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DNA BARCODING OF MACROFUNGI FROMTHE 2018 SMITH FORAY: NEW FUNGAL RECORDS FOR WISCONSINANDTHE UNITED STATES OFAMERICA

AldenDirks DepartmentofEcologyandEvolutionaryBiology UniversityofMichigan 1105NorthUniversityAvenue,4050BiologicalSciencesBuilding AnnArbor,MI48109

StephenD.Russell1 DepartmentofBotanyandPlantPathology PurdueUniversity WestLafayette,IN47907

ABSTRACT

Foraccurateevaluationoffungalconservation,modernbiodiversitysurveysbasedonvouchered specimensandDNAbarcodingareneededtoaugmentrecordsoffungaldistributionandphenology. Even relatively well studied and populated regions of the United States lack comprehensive informationonfungalbiodiversity, whichhampersourabilitytoswiftlyrespondtofungalpopulationdecline due to habitat loss, climate change, or other anthropogenic stressors. During the 2018 Smith Foray in Dane County, Wisconsin, we vouchered and DNAbarcoded 63 specimens of macrofungi. ThreespeciesconstitutedfirstrecordsfortheUnitedStates,and14additionalspecieswerereported forthefirsttimefromthestateofWisconsin.Furthermore,eightspecieswerenewreportsjustatthe county level, and barcode data for two species represented first records in GenBank, the national public repository for genetic information. Twenty-four specimens were assigned informal placeholdernamesdueto the lack of similar referencesin GenBankandarefertilegroundfor future taxonomic studies. While sequence-based identification requires caution due to inaccuracies in reference databases, the prevalence of multilocus genetic data in contemporary taxonomy facilitates globallinkagesinfungaldistributionandincreasinglytraceablebiodiversity assessments.

KEYWORDS: Dane County, fungal biodiversity inventory, mushrooms, North American My- cofloraProject,sequence-basedidentification

INTRODUCTION

Despite their enormous importance for global ecosystem functioning and as reservoirsofgeneticresources,fungiareoneofthemostunderstudiedgroupsof organisms, especially in the context of conservation (Hawksworth 2004; Heilmann- Clausenetal.2014;Willis2018).Mycologistsestimatethatthetotalnumber offungi on Earth may exceed5 million species,yet we have described only approximately150,000, or aboutthree percent ofthe estimatedtotal(Blackwell 2011;Willis 2018). Many regions, including ones with alonghistory ofprofes

1Author for correspondence (srussell@purdue.edu)

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sional mycological activity, lack comprehensive data on fungal biodiversity (Haelewaters et al. 2018). Such knowledge gaps in local funga (Kuhar et al. 2018)—undescribedfungalbiodiversityandpoorlyunderstooddistributionsand phenology—hamper our ability to assess and respond to population declines. Anthropogenicdisturbancessuchasclimatechange,habitatdestruction,andexcess nitrogen deposition threaten the well-being of fungi and the ecosystems whosefunctioningdependsonthem(Mueller2017;Andrewetal.2018;vander Lindeetal.2018),therebylendingurgencytoincreasedbiodiversitysurveysfor theaccurateevaluationoftheconservationstatusoffungalspecies.High-quality vouchersofmushrooms,ideallypairedwithdetailedphotographyandDNAbarcode data, serve as evidence of reproducing populations of macrofungi and as benchmarks for future assessments of fungal range shifts and population declines (Andrew et al. 2018).

In this paper, we report on macrofungi that were vouchered and DNA bar- coded from the 2018A.H. Smith Lake States Foray (commonly known as the “SmithForayâ€)onOctober4–7,2018nearMazomanie,Wisconsin.Thiswasthe 44thannualSmithForay,whichhasbeenheldeachyearsince1975tohonorthe mycological accomplishments of Dr. Alexander Smith (1904–1986) (Thiers 1987) and to foster interactions among mycologists in the Great Lakes region. Past foray locations have included sites in Indiana, Illinois, Michigan, Minnesota, andWisconsin (Mycological Society ofAmerica 2019).Attendees typically consist ofprofessional mycologists from the upper Midwest andtheir students, as well as other amateur andprofessional scientists who are interestedin fungi. This annual eventis an opportunityto enhance our knowledge of macrofungalbiodiversityfrom the GreatLakes states,and ultimatelytocontribute our understandingoftheresponseoffungitoglobalchange(Andrewetal.2018),by augmenting records of occurrence, distribution, and phenology of local mushroom taxa.

MATERIALSAND METHODS

Study-site Description

Fungi were collectedfrom four areas in Dane County, Wisconsin, duringthe 2018SmithForay (Figure1)—FestgeCountyPark, MazomanieBottoms State NaturalArea, MazomanieOakBarrens State NaturalArea, andWalkingIron CountyPark—as well as opportunisticallyfrom several other locations around Dane County. These locations exist on the eastern edge of the extensive Driftless Area,aregionsurroundingtheupperMississippiRiverinWisconsin,Minnesota,Iowa,andIllinois. Asindicatedbyalackofglacialtill,theDriftlessregionwasneverglaciatedduringthelastIceAge (Hobbs 1999). It is characterized by a topology of rolling hills that contrasts with an otherwise smooth Midwestern landscape. Special geological features and ecosystems such as algific talus slopes harbor unique and speciose funga (Hawksworth2010;Thompson andColbert2020).Ashort description of eachofthe fourprimarycollecting sitesisasfollows:

Festge County Park in Cross Plains (43.121744 N, –89.6829076W): Mature Carya spp. (hickory) and Quercus spp. (oak) are the primary tree species here. The steep topography of Festge County Park is evidence of the unglaciated history of this region and provides an overlook of the BlackEarthCreekValleyandBlueMoundStateParkin thedistance.

MazomanieBottomsStateNaturalAreainMazomanie(43.219381N,–89.818698W):Thissite encompassesalargeareaofWisconsinRiverfloodplainforest.TheforestisdominatedbyAcer saccharinum L.(silver maple),Ulmus spp.(elm),Tilia americana L.(American basswood),andFraxinus spp. (ash) and, in addition, contains occasional individuals of Quercus bicolor Willd. (swamp

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FIGURE 1. Map of the locations of the 2018 Smith Foray collection sites in Dane County, Wisconsin. “SNA†is an abbreviation for “State NaturalAreaâ€.

whiteoak),Populus deltoides W.BartramexMarshall(easterncottonwood),Salix spp.(willow),and Betula nigra L.(riverbirch).Elmmortalityhasresultedincanopyopeningsthatsupportadenseunderstoryofnativeandintroducedshrubs. Periodicfloodingresultsinsandbars,ephemeralpools,and river channelsrunningthroughtheforest.

MazomanieOakBarrensStateNaturalAreainMazomanie(43.242305N,–89.739786W):This siteisdecidedlydrierthantheMazomaniefloodplainforestssurroundingtheWisconsinRiver.Wisconsin’s native cactus, Opuntia cespitosa Raf. (eastern prickly pear cactus) is abundant at MazomanieOakBarrens. OakspeciessuchasQuercus macrocarpa Michx.(buroak),Quercus velutina Lam. (black oak), and Quercus alba L. (white oak) interspersed with xeric prairie dominate in this dry,sandy environment.

WalkingIronCountyParkinMazomanie(43.187734N,–89.8246248W):Thisparkpreservesa segment of the extensive prairie that extendedfrom the bottoms area ofthe Wisconsin River to the surrounding oak savanna. Most ofthe parkis sandy uplands coveredbygrasslands, including some unplowed, remnantprairie.The northarea has aridgethatdrops sharplydownto MarshCreek,one exampleofthemany cool,spring-fed streams foundin thispartofthestate.

Specimen Collection, Processing, and Vouchering

Macrofungal sporocarps were opportunistically collected atthe foraylocations by eventparticipantsandbroughttoacentralizedprocessingareaatHoofbeatRidgeCamps. Noeffortwasmadeto systematicallycoveracollectinglocation.Eachspecimenwastentativelyidentifiedbyalocalexpert and recorded into a central database along with metadata such as collection location. Sixty-three mushrooms from the event were selected for DNA sequencing. These specimens were generally speciesthatwerenewtotheSmithForay,ofparticularinteresttotheforayattendants,orthatlacked referencesequencedatainpublicrepositories.EachspecimenthatwasselectedforDNAsequencing was assigned a collection number, photographed, and uploaded to iNaturalist (2020). The selected specimenswerethoroughlydriedinadehydrator(Presto06301)at32°C.Driedspecimensweredepositedin the KriebelFungarium (PUL) atPurdue University and were digitally accessionedin the MycologyCollectionsdata Portal(MyCoPortal2020;Miller andBates 2017).

Molecular Methods

Mushroom tissue was extracted from the interior flesh or gill tissue of fresh specimens at the foray processing center utilizing sterile forceps. The tissue was placed in 2.0 mL screw-top micro- centrifuge tubes containing 600 μL of Promega Nuclei Lysis Solution (Promega Corp., Madison, Wisconsin). Each tube was labeled with the specimen’s collection number and transported to the Aime Lab at Purdue University in West Lafayette, Indiana for DNA extraction and amplification. DNAextraction was accomplished by macerating the tissue using a sterile pestle, heatingthe solu

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tion at65°Cfor 15 minutes, and centrifugingthe contents ofthe tube at21,000gfor three minutes. The supernatant was transferredto a1.5mLmicrocentrifugetube, 200μLofPromegaNucleiLysis Solution was added, and the tube was then vortexed for 20 seconds. The solution was centrifuged againat21,000gforsix minutesandthe supernatantwasaddedtoanew,sterile,1.5 mLmicrocentrifugetube. 600μLof100% isopropanolwasaddedtothe supernatanttoprecipitatetheDNA.The solutionwascentrifugedforoneminuteat21,000gandthesupernatantwaspouredoff, leavingthe DNApelletinthebottomofthetube.600μLof70% ethanolwasaddedtothetube,andthesolution wascentrifugedafinaltimeforoneminuteat21,000g.Theethanolwaspouredoutandthe1.5mL microcentrifugetubewasplacedupsidedownonaKimwipeovernight.Thefollowingday,30μLof water was addedtothetube,resultingin purifiedDNAforuse in PCRamplification.

PCRamplificationsoftheinternaltranscribedspacer(ITS)ribosomalDNA(rDNA)region—the universalDNAbarcodemarkeroffungi(Schochetal.2012)—werecarriedoutusingtheITS1FforwardprimerandtheITS4reverseprimer( Whiteetal.1990;GardesandBruns1993).EachPCRreactioncontained12.5μLPromegaPCRMasterMix, 9μLwater,1.25μLforwardprimer,1.25μLre- verseprimer,and1μLDNAtemplateforatotalPCRvolumeof25μL.ThefollowingPCRprotocol wasused:(i)initialdenaturationat9Cforoneminute;(ii)30cyclesofdenaturationat94°Cforone minute, annealing at51°Cfor one minute, and extension at72°Cfor one minute;(iii)hold at72°C foreightminutes.Electrophoresiswitha1% agarosegelwasusedtoverifysuccessfulamplification. PCR amplicons were sentto Genewiz(Genewiz, Inc., Boston, Massachusetts,USA)for sequencing ofboththeforwardandreverseDNAstrands.ThetworeadswereassembledusingSequencher5.0.1 (Gene Codes Corp.,AnnArbor, Michigan) and the consensus sequence was deposited in GenBank (Clarketal.2016).RawDNAsequencedata(tracefiles)areavailableatthe2018SmithForayMycoMapproject( MycoMap2018).

Species-level Determination

Identificationsweremadewithacombinationofmacroscopic,microscopic,and/orITSrDNAsequence analysis. For sequence-based identifications, consensus sequences were analyzed with the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST®) (NCBI 2020) using the “megablast†option and excluding “uncultured/environmental sample sequencesâ€. Species-level assignments were made based on a minimum percent identity threshold of 98.5% andquery coverage of80%.Ambiguous nucleotides in the consensus sequence were regarded as correctiftheymatchedthecorrect nucleotide in the reference alignment. Species- level identifications were not made if more than one specific epithet in the BLAST search corresponded withthe cutoff values above. However, highly similar sequences from type specimens and UNITE species hypotheses took precedence when available (Nilsson et al. 2019). Species names werecheckedforsynonymyandverifiedusingMycoBank(Robertetal.2013;MycoBank2020).To facilitate the tracking of operationaltaxonomic units for future taxonomic research andbiodiversity surveys, informalplaceholder names were utilized. For specimens with ≥ 98.5% similarity to Gen- Bank references with provisional specific epithets, those names were adopted here (e.g., taxa assignedanomen provisorum byAmanitaceae expertRodTulloss);inaddition,newplaceholderlabels (designatedwiththestateinitialsforWisconsin,WI)weregeneratedforspecimensforwhichnoreference sequences had≥98.5% similarityin GenBank(Table 1).These informal names serve to delineatelikelytaxaatthespecieslevelbutdonot necessarilyimplythat agivenspeciesistaxonomically novel; rather, additional research is required to obtain a sequence-supported identification for that specimen. Finally, to determine whether any identified specimens represented first records for DaneCounty,Wisconsin,orthe UnitedStates,thecurrentlyknown distribution of eachspecies was checkedin MyCoPortal(MillerandBates2017).

RESULTS

The identities and accession data of the 2018 Smith Foray macrofungi are listed in Table 1. Of the 63 specimens that were vouchered and sequenced, 35 (56%) were identified to officially described species, 24 (38%) were assigned

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species-level informal names, and the remaining four specimens (6%) could only be identified to the genus level due to conflicting reference information. Taxonomically,thesecollectionswerespreadacrossthephylaAscomycota(five specimens)andBasidiomycota(58specimens)andincluded44differentgenera. Twenty-five species represented new records for Dane County, 17 represented newrecordsforthestateofWisconsin,andthreerepresentednewrecordsforthe UnitedStates. New records are indicatedinTable 1.

DISCUSSION

Asmallportion ofthe total estimated number offungal species are currently described, and even fewer have been evaluated for their conservation status (Hawksworth and Lücking 2017; Mueller 2017). More surveys of macrofungi are desperately neededto advance our understanding offungalbiodiversity and distribution, especially in the tropics (Aime and Brearley 2012). In turn, these dataaidglobalchangebiologistsinassessingfungalrangeshiftsandpopulation declines. By opportunistically sequencing specimens collected during the 2018 Smith Foray in Mazomanie, Wisconsin, we significantly expanded the known rangesof17fungalspecies,includingthreespeciesthathadnotbeenpreviously reportedfromthe UnitedStates, andprovided novelgeneticbarcode datafor11 specimens of uncertain species-level taxonomic affinity (those with newly assigned “WI†informalplaceholder labels) .

While great care is required in accurately interpreting sequencing results (Haelewaterset al.2018;Hofstetteret al.2019), modern biodiversitysurveysof macrofungi using DNA barcoding routinely result in significant range expansions and uncover potentially novel species. For example, Haelewaters et al. (2018) discovered four new taxa, new fungal records for North America and Massachusetts, and a novel ecological interaction between a cheese mold (Chrysosporium sulfureum (Fiedl.) Oorschot & Samson) and woodlice (Crustacea: Malacostraca:Isopoda:Oniscidea)atapopularurban-islandnationalpark outside ofBoston. Hofstetter et al. (2019)documentedthe polypore Antrodiella stipitata H.S. Yuan & Y.C. Dai for the first time in Europe and recorded four other macrofungal species for the first time in Switzerland, not to mention numerous very rare and indicator taxa. Together, these studies reveal the great paucity ofinformation on fungalbiogeography andthe factthatfungi, even terrestrial macrofungi in populated areas, are an understudied reservoir of biodiversity. In the following paragraphs, we discuss several of the most interesting collections that were first records for the state of Wisconsin or for the entire UnitedStates.

Highlighted New Records forWisconsin

Cortinarius dolabratus Fr.hasbeenpreviouslydocumentedfromEuropeand North America, but in the United States it had only been collected in Alaska, California,andWashington(Liimatainenetal.2017).Ourcollection(iNaturalist

Page  196 TABLE1. List of vouchered andDNA-barcodedfungal specimens from the 2018SmithForayin Mazomanie,Wisconsin. Each row corresponds to asingle specimen and lists that specimen’s determination as well as its accession numbers for iNaturalist (photos and metadata), MyCoPortal (fungarium information), and GenBank (ITS rDNA sequence). Informal placeholder names are enclosed by quotation marks; these names have either been propagated from other sources (in which case the name is followed by a citation) or are new labels from this study (all WI labels). The last column indicates whether a specimen was a new geographic record for just Dane County (DC), for Wisconsin and Dane County (WI), or for the United States, Wisconsin, and Dane County inclusive (US), or whether the ITS rDNA sequence that was generated for that specimen was the first reference for that species on GenBank; if the specimen was not novel in any of these regards, the column is marked with a hyphen.

Species iNaturalist MyCoPortal GenBank New Record

Agaricus kriegeri Kerrigan 17333117 6596294 MK573882 WI Agaricus pallens L.A.Parra 17232000 6596228 MN989986 WI Amanita solaniolens H.L. Stewart &Grund 17338999 6596241 MK573911 WI Amanita sp.“longicuneusâ€(Tulloss andRodríguezCaycedo 2020) 17333556 6596284 MK573886 - Amanita sp.“texasorora†(Tullosset al. 2020) 17231768 6596227 MK573879 - Byssocorticium atrovirens (Fr.)Bondartsev &Singer 17333142 6596293 MN989989 - Chalciporus piperatus (Bull.)Bataille 17338127 6596254 MK573906 DC Clitocella sp.“WI-01†17340579 6596231 MK573922 - Clitocybe sp. 17340117 6596236 MK573913 - Clitopilus abortivus Berk.&M.A.Curtis 17340459 6596234 MK573919 - Collybia cookei (Bres.)J.D.Arnold 17231324 6596225 MK573873 DC Coprinellus sp. 17334230 6596275 MK573891 - Coprinellus sp.“IN-01â€(Russell2020) 17231181 6596224 MK573872 - Coprinellus sp.“IN-01â€(Russell2020) 17339226 6596244 MK573918 - Cortinarius dolabratus Fr.(Russell2020) 17336721 6596258 MK573902 - Cystoagaricus sp. “WI-01†17332756 6596282 MK573876 - Cystodermella sp.“IN-01â€(Russell2020) 17335122 6596272 MK573895 - Cystodermella sp.“IN-01â€(Russell2020) 17339256 6596243 MK573917 - Cystolepiota sp. 17334037 6596278 MK573889 - Echinoderma sp. “IN-01â€(Russell2020) 17335043 6596283 MK573894 - Entoloma psammophilohebes Vila &J. Fernández 17337073 6596255 MK573905 US Flammula sp.“WI-01†17338285 6596251 MK573909 - Fuscopostia fragilis (Fr.)B.K.Cui, L.L. Shen&Y.C. Dai 17333313 6596290 MK573885 - Galerina sp.“WI-01†17339978 6596238 MK573914 - Galerina triscopa (Fr.)Kühner 17338265 6596252 MK573908 WI Gerronema subclavatum (Peck)SingerexRedhead 17333993 6596279 MK573888 WI

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Hygrocybe cantharellus (Schwein.)Murrill 17340623 6596230 MK573923 - Hygrophorus sordidusVelen. 17333057 6596296 MK573880 DC Hymenoscyphus fructigenus (Bull.)Gray 17332929 6596297 MK573877 - Hymenoscyphus immutabilis (Fuckel)Dennis 17334099 6596277 MK573890 WI Hypomyces sp.“WI-01†17338188 6596253 MK573907 - Inocybe ericetorum Vauras&Kokkonen 17323865 6596229 MK573874 US Inocybe griseoscabrosa (Peck)Earle 17336824 6596256 MK573904 WI Lactarius imperceptus Beardslee&Burl. 17339056 6596247 MK573912 DC Lactarius sp. “IN-06â€(Russell2020) 17338937 6596250 MN989993 - Lentinellus ursinus (Fr.)Kühner 17335268 6596266 MK573897 - Lepiota castanea Quél. 17334272 6596273 MK573893 DC Lepiota clypeolaria (Bull.)P.Kumm. 17336609 6596260 MK573900 DC Lepiota umbrosa Morgan 17339931 6596239 MK573915 WI Lepista sp. 17333187 6596292 MK573883 - Limacella sp. “CMP0152â€(Tulloss 2020) 17336802 6596257 MK573903 WI Lycoperdon marginatum Vittad. 17231495 6596226 MK573878 - Lycoperdon sp.“IN-01†(Russell2020) 17340541 6596233 MN989996 - Mycena griseoviridis A.H.Sm. 17336556 6596261 MK573899 GenBank Mycena olida Bres. 17332707 6596281 MK573875 WI Mycena sp.“WI-01†17339784 6596242 MK573916 - Mycena sp.“WI-02†17332978 6596280 MN989988 - Mycetinis sp.“WI-01†17339146 6596245 MN989995 - Neofavolus sp. “SAV-10â€(SeelanSathiyaSeelanetal.2015) 17333225 NA MK573884 - Neottiella vivida (Nyl.)Dennis 17333411 6596288 MN989990 WI Otidea rainierensis Kanouse 17335858 6596263 MK573898 WI Phleogena faginea (Fr. &Palmquist)Link 17333436 6596287 MN989991 GenBank Pholiota highlandensis (Peck)Quadr.&Lunghini 17333602 6596286 MK573887 DC(WI,since 1967) Pholiota highlandensis (Peck)Quadr.&Lunghini 17334245 6596274 MK573892 DC(WI,since 1967) Ramaria sp.“WI-01†17339094 6596246 MN989994 - Rhodocollybia badiialba (Murrill)Lennox 17335179 6596271 MK573896 WI Russula sp.“WI-01†17340565 6596232 MK573921 - Singerocybe adirondackensis (Peck)ZhuL.Yang&J.Qin 17336637 6596259 MK573901 DC Tephrocybe sp.“WI-01†17334189 6596276 MN989992 - Tephrocybe sp.“WI-02†17332892 6596285 MN989987 - Tricholoma hemisulphureum (Kühner)A.Riva 17340511 6596237 MK573920 US Tricholoma saponaceum (Fr.)P. Kumm. 17333116 6596295 MK573881

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#17338950)isa100.0% matchtotheepitypecollectionfromSweden(GenBank #KX964309) and thus is the first representative east of the Mississippi River. Agaricus kriegeri Kerrigan was describedin 2016from Pennsylvania (Kerrigan 2016). Our specimen (iNaturalist #17333117) is a 99.72% match (with 94% query coverage) to the type collection and is only the second vouchered record ofthisspeciesasidefromthePennsylvaniatypecollections.InregardtoMycena olida Bres., even though numerous collections of M. olida were made by Alexander Smith, an expert on Mycena and other genera of agarics, these collections were restricted to Michigan. Nomenclature databases do not agree on the current name of this taxon. Index Fungorum (Index Fungorum Partnership 2020) lists Phloeomana minutula (Sacc.) Redhead as the currently accepted name for M. olida, but MycoBank does not list them as synonyms. In naming our collection (iNaturalist #17332707), we follow the lead of MycoBank and Telfer et al. (2015), as our specimen is a100% identity match with100% query coverage to their specimen under this name (GenBank#KT695358). Lastly, we used microscopy to identity a specimen growing in moss at a xeric oak barren (iNaturalist #17333411) as Neottiella vivida (Nyl.) Dennis. Sequence data later showeda99.28% matchtoGenBankaccession#MF066095fromtheCzechRepublic, which was identified to the same species. Microscopic details for the Wisconsin specimen can befound atMushroom Observer (2020).

New Records for the United States

InadditiontobeingnewrecordsforWisconsin,threespecieswithvouchered collectionsandDNAsequencedataarebelievedtobefirstrecordsfortheUnited States.Entoloma psammophilohebes Vila&J.Fernándezwasdescribedin2013 from acollection made in the Basque region ofSpain. Our specimen (iNaturalist #17337073) is a 99.37% match (91% query cover) to the type collection (GenBank#JX454912).Additional specimens with amatchingITS region were alsocollectedfromIndianaafewweeksaftertheWisconsincollectionandagain from Indiana in the fall of 2019. Images and metadata for these collections can befoundatiNaturalistunderaccession#18030457and#34805034,respectively. Inocybe ericetorum Vauras & Kokkonen was described in 2012 from Finland (KokkonenandVauras2012)andhadpreviouslyonlybeendocumentedineasternCanada. TheITSregionofourspecimenisa99.09% match(86% querycoverage) to the type collection (GenBank #NR_119994), expanding the range of this species into the Midwest. Finally, Tricholoma hemisulphureum (Kühner)A. Riva ex Bofelli was first described as Tricholoma sulphureum var. hemisulphureum Kühner in 1988 from France. Our Wisconsin specimen is a 99.84% match(87% query coverage)to aspecimen withthis name from Estonia, where the identity was determined to be appropriately applied for the morphological characters and sequence data present from the specimen (Heilmann-Clausen et al. 2017).There are two matching sequences from Florida which may represent the same species (GenBank #MF153041, #MF153084); however, they are currentlylisted under the name Tricholoma sulphureum in MyCoPortal.

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CONCLUSION

Manyofthenewrecordsreportedinthisstudyrepresentspeciesthatweredescribed only recently. Indeed, the increasing prevalence of multilocus genetic dataintaxonomicstudiesfacilitatesbiodiversityassessmentsbyaugmentingthe number of type specimens in reference databases. In turn, species that were thought to be isolated to confined geographic regions are discovered to exist acrosscontinentswithhighgeneticsimilarity.Inadditiontothepositivelydetermined specimens from the 2018 Smith Foray, the 24 specimens that were assigned informal placeholder names constitute fertile avenues for future taxonomic investigations. They may represent previously described species for whichnoITSbarcodedataexistinGenBankornovelspeciesthatawaitdetailed analysis. We hope that informal placeholder names will make species associations traceable across time, allowingfor the increased elucidation ofthe hidden biodiversitythatis so prevalentinWisconsin’s macrofungi.

ACKNOWLEDGMENTS

ThisworkwassupportedbyagrantfromtheNorthAmericanMycofloraProject.Wewouldlike tothankHalBurdsallfororganizingtheeventandcontributingtositedescriptions,aswellasHoofbeat Ridge Camps near Mazomanie for providing us ameetinglocation andlodging. Thankyou to allattendeeswhosharedtheircollections.WewouldalsoliketothankDr.M.CatherineAimeatPurdueUniversityfor allowingthemolecularworkto beconductedinher laboratory.

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