Tropic origins, a dispersal model for saprotrophic mushrooms in Agaricus section Minores with descriptions of sixteen new species

Agaricus section Minores contains the richest species diversity within the genus. Its Phylogeny is firstly presented by a Maximum Likelihood tree generated through DNA sequences from four gene regions of 91 species. Furthermore, a molecular dating analysis is conducted used those sequences, and it provided the divergence times of the clades within section Minores. Study showed section Minores has a tropical origin. Four main dispersal routes are proposed: (1) species from South Asia migrated through the Tibetan Plateau and reached Europe ca. 9–13 Ma; (2) species from out of South Asia dispersed to Europe in the earlier time of ca. 22 Ma; (3) species from South Asia dispersed through North Asia to Alaska, and reached West America around ca. 9 Ma; and (4) species from South Asia dispersed south and reached Oceania by at least three invading events about ca. 9, 12 and 16–18 Ma respectively. Those routes excepting the second route coincide with those of ectomycorrhizal mushrooms. To know whether the second route existed in the saprotrophic mushrooms requires further studies, and the fourth route may explain why the secotioid species occurring in Australia are morphologically similar but cluster in different phylogenetic clades. This study also demonstrates a great biodiversity of A. section Minores in China. Sixteen new species and three new records are introduced from China with morphological descriptions, illustrations, color photographs and phylogenetic analyses.

a comprehensive taxonomic system for Agaricus 9 . In that study, Agaricus was segregated into five subgenera and 20 sections, subgenus Minores was established and comprised three sections.
The epithet "Minores" was established in 1874 to accommodate a small group of fungi with small basidiomes 17 . Morphological and biochemical examination have shown that species in this section always have a strong positive KOH reaction and Schäffer's reaction, a single annulus, basidiomes which are flavescent on cutting and bruising, an odour of almond or anise. Most of the species in this section are saprobic and edible 4,9 . Historically, species of section Minores were known by their small basidiomes, but recent studies have shown that species from this section can also have large-sized basidiomes 8,18,19 , which provides the possibility of developing some species as cultivated mushrooms for food 10,18 .
The number of species in A. section Minores has been underestimated. Less than 20 species were known before the early 21 st century 12,20,21 . However, recently, 21 species from Europe 4 and 38 species from Greater Mekong Subregion 10 have been recognized. It was hypothesized that there are at least 200 species in this section worldwide 10 .
In this study, based on our Agaricus project over five years in China and previous published data of the section Minores from other continents and countries 1,4,10,18,[42][43][44][45] , we address the origin and dispersal of the saprotrophic mushroom genus Agaricus section Minores based on timing of evolutionary events. The species new to science are described in the taxonomic part of this paper with phylogenic analyses and morphological characteristics.

Results and Discussion
Phylogenetic analysis. A totally of 154 assembled multi-gene sequences were included for phylogenetic reconstruction, representing 97 species from the subgenus Minores, including 91 species of the section Minores, three species of the section Leucocarpi, three species of unnamed section 1; one species of subgenus Minoriopsis and the outgroup taxon A. campestris L. There are 2675 bp (base pairs) in the final alignment of each assembled sequence, of which 744 characters are from LSU, 541 characters from tef1-α, 776 characters from rpb2 and 614 characters from ITS.
Multi-gene trees generated from ML, MP and Bayesian analyses yielded highly similar topologies with some ungrouped taxa. The ML tree is shown in Fig. 1. In this tree, section Minores are supported by 57% BS and 1.0 PP values, separated from other two sections Leucocarpi and unnamed section 1 (Fig. 1) under the clade represented as subgenus Minores. Within section Minores, 15 clades are recognized in this study and named as Clades I-XV mainly based on the molecular dating analysis (Fig. 2), and those 15 clades also reflects in the ML tree (Fig. 1). In the ML tree ( Fig. 1), the clades II, VII, VIII, X, XI, XII, XIIII, and XV are well support with the PP/BS of 0.9-1/69-100%; clade I has a support of 1.0/-PP/BS; however the clades III-VI, IX and XIII are not supported because of failed to form the monophyletic groups or low statistical support. The proposed new species in this study are well-supported with values of 0.9/-to 1 Combined with the different climate types (temperate, sub-tropical and tropical) and relative isolated positions (Tibetan Plateau), we named the 15 phylogenetic clades as 11 types, which are abbreviated as EUP (Europe, temperate); NEA (Northeast Asia, temperate), NZL (New Zealand, temperate), NWA (Northwest America, temperate), ALK (Alaska, cold temperate), TBP (Tibet, plateau), AMT (America tropic), ASS (Asia, sub-tropic), AUS (Australia, tropic), AST (Asia, tropic) and AFT (Africa, tropic). We use mean stem age to represent the divergence times 9 of those clades. The established divergence times of those clades and ungrouped species are presented in Table 1 and Fig. 2.

Conclusions
Phylogeny of Agaricus section Minores. Agaricus section Minores belongs to A. subgenus Minores in the standardized taxonomic system which was established by evidence from analysis of combined multi-gene sequence data and divergence time analyses 9 . Twenty-one recognized species of this section have been well-documented in Europe 4 . Thirty-eight species from this section were reported from the Greater Mekong Subregion 10 (GMS: a region around the Mekong River basin in Southeast Asia, which includes Cambodia, Laos, Myanmar, Thailand, Vietnam, and Yunnan Province of China). In this study, we include all known species with molecular data, plus 105 Chinese samples and present a phylogeny of subgenus Minores, including 91 phylogenetic species of section Minores. The results using morphology and multi-gene sequences analysis resolved 58 species in this section, 38 of which can be found in China, including 16 species are new to science and three species are new records for China (for details see Taxonomy part). Sequences produced from this study are in blue. "T" refers to sequences from type specimen, and "T" in red refers to the sequences from type specimen and new to science from this study.
Our phylogenetic result shows a highly similar topology with those of Chen et al. 's work 10 . Chen et al. recognized eleven clades, which are identical to our clades I, V, VI, X, XI, XIIII and XV. The exceptions are clade IX which is a new clade in this study, and clades II, III, IV, VII, VIII, XII and XIII failed to form monophyletic lineages or had the statistical support of less than 0.8 PP. Morphological traits of those clades in section Minores vary and tend to be overlap 10 . However, with larger sampling and statistics of the morphology features in every species (see Table 2), we found that the fibrils color of the pilei is an informative phylogenetic trait in section Minores. For example, reddish-brown (such as pinkish-brown, and purplish-brown) is common in most species of this section. Species with yellowish-brown fibrils on the pileus surface are only found in clades III and IX, which are specifically from tropical and subtropical.

Phylogeography of Agaricus section Minores. Species of Agaricus section
Minores have a worldwide distribution 1,4,10,44 . The evolutionary history of section Minores and phylogenetically closely related sections have been thought to be heavily affected by geographical and climatic factors 1,10 . In this study, we include a large number of species of this section from different geographic area; these geographic areas are defined as 11 types (in Fig. 2, coded as EUP, NEA, NZL, NWA, ALK, TBP, ASS, AUS, AST, AFT and AMT) based on discrete units marked by the present limits of dispersal. To address the dispersal routes of species in A. section Minores, we defined the 15 phylogenetic clades (I-XV, in Figs 1-2) with its geographical origin (Fig. 2). Both phylogeny and molecular dating analysis (Figs 1-2) indicate that the distribution of same species or clades have the same or similar climatic distributions. On the contrary the different species or clades are generally in their isolated geography areas respectively. Combined with divergence times and geographic origin of those clades and those of ungrouped species could speculate the origin and dispersal routes of section Minores.
This study shows species in the basal clade of subgenus Minores are originated from tropic Africa, America and Asia, such as A. rufoaurantiacus Heinem., A. candidolutescens L.J. Chen & R.L. Zhao, and A. leucocarpus L.J. Chen, Callac, R.L. Zhao & K.D. Hyde, which agree with the previous study 1,9,10 . For the speciation time, tropical species originate between 1.98 to 8.93 Ma, which is generally older than most species origin from sub-tropical and temperate areas (forming at 1.5 to 7.7 Ma and 1.27 to 5.94 Ma respectively). Hence we conclude species section Minores is a tropic origin group.
Even several clades (IX and XIII) failed to establish divergence times due to the phylogenetic support values are less than 0.8 PP, the rest 13 clades are successfully dated. We then hypothesized that there are four routes which species section Minores spread to North America, Oceania and Europe from their tropic origin areas (Fig. 3). We speculate that species of section Minores from Europe are transmigrated through two routes in different times. This conclusion is based on: in both phylogenetic topologies (Figs 1-2 The third route is for species of section Minores from tropical Asia spread towards north, reach Northeast Asia around 9 Ma and through Alaska to the West America, their descendants reflect the species of clade VII (Figs 2-3).
The fourth route revealed from our study is species of section Minores from South Asia spread towards south and finally reach Oceania. There are four sequestrate (secotioid) species of section Minores that have been discovered [44][45][46] . In our analysis they are distributed in four clades (VIII, XII, XIII and XV) and in three different divergence times. Then we concluded there are at least three invading events occurred through this route that species The pattern of the dispersal routes revealed from this study are generally identical with ectomycorrhizal mushrooms 26 , wood-decaying mushrooms 47 , such as tropic origin, dispersed towards west, then reach Europe; towards north, reach Northeast Asia, then through Alaska to West America; towards south and reach Oceania. Moreover, our study suggests a new route for section Minores species that dispersed from out of South Asia to Europe directly, which different with the well-known route from tropic Asia through Tibetan plateau to Europe. Does this new dispersal route exist in saprotrophic mushrooms require further studies.
The ferocious arid climate in central Australia makes most species in section Minores evoluted into secotioid species. Four secotioid species can be found in Australia: Agaricus colpeteii T. Lebel Lebel 42,44,45 , and they are all located in four different phylogenetic clades with different divergence times. Different phylogenetic clades and divergence times represented different invading events. Our study indicates at least three invading events occurred from tropic Asia to Oceania. This is the reason why four of the five secotioid Agaricus species occur in Australia, and all cluster in different phylogenetic clades.  Taxonomy. Combined with the phylogenetic analysis and morphological characteristics, sixteen species new to science and three new record species from China are introduced. The diagnosis and morphological features of every species described in this study are summarized in Table 2 Original description: Pileus 13-28 mm in diam., plane, disc subumbonate; margin straight, slightly uplifted when mature; surface dry, covered by fibrils completely, and form fibrillose scales, triangular, appressed, denser at disc, scanty towards the margin, dark purple, purple brown; fibrils often rubbed by rain drops; background white or light gray. Context up to 1 mm thick, flesh, white. Lamellae 2 mm broad, far free, crowded, broad, light brown, dark brown in age, edge even. Annulus 1-2 mm in diam., fragile, membranous, single, white, pendant. Smooth at the both sides. Stipe 25-51 × 1-6 mm, white, cylindrical, hollow; surface dry, smooth. Odour of almonds. Basidiome flavescent when touching, bruising and cutting.
KOH reaction: positive yellow; Schäffer's reaction: positive, reddish orange on dry specimen.   Original description: Pileus 55 mm in diam., parabolic when young, then convex; disc slightly depressed; margin straight with appendiculate remains of universal veil; surface dry, completely covered by fine fibrils, and forms fibrillose scales, brown, mess, appressed, denser at disc, scattered towards the margin radially; background white. Context 6 mm thick, flesh, white. Lamellae 6 mm broad, free, crowded, pink, pinkish-brown, brown in age, edge white, even, intercalated with lamellulae. Annulus 8 mm in diam., membranous, single, white, pendant, smooth at both sides. Stipe 70 × 8 (15 at base) mm, white, cylindrical with bulbous base, narrow hollow; surface dry, fibrillose, white. Odour of strongly almonds. Basidiome strongly flavescent when touching, bruising or cutting (especially at the base of the stipe).
KOH reaction: positive yellow; Schäffer's reaction: positive, reddish orange on dry specimen.   (Figs 1-2). In the morphology, this new species is different from A. robustulus by its heavily fibrils on the surface of pileus, while those of A. robustulus is forming triangular scales; A. fimbrimarginatus has purplish fibrils on the cap, and those of A. bonussquamulosus is brown colored, also, the relative larger basidiospores of A. fimbrimariginatus (4.7 ± 0.11 × 3.2 ± 0.09) is another difference between these two species 10  Original description: Pileus 32-78 mm in diam., convex first, plane with age; disc umbonate, margin straight, also can be uplifted in age, margin slightly exceeding; surface dry, covered by plenty of fibrils at whole cap, fibrils can be rubbed by raindrop; background white or gray, get red in wet; fibrillose scales reddish brown or purplish brown, denser at disc, scanty obviously towards the margin; tiny fibrillose scales triangular-shaped, appressed, or erected. Context 3-4 mm thick, flesh, white or light gray. Lamellae 4-5 mm broad, free, crowded, edge even, pinkish brown to brown in age, intercalated with lamellulae. Annulus up to 10 mm in diam., fragile, membranous, single, white, pendant, smooth on both sides. Stipe 60 × 5-7 (10-11 at base) mm, white, hollow, cylindrical, slightly bulbous at base, surface dry, with white fibrils below the annulus. Odour of almonds. Basidiome flavescent when touching and bruising, then becoming orange brown after several minutes. No discoloration or slightly yellowish on cutting.
KOH reaction: positive yellow; Schäffer's reaction: positive, reddish orange on dry specimen.  Notes: In our phylogeny analysis (Figs 1-2), the proposed new species clusters with A. catenatus (another new species from this study) in clades IX under the support of 1.0 PP value. Compared with A. catenatus, they are sharing the same sized basidiome, similar basidiospores in shape and size, however the cheilocystidia of A. catenatus are various in shape and often in chains, and those of A. jingningensis is simple. In the field, they can also be easily separated from A. catenatus having a nearly white pileus. Agaricus pallens (J.E. Lange) L.A. Parra is another species which resembles this new species, because both of them have relative slender basidiome, similar characters in pileus and basidiospores. However, the cheilocystidia of A. pallens is septa at base or multiseptate mostly, which is different from A. jingningensis 4  Original description: Pileus 70-90 mm in diam., parabolic with flat top when young, then applano-convex, finally plane with umbo, margin eroded mostly, or straight, margin exceeding with white appendiculate elements of universal veil; surface dry covered by fibrils at whole cap; background white or light yellow, turn red in wet; fibrillose scales ochraceous-yellow, triangular, appressed, denser on disc, radially scattered towards the margin. Context 2-8 mm thick, flesh, white, brown in old. Lamellae 3-7 mm broad, free, crowded, pink or pinkish brown firstly, then brown, edge even, normal to ventricose, intercalated with lamellulae. Annulus 8-15 mm in diam., single, membranous, pendant, smooth on both sides, white, turn yellow when dry or old. Stipe 47-128 × 6-65  (Figs 1-2). In morphology, A. cerinupileus differs from A. fulvoaurantiacus by its heavily fibrils on the stipe surface below the annulus, while those of A. fulvoaurantiacus is weak 10 ; differs from A. luteoflocculosus by its capitate cheilocystidia and larger basidiospores, while those of A. luteoflocculosus are simple cheilocystidia and basidiospores 5.1-5.3 × 3.4-3.7 μm 4 . In summary, this new species is characterized by its pileus orchraceous-yellow, cheilocystidia capitate with long narrow stipe and containing yellow pigments.  Etymology: the epithet "minor" means small basidiome, and "purpureus" means purple fibrils on pileus. Holotype: Manda village, Mangao, Xishuangbanna, Yunnan Prov., China, 24 June 2010, collected by Zhao Rui-Lin ZRL2010058 (HMAS275776, holotype).
KOH reaction: positive yellow; Schäffer's reaction: positive, reddish orange on dry specimen.  Original description: Pileus 50 mm in diam., plane; disc slightly truncated; margin straight with white appendiculate remains of universal veil; surface dry, fibrillose, appressed, white generally excepting light brown to reddish brown at disc; background white or light gray, turning red in wet. Context 3 mm thick at disc, flesh, white. Lamellae up to 3 mm broad, far free, crowded, pink brown to brown. Annulus single, fragile, membranous, white, pendant, smooth on both sides. Stipe 55 × 6 mm, hollow, white, cylindrical; surface dry, smooth above the annulus, fibrillose below the annulus. Odour of almonds. Basidiome no discoloration or slightly flavescent when touching. Discoloration yellowish brown after several minutes on cutting.
KOH reaction: positive yellow. Schäffer's reaction: positive, reddish orange on dry specimen.  Original description: Pileus 16-42 mm in diam., convex first, plane with age, disc umbonate, or truncate umbonate, margin decurved when young, then straight with age, slightly exceeding; surface dry, covered by fibrils at the whole cap, forming fibrillose scales, light brown, ochre, or reddish brown, dense at disc and scattered towards the margin; background white or light yellow. Context 4 mm thick, flesh, white at pileus and yellowish brown at stipe. Lamellae 5-6 mm broad, free, crowded, broad, first white, pink or pinkish brown, then brown, edge even, intercalated with lamellulae. Annulus up to 3 mm in diam., fragile, membranous, single, white, pendant, smooth at both sides. Stipe 38-40 × 3-5 mm (6-7 mm at base), cylindrical, sometimes with a swallow base, hollow, surface dry, white, fibrillose below the annulus. Odour of almonds. Basidiome flavescent, orange when touching and bruising. Context turning flavescent first, then brown on exposure after few minutes.
KOH reaction: positive yellow. Schäffer's reaction: positive, reddish orange on dry specimen.  (Figs 1-2). In the morphology all of them have discoloration of distinct yellow on touching, similar basidiospores and cheilocystidia in size and shape. Even A. armandomyces are morphologically similar to the known species A. kerriganii and A. edmondoi, they can be molecular identified as different species. For example, there are six informative characters in ITS sequences between A. armandomyces, A. kerriganii (KF447893 from type specimen) and A. edmoindoi (KF447902 from type specimen) respectively (details see Original description: Pileus 11-62 mm in diam., parabolic first, then convex, finally plane with age; margin incurved when young, then decurved, finally can be uplifted in age, margin exceeding; surface dry, covered by reddish brown, purple fibrils against white background; fibrils often break into triangular fibrillose scales at disc, appressed; sometimes cracking; turn reddish black in wet. Context 5-8 mm thick, flesh, white. Lamellae 3-15 mm broad, free, crowded, pink or pinkish brown, then brown, edge even, intercalated with lamellulae. Original description: Pileus 22-30 mm in diam., convex, applanate with subumbo; margin straight, exceeding; surface dry, covered by fibrils, reddish brown, purplish brown at disc, and fading into white towards margin, appressed, turn reddish purple in wet. Context 1-2 mm thick, flesh, white. Lamellae 2-3 mm broad, free, crowded, pink or pinkish brown first, then brown in age, edge white, crenate, intercalated with lamellulae. Annulus 2-3 mm in diam., single, fragile, membranous, white, pendant, smooth on both sides. Stipe 50 × 2-3 (5-7 at base) mm, white, hollow, cylindrical, bulbous, surface above the annulus smooth, below fibrillose, white, always with rhizomorphs. Odour of almonds. Basidiome flavescent when touching, bruising and cutting.

Samples
Positions in the ITS alignment (713 nts)   Notes: This new species is represented by specimen ZRL2010056. In phylogeny, this species is sister to the unnamed specimen ZRL20151437 under the fully support of 1.0/100 PP/BS values in the clade IX (Figs 1-2). In the morphology, A. mangaoensis is easily separated from specimens ZRL20151437 because the latter has middle-sized basidiome. Some species of section Minores have tiny or small basidiome and reddish brown, purple brown caps, such as the known species A. gemloides 5 , A. purpurellus 4 and A. parvibicolor 19 , but their phylogenetic positions are far from A. mangaoensis, and nested in clade I and clade II respectively (Figs 1-2). In this study we introduce another new species A. minorpurpureus, which is morphologically similar to A. mangaoensis in the field. However, A. mangaoensis has elongate basidiospores and those of A. minorpurpureus are broadly ellipsoid (Q = 1.4-1.6). Based on the phylogenetic and morphological analysis, we proposed this species as new to the science. This new species is characterized by its small basidiome (less than 30 mm in diam. of pileus), reddish brown fibrils on the cap, stipe cylindrical with bulbous base and clavate cheilocystidia.  Original description: Pileus 18-26 mm in diam., convex, plane; disc slightly umbonate; margin straight, with appendiculate remains of universal veil; surface dry, covered by fibrils and forming fibrillose scales, denser at disc, scattered towards the margin, appressed, purple, reddish brown, fading into white towards margin. Context 1-2 mm thick, flesh, white. Lamellae 2-3 mm broad, free, crowded, pink or pinkish brown firstly, then brown, edge crenate, intercalated with lamellulae. Annulus 3 mm in diam., single, membranous, white, pendant, upper surface smooth, lower surface fibrillose. Stipe 30-66 × 2-3 (3-4 at base) mm, white, hollow, cylindrical, surface dry, surface below the annulus fibrillose. Odour of almonds. Basidiome strongly yellow then orange brown after several minutes when touching, bruising and cutting.
KOH reaction: positive yellow. Schäffer's reaction: positive, reddish orange on dry specimen.  (Figs 1-2). This new species is characterized by its slender basidiome, purple fibrillose scales on the cap and clavate cheilocystidia which contains yellow pigments. This combination of morphological characters make it similar to another introduced new species A. mangaoensis in this study and A. gemloides 5    Original description: Pileus 23-38 mm in diam., convex, plane; disc flat; margin straight; surface dry, covered by fibrils, white or slightly gray, forming fibrillose scales at disc, appressed or recovered. Context 2 mm thick, flesh, white or gray. Lamellae up to 2 mm broad, free, crowded, pink or grayish brown firstly, then brown in age. Annulus single, fragile, membranous, white, pendant, smooth, turn to yellow when bruised or dry. Stipe 23-50 × 3-3.5 (7-9 at base) mm, hollow, cylindrical, with short rhizomorphs, surface dry, white or gray, smooth above the annulus, below fibrillose. Odour of aniseed. Basidiome strongly yellow when touching, especially on the stipe and cap. Discoloration yellow firstly, then yellowish brown after few minutes on cutting.
KOH reaction: positive yellow. Schäffer's reaction: positive, reddish orange on dry specimen.  and phylogenetic features, we proposed A. pseudopallens as a new species, and this species is characterized by its nearly white and smooth pileus, elongate ellipsoid basidiospores and absence of cheilocystidia. 13 (Figs 1-2). This new species is characterized by its slender and related long stipe, weak fibrils on the cap and capitate cheilocystidia which contains yellow pigments. In the phylogeny, A. elongatestipes is sister to the unnamed specimen ZRLLD013 (Figs 1-2), but they are obviously different species because the cheilocystidia of ZRLLD013 are globose or broadly clavate.  Notes: Five specimens represented as A. neimengguensis and cluster together with the support of 1.0/100 PP/ BS values (Figs 1-2). Agaricus neimengguensis and the unnamed specimen Vellinga2360 composed of clade VII, which located at an isolated phylogenetic position (Figs 1-2). This new species is characterized by weak fibrils at the pileus and cheilocystidia always containing yellow pigments.
The following three species are introduced as new records for China.  Figure 20 Pileus 54 mm in diam., convex with slightly truncated disc, applanate; margin entire, slightly exceeding and with the appendiculate remains of universal veil; surface dry, covered by fibrils completely; forming fibrillose scales, reddish brown, triangular, appressed, denser at disc, scattered radially towards the margin. Context 5 mm thick, flesh, white. Lamellae 5 mm broad, free, crowded, pink or pinkish brown, edge even, normal, intercalated with lamellulae. Annulus 10 mm in diam., single, membranous, smooth on both sides, pendant, persistent, white. Stipe 62 × 5 (12 at base) mm, white, hollow, cylindrical with bulbous base, surface dry, above the annulus smooth, below slightly fibrillose. Odour of bitter almonds. Basidiome flavescent immediately on touching, bruising and cutting.
Notes: Agaricus patris recently described from Thailand 10 . The morphological characters of this specimen is identical with the original description 10 . In phylogenetic analysis (Figs 1-2), our specimen also nest with the type specimen of A. patris well. Here this species is firstly reported for China.  Figure 21 Pileus 23 mm in diam.; convex, margin straight, slightly exceeding; surface dry, covered by fibrils on whole cap; fibrils broken into fibrillose scales at disc, purple, reddish brown, tiny, triangular, fading and scattered towards the margin. Context 2 mm thick, flesh, white. Lamellae 2-5 mm broad, free, crowded, broad, pink or pinkish brown Cheilocystidia 15-24.5 × 7-16 μm, smooth, clavate to broadly clavate, 1-2 septa at base, hyaline. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 4.7-11.6 μm in diam., smooth, cylindrical, brown, slightly constricted at septa.
Notes: Agaricus megalosporus is firstly described from Thailand 18 . It has middle to large sized basidiome and larger basidiospores compared with other species of section Minores. Our specimen has almost all identical morphological characters with the original description, except of the terminal element of the cheilocystidia from this study is slightly wider than those of original description 18 . Furthermore our specimen has the identical ITS sequence with those of type specimen. Considering no more significant difference between them, we identified our specimen as A. megalosporus, which is new records for China.

Morphological examination. Every newly collected specimen was photographed in situ.
Macro-morphological characteristics and biochemical color reactions were recorded from fresh specimens. The specimens were dried in a food drier at 60 °C. Anatomical and cytological features including basidiospores, basidia, cystidia and pileipellis were observed under a Olympus CX31 microscope. At least 20 measurements were made. Data were recorded as follows: X = the mean of length by width ± SD; Q = the quotient of basidiospore length to width, and Q m = the mean of Q values ± SD. The protocol of morphological study and chemical reaction followed Largent's methodology 49 . Specimens are deposited at Herbarium Mycologicum Academiae Sinicae (HMAS).