er-reviewed open-access journal MycoKeys 54: 31-47 (2019) 6 doi: 10.3897/mycokeys.54.35386 RESEARCH ARTICLE Oo Mycokeys http://mycokeys.pensoft.net Launched to accelerate biodiversity research Reassessment of the generic limits for Hydnellum and Sarcodon (Thelephorales, Basidiomycota) Karl-Henrik Larsson'?, Sten Svantesson*?*, Diana Miscevic?, Urmas Kéljalg®, Ellen Larsson”? | Natural History Museum, University of Oslo, PO. Box 1172 Blindern, NO 0318 Oslo, Norway 2 Gothenburg Global Biodiversity Centre, PO. Box 461, SE 405 30 Goteborg, Sweden 3 Department of Biological and Environmental Sciences, University of Gothenburg, PO. Box 461, SE 405 30 Goteborg, Sweden 4 Royal Botanic Gardens Victoria, Birdwood Ave, Melbourne, Victoria 3004, Australia 5 Vastkuststiftelsen, Sandihamnsvigen 71, SE 434 94 Vallda, Sweden 6 Institute of Ecology and Earth Sciences, 40 Lai Street, 51005 Tartu, Estonia Corresponding author: Karl-Henrik Larsson (k.h.larsson@nhm.uio.no) Academic editor: Maria P Martin | Received 11 April 2019 | Accepted 21 May 2019 | Published 10 June 2019 Citation: Larsson K-H, Svantesson S, Miscevic D, Kéljalg U, Larsson E (2019) Reassessment of the generic limits for Hydnellum and Sarcodon (Thelephorales, Basidiomycota) MycoKeys 54: 31-47. https://doi.org/10.3897/ mycokeys.54.35386 Abstract DNA sequences from the nuclear LSU and ITS regions were used for phylogenetic analyses of Thelephorales with a focus on the stipitate hydnoid genera Hydnellum and Sarcodon. Analyses showed that Hydnellum and Sarcodon are distinct genera but that the current division, based on basidioma texture, makes Sarcodon paraphyletic with respect to Hydnellum. In order to make genera monophyletic several species are moved from Sarcodon to Hydnellum and the following new combinations are made: Hydnellum amygdaliolens, H. fennicum, H. fuligineoviolaceum, H. fuscoindicum, H. glaucopus, H. joeides, H. lepidum, H. lundellii, H.. martioflavum, H. scabrosum, H. underwoodii, and H. versipelle. Basidiospore size seems to separate the genera in most cases. Hydnellum species have basidiospore lengths in the range 4.45-6.95 um while the corresponding range for Sarcodon is 7.4-9 um. S. quercinofibulatus deviates from this pattern with an average spore length around 6 um. Neotropical Sarcodon species represent a separate evolutionary lineage. Keywords Phylogeny, stipitate hydnoid, taxonomy, Thelephorales, tooth fungi Copyright Karl-Henrik Larsson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 32 Karl-Henrik Larsson et al. / MycoKeys 54: 31-47 (2019) Introduction The order Thelephorales is a distinctive lineage of Agaricomycetes, well-known for its almost ubiquitous ectomycorrhizal life style (Tedersoo et al. 2010). Several species have stipitate hydnoid basidiomata (Fig. 1). They have traditionally been divided into four genera, Phellodon and Bankera with hyaline basidiospores, and Hydnellum and Sarcodon with yellow to brown tinted basidiospores (Maas Geesteranus 1975). In both cases the genera within each pair differ in basidioma structure, with Phellodon and Hydnellum being hard and dry, and Bankera and Sarcodon forming softer, fleshier basidiomata. This difference in texture is, however, difficult to assess and a series of recent molecular phylogenetic analyses, as outlined below, have indicated that the traditional, morphology-based generic limits are equivocal. In a recent comprehensive study of stipitate hydnoid species from south-eastern North America, Baird et al. (2013) found that Bankera could not be separated from Phellodon and the genera were hence combined into a more comprehensive Phellodon. The same study suggested that the generic limits of Sarcodon and Hydnellum need reassessment. Nitare and Hégberg (2012) examined the Nordic species of Sarcodon and included a preliminary molecular phylogeny for the species accepted in Sarcodon. Hydnellum species were also included in non-published test runs and found to be nested among Sarcodon species. They concluded that revisions of limits of both genera were probably necessary. Miscevic (2013) expanded on the results in Nitare and Hégberg (2012) by including more sequences for each species and by including a selection of Hydnellum species in published phylogenies. The results were in congruence with Baird et al. (2013) with regard to overall tree topology and again the conclusion was that the limits of Sarcodon and Hydnellum need further study. A recent phylogenetic overview of Thelephorales (Vizzini et al. 2016) and a study of Hydnellum from the Mediterranean region (Loizides et al. 2016) came to similar conclusions, although Vizzini et al. (2016) did not include sequences from several Neotropical Sarcodon species described by Grupe et al. (2015, 2016). In this paper we analyse ITS and nuclear LSU sequences from a wide selection of Thelephorales species with a focus on Hydnellum and Sarcodon in order to resolve the relationship between these two genera. We also make some nomenclatural changes that follow from the revision of genus circumscriptions. We demonstrate that Neotropical Sarcodon species do not cluster with temperate and boreal species and may be warranted as one or more new genera with more data. Methods For the phylogenetic analyses we compiled two datasets. ‘The first dataset consists of nuclear LSU sequences from most genera in Thelephorales and from a majority of the Hydnellum and Sarcodon species occurring in Europe. For our two target genera we chose only sequences generated for this study from recently collected basidiomata. Reassessment of the generic limits for Hydnellum and Sarcodon 33 Figure |. Fruiting bodies of Hydnellum and Sarcodon A Hydnellum suaveolens B H. aurantiacum C H. ferrugineum D Sarcodon imbricatus. We deliberately excluded sequences from specimens identified as H. concrescens or H. scrobiculatum since these names seem to cover more than just two species and it is currently unclear how the names should be applied (Ainsworth et al. 2010). Since this study is positioned as a revision of the genus limits we were more interested in sequence quality control than a complete coverage of all species reported from Europe. For our second dataset we chose a different strategy. Here we included ITS sequences from all Hydnellum and Sarcodon species represented among our own sequences and in GenBank as of December 1, 2018. The reason is that many species, and especially the recently described species from tropical regions, are only available as ITS sequences. However, we made no attempt to verify the identifications given in GenBank and do not endorse them as correct. DNA was extracted from recent dried collections of basidiomata from North Eu- rope. Voucher numbers, herbarium location, and GenBank numbers are given in Table 1. DNA extraction and PCR protocols follow Larsson et al (2018). Sequencing was ei- ther done in-house at University of Oslo, or as a commercial service by Macrogen Inc., South Korea. Assembly of chromatograms was done with Sequencher 5.2.4 (Gene Codes Co., Ann Arbor). Aligning was performed either manually using the editor in PAUP* 4.0a (Swofford 2002) or the software ALIVIEW 1.18 (Larsson 2014), or au- tomatically utilising the L-INS-i strategy as implemented in MAFFT v. 7.017 (Katoh and Standley 2013), followed by manual adjustment. 34 Karl-Henrik Larsson et al. / MycoKeys 54: 31-47 (2019) Table I. Specimens sequenced or downloaded from GenBank. Herbarium acronyms follow Thiers. Sequences generated for this study are marked in bold. Species Amaurodon aquicoeruleus Agerer Amaurodon viridis (Alb. & Schwein.:Fr.) J.Schrot Bankera fuligineoalba (J.C.Schmidt:Fr.) Pouzar Bankera violascens (Alb. & Schwein.:Fr.) Pouzar Boletopsis leucomelaena (Pers.:Fr.) Fayod Hydnellum aurantiacum (Batsch:Fr.) P.Karst. Hydnellum aurantiacum Hydnellum aurantiacum Hydnellum auratile (Britzelm.) Maas Geest. Hydnellum auratile Hydnellum auratile Hydnellum caeruleum (Hornem.:Fr.) P.Karst. Hydnellum caeruleum Hydnellum caeruleum Hydnellum complicatum Banker Hydnellum concrescens (Pers.) Banker Hydnellum cristatum (G.EAtk.) Stalpers Hydnellum cumulatum K.A.Harrison Hydnellum cyanopodium K.A.Harrison Hydnellum diabolus Banker Hydnellum dianthifolium Loizides, Arnolds & P.-A.Moreau Hydnellum earlianum Banker Hydnellum ferrugineum (Fr.:Fr.) P.Karst. Hydnellum ferrugineum Hydnellum ferrugineum Hydnellum ferrugipes Coker Hydnellum geogenium (Fr.) Banker Hydnellum geogenium Hydnellum geogenium Hydnellum gracilipes (P.Karst.) P.Karst. Hydnellum gracilipes Hydnellum mirabile (Fr.) P.Karst. Hydnellum mirabile Hydnellum mirabile Hydnellum peckii Banker Hydnellum peckii Hydnellum peckii Hydnellum pineticola K.A.Harrison Hydnellum piperatum Maas Geest. Hydnellum regium K.A.Harrison Hydnellum scleropodium K.A.Harrison Hydnellum scrobiculatum (Fr.) P.Karst. Hydnellum spongiosipes (Peck) Pouzar Hydnellum suaveolens (Scop.:Fr.) P.Karst. Hydnellum suaveolens Hydnellum suaveolens Hydnellum subsuccosum K.A.Harrison Lenzitopsis daii L.W.Zhou & Kéljalg Lenzitopsis oxycedri Malengon & Bertault Odontia fibrosa (Berk. & M.A.Curtis) Kéljalg Phellodon cf niger Phellodon tomentosus (L.:Fr.) Banker Pseudotomentella flavovirens (Hohn. & Litsch.) Svréek Sarcodon amygdaliolens Rubio Casas, Rubio Roldan & Catala Sarcodon aspratus (Berk.) S.Ito Voucher Agerer & Bougher KH Larsson 14947b E Larsson 400-13 MV 130902 M Krikorev 140912 RG Carlsson 08-105 E Bendiksen 177-07 O-F-295029 O-F-294095 O-F-242763 J Nitare 110926 O-F-291490 E Bendiksen 575-11 E Bendiksen 584-11 REB 71 K(M) 134463 REB 169 SE Westmoreland 69 SE Westmoreland 85 KAH 13873 ML61211HY REB 375 O-F-297319 E Larsson 356-16 E Larsson 197-14 REB 176 O-F-66379 O-F-296213 E Bendiksen 526-11 E Larsson 219-11 GB-0113779 RG Carlsson 11-119 E Larsson 170-14 S Lund 140912 S Svantesson 328 E Larsson 174-14 E Bendiksen 567-11 RB 94 REB 322 SE Westmoreland 93 REB 3 REB 78 REB 52 E Larsson 139-09 E Larsson 8-14 S Svantesson 877 REB 10 Yuan 2959 KH Larsson 15304 TU115028 E Larsson 35-14 E Bendiksen 118-10 KH Larsson 16190 SC 2011 Herb. GenBank number ITS LSU AM490944 AM490944 MKG602707 MKG602707 MK602708 MK602708 MKG602709 MK602709 MK602710 MK602710 MK602711 MK602711 MK602712 MK602712 MKG602713 MK602713 MK602714 MK602714 MK602715 MK602715 MK602716 MK602716 MKG602717 MKG602717 MKG602718 MK602718 MKG602719 MKG602719 KC571711 EU784267 JN135174 AY569026 AY569027 AF351863 KX619419 JN135179 MK602720 MK602720 MK602721 MK602721 MK602722 MK602722 KC571727 MK602723 MK602723 MK602724 MK602724 MK602725 MK602725 MK602726 MK602726 MK602727 MK602727 MK602728 MK602728 MK602729 MK602729 MK602730 MK602730 MK602731 MK602731 MK602732 MK602732 MK602733 MK602733 KC571734 JN135173 AY569031 JN135186 JN135181 JN135184 MK602734 MK602734 MK602735 MK602735 MK602736 MK602736 JN135178 JN169799 JN169793 MK602774 MK602774 MK602775 MK602775 MK602782 MK602782 MK602781 MK602781 MK602780 MK602780 JN376763 DQ448877 Reassessment of the generic limits for Hydnellum and Sarcodon Species Sarcodon atroviridis (Morgan) Banker Sarcodon atroviridis Sarcodon bairdii A.C.Grupe & Vasco-Pal. Sarcodon colombiensis A.C.Grupe & Vasco-Pal. Sarcodon fennicus (P.Karst.) P.Karst. Sarcodon fennicus Sarcodon fennicus Sarcodon fuligineoviolaceus (Kalchbr.) Pat. Sarcodon fuligineoviolaceus Sarcodon fuligineoviolaceus Sarcodon fuscoindicus (K.A.Harrison) Maas Geest. Sarcodon glaucopus Maas Geest. & Nannf. Sarcodon glaucopus Sarcodon glaucopus Sarcodon imbricatus (L.:Fr.) P.Karst. Sarcodon imbricatus Sarcodon imbricatus Sarcodon joeides (Pass.) Bataille Sarcodon joeides Sarcodon joeides Sarcodon joeides Sarcodon lepidus Maas Geest. Sarcodon lepidus Sarcodon lepidus Sarcodon leucopus (Pers.) Maas Geest. & Nannf. Sarcodon leucopus Sarcodon leucopus Sarcodon lundellii Maas Geest. & Nannf. Sarcodon lundellii Sarcodon lundellii Sarcodon martioflavus (Snell, K.A.Harrison & H.A.C.Jacks.) Maas Geest. Sarcodon martioflavus Sarcodon martioflavus Sarcodon pakaraimensis A.C.Grupe & T.W.Henkel Sarcodon pallidogriseus A.C.Grupe & Vasco-Pal. Sarcodon portoricensis A.C.Grupe & T.J.Baroni Sarcodon quercophilus A.C.Grupe & Lodge Sarcodon quercinofibulatus Pérez-De-Greg., Macau & J.Carbé Sarcodon rufobrunneus A.C.Grupe & Vasco-Pal. Sarcodon scabripes (Peck.) Banker Sarcodon scabrosus (Fr.) P.Karst. Sarcodon scabrosus Sarcodon scabrosus Sarcodon squamosus (Schaeff.) Quél. Sarcodon squamosus Sarcodon squamosus Sarcodon umbilicatus A.C.Grupe, T.J.Baroni & Lodge Sarcodon underwoodii Banker Sarcodon versipellis (Fr.) Nikol. Sarcodon versipellis Sarcodon versipellis Sistotrema brinkmannii (Bres.) J.Erikss. Steccherinum ochraceum (J.E.Gmel.:Fr.) Gray Thelephora caryophyllea (Schaeff.:Fr.) Pers. Thelephora terrestris Ehrh.:Fr. Tomentella stuposa (Link) Stalpers Tomentellopsis pulchella Kéljalg & Bernicchia Voucher REB 104 REB 61 Vasco 990 Vasco 2084 S Westerberg 110909 O-F-242833 O-F-204087 LA 120818 B Nylén 130918 A Molia 160-2011 OSC 113622 RG Carlsson 13-060 J Nitare 060916 A Edvinson 110926 S Svantesson 355 J Rova 140829-2 E Larsson 384-10 RG Carlsson 11-090 K Hjortstam 17589 J Nitare 110829 REB 270 E Grundel 110916 RG Carlsson 10-065 J Nitare 110829 O-F-296944 O-F-296099 P Hedberg 080811 L&A Stridvall 06-049 O-F-242639 O-F-295814 A Delin 110804 O-F-242435 O-F-242872 T Henkel 9554 Vasco 989 TG Baroni 8776 CFMR-BZ-3833 JC 20090718-2 Vasco 1989 REB 351 O-F-295824 O-F-292320 O-F-360777 O-F-177452 E Larsson 248-12 O-F-295554 TJ Baroni 10201 REB 50 RG Carlsson 13-057 RG Carlsson 11-085 E Bendiksen 164-07 KH Larsson 14078 KH Larsson 11902 E Larsson 89-09S E Larsson 295-13 Th-0764 KH Larsson 16366 Herb. TENN GenBank number ITS LSU JN135190 KC571768 KR698938 KP972654 MK602739 MK602739 MK602738 MK602738 MK602737 MK602737 MK602740 MK602740 MK602741 MK602741 MK602742 MK602742 EU669228 MK602743 MK602743 MK602744 MK602744 MK602745 MK602745 MK602748 MK602748 MKG602746 MKG602746 MKG602747 MK602747 MKG602749 MKG602749 MKG602750 MK602750 MK602751 MK602751 KC571772 MK602753 MK602753 MK602752 MK602752 MK602754 MK602754 MKG02756 MKG602756 MK602755 MK602755 MK602757 MKG602757 MK602758 MK602758 MK602759 MK602759 MK602760 MK602760 MKG602763 MK602763 MK602762 MK602762 MK602761 MK602761 KM668103 KR698939 KM668100 KM668101 JX271818 MK602773 KR698937 JN135191 MK602764 MK602764 MKG02766 MKG02766 MKG602765 MKG602765 MK602768 MK602768 MKG602767 MKG602767 MKG602769 MKG602769 KM668102 KC571781 MK602771 MK602771 MK602772 MK602772 MKG602770 MKG602770 KF218967 KF218967 JQ031130 JQ031130 MKG602776 MK602776 MK602777 MK602777 MK602778 MK602778 MK602779 MK602779 35 36 Karl-Henrik Larsson et al. / MycoKeys 54: 31-47 (2019) In the phylogenetic analyses we assumed the following minimal partitions for the nrDNA region: ITS1, 5.88, ITS2 and LSU (approximately 1200 bases of the 5’ end). Two datasets were analysed separately: an LSU dataset only including the LSU region, and an ITS dataset including ITS1, 5.8S and ITS2. We used the automated best-fit tests implemented in PAUP* 4.0a (Swofford 2002) to select optimal substitution models for each complete, non-partitioned dataset (PHYML) and optimal substitution model partitions for each minimal partition (BEAST). Models and partitions were chosen based on BIC score for the BEAST analysis and AICc score for the PHYML analysis. All tests were conducted using three substitution schemes and evaluated substitution models with equal and gamma- distributed among-site rate variation. The tests for the PHYML analysis also evaluated substitution models with invariant sites. The following partitions and models had the highest ranking, according to BIC: ITS1+ITS2 (GTR+G), 5.88 (K80+G), LSU (GTR+G). According to AICc the GT R+I+G model provided the best fit for both the ITS and the LSU datasets. To generate Bayesian phylogenetic trees (BI) from the alignments we used BEAST 2.4.7 (Bouckaert et al. 2014). We prepared the xml-files for the BEAST 2 runs in BEAUTI 2.4.7 (Bouckaert et al. 2014). We set the substitution model to GITR+G for the LSU run. In the ITS run we set it to HKY+G for 5.8S, since it is the most similar model to K80+G available in the program. Test runs revealed convergence problems due to insufficient data for some substitution rates in the GIR+G model initially used for the ITS1+ITS2 partition, and it was hence changed to HKY+G. In the ITS run the substitution rate of both partitions were estimated independently. We set the trees of the minimal nrDNA partitions as linked in this analysis and the clock models as un- linked. A lognormal, relaxed clock model was assumed for each partition, as test runs had shown that all partitions had a coefficient of variation well above 0.1 (i.e. implying a relatively high rate variation among branches). ‘The clock rate of each partition was estimated in the runs, using a lognormal prior with a mean set to one in real space. We set the growth rate prior to lognormal, with a mean of 5 and a standard deviation of 2. We ran the Markov Chain Monte Carlo (MCMC) chains of both datasets for 20 million generations with tree and parameter files sampled every 1,000 generations. The analyses all converged well in advance of the 10 % burn-in threshold, had ESS values well above 200 for all parameters, and chain mixing was found to be satisfac- tory as assessed in TRACER 1.6.0 (Rambaut et al. 2014). After discarding the burn-in trees, maximum clade credibility trees were identified by TREEANNOTATOR 2.4.7 (Bouckaert et al. 2014). To generate Maximum Likelihood (ML) gene trees we used PHYML 3.1 (Guin- don et al. 2010). We set the substitution model to GT R+I+G for both the ITS and LSU datasets. Tree topology search was conducted using NNI+SPR, with ten random starting trees. Non-parametric bootstrap analyses with 1000 replicates were performed on the resulting trees. Reassessment of the generic limits for Hydnellum and Sarcodon oF Results Seventy-five Thelephorales specimens from the genera Amaurodon, Bankera, Boletopsis, Hydnellum, Lenzitopsis, Phellodon, Pseudotomentella, Sarcodon, Thelephora, Tomentella, and Yomentellopsis, were sequenced for this study. In addition, 39 sequences were downloaded from public databases (GenBank, UNITE) including outgroup sequences of Steccherinum ochraceum (Polyporales) and Sistotrema brinkmannii (Cantharellales) included in the LSU dataset. The ITS analyses were rooted by the default method (BEAST) or left unrooted (PHYML). The aligned LSU dataset consisted of 1443 nucleotide positions. After exclusion of ambiguous regions 1377 positions remained for the analyses. BI returned a tree where the focus genera Hydnellum and Sarcodon are distributed over two strongly supported clades. The larger of these clades includes the type of Hydnellum, H. suaveolens, and an additional 17 species, all except one forming strongly supported terminal clades. Nine of these taxa are currently placed in Sarcodon. With a few exceptions the relationships within Hydnellum are not resolved. H. aurantiacum and H. auratile are recovered as a strongly supported group; Sarcodon scabrosus and S. fennicus are grouped with 0.97 posterior probability support; S. fuligineoviolaceus, S. glaucopus, and S. joeides form a subclade with 0.97 posterior probability support; and finally H. suaveolens and S. versipellis form a strongly supported clade. The type of Sarcodon, S. imbricatus, and three other species form the second main clade. The three sequences of S. imbricatus cluster together but the clade is unsupported. Hydnellum and Sarcodon are recovered as sister clades but the support for this arrangement is weak. For target taxa the ML tree is essentially similar to the BI tree with strong support for the similarly composed Hydnellum and Sarcodon clades (Fig. 2). As for the BI analysis the relationships among species within Hydnellum and Sarcodon are not resolved except for a weak to moderate support for grouping H. aurantiacum with H. auratile and H. suaveolens with S. versipellis. S. fuligineoviolaceus, S. glaucopus, and S. joeides also group together in the ML tree but without support. Again S. imbricatus does not get support and is not separated from S. quercinofibulatus. The aligned ITS dataset consisted of 1068 nucleotide positions of which 505 remained for the analyses after removal of ambiguous regions. Bayesian inference produced a tree with two strongly supported clades (Fig. 3). The smaller one, which we here informally call “Neosarcodon”, contains nine Sarcodon species, all with a distribution in the tropical and subtropical Americas. Remaining Hydnellum and Sarcodon taxa, including both type species, formed the other clade. Within the latter clade two subclades are visible, corresponding to the genera Hydnellum and Sarcodon, and with the same delimitation as in the LSU trees. Only the Sarcodon subclade has strong support. Within each larger clade several groups of taxa received moderate to strong support. The reader is referred to Fig. 2 for further details. The ML tree recovered the same two main clades with strong support but could not resolve the relationships within the larger Hydnellum/Sarcodon clade. In the ML 38 Karl-Henrik Larsson et al. / MycoKeys 54: 31-47 (2019) Hydnellum aurantiacum MK602711 0.99/87 Hydnellum aurantiacum MK602712 87 Hydnellum aurantiacum MK602713 Hydnellum auratile MK602713 Hydnellum auratile MK602714 Hydnellum auratile MK602715 99 Sarcodon scabrosus MK602764 Sarcodon scabrosus MK602766 Sarcodon scabrosus MK602765 0.97/- Sarcodon fennicus MK602739 ‘ Sarcodon fennicus MK602738 Sarcodon fennicus MK602737 Sarcodon lundellii MK602758 Sarcodon lundellii MK602759 Sarcodon lundellii MK602760 86 Sarcodon glaucopus MK602743 Sarcodon glaucopus MK602744 Sarcodon glaucopus MK602745 Sarcodon fuligineoviolaceus MK602740 0.97/- Sarcodon fuligineoviolaceaus MK602741 r 84 Sarcodon fuligineoviolaceus MK602742 Sarcodon joeides MK602749 Sarcodon joeides MK602750 91! Sarcodon joeides MK602751 Hydnellum mirabile MK602728 Hydnellum mirabile MK602729 Hydnellum mirabile MK602730 Sarcodon lepidus MK602753 Sarcodon lepidus MK602752 Sarcodon lepidus MK602754 Hydnellum gracilipes MK602726 Hydnellum gracilipes MK602727 Sarcodon martioflavus MK602763 Sarcodon martioflavus MK602762 Sarcodon martioflavus MK602761 Hydnellum caeruleum MK602717 Hydnellum caeruleum MK602718 Hydnellum caeruleum MK602719 Hydnellum geogenium MK602723 Hydnellum geogenium MK602724 Hydnellum geogenium MK602725 90 Hydnellum peckii MK602731 97 Hydnellum peckii MK602732 Hydnellum peckii MK602733 Hydnellum suaveolens MK602734 Hydnellum suaveolens MK602735 74 Hydnellum suaveolens MK602736 Sarcodon versipellis MK602771 Sarcodon versipellis MK602772 Sarcodon versipellis MK602770 0.93/- Hydnellum ferrugineum MK602720 . Hydnellum ferrugineum MK602721 Hydnellum ferrugineum MK602722 99 Sarcodon leucopus MK602755 Sarcodon leucopus MK602756 Sarcodon leucopus MK602757 Sarcodon quercinofibulatus MK602773 Sarcodon imbricatus MK602748 97 Sarcodon imbricatus MK602746 Sarcodon imbricatus MK602747 Sarcodon squamosus MK602768 Sarcodon squamosus MK602767 97' Sarcodon squamosus MK602769 Boletopsis leucomelaena MK602710 98 Lenzitopsis daii JN169799/JN169793 Lenzitopsis oxycedri MK602774 99 -/7) Thelephora caryophyllea MK602776 75 Thelephora terrestris MK602777 Tomentella stuposa MK602778 Odontia fibrosa MK602775 0.98/96 Pseudotomentella flavovirens MK602780 : 77,— Bankera fuligineoalba MK602708 98 Bankera violescens MK602709 Phellodon cf niger MK602782 -/82 Phellodon tomentosus MK602781 Tomentellopsis pulchella MK602779 Amaurodon viridis MK602707 Amaurodon aquicoeruleus AM490944 Steccherinum ochraceum JQ031130 Sistotrema brinkmannii KF218967 Hydnellum Sarcodon 0.1 Figure 2. Maximum likelihood analyses of LSU dataset for Thelephorales. Branches in bold have a posterior probability value of 1 in Bayesian inference and 100% bootstrap support in ML analysis, if not otherwise indicated by a figure. Lower support values on other branches are indicated by figures. Steccherinum ochraceum and Sistotrema brinkmannii are used as outgroup (branch lengths shortened). Reassessment of the generic limits for Hydnellum and Sarcodon -/70 1/99 0.99/- 1/76 1/99 0.98/- 1/98 1/96 0.99/78 1/94 1/100 0.97/- 0.96/61 0.98/74 = 0.92/na 0.97 1/na /52 1/87 1/98 1/69 0.98/81 0.94/61 1/100 1/98 0.05 length units Sarcodon fuligineoviolaceus MK602740 Sarcodon fuscoindicus EU669228 Sarcodon joeides KC571772 Sarcodon joeides MK602751 Sarcodon glaucopus MK602745 Hydnellum mirabile MK602728 Hydnellum peckii MK602733 Hydnellum regium AY569031 Hydnellum gracilipes MK602727 Hydnellum piperatum JN135173 Hydnellum cristatum JN1351 74 Sarcodon lundellii MK602760 Sarcodon versipellis MK602771 Hydnellum suaveolens MK602735 Hydnellum cumulatum AY569026 Hydnellum ferrugineum MK602721 Hydnellum spongiosipes JN135184 Hydnellum pineticola KC571734 Hydnellum diabolus AF351863 Hydnellum geogenium MK602723 Hydnellum cyanopodium AY569027 Hydnellum scleropodium JN135186 Hydnellum caeruleum MK602719 Hydnellum ferrugipes KC571727 Sarcodon martioflavus MK602762 Hydnellum concrescens EU/84267 Hydnellum subsuccosum JN135178 Hydnellum scrobiculatum JN135181 Hydnellum dianthifolium KX619419 Sarcodon lepidus MK602753 Sarcodon fennicus MK602737 Sarcodon scabrosus MK602766 Sarcodon amygdaliolens JN376763 Sarcodon underwoodii KC571781 Hydnellum aurantiacum MK602711 Hydnellum earlianum JN135179 Hydnellum auratile MK602714 Hydnellum complicatum KC571711 Sarcodon imbricatus MK602747 Sarcodon squamosus MK602767 Sarcodon quercinofibulatus JX271818 Sarcodon aspratus DQ448877 Sarcodon scabripes JN135191 Sarcodon leucopus MK602755 Sarcodon atroviridis JN135190 Sarcodon atroviridis KC571768 Sarcodon portoricensis KM668100 Sarcodon quercophilus KM668101 Sarcodon umbilicatus KM668102 Sarcodon rufobrunneus KR698937 Sarcodon pakaraimensis KM668103 Sarcodon columbiensis KP972654 Sarcodon bairdii KR698938 Sarcodon pallidogriseus KR698939 59 Hydnellum Sarcodon “Neosarcodon” Figure 3. Ultrametric default rooted BEAST tree of ITS dataset for Hydnellum and Sarcodon. Posterior probability values and bootstrap percent support from ML analysis are indicated by figures; na = not applicable. 40 Karl-Henrik Larsson et al. / MycoKeys 54: 31-47 (2019) tree the clade corresponding to Hydnellum in the LSU tree is correctly identified but not supported while the clade corresponding to Sarcodon appears polyphyletic. Based on these results we hereby revise the limits of the two genera by moving a number of species from Sarcodon to Hydnellum. Consequently the genus description for Hydnellum must be emended while the genus description for Sarcodon can remain unaltered. Taxonomy Hydnellum P.Karst., Meddn Soc. Fauna Flora fenn. 5: 41 (1879). Type species. Hydnellum suaveolens (Scop.:Fr.) P.Karst. (1879) Basionym. Hydnum suaveolens Scop.:Fr. (1772) Basidiomata with pileus and stipe, single or concrescent; pileus thin to thick, at first smooth and velutinous, when mature felted, fibrillose, scaly, ridged, or irregularly pitted and scrupose, mostly brownish but also with white, olive yellowish, orange, pur- plish or bluish colours, often concentrically zonate; stipe narrow to thick, solid, mostly short; hymenophore hydnoid, usually strongly decurrent; context from soft and brittle to corky or woody; hyphal system monomitic, septa with or without clamps, context hyphae inflated or not; cystidia lacking; basidia narrowly clavate, producing four ster- igmata; basidiospores with irregular outline, more or less lobed, verrucose, brownish. Terrestrial, forming ectomycorrhiza with forest trees. Hydnellum amygdaliolens (Rubio Casas, Rubio Roldan & Catala) E.Larss., K.H.Larss. & Kéljalg, comb. nov. MycoBank No.: MB830570 Basionym. Sarcodon amygdaliolens Rubio Casas, Rubio Roldan & Catala, Boln Soc. Micol. Madrid 35: 44-45. 2011. Holotype: Spain, Tamajén, Barranco la Jara. L. Rubio-Casas & L. Rubio-Roldan, AH 42113. Hydnellum fennicum (P.Karst.) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830571 Basionym. Sarcodon scabrosus var. fennicus P.Karst., Bidr. Kann. Finl. Nat. Folk 37: 104. 1882. Type: not indicated (neotype: H, designated by Maas Geesteranus & Nannfeldt 1969: 406) Reassessment of the generic limits for Hydnellum and Sarcodon 41 Hydnellum fuligineoviolaceum (Kalchbr.) E.Larss., K.H.Larss. & K6ljalg, comb. nov. MycoBank No.: MB830572 Basionym. Hydnum fuligineoviolaceum Kalchbr., in Fries, Hymenomyc. eur. (Upsaliae): 602. 1874. Holotype: Slovakia, Presovsky kraj, Olaszi. C. Kalchbrenner, UPS F-173546. Hydnellum fuscoindicum (K.A.Harrison) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830573 Basionym. Hydnum fuscoindicum K.A.Harrison, Can. J. Bot. 42: 1213. 1964. Holotype: USA, Washington, Olympic Nat. Park, A.-H. Smith. MICH 10847. Hydnellum glaucopus (Maas Geest. & Nannf.) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830574 Basionym. Sarcodon glaucopus Maas Geest. & Nannf., Svensk bot. Tidskr. 63: 407. 1969. Holotype: Sweden, Uppland, Borje par., J. Eriksson. UPS F-013955. Hydnellum joeides (Pass.) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830575 Basionym. Hydnum joeides Pass., Nuovo G. bot. ital. 4: 157. 1872. Holotype: Italy, Emilia-Romagna, Collecchio, G. Passerini. PAD. Hydnellum lepidum (Maas Geest.) E. Larss., K.H.Larss. & Kéljalg, comb. nov. MycoBank No.: MB830576 Basionym. Sarcodon lepidus Maas Geest., Verh. K. ned. Akad. Wet., tweede sect. 65: 105. 1975. Holotype: The Netherlands, Lochem, Ampsen, G. & H. Piepenbroek. L. Hydnellum lundellit (Maas Geest. & Nannf.) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830577 Basionym. Sarcodon lundellii Maas Geest. & Nannf., Svensk bot. Tidskr. 63: 421. 1969. Type: Sweden, Uppland, Storvreta, S. Lundell & J.A. Nannfeldt, distributed 42 Karl-Henrik Larsson et al. | MycoKeys 54: 31-47 (2019) in S. Lundell & J.A. Nannfeldt Fungi exs. suec. as number 252 (lectotype, designated here, UPS F-010975; MycoBank No.: MBT387081). The UPS herbarium has two copies of the exsiccate and the specimens of H. /undellii are registered as F-010975 and F-013956, respectively. From F-010975 an ITS2 sequence has been generated [Gen- Bank MK753037] and this specimen is here selected as lectotype). Hydnellum martioflavum (Snell, K.A.Harrison & H.A.C.Jacks.) E.Larss., K.H.Larss. & Kéljalg, comb. nov. MycoBank No.: MB830578 Basionym. Hydnum martioflavum Snell, K.A.Harrison & H.A.C.Jacks., Lloydia 25: 161. 1962. Holotype: Canada, Quebec, Ste Anne de la Pocatiére, H.A.C. Jackson & W.H. Snell 13 Sep. 1954, BPI 259438. Hydnellum scabrosum (Ft.) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830579 Basionym. Hydnum scabrosum Fr., Anteckn. Sver. Atl. Svamp.: 62. 1836. Type: not indicated (neotype: Sweden, Smaland, Femsjé, S. Lundell, UPS F-013954, designated by Maas Geesteranus & Nannfeldt 1969: 426) Hydnellum underwoodii (Banker) E.Larss., K.H.Larss. & Koljalg, comb. nov. MycoBank No.: MB830580 Basionym. Sarcodon underwoodii Banker, Mem. Torrey bot. Club 12: 147. 1906. Holotype: USA, Connecticut, NY 776131. Hydnellum versipelle (Fr.) E.Larss., K.H.Larss. & Koéljalg, comb. nov. MycoBank No.: MB830581 Basionym. Hydnum versipelle Fr., Ofvers. K. Svensk. Vetensk.-Akad. Férhandl. 18(1): 31. 1861. Type: not indicated (neotype: Sweden, Uppland, Danmark par., J. Eriksson & H. Nilsson, UPS F-013958, designated by Maas Geesteranus & Nannfeldt 1969: 430) Sarcodon Quél. ex P.Karst., Revue mycol., Toulouse 3 (no. 9): 20 (1881). Type species. Sarcodon imbricatus (L.:Fr.) P.Karst. (1881) Basionym. Hydnum imbricatum L.:Fr. (1753). Reassessment of the generic limits for Hydnellum and Sarcodon 43 Basidiomata with pileus and stipe, single or concrescent; pileus thin to thick, at first smooth and velutinous, when mature smooth or scaly, brownish; stipe thick, solid, mostly short; hymenophore hydnoid, usually strongly decurrent; context soft and brittle; hyphal system monomitic, septa with clamps, context hyphae inflated; cystidia lacking; basidia narrowly clavate, producing four sterigmata; basidiospores with irregular outline, more or less lobed, verrucose, brownish. Terrestrial, forming ectomycorrhiza with forest trees. Discussion In this paper we show that the current morphology-based concepts of Sarcodon and Hydnellum do not correspond to monophyletic subgroups within the Thelephorales. The characters traditionally used to separate the two genera do not reflect true relationships. These characters, however, are vague and open to subjectivity; hence it is not surprising that they have now been shown to be unreliable. Maas Geesteranus (1975) pointed to the context structure and consistency as the main differentiating character. For Hydnellum he describes the context as “... fibrillose, soft or tough, corky to woody, more or less duplex, zoned, ...” and hyphae are said to be “...usually not inflating ...”. In Sarcodon the same structures are described as “... fleshy, brittle, soft or firm (never corky or woody), not duplex, not zoned ...” and “...hyphae inflating ...”. While these morphological characteristics remain true for Sarcodon, the corresponding descriptions for Hydnellum had to be emended. Instead of context structure it seems that average basidiospore size may in most cases offer a possibility to separate a Sarcodon species from one belonging to Hydnellum. Table 2 summarizes basidiospore measurements from the literature. Average basidiospore lengths in Hydnellum fall between 4.45 and 6.95 um while the same figures for Sarcodon are 7.4 and 9 um, ornamentation excluded. However, S. quercinofibulatus clearly deviates from this pattern. According to measurements in the protologue (Pérez-de-Gregorio et al. 2011) and in Vizzini et al. (2013) average basidiospore length was measured to 6.95 and 7.0, respectively, but then included the ornamentation. Measurements excluding ornamentation would be approximately 1 pm less. Clearly, for S. guercinofibulatus basidiospore length alone will not be decisive for genus placement. Not all sequences from species described as Sarcodon spp. were recovered within either Sarcodon or Hydnellum. In our ITS-only analyses nine species formed a well- supported clade of their own, separated from Sarcodon sensu stricto and Hydnellum (Fig. 3). This clade, here informally called “Neosarcodon’”, contains species collected in tropical and subtropical regions of the Western Hemisphere and may represent one or several distinct genera. However, further analyses based on an expanded dataset using more conservative molecular markers would be required to definitely identify any new higher taxa in the group. The failure to generate support for Sarcodon and Hydnellum in the ITS-only analyses reflects the large genetical distances present among the species within this 44 Karl-Henrik Larsson et al. | MycoKeys 54: 31-47 (2019) Table 2. Basidiospore measurements for Hydnellum and Sarcodon from the literature. Sources: B = Baird et al. (2013), M = Maas Geesteranus (1975), J = Johannesson et al. (1999). All measurements exclude ornamentation. For species treated in this paper names follow our new classification. For other species names are according to cited authors. Species Measurements Mean length Hydnellum aurantiacum (M) (5.8-)6-6.7 x (4-)4.3-4.9 635 Hydnellum auratile (M) 4.9-5.8 x 3.6-4.5 5235 Hydnellum caeruleum (M) 5.4-6(-6.3) x 3.4-4.3 5.70 Hydnellum compactum (Pers.:Fr.) P. Karst. (M) 5.4-6.3 x 3.6-4.5 5.85 Hydnellum complicatum (B) 4-5 x 3-5 4.50 Hydnellum concrescens (M) 5.4-6.1 x (3.6-)4-4.5 dep Hydnellum cristatum (B) 5-6 x 4-5 5.50 Hydnellum cruentum K.A.Harrison (B) 4-5 x 3-4 4.50 Hydnellum cumulatum (M) 4.3-5.6 x 3.6-4.3 4,95 Hydnellum diabolus (B) 6-7 x 5-6 6.50 Hydnellum earlianum (B) 5=6:% 425 5.50 Hydnellum fennicum (M) 6.3-7.6 x 4.5-5.2 6.95 Hydnellum ferrugineum (M) (5.4-)5.8-6.3 x 3.6-4.5 6.05 Hydnellum ferrugipes (B) 5-7 x 5-6 6.00 Hydnellum fuligineoviolaceum (M) 5.4-6.5 x 4-4.7(-5.4) 5:95 Hydnellum geogenium (M) 4,5-5.2 x 3.1-3.6 4.85 Hydnellum glaucopus (M) (5-)5.4-5.8(-6.3) x (3.6-)4-4.5 5.60 Hydnellum gracilipes (M) 4.3-4.6 x 2.7-3.6 4.45 Hydnellum joeides (M) 5.4-5.8 x 3.6-4.2 5.60 Hydnellum lepidum (M) 5.8-6.3 x 3.6-4.3 6.05 Hydnellum lundellii (M) 4.9-5.8 x 3.6-4.2 5.35 Hydnellum martioflavum (M) 5-6.3 x 3.6-4.5 5.65 Hydnellum peckii (M) 4.9-5.4 x 3.8-4 5215 Hydnellum pineticola (B) 5-7 x 4-6 6.00 Hydnellum piperatum (B) 4-6 x 4-5 5.00 Hydnellum scabrosum (M) (5.4-)6.3-7.3 x (3.6-)4-5 6.80 Hydnellum scleropodium (B) 4-6 x 3-4 5.00 Hydnellum spongiosipes (B) 6-7 x 5-6 6.50 Hydnellum suaveolens (M) 4-5 x 3-3.6 4.50 Hydnellum subsuccosum (B) 5-6 x 4-6 5.50 Hydnellum versipelle (M) 4.5-5.5 x 3.5-4.5 5.00 Hydnellum underwoodii (B) 5-7 x 5-6 6.00 Sarcodon atroviridis (B) 8-9 x 7-8 8.50 Sarcodon excentricus R.E.Baird (B) 8-9 x 6-8 8.50 Sarcodon harrisonii R.E.Baird (B) 7-9 x 6-8 8.00 Sarcodon leucopus (M) (6.7-)7.2-7.6(-9) x 4.5-5.6 7.40 Sarcodon imbricatus (M) 7.2-8.2 x 4.9-5.4 7.70 Sarcodon scabripes (B) 8-10 x 7-9 9.00 Sarcodon squamosus (J) 7.2-8.2 x 4.9-5.4 7.70 marker. Our general experience with the ITS region for thelephoralean target genera is that species are extremely well separated and the internal variation surprisingly low, even when a large number of specimens from both Europe and America are considered. On the other hand, the genetical difference among species is moderate to high, making alignments difficult and prone to ambiguities. In our ITS analyses we chose to remove ambiguous regions, thus halving the number of nucleotide positions suggested by Reassessment of the generic limits for Hydnellum and Sarcodon 45 automatic alignment through MAFFT. This seems to have affected the ML analyses most. However, the ITS analyses only served to position neotropical Sarcodon species and the results clearly show that they belong to a separate lineage. Otto (1997) suggested that Aydnum auratile is a later synonym of Hydnum aurantiacum and that the species we now call Hydnellum aurantiacum should be named Hydnellum floriforme (Schaeff.) Banker. The name change is based on a reinterpretation of Batsch’s original illustration, which, according to Otto, clearly shows the same species as Hydnum auratile. In phylogenetic analyses H. aurantiacum and H. auratile are sister taxa and during our study we have sequenced several specimens identified as H. auratile that turned out to be H. aurantiacum. Thus separating these species can be hazardous and to interpret illustrations must be even harder. We currently do not accept this unfortunate name change. The present study will serve as the basis for further exploration of species limits within Hydnellum and Sarcodon. As has been demonstrated for the genera, many species interpretations are in need of revision. Over the years we have found numerous specimen misidentifications as well as specimens that could not be assigned to pre- existing names. A closer inspection of the ITS tree in Fig. 3, where we let the terminals retain the identifications given in GenBank, shows some examples. The American sequence of Sarcodon joeides (KC571772) does not cluster with the European representative of the same species (MK602751) and the American sequence named Hydnellum earlianum seems to be identical to what is in Europe called H. auratile. Considering that many stipitate hydnoid species are red-listed and used as indicators of forests in need of conservation (Ainsworth 2005, Nitare 2019), it is of utmost importance to sort out the taxonomy of these species. Acknowledgements This study was supported by grants from ArtsDatabanken, Norway, to KH Larsson (ADB54-09), from Artdatabanken, Sweden, to E Larsson (2014-152 4.3), and from Estonian Research Council to U Koljalg (IUT20-30). We also acknowledge support to S Svantesson from Kungliga Vetenskaps- och Vitterhetssamhallet i Goteborg and from Kapten Carl Stenholms donatationsfond. We are grateful to many dedicated mycologists in Norway, Sweden and Finland for sending valuable collections. 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