Agaricus macrochlamys, a New Species from the (Sub)tropical Cloud Forests of North America and the Caribbean, and Agaricus fiardii, a New Synonym of Agaricus subrufescens

Agaricus is a genus of fungi in the family Agaricaceae, with several highly priced edible and medicinal species. Here we describe Agaricus macrochlamys, a new species, in A. sect. Arvenses, sympatric and morphologically cryptic with the edible and medicinally cultivated mushroom, A. subrufescens. Phylogenetic analyses showed that A. macrochlamys is closely related to A. subrufescens, and that A. fiardii is a new synonym of A. subrufescens. Despite being morphologically cryptic, A. macrochlamys can be distinguished from A. subrufescens by several ITS and tef1α species-specific markers and a 4-bp insertion in the tef1α sequence. Furthermore, A. subrufescens is a cosmopolitan species, while A. macrochlamys distribution is so far restricted to Mexico, the Dominican Republic, and the United States.


Introduction
Agaricus L. (Agaricaceae, Basidiomycota) includes saprotrophic mushrooms of medicinal, nutraceutical and nutritional interest. The genus has a worldwide distribution comprising around 580 species [1][2][3][4][5][6]. According to Zhao et al. [7], the diversity of the genus Agaricus is clearly underestimated. Since 2000, taxonomic studies of the genus Agaricus have been very scarce in Africa, South America and Oceania, where only 17 new species have been described.
Despite its ecological and economic importance, this genus has scarcely been studied in Mexico and the Dominican Republic, unlike in the United States. Mata et al. [8] reported 32 species for Mexico; Medel et al. [9] concluded that in Mexico tropical cloud forest is  Table 1. The ITS dataset included 66 sequences, of which 24 sequences were newly generated from this study: 49 sequences of A. subrufescens from different geographical origins and bearing different ITS types, and 17 sequences of A. macrochlamys were used. Table 1. GenBank accession numbers and specimens used in the phylogenetic analyses. New species, new specimens, and new sequences are in bold. T = Type specimen. * CA1110 is the strain isolation of the holotype. ** A. fiardii, a new synonym of A. subrufescens introduced in this study.

Phylogenetic Analyses
The sequences were aligned by using MAFFT [32] for each region independently, then manually adjusted in BioEdit v. 7.0.4 [33]. Two datasets were prepared for different analyses.
The combined dataset was used for maximum likelihood and Bayesian analyses. The maximum likelihood (ML) analysis was performed in RAxMLHPC2 v. 8.2.4 [34], as implemented on the Cipres portal [35], under a GTRGAMMA model with one thousand rapid bootstrap (BS) replicates. The Bayesian inference (BI) analysis was performed with MrBayes v. 3.1.2 [36], under a partitioned model. The combined dataset was partitioned into ITS, tef1 intron and tef1 coding sites. The best substitution model for each partition was inferred with the program MrModeltest 2.2 [37]: HKY + I + G for ITS; GTR for tef1 intron sites; and SYM + I + G for tef1 coding sites. Two runs of six Markov chains were run for one million generations and sampled every 100th generations. Burn-in was determined by checking the likelihood trace plots in Tracer v. 1.6 [38] and subsequently discarded. The outputs were displayed in FigTree v 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 8 April 2022)).
The polymorphisms in the ITS region were found to be useful to characterize the species and varieties within Agaricus [31,39,40]; therefore, we used them to characterize the new collections made in this study. An unusual intraspecific variability is known in A. subrufescens. The different ITS types (A, B or C) were identified using the polymorphisms at nine ITS positions, as described by Chen et al. [17]. Briefly, types A and B, which differ at six polymorphic positions from each other (positions 39, 122, 130, 145, 146 and 200 in Table 2) are known in the samples from the Americas and Europe. Type C, which differs at three positions from A or B (positions 269, 466, and 475 in Table 2) are known from Hawaii and Southeastern Asia. For the heteromorphic sequences at these nine positions, haploid sequences were obtained by three different methods: (i) sequencing of homokaryotic spores for WC837 [16], which is of type AB; (ii) by PCR cloning for CA487, which is of type ABC [17]; or (iii), for CA864, by interpreting the two sequences which are superimposed with an offset in the electropherograms due to a length polymorphism [17]. The latter method was applied in the present study to the Mexican strain CA603, and revealed that it was an AB type, as in the French sample CA864 ( Table 2). The GenBank accession numbers that included the sequences of 57 heterokaryotic samples and nine haploid sequences are indicated in Table 2.
The ITS dataset was used for the unweighted pair group method with arithmetic mean (UPGMA) analysis. A UPGMA tree was constructed using MEGA11 [41] with default setting.
Lowercase letter in an ITS type indicates that the characteristic polymorphisms of the type are not all present. The parenthesis indicates that the haploid ITS sequences of these heterokaryons are unknown. 1 Haploid sequences, not deposited individually in GenBank, obtained by interpreting the two sequences switched in the heteromorphic electropherograms; 2 Haploid sequences of each type obtained by sequencing the clones of the heterokaryotic strain, only the haploid sequences are deposited in GenBank. Bold letters denote unique nucleotide substitutions for each ITS type. 'Type' as recorded here specifically refers to the genotype of the culture, which in heterokaryons will typically be comprised of two allelic copies (or rarely three) which may be homoallelic or heteroallelic, with each allele having its own molecular 'type' designated A, B, C or M.

Species-Specific ITS Markers
Comparisons were made between the 104 sequences (ITS1 + 5.8S + ITS2) of A. sect. Arvenses used for the phylogenetic analyses. The position of a unique nucleotide (nt) in the ITS sequence of a species is indicated as follows: "xxxxxXxxxxx @ position", where the capital letter represents the exclusive or informative character, and xxxxx represents the flanking characters. Square brackets were used for insertion or deletion (indel). It must be noted that the comparisons are made with all species of the section for which sequences are currently available; however, with more taxa, reassessment might be needed.

Phylogenetic Analyses
The final alignment of the combined dataset included sequences of 105 specimens and 1192 characters. The phylogenetic trees generated by the ML and BI methods were very similar in topologies. The maximum likelihood (ML) tree is shown in Figure 1. Agaricus sect. Arvenses is monophyletic and four major clades are recovered, as in Cao et al. [5], and except for clade I, which is poorly supported, the remaining clades are moderately (clade II) to well-supported (clade III and clade IV). The 24 new collections are clustered in clade IV: seven of them joined the subclade of A. subrufescens, including the type specimen of A. fiardii; the remaining 17 formed a well-supported sister clade to A. subrufescens, which represents the new species described in our taxonomic treatment.

ITS Types and Characteristic Polymorphisms
The ITS alignment of 66 sequences of A. subrufescens and the newly described species revealed 14 polymorphic positions, which are informative to distinguish between the different ITS types. The characteristic polymorphisms of three types of ITS (A, B and C) within A. subrufescens are shown in Table 2. The five samples (LAPAM77, LAPAM78, LAPAM100, LAPAM101 and LAPAM103) from the Dominican Republic have typical ITS type A; and the three Mexican samples have typical ITS type B (IE913) and AB (CA603 and LD201903). Additionally, 17 collections of the newly described species differ from all of the ITS sequences of A. subrufescens at five polymorphic positions (166, 196, 210, 494 and 518) and were unique to the newly described taxon (Table 2).
In addition to the 14 polymorphic positions characteristic of different ITS types showed in Table 2, there were 46 other polymorphic positions in the alignment. The variant alleles at these positions were mostly found in a single sample. In rare cases, where they were found in two or at most three samples, they always had the same ITS type.
In order to have a better representation of the relationships between these different ITS types, a UPGMA tree was constructed (Figure 2). The results showed that the different ITS types were clustered into two groups: the new species bearing the new ITS type M belonged to a distinct lineage, and geographically was restricted to North America; another group clustered samples of A. subrufescens with the ITS types A, B and C, or their combinations.
The sequences with ITS types A, B or AB were mainly found in the Americas or in Europe, except two samples that were from China, and sequences of the ITS type C were found in Hawaii or Asia. In the tree, three samples (CA918, NTF67 and CA487-c6) were not clustered according to their ITS types (per the assignments in Table 2), due to the presence of variant characters other than the characteristic polymorphic positions.

Phylogenetic Analyses
The final alignment of the combined dataset included sequences of 105 specimens and 1192 characters. The phylogenetic trees generated by the ML and BI methods were very similar in topologies. The maximum likelihood (ML) tree is shown in Figure 1. Agaricus sect. Arvenses is monophyletic and four major clades are recovered, as in Cao et al. [5], and except for clade I, which is poorly supported, the remaining clades are moderately (clade II) to well-supported (clade III and clade IV). The 24 new collections are clustered in clade IV: seven of them joined the subclade of A. subrufescens, including the type specimen of A. fiardii; the remaining 17 formed a well-supported sister clade to A. subrufescens, which represents the new species described in our taxonomic treatment.

ITS Types and Characteristic Polymorphisms
The ITS alignment of 66 sequences of A. subrufescens and the newly described other group clustered samples of A. subrufescens with the ITS types A, B and C, or the combinations. The sequences with ITS types A, B or AB were mainly found in the Amer cas or in Europe, except two samples that were from China, and sequences of the ITS typ C were found in Hawaii or Asia. In the tree, three samples (CA918, NTF67 and CA487-c were not clustered according to their ITS types (per the assignments in Table 2), due the presence of variant characters other than the characteristic polymorphic positions.  Etymology: the name is derived from classical Greek μακρός (macros) meaning large, and χλᾰμῠς (chlamys) meaning dress or mantle, referring to the ample annulus close to the stipe apex.      Macroscopic description (Figures 3 and 5): Pileus 3.9-13.7 cm diam., truncate-convex when young, then hemispherical to plano-convex, finally plane at maturity, usually with a slightly depressed center (never with prominent umbo); surface dull and dry, very variable in appearance and colors, mostly completely covered with brownish fibrils when  Macroscopic description (Figures 3 and 5): Pileus 3.9-13.7 cm diam., truncate-convex when young, then hemispherical to plano-convex, finally plane at maturity, usually with a slightly depressed center (never with prominent umbo); surface dull and dry, very variable in appearance and colors, mostly completely covered with brownish fibrils when young, becoming to fibrillose, slightly reddish or hazelnut-brown or violet-brown squamules over a white, grayish or beige background, denser at the disc, forming an entire brown to dark brown center, less towards the margin; in other cases, also completely white, whitish or very light cream with only the disc colored brown to dark brown; in these collections with pale or whitish pileus, the surface appears smooth or slightly fibrillose, in some collections, somewhat rough, never scaly. Margin always exceeding the lamellae. Lamellae free, crowed, up to 1 cm broad, intercalated with numerous lamellulae, white to pinkish when young, finally dark brown at maturity, with finely denticulate whitish edge. Stipe 4.6-14.9 × 0.5-1.6 cm, cylindrical, sometimes curved with bulbous to sub-bulbous base (1.5-3.5 cm wide), fistulose, with an annulus in its upper third, above the annulus smooth and below the annulus whitish or slightly rosaceous below, at first pruinose then smooth, with a pure white evanescent flocculosity towards the base, slightly yellowing on handling, sometimes with short white rhizomorphs at the base. Annulus attached to the upper part of the stipe, superous, pendent, very broad, fragile, membranous to fibrillose-araneose, double, upper surface white smooth to subtly striate radially, lower surface covered by 0.2-0.4 cm floccose-squamulose white or light brownish square patches (thicker toward the pileus margin, conspicuous in younger specimens). Context white, firm, when cut unchanging or discoloring slightly yellowish. Odor of almond or anise. Spore print dark brown.
Habit, habitat, occurrence and distribution: Solitary or gregarious, usually among the leaf litter in (sub)tropical broadleaf cloud forests, only one collection among pine needle Etymology: from the Latin compound subrufescens meaning slightly (sub-) reddening (rufescens), it should presumably refer to the pileus "varying in color from white to grayish to dull reddish-brown", described by Peck in the original description as etymology is not explained by this author.

Note:
The following morphological description is based on some unpublished collections of A. subrufescens sympatric with A. macrochlamys from Mexico and the Dominican Republic. Other sympatric collections of A. subrufescens with A. macrochlamys were also described previously in Parra et al. [11].
Macroscopic description ( Figure 7): Pileus 5.3-11 cm diam., conical, then hemispherical or trapezoid, finally plano-convex, sometimes depressed at the disc, background whitish covered by fine hazelnut-brown, reddish-brown, brown-violet fibrils becoming squamules toward the disk, forming an entire densely reddish-brown center. Surface dull and dry, slightly yellowing on handling. Margin exceeding the lamellae, even in mature basidiomata. Lamellae free, crowded, straight, 0.6-0.8 cm broad, intercalated with numerous lamellulae, at first white (pinkish on handling, resembling a Leucoagaricus sp.), then pale pink for a long time, finally dark brown in mature basidiomata, with even and concolors or finely denticulate edge. Stipe 9-16 × 0.7-1.4 cm, cylindrical, sometimes curved at the base, enlarged, bulbous or abruptly marginate bulbous at the base (1.6-2.6 cm), fistulose, with an annulus in its upper third; above annulus white and smooth, below annulus white, pruinose when young, frequently covered by a white floccosity particularly towards the base; slightly yellowing on handling. At times with a single thick rhizomorph at the base. Annulus superous, double, white, fragile and fine, upper surface smooth to fibrillose-cobweb, lower surface covered by white or ochre floccose to wooly-floccose radially arranged square patches. Context in pileus and stipe firm, when cut white, sometimes slightly yellowing, with strong odor of anise.
However, A. macrochlamys shows significant differences with A. subrufescens in the ITS (five positions) and tef1α regions (six positions and a 4-bps insertion), despite the fact Notes: From a morphological point of view, it was not possible to find any macroscopic or microscopic differences among A. macrochlamys, A. fiardii and A. subrufescens. Macroscopically, the three species are highly variable in the color of the pileus surface, ranging from white to purplish brown. The Caribbean collections with the white pileus of A. subrufescens were described in Parra et al. [11] for the Dominican Republic occurring at the same localities where A. macrochlamys was also collected, and were also described in A. fiardii by Pegler [18] at varietal rank as A. fiardii var. exalbidus Pegler, which only differs from the autonymic variety by "the white pileal surface". Collections of A. subrufescens having a white pileus surface have also been published in the United States [14], and the specimen of A. macrochlamys from Georgia (USA) included in this study also has a white pileus surface. The annulus is also variable in structure and consistency, varying from fibrillose-araneous and very fragile to membranous and more persistent, but always with the characteristic small floccose to woolly-floccose squamules covering the whole lower surface. Microscopically, in our microscopic examination of the type of A. fiardii (F353, K(M) 234316; Figure 9) the following measurements were obtained: Basidiospores 5-5.6-6.1 × 3.6-3.8-4 µm, Q = 1.39-1.47-1.55; basidia 15-25 × 6-9 µm with sterigmata up to 2.5 µm long; and cheilocystidia in short chains of two-four globose or ellipsoid elements 6.5-15 × 6-12 µm. Therefore, all of the available microscopic characters are very similar and overlap widely in the three species, and no relevant distinguishing microscopic characters were noticed among the three species.
For all of the above, the samples from the collections of the three species were sequenced to compare their molecular characters. We observed that the ITS sequence of the type specimen of A. fiardii could not be differentiated from A. subrufescens, and the former was therefore included under the synonymy of A. subrufescens in this work.
However, A. macrochlamys shows significant differences with A. subrufescens in the ITS (five positions) and tef1α regions (six positions and a 4-bps insertion), despite the fact that both species are sympatric in Mexico and the Dominican Republic and the unusual intraspecific variability in A. subrufescens. Within A. sect. Arvenses, A. macrochlamys has four species-specific ITS markers: tgtgaGagctt @ 172; atggaGtctc[-] @ 202; ttggaAtgtgg @ 502; ctt[-]tGyggkg @ 528. No specimens having allelic ITS sequences characteristic of both A. subrufescens and A. macrochlamys are known, strongly implying that they are reproductively isolated, as well as being phylogenetically distinct.

Discussion
The molecular data from twenty-four samples that were morphologically highly similar were identified as follows: two and five samples from Mexico and the Dominican Republic, respectively, belong to A. subrufescens; the remaining samples from Mexico (10), the Dominican Republic (6) and the USA (1) belong to the new species A. macrochlamys. In addition, A. fiardii is now considered a new synonym of A. subrufescens, based on the molecular phylogenetic analyses presented in this paper.
According to our combined phylogenetic analyses, the new species A. macrochlamys formed a full-supported sister clade to A. subrufescens. Because no significant morphological differences were observed between the two species, the molecular data remain essential for species identification. Indeed, in comparison with A. subrufescens, the new species has five distinguishing ITS nucleotide substitutions, of which four are unique within the A. sect. Arvenses. This level of genetic divergence is higher than is generally observed between the closely related species in the genus [42].
The biology of Agaricus macrochlamys remains to be studied in detail. Its geographical range in Mexico and the Dominican Republic seems to be restricted to cloud forests (an ecosystem characterized by temperate and tropical vegetation elements with a high rainfall and subtropical weather). However, the ARB1353 sample from Georgia, USA, was collected in an area with subtropical weather. The life cycles of A. macrochlamys need to be compared with those of A. subrufescens, which is known to be amphithallic (i.e., both heterothallic and pseudo-homothallic) and versatile [16,43]. Other important questions to be solved are whether this easily cultivated taxon indeed possesses culinary and/or medicinal or nutraceutical value.