Evolutionary relationships of Heimioporus and Boletellus (Boletales), with an emphasis on Australian taxa including new species and new combinations in Aureoboletus, Hemileccinum and Xerocomus

Nigel Fechner

Boletellus and Heimioporus, two genera of Boletaceae with ornamented basidiospores, are shown to be distinct genera on the basis of phylogenetic analyses of nuclear ribosomal large-subunit and translation elongation-factor 1α DNA sequences. Comparison of spore ornamentation type – longitudinally ribbed in Boletellus v. punctate, alveolate-reticulate in Heimioporus – are further evidence for distinction. Analyses of multiple accessions from the Americas, Asia and Australia support the monophyly of Heimioporus and a ‘core Boletellus’ clade, containing the type species, B. ananas (M.A.Curt.) Murrill, and approximately seven additional species. Tests of alternative phylogenetic topologies could not reject monophyly of a more inclusive group containing the core Boletellus clade and six other species. Heimioporus australis Fechner & Halling, H. cooloolae Fechner & Halling, Boletellus deceptivus Halling & Fechner, B. reminiscens Halling & Fechner and B. sinapipes Fechner, K.Syme, R.Rob. & ...

CSIRO PUBLISHING Australian Systematic Botany, 2015, 28, 1–22 http://dx.doi.org/10.1071/SB14049 Evolutionary relationships of Heimioporus and Boletellus (Boletales), with an emphasis on Australian taxa including new species and new combinations in Aureoboletus, Hemileccinum and Xerocomus Roy E. Halling A,H, Nigel Fechner B, Mitchell Nuhn C, Todd Osmundson D, Kasem Soytong E, David AroraF, Manfred BinderG and David Hibbett C A Institute of Systematic Botany, The New York Botanical Garden, Bronx, NY 10458-5126, USA. Queensland Herbarium, Mt Coot-tha Road, Toowong, Brisbane, Qld 4066, Australia. C Department of Biology, Clark University, Worcester, MA 01610-1477, USA. D Department of Biology, 3034 Cowley Hall, University of Wisconsin – La Crosse, 1725 State Street, La Crosse, WI 54601, USA. E Faculty of Agricultural Technology, King Mongkut’s Institute of Technology, Ladkrabang, Bangkok, Thailand. F PO Box 672, Gualala, CA 95445, USA. G Asam Strasse 50, D-93352 Rohr in Niederbayern, Germany. H Corresponding author. Email: rhalling@nybg.org B Abstract. Boletellus and Heimioporus, two genera of Boletaceae with ornamented basidiospores, are shown to be distinct genera on the basis of phylogenetic analyses of nuclear ribosomal large-subunit and translation elongation-factor 1a DNA sequences. Comparison of spore ornamentation type – longitudinally ribbed in Boletellus v. punctate, alveolatereticulate in Heimioporus – are further evidence for distinction. Analyses of multiple accessions from the Americas, Asia and Australia support the monophyly of Heimioporus and a ‘core Boletellus’ clade, containing the type species, B. ananas (M.A.Curt.) Murrill, and approximately seven additional species. Tests of alternative phylogenetic topologies could not reject monophyly of a more inclusive group containing the core Boletellus clade and six other species. Heimioporus australis Fechner & Halling, H. cooloolae Fechner & Halling, Boletellus deceptivus Halling & Fechner, B. reminiscens Halling & Fechner and B. sinapipes Fechner, K.Syme, R.Rob. & Halling are described as new species. Phylogenetic analyses also support the following new combinations: Aureoboletus projectellus (Murrill) Halling, A. mirabilis (Murrill) Halling, Hemileccinum subglabripes (Peck) Halling and the new name, Xerocomus tenax Nuhn & Halling. Additional keywords: biogeography, boletes, Boletineae, phylogeny, ribosomal DNA, translation elongation-factor 1a. Received 13 December 2014, accepted 17 February 2015, published online 10 September 2015 Introduction Boletellus, as originally conceived by Murrill (1909), included one species from the south-eastern USA, B. ananas (M.A.Curt.) Murrill, which was based on Boletus ananas (Curtis 1848) from South Carolina. Singer’s (1986) concept of the genus included 33 species in seven sections, with either smooth or variously ornamented basidiospores. Other features, including moisture content, scaliness of the pileus, or both, or the degree of stipe ornamentation, were used to deﬁne sections. Recent phylogenetic studies by Nuhn et al. (2013) and Wu et al. (2014) suggested that such characters may not delimit clades in Boletales. However, this apparent failure of morphological characters to reﬂect underlying phylogeny may instead be a consequence of loose application of generic concepts; once concept drift is corrected (Halling and Ortiz-Santana 2009), the morphological characters of described species can have a bearing on phylogenetic inference. Journal compilation CSIRO 2015 Heimioporus was proposed by Horak (2004) to replace Heimiella Boedijn non-Lohmann (1913). The original species included by Boedijn (1951) was Boletus retisporus, described by Patouillard and Baker (1918) from specimens gathered in Singapore. In the recent nomenclatural overview by Horak (2004), 16 species were placed in two subgenera distinguished by the type of spore ornamentation (alveolate v. pitted). However, one smooth-spored taxon was admitted (Boletus mirabilis (Murrill) Murrill). With the appearance of Corner’s (1972) book on Malaysian boletes, Heimioporus E.Horak (as Heimiella Boedijn) received recognition as a distinct genus with six species, based primarily on spore morphology (the lack of an adaxial patch distinguished it from Strobilomyces Berk.) and tube trama type (truly boletoid). In contrast, Singer (1945, 1986) classiﬁed the group as Boletellus sect. Retispori Singer. Interestingly, Corner (1972) relegated www.publish.csiro.au/journals/asb 2 Australian Systematic Botany Boletellus to a subgenus of Boletus L. s.l. by virtue of extreme variation in spore shape and ornamentation, basidiome stature and tube trama type. Watling and Gregory (1986) initiated a study on boletes of the Cooloola Sandmass in south-eastern Queensland, the ﬁrst installment of which covered those taxa with ornamented spores (Austroboletus (Corner) Wolfe, Heimiella, Boletellus). This latter publication has provided a starting point from which we have assembled more material with detailed observations. We studied multiple accessions morphologically assignable to Boletellus and Heimioporus from the Americas, Australia, Japan, South-east Asia and Zambia, to investigate the limits of Boletellus v. Heimioporus, with a particular emphasis on taxa occurring in Australasia. The preliminary analyses of Osmundson (2009), Nuhn et al. (2013) and Wu et al. (2014) indicated that additional material and loci would be appropriate in our dataset. Methods and materials Morphological datasets Macromorphological data were obtained from fresh specimens. General colour terms are approximations, and the colour codes (e.g. 7D8) are page, column and grid designations from Kornerup and Wanscher (1983). Microscopic structures were observed with an Olympus BHS compound microscope equipped with Nomarski differential interference contrast (DIC) optics, and measured from dried material revived in 3% KOH. The letter abbreviation Q refers to the mean length : width ratio measured from n basidiospores, and x refers to the mean length mean width. Scanning electron micrographs of the spores were captured digitally from a Hitachi S-2700 scanning electron microscope operating at 20 kV. Hymenophoral fragments were removed from dried basidiomata, mounted directly on aluminum stubs with carbon adhesive tabs, and coated with 10 nm of gold by using a Hummer II sputter coater. Herbarium codes (Thiers 2014) are cited for all collections from which morphological features were examined. Sampling and molecular datasets Taxon sampling focused on species that have been placed in or proposed to be closely related to Boletellus, or that have longitudinally ornamented spores. The sample of taxa was informed by prior molecular studies (Binder and Hibbett 2006; Halling et al. 2012a, 2012b; Nuhn et al. 2013), as well as preliminary maximum likelihood (ML) analyses (not shown) that included as many genera of Boletaceae as possible. Genera or species that are not pertinent to Boletellus were removed, e.g. all members of the Chromapes group as reported by Halling et al. (2012b). Newly generated sequences were submitted to GenBank (Table 1); sequence alignments and phylogenies were submitted to TreeBase (#16346, http://purl.org/phylo/treebase/phylows/ study/TB2:S16346, accessed September 2014). DNA extraction, polymerase chain reaction (PCR) ampliﬁcation, sequencing and alignment Genomic DNA was extracted using the phenol–chloroform method described by Lee and Taylor (1990), with modiﬁcations (Halling et al. 2012a, 2012b). Puriﬁed gDNA was suspended in 50 or 100 mL HPLC H2O. PCR ampliﬁcation of nuclear large-subunit R. E. Halling et al. rRNA (nuc-lsu) and translation elongation-factor 1-a (tef1) genes was performed with respective primer pairs LR0R/LR5 and TEF1-983F/TEF1-2218R. PCR protocols were as previously established (Halling et al. 2012a, 2012b; Nuhn et al. 2013). PCR products were puriﬁed using a series of ethanol-centrifuge washes and sequenced by using the BigDye 3.1 terminator sequencing kit (Applied Biosystems, Foster City, California, USA). In addition to the primers used in PCR reactions, two internal primers were used for both genes: LR3R and LR3 for nuc-lsu and TEF1-1577F and TEF1-1567R for tef1. Raw sequence ﬁles were edited and assembled into contigs using Geneious R7 v.7.1.2 (Biomatters, Auckland, New Zealand; https://www. geneious.com, accessed March 2014). Curated sequences were aligned using MAFFT v7.017, as implemented in Geneious R7 (Katoh et al. 2002). The G-INS-i settings were used for nuc-lsu and the E-INS-i settings were used for tef1. Thirty-nine publicly available DNA-directed RNA polymerase Subunit 1 (rpb1) sequences were included in the analyses and were aligned using the same methods as for tef1 (Nuhn et al. 2013). Phylogenetic analyses Individual gene alignments were analysed using the RAxML Blackbox server (http://embnet.vital-it.ch/raxml-bb/, accessed May 2014; Stamatakis et al. 2008). The resulting trees were manually compared for conﬂicts with bootstrap support (BS) values greater than 70% (trees not shown). No conﬂict was observed. Therefore, a supermatrix, including 130 nuc-lsu, 111 tef1 and 37 rpb1 sequences, was assembled and analysed. Maximum likelihood and Bayesian analyses of the supermatrix used the CIPRES Science Gateway (Miller et al. 2010). For all phylogenetic analyses, the supermatrix was partitioned by locus, and members of the Paxillaceae were used as the outgroup. The tree topology and BS values were estimated using ML, as implemented by RAxML HPC2 on XSEDE (Stamatakis 2014), with the following parameters: GTRGAMMA model of molecular evolution, 1000 rapid bootstrap replicates, and a simultaneous best-scoring ML tree search. Bayesian posterior probability (PP) values were estimated with MrBayes v3.2.2 (Huelsenbeck and Bollback 2001; Ronquist et al. 2012). The supermatrix was analysed with two runs of four Markov chain Monte Carlo (MCMC) chains using the GTR model, gamma distribution rates, and 30 million generations. Sampling occurred every 100 generations and a burn-in fraction of 0.3 was used. Convergence was checked using Tracer (Rambaut et al. 2014) and AWTY (Wilgenbusch et al. 2004). Alternative-topology testing The optimal topology recovered in ML analyses suggests that Heimioporus is nested within a weakly supported paraphyletic grade that contains most of the species referred to Boletellus (Fig. 1). An alternative topology that forces monophyly of Boletellus (excluding only Boletellus russellii (Frost) E.-J.Gilbert and B. shichianus (Teng & L.Ling) Teng), with Heimioporus as the sister group, was evaluated. Topology testing was performed using the following three methods: Bayesian analysis (Ronquist et al. 2012), the Shimodaira–Hasegawa (SH) test (Stamatakis 2014) Isolate ID 00-436 Pug1 MB 03-104 REH8717 REH9765 Xle1 NYBG13392 NYBG13393 AFTOL-713; MB03-118 DS626-07 AFTOL-450 ARB1223 REH7763 REH7924 REH8548 REH8613 REH8788 00-335 REH9484 REH9690 Syme 3794 REH8923 DD9852 134/96 3838 54/97 REH9015 REH8937 REH9017 REH9634 REH9653 REH9786 REH8943 REH9435 REH9466 REH9624 REH9688 REH9768 TMI22101 Species Afroboletus luteolus Aureoboletus gentilis Aureoboletus innixus Aureoboletus mirabilis Aureoboletus mirabilis Aureoboletus moravicus Aureoboletus projectellus Aureoboletus projectellus Aureoboletus projectellus Aureoboletus roxanae Aureoboletus thibetanus Boletellus ananas Boletellus ananas Boletellus ananas Boletellus ananas Boletellus ananas Boletellus ananas? Boletellus sp. Boletellus ananiceps Boletellus ananiceps Boletellus ananiceps Boletellus ananiceps Boletellus badiovinosus Boletellus betula Boletellus betula Boletellus chrysenteroides Boletellus chrysenteroides Boletellus chrysenteroides Boletellus deceptivus Boletellus deceptivus Boletellus deceptivus Boletellus deceptivus Boletellus deceptivus Boletellus dissiliens Boletellus dissiliens Boletellus dissiliens Boletellus dissiliens Boletellus dissiliens Boletellus dissiliens Boletellus elatus Chestnut Ridge Park, New York, USA Kunming, Yunnan, China Crooked River, Georgia, USA Alajuela Province, Costa Rica Cartago Province, Costa Rica Belize District, Belize Puntarenas Province, Costa Rica Chiang Mai Province, Thailand Zambia Fraser Island, Queensland, Australia Cooloola, Queensland, Australia Denmark, Western Australia, Australia Victoria, Australia Davies Creek, Queensland, Australia Orange County, North Carolina, USA Massachusetts, USA Towy Park, North Collins, New York, USA Massachusetts, USA Pennsylvania, USA Mount Baldy, Atherton State Forest, Queensland, Australia Fraser Island, Queensland, Australia Mount Baldy, Atherton State Forest, Queensland, Australia Springbrook National Park, Queensland, Australia Queen Mary Falls, Queensland, Australia Mount Baldy, Atherton State Forest, Queensland, Australia Fraser Island, Queensland, Australia Fraser Island, Queensland, Australia Davies Creek, Queensland, Australia Cooloola, Queensland, Australia Vicinity Canungra, Queensland, Australia Tottori, Japan Zambia Maindreieck, Germany Lincoln, Massachusetts, USA Humboldt County, California Mendocino, California, USA Maindreieck, Germany North Collins, New York, USA North Collins, New York, USA Cape Cod, Massachusetts, USA Location 18 Feb. 2011 6 Mar. 2011 15 Feb. 2012 11 Mar. 2012 12 Feb. 2013 6 Sep. 1996 14 Feb. 2013 21 Mar. 2007 2 Mar. 2012 7 Feb. 2009 21 Feb. 2012 25 Oct. 2006 – 19 Oct. 2012 27 June 1998 24 June 2000 14 Oct. 2003 11 June 2004 4 June 2006 2000 17 May 2011 12 Mar. 2012 25 May 2011 7 May 2010 14 Mar. 2007 28 Feb. 2013 31 Aug. 1996 10 Oct. 1995 21 Aug. 1997 10 Aug. 2008 21 Mar. 2007 3 Oct. 1995 3 Aug. 2014 15 Nov. 2005 10 Dec. 2012 19 Sep. 1998 20 Sep. 1987 23 Aug. 1986 14 Sep. 2003 Date R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling E. Nagasawa R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling E. Both Z.-L. Yang A.R. Bessette R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling D. Arora R.E. Halling R.E. Halling K. Syme N.H. Sinnot R.E. Halling D. Drehmel N. Arnold E. Both N. Arnold, H. Besl R.E. Halling R.E. Halling D. Arora J. Schreiner M. Binder R.E. Halling R.E. Halling J. Schreiner E. Both E. Both M. Binder Collector identiﬁer JX889674 KP327654 KP327657 KP327659 KP327662 KP327620 KP327663 KP327642 KP327658 KP327646 KP327619 KF030311 AY700189 KP327618 KP327626 KP327627 KP327628 KP327629 KP327636 KF030330 KP327655 KP327660 KP327667 KP327666 KP327640 AY612797 AF050642 KF030312 DQ534634 KP327645 KP327641 KF030238 DQ534635 KF030239 KF030299 KP327661 DQ534641 KP327622 KP327623 AY684158 nuc-lsu Table 1. Voucher information and GenBank accession numbers (new submissions in bold) for the specimens studied – – – – – – – – – – – KF030381 DQ435800 – – – – – – – – – – – – – – KF030383 – – – KF030392 – – – – – – – AY662660 rpb1 Australian Systematic Botany (continued next page) KP327673 KP327706 KP327707 JX889684 KP327694 KP327697 KP327672 KF030402 DQ029199 KP327671 KP327679 KP327680 KP327681 KP327682 KP327689 – KP327705 KP327708 – KP327712 KP327693 – – KF030432 – – – KF030397 KF030399 KF030400 – KP327709 KF030403 KP327675 KP327676 AY879116 tef1 Heimioporus and Boletellus in Australia 3 Isolate ID 06-204NR REH8734 REH8742 REH8808 REH8904 REH8741 REH8782 REH8905 REH9034 REH9038 REH9061 REH9284 DPL6698 MEN11-031 73887 AFTOL-532 REH9018 REH9408 REH9592 25511 VB4530 VB4460 4588 112605ba MEN11-0MB MB 07-001 2858 Bc1 MB 06-061 JAM 0607 Be1 MB 06-060 MB 06-044 AT2001087 8206 3959 DS615-07 DS 4514 9606 REH8721 4252 MB 06-059 4414 27882 4249 Species Boletellus elatus Boletellus emodensis Boletellus emodensis Boletellus emodensis Boletellus emodensis Boletellus obscurecoccineus Boletellus obscurecoccineus Boletellus obscurecoccineus Boletellus obscurecoccineus Boletellus obscurecoccineus Boletellus obscurecoccineus Boletellus reminiscens Boletellus russellii Boletellus russellii Boletellus russellii Boletellus shichianus Boletellus sinapipes Boletellus sinapipes Boletellus sinapipes Boletellus sinapipes Boletellus singeri Boletellus singeri Boletus abruptibulbus Boletus amygdalinus Boletus aurantioruber Boletus bicolor var. bicolor Boletus bicolor var. borealis Boletus calopus Boletus carminipes Boletus dupainii Boletus edulis Boletus ﬁrmus Boletus inedulis Boletus luridiformis Boletus morrisii Boletus peckii Boletus pseudosensibilis Boletus pulchriceps Boletus pulverulentus Boletus regineus Boletus rhodosanguineus Boletus roseopurpureus Boletus rufomaculatus Boletus subalpinus Boletus variipes var. fagicola 25 June 2005 7 Feb. 2009 15 Feb. 2011 17 July 2011 25 May 2011 5 Sep. 2012 27 Oct. 2009 1 Apr. 2005 26 Nov. 2005 12 July 1998 30 July 2006 6 Aug. 1997 10 Aug. 1968 Yunnan, China Fraser Island, Queensland, Australia Fraser Island, Queensland, Australia Denmark, Western Australia, Australia Denmark, Western Australia, Australia Veracruz, Mexico Veracruz, Mexico Cape San Blas, Florida, USA Mendocino, California, USA Cape Cod, Massachusetts, USA Chestnut Ridge Park, New York, USA Erie County, New York, USA Bavaria, Germany Erie County, New York, USA Butner, New York, USA Bavaria, Germany Chestnut Ridge Park, New York, USA Erie County, New York, USA Berkshire, England, UK Concord, Massachusetts, USA Erie County, New York, USA Chestnut Ridge Park, New York, USA Chiricahua Mountains., Arizona, USA West Newton, Massachusetts, USA Redwood National Park, Humboldt County, California, USA Chestnut Ridge Park, New York, USA Chestnut Ridge Park, New York, USA Chestnut Ridge Park, New York, USA Cheboygan County, Michigan, USA 2 Aug. 2006 4 Aug. 1995 7 July 1995 1 Aug. 91 9 June 2006 17 Nov. 2005 7 Sep. 1994 4 Aug. 2006 8 Mar. 2000 14 Sep. 1994 3 Aug. 2006 3 Aug. 2006 28 July 1995 14 Sep. 2006 4 Feb. 2006 9 Feb. 2006 11 June 2006 11 Mar. 2007 6 Feb. 2006 25 Feb. 2006 11 Mar. 2007 8 Feb. 2009 9 Feb. 2009 12 Feb. 2009 27 Mar. 2010 22 June 2005 3 July 2005 Date Table 1. (continued ) Tottori, Japan Davies Creek, Queensland, Australia Davies Creek, Queensland, Australia Chiang Mai Province, Thailand Davies Creek, Queensland, Australia Paluma, Queensland, Australia Paluma, Queensland, Australia Davies Creek, Queensland, Australia Fraser Island, Queensland, Australia Fraser Island, Queensland, Australia Fraser Island, Queensland, Australia Fraser Island, Queensland, Australia Texas Mount Wachusetts, Massachusetts, USA Location KF030252 KF030262 KF030248 KF030430 JQ327014 KP327621 KP327631 KP327633 KP327637 KP327638 KP327632 KP327634 KP327639 KP327648 KP327649 KP327650 KP327651 KF030325 KP327625 AF050651 AY647211 KP327647 KP327653 KP327656 KP327668 KP327669 KP327670 KF030302 JQ326996 KF030342 KF030370 JQ326998 AF456833 JQ327001 KF030251 AF050643 KF030278 JQ327013 JQ326995 KF030433 JQ326999 KF030257 KF030261 KF030313 KF030339 nuc-lsu KF030412 KF030410 KF030406 KF030427 JQ327017 KP327713 – KF030401 JQ327024 – KF030405 JQ327021 JQ327019 JQ327022 KF030413 JQ327018 KF030408 JQ327020 JQ327023 – JQ327026 KF030407 KF030409 KF030418 KF030426 KP327674 KP327684 KP327686 KP327690 KP327691 KP327685 KP327687 KP327692 KP327699 KP327700 KP327701 KP327702 – KP327678 – DQ408145 KP327698 KP327704 tef1 – KF030372 KF030369 KF030379 KF030378 – – – – – – – – – – – – – – – – – – – – – – KF030388 KF030360 – – – – KF030363 KF030361 – KF030368 KF030362 – – – – KF030376 KF030364 KF030377 rpb1 Australian Systematic Botany E. Both A. Taylor, M. Binder E. Both J. Trappe A. H. Smith Bill Neill R.E. Halling Z.-L. Wang R.E. Halling R.E. Halling R.E. Halling K. Syme V.M. Bandala V.M. Bandala E. Both B. Neill M. Binder E. Both E. Both N. Arnold M. Binder, E. Both E. Both M. Binder M. Binder M. Binder, E. Both A.F.S. Taylor B. Neill A.R. Clark, E. Both E. Both E. Nagasawa R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling R.E. Halling D. Lewis M.E. Nuhn Collector identiﬁer 4 R. E. Halling et al. HKAS73785 Pul1 Bap1 11265 MB 04-001 4302 DS4640-3 REH9502 238 Gl1 REH9288 REH9817 REH9852 REH8958 REH8962 REH8620 FRIM4636 REH8785 REH8074 MS6 Bim1 72206 Buf 4507 930809/1 MB 06-040 Ls1 MB 06-066 Pf1 Po1 Pv2 Pp1 Xpa1 202/97 DED 7873 Sf1 REH8514 78206 AT2001011 MB 06-053 Xch1 AT2005034 REH8724 MB 00-005 Xs1 REH6871 Borofutus dhakanus Buchwaldoboletus lignicola Butyriboletus appendiculatus Butyriboletus autumniregius Chalciporus piperatus Chalciporus pseudorubinellus Chalciporus rubinus Fistulinella prunicolor Fistulinella viscida Gyrodon lividus Heimioporus australis Heimioporus cooloolae Heimioporus cooloolae Heimioporus fruticicola Heimioporus fruticicola Heimioporus ivoryi Heimioporus mandarinus Heimioporus mandarinus Heimioporus retisporus Heimioporus retisporus Hemileccinum impolitum Hemileccinum subglabripes Leccinellum corsicum Leccinellum crocipodium Leccinum albellum Leccinum scabrum Paragyrodon sphaerosporus Paxillus ﬁlamentosus Paxillus obscurisporus Paxillus vernalis Phylloporus pelletieri Pseudoboletus parasiticus Retiboletus griseus Spongiforma thailandica Strobilomyces ﬂoccopus Strobilomyces sp. Tylopilus badiceps Tylopilus felleus Tylopilus ferrugineus Xerocomellus chrysenteron Xerocomellus cisalpinus Xerocomellus zelleri Xerocomus perplexus Xerocomus subtomentosus Xerocomus tenax 9 Aug. 1993 30 July 2006 14 Sep. 1995 22 Sep. 2006 21 Aug. 1995 21 Aug. 1998 31 Aug. 1997 9 Sep. 1995 9 July 1995 30 Aug. 1997 France Erie County, New York, USA Austria Iowa City, Iowa, USA Bavaria, Germany Bavaria, Germany Canada Bavaria, Germany Bavaria, Germany Massachusetts, USA Bronx, New York, USA 25 July 1995 5 Aug. 2000 10 Aug. 1995 7 July 2005 12 Aug. 1995 3 Oct. 2003 28 July 2006 17 Sep. 2001 3 Aug. 2006 8 Aug. 1995 30 July 2005 18 Nov. 2005 21 Sep. 1995 22 July 2006 Bavaria, Germany Jefferson, New Hampshire, USA Thailand Bavaria, Germany Cayo District, Belize Holliston, Massachusetts, USA Stadsskogen, Uppsala, Sweden Erie County, New York, USA Bavaria, Germany Uppsala, Uppland, Sweden Redwood National Park, Humboldt County, California, USA Rutland, Massachusetts, USA Bavaria, Germany 23 Mar. 2007 12 June 2004 19 June 2007 3 June 2006 16 Jan. 2001 11 Aug. 1995 28 Mar. 2010 22 Feb. 2013 28 Feb. 2013 22 Mar. 2007 18 May 2011 7 June 2011 9 Sep. 1995 10 Aug. 1995 26 Nov. 2005 28 Sep. 2004 14 Sep. 1998 Bavaria, Germany Fraser Island, Queensland, Australia Cooloola, Queensland, Australia Cooloola, Queensland, Australia Davies Creek National Park, Queensland, Australia Kuranda, Queensland, Australia Puntarenas Province, Costa Rica Sungai Kejar, Malaysia Chian Mai Province, Thailand Java, Indonesia Bangladesh Maindreieck, Germany Bavaria, Germany Mendocino County, California, USA Rutland, Massachusetts, USA Deer Meadows, Tulet, New York, USA Germany Fraser Island, Queensland, Australia M. Binder J. Enzmann, A. Bresinsky R.E. Halling A.F.S. Taylor E. Both M. Binder A.F.S. Taylor R.E. Halling G. Lannoy M. Binder M. Binder M. Binder L. Krieglsteiner Ch. Hahn T. Lohmeyer M. Kronfeldner A. Bresinksy N. Arnold, W. Helfer, W. Steglich D.E. Desjardin J. Enzmann R.E. Halling J. Schreiner B. Neill R.E. Halling R.E. Halling R.E. Halling R.E. Halling H. Besl, W. Helfer R.E. Halling R.E. Halling R.E. Halling R.E. Halling Md. Iqbal Hosen J. Schreiner J. Schreiner Bill Neill M. Binder E. Both J. Schreiner R.E. Halling KF030320 JQ003702 AF139716 EU685108 DQ534626 EU685109 KF030335 JQ326993 JQ326994 AF050647 KF030354 KF030271 KP327644 KP327630 KP327624 KP327635 KP327617 AF050650 AF139715 KF030303 KF030347 AF139694 JQ327007 AF139705 GU187593 AF167680 AY177256 AY645059 AF456818 AF050646 AF456834 JQ928615 JQ326997 AF456837 KF030267 DQ534648 KF030284 KF030283 J889648 AF456826 AF098378 KP327652 KP327664 KP327665 KP327643 KF030437 KF030438 JQ327035 KF030436 JQ327037 KF030398 KF030429 JQ327015 JQ327016 KF030415 KF030417 KF030416 KP327696 KP327683 KP327677 KP327688 – – JQ327034 KF030404 KF030435 KF030434 JQ327038 JQ327039 GU187737 GU187736 KF030442 DQ457629 JQ327036 KF030443 KF030414 JQ928577 JQ327040 JQ327025 KF030411 GU187690 KF030441 KF030440 JX889690 – GU187701 KP327703 KP327710 KP327711 KP327695 – – KF030391 KF030387 AY858963 – – KF030380 – KF030365 KF030367 KF030366 – – – – – – KF030375 KF030374 KF030389 – – – – – – – KF030390 KF030394 KF030373 JQ928585 – KF030359 – GU187453 – – – – GU187461 – – – – Heimioporus and Boletellus in Australia Australian Systematic Botany 5 6 Australian Systematic Botany Boletellus deceptivus REH8937 N QLD Boletellus deceptivus REH9786 Boletellus deceptivus REH9653 S QLD Boletellus deceptivus REH9634 N QLD 0.97 Boletellus deceptivus REH9017 S QLD 100 Boletellus emodensis REH8742 98 Boletellus emodensis REH8734 N QLD Boletellus emodensis REH8904 Boletellus emodensis REH8808 THAILAND 77 76 Boletellus 0.99 Bolet dissiliens REH9435 S QLD 76 Boletellus Bole dissiliens REH8943 N QLD 95 Boletellus Bolet dissiliens REH9466 S QLD 96 94 Boletellus dissiliens REH9768 Boletellus dissiliens REH9688 0.99 Boletellus dissiliens REH9624 N QLD 87 Boletellus ananiceps Syme WA 83 0.99 Boletellus ananiceps 3794 VIC 100 Boletellus ananiceps REH9690 92 S QLD 0.99 0.99 0.99 0.99 Boletellus ananiceps REH9484 99 Boletellus ananas REH8613 100 Boletellus ananas REH7763 COSTA RICA 76 Boletellus ananas REH7924 100 83 Boletellus ananas REH8548 BELIZE 0.98 0.96 Boletellus ananas ARB1223 Georgia, USA 79 Boletellus ananas? REH8788 THAILAND 0.99 Boletellus sp. ZAMBIA core Boletellus 96 Boletellus singeri VB4460 MEXICO 94 Boletellus singeri VB4530 0.99 Bo obscurecoccineus REH9038 100 Boletellus 100 Boletellus Bo obscurecoccineus REH9034 S QLD B Boletellus obscurecoccineus REH9061 100 85 Boletellus Bolet obscurecoccineus REH8905 100 Bole Boletellus obscurecoccineus REH8741 N QLD Bolet Boletellus obscurecoccineus REH8782 100 Boletellus sinapipes REH9592 WA 85 Boletellus sinapipes 25511 100 Boletellus sinapipes REH9018 0.99 QLD Boletellus sinapipes REH9408 Boletellus reminiscens REH9284 83 Boletellus chrysenteroides 3838 100 Boletellus chrysenteroides 54/97 USA 100 0.95 Boletellus chrysenteroides REH9015 Boletellus badiovinosus REH8923 98 Heimioporus fruticicola REH8962 0.99 Heimioporus fruticicola REH8958 QLD Heimioporus australis REH9288 100 Heimioporus cooloolae REH9852 Heimioporus cooloolae REH9817 Heimioporus mandarinus REH8785 THAILAND 71 Heimioporus Heimioporus mandarinus FRIM4636 MALAYSIA 87 99 Heimioporus retisporus REH8074 JAVA Heimioporus retisporus MS6 0.95 Heimioporus ivoryi REH8620 COSTA RICA Boletus morrisii 8206 100 Boletellus betula DD9852 USA Boletellus betula 134/96 100 Boletellus elatus 06-204NR JAPAN —Type Species Boletellus elatus TMI22101 0.96 100 Aureoboletus projectellus NYBG13393 —Type Species 100 Aureoboletus projectellus NYBG13392 collection nearest 99 Aureoboletus projectellus MB03-118 type locality 100 Aureoboletus mirabilis REH8717 USA Aureoboletus mirabilis REH9765 Boletellus russellii DPL6698 100 Boletellus russellii MEN11-031 Boletellus russellii AF050651 100 Aureoboletus roxanae 99 70 Aureoboletus moravicus Aureoboletus gentilis 0.99 Aureoboletus thibetanus Boletus abruptibulbus 75 Boletellus shichianus CHINA Aureoboletus innixus Xerocomus perplexus 98 86 Xerocomus tenax 100 Xerocomus subtomentosus Phylloporus pelletieri 100 Hemileccinum impolitum Hemileccinum subglabripes 97 Fistulinella viscida Fistulinella prunicolor 94 Boletus subalpinus 100 Boletus regineus 79 Boletus aurantioruber 0.99 100 Boletus edulis Boletus variipes var. fagicola Strobilomyces sp. 100 85 Strobilomyces floccopus 96 Afroboletus luteolus 100 Tylopilus ferrugineus 100 95 Tylopilus badiceps Tylopilus felleus 100 Xerocomellus chrysenteron 100 Xerocomellus zelleri Xerocomellus cisalpinus Leccinum albellum Leccinum scabrum 100 Leccinellum crocipodium 97 Leccinellum corsicum 100 99 Spongiforma p g thai thailandica 0.99 dhakanus Borofutus dhaka Retiboletus griseus 100 “Boletus bicolor var. bicolor” Boletus rufomaculatus 94 Boletus calopus 99 Boletus inedulis Boletus firmus 92 Boletus rhodosanguineus Boletus dupainii Caloboletus Boletus amygdalinus 0.97 Boletus luridiformis 76 Boletus peckii 100 100 0.99 99 Boletus pulchriceps 100 Butyriboletus autumniregius Butyriboletus appendiculatus 0.99/98 0.95 Boletus roseopurpureus 0.99 91 Boletaceae “Boletus bicolor var. borealis” 100 Boletus carminipes 100 75 Boletus pseudosensibilis 0.99 Boletus pulverulentus Pseudoboletus parasiticus Chalciporus rubinus 86 Chalciporus pseudorubinellus 100 0.99 Chalciporus piperatus Buchwaldoboletus lignicola 100 Paxillus obscurisporus Paxillaceae 100 Paxillus vernalis 100 Paxillus filamentosus Paragyrodon sphaerosporus Gyrodon lividus 0.04 substitutions R. E. Halling et al. B. betula & B. elatus Heimioporus Heimioporus and Boletellus in Australia and the ‘approximately unbiased’ (AU) test (Shimodaira and Hasegawa 2001). For the Bayesian analysis, two models were tested, one that forces the monophyly of Boletellus species in the indicated clade (see Fig. 1, inset), and another that forces the absence of this Boletellus clade. Unlike the analyses using the SH and AU tests, a sister-group relationship between Boletellus and Heimioporus was not enforced. Two MCMC analyses were performed, one for each model, using the same settings (e.g. number of generations, sampling) as described previously. Additionally, the arithmetic and harmonic mean for each MCMC analysis was calculated. For the SH and AU tests, a constraint tree was manually generated from the ML topology by swapping branches to force the sister relationship between Boletellus and Heimioporus, and collapsing all other ingroup nodes to a polytomy. For the SH test, 100 trees were generated using RAxML under the settings described previously, except that each inference was independent and constrained to have the sister-clade topology, and bootstrapping and ML optimisation were not performed (Stamatakis 2014). The 100 trees were then compared to the unconstrained ML tree using the default SH test settings (Stamatakis 2014). The AU test requires per site log-likelihood values, which were calculated using RAxML, under the GTRGAMMA model for all 101 trees (Shimodaira and Hasegawa 2001; Stamatakis 2014). Consel version 0.2 (http://en.shimolab.com/consel, accessed May 2014) was used to perform the AU test for all 101 trees, with default settings, and the resulting P-value for each tree was recorded. Results Phylogenetic analyses The concatenated alignment contains ~49% gaps or undetermined characters. The RAxML analysis reported 2354 unique alignment patterns in the concatenated alignment. A sample of 380 682 trees was used to estimate Bayesian PP. The Paxillaceae and Boletaceae were strongly supported (Fig. 1). As in prior analyses (Binder and Hibbett 2006; Halling et al. 2012a, 2012b; Nuhn et al. 2013), clades that largely correspond to genera are often supported, but backbone nodes between these clades and the early most diverging clades are generally weakly supported. Clades of non-target species that are strongly supported include Boletus sensu stricto, Tylopilus sensu stricto, and the clade containing Xerocomus sensu stricto and Phylloporus pelletieri (Lév.) Quél. (Fig. 1). Seventeen strongly supported clades containing only Boletellus species (including nested clades) were recovered, as well as a clade with 1.0 PP that contains Aureoboletus mirabilis Australian Systematic Botany 7 (Murrill) Halling and A. projectellus (Murrill) Halling (which were formerly placed in Boletellus sensu Singer 1945), Boletellus russellii, B. shichianus, A. gentilis (Quél.) Pouzar (the type of Aureoboletus) and others (Fig. 1). The most inclusive strongly supported group of Boletellus species (including the type species, B. ananas) is labelled as the ‘core Boletellus’ clade (Fig. 1). The core Boletellus clade is nested within a paraphyletic assemblage including three lineages that contain (1) Boletellus chrysenteroides (Snell) Snell and B. badiovinosus E.Horak, (2) B. reminiscens and (3) B. sinapipes. Monophyly of the core Boletellus clade plus B. chrysenteroides, B. badiovinosus, B. reminiscens and B. sinapipes is weakly supported. A clade containing six species of Heimioporus (including the type species, H. retisporus (Pat. & C.F.Baker) E.Horak) is strongly supported (BS = 99, PP = 1.0), with Boletus morrisii Peck as its sister group (PP = 0.95). The Heimioporus–B. morrisii clade is weakly supported as the sister group of the group that contains the core Boletellus clade (Fig. 1). Two strongly supported lineages containing two isolates each of Boletellus betula (Schwein.) E.-J.Gilbert and Boletellus elatus Nagasawa are weakly supported as sister taxa, and the B. betula–B. elatus group is weakly supported as the sister group of the clade containing Heimioporus and the core Boletellus clade (Fig. 1). Alternative-topology testing The Bayesian MCMC analysis that forced monophyly of Boletellus species (excluding B. russellii and B. shichianus) returned a harmonic mean average of both runs of –45 935.69, whereas the analysis that forced non-monophyly of Boletellus species had a harmonic mean average of –45 932.64 (a difference of –0.03172 log-likelihood units). In the SH test, the unconstrained optimised ML tree had a score of –49 784.032084, whereas all the constrained trees had higher likelihoods than did the unconstrained topology, with an average score of –49 743.86979, and none of the constrained trees could be rejected. P-values for all constrained trees were greater then 0.05. None of the constrained topologies could be rejected using the AU test (P = 0.361–0.756). Collectively, these results suggest that monophyly of the core Boletellus clade plus B. chrysenteroides, B. badiovinosus, B. betula, B. elatus, B. reminiscens and B. sinapipes cannot be rejected. Biogeography Boletellus and related taxa have complex biogeographic relationships. The core Boletellus clade contains taxa from North and Central America (USA, Mexico, Belize and Costa Fig. 1. Phylogenetic relationships between species of the genera Boletellus and Heimioporus within the Boletaceae. The topology was inferred from the nucLSU, tef 1-a, and rpb2 using maximum likelihood (ML) methods with likelihood optimization of the ﬁnal topology using RAxML. Bootstrap values 70% are reported, as are posterior probabilities (PP), generated using Mr Bayes, if PP0.95 but <1. Bold branches represent PP values 0.95. Closed circles indicate a clade that has an alternative topology that cannot be rejected based on an AU test, as implemented in Consel, and the SH test as implemented in RAxML. The topology recovered in the ML analysis results in a polyphyletic grouping of Boletellus species with Heimioporus species. The alternative topology (inset) places Boletellus and Heimioporus as sister clades; and clades that have different topologies are indicated. Names in bold represent the taxa of interest. Locations of Australian exemplars are abbreviated: QLD (Queensland), VIC (Victoria), WA (Western Australia). The polyphyletic nature of Caloboletus is indicated with a broken line uniting Boletus calopus, B. inedulis, B. ﬁrmus, and B. peckii, but not B. rhodosanguineus, B. dupainii, B. amygdalinus, and B. luridiformis. 8 Australian Systematic Botany Rica), Africa (Zambia), Thailand and Australia. Similarly, the Heimioporus clade contains taxa from Australia, Costa Rica, Java, Malaysia and Thailand. B. betula, collected in the USA, is the sister group to B. elatus, which was collected in Japan. A single Thailand collection of B. emodensis (Berk.) Singer is nested within a clade of Australian specimens. A regional geographical pattern of a north–south split was recovered for B. obscurecoccineus (Höhn.) Singer specimens collected in Queensland, Australia. Taxonomy Boletellus ananas (M.A.Curt.) Murrill, Mycologia 1: 10 (1909) (Figs 2A, 3A, 4A) Boletus ananas M.A. Curt., Amer. J. Sci. Arts 6: 351 (1848). =? Boletus coccineus Fr., Epic. Myc. 423 (1838), nom. illeg., non Boletus coccineus Bull., Hist. Champ. France 364 (1791). Strobilomyces coccineus Sacc., Syll. Fungorum 6: 50 (1888). Boletellus coccineus (Sacc.) Singer in Singer, García & Gómez, Beih. Nova Hedwigia 105: 6 (1992). = Boletus isabellinus Peck, Bull. Torrey Bot. Club 24: 146 (1897), nom. illeg., non Schweintiz, Schr. naturf. Ges. Leipzig 1: 96 (1822). Mycobank number: MB 100683. This iconic bolete, originally described from the south-eastern USA (South Carolina, Curtis 1848), is the type species of the genus and has been described, illustrated and characterised in more detail in several publications on North American boletes (Coker and Beers 1943; Singer 1945; Thiers 1963; Smith and Thiers 1971; Bessette et al. 2000). The species has also been collected, described and illustrated from Mexico, Central America and Colombia (Singer 1970; Singer et al. 1983, 1992; Halling and Mueller 2005; Ortiz-Santana et al. 2007). It was originally diagnosed as parasitic on pine trees (Murrill 1909), but this has since been discounted (Singer 1945; Thiers 1963). Without justiﬁcation, Corner (1972) went so far as to suggest that B. ananas, B. emodensis and B. dissiliens (Corner) Pegler & T.W. K. Young appeared not to be mycorrhizal. On the basis of our (REH, NF) observations of the latter two species in Queensland (including B. deceptivus and B. ananiceps (Berk.) Singer), these taxa can appear (but not always) attached to bark at the base of living trees or coming from logs on the ground. Thiers (1963) noted that this type of habit is ‘. . .the result of the activity of mycelium which has migrated from the soil to the outer bark. . .’ (p. 37). Corner (1972) summarised the reports of B. ananas known to him, and Horak (2011) later added information for the USA, South-east Asia and Australasia (Australia, New Zealand and New Caledonia). Heim and Perreau-Bertrand (1963) discussed possible synonymy of the ananas-group in their treatment of Boletellus from Madagascar and New Caledonia. Watling and Gregory (1986) compared B. ananas in some detail to concepts of Singer (1955), Corner (1972), Pegler and Young (1981) and Singer et al. (1983). Their commentary is largely based on Australian specimens lacking good ﬁeld notes, but they essentially conclude that B. ananas is an American species. With the possible exception of Halling 8788 from Thailand, we would agree that the reports of B. ananas from Australasia R. E. Halling et al. and South-east Asia actually describe any combination of B. emodensis, B. deceptivus or B. dissiliens, and possibly B. ananiceps if the spores were not examined for presence or absence of cross-striae. Similarly, Zeng and Yang (2011) noted that the occurrence of B. ananas in China has not yet been conﬁrmed. The best, detailed descriptions of B. ananas are those of Singer (1945, but excluding Strobilomyces pallescens Cooke & Massee as a synonym), Thiers (1963) and Ortiz-Santana et al. (2007, as B. coccineus (Sacc.) Singer). It is important to note here that these accounts feature the idiosyncrasies of the morphology of the pileus, stipe and context and the speciﬁc location of oxidation reactions. Although there have been descriptions published since, critical details on changes in the temporal integrity of pigments, squamules and oxidation reactions are often not recorded. This is likely to be due to the age of specimens or isolated environmental conditions when collected. In particular, the true colour of the context (both pileus and stipe before oxidation), and the speciﬁc colour and location of the oxidations are distinctive. As noted by the authors above, the colour of the pileus context is some shade of yellow at ﬁrst and is quickly cyanescent. The oxidation reaction quickly masks the true colour and makes it seem white. The stipe context is white (rarely yellowish-cream), reddening or cyanescent (sometimes slowly). These localised context colours and oxidation reactions are similar to those observed for B. ananiceps, B. deceptivus and B. dissiliens (see below). So far, in this group, only B. emodensis has yellow ﬂesh throughout and is cyanescent throughout. Material examined (among 47 specimens in NY) USA. Georgia. Camden County, Crooked River State Park, 19 Oct. 2012, A.R. Bessette 1223 (NY). North Carolina. Carteret County, Kohlmeyer property, Broad Creek, 6 July 1971, J.J. Kohlmeyer 3390 (NY). South Carolina. Society Hill, Aug. 1847, M.A. Curtis (FH); Santee Canal, Ravenel (M.A. Curtis 1649) (isotype: FH). BELIZE. Belize District. Western Highway: Foster Property, near Belize Zoo, 14 Oct. 2003, Halling 8548 (BRH, NY); 16 Oct. 2003, Halling 8560 (BRH, NY). COSTA RICA. Alajuela Province Grecia, Bosque del Niño, 27 June 1998, Halling 7763 (NY, USJ); Cartago Province Palo Verde, 4.5 km E of km 31 of Interamerican Highway near town of Palo Verde, Halling 7924 (NY, USJ); Guanacaste Province Area Conservación Guanacaste, Parque Nacional Rincon de la Vieja, sector Pailas, 18 June 2003, Halling 8391 (NY, USJ). Puntarenas Province Zona Protectora Las Tablas, Sitio Tinieblas, Halling 8613 (NY, USJ). THAILAND. Chiang Mai Province km 22 on Highway 1095, Halling 8788 (MFLU, NY). Boletellus ananiceps (Berk.) Singer, Sydowia 9: 423 (1955) (Figs 2B, 3B, 4E) Boletus ananaeceps Berk., J. Linn. Soc., Bot. 13: 161 (1873). Strobilomyces ananaeceps (Berk.) Sacc., Syll. Fung. 6: 50 (1888). Mycobank number: MB 810820. Singer (1955) was the ﬁrst to recognise this taxon as a Boletellus and concluded that it was similar to B. ananas but was distinguished by the lack of cross-striae on the ribbed spore ornamentation. Berkeley’s (1872) terse protologue described a specimen from Victoria, Australia, and included a macroscopic description with nine words that could describe any mushroom Heimioporus and Boletellus in Australia A Australian Systematic Botany 9 B C D E F Fig. 2. A. Boletellus ananas (Halling 8548). B. B. ananiceps (Halling 9893). C. B. deceptivus (Halling 9785). D. B. dissiliens (Halling 9022). E. B. emodensis (Halling 8761). F. B. reminiscens (Halling 9063). Scale bar: 1 cm. with squamose warts on the pileus. Since then, without a deﬁnitive macroscopic concept, the ribbed spores lacking cross-striae were the deﬁning feature of the species (but see Commentary). Watling and Gregory (1986) provided the best macroscopic description up to that time based on a specimen from New South Wales. They suggested that B. ananiceps was less closely related to B. ananas than the latter was to B. emodensis, precisely because the latter two feature cross-striae on the spore ornamentation. Boletellus ananiceps was treated by Bougher and Syme (1998) and illustrated via a watercolour of material from Western Australia. Halling and Fechner (2011b) brought the macroscopic concept forward with colour photographs that had been lacking previously. The key features are the pink to pale red colour that is conﬁned to a portion of the pileus that is beneath an overlying, ﬁne superﬁcial layer of hyphae. Later, those superﬁcial hyphae have coalesced and gained an ochre colour, and the pale red to pink pigmentation has remained below or between the developing squamae; the pigmentation may disappear with time and exposure. Finally, the squamae can become thick and coarse (but not always), ﬂattened on and near the disc, to somewhat so towards the margin, but are not, and never were, pigmented. In B. ananiceps (and the others in this group), the true colour of the pileus context is yellow, but is 10 Australian Systematic Botany A C E R. E. Halling et al. B D F Fig. 3. Scanning electron micrographs – Basidiospores. A. Boletellus ananas (Halling 8548). B. B. ananiceps (Halling 9276). C. B. deceptivus (holotype, Halling 9786). D. B. dissiliens (Halling 9805). E. B. emodensis (Lectotype). F. B. reminiscens (Halling 9063). Arrows point to lacunae. Scale bar: 5 mm. quickly obscured by the bluing so as to appear white. In the stipe, the context is white, but there is a slow change to a brownishorange or a pinkish-brown (a rufescence), especially towards the base, although this reaction can sometimes be localised. The fresher the basidiome, the more convincing and obvious is this reaction. The species has been noted in other publications on Australian boletes in a list compiled by May and Wood (1997). Zeng and Yang (2011) noted that reports of B. ananiceps from China represent specimens of B. emodensis. Commentary We would note here that the exemplars from Victoria and Western Australia are on a branch separate from those from Queensland, indicating some biogeographical structure. Further, on the basis of the description and habit illustration in Bougher and Syme (1998), the pink pigmentation is less obvious than but disposition of the pileus squamules is approximately the same as for the specimens found in Queensland. On an extremely Heimioporus and Boletellus in Australia Australian Systematic Botany A B C D 11 E Fig. 4. Differential interference contrast (DIC) light micrographs – Basidiospores of Boletellus. A. B. ananas (Halling 8515). B. B. deceptivus (Halling 9786). C. B. dissiliens (Halling 9816). D. B. emodensis (Halling 8734). E. B. ananiceps (Halling 9484). Scale bar: 5 mm. rare occasion, a spore of B. ananiceps can exhibit a few crossstriae, but these are never so obvious as those seen in B. ananas, B. emodensis, B. deceptivus and B. dissiliens. Furthermore, the ribs of the latter four species are thicker than in B. ananiceps. Material examined AUSTRALIA. Victoria. Wangaratta, 612 (holotype, isotype: K); Gippsland, 1880, Mrs. Campbell (3 specimens: K); Gippsland Plain, Mornington Peninsula, Buckley Nature Reserve (Myers and Balnarring roads), 38200 3200 S, 14550 4900 E, 7 May 2010, N.H. Sinnot 3794 (MEL2341322, NY). New South Wales. Paramatta, HMS Challenger Expedition (2 specimens: K; = B. emodensis). Queensland. Wide Bay District. Great Sandy National Park, Fraser Island, walking track S of Central Station, 25290 S, 153030 2700 E, 155 m, 3 June 2009, Halling 9139 (BRI, NY); Fraser Island, road from Lake McKenzie to Central Station, 25270 S, 153020 1700 E, 50–65 m, 6 June 2009, Halling 9178 (BRI, NY); 25240 0800 S, 153010 5400 E, 47 m, 28 Mar. 2010, Halling 9287 (BRI, NY); Fraser Island, Birrabeen Road, between Lake Boomanjin and Dillingham Road, 25340 0800 S, 153030 4300 E, 131 m, 9 June 2009 Halling 9212 (BRI, NY); Fraser Island, Lake Boomanjin, 25330 2500 S, 153030 3700 E, 68 m, 25 Mar. 2010, Halling 9276 (BRI, NY); Fraser Island, 4.8 km along Woralie Road, 25130 0700 S, 153130 2300 E, 171 m, 18 May 2010, Halling 9314 (BRI, NY); Fraser Island, between Pile Valley and Lake McKenzie, 25280 1900 S, 153040 2000 E, 33 m, 24 May 2010, Halling 9367 (BRI, NY); Fraser Island, Northern Road, 6.8 km N 12 Australian Systematic Botany R. E. Halling et al. of Cornwells Road, 25230 2600 S, 153040 5400 E, 129 m, 26 May 2010, Halling 9388 (BRI, NY); Fraser Island, road from Eurong to Central Station, 25300 0100 S, 153060 1900 E, 51 m, 17 May 2011, Halling 9484 (BRI, NY); Cooloola, near Frankis Gulch, 26020 5200 S, 153040 5800 E, 68 m, 11 May 2012, Halling 9690 (BRI, NY); Cooloola, Vic. Camp Milo, eastern ﬁrebreak, 25590 3800 S, 153040 3000 E, 58 m, Halling 9836 (BRI, NY); vicinity Rainbow Beach, on sand track between rubbish tip and Rainbow Beach Road, 25550 0000 S, 153050 0400 E, 16 m, 28 Feb. 2013, Halling 9847 (BRI, NY); Cape Hillsborough National Park, Twin Beach Lookout Track (Andrews Point track), 20550 4000 S, 149030 0600 E, 100 m, 13 Feb. 2014, Halling 9893 (BRI, NY). Western Australia. Vicinity of Denmark, 25 May 2011, K. Syme (NY); Denmark, Heritage Trail W of McLeod Road, 34580 48.300 , 117140 200 E, 3 June 2013, K. Syme 2852 MEL2372698, NY). Boletellus deceptivus Halling & Fechner, sp. nov. (Figs 2C, 3C, 4B, 5A–C, 6) Diagnosis Recalling others in sect. Boletellus but with deep red, erect squamules then coarsely squamose pileus, with tips of squamae usually whitish to pale ochraceous early on; stipe with scattered pale pink tones, soon white, with ﬂesh barely yellow and cyanescent above, white and rufescent below; spores longitudinally ribbed with cross-striae; associated with Myrtaceae, Allocasuarina. TYPE: AUSTRALIA. Queensland. Main Range National Park, Queen Mary Falls, 28200 29.500 S, 152220 16.900 E, 877 m, 14 Feb. 2013, Halling 9786 (holotype: BRI; isotype: NY). Etymology Deceptivus – deceptively similar to other coarsely squamose species in sect. Boletellus. Mycobank number: MB 811399 Pileus (2.5–)7–9 cm broad, hemispheric to convex to planoconvex, dry, medium to coarsely erect squamulose at ﬁrst, coarsely squamose scaly, subpyramidal on disc, subrepent to repent towards margin, sometimes with ﬂattened large scales, deep red to raspberry red to grayish-red to ruddy red to pinkishred (9,10B-C6,5,4), soon becoming dull ochraceous tan, or sometimes with scale tips dull and pale olive coloured, frequently retaining the reddish pigment between the scales or at the base of the scales, or with dull yellow between scales, with a sterile marginal veil covering tubes when young, breaking and shrinking with age, often with the scales continuous onto outer surface of veil, with a white band at edge of young veil; appendiculate veil remnants hanging from margin. Flesh yellow to pale yellow, immediately cyanescent when exposed, then appearing white and blue, with mild odour and taste. Tubes deeply depressed to adnate, bright yellow to greenish-yellow to olive brown (4E7), up to 2.5 cm deep, cyanescent, with pores D E A B F G C J I H Fig. 5. A–C. Boletellus deceptivus (Halling 9786). A, B. Pleurocystidia. C. Cheilocystidium. D–F. B. reminiscens (Halling 9063), D. Cheilocystidia. E. Caulocystidia. F. Pileipellis. G, H. B. sinapipes (Halling 9043), G. Pileipellis. H. Caulocystidia. I, J. B. sinapipes (Halling 9592). I. Pleurocystidia. J. Cheilocystidia. Scale bar: 10 mm for all, except 5 mm for F and G. Fig. 6. Boletellus deceptivus (holotype, Halling 9786). Context shows cyanescence masking yellow pigment in pileus (arrow). Stipe base context shows rufescence. Scale bar: 1 cm. Heimioporus and Boletellus in Australia 1 mm broad, concolourous becoming yellowish-brown (5E7) to dark brownish-red with age. Stipe 4–8(–12) cm long, (4–) 10–15 mm broad, equal, often bulbous at base, strict or curved, dry, pink to pale red to red at apex, white to tan to pale grayishbrown or with scattered pinkish to pale red tints below, white at base, ﬁbrillose striate to ﬁnely appressed ﬁbrillose, with interior pale yellow at apex, whitish below, cyanescent in upper one-third, white below to base with a slower orangish-brown oxidation, rarely with some immediate cyanescence in base, often basal cyanescence disappearing with orange–brown prevailing. Basidiospores dark olive brown in deposit, 15.4–17.5 (–18.9) 7–7.7(–8.4) mm, n = 20, x = 16.59 7.32 mm, Q = 2.27, subfusoid to fusoid to ellipsoid, longitudinally ribbed and cleft, shallowly cleft at apex, with ribs continuous or attenuated, ﬂattened on edges, cross-striate, melleous in KOH, inamyloid. Basidia 25–32 7–10 mm, clavate, four-sterigmate. Pleurocystidia uncommon, scattered, up to 50 mm long, fusoid to fusoid-ventricose, hyaline or rarely with granular melleous contents. Cheilocystidia 30–40 mm long, subclavate to clavate, hyaline, thin-walled. Hymenophoral trama bilateral, of the Boletus-subtype, with cells 4–9.8 mm broad, hyaline, with lateral strata gelatinised with age. Pileus trama interwoven with elements hyaline, smooth and thin-walled, 7–12 mm broad, inamyloid. Pileipellis hyphae suberect to repent, with elements composed of cylindrical hyphae in long chains, 7–17.5 mm broad, smooth, thinwalled, with pale red to pink plasmatic pigment, dissolving in KOH. Stipitipellis vertically oriented, hyaline, thin-walled, with rare, recurved, short endcells. Clamp connections absent. Australian Systematic Botany 13 NY); Springbrook National Park, Apple Tree Park, 2890 5200 S, 153150 3800 E, 547 m, 2 Mar. 2012, R.E. Halling 9653 (BRI, NY); Main Range National Park, Queen Mary Falls, 28130 2100 S, 152300 5200 E, 340 m, 14 Feb. 2013, R.E. Halling 9785, 9786 (BRI, NY); Mareeba Shire, Davies Creek National Park, Davies Creek Road, Davies Creek Falls car park, 8.6 km from Kennedy Highway, 1700 3500 S, 145340 600 E, 620 m, 1 Feb. 2015, R.E. Halling 9975 (BRI, NY); Davies Creek National Park, Davies Creek Road, ~12 km from Kennedy Highway, 1710 32.300 S, 145350 55.700 E, 678 m, 3 Feb. 2015, R. E. Halling 9983 (BRI, NY). Boletellus dissiliens (Corner) Pegler & T.W.K. Young, Trans. Brit. Mycol. Soc. 76: 113 (1981) (Figs 2D, 3D, 4C) Boletus dissiliens Corner, Boletus in Malaysia 98 (1972). Solitary to gregarious, in soil or sand or sometimes at base of living trees, in wet or dry sclerophyll forests, under Eucalyptus L.’Hér., Syncarpia Ten., Lophostemon Schott., Leptospermum J.R.Forester & G.Forster, Allocasuarina L.A.S. Johnson, Acacia Mill. So far, occurring in New South Wales to northern Queensland. Mycobank number: MB 112138. The species was well described and illustrated by Corner (1972), on the basis of material collected in Singapore. The distinctive features include the virtual lack of scales on the pileus and lack of red pigmentation in the pileus, although it can become dull pinkish-tan. In fact, the pileus is nearly white and matted subtomentose at ﬁrst, but breaks up into felt-like patches or rarely low, repent squamae. It is not squamose to squamulose like that of B. ananas, B. ananiceps, B. deceptivus or B. emodensis. Red pigments are absent, although, on rare occasions (in the Australian material examined), some very pale pinkish tints can appear with age. A further distinction is the colour of the unoxidised context of pileus and stipe. In B. dissiliens, the pileus context is yellow and immediately cyanescent. This bluing will quickly mask the true colour, making it appear white. The yellow pigments (and cyanescence) can appear in the upper-third of the stipe context. However, more commonly, the stipe context is white, not cyanescent, but rather rufescent to brownish-orange. This latter oxidation is slower to appear than the cyanescence and will typically appear on the stipe surface as well. Chiu’s (1948) description of B. ananas from Yunnan, especially in terms of the colours of the pileus and stipe, recalls B. dissiliens. Commentary Habit, habitat and distribution It is quite possible that this taxon has been confused with B. emodensis and B. dissiliens in Australia. It is also possible that it has been misidentiﬁed as B. ananas. However, the scales on the pileus are coarser than in B. emodensis and the stipe ﬂesh is white and rufescent. Although, B. dissiliens (see next) has rufescent, white ﬂesh in the stipe, the pileus is not red and has felt-like patches. Finally, B. ananas appears almost wholly restricted to the Americas and has a pinkish-coloured pileus that soon loses its colour. Solitary to gregarious in sand, or rarely on living tree bark or dead wood with Eucalyptus pilularis Sm., E. racemosa Cav., E. signata F.Muell., Melaleuca L., Leptospermum sp., Acacia sp., Allocasuarina sp., Syncarpia glomulifera (Sm.) Nied., S. hillii F. M.Bailey, Lophostemon confertus (R.Br.) Peter G.Wilson & J.T. Waterh., Callitris sp. and Xanthostemon sp. in Australia; under Quercus in Singapore. Additional material examined As noted by Corner (1972, p. 3), type specimens of his Malaysian boletes were retained at Cambridge, and whenever possible, a duplicate set of types were deposited at Kew. Pegler and Young (1981) provided two SEM micrographs from a specimen at Kew, which they cited as a type. This specimen (cited above) is marked type-duplicate on the packet. Horak (2011), however, cited a specimen from the same locality with same date, but with a number (Corner 26B) that is lodged in Edinburgh (E-00084564) and noted that it is the holotype preserved in alcohol–formalin. It may well be the holotype, because Corner (1972, p. 98) Habit, habitat and distribution AUSTRALIA. New South Wales. Tweed Shire, Route 97 (Nerang–Murwillumbah road), near New South Wales–Queensland border, 28160 1400 S, 153140 2600 E, 160 m, 2 June 2007, R.E. Halling 8978 (BRI, NY). Queensland. Atherton Shire, Atherton State Forest, Mount Baldy Road, 3.6 km from Atherton–Herberton highway, 17190 5800 S, 145250 300 E, 1140 m, 21 Mar. 2007, R.E. Halling 8937 (BRI, NY); 21 Feb. 2012, R.E. Halling 9634 (BRI, NY); Wide Bay District, Great Sandy National Park, Fraser Island, Central Station, 25280 3400 S, 15330 2000 E, 90 m, 7 Feb. 2009, R.E. Halling 9017 (BRI, Commentary 14 Australian Systematic Botany R. E. Halling et al. speciﬁcally states ‘(typus, Corner s.n. 26 March 1931, C & E)’ in the protologue. Presumably, ‘C’ is Cambridge and ‘E’ is Edinburgh. Pegler and Young (1981) gave the spore size as 13–18.5 5.5–7 mm, Q = 2.4, and indicated the presence of ﬁne striae. These measurements are in line with Corner’s observations (14–16(–17) 5.5–7 mm). Although Corner did not note the presence of cross-striae, he stated the spores are ‘. . .as in B. ananas but not so strongly ridged’ (Corner 1972, p. 98). Horak’s (2011) measurements fall within the ranges given by Corner and Pegler and Young, but he commented that there are ‘. . .numerous longitudinal and well-deﬁned clefts (not ridges!),. . .’ (p. 191) and cross-striae are absent. One of us (R. E. Halling) examined the Kew isotype and observed that when the spores are not so strongly ribbed they lack crossstriae; these are seemingly young. Spores that do possess crossstriae have broader ribs (with clefts in between), and with the following statistics: (14–) 4.7–17.5(–18.2) (4.9–)5.6–8.4 mm, (n = 11, x = 15.9 7.1 mm, Q = 2.23). See Figs 3D, 4C. cyanescence. The scales on the pileus are quite ﬁne from the beginning and sometimes become somewhat coarser with age, although not to the degree seen in B. deceptivus or B. ananiceps. According to Hooker’s notes (Berkeley 1851), the stipe is reddish-brown in the base, white or pinkish above, and slightly changing to blue when cut. One of us (R. E. Halling) has observed that the amount of yellow (and intensity of cyanescence) in the stipe ﬂesh decreases with age and degree of freshness. However, because of the rapidity with which the cyanescence occurs, the yellow colour is easily masked and appears white. In addition, as with closely related taxa (B. ananas, B. ananiceps, B. deceptivus and B. dissiliens), there is a slower but distinctive rufescence or brownish-orange oxidation that occurs in the base of the stipe. In contrast, the latter four species have a white context in the stipe that lacks the cyanescence (except rarely in the upper-third), and the brownish-orange oxidation is more pronounced upward from the base. All except B. ananiceps exhibit the transverse striations on the longitudinal ribs of the spores when viewed with a compound light microscope (Fig. 4). Material examined (from among 29 in NY) Habit, habitat and distribution AUSTRALIA. Queensland. Atherton State Forest, Mount Baldy Road, 5.8 km from Atherton–Herberton highway, 17180 5500 S, 145240 1500 E, 1027 m, 21 Mar. 2007, R.E. Halling 8943 (BRI, NY); Wide Bay District, Great Sandy National Park, Fraser Island, 3 km W of Central Station, Wanggoolba Creek Road, 25280 1600 S, 153020 1000 E, 24 m, 18 Feb. 2011, R.E. Halling 9435 (BRI, NY); Fraser Island, road from Pile Valley to Lake McKenzie, 25260 4200 S, 153020 5900 E, 107 m, 6 Mar. 2011, R.E. Halling 9466 (BRI, NY); Cooloola, Freshwater Road, 25570 4000 S, 153080 2500 E, 129–130 m, 11 Mar. 2012, R. E. Halling 9688 (BRI, NY); Mareeba Shire, Mareeba, Davies Creek National Park, Davies Creek Road, Campsite #5-6, 17010 3600 S, 145350 2300 E, 670 m, 15 Feb. 2012, R.E. Halling 9624 (BRI, NY); S of Canungra, along Beechmont Road, near Rhoades Road, 28030 4300 S, 153110 2800 E, 161 m, 12 Feb. 2013, R. E. Halling 9768 (BRI, NY). SINGAPORE. Reservoir Jungle, 26 Mar. 1931, Corner (isotype: K(M)141488). Solitary to gregarious on soil, in sand or rarely on living tree bark or dead wood; with Pinus, Dipterocarpaceae Blume (Thailand); with Fagaceae (China); with Eucalyptus, Acacia, Allocasuarina littoralis (Salisb.) L.A.S. Johnson, Leptospermum, Syncarpia, Lophostemon (Australia). Commentary Boletellus annamiticus (Pat.) E.-J. Gilbert, Bolets 107 (1931) There are three separate packets at Kew (K(M)141491, K(M) 164372, K(M)164373) that would appear attributable to Hooker’s type collection from India. K(M)164373 is in the best shape of the three, with a pileus 8.2 cm broad, with easily visible ﬁne squamules; the stipe is curved, 8.5 1.5 cm, and equal in width. The herbarium sheet is stamped in blue ink ‘Herbarium Hookerianum 1867’ inside a circle. Penciled on the sheet is ‘Boletus Emodensis, Berk. no. 100 Sikkim’. This latter specimen has been designated as the lectotype (above). The spores are costate with cross-striae (Fig. 4D) and some of the ribs appear not to be united at the apex (Fig. 3E) (smooth and rounded ﬁde Pegler and Young 1981). Horak (2011) also lists Boletus paradoxus Massee, Boletus porphyrius Pat. & C.F.Baker, and Boletellus ﬂoriformis Imazeki as synonyms of Boletellus emodensis. The ﬁrst two were described from Singapore and the last from Japan. = Strobilomyces pallescens Cooke & Massee in Cooke, Grevillea 18: 5 (1889). Material examined (among 43 specimens in NY) Boletellus emodensis (Berk.) Singer, Ann. Mycol. 40: 19 (1942) (Figs 2E, 3E, 4D) Boletus emodensis Berk., Hooker’s J. Bot. Kew Gard. Misc. 3: 48 (1851). INDIA. Sikkim, Darjeeling, Hooker 100 (Lectotype hic designatus: K(M)164373). = Strobilomyces annamiticus Pat., Bull. Soc. Mycol. France 25: 6 (1909). Boletellus pallescens (Cooke & Massee) E.-J. Gilbert, Bolets 107 (1931). Mycobank number: MB 472279. Zeng and Yang (2011) provided a detailed, modern description, with updated nomenclature and illustrations of Chinese materials referable to B. emodensis. The material cited below coincides in macro- and micromorphology. As noted by Zeng and Yang (2011), the species is well characterised on a macroscopic level by the purplish red to dull crimson colours of both pileus and stipe and a yellow context in both pileus and stipe, the true colour of which is quickly masked by the rapid AUSTRALIA. New South Wales. Boonoo State Forest, Junction Colongon Road and Lindrook Road, 28530 3800 S, 152130 800 E, 833 m, 2 Mar. 2011, R.E. Halling 9459 (BRI, NY). Queensland. Mareeba, Davies Creek National Park, Davies Creek Road, 1710 5300 S, 145360 2300 E, 700 m, 4 Feb. 2006, R.E. Halling 8734 (BRI, NY); Davies Creek Road, 1710 3100 S, 145350 5200 E, 720 m, 9 Feb. 2006, R.E. Halling 8742 (BRI, NY); 11 Mar. 2007, R.E. Halling 8904 (BRI, NY); Brisbane, Bailey 744 (isotype: Strobilomyces pallescens, NY); Brisbane, Ashgrove suburb, Ithaca Creek and Cooper’s Camp Road, 27270 600 S, 152580 4200 E, 41 m, 8 Mar. 2012, R.E. Halling 9676 (BRI, NY); Rainbow Beach, Bullock Point, near Innskip Point, 25490 4300 S, 15330 5500 E, 23 m, 13 Mar. 2012, R. E. Halling 9701 (BRI, NY); Girraween National Park, road from Bald Rock Heimioporus and Boletellus in Australia Creek to Dr Roberts Waterhole, 28490 3900 S, 151580 200 E, 979 m, 15 Feb. 2013, R.E. Halling 9789 (BRI, NY); Wide Bay District. Great Sandy National Park, Fraser Island, Central Station, 25280 3400 S, 15330 2000 E, 90 m, 24 Mar. 2010, R.E. Halling 9260 (BRI, NY); Cooloola, Freshwater Road, 25560 4800 S, 15370 4400 E, 133 m, 28 Feb. 2013, R.E. Halling 9850 (BRI, NY). THAILAND. Chiang Mai Province, km 22 on Highway 1095, 1970 3000 N, 98450 4700 E, 731 m, 11 June 2006, R.E. Halling 8808 (MFLU, NY). Boletellus reminiscens Halling & Fechner, sp. nov. (Figs 2F, 3F, 5D–F) Diagnosis Recalling B. obscurecoccineus, but lacking ﬁne scabers on the stipe, with cyanescent context, and costate spores often discontinuously tuberculate. Type AUSTRALIA. Queensland. Wide Bay District, Great Sandy National Park, Fraser Island, Pile Valley Walking track, from Central Station to Pile Valley, 25280 3400 S, 15330 2000 E, 100 m, 12 Feb. 2009, R.E. Halling 9063 (holotype: BRI; isotype: NY). Etymology Reminiscens, past participle of reminisci, to remember or recall, in reference to B. obscurecoccineus. Mycobank number: MB 811400. Pileus 2.5–4.5 cm broad, convex to plano-convex, dry, deep dark red, ﬁnely subtomentose to subvelutinous, mottled slightly with small dark yellow spots, becoming ﬁnely areolate and a pale olive when fading. Flesh pale yellow, cyanescent then obscurely white, with mild odour and taste. Tubes adnexed, bright yellow to dark yellow to greenish-yellow, cyanescent, with pores cyanescent then eventually pale brown. Stipe 3.5–5 cm long, 6–9 mm broad, strict or curved, equal or slightly broader below, rarely tapering to a blunt point, dry, whitish to pale greenishyellow at apex, otherwise uniformly deep red with a white base, heavily pruinose to subpruinose above, ﬁbrillose striate below, cyanescent, with interior yellow, cyanescent, deep reddish around larval tunnels. Basidiospores 12.6–14.7 4.2–5.6 mm, n = 20, x = 13.3 5.04 mm, Q = 2.64, ﬁnely ribbed with ribs shallowly and moderately spaced, with rounded edges and longitudinally intermittent-turberculate, melleous in KOH, inamyloid. Basidia 24–30 10–12.6 mm, clavate, 4-sterigmate, hyaline. Pleurocystidia rare, ventricose rostrate, more abundant near pores. Cheilocystida 20–45 10–12 mm, ventricose rostrate to subfusoid, hyaline, thin-walled. Hymenophoral trama bilateral, of the Boletus subtype, with cells 4–9.8 mm broad, hyaline. Pileus trama interwoven with elements hyaline, smooth and thin-walled, 7–14 mm broad, inamyloid. Pileipellis an erect trichodermium of cylindrical to slightly inﬂated subcylindrical elements, 7–21 mm broad, smooth thin-walled, inamyloid with pale red plasmatic pigment. Stipitipellis vertically oriented, hyaline, thin-walled, with recurved end cells, occasional isolated basidia, or clusters of clavate to nearly short-subfusoid caulocystidia, 20–25 mm long, usually with melleous contents. Clamp connections absent. Australian Systematic Botany 15 Habit, habitat and distribution Solitary to scattered in sand under Syncarpia, Lophostemon, Eucalyptus, Allocasuarina and Leptospermum. Currently, only seen on Fraser Island in the Great Sandy National Park of southeastern Queensland. Commentary At present, Boletellus reminiscens appears to be uncommon and restricted to the sand habitats of Fraser Island. Despite the deep red colours, it differs from B. obscurecoccineus in the cyanescent oxidation reaction, lack of scales on the stipe, and yellow context. Microscopically, the spores of B. reminiscens are ﬁnely longitudinally ridged as in B. obscurecoccineus, but have narrower costae that are sometimes discontinuous, forming isolated tubercules or short pustulate ribs (Fig. 3F). Additional material examined AUSTRALIA. Queensland. Wide Bay District, Great Sandy National Park, Fraser Island, Lake Garawongera Scenic Drive, W of lake, 25200 2400 S, 15390 800 E, 200 m, 27 Mar. 2010, R.E. Halling 9284 (BRI, NY); Fraser Island, Wanggoolba Creek Road, 25280 4600 S, 15320 4500 E, 35 m, 29 Mar. 2010, R.E. Halling 9305 (BRI, NY). Boletellus sinapipes Fechner, K.Syme, R.Rob., & Halling, sp. nov. (Figs 5G–J, 7B, D, 8A) Diagnosis Characterised by the brown colours nearly overall, with bright yellow, cyanescent tubes, and mustard brown-coloured tomentum on the stipe base. Type AUSTRALIA. Queensland. Fraser Island, road from Central Station to Lake McKenzie, 25280 4000 S, 15320 5100 E, 109 m, 9 Feb. 2009, R.E. Halling 9040 (holotype: BRI, isotype: NY). Etymology Sinapi- = mustard, + -pes = foot, in reference to the mustard brown-coloured tomentum at the base of the stipe. Mycobank number: MB 811401. Pileus 3–5(–9.4) cm broad, convex to plano-convex to plane, dry, brown (5F4) to dark brown with a subtle hint of red, velutinous, subtomentose to matted subtomentose, becoming minutely to ﬁnely areolate or sometimes cracked at margin. Flesh pale yellow and cyanescent at ﬁrst and then appearing white under the cyanescence, entirely red with age, with mild odour and taste. Tubes adnexed, bright yellow (2A5) to near chrome yellow (3A7,6), cyanescent, with pores bright yellow and cyanescent then slowly brown, becoming dark olive brown with age. Stipe 5–9(–10.5) cm long, (4–)7–15(–20) mm broad, strict, sometimes curved at base, equal to slightly broader below, dry, ﬁnely and obscurely subpruinose to ﬁnely subtomentose at apex, ﬁbrillose streaked to matted ﬁbrillose below, bright yellow at apex, dull pinkish-brown to dull brownish-red to dull red below, eventually entirely red, with conspicuous mustard brown (5E7) velvety tomentum at base, with interior pale yellow to dull yellow 16 Australian Systematic Botany at apex, whitish below, brownish-yellow in the base, intensely cyanescent at ﬁrst, completely red with age. Basidiospores olive brown in deposit, 11.9–15.4 5.6–7 mm, n = 20, x = 13.13 5.99 mm, Q = 2.19, subfusoid to fusoid to ellipsoid, longitudinally ribbed and cleft, sometimes shallowly cleft at apex, with ribs continuous or attenuated, rarely not continuous and isolated between apex and base, broadly rounded at ﬁrst then ﬂattened on edges, lacking cross-striae, melleous in KOH, inamyloid. Basidia 31–42 13–17 mm, short-clavate, hyaline to melleous, 4-sterigmate. Pleurocystidia up to 65 mm long, fusoid to fusoid-ventricose, hyaline. Cheilocystidia 30–40 mm long, subclavate to clavate or sometimes subrostrate, hyaline, A C R. E. Halling et al. thin-walled. Hymenophoral trama bilateral, of the Boletus subtype, with cells 4–9.8 mm broad, hyaline, ﬂeeting amyloid. Pileus trama interwoven with elements hyaline, smooth and thinwalled, 7–14 mm broad, inamyloid. Pileipellis hyphae a tangled trichodermium, with elements composed of cylindrical hyphae, 5–10 mm broad, with scattered, rusty brown, plaque-like encrusting pigment (in water and Melzer’s), thin-walled, with amorphous plasmatic melleous pigment, dissolving in KOH. Stipitipellis vertically oriented, hyaline, thin-walled, with recurved, short end cells or clusters of short-clavate to shortsubfusoid caulocystidia, 20–25 mm long. Clamp connections absent. B D E F Fig. 7. A. Heimioporus australis (Halling 9288). B. Boletellus sinapipes (Halling 9310). C. H. fruticicola (Halling 9775). D. B. sinapipes (Halling 9040). E. H. fruticicola (Halling 8970). F. H. cooloolae (Halling 9845). Scale bar: 1 cm. Heimioporus and Boletellus in Australia Habit, habitat and distribution Solitary to gregarious in sand, or rarely on living tree bark (Queensland) with Eucalyptus, Syncarpia and Lophostemon; or soil (Western Australia) with Eucalyptus patens Benth., E. diversicolor F.Muell., E. marginata Donn ex Sm., Corymbia calophylla (R.Br.) K.D.Hill & L.A.S.Johnson, Allocasuarina decussata (Benth.) L.A.S.Johnson and Agonis ﬂexuosa (Wild.) Sweet. Commentary The most distinctive feature of B. sinapipes is the mustard brown-coloured tomentum at the base of the stipe. Fig. 7B depicts a young basidiome, but even as the basidiomes mature (Fig. 7D) and become dark brown to reddish-brown, the colour of the basal tomentum is consistent. There is some biogeographic structure present here with western (REH 9592, 25511) and eastern (REH 9018, REH 9408) Australian components. Additional material examined AUSTRALIA. Queensland. Wide Bay District, Great Sandy National Park, Fraser Island, Central Station, 25280 3400 S, 15330 2000 E, 100 m, 7 Feb. 2009, R.E. Halling 9018 (BRI, NY); Fraser Island, near Central Station, 25280 3400 S, 15330 2000 E, 90 m, 9 Feb. 2009, R.E. Halling 9043 (BRI, NY); Fraser Island, road from Central Station to Lake Birrabeen, 25290 4200 S, 15330 5200 E, 160 m, 13 Feb. 2009, R.E. Halling 9071 (BRI, A C Australian Systematic Botany 17 NY); 25290 3600 S, 15330 1000 E, 113 m, 25 Mar. 2010, R.E. Halling 9278 (BRI, NY); Fraser Island, Pile Valley, 25280 2800 S, 15340 2000 E, 87 m, 30 Mar. 2010, R.E. Halling 9310 (BRI, NY); Fraser Island, road from Eurong to Central Station, 25280 4000 S, 153040 900 E, 77 m, 15 Feb. 2011, R.E. Halling 9408 (BRI, NY). Western Australia. Denmark Shire, vicinity of Denmark, 25 May 2011, K. Syme s.n. (NY), 34590 17.900 S, 117160 35.500 E, Heritage Trail W of Lights Road, 21 May 2013, K. Syme 2836 (MEL2371699, NY); Denmark Shire, Digby & Lyn Mercer property, Loc 3298 off Denmark–Nornalup Heritage Trail, 34580 4800 S, 117140 0300 E, 17 July 2011, R.E. Halling 9592 (NY); Denmark, Heritage Rail Trail W of Lights Road, 34590 17.900 S, 117160 35.500 E, 21 May 2013, K. Syme 2836 (MEL2371699, NY). Heimioporus australis Fechner & Halling, sp. nov. (Figs 7A, 8B) Diagnosis Differs from other species in Heimioporus by the shallowly lacerate-ridged stipe with a dense red pruina. Type AUSTRALIA. Queensland. Wide Bay District, Great Sandy National Park, Fraser Island, Kingﬁsher Bay, 25230 35.700 S, 153010 50.700 E, 8 m, 28 Mar. 2010, R.E. Halling 9288 (holotype: BRI; isotype: NY). B D Fig. 8. Scanning electron micrographs – Basidiospores. A. Boletellus sinapipes (holotype, Halling 9040). B. Heimioporus australis (holotype, Halling 9288). C. H. cooloolae (Halling 9446). D. H. fruticicola (Halling 9811). Scale bar: 5 mm. 18 Australian Systematic Botany R. E. Halling et al. Etymology Australis- adjective, southern. Mycobank number: MB 811402. This taxon was previously thought to be Heimioporus japonicus (Hongo) E.Horak by Halling and Fechner (2011a). After a careful re-evaluation of specimens, we now know that the surface morphology of the stipe surface on the specimen from Fraser Island was misinterpreted; it is shallowly lacerate-ridged with a dense pruina, but it is not beset with a true reticulum possessed by H. japonicus (Imazeki et al. 1988, Masai 2014). Illustrations and a full description are in Halling and Fechner (2011a). Habit, habitat and distribution Solitary to gregarious under Eucalyptus, Allocasuarina, Leptospermum and Melaleuca; so far known only from southeastern Queensland. Additional material examined AUSTRALIA. Queensland, Caloundra, Queens Street, Ben Bennett Bushland Reserve, 26470 5000 S, 15370 2000 E, 29 Jan. 2015, N. Fechner, AQ905831 (BRI, NY). Heimioporus cooloolae Fechner & Halling, sp. nov. (Figs 7F, 8C, 9A–E) Diagnosis Differing from other species of Heimioporus by a dull to pale red pileus; a white- or olive-coloured tomentum near the stipe base; A B C D E basidiospores with rugulose surface, a subtruncate, eroded apex, and crater-like pits. Type AUSTRALIA. Queensland, Wide Bay District, Great Sandy National Park, Cooloola, Freshwater Road, 25560 47.700 S, 15370 4400 E, 133 m, 28 Feb. 2013, R.E. Halling 9852 (holotype: BRI, isotype: NY). Etymology Cooloola- + e, genitive, of the Cooloola Sandmass. Mycobank number: MB 811403. Pileus 3.5–5(–7.5) cm broad, convex to plano-convex, becoming plane, dry, subtomentose to ﬁnely matted subtomentose, pink or with red tones mixed with yellow, but not quite orange, with even margin that slightly projects a sterile ﬂap. Flesh white with some yellow beneath pileus surface, erratic light bluing near tubes, with mild odour and taste. Tubes adnexed, greenish-yellow to olive, unchanging or with some subtle cyanescence, with pores developing some pale brown. Stipe 7–9 cm long, 1–1.5 cm broad, curved, equal and tapered at base, dry, yellow to bright yellow at apex, dull yellow to red below and rhubarb red towards base, subﬂoccose at apex, ﬁbrillose striate below, with low ridges at ﬁrst, becoming more pronounced with age and then ﬁnely red subscabrous, with some scattered olive–mustard-coloured tomentum at base, otherwise white at base, with interior white to pale yellow at apex, more yellow below and a bit of red in base, with subtle cyanescence in mid-portion. Basidiospores 9.8–13.3 4.9–7 mm, n = 20, x = 11.2 6.16, Q = 1.82, broadly ellipsoid to oblong-ovate to broadly subfusoid or somewhat amygdaliform in proﬁle, rugulose with scattered crater-like pits, often with a subtruncate, eroded apex, honey brown in KOH, imamyloid (rarely slightly dextrinoid). Basidia 25–31 8–11 mm, clavate, hyaline, 4-sterigmate. Pleurocystidia rare, up to 55 mm long, fusoid to fusoidventricose. Cheilocystidia 30–45 mm long, subclavate to clavate, hyaline, thin-walled. Pileus trama interwoven with elements hyaline, smooth and thin-walled, 7–12 mm broad, ﬂeeting amyloid. Hymenophoral trama bilateral, of the Boletus subtype, with cells 4–9.8 mm broad, hyaline, ﬂeeting amyloid. Pileipellis a trichodermium collapsing with age, with cells 5–10 mm broad, cylindrical to narrowly clavate-subcapitate, smooth, thin-walled, inamyloid, hyaline or with granular golden yellow content in KOH, with an amorphous, soluble, reddish, plasmatic pigment. Stipitipellis hyphae hyaline, smooth, thin-walled, with caulocystidia forming an interrupted hymeniform layer of clavate elements, 28–36 12–16 mm broad. Clamp connections absent. Habit, habitat and distribution Solitary to subcespitose in sand under Eucalyptus, Allocasuarina and Leptospermum; so far known only from the Great Sandy National Park (Cooloola section) in Queensland. Commentary Fig. 9. A–E. Heimioporus cooloolae (Halling 9852). A. Pleurocystidia. B. Cheilocystidia. C, D. Caulocystidia. E. Pileipellis. Scale bar: 10 mm. This species has a type of spore ornamentation similar to that of H. fruticicola (rugulose with crater-like holes, subtruncate, Heimioporus and Boletellus in Australia eroded apex), but the spores are generally shorter in length and with fewer craters. Also, H. cooloolae differs in the paler colours and an occasional presence of a scattered, olivecoloured tomentum at the stipe base. Sand grains adhering to the stipe base will obscure features in this area. Watling and Gregory’s (1986) analysis of Austroboletus sp. 2 may well be this species. Additional material examined AUSTRALIA. Queensland. Wide Bay District, Great Sandy National Park, Cooloola, Freshwater Road, 25560 4300 S, 15350 500 E, 34 m, 20 Feb. 2011, R.E. Halling 9446 (BRI, NY); Cooloola, Freshwater Road, 25570 1600 S, 15350 5100 E, 144 m, 22 Feb. 2013, R.E. Halling 9817 (BRI, NY); Cooloola, Camp Milo, eastern ﬁrebreak, 25590 4400 S, 15340 3100 E, 58–68 m, 27 Feb. 2013, R.E. Halling 9845 (BRI, NY). Heimioporus fruticicola (Berk.) E.Horak, Sydowia 56: 240 (2004) (Figs 7C, E, 8D) Boletus fruticicola Berk., London J. Bot. 7: 574 (1848). Suillus fruticicola (Berk.) Kuntze, Rev. Gen. Pl. 3(2): 535 (1898). Austroboletus fruticicola (Berk.) E.Horak, Sydowia 33: 76 (1980) Heimiella fruticicola (Berk.) Watling & Hollands, Notes Roy. Bot. Gard. Edinburgh 46: 420 (1990). Mycobank number: MB 368304. The species has been described and illustrated previously by Halling and Fechner (2011a). It differs from H. cooloolae in brighter red colours, lack of a coloured tomentum on the stipe base, and larger spores with more prominent crater-like pits. Fig. 7C depicts material from southern Queensland, and Fig. 7E from northern Queensland. Material examined AUSTRALIA. Tasmania. Penquite, Gunn 1775 (holotype: K). Queensland. Mareeba, Davies Creek National Park, Davies Creek Road, 19 Feb. 1992, Halling 6837 (PERTH E4709); 17010 3600 S, 145350 2300 E, 670 m, 22 Mar. 2007, R.E. Halling 8958 (BRI, NY); 17000 3500 S, 145340 5600 E, 620 m, 24 Mar. 2007, R.E. Halling 8970 (BRI, NY); Kuranda, Black Mountain Road, 5.3 km N of Kuranda, 16470 0400 S, 145370 2500 E, 450 m, 23 Mar. 2007, R.E. Halling 8962 (BRI, NY); Cooloola, Mutyi, 17 Sep. 1982, BRIP 9148 (BRI); 17 Sep. 1982, BRIP 9150 (BRI); near Rainbow Beach, 17 Sep. 1982, BRIP 9149 (BRI); Cooloola, on Fig Tree Point track, 14 Apr. 1966, leg. C. Sandercoe & J. Milne (JECA 86/66 = BRIP 19814) det. R. Watling (BRI); Springbrook National Park, Apple Tree Park, 2890 5200 S, 153150 3800 E, 547 m, 12 Feb. 2013, R.E. Halling 9775 (BRI, NY); Freshwater National Park, W of Deception Bay, 27100 5200 S, 152590 4800 E, 19 m, 21 Feb. 2013, R.E. Halling 9811 (BRI, NY). Victoria. Grampians. Victoria Range, Cultivation Creek, near Buandik camping ground, 37150 S, 142170 E, 1 Nov. 1992, May 816 (MEL 2030279). Heimioporus retisporus (Pat. & C.F. Baker) E. Horak, Sydowia 56: 239 (2005) Boletus retisporus Pat. & C.F. Baker, J. Straits Branch Roy. Asiatic Soc. 78: 72 (1918). Boletellus retisporus (Pat. & C.F. Baker) E.-J. Gilbert, Bolets, 108 (1931). Australian Systematic Botany 19 Heimiella retispora (Pat & C.F. Baker) Boedijn, Sydowia 5: 217 (1951). Mycobank number: MB 369467. The type specimen from Singapore (leg. C.F. Baker, no. 5000, FH) has been well studied by Singer (1945) and Horak (1968, 2011). The species is also well described and illustrated by Corner (1972). Updated distribution provided by Horak (2011) includes Singapore, Malaysia, China, Indonesia, Papua New Guinea, but doubtfully in Australia. So far, we have not seen any bona ﬁde specimens from Australia. The following new combinations and a new name are supported by the phylogenetic analysis. Aureoboletus mirabilis (Murrill) Halling, comb. nov. Ceriomyces mirabilis Murrill, Mycologia 4: 98 (1912). Boletus mirabilis (Murrill) Murrill, Mycologia 4: 217 (1912). Xerocomus mirabilis (Murrill) Singer, Rev. Mycol. (Paris) 5: 6 (1940). Boletellus mirabilis (Murrill) Singer, Farlowia 2: 129 (1945). Heimioporus mirabilis (Murrill) E.Horak, Sydowia 56: 240 (2004). Mycobank number: MB 811404. Aureoboletus projectellus (Murrill) Halling, comb. nov. Ceriomyces projectellus Murrill, Mycologia 30: 524 (1938). Boletus projectellus (Murrill) Murrill, Mycologia 30: 525 (1938). Boletellus projectellus (Murrill) Singer, Farlowia 2: 129 (1945). Mycobank number: MB 811405. Hemileccinum subglabripes (Peck) Halling, comb. nov. Boletus subglabripes Peck, Bull New York St. Mus. 2: 112 (1889), nom. nov. for Boletus ﬂavipes Peck, Ann. Rep. New York St. Mus. 39: 42 (1887), non Berk., Hooker’s J. Bot. Kew Gard. Misc 6: 135 (1854). Suillus subglabripes (Peck) Kuntze, Rev. Gen. Pl. 3: 536 (1898). Ceriomyces subglabripes (Peck) Murrill, Mycologia 1: 153 (1909). Leccinum subglabripes (Peck) Singer, Mycologia 37: 799 (1945). Mycobank number: MB 811406. Xerocomus tenax Nuhn & Halling, nom. nov. Boletus tenax A.H. Sm. & Thiers, Boletes of Michigan 249 (1971), nom. illeg., non Lightfoot, Fl. Scot. 2: 1031 (1777), non Bolton, Hist. Fung. Halifax 2: 75 (1788). Mycobank number: MB 811407. Discussion The results presented here are consistent with those of previous studies (with more limited sampling) that suggested that Boletellus sensu Singer (1986) is not monophyletic (Dentinger et al. 2010; Li et al. 2011; Nuhn et al. 2013; Wu et al. 2014). The expected division between the previously sampled species Aureoboletus projectellus, A. mirabilis, B. russellii and B. shichianus was recovered, but the majority of Boletellus species and Heimioporus are placed in a new lineage that remains weakly supported. Alternative-topology tests do not reject monophyly of 20 Australian Systematic Botany the core Boletellus clade plus B. chrysenteroides, B. badiovinosus, B. reminiscens, B. sinapipes, B. betula and B. elatus. Indeed, the SH and AU tests found that the likelihood of the constrained topology (forcing monophyly of most Boletellus species) was marginally higher than that of the initially recovered topology. These results do not warrant new generic placements for B. chrysenteroides, B. badiovinosus, B. reminiscens and B. sinapipes, but they do indicate that more data from additional genes or genomes of exemplar taxa are needed to increase conﬁdence in the higher-level relationships between Boletellus and Heimioporus. Alternative placements of B. russellii and B. shichianus were not evaluated, but the optimal topologies suggest that these species will need generic re-evaluation. Likewise, further data for B. betula and B. elatus are needed to assess their relationships to Heimioporus and Boletellus. For example, the former taxon was placed in Heimioporus with H. punctisporus (Corner) E. Horak because of the perforated spore wall (Horak 2004). A unique spore morphology for B. elatus was ﬁrst documented by Nagasawa (1984) and veriﬁed by Halling and Ortiz-Santana (2009) to also include B. jalapensis (Murrill) Singer. The core Boletellus clade is composed of two strongly supported sister clades, one including B. obscurecoccineus and the other containing the rest of the core Boletellus species. The B. obscurecoccineus clade is further divided into two groups, one from northern Queensland and one from southern Queensland, which suggests that there may be discrete taxa. Our concept used here for Australian B. obscurecoccineus is illustrated in Grey and Grey (2005) and Fuhrer (2005). That concept is the one employed for documenting the taxon as a Fungimap target species in Australia (Grey and Grey 2005). The images offered by Zeng and Yang (2011) illustrate a slenderer taxon from China, with a coarser hymenophore. Other than the type specimen from Java (in FH), recent material from that area is not available for molecular analysis. Clearly, expanded sampling is desired (Fig. 1). Most of the collections in the remainder of the core Boletellus clade are from Australia, including four collections identiﬁed as B. ananiceps, six collections of B. dissiliens, three collections of B. emodensis, and ﬁve collections of B. deceptivus, each of which is strongly supported as monophyletic. One collection from Thailand was identiﬁed as B. emodensis and is weakly supported as the sister group of the Australian B. emodensis group (Fig. 1). The presence of the Thai collection nested within the Australian clade is not unprecedented; other recent analyses (Halling et al. 2012a, 2012b) have shown similar results for South-east Asian exemplars. Even though the morphological concept for austral B. emodensis used in the present study compares favourably to one from China (Zeng and Yang 2011), recent exemplars for molecular analysis from the Himalaya (type locality) are lacking. A Wallacean land-bridge hypothesis put forth by Halling et al. (2008) can be considered here, and future studies may well show additional phylogenetic distinction. Obviously, continued sampling on both sides of Wallacea is needed to test this hypothesis further. New World specimens in the core Boletellus clade include two collections of B. singeri Gonz.-Velázq. & R.Valenz. from Mexico, and ﬁve collections of B. ananas from Belize (one collection), Costa Rica (three collections) and the USA R. E. Halling et al. (Georgia; one collection). The latter is close to the type locality of B. ananas, the type species of Boletellus. A collection questionably identiﬁed as B. ananas from Thailand is sister to the New World B. ananas materials. There is also one collection of Boletellus sp. from Zambia that is placed as sister to the clade containing B. ananas, the Australian species, and the Thai B. emodensis collection. Similar complex patterns of relationships among collections from New World tropics and temperate regions, South-east Asia, Australia and Africa were also observed in Sutorius and the Chromapes group (Halling et al. 2012a, 2012b). This repeated pattern could suggest that members of Boletaceae were widely distributed on Pangaea before its fragmentation began ~100 million years ago (Halling et al. 2008). However, knowledge of tropical species of Boletellus and other Boletaceae, particularly in Africa, is too limited at this time to warrant strong historical biogeographic inferences. Additional sampling could also help reﬁne the limits of hypothetically broadly distributed taxa, such as B. ananas, B. emodensis and B. obscurecoccineus. The oxidation reaction of exposed context needs to be observed much more closely than previously considered. The rapid cyanescence will obscure the true colour of the unoxidised context (Fig. 6, arrow). Also, the localisation of oxidations, to include a rufescence in stipe contexts (Fig. 6, base of stipe context), is another feature of macroscopic distinction. The presence and type of spore ornamentation has been a traditional microscopic feature for distinguishing genera in the Boletaceae (e.g. Pegler and Young 1981; Singer 1986). Recent molecular phylogenetic studies have suggested that spore ornamentation appears less reliable on its own merits than in the past (Nuhn et al. 2013; Wu et al. 2014; the present study); however, these studies have not resolved this question in regard to the longitudinally ridged and reticulate ornamentation characteristic of Boletellus and Heimioporus because of sparse taxon sampling and low phylogenetic resolution for these genera. Osmundson (2009) presented phylogenetic evidence that Boletellus and Heimioporus are distinct clades, although sampling was limited with all of the included taxa belonging to the core clades of these genera. In the present study, we include a signiﬁcantly larger taxon sample than in any previous phylogenetic study of these genera, including taxa formerly included in Boletellus but with doubtful status on the basis of their spore morphology (e.g. A. projectellus, A. mirabilis). In our analyses (Fig. 1), B. singeri ﬁts within the core Boletellus clade, whereas B. elatus is allied to B. betula in a distinct clade, suggesting that the presence of longitudinal ridges is homoplasious. In the alternative-topology tested and found to be statistically slightly better to that in Fig. 1 (inset), B. elatus would be closer to the core Boletellus clade; however, the placement of B. russellii would still suggest homoplasy for the presence of longitudinally ridged basidiospores. Therefore, it can be concluded that basidiospore ornamentation is an important characteristic for identifying members of the core Boletellus clade, yet does not appear to be a synapomorphy for a monophyletic genus Boletellus. In addition, acute observation of ridges, costae, intercostal space, edges of costae and apical morphology of spores is still important for distinguishing entities at the species level (e.g. B. elatus and B. singeri in Halling and Ortiz-Santana 2009). Heimioporus and Boletellus in Australia The presence of longitudinally ridged basidiospores, while homoplasious, appears to be restricted to only a very few taxa, all of which appear to be closely related to Boletellus. Therefore, although convergent evolution of this character is possible, it may also be the case that longitudinally ridged basidiospores represent the ancestral character state of the common ancestor of Boletellus, Heimioporus, and allied taxa; our analysis did not attempt to distinguish between these two hypotheses. Among core Boletellus taxa with squamose pilei, only B. ananiceps appears to lack cross-striae on the costae of the spores when viewed with the light microscope (Fig. 4E); they are very rarely present. These striae are invisible on the costae when spores are examined with the SEM for the other taxa (Fig. 3A, C–E). Perreau-Bertrand (1967) provided transmission electron micrographs (TEM) of sections through spores of B. chrysenteroides, illustrating that within the costae, there are electron-transparent lacunae. In Fig. 3C (arrow), there are holes on the sides of the costae, indicating the presence of lacunae in spores of B. deceptivus. There are indentations or holes evident in Fig. 3A (B. ananas), Fig. 3D (B. dissiliens) and Fig. 3E (B. emodensis), but absent in Fig. 3B (B. ananiceps). The lacunae would cause refraction patterns when spores are viewed with transmitted light and appear as the cross-striate bands on spore costae. Such patterns are especially evident for B. ananas, B. deceptivus, B. dissiliens and B. emodensis, whereas they are not apparent in B. ananiceps (Fig. 4). Acknowledgements The senior author is grateful to The National Science Foundation (USA) for funding under grants DEB #9972018, DEB #0414665 and DEB #1020421 (the latter awarded to REH, DSH and MB), and to the National Geographic Society Committee for Research and Exploration in grants #7341–02, #8457–08. The Queensland Herbarium (BRI) provided assistance and logistical support to REH for herbarium and ﬁeld studies while in Queensland. Collaboration of Dr J. Carranza at Universidad de Costa Rica in support of the Macrofungi of Costa Rica project (NSF grants DEB #9300798, #9972018, #9972027) with REH and G. Mueller is very much appreciated. K. Syme (Denmark, Western Australia) and R. Robinson (Manjimup, Western Australia) are thanked for input on, and access to, additional specimens of B. ananiceps and B. sinapipes. V. Bandala (Xalapa, Mexico) kindly provided exquisite material of B. singeri for analysis. Likewise, S.-S. Lee (FRIM, Malaysia) loaned specimens of Heimioporus and A. R. Bessette (Georgia, USA) sent fresh specimens of B. ananas for analysis. We are indebted to M. Baxter for expertise and access to the SEM facility at the CUNY-Lehman College Campus (Bronx, NY, USA). E. Nagasawa kindly provided expert advice on the true concept of H. japonicus. S. Morath and S. Mandava helped with extractions and PCR in the L. B. & D. Cullman Laboratory at The New York Botanical Garden. T. Baroni (NSF grant DEB #0103621) provided REH with a ﬁeld opportunity in Belize. The Queensland Parks and Wildlife Service offered accommodation and orientation on Fraser Island. Fungimap Inc. is gratefully acknowledged for including REH in their activities. The contribution of KS and King Mongkut’s Institute of Technology in providing REH with a Material Transfer Agreement to study Thai macrofungi specimens is gratefully appreciated. Two anonymous reviewers provided constructive suggestions for which we are grateful. 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