Small agarics in Taiwan: Mycena albopilosa sp. nov. and Gloiocephala epiphylla

Background Small agarics are poorly documented in Taiwan, with previously reported species either rudimentarily described or lacking a description or diagnosis in most cases. A survey on small agarics in a lowland forest of Taiwan revealed two species previously unrecorded. Results One agaric, which is characterized mainly by white hairs overlying the pileus, a conspicuous cup-shaped basal disc surrounding the stipe, and inamyloid basidiospores, fits the genus Mycena and appears undescribed. It readily produced abundant basidiomata in culture in three weeks. The other agaric is Gloiocephala epiphylla, being characterized by its reduced hymenium and conspicuous pileogloeocystidia. ITS sequences from the two agarics further corroborated the identifications. Their macroscopic and microscopic features and culture morphology are described. A dichotomous key is provided to the species of Mycena reported in Taiwan. Conclusion The Mycena species is newly described as M. albopilosa herein. Gloiocephala epiphylla is new to Taiwan, being the only species of the genus known on the island.


Background
Small agarics with a pileus less than 1 cm diam pose a challenge for mycobiotic surveys, because they are easily overlooked and, after collected, their delicate, fragile basidiomata need to be measured and recorded in a timely fashion. Most of these small agarics belong to Mycena (Pers.) Roussel, Marasmius Fr., and Galerina Earle, each of which contains hundreds of taxa. Some of the small agarics can be found in genera such as Gloiocephala Massee and Omphalina Quél. They are saprophytes or in close association with mosses (Davey et al. 2013), while a few of them have been reported as being mycorrhizal (Zhang et al. 2012) or parasitic (Dennis 1957;Baker and Holliday 1957;Sequeira 1958;Bayliss 1911). These overlooked small agarics are actually underestimated. Notable plant pathogenic species include Mycena citricolor (Berk. & M.A. Curtis) Sacc., which causes the well-known American leaf spot that decreased annual yields of coffee crops by 20% (Sequeira 1958;Rao and Tewari 1987), and Marasmius perniciosus Stahel, which causes witches' broom disease on cacao in South America (Baker and Holliday 1957;Evans 1980). Certain Marasmius species contain laccases and other enzymes capable of degrading aromatic compounds, lignin, and β-carotene (Dedeyan et al. 2000;Scheibner et al. 2008). Noticeably, more than 30 species of Mycena are known bioluminescent (Desjardin et al. 2007).
There are over 500 Mycena species described in the world (Desjardin et al. 2008). In Taiwan, 21 Mycena species have been reported, but, in most cases, a species is merely furnished with a rudimentary diagnosis or lacks a description/diagnosis entirely. Shih et al. (2014) described a bioluminescent species M. kentingensis Shih et al., which represents the only novel species of the genus documented in Taiwan thus far. Despite the great potential and special features that these small agarics may possess, without extraordinary characters such as bioluminescence and pathogenicity, their taxonomy hardly becomes a research interest for mycologists in Taiwan.
In the present study, Mycena albopilosa, which is characterized by a white, minute, hairy pileus and a conspicuous cup-shaped basal disc at the stipe, is described as new. It has characteristics of Mycena section Sacchariferae Kühner ex Sing. in general, but its basidiospores are inamyloid. In addition, Gloiocephala epiphylla, the type species of the genus, is reported and represents a newly recorded genus in Taiwan. Both M. albopilosa and G. epiphylla are tiny, growing on substrates, such as fallen leaves or twigs, in humid forests.

Specimen collecting and culturing
Basidiomata together with the attached substrate were brought back to the laboratory. The substrates were placed in the plastic chambers with high humidity maintained for further basidiomata production. Freshly produced basidiomata were placed onto 2% malt extract agar (MEA) for collecting basidiospores. Germinating basidiospores were transferred to fresh MEA. Cultures were subsequently transferred to potato dextrose agar (PDA), MEA, and oatmeal agar (OA). Growth rate and culture morphology were recorded every 3-5 days.
Both species were also cultured on a formulated soil, which was modified from the one used for Mycena kentingensis (Shih et al. 2014), with white rice bran replaced by black rice bran. The formula was as follows: 80% potting soil (Green Orchids Co. #521) thoroughly mixed with 20% black rice bran. The mixture was divided into 10 g per 9-cm Petri dish and autoclaved at 121 °C for 30 min before use. Mycelia were grown in the laboratory at 25 °C for 4 weeks. To maintain the water content, the humidity was kept approximately 50-70%, and 1 ml sterile water was sprayed every 4-7 days on each Petri dish.

Morphological study
Macro-and micro-morphological data of Mycena albopilosa were mainly based on cultured fruiting bodies due to exhaustion of original specimens. The morphology of specimens artificially cultivated was similar to those growing in natural habitat. Color terms and notations used for morphological and culture features were compared with Methuen Hand Book of Colour (Konerup and Wanscher 1978). Microscopic observations of Gloiocephala epiphylla were rehydrated with distilled water and stained with Melzer's reagent. Hyphal tissue organization was observed from resin fixation and sectioning of basidiomata. Basidiospores were measured with a Leica DM2500 microscope under 100× immersion oil objective and 10× ocular. Basidiospore statistics were calculated by Piximetre 5.8, including the arithmetic mean and range of the spore length and spore width (30 spores in a specimen); Q: the range of spore length to width quotient.

Cultures and anamorph
Colonies on formulated soil covering Petri plates at 4 weeks, tomentose, white; basidiomata produced in 4 weeks.
Colonies on PDA 20 mm at 1 week, 32 mm at 2 weeks, 45 mm at 3 weeks, white to cream (4A3), irregular, wrinkled, wooly to tomentose, with powdered patches in places, sometimes radially sulcated and creased, with diffuse margins slightly fimbriate to tentacle-like and submerged; aerial hyphae inconspicuous; advancing zone 1.7-2.3 μm, sometimes 3-5 hyphae aggregated into bundles, aseptate; submerged hyphae branched, with globular enlargement; clamp connections abundant; odor indistinctive. Basidiomata produced in 18 days, much as those produced on formulated soil but differing in being extremely crowded, more variable in shape and generally smaller 1-1.5 mm diam; partially developed basidiomata sometimes produced.
Colonies on MEA 11 mm at 1 week, 20 mm at 2 weeks, 31 mm at 3 weeks, whitish, plane and waxy, with wavy margins; microscopic features as those on PDA.
Colonies on OA 20 mm at 1 week, 40 mm at 2 weeks, 55 mm at 3 weeks, irregular, partly floccose, with aerial hyphae brush-like, 0.5-1 mm long × 0.1-0.3 mm broad, white, with entire margins; basidiomata sometimes produced in 3 weeks, 3-6 mm diam, with macroscopic and  HOLOTYPE, HAST 142292), the holotype material composed of basidiomata produced on formulated soil, which are much like those collected from natural substrate, because the material collected from natural substrate was used up during the study.

Commentary
The salient features of M. albopilosa are the agglutinated pileocystidia resembling animal's fur, conspicuous cup-like basal disc, lack of cheilocystidia, and inamyloid basidiospores. Mycena albopilosa is capable of producing abundant basidiomata on PDA within a short period of time, which may be proven useful for developmental studies of basidiomata. Mycena albopilosa can be placed in section Sacchariferae, which is characterized by a granular to floccose pileus surface and ascending lamellae (Maas Geesteranus 1992), and in stirps Adscendens Desjardin (1995), which possesses smooth caulocystidia but lacks cherocytes.
Twelve  Imazeki and Toki (1955) was originally described as having inamyloid basidiospores in the section Sacchariferae. However, the holotype specimen is in poor condition, and recollected specimens were reported to have amyloid basidiospores (Tanaka and Hongo 2003). Mycena albopilosa differs from M. cryptomeriicola in mostly lacking ventricose-rostrate cheilocystidia.
Mycena alphitophora (Berk.) Sacc. and its varieties have floccose pilei and thus resemble M. albopilosa in gross morphology. However, Mycena albopilosa possesses a basal disc at all stages, whereas M. alphitophora and its varieties lack a basal disc but instead have a swollen stem base covered with wooly hyphae. In addition, M. albopilosa differs M. alphitophora and its varieties in lacking cheilocystidia and having smooth rather than spinulose caulocystidia (Table 1).
Mycena albopilosa produces basidiomata in culture easily. In contrast to the basidiomata produced in nature, those formed in culture tend to be more variable in shape, but a great portion of them are normal and fully developed. For those deviating basidiomata, agglutinated hyphal tufts on the pileus and primordia, basal discs, and microscopic characteristics remain the same, but the basidiomata are smaller in diameter (1-1.5 mm) and more fragile. Malformed characters of the basidiomata produced on PDA include partially developed to undeveloped pilei with a wide size range and twisted, smoother stipes. Although primordia are generally normal in microscopic features, they are three to four times smaller than those produced on formulated soil. It should be noticed that a translucent, circular hymenium was found forming directly on the shortened, globular stipe with blackened upper half on OA and PDA. The deformed stipe shows the same tissue construction as the normal stipe and is dextrinoid. The hymenium is inamyloid and composed of only basidia and basidioles. This phenomenon has never been found in other agarics.
An NCBI MEGABLAST query with the ITS sequence of M. albopilosa did not show high similarities with the top matches. The top 10 BLAST matches closest to M. albopilosa did not bear a species epithet. The next match with a known identity was KF007948, deposited by Baird, R. E. as M. pura (query coverage = 100%, identities = 589/691 [85%], gaps = 51/691 [7%]), which did not share a high identity with M. albopilosa. Mycena pura is a significantly different species from M. albopilosa in having a large pileus size of 20-50 mm diam with a pale pink color, having amyloid spores, and lacking pileocystidia and acanthocysts.

Cultures and anamorph
Colonies on soil (commercial soil and black rice bran of 4:1 ratio) full at 3 weeks, white, silk-like; basidiomata not produced.
Colonies on MEA 33.5 mm at 1 week, 52.5 mm at 2 weeks, full at 3 weeks, plane, cream (4A3) to pinkish white, forming inconspicuous concentric rings, with irregular, plumose margins; aerial hyphae inconspicuous; microscopic features as those on PDA.
Colonies on OA 35 mm at 1 week, 50 mm at 2 weeks, full at 3 weeks, hyaline to white, with entire margins, covered with conspicuous white, evenly distributed aerial hyphae; microscopic features as those on PDA.
Gloiocephala epiphylla is a newly recorded genus and species in Taiwan, being characterized by lamellae reduced or lacking, basidiospores inamyloid, and possession of secretory pileogloeocystidia (Antonín 2007). The secretory pileogloeocystidia on the basidiomata are so conspicuous that they appear spine-like by hand lenses. In Taiwan, the basidiomata were found growing on fallen leaves, twigs or decaying fruits in low-latitude broadleaf forests in spring. Desjardin et al. (1992) recorded that G. epiphylla were commonly found along the coastal areas or watercourses. Our collection in Taiwan was made from upper bank forest floor. Nevertheless, the collecting site is humid, and the forest floor is constantly flooded by temporary water flow from the rain fall.