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1 66 2 67 3 68 4 69 5 70 6 71 7 journal homepage: www.elsevier.com/locate/funbio 72 8 73 9 74 10 75 11 Phylogenetic overview of the Boletineae 76 12 77 13 78 a,1 b,1 c,e d 14 Q10 Mitchell E. NUHN , Manfred BINDER , Andy F. S. TAYLOR , Roy E. HALLING , 79 15 David S. HIBBETTa,* 80 16 81 17 aDept. of Biology, Clark University, 950 Main St., Worcester, MA 01610, USA 82 18 bCBS-KNAW Fungal Biodiversity Centre, Evolutionary Phytopathology, Institute of the Royal Netherlands Academy of Arts and Sciences, 83 19 Uppsalalaan 8, 3584 CT Utrecht, Netherlands 84 c 85 20 The James Hutton Institute, Craigiebuckler, Aberdeen AB15 8QH, Scotland, UK 21 86 dInstitute of Systematic Botany, New York Botanical Garden, 2900 Southern Blvd., Bronx, NY 10458-5126, USA 22 87 eInstitute of Biological and Environmental Sciences, University of Aberdeen, Cruickshank Building, St Machar Dr., Aberdeen AB24 3UU, UK 23 88 24 89 25 90 article info abstract 26 91 27 92 28 Article history: The generic and sub-generic relationships in the Boletineae (Boletales) were studied using 93 29 Received 20 February 2013 nuclear large subunit (nuc-lsu), translation elongation factor 1-alpha (tef1), and DNA di- 94 30 Accepted 23 April 2013 rected RNA polymerase largest subunit (RPB1). The Boletineae, with the exclusion of Hydno- 95 31 Corresponding Editor: merulius pinastri, was strongly supported and the status of the families Boletaceae and 96 32 Martin I. Bidartondo Paxillaceae is discussed. Members of the genus Boletus are found throughout the phylogeny, 97 33 with the majority not closely related to the type species, Boletus edulis. Many of the tradi- 98 34 99 Keywords: tional, morphologically defined genera are not supported as monophyletic and additional 35 100 Boletaceae sampling and taxonomic revisions are needed. The majority of the Boletineae are confirmed 36 101 Boletineae or putatively ectomycorrhizal (ECM), but two putatively mycoparasitic lineages (one line- 37 102 38 Ecology age of Buchwaldoboletus lignicola and Chalciporus piperatus and the second Pseudoboletus para- 103 39 Paxillaceae siticus) are strongly supported. 104 40 Systematics ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. 105 41 Q2 Taxonomic review 106 42 107 43 108 44 109 45 110 46 111 Introduction Hibbett (2006). The generic-level classifications of Smith & 47 112 Thiers (1971) and Singer (1986) along with current genera of 48 113 The suborder Boletineae (originally considered Agaricales)as Boletineae are presented in Table 1. The systems of Smith & 49 114 50 a taxonomic rank was first used by Gilbert (1931), and included Thiers (1971) and Singer (1986) mainly used morphological 115 51 both poroid and gilled species. This was a step forward in bo- characters and chemical staining reactions, e.g. the colours 116 52 lete taxonomy, the first time gilled species were included in produced by placing KOH on the pileipellis, to define genera. 117 53 a concept of a ‘bolete’. Since then, the generic and species con- Singer (1986) also incorporated the results of chemotaxo- 118 54 cepts in the Boletineae have been dominated by those proposed nomic studies, which identified pigments responsible for col- 119 55 by Singer (1986), which follows the inclusion of gilled species ouration and staining reactions. Chemotaxonomic data (Besl 120 56 proposed by Gilbert (1931) and Smith & Thiers (1971). The et al. 1974, 1986; Besl & Bresinsky 1977, 1979, 1997; Steglich 121 57 broad outlines of the ‘modern’ Boletineae, based on multi- et al. 1977; Bresinsky & Besl 1978) were not available at the 122 58 123 locus phylogenetic analyses, were presented by Binder & time of Smith and Thiers’s (1971) work. 59 124 60 125 61 126 62 * Corresponding author. Tel.: þ1 5087937420. 127 63 E-mail address: [email protected] (D. S. Hibbett). 128 64 1 These authors contributed equally to the work. 129 65 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. 130 http://dx.doi.org/10.1016/j.funbio.2013.04.008

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2 M. E. Nuhn et al.

131 196 e 132 Table 1 (continued) 197 133 Boletaceae of Boletineae of Current 198 134 Table 1 e Generic concepts of the Boletineae sensu Smith & Smith & Thiers Singer (1986) 199 135 Thiers, Singer, and Current. (1971) 200 136 201 Boletaceae of Boletineae of Current Incertae sedis 137 202 Smith & Thiers Singer (1986) Hydnomerulius 138 (1971) 203 139 a Species of Boletus sensu Smith & Thiers (1971) are distributed 204 140 Agaricales Agaricales Boletales across four genera of Singer (1986). 205 141 Boletaceae Agaricaceae Boletineae b Currently classified in Sclerotermatineae. 206 142 Boletellus Incertae sedis Boletaceae c Species of Leccinum sensu Singer are distributed across Leccinum 207 a 143 Boletus Notholepiota Afroboletus and Tylopilus (pro parte) by Smith & Thiers (1971). 208 144 Fuscoboletinus Boletineae Aureoboletus d Species of Tylopilus sensu Smith & Thiers (1971) are distributed 209 Gastroboletus Boletaceae Austroboletus across Tylopilus and Porphyrellus sensu Singer (1986). Single Smith 145 b 210 Gyroporus Boletoideae Australopilus and Thiers. 146 c 211 Leccinum Austroboletus Boletellus e Currently classified in Suillineae. 147 f 212 Pulveroboletus Boletellus Boletochaete f No molecular data available. 148 213 Tylopilusc,d Boletochaete Boletus 149 Strobilomyces Boletusa Borofutus 214 150 Suilluse Chalciporusa Bothia 215 151 Paxillaceae Fistulinella Buchwaldoboletus 216 152 Gyrodon Gastroboletus Chalciporus Morphological characters used to delimit genera and spe- 217 153 Phylloporus Leccinumc Chamonixia cies in Boletineae include, but are not limited to: stipe orna- 218 154 Phylloboletellus Fistulinella mentation, pileipellis and stipitipellis structures, pore 219 d 155 Porphyrellus Gastroboletus surface colour, pore depth, pore mouth diameter, staining re- 220 156 Pulveroboletus Gastroleccinumf 221 actions of bruised tissues, and staining reactions of different 157 Tylopilusd Harrya 222 tissues (such as pileus context, stipe context, pileipellis, stip- 158 Veloporphyrellus Heimioporusf 223 a itipellis) to chemicals, typically KOH, 5 % ammonia solution, 159 Xanthoconium Heliogaster 224 160 Gyrodontoideae Hemileccinum and FeSO4. An overview of the presence, absence, and states 225 161 Gyrodon Leccinellum of key morphological characters of the genera of Boletineae is 226 162 Paragyrodon Leccinum presented in Table 2. Chemical analysis of pigment produc- 227 f 163 Meiogranum Mycoamaranthus tion in the boletes has also been used as a taxonomic charac- 228 Strobilomyceloideae Notholepiota 164 ter and allowed the placement of species not previously 229 Strobilomyces Paxillogasterf 165 Chamonixia 230 Xerocomoideae Phylloboletellus thought to be closely related to the boletes, e.g. 166 231 Phylloporus Phyllobolites (Boletineae) and Coniophora (Coniophorineae), and further 167 232 Tubosaeta Phylloporus strengthened the separation of Suillus (Suillineae) from Boletus 168 a 233 Xerocomus Porphyrellus (Steglich et al. 1977; Besl et al. 1986; Besl & Bresinsky 1997). 169 234 Paxillaceae Pseudoboletus Smith & Thiers (1971) placed only poroid fungi in the Bole- 170 235 Paxillus Pulveroboletus taceae and included members of the modern Suillineae and Scle- 171 Retiboletus 236 rodermatineae (Besl & Bresinsky 1997; Jarosch 2001; Binder & 172 Rhodactina 237 173 Rossbeevera Bresinsky 2002a). The modern members of the Boletineae 238 174 Royoungia (Table 1) that are gilled, Paxillus and Phylloporus, were placed 239 175 Rubinoboletus in the Paxillaceae (Smith & Thiers 1971). Singer’s (1986) concept 240 176 Sinoboletusf of the Boletineae is almost identical to the modern Boletales 241 177 Spongiforma (Singer 1986; Binder & Hibbett 2006). The modern Boletineae 242 Strobilomyces 178 members are distributed in the Paxillaceae and Boletaceae in 243 179 Sutorius 244 Singer’s (1986) classification. However, only one genus of 180 Tubosaeta 245 Singer’s Paxillaceae, Paxillus s.str. (not including Tapinella or 181 Tylopilus 246 Austropaxillus [Tapinellineae]), is included in the modern Boleti- 182 Xanthoconium 247 183 Xerocomellus neae (Binder & Hibbett 2006). 248 184 Xerocomus Smith and Thiers were more conservative than Singer 249 Zangia 185 when considering whether differences between morphologi- 250 Paxillaceae 186 cal features warranted a separate genus (Smith & Thiers 251 Alpova 187 252 Austrogasterf 1971; Singer 1986). This led Smith and Thiers to ‘lump’ species 188 253 Gyrodon into larger genera than those recognized by Singer, except for 189 Hoehnelogasterf the genus Leccinum (Smith & Thiers 1971; Singer 1986). Overall, 254 190 Meiorganumf Singer recognized 22 genera (not including families that have 255 191 256 Melanogaster no modern representatives) of Boletineae and Smith and Thiers 192 257 Paragyrodon recognized 12 genera (including genera in the modern Boleti- 193 Paxillus 258 194 neae that Smith and Thiers placed outside the Boletaceae), in- 259 195 cluding Suillus (Table 1). However, Smith and Thiers placed 260 Paragyrodon as a section of Suillus and stated that Gyrodon

Phylogenetic overview of the Boletineae 3 Q1

261 lividus was most closely related to Suillus; in fact, both Paragyr- 326 262 odon and G. lividus are members of the modern Boletineae (Pax- Materials and methods 327 263 328 illineae in Binder & Hibbett 2006) (see Table 1; Smith & Thiers 264 329 1971; Singer 1986; Binder & Hibbett 2006). Taxon samplingdA taxon sampling scheme was designed 265 330 Some aspects of generic limits and inter-generic relation- based on a preliminary analysis of 457 nuc-lsu sequences 266 331 267 ships in Boletineae have remained unclear. This is due, in representing 40 genera and 247 species (Binder & Hibbett 332 268 part, to alternative interpretations of relatedness based on 2006), including 42 unnamed species. The nuc-lsu sequences 333 269 morphology (as well as lack of resolution in molecular stud- were aligned manually using the PAUP editor and analyzed 334 270 ies). An example is provided by Boletus, which Smith and in the RAxML Blackbox server using the default settings with 335 271 Thiers regarded as ‘the most distinctive genus in the family’. maximum likelihood (ML) optimization (http://phylobench.vi- 336 272 However, Smith and Thiers’s concept of Boletus encompasses tal-it.ch/raxml-bb/; Stamatakis et al. 2008; Swofford 2002). 337 273 multiple genera recognized by Singer, e.g. Aureoboletus, Xeroco- Based on the resulting phylogenetic tree (not shown) a sam- 338 274 mus, Xanthoconium (Smith & Thiers 1971; Singer 1986). pling scheme was designed, including 93 individuals for 339 275 340 Analyses of chemical characters (pigments and colourless which genomic DNA was available, that represents, as broadly 276 341 compounds that are produced via secondary metabolisms) as possible, the phylogenetic diversity of the Boletineae as rep- 277 342 resented in that tree. Amplification of tef1 and RPB1 genes was 278 have been useful for separating large groups, or genera, but 343 279 overall have been unable to resolve intergeneric relation- attempted using the 93 selected gDNAs. Forty-seven new tef1, 344 280 ships among the Boletineae (Besl & Bresinsky 1977, 1979, forty new RPB1 sequences, and seven new nuc-lsu sequences 345 281 1997; Besl et al. 1986; Besl et al. 1974; Binder & Hibbett 2006; were generated. An additional 28 previously unpublished nuc- 346 282 Bresinsky & Besl 1978; Steglich et al. 1977). Analyses of DNA lsu sequences were used. Q3 347 283 sequences have also been informative, but the molecular Single-gene analyses were conducted to assess variability 348 284 studies of the last 15 y have failed to resolve generic relation- of the intron regions in tef1 and RPB1, and conflict among in- 349 285 ships in the Boletineae and have often focused on only one or dividual genes using bootstrapped RAxML searches (results 350 286 351 a few genera at a time (Binder & Bresinsky 2002b; Binder & not shown). The intron regions of tef1 and RPB1 were analyzed 287 352 Hibbett 2002; Desjardin et al. 2008, 2009; Dentinger et al. by eye and deemed conserved enough to warrant inclusion in 288 353 2010; Vizzini et al. 2010; Lebel et al. 2011; Halling et al. the analyses, and no well-supported conflict was detected be- 289 354 290 2012a). As more taxa have been sampled in molecular stud- tween topologies of the three genes. The nuc-lsu, tef1, and 355 291 ies, it has become clear that some morphology-based generic RPB1 genes were then combined into two multi-gene datasets: 356 292 concepts do not correspond to monophyletic groups, espe- a ‘core’ dataset containing 48 species, each of which is repre- 357 293 cially in the larger genera, such as Boletellus, Boletus, Tylopilus, sented by all three genes, and an ‘extended core’ dataset, con- 358 294 Xerocomellus,andXerocomus, but there has been no compre- taining all of the sequences from the core dataset, plus 31 359 295 hensive phylogenetic analysis of all the genera of Boletineae. species represented by nuc-lsu and tef1 sequences. Finally, 360 296 Instead, many studies have focused on finding limits of indi- a constrained analysis of 370 nuc-lsu sequences (a subset of 361 297 vidual genera or placing species into genera (Binder & Besl those used in the preliminary analysis), containing 249 se- 362 298 363 2000; Binder & Bresinsky 2002b; Desjardin et al. 2008, 2009, quences publicly available from GenBank and 96 unpublished 299 364 2011; Dentinger et al. 2010; Li et al. 2011; Halling et al. 2012a, sequences, was conducted using the ML topology from the ex- 300 365 tended core dataset as the backbone constraint because the 301 b; Hosen et al. 2012). 366 302 Most previous molecular studies used the nuclear large extended core dataset contains more species and provided 367 303 subunit (nuc-lsu), which has been shown to have little resolv- better resolution. 368 304 ing power in the Boletineae when used in isolation (Binder & 369 305 Hibbett 2006). Continued use of nuc-lsu maintains compatibil- PCR amplification and sequencing 370 306 ity between studies and allows placement of species into gen- 371 307 era or cryptic forms into a family (e.g. truffle-like species), but PCR primer pair EF1-983FdEF1-2218R (Rehner & Buckley 2005) 372 308 additional loci are needed to resolve higher-level relationships was used for amplification of the tef1 fragment (approx 373 309 374 in Boletineae (Binder & Besl 2000; Binder & Bresinsky 2002b; 900e1200 bp). A new Boletineae specific (with the exception of 310 375 Peintner et al. 2003; Binder & Hibbett 2006; Desjardin et al. Chalciporus species, which have a divergent sequence at this 311 376 2008, 2009; Drehmel et al. 2008; Binder et al. 2010; Dentinger region) external, forward RPB1 primer, RPB1-Bf 50-GAATGYA- 312 0 377 313 et al. 2010; Vizzini et al. 2010; Lebel et al. 2011; Li et al. 2011; TATGYGTWAACTGTGG-3 was designed for this study. This 378 314 Halling et al. 2012a, b). Here, we present a broad phylogenetic primer was designed to bind upstream of intron A, as an alter- 379 315 analysis of all the major genera of the Boletineae, using three native to RPB1-Af. RPB1-Bf was paired with RPB1-Cr (Matheny 380 316 genes: nuclear large subunit ribosomal RNA (nuc-lsu), transla- et al. 2002) for amplification of the RPB1 fragment (approx 381 317 tion elongation factor 1-alpha (tef1), and DNA directed RNA 900e1200 bp). 382 318 polymerase II largest subunit (RPB1). Tef1 and RPB1 were cho- A touchdown PCR protocol was used to amplify tef1 and 383 319 sen based on the potential they have been shown to have to RPB1. The following protocols are for tef1: (1) initial dena- 384 320 resolve genera in the Boletineae in previous studies (Binder turation 94 C for 2 min, (2) denaturation 94 C for 40 s, (3) 385 321 386 et al. 2010; Dentinger et al. 2010; Li et al. 2011; Halling et al. 60 C for 40 s, minus 1 C per cycle, (4) 72 C for 1 min, (5) 322 387 2012a, b; Hosen et al. 2012). The goal of the present study repeat from step 2 for nine cycles, (6) 94 C for 45 s, (7) 323 388 324 was to develop a phylogenetic framework to guide further 50 C for 1 min 10 s, (8) 72 C for 1 min 30 s, (9) repeat 389 325 exemplar-based sampling and an eventual generic-level from step 6 for 36 cycles, (10) 72 C for 10 min. For RPB1 390 reclassification of the Boletineae. PCR protocol see Justo & Hibbett (2011).Forsequencing,

4 M. E. Nuhn et al.

391 456 e 392 Table 2 Morphological features of selected Boletineae genera. Q7 457 393 Genera Spore ornamentation Stipe ornamentation Veil 458 394 459 395 Smooth Bacil- Longitu- Other Smooth Longitu- Reticu- Scabr- Other None Partial Double 460 396 late ninally dinally late ous 461 ridged striated 397 462 398 Boletaceae 463 399 Afroboletus BB C BB B B BC C B 464 400 Aureoboletus CB B BC B B BBCC** B 465 401 Austroboletus BB C BC C B BBCBB 466 402 Boletellus CB C CC B C BBCCB 467 403 Boletus CB B BC C C BBCBB 468 404 Bothia CB B BC B C BBCBB 469 405 Buchwaldoboletus CB B BC B B BBCBB 470 406 Chalciporus CB B BC B B BBCBB 471 CB B BC C C BBCB B 407 Gastroboletus * 472 CB B BB B B BCCBB 408 Hemileccinum 473 CB B BB B B CBCBB 409 Leccinellum 474 Leccinum CB B BB B B CBCBB 410 475 Notholepiota CB B BC B B BBCBB 411 476 Phylloporus CC B BC C B BBCBB 412 477 Porphyrellus CB B BC C B BBCBB 413 478 Pseudoboletus CB B BC B B BCCBB 414 Retiboletus CB B BB B C BBCBB 479 415 Royoungia CB B BB B B BBCBB 480 416 Spongiforma CB B BB B B BBCBB 481 417 Strobilomyces BB B CB B B BCBCB 482 418 Tylopilus CB B BC B C BBCBB 483 419 Xanthoconium CB B BC B C BBCBB 484 420 Xerocomellus CB B BC C B BBCBB 485 421 Xerocomus CC B BC C B BBCBB 486 422 487 423 Paxillaceae 488 424 Paragyrodon CB B BB B B BBBCC 489 425 Paxillus CB B BB B B BBCBB 490 426 Gyrodon CB B BB B B BBCBB 491 427 492 428 Incertae sedis 493 CB B BB B B BBBBB 429 Hydnomerulius 494 430 495 431 C e Feature present in at least one species in genus. 496 432 B e Feature absent in all species. 497 433 ? e unknown. 498 434 499 435 500 436 501 437 two additional internal tef1 primers; EF1-1577F and EF1- raw sequences were processed using Sequencher 4.7 (Gen- 502 438 1567F, and a different terminal reverse primer, EF1-2212R eCodes, Ann Arbor, Michigan). Expected fragment length 503 439 504 were used (Rehner & Buckley 2005). An additional four in- and alternative primers for all genes can be found at

Phylogenetic overview of the Boletineae 5

521 586 522 587 Hymeno- Fruiting body morphology Hypo- Clamp Cauloba- Caulo- Cystida 523 588 phore geal connec- sidium hymenium 524 589 tions 525 Lamel- Poroid Pileate- Seco- Gas- Resu- Caulo- Cheilo- Chryso- Pleuro- 590 526 late stipitate tioid teroid pinate cystida cystida cystidia cystida 591 Regularly Irregularly 527 592 poroid poroid 528 593 529 594 530 BC B CBBBBBB B BCBC 595 531 BC B CBBBBBB B BCCC 596 532 BC B CBBBBBB B BCBC 597 533 BC B CBBBBCB B BCBC 598 534 BC B CBBBBBB B BCBC 599 535 BC B CBBBBBB B BCBC 600 536 BC B CBBBBBB B BCBC 601 CB B CBBBBBB B BCBC 537 602 BC C BCBBBBB B BCBC 538 603 BC B CBBBBBC C B BC 539 604 BB B CBBBBBC C CCBC 540 605 BC B CBBBBBC C CCBC 541 606 BB C BCBBBBB B ?? ? ? 542 CB B CBBBBBB B BCCC 607 543 BC B CBBBBBB B BCBC 608 544 BC B CBBBBBB B BBBC 609 545 BC B CBBBBBB B BCCC 610 546 BB B BBCBCBB B BBBB 611 547 BB B BBCBBBB B Cystidia Present 612 548 BC B CBBBBBB B C C 613 549 CCBBBBBCCCCCC614 550 BC B CBBBBBB B C CC 615 551 BC C CBBBBBC C CCCC 616 552 BC C CBBBBBC C CCCC 617 553 618 554 619 555 BB C CBBBBCB B CCBC 620 556 CB B CBBBBCB B BCBC 621 BB C CBBBBCB B BCBC 557 622 558 623 559 624 BB B BBBCBCB B Cystidioles 560 625 561 626 562 627 563 628 564 Lartillot & Philippe 2006; Lartillot et al. 2007) for Bayesian anal- 629 565 ysis (BY). Alignments have been deposited in TreeBase Results 630 566 (# 14219). 631 567 ML analyses were run using ML optimization, 1000 boot- DNA extractions, PCR and sequencing 632 568 strap (BS) replicates, and the GTRCAT and gamma model. 633 569 634 MP analyses were performed using parameters published by Tef1, RPB1, and nuc-lsu had 68 %, 42 %, and 100 % success rate 570 635 Justo & Hibbett (2011). BY analysis was done with four for sequencing respectively. The low success rate for RPB1 am- 571 636 MCMC, chains using the CAT-GTR settings. BY analyses was pliﬁcation is probably due to primer mismatches. 572 637 573 run until convergence, indicated by a max difference output 638 574 of the ‘bpcomp’ command of less than 0.1. Nodes were consid- Alignment and phylogenetic analysis 639 575 ered well supported if the BS value was greater than 70 % for 640 576 ML and MP, and posterior probability (PP) for BY was greater See Table 3 for technical output of the nuc-lsu, tef1, and RPB1 641 577 than 0.95. ML core and extended analyses, the core and extended MP 642 578 For the constrained analysis, 380 nuc-lsu sequences were analyses, and the constrained analysis. Following single- 643 579 aligned using the G-INS-i strategy on MAFFT server (http:// gene analyses to assess conﬂict, the core nuc-lsu, tef1, and 644 580 mafft.cbrc.jp/alignment/server/). Ten duplicate sequences RPB1 alignments were merged, as were the extended nuc- 645 581 646 were removed, resulting in a 370 taxon matrix. The alignment lsu, tef1, and RPB1 datasets. The majority of nuc-lsu sequences 582 647 was analyzed using the extended dataset ML tree topology as were generated with the primer set LR0RdLR5; however, 583 648 584 the backbone. Constrained analysis was performed using some nuc-lsu sequences used were generated using 649 d 585 RAxML with ML optimization, 500 BS replicates, and the LR0R LR7 resulting in longer sequences, by approximately 650 GTRCAT and gamma model. 800 bp. These longer sequences were left untrimmed. Thus,

6 M. E. Nuhn et al.

651 716 e d 652 Table 3 GenBank accession numbers Missing. Q8 717 653 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 718 654 Identiﬁer 719 655 720 e 656 Afroboletus luteolus 00-436 Africa D. Arora KF030238 KF030397 KF030392 721 Aureoboletus Pug1 Maindreieck, Germany 3-Oct-95 J. Schreiner DQ534635 KF030399 e 657 722 gentilis 658 723 Aureoboletus MB 03-104 Lincoln, MA, USA Aug-03 M. Binder KF030239 KF030400 e 659 724 innixus 660 725 Aureoboletus Xle1 Maindreieck, Germany 19-Spt-98 J. Schreiner DQ534641 KF030403 e 661 moravicus 726 662 Aureoboletus DS626-07 Chestnut Ridge Park, NY, USA 25-Oct-06 E. Both KF030311 KF030402 KF030381 727 663 roxanae 728 664 Aureoboletus AFTOL-450 Kunming, Yunnan, China e Z.-L. Yang AY700189 DQ029199 DQ435800 729 665 thibetanus 730 666 Austroboletus 112/96 MA, USA 30-Aug-96 N. Arnold DQ534624 KF030425 KF030358 731 667 gracilis 732 668 Boletellus 3838 Towy Park, North Collins, NY 10-Oct-95 E. Both KF030312 KF030432 KF030383 733 669 chrysenteroides 734 670 Boletellus AFTOL-713 Cape Cod, MA, USA 14-Sep-03 M. Binder AY684158 AY879116 AY662660 735 671 projectellus 736 672 Boletellus AFTOL-532 Yunnan, China 2003 L. Wang AY647211 DQ408145 e 737 673 shichianus 738 674 Boletinellus AFTOL-575 Rock House, MA, USA 29-Aug-02 M. binder AY684153 DQ056287 DQ435803 739 675 merulioides 740 676 Boletus 4588 Cape San Blas, FL, USA 1-Apr-05 E. Both KF030302 KF030401 KF030388 741 abruptibulbus 677 742 Boletus aereus REH8721 Redwood National Park, Humbolt 17-Nov-05 R.E. Halling KF030339 KF030426 KF030377 678 743 Co., CA, USA 679 744 Boletus 112605ba Mendocino Co., CA, USA 26-Nov-05 B. Neill JQ326996 JQ327024 KF030360 680 745 amygdalinus 681 Boletus Bap1 Bavaria, Germany 10-Aug-95 J. Schreiner AF456837 JQ327025 KF030359 746 682 appendiculatus 747 683 Boletus bicolor var MB 07-001 Chestnut Ridge Park, NY, USA 28-Jul-95 E. Both KF030370 KF030405 KF030370 748 684 bicolor 749 685 Boletus bicolor var 2858 Erie Co., NY, USA e E. Both JQ326998 JQ327021 e 750 686 borealis 751 687 Boletus calopus Bc1 Bavaria, Germany 7-Sep-94 N. Arnold AF456833 JQ327019 e 752 688 Boletus carminipes MB 06-061 Erie Co., NY, USA 4-Aug-06 M. Binder, JQ327001 JQ327022 KF030363 753 689 E. Both 754 690 Boletus dupainii JAM 0607 Butner, NY, USA 8-Mar-00 E. Both KF030413 KF030413 KF030361 755 691 Boletus edulis Be3 Bavaria, Germany 14-Sep-94 M. Binder KF030282 GU187682 GU187444 756 692 Boletus ﬁrmus MB 06-060 Chestnut Ridge Park, NY, USA 3-Aug-06 M. Binder KF030368 KF030408 KF030368 757 693 Boletus inedulis MB 06-044 Erie Co., NY, USA 3-Aug-06 M. Binder, JQ327013 JQ327020 KF030362 758 694 E. Both 759 e e 695 Boletus luridiformis AT2001087 Berkshire, England, UK A. F. S. Taylor JQ326995 JQ327023 760 e 696 Boletus morrisii 8206 Concord, MA, USA 2-Aug-06 B. Neill KF030433 KF030433 761 Boletus pallidus 179/97 Bavaria, Germany ee AF457409 KF030424 KF030396 697 762 Boletus peckii 3959 Erie Co., NY, USA 4-Aug-95 A. R. Clark, JQ326999 JQ327026 e 698 763 E. Both 699 764 Boletus DS615-07 Chestnut Ridge Park, NY, USA 7-Jul-95 E. Both KF030257 KF030407 e 700 765 pseudosensibilis 701 Boletus pulchriceps DS 4514 Chiricahua Mnts, AZ, USA 1-Aug-91 e KF030261 KF030409 KF030376 766 702 Boletus 9606 West Newton, MA, USA 9-Jun-06 Bill Neill KF030313 KF030418 KF030364 767 703 pulverulentus 768 704 Boletus regius 11 265 Mendocino Co., CA, USA 26-Nov-05 Bill Neill KF030411 KF030411 e 769 705 Boletus 4252 Chestnut Ridge Park, NY, USA 12-Jul-98 E. Both KF030252 KF030412 e 770 706 rhodosanguineus 771 707 Boletus MB 06-059 Chestnut Ridge Park, NY, USA 30-Jul-06 A. Taylor, KF030262 KF030410 KF030372 772 708 roseopurpureus M. Binder 773 709 Boletus 4414 Chestnut Ridge Park, NY, USA 6-Aug-97 E. Both KF030248 KF030406 KF030369 774 710 rufomaculatus 775 711 Boletus PBM 3076 Arataki Visitor Center, Auckland, 6-May-09 P.B. Matheny KF030352 KF030430 KF030384 776 712 semigastroideus New Zealand 777 713 Boletus separans DPL 2704 Texas 2000 D. Lewis KF030329 KF030431 KF030385 778 ee 714 Boletus subalpinus 27 882 J. Trappe KF030340 KF030427 KF030379 779 715 780

Phylogenetic overview of the Boletineae 7

781 846 e 782 Table 3 (continued) 847 783 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 848 784 Identifier 849 785 850 Boletus 72 206 Jefferson, NH 22-Jul-06 B. Neill KF030303 KF030404 KF030374 786 851 subglabripes 787 852 Boletus tenax REH6871 New York Botanical Garden, 25-Jul-95 R.E. Halling KF030320 KF030437 e 788 853 NY, USA 789 854 Boletus variipes var 4249 Cheboygan Co., MI, USA 10-Aug-68 A. H. Smith JQ327014 JQ327017 KF030378 790 fagicola 855 791 Bothia castanella MB 03-053 Massachussetts 25-Jun-05 M. Binder DQ867117 KF030421 KF030382 856 792 Buchwaldoboletus Pul1 Maindreieck, Germany 9-Sep-95 J. Schreiner JQ326997 JQ327040 e 857 793 lignicola 858 794 Chalciporus rubinus DS4640-3 Germany e J. Schreiner KF030283 KF030440 e 859 795 Chalciporus MB 04-001 Rutland, MA, USA 28-Sep-04 M. Binder DQ534648 GU187690 GU187453 860 796 piperatus 861 797 Chalciporus 4302 Deer Meadows, Tulet, NY 14-Sep-98 E. Both KF030284 KF030441 e 862 798 pseudorubinellus 863 799 Gyrodon lividus Gl1 Bavaria, Germany 11-Aug-95 H. Besl, AF098378 GU187701 GU187461 864 800 W. Helfer 865 801 Hemileccinum Bim1 Bavaria, Germany 21-Sep-95 J. Schreiner AF139715 JQ327034 KF030375 866 802 impolitum 867 e 803 Hydnomerulius CFMR:MD312 USDA Forest Products 13-Nov-19 GU187580 GU187708 GU187462 868 804 pinastri Laboratory 869 805 Leccinellum corsicum Buf 4507 KF030347 KF030435 KF030389 870 Leccinellum 930809/1 France 9-Aug-93 G. Lannoy AF139694 KF030434 e 806 871 crocipodium 807 872 Leccinum albellum MB 06-040 Erie Co., NY, USA 30-Jul-06 M. Binder JQ327007 JQ327038 e 808 873 Leccinum scabrum Ls1 Austria 14-Sep-95 M. Binder AF139705 JQ327039 e 809 874 Paragyrodon MB 06-066 Iowa City, IA, USA 22-Spt-06 M. Binder GU187593 GU187737 e 810 sphaerosporus 875 811 Paxillus Pf1 Bavaria, Germany 21-Aug-95 L. Krieglsteiner AF167680 GU187736 e 876 812 filamentosus 877 813 Paxillus Po1 Bavaria, Germany 21-Aug-98 Ch. Hahn AY177256 KF030442 e 878 814 obscurisporus 879 815 Paxillus vernalis Pv2 Canada 31-Aug-97 T. Lohmeyer AY645059 DQ457629 e 880 816 Phylloporus pelletieri Pp1 Bavaria, Germany 9-Sep-95 M. Kronfeldner AF456818 JQ327036 KF030390 881 817 Porphyrellus MB 97-023 Bavaria, Germany 9-Sep-96 M. Beisenherz DQ534643 GU187734 GU187475 882 818 porphyrosporus 883 819 Pseudoboletus Xpa1 Bavaria, Germany 9-Jul-95 A. Bresinksy AF050646 KF030443 KF030394 884 820 parasiticus 885 821 Retiboletus griseus 202/97 MA, USA 30-Aug-97 N. Arnold, AF456834 KF030414 KF030373 886 822 W. Helfer, 887 823 W. Steglich 888 e 824 Royoungia boletoides 27 546 New South Wales, Australia 25-Oct-99 J. Trappe JX889655 JX889696 889 e 825 Royoungia boletoides AWC4137 Victoria, Australia 28-May-01 T. Lebel et al. DQ534663 JX889700 890 Spongiforma DED 7873 Thailand 7-Jul-05 D.E. Desjardin EU685108 KF030436 KF030387 826 891 thailandica 827 892 Strobilomyces Sf1 Bavaria, Germany 12-Aug-95 J. Enzmann DQ534626 JQ327037 AY858963 828 893 floccopus 829 894 Strobilomyces sp REH8514 Cayo District, Belize 3-Oct-03 R.E. Halling EU685109 KF030398 e 830 Tylopilus badiceps 78 206 Holliston, MA, USA 28-Jul-06 KF030335 KF030429 e 895 831 Tylopilus felleus AT2001011 Stadsskogen, Uppsala, Sweden 17-Sep-01 A. F. S. Taylor JQ326993 JQ327015 KF030380 896 832 Tylopilus MB 06-053 Erie Co., NY, USA 3-Aug-06 E. Both JQ326994 JQ327016 e 897 833 ferrugineus 898 834 Tylopilus MB 06-056 Chestnut Ridge Park, NY, USA 4-Aug-06 E. Both KF030350 KF030439 KF030395 899 835 plumbeoviolaceus 900 836 Xanthoconium 3518 Lake Mize, Gainsville, FL, USA 10-Aug-92 E. Both KF030353 KF030428 KF030386 901 837 stramineum 902 838 Xerocomellus Xch1 Bavaria, Germany 8-Aug-95 M. Binder AF050647 KF030415 KF030365 903 839 chrysenteron 904 840 Xerocomellus PBM 1331 West Coast, USA e P.B. Matheny KF030297 KF030420 e 905 841 rubellus cf West 906 842 Coast 907 843 (continued on next page) 908 844 909 845 910

8 M. E. Nuhn et al.

911 976 e 912 Table 3 (continued) 977 913 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 978 914 Identifier 979 915 980 Xerocomellus MB 03-033 Worcester, MA, USA 23-Jul-03 M. Binder KF030294 KF030419 KF030371 916 981 rubellus cf East 917 982 Coast 918 983 Xerocomellus zelleri REH8724 Redwood National Park, 18-Nov-05 R.E. Halling KF030271 KF030416 KF030366 919 984 Humbolt Co., CA, USA 920 Xerocomus badius MB 03-098a Rutland, MA, USA 19-Aug-03 M. Binder KF030355 KF030423 KF030393 985 921 Xerocomus badius Xb2 Bavaria, Germany Sep-95 A. Bresinksy KF030357 KF030422 e 986 922 Xerocomus AT2005034 Upsala, Uppland, Finland 30-Jul-05 A.F. S. Taylor KF030354 KF030417 KF030367 987 923 cisalpinus 988 924 Xerocomus perplexus MB 00-005 Rutland, MA, USA 5-Aug-00 M. Binder JQ003702 KF030438 e 989 925 Xerocomus Xs1 Bavaria, Germany 10-Aug-95 J. Enzmann, AF139716 JQ327035 KF030391 990 926 subtomentosus A. Bresinsky 991 927 992 928 Constrained data set taxa 993 929 Alpova diplophloeus 17685 eeJ. Trappe AF071454 994 930 Alpova diplophloeus Adi1 Canada Sep-87 W. Steglich, AF352035 995 931 B. Sfeffan 996 932 Alpova trappei 28 042 eeJ. Trappe KF030307 997 ee 933 Alpova trappei 16 394 J. Trappe AF071456 998 934 Aureoboletus 35/94 MA, USA 28-Aug-97 H. Besl DQ534636 999 935 auriporus 1000 Aureoboletus DD971 Orange Co, NC, USA ee AY612819 936 1001 auriporus 937 1002 Aureoboletus REH8719 Humbolt Co, CA, USA 16-Nov-05 R.E. Halling KF030298 938 1003 citrinoporus 939 1004 Auroboletus innixus 136 eeeKF030240 940 Aureoboletus DD972 Orange Co, NC, USA ee AY612805 1005 941 viridiflavus 1006 942 Austroboletus DPL7541 eeeKF030351 1007 943 flavidus 1008 944 Austroboletus TH6300 Pakaraima Mnts, Guyana ee AY612798 1009 945 mucosus 1010 946 Austroboletus M312 New Zealand 10-May-95 A. Bresinksy DQ534622 1011 947 niveus 1012 948 Austroboletus M50 New Zealand 17-Mar-95 A. Bresinksy DQ534623 1013 949 novaezealandicae 1014 950 Boletellus ananas TH8819 Guyana e T.W. Henkel HQ161853 1015 951 Boletellus ananas TH6264 Pakaraima Mnts, Guyana e T.W. Henkel AY612799 1016 952 Boletellus cf 00-335 Africa 2000 D. Arora KF030330 1017 953 ananiceps 1018 954 Boletellus betula 134/96 MA, USA 31-Aug-96 N. Arnold AF050642 1019 ee 955 Boletellus betula DD9852 Orange Co, NC, USA AY612797 1020 Boletellus 54/97 MA, USA 21-Aug-97 N. Arnold, DQ534634 956 1021 chrysenteroides H. Besl 957 1022 Boletellus TH8840 Guyana e T.W. Henkel HQ161852 958 1023 dicymbophilus 959 1024 Boletellus mirabilis CBS 136.60 eeeAF050652 960 Boletellus mirabilis REH8717 Big Lagoon Park, Humbolt Co, 15-Nov-05 R.E. Halling KF030299 1025 961 CA, USA 1026 962 Boletellus piakaii TH8077 Guyana e T.W. Henkel HQ161861 1027 963 Boletellus projectellus sn2 Hor eeeKF030300 1028 964 Boletellus russellii 12/96 MA, USA 6-Aug-96 N. Arnold AF050651 1029 965 Boletellus russellii DPL6698 Texas 2000 D. Lewis KF030325 1030 966 Boletellus sp TAA195080 Valle de Mai, Praslin, ee AM412293 1031 967 Seychelles 1032 968 Boletineae sp EcM L2481_Bol5 L’Abondance, Mahe, ee AM412263 1033 969 Vateriopsis Seychelles 1034 970 Boletus abieticola 26 763 eeJ. Trappe KF030268 1035 971 Boletus appendiculatus REH8720 Shasta/Trinity National 15-Nov-05 R.E. Halling KF030269 1036 972 Forest, Trinity Co, CA, USA 1037 973 Boletus atkinsonianus 4471 Bigelow Hollow State Park, 18-Aug-00 B. Neill, E. Both KF030241 1038 974 Conn, USA 1039 975 1040

Phylogenetic overview of the Boletineae 9

1041 1106 e 1042 Table 3 (continued) 1107 1043 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1108 1044 Identifier 1109 1045 1110 Boletus atkinsonianus 4557 Tucker Co, WV, USA 28-Aug-00 W.C. Woody, KF030242 1046 1111 E. Both 1047 1112 Boletus aurantioruber MEN11-0 MB Cape Cod, MA, USA M. Binder KF030342 1048 1113 Boletus bicolor TH6933 Watuaga Co., NC, USA ee AY612800 1049 1114 Boletus bicolor snOH Ohio, USA e D. Lewis KF030258 1050 Boletus bicolor MB 06-037 Chestnut Ridge Park, NY, USA 3-Aug-06 M. Binder KF030250 1115 1051 Boletus bicolor var 3818 North Collins Town Park, NY, 10-Aug-95 E. Both KF030247 1116 1052 subreticulatus USA 1117 1053 Boletus billieae 4558 Well Fleet, Cape Cod, MA, USA 25-Aug-01 B. Neill, E. Both KF030265 1118 1054 Boletus calopus 112606 Mendocino Co., CA, USA 26-Nov-05 B. Neill KF030279 1119 1055 Boletus campestris 5/96 Eastabrook Woods, MA, USA 9-Aug-96 N. Arnold DQ534640 1120 1056 Boletus carminipes 4591 Berea, Cuyalioga Co, OH, USA 22-Jul-01 P. Monk, E. Both KF030259 1121 1057 Boletus cf edulis e Turkey ee AF462355 1122 1058 Boletus cf edulis 197/84 China ee AF462357 1123 1059 Boletus cf luridus MAN 011 Costa Rica 1-Jun-04 M.A. Neves KF030288 1124 1060 Boletus coniferarum 7/94 CA, USA Oct-94 W. Steglich AF456827 1125 1061 Boletus eastwoodiae AT2003096 eeA.F. S. Taylor KF030253 1126 1062 Boletus eastwoodiae TDB-1000C Marin Co., CA, USA 28-Nov-87 T. D. Bruns AF071528 1127 eee 1063 Boletus edulis OSC FAC 1615 AF071457 1128 ee 1064 Boletus edulis No. 15 Yugoslavia AF462356 1129 ee 1065 Boletus edulis HN141 Watuaga Co., NC, USA AY612802 1130 Boletus edulis FO 46874 Germany ee AF291300 1066 1131 Boletus erthyropus 3131 Raquette Lake, NY, USA 21-Jul-98 E. Both KF030291 1067 1132 Boletus erythropus Ber1 Bavaria, Germany 1-Oct-95 J. Enzmann, AF139683 1068 1133 H. Besl 1069 1134 Boletus fechtneri AT2003097 eeA.F. S. Taylor KF030270 1070 Boletus fechtneri Bf1 Bavaria, Germany 1-Sep-94 M. Beisenherz AF456821 1135 1071 Boletus fibrillosus PBM1342 WA, USA e P.B. Matheny KF030343 1136 1072 Boletus fibrillosus Thiers 6995 Jackson Forest, Mendocino Co, 21-Nov-92 R.E. Halling, KF030344 1137 1073 CA, USA E. Both 1138 1074 Boletus floridanus BD368 Parque Nacional de Guanacaste, e B. Dentinger HQ161859 1139 1075 Area de Conservacion Guanacaste, 1140 1076 Costa Rica 1141 1077 Boletus gertrudiae 86/97 MA, USA 23-Aug-97 N. Arnold AF457407 1142 1078 Boletus glabellus 00-194 Boston Cemitary, Boston, MA, USA e B. Neill KF030318 1143 1079 Boletus gyrodontoides MS5 Malaysia 8-Apr-91 N. Arnold, DQ534651 1144 1080 H. Besl 1145 1081 Boletus hypocarcinus DPL6899 USA e D. Lewis KF030260 1146 1082 Boletus inedulis NCJ14 Orange Co, NC, USA e D. Dremel AY612803 1147 e 1083 Boletus inedulis sn2002 NY, USA E. Both KF030281 1148 1084 Boletus junquilleus Bju1 Keilberg-Waldmichbach, 9-Nov-96 J. Schreiner DQ534645 1149 1085 Salzbuckel, Germany 1150 Boletus leptospermi M23 New Zealand 15-Mar-95 A. Bresinksy DQ534632 1086 1151 Boletus longicurvipes 8/97 USA 14-Aug-97 N. Arnold AF139688 1087 1152 Boletus longicurvipes TH6944 Watuaga Co., NC, USA ee AY612812 1088 1153 Boletus luridus Bl2 Bavaria, Germany 14-Aug-95 M. Beisenherz AF139686 1089 1154 Boletus luteocupreus Blu1 Karlburg, Hagwald, Germany 8-Aug-95 J. Schreiner DQ534657 1090 Boletus minato- 4091 Chestnut Ridge Park, NY, USA 26-Jun-97 E. Both KF030243 1155 1091 olivaceus 1156 1092 Boletus modestus 229/97 MA, USA 1-Sep-97 N. Arnold DQ534659 1157 1093 Boletus nobilis BD239 (MIN) Duke Forest, Durham, NC, USA ee EU232002 1158 1094 Boletus oliveisporus DPL6823 Texas 2000 D. Lewis KF030254 1159 1095 Boletus pallidoroseus BD396 Pine Bend Bluff Scientific and e B. Dentinger HQ161860 1160 1096 Natural Area, MS, USA 1161 1097 Boletus pallidoroseus sn Hor Suffolk Co, NY, USA 2000 J. Horman KF030305 1162 1098 Boletus pinophilus No. 4 Bavaria, Germany 20-Jul-92 H. Besl AF462358 1163 1099 Boletus pinophilus 42/93 eeeAF462359 1164 110 0 Boletus pulcherrimus 00-291 Mendocino Co., CA, USA e A. Mohr, E. Both KF030256 1165 1101 Boletus quercophilus BDCR0417 (MIN) along Rio Savegre, San Gerardo ee EU232001 1166 1102 de Dota, San Jose, Costa Rica 1167 eee 1103 Boletus radicans Brad1 AF336241 1168 1104 Boletus regius Bre1 Bavaria, Germany 24-Aug-95 L. Krieglsteiner DQ534653 1169 1105 (continued on next page) 1170

10 M. E. Nuhn et al.

1171 1236 e 1172 Table 3 (continued) 1237 1173 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1238 1174 Identiﬁer 1239 1175 1240 Boletus rex-veris JFA13101 Chinook Pass, WA, USA ee EU232005 1176 1241 Boletus rhodopus 4590 Letchworth State Park, Wyoming 30-Jul-05 E. Both KF030289 1177 1242 Co, NY, USA 1178 1243 Boletus rhodoxanthus Brh1 USA Jul-94 W. Steglich DQ534647 1179 1244 Boletus roseipes 4515 Essex B. John’s Brook Lodge, NY, 8-Aug-93 E. Both KF030301 1180 USA 1245 1181 Boletus 3765 Chestnut Ridge Park, NY, USA 26-Jul-96 E. Both KF030264 1246 1182 roseopurpureus 1247 1183 Boletus 4497 Storrs, Uconn Campus, Conn, USA 10-Aug-96 E. Both KF030263 1248 1184 roseopurpureus 1249 1185 Boletus rubripes 8722 Redwood National Park, Humbolt 17-Nov-05 R.E. Halling, KF030280 1250 1186 Co., CA, USA E. Both 1251 1187 Boletus 3094 Auburn, Worcester Co, MA, USA 7-Aug-84 B. Both KF030266 1252 1188 rubroﬁbrillosus 1253 1189 Boletus rubropunctus MB 05-003 MA, USA e M. Binder KF030304 1254 1190 Boletus satanas Bs2 Germany e M. Binder AF336242 1255 1191 Boletus sensibilis var 3929 NTCPdd 8-Sep-91 E. Both KF030310 1256 1192 subviscidus 1257 1193 Boletus separans 183/86 USA 1986 W. Steglich AF457404 1258 1194 Boletus smithii 8727 Humbolt Co, CA, USA 19-Nov-05 R.E. Halling KF030244 1259 ee 1195 Boletus sp 4689 R.E. Halling KF030319 1260 Boletus sp 4696 eeR.E. Halling KF030331 1196 1261 Boletus sp 4698 eeR.E. Halling KF030324 1197 1262 Boletus sp cf bicolor 3921 Buffalo, NY, USA 29-Aug-95 E. Both KF030290 1198 1263 Boletus speciosus 13/96 Flints Pond, MA, USA 9-Aug-95 N. Arnold DQ534654 1199 1264 Boletus speciosus var 4258 Chestnut Ridge Park, NY, USA 15-Jul-98 E. Both KF030317 1200 brunneus 1265 1201 Boletus 4070 NCES, NY, USA 23-Sep-96 E. Both KF030341 1266 1202 subcaerulescens 1267 1203 Boletus subluridellus 3737 NCES, NY, USA 2-Jul-95 E. Both KF030249 1268 1204 Boletus subvelutipes RV98.102 Giles Co, VA, USA ee AY612804 1269 1205 Boletus tenax 6641 New York Botanical Garden, NY, Aug-91 R.E. Halling KF030321 1270 1206 USA 1271 1207 Boletus torosus Btor1 Garmisch, Bavaria, Germany 31-Jul-95 M. Beisenherz, DQ534466 1272 1208 L. Kieglsteiner, 1273 1209 J. Schreiner 1274 1210 Boletus variipes BD378 USA e B. Dentinger HQ161846 1275 1211 Boletus variipes BD245 (MIN) Duke Forest, Durham, NC, USA e B. Dentinger EU232003 1276 1212 Boletus vermiculosus 222/97 Flints Pond, MA, USA 1-Sep-97 N. Arnold DQ534646 1277 eee 1213 Boletus GMB501 AF457403 1278 1214 violaceofuscus 1279 e 1215 Chalciporus 8434 Germany J. Schreiner KF030285 1280 amarellus 1216 1281 Chalciporus Cam1 Bavaria, Germany 18-Sep-94 A. Bresinksy AF456835 1217 1282 amarellus 1218 1283 Chalciporus 27 620 Australia e J. Trappe DQ534652 1219 1284 ovalisporus 1220 Chalciporus Cp1 Bavaria, Germany 17-Sep-94 M. Binder AF336244 1285 1221 piperatus 1286 1222 Chalciporus NSL 15 New Zealand 20-Mar-95 A. Bresinksy DQ534464 1287 1223 piperatus 1288 1224 Chalciporus BN07 NH, USA 2005 B. Neill KF030286 1289 1225 pseudorubinellus 1290 1226 Chalciporus DS612 07 Rocky Pt. National Resources Mgmt. 30-Sep-02 J. Herman, KF030287 1291 1227 pseudorubinellus Area, Suffolk Co, NY, USA M. Herman, 1292 1228 E. Both 1293 1229 Chalciporus 191/81 ME, USA Aug-81 W. Steglich EU685106 1294 1230 rubinellus 1295 1231 Chamonixia 92/83 Bavaria, Germany 4-Oct-83 H. Besl AF336245 1296 1232 caespitosa 1297 ee 1233 Chamonixia sp Muroi361 J. Trappe DQ218598 1298 e 1234 Durianella AWW240 Selangor Provence, Malaysia A.W. Wilson EU293062 1299 1235 echinulata 1300

Phylogenetic overview of the Boletineae 11

1301 1366 e 1302 Table 3 (continued) 1367 1303 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1368 1304 Identiﬁer 1369 1305 1370 Fistulinella viscida 238 New Zealand 26-Apr-95 A. Bresinksy AF456826 1306 1371 Gastroboletus 19/95 WA, USA 18-Aug-95 N. Arnold, AF336248 1307 1372 turbinatus J. Ammirati 1308 1373 Gastroboletus 27 480 eeJ. Trappe KF030245 1309 1374 vividus 1310 Gyrodon lividus GlM1 Bavaria, Germany 16-Sep-98 A. Bresinksy AF167677 1375 1311 Gyrodon monticola 30/96 Colombia 27-Apr-96 Ch. Hahn AF352040 1376 1312 Harrya chromapes BD377 USA e B. Dentinger HQ161856 1377 1313 Harrya chromapes sn1 eeeAF139709 1378 1314 Harrya chromapes sn2 eeeAY612834 1379 1315 Heimioporus MS6 Malaysia 8-Apr-91 H. Besl AF050650 1380 1316 retispora 1381 1317 Heliogaster TNS-F-11696 Mt. Kuram, Kyoto Pref., Kyoto-shi, e T. Orihara EF183541 1382 1318 columellifera Sakyo-ku, Japan 1383 1319 Heliogaster TNS-F-11700 Aichi Pref., Okazaki-shi e T. Orihara EF183544 1384 1320 columellifera 1385 1321 Hemileccinum Bd1 Bavaria, Germany 11-Sep-94 A. Bresinksy AF139712 1386 1322 depilatum 1387 1323 Hydnomerulius 412 Canada 10-Oct-73 J.H. Ginns AF352044 1388 1324 pinastri 1389 1325 Hydnomerulius Z. Wang sn CA, USA 22-Aug-04 Z. Wang DQ534667 1390 pinastri 1326 1391 Leccinellum carpini 930808 France 8-Aug-93 G. Lannoy AF139691 1327 1392 Leccinellum 931101/1 GL France 1-Nov-93 G. Lannoy AF139693 1328 1393 corsicum 1329 1394 Leccinellum lepidum 110684 Italy 11-Jun-84 H. Besl AF139698 1330 Leccinum 8909241AE Belledonne 38, Seiglieres, France 24-Sep-84 A. Estades DQ534618 1395 1331 aerugineum 1396 1332 Leccinum albellum TH6968 Watuaga Co., NC, USA ee AY612811 1397 1333 Leccinum La1 Bavaria, Germany 11-Sep-94 M. Beisenherz AF139689 1398 1334 aurantiacum 1399 1335 Leccinum HN1573 Giles Co, VA, USA ee AY612810 1400 1336 aurantiacum 1401 1337 Leccinum GR92103 eeeAF139690 1402 1338 calltitrichum 1403 1339 Leccinum cerinum MK11800 eeeAF139692 1404 1340 Leccinum 880904/4 GL France 4-Sep-88 Wolfer AF139695 1405 1341 duriusculum 1406 1342 Leccinum 24/98 USA 23-Aug-98 N. Arnold AF139696 1407 1343 ﬂavostipitatum 1408 1344 Leccinum TDB-969 CA, USA 21-Nov-94 T. D. Bruns DQ534613 1409 1345 manzanitae 1410 Leccinum monticola BDCR14 Cerro de la Muerta summit, e B. Dentinger HQ161869 1346 1411 Costa Rica 1347 1412 Leccinum nigellum GPL4676 France 11-Sep-79 G. Redeuilh AF139699 1348 1413 ined 1349 1414 Leccinum palustre MK11107 Finland 2-Sep-92 M. Korhonen AF139701 1350 Leccinum 9210040 France 10-Apr-92 A. Estades AF139702 1415 1351 percandidum 1416 1352 Leccinum piceinum Lp1 Pruz in Tyrol, Austria 14-Sep-94 M. Binder, H. Besl DQ534614 1417 1353 Leccinum quercinum 196695 NGS Ludwigshain, Kelheim, 5-Sep-94 A. Reisinger DQ534612 1418 1354 Germany 1419 1355 Leccinum rigidipes 196696 Massif Belledonne 38, Prabert, 11-Oct-89 A. Estades DQ534617 1420 1356 France 1421 1357 Leccinum MK7676:251 Finland e M. Korhonen AF139704 1422 1358 rotundifoliae 1423 1359 Leccinum rugosiceps TH6967 Watuaga Co., NC, USA ee AY612813 1424 1360 Leccinum scabrum NCJ26 Watuaga Co., NC, USA ee AY612814 1425 1361 Leccinum 921024/1 GL 59 Waredin, Rost, France 24-Oct-92 G. Lannoy DQ534615 1426 1362 schistophilum 1427 1363 Leccinum variicolor Lvar1 Bavaria, Germany 19-Sep-95 J. Enzmann AF139706 1428 1364 Leccinum versipelle Lv2 Bavaria, Germany 6-Sep-94 H. Besl AF139707 1429 1365 (continued on next page) 1430

12 M. E. Nuhn et al.

1431 1496 e 1432 Table 3 (continued) 1497 1433 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1498 1434 Identifier 1499 1435 1500 Melanogaster Ma2 USA 13-Aug-89 R. Watling AF352046 1436 1501 ambiguus 1437 1502 Melanogaster Mbr1 Bavaria, Germany 28-Jun-95 M. Binder AF098383 1438 1503 broomeianus 1439 1504 Melanogaster 628 USA 11-Oct-83 J. Trappe AF167679 1440 tuberiformis 1505 1441 Melanogaster 9666 eeJ. Trappe AF074919 1506 1442 tuberiformis 1507 1443 Melanogaster REG 384 Germany 21-Jul-80 H. Besl DQ534668 1508 1444 variegatus 1509 1445 Octaviania Octa1 Bavaria, Germany 3-Sep-97 L. Krieglsteiner DQ534619 1510 1446 asterosperma 1511 1447 Octaviania tasmanica TL2329 Australia ee HQ647147 1512 1448 Paragyrodon TDB-420 Becker Co, MN, USA 22-Aug-80 T. D. Bruns AF071531 1513 1449 sphaerosporus 1514 1450 Paxillus filamentosus PfM1 Bavaria, Germany 12-Sep-95 H. Besl AF167681 1515 1451 Paxillus involutus Pi3 Bavaria, Germany 10-Sep-95 M. Binder AF167682 1516 1452 Paxillus involutus Pi2 Bavaria, Germany 3-Sep-95 M. Binder AF167683 1517 1453 Paxillus involutus Pi5 Bavaria, Germany 8-Oct-95 M. Binder AF167684 1518 1454 Paxillus involutus PiM2 Bavaria, Germany 20-Sep-98 A. Bresinksy AF167686 1519 1455 Paxillus involutus PiM1 Bavaria, Germany 22-Sep-98 M. Jarosch AF167701 1520 Paxillus involutus RV98.135 Watuaga Co., NC, USA ee AY612815 1456 1521 Phylloboletellus XAL 3388 Municipio Coatepec, El Grande, 5-Aug-01 V. Bandala DQ534658 1457 1522 chloephorus Vercruz, Mexico 1458 1523 Phylloporus arenicola 27 954 USA e J. Trappe JQ003704 1459 1524 Phylloporus bellus MCA559 eeeAY612817 1460 Phylloporus MAN 037 Costa Rica e M. A. Neves JQ003664 1525 1461 centroamericanus 1526 1462 Phylloporus alborufus MAN 022 Costa Rica e M. A. Neves JQ003678 1527 1463 Phylloporus MB 05-007 MA, USA e M. Binder KF030322 1528 1464 leucomycelinus 1529 1465 Phylloporus MAN 017 Costa Rica e M.A. Neves JQ003669 1530 1466 phaeoxanthus 1531 1467 Phylloporus MAN 050 Costa Rica e M.A. Neves JQ003672 1532 1468 purpureus 1533 1469 Phylloporus 161/96 MA, USA 1-Sep-96 N. Arnold DQ534631 1534 1470 rhodoxanthus 1535 1471 Phylloporus SAR 89/457 Durham, Co, NC, USA ee U11925 1536 1472 rhodoxanthus 1537 e 1473 Porphyrellus 00-348 Africa D. Arora KF030348 1538 1474 Porphyrellus M225 New Zealand 24-Apr-95 A. Bresinksy DQ534630 1539 1475 brunneus 1540 Porphyrellus Pop4 Bavaria, Germany 28-Aug-97 T. Wagner, KF030346 1476 1541 porphyrosporus M. Binder 1477 1542 Porphyrellus Pop1 Bavaria, Germany 9-Sep-96 M. Beisenherz DQ534642 1478 1543 porphyrosporus 1479 1544 Porphyrellus REH8716 Big Lagoon Park, Humbolt Co, 15-Nov-05 R.E. Halling EU685107 1480 pseudoscaber CA, USA 1545 1481 Porphyrellus 148/98 Estabrook Woods, MA, USA 28-Aug-98 N. Arnold DQ534644 1546 1482 sordidus 1547 1483 Porphyrellus sp 00-356 Africa e D. Arora KF030349 1548 1484 Pseudoboletus 151/97 Blue Valley, Macon, NC, USA 25-Aug-97 N. Arnold DQ534655 1549 1485 parasiticus 1550 1486 Pulveroboletus DD973 Orange Co, NC, USA ee AY612818 1551 1487 auriflammeus 1552 1488 Pulveroboletus TH6943 Watuaga Co., NC, USA ee AY612820 1553 1489 curtisii 1554 1490 Pulveroboletus 64/96 MA, USA 25-Aug-98 N. Arnold KF030306 1555 1491 ravenelii 1556 1492 Retiboletus retipes RV98.127 Giles Co, VA, USA ee AY612821 1557 1493 Pulveroboletus sp 00-317 Africa 2000 D. Arora KF030332 1558 1494 Pulveroboletus sp 00-428 Africa 2000 D. Arora KF030333 1559 1495 Pulveroboletus sp 00-507 Africa 2000 D. Arora KF030334 1560

Phylogenetic overview of the Boletineae 13

1561 1626 e 1562 Table 3 (continued) 1627 1563 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1628 1564 Identifier 1629 1565 1630 Retiboletus flavoniger REH7247 San Jose, Costa Rica 8-Jun-98 R.E. Halling AF456828 1566 1631 Retiboletus flavoniger REH7189 San Jose, Costa Rica 24-Nov-93 R.E. Halling AF456829 1567 1632 Retiboletus griseus Both sn NY, USA e E. Both KF030308 1568 1633 Retiboletus Tyni1 Japan 15-Jul-96 R. Marumoto AF456832 1569 1634 nigerrimus 1570 Retiboletus ornatipes 93/97 USA 23-Aug-97 N. Arnold, AF456825 1635 1571 W. Helfer, 1636 1572 W. Steglich 1637 1573 Retiboletus ornatipes 186/97 USA 30-Aug-97 N. Arnold, AF456819 1638 1574 W. Helfer, 1639 1575 W. Steglich 1640 1576 Retiboletus retipes 96/97 USA 23-Aug-97 N. Arnold, AF456830 1641 1577 W. Helfer, 1642 1578 W. Steglich 1643 1579 Retiboletus retipes Japan1 Japan 10-Jul-97 H. Shibata AF456807 1644 1580 Retiboletus retipes REH7567 Cartago, Costa Rica 28-May-96 R.E. Halling AF456808 1645 1581 Retiboletus retipes 55/97 USA 21-Aug-97 N. Arnold, AF456811 1646 1582 W. Helfer, 1647 1583 W. Steglich 1648 1584 Retiboletus sp MAN 053 Costa Rica 12-Jun-04 M.A. Neves KF030327 1649 ee 1585 Rossbeevera MEL2079350 New Zealand HQ647157 1650 pachyderma 1586 1651 Rossbeevera M42 New Zealand 15-Mar-95 A. Bresinksy DQ534620 1587 1652 pachyderma 1588 1653 Rossbeevera MEL2293673 Australia ee HQ647163 1589 1654 vittatispora 1590 Rossbeevera MEL2233755 Australia ee HQ647164 1655 1591 westraliensis 1656 1592 Rubinoboletus sp 01-590 Africa 2001 e KF030314 1657 1593 Tylopilus ballouii TH6385 Pakaraima Mnts, Guyana ee AY612823 1658 1594 Spongiforma LHFB14 Malaysia e T.D. Bruns, HQ724509 1659 1595 squarepantsii D. E. Desjardin 1660 1596 Spongiforma LHFB01 Malaysia e T.D. Bruns, HQ724510 1661 1597 squarepantsii D. E. Desjardin 1662 1598 Strobilomyces 72 106 eeeKF030345 1663 1599 dryophilus 1664 1600 Strobilomyces HN0027 Orange Co, NC, USA ee AY612824 1665 1601 floccopus 1666 1602 Strobilomyces sp 177/97 MA, USA 26-Aug-97 N. Arnold DQ534627 1667 1603 Sutorius eximius 40/97 MA, USA 20-Aug-97 N. Arnold AF139684 1668 1604 Tylopilus aff 01-513 Africa 2001 D. Arora JX889672 1669 1605 chromapes 1670 Tylopilus aff conicus 01-533 Africa 2001 D. Arora KF030328 1606 1671 Tylopilus aff eximius 01-528 Africa 2001 D. Arora JQ327002 1607 1672 Tylopilus aff virens 01-541 Africa 2001 D. Arora JX889677 1608 1673 Tylopilus alboater TH6941 Durham, Co, NC, USA ee AY612832 1609 1674 Tylopilus alboater TDB-1207 Duncombe, Co., NC, USA 3-Sep-88 T. D. Bruns AF139708 1610 Tylopilus WV Camp Creek State Forest, WV, 14-Aug-02 W.C. Woody, KF030293 1675 1611 atronicotianus USA E. Both 1676 1612 Tylopilus e Clarion Public Hunting Area, 18-Sep-04 W. Sturgeon, EU685110 1677 1613 atronicotianus Clarion Co, PA, USA E. Both 1678 1614 Tylopilus badiceps NCJ20 Watuaga Co., NC, USA ee AY612833 1679 1615 Tylopilus badiceps MB 03-052 MA, USA Aug-03 M. Binder KF030336 1680 1616 Tylopilus cf 01-549 Africa 2001 D. Arora JX889671 1681 1617 chromapes 1682 1618 Tylopilus felleus ee ee AF071466 1683 1619 Tylopilus felleus HKAS54926 Marburg, Germany ee HQ326933 1684 1620 Tylopilus indecisus 98/98 USA 26-Aug-98 N. Arnold, AF456820 1685 1621 W. Helfer 1686 1622 Tylopilus intermedius BD277 Nerstrand-Big Woods State Park, e B. Dentinger HQ161875 1687 1623 MN, USA 1688 ee 1624 Tylopilus rhoadsiae RV98.261 Giles Co, VA, USA AY612836 1689 1625 (continued on next page) 1690

14 M. E. Nuhn et al.

1691 1756 e 1692 Table 3 (continued) 1757 1693 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1758 1694 Identifier 1759 1695 1760 Tylopilus 152/98 MA, USA 28-Aug-98 N. Arnold DQ534629 1696 1761 rubrobrunneus 1697 1762 Tylopilus sp 00-311 Africa 2000 D. Arora KF030309 1698 1763 Tylopilus sp 190/83 USA 18-Sep-83 W. Steglich AF457405 1699 1764 Tylopilus sp 00-366 Africa 2000 D. Arora KF030337 1700 Tylopilus sp 00-388 Africa 2000 D. Arora KF030338 1765 1701 Tylopilus sp 204/97 MA, USA 30-Aug-97 N. Arnold AF456813 1766 1702 Tylopilus 9306tv Plymouth, MA, USA 3-Sep-06 B. Neill KF030315 1767 1703 variobrunneus 1768 1704 Tylopilus sn Hor eeeKF030316 1769 1705 variobrunneus 1770 1706 Tylopilus virens e Japan 1997 R. Marumoto DQ534621 1771 1707 Xanthoconium affine 78/94 USA 31-Aug-94 W. Steglich AF457406 1772 1708 Xanthoconium affine RV98.112 Giles Co, VA, USA ee AY612838 1773 1709 Xanthoconium affine BD217 Mnt. Lake Biological Station, e B. Dentinger HQ161854 1774 1710 var maculosum Mountain Lake, VA, USA 1775 1711 Xanthoconium sp MB 06-049 Dutch Grove, Erie Co, NY, USA 4-Aug-06 M. Binder KF030292 1776 1712 Xerocomus TH6304 Pakaraima Mnts, Guyana ee AY612839 1777 1713 amazonicus 1778 1714 Xerocomellus Xar1 Spessart, Germany 6-Sep-95 L. Krieglsteiner KF030295 1779 1715 armeniacus 1780 Xerocomus badius Xb1 Bavaria, Germany Sep-95 M. Binder AF050648 1716 1781 Xerocomus badius MB 03-098(ajb) Rutland, MA, USA 19-Aug-03 M. Binder KF030355, 1717 1782 KF030356 1718 1783 Xerocomus bubalinus MB 05-008 Beaver St., Worcester, 5-Oct-05 M. Binder KF030296 1719 1784 Worcester Co, MA, USA 1720 Xerocomellus cf PBM925 USA ee KF030272 1785 1721 chrysenteron 1786 1722 Xerocomellus cf MB 00-017 Woodland St., Worcester, 2-Aug-00 M. Binder KF030277 1787 1723 porosporus Worcester Co, MA, USA 1788 1724 Xerocomellus IB20000405 eeeAF514809 1789 1725 chrysenteron 1790 1726 Xerocomellus REH8723 Redwood National Park, 17-Nov-05 R.E. Halling KF030276 1791 1727 chrysenteron Humbolt Co., CA, USA 1792 1728 Xerocomellus TDB-635 Crawford, Co., MI, USA 8-Aug-83 T. D. Bruns AF071537 1793 1729 chrysenteron 1794 1730 Xerocomellus Xpor1 Bavaria, Germany 17-Sep-95 M. Binder AF050645 1795 1731 porosporus 1796 1732 Xerocomellus IB19880304 eeeAF514810 1797 1733 porosporus 1798 eee 1734 Xerocomellus IB19990957 AF514811 1799 1735 porosporus 1800 Xerocomellus Xpru1 Bavaria, Germany 21, Aug-98 L. Krieglsteiner AF050644 1736 1801 pruinatus 1737 1802 Xerocomellus IB19980368 eeeAF514827 1738 1803 pruinatus 1739 1804 Xerocomellus GR22465 eeeAF514818 1740 ripariellus 1805 1741 Xerocomellus IB19980360 eeeAF514816 1806 1742 ripariellus 1807 1743 Xerocomellus rubellus GS961 eeeAF514829 1808 1744 Xerocomellus rubellus IB19990917 eeeAF514828 1809 1745 Xerocomellus zelleri 27 888 eeJ. Trappe KF030275 1810 1746 Xerocomus JAM0539 Cantabria, Las Rozas, Spain 21-Sep-04 J.A. Munoz KF040544 1811 1747 chrysonemus 1812 1748 Xerocomus cisalpinus 5823-130998 Germany 13-Sep-98 J. Schreiner KF030274 1813 1749 Xerocomus cisalpinus IB20000701 eeeAF514812 1814 1750 Xerocomus dryophilus IB19990901 eeeAF514823 1815 1751 Xerocomus dryophilus IB19991057 eeeAF514822 1816 1752 Xerocomus fennicus H126 eeeAF514820 1817 eee 1753 Xerocomus fennicus RJ126 AF514821 1818 1754 Xerocomus hortonii 84/94 MA, USA 23-Aug-94 W. Steglich AF139713 1819 1755 1820

Phylogenetic overview of the Boletineae 15

1821 1886 e 1822 Table 3 (continued) 1887 1823 Species Isolate ID Location Date Collector/ nuc-lsu tef1 rpb1 1888 1824 Identifier 1889 1825 1890 Xerocomus illudens 64/98 USA 24-Aug-98 N. Arnold, AF139714 1826 1891 W. Helfer 1827 1892 Xerocomus illudens DD9854 Orange Co, NC, USA ee AY612840 1828 1893 Xerocomus lanatus MB 95-074 Bavaria, Germany 13-Sep-95 M. Binder DQ534633 1829 1894 Xerocomus JAM0612 Araba, Albania 9-Aug-05 J.A. Munoz KF030323 1830 silwoodensis 1895 1831 Xerocomus sp RV98.123 Giles Co, VA, USA ee AY612841 1896 1832 Xerocomus spadiceus OKM25919 Clallam Co, WA, USA ee AY612842 1897 1833 Xerocomus IB19980452 eeeAF514831 1898 1834 subtomentosus 1899 1835 Xerocomus IB19991000 eeeAF514832 1900 1836 subtomentosus 1901 1837 Xerocomus 63/97 USA 21-Aug-97 N. Arnold, DQ534625 1902 1838 truncatus H. Besl 1903 1839 Xerocomus truncatus REH8718 CA, USA e R.E. Halling KF030273 1904 1840 1905 1841 1906 1842 the proportion of gaps and undetermined characters in the Basionym: Gastroboletus subalpinus Trappe & Thiers Brittonia 1907 1843 nuc-lsu dataset is artificially inflated due to the use of differ- 21: 245 (1969). 1908 1844 ent primer pairs. TYPE: Oregon. Hood River Co., Cloud Gap, Trappe 607 (Holo- 1909 1845 For the BY analyses, maximum differences in split fre- type: OSC). 1910 1846 quency between runs dropped to zero after approximately MycoBank #: 804290. 1911 1847 51 000 cycles for the core dataset and 27 000 for the extended Boletus semigastroideus M. Nuhn, Manfr. Binder, A. F. S. Taylor, 1912 1848 dataset, and the chains were then stopped. The chains for Halling, Hibbett, nom. nov. for Secotium areolatum G. Cunn., 1913 1849 each dataset were analyzed with the readpb program, remov- New Zealand J. Sci. Technol. 23: 172B (1942), non Boletus areolatus 1914 1850 1915 ing 10 % of the samples as burn-in. A total of 91 789 trees were Berkeley, Hooker’s J. Bot. Kew Gard. Misc. 4: 138 (1852). 1851 1916 used to estimate PPs for the core dataset, and 49 367 trees for Synonym: Notholepiota areolata (G. Cunn.) E. Horak, New 1852 1917 1853 the extended dataset (Table 4). Zealand J. Bot. 9: 479 (1971). 1918 1854 TYPE: New Zealand. Auckland, Swanson, J. Dingley, IV. 1943 1919 (Holotype: PDD 4595). 1855 Taxonomy 1920 1856 MycoBank #: 804291. 1921 1857 1922 Boletus separans (Peck), Bull. Buffalo Soc. Nat. Sci. 1: 59.(1873). 1858 1923 1859 Synonyms: Boletus edulis spp. separans (Peck) Singer, Amer. Discussion 1924 1860 Midl. Nat. 37: 26 (1947). 1925 1861 B. edulis f. separans (Peck) Vassilkov, BekyiGrib: 15 (1966). Overview of phylogenetic relationships in Boletineae 1926 1862 Xanthoconium separans (Peck) Halling & Both, Bull. Buffalo Soc, 1927 1863 Nat. Sci. 36: 240 (1998). In both the three-gene core and extended datasets, the Boleti- 1928 1864 TYPE: New York. Greenbush, August 1872, C.H. Peck (NYS). neae and Boletaceae are upheld with maximum supported 1929 1865 MycoBank #: 226550. values in all analyses (BS ¼ 100 %, MP ¼ 100 %, PP ¼ 1.0, re- 1930 1866 Boletus subalpinus (Trappe & Thiers) M. Nuhn, Manfr. Binder, ported as [BS/MP/PP] hereafter), but the backbone of the Bole- 1931 1867 1932 A. F. S. Taylor, Halling, Hibbett, comb. nov. taceae remains poorly resolved. Nonetheless, there are 1868 1933 1869 1934 1870 1935 1871 Table 4 e Alignment Information from ML and MP analyses. 1936 1872 1937 Alignment ML MP 1873 1938 1874 Positions Alignment % Gaps and Characters Constant Uninformative Informative 1939 1875 patterns undetermined characters characters characters 1940 1876 characters 1941 1877 1942 nuc-lsu, core 1537 482 35 N/A N/A N/A N/A 1878 1943 tef1, core 1327 702 12 N/A N/A N/A N/A 1879 1944 RPB1, core 1490 1079 27 N/A N/A N/A N/A 1880 1945 nuc-lsu, extended 1952 702 35 N/A N/A N/A N/A 1881 1946 tef1, extended 1345 763 13 N/A N/A N/A N/A 1882 RPB1, extended Same as core N/A N/A N/A N/A 1947 1883 nuc-lsu, constrained 2000 858 56 N/A N/A N/A N/A 1948 1884 Three gene core N/A N/A N/A 4349 2434 543 1327 1949 1885 Three gene extended N/A N/A N/A 4759 2732 558 1469 1950

16 M. E. Nuhn et al.

1951 eleven strongly support, named clades shared in both the is identical for the core and extended analyses. Eleven 2016 1952 three-gene core and extended, and an additional seven sup- strongly supported sub-groups within the anaxoboletus, lecci- 2017 1953 2018 ported, named clades in the three-gene extended (see Figs 1 noid, and hypoboletus groups are discussed below. Except for 1954 2019 and 2 for clades; clades 1e11 are shared, clades 12e18 are the Paxillaceae, group names do not correspond to formal taxa. 1955 2020 only in the three-gene extended analysis). The named, sup- Group names were designated based on the most inclusive 1956 2021 1957 ported clades may provide the basis for an eventual generic clade with strong support and are only meant to facilitate 2022 1958 level reclassiﬁcation of the Boletaceae. The major groups, discussion. 2023 1959 which we informally label as anaxoboletus, leccinoid, hypobo- The anaxoboletus group unites the genera Afroboletus, Bole- 2024 1960 letus, bicolor, dupainii, regius, carminipes, chalciporus, and tus, Gastroboletus, Notholepiota, Porphyrellus, Tylopilus, Strobilo- 2025 1961 Paxillaceae are all upheld by at least two of the three analysis myces, Xanthoconium, Xerocomellus, and species referred to as 2026 1962 methods (above 70 % for BS and MP, and 0.95 for PP) in the Xerocomus but shown to not be closely related to Xerocomus 2027 1963 three-gene core and extended phylogenies. However, two subtomentosus, the type species of Xerocomus. Boletus, Gastrobo- 2028 1964 groups, chalciporus and Paxillaceae, are represented by single letus, Notholepiota, and Xanthoconium found in anaxoboletus 2029 1965 2030 taxa in the three-gene core analysis. The overall tree topology are members of the ‘porcini’ group as deﬁned by Dentinger 1966 2031 1967 2032 1968 2033 1969 Afroboletus luteolus 2034 -Type species of genus Stobilomyces/Afroboletus clade 1970 ?-Putative Mycoparasite Strobilomyces floccopus 2035 Seciotiod fruiting body Xanthoconium stramineum 1971 Gasteroid fruiting body 2036 1972 Mycoparasite Porphyrellus porphyrosporus 2037 Saprotroph Tylopilus plumbeoviolaceus 1973 Tylopilus clade 2038 Tylopilus felleus 1974 Boletus subalpinus 2039 1975 Boletus aerues 2040 1976 Boletus variipes var fagicola “Porcini” clade Anaxoboletus 2041 1977 Boletus edulis Group 2042 Boletus separans 1978 Notholepiota areolata 2043 1979 Boletus pallidus 2044 97/- badius clade 1980 Xerocoums badius 2045 1981 Xerocomellus zelleri 2046 Xerocomellus chrysenteron Xercomellus clade 1982 100/100 2047 Xerocomus cisalpinus 78/- 1983 Xerocomellus rubellus Rubellus group 2048 1984 Leccinellum corsicum Leccinum/Leccinellum clade Leccinoid 2049 1985 95/- Spongiforma thailandica Group 2050 1986 Retiboletus griseus 2051 1987 89/87 Aureoboletus thibetanus 2052 71/- Aureoboletus roxanae Aureoboletus clade 1988 98/- Boletus abruptibulbus 2053 1989 Boletellus projectellus Hypoboletus 2054 1990 Hemileccinum impolitum Hemileccinum clade Group 2055 1991 Boletus subglabripes 2056 Xerocomus subtomentosus 1992 2057 Phylloporus pelletieri Xerocomus clade 1993 Boletellus chrysenteroides 2058 1994 Bothia castanella 2059 1995 Austroboletus gracilis 2060 1996 Boletus rufomaculatus bicolor clade 2061 Boletus bicolor var bicolor 1997 B 2062 97/78 Boletus inedulis 1998 Boletus firmus dupainii clade 2063

1999 85/- Boletus dupainii 2064 Boletus amygdalinus 2000 A 93/90 2065 2001 98/98 Boletus appendiculatus 2066 Boletus pulchriceps regius clade 2002 85/- 100/100 2067 Boletaceae Boletus roseopurpureus 2003 Boletus carminipes 2068 90/85 carminipes calde 2004 Boletineae Boletus pulverulentus 2069 2005 Pseudoboletus parasiticus 2070 2006 Chalciporus piperatus Chalciporus group? 2071 Gyrodon lividus Paxillaceae 2007 Hydnomerulius pinastri 2072 2008 Boletinellus merulioides 2073 2009 2074 2010 Fig 1 e Phylogenetic relationships of the Boletineae inferred from nuc-lsu, tef1, and RPB1 using RAxML, PhyloBayes, and 2075 2011 2076 PAUP*. This is referred to as the three-gene core analysis in the text. The tree topology corresponds to the optimal ML tree. 2012 2077 Support values ‡70 % BS for ML and MP are displayed [ML/MP], and PPs ‡0.95 % are depicted by bold branches. Groups and 2013 2078 2014 clades that were recovered in the analysis are indicated, and three taxonomic ranks are indicated: Boletineae, Boletaceae and 2079 2015 Paxillaceae. Nodes indicated by A and B indicate the most inclusive and second most inclusive clades that lack a formal 2080 taxonomic rank, respectively. Numbered clades 1e11 are the clades the core analysis shares with the extended analysis.

Phylogenetic overview of the Boletineae 17

2081 et al. (2010) and is equivalent to Singer’s Boletus section Boletus groups, the bicolor and carminipes clades, respectively and 2146 2082 (Singer 1986) containing the type species: Boletus edulis. The are not, based on our results, the same species. 2147 2083 2148 leccinoid group unites the genera Leccinellum, Leccinum, Spongi- 2084 2149 forma, and Retiboletus. The hypoboletus group contains mem- Ecological diversity in Boletineae 2085 2150 bers of Aureoboletus, Boletellus, Boletus (non-porcini), 2086 2151 2087 Hemileccinum, Phylloporus, and Xerocomus (the clade with the The great majority of the species of the Boletineae are ectomy- 2152 2088 type species). The relationship between these three lineages corrhizal (ECM) (Binder & Hibbett 2006; Tedersoo et al. 2010). 2153 2089 is unresolved. Chalciporus spp. and Buchwaldoboletus lignicola However, Pseudoboletus parasiticus and species of the Chalcipo- 2154 2090 are supported as a clade, designated chalciporus group. rus group (Chalciporus piperatus and Buchwaldoboletus lignicola) 2155 2091 Two other nodes receive support, node A and node B (see appear to be mycoparasites (Modess 1941; Godbout & Fortin 2156 2092 Figs 1 and 2). Node A supports the sister group relationship be- 1985; Yamada & Katsuya 1995; Kasuya & Igarashi 1996; 2157 2093 tween the chalciporus group and all other members of the Hogberg€ et al. 1999; Taylor et al. 2003; Zeller et al. 2007; 2158 2094 Boletaceae (see Figs 1 and 2), and node B supports Pseudoboletus Tedersoo et al. 2010). Pseudoboletus parasiticus, which produces 2159 2095 2160 parasiticus as the sister group of the non-chalciporus group its fruiting bodies on those of its host, Scleroderma citrinum 2096 2161 Boletaceae. All other nodes with support correspond to groups (Sclerodermatineae), is well characterized as a mycoparasite, 2097 2162 2098 that could be considered genera. but evidence on the nutritional mode of Chalciporus piperatus 2163 2099 Diverse morphology-based generic concepts have been is not as clear-cut. 2164 2100 proposed in the Boletineae, with most genera containing a small Chalciporus piperatus was originally considered to be ECM, 2165 2101 or moderate (50) number of species (Pilat 1969; Smith & but several synthesis attempts failed to show formation of 2166 2102 Thiers 1971; Singer 1986; Binder & Besl 2000; Binder & ECM with tree species in vitro (Modess 1941; Godbout & 2167 2103 Bresinsky 2002b; Binder & Hibbett 2006; Sutara 2008; Halling Fortin 1985; Yamada & Katsuya 1995; Kasuya & Igarashi 2168 2104 et al. 2012a, b). Twenty-three genera are included in the core 1996; Zeller et al. 2007). Garrido (1988) reported successful 2169 2105 analysis, representing roughly half of the total genera of the ECM synthesis using C. piperatus, but Tedersoo et al. (2010) 2170 2106 2171 Boletineae that have molecular data available, and 28 genera urged caution, stating that the illustrations of Garrido (1988) 2107 2172 are included in the extended analysis. In the extended analy- do not resemble true ECM hyphal formations. Additionally, 2108 2173 sis, the following genera are recovered as monophyletic: Stro- isotopic analysis suggested that C. piperatus may be saprotro- 2109 2174 € 2110 bilomyces, Leccinum, Leccinellum, Royoungia, Chalciporus, and phic (Hogberg et al. 1999; Taylor et al. 2003). However, isotopic 2175 2111 Paxillus. However, the following genera are not monophyletic values for saprotrophic species, especially leaf litter decayers, 2176 2112 using current generic concepts: Aureoboletus, Boletellus, Boletus, and some mycoparasites can overlap with isotopic values for 2177 2113 Xanthoconium, Xerocomellus, and Xerocomus. ECM species (Hobbie et al. 1999; Hogberg€ et al. 1999; Hobbie 2178 2114 The most difficult taxonomic issue facing boletelogists et al. 2001). 2179 2115 concerns the genus Boletus. The type species of Boletus, Ecological observations suggest a mycoparasitic nutri- 2180 2116 B. edulis, is nested in the strongly supported ‘porcini clade’, tional mode for C. piperatus, which has been found in associa- 2181 2117 which is part of the anaxoboletus group, but many other spe- tion with its putative host, Amanita muscaria (Agaricales), in 2182 2118 2183 cies of ‘Boletus’ are distributed among the distantly-related bi- New Zealand and Australia (Robinson 2010). Pinus radiata is 2119 2184 color, dupainii, regius, and carminipes clades (Fig 2). Members a non-native species in New Zealand that forms ECM with 2120 2185 2121 of the ‘porcini,’ bicolor, dupainii, regius, and carminipes A. muscaria, which is thought to have been introduced along 2186 2122 clades all share smooth spores, a spore print colour reported with P. radiata. Amanita muscaria has made a host jump onto 2187 2123 as some hue of olive brown, tube trama of the boletoid type, the native Nothofagus, and C. piperatus has been observed fruit- 2188 2124 and other characters (Smith & Thiers 1971; Singer 1986; ing in proximity to A. muscaria associated with Nothofagus,im- 2189 2125 Bessette et al. 2000; Ladurner & Simonini 2003; Munoz~ 2005; plying that it followed A. muscaria’s mycorrhizal host shift 2190 2126 Dentinger et al. 2010). However, the bicolor, dupainii, regius, (Tedersoo et al. 2010; Dunk et al. 2012). 2191 2127 and carminipes clade species are distinguished from the ‘por- Buchwaldoboletus lignicola, which is the sister taxon of Chal- 2192 2128 cini’ clade by the lack of a white pore surface when young and ciporus, also appears to be mycoparasitic. Fig 3 depicts the re- 2193 2129 2194 absence of the ‘stuffed’ pore characteristic (Dentinger et al. sults of a confrontation experiment in which the hyphae of 2130 2195 2010). The present study lacks the necessary sampling to at- B. lignicola can be seen wrapping around hyphae of Phaeolus 2131 2196 tempt to resolve the Boletus spp. that are not members of Bole- schweinitzii, a known brown-rot tree pathogen. Buchwaldobole- 2132 2197 2133 tus s.str., which may represent additional independent clades. tus lignicola is associated with P. schweinitzii in the field and ap- 2198 2134 While the need for a reclassification of Boletus has been ev- pears to be dependent on the latter for growth (Szczepka & 2199 2135 ident for some time (Binder & Hibbett 2006), certain infragene- Sokol 1984), which contradicts suggestions that it is saprotro- 2200 2136 ric taxa within Boletus have been shown to be problematical. phic (Pantidou 1962; Pilat 1965). Collectively, the confrontation 2201 2137 For example, Boletus sect. Subtomentosus sensu Smith & analysis and association with P. schweinitzii of B. lignicola, and 2202 2138 Thiers (1971) is represented by members of the carminipes the isotopic and ecological evidence for C. piperatus support 2203 2139 clade and Boletus pallidus, this latter appears in the distantly a mycoparasitic nutritional mode for some members of the 2204 2140 related badius clade of the anaxoboletus group (Fig 2). Simi- Chalciporus group (Taylor et al. 2003; Zeller et al. 2007). Because 2205 2141 2206 larly, species of Boletus sect. Calopodes sensu Singer (1986) are the Chalciporus group and P. parasiticus are nested within the 2142 2207 distributed across the carminipes clade and the regius clade Boletineae (Figs 1 and 2), the genera may represent parallel 2143 2208 2144 (Fig 2). Even some putative species have proven to be polyphy- shifts in nutritional mode from the ECM nutritional mode 2209 2145 letic. Specifically, Boletus bicolor var. bicolor and B. bicolor var. shared by members of the Paxillaceae and all of the species 2210 borealis sensu Smith & Thiers (1971) occur in two separate above node B (Figs 1 and 2).

18 M. E. Nuhn et al.

2211 100/100 Tylopilus badiceps 2276 2212 99/- Tylopilus ferrugineus Tylopilus 2277 99/- 2213 Tylopilus felleus clade 2278 Tylopilus plumbeoviolaceus 2214 -Type species of genus Strobilomyces sp 2279 ?-Putative Mycoparasite 100/100 Strobilomyces Seciotiod fruiting body 100/95 Strobilomyces floccopus

2215 Anaxoboletus Group 2280 Gasteroid fruiting body clade 2216 Mycoparasite Afroboletus luteolus 2281 Saprotroph Xanthoconium stramineum 2217 Porphyrellus porphyrosporus 2282 2218 100/100 Boletus subalpinus 2283 2219 100/100 Boletus aereus 2284 100/100 Boletus variipes var fagicola “Porcini” 2220 2285 99/93 Boletus edulis clade 2221 100/100 Notholepiota areolata 2286 94/94 2222 Boletus separans 2287 100/100 Xerocomellus chrysenteron Xercomellus 2223 2288 Xerocomellus zelleri clade 2224 71/- Xerocomus cisalpinus 2289 2225 100/100 Xerocomellus rubellus cf East Coast rubellus 2290 Xerocomellus rubellus cf West Coast clade 2226 2291 100/100 Xerocomus badius Xb2 badius 2227 Xerocomus badius MB03-098 clade 2292 Boletus pallidus 2228 79/- 2293 100/100 Leccinum scabrum 2229 Leccinum 2294 100/100 Leccinum albellum clade Leccinellum crocipodium Leccinoid 2230 96/80 Leccinellum 2295 81/95 Leccinellum corsicum clade Group 2231 95/- 2296 Spongiforma thailandica 2232 Retiboletus griseus 2297 2233 100/100 Aureoboletus moravicus 2298 2234 Aureoboletus roxanae 2299 80/72 Aureoboletus gentilis 2235 Aureoboletus thibetanus Aureoboletus 2300 2236 75/89 Boletellus shichianus clade 2301 2237 95/77 Boletus abruptibulbus 2302 Aureoboletus innixus Hypoboletus 2238 Boletellus projectellus 2303 2239 100/100 Boletus subglabripes Hemileccinum Group 2304 2240 Hemileccinum impolitum clade 2305 Boletus morrisii 2241 89/73 2306 92/74 Boletellus chrysenteroides 2242 98/93 Xerocomus perplexus Xerocomus 2307 89/- Boletus tenax 2243 clade 2308 100/100 Xerocomus subtomentosus 2244 Phylloporus pelletieri 2309 2245 100/100 Royoungia sp Royoungia 2310 2246 Royoungia boletoides clade 2311 Bothia castanella 2247 Austroboletus gracilis 2312 2248 100/100 Boletus rufomaculatus bicolor clade 2313 2249 Boletus bicolor var bicolor 2314 Boletus firmus 2250 B 100/97 Boletus calopus 2315 2251 79/73 Boletus inedulis 2316 71/- 94/70 2252 Boletus dupainii dupainii clade 2317 Boletus rhodosanguineus 2253 Boletus amygdalinus 2318 Boletus luridiformis 2254 94/81 2319 2255 100/100 Boletus pulchriceps 2320 A 99/98 Boletus peckii 2256 100/100 Boletus appendiculatus regius clade 2321 2257 100/100 93/91 Boletus regius 2322 Boletus roseopurpureus 2258 2323 91/82 Boletus carminipes 2259 Boletaceae Boletus bicolor var borealis 2324 carminipes clade 100/100 Boletus pseudosensibilis 2260 82/- 100/100 2325 2261 Boletus pulverulentus 2326 Pseudoboletus parasiticus 2262 Chalciporus amarellus 2327 2263 Boletineae 98/96 Chalciporus pseudorubinellus Chalciporus Chalciporus 2328 100/100 100/100 2264 Chalciporus piperatus clade Group? 2329 Buchwaldoboletus lignicola 2265 100/100 Paxillus vernalis 2330 Paxillus 2266 100/100 Paxillus obscurisporus 2331 Paxillus filamentosus clade Paxillaceae 2267 100/97 2332 Gyrodon lividus 2268 Paragyrodon sphaerosporus 2333 2269 Hydnomerulius pinastri 2334 Boletinellus merulioides 2270 2335 2271 2336 2272 Fig 2 e Phylogenetic relationships of the Boletineae inferred from nuc-lsu, tef1, and RPB1 using RAxML, PhyloBayes, and 2337 2273 PAUP; all taxa have nuc-lsu and tef1, but not all taxa have RPB1.This is referred to as the three-gene extended analysis in the 2338 2274 text. The tree topology corresponds to the optimal ML tree. Support values ‡70 % BS for ML and MP are displayed [ML/MP], 2339 2275 and PPs ‡0.95 % are depicted by bold branches. Groups and clades that were recovered in the analysis are indicated, and 2340

Phylogenetic overview of the Boletineae 19

2341 2406 2342 2407 2343 2408 2344 2409 2345 2410 2346 2411 2347 2412 2348 2413 2349 2414 2350 2415 2351 2416 2352 2417 2353 2418 2354 2419 2355 2420 2356 2421 2357 2422 2358 2423 2359 2424 2360 2425 2361 2426 2362 2427 2363 2428 2364 2429 2365 2430 2366 2431 Fig 3 e Confrontation analysis between the hyphae of Buchwaldoboletus lignicola and Phaeolus schweinitzii. Initial hyphal 2367 2432 branching of Buchwaldoboletus lignicola is seen in the image on the left and indicated by blue arrows. In the image on the 2368 2433 2369 right, the black arrow indicates the P. schweinitzii hypha and where the hyphae of B. lignicola are wrapping around a hyphal 2434 2370 strand of P. schweinitzii, indicated by a white arrow. (For interpretation of the references to colour in this figure legend, the 2435 2371 reader is referred to the web version of this article.) 2436 2372 2437 2373 2438 2374 2439 2375 Clade-by-clade discussion of Boletineae considering the placement of Tylopilus chromapes outside the 2440 2376 clade containing the type species, Tylopilus felleus. Recently, 2441 2377 Because the topologies of the three-gene core and extended two new genera, Harrya and Australopilus, have been described 2442 2378 phylogenies are congruent, the following discussion refers to to accommodate the T. chromapes complex (Halling et al. 2443 2379 the more inclusive extended phylogeny. Discussion of the sys- 2012b). Despite these recent works, there still remain species 2444 2380 tematic and morphological features of the clades is confined called Tylopilus that are not supported as members of Tylopilus 2445 2381 to the taxa presented in Fig 2. The congruent Fig 2 clades in s.str. or lack enough molecular and morphological data to 2446 2382 the constrained analysis (Sup. Fig 1) are discussed only place in a different genus or erect a new genus:, e.g. Tylopilus 2447 2383 2448 when well-supported or otherwise noteworthy, but due to alboater, Tylopilus atronicotianus, Tylopilus badiceps, Tylopilus 2384 2449 the lack of support for nearly all nodes on the constrained rhoadsiae, Tylopilus variobrunneus, Tylopilus virens (see Sup. 2385 2450 Fig 1; Binder & Hibbett 2006). The Tylopilus clade containing 2386 tree (Sup. Fig 1), morphological and systematic features are 2451 2387 generally not discussed. For complete contents of the con- T. felleus in Li et al. (2011) corresponds to this study’s Tylopilus 2452 2388 strained analysis clades see Table 5 and Sup. Fig 1 for tree to- clade. The least inclusive clade containing all the species from 2453 2389 pology. anaxoboletus group [94/94/0.99]dAnaxoboletus the three-gene analysis receives no support in the constrained 2454 2390 comprises six clades: Tylopilus, Strobilomyces (including Afrobo- analysis; nevertheless it contains only species placed in 2455 2391 letus luteolus), Boletus sensu stricto, Xerocomellus, ‘rubellus,’ and Tylopilus. 2456 2392 ‘badius’ and two unresolved genera typified by Xanthoconium The traditional morphological concept of Tylopilus is that of 2457 2393 stramineum and Porphyrellus porphyrosporus. The relationships smooth spored species, with or without a reticulum on the 2458 2394 2459 between clades are not resolved. stipe, a spore print of pinkish to various brown hues, and 2395 2460 Tylopilus clade [99/-/1.0]dThe Tylopilus species in the ex- a pore surface that is frequently white when young and turns 2396 2461 tended analysis are recovered as monophyletic, with a place- pink, yellow, brown, grey or black with age (Bessette et al. 2000; 2397 2462 2398 ment within the tree that is similar to that in Dentinger et al. Singer 1986; Smith & Thiers 1971; Watling 2008). The variable 2463 2399 (2010). Li et al. (2011) reported Tylopilus as polyphyletic pore surface colour with age is one of the characters that 2464 2400 2465 2401 2466 2402 2467 three taxonomic ranks are indicated: Boletineae, Boletaceae and Paxillaceae. Nodes indicated by A and B indicate the most 2403 2468 e 2404 inclusive and second most inclusive clades that lack a formal taxonomic rank, respectively. Clade numbers 1 11 represent 2469 e 2405 the clades the extended analysis shares with the core analysis, numbers 12 18 represent clades in the extended analysis 2470 only.

Please cite this article in press as: Nuhn ME, et al., Phylogenetic overview of the Boletineae, Fungal Biology (2013), http:// dx.doi.org/10.1016/j.funbio.2013.04.008 2500 2506 2504 2509 2508 2505 2503 2502 2534 2532 2530 2529 2528 2526 2525 2523 2522 2520 2499 2498 2496 2495 2494 2493 2492 2490 2489 2488 2487 2486 2485 2484 2483 2482 2481 2480 2507 2535 2533 2524 2527 2497 2501 2491 2531 2521 2479 2478 2475 2477 2473 2472 2471 2476 2512 2516 2515 2514 2513 2510 2519 2474 2518 2517 2511 20 laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please e Table 5 Contents of Sup. Fig. 1. Q9 Group Clade Species in core dataset Additional species in extended Additional Species in constrained nLSU dataset dataset

Anaxoboletus Tylopilus Tylopilus plumbviolaceus MB06-056 Tylopilus badiceps 72806 Tylopilus badiceps MB06-053xx Tylopilus felleus AT2001011a Tylopilus ferrugineus MB06-053 Tylopilus indecisus AF456820 Tylopilus intermedius HQ161875 Tylopilus rubrobrunneus Tylopilus plumbeoviolaceus MB06-056? Tylopilus variobrunneus 9306 Tylopilus variobrunneus sn Hor Rubinoboletus 1590 28S Tylopilus ballouii AY612823 Tylopilus felleus sn Aurora Tylopilus felleus AF071466 Tylopilus alboater AF139708 Strobilomyces Strobilomyces floccopus Sf1 Strobilomyces sp 4515 Strobilomyces floccopus AY612824 Afroboletus luteolus 00436a Strobilomyces dryophilus 72106 Strobilomyces sp. DQ534627 Boletus sp. 4689 ‘Porcini’ clade Boletus subalpinus 27882 N/A Boletus edulis Boletus aereus 8721 Boletus pinophilus AF462358 Boletus variipes var. fagicola 4249 Boletus subcarulescens 4070 Boletus edulis Be1a Boletus pinophilus AF462356 Boletus semigastroides PBM3076a Boletus edulis AF462356 Boletus separans DPL2704 Boletus edulis AF291300 Boletus rex veris EU232005 Boletus fibrillosus Thiers 6995 Boletus fibrillosus PBM1342 Boletus cf edulis AF462357 Boletus cf edulis AF462355 Boletus quercophilus EU232001 Boletus edulis AY612802 Boletineae

Boletus gertrudiae AF457407 FUNBIO389_proof Boletus nobilis EU232002 Boletus violaceofuscus AF457403

uglBooy(03,http:// (2013), Biology Fungal , Boletus separans AF457404 Tylopius rhaodsiae AY612836 Boletus variipes BD378 HQ161846 Boletus variipes BD245 EU232003 Boletus aurantioruber MEN11-0 MB ■ ue2013 June 7 Xerocomellus Xerocomellus zelleri 8724 N/A Xerocomellus chrysenteron IM200000405 a

Xerocomellus chrysenteron Xch1 Xerocomellus cf porosporus MB00-017 Nuhn E. M. Xerocomellus cisalpinus AT2005034 Xerocomellus chrysenteron 8723 Xerocomellus porosporus IB19880304 Xerocomellus porosporus IB19990957 ■ 20/33 Xerocomus porosporus AF050645

Xerocomellus truncates al. et Octavania columelifera EF183541 2600 2544 2566 2564 2549 2548 2568 2569 2596 2595 2594 2593 2592 2590 2589 2588 2587 2586 2585 2584 2583 2582 2581 2580 2599 2598 2540 2539 2538 2536 2543 2542 2546 2545 2565 2563 2562 2560 2559 2558 2556 2555 2554 2553 2552 2550 2567 2597 2591 2579 2578 2575 2537 2561 2557 2570 2577 2547 2573 2572 2541 2571 2551 2576 2574 2644 2664 2649 2648 2606 2604 2665 2663 2662 2660 2646 2645 2643 2642 2640 2609 2608 2605 2603 2602 2659 2658 2656 2655 2654 2653 2652 2650 2639 2638 2636 2634 2632 2630 2629 2628 2626 2625 2623 2622 2620 2607 2635 2633 2624 2627 2661 2601 2657 2647 2637 2641 2631 2621 2651 2612 2616 2615 2614 2613 2610 2619 2618 2617 2611 hlgntcoeve fthe of overview Phylogenetic laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please Octavania columelifera EF183544 Xerocomellus fennicus H126 Xerocomellus fennicus RJ126 Xerocomellus ripariellus GR22465 Xerocomellus ripariellus IB19980360 Xerocomellus pruinatus AF050644 Xerocomellus pruinatus IB19980368 Xerocomellus cf. chrysenteron PBM925 Xerocomus truncates 8718 Xerocomus dryophilus IB19991057 Xerocomus dryophilus IB19990901 Xerocomellus zelleri 8724 Boletineae Xerocomus cisalpinus Xerocomus cisalpinus IB20000701 Xerocomellus chrysenteron AF071537 Xerocomus spadiceus AY612842 rubellus Xercomellus rubellus cf. East Coast Xerocomellus rubellus cf. West Coast Xerocomellus rubellus GS961 MB03-033 PBM1331 Xerocomellus rubellus IB19990917 Xerocomellus armeniacus Xerocomus bubalinus MB05-008 badius No representative Xerocomus badius Xb2 Xerocomus badius MB03-00 Xerocomus badius MB03-098 Boletus glabellus 00194 Boletus pallidus 179-97 Xerocomus badius AF050648 Tylopilus sp 00-311 Tylopilus sp 00-388 Tylopilus sp 00-366 No clade designated Xanthoconium strameniuma N/A Tylopilus atronicotianus WV Porphyrellus porphyrosporusa Tylopilus atronicotianus PA Tylopilus alboater AY612832 Porphyrellus sordidus DQ534644 Porphyrellus porphyrosporus MB97-023 Porphyrellus pseudoscaber EU685107 Porphyrellus porphyrosporus DQ534642 Boletineae Porphyrellus sp. 00-348 FUNBIO389_proof Vateriopsis sp. EcM L2481 AM412263 Xanthoconium affine var. maculosum HQ161845 Xanthoconium affine AY612838 uglBooy(03,http:// (2013), Biology Fungal , Xanthoconium affine AF457406 Xanthoconium sp. MB06-049 Tylopilus badiceps AY612833 ■

Leccinoid Leccinum No representative Leccinum scabrum Ls1 Leccinum manzanitae 2013 June 7 Leccinum albellum MB06-040 Leccinum aurantiacum AY612810 Leccinum cerinum AF139692 Leccinum piceinum Leccinum quercinum ■

Leccinum monticola HQ161869 21/33 Leccinum percandidum AF139702 Leccinum aurantiacum AF139689

(continued on next page) 21 2666 2668 2669 2689 2688 2687 2686 2685 2684 2683 2682 2681 2680 2700 2699 2667 2696 2695 2694 2693 2692 2690 2698 2679 2678 2675 2704 2706 2670 2677 2697 2703 2702 2705 2709 2708 2673 2672 2691 2707 2723 2722 2720 2726 2725 2730 2729 2728 2671 2724 2676 2701 2727 2721 2674 2712 2710 2716 2715 2714 2713 2719 2718 2717 2711 2759 2758 2756 2755 2754 2753 2752 2750 2795 2794 2793 2792 2790 2789 2788 2787 2786 2785 2784 2783 2782 2781 2780 2779 2778 2775 2739 2738 2736 2734 2732 2777 2770 2735 2733 2773 2772 2768 2766 2764 2757 2791 2771 2769 2765 2763 2762 2760 2767 2744 2737 2776 2751 2749 2748 2731 2746 2745 2743 2742 2740 2761 2774 2747 2741 22 laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please Table 5 e (continued) Group Clade Species in core dataset Additional species in extended Additional Species in constrained nLSU dataset dataset

Leccinum calltitrichum AF139695 Leccinum nigellum ined AF139699 Leccinum aerugineum Leccinum rigidipes Leccinum rotundifoliae AF139704 Leccinum versipelle AF139707 Leccinum schistophilum Leccinum scabrum AY612814 Leccinum ﬂavostipitatum AF139696 Leccinum variicolor AF139706 Leccinum palustre AF139701 Leccinellum Leccinellum corsicum BUF4507 Leccinellum crocipodium 9382809a Leccinellum corsicum AF139693 Leccinellum lepidum AF139698 Leccinellum carpini AF139691 Leccinum rugosiceps AY612813 Chamonixia sp DQ218598 Rossbeevera westraliensis HQ647164 Rossbeevera vittatispora HQ647163 Rossbeevera pachyderma HQ647157 Rossbeevera pachyderma DQ534620 Leccinum albellum AY612811 Chamonixia caespitosa AF336245 Boletus longicurvipes AF139688 Boletus longicurvipes AY612812 No clade designated Spongiforma thailandia DED7873a N/A Octaviania asterosperma Retiboletus griseus Both Octaviania tasmanica HQ547147 Austroboletus ﬂavidus DPL7541 Durianella echinulata EU293062 Tylopilus aff chromapes 01-513 Boletineae

Tylopilus aff chromapes 01-549 FUNBIO389_proof Tylopilus aff virens DQ534621 Tylopilus virens DQ534621

uglBooy(03,http:// (2013), Biology Fungal , Harrya chromapes HQ161856 Harrya chromapes AY612834 Harrya chromapes AF139709 Retiboletus retipes AF456808 Retiboletus sp MAN-053 ■ ue2013 June 7 Retiboletus retipes AF456830 Retiboletus retipes AF456807 .E Nuhn E. M. Retiboletus retipes AF456811 Retiboletus retipes AY612821 Retiboletus griseus Bothsn1 ■ 22/33 Tylopilus sp AF456813

Retiboletus nigerrimus AF456832 al. et Retiboletus flavoniger AF456829 2800 2844 2849 2848 2806 2804 2832 2830 2829 2834 2840 2839 2838 2836 2843 2842 2846 2845 2860 2859 2858 2856 2855 2854 2853 2852 2850 2805 2803 2802 2809 2808 2823 2822 2820 2826 2825 2828 2833 2835 2807 2824 2827 2796 2799 2798 2837 2857 2801 2831 2847 2797 2812 2821 2841 2851 2810 2816 2815 2814 2813 2819 2818 2817 2811 2900 2906 2904 2868 2866 2864 2909 2908 2905 2903 2902 2925 2923 2922 2920 2899 2898 2896 2895 2894 2893 2892 2890 2889 2888 2887 2886 2885 2884 2883 2882 2881 2880 2879 2878 2875 2869 2865 2863 2862 2907 2924 2867 2877 2870 2897 2873 2872 2871 2901 2891 2861 2921 2876 2912 2874 2916 2915 2914 2913 2910 2919 2918 2917 2911 hlgntcoeve fthe of overview Phylogenetic laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please Retiboletus flavoniger AF456828 Retiboletus ornatipes AF456819 Retiboletus ornatipes AF456825 Pulveroboletus curtisii AY612820 Spongiforma squarepantsii LHBF14 HQ724509 Spongiforma squarepantsii LHBF01 HQ724510 Hypoboletus Aureoboletus Aureobotelus thibetanus Aureoboletus moravicus Xleo1 Aureoboletus auriporus AY612819 Aureoboletus roxanae DS629-07 Aureoboletus gentilis Pug1a Aueroboletus viridiflavus Boletus abruptibulbus 4588 Boletellus shichianus AY612805 Boletellus projectellus Aureoboletus innixus MB03-101 Aureoboletus auriporus Boletus campestris Aureoboletus citrinoporus 8719 Boletineae Boletellus russellii AF050651 Boletellus russellii DPL6698 Aureoboletus innixus Aureoboletus innixus 136 Pulveroboletus auriflammeus AY612818S Boletellus shichianus Boletus atkinsonianus 4471 Boletus atkinsonianus 4557 Boletellus mirabilis AF050652 Boletellus mirabilis 8717 Boletellus projectellus sn2 Hor Hemileccinum Hemileccinum impolitum Bn1a N/A Hemileccinum depilaturm AF139712 a Boletus subglabripes 72206bs Boletus rubropunctus MB05-003 Xerocomus Xerocomus subtomentosus Xs1a Xercomus perplexus MB00-005 Boletus tenax 6641 Phylloporus pelletieri Pp1a Boletus tenax 6871 Xerocomus chrysonemus JAM0539 Xerocomus subtomentosus AF514831 Boletus leptospermi Xerocomus illudens AY612840 Xerocomus illudens AF139714 Xerocomus lanatus Xerocomus subtomentosus AF514832 Boletineae Xerocomous silwoodensis JAM0612 FUNBIO389_proof Phylloporus arenicola 27954 Phylloporus phaeoxanthus MAN-017 Phylloporus alborufus MAN-022 uglBooy(03,http:// (2013), Biology Fungal , Phylloporus leucomycelinus MB05-007 Phylloporus rhodoxanthus 16 196 Phylloporus rhodoxanthus U11925 ■

Phylloporus purpureus MAN-050 2013 June 7 Phylloporus centroamericanus MAN-037 Phylloporus bellus AY612817 Xerocomus amazonicus AY612839 Xerocomus sp AY612841 ■

Xerocomus hortonii AF139713 23/33 Boletus sp 4698 No clade designated Boletellus chrysenteroides 3838 Boletus morrisii 8206

(continued on next page) 23 2944 2966 2964 2968 2949 2948 2965 2963 2962 2960 2959 2958 2969 2926 2932 2930 2929 2928 2934 2940 2939 2938 2936 2943 2942 2946 2945 2950 2956 2955 2954 2953 2952 2990 2989 2988 2987 2986 2985 2984 2983 2982 2981 2980 2967 2933 2935 2927 2975 2979 2978 2961 2970 2977 2937 2957 2973 2972 2931 2947 2971 2941 2951 2976 2974 3000 3006 3004 3009 3008 3005 3003 3002 3044 3007 3049 3048 3046 3045 3043 3042 3040 3055 3054 3053 3052 3050 3039 3038 3036 3034 3032 3030 3029 3028 3026 3025 3023 3022 3020 3001 2999 2998 2996 2995 2994 2993 2992 3035 3033 3024 3027 2997 3047 3037 2991 3041 3031 3021 3051 3012 3016 3015 3014 3013 3010 3019 3018 3017 3011 24 laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please Table 5 e (continued) Group Clade Species in core dataset Additional species in extended Additional Species in constrained nLSU dataset dataset

Boletellus betula AF050642 Boletellus betula AY612797 Gastroboletus vividus 27480 Boletus smithii 8727 Boletus miniatoolivaceus 4091 Boletellus ananas AY612799 Boletellus ananas HQ161853 Cf Boletellus TAA195080 Intsia Boletellus cf ananiceps 00-335 Phophyrellus sp 00-356 Heimioporus retispora AF050650 Boletellus chrysenteroides Boletellus dicymbophilus HQ161852 Boletellus piakaii HQ161861 Boletus morrisii 8206 No group Royoungia No representative Royoungia boletoides 27 546a Royoungia sp 24836 No clade Bothia castanella MB03-053a N/A Tylopilus aff eximius 01528 Austroboletus gracilis 112-96 Boletus eximius AF139684 Austroboletus niveus Austroboletus novaezealandicae Austroboletus mucosus AY612798 Tylopilus aff conicus 01533 Fistulinella viscida AF456826 Pulveroboletus sp 00317 Pulveroboletus sp 00507 Pulveroboletus sp 00428 Boletus modestus No group Bicolor clade Boletus rufomaculatus4414 N/A Boletus subluridellus 3737 Boletineae

Boletus bicolor var. bicolor MB07-001 Boletus bicolor MB06-037 FUNBIO389_proof Boletus bicolor var subreticulatus 3818 Boletus sp bicolor 3921

uglBooy(03,http:// (2013), Biology Fungal , Boletus sensibilis var subviscidus 3929 No group Dupainii clade Boletus inedulis MB06-044 Boletus calopus Bc1 Boletus inedulis AY612803 Boletus ﬁrmus MB06-060 Boletus rhodosanguineus 4254 Boletus roseipes 4515 Boletus dupanii JAM0607 Boletus luridiformis AT2001807 Boletus coniferarum AF456827 Boletus amygdalinus 112605ba Pulveroboletus ravenelii 64-96 ■ ue2013 June 7 Boletus rubripes 8722 Boletus calopus 112606 .E Nuhn E. M. Boletus gyrodontoides MS5 Boletus inedulis sn2002 Chalciporus ovalisporus ■ 24/33 Boletus satanas AF336242

Porphyrellus brunneus DQ534630 al. et Boletus pulcherrimus 00291 3064 3066 3068 3063 3062 3060 3065 3069 3096 3095 3094 3093 3092 3090 3099 3098 3059 3058 3056 3067 3089 3088 3087 3086 3085 3084 3083 3082 3081 3080 3079 3078 3075 3097 3061 3070 3077 3091 3057 3073 3072 3071 3076 3100 3120 3074 3104 3106 3103 3102 3105 3109 3108 3107 3101 3112 3110 3119 3116 3115 3114 3113 3118 3117 3111 3144 3129 3128 3126 3125 3123 3122 3168 3166 3164 3149 3148 3124 3169 3165 3163 3162 3160 3159 3158 3156 3155 3154 3153 3152 3150 3146 3145 3143 3142 3140 3139 3138 3136 3134 3132 3130 3127 3167 3135 3133 3185 3184 3183 3182 3181 3180 3121 3179 3178 3175 3161 3157 3137 3177 3170 3147 3131 3173 3172 3141 3171 3151 3176 3174 hlgntcoeve fthe of overview Phylogenetic laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please Boletus eastwoodiae AT2003096 Boletues satanas AF071528 Boletus rodoxanthus Boletus erthyropus 3131 USA Boletus torosus Boletus sp 4696 Boletus rhodopus 4590 Boletus cf luridus MAN-011 Costa Rica Boletus luteocupreus Boletus luridus AF139686 Boletus erythropus AF139863 Boletus junquilleus Boletineae Regius clade Boletus appendiculatus Bap1 Boletus peckii 3959 Boletus regius Boletus pulchriceps DS4514 Boletus regius 11265 Boletus speciosus var brunneus 4258 Boletus roseopurpureus MB04-040 Boletus abieticola 26763 Boletus appendiculatus 8720 Boletus roseopurpureus 3765 Boletus roseopurpureus 4497 No clade N/A N/A Boletus speciosus Boletus fechtneri 2003097 Boletus fechtneri AF456821 Boletus ﬂoridanus HQ161859 No group Carminipes clade Boletus carminipes MB06-061 Boletus bicolor var borealis 2858 Boletus bicolor AY612800 Boletus pulverulentus 9606 Boletus pseudosensibilis DS615-07 Boletus pallidoroseus sn Hor Boletus subvelutipes AY612804 Boletus vermiculosus Gastroboletus turbinatus AF3362448 Boletus billieae 4588 Boletus rubroﬁbrillosus 3094 Phylloboletellus chloephorus Boletus bicolor snOH Boletus carminipes 4591 Boletus pallidoroseus HQ161860 Boletineae Boletus hypocarcinus DPL6899 FUNBIO389_proof Boletus oliveisporus 6823 No group No clade Pseudoboletus parasiticus Xpa1a N/A Pseudoboletus parasiticus 151a 1Chalciporus Chalciporus Chalciporus piperatus MB04-001a Chalciporus rubinus 4640-3 Chalciporus piperatus NSL-15a uglBooy(03,http:// (2013), Biology Fungal , Chalciporus pseudorubinellus 4302 Chalciporus piperatus AF336244a Chalciporus rubinellus Chalciporus amarellus AF456835 ■

Tylopilus tabacinus 6789 2013 June 7 Chalciporus pseudorubinellus DS612-07 Chalciporus pseudorubinellus BN07 Chalciporus amarellus 8434 No clade designated No representative Buchwaldoboletus lignicolaa N/A ■ a Pul1 25/33 Paxillus Paxillus No representative Paxillus involutus AF167683a Paxillus involutus AF167684a

(continued on next page) 25 3200 3244 3206 3204 3223 3222 3220 3226 3225 3232 3230 3229 3228 3234 3239 3238 3236 3250 3249 3248 3205 3203 3202 3209 3208 3224 3233 3235 3240 3243 3242 3246 3245 3207 3227 3237 3201 3221 3231 3247 3241 3212 3219 3196 3195 3194 3193 3192 3190 3199 3198 3210 3216 3215 3214 3213 3189 3188 3187 3186 3218 3197 3191 3217 3211 3300 3268 3266 3264 3269 3265 3263 3262 3260 3259 3258 3256 3255 3254 3253 3252 3299 3298 3296 3295 3294 3293 3292 3290 3289 3288 3287 3286 3285 3284 3283 3282 3281 3280 3306 3304 3267 3309 3308 3305 3303 3302 3297 3279 3278 3275 3307 3277 3270 3261 3257 3291 3273 3272 3301 3271 3251 3276 3274 3312 3315 3314 3313 3310 3311 26 laect hsatcei rs s unM,e l,Pyoeei vriwo the of overview Phylogenetic al., et ME, Nuhn as: press in article dx.doi.org/10.1016/j.funbio.2013.04.008 this cite Please Table 5 e (continued) Group Clade Species in core dataset Additional species in extended Additional Species in constrained nLSU dataset dataset

Paxillus vernalis Pv2 Paxillus involutus AF167682a Paxillus obscurisporus Po1 Paxillus involutus AY612815a Paxillus ﬁlamentosus Pf1 Paxillus involutus AF1677018 Paxillus involutus AF167686a Paxillus ﬁlamentosus AF167681 No clade designated Gyrodon lividus g11a N/A Gyrodon lividus AF167677 a Paragyrodon sphaerosporus MB06- Gyrodon monticola AF352040 066a Alpova trappei 28042 Alpova trappei AF071456 Melanogaster variegates Melanogaster broomeianus AF098383 Melanogaster tuberiformis AF167679 Melanogaster tuberiformis AF074919 Melanogaster ambiguous AF352046 Alpova diplophloeus AF071454 Alpova diplophloeus AF352035 Paragyrodon sphaerosporusa AF071531 No group No clade Hydnomerulius pinastria N/A Hydnomerulius pinastri AF352044a Hydnomerulius pinastri China Wanga Outgroup N/A Boletinellus merulioidesa N/A N/A

a Type species for genus. Boletineae FUNBIO389_proof uglBooy(03,http:// (2013), Biology Fungal , ■ ue2013 June 7 .E Nuhn E. M. ■ 26/33 tal. et 3344 3349 3348 3364 3366 3368 3363 3362 3360 3365 3369 3380 3326 3325 3323 3322 3320 3329 3328 3340 3343 3342 3346 3345 3359 3358 3356 3355 3354 3353 3352 3350 3367 3324 3332 3330 3334 3339 3338 3336 3327 3333 3335 3361 3375 3379 3378 3357 3370 3377 3321 3337 3347 3373 3372 3331 3341 3371 3351 3376 3374 3316 3319 3318 3317 FUNBIO389_proof ■ 7 June 2013 ■ 27/33

Phylogenetic overview of the Boletineae 27

3381 separate Tylopilus from Boletus s.str. The context is often bitter Xanthoconium stramineum and Porphyrellus porphyrospor- 3446 3382 tasting in the type, T. felleus and a few other species, but not usdBoth are type species of their respective genera, are 3447 3383 3448 everyone is able to detect this (personal experience; Arora strongly supported as members of the anaxoboletus group, 3384 3449 1986). but their placement within the anaxoboletus group in respect 3385 3450 The species in the three-gene extended Tylopilus clade are to the other members is not resolved. This is in conflict with 3386 3451 3387 members of either the stirps felleus or indecisus of Smith and the topology presented by Hosen et al. (2012), but there is no 3452 3388 Thiers, and Singer’s Tylopilus subsections Tylopilus or Oxyd- support for nodes along the backbone of that phylogeny 3453 3389 ables (Singer 1986; Smith & Thiers 1971). The morphological either. 3454 3390 concept of Tylopilus circumscribes a non-monophyletic group- In the constrained analysis, P. porphyrosporus forms a clade 3455 3391 ing of species, despite recent works that have removed several with 99 % BS support with five other Porphyrellus taxa. Xantho- 3456 3392 species previously assigned to either Boletus, Tylopilus or Lecci- conium stramineum forms a clade with 80 % BS containing four 3457 3393 num, and created the new genera Australopilus, Harrya, and other Xanthoconium taxa and Tylopilus badiceps, which may be 3458 3394 Sutorius (Halling et al. 2012a, b). Smith and Thiers’s (1971) mor- misidentified. The placement of the clade containing X. stra- 3459 3395 3460 phological concept of the stirps felleus and indecisus is species mineum and P. porphyrosporus in the three-gene analysis is to- 3396 3461 that have smooth spores that are pinkish to vinaceous in pologically similar to the results of other studies (Desjardin 3397 3462 3398 print, pileus that is violaceous, vinaceous-brown or tan-ish, et al. 2008, 2009; Dentinger et al. 2010). 3463 3399 with a reticulate stipe. Singer’s (1986) concept of the Tylopilus Morphologically, Xanthoconium was separated from Boletus 3464 3400 sections Tylopilus and Oxydables encompasses smooth spored based on unique golden yellow colour of the spores under mi- 3465 3401 species, with a spore print that is pinkish to a grey-brown col- croscopic view and in deposit, and the absence of certain pig- 3466 3402 our, a stipe that is typically reticulated, and a pileus colour ments, though Singer neither mentions nor provides a citation 3467 3403 that is highly similar to Smith and Thiers’s (1971) concept. Fur- for these different pigments (Singer 1944, 1947, 1986). Further- 3468 3404 ther sampling is needed to know if these stirps and sections more, Singer (1986) separated Xanthoconium from Tylopilus 3469 3405 are sufficient to circumscribe the members of the least inclu- based on the colour of the spores, the unchanging white, 3470 3406 3471 sive clade containing T. felleus. mild context, and the non-reticulate, non-pustulate-floccose 3407 3472 Strobilomyces clade [100/95/1.0]dThis placement of the stipe. Smith and Thiers’s (1971) concept did not recognize Xan- 3408 3473 Strobilomyces clade in relation to other Boletineae genera is thoconium as unique, instead placing the species in Boletus sub- 3409 3474 3410 novel. Afroboletus luteolus receives strong support as the sister section Versicolores strips Affinis. 3475 3411 group of Strobilomyces [100/95/1.0]. Afroboletus was first recog- Later works included reticulate species in an expanded 3476 3412 nized as unique by Singer (1945) and placed in Strobilomyces concept of Xanthoconium (Smith 1973; Wolfe 1987; Halling & 3477 3413 section Pterospori; the only other section in Strobilomyces be- Both 1998). Singer (1944) originally placed Xanthoconium strami- 3478 3414 sides section Strobilomyces. Later, Pegler & Young (1981) recog- neum and X. affine in Xanthoconium, both of which were de- 3479 3415 nized Afroboletus as a distinct genus based on unique spore scribed as having a glabrous stipe, but other authors have 3480 3416 morphology. As with Tylopilus, the clade containing all of the reported reticulation at the apex of the stipe of these two spe- 3481 3417 three-gene Strobilomyces clade members (including Afrobole- cies (Wolfe 1987, 1988, 1989; Halling & Both 1998; Bessette et al. 3482 3418 3483 tus) receives no support in the constrained analysis. However, 2000). Halling & Both (1998) placed Boletus separans in the ge- 3419 3484 a clade containing the three-gene Strobilomyces spp, and not nus, partially based on previous inclusions of reticulate spe- 3420 3485 3421 Afroboletus luteolus, receives 99 BS support in the constrained cies in the genus. Molecular data does not support the 3486 3422 analysis and includes S. dryophilus, a different S. floccopus ex- inclusion of B. separans in Xanthoconium (see Figs 1 and 2). Xan- 3487 3423 emplar and a different Strobilomyces sp. exemplar. thoconium separans is described as reticulate on at least the up- 3488 3424 The species of the Strobilomyces clade are morphologically per portion of the stipe or finely reticulate as in Boletus edulis, 3489 3425 distinct, with a veil and coarsely fibrillose to conspicuously while all members of Xanthoconium with molecular support, 3490 3426 scaly pileus with scales turning grey to black (Smith & X. stramineum and X. affine in this study and Xanthoconium pur- 3491 3427 Thiers 1971). Strobilomyces spores are globose, with reticulate pureum (¼Boletus purpureofuscus H.V. Smith & A.H. Smith) in 3492 3428 or variously echinate to cristate ornamentation (see Table 2), Dentinger et al. (2010), are described as glabrous or reticulate 3493 3429 3494 never having longitudinal costae and a dark rusty brown to only at the very apex of the stipe (Smith & Thiers 1971; 3430 3495 blackish brown spore print (Smith & Thiers 1971; Pegler & Singer 1986; Wolfe 1987, 1988; Bessette et al. 2000). Addition- 3431 3496 Young 1981). Pegler & Young (1981) moved members of ally, X. separans shares the ‘stuffed’ pore feature found in all 3432 3497 3433 Singer’s section Pterospori to Afroboletus based on spore shape non-secotioid members of the ‘porcini’ group (Smith & 3498 3434 and ornamentation, subglobose to ellipsoid, longitudinally Thiers 1971; Singer 1986; Dentinger et al. 2010). 3499 3435 costate, with a basal thickened rim (around apiculus), and ‘porcini’ Clade [99/93/1.0]d This group is recovered with 3500 3436 the restriction of these species to Africa. Because recent similar placement in the Boletaceae as reported by Dentinger 3501 3437 works have described both morphological affinities and et al. (2010) and Li et al. (2011). Following the terminology out- 3502 3438 clades containing a genus or closely related genera with spe- lined by Dentinger et al. (2010), the clade containing Boletus 3503 3439 cies from multiple continents, e.g. Sutorius spp., Harrya chro- edulis but not Xanthoconium separans is Boletus sensu stricto, 3504 3440 mapes and H. chromapes like species and T. ballouii, it seems and the clade containing X. separans but not B. edulis is ‘allobo- 3505 3441 3506 plausible that Afroboletus may be restricted to Africa and letus.’ Notholepiota areolata is placed in the ‘porcini’ clade for 3442 3507 could either be the sister genus of Strobilomyces or merely the first time. Notholepiota areolata was originally described 3443 3508 3444 a geographically isolated Strobilomyces lineage (Wolfe & as a secotioid member of the Agaricaceae by Horak (1971), 3509 3445 Bougher 1993; Osmundson & Halling 2010; Halling et al. but was later shown to have affinities with the Boletaceae 3510 2012a, b, 2008). (Vellinga 2004). Xanthoconium separans was previously placed

28 M. E. Nuhn et al.

3511 in Boletus and removed based on morphological characters, morphology, however, they form a clade that lacks support 3576 3512 but is not closely related to the type of Xanthoconium (see in our analyses. Members of these clades were distinguished 3577 3513 3578 Figs 1 and 2; Halling & Both 1998). from Boletus based on a hymenophore trama that is interme- 3514 3579 An equivalent ‘porcini’ clade is not supported in the con- diate between boletoid and phylloporoid types, a lateral stipe 3515 3580 strained analysis, but the two major clades inside the ‘porcini’ trama that is reduced, no thicker than 30e40 mm and non- 3516 3581 are supported in the constrained analysis, with 89 % BS for Bo- gelatinized or absent lateral stipe trama, and spores that are 3517 3582 3518 letus s.str. clade and 95 % BS for the ‘alloboletus’ clade, includ- longitudinally striate or smooth, never bacillate (Sutara 3583 3519 ing N. areolata. 2008). Boletus has spores that are always smooth, boletoid 3584 3520 Morphologically, Boletus s.str. has been united by a variety type hymenophore trama, and lateral stipe stratum that is 3585 3521 of characters: white (or pale yellow) unchanging context tis- 60e90 mm thick and often gelatinized (Sutara 2008). The gelati- 3586 3522 sue that is mild tasting, smooth spores that are yellow- nized trama appears as viscid fluid and/or partially dissolved 3587 3523 brown or olive-brown in deposit, stipe with enlarged base hyphae surrounding the normal hyphae in the trama. 3588 3524 and reticulum, a hyphal mat (cheilocystidia) that covers the Additionally, Sutara (2008) separated Xerocomellus from 3589 3525 3590 tubes of immature specimens, leading to the ‘plugged’ or ‘stuf- Xerocomus on the lack of bacillate spores, an intermediate 3526 3591 fed’ pore feature (Coker & Beers 1943; Smith & Thiers 1971; form of hymenophore trama that is never of the phylloporoid 3527 3592 3528 Singer 1986; Dentinger et al. 2010). The ‘stuffed’ pore feature trama type, and a pileipellis that is a palisadoderm that typi- 3593 3529 has been reported in species that are distantly related to Bole- cally is heavily encrusted; not a trichoderm with little or no 3594 3530 tus s.str., including a Phlebopus sp. (Sclerodermatineae)(Singer encrustations. See Table 1 for the overview of the morpholog- 3595 3531 1986; Miller Jr et al. 2000) and is mimicked in Xanthoconium ical features of Boletus, Xerocomellus, Xerocomus. 3596 3532 sensu Singer by compressed pore mouths in young specimens ‘leccinoid’ group [95/-/0.99]dThere are two clades, Lecci- 3597 3533 (Singer 1986). Despite variations in morphological features, num and Leccinellum, and two additional species, Spongiforma 3598 3534 a combination of white context, reticulated stipe, smooth thailandica (type specimen) and Retiboletus griseus in this group. 3599 3535 spores, pore surface that is white or nearly white when young, Leccinum clade [100/100/1.0]dThe clade contains Leccinum 3600 3536 3601 basal mycelium that is white, and a ‘stuffed’ pore appearance albellum and Leccinum scabrum. The clade in the constrained 3537 3602 due to a mycelium layer growing over the pore surface appear- analysis containing the two Leccinum species receives 100 % 3538 3603 unique to the ‘porcini’ clade (Coker & Beers 1943; Smith & BS support and contains 20 extra Leccinum taxa. A more inclu- 3539 3604 3540 Thiers 1971; Singer 1986; Dentinger et al. 2010). Dentinger sive clade in the constrained analysis contains Octaviania 3605 3541 et al. (2010) provide an in-depth overview of morphological asterosperma and Octaviania tasmanica, but only receives 60 % 3606 3542 traits in the ‘porcini’ group. BS support. 3607 3543 Xerocomellus clade [100/100/1.0]dThe placement of the Xero- Leccinellum cladedThe separation of Leccinellum corsicum 3608 3544 comellus clade in the ‘anaxoboletus’ is mirrored in Li et al. (2011), and the type species of Leccinellum, Leccinellum crocipodium, 3609 3545 though there is no support for a group corresponding to the from the genus Leccinum is supported in our analysis [81/95/- 3610 3546 ‘anaxoboletus’ in Li et al. (2011).TheXerocomellus clade contains ], and the clade containing both Leccinellum and Leccinum re- 3611 3547 the type species, Xerocomellus chrysenteron, and the division be- ceives maximum support in all analysis methods. 3612 3548 3613 tween Xerocomellus and Xerocomus subtomentosus based on mor- Previous studies support the division of Leccinum and Lecci- 3549 3614 phological features is supported by a hosteparasite study of the nellum, and the close relationship between the two genera 3550 3615 group and the phylogeny in Figs 1 and 2 (Douhan & Rizzo 2003; (Binder & Besl 2000; Bresinsky et al. 2003; Dentinger et al. 3551 3616 3552 Sutara 2008). Further, Peintner et al. (2003) support the division 2010). The branch leading to Leccinum and Leccinellum in the 3617 3553 of the Xerocomellus and ‘rubellus’ clades. The Xerocomus/Xeroco- constrained analysis receives 81 % BS support. There is no 3618 3554 mellus containing clades (Xerocomellus, ‘rubellus,’ and ‘badius’) support for a Leccinellum clade in the constrained analysis. 3619 3555 in the ‘anaxoboletus’ group do form an unsupported clade, Leccinum and Leccinellum species have been circumscribed 3620 3556 but are morphologically more similar to each other than other based on Singer’s (1986) broad classification, or Smith and 3621 3557 members of the ‘anaxoboletus’. Thiers’s (1971) more strict classification. Smith & Thiers 3622 3558 ‘rubellus’ clade[100/100/1.0]dTwo ‘rubellus’ specimens (1971) used the darkening stipe scabrosities and white context 3623 3559 3624 were included, one from Eastern USA and one from Western as the defining characteristic of the genus Leccinum. Sequence 3560 3625 USA. The two specimens form a clade separate from other and chemotaxonomic analysis supports a Leccinum concept 3561 3626 Xerocomellus species. Inclusion of ‘rubellus’ clade in the Xeroco- sensu Smith and Thiers over that of Singer’s (Singer 1986; 3562 3627 3563 mellus clade is only supported by 71 % BS support in the ML Binder & Besl 2000; Bakker & Noordeloos 2005; Binder & 3628 3564 analysis. Further, the constrained analysis gives 96 % BS sup- Hibbett 2006). Bresinsky et al. (2003) separated Leccinellum 3629 3565 port to the clade containing Xerocomellus rubellus species. from Leccinum based on a yellow context, a difference that 3630 3566 ‘badius’ clade [79/-/0.96]dThe two Xerocomus badius speci- Smith & Thiers (1971) noted as well. 3631 3567 mens form a clade with Boletus pallidus, albeit with weak sup- The placement of S. thailandica as sister to the clade con- 3632 3568 port. Boletus pallidus has been previously recovered in a close taining ‘leccinoid’ species is inconsistent with the studies of 3633 3569 relationship to X. rubellus (Desjardin et al. 2009). Inside this Desjardin et al. (2009), Li et al. (2011), and Hosen et al. (2012) 3634 3570 group the X. badius clade receives maximal support. Xerocomus where it is sister to Porphyrellus and Strobilomyces. However, 3635 3571 3636 badius species form a clade with 100 % BS support in the con- Desjardin et al. (2009) used only nuc-lsu sequence data, which 3572 3637 strained analysis including one non-Xerocomellus species, Bole- is too conserved to resolve the Boletineae. Despite this, Li et al. 3573 3638 tus glabellus 00-194. (2011) have support for a clade containing Spongiforma, Por- 3574 3639 3575 Sutara (2008) placed the members of the Xerocomellus, ‘bad- phyrellus, and Strobilomyces, and Hosen et al. (2012) have sup- 3640 ius’, and ‘rubellus’ clades in the genus Xerocomellus based on port for a clade containing Spongiforma and Porphyrellus.In

Phylogenetic overview of the Boletineae 29

3641 the constrained analysis S. thailandica forms a strongly sup- buff spore print as the defining characteristics. The classifica- 3706 3642 ported clade [100] with the only other species in the genus, tion of Smith & Thiers (1971) placed the majority of the Aureo- 3707 3643 3708 Spongiforma squarepantsii. Recently, a new genus, Borofutus, boletus clade in Boletus section Subtomentosi subsection 3644 3709 was shown to be related to Spongiforma (Hosen et al. 2012). Versicolores stirps Roxanae, but placed Boletellus projectellus in 3645 3710 Morphologically, Spongiforma species are distinct. The two the Subsection Mirabiles of section Subtomentosi. Singer’s 3646 3711 3647 species known are both gasteroid, appearing like a sponge (1986) concept placed B. projectellus in section Mirabilis of Bole- 3712 3648 and are unlike any other members of the Boletales. There is tellus, based on the smooth spores, not bluing but often stain- 3713 3649 no gross morphological similarity to place Spongiforma in the ing lemon yellow tissue, and association with Pinaceae and 3714 3650 ‘leccinoid’ clade, or in any other Boletineae clade. Fagaceae. This is in conflict with Smith & Thiers (1971) Boletel- 3715 3651 Retiboletus griseus is the sister group of the rest of the ‘lecci- lus concept, which excluded B. mirabilis and B. projectellus, 3716 3652 noid’ group, which is congruent with previous studies placing them in Boletus instead. However, here we use the 3717 3653 (Dentinger et al. 2010; Lebel et al. 2011; Li et al. 2011). In the con- Singer taxonomy as both species are shown to be distantly re- 3718 3654 strained analysis R. griseus is in a distinct, albeit unsupported, lated to B. edulis (Fig 2). There are no apparent morphological 3719 3655 3720 clade that contains all Retiboletus species in the analysis and characters uniting the Aureoboletus clade. Both smooth and 3656 3721 three taxa that are not Retiboletus (See Sup. Fig 1). ornamented (only Boletellus schichianus) spores occur in the 3657 3722 3658 Retiboletus species are highly reticulate along the entire clade; the hymenophore colour is variable: bright lemon yel- 3723 3659 stipe, but morphologically very similar to the classical de- low to ‘pale olive-fresh’; stipe ornamentation ranges from 3724 3660 scription of Boletus; e.g. smooth spores, olive or brownish non-reticulate, to longitudinally striated, to highly or coarsely 3725 3661 spore print (See Table 1; Smith & Thiers 1971; Singer 1986). reticulate; spore print colour is reported as a pale ochraceous 3726 3662 Retipolides, a unique pigment group, was reported in Retibo- buff to dark olive brown; gelatinization of the stipe and pileus 3727 3663 letu ornatipes/Retiboletu retipes (Herman 1980), and with addi- is found in some species; and non-veiled species and veiled 3728 3664 tional molecular analysis, the occurrence of this unique species are present: gelatinous veil, Aureoboletus thibetanus 3729 3665 pigment provided the description of Retiboletus (Binder & and typical veil, B. projectellus are present (Smith & Thiers 3730 3666 3731 Bresinsky 2002b). Not all Retiboletus spp. that were placed in 1971; Singer 1986). In depth taxon sampling is necessary to re- 3667 3732 the genus with molecular methods have been tested for the solve phylogenetic relationships in this clade. 3668 3733 occurrence of retipolides, but in all Retiboletus species chryso- Hemileccinum clade [100/100/1.0]dThis well-supported 3669 3734 3670 cystidia, which are rare or absent in Boletus, are present (Smith clade is sister to the Aureoboletus clade. It contains the type 3735 3671 & Thiers 1971; Singer 1986; Binder & Bresinsky 2002b; Watling species of Hemileccinum, Hemileccinum impolitum and Boletus 3736 3672 2008). subglabripes. This is the first analysis containing both H. impo- 3737 3673 While the type species of Leccinum is not included in core litum and B. subglabripes. Previously, B. subglabripes had been 3738 3674 analyses, the topology of the Leccinum, Leccinellum, and Retibo- placed as a close relative of Leccinellum, Leccinum and Chamo- 3739 3675 letus containing clade is identical to the topology of the com- nixia (Desjardin et al. 2008) and Rossbeevera (Lebel et al. 2011). 3740 3676 bined analysis in Dentinger et al. (2010). All members of the Desjardin et al. (2009) placed H. impolitum and Hemileccinum 3741 3677 ‘leccinoid’ clade (not including Spongiforma) have a boletoid depilatum as sister to a clade equivalent to the three-gene ex- 3742 3678 3743 type tube trama, and most have smooth spores. The exception tended analysis Xerocomus clade. Inclusion of H. depilatum in 3679 3744 is S. thailandica which has rough spores in SEM (Desjardin et al. this analysis would provide a more conclusive view of the 3680 3745 3681 2009). Hemileccinum clade. Hemileccinum species have been placed in 3746 d 3682 ‘hypoboletus’ group[89/73/0.99] The ‘hypoboletus’ group Boletus, Xerocomus, and Leccinum using different generic con- 3747 3683 contains four supported clades, three of which we have given cepts (Smith & Thiers 1971; Singer 1986; Binder & Besl 2000). 3748 3684 designations: Aureoboletus, Hemileccinum, and Xerocomus. Sutara (2008) erected Hemileccinum to accommodate species 3749 3685 Aureoboletus clade [95/77/0.99]dThe Aureoboletus clade con- that are smooth spored, with hymenophore trama that is 3750 3686 tains eight taxa, five Aureoboletus species, two species cur- boletoid, lateral stipe stratum that is gelatinous, and stipe 3751 3687 rently classified in the genus Boletellus by some authors, and scrabrosities. 3752 3688 one Boletus species. The type species, Aureoboletus gentilis is Xerocomus clade [100/100/1.0]dThis clade contains the type 3753 3689 3754 nested in the clade. The placement of Boletellus species within species of Xerocomus and X. subtomentosus. Previous studies 3690 3755 the Aureoboletus clade has been reported in previous studies have reported the distant relationship among the members 3691 3756 (Binder & Hibbett 2006; Desjardin et al. 2009; Dentinger et al. of the Xerocomus clade and the Xerocomellus, ‘badius,’ and 3692 3757 3693 2010). Dentinger et al. (2010) included the type species of Bole- ‘rubellus’ clades (Douhan & Rizzo 2003; Peintner et al. 2003). 3758 3694 tellus, Boletellus ananas, which is placed outside of the clade As previous studies have found, Singer’s view of the close re- 3759 3695 containing the Aureoboletus species in their analysis. The phy- lationship between Xerocomus and Phylloporus is upheld 3760 3696 logeny shown by Li et al. (2011), gives indirect support for this (Singer 1986; Binder & Hibbett 2006; Hosen et al. 2012; Neves 3761 3697 relationship, with Boletellus mirabilis and Boletellus projectellus et al. 2012). 3762 3698 as sister group to Xerocomus illudens and X. subtomentosus, a re- Smith and Thiers’s (1971) concept did not differentiate (at 3763 3699 lationship that has been recovered in the three-gene extended the generic level) between Xerocomus s.l. (Xerocomus and Xero- 3764 3700 analysis with taxon pruning. comellus including the ‘badius’ and ‘rubellus’ clades) and Bole- 3765 3701 3766 Aureoboletus was circumscribed by Pouzar (1957), but tus s.l., stating that the differences in the hypha and 3702 3767 Singer’s (1986) concept placed all the Aureoboletus spp. in- hymenophore trama, when combined with an ambiguous def- 3703 3768 3704 cluded in our analysis in the genus Pulveroboletus sect. Auripori, inition from other authors (including Singer), caused them to 3769 3705 based on the gelatinized pileus, voluminous cheilocystidia place Xerocomus s.l. species in the genus Boletus. Molecular 3770 with yellow sap in ammonia solutions, and an ochraceous work has helped to differentiate between the two genera,

30 M. E. Nuhn et al.

3771 but Sutara (2008) provides a clear morphological framework, by strong support [94/81/0.99]. Only Boletus calopus and Boletus 3836 3772 which is supported by molecular data, to separate the prob- inedulis species have been previously published in phyloge- 3837 3773 3838 lematic genera. netic studies (Binder & Hibbett 2006; Desjardin et al. 2009, 3774 3839 The key characteristics of Xerocomus are: a non-gelatinized 2008; Li et al. 2011; Halling et al. 2012a). A clade containing 3775 3840 lateral stipe stratum, bacillate ornamented spores, and hyme- both B. calopus and B. inedulis is supported in all analysis ex- 3776 3841 3777 nophore trama of the phylloporoid type (Sutara 2008). This is cept for Desjardin et al. (2008), which did not include B. inedulis. 3842 3778 different from Boletus s.str. which generally has a gelatinized Species relationships inside dupainii are poorly resolved. 3843 3779 lateral stipe stratum with hyphae that are distant from each regius clade [100/100/1.0]dInside this clade only Boletus 3844 3780 other, smooth spores, and a boletoid type hymenophore reguis and Boletus appendiculatus have been published in mo- 3845 3781 trama. Furthermore, it is separated from Xerocomellus based lecular studies (Binder & Hibbett 2006; Halling et al. 2012a). 3846 3782 on the presence of longitudinally striate or smooth spores carminipes clade [82/e/0.96]dThis group contains Boletus 3847 3783 that are never bacillate in Xerocomellus, hymenophore trama carminipes, B. bicolor var. borealis, Boletus pseudosensibilis, and 3848 3784 that is intermediate between boletoid and phylloporoid type, Boletus pulverulentus. It is a mixture of different sections of 3849 3785 3850 the lack of a lateral stipe stratum (or a very reduced lateral both Singer’s (1986) concept of the genus Boletus and the sec- 3786 3851 stipe stratum, 30e40 mM compared to 80e200 mMinXerocomus tions of Smith and Thiers’s (1971) concept of the genus Boletus. 3787 3852 s.str.), and a pileipellis constructed of hyphae with encrusted The above groups and clades are supported [79/73/0.99] as 3788 3853 3789 walls (Sutara 2008). a clade, and are more closely related to each other than the 3854 3790 Xerocomus s.str. is separated from Hemileccinum by having remaining members of the Boletineae, Pseudoboletus parasiticus, 3855 3791 bacillate spores, a hymenophore trama that is of the phyllop- the Chalciporus clade, and the Paxillaceae. 3856 3792 oroid type and a lack of stipe scabers or scabrousity that are Pseudoboletus parasiticusdPseudoboletus parasiticus is 3857 3793 typical of Hemileccinum (Sutara 2008). The morphological dis- placed as sister to the species indicated by node B in Figs 1 3858 3794 tinctions between Boletus, Hemileccinum, Xerocomellus, and and 2 [100/100/1.0]. This is consistent with the study of 3859 3795 Xerocomus are succinctly and sufficiently described by Sutara Desjardin et al. (2009). This bolete is distinctive and difficult 3860 3796 3861 (2008). to misidentify when found on its host, Scleroderma citrinum 3797 3862 Royoungia clade [100/100/1.0]dThe monotypic Royoungia (Order Boletales, Suborder Sclerodermatineae), and more rarely 3798 3863 clade contains two specimens of Royoungia boletoides and has on other Scleroderma spp (Smith & Thiers 1971; Singer 1986). 3799 3864 e 3800 previously been shown as closely related, without support, Pseudoboletus parasiticus is characterized by yellow brown fi- 3865 3801 to Harrya chromapes (as Tylopilius, Desjardin et al. 2009) and to brils similar in appearance to the scabers of Leccinum and Lec- 3866 3802 the new genus Zangia (Li et al. 2011). Li et al. (2011) demon- cinellum, an instant cinnabar-orange KOH reaction of the stipe, 3867 3803 strated a sister taxa relationship between Zangia species and orange-ochraceous KOH reaction of pileus, and Phylloporus- 3868 3804 T. chromapes using nuc-lsu, tef1, mitochondrial large subunit, type trama (Smith & Thiers 1971; Singer 1986; Sutara 1991). 3869 3805 mitochondrial small subunit, and atp6. However, the defining characteristic is growth from a fruiting 3870 3806 Recent work by Halling et al. (2012b) has shown strong sup- body from Scleroderma spp. 3871 3807 port for a clade containing Zangia, Royoungia, Harrya, Australo- This clade may contain multiple species. Singer (1986) 3872 3808 3873 pilus, and Tylopilus species which are not Tylopilus s.str. All mentions Xerocomus astraeicola as a possible sister species, 3809 3874 things equal, this clade would be sister to the clade B in and the fruiting bodies of P. parasiticus were collected near, 3810 3875 3811 Figs 1 and 2.The clade appears to be united by pigment in but not on, Scleroderma spp. formed ECM in culture with Pinus 3876 3812 the base of the stipe for stipitate species, and in the peridium resinosa, possibly indicating a new species (Richter & Bruhn 3877 3813 of Royoungia, though the chemical work to identify the pig- 1989). 3878 3814 ment to test for homology across the genera has not been per- Chalciporus group [100/100/1.0]dThe Chalciporus group is 3879 3815 formed. bicolor Clade [100/100/1.0]dThis clade contains two the earliest branching lineage of the family Boletaceae based 3880 3816 species, Boletus bicolor var. bicolor sensu Peck and Boletus rufo- on sampling used in this study, which is indicated at node A 3881 3817 maculatus. The two members of the clade are characterized on Figs 1 and 2. The Chalciporus group includes the monophy- 3882 3818 by distinct yellow pore surface, with minute pore openings, letic genus Chalciporus and Buchwaldoboletus lignicola. The 3883 3819 3884 and stipe colouration and are closely related based on mor- placement of the Chalciporus group in Figs 1 and 2is supported 3820 3885 phology (Bessette et al. 2000; Both 1998; Smith & Thiers by previous studies (Binder & Bresinsky 2002b; Binder & 3821 3886 1971). Peck’s B. bicolor is an illegitimate name; B. bicolor was Hibbett 2006; Desjardin et al. 2009; Li et al. 2011). 3822 3887 3823 originally described by Raddi in 1807, though the details of Singer (1986) defined Chalciporus as having a solid stipe, 3888 3824 what species Raddi named B. bicolor have been lost (Peck a hymenophore that is dull cinnamon red, carmine to wine 3889 3825 1870; Raddi 1807; Snell & Dick 1941). We have chosen to use Bo- red or pink, having basal mycelium that is bright yellow in 3890 3826 letus bicolor sensu Peck, despite the illegitimate naming, as two part or in all, and a taste that is either mild or acrid peppery. 3891 3827 of the three synonyms available would create a different poly- Furthermore, Singer (1986) describes Chalciporus as lacking 3892 3828 tomy in Xerocomus and Boletus and the third places the species clamp connections and having adnate to subdecurrent hyme- 3893 3829 in the now defunct Ceriomyces (Murrill 1909; Singer 1947 1986). nophore, but never arcuate. Smith & Thiers (1971) use similar 3894 3830 Correcting this taxonomic confusion necessitates the descrip- characters to describe Chalciporus species, but place them in 3895 3831 3896 tion of a new genus, but at this time it would be premature to the section Piperati of Boletus. 3832 3897 describe a new species based on the small amount of data on Buchwaldoboletus was described based on the lignicolous 3833 3898 3834 hand. habitat, decurrent and arcuate hymenophore, bluing, yellow 3899 d 3835 dupainii clade [71/ / ] While this clade has support in flesh and yellow basal mycelium (Pilat 1969). In addition, Buch- 3900 only ML methods, it is separated from residual regius clade waldoboletus spp. lack clamp connections in the basidocarp,

Phylogenetic overview of the Boletineae 31

3901 but clamp connections are present in hyphae of cultures (Pilat 3966 3902 1969; Singer 1986; Watling 2008). This is similar to the descrip- Acknowledgements 3967 3903 3968 tion of Chalciporus, and the two genera are united by the yellow 3904 3969 basal mycelium, lack of veil, and overall hymenophore struc- This work has been supported by grants from the National Sci- 3905 3970 ture (Pilat 1969); the respective type species of the genera may ence Foundation, including award DEB-1020421 to REH, DSH 3906 3971 3907 also share a mycoparasitic nutritional mode. and MB. We thank the many collaborators who provided spec- 3972 d 3908 Paxillaceae [100/97/0.99] The Paxillaceae comprises mem- imens and DNA, including Ernst Both and PB Matheny who 3973 3909 bers of the genus Paxillus, and the type species of Gyrodon provided gDNA for Boletus semigastroideus. We would like to 3974 3910 and the monotypic Paragyrodon sphaerosporus. Paxillus is thank Duccio Migliorini for preparing the interactions be- 3975 3911 monophyletic in the Paxillaceae[100/100/1.0] and the Paxillaceae tween Buchwaldoboletus lignicola and Phaeolus schweinitzii. 3976 3912 currently contains only ECM fungi. Alfredo Justo provided helpful comments on the manuscript. 3977 3913 The family includes morphologically diverse taxa. The ge- We thank the two anonymous reviewers who provided help- 3978 3914 nus Paxillus comprises only lamellate fungi, while Gyrodon liv- ful comments and suggestions. 3979 3915 3980 idus produces a tubular hymenophore resembling that of 3916 3981 Boletinellus merulioides (Sclerodermatineae, Boletales), and Para- 3917 Appendix A. Supplementary data 3982 3918 gyrodon sphaerosporus is one of the few species in the Boletineae 3983 3919 with a veil (Smith & Thiers 1971; Singer 1986; Binder & 3984 Supplementary data related to this article can be found at 3920 Bresinsky 2002a; Binder & Hibbett 2006). Despite the large 3985 3921 morphological differences found in the Paxillaceae, all mem- http://dx.doi.org/10.1016/j.funbio.2013.04.008. 3986 3922 bers in this study have clamp connections in the basidiocarp, 3987 3923 an uncommon occurrence in the Boletineae (Singer 1986; references 3988 3924 Binder & Bresinsky 2002a; Watling 2008). 3989 3925 Hydnomerulius pinastridThis resupinate, brown-rot fungus 3990 3926 3991 was considered as a member of the Boletineae incertae sedis 3927 Arora D, 1986. Mushrooms Demystified: a comprehensive guide to the 3992 (Binder & Hibbett 2006), and our analyses lack support for its 3928 fleshy fungi. Ten Speed Press, Berkeley. 3993 placement in both the three-gene core and extended analyses. 3929 Bakker HC, Noordeloos M, 2005. A revision of European species of 3994 3930 It is similar to the members of the Paxillaceae in spore shape Leccinum Gray and notes on extralimital species. Persoonia- 3995 leiden 18: 511. 3931 and ornamentation, and the presence of clamp connections 3996 Besl H, Bresinsky A, 1977. Notizen uber€ Vorkommen und sys- 3932 (Watling 2008). 3997 tematische Bewertung von Pigmenten in hoheren€ Pilzen (2). 3933 3998 Zeitschrift fur Pilzkunde 43: 311e322. 3934 3999 Besl H, Bresinsky A, 1979. Notizen uber€ Vorkommen und sys- 3935 Conclusions tematische Bewertung von Pigmenten in hoheren€ Pilzen (3). 4000 3936 Untersuchungen an Boletales aus Amerika. Zeitschrift fur Pilz- 4001 3937 The analyses presented here identify clades containing the kunde 45: 247e264. 4002 3938 4003 type species for the following genera: Aureoboletus, Boletus, Besl H, Bresinsky A, 1997. Chemosystematics of Suillaceae and 3939 Plant Systematics and 4004 Chalciporus, Hemileccinum, Leccinellum, Leccinum, Paxillus, Roy- Gomphidiaceae (suborder Suillineae). 3940 Evolution 206: 223e242. 4005 oungia, Strobilomyces, Tylopilus, Xerocomellus, and Xerocomus. 3941 Besl H, Bresinsky A, Kammerer€ A, 1986. Chemosystematics of the 4006 3942 The type species of Buchwaldoboletus, Gyrodon, Notholepiota, Coniophoraceae. Zeitschrift fur€ Mykologie 52: 277e286. 4007 3943 Phylloporus, Paragyrodon, Pseudoboletus, Spongiforma, and Xan- Besl H, Michler I, Preuss R, Steglich W, 1974. Pigments of fungi, 4008 3944 thoconium have also been sampled. Three new, strongly sup- XXII Grevillin D, the main pigment of Suillus granulatus, S. lu- 4009 € 3945 ported lineages within the Boletineae were recovered: the teus and S. placidus (Boletales). Zeitschrift fur Naturforschung 4010 e 3946 anaxoboletus, leccinoid, and hypoboletus groups. These re- 784 786. Q4 4011 Bessette A, Roody WC, Bessette A, 2000. Noth American Boletes: 3947 sults could form the basis for a genus-level reclassification 4012 a color guide to the fleshy pored mushrooms, 1st edn. Syracuse 3948 of the Boletineae. However, to achieve a comprehensive phylo- 4013 3949 University Press, Syracuse, New York. 4014 genetic classification of the Boletineae, many more taxa need to 3950 Binder M, Besl H, 2000. 28S rDNA sequence data and chemotax- 4015 be sampled with multiple loci, including taxa from the Xeroco- onomical analyses on the generic concept of Leccinum (Bole- 3951 4016 mellus/Xerocomus containing clades, the leccinoid group and tales) AMB, Italy. Centro Studi Micologici. Micologia 2000: 3952 4017 71e82. 3953 Aureoboletus clade. 4018 Binder M, Bresinsky A, 2002a. Derivation of a polymorphic lineage 3954 The genus Boletus should be restricted to the ‘porcini’ clade; 4019 of Gasteromycetes from boletoid ancestors. Mycologia 94 3955 this would reduce the magnitude of Boletus from approxi- 4020 85e85. Q5 3956 mately 300 to approximately 30 species (Kirk et al. 2008; Binder M, Bresinsky A, 2002b. Retiboletus, a new genus for a spe- 4021 3957 Dentinger et al. 2010). We return Boletus separans to the genus cies-complex in the Boletaceae producing retipolides. Feddes 4022 3958 from Xanthoconium, present Boletus subalpinus comb. nov, for- Repertorium 113:30e40. 4023 3959 mally transferring Gastroboletus subalpinus to Boletus, and Binder M, Hibbett DS, 2002. Higher-level phylogenetic relation- 4024 3960 transfer Notholepiota areolata from the Agaricales to Boletus ships of Homobasidiomycetes (mushroom-forming fungi) in- 4025 3961 ferred from four rDNA regions. Molecular Phylogenetics and 4026 s.str., Boletus semigastroideus nom. nov. By far the largest chal- 3962 Evolution 22:76e90. 4027 lenge to future phylogenetic studies in the Boletineae is resolv- 3963 Binder M, Hibbett DS, 2006. Molecular systematics and biological 4028 3964 ing the relationships of the roughly 270 species that have been diversification of Boletales. Mycologia 98 971e971. 4029 3965 classified in Boletus but are not likely to be placed in the ‘por- Binder M, Larsson KH, Matheny PB, Hibbett DS, 2010. Amylocor- 4030 cini’ clade using molecular markers. ticiales ord. nov. and Jaapiales ord. nov.: early diverging clades

32 M. E. Nuhn et al.

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Please cite this article in press as: Nuhn ME, et al., Phylogenetic overview of the Boletineae, Fungal Biology (2013), http:// dx.doi.org/10.1016/j.funbio.2013.04.008