Recognition of Mycenasect.Amparoina sect. nov. (Mycenaceae, Agaricales), including four new species and revision of the limits of sect. Sacchariferae

Abstract Phylogenetic reconstruction revealed that Mycena stirps Amparoina, which is traditionally classified in sect. Sacchariferae, should be treated at section level. Section Amparoina is characterised by the presence or absence of cherocytes, the presence of acanthocysts and spinulose caulocystidia. Eight species referred to Mycenasect.Amparoinasect. nov. are recognised in China. Of these taxa, four new species classified in the new section are formally described: M.bicystidiatasp. nov., M.griseotinctasp. nov., M.hygrophoroidessp. nov. and M.miscanthisp. nov. The new species are characterised by the absence of both cherocytes and a basal disc, along with the presence of acanthocysts on the pileus, spinulose cheilocystidia and caulocystidia. Descriptions of the new species, accompanied by illustrations of morphological characters and comparisons with closely related taxa, are provided. A multi-locus analysis utilising the ITS + nLSU + SSU regions was carried out using maximum likelihood and Bayesian Inference. A key to the 12 species of sect. Amparoinasect. nov. and sect. Sacchariferae that are found in China is provided.

. Mycena sect. Sacchariferae Kühner ex Singer, which is one of the largest sections in the genus, was first published as a nomen nudum by Kühner (1938), who defined the section to include members that possess a granulose or "sugar coated" pileus. In 1958, Singer erected the monotypic genus Amparoina Singer to house Marasmius spinosissimus Singer based on the collections from Argentina (Singer 1958). Later, Singer (1976) established Amparoinaceae with A. spinosissima (Singer) Singer as type species and introduced another species in Amparoina, A. heteracantha Singer. Meanwhile he suggested that Amparoina was similar to sect. Sacchariferae, but maintained the autonomy of the former due to inamyloid basidiospores and revised sect. Sacchariferae to be characterised by a pileipellis with acanthocysts, which remain as terminal cells overlaid by a universal veil (Singer 1976). The pileus of cherocytes and acanthocysts distinguish taxa of sect. Sacchariferae from all other Mycena species. Section Sacchariferae was subdivided by Desjardin (1995) into stirps Amparoina Desjardin, stirps Alphitophora Desjardin and stirps Adscendens Desjardin, with 55 epithets classified into 27 taxa, based on presence or absence of a basal disc, cherocytes, and diverse caulocystidia. Maas Geesteranus and de Meijer (1997) Desjardin (1995), has been widely adopted. However, no phylogenetic reconstruction of relationships in sect. Sacchariferae has been published to assess the validity of the infrasectional classification.
A phylogenetic reconstruction of Mycena was incongruous with the traditional classification of stirps Amparoina within sect. Sacchariferae and indicated that the taxonomic classification of the section should be reconsidered. During our ongoing research on Mycena, four new taxa without a basal disc and cherocytes, belonging to the new section, were found in southern China in Chongqing City, Guangdong Province, Henan Province, Hubei Province, Tibet Autonomous Region, Yunnan Province and Zhejiang Province. These species are described here. Based on the phylogenetic analyses, an identification key to the 12 species of sect. Sacchariferae and sect. Amparoina currently known from China is provided.

Morphological study
Macroscopic characters were described from fresh specimens following conventional taxonomic methods. Colour terms and notations refer to those of Kornerup and Wanscher (1978). Microscopic characters were observed from dried specimens rehydrated in 5% potassium hydroxide (KOH) and stained with Congo red, using a Nikon 80i light microscope. Melzer's reagent was used for testing amyloid and dextrinoid reactions of all tissues (Horak 2005). The spore shape quotient (spore length divided by spore width; Q = L/B) was calculated from 40 mature basidiospores; 90% of the numerical range is indicated outside parentheses and the 10% extreme values are enclosed in parentheses. Author abbreviations are based on those used in Index Fungorum (https://www.indexfungorum.org). Voucher specimens have been deposited in the Herbarium Mycology of Jilin Agricultural University (HMJAU).

DNA extraction, PCR amplification and DNA sequencing
Material for DNA isolation was taken from dried specimens. Genomic DNA was extracted from samples using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Company Limited, Beijing, China). The internal transcribed spacer (ITS) region was amplified with the primer pair ITS1 and ITS4 (White et al. 1990). The nLSU and SSU regions were amplified using the primers LROR/LR7 and MS1/ MS2, respectively (Ward et al. 1992;Hopple and Vilgalys 1999). The PCR cycling schedule for the ITS, nLSU and SSU region used a touchdown programme (Na and Bau 2018). All newly generated sequences were deposited in GenBank (Table 1).

Sequence alignment and phylogenetic analysis
A dataset, comprising sequences for the ITS + nLSU + SSU region from 96 accessions with taxonomic coverage of Europe, North America, Australia, Africa and Asia, was compiled and analysed. Sequences for 32 accessions were downloaded from GenBank and 64 newly generated sequences obtained in this study were aligned and adjusted manually using BioEdit 7.0.4.1 and Clustal X (Thompson et al. 1997; analysis was performed in raxmlGUI 1.5b1, with a rapid bootstrapping algorithm involving 1,000 replicates (Stamatakis et al. 2004). Topology support values greater than 75% bootstrap support (ML) 0.95 and Bayesian posterior probabilities (BPP) are shown at each branch node.
furfur-like scattered, margin entire first, then nearly plane and finally fissile. Context very thin and fragile, pure white. Lamellae 0.5 mm thick, narrowly adnate, off-white, concolorous with the sides. Stipe slender, 15-28 × 0.5-1.0 mm, cylindrical, hollow, fragile, pure white, densely pruinose on the whole surface, base swollen and not forming a basal disc, hirsute. Odour and taste inconspicuous.
Habit and habitat. Scattered to gregarious on litter layer in Quercus, Picea, Abies, Pinus mixed forests.
Remarks. Mycena griseotincta is considered a new species in sect. Amparoina stirps Alphitophora on account of the absence of both a basal disc and cherocytes on the pileal surface (Desjardin 1995). Five species have ellipsoid basidiospores, caulocystidia covered with excrescences and a universal veil composed of acanthocysts: M. alphitophora, M. brunneospinosa, M. depilata, M. hemitrichialis and M. incarnativelum. Mycena alphitophora most resembles M. griseotincta, but the former differs in having pure white lamellae, a white and shorter stipe (< 50 mm), sphaero-pedunculate or obovoid cheilocystidia and larger spores (8.1-9.7 × 4.5-5.5 μm), as reported in the original de- scription (Maas Geesteranus 1980, 1992b. Mycena brunneospinosa, a taxon named by Desjardin (1995), is readily identified by its dull brown or purplish-brown pileus, globose acanthocysts forming chains and broadly ellipsoid spores. Mycena incarnativelum is a unique species in sect. Sacchariferae, distinguished by the absence of cheilocystidia and deep pink basidiomata when young (Desjardin 1995). Mycena depilata is closely allied to M. griseotincta, but differs in the convex pileus less than 1 mm in diameter and short and broadly clavate caulocystidia (Singer 1989). Mycena hemitrichialis can be mistaken for M. griseotincta on account of its grey or pallid pileus and ellipsoid spores, but is distinguished from M. griseotincta by its white stipe, free lamellae and pilose stipe forming a flattened ring of mycelium (Desjardin 1995). Mycena corynephora is widely distributed worldwide and is recognised by its tiny basidiomata (pileus < 2.4 mm), absence of a basal bulb or basal disc and large globose to subglobose basidiospores, typical of stirps Alphitophora (Desjardin 1995;Aronsen and Laessøe 2016). The same spore shape occurs in M. yalensis of which the holotype was collected from Argentina (Singer 1973). Aravindakshan and Manimohan (2015) reported one new species and two others newly combined in Mycena, collected from India. The new taxon, M. roseotincta, differs from M. griseotincta in its pink pileus and universal veil, subcylindrical spores and smaller caulocystidia (Aravindakshan and Manimohan 2015). Mycena globispora and M. distincta are mainly distinguished in macromorphology from M. griseotincta by their white basidiomata and, in micromorphology, by the globose spores and subcylindrical spores, respectively (Aravindakshan and Manimohan 2015).
Remarks. The distinctive features of Mycena miscanthi include a white, granulose pileus, a pubescent stipe without forming a basal disc, narrow-ellipsoid spores, two types of acanthocysts and growth on dead stems of Miscanthus species. In combination, these features support the placement of M. miscanthi in sect. Amparoina stirps Alphitophora. Similar to M. miscanthi, M. alphitophora and M. depilata produce pure white basidiomata, cylindric spores and sphaero-pedunculate and spinulose cheilocystidia (Desjardin 1995;Aravindakshan and Manimohan 2015). However, the two types of acanthocysts and longer caulocystidia can be used to distinguish M. alphitophora and M. depilata from M. miscanthi (Desjardin 1995). Mycena hemitrichialis is closely allied to M. miscanthi, but differs in producing caulocystidia up to 400 μm in length that lack spinulae or with a few spinulae in the upper half (Singer 1989). Mycena distincta, which was originally described as M. alphitophora var. distincta, was elevated to species level by Manimohan and Leelavathy (1989). It differs from M. miscanthi in producing broadly ellipsoid spores and caulocystidia up to 300 μm in length (Aravindakshan and Manimohan 2015). The pigmented pileus present in M. brunneospinosa, M. incarnativelum and M. roseotincta readily distinguishes these species from M. miscanthi (Desjardin 1995;Aravindakshan and Manimohan 2015).

Discussion
The present phylogenetic analysis showed that sect. Amparoina formed a distinct clade independent from sect. Sacchariferae with high BPP and BS support. This finding suggests that the presence of caulocystidia with dense spines is the most important character to separate sect. Amparoina from sect. Sacchariferae. However, in the presence of a basal disc, the species of sect. Sacchariferae are similar to stirps Amparoina and, in the acanthocysts on the pileus sect. Amparoina stirps, Amparoina resembles sect. Sacchariferae. It can be concluded that the difference in caulocystidia can be used to distinguish sect. Amparoina and sect. Sacchariferae and the basal disc and cherocytes are the basis of an infrasectional classification of sect. Amparoina. Thus, the circumscription of sect. Sacchariferae should be revised, for which the diagnostic characters are a well-developed basal disc, cherocytes absent, pileipellis a cutis not overlaid by elements of a universal veil composed of acanthocysts and caulocystidia smooth overall.
In morphology, sect. Amparoina and sect. Sacchariferae are closely allied with sect. Polyadelphiae Singer ex Maas Geest. and sect. Basipedes (Fr.) Quél (Desjardin et al. 2003). Species of sect. Polyadelphiae lack both ornamented pileipellis elements and a stipe with a basal disc and thus differ from species classified in sect. Amparoina and sect. Sacchariferae. Section Basipedes shares the same habitat and a stipe forming a developed basal disc, but the cheilocystidia are covered with rounded and few excrescences. Morphological characters distinguish sect. Polyadelphiae and sect. Basipedes from sect. Amparoina and sect. Sacchariferae and only one ITS sequence for M. stylobates It is worth mentioning that the placement of M. echinocephala (G.F. Atk.) Desjardin and M. cylindrospora A.H. Sm. remains unclear. The species are tentatively placed in stirps Alphitophora because of the lack of a basal disc on the stipe, but their caulocystidia are extraordinary in being smooth, terminated by a spinulose apex or smooth with an amorphous apex (Atkinson 1902;Smith 1947;Desjardin 1993). Both species show obvious differences to the four newly described taxa. Furthermore, M. cryptomeriicola Imazeki & Toki is distinctive in producing inamyloid spores and a basal disc, which is unusual for specimens of sect. Sacchariferae from Japan (Imazeki and Toki 1995). An additional unusual species, M. minya Grgur., which lacks caulocystidia, was reported from Australia (Grgurinovic 2003). No species similar in morphology to M. cryptomeriicola and M. minya are classified in sect. Sacchariferae, so the two species are tentatively accepted in sect. Sacchariferae.