Updated description of Atheniella (Mycenaceae, Agaricales), including three new species with brightly coloured pilei from Yunnan Province, southwest China

Abstract An updated description of the genus Atheniella, combining macro- and micromorphological characters that elaborate on the original generic characterisation, is presented. Atheniella is characterised by a brightly coloured pileus, all tissues inamyloid and pileipellis covered with simple to branched excrescences. Previously, nine Atheniella species were known globally, of which three species were accepted in China. Three newly-recognised species classified in the genus are here formally described from Yunnan Province: Atheniella flavidasp. nov., A. rutilasp. nov. and A. taoyaosp. nov. The new species are characterised by a yellow, orange, pink or red pileus, fusiform cheilocystidia and pleurocystidia, non-smooth pileipellis, stipitipellis smooth or with cylindrical ornamentation, caulocystidia fusiform or subglobose, if present and all tissues inamyloid. Morphological descriptions, photographs, line drawings and comparisons with closely-related taxa are presented for the new species. A phylogenetic analysis of sequence data for the rDNA internal transcribed spacer region and nuclear large ribosomal subunit (ITS + nLSU) supported that Atheniella is resolved as monophyletic and also supported the taxonomic recognition of the new species. A key to the 12 species of Atheniella is also provided.

Three Atheniella species, namely A. adonis, A. aurantiidisca and A. flavoalba (Fr.) Redhead, Moncalvo, Vilgalys, Desjardin & B.A. Perry, were previously recognised in China (Bau and Liu 2011;Li et al. 2015;Na 2019). During our ongoing research on Mycena s.l., three new mycenoid species belonging to Atheniella were discovered in Yunnan Province, southwest China and are formally described here as A. flavida Q. Na & Y.P. Ge, A. rutila Q. Na & Y.P. Ge and A. taoyao Q. Na & Y.P. Ge. In addition, the generic morphological description of Atheniella is updated and a key for identification of the 12 species of Atheniella currently known is provided.

Morphological examination
Macroscopic descriptions were prepared, based on freshly-collected specimens, whereas micromorphological descriptions relied on dried material. In the descriptions, colour abbreviations follow Kornerup and Wanscher (1978). Microscopic observations were conducted on dried specimens mounted in 5% potassium hydroxide (KOH) and stained with Congo red when necessary. Melzer's Reagent was used to test whether spores and tissues were amyloid (Horak 2005). Twenty mature basidiospores from each basidiocarp were measured in lateral view and one or two basidiocarps were examined per specimen. The basidiospore dimensions were recorded; the notation [a/b/c] used at the beginning of each basidiospore description indicates that a basidiospores from b basidiocarps of c specimens were measured. Measured dimensions (length × width) are presented as (d) e-f-g (h) × (i) j-k-l (m), where d is the minimum length, e-g represents the range of at least 90% of values, f is the average length and h is the maximum length; width (i-m) is expressed in the same manner. In addition, Q is the length: width ratio of a spore and Q ± SD is the average Q of all basidiospores ± the sample standard deviation. Authority abbreviations follow those used in Index Fungorum (https://www.indexfungorum.org). Voucher specimens have been deposited in the Fungarium of the Fujian Academy of Agricultural Sciences (FFAAS), China.

DNA extraction, PCR amplification and DNA sequencing
Genomic DNA was extracted from tiny pieces of lamellae using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Co., Beijing, China). The internal transcribed spacer (ITS) region and the nuclear large subunit (nLSU) of rDNA were amplified with the primer pairs ITS1/ITS4 and LROR/LR7, respectively (White et al. 1990;Hopple and Vilgalys 1999). The PCR thermocycling protocol (for both ITS and nLSU) was 94 °C for 4 min, followed by 34 cycles of 94 °C for 45 sec, 52 °C for 45 sec and 72 °C for 1 min and final extension for 10 min at 72 °C. The new sequences were submitted to GenBank (Table 1). The nBLAST tools (http://blast.ncbi. nlm.nih.gov/Blast.cgi) were used to compare the sequence identity with sequences in the NCBI databases. The GenBank accession numbers for the ITS and nLSU sequences are as follows: Atheniella flavida (MW969653-MW969654; MW969665), A. rutila (MW969658-MW969659; MW969668) and A. taoyao (MW969655-MW969657; MW969666-MW969667).

Sequence alignment and phylogenetic analysis
A dataset comprising concatenated sequences for the ITS and nLSU regions from 45 accessions of three genera (Atheniella, Hemimycena Singer and Mycena) was compiled. A total of 112 sequences downloaded from GenBank and 11 sequences newly generated in this study were aligned and adjusted manually using BioEdit 7.0.4.1 and Clustal X (Thompson et al. 1997;Hall 1999). Gaps in the alignments were treated as missing data. The alignment was deposited with TreeBase (submission ID, 28111; study accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S28111). Hydropus scabripes (Murrill) Singer was chosen as the outgroup. The aligned dataset consisted of 836 ITS and 879 nLSU nucleotide sites (including gaps). The best-fit evolutionary model was determined using Modeltest 2.3 for each of the ITS and nLSU data partitions for Bayesian Inference (BI), which was implemented with MrBayes 3.2.6 ( Ronquist and Huelsenbeck 2003;Nylander 2004). Markov Chain Monte Carlo (MCMC) chains sampling every 100 th generation until the topological convergence diagnostic value was less than 0.01 (Ronquist and Huelsenbeck 2003). Maximum Likelihood (ML) analysis was performed using raxmlGUI 1.5b1 and topological support was assessed using the rapid bootstrapping algorithm with 1000 replicates (Stamatakis et al. 2004). Topology support values, greater than 75% bootstrap support (ML) and 0.95 Bayesian posterior probabilities (BPP), are shown for relevant branch nodes.

Phylogenetic analysis
The concatenated dataset comprised 45 taxa and 1715 sites. The GTR + G evolutionary model was selected for both ITS and nLSU regions. The optimal evolutionary model for the 5.8S and nLSU partitions was lset nst = 6, rates = invgamma and prset statefreqpr = dirichlet (1,1,1,1). The BI and ML phylogenetic reconstructions were consistent in topology and, thus, only the BI tree is presented (Fig. 1).
Habit and habitat. Saprophytic on grass, moss, rotten wood or plant debris (leaves, pine needles and twigs).
Habit and habitat. Scattered on rotten wood in evergreen broadleaf and Pinus mixed forest.

Atheniella taoyao
Etymology. Refers to the pinkish to reddish basidiomata. Tao Yao is a poem in the "The Book of Songs" that praises a young woman, whose beauty is compared to a flowering peach tree and who will be married and assume a new role in life.

Discussion
The present phylogenetic analysis showed that Atheniella formed a distinct clade independent of Hemimycena and Mycena with high BPP and BS support and, thus, supported segregation of the genus from the Mycenaceae (Moncalvo et al. 2002;Matheney et al. 2006). This finding also supported the view of Redhead et al. (2012) that Atheniella, formerly treated as Mycena sect. Adonideae, should be elevated to generic rank. Atheniella is more closely related to Mycena than to Hemimycena, based on genetic distance, in accordance with the greater similarity of Atheniella to Mycena spp. in morphological characters. The presence of pileocystidia and the morphological differences of the cheilocystidia, caulocystidia and stipitipellis can be used to distinguish Atheniella species from Hemimycena and Mycena.
Atheniella was originally established by Redhead et al. (2012) to accommodate four species: A. adonis, A. amabillissima, A. aurantiidisca and A. flavoalba. In recent years, the number of recognised species of Atheniella has increased to nine, but the description of the genus was incomplete and not detailed (Redhead et al. 2012;Gminder and Böhning 2016;Lehmann and Lüderitz 2019). With description of the new species in the present study, the generic description for Atheniella requires updating. Amongst Atheniella species, the bright colour of the pileus may be uniform or be tinted at the centre, but fades to white at the margin, the lamellae are adnate to decurrent and the stipe colour sometimes changes to yellow or pink towards the base. With regards to micromorphological characters, Atheniella produces globose to cylindrical spores, caulocystidia are present or absent and the stipitipellis is smooth or has projections.
Morphological and molecular evidence support classification of the three newlyrecognised species as members of Atheniella. The three species share white lamellae, a pruninose stipe base without a basal disc, inamyloid basidiopores, fusiform and thin-walled cheilocystidia, pileipellis covered with excrescences and are unreactive in Melzer's Reagent. In addition, the three species grow on rotten wood or other plant debris. A. flavida is mainly distinguished from A. taoyao and A. rutila by its distinctly yellowish-orange to yellow pileus and globose spores. The pinkish or reddish basidiomata support the inclusion of A. taoyao and A. rutila in Atheniella. Compared with A. rutila, A. taoyao is readily discriminated, based on the light pink basidiomata, narrow ellipsoid basidiospores and subglobose or fusiform caulocystidia. A. amabillissima shows the most morphological similarities to A. taoyao and A. rutila; however, A. amabillissima has a pileus which fades to white with age, smaller spores and longer cheilocystidia (Smith 1935b).
It is noteworthy that the taxonomic status of Mycena floridula remains unresolved (Josserand 1930;Kühner 1938;Aronsen and Laessøe 2016). This species was formerly classified in Mycena sect. Adonideae as a form of M. flavoalba with a pink pileus (Josserand 1930;Kühner 1938;Aronsen and Laessøe 2016). More recently, it was proposed that the name M. floridula was a synonym of A. adonis (Redhead et al. 2012). The phylogenetic reconstructions in our study and accessions of M. floridula indicated that M. floridula was closely related to A. flavoalba, with little genetic distinction between the two taxa. Therefore, we tentatively accept M. floridula as a pink form of A. flavoalba, but emphasise that a detailed appraisal of the morphological and molecular variation of A. flavoalba is required.