Multiple Cryptic Species with Divergent Substrate Affinities in the Serpula

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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 Multiple cryptic species with divergent substrate afﬁnities 76 12 Serpula himantioides 77 13 Q1 in the species complex 78 14 79 15 80 a, a b 16 Tor CARLSEN *, Ingeborg Bjorvand ENGH , Cony DECOCK , 81 17 Mario RAJCHENBERGc,Havard KAUSERUDa 82 18 83 19 aMicrobial Evolution Research Group (MERG), Department of Biology, University of Oslo, P.O. Box 1066, Blindern, N-0316 Oslo, Norway 84 20 bMycotheque de l’Universite Catholique de Louvain, Earth and Life Institute e Mycology, 1348 Louvain-la-Neuve, Belgium 85 c 86 21 Centro de Investigacion y Extension Forestal Andino Patagonico, C.C. 238, 9200 Esquel, Chubut, Argentina 22 87 23 88 24 article info abstract 89 25 90 26 91 Serpula himantioides 27 Article history: is a widespread saprotrophic morphospecies mainly colonising conifer- 92 28 Received 4 May 2010 ous wood in nature, but it appears frequently in buildings as well. From an earlier study, it 93 29 Received in revised form is known that at least three divergent lineages occur within the S. himantioides species 94 30 18 August 2010 complex. In this study, a broader sample of S. himantioides isolates has been analysed by 95 31 Accepted 15 October 2010 multi-locus sequencing, including new isolates from Asia, North and South America. Alto- 96 32 Corresponding Editor: gether ﬁve phylogenetical species (PS1e5) were detected, all recognised across indepen- 97 33 Karl-Henrik Larsson dent gene phylogenies. A new southern South American phylogenetic species (PS1) was 98 34 found, representing an early diverging lineage within the S. himantioides species complex. 99 35 100 Keywords: The two closely related PS2 and PS3 lineages included isolates from North America only, 36 101 Cryptic species and PS4 was also dominated by North American isolates. Most of the investigated isolates 37 102 Phylogenetic species (76 %) clustered into PS5, a lineage that has been found on most continents, including 38 103 39 Phylogeography North America. Overall, little phylogeographical structure was found in PS5, indicating fre- 104 40 Saprotrophic quent and recent long-distance dispersal events within this widespread lineage. Our anal- 105 41 Serpula himantioides yses indicate that South and North America are the centres of divergence for the 106 42 S. himantioides species complex. Some of the lineages seem adapted to various substrates, 107 43 but PS5 is able to decay a wide array of angiosperms and gymnosperms, which may have 108 44 facilitated the spread of this lineage throughout the world. 109 45 ª 2010 Published by Elsevier Ltd on behalf of The British Mycological Society. 110 46 111 47 112 48 113 49 114 50 Introduction differentiated lineages were detected within S. himantioides 115 51 across independent gene phylogenies (Kauserud et al. 2006). 116 52 Serpula himantioides (Fr.) P. Karst. is a saprotrophic morphospe- These three lineages also showed compatible mating within 117 53 118 cies with a wide geographical distribution, observed on all con- lineages, and incompatible mating between lineages. Thus, as 54 119 tinents except Antarctica. It is mainly found on dead wood of many other basidiomycetes (Nilsson et al. 2003; Geml et al. 55 120 56 various coniferous tree species, but do also commonly occur 2006; Kauserud et al. 2007a), S. himantioides seems to represent 121 57 in buildings. It produces thin, resupinate and brownish annual a species complex including multiple cryptic lineages. Espe- 122 58 basidiocarps and has a heterothallic tetrapolar mating system, cially when it comes to morphospecies that produces simple, 123 59 based on Northern Hemisphere specimens (Hwang 1955; resupinate fruiting structures, such as S. himantioides,itmight 124 60 Harmsen 1960). In a previous study, three genetically well- be problematic to detect species boundaries by morphological 125 61 126 62 127 63 * Corresponding author. Tel.: þ47 22854588; fax: þ47 22854726. 128 64 E-mail addresses: [email protected], [email protected], [email protected] 129 65 1878-6146/$ e see front matter ª 2010 Published by Elsevier Ltd on behalf of The British Mycological Society. 130 doi:10.1016/j.funbio.2010.10.004

2 T. Carlsen et al.

131 means (Taylor et al. 2006). Likewise, multiple cryptic species has analyses. For Bayesian analyses, the different regions were 196 132 also been detected in several Coniophora species that also pro- analysed with an independent model for each partition 197 133 198 duce simple and resupinate fruiting bodies (Ainsworth & (ITS ¼ GTR þ I, LSU ¼ GTR, tub ¼ GTR þ G, and hsp ¼ SYM þ G). 134 199 Rayner 1990; Kauserud et al. 2007a). Genealogical sorting index (GSI) statistics (Cummings et al. 135 200 Biogeographical studies show that fungi have complex his- 2008) were performed to test for phylogenetic correlation 136 201 137 tories of vicariance and dispersal in the same way as plants between isolates from different substrates, continents, and 202 138 and animals (Taylor et al. 2006; Matheny et al. 2009). Although habitats. Substrates were categorized as Pinus, Picea, Nothofa- 203 139 long-distance dispersal events may be rare, it is the best ex- gus, unknown conifer, and unknown hardwood. Continents 204 140 planation for the present day distribution of many fungal were categorised according to geography and habitat catego- 205 141 taxa (Hibbett 2001; James et al. 2001; Zervakis et al. 2004; rized as indoor or outdoor. 206 142 Hosaka et al. 2008; Moncalvo & Buchanan 2008). For some 207 143 fungi, the natural biogeographical patterns may be blurred 208 144 by modern spread by man (Brasier & Buck 2001; Coetzee Results and discussion 209 145 210 et al. 2001; Slippers et al. 2001; Kauserud et al. 2007b). 146 211 In the dry rot fungus Serpula lacrymans that is closely related In Fig 1, a multi-locus phylogeny of the 67 analysed isolates of 147 212 148 to S. himantioides, there seems to have happened a specializa- Serpula himantioides is shown, demonstrating that the isolates 213 149 tion towards growing in buildings (Kauserud et al. 2007b). group into five different lineages with high support. A high 214 150 Serpula himantioides is also known as a common destroyer of congruence in topology was observed across the four investi- 215 151 wooden constructions, but whether any adaptations towards gated DNA regions when these were analysed separately 216 152 this growing habit have happened in any of the S. himantioides (Supplementary Information, Fig S1AeC). The five lineages 217 153 lineages is not clear. were named PS1ePS5 corresponding to phylogenetic species 218 154 In this study, a broader sample of S. himantioides is included under the phylogenetic species recognition definition (Taylor 219 155 compared to Kauserud et al. (2006), including newly obtained et al. 2000; Kroken & Taylor 2001, 2009). This, together with 220 156 221 isolates from Asia and both of the American continents. The earlier mating experiments that has shown that there are 157 222 aims of this study were to (1) analyse whether even more phy- compatible matings within the lineages PS3, PS4, and PS5 158 223 logenetic species can be found within S. himantioides, (2) check but incompatible across lineages (Harmsen 1960; Kauserud 159 224 e 160 whether a biogeographical structure of various S. himantioides et al. 2006), indicate that PS1 PS5 represent different biologi- 225 161 lineages can be observed, and (3) analyse whether any sub- cal species. As there is only one sample in PS2, and no exper- 226 162 strate specialization has happened during the diversification imental crosses could be performed, it may be premature to 227 163 of the S. himantioides species complex. To illuminate these conclude with certainty that this is a distinct species. But as 228 164 topics, the isolates where analysed by multi-locus sequencing the genetic divergence between PS2 and PS3 is comparable 229 165 of three independent DNA regions. to the divergence to the other phylogenetic species within 230 166 S. himantioides, we will treat it as a separate entity here. 231 167 A recent dated molecular phylogeny of the family Serpula- 232 168 233 Material and methods ceae has dated the oldest divergence of the cryptic lineages of 169 234 S. himantioides to approx. 12 million years ago (Engh et al. in 170 235 171 The material included in this study is listed in Table 1. Com- prep). A similar time estimate was made for the split between 236 172 pared to Kauserud et al. (2006), 40 new isolates of Serpula Serpula lacrymans var. lacrymans and Serpula lacrymans var. 237 173 himantioides were included in this study. DNA was extracted shastensis that are still able to mate in vitro (Harmsen 1960). 238 174 from the new cultures following a 2 % CTAB (cetyltrimethy- Lineage PS1 is sister to the other lineages and includes five 239 175 lammonium bromide) miniprep method described by South American isolates. PS2, PS3, and PS4 include mainly 240 176 Murray & Thompson (1980) with minor modifications: DNA North American isolates. However, the sample size here is 241 177 m 242 was resuspended in 100 L distilled sterile H2O at the final too small to conclude that the lineages are mainly restricted 178 step of extraction. The four DNA markers ITS, LSU, hsp and to this continent. PS5 is a widely distributed lineage that has 243 179 244 tub, were PCR amplified and sequenced as outlined in been detected on all continents except for South America 180 245 Kauserud et al. (2006). All sequences have been accessioned and Antarctica. The lack of a distinct phylogeographical struc- 181 246 in GenBank (for accession nos. see Table 1). ture within PS5 presumably indicates recent long-distance dis- 182 247 183 Phylogenetic analyses were conducted using TNT (Goloboff persal events. It is well-known that many basidiomycetes have 248 184 et al. 2008). Heuristic searches were performed with 1000 ran- been spread by man on infected timber or plants (Coetzee et al. 249 185 dom addition sequences and TBR branch swapping. Jack-knife 2001; Gonthier et al. 2004; Linzer et al. 2008; Pringle et al. 2009) 250 186 analyses were performed with 10 000 replicates, 36 % removal and this could also be the case with S. himantioides. 251 187 probability, and absolute frequencies as output. Bayesian Within PS5, a high level of genetic variation is found 252 188 analyses were performed in MrBayes 3.1.2 (Huelsenbeck & among the isolates from East Asia and North America. Over- 253 189 Ronquist 2001; Ronquist & Huelsenbeck 2003) with models all, South and North America seem to have played an impor- 254 190 inferred from MrModeltest 2.3 (Posada & Crandall 1998). Two tant role during the evolution of the S. himantioides species 255 191 256 independent runs with five chains (four heated) were run for complex. The GSI analysis showed that there was a highly 192 257 10 000 000 generations and summarised after discarding significant grouping of the South American samples (GSI 193 258 < 194 25 % burn-in. Initially, each DNA region was run separately, 1.00 p 0.00001), but samples from other continents showed 259 195 but as there was no significant incongruence between data- no significant grouping. One might speculate that allopatric 260 sets, a concatenated dataset were used for all further speciation in South and North America may have happened

Please cite this article in press as: Carlsen T et al., Multiple cryptic species with divergent substrate affinities in the Serpula himantioides species complex, Fungal Biology (2010), doi:10.1016/j.funbio.2010.10.004 300 306 304 268 266 264 309 308 305 303 302 269 265 263 262 325 323 322 320 299 298 296 295 294 293 292 290 289 288 287 286 285 284 283 282 281 280 307 267 324 297 279 278 275 301 277 270 261 291 273 272 321 271 276 312 274 316 315 314 313 310 319 318 317 311 utpecytcseiswt iegn usrt affinities substrate divergent with species cryptic Multiple laect hsatcei rs s ale ta. utpecytcseiswt iegn usrt fnte nthe in affinities substrate divergent with species cryptic Multiple al., et T Carlsen as: press in himantioides article this cite Please

Table 1 e Specimens included in this study. Locality information, culture collection, strain number, substrate information, and GenBank numbers are given where they are

pce ope,Fna ilg 21) doi:10.1016/j.funbio.2010.10.004 (2010), Biology Fungal complex, species available. Code Locality Collector Culture Strain Country Substrate LSU ITS tub hsp collectiona number

SH16 Belgium: Louvain-la-Nueve G.L. Hennebert MUCL 30528 BEL Brick wall AM076531 AM076498 AJ518086 AM076442 SH17 British Columbia, N.a. MUCL 30795 CAN Picea glauca AM076527 AM076494 AJ557380 AM076438 Aleza Lake SH18 British Columbia, N.a. WFPL 49B CAN Callitropsis AM076532 AM076499 AJ557377 AM076443 Mt Revelstoke nootkatensis SH19 Bedgebury Pinetum N.a. WFPL 49C GBR Inner bark, AM076533 AM076500 AJ518087 AM076444 Pinus sp. SH20 British Columbia, N.a. WFPL 506A CAN Pseudotsuga AM076526 AM076492 AJ557382 AM076436 Cowichan Lake menziesii SH22 UK N.a. MUCL 30855 GBR Log, Larix sp. AM076535 AM076502 AJ518088 AM076446 SH23 Denmark N.a. MUCL 30856 DEN Pinus sp. post AM076536 AM076503 AJ557379 AM076447 SH24 Denmark N.a. MUCL 30857 DEN Pole; decayed AM076537 AM076504 AJ518089 AM076448 SH25 Antwerpen, Schooten (rue G.L. MUCL 31289 BEL Bathroom AM076538 AM076505 AJ518090 AM076449 Andre Ullenslei) Hennebert in house SH26 Manicaland, Chimanimani area, Pascal S., MUCL 38575 ZIM Dead trunk AM076539 AM076506 AJ557373 AM076450 Muguzo Forest Decock C. and litter, Pinus Research Station area sp. and Pinus litter SH27 Manicaland, Pascal S., MUCL 38576 ZIM Dead trunk and litter, AM076540 AM076507 AM076430 AM076451 Chimanimani area, Decock C. Pinus sp. and Pinus litter Muguzo Forest Research Station area SH28 UK J. Carrey MUCL 38935 GBR Soil AM076541 AM076508 AM076423 AM076452 SH29 Vosges, Col de la Decock C. MUCL 38979 FRA Dead standing AM076542 AM076509 AM076427 AM076453 Schlucht, Sentier des roches trunk, coniferous

SH30 Bruxelles, Decock C. MUCL 39729 BEL Cellar, house AM076543 AM076510 AM076425 AM076454 FUNBIO104_proof St. Josse SH31 Akershus, F. Roll-Hansen NFRI 82-90/7 NOR Picea abies AM076528 AM076495 N.a. AM076439 As, Slørstadskogen SH96 Maryland, R.W. Davidson FSC CFS- FSC-31 USA Abies sp. AM076529 AM076496 AM076825 AM076440 Beltsville Atlantic SH99 Bursfelde H Butin CBS 383.82 GER Picea abies AM076547 AM076514 AM076432 AM076458 SH100 Wilsede O. Schmidt Schmidt P218 GER Spruce stump AM076548 AM076515 AM076434 AM076459 ■ oebr2010 November 3 SH101 Putten, J.A. Stalpers CBS 302.82 NED Gymnosperm AM076549 AM076516 AM076416 AM076460 Schovenhorst wood SH103 Schleswig- O. Schmidt Schmidt P283 GER Spruce stump AM076550 AM076517 AM076424 AM076461 Holstein, Malente SH104 Schleswig-Holstein, O. Schmidt Schmidt P284 GER Spruce stump HM135711 HM135661 HM135575 HM135627 Malente ■ Serpula SH105 Schleswig-Holstein, O. Schmidt Schmidt P288 GER Spruce stump AM076551 AM076518 AM076418 AM076462 3/8 Malente

(continued on next page) 3 344 364 366 368 349 348 363 362 360 365 369 390 389 388 387 386 385 384 383 382 381 380 326 329 328 340 343 342 346 345 359 358 367 332 330 334 339 338 336 356 355 354 353 352 350 327 333 335 361 379 378 375 370 377 337 347 357 373 372 331 341 371 376 351 374 444 400 449 448 446 445 443 442 440 406 404 409 408 405 403 402 439 438 436 434 432 430 429 428 426 425 423 422 420 399 398 396 395 394 393 392 455 454 453 452 450 407 435 433 424 427 397 447 401 441 437 391 431 421 451 412 416 415 414 413 410 419 418 417 411 4 laect hsatcei rs s ale ta. utpecytcseiswt iegn usrt fnte nthe in afﬁnities substrate divergent with species cryptic Multiple al., et T Carlsen as: press in himantioides article this cite Please Table 1 (continued) Code Locality Collector Culture Strain Country Substrate LSU ITS tub hsp collectiona number

SH112 Ceska Trebova J. Volc CCBAS 110 CZE Bric house AM076553 AM076520 AM076419 N.a. pce ope,Fna ilg 21) doi:10.1016/j.funbio.2010.10.004 (2010), Biology Fungal complex, species cellar. Spruce beam SH113 Hedmark: Engerdal: H. Kauserud MUCL 46270 NOR Pinus sylvestris AM076530 AM076497 AM076414 AM076441 Kvemskjølen SØ. SH114 126 Paradise Valley Rd. Edwards NZFS 1436 NZL Ground AM076554 AM076521 AM076421 AM076465 SH115 Yunnan Province, Chu Xiong, Decock C. MUCL 47005 CHN Base of a HM135703 HM135653 HM135568 HM135620 Zi Xi Shan Nature Reserve, living trunk, 2400 m alt. Pinus sp. SH116 Yunnan Province, Chu Xiong, Decock C. MUCL 47007 CHN Base of a HM135712 HM135662 HM135576 HM135628 Zi Xi Shan Nature Reserve, living trunk, 2400 m alt. Pinus sp. SH117 Limal Decock C. MUCL 47155 BEL Timber, Pinus sp. HM135713 HM135663 HM135577 HM135629 SH120 Brabant Wallon, Ottignies- Hennebert G. MUCL 47228 BEL Dead wood in HM135714 HM135664 HM135578 HM135630 Louvain-la-Neuve garden, Chamaecyparis sp. SH125 Normandie, Le Tilleul Othon Gesquiere P. MUCL 47679 FRA House timber, HM135715 HM135665 HM135579 N.a. Pinus sp. SH127 Yunnan, Chu Xiong, Zi Xi Shan Decock C.) MUCL 47928 CHN Dead stump, HM135704 HM135654 HM135569 HM135621 Nature Reserve, 2400 m alt Pinus sp. SH129 Yunnan, Chu Xiong, Zi Xi Shan Decock C. MUCL 47930 CHN Dead stump, HM135701 HM135651 HM135566 HM135618 Nature Reserve, 2400 m alt Pinus sp. SH130 Yunnan, Chu Xiong, Zi Xi Shan Decock C. MUCL 47931 CHN Dead stump, HM135702 HM135652 HM135567 HM135619 Nature Reserve, 2400 m alt Pinus sp. SH133 British columbia, WFPL 506B CAN Pseudotsuga AM076544 AM076511 AM076415 N.a. Snooke menziesii SH134 Ontario, Lake Nipigon J.R. Hansbrough CFMR Bud-1908 CAN Picea glauca HM135716 HM135666 HM135580 N.a. SH135 Washington; Lewis; K.R. Shea CFMR Sh-17 USA Picea sitchensis, HM135717 HM135667 HM135581 HM135631 Centralia; Weyerhaeuser 17 y old Timber Co., Forestry FUNBIO104_proof Research Centre SH136 Nevada; Nye; G Tunnel, R.L. Gilbertson CFMR RLG-12941-Sp USA Pseudotsuga menziesii, HM135718 HM135668 HM135582 HM135632 Madison Drift Nuclear white rhizomorph near test site SH137 Ontario; Ft. William; G. Englerth CFMR Bud-205A CAN Picea glauca HM135719 HM135669 HM135583 HM135633

Lake Nipigon, on Black ■

Sturgion Conc. GLP 2010 November 3 SH139 Munchen, F.F. Lombard CFMR R-1 GER Picea, brown and red HM135720 HM135670 HM135584 HM135634 Forstbotanisches stain study

Institute Carlsen T. SH140 Vermont; Warren; P.V. Mook CFMR PVM-52B-R USA Picea rubens, 293 y old HM135721 HM135671 HM135585 N.a. Clay Brook area, Green Serpula

Mt. Nat’l Forest ■ 4/8 tal. et 500 496 495 494 493 492 490 499 498 504 506 464 466 468 489 488 487 486 485 484 483 482 481 480 503 502 505 509 508 520 463 462 460 459 458 456 465 469 497 507 467 491 501 461 457 475 479 478 470 477 473 472 471 476 512 474 510 516 515 514 513 519 518 517 511 544 568 566 564 549 548 585 584 583 582 581 580 569 565 563 562 560 559 558 556 555 554 553 552 550 546 545 543 542 540 539 538 536 534 532 530 529 528 526 525 523 522 567 535 533 524 527 579 578 575 561 557 537 577 570 547 531 521 573 572 541 571 551 576 574 utpecytcseiswt iegn usrt afﬁnities substrate divergent with species cryptic Multiple laect hsatcei rs s ale ta. utpecytcseiswt iegn usrt fnte nthe in afﬁnities substrate divergent with species cryptic Multiple al., et T Carlsen as: press in himantioides article this cite Please

Table 1 (continued) Code Locality Collector Culture Strain Country Substrate LSU ITS tub hsp pce ope,Fna ilg 21) doi:10.1016/j.funbio.2010.10.004 (2010), Biology Fungal complex, species collectiona number

SH141 Colorado, Bears Ears T.E. Hinds CFMR Colo-59-1650 USA Picea engelmannii HM135705 HM135655 HM135570 HM135622 Ranger District, Routt Nat’l Forest SH142 Oregon R. Graham CFMR MD-110 USA Alnus stake HM135722 HM135672 HM135586 N.a. SH144 New York; Warren; J.H. Ginns CFMR JHG-459-Sp USA Tsuga canadensis HM135723 HM135673 HM135587 HM135635 Warrensburg SH147 Arizona, Portal, Rustler Park, J.L. Lowe & R.L. CFMR L-9729-Sp USA Pinus HM135724 HM135674 HM135588 HM135636 Coronado Nat’l Forest Gilbertson SH148 Arizona, Palisades, R.L. Gilbertson CFMR RLG-11350-Sp USA Pinus ponderosa HM135725 HM135675 HM135589 HM135637 Santa Catalina Mts, Coronado Nat’l Forest SH150 Georgia, Athens, W.A. Campbell CFMR FP-94342-R USA Pinus echinata HM135726 HM135676 HM135590 HM135638 Oconee National Forest SH152 Maryland, Beltsville R.W. Davidson CFMR FP-97366-Sp USA Pinus HM135727 HM135677 HM135591 N.a. SH154 Maryland, Beltsville, R.W. Davidson CFMR FP-97367-R USA Pinus virginiana HM135728 HM135678 HM135592 N.a. Plant industrial station SH155 Maryland, Beltsville, R.W. Davidson CFMR FP-97439-Sp USA Pinus virginiana HM135729 HM135679 HM135593 HM135639 Plant industrial station SH156 Maine, Kittern J.D. Diller CFMR FP-97448-Sp USA Soil HM135730 HM135680 HM135594 HM135640 SH157 Maryland, Holiday beech H.H. McKay CFMR FP-104042-T USA Eucalyptus HM135731 HM135681 HM135595 HM135641 wood stakes SH158 Maryland, Beltsville J.D. Diller CFMR FP-104137-T USA Pinus leaves, HM135732 HM135682 HM135596 HM135642 twigs SH159 Maryland, Beltsville R.W. Davidson CFMR FP-104361-Sp USA Hardwood HM135733 HM135683 HM135597 HM135643 SH160 Maryland, Beltsville R.W. Davidson CFMR FP-104397-Sp USA Hardwood HM135734 HM135684 HM135598 N.a. SH161 Maryland, Beltsville R.W. Davidson CFMR FP-104405-Sp USA Hardwood HM135735 HM135685 HM135599 N.a. SH162 Maryland, Beltsville R.W. Davidson CFMR FP-104572-Sp USA Pinus HM135736 HM135686 HM135600 HM135644 FUNBIO104_proof SH163 Oregon, Hungry Horse, M.J. Larsen CFMR FP-134010-Sp USA Conifer HM135737 HM135687 HM135601 HM135645 Coram Expt Forest SH164 Alaska, North side N.a. CFMR HHB-17587-Sp USA Picea glauca AF518648 AM076493 AM076412 AM076437 of Kenai Lake, Kenai Peninsula

SH167 Yunnan, Chu Xiong, Decock C. MUCL 46914 CHN Dead wood, HM135738 HM135688 HM135602 HM135646 ■

Zi Xi Shan Nature Pinus sp. 2010 November 3 Reserve SH168 Concepcion Goetz Palfner CONC-F 323-2 CHI From house HM135699 HM135649 HM135564 HM135616 SH169 Concepcion Goetz Palfner CONC-F 324-1 CHI From house HM135700 HM135650 HM135565 HM135617 SH170 Concepcion Goetz Palfner CONC-F 324-2 CHI From house HM135739 HM135689 HM135603 N.a. SH171 Tierra del Fuego, A. Greslebin CIEFAP 388 ARG Fallen trunk of HM135740 HM135690 HM135604 N.a. ■

Serpula Depto. Ushaia, Nothofagus pumilio

El Valdez 5/8 (continued on next page) 5 644 600 649 648 640 643 642 646 645 604 606 626 625 623 622 620 639 638 636 634 632 630 629 628 650 603 602 605 609 608 596 595 594 593 592 590 589 588 587 586 599 598 624 635 633 607 627 597 637 647 601 591 621 631 641 612 619 610 616 615 614 613 618 617 611 FUNBIO104_proof ■ 3 November 2010 ■ 6/8

6 T. Carlsen et al.

651 716 652 717

653 New 718 ¼ 654 719 655 720 656 721 657 722

658 tub hsp 723 659 724 660 725

661 Fredericton Stock Culture Col- 726

662 ¼ 727 663 728 Western Forest Products Laboratory

664 ¼ 729 665 730

666 Centre For Forest Mycology Research, Forest 731 e catholique de Louvain, Belgium; NZFS ¼ 667 on, Chile; FSC 732 668 733 669 HM135741 HM135691 HM135605HM159424 N.a. HM146135HM159425 HM159426 HM146136AM076555 HM159422 HM159427 AM076522 HM159423 AM076428 AM076466 734 670 735 671 736 672 737 673 eque de l’Universit 738 674 739 ‘ From G. Palfner, Concepci 675 Mycoth 740 ¼ ¼

676 stump 741 677 742 678 Nothofagus betuloides guindo’ wood wood Picea 743 679 744 From O. Schmidt, Hamburg Germany; WFPL

680 ¼ 745 681 746

682 Country Substrate LSU ITS 747 683 748 684 749 onico, Argentinia; CONC-F 685 750 686 751 e

Strain Fig 1 Phylogenetic tree from a Bayesian analysis of

687 number 752 tef hsp 688 a combined dataset of ITS, LSU, , and sequences. 753

689 Culture Collection of Basidiomycetes, Pragua, Czech republic; CFMR Numbers below branches indicate posterior probability 754 ¼ 690 a values. Numbers above branches indicate parsimony 755 691 jack-knife support values (only values above 50 % is shown). 756 From N. Hallenberg, Gothenburg, Sweden; MUCL

692 ¼ The corresponding parsimony trees were of length 350 with 757 on Forestal Andino Patag

693 MUCLMUCL 52396 52397 MEX MEX Soil and buried Soil and buried [ [ 758 collection CI 0.763 and RC 0.674. 694 759 695 760 696 761 on y Extensi 697 in the ancestral lineage splitting into PS1 and the ancestral 762 698 lineage to PS2ePS5. Our analyses indicate furthermore that 763 699 764 PS2, PS3, and PS4 have a primary afﬁnity to North America, Hallenberg Hallenberg 2024PS DEN Valenzuela Valenzuela 700 A. Greslebin CIEFAP 1517 ARG Fallen trunk of 765 but with some northern European representatives. The al- 701 Norwegian Forest and Landscape Institute, Norway; Schmidt 766

¼ most cosmopolitan PS5 may have spread out from North 702 767 703 America to Eastern Asia and more recently obtained 768 Centro de Investigaci a world-wide distribution with the help of man. In Fig 2, 704 ¼ 769 705 the geographical distribution of the North American and Eu- 770 706 ropean isolates is shown. Although speculative, it may be 771 707 that PS5 has a more temperate distribution on the North 772 708 American continent while PS4 has a more boreal distribu- 773 ) 709 tion. In the European samples PS4 was only found in Norway 774 710 while the other European samples were all in PS5. 775 711 776 There is no evidence for a specialization towards growing Frøslev Plantage Depto. Ushaia, Estancia Moat 712 continued 777 Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; CCBAS on wooden constructions, as has apparently happened in S.

713 ¼ 778 714 lacrymans var. lacrymans (Bagchee 1954; Kauserud et al. 779 715 2007b; White et al. 2001). In the phylogenetic tree (Fig 1), iso- 780 SH175 DurangoSH176 DurangoSH181 S. Jutland. C. Decock and R. C. Decock and R. Table 1 ( CodeSH172 Tierra del Fuego, Locality Collectora CBS Products Laboratory, USA; CIEFAP Culture lection (Now Atlantic Forestry Centre, FrederictonZealand Canada); Hallenberg Forest Research(Now Institute; Forintec). NFRI lates derived from nature and buildings appear both in PS1

Multiple cryptic species with divergent substrate afﬁnities 7

781 846 782 847 783 848 784 849 785 850 786 851 787 852 788 853 789 854 790 855 791 856 792 857 793 858 794 859 795 860 796 861 797 862 798 863 799 864 800 865 801 866 802 867 803 868 804 869 805 Fig 2 e Approximate geographic distribution of the analysed isolates and cryptic species of Serpula himantioides in North 870 806 America and Europe. Google Maps mapping service. 871 807 872 808 873 809 874 810 875 811 and PS5 without any apparent structuring, indicating that (2006), enabling a better understanding of the biogeography 876 812 a constant influx of spores happens from nature to buildings and substrate preferences of the S. himantioides species complex. 877 813 and possibly vice versa. This is supported by GSI analysis that 878 814 showed that there was no grouping of outdoor (GSI 0.048 879 815 p ¼ 0.41) or indoor (GSI 0.051 p ¼ 0.13) isolates. Acknowledgments 880 816 There was however a slightly significant signal in the iso- 881 817 lates from Pinus substrates (GSI 0.0836 p ¼ 0.047). Isolates from Cecilie Mathiesen is acknowledged for molecular analyses and 882 818 883 Pinus substrates were only found in the S4 (one isolate) and S5 the Research Council of Norway for financial support. Inger 819 884 lineages. The two PS1 isolates that were found outdoors were Skrede is thanked for helpful comments on the manuscript. 820 885 ¼ ¼ The following persons and culture collections are acknowl- 821 obtained from Nothofagus,(GSI 0.239 p 0.011). The substrate 886 edged for help with assembling the study material and provid- 822 from the three indoor isolates in the same clade is unknown, 887 ing information about the isolates: Ladislav Homolka (CCBAS), 823 but it is not unlikely that these are also Nothofagus,asthisis 888 824 a common source of building material in the region (Martınez Lindsay Bulman (NZFS), Rita Rentmeester (FPL), Edward Hurley 889 825 Pastur et al. 2000). In isolates from PS2, PS3, and PS4, most iso- (FSC), Dian-Qing Yang (Forintec, WFPL), Volkmar Timmermann 890 826 lates were from Picea, and none of these were from buildings. and Gry Alfredsen (NFRI). MR is researcher of CONICET 891 827 In the PS5 lineage a lot of different substrates were found, (Argentina). 892 828 from Eucalyptus and Alnus to Abies and Pinus.Thisindicates 893 829 894 that the putative allopatric speciation event leading to the split 830 Supplementary material 895 between PS1 and the PS2e5 lineages was accompanied with 831 896 a host preference partition as well as a geographic split. In addi- 832 Supplementary data related to this article can be found online 897 833 tion, there seems to have been a switch from a predominantly at doi:10.1016/j.funbio.2010.10.004. 898 834 Abies host preference seen in the PS2 and PS4 lineages, to 899 835 a higher degree of non-specific host preference in the PS5 line- 900 836 age. Although PS5 may initially also have been a host specialist, references 901 837 PS5 has today a wider host range than the other lineages in ad- 902 838 dition to its much wider geographic distribution. The ability to 903 839 colonise a wide spectrum of substrates may have facilitated Ainsworth AM, Rayner ADM, 1990. Mycelial interactions and 904 840 the spread of this lineage throughout the world. outcrossing in the Coniophora puteana complex. Mycological 905 841 Research 94 e 906 This study underlines the importance of having a broad geo- : 627 634. 842 Bagchee K, 1954. Merulius lacrymans (Wulf) Fr. in India. Sydowia 907 graphic sample when analysing intraspecies variation and di- 843 8:80e85. 908 844 vergence. Undiscovered cryptic species may have a restricted Brasier CM, Buck KW, 2001. Rapid evolutionary changes in 909 845 distribution in unsampled areas. We have discovered two new a globally invading fungal pathogen (Dutch elm disease). 910 cryptic lineages in this study compared to Kauserud et al. Biological Invasions 3: 223e233.

8 T. Carlsen et al.

911 Coetzee MPA, Wingfield BD, Harrington TC, Steimel J, phylogeography of the fungal pathogen Heterobasidion anno- 963 912 Coutinho TA, Wingfield MJ, 2001. The root rot fungus sum, including evidence of interspecific horizontal genetic 964 913 Armillaria mellea introduced into South Africa by early Dutch transfer and of human-mediated, long-range dispersal. 965 914 settlers. Molecular Ecology 10: 387e396. Molecular Phylogenetics and Evolution 46: 844e862. 966 915 Cummings MP, Neel MC, Shaw KL, 2008. A genealogical approach Martınez Pastur G, Cellini JM, Peri PL, Vukasovic RF, 967 916 to quantifying lineage divergence. Evolution 62: 2411e2422. Fernandez MC, 2000. Timber production of Nothofagus pumilio 968 917 Geml J, Laursen GA, O’Neill K, Nusbaum HC, Taylor DL, 2006. forests by a shelterwood system in Tierra del Fuego 969 918 Beringian origins and cryptic speciation events in the fly (Argentina). Forest Ecology and Management 134: 153e162. 970 919 agaric (Amanita muscaria). Molecular Ecology 15: 225e239. Matheny PB, Aime M, Bougher N, Buyck B, Desjardin D, Horak E, 971 920 Goloboff PA, Farris JS, Nixon KC, 2008. TNT, a free program for Kropp B, Lodge D, Soytong K, Trappe J, Hibbett D, 2009. Out of 972 921 phylogenetic analysis. Cladistics e International Journal of the the paleotropics? Historical biogeography and diversification 973 922 Willi Hennig Society 24: 774e786. of the cosmopolitan ectomycorrhizal mushroom family Ino- 974 923 Gonthier P, Warner R, Nicolotti G, Mazzaglia A, Garbelotto MM, cybaceae. Journal of Biogeography 36: 577e592. 975 924 2004. Pathogen introduction as a collateral effect of military Moncalvo J-M, Buchanan PK, 2008. Molecular evidence for long 976 925 activity. Mycological Research 108: 468e470. distance dispersal across the Southern Hemisphere in the 977 926 Harmsen L, 1960. Taxonomic and cultural studies on brown Ganoderma applanatumeaustrale species complex 978 927 spored species of the genus Merulius. Friesia VI: 233e277. (Basidiomycota). Mycological Research 112: 425e436. 979 928 Hibbett D, 2001. Shiitake mushrooms and molecular clocks: Murray MG, Thompson WF, 1980. Rapid isolation of high 980 929 historical biogeography of Lentinula. Journal of Biogeography molecular weight plant DNA. Nucleic Acids Research 8: 981 930 28: 231e241. 4321e4325. 982 931 Hosaka K, Castellano M, Spatafora J, 2008. Biogeography of Nilsson RH, Hallenberg N, Norden B, Maekawa N, Wu S-H, 2003. 983 932 Hysterangiales (Phallomycetidae, Basidiomycota). Mycological Phylogeography of Hyphoderma setigerum (Basidiomycota) in 984 933 Research 112: 448e462. the Northern Hemisphere. Mycological Research 107: 645e652. 985 934 Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference Posada D, Crandall KA, 1998. MODELTEST: testing the model of 986 935 of phylogenetic trees. Bioinformatics 17: 754e755. DNA substitution. Bioinformatics 14: 817e818. 987 936 Hwang S-W, 1955. Compatibility and variability in Merulius Pringle A, Adams RI, Cross HB, Bruns TD, 2009. The ectomycor- 988 937 americanus. Mycologia 47: 317e328. rhizal fungus Amanita phalloides was introduced and is 989 938 James T-Y, Moncalvo J-M, Li S, Vilgalys R, 2001. Polymorphism expanding its range on the west coast of North America. 990 939 at the ribosomal DNA spacers and its relation to breeding Molecular Ecology 18: 817e833. 991 940 structure of the widespread mushroom Schizophyllum Ronquist F, Huelsenbeck JP, 2003. MrBayes 3: Bayesian phyloge- 992 941 commune. Genetics 157: 149e161. netic inference under mixed models. Bioinformatics 19: 993 942 Kauserud H, Stensrud Ø, Decock C, Shalchian-Tabrizi K, 1572e1574. 994 943 Schumacher T, 2006. Multiple gene genealogies and AFLPs Slippers B, Wingfield MJ, Coutinho TA, Wingfield BD, 2001. 995 944 suggest cryptic speciation and long-distance dispersal in the Population structure and possible origin of Amylostereum 996 945 basidiomycete Serpula himantioides (Boletales). Molecular Ecol- areolatum in South Africa. Plant Pathology 50: 206e210. 997 946 ogy 15: 421e431. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, 998 947 Kauserud H, Svegarden IB, Decock C, Hallenberg N, 2007a. Hibbett DS, Fisher MC, 2000. Phylogenetic species recognition 999 948 Hybridization among cryptic species of the cellar fungus Con- and species concepts in fungi. Fungal Genetics and Biology 1000 949 iophora puteana (Basidiomycota). Molecular Ecology 16: 389e399. 31:21e32. 1001 950 Kauserud H, Svegarden IB, Saetre G-P, Knudsen H, Stensrud Ø, Taylor JW, Turner E, Townsend JP, Dettman JR, Jacobson D, 2006. 1002 951 Schmidt O, Doi S, Sugiyama T, Hogberg€ N, 2007b. Asian origin Eukaryotic microbes, species recognition and the geographic 1003 952 and rapid global spread of the destructive dry rot fungus limits of species: examples from the kingdom Fungi. Philo- 1004 953 Serpula lacrymans. Molecular Ecology 16: 3350e3360. sophical Transactions of the Royal Society B: Biological Sciences 1005 954 Kroken S, Taylor JW, 2001. A gene genealogical approach to 361: 1947e1963. 1006 955 recognize phylogenetic species boundaries in the lichenized White NA, Dehal PK, Duncan JM, Williams NA, Gartland JS, 1007 956 fungus Letharia. Mycologia 93:38e53. Palfreyman JW, Cooke DEL, 2001. Molecular analysis of inter- 1008 957 Kroken S, Taylor JW, 2009. Phylogenetic species, reproductive specific variation between building and “wild” isolates of 1009 958 mode, and specificity of the green alga Trebouxia forming Serpula lacrymans and their relatedness to S. himantioides. 1010 959 lichens with the fungal genus Letharia. The Bryologist 103: Mycological Research 105: 447e452. 1011 960 645e660. Zervakis GI, Moncalvo J-M, Vilgalys R, 2004. Molecular phylogeny, 1012 961 Linzer RE, Otrosina WJ, Gonthier P, Bruhn J, Laflamme G, biogeography and speciation of the mushroom species 1013 962 Bussieres G, Garbelotto M, 2008. Inferences on the Pleurotus cystidiosus and allied taxa. Microbiology 150: 715e726. 1014