Molecular phylogeny and morphology reveal three new species ofCantharellus within 20 m of one another in western Wisconsin, USA

Matthew J. FoltzKathryn E. Perez1

Thomas J. VolkDepartment of Biology, University of Wisconsin-LaCrosse, 1725 State Street, La Crosse, Wisconsin 54601

Abstract: Three new species, Cantharellus phasmatis,Cantharellus flavus and Cantharellus spectaculus, allpreviously considered Cantharellus cibarius, are de-scribed in this study. The circumscription of thesethree species from C. cibarius and other Cantharellusspecies is supported by morphological differencesand nuclear DNA sequence data (nLSU, ITS, TEF1).All were found under Quercus spp. in a small plot inHixon Forest Park in La Crosse, Wisconsin, empha-sizing the need for further taxonomic study of evencommon and conspicuous genera in North America.In addition, a review of the current state of C. cibariussensu lato systematics is presented, including a reviewof the recent elevation of C. cibarius var. roseocanus tothe species rank. Taxonomic descriptions and photo-graphs are provided for the newly described species.

Key words: Cantharellales, Cantharellus cibarius,chanterelle, diversity, systematics

INTRODUCTION

Chanterelle mushrooms are considered choice edi-bles in many countries around the world because oftheir apricot odor and delicious flavor. As choiceedible mushrooms they are highly sought after andeconomically important (Watling 1997), and thetaxonomy of this genus recently has undergonemuch-needed revision (Feibelman et al. 1994, Dun-ham et al. 2003, Buyck and Hofstetter 2011). Thecommon yellow-golden chanterelle (Cantharelluscibarius Fr.) originally was described by Fries in 1821as having ‘‘a glabrous, egg-yolk colored pileus that isturned up at the margin … folds swollen, somewhatdistant … stipe solid and narrowing toward base …long lived … and having an overall stature somewhatcompact’’ (English translation from Latin by T. Volk).This description is not sufficiently detailed todistinguish this species from taxonomic names inmodern use outside Sweden, and no type specimenexists for Fries’ C. cibarius. Investigations using

morphological and DNA data have shown that C.cibarius in the United States is a species complex thatrequires further taxonomic attention (Feibelman etal. 1994, Dunham et al. 2003, Moncalvo et al. 2006,Arora and Dunham 2008, Buyck and Hofstetter2011). In this study we continue efforts to documentNorth American Cantharellus diversity.

In the past 50 years, several advancements intaxonomy of Cantharellus have been made frommorphological data. Smith (1968) described C.cibarius var. cibarius from Michigan, which hebelieved to be the same as C. cibarius from Europe.Some of the key diagnostic features from thisdescription are the ‘‘egg-yellow or paler’’ hymenium,the ‘‘pale-ochraceous’’ spore print and the incurved-margin becoming plane-to-wavy and finally broadlyinfundibuliform. Smith also described a variety inMichigan with the unique characteristics of a whitishstalk and a pale pink hymenium, which he called C.cibarius var. pallidifolius A.H. Sm. (later reiteratedand illustrated by Petersen [1976, 1979]). This varietyalso was unusual in that the spore print wasochraceous salmon (flushed pink). Another pink-spored chanterelle was mentioned in Coker’s (1919)description of the samples he examined. Sample 1168was described as having ‘‘Spores salmon pink, exactlyas in Craterellus cantharellus … Except for the sporecolor these plants are exactly Cantharellus cibarius.’’

Petersen (1969) initially reported finding twovarieties in the Appalachian Mountains. He found acream-spored variety that he considered a close matchto the European specimens and also a yellow-sporedvariety. He distinguished the yellow form by itsdeeper, more brightly colored gill folds, morecrowded and well developed gill folds, the moreeverted margin of the pileus, as well as the morebrightly colored and slightly smaller spores. He notedthat the cream-spored form closely matched thedescription of C. cibarius by Smith and Morse(1947) but that Coker’s (1919) description of C.cibarius would include both forms. Petersen (1969)also noted that in 1967 Smith had presented a paperat the Mycological Society of America meetingfeaturing an undescribed chanterelle exhibitingsalmon coloration across the entire basidiocarp.

Following extensive observation of European spec-imens, Petersen (1976) concluded that C. cibarius inEurope and C. cibarius in North America were notconspecific. He stated that across the two continents

Submitted 18 May 2012; accepted for publication 7 Aug 2012.1 Corresponding author. E-mail: perezke@gmail.com

Mycologia, 105(2), 2013, pp. 447–461. DOI: 10.3852/12-181# 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

447

the name C. cibarius was being applied to 8–10 taxaand probably many more (Petersen 1979). Hisconcept of these taxa was based on morphologicalcharacters such as spore print color, stipe color, gill-fold anastomosis (or lack thereof) and micromor-phological characters. Petersen said that in histentative keys he found at least three different taxain central Sweden, four in southern Germany and fivein the southern Appalachian Mountains, all passingunder the name C. cibarius. In this work he alsoillustrated C. cibarius var. pallidifolius, the varietySmith (1968) had described from Michigan, andrecognized it as having one of the largest basidiocarpsin the genus Cantharellus (Petersen 1979). Althoughhe described this chanterelle as common in NorthAmerica, especially in the east, Petersen stated that innorthern and western America, intermediate taxaoccur, making species delineation difficult and thatperhaps ‘‘several complexes in the genus have yet toevolve sufficiently to show discrete taxa.’’

In Bigelow’s (1978) description of C. cibarius in NewEngland, he described a single variety with a cream-buffspore print. He acknowledged Petersen’s varieties aswell as Corner’s and said he was not sure which varietieswere present in New England because his focus for thechanterelles was primarily on their edible nature.Homola (1993) suggested that spore deposit colorand ornamentation were more important for taxonomythan the shape of the basidiocarp or the structure ofthe hymenophore. Homola (1993) considered thesemacroscopic features to be non-diagnostic for system-atics and examples of convergent evolution. Feibelmanet al. (1997) used molecular data to test the utility ofmorphological characters, finding that the shape andtexture of the basidiomata was more important forseparating the genera than clamps, secondary septa,development or hymenial configuration, althoughthese characters still were informative for relationshipsamong species within a genus.

Buyck and Hofstetter (2011) described two newchanterelles from the southern United States, C.tenuithrix Buyck and C. altipes Buyck. These authorsprovide microscopic descriptions of terminal hyphalcells from the pileus, as well as basidia measurementsand spore attributes. They suggested that the lengthand cell-wall thickness of the terminal cells might bean important diagnostic character. Although thesecharacteristics may be diagnostic for species delinea-tion or to show subgenus relationships, they reportthat these attributes are ‘‘extremely delicate’’ andmay be difficult to use for identification without thesupport of molecular data.

Petersen (1971) summarized some of his conclu-sions about the morphology of fungi when he wrote‘‘… if anything is clear, it’s that gross morphology is

deceiving, and that additional characters must berelied on just as heavily in determining probablerelationships between groups of organisms.’’ The useof molecular techniques to provide independent linesof evidence has proved necessary to resolve some ofthese relationships and delineate species (Feibelmanet al. 1994, Feibelman et al. 1997, Buyck andHofstetter 2011, Tibuhwa et al. 2012).

Phylogenetic studies at genus and family ranksrelying on nuclear small subunit (nSSU) and nLSUsequences of cantharelloid fungi have been plaguedwith alignment difficulties due to an acceleratedmolecular evolution of the nuclear rDNA genes inthese taxa, resulting in their placement on distinc-tively long branches (Moncalvo et al. 2006). However,a study using nLSU to infer relationships amongCantharellus (Feibelman et al. 1997) was supported bya four-gene phylogeny of nLSU, nSSU, mitochondrialsmall subunit (mSSU) and RNA polymerase subunitII (RPB2) sequences (Moncalvo et al. 2006) and astudy of the translation elongation factor 1a (TEF1)region (Buyck and Hofstetter 2011). Species delinea-tion in Cantharellus has been assisted by the use of avariety of molecular markers. Arora and Dunham(2008) used RFLP data from ITS sequences toprovide molecular support for the distinction of C.californicus Arora & Dunham, a large yellow chante-relle from the western United States, from C. formosusCorner, C. subalbidus A.H. Sm. & Morse and C.cibarius var. roseocanus. ITS and nLSU were used todistinguish species of Cantharellus and Craterellus(Feibelman et al. 1997). Dunham et al. (2003) usedITS and nLSU sequences to describe a new speciesfrom the C. cibarius complex, C. cascadensis Dunham,O’Dell, & R. Molina, from the Pacific Northwest. Thisresult was supported by Moncalvo et al. (2006) whoalso found support for the specific status of C.cascadensis, C. formosus, C. subalbidus, C. persicinusR.H. Petersen, C. lateritius (Berk.) Singer andEuropean C. cibarius (Moncalvo et al. 2006). Whilethese studies successfully delineated species withinCantharellus, most found indications of more unde-scribed diversity.

Feibelman et al. (1994) in C. cibarius s.l. foundvariable lengths of the internal transcribed spacer(ITS) region of nuclear ribosomal DNA. This variationsuggested that C. cibarius could be a species complex(Feibelman et al. 1994). Dunham et al. (2003) notedthat some C. cibarius s.l. samples from North Carolinawere excluded from the study because their sequenceswere highly divergent and non-monophyletic, suggest-ing that more species exist in the C. cibarius complexin North Carolina (eastern United States). Followingup on these studies, Buyck and Hofstetter (2011) usedthe TEF1 locus to delineate two new species, C.

448 MYCOLOGIA

tenuithrix and C. altipes, within the C. cibarius complexin the southeastern United States. However, there arepotentially more undescribed species within the C.cibarius complex in the eastern, southern, midwesternand northern United States (Feibelman et al. 1994,Dunham et al. 2003, Moncalvo et al. 2006, Buyck andHofstetter 2011, Toby Feibelman pers comm, BartBuyck pers comm).

Our study was initiated when morphologicallydistinct Cantharellus specimens were found growingunder Quercus within 20 m of each other in a wellsampled, highly traveled city park (Hixon Forest Park,La Crosse, Wisconsin); the distinctive morphologysuggested the existence of more than one species. Inaddition, the morphology of these chanterelles didnot closely match each other or descriptions of C.cibarius. The purpose of our study was to (i) identifyconsistent morphological differences and groupchanterelles into morphotypes, (ii) test for differenc-es in nuclear gene sequence between morphotypesand (iii) describe any new species discovered.

MATERIALS AND METHODS

Fieldwork and herbarium materials.—Chanterelles werecollected (MJF and TJV) and grouped into morphotypesusing characters such as the color of the pileus, hymenium,stipe, spore print. The associated genus of plant wasrecorded. Collection sites were georeferenced with the aidof Google Earth (http://www.google.com/intl/en/earth/index.html). We also collected chanterelles from Idaho andColorado to compare with our samples and includedspecimens from other collectors and from herbaria (TABLE I).Fresh specimens were photographed, and tissue samples (2–3 mm3 fresh) from the pileus were digested in 50 mL filter-sterilized cell lysis solution (CLS; Lindner and Banik 2009)containing 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM EDTA and 2%

hexadecyltrimethylammonium bromide (CTAB) and frozenat 220 C. Tissue samples from dried specimens also weretaken from the pileus and digested in CLS. Spore prints weretaken from freshly collected specimens by setting them onlight blue paper and covering them with a beaker 12 h. Freshbasidiocarps were dehydrated at 35 C in a food dehydratorand stored for herbarium accession. Microscopic analyseswere performed with dried material reconstituted in 3%

KOH. Pileipellis hyphal extremities were examined as inBuyck and Hofstetter (2011). Twenty-four new collectionsfrom our study were deposited in the Field Museum ofNatural History (F).

Molecular data collection.—Tissue samples in 50 mL CLSwere ground with sterile plastic pestles fitted into an electricdrill. Four hundred fifty microliters CLS was added andtubes were incubated at 65 C for 2 h. Five hundredmicroliters chloroform/isoamyl alcohol (24 : 1) was added,and tubes were shaken vigorously for 5 min to form anemulsion. Samples were centrifuged at 13 000 RPM for12 min. Supernatants were transferred to new tubes, and

the previous two steps were repeated. Six hundredmicroliters of cold (220 C) isopropanol were added toeach reaction, and tubes were inverted and stored at 220 Cfor a minimum of 12 h. Reactions were centrifuged at13 000 RPM for 12 min, and alcohol was decanted. Theremaining DNA pellet was rinsed with 1 mL 70% EtOH andcentrifuged. Alcohol again was decanted and pellets wereallowed to air dry in a fume hood, then resuspended in50 mL TE buffer (8.0 pH) and stored at 220 C. Electro-phoresis was used to visualize DNA products on 1.5%

agarose gels in TAE buffer at 100 V for 45 min.PCR primers used for the nLSU locus were ITS4R (White et

al. 1990) and LR5 (Vilgalys and Hester 1990). For the ITSregion we used ITS1-F (Gardes and Bruns 1993) and ITS4(White et al. 1990). For the TEF1 locus we used TEF1F andTEF1R (Morehouse et al. 2003). PCR reactions (25 mL)contained 1 mL BSA [103], 1 mL MgCl2 [25 mM], 0.5 mL eachprimer [10 mM], 12.5 mL green GoTAQ Master Mix# [23],8.5 mL of nuclease-free dH2O, and 1 mL DNA [1 : 50]. FornLSU and ITS, thermal-cycler conditions were: denature at94 C for 30 min, 30 cycles of denature at 94 C for 1 min, annealat 53 C for 1 min, and extend at 72 C for 3 min, a finalextension at 72 C for 10 min and holding at 4 C. For TEF1,conditions were as described by Morehouse et al. (2003). FornLSU and TEF1, PCR cleanups were performed with QIAGENQIAquick PCR purification kits according to manufacturerprotocol. For ITS samples, to limit sequencing to the targetlocus, gel-extractions were performed followed by thestandard QIAGEN QIAquick PCR purification protocol.

Sequencing reactions were performed with BigDye termi-nator cycle sequencing (ABI Prism). Samples were se-quenced in both directions with the same primers used inPCR. Reactions were 10 mL and contained 2.4 mL purifiedPCR-product sample and 7.6 mL master mix (0.5 mL BigDye,2.0 mL BigDye Buffer, 0.8 mL primer [2.0 mM] and 4.3 mLdH2O). Thermal-cycler conditions consisted of an initialdenaturing step at 95 C for 3 min, 35 cycles of denaturing at95 C for 20 s, annealing at 45 C for 30 s, and extending at 60 Cfor 4 min, with a final extension at 72 C for 7 min and a 4 Cholding temperature. Reactions were brought to 20 mL withsterile nuclease-free dH2O and then cleaned with QIAGENDyeEx 2.0 spin columns according to manufacturer protocol.These samples were shipped to the University of WisconsinBiotechnology Center (Madison) for sequencing.

Sequence alignment and phylogenetic analyses.—Sequenceswere edited manually in Bioedit v.7.0.9 (Hall 2007) bycomparison of the ABI chromatographs and alignment withcontiguous sequences. Sequences accompanied with suffi-cient locality information from other published studiesfrom GenBank were included in our analysis (Dunham et al.2003, Buyck et al. 2011, Buyck and Hofstetter 2011). We alsoincluded ‘‘C. cibarius’’ sequence DQ200926, which wasfrom the assembling the fungal tree of life (AFTOL)analysis. Fifty sequences (860 bp) were analyzed at thenLSU locus. Thirty-seven sequences were produced in thisstudy (TABLE I; GenBank numbers JX030419–JX030455),and 13 sequences were obtained from GenBank. FourteenITS sequences (539 bp) were produced in this study(TABLE I; GenBank numbers JX030456–JX030469) and 15

FOLTZ ET AL.: CANTHARELLUS SYSTEMATICS 449

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sequences were obtained from GenBank. Sequences couldbe obtained only in one direction (with the ITS4 primer) andwere truncated before alignment. Fifty-seven sequences(1021 bp) were analyzed for the TEF1 locus. Five sequen-ces were produced in this study (TABLE I; GenBanknumbers JX030414–JX030418), and 52 sequences wereobtained from GenBank (Buyck et al. 2011, Buyck andHofstetter 2011). There was insufficient overlap amongsequenced individuals among any of the loci to perform acombined analysis. Therefore, sequence alignments for eachlocus were analyzed separately. Any Cantharellus sequencesfrom North American species available on GenBank wereincluded in each analysis to provide context and outgroups.Sequences were aligned with MUSCLE (executed by EMBL-EBI, http://www.ebi.ac.uk/). The nLSU sequence alignmentcontained gaps in a region of , 100 bp near the middle ofthe alignment in the outgroup taxa. The ingroup taxa wereconsistently aligned in that region. We analyzed the nLSUalignment both with and without that region and the treetopologies and support values were similar. Because thatregion adds information for the ingroup, it was retained inthe analysis. Evolutionary models were determined withjModelTest (Guindon and Gascuel 2003, Posada 2008).These analyses indicated a GTR + I + G model was the mostappropriate for our data. Maximum likelihood (ML) analyseswere performed with Garli 2.0 (Zwickl 2006) for 64-bitoperating systems. Garli also was used to perform 1000 MLbootstrap replicates, and bootstrap values were obtained withPAUP (Swofford 1993) via PaupUp (Calendini and Martin2007). To view trees, TreeView (Page 1996) was used.Mesquite 2.75 (Maddison and Maddison 2011) was used toview and edit trees and prepare data for TreeBASE. Taxaused for rooting trees were those consistently supported byFeibelman et al. (1997), Dunham et al. (2003), Moncalvo etal. (2006), Buyck and Hofstetter (2011). Taxonomic deter-mination of new species was made by designating the leastinclusive monophyletic groups that were supported bydistinct morphological characteristics.

RESULTS

Morphology.—Chanterelles resembling C. cibariusfound in La Crosse were categorized into three groupsbased on morphology. The first group, with a whitemorphotype, was defined by having white lamellae thatturn pink as they mature, an orange-yellow pileus withan incurved margin, a thick white stalk and a light pinkspore print (FIG. 1A, B). The second group, with ayellow morphotype, was distinguished by yellow lamel-lae, a yellow stalk, a yellow pileus with the margin ofteneverted at maturity, a yellow spore print and a moreslender and slightly smaller stature than the whitemorphotypes (FIG. 1C, D, E). The third group, with asalmon morphotype, features salmon lamellae, thepileus is a shade of orange/pink/salmon with a marginthat is often curled down when young, becoming planeand wavy with age, and an orange stalk that is whitish atthe base (FIG. 2A, B, C). The stature is smaller andT

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FOLTZ ET AL.: CANTHARELLUS SYSTEMATICS 451

FIG. 1. A, B. Cantharellus phasmatis sp. nov. type collection (C0171588F). C, D, E. Cantharellus flavus sp. nov. typecollection (C0171585F). Both were found in Hixon Forest Park in La Crosse, Wisconsin, July 2010. Scale bars are ,1 cm.

452 MYCOLOGIA

FIG. 2. A, B, C. Cantharellus spectaculus sp. nov. type collection (C0171590F) found in Hixon Forest Park in La Crosse,Wisconsin. Jul 2010. D, E. Cantharellus roseocanus found in Colorado. Scale bars are ,1 cm.

FOLTZ ET AL.: CANTHARELLUS SYSTEMATICS 453

more slender than the other morphotypes, and thespore print is salmon-pink. None of our species hadsimilar morphology to those from Idaho and Colora-do, which were much smaller, more squat and grewassociated with conifers (FIG. 2D, E).

nLSU analysis.—Maximum likelihood analysis of thenLSU locus resulted in a single tree with 2lnL 5

21989.6209 (FIG. 3). Alignments, trees and analyses aredeposited in TreeBASE at the following URL: http://purl.org/phylo/treebase/phylows/study/TB2:S12738.The salmon-morphotype chanterelles were separatedwith strong support from other C. cibarius-like chante-relles, with relatives from southern Illinois and Missouri(FIG. 3). Although indistinguishable from each other atthe nLSU locus, the white and yellow morphotypesformed a strongly supported clade that was distinct fromC. cibarius from Sweden, as well as from C. roseocanus.All of the samples in the C. roseocanus clade were foundbeneath coniferous trees (mainly Tsuga and Picea),whereas the white, yellow and salmon morphotypesamples were found beneath species of Quercus.

ITS analysis.—Maximum likelihood analysis of theITS region resulted in a single tree with 2lnL 5

2965.5421 (FIG. 4). The white and yellow morphotypeswere separate clades from C. cibarius and C. roseocanus,however the relationships among these taxa areunresolved. The white and yellow morphotypes alsoformed mutually exclusive monophyletic clades(FIG. 4). They formed clades distinct from C. cibariusand C. roseocanus, which group together in thisphylogeny, although with weak support.

TEF1 analysis.—Maximum likelihood analysis of theTEF1 locus resulted in a single tree with 2lnL 5

27127.1283 (FIG. 5). The topology of our TEF1phylogeny of Cantharellus is identical to the topologyfrom Buyck and Hofstetter (2011) with the additionof the five samples from this study. The TEF1phylogeny infers the salmon morphotype sister to C.amethysteus (Quel.) Sacc. from Europe (FIG. 5). Oursample of C. roseocanus from Colorado is sister to C.cibarius from Europe (100% bootstrap). The whiteand yellow morphotypes are sister taxa (64% boot-strap) that share a most recent common ancestor withtwo specimens of the newly described C. tenuithrixfrom the southern United States (100% bootstrap).The yellow morphotype sample forms a monophyleticgroup with one collection of C. tenuithrix describedin Buyck and Hofstetter (2011).

TAXONOMY

Cantharellus phasmatis M.J. Foltz & T.J.Volk, sp.nov. FIG. 1A, B, SUPPLEMENTARY FIGS. 1–3

MycoBank MB800425, C0171588F.Pileus yellow; hymenium pale cream to white,

becoming pink; stalk pale yellowish white, becomingyellow; tissues staining ochraceous brown whenbruised; spore print pink. Molecular data from ITSand TEF1 loci distinguish this species from all otherCantharellus species (FIGS. 4, 5).

Etymology: Named phasmatis meaning ‘‘ghostly’’ forthe distinctive ghostly white hymenium of youngspecimens of this chanterelle.

Holotypus: UNITED STATES. WISCONSIN: LaCrosse County, Hixon Forest Park, along the pathrunning parallel to Bliss Road, associated with oak inoak-hickory mixed deciduous forest. N43.8157uW91.2091u, 18-VII-2010, here designated Foltz C073,Collection CP002, C0171588F.

Pileus egg-yolk yellow or paler (especially insunlight or dry conditions), 6–12 cm diam, plano-convex, becoming broadly convex to depressed, oftenmottled at maturity, partly due to spore deposit,surface dry and covered in a thin layer of fibrils,staining ochraceous brown when bruised; marginincurved when young, regular to irregular, sometimeslobed, often wavy with age, context thick, white, firm;lamellae deeply decurrent, white when young, be-coming pinkish buff with age, often yellowish near themargin, often forking and anastomosing, sometimesalmost poroid in some specimens near the margin,bruising ochraceous brown; stipe white and solid,yellowing and peeling with maturity, bruising ochra-ceous brown, 4–8 cm long, 1–3 cm thick, contextwhite; spore deposit salmon-pink, (7)7.5–10(11) 3 4–6(7) mm, subglobose to ovate when immature, maturespores are obovate to oblong, sometimes reniform (n5 30); basidia (55)60–70(75) 3 (7)7.5–11(13) mm, 4–6 sterigmate, clavate, often undulate; pileus hyphaewith long terminal cells, 95–105 3 4.5–5.5 mm,sometimes with thickened walls; clamps found in alltissues; odor strong and pleasant, like apricots; flavormild at first, becoming peppery; KOH intensifyingcolor of all tissues. Habitat, habit and distribution:gregarious to scattered; associated with Quercus(Fagaceae) and Carya (Juglandaceae) (oak-hickory);often found growing in lines along roots; commonJul–Aug in La Crosse, Wisconsin, presumably morewidespread. Edibility: choice; these are the mostdelectable chanterelles we have eaten.

Additional specimens examined: UNITED STATES. WIS-CONSIN: La Crosse County, Hixon Forest Park, along thepath parallel to Bliss Road, associated with Quercus (oak) inoak-hickory mixed deciduous forest. 18-VII-2010, CP001,CP002, CP003.

Comments: This species can be distinguished fromother chanterelles by its white hymenium that becomespink with age, and by its pink spore print. This species is

454 MYCOLOGIA

FIG. 3. Molecular phylogeny of Cantharellus. Maximum likelihood phylogram of 50 nLSU sequences. Maximum likelihoodbootstrap values from 1000 replicates are near branches. ML 2lnL 5 21989.6209. GenBank number or collection numbersare indicated after the name of the collection, followed by an abbreviation of the collection locality if available.

FOLTZ ET AL.: CANTHARELLUS SYSTEMATICS 455

probably the same as C. cibarius var. pallidifolius describ-ed by Smith in 1968 (see DISCUSSION). We suggest thecommon name ‘‘ghost chanterelle’’ for this species.

Cantharellus flavus M.J. Foltz & T.J.Volk, sp. nov.FIG. 1C, D, E, SUPPLEMENTARY FIG. 4

Mycobank MB800426, C0171585F.

Pileus yellow; hymenium yellow; stalk yellow; sporeprint yellow. Molecular data from ITS and TEF1 locidistinguish this species from all other Cantharellus(FIGS. 4, 5).

Etymology: named flavus for the yellow stipe,hymenium, pileus and spore print.

Holotypus: UNITED STATES. WISCONSIN: LaCrosse County, Hixon Forest Park, along the path

FIG. 4. Molecular phylogeny of Cantharellus. Maximum likelihood phylogram of 29 ITS sequences. Maximum likelihoodbootstrap values from 1000 replicates are above branches. ML 2lnL 5 2965.5421. GenBank number or collection numbersare indicated after the name of the collection, followed by an abbreviation of the collection locality if available. Double lineindicates branch was shortened.

456 MYCOLOGIA

parallel to Bliss Road, approximately halfway up thepath when heading uphill, on right side, associatedwith Quercus (oak) in oak-hickory mixed deciduousforest, N43.8157u W91.2091u, 18-VII-2010, here desig-nated Foltz C066, Collection CF001, C0171585F.

Pileus egg-yellow or paler with age or exposure tolight, 6–9 cm diam, plano-convex when immature,becoming plane to wavy and depressed to broadly

infundibuliform; margin incurved, regular to irregular,usually everted when mature, sometimes lobed orsinuate on one side, context yellowish, thin, somewhatspongy and watery in texture; lamellae deeply decurrent,egg-yellow, often forking and anastomosing, not readilystaining when bruised, bruises appear ochraceousbrownish yellow in dried specimens; stipe yellow andsolid, sometimes patched with white and mottled in age,

FIG. 5. Molecular phylogeny of Cantharellus. Maximum likelihood phylogram of 57 TEF1 sequences. Maximum likelihoodbootstrap values from 1000 replicates are near branches. ML 2lnL 5 27127.1283. GenBank number or collection numbersare indicated after the name of the collection.

FOLTZ ET AL.: CANTHARELLUS SYSTEMATICS 457

3–8 cm long, 0.5–2 cm thick, context yellowish white;spore deposit yellow, (7.5)8–10(11) 3 (4)4.5–6 mm,subglobose to obovate when young, becoming oblongwith maturity (n 5 30); basidia (63)75–80(84) 3 7–9(10) mm, 4–6 sterigmate, clavate, often undulate; pileushyphae with a long terminal cell (78)85–95(100) 3 4.5–5.5(6) mm, sometimes with thickened walls; clampsfound in all tissues; odor fragrant like apricots; flavorslightly peppery; KOH intensifying color of all tissues.Habitat, habit and distribution: cespitose to gregarious;associated with Quercus (Fagaceae) and Carya (Juglan-daceae) (oak-hickory); on well drained soil, especially onhillsides; common Jul–Aug in western Wisconsin,presumably more widespread. Edibility: choice.

Additional specimens examined: UNITED STATES. WIS-CONSIN: La Crosse County, Hixon Forest Park, along thepath running parallel to Bliss Road, associated with oak inoak-hickory mixed deciduous forest, 18-VII-2010, CF001;UNITED STATES. WISCONSIN: La Crosse County, HixonForest Park, on hillside, behind the National WeatherService Station on County Highway FA, 18-VIII-2009, CF002.C0171586F.

Comments: This species is most easily distinguishedfrom other chanterelles by its yellow hymenium, stalk,and spore print. We suggest the common name‘‘Midwestern yellow chanterelle’’ for this species.

Cantharellus spectaculus M.J. Foltz & T.J.Volk, sp. nov.FIG. 2A, B, C, SUPPLEMENTARY FIG. 5

MycoBank MB800427, C0171590F.Pileus orange-salmon; hymenium salmon, some-

times with a purple hue; spore print salmon-pink andlarger than C. phasmatis. Molecular data from nLSUand TEF1 loci distinguish this species from all otherCantharellus (FIGS. 3, 5).

Etymology: named spectaculus for the unusualsalmon-pinkish, sometimes purple hymenium and orangestalk, a showy combination.

Holotypus: UNITED STATES. WISCONSIN: LaCrosse County, Hixon Forest Park, along the pathparallel to Bliss Road, approximately halfway up thepath on right side, associated with oak in oak-hickorymixed deciduous forest, N43.8157u W91.2091u, 18-VII-2010, here designated Foltz C081, Collection CS001,C0171590F.

Pileus orange/salmon, 4–8 cm diam when mature,usually circular but sometimes elongated, umbracula-form when young, becoming plano-convex to de-pressed/infundibuliform with maturity, pileus surfacetextured or subtomentose under a hand lens, contextthin, similar in color to pileus but more pale; marginincurled, becoming wavy with age, usually regular;lamellae salmon/pink, sometimes with purple huesunder certain light, often forking and sometimesanastomosing; stipe orange and solid, white at the

base, slender and usually as long or longer than thediameter of the pileus, context whitish, with cortextissue similar in color to the surface; spore depositsalmon-pink, 10–12(14) 3 5–7 mm, oblong-ellipticalwhen mature (n 5 30); basidia (104)110–120(125) 3

(9)10–11 mm, mostly 4(5)-sterigmate, clavulate, some-times with clamps at the base that stem new basidiadirectly from the clamp; pileus hyphae terminal cell(52)60–65 3 4.5–5.5 mm; clamps found in all tissues.Habitat, habit and distribution: cespitose to gregari-ous; associated with Quercus (Fagaceae) and Carya(Juglandaceae) (oak-hickory); Jul–Aug, rare in thetype locality in Wisconsin. Edibility: choice.

Additional specimens examined: None found beyond thetype collection.

Comments: This chanterelle can be distinguishedfrom other chanterelles by its salmon hymenium,orange stalk, lack of egg-yellow pileus, salmon-pinkspore print and larger spore. We suggest the commonname ‘‘Spectacular chanterelle’’ for this species basedon its unusual color combination.

DISCUSSION

Three new species, Cantharellus phasmatis, Canthar-ellus flavus and Cantharellus spectaculus, are proposedbased on phylogenetic analysis of molecular charac-teristics and comparison of morphological featuresthat distinguish them from other Cantharellus species.

Utility of morphological characters in Cantharellustaxonomy.—The morphological characteristics thatwere useful to delineate species included the hymeni-um and spore print colors, as well as the larger sporesof C. spectaculus. Geographic distribution and mycor-rhizal host-type associations are useful for separatingthe newly proposed species from C. cibarius sensustricto, which is known only from Europe (Buyck andHofstetter 2011), and from C. roseocanus, which hasbeen reported only under coniferous trees (Redheadet al. 1997, Dunham et al. 2003, Arora and Dunham2008, this study). Buyck and Hofstetter (2011) notedthat C. tenuithrix had characteristically long terminalhyphal cells on the surface of the pileus. We found thatboth C. phasmatis and C. flavus share this character-istic with C. tenuithrix.

Spore print color has been used to distinguishspecies in the Cantharellales. In this study, ITSsupports the spore-color discrimination hypothesisof Matheny et al. (2010) of differences between Cr.fallax A.H. Sm. and Cr. cornucopioides (L.) Pers., twospecies that were difficult to resolve with nLSU andhad been combined by Dalhman et al. (2000). Ourstudy provides additional support for spore-printcolor as an important diagnostic feature at species

458 MYCOLOGIA

rank and the use of the ITS locus to delineateCantharellus species.

Taxonomic details of the three new species.—Canthar-ellus phasmatis is probably C. cibarius var. pallidifoliusas described by Smith (1968) based on morphologicaldescription. We propose to recognize this taxon at therank of species. This distinction is supported by itsplacement in a monophyletic group, separate from C.cibarius in three gene phylogenies (FIGS. 3–5). Buyckand Hofstetter (2011) said that Petersen and Eyssar-tier had both concluded upon re-examination ofPeck’s and Smith’s type collections that they were notaccompanied by sufficient descriptions, illustrationsor molecular data to be useful. Petersen recognizedSmith’s C. cibarius var. pallidifolius as a valid taxon(1976) and he illustrated it himself (1979). Inaddition, we found Smith’s original description(1968) to be thorough, including adequate micro-scopic descriptions and a photograph. Arora andDunham (2008) attempted to sequence the typespecimen of C. cibarius var. pallidifolius but reportedthat DNA degradation in the type collection prevent-ed them from producing a sequence. Given thisuncertainty, we have decided to describe this specieswith a new epithet, C. phasmatis.

Cantharellus flavus is morphologically very similarto Smith’s (1968) description of C. cibarius var.cibarius with the one major exception of spore-printcolor. Smith described the spores as cream-buff,whereas our samples produced a bright yellow sporeprint. However, Petersen’s (1969, 1985) discussions ofa ‘‘yellow-spored chanterelle’’ are fitting and could bethe same taxon we have described here.

Among the species we examined, C. phasmatis and C.flavus are sister taxa sharing a close relationship with C.tenuithrix. One of the primary morphological featureswe used to distinguish between C. phasmatis and C.flavus was the hymenium color. The description of C.tenuithrix does not include the color of the hymenium.However, Buyck’s photograph (http://www.mtsn.tn.it/cantharellus-news/tx_photos.asp?index520008) showsan orange hymenium. One of the specimens of C.tenuithrix was a close sequence match (TEF1) to C.flavus. The other two samples of C. tenuithrix,including the holotype, were monophyletic with strongsupport. This suggests that one of the individuals called‘‘C. tenuithrix’’ in Buyck and Hofstetter (2011) is notthat species but instead is C. flavus. Our other maindistinguishing morphological character was spore-printcolor. The pink spore print of C. phasmatis and yellowspore print of C. flavus are distinct from the creamspore prints of C. tenuithrix. The full geographic rangeof the new species described here and those describedby Buyck and Hofstetter (2011) are not yet clear, but

these data suggest that the range of C. flavus extends tothe southern United States.

Cantharellus spectaculus is in a more distant cladeand represents the first representative in this clade inNorth America. Its closest relative from the TEF1dataset is C. amethysteus from Europe. In 1967, Smithpresented a paper at a meeting of the MycologicalSociety of America featuring an undescribed chante-relle exhibiting salmon coloration across the entirebasidiocarp (Petersen 1969), although Smith neverformally described this species. This salmon taxoncould be the one described here. In addition, wesequenced the nLSU region of two C. cibarius-likechanterelles from southern Illinois and Missouri thatrepresent two undescribed lineages that are closelyrelated to C. spectaculus in our nLSU gene phylogeny.These taxa require further investigation.

It is noteworthy that the three new Cantharellusspecies described here were found in a small forest plotwithin 20 m of one another. It is sometimes errone-ously assumed that most North American species ofmushrooms have been described, especially in con-spicuous genera like Cantharellus. That is not the case.This is a pattern being uncovered in North Americawith many common genera, such as Armillaria(Anderson and Ullrich 1979), Laetiporus (Banik et al.1998) and Morchella (Kuo et al. 2012), where manyspecies are found to be masquerading under onescientific name. Intraspecific variation in morphologyhas been attributed to widespread genetic variabilityand phenotypic plasticity. Molecular tools have givenmycologists the framework and the confidence todiscern morphological differences that separate thespecies. Sometimes these differences turn out to bestriking (What were we thinking?), but often they aremore subtle. Clearly much more systematic work needsto be done on North American fungi, even thecharismatic megamycota, to elucidate these species.

Cantharellus roseocanus.—The combination Canthar-ellus roseocanus (Redhead, Norvell & Danell) Red-head, Norvell & Moncalvo was made (Redhead 2012),but there was no explanation given for this onlineelevation to species rank. In all three gene phyloge-nies in this study C. roseocanus was distinct from C.cibarius and other Cantharellus species, supportingthe new combination of Redhead (2012). Our nLSUdata suggest this taxon may be the most widespreadchanterelle in North America, with a known rangeacross Washington, Oregon (Dunham et al. 2003),Idaho, Colorado, northern Michigan, Massachusetts(this study), New York (AFTOL), and Newfoundland,Canada (Greg Thorn pers comm Jul 2011). Our dataindicate that C. roseocanus might be found associatedwith the northern tier of conifers across the northern

FOLTZ ET AL.: CANTHARELLUS SYSTEMATICS 459

United States and into Canada. Cantharellus roseoca-nus appears to be ecologically separated from thenewly described species in this study in that it hasbeen found associated only with coniferous trees. Thismight have gone unnoticed because of morphologicaldifferences associated with geographic location. Forexample, the specimens we found in Colorado wereonly about half the size of the original description(Redhead et al. 1997), and the specimens fromMichigan were even smaller (pileus about 20 mmdiam), although they may have been immature. Ourdata support Redhead’s (2012) recognition of C.roseocanus at the species rank based on molecularphylogeny, ecological association and geographicseparation from C. cibarius, which is known onlyfrom Europe. Further analysis of ITS and TEF1regions eventually may show that C. roseocanus iscomposed of more than one geographically isolatedspecies. However, we see no support in our data forcontinuing to describe members of this clade as avariety of C. cibarius.

The genus Cantharellus is proving to be diverse,and, in addition to the three new species describedhere, our sequence data (some not shown) indicatesthe potential for many other undescribed lineagesand species complexes within North America. Exten-sive geographic sampling, ecological, morphologicaland molecular data will be needed to fully describethe diversity of the North American Cantharellus.

ACKNOWLEDGMENTS

We thank Daniel Lindner for providing the primary authorwith training in molecular techniques and assistance withour preliminary investigations. We also thank Bart Buyck,Toby Feibelman and Steve Rehner for helpful suggestionsand advice. Many collectors sent in their chanterellesamples to assist in our investigation (TABLE I), and theircontributions are appreciated. We also thank Greg Thornfor sharing unpublished information. Thank you to AnitaBaines, Meredith Thomsen and two anonymous reviewersfor helpful suggestions for this manuscript. This study wasconducted with financial support from a Graduate StudentResearch, Service and Educational Leadership grant pro-vided by the University of Wisconsin at La Crosse.

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