Three New Species of Cortinarius From Kashmir Himalayan Coniferous Forests Based On Morphological And Molecular Evidence

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 identied 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. 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 thin-walled, smooth, hyaline to slightly olivaceous brown in 5% KOH. Clamp connections present.

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 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.  Halme et al. 2012aHalme et al. , 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 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(Peintner et al. , 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% -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 25 L reaction mixture for PCR ampli cation contained 1 L template DNA, 3 L PCR buffer, 2.5 L of 2mM dNTP's, 2 L of each primer, 1.5 L of Mgcl 2 , 1 L of DMSO and 0.4 L 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 ℃ 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). Data analysis. An ITS data set comprising newly generated sequences and identical sequences retrieved from the fungal database were used in the analysis (  (Sievers et al. 2011) and the alignment was further re ned with BioEdit 7.0.9 (Hall 1999(Hall , 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

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. 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.
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.
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. Etymology. The name refers to its morphological a nity with C. brunneus.
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. 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. 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.