Morphological and Phylogenetic Evidence Reveal Five New Telamonioid Species of Cortinarius (Agaricales) from East Asia

Five new Cortinarius species, C. neobalaustinus, C. pseudocamphoratus, C. subnymphatus, C. wuliangshanensis and C. yanjiensis spp. nov., are proposed based on a combination of morphological and molecular evidence. Cortinarius neobalaustinus is characterized by a very weakly hygrophanous and yellowish-brown to brown pileus and small and weakly verrucose basidiospores. Cortinarius pseudocamphoratus can be characterized by a viscid pileus, a strongly unpleasant smell, amygdaloid to somewhat ellipsoid basidiospores and lageniform to subfusiform cheilocystidia. Cortinarius subnymphatus is identified by a strongly hygrophanous pileus that is reddish-brown with a black-brown umbo, a yellowish universal veil and ellipsoid to subamygdaloid basidiospores. Cortinarius wuliangshanensis is characterized by a moderately to strongly hygrophanous, translucently striated and yellowish to reddish-brown pileus and rather weakly and moderately verrucose basidiospores. Cortinarius yanjiensis is distinguished by a weakly to moderately hygrophanous and yellowish to brown pileus and moderately to rather strongly verrucose basidiospores. The phylogenetic analyses were performed with maximum likelihood and Bayesian inference methods based on the data set of nuc rDNA ITS1-5.8S-ITS2 (ITS), D1–D2 domains of nuc 28S rDNA (28S) and RNA polymerase II second largest subunit (rpb2), and the results show that C. neobalaustinus, C. wulianghsanensis and C. yanjiensis cluster in sect. Illumini, C. pseudocamporatus belongs to sect. Camphorati and C. subnymphatus belongs to sect. Laeti. In addition, a study of basidiospores under field emission scanning electron microscopy (FESEM) was conducted. An identification key for the five new species and related species from China is also provided.


Introduction
Cortinarius (Pers.) Gray is an important ectomycorrhizal agaric genus in Agaricales and few species have sequestrate basidiome morphologies [1][2][3]. Although Cortinarius is known to be the largest basidiomycete genus with more than 3000 species worldwide [4], there are many new species described every year [5][6][7]. This could be due to the application of a polyphasic approach that combines phylogenetic and morphological methods [8][9][10].
Telamonia (Fr.) Trog is a traditional Cortinarius subgenus characterized by a dry to more or less hygrophanous pileus and stipe, often without bright colours [3,11]. However, some species have a more or less bluish tinge and/or a brightly coloured universal veil [3,11]. Some phylogenetic studies have shown that this subgenus is polyphyletic, while many lower ranking taxa formed well-supported monophyletic clades [3,[12][13][14]. The subgenus is

Molecular Phylogeny
Total DNA was extracted from dried specimens, using a NuClean PlantGen DNA Kit (CWBIO, Taizhou, China). Primers ITS1F and ITS4 were used to amplify the nrDNA ITS1-5.8S-ITS2 (ITS) region [16,17]. The D1-D2 domains of nuc 28S rDNA (28S) were amplified with primers LR0R and LR7 [18]. The RNA polymerase II second largest subunit (rpb2) were amplified with primers bRPB2-6F and bRPB2-7.1R [19,20]. The PCR procedures are as follows: for the ITS region, initial denaturation at 95 • C for 5 min, followed by 35 cycles at 95 • C for 30 s, 48 • C for 30 s and 72 • C for 1 min, and a final extension of 72 • C for 10 min; for 28S, initial denaturation at 94 • C for 2 min, followed by 35 cycles at 94 • C for 30 s, 51 • C for 30 s and 72 • C for 1 min, and a final extension of 72 • C for 10 min; for rpb2, initial denaturation at 95 • C for 5 min, followed by 35 cycles at 95 • C for 30 s, 58 • C for 40 s and 72 • C for 1 min, and a final extension of 72 • C for 8 min. Sequencing was performed by Sangon Biotech (Shanghai, China) Co., Ltd. All newly generated sequences were deposited in GenBank (Table 1).
The taxon sampling strategy for the selection of sequences for phylogenetic trees was to choose related taxa based on a BLASTn search in GenBank within Cortinarius and based on Soop et al. [14]. Three partition datasets (ITS, 28S, rpb2) were separately aligned and manually adjusted with BioEdit 7.1.3.0 [21]. Phyutility 2.2 was used to concatenate the aligned datasets [22]. Cortinarius cyanites Fr. and C. boreicyanites Kytöv., Liimat., Niskanen & A.F.S. Taylor were selected as an outgroup for phylogenetic analyses of the combined dataset, following Xie et al. [7].
For phylogenetic analyses, Bayesian inference (BI) and maximum likelihood (ML) methods were used. For BI analysis, the best-fit model for each partition was estimated using the Akaike information criterion (AIC), implemented in MrModeltest 2.3 [23]. The BI analysis was performed with MrBayes 3.2.6 [24]. Four Markov chains were run for 2 runs from random starting trees for 500,000 generations, sampling every 100th generation. The first 25% of trees were discarded to build the 50% majority rule consensus tree. RAxML 8.2.12, implemented in raxmlGUI, was used for ML analysis, with a rapid bootstrapping algorithm of 1000 replicates [25,26]. All default parameters with the GTRGAMMA model were used in the ML analysis. For BI analysis, GTR + I + G, GTR + I + G and GTR + I were the best-fit model for ITS, 28S and rpb2 partitions, respectively.

Species
Voucher Number GenBank Accession Number New sequences are shown in black bold font.

Molecular Phylogeny
The ITS + 28S + rpb2 dataset for phylogenetic analyses included 68 samples, representing 50 species. The resulting alignments were deposited at TreeBASE (http://www.treeba se.org; submission ID S29164; accessed on 28 December 2021). The BI and ML trees showed similar topologies, and the ML tree was selected as the representative phylogeny ( Figure 1).
Basidiospores 5.6-6.8(-7.6) µm × 4.6-5.8(-6.8) µm, av. 5.9-6.3 µm × 4.9-5.4 µm, Q = 1.04-1.37, av. Q = 1. 18 Notes: Cortinarius neobalaustinus is characterized by the weakly hygrophanous yellowishbrown to brown pileus, small-sized basidiospores with finely verrucose and finely encrusted hyphae of the lamellar trama. It is a typical member of sect. Illumini. Cortinarius balaustinus shows similarity to the new taxon for its brown pileus and small basidiospores, but the basidiospores of C. balaustinus are moderately verrucose [27,28]. In addition, C. balaustinus is a widely distributed species in the Northern Hemisphere, usually under Betula, but less often with Carpinus, Corylus and Quercus trees [27,28]. Molecularly, (in ITS) based on the BLASTn, the most closely related species is C. balaustinus, which differs by 20 substitutions and indel positions, with a similarity of 96.5%. Table 2 provides the critical characteristics distinguishing new species and their similar species in sect. Illumini. Description: Pileus 26-55 mm diam., hemispherical, later plano-convex, viscid, not hygrophanous; greyish-white when young, with slightly violet tinge, later yellow; surface with veil remnants, especially on the margin. Lamellae emarginated, moderately crowded, violet when young, later bluish-brown to brown, edge paler, uneven. Stipe 50-75 mm long, 6-15 mm at above, 9-28 mm at the base, cylindrical to clavate, violet, especially at the apex, coated by abundant veil layer, more violet tinge visible when scraped. Universal veil whitish, then yellowish, very copious, forming incomplete girdles on the stipe. Context of the pileus whitish-yellowish, violet at the apex of the stipe, gradually transitioning to yellowish-grey at the base. Odour strong and unpleasant, typical of C. camphoratus.
Hypodermium developed, hyphae colourless to lightly olivaceous-brown, 5-25 μm wide, smooth. Trama hyphae colourless to lightly olivaceous-brown, smooth. Clamp connections present.     Notes: Cortinarius pseudocamphoratus is characterized by whitish with slightly violettinged basidiomata, viscid pileus, strong and unpleasant odour, amygdaloid to somewhat ellipsoid basidiospores, finely and densely verrucose and somewhat lageniform to subfusiform cheilocystidia, which correspond well to the circumscription of the sect. Camphorati. There are only five described species that belong to sect. Camphorati [14,30]. Three species are only distributed in Australasia, whereby C. dysodes and C. tasmacamphoratus are associated with Nothofagus and C. austrotorvus is a Eucalyptus-associated species. Another two species, C. camphoratus and C. putorius, are conifer-associated species and distributed in the Northern Hemisphere. However, C. camphoratus has a more violet and non-viscid pileus and the universal veil is lilac when young [28,31,32]. Cortinarius putorius, meanwhile, usually has a thin universal veil and the basidiospores are smaller, 8.8-9.5(-10) µm × 5-5.7 µm [30]. Phylogenetically, C. pseudocamphoratus cluster in sect. Camphorati and form a sister relationship with the clade of C. camphoratus and C. putorius. Molecularly, (in ITS) based on the BLASTn, the most closely related species is C. camphoratus, which differs by 35 substitutions and indel positions, with a similarity of 94.1%. Table 3 provides the critical characteristics distinguishing C. pseudocamphoratus and its similar species in sect. Camphorati. Notes: Cortinarius subnymphatus is characterized by a reddish-brown, mat and strongly hygrophanous pileus with a black-brown umbo, yellowish universal veil, ellipsoid to subamygdaloid basidiospores and encrusted hyphae of the lamellar trama. Morphologically, C. subnymphatus is similar to C. fulvescens and C. fulvescentoideus. Cortinarius fulvescens and C. fulvescentoideus, however, usually have bigger basidiospores, over 8 µm long [33]. Phylogenetically, C. subnymphatus clustered in sect. Laeti, sister to C. nymphatus. However, the pileus of C. nymphatus is redder, without the black-brown umbo, the basidiospores are usually less than 5 µm wide and associated with Pinus and Picea [33]. Molecularly, (in ITS) based on the BLASTn, the most closely related species is C. nymphatus, which differs by 10 substitutions and indel positions, with a similarity of 98.4%. Table 4 provides the critical characteristics distinguishing C. subnymphatus and its similar species in sect. Laeti. Etymology: The name refers to the type location, Wuliangshan Mountains. Description: Pileus 24-77 mm, hemispherical to convex at first, then becoming planar, slightly depressed at the centre, wavy and eroded at the margin in mature condition; surface medium hygrophanous, with a clear hygrophanous zone, slightly translucently striated and crenated at the margin, innately fibrillose, silvery-whitish when young, then strongly hygrophanous, with distinct translucent striations; yellowish-white to greyish-yellow at first, reddish-orange to reddish-brown at the centre with age. Lamellae emarginated, subdistant, greyish-yellow at first, then yellowish-brown to brown, edge even. Stipe 28-80 mm long, 4-12 mm thick at above, 6-25 mm thick at the base, bulbous at the base when young, then clavate, with white fibrils, whitish to lightly yellowish-white, yellow to yellowish-brown with age, basal mycelium white. Universal veil white, rather sparse. Context thin, yellowish-white when young, later yellowish-brown to brown at the stipe, hollow at the stipe when mature. Odour: radish.
Basidiospores 4.8-6.1 (-6.8) µm × 3.9-4.8 (-5.1) µm, av. 5.1-5.7 µm × 4.1-4.6 µm, Q = 1.09-1.41, av. Q = 1.23-1.26, subglobose to broadly ellipsoid, rare ellipsoid, rather finely verrucose, moderately dextrinoid. Basidia clavate, four-spored. Lamellar edges fertile, with narrow clavate cells. Lamellar trama hyphae lightly olivaceous-brown to olivaceous-brown, up to 18 µm wide, smooth. Pileipellis with a very thin epicuits, hyphae 3-7.5 µm wide, equal, colourless to lightly olivaceous-brown, smooth. Hypodermium developed, hyphae colourless to lightly olivaceous-brown, 6-20 µm wide, cylindrical to subcellular, smooth. Notes: Cortinarius wuliangshanensis is characterized by moderately to strongly hygrophanous, yellowish to reddish-brown and distinctly translucently striated pileus, bulbous at the stripe base when young, small-sized and rather finely verrucose basidiospores and smooth hyphae of lamellar trama. It is a typical member of sect. Illumini. Morphologically, it is similar to C. microglobisporus, sharing the yellowish pileus and white fibrillate stipe when young. However, the pileus of C. microglobisporus is without the distinct translucent striations and never reddish-brown and it is only distributed in the Mediterranean under Quercus cerris trees [29]. Molecularly, (in ITS) based on the BLASTn, the most closely related species is C. microglobisporus, which differs by 33 substitutions and indel positions, with a similarity of 94.8%. Table 2  Etymology: The name refers to the type location, Yanji county. Description: Pileus 20-70 mm, convex at first, then becoming plano-convex with a broad umbo; weakly to moderately hygrophanous, innately fibrillose; lightly yellow, yellowish-brown to brown, paler at the margin. Lamellae adnexed to subadnate, subdistant to moderately crowded, greyish-yellow at first, then yellowish-brown to brown, edge even at first, then uneven. Stipe 25-83 mm long, 4-14 mm thick at above, 7-23 mm thick at the base, cylindrical to slightly clavate, usually with slightly tapered base, white fibrils, white at first, somewhat yellow or yellowish spots at the upper part, basal mycelium white. Universal veil white, spares. Context rather thick, white at first, then yellowish to yellowish-brown at the stipe. Odour: radish. Basidiospores  Notes: Cortinarius yanjiensis is characterized by a weakly to moderately hygrophanous and yellowish to brown pileus, tapered base of the stipe, small-sized and rather strongly verrucose basidiospores, and smooth hyphae of the lamellar trama. It is a typical member of sect. Illumini. Morphologically, it is similar to C. balaustinus and C. neobalaustinus, sharing the weakly hygrophanous pileus. However, the lamellar trama hyphae of C. balaustinus and C. neobalaustinus are finely encrusted. The basidiospores of C. balaustinus are moderately verrucose [27,28] while those of C. neobalaustinus are finely verrucose. Molecularly, (in ITS) based on the BLASTn, the most closely related species is C. illuminus, which differs by 43 substitutions and indel positions, with a similarity of 93.1%. Table 2 provides the critical characteristics distinguishing C. yanjiensis and its similar species in sect. Illumini.

Discussion
In this study, five species, Cortinarius neobalaustinus, C. pseudocamphoratus, C. subnymphatus, C. wuliangshanensis and C. yanjiensis, are described from China as new species based on macro-, micro-and ultra-characteristics and multi-gene phylogeny. Phylogenetic analyses showed that the five new species clustered in the telamonioid clade, outside Telamonia s.s., which is consistent with other studies [3,[12][13][14].
Section Camphorati was described as a subgenus in a previous study [34] but was reduced to a section based on multi-gene phylogenetic analyses [14]. Section Camphorati has the typical characteristics of medium to large-sized basidiomata, with a blue to purple tinge when young, a strongly unpleasant odour, basidiospores that are amygdaloid to somewhat ellipsoid and cheilocystidia that are somewhat lageniform. There are five species in this section worldwide [14,29], and in the present study, C. pseudocamphoratus is considered to be the sixth species worldwide and the only confirmed species from China in this section.
Section Illumini was described as a subgenus based on a previous study [34] but was reclassified at section level based on multi-gene phylogenetic analyses [14]. Section Illumini has the typical characteristics of medium to large-sized basidiomata, a yellowish to brown or reddish-brown pileus, being more or less hygrophanous and innately fibrillose, its basidiospores globose, subglobose, ovoid to broadly ellipsoid and weakly to rather strongly verrucose. There are six species in this section worldwide, with one species described from China [14,35]. The present study confirms the placement of six species in sect. Illumini, including three new species from China.
Section Laetii species were previously included in subg. Telamonia or subg. Hydrocybe [36,37] and as a section within sect. Fulvescentes Melot, distinguished by the colour of the universal veil [3,33]. However, previous phylogenetic analyses showed that sect. Fulvescentes is a synonym of sect. Laetii [12][13][14]33]. Section Laeti has the typical characteristics of small to medium-sized basidiomata, a pileus that is more or less hygrophanous, its universal veil a bright colour, its basidiospores subglobose, ellipsoid to amygdaloid and weakly to moderately verrucose. In this study, we confirmed five species in sect. Laeti distributed in China, with the new species C. subnymphatus, sister to C. nymphatus, a European species [33].
Most of the Cortinarius species were originally described in Europe and North America, while little work has been done in Asia [38]. In recent years, we have been devoted to researching Cortinarius in China. The present study reports 12 telamonioid species, including five new species, confirming and clarifying the species component of sections Camphorati, Illumini and Laeti in China, enriching the diversity of Cortinarius.  Data Availability Statement: All resulting alignments were deposited in TreeBASE (http://ww w.treebase.org; accession number S29164; accessed on 28 December 2021). All newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accessed on 23 December 2021). All new taxa were linked with MycoBank (https://www.mycobank.org/; accessed on 23 December 2021).