcontainsLower Pleistocenefaunalremains.Thedifferentspeciesofthe genus Mimomys foundheresuggestachronologybetween MN17andMQ1forthissite(Asensio,1993).Similarly,the CovaBonicafilloriginatingfromthekarstplateauincludes arichnessofsmallvertebratespeciesandCercopithecidae remains.Theremainsrecoveredpresentatwo-stage chronology,MN15andMN17/MQ1(Delson,1971,1973, 1974;DelsonandNicolaescu-Plopsor,1975;CrusafontPairoandGolpe-Posse,1984;Agust ,1988;Blain,2009). CollSostrellisakarstfissureassociatedwiththeplateau.Its conglomerateandbrecciadepositscontainfaunalremains fromtheLowerPleistocene(Daura,2008).Finally,rhinocerosremainsassignedpreliminarilyto Stephanorhinus etruscus fromPoudelaRevolucioatsite2datethesite fromMN16tothebeginningoftheMiddlePleistocene (Guerin,1980;vanderMade,2010;vanderMadeand Grube,2010).J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 77

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Figure6.Site1,EsquerdadelesAlzines:A,stratigraphyofindividuallocations;B,assembledschematicprofilewithsuggested chronology.Site2,Alzines:C,stratigraphictestpitsdug;D,assembledschematicprofilewithsuggestedchronology.Site3,Avenc Marcel:E,shaftgeologicalcross-sectionoftheplateau;F,proposedevolutionarymodelfortheformationofthesiteduringPliocene andLowerPleistocenestages.KARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES78 N JournalofCaveandKarstStudies, August2014

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Table2.MainlayersidentifiedinthedolinesandshaftsontheGarrafplateau. Stage PlateauSitesaDescription Site1, DEAbSite2, DAcColl Sostrell Site3, Bonica Site4,Avenc Marcel 4 NNN Ongoingerosionprocess. CurrentSoil1A Roundedandsub-angularMesozoiclimestonegravelwithoutlutites.Current dolinesoilisnotadded.Lowercontactiscleanandclear.Thickness: 510cm. 3 B Compactlutitesandfewgravels.Thickness:About50cm.Holocenefaunal remains. B Compactlutitesandfewgravels.Thickness:About50cm. B1 Lutiteswithfewpebblesandcombustionsignals.BronzeAgepottery. DD-top Notverycompactedlutiteswithfewgravels.Thickness:50cm. D1 Reddish,yellow-reddishandyellowburntlutiteswithlargecharcoal accumulation.Gravelisalteredandthecolourvariesfromwhitishtolight greyorblueduetocombustion.Thickness:1m. D2 Mildlyburntlutitesandsubroundedpebbles.Thickness:50cm. 2 C2 Non-cementedlutiteswithoutgravels.LocatedinDEA-SectorIII,is equivalenttolayerCbutitisnotpossibletodemonstratecomplete stratigraphiccontinuityacrossAvencGran.SteepSW-NEslopewithlarge blocksatthebase.MiddlePalaeolithicartifacts.Soilaffectedbywildfire. C Non-cementedlutiteswithoutgravels.LocatedinDEA-SectorIandratherthin (ca.10cm),itcontainsMiddlePalaeolithicstonetools.Soilaffectedbywildfire. 1b H Compactedlutitesandsubroundedgravels.UppercontactwithlayerCand lowercontactwithlayerF.SlopessteeplytowardstheentranceofAvenc GrandelesAlzines.Thickness:1m. 1a J JReddishcompactedlutiteswithironoxidesinfiltredthroughkarsticfissures. FaunalremainsinCovaBonicaandAvencMarcelhavebeendocumented. EEKK KReddishcementedbreccia.Coarsefractionispredominant(30%)withsubangularmorphologiesanddog-toothspar.PlioceneandPlistocenefaunal remainsinDEA,CollSostrell,CovaBonicaandAvencMarcel. G1 CementedbrecciarecordedinAvencdelsArqueo`legs.Thickness:7m.Present attheentranceandpartlycollapsedinsidethecavity.Faunalremainsare removedinsideshaft. 1a II Highlycementedbrecciawithfewpebbles.DEA-SectorIIIhaslateralcontact withsedimentsoflayersDandD2.FaunalremainsarerecoveredinDEA andDA.Thickness:ca.30cmaLettersymbolsrefertoinfillinglayers.bDolinadelEsquerdadelesAlzines.cDolinaIIdelesAlzines.J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 79

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Table3.14Candoptically-stimulated-luminescenceagesforlayersB,C2,D,D1andD2fromtheDolinadelEsquerdadelesAlzinesandlayerEfromtheDolinaII delesAlzines. SiteaLayerMaterialLab #13C/12C ratio Archeolological dose(Gy) Annual dose (mGy/ Year) U (ppm) Th (ppm) K20 (%) H20 (%) Measured14CageBP14CageBP (1 s)Yearsago(1 s) DEABAntler ( Cervus elaphus ) Beta216702 2 20.5 % 1280 6 501212 6 56b/dDEACBurntflintMADN5989BIN 171.587.599.962.880.011.2 22744 6 1605 DEAC2topSediment (2m ) MAD-4959 62.723.550.1712.551.7413.59 18200 6 1689eDEADtopCharcoal ( Quercus ilex/ coccifera ) Beta-210946 2 26.8 % 1810 6 401754 6 50c/dDEAD1Sediment (2m ) MAD-4566 10.583.771.1712.70.63.4 3241 6 210eDEAD2Sediment (2m ) MAD5693rBIN 29.166.544.2911.581.5411.34 4458 6 366eDEAD2baseSediment (2m ) MAD5291SDA 25.353.450.0118.851.8413.55 7347 6 441eDAESediment: Neocarbonates (2m ) MAD-5807 488.715.013.816.5611.8 97526 6 6542eaDEA:Site1-DolinaEsquerdadelesAlzines;DA:Site2-DolinaIIdelesAlzines.bcalAD738 6 56.ccalAD196 6 50.dCalPalprogram(Weningeretal.,2008)andtheHuluagemodel(WeningerandJoris,2008)wasusedtoconvertconventional14Cagesintothecalendartimescale.eSedimentdateswereobtainedbyOSLattheRadiochemistryandDatingLaboratoryoftheUniversidadAutonomadeMadrid,usingtheadditivedosemethodandstandardprocedures(Fleming,1970;Zimmerman,1971; Aitken,1985;NambiandAitken,1986;Arribasetal.,1990).KARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES80 N JournalofCaveandKarstStudies, August2014

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Theoldestabsolutedateforthesedimentsatthebaseofthe dolineswasfoundinlayerEoftheDolinaIIdelesAlzines(site 2).Amulti-grainOSLaveragedateof97526 6 6542BPwas obtained.However,theresultshouldbetreatedwithsome cautiongiven,themethodused (notsingle-g rain)andthe taphonomyofthesediments. MorerecentlayershavebeenDatedusingother techniques.LayersCandC2atthetheDolinade lEsquerdadelesAlzines(site1)containlithicartifacts thatarechronologicallydatedtotheMiddleorUpper Palaeolithictechno-complex(Dauraetal.,2011).A thermoluminescencedateforburntflintfromlayerCgives Figure7.Verticaldistributionofthemaintaxaidentifiedatsite3,AvencMarcel(Asensio,1993;Daura2008),andsite4, CovaBonica.SmallmammalsfromCovaBonicaaredescribedhereforthefirsttime,followingtheidentificationcurrently listedinthecollectionsoftheMuseudeGeologiadeBarcelonaandAgust ,(1988)whereastheCercopithecidaehavebeen previouslydescribed(Delson,1971,1973,1974;DelsonandNicolaescu-Plopsor,1975;Crusafont-PairoandGolpe-Posse, 1984).Ageinmillionsofyears(Ma),MammalNeogeneUnits(MN)(Agust ,2001)andoxygenisotopecurve(Shackletonet al.,1995)ontheleft.Theverticallineshighlightthechronologicalrangesbasedoncurrentliteratureforlargemammals (PickfordandMorales,1994;vanderMade,2005;Mart nez-Navarroetal.,2010;RookandMart nez-Navarro,2010)and micromammals(Minwer-Barakatetal.,2004;deMarfa`,2009;Furio,2007;Garc a-Alixetal.,2007;Minwer-Barakatetal., 2008).Grayrectangleindicatesthechronostratigraphicrangeproposedforthesite.J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 81

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anageof22744 6 1605BP,andanOSLdateis18200 6 1689 BPforsedimentfromthetopoflayerC2. TheDEAcontinuedtobefilledthroughouttheUpper PleistoceneandHolocene,from 22.7kauntilthe present.However,severalwildfiresoccurredduringthis time,affectingdifferentlayersandalteringthesediments. Asaresult,reliabledirectOSLdatesarenoteasily obtained,asfirescanmakesedimentappearyounger, especiallybecauseofburnttreestumps.Nonetheless,these dates(Table3)areindicativeofan antequem ageforthe fillthatisolderthanthedatesobtainedbothbyOSL(7ka to3ka)andradiocarbondating(calAD738 6 56and 1966 50).ThepresenceofBronzeandIronAgepotteryin LayersB1andD1provideanapproximatechronologyfor theaccumulationofthesedepositsinaccordancewith OSLresults.AMODELOFLANDSCAPEDEVELOPMENT ANDKARSTIFICATIONAttemptsweremadetocorrelatethevariousdolines andshaftsonthebasisoftheirlayers,stratigraphic position,andchronology.Thiscorrelationremains tentativebecauseofthediscontinuitiesbetweenthe variousdepositsandtheshortcomingspresentedbyeach deposit.Ourstratigraphicstudyidentifiesfivestagesor episodesresultingintheformationofasingleunified sequence(Fig.8).Itsbackboneisformedbythesediments preservedintheDolinadelEsquerdadelesAlzinesand theDolinaIIdelesAlzines,whiletheCovaBonica,Avenc Marcel,andCollSostrellsitesrepresentsignificant depositsfordeterminingtheancientstageoftheGarraf plateau. Stage0correspondstotheformationoftheGarraf plateauduringtheUpperMiocenethatresultedinthe horizontalmodellingofthelandscape(Llopis,1943,1947; Montoriol,1950,1954;MontoriolandMuntan,1959, 1961).Assuch,theconditionsrequiredtotriggerthedoline formationprocesswerepresentduringthisstage,despite thefactthatnosedimenthadyetaccumulated.Theresult ofthisstageisthepresenceofsomemonadnock-shaped standingremains. Stages1aand1bcorrespondtotheoldestsediment accumulationsintheplateau,datingfrombetweenthe PlioceneandLowerPleistocene.Theprocessofdolineand shaftfillisconfirmedbythepresenceofthemostancient depositslocatedatthebaseofthedolinesandthe sedimentscollapsedintotheshafts.Intheearlierpartof thestage(1a),thefirstlayer(G1)ofsedimentsintheDEA liesatthebaseoftheAvencdelsArqueo`legsareaandhas partiallycollapsedintothesubterraneannetwork.The lowercontactisirregularandoverliestheCretaceous limestone.Inthelatterpartofthestage(1b),layersarealso foundintheAvenc(shaft)delEsquerdadelesAlzines (layerI)andinSectorIII-Watthebaseofthedolines center(layerE),aswellasintheDA,whichcontains mammalbones.Inthiscase,depositsarelocatednearPou delaRevolucio(Fig.3),andpartofthefillcollapsedinto theshaftthroughtheParetsdelsOssos. Infillingofsimilarmaterialthatprobablyoriginated fromacollapsedsurfacedepositisfoundadheredtothe wallsinViadelOssosoftheAvencMarcelshaft.The fillcontainsmammalbonesandhasbeenscattered throughoutthesubterraneannetworkfromwhich palaeontologicalremainswererecovered.Finally,the CovaBonicafilloriginatesfromsedimentinfiltration fromtheplateauthroughacombinationofkarst fissuresandkarren. Stages2and3correspondtothesecondlayersof depositstoaccumulate.Aftertheoldestsediment,thoseof stage1(layersE,G1,andI)developed,partsofthese sedimentswerealteredbytheevolutionaryprocess undergonebythedepression.Insomeareas,fillfromstage 1wascementedandpreservedattherimofthedolineor insideitsshafts,aswellasinlayerEoftheDEA(Fig.3 and6)andthebaseoftheDA.Inothercases,shafts receivedpartofthecollapsedsediment,asisthecaseinthe DA(Fig.6). Stage1sedimentsfoundatthedolinebasearealways cappedbystage2layers.Themostsignificantexamplesof stage2arelayersCandC2intheDEA.Theycontain UpperPalaeolithicartifactssuchasburins,denticulates, andscrapers,amongothers. Thefinalsedimentlayer(stage3)ischaracterizedby highlycompactlutiteswithfewpebblesandgravels.These havebeenalteredbywildfirecombustioninsectorsI,II, andIII-SWintheDEA,adistortioninthisareathatis onlylimitedbyagentleslope.LayersB,B1,andD1ofthis unitcontainprehistoricpottery.Onelayer,composed mainlyofroundedsub-angularlimestonegravels,correspondstothecurrentdolinesoil. Stage4correspondstotheongoingerosionprocessin whichthedolinesarebeingcontinuouslytransformedby agriculturalorspeleologicalactivities.DISCUSSIONANDCONCLUSIONSTheresultsobtainedfromtheGarrafcasestudy coincidewithproposalsmadeforotherEuropeankarst massifs(Piccinietal.,2003;Ufrecht,2008)thattheprimary karstdevelopmentappearstohavetakenplaceattheend oftheTertiaryandthebeginningoftheLowerPleistocene. ThemainkarstfeaturesaroundthewesternMediterranean wereprobablyinheritedfromtheendoftheMiocene (Messinianperiod),whenthesealeveldroppeddramaticallyduetotheclosingoftheAtlanticconnectionandthe subsequentdesiccationoftheMediterraneansea(Krijgsmanetal.,1999).Thedepthsandaltitudesofseveral conduitsandgalleriesinkarstareasaroundthewestern MediterraneanthatdonotmatchknownQuaternary glacio-eustaticsealevelsarelinkedtotheerosivefeatures observedduringtheMioceneandthePliocene(AudraetKARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES82 N JournalofCaveandKarstStudies, August2014

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al.,2004).Thesekarstpaleo-galleriesevolvedafterthe subsequentPliocenesealevelriseandtransgression, floodingtheformerkarstsystem.Lateroscillationsofthe watertable,forcedbytheQuaternaryeustaticsea-level changes,favoredtheevolutionofthekarstdepressionsand thesedimentaryinfilloftheformerkarstsystemabovethe watertable. Previousresearchhasarguedinfavorofageneral MioceneagefortheGarrafplateau(Llopis,1943,1947; Montoriol,1950,1954;MontoriolandMuntan,1959, 1961).Basedonthesedimentspreserved,wecanconfirm thatthedolineandshaftinfilloriginatingfromtheGarraf plateaucanbedatedtothebeginningofthePliocene(stage 1)and,hence,earlierkarstificationprocessesmusthave takenplaceduringtheMiocene(stage0),whenconditions weresuitedtotriggeringthedoline-formationprocess.The Garrafdeepkarstsystemwasprobablyformedduringthe Messinianperiod( 2 5.96to 2 5.32Ma)andwasreflooded duringthePliocenewhentheMediterraneansalinitycrisis terminated(Audraetal.,2004).Infact,thedischargepoint forwaterdrainingoutoftheGarrafmassifisLaFalconera shaft,atleast81mbelowthepresentsealevel,suggesting thatkarstificationoccurredduringthisperiodoflowersea level(Montoriol,1966;Custodio,1975;Cardona,1990). Figure8.CharacteristicstagesofGarrafdolineevolution,accordingonhypothesisbaseddataobtainedatthedifferent dolinesandshaftsstudiedandappliedtovariouspartsofsite1,DolinaEsquerdadelesAlzines,andsite3,AvencMarcel.The labelsonthesedimentlevelsarethoseinFigure6andthetext.J.DAURA,M.SANZ,J.J.FORNO S,A.ASENSIO,ANDR.JULIA `JournalofCaveandKarstStudies, August2014 N 83

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Palaeopalynologicalstudies(SucandCravatte,1982;Suc etal.,1995;Fauquetteetal.,1999)emphasizeasubtropical climateforthenorthwesternMediterraneanduringthe Pliocene,andtheseenvironmentalconditionscouldhave acceleratedkarstification. Palaeontologicalremainsfromstage1indicateanage ofMN15andMN17(Delson,1971,1973,1974;Delson andNicolaescu-Plopsor,1975;Crusafont-PairoandGolpePosse,1984;Agust ,1988;Asensio,1993;Daura,2008). Today,sedimentsfromthisstagearelocatedwithinthe deepestinnerpartoftheGarrafkarstandhavebeen erodedfromtheiroriginalpositionintheGarrafplateau. Inthissense,theCanalNegre-1(Guillen,2010)assemblage couldrepresenttheearliersedimentaccumulation,from stage1totheUpperMiocene(MN13). ThespeciesrichnessatthepaleontologicalsitesCova Bonica,AvencMarcel,andCanalNegre-1andtheresults ofthepollenanalyses(SucandCravatte,1982;Sucetal., 1995;Fauquetteetal.,1999)pointtoecologicaloptimain theGarrafplateauduringthePliocene,adifferentscenario fromthecurrenthabitabilityofthisarea.Thus,dolinesand collapsedcavitiesundertheplateauareausefulrecordfor Quaternarypaleoenvironmentalstudies. DuringtheMiddle-UpperPleistocene(stage2),significantsedimentdepositsaccumulatedinthedolinesand shaftsoftheGarrafkarstplateau.Infillofthesame chronologyiscommonbeyondtheplateauinotherkarst landforms,includingthecavesofCovadelGegant(Daura etal.,2010a),CovadelRinoceront(Dauraetal.,2005), andCovadelcollVerdaguer(Dauraetal.,2010b). However,inthekarstplateausedimenthascollapsed downintothesubterraneannetwork,suggestingthat archaeologicalandpaleontologicalrecordsmustbesought inthedeeperlevelsofthekarstsystem.Thisconclusionis supportedbyalargenumberofsitesintheIberian Peninsula,includingtheAlmondasystemintheMacic o CalcarioEstremenho(Zilhaoetal.,1993;Hoffmannetal., 2013),ElSidroninSurcoOviedo-Infiesto(Rosasetal., 2006),theSierradeAtapuerca(Rosasetal.,2001),and CuevadelA ngelintheSierradeAraceli(BarrosoRu zet al.,2011),amongothers. AmarkedgapshouldbenotedbetweenthePliocene LowerPleistoceneandtheMiddle-UpperPleistocene,with sedimentarydepositsfromthisperiodbeingunknowninthe GarrafplateauandinothercavesandshaftsoftheGarraf Massif.Therearethreepossibleexplanationsforthis:the sedimentscollapsedintotheinnerkarstsystemthrough verticalshafts,nosedimentaryprocessesoccurred,orthe phasehasyettobedocumented.However,wespeculatethat thesedimentsaccumulatedattheBigGalleryofAvencGran delesAlzinesinsite1andatthebaseofRampaSedimentsin site2couldbedatedtothisperiod. TheGarrafMassifandthefivestagesidentifiedherein itsevolutionpointtotheimportanceofkarstdevelopment andsedimentdisplacementforarchaeologicalandpaleontologicalstudies,especiallyforthedetectionandevaluation oftheviabilityofakarstplateauasanareaofhominid presence(Bourguignon,2004;Mosqueraetal.,2007) becauseofitsfaunalorwaterresources. Ourresults,basedongeomorphologicargumentsand sedimentdating,aremoreaccurate,webelieve,thanthe previouslyproposedestimatesoftheageofGarrafdoline andkarstlandforms(Llopis,1943,1947;Montoriol,1950, 1954;MontoriolandMuntan,1959,1961).Wehavebeen abletoconstructamodelofthegeomorphicevolutionof theGarrafMassifdolinesthatresultedinasingleand unifiedsequence(Fig.8).ThissequencespansthePliocene totheHoloceneandconstitutesthefirstchronological proposalfortheGarrafkarst.Ourstudyhasprovidednew examplesandimportantdataregardingthespeleogenetic processesandthechronologyofwesternMediterranean dolines,shaftsandkarstplateaus.ACKNOWLEDGEMENTSThispaperformspartoftworesearchprojects, Humans, Carn vorsimedinaturalalGarraf and ElPlistoce`Superiori lHoloce`aCatalunya ,supportedby2014SGR-108and 2009ACOM00090(GeneralitatdeCatalunya),HAR201126193,CGL2010-18616andCGL2009-07392projects(MICINN-FEDER).FieldworkwassupportedbyServei dArqueologiaiPaleontologia(GeneralitatdeCatalunya) andAjuntamentsdeValliranaiBegues.J.Daurahasbeen supportedbyapostdoctoralgrant(JuandelaCierva Subprogram,JCI-2011-09543)andM.Sanzbyapredoctoralgrant(FI)fromtheComissionatperaUniversitatsi RecercadelDepartamentdInnovacio,Universitatsi EmpresadelaGeneralitatdeCatalunya,andtheFons SocialEuropeu.WeareespeciallygratefultoH.Martins (UniversityofBristol),LianaM.Boop(UniversityofSouth Florida),andVanceT.Holiday(UniversityofArizona)for commentsonthemanuscript,theownersofthedolines(Can PaudelaFigueraestate)fortheircollaboration,andtothe GrupdEspeleologiaBadalonaandtheSeccioEspeleolo`gica OrdaldelCentreExcursionistadeValliranaforthe speleologicalwork.Theauthorswouldliketoextendtheir thankstoX.EsteveandM.Yuberoforthedigitizationof Figures1and2b,respectively,andtoeditorialstafffortheir constructivecomments.REFERENCESAgust ,J.,1988,Elscordats[excepteelsprimatshom nids], in Gallem ,J., cord.,RegistreFo`ssil.Histo`riaNaturaldelsPa sosCatalans: Barcelona,FundacioEnciclope`diaCatalana,v.15,p.389. Agust ,J.,2001,DefiningMN-unitsandmagnetobiostratigraphic correlationoftheSpanishsections, in Latal,C.,andPillar,W.E., eds.,EnvironmentalandEcosystemDynamicsoftheEurasian Neogene(EEDEN);Stratigraphy&PaleogeographyWorkshop March152001,Graz,Austria:Graz,BerichtedesInstitutesfu r GeologieundPalaontologiederKarl-Franzens-UniversitatGraz, no.4,p.23. Agust ,J.,Cabrera,L.,Garces,M.,Krijgsman,W.,Oms,O.,andPare s, J.M.,2001,AcalibratedmammalscalefortheNeogeneofWesternKARSTEVOLUTIONOFTHEGARRAFMASSIF(BARCELONA,SPAIN):DOLINEFORMATION,CHRONOLOGYANDARCHAEO-PALAEONTOLOGICAL ARCHIVES84 N JournalofCaveandKarstStudies, August2014

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BIOGENICITYANDCHARACTERIZATIONOFMOONMILK INTHEGROTTANERA(MAJELLANATIONALPARK, ABRUZZI,CENTRALITALY)PAOLACACCHIO1,GIANLUCAFERRINI2,CLAUDIAERCOLE1,MADDALENADELGALLO1,ANDALDOLEPIDI1Abstract: Observationsandhypothesesonthepossibleinfluenceofunidentified calcifyingbacteriaonmoonmilkspeleothemformationintheGrottaNeraarereported forthefirsttime.TheMajellaMassifhostsacomplexkarstsystemofseveralcaves;the accessibleGrottaNeraisthemostinterestingone.Despiteitsname,thecaveis characterizedbyparticularlyabundantivory-whitedepositsofmoonmilk.Twosamples ofmoonmilkwereanalyzedtodeterminethegeochemistry,fabric,depositionalsetting, andextentofbiogenicity.Forthis,wecombinedgeochemical,scanningelectron microscopic,microbiological,and invitro precipitationstudies.X-raydiffractionofthe moonmilkdepositsgaveclearevidenceforthepresenceofcalcite.Scanningelectron microscopyshowedthatmoonmilkintheGrottaNeraconsistsofanetworkofcalcite fibersorientedinalldirections,resemblingafeltedmat.Thecultivationonspecific mediumofmoonmilkanddrip-watersamplesshowedthepresenceoffungi, actinomycetes,andotherbacteria,butthedominantcultivablemicroorganismswere bacteria,whichproducedsignificantcrystallization.ExaminationofGram-stained smearstakenfromthefifteendifferentcolonytypesshowedthatthemajority(66.7%)of thebacterialisolateswereGram-negative.Singlesmallrodsandrodchainswerethemost commonbacteriaisolatedfromtheGrottaNera.Noneofthemoldsisolatedfromthe GrottaNerasampleswereabletoprecipitateCaCO3crystals,suggestingamajor bacterialcontributiontomoonmilkdepositioninthecave.Bacteriawerecapableof precipitatingCaCO3onB-4solidmediumat15(cavetemperature),22,and32 u C.The calcifyingbacteriaisolatedfromtheGrottaNerashowedagreatercapabilityto solubilizeCaCO3thanthoseassociatedwithconsolidatedstalactitessampledfrom previouslystudiedcaves.Theelectronmicroscopyandmicrobiologicalevidences, togetherwiththegeochemistryandenvironmentaldata,allowedustopostulatethe biogenicnatureofthemoonmilkintheGrottaNeraCave.INTRODUCTIONMoonmilkisawhitishmaterialdescribedassoftand pasty,resemblingcream-cheese,whenwet,andcrumbly andpowdery,likechalk,whendry(Fisher,1988;Hilland Forti,1997;NorthupandLavoie,2001;Canaverasetal., 2006).Onaging,themoonmilkbecomesdryandmore rigidandcompact,buttheexternalmorphologystays unchanged(Gradzinskyetal.,1997).Itisamicrocrystallineaggregate,typicallyfoundontheceilings,floors,and wallsofcarbonatecavesand onspeleothems.Moonmilk depositshavebeenreportedinnumerouscavesworldwide,inavarietyofdifferentcountriesandinclimates fromalpinetotropical(OnacandGhergari,1993;Hill andForti,1997;Chirienco,2002;Lacelleetal.,2004; FordandWilliams,2007;BlythandFrisia,2008;Richter etal.,2008;Curryetal.,2009).Frequently,moonmilkis theonlyspeleothempresentincold,high-altitudeor high-latitudecaves,wherem assivecalcitespeleothems suchasstalagmitesdonotform(OnacandGhergari, 1993;HillandForti,1997;Borsatoetal.,2000;Lacelleet al.,2004).Moonmilkiscomposedofwaterandsmall crystalsofmineralssuchasCaCO3polymorphs(calcite, aragonite,vaterite),monohydrocalcite(CaCO3? H2O),magnesite(MgCO3),hydromagnesite(Mg5(OH)2(CO3)4? 4H2O), dolomite(CaMg(CO3)2),nesquehonite(MgCO3? 3H2O), huntite(Mg3Ca(CO3)4),andgypsum(CaSO4? 2H2O)(Onac andGhergari,1993;HillandForti,1997;Northupand Lavoie,2001;Lacelleetal.,2004).Thisarrayofminerals relatestovarioushostlithologies(Gradzinskietal.,1997) andwaterchemistriesassociatedwitheachcave. About95%ofmoonmilkdepositsarecarbonatic,and itsmostcommontypeiscalcitemoonmilkwithgreater than90%calciteinitssolidphase(Fisher,1992,1993).The watercontentofactivemoonmilkvariesconsiderably. Underitshydratedphase,itswatercontentrangesfrom40 to70%byweight(HillandForti,1997;Lacelleetal., 2004).AccordingtoIstvanetal.(1995),thewaterretaining 1DepartmentofLife,Health&EnvironmentalSciences,MicrobiologyLaboratory, UniversityofLAquila,Coppito,67010LAquila,Italypaolacacchio@yahoo.it2DepartmentofLife,Health&EnvironmentalSciences,GeologyLaboratory, UniversityofLAquila,Coppito,67010LAquila,ItalyP.Cacchio,G.Ferrini,C.Ercole,M.DelGallo,andA.LepidiBiogenicityandcharacterizationofmoonmilkintheGrottaNera (MajellaNationalPark,Abruzzi,centralItaly). JournalofCaveandKarstStudies, v.76,no.2,p.88.DOI:10.4311/2012MB027588 N JournalofCaveandKarstStudies, August2014

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capacityofactivemoonmilkcanbeattributedtoitsporous networkofcalcitefibers. Themorphologyofmoonmilkisasvariedasits composition(Curryetal.,2009).Microscopically,the mostdiagnosticcalciticmoonmilkfeatureisneedle-shaped orfibrouscrystalmorphologythatappearsasunstructured aggregatesofmicrometer-tonanometer-sizedcrystalswith noapparentpreferredorientation(OnacandGhergari, 1993;Gradzinskietal.,1997;HillandForti,1997;Borsato etal.,2000;Canaverasetal.,2006).Moonmilkthatis biologicallyactivealsocontainssignificantamountsof cells,filaments,andapparentbiofilms(Gradzinskietal., 1997;Bostonetal.,2001;Curryetal.,2009).Theoriginof moonmilkhaslongbeendiscussed,andmanyhypotheses foritsgenesishavebeenproposedsinceitsfirstdescription wasmadebyNicolasLanghin1708(Bernasconi,1976). Bioticandabioticprocesseshavebeenpostulated(Gradzinskyetal.,1997;Borsatoetal.,2000;Forti,2001; NorthupandLavoie,2001).Someevidencesofmicrobial activitiesrelatedwithcalcitemoonmilkdepositshavebeen reported(Bertouille,1972;Jamesetal.,1982;Callotetal., 1985;Gradzinskietal.,1997;BartonandNorthup,2007; Braissantetal.,2012).Moonmilkisthecavedepositmost commonlyassociatedwithbiogeniccalciteprecipitation, eitherbydirectprecipitationbymicroorganisms(fungi, algae,bacteria,andarchaea)(Castanieretal.,1999;Barton andNorthup,2007;Ercoleetal.,2012)orbypassive precipitationinwhichmicroorganismsthemselvesactas nucleationsurfacesonwhichmineralsprecipitate(Jones andKahle,1993;BlythandFrisia,2008).Thereisan ongoingdebateabouttheextentofthemicrobialroleinthe formationofmoonmilk.Someresearchershaveclaimedto haveidentifiedmicrobialstructuresassociatedwiththe crystalmatrix,includingcalcifiedcellsandfilaments (Gradzinskyetal.,1997;Canaverasetal.,2006),whereas others,citingthelackofunequivocalevidenceofbioprecipitation,haveattributedmoonmilkformationtopredominantlyinorganicprocesses(Bernasconi,1961;Melon andBourguignon,1962;Ge`ze,1976;OnacandGhergari, 1993;HillandForti,1997;MooreandSullivan,1997; Borsatoetal.,2000).Inadditiontobeingprecipitated inorganicallyorbymicrobes,moonmilkcanalsobe formedbyspeleothemweathering(Sweeting,1973;Hill andForti,1997).Thisprocessisalsothoughttobe biologicallymediated;forexample,itcanresultfrom biochemicalcorrosionofbedrockbyorganicacids producedbymicrorganisms(CaumartinandRenault, 1958).Morerecently,moonmilkhasbeenattributedtoa combinationofbothphysico-chemicalandbiogenic processes(OnacandGhergari,1993;Basillais,1997). Todeterminetheroleofcalcifyingbacteriaas geologicalagentsinthegenesisofmoonmilkspeleothems fromtheGrottaNeraintheAbruzziRegionofItaly,a combinationofstudieshavebeencarriedout:microscopic, microbiological, invitro precipitation,andgeochemical investigations.BecauseDNAanalysisofbacteriaprovides noinformationonthemetabolism,thephysiology,the ecology,thebiochemistry,orthegeomicrobiologyofa strain,laboratory-basedcultureexperimentsandgeochemicaltechniqueswereusedtodeterminetheirabilitytoalter thechemistryoftheirmicroenvironmentandproduce biominerals.Thispaperreportstheresultsofapreliminary identificationandcharacterizationofmicroorganisms isolatedfrommoonmilkanddrip-watersamplesfromthe GrottaNera,Gramstainingandcellmorphologyanalysis bylightmicroscopy,chemicalanalysisbyX-raydiffraction andscanningelectronmiscroscopyofmoonmilkand calciumcarbonatecrystalsobtained invitro inthepresence ofbacterialisolates,and invitro solubilizationtestsof calciumcarbonatebythecalcifyingbacteria.Theresults provideadditionalargumentsforthesignificantroleof bacteriaasgeochemicalagents.SITEANDENVIRONMENTALPARAMETERSGrottaNera(129AinCatastoGrotteAbruzzo,sourceof thesedata) Elevation:m1380a.s.l. Totallength:110mVerticalextent:10m Maps:CartadItaliaIstitutoGeograficoMilitare(1:25000) sheet147IIINE(Pennapiedimonte),geologicalmapof Abruzzi(1:100,000)(VezzaniandGhisetti,1993) TheGrottaNeraisnotonlyoneofthemostfamous andpeculiarcavesintheAbruzziregionincentralItaly (Fig.1),butitalsohasthemostimpressiveexamplesof moonmilkspeleothemsthathavebeendescribedinItaly (FortiandRossi,2003).Itislocatedintheheartofthe MajellaNationalPark,intheFeudoUgniNatural Reserve,adensemixed-deciduousforestcharacterizedby largeseasonaltemperaturevariationsandrelativelyhigh precipitation.Thisparkwasestablishedinparttoprotect thispeculiarhigh-altitudekarstlandscape.Inthisarea, surfacekarstfeatureshavebeenaffectedbyimportant signsofglacialerosion.TheMajellaMassifhostsa complexkarstsystemofseveralcaves(Burri,2003),some ofwhichareshowcaves,themostfamousofwhichisthe GrottadelCavallone. AccesstotheGrottaNerahasbeenstrictlyregulated formanyyearsandisonlyallowedforscientificpurposes topreservethepeculiarconcretionsforwhichthecaveis famous.Thecaveischaracterizedbyawideentrance (Fig.1).Thisopensdirectlyonthecliffandisaccessibleby arockyroutethatismadeeasierbyafewcutstepsanda safetywirerope.Thistrailleadsintoalargeroomwithits floorcompletelycoveredbycollapsedmaterial,including angularboulders,someofthemcoveredbytheremainsof brokenstalactites(Figs.1and2).Thisfirstlargespace leadsontoanarrowpassagethatislinedwithstalagmite columnsandprotectedbyagate.Thisistheentrancetothe mainroomofthecave,whereitspeculiarspeleothemsare concentrated(Figs.3and4a,b,c).Inthisinner,bell-shaped room,extensiveivory-whitedepositsofmoonmilkasP.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 89

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stalactites,stalagmites,andcavepearlscoverthewhole cavity(Fig.4).Reallyimpressivestalactiteshangfromthe 10-mhighceilingandshowapeculiarshapecharacterized byanapexdiameterthatislargerthanthatoftheroot andadevelopmentnotvertical(Fig.3).Thesebeautiful roundedstalactitesareconcentratedmostlyinasmall portionoftheroom(Fig.2b)andarehuge,withan averagelengthofabout1.5m.Theirsize,andconsequently theirweight,playakeyroleintheirgeneraldevelopment. Infact,inthecenteroftheroomthereisabuildupof brokenorfallenstalactitesthathavebeenre-cementedby flowsofcoatingmaterial(Fig.4g).Numerousactivegours withgrowingpisolitesinsidearealsopresentonthe flowstonefloor(Fig.4i). Thesepeculiarmoonmilkformationsweredescribedby FortiandRossi(2003)as trays whosedevelopmentwas inferredtoberelatedtostrongevaporationcausedby complexairflowswithintheroom(FortiandRossi,2003; Savini,2004).Traysareflat-bottomedspeleothemsthat endinaflat,horizontalsurface(HillandForti,1986).The mechanismoftheirformationisstillnotwellunderstood. Martini(1986)wasthefirsttospeculateontheabiotic originofcarbonate(calcite-aragonite)trays.Calaforraand Forti(1994)haveproposedanabioticmechanismforthe growthofgypsumtrays.Biogenictrayswerefoundinthe submarinecaveLuLampiu`ne,oneofthemostcomplex andlargestcavesintheSalentocoastofsoutheasternItaly. Thosestructureshangabundantlyfromtheroofand lateralwallsofthecave,wheretheyshowaslanted,nonverticalorientation(Onoratoetal.,2003).Thetraysinthe GrottaNera(Fig.3)arethebiggestsofardescribedin Italy;theyhaveanasymmetricenlargementatthebottom andaresimilartoatongue(FortiandRossi,2003). Asclearlyexposedinthegeologicalmapofthecave (Figs.2,5),thedevelopmentofthecavewasstrongly controlledbyseveralvariouslyorientedfaults.Oneofthese faultscontrolstheshapeoftheroomwherethemoonmilk traysgrowandwhereaclearfaultplaneisoutcropping Figure1.GrottaNeraentrancefrominside.Intheforegroundafallenbigbouldercluttersthepassage.Intheinset,thered dotshowstheapproximatelocationofthecave.BIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)90 N JournalofCaveandKarstStudies, August2014

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Figure2.MapandgeologyofGrottaNera(Abruzzi,centralItaly).Basedonafirstsurveyin1969byE.Burri,E. Bevilacqua,andG.DiIoriodrawnbyE.Burriandasecondsurveyin2005byG.DiPrinzioandG.Ferrinidrawnby G.Ferrini.P.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 91

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(Fig.2b);severalplantrootsareleaningalongthefault plane,showingthattheceilingisrelativelythin. Fromaspeleogenesispointofview,thecavitycanbe seenasarelictgalleryundergoinganimportantgravity reshaping,especiallyinthegreatentranceantechamber; herethebigbouldersclutteringthepassagecouldbe relatedtocollapselinkedtothelastHoloceneglacial period.Todate,nowaterflowsorwater-transported depositshavebeenreportedinthecave;onlyamoderate drippingintheinnerportionhasbeennoted. Apreliminarysurveyofair-temperaturedistribution wascarriedouttohighlighttherelationshipbetweenthe caveandtheoutsidetemperatures,measureanythermal gradientsinsidethecaveandtheirinfluenceonair movementinthecave,andthecavesmicroclimate.Inlate Figure3.Moonmilkstalactites(trays)hangingfromthe ceilingoftheinnerroomoftheGrottaNera.Notethepeculiar shapeandthedevelopmentdifferingfromtheverticalaxis. Figure4.Moonmilkdecoratingtheceiling(a,b,c),thewalls(d,e,f)andthefloor(g,h,i)oftheinnerroomofGrottaNera. Notethehugemoonmilkstalactiteswithanaverageheightofabout1.5m(a),roundandsmoothintheirform(a,b,c),witha non-verticaldevelopment(b,c);amoundformedbyfallenstalactites(g);thepresenceofabat(h);thepresenceofpisolites(i).BIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)92 N JournalofCaveandKarstStudies, August2014

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spring,earlysummer,andautumntheairtemperaturewas monitoredinsevensites,threeinthefirstroomandfourin theinnerone(Fig.2a).Thecollecteddatashowedthatthe airtemperatureinsidethelargeentranceroomisstrongly affectedbytheclimaticconditionsoutside,whichare characterizedbystrongseasonalvariability.Thesituation isquitedifferentfortheinnerroom.Regardlessofthe season,atemperaturepatternshowedrelativelysmall differencesacrosstheinnerroom.Itwasnotedthatfor mostoftheyearthetemperaturesrecorded(anaverageof 15 u C)werehigherthanthoseontheoutside.Thehigh temperaturemeasuredintheinnerroomcouldbeduetoa loworlackofwatercirculationduringthelastperiodof thecavesevolutionandtothegeometryofthecave.The GrottaNeraisanascendingcavitywithasingleinput,the entrancebeinginthelowerpartofthecave,suggestingthat itsinnerroommaybehaveasahottrapwhereairis trappedduringthewinter(Crammer,1899).Duringthe summerinahottrap,theair,whichiscolderthanthe outsideatmosphere,flowsdownward,whereasinthe winter,thecaveair,whichiswarmerthantheoutside atmosphere,remainsinequilibrium,andthereisonlya limitedcirculationattheentrance(Crammer,1899). AirflowswithintheinnerroomoftheGrottaNerawere assessedbysmokemovement,andbythethree-dimensional patternoftemperatures(Fig.5)atvaryingdistancesinthe caveandatvaryingheightsabovethecavefloor.Circulation insidetheinnerroomseemsnottobelinkedtochangesin barometricpressure,whichhasnoinfluenceontheinsignificanttotalvolumeofthecave,butitmightpossiblybedueto aninternalconvectiveflowgeneratedbytemperature gradientsandbyexchangeswithexternalair.Duringthe summer,hotaircomesinfromoutsideandflowsintothe innerroom,therebyactivatin gacirculation.Duringthecold season,coldairpenetratesinfromtheexteriorthroughthe thinceilingandgeneratesthesametypeofaircirculation. Inconclusion,theinnerroomhasitsownmicroclimate that,ingeneral,istypicalofthehottrapwiththe peculiaritiesthatarecausedbythepresenceofaverythin layerofuppersoilsothattheinternalcirculationinthis roomofthecaveisnotlimitedtotheentrance,according toCrammer(1899)butisamorecomplexcirculation (FortiandRossi,2003).MATERIALSANDMETHODSSAMPLELOCATIONANDSAMPLINGThestudiedsampleswerecollectedintheinnerroomof theGrottaNera(Fig.2a).Twomoonmilksamples(MI andMII)werecollectedrespectivelyfromsite1,located 74mfromtheentrance,andsite2,locatedinamore distantportionofthecave(96m);asampleofpercolating water(W)wastakeninasteriletubeatsite3.Moonmilk samplesweretakenasepticallyinsteriletubesfromsmall stalactitesapproximately15to20cminlengthand3to 4cmindiameter.MIwasaportionofawhitesolid stalactitewithoutaninternalfeedingtube.MIIwastaken fromastalactite,coloredbrownduetoplantroots,witha largeinternaltube.Themoonmilkandpercolatingwater sampleswerestoredatroomtemperature(18 u C)forabout 18hoursbeforemicrobiologicalanalysiswascarriedout.MOONMILKPHYSICOCHEMICALANALYSESMoonmilksampleswereanalyzedbyX-raydiffraction andX-rayfluorescence.X-raydiffractionwasusedto determinethemineralsinthemoonmilkdepositsandthe crystalsdeposited invitro .Measurementsweremadeby usingatwo-circle h /2 h diffractometerwithaCuradiation source,secondarygraphitemonochromator,andscintillationdetector(SeifertMZIV).Thesupplyvoltageofthe X-raytubewassetat50kVand30mA.The2 h -scanrange wasbetween22and50 u ;eachscanwasdoneatstepsof Figure5.TemperaturedistributioninGrottaNerainlateautumn.P.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 93

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0.05 u .Dependingonthesampledensity,acountingtime betweenoneandtensecondsperstepwasselected.The crystallinephaseswereidentifiedusingdatabasecardsfrom theInternationalCenterforDiffractionData. X-rayfluorescencespectroscopyisanon-destructive analysistechniquefromwhichitispossibletoobtainthe elementalcompositionofasamplethroughthestudyof fluorescenceradiation.WeperformedfluorescencespectroscopyonbothsamplesofmoonmilktakenintheGrotta Neraatsites1and2utilizingaSPECTRO(mod.XEPOS 2000)spectrophotometer.Fourgramsofmoonmilk powderweregrounddownto 100mm,wellhomogenized with0.9gofHoechstwax,andthenpressedwith12tonsto a32-mmpellet.ISOLATIONANDCHARACTERIZATIONOFCULTIVABLEHETEROTROPHICCALCIFYINGBACTERIAToisolatethecultivableheterotrophicbacterialmicroflorathatisassociatedwiththemoonmilksamplesfrom theGrottaNera,10gofeachsolidsamplewasgroundtoa powderusingasterilemortarandpestle.Thispowderwas thensuspendedin90mLofsalinesolution(0.9%NaCl). TriplicateB-4agarplates(Boquetetal.,1973)were inoculatedwithmoonmilksampledilutionsrangingfrom 102 1to102 6.Thedrip-watersamplewassimilarlyplated, undilutedordilutedto102 5.SolidB4mediumwas composed(perliter)of2.5-gcalciumacetate,4.0-gyeast extract,10.0-gglucose,and18.0-gagar.ThefinalpHwas adjusted,aftersterilization,to8.0usingNaOH.The inoculatedplateswereincubatedat22 u C,ahigher temperaturethaninthecavefromwherethesampleswere obtained,forfourweeksinordertoisolateslowlygrowing strains.Previousstudieshavedemonstratedthatcolonies fromcavesamplesgrowveryslowlyatcavetemperature andthatthediversityofthecultivablegeneraobservedwas similarwhetherthebacteriaweregrownat13 u C(cave temperature)orat28 u C(Grothetal.,2001;Laizetal., 2003).Individualcolonieswereselectedandpurifiedby streakingonB-4agar.Therelativeabundanceofeach isolate,withrespecttothetotalcultivablebacterial microflora,wasdeterminedbydirectcountsonB-4agar plates.Forshort-termpreservation,theisolateswere streakedonB-4agarslantsandstoredat4 u C,butfor long-termmaintenance,purecalcifyingisolateswerestored inliquidnitrogenat 2 196 u C.Cellandaggregate morphologywasstudiedunderalightmicroscope(LeitzBiomed),andGram-stainingwasperformedwiththeColor Gram2kit(bioMerieux,Marcy-lEtoile,France).CALCIUMCARBONATEPRECIPITATIONANDDISSOLUTION BYCALCIFYINGBACTERIAWeassessedthecalciteproductionofisolatesbyculturing themonB-4agarplates,asdescribedabove.Thebacterial isolateswerespreadintriplicateonthesurfaceofagarplates andwerethenincubatedaerobicallyat15,22,or32 u C.For upto30daysafterinoculation,tofollowcrystalproduction, allplateswereexamineddailyunderalightmicroscope (Leitz-Biomed).Withrespecttonegativecontrols,wechecked forthepresenceofcrystalsinasterilemediumandina mediuminoculatedwithautoclavedbacteria. Sinceithasbeenestablishedthatmicrobiallymediated reactionscangenerateconsiderableamountsofH+ions thatcandissolvethecavewallsorthespeleothemsandthat moonmilkcanalsobeformedbyspeleothemdecay (Sweeting,1973;HillandForti,1997),theabilityof calcifyingbacteriatodissolvecalciumcarbonatewasalso tested.CalcifyingisolatesweregrownonDeveze-Bruni medium(pH6.8)containing0.14or2.5%CaCO3at15 u C (NormalCommision,1990).Carbonatesolubilizationafter 7,15,and30dayswasquantifiedbymeasuringthe diameteroftheclearhalothatsurroundedeachcolonyin responsetodecreasedpH(Martinoetal.,1992).SEMANALYSISMorphologicalcharacteristicswerestudiedbyscanning electronmicroscopy.Culturedsolidmediasampleswere driedat37 u C;agarmediumwascutintoflatblocks,goldshadowed,andobservedwithaPhilipsscanningelectron microscopeXL30CP. Forcrystallite-poorsamples,themethodofRivadeneyraetal.(1998)wasapplied.Thecrystalsproducedby culturedbacteriawereremovedfromthemediumby cuttingoutagarblocksandplacingtheminboilingwater untiltheagardissolved.Thesupernatantsweredecanted andthesedimentwasresuspendedandwashedindistilled wateruntilthecrystalswerefreeofimpurities.Thewashed crystalswereair-driedat37 u CandthenusedforSEM analysis.Toobservetheinnerportionofthebioliths, crystalswerefirstpowderedbyusingamortarandpestle.RESULTSANDDISCUSSIONMOONMILKPHYSICOCHEMICALANALYSESX-raydiffractionrevealedthatbothsamplesofmoonmilktakenattheGrottaNeraconsistofasinglemineral phaseofcalciumcarbonate,i.e.,calcite.Ontheotherhand, accordingtopreviousliterature,moonmilkisusually composedofcalcite(Fisher,1992,1993),eventhoughthe presenceofothercarbonates,aswellassulfatesand phosphatesinmoonmilk,hasbeenreportedbyseveral authors(OnacandGhergari,1993;HillandForti,1997; MooreandSullivan,1997;Borsatoetal.,2000;Lacelleet al.,2004).X-rayfluorescenceanalysisshowedthatthe moonmilkconsistsprimarilyofCaO(60.87%atsite1, sampleMI,and60.18%atsite2,sampleMII),whileother constitutiveelementssuchasMgOandAl2O3never reached1%(Table1).MICROBIALCULTURESFROMMOONMILKANDDRIPPINGWATERTheculturesfrombothmoonmilkdepositsand drippingwateryieldedavisiblegrowthforcommonsoilBIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)94 N JournalofCaveandKarstStudies, August2014

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microfloragroups(fungi,bacteria,andactinomycetes bacteria)(Fig.6),butthedominantcultivablemicroorganismswerebacteriathatcausedsignificantcrystallization.Awiderangeofmicrobes,particularlybacteriaand streptomycetesbacteria,butalsofungi,algae,andprotozoa,canalsobeculturedfrommoonmilk,ofteninvery highdensities(Northupetal.,2000). Byusingthetraditionalcultivationtechniques,an abundantcultivableheterotrophicbacterialmicroflora fromthemoonmilksamplesMI(2.13104colony-forming unitspergramofdryweight)andMII(6.03104cfu/gd.w.) wasisolated,suggestingthatbacteriapresencewas probablynotaccidental.Asimilarbacterialdensityhas beenreportedbyBaskaretal.(2011)formoonmilk depositsfromKremMawmluhCave,India.Asimilar bacterialdensityforthecalcareousspeleothemsfromthe StiffeandCervoCavesandalsoforanunusualnewly describedcalcitespeleothemfromGraveGrubboCave werereportedinourpreviousstudies(Cacchioetal.,2003, 2004,2012).ThehigherbacterialdensityinsampleMII mayberelatedtoagreaterpresenceoforganicmatter releasedfromplantrootsandtoagreaterwatercontent. TheactivemoonmilksamplestakenfromtheGrottaNera hadawatercontentof74%(MI)and78%(MII)in summer. Basedonthecellandcolonymorphologiesofthe isolates(Table2),itwasconcludedthatthesampleMI containedninecultivablemicrobialstrains(eightbacterial isolatesnumberedfromM1toM8andonemold),while themoonmilksamplenamedMIIcontainedeightcultivablemicrobialstrains(fourbacterialisolatesnumberedfrom M9toM12andfourmolds).Thecultivableheterotrophic bacterialdensityfoundinthesampleofpercolatingwater, wasoftwoordersofmagnitudelowerthanthatfoundin moonmilk(4.13102cfu/mL).However,thedrippingwater containedaconsiderablenumberofbacteria,andthey couldnothavefilteredthroughthethinlayerofrockthat coversthecave,duetothefactthattheGrottaNeraisina mixed-deciduousforest.Fromthissamplefourstrainswere isolated,threebacterialstrainsnamedfromW1toW3and onemold.RELATIVEABUNDANCEANDPRELIMINARYCHARACTERIZATIONOFBACTERIALISOLATESThemostabundantstrainswerethefollowing:isolate M8fromthemoonmilksampleMII,isolateM9fromthe moonmilksampleMII,andtheW2strainfromthedripwatersample.Thesebacterialstrainsrepresented40.00%, 73.12%,and68.05%oftheirsamplesrespectivebacterial populations.ThestrainsM2andM3represented22.90% Table1.Chemicalcontentsofmoonmilkfromsite1(sample MI)andsite2(sampleMII)inGrottaNera. Mineral Constituent Sites Site1 Site2 CaO 60.87 60.18 MgO 0.83 0.83 Al2O3, 0.19 0.19 P2O50.18 0.19 SiO20.05 0.06 SO30.05 0.04 Fe2O30.01 0.02 MnO 0.01 0.01 K2O 0.01 0.01 Figure6.Distributionofactinomycetes,otherbacteria,andfungi(percentageofstrains)inthecultivableheterotrophic microflorafrommoonmilk(MIandMII)andpercolatingwater(W)samplescollectedfromGrottaNera.P.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 95

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and17.89%oftheMImoonmilkbacterialpopulation, respectively.ThestrainM11represented14.13%oftheMII sample,whilethestrainW1represented29.51%ofthe bacterialpopulationinthepercolatingwatersample.The contributionsoftheremainingisolatestotheoverall respectivebacterialpopulationrangedfrom2.13%to 7.42%. Theactinomycetescomponentdidnotexceed7.42%of thetotalbacterialpopulationintheMIImoonmilksample, takeninthepresenceofplantroots,and2.44%inthedripwatersample.InsampleMIfromthefullymoonmilk speleothem,theactinomycetescomponentwasabsent (Table2).Actinomycetesarecommonincaves,where theirgrowthisrelatednotonlytoparticularenvironmental conditions,suchastemperaturerangingfrom10to15 u C andhighrelativehumidity,butalsototheinputof refractoryorganicmatterindrippingwater(Grothand Saiz-Jimenez,1999).Dissolvedorganicmatterfromsoil, whichistheoriginoftheorganiccarbonfoundindripping waters,containsaliphaticorganicacidsandphenolic compoundsproducedbylignocellulosedegradation(Guggenbergetal.,1994;Saiz-JimenezandHermosin,1999). Bothlignocelluloseandhumicmaterialsarealmost selectivelydegradedbyactinomycetes,whicharewell knownfortheirabilitytogrowonverypoormediaand touserecalcitrantorganicmatter(Crawfordetal.,1983; McCarthy,1987;GrothandSaiz-Jimenez,1999).The findingsofthisresearchnotonlyconfirmtheimportance oftemperatureandrelativehumiditytodetermineactinomycetescolonizationandlong-termgrowthoncave surfaces,butalsounderlinestheroleofrecalcitrantorganic matterthat,accordingtoourresults,seemstobethemain determiningfactor.Infact,theproportionofactinomycetesamongthebacterialmicrofloraishigherinthesample MII,whichreceivesorganicmatterfrompercolatingwater andplantroots,thaninthedrip-watersample,andthey disappearentirelyinthestalactiteofmoonmilkthatdoes notreceiveorganicmatterfromdrippingwaterandroot exudates. Theseresultsareinteresting,butmaynotreflectthe actualmicrobialactivitytakingplaceinthespeleothem becausecultivationtechniquesarethoughttogreatly underestimatemicrobialdiversityduetothenon-culturabilityofthelargemajorityofmicroorganisms(Dojkaet al.,2000).Inthisstudy,singlesmallrodsandrodchains predominateinthestrainsisolated(Table2).AnexaminationofGram-stainedsmearstakenfromthefifteen differentcolonytypesfrommoonmilkdepositsanddripwatershowedthatthemajority(66.7%)oftheisolated strainswereGram-negative(Table2).Therelativeabundancesofeachisolatewithrespecttothetotalcultivable bacterialmicroflorashowedthat(Fig.7andTable2)the mostabundantstrains(M2andM8strainsintheMI sample,M9andM11strainsintheMIIsample)inboththe studiedmoonmilksampleswereGram-negative,aspreviouslyfoundbysomeauthorselsewhere(Danielliand Edington,1983;Mulecetal.,2002).TheGrottaNerais locatedinamixed-deciduousforest,anditisknownfrom previousliteraturethatGram-negativebacteriatendtobe moreabundantinrhizospheresoilcomparedtothebulk soil(Schlegel,1993;SteerandHarris,2000):Gram-negative bacteriabiomassincreaseswhenrapidlydecomposable Table2.BacterialstrainsisolatedfrommoonmilkandwatersamplescollectedfromGrottaNera,Abruzzi,Italy,withtheir colonyandcellmorphologies,Gramresult,andrelativeabundances. Isolate ColonyMorphologyCellMorphology GramRelativeAbundance MIMoonmilkSample M1 Mediumcreamy Rodchain G 2 2.13 M2 Mediumcreamy Singlerod G 2 22.90 M3 Mediumwhite Singlerod G 2 17.89 M4 PunctiformcreamySinglerod G 2 2.18 M5 Bigcreamy Singlerod G 2 4.26 M6 Bigwhite Rodchain G + 4.26 M7 MediumyellowishSinglerod G 2 6.38 M8 Mediumwhite Singlerod G 2 40.00 MIIMoonmilkSample M9 Punctiformpink Rodchain G 2 73.12 M10 Mediumblack Actinom. G + 7.42 M11 Smallcreamy Rodchain G 2 14.13 M12 Bigcreamy Rodchain G + 5.33 WaterSample W1 PunctiformcreamySinglerod G + 29.51 W2 Smallyellowish Singlerod G 2 68.05 W3 PunctiformwhiteActinom. G + 2.44BIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)96 N JournalofCaveandKarstStudies, August2014

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carboncompounds,suchassugarsandorganicandamino acids,areavailable(MarilleyandAragno,1999),whereas higherproportionsofGram-positivebacteriaareusually foundinresourcelimitedareas(AtlasandBartha,1998; Kourtevetal.,2002).Infact,theratioofGram-negativeto Gram-positivebacteriainmoonmilksamplesMI(95.74%) andMII(87.25%)wassignificantlyhigherthaninpreviously studiedconsolidatedcalcareousspeleothemandsoilsamples (Cacchioetal.,2003,2004,2012)fromwhichnearlyall strainsisolatedwereGram-positive.Ahighproportion (68.05%)ofGram-negativebacteriawasalsofoundinthe percolatingwatersample.Ingeneral,cultivablebacterial communitiesfromgroundwaterhavelowproportionsof Gram-positivebacteria(Laizetal.,1999).Noattemptswere madetoidentifythevariousbacterialspeciespresent,as attentionwasdirectedontheprocessesofcarbonate depositionratherthanontaxonomy.INVITROBACTERIALPRECIPITATIONANDDISSOLUTION OFCALCIUMCARBONATESinceallthemoldsisolatedfrommoonmilkandwater sampleswereincapableofprecipitatingCaCO3crystals, thissuggestsamajorparticipationofbacteriainthe biomineralizationprocessesinvolvedinthemoonmilk stalactiteformation.Somepreviousstudieshavesuggested fungiasthemajorparticipantsintheprocess(Callotetal., 1985),althoughmorerecentinvestigationshaveproposed bacteriaasthemajorinducersofcarbonatedeposition formingmoonmilkincaves(Canaverasetal.,2006;Barton andNorthup,2007;PortilloandGonzales,2011).Bacterial CaCO3precipitationonB-4solidmediumoccurredatall thetemperaturestested,15 u C(cavetemperature),22 u C, and32 u C.Underlaboratoryconditions,itwasfoundthat allofthebacterialisolatesassociatedwiththehollow stalactite(sampleMII)andthedrip-watersamplewere capableofformingcrystallinecalciumcarbonate.This confirmsthehypothesisthatinappropriateconditions, especiallyincarbonate-richenvironmentssuchaslimestonecaves,manybacteriacanformcalciumcarbonate crystals(Boquetetal.,1973;Cacchioetal.,2003,2004, 2012).Notallofthebacterialstrainsisolatedfromthesolid moonmilkstalactite(sampleMI)werecalcifying;thehigh relativeabundance(40%)ofstrainM8,whichwasnot capableofprecipitatingcrystals,isconsistentwiththeless intensecalcificationofthisstalactite.Thisisconsistentwith ourprevioushypothesis(Cacchioetal.,2004),accordingto whichdrip-watermayselectcalcifyingbacteria,andhence theintensityofcalcifyingactivityisgreaterinyounger stalactites.Itisworthnotingthatintubularstalactites, calcificationalsotakesplaceintheinnersurface,givingrise tofillingofthespace(MooreandSullivan,1997).Byusing aknock-outinthe cha Acalciumantiporterprotein,Banks etal.(2010)havesuggestedthatcalciumtoxicityprovides boththephysiologicalbasisandselectionpressureforthe calcificationphenotype.AlltheGram-positivestrains isolatedfromtheGrottaNerawerecapableofdepositing crystalsonB4agarplatesatallthestudiedtemperatures. Thecomparisonofthepercentagesofcalcifyingbacteria isolatedfromthepreviouslystudiedcaves(Cacchioetal., 2003,2004,2012)hasshownthatthetemperaturefactor playsakeyroleintheextentofcalciumcarbonate depositionbybacteria.Infact,inStiffeandCervoCaves, thetemperaturerangesfrom10to12 u C,whereasinGrave GrubboCave,locatedinsouthItaly,wheretherangeis14 to15 u Cthebiogenicactivityis,asintheGrottaNera, muchmoreimpressive.Hence,itcanbesuggestedthat15 u Cisthethresholdoftwobiologicallydifferentcalcificationenvironments. Carbonateprecipitationbycalcifyingisolatesat22and 32 u Cstartedwithsomedelaycomparedtothatat15 u C. Allthecalcifyingbacteriabegantoprecipitatecarbonate afterthreedaysat15 u C,afteroneweekat22 u C,andafter Figure7.Gram-negative/Gram-positiveproportionsinthecultivableheterotrophicbacterialmicrofloraofmoonmilk(MIand MII)andpercolatingwater(W)samplescollectedfromGrottaNera,byrelativeabundance(seeTable2).P.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 97

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fourweeksat32 u C(Fig.8).Inourpreviousstudies,itwas foundthatcalcificationtookplacemorequicklyat32 u C thanatlowertemperatures,includingthetemperatureof thestudiedcaves(Cacchioetal.,2003,2004,2012).Infact, thebacteriaisolatedfrommoonmilkdepositsaremuch morerapidintheprocessofcalcificationinthecavethan thestrainsisolatedfromcavespeleothemsnotconsistingof moonmilk(Cacchioetal.,2003,2004,2012).Toexplain thissignificantdifference,aworkinghypothesisisadopted thatmicrobesinhabitingtheGrottaNerahavebeen exposedtoalongerperiodofevolutionataconstant temperatureof15 u C,hencestrengtheningtheircalcificationcapabilityatsuchatemperature.Intheotherstudied caves,whichhaverunningwater,thetemperaturetendsto changethroughouttheyearaccordingtothedischarge changesandperiodsofdrought. ThecalcitenatureofthecrystalsdepositedonB4agar plateswasconfirmedbyX-raydiffractionanalysis.No carbonateprecipitationtookplaceinthecontrolssince metabolicactivityisnecessaryforprecipitation. Thecorrosionbehaviorofthecalcifyingisolateswas alsostudiedshowingthat13%ofthecalcifyingbacteria isolatedfromtheMIImoonmilkspeleothemsolubilized calciumcarbonatewhengrownonagarplatescontaining 0.14%and2.5%CaCO3aftertwoweeksat15 u C.This percentageincreasedto32%forthecalcifyingbacteria isolatedfromthedrippingwater.Withrespecttothe solubilizationactivityofthecalcifyingbacteriaassociated withtheMImoonmilkspeleothem,thiswasatleast32% afteroneweekat15 u C.However,thecalcifyingbacteria isolatedfromtheGrottaNeraCaveshowedagreater abilitytodissolvecalciumcarbonatethanthoseassociated withconsolidatedstalactitessampledfrompreviously studiedcaves(Cacchioetal.2003,2004and2012).SEMANALYSISScanningelectronmicrographsofthecrystalsdeposited onB-4agarplatesrevealedsignificantamountsofbacterial cellsontheinnersurfaceofthecrystals(Fig.9a,b), calcifiedbacterialcells(Fig.9e,f)andtheirimprints (Fig.10c,d,e,f);newlyformed(Fig.9a,b,c,d)orcalcified biofilms(Fig.9e,f)cementingthecarbonategrains;andthe presenceofcrystalsthatvaryinsizeandshape(Fig.10a,b). SEMstudiesoftheextensivemoonmilkdepositsinthe GrottaNerarevealedthattheyweremainlycomposedofa networkoffibercalcitecrystalsandfilaments(Fig.10g,h). TheresultsofSEMexaminationpointtowardasignificant bacterialinfluenceinthegenesisofmoonmilkintheGrotta Nera.CONCLUSIONSCavesareextremeandspecializedhabitatsforterrestriallifethatsometimescontainmoonmilk.Therearemany cavesaroundtheworldwithimpressiveamountsof moonmilk(OnacandFarcas,1992;Chirienco,2002).In Italy,calcitemoonmilkisfoundinmanycavesinthe ItalianAlps(Borsatoetal.,2000).TheCesareBattistiCave containsmoonmilkdepositsupto0.5mthickand120m longthatdevelopedunderseveralseepages(Borsatoetal., 2000)andamoonmilkflowstoneonthewalloftheCripta Chamberthathasdevelopedtoathicknessof40to50cm. IntheBusdelToniCave,thereareextensivecurtainsof moonmilk(MiorandiandBorsato,2005) Figure8.Relationshipbetweenpercentageofcalcifyingstrainsshowingproductionofcalcitebytheindicateddurationof culturingonB-4agarculturesat156C,226C,and326C.Notenon-linearXaxis.BIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)98 N JournalofCaveandKarstStudies, August2014

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Figure9.Scanningelectronmicrographs:(a)CalcifyingbacterialcellsisolatedfrommoonmilkcollectedfromGrottaNeraof theunidentifiedstrainM5ontheinnersurfaceofaCaCO3crystalprecipitatedonB-4agar,after30daysat226C;scalebar 20mm.(b)Highermagnificationviewof(a);scalebar10mm.Observethesignificantnumberofbacterialcellsandthepresence ofbiofilm.(c)BacterialcellsincludedinabiofilmthatbridgescalcitecrystalsdepositedonB-4agarplatesafter30daysof incubationat326CofthestrainW1isolatedfromasampleofdrip-watercollectedinGrottaNera;scalebar20mm.(d)Higher magnificationviewof(c);arrowpointstosomeofthecells.Scalebar10mm.(e)Cementedbiofilmsthatincorporatethe calcifyingcellsoftheM9strainisolatedfrommoonmilkcollectedinGrottaNera,observedafter30daysofincubationonB-4 agarat326C;scalebar20mm.(f)Highermagnificationviewof(e);scalebar20mm.Observethesignificantthicknessofthe calciumcarbonatelayerproducedbyM9,themostabundantstrainintheMIImoonmilksample;comparewith(c).P.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 99

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IntheGrottaNera,moonmilkentirelycoversthewalls, theceiling,andtheflooroftheroom,generating exceptionalexamplesofdecorations,includingstalactites, stalagmites,andcavepearls.EventhoughinItalyitisnot commontofindextensivemoonmilkdeposits,theextraordinaryscientificimportanceofthiscaveismainlylinkedto thepresenceofunusualspeleothemsclassifiedas trays whosesizehasnoequalinothercavesinItaly(Fortiand Rossi,2003). Ourpreliminarystudyofcultivablemicrobialpopulationsintwosamplesofmoonmilkandoneofdripping waterfromGrottaNerawascarriedouttoestimatethe concentrationofcolony-formingunits;noidentificationof generaandspecieswasprovided.Thisstudyrevealedhigh Figure10.ScanningelectronmicrographsofCaCO3depositedonB4agarplatesbythestrainM9,isolatedfrommoonmilk sampleMIIcollectedinGrottaNera.Spherulitecrystalsandcalciteaggregates;(a)scalebar50mm,(b)scalebar50mm. Bacterialimprintsintheinnerportionofthecrystals;(c)scalebar50mm,(d)scalebar10mm,(e)scalebar20mm,(f)scalebar 10mm.Calcitemoonmilkfiber;(g)scalebar10mm,(h)scalebar10mm.BIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)100 N JournalofCaveandKarstStudies, August2014

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cultivableheterotrophicpopulationdensitiesandadiverse microbialcommunity,includingfilamentousbacteria, associatedwiththemoonmilk.Moonmilkanddrip-water bacterialcommunitiestendtohavelowproportionsof Gram-positivebacteriaandaremainlycomposedofGramnegativesinglerodsandrodchains.Thiscanbeascribedto thefactthatGram-positivebacteriaarelikelytobemore successfulinresource-limitedareas,suggestingthatthe associationofmoonmilkwithGram-negativebacteriaisat leastpartiallydrivenbythecharacteristicsofthesoiland overlyingvegetation.Mostofthebacteriaisolatedhavethe abilitytoprecipitatecalcitecrystalswhenculturedusingB4agar,andduetotheirmetabolicactivitythisprobably happensalsoinnaturalhabitats.Incarbonatecaves,the metabolismofmicroorganismscanaltertheirmicroenvironment,ifproductsofmicrobialactivityresultinapH increaseintheenvironment.Anincreaseinmetabolicend products,suchascarbonateions,canincreaseprecipitation ofcalciumcarbonateincaves(Braissantetal.,2002; BartonandNorthup,2007;Portilloetal.,2009).The differentmorphologiesoftheprecipitatesformedbythe differentcalcifyingisolatesconfirmedthatcrystalmorphologywasspecies-specific,andthissuggeststhatthe bacteriaplayamajorroleintheprecipitationprocess. Whenexaminedbyscanningelectronmicroscope,the GrottaNeramoonmilksamplesexhibitedafeltedmatof fibers;X-raydiffractionanalysisofthespeleothemsgave clearevidenceforcalcite. Theelectronmicroscopicandmicrobiologicalevidences,togetherwiththegeochemistryandenvironmentaldata obtainedinthisstudy,indicatethatmoonmilkfromthe GrottaNerastalactitesisofbiogenicorigin.Itistherefore possibletoinferthattherearetwodifferentbacterial contributionstothebiogenicmoonmilkhostedinthe GrottaNera,activeprecipitationofmoonmilkbybacteria andbacterialbiochemicalcorrosionofthebedrockby organicacid,assuggestedbythepresenceofnon-calcitic mineralinclusionsintothemoonmilkandbythesolubilizationactivityofthecalcifyingbacterialstrains. TheextensivepresenceofbiogenicmoonmilkinGrotta NeramayberelatedtothepeculiarCaCO3precipitation environment,i.e.,itsmiddleelevation,mixed-deciduous vegetationcover,andlocalmicroclimate.Thereforea conclusionofthisstudyisthatmicrobialactivityata constantandoptimumtemperatureappearstobeakey factorpromotingcalciteprecipitationandmoonmilk formationinGrottaNera.ACKNOWLEDGEMENTSWethankM.GiammatteoandL.Arrizzaforassistance withSEM,G.CappuccioandF.Ferrantefortheir collaborationinchemical-physicalanalyses,G.DiPrinzio forherassistanceinthefieldandduringlaboratorywork. WealsothanktheMajellaNationalParkauthorityfor givinguspermissionforsamplingintheGrottaNeraand theCorpoForestaledelloStatoofPescaraforguidingus throughthecave.Finally,wethankR.DiStefanoandM. RomaforassistancewiththeAbruzzimapandC.DeRose forgraphicassistanceandE.Burriforthephotoin Figure4.REFERENCESAtlas,R.M.,andBartha,R.,1998,MicrobialEcology:Fundamentals andApplications,4thedition:SanFrancisco,BenjaminCummings, 640p. 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Gradzinski,M.,Szulc,J.,andSmyk,B.,1997,Microbialagentsof moonmilkcalcification, in Jeannin,P.-Y.,ed.,Proceedingsofthe12th InternationalCongressofSpeleology,vol1:LaChaux-de-Fonds, SwissSpeleologicalSociety,p.275. Groth,I.,andSaiz-Jimenez,C.,1999,Actinomycetesinhypogean environments:GeomicrobiologyJournal,v.16,p.1.doi:10.1080/ 014904599270703. Groth,I.,Schumann,P.,Saiz-Jimenez,C.,Laiz,L.,Sanchez-Moral,S., Canaveras,C.J.,andSaiz-Jimenez,C.,2001,Geomicrobiological studyoftheGrottadeiCervi,PortoBadisco,Italy:Geomicrobiology Journal,v.18,p.241.doi:10.1080/01490450152467778. Guggenberg,G.,Zech,W.,andSchulten,H.-R.,1994,Formationand mobilizationpathwaysofdissolvedorganicmatter:evidencefrom chemicalstructuralstudiesoforganicmatterfractionsinacidforest floorsolutions:OrganicGeochemistry,v.21,p.51.doi:10.1016/ 0146-6380(94)90087-6. Hill,C.A.,andForti,P.,1986,CaveMineralsoftheWorld:Huntsville, NationalSpeleologicalSociety,238p. Hill,C.A.,andForti,P.,1997,CaveMineralsoftheWorld,second edition:Huntsville,NationalSpeleologicalSociety,463p. Istvan,D.,Manescu,S.,andJurca,M.,1995,Studyonmoonmilkfrom PesteraMare,PiatraMolosnaia(Repedea,MaramuresMountains, Romania):TheoreticalandAppliedKarstology,v.8,p.69. James,J.M.,Jennings,J.N.,andDyson,H.G.,1982,Mineraldecoration andweatheringofthecaves, in Dyson,H.J.,Ellis,R.,andJames, J.M.,eds.,WombeyanCaves:Sydney,SydneySpeleologicalSociety, p.121. Jones,B.,andKahle,C.F.,1993,Morpho logy,relationship,andoriginoffiber anddendriticcalcitecrystals:JournalofSedimentaryPetrology,v.63, p.101831.doi:10.1306/D4267C85-2B26-11D7-8648000102C1865D. Kourtev,P.S.,Ehrenfeld,J.G.,andHaggblom,M.,2002,Exoticplant speciesalterthemicrobialcommunitystructureandfunctioninthesoil: Ecology,v.83,p.315266.doi:10.1890/0012-9658(2002)083[3152: EPSATM]2.0.CO;2. Lacelle,D.,Lauriol,B.,andClark,I.D.,2004,Seasonalisotopicimprint inmoonmilkfromCavernedelOurs(Quebec,Canada):implications forclimaticreconstruction:CanadianJournalofEarthSciences,v.41, p.1411.doi:10.1139/e04-080. Laiz,L.,Gonzalez-Delvalle,M.,Hermosin,B.,Ortiz-Martinez,A.,and Saiz-Jimenez,C.,2003,Isolationofcavebacteriaandsubstrate utilizationatdifferenttemperatures:GeomicrobiologyJournal,v.20, p.479.doi:10.1080/713851125. Laiz,L.,Groth,I.,Gonzales,I.,andSaiz-Jimenez,C.,1999,MicrobiologicalstudyofthedrippingwatersinAltamiracave(Santillanadel Mar,Spain):JournalofMicrobiologicalMethods,v.36,p.129. doi:10.1016/S0167-7012(99)00018-4. Marilley,J.,andAragno,M.,1999,Phylogeneticdiversityofbacterial communitiesdifferingindegreeofproximityof Loliumperenne and Trifoliumrepens roots:AppliedSoilEcology,v.13,p.127. doi:10.1016/S0929-1393(99)00028-1. Martini,J.,1986,Thetray:anexampleofevaporation-controlled speleothems:BulletinoftheSouthAfricanSpeleologicalAssociation, v.27,p.46. Martino,T.,Salamone,P.,Zagari,M.,andUrz`,C.,1992, AdesioneasubstratisolidiesolubilizzazionedelCaCO3qualemisura dellacapacita`deteriorantedibatteriisolatidalmarmoPentelico: ItalianSocietyofGeneralMicrobiologyandMicrobialBiotechnology (SIMGBM)XIMeeting,Gubbio,p.249. McCarthy,A.J.,1987,Lignocellulose-degradingactinomycetes:FEMS MicrobiologyLetters,v.46,p.145.doi:10.1016/0378-1097(87) 90061-9. Melon,J.,andBourguignon,P.,1962,E tudedemondmilchdequelques grottesdeBelgique:BulletindelaSocie te FrancaisedeMineralogieet Cristallographie,v.85,p.234. Miorandi,R.,andBorsato,A.,2005,Ambientediformazioneditufo calcareoelattedimonteingrottedelTrentinoconparticolare riferimentoalGruppodiBrentaePaganella:StudiTrentinidiScienze NaturaliActaGeologica,v.82,p.225. Moore,G.W.,andSullivan,N.,1997,Speleology:CavesandtheCave Environment:St.Louis,CaveBooks,176p. Mulec,J.,Zalar,P.,Hajna,N.Z.,andRupnik,M.,2002,Screeningfor culturablemicroorganismsfromcaveenvironments(Slovenia):Acta Carsologica,v.31,no.2,p.177. NORMALCommission,1990,NORMAL9/88recommendations. Autotrophicandheterotrophicmicroflora:isolationinculture:Rome, CNR/ICR. Northup,D.E.,Dahm,C.N.,Melim,L.A.,Spilde,M.N.,Crossey,L.J., Lavoie,K.H.,Mallory,L.M.,Boston,P.J.,Cunningham,K.I.,and Barns,S.M.,2000,Evidenceforgeomicrobiologicalinteractionsin Guadalupecaves:JournalofCaveandKarstStudies,v.62,p.80. Northup,D.E.,andLavoie,K.H.,2001,Geomicrobiologyofcaves:a review:GeomicrobiologyJournal,v.18,p.199.doi:10.1080/ 01490450152467750. Onac,B.P.,1995,Mineralogicaldataconcerningmoonmilkspeleothemsin fewcavesfromNorthernNorway:ActaCarsologica,v.24,p.4297. Onac,B.-P.,andGhergari,L.,1993,Moonmilchmineralogyinsome RomanianandNorwegianCaves:CaveScience,v.20,p.107.BIOGENICITYANDCHARACTERIZATIONOFMOONMILKINTHEGROTTANERA(MAJELLANATIONALPARK,ABRUZZI,CENTRALITALY)102 N JournalofCaveandKarstStudies, August2014

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Onac,B.P.,andFarcas,T.,1992,LemoonmilkdesgrottesdeTrollkjerka etdeReshellarn(Lavangsmarka,Nordland,Norve`ge):Travauxde lInstitutedeSpeologieEmileRecovitza,v.31,p.133. Onorato,R.,Forti,P.,Belmonte,G.,Poto,M.,andCostantini,A.,2003, LaGrottasottomarina luLampiu `ne :novita`esplorativeeprime indaginiecologiche:ThalassiaSalentina,v.26Suppl.,p.55. Portillo,M.C.,andGonzales,J.M.,2011,Moonmilkdepositsoriginate fromspecificbacterialcommunitiesinAltamiraCave(Spain): MicrobialEcology,v.61,p.182.doi:10.1007/s00248-010-9731-5. Portillo,M.C.,Porca,E.,Cuezva,S.,Canaveras,J.C.,Sanchez-Moral,S., andGonzales,J.M.,2009,Istheavailabilityofdifferentnutrients acriticalfactorfortheimpactofbacteriaonsubterraneous carbonbudgets?:Naturwissenshaften,v.96,p.1035.doi:10. 1007/s00114-009-0562-5. Richter,D.K.,Immenhauser,A.,andNeuser,R.D.,2008,Electron backscatterdiffractiondocumentsrandomlyorientatedc-axesinmoonmilkcalcitefibres:evidenceforbiologicallyinducedprecipitation: Sedimentology,v.55,p.487.doi:10.1111/j.1365-3091.2007.00915.x. Rivadeneyra,M.A.,Delgado,G.,Ramos-Cormenzana,A.,andDelgado, R.,1998,Biomineralisationofcarbonatesby Halomonaseurihalina in solidandliquidmediawithdifferentsalinities:crystalformation sequence:ResearchinMicrobiology,v.149,p.277.doi:10.1016/ S0923-2508(98)80303-3. Saiz-Jimenez,C.,andHermosin,B.,1999,Thermallyassistedhydrolysis andmethylationofdissolvedorganicmatterindrippingwatersfrom theAltamiraCave:JournalofAnalyticalandAppliedPyrolysis,v.49, p.337.doi:10.1016/S0165-2370(98)00112-0. Savini,N.,2004,LeGrottedellaMontagnaSacra:IlForestale,no.25, 28p. Schlegel,H.G.,1993,GeneralMicrobiology,seventhedition:Cambridge, CambridgeUniversityPress,655p. Steer,J.,andHarris,J.A.,2000,Shiftsinthemicrobialcommunityin rhizosphereandnonrhizospheresoilsduringthegrowthof Agrostis stolonifera: SoilBiologyandBiochemestry,v.32,p.869.doi:10. 1016/S0038-0717(99)00219-9. Sweeting,M.M.,1973,KarstLandforms:NewYork,ColumbiaUniversityPress,362p. Vezzani,L.,andGhisetti,F.,1993.CartageologicadellAbruzzo.Regione Abruzzo-Settoreurbanistica,beniambientaliecultura.S.E.L.C.A., Firenze.P.CACCHIO,G.FERRINI,C.ERCOLE,M.DELGALLO,ANDA.LEPIDIJournalofCaveandKarstStudies, August2014 N 103

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ADAPTATIONSOFINDIGENOUSBACTERIATOFUEL CONTAMINATIONINKARSTAQUIFERSIN SOUTH-CENTRALKENTUCKYTOMD.BYL1,2*,DAVIDW.METGE3,DANIELT.AGYMANG2,MIKEBRADLEY1,GREGGHILEMAN1,ANDRONW.HARVEY3Abstract: ThekarstaquifersystemsinsouthernKentuckycanbedynamicandquickto change.Microorganismsthatliveintheseunpredictableaquifersareconstantlyfaced withenvironmentalchanges.Theirsurvivaldependsuponadaptationstochangesin waterchemistry,takingadvantageofpositivestimuliandavoidingnegative environmentalconditions.TheU.S.GeologicalSurveyconductedastudyin2001to determinethecapabilityofbacteriatoadaptintwodistinctregionsofwaterqualityina karstaquifer,anareaofclean,oxygenatedgroundwaterandanareawherethe groundwaterwasoxygendepletedandcontaminatedbyjetfuel.Watersamples containingbacteriawerecollectedfromonecleanwellandtwojetfuelcontaminated wellsinaconduit-dominatedkarstaquifer.Bacterialconcentrations,enumerated throughdirectcount,rangedfrom500,000to2.7millionbacteriapermLintheclean portionoftheaquifer,and200,000to3.2millionbacteriapermLinthecontaminated portionoftheaquiferoveratwelvemonthperiod.Bacteriafromthecleanwellrangedin sizefrom0.2to2.5mm,whereasbacteriafromonefuel-contaminatedwellweregenerally larger,ranginginsizefrom0.2to3.9mm.Also,bacteriacollectedfromthecleanwellhad ahigherdensityand,consequently,weremoreinclinedtosinkthanbacteriacollected fromcontaminatedwells.Bacteriacollectedfromthecleanportionofthekarstaquifer werepredominantly( 95%)Gram-negativeandmorelikelytohaveflagellapresentthan bacteriacollectedfromthecontaminatedwells,whichincludedasubstantialfraction ( 30%)ofGram-positivevarieties.Theabilityofthebacteriafromthecleanportionof thekarstaquifertobiodegradebenzeneandtoluenewasstudiedunderaerobicand anaerobicconditionsinlaboratorymicrocosms.Therateoffuelbiodegradationin laboratorystudieswasapproximately50timesfasterunderaerobicconditionsas comparedtoanaerobic,sulfur-reducingconditions.TheoptimumpHforfuel biodegradationrangedfrom6to7.Thesefindingssuggestthatbacteriahaveadapted towater-saturatedkarstsystemswithavarietyofactiveandpassivetransport mechanisms.INTRODUCTIONApproximately40%oftheUnitedStateseastofthe MississippiRiverisconsideredkarstterrain(Quinlan, 1989).Over55%ofTennesseeandKentuckyisunderlain bycarbonaterocksandexhibitsclassickarstfeaturessuch assinkholes,disappearingstreams,andcavesystems (Wolfeetal.,1997;Floreaetal.,2002).Thekarstconduit systemsprovidehabitattoadiversefauna(Schneiderand Culver,2004)andcanbeanimportantsourceofwaterfor humans(Hutson,1995)andtheecosystem.Despitethe commonoccurrenceandimportanceofkarstaquifers,very littleisreportedinthescientificliteratureaboutthe adaptationsofindigenousbacteriafoundinwater-saturatedkarstconduits(Byletal.,2002).Karstaquifersprovide distincthydrologicandchemicalenvironmentscompared tounconsolidatedsandyaquifers.Forexample,karst conduitstypicallyoffermuchsmallersurfaceareasfor biofilmdevelopmentpervolumeofwaterrelativeto granularaquifers.Itisthereforereasonabletoassumethat therearedifferentadaptationsanddistributionsinkarst microbialcommunitiesascomparedtothosereportedfor sandyaquifers(Haacketal.,2012;Harveyetal.,1984; Harveyetal.,1997;Kolbel-Boelkeetal.,1988;Bartonand Northup,2007). Therearedifferenttypesofkarstsystemsthroughout theUnitedStatesandtheworld(White,2002).Thekarst aquifersofnorth-middleTennesseeandsouthernKentucky maytraplargevolumesofwaterinfracturesalongbedding planesandotherfeaturesisolatedfromactivegroundwater flowpaths(Wolfeetal.,1997).Inessence,thereare dissolutionopeningswithactivelyflowingwaters,aswell asstagnantwater-filledopeningswithsubstantiallylonger *CorrespondingAuthor:tdbyl@usgs.gov1U.S.GeologicalSurvey,Nashville,TN,372112TennesseeStateUniversity,Civil&EnvironmentalEngineeringDept.,Nashville, TN,372093U.S.GeologicalSurvey,Boulder,CO,80303T.D.Byl,D.W.Metge,D.T.Agymang,M.Bradley,G.Hileman,andR.W.HarveyAdaptationsofindigenousbacteriatofuel contaminationinkarstaquifersinsouth-centralKentucky. JournalofCaveandKarstStudies, v.76,no.2,p.104.DOI:10.4311/ 2012MB0270104 N JournalofCaveandKarstStudies, August2014

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residencetimes.Inthesestagnantareasisolatedfromthe majorgroundwaterflowpaths,thebacteriaandwatermay residelongenoughtohaveasubstantialeffectonthe geochemistryinthatpartoftheaquifer(BylandWilliams, 2000;Byletal.,2001).Thegeochemistryintheseconduits canrangefromaerobictoanaerobicandisaffectedby rechargeevents. Thepotentialforcontaminationtoenterandspread rapidlythroughakarst-conduitsystemishigh,butthe responseofindigenousbacteriatodissolvedcontaminants inthesewater-filledconduitsystemsispoorlyunderstood. Barton(2006)mentionsabacteriumidentifiedinCarlsbad Caverns,NewMexico,thatcandegradecomplexaromatic compoundslikebenzothiazoleandbenzenesulfonicacid. Otherbacteria,collectedinLechuguillaCave,New Mexico,possesstheabilitytofixnitrogen,metabolize complexaromaticcompounds,turnoverlipids,and scavengescarcenutrients(BartonandJurado,2007). NorthrupandLavoie(2001)conductedastudythat showedbacteriathriveinkarstsystemsunderavarietyof redoxconditions,thusdemonstratingthattheyhave adaptedtousingavarietyofterminalelectronacceptors. Inapreviousstudyinsouth-middleTennessee,Byland Williams(2000)demonstratedthatbacteriainakarst aquifercanbiodegradetrichloroethylenebyreductive dechlorination.Itisclearthatkarstbacteriahaveadapted tobiodegradeavarietyofcompoundsunderabroadrange ofgeochemicalconditions. ThekarstaquifersofsouthernKentuckyareknownfor beinghydrologicallyresponsivetorainevents(Ryanand Meimen,1996;VesperandWhite,2006).Consequently,it wouldbeexpectedthatsomeoftheevolutionaryadaptations withinthenativemicrobialcommunitieswouldfacilitate copingwithsuddenchanges.In2001,theU.S.Geological Surveyconductedastudytoexamineadaptationsofthe indigenousbacteriafoundinwater-filledkarstconduit systemsbymonitoringbacterialresponsetojetfuel contaminationandpHchanges.Thescopeofthestudy includedthecollectionofbacterialcommunitiesfromclean andfuelcontaminatedportionsofthesameaquiferandthe comparisonoftheirpopulationsizes,buoyantdensities, Gramtypes,andaveragecellsizes.Thispaperexamines adaptations,sizeandquantityofbacteria,andratesoffuel biodegradationunderbothaerobicandanaerobicconditions.STUDYAREADESCRIPTIONThreewellslocatednearanairfieldinsouth-central Kentuckywereselectedforthisstudybecausethey representedtwodistinctareasofwaterqualityinthekarst aquifer:anareacontaininguncontaminatedgroundwater andanareawherethegroundwaterhadbeencontaminated byjetfuel(Fig.1aand1b).Twofuel-contaminated bedrockwells(MW-1andMW-2)werelocateddowngradientfromanuncontaminatedbedrockmonitoringwell (MW-3)locatedapproximately100feetupgradientfrom MW-1and150feetupgradientfromMW-2(Fig.1a). Quarterlysamplinghaddetectednopetroleumhydrocarbonsinthecleanwellduringtheprevious10years(Dames andMoore,Inc.,2001).Theairfieldhadseveralreported fuelleaksoverseveraldecadesofintensiveuse.Wells drilledandscreenedinthebedrockduringthe1980s confirmedthepresenceofdissolvedfuelsinspecificareas ofthekarstaquifer(Ewersetal.,1992).ThepHfluctuated between6.4and7.8inthecleanwellandcontaminatedwell MW-2.Thewellswerecasedwith4-inchPVCpipeand screenedintheupperbedrockat120to136feetbelow groundsurface(Fig.1b).METHODSANDMATERIALSWatersamplesforthisstudywerecollectedinMay, June,September,andDecemberof2001fromwellsMW-3 andMW-1.WellMW-2wasaddedafterasuddenincrease inpHwasobservedinMW-1.ThepHchangefrom approximately7to12wasduetotheover-drillingofa 31-m(100-ft)deepwellfilledwithashmaterialthatwas initiallydesignedtobeusedasananodetoreducefuel-pipe corrosionaroundtheairfield(Fig.1a).Theanodewellwas convertedtoanextractionwell,butintheprocessofoverdrillingandgroutingthenewwell,thepHofthe surroundingaquiferjumpedto12.Thisextremeincrease inpHprovidedauniqueopportunitytoobservehowthe bacteriainthepH-alteredpartoftheaquiferwould respondtoasuddenandprolongedchangefromneutralto stronglyalkalineconditions.ThepHinthealternatewells MW-2andMW-3didnotincreaseabovepH7.8,andit appearedtobeunaffectedbythepH-alteringactivities closetowellMW-1.COLLECTIONOFGROUNDWATERANDBACTERIAWatersampleswerecollectedbyloweringtheintakeof acleaned,decontaminatedGrundfosjetpumpinthewells tospecifieddepthsequivalenttotheconduitopenings(Any useoftrade,firm,orproductnamesisfordescriptive purposesonlyanddoesnotimplyendorsementbytheU.S. Government).Forexample,inwellMW-3,thepump intakewasloweredto40mbelowthetopofcasingbecause geophysicalloggingindicatedtherewasa5cmwater-filled conduitopeningatthislevel.Thewaterwaspumpedata rateof1.5to3Lmin2 1untilspecificconductanceand temperatureremainedsteadyforfifteenminutesormore. Thepurginggenerallytookthirtytofortyminutes.Water levelsinthewelldidnotfluctuateduringpumping, indicatingthatthewaterbeingcollectedwasprimarily drawnfromthebedrockopeningsandnotfromthe stagnantwell-casingwater.Thegroundwatercollected rangedfromcleartoslightlyturbid,withasteady temperatureof14.7 u C.Thespecificconductanceranged from340to440mScm2 1inthecleanwelland340to 600mScm2 1inthecontaminatedwells,dependingon recentrainevents.T.D.BYL,D.W.METGE,D.T.AGYMANG,M.BRADLEY,G.HILEMAN,ANDR.W.HARVEYJournalofCaveandKarstStudies, August2014 N 105

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Thejetpumpusedtocollectwatersampleswas relativelynewandhadaTeflonhose.Betweenuses,the pumpwascleanedwithaweak0.3%bleachsolutionand rinsedwithheat-sterilizeddistilledwatertoremove hypochloriteresidue.Qualitycontroltestsconductedon thedecontaminatedpumpsystemfoundlessthan10 bacteriaperLwater,indicatingtherewasinsignificant microbialcross-contaminationduetothepumpsystem. Thegroundwatersamplesforgeochemicalandbacterial analyseswerecollectedinclean,sterile,1Lbrownglass bottles.Samplesforanalysisofvolatileorganiccompounds (VOCs)werecollectedinclean,40mLbrownglassbottles. Allbottleswereover-filledtoavoidheadspaceandprevent samplevolatilizationandsealedwithasterilecap.The bottleswereplacedoniceandtransferredtothelaboratory atTennesseeStateUniversityinNashville,Tennessee,for microbial,VOC,andgeochemicalanalysis.VOCanalysis wasdoneonaSyntexgaschromatographequippedwitha Figure1.Mapofsiteinvestigationarea,planview(a),andaconceptualcrosssectionofthewellsusedinthestudy(b).Well MW-2waspollutedwithfuel,andwellMW-3wasclean.ThewaterinwellMW-1changedduringthestudyduetothedrilling ofnearbywellANODE.ADAPTATIONSOFINDIGENOUSBACTERIATOFUELCONTAMINATIONINKARSTAQUIFERSINSOUTH-CENTRALKENTUCKY106 N JournalofCaveandKarstStudies, August2014

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purge-and-trapsystem,30mby0.32mm,1.8mmsilica-film capillarycolumn,argoncarryinggas,andmicro-argon ionizationdetector.Thelowerdetectionlimitfortoluene andbenzeneonthisGCwas0.5microgramperliter (mgL2 1).Everyfourthsamplewaseitheraduplicate sampleorastandardofknowntolueneconcentration.A completecalibrationcurveofbenzene,toluene,ethylbenzene,andxyleneswasrunpriortoandafterthemicrocosm samples.Benzeneandtolueneweretheonlymonoaromaticringcompoundsidentifiedinthewater.Additionalunidentifiedpeakswerepresentinthesamples. ResultsoftheVOCanalysiswereorganizedandgraphed onaspreadsheet.Bacterialsamplesthatwereanalyzedfor buoyantdensity,size,andmicroscopedirectcountswere packedoniceandshippedovernighttotheUSGS laboratoryinBoulder,Colorado.BACTERIALQUANTIFICATION,FLAGELLA,ANDAVERAGESIZETwomethodswereusedtoquantifythebacteria,a directcountonthemicroscopeandaliquidculturing method.Thedirectcountmethodcountsallbacteria,even thosethataredormant,whereastheliquidculturing methodprovidesapopulationestimatebasedonthe bacteriathatareactivelydividingandgrowing.Additional microscopicexaminationwasdonetoassessthepresence orabsenceofflagellaandtheGram-stainingcharacteristics ofthebacteria.TraditionalGramstainingwasconducted, andtheresultwasviewedunderabright-fieldmicroscope (Beveridge,2001).Bacteriaflagellastainingandviewing wasdoneasdescribedinGrossartetal.(2000). Directcountandsizefrequencyanalysesoffree-living bacteriaweredoneusingaNikonOptiphotIIepifluorescencemicroscopeandanITCimageprocessorconnected toapersonalcomputer,aDageSIT66blackandwhite camera,andaSonyblackandwhitemonitor.Theimage systemwasoptimizedtoanalyzeandcalculatelength, width,area,andperimeteroffluorescent-stained(acridine orange)bacteriainsamplespreviouslyanalyzedfor bacterialabundance.Measurementsfromtheimagesystem werestandardizedusingfluorescent-stained0.45,0.95,and 1.07mmmicrospheresinordertoconvertpixelmeasurementstomicrometers.Allanalyseswereperformedat microscopemagnificationsof788to1260. BiologicalActiveReactionTests(BART)wereconductedtoestimatetheculturableheterotrophicaerobic bacteriainthegroundwater.TheBARTassaysuse selectivemediatogrowparticulargroundwaterbacterial types(DBI,2004).TwentymLofgroundwaterwere transferredfromasamplebottle,havingbeenvigorously shakentore-suspendthebacteria,intotheBARTvials, sealed,andincubatedinthedarkat22 u Cfor7days.The vialswerecheckedevery24hoursforvisiblesignsof bacteriagrowth,whichwasevidentbydye-colorchanges andcloudiness,asdescribedbyCullimore(2008,chapter 9).Growthpatternswererecordedandcomparedtothe growthchartstoestablishanestimateoftheculturable bacterialconcentrationinthegroundwater.Resultswere reportedasbacteriapermL.BUOYANT-DENSITYANDDENSITY-GRADIENTDETERMINATIONSBuoyant-densitydeterminationswereperformedusing themethoddescribedbyHarveyetal.(1997).Approximately2Lofuncontaminatedgroundwateror1Lof contaminatedgroundwaterwasfilteredthrough47mm (diameter),0.2mm(poresize)polycarbonatemembrane filtersat 2 0.3atmospheretransmembranepressure. Bacteriaretainedbythefiltersweregentlywashedwith sterilesalinesolution(2mMNaCl,pH6.8)and resuspendedinsterile0.15MNaCltoprovideacloudy suspensionconsistingof1to400cellspermL.Microscopic examinationrevealedthatthevastmajorityofbacteria weresingleandnotattachedtoparticles.Fewnonbacterialcolloidswereobserved.Densitygradientswere createdwithintransparent,50-mLOakRidgeor10mL polycarbonatecentrifugetubesusingPercollIsolution (1.131gcm2 3,SigmaChemicalCompany,St.Louis, Missouri),acolloidalsilicasuspension,dilutedwith0.15M NaCl.Thetubeswerethenspunfor30minutesat15,000G inaSorvallRC-5Brefrigeratedcentrifuge.Theresulting gradientformedsymmetricallyoneithersideofthestarting densityof1.100gcm2 3.Brightlycoloreddensitymarker beadsobtainedfromSigmaChemicalCompanywereused toindicatespecificbuoyant-densityvaluesalongthe longitudinalaxisofthetubes.Aliquots(2.5mL)ofthe bacterialsuspensionswerecarefullylayeredonthetopof thepre-formedgradients.Gradienttubescontainingthe bacteriawerespunat15,000Gfor1hour.Theequilibrium positionsofthebacterialpopulationswereobservedas distincttranslucentbands.Thebuoyantdensitiesofthe populationswereindicatedbythepositionofthebacterial bandsrelativetothoseofthemarkerbeads.Theband thickness(inmm)providedasemi-quantitativemeasureof themicrobialpopulationcorrespondingtoaparticular buoyantdensity.Thetotalthicknessofthebandsequals 100percent,andeachbandrepresentsasubsetor percentageofthattotalbandthickness.BATCHMICROCOSMSFORBIODEGRADATIONSTUDIESTherateatwhichtheculturedbacteriadegradedfuels underaerobicoranaerobicconditionsandunderdifferent pHconditionswasthesubjectofmicrocosmstudies. Stagnantbatchmicrocosmswereestablishedusingraw, unfilteredwatercollectedfromeitherwellMW-3orwell MW-2.WaterfromwellMW-3wasusedtosetupthe aerobicmicrocosmsbecausethewatersconsistently haddissolvedoxygenreadingsof3mgL2 1orhigher. Anaerobicmicrocosmswereestablishedusingwaterfrom wellMW-2,whichconsistentlyhadlessthan0.5mgL2 1dissolvedoxygen.Themicrocosmsconsistedof300-mL brown,biological-oxygen-demand(BOD)glassbottlesT.D.BYL,D.W.METGE,D.T.AGYMANG,M.BRADLEY,G.HILEMAN,ANDR.W.HARVEYJournalofCaveandKarstStudies, August2014 N 107

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incubatedat25 u C,whichisapproximately10degrees warmerthannaturalconditionsintheconduit.Microcosmsweresampledatfivedifferentharvesttimeswith threereplicatesperharvest,aerobicandanaerobicconditions,foratotalofthirtymicrocosms.Aerobicmicrocosms wereharvestedondays0,1,3,5,and7.Anaerobic microcosmswereharvestedondays0,2,5,7,and10.The dissolvedoxygenlevelsweremeasuredineachmicrocosm uponharvesttoconfirmaerobicandanaerobicconditions usingYSI-5100BODmeters.Thedatafromthefifth harvestfortheaerobicmicrocosmswerenotusedbecause theoxygenhadbeenconsumed,andthemicrocosmshad goneanaerobic.AdditionalsterileandpH-alteredmicrocosmswereestablishedforcomparison.ThepHwas adjustedfrom6.85withsodiumhydroxideorhydrochloric acidtoachievepHof2,5,7,9,and12.RESULTSBACTERIALQUANTIFICATION,FLAGELLA,SIZE,ANDBUOYANTDENSITYThemicrobialpopulationinthethreesampledwells rangedfrom200,000toalmost4,000,000bacteriapermL (Fig.2).Populationgrowthmeasuredbydirectcountsand BARTassayshadasimilarpatternwiththeexceptionof contaminatedwellMW-1.Asmentionedearlier,thepHin MW-1increasedfrom7to12asaresultofnearbydrilling activitiesinlateMay,betweenthefirstandsecond samplingevents.Alkalineconditionswilloftenhamper microbialmetabolismandpromotedetachmentofbacteria fromaquifersurfaces(Harveyetal.,2010).Thesharp increaseinpHatMW-1resultedinanincreaseinbacteria usingthedirect-countmethodintheJune2001sample,but a99.99%dropinviablebacteriadeterminedbytheBART method.Enumeratingenvironmentalbacteriausingtraditionalculturingtechniques,suchasagarmediaplates,can resultinquestionablebacterialcountsduetoalow cultivatablepercentage(Bartonetal.,2004).Butthe BARTtests,whicharealsogrowth-basedassays,did provideimportantinformationconcerningtheviabilityof thebacteria.Usingthedirectcountmethodalonewould haveprovidedafalsesensethatthispartofthekarst aquiferhad3-millionbacteriapermLinJune,whenthe viablebacteriacountreallydroppedfrom 1millionto 100bacteriapermL. WiththeexceptionofthepH-shockedwell(MW-1), therewasgenerallygoodagreementbetweenthedirect countsandtheBARTestimates.Therewasariseandfall inbacterianumbersduringthesamplingperiod.Unfortunately,therewereinsufficientdatatoconcludewhether populationfluxwasduetoseasonalpatternsorrecent weatherevents.Itisclear,however,thatbacterial concentrationsinthekarstconduitwatercanfluctuate anorderofmagnitudeoverseveralmonths.TheBART methodgenerallyprovidedlowerpopulationestimates thanthedirectcountmethod,indicatingthatsomebacteria typesdidnotgrow,andwerenotmeasured.However,the BARTbacterialestimateswereclosertothedirectcounts thantraditionalheterotrophicplatecounts,whichwerein thehundredstothousandsofcolonyformingunitspermL (datanotshown).TheBARTassayisdifferentfrom conventionalplate-countmethodsinseveralaspects.The BARTsystemallowsthewater-bornebacteriatoremain free-livinginthewaterortoattachtoasurfaceand maintainsymbioticassociations.TheBARTvialsprovide nutrientandoxygengradientsinthetestwaters,which offerdifferenttypesofbacteriatheiroptimalhabitatzones. MicrobialsampleswereGramstainedandviewedusing bright-fieldmicroscopy.Gramstainhastraditionallybeen usedbymicrobiologiststohelpidentifybacteriabasedon cell-wallmake-up.Aquiferbacteriacanaltertheircell-wall chemistryunderdifferentenvironmentalconditions(Harveyetal.,2011),therebyaffectingtheirabilitytoholdthe Gramstain(Beveridge,2000).Thus,theobjectiveofthis Gramstainingeffortwastoprovideinformationabout relativedifferencesintheaveragecompositionofthecell envelopeincleanversuscontaminatedconduits.Bacteria collectedfromthecleanwell,MW-3,wereapproximately 95%Gram-negativerods,whereasbacteriacollectedfrom MW-1andMW-2were68%Gram-negative.Theincrease inGram-positivebacteriainthepresenceoffuelhasbeen observedbyotherinvestigators(Fahyetal.,2008;Ramos etal.,2002).TheyfoundGram-positivebacteriawere bettercompetitorsthanGram-negativeorganismsathigh benzeneconcentrations,whichsuggeststhatsomeGrampositivebacteriaaretolerantofhighfuelconcentrations andcanplayaroleinthenaturalattenuationoffuel. Seguraetal.(1999)listthreemechanismsusedbyGrampositivebacteriatoleranttoaromatichydrocarbons, metabolizingthetoxichydrocarbons,whichcancontribute totheirtransformationintonon-toxiccompounds,rigidFigure2.Bacteriaquantifiedinwaterscollectedfromthree wellsintersectingawater-filledconduitinsouth-central Kentucky,asmeasuredbymicroscopecountingandby BARTanalyses.ThepHofwellMW-3changedfollowing May2001.ADAPTATIONSOFINDIGENOUSBACTERIATOFUELCONTAMINATIONINKARSTAQUIFERSINSOUTH-CENTRALKENTUCKY108 N JournalofCaveandKarstStudies, August2014

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ifyingthecellmembranebyalteringthecompositionof phospholipids,andsecretingthetoxiccompoundinan energy-dependentprocess. Groundwaterbacteriahavebeenknowntousedifferent methodsoflocomotion.Groundwaterbacteriamayuse directedlocomotioninthepresenceofchemicalgradients (chemotaxis)toactivelypursueenergysources,thus facilitatingbiodegradation(FordandHarvey,2007).Many aquaticbacteriapropelthemselvesusingflagella(Harschey, 2003),glidebyoneofseveralmethods(McBride,2001),or modifytheirbuoyantdensityandfloatwiththegroundwater(Harveyetal.,1997).Theuseofflagellaforlocomotionis energyintensiveandmaybelimitedinanaerobicenvironments,whererapidexpenditureofenergyiscostlytothe organism.Alternatively,modifyingtheorganismsbuoyant density,therebyallowingthebacteriumtofloatinthewaterfilledconduitopenings,wouldbealow-energymethodof locomotion.Thisstudytookasimplelookatbacteria flagellaandbuoyancymechanismscollectedfromtheclean andfuel-contaminatedpartsoftheaquifer.Glidingmechanismswerenotpartofthisstudy. Flagellaaredifficulttoobservebecausetheyaresmall andbacteriahavedevelopedmechanismstorapidlydrop theirflagellawhenenvironmentalconditionschange(Ford andHarvey,2007;Harschey,2003).Nonetheless,we observedareasonablenumberofflagellaattachedto bacteriacollectedfromcleanwellMW-3(Fig.3a).Detached flagellawerealsoobserved.Bycomparison,therewereno flagellaobserved,attachedordetached,tothebacteria collectedfromfuel-contaminatedwells(Fig.3b).These resultswerenotquantitative,buttheydosuggestthata discernablefractionofthebacteriainMW-3useflagellato moveinthewatercolumn.Alternatively,thesefindings suggestthatflagellaarenotacommonmodeofmotilityin thefuel-contaminatedpartoftheaquiferlikelybecauseof theanaerobicenvironment,whichisnotconducivetothe oxygen-dependentphosphorylationenergypathway. BacterialivinginwaterfromwellsMW-1orMW-2 appeartoavoidmotilitymechanismsthatrequireoxygendependentrespirationbecausetheaquiferwasdissolvedoxygenpoor(lessthan1mgL2 1throughouttheyear-long study).ThepartoftheaquiferintersectedbywellMW-3 haddissolved-oxygenlevelsrangingfrom3to8mgL2 1.It isreasonabletoassumethebacteriafromoxygen-poor partsofthekarstaquiferhaveadaptedamorepassive methodtomoveaboutintheirenvironment. Animportantpropertygoverningmicrobialmobilityin groundwaterenvironmentsisbuoyantdensity,alsoknown asspecificgravity.Figure4depictsthebuoyant-density distributionofthebacteriafromeachwellasarelative percentagederivedfromthebacteriabandthicknessafter thedensity-gradientcertifugation.Onehundredpercentof thebacterialpopulationcollectedfromMW-1onMay14 hadaverylightspecificgravityof1.02to1.03gcm2 3.This microbialpopulationwouldberelativelybuoyantand inclinedtofloatinthewatercolumn.Theconstructionof awellnearbyinthelastweekofMay,subsequentlyraising thepHto12,coincidedwithashiftinbuoyantdensity.The bacteriabecamedenserandapttodescendinthewater column.ThemicrobialpopulationinwellMW-2hada slightlylighterbuoyantdensity(1.02to1.04gcm2 3) comparedtothebacteriacollectedfromcleanwellMW-3. BacteriacollectedfromMW-3hadamoderatebuoyant densitythroughoutthesamplingperiod.Thedatagenerated bythismethodwerenotsuitableforstatisticalanalysis. Thesizedistributionofthecollectedbacteriafromwells MW-1andMW-3(Fig.5)rangedfrom0.2to4.0mm. Therewasasubtleexcessinlengthinthebacteriacollected fromMW-1priortothepHshiftwhencomparedto bacteriacollectedfromMW-3.Themedianlengthofthe bacteriacollectedfromMW-3was0.6mm.Themedian bacteriallengthfoundinMW-1was0.8mm.However, becausetherewasalargerangeofbacterialsizesinthe cleanandcontaminatedwells,ananalysisofvarianceFtestdidnotfindthemedianlengthstobesignificantly different.BATCHMICROCOSMSFORBIODEGRADATIONSTUDIESBacteriacollectedfromthewellsinthisstudyappearto haveadaptationsthatpermitthemtoswimincleanwaters andfloatinanaerobicfuel-contaminatedwaters.These adaptationsallowthebacteriatoovercomethelimited surfaceareaforbiofilmdevelopment.Becausetheyhave adaptedmechanismsthatenhancetheirabilitiestolive unattachedtorocksurfaces,thereisalsoincreased opportunityforbacteriatoconsumedissolvedconstituents suchasbenzeneandtolueneinthewatercolumn.Results fromthemicrocosmstudyinthelaboratoryindicatedthat theaerobicandanaerobicbiodegradationratesofbenzene andtoluenewerepseudo-firstorderasexponential equations.Thefirst-orderrateconstantforaerobic biodegradation(Fig.6)was0.64forbenzene( R25 0.97) and1.03fortoluene( R25 0.93).Underaerobicconditions, Figure3.Rod-shapedbacteriafromcleanwellMW-3with polarflagella(arrow)(a),androd-shapedbacteriafrom contaminatedwellMW-1orMW-2(b).Originalmagnification10003;lineis1mmlong.T.D.BYL,D.W.METGE,D.T.AGYMANG,M.BRADLEY,G.HILEMAN,ANDR.W.HARVEYJournalofCaveandKarstStudies, August2014 N 109

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toluenewasbiodegradedslightlyfasterthanthebenzene (Fig.6),buttherewasnotastatisticallysignificant difference.Thesterilecontrolslostlessthan3%overthe sametimeperiod. Thefirst-orderanaerobicrateconstantsfordegradation ofbenzeneandtoluenewereessentiallythesame,0.027and 0.031,respectively( R25 0.98forboth)(Fig.7),which demonstratedthatbacteriacollectedfromthethreewellsin thisstudyarecapableofbiodegradingfuelcompoundsunder aerobicandanaerobicconditions.Wedocumentedboth aerobicandanaerobicconditionsinthebedrockaquiferat thissite.ConditionsinwellsMW-1andMW-2were anaerobic,withlessthan1mgL2 1dissolvedoxygen,which wasprobablyduetouseofoxygenasthefinalelectron acceptorduringtheconsumptionofthedissolvedfuel.OPTIMUMPHFORFUELBIODEGRADATIONAllenzymes,includingthoseinvolvedinbiodegradation offuels,haveanoptimumpH.Someenzymescanfunction inabroadrangeofpHvalues.Othersrequireanarrowand Figure5.Bacterialsizedistributionformicrobescollectedfromaclean(MW-3)andfuel-contaminated(MW-1)karstaquifer insouthernKentucky.Notelogarithmicscale.Thedifferenceinthemediansisnotstatisticallysignificant. Figure4.Buoyantdensitiesofbacteriacollectedfromthethreekarstwellsshownasapercentofthetotalpopulationat sampledatesin2001.MW-1,fuel-contaminatedwellthatexperiencedpHshiftto12priortoJune;MW-2,fuel-contaminated welladdedtothestudyafterJune;MW-3,cleanreferencewell.ADAPTATIONSOFINDIGENOUSBACTERIATOFUELCONTAMINATIONINKARSTAQUIFERSINSOUTH-CENTRALKENTUCKY110 N JournalofCaveandKarstStudies, August2014

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controlledpHrangeinordertofunction.Aerobicsamples fromcleanwellMW-3weretreatedwithbenzeneandtoluene levelsof4.8and15.3mgL2 1andhadpHadjustedtovarious levelsasdescribedinthemethodssection.Resultsfromthis microcosmstudyshowthatthemicrocosmswithpH2and12 hadnonoticeableconsumptionofdissolvedoxygenovera 12-dayperiod;whereastheothermicrocosms(pHvaluesof5, 7,and9)becameanaerobicaroundday5(Fig.8). Thebenzeneandtolueneinthemicrocosmswere measuredandthedataweretransformedtopercent benzeneandtolueneremainingonday5(Fig.9).The twomicrocosmtreatmentswithpH2and12waterdidnot haveanytolueneorbenzeneremovaloverthefive-day period.Thegreatestamountofremovalwasfoundin microcosmswithneutralpH,whichindicatedthatthe optimumpHforaerobictolueneorbenzenebiodegradationwasbetweenpH6and7.DISCUSSIONInspiteoftheaustereconditionspresentinkarst aquifersinsouth-centralKentucky,thisstudyshowedthat theseenvironmentscanharborlargenumbersofbacteria, from200,000to3,000,000bacteriapermL.Manyofthe bacteriaintheclean,oxygenatedgroundwaterpossessed flagellaformotility,whereasbacteriafromtheoxygendepleted,fuel-contaminatedzoneswerelesslikelytohave flagellaandmorelikelytoaltertheirbuoyantdensityto facilitatetheiradvectivetransport.Amicroorganisms buoyantdensityaffectsitsfrequencyofcollisionwith conduitsurfacesbecauseitaffectstherateofsettling. Buoyantdensityofbacteriacollectedinthesethreewells rangedfromlight(1.02gcm2 3)todense(1.08gcm2 3).A similardifferenceinsizeandbuoyantdensitiesof groundwaterbacteriaobservedinpristineversuscontaminatedzonesofasandyaquiferinCapeCod,Massachusetts,resultedinanestimated64-folddifferenceintheir settlingrates(Harveyetal.,1997).However,therelationshipbetweenbacteriashape,size,andsedimentation velocityisnotfullyunderstood,andotherfactorsmay affectsedimentationrate.Thebacteriainthefuelcontaminatedpartswere,onaverage,largerthanthose Figure6.Aerobicbiodegradationofmono-aromaticfuel compoundsbenzeneandtoluenebybacteriacollectedfrom cleanwellMW-3,comparedtosterilecontrolsamples.Error barsareonestandarddeviationofthreereplicates. Figure7.Anaerobicbiodegradationofmono-aromaticfuel compoundsbenzeneandtoluenebybacteriacollectedfrom pollutedwellMW-2,comparedtosterilecontrolsamples. Errorbarsareonestandarddeviationofthreereplicates. Figure8.Dissolvedoxygenlevelsinthestaticmicrocosms incubatedovera12-dayperiodestablishedusingrawwater fromwellcleanwellMW-3withadjustedpH.Sampleswith near-neutralpHwereanaerobicbyday5.T.D.BYL,D.W.METGE,D.T.AGYMANG,M.BRADLEY,G.HILEMAN,ANDR.W.HARVEYJournalofCaveandKarstStudies, August2014 N 111

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collectedfromacleanpartoftheaquifer.However,the mediandifferenceinsizewasnotstatisticallysignificant duetothewiderangeofsizesinbothcleanandfuelcontaminatedpartsoftheaquifer. Bacteriafromtheclean,aerobicsectionandthe anaerobic,fuel-contaminatedsectionoftheaquiferwere abletobiodegradebenzeneandtoluene.Thehalf-livesfor aerobicbenzeneandtoluenebiodegradationat25 u Cwere 1.1and0.7days,respectively.Theaquiferinthisstudywas asteady14.7 u Coverthecourseofthisstudy.Assuming theArrheniusprincipleappliestothebiodegradation kinetics,whichstatesthatforevery10 u Cincreasethere isanapproximatedoublingofthereactionrate(Laidler, 1984),thehalf-lifeofbenzeneandtoluene insitu shouldbe closerto1.5to3days.Bycomparison,underanaerobic conditions,thebenzeneandtoluenehadlaboratoryhalflivesof23to26days,whichshouldbecloserto50daysin thenaturalanaerobicaquiferenvironment.Theoptimum pHobservedinthelaboratorymicrocosmsunderaerobic conditionswasbetween6and7.ThispHrangeis reasonable,consideringthisaquifersystemgenerallyhad apHrangingfrom6.4to7.8,withtheexceptionofwell MW-1,whichwasinfluencedbydevelopmentofanewwell incloseproximity.ThepHinwellMW-1wasinitiallypH 7butincreasedtopH12.Thisstudyshowedthatthereare largenumbersofbacteriainthewater-saturatedkarst conduitsofsouth-centralKentuckythatareadaptedtotheir environmentandfullycapableofdegradinglightfuels.ACKNOWLEDGEMENTSTheauthorswishtothankDr.L.King-Thomas,J. Cartwright,H.Welch,S.Cooper,andtwoanonymous journalreviewersfortheirconstructivereviewcomments andsuggestions.REFERENCESBarton,H.A.,Taylor,M.R.,andPace,N.R.,2004,Molecularphylogeneticanalysisofabacterialcommunityinanoligotrophiccave environment:GeomicrobiologyJournal,v.21,p.11.doi:10.1080/ 01490450490253428. Barton,H.A.,2006,Introductiontocavemicrobiology:Areviewforthe non-specialist:JournalofCaveandKarstStudies,v.68,no.2, p.43. Barton,H.A.,andJurado,V.,2007,Whatsupdownthere?Microbial diversityincaves:Microbe,v.2,no.3,p.132. Barton,H.A.,andNorthup,D.E.,2007,Geomicrobiologyincave environments:past,currentandfutureperspectives:JournalofCave andKarstStudies,v.69,no.1,p.163. Beveridge,T.J.,2001,UseoftheGramstaininmicrobiology:Biotechic& Histochemistry,v.76,no.3,p.111. Byl,T.D.,andWilliams,S.D.,2000,Biodegradationofchorinatedethenes atakarstsiteinMiddleTennessee:U.S.GeologicalSurveyWaterResourcesInvestigationsReport99-4285,58p. Byl,T.D.,Hileman,G.E.,Williams,S.D.,andFarmer,J.J.,2001, Geochemicalandmicrobialevidenceoffuelbiodegradationina contaminatedkarstaquiferinsouthernKentucky,June1999, in Kuniansky,E.L.,ed.,U.S.GeologicalSurveyKarstInterestGroup Proceedings,St.Petersburg,Florida,February13,2001:U.S. GeologicalSurveyWater-ResourcesInvestigationsReport01-4011, p.151. Byl,T.D.,Hileman,G.E.,Williams,S.D.,Metge,D.W.,andHarvey, R.W.,2002,Microbialstrategiesfordegradationoforganiccontaminantsinkarst, in Aikens,G.R.,andKuniansky,E.L.,eds.,U.S. GeologicalSurveyArtificialRechargeWorkshopProceedings,Sacramento,California,April2,2002:U.S.GeologicalSurveryOpenFileReport02-89,p.61. Cullimore,D.R.,2008,PracticalManualofGroundwaterMicrobiology, secondedition:BocaRaton,Florida,TaylorandFrancisGroup,376p. DamesandMoore,Inc.,2001,RemedialInvestigationsReport,Fort Campbell,Kentucky:preparedforU.S.ArmyToxicandHazardous MaterialsAgency,AberdeenProvingGround,Maryland,contract number15-88-D-0008,2volumes. DBI,2003,BARTUserManual,2004edition,DroyconBioconceptsInc, Regina,Saskatchewan,54p.,http://www.dbi.ca/BARTs/Docs/Manual. pdf[accessedMarch1,2013] Ewers,R.O.,Duda,A.J.,Estes,E.K.,Idstein,P.J.,andJohnson,K.M., 1992,Thetransmissionoflighthydrocarboncontaminantsin limestonekarstaquifers, in ProceedingoftheThirdConferenceon Hydrogeology,Ecology,Monitoring,andManagementofGround WaterinKarstTerranes:Dublin,Ohio,WaterWellJournal Publishing,p.287. Fahy,A.,Ball,A.S.,Lethbridge,G.,McGenity,T.J.,andTimmis,K.N., 2008,HighbenzeneconcentrationscanfavourGram-positivebacteria ingroundwatersfromacontaminatedaquifer:FEMSMicrobiolial Ecology,v.65,p.526.doi:10.1111/j.1574-6941.2008.00518.x. Florea,L.J.,Paylor,R.L.,Simpson,L.,andGulley,J.,2002,KarstGIS advancesinKentucky:JournalofCaveandKarstStudies,v.64, no.1,p.58. Ford,R.M.,andHarvey,R.W.,2007,Roleofchemotaxisinthetransport ofbacteriathroughsaturatedporousmedia:AdvancesinWater Resources,v.30,p.160817.doi:10.1016/j.advwatres.2006.05.019. Grossart,H.-P.,Steward,G.F.,Martinez,J.,andAzam,F.,2000,A simple,rapidmethodfordemonstratingbacterialflagella:Applied Figure9.Aerobicbiodegradationofbenzeneandtoluenein microcosmswithadjustedpHusingrawwaterfromwell MW-3.They-axisdisplaysthepercentbenzeneortoluene remaininginthemicrocosmsafter5days.Nodegradationof theorganiccompoundswasobservesattheextremesofpH. Sterilecontrolslostlessthan2%overthe5daysof incubation.Threereplicatespertreatment;errorbars representcoefficientofvariation.ADAPTATIONSOFINDIGENOUSBACTERIATOFUELCONTAMINATIONINKARSTAQUIFERSINSOUTH-CENTRALKENTUCKY112 N JournalofCaveandKarstStudies, August2014

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andEnvironmentalMicrobiology,v.66,p.3632.doi:10.1128/ AEM.66.8.3632-3636.2000. Haack,S.K.,Metge,D.W.,Fogarty,L.R.,Meyer,M.T.,Barber,L.B., Harvey,R.W.,LeBlanc,D.R.,and Kolpin,D.W.,2012,Effectson groundwatermicrobialcommunitiesofanengineered30-day in-situexposuretotheantibiotic sulfamethoxazole:Environmental ScienceandTechnology,v.46,no.14,p.74787486.doi:10.1021/ es3009776. Harschey,R.M.,2003,Bacterialmotilityonasurface:manywaystoa commongoal:AnnualReviewofMicrobiology,v.57,p.249. doi:10.1146/annurev.micro.57.030502.091014. Harvey,R.W.,Smith,R.L.,andGeorge,L.,1984,Effectoforganic contaminationuponmicrobialdistributionsandheterotrophicuptake inaCapeCod,Mass.,aquifer:AppliedandEnvironmental Microbiology,v.48,p.1197. Harvey,R.W.,Metge,D.W.,Kinner,N.,andMayberry,N.,1997, Physiologicalconsiderationsinapplyinglaboratory-determinedbuoyantdensitiestopredictionsofbacterialandprotozoantransportin groundwater:Resultsofin-situandlaboratorytests:Environmental ScienceandTechnology,v.31,p.289.doi:10.1021/es960461d. Harvey,R.W.,Metge,D.W.,Barber,L.B.,andAiken,G.R.,2010,Effects ofalteredgroundwaterchemistryuponthepH-dependencyand magnitudeofbacterialattachmentduringtransportwithinan organicallycontaminatedsandyaquifer:WaterResearch,v.44, p.106271.doi:10.1016/j.watres.2009.09.008. Harvey,R.W.,Metge,D.W.,Mohanram,A.,Gao,Xiaodong,and Chorover,J.,2011,Differentialeffectsofdissolvedorganiccarbon uponre-entrainmentandsurfacepropertiesofgroundwaterbacteria andbacteria-sizedmicrospheresduringtransportthroughacontaminated,sandyaquifer:EnvironmentalScienceandTechnology,v.45, p.325259.doi:10.1021/es102989x. Hutson,S.S.,1995,Ground-waterUsebyPublic-SupplySystemsin Tennesseein1990:U.S.GeologicalSurveyOpen-FileReport94-483, 1sheet. Kolbel-Boelke,J.,Anders,E.-M.,andNehrkorn,A.,1988,Microbial communitiesinthesaturatedgroundwaterenvironment,II.Diversityof bacterialcommunitiesinaPleistocenesandaquiferandtheirinvitro activities:MicrobialEcology,v.16,p.3148.doi:10.1007/BF02097403. Laidler,K.,1984,ThedevelopmentoftheArrheniusequation:Journalof ChemicalEducation,v.61,p.494.doi:10.1021/ed061p494. McBride,M.J.,2001,Bacterialglidingmotility:multiplemechanismsfor cellmovementoversurfaces:AnnualReviewofMicrobiology,v.55, p.49.doi:10.1146/annurev.micro.55.1.49. Northup,D.E.,andLavoie,K.H.,2001,Geomicrobiologyofcaves:a review:GeomicrobiologyJournal,v.18,p.199.doi:10.1080/ 01490450152467750. Quinlan,J.F.,1989,Ground-watermonitoringinkarstterranes: recommendedprotocolsandimplicitassumptions:LasVegas, Nevada,U.S.EnvironmentalProtectionAgency,Environmental MonitoringSystemsLaboratory,EPA/600/X-89/050,88p. Ramos,J.L.,Duque,E.,Gallegos,M.-T.,Godoy,P.,Ramos-Gonza lez,M.I., Rojas,A.,Tera n,W.,andSegura,A.,2002,Mechanismsofsolvent toleranceinGram-negativebacteria:AnnualReviewofMicrobiology, v.56,p.743.doi:10.1146/annurev.micro.56.012302.161038. Ryan,M.,andMeimen,J.,1996,Anexaminationofshort-termvariations inwaterqualityatakarstspringinKentucky:GroundWater,v.34, no.1,p.23.doi:10.1111/j.1745-6584.1996.tb01861.x. Schneider,K.,andCulver,D.C.,2004,Estimatingsubterraneanspecies richnessusingintensivesamplingandrarefactioncurvesinahigh densitycaveregioninWestVirginia:JournalofCaveandKarst Studies,v.66,p.39. Segura,A.,Duque,E.,Mosqueda,G.,Ramos,J.L.,andJunker,F.,1999, MultipleresponsesofGram-negativebacteriatoorganicsolvents: EnvironmentalMicrobiology,v.1,no.3,p.191.doi:10.1046/ j.1462-2920.1999.00033.x. Vesper,D.J.,andWhite,W.B.,2006,Comparativestormresponseof contaminantsinacarbonateaquifer,FortCampbell,KentuckyTennessee, in Harmon,R.S.,andWicks,C.,eds.,Perspectiveson KarstGeomorphology,Hydrology,andGeochemistryATribute VolumetoDerekC.FordandWilliamB.White:GeologicalSociety ofAmericaSpecialPaper404,p.26774.doi:10.1130/ 2006.2404(22). White,W.B.,2002,Karsthydrology;recentdevelopmentsandopen questions:EngineeringGeology,v.65,p.85.doi:10.1016/S00137952(01)00116-8. Wolfe,W.J.,Haugh,C.J.,Webbers,A.,andDiehl,T.H.,1997, Preliminaryconceptualmodelsoftheoccurrence,fate,andtransport ofchlorinatedsolventsinkarstaquifersofTennessee:U.S.Geological SurveyWater-ResourcesInvestigationsReport97-4097,80p.T.D.BYL,D.W.METGE,D.T.AGYMANG,M.BRADLEY,G.HILEMAN,ANDR.W.HARVEYJournalofCaveandKarstStudies, August2014 N 113

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AEROSOLIZEDMICROBESFROMORGANICRICH MATERIALS:CASESTUDYOFBATGUANOFROMCAVES INROMANIADANIELAR.BORDA1,RUXANDRAM.NASTASE-BUCUR1*,MARINASPI NU2,RALUCAURICARIU2,ANDJANEZMULEC3Abstract: Caveair,batguano,andswabsofbatfurfromcaveswithbatguanoin RomaniawereanalyzedbyusingRIDA HCOUNTcultivationplatesandstandard selectivemediafor Staphylococcus and Streptococcus .Thesamplesofguanovaried inconcentrationofcultivablechemoheterotrophicbacteria(max.1.931010CFU/g), coliforms(max.2.23108CFU/g), Escherichiacoli (max.1.03108CFU/g),andyeastsand molds(max.1.73107CFU/g).Thegravity-settlingprinciplewasappliedtosampleairborne microorganisms,andanewmethodwasdevelopedforevaluationofaerosolization potential.Incaveair,theconcentrationoftotalbacteriawashigherthanyeastandmolds. Inadditiontocoliforms,enterobacteria, E.coli ,andunidentifiedcultivablebacteriainthe airsamples,wealsoidentified Chryseomonasluteola Klebsiellapneumoniae Micrococcus Salmonella Staphylococcus ,and Streptococcus .Intheexperimentthatprovokedmicrobial aerosolizationfromguano,3.35%oftotalcultivablefungiwereaerosolized,upto0.10%of bacteria,and0.00%of E.coli .Theconcentrationof Staphylococcus intheairexceeded countsof Streptococcus. Thehighestconcentrationsofairbornemicroorganismswereon thegroundlevel.Usingcultivationplatesasarobustmethodwedemonstratedthatthe relativeproportionofmicrobialsubgroupsintheairremainedconstantindifferent seasons,withlowerconcentrationsofairbornemicrobiotaintheautumn.Cavesas simplifiednaturalsystemsdemonstratedcomplexrelationshipsbetweenatmospheric parametersandmicroorganisms.Batsintroduceintocavesvarying,butnotnegligible, concentrationsofmicrobesontheirfur.Caveswithguanohadrelativehighconcentration ofairbornemicrobesthatmayrepresentabiohazardforanimalsandhumans.INTRODUCTIONAirisanimportanthabitatformetabolicallyactiveand reproducingmicrobes(Womacketal.,2010)andavehicle forthetransportofdifferentmicroorganisms.When airborne,microorganismscantravelreasonabledistances. Forsomemicrobescertainairconditions,suchas desiccation,extremetemperature,UVradiation,orchemicalandradioactivestressors,canbelethal. Aerosolization,theproductionofanaerosol,resultsina finemistorspraycontainingminuteparticlesthatcontain biologicalparticles.Therearedifferenttypesofbioaerosol formationcausedbywind,animalsandhumans,orsplashing water(Mulecetal.,2012c).Itoccursinnaturalandmanmadeenvironments.Humanexposuretoaerosolsoforganicrichmaterialsgeneratesapotentialriskandcancause differenttypesofinfection.Thehealthhazardsofpoorair qualitycanbeassociatedwithairbornemicrobes,and exposuretoelevatedconcentrationsofmicroorganismscan leadtonumerousrespiratoryanddermatologicalinfections, allergies,andotherproblems(Fabianetal.,2005). Thestudyofbioaerosolsincontrolledlabconditions providesvaluableinformation,buttrialsundernatural conditionsprovideabetterinsightintothefateofminute biologicalparticles.Duetochangeableatmospheric conditionsandinterrelatedenvironmentalstressorssuch aswind,UV,andhumidity,somenaturaloutdoor environmentsprovidecomplexstudyconditions.Anatural systemthatislowinenvironmentalstressorsandrichin organicmaterialiskarstcavesharboringpilesofbat guano.Cavesaregenerallynaturallight-freeenvironments connectedwiththeoutsidebyoneormoreentries,and withhighrelativeairhumidity,constanttemperature,and lowornegligibleairmovements(Simon,2012;Whiteand Culver,2012).PoulsonandLavoie(2000)consideredbat guanooneofthemostimportantenergyinputsforcavesin temperateclimatezones.Guanoisanimportanthabitat,a sourceformicrobialaerosolization,andabiohazardfactor forhumansandbats,e.g. Histoplasmacapsulatum (Alteras, 1966;Julgetal.,2008). Theobjectivesofthisstudyweretodefinetherelations betweenatmosphericparametersandairbornemicroorganismsthatderivefrom insitu organicmatterincaves. *Correspondingauthor:ruxandra.nastase.bucur@academia-cj.ro1EmilRacovitaInstituteofSpeleology,DepartmentofCluj-Napoca,Romanian Academy,ClinicilorSt.5,POBox58RO-400006Cluj-Napoca,Romania2UniversityofAgriculturalScienceandVeterinaryMedicine,FacultyofVeterinary Medicine,CaleaMa nas tur3-5,RO-400372Cluj-Napoca,Romania3KarstResearchInstitute,ResearchCentreoftheSlovenianAcademyofSciences andArts,Titovtrg2,SI-6230Postojna,SloveniaD.R.Borda,R.M.Na stase-Bucur,M.Spnu,R.Uricariu,andJ.MulecAerosolizedmicrobesfromorganicrichmaterials:casestudy ofbatguanofromcavesinRomania. JournalofCaveandKarstStudies, v.76,no.2,p.114.DOI:10.4311/2013MB0116114 N JournalofCaveandKarstStudies, August2014

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CavesinRomaniawithbatguanowerestudiedtoobserve seasonalandair-stratificationeffectsonconcentrationof bioaerosols,aswellastoestimatethelevelofaerosolized microbesfromguanoandtheroleofbatsasvectorsfor microbialtransmission.Toestimatethepercentageof aerosolization,anewmethodtoinduceaerosolizationwas developedandtestedincaves.Resultsfromthestudyare usefulforcomparisonwithotherorganic-richenvironmentsthatcontainbiohazardmicrobes,suchasfarms, landfills,andwastewaterorcomposttreatmentplants.MATERIALSANDMETHODSSTUDYSITESSevencavesthatarerichinguanodepositsand populatedbybatsinthreebiogeographicandclimate regionsinRomaniawereselected:AvenulluiAdam(Adam Cave),PesteraCeta t eauaMaredinCheileTurzii(Ceta teauaMareCavefromGorgesofTurda,referredtointhe textandtablesasCheileTurziiCave),PesteraFusteica (FusteicaCave),PesteraLiliecilordelaGuraDobrogei (BatCavefromGuraDobrogei,referredtoasGura DobrogeiCave),PesteraMeziad(MeziadCave),Pesterade laRastoci/PesteraMagurici(RastociCave),andPestera Topolnita(TopolnitaCave)(Table1).ApartfromCheile TurziiCave,whichcontainsamaternityroost,the othercaveshostbatsallyearround,includingmaternity andhibernationcolonies.Themostprevalentbatspecies andnumberofspeciesineachcavearepresentedin Table2. AdamCaveislocatedinBaileHerculaneareainthe Domogled-CernaValleyNationalPark,whichhas700to 750mmofannualprecipitation(Munteanu,2011).The caveentranceisvertical,startingwithan11mshaft. Temperatureisconstantyearround,withanaverageof 27 u Cthatisattributedtointermittentsteamvapors;water Table1.Cavesstudied,withabbreviationsusedinthefigures.Thedistancefromthecaveentrancetothemostremote bioaerosolsamplingsiteisincluded.LithologyafterBandraburandRadu,1994,Bleahuetal.,1976,andTodoranandOnac 1987;biogeographicalregionsafterANPM,2013. Cave Biogeographical Region Entrance a.s.l.(m) Lithology StudiedDistance/ LengthofCave(m) AdamCave,AC Continental295Jurassic/Cretaceouslimestone25/169 CheileTurziiCave,CTAlpine 552Jurassiclimestone 36/120 FusteicaCave,FC Continental200Jurassic/Cretaceouslimestone47/1270 GuraDobrogeiCave,GDSteppe 46Jurassiclimestone 125/500 MeziadCave,MC Alpine 440Triassiclimestone 375/4750 RastociCave,RC Continental319Eocene/Oligocenelimestone 87/507 TopolnitaCave,TC Continental434Jurassic/Cretaceouslimestone320/20500 Table2.Batsinstudiedcaves;datafromBorda,2002a,b;Bordaetal.,2004;Burghele-Ba la cescuandAvram,1966;Carbonnel etal.,1996;Coroiuetal.,2007;PocoraandPocora,2011. Cave Presence Numberof Individuals Numberof BatSpeciesMainGuanoContributors AdamCave Allyear5000 8 Miniopterusschreibersii Myotiscapacinii Myotismyotis Rhinolophuseuryale CheileTurziiCaveSummer1500 3 Miniopterusschreibersii Myotismyotis / M.oxygnathus FusteicaCave Allyear1500 7 Miniopterusschreibersii Myotiscapacinii Myotismyotis / M.oxygnathus GuraDobrogeiCaveAllyear300 10 Miniopterusschreibersii Myotisdaubentonii Myotismyotis / M.oxygnathus MeziadCave Allyear50000 10 Miniopterusschreibersii Myotismyotis / M.oxygnathus RastociCave Allyear200 5 Myotismyotis / M.oxygnathus TopolnitaCave Allyear1500 10 Miniopterusschreibersii Myotismyotis / M.oxygnathusD.R.BORDA,R.M.NA STASE-BUCUR,M.SPI NU,R.URICARIU,ANDJ.MULECJournalofCaveandKarstStudies, August2014 N 115

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temperatureisaround42 u Candburstsofvaporat44.5to 53 u C(Povaraetal.,1972).Becauseoftheseparticularities, theAdamCaveisuniqueinRomania,beingatropical-type cave,distinctfromallothercavesfromRomania.Dueto theintermittentsteamemissions,airmixingiscausedby theascendingwarmanddescendingcolderair.Theair circulationismoreobviousinthewinter,whentemperaturedifferencesbetweencaveandexternalairaremore pronounced.Thewarmairaccumulatinginthecave becomesaperfectshelterformaternitycoloniesofbats. ThelargestguanoheapintheGuanoGalleryisupto2.5m high(Carbonneletal.,1996). CheileTurziiCaveisasmallcavelocatedontheright slopeofHasdateValleyintheTrascauMountainswhere theaverageannualprecipitationisaround600mm (Beldean,2005).Inthepast,thecaveofferedshelterfor thelocalpeopleagainstinvasions.Theentrancestill containspartofafortificationwallthatpartiallyobstructs normalairventilation. FusteicaCaveislocatedintheVlcanMountainswhere theannualaverageprecipitationisapproximately800mm (Costache,2011).Thisisanactivecavewithlargeguano depositsthatarepartiallyflushedawaybyanintermittent subterraneanstreamthatdrainstotheIsvarnaRiver (Burghele-Ba la cescuandAvram,1966).Bothmaternity andhibernationbatcoloniesareshelteredinthecave. GuraDobrogeiCaveislocatedinDobrogeain southeasternRomania.Annualprecipitationintheregion is350to450mm(Lungu,2008).IntheFossilsGallerythere arelargequantitiesofoldandfreshdepositsofguano. MeziadCaveispartlyatouristcaveandislocatedin thePadureaCraiuluiMountains,partoftheApuseni Mountains.Theannualaverageofprecipitationinthearea is720mm(Moza,2008).Thecaveisabigsubterranean cavitydevelopedonthreelevels,themaingallery,thefirst floor,andthesecondfloor.MeziadCavewasincludedin thestudybecauseofbigmaternityandmatingcoloniesof batsandimportantfreshguanodeposits.Duringour researchactivitiestherehasbeennoelectrificationandno constructionofnewtrails;thisresultedinminimalhuman visitsandpreservationofthebatpopulation.Thecavehas afrontgateembeddedinanartificialstonewallthree metershigh. RastociCaveisoneofthemaincavesoftheSomes an PlateauofTransylvania,anditistheonlycaveinthatarea thatsheltersaconsiderablenumberofbats.Theannual averageprecipitationintheSomesanareaforthelast hundredyearsis635mm(SorocovschiandVoda,2009). TopolnitaCaveisalargecavelocatedintheMehedinti Mountainswhereprecipitationisbetween900and 1,000mmperyear(Robu,2009).Thegallerynetworkof thecaveisdevelopedonfourlevels,twodry(onelowerand oneupper),onesemi-active,andthelowestoneactive.We investigatedonlytheupperinactivepartofthecave,where H.capsulatum wasdetectedforthefirsttimeinEurope (Alteras,1966).Agateinstalledin1960srestrictedthe accessofbatsinthispartofthecaveandprobablyinduced changesinatmosphericconditions.In1996twonew openingsforbatswerecutinthewall,whichallowedfor re-colonizationbybats.MICROBIOLOGICALMEDIAANDREADINGRESULTSDuetotheprovenversatilityofRIDA HCOUNT cultivationplatesincaves(Mulecetal.,2012a,b)and inorganic-richenvironments(Oargaetal.,2012),the followingvarietiesofthisproductwereused:fortotal bacterialcounts(RIDA H COUNTTotalAerobicCount), for Escherichiacoli andcoliforms(RIDA HCOUNT E.coli / Coliform),forenterobacteria(RIDA H COUNTEnterobacteriaceae),andforyeastandmolds(RIDA HCOUNT Yeast&MoldRapid).After24and48hoursofcultivation at37 u C,readingsofbacterialgrowthwerescored.For yeastsandmolds,readingsweretakenafter48and72hours ofcultivationatthetemperatureof25 u C.Thecountsof bacteriaafter48andforfungiafter72hourswere consideredforstatisticalanalyses.Prolongedincubation for24hoursgivesamorerealisticviewofthemicrobial communities,assomecavemicrobeshavedemonstrated slowgrowthonRIDA HCOUNTmedia(Mulecetal., 2012a).Isolatesthatexhibited-D-glucuronidaseand-DgalactosidasebiochemicalactivitiesonRIDA HCOUNT E. coli /Coliformplateswereconsideredindicativeof E.coli (R-BiopharmAG,Germany). TosupplementRIDA HCOUNTreadingswithadditionalindicatorsofpotentialpathogenicmicrobes,weused thestandardselectivemediafor Streptococcus (Azide bloodagarmedium;HolmesandLermit,1955)and Staphylococcus (Mannitolsaltagarmedium;Chapman, 1945).OpenPetriplateswiththesemediawereplaced paralleltoRIDA HCOUNTplatesinMeziadCaveand CheileTurziiCave.Theplateswereincubatedat37 u C,and colonieswerecountedafter24and48hours.Thisapproach isfrequentlyusedinRomaniatoquantifyairborne microbiotainorganic-richenvironmentssuchasdomestic farms(Draghicietal.,2002),zoos(Bordaetal.,2012),and caveswithbatguano(Bordaetal.,2004;2009;Bordaand Borda,2004). Forsubsequentidentificationofbacterialisolatesfrom AdamCaveandTopolnitaCave,themorphologically distinctcoloniesdevelopedonTotalAerobicRIDA H COUNTwereplatedonglucosenutrientagar(Oxoid, UK)andMacConkeyagar(Sigma-Aldrich).AfterGram staining,API H strips(Biomerieux,France)wereusedto identifyisolates,API20Eforentericbacteria,API20NEfor non-entericGram-negativebacteria,andAPIStaphfor identificationofstaphylococciandmicrococci.Theresults wereinterpretedusingAPIwebsoftware.SAMPLINGPROCEDUREANDATMOSPHERICPARAMETERSFourdifferenttypesofsampleswereanalyzed:bat guano,caveair,caveairinoculatedwithguanobyour in situ aerosolizationprocedure,andswabsofbatsfur.ForAEROSOLIZEDMICROBESFROMORGANICRICHMATERIALS:CASESTUDYOFBATGUANOFROMCAVESINROMANIA116 N JournalofCaveandKarstStudies, August2014

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airsamples,resultsarereportedascolony-formingunits (CFU)persurfaceinadefiniteperiodofsampling,e.g., CFU/20cm2/20min,forguanoasCFUpergram(w/v), andforswabsasCFUpersurface,e.g.CFU/20cm2. Freshguanoandguanoaccumulatedincavesin previousyearswereincludedinthestudy.Formicrobiologicalanalysis,guanoheapswereasepticallysampledwith aspooninarangefrom0to5cmdepth.Aftersampling, characteristicsofguanoheapsweremeasured:volume, surfacearea,andpH.pHwasmeasuredatthesiteusing pHindicatorstrips(EMDChemicals,Germany)aftera sampleofguanowashomogenizedwithanequalpartof steriledeionisedwater.Samplesformicrobiologicalanalysesweretransportedinacoolboxtothelabassoonas possible.Afterestablishingtheweightofaguanosamples, generallyaround2g,15mLofsterilephysiologicalsaline wasaddedtothesamplesandrigorouslyvortexed.This mixturewasseriallydilutedupto102 8.Onemilliliterof theseserialdilutionswasappliedontoRIDA H COUNTtest plates.Microbialcountswereexpressedascolony-formingunitspergram. Tosampleairbornemicroorganismsthegravity-settling methodwasadopted.Openplateswithmediawereexposed tothecaveatmospherefor20minutesand,after cultivationinlaboratoryconditions,microbialcountswere expressedasCFUpersurfaceunit(Bordaetal.,2004; Mulec,2008;Mulecetal.,2012a,b).Incaves,bioaerosol wassampledonthegroundflooratdifferentdistances fromthecaveentranceandguanoheaps.Besidesthe horizontalgradientsampling,averticalgradientof bioaerosolswasalsosampledbyusinga1.5-mstanding rack.Todiminishtheinfluenceofresearcherspresence upontheairquality,samplingwasperformedconsecutively;thefirstsamplewastakenatthecaveentrance,andthe nextoneslateratpreviouslyselectedsites,uptothelast samplingsitesataguanoheapinthecave.Ifcave morphologyataparticularsiteallowed,samplingwas performedinduplicateortriplicate,andtheaverageCFU valuewasusedinsubsequentanalyses.Aftersamplingwas finished,thedistancesfromthecaveentranceandguano heapforeachindividualsamplingsitewasmeasured.In somecaves(AdamCave,FusteicaCave,GuraDobrogei Cave,RastociCave,TopolnitaCave)aerosolswere sampledonceperyear,whileinCheileTurziiCaveand MeziadCavewealsosampledindifferentperiodsofthe yearinordertoobserveseasonalvariability.Thesummer investigationsinthesetwocavestookplaceoncethefresh guanowasdeposited. Inselectedcaves(CheileTurziiCave,GuraDobrogei Cave,andMeziadCave)weperformedartificiallyinduced aerosolizationtoestimatethemaximumnumbersof cultivablemicroorganismsfromaerosols.Aspoonof guanowasasepticallytransferredtoasterilebeaker (diameter7cm,height9cm,withtotalvolumeof 0.346liter).Toprovoke insitu aerosolizationofmicrobes inguano,thebeakerwithguanowastemporarysealedwith aplasticbagandmanuallyshakenfortenseconds.Big particlesofguanowereshakenoffandremovedfromthe beaker,andthebeakerwasplacedwiththeopeningover theRIDA HCOUNTmediafor20minutes,sotheairborne microbescouldsettle(Fig.1).TheinoculatedRIDA H COUNTmediawaslatertransferredinthelaboratoryand incubatedaspreviouslydescribed. Duringnaturalaerosolsamplingwemeasuredatmosphericparameters,temperature,relativehumidity(RH), andairpressurewithaKestrel4500PocketWeather Tracker.ThecarbondioxideconcentrationintheatmospherewasmeasuredwithaMI70VaisalaCO2meter. Swabsofbatcoatsweresampledtogetanideaofhow manycultivablemicrobesbatshostontheirbodysurfaces andhowmuchbatscontributetospreadingofmicrobes. Amoistenedsterilecottonswabwasusedtoswabthe bodyofabat.Thecottonswabwasplacedintoatube with4mLofphysiologicalsalineandthoroughlyshaken. Finally,onemLofthesuspensionwasspreadon RIDA H COUNTtestplates(TotalAerobicCount, E. coli /Coliform,Yeast&MoldRapid).Cultivationconditionswereasdescribedabove.Microbialcountsfromthe furcoatwereexpressedasCFUpersurface.Two individualsof Rhinolophusferrumequinum and R.hipposideros wereswabbedatthebeginningofhibernation (October-November2010).STATISTICALEVALUATIONSCanonicalcorrespondenceanalysiswasusedtocorrelatedataofabundanceofallcultivablemicrobialgroups, E.coli (EC),nonE.coli coliforms(NECCO),non-coliform bacteria(NCOBA),andyeastsandmolds(Y&M)with environmentalvariables.TheNECCOcountwascalculatedasthenumberof E.coli coloniessubtractedfromthe totalcoliformcounts,andtheNCOBAcountrepresented allbacteriaexcludingcoliforms(Oargaetal.,2012). Parametricmultivariateanalysiswasrunbytheprogram packageCANOCO4.5(terBraakandS milauer,2002). Thesignificanceofenvironmentalvariablesintheanalysis wastestedbyaMonteCarlopermutationtest.No transformationsoftheenvironmentaldatawereapplied.RESULTSANDDISCUSSIONBATGUANOAnimalexcrementisanimportantsourceofnutrients, includingbatguanoincaves(Deharveng,2005).Guano containsdiversemicrobiota(Chron akovaetal.,2009)and isasourceformicrobialaerosolization,whichrepresentsa potentialbiohazardforhumansandbats,(e.g.,Alteras, 1966;Julgetal.,2008).Freshguanoisbasic,andolder guanobecomesacidic(Moulds,2006).BasedonmacroscopicobservationandpHmeasurements,guanosamples wereassignedasfreshiftheywereuptooneseasonoldand neutraltoalkaline.Nocorrelationswereobservedbetween physicalguanoparameters(pH,volume,surface)andD.R.BORDA,R.M.NA STASE-BUCUR,M.SPI NU,R.URICARIU,ANDJ.MULECJournalofCaveandKarstStudies, August2014 N 117

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microbialgroups.ThebiggestguanoheapwasinAdam Cave(Table4).Thesamplesofguanovariedinconcentrationofbacteria(upto1.931010CFU/g),coliforms(up to2.23108CFU/g), E.coli (upto1.03108CFU/g),and yeastsandmolds(upto1.73107CFU/g).Theconcentrationsoffungivariedconsiderably;interestinglyno isolateswereretrievedfromoldguanosamplesfromGura DobrogeiCave(Table5).Coliformsand E.coli werenot detectedinoldanddryguanosamplesfromGura DobrogeiCaveandTopolnitaCave.Allguanosamples positiveon E.coli -RIDA HCOUNTspecificplatewere fresh,afewweeksorafewmonthsold.ByusingtheAPI identificationscheme, Chryseomonasluteola (99.9%ID) and Burkholderiacepacia (99.9%ID)wereidentifiedin freshguanofromAdamCave,andinaddition, B.cepacia waspresentinoldguanofromTopolnitaCave.Ina previousstudyofmicrobialquantificationfromguano fromTopolnitaCave,Hodorogea(1972)showedapproximatelya150-folddecreaseinnumberofbacteriainthe 10cmlayercomparedwiththesurfacelayer(1cm), andfungalconcentrationwasapproximately100-fold higherinthelowerlayercomparedtotheguanosurface (Hodorogea,1972).ATMOSPHERICCONDITIONSINCAVESThehighesttemperaturedifferencesamongsampling sitesinthesamecavewereobservedinMeziadCaveduring samplinginOctober2011(6.6 u C),andinCheileTurzii CaveinJuly2011(4.1 u C)(fortemperaturerangessee Table3).Inbothcaves,evenincoldermonths,slightly highertemperatureswereobservedinthesectorswiththe biggestguanoheaps.Temperaturedifferencesbetween samplingsiteswerehighinMeziadCavealsoduring samplinginNovember2010,2.7 u C,andinFusteicaCave inOctober2010,2.1 u C.Inothercaves,temperature differencesweresmaller.Thehighestairtemperatureswere measuredinAdamCave(26.0to27.3 u C),withadifference of1.3 u Cbetweensamplingsites.Thebiggestdifferencesin relativehumidityamongsitesinthesamecavewerein MeziadCaveinOctober2011at27.5%andinNovember 2010at14.4%.Thenexthigherdifferencesamongsites wereinFusteicaCave(20%),followedbyCheileTurzii Cave(8.4%);thiscavehadalsothelowestmeasured relativehumidity(42.8to51.2%).WhenCO2concentration wasmeasured,thebiggestdifferencesamongsamplingsites wereinMeziadCaveduringNovember2010sampling (175ppm),followedbyRastociCave(133ppm)andAdam Cave(103ppm).InAdamCave,theconcentrationwasthe highestamongallcaves,rangingbetween1,307and 1,410ppm(Table3).AIRBORNEMICROBESExceptinFusteicaCaveandMeziadCave,thetotal concentrationofairbornebacteriawashigherthanthe totalconcentrationofairborneyeastandmolds(Fig.2).In AdamCave,theconcentrationofairbornebacteriawasthe Figure1.Schematicrepresentationofaerosolizationexperiment.A,beakerwithminuteparticlesrepresentedasdots;B, RIDA HCOUNTmicrobiologicalmedium;C,flexiblesupportwithattachedmicrobiologicalmedium.AEROSOLIZEDMICROBESFROMORGANICRICHMATERIALS:CASESTUDYOFBATGUANOFROMCAVESINROMANIA118 N JournalofCaveandKarstStudies, August2014

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highestcomparedtoothercaves,probablyduetothelarge volumeandcontactsurfaceofguano(Table4).High temperatureandhighhumidity(96.5%to99.8%)in combinationwithvaporizedcompoundsfromguanothat canserveasnutrientsformicrobialmultiplicationcreatean excellentairhabitatformicrobes.Inaddition,intermittent thermalemanationsinthiscavecanintroduceadditional compoundsintocaveatmosphereandenhancethe aerosolizationeffect.Thelowestconcentrationofairborne microbeswasinFusteicaCave,whereguanoheapswere locatedalongbothsidesoftheriverbed.Thelow concentrationcanberelatedtothepermanentflowofthe undergroundriverthatconstantlywashesthecentralpart oftheguanodepositandcreatescontinualwatersaturation oftheair,sothataerosolparticlessettlerelativelyquickly. Airbornemicroorganismshadpreviouslybeenstudied inRomaniancaves(BordaandBorda,2004;Bordaetal., 2004)withdifferentrangesofcultivableaerobicbacteria (56to1,021CFU/m3)andfungi(52to22,373CFU/m3). Whencaveswererichinguano,totalaerobicbacteriaofup to11,317CFU/m3weredetected.Adirectcomparisonwith thisstudyisnotappropriateduetotheuseofdifferent samplingmethodsandcultivationmedia;inthisstudy RIDA HCOUNTcultivationplateswereusedandresults arereportedasCFU/20cm2/20min. Coliformbacteriaand E.coli ,representativeoffecal enterobacteriaandcommonsourcesofentericinfections (Guentzel,1996),werescreenedtoobservemicrobialload thatprobablyoriginatedfromfeces.Concentrationsof airbornecoliformsvariedreasonably.Typical E.coli coloniesonRIDA HCOUNT E.coli /Coliformplateswere notdetectedintheairinAdamCave,CheileTurziiCave, andFusteicaCave(Fig.3).Furthermore,despitehigh concentrationofairbornecoliformsandtotalbacteriain AdamCaveandCheileTurziiCave,notypical E.coli colonieswereretrievedonthemedia,whichindicatesthat E.coli isshort-livedwhenairborneanditspresenceinthe caveairismoreorlessrandom.Additionally,theresultsof insitu aerosolizationshowedthat E.coli ishardtofindin aerosols(Fig.6). E.coli doesnotsurvivegenerallymore thantwoorthreeweeksinlow-nutrientenvironmentsina viableandcultivablestate(Neidhardtetal.,1996),and indicationsofitspresenceinnutrient-poorcavehabitats shouldbecarefullyexamined(BartonandPace,2005). Airbornefecalcoliformsgenerallydonotsurvivelong outdoors,sotheprobabilityofcausinginfectionsforwildlife andhumansislow(Hughes,2003).Similarconclusioncould bedrawnfortheundergroundenvironment. DistinctbacterialcoloniesfromAdamCaveand TopolnitaCavethatdevelopedontheRIDA HCOUNT TotalAerobicwereidentifiedusingAPIas Klebsiella pneumoniae ssp. ozaenae,Salmonellaarizonae ,and Salmonella spp.Thesourceofthesemicrobesisverylikelybats intestines(e.g.,Adesiyunetal.,2009;DiBellaetal.,2003). Thesemicrobesarealsopresentinhumanintestines (Guentzel,1996).IntheairinTopolnitaCave,anonEnterobacteriaceaeisolate, Chryseomonasluteola (99.9% ID),wasalsofound. Table4.Physicalcharacteristicsofstudiedguanoheapsintheinvestigatedcaves. Cave StudiedGuanoHeapspH Volume,m3Surface,m2AdamCave 1 4.4 32.710 43.90 CheileTurziiCave 1 5.8.5 0.0100.400 0.25.00 FusteicaCave 3 4.0.5 0.0010.058 0.30.00 GuraDobrogeiCave 4 4.8.2 0.0010.610 0.09.51 MeziadCave 2 4.7.8 0.0151.360 1.54.60 RastociCave 1 4.5 0.049 0.98 TopolnitaCave 2 4.0.8 0.3759.600 3.75.00 Table3.Rangesofatmosphericparametersduringsampling. Cave Date(mm/dd/yy)Temperature( u C)RelativeHumidity(%)CO2(ppm) AdamCave 10/29/10 26.07.3 96.49.8 13070 CheileTurziiCave 07/15/11 14.88.9 10/16/11 8.4.3 42.81.2 410 FusteicaCave 10/28/10 8.2.3 80.000.0 421 GuraDobrogeiCave10/23/11 11.02.8 MeziadCave 11/02/10 11.54.2 84.38.7 397 07/26/11 13.34.6 10/17/11 7.5.1 62.90.4 370 RastociCave 10/25/10 8.1.8 96.000.0 418 TopolnitaCave 10/30/10 12.73.6 96.000.0 488D.R.BORDA,R.M.NA STASE-BUCUR,M.SPI NU,R.URICARIU,ANDJ.MULECJournalofCaveandKarstStudies, August2014 N 119

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Screeningforthepresenceof Streptococcus and Staphylococcus wasperformed;theyareanimportantpart ofindooratmospheres(MandalandBrandl,2011; Hospodskyetal.,2012). Staphylococcus isusuallyusedas anindicatorformicrobiotafromskinandmucous membranes(Aydogduetal.,2005;Schulzetal.,2004), and Streptococcus indicatesoral,pharyngeal,andskin bacterialbiotaandevenfaecalsoilpollution(Kibbeyetal., 1978).ExceptforsamplinginMeziadCaveinJuly2011, theconcentrationof Staphylococcus intheairexceeded countsof Streptococcus (Fig.4).Concentrationofstaphylococciandstreptococciarefrequentlyelevatedinthe proximityofguanoheapsduringthesummerwhenbatsare activeandflyintheirundergroundroosts(Bordaetal., 2004).ThebiochemicalprofileoftwoisolatesfromAdam Caveatmosphererevealed Staphylococcus and Micrococcus (99.8%ID).Micrococciarenotascommonasstaphylococci;however,botharefrequentlypresentinbatguano (Mohod,2011;Vandzurovaetal.,2013).Thisgroupof microbescansurviveintheairforalongtime;forexample, Staphylococcusaureus cansurviveseveralmonthsonfabric ordustparticles(MitscherlichandMarth,1984).Under naturalcaveconditionsairborne Staphylococcus and Streptococcus areexpectedtobeviablemuchlongerthan E.coli Incaveswithbatguano,elevatedconcentrationsof airbornebacteria,andtoalesserextent,fungiweredetected inourstudy.Weidentified Chryseomonasluteola E.coli Klebsiellapneumoniae ssp. ozaenae Micrococcus Salmonella Staphylococcus ,and Streptococcus ,aswellasunidentified cultivablebacteria,coliforms,andenterobacteria.Allthese microbescanbeindicativeforbatsandguano,andincaves, theymightrepresentabiohazardbecausetheycansurvive longerasairbornetherethaninotherorganic-rich environmentswithmoreenvironmentalstressors.High microbialconcentrationsinguanodidnotalwayscorrespondtohighconcentrationofairbornemicrobesatthe sameguanoheap(compareFig.2andTable5). Figure2.Rangesofconcentrationsofairbornemicrobes(grey,totalbacteria;black,yeastsandmolds)intheairofAdam Cave(AC),FusteicaCave(FC),MeziadCave(MC),RastociCave(RC),andTopolnitaCave(TC)inautumnof2010,andin CheileTurziiCave(CT),GuraDobrogeiCave(GD),andMeziadCave(MC)inautumnof2011. Table5.Microbialcountsinguanosamplesexpressedascolony-formingunitspergramafter48hoursofincubationfor bacteriaand72hoursforyeastsandmolds. Cave Bacteria(CFU/g)Coliforms(CFU/g) E.coli (CFU/g)YeastandMolds(CFU/g) AdamCave 2.13108.131083.373105.531051.43104.231052.53106.73107CheileTurziiCave6.53107.9310101.73106.031081.23106.031085.93103.73107FusteicaCave 8.83104.731061.83103.831040.0 1.03105.33106GuraDobrogeiCave9.33103.931050.0.331040.0.131030.0.43104MeziadCave 3.13105.231083.93103.831070.0.131067.23101.63106RastociCave 1.231052.231030.0 6.83104TopolnitaCave3.63103.131090.0.231080.0.331049.23104.93105AEROSOLIZEDMICROBESFROMORGANICRICHMATERIALS:CASESTUDYOFBATGUANOFROMCAVESINROMANIA120 N JournalofCaveandKarstStudies, August2014

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AIRBORNEMICROBESANDATMOSPHERICPARAMETERSCanonicalcorrespondenceanalysiswasperformedon thebasisofsixvariables,ifavailable:temperature,relative humidity,airpressure,CO2,distancetothecaveentrance fromeachindividualsamplingsite,anddistancetothe guanoheapfromeachindividualsamplingsite.Onlya smallportionofmeasuredenvironmentalvariablesexplainedthevariances(Table6).Measuredphysicalparametersexplainedthehighestvarianceinthestructureof microbialcommunitiesintheautumninGuraDobrogei Cave(October2011)by0.31andinMeziadCave (November2011)by0.12.Inothercavesinautumnand alsoinsummertheyexplainedlessthan0.09.Temperature hadasignificantimpact(p 0.05)onthebioaerosol abundanceinMeziadCaveinNovember2011andRHin TopolnitaCaveinOctober2010. InastudyfromPostojnaCave,Slovenia,(Mulecetal., 2012c)wheretheimpactingsamplingmethodwasused,in thetransitionperiods,i.e.,springandautumn,physical parametersexplainedvariancesless(winter0.62,spring 0.25,summer0.49,autumn0.08).Thesamplingmethod usedinthisstudyisbasedongravitysettlingandisnot directlycomparable.Themethodbasedonsettlingisvery sensitivetoanyairdisturbancesthatcancausetheparticles todeviatefromtheirverticalsettlingroute.Otherfactors thatinfluencesettlingbehavior,inadditiontoair movements,pressure,temperature,andhumanandanimal movements,arecharacteristicsofanindividualparticle, Figure4.Rangesofconcentrationsofairborne Staphylococcus (grey)and Streptococcus (black)inMeziadCave(MC)and CheileTurziiCave(CT)inJuly(7)andOctober(10)of2011. Figure3.Rangesofconcentrationsofcoliforms(grey)and E.coli (black)intheairofAdamCave(AC),FusteicaCave(FC), MeziadCave(MC),RastociCave(RC),andTopolnitaCave(TC)inautumnof2010,andinCheileTurziiCave(CT),Gura Dobrogeicave(GD),andMeziadCave(MC)inautumnof2011.D.R.BORDA,R.M.NA STASE-BUCUR,M.SPI NU,R.URICARIU,ANDJ.MULECJournalofCaveandKarstStudies, August2014 N 121

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suchasitssizeandmass,andmorphologicalcharacteristics ofaspacethat,forexample,enablescreatingofairgaps. Interestingly,distancesfromguanodidnotalways contributetothecommonvarianceduringautumn samplings,butduringbothsummersamplingsinCheile TurziiCaveandinMeziadCavetheydid.Thiscanbe attributedasaseasonaleffectconnectedwiththepresence ofbatsandveryfreshguanodroppingsanditsaerosolization.Inthatperiod,apronouncedgradientofairborne microbescanbeformedradiatingfromasiteofabat colonyanditsguanoheap,butmoredataareneededto confirmthat.Nevertheless,conditionsincaveatmospheres arecomplexanddynamicandbecomemorecomplicated whenotherfactorsareinvolved,suchasaerosolized microbes/particlesfromanimalexcrements.Sampling positioninthespacehasalsoveryimportantinfluenceon thedetectionofbioaerosolsignals.VERTICALMICROBIALGRADIENTANDEFFECT OFSEASONALITYConcentrationofairbornemicroorganismsatdifferent heightsabovethegroundvaried,indicatingthatselection ofbioaerosolssamplingpositionsisimportantandshould beclearlyreported(Fig.5).Theconcentrationofairborne microorganismswasgenerallyhigherwhenbioaerosols weresampledatthegroundlevel,inCheileTurziiCaveon average3.4ordersofmagnitudeandinMeziadCaveon average1.3orders.InbothCheileTurziiCaveandMeziad Cave,thetotalmicrobialcountswerehigherinsummer thaninautumn.Totalmicrobialcountsweredefinedasa sumofcountsof E.coli (EC),nonE.coli coliforms (NECCO),non-coliformbacteria(NCOBA),andyeasts andmolds(Y&M)(Fig.5).Coliformbacteriaotherthan E.coli weredetectedinbothcavesinthesummerperiod.In addition,inMeziadCave E.coli wasdetectedairborneon E. coli -specificplates,whileinCheileTurziiCavethesebacteria werenotobservedintheairatall.SamplingsiteMC2in MeziadCaveislocatedinabigchamber(PyramidRoom, approx.220,000m3)with0.015m3ofguano,andsampling siteMC3islocatedinasmallerchamber(BatRoom, approx.37,500m3)withagreaterquantityofguano ( 1.36m3)thatismorescatteredinthespace.TheBat Room,whichhadahigherconcentrationofairborne microbes,sheltersfromMaytoAugustabignurserycolony ofaboutfivetoseventhousandindividualsof Myotis myotis/M.oxygnathus mixedwith Miniopterusschreibersii Table6.Summaryofcanonicalcorrespondenceanalysisanalysisusingforwardselectionforexplanationofvarianceby selectedvariable.Dist_Ent,distancefromtheclosestcaveentrance;Dist_Gua,distancetotheclosestguanoheap.AC,Adam Cave;CT,CheileTurziiCave;FC,FusteicaCave;GD,GuraDobrogeiCave;MC,MeziadCave;RC,RastociCave;TC, TopolnitaCave.Inbold, p 0.05. Parameter Cave/Period AC/ Oct10 CT/ Jul11 CT/ Oct11 FC/ Oct10 GD/ Oct11 MC/ Nov10 MC/ Oct11 MC/ Oct11 RC/ Oct10 TC/ Oct10 Temperature p 1.001.00 1.000.240.051.000.141.001.00 var. 0.080.02 0.010.020.100.020.050.010.00 RelativeHumidity p 1.00 1.00 0.03 var. 0.01 0.00 0.06 Pressure p 0.171 0.430.37 var. 0.05 0.020.03 CO2p 0.380.12 var. 0.020.00 Dist_Ent p 0.22 0.36 var. 0.05 0.03 Dist_Gua p 0.35 0.090.200.43 var. 0.02 0.290.010.01 Varianceexplained0.090.040.050.010.310.120.080.070.060.09 Sumofalleigenvalues0.080.050.050.010.310.120.080.070.060.09AEROSOLIZEDMICROBESFROMORGANICRICHMATERIALS:CASESTUDYOFBATGUANOFROMCAVESINROMANIA122 N JournalofCaveandKarstStudies, August2014

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Theproportionofmicrobialsubgroupsseemstobethe sameateachtestedsite,withgenerallysmallerconcentrationsintheautumnwhenairchangeswiththesubsurface aresuppressedandmaternitycoloniesofbatsare dispersed.Theconcentrationsofairbornefungiwere ratherlowcomparedtobacteria(Fig.5).Itisimportant tounderlinethatsamplingat1.5mabovethegroundwas chosenasthelevelofhumanbreathingtopointoutthe possibilityofgettingincontactwithbioaerosols.AEROSOLIZATIONOFMICROBESFROMGUANOPercentageofaerosolizedmicrobialgroupsfrom samplesfromCheileTurziiCave,GuraDobrogeiCave, andMeziadCavearesummarizedinFigure6.Total bacterialcountsrangedfrom0.00to0.10%,coliformsfrom 0.00to0.16%, E.coli 0.00%,enterobacteriafrom0.00to 0.03%,andyeastsandmoldsfrom0.00to3.35%.Inthe threeindependentsamplingcampaigns,aerosolizationof E.coli wasunsuccessful,althoughconcentrationofthis microbeindifferenttestedguanosvariedfrom0.0to1.73107CFU/g(Table5).Ontheotherhand,successfulaerosolizationofcoliformbacteriaandenterobacteriaindicatedthat aerosolizationof E.coli maybepossible.Transmissionof E. coli viacaveairisprobablyquitelimited. Artificiallyinducedaerosolizationresultedinalarge amountofaerosolizedfungifromguano.Aerosolizationof microbesincavescanbeeasilyenhancedbydisturbing guanosurfaceswhilewalking.Fromothermicrobes-rich materialsindifferentenvironments,aerosolizationcanalso becausedbywindandanimals.Duetotheirsmallsize, microbescanpersistlongintheair.Forexample,droplets morethan5mmindiametersedimentmorequicklyonthe groundthandropletsthatarelessthan5mm,whichcan remainsuspendedintheairforalongtime(Darcyetal., 2012).AsbatguanoinTopolnitaCavewasalreadyreported tobethesourceof H.capsulatum (Alteras,1966),the estimationthatmorethan3%ofcultivablefungimaybe aerosolizedgivesanimportantwarningtoavoidcontactwith potentialfungalpathogensfromguano.Highaerosolization potentialoffungalsporesfromguanocanbetheanswerfor manycasesofguano-associatedhistoplasmosesreportedin landfill,bridge,andwagon-trainworkers(Gustafsonetal., 1981;Huhnetal.,2005),duringhomerenovations(Schoenbergeretal.,1988),oramongcavers(Ashfordetal.,1999).BATSASVECTORSFORMICROORGANISMSBatswereswabbedtogetanestimationontransmission ofmicrobesontheirbodies.Swabbingof Rhinolophus Figure5.EffectofseasonandsamplingpositiononconcentrationofairbornemicrobesatCheileTurziiCave(CT)sampling sites2and3andatMeziadCave(MC)samplesites2and3.SamplingwasperformedinJuly(7)andOctober(10)2011. Samplestaken1.5metersabovethegroundaredesignatedbyanasterisk;othersamplesweretakenatgroundlevel.EC, E. coli ;NECCO,othercoliforms;NCOBA,non-coliformbacteria;Y&M,yeastsandmolds.D.R.BORDA,R.M.NA STASE-BUCUR,M.SPI NU,R.URICARIU,ANDJ.MULECJournalofCaveandKarstStudies, August2014 N 123

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ferrumequinum coatsproducedhighernumbersofmicroorganismsthan R.hipposideros coats(Table7).Although theirbodysizesarequitedifferent, R.ferrumequinum being almosttwicethatof R.hipposideros (57to71mmversus37 to45mmheadandbody;350to400mmversus192to 254mmwingspan),itispossiblethatbehaviorismore importantformicrobialcontaminationofcaveair.Inthe samplingperiodatthebeginningofNovember,individuals of R.hipposideros werealreadypreparedtohibernateand theirdailytorporwasalreadyverydeep,incontrastto R. ferrumequinum ,whichwerestillactive.Socialbehaviorof batscouldalsoplayanimportantroleinspreadingof microorganisms;solitarybatsrarelygetincontactwith otherbats. R.hipposideros usuallyhangfreeasisolated individualsandduringhibernationwrapthemselves completelyintheirwings,butindividualsof R.ferrumequinum hibernateinbigclusters,wherebatscoatsgetin contactbecauseofincompletewingwrapping.Theroleof batspeciesinthepropagationandspreadoffungiis alreadyknown.Ononehandtheirfecesserveasasourceof nutrientsformicrobes,andontheotherhandtheycanbe activedisseminatorsoffungiintheenvironment(Hoffand Bigler,1981).CONCLUSIONSElevatedconcentrationsofbacteria,includingthoseof entericoriginandtoalesserextentoffungi,weredetected in,andaround,batguano.Favorableatmospheric conditionsformicrobialmultiplication,suchashigh temperatureandvaporizedcompoundsfromorganic matter,producedthehighestcountsofairbornemicroorganisms.Acaverichwithguano,withhighrelative humidity,andwithaflowingriverhadthelowest concentrationsofairbornemicrobes.Concentrationsof airbornemicroorganismswerehigheratgroundlevel comparedtotheconcentrationsat1.5mabovetheground. Coliformbacteriawerefrequentlydetectedinairinareas withhighorganicmatterandanimalexcrements. E.coli wasrarelyfoundinair,anditssuccessfulaerosolization Table7.Swabanalysesoffurcoatof Rhinolophus fromMeziadCaveexpressedasCFU/20cm2after48hoursofplate incubationforbacteriaand72hoursforyeastandmolds. BatSpecies TotalBacteria (CFU/20cm2) E.coli (CFU/20cm2) Coliforms (CFU/20cm2) YeastandMolds (CFU/20cm2) R.ferrumequinum 1 821 0 0 1067 R.ferrumequinum 2 2005 0 135 1030 R.hipposideros 10002 1 R.hipposideros 25007 Figure6.Percentageofaerosolisedmicrobialgroupsfrombatguano.AEROSOLIZEDMICROBESFROMORGANICRICHMATERIALS:CASESTUDYOFBATGUANOFROMCAVESINROMANIA124 N JournalofCaveandKarstStudies, August2014

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wasnotachieved.Microbialaerosolizationratefrom guanowasforbacteriaupto0.10%,andforfungiupto 3.35%. Staphylococcus and Streptococcus werefoundinair closetobatguano.Inthesummer,microbialloadinthe caveairwaselevated,whichweattributetothepresenceof bats.Theproportionofmicrobialgroupswaspreservedin thesummerandautumnperiods.Batsandespeciallytheir socialbehaviorarecrucialforintroductionandspreadof airbornemicrobes.Airbornemicrobesindicativeofbats andguanoandespeciallyahighpotentialoffungal aerosolizationrepresentabiohazardforanimalsand humansincaves.Activitiesthatleadtoaerosolformation fromanimalexcrementsshouldbeavoided.Incomparison tootherexternalenvironments,cavesprovideasimplified systemofstudyingbioaerosols,althoughevenincaves complexrelationshipsbetweenatmosphericandmicrobiologicalparametersco-exist.ACKNOWLEDGEMENTSThestudywassupportedbytheResearchProgramme P6-0119KarstResearchandPNII-MIII(BI-RO/10-11012)ResearchBilateralProjectbetweenSloveniaand Romania.AuthorsaregratefultoAncaDraguforher helpduringfieldandlabwork,andalsotoAndreeaOarga forassistingduringfieldwork.WethankSorinSosu,who facilitatedtheaccessinTopolnitaCave,andtoEmilia Liddellforlanguageassistance.REFERENCESAdesiyun,A.A.,Stewart-Johnson,A.,andThompson,N.N.,2009,Isolation ofentericpathogensfrombatsinTrinidad:JournalofWildlifeDiseases, v.45,no.4,p.952961.doi:10.7589/0090-3558-45.4.952. Alteras,I.,1966,FirstRomanianisolationof Histoplasmacapsulatum fromthesoil:InternationalJournalofDermatology,v.5,no.2, p.69.doi:10.1111/j.1365-4362.1966.tb05188.x. ANPM,2013,Agentianationala pentruprotect iamediului:Harta delimitariiregiunilorgeografice(mapofRomaniangeographicregions), http://www.anpm.ro/upload/12561_Anexa2_harta_regbiogeografice. pdf[accessedMay15,2013]. Ashford,D.A.,Hajjeh,R.A.,Kelley,M.F.,Kaufman,L.,Hutwagner,L., andMcNeil,M.M.,1999,Outbreakofhistoplasmosisamongcavers attendingtheNationalSpeleologicalSocietyAnnualConvention, Texas,1994:TheAmericanJournalofTropicalMedicineand Hygiene,v.60,no.6,p.899. Aydogdu,H.,Asan,A.,Otkun,M.T.,andTure,M.,2005,Monitoringof fungiandbacteriaintheindoorairofprimaryschoolsinEdirneCity, Turkey:IndoorandBuiltEnvironment,v.14,p.411.doi:10. 1177/1420326X05057539. 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Hospodsky,D.,Qian,J.,Nazaroff,W.W.,Yamamoto,N.,Bibby,K., Rismani-Yazid,H.,andPeccia,J.,2012,Humanoccupancyasa sourceofindoorairbornebacteria:PLoSONE,v.7,no.4,e34867. http://www.plosone.org/article/i nfo%3Adoi%2F10.1371%2Fjournal.pone. 0034867,[accessedMay15,2013].doi:10.1371/journal.pone.0034867. Hughes,K.A.,2003,Influenceofseasonalenvironmentalvariablesonthe distributionofpresumptivefecalColiformsaroundanAntarctic researchstation:AppliedandEnvironmentalMicrobiology,v.69,no. 8,p.4884.doi:10.1128/AEM.69.8.4884-4891.2003. Huhn,G.D.,Austin,C.,Carr,M.,Heyer,D.,Boudreau,P.,Gilbert,G., Eimen,T.,Lindsey,M.D.,Cali,S.,Conover,C.S.,andDworkin, M.S.,2005,Twooutbreaksofoccupationallyacquiredhistoplasmosis: morethanworkersatrisk:EnvironmentalHealthPerspectives,v.113, no.5,p.585.doi:10.1289/ehp.7484. 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GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDY OFGLACIOLACUSTRINELITHOFACIESINCAVES, HELDERBERGPLATEAU,CENTRALNEWYORKJEREMYM.WEREMEICHIKANDJOHNE.MYLROIEDepartmentofGeosciences,MississippiStateUniversity,MS39762-5448,USA,jmw868@msstate.eduAbstract: TheglaciallyderangedkarsttopographyoftheHelderbergPlateau,central NewYork,containsglaciolacustrinelithofaciesdepositedattheendoftheWisconsin glaciation.Eightpre-glacialcaves(BarrackZourieCave,McFailsCave,Howe Caverns,SecretCaverns,BensonsCave,GageCaverns,SchoharieCaverns,and CabooseCave),containingauniquesedimentsection,arelocatedwithinthefootprint ofGlacialLakeSchoharie,SchoharieCounty.Thelithofaciesconsistofthreeindividual facies,stratigraphicallyuniform,withthemiddlefaciesinsharpcontactwiththefacies directlyaboveandbelow.Thisassemblagedisplaysasimilarstratigraphicsequence frombottomtotop:tan/whitetolight-grey,verythinlybedded,siltsandclays,richin calcite,overlainbypoorlysorted,matrix-supportedgravels,inturnoverlainbydarkbrownverythinlybeddedsiltsandclays.Apost-glacialcavewithinthelakesfootprint (WestfallSpringCave)andanearbypre-glacialcaveoutsidethefootprint(KnoxCave) werefoundtolacktheselithofacies.Thetan/whitetolight-greysedimentfaciesis interpretedtobeaglacialrockflourdepositedunderstagnantlakeconditionsthat limitedfine-grainedcalciteparticledissolution.Theoverlyinggravelfacieswere emplacedduringlaketerminationandreestablishmentofturbulentepigenicflowinthe eightstreamcaves.Themorerecentdark-brownfaciesisperhapssoil-lossdeposition followingEuropeansettlement.Initialinterpretationshypothesizedthatthedeposits werelaiddownunderice-coverconditions,butsimilardepositswerenotfoundinother glaciatedcavesettingsinNewYork.Theresultspresentedhereexplainwhytheunusual tan/whiteandlight-greyglaciolacustrinefaciesarenotfoundinothercavesinthe glaciatedcentralNewYorkregion,asthoseareaswerenotsubjecttoinundationby glaciallakewater.INTRODUCTIONAsglaciersadvancedandretreatedacrossnortheastern USAduringthelatePleistocene,sedimentandexposed bedrockwerestrippedfromthecave-richHelderberg PlateauincentralNewYorkState(Fig.1)andsubsequentlycoveredbyallochthonousglacialsediment.The sedimentwasdepositedonthesurfaceoftheplateauandin caveswithintheplateau.Theglacialsedimentdeposited withinthecaveshasbeenshelteredfromsurficialweatheringanderosion,perhapsallowingforamoreaccurate recordtobepreserved.Interpretationandanalysisofcavesedimentsamplescanassistinreconstructingtheglacial surficialenvironment.Inmostglaciallyderangedlandscapes,surficialdepositscanbescarceanddifficultto identify;thisstudyfocusedandreliedonsamplescollected fromwithincaves.Specifichorizonsofsedimentfound withinthecavesintheareaarethoughttobeassociated withtheexistenceofaglacialevent(Mylroie,1984;Palmer etal.,1991;Palmeretal.,2003).Theworkpresentedhere re-interpretsthoseearlierstudiesandclassifiestheunique cavesedimentsasbeingtheresultofaglaciallake inundatingthecaves.Thislake,referredtoasGlacial LakeSchoharie,isbelievedtohaveexistedduringtheLate Wisconsinglacialperiodapproximately23.02.0kainthe present-daySchoharieValleyincentralNewYork(e.g., Dineen,1986). Todeterminethenatureofthesurficialenvironmentin theSchoharieValleyduringtheLateWisconsinglacial period,multipleresearchtripsweretakentoselectcaves locatedintheHelderbergPlateau.Fromwesttoeast,the cavesinthisstudyinclude:BarrackZourieCave(1in Fig.1),McFailsCave(2),HoweCaverns(3),theSecretBensonCaveSystem(4and5),GageCaverns(6),Westfall SpringCave(7),SchoharieCaverns(8),CabooseCave(9), andKnoxCave(10)(Figs.1and2).Asdocumentedby LauritzenandMylroie(2000),U/Thdatingofstalagmites demonstratesthatthecavesoftheSchoharieValleyare olderthantheonsetofthemostrecentglaciationand,in somecases,severalglaciationsreachingback350ka. Thepurposeofthisstudywastoreconstructthepaleoenvironmentofaproglaciallake,GlacialLakeSchoharie, locatedprimarilywithinSchoharieCounty,NewYork. Theglaciallakeisthoughttohaveenduredatleastfour readvancesoftheMohawkandHudsonglaciallobes duringtheWoodfordianSubstageoftheLateWisconsinJ.M.WeremeichikandJ.E.MylroieGlacialLakeSchoharie:aninvestigativestudyofglaciolacustrinelithofaciesincaves,Helderberg Plateau,centralNewYork. JournalofCaveandKarstStudies, v.76,no.2,p.127.DOI:10.4311/2013ES0117JournalofCaveandKarstStudies, August2014 N 127

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glaciation;seeDineen(1986)formoredetailonthenature ofthereadvances.Themultiplereadvancescausedthe shorelineofthelake,andhenceitsfootprint,tobe modifiedmultipletimesthroughoutitsexistence.The cavesselectedforinvestigationwerechosenbecauseof theknownorsuspectedexistenceofwhathadbeen presumedtobeglaciallydepositedclastics,inparticulara characteristicwhiteortanclaythatissometimesvarved (e.g.,Mylroie,1984;Dumont,1995)(Figs.3and4).Itwas alsothepurposeofthisstudytodeterminethecomposition ofthewhiteclayhorizon,aswellasthecompositionof otherassociatedsedimenthorizons(Fig4). InitialinterpretationofMylroie(1984)wasthatthe sedimentsfoundinthecaveswerecausedbystagnantsubiceconditionsduringthelastglacialmaximum.Under theseconditions,Mylroie(1984)thoughtthatthestagnant waterwouldsoonsaturatewithCaCO3andthatany furtherfine-grainedparticulateCaCO3introducedtothe caveswouldnotdissolveandcouldcollectasasediment deposit.Therewasnodisagreementintheliteratureabout Figure1.MapshowingthegenerallocationoftheHelderbergPlateauineast-centralNewYorkaswellasthelocationsof thecavesincludedinthisstudy.1 = BarrackZourieCave,2 = McFailsCave,3 = HoweCaverns,4 = SecretCaverns, 5 = BensonsCave,6 = GageCaverns,7 = WestfallSpringCave,8 = SchoharieCaverns,9 = CabooseCave,10 = KnoxCave. Re-drawnandmodifiedfromDineen(1986).ThetopographicmapisaUSGSDigitalRasterGraphicoftheBinghamton Quadrangle(1:250,000scale).GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDYOFGLACIOLACUSTRINELITHOFACIESINCAVES,HELDERBERGPLATEAU,CENTRALNEWYORK128 N JournalofCaveandKarstStudies, August2014

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Figure2.A:MapofthekarstsystemsandflowroutesoftheCobleskillPlateau.TheburiedvalleyislocatedbetweenBarrack ZourieCaveandMcFailsCave(re-drawnfromDumont,1995).B:MapofthekarstsystemsandflowroutesofBartonHill (re-drawnfromMylroie,1977).J.M.WEREMEICHIKANDJ.E.MYLROIEJournalofCaveandKarstStudies, August2014 N 129

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thisinterpretation(e.g.,Palmeretal.,2003),butitwas recognizedthatcavesinotherareasofthestatelacked theseglacialsediments.Thequestionbecame,whatwas uniqueaboutthecavesintheSchoharieValley.The presenceofaglaciallakecouldcreatethesamestagnantwaterconditionsintheunderlyingcaves,andthelakes footprintwouldexplaintheuniqueclusterofcaves containingtheglacialsediment.CAVESOFTHEHELDERBERGPLATEAUThecaveslocatedintheHelderbergPlateauformedin theUpperSilurianandLowerDevonianlimestonesofthe HelderbergGroup.Themajorcavesandcavesystems withintheplateau,includingthecavesmentionedinthis study,primarilyformedwithinthethick-beddedCoeymans LimestoneandthethinlybeddedManliusLimestone (Fig.5).Therehasalsobeensomecaverndevelopment withintheRondoutDolomite,asatKnoxCaveand BarytesCave(Mylroie,1977;Palmer,2009),butcavern developmentwithinthisparticularunitisusuallylimitedto conduitswithsmallcross-sectionalareas. ThecavesandkarstfeaturesoftheHelderbergPlateau werecreatedbyepigenicprocessesprovidingsurfacerunoff wateranalternativetoover-landflowpaths,allowing watertotravelmoredirectly.Thekarstfeaturesofthe HelderbergPlateauhavebeendescribedasoneofthe finestexamplesofglaciatedkarstinthecountry(Palmer etal.,1991,p.161). Therehasbeenextensivepublishedworkregardingthe caveslocatedintheCobleskillPlateauandadjoiningareas, suchasthatofDumont(1995),Kastning(1975),Mylroie (1977),andPalmeretal.(2003).Theglacialdepositswithin thecaveswerediscussedbytheseauthors,butthelinkof thesesedimentstoapostulatedglaciallakeinthisareahad notbeenthoroughlyinvestigated. WestfallSpringCavewasincludedinthisstudybecause itsgeologiccontextsuggesteditwaspost-glacialinorigin; andtherefore,itshouldnothaveaglacialsediment signature.KnoxCavewasincludedbecauseitisoutside thefootprintofGlacialLakeSchoharie.Thesetwocaves actedascontrolsforthesedimentstudy.THEWISCONSINGLACIATIONINTHEHELDERBERGPLATEAUThelastmajorPleistoceneglaciationtooccurinNew YorkwastheLateWisconsinglaciation.TheHelderberg Plateauwascoveredbythreelobesofglacialice,the Mohawklobe,theHudsonlobe,andtheSchohariesublobe,duringtheonsetoftheLateWisconsinglaciation (Dineen,1986).Thelobesthatcoveredtheplateauentered Figure3.Distinctwhiteclaywasfoundtoexistinmultiplecavesselectedforthisstudy.NoteinB,D,andEtheabrupt transitionfromthefinelylaminatedlight-coloredmaterialatthebottomofthesectiontotheverycoarsegravellylayerabove. Thedarkercoloredfine-graineddepositthatcommonlycapsthesequenceisbestseeninB.GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDYOFGLACIOLACUSTRINELITHOFACIESINCAVES,HELDERBERGPLATEAU,CENTRALNEWYORK130 N JournalofCaveandKarstStudies, August2014

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theregionfromthenortheast,asshownbydrumlinsand bedrockstriationsfoundinthestudyareathathaveaclear northeast-southwesttrend(MylroieandMylroie,2004). Theglaciallobesderangedthelandscape,alteringit greatly.Thelandscapeseentodayiscoveredbydrumlins, kames,glaciokarst,andburiedglacialdrainagebasins,as wellasotherpaleoglaciallandforms.DineenandHanson (1985)proposedtheLateWisconsinglaciationendedinthe regionapproximately12,300yearsago,buttheexactdate isstillhighlydebated.AccordingtoMullerandCalkin (1993),theWisconsinisbrokenupintoEarly( 117.0 64.0ka),Middle( 64.0.0ka),andLate( 23.01.9ka) episodes.GLACIALLAKESCHOHARIEAglaciallakeisabodyoffreshwaterthatisconfined partlyorentirelybyaglacierorageomorphicfeature producedbyaglacier(LaFleur,1976).Asmentioned earlier,therewereanumberofadvance,retreat,and readvancephasesassociatedwiththeLateWisconsin glaciationinNewYork,resultingintheformationof multipleglaciallakes. TheinstabilityofglacialicecausedGlacialLake SchoharietohaveshorelinesatvaryingelevationsthroughouttheLateWisconsinglaciation.TheWoodstockice marginwasestablishedbyahaltiniceretreatfrom18.2 17.4ka,accordingtoRidge(2004).Followingthe establishmentoftheicemargin,glacialmeltwaterbegan tofloodtheSchoharieValley.Asthestagnatedglacialice continuedtomeltandretreattowardthenortheast,water levelscontinuedtoriseuntilaglaciallakewasestablished withashorelineatanelevationof213m(700ft)abovesea level(Dineen,1986)(Fig.6).TheestablishmentofGlacial LakeSchohariewillbecalled stageone ofthreeknown stagesoftheglaciallakesdevelopment. WiththeonsetoftheMiddleburgreadvancementat 17.4ka,basedonRidge(2004),advancingicere-entered theSchoharieValleyandthegreaterHelderbergareauntil theicereachedtheCatskillFront(CatskillMountains). AfterreachingtheCatskillFront,theglacierstagnatedand onceagainbegantoretreatnorthward.Whileretreating, theglacierproducedavastamountofmeltwater,resulting intheenlargementofseveralproglaciallakes(Dineen, 1986).GlacialLakeSchoharieenlargedconsiderablyand establishedashorelinebetween354and366m(1,160and 1,200ft)abovesealevel(Fig.7),reaching stagetwo WiththeestablishmentoftheDelmaricemarginat 16.2ka(Ridge,2004),waterfromGlacialLake Schohariedrainedtothenortheast,throughwhatisknown Figure4.Examplesofthesedimentlayers,instratigraphicorder,encounteredwithinthecavesinthisstudy.J.M.WEREMEICHIKANDJ.E.MYLROIEJournalofCaveandKarstStudies, August2014 N 131

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astheDelansonspillway(LaFleur,1969).Thespillwayfed theDelansonRiver,whicheventuallyemptiedintoGlacial LakeAlbany(LaFleur,1976).The stagethree shorelineof GlacialLakeSchohariewasestablishedat256to213m (840to700ft)abovesealevel(LaFleur,1969;Dineenand Hanson,1985)(Fig.8).METHODSAtotalof63sampleswerecollectedandstoredin sterilizedplastic35mmfilmcanistersforthisstudy;three additionalsampleswerecollectedfromBarrackZourie CavebyKevinDumontin1995.Eachsamplewaslabeled Figure5.GeneralizeddiagramdepictingtheSilurianandDevonianrocksinwhichthecavesofthisstudyformed.Thegrey unitisshale,blueunitisdolomite,andthetanunitsarelimestone.GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDYOFGLACIOLACUSTRINELITHOFACIESINCAVES,HELDERBERGPLATEAU,CENTRALNEWYORK132 N JournalofCaveandKarstStudies, August2014

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withthecave,thelocationinthecave,andthestratumin theoutcropatthatlocation. Thesamplescollectedforthisstudycomefromawider suiteofcavesthanthoseusedinMylroie(1984),andmore sophisticatedanalysistechniqueswereconductedto determinehowreliableMylroiesresultswereandhow thesamplescomparetohisdatafromCabooseCave.The sampleanalyseswerenotintendedtobediagnostic,butto provideareconnaissancebaselinetoguidefurtherresearch. Todetermineageneralmineralogicalcontentofthe samplescollected,x-raydiffraction(XRD)wasutilized becauseofitsabilitytoprovidequalitativeresultsinacosteffectiveandtime-efficientmanner.AllXRDanalysesof powderedsampleswereconductedusingtheRigakuUltima IIIX-raydiffractometerandwereinterpretedusingtheMDI Jade8program.TheXRDpatternforeachsamplewas obtainedusingCuK a radiationwithawavelengthof 1.541867A .Scanspeedwassetfor2degreesaminutewith ascanstepof0.02degrees,ascanaxisof2-theta/theta,and aneffectivescanrangeof3.00.00degrees.RESULTSThelaboratoryanalysesofsamplestodeterminethe massofwaterineachsample,themassofcarbonates,and themassoforganicswiththepurposeofdiscerninga patternamongindividualclasticunitsrecoveredfromthe tencavesinthisstudywasinconclusiveintermsofa recognizablepatternandcanbeseeninWeremeichik (2013). Althoughthelaboratoryresultswereinconclusive,Xraydiffractionyieldedmoreconclusiveinformation.The XRDdatawerenotusedtodetermineactualamountsof materialsinagivensample;itwasanassayofpresenceor absence.Table1showsthefrequencyofmineralcontent foundtoexistineachtypeofsample.Figure4shows typicalexamplesoftheverticalsequenceofthesediment typesfoundinthecavesandusedinTable1.Forexample, thedark-grey/dark-brownclayunithadcalcitein62%of thesamples,andtheallogenicoutwashunithadcalcitein 40%ofthesamples.Together,thesepost-glaciallake sedimentshad56%calciteoccurrence.Thelight-greyclay unithadcalcitein100%ofthesamples,andthetan whiteclayunithadcalcitein75%ofthesamples. Together,thesupposedglaciallakesedimentshadcalcitein 81%ofthesamples.TheKnoxCaveandWestfallCave sedimentcontrolsamples,becausethosecavedidnotlie underthelakeorpostdatedit,hadcalciteinonly33%of thesamples.Brushiteshowsadifferenttrend,beingmore commoninthepost-glacialsediments. Figure6.Topographicmapshowingthelocationofthecavesinrelationtothe213m(700ftorstageone)shorelineofGlacial LakeSchoharieoutlinedinorange,createdusinginformationprovidedbyDineen(1986).CavesarenumberedasinFigure1. Thegridis6.4by6.4km(4by4miles.)J.M.WEREMEICHIKANDJ.E.MYLROIEJournalofCaveandKarstStudies, August2014 N 133

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DISCUSSIONDuring stageone ofGlacialLakeSchohariesdevelopment,therewouldnothavebeenanyoutletforthewaterto escapebywayoftheSchoharieValley.However,itwould havebeenpossibleforthewaterinGlacialLakeSchoharie todrainnorthtowardwhatisknowntodayastheMohawk Valley.Butthereisaproblemwiththisidea,because duringtheLateWisconsintheMohawkValleywas occupiedbytheactiveMohawkglaciallobe.TheMohawk glaciallobe,alsoreferredtolocallyastheMohawkIce Block,filledtheareabetweentheneighboringCobleskill andBartonHillplateausandactedasaplug,trapping glacialmeltwaterintheSchoharieValley(LaFleur,1969). NearthecloseoftheWisconsinglaciation,atleast50%of GlacialLakeSchohariewouldhavebeencoveredbyactive glacialicebelongingtotheSchoharieglacialsub-lobe (Dineen,1986).AsseeninFigures6and9,during stage one (213m)therewouldnothavebeenasufficientamount ofwaterinGlacialLakeSchoharietoevenpartially inundatethecavesoftheCobleskillPlateauandBarton Hillincludedinthisstudy. Figure7.GlacialLakeSchoharieshorelineat354m(1,160,200ftorstagetwo)abovesealeveloutlinedinpurple. NotethereductionofscaletoportrayamuchlargerlakeandthelakesextensioneastwardintoSchenectadyandAlbany Counties.CavesarenumberedasinFigure1.Thegridis6.4by6.4km(4by4mi).GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDYOFGLACIOLACUSTRINELITHOFACIESINCAVES,HELDERBERGPLATEAU,CENTRALNEWYORK134 N JournalofCaveandKarstStudies, August2014

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InFigures7and9itcanbeseenthatnearlyallof thecavesintheCobleskillPlateauandBartonHillare completelyinundatedbywaterfromGlaciallakeSchoharie during stagetwo ( 360m).Notethatalthoughtheentrances tobothMcFailsCaveandGageCavernswerenot inundated,themajorityofthecavepassagesareover30m belowthesurfaceandwouldhavebeeninundatedbasedon theirelevationrelativetosealevelandthe stagetwo lake level.Thiswouldincludeinundationoflocationswhere sampleswerecollectedforthestudy.Theupperpassages wheresampleswerecollectedinKnoxCavewouldnothave beeninundatedbyglaciallakewaterduetotheirelevation, evenifthelakehadextendedthatfareastward,andsothese samplesactedasacontrol.WestfallSpringCave,beingpostglacialinorigin,isnotincludedinFigure9. Theclaysedimentsencounteredinthecavesfitthe descriptionofavarvedsequence(Fig.3).Varvesare usuallycoupletsoffineandcoarsegrainedmaterial (Neuendorfetal.,2011).Thesesedimentsdonotshow thealternatingcoarse-to-finesequencing;theyappearto onlycontainthefinesediments.Apossiblereasonthe sedimentsthatcomposetheunitsaresouniformisthatthe insurgencesandresurgencesofthecavesweremostlikely chokedwithglaciallytransportedmaterial,soonlythe smallestofsedimentswouldbeabletoslowlypercolate throughthedebris.Thesedepositsareconsistentwitha laminar-flowregimeexpectedfordepositiondeepincave passagesbelowaglaciallake.Thewhitetotanclay depositshaveanabruptcontactwithoverlyingsandand graveldeposits(Fig.3).Mylroie(1984)interpretedthese coarse-grainedsedimentstobetheresultoficeretreatand re-establishmentoftheepigenicturbulentflowsystemin thesecaves.Thesamethingwouldhaveoccurredduring thedrainingofGlacialLakeSchoharie.Thedark-brown claywashypothesizedbyMylroie(1984)torepresenta surgeofincomingsedimentassociatedwithclear-cutting Figure8.GlacialLakeSchohariewithashoreline,outlinedindarkblue,at256m(840ftorstagethree)abovesealevel, createdusinginformationprovidedbyLaFleur(1969)andDineenandHanson(1985).Theredlinesindicateaspillway,andthe light-bluelinesindicatetheboundarybetweenglaciallakewaterandactiveglacialice.CavesarenumberedasinFigure1.The gridis6.4by6.4km(4by4mi).J.M.WEREMEICHIKANDJ.E.MYLROIEJournalofCaveandKarstStudies, August2014 N 135

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followingEuropeansettlementinthe1700s.Theresults herecanneitherprovenordisprovethatspeculation,but thedark-brownclaysdorepresentwhatisbeingdeposited inthecavestodayduringfloodcycles.Thesequenceof eventsthatproducedthesedimentcolumnofFigure4is presentedinFigure10. TheHoweCavernssedimentsectionisespecially instructiveandisthethickestofallsuchsequences.While Table1.X-raydiffractionresults.Thefiguresarethepercentagesofsamplesofthegivenstratathatshowedqualitativelythe presenceofthemineral.Thetotalsarenotnumericalaveragesofthevaluestotheirleftbecausetherewerenotequalnumbers ofsamplesofeachtype.ThecontrolsampleswerefromcavesnotunderGlacialLakeSchoharie. Mineral,% GlacialLakeSediment,% PostGlacialSediment,% Control Samples,% LightGrey Clay TanWhite ClayTotal Dark BrownClay AllogenicGlacial OutwashTotal Calcite 100 75 8162 40 56 33 Dolomite? 0 0 0 4 0 3 0 Quartz 100100100100 100 100100 Muscovite 60 69 6773 80 75 83 Phlogopite 40 19 2431 40 33 67 Chlorite 0 0 0 0 0 0 0 Montmorillonite0 0 0 0 0 0 0 Albite(lowtemp)20 44 3858 50 56 67 Enstatite? 0 6 5 0 0 0 0 Brushite 40 31 3365 40 58 83 Nacrite 20 0 515 0 11 17 Carbon 0 25 19 8 30 14 67 Figure9.TheelevationsoccupiedbyeachcaveinrelationtotheshorelineelevationsofGlacialLakeSchoharieduringstage one(213m,Fig.6),stage2( 360m,Fig.7),andstage3(256m,Fig.8).GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDYOFGLACIOLACUSTRINELITHOFACIESINCAVES,HELDERBERGPLATEAU,CENTRALNEWYORK136 N JournalofCaveandKarstStudies, August2014

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mostcaveshavelessthanorequalto1mofthelight-grey andtanwhiteclay,HoweCavernshasover2mof section.ThisgreaterthicknessistheresultofHowe Cavernsmainstreampassagebeingthelowestinelevation ofallthecavepassagesstudied,byapproximately30meters (Fig.9).Therefore,whilelakesurfaceelevationsshifted vertically,HoweCavernsspentmoretimeunderGlacial LakeSchohariethananyothercaveinthestudy,being inundatedevenduring stagethree .Inaddition,Figure3 showsaninterestingtransitionfromaveryamorphous whiteclaydepositatthebase(nexttotheknife)toa progressivelybetterlayeredlight-greyclayinwhichthe individuallayersgetthickerupwardstothecontactwith moreordinarycavesediments.Thistransitioncanbe interpretedtoindicateinitialclaydeposition stagetwo whentheHoweCavernsstreampassagewouldhavebeen 100mbelowthelakessurfaceat 360m.Sediment transportbylaminarflowintothecavewouldhavebeen slowandquiteisolatedfromseasonalchanges,indicatedby thelackofrhythmicallayeringinthewhiteclaydeposit. Aslakelevelloweredtothe256mlevelduring stagethree theHoweCavernsmainstreampassagewouldhavebeen merelymetersbelowthelakessurfaceandmorelikelyto recordtheseasonalchangesinwaterandsedimentaddition tothelake,asdemonstratedbythelight-greyclay.The upwardthickeningmayrecordthefinaltransitionofHowe Cavernsoutofthelakefootprintasthelakedrainedaway. Thesedimentanalyseswereforthemostpartinconclusive.BasedontheX-raydiffractionresults,theglacial sedimentsaremorelikelytohavecalciteinthem,whichis consistentwiththestagnantwaterconditionsproposedby Mylroie(1984).Themass-lossexperimentswereless convincing,withagreatdealofvariationwithinthedata andnoconsistentpattern.Mylroie(1984)hadreporteda veryhighsolublescontentfortheCabooseCavewhiteclay, andwhilethisstudydidreplicatethatresulttoanextent,the highsolublescontentwasnotconsistentacrosstheother cavesinthestudy.ItisusefultonotethatKnoxCave,acting asapre-glacialcontrolcave,hasmuchlessvariationinits samplesthanthecavesundertheGlacialLakeSchoharie footprint.Thesedimentanalysiswasdoneasareconnaissance,todetermineifmoreworkwouldbeworthwhileinthe future,anditwasnotcentraltothefinalinterpretationof thesedimentsglaciolacustrineorigin.CONCLUSIONSThecavessuspectedtohavebeeninundatedbyglacial lakewaterand,therefore,tohavecollectedfine-grained lakesedimentdonotshowanystatisticalcorrelation betweensamplescollected(seeWeremeichik,2013).But,as seeninFigure3,itisapparentthatphysicalsimilarities betweenthesamplescollectedexist.Thesephysical similaritiescanbecorrelatedwiththeirmineralscolor andgrainsize.Itwasoriginallyhypothesizedthatthe sedimentinthecavesmayhavebeendepositedduringa retreatphaseofglaciationresultingfromstagnanticecoveredconditions(e.g.,Mylroie,1984).Thishypothesis wasthoughttobetruebecauseitexplainshowthefinegrainedsedimentwasdepositedinthecaves.Thiscouldnot havehappenediftherehadbeenturbidoreventransitionallylaminarflowthroughthecaves.Icecoverwouldhave createdthenecessarystagnantconditions.Theglacial-lake hypothesispresentedherealsowouldcreatestagnant conditions,butinanenvironmentwheretheassociated fine-grainedsedimentcouldmoreeasilyenterthecave. Figure10.Cartoondepictingthecaveshistoryduringaglacialcycle,withtimeprogressingfromlefttoright.J.M.WEREMEICHIKANDJ.E.MYLROIEJournalofCaveandKarstStudies, August2014 N 137

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GlacialLakeSchoharieenduredmultipleretreatsand readvancesofglacialice,inpart,bybeinginsulatedand protectedbyalayerofstagnatedglacialice.Duringretreat phasesofglacialactivity,newglacialmeltwatercarrying glacial-derivedsedimentmusthavebeendeliveredtothe lake,whichsubsequentlyfilteredintothecavesbelow. Theanalysesofthesedimentsthemselvesareconsistent withtheglacial-lakehypothesis.Theyareextremelyfinegrained,verylowinorganics(Weremeichik,2013),and withameasurablesolublecontentofcalcite.Theyare visuallystrikingwhenobservedinthefieldandareeasily recognized.Theyare,todate,knownonlyfromwithinthe footprintofGlacialLakeSchoharie.Thisfinalaspectis important,asthedepositswereoriginallyconsideredby earlierworkers(e.g.,Mylroie,1984)tobesub-icedeposits. Thefailuretofindsuchdepositselsewhereinthe HelderbergPlateauorinotherglaciatedkarstregions wasveryproblematic.Everyonewhosawthedeposits in situ agreedwiththeirglacialrock-flourorigin(e.g.,Palmer etal.,2003).TheuseoftheGlacialLakeSchoharie footprinttoexplainthesedepositsasnotsub-ice,butsublakedeposits,explainsthefailuretofindsuchdepositsin otherglaciatedkarstlocalesintheregion.REFERENCESDineen,R.J.,1986,DeglaciationoftheHudsonValleyBetweenHyde ParkandAlbany,NewYork, in Cadwell,D.H.,ed.,TheWisconsinan StageoftheFirstGeologicalDistrict,EasternNewYork,Albany, NewYorkStateMuseumBulletin455,p.89. Dineen,R.J.,andHanson,E.L.,1985,DeglaciationoftheMiddle MohawkandSacandagaValleys,orataleoftwotongues, in Lindemann,R.H.,ed.,FieldTripGuidebookoftheNewYorkState GeologicalAssociation57thAnnualMeeting,SaratogaSprings,New York,p.250. Dumont,K.A.,1995,KarstHydrologyandGeomorphologyofthe BarrackZourieCaveSystem,SchoharieCounty,NewYork[M.S. thesis]:MississippiState,Miss.,MississippiStateUniversity,reprinted asNewYorkCaveSurveyBulletin5,70p.,mapplate. Kastning,E.H.,1975,CavernDevelopmentintheHelderbergPlateau, East-CentralNewYork[M.S.thesis]:Storrs,Conn.,Universityof Connecticut,reprintedasNewYorkCaveSurveyBulletin1,194p. plusmapplates. Lauritzen,S-E.,andMylroie,J.E.,2000,ResultsofaspeleothemU/Th datingreconnaissancefromtheHelderbergPlateau,NewYork: JournalofCaveandKarstStudies,v.62,p.20. LaFleur,R.G.,1969,GlacialgeologyoftheSchoharieValley, in Bird, J.M.,ed.,61stAnnualMeetingoftheNewEnglandIntercollegiate GeologicalConferenceGuidebookforFieldTripsinNewYork, Massachusetts,andVermont,p.(5-1)(5-20). LaFleur,R.G.,1976,GlacialLakeAlbany, in Rittner,D.,ed.,ThePine BushAlbanysLastFrontier:Albany,N.Y.,PineBushHistoric PreservationProject,p.1. Muller,E.H.,andCalkin,P.E.,1993,TimingofPleistoceneglacialevents inNewYorkState:CanadianJournalofEarthSciences,v.30, p.1829.doi:10.1139/e93-161. Mylroie,J.E.,1977,SpeleogenesisandKarstGeomorphologyofthe HelderbergPlateau,SchoharieCounty,NewYork[Ph.D.dissertation]:Troy,N.Y.,RensselaerPolytechnicInstitute,reprintedasNew YorkCaveSurveyBulletin2,336p. Mylroie,J.E.,1984,Pleistoceneclimaticvariationandcavedevelopment:NorskGeografiskTidsskrift,v.38,p.151.doi:10.1080/ 00291958408552119. Mylroie,J.E.,andMylroie,J.R.,2004,Glaciatedkarst:Howthe HelderbergPlateaurevisedthegeologicperception:Northeastern GeologyandEnvironmentalSciences,v.26,no.1,p.82. Neuendorf,K.K.E.,Mehl,J.P.Jr.,andJackson,J.A.,2011,ed.,Glossary ofGeology,5threvisededition:Alexandria,Virginia,American GeosciencesInstitute,800p. Palmer,A.N.,2009,Caveexplorationasaguidetogeologicresearchin theAppalachians:JournalofCaveandKarstStudies,v.71,no.3, p.180. Palmer,A.N.,Rubin,P.A.,andPalmer,M.V.,1991,Interactionbetween karstandglaciationintheHelderbergPlateau,SchoharieandAlbany Counties,NewYork, in Ebert,J.R.,ed.,NewYorkStateGeological AssociationFieldTripGuidebook63rdAnnualMeeting,p.161. Palmer,A.N.,Rubin,P.A.,Palmer,M.V.,Engel,T.D.,andMorgan,B., 2003,KarstoftheSchoharieValleyandlanduseanalysis,Schoharie County,NewYorkState, in Johnson,E.L.,ed.,NewYorkState GeologicalAssociationFieldTripGuidebook75thAnnualMeeting, p.141. Ridge,J.C.,2004,TheQuaternaryglaciationofwesternNewEngland withcorrelationstosurroundingareas, in Ehlers,J.,andGibbard, P.L.,eds.,QuaternaryGlaciationsExtentandChronologyPartII: NorthAmerica:Amsterdam,Elsevier,DevelopmentsinQuaternary Sciencesseries2,p.169.doi:10.1016/S1571-0866(04)80196-9. Weremeichik,J.M.,2013,Paleoenvironmentalreconstructionbyidentificationofglacialcavedeposits,HelderbergPlateau,SchoharieCounty, NewYork[M.S.thesis]:MississippiState,MS,MississippiState University,191p.GLACIALLAKESCHOHARIE:ANINVESTIGATIVESTUDYOFGLACIOLACUSTRINELITHOFACIESINCAVES,HELDERBERGPLATEAU,CENTRALNEWYORK138 N JournalofCaveandKarstStudies, August2014

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DIETANALYSISOF LEOPOLDAMYSNEILLI ACAVE-DWELLINGRODENTINSOUTHEASTASIA, USINGNEXT-GENERATIONSEQUENCINGFROMFECESALICELATINNE1,2,MAXIMEGALAN3,SURACHITWAENGSOTHORN4,PRATEEPROJANADILOK5, KRAIRATEIAMAMPAI6,KRIANGSAKSRIBUAROD7,ANDJOHANR.MICHAUX1,3Abstract: Leopoldamysneilli isaMurinaerodentendemictolimestonekarstof ThailandandtheLaoPDR,butitsecologyandthereasonsofitsendemismtokarstare stilltotallyunknown.Theaimofthispilotstudywastoexaminetheplantcomposition ofthedietof L.neilli attheleveloforderandfamilyusingDNAformolecular identificationandtocompareitwithtwootherforest-dwelling Leopoldamys species, L. herberti and L.sabanus .A202bpfragmentofthe rbc Lgenewasamplifiedandsequenced fortwenty-threefecalsamplesofthethreespeciesusing454pyrosequencing.We successfullyidentifiedatotalofseventeenordersandtwenty-oneplantfamilies, correspondingtothirty-threeputativespecies,inthefecesofthesethree Leopoldamys species.Solanaceaewerethemostcommonplantsinthedietof L.neilli regardlessofthe regionandsamplingseason,andtheywerealsopresentinfecesofboth L.herberti and L. sabanus .TheAraceae,Fabaceae,andApocynaceaefamilieswerealsoidentifiedinfeces of L.neilli collectedinvariousregionsofThailandandatdifferentseasons.Plantsofthe Oleaceaefamilyareconsumedbyboth L.herberti and L.sabanus butwerenotfoundin thedietof L.neilli .Furtherimprovementsofthestudy,suchastheuseofadditional genes,thecreationofareferencecollection,themicrohistologicalexaminationofplant fragmentstodeterminewhichpartsoftheplantareconsumed,andtheanalysisofthe animaldietof Leopoldamys aresuggestedtoenhancethequalityandaccuracyofthe resultsobtained.INTRODUCTIONSeveralMurinaerodentsendemictolimestonekarst havebeendescribedinSoutheastAsia,buttheirecologyis stillpoorlyknown. Niviventerhinpoon (Marshall,1977)is foundinThailand, Saxatilomyspaulinae (Musseretal., 2005)intheLaoPDR,and Tonkinomysdaovantieni (Musseretal.,2006)inVietnam,while Leopoldamysneilli (Marshall,1977)hasbeendescribedinThailandbuthas alsorecentlybeendiscoveredintheLaoPDR(Balakirev etal.,2013;Latinneetal.,2013a).Recentphylogeographic studiesof L.neilli revealedadeepgenealogicaldivergence amonggeographicallycloselineagesofthisspeciesin Thailandandahighpopulationfragmentationrelatedto thepatchydistributionoflimestonekarst(Latinneetal., 2011;Latinneetal.,2012).Suchstrongphylogeographic structureisnotobservedforotherMurinaerodentsin Thailandthatarecharacterizedbylowerhabitatspecialization(Latinne,2012).Theseresultssuggestedthatthe spatialisolationofkarstareaspreventsmigrationamong lineagesof L.neilli andindicatedacloseassociationofthis specieswiththishabitat.However,ecologicaldataon L. neilli arelacking,andthereasonsofitsendemismto limestonekarstarestilltotallyunknown.Abetter knowledgeoftheecologyof L.neilli ,notablyitsfeeding habits,isthusnecessaryfordeterminingifdietcontributes tothehabitatspecializationanddistributionallimitsofthis species,aswellasforunderstandingitsfunctionalrolein karstecosystems. Rodentsandothersmallmammalslivinginforestsof SoutheastAsiaaregenerallyconsideredtobeomnivorous (Emmons,2000;Langham,1983;Lim,1970),andtheyplay akeyroleinthefoodchain,bothasconsumersofplants andsmallinvertebrates,andasfoodresourcesforlarger predators.Rodentsmayalsoplayanimportantroleinthe frugivorescommunityasseeddispersersorseedpredators, andithasbeensuggestedthatsome Leopoldamys species mightbenefitseedrecruitmentofseveraltreespeciesby seedhoardingorseedingestioninSoutheastAsiaand China(Chengetal.,2005;Wellsetal.,2009;Zhangetal., 2008).Howeverdetailedinformationontheexactdiet compositionofSoutheastAsianrodentsremainsscarce andshouldbeimprovedtobetterunderstandthetrophic 1ConservationGeneticsUnit,InstitutdeBotanique,UniversityofLie`ge,4000Lie`ge, Belgium,alice.latinne@gmail.com2DepartmentofParasitology,FacultyofVeterinaryMedicine,KasetsartUniversity, Bangkok10900,Thailand3CBGP(CentredeBiologieetdeGestiondesPopulations),UMRINRA/IRD/ Cirad/MontpellierSupAgro,CampusinternationaldeBaillarguet,CS30016,34988 Montferrier-sur-LezcedexFrance4EnvironmentandResourcesTechnologyDepartment,ThailandInstituteof ScientificandTechnologicalResearch,35Mu3TambonKhlongHa,Amphoe KhlongLuang,ChangwatPathumThani12120,Thailand5DoiChiangDaoWildlifeResearchStation,ChiangMai,Thailand6BungBoraphetWildlifeResearchStation,NakhonSawan,Thailand7KhlongSaengWildlifeResearchStation,SuratThani,ThailandA.Latinne,M.Galan,S.Waengsothorn,P.Rojanadilok,K.Eiamampai,K.Sribuarod,andJ.R.MichauxDietanalysisof Leopoldamysneilli,acave-dwellingrodentinSoutheastAsia,usingnext-generationsequencingfromfeces. JournalofCaveandKarst Studies, v.76,no.2,p.139.DOI:10.4311/2013LSC0100JournalofCaveandKarstStudies, August2014 N 139

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relationshipsinSoutheastAsianecosystemsandthe functionalroleofrodentsinthesebiologicalcommunities, aswellastheresourcepartitioningamongcompeting species. Directobservationsofforagingandfeedingbehaviors aregenerallytime-consuming,andtheyareparticularly difficulttoobtainforsmallnocturnalmammalslivingin karsthabitats.Fecesanalysisrepresentsanefficientand non-invasivealternativetocircumventthisproblem. Microhistologicalexaminationofplantandinvertebrate fragmentsinfecalsampleshasbeentraditionallyused,but thismethodrequiresalotoftimeandtraining,andits resultsareoftenimprecise(Soininenetal.,2009;Emmons, 2000).Morerecently,moleculartechniquesusingDNA barcodinghavebeendevelopedtosuccessfullyanalyzethe dietofwildherbivoresfromfeces(Bradleyetal.,2007;Kim etal.,2011;Soininenetal.,2009;Valentinietal.,2009). Thesemethodsaimtoamplifysmall,buthighlyvariable, DNAfragmentscontainedinthefeceswithuniversal primersandusethemasbarcodestoidentifytheplanttaxa thathavebeeneaten.SeveralDNAregionshavebeenused forthispurposeintheliterature,andthechoiceofthe targetsegmentresultsfromacompromiseamonga minimalsize,amaximalgeneticdistancebetweenspecies, aminimalgeneticdiversitywithinspecies,andtheexistence ofanadequatereferencecollection(Bradleyetal.,2007). AsfecescontainonlyhighlydegradedDNA,thelengthof fragmentsthatcanbeamplifiedisusuallyshorterthan200 basepairs(bp). Usinga202bpshortsegmentoftheribulose-bisphosphatecarboxylase( rbc L)geneofthechloroplastgenome asabarcoderegion,thepresentstudywasdesignedasa pilotstudytoassesstheperformanceofthismethodin analyzingtheplantcompositionofthedietof L.neilli at theleveloforderandfamily.Anotherobjectiveofthis studywastocomparethedietof L.neilli withtwoother forest-dwelling Leopoldamys speciesalsofoundinThailand butnon-endemictolimestonekarst, L.sabanus and L. herberti. ( L.herberti waspreviouslythoughttobelongto L. edwardsi ,butseveralrecentstudieshaveshownthatit shouldberegardedasadistinctspeciesfrom L.edwardsi (Balakirevetal.,2013;Latinneetal.,2013a).METHODSTwenty-sixfecalsamplesfromthethree Leopoldamys specieswerecollectedfromnineteenlocalities(Fig.1) belowtraps,baitedwithripebanana,wheretheanimals werecaughtduringasurveyoftherodentdiversityinThai limestonekarst.Thesampleswerepreservedinsilicagel. Twomitochondrialgenesweresequencedforalltrapped animalsusingtissuebiopsyfromtheeartoreliablyidentify thematthespecieslevel(Latinneetal.,2013b).Thespecific statusoftheseindividualswasalsoconfirmedbyan independentmolecularanalysisusingamitochondrial mini-barcodefromfeces(Galanetal.,2012).DNAwas extractedfromfecesusingtheQIAampDNAStoolKit (Qiagen)andfollowingtheprotocoldesignedforthe isolationofDNAfromhumanstool. A202bpfragmentofthe rbc Lgenewasamplifiedfor eachsampleusinguniversalprimersZ1aFandhp2R (Hofreiteretal.,2000),modifiedbytheadditionofa specifictagonthe5 9 end,followingthetaggingand multiplexingmethodforthe454pyrosequencingdeveloped byGalanetal.(2010).Thistagconsistsofashort7bp sequencetoallowtherecognitionofthesequencesafterthe pyrosequencingwhereallthePCRproductsfromthe differentsamplesaremixedtogetheranda30bpTitanium adaptorrequiredfortheemPCRand454GS-FLX pyrosequencingusingLib-LTitaniumSeriesreagents.Six andfivedifferenttagsweredesignedfortheforwardand thereverseprimers,respectively.Thisgivesthirtyputativelyuniquecombinationsofforwardandreversetags, andthus,allowstagginguptothirtydifferentamplicons. PCRswerecarriedoutina10mLreactionvolumeusing 5mLof2xQIAGENMultiplexKit(Qiagen),0.5mMof eachprimer,and2mlofDNAextract.ThePCRstartedby aninitialdenaturationstepat95 u Cfor15min,followed byfortycyclesofdenaturationat94 u Cfor30s,annealing at45 u Cfor45s,andextensionat72 u Cfor30s,followed byafinalextensionstepat72 u Cfor10min. PositivePCRproductswerethenpooledtogetherfor 454pyrosequencingusing3mLperstrongPCRamplificationproductsor7mLperlighterones.ThePCRpoolwas processedbyBeckmanCoulterGenomics(Danvers, Massachusetts).Ampliconsweresequencedafterthe emPCRona454GenomeSequencerFLX(Roche)in 1/4thoftitaniumpicotiterplate. ThesoftwareSESAME1.1B(Megleczetal.,2011)was usedtosortthesequences.Thankstothetagcombinations, thesequenceswereassignedtothefecalsamplefromwhich thePCRampliconwasobtained.Artifactualvariantsdue tosequencingerrorsduringPCR,emPCR,and454 sequencingwerediscardedasdescribedinGalanetal. (2012). Thevalidatedvariantsof rbc Lsequencesobtainedwere comparedwithpublished rbc Lsequencesavailableon GenBankusingNCBIsBLASTNprogram(Zhangetal., 2000)andwereassignedtoorderandfamilyoftheclosest sequences(withatleast98%ofidentityand100%ofquery coverage)followingtheAPGIIIclassification(Bremer etal.,2009).RESULTSOutofthe26 Leopoldamys fecesanalyzedinthisstudy, 23weresuccessfullyamplified(Table1)andatotalof392 rbc Lsequences,including112distinctvariantscorrespondingto33validatedvariants,wereobtainedwithameanof 15sequencespersamples.Eachvariantwasassigned unambiguouslytooneplantfamily,withtheexceptionof foursequencesoftheZingiberalesorderthatcouldbelongDIETANALYSISOFLEOPOLDAMYSNEILLI,ACAVE-DWELLINGRODENTINSOUTHEASTASIA,USINGNEXT-GENERATIONSEQUENCINGFROMFECES140 N JournalofCaveandKarstStudies, August2014

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Figure1.Locationsof Leopoldamys fecalsamplesanalyzedinthisstudy.Theprovinceabbreviationsarespelledoutin Table1.A.LATINNE,M.GALAN,S.WAENGSOTHORN,P.ROJANADILOK,K.EIAMAMPAI,K.SRIBUAROD,ANDJ.R.MICHAUXJournalofCaveandKarstStudies, August2014 N 141

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eithertoMarantaceaeorMusaceaefamilies(Table2). Several rbc Lvariantswereassignedtothesamefamilyand couldrepresentdifferentplantspeciesifeachvariant belongstoadifferentspecies,butthisassumptionshould beconfirmedbyfurtheranalyses.Atotalof17ordersand 21plantfamilies,correspondingto33putativespecies, wereidentifiedinthefecesofthe Leopoldamys species (Table2). Thedietof Leopoldamysneilli isquitediversified,with seventeenordersandnineteenfamiliesidentifiedwithin fecesofthisspecies.PlantsbelongingtoSolanaceae (correspondingtoasinglevalidatedvariant)andMarantaceae/Musaceae(correspondingtofourvalidatedvariants)wereidentifiedintenoutofthenineteenfecesof L. neilli analyzed(53%).Solanaceaearealsoidentifiedinthe twofecesof L.sabanus (100%)andoneofthetwofecesof L.herberti (50%).PlantsoftheOleaceaefamilyare consumedbyboth L.herberti and L.sabanus butwere notfoundtobeconsumedby L.neilli SolanaceaeandMarantaceae/Musaceaewerehighly commoninthedietof Leopoldamysneilli astheywereeaten byspecimensinallsampledregions(Fig.2).Mostofthe plantfamiliesidentifiedinthisstudy(14/19)wereencountered inonlyone L.neilli sample,buttheAraceae,Fabaceae,and Apocynaceaefamilieswereidentifiedinsamplescollectedin variousregionsofThailandandatdifferentseasons.DISCUSSIONThispilotstudyisthefirststudyofthedietcomposition of Leopoldamysneilli thatremainedtotallyunknownupto now.Wesuccessfullyidentifiedatotalofseventeenorders andnineteenplantfamilies,correspondingtothirty putativespecies,inthefecesofthislong-tailedgiantrat endemictolimestonekarstofThailandandtheLaoPDR. Theplantdiversityobservedinthe L.neilli fecesishighand similartotheonedescribedforlargeherbivoresspecies usingsimilarmethodsofmolecularidentification(Bradley etal.,2007;Kimetal.,2011;Valentinietal.,2009).Plants identifiedinthedietof Leopoldamys speciesareall floweringplants(angiosperms),andmostoftheseplant familieshavebeenobservedinthefloraoflimestonekarst insouthernVietnam(InternationalFinanceCorporation, 2002).Eventhougharecentstudyshowedthattheprimers Z1aFandhp2Rusedinourstudyalsoallowthe amplificationofsequencesbelongingtofernsormosses (Kimetal.,2011),nofernormosswasdetectedinthefeces ofthethreestudied Leopoldamys species. Table1.Samplelocations,regions,andseasonsforthe Leopoldamys fecalsamplesinthisstudy.SeeFigure1foramapof thelocations. Species SampleIDProvince(Locality) RegionSeasonPCRSuccess Leopoldamysneilli F161Kanchanaburi(KAN1) West Dry Yes F567Kanchanaburi(KAN1) West RainyYes F172Kanchanaburi(KAN2) West Dry Yes F565Kanchanaburi(KAN2) West RainyYes F191Kanchanaburi(KAN3) West Dry Yes F577Kanchanaburi(KAN3) West RainyYes F554UthaiThani(UT) West RainyYes F313Chaiyaphum(CHAI) NortheastDry Yes F327KhonKaen(KK) NortheastDry Yes F331KhonKaen(KK) NortheastDry Yes F418Petchabun(PET1) NortheastDry Yes F399Nan(NAN) North Dry Weak F406Nan(NAN) North Dry Weak F391Phrae(PHR) North Dry Yes F441ChiangRai(CHR) North Dry Yes F505Saraburi(SARA1) CentreRainyWeak F508NakhonRatchasima(NKR)CentreRainyWeak F534Saraburi(SARA2) CentreRainyWeak F538Lopburi(LOP) CentreRainyYes Leopoldamysherberti F420Petchabun(PET2) NortheastDry Yes F424Petchabun(PET2) NortheastDry No F430Petchabun(PET2) NortheastDry Yes Leopoldamyssabanus F254Krabi(KRA) South RainyYes F298SuratThani(SUR) South RainyNo F445PrachuapKhiriKhan(PRA)South RainyYes F477Chumphon(CHUM) South RainyNoDIETANALYSISOFLEOPOLDAMYSNEILLI,ACAVE-DWELLINGRODENTINSOUTHEASTASIA,USINGNEXT-GENERATIONSEQUENCINGFROMFECES142 N JournalofCaveandKarstStudies, August2014

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SpeciesoftheSolanaceaeandMarantaceae/Musaceae familiesarethemostcommonplantsidentifiedinthediet of Leopoldamysneilli regardlessoftheregionandseason. However,trapsusedinthisstudywerebaitedwith ripebanana( Musa sp.,agenusoftheMusaceaefamily), andthesebananaswereprobablyeatenbytrapped ratsseveralhoursbeforethecollectionoffeces,because thefeceswerecollectedatleasttwelvehoursafter trapsetup.Directcontactoffecalsampleswithbanana wasalsopossibleinthetrap.Thefrequentpresenceof Musaceaeinthefecesof L.neilli couldthusrepresentabias duetothebaitused,ratherthantherealdietofthisspecies. ThereforetheMarantaceae/Musaceaefamiliesshouldnot beincludedpositivelyinthedietof L.neilli withoutfurther verification. Asthenumberoffecalsamplesanalyzedsuccessfully for Leopoldamysherberti and L.sabanus wasmuchlower thanfor L.neilli ,itisnotpossibletocomparerigorously thedietcompositionofthesethreespecies.Despitethe smallnumberofsamples,Solanaceaewerealsoidentified infecesofboth L.herberti and L.sabanus .Thereforethis plantfamilyseemstobeverycommoninthedietofallthe Leopoldamys speciesinThailand.SolanaceaearerepresentedinSoutheastAsiabytheSolanoideaesubfamilyand maytaketheformofherbs,shrubs,orsmalltreesinthis region,butthelackofresolutionatthespecieslevelofthe rbc Lfragmentthatweuseddoesnotallowustogetmore informationonthetypeofSolanaceaeconsumedbythe Leopoldamys species.CONCLUSIONDespitethelimitationsandsmallsamplesizeofthis pilotstudy,thesepreliminaryresultsconfirmthatDNA barcodingfromfecesisapromisingtooltobetter understandthefeedinghabitsof Leopoldamysneilli .We suggestsomeimprovementsforfuturestudiestoenhance thequalityandaccuracyoftheresults. First,abetterknowledgeofthefloraofThailimestone karstisabsolutelyneededtoallowplantidentificationat lowertaxonomiclevelthanorderandfamily.Thecreation ofareferencecollectionbysampling,identification,and DNAsequencingofthemostcommonplantsofThai limestonekarstwouldhelptoassessmoreaccuratelythe dietofthesespeciesandallowmorepreciseidentifications ofthesequencesobtainedfromfecesthandatanowin publicdatabasessuchasGenBank(Valentinietal.,2009). WealsosuggestusingotherhighlyvariableDNAregions suchas trnH,psbA (KressandErickson,2007), matK (Hollingsworthetal.,2009), trn L(Taberletetal.,2007; Valentinietal.,2009),or ITS-2 (Bradleyetal.,2007)as DNAbarcodesinassociationwith rbc Ltoobtainmore Table2.Plantfamiliesidentifiedinthefecesofthree Leopoldamys speciesinThailand,withnumberandfrequency ofoccurrence. Order Family Numberof Validated Variants Frequency L.neilli (n 5 19) L.herberti (n 5 2) L.sabanus (n 5 2) AlismatalesAraceae 2 3(16%) Brassicales Brassicaceae 1 1(5%) CommelinalesCommelinaceae1 1(5%) CucurbitalesCucurbitaceae 1 1(5%) DioscorealesDioscoreaceae 1 1(5%) Fabales Fabaceae 5 4(21%) Fagales Fagaceae 1 1(5%) GentianalesApocynaceae 3 3(16%) Lamiales Lamiaceae 1 1(5%) Oleaceae 2 1(50%) 1(50%) MalpighialesPhyllanthaceae 1 1(5%) Putranjivaceae 1 1(50%) Malvales Malvaceae 1 1(5%) Poales Poaceae 1 1(5%) Piperales Aristolochiaceae1 1(5%) Rosales Rhamnaceae 1 1(5%) Sapindales Burseraceae 1 1(5%) Sapindaceae 2 1(5%) 1(50%) Solanales Convolvulaceae1 2(10%) Solanaceae 1 10(53%) 1(50%) 2(100%) ZingiberalesMarantaceaeor Musaceaea4 10(53%) 2(100%)aPossiblecontaminationbythebait.A.LATINNE,M.GALAN,S.WAENGSOTHORN,P.ROJANADILOK,K.EIAMAMPAI,K.SRIBUAROD,ANDJ.R.MICHAUXJournalofCaveandKarstStudies, August2014 N 143

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preciseresults.Checkingtrapsforcapturesmorefrequently andcollectingfecesmorerapidlyaftertrapsetupwould preventbaitcontaminationofthefeces.Theuseof differentbaitsorbaitsdistinctfromallplantspecies knowntooccurinthestudiedregionwillalsohelpto determinewhetherMusaceaeispartofthenaturaldietof Leopoldamysneilli ornot. Moreover,combiningDNA-basedanalysisoffeceswith microhistologicalexaminationofplantfragmentsinfecal sampleswouldhelptodeterminewhichpartsoftheplant areconsumedby L.neilli andother Leopoldamys species, asthisinformationremainsunknownwhenusingDNA barcoding.Inparticular,thestudyofthediversity, quantity,andviabilityofseedsdefecatedbytheselongtailedgiantratsisneededtobetterassesstheirpotential roleasseeddispersersinSoutheastAsianecosystemsvia seedingestionandsubsequentdefecation,asalready suggestedfor L.sabanus byWellsetal.(2009). ItcouldalsobeveryinterestingtoperformsuchDNA barcodinganalysisusingunivers alprimersdesignedtoamplify animalDNA,becausethe Leopoldamys speciesalsoeatsmall preyssuchasinsectsorsnails(Langham,1983;Lim,1970).A smallfragmentofthecytochrome c oxidaseIgene(Hajibabaei etal.,2011)couldbetheidealmarkerforthispurpose. Finally,mostoftheplantfamiliesidentifiedwithinour datasetwereencounteredinonlyonesample.This observationstrengthenstheimportanceofstudyingalarge numberofsamplestoobtainanexhaustivelistoftheplant compositionofthe Leopoldamys diettobettercomprehend thewholediversityoffoodresourcesconsumedbythese long-tailedgiantratsandhowitmayvaryinspaceand timeandamongspecies.ACKNOWLEDGEMENTSWeareindebtedtoB.Tontanforhisvaluablehelp duringourfieldworkandtoS.Jittapalapongforhis administrativesupport.Wealsothanktwoanonymous reviewersfortheirvaluablecommentsthatledto improvementinthemanuscript.Thisworkwassupported byaBelgianFRS-FNRS(FondsdelaRecherche Scientifique)fellowshiptoA.Latinne(mandataspirant) andtoJ.R.Michaux(mandatmatrederecherches),by afinancialgrantfromtheBelgianFRS-FNRS(credits pourbrefssejoursa`letranger)toA.LatinneandJ.R. Michaux,andcreditsfromtheFondsdelaRecherche FondamentaleCollective(FRFC)toJ.R.Michaux,from theUniversityofLie`ge(Patrimoine),fromtheCommunautefrancaisedeBelgique(boursedevoyage),andfrom theInstitutNationaldelaRechercheAgronomique(Projet innovantdudepartementEFPA2011-www4.inra.fr/efpa). A.LatinneiscurrentlyfundedbyaMarieCurieCOFUND Figure2.Numbersofsamplesoffecesof Leopoldamysneilli outoftotalofnineteenshowingplantfamilies,withsamples codedforseasonandregion.DIETANALYSISOFLEOPOLDAMYSNEILLI,ACAVE-DWELLINGRODENTINSOUTHEASTASIA,USINGNEXT-GENERATIONSEQUENCINGFROMFECES144 N JournalofCaveandKarstStudies, August2014

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postdoctoralfellowship.ThisstudyispartoftheCERoPathproject(CommunityEcologyofRodentsandtheir PathogensinSouth-EastAsia:effectsofbiodiversity changesandimplicationsinhealthecology),ANR BiodiversityANR07BDIV012,fundedbytheFrench NationalAgencyforResearch.REFERENCESBalakirev,A.E.,Abramov,A.V.,andRozhnov,V.V.,2013,Revisionof thegenus Leopoldamys (Rodentia,Muridae)asinferredfrom morphologicalandmoleculardata,withaspecialemphasisonthe speciescompositionincontinentalIndochina:Zootaxa,v.3640,no.4, p.521.doi:10.11646/zootaxa.3640.4.2. Bradley,B.J.,Stiller,M.,Doran-Sheehy,D.M.,Harris,T.,Chapman, C.A.,Vigilant,L.,andPoinar,H.,2007,PlantDNAsequencesfrom feces:Potentialmeansforassessingdietsofwildprimates:American JournalofPrimatology,v.69,no.6,p.699.doi:10.1002/ajp. 20384. 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Zhang,Zheng,Schwartz,S.,Wagner,L.,andMiller,W.,2000,Agreedy algorithmforaligningDNAsequences:JournalofComputational Biology,v.7,no.1,p.203.doi:10.1089/10665270050081478.A.LATINNE,M.GALAN,S.WAENGSOTHORN,P.ROJANADILOK,K.EIAMAMPAI,K.SRIBUAROD,ANDJ.R.MICHAUXJournalofCaveandKarstStudies, August2014 N 145

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MICROCLIMATEEFFECTSONNUMBERAND DISTRIBUTIONOFFUNGIINTHEWODARZ UNDERGROUNDCOMPLEXINTHEOWLMOUNTAINS (GO RYSOWIE),POLANDRAFAOGO REK1,WOJCIECHPUSZ1*,AGNIESZKALEJMAN2,ANDCECYLIAUKLAN SKA-PUSZ3Abstract: InJuly2013westudiedtheoccurrenceoffungiinanundergroundcomplex namedWodarz,locatedinsidethemassifofWodarz,withintheOwlMountains,Lower Silesia,Poland.ThestudyisthefirstmycologicalevaluationoftherocksintheWodarz undergroundcomplexandtheairinsideandoutsideofit.Toexaminetheair,theAir Ideal3PsamplerandPDAmediumwereused.Microbiologicalevaluationoftherocks insidetheaditwasperformedusingtwomethods,swabsamplingandrinsesampling. TheresultswereanalyzedbyANOVA,andmeanswerecomparedusingFishersleast significantdifference(LSD)testat a # 0.05.Eleventaxaoffilamentousfungiwere isolatedfromtheairsampledoutsidetheWodarzadit,andfifteenfromtheairinside. Between65.5and1003colony-formingunitsoffungiperm3ofairwereisolatedfromthe airsampledintheaditandabout1115CFUfromtheairsampledoutsideofit;the differencesarestatisticallysignificant.Themajorityoftheairbornefungiwereisolated fromoutsidetheaditandfromtheventilationshaftcontainingawaterfall,probablydue toairmovement.Fromtherockwallsoftheshaftsseventaxaoffungiwereisolated, whereasfromtherockdebrisontheaditsfloor,onlysixtaxa.Thedensitiesoffungi obtainedfromtheresearchlocationsarestatisticallysignificant,andthemostdense fungusisolatedfromtheairoutsideandinsidetheaditwas Cladosporiumcladosporioides followedby C.herbarum atonelocationsintheadit.Taxaofthe Aspergillusniger group weremostcommonontherockdebrisandwallrocksexceptforonelocationwhere Penicilliumchrysogenum wasmostcommonontherockdebrisandonelocationwhere Cladosporiumcladosporioides wasmostcommonfromtherockswalls.INTRODUCTIONTheWodarzundergroundcomplexisasystemofadits andshaftsconstructedinsidetheupperpartofthemassif ofWodarz(inGerman,Wolfsberg),partoftheOwl Mountains(GorySowie)inCentralSudetes,LowerSilesia, Poland.Thecomplexislocatedwithinthenortheastern slopeofthemassif.Themountainrangeextendsinthe CentralSudetesalonganorthwest-southeastaxis,butfrom thepointofviewofgeologyandtectonics,thematerialis distinctanddescribedastheOwlMountainsgneiss. TheWodarzcomplexisoneofthecomponentsofa tunnelcomplexcode-namedRieseconstructedbytheNazis beginningin1943.Thelocationhadbeenchosenfortwo mainreasons.Theareawaslocatedawayfromthefront lines,andthestrengthoftherockandthestabilityofthe mountainswasadequateprotectionagainstpossibleair strikes.Thetunnelsweredrilledusingminingtechniques; prisonersboredholesintotherocksubstratethatwerethen loadedwithexplosives.Theworkforceusedwereprisoners fromtheNaziconcentrationcampGross-Rosen.Currently theinteriorofthecomplexcanonlybeaccessedbyoneof thefourexistingtunnels.Oftheremainingthree,two tunnelsarenowfloodedandcollapsed(Fig.1).Itis assumedthatthetargetnumberoftunnelswassix,butno previousstudieshaveconfirmedtheassumption.The entrancestothetunnelsarelocatedatanaltitudebetween 585and590mmsl,andthetunnelsruninthenortheastsouthwestdirectioninsidethetop-mostpartofWodarz Massif.Thetotallengthoftheknownandmapped excavationsisabout3100m,withatotalfloorsurfaceof approximately10,710m2andavolumeof42,000m3(Kasza2012).TheWodarzcomplexwasopenedfor touristsbeginningin2004,withaboutfortythousand visitorsayear,makingitoneofthemostpopularsitefor touristvisitsinLowerSilesia. Thepresenceoftouristsincavesandinotherundergroundplacesiscapableofchangingthemicroclimate,the biogeochemistry,andthebalanceoforganicmatterinthem. Itmayhenceindirectlyimpactautochthonousmicrobial *CorrespondingAuthor:wojciech.pusz@up.wroc.pl1WrocawUniversityofEnvironmentalandLifeSciences,DepartmentofPlant Protection,DivisionofPhytopathologyandMycology,pl.Grunwaldzki24a,50-363 Wrocaw,Poland3WrocawUniversityofEnvironmentalandLifeSciences,Departmentof Horticulture2WrocawUniversityofEnvironmentalandLifeSciences,Departmentof AgroecosystemsandGreenAreasManagementR.Ogorek,W.Pusz,A.Lejman,andC.Uklan ska-PuszMicroclimateeffectsonnumberanddistributionoffungiintheWodarz undergroundcomplexintheOwlMountains(GorySowie),Poland. JournalofCaveandKarstStudies, v.76,no.2,p.146.DOI: 10.4311/2013MB0123146 N JournalofCaveandKarstStudies, August2014

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communities,suchasthoseoffungi.Seenfromthat perspective,thevisitsarealwaysundesirable,andtherefore,thepresenceofmicroorganismsshouldbemonitored inallcaveandundergroundecosystems.Theidentificationofreservoirsofpotentiallypathogenicfungiandthe elucidationofthedistributionofthesecommunitiesand theircomponentsareimportantnotonlyinpreventing potentialhealthproblemsintourists(Ferna ndez-Corte set al.,2011;Sa z-Jime nez,2012),butalsoinmaintainingthe stabilityofanyundergroundecosystem.Fungicanbe dangerousforhumansandalsoaffectthewallsofcaves andadits.Inhumans,theycancauseinfectionsand allergies.Forexample, Aspergillus spp.cancauseaspergillosisofthelungs,sinuses,cornea,orbit,skin,nails,and earcanal. Rhizopus spp.cancausegeneralizedmucormycosis,aswellasinfectionsoflungsandsinuses.Infection by Fusarium spp.canresultingeneralizedfusariosis (Adamskietal.,2008).Fungisuchas Fusarium spp.in cavesandaditsmaycausebiodeteriorationofnatural stones(Guetal.,1998),andfungisuchas Epicoccum spp.cancontaminatestoneswithpigments(Lietal., 2008). Ourresearchfocusedontwogoals:1)themycological analysisofthespeciescompositionofthefungifoundin theairinsideandoutsideoftheundergroundWodarz complexandonrocksinit,and2)toquantifytheir concentrations.MATERIALSANDMETHODSTheairsamplesweretakenonJuly5,2013fromone locationoutsidetheadit(nearentrance4,samplelocationI inFig.1)andfromthreelocationsinsideit,theguard house(II),thecrossing(III),andtheventilationshaft(IV). Theairtemperatureandrelativehumidityweremeasured usingathermohygrometerLB-522(LAB-EL).Thecarbon dioxideconcentrationwasmeasuredusingaCO2meter pSenseRH(Gazex).Moisturecontentofrockswas measuredwiththeTesto606-1(Testo)hygrometer,and thewindspeedbyaerometerTesto410-1(Testo).MEDIAThefollowinggrowthmediawereused:PotatoDextroseAgar(PDA,Biocorp),Czapek-DoxAgar(1.2%agar, Biocorp)andMaltExtractAgar(MEA,Biocorp).PDA mediumwasusedfortheisolationoffungifromtheairand therocksandfortheidentificationofsomespecies. Czapek-DoxagarmediumandMEAmediumwereused foridentificationofthefungiof Penicillium and Aspergillus genera.FUNGALIDENTIFICATIONThefungalcoloniesgrownonallthePetridisheswere countedandidentified.Thespecificidentificationofthe sampledfungiwasperformedusingmacro-andmicroscopic Figure1.Theplan,basedonKasra(2012),oftheWodarzundergroundcomplexoftunnels,withthesamplelocations indicated.I,theentrance;II,theguardhouse;III,thecrossroads;andIVtheventilationshaft.R.OGO REK,W.PUSZ,A.LEJMAN,ANDC.UKLAN SKA-PUSZJournalofCaveandKarstStudies, August2014 N 147

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observationsofhyphae,conidia,andsporangiaandcolony morphologyaccordingtothecommonlyacceptedmethods usedinmycologicallaboratories.Thefungiwereidentified usingdiagnostickeys(RaperandFennell1965;Raperand Thom1968;Ellis1971;ZychaandSiepmann1973;Arx 1974).MYCOLOGICALEVALUATIONOFTHEAIRWeusedPDAmediumtoexaminethefungalloadof air.Thesampler(AirIdeal3P)wasprogrammedforair samplevolumesof50L,100L,and150L.Measurements ineachlocationwereperformedinsixreplicatesforeach volume.Thesamplerwaspositioned1.5mabovethelevel ofthecavefloor.Theincubationofthecultureswascarried outatroomtemperature(22 u C)for2to7days,in darkness.Aftertheincubation,thefungiwereidentified andthenumbersofcolonyformingunitsperm3ofairwere calculated.MYCOLOGICALEVALUATIONOFTHEROCKSMycologicalevaluationoftherocksinsidetheaditswas performedusingtwomethods,swabbingofthewalland rinsingofrockdebris. AtsamplelocationsII,III,andIVinsidethecomplex (Fig.1)swabsofthewallweremadeusingsterileswabsin transporttubes(sterile15cmviscoseswab).Materialfrom everylocationwassampledwiththreeswabsfromasurface areaof1cm2.Thesamplesweretakenfromthecavewalls attheheightof1.5mabovethefloor.Onthesameday,the collectedsampleswereshakenfor20minutesin50-ml Erlenmayerflaskscontaining10mlofsterilewater.After shaking,thesampleswereplacedinPetridishesontothe PDAagarusingserialdilution.Theincubationofcultures wasatroomtemperature(22 u C)indarkness,for2to 7days.Afterincubation,thefungiwereidentifiedandthe numbersofCFUpercm2ofrocksurfacewerecalculated. AtsamplinglocationsII,III,andIV(Fig.1),four samplesofrockdebrisfromthefloorwerecollectedin sterilesamplingbags(114by229mm).Onthesameday, eachsample(ca.50g)wasshakenfor20minutesina250mLErlenmeyerflaskcontaining100mLofsterilewater. Aftershaking,thesampleswereplatedontoPDAagar usingserialdilution.Theincubationofcultureswasfor2 to7days.Aftertheincubation,thefungaltaxawere identifiedandthenumbersofCFUper50gofrockdebris werecalculated.STATISTICALANALYSISTheresultswereanalyzedbyANOVA,usingStatistica 9.0package.MeanswerecomparedusingFishersleastsignificant-differencetestat a # 0.05.RESULTSTheresultsoftheenvironmentalmeasurementsarein Table1.TheairtemperatureduringthestudyinWodarz complexwas24.6 u Coutsidetheaditandapproximately 11 u Cinsideit.Thewindattheentrance(SiteI)was 0.8ms2 1.Insideairmovementwasonlyobservedatthe ventilationshaftwiththewaterfall(SiteIV),andthereit wasonly0.2ms2 1.Thehighestconcentrationofcarbon dioxidewasobservedinsidetheaditatSiteIII,andthe lowestwasattheentrance.Rockmoisturewashigherin wallrockthanindebrisontheaditsfloor. Eleventaxaoffilamentousfungiwereculturedfromthe airsampledoutsidetheWodarzcomplexandfifteenfrom theinsideair.Fromtherockwallsweculturedseventaxa offungi,butonlysixtaxafromthefloordebris. Aspergillus niger groupand Mucor spp.werepresentonlyontherock, whereas Acremoniumstrictum Alternariaalternata Botrytiscinerea Cladosporiumherbarum Epicoccumnigrum Fusariumavenaceum Fusariumequiseti Penicilliumcitrinum Penicilliumwaksmanii Sordariafimicola ,and Ulocladiumalternariae werefoundexclusivelyintheair(Table2). Thequantitiesoffilamentousfungitaxaisolatedfrom theairinsideandoutsidetheWodarzaditrangedbetween 65.5to1115.9colonyformingunitsperm3ofair(Table3). Themosttaxaoffilamentousfungiwereisolatedfromthe airoutsidethecomplex,whereasfromtheairinside,the highestnumberofspecieswereisolatedfromtheventilationshaftwithwaterfall(SiteIV)andthesmallestnumber ofspeciesfromthisgroupwasobservedintheguardhouse (Table3).Thehighestdensityofcolonies,bothfromthe rockwallsandfromtherockdebrisontheaditsfloor, wereculturedfromtheventilationshaftwithwaterfall (Tables4and5).ThesmallestnumberofCFUwas Table1.MicroclimateconditionsintheWodarzundergroundcomplexonJuly5,2013. MeasurementLocation Air WindSpeed, ms2 1CO2,ppm MoistureContent,% Temperature, u C Relative Humitidy,% Rock Wall Rock Debris Neartheentrance(SiteI)24.6 50.2 0.8390.0 ??? ??? Guard-house(SiteII) 11.0 61.4 0.0560.039.421.2 Crossroads(SiteIII) 10.6 67.4 0.0608.036.021.0 Ventilationshaftwithwaterfall (SiteIV) 12.8 98.0 0.2400.039.932.1MICROCLIMATEEFFECTSONNUMBERANDDISTRIBUTIONOFFUNGIINTHEWODARZUNDERGROUNDCOMPLEXINTHEOWLMOUNTAINS(GO RYSOWIE),POLAND148 N JournalofCaveandKarstStudies, August2014

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observedintheguardhouseforrockdebris,whileforrock wallboththeguardhouseandthecrossroadssiteswere smallestandstatisticallysimilar.(Tables4and5). Thefungusmostfrequentlyculturedfromtheair outsideandinsidetheaditwas Cladosporiumcladosporioides ,exceptintheguardhouse,where C.herbarum was mostcommon.Theleastcommonfungusspeciesinthe outsideairwas Acremoniumstrictum ,buttheabundanceof thisspeciesdonotdiffersignificantlyfromthatofsixother species.Thespecies Epicoccumnigrum Fusariumculmorum Penicilliumcitrinum Sclerotiniasclerotiorum ,and Ulocladiumalternariae weretheleastcommonintheguard house, F.culmorum wasleastcommoninthecrossroads samples,and F.culmorum and F.oxysporum wereleast commonattheventilationshaftsite(Table3). Thespeciesmostcommoninculturedfromtherock wallsandtherockydebriswasthe Aspergillusniger group intheguardhouseandcrossroadslocations,whileatthe ventilationshaft Cladosporiumcladosporioides wasmost commonontherockwallsand Penicilliumchrysogenum in rockyfloordebris(Tables4and5).DISCUSSIONThetotalnumbersoffungiculturedfromtheairatthe varioussamplingsitesdifferedsignificantly(Table3).The mostairbornefungiwerefoundoutsidethecomplex(SiteI inFig.1)andintheventilationshaftwithwaterfall(Site IV).Thesituationisprobablyduetotheairmovementat thoselocations(Table1).Moreover,thecomplexis populatedbybatcoloniesandaccessibletotourism (Martini,1963;ShapiroandPringle,2010;Porcaetal., 2011;Ogoreketal.,2013).Mulec(2008)reportedthat microbesarepassivelytransportedbyairflows,which dependontheseasonandrepresentanimportantmodeof spreadinginoculumtodifferentpartsofcaves.Dripping andseepingwater,asintheWodarzcomplexatthe ventilationshaft,maybeanothercarrierofmicrobes.Hsu andAgoramoorthy(2001),Kuzminaetal.(2012),and Mulecetal.(2012)notedthatmicroorganismsshow decreasingbiodiversityandbiomassstartingfromthe entrancetowardsthedeepzonesincaves,agradientthat theyfoundveryimportantforsurvivalanddevelopmentof fungi. AccordingtoNieves-Rivera(2003),Novakova(2009), andVanderwolfetal.(2013),themostabundantfungi incavesare Aspergillus Penicillium Mucor Fusarium Trichoderma ,andthoseof Cladosporium genus.Ourresults agreewiththeirs.Themostcommonairbornefungiinthe Wodarzcomplexwere Cladosporium fromair, Aspergillus and Cladosporium fromrockwalls,and Aspergillus and Penicillium fromrockydebris.Theseresultswerestatisticallysignificant.Theworkofotherresearcherssurveying caveswithrespecttofungalsporessupportsthesefindings. Table2.SpeciesoffilamentousfungiculturedfromtheoutsideandinsideairandfromtherocksintheWodarzcomplex.A + indicatesthespecieswasfound. Taxa Air Rock OutsideInsideWallDebris 1 Aspergillusniger group 22 ++ 2 Acremoniumstrictum + 222 3 Alternariaalternata + 222 4 Botrytiscinerea 2 + 22 5 Cladosporiumcladosporioides ++++ 6 Cladosporiumherbarum ++ 22 7 Epicoccumnigrum ++ 22 8 Fusariumavenaceum 2 + 22 9 Fusariumculmorum 2 ++ 2 10 Fusariumequiseti + 222 11 Fusariumoxysporum +++ 2 12 Mucor spp. 22 ++ 13 Penicilliumchrysogenum 2 + 2 + 14 Penicilliumcitrinum ++ 22 15 Penicilliumexpansum 2 ++ 2 16 Penicilliumwaksmanii 2 + 22 17 Rhizopus spp. ++++ 18 Sclerotiniasclerotiorum 2 + 2 + 19 Sordariafimicola ++ 22 20 Ulocladiumalternariae ++ 22 S species 11 15 7 6R.OGO REK,W.PUSZ,A.LEJMAN,ANDC.UKLAN SKA-PUSZJournalofCaveandKarstStudies, August2014 N 149

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Table3.FilamentousfungiculturedfromtheairattheWodarzundergroundcomplex,withmeansofcolonyformingunitsper m3forsixreplicatedairsamples. SamplingLocation Taxa Air(CFU/m3) Name Percent NearEntrance(SiteI) Alternariaalternata 0.3 3.3d Acremoniumstrictum 0.3 3.1d Botrytiscinerea 29.8 333.0b Cladosporiumcladosporioides 58.2 650.0a Cladosporiumherbarum 3.6 40.0c Epicoccumnigrum 0.6 6.7d Fusariumequiseti 0.3 3.0d Fusariumoxysporum 0.6 6.7d Penicilliumcitrinum 2.1 23.3cd Rhizopus spp. 3.0 33.0c Sclerotiniasclerotiorum 0.6 6.7d Ulocladiumalternariae 0.6 7.1d 1115.9A GuardHouse(SiteII) Cladosporiumcladosporioides 8.1 5.3d Cladosporiumherbarum 42.6 28.0a Epicoccumnigrum 2.3 1.5e Fusariumculmorum 2.0 1.3e Penicilliumchrysogenum 12.2 8.0b Penicilliumcitrinum 1.7 1.1e Penicilliumexpansum 8.1 5.3d Penicilliumwaksmanii 10.2 6.7c Rhizopus spp. 8.4 5.5cd Sclerotiniasclerotiorum 2.0 1.3e Ulocladiumalternariae 2.4 1.6e 65.5D Crossroads(SiteIII) Cladosporiumcladosporioides 33.8 25.0a Cladosporiumherbarum 19.6 14.4c Fusariumculmorum 1.5 1.1f Penicilliumchrysogenum 6.8 5.0e Penicilliumcitrinum 7.5 5.6e Penicilliumexpansum 21.8 16.1b Sclerotiniasclerotiorum 9.0 6.7d 73.9B VentilationShaftwith Waterfall(SiteIV) Botrytiscinerea 1.1 11.0e Cladosporiumcladosporioides 84.8 851.0a Cladosporiumherbarum 6.0 60.0b Epicoccumnigrum 0.7 6.7fg Fusariumavenaceum 0.5 5.1gh Fusariumculmorum 0.3 3.0h Fusariumoxysporum 0.3 3.3h Penicilliumexpansum 2.0 20.0d Sclerotiniasclerotiorum 3.3 33.3c Sordariafimicola 1.0 9.6ef 1003.0CForeachlocation,concentrationsfollowedbythesameletterarenotstatisticallydifferentatthe a # 0.05levelaccordingtoFishersleast-significant-differencetest;othersare. Smalllettersmarktheeffectoflocationsonisolatestaxafungi.Capitallettersmarktheeffectofaparticularlocationontotalfungalisolates.MICROCLIMATEEFFECTSONNUMBERANDDISTRIBUTIONOFFUNGIINTHEWODARZUNDERGROUNDCOMPLEXINTHEOWLMOUNTAINS(GO RYSOWIE),POLAND150 N JournalofCaveandKarstStudies, August2014

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AcaveinSpainexaminedbyDocampoetal.(2011)turned outtohostseveralfungalspecies,with Penicillium and Cladosporium generabeingthemostnumerous.Interestingly,inourairsamplingbothneartheentranceandinside wefoundmostlythesporesof Cladosporium atthe Wodarzcomplex.Ourresultsgenerallyagreewiththose ofPorcaetal.(2011)andFernandez-Cortesetal.(2011). Theyclaimedthatthemostabundanttypeofsporeinthe Table4.FilamentousfungiculturedfromswabsamplingthewallrockintheWodarzundergoundcomplex,meansofthree replicatesamples. SamplingLocation Taxa RockSurface (CFU/cm3) Name Percent Guard-House(SiteII) Aspergillusniger group 42.8 43.7a Cladosporiumcladosporioides 9.2 9.4d Mucor spp. 22.9 23.4b Penicilliumchrysogenum 5.5 5.6e Rhizopus spp. 19.7 20.1c 102.2BB Crossroads(SiteIII) Aspergillusniger group 55.4 57.3a Mucor spp. 20.7 21.4c Rhizopus spp. 23.9 24.7b 103.4B VentilationShaftwith Waterfall(SiteIV) Aspergillusniger group 14.0 25.0c Cladosporiumcladosporioides 37.1 66.0a Fusariumculmorum 6.2 11.0f Fusariumoxysporum 4.5 8.0g Mucor spp. 16.3 29.0b Penicilliumexpansum 9.6 17.0e Rhizopus spp. 12.4 22.0d 178.0AForeachlocation,concentrationsfollowedbythesameletterarenotstatisticallydifferentatthe a # 0.05levelaccordingtoFishersleast-significant-differencetest;othersare. Smalllettersmarktheeffectoflocationsonisolatestaxafungi.Capitallettersmarktheeffectofaparticularlocationontotalfungalisolates. Table5.FilamentousfungiculturedfromrinsesamplingthefloorrockdebrisintheWodarzundergoundcomplex,meansof fourreplicatesamples. SamplingLocation Taxa Rock (CFU/50g) Name Percent Guard-House(SiteII) Aspergillusniger group 55.7 76.0a Mucor spp. 28.6 39.0b Penicilliumchrysogenum 7.5 10.2c Sclerotiniasclerotiorum 8.3 11.3c 136.5C Crossroads(SiteIII) Aspergillusniger group 62.8 104.2a Cladosporiumcladosporioides 3.1 5.1d Mucor spp. 24.1 40.0b Rhizopus spp. 10.0 16.5c 165.8B VentilationShaftwith Waterfall(SiteIV) Aspergillusniger group 4.3 16.3d Cladosporiumcladosporioides 17.3 66.0c Mucor spp. 26.2 100.0b Penicilliumchrysogenum 52.3 200.0a 382.3AForeachlocation,concentrationsfollowedbythesameletterarenotstatisticallydifferentatthe a # 0.05levelaccordingtoFishersleast-significant-differencetest;othersare. Smalllettersmarktheeffectoflocationsonisolatestaxafungi.Capitallettersmarktheeffectofaparticularlocationontotalfungalisolates.R.OGO REK,W.PUSZ,A.LEJMAN,ANDC.UKLAN SKA-PUSZJournalofCaveandKarstStudies, August2014 N 151

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externalenvironmentwas Cladosporium ,whereasthe sporesmostwidelyrepresentedinsidethecavebelonged to Aspergillus and Penicillium .Accordingtootherauthors, themostabundantfungaltaxaisolatedfromrocks, especiallyfromgranite,werefromthefamily Mucorales andthegenera Penicillium Phoma Auerobasidium ,and Trichoderma (Hirschetal.,1995;Burfordetal.,2003a,b; Brunneretal.,2011).Ourresultsshowstatisticaldifferencesinthenumbersoffungiisolatedfromtherocks,and themostabundantfungiculturedfromgraniteinsidethe Wodarzcomplexwerethe Aspergillusniger groupfrom SitesIandIIand Cladosporiumcladosporioides fromrock wallsand Penicilliumchrysogenum fromrockdebrisfrom SiteIV.Highconcentrationsof Cladosporium spp.onthe wallsattheventilationshaftareprobablycausedbythe movementsofairandwaterfromtheoutsideintothe complexthere.Theventilationshaftprovidesaconstant bidirectionalexchangeofairfromtheexternalenvironmentthatwasdominatedfungiofthegenus Cladosporium Duringthepresentstudy,thehumidityoftheairand therockmoistureinsideandoutsidethecomplexwere conduciveforthesurvivalanddevelopmentoffungi.The airtemperatureoutside,butnotinside,wasfavorableto theirdevelopment.Krzysztofik(1992)reportedthatthe mostimportantfactorsaffectingthesurvivaloffungiin theenvironmentaretemperatureandhumidity.Nevertheless,thatauthorhadobservedthattheincidenceof fungiintheinvestigatedshaftshadbeendependenton airflow.Thisisconsistentwiththenumbersoffungi culturedfromtheairinsideourcomplexandfromrocks there.Themostfungiwereisolatedfromlocationswithair movement,includinganinterioroneconnectedwiththe externalenvironment. Thehighconcentrationsofcarbondioxideinsidethe complexmaybebroughtaboutbytouristsorbatsorby fungithatdecomposeorganicmatter.Hoyosetal.(1998) reportedthatopeningacavetotouristscanresultin changesinitsmicroclimaticconditionsandthefoodweb, asthemassofvisitorsincreasesthecavetemperature,CO2concentration,andtheamountofwatervaporinthecaves atmosphere.AstheWodarzundergroundcomplexisfairly popularfortouristvisits,thepossibleinfluenceofvisitors ontheconditionsinthecaveseemsquitelikelytoaffectthe fungalpopulationshostedbyit.AlthoughWellsandUota (1970)reportthatsomefungispeciesdecreaseinabundancewherethehighestvaluesofcarbondioxideare observed,itappearsthatinsomesituationshighercarbon dioxideconcentrationsmaystimulatefungitomore intensivegrowth.CONCLUSIONSMycobiotaoftheartificialundergroundaregenerally similartothoseofnaturalcaves.Theinternalmicroclimate andtheairflowinaditsproducestatisticallydistinct concentrationsandspeciescompositionsoffilamentous fungiinthem.MostfungiintheWodarzunderground complexoccurinplaceswheretheymayhavemigrated fromtheexternalenvironmentduetoairflow.Themost frequentlyculturedfungifromtheairoutsideandinside thecomplexwere Cladosporium spp.The Aspergillusniger groupweremostcommonlyfoundontherockswallsand rockdebrisexceptforonelocation,where Penicillium chrysogenum wasthespeciesmostoftenisolatedfromthe bottomsubstraterocksand C.cladosporioides fromthe rockwalls.Theincidencelevelofthefungiisolatedfrom internalairoftheaditconstitutesnothreattothehealthof thevisitingtourists,thoughitmaybeproblematicdueto possibleeffectsonhistoricalobjectslikeminingtrolleysor uniforms.Therefore,itseemsthatspeleomycological researchandmonitoringarenotonlyimportantforthe undergroundecosystemsthemselves,butalsoforprotectionofhistoricalmemorabilia.ACKNOWLEDGEMENTSWethanktheManagementStaffofWodarzcomplex fortheirpermittingourresearchintheWodarzundergroundcomplex.REFERENCESAdamski,Z.,Henke,K.,Zawirska,A.,andKubisiak-Rzepczyk,H.,2008, Grzybicenarzadowe(Fungalinfectionsoftheorgans), in Baran,E., ed.,Mykologiaconowego?(MycologyWhatsNew?),Wrocaw, Cornetis,p.189. Arx,J.A.von.,1974,TheGeneraofFungiSporulatinginPureCulture, Berlin,J.Cramer,315p. Brunner,I.,Plotze,M.,Rieder,S.,Zumsteg,A.,Furrer,G.,andFrey,B., 2011,PioneeringfungifromtheDammaglacierforefieldintheSwiss Alpscanpromotegraniteweathering:Geobiology,v.9,p.266. doi:10.1111/j.1472-4669.2011.00274.x. Burford,E.P.,Fomina,M.,andGadd,G.M.,2003a,Fungalinvolvementin bioweatheringandbiotransformationofrocksandminerals:MineralogicalMagazine,v.67,p.1127155.doi:10.1180/0026461036760154. Burford,E.P.,Kierans,M.,andGadd,G.M.,2003b,Geomycology:fungi inmineralsubstrata:Mycologist,v.17,no.3,p.98.doi:10.1017/ S0269915X03003112. Docampo,S.,Trigo,M.M.,Recio,M.,Melgar,M.,Garca-Sanchez,J., andCabezudo,B.,2011,Fungalsporecontentoftheatmosphereofthe CaveofNerja(southernSpain):diversityandorigin:ScienceoftheTotal Environment,v.409,p.835.doi:10.1016/j.scitotenv.2010.10.048. Ellis,M.B.,1971,DematiaceousHyphomycetes:Kew,Surrey,CommonwealthMycologicalInstitute,608p. Fernandez-Cortes,A.,Cuezva,S.,Sanchez-Moral,S.,Canaveras,J.C., Porca,E.,Jurado,V.,Martin-Sanchez,P.M.,andSa z-Jimenez,C., 2011,Detectionofhuman-inducedenvironmentaldisturbancesina showcave:EnvironmentalScienceandPollutionResearch,v.18, p.1037.doi:10.1007/s11356-011-0513-5. Gu,Ji-Dong,Ford,T.E.,Berke,N.S.,andMitchell,R.,1998, Biodeteriorationofconcretebythefungus Fusarium :International BiodeteriorationandBiodegradation,v.41,p.101.doi:10.1016/ S0964-8305(98)00034-1. Hirsch,P.,Eckhardt,F.E.W.,andPalmer,R.J.Jr.,1995,Fungiactivein weatheringofrockandstonemonuments:CanadianJournalof Botany,v.72,no.S1,p.138490.doi:10.1139/b95-401. Hoyos,M.,Soler,V.,Canaveras,J.C.,Sanchez-Moral,S.,andSanzRubio,E.,1998,Microclimaticcharacterizationofakarsticcave: humanimpactonmicroenvironmentalparametersofaprehistoric rockartcave(CandamoCave,northernSpain):Environmental Geology,v.33,p.231.doi:10.1007/s002540050242.MICROCLIMATEEFFECTSONNUMBERANDDISTRIBUTIONOFFUNGIINTHEWODARZUNDERGROUNDCOMPLEXINTHEOWLMOUNTAINS(GO RYSOWIE),POLAND152 N JournalofCaveandKarstStudies, August2014

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Hsu,MinnaJ.,andAgoramoorthy,G.,2001,Occurrenceanddiversityof thermophiloussoilmicrofungiinforestandcaveecosystemsof Taiwan:FungalDiversity,v.7,p.27. Kasza,D.,2012,Moz liwosciwykorzystaniaaplikacjiGISdoprac zwiazanychzkartowaniemgeologicznymnaprzykadziepodziemnego obiektuWodarzwGorachSowich:PraceNaukoweInstytutu GornictwaPolitechnikiWrocawskiej,no.135,p.23.doi:10.5277/ gig121802. Krzysztofik,B.,1992,Mikrobiologiapowietrza,Warsaw,Wydawnictwa PolitechnikiWarszawskiej,Warsaw,198p. Kuzmina,L.Y.,Galimzianova,N.F.,Abdullin,S.R.,andRyabova,A.S., 2012,MicrobiotaoftheKinderlinskayaCave(SouthUrals,Russia): Microbiology,v.81,no.2,p.251.doi:10.1134/S0026261712010109. Li,Xianshu,Arai,H.,Shimoda,I.,Kuraishi,H.,andKatayama,Y.,2008, Enumerationofsulfur-oxidizingmicroorganismsondeteriorating stoneoftheAngkormonumentsCambodia:MicrobesandEnvironments,v.23,p.293.doi:10.1064/jsme2.ME08521. Martini,A.,1963,Yeastsincavernenvironments:ArchivfurMikrobiologie,v.45,p.111.doi:10.1007/BF00408431. Mulec,J.,2008,Microorganismsinhypogeon:examplesfromSlovenian karstcaves:ActaCarsologica,v.37,no.1,p.153. Mulec,J.,Vaupotic ,J.,andWalochnik,J.,2012,Prokaryoticand eukaryoticairbornemicoorganismsastracersofmicroclimatic changesintheunderground(PostojnaCave,Slovenia):Microbial Ecology,v.64,p.654.doi:10.1007/s00248-012-0059-1. Nieves-Rivera,A .M.,2003,MycologicalsurveyofRioCamuyCaves Park,PuertoRico:JournalofCaveandKarstStudies,v.65,no.1, p.23. Novakova,A.,2009,MicroscopicfungiisolatedfromtheDomicaCave system(SlovakKarstNationalPark,Slovakia).Areview:InternationalJournalofSpeleology,v.38,p.71.doi:10.5038/1827806X.38.1.8. Ogorek,R.,Lejman,A.,andMatkowski,K.,2013,Fungiisolatedfrom Niedz wiedziaCaveinKletno(LowerSilesia,Poland):International JournalofSpeleology,v.42,p.161.doi:10.5038/1827-806X. 42.2.9. Porca,E.,Jurado,V.,Martin-Sanchez,P.M.,Hermosin,B.,Bastian,F., Alabouvette,C.,andSaiz-Jimenez,C.,2011,Aerobiology:an ecologicalindicatorforearlydetectionandcontroloffungal outbreaksincaves:EcologicalIndicators,v.11,p.159498. doi:10.1016/j.ecolind.2011.04.003. Raper,K.B.,andFennell,D.I.,1965,TheGenusAspergillus:Baltimore, WilliamsandWilkinsCompany,686p. Raper,K.B.,andThom,C.,1968,AManualofthePenicillia:NewYork, HafnerPublishingCompany,876p. Sa z-Jimenez,C.,2012,Microbiologicalandenvironmentalissuesinshow caves:WorldJournalofMicrobiologyandBiotechnology,v.28, no.7,p.245364.doi:10.1007/s11274-012-1070-x. Shapiro,J.,andPringle,A.,2010,Anthropogenicinfluencesonthe diversityoffungiisolatedfromcavesinKentuckyandTennessee: AmericanMidlandNaturalist,v.163,no.1,p.76.doi:10.1674/ 0003-0031-163.1.76. Vanderwolf,K.J.,Malloch,D.,McAlpine,D.F.,andForbes,G.J.,2013, Aworldreviewoffungi,yeasts,andslimemoldsincaves: InternationalJournalofSpeleology,v.42,no.1,p.77. doi:10.5038/1827-806X.42.1.9. Wells,J.M.,andUota,M.,1970,Germinationandgrowthoffivefungiin low-oxygenandhigh-carbondioxideatmospheres:Phytopathology, v.60,no.1,p.50.doi:10.1094/Phyto-60-50. Zycha,H.,Siepmann,R.,andLinnemann,G.,1973,KeystotheFamilies, GeneraandSpeciesoftheMucorales:Berlin,J.Cramer,49p.R.OGO REK,W.PUSZ,A.LEJMAN,ANDC.UKLAN SKA-PUSZJournalofCaveandKarstStudies, August2014 N 153

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BOOKREVIEW SourcesetSitesdesEauxKarstiques (Karstspringsandtheirsettings) JeanNicod,2012,Mediterranee(Geographicalrevueof Mediterraneancountries),Specialedition,PressesUniversitairesdeProvence,29ave.RobertSchuman,13621Aixen-Provence,Cedes01,France,277p.,8.2311.5in.,ISBN 978-2-85399-810-9,soft-cover, J 30( $40). FewreadersofthisjournalarefluentinFrench,but whenthisbookarrivedforreviewitshowedpromisebeyond itsimmediategoalofsummarizingthesignificantkarstin thecountriesborderingtheMediterranean.Prof.Nicod,one oftheworldspremierkarstresearchers,hasassembleda lifetimeofinformationaboutthisimportantregion.In condensedfashion,hesummarizeshisownresearchandthat ofmanyothers.Itispurposelynotcomprehensive,but containsthemostrepresentativeandinstructiveexamples. Karstgeomorphologyandlandusearethemajortopics. Theauthoradmitstoanold-schoolapproach,withmuch attentiontothephysicalsettingandhowpeoplehave adaptedtoit.Mostoftheemphasisisonthesouthern Europeancountries,lesssoontheLevantandnorthern Africa,butthegeographicrangeextendsoutwardasfaras Germany.Manyobservationsspanseveraldecades,givinga historicalperspective.Surfacefeaturesreceivemoreemphasisthancaves. Thebookcontainsfourmajorsections:(1)karstsprings andtheirgeographiccontext,(2)karstlakes,(3)water supply,dams,andaqueductsinkarst,and(4)travertine sites.Thesearedividedintoatotalofeighteenchapters, eachcontainingnumerousindividualtopics.Thereare manysidebarswithspecialexamplesandcasehistories, manyofthemclassics.Inthereararefifteenpagesofcolor photosandmaps,includingadetailedtwo-pagespreadof theregioncovered. Descriptionsareshortandtothepoint,butwithroom forinstructiveanecdotes.Morethanhalfthespaceis devotedtomonochromephotos,diagrams,maps,sketches, andtables.Geologicmapsandcrosssectionsaredetailed, butmanyofthecaveprofilesaresimplified.Manyshow theiragebytheirinformalbutpersonalizedhand-drawn style.Eachchapterendswithalistofpertinentreferences. Casehistoriesprovideexamplesandoddanecdotesof theuse(andmisuse)ofkarstwater,suchasvanishinglakes, reservoirsthatneverfilled,andpollution.Forexample,one learnsthatmuchoftheupperpartoftheDanubeRiver disappearsundergroundandemergesinadistantkarst springintheRhinebasin,toreachtheoceanattheopposite endofEuropefromwhereitwasoriginallyheaded.Have youheardoftheaccidentalwater-tracewithabsinthe?Or thedeepspringsthataretheindirectresultoftheMessinian CrisiswhentheMediterraneanSeadriedupafewmillion yearsago?Orwhatlifeislikeinahomecarvedoutof travertinedepositedbykarstsprings?OrSt.JohnofTufa? WhatifyoudontreadFrench?Firstfindaninteresting mapordiagramorexclamationpointsinthetextandenter theaccompanyingsentencesintooneofthefreeon-line translationsites.Opticalcharacterrecognitiononyour scannercanevenbypasstheneedfortyping.The translationwillnotbepoetic,butanyonefamiliarwith karstwillunderstandit. Prof.Nicoddedicatesthisbookasatributetohis colleagues,pastandpresent.Herecognizesthattheir originalmaterialisscatteredandincreasinglydifficultto find,andthatmodernresearchersrarelyprobetheolder literature.Thesestatementscanbereadasaninvitation: Researcherswithalife-longpassionhavemuchtosay,and theyareencouragedtosummarizethedevelopmentsthey havewitnessedintheirfieldssothattherecordispreserved.ReviewedbyArthurN.Palmer,Dept.ofEarthandAtmosphericSciences, StateUniversityofNewYork,Oneonta,NY13820-4015,(palmeran@ oneonta.edu). DOI:10.4311/2013BR0103154 N JournalofCaveandKarstStudies, August2014

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GUIDE TO AUTHORS The Journal of Cave and Karst Studies is a multidisciplinary journal devoted to cave and karst research. The Journal is seeking original, unpublished manuscripts concerning the scientic study of caves or other karst features. Authors do not need to be members of the National Speleological Society, but preference is given to manuscripts of importance to North American speleology. LANGUAGES: The Journal of Cave and Karst Studies uses American-style English as its standard language and spelling style, with the exception of allowing a second abstract in another language when room allows. In the case of proper names, the Journal tries to accommodate other spellings and punctuation styles. In cases where the Editor-in-Chief nds it appropriate to use nonEnglish words outside of proper names (generally where no equivalent English word exists), the Journal italicizes them. However, the common abbreviations i.e., e.g., et al., and etc. should appear in roman text. Authors are encouraged to write for our combined professional and amateur readerships. CONTENT: Each paper will contain a title with the authors names and addresses, an abstract, and the text of the paper, including a summary or conclusions section. Acknowledgments and references follow the text. ABSTRACTS: An abstract stating the essential points and results must accompany all articles. An abstract is a summary, not a promise of what topics are covered in the paper. STYLE: The Journal consults The Chicago Manual of Style on most general style issues. REFERENCES: In the text, references to previously published work should be followed by the relevant authors name and date (and page number, when appropriate) in parentheses. All cited references are alphabetical at the end of the manuscript with senior authors last name rst, followed by date of publication, title, publisher, volume, and page numbers. Geological Society of America for mat should be used (see http://www.geosociety.org/pubs/geoguid5. htm). Please do not abbreviate periodical titles. Web references are acceptable when deemed appropriate. The references should follow the style of: Author (or publisher), year, Webpage title: Publisher (if a specic author is available), full URL (e.g., http://www. usgs.gov/citguide.html) and date when the web site was accessed in brackets; for example [accessed July 16, 2002]. If there are specic authors given, use their name and list the responsible organization as publisher. Because of the ephemeral nature of websites, please provide the specic date. Citations within the text should read: (Author, Year). SUBMISSION: Effective February 2011, all manuscripts are to be submitted via Peertrack, a web-based system for online submission. The web address is http://www.edmgr.com/jcks. Instructions are provided at that address. At your rst visit, you will be prompted to establish a login and password, after which you will enter information about your manuscript (e.g., authors and addresses, manuscript title, abstract, etc.). You will then enter your manuscript, tables, and gure les separately or all together as part of the manuscript. Manuscript les can be uploaded as DOC, WPD, RTF, TXT, or LaTeX. A DOC template with additional manuscript specications may be downloaded. (Note: LaTeX les should not use any unusual style les; a LaTeX template and BiBTeX le for the Journal may be downloaded or obtained from the Editor-inChief.) Table les can be uploaded as DOC, WPD, RTF, TXT, or LaTeX les, and gure les can be uploaded as TIFF, EPS, AI, or CDR les. Alternatively, authors may submit manuscripts as PDF or HTML les, but if the manuscript is accepted for publication, the manuscript will need to be submitted as one of the accepted le types listed above. Manuscripts must be typed, double spaced, and single-sided. Manuscripts should be no longer than 6,000 words plus tables and gures, but exceptions are permitted on a case-bycase basis. Authors of accepted papers exceeding this limit may have to pay a current page charge for the extra pages unless decided otherwise by the Editor-in-Chief. Extensive supporting data will be placed on the Journals website with a paper copy placed in the NSS archives and library. The data that are used within a paper must be made available. Authors may be required to provide supporting data in a fundamental format, such as ASCII for text data or comma-delimited ASCII for tabular data. DISCUSSIONS: Critical discussions of papers previously published in the Journal are welcome. Authors will be given an opportunity to reply. Discussions and replies must be limited to a maximum of 1000 words and discussions will be subject to review before publication. Discussions must be within 6 months after the original article appears. MEASUREMENTS: All measurements will be in Systeme Internationale (metric) except when quoting historical references. Other units will be allowed where necessary if placed in parentheses and following the SI units. FIGURES: Figures and lettering must be neat and legible. Figure captions should be on a separate sheet of paper and not within the gure. Figures should be numbered in sequence and referred to in the text by inserting (Fig. x). Most gures will be reduced, hence the lettering should be large. Photographs must be sharp and high contrast. Color will generally only be printed at authors expense. TABLES: See http://www.caves.org/pub/journal/PDF/Tables. pdf to get guidelines for table layout. COPYRIGHT AND AUTHORS RESPONSIBILITIES: It is the authors responsibility to clear any copyright or acknowledgement matters concerning text, tables, or gures used. Authors should also ensure adequate attention to sensitive or legal issues such as land owner and land manager concerns or policies. PROCESS: All submitted manuscripts are sent out to at least two experts in the eld. Reviewed manuscripts are then returned to the author for consideration of the referees remarks and revision, where appropriate. Revised manuscripts are returned to the appropriate Associate Editor who then recommends acceptance or rejection. The Editor-in-Chief makes nal decisions regarding publication. Upon acceptance, the senior author will be sent one set of PDF proofs for review. Examine the current issue for more information about the format used. ELECTRONIC FILES: The Journal is printed at high resolution. Illustrations must be a minimum of 300 dpi for acceptance.The Journal of Cave and Karst Studies (ISSN 1090-6924, CPM Number #40065056) is a multi-disciplinary, refereed journal published three times a year by the National Speleological Society, 2813 Cave Avenue, Huntsville, Alabama 35810-4431 USA; Phone (256) 852-1300; Fax (256) 851-9241, email: nss@caves.org; World Wide Web: http://www.caves.org/pub/journal/. Check the Journal website for subscripion rates. Back issues and cumulative indices are available from the NSS ofce. POSTMASTER: send address changes to the Journal of Cave and Karst Studies, 2813 Cave Avenue, Huntsville, Alabama 35810-4431 USA. The Journal of Cave and Karst Studies is covered by the following ISI Thomson Services Science Citation Index Expanded, ISI Alerting Services, and Current Contents/Physical, Chemical, and Earth Sciences. Copyright 2014 by the National Speleological Society, Inc. Front cover: Moonmilk stalatites in Grotta Nera, Italy. See Cacchio et al., in this issue.Published By The National Speleological SocietyEditor-in-Chief Malcolm S. FieldNational Center of Environmental Assessment (8623P) Ofce of Research and Development U.S. Environmental Protection Agency 1200 Pennsylvania Avenue NW Washington, DC 20460-0001 703-347-8601 Voice 703-347-8692 Fax eld.malcolm@epa.govProduction EditorScott A. EngelCH2M HILL 2095 Lakeside Centre Way, Suite 200 Knoxville, TN 37922 865-560-2954 scott.engel@ch2m.comJournal Copy EditorBill MixonJOURNAL ADVISORY BOARD Penelope Boston Gareth Davies Luis Espinasa Derek Ford Louise Hose Leslie Melim Wil Orndorf Bill Shear Dorothy Vesper BOARD OF EDITORS AnthropologyGeorge Crothers University of Kentucky211 Lafferty Hall george.crothers@uky.eduConservation-Life SciencesJulian J. Lewis & Salisa L. LewisLewis & Associates, LLC. lewisbioconsult@aol.comEarth SciencesBenjamin SchwartzDepartment of Biology Texas State University bs37@txstate.eduRobert BrinkmanDepartment of Geology, Environment, and Sustainability Hofstra University robert.brinkmann@hofstra.eduMario PariseNational Research Council, Italy m.parise@ba.irpi.cnr.itExplorationPaul BurgerCave Resources Ofce National Park Service Carlsbad, NM paul_burger@nps.govMicrobiologyKathleen H. LavoieDepartment of Biology State University of New York, Plattsburgh, lavoiekh@plattsburgh.eduPaleontologyGreg McDonaldPark Museum Management Program National Park Service, Fort Collins, CO greg_mcdonald@nps.govSocial SciencesJoseph C. DouglasHistory Department Volunteer State Community College joe.douglas@volstate.eduBook ReviewsArthur N. Palmer & Margaret V. PalmerDepartment of Earth Sciences State University of New York, Oneonta palmeran@oneonta.edu

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Journal of Cave and Karst Studies Volume 76 Number 2 August 2014Article 69Karst Evolution of the Garraf Massif (Barcelona, Spain): Doline Formation, Chronology and Archaeopalaeontological Archives John Daura, Montserrat Sanz, Joan Josep Forns, Antoni Asensio, and Ramon JuliArticle 88Biogenicity and Characterization of Moonmilk in the Grotta Nera (Majella National Park, Abruzzi, Central Italy) Paola Cacchio, Gianluca Ferrini, Claudia Ercole, Maddalena Del Gallo, and Aldo LepidiArticle 104Adaptations of Indigenous Bacteria to Fuel Contamination in Karst Aquifers in South-Central Kentucky Tom D. Byl, David W. Metge, Daniel T. Agymang, Mike Bradley, Gregg Hileman, and Ron W. HarveyArticle 114Aerosolized Microbes from Organic Rich Materials: Case Study of Bat Guano From Caves in Romania Daniela R. Borda, Ruxandra M. Na stase-Bucur, Marina Spnu, Raluca Uricariu, and Janez MulecArticle 127Glacial Lake Schoharie: An Investigative Study of Glaciolacustrine Lithofacies in Caves, Helderberg Plateau, Central New York Jeremy M. Weremeichik and John E. MylroieArticle 139Diet Analysis of Leopoldamys Neilli, A Cave-Dwelling Rodent In Southeast Asia, Using Next-Generation Sequencing From Feces Alice Latinne, Maxime Galan, Surachit Waengsothorn, Prateep Rojanadilok, Krairat Eiamampai, Kriangsak Sribuarod, and Johan R. MichauxArticle 146Microclimate Effects on Number and Distribution of Fungi in the Wodarz Underground Complex in the Owl Mountains (Gry Sowie), Poland Rafa Ogrek, Wojciech Pusz, Agnieszka Lejman, and Cecylia Uklan ska-PuszBook Review 154Sources et Sites des Eaux KarstiquesJournal of Cave and Karst StudiesVolume 76 Number 2 August 2014 Journal of Cave and Karst Studies Distribution Changes During the November 9, 2013, Board of Governors meeting, the BOG voted to change the Journal to electronic distribution for all levels of membership beginning with the April 2014 issue. Upon publication, electronic les (as PDFs) for each issue will be available for immediate viewing and download through the Member Portal on www.caves.org. For those individuals that wish to continue to receive the Journal in a printed format, it will be available by subscription for an additional fee. Online subscription and payment options will be made available through the website in the near future. Until then, you can arrange to receive a print subscription of the Journal by contacting the NSS ofce at (256) 852-1300. August 2014 Volume 76, Number 2 ISSN 1090-6924 A Publication of the National Speleological Society JOURNAL OF CAVE AND KARST STUDIES rf


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