The Northeast Chinese species of Psathyrella (Agaricales, Psathyrellaceae)

Abstract Twenty seven species of Psathyrella have been found in Northeast China. Amongst them, P. conica, P. jilinensis, P. mycenoides and P. subsingeri are described as new species, based on studying morphological characteristics and phylogenetic analyses. Detailed morphological descriptions, line drawings and photographs of the new species are presented. Phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS) region and an identification key to the 27 Psathyrella species occuring in Northeast China are provided.


Introduction
Psathyrella (Fr.) Quél. is one of the large genera of Agaricales Underw. which consists of 1,030 records in Index Fungorum (http://www.indexfungorum.org), comprising approximately 500 species (Smith 1972;Kits van Waveren 1985;Örstadius and Kundsen 2012;). It is characteristic of fragile basidiomata, hygrophanous pileus, brown to Specimens are deposited in the Herbarium of Mycology, Jilin Agricultural University (HMJAU). Macroscopic characteristics were recorded from fresh specimens. Colour codes are from Kornerup and Wanscher (1978). Samples for microscopic examination were mounted in water and 5% aqueous KOH. Amyloid reactions were diagnosed in Melzer's reagent. Thirty basidiospores, cystidia and basidia were measured for each collection. The basidiospores quotient (Q=L/B) was calculated from measurements of basidiospores.

DNA extraction and sequencing
The NuClean Plant Genomic DNA kit (CWBIO) was employed for DNA extraction and PCR am plification from dried specimens. PCR was performed using a touchdown programme (Yan and Bau 2017) and the ITS region was amplified with the primer pair ITS1 and ITS4 (White et al. 1990). The details of sequenced specimens are given in Table 1. The DNA sequencing was done by Comate Bioscience Co., Ltd., Changcun City, China.

Data analyses
ITS1+5.8S+ITS2 sequences of 27 species were tested with BLAST in GenBank. Fifty five sequences were downloaded from GenBank, including 21 type species of Psathyrella, based on BLAST results and referred to the recent studies (Nagy et al. 2013;von Bonsdorff et al. 2014;Örstadius et al. 2015;Yan and Bau 2017). A total of 103 ITS sequences were aligned using MAFFT 7.205 (Katoh and Standley 2013). The aligned ITS dataset consisted of 643 nucleotide sites (including gaps). The best model (GTR+I+G) was selected by AIC in MRMODELTEST 2.3 (Nylander 2004). Bayesian Inference (BI) was performed with MRBAYES 3.2.6 and four Markov Chains (MCMC) were run for three million generations, sampling every 300th generation. The first 25% trees were discarded (Ronquist and Huelsenbeck 2003). Maximum likelihood analysis was performed with IQTREE 1.5.6 (Nguyen et al. 2014).

Results
The phylogenetic tree ( Figure 1) shows that all studied materials fall into Psathyrella, with a high statistical support value (BPP=1). It is divided into 14 clades. Most of them have a high statistical support value (BPP≥0.95, Bootstrap≥75), except /fibrillosa I and /fibrillosa II. Four new species are separated into individual lineages (BPP=1, Bootstrap=100) and are independent from the close taxa. Psathyrella conica forms a distinct lineage in /fibrillosa II; P. jilinensis belongs to /fibrillosa II and groups together with P. borealis; P. mycenoides belongs to /prona and is closely related to P. lilliputana Örstadius & E. Larss.; and P. subsingeri forms a distinct lineage in /candolleana. The positions of some species are firstly supplemented: P. amaura belongs to /pygmaea and is very close to P. olympiana A.H. Sm.; P. borealis belongs to /fibrillosa II. P. mammifera belongs to /spadiceogrisea; P. singeri A.H. Sm. belongs to /candolleana; and P. subterrestris belongs to /noli-tangere. Diagnosis. Pileus campanulate to conical, with a subacute to obtuse umbo in early stage. Lamellae 3.0-5.0 mm broad, close. Basidiospores 7.8-8.8 × 4.0-4.5(-5.0) μm, germ pore indistinct or absent. Pleurocystidia numerous, narrowly utriform, with obtuse to broad obtuse or slightly subcapitate at apex. Cheilocystidia scattered.
Habit and habitat. Solitary to scattered on humus in mixed forests. Other specimens examined. CHINA. Jilin Province, Changchun City, Jilin Agricultural University, 12 Sep 2016, HMJAU 37993.
Habit and habitat.

Discussion
These phylogenetic results are very much in congruence with the study of Larsson and Örstadius (2008) and Örstadius et al. (2015), except /fibrillosa, which separates to two lineages (/fibrillosa I and /fibrillosa II). As only ITS sequences were analysed in this study, this accounts for the difference and the very low support value (BPP<0.3). Four new species are separated into individual lineages (BPP=1) and distinct from other closely related taxa.
Psathyrella conica is a distinct lineage in fibrillosa II, which is independent from any other related taxa. Morphologically, it can be classified in subsection Spadiceogriseae (Kits van Waveren 1985). Only P. clivensis (Berk. & Broome) P. D. Orton does not have a germ pore in this subsection, but basidiospores of P. clivensis are obviously broader, 8-10 × 5.5-6.5 μm and ellipsoid to ovoid (Kits van Waveren 1985). It can also be classified in section Fatuae (Smith 1972), some species having sturdy stipe and utriform cystidia, but they can be clearly distinguished from P. conica by other micromorphology. Psathyrella acadiensis A.H. Sm. has smaller basidiospores, which are only up to 6.0 μm long; P. albocinerascens A.H. Sm. has an obvious germ pore and white pileus in the early stage; P. amarella A.H. Sm. and P. spadiceogrisea (Schaeff.) Maire have an obvious germ pore; P. vesiculocystis A.H. Sm. has pedicellate-pleurocystidia (Smith 1972). Furthermore, P. terrestris Natarajan has aspects of P. conica, whose pileus is umbonate, but it has broadly utriform pleurocystidia and its basidiospores are dark brown, subglobose and up to 8.5 μm broad (Natarajan 1978).
Psathyrella jilinensis grouped together with P. borealis in /fibrillosa II. However, P. borealis has an obvious germ pore. Morphologically, it can be classified in section Hydrophilae by basidiospores rarely exceeding 7.5 μm and the presence of pleurocystidia. There are hardly any other species in the section that match the characteristics of P. jilinensis.  (Smith 1972) and P. pennata grows on burnt soil, its basidiospores being larger and narrowly amygdaloid (Örstadius and Kundsen 2012).