A new species of Psathyrella (Psathyrellaceae, Agaricales) from Italy

Abstract Sporophores of a new Psathyrella species have been reported for the first time as growing at the base of Cladiummariscus culms in the Botanical Garden of the University of Calabria, Rende, Cosenza, southern Italy. The fungus was initially identified as P.thujina (= P.almerensis) by means of both ecology and macro- and microscopic characteristics of the basidiomes, then referred to P.cladii-marisci sp. nov. after extraction, amplification, purification and analysis of the rDNA ITS region. We came to this conclusion after comparing our specimen with the descriptions of the taxa available in the literature for the genus Psathyrella.


Introduction
Within the cosmopolitan fungal genus Psathyrella (Fr.) Quél. (Agaricales, Psathyrellaceae), about one hundred species have traditionally been recognised in Europe, almost all saprotrophs and found in many and diverse environments. Either terrestrial or lignicolous, they grow mainly on organic debris from various origins, such as dung, post-fire locations and dead stems of larger herbaceous plants (Vesterholt and Knudsen 1992). Psathyrella basidiomes are pileate, stipitated and exannulate or, at most, with a fugacious ring and the hymenophore is gilled, pale pink when young, turning brown with age due to a dark spore print. Moreover, they have, as the etymology indicates, a very fragile and ephemeral consistency. Despite these common macroscopic characters of the basidiomes, a recent phylogenetic analysis revealed the extremely complex origin of this genus, recognising species as belonging to a Psathyrella sensu stricto group or to P. sensu lato complex, the former including 19 clades and the latter involving eight genera (Coprinellus, Kauffmania, Cystoagaricus, Typhrasa, Lacrymaria, Homophron, Coprinopsis, Parasola), thus consistently widening the list of such "psathyrelloid" basidiomycetes (Örstadius et al. 2015).
During an investigation on the mycoflora of the Botanical Garden at the University of Calabria (Rende, Cosenza, Italy), basidiomes of an apparently "psathyrelloid" fungus were detected at the base of a fen-sedge [Cladium mariscus (L.) Pohl (Cyperaceae)], a cosmopolitan-distributed plant species (Lansdown et al. 2018) occurring in marshy places of most Italian regions (Bartolucci et al. 2018), but rarely in southern Italy.
The aim of this work was therefore to identify our basidiomes by using both morpho-ecological and biomolecular tools. This was highly encouraged by the habitat peculiarity and the close relationship with a plant species with which no species of Psathyrellaceae had ever been found associated.

Materials and methods
Eight basidiomes of the above "psathyrelloid" fungus were observed and collected on 10 April 2018, as gregarious all around and at the base of Cladium mariscus cut culms (Fig. 1). In 2012, that plant had been removed, together with the whole clump of mud attached to its roots, from a natural marsh named Lago dell'Aquila (Laureana di Borrello, Reggio Calabria, southern Italy) and transplanted to the Botanical Garden at the corner of a 90 × 37 cm-wide and 30 cm-deep concrete tank, which had permanently been kept full to the brim with water. Since then, some leaves of water lily (Nymphaea alba L.) have been introduced to float on the water surface inside the tank and the mud mass has been increasing, while the C. mariscus plant has been expanding and producing new culms that are cut every year.

Morphology
The basidiomes were first macroscopically examined for features, colours, sizes, hymenophore shape, pileus and stipe ornamentations, smell and taste. Then, the structures of the basidiome were microscopically inspected for cheilo-and pleurocystidia occurrence and features, presence of clamp connections, basidia and spore features. These observations were carried out under a light microscope (Axioplan 2 Imaging Microscope, Carl Zeiss, Germany) at 400 and 1,000 magnifications on fragments of pileipellis and gills placed on slides in 10% NH 4 OH. The results were compared with those published in the morphological keys for the Psathyrella species and, more specifically, with those species reported as the closest, according to morphology and ecological site conditions, i.e. P. thujina, P. typhae and P. lutensis (Kits van Waveren 1985, Vesterholt and Knudsen 1992, Christan et al. 2017, Henrici 2017.

DNA Extraction, Amplification and Sequencing
One of the basidiomes was dehydrated at room temperature and destroyed for molecular analysis: DNA extraction, amplification, purification and sequencing of the nuc rDNA internal transcribed spacer region (ITS). DNA extraction was implemented by using CTAB protocol (Doyle and Doyle 1987) and the ITS region was amplified using the primer combination ITS1F/ITS4 (White et al. 1990). The polymerase chain reaction (PCR) was performed in a 25-µl reaction volume containing 1.0 µl DNA, 2.5 µl 10 × 5-Prime-MasterMix Buffer (Thermo Fischer Scientific, Waltham, Massachusetts, USA) and 1.25 µl of each primer (10 µM/µl). The PCR was carried out according to the following amplification programme: 3 min initial denaturation at 94 °C, 35 cycles (30 s denaturation at 94 °C, 1 min annealing at 55 °C, 45 s extension at 72 °C) and a 10 min final extension at 72 °C. This programme was carried out in a T1000 Thermocycler (Biometra, Goettingen, Germany). The PCR products were purified using a QIAquick PCR purification kit (Qiagen Inc., Valencia, California, USA). Sequencing was performed by means of a Bigdye terminator cycle sequencing kit (Applied Biosystems, Foster City, California, USA). The sequencing reaction was run by BMR Genomics (Padua, Italy) on a 96-capillaries ABI 3730XL DNA Sequencer.
Forward and reverse DNA fragment electropherograms were checked by means of the CHROMAS 2.6.5 software (technelysium.com.au) for a complete reconstruction of the ITS1, ITS2 and 5.8 gene fragments. Ambiguous regions at the start and the end of the alignment were deleted and gaps were manually adjusted to optimise the alignment. The sequence generated for this study is deposited in GenBank with the code MK080112.

Alignment and Phylogenetic Analysis
Consensus sequences were generated from both forward and reverse primer reads in the BioEdit sequence alignment editor, version 7.2.5 (Hall 1999), then homology searches were performed at the National Centre for Biotechnology Information (NCBI) Web site using BLAST. This sequence was then compared with those of the Psathyrella species deposited in GenBank on which the phylogenetic analysis had recently been performed (Padamsee et al. 2008, Battistin et al. 2014, Örstadius et al. 2015, Yan and Bau 2018. A total of 45 ITS sequences, including three Coprinellus spp. (Table 2) were aligned using MAFFT with the L-INS-i option (Katoh et al. 2017). The aligned ITS dataset consisted of 702 nucleotide sites (including gaps). FASTA alignments from MAFFT were loaded in IQ-TREE 1.5.6 (Nguyen et al. 2014) to perform Maximum Likelihood Analysis. Clade robustness was assessed using a bootstrap (BT) analysis with 1,000 replicates (Felsenstein 1985). Phylogenetic trees were visualised using the FigTree v1.3.1 (Rambaut 2009).

Morphology
The macro-and micro-morphological features of the basidiomes collected at the base of the fen-sedge plant in the Botanical Garden are shown in Figures 2, 3. At first sight, by observing the macro-level characters, i.e. the small-medium size, the extreme fragility at handling and the brown-blackish spore print, the basidiomes were easily assigned to the Psathyrella genus (Vesterholt and Knudsen 1992). Secondly, the occurrence of sphaeropedunculate and clavate cells along the gill edge and the utriform shape of some cheilo and pleurocystidia seemed to direct them to the Section Spadiceogriseae Kits van Wav., subsection Spadiceogriseae (Romagn.) ex Kits van Wav. (Kits van Waveren 1985).
If we compare the morphological features of our specimens with those belonging to the closest Psathyrella species, a number of differences emerge (Table 1). Our specimens appeared to be more similar to P. thujina (Henrici, 2017), previously described as P. almerensis (Kits van Waveren 1985, Vesterholt andKnudsen 1992), except for Table 1. Main differences between our Psathyrella sp. and the closest species, according to the morphological characteristics of basidiomes and mycelium, and ecology. (Differences from our specimen are in bold characters).   the pileus diameter reaching 3.5 cm in our specimens, but never exceeding 2.5 cm in this species. Furthermore, our Psathyrella revealed versiform-shaped cheilocystidia, while those reported for P. thujina are only utriform. P. typhae was also divergent for the pileus diameter, not exceeding 2.5 cm, but even for pileus and stipe colours and for lacking pleurocystidia. On the other hand, the mucoid deposits, characterising the pleurocystidioid cheilocystidia of P. lutensis, were absent in our specimens. In addition, the spore length range was wider in our specimens than in P. thujina and P. lutensis and all the closest three species, which showed larger spores on average. As for ecology, the plant genus Cladium Browne has never been reported as a substrate to any other Psathyrella, although P. thujina and P. typhae are commonly found on the remnants of ecologically similar plants (Kits van Waveren 1985, Vesterholt and Knudsen 1992, Örstadius et al. 2015, Henrici 2017. Furthermore, the genus Cladium was not mentioned in the unique Italian report of P. thujina, which was found "in open sites, close to any hygrophilous plants" (Voto 2016), in accordance with Henrici (2017) who refers this species to reed-beds and generic damp marshy habitats. Finally, our specimen was collected in the spring, whereas the above three other Psathyrella species seem to occur in other seasons.

DNA Analysis
The obtained nrDNA sequence was 702 bp long. By comparing it with those published in GenBank, we obtained a data matrix composed of 44 taxa and 710 characters, 276 gap-free sites and 240 conserved sites. The highest homology (99%) was observed with P. candolleana (Fr.) Maire, which was confirmed by the phylogenetic analysis (Fig. 4). Indeed, the phylogenetic tree shows that our specimen falls into the "candolleana" clade, such a heterogeneous group, including taxa from different morphology, ecology and geographic provenance and, amongst them, the above-cited P. typhae (Battistin et al. 2014, Örstadius et al. 2015, Yan and Bau 2018.

Discussion and conclusions
Based on results from both morphological and molecular analysis, our collection cannot be assigned to a known species. According to morphology, our Psathyrella should be closer to P. thujina (Section Spadiceogriseae). By contrast, the DNA ITS sequence would undoubtedly include it in the "candolleana" clade, where each species showed up to a 99% ITS sequence similarity with our sample. The most widespread and known species in this clade, P. candolleana and P. leucotephra (Berk. & Broome) P.D. Orton, both commonly occurring in Europe, too, are however morphologically very different from our specimen, by forming large pilei (diameter up to 8.0 cm) and lacking pleurocystidia; furthermore, the latter frequently even shows a torn annulus in the upper part of the stem, which we did not observe in our Psathyrella (Kits van Waveren 1985,   Vesterholt andKnudsen 1992, Consiglio 2005). The "candolleana" clade encompasses two more European species according to two recent phylogenetic analyses (Nagy et al. 2011, Battistin et al. 2014: P. sulcatotuberculosa (J. Favre) Einhell., previously regarded as a variety of P. typhae (Kits van Waveren 1985), which mainly differs from our Psathyrella and from P. typhae itself with a partially-sulcate and -tuberculate pileus surface, and P. badiophylla (Romagn.) Bon which forms spores normally exceeding 10-11 µm in length (Kits van Waveren 1985, Vesterholt andKnudsen 1992); in addition, both also lack pleurocystidia, which was considered to be such a morphologically relevant character to induce the establishment of the Section Spintrigerae within the subgenus Psathyra (Fr.) Sing. ex Kits van Wav. (Kits van Waveren 1985). Moreover, except for P. typhae, which is the only Psathyrella ecologically comparable to our collection, all the above species are reported to grow in diverse site conditions, i.e. close to stumps of trees or on branches, on moist ground, in grass, on mossy woods or on various other vegetable matter (Kits van Waveren 1985, Vesterholt andKnudsen 1992). Finally, as far as we know, other species in the "candolleana" clade are even geographically more distant, each colonising a different kind of organic debris (Padamsee et al. 2008, Örstadius et al. 2015, Yan and Bau 2018. Therefore, within this framework, the placement of our fungus into the "candolleana" clade, together with other species showing strong differences for geographic and ecologic reasons, should not prevent the recognition of a new Psathyrella species. Anyhow, more and more scientific contributions are remarking that the genetic analysis of a fungus aiming at taxonomic purposes can alone generate artefacts, i.e. "false positive" or "chimeras", especially when such analysis is implemented by using a unique gene (Thines et al. 2018, Lücking et al. 2018. A polyphasic approach, i.e. based on the combination and integration of all the available informative data (Colwell 1970), is becoming more and more desirable for taxonomic research in mycology, whereas the ITS rDNA region is still considered as the universal genetic marker for fungi (Schoch et al. 2012).
On the basis of the outcomes deriving from the morphologic, ecologic and biomolecular characteristics which we have identified in this note, we are therefore inclined to establish a new species of Psathyrella. Description. Habit psathyrelloid. Pileus up to 3.5 cm diam., conical-convex when young, hemispheric to applanate at maturity, with a deeply striate margin, hazelnut in colour, turning to pale beige when dry. Pileipellis with evident concentric arachnoid fibrils of velar origin, whitish and easily removable, often exceeding the cuticle margin. Lamellae distant, ventricose, adnate, intermingled with numerous lamellulae, initially pale pink, then intensely brown-purplish. Lamella edge whitish with numerous sphaeropedunculate cells. Stipe, very fragile, cylindrical, white, exannulate with a diffuse fibrillosity especially on the basal surface, apical surface pruinose. Basidiospores 7.2-11.8 × 4.3-6.0 µm (n = 100), ellipsoid to ovoid-ellipsoid, with a thick and smooth wall, adaxially flattened with a central 2µm-wide germ pore and a distinct hilar appendix. Spore-print dark brown. Basidia clavate, 4-spored. Cheilocystidia versiform, often utriform, seldom cylindrical to clavate. Pleurocystidia utriformshaped. Mycelium septate and clamped. Context with apparently no smell, taste mild.

Psathyrella cladii-marisci
Habit, habitat and distribution. In small groups (gregarious), on the culm remnants of Cladium mariscus. So far, known only from the type locality.

Conclusions
This probably rare and, apparently, never before detected species could occur more commonly if further surveys confirmed a sort of preference for C. mariscus as a growing substrate for the fungus. This plant was observed all over Italy (Bartolucci et al. 2018), although becoming more and more scattered due to the progressive surface reduction of its natural growing environment, i.e. marshes and wet sites quite close to the sea at mid-low altitudes. These sites have been long subjected to draining and other forms of anthropogenic land uses. Since human activities have been causing a deep influence and restriction on density and distribution of the spontaneous flora, including C. mariscus, the gradual depletion of plant biodiversity in such sites could also result in negative effects on fungal diversity, thus rendering even more scarce the occurrence of basidiomes of such taxa as P. cladii-marisci in Italy.