Mycol. Res. 107 (4): 485–494 (April 2003). f The British Mycological Society 485 DOI: 10.1017/S0953756203007585 Printed in the United Kingdom. First records of ectomycorrhizal Cortinarius species (Agaricales, Basidiomycetes) from tropical India and their phylogenetic position based on rDNA ITS sequences Ursula PEINTNER1*, Meinhard M. MOSER#, K. Agretious THOMAS2 and P. MANIMOHAN2 1 Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria. Department of Botany, Calicut University, Kerala, 673 635, India. E-mail : ursula.peintner@uibk.ac.at 2 Received 17 April 2002; accepted 13 February 2003. Three new Cortinarius species, Cortinarius conopileus, C. keralensis, and C. phlegmophorus spp. nov., are described from Kerala State in southern India. This is the first record of ectomycorrhizal Cortinarius spp. in the tropical part of India. In addition to distinct morphological characters, the comparative analysis of rDNA ITS sequences of the collections from India and morphologically similar species support the recognition of these taxa as new species. Phylogenetic analyses demonstrate that the three Indian Cortinarius spp. belong to both larger subclades of the genus Cortinarius, clade/cortinarius and clade/telamonia. As supported by morphological and molecular data, C. phlegmophorus belongs to Cortinarius subgen. Myxacium sect. Defibulati. Based on classical morphological characters, both C. keralensis and C. conopileus are representatives of subgen. Telamonia. However, C. conopileus belongs to clade/obtusi, which is a well-supported subclade of clade/cortinarius. Thus, in contrast to classical taxonomy, the clade/obtusi represents an independent evolutionary origin of telamonioid taxa. This result is also reflected by the distinct morphological characters of taxa of clade/obtusi, namely the lamellar trama with ellipsoid inflated hyphae and the presence of cystidia. In contrast, C. keralensis is a typical member of clade/telamonia. Within/telamonia, only relationships of closely related taxa are resolved due to the low genetic divergence found in ITS sequences. Based on morphological and molecular criteria, C. keralensis is a distinct taxon of sect. Saturnini. INTRODUCTION The ectomycorrhizal genus Cortinarius occurs predominantly in temperate to subarctic-alpine climates in both Hemispheres. Until the recent past it had been postulated (Singer et al. 1983) that ectomycorrhizas are the exception in the tropics. In the last two decades quite a lot of information has been accumulated, demonstrating that several ectomycorrhizal genera are well represented in the tropics (e.g. Russulales, Boletales ; Fassi & Moser 1991, Buyck et al. 1996, Buyck & Horak 1999, Henkel, Aime & Miller 2000). Moreover, ectomycorrhizal associates of leguminous host trees in the genus Dicymbe (Caesalpiniaceae) have been studied extensively in the Guyana’s Pakaraima Mountains (Henkel et al. 2000, Henkel 2001, Henkel, Terborgh & Vilgalys 2002). However, the occurrence of Cortinarius in tropical areas is indeed rather the exception. A few species are described from Brazil (Singer et al. 1983) and Mexico (Murrill 1912), several * Corresponding author. # Deceased; see Mycological Research 107 (4): 506–508 (April 2003). (partly undescribed) species are known from Congo (Beeli 1928, unpubl. collections from Mme. GoossensFontana), and several Cortinarius spp. occur in Guyana (unpubl. species of Terry Henkel). In the latter two cases, these Cortinarius spp. are associated with leguminous plants. All other reports of Cortinarii in tropical areas are from mountainous habitats. From tropical India, as yet no representatives of this genus were known. Here three taxa from southern India, assumed to be associated with Hopea, Dipterocarpaceae, are described. Dipterocarpaceae are reported to be ectomycorrhizal in Malaysia and the Philippines (Watling 1994, 1995). Moreover, in vitro synthesis has been achieved between ectomycorrhizal fungi and root organs of tropical Dipterocarp species (Louis & Scott 1987), but as yet no Cortinarii have been reported in association with this plant family. In Kerala State, the climate is equable and varies little from season to season. The temperature normally ranges 24–37.5 xC in the plains and 10–32 x in the hills. The state gets its due share of both the southwest as well as the north-east monsoons, and the rainfall is heavy, averaging around 3000 mm p.a. In the Ectomycorrhizal Cortinarius species from tropical India Cortinariaceae, all three new species, as well as the recently described monotypic agaric genus Anamika (Thomas et al. 2002) were from a single locality in the Wayanad District of Kerala State. Floristically this locality is a transitional zone between wet evergreen and teak (deciduous) forests. All the collections were from a riverbank where the riparian flora is predominantly evergreen. Several plants of this locality, including the Hopea spp. and other trees with which these agarics are thought to be associated, are endemic to peninsular India. In addition to the endemics, elements or relatives of the Sri Lankan, Madagascarian, African, and Malayan biotas are also seen in this region. Due to the special habitat of these new Cortinarius species, we attempted to clarify their phylogenetic position. We used nuclear ribosomal DNA sequences of the internal transcribed spacers (ITS-rDNA) to investigate the taxonomic position of these taxa. These sequences have proved to be most useful for molecular systematics of Cortinarius (Rogers & Ammirati 1997, Høiland & Holst-Jensen 2000, Seidl 2000, Moser et al. 2001, Peintner et al. 2001) and related genera (Aanen et al. 2000, Moncalvo et al. 2000, 2002). For the phylogenetic placement of these species, numerous sequences are available for comparison in GenBank or in the Cortinarius database in Rytas Vilgalys’ laboratory (Duke University, Durham, NC). Our goal was to report three new taxa and to address the question of the phylogenetic relationships between these and morphologically similar Cortinarius spp. MATERIALS AND METHODS Macro- and microscopical characterisation Colours of basidiomes are described based on the codes of Kornerup & Wanscher (1978). The specimens were examined by standard microscopic techniques in 3% KOH. Microscopic data were documented by video prints produced with a Sony video camera SASC-C350P and a Sony video printer UP 910. All measurements were on prints at 2900r. For statistical evaluation, 35 spores were measured. If not otherwise stated values are given as min-max. Single extraordinarily high or low values are in brackets. For scanning electron microscopy (SEM), dried fragments of lamellae were rehydrated in 25 % ammonia solution for 1 h, dehydrated in 70 % aqueous ethanol for 1 h, fixed in pure 1,2-dimethoxy-methane for 1.5 h and then immersed in pure acetone for at least 2 h. After critical-point drying, they were mounted on aluminium porters and sputtered with gold. Micrographs were prepared using a Zeiss DSM-950 SEM. Molecular techniques In order to establish the phylogenetic relationships of the three new Cortinarius spp., ITS-rDNA sequences 486 Table 1. Species used in this study with their GenBank ITS sequence accession nos. When available, reference collection numbers are given. About half the sequences were generated in earlier studies (Liu et al. 1997, Aanen et al. 2000, Høiland & Hoist-Jensen 2000, Seidl 2000, Peintner et al. 2001). Species Cortinarius C. acutovelatus C. acutovelatus C. acutus C. albocanus C. alnetorum C. alnetorum C. atrocoeruleus C. cedriolens C. cinereobrunneus C. collinitus C. conopileus (holotype) C. decipiens C. evernius C. helvelloides C. heterosporus C. hinnuleus C. hinnuleus C. keralensis (holotype) C. laniger Fr. C. laniger C. leucopus C. lividoochraceus C. miniatopus C. mucifluus C. mucosus C. muscigenus C. obtusus C. paleaceus C. paleaceus C. paveleckii C. phlegmophorus (holotype) C. porphyroideus C. porphyroides C. pseudosalor C. pulchellus C. pulchellus C. saturninus C. trivialis C. umbilicatus C. umidicola C. urbicus C. vanduzerensis Hebeloma H. circinans H. fastibile H. mesophaeum Collection IB19980108 IB19930722 IB19980137 Halling5832 IB19920217 IB19970382 IB20000132 IB20000045 IB19630258 IB19890462 IB19990302 IB19740451 IB19930140 IB19960139 IB19930113 IB19990305 IB19740251 IB19990511 IB19630078 IB19960258 IB19940186 IB19980139 IB19990308 IB19960086 IB20000025 IB19980189 IB19890415 IB19970431 IB19940036 GenBank accession no. AY083174 AY083175 AF325578 AF325599 AY083176 AY083177 AY083178 AY083179 AF325600 AY083181 AY083187 AY083180 AJ236077 AY083182 AF268894 AY083183 AY083184 AY083188 AF325591 AF325592 AF325593 AF325565 U56029 AF182795 AF182801 AY083185 AJ238035 AF389156 AJ236078 AF325564 AY083186 AF325576 AF325577 AF182792 AF389155 AY083192 AY083189 AJ236066 U56032 AY083191 AY083190 AF182794 AF124716 AF325643 AF126100 were prepared from the type material of each taxon. Sequences of morphologically similar taxa were used to investigate the phylogenetic relationship. They were either created for this study, or retrieved from GenBank or from the Vilagalys Cortinarius database. The fungal collections used are listed in Table 1, with their GenBank accession nos. In order to distinguish between names of clades and names of taxa, clade names are marked with a slash and not written in italics (e.g. clade/telamonia). U. Peintner and others DNA was isolated from dried herbarium material following standard protocols (Zolan & Pukkila 1986). Primers used for the ITS region were ITS1 and ITS4 (White et al. 1990). Amplifications were carried out in a 25 ml reaction mix under standard conditions (Vilgalys & Hester 1990) in a Perkin-Elmer 9600 thermocycler (Perkin-Elmer, Foster City, CA). Sequencing was performed using fluorescent dye terminator chemistries following the manufacturer’s instructions (Perkin-Elmer) on automated sequencers (ABI 373A, ABI 377, Perkin-Elmer, Norwalk, CN). Sequence chromatograms were compiled with Sequencher software version 2.0 (Gene Codes, Ann Arbor, MI) or Sequence Navigator (Perkin-Elmer, Foster City, CA). Sequences were submitted to GenBank, and alignments are deposited in TreeBASE (http://www. treebase.org/treebase//) under study accession no. S799 and the matrix accession no. M1265. Phylogenetic analyses The three Cortinarius spp. would be expected to belong to different subgenera based on morphological characters and classical systematics. Therefore, we divided the analyses into three steps : First, to place the sequences in a broader phylogenetic context, BLAST searches were made in GenBank. Then, the ITS sequences of the three taxa from India were manually added to the alignment of the large Vilgalys Cortinarius database in the data editor of PAUP* 4.0b8 (Swofford 1998). The relationships of the new taxa were first assessed by a fast bootstrap analysis (Felsenstein 1985) of the large dataset. Based on these results, the final analysis was restricted to 35 ingroup species of Cortinarius (42 sequences), and three Hebeloma spp. as an outgroup. The alignment for this large data matrix with 45 sequences was 840 characters long. Areas of ambiguous alignment and gapped areas were excluded; thus 461 characters remained for analysis : 288 characters were constant, 43 were parsimony uninformative, and 130 characters were parsimony informative. The exluded areas are indicated in the TreeBASE file. Phylogenetic analyses were performed with maximum parsimony (MP) and maximum likelihood (ML) in PAUP* 4.0b8 (Swofford 1998). MP was used to search for optimal trees, with maxtrees set to 20 000 and employing the default PAUP* settings (i.e. mulpars=on and steepest descent not in effect). MP analysis was conducted with a step matrix giving transitions twice the weight of transversions. Analyses were carried out with tree-bisection-connection (TBR) branch swapping, and with gaps treated as ‘ missing ’. Most parsimonious trees (MPT) were found with starting trees obtained by stepwise addition, using 100 replicates holding one tree at each step, and saving only ten trees with score f1. In a second heuristic search these MPT were used as starting trees and swapped with a reconnection limit of ten. To evaluate branch robustness of trees generated by parsimony-based 487 methods, bootstrap analyses (Felsenstein 1985) were applied. Bootstrap analyses were conducted using 1000 replications, carried out with random addition sequences, each with ten replicates and TBR branch swapping. The program Modeltest version 3.06 (Posada & Crandall 1998) was used to choose an optimal model of DNA substitution. The ML analysis under the GTR+G model was performed with six substitution types and a user-specified substitution rate matrix, nucleotide frequencies A=0.26153, C=0.22576, G= 0.20619, T=0.30652, proportion of invariable sites=0, rate heterogeneity following the discrete gamma approximation with four categories and a shape parameter a=0.30743. The heuristic search with TBR branch swapping used the first MPT as starting tree. In addition, one hundred bootstrap replicates were run with ML, starting tree(s) obtained via stepwise addition, random addition sequence, one replicate, one tree held at each step during stepwise addition and TBR. In a second step of analysis, we separately analysed each of the new Cortinarius species with their most closely related species. With this approach, more informative characters could be included in the analysis. The data matrix created for the phylogenetic analysis of C. keralensis was restricted to 27 sequences (22 taxa). 375 out of the 840 nucleotide positions were excluded, of the remaining 465 characters 337 characters were constant, 51 variable characters were parsimony uninformative, and 77 characters were parsimony informative. Most parsimonious trees were searched with the settings described above. For the phylogenetic placement of C. conopileus, a data matrix with 819 nucleotide positions was created for the seven sequences. After exclusion of areas with gaps or ambiguous alignment, 585 nucleotide positions remained for analysis : 495 characters were constant, 49 variable characters were parsimony uninformative and 41 characters were parsimony informative. An exhaustive search was carried out. The data matrix created for C. phlegmophorus included ten ingroup taxa ; two collections of C. paveleckii were used as outgroup. The alignment was 840 nucleotide positions long. After the exclusion of areas with gaps, 610 nucleotide positions were used for analysis : 504 characters were constant, 32 variable characters were parsimony uninformative, and 74 characters were parsimony informative. An exhaustive search was carried out. TAXONOMY Cortinarius (subgen. Myxacium) phlegmophorus K. A. Thomas, M. M. Moser, Peintner & Manim., sp. nov. (Figs 1, 4–7) Etym. : phlegmophorus (Greek), slime bearing. Ectomycorrhizal Cortinarius species from tropical India 488 Figs 1–3. Basidiomata. Fig. 1. Cortinarius phlegmophorus. Fig. 2. C. conopileus. Fig. 3. C. keralensis. Bar=1 cm. Pileo 20–60 mm lato, conico-convexo, dein plano-convexo, interdum late umbonato, glutinoso, hygrophano, primo obscure brunneo, dein griseo-aurantiaco vel brunneo-aurantiaco, glabro, sicco ruguloso. Lamellis adnatis primo griseo-violaceis, interdum obscure violaceis, maturitate pallide brunneis acie pallidiore sive albidula, serrulata sub lente, subconfertis vel confertis, usque ad 5 mm latis, lamellulis praesentibus, stipite 45–75r5–10 mm, cylindraceo, fere aequali vel attenuato apicem versus, compacto dein fistuloso, superficie griseoviolaceo vel obscure violaceo, aetate brunneo-aurantiaco vel griseo-brunneo, obtecto e velo glutinoso, abido-fibrillosofloccoso, carne albida odore insignificante, sporis in cumulo brunneis. Basidiosporis 10.5–14.5r6–8 mm, amygdaliformibus usque limoniformibus, verrucosis, basidiis (2)–4-sporigeris, 32–48r10–14 mm, clavatis, cheilocystidiis 13–55r 9–32 mm vesiculosis, clavatis. Fibulis absentibus. Typus: India: Kerala State : Wayanad District, Ponkuzhy, ad terram, sparsis vel gregariis, sub Hopea (Dipterocarpaceae) 28 Oct. 1998, A. Thomas T344 (IB19990308 – holotypus). Pileus 20–60 mm diam, conico-convex becoming planoconvex, sometimes with a broad blunt umbo, margin somewhat incurved, becoming decurved, slightly appendiculate when young, becoming entire with maturity, sometimes eroded or fissile; surface viscid when wet, initially dark brown (6F8, 7F8), turning brown (6E8, 7E8), at the centre and almost greyish orange (5B3) or brownish orange (5C3, 6C4) towards margin, hygrophanous and becoming grayish orange (5B3), glabrous, smooth, becoming dry and wrinkled. Context in pileus white, up to 5 mm thick at the centre. Lamellae adnate, initially violet grey (16D2) or sometimes dull violet (16D3), later light brown (6D6), subcrowded to close, up to 5 mm wide, with lamellulae of different lengths, edges white or paler than the sides, finely lacerate under a lens. Stipe 45–75r5–10 mm, central, cylindric, almost equal or tapering apically, solid, becoming fistulose; surface violet gray (16D2) or dull violet (16D3) when young, changing to brownish orange (5C3) or greyish brown (5D3) with maturity, covered with a glutinous white, fibrillose-floccose veil. Context white to whitish. Odour not distinctive. When the basidiomata mature, the pileus consistently and naturally becomes detached from the stipe and falls down. Spore print greyish brown to light brown (6D4). Basidiospores 10.5–14.5r6–8 mm, average (av) ¡standard deviation (SD)=12.2¡0.9r6.8¡0.5 mm, Q=1.5–2.0, av¡SD=1.8¡0.1, vol.=217–408 mm av¡ 3 SD=299¡56 mm , amygdaliform to sublimoniform with apicular callus, yellowish brown, thin- to slightly thickwalled, strongly verrucose with irregular warts, apiculus smooth, with suprahilar pseudoplage. Basidia (2)– 4-spored, 32–48r10–14 mm, clavate, sterigmata up to 6.5 mm long. Edge of lamellae crowded with baloonshaped, thin-walled, colourless cheilocystidia, 13–55r 9–32 mm. Pleurocystidia none. Lamellar trama regular, hyphae in lateral stratum with a diameter of 2–9 mm, in mediostratum up to 24 mm, thin-walled, sometimes faintly encrusted, colourless. Pileus surface with a gelatinous layer of 2–5 mm wide, repent, thin-walled sometimes encrusted, colourless hyphae. Epicutis consisting of two to three layers of colourless, 6–10 mm wide hyphae. Hypocutis with ellipsoid-inflated hyphae at the base, segments 20–45 (–60)r11–25 mm, in upper part nearly colourless, in deeper layers brown encrusted, partly with platelets, followed by a strongly pigmented layer of hyphae with a diam of 5–9 (–10) mm, encrusted with platelets, with some dark brown oleiferous hyphae. Stipe context composed of 2–24 mm wide parallel hyphae, thin-walled, colourless, with some oleiferous hyphae (brown content). Stipe cortex composed of 6–13 mm wide hyphae, encrusted, pale yellow to colourless, covered by a gelatinous layer consisting of 2–4.5 mm wide colourless hyphae. Clamp connections not observed in any tissue. U. Peintner and others Fig. 4. Basidiospore of Cortinarius phlegmophorus (SEM). Bar=2 mm. Figs 5–6. Cortinarius phlegmophorus (holotype). Fig. 5. Basidiospores. Bar=10 mm. Fig. 6. Cheilocystidia. Bar=20 mm. Habitat : On the ground, attached to underground roots, scattered or sometimes in groups of two in the vicinity of Hopea spp. (Dipterocarpaceae), Meiogyne pannosa (Annonaceae) and Cansjera rheedii (Opiliaceae). Other specimens examined: India : Kerala State : Wayanad District, Ponkhuzy, 31 Oct. 1999, A. Thomas T344b (IB19990309). Cortinarius (subgen. Telamonia) conopileus K. A. Thomas, M. M. Moser, Peintner & Manim., sp. nov. (Figs 2, 8–11) Etym. : conopileus (Latin), conical pileus. 489 Fig. 7. Longitudinal section through the pileus surface of Cortinarius phlegmophorus (holotype) : (a) gelatinous layer ; (b) epicutis ; (c) hypocutis ; and (d) uppermost layer of trama with oleiferous hyphae. Bar=60 mm. Pileo 4–15 mm lato, primo fere campanulato, conico vel conico-convexo, dein convexo usque plano-convexo umbone obtuse acuteve praedito, hygrophano, brunneo vel pallide brunneo, sicco griseo-aurantiaco, glabro, iove udo leviter translucente striato, lamellis adnatis usque adnexis, brunneoaurantiacis, dein pallide brunneis, confertis, lamellulis praesentibus, usque ad 2 mm latis, acie plus minusve erosa (sub lente) ; stipite 25–60r1.5–3 mm, tereto, apice leviter attentuato, brunneo-aurantiaco usque pallide brunneo, glabro. Odore non significante. Basidiosporis 6–8.5 (–10)r3.5– 4.5 mm, elongatis, ellipsoideis usque subamygdaliformibus, punctatis (subtiliter verrucosis), basidiis 25–32r6–9 mm, clavatis, tetrasterigmatis, cheilocystidia 11.5–51.5r4.5–15.5 mm frequentis, clavatis, vesiculosis, rarior lageniformibus vel fusoideis, trama lamellarum e hyphis ellipsoideo-inflatis, segmentis 30–60r15–28 mm, fibulis praesentibus. Typus: India: Kerala State : Wayanad District, Ponkuzhy, ad terram in silvis, fasciculatis vel dispersis, sub Hopea (Dipterocarpaceae), 28 Oct. 1999, A. Thomas T341b (IB19990302 – holotypus). Pileus 4–15 mm diam, initially almost campanulate, conic or conic-convex, becoming convex to planoconvex, with a blunt to almost pointed umbo ; surface dark brown (6F8, 6F7) or brown (6E8) towards the centre, brown (6E7) or light brown (6D6, 6D7) elsewhere, hygrophanous and eventually entirely turning grayish orange (5B3), glabrous, dull, faintly translucently striate when moist, margin decurved, entire to almost crenate, sometimes fissile. Lamellae adnate to adnexed, brownish orange (6C6) becoming light brown (6D7), close, up to 2 mm wide, with lamellulae of 3 lengths; edge pale, scalloped or eroded under a lens. Stipe 25–60r1.5–3 mm, central, terete, slightly tapering apically, solid ; surface brownish orange (6C6) Ectomycorrhizal Cortinarius species from tropical India 490 an epicutis of hyphae between (2–) 3–6 (–7.5) mm, thick, almost colourless to yellow brown, encrusted with granules to platelets ; in hypocutis hyphae inflated up to 15–20 mm diam, yellow brown, encrusted. Pileal context consisting of parallel 2–25 mm thick hyphae, pale yellow to yellowish brown, faintly encrusted. Stipe cortex consisting of hyphae of 2–7.5 mm diameter, colourless to pale yellowish brown, faintly encrusted. Hyphae of the stipe context 2–16.5 mm wide, parallel, faintly encrusted, yellowish to yellowish brown. Clamp connections present on all hyphae. Habitat : on the forest floor, mostly in clusters or scattered, attached to small subterranean roots, probably mycorrhizal with nearby trees : Hopea sp. (Dipterocarpaceae), Meiogyne pannosa (Annonaceae) and Cansjera rheedii (Opiliaceae). Other specimen examined : India : Kerala State : Wayanad District, Ponkuzhy, 17 Oct. 1999, A. Thomas T341 (IB19990302); 31 Oct. 1999, A. Thomas T341c (IB19990304). Fig. 8. Basidiospores of Cortinarius conopileus (SEM). Bar=2 mm. Cortinarius (subgen. Telamonia) keralensis K. A. Thomas, M. M. Moser, Peintner & Manim., sp. nov. (Figs 3, 12–14) Etym. : keralensis (Latin), pertaining to Kerala. Figs 9–11. Cortinarius conopileus. Fig. 9. Basidiospores of IB19990302 (paratype). Fig. 10. Basidiospores of IB19990302 (holotype). Bar=10 mm. Fig. 11. Cheilocystidia. Bar=20 mm. to light brown (6D7). Odour not distinctive. Basidiospores 6–8.5 (–10)r3.5–4.5 mm, av¡SD=7.6¡0.3r 4.2¡0.1, Q=1.5–2.2, av¡SD=1.8¡0.1, vol.=44– 102 mm3, av¡SD=72¡11 mm3, slender amygdaliform to subfusoid, yellowish brown, slightly thick-walled, punctate. Basidia 25–32r6.9 mm, 4-spored, clavate, sometimes with yellowish content, deformed with age, sterigmata up to 4.5 mm long. Cheilocystidia 11.5– 51.5r4.5–15.5 mm, numerous, versiform, clavate to vesiculose, fusoid or sublageniform, thin-walled, colourless, sometimes faintly encrusted. Pleurocystidia none. Lamellar trama regular, hyphae in lateral stratum 2.5–6 mm wide, in the mediostratum up to 28 mm thick, ellipsoid-inflated, segments 30–60r15–28 mm, walls very pale ochraceous, buff, to pale yellowish brown, some hyphae finely encrusted. Pileipellis with Pileo 15–80 mm lato, primo subgloboso, dein convexo, margine primo incurvato, maturitate saepe depresso, sicco, hygrophano, in statu humectato unicolore obscure brunneo, margine subtiliter translucente striato, in statu sicco brunneoaurantiaco, glabro, lamellis late adnatis vel dente decurrentibus, cinnamomeo-brunneis vel leviter obscurioris, subconfertis usque confertis, lamellulis praesentibus, usque ad 7 mm latis, acie pallidiore. Stipite 35–65r5–13 mm, apice leviter attenuato, basin versus leviter inflato, albida, apice basique interdum griseo-purpuream tincto, cortina abundate, zonam annuliformem formante, interdum peronato. Carne pallide brunnea. Odore non significante. Sporis in cumulo ferrugineis. Basidiosporis 7.5–10r4–5 mm, amygdaliformibus, verrucosis, basidiis 25–47.5r6.5–10 mm, clavatis usque subcylindraceis, (2)–4-sporigeris, cheilocystidiis praesentibus, 13–54.5r4.5–13 mm, clavatis, interdum lageni- vel uteriformibus. Typus: India : Kerala State : Wayanad District, Ponkuzhy: in silvis ad terram in fasciculis vel dispersis, sub Hopea spp. (Dipterocarpaceae), Calophyllum sp., Meiogyne sp., 2 Sept. 1999, A. Thomas T118 (IB19990305 – holotypus). Pileus 15–80 mm diam, initially subspherical, becoming convex, with or without a central depression when mature, margin incurved when young, becoming decurved, almost entire, at times becoming fissile, surface dark brown when moist, (7F6, 7F7, 8F6, 8F7), unicolorous, slowly becoming brownish orange (6C6 or 6D6) smooth and glabrous, hygrophanous, faintly translucently striate and slightly lubricous when moist. Context in pileus pale brown. Lamellae broadly adnate to adnate with a decurrent tooth, cinnamon brown (6D6) or slightly darker, subcrowded to close, to 7 mm wide, with lamellulae of 3 lengths; edges paler than the lamellar faces or white, finely torn. Stipe 35–65r 6–13 mm, central, terete, slightly tapering apically and U. Peintner and others 491 mostly clavate, sometimes utriform or lageniform, thin-walled, hyaline, sometimes encrusted. Without pleurocystidia. Lamellar trama regular, hyphae in subhymenium 1.5–3 mm wide, in lateral stratum 4–8 mm wide, in mediostratum 11–16–24 mm wide and inflated, thin-walled, yellowish brown, slightly encrusted. Epicutis consisting of hyphae of 4–8 mm diam, pale brown, not or slightly encrusted. Hypocutis hyphae up to 12 mm thick. Pileus context hyphae 2–24 mm wide, inflated, parallel to interwoven, thin-walled, encrusted, yellowish brown. Stipe context of 2–20 mm wide hyphae, parallel, thin-walled, faintly encrusted, colourless to pale yellow. Stipe cortex of 2–7.5 mm wide hyphae, thinwalled, pale yellow to almost colourless. Veil hyphae 2–4 mm. Clamp connections present on all hyphae. Habitat : on the forest floor, in clusters or scattered, under Hopea spp. (Dipterocarpaceae), Calophyllum sp. (Clusiaceae), Meiogyne sp. (Annonaceae) and Cansjera rheedii (Opiliaceae). Fig. 12. Basidiospores of Cortinarius keralensis (SEM). Bar=2 mm. Other specimens examined: India : Kerala State : Wayanad District, Ponkuzhy, 17 Oct. 1999 A. Thomas T118b (IB19990306); 28 Oct. 1999, A. Thomas T118c (IB19990307). RESULTS Figs 13–14. Cortinarius keralensis (holotype). Fig. 13. Basidiospores. Bar=10 mm. Fig. 14. Cheilocystidia. Bar=20 mm. with a slight swollen base, solid. Surface somewhat white, apically and sometimes basally with a purplish gray (13C2) shade, initially with a well developed veil which leaves annular remnants sometimes with a fibrillose-peronate sheath but not a volva. Context pale brown. Without distinctive odour. Spore print rustbrown (6E8). Basidiospores 7.5–10r4–5 mm, av¡SD= 8.8¡0.6r4.5¡0.2 mm, Q=1.7–2.1, av¡SD=1.9¡0.1, vol.=68–130 m3, av¡SD=94¡14 mm3, elongate almond-shaped, punctate verrucose, yellowish brown, thin to slightly thick-walled. Basidia 25–47.5r6.5– 10 mm, subcylindric to clavate, (2)–4 spored, mature basidia sometimes contain yellowish brown plasmatic pigment, sterigmata up to 4.5 mm long. Lamella-edge heteromorphous to almost sterile with numerous cheilocystidia. Cheilocystidia 13–54.5r4.5–13 mm, The parsimony analysis of the large dataset including 43 ITS sequences of all Cortinarius spp. treated resulted in 3370 MPT with tree length of 397 steps, a consistency index (CI) of 0.6121, a retention index (RI) of 0.7858 and a rescaled consistency index (RC) of 0.4810 (Fig. 15). The phylogenetic analysis with maximum likelihood resulted in the MLT with a xln likelihood of 2718.47573., which is in agreement with the MPT. Based on both phylogenetic analyses, the ingroup clade representing the genus Cortinarius is supported with bootstrap values of 100%, the given outgroup clade is Hebeloma. Within the genus Cortinarius, one major clade is supported by higher bootstrap values (f70%) and congruent with all MPTs and the MLT : clade/cortinarius. Within clade/cortinarius, three monophyletic groups are well resolved (bootstrap values >60 %) : clade/obtusi (i.e. subgenus Telamonia sect. Obtusi ), clade/defibulati (i.e. subgen. Myxacium sect. Defibulati) and clade/myxacium (i.e. subger. Myxacium sect. Myxacium). The latter two clades have sistergroup relationships. The basal clade/telamonia recovered by MP analysis is not supported by bootstrap analyses and represents a basal polytomy in the MLT. This group is characterised by short branches in both analytical approaches, closely related taxa fall into wellsupported clades (e.g. C. atrocoeruleus and C. decipiens) but basal relationships are not resolved. The Cortinarius taxa from tropical India fall into both subclades of the genus Cortinarius, clade/telamonia and clade/cortinarius. Cortinarius keralensis has an independent position within the /telamonia clade, and a close relationship to C. saturninus or to any other included taxon can be excluded based on molecular data. C. conopileus belongs to clade/obtusi. C. phlegmophorus Ectomycorrhizal Cortinarius species from tropical India Fig. 15. One of the 3370 most parsimonious trees resulting from the analysis of 42 ITS sequences. Bold branches are present in the strict consensus tree. Bootstrap values higher than 50 % are shown above the respective branches, asterisks* stand for bootstrap values >50. The three new Cortinarius spp. (bold) belong to three different clades: C. phlegmophorus is basal to clade/defibulati, C. conopileus belongs to clade/obtusi and C. keralensis has an isolated position within clade/telamonia. 492 U. Peintner and others has an independent, basal position in clade/defibulati. Based on these results, separate analyses were carried out for each species from India. The MP analysis carried out with the second dataset created for C. keralensis yielded 494 MP trees with a tree length of 250 steps, CI=0.672, RI=0.624, RC= 0.419. After reweighting characters by maximum value of rescaled consistency indices, 37 MPT were found with a tree length of 141.30376 steps, CI=0.8618, RI=0.8132, RC=0.7008 (not shown). The best fitting ML model selected by Modeltest was the GTR+G+I model (number of substitution types=6, nucleotide frequencies: A=0.26450, C=0.20990, G=0.21110, T=0.31450, proportion of invariable sites=0.44, gamma shape parameter=0.7315. The ML search resulted in 2 ML trees with xln likelihood=1995.484 (not shown). There is no conflict between the tree resulting from ML analysis and the MPT. Basal relationships were not better resolved with this smaller dataset than in the analysis of the large dataset. Relationships of closely related species could be resolved in all three analytical approaches (e.g. C. atrocoeruleus – C. decipiens), demonstrating that C. keralensis is not closely related to any of the involved taxa. The analysis of the data matrix created for C. phlegmophorus resulted in four MP trees with a tree length of 130 steps, CI=0.8846, RI=0.9272, RC=0.8202 (tree not shown). During the exhaustive search, 654729075 trees were evaluated. The tree topology is identical with the one resulting from the large dataset (Fig. 15): C. phlegmophorus has in isolated position in the clade/defibulati. The exhaustive search of the clade/obtusi evaluated 945 trees. The score of the best tree was 102. One tree was retained with CI=0.9314. HI=0.1373, RI= 0.8814, RC=0.8209 (tree not shown). As already resulting from the large dataset (Fig. 15), the clade/obtusi falls into two subclades, clade/acuti and clade/obtusi. Cortinarius conopileus has an independent position in the subclade/acuti. DISCUSSION Based on molecular and morphological data, Cortinarius conopileus fits well in Cortinarius subgenus Telamonia sect. Obtusi subsect. Acuti (Moënne-Loccoz, Reumaux & Henry 1990). This is corroborated by the shape of the basidiomata, by the colours, by the presence of numerous clavate to vesiculose or fusoid cheilocystidia and by the typical structure of the lamellar trama consisting of wide, ellipsoid inflated hyphae. It differs from all other taxa of this group in the slender amygdaliform, often subfusoid spores with an average Q-value of 1.8. The independent position within the Acuti is also supported by molecular data. Clade/obtusi is a well supported subclade of clade/cortinarius. Thus, in contrast to classical taxonomy, the clade/obtusi represents an evolutionary origin independent of taxa 493 belonging to clade/telamonia, which is also reflected by morphological characters (basidiome habit, lamellar trama, cystidia). Other phylogenetic rDNA analyses (Høiland & Holst-Jensen 2000, Peintner et al. 2001) suggest that the clade/telamonia (without /obtusi) is a natural group, a result supported also by our MP analysis, but not by our MLT. Within /telamonia, relationships of closely related taxa are resolved by analysis of ITS sequences, however, no larger groups can be resolved due to the low genetic divergence found in ITS sequences. Based on morphological criteria, C. keralensis is a typical Telamonia, belonging to sect. Saturnini. This section circumscribes species with larger or medium sized basidiomes with medium to chocolate brown colours, dark red brown to chocolate lamellate, a white veil and medium sized ellipsoid spores (MoënneLoccoz, Reumaux & Henry 1990). Macro- and microscopically the most closely related species seems to be C. saturninus, which differs in the somewhat darker colours, red brown to chocolate lamellae and different ITS sequences. Other similar taxa are C. castaneus and C. assiduus. C. castaneus is smaller with darker colours and occurs mostly with conifers. C. assiduus differs by a thin, fugaceous veil, violaceous lamellae when young and a raphanoid smell (Mahiques, Ortega & Bidaud 2001). Based on both morphological and molecular data C. phlegmophorus can be assigned to subgen. Myxacium. The lack of clamp connections places it in sect. Defibulati (Moser 1969). As shown in our study and other phylogenetic rDNA analyses (Seidl 1999), sects Myxacium and Defibulati are sistergroups within subgen. Myxacium, and both represent an independent evolutionary lineage. Morphologically, C. phlegmophorus is somewhat reminiscent of C. pseudosalor, but differs in the brighter, more orange-brown pileus, and the slightly smaller and narrower spores. The results of the ITS data analysis confirm the distinctness of the two taxa. Our morphological and phylogenetic analyses demonstrated that the three Indian Cortinarius spp. belong to three different clades, each of them constituting an independent evolutionary lineage. This result disproves the hypothesis of geographically determined radiating clades of Cortinarius related to geographic isolation, the establishment of mycorrhizal interactions, and host specifity. The geological history of peninsular India could indicate a possible origin of these endemic taxa from Gondwanan ancestors. Peninsular India is a part of the Gondwanaland landmass, with a geological lineage of great antiquity. It is assumed that the Indian Plate of Gondwanaland detached itself during Middle Eocene and moved northwards from its original position near Madagascar in southern latitudes to crash against Laurasia in the northern tropical latitudes during the late Cretaceous (Ahmadullah & Nayar 1986). 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