In Press at Mycologia, preliminary version published on August 3, 2015 as doi:10.3852/14-351
Short title: Four new species of Leucoagaricus Four new species in Leucoagaricus (Agaricaceae, Basidiomycota) from Asia Zai-Wei Ge1 Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China Tayyaba Qasim Fungal Biology and Systematics Lab, Department of Botany, University of the Punjab, Quaid e Azam Campus, Lahore, Pakistan Zhu L. Yang1 Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China R. Nawaz A.N. Khalid Fungal Biology and Systematics Lab, Department of Botany, University of the Punjab, Quaid e Azam Campus, Lahore, Pakistan Else C. Vellinga 111 Koshland Hall 3102, University of California at Berkeley, Berkeley, California 94720-3102 Abstract: The genus Leucoagaricus has been well studied in Europe. However, species diversity of Leucoagaricus in Asia remains poorly known, especially in the mountains of southwestern China, a hot spot for biodiversity. Based on morphological characters and molecular data, four new species are described, La. asiaticus, La. subcrystallifer, La. subpurpureolilacinus and La. truncatus. Detailed morphological descriptions, drawings of microstructures for novel taxa and comparisons with closely allied taxa are provided. Phylogenetic analyses inferred from internal transcribed spacer (ITS) and region 6–7 of the
Copyright 2015 by The Mycological Society of America.
gene for the second largest subunit of RNA polymerase II (rpb2) sequences show that the novel taxa are nested within a well-supported clade jointly formed by members of Leucoagaricus section Rubrotincti and subgenus Sericeomyces. Key words: Agaricales, Leucoagaricus, Leucocoprinus, new taxa, taxonomy INTRODUCTION The genus Leucoagaricus Locq. ex Singer, typified by La. macrorhizus Locq. ex Singer (= La. barssii [Zeller] Vellinga) (Singer 1948, Vellinga 2000), contains approximately 90 species according to Kirk et al. (2008). Members of this genus are characterized by their fibrillose to squamulose or silky pilea surface, white to cream or pink spore print, dextrinoid spores that are metachromatic in cresyl blue and hyphae without clamp connections (Singer 1986, Vellinga 2001). Leucoagaricus is morphologically distinguished from Leucocoprinus Pat. in that species of the latter genus have a pileus with notable striations, spores with apical germ pores and hymenium with pseudoparaphyses (Singer 1986). Molecular phylogenetic studies (Vellinga 2004, Vellinga et al. 2011) have shown that Leucoagaricus is a heterogeneous group with its species intermixed with Leucocoprinus. However, it is premature to split the Leucoagaricus/Leucocoprinus clade into distinct, monophyletic genera because an extensive morphological and molecular analysis of described taxa is still lacking. Considering the above-mentioned morphological differences, mycologists usually retain the separate generic status of Leucoagaricus and Leucocoprinus when describing novel species within these groups (e.g. Kumar and Manimohan 2009; Muñoz et al. 2012, 2014; Malysheva et al. 2013). Leucocoprinus has been restricted to taxa with a plicate pileus and pseudoparaphyses in the hymenium. The species in this study lack these characteristics, and the genus name Leucoagaricus is used herein for them. The genus Leucoagaricus is widely distributed in both northern and southern hemispheres and has been well studied in Europe (Candusso and Lanzoni 1990, Bon 1996, Vellinga 2001),
to a lesser extent in North America (Kauffman 1924, Smith 1954, Sundberg 1967 [all under Lepiota], Akers 1997, Vellinga 2007, Vellinga and Davis 2007, Vellinga 2010, Vellinga et al. 2010) and Asia. However, the classical work by Berkeley and Broome (1871) and Pegler's study of the specimens from Sri Lanka (1972, 1986) laid the basis for further research, and several species have been described from Asia (Kumar and Manimohan 2009, Ge 2010, Liang et al. 2010, Malysheva et al. 2013, Yuan et al. 2014). Here we focus on the mountains of southwestern China, one of the world's biodiversity hotspots (Myers et al. 2000), and the under-investigated lowland, northeastern Pakistan. During trips in these regions, interesting collections of the genus Leucoagaricus were made. We discovered that the genus is present at elevations, above 3000 m, which is unusual for the known species in the genus. Based on morphological observations and molecular phylogenetic analyses, this study describes four new species from China and Pakistan. MATERIALS AND METHODS Morphological characters.—Specimens examined were deposited in the Herbarium of Cryptogams of Kunming Institute of Botany, Chinese Academy of Sciences (HKAS) and the Herbarium of University of the Punjab, Lahore, Pakistan (LAH), respectively. Macroscopic characters were recorded based on fresh material. Color notations indicated in the descriptions for the Chinese species are from Kornerup and Wanscher (1978) and follow Munsell's Soil Color Charts (1975) for the Pakistan species. Microscopic characters were observed from material rehydrated in 5% aqueous KOH (w/v). Pilea structure, cheilocystidia and basidia were observed and measured in 5% KOH, with 0.5% aqueous Congo Red (w/v). Melzer's reagent was used to test the amyloidity of spores. Spores and basidia were taken from lamella fragments of mature basidiomata. Basidium measurements do not include sterigmata. At least 20 spores were measured in side view for each mature basidioma, and measurements were rounded to the nearest 0.5 µm. Dimensions for basidiospores are reported as (a–)b–c(–d). The range b–c contains a minimum of 90% of the measured values. Extreme values (a and d) are in parentheses. The mean value for spore length and width, the Q value (ratio of the mean spore lengths divided by the mean spore widths) and the average Q (avQ) of all basidiospores ± sample standard deviation were calculated. DNA extraction, PCR and sequencing.—Genomic DNAs were isolated from pieces of lamellae following a
modified CTAB method (Doyle and Doyle 1987). PCR was performed on an ABI 2720 thermal-cycler (Applied Biosystems, Foster City, California). Primer sets ITS1F / ITS4 and RPB2-6F / RPB2-7R (Gardes and Bruns 1993, Liu et al. 1999) were used, respectively, to amplify the nuc rDNA ITS1-5.8S-ITS2 region (ITS) and the region between the conserved domains 6 and 7 of the gene for the second largest subunit of RNA polymerase II (rpb2). PCR reaction parameters, sequencing reactions and methods for editing the sequences are the same as described in Ge et al. (2014). The sequences produced in this study have been deposited in GenBank (SUPPLEMENTARY TABLE I). Phylogenetic analyses.—The initial BLAST results revealed that the most similar sequences to the novel taxa are sequences of species within La. sect. Rubrotincti Singer and La. subgen. Sericeomyces (Heinem.) Bon; thus ITS and rpb2 sequences from species within these groups were included in the phylogenetic analysis. Besides representative sequences from different sections within Leucoagaricus, additional sequences of representative genera within Agaricaceae also were included in the datasets. Sequences generated from Asian collections, especially from type specimens, are included wherever possible. Multiple sequence alignments were performed with Mafft 6.8 (Katoh et al. 2008), followed by minor improvements. The resulting alignments can be accessed at TreeBASE.org with submission ID assigned as S16873 (http://purl.org/phylo/treebase/phylows/study/TB2:S16873). Coprinus comatus (O.F. Müll.) Pers. was designated as outgroup for the dataset. To infer phylogenetic relationships, maximum likelihood (ML) analyses were conducted with RAxML 7.2.3-WIN with default settings under the GTRGAMMA model (Stamatakis et al. 2008), and 1000 bootstrap partitions with the rapid bootstrap option were performed to assess the bootstrap support (BS) for branches.
RESULTS The ITS dataset included 143 sequences, and the resulting alignment was 907 bp long. After the exclusion of ambiguously aligned regions due to the huge divergence of ITS1 and ITS2 across the Agaricaceae, the resulting alignment for analyses was 584 bp. The final ML optimization likelihood obtained for ITS tree is −14790.048016. In the ITS phylogram (FIG. 1), the novel taxa are nested within a well-supported clade (shaded in gray; with a bootstrap value of 91%). This clade, consisting of species with white or colored basidiomata, is jointly formed by members of La. sect. Rubrotincti (typified by La. rubrotinctus [Peck] Singer) with a colored pileus and subgenus Sericeomyces (typified by La. serenus [Fr.] Bon & Boiffard)
that typically has white basidiomata. It should be noted that the group of La. sericifer falls outside this clade. The novel taxon La. subpurpureolilacinus is sister to an undescribed species, Leucoagaricus sp. Thiele 2646 from Australia, forming a clade with 95% bootstrap support. The other novel taxa with colored pilei, La. subcrystallifer and La. asiaticus, form part of a well-supported clade (99%) with white taxa (La. menieri [Sacc.] Singer, La. subvolvatus [Malençon & Bertault] Bon, and La. crystallifer Vellinga). The fourth novel taxon, La. truncatus, forms a sister taxon to La. purpureolilacinus Huijsman with 93% bootstrap support. The rpb2 dataset included 88 sequences, and the resulting alignment was 653 bp long. All sites were included in the analyses because all sites can be unambiguously aligned. The final optimized ML Likelihood for the rpb2 dataset is −12341.252407. Similar to the ITS phylogeny the novel taxa are nested within a strongly supported (100%) clade formed by members of La. sect. Rubrotincti and subgen. Sericeomyces in the rpb2 phylogram (FIG. 2). Species within this strongly supported clade share some morphological characters such as the cutis-like pileipellis and shape of the spores and the cystidia. For instance the pileus coverings of these species are composed of subcylindrical, smooth, repent, rarely differentiated hyphae, more often radially arranged; ovoid to amygdaliform basidiospores, more or less constricted near the apex without a germ pore. TAXONOMY Leucoagaricus asiaticus T. Qasim, R. Nawaz and A. N. Khalid, sp. nov.
FIG. 3A, 4
MycoBank MB810821 Typification: PAKISTAN. PUNJAB: Lahore, Botanical Garden, University of the Punjab, 217 m, 10 Sep 2012, A. N. Khalid (holotype LAH 10012012). Etymology: The specific epithet “asiaticus” (L.) refers to the continent from where it is collected.
Pileus 23–60 mm diam, more or less plano-concave when mature, obtusely umbonate with dark brown to reddish brown disk (7.5YR3/4), brownish gray striations on white background, continuous at first then split radially 5 mm before margin; margins uplifted and split at maturity. Lamellae free, close, white (7.5PB9/2), thin, with lamellulae in 1–2 tiers, with entire edge. Stipe 39–65 × 2–10 mm, central, unequal, sometimes ridged, attenuating toward pileus, smooth, with bulbous base. Annulus thin, white, superior. Context white, fleshy, not changing in color upon bruising. Flavor and odor not recorded. Basidiospores (7)8–10(11) × (5)6–7 µm (Q = 1.14–1.6(2.0), avQ = 1.37 ± 0.18), oblong to ellipsoid, some slightly amygdaliform in side view, pale yellowish in KOH, guttulate, apiculate, thin-walled, smooth, dextrinoid, without germ pore. Basidia 17–22 × 9–12 µm, clavate, hyaline in KOH, thin-walled, 2–4-spored; sterigmata up to 3 µm long. Cheilocystidia 33–40 × 11–17 μm, subclavate, utriform, ventricose or fusoid, hyaline in KOH, thin- to thick-walled, with fine crystals or granules at apex. Pleurocystidia absent. Pileipellis a cutis made up of 4–11 µm wide branched, septate, thin-walled hyphae, pale brown in KOH. Clamp connections absent. Habitat and distribution: Saprotrophic and solitary to scattered on nutrient-rich loamy soil under planted angiospermic vegetation. So far only known from lowland northeastern Pakistan. Additional specimen examined: PAKISTAN. PUNJAB: University of the Punjab, Lahore, 217 m, scattered on rich loamy soil of the garden, 6 Jul 2011. A.N. Khalid MCR58 (LAH 5872011). Comments: Leucoagaricus asiaticus has a combination of striking features including a centrally depressed white pileus with dark brown disk and grayish brown striations, bulbous stipe base, oblong to ellipsoid spores, cheilocystidia with crystals at the apex, and the pileipellis is a cutis composed of pale brown, branched, thin-walled hyphae, 4–11 µm wide.
According to the phylogenetic analysis (FIG. 1), the closest relatives are the white species Leucoagaricus crystallifer, La. menieri and La. subvolvatus, and La. subcrystallifer which features a colored pileus. Leucoagaricus crystallifer resembles La. asiaticus in its bulbous stipe base, ellipsoid to oblong spores of approximately the same length and crystal-bearing cheilocystidia. But La. crystallifer is a white species, known from deciduous woods in temperate Europe (Vellinga 2001). Two other European species with white basidiocarps, La. menieri and La. subvolvatus, and the newly described species La. subcrystallifer from China, have similar shaped basidiospores and cheilocystidia with crystals at the apex. However, La. menieri has fragile, shining, sericeous basidiomata and La. subvolvatus has fleshy basidiomata with a white to cream pileus (Candusso and Lanzoni 1990). Differences between La. asiaticus and La. subcrystallifer will be discussed under La. subcrystallifer Comments. Other species resembling La. asiaticus include: La. sublittoralis (Kühner ex Hora) Singer, which resembles La. asiaticus in having approximately the same spore size (6.5–10 × 3.5–5 µm) and apical crystals on the cheilocystida but differs in having pinkish brown pileus and annulus edge and spores with apical papilla. In addition to these morphoanatomical differences, the sequences of both taxa have nucleotide polymorphisms at 28 positions. Leucoagaricus vassiljevae E.F. Malysheva, T. Yu. Svetasheva & E.M. Bulakh is characterized by the plano-convex pileus with red-brown appressed fibrils, crowded lamellae, larger spores (8)8.4–11.5(13) × 5–6 µm and cheilocystidia without apical crystals (Malysheva et al. 2013). Leucoagaricus wichanskyi (Pilát) Bon & Boiffard possesses larger pilei with pink-brown or purplish brown covering; cheilocystidia are narrow, rarely with crystals at top (Vellinga 2001). Leucoagaricus purpureolilacinus is characterized by dark purplish pileus, bigger spores than La. asiaticus and cheilocystidia crowned with abundant crystals (Vellinga 2001). Leucoagaricus subcrystallifer Z.W. Ge & Zhu L. Yang, sp. nov.
FIG. 3B, 5
MycoBank MB810791 Typification: CHINA. SICHUAN PROVINCE: Luhuo County, about 25 km from Luhuo to Wengda, 3440 m, 12 Aug 2005, Z.W. Ge 878 (holotype HKAS 49373). Etymology: from sub-(L.) “almost” + “crystallifer” (Gr.), bearing tiny crystals on the
cheilocystidia. Pileus 30–50 mm diam, ovoid when young, becoming convex with age, sometimes margin slightly reflexed, with radially arranged greenish gray (1B2-1C2-1D3) to yellow gray (2B2-2C2-3C2), fibrillous squamules on the white background, squamules usually with purplish tinge; umbo inconspicuous, olive gray (1E2-1F2) to dark gray (29F4-29F5-29F6). Lamellae free, crowded, white; edge finely scalloped. Stipe 50–70 × 4–8 mm, clavate, attenuate upward, white, smooth. Annulus simple, located at the middle to upper part of the stipe, white, membranous. Context thin, white, not changing color with handling. Odor weak, indistinct; flavor mild. Spore print white. Basidiospores (7)7.5–8.5(9) × 5–5.5(6) µm (Q = [1.33] 1.45–1.7 [1.8], avQ = 1.56 ± 0.08), amygdaliform to ovoid in side view, ovoid in front view, hyaline, smooth, slightly thick-walled, dextrinoid, metachromatic, without germ pore. Basidia 22–27 × 9–11 µm, clavate, four-spored, rarely two-spored. Cheilocystidia 25–46 × 7–10 µm, narrowly fusiform, at times clavate, slightly thick-walled, apex with fine crystals, hyaline. Pleurocystidia absent. Pileipellis a cutis of radially arranged repent hyphae, 4–7 µm diam, thin-walled, with yellowish to pale brownish vacuolar pigments. Clamp connections not observed. Habitat and distribution: Saprotrophic and solitary to scattered, terrestrial in Picea spp. forest above 3000 m. Only known from Sichuan province in southwestern China. Additional specimen examined: CHINA. SICHUAN PROVINCE: Seda County, Wengda, 3090 m, 7 Aug 2005, Z. W. Ge 796 (HKAS 49291).
Comments: Leucoagaricus subcrystallifer is distinguished from other species by its small to medium-sized pileus with an indistinct dark gray umbo and greenish gray to yellow gray squamules with purplish tinge, amygdaliform basidiospores, narrowly fusiform cheilocystidia with crystals at the apex, and pileipellis composed of repent filamentous hyphae 4–7 µm wide. The ITS phylogeny (FIG. 1) shows that La. subcrystallifer is close to the white species La.
menieri and La. subvolvatus. Morphologically these species have similarly shaped basidiospores and cheilocystidia with crystals at the apex. However, La. menieri has fragile, shining, sericeous basidiomata and the basidiospores are smaller (6–7 × 4.7–5 µm; Candusso and Lanzoni 1990, Bon 1996) and La. subvolvatus has stout, fleshy basidiomata with a white to cream pileus, roundish stipe base that is somewhat volvate (Candusso and Lanzoni 1990, Bon 1996). Leucoagaricus subcrystallifer also is similar to another white species, La. crystallifer, a species originally described from the Netherlands. Both species have similarly shaped spores and crystals at the apex of cheilocystidia. However, basidiomata of La. crystallifer are white to whitish and its basidiospores are smaller, 6.3–7.7 × 3.9–4.2 µm (Vellinga 2001). Leucoagaricus purpureolilacinus, originally described from Netherlands, is also similar to L. subcrystallifer in the shape of the basidiomata and the presence of crystals at the apex of the cheilocystidia. However, La. purpureolilacinus has a pinkish brown pileus with a dark purple brown disk, a stipe attenuating toward the base, amygdaliform spores and clavate cheilocystidia often with constrictions in the middle (Vellinga 2001). Leucoagaricus asiaticus also is similar to La. subcrystallifer in the overall appearance of basidiomata and cheilocystidia with crystals. However, the pilei of La. asiaticus are brownish gray with a dark brown disk and the cheilocystidia are wider than those of La. subcrystallifer. Leucoagaricus subcrystallifer was found in alpine coniferous forests at 3090–3440 m, whereas La. asiaticus was collected in lowlands in Pakistan under angiospermic vegetation at 217 m. Leucoagaricus crystalliferoides T.K.A. Kumar & Manim., originally described from India, also has fine, granular crystals on the variably shaped cheilocystidia. However, this species can be distinguished from La. subcrystallifer by these characters: small (9–19 mm) pileus covered with fibrillose brown squamules, lamellae and stipe turning light brown to brown on
bruising, spores with an apical germ pore and hyphae of the pileipellis having plasmatic pigment (Kumar and Manimohan 2009). Leucoagaricus subpurpureolilacinus Z.W. Ge & Zhu L. Yang, sp. nov.
FIG. 3C, 6
MycoBank MB810792 Typification: CHINA. YUNNAN PROVINCE: Kunming City, Heilongtan, Kunming Botanical Garden, 1980 m, 22 Jun 2005, Z.W. Ge 406 (holotype HKAS 48285). Etymology: the prefix “sub-” (L.) means “almost, close”, and “purpureolilacinus” refers to the epithet of the species that this novel taxon resembles. Pileus 30–70 mm diam, at first ovoid to ellipsoid, expanding to convex to applanate, light brown (6D4), brown (6E4) to brown gray (5E2) fibrillose to silky fibrillose, often with purplish hues; umbo broad, brown (6E4) to dark ruby (12F3–12F4); sometimes radially cracking toward margin into non-imbricate squamules, exposing the white background. Lamellae free, white, crowded, up to 5 mm tall, with 2–3 tiers of lamellulae; edge finely scalloped. Stipe 70–115 × 3–10 mm, subclavate, attenuate upward, white, smooth, hollow; annulus white, membranous, persistent on the upper stipe, edge of the upper surface brown to dark brown. Context white in pileus and stipe, not discoloring when bruised. Odor indistinct. Faste mild. Spore print white. Basidiospores (7.5)8–9(11.5) × 4.5–6 (7) µm (Q = [1.38] 1.55–1.78 [1.89], avQ = 1.65 ± 0.08), amygdaliform to oblong ovoid (occasionally citriniform) in side view, ovoid in front view, with a constriction near apex, without germ pore, hyaline in 5% KOH, smooth, slightly thick-walled (about 0.5 µm), strongly dextrinoid, metachromatic. Basidia clavate, 16–25 × 6–9 µm, hyaline, four-spored. Cheilocystidia 30–53 × 10–16 µm, hyaline, thin-walled, forming clusters, clavate, at times fusiform; apex with gelatinous covering intermixed with crystals. Pleurocystidia abscent. Pileal squamules a cutis with bundles of radially arranged repent hyphae, (5)6–8(10) µm diam, thin-walled, with brownish yellow pigments on the
brownish yellow hyphal wall, sometimes with brownish yellow to yellow brown vacuolar pigments. Clamp connections not observed. Habitat and distribution: Saprotrophic and solitary to scattered, terrestrial in Cedrus forest. So far known only from Kunming, Yunnan province in southwestern China. Additional specimens examined: CHINA. YUNNAN PROVINCE: Kunming Botanical Garden, 1980 m, 27 Jun 1978, X.L. Wu 998 (HKAS 998); same locality, 20 Jun 1990, Z.L. Yang 994 (HKAS 22658); same locality, 10 Jul 1991, Z.L. Yang 1087 (HKAS 24837), same locality, 12 Jun 2003, H.C. Wang 264 (HKAS 44354); same locality, 17 Jun 2004, X.H. Wang 964 (HKAS 36725); same locality, 28 Jun 2004, Z.L. Yang 3959 (HKAS 45050).
Comments: Leucoagaricus subpurpureolilacinus is characterized by the broad, brown to dark ruby umbo, light brown to brown fibrillose to silky fibrillose squamules with purplish hues, amygdaliform to oblong ovoid basidiospores with a constriction near apex, clavate cheilocystidia with gelatinized covering intermixed with crystals, and the pileipellis composed of a cutis of repent hyphae which sometimes have brownish yellow pigments on the cell wall. Leucoagaricus subpurpureolilacinus is similar to La. purpureolilacinus with regard to the appearance of the basidiomata and shape of the cheilocystidia. However, in the latter the pileus is pink brown, the stipe attenuates toward the base, and cheilocystidia have crystals but no gelatinized covering (Vellinga 2001). Phylogenetically La. purpureolilacinus clusters with La. truncatus, but not with La. subpurpureolilacinus (FIG. 1). Leucoagaricus sublittoralis and La. crystallifer also have cheilocystidia covered with crystals and similar-sized basidiospores. However, La. sublittoralis differs in the pale pink-brown pileus and its cheilocystidia are more variable, from clavate to narrowly clavate or narrowly fusiform, while La. crystallifer has white to whitish basidiomata with a cream to ochraceous disk, and cheilocystidia vary from clavate to fusiform or narrowly clavate (Vellinga 2001).
Leucoagaricus japonicus (Kawam. ex Hongo) Hongo and La. rubrotinctus are similar in the shape of the spores and cheilocystidia. However, La. japonicus has a testaceous scaly pileus and La. rubrotinctus a pinkish to reddish pileus, and crystals have not been observed on cheilocystidia of either taxon (Kauffman 1924, Hongo 1959, Yang 2000). Lepiota erythrogramma (Berk. & Broome) Sacc., a reddish brown to reddish purple species from Sri Lanka, differs from the current species in having inconspicuous clamp connections at the septa and brown cheilocystidia without crystals (Pegler 1986). Leucoagaricus glabridiscus (Sundb.) Wuilb. and Lepiota rubrotinctoides Murrill, originally described from western USA (Murrill 1912, Smith and Sundberg 1979), are similar. However, La. glabridiscus has smaller (15–42 mm), fragile reddish brown to orange brown basidiomata, interwoven and double-layered cuticle, and slender basidiospores (6.3)7–9.5(10.3) × 3.9–4.8(5.5) µm (Smith and Sundberg 1979); L. rubrotinctoides differs from La. subpurpureolilacinus by relatively big basidiomata, pink to orange, and a strongly contrasting darker pileus center, and a smoother aspect of the pileus due to the absence of scales on the pileus surface (Murrill 1912). Moreover, the ITS phylogeny (FIG. 1) supports the recognition La. subpurpureolilacinus as distinct from L. rubrotinctoides. Three species described from the Russian Far East also somewhat similar to La. subpurpureolilacinus in having reddish brown to red brown basidiomatata and subamygdaliform basidiospores. However, the ITS phylogeny shows clear differences among these species (FIG. 1). Morphologically La. rubrobrunneus E.F. Malysheva, T. Yu. Svetasheva & E.M. Bulakh differs from La. subpurpureolilacinus by having red-brown basidiomatata and smaller basidiospores (5–5.7 × 3.2–4.3 µm); La. vassiljevae differs in having red-brown appressed fibrils on the pileus and cheilocystidia without crystals at the apex and La. lateritiopurpureus (Lj. N. Vassiljeva) E.F. Malysheva, T. Yu. Svetasheva & E.M. Bulakh differs in having smaller and slender basidiomatata with pilei less than 20 mm wide and
smaller basidiospores, 5.7–8 × (3)3.2–4.3(4.6) µm (Malysheva et al. 2013). Leucoagaricus truncatus Z.W. Ge & Zhu L. Yang, sp. nov.
FIG. 3D, 7
MycoBank MB810793 Typification: CHINA. SICHUAN PROVINCE: Seda County, on the way from Wengda to Maerkang, 3090 m, 7 Aug 2005, Z.W. Ge 793 (holotype HKAS 49288). Etymology: truncatus (L.), referring to the shape of the pileus apex. Pileus 40–80 mm diam, truncated campanulate in early stage, becoming truncated convex to broadly truncated convex upon opening, background white, covered with orange-white (6A2), pale orange (6A3) to gray-orange (5B3-6B3) furfuraceous squamules. Lamellae free, white, crowded, 6 mm tall, with 1–2 lamellulae; edge finely scalloped. Stipe 50–70 × 4–1.5 mm, subclavate, thickening toward base, white, smooth, hollow; annulus white, membranous, persistent on the middle part of the stipe. Context white, not discoloring when bruised. Odor indistinct; flavoraste mild. Spore print white. Basidiospores (8)8.5–9.5 × (5.5)6–6.5(7) µm (Q = [1.31]1.36–1.50[1.58], avQ = 1.45 ± 0.06) broadly amygdaliform in side view, ovoid in front view, hyaline, smooth, without germ pore, slightly thick-walled, dextrinoid, metachromatic. Basidia 22–28 × 9–11 µm, clavate, four-spored, occasionally two-spored. Cheilocystidia 24–40(80) × 12–18(25) µm, clavate to narrowly clavate, hyaline, thin-walled; some cheilocystidia from young basidiomatata with crystals at the apex. Pleurocystidia not observed. Pileipellis a cutis 130–210 µm thick composed of appressed, thin-walled filamentous hyphae, sometimes slightly interwoven, hyaline to pale yellowish, 2.5–5.5 µm diam. Clamp connections not observed. Habitat and distribution: Saprotrophic and terrestrial on clay soil in Picea forest at approximately 3000 m, scattered. So far known only from Sichuan province in southwestern China. Comments: Macroscopically the distinctive delimitating characters of La. truncatus are
medium to large basidiomata with a truncated pileus apex, and the orange-white to gray-orange furfuraceous squamules on the pileus. Leucoagaricus truncatus is close to La. purpureolilacinus and La. sp. “Rogers 6184” in the ITS phylogeny (FIG. 1). However, La. purpureolilacinus has a pink-brown pileus, a stipe attenuating toward the base, and cheilocystidia with crystals (Vellinga 2001). Leucoagaricus truncatus resembles La. serenus and La. crystallifer in the shape and size of basidiospores and cheilocystidia. However, the latter two species have small, white to whitish basidiomata with a pileus that is mostly smooth with obvious striations near the margin, and an obtuse umbo, which is white to cream, while La. truncatus forms medium-sized to large basidiomata with a truncated pileus apex, and its pileus is covered with distinct pale orange to gray-orange furfuraceous squamules. In addition, the cheilocystidia of La. crystallifer are clavate to narrowly fusiform, with obvious crystals on the surface (Vellinga 2000, 2001), while the cheilocystidia of La. truncatus are clavate and only in young basidiomata have some crystals at the apex. The pileus of La. truncatus is similar to that of Macrolepiota mastoidea (Fr.) Singer, but it differs in microscopic characters including the structure of the pileipellis. Species of Macrolepiota have an umbonate pileus, large, ellipsoid, thick-walled basidiospores with an apical germ pore, the pileipellis is a trichodermal palisade, and clamp connections are present at the base of the basidia (Vellinga 2001, Ge et al. 2010). DISCUSSION According to our preliminary molecular and morphological studies on the specimens collected from Asia, the species diversity of Leucoagaricus in Asia is underestimated and many more new species remain to be described. Even within La. sect. Rubrotincti, three more collections (Ge 2044, Ge 2104 and JX827166 in FIG 1) that represent novel taxa were collected in the mountains of southwestern China. This mountainous region harbors a rich
fungal diversity due to the diverse ecological niches caused by the unique geological history (Yang 2005, Ge et al. 2014), as revealed by recent studies focusing on other groups of larger fungi in this region, such as the genera Amanita, Boletus, Cudonia, Flammulina, Morchella and Spathularia (Du et al. 2012; Feng et al. 2012; Ge et al. 2008, 2014; Cai et al. 2014). This is probably also true for the genus Leucoagaricus. Pakistan also comprises a variety of habitats that range from coastal areas along Arabian Sea to arid scrubland in Punjab province and to the high altitude forest and tundra of Himalaya. These ecological variations assist a wide variety mycoflora to appear on grassy lands as well as in deserts (Yousaf et al., 2012). Previously only two species of genus Leucoagaricus (viz. L. leucothites [Vittad.] Wasser and L. serenus [Fr.] Bon) have been reported from Pakistan (Ahmad et al. 1997). The mycoflora of Pakistan has not exhaustively been explored and much remains to be discovered. The name La. rubrotinctus has been applied to collections from France (Balot 2002), Switzerland (Breitenbach & F. Kränzlin 1995), India (Kumar & Manimohan 2009) and Japan (Imazeki et al. 1988). Several ITS sequences (one each from Costa Rica and Russia and four from China) downloaded from GenBank are labeled Leucoagaricus rubrotinctus. However, La. rubrotinctus originally was described by Peck (1884) from the Helderberg Mountains and East Worcester in Massachusetts in the northeastern USA, and in this study we have incorporated two ITS sequences generated from specimens from USA, one of which (157_MN in FIG 1) from Minnesota is similar to a sequence from a collection from Worcester County in Massachusetts (Justo pers comm near the type locality), thus very likely is true La. rubrotinctus. Those collections from China, Costa Rica and Russia belong to different species, according to the ITS phylogeny. The putative novel taxa from China, labeled “La. rubrotinctus” due to their more or less reddish pileus, are represented only by a single collection each, thus we do not propose these as new in the present paper. With additional field work and more collections available more
new taxa will be disclosed in soon. ACKNOWLEDGMENTS Alfredo Justo was kind to provide us with an unpublished ITS sequence of La. rubrotinctus. This study was supported by the National Natural Science Foundation of China (Nos. 31270074, 31461143031), and the project was sponsored by the scientific research foundation for the overseas Chinese scholars of the Ministry of Education. T. Qasim, R. Nawaz and A.N. Khalid are financially supported by University of the Punjab, Lahore, Pakistan.
LITERATURE CITED Ahmad S, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of Pakistan, Lahore. 248 p.
Akers BP. 1997. The family Lepiotaceae (Agaricales, Basidiomycetes) in Florida [doctoral thesis. Carbondale, Illinois: Southern Illinois Univ. Press. 253 p.
Balot G. 2002. Récoltes de Leucoagaricus rubrotinctus (Peck) Singer dans les Pyrénées-méditerranéennes. Bull FAMM, n.s. 21:19–23.
Berkeley MJ, Broome CE. 1871. The fungi of Ceylon (Hymenomycetes, from Agaricus to Cantharellus). J Linn Soc, Bot 11:494–567.
Bon M. 1996. Die Großpilzflora von Europa 3 Lepiotaceae. Eching, Germany: IHW-Verlag. 153 p.
Breitenbach J, Kränzlin F. 1995. Pilze der Schweiz. Band 4. Blätterpilze 2. Teil. Luzern, Switzerland: Verlag Mykologia. 368 p.
Cai Q, Tulloss RE, Tang LP, Tolgor B, Zhang P, Chen ZH, Yang ZL. 2014. Multilocus phylogeny of lethal amanitas: implications for species diversity and historical biogeography. BMC Evol Biol 14:1–31. doi: 10.1186/1471-2148-14-143
Candusso M, Lanzoni G. 1990. Fungi Europaei 4. Lepiota s.l. Saronno, Italy: Giovanna Biella. 743 p.
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf material. Phytochem Bull 19:11–15.
Du X-H, Zhao Q, O'Donnell K, Rooney AP, Yang ZL. 2012. Multigene molecular phylogenetics reveals true morels (Morchella) are especially species-rich in China. Fungal Genet Biol 49:455–469.
Feng B, Xu J, Wu G, Zeng N-K, Li Y-C, Tolgor B, Kost GW, Yang ZL. 2012. DNA Sequence analyses reveal abundant diversity, endemism and evidence for Asian origin of the Porcini mushrooms. PloS one 7, e37567, doi: 10.1371/journal.pone.0037567
Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for Basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118, doi: 10.1111/j.1365-294X.1993.tb00005.x
Ge ZW. 2010. Leucoagaricus orientiflavus, a new yellow lepiotoid species from southwestern China. Mycotaxon 111:121–126.
———, Yang ZL, Pfister DH, Carbone M, Bau T, Smith ME. 2014. Multigene molecular phylogeny and biogeographic diversification of the earth tongue fungi in the genera Cudonia and Spathularia (Rhytismatales, Ascomycota). PLoS ONE 9: e103457, doi: 10.1371/journal.pone.0103457
———, ———, Vellinga EC. 2010. The genus Macrolepiota (Agaricaceae, Basidiomycota) in China. Fung Divers 45:81–98, doi: 10.1007/s13225-010-0062-0
———, ———, Zhang P, Matheny PB, Hibbett DS. 2008. Flammulina species from China inferred by morphological and molecular data. Fungal Divers 32:59–68.
Hongo T. 1959.The Agaricales of Japan I. Mem Shiga Univ Nat Sci 10:47–94.
Imazeki R, Otani Y, Hongo T. 1988. Fungi of Japan. Tokyo: Yama-Kei Publishers Co. 623 p.
Katoh, K, Toh H, 2008. Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298, doi: 10.1093/bib/bbn013
Kauffman CH. 1924. The genus Lepiota in the United States. Pap Michigan Art Academy. Arts Lett 4:319–344.
Kirk PM, Cannon PF, Minter DW, Stalpers JA, eds. 2008. Dictionary of the Fungi. 10th ed. CABI. 771 p.
Kornerup A, Wanscher JH. 1978. Methuen handbook of colour. 3rd ed. London: Eyre Methuen Ltd. 252 p.
Kumar TKA, Manimohan P. 2009. The genera Leucoagaricus and Leucocoprinus (Agaricales, Basidiomycota) in Kerala state, India. Mycotaxon 108:385–428.
Liang JF, Yang ZL, Xu J, Ge ZW. 2010. Two new unusual Leucoagaricus species (Agaricaceae) from tropical China with blue-green staining reactions. Mycologia 102:1141–1152, doi: 10.3852/09-021
Liu YJ, Whelen S, Hall BD. 1999. Phylogenetic relationships among Ascomycetes: evidence from an RNA polymerse II subunit. Mol Bio Evol 16:1799–1808, doi: 10.1093/oxfordjournals.molbev.a026092
Malysheva EF, Svetasheva TY, Bulakh EM. 2013. Fungi of the Russian Far East I. New combination and new species of the genus Leucoagaricus (Agaricaceae) with red-brown basidiomata. Mikol Fitopatol 47:169–179.
Munsell™. 1975. Soil color charts. Baltimore, Maryland. 34 p.
Muñoz G, Caballero A, Contu M, Vizzini A. 2012. A new Leucoagaricus species of section Piloselli (Agaricales, Agaricaceae) from Spain. IMA fungus 3:117–123.
———, ———, ———, Ercole E, Vizzini A. 2014. Leucoagaricus croceobasis Agaricales, Agaricaceae, a new species of section Piloselli from Spain. Mycol Prog 133:649–655.
Murrill WA. 1912. The Agaricaceae of the Pacific Coast II. Mycologia 4:231–262.
Myers M, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403:853–858.
Peck CH. 1884 [1882]. New York species of Lepiota. Ann Rep NY State Mus Nat Hist 35:150–164.
Pegler DN. 1972. A revision of the genus Lepiota from Ceylon. Kew Bull 27:155–202.
———. 1986. Agaric flora of Sri Lanka. Kew Bull Add Ser 12:1–519.
Singer R. 1948. Diagnoses fungorum novorum agaricalium. Sydowia 2:35–36.
———. 1986. The Agaricales in modern taxonomy. 4th ed. Koenigstein, Germany: Koeltz Scientific Books. 981 p.
Smith HV. 1954. A revision of the Michigan species of Lepiota. Lloydia 17:307–328.
———, Sundberg WJ. 1979. Studies on the Lepiotaceae of the Pacific Coast region I. Two new species. Mycotaxon 8:446–452.
Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 57:758–771, doi: 10.1080/10635150802429642
Sundberg WJ. 1967. The family Lepiotaceae in California (master's thesis). San Francisco State Univ. Press. 219 p.
Vellinga EC. 2000. Notes on Lepiota and Leucoagaricus. Type studies on Lepiota magnispora, Lepiota barssii and Leucogaricus americanus. Mycotaxon 76:429–438.
———. 2001 [2000]. Notulae ad Floram Agaricinam Neerlandicam—XXXVIII. Leucoagaricus subgenus Sericeomyces. Persoonia 17:473–480.
———. 2001. Leucoagaricus. In: Noordeloos ME, Kuyper ThW, Vellinga EC, eds Lisse, the Netherlands: A.A. Balkema Publishers. Flora Agaricina Neerlandica 5:85–108.
———. 2004. Genera in the family Agaricaceae: evidence from nrITS and nrLSU sequences. Mycol Res 108:354–357, doi: 10.1017/S0953756204009700
———. 2007. Lepiotaceous fungi in California, USA 3. Pink and lilac species in Leucoagaricus sect. Piloselli. Mycotaxon 98:213–224.
———. 2010. Lepiotaceous fungi in California, USA Leucoagaricus sect. Piloselli. Mycotaxon 112:393–444.
———, Contu M, Vizzini A. 2010. Leucoagaricus decipiens and La. erythrophaeus, a new species pair in sect. Piloselli. Mycologia 102:447–454.
———, Davis RM. 2007. Lepiotaceous fungi in California, USA 1. Leucoagaricus amanitoides. Mycotaxon 98:197–204.
———, Sysouphanthong P, Hyde KD. 2011. The family of Agaricaceae: phylogenies and two new white-spored genera. Mycologia 103:494–509, doi: 10.3852/10-204
Yang ZL. 2000. Type studies on agarics described by N. Patouillard (and his co-authors) from Vietnam. Mycotaxon. 75:431–476
———. 2005. Diversity and biogeography of higher Fungi in China (p. 35–62). In: Xu J, ed. Evolutionary Genetics of Fungi. Norfolk, UK: Horizon Bioscience. 350 p.
Yousaf N, Niazi AR, Khalid AN. 2012. New records of noteworthy gasteroid fungi from Pakistan. Mycotaxon 119:261–267.
Yuan Y, Li YK, Liang JF. 2014. Leucoagaricus tangerinus, a new species with drops from southern China. Mycol Prog 13:893–898, doi: 10.1007/s11557-014-0974-2
LEGENDS FIG. 1. ML tree inferred from the nrITS alignment. Bootstrap values above 50% are indicated along nodes. The clade where new taxa are nested is highlighted in gray. New taxa and sequences retrieved from GenBank, labeled Leucoagaricus rubrotinctus, are in boldface. The following abbreviations are used: A. (Agaricus), Chl. (Chlorophyllum), L. (Lepiota), La. (Leucoagaricus), Lc. (Leucocoprinus), Ly. (Lycoperdon), M. (Macrolepiota). FIG. 2. ML tree inferred from the rpb2 dataset. Bootstrap values (> 50) are indicated along nodes. The clade where new taxa are nested is highlighted in gray, and new taxa are in boldface. FIG. 3. A. Basidiomata of Leucoagaricus asiaticus. B. Leucoagaricus subcrystallifer. C. Leucoagaricus subpurpureolinacinus. D. Leucoagaricus truncatus. FIG. 4. Micromorphological features of Leucoagaricus asiaticus (LAH 10012012). A. Pileipellis elements (squamules on pileus). B. Basidiospores. C. Basidia. D. Cheilocystidia. FIG. 5. Micromorphological features of Leucoagaricus subcrystallifer (HKAS 49373). A. Pileipellis elements (squamules on pileus). B. Basidiospores. C. Basidia. D. Cheilocystidia. FIG. 6. Micromorphological features of Leucoagaricus subpurpureolinacinus (HKAS 48285). A. Pileipellis elements (squamules on pileus). B. Basidiospores. C. Basidia. D. Cheilocystidia. FIG. 7. Micromorphological features of Leucoagaricus truncatus (HKAS 49288). A. Pileipellis elements (squamules on pileus). B. Basidiospores. C. Basidia. D. Cheilocystidia.
FOOTNOTES Submitted 24 Dec 2014; accepted for publication 26 May 2015. 1
Corresponding authors. E-mail:
[email protected],
[email protected]
59
La. adelphicus ecv3153 La. pilatianus MCVE9558 La. hesperius ecv3515 La. ionidicolor ecv2280 La. decorata ecv2810 La. cupresseus ecv3204 La. dyscritus ecv3956 La. pardalotus ecv3313 La. variicolor GM2486 La. badhamii MCVE03047 94 L. fuliginescens ecv3219 L. fuliginescens ecv3223 61 La. georginae ecv2238 La. brunnescens Balsley La. atroazureus HKAS48453 La. jubilaei Guinberteau 100 L. roseolivida ecv2990 La. marriagei ecv2005 La. pyrrhophaeus ecv3251 83 La. flammeotinctoides ecv3304 La. pyrrhulus ecv3306 97 La. decipiens Contu La. erythrophaeus ecv3254 La. barssii ecv3126 La. sp. Thiele2645 La. croceobasis GM2465 100 La. sp. ecv2746 L. flammeotincta ecv3967 100 La. viridiflavus AK80a La. flavovirens HKAS50024 100 La. tangerinus Zhang623 66 La. tangerinus RITF2279 La. tener ecv2261 64 La. melanotrichus ecv2262 Lc. brebissonii JJ91 La. sp. EF080869 La. croceovelutinus ecv2243 83 Lc. cepaestipes EFM518 La. cinerascens PBM1830 97 Lc. brebissonii ecv1784 Lc. longistriatus JJ142 65 La. leucothites Qiu La. nympharum Ge379 Lc. straminellus ecv2080 Lc. birnbaumii EFM549 Lc. fragilissimus PA250 57 La. sericatellus ecv2013 La. sericatellus Bas8842 100 La. sericifer ecv2116 La. sp. ecv2607 subpurpureolilacinus r ure r olilacinus Yang3959 g 95 100 La. subpurp La. subpurp subpurpureolilacinus p rpure r olilacinus Ge40 Ge406 6 56 6 96 La. sp. Thiel Thiele2646 e26 4 6 6 La. ru rrubrotinctus bro r ti t nctus xsd08140 China La. ru rrubrotinctus bro r ti t nctus JX827166 China 74 4 La. ru rrubroconfusus bro r confu f sus zt13003 75 La. a ru rrubrotinctus ubro r ti t nctus (cf) Ge2044 China 75 100 1 00 La. lahore lahorensis r nsis KJ701794 lahorensis La. lahore r nsis KJ701795 61 La. ru rrubrotinctus bro r ti t nctus JX133167 Russia 77 7 La. ru rrubrotinctus bro r ti t nctus ((cf)) Ge2104 China 99 La. ru rrubrotinctus bro r ttinctus JJ100 Costa Rica 92 La. ru rrubrotinctus bro r ti t nctus gr g 0557 TN USA La. sublittora sublittoralis r lis ecv2 ecv2235 235 51 5 1 L. ru rrubrotinctoides bro r ti t nctoides ecv3920 La. ru rrubrotinctus bro r ti t nctus g gr 157 MN USA vassiljevae 94 v va ssil iljeva v e LE289432 LE289432 91 1 La. La. va vassiljevae v ssil iljeva v e LE289433 99 9 9 La. sub subcrystallifer cry rysta t llife f r Ge878 87 7 La. subcrystallifer subcry rysta t llife f r Ge796 99 9 La. subvo subvolvatus v lva v tus 6140 h 87 La. menieri r cf 6275 99 La. cry crystallifer rystallife f r Huijser j 100 0 La. asiati asiaticus t cus T0 T01 1 La. asiati asiaticus t cus R58 littoralis littora r lis MCVE702 100 La. littoralis La. littora r lis MCVE856 La. wy w wychanskyi ychansky kyi Huijser La. tr ttruncatus runcatus G Ge793 e793 98 93 La. p purp purpureolilacinus rpure r olil i acinus ecv2291 La. sp. Rogers6184 100 La. pro proximus r xi x mus LE262861 99 proximus La. pro r xi x mus LE262855 91 56 La. lateri r ti t opurp r ure r us VLA. M472 lateritiopurpureus 100 10 00 La. lateri lateritiopurpureus 97 r ti t opurp p rpure r us LE289361 La. cf sere serenus r nus Liang100 g La. ru rrubrobrunneus bro r bru r nneus LE262863 100 La. ori orientiflavus r enti t fl f avus Ge2068 100 r enti t fl f avus Ge2063 La. ori orientiflavus 100 serenus r nus ecv1930 100 1 00 La. sere La. sere serenus r nus AY176420 A 176420 AY 54 La. sp. ecv2457 100 La. bru brunneocanus r nneocanus Yang3972 r nneocanus Ge97 La. bru brunneocanus La. sp. ecv2561 79 La. americanus ecv2454 87 La. bresadolae AF295929 55 La. meleagris ecv1990 La. sinicus HMAS60647 89 Lc. cretaceus Lc. zamurensis PA415 100 La. viriditinctus HKAS50033 71 La. viriditinctus AF482873 La. irinellus AY243648 A. campestris OKM25665 89 100 A. aff campestris PBM2580 A. subrufescens WC837 100 51 A. pocillator J173 A. rotalis ecv3768 56 A. bisporus RWK1885 100 Clarkeinda trachodes ecv3550 Clarkeinda trachodes ecv3838 Heinemannomyces splendidissimus ecv3586 99 Hymenagaricus epipastus zrl3045 99 Micropsalliota sp. Laessoe6025 Hymenagaricus taiwanensis 100 100 Eriocybe chionea ecv3616 Eriocybe chionea ecv3560 100 Coniolepiota spongodes png012 Coniolepiota spongodes ecv3613 94 Chl. rachodes ecv2106 99 Chl. olivieri ecv2230 63 62 Chl. brunneum Manson8 83 Chl. molybdites JJ162 Chl. agaricoides Brotzu Micropsalliota sp. ecv3638 100 M. dolichaula Yang4660 88 M. dolichaula Ge1 100 M. procera Zhang517 98 83 M. procera Zhang381 M. velosa Zeng 562 99 L. clypeolaria Ge629 92 L. clypeolaria Ge1960 100 L. spheniscispora ecv2256 84 L. magnispora Ge985 77 L. magnispora Ge984 56 L. fuscovinacea Huijser6281 Melanophyllum haematospermum ecv2249 84 Bovista plumbea MJ4856 100 Bovista plumbea NYGD01 61 Ly. pyriforme DSH96054 Ly. perlatum 5TENN58040 Coprinus comatus ecv3198 0.1
100
L. furfuraceipes ecv3621 L. cf. furfuraceipes ecv3647 L. cf. atrodisca ecv3107 99 L. cf. atrodisca ecv3261 La. sp. ecv3568 75 100 Lc. cepistipes ecv3741 Lc. cepistipes ecv3759 58 92 La. leucothites Qiu La. nympharum Ge379 99 La. croceovelutinus Huijser6274 80 L. castanescens ecv3131 Lc. brebissonii Huijser6279 100 64 99 Lc. brebissonii Huijser6278 Lc. birnbaumii ecv3746 La. sp. deh2762 La. erythrophaeus ecv3254 63 La. pardalotus ecv3313 100 La. flammeotinctoides ecv3304 La. flammeotinctoides ecv3247 La. adelphicus ecv3153 L. roseolivida ecv2290 91 97 L. fuliginescens ecv3219 96 L. fuliginescens ecv3223 99 La. badhamii Huijser6272 100 La. badhamii Huijser6273 La. barssii ecv3126 99 La. cf. japonicus j ponicus Maruyama ja 97 La. sublittora sublittoralis r lis Huijser6277 75 La. sp. ecv3137 99 subpurpureolilacinus r ure r olilacinus Ge406 100 La. subpurp 86 La. subpurp subpurpureolilacinus r ure r olilacinus Yang3959 65 La. ru rrubroconfusus bro r confu f sus zt13003 La. subcry subcrystallifer rysta t llife f r Ge878 La. bru brunneocanus r nneocanus Ge97 La. sp. ecv3136 100 La. sp. ecv3218 La. tr ttruncatus runcatus Ge793 100 La. sp. ecv3767 92 100 La. ori orientiflavus r enti t fl f avus Ge2068 89 La. ori r enti t fl f avus Ge2063 orientiflavus 95 La. cf. sere serenus r nus Liang100 La. sp. ecv3745 La. sp. ecv3754 100 100 La. sp. ecv3757 L. flammeotincta ecv3315 63 La. sp. ecv3634 99 Micropsalliota sp. ds06156 69 Micropsalliota sp. ecv3638 66 53 La. meleagris Young La. americanus Sheine 100 Coniolepiota aff. spongodes ecv3613 88 Coniolepiota spongodes png012 Heinemannomyces splendidissimus Clarkeinda trachodes ecv3350 62 A. subrufescens WC837 94 89 A. bisporus OMF A. rotalis ecv3768 Eriocybe chionea ecv3616 100 95 Eriocybe chionea ecv3560 100 Chl. olivieri Huijser6271 52 Chl. olivieri Huijser6270 Chl. brunneum Rowe6230 100 100 Chl. brunneum Lange Chl. rachodes ecv3210 100 M. dolichaula Ge1 69 95 M. dolichaula Yang4660 M. procera Zhang517 100 50 M. procera Zhang381 M. velosa png015 Podaxis pistillaris Bethker623 100 Ly. sp. ecv3825 59 Ly. perlatum Stevens6257 Bovista plumbea NYGD01 79 100 Ly. sp. DSH96054 Bovista dermoxantha Stevens625 96 L. magnispora Ge985 L. magnispora Ge984 100 L. spheniscispora ecv2817 85 L. clypeolaria Ge629 51 L. clypeolaria Ge1960 58 L. fuscovinacea Huijser6281 74 Mel. haematospermum Bender6280 82 Cystolepiota sp. ds06004 L. aff. pilodes ecv3234 98 L. aff. subincarnata ecv3259 Tulostoma sp. ecv3117 100 Tulostoma sp. 6520 Coprinus comatus ecv3198 50
0.1
