Microbial
Ecology
Soil Fungal Communities Underneath Willow Canopies on a
Primary Successional Glacier Forefront: rDNA Sequence Results
Can Be Affected by Primer Selection and Chimeric Data
Ari Jumpponen
Division of Biology, Kansas State University, 125 Ackert Hall, Manhattan, KS 66506, USA
Received: 7 January 2004 / Accepted: 9 March 2004 / Online publication: 3 November 2006
Abstract
Soil fungal communities underneath willow canopies
that had established on the forefront of a receding glacier
were analyzed by cloning the polymerase chain reaction
(PCR)-amplified partial small subunit (18S) of the
ribosomal (rRNA) genes. Congruence between two sets
of fungus-specific primers targeting the same gene region
was analyzed by comparisons of inferred neighbor-joining
topologies. The importance of chimeric sequences was
evaluated by Chimera Check (Ribosomal Database Project) and by data reanalyses after omission of potentially
chimeric regions at the 50- and 30-ends of the cloned
amplicons. Diverse communities of fungi representing
Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota were detected. Ectomycorrhizal fungi comprised a
major component in the early plant communities in
primary successional ecosystems, as both primer sets
frequently detected basidiomycetes (Russulaceae and
Thelephoraceae) forming mycorrhizal symbioses. Various
ascomycetes (Ophiostomatales, Pezizales, and Sordariales) of uncertain function dominated the clone libraries
amplified from the willow canopy soil with one set of
primers, whereas the clone libraries of the amplicons
generated with the second primer set were dominated by
basidiomycetes. Accordingly, primer bias is an important
factor in fungal community analyses using DNA extracted
from environmental samples. A large proportion (930%)
of the cloned sequences were concluded to be chimeric
based on their changing positions in inferred phylogenies
after omission of possibly chimeric data. Many chimeric
sequences were positioned basal to existing classes of
fungi, suggesting that PCR artifacts may cause frequent
discovery of new, higher level taxa (order, class) in direct
PCR analyses. Longer extension times during the PCR
Correspondence to: Ari Jumpponen; E-mail: ari@ksu.edu
DOI: 10.1007/s00248-004-0006-x
& Volume 53, 233–246 (2007) & *
amplification and a smaller number of PCR cycles are
necessary precautions to allow collection of reliable
environmental sequence data.
Introduction
Fungi perform important ecosystem functions by participating in the decomposition of dead tissues as well as
plant uptake of water and nutrients [6, 34]. Assessment
of fungal community composition is difficult because of
unreliable and ephemeral production of identifiable
macroscopic fruiting bodies [11, 27, 35]. Many fungi
also produce microscopic, sexual or asexual fruiting
structures or fruit below ground escaping detection in
assessments relying exclusively on the collection of
epigeous fruiting bodies. Pure culture techniques allow
fungal community assays of soil and tissue samples in the
absence of identifiable macroscopic fruiting bodies.
However, similar to bacteria [38], it is likely that large
numbers of fungi would be missed in such pure culture
assays (see [31, 41]). To overcome these problems in
fungal community analysis, molecular means specifically
targeting fungi in environmental samples have been
developed [3, 9, 14, 25, 28, 32, 33, 40].
Direct molecular assessment of the fungal communities allows analyses without relying on whether or not
the fungi can be grown in pure culture or produce
fruiting bodies. However, polymerase chain reaction
(PCR) artifacts, such as chimeric sequences resulting
from amplification of more than one template, can cause
problems in environmental samples with unknown
sources of diverse initial template DNA [13, 19, 24, 42,
43]. Various coextracted substances and low concentrations of the target template in the presence of highly
similar competing target and nontarget templates may
further influence the fidelity of PCR reactions [42].
Springer Science + Business Media, Inc. 2006
233
Chimera at
RDP
Yes (G20)
Yes (G40)
Yes (G80)
Yes (G40)
Yes (G20)
Yes (G20)
Yes (G20)
Yes (G20)
Yes (G20)
Yes (G40)
No
Yes (G100)
Yes (G40)
No
Yes (G80)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G40)
No
Yes (G20)
Yes (G40)
Yes (G40)
Yes (G80)
Yes (G40)
Yes (G40)
Yes (G40)
Yes (G80)
Yes (G20)
Yes (G20)
No
Environmental clone
B_Canopy_300_01_08 [AY382401]
B_Canopy_300_01_14 [AY382402]
B_Canopy_300_01_16 [AY382403]
B_Canopy_300_01_18b [AY382404]
B_Canopy_300_02_04b [AY382405]
B_Canopy_300_02_05 [AY382406]
B_Canopy_300_02_06 [AY382407]
B_Canopy_300_02_10 [AY382408]
B_Canopy_300_02_12 [AY382419]
B_Canopy_300_02_14b [AY382410]
B_Canopy_300_03_06 [AY382411]
B_Canopy_300_03_12b [AY382412]
B_Canopy_300_03_17 [AY382413]
B_Canopy_300_03_19b [AY382414]
B_Canopy_450_01_02 [AY382415]
B_Canopy_450_01_06 [AY382416]
B_Canopy_450_01_13 [AY382417]
B_Canopy_450_01_14 [AY382418]
B_Canopy_450_01_18 [AY382419]
B_Canopy_450_02_02 [AY382420]
B_Canopy_450_02_13 [AY382421]
B_Canopy_450_03_02 [AY382422]
B_Canopy_450_03_05 [AY382423]
B_Canopy_450_03_07 [AY382424]
B_Canopy_450_03_14 [AY382425]
B_Canopy_750_01_01b [AY382426]
B_Canopy_750_01_07b [AY382427]
B_Canopy_750_01_10b [AY382428]
B_Canopy_750_01_15b [AY382429]
B_Canopy_750_02_13 [AY382430]
B_Canopy_750_02_15b [AY382431]
B_Canopy_750_02_19b [AY382432]
B_Canopy_750_03_03b [AY382433]
B_Canopy_750_03_04 [AY382434]
B_Canopy_750_03_08b [AY382435]
B_Canopy_750_03_11 [AY382436]
Spilocaea oleaginea [AF338393] (Chaethothyriales/Dothidiales)
Spilocaea oleaginea [AF338393] (Chaethothyriales/Dothidiales)
Hymenoscyphus ericea [AY228753] (Helotiales)
Inocybe geophylla [AF287835] (Agaricales)
Dark septate endophyte DS16b [AF168167] (Unknown)
Peziza griseorosea [AF133150] (Pezizales)
Peziza griseorosea [AF133150] (Pezizales)
Tetracladium marchalianum [AY204613] (Incertae sedis)
Spilocaea oleaginea [AF338393] (Chaethothyriales/Dothidiales)
Oidiodendron tenuissimum [AB015787] (Onygenales)
Prismatolaimus intermedius [AF036603] (Enoplida; Prismatolaimidae)
Cladonia sulphurina [AF241544] (Lecanorales)
Hypoxylon submonticulosum [AF346544] (Xylariales)
Neobulgaria premnophila [U45445] (Helotiales)
Pulvinula archeri [U62012] (Pezizales)
Hypomyces chrysospermus [AB027339] (Hypocreales)
Oidiodendron tenuissimum [AB015787] (Onygenales)
Oidiodendron tenuissimum [AB015787] (Onygenales)
Oidiodendron tenuissimum [AB015787] (Onygenales)
Rhizoctonia solani [D85643] (Ceratobasidiales)
Hypomyces chrysospermus [AB027339] (Hypocreales)
Connersia rilstonii [AF096174] (Eurotiales)
Raciborskiomyces longisetosum [AY016351] (Chaetothyriales)
Herpotrichia juniperi [U42483] (Pleosporales)
Mycosphaerella mycopappi [U43463] (Chaetothyriales)
Inocybe geophylla [AF287835] (Cortinariaceae)
Peziza griseorosea [AF133150] (Pezizales)
Anamylopsora pulcherrima [AF119501] (Agyriales)
Pulvinula archeri [U62012] (Pezizales)
Hypomyces chrysospermus [M89993] (Hypocreales)
Ophiostoma piliferum [AJ243294] (Ophiostomatales)
Hypomyces chrysospermus [M89993] (Hypocreales)
Sarcinomyces petricola [Y18702] (Chaetothyriales)
Laccaria pumila [AF287838] (Agaricales)
Laccaria pumila [AF287838] (Agaricales)
Peziza griseorosea [AF133150] (Pezizales)
BLAST match [accession number] (Order)
Ascomycota
Ascomycota
Ascomycota
Basidiomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Contaminant
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Basidiomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Basidiomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Ascomycota
Basidiomycota
Basidiomycota
Ascomycota
Phylum
98
96
95a
97
98
99
98
99a
98
97
97
93
96
98
94
96
98
97
98
95
96
99/99a,c
99
97
98
97
99
97
97
96
97/97c
95
95
98
96
99
Similarity
0.60
0.20
0.10
0.10
0.22
0.11
0.11
0.11
0.33
0.11
0.08
0.15
0.31
0.46
0.27
0.20
0.07
0.33
0.13
0.06
0.94
0.14
0.14
0.43
0.29
0.20
0.40
0.20
0.20
0.33
0.44
0.22
0.14
0.29
0.14
0.43
Frequency
Table 1. BLAST and RDP analyses of the environmental sequences obtained from underneath the willow canopies established on the forefront of a receding glacier
234
A. JUMPPONEN: FUNGI
IN THE
WILLOW CANOPY SOIL
ON A
GLACIER FOREFRONT
Yes
Yes
No
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
(G100)
(G40)
(G80)
(G160)
(G100)
(G40)
(G20)
(G100)
(G80)
(G60)
(G60)
(G20)
(G20)
(G20)
(G40)
(G160)
(G100)
(G20)
(G40)
(G40)
(G20)
(G20)
(G40)
(G40)
(G80)
(G40)
(G80)
(G40)
(G60)
(G80)
(G40)
(G40)
Russula compacta [AF026582] (Agaricales)
Hypomyces chrysospermus [AB027339] (Hypocreales)
Russula compacta [AF026582] (Agaricales)
Oidiodendron tenuissimum [AB015787] (Onygenales)
Chaetomium elatum [M83257] (Sordariales)
Thelephora sp. [AF026627] (Thelephorales)
Dark septate endophyte DS16b [AF168167] (Unknown
Pulvinula archeri [U62012] (Pezizales)
Hypomyces chrysospermus [AB027339] (Hypocreales)
Polyporoletus sublividus [AF287840] (Cantharellales)
Thelephora sp. [AF026627] (Thelephorales)
Bulgaria inquinans [AJ224362] (Helotiales)
Inocybe geophylla [AF287835] (Agaricales)
Bulgaria inquinans [AJ224362] (Helotiales)
Mortierella chlamydospora [AF157143] (Mucorales)
Bulgaria inquinans [AJ224362] (Helotiales)
Limnoperdon incarnatum [AF426952] (Aphyllophorales)
Panellus serotinus [AF026590] (Agaricales)
Spizellomyces acuminatus [M59759] (Spizellomycetales)
Laccaria pumila [AF287838] (Agaricales)
Byssoascus striatosporus [AB015776] (Onygenales)
Entoloma strictius [AF287832] (Agaricales)
Bulgaria inquinans [AJ224362] (Helotiales)
Ophiostoma stenoceras [M85054] (Ophiostomatales)
Thelephora sp. [AF026627] (Thelephorales)
Inocybe geophylla [AF287835] (Agaricales)
Inocybe geophylla [AF287835] (Agaricales)
Sporothrix schenkii [M85053] (Ophiostomatales)
Dendrocorticium roseocarneum [AF334910] (Aphyllophorales)
Bulgaria inquinans [AJ224362] (Helotiales)
Termitomyces sp. [AB051891] (Agaricales)
Inocybe geophylla [AF287835] (Agaricales)
Laccaria pumila [AF287838] (Agaricales)
Cyathrus striatus [AF026617] (Nidulariales)
Inocybe geophylla [AF287835] (Agaricales)
Russula compacta [U59093] (Agaricales)
Thelephora sp. [AF026627] (Thelephorales)
Basidiomycota
Ascomycota
Basidiomycota
Ascomycota
Ascomycota
Basidiomycota
Ascomycota
Ascomycota
Ascomycota
Basidiomycota
Basidiomycota
Ascomycota
Basidiomycota
Ascomycota
Zygomycota
Ascomycota
Basidiomycota
Basidiomycota
Chytridiomycota
Basidiomycota
Ascomycota
Basidiomycota
Ascomycota
Ascomycota
Basidiomycota
Basidiomycota
Basidiomycota
Ascomycota
Basidiomycota
Ascomycota
Basidiomycota
Basidiomycota
Basidiomycota
Basidiomycota
Basidiomycota
Basidiomycota
Basidiomycota
96
97
98
99
98
99
98
97
94
94
98
98
98
95
97
96
94
94
97
97
94
93
98
95
97/94c
96
98
93
92
95
94
97
95
97
98
97
97
0.25
0.13
0.63
0.09
0.18
0.55
0.09
0.09
0.75
0.13
0.13
0.11
0.89
0.14
0.29
0.14
0.14
0.29
0.67
0.33
0.25
0.25
0.50
1.00
0.40
0.40
0.20
0.92
0.08
0.14
0.43
0.14
0.14
0.14
0.77
0.23
1.00
IN THE
WILLOW CANOPY SOIL
ON A
Chimera Check scores in parentheses. Frequency refers to the occurrence of a clone in the library obtained from one sample.
RDP = Ribosomal Database Project; BLAST = basic local alignment search tool.
a
Sequence omitted from the neighbor-joining analyses because of a large insert.
b
Sequence determined chimeric in analyses after omission of data beyond chimera points.
c
BLAST matches were partial and did not span over the entire cloned sequence.
B_Canopy_900_01_03b [AY382437]
B_Canopy_900_01_16 [AY382438]
B_Canopy_900_01_19 [AY382439]
B_Canopy_900_02_02 [AY382440]
B_Canopy_900_02_04 [AY382441]
B_Canopy_900_02_06 [AY382442]
B_Canopy_900_02_10 [AY382443]
B_Canopy_900_02_12b [AY382444]
B_Canopy_900_03_09 [AY382445]
B_Canopy_900_03_11b [AY382446]
B_Canopy_900_03_17 [AY382447]
S_Canopy_300_01_01 [AY382448]
S_Canopy_300_01_07 [AY382449]
S_Canopy_300_02_01b [AY382450]
S_Canopy_300_02_11 [AY382451]
S_Canopy_300_02_13b [AY382452]
S_Canopy_300_02_14b [AY382453]
S_Canopy_300_02_19b [AY382454]
S_Canopy_300_03_04 [AY382455]
S_Canopy_300_03_18 [AY382456]
S_Canopy_450_01_02b [AY382457]
S_Canopy_450_01_07 [AY382458]
S_Canopy_450_01_19 [AY382459]
S_Canopy_450_02_05 [AY382460]
S_Canopy_750_01_10 [AY382461]
S_Canopy_750_01_11 [AY382462]
S_Canopy_750_01_18 [AY382463]
S_Canopy_750_02_09 [AY382464]
S_Canopy_750_02_17b [AY382465]
S_Canopy_750_03_01b [AY382466]
S_Canopy_750_03_13 [AY382467]
S_Canopy_750_03_15 [AY382468]
S_Canopy_750_03_18b [AY382469]
S_Canopy_750_03_19 [AY382470]
S_Canopy_900_01_06 [AY382471]
S_Canopy_900_01_11 [AY382472]
S_Canopy_900_03_02 [AY382473]
A. JUMPPONEN: FUNGI
GLACIER FOREFRONT
235
236
A. JUMPPONEN: FUNGI
Furthermore, primer sets designed to obtain broad
specificity to a target group (e.g., fungi) may have biases
and preferentially amplify one target group but not
another [2, 36].
The overall goal of the presented studies was to
characterize fungal community composition within
established willow (Salix spp.) canopies on the forefront
of a receding glacier. The nuclear small subunit (18S) of
the ribosomal RNA gene (rDNA) was amplified with two
different sets of fungus-specific primers to estimate the
influence of primer selection on the observed community
structure. To evaluate the influence of chimeric amplicons on the obtained 18S phylogenies, data sets were
reanalyzed after omission of the chimeric regions
identified using Chimera Check software of the Ribosomal Database Project (RDP, version 2.7 [26]). The
results indicate that diverse fungal communities exist
within the willow canopies, that primer selection strongly
influences the observed fungal community structure, and
that chimeras are a serious concern in direct PCR
applications targeting fungi in environmental samples.
Methods
Lyman Glacier (48-1005200N, 120-5308700W)
is located in the Glacier Peak Wilderness Area in the
North Cascade Mountains (Washington, USA). The site
has been utilized in several studies on early plant
community assembly in recently deglaciated substrate
(e.g., [20, 23]). Similarly, it has been a focus of studies
aiming to examine fungal community assembly in such
an environment [19, 21, 22]. The elevation of the present
glacier terminus is about 1800 m. The deglaciated forefront is approximately 1000 m long over an elevation drop
of only 60 m with no distinctive recessional moraines
[4, 20]. The glacier has receded since the 1890s, opening
the forefront to colonization by plants and fungi.
Periodic photographs and snow survey data have allowed
the reconstruction of the glacier retreat over the last
century [20].
Study Site.
Sampling and DNA Extraction.
Shrub willows
(Salix commutata and S. planifolia) comprise the early
perennial plant communities and are the largest plant
individuals during early vegetation development [22].
Twelve shrub canopies–three of approximately equal size
at distances of 300, 450, 750, and 900 m from the glacier
terminus–were selected, and 200-mL soil samples were
IN THE
WILLOW CANOPY SOIL
ON A
GLACIER FOREFRONT
collected in August 2001. Samples were stored on ice
until processed. In the laboratory, roots were handpicked
from soil, and soil was homogenized manually in plastic
bags. Approximately 0.25 g of soil was transferred to
the extraction buffer, and DNA was extracted using
UltraClean Soil kit (Molecular Biology Laboratories
Inc., Carlsbad, CA) following manufacturer’s protocol.
Extracted DNA was stored frozen (_20-C) until further
processing.
PCR Amplification of the Fungal DNA.
A partial
sequence of the 18S of the fungal rDNA was amplified
with two different primer sets in 50-2L PCR reaction
mixtures. First, the reaction to collect data set B
contained final concentrations or absolute amounts of
reagents as follows: 400 nM of each of the forward and
reverse primers (nu-SSU-0817-50 and nu-SSU-1536-30
[3]), 2 2L of the extracted template DNA, 200 2M of
each deoxynucleotide triphosphate, 2.5 mM MgCl2, 1 U
of Taq DNA polymerase (Promega, Madison, WI), and
5 2L of manufacturer’s PCR buffer. The PCR cycle
parameters consisted of an initial denaturation at 94-C
for 3 min, then 40 cycles of denaturation at 94-C for
1 min, annealing at 56-C for 1 min and extension at
72-C for 1 min, followed by a final extension step at 72-C
for 10 min. Second, the reaction to collect data set S
contained final concentrations or absolute amounts of
reagents as follows: 300 nM of each of the forward and
reverse primers (EF4 and EF3 [32]), 2 2L of the extracted
template DNA, 200 2M of each deoxynucleotide
triphosphate, 1.7 mM MgCl2, 2 U of Taq DNA polymerase (Promega), and 5 2L of manufacturer’s PCR
buffer. The PCR cycle parameters consisted of an initial
denaturation at 94-C for 3 min, then 40 cycles of denaturation at 94-C for 1 min, annealing at 48-C for 1 min
and extension at 72-C for 1 min, followed by a final
extension step at 72-C for 10 min. All PCR reactions
were performed in a Hybaid OmniCycler (Hybaid Ltd.,
Middlesex, UK). Possible PCR amplification of airborne
and reagent contaminants was determined using a blank
sample ran through the extraction protocol simultaneously with the actual samples and a negative PCR
control in which the template DNA was replaced with
ddH2O. These remained free of PCR amplicons in all
trials.
Small-Subunit rDNA Clone Library Construction and
Primers specific to fungi and stringent PCR
Analysis.
conditions resulted in amplicons of expected size (about
Figure 1. Neighbor-joining analysis of environmental partial 18S sequences (see Table 1 for accession numbers; AY382401–AY382473)
0
0
obtained with primer set B (nu-SSU-0817-5 and nu-SSU-1536-3 [3]) from willow canopy soil on the forefront of a receding glacier.
Accession numbers of the GenBank-obtained sequences are shown in parentheses. Sequence data were aligned in Sequencher and
neighbor-joining analyses performed in PAUP* [37]. Numbers above the nodes refer to the occurrence of that node in 1000 bootstrap
replicates. Values 9 50% are shown.
A. JUMPPONEN: FUNGI
IN THE
WILLOW CANOPY SOIL
ON A
GLACIER FOREFRONT
237
238
780 bp in set B and about 1400 bp in set S) when the
PCR products were visualized on 1.5% agarose gels. The
mixed populations of PCR products were ligated into a
linearized pGEM-T vector (Promega). The circularized
plasmids were transformed into competent JM109 cells
(Promega) by heat shock, and the putative positive
transformants were identified by !-complementation
[30].
Twenty putatively positive transformants from each
clone library were randomly sampled, and the presence
of the target insert was confirmed by PCR amplification
in 15-2L reaction volumes under the same reaction
conditions as described above. To select different plasmids for sequencing, these PCR products were digested
with endonucleases (HinfI, AluI; New England BioLabs,
Beverly, MA) and were resolved on 3% agarose gels [15].
The PCR screening of clone libraries combined with
restriction fragment length polymorphisms (RFLP) enabled the selection of different RFLP phenotypes for
sequencing. Sequences from each different RFLP phenotype in all clone libraries were obtained by use of
fluorescent dideoxy-terminators (ABI Prism\ BigDyei
Applied Biosystems, Foster City, CA) and an automated
ABI Prism\ 3700 DNA Analyzer (Applied Biosystems)
at the DNA Sequencing and Genotyping Facility at
Kansas State University (GenBank accession numbers
AY382401–AY382473). Vector contamination was removed with the automated vector trimming function in
Sequencher (Version 4.1, GeneCodes, Ann Arbor, MI).
The similarities to existing rDNA sequences in the
GenBank database were determined at the National
Center for Biotechnology Information (http://www.
ncbi.nlm.nih.gov/BLAST/ [1]) by standard nucleotide
basic local alignment search tool (BLAST, version 2.2.1)
without limiting queries and Sequence Match (version
2.7) at the RDP (http://rdp.cme.msu.edu/html/ [26]).
The environmental sequences and sequences from
GenBank were aligned in 830 positions (data set B) and
in 1623 positions (data set S) using Sequencher and were
manually adjusted to maximize conservation. Regions
adjacent to the priming sites were omitted because of
high frequency of ambiguous sites. Data set B contained
one nontarget contaminant (B_Canopy_300_03_06 most
similar to Prismatolaimus intermedius, Enoplida, in
BLAST searches; Table 1) and three clones that contained
large insertions and were unalignable with other fungal
sequences (B_Canopy_300_01_16, B_Canopy_300_
02_10, and B_Canopy_450_03_02; Table 1). Although
A. JUMPPONEN: FUNGI
IN THE
WILLOW CANOPY SOIL
ON A
GLACIER FOREFRONT
large insertions have been observed in the rDNA of
Helotiales, Lecanorales, and Onygenales (see [3, 16, 17,
29]), the unalignable sequences were omitted because
true insertions and chimeric PCR products could not be
identified reliably. The taxonomic relationships among
the fungal sequences were inferred by neighbor-joining
(NJ) analyses in phylogenetic analysis using parsimony
(PAUP*) [37]. A chytridiomycetous fungus (Monoblepharis hypogyna) was selected for the outgroup. Data
matrices were left uncorrected, rates for variable sites
were assumed equal, and no sites were assumed invariable. Sites with missing data, ambiguous nucleotides, or
gaps, were randomly distributed among taxa. The
robustness of the inferred NJ topologies was tested by
1000 bootstrap replicates. The most parsimonious trees
were obtained using random addition sequence and a
branch-swapping algorithm with tree bisection reconnection. The number of equiparsimonious trees was
expected to be high attributable to several closely related
sequences in the clone libraries. As a result, the maximum number of retained trees was restricted to 1000.
The consensus (50% majority rule) and NJ topologies
placed the environmental sequences similarly (data not
shown).
Detection and Analysis of Chimeric Sequences.
Chimeric sequences may be frequent in environmental
samples with diverse, mixed populations of competing
templates [19, 24, 42]. To identify the most likely
chimera breakpoints, all sequenced clones were analyzed
by the Chimera Check program of the RDP (version 2.7
[26]). To test the effects of the chimeric sequences on the
placement of the environmental clones in the obtained
NJ topologies, the data were reanalyzed after exclusion of
data upstream and downstream of the most commonly
encountered chimera breakpoints (positions 1–391 and
502–830 in data set B alignment and positions 1–730 and
902–1623 in data set S). The obtained topologies were
compared to detect clones that clearly changed positions
in different analyses.
Results
Fungal Community Analyses.
A total of 480 rDNA
clones in 24 libraries were screened, and unique RFLP
phenotypes were identified and sequenced to assay fungal
community composition within established Salix spp.
canopies in a primary successional ecosystem. After
Figure 2. Neighbor-joining analysis of environmental partial 18S sequences (see Table 1 for accession numbers; AY382401–AY382473)
obtained with primer set S (EF4 and EF3 [32]) from willow canopy soil on the forefront of a receding glacier. Accession numbers
of the GenBank-obtained sequences are shown in parentheses. Sequence data were aligned in Sequencher and neighbor-joining
analyses performed in PAUP* [37]. Numbers above the nodes refer to the occurrence of that node in 1000 bootstrap replicates.
Values 9 50% are shown.
A. JUMPPONEN: FUNGI
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240
exclusion of likely chimeric sequences, data set B
contained 24 and data set S 18 unique clones. BLAST
(Table 1) and NJ analyses (Figs. 1 and 2) placed the
cloned environmental sequences into the kingdom Fungi.
The target sequences broadly represented fungi including
Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota. Overall, the cloned sequences indicated
the presence of various groups of fungi in the soil
underneath the willow canopies at the receding glacier
forefront. Nontarget contaminants were rare; one clone
was determined to be a nematode (P. intermedius). Three
additional sequences were omitted because they contained
large unalignable inserts whose origin could not be
confirmed to be fungal.
The majority of clones obtained with both primer
pairs were placed among hymenomycetes and filamentous ascomycetes. The clones included taxa with likely
affinities within the ascomycetous Sordariomycetes and
basidiomycetous Russulales and Thelephorales (Figs. 1
and 2; Table 1). Two general points are noteworthy.
First, various basidiomycete clones likely represent
ectomycorrhizal fungi. Clones in data sets B and S had
well-supported affinities within Russulaceae (B_Canopy_900_01_19 in data set B and S_Canopy_900_01_11 in
data set S) and Thelephoraceae (B_Canopy_900_02_06
and B_Canopy_900_03_17 in data set B and S_Canopy_750_01_10 and S_Canopy_900_03_02 in data set S).
Second, some ascomycete clones, similarly, are likely to
form associations with willow roots. Both data sets
contained clones with well-supported affinities to Sordariales (B_Canopy_900_02_04 in data set B and
S_Canopy_450_02_05 and S_Canopy_750_02_09 in data
set S). These sordarialean fungi are likely similar to those
forming ectomycorrhizas with willows as reported earlier
by Trowbridge and Jumpponen [39].
Most clone libraries were dominated by a single
sequence type (Table 1). In two cases (samples S_Canopy_
450_2 and S_Canopy_900_03), the libraries contained
only one sequence type. These libraries were unlikely
to be representative because data set B contained more
than one sequence type in those samples. The dominant,
nonchimeric sequence types in data set B were not
identical with those in data set S suggesting primer bias
(see below).
Congruence in Fungal Community Composition
Analysis of the 18S
Among the Two Data Sets.
rDNA with two different primer sets designed to be
specific to fungi congruently identified several groups.
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These included well-supported groups with affinities
within Sordariales, Russulaceae, and Thelephoraceae.
However, after exclusion of all suspected chimeric data,
several incongruences were also evident (Figs. 3 and 4).
Data set B (20 ascomycete clones of the 24 total clones)
contained a larger number of ascomycete sequences than
did data set S (4 ascomycete clones of the 18 total
clones). Many of the groupings were not supported in
bootstrap analyses, but three ascomycete groups exemplify the more abundant detection of ascomycetes in data
set B. First, two clones (B_Canopy_450_03_14 and
B_Canopy_450_03_17) were placed among Dothideomycetes with reasonably high bootstrap support in NJ
analyses (Fig. 1). Second, three clones (B_Canopy_300_
02_05, B_Canopy_300_02_06, and B_Canopy_750_
03_11) were grouped with Peziza griseorosea with 100%
bootstrap support, strongly indicating an affinity within
Pezizaceae. Third, five clones (B_Canopy_300_03_17,
B_Canopy_450_01_06, B_Canopy_450_02_13, B_Canopy_750_02_13, and B_Canopy_900_03_09) from five
different samples were placed on a sister clade to
Ophiostomatales. None of these well-supported groups
occurred in data set S.
Data set S contained well-supported groups within
Chytridiomycota (S_Canopy_300_03_04; Fig. 2) and
Zygomycota (S_Canopy_300_02_11; Fig. 2). In contrast,
data set B contained no clones representing lower fungi.
This result was not attributable to mere exclusion of
chimeric data, as no lower fungi were detected in data set
B in BLAST analyses. Data set S also included a large
group of basidiomycetes with likely affinities within
Cortinariaceae representing at least two distinct taxa
(Cortinarius sp. and Inocybe sp.). No clones had wellsupported affinities to Cortinariaceae in data set B,
although at least three sequences were determined most
similar to Inocybe geophylla in BLAST analyses.
Detection and Importance of Chimeric Sequences.
A majority of the environmental sequences were determined to be likely chimeric by Chimera Check of the
RDP. Further testing by reanalyses identified 17 chimeras
in data set B and 8 in data set S (Figs. 3 and 4).
Exceptionally high scores (980) in Chimera Check were
always confirmed chimeric in the reanalyses. Lower
scores did not indicate nonchimeric origin of a
sequence, but many sequences could be confirmed
chimeric in the NJ analyses (Table 1). Many of the
chimeric sequences were likely a result of combined PCR
products of templates representing fungi from different
Figure 3. Reanalyses of data set B. Phylogram obtained by neighbor-joining analysis after the omission of potentially chimeric upstream
data (positions 502–830) as identified by Chimera Check. Arrows on the right show the new placement of environmental sequences
after the omission of potentially chimeric downstream data (positions 1–391). The environmental sequences with unstable placements
in these reanalyses were concluded to be chimeric and were excluded from analyses shown in Fig. 1.
A. JUMPPONEN: FUNGI
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241
242
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A. JUMPPONEN: FUNGI
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divisions as indicated by placement among Ascomycota
in analyses utilizing only 50-end of the sequences and
among Basidiomycota in analyses utilizing only 30-end
of the sequences (e.g., B_Canopy_900_02_12 and
B_Canopy_750_03_03 in data set B–Fig. 3; and
S_Canopy_750_02_17 in data set S–Fig. 4). Data set S
was expected to have a greater proportion of chimeras, as
their likelihood was anticipated to increase with
increasing amplicon length. Surprisingly, data set B
contained 40% (17/43) chimeric sequences, whereas
data set S contained only 31% (8/26) chimeras.
Discussion
Fungal Communities Within Willow Canopies in the
Fungal PCR amplicons were
Glacier Forefront Soil.
successfully obtained from environmental soil samples
collected at the forefront of a receding glacier. A large
proportion of the sequences was determined to be
chimeric by the Chimera Check software of the RDP.
Analyses conducted after exclusion of the sequence data
potentially obtained from another target organism
confirmed many chimeras, but the placement of most
cloned sequences was insensitive to the exclusion of the
potentially chimeric data. In other words, the placement
of a majority of the cloned sequences was similar whether
or not the data identified as possibly chimeric by
Chimera Check were included in the analyses.
After exclusion of chimeric data, 24 and 18 environmental sequences were analyzed in the two data sets.
Most of the basidiomycetes detected in these analyses
likely represented ectomycorrhizal associates of the
willow plants. Earlier studies on sporocarp occurrence
have indicated that Cortinariaceae (Inocybe spp. and
Cortinarius spp.) and Tricholomataceae (Laccaria spp.)
are common throughout the primary successional glacier
forefront [21, 22]. Neither primer set produced cloned
sequences that would find strongly supported affinity to
Laccaria spp. in the NJ analyses, although both data sets
contained nonchimeric sequences that were deemed
similar to Laccaria pumila in BLAST analyses. The
absence of support in NJ analyses is likely because of
the poor resolution within the Agaricales that the 18S
rDNA data provide. Several sequences similar to Cortinariaceae were detected in both data sets, although only
data set S had well-supported affinities to Cortinarius
iodes and I. geophylla. Additional infrequently fruiting
ectomycorrhizal fungi exclusive to areas adjacent to the
terminal moraine (Russulales representing genera Lactar-
243
ius and Russula [21, 22]) were detected in the soil
samples collected 900 m from the glacier terminus by
both primer sets. Finally, ectomycorrhizal fungi with
inconspicuous fruiting bodies (Thelephoraceae) were
detected within the willow canopies furthest from the
glacier terminus by both primers.
Although functional roles of the ectomycorrhizal
basidiomycetes are often simple to decipher from their
affinities to taxa available in sequence databases, the
function of a majority of ascomycetes detected in these
analyses remain unclear. Data set B contained clones
with affinities to Pezizales (P. griseorosea), and both data
sets contained clones with well-supported affinities to
Sordariales. Several taxa within Pezizales have various
associations ranging from pathogenicity to mycorrhizal
symbiosis with ectomycorrhizal hosts [7, 8, 10]. Recent
studies at the Lyman glacier site have suggested that taxa
with affinities to Sordariales may, unexpectedly, be
common mycorrhizal associates of the shrub willows
[39]. Although it is very likely that many cloned ascomycetes represent these (facultative) biotrophic associations,
various groups of the detected ascomycetes (e.g., taxa
with affinities to Dothideales) are soil-inhabiting saprobes.
Congruence in Fungal Community Composition
Differential PCR ampliAmong the Two Data Sets.
fication may be a result of various factors including
template concentration, numbers of template molecules,
GC content of the template molecules, efficiency of
primer-template hybridization, polymerase extension
efficiency for different templates, relative substrate exhaustion for different templates, and primer specificity
[5, 12, 36, 42, 44]. The presented results of rDNA
analyses using two sets of primers confirmed predicted
EF4–EF3 primer bias toward basidiomycetes and lower
fungi [2, 32]. Only 4 of the 18 nonchimeric clones in
data set S were ascomycetous, whereas ascomycetes
comprised a majority of nonchimeric clones in data set
B (20 ascomycetes of the total of 24 nonchimeric
sequences). Although not observed in the present study,
primers for data set B do amplify chytridiomycetes
and zygomycetes from environmental samples [3, 19].
The observed incongruences are therefore likely to
have resulted either from true primer bias or from
stochastic variation within an environmental DNA extract. However, the two different fungus-specific primers
congruently identified several groups. These included
well-supported groups with affinities within Sordariales,
Russulaceae, and Thelephoraceae. The congruence among
Figure 4. Reanalyses of data set S. Phylogram obtained by neighbor-joining analysis after the omission of potentially chimeric upstream
data (positions 902–1623) as identified by Chimera Check. Arrows on the right show the new placement of environmental sequences
after the omission of potentially chimeric downstream data (positions 1–730). The environmental sequences with unstable placements
in these reanalyses were concluded to be chimeric and were excluded from analyses shown in Fig. 2.
244
A. JUMPPONEN: FUNGI
the data sets could possibly have been improved by
increasing the number of clones sampled from each
library. However, there is often a compromise between
the number of clones sampled from each library and the
number of samples to be processed. Clearly, choice of
primers and the number of sampled transformants within
the clone libraries have a pivotal importance on the observed
community structure. Comparisons among multiple
extracts of the same sample, two or more primer sets, as
well as multiple replicate samples may be necessary to obtain
a more comprehensive view of the fungal communities.
IN THE
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after the omission of potentially chimeric regions
included such groups. Both B and S data sets included
cloned sequences that were basal to ascomycetous
Saccharomycetales (e.g., B_Canopy_750_02_19 in Fig. 3
and S_Canopy_300_02_01 in Fig. 4) and basidiomycetous hymenomycetes (e.g., B_Canopy_300_01_18 and
S_Canopy_750_03_18). Data set S included a sequence
(S_Canopy_300_02_19) that was positioned basal to
higher fungi (i.e., Ascomycota and Basidiomycota). None
of the sequences placed in these basal positions were
consistent in the reanalyses of the partial data sets and
were therefore concluded to be PCR artifacts.
Detection and Importance of Chimeric Sequences.
Chimera Check overestimated the number of chimeric
sequences as determined in confirmatory NJ analyses.
However, sequences with high scores (980) in the
Chimera Check were always confirmed chimeric. Lower
scores included sequences that were determined chimeric
and many that appeared stable in their position in
confirmatory NJ analyses.
The NJ analyses presented here aimed to identify and
detect sequences whose positions in the obtained topologies were inconsistent when only 50-ends or only 30-ends
of the sequences were utilized. Reanalyses of partial data
sets identified 17 chimeras in data set B and 8 in data set S,
more than 30% of all analyzed sequences. Similar chimera
frequencies have been observed in bacterial community
analyses [43] and analyses of somatic mutations [13].
Chimeric sequences are particularly frequent if sequence
similarity among the competing templates and the
number of PCR cycles are high [13, 43]. Accordingly,
simple precautionary measures, such as longer extension
times and fewer PCR cycles [42, 43], to minimize the
generation of chimeras seem necessary.
It was hypothesized that longer target amplicons
would be more susceptible for chimera formation.
Unexpectedly, the data set with shorter target amplicon
had greater number of identified chimeras. This observation may be a result of the larger number of competing
templates with fairly high similarity when primers with
lesser bias were used (data set B; see [13, 43]). Overall,
more data (longer amplicons) are usually beneficial, as
they often allow better resolution in inferred topologies
[18]. This is especially important when using conserved
gene regions such as the 18S of the rDNA. It appears that
the generation of chimeras is stochastic, and that
targeting shorter amplicons may be unnecessary in fear
of poor-quality environmental sequence data if steps to
minimize chimera formation have been taken.
Recent studies that utilize direct PCR from environmental samples have suggested frequent occurrences of
novel fungal phyla, which find positions basal to
filamentous ascomycetes or hymenomycetes [31, 41].
The preliminary analyses conducted prior to exclusion of
chimeras as well as the analyses using partial sequences
Conclusions
The results indicate that ascomycetous and basidiomycetous ectomycorrhizal fungi comprise a substantial
component in the fungal communities associated with
the established willow canopies in primary successional
ecosystems on the forefront of a receding glacier. Use of
different primers yielded different results and supported
different conclusions. It seems therefore necessary to
view the results of direct molecular assessments with
some caution. Finally, chimeras seem to comprise a large
proportion of the environmental sequence data as
determined by the Chimera Check of RDP and data
reanalyses. Many of the chimeric reads appeared to
comprise novel taxa at least on the level of an order.
However, because it is possible that these sequences may
be but PCR artifacts, the discovery of novel taxa without
microscopic or culture-based confirmation may be
premature.
Acknowledgments
This work was supported by Kansas State University
BRIEF program, National Science Foundation EPSCoR
Grant No. 9874732 with matching support from the
State of Kansas, and National Science Foundation Grant
No. OPP-0221489. I am grateful to Dr. Francesco T.
Gentili, Nicolo Gentili, Anna Jumpponen, and Dr. James
M. Trappe for their assistance during sample collection,
transport, and preparation in August 2001 and to Emily L.
King and Justin Trowbridge for their assistance in clone
library screening and plasmid preparation. Dr. Charles L.
Kramer, Nicholas B. Simpson, and Dr. James M. Trappe
provided helpful comments on early drafts of this manuscript. Nicholas B. Simpson edited the manuscript.
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