﻿Four new species of Mycenasect.Calodontes (Agaricales, Mycenaceae) from northeast China

﻿Abstract Species of Mycenasect.Calodontes are representative of the Mycena genus as a whole and are easily recognised by the pinkish, reddish, purplish to brownish pileus and larger basidiomata. Furthermore, the colour of the pileus in the species of sect. Calodontes often has a transition or changes in different stages and the combination of the colour of the pileus with cystidia and basidiospores can be used to recognise taxa within this section. To date, 19 species of Mycenasect.Calodontes have been reported worldwide. Including our recent description of M.yuezhuoi, five species of sect. Calodontes have been recorded in China. During examination of specimens collected in coniferous forests or mixed broadleaf-conifer forests in temperate regions of China, additional taxa assigned to sect. Calodontes were identified. Four new species are recognised, based mostly on characters of the pileus and cystidia. Phylogenetic analysis of sequence data from multiple DNA regions (ITS + rpb1 + tef1) supported the morphological evidence. Here, we propose M.polycystidiata, M.rufobrunnea, M.shengshanensis and M.subulata as new species in Mycenasect.Calodontes. Morphological descriptions, line drawings, habitat photos and comparisons with closely-related taxa are provided. A key to the 23 known species of sect. Calodontes is presented.


Introduction
sect. Calodontes from the temperate zone of northeast China. The four new species share a unique set of striking morphological characters and contribute to an improved understanding of the classification of sect. Calodontes.

Morphological observations
Thirteen fungal specimens were examined in this study, which were mainly collected in coniferous forests and some from mixed broadleaf-conifer forests in 2021. Macrocharacters were recorded from fresh specimens. Colour codes in descriptions follow those of Kornerup and Wanscher (1978). Microcharacters were observed from tissues sampled from dried specimens and rehydrated with 5% potassium hydroxide (KOH) and stained with Congo red (1% [w/v] aqueous solution), if necessary, using a Lab A1 microscope (Carl Zeiss AG, Jena, Germany). The amyloid reaction was tested with Melzer's Reagent (Clémençon et al. 2004;Horak 2005). Twenty basidiospores were measured per specimen. For the holotype, 40 basidiospores from different basidiomata were selected for measurement. Basidiospore statistics are expressed as (a/b/c) (d)e-fg(h) × (i)j-k-l(m) μm [Q = (n)o-p(q), Q = r ± s], where a-c represent a basidiospores of b basidiomata from c specimens measured; d and h are the minimum and maximum length (5% extremum), respectively, e and g indicate the range of values for the remaining 90% of the spores and f is the average length; width (i-m) and Q values (n-q) are expressed in a similar manner; and r and s are the average Q value and its standard deviation, respectively Liu et al. 2021;Na et al. 2021;Na et al. 2022). The measurement of basidia, cystidia and other characters were each based on 20 observations. All specimens have been deposited in the Fungarium of the Fujian Academy of Agricultural Sciences (FFAAS).

DNA extraction, PCR, cloning and DNA sequencing
The Plant Genomic DNA Kit (CoWin Biosciences, Beijing, China) was used to isolate total genomic DNA from dried specimens in accordance with the manufacturer's instructions. Three nuclear loci were sequenced, comprising the internal transcribed spacer (ITS), RNA polymerase II largest subunit (rpb1) and translation elongation factor-1 alpha (tef1). The primer pairs ITS1/ITS4, rpb1Mp_f1/rpb1Mp_r1 and tEF-Mp_f2/ tEFMp_r2 were selected to amplify ITS, rpb1 and tef1, respectively (White et al. 1990;Harder et al. 2013;Yu et al. 2020). The PCR reactions were performed in a total volume of 25 μl containing 2 μl DNA template, 1 μl for each primer, 8.5 μl nuclease-free H 2 O and 12.5 μl 2× Utaq PCR MasterMix (ZomanBio, Beijing, China). The PCR protocol for amplification of the ITS region was as follows: 94 °C for 4 min, then 34 cycles of 94 °C for 45 s, 52 °C for 45 s and 72 °C for 1 min, with a final extension of 72 °C for 10 min (Na et al. 2022). The PCR protocol for amplification of the rpb1 and tef1 regions followed that of Harder et al. (2013): 94 °C for 60 s, then 10 cycles of 94 °C for 35 s, 53 °C for 45 s and 72 °C for 45 s; then 25 cycles of 94 °C for 35 s, 56 °C for 45 s, 72 °C for 45 s and final extension of 72 °C for 10 min. The PCR products were purified by gel electrophoresis or filter membrane and subjected to Sanger dideoxy sequencing by the Beijing Genomics Institute (Beijing, China).

Phylogenetic analysis
A combined ITS, rpb1 and tef1 dataset was analysed to infer relationships of the new taxa with other members of sect. Calodontes. We used sequences included in previous studies of sect. Calodontes and from members of the most closely-related section deposited in the GenBank database, which were mainly submitted by Harder et al. (2013), Osmundson et al. (2013), Chew et al. (2014) and Liu et al. (2021). For the analysis, representative species of Mycena sect. Supinae Konrad & Maubl., which is closely related to sect. Calodontes, were selected as the outgroup (Osmundson et al. 2013;Na and Bau 2019). Sequences for each DNA region (ITS, rpb1 and tef1) were aligned in MAFFT version 7 online and the aligned matrices were manually checked with BIOEDIT 7.2.5.0 (Hall 1999;Kuraku et al. 2013;Katoh et al. 2019). The best-fit substitution model for each gene partition was determined with MODELTEST 2.3, based on the Akaike Information Criterion (Posada and Crandall 1998). Maximum Likelihood (ML) analysis was conducted by raxmlGUI 2.09 (Edler et al. 2020). The phylogenetic analysis was performed by a single analysis with six partitions (ITS1, 5.8S, ITS2, rpb1 exons, tef1 exons, intron of rpb1 + introns of tef1), using the GTR-GAMMA model and 1,000 rapid bootstrap (BS) replicates. For Bayesian Inference (BI), two runs of six chains were run for 15,000,000 generations and sampled every 10,000 generations by MrBayes 3.2.6. At the end of the run, the average deviation of split frequencies was 0.007821, ESS (effective sample size) was 1300.3 and the average Potential Scale Reduction Factor (PSRF) parameter values (excluding NA and > 10.0) = 1.000 and the "sump" and "sumt" commands were used to summarise sampled parameters with 25% burn-in (Ronquist and Huelsenbeck 2003).
Known distribution. Heilongjiang Province, China. The pleurocystidia of M. polycystidiata varied in shape amongst specimens (Fig.  5). In all four specimens, most pleurocystidia were lanceolate and with round to blunt apices, but pleurocystidia with lanceolate and acute apices, elliptical and ovate and acute apices were also observed in FFAAS0417 (Holotype) and FFAAS0418, while elongated lageniform-lanceolate or round apices ovate were detected in FFAAS0421 and FFAAS0422. The multi-shaped pleurocystidia may show a morphological continuum that changes between developmental stages. Nevertheless, the multi-shaped pleurocystidia are unquestionably diagnostic for identification of this species. Etymology. Refers to reddish-brown pileus.
Habitat Notes. Cheilocystidia with a long and narrow protuberance is the key microscopic character that distinguishes M. subulata and is uncommon in sect. Calodontes (Smith 1947;Maas Geesteranus 1992a, 1992bGrgurinovic 2003;Robich 2003;Chew et al. 2014;Aravindakshan and Manimohan 2015;Aronsen and Laessøe 2016;Na 2019;. Mycena lammiensis Harmaja and M. pelianthina (Fr.) Quél. have similar cheilocystidia, but differ from M. subulata by their broader cheilocystidia with purplish-brown contents and having pleurocystidia (Smith 1947;Robich 2003;Aronsen and Laessøe 2016). The cheilocystidia of M. subcorticalis (Cooke & Massee) Sacc. with a protuberance are similar to those of M. subulata. However, M. subcorticalis has larger and inamyloid spores, a gelatinised pileipellis and a stipitipellis with sparse excrescences (Grgurinovic 2003). More rarely, mucronate cheilocystidia and absence of pleurocystidia have been described for M. pearsoniana and its clay pink pileus is similar to that of M. subulata, but M. pearsoniana differs in having a slightly glutinous pileus when wet and inamyloid spores (Aronsen and Laessøe 2016;Na 2019). Other species that are macroscopically similar to M. subulata, namely M. luteovariegata, M. nullawarrensis and M. pura, can be distinguished by cheilocystidia shape and presence of pleurocystidia (Perry 2002;Robich 2003;Grgurinovic 2003;Harder et al. 2013;Aronsen and Laessøe 2016;Na 2019 (Harder et al. , 2012(Harder et al. , 2013. Chew et al. (2014) supported the views of Harder et al. (2010Harder et al. ( , 2012 and the new taxa proposed by the former authors were not assigned to a subsection. Additionally, subsect. Purae was proved to be polyphyletic in our combined analysis of ITS, rpb1 and tef1 dataset, which also supported analysis, based on single gene region (Harder et al. 2013). The five taxa of Mycena sect. Calodontes recorded from China show obvious differences in pileus colour and in the shapes of cheilocystidia and pleurocystidia (if present) . The colour of the pileus includes greyish-rose, reddish-grey, purple, reddish-brown and violet-brown and most show a gradual transition with age. Clavate, obclavate, utriform and fusiform cheilocystidia with a long, narrow protuberance are observed, but pleurocystidia are present only in M. polycystidiata. Forms and variations within M. pura complex had a wide range of pileus colour, but the shape of cheilocystidia was highly similar and could be clearly distinguished from the four new taxa (Robich 2003).
In our phylogenetic analysis, four new species all formed separate clades with high support and had obvious genetic distance from other species in sect. Calodontes. Mycena rufobrunnea is more closely related to the phylogenetic species within M. pura complex by Harder et al. (2013). While the other three new species are significantly more distant from M. pura complex genetically, M. shengshanensis and M. subulata, formed a sister relationship with high support from M. pearsoniana; M. polycystidiata clustered with M. diosma, but is poorly supported.
Based on extensive field work in China, most specimens of sect. Calodontes have been observed in coniferous forests or mixed coniferous-broadleaved forests in early autumn (Na 2019;Liu et al. 2021). Specimens of the four new taxa described in the present study were collected from Changbai Mountain and the Lesser Khinggan Mountains in northeast China from mixed broadleaf-Korean pine (Pinus koraiensis) forests (Zhao et al. 2004;Wang and Guo 2016). In particular, M. polycystidiata and M. subulata were both distributed in the Liangshui National Nature Reserve on the Lesser Khinggan Mountains, where the dominant forest species is P. koraiensis, mixed with fewer Betula, Tilia, Quercus and Picea individuals (She et al. 2022). Moreover, more specimens were located in the northern region of China with an average temperature not more than 20 °C in August. For example, the average temperature is 16.4 °C in Liangshui National Nature Reserve and 16.3 °C in Shengshan National Nature Reserve (Liu 2017). Therefore, we speculate that members of this section in China prefer the climate types Dwa, Dwb and Dwc according to the Köppen climate classification (Kottek et al. 2006;Wang et al. 2020).