High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium.

Abstract

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.

Full text

Page 1

Molecular Ecology (2005)
14
, 613–626doi: 10.1111/j.1365-294X.2005.02424.x
© 2005 Blackwell Publishing Ltd
Blackwell Publishing, Ltd.
High specificity generally characterizes mycorrhizal
association in rare lady’s slipper orchids, genus
Cypripedium
RICHARD P. SHEFFERSON,
*
Department of Integrative Biology, University of California, 3060 Valley Life Sciences Building, #3140, Berkeley, California 94720
USA,
†
Botanisches Institut, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany,
Botany, Estonian Agricultural University, Riia 181, Tartu 51014 Estonia,
I Building, 902 N. Koyukuk Drive, Fairbanks, Alaska 99775–7000 USA
*¶ MICHAEL WEIß,† TIIU KULL‡ and D. LEE TAYLOR§
‡
Institute of Zoology and
§
Institute of Arctic Biology, University of Alaska, 311 Irving
Abstract
Lady’s slipper orchids (
the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for
germination and seedling nutrition. The nutritional relationships of adult
mycorrhizae are unclear; however,
Cypripedium
rhizal specificity, whether this specificity occurs only during the seedling stage or carries
on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100
Cypripedium
plants, and successfully did so with two
californicum
, six
Cypripedium candidum
, 16
Cypripedium fasciculatum
guttatum
, 12
Cypripedium montanum
, and 11
of 44 populations in Europe and North America, yielding fungal nuclear large subunit and
mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism)
data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting
structures, we assessed fungal identity through direct PCR (polymerase chain reaction)
amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic ana-
lysis revealed that the great majority of
Cypripedium
clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include
members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus,
C. californicum
was the only orchid species with apparently low specificity, as it associated
with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our
results add support to the growing literature showing that high specificity is not limited to
nonphotosynthetic plants, but also occurs in photosynthetic ones.
Cypripedium
spp.) are rare terrestrial plants that grow throughout
Cypripedium
distribution may be limited by mycor-
Cypripedium calceolus
, 10
Cypripedium
Cypripedium
plants from a total
, two
Cypripedium parviflorum
mycorrhizal fungi are members of narrow
Phialophora
.
Keywords
: Ceratobasidiaceae, myco-heterotrophy,
Rhizoctonia
, Sebacinaceae, specificity, Tulasnellaceae
Received 31 August 2004; revision received 4 November 2004; accepted 4 November 2004
Introduction
Symbiotic relationships are important to the evolution and
conservation of many, perhaps all, organisms. For plants,
symbioses are particularly important for sexual repro-
duction, as when animals act as pollinators or seed dis-
persers in return for nectar or a portion of the seed crop
(Jordano 1993; Pellmyr
et al
. 1996; Kawakita & Kato 2004).
At least as important to plants are microbial symbioses
resulting in significant nutritional advantage. Primary
among these is the mycorrhiza, a plant-fungus symbiosis
from which most plants gain the majority of their
nutrients, including those limiting their growth (Smith &
Read 1997). Mycorrhizal fungi are thought to have evolved
at the same time as land plants, allowing the latter to
colonize and radiate on land (Wilkinson 2001; Brundrett
2002). Most plants respond mutually to such gains by
allowing the fungus a share of photosynthetically-fixed
carbon, which limits fungal growth (Smith & Read 1997).
However, some plants, including the entire orchid family,
Orchidaceae, have evolved a different kind of mycorrhizal
Correspondence: Richard P. Shefferson, ¶Microbial Ecology
Laboratory, Forestry and Forest Products Research Institute,
Tsukuba 305-8687, Japan, Fax: (+81) 29 859 0644;
E-mail: dormancy@gmail.com

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R. P. SHEFFERSON
ET AL
.
© 2005 Blackwell Publishing Ltd,
Molecular Ecology
, 14, 613–626
relationship in which carbon is not supplied to the fungus
(Taylor
et al
. 2002). Orchid mycorrhizae have been studied
for nearly a century now, and yet the notoriously cryptic
fungi involved in these symbioses have limited even our
understanding of their taxonomic identities and evolu-
tionary relationships (Rasmussen 1995). Of all orchids
that have been studied, few have been the object of mycorrhizal
inquiry for as long as the primary genus of temperate
lady’s slipper orchids,
Cypripedium
Mycorrhizal infection is a prerequisite for the germina-
tion and growth of all orchid seeds in the wild (Bernard
1904). For
Cypripedium
, life begins as a ‘dust seed’, a seed so
small as to be almost microscopic (Curtis 1943; Rasmussen
1995; Kull 2002), with recruitment estimated at less than
0.06% in
Cypripedium calceolus
by a compatible mycorrhizal fungus, the seed germinates
and develops into a seedling that consumes fungal sugars,
a condition known as ‘myco-heterotrophy’ (Beau 1920;
Smith 1967; Alexander & Hadley 1985; Leake 1994;
Rasmussen 1995). The myco-heterotrophic orchid seed-
ling may grow for several years prior to developing into
a ‘green’ seedling (Rasmussen 1995; Kull 1999). As
adults,
Cypripedium
species undergo ‘adult dormancy’,
periods of one or more years during which no sprouts
are produced and no photosynthesis takes place (Lesica
& Steele 1994; Shefferson
et al
these plants may retain myco-heterotrophy into adulthood
(Gill 1989).
Efforts to understand mycorrhizal associations in this
group generally focus on attempts at seed germination
and propagation with various species of the fungal genus
Rhizoctonia
. However, since MacDougal’s (1899) classic
description of
Cypripedium
mycorrhizal morphology, studies
of this interaction have either failed to yield germination
of
Cypripedium
seeds or have led to inconsistent results
(Curtis 1939; Zelmer
et al
. 1996; Ramsay & Stewart 1998).
This may be the result of confusion surrounding this
fungal genus.
Rhizoctonia
is often noted for its associations
with most other orchids (Rasmussen 1995; Case
Ramsay
et al.
1998). It is polyphyletic, including fungi from
across the fungal division Basidiomycota, particularly
from the families Tulasnellaceae, Sebacinaceae, and
Ceratobasidiaceae (Andersen 1996; Muller
However, because
Rhizoctonia
based on asexual stages, the identities of and phylogenetic
relationships among
Rhizoctonia
be evaluated now with the dawn of molecular phyloge-
netics (Andersen 1996; Muller
et al
. 2003).
If
Cypripedium
species specialize on particular mycor-
rhizal fungi, then these fungi may limit their distribution.
Soil microbial diversity plays important roles in determining
plant abundance (Klironomos 2002), and seed germina-
tion in some orchids only occurs near adults (Batty
.
(Kull 2002). Upon infection
. 2003), suggesting that
et al
. 1998;
et al
. 1998).
is an artificial genus
spp. have only begun to
et al
. 1998; Taylor 2000; Ma
et al
.
2001). Some have suggested that
be mycorrhizal generalists because of inoculation trials
showing germination of their seeds by fungi isolated from
distantly related orchids (Curtis 1939; Hadley 1970; but see
Tomita & Konno 1998). However, orchid seeds form mycor-
rhizae with a greater breadth of fungi
wild (Masuhara & Katsuya 1994). By current standards, in
which specificity is defined not by the number of fungal
species that a plant can associate with but by the phylo-
genetic breadth of symbionts (Molina
et al
. 2002; McCormick
et al
. 2004), the mycorrhizal specificity
of
Cypripedium
has never been rigorously assessed.
We assessed the identity and breadth of mycorrhizal
fungi that associate with seven
North America and Europe. We directly PCR (polymerase
chain reaction)-amplified nuclear and mitochondrial genes
previously established as useful for this purpose directly
from mycorrhizal tissue. Mycorrhizal identity and spe-
cificity were assessed by sequencing these genes and
performing phylogenetic analyses. We further examined
whether these associations differed with
species and with geography.
Cypripedium
spp. must
in vitro
than in the
et al
. 1992; Taylor
Cypripedium
species from
Cypripedium
Materials and methods
Sample collection
Seven
based on their accessibility and geographical distribution.
From least to most widely distributed, according to
distribution maps provided in Cribb (1997), they are:
Cypripedium californicum
A. Gray,
Kellogg ex S. Watson,
Cypripedium candidum
ex Willd.,
Cypripedium montanum
Cypripedium guttatum
Sw.,
Cypripedium parviflorum
and
Cypripedium calceolus
L. While taxonomic controversy
surrounds the unusually high levels of genetic variation
in
C. calceolus
(Case 1994; Brzosko
tainty over the taxonomic status of
to this day (Cribb 1997), all current phylogenetic hypotheses
suggest that the seven species used in this study are at the
very least distinct from one another (Cox
1997). Although the entire genus is listed on CITES
(Convention on International Trade of Endangered Species)
Appendix 2 (World Conservation Union 1963), the distri-
butions vary, and the first four species are exceptionally
restricted geographically.
C. californicum
west range, occurring only in northern California and
southern Oregon (Cribb 1997).
the same range as
C. californicum
of Washington, British Columbia, and the central Rocky
Mountains.
C. candidum
occurs only in the northern central
United States and southernmost Ontario.
occurs only in the Pacific Northwest of the United States
Cypripedium
species were chosen for this study
Cypripedium fasciculatum
Mühl
Douglas ex Lindl.,
Salisb.,
et al
C. parviflorum
. 2002), and uncer-
continues
et al
. 1997; Cribb
has the narro-
C. fasciculatum
, but also includes parts
occurs over
C. montanum

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CYPRIPEDIUM MYCORRHIZAE
615
© 2005 Blackwell Publishing Ltd,
Molecular Ecology
, 14, 613–626
and Canada, extending from northern California to the
Alaskan Panhandle. At the other extreme,
sole European species, occurs across the widest territory,
from Great Britain to Japan, and from Spain to Scandinavia.
C. parviflorum
is the most widespread North American
species, occurring throughout Canada, the eastern and
central United States, and in pockets in the American
Rocky Mountains and Pacific Coast.
unusual distribution including a small pocket in eastern Alaska,
but extending throughout central Siberia, northeastern and
south-central China, Japan, and North Korea.
Sampling was conducted between May and October
every year from 2000 to 2003, with a total of 100 plants sam-
pled across four US states and in Estonia (Table 1). As
many plants were sampled from as many populations as
regional managers and landowners allowed, although most
populations were too small to offer more than a limited
number of sampled plants. When possible, we chose
plants representing a range of life stages, from small,
vegetative sprouts to large, multistemmed and flowering
individuals. Between two and six roots representing a
range of root ages were taken per plant. Two or three
whole plants, including adults and photosynthetic seed-
lings, were also collected from each study species. All roots
were surface-sterilized using 10% to 20% bleach solution
(Taylor & Bruns 1997). Using a compound microscope, we
sampled
Cypripedium
roots in 0.5–1.0 cm intervals looking
for the presence of pelotons, mycorrhizal hyphal coils
growing within orchid root cortical cells (Smith & Read
1997), resulting in five to 20 mycorrhizal root samples per
plant.
C. calceolus
, the
C. guttatum
has an
Molecular methodology
Characterization of
extraction of fungal and plant DNA from mycorrhizal
plant tissue, (ii) amplification of fungal genomic regions
useful in determining fungal identity, (iii) DNA sequencing,
and (iv) phylogenetic analysis for identification of mycor-
rhizal fungi and assessment of specificity.
Fungal and plant DNAs were extracted from mycor-
rhizal root samples using the QIAGEN DNeasy Plant Mini
DNA kit (QIAGEN). Some sections containing either no
morphological evidence of mycorrhizal infection, or mor-
phology suggesting parasitic or other fungal infections,
were also taken to provide controls.
To assess candidate groups of mycorrhizal fungi,
we first attempted to PCR amplify DNA extracts using
primers from the internal transcribed spacer, hereafter
ITS: ITS1F–ITS4 (White
et al
. 1990; Gardes & Bruns 1993),
ITS1–ITS4B (Gardes & Bruns 1993), and ITS1F–ITS4-Tul
(Taylor 1997). Only the ITS1F–ITS4 combination yielded
PCR product, so we used the fungal-specific primers ITS1F
and cNL2F, the latter allowing us to amplify an
portion of the highly conserved 28S nuclear large subunit
ribosomal gene, hereafter nucLSU (Taylor
currently, we amplified the mitochondrial large subunit
ribosomal gene (hereafter, mtLSU) of all samples with the
primer pairs ML5–ML6 and MLIN3–ML6 (Bruns
1998). Although both mitochondrial primer pairs ampli-
fied DNA, the primer combination MLIN3–ML6 produced
sequences strikingly similar to plant chloroplast DNA, and
was not used further. PCR involved 35 cycles with an
Cypripedium
mycorrhizae involved: (i)
c.
300 bp
et al
. 2003). Con-
et al
.
Table 1 List of surveyed Cypripedium species, regions and locales sampled, years harvested, and numbers of populations and individuals
sampled at each locale. Numbers in parentheses under the ‘# Pops sampled’ and ‘# Plants sampled’ columns indicate the number of
populations and plants, respectively, yielding PCR product with fungal nucLSU or mtLSU primers. All plants exhibited at least a small
amount of peloton-containing root tissue

Cypripedium species
Country or
US StateRegion (county/park/forest) Year sampled
# Pops
sampled
# Plants
sampled
calceolus
californicum
Estonia
California
Baltic coast
Sonoma County
Mendocino National Forest
Del Norte National Forest
Lake County
Hardin County
Klamath National Forest
Mendocino National Forest
Plumas National Forest
Six Rivers National Forest
Fairbanks
Klamath National Forest
Lake County
Hardin County
Powell County
2003
2002
2002
2002
2001
2001
2002
2002
2002
2002
2003
2000–2002
2001
2001
2001
5 (2)
6 (3)
1 (1)
3 (1)
1 (1)
2 (2)
2 (2)
2 (1)
2 (2)
2 (2)
2 (2)
9 (8)
1 (1)
4 (4)
2 (2)
44 (34)
11 (2)
9 (4)
3 (3)
7 (3)
2 (2)
5 (4)
3 (3)
3 (1)
8 (6)
8 (6)
5 (2)
23 (12)
7 (5)
4 (4)
2 (2)
100 (59)
candidum
Illinois
Kentucky
California
fasciculatum
guttatum
montanum
parviflorum
Alaska
California
Illinois
Kentucky
Total

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R. P. SHEFFERSON
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.
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Molecular Ecology
, 14, 613–626
annealing temperature of 55
cycler (MJ Research). Representative samples were chosen
for each plant via RFLP (restriction fragment length poly-
morphism) analysis of ITS-nucLSU PCR product using the
restriction enzymes
Hin
fI and
In some cases, RFLP analysis revealed the presence of
multiple fungi in a root section. These PCR products were
cloned using Stratagene XL-1 Blue Supercompetent cells
(Stratagene) and the pDrive cloning vector (QIAGEN), and
blue-white screened on LB medium infused with X-gal,
ampicillin, and kanamycin. Plasmid DNAs were purified
with the QIAprep Spin Miniprep Kit (QIAGEN), after
which the cloned PCR product was re-amplified with
primers ITS1F and cNL2F and screened with RFLP analysis
to assess the diversity of ITS-nucLSU PCR products per
plate. Clones representative of the major RFLP-types found
in each sample were chosen for sequencing, as were some
duplicates and all ITS-nucLSU and mtLSU products from
samples not requiring PCR cloning.
Sequencing began with purification of PCR samples
with the MinElute PCR Purification Kit (QIAGEN). We
cycle sequenced each PCR sample with BigDye version
3.1 chemistry (Applied Biosystems) modified with Better-
Buffer (The Gel Company). Cycle sequenced samples
were electrophoresed with an ABI 377–96 Genetic Analyser
(Applied Biosystems). The resulting sequences were edited
in
sequencher
(Gene Codes) and analysed with
(Altschul
et al
. 1997) against the NCBI sequence database
(National Center for Biotechnology Information, GenBank:
http://www.ncbi.nlm.nih.gov) to detect similar sequences
of known phylogenetic placement.
°
C, using an MJ PTC-200 Thermo-
Mbo
I (Gardes & Bruns 1996).
blast
Phylogenetic analysis
We began by grouping sequences by family association
as determined in
blast
, and conducting phylogenetic
analyses with representatives of each endophyte family.
Initial phylogenetic analysis involved adding nucLSU
(ITS4 to cNL2F) sequences into an alignment representing
the major plant-associated groups of Basidiomycetes and
Ascomycetes. Next, a similar analysis was conducted
focusing on Basidiomycetes. The poor fit of the major
fungal clade in the Basidiomycete nucLSU tree led us
to take representative samples from this clade for further
amplification and sequencing of an
nucLSU using the primer set ITS1F–TW14 (White
1990), followed by phylogenetic analysis as before. Further
analyses involved adding sequences to alignments repres-
enting narrower phylogenetic breadth. After nucLSU analysis,
mtLSU sequences were added to an alignment of mycorrhizal
fungal species (Bruns
et al
. 1998).
Sequences were aligned using
et al
. 1997). Phylogenetic analysis involved maximum like-
lihood searches in
metapiga
1.0.2b for Windows XP (Lemmon
c.
600 bp region of the
et al
.
clustalx
(Thompson
& Milinkovitch 2002) using the HKY85 (Hasegawa-Kishino-
Yano) nucleotide substitution model (Hasegawa
1985), with rate heterogeneity and invariant sites allowed,
transition : transversion rates estimated from the data, and
eight populations and eight trees per population with ran-
dom starting trees in the metapopulation search algorithm.
Branch support was estimated via 250 maximum like-
lihood replicates in
metapiga
four trees per population, yielding 1000 trees per run, and
all other parameters as before (Lemmon & Milinkovitch
2002). These support values are considered estimates of
the probabilities that the respective clades are monophy-
letic given the sequence data (Lemmon & Milinkovitch 2002).
Rarely encountered fungi with strong
not phylogenetically analysed, although they are presented
with
blast
results in this study. Sequences generated in this
study have been deposited in GenBank under accession nos.
AY578184–AY578251, AY578268–AY578284, AY585831,
AY674054-AY674056 and AY682107.
Inference of mycorrhizal interaction from direct DNA
amplification is complicated by the high likelihood of
encountering nonmycorrhizal fungi in root tissue found in
nature. For example, peloton-containing root tissue may
also include other endophytes. Absolute proof of the inter-
action is perhaps impossible in this system because experi-
mental tests of mycorrhizal colonization are precluded by
the 10–16 years required for the plant to grow to maturity
(Shefferson
et al
. 2001), by the rarity of the plants (Cribb
1997), because cultivation is notoriously difficult and often
unsuccessful (Cribb 1997), and because fungal isolations
from members of this genus generally fail (Ramsay
1998). We assumed that fungal groups were more likely to
be mycorrhizal when they included uncloned sequences,
because, if they contain pelotons, the resulting sequences are
most likely to have come from the peloton-forming fungi.
We also assumed that large clades of sample sequences
represent fungi more likely to be mycorrhizal than single
sequences spread widely across the Kingdom Fungi. Ampli-
fication of multiple fungi would result in greater difficulty
in parsing out which of the groups are mycorrhizal, after
all. Lastly, a greater potential for mycorrhizal status was
suggested with fungal groups known to form such associ-
ations with other orchids.
et al
.
with four populations and
blast
support were
et al.
Results
In contrast to previous research suggesting that evidence
of mycorrhizal infection can only be found in proto-
corms and young plants (Vinogradova & Andronova 2002),
we found partially digested, brownish pelotons in all
Cypripedium
plants in the study (Fig. 1). Roots, rather than
rhizomes, were mycorrhizal in adults, although rhizomes
were sometimes colonized in seedlings. Mycorrhizal colon-
ization was observed at sparse, irregular intervals along

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the length of the root system, with young roots usually
devoid of mycorrhizae, old roots generally decaying, and
intermediate aged roots displaying the most colonization.
No completely undigested pelotons were observed, al-
though Cypripedium guttatum pelotons appeared most
intact. Isolations were not attempted, as a result of a long
history of unsuccessful attempts by other researchers
(Rasmussen 1995). Furthermore, root sampling conducted
throughout the growing season across all years of study
failed to reveal the annual time of mycorrhizal establish-
ment. PCR amplification of mycorrhizal tissue revealed
that although ITS, nucLSU, and mtLSU could be amplified
for all six North American species, only the mtLSU could
be amplified for Cypripedium calceolus, and PCR product
could be obtained for a total of 59 out of 100 sampled plants
(Table 1). Amplification failures could have resulted from
a combination of spotty colonization, digested pelotons, and
small root samples, which could not be avoided because
of the threatened status of this genus. Lack of nucLSU
amplification in C. calceolus may have also resulted from
inappropriate primer sets. Negative controls in PCRs,
utilizing ultra-filtered water further sterilized with ultra-
violet light, surface-sterilized nonmycorrhizal root tissue
without noticeable fungal endophytes, surface-sterilized
stem tissue, and leaf tissue rinsed with ultra-filtered water,
consistently yielded no PCR product. When checked on
1.5% agarose gels, PCR tests of root tissue appearing
infected by nonmycorrhizal fungi yielded multiple bands
and smears inconsistent with mycorrhizal PCR product.
Mycorrhizal symbionts and other endophytes
Analysis of nucLSU and mtLSU sequences congruently
suggested that mycorrhizal tissue was dominated by fungi
from the family Tulasnellaceae (clades snLT1 and 2, Fig. 2;
clade mT1, Fig. 3), a result confirmed in an analysis
involving twice-longer sequences generated using primers
ITS1F–TW14 and reference sequences from a broad
phylogenetic scope of Hymenomycetes (not shown).
When all samples were accounted for, this clade accounted
for the vast majority of mycorrhizal infections in Cypri-
pedium root tissue, with 82.7% of basidiomycetous nucLSU
PCR product. The only fungal group found in all Cypripedium
species, it was also found in 39 of the 59 total plants (66.1%)
that yielded fungal nucLSU and/or mtLSU PCR product,
more than any other endophyte (Table 2).
Two tulasnelloid clades were identified. Of these, clade
snLT1, sister to all other taxa of Tulasnellaceae in our
Fig. 1 Cross-section of mycorrhizal hyphal coils (i.e. pelotons)
observed in Cypripedium guttatum root cortical cells. Arrows
indicate mycorrhizal fungal hyphae, separated from the intact
peloton (A), and still within an intact peloton (B). Scale bar
represents 100 µm.
Table 2 Fungal root endophytes in Cypripedium. Fungal groups represent closest-related taxa from nucLSU and mtLSU phylogenies of
Basidiomycota and Ascomycota, and from NCBI blast results. Each row lists the corresponding numbers of plants associated with each
fungal group. Numbers of plants yielding multiple fungi, and hence requiring cloning of PCR products, are listed in parentheses. Total
numbers of plants of each species yielding PCR product are listed in the final row, and total numbers of Cypripedium plants yielding each
fungal group are listed in the final column. Column sums may be greater than totals listed in the final row, because some plants contained
multiple fungi and are listed multiple times per column


Fungal group
calceoluscalifornicum candidumfasciculatumguttatummontanum parviflorum
Total Plants
Tulasnellaceae
Sebacinaceae
Ceratobasidiaceae
Thelephoraceae
Russula
Agaricales
Phialophora
Glomus
Total Plants
2 (0)
—
—
—
—
—
—
—
2
3 (1)
2 (2)
3 (0)
—
—
—
3 (3)
1 (1)
10
2 (1)
—
—
1 (1)
—
—
4 (4)
—
6
14 (7)
1 (1)
—
—
—
—
3 (3)
—
16
0 (2)
—
—
—
—
—
—
—
2
10 (7)
—
—
1 (1)
—
—
4 (4)
—
12
6 (4)
1 (1)
—
—
1 (1)
1 (1)
4 (4)
1 (1)
10
39
4
3
2
1
1
18
2

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Fig. 2 Fungal nuclear large subunit phylogeny of the Tulasnellaceae, showing the overwhelming majority of Cypripedium mycorrhizal
fungi which fall within this family. Of two clades, the majority of fungi belong to clade snLT1 (short for ‘short nuclear large tulasnelloid
1’), which includes fungi from all Cypripedium species sampled except Cypripedium califorinicum, Cypripedium candidum and Cypripedium
calceolus, and is sister to the remaining Tulasnellaceae. Clade snLT2, smaller than snLT1, falls firmly within the Tulasnellaceae and includes
fungi from Cypripedium califorinicum and C. candidum. Phylogeny constructed using a 295 bp alignment of the 28S nucLSU rDNA region,
and rooted with Auricularia auricula-judae. The best tree resulting from heuristic maximum likelihood analysis is presented, with support
values derived using maximum likelihood replicates (only values ≥ 50% shown); for details, see text. Sequences generated from fungal
symbionts in this study are noted by a one-letter code designating the source Cypripedium species: F—Cypripedium fasciculatum, G—
Cypripedium guttatum, L—Cypripedium californicum, M—Cypripedium montanum, N—Cypripedium candidum, and P—Cypripedium parviflorum.
This is followed by parentheses containing the plant number (given as ‘pXXX’), clone number (given as ‘cXX’, if applicable; if clone number
is missing, then no cloning was performed), and an identifier for the geographical location of the plant. Geographic codes are as follows:
DCa—Del Norte National Forest, California, USA; FAk—Fairbanks, Alaska, USA; HKy—Hardin Co., Kentucky, USA; KCa—Klamath
National Forest, California, USA; LIl—Lake County, Illinois, USA; PCa—Plumas National Forest, California, USA; PKy—Powell Co.,
Kentucky, USA; SCa—Sonoma Co., California, USA; and 6 Ca—Six Rivers National Forest, California, USA. Sources for reference sequences
used in this figure: (a)—Bidartondo et al. (2003), (b)—Taylor et al. (2003), (c)—Kottke et al. (2003), (d)—Langer, unpublished, (e)—Hibbett et
al. (2000), and (f)—Weiß & Oberwinkler (2001).

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CYPRIPEDIUM MYCORRHIZAE 619
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Fig. 3 Fungal mitochondrial large subunit phylogeny supporting the hypothesis that the main fungi mycorrhizal with Cypripedium are
primarily within the family Tulasnellaceae. Clade mTR1 (short for ‘mtLSU tulasnelloid reference 1’) and Gloeotulasnella cystidiophora include
all tulasnelloid mtLSU sequences on GenBank as of the writing of this manuscript. This clade is basal to clade mT1, which includes all
mtLSU samples in this study. Phylogeny constructed using a 282 bp alignment of the mtLSU, rooted with Auricularia auricula-judae. The
best tree resulting from heuristic maximum likelihood analysis is presented, with support values derived using maximum likelihood
replicates (only values ≥ 50% shown); for details, see text. Sequences generated from fungal symbionts in this study are noted by a one-letter
code designating the source Cypripedium species: C—Cypripedium calceolus, F—Cypripedium fasciculatum, G—Cypripedium guttatum, L—
Cypripedium californicum, M—Cypripedium montanum, N—Cypripedium candidum, and P—Cypripedium parviflorum. This is followed by
parentheses containing the plant number (given as ‘pXXX’), clone number (given as ‘cXX’, if applicable; if clone number is missing, then
no cloning was performed), and an identifier for the geographical location of the plant. Geographic codes are as follows: Est—Estonia; FAk—
Fairbanks, Alaska, USA; HKy—Hardin Co., Kentucky, USA; KCa—Klamath National Forest, California, USA; LIl—Lake County, Illinois,
USA; SCa—Sonoma Co., California, USA; and 6 Ca—Six Rivers National Forest, California, USA. Sources for reference sequences used in
this figure are as follows: (a)—Kristiansen et al. (2004), (b)—Kristiansen et al. (2001), (c)—Hibbett & Binder (2002), (d)—Bruns et al. (1998),
(e)—Binder & Hibbett (2002), and (f)—Lilleskov, Fahey, Horton, and Lovett, unpublished.

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620 R. P. SHEFFERSON ET AL.
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analysis, included 63.5% of all Basidiomycete nucLSU
sequences (Fig. 2). Clade snLT2 was smaller but was less
phylogenetically distinct from reference Tulasnellaceae
taxa. This clade included the only tulasnelloid sequences
from Cypripedium candidum and Cypripedium californicum,
as well as some from Cypripedium montanum and Cypripedium
parviflorum (Fig. 2). Although the nuclear ribosomal loci
in this family evolve at a faster rate than those of other
Basidiomycetes (Taylor 1997), distances among tulasnelloid
mycorrhizal samples within these two clades were small,
suggesting high specificity to these clades rather than to
other tulasnelloid groups.
Apart from these basidiomycetous fungi, sequences of
ascomycetous endophytes corresponding to the poly-
phyletic genus, Phialophora, were commonly recovered.
These were found in representatives of all Cypripedium
species studied except C. guttatum and C. calceolus, and
were associated with 18 of 59 plants (30.5%; Fig. 4; Table 2).
All of these fungi were most closely related to parasitic and/
or saprotrophic groups of Phialophora (Fig. 4), especially
Phialophora gregata, the causal disease agent of soybean brown
stem rot (Malvick et al. 2003), the root- and stem-rotting
fungus Phialophora malorum (Sugar & Spotts 1992; Blok et al.
1995), and the obscure rot-fungus Phialophora melinii (Shigo
1974). A small group was associated with Chalara sp. and
Xenochalara juniperi (Fig. 4), both relatively obscure species
(Coetsee et al. 2000). In no cases did these Ascomycetes
appear closely related to the ectendomycorrhizal species
Phialophora finlandia (Harney et al. 1997; Vrålstad et al.
2002). Because of this evidence, because all Phialophora
sequences were clones from a small subset of Cypripedium
plants associating with tulasnelloid mycorrhizal fungi, and
because this association was minor in the well-sampled
species Cypripedium fasciculatum and Cypripedium montanum
(Table 2), we suggest that these fungi are most likely either
parasites of the orchids or of their basidiomycetous mycor-
rhizal fungi, or rotters of dead or dying root tissue.
Other fungi were also occasionally found (Table 3).
Fungi from family Ceratobasidiaceae associated with C.
californicum plants from the Del Norte National Forest, but
no other plants (Table 3). Although ceratobasidioid associ-
ations were observed in Goodyera oblongifolia plants sym-
patric with one C. fasciculatum population at the Plumas
National Forest, California (Table 3), the latter associated
only with tulasnelloid fungi. C. californicum was the only
well-sampled species to exhibit mycorrhizal patterns counter
to the general patterns we observed, with most uncloned
nucLSU sequences belonging to the Ceratobasidiaceae,
and approximately equal numbers of cloned sequences
belonging to the Tulasnellaceae and Sebacinaceae (Tables 2
and 3). Other endophytes, belonging to Thelephorales,
Russulales, Agaricales, and Glomeromycota, were found
in one or two plants each, and only in samples co-occurring
with other fungi (i.e. requiring PCR cloning; Table 2).
Discussion
Identity of the mycorrhizal fungi
The primary mycorrhizal symbionts of Cypripedium are
within the family, Tulasnellaceae. This obscure family
includes many known orchid mycorrhizal fungi (Warcup
Table 3 Nuclear large subunit blast search results for fungi occurring rarely in Cypripedium mycorrhizal tissue. Only potentially
mycorrhizal fungi are listed. The nearest taxon listed is as described in the GenBank accession. Overlap length corresponds to the number
of base pairs in the sample sequence that fit a pairwise alignment with the nearest taxon accession in GenBank. The last column gives the
number of matching base pairs divided by the length of the overlap with the nearest taxon


Orchid host species
Sample
accession
Sequence
length Nearest taxon
Accession of
nearest taxon
Overlap
length% match
Cypripedium
californicum
C. californicum
C. californicum
C. californicum
C. californicum
C. californicum
C. californicum
C. candidum
C. fasciculatum
C. montanum
C. parviflorum
C. parviflorum
C. parviflorum
C. parviflorum
Goodyera oblongifolia
AY578184249 Uncultured Glomus sp. AY138144244 98%
AY578185
AY578228
AY578229
AY578233
AY578234
AY674054
AY578230
AY578232
AY578231
AY578186
AY578235
AY674055
AY674056
AY578227
249
252
251
252
252
251
254
252
254
250
251
256
257
254
Glomus clarum
Ceratobasidium sp.
Ceratobasidium sp.
Sebacina vermifera
Sebacina vermifera
Ceratobasidium sp.
Thelephoraceous ectomycorrhiza
Sebacinaceous endomycorrhiza
Thelephoraceous ectomycorrhiza
Glomus mosseae
Sebacinaceous mycorrhiza
Russula sardonia
Pluteus ephebeus
Thanatephorus cucumeris
AJ510242
AY293171
AY293171
AF291366
AF291366
AY293171
AF430277
AF440652
AF430277
AF396788
AF298948
AF325318
AF261574
AF354119
245
253
252
248
247
252
250
252
255
256
252
256
257
254
94%
99%
98%
97%
98%
99%
98%
99%
98%
86%
91%
99%
92%
99%

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CYPRIPEDIUM MYCORRHIZAE 621
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& Talbot 1967; Warcup & Talbot 1971; Rasmussen 1995),
and even some liverwort symbionts (Bidartondo et al. 2003;
Kottke et al. 2003). This group has been noted as a primary
mycorrhizal symbiont of Neuwiedia veratrifolia (Kristiansen
et al. 2004), a member of the Apostasioideae, an orchid
subfamily immediately basal to the Cypripedioideae and
sister to the remaining Orchidaceae, suggesting that family
Tulasnellaceae may be the ancestral family of mycorrhizal
fungi in the Orchidaceae. However, Cypripedium-mycorrhizal
tulasnelloids are not similar enough to sequences from
other Tulasnellaceae in GenBank to infer a genus-level
classification at this time. Rhizoctonia sclerotica, previously
isolated from Cypripedium reginae, and Rhizoctonia subtilis,
previously isolated from both Cypripedium candidum and
Fig. 4 Fungal internal transcribed spacer phylogeny suggesting that ascomycetous Cypripedium root endophytes are likely saprotrophic or
parasitic members of the polyphyletic genus Phialophora. Clade IP1 (short for ‘ITS phialophoroid 1’) includes Phialophora gregata, Phialophora
melinii, and Phialophora malorum, all rot fungi. Clades IP2 and IP3 include Xenochalara juniperi and Chalara sp., which are not well-described
fungi. Clade IPR1 (short for ‘ITS phialophoroid reference 1’) includes the only mycorrhizal species, Phialophora finlandia, which appears
relatively unrelated to any Cypripedium root endophyte. Tree constructed using a 454 bp alignment of the internal transcribed spacer (ITS),
and rooted with Penicillium dendriticum (LaBuglio et al. 1993). The best tree resulting from heuristic maximum likelihood analysis is
presented, with support values derived using maximum likelihood replicates (only values ≥ 50% shown); for details, see text. Sequences
generated from fungal symbionts in this study are noted by a one-letter code designating the source Cypripedium species: F—Cypripedium
fasciculatum, L—Cypripedium californicum, M—Cypripedium montanum, N—Cypripedium candidum, and P—Cypripedium parviflorum. This is
followed by parentheses containing the plant number (given as ‘pXXX’), clone number (given as ‘cXX’, if applicable; if clone number is
missing, then no cloning was performed), and an identifier for the geographical location of the plant. Geographic codes are as follows:
HKy—Hardin Co., Kentucky, USA; KCa—Klamath National Forest, California, USA; LIl—Lake County, Illinois, USA; MCa—Mendocino
National Forest, California, USA; PCa—Plumas National Forest, California, USA; and SCa—Sonoma Co., California, USA. Sources
for reference sequences used in this figure are as follows: (a)—McKenry, Rogers, and Wang, unpublished (b)—Harrington et al. (2000),
(c)—Kennedy et al. (2003), (d)—Saenz & Taylor (1999), (e)—Tedersoo et al. (2003), (f)—Golonka, unpublished, (g)—Coetsee et al. (2000),
(h)—Mostert et al. (2003), (i)—Abliz et al. (2003), (j)—Yan et al. (1995), and (k)—LaBuglio et al. (1993).

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622 R. P. SHEFFERSON ET AL.
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Cypripedium parviflorum (Curtis 1939), may be members of
this group, although no molecular data on these cultures
exist (Andersen 1996; Shan et al. 2002).
Some other orchid-associating fungi were also observed.
Fungi in family Sebacinaceae have been isolated from a
broad range of orchid species, including Caladenia spp.,
Elythranthera spp., Eriochilus spp., Glossodia major, Microtis
unifolia, and Platanthera orbiculata (Warcup & Talbot 1967;
Warcup 1971; Warcup 1981; Currah et al. 1990). Sebaci-
noids are the mycorrhizal symbionts of the achlorophyl-
lous orchids Neottia nidus-avis and Hexalectris spicata
(McKendrick et al. 2002; Selosse et al. 2002b; Taylor et al.
2003), which utilize this fungal group for carbon nutrition.
They are also the rhizoctonias isolated from the Australian
green orchids Cyrtostylis reniformis and Microtis uniflora,
having been assigned to the broad species complex of
Sebacina vermifera by induction of their perfect stages
(Warcup 1981; Warcup 1988; Weiß et al. 2004). Fungi in
family Ceratobasidiaceae, which associate with Cypripedium
californicum in Del Norte National Forest of northern
California, have been previously isolated from the Australian
orchid Acianthus reniformis (Warcup & Talbot 1967). Related
fungi have been found in a wide variety of orchid species
around the world (Warcup 1981; Otero et al. 2002). Mem-
bers of the Russulaceae family have been observed in
Corallorhiza maculata and Corallorhiza mertensiana, and the
Thelephoraceae have been observed in Cephalanthera
austinae (Taylor & Bruns 1997; Taylor & Bruns 1999). Lastly,
Phialophora endophytes may be related to Phialocephala
victorinii, previously observed as a root endophyte in some
populations of C. parviflorum (Vujanovic et al. 2000), although
the phylogenetic relationships among Phialophora and
Phialocephala species and their ecological functions are not
clear (Jacobs et al. 2003).
Tulasnelloid fungi may serve to transfer carbon to the
mature Cypripedium plant. Ectomycorrhizal members
of the Tulasnellaceae have been found to transfer carbon
from birch seedlings to the nonphotosynthetic liverwort
Cryptothallus mirabilis (Bidartondo et al. 2003). Even some
sebacinoids are ectomycorrhizal (Warcup 1988; Glen et al.
2002; Selosse et al. 2002a; Urban et al. 2003; Weiß et al. 2004),
suggesting that even C. californicum may always associate
with fungi capable of contributing carbon. 13C and 15N
isotope ratios assessed in green orchids in comparison to
their achlorophyllous counterparts suggest that some green
orchids obtain some of their carbon from ectomycorrhizal
sources (Gebauer & Meyer 2003; Bidartondo et al. 2004).
Specificity and distribution
Cypripedium mycorrhizal specificity appears generally
high. We suggest, however, that habitat characteristics
may be as important limiters of Cypripedium distribution as
mycorrhizal fungi. C. californicum, for example, is not only
the least mycorrhizal-specific orchid in this study, but the
only one limited to serpentine sites with standing or running
water (Coleman 1995; Cribb 1997). Associations with multi-
ple families of fungi suggest that C. californicum species may
switch among mycorrhizal fungal families relatively easily.
Cypripedium montanum and Cypripedium fasciculatum,
well-sampled species that associated only with the Tulas-
nellaceae, may indeed be limited by the distribution of
their fungal partners. C. candidum associated with only a
subset of the fungi associating with sympatric C. parviflo-
rum, and was rarer in these sites (the former associating
with clade snLT2, and the latter with both clades snLT1
and 2; Fig. 2). Even common and widespread temperate,
terrestrial orchids, such as Goodyera repens, are thought to
be fungus-limited (Rasmussen & Whigham 1994). Many
Tulasnella species are considered rare enough to warrant
inclusion on European Red Lists (Arnolds & de Vries
1993), lending support to the suggestion that mycorrhizae
are likely to be important factors affecting Cypripedium
distribution, although this fungal genus has been largely
ignored and may be more common than thought. However,
unlike other Cypripedium species, C. calceolus, C. guttatum
and C. montanum were only sampled in one region each.
Furthermore, the former two species are represented
by symbionts from only two plants each (Table 1). Thus,
the high specificity that we report for these taxa may be
artefactual.
C. californicum’s lack of specificity appears to be anoma-
lous. Given that C. californicum is generally limited to wet,
serpentine sites, the toxicity of serpentine sites and the abil-
ity of some mycorrhizal fungi to reduce plant uptake of
toxic elements (Joner et al. 2000; Meharg & Cairney 2000)
may result in different mycorrhizal needs for the plant,
perhaps even resulting in a need for a greater breadth of
fungi. Such interactions between stratum and specificity
have been explored in epiphytic orchids by Otero et al.
(2004), but have not been well-explored in terrestrial orchids.
Patterns of association and specificity closely resemble
previously reported results for other orchids, in that spe-
cificity is often, although not always, high. In a study of
three varieties of the nonphotosynthetic orchid Hexalectris
spicata, the primary association was with the Sebacinaceae,
although a ceratobasidioid fungus was also found associ-
ated with one variety (Taylor et al. 2003). Dactylorhiza majalis
associates primarily with the Tulasnellaceae, but also
forms occasional associations with members of the genus
Laccaria, a rather unrelated group (Kristiansen et al. 2001).
McCormick et al. (2004) found a range in specificity among
unrelated photosynthetic orchids, each with a different
ecology, and inferred a lack of correspondence to any eco-
logical characteristics. Otero et al. (2002) and Otero et al.
(2004), in examining tropical, epiphytic orchids, found
some, like Tolumnia variegata, to be generalists, while
others, including Ionopsis utricularioides, a close relative of

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CYPRIPEDIUM MYCORRHIZAE 623
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Tolumnia variegata, exhibited evidence of high specificity.
Our results also concur with those of other green orchids,
including Caladenia, Dendrobium, and Pterostylis (Warcup
1981), and Liparis (McCormick et al. 2000).
Conclusion
Mycorrhizal dynamics in photosynthetic orchids have
long been controversial. In vitro and in situ approaches
to mycorrhizal specificity often lead to conflicting results
(Masuhara & Katsuya 1994), and isolations are inherently
biased by choice of and response to growth medium (Allen
et al. 2003; McCormick et al. 2004). The growing consensus
on mycorrhizal specificity in mature, green orchids seems
to be that they may run the gamut of specialization. Some
widespread green orchids are less specific and associate
with a wider array of fungal species than nonphotosynthetic
orchids, but others are more specific (McCormick et al.
2000). The genus, Cypripedium appears to be predominantly
specialist, although Cypripedium californicum appears to
associate with three fungal families. However, our infer-
ence was limited by the difficult life histories of these plants,
which precludes laboratory experimentation with mature
individuals.
Because mycorrhizal specificity does not correspond
with geographical distribution (i.e. no gradient in speci-
ficity correlates with geographical extent across species), we
must ask what this gradient in specificity means ecolo-
gically. The possible influence of habitat on the distribution
of C. californicum suggests that nutrient conditions and per-
haps the presence of toxic elements may play a large role.
Furthermore, some green orchids exhibit nitrogen and
carbon stable-isotope ratios suggesting that fungal carbon
may provide them with energy throughout their lives
(Bidartondo et al. 2004), which proposes that carbon
requirements, or other nutrient requirements related to
mycorrhizal fungi, may govern mycorrhizal associations
in Cypripedium. In particular, although Cypripedium plants
are photosynthetic and do not appear to need external car-
bon sources, do times of adult dormancy translate to times
of mycoheterotrophy (Lesica & Steele 1994; Rasmussen
1995; Shefferson et al. 2001; Kull 2002; Shefferson et al.
2003)? Further research should elaborate on these possible
connections between mycorrhizal association, habitat con-
ditions, life history stages, and demographic trends.
Acknowledgements
The authors thank M. Bidartondo, T. Bruns, I. Herriott, A. Izzo, J.
Nagata, and E. Simms for their valuable support while conducting
this project. Special thanks to T. Bruns and the Mycorrhizal Dis-
cussion Group at the University of California, Berkeley, to Brian
Charlesworth and three anonymous reviewers for their extra
efforts in providing comments and helpful critiques on earlier
drafts of this manuscript, as well as to Dr Harry Smith for his
helpful advice. Access to sites was provided by the Estonian Agri-
cultural University, Illinois State Nature Preserves Commission,
Kentucky State Nature Preserves Commission, the Kentucky
State Parks, the Lake County Forest Preserve District, the Nature
Conservancy, Tartu University, the United States Forest Service
Region 5, and the University of Alaska at Fairbanks. Logistical
support during this study was provided by the Department of
Integrative Biology and the University of California Botanical
Garden at the University of California, Berkeley, as well as the
Forestry and Forest Products Research Institute in Tsukuba,
Japan. This work was funded by the Budweiser Conservation
Scholarship from the Anheuser-Busch Corp. and the National Fish
and Wildlife Foundation, a cost-sharing agreement with the
USDA Forest Service, the Furniss Foundation/American Orchid
Society Graduate Fellowship, Grants-In-Aid from the Sigma Xi
Scientific Society, Estonian SF grant #4833, and fellowships
from the University of California Botanical Garden and the
Department of Integrative Biology at the University of California,
Berkeley.
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This research was conducted as part of the Ph.D. dissertation of
Richard Shefferson, and is part of a collaborative effort to deter-
mine the causal agents of and factors affecting adult dormancy in
geophytes. Michael Weiß focuses on the systematics and phylo-
genetics of basal hymenomycetes. Tiiu Kull studies the population
ecology of rare plants, particularly terrestrial orchids. D. Lee Taylor
studies microbial-plant symbioses, both mycorrhizal and rhizobial.