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Sylvan Research, 198 Nolte Drive, Kittanning, Pennsylvania 16201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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Agaricus subrufescens Peck was cultivated first in the late 1800s in eastern North America. The type consists partly of cultivated material and partly of field-collected specimens. Once a popular market mushroom, the species faded from commerce in the early 20th century. More recently, a mushroom species growing wild in Brazil has been introduced into cultivation in Brazil, Japan and elsewhere. This Brazilian mushroom has been referred to by various names, most commonly as A. blazei Murrill (sensu Heinemann) and most recently as A. brasiliensis Wasser et al. The author first cultivated A. subrufescens in 1981 and has grown and studied Brazilian isolates since 1992. The species has an amphithallic pattern of reproduction. Based on DNA sequence analysis of the rDNA ITS region and on mating studies and genetic analysis of hybrid progeny, there is a strong case for conspecificity of the Brazilian mushrooms with A. subrufescens. Based on a study of the type and other data, the recent lectotypification of A. subrufescens is accepted. Data are presented on mushrooms of diverse geographical origins, including A. rufotegulis Nauta from western Europe, another apparent con-specific. A possible role for interpopulational hybridization in current populations of A. subrufescens is proposed. The agronomic history of the species is reviewed.
Key words: ABM, Cogumelo do Sol, Himematsutake, interpopulational hybridization, phylogeography, Royal Sun Agaricus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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; Falconer to Peck, in lit. [NYS]). The mushroom, called the "almond mushroom" or "almond-flavored mushroom" due to its fragrance and taste, was widely cultivated, sold and eaten in the Atlantic states of the United States from at least Massachusetts to Washington D.C., from the late 19th century into the 20th (Falconer 1894a, b; Anon. 1904; Anonymous 1909; Kauffman 1918). Spawn (inoculum culture for farming) of A. subrufescens was even offered for sale (Falconer 1894a, b; Anonymous 1909). As late as 1918 Kauffman (1918) reported it to be in cultivation. Commercial production of A. subrufescens subsequently declined as market trends changed; soon the related "button mushroom" species Agaricus bisporus (J.E. Lange) Imbach appears to have been the only mushroom species being regularly cultivated in the United States (see Duggar 1920, Charles 1931).
Agaricus subrufescens often occurs in domesticated or semidisturbed habitats, including leaf piles. It has been recognized occasionally growing "wild" outside northeastern North America, for example in California (Kerrigan 1982, 1983a, 1986; Kerrigan and Ross 1987a), Israel (R. Kenneth, personal communication 1984–85), Taiwan (Tu and Lin 1981) and Hawaii, where it grows under forest trees (Peterson et al 2000). Brazilian examples in the past three decades have entered commerce and (not having been recognized as A. subrufescens) raised questions, discussed below, about nomenclature and identity. In addition, the recently described A. rufotegulis Nauta from the Netherlands, the United Kingdom and Portugal (Nauta 1999, Hausknecht 2002) is also extremely similar and considered here to be conspecific with A. subrufescens.
The history of A. subrufescens was reviewed by Kerrigan (1983a); updated and extended information follows. A discussion of its properties as an easily cultivated mushroom was presented by Kerrigan (1983a, 1984); notably, very similar outdoor methods are typically employed in Brazil today. A culture (RWK 1185; voucher at SFSU) of A. subrufescens was isolated by me from basidiomata growing in rich compost covered with sandy soil for raspberry culture in California in 1981. This culture was sold commercially to hobbyist mushroom growers (it recently has been cultivated at commercial scale in the United States and, based on sequence and other data, abroad (see below)). Reproductive micromorphology and genetic behavior of this strain were investigated by Kerrigan and Ross (1987a, b, unpublished).
Mushrooms originating in the Atlantic region of Brazil and agreeing closely with Agaricus subrufescens have begun in recent decades to be cultivated on a broad scale as "medicinal mushrooms" that are marketed primarily in Japan (Kerrigan 1983a, Wasser et al 2002). However, in this context the mushroom typically is referred to (incorrectly) as A. blazei Murrill, "A. blazei ss Heinem." (see Wasser et al [2002]) or, in some commercial literature and product packaging, "A. sylvaticus".
The conventional account of the origin of ‘Agaricus blazei Murrill sensu Heinemann’ and its arrival in Japan is related by Wasser et al (2002). It is consistent with the account given to me by Mr Shusuke Minoura in Hiroshima, Japan, in 1981. His account and my conclusion that the Brazilian Agaricus then cultivated in Japan was "almost certainly" A. subrufescens were published shortly thereafter with one of Minoura’s photographs (Kerrigan 1983a). The conventional history also agrees with a specific and plausible account by Mr. B.-A. Eckart (personal communication) of Brazil and Germany, related to him by Mr Ernesto Noburo, which attributes the discovery of the Brazilian mushroom and its distribution to Japanese researchers to the late Mr Takatoshi Furumoto, an immigrant from Japan to Piedade, Brazil. Details in the report of Heinemann (1993) indicating that a culture was isolated from a collection made at São Paulo, Piedade, Brazil, in Feb 1973, and cultivated in Japan by Iwade, from which specimens (at BR) were preserved by Hongo, are also concordant. See also Souza Dias et al (2004).
Controversy has existed regarding the correct name of the Brazilian species. A search of the World Wide Web and a review of diverse commercial product literature indicated that association of the name A. blazei with the Brazilian mushroom is attributed to P. Heinemann. Minoura used this name in 1981; the published taxonomic determination appears in Heinemann (1993). The name A. sylvaticus Schaeff. sometimes is associated with the species, and this is said to have resulted from a determination of Brazilian cultivated material made by a European mycologist at the request of a Brazilian producer (R. Maziero personal communication). Agaricus sylvaticus customarily is placed in section Sanguinolenti (F.H. Møller & Jul. Schäffer) Singer, while A. subrufescens (under any name and including Brazilian material) is certainly a member of section Arvenses Konrad & Maubl. (Kerrigan 1982, 1986).
Wasser et al (2002) rejected the name A. blazei for the Brazilian mushroom, correctly in this author’s opinion, and recognized the latter as a new species, A. brasiliensis Wasser et al. Wasser et al also rejected conspecificity between Brazilian mushrooms and A. subrufescens, based on features of spores and cheilocystidia, although they wrote that the two species seemed to be each other’s closest relatives.
However, new data presented below indicate that the medicinal mushroom from Brazil and Japan is biologically and phylogenetically the same species as A. subrufescens from North America. I will review features that have been emphasized in the literature on the taxa involved. Because Peck’s name A. subrufescens is older than A. brasiliensis, it has priority and therefore is the correct name of the species. A. rufotegulis Nauta equally belongs to A. subrufescens, based on overall morphological and sequence criteria. The rDNA ITS1 + 2 sequences from Hawaiian specimens of A. subrufescens are the most divergent of those studied.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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, Kerrigan et al 1996). Samples evaluated are given (TABLE I). For microscopy, excised dried material was wetted in 95% ethanol, then mounted and measured in 3% KOH; spore length excludes the apiculus. Cultures of commercial samples of "medicinal agaricus" of Chinese and Japanese origin were obtained from spores aseptically washed from lamellae of dried mushrooms, in the form of single spore isolates (SSIs). The karyotic status (n versus n + n) of each SSI was determined from potato-dextrose yeast broth (PDYB) grown samples using allozyme markers including peptidase and esterase. Heteroallelic SSIs were assumed to be heterokaryotic, while homoallelic SSIs were of uncertain status. Crosses were attempted between homoallelic SSIs on PDA, transferred to grain spawn medium, then compost (for cropping); successful hybrid cultures were isolated and cultured in PDYB for genetic analysis.
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).
Amplification of the ITS1 + 2 region of the rDNA, sequencing of the products and subsequent alignment and analysis was done as described in Challen et al 2003. Normally primers ITS1 and ITS4 (White et al 1990) were used to produce a full-length ITS1 + 2 PCR product; however, some herbarium material provided DNA that only produced shorter PCR products, using primers ITS1 and ITS2, or ITS3 and ITS4. In this project it was routine in sequencing full-length products to use internal primers ITS2 and ITS3 in addition to terminal primers ITS1 and ITS4 due to length heterogeneity which often was present in both ITS segments 1 and 2 in this species. The sequence analyzed begins with ggaaggat in the 18S gene and ends with gaacttaa in the 25–28S gene. Sequencing was performed on the most current equipment available at the time at either the Pennsylvania State University or at the University of Pittsburgh. Output was ABI trace files; these were inspected, corrected and assembled using the Seqman module of the Lasergene version 5 package (Dnastar). A sequence from A. urinascens ( Jul. Scháffer & F.H. Møller) Singer (= A. macrosporus [F.H. Møller & Jul. Schäffer] Pilát, nom. illeg.; non A. macrosporus Mont. [1837], per Nauta [2000]) (GenBank AF432878
[GenBank]
) was used as outgroup. Alignment was done using the Clustal W algorithm of the Megalign module of Lasergene, followed by inspection and manual correction. All sequences comprise data from both strands, although in the 18S, 5.8S and 25–28S genes short regions of single strand data are present. A few 5.8S sequences (which are almost invariant within Agaricus) are incomplete. Sequences were deposited in GenBank (AY818646
[GenBank]
-AY818660).
It was apparent that the distance matrix generated from the alignment by Megalign was treating heteromorphic characters as ambiguous data, which then were excluded from similarity calculations. The DNADIST program of PHYLIP also ignores heteromorphic data. Consequently, I manually constructed a pairwise distance matrix in which each pair of identical characters was scored as 0.0, each pair of fully dissimilar characters was scored, as 1/n, where n = character positions scored, and a pair consisting of one heteromorphic character and one of its constituent characters was scored as 0.5/n. Both character and length heteromorphisms were scored in this way. Total pairwise distance was the sum of scores over all (nominally 711) positions. The matrix file was evaluated using both FITCH and the UPGMA method in NEIGHBOR (PHYLIP; Felsenstein 2004) to produce a graphical representation of similarity, rather than a phylogenetic hypothesis, because the presence of numerous heteromorphisms and the hypothesis of population-level hybridization in this species (see below) are not compatible with the usual assumptions of character state evolution and radiating phylogenetic lineages.
To evaluate the phylogenetic unity of A. subrufescens and putatively conspecific taxa, the sequences described above were compared to others of species in several sections of Agaricus, primarily section Arvenses. Sequences from GenBank included AB113576
[GenBank]
.1, AF161013
[GenBank]
, AJ131126
[GenBank]
.1 and AY484697
[GenBank]
.1 (as A. blazei, deposited by four research groups), AY484671
[GenBank]
.1, AY474672.1 (as A. augustus), AY484690
[GenBank]
.1 (as A. arvensis), AF482834
[GenBank]
.1 (as E. depressum, = A. inapertus), AY484670
[GenBank]
.1 (as A. nivescens), AY 484686.1 (as A. macrocarpus), and AY484675
[GenBank]
.1 (as A. albolutescens), all in section Arvenses. Names associated with GenBank deposits were accepted provisionally. Other sections were represented as follows: Agaricus: A. campestris: W1H (M. Challen, HRI: includes AJ418775
[GenBank]
); Xanthodermatei: A. xanthodermus W3I (M. Challen, HRI: includes AJ418776
[GenBank]
); Duploannulati: A. bisporus RWK 1885 (AF432886
[GenBank]
); Sanguinolenti: A. pattersonae RWK 1415 (includes AJ418715
[GenBank]
); other: A. subrutilescens (in or near section Spissicaules) RWK 1940. The alignment file was prepared as described above and was evaluated under maximum parsimony using PAUP* version 4.0b8 (Swofford 2000).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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). Segregation of alleles in offspring occurred at the PEP1 and PEP2 loci (Kerrigan et al 1996, Royse and May 1982) in progeny of I-101 and at an esterase locus in SBRFG. This demonstrates that meiosis, recombination and partitioning of recombinant nuclei into spores is occurring in these isolates of A. subrufescens, therefore the species is not homothallic. Furthermore, some SSIs had heteroallelic genotypes, proving that multiple nuclei were present in heterokaryotic spores, while other spores were homoallelic, implying that they could be homokaryotic. All these observations are consistent with the presence of a basic system of amphithallic reproduction in which both uniparental reproduction (via intramixis) and outcrossing (heteromixis) is possible. The tendency of at least some strains of A. subrufescens to produce substantial numbers of bi- and tri-sporic basidia under some conditions also is consistent with the presence of an amphithallic life cycle (Kerrigan and Ross 1987a, see also comments in Heinemann 1993).
An interesting property of SSIs of SBRFG is that they varied with respect to reproductive ability (as expected among amphithallic offspring). Although the SBRFG parent had a pigmented pileus, some fertile SSI offspring produced basidiomata with white pilei. The latter observation could be explained by the presence of a Mendelian determinant for pileus color located at sufficient distance from the centro-mere to allow frequent crossing over, and the presence of one recessive allele in the parent, leading to a minority of homoallelic recessive heterokaryotic offspring (cf. Kerrigan et al 1993). These observations were first noted in work at UCSB in 1986 (Kerrigan and Ross unpublished). Some SSIs of I-101 (e.g., -s1) also were fertile.
Interfertility and hybrid analysis.— – All tested SSIs from the BS1 (Brazilian commercial) and CS4 (Chinese commercial) samples were heteroallelic in allozyme analysis, so no crosses from these SSIs were attempted. Homoallelic SSIs from the I-101 and SBRFG stocks were selected to be the progenitors of a series of hybrids. Hybrid cultures were isolated for each pairing. The hybrid between SBRFG-s1 and I-101-s1, called H1X1, will serve as an example.
The hybrid status of the putative new hybrids was verified by allozyme analysis. In the case of the hybrid H1X1, the progenitor I-101-s1 carries allele Pep2-s, while progenitor SBRFG-s1 carries allele Pep2-f. Both SSIs are fertile, therefore either or both might be heterokaryotic. The hybrid H1x1 between these two SSIs has the expected genotype Pep2-s/f, demonstrating that this isolate incorporates DNA from each parent. The simplest and most conventional explanation is that H1x1 received one nucleus from I-101-s1 and another nucleus from SBRFG-s1.
A set of 25 A. subrufescens hybrids between parents I-101 and SBRFG were grown on small containers of compost under standard conditions, with the SBRFG parent also present as a control. The phenotype of H1x1 serves as an example of how morphological and cultural traits may be inherited from the progenitors of such a hybrid.
Parent SBRFG had a thick-fleshed, wavy pileus with a brown pigmented surface and robust basidiocarps (FIG. 1). SSI SBRFG-s1, progenitor of the hybrid, was similar but white and fruited a few days later than its parent (FIG. 2). SSI I-101-s1 had a thin-fleshed, narrowly convex pileus with a brown pigmented surface and gracile basidiocarps that fruited about 10 d later than SBRFG (FIG. 3). H1x1 produced robust mushrooms that fruited concurrently with those of SBRFG, with brown-pigmented pilei that were convex and not (or only obscurely) wavy (FIG. 4). The several traits described here were inherited and expressed in different modes from the two SSIs; for example the pileus color trait exhibited classic Mendelian dominant/recessive behavior.
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), the sequences of the ITS1+2 regions of the nuclear rDNA were aligned and compared. The samples studied had an rDNA ITS1+2 sequence with nominal length of 711 nt; however, due to frequent length polymorphisms at either of two positions, the actual sequence lengths ranged from 710 to 712 or 713 nt and a majority of samples yielded sequences of more than one length. These length polymorphisms occurred at positions 49–51 (ttt versus tttt) and at or near position 485 (tttt versus tttttt versus ttttttt) in the nominal 711 nt sequence. They were commonly heteromorphic within single isolates (downstream from the indel, two distinct peaks were superimposed at each position when the output trace file was examined), which interfered with sequencing and necessitated bidirectional sequencing using four primers. The sequences were otherwise identical except at 16 other variable positions (TABLE II).
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). The term heteromorphism should not be equated automatically with heteroallelism (because the ITS region is moderately or highly repeated), although equivalence in this instance would be the simplest possibility and is supported by the sequence of H1x1. The sample from the UK, identified by M.M. Nauta as A. rufotegulis, fits this sequence pattern, although no heteromorphisms were present. The three successfully amplified sequences from Hawaii (DEH 1073, 513; KRP 070) were identical among themselves and were similar to non-Hawaiian sequences but had slightly different sequence characteristics. Like A. rufotegulis, Hawaiian sequences lacked both compositional and length heteromorphisms. They had two sequence characters, at positions 281 and 478, not found in any of the other non-Hawaiian sequences. Finally, their lengths were one nucleotide shorter than all other "nominal" sequences, at a third position (~485), where other sequences either were nominal or one nucleotide longer. DNA extracts from three other Hawaiian specimens of A. subrufescens (DEH 337, 527, 1452 [SFSU]) unfortunately failed to amplify.
Excluding several GenBank sequences identified as Agaricus blazei, mainly deposited by laboratories in Asia, the public sequences with the greatest affinity to the A. subrufescens samples all belong to taxa in section Arvenses. One of these is GenBank AF432878 [GenBank] , deposited as A. macrosporus RWK 1925 but correctly named A. urinascens. The A. urinascens sequence was 2.9–3.2% divergent from the A. subrufescens samples (using Megalign), while distances among A. subrufescens samples ranged from 0 to 0.27% (hand calculated), with Hawaiian isolates having the highest divergence scores.
In the FITCH and UPGMA trees derived from the distance matrix, Brazilian sequences always were interspersed with those from other regions (FIG. 5). No phylogenetic signals associated either with geographical regions (other than Hawaii) or with recently proposed taxa were evident. In contrast, in an MP analysis of A. subrufescens sequences within Agaricus, particularly within section Arvenses, all A. subrufescens sequences (including several deposited in GenBank as ‘A. blazei’) formed a monophyletic unit within Arvenses (FIG. 6). Bootstrap values for the A. subrufescens clade and the Arvenses sectional clade were both 100%. Within A. subrufescens, a clade formed by the three identical Hawaiian sequences received 88% bootstrap support. Taken together results from both distance and parsimony analyses indicate the presence of a single phylogenetic species.
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. Referring to their photograph, I was able to segregate and label specimens and fragments assigned by Didukh et al as "type A" and "type B", with an additional upper stipe fragment now assigned to element B. Element A comprises two basidiomata, in agreement with Falconer’s letter to Peck (21 Oct 1892 [NYS]), which are relatively gracile and have dark brown pileus pigment. Lamellae of these basidiomata are fused and their cellular structure is collapsed and indistinct. Element B comprises four basidiomata, based on stipe apices, not two as reported in Didukh et al (2003), in fragments. The B specimen fragments are in better condition and have a slightly more robust aspect and less pronounced pileus pigmentation, relative to the A specimens. Spores of the B element are larger, on average, than those of A (TABLE III), as reported by Didukh et al (2003). However, in both elements the spore lengths span similarly broad ranges (5.1–7.5 or –8.3 µm). The distribution of lengths in element A is clearly bimodal, and it appears to be bimodal or multimodal in element B (FIG. 7). Element B has a greater proportion of large spores (ca. 50%) relative to A (
10%). Developing spores were observed on the hymenium of the B element; while spore tetrads were most common; triads, diads and occasional monads also were observed at progressively lower frequencies. Cheilocystidia could not be observed in element A, as expected given the condition of the lamellae. Irregular to semiglobose, catenulate cheilocystidia ca. 7.5–13.5 µm broad were observed in element B. Inflated elongate cells and occasional subglobose elements 10–26 µm broad were observed among narrower hyphae of the pileal disc of element B. No velar material could be identified with certainty in any part of the holo-type. One loose lamellar fragment that fell from one of the basidiomata of element A had a spore size distribution like that of element B, indicating at least minor cross-contamination of the two formerly mixed elements due to the large number of loose fragments present.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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The data presented here further show that the DNA sequences of the ITS1+2 regions of geographically diverse members of this species are very similar. Three Hawaiian samples had no heteromorphisms but three otherwise unique polymorphisms, while one UK sample also had no heteromorphisms. Heteromorphisms always are present and frequent in the North and South American isolates and specimens studied to date. These data suggest an interpretation, developed below, based on the possibility of interpopulational hybridization; this hypothesis is speculative but useful in providing guidance for further studies.
No geographical population (with the exception of the Hawaiian samples) could be distinguished uniquely by sequence characters; the data provide no justification for recognizing distinct North American, South American or European taxa (some heteromorphisms were unique to the California + "Chinese" samples, but because only a single genotype might be represented, the significance of those characters cannot be assessed). FITCH and UPGMA phenograms derived from the distance matrix showed American, Brazilian and European sequences intermingled, while the Hawaiian sequences formed a distinct branch (FIG. 5).
With respect to Hawaii, the question of how levels of sequence divergence relate to taxonomic rank-relationships arises. Some rough benchmarks for taxonomic rank relationships in Agaricus, appropriate to various degrees of ITS1+2 sequence divergence, are beginning to emerge. Two varieties of A. bisporus, distinguished by morphological characters and reproductive behaviors, each exhibited single unique sequence character differences from var. bisporus (Callac et al 1993, 2003). An alpine relative of A. devoniensis P.D. Orton that has two unique sequence characters currently is considered to be a subspecies (Challen et al 2003, Kerrigan unpublished). The homothallic "highland" and "lowland" entities (Kerrigan et al 1999) of A. subfloccosus ( J. Lange) Hlavác ek s. l., arguably either subspecies or sister species, differ at only one position in the ITS1+2 sequence (Challen et al 2003, Kerrigan unpublished). Other "sister species" have greater ITS1+2 divergence (e.g., about 1.4–1.9% in A. bisporus versus A. subfloccosus, which corresponds to about 10–14 character differences. However some species in section Xanthodermatei Sing. might differ by smaller numbers of ITS1+2 sequence characters (Kerrigan et al unpublished, Callac et al unpublished) By comparison with these observations any taxon that might be proposed for the Hawaiian A. subrufescens could be argued to deserve either infraspecific or specific rank, presumably depending upon other observations that hopefully would include interfertility data.
Phylogeographic structure and dispersal.— –
The distribution of A. subrufescens is documented only patchily, because the species appears not to have been well recognized in some regions (Europe, South America). However, it appears to have a tropical or subtropical to temperate zone distribution. Agaricus subrufescens is the only species of Agaricus known to be harmed or killed by prolonged exposure to temperatures of ca. 4 C or lower (Kerrigan unpublished, cf. A. brasiliensis in Wasser et al 2002). Wasser et al understandably were unaware of this trait of A. subrufescens when they attempted to use it to distinguish A. subrufescens from A. brasiliensis. It is also remarkably tolerant of high temperatures (Kerrigan 1983a). It is unusually versatile nutritionally, having been observed to have spontaneously colonized a bag of wet sawdust on one occasion (Kerrigan unpublished). The most "natural" habitat reported for the species is piles of leaves.
The Hawaiian Islands are geographically isolated. Relatively more sequence divergence might be expected within small island populations ( Johnson and Seger 2001). The UK is less isolated but might be near the limit of the natural range of the species in the Eastern Hemisphere, which currently remains unclear. The samples from these localities might represent "pure" members of relatively isolated or marginal populations. Their lack of sequence heteromorphism is consistent with this theory. Conversely, samples from Brazil (and Japan by extension) and California are highly heteromorphic. Some sequences in GenBank (e.g., AB113576
[GenBank]
.1) also exhibit this characteristic. The Americas samples might represent examples of natural or accidental hybrids between formerly "pure" populations, assuming that the dual peaks and sequence length differences arise from allelic variations at the haplotype level (which is supported by the implicit allelic segregation needed to produce the H1x1 sequence) (TABLE II). The observation that heteromorphisms including length polymorphisms are relatively uncommon among species of section Duploannulati Wasser emend. Challen et al (2003) supports this hypothesis (TABLE IV). A. devoniensis is the exception to this pattern, appearing to exhibit diverse aspects of recent population level hybridization (Callac et al unpublished).
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Contemporary documents show that spawn of A. subrufescens was being sold commercially, sometimes as ‘A. fabaceus’, about a century ago (Falconer 1894a, b; Anonymous 1909). At that time the two species names were considered to be synonymous by B.M. Duggar, a USDA BPI collaborator involved with the developing mushroom spawn industry in the USA (Duggar 1905, 1920). A. fabaceus is an older American name dating from 1847. It was described having a pileus that was white becoming yellow, and viscid when moist. It appears unlikely to be the same species as A. subrufescens. The type of A. fabaceus is at Kew. Spawn distribution is a documented route for geographically broad dispersal of Agaricus germ plasm into the environment (Kerrigan et al 1999). In addition, any organism that can use composted manure and wood as a substrate, and is thermotolerant, potentially could cross oceans on a traditional wooden sailing ship. When the vigor and amphithallic reproductive versatility (including single-spore fertility) of A. subrufescens is factored in, dispersal, establishment and gene flow in this species is not surprising.
A quote from a Boston Mycological Club bulletin (Anonymous 1904) illustrates the reproductive potential of this species: "Its spontaneous appearance in various greenhouses in widely separate localities has brought it to notice at various times because it forced itself so persistently and abundantly on the owners that they have sent in specimens to the Club exhibitions in order to learn whether the visitation was to be regarded as a curse or a blessing. . . . When it appears it comes with a rush, having previously, with its strong growing, strand like mycelium, taken possession of some rich portion of soil, and then it sends its abundant fruits to the light, undaunted by any overlying difficulties."
In Santa Barbara, California, I encountered A. subrufescens growing on a lawn about 200 m from a laboratory where I had cultivated it the previous year (Kerrigan and Ross 1987a). The possibility that this represented an escape from cultivation was not contradicted by allozyme data I obtained from both isolates (Kerrigan unpublished). This was the second and only other time I ever encountered the species in nature.
Morphology of basidiomata.— – Although the present study did not primarily emphasize morphological criteria, a discussion of some points is in order. The basidiomatal morphology of A. subrufescens is somewhat variable. This would not be surprising in a situation where populations had diverged in isolation and were now interbreeding. Sporocarps can be robust or gracile, both due to genotype (see above) and environmental influences (Kerrigan unpublished).
Cuticle pigmentation and background yellowing are also variable (the degree of analogous reddening of the tissues of A. bisporus is under genetic control and both traits are managed actively in mushroom breeding programs [Kerrigan 2004, unpublished]). Although Peck noted that the lamellae could be yellowish-white, he reported the flesh to be "white, unchangeable". Wasser et al have emphasized this as a point of distinction between A. subrufescens and A. brasiliensis. However, this variation does occur naturally within member species of section Arvenses (Kerrigan 1982, unpublished). Furthermore, the specimens Peck received had traveled more than 300 km (the straight-line distance between Glen Cove and Albany) in 1892 before he saw them and did not arrive in the best condition— "Dear Sir: I am glad you rec’d the mushrooms, but sorry they were so long delayed and in such poor condition. I sent them to Dr L(-) to forward to you as I did not know your proper address." (Falconer to Peck, 21 Oct 1892 [see also 15 Oct 1892] [NYS]). Even the second shipment apparently suffered. Peck (1897) wrote that "From some of these specimens kindly sent me by the discoverer the original description was derived, but the specimens were not in satisfactory condition to figure." Kauffman (1918) wrote that "The original description was made from old material. . . . Peck’s description of this species is, therefore, misleading." Color changes in Agaricus diminish in time after harvest as well as with age and deteriorating condition of basidiomata. Finally, the man who obtained and sent the type material of A. subrufescens to Peck, Mr W. Falconer (editor of the journal "Gardening" and subsequent author of the 1897 USDA Farmer’s Bulletin 53, "How To Grow Mushrooms"), reported that the species had a "lemon-tinted neck", indicating that Peck’s description was at least incomplete (Falconer 1894a).
In my experience the cap shape also is variable, ranging from narrowly convex through cuboidal to broadly convex. However, two characters unusual in other species may sometimes be present: (i) a transient concave shoulder or bell-shape to the pileus as expansion in young pilei begins first near the margin; (ii) radially oriented folds or "pleats" of the pileus. Both features are illustrated in Kerrigan (1986). Falconer even included a tiny sketch of a pleated pileus in his first letter to Peck (15 Oct 1892). The elastic veil, which usually remains attached to the pileus margin during expansion, stretching while the lower layer breaks up into a large number of small cottony floccules, is a fairly consistent feature of the species.
Agaricus subrufescens is a widely illustrated species. The range of gross morphologies can be seen in these references: Peck 1897; Anonymous 1904; Anonymous 1909; Duggar 1905, 1920 (Pl. VI); Kauffman 1918; Tu and Lin 1981; Kerrigan 1983b, 1984, 1986; Nauta 1999; Hausknecht 2002; Wasser et al 2002; Peterson et al 2000. In its heyday the mushroom was described as "uncouth looking" (Falconer 1894a). The figure and description furnished by Hotson and Stuntz (1938) probably refer to A. augustus Fr.
Microscopic features have been described by Kerrigan (1982, 1986), Heinemann (1993), Nauta (1999), Peterson et al (2000), Hausknecht (2002) and Wasser et al (2002), among others. The type of A. subrufescens has been examined and described by Freeman (1979), Wasser et al (2002) and by Smith (1940), who could not "add any information to that found in Kauffman’s account." Some authors, particularly Wasser et al, have emphasized points of distinction among the entities they recognize. Spore size variability deserves some discussion. Heinemann (1993) found the sizes of spores to be heterogeneous within single collections of cultivated Brazilian stock. He suspected the presence of bisporic basidia among the tetrasporic ones but was unable to verify this. Kerrigan and Ross (1987a, b) did document the presence of dynamically varying numbers of bi-, tri- and tetrasporic basidia in field and cultivated specimens of A. subrufescens, leading to an expectation of variation among spore size measurements. Kerrigan (1982, see A. subrufescens and A. augustus) also presented data on variation in spore size from single sporocarps at different developmental stages. Occasional North American collections of A. subrufescens have been observed to have relatively longer spores (e.g., Smith 6789 [MICH], Rives, Washington D.C. [NYS] (TABLE III); the latter specimens were illustrated by Peck [1897]). We should expect considerable variation in spore lengths within and among collections of A. subrufescens, given the bimodality or multimodality observed in the distributions of spore sizes in the two elements of the holotype and the known dynamic variability in proportions of n-spored basidia that results from temperature shifts or other diurnal cues.
Cheilocystidia in A. subrufescens are usually reported to be variable: cylindrical to clavate or swollen, with few to many catenulate-globose cells (Kerrigan 1982, Heinemann 1993, Nauta 1999, Wasser et al 2002, Hausknecht 2002). However, Wasser et al (2002) did not observe cheilocystidia in element A of the type of A. subrufescens. Freeman (1979) also reported cheilocystidia to be absent in the type. By contrast, Kauffman’s (1918) description of Michigan collections noted numerous subcylindrical sterile cells on edges of gills, and Peck authenticated at least some of Kauffman’s A. subrufescens material. Typical cheilocystidia are present in element B of the holo-type. Unfortunately, given the condition of the element A type specimens (discussed above), it is unlikely that cheilocystidia will be observed therein.
Two observations on this species argue against overemphasizing morphological features evaluated from dried specimens. First, observations and experience show that morphology within the interfertile group is rather diverse because of underlying genetic variation and is additionally plastic in response to environmental influences. Second, this is perhaps the easiest species of Agaricus to cultivate and, as shown above, is amenable to outcrossing. There are compelling advantages to studying the expression of morphological traits in living material under standard (and possibly nonstandard) conditions and to investigating trait expression when genomes from putatively distinct taxonomic entities share a common cytoplasm. For example, distinctive cells of the veils and/or cuticle of this species have been discussed by Nauta (1999), Heinemann (1993), and Wasser et al (2002). I have observed that SBRFG has sausage-like chains of cells in the universal veil of the annulus, but not in velar patches on the pileus. Similar cells have been observed in A. subrufescens from eastern North America (O.K. Miller personal communication). The best way to assess the taxonomic and/or phylogenetic significance of any differences in velar cells among populations would be to study expression of the trait in hybrids and their offspring.
If, as in studies of some other basidiomycetes, barriers to gene flow were discovered within the A. subrufescens phylogenetic unit, that might indicate that a more elaborate taxonomic arrangement could be appropriate. For these reasons living cultures from European, Hawaiian and other populations will be of great interest to future studies.
Other notable reports.— –
Agaricus subrufescens first was reported from Brazil by J. Rick (1930). However, Rick gave the spore size of material from São Leopoldo as 5 x 3 µm, so Peck’s name is unlikely to apply to these specimens. Later, Rick (1939) suggested the existence of a ‘varietas microspora’ with spores 3 x 2 µm; however this is not a validly published name and is unlikely to be phylogenetically congruent with the species.
In the photographic collection of the late W.F. Isaacs, one transparency of A. subrufescens is labeled "from Argentine". The slide, which shows the mushrooms growing indoors in wooden boxes, was processed in the USA in Mar 1963. This suggests that some aspects of the cultural history of A. subrufescens in South America remain incompletely known.
A European culture identified as A. purpurascens (Cooke) Pilát (nom. illeg., non A. purpurascens Fr.; see A. porphyrizon Orton 1960) was cultivated experimentally at INRA Bordeaux, France (Brian et al 1981). Based on experience and on published and unpublished photographs, these mushrooms agree with A. subrufescens ( J. Guinberteau and P. Callac personal communication). This report indicates that European strains should be amenable to cultural and hybridization studies.
Nomenclature and typification.— –
Peck’s publications and correspondence from Falconer to Peck at NYS make clear that the holotype of A. subrufescens comprises two separate collections. Under the circumstances lectotypification of A. subrufescens is appropriate. Didukh et al (2003) concluded from a study of the type that two different elements were present. They also concluded that two different taxa were present; however I disagree. Elements A and B fall within the range of variation known for A. subrufescens. This can be seen in the spore-size data (TABLE III). Didukh et al formally designated element B to be the lectotype. Peck (1897) indicated that the description of the species was based on specimens from Falconer’s "compost heap composed chiefly of decaying leaves." Based on the numbers and condition of basidiomata in the two elements of the type, Peck was referring to the B element as the basis for his concept. Although spores from the B element are long relative to Peck’s description (and spores from the A element are short), both elements have spore length ranges that bracket Peck’s measurement. Didukh et al have selected the best element for the lectotype of A. subrufescens. The A element has now been segregated and should be considered an authenticated collection of the species. The synonymy of the species is:
Agaricus subrufescens Peck New York State. Mus. Ann. Rep. 46:105. 1893
Psalliota subrufescens Kauff. The Agaricaceae of Michigan 239. 1918.
= Agaricus rufotegulis Nauta Persoonia 17:230. 1999.
= Agaricus brasiliensis Wasser, Didukh, de Amazonas & Stamets. Int. J. Med. Mush. 4:274. 2002.
Misapplied names:
– Agaricus blazei Murrill sensu Heinemann Bull Jard Bot Belgium 62:365–368. 1993.
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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ACKNOWLEDGMENTS |
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1 E-mail: rwk{at}sylvaninc.com
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LITERATURE CITED |
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