Two new psathyrelloid species of Coprinopsis (Agaricales, Psathyrellaceae) from China

Abstract In this study, Coprinopsisjilinensis and Coprinopsispusilla were introduced, based on their morphological characteristics, the internal transcribed spacer (ITS) and large subunit ribosomal (LSU) region sequences of nrDNA. These new psathyrelloid species were found in Jilin Province, China. Coprinopsisjilinensis has brown pileus, utriform pleurocystidia, brown, smooth, dextrinoid basidiospores and tiny pore. It mainly grows on humus. Coprinopsispusilla has small basidiomata, greyish-white pileus, thick and distinct veil at edges, subcolourless and verrucose basidiospores. It is poreless and it grows on the decaying wood of broad-leaved trees. Both of them belong to the C.sect.Melanthinae. A supplementary description of C.sect.Melanthinae was given in combination with the newly-discovered taxa and an identification key to the fourteen psathyrelloid species of Coprinopsis is provided. Coprinopsissect.Canocipes and C.sect.Quartoconatae were evaluated and the phylogenetic position of the psathyrelloid species of Coprinopsis was discussed. Psathyrellasubagraria, as a confusing species, was also discussed in this study.


Introduction
Coprinoid mushrooms are fascinating fungal taxa with the characteristic of deliquescent lamellae. Coprinus sensu lato is not monophyletic (Johnson and Vilgalys 1998;Hopple and Vilgalys 1999;Park et al. 1999aPark et al. , 1999bMoncalvo et al. 2002). Based on molecular studies, Redhead et al. (2001) subdivided Coprinus s. l. into four genera, The morphological description referred to Largent et al. (1977 and1978). The surface of the basidiospores was observed and photographed under a scanning electron microscope (SEM) (Hitachi SU8000) at 2.0 kV, with a working distance of 8 mm. The following symbols were used in the description: [n/m/p] indicates that 'n' randomly selected basidiospores from 'm' basidiomata of 'p' collections were measured, 'avl' means the average length of basidiospores, except the extreme values, 'avw' means the average width of the basidiospores, except the extreme values, 'Q' represents the quotient of the length and width of a single basidiospores in side view, 'Q m ' refers to the average Q value of all basidiospores ± standard deviation. Dimensions for basidiospores are given as (a) b-c (d). The range of b-c contains a minimum of 90% of the measured values. Extreme values (i.e. a and b) are given in parentheses.

Research methods of molecular systematics
The total DNA of the specimens was extracted by the new plant genomic DNA extraction kit from Jiangsu Kangwei Century Biotechnology Company Limited. The amplification primers of LSU nrDNA (LSU) were LROR and LR5 (Vilgalys and Hester 1990), the ITS nrDNA (ITS) regions were ITS1 and ITS4 (White et al. 1990; Gardes and Bruns 1993). The amplification reactions were carried out in a 25 μl system and the total amount of the reactions was as follows: ddH 2 O 13.5 μl, 10×Taq Buffer 5 μl, 10 mM dNTPs 1 μl, 10 mM upstream primer 1 μl, 10 mM downstream primer 1 μl, DNA sample 2 μl, 2 U/μm Taq Polymerase 1.5 μl. The cycle parameters were as follows: 4 min at 94 °C for 1 cycle; 40 s at 94 °C, 40 s at 54 °C, 1 min at 72 °C for 35 cycles; 10 min at 72 °C for 1 cycle; storage at 4 °C. The PCR product was subjected to 0.5% agarose gel electrophoresis to test strips. The sequencing work was entrusted to Shenggong Bioengineering (Shanghai) Company Limited and the sequencing results were clipped with Seqman 7.1.0 (Swindell and Plasterer 1997) and then submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The newly-obtained sequences are shown in Table 1. The sequences of relevant taxa were downloaded from GenBank and from the related articles (Larsson and Örstadius 2008;Nagy et al. 2010;Nagy 2012;Örstadius et al. 2015;Crous 2017;Melzer et al. 2017).
The 'auto' strategy and normal alignment mode of MAFFT (Katoh et al. 2005) were used for Sequence alignment and Gblocks (Castresana et al. 2000;Talavera and Castresana 2007) was used to obtain the conservative segments of sequences with the following parameters: the minimum number of sequences for a conserved/flank position (12/12), the maximum number of contiguous non-conserved positions (8), minimum length of a block (10) and allowed gap positions (with half ). ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit models using the Bayesian Information Criterion (BIC). The Maximum Likelihood (ML) analyses were performed in IQTree 1.6.8 (Nguyen et al. 2015) and the Bayesian Inference phylogenies were performed in MrBayes 3.2.6 (Ronquist et al. 2012) (2 parallel runs, 2000000 generations), in which the initial 25% of sampled data were discarded as burn-in. The above software was integrated into PhyloSuite 1.2.2 (Zhang et al. 2020). The neighbour-joining (NJ) tree was carried out in Mega X (Kumar et al. 2018). The ML and NJ trees were evaluated by bootstrap analysis with 1000 replicates and the best models are shown in Table 2.

BLASTn results
In the BLASTn alignment, based on ITS sequences, Coprinopsis pusilla and C.

Phylogenetic analyses
After Gblocks clipping, the ITS data matrix included 31 sequences of 588 nucleotide sites from 16 taxa and the data matrix included 20 sequences of 921 nucleotide sites from 14 taxa (gaps included). In the ITS and LSU phylogenetic trees (Figs 1, 2), Coprinopsis pusilla and C. jilinensis both belong to C. sect. Melanthinae. Coprinopsis pusilla and C. melanthina formed a sister clade, C. jilinensis and C. uliginicola formed a sister clade, both of which were strongly supported. In the phylogenetic trees, based on ITS sequences (Fig. 1), C. submicrospora and C. marcescibilis/C. pseudomarcescibilis/C. musae/C. udicola formed a sister clade in the ML and BI trees (MLBP/BIPP: 34/0.5), but C. submicrospora and C. canoceps/C. pannucioides formed a sister clade in the NJ tree (NJBP: 39). In the phylogenetic trees, based on LSU nrDNA sequences (Fig. 2), C. marcescibilis and C. pseudomarcescibilis formed a sister clade with strong support in the NJ and BI trees (NJBP/BIPP: 99/0.8). However, in the ML tree, C. marcescibilis formed a sister clade with all taxa, except the outgroups (MLBP: 100) and C. pseudomarcescibilis formed a sister clade with all taxa, except the outgroups and C. marcesci-

Coprinopsis pusilla MZ398067
Coprinopsis marcescibilis FM876277 Etymology. The epithet "jilinensis" refers to this species that was first discovered in Jilin Province, China.
Description. Basidiomata small to medium-sized. Pileus 33-52 mm broad, conical to convex, dark brown or clay brown, densely covered with white hairs, not sticky when dry or wet, not hygrophanous, veil remnants flocculent at edges. Lamellae close or crowded, grey-white to fleshy brown, brownish-black after drying, sinuate or adnexed, not the same length and width, edges slightly toothed, concolorous, not deliquescent. Stipe 80-95 × 5-9 mm, white to milky white, cylindrical, down slightly rough, fibrous, a little fragile, hollow, the base with white mycelium, dense or sparse, close to the stipe surface covered with brownish-yellow pubescent, no ring. Spore print without record.
Morphologically and phylogenetic similar to Coprinopsis jilinensis, C. uliginicola is characterised by long basidiospores of 10-12(-15) μm, pleurocystidia absent and caulocystidia present, pileipellis no encrusting pigment (Smith 1972). Other similar species, C. cineraria is characterised by grey, hygrophanous and striate pileus, little short basidiospores (6.5-8.5 μm), pleurocystidia absent, pileipellis an epithelium (Takahashi 2000); C. melanthina is characterised by little long and subcolourless basidiospores (avl × avw = 10.5 × 5.8 μm), pleurocystidia absent (Kits van Waveren 1985); C. pusilla has small basidiomata, grey pileus, subcolourless and verrucose basidiospores (this study) and Psathyrella subagraria has hygrophanous pileus, thick flesh and caulocystidia present (Smith 1972), both of which could be clearly distinguished from C. jilinensis in terms of morphology.  Description. Basidiomata very small to small. Pileus 21-29 mm broad, bellshaped to hemispherical when young, then convex, flat to slightly reflexed at edges, with inconspicuous bulge at the middle, grey or greyish-white when dry, no record when wet, densely covered with flocculent hairs, sometimes central with blackish-grey squamous tapering to the edges, not slime, sometimes the edges crack, hygrophanous no record, veil remnants dense at edges, triangular, subtriangular or massive, not easily disappearing. Lamellae close or crowded, subwhite, greyish-white or coffee brown, flesh blond after drying, sinuate or adnexed, sometimes with vertical teeth, edges slightly toothed, concolorous, not deliquescent. Stipe 35-57 × 3-7 mm, cylindrical, subcylindrical, subequal or a little rough towards the base, white, cream white, hollow, a little fragile, not easy to detach from the cap, densely covered with white and flocculent hairs, brown, brownish-grey to brownish-yellow near the base, veil present at the stalk and cap joints, easily disappearing, no ring, the base with white mycelium. Spore print without record.
Habitat and distribution. On the dead and rotten wood of broad-leaved forest or coniferous and broad-leaved mixed forests in autumn.
Notes. Coprinopsis pusilla has a variable macromorphology, but stable micromorphology, which is characterised by small basidiomata, greyish-white pileus, thick and distinct veil remnants at edges, subcolourless and verrucose basidiospores, no pore, the habitat on the decaying wood of broad-leaved trees. C. pusilla forms a stronglysupported independent clade in both ITS and LSU phylogeny trees (Figs 1, 2).
According to the results of BLASTn analyses, ITS showed higher interspecific variability, so the ITS sequence was more advantageous in reflecting the interspecific relationship of Coprinopsis than the LSU sequence. The sequence identity between C. jilinensis from Jilin Province, China and C. uliginicola MG712323 from Hubei Province, China, was 99.27%. Based on the molecular sequence alone, C. uliginicola MG712323 and C. jilinensis could be the same species and subsequent re-examination of this specimen is recommended.
In this study, some sequences of Coprinopsis were selected, which belong to C. sect. Melanthinae, C. sect. Canocipes and C. sect. Quartoconatae in the grouping system proposed by Wächter and Melzer (2020). In the phylogenetic analyses (Figs 1, 2), whether in the phylogenetic trees, based on ITS or LSU, the tree shape of C. sect. Melanthinae obtained was consistent and the branches were stable. However, C. sect. Canocipes and C. sect. Quartoconatae were somewhat different from the grouping described by Wächter and Melzer (2020), based on different sequences and analyses. In the phylogenetic trees, based on ITS, C. udicola and C. submicrospora, which originally belonged to C. sect. canocipes, formed a branch with three species belonging to C. sect. Quartoconatae in ML and BI trees. However, in the NJ tree, C. udicola formed a sister clade with three species belonging to C. sect. Quartoconatae. In the phylogenetic trees, based on LSU, C. sect. Canocipes and C. sect. Quartoconatae were well separated in the NJ tree, while in ML and BI trees, C. musae belonging to C. sect. Quartoconatae formed a sister clade with four species of C. sect. Canocipes. The branching relationships of phylogenetic trees, based on different molecular sequences and analyses may vary. Over-subdivision of sections would cause intersection. Subdivision of C. sect. Canocipes and C. sect. Quartoconatae may need to be reconsidered.
Coprinopsis sect. Melanthinae has a relatively clear systematic differentiation (Wächter and Melzer 2020), which is consistent with the results of this study. Currently, there are only six species in this section, including the C. lignicola nom. prov. (GenBank no.: MG966286 and MF163181). Combined with the two newly-discovered species, the expression of this section is modified as follows:
Coprinopsis pusilla has a variety of macroscopic morphology, including the shape and colour of the pileus, the shape of the veil and the thickness and length of the stipe and so on, which makes it difficult to determine whether it is the same species during the collection process, when the size of the basidiomata and ecological habits seem to be stable. The microscopic morphology of C. pusilla is relatively stable, including the size, shape, colour and decoration of basidiospores, the type of pileipellis and the presence or absence of cystidia. Interestingly, Coprinoid fungi are thought to be a taxon of dark-coloured basidiospores (Noordeloos et al. 2005), but C. pusilla, including C. melanthina, which is closely related to C. pusilla, have subcolourless basidiospores. Collection HMJAU 58779 and collection HMJAU 58780 were collected at approximately the same time in the same forest, which were not far apart from each other. However, due to the significant difference in macroscopic morphology, they were made into two collections. The macroscopic morphology of Collection HMJAU 58781, collected later, was also significantly different from the two specimens collected previously. Through the observation of the microscopic morphology of these collections and the phylogenetic analysis, combined with ITS and LSU molecular sequences, the results showed that the three collections were the same species.
Pleurocystidia is present in many species of Coprinopsis, such as C. cinerea, C. jonesii and C. pseudoradiata (Redhead et al. 2001), but in psathyrelloid species of Coprinopsis, spe-cies with pleurocystidia are rare. The pleurocystidia are sparsely distributed in the hymenium of C. jilinensis, but not present in C. uliginicola, which is closely related to C. jilinensis. In psathyrelloid species of Coprinopsis, only C. pannucioides (Larsson and Örstadius 2008), C. udicola (existing but rare) (Örstadius et al. 2015) and C. jilinensis have pleurocystidia.
Psathyrella subagraria is a confusing species, described by Smith (1972) as it is very similar to P. uliginicola morphologically, with the main difference being that this species has pleurocystidia, which are mainly growing on humus. Since Smith (1972) introduced P. subagraria, no further reports have been made. Örstadius et al. (2015) moved P. uliginicola to Coprinopsis, based on molecular studies, the taxonomic status of P. subagraria being questionable. There are some differences between P. subagraria and C. jilinensis in macroscopic morphology, but they are similar in microscopic morphology. Smith (1972) described P. subagraria as having two basidiospores sizes [8-10 × 4-5 (10-12 × 4.5-5.5) μm], one of which was very close to the size of C. jilinensis [(8) 8.5-10 (10.2) × 4.5-5.9 (6) μm], requiring re-examination of the type specimens of P. subagraria in future studies.