Two new species of Xanthagaricus and some notes on Heinemannomyces from Asia

Abstract Xanthagaricus flavosquamosus and X. necopinatus, two new species of Agaricaceae, are described and illustrated from Asia. Macroscopically, both species are closely related to each other, but there are obvious micromorphological and molecular differences between them. Morphological and phylogenetic data showed that the two new species are distinct from other known species of the genus Xanthagaricus. Xanthagaricus flavosquamosus from China is characterized by its small, yellow basidiomata, short clavate cheilocystidia, epithelial pileipellis, and verrucose basidiospores measuring 5–5.5 × 3–3.5 μm. Xanthagaricus necopinatus from Bangladesh is characterized by having small, yellow basidiomata, a fugacious annulus, clavate to narrowly clavate cheilocystidia, epithelial pileipellis, and rugulose-rough basidiospores measuring 4–5 × 2.7–3.2 μm. In addition to the new species, a Heinemannomyces collection from China is reported. Morphological data and molecular phylogenetic analyses fully support the Chinese collection being Heinemannomyces splendidissimus, a species of Agaricaceae, originally described from Southeast Asia. Detailed descriptions, color photos and illustrations of the three species are presented. A key to the genus Xanthagaricus occurring in Bangladesh and China is provided.


Introduction
Xanthagaricus (Heinem.) Little Flower, Hosag. & T.K. Abraham is mainly characterized by small basidiomata with squamulose pileus, epithelial pileipellis, and more or less yellow-colored basidiospores (Little Flower et al. 1997;Hosen et al. 2017). This genus was originally placed in Hymenagaricus Heinem. subgenus Xanthagaricus Heinem. by Heinemann and Little Flower (1984) with the type species X. flavidorufus (Berk. & Broome) Heinem. & Little Flower. Heinemann (1981) erected Hymenagaricus as an independent genus in the family Agaricaceae with the type species H. hymenopileus (Heinem.) Heinem., and is characterized by a squamulose pileus, a hymeniform pileipellis, and brown basidiospores (Heinemann and Little Flower 1984, Ge et al. 2008, Hosen et a. 2017. The epithelial pileipellis and yellow basidiospores of the subgenus Xanthagaricus did not fit completely with the genus circumscription of Hymenagaricus. Subsequently, the subgenus Xanthagaricus was elevated to the genus level in Agaricaceae (Little Flower et al. 1997).
Species in the genus Xanthagaricus are saprotrophic, and mainly distributed in Asia and South Africa. For instance, 11 species from India (Heinemann andLittle Flower 1984, Little Flower et al. 1997), four species from Sri Lanka (Heinemann and Little Flower 1984, Pegler 1986, Little Flower et al. 1997, two species from Africa Eicker 1995, 1998), one species each from mainland China (Hosen et al. 2017), Pakistan (Hussain et al. 2017), and Taiwan, China (Ge et al. 2008) have so far been validly reported. However, Asia has more than the currently known species of Xanthagaricus, as sequences of several species of this genus are available in GenBank, especially from Thailand and Malaysia. Index Fungorum (http://www.indexfungorum.org/Names/ Names.asp) lists 12 taxa of Xanthagaricus. However, a recent study by Hussain et al. (2017) has transferred six species to Xanthagaricus from Hymenagaricus. Watling (1998) circumscribed Heinemannomyces as an independent monotypic genus in the family Agaricaceae from specimens collected in Malaysia and Thailand. Since then, no additional species with detailed descriptions and geographical extensions of the genus Heinemannomyces have been reported so far. Heinemannomyces is distinguished by its medium-sized basidiomata, extremely woolly-arachnoid veils on the pileus surface composed of cylindrical cells, leaden gray to dark blue lamellae, and reddening context when injured (Watling 1998). Phylogenetically, Heinemannomyces is closely related to Hymenagaricus, but can be differentiated by its morphology.
In this study, three collections of Xanthagaricus and Heinemannomyces from China, and one collection of Xanthagaricus with several basidiomata from tropical Bangladesh were examined. Based on macromorphology, both East Asian and South Asian Xanthagaricus collections could be the same species. However, careful microscopic observations along with molecular data revealed that they are not conspecific, but represent undescribed species within Xanthagaricus. In addition, a brief description from the Chinese collection of Heinemannomyces is provided along with molecular data. With the inclusion of the two new species of Xanthagaricus in this study and another two recently described new species, namely X. caeruleus Iqbal Hosen, T.H. Li & Z.P. Song (Hosen et al. 2017) and X. pakistanicus Hussain, Afshan & Ahmed (Hussain et al. 2017), the number of known species of this genus increases to 22.

Morphological studies
Specimens of Xanthagaricus and Heinemannomyces were collected from south China and Bangladesh (Xanthagaricus). The examined specimens were deposited in the Fungal Herbarium of the Guangdong Institute of Microbiology (GDGM), Guangzhou, China, and in the private herbarium (PHI) of the first author. Macromorphological descriptions were based on the field notes and photographs. Color codes and names follow Kornerup and Wanscher (1978).
Micromorphological observations were made from the dried specimens. Line drawings were freehand. Water, 5% aqueous KOH (w/v), and Congo Red were used as mounting media; Melzer's solution was used to check any amyloid reaction of basidiospores and tissues. In the descriptions of basidiospore measurements, the notation [n/m/p] was used, which means n basidiospores from m basidiomata of p collections. Dimensions for basidiospores are given as (a-)b-c(-d), in which 'b-c' contains a minimum of 90% of the measured values and extreme values 'a' and 'd' are given in parentheses, whenever necessary. Q denotes the length/width ratio of a measured basidiospore, Q m denotes the average of n basidiospores and SD is their standard deviation. Results are presented as Q m ± SD. Basidiospores were also observed using a scanning electron microscope (SEM) following the protocol of Hosen et al. (2013).

Molecular studies
Protocols for genomic DNA extraction, PCR amplification, and sequencing followed Hosen et al. (2013). ITS1-F/ITS4 (White et al. 1990) and LROR/LR5 (Vilgalys and Hester 1990) primer pairs were used for the amplification of the nuclear ribosomal internal transcribed spacer (ITS) region and partial sequence of nuclear ribosomal large subunit (28S) domains D1 and D2.
A total of 52 sequences (36 for ITS and 16 for 28S, Table 1) of Agaricaceae was retrieved from GenBank based on NCBI blast search results and recent publications (Ge et al. 2008, Vellinga et al. 2011, Ge and Yang 2017, Hosen et al. 2017, Hussain et al. 2017, and then combined with the newly generated ITS and 28S sequences of Clarkeinda trachodes (Berk.) Singer, Xanthagaricus and Heinemannomyces. Each individual dataset, either ITS or 28S, was aligned in MAFFT v.6.8 (Katoh et al. 2005)  Highlighted in bold are newly generated sequences in this study. * holotype, # isotype separately with default settings, and manually edited in BioEdit v.7.0.9 (Hall 1999). ITS and 28S datasets was then concatenated using Phyutility (Smith and Dunn 2008) for further phylogenetic analyses, and treated here as a ITS-28S dataset. The combined dataset (ITS-28S) was used for the recognition of the new species in Xanthagaricus and to find out their relationships with allied genera in Agaricaceae. Maximum Likelihood (ML) was performed using RAxML v.7.2.6 (Stamatakis 2006). As RAxML only supports the GTR model of nucleotide substitution, the GTRGAMMAI model was used for phylogenetic analyses, and statistical support values were obtained using nonparametric bootstrapping (BS) with 1000 replicates. Chlorophyllum rachodes (Vittad.) Vellinga was chosen as the outgroup.

Molecular phylogenetic results
A total of 10 nuclear ribosomal RNA gene sequences (five each for ITS and 28S) was generated from the newly collected materials of C. trachodes, Heinemannomyces and Xanthagaricus, and deposited in GenBank (Table 1). In the aligned ITS-28S dataset, sequences of the 43 samples were included with 1663 nucleotide sites (784 for ITS and 879 for 28S, gaps included) for each sample, of which 1186 were constant characters, 384 were parsimony informative characters, and 93 were parsimony uninformative characters. The resulting aligned dataset has been deposited in TreeBASE (http://purl. org/phylo/treebase/phylows/study/TB2:S21521). In the combined ITS-28S ML tree ( Fig. 1), the proposed two new species are distinct, the collection from Bangladesh is a close relative to X. pakistanicus with strong BS support value (97% ML BS), while the Chinese collection is not sister to any single species. Both clustered together with X.  Etymology. The species epithet "flavosquamosus" (Lat.) refers to the yellow squamules on the pileus surface.
Habit, habitat and distribution. Gregarious to scattered, ground covered with fallen needles or debris of Pinus sp., currently only known from Jiangxi Province of China.  Diagnosis. Morphologically similar to X. flavosquamosus but differs in the presence of a fugacious annulus, smaller and denser squamules, comparatively smaller basidiospores with rugulose-rough surface, clavate to narrowly clavate cheilocystidia.
Etymology. The species epithet "necopinatus" (Lat.) means unexpected, refers to the unexpected, surprising habitat of the collection, which was found on a concrete wall.
Habit, habitat and distribution. Scattered, clustered on a concrete wall, currently only known from Bangladesh. Description. Basidiomata medium-sized to large. Pileus 35-65 mm broad, at first hemispherical, then convex to applanate with age, sometimes depressed at disc, pileus surface covered by snuff brown, chestnut brown, purple-brown or grayish red (9B4, 9C4, 10CD4), woolly-floccose or woolly arachnoid velar remnants, usually darker at center, outer zone showing dull white to whitish background when the velar remnants vanish; margin incurved with slightly appendiculate velar remnants, often slightly lacerated; context 3-4 mm thick at pileus centre, elsewhere thin, changes from white to slightly reddening when cut or injured. Lamellae free, depressed around the stipe, broadly ventricose, bluish gray to leaden gray (19B3, 18C3) when young, becoming dark blue (19E4-7) to bluish gray (19D3-5) when mature; 3-4 tiers of lamellulae. Stipe 50-60 × 5-6 mm, central, cylindrical, slightly tapering towards the base,  floccose-squamulose all over the stipe, often vanish from handling or rain, with lighter shade of the pileus color, fistulose; stipe context slightly reddening when cut or injured. Annulus delicate, fugacious. Odor and taste unknown.
Habit, habitat and distribution. Solitary, scattered on the ground; known from Malaysia, Thailand, and now China.

Discussion
Little Flower et al. (1997) defined Xanthagaricus to include taxa with "basidiomata small, pileus with characteristic woolly squamules and appendiculate margin; lamellae free, brown when mature; stipe cylindrical, slender, fistulose, slightly broader at the apex, veil absent; context thin, sometimes becoming vinaceous red on bruising; spore print brown, spores subglobose to ellipsoid, smooth, thick-walled, brown with yellowish tinge; lamellar edge heteromorphous; cheilocystidia present; hymenophoral trama regular to subregular; pileal surface a disrupted epicutis of radial hyphae with plenty of spherical or subspherical cells at the scales; clamp connections absent". In spite of that, some species of Xanthagaricus do not warrant the generic circumscription on account of lamellae color (ink-blue in X. caeruleus), presence of an annulus (tiny and fugacious annulus in X. necopinatus), basidiospore color (greenish gray to grayish brown in X. caeruleus) with either smooth (X. caeruleus) or ornamented basidiospores (X. flavosquamosus, X. necopinatus, X. epipastus, X. subepipastus, etc.), but share the common features like small basidiomata, squamulose pileus, epithelial pileipellis with globose to subglobose terminal elements, the presence of cheilocystidia, the absence of pleurocystidia, more or less yellow to brownish yellow basidiospores, and the absence of clamp connections. It should be noted that most species of the genus Xanthagaricus have ornamented basidiospores (Figs. 15c-h, 16 in Little Flower et al. 1997) but it was not mentioned in the genus circumscription by the authors (1997).
Xanthagaricus taiwanensis (=Hymenagaricus taiwanensis Zhu L. Yang, Z.W. Ge & C.M. Chen), originally described from Taiwan, China is distinguished from X. flavosquamosus by having a yellow-brown pileus covered with fuscous brown-black squamules, a white membranous annulus, and comparatively wider basidiospores 5-5.5 × 3-4 μm (Ge et al. 2008). Xanthagaricus caeruleus, a recently described species from China, can also be distinguished from X. flavosquamosus by its grayish lilac to grayish violet squamules on pileus, ink-blue lamellae, and comparatively larger and smooth basidiospores 5-6 × 3-3.5 μm (Hosen et al. 2017). On the other hand, X. necopinatus is distinguished from all closely related species of this genus (see above), and the first contribution to the genus Xanthagaricus for Bangladesh.
It is interesting to note that Xanthagaricus appears to be a monophyletic genus and close sister to Pseudolepiota Z.W. Ge, a monotypic genus, recently described from China, with strong BS support value (85% ML BS). The latter genus is distinguished in having white color of the lamellae, hyaline basidiospores, and a subcutis pileipellis made up of slightly interwoven cylindrical hyphae (Ge and Yang 2017). However, the synapomorphic features of the two genera are the squamulose pileus, the absence of pleurocystidia, and the absence of clamp connections. Though a recent molecular study by Hosen et al. (2017) recovered Xanthagaricus as a close sister genus to Hymenagaricus, lacked significant BS support value while using ITS data. With the inclusion of eight species of Xanthagaricus including two new species based on ITS-28S phylogeny, the monophyly of the genus is resolved with the close evolutionary relationship to Pseudolepiota, and distinct from Hymenagaricus (Fig. 1).
The collection of Heinemannomyces made from south China matches well with the salient features (woolly-arachnoid veil on pileus, leaden gray lamellae, and a reddening context) of H. splendidissimus reported in the protologue by Watling (1998). However, the Chinese material slightly deviates from the original description in having comparatively larger basidiomata (up to 65 mm broad), and the absence of clamp connections. The authors were unable to include the type material of Heinemannomyces in the present study, but several sequences of H. splendidissimus from the type locality and its adjacent areas (Thailand) are available in the public accessible database (GenBank) to compare with the Chinese material. Sequences of Heinemannomyces from Thailand fall in the same clade with those from China, and are closely related to Hymenagaricus (Fig. 1). However, morphologically, Heinemannomyces differs from Hymenagaricus by having woolly-arachnoid veil remnants on the pileus surface, becoming brown, a reddening context when cut or injured, leaden gray lamellae, and a pileipellis composed of cylindrical hyphae (Watling 1998). It should be noted that three species of Hymenagaricus (GenBank voucher numbers. C.M. Chen 3636, T. Laessoe 6025 and ecv3807) used in this study (Xanthagaricus clade, Fig. 1) were also grouped together with strong BS support value in the molecular study of Vellinga et al. (2011) based on either ITS or multigene phylogeny, and Heinemannomyces was separated from them, and formed an independent lineage (Figs. 2 and 3 in Vellinga et al. 2011). However, no additional species of Hymenagaricus from the Hymenagaricus-Heinemannomyces clade (Fig.1) was included in the study of Vellinga et al. (2011). In the present analysis, more species of Hymenagaricus including some other close relative taxa of Agaricaceae were included, and Heinemannomyces showed a close affinity to Hymenagaricus (Fig. 1). One possible classification would be to collapse Heinemannomyces into a single genus Hymenagaricus or separate it into a subgenus/section. However, there are some remarkable morphological characteristics for Heinemannomyces and Hymenagaricus, which supports separating them into different genera. Further exploration of Hymenagaricus/Heinemannomyces species diversity and historical biogeography from Asia/South Africa, which seems to be species rich in these genera, could help to confirm or refute the hypothesis of monophyly/paraphyly of Hymenagaricus.
Key to the taxa of Xanthagaricus known from Bangladesh and China