﻿Three new species of Favolaschia (Mycenaceae, Agaricales) from South China

﻿Abstract The genus Favolaschia within the family Mycenaceae is characterised by the gelatinous basidiomata with poroid hymenophore and most species inhabit monocotyledonous plants. In this study, many samples covering a wide geographic range in China were examined morphologically and phylogenetically using concatenated ITS1-5.8S-ITS2-nLSU sequence data. Three new species clustering in Favolaschiasect.Anechinus, namely Favolaschiaimbricata, F.miscanthi and F.sinarundinariae, are described. Favolaschiaimbricata is characterised by imbricate basidiomata with pale grey to greyish colour when fresh and broadly ellipsoid basidiospores measuring 7–9 × 5–6.8 µm; F.miscanthi is characterised by satin white basidiomata when fresh, broadly ellipsoid basidiospores measuring 7.5–10 × 5.5–7 µm and inhabit rotten Miscanthus; F.sinarundinariae is characterised by greyish-white basidiomata when fresh, dark grey near the base upon drying, broadly ellipsoid to subglobose basidiospores measuring 7–9 × 5–7 µm and inhabit dead Sinarundinaria. The differences amongst the new species and their morphologically similar and phylogenetically related species are discussed. In addition, an updated key to 19 species of Favolaschia found in China is provided.

A total of 119 records of Favolaschia are listed in Index Fungorum (http://www.indexfungorum.org) and around 60 species are accepted (Johnston et al. 2006;Gillen et al. 2012;Magnago et al. 2013).The name "Favolaschia" was first introduced by Patouillard (1887) as a section of Laschia Fr and treated at a generic level later (Patouillard and Lagerheim 1892).The taxonomic history of the genus has been systematically compiled by Singer (1974) and Johnston et al. (2006).Singer (1974) divided Favolaschia into two sections, based on morphological studies, namely section Favolaschia Singer and section Anechinus Singer.Amongst them, taxa of F. sect.Favolaschia is characterised by having mostly orange, yellow, red, lateritious or pink pileus and the presence of acanthocytes in pileipellis.Inversely, taxa of F. sect.Anechinus are characterised by lacking acanthocystidia or replaced by diverticulate hyphae.Later, Johnston et al. (2006), Gillen et al. (2012) and Magnago et al. (2013) confirmed the two sections by phylogenetic analyses.
Recently, the systematic study of Favolaschia in China has gradually increased.Tolgor et al. (2021) investigated the phylogenetic relationships of Favolaschia and its related genera in Mycenaceae and reported four species distributed in China.Zhang and Dai (2021) investigated phylogenetic analysis of the genus Favolaschia, based on a large sample and revealed four new species in the F. calocera complex.Until Zhang et al. (2023) re-summarised the species diversity, phylogenetic relationships, divergence time and potential geographic distribution using a large number of samples covering a wide geographic range in China.To date, 16 Dai and F. tonkinensis (Pat.)Kuntze; the first four species belong to the F. calocera complex of the F. sect.Favolaschia; while the others belong to the F. sect.Anechinus (Zhang and Dai 2021;Guo et al. 2022;Ma et al. 2022;Wu et al. 2022a;Dong et al. 2023;Zhang et al. 2023).
During a study on wood-rotting fungi from south China, several samples belonging to Favolaschia were collected and three unknown species were morphologically distinguished.To confirm the affinity of the taxa, phylogenetic analysis was performed, based on a combined sequence dataset of ITS1-5.8S-ITS2-nLSU.

Morphological studies
The specimens were collected from Guangxi Autonomous Region, Guangdong and Sichuan Provinces in south China and the specific locations were marked in Fig. 1.They were deposited in the Fungarium of the Institute of Microbiology, Beijing Forestry University (BJFC), Beijing, China.Macro-morphological descrip-tions were based on field notes and dried specimens.Microscopic features were examined and described in 5% KOH (potassium hydroxide) and 2% phloxine B (C 20 H 2 Br 4 Cl 4 Na 2 O 5 ) with a magnification of up to 1,000× using a Nikon Eclipse 80i microscope and phase contrast illumination.Colour terms followed Kornerup and Wanscher (1978) and Petersen (1996).A Nikon Digital Sight DS-L3 camera was used to photograph microscopic structures.Other reagents, including Cotton Blue and Melzer's reagents were used to observe the micromorphology structures following Zhang et al. (2023).To show the variation in spore sizes, 5% of measurements were excluded from each end of the range and shown in parentheses.Thirty basidiospores from each specimen were measured.The following abbreviations are used: IKI = Melzer's reagent, IKI+ = amyloid; CB = Cotton Blue, CB-= acyanophilous in Cotton Blue; L = arithmetic average of basidiospores length, W = arithmetic average of basidiospores width, Q = L/W ratios, (n = x/y) = the number of spores (x) measured from a given number of specimens (y).

DNA extraction and sequencing
A cetyl trimethylammonium bromide (CTAB) rapid plant genome extraction kit (Aidlab Biotechnologies, Co., Ltd., Beijing, China) was used to extract DNA (Wu et al. 2020(Wu et al. , 2022b)).The ITS1-5.8S-ITS2 region was amplified with the primer pair ITS5/ITS4 (White et al. 1990) using the following protocol: initial denaturation at 95 °C for 4 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for 45 s and 72 °C for 1 min and final extension at 72 °C for 10 min.The nLSU region was amplified with the primer pair LR0R/LR7 (White et al. 1990) using the following protocol: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1.5 min and final extension at 72 °C for 10 min.The PCR products were purified and sequenced by the Beijing Genomics Institute (BGI), China with the same primers.The newly-generated sequences in this study have been deposited in GenBank and are listed in Table 1.
Maximum Likelihood (ML) analyses and Bayesian Inference (BI) were carried out by using RAxML v.8.2.10 (Stamatakis 2014) and MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003), respectively.In ML analysis, statistical support values were obtained by using rapid bootstrapping with 1000 replicates, with default settings for other parameters.For BI, the best-fit partitioning scheme and substitution model were determined by using ModelFinder (Kalyaanamoorthy et al. 2017) via the "greedy" algorithm, branch lengths estimated as "linked" and AICc.Four Markov chain Monte Carlo chains (one cold) were constructed for 5,000,000 generations, with sampling every 1000 generations.Convergence was assessed as the standard deviation of split frequencies < 0.01.The first quarter of the trees, which represented the burn-in phase of the analyses, were discarded and the remaining trees were used to calculate posterior probabilities (BPP) in the majority rule consensus tree.
Phylogenetic trees were visualised by using FigTree version 1.4.4 (Rambaut 2018).Branches that received bootstrap supports for ML (≥ 75%) and BPP (≥ 0.95) were considered as significantly supported.The best topologies from ML analyses are shown in this study and the final alignments and the retrieved topologies were deposited in TreeBASE (http://treebase.org/treebase-web/home.html),under accession ID: 30973.

Phylogenetic analyses
In this study, the combined ITS1-5.8S-ITS2-nLSUdataset included sequences from 113 specimens, representing 34 species of Favolaschia and one species of Mycena (Pers.)Roussel as the outgroup (Table 1, Fig. 2).ModelFinder suggested models were HKY+F+G4 for ITS1+ITS2, K2P+G4 for 5.8s and K2P+G4 for nLSU, for the Bayesian analysis.The BI analysis resulted in a concordant topology with an average standard deviation of split frequencies of 0.008584.The ML and BI analyses resulted in nearly identical topologies and only the ML  tree is presented with the bootstrap supports for ML and BPP not less than 50% and 0.90, respectively.The phylogeny is similar to those of Johnston et al. (2006), Magnago et al. (2013) and Zhang et al. (2023).All Favolaschia samples are clustered within two groups (A and B), which correspond to the sections proposed by Singer (1974): Favolaschia sect.Favolaschia (group A) and F. sect.Anechinus (group B), except F. manipularis.In this study, three new lineages with high support (100/1.00,100/1.00 and 71/1.00,respectively) nest in F. sect.Anechinus (group B).Amongst them, five specimens from Guangdong and Guangxi formed two lineages and clustered into a clade with strong support (100/1.00),namely F. imbricata and F. miscanthi.Eight specimens from Sichuan formed a support lineage, namely F. sinarundinariae, sister to Favolaschia tephroleuca.Furthermore, similarity searches with the ITS barcoding from the three new species were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) from NCBI and showed less than 95% identity, respectively, in the genus Favolaschia, which demonstrated the obvious nucleotide differences of the three species with other species in the genus.Macrostructures.Basidiomata annual, gregarious, gelatinous when fresh and dry.Pilei 2-5 × 1.5-3 mm, conchoid, semicircular to subcircular; pileal surface pale grey to greyish-white (B1) when fresh, dark grey or mouse-grey (1D1-1F1) upon drying, convex with a reticulate pattern matching the pores below, faintly pruinose when dry; margin incurved, entire; context thin.Hymenophore paler than pileal surface, greyish-white (B1) when fresh and ash-grey (1B1-1C1) when dry, poroid, about 22-55 pores per basidiomata; mature pores 0.2-0.6 mm diam., polygonal, larger near the base and smaller near the edge, the marginal pores often incomplete; tubes up to 0.3 mm long.Stipe absent.
Distribution and ecology.Favolaschia imbricata is distributed in subtropical area of Guangdong Province, China; it grows on dead bamboo and causes a white rot.Etymology."miscanthi" (Lat.):refers to the species inhabiting rotten Miscanthus.

Discussion
The earliest records of Favolaschia species in China date back to the 20 th century (Liu 1994;Liu and Yang 1994).Recently, with the application of molecular systematics, the species of Favolaschia in China have been continually updated (Liu 2020;Tolgor et al. 2021;Zhang and Dai 2021;Zhang et al. 2023).Morphological examination and phylogenetic analyses identified 16 species of Favolaschia in China (Zhang and Dai 2021;Zhang et al. 2023).In this study, three new species of Favolaschia are identified in south China: Favolaschia imbricata, F. miscanthi and F. sinarundinariae.
Phylogenetic studies of Favolaschia largely support the section classification, based on morphology proposed by Singer (1974).Morphologically, the F. sect.Anechinus includes two subsections, F. subsect.Rubrinae and F. subsect.Depauperatae, the former lacks gloeocystidia and gloeoporous hyphae (with granular contents); the latter has these structures, while the two subsections are not well distinguished in the phylogenetic tree.The three new species, Favolaschia imbricata, F. miscanthi and F. sinarundinariae, clustered within section Anechinus (group B), which is characterised by the lack of acanthocysts, gloeocystidia and gloeoporous hyphae replaced by irregular cystidia at the dissepiment edge or pileipellis.
Eight specimens from Heizhugou and Micangshan National Forest Park in Sichuan Province formed a support lineage (71/1.00)and there are two base pairs differences between them in the ITS regions and no differences in morphology.We described these as a new species, namely Favolaschia sinarundinariae.Phylogenetically, Favolaschia sinarundinariae and F. tephroleuca cluster together in a supported clade (57/1.00)within Favolaschia sect.Anechinus, with each forming highly-supported branches.However, F. tephroleuca is readily distinguished from F. sinarundinariae by its grey to dark grey pileal surface, when fresh and fewer pores (up to 40 vs. up to 80).Morphologically, Favolaschia flabelliformis is similar to F. sinarundinariae by sharing a similar pileal surface, the absence of stipe and almost the same size basidiospores (Zhang et al. 2023), but the former species has smaller basidiomata (2-3 mm vs. 4-7 mm), smaller pores (0.2-0.25 mm vs. 0.5-1 mm) and fewer pores (10-40 vs. up to 80, Zhang et al. (2023)).In addition, F. flabelliformis and F. sinarundinariae are phylogenetically distantly related.
During the investigations of Favolaschia, the information on distribution areas is obtained (Fig. 1).Regarding geographical distribution, Favolaschia is distributed in south China (the south Qinling-Huai River Line).In addition, an updated key to 19 species of Favolaschia is provided in the following.

Figure 1 .
Figure 1.The geographical locations of Favolaschia species distributed in China (the base map of China was obtained from the Map Technology Review Center, Department of Natural Resources, http://bzdt.ch.mnr.gov.cn/).

Table 1 .
Names, specimen numbers, locality and corresponding GenBank accession numbers of the taxa used in this study.