A second record of Entoloma azureoviride (Agaricales, Basidiomycota) from Brazilian Amazon

(A second record of Entoloma azureoviride (Agaricales, Basidiomycota) from Brazilian Amazon). A new material collected and the holotype of Entoloma azureoviride were studied. The analysis of these specimens showed some discrepancies with the protologue which are discussed here. The diagnostic characters of this species are the following: fibrillose pileus, blue lamellae, cuboid spores, and abundant oleiferous hyphae in the lamellae trama. It was also observed a peculiar pseudoparenchymatous pileus trama consisting of inflated elements up to 25 μm in diam., that become more elongated towards the hymenium. A comparison of E. azureoviride with other species with blue tints in the subgenus Inocephalus and a discussion about its morphological peculiarity are provided.


Introduction
Entoloma (Fr.) P. Kumm. is a species-rich genus in the order Agaricales and is easily recognized by its pink spore print and angular basidiospores (Noordeloos 1981, Singer 1986).More than 1500 species are currently known (e.g.Co-David et al. 2009, Morgado et al. 2013), of which at least 271 species are from Central and South America (Coimbra 2014).
The species treated here belongs to Entoloma subg.Inocephalus Noordel.There are different opinions about the taxonomic status of Inocephalus.Some authors (e.g.Baroni & Halling 2000, Largent et al. 2008, 2013, Karstedt & Capelari 2013) prefer to accommodate all taxa with mycenoid stature, conical pileus with radially fibrillose surface, nearly free lamellae, intracellular pigments and cuboid spores, in the segregated genus Inocephalus (Nopordel.)P.D. Orton.On the other hand, Pegler (1983) used the name Inopilus (Romagn.)Pegler, in accordance with the original concept of Romagnesi (see Noordeloos 1979) to accommodate the taxa with these characteristic and (sub)isodiametric basidiospores and inflated cystidia, with I. versatilis (Gillet) Pegler as the type species.However, since Romagnesi equivocally changed the type of the subgenus Inopilus from Rhodophyllus versatilis [type of the subgen.Pouzarella (Mazzer) Noordel.]into E. inocephalum without following the rules of the International Botanical Code (Noordeloos 1981), the name Inopilus is illegitimate and cannot be used for this group of species (Noordeloos 1979).So, E. inocephalum is the type species of the genus Inocephalus.Recent molecular studies (Co-David et al. 2009, Morgado et al. 2013), however, showed that Inocephalus forms an integral part of a monophyletic genus Entoloma, and is a morphologically highly specialized group, characterized by very abundant tramal granules and almost exclusively cuboid spores (Noordeloos 1981).
Pseudoparenchymatous pileitrama among Entoloma sensu lato is not well studied.Noordeloos (1981) already cited that the pileus context of this genus is regular, in general made up of the same type of elements as found in the lamella trama.However, in some thick-fleshed specimens the centre of pileus can be more irregular or even pseudoparenchymatous.
We examine a new collection of Entoloma azureoviride, a blue tinted species with mycenoid/ inocybvoid habit bearing cuboid basidiospores, and studied the holotype for comparison and provide an account of an interesting anatomical characteristic of Entoloma subg.Inocephalus.

Material and methods
The basidiome was collected from Reserva Ducke, located in the outskirts of Manaus, Brazil (02°55'S, 59°59'W).The reserve protects 10,000 ha (10 × 10 km) of "terra-firme" forests but is being isolated by the expansion of Manaus city.A detailed description of the collection site was given in Braga-Neto et al. (2008).
We followed the traditional methodology for the study of agarics (Singer 1986) and the colors of the basidiome were named using the British Fungus Flora Color Chart (Henderson et al.1969).Basidiospores measurements follow the style proposed by Baroni & Lodge (1998), on which the cuboid basidiospores are measured using the flat sides of the 4-sided spores, from the apex to the base and from the adaxial to the abaxial facet while the spore was in lateral (profile) view.

Clamp connections abundant.
Habitat: solitary on soil of mature tropical 'terrafirme' moist forest, along plateaus with clayish soils and sparse leaf litter layer.Distribution: known from the type locality in Amazon and Atlantic Forest of southeast Brazil (Karstedt & Capelari 2013).
Remarks: Entoloma azureoviride belongs to the subgenus Inocephalus due to the distinctly fibrillose pileus and the cuboid basidiospores (Noordeloos 1981(Noordeloos , 1987)).It is easily recognized in the field by its ochraceous brownish pileus and blue lamellae (although reported bluish then ochre-brown by Horak 1982 andKarsted &Capelari 2013).Microscopically, the cuboid basidiospores, presence of pseudocystidia and the relatively pseudoparenchymatous pileitrama are remarkable characters.
Although well delimited, the protologue of E. azureoviride was described with blue fading to green or ochre-green pileus and stipe, concolorous lamellae, a smooth and hygrophanous pileus and smaller basidiospores 5-8.5 µm (Horak 1982).At first, we thought that our specimen was a new species, but Karstedt & Capelari (2013) already reviewed the holotype and no microscopic differences among these materials as well the material analyzed by us.
Our examination of the holotype of E. azureoviride revealed discrepancies in comparison to the protologue and suggested a closer relationship with our specimen: (1) somewhat larger basidiospores than reported in the protologue (6.5-)7-9.5(-10)µm (average 8 µm), with four but only very occasionally with five angles; (2) a pseudoparenchymatous pileitrama; and (3) a transitional pileipellis between a cutis and trichoderm (trichocutis).We also have observed hyphoid pseudocystidia, which frequently arise from the lamellar trama, but only infrequently higher than the rest of the structures of the hymenium.Although not mentioned in the protologue, Horak (1982, legend of fig. 3L) referred to these structures as 'pleurocystidia'.Recent studies by Karstedt & Capelari (2013) who provided an excellent description, also reported similar features in their type study, although they described the pileus trama of differtent way: "composed of radially arranged and parallel hyphae, with hyphae 5-26 mm diam., cylindrical or slightly inflated…" (Karstedt & Capelari (2013).
Before comparing this Amazonian fungus with other species, we need to comment on its remarkable pileitrama.The pileitrama of E. azureoviride consists of mostly inflated elements that give an appearance of a pseudoparenchymatous tissue in the middle of the pileitrama, that gradually become more elongate in the direction to top of lamellae (figure 3).Although this specialized trama is more frequent in thick-fleshed species (Noordeloos 1981: 130), it is an interesting new anatomical finding in this thin-flesh species.In the case of the holotype and our specimen, these rounded cells occur in almost all pileitrama.
Compiling our observations and those of Karstedt and Capelari (2013), E. azureoviride can be characterized by a brownish to ochraceous brown pileus fading to blue with greenish tints at maturity,   Several other taxa with blue lamellae, mycenoid stature, cuboid spores and brownish pigmented cystidia are known.They can be primarily segregated from the Amazonian taxon by the shape of the cystidia as well as the pileitrama features in some cases: The type of E. virescens (Berk.& M.A. Curtis) E. Horak ex Courtec.nom illegit.(non Schaeff.1774), originally known from Bonin Island off Taiwan, and was described as having a conic pileus with a conic papilla and cheilocystidia strangulated at the apex and 40-120 × 6-20 µm in size [somewhat rostrate in our interpretation of Horak's (1976, p. 201, fig. 19g) figure].It is also reported from Japan, Papua New Guinea, New Zealand, Malaya, Sri Lanka and Madagascar (Horak 1976(Horak , 1980)).In addition, it was described as having a blue color when young, fading to blue greenish or yellow-blue greenish, smooth to radially fibrillose and striate pileus, and basidiospores that are (7-) 8-10 µm in size (Horak 1976).Additionally, Noordeloos and Hausknecht (2007) described "E.virescens sensu lato" as being only slightly translucently striate with age when moist and vivid light blue to grayish blue unchanging with age.Later, Courtecuisse (1986) and Pegler [1997 as Inopilus virescens (Berk. and M.A. Curtis) Pegler] also reported somewhat larger spores: 10.5-12.5 × 10.5-12 µm, 8.8-11 µm and 10-12 (-14) × 9-11 µm respectively.
Studies by Noordeloos & Hauksnecht (2007) concluded that E. virescens sensu lato probably has a very wide geographical distribution, from the paleotropics to the Neotropic, including the temperate regions of Japan, New Zealand and Australia.However, Largent & Abell-Davis (2011) and Karstedt & Capelari (2013) claimed that only careful morphological and molecular studies will clarify whether the degree of morphological variation can be used for species delimitation.
Entoloma hochstetterii (Reichert) G. Stev.was recently reviewed by Horak (2008) and he reported cheilocystidia with strangulate-rostrate apex and larger basidiospores (11-15 × 11-14 µm).Unfortunately, no conclusions were taken for this species owing to the lack of type material and poorly preserved authentic specimens.Stevenson (1962), on the other hand, did not review the type.

Figure 3 .
Figure 3. Pileitrama of Entoloma azureoviride and the top of the lamellae near midratio presenting more elongate elements towards the lamellae trama.Scale bar 10 µm.