A New Tropical Species of Psathyrella (Psathyrellaceae, Agaricales) From the Cúc Phương National Park, Vietnam


 Basidiocarps of a hitherto undescribed Psathyrella species were collected during a sampling excursion in North Vietnam. They grew on deciduous deadwood in the Southeastern part of the Cúc Phương National Park (Vietnamese: Vườn quốc gia Cúc Phương), Ninh Bình Province. Herein, we describe Psathyrella euryspora sp. nov. characterized by broadly ellipsoid to broadly ovoid basidiospores (5.5–7.0 × 4–5(–6)) µm without visible germ pore, utriform to ventricose-clavate cheilocystidia, heteromorphic caulocystidia and the absence of pleurocystidia and pileocystidia. Based on an isolated pure culture, the genome was sequenced and a full ribosomal RNA gene cistron including 18S, internal transcribed spacer 1 (ITS), 5.8S, ITS2 and 28S rRNA annotated. Phylogenetic analysis of P. euryspora revealed its close affiliation to the Psathyrella sect. Candolleanae species clade.


Introduction
The dark spored agaric genus Psathyrella (Fr.) Quél. is ubiquitously distributed across the world (Hoashi 2008;Singer 1978;Van Waveren 1985). Thus, Psathyrella species can be found in arctic or alpine regions but in the subtropics and tropics as well (Pegler 1983(Pegler , 1977Smith 1972). Although, over 900 species have been described so far (http://www.mycobank.org), the number of which is steadily increasing Species of the genus are usually following saprotrophic lifestyles and thus, grow on deadwood, plant debris, dung, leaf-litter or humus-rich soil. The genus' name refers to the fragile or brittle constitution of fruiting bodies, which all Psathyrella species have in common (diminutive of Psathyra, from Old Greek ψαθυρος = friable, cumbling).
Recent molecular studies have indicated closer relation of the genus to other psathyrelloid genera, such as Coprinellus P. Karst. and Coprinopsis P. Karst. (Matheny et al. 2006;Nagy et al. 2009;Örstadius et al. 2015; Redhead et al. 2001). In consequence, these genera were included into the family of Psathyrellaceae, which, according to Örstadius et al. (2015), now includes Coprinopsis, Parasola Redhead (Örstadius et al. 2015), of which /candolleana is well supported and the closest relative to Coprinellus. A commonly distributed morphologic feature within /candolleana is the absence of pleurocystidia.
A Psathyrella basidiocarp resembling the recently described species P. aberdarensis A. Melzer, Kimani & R. Ullrich was collected during a sampling excursion with focus on macrofungi in the Cúc Phương National Park (Vietnamese: Vườn quốc gia Cúc Phương, Ninh Bình Province, North Vietnam) in November 2018. This specimen, from which a strain was isolated, turned out to microscopically deviate from all known Psathyrella spp. and therefore, shall be described herein. In addition, the strain was sequenced and its full genome sequence providing relevant data for further phylogenetic and biotechnological analyses deposited at NCBI (National Centre for Biotechnology Information) Bioproject PRJNA647680.
Although, Vietnam is without doubt a hotspot of biodiversity, only little information is currently available on the funga of this country (de Queiroz et al. 2013). For example, the Global Biodiversity Information Facility (https://www.gbif.org) does not list any Vietnamese fungal herbarium or culture collection entry. On the other hand, the enormous diversity of large uncharted regions in Vietnam (Collen et al. 2014) will surely lead to the discovery of more fungal species, as it has recently been reported in a survey on comparable Chinese regions (Yan and Bau 2018).

Materials And Methods
A twig bearing two fresh basidiomata was collected in Cúc Phương National Park and transferred to Zittau (Germany) for further cultivation and examination. The in situ collected twig was placed under saturation vapor at 23 °C in an incubation chamber. After about ve weeks three basidiocarps grew on the twig consecutively within a month. An axenic pure fungal strain was isolated from this material using 2% malt-agar plates with antibiotic additions (50 µg/ml streptomycin, penicillin, chloramphenicol, benomyl and 40 µg/ml nystatin). The strain was deposited at the Vietnam Type Culture Collection (VTCC, Hanoi, Vietnam) under number VTCC 930004.

Morphology
Dried specimen voucher are deposited at the Herbarium Senckenbergianum Görlitz, Germany (GLM-F126263). All macroscopic characteristics were recorded based on fresh material. Photographs of the cultivated basidiomata were taken and color codes are based on the RGB color model (Poynton 2003).
Microscopic characteristics were analyzed based on hand sections of fresh and revived material (Zeiss, Axio Scope A1, Oberkochen, Germany; Canon EOS 60 D, Tokyo, Japan). The spore size was measured in water using spores from a spore print at the stipe apex. Color of spores was assessed in water, ammonia solution (10%) and potassium hydroxide solution (5% w/v KOH). Cystidia and other microscopic structures were studied in 5% KOH. DNA Extraction, Genome Sequencing and ribosomal RNA gene retrieval Biomass of a culture plate of Psathyrella euryspora was scraped off to extract genomic DNA by a standard acetyltrimethylammonium bromide (CTAB)-based method. Genomic DNA was sonographically sheared with a S2 ultrasonicator (Covaris, Woburn, MA, USA) to subsequently construct a 400 bp library using the Ion Plus Fragment Library Kit (Thermo Fisher, Darmstadt, Germany). The library was sequenced on an Ion GeneStudio™ S5 System using the Ion 530 Chip Kit. The resulting 11.3 million reads were ltered to include lengths between 300 and 625 bp and were assembled using MIRA 4.0 (minimum reads per contig = 100, mode = accurate; Chevreux et al., 1999). Overlapping contigs were joined and duplicate contigs were ltered using, in a second step, the Geneious assembler R11.4.1 (parameter: highest sensitivity/slow; Kearse et al., 2012). For complete annotation of the full ribosomal RNA gene cistron including 18S (SSU), internal transcribed spacer 1 (ITS), 5.8S, ITS2 and 28S (LSU) rRNA gene regions and spacer, the contig was manually identi ed comparing references obtained from NCBI nr-Database to the full assembly including all contigs. The full ribosomal cistron including SSU, LSU and 5.8S rRNA gene regions are available at GenBank under accession number MT651560. The IonTorrent raw reads have been deposited at the Sequence Read Archive (NCBI) under accession number SRR12301154.

Alignment and Phylogenetic Analysis
In a rst step, the identi ed P. euryspora ITS and 28S rRNA (D1-D3) gene regions were used to nd similar sequences of the genus Psathyrella (sensu stricto, outgroup generated) and speci cally of the P.
candolleana subclade in NCBI GenBank using a blastn search (E-value 1e − 1 ). Phylogeny of the taxonomic classi cation of the clades is given in supplement S1. Additional information on β-tubulin and the translation elongation factor (tef)1α can be accessed in supplement S2 (i.e. gene and protein).
Furthermore, a set of sequence data including ITS region and the 28S rRNA gene region (as far as available) of the P. candolleana clade was composed and is listed in Table 1. Alignments were calculated using MUSCLE 3.8.425 (Edgar 2004) with default settings integrated in Geneious R11. Phylogenies were constructed using MEGA 10.0.5 (Kumar et al. 2018). For both sets, a maximum likelihood approach after a model selection was used (Guindon and Gascuel 2003). The substitution model with the lowest Bayesian Information Criterion (BIC) score was chosen (K2 + G + I, Kimura, 1980). Branch support was estimated by using a bootstrap approach using 1,000 replicates. Furthermore, a Bayesian approach using  aberdarensis. A detailed analysis of the /candolleana clade was performed including ITS and 28S rRNA regions. (Fig. 1). As the outcome, P. sulcatotuberculosa and P. aberdarensis, the new Vietnamese specimen and the sequence of a specimen that is regarded as P. singeri form a well-supported subclade (n = 1,000 bootstrap), namely /sulcatotuberculosa.  (Fig. 2). Lamellae medium spaced to crowded, L = 28-30, interspersed with 1-3 lamellulae, adnexed to adnate, dark-cream to brownish, nally becoming brown at age, lamellar edge white, pruinose (lens). Stipe up 15-20 mm × 1 mm, cylindrical, white in the upper part, downwards somewhat brownish-creamy, tip pruinose from caulocystidia, base white tomentose.
Habit and habitat.
Solitary on deadwood (fallen twigs) of broad-leaved trees. Humid and near-ground habitat.

Discussion
Psathyrella euryspora is characterized by its small sporocarps, its occulose yet relatively persistent veil consisting of rather thin walled and moderately encrusted, cylindric hyphae as well as by its small, broad (QAV. <1.5) and pale spores without a visible germ pore.
According to DNA-analysis, P. euryspora is closely related to P. aberdarensis and P. sulcatotuberculosa. The latter, seemingly rare species had been listed as a variety of P. typhae (Kalchbr.) A. Pearson & Dennis for a long time and was later placed into the /candolleana clade (Battistin et al. 2014;Matheny et al. 2006). Both species, however, can bear lageniform cheilocystidia and seem to be restricted to humid/wet habitats, which is in contrast to P. euryspora (Battistin et al. 2014; Ludwig 2007).
Another closely related species is Psathyrella singeri A. H. Sm (Melzer et al. 2018), which was described as a twig-dwelling fungus from Florida (Smith 1972). Smith did not mention any veil and described the lamellae being crowded. The ITS sequence of a specimen collected in China and determined as P. singeri was deposited in GenBank (compare Table 1) and differs from sequences of P. euryspora. It is unclear, however, whether this specimen is in fact P. singeri (Melzer et al. 2018). Nevertheless, all mentioned species have very pale spores and share the same branch /sulcatotuberculosa within the Candolleanae clade (compare Fig. 3). However, P. euryspora differs from other species within /sulcatotuberculosa by having smaller and more rotund spores. Obviously, there are two different veil types within /sulcatotuberculosa. The veil of P. aberdarensis consists of two types of cells, i.e. (i) diverticulate, often thick walled and brownish pigmented cells and (ii) globose elements (Melzer et al. 2018). In contrast, the veil elements of P. euryspora consist of cylindrical, subhyaline to pale brown and -in some parts -slightly encrusted hyphae, and therefore resemble the veil hyphae of P. sulcatotuberculosa (Battistin et al. 2014).
Other species with more or less pale spores, lacking or indistinct germ pore and without pleurocystidia were already discussed in Melzer et al. (2018) but shall be mentioned here again.
Psathyrella glaucescens Dennis has olive shades on the cap as well but differs by a larger pileus (1.5-5 cm), an only slightly translucently striate margin, fugacious veil made of ventricose elements and by truncate spores with a porus (Dennis 1961;Pegler 1977).
Psathyrella pusilla Pegler has only clavate cheilocystidia, no veil and spores that are apically truncate by a germ pore (Pegler 1977).
Psathyrella subsingeri T. Bau & J.Q. Yan is a slightly larger species with longer and more slender spores (Q = 1.4-2.0) and differs in ITS sequence (compare Fig. 1

Competing interests
The authors declare that they have no competing interests.   (Kimura 1980). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sampling from the ML tree of the existing molecular data using the reconstruction method MCMCMC (Geyer 1991) using MrBayes, values are contained in square brackets. The tree is rooted using Coprinellus spp. as outgroup.