Global diversity of the Tylopilus alboater complex (Boletaceae, Boletales): new genus and species, and typification of the name Boletus alboater

1 Global diversity of the Tylopilus alboater complex (Boletaceae, Boletales): new genus and species, and typification of the name Boletus alboater Jin Li1,2,3 , Roy E. Halling4, Todd W. Osmundson5, Zhu L. Yang1,2 , Yan-Chun Li1,2 1 Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences, Lanhei Road, Kunming, 650201, China 2 Yunnan Key Laboratory for Fungal Diversity and Green Development, Lanhei Road, Kunming, 650201, China 3 University of Chinese Academy of Sciences, Yanqi Lake East Road, Beijing 100049, China 4 Center for Diversity & Evolution, The New York Botanical Garden, Bronx, NY 10458-5126, USA 5 Department of Biology, University of Wisconsin–La Crosse, 1725 State Street, La Crosse, WI 54601, USA Corresponding authors: Yan-Chun Li ([email protected]); Zhu L. Yang ([email protected]) Copyright: © Jin Li et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Recognition of Tylopilus alboater once relied heavily on the morphological features of the basidiomata, which resulted in considerable confusion due to morphological stasis and plasticity. In this study, we examined specimens morphologically identified as T. alboater from Asia, North America, and Australia using phylogenomics and multi-locus sequence data. To clarify its phylogenetic placement within Boletaceae, we conducted a phylogenomic analysis based on 45 genomes (including three newly sequenced T. alboater genomes) comprising representatives from the eight subfamilies of Boletaceae. Additionally, we constructed a concatenated dataset (nrLSU + tef1-α + rpb1 + rpb2), incorporating representative species from all genera within the subfamily to which T. alboater belongs, to infer its phylogeny. Our phylogenomic analysis revealed that specimens morphologically identified as T. alboater exhibit polyphyly, clustering entirely within the subfamily Boletoideae. Our multi-locus phylogenetic analyses further indicated that these specimens represent eight distinct species distributed across five generic lineages within the subfamily Boletoideae, including one new genus, Neoporphyrellus, proposed in this study, as well as one new species, N. sinoalboater, and two new combinations, N. alboater and N. atronicotianus. The remaining five species are nested in four known genera, including two new species of Abtylopilus, Ab. indonesiensis and Ab. australiensis, and three known species belonging to Anthracoporus, Indoporus, and Tylopilus, respectively. These findings highlight the taxonomic complexity of the T. alboater complex and emphasize the importance of integrating phylogenomic and multi-locus approaches for accurate fungal systematics. Color photos of fresh basidiomata, line drawings of microscopic features, and detailed descriptions of the new taxa are presented. Key words: Boletes, morphology, new taxa, phylogeny, taxonomy Academic editor: Aída Vasco-Palacios Received: 21 May 2025 Accepted: 10 October 2025 Published: 31 October 2025 Citation: Li J, Halling RE, Osmundson TW, Yang ZL, Li Y-C (2025) Global diversity of the Tylopilus alboater complex (Boletaceae, Boletales): new genus and species, and typification of the name Boletus alboater. IMA Fungus 16: e159676. https://doi. org/10.3897/imafungus.16.159676 IMA Fungus 16: e159676 (2025) DOI: 10.3897/imafungus.16.159676 2 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Introduction The genus Tylopilus P. Karst. was proposed by Karsten (1881) and typified by T. felleus (Bull.) P. Karst. Most species traditionally attributed to this genus have white, pinkish, purplish pink, reddish, reddish brown, or brownish hymenophores with pink to pinkish brown spore prints. With the development and utilization of molecular techniques, phylogenetic analyses based on multigene sequences indicated that the original concepts of Tylopilus were polyphyletic, and many new genera were proposed based on species formerly placed in this genus, viz. Zangia Yan C. Li & Zhu L. Yang, Harrya Halling, Nuhn & Osmundson, Sutorius Halling, Nuhn & N.A. Fechner, Pseudoaustroboletus Yan C. Li & Zhu L. Yang, Chiua Yan C. Li & Zhu L. Yang, Hymenoboletus Yan C. Li & Zhu L. Yang, Tylocinum Yan C. Li & Zhu L. Yang, Indoporus A. Parihar, K. Das, Hembrom & Vizzini, Anthracoporus Yan C. Li & Zhu L. Yang, Abtylopilus Yan C. Li & Zhu L. Yang, Brasilioporus A.C. Magnago, Alves-Silva & T.W. Henkel, Nevesoporus A.C. Magnago & T.W. Henkel, and Kgaria Halling, Fechner & Davoodian (Li et al. 2011, 2014; Halling et al. 2012a, 2012b; Wu et al. 2016a; Parihar et al. 2018; Li and Yang 2021; Magnago et al. 2022; Halling et al. 2023). Among these genera, six of them—viz., Abtylopilus, Anthracoporus, Brasilioporus, Nevesoporus, Kgaria, and Indoporus—have common morphological characteristics: dark-colored basidiomata and initially reddish, then blackish, discoloration of the context when injured. Such traits are also shared by Tylopilus alboater (Schwein.) Murrill. Tylopilus alboater was originally described as Boletus alboater Schwein. from North America by Schweinitz (1822). Kuntze (1898) transferred it to Suillus Gray, and Gilbert (1931) then transferred it to Porphyrellus based on its umber basidiomata. Murrill (1909) transferred it to Tylopilus based on its dull pink or flesh-colored hymenophore and rosy to flesh-colored basidiospores, a treatment followed by Singer (1947) and Smith and Thiers (1971). Due to morphological stasis and plasticity, T. alboater was regarded as a single polymorphic species distributed in Asia, North America, and Australia (GBIF: https://www.gbif.org/, data accessed 10 April 2025). However, whether the samples identified as T. alboater from such a vast geographic scope represent a single species has not been specifically addressed. In this study, eleven samples identified as T. alboater from Asia, Australia, and North America were analyzed based on molecular phylogenetic analyses using nuclear genes: the nuclear ribosomal large subunit (nrLSU), the translation elongation factor 1-α gene (tef1-α), the largest subunit of RNA polymerase II (rpb1), and the second-largest subunit of RNA polymerase II (rpb2), along with morphological and ecological data. A new genus, Neoporphyrellus, including one new species and two new combinations, and two new species belonging to Abtylopilus are proposed. The aims of this paper are to (1) investigate the phylogenetic position of species identified as T. alboater from Australia, East Asia, and North America; (2) evaluate the relationships among species in Neoporphyrellus; and (3) compare the morphological features of the new genus Neoporphyrellus with similar genera Abtylopilus, Anthracoporus, Brasilioporus, Nevesoporus, and Indoporus. 3 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Materials and methods Morphological studies Macromorphological characteristics were derived from sample records and photographs of the fresh basidiomata. Color codes used for the newly described taxa are based on Kornerup and Wanscher (1981). Microscopic characteristics were examined under light microscopy after sectioning and mounting tissues in 5%–10% KOH solution. The features assessed included the structure of the pileipellis, the morphology of the basidia and cystidia, and the morphology and ornamentation of the basidiospores. The amyloidity and dextrinoidity of the basidiospores were tested in Melzer’s reagent. All microscopic structures illustrated here were drawn freehand from rehydrated materials. In the descriptions of basidiospores, the abbreviation n/m/p means n basidiospores measured from m basidiomata of p samples. The notation of the form (a) b–c (d) stands for the dimensions of the basidiospores; the range b–c contains 90% of the measured values, and a or d, given in parentheses, represents the extreme values. Q refers to the length/width ratio of a basidiospore in profile view, and Qm is the average Q of all basidiospores ± sample standard deviation. Voucher specimens from our field investigations are deposited in the fungal herbarium of the Herbarium KUN and compared with specimens in NY, BPI, FH, K, PH, and UPS. Herbarium codes follow Thiers (2025). Genome sequencing, assembly, ortholog extraction, and phylogenomic analysis To clarify the phylogenetic placement of “Tylopilus alboater” within Boletaceae, a phylogenomic analysis was conducted. Three samples identified as Tylopilus alboater were newly sequenced on an Illumina HiSeq platform, following the protocols described in Zeng et al. (2018). Raw data quality was controlled using Fastp v0.23.4 (Chen et al. 2018). Genome assembly was performed with SPAdes v3.15.5 (Bankevich et al. 2012) using automatic k-mer selection based on reads length. According to the phylogenomic study of Tremble et al. (2024), we selected 45 genomes representing taxa of the eight subfamilies of Boletaceae, combined with the three newly sequenced genomes of T. alboater, for phylogenomic analysis. Two genomes of Suillus (Suillaceae) and two genomes of Paxillus (Paxillaceae) were selected as outgroup taxa. Assembly completeness and ortholog extraction of all genomes were performed using BUSCO v5.8.2 (Manni et al. 2021a, b) with the “basidiomycota_odb12” dataset, following the extraction protocols of Tremble et al. (2024) and Wang et al. (2024). Selected orthologs were aligned using MAFFT v7.525 (Katoh et al. 2019) with the L-INS-i strategy, and conserved sequences were extracted using Gblocks v0.91b (Castresana 2000) with default parameters. Phylogenomic maximum-likelihood gene trees were inferred using IQ-TREE v2.4.0 (Minh et al. 2020) with ultrafast bootstrapping (Hoang et al. 2018) of 1,000 replicates. The model for each gene was selected automatically in ModelFinder (Kalyaanamoorthy et al. 2017). Sample information is provided in Suppl. material 1. 4 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Molecular procedures and phylogenetic analyses Total DNA was extracted from silica gel–dried or herbarium materials using the CTAB method (Doyle and Doyle 1987). PCR, sequencing, and sequence alignment followed those described in Liu et al. (2021), Orihara et al. (2021), Mao et al. (2023), Shen et al. (2023), Tang et al. (2023), and references therein. The phylogenetic analyses were based on four nuclear loci: nrLSU, tef1-α, rpb1, and rpb2. The common primer pairs LROR/LR5, 983F/1567R, rpb1-Af/rpb1-Cr, and rpb2-6F/rpb2-7R were used to amplify the above gene fragments, respectively. DNA sequences were compiled with SeqMan (DNASTAR Lasergene 9). Alignments were constructed separately for each of the gene fragments using MAFFT v7.310 (Katoh and Standley 2013) with the L-INS-i strategy. Suboptimal gap placements, homology displacement, chimeric artifacts, or sequencing-induced mutations were manually corrected. The alignments were then concatenated using PhyloSuite v1.2.3 (Zhang et al. 2020), with unsampled gene regions coded as missing data. Sequences newly generated in this study have been submitted to GenBank. Detailed information on voucher specimens, including GenBank accession numbers, is provided in Table 1. Genera are abbreviated as follows: Ab. for Abtylopilus, An. for Anthracoporus, Af. for Afroboletus Pegler & T.W.K. Young, Afr. for Afrocastellanoa M.E. Smith & Orihara, B. for Boletus, Br. for Brasilioporus, Bu. for Butyriboletus D. Arora & J.L. Frank, E. for Erythrophylloporus Ming Zhang & T.H. Li, G. for Guyanaporus T.W. Henkel & M.E. Sm., H. for Hortiboletus Simonini, Vizzini & Gelardi, I. for Indoporus, Im. for Imleria Vizzini, J. for Jimtrappea T.W. Henkel, M.E. Smith & Aime, K. for Kgaria, N. for Neoporphyrellus, Ne. for Neotropicomus A.C. Magnago, Alves-Silva & T.W. Henkel, Nev. for Nevesoporus A.C. Magnago & T.W. Henkel, P. for Porphyrellus E.-J. Gilbert, Pa. for Parvixerocomus G. Wu & Zhu L. Yang, Pax. for Paxilloboletus Furneaux, De Kesel & F.K. Khan, S. for Strobilomyces Berk., T. for Tylopilus, Te. for Tengioboletus G. Wu & Zhu L. Yang, and X. for Xanthoconium Singer. Forty-six sequences (14 for nrLSU, 13 for tef1-α, 7 for rpb1, and 12 for rpb2) from thirteen samples were newly generated in this study and aligned with selected sequences from GenBank and previous studies (Desjardin et al. 2009; Dentinger et al. 2010; Nuhn et al. 2013; Wu et al. 2014, 2016a, 2016b; Chakraborty et al. 2018; Han et al. 2017, 2018; Parihar et al. 2018; Li and Yang 2021; Magnago et al. 2022) (Table 1). Butyriboletus roseoflavus (Hai B. Li & Hai L. Wei) D. Arora & J.L. Frank and E. cinnabarinus Ming Zhang & T.H. Li were chosen as outgroup taxa (Wu et al. 2016; Zhang and Li 2018). For protein-coding genes, tef1-α was divided into five blocks (three exons and two introns), rpb1 into four blocks (two exons and two introns), and rpb2 into three blocks (two exons and one intron). The nrLSU was treated as a single block. Thus, the dataset was partitioned into 13 partitions. The combined nuclear dataset was analyzed using Maximum Likelihood (ML) and Bayesian Inference (BI). The Approximately Unbiased (AU) test was performed with IQ-TREE v2.2.6 using parameters “-m MFP+MERGE -bb 1000 -zb 10000” to evaluate topological incongruence among the four genes (Shimodaira 2000; 5 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Minh et al. 2020). Conflicts were observed among gene trees (Suppl. material 2), manifested as topological conflicts in individual loci. Nevertheless, concatenated analysis was conducted because multi-locus data integration can statistically compensate for single-gene discordance, enhance reconstruction of species divergence history, and resolve phylogenetic relationships with much stronger nodal support (Xi et al. 2013; Wascher and Kubatko 2021). The model for each partition was selected independently. The ML analysis was performed with IQ-TREE v2.2.6, with automatic model selection in ModelFinder for each partition (Kalyaanamoorthy et al. 2017) and ultrafast bootstrapping (Hoang et al. 2018) with 1,000 replicates. The best model for each partition is provided in Suppl. material 3. The BI analysis was performed with MrBayes v3.2.7a, running 5 million bootstrap replicates combined with a BI search (Ronquist et al. 2012). The parameter model was selected by the Akaike Information Criterion (AIC) as the best-fit likelihood model in ModelFinder (Kalyaanamoorthy et al. 2017; Zhang et al. 2020). The models employed for the four genes in BI analyses were GTR + F + I + G4 for nrLSU and SYM + I + G4 for tef1-α, rpb1, and rpb2. Subsequently, the sampled trees were summarized after omitting the first 25% of trees as burn-in using the ‘sump’ and ‘sumt’ commands implemented in MrBayes. For BI analyses, the average standard deviation of split frequencies was 0.007272, and all ESS values were > 200 (detailed in Suppl. material 4). Abbreviations AIC the Akaike Information Criterion AU test Approximately Unbiased test BI Bayesian inference BP bootstrap proportion BPI USDA United States National Fungus Collections CTAB hexadecyl trimethyl ammonium bromide, cetyltrimethylammonium bromide DNA deoxyribonucleic acid FH Harvard University, Farlow Herbarium ILD the incongruence length difference K Royal Botanic Gardens,Kew KUN-HKAS The Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences ML Maximum likelihood nrLSU the nuclear ribosomal large subunit NY New York Botanical Garden PCR Polymerase chain reaction PH Academy of Natural Sciences of Drexel University PP posterior probabilities rpb1 largest subunit of RNA polymerase II rpb2 second-largest subunit of RNA polymerase II tef1-α translation elongation factor 1-α gene UPS Uppsala University, Museum of Evolution 6 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Table 1. Information on the taxa used in the phylogenetic analyses. Names, voucher numbers, localities, and their corresponding GenBank accession numbers are listed here. Sequences obtained in this study are shown in bold. “–” represents missing data. Species Voucher Locality Accession References nrLSU rpb1 rpb2 tef1-α Abtylopilus alborubellus HKAS 99704 type China MT154713 – – – Li and Yang 2021 Ab. australiensis NY 2049854 Australia OR122506 OR122507 OR130499 OR130500 OR130501 OR130502 OR130503 OR130504 This study Ab. indonesiensis NY 1393740 Indonesia OQ690002 –OQ689705 OQ689702 This study Ab. scabrosus HKAS 59826 China KT990558 MT110379 –MT110336 Li and Yang 2021 Ab. scabrosus HKAS 50211 type China KT990552 KT990920 KT990389 KT990752 Li and Yang 2021 Afroboletus luteolus Yorou 3141 Zambia – KY553114 KY553124 KY553134 Han et al. 2017 Af. luteolus Yorou 4599 Madagascar – KY553115 KY553125 KY553135 Han et al. 2017 Af. multijugus PC 0723572 Burundi – KY553118 KY553128 KY553138 Han et al. 2017 Af. multijugus PC 0723578 Madagascar – KY553117 KY553127 KY553137 Han et al. 2017 Afrocastellanoa ivoryana OSC 150014 type Zimbabwe KX685720 KX685715 – – Oriharaa and Smith 2017 Anthracoporus cystidiatus HKAS 55375 China KT990622 KT990969 MT110410 KT990816 Li and Yang 2021 An. cystidiatus NY 2049837 Thailand OQ690003 – – – This study An. cystidiatus NY 2072261 Thailand OQ690001 –PV189435 OQ689700 This study An. cystidiatus MHHNU 7312 type China MT154710 MT110377 MT110411 – Li and Yang 2021 An. holophaeus HKAS 59407 China KT990708 KT991030 KT990506 KT990888 Wu et al. 2016a An. holophaeus HKAS 50508 China – KX869634 KX869506 KX869376 Han et al. 2018 An. nigropurpureus HKAS 52685 China KT990627 KT990973 –KT990821 Wu et al. 2016a An. nigropurpureus HKAS 53370 China KT990628 KT990974 KT990460 KT990822 Wu et al. 2016a Boletus edulis HMJAU 4637 China KF112455 KF112586 KF112704 KF112202 Wu et al. 2014 B. reticuloceps HKAS 51232 China KT990537 KT990906 KT990376 KT990739 Wu et al. 2016a B. reticuloceps HKAS 57671 China KF112454 KF112648 KF112703 KF112201 Wu et al. 2014 Brasilioporus olivaceoflavidus VIES 9901322 type Brazil NG088318 OM160565 OM160576 OM160555 Magnago et al. 2022 Br. olivaceoflavidus VIES 9901323 Brazil OM068913 OM160566 –OM160556 Magnago et al. 2022 Br. rufonigricans TH 6376 Guyana AY612835 – – – Magnago et al. 2022 Br. simoniarum VIES 9901327 type Brazil NG088319 OM160567 OM160577 OM160557 Magnago et al. 2022 Butyriboletus roseoflavus HKAS 53405 China KF739666 KF739780 KF739742 KF739704 Wu et al. 2016a Bu. roseoflavus HKAS 54099 China KF739665 KF739779 KF739741 KF739703 Wu et al. 2016a Erythrophylloporus cinnabarinus GDGM 44440 China MH374032 MH378801 MH374029 MH374033 Zhang and Li 2018 E. cinnabarinus GDGM 70536 type China MH374045 MH378802 MH374031 MH374035 Zhang and Li 2018 Guyanaporus albipodus Henkel 8848 type Guyana HQ161868 –LC043082 LC043083 Henkel et al. 2016 Hortiboletus amygdalinus HKAS 54242 China KT990580 –KT990415 KT990776 Wu et al. 2016a H. amygdalinus HKAS 54166 type China KT990581 KT990933 KT990416 KT990777 Wu et al. 2016a H. subpaludosus HKAS 68158 China KT990583 KT990934 KT990418 KT990779 Wu et al. 2016a H. subpaludosus HKAS 59608 China KF112371 KF112551 KF112696 KF112185 Wu et al. 2014 Indoporus shoreae AP 6693 type India MK123973 –MK243367 – Parihar et al. 2018 I. shoreae AP 6697 India MK123976 –MK243368 – Parihar et al. 2018 I. squamulosus HKAS 107153 China MT154708 MT110375 –MT110334 Li and Yang 2021 I. squamulosus HKAS 76299 type China MT154709 MT110376 MT110409 MT110335 Li and Yang 2021 I. squamulosus NY 2049839 Thailand OQ690004 –OQ689704 OQ689701 This study Jimtrappea guyanensis Henkel 9163 Guyana LC053660 –LC053661 – Smith et al. 2015 Kgaria cyanogranulifer NY 1194066 Australia JX889646 JX889688 –OR263680 Halling et al. 2023 K. cyanogranulifer NY 1194065 Australia JX889647 JX889689 –OR263681 Halling et al. 2023 K. similis NY 1193839 Australia OR063867 OR113660 – OR263685 Halling et al. 2023 K. similis NY 1193840 Australia OR063869 – – OR263686 Halling et al. 2023 Imleria badia Xb2 Germany KF030357 – – KF030422 Nuhn et al. 2013 Im. badia HKAS 74714 China KF112375 KF112609 – – Wu et al. 2014 7 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Species Voucher Locality Accession References nrLSU rpb1 rpb2 tef1-α Im. parva HKAS 55341 type China KC215216 KC215229 KC215238 KC215252 Zhu et al. 2014 Im. parva HKAS 59437 China KC215215 KC215228 KC215237 KC215250 Zhu et al. 2014 Neoporphyrellus alboater TH 6941 USA AY612832 – – – Drehmel et al. 2008 N. alboater NY 1193926 USA OP771514 OP765296 OP762644 OP750052 This study N. atronicotianus snWV USA KF030293 – – – Nuhn et al. 2013 N. atronicotianus CFMR 160. USA EU685110 – – – Desjardin et al. 2009 N. atronicotianus NY 815170 paratype USA OQ642297 – – PV189431 This study N. atronicotianus NY 815171 paratype USA OQ642298 –OQ689703 OQ689699 This study N. sinoalboater HKAS 107186 China OP771513 OP765295 OP762643 PV189432 This study N. sinoalboater HKAS 78815 China OP771512 PV189433 PV189438 OP750051 This study N. sinoalboater HKAS 145312 China PQ725682 –PV189436 PV189429 This study N. sinoalboater HKAS 145313 China PQ725682 PV189434 PV189437 PV189430 This study Neotropicomus australis VIES 9901329 Brazil OM068917 – – – Magnago et al. 2022 Ne. australis ICN 202156 Brazil OM068916 OM160572 – – Magnago et al. 2022 Ne. parvogracilis TH 9209 type Guyana NG059946 – – – Magnago et al. 2022 Nevesoporus exiguus TH 9549 Guyana KT339205 – – – Magnago et al. 2022 Nev. nigrostipitatus VIES 9901383 type Brazil NG149037 – – OM160562 Magnago et al. 2022 Nev. nigrostipitatus VIES 9901384 Brazil OM068919 – – – Magnago et al. 2022 Parvixerocomus aokii HKAS 59812 China KF112378 – – KF112266 Wu et al. 2014 Pa. pseudoaokii HKAS 52633 China KF112379 KF112598 KF112736 KF112267 Wu et al. 2014 Pa. pseudoaokii HKAS 80480 type China KP658468 KP658472 KP658470 – Wu et al. 2016b Pa. pseudoaokii HKAS 77032 China KP658467 KP658471 KP658469 – Wu et al. 2016b Paxilloboletus africanus SAB 0716 type Guinea MZ702479 MZ707865 MZ707878 MZ707869 Badou et al. 2022 Pax. latisporus ADK 5072 type DR Congo MZ702481 MZ707866 MZ707879 MZ707870 Badou et al. 2022 Porphyrellus cyaneotinctus HKAS 80183 China MT154718 – – MT110340 Li and Yang 2021 Po. cyaneotinctus HKAS 80192 China MT154719 – – – Li and Yang 2021 Po. porphyrosporus HKAS 48585 China KT990543 KT990911 KT990382 KT990745 Wu et al. 2016a Po. porphyrosporus HKAS 49182 China KT990544 KT990912 KT990383 KT990746 Wu et al. 2016a Strobilomyces seminudus HKAS 82848 China – KT990985 KT990472 KT990835 Wu et al. 2016a S. seminudus HKAS 80400 China – APA20757 APA20446 APA20607 Wu et al. 2016a S. verruculosus HKAS 59637 China – – APA20450 APA20611 Wu et al. 2016a S. verruculosus HKAS 77026 China – APA20761 APA20450 APA20611 Wu et al. 2016a Tylopilus felleus HKAS 54926 China KF112411 KF112575 KF112737 HQ326866 Wu et al. 2014 T. himalayanus DC 17-25 India MG799328 –––Chakraborty et al. 2018 T. himalayanus HKAS 91278 China MT154742 MT110392 MT110427 MT110354 Li and Yang 2021 T. jiangxiensis HKAS 107152 type China MT154731 – – – Li and Yang 2021 T. jiangxiensis HKAS 105250 China MN304779 MN304785 MN304791 MN304797 Zhao et al. 2020 T. rubrobrunneus HKAS 19069 USA OQ819424 OQ828460 OQ828460 OQ828458 This study T. rubrobrunneus BD 329 USA HQ161876 –HQ161845 – Dentinger et al. 2010 Tengioboletus glutinosus HKAS 53425 type China KF112341 KF112578 KF112800 KF112204 Wu et al. 2014 Te. glutinosus HKAS 53452 China KT990655 KT990994 KT990480 KT990844 Wu et al. 2016a Te. reticulatus HKAS 53426 China KF112491 KF112649 KF112828 KF112313 Wu et al. 2014 Te. reticulatus HKAS 53453 type China KT990656 –KT990482 KT990846 Wu et al. 2016a Xanthoconium purpureum BD 228 USA HQ161864 HQ16183 – – Dentinger et al. 2010 X. purpureum NY 00720964 USA KT990663 KT991001 –KT990852 Wu et al. 2016a X. sinense HKAS 80118 China KT990666 KT991004 KT990490 KT990855 Wu et al. 2016a X. sinense HKAS 77758 type China KT990665 KT991003 KT990489 KT990854 Wu et al. 2016a Xerocomellus chrysenteron HKAS 56494 China KF112357 KF112526 KF112685 KF112172 Wu et al. 2014 Xe. corneri HKAS 90206 China KT990669 KT991007 KT990493 KT990857 Wu et al. 2016a Xe. corneri HKAS 52503 China KT990668 KT991006 KT990492 KT990856 Wu et al. 2016a Xe. roseonigrescens GDGM 43238 type China KT220588 KT220591 KT220593 KT220595 Gelardi et al. 2015 Xe. roseonigrescens ZT 13553 China KT220589 KT220592 KT220594 KT220596 Gelardi et al. 2015 8 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Results Phylogenomic analysis Our phylogenomic analysis incorporated 52 high-quality genomes, including 48 representing 42 species from 27 genera across all eight Boletaceae subfamilies. Additionally, two Suillus of Suillaceae and two Paxillus of Paxillaceae were included as outgroup taxa. All selected genomes met the quality threshold of ≥ 80% BUSCO completeness scores (detailed metrics provided in Suppl. material 1). Using BUSCO assessments, we identified 259 single-copy orthologous genes present in all 52 genomes, which were subsequently selected for phylogenomic analysis. The final alignment comprised 89,095 amino acid sites. Our analysis yielded a robust phylogeny of Boletaceae (Fig. 1), unequivocally validating the eight-subfamily classification framework previously proposed by Tremble et al. (2024) and Wu et al. (2023, preprint). However, a minor discrepancy was observed in the phylogenetic placement of Leccinoideae, potentially attributable to either the limited number of single-copy orthologous genes analyzed or methodological constraints in phylogenetic reconstruction. Notably, our phylogenomic analysis demonstrated that specimens morphologically identified as T. alboater exhibit polyphyly and all clustered within the subfamily Boletoideae. A particularly significant finding was the identification of a strongly supported (BP = 100%) monophyletic lineage comprising three specimens: one from New York (NY 1034447) and two from China (HKAS 107186, HKAS 78815). This distinct lineage likely represents a previously unrecognized genus within Boletoideae. Additionally, another New York specimen (HKAS 19069) was found to cluster within Tylopilus. To comprehensively resolve the phylogenetic relationships of species identified as T. alboater, we performed a multi-locus phylogenetic analysis based on four commonly used genes (nrLSU, tef1-α, rpb1, and rpb2), with representatives from all 23 genera of Boletoideae to which the T. alboater complex belongs, based on our study and that of Tremble et al. (2024). Phylogenetic analysis The combined dataset (nrLSU + tef1-α + rpb1 + rpb2), including 100 sequences representing all 23 genera of Boletoideae and two genera of Suillelloideae, consisted of 3,056 nucleotides (including gaps) with the following gene lengths: nrLSU, 902 bp; tef1-α, 640 bp; rpb1, 806 bp; and rpb2, 708 bp (Suppl. material 5). The alignment was submitted to TreeBASE (S30298). In our multi-locus phylogenetic analyses, both ML and BI approaches produced highly congruent tree topologies, with minimal discrepancies observed in branch support values; thus, only the ML tree was selected for display (Fig. 2). Our multi-locus phylogenetic analyses revealed topological discrepancies compared to both the phylogenomic results of Tremble et al. (2024) and our current study. Specifically, the terminal genus-level branches received statistical support, whereas several backbone branches lacked sufficient support. This is potentially attributable to the limited number of genes analyzed. Thirteen samples were studied, including two (NY 815170, NY 815171) of T. atronicotianus, both from North America, and eleven labeled as T. alboater from Australia, China, Indonesia, Thailand, and the USA. Sequences of these samples represent eight species and are nested within five generic lineages of the 9 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Figure 1. Phylogenomics of the family Boletaceae. The tree was inferred from a matrix containing 259 single-copy orthologous genes based on the maximum-likelihood (ML) analysis. The eight subfamilies are highlighted in different colors. Specimens identified as “Tylopilus alboater” are in bold and highlighted in red. 0.04 Boletoideae Brasilioporus rufonigricans TH10115 Strobilomyces dryophilus NY2685980 Nevesoporus exiguus TH10119 100 93 100 100 "Tylopilus alboater" HKAS78815 Tylopilus alboater NY1034447 Strobilomyces echinocephalus NY2342738 "Tylopilus alboater " HKAS107186 Neotropicomus parvogracilis TH9209 100 100 Boletus barrowsii UT480 Boletus edulis BD747 utah Xanthoconium separans NY815401 Xanthoconium separans UT517 Tylopilus intermedius MICH12344 "Tylopilus alboater" HKAS19069 Tylopilus felleus MICH12343 Imleria badia 84.06 Imleria badia NY796215 Boletus coccyginus Bolcoc1 Xerocomellus chrysenteron MICH10003 Hortiboletus rubellus MICH10035-100 Bothia castanella NY817394 Fistulinella cinereoalba TH8030 Veloporphyrellus conicus NY796126 Austroboletus festivus TH9250 Fistulinella prunicolor NY2686046 Butyriboletus roseoflavus LA02 Butyriboletus roseoflavus MG29 Sutorius australiensis NY2686008 Sutorius aff. eximius TH9249 Harrya chromipes MICH254595 Harrya chromipes NY2072388 Chiua virens LA06 Zangia roseola NY2342733 Xerocomus potaroensis TH9260 Xerocomus amazonicus TH10052 Boletellus pseudochrysenteroides MICH4994 Boletellus deceptivus NY2072458 Leccinum snellii MICH1128 Rossbeevera mucosa NY1194094 Leccinum boreale MICH11247 Rossbeevera pachydermis NY2072539 Tylopilus vinaceipallidus TH10094 Phylloboletellus chloephorus MICH11739 Chalciporus sphaerocephalus FLAS-F-68321 Chalciporus piperatus UT-M0002063 Chalciporus piperatus UT-M0000116 Chalciporus chontae NY817307 Paxillus rubicundulus Ve08.2h10 Suillus decipiens EM49 Paxillus involutus ATCC200175 Austroboletoideae Suillelloideae Zangioideae Xerocomoideae Leccinoideae Phylloboletelloideae Chalciporoideae Boletaceae 99 67 67 72 75 87 99 98 93 95 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Suillus americanus EM31 100 100 100 100 Xerocomellus dryophilus PAM 100 100 100 16 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) the USA, so it is highly unlikely that the identification is correct. Also, based on current knowledge, B. alboater has not been documented in Europe. According to information listed on MycoPortal.org, another specimen in PH (00078756) listed as Boletus sp., determined by Schweinitz, with an “ID Remarks” as “Boletus alb.” The collector is listed as unknown, there are no locality data, and a verbatim date of “1805-01-02” is cited without a known collector number. Thus, due to a lack of valid original material, we have proposed here a neotype in NY with an isoneotype in KUN. As we noted above, the NYBG site is only ± 150 km east of the type locality cited later by Schweinitz (1832). Type. (Neotype designated here, Figs 3, 7a, b, Mycobank No.: MBT 10026353): USA • New York, Bronx Co., Bronx, New York Botanical Garden, east end of eastern Twin Pond, 23 August 2011, Halling 9601 (Neotype: NY1193926, GenBank Acc. Nos. OP771514 for nrLSU, OP765296 for rpb1, OP762644 for rpb2, OP750052 for tef1-α; Isoneotype: KUN-HKAS147016). Notes. Neoporphyrellus alboater is characterized by its black to dark gray to dark brown pileus, which is areolate with age; white context slowly turning light pinkish orange or red to pinkish gray, then black when injured; finely subpruinose stipe, which is white above and black below at first and eventually black overall; white to pinkish vinaceous hymenophore becoming pinkish orange to reddish brown, then black when bruised; smooth basidiospores; and trichoderm pileipellis (Coker and Beers 1943; Singer 1947; Snell and Dick 1970; Smith and Thiers 1971; Roody 2003; Phillips 2005; Moore et al. 2007; Bessette et al. 2024). This species shares basidiospore size and the same discoloration reaction when injured with An. cystidiatus (Li and Yang 2021). However, An. cystidiatus differs from N. alboater in its grayish red to brownish red or ruby red pileus, which is slightly darker in the center, and its epithelial pileipellis composed of 8–21 μm wide inflated concatenated cells. The white hymenophore when young and dull pink when mature and the sometimes slightly reticulated stipe apex of N. alboater are similar to those of N. atronicotianus. Moreover, both of these species are phylogenetically related. However, N. atronicotianus has a finely tomentose pileus and stipe, a bright brown hymenophore, a reddish-brown spore print, relatively large basidia (31–46 × 7.5–9.5 μm), and narrow pleurocystidia and cheilocystidia (9–12 μm and 6–9 μm wide, respectively). Neoporphyrellus atronicotianus (Both) Yan C. Li, J. Li, Halling, Osmundson & Zhu L. Yang, comb. nov. MycoBank No: 858941 Figs 4, 8c, d Basionym. Tylopilus atronicotianus Both, Bull. Buffalo Soc. Nat. Sci. 36: 216. 1998 Description. Basidiomata medium to large sized. Pileus 7.5–20 cm in diameter, hemispherical to broadly convex or flattened; surface dry, smooth, light brown to olive-brown, becoming darker in color when matured; context whitish, staining pink to pinkish red at first, and then becoming black when injured. Hymenophore surface initially whitish, staining red at first and then blackish when injured; pores angular, up to 1.5 mm wide; tubes up to 8 mm long, bright brown, staining reddish at first and then black when injured. Stipe 6–12 × 1.5–4.5 cm, clavate to subcylindrical, solid, grayish to dark brown, almost black at the base, 17 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) surface finely tomentose, sometimes finely reticulate near apex; context white, pink to pinkish-red at first, and then becoming black when injured. Spore print reddish-brown (Both 1998; Bessette et al. 2000, 2024; and our observation). Basidia 31–46 × 7.5–9.5 μm, clavate, thin-walled, 4-spored, hyaline to yellowish in KOH. Basidiospores [40/2/2] 7.5–10.5 × 3.5–5 μm [Q = 1.78–2.38, Qm = 2.14 ± 0.09], subfusiform in profile view with slight suprahilar depression, elongated to fusiform in ventral view, smooth, slightly thick-walled (up to 0.5 μm), hyaline to brownish in KOH, brown to yellowish brown in Melzer’s reagent. Hymenophoral trama boletoid; hyphae cylindrical, hyaline to yellowish in KOH, yellowish to yellow in Melzer’s reagent. Cheilocystidia 32–47 × 6–9 μm, fusiform or subfusiform, thin-walled, yellowish brown to brownish in Figure 4. Microscopic features of Neoporphyrellus atronicotianus (NY 00815170, paratype). a Basidiospores b Basidia, basidioles and pleurocystidium c Cheilocystidia d Pleurocystidia e Pileipellis. Scale bars: 20 μm. a b cd e 18 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) KOH, yellow-brown to brown in Melzer’s reagent; surface without encrustations. Pleurocystidia morphologically similar to cheilocystidia but much bigger, 38– 76 × 9–12 μm. Pileipellis a trichoderm, composed of 3.5–7 μm wide filamentous interwoven hyphae, yellowish brown to brownish in KOH, and brown to yellow-brown in Melzer’s reagent; terminal cells 11.5–76 × 3.5–5.5 μm, clavate to subcylindrical, thin-walled. Pileal trama composed of thin-walled hyphae; hyphae 4.5–9 μm wide, hyaline to yellowish in KOH, yellow to yellowish brown in Melzer’s reagent. Clamp connections absent in all tissues. Habitat and distribution. Solitary on the ground of deciduous forest dominated by red oak (Quercus rubra), beech (Fagus sp.), and hemlock (Tsuga sp.); currently known in the United States from New York to West Virginia. Specimen examined. USA • New York, Erie Co., North Collins, Town Park, alt. ca 1319 m, 42.5953°N, 78.9411°W, August 1983, E.E. Both 2480 (NY 815170, paratype); New York, Erie Co., North Collins, Ukrainian Camp, alt. ca 1319 m, 42.5953°N, 78.9411°W, 18 September 1981, B. Both 2358 (NY 815171, paratype). Notes. Neoporphyrellus atronicotianus was originally described from New York and is currently known from the eastern USA (Both 1998; Bessette et al. 2000, 2024). It is characterized by its light brown to olive-brown pileus, whitish context staining pink to pinkish-red and then becoming black when injured, whitish hymenophore staining red at first and then blackish when injured, smooth basidiospores, and trichodermium pileipellis (Both 1998; Bessette et al. 2000, 2024; and our observation). This species is morphologically similar to T. alpinus Yan C. Li & Zhu L. Yang, as both of them have an olive-brown pileus, whitish context, whitish hymenophore, and reticulum at the upper part of the stipe. However, T. alpinus differs from N. atronicotianus in its context staining pale red to grayish red but without any black or blackish tinges when injured, hymenophore staining a brownish red to grayish red or orange-brown tinge when bruised, and relatively long basidiospores (13–14.5 μm) (Li and Yang 2021). Neoporphyrellus atronicotianus is phylogenetically closely related to N. alboater and N. sinoalboater. However, N. alboater has a black to dark gray to dark brown pileus, which is areolate with age, a finely subpruinose stipe, a white to pinkish vinaceous hymenophore, and relatively broad cheilocystidia measuring 24–46 × 8–18 μm and pleurocystidia measuring 39–56 × 13–17 μm. While N. sinoalboater differs from N. atronicotianus in its gray to brownish gray pileus and relatively small pleurocystidia (34–55 × 8–14 μm). Neoporphyrellus sinoalboater Yan C. Li, J. Li, Halling, Osmundson & Zhu L. Yang, sp. nov. MycoBank No: 858942 Figs 5, 8e, f Etymology. sino (Latin) = China, reflecting that the basidiomata were collected from China + alboater for the similarity of the basidiomata to T. alboater. Type. CHINA • Yunnan Province, Lijiang City, Liming Town, Laojunshan, alt. ca 2516 m, 26.8470°N, 99.8502°E, 31 August 2024, Y.C. Li 6989 (KUN-HKAS 145312). Diagnosis. Neoporphyrellus sinoalboater differs from other species of Neoporphyrellus in its gray to brownish gray pileus, whitish to pallid context, whitish to cream and then dirty white or grayish hymenophore, grayish to blackish 19 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) brown stipe, a trichoderm pileipellis composed of 3.5–7 μm wide filamentous interwoven hyphae. Description. Basidiomata medium to large sized. Pileus 3–4.5 cm in diameter, subhemispherical to applanate; surface dry, smooth, gray (5B1) to brownish gray (5B2), staining reddish brown when bruised; margin slightly extended; context whitish (1A2) to pallid (1A3), changing to reddish (8B4) or brownish red (8C4) quickly, and then slowly becoming blackish (19F5) when injured. Hymenophore adnate to slightly decurrent, or sometimes slightly depressed around apex of stipe when mature; surface initially whitish (1A2) to cream (1A3) or dirty white to grayish (4B1); pores angular to roundish, up to 1 mm wide, tubes up to 6 mm long, concolorous or a little paler than hymenophoral surface, changing to reddish brown (8C3) at first and then becoming blackish when injured. Stipe 5–8.3 × 1–4 cm, clavate to subcylindrical, flexuous, solid, grayish (5C3) to blackish brown (5E2), dark in color downwards, staining reddish brown at first and then blackish when bruising; context white (1A1) to cream (1A2), changing to brownish red (8C4) quickly, then slowly becoming blackish (19F5) when injured; basal mycelium white (1A1). Taste and odor mild. Basidia 25–34 × 9–10 μm, clavate, thin-walled, 4-spored, hyaline or yellowish brown in KOH. Basidiospores [80/4/4] (7) 7.5–9.5 (10) × (3.5) 4–4.5 (5) μm, [Q = (1.67) 1.75–2.25 (2.38), Qm = 1.99 ± 0.13], subfusiform in profile view with slight suprahilar depression, elongated to fusiform in ventral view, smooth, slightly thick-walled (up to 0.5 μm), yellowish or brownish in KOH, brown to yellowish brown in Melzer’s reagent. Hymenophoral trama boletoid; hyphae cylindrical, 4–10 μm wide, hyaline to yellowish in KOH, yellow to yellowish brown in Melzer’s reagent. Cheilocystidia 34–55 × 8–14 μm, fusiform or subfusiform, thin-walled, hyaline to brownish in KOH, yellowish brown to brownish in Melzer’s reagent; surface without encrustations. Pleurocystidia morphologically similar to cheilocystidia. Pileipellis a trichoderm, composed of 3.5–7 μm wide filamentous interwoven hyphae, yellowish brown to brownish in KOH and brown to dark brown in Melzer’s reagent; terminal cells 15–88 × 3.5–7 μm, clavate to subcylindrical or fusiform, thin-walled. Pileal trama composed of thin-walled hyphae, 3.5–6 μm wide, hyaline or yellowish in KOH, brownish in Melzer’s reagent. Clamp connections absent in all tissues. Habitat and distribution. Solitary on the ground under Quercus semicarpifolia; currently known from central and southwestern China. Additional specimens examined. CHINA • Hubei Province, Shennongjia, Muyu Town, alt. ca 1800 m, 31.4689°N, 110.3663°E, 16 July 2012, Q. Zhao1556 (KUN-HKAS 78815); Yunnan Province, Dali, Xiangyun Town, Dasongping Village, alt. ca 2040 m, 25.6653°N, 100.6955°E, 10 July 2009, N.K. Zeng 297 (KUN-HKAS 107186); Lijiang City, Liming Town, Laojunshan, alt. ca 2516 m, 26.8470°N, 99.8502°E, 31 August 2024, Y.C. Li 7010 (KUN-HKAS 145313). Note. Neoporphyrellus sinoalboater is characterized by its gray to brownish gray pileus and stipe staining reddish brown at first and then becoming blackish when bruised; white to cream hymenophore usually staining reddish and then blackish when bruised; white to pallid context changing to reddish or brownish red initially and then slowly becoming blackish when injured; smooth basidiospores; and trichoderm pileipellis. All these features are very similar to those of Abtylopilus scabrosus Yan C. Li & Zhu L. Yang and Abtylopilus alborubellus Yan C. Li & Zhu L. Yang. However, the latter two species 20 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) have a glabrous pileus, a white to cream or grayish and then grayish pink hymenophore, relatively long basidiospores (up to 11 μm), and a palisadoderm pileipellis composed of broad (up to 9 μm) vertically arranged hyphae (Li and Yang 2021). Neoporphyrellus alboater and N. atronicotianus share the same discoloration as N. sinoalboater when injured, but N. alboater has a black to dark gray to dark brown pileus becoming gray with age; a dull pinkish or flesh-colored hymenophore; fine hymenophoral pores (up to 0.5 mm wide); relatively large basidiospores measuring 8–11 × 4–5 μm; and wide hymenial cystidia measuring 39–56 × 13–17 μm (Singer 1947). Neoporphyrellus atronicotianus has a light brown to olive-brown pileus, a grayish to dark brown stipe with the base almost black, relatively long tubes (up to 22 mm), and relatively large basidia measuring 31–46 × 7.5–9.5 μm (Singer 1947; Bessette and Bessette 2000; Bessette and Bessette 2001; Roody 2003). Figure 5. Microscopic features of Neoporphyrellus sinoalboater (KUN-HKAS107186). a Basidiospores b Basidia, basidioles and pleurocystidia c Pleurocystidia d Cheilocystidia; e Pileipellis. Scale bars: 20 μm. a b e c d 21 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Abtylopilus Yan C. Li & Zhu L. Yang, The Boletes of China: Tylopilus s. l. (Singapore): 39 (2021) Notes. Abtylopilus was proposed by Li and Yang (2021) as a new genus based on morphological and multi-locus phylogenetic studies. This genus is characterized by its nearly glabrous pileus, white to cream hymenophore, fine hymenophoral pores (0.3–1 mm wide), initially red and then black discoloration when injured, and palisadoderm pileipellis. Two species were described from China, viz., Ab. alborubellus and Ab. scabrosus. Here two additional new species from Indonesia and Australia are described and documented in detail. Abtylopilus australiensis Yan C. Li, J. Li, Halling, Osmundson & Zhu L. Yang, sp. nov. MycoBank No: 859185 Figs 6, 8g Etymology. australiensis referring to the species being found in Australia. Type. AUSTRALIA • Queensland, Tablelands, Mareeba, Barron Gorge National Park, Wright’s Lookout, 18.8401°S, 145.6427°W, ca 367 m, 4 Feb 2006, T.W. Osmundson 1080 (Holotype: BRI AQ0796293, Isotype: NY 2049854). Diagnosis. Abtylopilus australiensis differs from other species of Abtylopilus in its reddish-brown pileus, reddish to orange-red hymenophore staining dark red initially then blackish when bruised, orange-red, brownish red to blackish stipe, and a trichoderm pileipellis composed of 3–4 μm wide vertically arranged to slightly interwoven hyphae. Description. Basidiomata medium to large sized. Pileus 3.4–5.6 cm in diameter, convex to plane; surface subtomentose, dry, reddish brown(7E7-7F7); context whitish, staining dark red at first, and then becoming black when injured. Hymenophore depressed around apex of stipe; surface cream (4A3-4A4) to reddish (6A3-6A4), becoming orange-red (7A6-7A7) in age, staining dark red initially then blackish when bruised; pores nearly round, 0.3–1 mm wide, pale cream(4A2), becoming pale pinkish brown (7D5) to nearly black(4F6) in age; tubes up to 4 mm long, concolorous with hymenophoral surface. Stipe 4–5.9 × 0.7–1.1 cm, equal, solid, cream (4A2) to yellowish (4A4) when young, orange-red (7A6) to brownish red (7D8) at apex and reddish brown (6D8-6E8) to blackish (4F6) downward when mature; surface densely covered with minute-pruinose squamules; context whitish, staining dark red at first, and then becoming black when injured. Basidia 25–30 × 8–12 μm, clavate to narrowly clavate, thin-walled, 4-spored, hyaline to yellowish in KOH. Basidiospores [60/2/1] (8.0) 8.5–10.5 (11) × (3.0) 3.5–4.5 (5) μm [Q = (1.78) 2.11–2.86 (3.17), Qm = 2.47 ± 0.23], subcylindrical or subfusiform and inequilateral in profile view with slight suprahilar depression, oblong to fusiform in ventral view, smooth, yellowish to brownish in KOH, yellow to yellow-brown in Melzer’s reagent. Hymenophoral trama boletoid; hyphae cylindrical, hyaline to yellowish in KOH, yellowish to yellow in Melzer’s reagent. Cheilocystidia 45–56 × 12–13 μm, broadly subfusiform to fusoid-ventricose, thin-walled, yellowish to brownish in KOH, yellow to yellow-brown in Melzer’s reagent; surface without encrustations. Pleurocystidia morphologically similar to cheilocystidia. Pileipellis a trichoderm, composed of 3–4 μm wide vertically 22 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) arranged to slightly interwoven hyphae, yellowish brown to brown in KOH and yellow-brown to dark brown in Melzer’s reagent; terminal cells 17–27 × 2–3 μm, subfusiform to cystidioid. Pileal trama composed of thin-walled hyphae; hyphae 2.5–4 μm wide, hyaline to yellowish in KOH and yellowish to yellow in Melzer’s reagent. Clamp connections absent in all tissues. Habitat and distribution. Gregarious on soil in rainforest; currently known in Australia. Notes. Abtylopilus australiensis is characterized by its reddish-brown pileus; white context staining dark red at first and then becoming black when injured; reddish to orange-red hymenophore staining dark red initially and then blackish when bruised; orange-red, brownish red to blackish stipe; smooth basidiospores; and trichoderm pileipellis. Phylogenetically, Ab. australiensis clusters with Ab. scabrosus and forms a sister group with Ab. indonesiensis, and they are morphologically similar to each other. However, Ab. scabrosus differs from Ab. australiensis in its grayish red to brownish red pileus covered with tomentose squamules; gray to grayish pink hymenophore; white to dingy white stipe covered with dark scabrous squamules; and relatively large basidia measuring 28–55 × 16–17 μm (Li and Yang 2021). Abtylopilus indonesiensis differs from Ab. australiensis in its chocolate-brown to black pileus, pinkish hymenophore, pinkish orange stipe that is deep purple to black toward the base, and relatively small basidia measuring 26–40 × 10–11 μm (Li and Yang 2021). Figure 6. Microscopic features of Abtylopilus australiensis (NY 2049854, type). a Basidiospores b Basidia and pleurocystidium c Cheiloand pleurocystidia d Pileipellis. Scale bars: 20 μm. a b c d 23 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Abtylopilus indonesiensis Yan C. Li, J. Li, Halling, Osmundson & Zhu L. Yang, sp. nov. MycoBank No: 859186 Figs 7, 8h Etymology. indonesiensis referring to the species being found in Indonesia. Type. INDONESIA • Java, Haurbentes Park, alt. ca 300 m, 6.5442°S, 106.438°E, 16 Jan 2001, Halling 8070 (Holotype: BO; Isotype: NY 1393740). Diagnosis. Abtylopilus indonesiensis differs from other species of Abtylopilus in its a chocolate-brown to black pileus becoming a pale pinkish brown with age, a pinkish orange stipe but deep purple to black downward, a pinkish hymenophore usually, a whitish context changing to orange-red at first and then black when injured, and trichoderm pileipellis composed of 1.5–3.5 μm wide vertically arranged hyphae. Description. Basidiomata medium to large sized. Pileus 4–10 cm in diameter, convex to plano-convex, subvelutinous to tomentose, dry, chocolate-brown (7C3-7C4) to black (4F6), fading to a pale pinkish brown; context 1–1.5 cm thick, whitish, changing to orange-red then blackish when injured. Hymenophore adnate to adnexed; surface pinkish (7A2-7A3), staining orange-red at first and then black when bruised; tubes up to 8 mm long, white initially then pinkish to flesh colored when matured, staining orange-red at first and then black when bruised; pores angular, concolorous with hymenophoral surface and staining likewise. Stipe 4–6 × 1.5–2 cm, equal, dry, subpruinose, white to pinkish orange (7A3-7B3) at apex, deep purple (13E5-13E7) to black (4F6) downwards, staining red initially then blackish when injured. Odor and taste mild. Basidia 26–40 × 10–11 μm, clavate, thin-walled, 4-spored, hyaline to yellowish in KOH. Basidiospores [60/3/1] 9.0–10.5 × 4–5 μm [Q = (1.90) 2.00–2.71 (2.86), Qm = 2.31 ± 0.22], subfusiform in profile view with slight suprahilar depression, elongated to fusiform in ventral view, smooth, slightly thick-walled (up to 0.5 μm), hyaline to yellowish in KOH, yellowish to yellowish brown in Melzer’s reagent. Hymenophoral trama boletoid; hyphae cylindrical, hyaline to yellowish in KOH, yellowish in Melzer’s reagent. Cheilocystidia 32–55 × 11–14 μm, fusiform or subfusiform, thin-walled, brownish to yellowish brown in KOH, yellow to yellow-brown in Melzer’s reagent. Pleurocystidia morphologically similar to cheilocystidia. Pileipellis a palisadoderm, composed of 1.5–3.5 μm wide vertically arranged hyphae, yellowish brown to brownish in KOH and yellow-brown to dark brown in Melzer’s reagent; terminal cells 15–35 × 2–3 μm, subfusiform to cystidioid, thin-walled. Pileal trama composed of thin-walled hyphae; hyphae 2–3 μm wide, hyaline to yellowish in KOH and yellowish to yellow in Melzer’s reagent. Clamp connections absent in all tissues. Habitat and distribution. Gregarious on the ground of dipterocarp forest (Shorea, Dipterocarpus, Hopea); currently known from Indonesia. Notes. Abtylopilus indonesiensis is characterized by a chocolate-brown to black pileus becoming pale pinkish brown with age; a pinkish orange stipe that is deep purple to black toward the base, staining reddish brown at first and then becoming blackish when bruised; a pinkish hymenophore usually staining reddish and then blackish when bruised; a whitish context changing to orange red at first and then black when injured; smooth basidiospores; and trichoderm pileipellis. Abtylopilus indonesiensis is phylogenetically related 24 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) to Ab. scabrosus and Ab. australiensis. However, Ab. scabrosus differs from Ab. indonesiensis in its grayish red to brownish red or ruby pileus, white to dingy white stipe densely covered with dark scabrous squamules, and relatively large basidia measuring 28–55 × 16–17 μm (Li and Yang 2021). Abtylopilus australiensis differs from Ab. indonesiensis in its reddish-brown pileus, reddish to orange-red hymenophore, and reddish brown to blackish stipe densely minute-pruinose (Li and Yang 2021). Discussion According to our morphological studies and phylogenetic analyses, specimens from North America, Asia, and Australia labeled as “Tylopilus alboater” represent eight species belonging to four known genera—Abtylopilus, Anthracoporus, Indoporus, and Tylopilus—and one new genus, Neoporphyrellus. Specimens from East and Southeast Asia represent four species, including two known species, viz. I. squamulosus and An. cystidiatus, and two new species, N. sinoalboater and Ab. indonesiensis. Specimens from America represent three species, including two new combinations, viz. N. alboater and N. atronicotianus, and one known species, viz. T. rubrobrunneus. A specimen from Australia represents a new species of Abtylopilus, viz., Ab. australiensis. Figure 7. Microscopic features of Abtylopilus indonesiensis (NY 1393740, type). a Basidiospores b Basidia c Cheiloand pleurocystidia d Pileipellis. Scale bars: 20 μm. a c b d 25 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Figure 8. Basidiomata of “Tylopilus alboater” from different regions. a, b N. alboater from North America (NY 1193926, images by Halling) c, d N. atronicotianus from North America (NY 815171, images by Both) e, f Neoporphyrellus sinoalboater from China (e KUN-HKAS 145312, type; f KUN-HKAS 145313) g Abtylopilus australiensis from Australia (NY 2049854, image by Osmundson) h Ab. indonesiensis from Indonesia (NY 1393740, image by Halling) (c–h from https://sweetgum.nybg.org/science/vh/specimen-list/). 32 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Supplementary material 4 Bayes.log Authors: Jin Li, Roy E. Halling, Todd W. Osmundson, Zhu L. Yang, Yan-Chun Li Data type: txt Explanation note: The log file of MrBayes, the ESS values are listed here. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.159676.suppl4 Supplementary material 5 Boletoideae lsu, tef1, rpb1, rpb2 Authors: Jin Li, Roy E. Halling, Todd W. Osmundson, Zhu L. Yang, Yan-Chun Li Data type: fas Explanation note: The combined dataset (nrLSU + rpb1 + rpb2 + tef1-α) used in ML and MB phylogenetic analyses including 100 sequences (representing 23 genera of Boletoideae and two genus of Suillelloideae) consisted of 3056 nucleotides. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.159676.suppl5 Supplementary material 6 nrLSU gene trees Authors: Jin Li, Roy E. Halling, Todd W. Osmundson, Zhu L. Yang, Yan-Chun Li Data type: jpg Explanation note: The tree of the nuclear ribosomal large subunit (nrLSU). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.159676.suppl6 33 IMA Fungus 16: e159676 (2025), DOI: 10.3897/imafungus.16.159676 Jin Li et al.: Global diversity of Tylopilus alboater complex (Boletaceae, Boletales) Supplementary material 7 tef1-α gene tree Authors: Jin Li, Roy E. Halling, Todd W. Osmundson, Zhu L. Yang, Yan-Chun Li Data type: jpg Explanation note: The tree of the translation elongation factor 1-α gene (tef1-α). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.159676.suppl7 Supplementary material 8 rpb1 gene tree Authors: Jin Li, Roy E. Halling, Todd W. Osmundson, Zhu L. Yang, Yan-Chun Li Data type: jpg Explanation note: The tree of the largest subunit of RNA polymerase II (rpb1). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.159676.suppl8 Supplementary material 9 rpb2 gene tree Authors: Jin Li, Roy E. Halling, Todd W. Osmundson, Zhu L. Yang, Yan-Chun Li Data type: jpg Explanation note: The tree of the second-largest subunit of RNA polymerase II (rpb2). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.159676.suppl9