Mycol Progress (2012) 11:449–462 DOI 10.1007/s11557-011-0760-3

ORIGINAL ARTICLE

Three new Tomentella species from West Africa identified by anatomical and molecular data Nourou Soulemane Yorou & Sebastian Gardt & Marie-Laure Guissou & Moussa Diabaté & Reinhard Agerer

Received: 7 January 2011 / Revised: 18 March 2011 / Accepted: 1 April 2011 / Published online: 20 April 2011 # German Mycological Society and Springer 2011

Abstract We used a combination of molecular-phylogenetic inference of 82 ITS rDNA sequences and anatomical approach to describe three new west African thelephoroid species, namely Tomentella afrostuposa, T. guineensis and T. guinkoi. Anatomically, T. afrostuposa is reminiscent of T. stuposa with globose to broadly ellipsoid large basidiospores of 8–14 μm, long aculei of up to 3 μm and prominent apiculi of 2 μm width. Molecular-phylogenetically, it falls within the T. stuposa complex. However, T. afrostuposa deviates by at least 7.80–10.74% from T. stuposa in regard with the ITS rDNA sequences. Tomentella guineensis is characterised by long (up to 85 μm) utriform basidia, the presence of reniform basidiospores in lateral view (up to 9 μm) with aculei not exceeding 1 μm and a strong cyanescent reaction of the subhymenial hyphae and basidia in 2.5% KOH. It forms a sister species of the newly described species Tomentella maroana; however, deviating from the last species by at least 9.75–10.04%. The very short, inflated (up to 14 μm) and thick-walled septate (septa up to 1.5 μm) subhymenial hyphae combined with ellipsoid basidiospores N. S. Yorou (*) : S. Gardt : R. Agerer Department for Biology I, Division of Organismic Biology: Mycology, Ludwig-Maximilians-Universität München, Menzinger Str. 67, 80638 Munich, Germany e-mail: [email protected] M.-L. Guissou École Normale Supérieure, Université de Koudougou, BP 376 Koudougou, Burkina Faso M. Diabaté Herbier Serg. de Guinée, Centre Régional de Recherche Agronomique pour la Guinée Forestière CRRA Sérédou / IRAG/CRA, BP 1523 Conakry, Guinée

(up to 8 μm) and short aculei not exceeding 0.5 μm characterise Tomentella guinkoi. Anatomically, T. guinkoi recalls T. ellisii. Genetic distance between both species ranges from 12.67 to 13.73% according to ITS rDNA sequences analyses. Tomentella guinkoi forms a sister species of the group composed of T. ellisii, T. hjortstamiana and T. pisoniae. Detailed anatomical comparisons between the newly described species and their close relatives are given. Keywords Anatomy . Sequences analyses . Molecular-phylogeny . SEM micrographs . Species concept . Tomentella

Introduction Resupinate Thelephorales (Amaurodon J. Schröt., Tomentellopsis Hjortstam, Pseudotomentella Svrček and Tomentella Persoon ex. Pat. (Kõljalg 1996) belong to the Class Agaricomycetes (Basidiomycota, Fungi) and have a worldwide distribution (Cunningham 1957; Kõljalg 1996; Larsen 1965, 1967, 1968, 1974; Malençon 1952; Martini and Hentic 2002, 2005; Melo et al. 1998, 2000, 2002, 2003, 2006; Patouillard 1897; Stalpers 1993; Wakefield 1966, 1969; Yorou 2008). However, there is still no record of the genus Tomentellopsis from tropical Africa and its affiliated islands (Yorou 2008; Suvi et al. 2010). Martini and Hentic (2002) reported on one Pseudotomentella species from Gabon (Central Africa) whilst numerous Tomentella species have been documented from West Africa (Yorou et al. 2007; Yorou and Agerer 2007, 2008; Yorou et al. 2011a,b) and from the Seychelles (Suvi et al. 2010). Resupinate Thelephorales count many ectomycorrhizal species worldwide (Agerer 1987; Agerer 2006; Agerer 2006; Kõljalg et

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al. 2000, 2001; Suvi et al. 2010; Erland and Taylor 1999; Yorou et al. 2008a,b; Diedhiou et al. 2004, 2010). As tropical Africa hosts ectomycorrhizal tree species that are different from temperate and boreal ones, it is more likely that many new thelephoroid species still remain to be described from this area. The present paper reports on three new Tomentella species collected in northern Guinean seasonal forests of Burkina Faso and Guinea (West Africa). Not only does it complete recent documentations on resupinate Thelephorales from Benin (Yorou 2008) and Togo (Yorou et al. 2011a) but it confirms the commonness of this fungal group in tropical Africa.

Materials and methods Specimen sampling, light microscope studies and drawings Investigated specimens were collected in summer 2009 in different vegetation types of the northern Guinean seasonal forests (White 1983) of Burkina Faso and Guinea (West Africa). Preliminary notes are recorded using fresh material, which was dried by mean of a propane gas-heated field dryer (De Kesel 2001). Type materials were then deposited under following herbarium labels SYN 2235, SNY 2292 and SYN 2331. Colour codes of the dried basidiocarps are given according to Kornerup and Wanscher (1978). All specimens used for descriptions are deposited in M (Holmgren et al. 1990). We refer to Yorou and Agerer (2007, 2008, 2011a, b), Yorou et al (2011a, b) for protocols of light microscopy and scanning electron microscopy. DNA Extraction, amplification and sequencing Total DNA was extracted from tiny pieces (about 0.5 mm × 0.5 mm) of dried basidiocarps using 1:10 scale of the ChargeSwitch® gDNA Plant Kit (Invitrogen, Darmstadt, Germany) as recommended by the manufacturer. The ITS region (ITS1, 5.8 S, and ITS2 rRNA gene) was amplified using the primer pairs ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). Incredients were added as recommended by the manufacturer of the Taq polymerase (Invitrogen) and sterile distilled water was added to obtain a final reaction volume of 25 μL. The PCR started by an initial denaturation step (94°C, 2 min). This first step was followed by 5 cycles of denaturation (94°C, 0.5 min), primer annealing (52°C, 1 min), elongation (72°C, 2 min), another 30 cycles with a decreased annealing temperature (50°C), and a final elongation (72°C, 5 min). Addition of 17.5 μL 100% isopropanol and 2.5 μL 5 M NaCl served to precipitate the amplicons overnight at room temperature. Following centrifugation and re-suspension, the ITS region

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was sequenced through the sequencing service of the Division of Genetics (Department of Biology, LudwigMaximilians-University, München), using an ABI 3730 capillary sequencer. A consensus sequence of the forward and reverse strands, obtained by applying the PCR primers, was created by using the Staden software package (Staden 1996). All three generated sequences are deposited in GenBank (NCBI) under accession numbers JF520430, JF520431 and JF520432. Sequence edition and phylogenetic analyses The sequences were edited and analysed with BioEdit v7.0.5 (Hall 2005) as described in Yorou et al (2011a, b). The most similar sequences of each generated consensus sequence were searched for in UNITE (Abarenkov et al. 2010; Kõljalg et al. 2005, http://unite.ut.ee) using the “BLASTN” search option. The search was extended to the GenBank EMBL and DDJB by activating the UNITE+ INSD. option. The 15 best matches identified to species level (unidentified environmental samples are disregarded) were downloaded. Similar search has been undertaken in the GenBank NCBI (http://www.ncbi.nlm.gov) using the “Megablast search option” (Zhang et al. 2000). The top 100 best matches have been examined. The most similar sequences of completely identified taxa obtained from both UNITE and NCBI were completed by that one published by Yorou and Agerer (2007, 2008), Yorou et al. (2007, 2011a, b) from West Africa and those from the Seychelles (Suvi et al. 2010). Sequences were automatically aligned in BioEdit v7.0.5 and afterwards manually optimised. The final dataset included a total of 82 ITS rDNA thelephoroid sequences with a length of 500 characters. The best scoring tree was obtained by running a fast bootstrap search of 100 pseudoreplicates using the program RAxML v.7.0.4 (Stamatakis et al. 2008). Details of the molecularphylogenetic analyses are given in Yorou et al (2011a,b).

Results ITS rDNA nucleotides-based analyses of the new species According to BLASTN search in UNITE + INSD., Tomentella stuposa (Link) Stalpers is the most similar species to T. afrostuposa sp. nov. Eight sequences from the 15 best matches are composed of T. stuposa. At the opposite, the 15 best matches of Tomentella guineensis sp. nov. are unidentified/uncultured Tomentella/Thelephoraceae. The highest similarity rate (96%, query coverage 100%) is obtained with an unidentified ectomycorrhizae (accession number AM113445) sampled in Guinea. As far as specimen Tomentella guinkoi sp. nov. is concerned, 12 of

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the 15 most similar sequences are composed of unidentified thelephoroid species. Three sequences (DQ974775, DQ068971 and UDB000219) identified as T. ellisii (Sacc.) Juel & Stalpers range among the 15 best matches. The BLASTN searches in NCBI display similar results. Among the 100 best matches, T. stuposa (two sequences EU819501 and EU819523) is the unique identified most similar species (similarity=92%, query coverage 100%, E-value= 0.0) to T. afrostuposa. For T. guinkoi, highest similarity rates (93%, query coverage 100%, E-value =0.0) are obtained with two unidentified thelephoroid taxa (AB444649, FJ013054). For this specimen, the most similar taxa completely identified are Thelephora terrestris Ehrh. (92% similarity, query coverage 100%, E-value=0.0) and two sequences of T. ellisii (AF272913 and DQ068971) with identity rates of 89%. Three sequences (EF507250, EF507251 and EF507252) assigned to T. maroana Yorou from Benin (Yorou et al. 2011a) present identity rates of 90%. T. maroana is followed by four additional representatives of Thelephora terrestris (EU427330, DQ068970, FJ809999 and GQ267490) with identity rates lower than 90%. Phylogenetic placement of the new species The proportion of gaps in the final dataset is estimated at 1.01%. The best scoring tree found has a maximal likelihood of ML = −2,332.397746 (tree length = 1.687930). Substitution rates have been estimated as follow: A↔C: 1.199787, A↔G: 2.450885, A↔T: 1.419037, C↔G: 1.080095, C↔T: 4.390512, G↔T: 1.000000 with following base frequencies: freq (A): 0.257810, freq (C): 0.246679, freq (G): 0.255337 and freq (T): 0.240175. The topology and bootstrap supports of major clades of the best scoring tree (Fig. 1) are very similar to the ones published in Yorou et al (2011a, b). Phylogenetically, Tomentella guinkoi forms a sister species of the group comprising T. ellisii (DQ974775 and UDB 000219), Tomentella hjortstamiana Suvi & Kõljalg (AM412303) and T. pisoniae Suvi & Kõljalg (FM244908). All four species form a well-supported monophyletic group with a strong bootstrap support of 95% whilst the clade composed of T. ellisii, T. hjortstamiana and T. pisoniae is supported by 84% bootstrap value. Based on sequence analyses and detailed anatomical comparison, T. guinkoi is described below as a new species. Tomentella guineensis forms with T. maroara a well-supported clade with 84% bootstrap value. In this clade, all three sequences assigned to T. maroana cluster together with 99% bootstrap support. Detailed sequence analyses and anatomical comparison between both species suggested the description of Tomentella guineensis as a new species. As far as Tomentella afrostuposa is concerned, it falls within the T. stuposa

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complex, forming a terminal clade with one sequence assigned to T. stuposa (EU819523) with a good bootstrap support of 87%. A sister clade of both specimens is composed of four representatives of T. stuposa that all cluster together with 85% bootstrap value. The description of T. afrostuposa as a new species is justified by sequence analyses and anatomical comparison (see discussion). Description of the new species Tomentella afrostuposa Yorou sp. nov. (Figs. 2, 3 and 4) Mycobank MB560135, GenBank NCBI, accession number JF520431 Basidiocarpa resupinata, crassa, ad 1 mm alta, separabilia, arachnoidea, continua. Hymenium subtile granulosum, continuum, badium usque ad obscurobadium. Subiculum arachnoideum, coloratum ut hymenium, marginibus sterilibus indeterminatis, coloratis ut subiculum. Rhizomorphae absentes. Hyphae subiculi fibuligerae, 3– 5.5(6) μm in diametro, in aqua et in 2.5% KOH nunnullae densis incrustationibus, cyanescentes. Hyphae subhymenii fibuligerae, 5–6(8) μm in diametro, interdum crassitunicatae (0.5–1 μm). Cystidia absentia. Basidia fibuligera, 45– 65(70) μm longa, apicibus 9–14(16) μm, basibus 6.5– 7.5 μm, utriformia, non stipitata, plerumque sinuosa, 4sterigmatica, sterigmatibus 9–15 μm longis, basaliter 2– 4 μm latis. Basidiosporae (8)9–13(14) × (8)9–12(13) μm in aspectu frontali, (8.5)9–12(13) × (8)9–12(13) μm in aspectu laterali, globosae usque ad late ellipsoideae in aspectu frontali, globosae, infrequenter elongatae in aspectu laterali, echinulatae; aculeis longissimis, conicis, interdum basibus furcatis, apiculo magno, 2 × 2 μm. Chlamydosporae absentes. Basidiocarp resupinate, thick, up to 1 mm, separable from the substrate, arachnoid, continuous. Hymenophore finely granulose, continuous, hymenium brown to dark brown (7 F6 to 7 F7), subiculum arachnoid, concolorous with the hymenium, sterile margin indeterminate, concolorous with the subiculum. Rhizomorphs absent. Subicular hyphae clamped, simple septa absent, 3–5.5(6) μm wide, usually regular, commonly sinuous in 2.5% KOH, rarely inflated in 2.5% KOH, cross-shape branching and anastomoses rare, some thin-walled (0.2–0.5 μm), others thick-walled (0.5–1 μm), light brown to brown in water and in 2.5% KOH, some strongly encrusted in water and in 2.5% KOH, cyanescent, not congophilous, not cyanophilous, not amyloid. Subhymenial hyphae clamped, 5–6(8) μm wide, short not inflated, usually thin- (0.2–0.5 μm) sometimes thickwalled (0.5–1 μm), with internal walls detaching from the outer one, then up to 2 μm thick, some strongly encrusted in water and in 2.5% KOH, colourless to pale brown in

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Tomentella guinkoi JF520430 (Burkina Faso)

Tomentella guineensis JF520432 (Guinea)

Tomentella afrostuposa JF520431 (Guinea)

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ƒFig. 1

The Maximum Likelihood tree showing the placement of the new species within Tomentella species. Bootstrap values higher than 50% are shown above the branches. Databank (UNITE or NCBI) accession numbers and country of origin of selected species are indicated after species names. Black triangles indicate branches that have been collapsed in order to reduce the span of the tree

water and in 2.5% KOH, not cyanescent, slightly congophilous, not cyanophilous, not amyloid. Cystidia absent. Basidia clamped at base, 45–65(70) μm long, 9–14(16) μm at apex, 6.5–7.5 μm at base, utriform, not stalked, commonly sinuous, rarely with transverse septa, vesiclelike elements present, young basidia sometimes very large, up to 20 μm diam., immature basidia sometimes strongly encrusted in water and in 2.5% KOH (Fig. 2), basidia commonly with numerous oil droplets, colourless in water and in 2.5% KOH, not cyanescent, congophilous, cyanophilous, not amyloid, 4-sterigmate, sterigmata long and very large, 9–15 μm long and 2–4 μm at base (Fig. 3). Basidiospores (8)9–13(14) × (8)9–12(13) μm in frontal face, (8.5)9–12(13) × (8)9–12(13) μm in lateral face, globose to broadly ellipsoid in frontal view, globose, rarely elongated in longitudinal axis view (Fig. 4), echinulate, aculei very long, 1.5–3 μm, conical, rarely forked at their basis, apiculus large, 2 μm long and 2 μm large, basidiospores pale brown to brown in water and in 2.5% KOH, not cyanescent, not congophilous, not cyanophilous, not amyloid. Chlamydospores absent.

Fig. 2 Tomentella afrostuposa. Optical section of young basidia (from SYN 2292). Bar 10 μm

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Material studied and habitat Guinea, upper Guinea, Province of Kakan, Prefecture of Kouroussa, Forest Reserve of Baro, along the road between Kouroussa and Kankan, 10°57′0.92″N, 009°63′60.0″W, forest dominated by Caesalpiniaceae (Afzelia africana Smith and Anthonota crassifolia Baill.), Euphorbiaceae (Uapaca guineensis Müll. Arg.) and Dipterocarpaceae (Monotes kerstingii Gilg.), on dead valves of Afzelia africana, leg. and det. NS. Yorou, 18.07.2009, herb. holotype SYN 2292 (M), GenBank NCBI, accession number JF520431. Etymology In reference to the anatomical reminiscence of T. stuposa, a common ectomycorrhizal former in temperate and boreal forest ecosystems. Tomentella guineensis Yorou sp. nov. (Fig. 5) Mycobank MB560136, GenBank NCBI, accession number JF520432 Basidiocarpa resupinata, crassa, usque ad 1.5 mm alta, adherentia, crustosa, continua. Hymenium laeve usque ad minute granulosum, continuum, cinereobrunneum. Subiculum arachnoideum, tenebricius quam hymenium, subbrunneum usque ad subnigrum, marginibus sterilibus subindeterminatis, farinosis et albidis. Rhizomorphae absentes. Hyphae subiculi fibuligerae, 3.5–5.5(6) μm in diametro, plerumque crassitunicatae (0.5–1 μm), subcongophilae. Hyphae subhymenii fibuligerae, 3.5–6 μm in diametro, tenuitunicatae (0.2–0.5 μm), interdum cyanes-

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Fig. 3 Tomentella afrostuposa. a Section through the basidiocarp. b Basidiospores in frontal view. c Basidiospores in lateral view (from SYN 2292). Bar 10 μm

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Fig. 4 Tomentella afrostuposa. SEM of basidiospores (from SYN 2292). a Basidiospore in lateral view. b Basidiospores in a nearly frontal view. c Basidiospore in frontal view. d Basidiospore in proximal view. Bar 1 μm

centes, subcongophilae, subcyanophilae. Cystidia absentia. Basidia fibuligera, 40–75(85) μm longa, apicibus 6.5–9 (10) μm, basalibus 5.5–6 μm, utriformia, interdum cyanescentiora, congophila, cyanophila, 4-sterigmatica, sterigmatibus 5–8(10) μm longis, basaliter 1.5–2 μm latis. Basidiosporae (6.5)7–8.5(9) × (5)6–7(8) μm in aspectu frontali, (6.5)7–8.5(9) × (6) 6.5–7.5(8) μm in aspectu laterali, triangulares in aspectu frontali, ellipsoideae usque ad reniformes in aspectu frontali, echinulatae, aculeis brevibus, 0.2–0.8(1) μm altis. Chlamydosporae absentes. Basidiocarp resupinate, thick, up to 1.5 mm, adherent to the substrate, crustose continuous. Hymenophore smooth to finely granulose, continuous, hymenium greyish brown (7D3 to 8D3), subiculum arachnoid, darker than the hymenium, brownish to blackish, sterile margin slightly determinate, farinaceous, whitish. Rhizomorphs absent. Subicular hyphae clamped, simple septa rare, 3.5–5.5(6) μm wide, usually regular, sometimes sinuous in 2.5% KOH (Fig. 5), cross-shaped branching and anastomoses rare,

commonly thick-walled (0.5–1 μm), brown in water and in 2.5% KOH, without encrustations in water and in 2.5% KOH, not cyanescent, slightly congophilous, not cyanophilous, not amyloid. Subhymenial hyphae clamped, 3.5–6 μm wide, neither short nor inflated, thin-walled (0.2–0.5 μm), light brown to brown in water and in 2.5% KOH, without encrustations in water and in 2.5% KOH, sometimes cyanescent, slightly congophilous, slightly cyanophilous, not amyloid. Cystidia absent. Basidia clamped at base, 40–75(85) μm long, 6.5–9(10) μm at apex, 5.5–6 μm at base, utriform, not stalked, sinuous, rarely with transverse septa, colourless in water and in 2.5% KOH, commonly with numerous guttulae, sometimes strongly cyanescent, congophilous, cyanophilous, not amyloid, 4-sterigmate, sterigmata 5–8(10) μm long and 1.5–2 μm at base. Basidiospores 6.5(7)–8.5(9) × 5(6)–7(8) μm in frontal face, (6.5)7–8.5(9) × (6) 6.5–7.5(8) μm in lateral face, triangular in frontal, ellipsoid to reniform in lateral view,

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echinulate, aculei short 0.2–0.8 (1) μm, brown in water and in 2.5% KOH, not cyanescent, not congophilous, not cyanophilous, not amyloid. Material studied and habitat Guinea, upper Guinea, Province of Kankan, Prefecture of Kouroussa, forest reserve of Moussaya, along the road between Kouroussa and Dabola, 10°70′76.7″N, 009°99'76.1″W, forest composed of Uapaca guineensis (Eurphorbiaceae) and Anthonota crassifolia (Caesalpiniaceae), on dead logs, under A. crassifolia, leg. and det. NS. Yorou, 19.07.2009, herb. holotype SYN 2331 (M), GenBank NCBI, accession number JF540232. Guinea, middle Guinea, region of Mamou, on the road between Mamou and Farannah, about 2 km from Mamou, 10°34′ 44.0″N, 012°04′00.5″W, riparian forest of Uapaca esculanta A. Chev. (Euphorbiaceae), Afzelia africana and Anthonota crassifolia (Caesalpiniaceae), on dead logs, leg. and det. NS. Yorou, 21.07.2009, herb. SYN 2377 (M). Etymology In reference to the Guinean origin of the type material. Tomentella guinkoi Yorou sp. nov.(Figs. 6, 7 and 8) Mycobank MB560137, GenBank NCBI, accession number JF520430 Basidiocarpa resupinata, crassa, usque ad 1 mm alta, adherentia, crustosa, continua. Hymenium laeve usque ad minute granulosum, granulis in acervulis aggregatis, discontinuum, si recens cinereum, si siccum cinereobrunneum. Subiculum compactum, arachnoideum, tenebricius quam hymenium, marginibus sterilibus indeterminatis. Rhizomorphae absentes. Hyphae subiculi plerumque fibuligerae, 3.5–6 μm in diametro, in 2.5% KOH interdum sinuosae, vel inflatae ad septa, ibi usque ad 9(10) μm, crassitunicatae (1–1.5 μm). Hyphae subhymenii fibuligerae, 6–12(14) μm in diametro, semper brevibus cellis, compacte aggregatae, crassitunicatae (1–1.5 μm), in aqua et in 2.5% KOH nigris. Cystidia absentia. Basidia fibuligera, (30)35–60(65) μm longa, apicibus (6.5)7–8.5 (10) μm, basalibus 5.5–7 μm, utriformia, congophila, cyanophila, 4-sterigmatica, sterigmatibus 6–9 μm longis, basaliter 2–3.5 μm latis. Basidiosporae (6)6.5–8(8.5) × (5) 5.5–7(7.5) μm in aspectu frontali, (6)6.5 – 7.5(8) × (5)5.5 – 7(7.5) μm in aspectu laterali, ellipsoideae in aspectu frontali et in aspectu laterali, echinulatae, aculeis isolatis, brevissimis (0.2–0.5 μm altis). Chlamydospores absentes. Basidiocarp resupinate, thick, up to 1 mm, adherent to the substrate, crustose, continuous. Hymenophore smooth to granulose in tiny humps, discontinuous, grey in fresh condition, greyish brown (6D3) in dry condition, subiculum

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compact, arachnoid, darker than the hymenium, sterile margin indeterminate. Rhizomorphs absent. Subicular hyphae usually clamped, simple septa present, 3.5–6 μm wide, usually regular, sometimes either sinuous in 2.5% KOH, or with inflations at the septa, then up to 9 (10) μm, cross-shaped branching and anastomoses rare, always thick-walled (1–1.5 μm), yellow-brown to redbrown in water, brown to dark brown in 2.5% KOH, without encrustations in water and in 2.5% KOH, not cyanescent, not congophilous, not cyanophilous, not amyloid. Subhymenial hyphae clamped, 6–12(14) μm wide, always short and inflated (Fig. 6), stubbed and compactly arranged, thick-walled (1–1.5 μm), commonly with thick septa, septa appearing black in water and 2.5% KOH, subhymenial hyphae yellow-brown in water, light brown to dark brown in 2.5% KOH, without encrustations in water and in 2.5% KOH, not cyanescent, not congophilous, not cyanophilous, not amyloid. Cystidia absent. Basidia clamped at base, (30)35–60(65) μm long, (6.5)7–8.5(10) μm at apex, 5.5–7 μm at base, utriform (Fig. 7), not stalked, sometimes sinuous, without transverse septa, colourless in water and in 2.5% KOH, basidia congophilous, cyanophilous, not cyanescent, not amyloid, 4-sterigmate, sterigmata 6–9 μm long and 2–3.5 μm at base. Basidiospores (6)6.5–8(8.5) × (5)5.5–7(7.5) μm in frontal face, (6.)6.5 – 7.5(8) × (5)5.5 – 7(7.5) μm in lateral face, ellipsoid in both frontal and lateral views, (Fig. 8), commonly with oil drops, echinulate, aculei isolate, very short, 0.2–0.5 μm, basidiospores pale brown in water, brown in 2.5% KOH, not cyanescent, not congophilous, not cyanophilous, not amyloid. Chlamydospores absent. Material studied and habitat Burkina Faso, South Western part, region of BoboDioulasso, forest reserve of Mouhoun, along the national road between Orodara and Banfora, riparian forest dominated by Berlinia grandiflora (Vahl) Hutch. & Dalziel (Caesalpiniaceae), 10°52′12.1″N, 004°50′1.07″W, about 440 ma.s.l., on dead logs, leg. and det. NS Yorou, 06. 07. 2009, herb. holotype SYN 2235 (M), GenBank NCBI, accession number JF520430; SYN 2241 (M). Etymology In honour of Prof. emeritus Sita Guinko (from Burkina Faso) for his important contribution to the knowledge of the vegetation and flora of Burkina Faso.

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Fig. 5 Tomentella guineensis. a Section through the basidiocarp. b Basidiospores in frontal view. c Basidiospores in lateral view (from SYN 2331). Bar 10 μm

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Fig. 6 Tomentella guinkoi. Optical section through subhymenial hyphae (from SYN 2235). Bar 10 μm

Discussion The most remarkable anatomical features of T. afrostuposa are the large globose to broadly ellipsoid brown basidiospores with very long conical aculei (up to 3 μm). In addition, the long basidia and sterigmata confirm the identification of this species. Anatomically, this species is reminiscent of T. stuposa and T. gigaspora Hjortstam & Ryvarden which present subglobose to globose basidiospores with very long aculei (Kõljalg 1996; Dämmrich 2006; Stalpers 1993; Yorou and Agerer 2011a). Tomentella stuposa has been proposed by Stalpers (1984) as synonymous with T. ruttneri Litsch. (Litschauer 1932). Kõljalg (1996) enlarged the species concept and included T. bresadolae (Brinkmann) Höhnel and Litschauer. Many anatomical similarities in regard with the shape and ornamentation of the basidiospores and the shape of subicular hyphae in KOH (Larsen 1968, 1974; Litschauer 1932; Stalpers 1993; Kõljalg 1996) could be observed for both T. afrostuposa and T. stuposa. However, subicular hyphae of T. afrostuposa are thinner (4–6 μm) unlike those in T. stuposa (up to 8 μm; see Stalpers 1993; Larsen 1974). In addition, encrustations are also absent on subicular or subhymenial hyphae of T. stuposa (Larsen 1974; Stalpers 1993; Kõljalg 1996; Yorou and Agerer 2011a). Subicular, subhymenial and young basidia of T. afrostuposa are strongly encrusted in water and in 2.5% KOH. Though not common in the genus Tomentella, Yorou and Agerer (2011a) reported on the presence of internal hyphae in the basidia of T. stuposa. We were not able to observe internal hyphae in the basidia of T. afrostuposa. As far as T.

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gigaspora is concerned, it deviates from T. afrostuposa by its distinctly larger basidiospores (14–17 μm) with dense individual aculei. The phylogenetic placement of T. afrostuposa in the T. stuposa clade is supported by many anatomical similarities between the two species (see above). The deeper clade composed of T. afrostuposa and five representatives of T. stuposa is supported by a bootstrap value of 81%. This clade forms a sister clade of the group comprising T. bryophila (Pers.) M. J. Larsen and T. agbassaensis Yorou (Yorou et al. 2011a). Similar molecular-phylogenetic analyses have reported on the proximity between T. stuposa complex and T. bryophila (Kõljalg et al. 2000, 2001; Yorou et al. 2007, 2011a, b). Though T. afrostuposa clusters with one representative of T. stuposa (EU819523), genetic distance between both sequences is estimated as 7.80%. In addition, many other representative sequences of T. stuposa (from Fig. 1 and additional sequences from GenBanks UNITE and NCBI) show a genetic deviation of at least 7.81–10.74% with regard to the ITS rDNA nucleotides. Tomentella stuposa is supposed to have a worldwide distribution (Kõljalg 1996). It is a common ectomycorrhizal former in temperate and boreal forests (Jakucs et al. 2005; Kõljalg et al. 2000, 2001) where it is associated with gymnosperms as well as angiosperms (Stalpers 1993; Kõljalg 1996; Yorou and Agerer 2011a). T. afrostuposa has been collected in Caesalpinioid-dominated forests in the upper Guinea with quite different tree composition in comparison to temperate and boreal forests, supporting it as a new species different from T. stuposa. Crustose, adherent basidiocarps and the contrasting colours between the hymenium (greyish brown) and the subiculum (brownish to blackish) are remarkable morphological characters for T. guineensis. Anatomically, the long basidia (up to 85 μm), the presence of reniform (in lateral face) basidiospores with short aculei (maximum of 1 μm) and the strongly cyanescent reaction of subhymenial and basidia are important identification features for this species. Such a combination of anatomo-morphological characters is known in T. atramenteria Rostr. (Dämmrich 2006; Kõljalg 1996; Yorou and Agerer 2011a). Morphologically, T. guineensis deviates from T. atramenteria by its brownish to blackish subiculum whilst the subiculum of T. atramenteria is concolorous with the hymenium. Anatomical differences include the larger basidiospores (up to 13 μm against 9 μm in T. guineensis). Molecular-phylogenetically, both species fall in two distant clades; T. atramenteria clustering with T. badia (congruent with previous molecular-phylogenetic investigations; see Yorou et al. 2011a, b; Kõljalg et al. 2000, 2001; Jakucs et al. 2005) and T. guineensis forming a sister species of T. maroana. The molecular-phylogenetic clustering of T. guineensis and T. maroana as sister species is supported by many

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Fig. 7 Tomentella guinkoi. a Section through the basidiocarp. b Basidiospores in frontal view. c Basidiospores in lateral view (from SYN 2235). Bar 10 μm

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Fig. 8 Tomentella guinkoi. SEM of basidiospores (from SYN 2235). (a–c Basidiospores in lateral view. d Basidiospore in frontal view. e General view of basidiospores. Bars (a–d) 1 μm, (e) 5 μm

morphological similarities, among them the crustose adherent and the dark brown to black colour of the subiculum. The anatomical similarities between both species are the

thick-walled subicular hyphae with similar size (3.5–6 for T. guineensis and 4–7 for T. maroana) and the short size of the aculei not exceeding 1 μm in both species. However,

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the complete lack of rhizomorphs in T. guineensis (against slightly differentiated rhizomorphs in T. maroana) and the size of basidiospores (up to 9 μm in T. guineensis against 12 μm in T. maroana) make the discrimination of both species easy. Sequence analyses highlighted a genetic distance of at least 9.75–10.04% (with regard to the ITS rDNA nucleotides) between the two species. Another genetically related species is Thelephora terrestris according to BLASTN search in NCBI. However, detailed sequence analyses revealed a genetic distance of at least 10.01–10.78% between T. guineensis and Thelephora terrestris. Anatomically, T. guinkoi is characterised by the very short, inflated stubbed and compactly arranged thickseptate subhymenial hyphae. These features, coupled with the ellipsoid (up to 8 μm) basidiospores, helps with its identification. So far, similar combinations of anatomical features have been reported in T. ellisii (Dämmrich 2006; Yorou and Agerer 2011a; Kõljalg 1996). Basidiospores in both species are triangular to ellipsoid in frontal view, though reniform basidiospores are observed in T. ellisii (Kõljalg 1996; Dämmrich 2006; Yorou and Agerer 2011a). Additional anatomical similarities include the cyanescent reaction of some subhymenial hyphae and basidia in 2.5% KOH (Dämmrich 2006; Yorou and Agerer 2011a). However, many morphological and anatomical divergences between the two species can be noted. Morphologically, T. ellisii presents a smooth to finely granulose hymenophore, dull-red (8D3), brown (7E5) to red-brown (8E4) hymenium with a farinaceous, dirty white to light yellow sterile margin (Yorou and Agerer 2011a; Dämmrich 2006; Kõljalg 1996). It may present monomitic rhizomorphs in the subiculum (Kõljalg 1996; Dämmrich 2006). In T. guinkoi, the hymenophore is smooth with numerous small humps and the hymenium is rather grey-brown (6D3). Tomentella guinkoi lacks rhizomorphs. Anatomical differences between the two species include the width of the subicular hyphae which is larger in T. ellisii (4–10 μm; see Dämmrich 2006; Yorou and Agerer 2011a) than in T. guinkoi (3–6 μm). Similarly, the basidiospores are bigger in T. ellisii with a size range of 7–10 μm, sometimes up to 13 μm (aculei up to 1 μm), against 6–7.5 μm (aculei up to 0.5 μm) in T. guinkoi. The subhymenial hyphae of T. guinkoii are thick-walled (1–1.5 μm), with septa appearing dark in KOH. Subhymenial as well as subicular hyphae of T. guinkoi are thin-walled (Kõljalg 1996; Dämmrich 2006; Yorou and Agerer 2011a). The genetic distance between T. guinkoi and T. ellisii (DQ974775, DQ068971 and UDB000219) ranges from 12.67 to 13.73% with regard to the ITS rDNA nucleotides. Such sequence deviations between the two species coupled with anatomomorphological dissimilarities highlighted in previous section, support the description of T. guinkoi as a new species

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different from T. ellisii. Molecular-phylogenetically, Tomentella guinkoi forms a sister species of the group composed of T. ellisii, T. hjortstamiana and T. pisoniae in a strongly supported (bootstrap of 95%) clade. Anatomical comparison between T. ellisii, T. hjortstamiana and T. pisoniae is provided in Yorou et al (2011a). Acknowledgments Financial support was provided by the Deutsche Forschungsgemeinschaft (DFG, Project Ag7/19-1) to whom we address our sincere gratitude. Special thanks to Frank Van Caekenberghe (National Botanic Garden of Belgium, Meise) for SEM micrographs.

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