Mycol Progress DOI 10.1007/s11557-013-0927-1

ORIGINAL ARTICLE

Transatlantic disjunction in fleshy fungi. I. The crispa complex

Karen W. Hughes & Ana Reboredo Segovia & Ronald H. Petersen

Received: 8 July 2013 /Revised: 22 August 2013 /Accepted: 23 August 2013 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2013

Abstract Phylogenies based on ITS and LSU sequences There, it apparently shares a bipolar mating system (Martin show that the complex comprises several and Gilbertson 1976) and brown-rot physiology (Martin and monophyletic clades, in some cases co\rresponding to named Gilbertson 1976; Hibbett and Donoghue 2001) with the other taxa (i.e. S. crispa, S. radicata), but others lacking names (i.e. members of the family. eastern and southwestern North American S. “crispa”). In our Sparassis can be conveniently divided into two complexes. study, morphological examination of numerous collections In one, the S. crispa complex, the ultimate branches of also distinguished subtle differences correlated with geo- basidiomata are curled or crisped (Fig. 1). Included species graphic distribution. Underlying these problems, several taxa are S. crispa Wulfen ex Jacquin: Fries (typus generis), S. lacked type specimens for taxonomic analysis. In this paper, radicata Weir, and S. latifolia Y.C. Dai and Zheng Wang. In appropriate epitypes are designated and names assigned. the second, the branches are erect, stiff, blade-like and without Extensive sexual compatibility experiments, described within, curled or crisped margins. Included are S. spathulata Schw.: indicate that monokaryon, haploid isolates of collections from Fr., S. brevipes Krombholz, S. laminosa Fr. and some other North America and Europe are consistently sexually compat- names. Three epithets (S. crispa, S. spathulata, S. brevipes) ible to some degree. Inherent in the study, different “species are commonly used to summarize the Euro-American mem- concepts” were tested, with the “biological species concept,” bers of the (see Wang et al. 2004 for Citation of such based on sexual compatibility, being the least restrictive. We summaries), to which S. laminosa may be added (see Kreisel propose two new taxa, S. americana and S. americana f. 1983: 678; Breitenbach and Kränzlin 1986: 368; Courtecuisse arizonica. and Duhem 2008:61). Some characteristics previously attributed to Sparassis Keywords Cauliflower mushrooms . Mating studies . require correction (correctly reported by Kreisel 1983; Wang Biogeography . Hybrids et al. 2004). In particular, the presence/absence of clamp connections (Wang et al. 2004; Light and Woehrel 2009)have been incorrectly reported, and amphigenous hymenium Introduction (Wang et al. 2004; Desjardin et al. 2004) is not the case except for individual flabelli/lamina in which orientation is precisely The genus Sparassis has been placed in the small family vertical (Cotton 1907). Herter (Jülich 1981) and in the order Although J.C. Schaeffer (1763) furnished Latin and German (Binder et al. 2005; Cannon and Kirk 2007). descriptions of a ramose Elvela, only later did he (Schaeffer 1774) use the binomial Elvela ramosa for it. Schaeffer’s(1774: plate 163) illustration, while somewhat phantasmagoric, surely Electronic supplementary material The online version of this article shows a Sparassis.Wulfen(1781: 100) produced a description (doi:10.1007/s11557-013-0927-1) contains supplementary material, ’ which is available to authorized users. and illustration for his Clavaria crispa.Wulfens(1781:Tab.14 Fig. 1) illustration is, if anything, less realistic of a Sparassis than : : * K. W. Hughes A. R. Segovia R. H. Petersen ( ) Schaeffer’s, with the ultimate margins of branches distinctly a Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996-1100, USA darker caramel color than the lamina, and fimbriate (as could e-mail: [email protected] represent a thelephoroid form). Fries (1821), having introduced Mycol Progress

Fig. 1 Basidiomata of Sparassis crispa complex. a S. crispa, Europe, courtesy of Wikipedia. b S. americana f. americana TENN65974. c S. americana f. arizonica, courtesy of Flickr. c S. radicata, TENN67999-SAT301- 01, courtesy S. Trudell. Bars=10 cm

the genus name Sparassis previously (Fries 1819), recombined (especially the Genealogical Concordance Phylogenetic Species Wulfen’s epithet into Sparassis,withElvela ramosa Schaeffer as Recognition criteria), have been discussed by Cai et al. (2011) a synonym. This adoption of Sparassis crispa constitutes sanc- and Yang (2011). As is common, however, previous taxo- tion and thus nomenclatural protection. nomenclatural foundations for our investigation were missing Analyses based on a combined ribosomal ITS + LSU (internal or poorly constructed. Typification, especially for DNA extrac- transcribed spacer–large subunit) data set (Wang et al. 2004) tion exemplars, was missing, and when type specimens were suggested that S. crispa from Europe actually consisted of two extant, they had rarely been carefully reported (see Hawksworth closely related clades, and that collections under the same name 2012). These tasks have also been taken up here. from eastern North America formed a separate clade. In the current study, morphological examination of numerous speci- mens indicated that the North American basidiomata could be Materials and methods separated microscopically from those of European S. crispa. Martin and Gilbertson (1976), seeking to clarify the relationships DNA extraction, PCR amplification, cloning and Sanger of their concept of S. radicata to other Sparassis taxa, reported Sequencing DNAs were extracted as described in Hughes on sexual compatibility experiments that showed that their west- et al (2013). The ITS region of the ribosomal RNA repeat ern North American isolates (which they grouped under S. was amplified with primers ITS1F (Bruns and Gardes 1993) crispa) were compatible with European and Asian isolates under and ITS4 (White et al. 1989). The ribosomal large subunit the same name. Collections of their concept of “…the southeast- gene was amplified with primers LR0R and LR5 (Moncalvo ern Sparassis…,” seemingly pointing to S. spathulata,were et al. 2002). Base-pair heterozygosity was inferred from the apparently incompatible with their representatives of S. “crispa.” presence of double peaks on a chromatogram. Indels were Our aim in the current study has been to ascertain relation- inferred when peaks on a chromatogram abruptly went out of ships among Sparassis crispa-like populations from Europe and phase. For a simple 1–2-bp indel, a comparison of forward and North America, with a focus on transatlantic distribution. Three reverse sequences allowed for the determination of haplo- approaches were utilized: (1) phylogenetic placements using ITS types. When heterozygosity due to overlapping indels was and LSU sequences, (2) morphological examination, especially impossible to read, the ITS region was cloned. PCR products of specimens whose sequences appeared in phylogenies, and (3) were cloned following the manufacturer’s instructions a series of intercollection (intercontinental) sexual compatibility (pGEM-T easy kit with JM109 Competent cells, Promega pairings to test the cohesiveness of the “biological species con- Corporation, 2800 Woods Hollow Road, Madison, WI cept” as applied to this complex. These “species concepts” 53711 USA). Between five and 10 clones were sequenced (Petersen and Hughes 1999), as opposed to “species criteria” for each fruitbody. Clone consensus sequences were not Mycol Progress determined. Sequencing was performed with an automated Morphology/specimens Examination of fresh and herbarium ABI 3100 DNA sequencer (ABI Prism Dye Terminator cycle specimens was undertaken with and without magnification. sequencing, Perkin-Elmer, Inc) using primers ITSIF, ITS4, Macromorphological features included dimensions of LR0R and LR5. Sequences were deposited with GenBank basidiomata and their parts, colors (if fresh), distinction be- and are given in Table 1. tween hymenial and abhymenial surfaces, and basal basidiome size and consistency. Micromorphological exami- Phylogenetic analyses Sequences were aligned using the nation was accomplished under phase contrast (PhC) and PileUp program in the GCG software suite and adjusted bright field microscopy (BF) with no stains applied. manually. Phylogenetic relationships within a species were Particular attention was given to presence and location of estimated by maximum parsimony and Bayesian analysis. clamp connections, extent and morphology of “freeform” For parsimony analysis implemented in PAUP* 4.0b10 cells of the outer flabellar trama and of paraphyses on the (Swofford 2002), gaps were treated as a fifth base and char- abhymenial flabellar surface, and to dimensions and shapes of acters were unordered and unweighted. Heuristic searches basidiospores. Fresh colors within quotation marks are from were conducted under the following conditions: the starting Ridgway (1912), matched in Kornerup and Wanscher (1967) tree was obtained via stepwise addition, and the branch- and cited alphanumerically. Latitude/longitude readings were swapping algorithm was tree-bisection-reconnection (TBR). obtained using label data coupled with Google Earth. Branch robustness was evaluated by 1,000 bootstrap repli- Herbarium acronyms follow Index Herbariorum (http:// cates (Felsenstein 1985) using the same conditions as above. sciweb.nybg.org/science2/IndexHerbariorum.asp). A model test (Posada and Crandall 1998) was used to estimate the appropriate model of nucleotide substitution for Bayesian Abbreviations GSMNP = Great Smoky Mountains National analysis using MrBayes v. 3.0 (Huelsenbeck and Ronquist Park (TN and NC): KWH and RHP = initials of first and last 2000). If the selected model could not be implemented in authors; TFB = Tennessee Field-book number, assigned to MrBayes 3.0, the closest model with equivalent parameters fresh specimens for early association; TENN = Tennessee or more relaxed parameters was selected. A Markov chain Herbarium number; permanent tracking number for preserved Monte Carlo (MCMC) search was run with four chains for specimens. Acronyms for states within United States follow 500,000 generations with sampling every 100 generations. US postal abbreviations. In culture procedures, CZ = contact The first 1,000 trees were discarded based on preliminary zone between two haploid donor blocks; SBI = single- analyses showing that likelihood values had reached stability basidiospore isolate; barrage = a CZ zone differentiation in with the first 1,000 trees. Posterior probabilities were estimat- which a strip of congested, usually submerged hyphae pre- ed by sampling trees generated after likelihood values di- dominates; flat = a CZ differentiation in which hyphal growth verged. A 50 % majority-rule consensus tree was generated. into the CZ is inhibited, often accompanied by juxtaposed ITS and LSU regions were analyzed separately and as a zones of raised, congested hyphae. combined data set. Aligned sequences were deposited in TreeBASE number 14218: SplitsTree4 (Huson and Bryant Cultures Cultures from the Forest Products Laboratory, 2006) was used to generate unrooted phylogenetic networks. Madison, WI (CFMR) sequenced for ITS/LSU and appearing Preliminary analyses were performed to determine appro- in the American “crispa” clades were: JPL 187, Arizona, Pima priate outgroups for European and North American Sparassis Co., Santa Catalina Mts.; KJM 274, Arizona, Pima Co., Santa afn crispa collections using S. brevipes and S. spathulata as Catalina Mts., Bear Wallow; KJM 279, Arizona, Pima Co., outgroups. These analyses placed S. latifolia as basal to Santa Catalina Mts.; KJM 391, Arizona, Mt. Lemmon; KJM European and North American Sparassis afn crispa collec- 450, Arizona, Coconino Co., Kaibab Plateau; KJM 468, tions. Subsequent analyses used S. latifolia as the outgroup. Arizona, Pima Co., Santa Catalina Mts.; RLG 8240, Arizona, no further information; FP 135343, West Virginia; Phylogenetic networks NeighborNet (Bryant and Moulton no further information; OKM 7058, Maryland, no further 2003) phylogenetic networks were visualized in information. Portions of most of the Arizona collections are SplitsTrees4 (Huson and Bryant 2006), which provides also at ARIZ but were not made available for this study. Two an implicit representation of evolution and is less sensitive cultures produced a crust in culture with a spore-bearing to hybridization and recombination than standard tree hymenium. Single-spore isolates were obtained by inverting programs. these Petri dishes over a sterile Petri dish containing malt agar to obtain a spore drop. Germinating spores were isolated and Tests for recombination Recombination was detected statisti- subcultured for crossing experiments. cally using Φω (Bruen et al. 2006), as implemented in the program SplitsTree4 (Desjardin et al. 2004)usingawindow Crosses In order to test sexual (in-)compatibility across a size of 100 bp. wide geographic range, single-basidiospore isolates (SBIs) Mycol Progress

Table 1 Specimens used in molecular phylogenetic analyses and GenBank accession numbers

Collection number Code on Herbarium GenBank number GenBank number Geographic location phylogeny designation rDNA ITS rDNA LSU

Sparassis latifolia – Asia DAI 2441 DAI 2441 JQ743075 JQ743084 China, Jilin HMJAU5301 Isotype HKAS 55938 HK55938 HKAS 55938 JN387098 JN387109 China HKAS 59854 HK59854 HKAS 59854 JQ743071 JQ743081 Japan HMJAU 2955 HMJAU HMJAU 2955 JQ743073 JQ743083 Russia HKAS 55435 HK55435 HKAS 55435 JN387099 JN387110 China HKAS 43721 AFTOL703 AfTol703 DQ250597 AY629321 China HKAS 17477 CHN21S HKAS 17477 AY218438 AY218400 China, South HKAS 32363 CHN20M HKAS 32363 AY218437 AY218399 China, Middle HKAS 15728 CHN19M HKAS15728 AY218436 AY218398 China, Middle YCDA 12470 CHN2 YCD2470 AY218424 AY218386 China YCDA 12145 CHN1N YCDA 12145 AY218423 AY218385 China Sparassis crispa – Europe Not given FR686581 NA Germany RB9/6/87/ss23 GER23 AY218440 AY218402 Germany MBUH-PIRJO FRA5 AY218427 AY218389 France &ILKKA94-1587/ss5 FP-135343 c1, c5, c7 CMFR: KC987565 KF053376 Unknown FP-135343 [ITS Sequence is European] CBS120826 h1,h2 CBS120826h1 KC987552-53 KF053374 Germany CBS120826h2 CBS423.51 c2, c7 CBS423c2 KC987554-55 KF053375 Germany CBS423c7 CBS470.48 CBS470 KC987556 KF053379 Unknown CBS716.94 c1,c3,c4,c5, CBS716c3 KC987557-62 KF053377 Netherlands c6,c7 CBS830.91 CBS830 KC987563 KF053381 Netherlands (AF308852 & AY156936) resequenced FPRL312A LM312A CMFR: FPRL312A KC987579 KF053383 England IB75-357 IB75357 KC987583 KF053384 Austria IB98-129 IB98129 KC987584 KF053382 Sweden LE0432 LE043 KC987585 KF053380 Germany MBUH-DorisSlaber/ss25 GER25 AY218442 AY218404 Germany CBS WY212 c2,c3, c5,c8 WY212 CMFR: CBS KC987568-71 KF053378 Unknown c2,c3,c5,c8 WY212 BMS2857/ss10 (Wang) BMS2857 AY218431 AY218394 England Not given FR686581 NA Germany MO190754 KC987586 NA Germany, Waldkrai-burg Sparassis americana f. americana – North America SAT11-178-06 TN66311 TENN66311 KC987595 KF053397 USA, Tennessee GSMNP CIF 2005-277 FCME Cif2005-277 KC987564 NA USA, Tennessee GSMNP OKM7058 OKM7058 CMFR: OKM7058 KC987581 KF053389 USA, Maryland SAT11-180-08 TENN66366 TENN66366 KC987594 KF053388 USA, Tennessee GSMNP REB44575 TENN44575 TENN44575 KC987590 KF053392 USA, Georgia Mycol Progress

Table 1 (continued)

Collection number Code on Herbarium GenBank number GenBank number Geographic location phylogeny designation rDNA ITS rDNA LSU

(AY218445) AY218407 resequenced resequenced ASM13154 ASM13154 EIEU ASM13154 KC987551 KF053390 USA, Tennessee GSMNP TFB13005 TENN60930 KC987541 NA USA, Tennessee GSMNP TFB13888 TENN65584 TENN65584 KC987542 KF053395 USA, North Carolina TFB4067 TENN50258 TENN50258 KC987549 KF053385 USA, Tennessee GSMNP TFB4076 TENN50232 TENN50232 KC987550 KF053396 USA, North Carolina (AY218449) (AY218410) resequenced resequenced TFB14027 h1, h2 TENN65971 TENN65971 KC987543-44 KF053386-87 USA, Tennessee, TFB14103 TENN67852 TENN67852 KC987547 KF053394 USA, North Carolina TFB14032 TENN65974 TENN65974 KC987546 KF053391 USA, Tennessee, GSMNP TFB14028 TENN65972 TENN65972 KC987545 KF053393 USA, Tennessee Sparassis americana f. arizonica – North America RLG8240 RLG8240 CMFR: RLG8240 KC987582 KF053398 USA, Arizona KJM468 KJM468 CMFR: KJM468 KC987578 KF053401 USA, Arizona KJM274 KJM274 CMFR-KJM274 KC987575 KF053399 USA, Arizona KJM450 KJM450 CMFR-KJM450 KC987577 KF053402 USA, Arizona KJM279 KJM279 CMFR-KJM279 KC987576 KF053404 USA, Arizona FP JPL187 JPL187 CMFR-JPL187 KC987574 KF053400 USA, Arizona KJM391h1,h2 KJM391 CMFR-KJM391 KF145158 KF053403 USA, Arizona KF145159 Sparassis radicata – Western North America TFB5727 TENN52558 KC987592 KF053405 USA, Washington AY218450 AY218411 TFB9540 TENN56253 KC987593 KF053410 USA, California AY218446 (AY208408) resequenced UBC F1264 UBC F1264 AY218443 AY218405 Canada, BC UBC F19728 UBC F19728 HQ604825 NA Canada, BC ASM3359 ASM3359 TENN45811 KC987591 KF053406 USA, Washington AY218444 AY218406 resequenced resequenced OKM4756 OKM4756 CMFR: OKM4756 KC987580 KF053407 USA, Idaho FP-133458 FP133458 CMFR: KC987572 KF053408 USA, Oregon FP-133458 FP-133489 FP133489 CMFR: KC987573 KF053409 USA, Oregon FP-133489 SAT295-01 TENN67997 TENN67997 KC987587 KF053411 USA, Washington SAT295-02 TENN67998 TENN67998 KC987588 KF053412 USA, Washington SAT301-01 TENN67999 TENN67999 KC987589 KF053413 USA, Washington TFB14183 TENN67982 TENN67982 KC987548 KC053414 USA, Washington h1 and h2 refer to haplotypes determined by reading through indels GenBank accession numbers in bold were generated by this study c clone number, TFB Tennessee Field Book and culture numbers, LE Komerov Botanical Institute, St Petersburg, CBS Centraalbureau voor Schimmelcultures, Royal Netherlands Academy of Arts, IB Innsbruck, FP CMFR: Forest Products Laboratory, Madison, Wisconsin, GSMNP Great Smoky Mountains National Park Mycol Progress were established from fresh basidiomata or spore prints, as Collections of Sparassis crispa from Europe clustered into follows: TENN65974 (TN), TENN65584 (NC; E-US S. two broad clades with a number of between-clade hybrids, and “crispa” hybrid between clades I and II), TENN65971 (TN), evidence of mating and recombination (Figs. 2 and 3)based OKM 7058 (MD, dikaryon culture from CFMR; E-US S. on both Bayesian and parsimony analyses of the ITS region “crispa” clade II); KJM 279 (AZ; spontaneously fruiting alone and in combination with the ribosomal LSU region. The dikaryon culture from CFMR); TENN67997-SAT 295-01 Φω test for recombination was highly significant (WA; S. “radicata”), TENN67999-SAT 301-01 (OR; S. (p =4.384E−4). A phylogenetic network employing “radicata”), LE 043 (Germany, S. crispa,dikaryonculture SplitsTree4 shows alternate phylogenetic relationships within from the Komarov Botanical Institute, St. Petersburg, Russia). European collections and graphically demonstrates the exten- Collections from temperate moist forests of western North sive recombination and re-assortment occurring within America (under S. “radicata”) were singled out for a separate European S. crispa (Fig. 4). intercollection sexual compatibility experiment in order to test Several sources of DNA from Europe produced complex the “biological” cohesiveness of this entity. Collections sequences requiring cloning to ascertain haplotypes. This was employed were as follows: TENN49093 (BC); TENN56253 especially true of cultures from Centraalbureau voor (CA); TENN67982 (WA); TENN67985 (WA); TENN67997- Schimmelcultures (CBS 423.51-Germany and CBS 716.94- SAT 295-01 (WA); TENN67999- SAT 301-01 (OR). Netherlands), and Forest Products Lab Madison (FP13543 In all cases, numerous SBIs were established, and all SBIs and FP CBS WY212, both of unknown origin but with were surveyed for the presence of clamp connections as a European sequence signatures). In the cloned material, some proxy for dikaryon conditions. Only clampless isolates were novel genotypes were detected suggesting either lack of ho- selected for pairing experiments. Four SBIs were selected mogenization of the ribosomal repeat or clone errors. Within based on growth rate and colony vigor (“random” SBIs) or the European S. crispa clade, GenBank number JX566462 based on their mating types from a self-cross (“tester strains”). (Ryoo et al. 2013) ostensibly originated from American ma- Using these SBIs, pairing experiments were performed in terial. Ka (pers. comm.) confirmed this, and an explanation of which SBIs of all collections were paired in random, n =4 an “American” sequence in an otherwise European clade pairings. Pairings were performed on potato dextrose agar (present study) is not clear. (Difco Potato Dextrose broth, 24 g/L; Difco Bacto-agar, 20 g/ In North America, two major clades were resolved with L) as described in Petersen (1992). Donor colonies were parsimony analysis of the ITS region (Fig. 2), of the LSU allowed to grow for approximately one month, at which region (data not presented) and the combined ITS + LSU data time they had overgrown each other and had established set (Fig. 3). Bayesian analyses of the ITS region, however, a definable CZ. placed the S. radicata clade as a long branch within Eastern All pairings were examined macro- and microscopically North American collections (not shown). The ribosomal LSU for the following characteristics: (1) CZ morphology (i.e., region contains insufficient information to produce well- “barrage,”“flat”), (2) presence/absence of clamp connections supported phylogenies for Sparassis by itself. Bayesian anal- (including false clamps) on CZ hyphae (i.e., using a standard ysis of the LSU region combined European collections with of absent, occasional, common, abundant), (3) other hyphal North American collections, separating only S. radicata as a differentiation [i.e., chains of thick-walled, swollen, turbinate discrete clade (data not presented). Sparassis radicata seems to cigar-shaped cells (PhC); refringence (PhC), “vascular hy- restricted to the moist forests of the northwest USA and phae;” slender, thin-walled, crumpled hyphae], (4) evident southwest Canada. hyphal growth on donor blocks (i.e., well-defined, compact The second clade includes representatives from the eastern “blisters;” felty-fimbriate white mycelium; etc.), and (5) pres- USA and high-altitude forests of the southwestern USA, and ence and location of crystal formation (i.e., submerged red is here named S. americana. Two subgroups of S. americana crystal clusters; hyaline aerial crystals). are resolved, ENA clade II with eastern US and Arizonan representation and ENA clade I, which is restricted to the southern Appalachians (collections TENN65971h1, TENN67852, CIF2005 277, TENN66366, FP OKM7058: Results Figs. 2, 3 and 5). Falling within Clade II (Figs. 2 and 3)are

collections which are actually F1 hybrids between ENA clades Phylogenetic reconstructions Parsimonyanalysesofthecom- I and II. There is no evidence of recombination between ITS bined European and North American data set for the ITS sequences of ENA clade I or clade II in nature. A NeighborNet region and the combined ITS+LSU data set are given in phylogeny (Fig. 5) shows two parents and hybrids be- Figs. 2 and 3. Both parsimony and Bayesian analyses (not tween them but no evidence for alternate trees resulting shown) clearly separate European from North American from recombination. The Φω test shows no evidence collections. for recombination (p =1.0). Mycol Progress

Fig. 2 ITS phylogeny of the Sparassis crispa complex. One of the 1,000 Great Smoky Mountains National Park. C clone number. All characters most parsimonious trees of length 141 based on the ribosomal ITS gene were unordered, of equal weight and gaps were treated as a fifth base. Of region. Bootstrap support values greater than 70 % are followed by 600 characters, 37 variable characters were parsimony uninformative and Bayesian probability values greater than 0.70. Locations in the United 54 characters were parsimony informative. European and North Ameri- States are given as state abbreviations. Unk unknown location. GSMNP can clades were rooted with S. latifolia

Sexual compatibility experiments Delatour (1975) and Martin nuclear staining with DAPI under ultraviolet epifluorescent light and Gilbertson (1976) judged that Sparassis was bipolar (i.e., microscopy. Although some light microscopy (PhC) readings the single mating-type gene, A, governing sexual compatibility, were corrected, results were as noted above. and mating types represented as A1 and A2). In the course of the Intercollection (including intercontinental) pairing experi- present study, eight self-cross experiments were performed in an ments were performed to determine the ability of geographical attempt to accurately report the mating system of the S. crispa populations to interbreed (Online materials ESM1,ESM2): complex. Results were as follows: bipolar = OKM 7058 (MD); (1) pairings between S. americana clade I (TENN65974) and TENN50258 (TN); TENN65584 (NC-hybrid); KJM 279 (AZ); clade II (OKM7058) show generally strong compatibility, (2) TENN56253 (CA, ambiguous, probably bipolar); terapolar = pairing involving F1 hybrids, especially collection TENN65971 (TN-hybrid); LE 043 (Germany); ?amphithallic = TENN65584, showed reduced compatibility, (3) pairings unnumbered (Sweden), ambiguous. Thus, bipolarity was indi- among S. americana clade I×f. arizonica were fully compat- cated in five of eight self-crosses (including 9540), while ible, (4) S. americana clade I and clade II appeared full tetrapolarity was indicated in two, with probable amphithallism compatibility with S. crispa, and (5) S. radicata clade repre- in one. Subsequently, all self-cross pairings were subjected to sentatives showed reduced compatibility within the clade and Mycol Progress between S. radicata and S. americana clade I and clade II, but 5* North America from Atlantic coast to forests of appeared compatible with S. crispa (Germany) and with f. the southwest (but not temperate rain forests of western states arizonica. Thus, considering the small sample from Europe and southwest Canada); ultimate flabelli 10–20 mm broad, and Arizona, all tested representatives appeared sexually curled or crisped, entire or fluted or frilled; freeform outer intercompatible to a greater or lesser extent across geograph- tramal cells usually puzzle-shaped; paraphyses 25–50 μm ical regions. long, usually clamped only at point of origin; usually associ- ated with ..…...... 6 6 Eastern North America; basidiomata off-white to cream- colored; ultimate flabelli usually 12–18 mm broad, entire; usually associated with pine (P. strobus , P. taeda ; P. palustris); two discrete, morphologically indistinguishable Key to taxa of the Sparassis crispa complex ITS clades …………………………..…...... S. americana 1 Cystidia present in hymenium, 100–144×(6.4-)7–11 μm; 6* High-altitude pine forests of American southwest (AZ, basidiospores (6.5-)7–9(−9.5)×(5.5-)6–7(−7.5) μm; NM, S-CA); basidiomata cream-colored, usually with pinkish Southeast Asia ……………….....………….... S. cystidiosa* or cantaloupe hue; ultimate flabelli usually 15–22 mm broad, 1* Cystidia absent; basidia and basidiospores variable; entire or usually fluted, frilled or lobate; usually associated Asia, Europe, North America ……………..……...... 2 with Pinus ponderosa; a discrete ITS clade ...... S. 2 Southwestern China; basidiospores (6.5-)7– americana f. arizonica 9(9.5)×(5.5-)6–7(−7.5) μm; ultimate flabelli fan-shaped, * = not covered in this paper flabelliform; ITS sister clade with S. cystidiosa ….……...... ….. S. cystidiosa f. flabelliformis* 2* Temperate Asia, Europe, North America; basidiospores Taxonomic descriptions smaller ……………………………………...... 3 3 Widespread in temperate Asia; ultimate flabelli curled or Sparassis crispa Wulfen ex Jacquin: Fries. 1821. Syst. crisped, entire; basidomata off-white to cream-colored; mycol. 1: 465 basidia 55–68×5–7 μm; basidiospores (4-)4.5– Figs. 1a, 6 and 11a 5.5(−6)×(3-)3.5–4(−5.4) μm; forming a discrete ITS clade ≡ Clavaria crispa Wulfen ex Jacquin. 1781. Misc. …………………………………………...... S. latifolia* Austriaca Bot., Chem., Hist. Nat. 2: 100. 3* Europe, North America; ultimate flabelli curled, ≡ Masseeola crispa (Wulfen ex Jacquin) Kuntz. 1891. crisped, usually entire but sometimes with frilled, fluted or Revisio. Gen. Pl. 2: 859. lobate margin; basidiomata complex, off-white, cream- Holotype Not designated. colored, yellowish, yellow, pinkish or pallid cantaloupe; Lectotype (iconotype) Wulfen in Jacquin. 1781. Misc. basidia and basidiospores variable ………………...... ….. 4 Austriaca Bot., Chem., Hist. Nat. 2: Tab. 4, Fig. 1. 4 Europe (from Atlantic coast to central Russia); usually “Neotype” (design. Burdsall and Miller, 1988 Mycotaxon associated with Pinus sylvestris;ultimateflabelliusually8– 31: 592): GERMANY, Etterzhausen, 20.IX.1976, leg. Besl, 15 mm broad, curled, crisped, rarely frilled; basidiomata com- nr. 23 (REG). plex, off-white to cream-colored; two distinct but closely Epitype (hic. design.): AUSTRIA, Opferholz, Viktring related ITS clades not morphologically separable [Klagenfurt am Wörthersee], Kärnten, N 46° 35′ 22.60′,E ...... S. crispa 14° 16′ 06.77″, 3.X.1975, coll M. Moser, 75/357 (IB 4* North America, including western rain forests, south- 18750357) western pine forests and eastern mixed forests; margin of = Elvela ramosa Schaeffer. 1774; Index 106. [non-sanc- ultimate flabelli entire (east, northwest) or frilled or fluted tioned name] (southwest); basidiomata complex, off-white, cream-colored Basidiomata (Fig. 1a) arising from vegetative hyphal mass (east) to yellow (western rain forests) to pinkish or pastel in buried wood (including living or dead roots), expanding cantaloupe (southwest) …………………………...... 5 upward through indefinite growth (enveloping bits of sub- 5 North American western moist forests (BC, WA, OR, N- strate), emerging at soil (or litter) surface. portion almost CA, ID); ultimate flabelli fan-shaped, large (usually 15– absent to extensive, in older specimens sometimes becoming 25 mm broad), distinctly yellow or yellowish; freeform gelatinized. Upper basidiome portion generally hemispheri- outer tramal cells usually 4–5 times longer than broad; cal, composed of irregular, somewhat flattened, often anasto- abhymenial paraphyses 40–70 μm long, often internally mosed, curled plates (flabelli), eventually curled in all planes clamped; basidiomata usually associated with Pseudotsuga (crisped), cream colored, near “light ochraceous buff” (5A4) menziesii or Picea sitchensis; a discrete ITS clade or “warm buff” (5A4), occasionally off-white; flabelli ……..…...... S. radicata exhibiting unilateral hymenium, drying delicately waxy, Mycol Progress

(rarely subdivided, frilled or delicately lobate), variable in size [(3-)4–12(−14) mm broad], usually somewhat discolored and subcartilaginous when dried; abhymenial surface off-white [“pale ochraceous buff” (4A2), “light ochraceous buff” (5A4)], minutely silky to delicately radially undulate. Spore print white. Edible (choice) with little odor or taste (fresh). Habitat and phenology: Europe, from the Balkans and Caucasus to Scandinavia and the Ural Mountain chain; appar- ently usually associated with Pinus sylvestris, although re- ported from other tree associations. Late summer through autumn. Medullary tissue of lower stipe (Fig. 6a–c): Hyphal con- struction monomitic; hyphae generative, of at least three types: (1) 4–6.5 μm diam, slender, easily disarticulated, thin- walled, with occasional clamp connections; (2) skeletalized generatives 5–16 μm diam, easily disarticulated into short fragments, thick-walled [wall −2.5(−6.5) μm thick], hyaline, with occasional clamp connections, occasionally expanded into several branches; clamps often asymmetrical; (3) “hairy” generatives, 4–13 μm diam, thick-walled (wall −2.5 μm thick), with outer wall appearing “hairy” (i.e., with very fine beard of minute “cilia,” -1.5 μm high in profile), which, in older or larger hyphae, are gathered into rough bands (PhC), Fig. 3 Unrooted phylogeny, ITS + LSU, Sparassis crispa complex. One often with a surrounding slightly “cleared” area as though of the 1,000 most parsimonious trees of length 162 based on the ribo- subviscid; and (4) gloeoplerous hyphae, 5–10 μmdiam, somal ITS + LSU gene region. Bootstrap support values greater than 70 % are followed by Bayesian probability values greater than 0.70. All subrefringent (PhC), sometimes branched, thick-walled characters were unordered, of equal weight and gaps were treated as a (wall −1.5 μm thick), often clamped, apparently aseptate. fifth base. Of 1,438 characters, 39 variable characters were parsimony Tramal tissue of upper flabellum. Hyphal construction uninformative and 66 characters were parsimony informative monomitic, of three types of generative hyphae: (1) 2– 10 μm diam, more or less equal, thin- to thick-walled neutral brown-tan (“clay color” 5C4), occasionally exhibiting (wall −2.5 μm diam), occasionally but conspicuously one or more transverse, zonate watermarks, especially toward clamped, apparently persistent in KOH; (2) 5–32 μmdiam, margin; ultimate flabellar margins usually more or less entire irregularly inflated from subspherical (apparently hyphal

Fig. 4 A NeighborNet phylogenetic network of Sparassis crispa collections (Europe) computed in SplitsTree4 showing alternate phylogenies. unk unknown location. C clone number Mycol Progress

Fig. 5 A NeighborNet phylogenetic network of southeastern and Arizonan Sparassis americana collections computed in SplitsTree4 showing two eastern North American clades and F1 hybrids between the clades and the relationship of Arizonan clades to Eastern North American clades. TENN65971h1 and h2 represent the two haplotypes of an F1 hybrid

termini or juxtaposed to septa) to figure eight-shaped (articu- dendritic (i.e., irregularly branched in 1–3ranks,with lated into a three-dimensional “dryophila structure;” Fig. 6d), branches somewhat gnarled), bearing 1–2 conspicuous thick-walled (wall −2 μm thick), with wall sometimes clamp connections. slowly gelatinizing in KOH; swollen cells apparently Commentary While Burdsall and Miller (1988b) more scattered in ultimate flabelli than in lower neotypified S. crispa with a specimen collected in the general branches where they are more concentrated nearer to topotype of Schaeffer’s original protologue of Elvella the branch surface; and (3) “vascular hyphae” 3– ramosa,theyalsoopined:“Because Fries (1821)sanctioned 6.5 μm diam, subrefringent (PhC), as intercalary lengths the name proposed by Wulfen, a specimen from Carinthia (never seen with terminal septa). would be most appropriate to serve as neotype.” Wulfen (in Hymenium apparently thickening (i.e., fertile basidia occa- Jacquin), as part of the protologue, noted the topotype of S. sional, with basidioles densely interspersed), distinctly unilat- crispa as Klagenfurt in today’s southeastern Austria eral (on darker, waxy undersides of the upper flabelli). (Carinthia). The epitype specimen designated above Basidioles 1.5–2.5 μm diam, abundant, usually unbranched serves two purposes. (1) It provides DNA sequences but occasionally branched from a conspicuous clamp connec- with which to characterize and anchor the taxon; and tion, sometimes irregularly subcatenulate. Basidia (Fig. 6f) (2) its herbarium data places its origin near Klagenfurt, 37–48(−67)×(5.5-)7–8.5 μm, elongate-clavate, 4-sterigmate, and furthermore links the specimen to M. Moser, an arising from a clamp connection; contents more or less important source of European taxonomic judgment. homogeneous. The epitype specimen has been damaged somewhat by Basidiospores (Fig. 11a)4.5–5(−7)×3.5–4(5) μm, broadly insects (frozen and re-dried during this study), but presents ellipsoid, smooth, thin-walled, subrefringent (PhC). more than adequate basidiome tissue for subsequent morpho- inamyloid. logical study if needed. Abhymenial region composed of outer medullary tis- In all examined specimens, the abhymenial surface of the sue of irregularly inflated and typically slender hyphae paraphyses invariably is buried in a thick spore deposit, prob- (see above), producing side branches which narrow into ably caused by close proximity to the fertile hymenium of paraphyses. Paraphyses (Fig. 6e) forming a loose thatch an adjacent flabellum. Whether this creates an environ- usually immersed in spore deposit, hyphal, slender, 2– ment for paraphysis proliferation (i.e., moist or “chem- 3.5 μm diam, simple and then irregularly gnarled to ical-friendly” environment) remains unknown. Mycol Progress

The medullary tissue composed of loosely articulated, ir- Thasoul, [circum 50°00′00.27″N, 5°42′51.40″E = Bastogne], regularly inflated and perhaps gelatinizing “dryophila struc- 22.X.1972, coll RHP, no. B9 (TENN36897; ex BR); prov. ture” was unexpected (by RHP). These inflated, often thick- Brabant, Uccle [50°48′01.14″N,4° 20′00.90″E], 2.X.1965, coll. walled, elongated cells become uncommon deep in the trama, P. Heinemann, US0 293138 (BPI). CZECH REPUBLIC, but more common outward toward the hymenial or “Fungi Bohemici,” Tábor [49°24′51.37″N, 14°39′28.95″E], abhymenial surfaces. Hyphae of European specimens often Dobrovici, 24.IX.1904, leg. F. Bubák, s.n. (USO 293083; BPI); exhibit almost spherical shapes, usually juxtaposed to septa, Teplitz [50°38′24.27″N, 13°49′29.06″E], “Fungi Eichleriani,” never seen in American specimens. The presence of these VIII.1902, leg. G. Eichler, no. 148 (USO 293091 BPI). inflated cells seems to be diagnostic of S. crispa and are easily GERMANY, location unknown, 16.IX.1972, coll RHP, no. observed. Annotations by K.J. Martin (Martin and Gilbertson D14 (TENN36739); Brandenburg: Sophienstadt [52°48′42.53″ 1976) indicate his recognition of such unique cells. N, 13°35′18.65″E] bei Ruhlsdorf, Kreis Nieder-Barnim, The presence of clamp connections at basidium and pa- 2.IX.1916, leg. P. Sydow, “Mycotheca germanica,” no. 1439 raphysis bases (and often distal in paraphyses as well) is not (USO 293077; BPI); Saxony, Königstein [50°10′18.40″N, diagnostic of S. crispa, as was sometimes concluded in the 8°27′57.04″E], IX.1893, leg W. Krieger “Fungi Saxonici” (as past. The same structures in the same places in S. brevipes, S. ramosa), Krieger 858 (USO 293090; BPI); Oberpfalz, often considered clampless, render their presence useless as a Schwarzenberg, [50°32′37″N, 12°46′42″E], 2.IX.1905, leg. K. diagnostic element. Conversely, the tramal tissues of S. Starcs, no. 5.127, 0190765 (M); vic. Regensburg, [49°01′00″N, brevipes conspicuously lack clamp connections, which are 012°06′45″E], X.1950, leg. S. Killermann (as S. ramosa), commonly present in tramal tissue of S. crispa. 0190763 (M); Upper Palatinate, Leuchtenberg, [49°36′26″N, The refringent “gloeoplerous hyphae” in the stipe tissue 012°15′16″E], IX.1909, leg. S. Killermann, 10190764 (M); vic. also seem somewhat unique—not merely the hyphae them- Regensburg, [49°01′00″N, 012°06′45″E], X.1950, leg. S. selves, but their distinctive refringence under PhC. They are Killermann (as S. ramosa), 0190766 (M); Bavaria, vic. also found in the stipe tissue of S. brevipes. Their more Trauchgau (modern Halblech), [47°37′57″N, 10°49′16″E], specific contents and ability to stain remain unidentified. VIII.1901, leg. A. Allescher (as S. ramosa), ex Sydow, It is my (RHP) impression that the ultimate crisped flabelli Mycotheca germanica, no. 104 (as S. crispa), 0190767 (M); of European specimens are smaller, thinner and more delicate Waldkraiburg, [48°12′32″N, 12°24′05″E], 4.X.1975, coll. H. than those of American basidiomata (i.e., S. americana, S. Marschner (as S. nemecii), HM no. 557, 0190754 (M); radicata). This is a difficult metric to measure, especially in Saxony, Königstein, [50°10′19″N, 8°27′52″E], IX.1893, leg W. dried material. Photos labeled as S. crispa on the web usually Krieger, ex Krieger, Fungi Saxonici (as S. ramosa), no. 858, do not bear any geographic origin, so drawing conclusions on 0190756 (M); Saxony, Königstein, [50°10′19″N, 8°27′52″E], this impression is impossible. IX.1893, leg W. Krieger, ex Krieger, Fungi Saxonici (as S. C.G. Lloyd received some specimens of European ramosa), no. 858, 0190756 (M); Waldkraiburg, [48°12′32″N, Sparassis, but professed indecision on their identity. An an- 12°24′05″E], 4.X.1975, coll. H. Marschner (as S. nemecii), HM notation slip with one specimen (United Kingdom, US no. 557, 0190754 (M). SWEDEN, S. Smoland, Femsjö [56°54′ 0333334; as S. laminosa; BPI) reads: “This agrees with our 00.49″N, 13°19′00.45″E], Dullaberget, 13.IX.1998, leg & det M. recollection of the specimen. Sparassis laminosa is not alto- Moser, no. 1998/0129 (IB). THE NETHERLANDS,prov. gether clear to me as I have seen forms which connect it with Utrecht, Amerongen [52°00′00.00″N, 5°27′00.30″E], [S.] crispa and which are difficult to place. I should like to see 16.X.1960, coll. & det. R.A. Maas Geesteranus (as Masseeola it again typical, and in a fresh shale [sic].” crispa), MG no. 13473 (USO 293118; BPI, ex L). UNITED Delatour (1975) reported a bipolar mating system KINGDOM, location unknown, date unknown, ex Kew, ex employing Europe strains. For more on mating systems of herb. Lloyd (as S. laminosa), cat. No. 32608 (USO 333334; Sparassis, see under S. americana. BPI). Specimens examined for morphology AUSTRIA, Sparassis americana [f. americana] R.H. Petersen, sp. Treimischerteich bei Viktring [Klagenfurt am Wörthersee], nov. Mycobank no. 804647 Kärnten [46°35′22.60″N, 14°16′06.77″E], 7.X.1978, coll M. Figs. 1b, 7 and 11b. Moser, nr. 78/414 (IB 19780414); Opferholz, Viktring Holotype United States, Tennessee, Blount Co., GSMNP, [Klagenfurt am Wörthersee], Kärnten [46°35′22.60″N, 14°16′ Cades Cove, picnic area near campground, 22.VI.2012, coll 06.77″E], 3.X.1975, coll M. Moser, 75/357 (IB 18750357; RE Baird, s.n., TFB14032 (TENN65974). epitype); Lower Austria, Vienna, Hütteldorf [48°12′35.03″N, Diagnosis Occurring in eastern North America; phyloge- 16°16′16.35″E], 19.VIII.1985, coll M. Haberhofer, no. 7763/4 netically separate from S. crispa; colors of upper branches and (WU 4791); Öber Austria, Grieskirchen, Natternbach [48°23′ flabelli muted (pallid cream to dull grayish cream); subterra- 49.01″N,13°44′55.78″E], 11.IX.1994, coll. H. Voglmayr, no. nean stipe often present; stipe trama dry or marbled; stipe 7548/3 (WU 19887). BELGIUM, eastern Belgium, ?Kyleet- tramal hyphae thick-walled. Mycol Progress

ƒFig. 6 Sparassis crispa. Tramal hyphae, origin of abhymenial paraphyses and basidia. a Rough-walled deep tramal (only a portion shown as rough-walled). b Deep tramal hyphae of middle branches, showing clamp connections. c Articulation of deep and shallow tramal hyphae of upper branches. d Inflated, thick-walled freeform cells of “dryophila structure.” e Paraphysoid termini. f Basidia. a–c, f 75/357 (IB). d, e 1998/0129 (IB) Bars=20 μm

Basidioma (Fig. 1b) −130(−200) mm high, −140(−350) mm broad, arising on a single stipe of variable dimensions (i.e., sometimes shallowly pinched and ending just below soil level, sometimes forming a considerable subterranean exten- sion). Stipe externally appearing single, white, smooth, usu- ally longitudinally channeled; in longitudinal section, stipe flesh white, moist but not gelatinous, sometimes delicately marbled, sometimes with coarsely longitudinal, water-soaked (not gelatinous) bands or veins (“tilleul buff” 7B2), where submerged stipe branches are compressed. Upper basidiome portion appearing coarsely and irregularly (not dichotomous- ly) branched, rebranched to produce expanded petaloid flabelli. Flabelli crisped (i.e., curling and recurling), usually entire but sometimes lobate to finely dissected; hymenial surface usually radially undulate, uniformly “ochraceous buff” (5A5) to “capucine buff” (4A3) with ultimate margins drying more or less cartilaginous and reddish-brown (“russet” 7D6) to brown (“clove brown” 6F5), when fresh appearing waxy (50×) (similar to lamellar surface of some hygrophoroid mushrooms), retaining this appearance when dried; abhymenial surface paler, “light ochraceous buff” (5A4) “pale ochraceous buff” (4A2), off-white, matte, gently longitudinal- ly undulate, not appearing waxy (at 75× appearing plushy). Odor negligible; taste negligible. Habitat and phenology: apparently associated with Pinus spp., perhaps as root parasite; from New England (NH, MA) through the Appalachian Mountains to (at least) central GA, west to MO, AR, apparently missing in FL and the Gulf Coast; mid-summer through mid-autumn. Stipe trama: hyphal construction monomitic, hyphae of three types (Fig. 7a–c). (1) Diameter 2.5–5 μm, thick-walled (wall −1.5 μm thick), sometimes leaving only a capillary lumen, commonly and conspicuously clamped, well-defined, without ornamentation, making up the “ground tissue;” (2) 4– 10(−12) μm diam, thick-walled (wall −1.5 μm thick, ill- defined), in profile appearing hairy or ciliate (1250×, PhC), in face view appearing minutely gritty, occasionally conspic- uously clamped, with cells −750 μm long between clamps; clamps modified tibiiform (see Fig. 7); (3) well-defined gloeoplerous hyphae, refringent, well-defined, 9.5–14 μm diam, not noticeably “puffed,” not noticeably septate, appar- ently easily disarticulated into lengths but meandering through the ground tissue (probably “vascular hyphae” described for dried material; see Reid 1965). Mycol Progress

ƒFig. 7 Sparassis americana [f. americana]. Tramal hyphae of lower branches and stipe, origin of abhymenial paraphyses, basidia. a Rough- walled deep tramal hypha (only a portion shown as rough-walled). b Deep tramal hyphae of middle branches, showing clamp connections. c Thin-walled “vascular” hypha. d Freeform hyphae of “dryophila structure.” e Paraphyses arising from subabhymenial cells. f Basidia. a, b, e TENN65974 (clade II). c TENN50258 (clade II). d, f TENN65584 (hybrid). Bars=20 μm

Outer flabellar trama monomitic, of two hyphal types (Fig. 7d): (1) irregularly inflated hyphae in lobate to tibiiform shapes, loosely integrated into a three-dimensional “dryophila structure;” and (2) 4–15 μm diam, firm-walled with conspic- uous, medallion or tibiiform clamps, producing less-inflated cells which produce hymenium or paraphyses. Hymenial surface of ultimate flabellum: outer flabellar tramal cells give rise to densely packed, crushed, thin- walled, clamped subhymenium, which seems to gelatinize as hymenium thickens. Hymenium significantly thickened; basidia (Fig. 7f) (40-)50–66(−72)×(4.5-)6–7(−8) μm, narrow- ly clavate, clamped, (2-)4-sterigmate (sterigmata −6.5 μm long, slender); contents homogeneous when immature, be- coming multigranular to few-guttulate by maturity. Basidiospores (Fig. 11b)4.5–6(−7.0)×(3.0)3.5–4.0(−4.5) μm, smooth, broadly ellipsoid, flattened axially, non-amyloid, thin-walled; contents uniguttulate, refringent (PhC). Abhymenial surface of ultimate flabellum: paraphyses (Fig. 7e) −75×2–3.5 μm, thin-walled, conspicuously clamped, often branched, always gnarled to some extent, rarely with one or more small guttules. Commentary: Two prior names could have been candi- dates for the American “crispa:” Stereum carolinensis Cooke and Ravenel, and Sparassis tremelloides Berkeley. According to Reid (1962, 1965) and our examination of the type specimen of Stereum carolinense, it matches S. spathulata and according to Burdsall and Miller (1988a)and our examination of the type of S. tremelloides,itistrulya tremelloid . Thus there seems to be no prior name for the American entity. The thickening of the hymenium is notable and is one of the characters that traditionally placed Sparassis in the Clavarioids and Aphyllophorales. As the hymenium thickens, basidia tend to be somewhat longer, so basidial length can be indicative of hymenial age. Basidia are often perceived as sorting into two lengths, and longer, immature basidia may be interpreted as cystidia (Desjardin et al. 2004). The extent of dissection of ultimate flabelli is difficult to describe. A form with rather large, coarse ultimate flabelli is exemplified by TENN65584 (NC-hybrid) and TENN60930 (NC), while TENN50258 (TN) shows much more delicately dissected flabelli. An intermediate form is represented by TENN65972 (TN) and TENN44575 (GA). Mycol Progress

ƒFig. 8 Sparassis americana f. arizonica. Thick-walled, deep tramal hyphae, subabhymenial cells. a Tramal hypha with outer wall roughened. b Tramal hypha with smooth outer wall; inner wall roughened. c Irregular gelatinous sheath surrounding tramal hypha; d Tramal hyphae with smooth outer and inner walls, showing clamp connections. e “Vascular hypha” of tramal tissue, showing banded ornamentation. f Freeform subabhymenial cells. a–d.KJM274 (CFMR). e, f. KJM 468 (CFMR). Bars=20 μm

A recent report (Light and Woehrel 2009) also speculates on an association of S. americana (as S. “crispa”) with . Herbarium specimens with notes on habitat almost uniformly cite Pinus, often with language like “at the base of pine.” TENN44575 is one of two collections (the other from Massachusetts) which Wang et al. (2004) placed in a small clade. They would appear to be the sole representatives of S. americana in that phylogeny, and morphologically, TENN44575 seems typical of eastern S. americana. This specimen is a representative of ENA clade II, which seems to be limited to the southern Appalachian Mountains. There is also some discrepancy in spore germination among eastern S. americana collections, but so far, no con- nection between this character and morphological characteri- zation has been made. In one strain (i.e. TENN65971 hybrid), spore germination on agar began within 96 h, and germlings produced “seeker” hyphae. Putative dikaryons formed widely-spread hyphae and young colonies were arachnoid. In the other strains [i.e. TENN65972 (ENAI), TENN65974 (ENAI)], germination occurred in about 2 weeks, with slow germ tube growth. Once dikaryotization had taken place, growth was slow and colonies were rather dense. Wang et al. (2004) placed TENN50232 from North Carolina in clade radicata, otherwise from the west coast. We resequenced this specimen, and both ITS and LSU place this specimen in S. americana clade I, as expected from its geograph- ical origin. It would be convenient to characterize S. americana using some anatomical features of the ultimate flabelli, rather than the anatomy of stipe tissue. Most herbarium specimens, how- ever, consist of upper basidiome parts without basal tissue. Stipe anatomy might segregate the eastern S. americana from the S. crispa, but stipe micromorphology cannot be ascertained from most herbarium material. ITS phylogenies show that S. americana isnotthesameasS. crispa as traditionally thought (it is also separated from S. radicata). In fact, eastern S. americana populates two separate but closely related clades, distinct from the southwestern and the upper west coast entities. Thus far, we have been unable to morphologically distinguish members of these two clades of There seems to be no micromorphological difference be- eastern S. americana, and pairing experiments among the vari- tween the white and water-soaked stipe flesh. The water- ous collections show predominant sexual intercompatibility. soaked appearance may be caused by compression among Specimens examined for morphology GEORGIA,Murray basidiome initials from being squeezed as development occurs. Co., Fort Mountain State Park, 34° 45′ 43″ N, 84° 42′ 10″ W, Mycol Progress

13.IX.1983, coll. RE Baird, s.n. (TENN44575). NORTH ponderosa and Abies concolor, “primarily during the rainy CAROLINA, Macon Co., Highlands, 6.VII.1991, coll. un- season in July, August and September.” Reported by Martin known, TFB4076 (TENN50232). TENNESSEE, Blount Co., and Gilbertson (1976) from southern Arizona, western New GSMNP, Cades Cove, picnic area near campground, Mexico, and doubtfully from Marin Co., California. 22.VI.2012, coll. RE Baird, s.n., TFB14032 (TENN65974); Hyphae of the pseudosclerotium compact, of two types: (1) Blount Co., GSMNP, Cades Cove, 14.VII.2004, coll. S. 2–4 μm diam, thin-walled, contorted, with clamp connections; Cantrell, leg. J. Caranza, JC 1 (TENN60165); Blount Co., (2) 6–12 μm in diam, thin-walled; walls highly refractive GSMNP, Cades Cove, 28.VI.1991, coll. RHP, TFB4067 (PhC, BF); contents staining deeply in KOH-phloxine, with (TENN50258); Blount Co., GSMNP, Cades Cove, 4.X.2010, clamp connections and occasional simple septa. Lower branch coll. M.G. Wood, MGW 798 (TENN65374); Cumberland Co., trama hyphal construction is monomitic. Tramal hyphae Crossville, Rinnie Comm., 10.VI.2012, coll. Steve Roberts, (Fig. 8a–e)6–11 μm diam, thick-walled (wall often occluding TFB14028 (TENN65972); Rock Island Co., Rock Island State the cell lumen), occasionally but conspicuously clamped, Park, N 35° 47′ 56.17″,W85°37′ 39.79″, 17.VI.2012, coll. often roughened on the outer wall surface and then sometimes Christina Braaten, TFB14027 (TENN65971); Sevier Co., with subtle transverse banding (PhC), seemingly embedded in GSMNP, Trail to Albright Grove, Madron Bald Trailhead, a gelatinous or mucoid matrix which tends to obscure the 19.X.2005, coll KWH, TFB13005 (TENN60930); GSMNP, outer wall definition. Outer tramal cells and sub(ab-)hymenial Pitman Center, Greenbrier, UT Biology Field Station, hyphae (Fig. 8f) freeform, compactly arranged into a tissue 27.VII.2009, coll. A. Wolfenbarger, S. Trudell, PB Matheny (as similar to a “dryophila structure,” 4–16(−30) μm diam, thin- S. “crispa”), PBM3247 (TENN64565). to thick-walled (wall −1.0 μm thick), not clearly differentiated Sparassis americana f. arizonica R.H. Petersen, f. nov. from contextual hyphae, with often conspicuous clamp con- Mycobank no. 804648 nections; gloeoplerous hyphae scattered throughout, aseptate, Figs. 1c, 8 and 11d. thin-walled, 6–12 μm diam, most abundant towards the base Holotype United States, Arizona, Pima Co. Coronado Nat. of the basidioma. Forest, Santa Catalina Mts., Mt. Bigelow, 32° 24′ 54.5″ N, 110° 42′ 52.4″ W, 31.VIII.1972, coll K.J. Martin (as S. radicata), KJM279 (CFMR, with culture; probably also AZ) Diagnosis Occurring in high-altitude conifer forests of southwest United States, chiefly Arizona; phylogenetically separate from S. americana f. americana; margins of ultimate flabelli often frilled or digitate; basidiomata usually with pinkish tint. Basidiomata (Fig. 1c) perennial, producing above-ground fruitbodies more or less annually from a persistent pseudosclerotium. Pseudosclerotium is a mass of soil and humus particles held together by white mycelium in a firm, subgelatinous matrix, ~700 mm long and −100 mm broad, attached to underground roots of living or dead conifers or sometimes growing directly out of wood and lacking a pseudosclerotium. Above-ground basidioma becoming quite large, mostly 100–300 mm broad, 100–200 mm high, creamy yellow to yellowish brown, with multiple meristematic points forming a rounded mass of numerous anastomosing and subdivided, negatively geotropic petaloid branches becoming thinner distally, with ultimate branchlets narrow (usually 6– 8 mm broad) thin, crisped or curled flabelli with usually frilled or delicately lobate, wavy margins, often exhibiting some pinkish tints; hymenium mostly unilateral. Odor reportedly strong, becoming slightly disagreeable, “somewhat like smoke-cured bacon” (notes with specimens at CFMR); taste (fresh) bland. Habitat and phenology: Conifer forests of southeastern Fig. 9 Sparassis americana f. arizonica. a Paraphyses arising from Arizona and adjacent New Mexico. Chiefly associated with subabhymenial cells. b Paraphyses. c Basidia and basidioles. a, c KJM Douglas fir (Pseudotsuga menziesii)butalsoPinus 468. (CFMR). b KJM 274 (CFMR). Bar=20 μm Mycol Progress

ƒFig. 10 Sparassis radicata. Microscopic structures. a, b Thick-walled tramal hyphae. c Inflated, thick-walled tramal hyphal segments. d “Vascular” hypha. e Freeform subabhymenial cells; f, g Paraphyses. h Basidia. a, c TENN34723-RW004675. b, d TENN67998-SAT295-02. e, g, h TENN52558. f TENN56253. Bar=20 μm

The hymenial layer distinct, thickening, mostly unilateral depending on the position of the flabellum; basidia (Fig. 9c) closely packed, clavate, 4-sterigmate, 40–60×4–8 μm. Basidiospores (Fig. 11d) (4-)5–6(−7)×(3-)3.5–4(−4.5) μm, ellipsoid, hyaline, smooth, inamyloid, flattened some- what axially; contents 1-guttulate (fresh); spore print white. Abhymenial tramal cells (Fig. 8f) freeform, usually signif- icantly longer than broad, in a loose “dryophila structure,” producing side branches as paraphyses. Paraphyses (Fig. 9a, b) digitate to hyphal, firm-walled, 2.5–3.5 μm diam, arising from a clamp connection. Commentary The description of S. “radicata” by Martin and Gilbertson (1976), with the exception of the type speci- men from Idaho, was based on southwestern North American collections (AR, CA, NM). It is possible, therefore, to use their description together with new observations (as above) to describe this taxon. Their two photographs of basidiomata are numbered in their legend and represent Arizonan material (not Pacific Northwest), In addition, they reported: “Associated with a yellowish-brown, carbonizing butt and root rot of living conifers. Apparently rather common in southern Arizona. The basidiocarps are associated with large, hypogeous pseudo- sclerotia.” Host trees were listed as: Abies concolor, Pinus ponderosa, P. s tro bi fo rm is and Pseudotsuga menziesii. Molecular phylogenies (this study) indicate that identifica- tion of southwestern specimens as S. radicata (Gilbertson et al. 1974; Martin and Gilbertson 1976) was incorrect. Southwestern collections seem to be closer to eastern S. amer- icana than to true S. radicata. In fact, morphologically, southwestern specimens are difficult to distinguish from east- ern S. amricana, except as noted above. Specimens examined for this study (CFMR not ARIZ) from southern Arizona seem to describe the high-altitude forests known as the “Sky Islands,” mycologically described by Gilbertson and Bigelow (1998). A somewhat expanded description and illustration of f. arizonica (as S. radicata)was furnished by Gilbertson et al (1974). Photos of basidiomata (i.e., Martin and Gilbertson 1976:Fig.1c)seemtoshowthe ultimate flabelli of southwestern United States specimens as smaller and more crowded than those of eastern S. america- na, and often with frilled or delicately lobate margins. This is also true of some (not all) of the specimens examined for this paper. Microscopically, tramal tissues of upper stipe and lower branches seem to involve a gelatinous or mucoid matrix, obscuring hyphal walls, and subabhymenial, freeform cells Mycol Progress

Basidiomata (Fig. 1d) up to 250 mm broad, up to 300 mm high, composed of two prominent portions: (1) subterranean, pseudosclerotial stipe (usually missing from herbarium spec- imens), tapering downward, misshapen, with small lacunose holes from complex branches being compressed in various ways; context subgelatinous to gelatinous to varying extents, when dried off-white and minutely (25×) to extensively marbled-cartilaginous; (2) above-ground fertile structure com- posed of complex, often anastomosed lacunose branches, with ultimate flabelli thin, parchment-like in well-dried specimens, curled or crisped, varying considerably in size (up to 25 mm broad) but usually broader than those of eastern North American basidiomata. Hymenial and abhymenial surfaces Fig. 11 Basidiospores of Sparassis crispa complex. a S. crispa (Eu- minimally distinguishable when fresh; hymenial surface uni- rope). b S. americana [f. americana]. c S. radicata. d S. americana f. lateral except on vertical flabelli in basidiome center, pallid arizonica. Solid lines represent 90 % of spore dimensions and shape. cream color to pastel yellow when fresh, drying near “cinna- Stippled lines represent largest 10 % of spore dimensions and shape. mon buff” (6B4) appearing waxy under lens (40×); Bar=5 μm abhymenial surface off-white (“pale pinkish cinnamon” 6A2), minutely matte, usually finely radially undulate. Odor negligible; taste negligible. often have minutely roughened walls with very subtle Habitat and phenology: associated with roots of coniferous fragmented, superficial banding. trees (chiefly Pseudotsuga) in temperate rainforests of North Specimens examined for morphology United States; all America (northern CA, OR, WA, northern ID, British Arizona; Cochise Co., Coronado Nat. Forest, Chiricahua Columbia; autumn, September—mid-November. Mts, Tub Spring, [31°52′46.87″N, 109°54′26.66″W], Stipe flesh hyphal construction is monomitic; generative 27.VIII.1968, coll R.L. Gilbertson, RLG 8240 (CFMR); hyphae (Fig. 10a–d) of two types: (1) (2.5-)3.5–13 μmdiam, Graham Co., Coronado Nat. Forest, Pinaleno Mts, firm- to thick-walled (wall −1.5 μm thick), often branched Moonshine Creek, [32°40′50.50″N, 109°54′26.66″W], (irregularly), conspicuously clamped, loosely interwoven, rel- 31.VIII.1973, coll K.J. Martin (as S. radicata), KJM 469A atively well-defined, embedded in gelatinous or mucoid ma- (CFRM); same locality, same date, coll. K.J. Martin (as S. trix, forming the “ground tissue;” and (2) 5–11(−16) μmdiam, radicata), KJM 468 (CFMR); Pima Co. Coronado Nat. refringent to subrefringent (and then appearing as “vascular Forest, Santa Catalina Mts., Mt. Bigelow, [32°24′54.5″N, hyphae”), thin- to thick-walled (wall −1 μm thick), rarely 110°42′52.4″W], 31.VIII.1972, coll K.J. Martin (as S. conspicuously clamped. radicata), KJM 279 (CFMR); Upper Bear Wallow, [32°25′ Flabellar tramal tissue juxtaposed to subhymenium com- 27.6″N, 110°44′23.5″W], 9.VIII.1973, coll J.P. Lindsey (as S. posed of irregularly inflated (5–28 μm diam), thin-walled, radicata), JPL 188 (CFMR); same locality, same date, coll tibiiform or vermiform, loosely articulating cells (Fig. 10e) K.J. Martin (as S. radicata), KJM 389 (CFMR); Mt. appearing “empty,” intricately interwoven into a “dryophila Lemmon, [32°26′27″N, 110°45′27″W], 12.IX.1973, coll structure,” sometimes resembling a palisade, obscurely K.J. Martin (as S. radicata), KJM 202 (CFMR); Bear clamped, perhaps with some mucoid or gelatinoid matrix, Wallow, [32°25′27.6″N, 110°44′23.5″W], 11.VIII.1974, coll producing branches which form the subhymenium; K.J. Martin, KJM-274 (CFMR); same locality, 11.VIII.1973, subhymenium of tightly interwoven, extremely thin-walled, coll K.J. Martin (as S. radicata), KJM-392 (CFMR); Mt. perhaps semi-gelatinized hyphae from which the hymenium Lemmon, [32°26′27″N, 110°45′27″W], 9.VIII.1973, coll arises. K.J. Martin, KJM 391 (CFMR). All collections with probable Hymenium of basidia and basidioles arising from clamp duplicates at ARIZ. connections. Basidioles paraphysoid to narrowly clavate; Sparassis radicata Weir 1917. Phytopathology 7: 166. basidia (Fig. 10h)38–60(−85)×(5.5-)6.5–8 μm, clavate, con- Figs. 1d, 10 and 11c. spicuously clamped, (2-)4-sterigmate (sterigmata −6.5 μm Holotype UNITED STATES, Idaho, Priest River, location long); contents minutely granular, never guttulate. unknown, IX.1916, Weir no. 9822 (CFMR s.n.!). Basidiospores (Fig. 11c)5–6.5×3.5–4(−5) μm, broadly Epitype (hic design.) WASHINGTON, King Co., vic. ellipsoid to ellipsoid, flattened axially, smooth, hardly Enumclaw, Federation Forest State Park, N 47° 09′ 20.21″, refringent (PhC), inamyloid. W 121° 42′ 10.98″, 3.X.1992, coll. RHP, TFB5727 Abhymenial surface: outer tramal tissue and suhymenial (TENN52558). hyphae similar to those of hymenial surface, thick-walled Mycol Progress

(wall −1.5 μm thick), with disrupted gelatinous or mucoid western fungus. Contextual hyphae of the southeastern matrix, producing paraphyses; paraphyses (Fig. 10f, g) Sparassis are mostly thick-walled and simple-septate and −145 μm long, 2.5–3(−5) μm diam, occasionally branched those of the western S. radicata are thin-walled with clamps.” (not usually), often with intercalary clamp connections, with Cultures of their western S. radicata were “not different” from demonstrable disrupted gelatinous or mucoid matrix. cultures from European and Asian S. crispa, but differed from Sexual compatibility experiments In the intercollection “the southeastern USA” fungus. In every case, their descrip- pairing experiment limited to northwestern North American tions of the southeastern fungus point to S. spathulata. collections (i.e., S . “radicata”)(see“Materials and Burdsall and Miller (1988a)merelydismissedS. radicata methods”), two pairings (TENN56253 × SAT 301-01; as a synonym under S. crispa, which was considered to be TENN67985 × SAT 295-01) were predominantly incompati- distributed in Europe and across North America. They repeat- ble (1/4), with all others either 3/4 or 4/4. The general indica- ed that Martin and Gilbertson’s(1976) Arizona isolates of S. tion was that all collections represented a single “biological “crispa” were sexually compatible with European S. crispa taxon” (the two predominantly negative pairings did not show isolates. Our phylogeny indicates that the Arizona organism a pattern) (Online Materials Fig. ESM1). hardly differs molecularly from S. crispa and S. americana. The second intercollection pairing experiment involved S. In side-by-side comparisons of herbarium specimens, the radicata paired with collections from eastern North America, ultimate flabelli of the Pacific Northwest organism may be Arizona and Europe. In this experiment, all collections were somewhat larger and less intricately-lobed than those of east- predominately intercompatible, with no indication that S. ern S. americana or S. crispa, but some basidiomata have radicata was a separate “biological taxon.” Single basidial been observed to vary significantly across the fertile surfaces. isolates of S. radicata (SAT 295-01; SAT 301-01) were paired Microscopically, abhymenial paraphyses of S. radicata ap- with eastern North American S. americana (clade I-II hybrid pear longer and less branched than those of S. americana and = TENN65584; clade 2 = TENN65974; OKM7058; frequently exhibit more than one clamp connection. TENN65971). Uniform sexual compatibility was observed The irregularly swollen outer tramal hyphal cells of S. (Online Materials Fig. ESM2). radicata, while similar in shape to those of S. americana, Commentary Weir (1917) diagnosed his S. radicata based might be somewhat smaller (i.e., smallest and largest diame- on a few characters: (1) ultimate flabellum thinner than those ters less). In both species, however, the gelatinous or mucoid of other taxa; (2) presence of a prominent subterranean matrix of shallow subabhymenial hyphae and paraphyses pseudosclerotial mass; (3) parasitic association with roots of seems insoluble in KOH. They appear as short, curved, lam- conifers; and (4) production of cuboidal brown rot of vascular inar “flakes” adhered to the outside of these structures (PhC). tissues of large roots and adjacent trunks of host trees. Observation of significantly longer basidia than is typical Weir (1917) wrote extensively of the sclerotial mass he may be a function of a thickening hymenium. Such long found at the base of basidiomata of several collections basidia, if observed in immaturity, may account for the throughout the Pacific Northwest, comparing it to similar “cystidia” in S. cystidiosa Desjardinetal(2004). In several structures scattered among the fleshy fungi. The current term instances in S. radicata, such basidia exhibited normal sterig- for such a structure would be a pseudosclerotium, in which mata. Basidia are conspicuously clamped, with clamps typical substrate is enveloped in compact fungal mycelium to form a of the medallion type. New basidial initials are produced from tuber-like body. Apparently, this pseudosclerotium has been the basal clamp, and if observed when newly initiated, the overlooked by more recent collectors, perhaps because its clamp appears “beaked.” presence and size are quite variable, obscure and underground, Specimens examined for morphology CANADA, British but also because harvest of the pseudosclerotium is not nec- Columbia, vic. Whistler, [50°07′N, 122°57′W], 6.X.1990, essary for use in cuisine. At any rate, the pseudosclerotium is coll. NAMA, TFB3400/19 (TENN49093). UNITED missing from most herbarium specimens collected in the STATES, California, Monterey Co., Pacific Grove, appropriate range, although a stipe portion is sometimes Asilomar Conference Center, [36°37′59.20″N, 121° 5′ 8.57″ included. W], 13.II, 1998, coll RHP & KWH, TFB9540 (TENN56253). Martin and Gilbertson (1976) drew distinctions between Idaho, Bonner Co., Priest River, location unknown, IX.1916, their concept of S. radicata, drawn largely from southwestern Weir no. 9822 (holotype; CFMR s.n.); Valley Co., McCall, collections, “…the southeastern USA Sparassis …,” and [44°54′40.90″N, 116°05′54.55″W], 4.X.1970, coll. Ralph other United States collections “labeled as S. crispa.” Their Warner, RHP 4675 (TENN34723). Oregon,CoosCo.,Coos diagnosis of “the southeastern Sparassis” was: "Basidiocarp Head, 29.XII.1940, coll M.S. Doty, Doty no. 3122, USO lobes of the southeastern fungus are larger, thicker, darker in 293082 (BPI); Lane Co., Eugene, [44°03′N, 123°05′W], color and more vertically oriented than those of the western S. X.1902, coll A.R. Sweetzer, det Peck (as S. herbstii), s.n. radicata. They are also zonate with a white margin in contrast (NYS); “Oregon Coast,” 10.XI.2012, Eugene Mushroom to the azonate, uniformly cream colored to buff lobes of the Club display, SAT 301-01 (TENN68003); vic. Carlton, Mycol Progress

16.X.2012, coll P. Whitney, misit S. Kristic, s.n. (TENN68006). reports. The complex appears to exhibit sexual bipolarity but Washington ,Gray’sHarborCo.,OlympicNat.Forest,vic.Lake with some ambiguity. Quinault, 21.X.2012, coll. R. Blain & C. Herrera, leg. S.A. Pairing experiments in which n =4 present results that are Trudell, SAT 12-295-02 (TENN67998); same data, SAT 12- statistically less than robust. Moreover, given unpredictable nu- 295-01 (TENN67997); same data, SAT 12-295-03 clear behavior (see Nieuwenhuis et al. 2013), underlying causes (TENN67997); Jefferson Co., Lake Quinault, North Shore of apparent incompatibility cannot be traced in a study such as Drive, [47°29′01″N, 123°53′20″W], 4.X.1984, coll A.S. ours. Adequate, however, is the understanding that clamp con- Methven (as S. crispa), ASM 3359 (TENN45811); Mt. Rainier nection production may be accepted as a proxy for sexual Nat. Park, 46° 56′ N, 121° 41′ W, approx. 0.8 km from park compatibility, while apparent incompatibility can result from entrance, 26.X.1967, coll RHP (as S. radicata), RHP (not TFB) cytogenetic as well as sexual causes. With preponderant 2727 (TENN32933); Hansville, vic. Buck Lake, 20.X.2012, coll. intercompatibility among all tested collections in this study, using K. Dobson, TFB14186 (TENN67965); King Co., vic. clamp connection production as proxy for sexual compatibility, Enumclaw, Federation Forest State Park, N 47° 09′ 20.21″,W we conclude that the two S. americana clades from eastern 121° 42′ 10.98″, 3.X.1992, coll. RHP, TFB5727 (TENN52558); North America and Arizona (S. americana clade I; S. americana ?King Co., ?Seattle, 30.IX.2012, leg D. Miller, TFB14183 cryptic taxon), the S. radicata cladeandthesolecollectionofS. (TENN67940); Seattle, 10.X.2012, misit B. Luther, TFB14189 crispa all belong to a single “biological species.” This conclusion (TENN68005); Seattle, 11.X.2012, coll M. Harding, misit B. wasalsoreportedbyMartinandGilbertson(1976). Luther, TFB14190 (TENN68004); Whatcom Co., Bellingham, This conclusion appears counter to the recognition of these Stimson Nature Reserve, 10.XI.2012, coll D. Viney, misit B. separate clades based on molecular data and recognition of McAdoo, TFB14187 (TENN67968). distinct macro- and micromorphotypes. Of the three “species concepts” tested in this study, two concepts, molecular and morphological, appear to be more restrictive (especially when General discussion numerous collections are examined), while the “biological spe- cies concept” appears more permissive. This conclusion agrees Several previous reports (Desjardin et al. 2004;Wangetal. with previous reports on transatlantic disjuncts in such genera as 2004; Dai et al. 2006;Zhaoetal.2013) have distinguished Collybia (= Gymnopus)(Mataetal.2007; Vilgalys 1986, 1991; two major clades within Sparassis: (1) the S. crispa complex, Vilgalys and Miller 1983, 1987a, b).) and Marasmius (Gordon including S. crispa, S. latifolia and S. radicata;and(2)theS. and Petersen 1997, 1998), but cannot be extended to taxonomic spathulata clade, including S. spathulata, S. brevipes, S. complexes for which only inadequate material (i.e., without laminosa, S. minoensis, S. nemecii, S. subalpina and S. monokaryon SBIs, etc.) exists. Strong conservation of the sexual simplex Reid (1958) (B. Dentinger, pers. comm.). Desjardin recognition system seems reflective of origins and of the evolu- et al. (2004) indicated that S. cystidiosa belonged to neither tion of the system itself (Heitman et al. 2013). clade, but appeared to be basal to both, and Dai et al. (2006) There are few diagnostic synapomorphies in the ribosomal found S. cystidiosa f. flabelliformis to be placed in the same ITS and LSU regions in Sparassis. It was a surprise, therefore, basal clade. The present study does not cover the S. spathulata to find that eastern North American S. americana contained complex, and places S. latifolia using only sequences depos- two well-supported clades that were only three base pairs ited by previous authors in GenBank. different in the ITS region (0.05 %) plus F1 hybrids between Our phylogeny (Fig. 2) includes several LSU sequences from the two clades. Pairing experiments demonstrated that clade I, GenBank, deposited by Wang et al. (2004) as a component of clade II and hybrids were capable of dikaryotization in vitro. their phylogenies. There, the sequences were indexed (their However, with no evidence for F2 progeny or recombination Table 1)as“S.cf.crispa” and in their compound phylogeny between these two clades in nature, it would appear that clade I is as “Asian crispa clade.” Later,Daietal.(2006) described S. a cryptic endemic taxon and is reproductively isolated from clade latifolia and included under that name sequences previously II by some kind of post-zygotic barrier. Suggestions of a barrier labeled as “Asian crispa clade” by Wang et al (2004). can be observed in reduced compatibility in crosses involving the

In a more recent paper by overlapping authors (Zhao et al. hybrid F1 progeny, but it is unknown if other types of post- 2013), several ITS and LSU sequences were listed with zygotic barriers exist as well. A similar pattern was seen with GenBank numbers, indexed (their Table 1)asS. latifolia (n. Gymnopus dichrous clades I and II from the southern sp.) and in their compound phylogeny under HKAS numbers Appalachians and within the Hygrocybe chlorophana/flavescens as S. latifolia. None of the prior sequences (Wang et al. 2004; group but genetic distances in those cases were much greater Dai et al. 2006) were included. (Hughes et al. 2013). Thus, it would appear that cryptic specia- Sexual compatibility experiments Unfortunately, we can- tion processes may occur without significant genetic differenti- not report definitively on the mating system in the S. crispa ation in some cases and in spite of retention of ability for in vitro complex, although our results are consistent with previous dikaryotization. Although the principle has been treated Mycol Progress previously (Taylor et al. 2000;Hawksworth2006; Bickford References et al. 2007;Nilssonetal.2008; Hughes et al. 2009;), similar situations are being increasingly reported (Osmundson et al. Ainsworth AM, Parfitt D, Rogers HJ, Boddy L (2010) Cryptic 2005; Ainsworth et al. 2010;Harderetal.2010; Carlsen et al. taxa within European species of Hydnellum and Phellodon 2011; Hibbett et al. 2011;Vincenotetal.2011; Van de Putte revealed by combined molecular and morphological analysis. Fung Ecol 3:65–80 et al. 2012;Sheedyetal.2013). Bickford D, Lohman DJ, Sodhi NS, Ng PKI, Meier R, Winker K, Ingram Collections from Arizona are genetically divergent from KK, Das I (2007) Cryptic species as a window on diversity and each other and although related to S. americana clade II, they conservation. Trends Ecol Evol 22:148–155 have a distinct phenotype. While in vitro interbreeding is Binder M, Hibbett DS, Larsson K-H, Larsson E, Langer E, Langer G (2005) The phylogenetic distribution of resupinate forms across the possible between Arizonan collections and both S. radicata major clades of mushroom-forming fungi (Homobasidiomycetes). and S. americana, there is no evidence from natural collec- Syst Biodiv 3:1–45 tions of intermixing and recombination. In order to determine Breitenbach J, Kränzlin F (1986) Fungi of Switzerland Vol 2: Nongilled if Arizonan collections constitute a separate entity, further Fungi Bruen TC, Philippe H, Bryant D (2006) A simple and robust test for collection across the southern USA will be necessary. The detecting the presence of recombination. Genetics 172:2665–2681 unusual divergence of Arizonan collections compared with Bruns TD, Gardes M (1993) Molecular tools for the identification of other geographical areas may reflect island biogeography with ectomycorrhizal fungi−taxon-specific oligonucleotide probes for – populations isolated from each of the “sky islands” of the suilloid fungi. Mol Ecol 2:233 242 Bryant D, Moulton V (2003) Neighbor-net, an agglomerative method for the Chiricahua Mountains. This region is one of the most biolog- construction of phylogenetic networks. Mol Biol Evol 21:255–265 ically diverse areas in North America and like the Great Burdsall HH, Miller OK (1988a) Type studies and nomenclatural con- Smoky Mountains, it is an area where temperate and tropical siderations in the genus Sparassis. Mycotaxon 31:199–206 zones meet. Burdsall HH, Miller OK (1988b) Neotypification of Sparassis crispa. Mycotaxon 31:591–593 Given significant data from various analyses, problems Cai L, Giraud T, Zhang N, Begerow D, Cai G, Shivas RG (2011) The remain concerning names and ranks for the North American evolution of species concepts and species recognition criteria in members of the S. crispa clade, i.e., whether to base names plant pathogenic fungi. Fungal Divers 50:121–134 and ranks on morphological (macro- or micro-) distinctions, Cannon PF, Kirk PM (2007) Fungal families of the world. Centre for Agricultural Bioscience International, Amsterdam phylogenetic clade monophyly or sexual intercompatibility. Carlsen T, Engh IB, Decock C, Rajchenberg M, Kauserud H (2011) We have chosen to limit the basionymic species epithet to only Multiple cryptid species with divergent substrate affinities in the distributions of phylogenetic S. crispa, which appears to Serpula himantioides species complex. Fung Biol 115:54–61 circumscribe Europe, from Scandinavia to the Caucasus and Cotton AD (1907) On the structure and systematic position of Sparassis. Trans Brit Mycol Soc 3:333–339 east to the Ural Mountain chain, although two closely related Courtecuisse R, Duhem B (2008) Guide des champignons de France et European clades are molecularly identifiable but not morpho- d’Europe. Delachaux et Niestle. 476 pp logically separable. The organism collected in the moist Dai Y-C, Wang Z, Binder M, Hibbett DS (2006) Phylogeny and a new forests of western North America, already bearing a species species of Sparassis (Polyporales, ): evidence from mitochondrial atp6, nuclear rDNA and rpb2 genes. Mycologia 98: epithet and separable using ITS phylogenies and morpho- 584–592 logical distinctions (although not so on sexual compatibility), Delatour C (1975) Comportement in vitro du Sparassis crispa remains as S. radicata. The remaining North American rep- Wulf. ex Fr. et su Sparassis laminosa Fr. Eur J For Path 5: resentatives, although sexually intercompatible with S. 240–247 Desjardin DE, Wang Z, Binder M, Hibbett DS (2004) Sparassis radicata and European S. crispa, represent distinct clades cystidiosa sp. nov. from Thailand is described using morphological and subtle morphological disparities. We have chosen to pro- and molecular data. Mycologia 96:1010–1014 pose S. americana [f. americana] for the taxon collected from Felsenstein J (1985) Confidence limits on phylogenies: an approach using eastern Canada to the Gulf Coast and to recognize an atypical the bootstrap. Evolution 39:783–791 Fries EM (1819) Novit. flora svecica 5(cont.): 80 form, S. americana f. arizonica, for separable collections from Fries EM (1821) Systema mycologicum. Lundae 1:1–520 high-altitude conifer forests in the southwest United States. Gilbertson RL, Bigelow DM (1998) Annotated checklist of wood-rotting basidiomycetes of the Sky Islands in southeastern Arizona. J Acknowledgments Dr. Steve Trudell (University of Washington) and Arizona-Nevada Acad Sci 31:13–36 Mr. Brian Luther organized and furnished specimens and spore prints of Gilbertson RL, Martin KJ, Lindsey JP (1974) Annotated checklist and S. radicata; Dr. Nadezhda Psurtseva furnished dikaryon cultures from the host index for Arizona wood-rotting fungi. Arizona Exp Stat Tech Komarov Botanical Institute, St. Petersburg, Russia. Ms. Rita Bull 209:1–48 Rentmeester expedited dikaryon cultures from CFMR, which represented Gordon SA, Petersen RH (1997) Infraspecific variation among geograph- several collections cited by Martin and Gilbertson (1976), and Dr. Beatriz ically separated collections of Marasmius androsaceus. Mycol Res Ortiz-Santana arranged a loan of specimens from CFMR. Dr. Egon Horak 101:365–371 investigated the herbarium of IB and identified specimens of S. crispa Gordon SA, Petersen RH (1998) Infraspecific variation among geograph- from the topotype area. Research was supported by a US National ically separate collections of Marasmius scorodonius.Mycotaxon Science Foundation Grant to RHP and KWH. 69:453–466 Mycol Progress

Harder CB, Laessøe T, Kjøller R, Frøslev TG (2010) A comparison Posada D, Crandall KA (1998) Modeltest: testing the model of DNA between ITS phylogenetic relationships and morphological species substitution. Bioinformatics 14(9):817–818 recognition within Mycena sect. Calodontes in northern Europe. Reid DA (1958) New or interesting records of British Hymenomycetes. Mycol Prog 9:395–405 II. Trans Brit Mycol Soc 41:419–445 Hawksworth DL (2006) Pandora’s mycological box: molecular se- Reid DA (1962) Notes on fungi which have been referred to the quences vs. morphology in understanding fungal relationships and Thelephoraceae sensu lato. Persoonia 2:109–170 biodiversity. Rev Iberian Micol 23:127–133 Reid DA (1965) A monograph of the stipitate stereoid fungi. Nova Hawksworth DL (2012) Addressing the conundrum of unavailable name- Hedwig Beih 18:1–382 bearing types. IMA Fungus 3:155–157 Ridgway R (1912) Color standards and color nomenclature. Publ. Priv., Heitman J, Sun S, James TY (2013) Evolution of fungal sexual repro- Washington DC duction. Mycologia 105:1–27 Ryoo R, Sou H-D, Ka K-H, Park H (2013) Phylogenetic relationships of Hibbett DS, Donoghue MJ (2001) Analysis of character correlations Korean Sparassis latifolia based on morphological and ITS rDNA among wood decay mechanisms, mating systems, and substrate characteristics. J Microbiol Biotech 51:43–48 ranges in homobasidiomycetes. Syst Biol 49:215–242 Schaeffer JC (1763) Fungorum qui in Bavaria et Palatinu circa Hibbett DS, Ohman A, Glotzer D, Nuhn M, Kirk P, Nilsson RH (2011) Ratisbonam nascuntur. Vol. II Progress in molecular and morphological taxa discovery in fungi Schaeffer JC (1774) Fungorum qui in Bavaria et Palatinu circa and options for formal classification of environmental sequences. Ratisbonam nascuntur. Vol IV. Index primus. Synonyma et auctores Fungal Biol Rev 23:38–47 Sheedy EM, Van de Wouw APV, Howlett BJ, May T (2013) Multigene Huelsenbeck J, Ronquist F (2000) MrBayes: Bayesian inferences of sequence data reveal morphologically cryptic phylogenetic species phylogeny (software). University of California, San Diego within the genus Laccaria in southern Australia. Mycologia 105: Hughes KW, Petersen RH, Lickey EB (2009) Using heterozygosity to 547–563 estimate a percentage DNA sequences similarity for environmental Swofford D (2002) PAUP* 4.0b10: phylogenetic analysis using parsimo- species delimitation across basidiomycete fungi. New Phytol 189: ny (*and Other Methods). Sunderland, MA, Sinauer Associates 795–798 Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geisser DM, Hibbett DS, Hughes KW, Petersen RH, Lodge DJ, Bergemann S, Baumgartner K, Fisher MC (2000) Phylogenetic species recognition and species Tulloss RT, Lickey EB, Cifuentes-Blanco J (2013) Evolutionary concepts in fungi. Fung Genet Biol 31:21–32 consequences of putative intra- and interspecific hybridization in Van de Putte K, Nuytinck J, Das K, Verbeken A (2012) Exposing hidden agaric fungi. Mycologia. doi:10-3852/13-041 diversity by concordant genealogies and morphology - a study of the Huson DH, Bryant D (2006) Application of phylogenetic networks in Lactifluus volemus (Russulales) species complex in Sikkim evolutionary studies. Mol Biol Evol 23:254–267 Himalaya (India). Fung Divers 55:171–194 Jülich W (1981) Higher Taxa of Basidiomycetes. Vaduz, J. Kramer Vilgalys R (1986) Phenetic and cladistic relationships in Collybia sect. Kornerup A, Wanscher JH (1967) Methuen handbook of colour. Methuen Levipedes (Fungi: Basidiomycetes). Taxon 35:225–233 & Co., London Vilgalys R (1991) Speciation and species concepts in the Collybia Kreisel H (1983) Zur taxonomie von Sparassis laminosa Fr. s.l. Fed dryophila complex. Mycologia 83:758–773 Report 94:675–682 Vilgalys R, Miller OK (1983) Biological species in the Collybia Light W, Woehrel M (2009) Classification of the nomenclatural confu- dryophila group in North America. Mycologia 75:707–723 sion of the genus Sparassis [Polyporales: Sparassidaceae] in North Vilgalys R, Miller OK (1987a) Morphological studies on the Collybia America. Fungi 2:10–15 dryophila group in Europe. Trans Brit Mycol Soc 88:461–472 Martin KJ, Gilbertson RL (1976) Cultural and other morphological studies of Vilgalys R, Miller OK (1987b) Mating relationships within the Sparassis radicata and related species. Mycologia 68:622–639 Collybia dryophila group in Europe. Trans Brit Mycol Soc Mata JL, Hughes KW, Petersen RH (2007) An investigation of/ 89:295–300 omphalotaceae (Fungi: Euagarics) with emphasis on the genus Vincenot L, Nara K, Sthultz C, Labbé J, Dubois M-P, Tederson L, Martin Gymnopus. Sydowia 58:191–289 F, Selosse M-A (2011) Extensive gene flow over Europe and pos- Moncalvo JM, Vilgalys R, Redhead SA, Johnson JE, James TY, Aime sible speciation over Eurasia in the ectomycorrhizal basidiomycete CA, Hofstetter V, Verduin SJW, Larsson E, Baroni TJ, Thorn RG, Laccaria amethystina complex.MolEcol21:281–299 Jacobsson S, Heinz Clémençon H, Miller OK (2002) One hundred Wang Z, Binder M, Dai Y-C, Hibbett DS (2004) Phylogenetic relation- and seventeen clades of Euagarics. Mol Phylogen Evol 23:357–400 ships of Sparassis inferred from nuclear and mitochondrial ribo- Nieuwenhuis BPS, Debets AJM, Aanen DK (2013) Fungal fidelity: somal DNA and RNA polymerase sequences. Mycologia 96:1015– nuclear divorce from a dikaryon by mating or monokaryon regen- 1029 eration. Fung Biol 117:261–267 Weir JR (1917) Sparassis radicata, an undescribed fungus on the roots of Nilsson RH, Kristinansson E, Ryberg M, Hallenberg N (2008) conifers. Phytopath 7:166–177 Intraspecific ITS variability in the kingdom Fungi as expressed in White TJ, Arnheim N, Erlich HA (1989) The polymerase chain reaction. the international sequence databases and its implications for molec- Trends Genet 5:185–189 ular species identification. Evol Bioinform 4:193–201 Wulfen FX (1781) Plantae rariores Carinthiacae. In: Jacquin, 2. Misc. Osmundson TW, Cripps CI, Mueller GM (2005) Morphological and Austriaca Bot Chem Hist Nat 2:25–138 molecular systematics of Rocky Mountain alpine Laccaria. Yang ZL (2011) Molecular techniques revolutionize knowledge of basid- Mycologia 97:949–972 iomycete evolution. Fungal Biodiv 50:47–58 Petersen RH (1992) Mating systems in three New Zealand agarics. N Z J Zhao Q, Feng B, Yang Z-L, Dai Y-C, Wang Z, Tolgor B (2013) New Botany 30:189–197 species and distinctive geographical divergences of the genus Petersen RH, Hughes KW (1999) Species and speciation in mushrooms. Sparassis (Basidiomyota): evidence from morphological and mo- Bioscience 49:440–452 lecular data. Mycol Prog 12:445–454