Research Article
Print
Research Article
Two new species of Xanthagaricus and some notes on Heinemannomyces from Asia
expand article infoMd. Iqbal Hosen, Zong-ping Song§|, Genevieve Gates, Samantha C. Karunarathna#, M. Salahuddin M. Chowdhury¤, Tai-Hui Li
‡ Guangdong Institute of Microbiology, Guangzhou, China
§ University of Chinese Academy of Sciences, Beijing, China
| South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China
¶ University of Tasmania, Tasmania, Australia
# Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
¤ Sher-e-Bangla Agricultural University, Dhaka, Bangladesh
Open Access

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.

Keywords

Hymenagaricus, molecular phylogeny, monophyly, South Asia, taxonomy

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 and Little 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 (Reid and 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.

Materials and methods

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, Qm denotes the average of n basidiospores and SD is their standard deviation. Results are presented as Qm ± 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) 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.

List of fungal taxa of Agaricaceae and their GenBank accession numbers used in molecular phylogeny.

Name of the species Voucher/collection no. Country GenBank accession no.
ITS 28S
Agaricus aff. campestris Murphy 6242 USA HM488744
Agaricus bisporatus Contu1 AF432882
Agaricus bohusii LAPAG562 KR006613 KR006613
Agaricus deserticola S. Smith USA HM488747
Agaricus diminutivus Vellinga 2360 USA AF482831 AF482877
Agaricus megacystidiatus MFLU 12–0137 Thailand NR_119953
Agaricus rotalis ecv3768 USA HM488746 HM488792
Agaricus sp. BAB–5059 India KR155104
Agaricus sp. CA833 Thailand JF727858
Agaricus sp. C3182 Togo KJ540956
Agaricus sp. NTS113 Thailand JF514531
Chlorophyllum rachodes Vellinga 2106 Netherlands AF482849
Clarkeinda trachodes ecv3838 Thailand M488750 HM488771
Clarkeinda trachodes Iqbal 806 Bangladesh MG462712
Coniolepiota spongodes ecv3898 Thailand HM48875
Coniolepiota spongodes HKAS 77574 Bangladesh KC625531 KC625530
Eriocybe chionea ecv3560 Thailand HM488752 HM488773
Heinemannomyces splendidissimus ecv3586 Thailand HM488760 HM488769
Heinemannomyces splendidissimus zrl3043 Thailand JF691559
Heinemannomyces splendidissimus GDGM 46633 China MF621038 MF621039
Heinemannomyces splendidissimus GDGM 46633 China MF621040
Hymenagaricus ardosiicolor LAPAF9 Togo JF727840
Hymenagaricus ardosiicolor isolateZ4 Tanzania KM360160
Hymenagaricus cf. kivuensis BR6089 Burundi KM982454
Hymenagaricus sp. CA833 Thailand JF727858
Hymenagaricus sp. zrl3103 Thailand KM982450 KM982452
Hymenagaricus sp. CA801 Thailand JF727859
Hymenagaricus sp. LD2012186 Thailand KM982451 KM982453
Pseudolepiota zangmui Ge2106* China KY768927
Pseudolepiota zangmui MFLU100515 Thailand KX904355
Xanthagaricus caeruleus GDGM 50651* China MF039088 MF039086
Xanthagaricus caeruleus GDGM 50794 China MF039089 MF039087
Xanthagaricus epipastus zrl 3045 Thailand HM436649 HM436609
Xanthagaricus flavosquamosus GDGM 50913 China MF351627
Xanthagaricus flavosquamosus GDGM 50918* China MF351629 MF351631
Xanthagaricus flavosquamosus GDGM 50924 China MF351628
Xanthagaricus necopinatus Iqbal–821 (GDGM 46632*, PHI–12#) Bangladesh MF351626 MF351630
Xanthagaricus pakistanicus LAH SH 207 Pakistan KY621555
Xanthagaricus pakistanicus HUP SH 315 Pakistan KY621556
Xanthagaricus sp. TL6025 Malaysia AF482835 AF482879
Xanthagaricus sp. ecv3807 Thailand HM488761 HM488770
Xanthagaricus taiwanensis HKAS 42545 Taiwan, China DQ490633 DQ089016
Xanthagaricus taiwanensis C.M. Chen 3636* Taiwan, China DQ006271 DQ006270

Results

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. epipastus (Berk. & Broome) Hussain, X. taiwanensis (Zhu L. Yang, Z.W. Ge & C.M. Chen) Hussain, X. caeruleus, and two unnamed species of the same genus. The result of the phylogenetic analysis is presented in Fig. 1.

Figure 1. 

Phylogenetic relationships of Xanthagaricus species and its allied genera inferred from ITS-28S data using ML method. RAxML bootstrap support values (ML BS, ≥50%) are indicated on the branches at nodes. The two new species of Xanthagaricus from Bangladesh and China, and Heinemannomyces splendidissimus from China, are highlighted in bold on the tree. Herbarium or voucher specimen numbers and country names are provided after the species name. Chlorophyllum rachodes is rooted as the outgroup. Bar: indicates 0.1 expected change per site per branch.

Taxonomy

Xanthagaricus flavosquamosus T.H. Li, Iqbal Hosen & Z.P. Song, sp. nov.

MycoBank No: 822730
Figs 2a–b, 3, 5b

Diagnosis

Closely related to X. epipastus and X. subepipastus but differs in having larger basidiospores with verrucose surface, short but broadly clavate cheilocystidia, and found on the ground covered by fallen needles or debris of Pinus sp.

Typification

CHINA, Jiangxi Province, Jiulong Provincial Forest Park, 25 August 2015, Ming Zhang, Jun Ping Zhou & Hao Huang (GDGM 50918, holotype).

Etymology

The species epithet “flavosquamosus” (Lat.) refers to the yellow squamules on the pileus surface.

Description

Basidiomata small-sized. Pileus 8–13 mm broad, at first hemispherical to convex, then plano-convex to nearly applanate with age, yellow (2A4–7) to vivid yellow (2A8), lemon yellow or mustard yellow (3B8, 3B6), more or less yellow-brown to grayish brown at centre, concentrically fibrillose-squamulose, sometimes woolly to matted squamulose on the surface, more densely and darker at centre; margin incurved with appendiculate, often lacerated velar remnants, concolorous with the squamules; context 0.8 mm thick at the pileus center, elsewhere thin, no color change when cut or injured. Lamellae free, depressed around the stipe, broadly ventricose, yellowish white (3A2) to light pinkish white (10A2), with crenulate margin; 3–4 tiers of lamellulae. Stipe 20–30 × 1.5–2 mm, equal, central, cylindrical, slightly curved, fistulose, pale yellow (3A3) to slightly grayish yellow (3B3), some scattered squamules or scales on surface, with white mycelial tufts at base. Annulus absent. Odor and taste unknown.

Basidiospores [60/3/3] 5–5.5(–6) × 3–3.5 µm, [mean length = 5.38 µm, mean width = 3.25 µm, Q = (1.51–)1.61–1.68(–1.71), Qm = 1.65 ± 0.052], ellipsoid to broadly ellipsoid, slightly thick-walled (0.5 µm), smooth under light microscope but minutely verrucose or warty under SEM, pale yellow to yellowish brown in H2O and 5% KOH, inamyloid. Basidia 10–12 × 5–6 µm, clavate, pale yellow in H2O, hyaline, thin-walled, 4-spored, with sterigmata up to 3 µm long. Lamellar trama regular to subregular, composed of thin-walled cylindrical hyphae 4–8 µm wide. Cheilocystidia 7–15 × 6–9 µm, abundant, clavate to broadly clavate, sometimes slightly fusoid to obovate, smooth, hyaline, thin-walled. Pleurocystidia absent. Pileipellis (squamules on pileus) epithelial, composed of agglutinated globose to subglobose, rarely clavate to ellipsoidal thin-walled cells, terminal cells 6–12 × 6–10 µm, slightly encrusted, with some vacuolar pigments when observed in KOH or H2O. Caulocystidia not found. Stipe trama composed of parallel hyphae 3–8 µm wide, yellowish brown in mass but pale yellow or subhyaline individually. Clamp connections absent in all tissues.

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.

Additional specimens examined

CHINA, Jiangxi Province, Jiulong Provincial Forest Park, 26 Aug 2015, Ming Zhang, Jun Ping Zhou & Hao Huang (GDGM 50924); same location, 26 Aug 2015, Ming Zhang, Jun Ping Zhou & Hao Huang (GDGM 50613b).

Figure 2. 

Basidiomata of Xanthagaricus species. a, b Basidiomata of X. flavosquamosus (a, b GDGM 50918, holotype) c, d Basidiomata of X. necopinatus (GDGM 46632, holotype; PHI-12, isotype). Scale bars: 5 mm.

Xanthagaricus necopinatus Iqbal Hosen, T.H. Li, & G.M. Gates, sp. nov.

MycoBank No: 822731
Figs 2c, d, 4, 5b

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.

Typification

BANGLADESH, Dhaka Division, Sher-e-Bangla Nagar, Chondrima Uddan, 21 Aug 2014, Iqbal 821 (GDGM 46632, holotype; PHI-12, isotype).

Etymology

The species epithet “necopinatus” (Lat.) means unexpected, refers to the unexpected, surprising habitat of the collection, which was found on a concrete wall.

Figure 3. 

Microscopic features of Xanthagaricus flavosquamosus (GDGM 50918, holotype). a Basidia with basidioles b Basidiospores c Epithelial pileipellis with encrusted wall. d Cheilocystidia. Scale bars: 10 µm.

Description

Basidiomata small-sized. Pileus 10–15 mm broad, hemispherical, convex to plano-convex, yellow (2A4–7) to vivid yellow (2A8), maize yellow (4A6), light olive yellow (3D3–4) to pale brown (5D4) at disc, with yellow (3A4) to yellowish brown (5D8, 5E8) squamulose or finely fibrillose squamules on the surface, more concentrated and darker at center but scattered elsewhere; margin incurved with appendiculate velar remnants, concolorous with the pileus squamules; context 0.7 mm thick at pileus center, elsewhere thin, unchanged when cut or injured. Lamellae free, depressed around the stipe, yellowish white (3A2) to pinkish white (10A2), light brownish gray (6C3, 6D3), with crenulate edge, broadly ventricose; lamellulae commonly with 3–4 tiers. Stipe 18–28 × 1.5–2 mm, equal to slightly attenuated towards base, central, cylindrical, slightly curved, fistulose, yellowish brown (5D4) to dull brown (5C2), with some scattered squamules on surface; squamules more concentrated toward apex. Annulus very thin and tiny, superior, fugacious, often gone due to handling or with age. Odor and taste unknown.

Basidiospores [60/3/1] 4–5 × 2.7–3.2 µm, [mean length = 4.45 µm, mean width = 2.98 µm, Q = (1.31–)1.41–1.64(–1.72), Qm = 1.49 ± 0.064], ellipsoid to ovoid-ellipsoid, slightly thick-walled (0.5 µm), inamyloid, smooth under light microscope but rugulose-rough surface under SEM, yellow to yellowish brown in H2O and 5% KOH. Basidia 13–17 × 5–6 µm, clavate to narrowly clavate, pale yellow in H2O, thin-walled, 4-spored, with sterigmata up to 2 µm long. Lamellar trama regular to subregular, composed of thin-walled cylindrical hyphae, 4–8 µm wide. Cheilocystidia 15–20 × 4–6 µm, abundant, clavate to narrowly clavate, sometimes narrowly fusoid, smooth, hyaline, thin-walled. Pleurocystidia absent. Pileipellis (squamules on pileus) epithelial, composed of agglutinated globose, subglobose to broadly ellipsoid, rarely clavate cells, terminal cells measuring 9–15 × 6–10 µm, slightly encrusted, with some vacuolar pigments when observed in KOH or H2O. Caulocystidia sometimes present, cylindro-clavate to narrowly clavate measuring 18–25 × 5–7 µm, thin-walled, smooth, hyaline. Stipe trama composed of parallel hyphae 4–10 µm wide, yellowish brown in mass but pale yellow to subhyaline individually. Clamp connections absent in all tissues.

Habit, habitat and distribution

Scattered, clustered on a concrete wall, currently only known from Bangladesh.

Figure 4. 

Microscopic features of Xanthagaricus necopinatus (GDGM 46632, holotype; PHI-12, isotype). a Basidia with basidioles b Basidiospores c Epithelial pileipellis with encrusted wall d Cheilocystidia. Scale bars: 10 µm.

Figure 5. 

SEM of basidiospores of Xanthagaricus spp. a SEM basidiospores of X. flavosquamosus (GDGM 50918, holotype) b SEM of basidiospores of X. necopinatus (PHI-12, isotype). Scale bars: 5 µm.

Heinemannomyces splendidissimus Watling, Belg. J. Bot. 131(2): 135 (1998)

Figs 6, 7

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.

Basidiospores [60/3/3] (5.5–)6–6.5(–7) × 3.5–4.5(–5) µm, [mean length = 6.25 µm, mean width = 4.15 µm, Q = 1.42–1.55(–1.63), Qm = 1.50 ± 0.043], ellipsoid to ovoid-ellipsoid, inamyloid, slightly thick-walled (0.5 µm), smooth, dark brown, grayish brown to slightly leaden gray in H2O and 5% KOH. Basidia 13–19 × 7–9 µm, clavate to broadly clavate, colorless or pale yellow in H2O and KOH, 4-spored, rarely 2-spored, with sterigmata up to 2 µm long. Lamellar trama regular to subregular, composed of thin-walled cylindrical hyphae 4–8 µm wide. Cheilocystidia 15–22 × 6–10 µm, abundant and scattered colorless, clavate to cylindro-clavate, sometimes narrowly fusoid, smooth, hyaline, thin-walled. Pleurocystidia absent. Pileus hyaline or pale yellow, 4–10 wide hyphae; refractive hyphae very common, 5–8 µm wide. Pileipellis (woolly-floccose squamules on pileus) a complex of hyphal types, interwoven, loosely arranged, brick red in mass but hyaline to light red or pale red individually when observed in H2O and KOH, sometimes slightly puffy or swollen in some portion of some hyphae, smooth, thin-walled, cylindrical hyphae 4–10 µm wide; terminal elements measuring 20–50 × 4–10 µm. Stipitipellis similar to pileipellis but with paler color and slightly narrower hyphae measuring 3–8 µm wide. Stipe trama composed of parallel hyphae 4–9 µm wide, hyaline; refractive hyphae sometimes present 3–5 µm wide. Clamp connections not found in any tissue.

Habit, habitat and distribution

Solitary, scattered on the ground; known from Malaysia, Thailand, and now China.

Specimens examined

CHINA, Guangdong Province, Shantou City, Nanao Island, 8 May 2015, Iqbal, Tai-Hui Li & Ting Li (GDGM 46633); same location, 9 May 2015, Iqbal, Tai-Hui Li & Ting Li (GDGM 46634, GDGM46635).

Figure 6. 

Basidiomata of Heinemannomyces splendidissimus. a Basidiomata showing leaden-blue lamellae and floccose pileus surface (GDGM 46633) b Basidiomata showing dark blue lamellae and floccose pileus surface (GDGM 46633) c Basidiomata showing blue lamellae and slightly depressed pileus disc (GDGM 46634) d Basidiomata showing pileus surface and a reddening context when cut (GDGM 46635). Scale bars: 20 mm.

Figure 7. 

Microscopic features of Heinemannomyces splendidissimus (GDGM 46633). a Basidiospores b Basidia c Elements from stipe surface d Pileipellis e Cheilocystidia. Scale bars: 10 µm.

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).

Macromorphologically, both new species are superficially close to each other, and could be confused in the field, although X. flavosquamosus has relatively larger squamules, no annulus and slightly lighter color that X. necopinatus. However, they can be separated microscopically. Xanthagaricus flavosquamosus has short and broadly clavate cheilocystidia, comparatively larger and wider basidiospores with a verrucose surface under SEM, while X. necopinatus has narrowly clavate cheilocystidia and shorter basidiospores with a rugulose-rough surface under SEM. Furthermore, these two species are in different clades in the phylogeny. Xanthagaricus flavosquamosus creates a new phyletic line with weak support, while X. necopinatus is a close relative to X. pakistanicus with strong BS support value (93% ML BS, Fig. 1). However, morphologically, X. pakistanicus has a light orange-yellow to moderate orange-yellow pileus, and globose to subglobose basidiospores measuring 7–7.5 × 6.5–7.0 µm (Hussain et al. 2017).

Some morphologically closely related species to be compared to X. necopinatus and X. flavosquamosus are X. epipastus, X. ochraceoluteus (D.A. Reid & Eicker) Hussain, X. subepipastus (Heinem. & Little Flower) Little Flower, Hosag. & T.K. Abraham, and X. viridulus (Heinem. & Little Flower) Little Flower, Hosag. & T.K. Abraham. The latter two species differ from X. necopinatus in having comparatively larger and wider basidiospores (Heinemann and Little Flower 1984, Daniëls et al. 2015). Xanthagaricus epipastus has an olive yellow to olivaceous squamulose pileus, and slightly wider basidiospores (3.7–4.7 × 2.8–3.4 µm) with lower Q value (avg. 1.36) (Heinemann and Little Flower 1984). Xanthagaricus ochraceoluteus differs from X. necopinatus in having olive-buff lamellae, no annulus, and variable cystidia. Xanthagaricus flavosquamosus has comparatively larger and wider basidiospores (5–5.5× 3–3.5 µm) than those of X. epipastus (see above), X. subepipastus (3.7–4.7 × 2.8–3.4) and X. viridulus (3.8–5.0 × 2.9–3.6 µm) (Heinemann and Little Flower 1984, Heinemann and Rammeloo 1986, Daniëls et al. 2015). Furthermore, X. epipastus has a pileus covered by olive yellow to olivaceous squamules (Heinemann and Little Flower 1984). Moreover, X. subepipastus and X. viridulus, originally described from the Kerala state of India, differ in having lageniform cheilocystidia (Heinemann and Little Flower 1984). Xanthagaricus viridulus also has an umbonate, brown, floccose-squamulose pileus, and lageniform to clavate cheilocystidia (Heinemann and Little Flower 1984). Xanthagaricus ochraceoluteus has olive-buff adnexed lamellae (Reid and Eicker 1998). Unfortunately, molecular data for the Indian collections and other comparable species are unavailable to include in this study.

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

1 Basidiomata small (8–15 mm broad) 2
Basidiomata small (15–35 mm broad), with yellow brown pileus, covered with fuscous black squamules, lamellae pink becoming grayish pink, basidiospores 5–5.5 × 3–4 µm, smooth, pileipellis epithelial with encrusted wall, found in Taiwan, China X. taiwanensis
2 Basidiomata small (10–15 mm broad), with dull lilac to grayish lilac or grayish violet squamules, lamellae white becoming light blue to blackish blue, basidiospores 5–6 × 3–3.5 µm, smooth surface under SEM, pileipellis epithelial without encrusted wall but pigmented, found in China X. caeruleus
Basidiomata small (8–15 mm broad), with yellow to yellowish brown, lamellae yellowish white to light pinkish white, pileipellis epithelial with encrusted wall, basidiospores 4–5.5 × 2.7–3.5 µm, ornamented under SEM 3
3 Basidiomata 8–13 mm broad, basidiospores 5–5.5 × 3–3.5 µm, ornamented with verrucose surface under SEM, fugacious annulus absent, found in China X. flavosquamosus
Basidiomata 10–15 mm broad, basidiospores 4–5 × 2.7–3.2 µm, ornamented with rugulose-rough surface under SEM, fugacious annulus present, found in Bangladesh X. necopinatus

Acknowledgments

The authors are very grateful to Dr. David Ratkowsky (University of Tasmania, Australia) for checking the English in the manuscript. The first author thanks Shah Hussain (University of Swat, Pakistan) for providing invaluable information on Xanthagaricus pakistanicus prior to formal publication. Dr. Zai-Wei Ge (Kunming Institute of Botany, China) is acknowledged for providing sequences data of Pseudolepiota zangmui. Dr. Ming Zhang, Mr. Jun Ping Zhou and Mr. Hao Huang (Guangdong Institute of Microbiology, China) are acknowledged for providing voucher specimens. This work was supported by the NSFC Research Fund for International Young Scientists (No. 31750110476) and China Postdoctoral Science Foundation Grant (No. 2017M610514, 61st Phase) to the first author. This study was also partially supported by the Science and Technology Program of Guangzhou, China (No. 201607020017), the National Natural Science Foundation of China (No. 31670029), and the Ministry of Science and Technology of China (Nos. 2013FY111500 and 2013FY111200).

References

  • Daniëls PP, Hama O, Fernández AF, García-Pantaleón FI, Barage M, Ibrahim D, Alcántara MR (2015) First records of some Asian macromycetes in Africa. Mycotaxon 130: 337–359. https://doi.org/10.5248/130.337
  • Ge ZW, Chen CM, Yang ZL (2008) A new species of the genus Hymenagaricus (Basidiomycota) from Taiwan and its phylogenetic position inferred from ITS and nLSU sequences. Cryptogamie, Mycologie 29: 259–265.
  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Heinemann P (1981) Hymenagaricus Heinem. gen. nov. (Agaricaceae). Bulletin du Jardin Botanique National de Belgique 51: 465–466. https://doi.org/10.2307/3668081
  • Heinemann P, Little Flower S (1984) Hymenagaricus (Agaricaceae) de Kerala (Inde) et de Sri Lanka. Bulletin du Jardin Botanique National de Belgique 54: 151–182. https://doi.org/10.2307/3667871
  • Heinemann P, Rammeloo J (1986) Flore illustrée des champignons d’Afrique central: Agariceae (Agaricaceae), Paxillaceae (Boletineae). Vol. 12.
  • Hosen MI, Feng B, Wu G, Zhu XT, Li YC, Yang ZL (2013) Borofutus, a new genus of Boletaceae from tropical Asia: phylogeny, morphology and taxonomy. Fungal Diversity 58: 215–226. https://doi.org/10.1007/s13225-012-0211-8
  • Hussain S, Afshan NS, Ahmed H, Sher H, Khalid AN (2017) Xanthagaricus pakistanicus sp. nov. (Agaricaceae), the first report of the genus from Pakistan. Turkish Journal of Botany. https://doi.org/10.3906/bot-1705-21
  • Katoh K, Kuma K, Toh H, Miyata T (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: 511–518. https://doi.org/10.1093/nar/gki198
  • Kornerup A, Wanscher JH (1978) Methuen Handbook of Colour (3rd edn). Eyre Methuen, London, 252 pp.
  • Little Flower S, Hosagoudar VB, Abraham TK (1997) Xanthagaricus, a new generic name in the family Agaricaceae. New Botanist 24: 93–100.
  • Pegler DN (1986) Agaric flora of Sri Lanka. Kew Bulletin Add Ser 12. HMSO, London.
  • Reid DA, Eicker A (1998) South African Fungi 8. Three new species of Hymenagaricus from South Africa, with a revised key to South African taxa. South African Journal of Botany 64: 356–360. https://doi.org/10.1016/S0254-6299(15)30924-8
  • Vellinga EC, Sysouphanthong P, Hyde KD (2011) The family Agaricaceae: phylogenies and two new white-spored genera. Mycologia 103: 494–509. https://doi.org/10.3852/10-204
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
  • Watling R (1998) Heinemannomyces: a new lazuline-spored agaric genus from South East Asia. Belgian Journal of Botany 131: 133–138.
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1