New and rare taxa in Agaricus section Bivelares ... - Mycologia
New and rare taxa in Agaricus section Bivelares ... - Mycologia
New and rare taxa in Agaricus section Bivelares ... - Mycologia
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<strong>Mycologia</strong>, 100(6), 2008, pp. 876–892. DOI: 10.3852/08-019<br />
# 2008 by The Mycological Society of America, Lawrence, KS 66044-8897<br />
<strong>New</strong> <strong>and</strong> <strong>rare</strong> <strong>taxa</strong> <strong>in</strong> <strong>Agaricus</strong> <strong>section</strong> <strong>Bivelares</strong> (Duploannulati)<br />
Richard W. Kerrigan 1<br />
Sylvan Research, 198 Nolte Drive, Kittann<strong>in</strong>g,<br />
Pennsylvania 16201<br />
Philippe Callac<br />
INRA, MYCSA (Mycologie et sécurité des aliments) BP<br />
81, 33883 Villenave d’Ornon cedex, France<br />
Luis A. Parra<br />
Avenida Padre Claret 7, 5th G, 09400 Ar<strong>and</strong>a de<br />
Duero, Burgos, Spa<strong>in</strong><br />
Abstract: Ongo<strong>in</strong>g field <strong>and</strong> laboratory studies have<br />
led to our recognition of new <strong>taxa</strong> <strong>in</strong> <strong>Agaricus</strong> <strong>section</strong><br />
<strong>Bivelares</strong>, a recent comb<strong>in</strong>ation <strong>and</strong> now the earliest<br />
synonym <strong>and</strong> correct name of <strong>section</strong> Duploannulati.<br />
<strong>Agaricus</strong> cupressophilus <strong>and</strong> A. tlaxcalensis, <strong>in</strong> the new<br />
<strong>Agaricus</strong> sub<strong>section</strong> Cupressorum, <strong>and</strong> A. agr<strong>in</strong>ferus,<br />
A. devoniensis subsp. bridghamii, <strong>and</strong> A. subsubensis <strong>in</strong><br />
<strong>Agaricus</strong> sub<strong>section</strong> Hortenses, are described. <strong>Agaricus</strong><br />
subfloccosus is lectotypified. Phylogeny reconstruction<br />
methods with ITS1+2 DNA sequences were used to<br />
determ<strong>in</strong>e appropriate placements of the new <strong>taxa</strong>.<br />
Collectively these new <strong>taxa</strong> <strong>and</strong> phylogenetic associations<br />
represent a substantial augmentation <strong>and</strong><br />
clarification of our knowledge of <strong>section</strong> <strong>Bivelares</strong>;<br />
described, sequenced species-level <strong>taxa</strong> <strong>in</strong> the northern<br />
hemisphere are <strong>in</strong>creased from six to 10, a<br />
dist<strong>in</strong>ct sub<strong>section</strong>al l<strong>in</strong>eage is revealed <strong>and</strong> <strong>in</strong>fraspecific<br />
resolution with<strong>in</strong> A. devoniensis is improved. An<br />
anomalous ITS1+2 sequence is documented <strong>in</strong> one<br />
collection of A. subsubensis. Prelim<strong>in</strong>ary data on<br />
another novel member of <strong>Bivelares</strong> from France also<br />
are provided. Several of these <strong>taxa</strong> are <strong>rare</strong>, highlight<strong>in</strong>g<br />
opportunities <strong>and</strong> challenges for document<strong>in</strong>g<br />
biodiversity <strong>in</strong> this group. Additional comments<br />
on related <strong>taxa</strong> treated <strong>in</strong> recent publications are also<br />
provided.<br />
Key words: button mushroom, cultivation,<br />
Cupressus, ITS sequence evolution, phylogeny,<br />
taxonomy<br />
INTRODUCTION<br />
The familiar cultivated button mushroom species<br />
<strong>Agaricus</strong> bisporus ( J.E. Lange) Imbach <strong>and</strong> the<br />
cosmopolitan A. bitorquis (Quél.) Sacc. belong to a<br />
relatively small <strong>in</strong>frageneric group. Several <strong>section</strong>al<br />
Accepted for publication 25 August 2008.<br />
1 Correspond<strong>in</strong>g author. E-mail: rwk@sylvan<strong>in</strong>c.com<br />
876<br />
<strong>and</strong> sub<strong>section</strong>al names have been applied to this<br />
group, most often with A. bitorquis (or its heterotypic<br />
synonym Psalliota rodmanii) or A. hortenses (a<br />
heterotypic synonym of A. bisporus) as the type<br />
species (see Challen et al 2003).<br />
<strong>Agaricus</strong> <strong>section</strong> Duploannulati Wasser ex Wasser<br />
(1980), typified by A. bitorquis, has been the subject of<br />
two recent phylogenetic reassessments based on<br />
sequence analysis of the ITS1+2 region of the nuclear<br />
rDNA. Challen et al (2003) evaluated six species-level<br />
<strong>taxa</strong> <strong>and</strong> several <strong>in</strong>fraspecific entities <strong>and</strong> restricted<br />
the <strong>section</strong> to a clade conta<strong>in</strong><strong>in</strong>g the type species, A.<br />
bitorquis, while exclud<strong>in</strong>g species that we believe<br />
should be placed <strong>in</strong> <strong>section</strong> Chitonioides Romagn.<br />
Didukh et al (2005) extended this analysis with an<br />
emphasis on <strong>taxa</strong> from Israel, accord<strong>in</strong>g to Wasser’s<br />
(1980; see also Wasser et al 1976) orig<strong>in</strong>al, broader<br />
concept of Duploannulati.<br />
Given S<strong>in</strong>ger’s (1975) selection of P. rodmani<br />
(Peck) Kauffman, a synonym of A. bitorquis, as the<br />
type of Kauffman’s (1919) Psalliota <strong>section</strong> <strong>Bivelares</strong>,<br />
<strong>Bivelares</strong> is the oldest epithet for the <strong>section</strong><br />
conta<strong>in</strong><strong>in</strong>g A. bitorquis, with priority from 1919.<br />
Consequently a new comb<strong>in</strong>ation <strong>in</strong> <strong>Agaricus</strong> has<br />
been created at the <strong>section</strong>al level (Parra 2008).<br />
We have cont<strong>in</strong>ued to collect <strong>and</strong> evaluate both<br />
field material <strong>and</strong> cultures hav<strong>in</strong>g unresolved aff<strong>in</strong>ities<br />
or status. As a result three new species are<br />
described here: <strong>Agaricus</strong> cupressophilus <strong>and</strong> A.<br />
tlaxcalensis, both belong<strong>in</strong>g to the new <strong>Agaricus</strong><br />
sub<strong>section</strong> Cupressorum, <strong>and</strong> A. subsubensis, <strong>in</strong><br />
<strong>Agaricus</strong> sub<strong>section</strong> Hortenses He<strong>in</strong>em. (1956). In<br />
addition we formally treat two entities that were<br />
discussed <strong>in</strong> our earlier publications, segregat<strong>in</strong>g the<br />
new species A. agr<strong>in</strong>ferus from the accumulated<br />
concept of A. subfloccosus ( J.E. Lange) Hlaváček<br />
<strong>and</strong> recogniz<strong>in</strong>g A. devoniensis subsp. bridghamii. Yet<br />
another new member of <strong>Bivelares</strong> from France<br />
(collection CA103 [INRA]) is given a prelim<strong>in</strong>ary<br />
report here. The latter three new <strong>taxa</strong> (<strong>and</strong> the<br />
unnamed entity) are placed <strong>in</strong> sub<strong>section</strong> Hortenses.<br />
F<strong>in</strong>ally we present comments <strong>in</strong>tended to supplement<br />
<strong>in</strong>formation provided <strong>in</strong> Challen et al (2003).<br />
MATERIALS AND METHODS<br />
Collection, preservation <strong>and</strong> study methods, <strong>and</strong> cultural<br />
practices, were rout<strong>in</strong>e <strong>and</strong> have been described elsewhere<br />
(Kerrigan et al 1999). Collection data are given below.<br />
North American material is deposited at SFSU unless<br />
otherwise <strong>in</strong>dicated. CA collections <strong>and</strong> cultures are housed
at the Collection du Germplasm des Agarics à Bordeaux<br />
(CGAB), France. Cultures were deposited at the American<br />
Type Culture Collection (ATCC) or at CGAB. DNA<br />
sequenc<strong>in</strong>g <strong>and</strong> sequence analysis also has been described<br />
elsewhere (Challen et al 2003). Briefly, PCR amplification of<br />
the ca. 710 nt ITS1+2 nuclear genomic rDNA region, here<br />
def<strong>in</strong>ed as beg<strong>in</strong>n<strong>in</strong>g at ‘‘position 1’’ with 59-ggaaggat <strong>in</strong> the<br />
SSU-rRNA gene <strong>and</strong> end<strong>in</strong>g with gaacttaa-39 <strong>in</strong> the LSUrRNA<br />
gene, was performed with primers ITS1 or ITS5 <strong>and</strong><br />
ITS4, or ITS2 <strong>and</strong>/or ITS3 as needed (White et al 1990).<br />
Sequences for both str<strong>and</strong>s were determ<strong>in</strong>ed with an<br />
automated DNA sequencer either at the Nucleic Acid<br />
Facility-Pennsylvania State University, University of Pittsburgh,<br />
McMaster University or INRA <strong>and</strong> were deposited <strong>in</strong><br />
GenBank (accession numbers are given below). Sequences<br />
were aligned with the Clustal W rout<strong>in</strong>e followed by manual<br />
<strong>in</strong>spection <strong>and</strong> correction <strong>and</strong> the f<strong>in</strong>al alignment was<br />
deposited <strong>in</strong> TreeBASE (study accession number 5 S2128;<br />
matrix accession number 5 M4027). In the special case of<br />
A. subsubensis JB 101 (discussed below) the ITS1+2<br />
sequence, <strong>in</strong>dependently amplified twice <strong>and</strong> sequenced<br />
twice for verification, was divided <strong>in</strong>to three parts; the first<br />
<strong>and</strong> third segments were aligned <strong>in</strong>dependently for<br />
comparisons but were not used <strong>in</strong> tree-build<strong>in</strong>g. Both<br />
maximum parsimony (MP) trees (FIG. 1; PAUP* v 4.0b8:<br />
Swofford 2000) <strong>and</strong> a neighbor-jo<strong>in</strong><strong>in</strong>g (N-J) tree (FIG. 2;<br />
MegAlign, Lasergene v. 6; unweighted nucleotide substitution<br />
model) were generated. Evaluation of the nonredundant<br />
sequences, <strong>in</strong>clud<strong>in</strong>g some that were too short for N-J<br />
analysis, under MP was performed as per Challen et al<br />
(2003) with the follow<strong>in</strong>g heuristic search sett<strong>in</strong>gs: r<strong>and</strong>om<br />
addition sequence, one tree held at each step dur<strong>in</strong>g<br />
stepwise addition us<strong>in</strong>g the tree-bi<strong>section</strong>-reconnection<br />
algorithm, branch collaps<strong>in</strong>g if maximum branch length<br />
was zero, gaps treated as miss<strong>in</strong>g data, stepwise descent<br />
option not <strong>in</strong> effect, topological constra<strong>in</strong>ts not enforced,<br />
bootstrapp<strong>in</strong>g (n 5 1 000 000) <strong>and</strong> MAXTREES 5 100.<br />
Dist<strong>in</strong>ctive DNA sequence characters are reported with<br />
position numeration (see above) specific to <strong>in</strong>dividual<br />
sequences. We report such characters when they appear to<br />
be useful <strong>in</strong> the context of available data; as with any<br />
taxonomic character, new data might lead to a reassessment.<br />
Nomenclature follows Kerrigan et al (2006: 1299) <strong>in</strong><br />
agreement with ICBN-2006 (Vienna Code). Rank relationships<br />
presented here<strong>in</strong> are hypotheses based on all available<br />
data. No s<strong>in</strong>gle criterion is sufficient to the task; however <strong>in</strong><br />
general we have observed <strong>in</strong> earlier studies (Challen et al<br />
2003, Kerrigan et al 2005) <strong>and</strong> <strong>in</strong> ongo<strong>in</strong>g work that<br />
conventionally accepted species of <strong>Agaricus</strong> generally differ<br />
by two or more ITS DNA characters, whereas <strong>in</strong>fraspecific<br />
<strong>taxa</strong> differ by two or fewer ITS characters. This emergent,<br />
rough phenetic st<strong>and</strong>ard cannot serve as a sole criterion for<br />
def<strong>in</strong><strong>in</strong>g taxon boundaries <strong>and</strong> was considered as only a<br />
s<strong>in</strong>gle l<strong>in</strong>e of evidence. In select<strong>in</strong>g the most appropriate<br />
rank hypothesis we also considered, as available, DNA<br />
sequences from other genes (e.g. from manuscripts <strong>in</strong><br />
preparation; see A. subsubensis), morphology (see A.<br />
tlaxcalensis <strong>and</strong> A. cupressophilus), physiology, cultural<br />
behavior, reproductive mode (for all see A. agr<strong>in</strong>ferus),<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 877<br />
mat<strong>in</strong>g studies, other molecular data, ecological <strong>and</strong><br />
geographical separation of populations/<strong>taxa</strong> <strong>and</strong> any<br />
patterns of association of ITS or other characters among<br />
the isolated populations, evidence for hybrids among<br />
putatively differentiated entities (see A. cupressicola, below,<br />
contra A. xanthodermus <strong>and</strong> A. moelleri <strong>in</strong> Kerrigan et al<br />
2006), the state of knowledge <strong>and</strong> complexity of the<br />
immediate group (see the A. devoniensis clade, below),<br />
nomenclatural practice <strong>and</strong> precedents, our preference for<br />
conservative rank placements, <strong>and</strong>, to a lesser (or greater)<br />
extent, the unexpectedly adamant guidance of reviewers.<br />
<strong>New</strong> names were recorded <strong>in</strong> MycoBank (http://www.<br />
mycobank.org) <strong>and</strong> the assigned unique identifiers are<br />
presented below after the Lat<strong>in</strong> diagnoses <strong>in</strong> the format MB<br />
######.<br />
DESCRIPTIONS OF TAXA<br />
<strong>Agaricus</strong> <strong>section</strong> <strong>Bivelares</strong> (Kauffman) L.A. Parra<br />
2008. Fung. Europ. 1(1):157.<br />
Type: Psalliota rodmanii (Peck) Lloyd, designated<br />
by S<strong>in</strong>ger, The Agaricales <strong>in</strong> Modern Taxonomy. 3rd<br />
ed.: 461. 1975.<br />
; Psalliota <strong>section</strong> <strong>Bivelares</strong> Kauffman, Agaric.<br />
Michigan: 234. 1919 [‘‘1918’’]. [basion.]<br />
5 <strong>Agaricus</strong> <strong>section</strong> Duploannulati Wasser ex Wasser,<br />
Fl. Fung. RSS Ucra<strong>in</strong>.: 125. 1980. [‘‘Du-<br />
Duploannulatae’’] [nom. illeg., nom. superfl.;<br />
art. 52.1 s<strong>in</strong>ce Psalliota rodmanii (Peck) Lloyd,<br />
type of the <strong>section</strong> <strong>Bivelares</strong> Kauffman (1919) is<br />
cited as a synonym of <strong>Agaricus</strong> bitorquis (Quél.)<br />
Sacc., the type of the <strong>section</strong> Duploannulati<br />
designated by Wasser (1980: 125)]<br />
5 <strong>Agaricus</strong> <strong>section</strong> Bitorques (He<strong>in</strong>em.) Bon &<br />
Cappelli, Doc. Mycol. 13(52): 16. 1983. Type:<br />
<strong>Agaricus</strong> bitorquis (Quél.) Sacc., designated by<br />
He<strong>in</strong>emann, Sydowia 30: 13. 1978.<br />
; <strong>Agaricus</strong> sub<strong>section</strong> Bitorques He<strong>in</strong>em.,<br />
Sydowia 30: 13. 1978.<br />
Orig. diag.: Annulus double, with thick flocculose<br />
patches on under side. MB 512252.<br />
Delimitation of the <strong>section</strong>: Schäffer’s reaction<br />
negative, KOH reaction negative (at least away from<br />
the disk), o-tolid<strong>in</strong>e generally blue or negative on<br />
peripheral tissues (pelles, veils, exposed stipe base)<br />
while violet-purple or negative on exposed <strong>in</strong>terior<br />
tissues, a-naphthol generally violet-purple (or <strong>rare</strong>ly<br />
negative) on the basal stipe context while orange to<br />
p<strong>in</strong>k on exposed <strong>in</strong>terior tissues. Exposed context<br />
rufescent: becom<strong>in</strong>g p<strong>in</strong>k or reddish, <strong>rare</strong>ly blood red<br />
or reddish-orange or sordid, or sometimes unchang<strong>in</strong>g<br />
or almost so. Veils form<strong>in</strong>g various types of annuli or<br />
occasionally volva-like basal structures. Stipe cyl<strong>in</strong>drical,<br />
clavate or slightly bulbous, usually not taper<strong>in</strong>g at<br />
the base, stuffed-hollow (hav<strong>in</strong>g a pith-filled central<br />
cavity) except for <strong>Agaricus</strong> bitorquis <strong>in</strong> which it is
878 MYCOLOGIA<br />
FIG. 1. One of six most parsimonius trees from a run of 1 000 000 iterations on a set of nonredundant ITS1+2 sequences<br />
from <strong>taxa</strong> of <strong>Bivelares</strong> <strong>and</strong> other <strong>section</strong>s of <strong>Agaricus</strong>. Three trees had the topology shown for the Cupressorum clade, while the<br />
other three trees had the alternative topology shown at the top of the figure. Abbreviations: BERN: A. bernardii; CPCL: A.<br />
cupressicola; CPHL: A. cupressophilus; TLAX: A. tlaxcalensis; DEVO: A. devoniensis (<strong>and</strong> variants); FRGV: A. fragilivolvatus;<br />
BRID: A. devoniensis ssp. bridghamii; SBPR: A. subperonatus (misapplied); BISP: A. bisporus; BISP BURN: A. bisporus var.<br />
burnettii; BISP EROT: A. bisporus var. eurotetrasporus; SFLC: A. subfloccosus; AGRN: A. agr<strong>in</strong>ferus; BITQ: A. bitorquis; CAPL: A.<br />
capellianus; SBSB: A. subsubensis; XANT: A. xanthodermus; SRTL: A. subrutilescens; SBRF: A. subrufescens; CAMP: A. campestris;<br />
PATR: A. pattersonae. Bootstrap values are based on 1000 replications.<br />
usually about solid. Basidiomata with mild or <strong>in</strong>dist<strong>in</strong>ct<br />
odors, most often like that of <strong>Agaricus</strong> bisporus, or<br />
occasionally more complex, as <strong>in</strong> some species of<br />
<strong>section</strong> Sangu<strong>in</strong>olenti Jul. Schäff. & F.H. Møller ex L.A.<br />
Parra (a fruity/spicy odor follow<strong>in</strong>g Kerrigan [1986]).<br />
Cheilocystidia usually present. Spores lack<strong>in</strong>g a rudimentary<br />
apical pore. Culture on rout<strong>in</strong>e media such as<br />
PDA or compost extract agar produces ample growth
(relative to cultures of Sangu<strong>in</strong>olenti). Characteristic<br />
ITS polymorphisms (Challen et al 2003): cttt(k/-)<br />
tCAggta @ 120–121, at (r) tAag (r) a @ 167, ttat (Y )<br />
atac @ 311, attaTattc @473, c(y)c(y) (R) atac @488,<br />
(y)cgtCtgcg @580, <strong>and</strong> tnag [-8-]gaca @663–670.<br />
We are aware that the <strong>section</strong>al name <strong>Bivelares</strong> has<br />
not been used for many years. However under the<br />
rules of nomenclature <strong>Bivelares</strong>, as typified by S<strong>in</strong>ger<br />
(1975) has priority over Duploannulati <strong>and</strong> Bitorques<br />
<strong>and</strong> is therefore the correct name of the <strong>section</strong> to<br />
which A. bitorquis belongs. A more complete discussion<br />
is provided by Parra (2008).<br />
Two closely related <strong>section</strong>s are sect. Xanthoderma-<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 879<br />
FIG. 2. Neighbor-jo<strong>in</strong><strong>in</strong>g tree of all ITS1+2 sequences evaluated <strong>in</strong> the present study, except (i) the too short sequences for<br />
the <strong>New</strong> Zeal<strong>and</strong> A. devoniensis variant (GenBank AF059225: ITS1 data are miss<strong>in</strong>g) <strong>and</strong> CA 504 / A/9.1.2, the type of A.<br />
fragilivolvatus (which matches that of DEVO CA 100 / Dv2), <strong>and</strong> (ii) the highly modified, nonalign<strong>in</strong>g sequence from SBSB<br />
JB-101. The Clustal W alignment was h<strong>and</strong> corrected. Abbreviations are as <strong>in</strong> FIG. 1.<br />
tei <strong>and</strong> sect. Chitonioides. As <strong>in</strong> sect. <strong>Bivelares</strong> these two<br />
<strong>section</strong>s also have negative Schäffer’s reactions<br />
(although <strong>in</strong> a lapsus <strong>in</strong> an earlier paper [Challen et<br />
al 2003], those authors wrote that Xanthodermatei is<br />
characterized by positive Schäffer’s reactions, when of<br />
course ‘‘negative’’ was meant [see also Kerrigan et al<br />
2006]).<br />
KEY TO SUBSECTIONS<br />
Pileus marg<strong>in</strong> typically not strongly <strong>in</strong>rolled <strong>in</strong><br />
young basidiomata, with a th<strong>in</strong> sterile marg<strong>in</strong>al rim<br />
not (or barely) exceed<strong>in</strong>g the lamellae; annulus
880 MYCOLOGIA<br />
th<strong>in</strong>, ‘‘simple’’, pendant or rema<strong>in</strong><strong>in</strong>g appended to<br />
the pileus marg<strong>in</strong>, stipe without conspicuous velar<br />
remnants below the annulus; habitat typically<br />
under Cupressaceae, occasionally under other<br />
trees .................. sub<strong>section</strong> Cupressorum.<br />
Pileus marg<strong>in</strong> strongly <strong>in</strong>rolled <strong>in</strong> young basidiomata<br />
(often even <strong>in</strong> mature basidiomata) with a<br />
thick, sometimes ‘‘swollen’’ rim conspicuously<br />
exceed<strong>in</strong>g lamellae, seldom appendiculate; annulus/veils<br />
various: usually not th<strong>in</strong>, usually <strong>in</strong>termediate,<br />
or peronate or occasionally pendant, but if<br />
appear<strong>in</strong>g pendant then sometimes with an ornamented<br />
undersurface or with some velar remnants<br />
towards the base; habitat various <strong>in</strong>clud<strong>in</strong>g under<br />
Cupressaceae. ............. sub<strong>section</strong> Hortenses.<br />
See also dist<strong>in</strong>guish<strong>in</strong>g molecular characters (below).<br />
Another helpful field character is a tendency<br />
(only) toward a slender stature <strong>in</strong> most species of<br />
Cupressorum (or not, <strong>in</strong> A. cupressicola) vs. a<br />
nonslender stature (with the exception of some A.<br />
devoniensis) <strong>in</strong> Hortenses. Generally speak<strong>in</strong>g the<br />
pendant annulus <strong>in</strong> <strong>Agaricus</strong> (see Kerrigan 1982)<br />
corresponds to the descend<strong>in</strong>g (Nauta 2001) or<br />
superous (see Boisselet 1990, Parra 2003, Cappelli<br />
1984) veils of European authors. These terms all<br />
apply to the veils of the new sub<strong>section</strong> Cupressorum,<br />
but <strong>in</strong> their strict senses are not typical of sub<strong>section</strong><br />
Hortenses.<br />
<strong>Agaricus</strong> [<strong>section</strong> <strong>Bivelares</strong>] sub<strong>section</strong> Cupressorum<br />
Callac & Kerrigan, subsect. nov.<br />
Stipes cyl<strong>in</strong>dricus ad clavatum. Margo pilei non valde<br />
<strong>in</strong>volutus. Annulus superus pendulus, tenuis, simplex. Caro<br />
fracta aurantiascens vel rubescens. Odor <strong>in</strong>conspectus vel<br />
Agarico bisporo similis. Cheilocystidia praesentia. Sporae<br />
apicis poro destitutae. Cultura <strong>in</strong> PDA bona. Polymorphismi<br />
propii regionis ITS: <strong>in</strong> positionibus: ctttGtcag @ 115,<br />
tattgGgAgaag @ 126, 128, ccttCgctg @ 152, tgcaTtgtg @<br />
173, gaaaGcGgtgc @ 181, 183, tgtaGagga @ 491, atctGcact @<br />
600, aggaTtacc @ 689 (ex typi Agarici cupressicolae<br />
sequentia positiones captae, L 5 705 nt). MB 512253.<br />
Typus: <strong>Agaricus</strong> cupressicola Bon & Grilli <strong>in</strong> Bon, Doc.<br />
Mycol. 17(67):11. 1987.<br />
Stipe cyl<strong>in</strong>drical, clavate, neither clearly bulbous<br />
nor taper<strong>in</strong>g at the base. Pileus marg<strong>in</strong> not strongly<br />
<strong>in</strong>rolled, sometimes at least partially appendiculate<br />
with velar remnants. Annulus simple, pendant-superous.<br />
Context becom<strong>in</strong>g orange to red. Odor <strong>in</strong>dist<strong>in</strong>ct<br />
or similar to <strong>Agaricus</strong> bisporus. Cheilocystidia usually<br />
present. Spores lack<strong>in</strong>g an apical pore. Growth on<br />
PDA good. Schäffer reaction negative; KOH usually<br />
negative, or yellow on disk. Dist<strong>in</strong>ctive ITS1+2 region<br />
characters: ctttGtcag @ 115, tattgGgAgaag @ 126, 128,<br />
ccttCgctg @ 152, tgcaTtgtg @ 173, gaaaGcGgtgc @<br />
181, 183, tgtaGagga @ 491, atctGcact @ 600,<br />
aggaTtacc @ 689 (<strong>in</strong> the LSU) (positions taken from<br />
the A. cupressicola type sequence, L 5 705 nt). Type<br />
species: A. cupressicola Bon & Grilli <strong>in</strong> Bon, Doc.<br />
Mycol. 17(67):11. 1987. Also <strong>in</strong>cluded: A. tlaxcalensis<br />
<strong>and</strong> A. cupressophilus, described below.<br />
<strong>Agaricus</strong> has a rich association with Cupressaceae.<br />
Several dozen <strong>Agaricus</strong> species are found under<br />
Cupressus, most particularly C. macrocarpa; more than<br />
30 species have been recorded from a s<strong>in</strong>gle hectare<br />
of this habitat (Kerrigan 1982). Many <strong>Agaricus</strong> spp.<br />
from California are known only from groves or<br />
plantations of Monterey cypress, possibly imply<strong>in</strong>g a<br />
specific habitat association. On the other h<strong>and</strong>, where<br />
Cupressus has been <strong>in</strong>troduced, <strong>Agaricus</strong> usually soon<br />
arrives, <strong>and</strong> novel, apparently <strong>in</strong>digenous species are<br />
encountered (Orton 1960, Callac 1994). Consider<strong>in</strong>g<br />
the extensive cohabitation of <strong>Agaricus</strong> (<strong>and</strong> other<br />
genera of Agaricaceae <strong>in</strong>clud<strong>in</strong>g Leucoagaricus, Lepiota,<br />
Macrolepiota, Chlorophyllum, Cystoagaricus <strong>and</strong><br />
Tulostoma) with Cupressaceae, we speculate that the<br />
ecological association might be ancient.<br />
Thus far three cypress-l<strong>in</strong>ked species are known to<br />
belong to sub<strong>section</strong> Cupressorum (FIGS. 1–2). All are<br />
of slender to medium stature (none have very short<br />
stems), with pendant annuli <strong>and</strong> rufescent flesh.<br />
<strong>Agaricus</strong> cupressicola, the type of Cupressorum, was<br />
orig<strong>in</strong>ally (Grilli 1988) placed <strong>in</strong> <strong>section</strong> Sangu<strong>in</strong>olenti<br />
(type species: A. sylvaticus Schaeff.), <strong>and</strong> by casual<br />
criteria (simple pendant annulus, red discolor<strong>in</strong>g) A.<br />
tlaxcalensis <strong>and</strong> A. cupressophilus could be placed<br />
there as well. However we noted that mycelia of these<br />
three species grow well <strong>and</strong> are white on compost<br />
extract agar or PDA, while A. sylvaticus <strong>and</strong> other<br />
species of Sangu<strong>in</strong>olenti grow very slowly <strong>and</strong> become<br />
brown as the medium around them becomes browner.<br />
We have successfully cultivated A. cupressicola <strong>and</strong><br />
A. cupressophilus (A. tlaxcalensis has not been tried).<br />
Phylogenetic analyses <strong>in</strong>dicate that these latter<br />
three species are not close to Sangu<strong>in</strong>olenti but<br />
<strong>in</strong>stead belong to a s<strong>in</strong>gle basally jo<strong>in</strong>ed clade with<strong>in</strong><br />
<strong>section</strong> <strong>Bivelares</strong>. In MP bootstrap analysis, a clade<br />
conta<strong>in</strong><strong>in</strong>g Cupressorum with<strong>in</strong> <strong>Bivelares</strong> had 96%<br />
support; Cupressorum itself had 99% bootstrap support<br />
(FIG. 1). A neighbor-jo<strong>in</strong><strong>in</strong>g tree also <strong>in</strong>dicated a<br />
sister relationship between sub<strong>section</strong>s Cupressorum<br />
<strong>and</strong> Hortenses (FIG. 2). On balance we prefer to<br />
recognize these two sub<strong>section</strong>s with<strong>in</strong> <strong>Bivelares</strong>, while<br />
not<strong>in</strong>g that three of six MP trees obta<strong>in</strong>ed did not<br />
<strong>in</strong>clude a unified clade conta<strong>in</strong><strong>in</strong>g both sub<strong>section</strong>s.<br />
Comments on A. cupressicola.—<strong>Agaricus</strong> cupressicola<br />
was first described under Cupressus sempervirens, <strong>in</strong><br />
the Abruzzi, <strong>in</strong> central Italy (Grilli 1988). It is also<br />
relatively frequent on the French Atlantic coast under<br />
C. macrocarpa (Gu<strong>in</strong>berteau et al 1998) <strong>and</strong> has been<br />
found by Callac under Juniperus phoenicea along the<br />
coast <strong>in</strong> Portugal. The latter small tree produces a
litter of small branches very similar to those of<br />
Cupressus spp.; such trees might have represented<br />
an ecological niche for cypress-lov<strong>in</strong>g species of<br />
<strong>Agaricus</strong> dur<strong>in</strong>g the absence of cypresses <strong>in</strong> cont<strong>in</strong>ental<br />
Europe after their local ext<strong>in</strong>ction dur<strong>in</strong>g the<br />
glacial period, before their (re)<strong>in</strong>troduction by<br />
humans. Contu (1992) also has encountered <strong>Agaricus</strong><br />
cupressicola <strong>in</strong> coastal s<strong>and</strong> dunes under Juniperus<br />
phoenicea, <strong>and</strong> Contu <strong>in</strong>dicated that He<strong>in</strong>emann<br />
(pers comm) had found it <strong>in</strong> Belgium under<br />
broadleaf trees with Taxus baccata. Gu<strong>in</strong>berteau et<br />
al (1998: 26, pl. 3, photo 5) reported it beneath<br />
Rob<strong>in</strong>ia. Lanconelli (2003) cited it under Cedrus<br />
atlantica <strong>and</strong> <strong>in</strong>dicated that Grilli (pers comm) had<br />
found it under Populus. <strong>Agaricus</strong> cupressicola has<br />
never been found <strong>in</strong> North America. (Similarly A.<br />
cupressophilus, collected ca. 100 km from the 19th<br />
century Californian refuge area of C. macrocarpa<br />
around Carmel Bay, is not known from Europe <strong>and</strong><br />
apparently has not followed the <strong>in</strong>troduction of this<br />
tree.)<br />
We have observed a moderate degree (3/705<br />
characters) of ITS sequence differentiation between<br />
(i) the Italian type specimen (coll. 84.112 deposited<br />
<strong>in</strong> Grilli’s private herbarium) (GenBank EU363031)<br />
of A. cupressicola <strong>and</strong> (ii) two collections of French<br />
(CA554: France, Budos, Laulan, 30 Sep 2006, under<br />
Cupressocyparis leyl<strong>and</strong>ii, leg. & det. J. Gu<strong>in</strong>berteau:<br />
[GenBank EU363030]; CA72 [5Cp1; GenBank<br />
AJ418769: Challen et al 2003]) <strong>and</strong> (iii) Greek<br />
(CA99 [5Cp2; GenBank AJ418770: Challen et al<br />
2003]) orig<strong>in</strong> (vouchers <strong>and</strong>/or stra<strong>in</strong>s at CGAB). A<br />
collection from Portugal (CA548: Portugal, Odeceixe,<br />
Monte Novo, 29 Oct 2006, under Juniperus phoenicea<br />
<strong>in</strong> paleodunes at the top of a coastal cliff (leg. & det.<br />
P. Callac <strong>and</strong> A. Tancrède [CGAB]: GenBank<br />
EU363029) was heteromorphic for all three of the<br />
sequence characters differ<strong>in</strong>g between the French/<br />
Greek <strong>and</strong> Italian material, suggest<strong>in</strong>g that <strong>in</strong>terbreed<strong>in</strong>g<br />
might be occurr<strong>in</strong>g with<strong>in</strong> a s<strong>in</strong>gle, somewhat<br />
subdivided species. Us<strong>in</strong>g these (nonredundant)<br />
sequences, an A. cupressicola clade had 58% bootstrap<br />
support. In N-J trees <strong>and</strong> <strong>in</strong> half of the nonredundant<br />
MP trees our A. cupressicola sequences group together.<br />
A speciation process may be <strong>in</strong> flux among<br />
Mediterranean A. cupressicola.<br />
Two new species <strong>in</strong> sub<strong>section</strong> Cupressorum are now<br />
described from North America:<br />
<strong>Agaricus</strong> tlaxcalensis Callac et G. Mata, sp. nov.<br />
FIGS. 3–7<br />
Figura saepe gracilis. Pileus 2–8 cm pallide brunneogriseus;<br />
primum convexo-cuboideus, conico-campanulatus<br />
fit, de<strong>in</strong>de plano-convexus cum p<strong>and</strong>itur, modice umbonatus,<br />
radiose fibrillosus, nonnumquam fibrillo-squamulosus,<br />
tactu rufescens. Stipes saepe curvus vel flexuosus (4)5–8(12)<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 881<br />
FIG. 3. Cheilocystidia of A. tlaxcalensis (Holotype,<br />
MATA 681).<br />
cm 3 5–15 mm. Simplex annulus membranaceus ac<br />
descendens. Caro a roseo ad rutilum admodum transit,<br />
de<strong>in</strong>de siccati sangu<strong>in</strong>is colorem ducit. Agaricum bisporum<br />
olet. Sporae ellipsoideae, forte ovato-ellipsoideae, (4.5–)<br />
4.8–6.3(–6.9) 3 (3.4–)3.6–4.8(–5.5) mm. Basidia tetrasporigera<br />
(21–27 3 6–8 mm). Cheilocystidia articulosa cyl<strong>in</strong>droutriformiaque<br />
18–30 mm 3 6–12 mm. Sub cupressis lectus.<br />
In septentrionalis Americae superioribus locis situs. HO-<br />
LOTYPUS: ‘‘Mexico, 11 October 2003, MATA 681’’ <strong>in</strong><br />
herbario XAL depositur. MB 512234.<br />
Pileus at first cuboidal-convex, becom<strong>in</strong>g conicocampanulate,<br />
later exp<strong>and</strong><strong>in</strong>g to plano-convex with a<br />
low umbo, 2–8 cm diam; pileipellis beige-gray, exceed<strong>in</strong>g<br />
whitish marg<strong>in</strong>, radially fibrillose, sometimes<br />
fibrillose-squamulose, the background layer concolorous,<br />
rufescent on h<strong>and</strong>l<strong>in</strong>g. In drier weather the<br />
surface can become smooth to polished, with concentric<br />
cracks. Lamellae free, ca. 10 per cm at 1 cm<br />
from stipe, at first v<strong>in</strong>aceous p<strong>in</strong>k, rufescent on<br />
h<strong>and</strong>l<strong>in</strong>g, later chocolate brown; marg<strong>in</strong> not dist<strong>in</strong>ctive.<br />
Stipe subequal, progressively thicker toward the<br />
base, sometimes curved or flexuous, sp<strong>in</strong>dly, with a<br />
length up to 2.5 times the width of the pileus, (4)5–<br />
8(12) cm long 3 5–15 mm thick; surface longitud<strong>in</strong>ally<br />
fibrillose, silky, partly rugulose, white, quickly<br />
becom<strong>in</strong>g sordid brown; basal bulb absent but<br />
rhizomorph often present at the base; context white,<br />
becom<strong>in</strong>g strongly red-orange when <strong>section</strong>ed, then<br />
v<strong>in</strong>aceous beige-gray, <strong>and</strong> f<strong>in</strong>ally dried-blood colored.<br />
Odor fa<strong>in</strong>t, typical of <strong>Bivelares</strong>. Veils dist<strong>in</strong>ctive, th<strong>in</strong>,<br />
membranous, sometimes rema<strong>in</strong><strong>in</strong>g attached to the<br />
pileus marg<strong>in</strong>, form<strong>in</strong>g an apical descend<strong>in</strong>g (pendant)<br />
annulus, white then v<strong>in</strong>aceous brown, persistent.<br />
Spores dark brown at maturity, ellipsoid to ovoideoellipsoid,<br />
(4.5–)4.8–6.3(–6.9) 3 (3.4–)3.6–4.8(–5.5)<br />
mm, mean 5 5.3 3 4.2 mm (N5 240), L/W 5 1.28;<br />
hilar appendix prom<strong>in</strong>ent. Basidia tetrasporic, cyl<strong>in</strong>dro-clavate,<br />
21–27 3 6.0–8.0 mm; sterigmata 2–3 mm<br />
long. Cheilocystidia present, often <strong>in</strong> cha<strong>in</strong>s of
882 MYCOLOGIA<br />
FIGS. 4–12. Basidiomata. 4–7. Four collections of <strong>Agaricus</strong> tlaxcalensis under Cupressus benthamii <strong>in</strong> Tlaxcala State, Mexico;<br />
bars 5 10 cm, to demonstrate variation <strong>in</strong> proportions. 4–6. In Rancho el Tejocote. 4. 19/07/2001 (not accessioned); 5. 02/<br />
10/2002, GANDARA 377; 6. 10/10/2003, MATA 675. 7. In the National Park of the Mal<strong>in</strong>che, MATA 6817, 11/10/2003:<br />
Holotype. 8. A. cupressophilus. FS-22*: Holotype. Specimens cultivated from culture FS-22, prepared from material from San
oadly cyl<strong>in</strong>drical elements, the term<strong>in</strong>al element<br />
be<strong>in</strong>g cyl<strong>in</strong>drical-utriform; 18–30 mm 3 6–12 mm.<br />
Chemistry: not determ<strong>in</strong>ed.<br />
ITS1 + 2 sequence (GenBank EU363032–<br />
33): L 5 705 nt (estimated). Dist<strong>in</strong>ctive characters:<br />
agagTttggaCtggt @ 486 & 492. The first might be<br />
unique <strong>in</strong> <strong>Agaricus</strong>, the second is unique with<strong>in</strong><br />
<strong>Bivelares</strong>.<br />
Habit, habitat, known distribution, occurrence:<br />
Isolated, gregarious or cespitose <strong>in</strong> litter under<br />
planted Cupressus benthamii, at more than 2400 m<br />
altitude <strong>in</strong> the state of Tlaxcala (Mexico); possibly<br />
frequent.<br />
Specimens exam<strong>in</strong>ed: MEXICO. Tlaxcala state: National<br />
Park of the Mal<strong>in</strong>che (near the restaurant of the park),<br />
under Cupressus benthamii, 11 Oct 2003, MATA 681, P.<br />
Callac <strong>and</strong> G. Mata, HOLOTYPE (XAL); Rancho El<br />
Tejocote at 2 km from Cuapiaxtla, under Cupressus<br />
benthamii, 19 Jul 2001, lost specimen, P. Callac <strong>and</strong> G.<br />
Mata; 02 Oct 2002, GANDARA 377 (XAL), G. Mata; 10 Oct<br />
2003, MATA 675 (XAL), P. Callac <strong>and</strong> G. Mata; Mycelium<br />
cultures of MATA 675 <strong>and</strong> MATA 681 both are available at<br />
MYCSA-INRA Bordeaux (CGAB: stra<strong>in</strong>s CA 219 <strong>and</strong> CA 221<br />
respectively).<br />
Etymology: from Tlaxcala state, Mexico.<br />
<strong>Agaricus</strong> tlaxcalensis is most closely related to A.<br />
cupressicola <strong>and</strong> A. cupressophilus. It resembles A.<br />
cupressicola <strong>in</strong> the small size of the sporocarp, the dull<br />
color of the pileus, the simple membranous descend<strong>in</strong>g<br />
annulus, the red discolor<strong>in</strong>g when <strong>section</strong>ed, the<br />
spore size, <strong>and</strong> the cypress habitat. It differs by its<br />
more slender, elongated silhouette with a stipe often<br />
curved, the stronger discolor<strong>in</strong>g when <strong>section</strong>ed or<br />
touched, the larger basidia (20–25 3 5–6 mm for A.<br />
cupressicola, per Bon [1987]), the cheilocystidia <strong>in</strong><br />
cha<strong>in</strong>s, which are rather cyl<strong>in</strong>drical-utriform than<br />
broadly clavate (see FIG. 4), <strong>and</strong> the highl<strong>and</strong><br />
Mexican geographic distribution. A. tlaxcalensis<br />
differs from A. cupressophilus by its substantially<br />
longer spores <strong>and</strong> larger cystidia, by its cap that tends<br />
to rema<strong>in</strong>s umbonate at maturity, while the cap of A.<br />
cupressophilus tends to become depressed, <strong>and</strong><br />
possibly by its geographic distribution because A.<br />
cupressophilus thus far is known only from under<br />
Monterey cypress <strong>in</strong> coastal California. ITS1+2 sequences<br />
of two A. tlaxcalensis collections consistently<br />
differ from A. cupressicola by seven polymorphisms<br />
<strong>and</strong> from A. cupressophilus by two polymorphisms. In<br />
r<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 883<br />
view of all of the data A. tlaxcalensis <strong>and</strong> A.<br />
cupressophilus are best treated as separate species.<br />
Edibility of A. tlaxcalensis is unknown.<br />
<strong>Agaricus</strong> cupressophilus Kerrigan, sp. nov. FIG. 8<br />
Figura gracilis. Pileus 1–7 cm latus, pallide griseus,<br />
mutabilis, brunnescens. Stipes plerumque aequalis, e farcto<br />
cavus, cavitum amplo, rufescens. Sporae ellipsoideae, (4.2–)<br />
4.6(–4.9) 3 (3.4–)3.7–3.9(–4.2) mm, basidia tetrasporigera,<br />
19–23 3 6–7 mm, cheilocystidia fasciculata, 16–22 3 6–7.5<br />
(–8.0) mm, plerumque cyl<strong>in</strong>dro-clavata. HOLOTYPUS: FS-<br />
22*, <strong>in</strong> herbario SFSU depositur. MB 512229.<br />
Pileus (1–) 4–7 cm diam., convex to broadly<br />
convex, center slightly depressed, marg<strong>in</strong> slightly<br />
<strong>in</strong>curved; pileipellis glabrous to m<strong>in</strong>utely appressedsquamulose,<br />
the fibrils grayish to gray-brown to buff<br />
or light tan, potentially develop<strong>in</strong>g dark brown sta<strong>in</strong>s<br />
hours after h<strong>and</strong>l<strong>in</strong>g; context (3–)5–8 mm thick,<br />
white becom<strong>in</strong>g sordid v<strong>in</strong>aceous-flesh color, odor<br />
<strong>in</strong>dist<strong>in</strong>ct to spicy. Lamellae free, close, ca. 13 per cm<br />
at 1 cm from stipe, 2–4 mm broad, marg<strong>in</strong> concolorous<br />
or fa<strong>in</strong>tly paler, dull flesh-colored becom<strong>in</strong>g dark<br />
brown. Stipe subequal to subclavate, (5–)10–12 mm<br />
above, (7–)11–20 mm below 3 (1.5–)4–6 cm long;<br />
stipitipellis buff to tan, lustrous-fibrous, color<strong>in</strong>g as<br />
context or more reddish when <strong>in</strong>jured, potentially<br />
darken<strong>in</strong>g toward the base <strong>in</strong> age or long after<br />
h<strong>and</strong>l<strong>in</strong>g, lack<strong>in</strong>g velar remnants; context lustrousfibrous,<br />
rapidly becom<strong>in</strong>g sordid v<strong>in</strong>aceous-flesh<br />
color near cuticle <strong>and</strong> base when exposed, stuffedhollow,<br />
the cavity relatively broad (to 10 mm), filled<br />
with white, unchang<strong>in</strong>g pith. Veils form<strong>in</strong>g a th<strong>in</strong>,<br />
whitish, subapical to supramedian pendant annulus,<br />
often tend<strong>in</strong>g to rema<strong>in</strong> partially attached to the<br />
pileus marg<strong>in</strong>, <strong>and</strong>/or tear<strong>in</strong>g <strong>in</strong>to radial str<strong>and</strong>s <strong>and</strong><br />
fibers, readily collaps<strong>in</strong>g.<br />
Spores dark brown at maturity, ellipsoid, (4.2–)<br />
4.6(–4.9) 3 (3.4–)3.7–3.9(–4.2) mm, mean 5 4.6 3<br />
3.8 mm (N5 30, C 5 1); hilar appendix moderately<br />
prom<strong>in</strong>ent; apical pore not evident. Basidia tetrasporic<br />
(some bisporic), cyl<strong>in</strong>dro-clavate, 19–23 3 6–7 mm;<br />
sterigmata 3–4 mm long. Cheilocystidia present <strong>in</strong><br />
occasional fascicles, mostly about cyl<strong>in</strong>drical to<br />
clavate, sometimes broader, often irregular to somewhat<br />
contorted, 16–22 3 6–7.5 mm, apices sometimes<br />
mucronate, occasional cells to 12–16 mm broad<br />
present; elongated cells arranged parallel to the<br />
marg<strong>in</strong> generally predom<strong>in</strong>ate.<br />
Mateo County, California. Scale ca. 0.45 3. 9–10. A. subsubensis. 9. Field collection JB-101, Riverside County, California, which<br />
was cultured <strong>and</strong> from which holotype JB-101* was cultivated. Scale ca. 0.4 3. 10. A. subsubensis RWK 1789*, cultivated from a<br />
field spore sample from Monterey County, California. Scale ca. 0.7 3. 11. A. agr<strong>in</strong>ferus RWK 1397, Monterey County,<br />
California: Holotype. Scale ca. 0.3 3. 12. A. devoniensis ssp. bridghamii RWK 1899, Clear Creek County Colorado: Holotype.<br />
Credit: 4, 5, 7 P. Callac; 6 G. Mata; 8–12: R.W. Kerrigan. U.S. penny is 18.5 mm diam.
884 MYCOLOGIA<br />
Chemistry: KOH yellow on disk (only); anil<strong>in</strong>e 3<br />
GAA (glacial acetic acid) negative.<br />
ITS1+2 sequence (GenBank EU258676): L 5<br />
708 nt. Dist<strong>in</strong>ctive characters: gagg[TG]ggat @ 612–<br />
613.<br />
Habitat, known distribution, occurrence; material<br />
studied: Known only from collection FS-22 (no longer<br />
extant), under Cupressus macrocarpa, USA, CA, San<br />
Mateo County, Pacifica, Skyl<strong>in</strong>e Blvd., Fred Stevens,<br />
26 Aug 1990. HOLOTYPE: FS-22*, basidiomata<br />
cultivated from tissue culture ARP 042 of FS-22<br />
(5MYA-4431 at ATCC) at Sylvan Research, Nov 2005<br />
[SFSU]; paratype (second gather<strong>in</strong>g from crop that<br />
produced holotype) [NY]; paratype 5 preserved<br />
culture MYA-4431, ATCC.<br />
Etymology: A cypress-lov<strong>in</strong>g species.<br />
The ITS sequence of A. cupressophilus is most<br />
similar to that of the Mexican A. tlaxcalensis, followed<br />
by the European A. cupressicola. The collector of the<br />
only known field specimens, Dr F. Stevens, provisionally<br />
identified the fresh FS-22 material as A. bisporus.<br />
He cultured this material but unfortunately no<br />
voucher was preserved. RFLP analysis done on the<br />
culture by RWK while <strong>in</strong> the J.B. Anderson lab,<br />
University of Toronto at Er<strong>in</strong>dale, demonstrated that<br />
FS-22 was not A. bisporus or any other RFLPcharacterized<br />
species. An opportunity to obta<strong>in</strong> the<br />
ITS1+2 DNA sequence later arose, thanks to J.-P. Xu<br />
of McMaster University. Availability of the unique<br />
sequence allowed placement of this entity with<strong>in</strong> the<br />
emergent Cupressorum clade <strong>and</strong> <strong>in</strong>spired the successful<br />
attempt to cultivate specimens, from which the<br />
description of the basidiomata was prepared.<br />
The yellow color appear<strong>in</strong>g with KOH on the disk is<br />
unusual, not normally reported for <strong>section</strong> <strong>Bivelares</strong>.<br />
This is likely to have been associated with velar<br />
hyphae, which can rema<strong>in</strong> as a substantial but obscure<br />
presence on the disk. We would not be surprised if<br />
this reaction were not observed on other material that<br />
had developed under different (e.g. less humid)<br />
conditions. No other tissue exhibited a yellow<strong>in</strong>g<br />
reaction with KOH.<br />
For a comparison with A. tlaxcalensis see that<br />
species. Edibility is unknown.<br />
<strong>Agaricus</strong> [<strong>section</strong> <strong>Bivelares</strong>] sub<strong>section</strong> Hortenses<br />
He<strong>in</strong>em., Bull. Jard. Bot. Etat 26:22. 1956.<br />
; <strong>Agaricus</strong> <strong>section</strong> Hortenses (He<strong>in</strong>em.) Bon, Doc.<br />
Mycol. 15(60):9. 1985.<br />
Orig. diag.: Vela complexa. Acies lamellarum semper<br />
cheilocystidiis praedita. Sporae breviter ellipticae, crasse<br />
tunicatae, poro non praeditae. Species ruderales, <strong>in</strong>terdum<br />
halophilae.<br />
Type: <strong>Agaricus</strong> hortensis (J.E. Lange) S. Imai<br />
[‘‘<strong>Agaricus</strong> hortensis (Cooke) Pilát’’], designated by<br />
He<strong>in</strong>emann, Bull. Jard. Bot. Etat 26:22. 1956.<br />
We regard A. hortensis ( J.E. Lange) S. Imai 1938<br />
(nom. illeg., art. 53.1 non A. [Clitocybe] hortensis<br />
Pers. : Fr., 1801), also known <strong>in</strong> literature as A.<br />
hortensis (Cooke) Pilát, 1951, to be a heterotypic<br />
synonym of A. bisporus ( J.E. Lange) Imbach 1946.<br />
The correct name for the sub<strong>section</strong> <strong>in</strong>clud<strong>in</strong>g A.<br />
bisporus is therefore <strong>Agaricus</strong> subsect. Hortenses He<strong>in</strong>em.<br />
(1956). This sub<strong>section</strong> is dist<strong>in</strong>guished from<br />
subsect. Cupressorum as noted above: the common<br />
tendency toward complex <strong>and</strong>/or thickened, even<br />
cottony or somewhat rubbery, often <strong>in</strong>termediate to<br />
occasionally peronate velar structures, <strong>and</strong> the pileus<br />
edge strongly <strong>in</strong>volute at least <strong>in</strong> young basidiomata <strong>in</strong><br />
Hortenses are likely to be the most useful macroscopic<br />
characters <strong>in</strong> this regard. Stature <strong>and</strong> habitat also can<br />
be useful. DNA ITS1+2 characters have been noted<br />
above. Three new <strong>taxa</strong> are described:<br />
<strong>Agaricus</strong> subsubensis Kerrigan, sp. nov. FIGS. 9–10<br />
Pileus 1–9 cm latus, convexus vel undulato-planus, laxe<br />
<strong>in</strong>tertextus, albus, mutabilis, aetate rufobrunneis; contextus<br />
ex albo obscure rufescens vel v<strong>in</strong>aceus; stipes 5–15(–25) mm<br />
crassus, 0.5–4.5 cm longus, plerumque aequalis, plerumque<br />
solidus; vela alba, friabilis; sporae (5.7–)6.2–6.7(–7.4) 3<br />
(4.3–4.9–)5.3–5.6(–6.2) mm; basidia 24–30 3 7.4–9.2 mm,<br />
bi-, tri-, et tetraspora; cheilocystidia nulla vel cyl<strong>in</strong>drica vel<br />
clavata. HOLOTYPUS: JB-101*, <strong>in</strong> herbario SFSU depositur.<br />
MB 512226.<br />
Description of JB-101: Pileus broadly convex,<br />
uneven, with <strong>in</strong>rolled marg<strong>in</strong>, becom<strong>in</strong>g approximately<br />
plane, wavy, 1–9 cm broad; cuticle somewhat loosely<br />
<strong>in</strong>terwoven, more scurfy than glabrous, with concolorous<br />
velar patches toward <strong>and</strong> on disk, obscurely rimose<br />
toward marg<strong>in</strong>, whitish, becom<strong>in</strong>g light brown (7C4 or<br />
lighter) when dried, or if bruised when young, quickly<br />
turn<strong>in</strong>g reddish brown (8D-E8, 9F5), then dry<strong>in</strong>g a<br />
mahogany brown (9F6); context semifirm, whitish,<br />
unchang<strong>in</strong>g <strong>in</strong> a V-shaped zone beneath disk, becom<strong>in</strong>g<br />
sordid above lamellae <strong>and</strong> p<strong>in</strong>kish above stipe <strong>and</strong><br />
at boundary of sordid <strong>and</strong> unchang<strong>in</strong>g zones, odor<br />
mild, <strong>in</strong>dist<strong>in</strong>ct, like that of A. bisporus. Lamellae free,<br />
not close, ca. 10–11 per cm at 1 cm from stipe. Stipe<br />
about equal, 5–15 mm 3 0.5–4.5 cm long; cuticle<br />
semismooth beneath velar remnants; context whitish,<br />
almost unchang<strong>in</strong>g or becom<strong>in</strong>g sordid, except<br />
becom<strong>in</strong>g p<strong>in</strong>kish at cuticles above <strong>and</strong> below, <strong>and</strong><br />
<strong>in</strong> basal context, with an <strong>in</strong>dist<strong>in</strong>ctly def<strong>in</strong>ed central<br />
region of white pith, especially evident <strong>and</strong> tend<strong>in</strong>g to<br />
become hollow near apex, base potentially subtended<br />
by copious str<strong>in</strong>gy mycelia (at least <strong>in</strong> culture). Veils<br />
form<strong>in</strong>g a narrow, <strong>in</strong>dist<strong>in</strong>ct, b<strong>and</strong>-like, supramedian,<br />
subperonate annulus, more obviously form<strong>in</strong>g a series<br />
of th<strong>in</strong> broken r<strong>in</strong>g-like remnants on the lower twothird<br />
of stipe, <strong>and</strong> sometimes velar patches on pileus<br />
(disk).<br />
Spores dark brown at maturity, broadly ellipsoid,<br />
(5.7–)6.2–6.6(–7.2) 3 (4.9–)5.3–5.6(–6.2) mm, mean
5 6.4 3 5.5 mm (N5 30, C 5 1); hilar appendix<br />
unremarkable; no apical pore evident. Basidia tetrasporic,<br />
cyl<strong>in</strong>dro-clavate, 24–30 3 7.4–9.2 mm; sterigmata<br />
4–5 mm long. Cheilocystidia absent or basidiolelike,<br />
the lamellar edge bear<strong>in</strong>g basidia <strong>and</strong> basidiolelike<br />
cells <strong>in</strong> regions one to several mm long, otherwise<br />
naked, with exposed hyphae oriented parallel to the<br />
edge.<br />
Chemistry: KOH negative; anil<strong>in</strong>e 3 GAA negative;<br />
o-tolid<strong>in</strong>e blue on cuticles <strong>and</strong> veils, stipe <strong>and</strong> disk<br />
context near cuticle, stipe pith <strong>and</strong> basal zone, purple<br />
on lamellae <strong>and</strong> somewhat obscurely so above basal<br />
context, otherwise unchang<strong>in</strong>g; alpha-naphthol<br />
brownish to scarlet on pileus cuticle, unchang<strong>in</strong>g or<br />
slightly orangish on pileus <strong>and</strong> stipe context except<br />
becom<strong>in</strong>g purple <strong>in</strong> portions of lower stipe.<br />
ITS1+2 sequence of the type (GenBank EU131641):<br />
L 5 661 nt, anomalous, with major deletions, substitutions<br />
<strong>and</strong>/or modified sequence from pos 124<br />
(gggtatA.) to pos 171 (.Gcctgtc), with up to 28<br />
other possible recently modified characters throughout<br />
the ITS. See discussion below, <strong>in</strong>clud<strong>in</strong>g ITS1+2<br />
sequence of RWK 1789 (GenBank EU257802).<br />
Habit, habitat, known distribution, occurrence;<br />
material studied: USA. California. Riverside County<br />
circa Thermal: JB-101, R.W. Kerrigan 7 Mar 1992<br />
(paratype, SFSU); gregarious, fascicled to cespitose,<br />
<strong>in</strong> s<strong>and</strong>y soil <strong>in</strong> sparsely l<strong>and</strong>scaped area near<br />
dwell<strong>in</strong>g, Sonoran Desert, elevation 26 m [5type<br />
locality]; specimen designated JB-101* (HOLOTYPE,<br />
SFSU; isotypes: NY, MA), cultivated at Sylvan Research,<br />
Kittann<strong>in</strong>g, Pennsylvania, from MYA-2981<br />
(paratype [preserved culture], ATCC), a culture<br />
derived from JB-101; Monterey County, Pacific Grove:<br />
RWK 1789* (SFSU), cultivated at Sylvan Research,<br />
Kittann<strong>in</strong>g, Pennsylvania, from MYA-4432 (preserved<br />
culture, ATCC), derived from RWK 1789 (no longer<br />
extant; collected from soil <strong>and</strong> Cupressus litter at<br />
Esplanade Park by R.W. Kerrigan).<br />
Etymology: The epithet ‘‘subsubensis’’ honors the<br />
tradition <strong>in</strong> <strong>Agaricus</strong> taxonomy of prefix<strong>in</strong>g epithets of<br />
the less-dist<strong>in</strong>ctive species with ‘‘sub’’. It is tempt<strong>in</strong>g to<br />
say that this species is noteworthy for its lack of<br />
dist<strong>in</strong>guish<strong>in</strong>g macroscopic features.<br />
Specimens from the desert were easily cultivated;<br />
they fruit prolifically <strong>and</strong> are therefore better known<br />
to us. These basidiomata tend to be small <strong>and</strong><br />
irregularly shaped, with an <strong>in</strong>dist<strong>in</strong>ct pileipellis. They<br />
are unlikely to be deemed attractive. The <strong>in</strong>delible<br />
rubro-brunnescence of the pileus surface observed <strong>in</strong><br />
sound young specimens is an unusual feature<br />
sometimes seen <strong>in</strong> a few species of sect. <strong>Agaricus</strong><br />
<strong>and</strong> sect. Sangu<strong>in</strong>olenti (Kerrigan 1986).<br />
RWK 1789 was not recognized <strong>in</strong>itially as conspecific<br />
with JB-101; <strong>in</strong> addition to its very different<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 885<br />
habitat, it is not easy to cultivate <strong>and</strong> only a few<br />
basidiomata were obta<strong>in</strong>ed from the orig<strong>in</strong>al culture.<br />
The two collections differ most remarkably <strong>in</strong> the<br />
divergence of their ITS sequences. The ITS1+2<br />
sequence of JB-101 was obviously anomalous with<strong>in</strong><br />
its group, <strong>and</strong> reproducibly so: two <strong>in</strong>dependent<br />
amplifications of genomic DNA obta<strong>in</strong>ed from<br />
mycelia grown <strong>in</strong> broth yielded a s<strong>in</strong>gle shared<br />
sequence with multiple deletions <strong>and</strong> other alterations.<br />
One segment of the ITS1 is unalignable with<br />
other <strong>Agaricus</strong> ITS1 sequences. At 661 nt the JB-101<br />
ITS1+2 is much shorter, by about 40–50 nt, than <strong>in</strong><br />
any other known <strong>Agaricus</strong>; whereas <strong>in</strong> RWK 1789 this<br />
DNA segment is unremarkable at 707 nt long. Even<br />
the alignable portions of JB-101 <strong>and</strong> RWK 1789 have a<br />
great number of differences between them but also<br />
some unusual shared unique characters. Both sequences<br />
have characteristics of <strong>section</strong> <strong>Bivelares</strong>, for<br />
example an 8 nt deletion about 55 nt 59 of the 39 end<br />
of the ITS1+2 region (as we def<strong>in</strong>e it). The<br />
phylogenetic situation became clear when uniquely<br />
match<strong>in</strong>g sequences from the LSU, EF1-a, <strong>and</strong> RPB2<br />
genes were obta<strong>in</strong>ed from the two cultures (Kerrigan<br />
<strong>and</strong> Xu unpubl; GenBank EU284015–20). Thus the<br />
ITS1+2 region of JB101 evidently has been altered <strong>in</strong><br />
a major way by an event or process that violates the<br />
assumptions of nucleotide substitution based phylogenetic<br />
analysis. While <strong>in</strong>terest<strong>in</strong>g, such a sequence<br />
cannot be used <strong>in</strong> a mean<strong>in</strong>gful tree-build<strong>in</strong>g<br />
exercise. The phylogenetic <strong>and</strong> geographical scope<br />
of the altered sequence is not known. However, based<br />
on comparisons of other gene sequences from the<br />
two cultures, we regard it to be an <strong>in</strong>traspecies event,<br />
hypothetically mark<strong>in</strong>g disjunct populations.<br />
This <strong>rare</strong> species has been collected once (among<br />
an abundance of A. bisporus var. burnettii) near Indio,<br />
California, <strong>in</strong> desert s<strong>and</strong> below sea level, <strong>and</strong> once<br />
(among an abundance of A. bisporus var. bisporus) <strong>in</strong><br />
Pacific Grove, California, with<strong>in</strong> meters of the coast<br />
(see Kerrigan 1995, FIG. 3, for site map <strong>and</strong> position<br />
of RWK 1789). Because the JB-101 material is much<br />
better documented, <strong>in</strong> part because of its cultivability,<br />
it therefore was selected as the type of the species.<br />
The significance of its anomalous ITS sequence is<br />
unknown; the species concept <strong>in</strong>cludes both of the<br />
known ITS sequences. Given the cultural <strong>and</strong> habitat<br />
differences between the two collections, while keep<strong>in</strong>g<br />
<strong>in</strong> m<strong>in</strong>d the ITS sequence differences, it rema<strong>in</strong>s<br />
possible that two divergent populations or even <strong>rare</strong><br />
sister <strong>taxa</strong> are <strong>in</strong>volved. For that reason a separate<br />
description of cultivated RWK 1789 (FIG. 10) is<br />
provided:<br />
Pileus 4–6.5 cm diam, oval <strong>in</strong> top view, convex to<br />
broadly convex, becom<strong>in</strong>g subplane or with two<br />
uplifted sides, center slightly depressed, the marg<strong>in</strong>
886 MYCOLOGIA<br />
<strong>in</strong>curved until maturity, ultimately with semishallow<br />
notched radial fissures; pileipellis glabrous to broadly,<br />
<strong>in</strong>dist<strong>in</strong>ctly appressed-squamose, the squamae large,<br />
obscurely imbricate, apparently due to velar separations,<br />
the surface white when young, becom<strong>in</strong>g<br />
unevenly v<strong>in</strong>aceous-brown from button stage through<br />
maturity; context (3–)5–8 mm thick, white or whitish<br />
becom<strong>in</strong>g salmon p<strong>in</strong>k-orange to darker v<strong>in</strong>aceous <strong>in</strong><br />
places when cut, odor <strong>in</strong>dist<strong>in</strong>ct, mushroom–like (as<br />
<strong>in</strong> A. bisporus), of sweet chicory at pileus after velar<br />
rupture. Lamellae free, close (distally) or not (proximally),<br />
with a pronounced bifurcat<strong>in</strong>g zone about<br />
1 cm from stipe, ca. 15 per cm at 1 cm from stipe, 1–<br />
2 mm broad, marg<strong>in</strong> concolorous, dull grayish fleshcolored<br />
becom<strong>in</strong>g medium brown. Stipe subequal,<br />
taper<strong>in</strong>g toward base, oval <strong>in</strong> cross-<strong>section</strong>, 8–13 3<br />
15–25 mm above, 7–8 mm below 3 3.5–4.5 cm long;<br />
stipitipellis whitish, semilustrous, adorned above the<br />
annulus with broad, appressed, dentate velar patchscales,<br />
these very light buff color, color<strong>in</strong>g as context<br />
or more orangish when <strong>in</strong>jured; context dull, whitish,<br />
rapidly becom<strong>in</strong>g salmon-orangish to v<strong>in</strong>aceous color<br />
<strong>in</strong> places <strong>and</strong> more sordid overall near base when<br />
exposed, stuffed-hollow, the cavity relatively broad (to<br />
11 mm), wedge-shaped, filled with white, rufescent<br />
pith. Veils form<strong>in</strong>g a short, narrow, submedian<br />
b<strong>and</strong>like <strong>in</strong>termediate annulus, whitish or becom<strong>in</strong>g<br />
brownish on the edge <strong>in</strong> age, the edge a two-limbed<br />
groove of 1–2 mm width when young, this <strong>in</strong>dist<strong>in</strong>ct<br />
<strong>in</strong> age, upper <strong>and</strong> lower annular surfaces smooth,<br />
rema<strong>in</strong><strong>in</strong>g white.<br />
Spores dark brown at maturity, ellipsoid, sometimes<br />
subglobose, occasionally narrowed toward one end,<br />
(5.7–)6.4–6.7(–7.4) 3 (4.3–4.9–)5.3–5.4(–6.0) mm,<br />
mean 5 6.5 3 5.4 mm (N 5 40, C 5 1); hilar<br />
appendix prom<strong>in</strong>ent; no apical pore evident. Basidia<br />
predom<strong>in</strong>antly tri- <strong>and</strong> bisporic, cyl<strong>in</strong>dro-clavate,<br />
24.5–27.5 3 (5.5–)7.5–8.5 mm; sterigmata 3–4 mm<br />
long. Cheilocystidia absent; lamellar marg<strong>in</strong> fertile.<br />
Chemistry: KOH gray-green on pileus surface,<br />
elsewhere <strong>in</strong>dist<strong>in</strong>ct; anil<strong>in</strong>e 3 GAA negative.<br />
ITS1+2 sequence (GenBank EU257802): L 5<br />
707 nt, unique characters: ctgtTcttg @ pos. 191.<br />
Description is of basidiomata cultivated from a<br />
spore culture of RWK 1789 at Sylvan Research, Nov<br />
2005.<br />
This unique collection was taken along with many<br />
others dur<strong>in</strong>g an unexpected collect<strong>in</strong>g opportunity<br />
under challeng<strong>in</strong>g travel conditions. It was only<br />
possible at the time to collect <strong>and</strong> process air-dried<br />
lamellae bear<strong>in</strong>g viable spores, which later were<br />
germ<strong>in</strong>ated, <strong>and</strong> to record spatial locations with<strong>in</strong><br />
the site. Growth <strong>and</strong> basidiomatal development are<br />
slow <strong>in</strong> this culture. Superficially RWK 1789 bears<br />
some resemblance to A. pequ<strong>in</strong>ii, a member of <strong>section</strong><br />
Chitonioides; the latter species has a peronate veil,<br />
solid stipe, deeply redden<strong>in</strong>g stipe context, <strong>and</strong><br />
abundant cheilocystidia.<br />
<strong>Agaricus</strong> agr<strong>in</strong>ferus Kerrigan et Callac, sp. nov.<br />
FIG. 11<br />
Pileus 5–8(–10) cm latus, late convexus, mediocriter fuscus<br />
vel raro subalbidus; pileipellis implexa, levis vel paulum<br />
squamosa, cont<strong>in</strong>uo <strong>in</strong>tegra <strong>in</strong> marg<strong>in</strong>e, cetera autem parte<br />
sordide aurantiaca evadente; stipes subaequalis, 3–8 cm<br />
longus 3 1–2 cm latus, externa parte levi, cetera autem parte<br />
paulum fibrata atque medullosa <strong>in</strong> medio, colorem sordide<br />
aurantiacum ducit; sporae circa 5.7–6.5 3 4.4–4.7 mm, late<br />
ellipsoideae vel tantum ellipsoideae, basidia tetrasporigera,<br />
cheilocystidia cyl<strong>in</strong>dracea forte etiam claviformia, 15–33 3 3–<br />
15 mm. Dispersus sub coniferis crescit <strong>in</strong> litoris regionibus<br />
parva altitud<strong>in</strong>e. HOLOTYPUS: Po<strong>in</strong>t Lobos State Reserve,<br />
Monterey County, California, USA: RWK 1397, 10 Jan 1986, <strong>in</strong><br />
herbario SFSU depositur. MB 512235.<br />
Pileus 5–8(–10) cm broad, at first broadly convex,<br />
with moderately <strong>in</strong>rolled marg<strong>in</strong>, becom<strong>in</strong>g plane,<br />
disk sometimes slightly depressed; surface dry, glabrous<br />
on disk, elsewhere <strong>in</strong>nately fibrillose, color<br />
pallid to sordid (when young) or medium brown, if<br />
brown then becom<strong>in</strong>g appressed fibrillose-squamose,<br />
squamae ca. 10 mm long 3 5–15 mm broad, background<br />
color whitish to pale buff; context white,<br />
becom<strong>in</strong>g reddish near lamellae <strong>and</strong> stipe apex when<br />
<strong>section</strong>ed, firm, up to 10–16 mm thick, odor of A.<br />
bisporus plus fruity/spicy as <strong>in</strong> some species of<br />
Sangu<strong>in</strong>olenti after exposure. Lamellae free, close,<br />
up to 10 mm broad, at first pallid to d<strong>in</strong>gy p<strong>in</strong>kish,<br />
f<strong>in</strong>ally dark blackish-brown, marg<strong>in</strong> somewhat pallid.<br />
Stipe 3–8 cm long 3 1–2 cm broad, equal to<br />
subclavate, occasionally ventricose (<strong>in</strong> culture), <strong>in</strong>terior<br />
white, quickly becom<strong>in</strong>g orange-red, ultimately<br />
red after longitud<strong>in</strong>al <strong>section</strong><strong>in</strong>g, <strong>in</strong> median transverse<br />
<strong>section</strong> becom<strong>in</strong>g orangish, stuffed-hollow;<br />
surface glabrous, or m<strong>in</strong>utely floccose or with<br />
fibrillose zones below, white, color<strong>in</strong>g as above when<br />
<strong>in</strong>cised; base firmly rooted <strong>in</strong> litter <strong>and</strong>/or soil. Veils<br />
form<strong>in</strong>g a supramedian, white <strong>in</strong>termediate-type<br />
annulus with a pendant aspect, sheath<strong>in</strong>g upward<br />
<strong>and</strong> with a broadly flar<strong>in</strong>g limb, unroll<strong>in</strong>g with the<br />
pileus marg<strong>in</strong>, upper surface smooth to striate, lower<br />
surface <strong>in</strong>terwoven to scurfy, sometimes subtended by<br />
one or two small limbs, marg<strong>in</strong> th<strong>in</strong>, uneven;<br />
universal veil also sometimes leav<strong>in</strong>g a scale r<strong>in</strong>g<br />
above the base of the stipe. Spores (4.5–5.3–)5.7–<br />
6.5(–7.1–7.5) 3 (4.1–)4.4–4.7(–5.3–6.0) mm, dark<br />
brown, broadly ellipsoid to ellipsoid, hilar appendix<br />
semiprom<strong>in</strong>ent, no apical pore evident. Basidia 17–<br />
28.5 3 (4–)7–9(–12) mm, clavate to cyl<strong>in</strong>dro-clavate,<br />
tetrasporic; sterigmata 1–4 mm long. Cheilocystidia<br />
15–33(–45) 3 3–15 mm, clustered, not abundant;<br />
lamellar marg<strong>in</strong> primarily composed of hyphae<br />
oriented parallel to the marg<strong>in</strong>, sterile.
Chemistry: KOH negative; anil<strong>in</strong>e 3 HNO3 negative;<br />
o-tolid<strong>in</strong>e blue on basal context of stipe,<br />
elsewhere violet; alpha-naphthol purple on basal<br />
context of stipe, elsewhere p<strong>in</strong>kish-orange to orange,<br />
weakly so on pileus context.<br />
Habit, habitat, known distribution, occurrence:<br />
Solitary to subgregarious under Cupressus macrocarpa<br />
<strong>in</strong> California, <strong>and</strong> under that tree <strong>and</strong> others <strong>in</strong> low<br />
elevation coastal areas of the U.K. <strong>and</strong> France. Fall–<br />
w<strong>in</strong>ter.<br />
ITS1+2 sequence (GenBank EU257801 <strong>and</strong><br />
AF432888 for RWK 1397 <strong>and</strong> FS-5 respectively;<br />
see also ITS1 sequences AJ418738-42, for RWK<br />
1397, Sf5, FS-10, FS-13, <strong>and</strong> WAT1 respectively:<br />
Challen et al 2003): L 5 703 nt. Dist<strong>in</strong>ctive characters:<br />
atgtTattg @ pos 259, vs C for A. subfloccosus, is<br />
unique with<strong>in</strong> <strong>Bivelares</strong>.<br />
Material exam<strong>in</strong>ed: USA. California. San Francisco<br />
County: RWK 738, 770b, 770c, 770*, 775, 884, 1117, 1140,<br />
1040*, 1153, 1175, FS-2 (5ARP 003, 5ATCC 66250), FS-5<br />
(5ARP 006, 5ATCC 66253), FS-13 (5ARP 030); San Mateo<br />
County: RWK 969, 1192, FS-10 (5ARP 020, 5ATCC 76561),<br />
FS-29 (5ARP 073, 5ATCC 200200); Monterey County: RWK<br />
1030, 1397 (TYPE: SFSU; Paratype: ATCC MYA-3441<br />
[preserved culture]), 1819, [1910 or 1911], NW-1 (5ARP<br />
205); UK. Scotl<strong>and</strong>: ATCC 34842; Engl<strong>and</strong>: W4 II* (5ATCC<br />
200593), W4 IV*, A 119* (5ATCC 200592), A 11*; France:<br />
Sf2 (5CA47), Sf5 (5CA70). (See also Kerrigan et al 1999,<br />
Challen et al 2003.) All California collections were made<br />
under Cupressus macrocarpa.<br />
Etymology: ager/agri: territory, l<strong>and</strong>; <strong>in</strong>ferus: lower.<br />
The epithet refers to the distribution of the species<br />
at elevations of about 5–200 m at middle northern<br />
latitudes.<br />
This mushroom was discussed <strong>in</strong> detail <strong>and</strong> figured<br />
by Kerrigan et al (1999). At that time we showed that,<br />
based on morphological, cultural, <strong>and</strong> molecular<br />
(RFLP, isozyme) characters, this lowl<strong>and</strong> entity was<br />
phylogenetically dist<strong>in</strong>ct from the highl<strong>and</strong>, spruce/<br />
fir/p<strong>in</strong>e associated form of A. subfloccosus (see<br />
Cappelli 1984, Parra <strong>and</strong> Suárez 2002, Kerrigan et al<br />
1999; this association is frequent but not absolute).<br />
Both entities have been referred to A. subfloccosus by<br />
various authors. We also <strong>in</strong>dicated that the two<br />
entities might be sister species, however without<br />
sequence data it was difficult to be confident of the<br />
best rank-relationship for them.<br />
We further noted the problem presented by the<br />
lack of a type specimen for Lange’s taxon. Lange<br />
(1926) mentioned only this syntype: ‘‘Hollufga˚rd<br />
form<strong>in</strong>g large fairy-r<strong>in</strong>gs <strong>in</strong> naked ground <strong>in</strong> plantation<br />
of Picea, Aug. 1915’’. No orig<strong>in</strong>al specimens of<br />
Psalliota subfloccosa exist <strong>in</strong> the Copenhagen Natural<br />
History Museum (H. Knudsen pers comm; Kerrigan<br />
et al 1999), therefore a lectotype designation is <strong>in</strong><br />
order:<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 887<br />
<strong>Agaricus</strong> subfloccosus (J.E. Lange) Hlaváček, Mykol.<br />
Sborn. 28(4–6):67. 1951 [‘‘subflocosus’’]<br />
Lectotype (hic designatus): [icon] ‘‘Psall. hortensis<br />
v. subfloccosa JEL.’’, <strong>in</strong> Lange, Danmarks Agaricaceer:<br />
plate 647 (unpubl), deposited <strong>in</strong> the Copenhagen<br />
Natural History Museum.<br />
Remarks: Plate 647 was reproduced by Lange<br />
(1939) as plate 139 D as Psalliota subfloccosa <strong>and</strong><br />
the orig<strong>in</strong>al plate is currently shown on the Copenhagen<br />
Botanical Museum web site at http://130.225.211.<br />
158/agaric<strong>in</strong>a/FMPro?-db5agaric<strong>in</strong>a.fp5&-format5<br />
flagar-storbillede.htm&-sortfield5fadtavle&-op5cn&<br />
FADogCurrent5<strong>Agaricus</strong>%20subfloccosus&-max5<br />
15&-recid533495&-f<strong>in</strong>d5<br />
Briefly the key field characters useful <strong>in</strong> differentiat<strong>in</strong>g<br />
A. agr<strong>in</strong>ferus from A. subfloccosus are: the<br />
former has an <strong>in</strong>terwoven pileipellis that is usually<br />
not pronouncedly scaly nor rimose toward the marg<strong>in</strong><br />
(as <strong>in</strong> the latter) <strong>and</strong> is most often a somewhat darker<br />
medium-brown (except <strong>in</strong> <strong>rare</strong> specimens with a<br />
nearly white pileus when young) than the pallid to<br />
lighter brown of the latter; A. agr<strong>in</strong>ferus has a<br />
relatively uniform stipe context, vs. the more fibrous<br />
stipe context of A. subfloccosus; the two species are<br />
separated by habitat <strong>and</strong> by elevation differences<br />
reach<strong>in</strong>g hundreds or thous<strong>and</strong>s of meters at middle<br />
latitudes <strong>in</strong> the northern hemisphere.<br />
Although these two l<strong>in</strong>eages differ at only one<br />
position <strong>in</strong> their ITS sequences, the rank of species<br />
was chosen for A. agr<strong>in</strong>ferus because both species are<br />
homothallic <strong>and</strong> their genomes <strong>and</strong> l<strong>in</strong>eages therefore<br />
are phylogenetically isolated, as <strong>in</strong>dicated by the<br />
nonreticulat<strong>in</strong>g pattern of RFLP markers with<strong>in</strong> <strong>and</strong><br />
between the two species (Kerrigan et al 1999); they<br />
are homomictic analogues of asexual species. The<br />
s<strong>in</strong>gle ITS character difference is consistent among<br />
studied stra<strong>in</strong>s: compare with GenBank AJ418743,<br />
AJ418745–47, AF432887, <strong>and</strong> EU131638–40 for A.<br />
subfloccosus collections SUB1, RWK 1542, 21552,<br />
21553, 21441, LAPAG 113, RWK 1568, 21993 <strong>and</strong><br />
21994 respectively. The two phylogenetic l<strong>in</strong>eages<br />
however are much more substantially diverged: the<br />
observed extent of RFLP molecular marker dissimilarity<br />
between the two sister species is 92% 6 2% SE<br />
(Kerrigan et al 1999). Abundant diverg<strong>in</strong>g cultural<br />
<strong>and</strong> molecular features, as well as the agreement of<br />
characters with<strong>in</strong> each species found on both sides of<br />
the Atlantic, also were evaluated (Kerrigan et al<br />
1999).<br />
Morphologically the two species are quasicryptic<br />
under some conditions. Because both species are<br />
homothallic, <strong>and</strong> each comprises multiple nonrecomb<strong>in</strong><strong>in</strong>g<br />
subl<strong>in</strong>eages (genets) (Kerrigan et al 1999), the<br />
process of homogenization that occurs with<strong>in</strong> an outcross<strong>in</strong>g<br />
species via <strong>in</strong>dependent association of most
888 MYCOLOGIA<br />
traits <strong>in</strong> each generation is absent. Instead, homothallic<br />
(homomictic) species can diverge <strong>in</strong>to genets<br />
that <strong>in</strong>creas<strong>in</strong>gly vary among themselves with respect<br />
to the presence of various traits that might useful for<br />
recognition of the species (e.g. absent <strong>in</strong> one vs.<br />
present or absent <strong>in</strong> the other); hence we describe<br />
‘‘tendencies’’ when present<strong>in</strong>g characteristics of<br />
these species (see Stebb<strong>in</strong>s 1950).<br />
We believe that Lange (1926) described the<br />
P<strong>in</strong>aceae-associated entity as P. hortensis subfloccosa,<br />
the basionym of A. subfloccosus. His material was<br />
collected from ‘‘large fairy-r<strong>in</strong>gs’’ <strong>in</strong> a ‘‘plantation of<br />
Picea’’ <strong>in</strong> Denmark. Danish material that we exam<strong>in</strong>ed<br />
had the nuclear RFLP genotype of the P<strong>in</strong>aceaeassociated<br />
entity, confirm<strong>in</strong>g its existence <strong>in</strong> the<br />
region of the type locality (Kerrigan et al 1999). In<br />
Denmark spruce will grow near sea level, while to the<br />
south it occurs, along with this highl<strong>and</strong> mushroom,<br />
<strong>in</strong> montane sett<strong>in</strong>gs. F<strong>in</strong>ally, although macroscopic<br />
differences between the two entities are subtle <strong>and</strong><br />
<strong>in</strong>consistent, Lange’s (1939) figures of Psalliota<br />
subfloccosa more closely resemble material of the<br />
P<strong>in</strong>aceae-associated entity, particularly with respect to<br />
the more fibrous appearance of the stipe context. We<br />
accept this <strong>in</strong>terpretation.<br />
In contrast Bohus (1994) published the illegitimate<br />
name A. cappellianus Bohus (;A. bohusianus L.A.<br />
Parra 2005) (non A. cappellianus Hlaváček 1987),<br />
based on an Hungarian type believed to correspond to<br />
A. subfloccosus ss. Cappelli (1984, Pl. 26). Both Cappelli<br />
<strong>and</strong> Bohus placed their mushroom(s) <strong>in</strong> <strong>section</strong><br />
Sangu<strong>in</strong>olenti, <strong>and</strong> our study (Parra unpubl) of the<br />
relevant material illustrated by Cappelli (1984, Pl. 26)<br />
<strong>in</strong>dicates that it is a pale collection of A. sylvaticus. Our<br />
attempt to study the type material of Bohus (BP) was<br />
unsuccessful; however his description, particularly the<br />
spore size of 5.2–7 3 3.5–4.5 mm, is a better fit to A.<br />
sylvaticus than to Lange’s A. subfloccosus. Therefore it<br />
appears that no new name actually has been typified by<br />
specimens of the montane, P<strong>in</strong>aceae-associated A.<br />
subfloccosus, simplify<strong>in</strong>g the correct application of the<br />
orig<strong>in</strong>al name to such material.<br />
In much of Europe south of Sc<strong>and</strong><strong>in</strong>avia <strong>and</strong> <strong>in</strong><br />
North America A. subfloccosus occurs from 500 m to<br />
more than 1700 m above sea level, typically with Picea<br />
(Italy, Switzerl<strong>and</strong>, Canada, USA) or other P<strong>in</strong>aceae<br />
(Spa<strong>in</strong>). It can be common <strong>in</strong> the bottoms of<br />
glaciated valleys <strong>in</strong> Alberta, Canada (e.g. Banff ca.<br />
1400 m <strong>and</strong>, farther north, Jasper ca. 1100 m), where<br />
it may form arcs (partial fairy-r<strong>in</strong>gs). In Colorado it<br />
occurs with spruce or with broadleaf trees <strong>and</strong> shrubs<br />
<strong>in</strong> urban parks at 1600 m (Denver: M. Shaw, G.<br />
Pickett <strong>and</strong> R. Kerrigan, pers obs). Parra <strong>and</strong> Suárez<br />
(2002) report it with P<strong>in</strong>us <strong>and</strong> Populus at 1500 m <strong>in</strong><br />
Spa<strong>in</strong>.<br />
In contrast the lowl<strong>and</strong> entity that is known from<br />
Scotl<strong>and</strong> <strong>in</strong>to France <strong>and</strong> from California usually<br />
occurs near the coast <strong>and</strong> is most commonly<br />
associated with Cupressus. We describe this entity<br />
here as A. agr<strong>in</strong>ferus. Like A. subfloccosus it consists of<br />
homothallic genets; however each genet may be more<br />
widely distributed <strong>in</strong> A. agr<strong>in</strong>ferus (Kerrigan et al<br />
1999).<br />
<strong>Agaricus</strong> agr<strong>in</strong>ferus has sometimes been (<strong>in</strong>correctly)<br />
called A. subperonatus ( J.E. Lange) S<strong>in</strong>g. (see<br />
Kerrigan et al 1999 for literature review). The latter<br />
species, recently proposed to be synonymous with A.<br />
cappellianus Hlaváček (;A. vaporarius [Pers.] Imbach<br />
1949, nom. illeg., non A. vaporarius Schrank<br />
1789) (e.g. Nauta 2001; Parra 2003, 2005, 2008), has<br />
been consistently illustrated with radially oriented<br />
brown fibrils <strong>in</strong> appressed scales on the pileus surface<br />
(Lange 1939, Essette 1964, Bon 1988, Cortecuisse <strong>and</strong><br />
Duhem 1994). Close <strong>in</strong>spection of fresh A. agr<strong>in</strong>ferus<br />
reveals that the fibrils of the pileipellis are highly<br />
<strong>in</strong>terwoven, not radially arranged (Kerrigan 1986); it<br />
is a dist<strong>in</strong>ct entity. Based on our review of its ITS1<br />
sequence, stra<strong>in</strong> WC721 from the PSUMCC (provided<br />
by RWK as FS-2/ARP003, from a collection by F.<br />
Stevens <strong>in</strong> San Francisco, California), deposited by<br />
Geml et al (2004) <strong>in</strong> GenBank (AY484698), belongs<br />
to A. agr<strong>in</strong>ferus, consistent with our own evaluation of<br />
this culture (Kerrigan unpubl).<br />
<strong>Agaricus</strong> devoniensis subsp. bridghamii Kerrigan,<br />
subsp. nov. FIG. 12.<br />
Pileus 3–7.5 cm latus, convexus demum late convexus,<br />
fibrillis adpressis, albus demum sordidus; contextus albus,<br />
immutabilis; stipes 5–8 cm longus, 1–1.5(–2.0) cm crassus,<br />
subaequalis, e farcto cavus; contextus albus; velum peronatum,<br />
<strong>in</strong>ferus; sporae (4.8–5.3–)6.0–6.6(–7.3–7.4) 3 (4.3–<br />
4.8–)5.1–5.5(–5.9) mm; basidia 19–23(–29) 3 9–10 mm,<br />
tetrasporigera; cheilocystidia (12.5–)20–31 3 11–12.5(–15–<br />
19) mm, equalis vel clavata vel subglobosa. HOLOTYPUS:<br />
RWK 1899: Colorado, Clear Creek Picnic Area, Clear Creek<br />
County, 16 Jul 1995: <strong>in</strong> herbario SFSU depositur. MB<br />
512227.<br />
Pileus broadly convex, 3–7.5 cm broad, marg<strong>in</strong><br />
<strong>in</strong>itially <strong>in</strong>rolled, disk slightly depressed <strong>in</strong> age;<br />
surface glabrous or obscurely appressed-fibrillose,<br />
color whitish or with irregular sordid brownish areas;<br />
context white, virtually unchang<strong>in</strong>g when cut, later<br />
(or <strong>in</strong> age) becom<strong>in</strong>g brownish near lamellae <strong>and</strong><br />
stipe, to 1.5 cm thick, odor mushroomy, unremarkable.<br />
Lamellae free, close, drab when young, obscurely<br />
if at all marg<strong>in</strong>ate (paler), later dark blackishbrown.<br />
Stipe approximately equal, slightly broader at<br />
base <strong>and</strong> sometimes at pileus, 5–8 3 1–1.5(–2.0) cm,<br />
surface f<strong>in</strong>ely erect-fibrillose above veils, glabrous or<br />
<strong>in</strong>nately fibrillose below, basal 5 mm covered with<br />
substrate; Veils submedian, peronate, sheath<strong>in</strong>g, with<br />
a flar<strong>in</strong>g upper marg<strong>in</strong> <strong>and</strong> a flar<strong>in</strong>g, or appressed, or
obscure lower marg<strong>in</strong>, all weak <strong>and</strong> soon collaps<strong>in</strong>g;<br />
context white, or obscurely rufescent <strong>in</strong> places,<br />
becom<strong>in</strong>g brownish near pileus, with a 3–5 mm broad<br />
pith-filled central cavity.<br />
Spores dark brown, broadly ellipsoid (Q 5 1.13–<br />
1.16 to 1.24–1.26), (4.8–5.3–)5.9–6.6(–7.3–7.4) 3<br />
(4.3–4.8–)5.0–5.5(–5.9) mm, the mean 6.21 3<br />
5.20 mm, (N 5 120, C 5 3); hilar appendix not<br />
prom<strong>in</strong>ent; apical pore not evident. Basidia clavate or<br />
cyl<strong>in</strong>dro-clavate, 19–23(–29) 3 9–10 mm, mostly tetrasporic,<br />
the sterigmata acute, to 4 mm long. Cheilocystidia<br />
<strong>in</strong> scattered to semicont<strong>in</strong>uous fascicles, mostly<br />
broadly clavate to subglobose or almost pyriform,<br />
otherwise quasi-cyl<strong>in</strong>drical, 20–27 3 11–12.5(–15) mm.<br />
Chemistry: unavailable.<br />
ITS1+2 sequence (GenBank AF432890–<br />
91, EU131637): L 5 707 nt. Dist<strong>in</strong>ctive characters:<br />
gtgaTaaca @ 180 <strong>and</strong> actcActtg @ 530 dist<strong>in</strong>guish the<br />
subspecies.<br />
Habit, habitat, known distribution, occurrence:<br />
Solitary or <strong>in</strong> small groups, under Picea, <strong>in</strong> fairly level<br />
areas <strong>in</strong> valley bottoms, at middle to high elevations <strong>in</strong><br />
the eastern Rocky Mounta<strong>in</strong>s of Colorado <strong>and</strong> Alberta.<br />
Material studied: USA. Colorado. Clear Creek County<br />
Near Empire: Clear Creek Picnic Area, elev. 2900 m. Under<br />
Picea, 16 Jul 1995, RWK 1899 (HOLOTYPE [SFSU];<br />
paratypes: preserved culture MYA-2671 [ATCC]); Mizpah<br />
Campground, elev. 3000 m. Under Picea, 16 Jul 1995, RWK<br />
1900 (NY), preserved culture 5 MYA-2672 [ATCC]);<br />
CANADA. Alberta. Canmore. Bow River Recreation Area<br />
campground, elev. 1300 m. Under Picea, 5 Aug 1990, RWK<br />
1664 (SFSU).<br />
Etymology: The name honors Joseph Bridgham,<br />
one of 19th century North America’s foremost<br />
illustrators of fungi <strong>and</strong> other natural subjects, who<br />
illustrated several <strong>Agaricus</strong> spp. for the Icones Farlowianae<br />
(Farlow 1929).<br />
Two ITS characters unify subsp. bridghamii contra<br />
devoniensis; one of these characters is unique with<strong>in</strong><br />
the <strong>section</strong> <strong>and</strong> possibly the genus. This level of<br />
ITS1+2 sequence divergence of subsp. bridghamii<br />
from A. devoniensis P.D. Orton subsp. devoniensis,<br />
which itself is somewhat heterogeneous as presently<br />
constituted (Challen et al 2003, Callac et al 2006; see<br />
FIG 1), is <strong>in</strong> the rank-range of both established species<br />
<strong>and</strong> varieties <strong>in</strong> the <strong>section</strong>. Because the morphological<br />
differention of the two entities is slight <strong>and</strong><br />
because subsp. devoniensis at least comprises one or<br />
more heterothallic, out-cross<strong>in</strong>g populations (Callac<br />
et al 2006), <strong>and</strong> furthermore because the ‘‘devoniensis<br />
clade’’ is a complex group with additional undescribed<br />
entities of uncerta<strong>in</strong> rank, we believe that it is<br />
more conservative <strong>and</strong> appropriate at present to<br />
recognize bridghamii at subspecific (vs. species) rank.<br />
Consider<strong>in</strong>g the other extreme, the physiological<br />
differentiation of the two subspecies is greater than<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 889<br />
what is seen among isolated populations or even<br />
varieties <strong>in</strong> related <strong>Agaricus</strong> species (cf. Callac et al<br />
1993). To underscore this po<strong>in</strong>t we also considered<br />
partial RPB2 gene sequence from RWK 1899 <strong>and</strong><br />
from RWK 1800 (A. devoniensis subsp. devoniensis<br />
from California) obta<strong>in</strong>ed for a separate study<br />
(Kerrigan <strong>and</strong> Xu unpubl; GenBank EU285571–72).<br />
We found 1.5% sequence divergence between the two<br />
sequences, whereas for this gene segment A. subrufescens<br />
Peck from California <strong>and</strong> Spa<strong>in</strong> diverged by<br />
0.6%, two A. placomyces Peck sequences differed by<br />
0.3%, <strong>and</strong> two A. depauperatus (F. H. Møller) Pilát<br />
sequences differed by 0.4% (Kerrigan <strong>and</strong> Xu<br />
unpubl).<br />
Morphologically subsp. bridghamii tends to have a<br />
more compact shape, with a stipe length/width ratio<br />
of 1.2–3.0 (vs. (2.4–)3.4–8.3 <strong>in</strong> subsp. devoniensis, per<br />
auct.), <strong>and</strong> shorter cheilocystidia (for subsp. devoniensis,<br />
36–50 mm, per Møller (1952), although LAP<br />
notes that the term<strong>in</strong>al elements <strong>in</strong> the type are often<br />
only 15–26 3 9–14 mm (we also note that subsp.<br />
devoniensis collection RWK 1800 from California<br />
lacked cheilocystidia).<br />
The habitat <strong>and</strong> range differences (s<strong>and</strong> dunes <strong>and</strong><br />
Cupressus macrocarpa groves near sea level for subsp.<br />
devoniensis, vs. montane Picea forests above 1300 m<br />
for subsp. bridghamii) are of uncerta<strong>in</strong> value <strong>in</strong><br />
delimit<strong>in</strong>g the <strong>taxa</strong>; however, the physiological<br />
divergence implied by the differ<strong>in</strong>g abilities of<br />
cultures of the two subspecies to use mushroom<br />
compost (RWK 1899 <strong>and</strong> RWK 1900 grow poorly <strong>and</strong><br />
ultimately unsuccessfully on commercial <strong>Agaricus</strong><br />
mushroom compost, while Californian, French <strong>and</strong><br />
Greek subsp. devoniensis grow <strong>and</strong> fruit well on it) is<br />
dist<strong>in</strong>ctive. In a contrast<strong>in</strong>g example <strong>in</strong>dividuals from<br />
all populations of A. bisporus, <strong>in</strong>clud<strong>in</strong>g those from<br />
coastal cypress <strong>and</strong> montane spruce forests, use<br />
compost approximately equally well (Kerrigan <strong>and</strong><br />
Callac unpubl). Consider<strong>in</strong>g the degree of sequence<br />
divergence, <strong>and</strong> the morphological, cultural, <strong>and</strong><br />
ecological attributes of the Rocky Mounta<strong>in</strong> organism,<br />
subspecific rank with<strong>in</strong> A. devoniensis appears to<br />
be most appropriate for this entity. Only certa<strong>in</strong> other<br />
isolates of subsp. devoniensis show clear mat<strong>in</strong>g<br />
reactions, so the negative results of mat<strong>in</strong>g confrontations<br />
attempted by Callac between the subspecies<br />
are ambiguous at this time (Callac et al 2006).<br />
Sordid discolorations on pilei of A. devoniensis<br />
subsp. bridghamii also were reported for A. litoralis<br />
(Wakef. & A. Pearson) Pilát, a species sometimes<br />
l<strong>in</strong>ked with A. devoniensis (;Psalliota arenicola<br />
Wakef. & A. Pearson; Cappelli (1984) provides a<br />
good summary <strong>and</strong> discussion of variation reported<br />
for European A. devoniensis <strong>and</strong> its purported relative<br />
A. litoralis) but now considered to be a synonym of A.
890 MYCOLOGIA<br />
spissicaulis F.H. Møller, which is the type of <strong>section</strong><br />
Spissicaules (He<strong>in</strong>em.) Kerrigan (Nauta 2001; Parra<br />
2005, 2008). Sta<strong>in</strong>s have not been noted otherwise on<br />
A. devoniensis proper.<br />
The variation RWK has observed <strong>in</strong> spore length/<br />
width ratios for the two Colorado collections strengthens<br />
our sense that members of the ‘‘devoniensis<br />
clade’’ may be morphologically plastic <strong>and</strong> correspond<strong>in</strong>gly<br />
difficult to delimit <strong>and</strong>/or identify. The<br />
entire ‘‘A. devoniensis clade’’ rema<strong>in</strong>s challeng<strong>in</strong>g, as<br />
discussed below.<br />
S<strong>in</strong>ce 2003 we also have also studied additional material<br />
of A. devoniensis not noted above. Collection data on this<br />
material is: CA 504 (5A. fragilivolvatus holotype): Italy,<br />
Sardegna, prov. Cagliari, Serramanna, under Eucalyptus<br />
camalduensis, 23 Oct 1992, leg. M. Contu <strong>and</strong> P. Dessi, coll.<br />
M. Contu 92/60 (CAG) (GenBank EU363035); CA 445<br />
(orig<strong>in</strong>ally determ<strong>in</strong>ed as A. fragilivolvatus): Italy, Sardegna,<br />
prov. Sassari, Golfo Araui, La Mar<strong>in</strong>ella, under<br />
Eucalyptus sp., 24 Nov 2004, leg. & det. M. Contu (without<br />
collection number, private herbarium) (EU363036);<br />
CA457: Spa<strong>in</strong>, La Rioja, Calahorra, under P<strong>in</strong>us halepensis,<br />
28 Nov 1997, leg. & det. Agustín Caballero, coll. AC2160<br />
(private herbarium) (EU363034); CA 568 (5Dv9; GenBank<br />
EU258677): Greece, Papakosta (close to Agiokampos),<br />
Thessalie, under C. sempervirens on the coast, 17 Oct<br />
2000, leg. & det. I. Theochari, P. Callac, & R. Kerrigan<br />
(CGAB); CA 117 (5Dv7; GenBank AF432895): Greece,<br />
Feres, Velest<strong>in</strong>o, under C. sempervirens, 13 Jan 1999, leg. I.<br />
Theochari, det. P. Callac (CGAB).<br />
DISCUSSION<br />
It is fair to say that <strong>section</strong> <strong>Bivelares</strong> encompasses a<br />
number of <strong>rare</strong>ly seen species, some of which rema<strong>in</strong><br />
undescribed. Collect<strong>in</strong>g <strong>and</strong> describ<strong>in</strong>g ultra<strong>rare</strong><br />
species of agarics present special challenges. We<br />
realize that we presently can provide only partial<br />
<strong>in</strong>formation on the ranges <strong>and</strong> variation of some of<br />
the <strong>taxa</strong> described here. Some conjecture rema<strong>in</strong>s,<br />
for example about the relationship of the two known<br />
collections of A. subsubensis. Particularly with the<br />
advent of molecular data, especially DNA sequences,<br />
it is possible to provide robust markers for biological<br />
novelty even when material for study is limited. We<br />
th<strong>in</strong>k it is important to place such data <strong>in</strong> the public<br />
record. Our <strong>in</strong>tent is to document such species as well<br />
as possible at present, <strong>in</strong> aid of encourag<strong>in</strong>g <strong>and</strong><br />
improv<strong>in</strong>g the recognition, preservation <strong>and</strong> report<strong>in</strong>g<br />
of future collections.<br />
Cultures of JB-101, FS-22, <strong>and</strong> RWK 1789 date from<br />
1990–1992. As far as we know these species have not<br />
been encountered otherwise <strong>in</strong> nature. Their novelty<br />
has long been evident from RFLP <strong>and</strong> other<br />
molecular data. Unfortunately the latter two collections<br />
were made under conditions that precluded<br />
voucher preservation; only a culture or viable spores<br />
were obta<strong>in</strong>ed. Both A. subsubensis <strong>and</strong> A. cupressophilus<br />
have now been cultivated successfully from orig<strong>in</strong>al<br />
cultures, which have been deposited at ATCC. The<br />
French CA103 (INRA), sequenced by Callac from a<br />
culture (available at MYCSA) from mushrooms obta<strong>in</strong>ed<br />
by J. Gu<strong>in</strong>berteau under cypress on Oléron<br />
Isl<strong>and</strong>, France, is also a unique entity that, lack<strong>in</strong>g an<br />
extant voucher, is <strong>in</strong> need of recollection. A. agr<strong>in</strong>ferus<br />
is uncommon <strong>in</strong> the Monterey cypress habitat of<br />
coastal California, the only place it is known to occur <strong>in</strong><br />
North America, where it has perhaps one-tenth the<br />
abundance of A. bisporus (Kerrigan 1982). <strong>Agaricus</strong><br />
tlaxcalensis is presently known only from scattered sites<br />
planted with C. benthamii <strong>in</strong> Tlaxcala, Mexico.<br />
The A. devoniensis clade is challeng<strong>in</strong>g not only<br />
due to rarity but also due to apparently recent<br />
speciation processes, which <strong>in</strong> the case of subsp.<br />
devoniensis <strong>in</strong> Europe might <strong>in</strong>clude re-<strong>in</strong>tegration of<br />
diverged populations (cf. Callac et al 2006 <strong>and</strong><br />
unpubl). On one h<strong>and</strong>, because we have observed<br />
no differences <strong>in</strong> the types of both species, <strong>Agaricus</strong><br />
fragilivolvatus Contu is considered here a synonym of<br />
A. devoniensis. The type of A. devoniensis has spores<br />
6–7 3 5–5.5 mm <strong>and</strong> clavate cheilocystidia 16–26 3 9–<br />
14 mm, while from the type of A. fragilivolvatus we<br />
measured spores <strong>and</strong> cheilocystidia respectively as 6–7<br />
3 (4.5–)4.8–5.5 mm <strong>and</strong> 17–34 3 8–13 mm. The<br />
cheilocystidia <strong>in</strong> the A. fragilivolvatus type were<br />
clavate to pyriform <strong>and</strong> not vesiculose as described<br />
by Contu. Compare also GenBank sequences<br />
AF432892, –93, –94, –96, –97 <strong>and</strong> EU363034 with<br />
EU363035–36) (FIGS. 1, 2).<br />
On the other h<strong>and</strong>, some real heterogeneity exists.<br />
A. devoniensis subsp. bridghamii has been found twice<br />
<strong>in</strong> Colorado <strong>and</strong> once <strong>in</strong> Alberta. Its range is<br />
therefore considerable; however <strong>in</strong> our experience it<br />
is <strong>rare</strong> or at least uncommon. Another member of the<br />
clade, known only from two collections (see CA 117,<br />
CA 568) from Greece has dist<strong>in</strong>ctly divergent ITS1+2<br />
sequences <strong>and</strong> might deserve taxonomic recognition<br />
after further study (see GenBank AF432895 <strong>and</strong><br />
EU258677).<br />
Two additional members of this clade (see Gen-<br />
Bank AF432889, AF059225) <strong>in</strong> need of further study<br />
have been reported from <strong>New</strong> Zeal<strong>and</strong> by Mitchell<br />
<strong>and</strong> Bres<strong>in</strong>sky (1999), <strong>in</strong>dicat<strong>in</strong>g a broad geographical<br />
scope for this phylogenetic radiation. We discussed<br />
<strong>in</strong> 2003 our sequence data from material<br />
determ<strong>in</strong>ed by Mitchell <strong>and</strong> Bres<strong>in</strong>sky as A. subperonatus<br />
but which <strong>in</strong>stead is closely related to A.<br />
devoniensis (Challen et al 2003). Based on our review<br />
<strong>and</strong> <strong>in</strong> comparison with European material, the ITS2<br />
sequence deposited by Mitchell <strong>and</strong> Bres<strong>in</strong>ski (1999)<br />
as A. devoniensis from <strong>New</strong> Zeal<strong>and</strong> also appears to
elong to a related but dist<strong>in</strong>ct entity <strong>in</strong> the ‘‘A.<br />
devoniensis complex’’ (the unresolved phylogenetic<br />
clade encompass<strong>in</strong>g A. devoniensis <strong>and</strong> any variants<br />
<strong>and</strong> closely related putative species) (FIG. 2).<br />
Dur<strong>in</strong>g this study we have evaluated some sequences<br />
present <strong>in</strong> GenBank. We note that ‘‘A. padanus<br />
HAI 0312’’ (GenBank AJ884647) recorded by Didukh<br />
(2004) <strong>and</strong> sequenced by Didukh et al (2005) is a very<br />
close match for A. bitorquis. We also have sequenced<br />
the type of A. padanus, <strong>and</strong> it has a dist<strong>in</strong>ct sequence<br />
similar to those of <strong>section</strong> Sangu<strong>in</strong>olenti (unpubl). We<br />
therefore regard GenBank AJ884647 to be of A.<br />
bitorquis.<br />
Some comments on our earlier report with M.<br />
Challen on Duploannulati (5<strong>Bivelares</strong>) (Challen et al<br />
2003) are <strong>in</strong> order. Nomenclature of A. subfloccosus<br />
s.l. <strong>and</strong> A. vaporarius are discussed above, as is the<br />
challenge presented by <strong>Agaricus</strong> devoniensis <strong>and</strong> its<br />
associates. The possibility of the existence of a<br />
dist<strong>in</strong>ct, <strong>rare</strong> A. subperonatus elicits a range of<br />
op<strong>in</strong>ions but cannot be fully resolved at present.<br />
M.M. Nauta (pers comm) has noted that our<br />
statement concern<strong>in</strong>g the lack of rufescence <strong>in</strong> A.<br />
bitorquis (Quél.) Sacc. is at least an overgeneralization.<br />
We are <strong>in</strong>cl<strong>in</strong>ed to agree, <strong>in</strong> view of our<br />
emerg<strong>in</strong>g belief that A. bitorquis var. validus (F.H.<br />
Møller) Bon & Cappelli is most likely to be a<br />
phylogenetically irrelevant name applied to occasional<br />
<strong>in</strong>dividuals of A. bitorquis exhibit<strong>in</strong>g discernible<br />
rufescence, a trait hypothetically (for example) due to<br />
a s<strong>in</strong>gle gene change result<strong>in</strong>g <strong>in</strong> over-expression of<br />
tyros<strong>in</strong>ase enzyme activity (see van Leeuwen <strong>and</strong><br />
Wichers 1999). We sequenced the ITS1+2 region of<br />
one such <strong>in</strong>dividual (LAPAG 517: duplicate from P.<br />
Arrillaga, garden, Zarauz, Guipúzcoa, Spa<strong>in</strong>, 8 Apr<br />
1996 [coll. 501-8/4/96 deposited <strong>in</strong> Arrillaga’s private<br />
herbarium] Arrillaga 2004) (GenBank EU257804)<br />
<strong>and</strong> found no differences from var. bitorquis sequences<br />
other than one heteromorphism. We also have<br />
added new A. bitorquis sequences to GenBank from<br />
CLO 4465 (Clarke Ovrebo, Oklahoma [OKL];<br />
EU258675) <strong>and</strong> JWS-1 ( John Sparks, <strong>New</strong> Mexico<br />
[SFSU]; EU258674) for comparison. Accord<strong>in</strong>g to<br />
Nauta the rufescent phenotype is not uncommon <strong>in</strong><br />
Europe, but <strong>in</strong> our own experience it is not common<br />
<strong>and</strong> less so <strong>in</strong> North America. At best the rufescence<br />
of typical A. bitorquis is limited to an <strong>in</strong>dist<strong>in</strong>ct sordid<br />
discoloration of the stipe <strong>and</strong> of pileus context near<br />
the lamellae, primarily <strong>in</strong> mature specimens.<br />
There are now 10 named species <strong>and</strong> several<br />
<strong>in</strong>fraspecific <strong>taxa</strong> formally placed <strong>in</strong> <strong>section</strong> <strong>Bivelares</strong>.<br />
Two sub<strong>section</strong>s are recognized. Entities need<strong>in</strong>g<br />
names, after adequate further study, are known from<br />
France, Greece <strong>and</strong> <strong>New</strong> Zeal<strong>and</strong>. Additional discoveries<br />
would not be surpris<strong>in</strong>g.<br />
KERRIGAN ET AL: NEW TAXA IN AGARICUS SECTION BIVELARES 891<br />
ACKNOWLEDGMENTS<br />
We thank D. Arora, P. Arrillaga, P. Boisselet, A. Caballero, K.<br />
Chadwick, M. Contu, J. Gu<strong>in</strong>berteau, G. Maddalena, G.<br />
Mata, A.D. Mitchell, M. Mo<strong>in</strong>ard, D. Murietta, A. Mua, C.<br />
Ovrebo, K. Peterson, G. Pickett, S. Rextoueix, M. Shaw, J.<br />
Sparks, F. Stevens <strong>and</strong> A. Tancrède, <strong>and</strong> the Colorado<br />
Mycological Society, for specimens, cultures <strong>and</strong>/or collect<strong>in</strong>g<br />
assistance. J. Gu<strong>in</strong>berteau provided FIG. 3 <strong>and</strong> other<br />
assistance. Advice on Lat<strong>in</strong> <strong>and</strong> translations were provided<br />
by G. Heucl<strong>in</strong>, R. Hall<strong>in</strong>g <strong>and</strong> J. Santos. Some sequences<br />
were k<strong>in</strong>dly provided by M. Challen <strong>and</strong> J.-P. Xu. P. Callac<br />
acknowledges ECOS-Nord <strong>and</strong> ANUIES (M06A01 Franco-<br />
Mexican program) <strong>and</strong> the BRG (Bureau des Ressources<br />
Génétiques, program No. 51, 2007/2008) for f<strong>in</strong>ancial<br />
support.<br />
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