Three New Species of Cortinarius From Kashmir Himalayan Coniferous Forests Based On Morphological And Molecular Evidence Sheikh Sajad Ahmed (  sheikhsajad.scholar@kashmiruniversity.net ) University of Kashmir https://orcid.org/0000-0003-4520-6734 Zafar A. Reshi University of Kashmir Bushra Jan University of Kashmir Khurshid I. Andrabi University of Kashmir Research Article Keywords: DNA barcoding, ectomycorrhiza, Mushrooms, Macrofungi, 3 new taxa DOI: https://doi.org/10.21203/rs.3.rs-585421/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract Cortinarius is the largest genus of mushroom forming fungi with several subgenera having ectomycorrhizal associations with coniferous trees and other plants. In view of limited studies on this speciose genus from the Himalayan region, a morpho-molecular phylogenetic approach was employed to study this taxon. Phylogenetic analysis and Bayesian inference of nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) separated these sequences along with the identical sequences from Gene bank into three distinct clads with high bootstrap values suggesting the possibility of new taxa. The new species were found to possess some diagnostic features that separated them from other closely related species in each section. Based on our study in the Kashmir Himalayan forests, we report three new species of Cortinarius from the Indian subcontinent. The identi ed species, C. cibum, C. neocephalixus, and C. nigricans belong to subgenera Myxacium, Phlegmacium and Telamonia. A taxonomic description of morphological characters is also provided for each new taxon. This study marks the beginning in studying the Cortinarius genus in Kashmir Himalaya using a combination of nuc rDNA ITS barcode approach together with morphological characters and microscopic spore analysis. The current study will help in lling the knowledge gaps in the study of Cortinarius and will further enrich the public DNA database for ease of comparative studies throughout the world. Introduction Cortinarius (Pers.) Gray is one of the largest genera in the order Agaricales, with reportedly 2250 species distributed throughout the world (He et al. 2019). The species of this genus form obligate ectomycorrhizal associations with diverse seed plants across various ecosystems (Garnica et al. 2016). The genus can be easily distinguished from other genera on the basis of presence of Cortina in young fruiting bodies and large number of rusty brown spores which provide the gills a brown color at maturity (Peintner et al. 2001). However, considerable phenotypic plasticity and convergence in morphological traits displayed by the species of this genus pose considerable di culty in correct identi cation of several species based on conventional morphological approach (Garnica et al. 2016; Badotti et al. 2017). Most of the Cortinarius species have been described from Europe and North America, while only few have been reported from other regions particularly from Asia (Peintner et al. 2004; Frøslev et al. 2005; Garnica et al. 2005; Frøslev et al. 2007; Teasdale et al. 2013; Garnica et al. 2016). The diversity of this genus from the Indian subcontinent remains poorly understood. The ectomycorrhizal Cortinarius species viz., Cortinarius conopileus, C. keralensis, and C. phlegmophorus spp. nov. were reported for the rst time from south India (Peintner et al. 2003). Cortinarius species were also reported in some earlier studies from Kashmir (Abraham 1993). However, only few reports of Cortinarius species associated with coniferous forests of Kashmir Himalaya are based on molecular phylogeny (Itoo et al. 2014, 2015). The Kashmir Himalayan region harbors a rich diversity of ectomycorrhizal fungi forming mutualistic association with the coniferous forest trees (Pande et al. 2004; Itoo et al. 2014). Earlier studies on macrofungal diversity have mostly focused on morphological characters and spore structure for the Page 2/23 identi cation of fungi (Watling and Abraham 1992; Abraham 1993; Kumar and Sharma 2011; Pala and WANI et al. 2011). Recently, the use of DNA sequences as successful molecular markers has improved the accuracy in identi cation of fungi. Several DNA barcodes have been employed for the delimitation of various living organisms up to species level. Among them, the nuc rDNA ITS1-5.8S-ITS2 region has been found to be a suitable molecular marker for the correct identi cation of fungi by the consortium of international barcode (Seifert 2009; Schoch et al. 2012; Schoch et al. 2014). Several studies have found that ITS barcode can prove signi cantly effective in distinguishing Cortinarius species (Garnica et al. 2016; Brandrud et al. 2018). Molecular approach together with morphological analysis can signi cantly improve the accuracy in identi cation of such fungi. In view of paucity of information about the genus Cortinarius in the Kashmir Himalaya and absolute reliance on the morphological characters for species identi cation in earlier studies, extensive surveys in several coniferous forests of this region we undertaken. Further, a combined morphological and molecular approach was employed for characterization of the specimens belonging to genus Cortinarius in order to ll the knowledge gap that exists at present regarding the ectomycorrhizal fungi in general and Cortinarius in particular. Materials And Methods Study area.– The study area included the coniferous forests of Kashmir Himalaya and the areas selected for survey included Sonmarg, Naranag, Yusmarg, Gulmarg, Pehelgam, Sinthan-Daksun, Hirpora, Lolab, Bungus-Nildori and Sadnah, spanning the entire Kashmir Himalayan region. The temperate climate of Kashmir Himalaya allows for dominance of evergreen coniferous forests with four distinct seasons of winter (December to February), spring (March to May), summer (June to August) and autumn (September to November). This Himalayan region receives major part of its precipitation in winter and spring seasons in the form of snow and occasional rains from western disturbances with an average mean maximum and minimum temperatures of Kashmir valley as 19.27 and 7.29 ℃, respectively, while as the average rainfall is 84 cm. The temperature starts to gradually rise from the spring season, after which the growth of macrofungal fruiting bodies is expected and collection is started. Sporocarp collection.– The sporocarps of all the available Basidiomycete fungi commonly called mushrooms were collected from the coniferous forests of Kashmir Himalaya dominated mainly by Cedrus deodara, Pinus wallichiana, Abies pindrow, and Picea smithiana along with Betula utilis occupying the tree line. With the onset of growing season, regular eld surveys were conducted after every fortnight for three consecutive years (2016–2018) during the entire growing season from early March to October. The eld surveys and sporocarp collections were carried out following the standard methods referred to as ‘Opportunistic sampling’ (Mueller et al. 2004; Atri 2005; Schmit and Lodge 2005; Halme et al. 2012a, 2012b). The sporocarps were photographed in the eld using digital camera and later dug out carefully using a knife and collected in separate bags. Fragile specimens were collected in separate containers to retain their structural integrity. The samples were taken to the laboratory, labelled Page 3/23 and the specimens stored under deep freeze conditions for further molecular investigations. The dried voucher specimens have been deposited in Kashmir University Botanical Garden Herbarium (KASH). Morphological analysis.– Fresh sporocarps were used for describing macroscopic characters like color, size, shape and detailed anatomical features of various parts. The color of the specimens was ascribed on the basis of color codes Kornerup and Wanscher (1978). The microscopic characters were described from thin sections of tissue mounted in water, 5% KOH, Melzer’s reagent, and Congo red using Light microscope with 100X resolution and tted with Amscope camera (Garnica et al. 2003; Niskanen et al. 2012). The spore measurements and morphology was based on several mature spores observed under light microscope. The Pileipellis structure was studied from sections cut halfway through the Pileus. A key to the new Cortinarius species from Kashmir Himalaya was developed based on the diagnostic characters. In this regard, we have referred to the morphology of Cortinarius taxa published by (Peintner et al. 2001, 2004). Molecular analysis.– DNA extraction. DNA was extracted from fresh sporocarp specimens using standard cetyltrimethylammonium bromide (CTAB)-chloroform method with few modi cations (Rogers and Bendich 1994; Porebski et al. 1997). For this, a small piece of fresh pileus was taken and ground into ne powder in liquid nitrogen using pestle and motor. The powdered material was transferred to 2 ml centrifuge tubes and 1ml of pre-warmed CTAB buffer (1M Tris HCl pH 8.0, 5M NaCl, 0.5M EDTA PH 8.0, CTAB, 2% 𝛽-mercaptoethanol) was added to it. The centrifuge tubes were incubated at 65 ℃ for 1 hour with periodic inversion. This was followed by centrifugation of these tubes at 12,000 rpm for 10–15 minutes in order to remove the cell debris. The supernatant from each of these tubes was transferred to new 1.5ml centrifuge tube and C.I mix (chloroform: isoamyl-alcohol in the ratio of 24:1) was added after which the tubes were centrifuged again at 12,000 rpm. This step was repeated twice and in between the RNA’ase treatment was given to the content in the tubes and incubated for 15 minutes. The upper layer was again separated, treated with 1 ml isopropanol and kept overnight at -20 ℃. Next day, the tubes were centrifuged again at 10,000 rpm for 10–20 minutes. The supernatant was discarded and the pellet was washed twice with 70% ice cold ethanol and spinned at 8,000 rpm for 5 minutes. The pellet was dried for 30 minutes in an incubator, re-dissolved in elution buffer, and stored at -20 ℃. The puri ed DNA was checked on 1% Agarose gel by staining with ethidium bromide and concentration was measured using Nano-Drop spectrophotometer. PCR ampli cation and puri cation. The isolated DNA was used as template and complete sequences of ITS1, 5.8S rRNA gene and ITS2 with partial sequences of 18S and 28S rRNA gene were ampli ed by Polymerase chain reaction in Applied Biosystems 2720 Thermal Cycler using four previously reported and widely used primers ITS1/ITS4, ITS1F/ITS4B (Gardes and Bruns, 1993). The 25𝜇L reaction mixture for PCR ampli cation contained 1𝜇L template DNA, 3 𝜇L PCR buffer, 2.5 𝜇L of 2mM dNTP’s, 2𝜇L of each primer, 1.5𝜇L of Mgcl2, 1 𝜇L of DMSO and 0.4𝜇L of Taq DNA polymerase. Ampli cations were performed in a thermal cycler with an initial denaturation step of 94 ℃ for 5 minutes followed by 35 cycles of 94 ℃ Page 4/23 for 1 minute, 52 ℃ for 30 seconds, and 72 ℃ for 1 minute and a nal extension of 72 ℃ for 10 minutes. PCR products were loaded on 1.5 % agarose gel, separated by electrophoresis (90 V, 180 mA, 50 min.), stained with ethidium bromide, and viewed in a gel documentation system. The PCR products were puri ed using GenElute™ PCR Clean-Up Kit (Sigma) according to manufacturer’s protocol and the DNA concentration was con rmed using NanoDrop (Spectrophotometer). The puri ed PCR products of the ITS ampli ed region were directly sequenced in both directions using the ITS1 and ITS4, ITS1F and ITS4B pair of ampli cation primers in Sci-Genome and Xcelris labs). Chromatograms were analyzed using chromas version 2.33 (Technelysium Pty Ltd). Phylogenetic analysis.– Sequence alignment. A BLAST (Basic Local Alignment Search Tool) search was carried out for the sequences using the National Center for Biotechnology Information (NCBI) USA (https://www.ncbi.nlm.nih.gov/) and UNITE fungal database (https://unite.ut.ee/). The sequences were matched in terms of the widely used ≥ 97% sequence similarity cut-off point for fungal species delimitation (Nilsson et al. 2008; Hibbett et al. 2011). In case of Cortinarius genus, the sequences that matched correctly having percentage identi cation > 99% with the sequences from database and showing consistency downstream, were assigned a species name with certainty while those with percentage identi cation < 99% were assigned a generic name (Garnica et al. 2016). The sequences were later deposited in NCBI database with accession numbers listed in Table 1. The rst hit on the blast results list was assumed to represent the best match, the next fully identi ed, and insu ciently identi ed matches were used to check for consistency. The specimens that showed ambiguity due to low sequence similarity were further con rmed using UNITE, and Mycobank database (http://www.mycobank.org/). The species names were further con rmed from Index Fungorum (http://www.indexfungorum.org). Page 5/23 Table 1 ITS sequences used in the phylogenetic analyses. Sequence name Voucher Accession No. Geographic origin Reference Cortinarius sp.nov K.HIM.1.1 MZ203578 Kashmir Himalaya, India New species Cortinarius sp.nov K.HIM.1.2 MZ203579 Kashmir Himalaya, India New species Cortinarius sp.nov K.HIM.1.3 MZ203580 Kashmir Himalaya, India New species Cortinarius sp. ANT275-HRL2130 MN992357.1 Canada Unpublished Cortinarius aff. pallidifolius ANT273-HRL2129 MN992334.1 Canada Unpublished Cortinarius olidus KS CO1159 KJ421068.1 Germany (Garnica et al. 2016) Cortinarius cephalixus TUB 011444 AY174784.1 Germany (Garnica et al. 2003) Cortinarius sp. 'squameopercomis' TEB397-16 MK358111.1 Hungary (Soop et al. 2019) Cortinarius pseudocephalixus IK98-1842 KF732634.1 Sweden (Liimatainen et al. 2014) Cortinarius aff. pallidifolius ANT082-QFB28694 MN992333.1 Canada: Quebec Unpublished Cortinarius herculeus TUB 019805 KJ421098.1 Germany Unpublished Cortinarius olidoamarus TUB 019787 KJ421042.1 Germany Unpublished Cortinarius misermontii IK872172 KF732622.1 Spain (Liimatainen et al. 2014) Cortinarius sp.nov K.HIM.2.1 MZ203581 Kashmir Himalaya, India New species Cortinarius sp.nov K.HIM.2.2 MZ203582 Kashmir Himalaya, India New species Cortinarius sp. OTA:60173 MN846506.1 New Zealand Unpublished Cortinarius sp. OTA:60187 MN846508.1 New Zealand Unpublished Cortinarius sp. OTA:60313 MN846514.1 New Zealand Unpublished Page 6/23 Sequence name Voucher Accession No. Geographic origin Reference Cortinarius sp. OTA:61935 MN846523.1 New Zealand Unpublished Cortinarius sp. PDD 103885 PDD:103885 KF727393.1 New Zealand Unpublished Cortinarius vibratilis F16046 FJ157098.1 Canada (Harrower et al. 2011) Cortinarius sp. CM13_090 KY774102.1 New Caledonia Unpublished Cortinarius sp. FC1752 MG553130.1 Australia Unpublished Cortinarius croceocristallinus PAM08082212 JQ749630.1 France Unpublished Cortinarius vibratilis DAVFP 26240 EU821696.1 Canada (Harrower et al. 2011) Cortinarius croceocaeruleus TUB 011833 AY669590.1 Germany (Garnica et al. 2005) Cortinarius sp. PERTH:06641814 FC748 MG553035.1 Australia Unpublished Cortinarius sp K.HIM.3.1 MZ203583 Kashmir Himalaya, India Unpublished Cortinarius sp K.HIM.3.2 MZ203584 Kashmir Himalaya, India Unpublished Cortinarius varius TUB 011392 AY174792.1 Germany (Garnica et al. 2003) Cortinarius sp. Daniel 1 FJ717603.1 Canada (Harrower et al. 2011) Cortinarius variosimilis VMS1 FJ717598.1 Canada (Harrower et al. 2011) Cortinarius variosimilis VMS26 FJ717596.1. Canada (Harrower et al. 2011) Cortinarius sp. 061708_2015_LMF1B KY510816.1 USA: WA, Yakima Unpublished Cortinarius variosimilis F16580 GQ159915.1 Canada (Harrower et al. 2011) Cortinarius varius TUB 019715 KJ421143.1 Germany (Garnica et al. 2016) Page 7/23 Sequence name Voucher Accession No. Geographic origin Reference Cortinarius caesiostramineus TUB 019702 KJ421179.1 Germany (Garnica et al. 2016) Cortinarius varius TUB 019761 KJ421000.1 Germany (Garnica et al. 2016) Cortinarius sp.nov K.HIM.4.1 MZ203585 Kashmir Himalaya, India New species Cortinarius sp.nov K.HIM.4.2 MZ203586 Kashmir Himalaya, India New species Cortinarius sp.nov K.HIM.4.3 MZ203587 Kashmir Himalaya, India New species Cortinarius brunneovernus PNWKC-07-125-12 KC608577.1 USA: Washington (Liimatainen et al. 2014) Cortinarius brunneovernus DM05-14 KC608580.1 USA: Washington (Liimatainen et al. 2014) Cortinarius brunneovernus JLF8693 MW341327.1 USA: Jackson County Unpublished Cortinarius brunneovernus JLF8670 MW341323.1 USA: Jackson County Unpublished Cortinarius brunneovernus K.HIM.KU.8.1 MW040382.1 Kashmir Himalaya Unpublished Cortinarius brunneovernus K.HIM.KU.8 MW040381.1. Kashmir Himalaya Unpublished Cortinarius brunneovernus WTU:J.F. Ammirati 13331 NR_131826.1 USA: Washington (Liimatainen et al. 2014) Cortinarius subcaesiobrunneus HMJAU:44434 MK234565.1 China (Xie et al. 2020) Cortinarius brunneovernus 110501-1 KC608579.1 USA: Washington (Liimatainen et al. 2014) AF124699.1 Netherlands (Aanen et al. 2000) AF325643.1 USA (Peintner et al. 2001) Hebeloma circinans Hebeloma fastibile IB 19940036 Page 8/23 Data analysis. An ITS data set comprising newly generated sequences and identical sequences retrieved from the fungal database were used in the analysis (Table 1). The sequences of genus Hebeloma were selected as outgroups following (Danks et al. 2010; Pastor et al. 2019). DNA sequences that were of insu cient quality or < 350 base pairs in length were discarded from the current analysis. The initial sequence alignment was performed for all the sequences using Muscle program of ClustalW (http://www.clustal.org/) (Sievers et al. 2011) and the alignment was further re ned with BioEdit 7.0.9 (Hall 1999, 2004). Maximum likelihood (ML) analysis was performed using phyML and MEGA6 (Tamura et al. 2013) and Branch support was assessed with Bootstrap analysis run with 1000 replicates (Felsenstein 1985). Bayesian inference (BI) analysis was performed using MrBayes 3.2.2 (Ronquist et al. 2012). A threshold dissimilarity value of 0.5% for species hypothesis was used to assess the diversity of Cortinarius in order to improve the accuracy and ease of comparison (Kõljalg et al. 2013). A value of 1% is considered signi cant to distinguish species of Cortinarius using ITS region (Garnica et al., 2016). Results Phylogenetic analysis.– The ITS sequences belonging to the Cortinarius species along with the homologous sequences obtained from Genebank database comprised the nal dataset of 52 sequences, that were subjected to phylogenetic analysis, in which sequences of two Hebeloma species were selected as an out-group (Table 1). These included ITS sequences representing Cortinarius species from Europe, N. America, South America and Asia with original sequences ranging from 600 to 700 base pairs. The best t model for the dataset was chosen as GTR + 1 using jModelTest program. A Phylogenetic tree depicting the phylogenetic position of different species was generated using Maximum Likelihood method in phyML and MEGA X software, which generated comparatively similar trees with high statistical support values as shown (Fig. 1). The ITS sequences were found to be highly species speci c in case of Cortinarius species, but in some of the cases, the NJ trees constructed from these sequences did not share similar topology possibly because of dissimilarities in length of ampli ed fragment used in the study. The inter-speci c and intra-speci c distances were calculated and were found to be ideal for the DNA barcodes with the interspeci c distances exceeding the intraspeci c distances. The Bayesian inference values calculated for the aligned sequences were > 0.95 which was in conformity with the obtained results as shown in the Phylogram. The resulting Phylogram of the ITS sequences of Cortinarius grouped them into several clearly distinct clads with moderate to high bootstrap values. However, the basal relationships of the clads were poorly resolved possibly because of the low resolution capacity of ITS sequences in determining the basal relationships within Cortinarius. The sequences of three new specimens clustered individually into separate clads from the other sequences obtained from the gene bank with high statistical support values thus conforming the identity of new taxa (Fig. 1). This approach differentiated three novel species belonging to Cortinarius genus. Page 9/23 Taxonomy Cortinarius cibum S.S. Ahmed and Z.A. Reshi, sp. nov. Diagnosis. Pileus conical to hemispherical and slimy with lustrous brown color and light pink gills. Universal veil sparsely present. Basidiospores ellipsoid and irregularly ornamented. Found mostly on wet ground surrounded by Sphagnum grass. The ITS sequence differs from other sequences by at least 10 insertions and 13 substitutions. Holotype. India. Union Territory of Jammu and Kashmir, District Kupwara, Town Handwara, Mawer valley, Bungus-Nildori, Coniferous forest (Dominated by Abies pindrow), 34º27' 19'' N, 74º23' 48'' E, Altitude 2715m, 6 July 2017, S.S. Ahmed, Genebank acc. No. MZ203581. Etymology. The name refers to its brown meat color of pileus. Description. Pileus 2.2–3.7 cm in diam., conical when young, campanulate with slight dome in center at maturity, lustrous brown (6D3–6) with slightly paler margins and dark at the center, glutinous, glabrous with smooth surface, margins entire when young, discontinuous breaking into three or more parts at maturity. Lamellae adnexed, subdistant to moderately spaced, light pink (4D3–5), pale yellow (2C3–4) when young, turning brown at maturity, margins entire, smooth. Stipe 4.3–6.2 cm long, 1.7–2.4 cm thick at apex to base, cylindrical to slightly bulbous and tapering at base, pale white to greyish brown (6C3–5) when young turning pale yellow at maturity, surface with white brils allover. Universal veil absent in mature sporocarps. Context greyish white to pale yellow. Odor indistinct, taste bitter. Exsiccata brown (5F8) to black brown (7F5) in color. Basidiospores 6.2–7.3 × 5.3–4.9 µm, Q = 1.11–1.37, X = 6.3– 7.6 × 6.4– 6.9µm, Q = 1.44–1.65 (30 spores), ellipsoid, slightly moderately verrucose. Basidia 4-spored, cylindrical to clavate, 23–44 × 6–11 µm, thin-walled, slightly hyaline to olivaceous brown in 5% KOH. Lamellar edges sterile, sterile cells cylindrical to clavate, 13–21 × 4.1–8.6 µm, thin-walled, hyaline in 5% KOH. Lamellar trama hyphae irregular, smooth, pale olivaceous to light yellow in 5% KOH. Pileipellis– epicutis hyphae cylindrical to elongated, 2–5 µm wide, pale yellow to olivaceous brown in 5% KOH, smooth, hypocutis well developed, hyphae 11–29 µm wide, smooth, sub-cellular, thin-walled, hyaline in 5% KOH. Pileus trama hyphae thinwalled, smooth, hyaline to slightly olivaceous brown in 5% KOH. Clamp connections present. ITS sequence. The ITS sequence of two specimens of C. cibum are 650–680 bp long (3 collections, Table 1). All the three sequences (MZ203581, MZ203582 and MW547496) are identical with no indels. The ITS sequence of C. cibum (Holotype) differs from other sequences in the section Vibratilis by at least 13 substitutions and 10 insertions. Ecology and distribution. Mycorrhizal with conifers; mostly growing scattered and very rarely sighted, found in summer months, growing in the mid altitudinal gradients of Kashmir Himalaya. Page 10/23 Additional specimens examined. India. Union Territory of Jammu and Kashmir, District Kupwara, Town Handwara, Mawer valley, Bungus-Nildori, Coniferous forest (Dominated by Abies pindrow with scattered Pinus sp.), 34º35' 44'' N, 74º32' 11'' E, Altitude 2732m, 6 July 2017, S.S. Ahmed, Genebank acc. No. MZ203581; 34º38' 25'' N, 74º24' 16'' E, Altitude 2635m, 6 July 2017, S.S. Ahmed Genebank acc. No. MZ203582; 34º35' 22'' N, 74º42' 33'' E, Altitude 2629m, 6 July 2017, S.S. Ahmed Genebank acc. No. MW547496. Comments. Cortinarius cibum can be easily distinguished from other species by the distinct lustrous surface and deep brown color if pileus. The gills are pink in young specimens and spores are much smaller in size compared to other species. The ITS sequence shows similarity of less than 92 percent from other closely related sequences and separates out as a separate cluster in the phylogenetic analysis. Cortinarius neocephalixus. S.S. Ahmed and Z.A. Reshi, sp. nov. Diagnosis. Pileus 3.6–5.4cm in Diam., glutinous, glabrous with brownish universal veil remnants scattered over the surface. Lamellae moderately crowded, pale yellow. Stipe clavate, grayish, tapering towards the end. Basidiospores 8.7–10.3 × 5.5–6.4 µm, Amygdaliform. The ITS sequences differ from other species by at least 11 substitutions and 7 indel positions. Holotype. India. Union Territory of Jammu and Kashmir, District Kupwara, Town Handwara, Mawer valley, Bungus-Nildori, Coniferous forest (Dominated by Abies pindrow), 34º33' 35'' N, 74º11' 10'' E, Altitude 2635m, 5 July 2017, S.S. Ahmed, Genebank acc. No. MZ203580 Etymology. The name refers to its a nity with C. cephalixus. Description. Pileus 3.6–5.4 cm in diam., hemispherical to convex when young, becoming broadly convex at maturity, ochraceous yellow (4B7–8), reddish brown (9E6–8) at the centre, pale yellow (4A4) towards margins, glutinous, glabrous, with very small scales in the center of the pileus, margins smooth and incurved. Universal veil remnants usually abundant, forming brown, loose scales or patches on the upper surface of pileus. Lamellae 6–9 mm broad, adnate, moderately crowded, pale yellow to grayish white (4A2) when young, turning brown (5F8) at maturity, crenulate margins. Stipe 3.5–5.4 long, 0.7–.09 cm thick at apex, 1.5–2.5 cm thick near base, base cylindrical to clavate, thick girdled to brillose of yellowbrown (6C–4) to more rarely whitish (4A3) veil, color grayish white (2A2), ochraceous white (2A1–2) towards base, often completely brownish in lower part from veil remnants; Universal veil prominent, ochraceous brown (9F6) to rather dark brown (8F6–8), rendering the lower part of stipe distinctly girdledoccose, sometimes more brillose-peronate. Context white, more greyish in stipe apex when young. Odor and taste not distinct, yeast like. Reaction to 3% KOH– negative to light brown at pileus, stipe and base. Exsiccata brown (6E5) to dark brown (6F6) in color. Basidiospores 7.9–10.3 × 5.5–6.4 µm, Q = 1.31–1.67, X = 9.2– 9.8 × 6.3– 6.6µm, Q = 1.37–1.48 (30 spores, 3 collections), Amygdaliform, distinctly and usually fairly densely verrucose. Basidia 4-spored, Page 11/23 cylindrical to clavate, 24–46 × 5–9 µm, moderately thin-walled, yellowish brown to olivaceous brown in 5% KOH. Lamellar edge sterile, sterile cells cylindrical-clavate, 9–22 × 3–8 µm, thin-walled and slightly hyaline in 5% KOH. Lamellar trama hyphae regular, smooth, olivaceous in 5% KOH. Universal veil having thin-walled hyphae, often with well-developed, yellow-brown, encrusted, parietal and intracellular pigment, hyaline to olivaceous yellow in 5% KOH. Gelatinous layer composed of various hyphal strata; Pileipellis duplex, with a distinctly subcellular hypoderm, elements often irregular, almost isodiametric, and imbedded in a brown, amber-like matrix, basal epicutis often with distinct, brown, encrusted pigment. ITS sequence. The ITS sequence of C. neocephalixus are 590–690 bp long (3 collections, Table 1). All the three sequences (MZ203578, MZ203579 and MZ203580) are identical with no indels. The ITS sequence of C. neocephalixus (Holotype) differs from other sequences in the section Phlegmacium by at least 11 substitutions and 7 indel positions. Ecology and distribution. Mycorrhizal with conifers, mostly growing scattered and rare, found in summer and early fall, growing in the mid elevational gradients of Kashmir Himalaya. Additional specimens examined. India. Union Territory of Jammu and Kashmir, District Kupwara, Town Handwara, Mawer valley, Bungus-Nildori, Coniferous forest (Dominated by Abies pindrow), 34º34' 39'' N, 74º13' 12'' E, Altitude 2622m, 5 July 2017, S.S. Ahmed, Genebank acc. No. MZ203578; 33º28' 22'' N, 74º18' 16'' E, Altitude 2645m, 6 July 2017, S.S. Ahmed Genebank acc. No. MZ203579; 34º26' 25'' N, 73º28' 11'' E, Altitude 2638m, 6 July 2017, S.S. Ahmed Genebank acc. No. MZ203580. Comments. Morphologically, Cortinarius neocephalixus shares close a nity with Cortinarius cephalixus but a closer examination of various structures reveals contrasting differences like the glabrous pileus with scattered universal veil remnants, short pear shaped stipe and the size and structure of basidiospore. Molecular phylogenetic analysis also revealed that the ITS sequences of C. neocephalixus showed similarity of less than 97 percent with other closely related sequences and clustered separately in the Phylogram with high bootstrap values. Cortinarius nigricans S.S. Ahmed and Z.A. Reshi, sp. nov. Diagnosis. Pileus 4.3–6.9 cm in diam. Surface brillose, having radiating brils from the centre with strongly incurved margins, dark brown in color. Lamella adnate, close to subdistant. Stipe with slight subapical bulb with spongy tissue surrounding the base and a pointed end. Basidiospores 6.6–9.3 × 6.1–9.8 µm, ellipsoid-subamygdaloid. The ITS sequence of the Holotype differs from other closely related species in the section by at least 8 substitutions and 6 indels. Holotype. India. Union Territory of Jammu and Kashmir, District Kupwara, Town Handwara, Mawer valley, Bungus-Nildori, Coniferous forest (Dominated by Abies pindrow), 34º38' 25'' N, 72º28' 14'' E, Altitude 2475m, 20 July 2018, S.S. Ahmed, Genebank acc. No. MZ203585 Etymology. The name refers to its morphological a nity with C. brunneus. Page 12/23 Description. Pileus 4.3–6.9 cm in diam., umbonate to broadly umbonate, becoming uplifted to undulate when mature sporocarps, with incurved to decurved edges, surface nely innately and radially brillose, reddish brown (5D5–6) to dark brown all over (5E7–8), surface moderately hygrophanous. Universal veil remnants very sparsely present on pileus or entirely absent in mature sporocarps. Lamellae adnate, moderately dense, close to subdistant, 6–8 mm broad; thick, brown, turning dark brown (5D4–6) at maturity. Stipe 3.8–4.2 × 1.1–2.1 cm thick at apex; cylindrical to subbulbous, with Ocher ring-like velum zones around the base, at rst whitish to pallid silky brillose with watery brown streaks, soon developing brownish tones and darker brown colors all over the surface. Universal veil persistent, thin, sheathing the base of young stipe. The lower part of basal bulb surrounded by thick network of grayish-white mass of hyphae. Context in pileus and stipe uniformly dark brown, hygrophanous. Odor and taste not distinct, earthy. Reaction to KOH–Negative reaction of pileus, stipe and base. Exsiccata dark grayish brown (10YR 4–2) to brown (10YR 4–3). Basidiospores 7.2–8.7 µm × 5.4–6.7 µm, Q = 1.21–1.42, X = 8.3– 8.8µm × 6.3– 6.6µm, Q = 1.32– 1.37, subglobose to broadly ellipsoid, moderately to strongly verrucose, strongly ornamented at the apex, dextrinoid. Basidia 4–spored, clavate, 33–37 × 9–11 µm, hyaline having yellow granulose contents in 5% KOH. Lamellar trama hyphae irregular, smooth-walled, olivaceous to olivaceous brown in 5% KOH. Pileipellis duplex, pale olivaceous yellowish brown, epicutis thin to moderately thick, hyphae 2.5–10 µm wide, with yellowish brown granular contents or sometimes hyaline, smooth. Hypoderm distinct, thick, elements 25–55 µm × 15–25 µm, hyaline or walls with brown pigment, smooth. ITS sequence. The ITS sequence of C. nigricans are 560–600 bp long (3 collections, Table 1). All the three sequences (MZ203585, MZ203586, and MZ203587) are identical with no indels. The ITS sequence of C. nigricans (Holotype) differs from other sequences in the section Telamonia by at least 8 substitutions and 6 indels. Ecology and distribution. Mycorrhizal with conifers; mostly growing scattered, found in summer, growing in the mid altitudinal gradients of Kashmir Himalaya. Additional specimens examined. India. Union Territory of Jammu and Kashmir, District Kupwara, Town Handwara, Mawer valley, Bungus-Nildori, Coniferous forest (Dominated by Abies pindrow), 33º34' 26'' N, 74º27' 21'' E, Altitude 2721m, 20 July 2018, S.S. Ahmed, Genebank acc. No. MZ203585; 33º34' 23'' N, 74º17' 15'' E, Altitude 2643m, 20 July 2018, S.S. Ahmed Genebank acc. No. MZ203586; 34º22' 25'' N, 74º28' 17'' E, Altitude 2676m, 20 July 2018, S.S. Ahmed Genebank acc. No. MZ203587. Comments. Cortinarius nigricans is morphologically similar to C. brunneus with slight differences in the color and shape of stipe and a white cottony mass around its base. However, the Molecular analysis of ITS sequences of C. nigricans reveals a distinct difference from other related species by clustering separately in Phylogram and shares a similarity of only 96 percent with those sequences. KEY TO NEW CORTINARIUS SPECIES OF KASHMIR HIMALAYA AND MORPHOLOGICALLY SIMILAR SPECIES IN EACH SECTION Page 13/23 1. Pileus and sometimes stipe also viscid, at least in young specimens…….…….….… 2 1฀. Pileus and stipe dry, hygrophanous, stem peronate or annulate from the remnants of the veil in addition to the cortina …………………………………….….…… (Telamonia) 5 2. Pileus viscid, stipe dry, gills bluish grey to violet, or with violet edges...…………………………....………………………….………… (Phlegmacium) 3 2฀. Pileus and stipe viscid, taste bitter, spores less than long, punctate to almost smooth, if subglobose, smaller ………………………………………………….…… (Myxacium) 4 3. Pileus viscid, glabrous, orange brown, yellow brown towards disk margins, slightly granulose with very small scales towards the center of the pileus, Lamellae pale ochraceous to pale argillaceous, adnate to emarginate, crowded, edges uneven. Stipe clavate, apex whitish, pallid, Universal veil persistent below cortina forming thick yellow brown occose ring-zone near apex. Spores marbled to nely verrucose, slender, almond shaped, 8.8–11.7 × 5–5.9 µm……………………………………………..................Cortinarius cephalixus 3฀. Pileus glutinous, glabrous, ochraceous yellow, darker towards centre, with brownish universal veil remnants scattered over the surface and towards margins. Lamellae pale yellow-greyish white, adnate, moderately crowded. Stipe clavate, pale-grayish white, tapering towards the end with brown veil remnants scattered over the surface. Basidiospores amygdaliform, distinctly and usually fairly densely verrucose, 7.9–10.3 × 5.5–6.4 µm..........................................................................................Cortinarius neocephalixus 4. Pileus yellow to orange yellow, slimy when fresh with pale and entire margins. Stipe cylindrical to clavate and slightly tapering at base. Spores ellipsoid, verrucose. Universal veil white surrounding the base of stipe. Taste of esh and cuticle very bitter, spores ellipsoid small at most 8–10 µm long…………………………………….…Cortinarius vibratilis 4฀. Pileus conical when young, lustrous brown glutinous, glabrous, margins entire when young, discontinuous breaking into three or more parts at maturity. Stipe cylindrical to slightly bulbous and tapering at base. Universal veil absent in mature sporocarps. Taste of esh bitter. Spores ellipsoid, slightly moderately verrucose 6–7 µm…Cortinarius cibum 5. Pileus hygrophanous and not viscid, surface nely innately rivulose, margin light brown to brownish tan. Lamellae subdistant to distant, adnexed with decurrent line to deeply adnexed. Stipe clavate to subbulbous. Universal veil: whitish, thinly sheathing stipe at rst. Spores subglobose, ellipsoid to broadly ovoid, thick-walled, moderately to strongly verrucose, somewhat more strongly ornamented at the apex, strongly dextrinoid……………………………………………………Cortinarius brunneovernus 5฀. Pileus slightly hygrophanous, surface innately and radially brillose, entirely dark brown. Lamellae adnate, close to subdistant. Stipe cylindrical to subbulbous, with Ocher ring-like velum zones around the base. Universal veil persistent, thin, sheathing the base of young stipe. The lower part of basal bulb Page 14/23 surrounded by thick network of grayish-white mass of hyphae. Spores subglobose to broadly ellipsoid, strongly ornamented………………………………………………………..Cortinarius nigricans Discussion Cortinarius is a relatively large and species rich genus of Agaricales with most of the species described from Europe and North America (Garnica et al. 2003; Liimatainen et al. 2014). The diversity exploration studies of this genus in Asian region have only recently been initiated, particularly in China, while only few studies have been carried out in Indian sub-continent (Xie et al. 2020). The Kashmir Himalayan region has a temperate climate that shares similar a nities with the temperate regions of Europe and N. America, supporting a rich diversity of ora with which diverse fungi form several associations. Some of the fungi reported earlier from this region are also present in the European and N. American forests having similar ITS sequence homologies (Itoo et al. 2015). These species form mycorrhizal associations with conifers as reported in studies from Europe and N. America. The Cortinarius species were reported for the rst time from south India (Peintner et al. 2003). So far only few studies have been conducted on Cortinarius genus associated with the conifers in Himalayan region. Further studies are required in order to assess the diversity of this genus in Himalaya. The present study mainly focused on the diversity of Cortinarius species present in the coniferous forests of Kashmir Himalaya. Morphological observations of several specimens of each species placed them in particular taxonomic groups, but revealed some contrasting characters that separated these taxa from earlier recorded species. This was followed by molecular characterization using rDNA ITS barcode that revealed many differences at several DNA loci and showed lower similarity values with already known sequences in Genebank. Thus, using a combination of morphological characters, spore analysis and molecular phylogenetic analysis, four novel taxa were identi ed. Among the studied species, two belonged to subgenus Phlegmacium, which is a diverse group distributed throughout the Northern Hemisphere. Another species belonged to subgenus Myxacium having several species recorded from Europe and N. America, while one species belonged to subgenus Telamonia. The Cortinarius genus has been further subdivided into several sections and subsections by several authors (Garnica et al. 2003); but the delimitation of such groups remains an active area of debate, hence has been excluded from the current study. The ITS analysis employed in the current study was able to delimit the species more accurately which further con rmed the reliability of ITS as a suitable barcode for Cortinarius species as found in previous studies (Liimatainen et al. 2014; Garnica et al. 2016). All the four taxa that were discovered from the Indian subcontinent were novel species reported for the rst time, thus con rming the knowledge gaps and the need for further taxonomic studies on this mycorrhizal genus. Thus, further studies focusing on the diversity of Cortinarius genus in Kashmir Himalaya will help us in understanding the mycorrhizal associations formed by these species with conifers. This study will partially ll the knowledge gap and will further help in identifying the environmental unknowns obtained from Metagenomic studies of environmental samples by sequence similarities in public data bases. The molecular studies on this genus will further enrich the public DNA databases thus helping in comparing the sequences as well as associated preserved specimens throughout the world for futuristic studies. Page 15/23 Declarations ACKNOWLEDGEMENT The authors are grateful to the Head, Department of botany, University of Kashmir, Srinagar for providing laboratory facilities and to University Grants Commission, New Delhi for nancial support under its scheme, namely “Centre with Potential for Excellence in Particular Areas”. Funding: The work was partially funded by UGC under its CPEPA programme. Con icts of interest/Competing interests: The authors declare that there was no con ict of interest with any person, organization or institution. Availability of data and material: The ITS data is available in the Genebank public database and the specimens have been deposited in the KASH herbarium. Code availability: Not applicable. Authors' contributions All the authors have contributed in the study and in the preparation of the manuscript. Mr. Sheikh Sajad Ahmed has worked on the laboratory analysis, initial preparation and further drafting of nal manuscript. Prof. Zafar A. Reshi has supervised the whole process from conceptualization, methodology, and laboratory analysis to review and editing the nal manuscript. Ms. Bushra Jan has contributed to the taxonomic study of the specimens and commented on various sections of the previous versions of draft. Prof. Khurshid I. Andrabi has supervised the laboratory analysis of applied molecular methods. References 1. Abraham SP (1993) Larger fungi from Kashmir-X. Indian Journal of Forestry. 16:204-13. 2. Atri NS (2005) Wild mushrooms-collection and identi cation. Frontier in mushroom biotechnology. 926. 3. Badotti F, de Oliveira FS, Garcia CF, Vaz AB, Fonseca PL, Nahum LA, Oliveira G, Góes-Neto A (2017) Effectiveness of ITS and sub-regions as DNA barcode markers for the identi cation of Basidiomycota (Fungi). BMC microbiology. 17:1-2. https://doi.org/10.1186/s12866-017-0958-x 4. Brandrud TE, Schmidt-Stohn G, Liimatainen K, Niskanen T, Frøslev TG, Soop K, Bojantchev D, Kytövuori I, Jeppesen TS, Bellù F, Saar G (2018) Cortinarius sect. Riederi: taxonomy and phylogeny of Page 16/23 the new section with European and North American distribution. Mycological Progress. 17:13231354. https://doi.org/10.1007/s11557-018-1443-0 5. Danks M, Lebel T, Vernes K (2010) ‘Cort short on a mountaintop’–Eight new species of sequestrate Cortinarius from sub-alpine Australia and a nities to sections within the genus. Persoonia: Molecular Phylogeny and Evolution of Fungi. 24:106. 10.3767/003158510X512711 . Felsenstein J (1985) Con dence limits on phylogenies: an approach using the bootstrap. Evolution. 39:783-91. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x 7. Frøslev TG, Jeppesen TS, Læssøe T, Kjøller R (2007) Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe. Molecular phylogenetics and evolution. 44:217-27. https://doi.org/10.1016/j.ympev.2006.11.013 . Frøslev TG, Matheny PB, Hibbett DS (2005) Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies. Molecular Phylogenetics and Evolution. 37:602-18. https://doi.org/10.1016/j.ympev.2005.06.016 9. Gardes M, Bruns TD (1993) ITS primers with enhanced speci city for basidiomycetes‐application to the identi cation of mycorrhizae and rusts. Molecular ecology. 2:1138. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x 10. Garnica S, Schön ME, Abarenkov K, Riess K, Liimatainen K, Niskanen T, Dima B, Soop K, Frøslev TG, Jeppesen TS, Peintner U (2016) Determining threshold values for barcoding fungi: lessons from Cortinarius (Basidiomycota), a highly diverse and widespread ectomycorrhizal genus. FEMS Microbiology Ecology. 92. https://doi.org/10.1093/femsec/ w045 11. Garnica S, Weiß M, Oertel B, Oberwinkler F (2005) A framework for a phylogenetic classi cation in the genus Cortinarius (Basidiomycota, Agaricales) derived from morphological and molecular data. Botany. 83:1457-77. https://doi.org/10.1139/b05-107 12. Garnica S, Weiß M, Oertel B, Oberwinkler F (2003) Phylogenetic relationships of european Phlegmacium species (Cortinarius, Agaricales). Mycologia. 95:115570. https://doi.org/10.1080/15572536.2004.11833025 13. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. InNucleic acids symposium series. 41: 95-98. 14. Hall T (2004) BioEdit version 7.0. 0 Distributed by the author, website: www mbio ncsu edu. BioEdit/bioedit html. 15. Halme P, Heilmann-Clausen J, Rämä T, Kosonen T, Kunttu P (2012) Monitoring fungal biodiversity– towards an integrated approach. Fungal Ecology. 5:75058. https://doi.org/10.1016/j.funeco.2012.05.005 1 . Halme P, Kotiaho JS. 2012. The importance of timing and number of surveys in fungal biodiversity research. Biodiversity and Conservation. 21:205-19. https://doi.org/10.1007/s10531-011-0176-z 17. He MQ, Zhao RL, Hyde KD, Begerow D, Kemler M, Yurkov A, McKenzie EH, Raspe O, Kakishima M, Sanchez-Ramirez S, Vellinga EC (2019) Notes, outline and divergence times of Basidiomycota. Fungal Diversity. 99:105-367.https://doi.org/10.1007/s13225-019-00435-4 Page 17/23 1 . Hibbett DS, Ohman A, Glotzer D, Nuhn M, Kirk P, Nilsson RH (2011) Progress in molecular and morphological taxon discovery in Fungi and options for formal classi cation of environmental sequences. Fungal biology reviews. 25:38-47. https://doi.org/10.1016/j.fbr.2011.01.001 19. Itoo ZA, Basharat Q, Majeed ST, Andrabi KI, Reshi ZA (2014) Ectomycorrhizal fungal species of Kashmir Himalaya: identi cation and characterization by ITS analysis. Brazilian Journal of Botany. 37:531-42. https://doi.org/10.1007/s40415-014-0081-2 20. Itoo ZA, Reshi ZA, Basharat Q, Majeed ST, Andrabi KI (2015) Identi cation and characterization of Ectomycorrhizal Cortinarius species (Agaricales, Basidiomycetes) from temperate Kashmir Himalaya, India, by ITS barcoding. Advances in Molecular Biology. 2015. https://doi.org/10.1155/2015/507684 21. Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AF, Bahram M, Bates ST, Bruns TD, Bengtsson‐ Palme J, Callaghan TM, Douglas B (2013) Towards a uni ed paradigm for sequence‐based identi cation of fungi. Molecular Ecology. 22: 5271-5277. https://doi.org/10.1111/mec.12481 22. Kumar SA, Sharma YP (2011) Diversity of wild mushrooms from Jammu and Kashmir (India). InProceedings of the 7th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7). 568-77. 23. Liimatainen K, Niskanen T, Dima B, Kytövuori I, Ammirati JF, Frøslev TG (2014) The largest type study of Agaricales species to date: bringing identi cation and nomenclature of Phlegmacium (Cortinarius) into the DNA era. Persoonia: Molecular Phylogeny and Evolution of Fungi. 33:98. 10.3767/003158514X684681 24. Mueller GM, Schmit JP, Ryvarden SM, O'Dell TE, Lodge DJ, Leacock PR, Mata M, Umania L, Czederpiltz DL (2004) Recommended protocols for sampling macrofungi. Biodiversity of fungi: inventory and monitoring methods. Amsterdam; Boston: Elsevier Academic Press. 168-172. https://doi.org/10.1016/B978-0-12-509551-8.X5000-4 25. Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson KH (2008) Intraspeci c ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identi cation. Evolutionary bioinformatics. 4:EBOS653. https://doi.org/10.4137/EBO.S653 2 . Niskanen T, Laine S, Liimatainen K, Kytövuori I (2012) Cortinarius sanguineus and equally red species in Europe with an emphasis on northern European material. Mycologia. 104: 24253. https://doi.org/10.3852/11-137 27. Pala SA, WANI AH, Bhat MY (2011) Six hitherto unreported Basidiomycetic macrofungi from Kashmir Himalayas. Nusantara Bioscience. 3. https://doi.org/10.13057/nusbiosci/n030206 2 . Pande V, Palni UT, Singh SP (2004) Species diversity of ectomycorrhizal fungi associated with temperate forest of Western Himalaya: a preliminary assessment. Current Science. 25:1619-23. 29. Pastor N, Chiapella J, Kuhar F, Mujic AB, Crespo EM, Nouhra ER (2019) Unveiling new sequestrate Cortinarius species from northern Patagonian Nothofagaceae forests based on molecular and morphological data. Mycologia. 111:103-17. https://doi.org/10.1080/00275514.2018.1537350 Page 18/23 30. Peintner U, Moncalvo JM, Vilgalys R (2004) Toward a better understanding of the infrageneric relationships in Cortinarius (Agaricales, Basidiomycota). Mycologia. 96:104258. https://doi.org/10.1080/15572536.2005.11832904 31. Peintner U, Moser MM, Thomas KA, Manimohan P (2003) First records of ectomycorrhizal Cortinarius species (Agaricales, Basidiomycetes) from tropical India and their phylogenetic position based on rDNA ITS sequences. Mycological Research. 107:48594. https://doi.org/10.1017/S0953756203007585 32. Peintner U, Bougher NL, Castellano MA, Moncalvo JM, Moser MM, Trappe JM, Vilgalys R (2001) Multiple origins of sequestrate fungi related to Cortinarius (Cortinariaceae). American Journal of Botany. 88:2168-79. https://doi.org/10.2307/3558378 33. Porebski S, Bailey LG, Baum BR (1997) Modi cation of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant molecular biology reporter. 15:815. https://doi.org/10.1007/BF02772108 34. Rogers SO, Bendich AJ (1994) Extraction of total cellular DNA from plants, algae and fungi. In Plant molecular biology manual, Springer, Dordrecht. 183-190. https://doi.org/10.1007/978-94-011-05118_12 35. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: e cient Bayesian phylogenetic inference and model choice across a large model space. Systematic biology. 61: 539-42. https://doi.org/10.1093/sysbio/sys029 3 . Schmit JP, Lodge DJ (2005) Classical methods and modern analysis for studying fungal diversity. Mycology Series. 23:193. 37. Schoch CL, Robbertse B, Robert V, Vu D, Cardinali G, Irinyi L, Meyer W, Nilsson RH, Hughes K, Miller AN, Kirk PM (2014) Finding needles in haystacks: linking scienti c names, reference specimens and molecular data for Fungi. Database. 2014. https://doi.org/10.1093/database/bau061 3 . Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences. 109: 62416. https://doi.org/10.1073/pnas.1117018109 39. Seifert KA (2009) Progress towards DNA barcoding of fungi. Molecular ecology resources. 9: 839. https://doi.org/10.1111/j.1755-0998.2009.02635.x 40. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD (2011) Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Molecular systems biology. 7: 539. https://doi.org/10.1038/msb.2011.75 41. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution. 30: 272529. https://doi.org/10.1093/molbev/mst197 42. Teasdale SE, Beulke AK, Guy PL, Orlovich DA (2013) Environmental barcoding of the ectomycorrhizal fungal genus Cortinarius. Fungal Diversity. 58: 299-310. https://doi.org/10.1007/s13225-012-0218-1 Page 19/23 43. Watling R, Abraham SP (1992) Ectomycorrhizal fungi of Kashmir forests. Mycorrhiza. 1992 2: 817. https://doi.org/10.1007/BF00203254 44. Xie ML, Wei TZ, Fu YP, Li D, Qi LL, Xing PJ, Cheng GH, Ji RQ, Li Y (2020) Three new species of Cortinarius subgenus Telamonia (Cortinariaceae, Agaricales) from China. MycoKeys. 69:91. 10.3897/mycokeys.69.49437 Figures Page 20/23 Figure 1 Maximum likelihood tree inferred from ITS sequences. The tree is rooted with Hebeloma species treated as outgroup. Newly reported species are shown in Bold. The ML bootstrap values are shown on each branch. The Bayesian posterior probabilities were calculated and found to be signi cant. Page 21/23 Figure 2 a, b. Sporocarp of Cortinarius cibum. Figure 3 a, b. Sporocarp of Cortinarius neocephalixus Figure 4 Page 22/23 a, b. Sporocarp of Cortinarius nigricans. Figure 5 a–c. Microscopic structure of spores. Page 23/23