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Laccaria Berkeley and Broome is a cosmopolitan
genus of mushrooms (Agaricales) collected frequently
throughout North America. Its taxa make up a sizeable part
of the world's mycota and have been reported from every
continent except Antarctica.
Individuals of most Laccaria species have been
reported to form ectomycorrhizal associations with numerous
tree species including many that are economically important
in North America [e.g., species of Quercus and other
Fagaceae, many species of Pinus, Pseudotsuga
menziesii (Mirb.) Franco and Tsuga
heterophylla (Raf.) Sarg.] (Trappe, 1962).
Singer and Moser (1965) and Watling (1977) have reported
that some taxa of Laccaria can act as pioneer
species. Other studies have supported this contention by
reporting that basidiomata of at least some Laccaria
are frequently found in recently disturbed sites and young
forest stands but not in mature forests (e.g., Danielson,
1984; Dighton and Mason, 1985; Dighton et al., 1986).
Thus Laccaria, at least in some situations, may play
an important role in primary and secondary succession.
Because of the relative ease with which some species of
Laccaria can be manipulated in the laboratory,
several taxa including L. bicolor (Maire) Orton,
L. laccata (Scop.: Fr.) Cooke and L. proxima
(Boud.) Pat. are being used actively in applied and basic
research on ectomycorrhizae (see Kropp and Langlois, 1990).
Laccaria has also proved useful in studies on the
biology of fungi which form ectomycorrhizae (e.g., Fries,
1983a; Fries and Mueller, 1984; Kropp and Fortin, 1988;
Barrett et al., 1989, 1990; Armstrong et al.,
1989; Doudrick and Anderson, 1989; Gardes et al.,
1990, 1991a, 1991b; Mueller and Gardes, 1991; Mueller,
1991c).
Stabilization of the classification of Laccaria,
therefore, would have applied implications because of the
ecological and potential economic importance of many of its
taxa in addition to adding to our basic knowledge of fungi
which form ectomycorrhizae.
Although most modern systematists consider Laccaria
to be an autonomous genus, easily segregated from other
members of the family Tricholomataceae (Singer, 1986),
delimitation of infrageneric taxa is difficult in many
instances. Much of this problem is due to the relative
morphological simplicity of its taxa which provides few
suites of systematically informative characters coupled with
a high degree of phenetic plasticity within certain
Laccaria taxa, especially those which have an
apparently wide geographic range. Continuing nomenclatural
confusion has exasperated this problem.
A large discrepancy exists in the number of recognized taxa
in the genus for these reasons. Although nearly 100 species
epithets have been used for Laccaria worldwide,
Singer (1986) recognized only 18 clearly defined species
while McNabb (1972) indicated that there may be as many as
43 species worldwide. There also has been much confusion
concerning the circumscription of several taxa, and
classification in this genus remains in a state of flux
(e.g., Singer, 1967, 1977, 1986; Bon, 1983; Moser,
1983; Clémençon, 1984; Ballero and Contu,
1987, 1989; Mueller, 1991a).
This study was undertaken in an attempt to resolve
systematic and nomenclatural problems, to determine species
composition and distribution for the genus Laccaria
in North America north of Mexico, and to add to our
knowledge of its biology. Because of the problems stated
above, it was necessary to examine extralimital taxa as well
as all available extant type collections, to designate
lectotypes, neotypes or representative specimens when
applicable, and to examine critically characters from
various stages of the life cycle. In addition to
morphological characters of basidiomata and somatic culture
mats, data from intra- and interstock pairing analyses,
restriction fragment length polymorphisms of mitochondrial
and ribosomal DNA, cytological studies, and in vitro
ectomycorrhizal synthesis attempts were incorporated into
this multifaceted study.
Taxa were delimited so that they are presumably monophyletic
and are diagnosable by a unique combination of character
states. Attempts were made to develop a classification for
the genus that reflects the evolutionary history of the
group.
This manuscript is the result of over 12 years of study and
consists of a synthesis of new and previously published data
(Mueller and Sundberg, 1981; Fries and Mueller, 1984;
Mueller, 1984, 1985, 1987, 1991a, 1991b, 1991c; Mueller and
Vellinga, 1986, 1990; Vellinga and Mueller, 1987; Gardes
et al., 1990, 1991a, 1991b; Mueller and Gardes,
1991).
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Taxonomic and Nomenclatural History
Europe and other extralimital areas. The name
Laccaria was first proposed by Berkeley and Broome in
1883 to accommodate species of Agaricus subgenus
Clitocybe which produced globose basidiospores that
often formed a white pulverulescence on the thick, attached
lamellae. Proposed for transfer to the new genus were
Agaricus laccatus Scopoli, A.
bellus Persoon and several unnamed species from
Ceylon and Europe. No new combinations were made until the
following year, however, when Cooke (1884) formally
transferred eight species to the genus.
Over the intervening century, the presence of clamp
connections and echinulate, multinucleate basidiospores have
become important characters in the modern generic concept of
Laccaria (Kühner, 1980, 1984; Singer, 1986).
The pre-Friesian history of the genus can be summarized as
follows. Scopoli (1772), describing fungi from the Tyrol of
southern Austria, was the first to use the binomial,
A. laccatus, for a member of the eventual
genus Laccaria. Two more species, A.
amethystinus and A. farinaceus, were
described in 1778 by Hudson from the environs of London
(Hudson, 1798). Hudson placed A. laccatus in
synonymy with A. farinaceus and thus A.
farinaceus is a superfluous name. Agaricus
tortilis was described from Halifax by Bolton (1788).
Finally, Persoon (1801) named A. bellus and
two varieties of A. farinaceus: var.
rosellus and var. tortilis.
Fries (1821) placed A. laccatus and A.
bellus in tribe VIII Clitocybe, Subtribe 4
Oseypii. He further divided A. laccatus
into variety "a" ("Pileo rufo 1.
carneo, sicco subochraceo") and variety
"b" ("Pileo amethystino, sicco
canescente"). Additionally, he listed A.
tortilis and A. pachyphyllus Fries
(1815) as species which needed to be further examined.
Between 1821 and 1884, a number of workers described new
species destined later to be incorporated in Laccaria
(e.g., Fries, 1836-1838, 1874; Berkeley, 1845, 1856;
Montagne, 1856; Berkeley and Curtis, 1859; Berkeley and
Broome, 1871; Ellis, 1874; Karsten, 1876; Spegazzini, 1880;
Boudier, 1881) .
The genus Russuliopsis was proposed by Schroeter
(1889) for all the taxa previously included in
Laccaria. Because Laccaria and its included
taxa were validly published, Russuliopsis is a later
typonym and thus a superfluous name.
Laccaria was recognized by both Fayod (1889) and
Patouillard (1900) in their attempts to develop a more
natural classification system for the Hymenomycetes. Peck
(1912) was the first worker in the United States to
recognize the genus and one of the first in the world to
publish a paper exclusively on the group.
It was not until Singer (e.g., 1943b, 1949, 1962, 1967,
1973, 1975, 1977) became interested in the genus, however,
that any attempt was made to organize and formulate a
coherent classification for Laccaria based on the
world mycota. To date, Singer's publications on the genus
are the most comprehensive and influential. His
classification has been the starting point for this study
and for most other systematic research on the genus since
the 1940's.
Several alternative classifications have been published
including those of Bon (1983), Clémençon
(1984) and Ballero and Contu (1989). Additionally, numerous
mycological surveys which included Laccaria have been
published by various authors for most areas of the world.
Some of these publications include: Rea (1922), Kühner
and Romagnesi (1953), Dennis et al. (1960), Orton
(1960), Phillips (1981); Moser (1983), Dennis (1986),
Ballero and Contu (1987), Watling (1987), Mueller (1991a)
for temperate Europe; Möller (1945), Lange (1955),
Kobayasi et al. (1967), Miller et al. (1982),
Gulden and Jenssen (1988) for the Arctic; Vellinga (1986)
for India; Malençon and Bertault (1975) for North
Africa; Heinemann (1964, 1966), Pegler (1977) for East
Africa; Binyamini (1973, 1976) for Israel; Stevenson (1964),
McNabb (1972) for New Zealand; Imai (1938), Hongo (1959,
1971) for Japan; Singer (1952, 1953), Singer and Digilio
(1952), Singer and Moser (1965), Horak (1979) for South
America; Singer (1957), Aguirre-Acosta and
Pérez-Silva (1978), Montoya-Bello et al.
(1987) for Mexico.
North America north of Mexico. Schweinitz (1822,
1834) was the first American mycologist to publish on the
North American mycota. In his list of all the then known
fungi from America, Schweinitz (1822) included
Agaricus (Omphalia) bellus and
A. (Omphalia) farinaceus. In 1834 he
included A. (Clitocybe) laccatus,
A. (Clitocybe) amethystinus and
A. (Clitocybe) bellus. Several
collectors working in North America, including Curtis, Lea,
Sullivant and Wright sent specimens and notes on American
fungi to various workers in Europe, especially Berkeley, and
several new species (e.g., A. ohiensis
Montagne, and A. ochropurpureus Berkeley) were
discovered and published in this manner.
Ellis (1874) described A. (Clitocybe)
trullissatus from the sand dunes of New Jersey, and
thus became the first North American to designate a new
species in this group. Charles H. Peck was the first North
American worker who studied and published on the group in
some detail (Peck, 1890, 1893, 1895, 1897, 1903, 1907,
1912). Laccaria amethystina Cooke, L.
laccata, L. ochropurpurea (Berk.) Peck,
L. striatula (Peck) Peck, and L.
tortilis (Bolt.) Cooke were included in the North
American Flora (Murrill, 1914). These same taxa were
included in a study of Laccaria in North Carolina
(Coker and Beardslee, 1922).
Little systematic work was done on the genus again until
Singer described L. tetraspora from Florida and L.
calospora and L. laccata var. carbonicola
from Massachusetts (Singer, 1946, 1967, 1973, respectively).
Mueller and Sundberg (1981) presented a treatment for the
genus from southern Illinois. Lahaie undertook a study of
the genus from eastern Canada for his Masters Degree thesis
(1981) but his results have not been published. Mueller
(1984, 1991b) described 6 new species from the continental
United States and Canada.
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Morphological Analyses of
Basidiomata
Extensive collecting was undertaken throughout the United
States and parts of eastern and western Canada. The
following states and provinces were sampled: British
Columbia, California, Colorado, Florida, Georgia, Idaho,
Illinois, Kentucky, Louisiana, Massachusetts, Michigan,
Minnesota, Mississippi, New York, North Carolina, Nova
Scotia, Ohio, Ontario, Oregon, South Carolina, Tennessee,
Texas, Virginia, West Virginia, Washington, Wisconsin, and
Wyoming. Field work was also undertaken in Sweden, Mexico,
Costa Rica, and much of South America as part of an ongoing
project to produce a world monograph of the genus and to
obtain comparative material. Dried specimens on loan from
numerous herbaria including most extant type specimens were
also examined.
Collections were made and assembled using standard
techniques (Smith, 1949; Largent, 1977). Descriptive terms
were taken from Snell and Dick (1971) and Largent (1977).
Unless otherwise noted, color names within parentheses and
quotation marks are from Ridgway (1912), colors from
Kornerup and Wanscher (1978) are listed by (page-column-row)
while color names outside of parentheses are
author-generated. Color names followed by M&P; were
taken from Maerz and Paul (1930).
Basidiomata were preserved by warm-air drying and deposited
in either F, TENN, UPS or WTU (Holmgren et al.,
1981). Most examinations were made directly using either a
Nikon Model S-Kt research microscope, a Zeiss Universal
photomicroscope, or a Zeiss RA standard microscope equipped
with both bright field and phase contrast optics. Recent
data acquisition and remeasurements were made using JAVA
1.31 (Jandel Video Analysis Software, 1989) running on an
AT&T; 6386 computer from images captured through an
Olympus BH-2 microscope with Nomarski differential
interference optics. Illustrations of micromorphological
characters were made with the aid of a drawing tube. All
measurements were taken on material mounted in 3% KOH.
Iodine reactions were determined in Melzer's reagent and the
cyanophilic reaction was determined in cotton blue (Kotlaba
and Pouzar, 1964; Singer, 1972; Largent et al.,
1977). Descriptive terminology was taken from Snell and Dick
(1971) and Largent et al. (1977).
Micromorphological data used in taxon descriptions were
based on a complete, detailed examination of at least five
(when possible) representative collections per taxon. The
number of collections examined was dependent upon the
availability of material and the amount of variability
encountered within the taxon. All specimens listed in
Specimens Examined (Appendix A) were examined, and any
deviations from the norm were noted. Measurements and
observations were taken from several basidiomata per
collection to check for uniformity. At least 10 randomly
sampled cheilocystidia, pleurocystidia, and terminal cells
of cuticular hyphae, and 15 randomly sampled basidia were
measured per collection. Width and diameter measurements of
these elements were taken at the widest point and rounded to
the nearest 0.5 µm. Arrangement of hyphae comprising
the pileipellis was observed both in radial and scalp
sections.
All basidiospore measurements were taken from hymenial
tissue and not from spore prints to treat all specimens
equally. The number of basidiospores measured and the number
of collections examined for calculating mean size (= ) and
length/width ratio (= Q) were included in brackets with
basidiospore size data to give some indication of
reliability of these data (Bas, 1974). When available, data
on basidiospores from additional collections were included
in basidiospore size range data. Ranges of collection means
(, ) for basidiospore data, rather than overall mean values
for the taxon, are provided to give a better indication of
intraspecific variation. Basidiospore size data are always
given without ornamentation and hilar appendix, with the
hilar appendix in profile. Means and other descriptive
statistics were obtained using SAS for Personal Computers
version 6.03 (SAS, 1985).
Scanning Electron Microscope
Analyses
Lamellar fragments from air-dried collections were
rehydrated in an acetone series, fixed in 2.5%
glutaraldehyde in phosphate buffer for 3-4 hrs at 4°C,
dehydrated in an acetone series and critical point dried in
a Balzers CPD 030 apparatus with CO2 as the transition fluid
(Cheeseman and Grund, 1985). Samples were then attached to
aluminum mounts with double-stick tape and coated with gold
in a Denton Vacuum Desk II sputter coater. Basidiospores
were examined and micrographs were taken at 20 kV with an
Amray 1810 scanning electron microscope.
Somatic Culture Mat
Analyses
The following procedure was employed to obtain
heterokaryotic tissue cultures of Laccaria. Small
pieces of tramal tissue excised from the pileus-stipe
interface were aseptically placed on modified Melin Norkrans
Medium (=MMN) plus benomyl (10 mg/1) in disposable test
tubes (Marx, 1969; Molina and Palmer, 1982; Mueller, 1984).
The benomyl was added to reduce ascomycetous contamination.
Six to ten replicates were taken for each collection
utilized. Subculturing of each resulting isolate was
undertaken until a pure culture was obtained. Stock isolates
were then transferred to tubes containing modified MMN or
N6:5 (Fries, 1983a) and stored in the dark at 2-4° C.
Voucher herbarium material of all specimens used for tissue
cultures were described and deposited in F, TENN, UPS and
WTU. All isolates are housed in the mycological culture
collection at Field Museum of Natural History.
Culture mat analyses were based on the classic work of
Nobles (1948, 1965). Five millimeter round plugs of agar
containing hyphal tips taken from the advancing zone of each
of 2-week-old "stock" plates were transferred to the edge
(mycelium side down) of a Petri plate containing 15-20 ml of
either MMN, Malt Extract Agar (= MEA), or Potato Dextrose
Agar (= PDA). Seven replicates of each medium for each
isolate were inoculated and placed in a dark incubator at
24°C. Macromorphological descriptions were taken during
the third and the sixth weeks. Photographs of representative
isolates were taken during the fourth week, and
micromorphological characters were examined during the sixth
week.
Macromorphological characters noted included: (a) radius of
the culture mat, (b) form and character of the advancing
zone, (c) mat color and topography, and (d) the presence or
absence of exudates. Terminology used was taken from Nobles
(1948, 1958b, 1965).
Micromorphological characters were observed by mounting
hyphae from the advancing zone, mat, and plug of each
isolate in 3% KOH and examining the slide under phase
contrast. Micromorphological characters examined included
the presence or absence of: (a) modified hyphae, (b)
chlamydospores or oidia, etc., and (c) basidiomata or
hymenial structures such as basidia or cystidia along the
mat surface. Descriptive terminology used was that of Nobles
(1948, 1965).
Extracellular oxidase activity of each isolate was tested
using both the Bavendamm (Davidson et al., 1938, 1942) and
gum guaic (Nobles, 1958a, 1965) tests (Mueller, 1984).
Intra- and Intercollection Pairing
Analyses
Homokaryotic (single basidiospore) and heterokaryotic
isolates derived from multiple-germinated basidiospores were
obtained following the techniques of Fries (1983a, 1983b)
and Fries and Mueller (1984). Homokaryotic isolates
originating from one basidioma were assigned a stock number.
Within a stock, each isolate received a unique extension
number. All isolates are stored at 2-4°C on N6:5 medium
in the mycological culture collection at Field Museum of
Natural History.
Intra- and interstock pairing analyses were carried out
following the procedures outlined in Mueller and Gardes
(1991). Isolates to be tested were placed * 10 mm apart on
N6:5 plates and allowed to grow together (2-4 wks). After an
additional one week or more, plugs of tissue were cut from
the interface and placed on fresh N6:5 plates. Mycelium
growing from these plates were checked for the presence or
absence of clamp connections by examining them through the
bottom of the inverted Petri plate at 200 x magnification.
Pairings that resulted in hyphae that bore clamp connections
were considered positive while those that did not yield
clamped hyphae were scored as negative. Two or more testers
for each stock, each containing different mating type
alleles, were used, when possible. As in Mueller and Gardes
(1991) and Mueller (1991c), the terms noncompatible and
intracompatible are restricted to intrastock pairings. I
used the terms intersterile for intercollection pairings
that did not form clamp connections and intercompatible
(rather than interfertile) for positive intercollection
matings since data regarding fruiting and progeny analysis
were not available.
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Many Laccaria taxa appear similar upon superficial
examination. Closer examination of the macro- and
micromorphological variation, when correlated with cultural
and molecular data, allows for the segregation of taxa in
this phenotypically variable group. The following discussion
on the delimiting characters within Laccaria is
restricted to North American taxa of which 18 of the 20 taxa
have been collected, photographed and described during the
course of this study.
Basidioma Macromorphology
Color. Basidioma and lamellar colors are the most
diagnostic macromorphological characters. A major problem
with this suite of characters is that the pigments
responsible for these colors are unknown in many groups of
agarics, including Laccaria [e.g., W.
Steglich, Univ. Bon, Germany (personal communication)
informed me that the pigments in L. amethystina
oxidize very readily upon extraction and would take special
care to analyze]. Currently, it is not possible to determine
homology between the pigment(s) in either the orange-brown
or violet- colored Laccaria or to determine the
primitive condition via comparison with presumably related
genera (outgroup comparison). As mentioned in the section on
Phylogenetic Considerations, I have made the assumption that
the pigment(s) are homologous within these two groups of
Laccaria (orange-brown vs. violet). Assumptions of
homology between the pigments in Laccaria and
pigments in potential outgroup taxa could not be made.
Because all members of the genus are hygrophanous,
especially those taxa with violaceous to purple basidiomata,
it is important to note the color of the pileus both when
fresh and in successive stages of fading. This is most
important in L. amethysteo occidentalis G. M.
Mueller, L. amethystina and L.
vinaceobrunnea G. M. Mueller. Basidiomata of all
three are bright violet to purple when young and fresh but
differ markedly in their color changes associated with age
(see descriptions). Laccaria ochropurpurea and
L. trullissata (Ellis) Peck are light
violaceous when very young but soon become buff or red-brown
at maturity.
Basidiomata of specimens of most other Laccaria are
some shade of orange brown to flesh color. Due to the large
amount of color variation found within these taxa (from
light buff to a strong orange-brown or red-brown), basidioma
color can rarely be used to delimit taxa in this large
group.
North American species of Laccaria exhibit either
pinkish flesh color to buff color or violaceous to purple
lamellae. All of the taxa with violaceous to purple
basidiomata plus L. ochropurpurea and L.
trullissata have bright violet to dark purple
lamellae. Basidiomata of L. bicolor (Maire)
Orton, L. nobilis G. M. Mueller and, possibly, L.
maritima (Teodorowicz) Singer ex Huhtinen have
light pinkish violet to wine-colored lamellae that
occasionally fade to a pinkish color with age. All other
North American taxa have flesh-colored lamellae. In some
instances the lamellae of large, older specimens may develop
a vinaceous appearance but an examination of younger
specimens should alleviate any confusion that this may
cause. Basidiomata of L. fraterna (Cooke &
Massee: Saccardo) Pegler, which probably is an introduced
taxon, have rosy-pink lamellae when fresh.
The color of the mycelium at the base of the stipe (= basal
mycelium) is also systematically important in
Laccaria. The basal mycelium can be either violet or
white. In addition to L. trichodermophora G.
M. Mueller and L. oblongospora G. M. Mueller, all of
the taxa with violet lamellae consistently have a violet
basal mycelium. In all of these taxa, however, the basal
mycelium occasionally fades to white with age.
Basidioma size. Although often highly variable due to
differences in age and environmental conditions, the size of
the basidioma can be diagnostic in some instances. Most taxa
have mature basidiomata of moderate size (pileus 20-50 mm
broad, stipe up to 60 mm long), so those taxa which
consistently have larger or smaller basidiomata are
noteworthy. Laccaria montana Singer, L.
ohiensis, L. pumila Fayod and L.
tortilis are characterized by being small (pileus
rarely up to 30 mm broad) while L. amethysteo
occidentalis, L. nobilis, L.
ochropurpurea, and L. trullissata often
have pilei greater than 60 mm broad.
The stature of the basidioma can also be systematically
important. Laccaria maritima, L.
ochropurpurea and L. trullissata are
usually robust (stipe diameter > 7 mm at apex), while all
of the taxa characterized by having small basidiomata plus
L. striatula (Peck) Peck, are generally
gracile (stipe diameter * 4 mm at apex).
Stipe and pileus ornamentation characteristics are often
associated with size. Most of the small taxa appear glabrous
to finely fibrillose when fresh while taxa with large
basidiomata, such as L. nobilis, often have
pilei which become scaly to squamulose due to cuticular
diffraction. Additionally, large basidiomata frequently have
stipes clothed with pronounced longitudinal striations which
can form reticulations or become scaly near the stipe apex
(e.g., L. amethysteo-occidentalis, L.
nobilis, L. ochropurpurea, and
L. trullissata).
Basidiospore color in mass. Except for L.
amethystina and L. ochropurpurea, which
can have either white or light violet basidiospore deposits,
all North American taxa have white spore prints. The color
of the print will occasionally yellow with age.
Additional macromorphological characters. Characters
such as type and degree of pileus striations, pileus shape,
lamellar attachment and thickness, pileus and stipe context,
or odor and taste are not systematically significant within
Laccaria. Except for color and, in some instances,
size, one is required to use micromorphological and cultural
characters to differentiate taxa.
Basidioma Micromorphology
Basidia. The number of basidiospores borne per
basidium is significant in Laccaria. Although many
other genera of agarics have species with varying number of
sterigmata in one basidioma (e.g., Hesler and Smith, 1963;
Singer 1986), the character appears to be consistent in
Laccaria. In all of the material examined, the vast
majority of the basidia observed in a mount had the same
number of sterigmata. Basidia examined from material of
L. fraterna, L. pumila, and L. tortilis
bore 2(-3) basidiospores. All of the rest of the North
American taxa had 4-sterigmate basidia. Bisterigmate basidia
generally had longer and stouter sterigmata. No other
basidial characters appeared to have systematic
significance.
Basidiospores. Basidiospore shape is of critical
importance in designating species. Basidiospore shape terms
used in this study are based on Bas (1969) with basidiospore
shape described in terms of length-width ratios (Q): globose
= 1.0-1.05, subglobose = 1.06-1.15, broadly ellipsoid =
1.16-1.23, ellipsoid or amygdaliform = 1.24 1.6, oblong =
1.65-2.0, cylindrical or subfusiform > 2.0.
Specimens of most North American taxa have basidiospores
which are subglobose to broadly ellipsoid. Laccaria
proxima (Boudier) Patouillard and L.
oblongospora are characterized by having ellipsoid to
oblong basidiospores while basidiospores in specimens of
L. trullissata and L. maritima are
oblong to subfusiform. Globose basidiospores are found in
specimens of L. amethystina, L. ochropurpurea,
L. ohiensis, L. striatula and L.
tortilis.
Basidiospore size is also an important systematic character
in some instances. Individuals of most taxa have overall
mean basidiospore lengths of 8-9 µm. However,
Laccaria bicolor, L. longipes G. M. Mueller,
L. nobilis and L. trichodermophora are
characterized by having relatively small basidiospores (L
< 8 µm) while specimens of L. fraterna, L.
montana, L. pumila and L. tortilis have
larger basidiospores (L = 9.5-13 µm). The basidiospores
of L. maritima and L. trullissata are
greater than 13 µm in length.
Except for L. trullissata which has finely
roughened basidiospores (Figs. 41a, 57b-d, 72d), all species
of Laccaria have echinulate basidiospores with a
plage near the hilar appendix. Echinulae length and width at
attachment are important systematic characters within the
genus.
Electron microscopic examinations of basidiospores by
surface replication (Bigelow and Rowley, 1968; Pegler and
Young, 1971), transmission electron microscopy (= TEM)
(Besson and Kühner, 1971), and scanning electron
microscopy (= SEM) (Pegler and Young, 1971; Mueller and
Sundberg, 1981; Irving et al., 1985; Mueller, 1991c;
and in this paper, Figs. 53-58) have shown details of the
surface and ornamentation characteristics. At my request,
Dr. A. von Hofsten (Institute of Physiological Botany,
University of Uppsala, Uppsala, Sweden) examined
basidiospores from collections of several Laccaria
species (L. bicolor, L. maritima and L.
proxima) and from a collection of Hydnangium
carneum Wallroth apud Klotzsch under the TEM (von
Hofsten, unpublished). The results of this study were
concordant with the findings of Besson and Kühner
(1971). The basidiospore echinulae of all Laccaria
species examined to date, plus the gasteroid relative
Hydnangium carneum, are composed of microtubials
which run perpendicular to the epispore (Besson and
Kühner, 1971; Kühner, 1980). This type of
echinulae ultrastructure appears to be unique to
Laccaria, Hydnangium and probably
Podohydnangium Beaton, Pegler and Young (not examined
under TEM). Echinulae ultrastructure is the primary
synapomorphy that supports the recognition of these three
genera as a monophyletic group (see PHYLOGENETIC
CONSIDERATIONS).
The basidiospores in all taxa are nonamyloid and
acyanophyllic.
Pileipellis. Three types of hyphal arrangements were
found in Laccaria (Fig. 1a-c): a trichodermium,
observed in most collections of L.
trichodermophora; interwoven as in collections of
L. ochropurpurea; or interwoven with scattered
nearly perpendicular fascicles of hyphae (the most common
pileipellis encountered). In L.
vinaceobrunnea, and some collections of L.
amethystina, the hyphae of the pileipellis are
interwoven with very numerous, long, individual hyphae that
are arranged nearly perpendicular to the pileus surface. If
the erect hyphae were aggregated closer together, the
pileipellis could be described as a palisadoderm.
Pileipellis hyphae were often encrusted with a light to
moderate yellowish brown pigment. In young specimens of
L. amethystina, L.
amethysteo-occidentalis and L.
vinaceobrunnea, the hyphae occasionally appeared
vinaceous brown in mass.
Although data on the size of the terminal cells are
presented in the species description, the size and shape of
these cells appeared to have little systematic significance.
Additionally, the underlying tramal layer was
morphologically undifferentiated and systematically
uninformative.
Cheilocystidia. The cheilocystidia of most
Laccaria species, if present, are little more than
elongate, filamentous hyphae which extend beyond the basidia
and basidioles. Only in specimens of L. amethystina,
L. amethysteo-occidentalis, and L.
vinaceobrunnea were large (overall mean dimensions up to
58 x 10 µm), clavate to strangulate, abundant
cheilocystidia observed. In many collections these
cheilocystidia formed a nearly sterile layer. The uniqueness
and consistent presence of large cheilocystidia in these
three Laccaria taxa was a good diagnostic character
when dealing with herbarium material that lacked
macromorphological notes.
Pleurocystidia were not seen in any North American material
of the genus. Morphologically undifferentiated caulocystidia
are present in some taxa.
Additional micromorphological characters. The
constant occurrence of clamp connections at virtually every
septum along with a parallel or rarely subparallel lamellar
trama are important generic characteristics of
Laccaria. Additionally, all hyphae were nonamyloid
and, unless otherwise specified, hyaline in KOH.
The hyphae comprising the basal mycelium were tightly
interwoven and either morphologically undifferentiated or
barrel-shaped. The diameter of these hyphae appeared to vary
as much within a taxon as between taxa.
Basidiospore and Basidium Cytology
Kühner (1980, 1984) emphasized the occurrence of
multinucleate basidiospores in Laccaria in his
decision to treat Laccaria in a family separate from
other genera traditionally placed in the Tricholomataceae.
To determine the significance of basidiospore and basidium
cytology in the systematics of Laccaria, cytological
studies on several species of Laccaria were carried
out in Dr. J. F. Ammirati's laboratory (Department of
Botany, University of Washington, Seattle). Detailed results
will be published in a separate manuscript (G. J. Mueller,
G. M. Mueller, L. Shih and J. F. Ammirati, in prep).
Two questions were addressed during the course of this
study. First, is the occurrence of multinucleate
basidiospores a characteristic of the genus, or is this
feature restricted to a few taxa? Second, where does the
postmeiotic mitosis occur in taxa with multinucleate
basidiospores?
Basidia and basidiospores of representative specimens of
each of the 11 Laccaria species and Hydnangium
carneum listed in Table 1 were examined using
fluorescent microscopy. Glutaraldehyde-fixed and nonfixed
tissues were mounted in Hoechts fluorescent dye and examined
using a Zeiss fluorescence microscope with 365 nm excitation
and 480 nm emission filters.
Multinucleate basidiospores appear to be a constant feature
within Laccaria since they occur throughout the genus
from the putatively primitive L. proxima to diverged
taxa such as L. tortilis, L.
amethysteo-occidentalis and L. trullissata (Table
1). These data also are concordant with the hypothesis of
the close relationship of Laccaria to
Hydnangium. According to Kühner (1980, 1984),
the occurrence of multinucleate basidiospores is rare in the
Tricholomatales sensu Kühner (±
Tricholomataceae sensu Singer).
The following is a summary of results obtained during
studies of tetra- and bisterigmate Laccaria taxa
(L. bicolor, L. galerinoides Singer, L.
laccata var. pallidifolia, L. montana,
L. proxima, L. proximella Singer, L.
pumila, L. tortilis and L. vinaceobruunea)
using Giemsa stained material examined under bright field
microscopy. The dikaryotic basidium of both tetra- and
bisterigmate Laccaria is more or less cylindrical in
shape. The two nuclei move to the center of the basidium and
fuse to form the diploid nucleus, during which time the
basidium gradually enlarges and becomes clavate in shape.
The diploid nucleus then moves to the apical region of the
basidium, where meiosis I and II occur. These divisions are
chiastic and the resulting four nuclei migrate to the center
of the basidium. During these events the basidium enlarges
to its mature size and shape, and sterigmata with developing
basidiospores are formed. Once the basidiospores are fairly
well developed, the nuclei begin to move through the
sterigmata and into the basidiospores. Basidiospores from
tetrasterigmate basidia each receive one nucleus, while
those with bisterigmate basidia each receive two nuclei. The
(nucleus) nuclei in the basidiospores then undergo a mitotic
division so that basidiospores from tetrasterigmate basidia
are binucleate and those of bisterigmate basidia are
tetranucleate.
Tommerup and colleagues reported similar nuclear behavior
for L. fraterna (Tommerup et al., 1991).
According to them, however, nuclear behavior in
Hydnangium carneum differs by having the postmeiotic
mitosis occur in the basidium prior to migration into the
basidiospores. Additional information on nuclear behavior in
Hydnangium and Podohydnangium is necessary
before it will be possible to determine if multinucleate
basidiospores in Laccaria and these two genera are
homologous. The Hydnangium used by Tommerup et
al. (1991) is reported to have tetrasterigmate basidia
and the illustrated basidiospores are broadly elliposid and
finely ornamented. This is in contrast to my concept of
H. carneum. All of the collections that I have
examine which are referable to this taxon have bisterigmate
basidia which bear strongly echinulate, globose
basidiospores. Similarily, published illustrations of
basidiospores and basidia for this taxon usually depict
globose, strongly echinulate basidiospores and bisterigmate
basidia (e.g., Pegler and Young, 1979, Beaton et al.,
1984; Castellano et al., 1989). Until a survey of
other taxa in the genus is undertaken, and until
phylogenetic relationships within the genus are resolved, it
is not possible to determine the pleisiomorphic condition
for nuclear behavior in Hydnangium.
Table 1. Number of nuclei observed in the basidiospores of
Laccaria and Hydnangium visualized with
Hoechts fluorescent dye.
|
Taxon
|
# spores/basidium
|
# nuclei/spore
|
|
L. amethysteo-occidentalis
L. bicolor
L. fraterna
L. laccata var. pallidifolia
L. montana
L. ochropurpurea
L. proxima
L. pumila
L. tortilis
L. trullissata
L. vinaceobrunnea
H. carneum
|
4
4
2
4
4
4
4
2
2
4
4
2
|
2
2
4
2
2
2
2
4
4
2
2
4
|
Somatic Culture Mat Morphology
Somatic culture mat studies based on the classic work of
Nobles (1948, 1958b, 1965) were undertaken to obtain
additional informative characters. Although becoming almost
routine in studies of the wood-rotting Aphyllophorales and
many groups of saprobic Agaricales, utilization of somatic
culture mat data has only infrequently been used in
systematic studies of fungi which form ectomycorrhizae. At
least two factors are responsible for this. First, many of
these fungi are recalcitrant to domestication (but see
Hutchinson, 1990a, 1990b; Hutchinson and Summerbell, 1990).
Secondly, early work indicated that cultures of fungi that
form ectomycorrhizae exhibited few morphological differences
(e.g., Zak and Bryan, 1963; Zak and Marx, 1964).
Fries and Mueller (1984) reported no differences in
morphology (except for the presence or absence of clamp
connections) between homokaryotic and heterokaryotic
isolates. Occasional differences in growth rate, color
intensity and other features have since been detected among
heterokaryotic isolates obtained via tissue culture, those
originating as polysporous cultures and homokaryotic
isolates (Kropp et al., 1986; Kropp and Fortin, 1988;
Mueller, unpublished). While overall similarities are
normally observed, care must be used when employing isolates
of different origin for comparative studies. The following
data are based primarily on isolates of tissue culture
origin.
Macromorphology. The two primary diagnostic
characters were the color of the culture mat on MMN and PDA
(all isolates were white on MEA) along with the rate of
growth (expressed as the radius of the culture mat at week 3
and week 6) on each of the three media employed. Photographs
of representative isolates are included as part of the
description for a number of North American taxa (Figures 7,
10, 13, 31, 34, 37, 44).
Taxa that had white to off-white culture mats on all three
media were L. fraterna, L. laccata,
L. longipes, L. montana, L. ohiensis, L.
proxima, L. pumila and L.
striatula. No isolates were obtained of L.
maritima or L. tortilis. Isolates of all other
North American taxa were violet to purple on PDA and MMN. To
date there is a 100% correlation between basal mycelium
color and somatic culture mat color. This supports the
utility of basal mycelium color as a good delimiting field
character.
Although a slight bleaching of color was often noted on the
reverse side of the culture mat, no significant color
changes occurred. Additionally, no exudates were
apparent.
Along with color, the rate of growth could be used to
delimit taxa. Isolates of most taxa grew at a moderate rate
on all three media (20-50 mm on PDA, 30-70 mm on MMN, and
40-70 mm on MEA--all at week 6). Isolates of L.
amethysteo-occidentalis, L. montana and L.
ohiensis grew much slower (often only 10-15 mm after 6
weeks growth). Conversely, isolates of L. bicolor,
L. nobilis, L. oblongospora, and L.
trichodermophora grew more rapidly.
Terminology used in discussing mat and margin texture was
from Nobles (1948, 1965).
Except for an occasional pruinose, aerial layer of hyphae
observed in older cultures of many taxa, the mycelium of all
of the isolates grew tightly appressed to the agar surface.
The transparency of the culture mat was an outcome of the
thickness and intricacy of the interwoven mat. All isolates
exhibited a felty, thick culture mat on PDA. The mat texture
on both MMN and MEA varied from silky to subfelty to felty.
Often the mat was thickest near the inoculation plug. In
most cases, culture mats were of a uniform thickness from
the plug to the margin. In some taxa, however, variously
distributed thicker zones were observed. These thicker zones
could be scattered, small sectors (e.g., some L.
trichodermophora isolates on MMN and L.
bicolor on MEA), 2-3 concentrically arranged bands
(e.g., L. amethysteo-occidentalis on MEA), or
radially arranged pie-shaped or dendritic sectors radiating
away from the inoculation plug (e.g., L.
trichodermophora on PDA).
When describing the margin texture and color, I refer to the
advancing zone of the culture mat. This zone is generally
somewhat thinner than the rest of the mat and can be either
a discrete, easily recognizable area or not well
differentiated. On all three media, this zone was always
silky to subfelty and thus presented little systematically
informative data.
All of the isolates had the same musty odor on all three
media.
Growth or diffusion zones were not obtained on gallic acid
agar with any of the isolates. Immediate reaction to the gum
guaiac drop test was observed only in some isolates of L.
bicolor. I did not attempt other tests for the presence
or absence of extracellular enzymes because of these
basically negative results. Hutchison (1990a), however,
employing drop tests of l-Naphthol and p-Cresol on numerous
isolates of ectomycorrhizal fungi growing on several
different media, reported the presence of tyrosinase
activity in all of the Laccaria that he tested
(i.e., L. bicolor, L. laccata, L.
ochropurpurea and L. proxima). Only L.
bicolor showed any indication of laccase activity
(Hutchison, 1990a).
Hutchison (1990b) investigated enzymatic degradation of
various carbon and nitrogen compounds by fungi that form
ectomycorrhizae. Using the same taxa listed in the previous
paragraph, he reported that Laccaria did not degrade
pectin, lipid, amylose or gelatin but showed various levels
of degradation of casamino acids and urea. Hutchison and
Summerbell (1990) reported that these same isolates gave red
to violet reactions to Diazonium Blue B when treated with
cold KOH and yellow reactions without cold KOH
treatment.
Micromorphology. There was little hyphal
differentiation observed within and among the
Laccaria isolates examined during this study nor were
any spores formed in culture.
In all isolates, the vast majority of hyphae were
morphologically undifferentiated with clamp connections at
nearly all septa (Figure 2a). Scattered among these sparsely
branched hyphae were subcoralloid to coralloid hyphae
(Figures 2b-c) and/or irregular swollen hyphae (Figure 2d).
All hyphae were hyaline in KOH, except where noted.
Localized, slightly thick walled swellings were often
observed in plates of all taxa (Figure 2e). These swellings
could either be terminal or intercalary and could be found
in chains of 2-4 and are likely a response to water stress
as they become more abundant when the agar in Petri plates
loose moisture. Hutchison (1989) reported similar swellings
from numerous isolates of ectomycorrhizal fungi.
Pantidou et al. (1983) reported the presence of
holoblastic conidia from a proported culture of Laccaria
laccata. Hutchison (1989), however, reported that these
structures were secretory cells of a Pleurotus
sp. and that the isolate used by Pantidou et
al. (1983) was from a species of that genus, not L.
laccata.
Intra- and Intercollection Pairing
Reactions
Information on intra- and intercollection pairing reactions
for systematic and biological studies of fungi that form
ectomycorrhizae has only recently been employed (Fries,
1987). Fries (1977) first reported the successful
germination of basidiospores from collections of
Laccaria. Several years later he reported the
occurrence of several incompatibility groups within L.
laccata sensu lato (Fries, 1983a). Fries and Mueller
(1984) later determined that these groups were referable to
separate species. They documented a good correlation between
species based on morphological characters and intersterility
groups with Swedish isolates of L. amethystina, L.
bicolor, L. laccata, and L. proxima.
Intraspecific pairings of these Swedish isolates revealed a
high incidence of intercompatibility in three of these
species (all but L. laccata). Two intersterility
groups were detected within Swedish isolates from
morphologically similar collections identified as L.
laccata. The two intersterility groups within L.
laccata were treated as sibling species since they could
not be delimited on morphological characters (Fries and
Mueller, 1984; Mueller and Vellinga, 1986). Studies by Kropp
and Fortin (1988) and Doudrick and Anderson (1989)
documented the occurrence of two or more intersterility
groups within the North American population of L.
bicolor.
When used in conjunction with data from other analyses,
information on intercollection pairing reactions has proven
useful for circumscribing taxa (Mueller and Gardes, 1991;
Mueller, 1991c). Mueller and Gardes (1991) reported three
intersterility groups within the examined material of North
American L. bicolor. Intragroup intercompatibility
was high and most intergroup pairings were intersterile.
These groups could be circumscribed on both morphological
and molecular characters and consisted of isolates of L.
bicolor sensu stricto, L. nobilis and L.
trichodermophora. Questions remain as to the
relationship of Swedish material, morphologically similar to
North American material of L. bicolor sensu stricto,
to the three North American taxa. The two isolates of
Swedish origin used as testers were 100, 57 and 21%
intercompatible with North American isolates of L.
bicolor, L. nobilis and L.
trichodermophora, respectively.
Interstock pairing reactions were also useful in a study of
the L. laccata complex (Mueller, 1991c). However,
because isolates of L. laccata var. laccata
were not available for inclusion in these analyses, some of
the systematic conclusions remain tentative. Several North
American intersterility groups were detected and all tested
North American isolates were intersterile with both of the
intersterility groups reported from Sweden by Fries and
Mueller (1984). Most, but not all, of these groups could be
delimited morphologically. Molecular divergence (detected
through analyses of restriction fragment length
polymorphisms of mitochondrial and nuclear ribosomal DNA)
was also detected between several of these intersterility
groups (Gardes et al., 1990, 1991a; see below).
Intersterility groups that could be delimited on
morphological characters were recognized at the species
level (i.e., L. longipes, L. montana,
L. ohiensis, and L. striatula).
As in the study on the L. bicolor complex (Mueller
and Gardes, 1991), data obtained to date do not resolve
questions concerning potential gene exchange between
geographically distant populations of some putatively
cosmopolitan species in the L. laccata complex
(Mueller, 1991c). The most commonly collected North American
taxon in the this complex is L. laccata var.
pallidifolia. Mueller and Vellinga (1986) and Mueller
(1991a) reported this taxon as being abundant in Europe.
However, North American and Swedish isolates referable to
this taxon are intersterile (Mueller, 1991c). Unfortunately,
representative Swedish material of this taxon was not
included in the studies of Gardes et al. (1990,
1991a, 1991b) so data are not available on molecular
divergence between the two populations. It is not possible
to delimit collections from the two populations on
morphology (Mueller, 1991c). For now, therefore, I treat
these two potentially intersterile populations as
contaxic.
Tested isolates of L. amethysteo-occidentalis, L.
amethystina, L. proxima, L. vinaceobrunnea
were intersterile in all attempted interspecific pairings.
Intraspecific pairings were nearly completely
intercompatible within these species. However, homokaryotic
isolates were available from only 2-3 stocks for most of
these species so it was not possible to rigorously test the
degree of intraspecific intercompatibility.
In this study I have delimited species so that they are
presumably monophyletic and are diagnosable by a unique
combination of character states. Data from intercollection
pairings were used in conjunction with other data sets and
did not outweigh information from the other analyses. Since
data on morphology, breeding and ecology are not always
concordant, it is inadvisable to rigorously adhere to the
biological species paradigm (see discussions in Mishler and
Donoghue, 1982; Donoghue, 1985; de Queiroz and Donoghue,
1988, 1990; Cracraft, 1990; Vilgalys, in press).
Restriction Fragment Length
Polymorphisms of rDNA and mtDNA
It is not yet possible to routinely obtain in vitro
basidioma production for any species of Laccaria, or
most other fungi that form ectomycorrhizae. One consequence
of this inability to obtain the complete life cycle for
these organisms is that it is impossible to carry out
genetic analyses or to determine if intercompatible isolates
are fertile (would produce viable progeny). This problem,
coupled with the fact that the ability to mate is a
pleisiomorphic character state (e.g., Donoghue, 1985), makes
it necessary to have data from other analyses to
substantiate hypotheses of gene exchange between
populations. Gardes et al. (1990, 1991a) obtained
data on restriction fragment length polymorphisms (RFLPs)
for both nuclear ribosomal DNA (rDNA) and mitochondrial DNA
(mtDNA) to: 1) investigate possible genetic divergence
between species and populations of the same putative
species; 2) determine concordance of data obtained through
morphological analyses and pairing analyses; and 3) evaluate
molecular markers for isolate typing. The resulting data
could not be used for phylogenetic reconstructions because
the numerous length mutations that were detected within the
segments of DNA analyzed prevented making the necessary
assumptions of homology.
Comparable results were obtained with both mtDNA and rDNA
for L. amethystina and L. laccata sensu lato
(Gardes et al., 1990, 1991a; Mueller, 1991c) in which
divergence was detected between North American and Swedish
isolates of these taxa. Divergence was also detected between
the tested intersterility groups of the North American L.
laccata complex.
Conflicting results were obtained with mtDNA and rDNA within
the L. bicolor complex (Gardes et al., 1990,
1991a; Mueller and Gardes, 1991). Divergence was detected
between the North American and Swedish populations but was
not observed between North American intersterility groups
using rDNA. Conversely, divergence was detected between each
of the North American intersterility groups but was not
detected between North American and Swedish populations
based on mtDNA polymorphisms. Additional European material
needs to be examined to attempt to resolve this discrepancy.
Only two Swedish isolates were utilized by Gardes et
al. (1990, 1991a) so it is not possible to compare the
amount of heterogeneity of either mtDNA or rDNA within North
American and European populations. A possible explanation
for these conflicting results is that Swedish L.
bicolor migrated from a large North American population
which contained a pool of mtDNA variation (J. W. Taylor,
University of California, Berkeley, personal communication).
The Swedish population would, therefore, only contain a
subset of the mtDNA variation. Following migration, the
North American population underwent divergence of
morphological and pairing alleles resulting in L.
nobilis and L. trichodermophora. Some of the
mtDNA variation within the original North American pool has
subsequently been lost as evidenced by the data on RFLPs
which uncovered more intraspecific than interspecific
similarity in mtDNA for the three North American taxa. This
hypothesis can not be tested until a robust phylogeny for
the group is obtained.
In Vitro Ectomycorrhizal
Synthesis Results
Isolates of Laccaria are frequently used in applied
and basic studies on ectomycorrhizae (see Kropp and
Langlois, 1990). In vitro mycorrhizal synthesis
studies employing the growth pouch technique of Fortin et
al. (1983) are ongoing as part of my studies on
Laccaria. Experiments were run in a plexiglass
mycorrhizal synthesis chamber, the design for which was
modified from plans given us by Dr. Steve Miller (University
of Wyoming, Laramie; personal communication). The primary
goals of these studies have been to further characterize
species of Laccaria as well as document the ability
of particular species of Laccaria to form
ectomycorrhizae with select tree species in the
laboratory.
To date we have synthesized ectomycorrhizae between several
Laccaria (L. amethysteo-occidentalis, L.
bicolor, L. laccata var. pallidifolia,
L. proxima and L. striatula) and the following
North American trees: Picea sitchensis (Bong.) Carr.,
Pinus ponderosa Laws, P. resinosa Ait., and
Pseudotsuga menziesii (Mirb.) Franco. Ectomycorrhizae
have also been synthesized between L. trullissata and
Pinus resinosa and between several South American
isolates of L. ohiensis and seedlings of the southern
beech, Nothofagus obliqua (Mirbel) Oerst.
While macromorphology of the ectomycorrhizae varies with the
species of host tree (e.g., degree of branching), their
micromorphology is similar. In all material examined to
date, a well-defined, 15-60 µm thick, tightly
interwoven mantle of clamped, morphologically
undifferentiated hyphae was formed. No cystidia-like
elements have been observed. All infected short roots had a
well-developed Hartig net that extended through 70-100% of
the cortex. These data coincide with published data on
Laccaria ectomycorrhizae.
|