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Home , Agaricales, Agaricomycetes, Aureoboletus auriporus, Boletaceae, Boletales, Boletellus russellii

Molecular and Evolution 57 (2010) 1276–1292

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Molecular Phylogenetics and Evolution

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Molecular phylogenetics of porcini ( section Boletus) ⇑ Bryn T.M. Dentinger a,b,c,n, , Joseph F. Ammirati d, Ernst E. Both e, Dennis E. Desjardin f, Roy E. Halling g, Terry W. Henkel h, Pierre-Arthur Moreau i, Eiji Nagasawa j, Kasem Soytong k, Andy F. Taylor l, Roy Watling m, Jean-Marc Moncalvo b, David J. McLaughlin a a Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA b Department of Natural History, Royal Ontario Museum, Toronto, ON, Canada M5S 2C6 c Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK d Department of Biology, University of Washington, Seattle, WA 98195, USA e Buffalo Museum of Science, Buffalo, NY 14211, USA f Department of Biology, San Francisco State University, San Francisco, CA 94132, USA g Institute of Systematic Botany, New York Botanical Garden, Bronx, NY 10458, USA h Department of Biological Science, Humboldt State University, Arcata, CA 95521, USA i Département de Botanique, Faculté des sciences pharmaceutiques et biologiques, Université Lille Nord de France, F-59006 Lille, France j Tottori Mycological Institute, 211, Kokoge, Tottori 689-1125, Japan k Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, l Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen, AB15 8QH Scotland, UK m Caledonian Mycological Enterprises, 26 Blinkbonny Avenue, Edinburgh, EH4 3HU Scotland, UK n Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada M5S 3B2 article info abstract

Article history: Porcini (Boletus section Boletus: : : ) are a conspicuous group of wild, edible Received 12 May 2010 mushrooms characterized by fleshy fruiting bodies with a poroid hymenophore that is ‘‘stuffed’’ with Revised 17 September 2010 white hyphae when young. Their reported distribution is with ectomycorrhizal plants throughout the Accepted 11 October 2010 . Little progress has been made on the systematics of this group using modern Available online 21 October 2010 molecular phylogenetic tools because sampling has been limited primarily to European and the employed were insufficient to resolve the phylogeny. We examined the evolutionary history Keywords: of porcini by using a global geographic sampling of most known species, new discoveries from little Molecular systematics explored areas, and multiple genes. We used 78 sequences from the fast-evolving nuclear internal tran- Biogeography scribed spacers and are able to recognize 18 reciprocally monophyletic species. To address whether or not Boletales porcini a monophyletic group, we compiled a broadly sampled dataset of 41 taxa, including other Evolution members of the Boletineae, and used separate and combined phylogenetic analysis of sequences from Synapomorphy the nuclear large subunit ribosomal DNA, the largest subunit of RNA polymerase II, and the mitochondrial ATPase subunit six . Contrary to previous studies, our separate and combined phylogenetic analyses ITS support the monophyly of porcini. We also report the discovery of two taxa that expand the known dis- RPB1 tribution of porcini to Australia and Thailand and have ancient phylogenetic connections to the rest of the ATP6 group. A relaxed molecular clock analysis with these new taxa dates the origin of porcini to between 42 Stuffed pores Sustainable non-timber product and 54 million years ago, coinciding with the initial diversification of angiosperms, during the Eocene Conservation epoch when the climate was warm and humid. These results reveal an unexpected diversity, distribution, and ancient origin of a group of commercially valuable mushrooms that may provide an economic incen- tive for conservation and support the hypothesis of a tropical origin of the ectomycorrhizal . Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Boa, 2004; Sitta and Floriani, 2008), yet empirical estimates of their an- nual net worth or the degree to which they are used as an ingredient in Porcini1 ( and allies) are some of the most widely and foods do not exist. The economic value of wild harvested porcini and commonly collected edible mushrooms in the world (Singer, 1986; allied species is clearly substantial, since an estimated 20,000– 100,000 metric tons are consumed annually and the median wholesale price in the U.S. for fresh mushrooms in 2009 was USD$60/kg2 and ⇑ Corresponding author. Present address: Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK. E-mail address: [email protected] (B.T.M. Dentinger). 2 Calculated from data collected by the Fruit and Vegetable Marketing News, 1 Porcini is the plural form of the Italian word porcino, but in common English usage Agricultural Marketing Service, United States Department of Agriculture (http:// it serves as both singular and plural (D. Arora, pers. comm.). marketnews.usda.gov/portal/fv).

1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.10.004 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 1277 can reach USD$200/kg (Hall et al., 1998). Porcini are not only one of the the diversity of porcini is found on other continents, especially most popular wild mushrooms in (Arora, 2001; Sitta and , they have not shed much light on the global diver- Floriani, 2008), but they are an important source of revenue for rural sity and origin of porcini. Importantly, no previous studies have ad- economies in several regions of the world (Boa, 2004; de Román and dressed the issue of whether or not porcini are monophyletic. Boa, 2004) and are becoming a substantial economic resource in wes- Not all taxonomic treatments of porcini treat the ‘‘stuffed pore’’- tern North America (Arora, 2008). Notably, their local value can exceed partial veil as a unifying trait for this group. In fact, although it that of timber, which should provide an economic motivation for con- is a feature often emphasized in field guides as a key character in servation of native (Oria-de-Rueda et al., 2008). Moreover, por- the diagnosis of porcini, it also has been reported from distantly re- cini are nutritionally complete, rivaling the content of meats lated taxa including beniensis (Miller et al., 2000), Boletus (Manzi et al., 2001), and they may have anti-cancer properties (Lucas auripes, and B. aureissimus (Singer, 1947; pers. obs.), indicating that et al., 1957; Ying et al., 1987; Hobbs, 1995). Despite their potential va- this trait may have evolved more than once or that it may be sym- lue as foods, sustainable non-timber forest products, and medicines, plesiomorphic. The lack of clearly identified traits unique to the the true diversity and evolutionary history of these large, conspicuous group has resulted in a great amount of confusion in the classifica- mushrooms remains poorly known. tion of allied taxa, with species placed in or transferred to other The porcini group (Boletus section Boletus; Singer, 1986; hereaf- genera (e.g., reticuloceps; Zang et al., 1993; Wang ter known as ‘‘porcini’’, which refers to the official commercial and Yao, 2005; and separans (Peck) Halling and name for this group in ; Sitta and Floriani, 2008) is a complex Both). of at least 25 species with a known distribution throughout the The recent transfer of Boletus separans Peck to the Xantho- Northern Hemisphere from the subarctic regions to possibly near conium illustrates the broader issue implicit in the taxonomic con- the equator, with only a few reports from Malaysia (1–2 spp.; Cor- fusion of porcini of whether or not they are monophyletic. This ner, 1972) and Colombia (1 sp., Halling, 1996). The distribution of species was reclassified based on a subset of morphological fea- porcini is more extensive than is generally known and there exist tures it shares with some species in Xanthoconium, including a undescribed species, especially in the tropics, that are often con- macrochemical reaction to ammonium hydroxide, the shape and sumed in their native regions, such as Thailand (D. Arora, pers. color of the , and the tendency to develop a sour, putrid comm.) and Vietnam (Dentinger, pers. obs.). The biogeographic his- scent in age or drying (Halling and Both, 1998). Because all other tory of the group is unknown but is generally assumed to be of species of Xanthoconium do not have a partial veil, but instead have north-temperate origin because the vast majority of known species pore mouths that are compressed when young (Singer, 1986), this come from these regions. Most of the known diversity is centered in transfer implicitly demoted the potential unifying role of this trait. eastern (12–18 spp.; Peck, 1889; Snell and Dick, 1970; Grand and However, this proposed reclassification was not presented in a Smith, 1971; Smith and Thiers, 1971; Singer, 1986; Both, 1993; Bes- phylogenetic framework and the morphological homologies em- sette et al., 2000) and western North America (7 spp.; Thiers, 1975; ployed for justifying the transfer can be considered only as a Arora, 2008). Porcini are found in the vicinity of ectotrophic plants hypothesis for phylogenetic relatedness (Patterson, 1982; Brower in the families , Betulaceae, Salicaceae, , Pinaceae and Schawaroch, 1996). On the other hand, no molecular phyloge- (Singer, 1986; Lavorato, 1991; Milne, 2002; Oria-de-Rueda and netic study including multiple species of porcini has found support DÌez, 2002; Águeda et al., 2006, 2008; Oria-de-Rueda et al., 2008), for a single common ancestor of X. separans and other species of and possibly Dipterocarpaceae in Malaysia (Corner, 1972). Based section Boletus (Binder, 1999; Simonini et al., 2001; Mello et al., on the confirmed ectomycorrhizal (ECM) status of several species 2006; Sitta and Floriani, 2008), seeming to indicate that X. separans (Agerer and Gronbach, 1990; Águeda et al., 2008; Smith and Read, is indeed distinct from all other porcini. 2008), the restriction of all known species to the vicinity of ectotro- Our goal in this study was to generate a globally representative, phic plants, their large size (indicating a robust source of carbon) molecular phylogeny for porcini using multiple, independent gene and terrestrial habit (most common habit for ECM fungi), all porcini regions that could be used to evaluate their monophyly and to esti- are assumed to be obligate ECM mutualists of plants. Hence, their mate their date of origin and diversification. Our approach in- global distribution appears to be limited to wherever their photo- volved two steps: (1) we first generated and evaluated sequences synthetic partners occur. from the nuclear internal transcribed spacer regions of the ribo- These morphologically similar fleshy, pored mushrooms (‘‘bo- somal DNA repeat (ITS) for as many broadly distributed samples letes’’) are traditionally united by a combination of several pheno- of porcini as we could reasonably acquire, (2) we then compiled typic traits including white (or pale yellow), mild-tasting flesh that a representative sampling of porcini, based on the results of the does not change color when exposed to air, spores that are either ITS analysis, to evaluate monophyly and date of origin with maxi- yellow-brown or olive-brown in deposit, a stalk with an enlarged mum likelihood and Bayesian methods, with and without a relaxed base and a raised netted pattern at least over the uppermost por- molecular clock and using both nuclear and mitochondrial loci. We tion, and a layer of tangled white hyphae that covers the immature selected three loci for this analysis: (1) The nuclear large subunit tubes (equivalent to a partial veil), a feature that effectively ‘‘plugs’’ (LSU) of the ribosome was chosen because of its traditional use their pores and has often been referred to misleadingly as ‘‘stuffed in fungal phylogenetics and its ease of amplification using univer- pores’’ (Fig. 1; Coker and Beers, 1943; Smith and Thiers, 1971; sal primers. (2) The largest subunit of the nuclear gene encoding Singer, 1986). Most of these features are not unique to porcini RNA polymerase II (RPB1) was chosen because it is a single-copy and can vary considerably among individuals, which at least partly protein-coding gene that is easily aligned across divergent taxa, explains the history of extensive taxonomic confusion in the group. and has been demonstrated to provide more phylogenetically For example, (www.indexfungorum.org) lists 54 informative characters than the LSU (Matheny et al., 2002; synonyms, , forms, and varieties just for the ‘‘King Matheny, 2005; Frøslev et al., 2005; Garnica et al., 2009). (3) The ’’, B. edulis. Recent molecular phylogenetic studies have mitochondrial ATPase subunit 6 (ATP6) was selected because it is shown that only four species of porcini can be distinguished in Eur- physically independent of the nuclear loci, it can be easily aligned ope (B. edulis, B. aereus, B. reticulatus, and B. pinophilus), but that across divergent taxa, it showed promise for phylogenetics in the substantial phenotypic variation exists, particularly in B. edulis, Boletales, and universal primers were available (Kretzer and Bruns, which has sometimes lead to incorrect segregation of independent 1999; Binder and Hibbett, 2006). This study provides the first glob- species (Leonardi et al., 2005; Beugelsdijk et al., 2008). Yet because ally relevant, in-depth analysis of the diversity and evolutionary these studies were limited to taxa present in Europe, while most of history of porcini. 1278 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292

Fig. 1. Representatives of porcini sensu stricto. All photos are by BTMD and copyright of the Bell Museum except when other ownership is indicated. All photos are used with permission. (A) Boletus (‘‘Alloboletus’’) nobilis (BD239, North Carolina, USA). (B) Xanthoconium (‘‘Alloboletus’’) separans (BD246, Arkansas, USA). (C) Boletus subcaerulescens (BD264, Minnesota, USA). (D) Boletus rex-veris (JFA13101, Washington, USA; photo by J. Ammirati). (E) Boletus edulis var. clavipes (BD270, Minnesota, USA). (F) Boletus edulis (BD380, , USA). (G) (AT2005068, Sweden; photo by A. Taylor). (H) Boletus fibrillosus (BD502, Oregon, USA). (I) Boletus quercophilus (BDCR0417, San Gerardo de Dota, Costa Rica). (J) Boletus ‘‘edulis’’ (Yunnan, ; photo by Xiang-Hua Wang). (K) (France; photo by P.-A. Moreau). (L) Boletus cf. variipes var. fagicola (BD369, Guanacaste, Costa Rica). (M) Boletus reticuloceps (Yunnan, China; photo by Xiang-Hua Wang). (N) Boletus hiratsukae (holotype collection; photo by E. Nagasawa). (O) Boletus (= ) subalpinus (Washington, USA; photo by Mike Beug). (P) ‘‘Obtextiporus’’ sp. and gen. nov., nom. prov. (Isaan, Thailand; photo by ). (Q) Close-up of layer of woven hyphae covering the pores of B. nobilissimus. (R) Longitudinal section through tubular hymenophore illustrating the woven hyphal layer covering the pores of B. nobilissimus.

2. Materials and methods taxonomically and geographically, of most known species of por- cini sensu stricto (sensu Singer, 1986; modified by Dentinger, 2.1. sampling 2007; Leonardi et al., 2005; Beugelsdijk et al., 2008; Table 1). Spec- imens used for this dataset include new and preserved collections We compiled two datasets to (1) analyze the species-level from throughout North America, Belize, Costa Rica, China, Japan, diversity of porcini and (2) to evaluate the evolution of porcini Europe, Morocco, and the Philippines. Type specimens were used within a broader phylogenetic context of the Boletineae. Nomen- when available. clature follows Index Fungorum, accessed on 30 April 2010, except For the second dataset, hereafter referred to as ‘‘Boletineae’’, our in cases where recent phylogenetic evidence contradicts the most goal was to sample broadly, both geographically and taxonomi- recent generic affinities in the database. The first dataset, hereafter cally, within the Boletineae (sensu Binder and Hibbett, 2006; Basid- referred to as ‘‘Porcini s.s.’’, includes a thorough sampling, iomycota:::Boletales). We used Table 1 Taxon and voucher information, collection localities, and GenBank accession numbers for specimens used in this study.

GenBank accession numbers Taxon Collection ID Location LSU RPB1 ATP6 ITS (Peck) Pouzar [= Boletus BDCR0431 On ground under Quercus copeyensis and Q. seemannii , Quebrada Trail, Savegre, Albergue de HQ161871 HQ161840 HQ161808 – atkinsonianus (Murr.) Sacc. and Trott. sensu Montaña, San Gerardo de Dota, San José, Costa Rica Halling] Aureoboletus thibetanus (Pat.) Hongo and Nagas. HKAS41151 China AY700189 DQ435800 DQ534600 – (AFToL-ID 450) ananas var. ananas (Curt.) Murr. TH8819 On humic deposits on trunks of corymbosa , near base camp at 5 ° 180 04.800 N; 59° 540 40.400 HQ161853 HQ161822 HQ161790 – W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Boletellus chrysenteroides (Snell) Snell BD394 In soil under Quercus macrocarpa and Q. ellipsoidalis with Betula and Populus nearby, along bank of HQ161867 HQ161836 HQ161804 – Beaver Pond, east side of Field D, Burn Unit 106, Cedar Creek Ecosystem Science Reserve, Anoka

County, MN, USA 1276–1292 (2010) 57 Evolution and Phylogenetics Molecular / al. et Dentinger B.T.M. Boletellus dicymbophilus Fulgenzi and T.W. TH8840 On humic deposits on trunks of Dicymbe corymbosa , near base camp at 5 ° 180 04.800 N; 59° 540 40.400 HQ161852 HQ161821 HQ161789 – Henkel W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Boletellus exiguus T.W. Henkel and Fulgenzi TH8809 On humic deposits on trunks of Dicymbe corymbosa , near base camp at 5 ° 180 04.800 N; 59° 540 40.400 HQ161862 HQ161831 HQ161799 – W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Boletellus piakaii T.W. Henkel and Fulgenzi TH8077 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161861 HQ161830 HQ161798 – N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Boletellus projectellus (Murr.) Singer MB 03-118 ‘‘Under Pinus rigida , USA: Cape Cod, Audubon Sanctuary, South Wellfleet, MA’’ AY684158 AY788850 DQ534604 – (AFToL-ID 713) (Frost) E.-J. Gilbert BD391 In soil under Quercus macrocarpa and Q. ellipsoidalis , in the southeast corner of Field D, Burn Unit HQ161874 HQ161843 HQ161811 – 106, Cedar Creek Ecosystem Science Reserve, Anoka County, MN, USA Boletellus sp. nov. BD366 On ground under Quercus oleoides , ‘‘Finca Jenny’’, sector Santa Rosa, 216 m elev., Parque Nacional HQ161857 HQ161826 HQ161794 – de Guanacaste, Area de Conservación Guanacaste, Guanacaste, Costa Rica merulioides (Schwein.) Murr. MB 02-199 ‘‘Populus sp. nearby and Quercus spp., USA: Rockhouse, MA elevation: 295 m’’ AY684153 DQ435803 DQ534601 – (AFToL-ID 575) Bull. GS1197 On ground under Quercus cerris , Pulpiano, Viano, RE, Italy – – – UNITE# UDB000945 Boletus aereus Bull. AT1998017 On ground in Quercus forest, Unter den Eichen, Wiesbaden, Germany – – – UNITE# UDB000940 Boletus aereus Bull. AT2000198 On ground in forest, Calangianus, Catala, Sardinia, Italy – – – UNITE# UDB000943 Boletus aereus Bull. Baer1585 Gallice, Cosenza, Italy – – – AY680962 Boletus aereus Bull. BaerZac2C Civitella Roveto, L’Aquila, Italy – – – AY680955 Boletus cf. atkinsonii Peck BD304 On ground under Quercus ellipsoidalis , with Prunus and Q. macrocarpa nearby, Afton State Park, – – – EU231950 Washington Co., MN, USA Boletus cf. barrowsii Thiers and A.H. Sm. JFA21519 In mowed grass under Tilia and Quercus cerris , Mt. Baker, Seattle, WA, USA – – – EU231945 Boletus chippewaensis A.H. Sm. and Thiers AHS71914 a MI, USA – – – EU231976 Boletus edulis Bull. AT2001091 On ground in mixed forest, Stadsskogen, Uppsala, Sweden – – – UNITE# UDB000944 Boletus edulis Bull. AT2004287 On ground in Tilia forest, Folkhögskola near Örmsköldsvik, Ångermanland, Sweden – – – UNITE# UDB001114 Boletus edulis Bull. BD186 On bank of reservoir under , Reservoir No. 3, west of Bradford on Hwy. PA-346, PA, USA – – – EU231974 Boletus edulis Bull. BD195 Under Picea abies east of Clear Lake, Concord Co., NY, USA – – – EU231975 Boletus edulis Bull. BD380 Under , Bellview Mtn., near Gothic, CO, USA HQ161848 HQ161817 HQ161781 EU231984 Boletus edulis Bull. Be1F Associated with Fagus silvatica , La Morricana, Teramo, Italy – – – AY680982 Boletus edulis Bull. Be2173 On sea sands, Pattanella, Grosseto, Italy – – – AY680987 Boletus edulis Bull. Chu11 Yunnan, China – – – DQ397949 1 Boletus edulis Bull. DJM1345 Under Pinus contorta , along road ca. =4 mi. from Sutton Creek Overlook, Siuslaw National Forest, – – – EU231982 north of Florence, OR, USA Boletus edulis Bull. HKAS39145 Under Pinus yunnanensis , Lion Mtn., 2400 m elev., Wuding Region, Yunnan Province, China – – – EU231966 Boletus edulis Bull. HKAS47563 Chuching Region, Yunnan Province, China – – – EU231965

Boletus edulis Bull. IRTA702 Associated with ladanifer, (?) – – – DQ002921 1279

(continued on next page) 1280 Table 1 (continued)

GenBank accession numbers Taxon Collection ID Location LSU RPB1 ATP6 ITS Boletus edulis Bull. OKM22130 Under Abies sp., Korea – – – EU231964 Boletus edulis Bull. PAM06- Fixed acidic dune, with Halimium alyssoides , near Monacia-d’Aullène, , France – – – EU231946 112612 Boletus edulis Bull. RK03.03 – – – UNITE# UDB000759 Boletus edulis Bull. TDB2997 Along trail under young Pinus muricata , originating from the 1995 fire, growing as a dense thicket, – – – EU231981 trailhead at the top of Limantour Rd. on the west side, Pt. Reyes Natl. Seashore, near Bayview, CA, USA Boletus edulis Bull. Trudell-03- Occasional on soil in coastal forest with Sitka spruce, western hemlock, and red alder, Kalaloch, EU232006 EU231999 HQ161784 EU231983 287-09 Jefferson Co., WA, USA Boletus edulis subsp. aurantioruber E.A. Dick and JFA2004 Harlow Creek area, road 550, Marquette Co., MI, USA – – – EU231978 Snell 1276–1292 (2010) 57 Evolution and Phylogenetics Molecular / al. et Dentinger B.T.M. Boletus edulis var. clavipes Peck BD298 On ground in mixed woods dominated by Populus, Trails End Campground, Gunflint Trail, Superior EU232004 EU231997 HQ161785 EU231985 National Forest, Cook Co., MN, USA Boletus edulis var. clavipes Peck BD300 In mixed woods, Kakabeka Falls Prov. Park, near Thunder Bay, ON, Canada – – – EU231980 Boletus edulis var. ochraceus A.H. Sm. and Thiers JFA2013 In mixed woods, near Forestville, Marquette Co., MI, USA – – – EU231977 Boletus edulis var. pusteriensis Bepust6 Montminal, France – – – AY680985 Boletus fibrillosus Thiers HDT8907 a Scattered in humus along cut in road, Jackson State Forest, Mendocino Co., CA, USA – – – EU231970 Boletus fibrillosus Thiers TDB1653 Rich mesic mixed redwood forest with Douglas-fir, western hemlock, grand fir, -, – – – EU231972 madrone, and others, about a 1/2 mile north of the 408/409 Junction, Jackson State Forest, Mendocino Co., CA, USA Boletus fibrillosus Thiers TDB1658 Rich mesic mixed redwood forest with Douglas-fir, western hemlock, grand fir, tanbark-oak, – – – EU231973 madrone, and others, about a 1/2 mile north of the 408/409 Junction, Jackson State Forest, Mendocino Co., CA, USA Boletus floridanus (Singer) Murr. BD368 On ground under Quercus oleoides , ‘‘Firebreak’’, sector Santa Rosa, 216 m elev., Parque Nacional de HQ161859 HQ161828 HQ161796 – Guanacaste, Area de Conservación Guanacaste, Guanacaste, Costa Rica Boletus frostii J.L. Russell BDCR0418 On ground under Quercus copeyensis and Q. rapurahuensis , in cow pasture along Río Savegre, San HQ161855 HQ161824 HQ161792 – Gerardo de Dota, San José, Costa Rica Boletus hiratsukae Nagasawa TMI-17481 Under Abies firma Sieb. and Zucc., in mixed woods of A. firma and Castanopsis cuspidata (Thunb.) – – – EU231959 Schottky, Ochidani, Tottori City, Tottori Pref., Japan Boletus hiratsukae Nagasawa TMI-18352 a In Abies firma –Castanopsis cuspidata forest, Ochidani, Tottori City, Tottori Pref., Japan – – – EU231960 Boletus mamorensis Redeuilh CL.M.03.715 Supermarché Auchan, Noyelles-Godault, France – – – EU231947 Boletus mamorensis Redeuilh PAM06- On sandy subarid soil under Quercus suber , Kenitra, Morocco – – – EU231948 112001 Boletus (‘‘Alloboletus ’’) nobilis Peck BD239 In sandy soil along creek under Ostrya, Acer, and Liquidambar , near gate #26, Duke Forest, Durham, EU232002 EU231993 HQ161813 – NC, USA Boletus nobilissimus Both and R. Riedel BD306 On ground in mixed oak woods, Populus and Betula nearby, Afton State Park, Washington Co., MN, – – – EU231949 USA Boletus nobilissimus Both and R. Riedel BD58 Near Quercus ellipsoidalis #436, outside of plot 24, Burn Unit 104, Cedar Creek Ecosystem Science – – – EU231951 Reserve, Anoka Co., MN, USA Boletus nobilissimus Both and R. Riedel BOTH4244 a Under Quercus rubra with young Pinus strobus present, Letchworth State Park, Wyoming Co., NY, – – – EU231954 USA Boletus pallidoroseus Both BD396 On ground under Quercus macrocarpa and Q. ellipsoidalis with Betula and Populus nearby, Bend HQ161860 HQ161829 HQ161797 – Bluffs SNA, Inver Grove Heights, Dakota County, MN, USA Boletus persoonii Bon Be2297 Associated with , Baselica, , Italy – – – AY680986 Boletus pinophilus Pilát and Dermek AT1997033 On ground in Pinus silvestris forest, Eggåsen, Sweden – – – UNITE# UDB000939 Boletus pinophilus Pilát and Dermek AT20000112 On ground in Pinus silvestris forest, Riddarhyttan Research plots, Skinnskatteberg, Västmanland, – – – UNITE# Sweden UDB000942 Boletus pinophilus Pilát and Dermek Bpi2F Associated with Fagus silvatica , Chiarino, Teramo, Italy – – – AY680974 Boletus pinophilus Pilát and Dermek BpiA Associated with Abies alba, Ceppo, Teramo, Italy – – – AY680972 Boletus pinophilus Pilát and Dermek MA-Fungi Spain – – – AJ419190 47700 Boletus quercophilus Halling and G.M. Mueller BDCR0417 On ground under Quercus copeyensis and Q. rapurahuensis , in cow pasture along Río Savegre, San HQ161849 HQ161818 HQ161782 EU231953 Gerardo de Dota, San José, Costa Rica Boletus regineus Arora and Simonini DED7034 Solitary under , Pseudotsuga, , and Sequoia, Bohemian Grove, Monte Rio, Sonoma – – – EU231992 Co., CA, USA Boletus regineus Arora and Simonini Sweringen Under oak, near Oakland Zoo, Oakland, Alameda Co., CA, USA – – – EU231991 12.11.99 Boletus regineus Arora and Simonini WILLITS#1 Under Quercus, Willits, Mendocino Co., CA, USA – – – EU231990 Boletus reticulatus Schaeff. [= B. aestivalis 1640 isolate Italy(?) – – – AY278764 (Paulet) Fr.] Bre1 Boletus reticulatus Schaeff. [= B. aestivalis AT2000076 On ground in Quercus robur forest, Quercus Avenue, Ultuna, Uppsala, Sweden – – – UNITE# (Paulet) Fr.] UDB000941 Boletus reticulatus Schaeff. [= B. aestivalis AT2004040 On ground in Quercus forest, Dalskarret near Hammarskog, Uppsala, Sweden – – – UNITE# (Paulet) Fr.] UDB001113 Boletus reticulatus Schaeff. [= B. aestivalis Baest1914 Associated with Abies alba, Picea abies , Pinus silvestris , Lago Scuro, Parma, Italy – – – AY680969 (Paulet) Fr.] Boletus reticulatus Schaeff. [= B. aestivalis Baest6F Associated with Fagus sylvatica on rendzine soil, Cascine, L’Aquila, Italy – – – AY680966 (Paulet) Fr.] ...Dnigre l oeua hlgntc n vlto 7(00 1276–1292 (2010) 57 Evolution and Phylogenetics Molecular / al. et Dentinger B.T.M. Boletus reticulatus Schaeff. [= B. aestivalis OKM21579 In Castanea forest, Italy – – – EU231944 (Paulet) Fr.] Boletus reticuloceps (M. Zang, M.S. Yuan, and HKAS39222 Laojin Mtn., Lijong Region, Yunnan Province, China – – – EU231968 M.Q. Gong) Q.B. Wang and Y.J. Yao Boletus rex-veris D. Arora and Simonini JFA13101 Gregarious to scattered in soil and litter in coniferous forest with Pseudotsuga menziesii , Picea EU232005 EU231998 HQ161783 EU231969 engelmannii , , Abies sp., Hwy410, mile 84, Chinook Pass (1655 m elev.), WA, USA Boletus rex-veris Arora and Simonini SNF-326 Mixed forest with ponderosa pine, pine, lodgepole pine, white fir, bordering Dinkey – – – EU231971 Creek, Dinkey Creek Work Center, Sierra National Forest, Fresno Co., CA, USA Boletus (‘‘Obtextiporus ’’) sp. nov. REH8790 Thailand HQ161879 HQ161877 HQ161878 – Boletus (‘‘Inferiboletus ’’) sp. nov. REH8969 Vegetation: Eucalyptus spp. S 17° 00 3500, E 145° 340 5600, 620 m elev. 5 km from Kennedy Highway, HQ161847 HQ161816 HQ161780 – Davies Creek Road, Davies Creek National Park, Mareeba Shire, Mareeba, Queensland, Australia Boletus subcaerulescens (E.A. Dick and Snell) BD264 On ground in Pinus resinosa , east side of entrance to McCormick Lake Day Use Area, – – – EU231987 Both, Bessette and A.R. Bessette General C.C. Andrews State Forest, near River, Pine Co., MN, USA Boletus subcaerulescens (E.A. Dick and Snell) BD383 On ground near Picea glauca , Abies balsamea , Pinus strobus , Betula sp., along Amity Creek, Lester – – – EU231986 Both, Bessette and A.R. Bessette Park, Duluth, St. Louis Co., MN, USA Boletus subcaerulescens (E.A. Dick and Snell) BOTH3820 a Under , North Collins Elementary School Woods, North Collins, Erie Co., NY, USA – – – EU231988 Both, Bessette, and A.R. Bessette Peck BD201 Under oak in open grassy area, Ridge County Park, Erie Co., NY, USA – – – EU231961 Boletus variipes Peck BD245 In sandy soil on river bank under Fagus grandifolia , Duke Forest, Durham, NC, USA EU232003 EU231996 HQ161786 EU231958 Boletus variipes Peck BD378 On ground along a dirt road in mixed woods with Quercus, Fagus, and Thuja, north of Warren, PA, HQ161846 EU232007 HQ161779 – USA Boletus variipes Peck Watling25598 With Pinus kesiya , near Baguio, Bokod, Philippines – – – EU231967 Boletus variipes var. fagicola A.H. Sm. and Thiers AHS75914 a Scattered to gregarious under - and some aspen trees, Creek, near Wolverine, – – – EU231962 Cheboygan Co., MI, USA Boletus variipes var. fagicola A.H. Sm. and Thiers BD190 On bank of Linn Creek(?), under hemlock, red oak, and beech, off Hwy PA-3121, south of PA-346, – – – EU231963 Allegheny National Forest, McKean Co., PA, USA Boletus variipes var. fagicola A.H. Sm. and Thiers REH7756 Gregarious on soil under Quercus oleoides , Sendero a Agua Termales, Rincón de la Vieja, sector Santa – – – EU231955 [= phaeocephalus (Pat. and C.F. María, Aréa de Conservacion Guanacaste, near Liberia, Guanacaste, Costa Rica Baker) Singer] Boletus variipes var. fagicola A.H. Sm. and Thiers REH8527 Solitary on sandy soil under Quercus oleoides , British Military Swamp, Douglas DaSilva, Mtn. Pine – – – EU231956 [= Xerocomus phaeocephalus (Pat. and C.F. Ridge, Cayo District, Belize Baker) Singer] Boletus variipes var. fagicola A.H. Sm. and Thiers REH8545 On sandy soil under Quercus oleoides and Pinus caribaea , near Belize Zoo, Foster Property, Western – – – EU231957 [= Xerocomus phaeocephalus (Pat. and C.F. Highway, Belize District, Belize Baker) Singer] Boletus venturii Bvent2226 Kardich, Austria – – – AY680989 Gastroboletus subalpinus Trappe and Thiers Trappe607 a Solitary to gregarious under Pinus albicaulis, Cloud Cap, 5800 m elev., Mt. Hood, OR, USA – – – EU231989 chromapes (Frost) Singer BD377 On ground along a dirt road in mixed woods with Quercus, Fagus, and Thuja, north of Warren, PA, HQ161856 HQ161825 HQ161793 – USA Leccinum crocipodium (Letell.) Watling RC/F94.103 Along a lake, under Quercus spp., Piney, Dept. Aube, France HQ161870 HQ161839 HQ161807 – Leccinum monticola Halling and G.M. Mueller BDCR14 On ground in leaf litter under Comarostaphylis arbutoides in páramo vegetation, summit of Cerro de HQ161869 HQ161838 HQ161806 – la Muerte, 3491 m elev., San José, Costa Rica

Phylloporus rhodoxanthus (Schwein.) Bresadola BD374 On ground along a dirt road in mixed woods with Quercus, Fagus, and Thuja, north of Warren, PA, HQ161851 HQ161820 HQ161788 – 1281 USA

(continued on next page) 1282 Table 1 (continued)

GenBank accession numbers Taxon Collection ID Location LSU RPB1 ATP6 ITS porphyrosporus (Fr. and Hök) E.-J. DJM1332 On ground under Picea sitchensis , Tyee Campground, Oregon Dunes National Recreation Area, HQ161850 HQ161819 HQ161787 – Gilbert Westlake, OR, USA griseus (Frost) Binder and Bresinsky BD210 On ground in mixed oak woods, Confederate Breastworks Interpretive Trail, Shenandoah HQ161858 HQ161827 HQ161795 – Mountain, George Washington National Forest, Augusta County, VA, USA strobilaceus (Scop.) Berk. [= S. MB 03-102 Not available AY684155 AY858963 DQ534607 – floccopus (Vahl) P. Karst.] (AFToL-ID 716) pictus (Peck) A.H. Sm. and Thiers MB 03-002 Not available AY684154 AY858965 DQ534608 – (AFToL-ID 717) ballouii (Peck) Singer (= Rubinoboletus TH8409 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161873 HQ161842 HQ161810 – ballouii (Peck) Heinem. and Rammeloo) N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana 1276–1292 (2010) 57 Evolution and Phylogenetics Molecular / al. et Dentinger B.T.M. Tylopilus ballouii (Peck) Singer (= Rubinoboletus TH8593 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161872 HQ161841 HQ161809 – ballouii (Peck) Heinem. and Rammeloo) N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Tylopilus intermedius A.H. Sm. and Thiers BD277 On ground in mixed hardwoods, Nerstrand-Big Woods State Park, Rice County, MN, USA HQ161875 HQ161844 HQ161814 – Tylopilus rubrobrunneus Mazzer and A.H. Sm. BD329 On ground near Quercus ellipsoidalis in mixed hardwood forest dominated by Acer and Tilia, HQ161876 HQ161845 HQ161815 – Wolsfeld Woods SNA, Hennepin Country, MN, USA Xanthoconium affine var. maculosum (Peck) BD217 On ground under Quercus, Betula, and Hamamelis, along hiking trails, Mountain Lake Biological HQ161854 HQ161823 HQ161791 – Singer Station, Mountain Lake, Giles County, VA, USA Xanthoconium purpureum Snell and E.A. Dick BD228 On ground at base of Thuja, in a campsite at Standing Indian Area campground, Nantahala National HQ161864 HQ161833 HQ161801 – Forest, near Franklin, Macon County, NC, USA Xanthoconium (‘‘Alloboletus ’’) separans (Peck) BD243 On ground in mixed hardwoods, Duke Forest, Durham, NC, USA EU232000 EU231994 HQ161812 – Halling and Both Xerocomus sp. TH8408 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161863 HQ161832 HQ161800 – N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Xerocomus sp. TH8821 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161866 HQ161835 HQ161803 – N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Xerocomus sp. TH8848 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161868 HQ161837 HQ161805 – N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana Xerocomus sp. TH8850 Terrestrial on root mat in monodominant Dicymbe corymbosa forest, near base camp at 5 ° 180 04.800 HQ161865 HQ161834 HQ161802 – N; 59° 540 40.400 W, Upper Potaro River Basin, Region 8 Potaro-Siparuni, Guyana

a Type specimen. B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 1283 the recent phylogenetic overview of the Boletales (Binder and identified using the PrimerQuestSM tool available through the web- Hibbett, 2006) and the traditional classification of three subfami- site of Integrated DNA Technologies, Inc. (www.idtdna.com/Sci- lies (Boletoidae, Strobilomycetoidae, and Xerocomoidae; Singer, tools/Applications/Primerquest/). The forward primer ‘‘bol-RPB1- 1986) as guides. Two taxa, one from the Suillineae (Suillus pictus) Afor’’ (50-AYTWAAGGCHGAYATCGTGAGTC-30) and reverse primer and one from the (), ‘‘bol-RPB1-Crev’’ (50-CARCAACTGCTCAAACTCCGTGAT-30) were were used as outgroups. Most of the samples for the Boletineae newly designed. These primers correspond to positions 65–80 dataset were taken from well-preserved, recently collected speci- (bol-RPB1-Afor) in exon region ‘‘A’’ and 779–803 (bol-RPB1-Crev) mens in to facilitate amplification of single copy nuclear pro- in exon region ‘‘C’’ according to the full annotated sequence of tein-coding genes. For this dataset, 11 taxa representing the known the Boletaceae representative Boletellus projectellus in GenBank phylogenetic diversity of the traditionally recognized porcini were (AFToL-ID 713; GenBank Accession No. AY788850). Mitochondrial selected based on preliminary results of the species-level study ATP6 was amplified and sequenced using primers ATP6-1 and with ITS sequences (7 spp.), newly discovered taxa (2 spp.), and ATP6-2 following the protocol of Kretzer and Bruns (1999), except divergent taxa according to previous studies (2 spp.). New collec- in most cases sequential PCR was necessary to obtain enough tem- tions were obtained from Australia, Costa Rica, Guyana, France, plate for direct sequencing. Amplifications were completed using a Thailand, and USA (Table 1). Dried voucher specimens are depos- touchdown program (Dentinger et al., 2010) on a MJ Research DNA ited in the University of Minnesota Herbarium (MIN), University Engine (PTC-200) Peltier Thermal Cycler (Bio-Rad Laboratories, of Guyana, Georgetown (BRG), Humboldt State University (HSC), Waltham, Massachusetts, USA) or an Eppendorf MasterCycler Clinton Herbarium, Buffalo Museum of Sciences (BUF), Université (Model 5345). PCR amplification was achieved using standard con- de Lille, Faculté des Sciences Pharamaceutiques et Biologiques Her- ditions according to the original citations for the loci and primers barium (LIP), HD Thiers Herbarium, San Francisco State University (listed above) or following that of Dentinger and McLaughlin (SFSU), University of at Berkeley Herbarium (UC), Uni- (2006) and Dentinger et al. (2010). PCR products were cleaned versity of Washington Herbarium (WTU), Kunming Institute of prior to sequencing by using either a QIAquick PCR Purification Botany, Chinese Academy of Sciences (KUN), Tottori Mycological Kit (Qiagen Inc., Valencia, CA) or by using one of the methods in Institute (TMI), Royal Botanic Garden, Edinburgh (E), Herbarium Dentinger et al. (2010). Unidirectional dye-terminated sequencing of the University of San Jose, Costa Rica (USJ), and New York Botan- for the forward and reverse reactions using the same primer com- ical Garden (NY). binations for PCR was conducted with an ABI PRISM™ 3700 DNA Analyzer (Foster City, California, USA) at the BioMedical Genomics 2.2. DNA extraction, PCR, and DNA sequencing Center, University of Minnesota and an ABI PRISM™ 3100 DNA Analyzer in the Department of Natural History at the Royal Ontario A total of four loci were sequenced for this study, including Museum. Contiguous sequences were made in Sequencher 3.0 three nuclear and one mitochondrial gene. For the Porcini s.s. data- (Gene Codes Corp., Ann Arbor, Michigan, USA) by overlapping the set, full and partial sequences of the nuclear internal transcribed unidirectional reads. In addition to new collections, 10 publicly spacers (ITS) of the ribosomal DNA repeat were generated. Three available sequences representing five additional taxa were ob- independent loci were sequenced for the Boletineae dataset: (1) tained from GenBank or the AFToL Molecular Database. the 5 region of the large subunit (LSU) of the ribosomal DNA repeat, (2) the 50 region of the nuclear gene coding for the largest subunit of RNA polymerase II (RPB1; Matheny et al., 2002; Matheny, 2005), 2.3. Sequence alignment and phylogenetic analysis (3) the 5 region of mitochondrial ATPase subunit 6 (ATP6; Kretzer and Bruns, 1999). Genomic DNA was extracted using a modified 2.3.1. Porcini s.s. dataset CTAB/SDS extraction (Dentinger and McLaughlin, 2006) or newly No taxa could be selected as an outgroup for this dataset be- proposed protocols for high-throughput DNA sequencing cause the ITS region is too divergent between porcini s.s. and taxa (Dentinger et al., 2010). PCR of the full or partial ITS region fol- outside of this group, including ‘‘Inferiboletus’’, to align unambigu- lowed Gardes and Bruns (1993) using primers ITS1F and ITS4/ ously. Sequences were aligned manually in the editor window of ITS4B (White et al., 1990; Gardes and Bruns, 1993) and/or primers PAUPv4b10 (Swofford, 2002). Maximum likelihood (ML) and ITS8F and ITS6R following the protocol of Dentinger et al. (2010). Bayesian (BA) methods were used for all analyses. Models of evo- For some taxa, only the ITS1 region could be amplified. For these lution were determined using the Akaike Information Criterion short fragments, PCR employed the same forward primers as implemented in ModelTest v3.7 (Posada and Crandall, 1998). ML the full ITS amplifications with the reverse primer 5.8S (Vilgalys analysis was performed using the Mac OS X GUI version of GARLI lab) or a new reverse primer, ‘‘bol-5.8Srev’’ (50-AGCGCAAGGTG v0.951 (Zwickl, 2006) with the factory defaults and estimating all CGTTCAAAGATTC-30), which was designed to complement a region model parameter values except in cases where it was necessary of the 5.8S based on an alignment of ITS sequences of B. edulis, B. to supply these values to invoke the appropriate model. Each ML aereus, B. pinophilus, and B. aestivalis downloaded from GenBank. search was conducted five times to avoid an aberrant result from The first ca. 900–1400 bp of double-stranded DNA was amplified stochastic effects (Zwickl, 2006). Branch support was assessed with and sequenced for the LSU using the forward primer LROR (Vilgalys 100 nonparametric bootstrapping replicates using model parame- and Hester, 1990) or a new forward primer designed for enhanced ters settings that were fixed to the average values from the previ- specificity to Agaricomycetes, LROR-A (50-CAAGGATTCCCCTAC- ous five independent ML runs. Partitioned Bayesian analyses using TAACTGC-30), in combination with the reverse primer LR5 (Vilgalys mixed models were conducted using the parallel CVS version of and Hester, 1990)orTW14(White et al., 1990). Initial attempts to MrBayes v3.2 (Ronquist and Huelsenbeck, 2003; Altekar et al., amplify RPB1 using previously published primers designed for fun- 2004; Ronquist et al., 2005) at the University of Minnesota Super- gi (gRPB1-Afor, fRPB1-Crev; Matheny et al., 2002) resulted in weak computing Institute. Separate partitions were used for the ITS1, amplification or non-specific amplification. These difficulties were 5.8S, and ITS2 regions to allow for different rates of evolution in overcome by sequential PCR or by using newly designed primers these three regions. All Bayesian analyses used mixed models, with enhanced specificity for . New primers were designed one for each partition, with model parameter values estimated using an alignment of all publicly available RPB1 sequences from for each partition independently. The BA analyses used two parallel Boletales taxa in the AFToL Molecular Database (aftol.biol- independent runs of 2 106 generations sampling trees every 100 ogy.duke.edu). Priming regions and primer sequences were generations. The first 2500 sampled trees from each run were 1284 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 discarded as the burn-in and the remaining 17,500 trees from each neoformans), Dacrymycetales (2 spp.), (2 spp.), run were combined to build a 50% majority rule consensus using (2 spp.), (2 spp.), (2 spp.), the ‘sump’ and ‘sumt’ commands in MrBayes. Analysis statistics and Boletales (10 spp.), including six species of porcini. The align- were used to diagnose MCMC chain convergence (e.g., sufficient ment was made in MacClade v4.08 (Maddison and Maddison, swapping among chains, standard deviation of split frequencies 2003) by combining the porcini sequences (with only a single rep- <0.05, potential scale reduction factors near 1; Ronquist et al., resentative of B. edulis) with selected sequences retrieved from the 2005). RPB1 and LSU alignments in the AFToL database (http://aftol1.biol- ogy.duke.edu/pub/alignments/download_alignments) used by 2.3.2. Boletineae dataset James et al. (2006). We excluded the same ambiguously aligned re- Sequences were aligned initially using MUSCLE v3.7 (Edgar, gions as James et al. (2006) prior to analysis. ModelTest v3.7 was 2004) and optimized manually. Individual loci were analyzed sep- used to select the best models of evolution for each gene using arately and combined. All analyses used RAxML v7.0.4 (Stamatakis, the hierarchical likelihood ratio test. The date of origin of porcini 2006) executed on the CIPRES server (Miller et al., 2009) and the was estimated using a relaxed molecular clock analysis parallel version of MrBayes v3.1.2 executed remotely on an Apple (Drummond et al., 2006) in BEAST v1.5.4 (Drummond and Ram- XServe in the Thornton lab at the University of Oregon. The best baut, 2007) with separate models and model parameters estimated model of evolution for each locus was selected using the Akaike independently for each gene partition. We used two different Information Criterion in ModelTest v3.7. Branch support was eval- internal calibrations for the divergence time between the Ascomy- uated using rapid nonparametric bootstrapping with 1000 repli- cota and the based on the estimates of 452 million cates in RAxML (Stamatakis et al., 2008) and using posterior years ago (Mya) (Taylor and Berbee, 2006) and 500–665 Mya probabilities from the MrBayes runs. Both the RAxML and MrBayes (Lucking et al., 2009). These dates reflect both a traditional, more analyses of the combined data used partitioned mixed models conservative estimate and a new estimate based on updated phy- allowing for model parameters to be estimated independently for logenetic information and improved molecular clock analysis. We each gene. The MrBayes analyses were automatically terminated constructed the BEAST input files using BEUti v1.5.4 (Drummond using the stoprul and stopval commands when the standard devia- and Rambaut, 2007) and implemented the calibrations by setting tion of the split frequencies fell below 0.01. Chain convergence was a uniform prior for the tMRCA parameter at the node leading to further verified by ensuring potential scale reduction factors the and Basidiomycota. For the first calibration point, neared 1 and using Tracer v1.5 to confirm sufficiently large ESS val- the prior was distributed normally about the mean of 452 Mya ues. Burn-ins were determined by visually inspecting the ln L with a standard deviation of 31.6 Mya, corresponding to a central trace plot in Tracer. Posterior probabilities from the Bayesian runs 95% range of 500–400 Mya. The mean of the prior is the date esti- were calculated by building majority-rule consensus trees from the mated by Taylor and Berbee (2006) for the Ascomycota/Basidiomy- posterior distributions of the MCMC chains in PAUP. For the ATP6 cota split. The standard deviation was chosen so that the minimum dataset, the first 10,000 trees were discarded as the burn-in and date in the central 95% range was 400 Mya, corresponding to the the last 70,410 trees from each run (140,820 total) were used to minimum age of the Ascomycota determined by the fossil Paleopy- build the consensus tree; for the LSU dataset, the first 5000 trees renomycites devonicus from the early Rhynie chert were discarded as the burn-in and the last 22,722 trees from each (Taylor et al., 2005). The second calibration used 500–665 Mya run (45,444 total) were used for the consensus tree; for the RPB1 for the divergence of the Ascomycota and Basidiomycota based dataset, the first 5000 trees were discarded as the burn-in and on Lucking et al. (2009). We used the mean of this range the last 31,012 trees from each run (62,024 total) used for the con- (582.5 Mya) for the prior with a normal distribution and a standard sensus tree; for the combined dataset, the first 2040 trees were dis- deviation of 50.15 Mya, which produces a central 95% range of carded as the burn-in and the last 18,371 trees from each run 500–665 Mya, corresponding to the range of dates reported by (36,742 total) used for the consensus tree. Lucking et al. (2009) for this node. For both calibrations, two inde- pendent runs were executed in BEAST for 1 107 generations, 2.4. Hypothesis testing sampling trees every 100 generations. Tracer v1.5 was used to diagnose MCMC chain convergence (based on ESS values >200) The Shimodaira–Hasegawa test (SHtest) was used to evaluate and, after discarding the first 1 107 trees (10%) as the burn-in alternative topological hypotheses for each gene (Shimodaira and from each run, the mean ± standard error and the 95% highest pos- Hasegawa, 1999; Goldman et al., 2000). GARLI v1.0 (Zwickl, terior density (HPD) interval for the dates of divergence in Mya 2006), which allows for positive topological constraints to be en- were calculated from the combined BEAST log files. forced, was used to identify maximum likelihood trees from searches that were either unconstrained or constrained to recover porcini as monophyletic. SHtest was conducted in PAUPv4b10 3. Results using the best trees from the ML searches in GARLI. Maximum like- lihood values were estimated for all model parameters in PAUP 3.1. Porcini s.s. (Fig. 2) and SHtest was conducted using RELL approximation with 1000 bootstrap replicates. The data matrix consisted of 74 complete ITS1-5.8S-ITS2 se- quences from at least 16 morphospecies (Table 1), and ITS1 only 2.5. Dating the origin of porcini sequences for four taxa. Sequences were aligned in 1010 positions: 182 were parsimony informative, 761 were constant, and 67 were An alignment of 27 RPB1 and LSU sequences representing the autapomorphic. (2 spp.), Ascomycota (2 spp.), and Basidiomycota Maximum likelihood analysis recovered five trees with ln L (23 spp.), was used to infer the date of origin for porcini, using Rhi- scores of 3461.7639–3461.7890 (Fig. 2), which differed only in zopus oryzae (: ) as the outgroup. The the position of Boletus ‘‘variipes’’ (Watling25598). One tree recov- Basidiomycota included taxa representing the ered B.‘‘variipes’’ sharing a most recent common ancestor (MRCA) (Agaricostilbum hyphaenes), (Ustilago maydis) with the containing B. aereus, B. mamorensis, B. sp. nov. 2 and and Agaricomycotina (21 spp.). Within the Agaricomycotina, taxa 3, and B. reticulatus. The other four trees recovered B. ‘‘variipes’’ were selected to represent the Filobasidiales (Cryptococcus sharing a MRCA with a clade containing the previous four taxa plus B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 1285

Fig. 2. Phylogram of the best ML tree using 78 ITS sequences from five independent searches using GARLI. All taxa are indicated by the specific epithet and are from the genus Boletus unless a different generic name is supplied. Numbers above branches are ML nonparametric bootstrap values and numbers below branches are posterior probabilities from the BA analysis. Support values are provided only for nodes that received either P60% ML bootstrap and/or P.90 Bayesian posterior probability. that correspond to species are shaded and labeled with the specific epithet on the right. Geographic range of each species is indicated at right.

B. cf. barrowsii, B. quercophilus, and B. nobilissimus. The BA analysis the triplet CGA, coding for the arginine, to the stop co- was identical to the ML tree at all nodes receiving significant boot- don TGA. Although it is possible that TGA is an alternate codon for strap (BS) and/or posterior probability (PP) support. arginine in this taxon, the additional features that distinguish this sequence from all of the other RPB1 sequences suggest that the 3.2. Boletineae (Figs. 3 and 4) RPB1 gene we sequenced from this specimen is not a functioning copy. The multi-gene Boletineae dataset consisted of 43 taxa. All data- Phylogenetically informative characteristics of each dataset and sets had complete sequence data except for one taxon (Xanthoconi- measures of homoplasy in each of the best trees from the ML um purpureum) that was represented by an incomplete RPB1 analyses of the separate and combined datasets are summarized sequence restricted to the first 269 bp of the alignment. One spec- in Table 2. ModelTest v3.7 selected the GTR+G+I model for all loci. imen was ultimately eliminated from the study (Xerocomus cf. spa- The LSU and ATP6 datasets had modest phylogenetic signal (26% diceus var. gracilis) because the RPB1 sequence we obtained using parsimony-informative sites) with relatively high levels of homo- primers bolRPB1-Afor and bolRPBP1-Crev exhibited characteristics plasy compared to the RPB1 dataset (38% parsimony-informative of a pseudogene. This sequence had two 3-bp indels that did not sites), and produced trees with a small proportion of supported interrupt the reading frame, seven autapomorphic nonsynony- nodes: 16% and 14% of branches received bootstrap values mous substitutions, a single bp indel that did interrupt the reading P70%, respectively, compared to the RPB1 dataset (20%). Maxi- frame, and two first-position C ? T substitutions that converted mum likelihood analysis of both LSU and ATP6 did not recover 1286 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292

Fig. 3. Phylograms of the best trees from separate ML searches of each gene using RAxML. Labels at the top left of each tree indicate the gene used for the analysis. Pie charts underneath each label are the percent of total characters in the alignment that are parsimony-informative. Taxa in red are traditionally classified as porcini. Numbers above branches are ML nonparametric bootstrap values and numbers below branches are posterior probabilities from the BA analysis. Support values are provided only for nodes that received either P60% ML bootstrap and/or P.90 Bayesian posterior probability. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.) porcini as a monophyletic group, in contrast to the RPB1 dataset, identification of species in this group is plagued by misapplication which recovered porcini as monophyletic with both nonparametric of names, particularly when collections were named as taxa de- bootstrap (74%) and Bayesian posterior probability (1.0) supports. scribed from disjunct regions, such as the identification of B. edulis However, the Shimodaira–Hasegawa test did not reject the (described from Europe) in eastern (HKAS39145, HKAS47563, hypothesis that porcini form a monophyletic group for the LSU DQ397949, OKM22130) and B. variipes (described from eastern or ATP6 data (Table 3). USA) in the Philippines (Watling25598). It is also clear that there The three genes were combined into a single dataset because no are still porcini taxa masquerading under other names that have nodes with statistical support in each of the separate analyses were not been sampled here, such as B. reticulatus from Japan in conflict with each other (Fig. 3), indicating that the data could be (Nagasawa, pers. obs). Compounding this problem, all of the infra- combined (Mason-Gamer and Kellogg, 1996; Reeb et al., 2004). The specific taxa and segregated species of B. edulis (with the exception ML tree produced from the combined analysis was similar to that of B. subcaerulescens and B. pinophilus) appear to be phenotypic recovered from the single gene RPB1 dataset (Figs. 3 and 4). variants of a single widespread panmictic population, confirming Twenty branches received strong nonparametric bootstrap support the results of Leonardi et al. (2005) and Beugelsdijk et al. (2008). (P70%) while only 15 of these received significant Bayesian pos- However, the ITS may simply not evolve fast enough to detect re- terior probabilities (P0.95). One branch received a significant pos- cently diverged taxa, in which case the stable forms of B. edulis that terior probability but not significant nonparametric bootstrap occur throughout its range may in fact be reproductively isolated support. Two branches had lower support in the combined dataset from each other. Confident diagnosis of species limits in B. edulis than the RPB1 dataset, including the node leading to porcini and sensu stricto and its segregated forms will require the addition of the internal node leading to ‘‘Inferiboletus’’ (REH8969) plus ‘‘Boletus faster-evolving genetic markers and population genetic studies s.s’’. with thorough sampling throughout its reported range in Europe, Asia, and North America. 3.3. Dating the origin of porcini (Table 4, Fig. 5) Because the ITS-based phylogram could not be confidently rooted, it is difficult to infer biogeographic and morphological evo- Divergence time estimates calibrated at the Ascomycota/Basid- lution in porcini sensu stricto at this time. Nonetheless, our phylog- iomycota node are summarized in Table 4. From the chronogram in eny shows several interesting patterns. First, Gastroboletus Fig. 4, mean divergence between ‘‘Inferiboletus’’ and Boletus s.s. is subalpinus is sister to B. regineus from the western USA and is de- estimated at 34.1 (95% HPD: 16.1–53.6) Mya and the divergence rived within the clade containing B. pinophilus and related taxa, between ‘‘Obtextiporus’’ and ‘‘Alloboletus’’ is estimated at 30.7 indicating that this taxon should be transferred to the genus Bole- (95% HPD interval: 13.5–50.3) Mya. tus as was proposed for the transfer of Gastrosuillus spp. to Suillus (Kretzer and Bruns, 1997). Second, the sister lineage to the best 4. Discussion known species of porcini, B. edulis,isBoletus reticuloceps, an infre- quently documented species known only from the mountainous 4.1. Diversity of porcini regions of Yunnan, China. This darkly pigmented species is charac- terized by a pitted, reticulated cap surface (Fig. 1; Zang et al., 1993; Eighteen clades of porcini s.s. are recognized in our analyses, Wang and Yao, 2005), reminiscent of the description of the rare, although not all of these received significant support (Fig. 2). Each relatively unknown Boletus mottiae from the Sierra Nevada in Cal- clade putatively represents a distinct species. However, the ifornia (Thiers, 1975). A second notable pattern is that none of the B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 1287

Fig. 4. Phylogram of the best tree from a ML search of the combined data using RAxML. Taxa in red are traditionally classified as porcini. Numbers above branches are ML nonparametric bootstrap values and numbers below are Bayesian posterior probabilities. Support values are provided only for nodes that received either P60% ML bootstrap and/or P.90 Bayesian posterior probability. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Table 2 Dataset characteristics and measures of homoplasy based on the best trees from a maximum likelihood search in RAxML.

Dataset Total number of aligned Number of parsimony-informative Tree Consistency Retention Rescaled consistency positions characters length index index index LSU 809 209 1159 .373 .450 .168 ATP6 638 166 693 .453 .523 .237 RPB1 498 191 1269 .284 .439 .125 Combined 1903 550 3054 .354 .441 .156 major clades are restricted to any one region; all appear to consist of porcini sensu stricto that has given rise to its current distribution. of mixtures of North American, Central American, European, and However, our sampling of B. edulis sensu stricto only includes spec- Asian taxa, suggesting a complex history of migration and dispersal imens from Europe and North America and the same is true for the 1288 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292

Table 3 erosion of the node leading to porcini in the combined dataset as Hypothesis testing using the Shimodaira–Hasegawa test for comparing two topolo- indicating the presence of only a few characters that support por- gies. SHTest was executed using RELL approximation with 1000 replicates on the best cini as monophyletic in the face of increasing levels of homoplasy trees from unconstrained and constrained maximum likelihood searches in GARLI after branch lengths were optimized in PAUP. as more and more data are added. Overall, branch support for the deepest nodes in the Boletineae, which includes the ancient rela- Best ln L from Best ln L from Diff ln Lp- tionships among porcini, was scarce in all analyses. unconstrained search constrained search Value Of the three genes used in this study, RPB1 showed the greatest LSU 6252.96636 6268.97852 16.01216 0.098 phylogenetic signal. Matheny et al. (2002) found RPB1 to improve ATP6 4257.64783 4259.22327 1.57544 0.452 the resolution and support of phylogenetic analysis of another ectomycorrhizal genus, , when used in combi- nation with LSU sequences. Frøslev et al. (2005) and Garnica clade containing Boletus pinophilus, a group that is most diverse in et al. (2009) have also shown RPB1 to provide substantial phyloge- western North America. Whether or not our sampling reflects the netic signal within . Our results show that sequence data true distribution of these taxa is yet to be determined. Finally, Bole- from LSU and ATP6 did not produce well-supported trees and did tus variipes and its darkly pigmented fagicola, described not improve the resolution of the Boletineae over earlier studies originally from eastern North America, apparently consists of two (Binder, 1999; Binder and Bresinsky, 2002; Binder and Hibbett, divergent taxa separated by Boletus hiratsukae, which is known 2006), even in combination with RPB1 and despite our broad tax- only from Japan. Interestingly, these two cryptic taxa of Boletus onomic sampling and inclusion of specimens from largely ne- variipes do not correspond to the varieties recognized by the origi- glected geographic areas (i.e. tropical). However, RPB1 only nal authors (Smith and Thiers, 1971), but show a distinct geo- marginally improved the level of resolution and branch support graphic signature: One clade occurs from North Carolina south for relationships within the Boletineae relative to the LSU and into Costa Rica and the other occurs farther north. However, sam- ATP6 datasets (Table 2, Fig. 3). In our datasets, 39% of aligned pling of these cryptic taxa throughout their entire ranges is incom- positions in RPB1 were parsimony-informative, compared to plete so this pattern may not hold under greater scrutiny. 26% in both LSU and ATP6 (Table 2). The third position of the RPB1 exons exhibited the greatest number of parsimony-informa- tive substitutions (148), followed by the first position (24) and sec- 4.2. Porcini, Boletineae and phylogenetic utility of RPB1 ond position (14). In contrast, in Inocybe, 32% of aligned positions in the RPB1 exons were parsimony-informative compared to only In this study, porcini are recovered as a monophyletic group for 10% of LSU characters (Matheny et al., 2002). This discrepancy the first time using RPB1 and a combined dataset (Figs. 3 and 4), between the greater number of parsimony-informative characters supporting the hypothesis of a single origin of the ‘‘stuffed pore’’- in the Boletineae dataset and the weak phylogenetic signal from type partial veil in this dataset. In contrast, both the LSU and these loci compared to the Inocybe dataset suggests a high degree ATP6 failed to recover this relationship (Fig. 3). Yet, the splitting of homoplasy, particularly in the third codon position. Even when of porcini into two unrelated groups in the ATP6 and LSU datasets error associated with multiple hits in the third codon position was did not receive statistical support. Moreover, for both the ATP6 and corrected for by applying a model of evolution in a maximum like- LSU datasets, alternative topologies where porcini were con- lihood framework, these genes did not provide robust phylogenetic strained to be monophyletic were no worse than the best ML trees information for the Boletineae. These observed differences in that did not recover this clade (Table 3). However, the branch sup- molecular evolution between two widespread ECM taxa, the Bolet- port for the node leading to a monophyletic porcini in the RPB1 ineae and Inocybe, might reflect differences in their relative ages dataset (Fig. 3; 74% BS, 1.0 PP) was eroded when the genes were and/or life histories. analyzed in combination (Fig. 4; 66% BS, <0.5 PP). While this might suggest some underlying conflict in phylogenetic signals from each 4.3. and nomenclature of the genes, no branches receiving statistical support in each of the single gene analyses were in conflict with each other, indicat- One proposed advantage of phylogenetic taxonomy is the abil- ing that there are no strongly contradictory phylogenetic patterns ity to recognize taxa of equal divergence (proxy for age) at equal between them (Mason-Gamer and Kellogg, 1996). We interpret the ranks (Hennig, 1965; Berbee and Taylor, 1994; Avise and Johns,

Table 4 Estimated divergence dates of selected Fungi using a relaxed molecular clock. Nodes are labeled according to Fig. 5. Estimated dates from two different calibrations are listed for each node. Under each calibration heading, the left column lists the mean ± standard error and the right column lists the 95% highest posterior density interval for each node.

452 Mya calibration Taylor and Berbee (2006) 582 Mya calibration Lucking et al. (2009) Node Mean ± standard error in Mya 95% HPD Mean ± standard error in Mya 95% HPD Root 533.7 ± 0.5 409.7–678.1 682.4 ± 0.8 509.1–884.2 Symbiomycota 476.2 ± 0.3 393.7–564.0 608.8 ± 0.4 483.5–735.5 Ascomycota/Basidiomycota 445.0 ± 0.1 383.2–508.4 568.8 ± 0.2 470.4–668.8 Pucciniomycotina/Basidiomycota 357.7 ± 0.5 275.6–439.2 456.8 ± 0.7 343.2–571.8 Ustilaginomycotina/Agaricomycotina 336.2 ± 0.5 253.0–417.4 429.4 ± 0.7 314.2–542.2 /Agaricomycotina 302.0 ± 0.6 222.3–380.8 386.0 ± 0.8 277.3–494.9 /Agaricomycetes 281.6 ± 0.7 206.8–359.1 359.9 ± 0.8 257.7–465.8 and /Agaricomycetes 222.0 ± 0.7 154.1–290.2 283.2 ± 0.8 194.2–375.5 Hymenochaetales/Agarcomycetes 189.7 ± 0.7 130.5–252.9 242.3 ± 0.8 163.6–324.1 Russulales/Agaricomycetes 174.2 ± 0.7 118.4–234.0 222.7 ± 0.8 148.6–300.3 Agaricales/Boletales 139.9 ± 0.6 91.8–191.6 178.8 ± 0.8 114.2–246.6 /Boletales 109.0 ± 0.6 68.1–152.3 140.0 ± 0.7 87.3–198.4 Suillus/Boletales 102.6 ± 0.6 63.6–144.6 132.0 ± 0.7 79.8–186.0 Boletinellus/Boletineae 71.6 ± 0.4 41.7–103.8 91.4 ± 0.5 52.7–133.5 Porphyrellus/porcini 41.9 ± 0.3 23.2–62.2 53.7 ± 0.3 29.8–80.8 Initial diversification of porcini 34.4 ± 0.2 18.8–51.2 44.0 ± 0.3 23.9–66.0 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 1289

Fig. 5. Chronogram used for the relaxed molecular clock analysis. Arrow indicates the node used for the calibration. Shaded boxes delineate taxonomic groups, labeled at top. Scale bar at bottom is in millions of years. Estimated divergence times for most nodes are listed in Table 4.

1999). Therefore, to acknowledge the difference between the rela- um, we have observed extensive divergence between the ITS se- tively minor divergence among the many taxa of porcini closely al- quences of X. stramineum and ‘‘Alloboletus’’ (unpubl.), suggesting lied to the type species, B. edulis (clade ‘‘Boletus sensu stricto’’, that they are not closely related. Moreover, X. stramineum, X. affine, Fig. 4), and the much greater divergence between the group these and X. purpureum have immature pores that appear ‘‘closed’’, not taxa form and the phylogenetically allied lineages of porcini sensu covered by partial veil (Singer, 1947, 1986; Watling, pers. obs.), lato, we have chosen to recognize the several distinct, ancient there is no reticulum on the , and they lack the characteristic divergences at the rank of genus. Hence, in addition to the afore- aroma and flavor of porcini (Dentinger, D. Arora, pers. obs.). None- mentioned ‘‘Boletus sensu stricto’’, we used three new provisional theless, we acknowledge that future work may show that X. strami- generic names (Fig. 4): (1) ‘‘Inferiboletus’’ (inferi – meaning ‘‘south- neum is closely related to X. separans, thereby rendering ern’’) is represented by the single new species from Australia ‘‘Alloboletus’’ obsolete. (REH8969) and the only known representative of porcini native to the southern hemisphere; (2) ‘‘Obtextiporus’’ (meaning ‘‘woven 4.4. Molecular clock estimates of porcini origin and divergence over pores’’, in reference to the ‘‘stuffed pore’’-type partial veil) is represented by the single new species from Thailand; (3) ‘‘Allobol- Using two different calibration dates, our relaxed molecular etus’’ (allo – meaning ‘‘other’’ in reference to the previous recogni- clock analyses suggest that porcini originated sometime in the tion that it was distinct from Boletus) corresponds to the divergent early Tertiary period during the late Eocene epoch, 41.9– group that was traditionally treated with porcini but was recently 53.7 Mya (Table 4, Fig. 5). Our estimates for the origin of porcini, recognized as morphologically distinct by Halling and Both (1998). and its initial radiation (34.4–44.0 Mya), are nearly identical to The new names are provisional because we feel it would be prema- the range of dates calculated by Bruns et al. (1998) for the radiation ture to formally describe new genera based on the few specimens of the ‘‘Boletoid’’ lineage (approximately the Boletineae here) while currently available. our date for the divergence of Suillus from the Boletales (102.6– Our recognition of ‘‘Alloboletus’’, instead of Xanthoconium as 132 Mya) exceeds the minimum age of the Suilloid clade proposed by Halling and Both (1998), is supported by the para- (50 Mya) based on fossil evidence (LePage, 1997). Interestingly, phyly of Xanthoconium in our phylogeny (Fig. 4). Although we have the new taxon ‘‘Inferiboletus’’ (REH8969) is the first documented not included X. stramineum, the nomenclatural type of Xanthoconi- porcini native to the southern hemisphere, which could be a relict 1290 B.T.M. Dentinger et al. / Molecular Phylogenetics and Evolution 57 (2010) 1276–1292 of an ancient, widespread distribution. But because our range of Arora, Robert Fogel, Josh Grinath, James Groth, Jeffrey Klemens, dates for the divergence between this taxon and the rest of porcini Christophe Lécuru, Daniel Mousain, and Tim Whitfeld. BTMD is is too young for porcini to have originated in Pangea or Gondwana- grateful to the University of Michigan Biological Station, Mountain land (Scotese and Golonka, 1997), its presence in Australia sug- Lake Biological Station, Ouachita Mountains Biological Station, and gests a more recent colonization of the continent, possibly from especially Rytas Vilgalys, Jason Thacker, and Tim James for hosting episodic, long-distance dispersal events, in agreement with several him during his 2003 collecting expedition. BTMD also thanks The other recent studies in mushrooms (Moyersoen et al., 2003; Quetzal Education and Research Center (especially Zana Finken- Moncalvo and Buchanan, 2008). In any case, the Eocene was char- binder), INBIO (especially Milagro Mata), Julieta Carranza (Univer- acterized by a warm, humid climate when the angiosperms diver- sity of Costa Rica) and Roger Blanco Segura for help with depositing sified, and porcini may have originated and diversified at a time specimens and securing permits for work in Costa Rica. We thank when the possibilities for ECM coevolution were great (Bruns the Minnesota Supercomputing Institute for Advanced Computa- et al., 1998). Our molecular clock dating and the discovery of ‘‘Infe- tional Research and Victor Hanson-Smith and the Thornton Lab riboletus’’ are also consistent with a Paleotropical origin of porcini, at the University of Oregon for access to computing resources. which has been suggested for (Horak, 1983) and the ECM The contribution of K.S. and King Mongkut’s Institute of Technol- lineages (Pirozynski, 1983; Buyck, 1989), Hysterangiales ogy in providing Roy Halling with a Material Transfer Agreement (Hosaka et al., 2008), and (Matheny et al., 2009). to study Thai bolete specimens is gratefully appreciated. PAM It is possible that our estimate for the origin of porcini is too thanks the Office de l’Environnement de la Corse for facilitating young. For example, ancient relicts of porcini from sub-Saharan fieldwork in Corsica and REH is supported by NSF Grants DEB- and/or could add deep nodes that may push 0414665 and DEB-9972018. Portions of this research were sup- the date of origin back. Recent fieldwork in south-central Africa has ported by the Mycological Society of America Backus Award, North revealed a speciose assemblage of Boletineae (Watling, 1993) American Mycological Association Memorial Fellowship, three fel- including at least one species with a ‘‘stuffed pore’’-type partial veil lowships from the University of Minnesota Graduate School (Alex- (D. Arora, pers. comm.). Furthermore, the recent discovery of the ander and Lydia Anderson Fellowship, Carolyn Crosby Fellowship, two species from Australia and Thailand illustrates that intensive Doctoral Dissertation Fellowship), and a Simons Fellowship in Sys- fieldwork can uncover previously unknown taxa from mycologi- tematic Biology and Dayton and Wilkie Natural History Funds from cally underexplored areas. 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