Six New Species of Leucoagaricus (Agaricaceae) from Northeastern China

: Sixnewspecies, Leucoagaricus albosquamosus, Leucoagaricus atroviridis, Leucoagaricus aurantioruber, Leucoagaricus candidus, Leucoagaricus centricastaneus and Leucoagaricus virens , collected from northeastern China are described based on morphological characters and molecular evidence. Illustrations of fresh basidiomata and line drawings of key anatomical characters are provided. A phylogenetic tree inferred from internal transcribed spacer (ITS) region and large subunit ribosomal RNA gene (LSU) sequences shows that three of the new taxa are nested within the section Leucoagaricus and two of the new taxa are in the subgenus Sericeomyces , whereas the other new taxus is clustered with Leucoagaricus viriditinctus and Leucoagaricus irinellus , forming a clade that does not ﬁt in any known section.


Introduction
Leucoagaricus Locq. ex Singer is widely distributed [1] and contains approximately 135 species [2], with more tropical species than temperate species [3]. As Leucoagaricus barssii (Zeller) Vellinga was an invalid name when Singer designated it as the type species of Leucoagaricus, Redhead designated Leucoagaricus rubrotinctus (Peck) Singer as the lectotype for this genus instead [4].
Members of Leucoagaricus have the following morphological characters: basidiomata slender and fragile to stout fleshy, a smooth or scaly pileal surface with or without an indistinct radiate fringe at the margin, free lamellae, simple annulus, dextrinoid and metachromatic basidiospores, with cheilocystidia, and hyphae without clamp connections [5,6].
Molecular phylogenetic studies show that Leucoagaricus is not monophyletic, as species within it are intermixed with those of Leucocoprinus Pat. and Micropsalliota Höhn. [7][8][9][10]. Morphologically, Leucoagaricus differs from Leucocoprinus in that the pileus of Leucocoprinus has obvious striations, and its hymenium has pseudoparaphyses [5]. However, phylogenetic relationships of some species in Leucoagaricus and Leucocoprinus remain unclear due to a lack of extensive morphological and molecular data [9][10][11].
Based on morphological observations and molecular phylogenetic analyses, in this study, we describe six new species of Leucoagaricus from China.

DNA Extraction, PCR and Sequencing
Total DNA was extracted from dried basidiomata by CTAB method [14]. The phylogenetic tree was reconstructed using internal transcribed spacer (ITS) and large subunit ribosomal RNA gene (LSU) markers [15][16][17]. Polymerase chain reaction (PCR) was implemented on an ABI 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR amplification and sequencing procedure followed procedures described in [18]. Specifically, each PCR reaction mixture contained of 1 µL (25 mmol/L) of each primer, 1 µL DNA template, 12.5 µL Biotaq master mix (BioTeke, Beijing, China) and ddH 2 O up to 25 µL. The PCR program was set as follows: pre-denaturation at 95 • C for 5 min; 32 cycles of denaturation at 95 • C for 50 s, annealing at appropriate temperature and time (ITS 52 • C and LSU 50 • C) and extension at 72 • C for 1 min, followed by a final extension at 72 • C for 8 min. The PCR products were purified using a PCR purification combo kit (BioTeke, Beijing, China) and sequenced on an ABI-3730-XL DNA analyzer (Applied Biological Systems, Foster City, CA, USA) using the same primers. The consensus sequences were obtained from forward and reverse primers using SeqMan v. 7.1.0 (DNASTAR, Madison, WI, USA).

Sequence Alignment and Phylogenetic Analyses
Preliminary analysis based on extensive representatives of Leucoagaricus and Leucocoprinus indicated that the putative new taxa are closely related to taxa in section Leucoagaricus and subgenus Sericeomyces. Thus, we built the data matrix by incorporating the newly generated sequences with sequences of taxa belonging to the section Leucoagaricus and subgenus Sericeomyces, as indicated by previous studies [10,11,[19][20][21], along with sequences of representative taxa in other Leucoagaricus sections, as well as sequences from representatives of Leucocoprinus Pat. and Micropsalliota Höhn. Cystolepiota seminuda (Lasch) Bon and Cystolepiota aff. seminuda were designated as outgroup taxa. The sequences were aligned using MAFFT v7.453 [22] and inspected and manually corrected using BioEdit v.7.0.9 [23]. TrimAl v.1.4.rev15 [24] was used to remove sites that vaguely aligned. MrModeltest v2.3 [25] predicted that GTR+GAMMA+I was the best model for nucleotide substitution under the Akaike information criterion (AIC). The phylogenetic tree was reconstructed using the Maximum likelihood (ML) and Bayesian inference (BI) methods of RAxML 7.2.6 [26] and MrBayes 3.2.3 [27].
The phylogenetic trees of ITS and LSU were reconstructed, and no potential topology conflicts were found. Thus, phylogenetic analysis was then performed based on the concatenated ITS-LSU dataset using PhyloSuite [28]. For ML analyses, statistical support was calculated with 1000 bootstrap replicates. For BI analyses, we used the GTR+G+I model, and three chains were run for 10 million generations, sampling trees every 100 generations.

Phylogenetic Analyses
The combined dataset included 125 ITS and 85 LSU sequences. A total of 60 new sequences were generated in this study, including 12 ITS and 11 LSU sequences of the new taxa (Table 1). The final dataset included 1470 sites, with 605 from ITS and 865 from LSU. The results from RAxML and Bayesian analyses were almost topologically congruent, so only the phylogenetic tree inferred from the ML analyses is shown (Figure 1).  Based on the phylogenetic analyses of the combined dataset, the genera Leucoagaricus, Leucocoprinus and Micropsalliota together form a monophyletic clade (BS = 100%, PP = 1.0), consistent with the results of previous studies [10,11,[19][20][21]. However, members of the /leucoagaricus clade (Leucoagaricus sensu stricto) and /sericeomyces clade (with the type species of Subgen. Sericeomyces nested within) do not mix with members of Leucocoprinus or Micropsalliota (Figure 1). Together, the /leucoagaricus clade and the /sericeomyces clade form a strongly supported monophyletic clade (BS = 91%, PP = 0.95). Among the five new species, Leucoagaricus albosquamosus, Leucoagaricus atroviridis and Leucoagaricus centricastaneus are nested within the /leucoagaricus clade, and each species had strong bootstrap supports on its own (both BS = 100%, PP = 1.0). Leucoagaricus aurantioruber and Leucoagaricus candidus are nested in the /sericeomyces clade (Figure 1 consistent with the results of previous studies [10,11,[19][20][21]. However, memb /leucoagaricus clade (Leucoagaricus sensu stricto) and /sericeomyces clade (wit species of Subgen. Sericeomyces nested within) do not mix with members of Leu or Micropsalliota (Figure 1). Together, the /leucoagaricus clade and the /sericeom form a strongly supported monophyletic clade (BS = 91%, PP = 0.95). Among th species, Leucoagaricus albosquamosus, Leucoagaricus atroviridis and Leucoagaricus c neus are nested within the /leucoagaricus clade, and each species had strong supports on its own (both BS = 100%, PP = 1.0). Leucoagaricus aurantioruber and L cus candidus are nested in the /sericeomyces clade (Figure 1), in which Leucoag rantioruber formed a distinct clade (BS = 100%, PP = 1.0) sister to Leucoagaricus neus, whereas the two collections of Leucoagaricus candidus were clustered toge strong bootstrap supports (BS = 100%, PP = 1.0), forming a clade close to an un Leucoagaricus species from Italy. The other species, Leucoagaricus virens, repre two collections that clustered together with strong bootstrap supports (BS = 1 1.0), is close to Leucoagaricus viriditinctus (Berk. & Broome) J.F. Liang, Zhu L. Ya and Leucoagaricus irinellus Chalange, forming a clade that does not fit in any know ( Figure 1).      Comments: The somewhat plicate pileus of La. albosquamosus may suggest that this species be placed in Leucocoprinus. Considering La. albosquamosus is nested within a strongly supported clade that corresponds to section Leucoagaricus, we placed this species within Leucoagaricus rather than Leucocoprinus. According to phylogenetic analysis (Figure 1), Leucoagaricus albosquamosus is a sister taxon to Leucoagaricus nivalis (W.F. Chiu) Z. W. Ge & Zhu L. Yang. Together, these two taxa form a sister clade to a clade jointly formed by Leucoagaricus umbonatus S. Hussain, H. Ahmad & Afshan and an undescribed species of Leucoagaricus [19,30,31].
Leucoagaricus sericifer (Locq.) Vellinga is also similar to La. albosquamosus in its white pileus and the shape of the basidiospores. However, La. sericifer has oblong amygdaliform to cylindrical amygdaliform basidiospores, as well as more or less lageniform cheilocystidia [6]. Considering the somewhat plicate pileus with squamules, La. albosquamosus is somewhat similar to Leucocoprinus cepistipes (Sowerby) Pat. However, Leucocoprinus cepistipes has small beige-brown to ochraceous brown squamules and lageniform to clavate cheilocystidia, often with apical excrescence [6].  Comments: The somewhat plicate pileus of La. albosquamosus may suggest that this species be placed in Leucocoprinus. Considering La. albosquamosus is nested within a strongly supported clade that corresponds to section Leucoagaricus, we placed this species within Leucoagaricus rather than Leucocoprinus. According to phylogenetic analysis ( Figure  1), Leucoagaricus albosquamosus is a sister taxon to Leucoagaricus nivalis (W.F. Chiu) Z. W. Ge & Zhu L. Yang. Together, these two taxa form a sister clade to a clade jointly formed by Leucoagaricus umbonatus S. Hussain, H. Ahmad & Afshan and an undescribed species of Leucoagaricus [19,30,31].
Leucoagaricus sericifer (Locq.) Vellinga is also similar to La. albosquamosus in its white pileus and the shape of the basidiospores. However, La. sericifer has oblong amygdaliform to cylindrical amygdaliform basidiospores, as well as more or less lageniform cheilocystidia [6]. Considering the somewhat plicate pileus with squamules, La. albosquamosus is somewhat similar to Leucocoprinus cepestipes (Sowerby) Pat. However, Leucocoprinus cepestipes has small beige-brown to ochraceous brown squamules and lageniform to clavate cheilocystidia, often with apical excrescence [6].    Comments: Macroscopically, Leucoagaricus atroviridis is characterized by medium to large basidiomata, stipe with a bulbous base and bottle-green to dark brown fibrils.
Phylogenetically, Leucoagaricus atroviridis is close to Leucoagaricus truncatus Z. W. Ge & Zhu L. Yang and Leucoagaricus purpureolilacinus Huijsman (Figure 1). However, La. purpureolilacinus, originally described in the Netherlands, has a purplish brown pileus and clavate to subfusiform cheilocystidia [6]. La. truncatus, originally described in Sichuan, southwestern China, differs in having a truncated pileus with orange-white to gray-orange squamules and larger basidiospores [11]. Comments: Macroscopically, Leucoagaricus atroviridis is characterized by medium to large basidiomata, stipe with a bulbous base and bottle-green to dark brown fibrils.
Leucoagaricus rubroconfusus Migl. & Coccia resembles La. aurantioruber in having orange fibrillose squamules and ellipsoid to ovoid basidiospores of approximately the same length. However, La. rubroconfusus has smaller cheilocystidia without crystals on the apex.
The new species is also similar to La. japonicus (Kawam. ex Hongo) Hongo in having ellipsoid or ovoid basidiospores. However, La. japonicus has testaceous, scaly pileus; longer basidiospores; and clavate to fusiform cheilocystidia without crystals [5,32,34]. ange fibrillose squamules and ellipsoid to ovoid basidiospores of approximately the same length. However, La. rubroconfusus has smaller cheilocystidia without crystals on the apex.
Two white European species, La. subvolvatus (Malencon & Bertault) Bon and La. menieri (Sacc.) Singer, are also similar to La. candidus in the overall white basidiomata and similar basidiospores. However, La. subvolvatus has white to cream pileus and cheilocystidia with crystals at the apex [32]; La. menieri has fragile and sericeous pileus and cheilocystidia with crystals at the apex [32]. Comments: Leucoagaricus candidus is morphologically similar to Leucoagaricus nivalis, Leucoagaricus serenus (Fr.) Bon & Boiffard and Leucoagaricus sericifer on the overall whitish to white basidiomata. However, La. candidus differs from them by having ellipsoid to broadly ellipsoid basidiospores and a pileus surface composed of wider hyphae (5.0-13.0 μm). In addition, both La. serenus and La. sericifer were originally described in Europe and are widespread in Europe [6,31,35].

Discussion
Based on morphological and molecular data, in this study, we described six new species of Leucoagaricus from northeastern China, with three species in section Leucoagaricus, two in subgenus Sericeomyces and one within a clade not belonging to any currently recognized section. The taxonomy of Leucoagaricus has been a focus worldwide in recent years, with many new taxa described around the world [11,[19][20][21]37,39,40]. However, as shown in Figure 1, there are putative new species to be described that are represented by a single collection (e.g., HKAS 123028, HKAS 105542, HKAS 123030 and HKAS 116134). With additional field work carried out and additional collections documented, more new taxa are expected to be described.