Three new species of Cortinarius subgenus Telamonia (Cortinariaceae, Agaricales) from China

Abstract Cortinarius is an important ectomycorrhizal genus that forms a symbiotic relationship with certain trees, shrubs and herbs. Recently, we began studying Cortinarius in China and here we describe three new species of Cortinarius subg. Telamonia based on morphological and ecological characteristics, together with phylogenetic analyses. Cortinarius laccariphyllussp. nov. (section Colymbadini) is associated with broadleaf trees, with strongly hygrophanous basidiomata, special Laccaria-like lamellae and white and extremely sparse universal veil. Cortinarius neotorvussp. nov. (section Telamonia) is associated with broadleaf trees and is easily confused with C. torvus, but can be distinguished by the colour of the fresh basidiomes and the stipe usually somewhat tapering towards the base. Cortinarius subfuscoperonatussp. nov. (section Fuscoperonati) is associated with coniferous trees, with subglobose to broadly ellipsoid spores and is closely related to C. fuscoperonatus. A key to the new species and similar species in sections Colymbadini, Telamonia and Fuscoperonati is provided.


Introduction
Cortinarius (Pers.) Gray is one of the most species-rich agaric genera, with reportedly more than 2250 species worldwide (He et al. 2019). While most of the Cortinarius species were described from Europe and North America, there are also some species described from Oceania (e.g. Bougher and Hilton 1989;Soop 2005;Gasparini and Soop 2008), South America (e.g. Valenzuela and Esteve-Raventos 1994;Garnica et al. 2003;San-Fabian et al. 2018) and Asia (e.g. Miyauchi 2001;Peintner et al. 2003;Xie et al. 2019). It was assumed that more than 900 species occur in northern European countries, based on phylogenetic studies (Niskanen et al. 2012). At least 500 Cortinarius species were reported in North America (Bessette et al. 1997). Only 229 Cortinarius species have been reported in China (Teng 1963;Tai 1979;Shao and Xiang 1997;Wei and Yao 2013;Li et al. 2015;Xie 2018;Xie et al. 2019;Cheng et al. 2019;Wei and Liu 2019). Recently, many new species have been described, based on the phylogenetic analyses, together with morphological and ecological data (e.g. Bojantchev and Davis 2011;Wei and Yao 2013;Harrower et al. 2015;Brandrud et al. 2018a). Many studies showed that nrDNA ITS barcodes are typically effective in distinguishing Cortinarius species (e.g. Liimatainen et al. 2014;Garnica et al. 2016;Schmidt-Stohn et al. 2017;Brandrud et al. 2018b).
The diverse ecosystems in China provide a conducive environment for the growth of Cortinarius species. Research, dedicated to the phylogeny and taxonomy of Chinese Cortinarius, was initiated in recent years. During field trips in the past years, many specimens of Cortinarius were collected from China. However, only two new Cortinarius species have been described and reported, based on Chinese specimens until now (Wei and Yao 2013;Xie et al. 2019). There are still many species that have never been reported according to the phylogenetic analyses, based on our materials. Further efforts are necessary to describe these species and reveal the species diversity of Cortinarius in China. In this study, three new species of the subgenus Telamonia sensu stricto were described, based on morphological and ecological characteristics, as well as phylogenetic analyses. An identification key to the new species and similar species in sections Colymbadini, Telamonia and Fuscoperonati is provided.

Sampling and morphological studies
We collected specimens from northeast China and northwest China, two important floristic areas of China. Fresh basidiomata were photographed and noted under daylight in the field, dried in an oven at about 50 °C and deposited in the Herbarium of Mycology, Jilin Agricultural University (HMJAU).
The macroscopic characters were described from fresh basidiomata. Colour codes were taken from Kornerup and Wanscher (1978). The microscopic characters were examined from dried specimens mounted in 5% aqueous potassium hydroxide (KOH) and Melzer's reagent using a Zeiss AX10 light microscope with a high-resolution 100× objective. Twenty to thirty mature basidiospores were measured (excluding apiculus and ornamentation) from each collection. The length/width ratio (Q) was calculated for individual spores. `X and `Q refer to the average value of basidiospores of each specimen. The basidia (ten basidia per collection), sterile cells of lamellar edge (20 sterile cells per collection) and hyphae of the lamellar trama were examined and measured from the pieces of lamellae. The pileipellis structure was studied from radial sections half-way from the pileus centre. Basidiospores, lamellar margin cells of this new species were photographed.

DNA extraction, PCR amplification and sequencing
We extracted the DNA from fresh tissue dried in silica gel by the NuClean PlantGen DNA Kit (CWBIO, China) and amplified the ITS region with primers ITS1F and ITS4 (White et al. 1990;Gardes and Bruns 1993).The PCR amplification progress followed Xie et al. (2019) and was sequenced by Sangon Biotech (Shanghai) Co. Ltd. The newly generated ITS sequences have been submitted to GenBank.

Data analysis
BLAST searches with the newly-generated ITS sequences were performed against NCBI (https://www.ncbi.nlm.nih.gov/) and UNITE (https://unite.ut.ee/) databases to retrieve similar sequences for the phylogenetic analyses (Table 1) (Niskanen 2008;Niskanen et al. 2011). All ITS sequences were aligned and edited with BioEdit 7.0.9 (Hall 1999). ITS1 and ITS2 were delimited by comparison with the sequence KC608590, which is fully an-  Bidaud et al. (2017) notated in GenBank. For phylogenetic analyses, both Bayesian Inference (BI) and Maximum Likelihood (ML) methods were used. The analyses were performed with two partitions, one including ITS1 and ITS2, the other including coding sequences (SSU, 5.8S and LSU). The two partitions alignments were concatenated using Phyutility 2.2 (Smith and Dunn 2008). Exactly identical sequences were removed from the data matrix (Vadthanarat et al. 2017). For BI analysis, the best-fit model for each partition was determined using the Akaike Information Criterion (AIC), implemented in MrModeltest 2.3 (Nylander 2004). BI analysis was performed with MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003). Markov Chain Monte Carlo (MCMC) chains were run for 200,000 generations, sampling every 100th generation at which point the average standard deviation of split frequencies was 0.00594. The first 25% of trees were discarded to build the 50% majority rule consensus tree. ML analysis was performed with RAxML (Stamatakis 2014) and implemented in raxmlGUI (Silvestro and Michalak 2012). All parameters in the ML analysis were kept as defaults, except for choosing GTRGAMMAI as the model of sequence evolution. For testing the support of the branches, rapid bootstrap analysis with 1,000 replicates was chosen. The resulting phylogenies were visualised in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).
ITS sequence. The ITS sequences of C. subfuscoperonatus are 524-525 bp long (5 collections, Table 1) and distinct from other members of section Fuscoperonatus. The ITS sequence of C. subfuscoperonatus (MK552387, holotype) differs from C. fuscoperonatus by six substitutions and six indels. Comments. Cortinarius subfuscoperonatus corresponds well to the characteristics of section Fuscoperonati, with weak hygrophanous pileus, an annular band on the middle stipe and distinct belts or zones lower down, large spores (> 10 μm long) and grow in coniferous forests. Cortinarius fuscoperonatus was previously placed in section Bovini M.M. Moser (Bidaud et al. 2009;Soop 2014) and Armillati (Brandrud et al. 1992), until Niskanen et al. (2015 placed it in section Fuscoperonati. Cortinarius subfuscoperonatus has remarkably similar morphological characteristics to C. fuscoperonatus, apart from the spores of C. fuscoperonatus being narrower (9.7-11.6 × 6.6-7.7 μm), the pileus being chocolate brown to blackish-brown and being fine fibrous to fine scaly (Schmidt-Stohn et al. 2017). In addition, C. subfuscoperonatus formed a sister relationship with C. fuscoperonatus and was well separated according to the phylogenetic analyses. C. subfuscoperonatus could be considered as the second species in section Fuscoperonati.

Discussion
Cortinarius is the most species-rich genus of Agaricales, with most of the described species distributed in the Northern Hemisphere. However, so far, little has been done on Cortinarius taxonomy in north-eastern Asia or even in the whole of Asia, leaving an important gap in our knowledge of this genus (Horak 1983). The flora of northern China has a strong affinity shared with the circumboreal areas of Europe and western North America but also harbours some floristic elements with a tropical and subtropical affinity (Wu 1979 In this study, we described the phylogenetic relationships amongst the three new species and other species, based on the ITS sequences. However, multiple genes should be used in future studies to describe more complex phylogenetic relationships in Cortinarius, which some mycologists have conducted. Peintner et al. (2002) assessed the phylogenetic relationships of Rozites, Cuphocybe and Rapacea by molecular phylogenetic approaches, based on ITS and LSU. Frøslev et al. (2005) analysed the phylogeny of Cortinarius subgenus Phlegmacium, a taxonomically difficult group, based on ITS, RPB1 and RPB2. They speculated that the sequences from RNA polymerase II genes have the potential for resolving the phylogenetic problems of Cortinarius. Later, the study of Frøslev et al. (2007) showed that the delimitation of species, based on ITS sequences, is more consistent with a conservative morphological species concept and there is considerable potential for using ITS sequence data as a barcode for section Calochroi. Soop et al. (2019) studied the global supraspecific taxonomy of Cortinarius by the phylogenetic approach, based on ITS, LSU, RPB1 and RPB2. Both ITS and LSU datasets and ITS, LSU, RPB1 and RPB2 datasets showed satisfactory results. Although phylogenetic analyses of Cortinarius have made significant progress in Europe, North America and even in Australasia, few phylogenetic analyses of Cortinarius, based on Chinese materials have been carried out. According to our analysis of ITS data, there are presently less than 200 accessions (excluding sequences obtained from mycorrhiza) from China in GenBank. Thus, the dedicated collection of specimens and studying the phylogeny of Cortinarius, based on the ITS or, preferably, multiple genes, are important contributions to the global phylogenetic framework of Cortinarius.