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2020 THE GREAT LAKES BOTANIST
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
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
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 (email@example.com)
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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)â€”undescribedfungalbiodiversityandpoorlyunderstooddistributionsand phenologyâ€”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 â€œSmithForayâ€)onOctober4â€“7,2018nearMazomanie,Wisconsin.Thiswasthe 44thannualSmithForay,whichhasbeenheldeachyearsince1975tohonorthe mycological accomplishments of Dr. Alexander Smith (1904â€“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.
Fungi were collectedfrom four areas in Dane County, Wisconsin, duringthe 2018SmithForay (Figure1)â€”FestgeCountyPark, MazomanieBottoms State NaturalArea, MazomanieOakBarrens State NaturalArea, andWalkingIron CountyParkâ€”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, â€“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,â€“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. â€œSNAâ€ is an abbreviation for â€œState NaturalAreaâ€.
whiteoak),Populus deltoides W.BartramexMarshall(easterncottonwood),Salix spp.(willow),and Betula nigra L.(riverbirch).Elmmortalityhasresultedincanopyopeningsthatsupportadenseunderstoryofnativeandintroducedshrubs. Periodicfloodingresultsinsandbars,ephemeralpools,and river channelsrunningthroughtheforest.
MazomanieOakBarrensStateNaturalAreainMazomanie(43.242305N,â€“89.739786W):This siteisdecidedlydrierthantheMazomaniefloodplainforestssurroundingtheWisconsinRiver.Wisconsinâ€™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,â€“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)at32Â°C.Driedspecimensweredepositedin the KriebelFungarium (PUL) atPurdue University and were digitally accessionedin the MycologyCollectionsdata Portal(MyCoPortal2020;Miller andBates 2017).
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 Î¼L of Promega Nuclei Lysis Solution (Promega Corp., Madison, Wisconsin). Each tube was labeled with the specimenâ€™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 at65Â°Cfor 15 minutes, and centrifugingthe contents ofthe tube at21,000gfor three minutes. The supernatant was transferredto a1.5mLmicrocentrifugetube, 200Î¼LofPromegaNucleiLysis Solution was added, and the tube was then vortexed for 20 seconds. The solution was centrifuged againat21,000gforsix minutesandthe supernatantwasaddedtoanew,sterile,1.5 mLmicrocentrifugetube. 600Î¼Lof100% isopropanolwasaddedtothe supernatanttoprecipitatetheDNA.The solutionwascentrifugedforoneminuteat21,000gandthesupernatantwaspouredoff, leavingthe DNApelletinthebottomofthetube.600Î¼Lof70% ethanolwasaddedtothetube,andthesolution wascentrifugedafinaltimeforoneminuteat21,000g.Theethanolwaspouredoutandthe1.5mL microcentrifugetubewasplacedupsidedownonaKimwipeovernight.Thefollowingday,30Î¼Lof water was addedtothetube,resultingin purifiedDNAforuse in PCRamplification.
PCRamplificationsoftheinternaltranscribedspacer(ITS)ribosomalDNA(rDNA)regionâ€”the universalDNAbarcodemarkeroffungi(Schochetal.2012)â€”werecarriedoutusingtheITS1FforwardprimerandtheITS4reverseprimer( Whiteetal.1990;GardesandBruns1993).EachPCRreactioncontained12.5Î¼LPromegaPCRMasterMix, 9Î¼Lwater,1.25Î¼Lforwardprimer,1.25Î¼Lre- verseprimer,and1Î¼LDNAtemplateforatotalPCRvolumeof25Î¼L.ThefollowingPCRprotocol wasused:(i)initialdenaturationat9Cforoneminute;(ii)30cyclesofdenaturationat94Â°Cforone minute, annealing at51Â°Cfor one minute, and extension at72Â°Cfor one minute;(iii)hold at72Â°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).
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 (BLASTÂ®) (NCBI 2020) using the â€œmegablastâ€ option and excluding â€œuncultured/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 â‰¥ 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 hadâ‰¥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).
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.
Asmallportion ofthe total estimated number offungal species are currently described, and even fewer have been evaluated for their conservation status (Hawksworth and LÃ¼cking 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 â€œWIâ€ 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 specimenâ€™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.â€œlongicuneusâ€(Tulloss andRodrÃguezCaycedo 2020) 17333556 6596284 MK573886 - Amanita sp.â€œtexasororaâ€ (Tullosset al. 2020) 17231768 6596227 MK573879 - Byssocorticium atrovirens (Fr.)Bondartsev &Singer 17333142 6596293 MN989989 - Chalciporus piperatus (Bull.)Bataille 17338127 6596254 MK573906 DC Clitocella sp.â€œWI-01â€ 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.â€œIN-01â€(Russell2020) 17231181 6596224 MK573872 - Coprinellus sp.â€œIN-01â€(Russell2020) 17339226 6596244 MK573918 - Cortinarius dolabratus Fr.(Russell2020) 17336721 6596258 MK573902 - Cystoagaricus sp. â€œWI-01â€ 17332756 6596282 MK573876 - Cystodermella sp.â€œIN-01â€(Russell2020) 17335122 6596272 MK573895 - Cystodermella sp.â€œIN-01â€(Russell2020) 17339256 6596243 MK573917 - Cystolepiota sp. 17334037 6596278 MK573889 - Echinoderma sp. â€œIN-01â€(Russell2020) 17335043 6596283 MK573894 - Entoloma psammophilohebes Vila &J. FernÃ¡ndez 17337073 6596255 MK573905 US Flammula sp.â€œWI-01â€ 17338285 6596251 MK573909 - Fuscopostia fragilis (Fr.)B.K.Cui, L.L. Shen&Y.C. Dai 17333313 6596290 MK573885 - Galerina sp.â€œWI-01â€ 17339978 6596238 MK573914 - Galerina triscopa (Fr.)KÃ¼hner 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.â€œWI-01â€ 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. â€œIN-06â€(Russell2020) 17338937 6596250 MN989993 - Lentinellus ursinus (Fr.)KÃ¼hner 17335268 6596266 MK573897 - Lepiota castanea QuÃ©l. 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. â€œCMP0152â€(Tulloss 2020) 17336802 6596257 MK573903 WI Lycoperdon marginatum Vittad. 17231495 6596226 MK573878 - Lycoperdon sp.â€œIN-01â€ (Russell2020) 17340541 6596233 MN989996 - Mycena griseoviridis A.H.Sm. 17336556 6596261 MK573899 GenBank Mycena olida Bres. 17332707 6596281 MK573875 WI Mycena sp.â€œWI-01â€ 17339784 6596242 MK573916 - Mycena sp.â€œWI-02â€ 17332978 6596280 MN989988 - Mycetinis sp.â€œWI-01â€ 17339146 6596245 MN989995 - Neofavolus sp. â€œSAV-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.â€œWI-01â€ 17339094 6596246 MN989994 - Rhodocollybia badiialba (Murrill)Lennox 17335179 6596271 MK573896 WI Russula sp.â€œWI-01â€ 17340565 6596232 MK573921 - Singerocybe adirondackensis (Peck)ZhuL.Yang&J.Qin 17336637 6596259 MK573901 DC Tephrocybe sp.â€œWI-01â€ 17334189 6596276 MN989992 - Tephrocybe sp.â€œWI-02â€ 17332892 6596285 MN989987 - Tricholoma hemisulphureum (KÃ¼hner)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.FernÃ¡ndezwasdescribedin2013 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 (KÃ¼hner)A. Riva ex Bofelli was first described as Tricholoma sulphureum var. hemisulphureum KÃ¼hner 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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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 prevalentinWisconsinâ€™s macrofungi.
ThisworkwassupportedbyagrantfromtheNorthAmericanMycofloraProject.Wewouldlike tothankHalBurdsallfororganizingtheeventandcontributingtositedescriptions,aswellasHoofbeat Ridge Camps near Mazomanie for providing us ameetinglocation andlodging. Thankyou to allattendeeswhosharedtheircollections.WewouldalsoliketothankDr.M.CatherineAimeatPurdueUniversityfor allowingthemolecularworkto beconductedinher laboratory.
Aime, M. C. and F. Q. Brearley. (2012). Tropical fungal diversity: Closing the gap between species estimates and species discovery. Biodiversity and Conservation 21: 2177â€“2180. https://doi.org/10.1007/s10531-012-0338-7
Andrew, C., J. Diez,T.Y. James, andH. Kauserud. (2018). Fungariumspecimens:Alargelyuntapped source in global change biology and beyond. Philosophical Transactions of the Royal Society B: Biological Sciences 374: 1â€“11. https://doi.org/10.1098/rstb.2017.0392
Blackwell, M. (2011).The fungi: 1, 2, 3... 5.1 million species?American Journal of Botany 98:426â€“ 438. https://doi.org/10.3732/ajb.1000298 Clark, K., I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, and E. W. Sayers. (2016). GenBank. Nucleic Acids Research 44: D67â€“D72. https://doi.org/10.1093/nar/gkv1276 Gardes, M. and T. D. Bruns. (1993). ITS primers with enhanced specificity for basidiomycetesô€€€application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113â€“118. Haelewaters, D.,A. C. Dirks, L.A. Kappler, J. K. Mitchell, L. Quijada, R.Vandegrift, B. Buyck, and
D. H. Pfister. (2018).A preliminary checklist of fungi at the Boston Harbor Islands. Northeastern Naturalist 25: 45â€“76. Hawksworth, D. L. (2004). Fungal diversity and its implications for genetic resource collections. Studies in Mycology 50: 9â€“18. https://doi.org/10.5598/imafungus.2011.02.01.14 Hawksworth, D. L. (2010). Funga and fungarium. IMA Fungus 1: 9â€“9. https://doi.org/10.1007/ BF03449321 Hawksworth, D. L. and R. LÃ¼cking. (2017). Fungal diversity revisited: 2.2 to 3.8 million species. Pp. 79â€“95 in The Fungal Kingdom. J. B. Heitman, J. Howlett, P. W. Crous, E. H. Stukenbrock, T. Y. James, and N. A. R. Gow, editors. ASM Press, Washington, D.C. https://doi.org/10.1128/ 9781555819583.ch4
Heilmann-Clausen, J., E. S. Barron, L. Boddy, A. Dahlberg, G. W. Griffith, J. NordÃ©n, O. Ovaskainen, et al. (2014).Afungalperspective on conservationbiology. ConservationBiology29: 61â€“68. https://doi.org/10.1111/cobi.12388
Page 200 200 THE GREAT LAKES BOTANIST Vol. 59
Heilmann-Clausen, J., M. Christensen, T. G. FrÃ¸slev, and R. KjÃ¸ller. (2017). Taxonomy of Tricholoma in northern Europe based on ITS sequence data and morphological characters. Persoonia 38: 38â€“57. https://10.3767/003158517X693174
Hobbs, H. (1999). Origin of the Driftless Area by subglacial drainageâ€”a new hypothesis. Pp. 93â€“ 102inGlacialProcessesPastandPresent. D. M. MickelsonandJ.W.Attig, editors. GeologicalSociety ofAmerica Special Paper 337.
Hofstetter, V., B. Buyck, G. Eyssartier, S. Schnee, and K. Gindro. (2019). The unbearable lightness of sequenced-based identification. Fungal Diversity 3: 243-284. https://doi.org/10.1007/s13225019- 00428-3
Index Fungorum Partnership. (2020). Index Fungorum.Available athttp://www.indexfungorum.org/.
iNaturalist. (2020). iNaturalist.Available at https://www.inaturalist.org/.
Kerrigan, R. W. (2016). Agaricus of NorthAmerica. NewYork Botanical Garden Press, Bronx, N.Y.
Kokkonen, K. and J. Vauras. (2012). Eleven new boreal species of Inocybe with nodulose spores. Mycological Progress 11: 299â€“341.
Kuhar, F., G. Furci, E. R. Drechsler-Santos, and D. H. Pfister. (2018). Delimitation of Funga as a valid term for the diversity of fungal communities: The Fauna, Flora & Funga proposal (FF&F). IMA Fungus 9:71â€“74.
Liimatainen, K., X. Carteret, B. Dima, I. KytÃ¶vuori, A. Bidaud, P. Reumaux, T. Niskanen, et al. (2017). Cortinarius section Bicolores and section Saturnini (Basidiomycota, Agaricales), a morphogenetic overview of European and North American species. Persoonia 39: 175â€“200. https://doi.org/10.3767/persoonia.2017.39.08
Miller,A. N. andS.T. Bates. (2017).The mycology collections portal(MyCoPortal). IMAFungus 8: 65â€“66.
Mueller, G. M. (2017). Progress inconservingfungi:Engagementandredlisting. BGJournal14:30â€“ 33.
Mushroom Observer. (2020). Accession #337425. Available at https://mushroomobserver.org/ 337425.
MycoBank. (2020). MycoBankDatabase: Fungal Databases, Nomenclature & Species Banks. Available at http://www.mycobank.org.
Mycological Society of America. (2019). Smith Foray Report â€“ Mycological Society of America. Available at http://msafungi.org/smith-foray-report/
MycoMap. (2018). Smith Foray 2018.Available at https://mycomap.com/projects/smithforay2018.
MyCoPortal. (2020). Mycology Collections Portal.Available at https://mycoportal.org.
NCBI. (2020). BLAST. Available at https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn& PAGE_TYPE=BlastSearch&LINK_LOC=blasthome.
Nilsson, R. H., K. H. Larsson, A. F. S. Taylor, J. Bengtsson-Palme, T. S. Jeppesen, D. Schigel, P. Kennedy, et al. (2019). The UNITE database for molecular identification of fungi: Handling dark taxa and parallel taxonomic classifications. NucleicAcids Research 47(D1): D259â€“D264.
Robert,V., D.Vu,A. B. H.Amor, N. van deWiele, C. Brouwer, B. Jabas, S. Szoke, et al. (2013). MycoBank gearing up for new horizons. IMA Fungus 4: 371â€“379. https://doi.org/10.5598/imafungus. 2013.04.02.16
Russell, S. D. (2020). Unpublished raw data. https://mycomap.com/projects/mycoflora-of-indiana.
Schoch, C. L., K. A. Seifert, S. Huhndorf, V. Robert, J. L. Spouge, C. A. Levesque, W. Chen, and FungalBarcodingConsortium. (2012). Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proceedings of the National Academy of Sciences 109: 6241â€“6246. https://doi.org/10.1073/pnas.1117018109
Seelan Sathiya Seelan, J., A. Justo, L. G. Nagy, E. A. Grand, S. A. Redhead and D. Hibbett. (2015). Phylogenetic relationships and morphological evolution in Lentinus, Polyporellus and Neofavolus, emphasizing southeasternAsian taxa. Mycologia 107: 460â€“474.
Telfer, A. C., M. R.Young, J. Quinn, K. Perez, C. N. Sobel, J. E. Sones, V. Levesque-Beaudin, et al. (2015). Biodiversity inventories in high gear: DNA barcoding facilitates a rapid biotic survey of a temperate nature reserve. Biodiversity Data Journal 3: 1â€“176. https://3897/BDJ.3.e6313
Thiers, H. D. (1987). Alexander H. Smith, 1904â€“1986. Mycologia 79: 811â€“818. https://doi.org/10.1080/00275514.1987.12025468
Thompson K. M. and J. T. Colbert. (2020). Lichens of Iowaâ€™s White Pine Hollow State Preserve. Evansia 37:31â€“49.
Page 201 2020 THE GREAT LAKES BOTANIST
Tulloss, R. E. (2020). Limacella sp-CMP0152. In Amanitaceae studies.Tulloss, R. E. andZ. L.Yang, editors. Available at http://www.amanitaceae.org?Limacella+sp-CMP0152
Tulloss, R. E., L. V. Kudzma, D. P. Lewis, and N. R. Goodman. (2020). Amanita texasorora. In Amanitaceae studies. Tulloss, R. E. and Z. L. Yang, editors. Available at http://www.amanitaceae. org?Amanita+texasorora
Tulloss, R. E. and C. RodrÃguez Caycedo (2020). Amanita longicuneus. In Amanitaceae studies.Tulloss, R. E. andZ. L.Yang, editors.Available athttp://www.amanitaceae.org?Amanita+longicuneus
van der Linde, S., L. M. Suz, C. D. L. Orme, F. Cox, H. Andreae, E. Asi, B. Atkinson, et al. (2018). Environment and host as large-scale controls of ectomycorrhizal fungi. Nature 558: 243â€“248. https://doi.org/10.1038/s41586-018-0189-9
White,T.J.,T.Bruns, S.J.W.T.Lee, andJ. L.Taylor.(1990).Amplificationanddirect sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315â€“322 in PCR protocols: A guide to methodsandapplications.M.A.Innis,D.H.Gelfand,J.J.Sninsky,andT.J.White,editors.AcademicPress, Cambridge,Massachusetts.
Willis, K. J. (2018). State of the Worldâ€™s Fungi. Kew Royal Botanical Gardens, London, United Kingdom.