Content uploaded by Ned Klopfenstein
Author content
All content in this area was uploaded by Ned Klopfenstein on Oct 20, 2017
Content may be subject to copyright.
Characterization of North American Armillaria species: genetic
relationships determined by ribosomal DNA sequences and
AFLP markers
By M.-S. Kim
1
,N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
USDA Forest Service, RMRS, 1221 S. Main St., Moscow, ID 83843, USA.
1
E-mail: mkim@fs.fed.us
(for correspondence)
Summary
Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed
using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal
transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and
amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU
region is less variable among Armillaria species than the ITS + 5.8S and IGS-1 regions
(nLSU < ITS + 5.8S < IGS-1). Phylogenetic analyses of the rDNA sequences suggested Armillaria
mellea,A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A.
ostoyae,A. gemina,A. calvescens,A. sinapina,A. gallica, NABS X and A. cepistipes). Several Armillaria
species (A. calvescens,A. sinapina,A. gallica, NABS X and A. cepistipes) clustered together based on
rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on
IGS-1, ITS + 5.8S, and/or D-domain/3¢ends of nLSU are not reliable for distinguishing A. calvescens,
A. sinapina,A. gallica and A. cepistipes. However, AFLP data provided delineation among these
species, and AFLP analysis supported taxonomic classification established by conventional methods
(morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential
for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in
future biological, ecological and taxonomic studies.
1 Introduction
Armillaria is a genus that comprises over 30 species of wood-decaying fungi. These species
are primarily recognized for their association with root and butt rot of woody plants, but
they are also important decomposers within many forested environments (e.g. natural,
urban and agroforestry). In addition, Armillaria species are ecologically diverse, with
variable pathogenicity, host specificity and other environmental requirements. For example,
Armillaria ostoyae (Romagn.) Herink is a primarily aggressive pathogen on conifers,
A. mellea (Vahl: Fr.) Kummer is a primarily aggressive pathogen on hardwoods, and
A. gallica Marxmu
¨ller & Romagn. is predominantly saprophytic (Shaw and Kile 1991).
Based on morphology and in vitro compatibility of isolates, Armillaria species in North
America were initially grouped into 10 North American Biological Species (NABS)
(Hintikka 1973; Korhonen 1978; Anderson and Ullrich 1979; Motta and Korhonen
1986; Termorshuizen and Arnolds 1987, 1997; Be
´rube
´and Dessureault 1988; Banik
et al. 1996; Volk et al. 1996). The absence of an annulus separates A. tabescens (Scop.) Emel
from other North American species of Armillaria (A. ostoyae,A. gemina Be´rube´ & Dessur.,
A. calvescens Be´rube´ & Dessur., A. sinapina Be´rube´ & Dessur., A. mellea,A. gallica,
A. nabsnona Volk & Burdsall, NABS X and A. cepistipes Velen.). In addition to mating tests,
the other Armillaria species, except NABS X, have been formally described on a basis of
macro-/microscopic and distributional characters (Volk and Burdsall 1995). However,
For. Path. 36 (2006) 145–164
2006 The Authors
Journal compilation 2006 Blackwell Verlag, Berlin
Received: 11.05.2005; accepted: 4.10.2005; editor: C. G. Shaw
www.blackwell-synergy.com
basidiospores are unavailable for most isolates of Armillaria species, and it can be
impractical or unreliable to identify species using other characteristics. Basidioma
morphology has also been used to identify Armillaria species (Motta and Korhonen
1986; Termorshuizen and Arnolds 1987, 1997; Be
´rube
´and Dessureault 1988), but
basidioma production can be unpredictable or rare.
Several molecular methods were developed to augment identification of Armillaria
species and determine phylogenetic relationships among Armillaria species (Anderson
et al. 1987, 1989; Jahnke et al. 1987; Smith and Anderson 1989; Smith et al. 1990;
Anderson and Stasovski 1992; Miller et al. 1994; Harrington and Wingfield 1995;
Schulze et al. 1995, 1997; Chillali et al. 1998; Piercey-Normore et al. 1998;
Terashima et al. 1998; White et al. 1998; Coetzee et al. 2000, 2001, 2003; Kim et al.
2000; Otieno et al. 2003; Gezahgne et al. 2004; Pe
´rez-Sierra et al. 2004). Anderson
and Stasovski (1992) determined the DNA sequences of intergenic spacer (IGS-1) and
assessed phylogenetic relationships among the NABS of Armillaria. In addition, Piercey-
Normore et al. (1998) compared anonymous nucleotide sequences from eight NABS of
Armillaria to evaluate their phylogeny. Previous molecular phylogenetic studies indicated
that A. calvescens,A. sinapina,A. gallica,A. nabsnona, NABS X and A. cepistipes represent
a closely related group that is well separated from A. ostoyae,A. gemina,A. mellea and
A. tabescens (Anderson and Stasovski 1992; Piercey-Normore et al. 1998). Subse-
quently, Coetzee et al. (2000, 2001, 2003) addressed the phylogenetic relationships among
isolates of A. mellea s.s. and other Southern Hemisphere Armillaria species based on DNA
sequences from the IGS-1 as well as the internal transcribed spacer (ITS) regions.
The 5¢end of the nuclear large subunit (nLSU) rDNA gene (proximal to ITS2), which
comprises the divergent domains D1–D3 region (Michot et al. 1984, 1990), has been used
to study phylogenetic relationships of agaric fungi (Hopple and Vilgalys 1999;
Moncalvo et al. 2000, 2002). This region contains the most phylogenetically informative
sites in the nLSU rDNA gene (Kuzoff et al. 1998; Hopple and Vilgalys 1999; Fell et al.
2000; Weiß and Oberwinkler 2001). However, this region has not been applied to the
study of phylogenetic relationships among the NABS of Armillaria.
The objectives of this study were to: (i) determine phylogenetic relationships among
10 NABS of Armillaria using rDNA (IGS-1, ITS + 5.8S and nLSU) sequence data and
(ii) assess genetic relationships among NABS of Armillaria, especially within the closely
related group that includes A. calvescens,A. sinapina,A. gallica,A. nabsnona, NABS X and
A. cepistipes, using high-resolution amplified fragment length polymorphism (AFLP)
markers. Attaining these objectives will also provide a better characterized set of diploid
tester strains (Kim et al. 2000) for use in biological, ecological and taxonomic studies of
Armillaria species.
2 Materials and methods
2.1 Fungal isolates
Tested species included A. ostoyae,A. gemina,A. calvescens,A. sinapina,A. mellea,
A. gallica,A. nabsnona, NABS X, A. cepistipes and A. tabescens. Three isolates representing
each of the 10 North American Armillaria species were included in the study, most of
which were previously characterized for nuclear DNA content (Kim et al. 2000). Isolates
originated from basidioma (stipe or context) tissue or mass-spore cultures, and were
previously identified by various investigators using haploid ·haploid mating or hap-
loid ·diploid pairing tests (Table 1). To ensure that each isolate represented a distinct
genet (vegetative clone), isolates were selected from different geographical regions or were
tested by somatic pairing (Anderson and Kohn 1995; Kim et al. 2000). All isolates used in
146 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
Table 1. Armillaria isolates used in phylogenetic analysis
Species Collection Isolate
1
Origin Source tissue
GenBank accession No.
ITS-1, 5.8S, ITS-2 and partial nLSU IGS-1 and partial nLSU
A. ostoyae DMR20
3
ST1 New Hampshire, USA Multisporous AY213552 AY509154
AMM9067
3
ST2 Washington, USA Basidioma AY213553 AY509155, AY509156
P1404
4
P1404 Idaho, USA Basidioma AY213554 AY509157
A. gemina JJL153
5
ST8 New York, USA Basidioma AY213555 AY509158, AY509159
JJW64
5
ST9 New York, USA Basidioma AY213556, AY213557 AY509160, AY509161
MIELKE
3
ST11 West Virginia, USA Unknown AY213558 AY509162
A. calvescens JB56A
6
ST3 Quebec, Canada Basidioma AY213559 AY509163
PR-3
3
ST17 Michigan, USA Basidioma AY213560, AY213561 AY509164
FFC-7
3
ST18 Michigan, USA Basidioma AY213562 AY509165, AY509166
A. sinapina SP81-1
7
M50 British Columbia, Canada Basidioma AY213563, AY213564 AY509167
AMM9065
3
ST12 Washington, USA Basidioma AY213565 AY509168
CF-2
3
ST13 Michigan, USA Multisporous AY213566, AY213567 AY509169, AY509170
A. mellea GB934
3
ST5 Virginia, USA Multisporous AY213584, AY213585 AY509185, AY509186
A3
3
ST20 Wisconsin, USA Basidioma AY213586 AY509187
TCH-2
3
ST21 New Hampshire, USA Multisporous AY213587 AY509188
A. gallica SP81-29
7
M70 British Columbia, Canada Basidioma AY213568 AY509171
EL-1
3
ST22 Michigan, USA Basidioma AY213569, AY213570 AY509172
MA-1
3
ST23 Wisconsin, USA Basidioma AY213571 AY509173
A. nabsnona C21
4
C21 Idaho, USA Basidioma AY213572 AY509174, AY509175
M90
7
M90 British Columbia, Canada Basidioma AY213573 AY509176, AY509177
SHAW,C.C
3
ST16 Alaska, USA Multisporous AY213574 AY509178
NABS
2
X 837
4
837 Idaho, USA Basidioma AY213575, AY213576 AY509179
D82
4
D82 Idaho, USA Basidioma AY213577, AY213578 AY509180
POR100
4
POR100 Idaho, USA Basidioma AY213579, AY213580 AY509181
A. cepistipes SP82-14
7
M110 British Columbia, Canada Basidioma AY213581 AY509182
SP83-07
7
S20 British Columbia, Canada Basidioma AY213582 AY509183
HH14867
3
W113 Washington, USA Basidioma AY213583 AY509184
A. tabescens AT-MU-S2
8
AT-MU-S2 South Carolina, USA Stipe AY213588 AY509189, AY509190
OOI-99
8
OOI-99 Georgia, USA Basidioma AY213589 AY509192
OOI-210
8
OOI-210 Georgia, USA Basidioma AY213590 AY509191
1
Identification code in this paper.
2
NABS ¼North American Biological Species.
3
Identified by the USDA Forest Service Center for Forest Mycology Research, Madison, WI, USA.
4
Identified by G. I. McDonald.
5
Identified by J. Worrall.
6
Identified by J. Be´ rube´.
7
Identified by D. Morrison.
8
Identified by G. Schnabel.
147Characterization of North American Armillaria spp.
this study are maintained as archival cultures at the USDA Forest Service, Forestry
Sciences Laboratory in Moscow, ID, USA.
2.2 DNA sequencing
For use in DNA sequence analysis, isolates were maintained in Petri dishes on 3% malt-agar
medium (3% malt extract, 1.5% peptone, 3% glucose, 1.5% agar) and incubated at 22Cin
the dark. The IGS-1, ITS + 5.8S and 5¢(divergent domains D1, D2 and D3; Michot et al.
1984, 1990) + 3¢(ca 207 nucleotides upstream from IGS-1) ends of nLSU rDNA regions
were amplified using polymerase chain reaction (PCR). Template DNA was derived from
scrapings of actively growing mycelial cultures (1–2 weeks old). Primers LR12R/O-1
(Veldman et al. 1981; Duchesne and Anderson 1990), ITS-1F/ITS-4 (White et al. 1990;
Gardes and Bruns 1993) and 5.8SR/LR7 (Moncalvo et al. 2000) were used to amplify the
3¢nLSU + IGS-1, ITS + 5.8S rDNA, and nLSU D-domains, respectively (Fig. 1). Each 50-
ll reaction mixture contained template DNA obtained from scraping cultured mycelia (or
no DNA template for negative control), 2.5 U Taq polymerase (Applied Biosystems, Inc.,
Foster City, CA, USA), PCR reaction buffer (supplied with Taq enzyme), 4 mmMgCl
2
,
200 lmdNTPs, and 0.5 lmof each primer. The PCR conditions were as follows: (i) for 3¢
nLSU + IGS-1, 95C for 1 min 30 s, 35 cycles of 95C for 1 min 30 s, 60C for 40 s, and
72C for 2 min, and finally 72C for 10 min; (ii) for ITS + 5.8S, 94C for 2 min 30 s, 35
cycles of 94C for 1 min, 48C for 1 min, and 72C for 1 min 30 s, and finally 72C for
10 min; and (iii) for nLSU-D domains, 94C for 3 min, 35 cycles of 94C for 1 min, 55C for
30 s, and 72C for 2 min, and finally 72C for 5 min. All PCRs were conducted by MJ PTC-
200 thermocycler (Bio-Rad Laboratories, Waltham, MA, USA). Intact PCR products were
electrophoresed in 1.5% agarose gels using 0.5X TBE (89 mmTris, 89 mmboric acid and
2mmEDTA) buffer. Gels were stained with ethidium bromide (0.5 lg/ml) and bands were
visualized using UV light. PCR products were treated with ExoSAP-IT (USB Corporation,
Cleveland, OH, USA), then sequenced with an ABI 377 or ABI 3700 DNA sequencer at the
Davis Sequencing Facility (Davis, CA, USA). IGS-1 and ITS + 5.8S regions were sequenced
with the same primers used for initial amplification, while the nLSU-D domains were
sequenced using the LR0R, LR15 and LR5 primers (Fig. 1) (Moncalvo et al. 2000).
2.3 Sequencing editing and alignment
Sequences were edited and aligned manually with bioedit software (Hall 1999). Sequence
editing was performed independently by two separate researchers to minimize errors. An
additional precaution was to sequence rDNA regions in both directions and duplicate all
steps (from PCR to DNA sequencing) for all of the Armillaria isolates we tested. Careful
attention was paid to sequences containing heterogeneous products. Polymorphisms were
coded with the IUPAC codes for ambiguous nucleotides. These polymorphisms were not
regarded as ambiguous but rather the result of heterogeneous products because of variation
within rDNA repeat units (Crease and Lynch 1991). In this study, heterogeneity was
detected when a chromatogram contained either single nucleotide polymorphisms
Fig. 1. Diagram of rDNA repeat. The arrows correspond to the annealing sites and direction for
several primers
148 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
(represented by a double peak occurring at a single nucleotide position) or a Ôframe-shiftÕ
(overlapping peaks due to length variation among the rDNA repeat units). When possible,
heterogeneous sequences were split into homogeneous sequence representations by one of
the three methods described in Hanna (2005) before phylogenetic analyses. The different
sequence types from an individual isolate were given a letter code (e.g. A or B) after genet
names (Figs 2 and 3; Table 1). For example, A. gemina isolate ST9 has two IGS-1 sequence
types, ST9-A and ST9-B, respectively, whereas A. gemina isolate ST11 produced a
homogenous sequence in the IGS-1 region, and has only one sequence type. All sequences
used in phylogenetic analyses have been deposited into GenBank (AY213552–AY213590;
AY509154–AY509192) (Table 1).
2.4 Phylogenetic analyses of IGS-1, ITS + 5.8S and nLSU sequences
Phylogenetic analyses were performed for each of three rDNA regions (IGS-1, ITS + 5.8S
and 5¢/3¢ends of nLSU) using neighbor-joining (NJ) (Saitou and Nei 1987), Parsimony
and Bayesian inference methods. Most of the genets of this study contained unique
sequence types; however, any duplicate sequence types were removed from the dataset
before phylogenetic analyses.
Any gaps in sequence alignments were treated as missing and coded using a simple
gap-coding method (Simmons and Ochoterena 2000). NJ analysis was performed using
paup* (4.0b10) (Swofford 2001) with 1000 bootstrap replicates (Felsenstein 1985).
Parsimony analysis was performed using paup* (4.0b10). The bootstrap method
(Felsenstein 1985) with heuristic search was used with 1000 bootstrap replicates on
each dataset to obtain 50% bootstrap majority-rule consensus trees. Multistate taxa were
interpreted as polymorphisms, starting trees were obtained via stepwise addition with
random addition sequence of 10 replicates, tree-bisection-reconnection was used for
keeping one tree at each step, and the steepest descent option was not in effect. The
analysis of the nLSU was performed with MaxTrees set to auto increase, while the IGS-1
and ITS + 5.8S regions were set to a maximum of 10 000 trees because of computational
runtime limitations.
Bayesian analysis was performed by MrBayes v3.0B4 (Huelsenbeck and Ronquist
2001). Bayesian inference of phylogeny calculates the posterior probability of phylogenetic
trees. To select appropriate evolutionary models to use in Bayesian analysis, MrModel-
test 1.0b (Nylander 2003) was used on each data set. For the nLSU and ITS + 5.8S data
sets, Hasegawa-Kishino-Yano (HKY) model (Hasegawa et al. 1985) was selected. And for
the IGS-1 data set, HKY + G (C;Yang 1993) model was selected. Four chains were run
for 3 ·10
6
generations generating files with 30 001 trees, the first 6000 of these trees were
discarded as the Ôburn-inÕof the chains. The remaining 24 001 trees were used to make 95%
majority consensus trees using paup* (4.0b10).
Ambiguous alignment of sequences due to overlapping indels of different lengths may
lead to misleading phylogenies (Hall 2001); therefore, we used a conservative approach
(Eriksson et al. 2003) and refrained from comparing sequences that resulted in ambiguous
alignment. In the IGS-1 and ITS + 5.8S regions, A. mellea and A. tabescens would not align
with the other species without ambiguity. Preliminary analysis suggested that the
phylogenetic placement of A. mellea and A. tabescens would be significantly resolved
using the nLSU D-domains and 3¢ends of nLSU; therefore, these species were excluded
from the IGS-1 and ITS + 5.8S datasets to remove ambiguity and give greater resolution to
the comparison of the more closely related individuals/species. Armillaria ostoyae and
A. gemina were used as outgroups for the IGS-1 and ITS + 5.8S regions, and A. mellea was
used as an outgroup for the nLSU region.
All phylogenetic trees generated from rDNA sequence data have been deposited into
TreeBASE (study accession number ¼S1452).
149Characterization of North American Armillaria spp.
2.5 AFLP markers
To generate biological material for AFLP studies, isolates were cultured on 0.2-lm pore,
nylon filters (Millipore Corp., Billerica, MA, USA) that overlayed the 3% malt-agar
medium in Petri dishes, then incubated at 22C in the dark for 2 weeks. Approximately
100 mg fresh mycelia was scraped from the nylon filter and used for DNA extraction.
DNeasy Plant Mini DNA extraction kits (Qiagen Inc., Valencia, CA, USA) were used to
extract and purify all DNA samples following the protocol of the manufacturer. DNA was
quantified for AFLP analysis using a TD-360 fluorometer (Turner Designs, Sunnyvale,
CA, USA).
Amplified fragment length polymorphism analyses were performed following the
protocol of Vos et al. (1995). For restriction digests, 350 ng of genomic DNA was digested
with EcoRI and MseI (New England BioLabs, Inc., Beverly, MA, USA) to serve as the
template. Resulting DNA fragments were ligated to adapters and diluted 1 : 10 with sterile
distilled water prior to pre-amplification. Pre-amplification reaction mixtures (total 30 ll)
contained 6 ll of diluted restriction/ligation mixture as template, 10X PCR buffer (Applied
Biosystems, Inc.), 3 mmMgCl
2
, 200 lmdNTPs, 300 nmof each pre-selective primer (EcoRI-
no extension and MseI-C), and 1.5 U AmpliTaq
DNA polymerase (Applied Biosystems,
Inc.). The PCR conditions for pre-amplification were as follows: 72C for 2 min, 20 cycles of
94C for 40 s, 56C for 1 min, and 72C for 3 min, and finally 72C for 10 min.
For the selective amplification, we screened 24 different primer combinations using
DNA from 10 isolates of Armillaria species. Among several promising selective primer
combinations, we choose three (EcoRI-AA/MseI-CTG, EcoRI-AG/MseI-CAC, and
EcoRI-AG/MseI-CTT) that produced the most manageable band number and interpretable
banding pattern. Selective amplification mixtures (total 25 ll) contained 5 ll of diluted pre-
amplification products [1 : 20 with low TE buffer (10 mmTris-HCl, 0.1 mmEDTA, pH
8.0)] as a template, 10X PCR buffer (Applied Biosystems, Inc.), 2.4 mmMgCl
2
, 300 lm
dNTPs, 100 nmof EcoRI-AA or -AG primers, 300 nmof MseI-CTG, -CAC, or -CTT
primers, and 1.25 units of AmpliTaqGold polymerase (Applied Biosystems, Inc.).
Amplifications were performed using the following PCR conditions: 94C for 2 min, 14
cycles of 94C for 30 s (1C per second to 65C), 65C for 30 s ()0.7C per cycle), and
72C for 2 min, 20 cycles of 94C for 30 s, 56C for 30 s, and 72C for 2 min, and finally
72C for 10min. All PCR were conducted using a MJ PTC-200 thermocycler (Bio-Rad
Laboratories).
Selective amplification products were separated in an ABI 3700 DNA automated
sequencer (Applied Biosystems, Inc.) at the University of Wisconsin – Biotechnology
Center (Madison, WI, USA). genotyper 3.7 NT (Applied Biosystems, Inc.) was used to
identify peaks with a fluorescent intensity greater than the threshold value (ca 300 units) in
at least one sample. Categories were made from these identified peaks for the scoring of all
samples.
2.6 AFLP data analysis
The AFLP bands were scored as present or absent using genescan 3.7 NT (Applied
BioSystems, Inc.) and a binary matrix was developed with molecular sizes ranging from 71
to 616 bp. The resulting binary matrix was used to calculate genetic distances (Nei and Li
1979) among Armillaria genets and distances were graphically displayed by unweighted
paired-group method with arithmetic means (upgma) using paup* (4.0b10). The upgma
dendrograms were constructed with confidence estimates assigned to its topology based on
1000 bootstrap replicates.
The upgma tree generated from AFLP marker data has been deposited into TreeBASE
(study accession number ¼S1452).
150 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
3 Results
3.1 Sequence data from IGS-1, ITS + 5.8S and nLSU rDNA regions
The IGS-1 data set consisted of 29 unique sequences that each contained 606 total
characters, of which 147 (24%) varied and 85 (14%) were parsimony informative. An
optimum sample tree from the heuristic search (limited to 10 000 trees) yielded a total
length of 526 steps with consistency index (CI) of 0.992, a retention index (RI) of 0.992,
and a rescaled consistency index (RC) of 0.984. The resulting 50% majority-rule bootstrap-
consensus tree from the parsimony analysis and the 95% majority-rule consensus tree
(based on posterior probability) from the Bayesian analysis showed nearly identical
topologies (Fig. 2).
The ITS + 5.8S data set consisted of 26 unique sequences that each contained 792 total
characters, of which 55 (6.9%) varied and 19 (2.4%) were parsimony informative. An
optimum sample tree from the heuristic search (limited to 10 000 trees) yielded a total
length of 93 steps (CI ¼0.968, RI ¼0.952 and RC ¼0.921). The resulting 50% majority-
rule bootstrap-consensus tree from the parsimony analysis and the 95% majority-rule
consensus tree (based on posterior probability) from the Bayesian analysis showed
identical topologies (Fig. 3).
The nLSU D-domains and 3¢ends data set consisted of 22 unique sequences that each
contained 1197 total characters, of which 82 (6.9%) varied and 42 (3.5%) were parsimony
informative. With optimality criterion set to parsimony, 198 equally parsimonious trees
were revealed. An optimum sample tree from the heuristic search yielded a total length of
1436 steps with CI ¼0.999, RI ¼0.979 and RC ¼0.978. The resulting 50% majority-rule
bootstrap-consensus tree from the parsimony analysis and the 95% majority-rule
consensus tree (based on posterior probability) from the Bayesian analysis showed a high
degree of congruence (Fig. 4).
3.2 Phylogenetic analyses from rDNA sequence data
Bayesian, Parsimony and NJ (data not shown) analyses showed remarkable congruence for
all three regions analysed, as exemplified by the Bayesian and Parsimony trees shown in
Figs 2–4.
Phylogenetic analyses of the IGS-1 region revealed two major clades: (i) A. nabsnona
(posterior probability 100%, bootstrap support 100%), and (ii) the other Armillaria species
that were analysed, NABS X, A. gallica,A. calvescens,A. sinapina and A. cepistipes (100%,
100%). Within the second clade, a cluster of NABS X species appears separate (96%, 68%)
(Fig. 2). In general, A. ostoyae and A. gemina were well separated from each other as
outgroups. One genet (ST9) of A. gemina possessed two IGS-1 sequence types (ST9-A and
ST9-B), one of which clustered with A. ostoyae (Fig. 2).
Phylogenetic analyses of the ITS + 5.8S region provided two major clades and several
sequence types: (i) A. nabsnona and NABS X sequence type A (837-A, D82-A and POR100-
A) (96%, 88%), (ii) A. cepistipes and one genet of A. sinapina (M50) (100%, 68%), and (iii) the
remaining unclustered Armillaria species/sequences that were analysed, NABS X sequence
type B (837-B, D82-B and POR100-B), A. sinapina,A. gallica and A. calvescens (Fig. 3).
Phylogenetic analyses of the nLSU rDNA region demonstrated two major clades:
(i) A. tabescens (97%, 93%), and (ii) the remaining Armillaria species, A. ostoyae,
A. gemina, NABS X, A. gallica,A. calvescens,A. sinapina,A. nabsnona and A. cepistipes
(100%, 89%) (Fig. 4). A separate cluster of A. ostoyae (ST2 and P1404) species was
apparent (99% posterior probability) with moderate bootstrap support (62%) (Fig. 4). An
A. nabsnona cluster also appeared separate from other Armillaria species with moderate
bootstrap support (64%), but no support from Bayesian probability (Fig. 4).
151Characterization of North American Armillaria spp.
Fig. 2. Bayesian and parsimony trees generated from intergenic spacer (IGS-1) rDNA sequences in
Armillaria species. To the left, a 95% majority-rule posterior probability-consensus tree based on
24 001 trees from the Bayesian inference analysis. Numbers above branches indicate their respective
posterior probabilities. To the right, a 50% majority-rule bootstrap-consensus tree from the parsimony
analysis. Bootstrap supports are indicated above branches based on 1000 bootstrap replicates
152 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
Fig. 3. Bayesian and parsimony trees generated from internal transcribed spacer including 5.8S
(ITS + 5.8S) rDNA sequences in Armillaria species. To the left, a 95% majority-rule posterior
probability-consensus tree based on 24 001 trees from the Bayesian inference analysis. Numbers above
branches indicate their respective posterior probabilities. To the right, a 50% majority-rule bootstrap-
consensus tree from the parsimony analysis. Bootstrap supports are indicated above branches based on
1000 bootstrap replicates
153Characterization of North American Armillaria spp.
Fig. 4. Bayesian and parsimony trees generated from nuclear large subunit rDNA (nLSU) rDNA
sequences in Armillaria species. To the left, a 95% majority-rule posterior probability-consensus tree
based on 24 001 trees from the Bayesian inference analysis. Numbers above branches indicate their
respective posterior probabilities. To the right, a 50% majority-rule bootstrap-consensus tree from the
parsimony analysis. Bootstrap supports are indicated above branches based on 1000 bootstrap
replicates
154 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
Table 2. Description of unique amplified fragment length polymorphism (AFLP) markers among North American Armillaria species
Primer
combination
1
Polymorphic
loci
2
A. ost
loci
3
A. gem
loci
A. cal
loci
A. sin
loci
A. mel
loci
A. gal
loci
A. nab
loci
NABS X
loci
A. cep
loci
A. tab
loci
Eaa/Mctg 139 aactg448 aactg180 aactg398 aactg234 aactg240 aactg323 aactg276 aactg298 aactg153
aactg575 aactg224 aactg282 aactg405 aactg278 aactg256
aactg355
aactg475
aactg569
Eag/Mcac 69 agcac226 agcac181 agcac269 agcac231 agcac119
agcac256 agcac130
agcac330
Eag/Mctt 96 agctt167 agctt298 agctt108 agctt408 agctt282 agctt136 agctt163
agctt293 agctt244 agctt178
agctt300 agctt362 agctt193
agctt194
agctt201
agctt267
agctt506
Total 304 2 5 3 5 1 4 3 5 15
A. ost ¼A. ostoyae;A. gem ¼A. gemina;A. cal ¼A. calvescens;A. sin ¼A. sinapina;A. mel ¼A. mellea;A. gal ¼A. gallica;A. nab ¼A. nabsnona;
NABS ¼North American Biological Species; A. cep ¼A. cepistipes;A. tab ¼A. tabescens.
1
AFLP-selective +2/+3 primer combination – Eaa/Mctg (EcoRI-AA/MseI-CTG), Eag/Mcac(EcoRI-AG/MseI-CAC), and Eag/Mctt (EcoRI-AG/MseI-
CTT).
2
The number of polymorphic markers found among all of the Armillaria genets.
3
Markers that are unique to species.
155Characterization of North American Armillaria spp.
3.3 AFLP marker data
The three selective primer combinations amplified a total of 307 fragments, of which 304
(99%) were polymorphic. Each of the 30 Armillaria genets had a unique AFLP phenotype.
However, some AFLP markers appeared to be species specific and were shared by all three
isolates of an individual Armillaria species, with the exception of A. calvescens (Table 2).
Of the 304 polymorphic AFLP loci evaluated, two were unique to A. ostoyae, five unique
to A. gemina, three unique to A. sinapina, five unique to A. mellea, one unique to A. gallica,
four unique to A. nabsnona, three unique to NABS X, five unique to A. cepistipes, and 15
unique to A. tabescens (Table 2). For example, loci aactg448 (selective primer combination
EcoRI-AA/MseI-CTG) and aactg575 were only found in the three genets of A. ostoyae.
3.4 Genetic analysis of AFLP marker data
A cluster analysis disclosed three major clades: A. tabescens (bootstrap support 100%),
A. mellea (100%), and the remaining North American Armillaria species (81%) (Fig. 5).
The third clade can be further separated into two groups: (i) A. ostoyae and A. gemina
(83%), and (ii) A. sinapina,A. cepistipes, NABS X, A. nabsnona,A. calvescens and A. gallica
(98%) (Fig. 5). Within the second major group, A. sinapina and A. cepistipes were
contained within a cluster (81%) that was separate from other Armillaria species (NABS X,
A. nabsnona,A. calvescens and A. gallica) (Fig. 5). Overall, genetic analysis of AFLP
marker data indicated strong support for intraspecific clustering (Fig. 5).
4 Discussion
Species of Armillaria in North America can be placed into three to four major groups based
on morphological, biological and molecular evidence (Anderson and Stasovski 1992;
Korhonen 1995; Piercey-Normore et al. 1998). Korhonen (1995) divided the Northern
Hemisphere Armillaria species into four clusters on the basis of morphological and
biological features. He designated A. mellea as a single species cluster and A. tabescens and
A. monadelpha (Morgan) Guillaumin & Mohammed as a two-species cluster. Armillaria
borealis,A. gemina and A. ostoyae were named the A. ostoyae cluster. A fourth group, the
A. gallica cluster, included A. calvescens,A. cepistipes,A. gallica,A. sinapina,A. nabsnona
and NABS X. Anderson and Stasovski (1992) concluded that three major groups of
Armillaria species could be separated with respect to IGS-1 region sequences: (i) A. borealis,
A. gemina and A. ostoyae, (ii) A. calvescens,A. cepistipes,A. gallica,A. sinapina,A. nabsnona
and NABS X, and (iii) A. mellea and A. tabescens. Recently, Piercey-Normore et al. (1998)
obtained anonymous nucleotide sequences (i.e. ramdom amplified polymorphic DNA
products) from eight NABS of Armillaria. They proposed a phylogeny of Armillaria species
that is very similar to that proposed by Anderson and Stasovski (1992). Overall, our
results from DNA sequences support previous results on the phylogenetic relationships
among Armillaria species; however, each rDNA region (i.e. IGS-1, ITS + 5.8S, and nLSU)
provided different levels of phylogenetic signal. Sequence data from the IGS-1 region in this
study can separate A. nabsnona and NABS X from the rest of KorhonenÕs (1995) group 4 –
A. gallica cluster and Anderson and StasovskiÕs (1992) group 2. Of particular note is that A.
calvescens,A. sinapina,A. gallica and A. cepistipes could not be separated on the basis of IGS-
1 or ITS + 5.8S sequences. Thus, it appears that these four species cannot be identified using
any techniques based on the IGS-1, ITS + 5.8S, or D-domain/3¢ends of nLSU.
Although basidioma morphology can be used to reliably separate A. mellea and
A. tabescens, the evolutionary relationship between these two species is not well resolved
(Chillali et al. 1998). The nLSU was the only rDNA region that allowed unambiguous
alignment of A. mellea and A. tabescens sequences with sequences of the other eight North
156 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
American Armillaria species. When Pleurotus sp. and Xerula sp. were used as outgroups
for preliminary analysis of nLSU sequences, A. mellea was placed basal to the other of nine
North American species, and A. tabescens was placed basal to the other eight species
(unpublished data). However, more isolates from diverse geographical regions should be
examined to confirm the phylogenetic relationship of A. mellea and A tabescens.Armillaria
mellea was previously considered the most divergent of the North American species
(Be
´rube
´and Dessureault 1988; Anderson et al. 1989; Smith and Anderson 1989;
Anderson and Stasovski 1992). Phylogenetic analysis of IGS-1 and ITS regions showed a
distinct split between isolates from eastern and western North America, although the
Fig. 5. upgma cluster of Armillaria species and genets generated from amplified fragment length
polymorphism (AFLP) data. Bootstrap supports are indicated above branches based on 1000 bootstrap
replicates
157Characterization of North American Armillaria spp.
definitive relationships among the geographical lineages remain undetermined (Coetzee
et al. 2000). The previous analysis of A. mellea also showed well-supported clades among
Europe, Asia and North America (Coetzee et al. 2000). In our study, three isolates of
A. mellea derived from eastern/mid-western North America (Table 1) also showed
divergent nLSU sequences (Fig. 4).
It has been proposed that the A. ostoyae–A. gemina group is closely related, but
divergence has occurred (Anderson and Stasovski 1992; Piercey-Normore et al.
1998). Piercey-Normore et al. (1998) indicated the position of A. gemina as ancestral
to A. ostoyae, but other studies have suggested that A. gemina was sympatrically
derived from A. ostoyae (Anderson et al. 1989; Anderson and Stasovski 1992).
Previous studies also showed similar nuclear DNA content for A. ostoyae and
A. gemina (Kim et al. 2000). Our results confirmed previous studies that these two
species are very closely related, but the phylogenetic relationships between them remain
unresolved (Figs 2–4). Based on IGS-1 sequence analyses, a heterogeneous combination
of sequence types was present in one A. gemina isolate (ST9) (Fig. 2): one sequence
type (ST9-A) from the A. gemina isolate ST9 clustered with A. gemina genets (ST8-A,
ST8-B and ST11) and the other sequence type (ST9-B) was clustered with A. ostoyae
genets (ST1, ST2-A, ST2-B and P1404). This possible hybridization between A. ostoyae
and A. gemina is strongly supported by both phylogenetic analyses (posterior
probability 100%, bootstrap support 100%) (Fig. 2). However, a hybridization
signature is not readily apparent in other rDNA regions because differences are
insufficient to allow meaningful comparisons between A. gemina and A. ostoyae. All
A. ostoyae isolates had 12 AFLP markers that were not shared by A. gemina isolates
ST8 and ST11, and A. gemina isolates ST8 and ST11 had 18 AFLP markers that were
not shared by any of the A. ostoyae isolates. Of these AFLP markers, A. gemina isolate
ST9 shared one A. ostoyae marker and 16 A. gemina markers. These results may reflect
a common ancestry of A. ostoyae and A. gemina. A polymorphism may have originated
in the IGS region of A. gemina, and evolutionary processes may have rendered this
region homogeneous in some lineages and heterogeneous in other lineages. Alternat-
ively, A. gemina isolate ST9 may be derived from a past hybridization event, followed
by introgression with A. gemina.
The close phylogenetic relationships hypothesized by Anderson and Stasovski (1992)
and Piercey-Normore et al. (1998) among A. calvescens,A. sinapina,A. gallica,
A. nabsnona, NABS X and A. cepistipes were supported by this study. However, we
found support for a more distinct separation of A. nabsnona from these species based on
sequence data from all three rDNA regions (Figs 2–4). Kim et al. (2000) reported that the
nuclear content of A. nabsnona (0.133 pg per nucleus) is significantly smaller than A.
calvescens (0.153 pg per nucleus), A. sinapina (0.150 pg per nucleus), A. gallica (0.168 pg
per nucleus), NABS X (0.152 pg per nucleus), and A. cepistipes (0.152 pg per nucleus),
while it is larger than A. ostoyae (0.113 pg per nucleus), A. gemina (0.120 pg per nucleus),
and A. mellea (0.122 pg per nucleus). Of additional interest is that A. gallica (0.168 pg per
nucleus) was previously shown to have a nuclear DNA content that was significantly larger
than other North American Armillaria species (Kim et al. 2000), thereby providing
evidence that A. gallica is a very distinct species in spite of any genetic relationships to
other species.
One striking example of interspecific relationships is evident in isolates of NABS X,
which contained heterogeneous sequence types within the ITS + 5.8S (Fig. 3). For each
individual of NABS X tested, one sequence type (837-A, D82-A and POR-100A) clustered
with A. nabsnona, while the other type (837-B, D82-B and POR-100B) remained separate
from A. nabsnona (Fig. 3). However, IGS-1 sequence data showed that NABS X grouped
with A. calvescens,A. sinapina,A. gallica and A. cepistipes (Fig. 2).
158 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
Of particular interest is that NABS X (0.152 pg per nucleus) has an intermediate nuclear
DNA content between A. gallica (0.168 pg per nucleus) and A. nabsnona (0.133 pg per
nucleus) (Kim et al. 2000). All A. gallica isolates had 11 AFLP markers that were not shared
by any A. nabsnona isolates, and all A. nabsnona isolates had 16 AFLP markers that were
not shared by any A. gallica isolates. Of these AFLP markers, NABS X shared 55% (six of
11) A. gallica markers and 25% (four of 16) A. nabsnona markers. The combined evidence
from ITS + 5.8S sequences, IGS-1 sequences and AFLP markers suggests that NABS X
may have originated by hybridization, although apparent phylogenetic implications differ
according to rDNA regions. It is of additional interest to note that A. calvescens (0.153 pg
per nucleus), A. sinapina (0.150 pg per nucleus) and A. cepistipes (0.152 pg per nucleus) also
have intermediate nuclear DNA content (Kim et al. 2000). Of markers that distinguish A.
gallica from A. nabsnona,A. calvescens shared 46% (five of 11) A. gallica markers and 6%
(one of 16) A. nabsnona markers; A. sinapina shared 27% A. gallica markers and 6%
A. nabsnona markers; and A. cepistipes shared 73% A. gallica markers and 25% A.
nabsnona markers. More studies are needed to determine the potential role of hybridiza-
tion in the creation and evolution of Armillaria species. It has been previously
hypothesized that narrowly distributed A. nabsnona and NABS X are derived from more
widely distributed A. sinapina (Piercey-Normore et al. 1998); however, this hypothesis is
not supported by our present DNA and AFLP data. Hybridization events among A.
calvescens,A. sinapina,A. gallica,A. nabsnona, NABS X and A. cepistipes are difficult to
confirm because (i) these species have common ancestry, (ii) the species are genetically
closely related, (iii) hybridization events may have occurred in the distant past and derived
organisms have likely undergone selection pressure and introgression, and/or (iv) the
hybridization events almost certainly involved isolates that were not included in this study.
Analyses of genetic relationships is further complicated by potential hybridization and
gene flow among closely related Armillaria species. A previous study used a combination of
molecular tools (PCR-RFLP and flow cytometry) to confirm interspecific mating in culture
among biological species of Armillaria (A. cepistipes,A. sinapina and NABS X) (Kim et al.
2001). Heterogeneous rDNA sequences from individual genets also suggest that hybrid-
ization may occur in nature. Thus, limited gene flow may occur among closely related
Armillaria species, and thereby cloud resolution of phylogenetic and genetic relationships.
Hybridization between pathogens is currently a major concern due to the potential for
hybrid individuals to possess new pathogenic traits (Brasier 2000); however, more studies
are needed to fully understand the impacts of hybridization among Armillaria species.
Repetitive DNA sequences like rDNA gene families are known to represent the result of
concerted evolution (Elder and Turner 1995). However, our study showed that
intraspecific homogenization of rDNA sequences is not the rule among the Armillaria
species that we tested. Heterogeneous PCR products indicating intraspecific and
intragenomic variations were common in all regions analysed, with the exception of the
5.8S rDNA. Direct PCR has been shown to detect 90% of the heterogeneous rDNA
products in an individual and the relative peak height seems to reflect relative
concentrations (Rauscher et al. 2002). This heterogeneity of IGS and ITS2 regions within
single genomes or among strains has been reported in several groups of fungi (Martin
1990; O’Donnell 1992; Appel and Gordon 1996; O’Donnell and Cigelnik 1997;
Kauserud and Schumacher 2003). Currently, studies of fungal evolution are beginning to
focus on protein-coding genes, such as elongation factor 1-a,b-tubulin, ribosomal
polymerase B, mitochrondrial ATPase 6, mating-type genes, and chitin synthase genes, due
to ease of alignment (Bruns and Shefferson 2004) and lack of heterogeneity. However,
these single-copy genes are not as conserved as rDNA genes because selection is on the
translated gene product, rather than the gene itself (Bruns and Shefferson 2004). Bruns
and Shefferson (2004) suggested that structural ribosomal genes, such as nLSU rDNA,
can be very useful for identification at high taxonomic levels due to a huge and growing
159Characterization of North American Armillaria spp.
data set and protein-coding genes, such as mating type and synthase genes, hold great
promise at lower taxonomic levels within fungi.
Sequences and variation of rDNA regions potentially contain very powerful phylo-
genetic information, which is typically unreported or underutilized. For precise phylo-
genetic analysis, it is essential that DNA sequence information is determined as accurately
as possible. In this study, we maximized the phylogenetic signal from rDNA sequences
through vigilant sequence editing, techniques to decipher heterogeneity within individuals,
and elimination of ambiguity within data sets (Hanna 2005).
Compared with rDNA sequence information, AFLP marker data provided generally
strong support for intraspecies grouping of Armillaria species. Genetic analyses of AFLP
data from our selected isolates support the currently defined Armillaria species, based
largely on the biological species concept. In addition, our data suggest that NABS X should
be considered for elevation to formal species status. AFLP data can be used to discriminate
among very closely related Armillaria species (i.e. A. calvescens,A. sinapina,A. gallica and
A. cepistipes) that were difficult to identify using IGS-based PCR-RFLP methods
(Harrington and Wingfield 1995; Kim et al. 2000). The increased resolution of AFLP-
based analyses is expected because AFLP markers allow for simultaneous screening of
multiple loci within the entire genome and reflect multiple sites within the genome that
might be associated with phenotypes, pathogenicity, environmental adaptation, etc. In
contrast, sequencing of one or more regions within the genome can provide higher order
phylogenies of populations by comparing the evolutionary rates of individual genotypes.
The results from this study suggest that AFLP data can be used to discriminate among 10
NABS of Armillaria. Several AFLP genetic markers offer potential for distinguishing
currently available NABS of Armillaria in future biological, ecological and taxonomic
studies. Unique loci associated with each North American Armillaria species could be
sequenced and used to develop primers for identifying biological species. However, more
AFLP markers are needed to develop species-specific probes for discriminating between
A. calvescens and A. gallica because of their close genetic relationship. To our knowledge,
this is the first study to apply AFLP data for analysis of North American Armillaria species
in comparison with data inferred from rDNA sequences.
An overriding goal of this study was to further characterize a set of diploid tester strains
(Kim et al. 2000) for use in future studies to help characterize additional isolates of
Armillaria. Our study provides the foundation to examine further questions about
intraspecific variation, global variation, interspecific relationships, intraspecific relation-
ships, etc. Such efforts should contribute to a better understanding of biological, ecological
and taxonomic relationships among Armillaria species.
Acknowledgements
This paper was supported by the USDA Forest Service, RMRS, RM-4552 (Microbial Processes as
Ecosystem Regulators in Western Forests) and the Sustainable Forestry component of Agenda 2020, a
joint effort of the USDA Forest Service Research & Development and the American Forest & Paper
Association with Forest Capital (formerly Boise Corporation) as a research partner. We thank Dr Zaid
Abdo, Bryce A. Richardson, and Jonalea R. Tonn for their comments on an earlier version of this
manuscript. We also thank Jane E. Stewart, James B. Donley and Joanne M. Tirocke for their technical
assistance. Use of trade names in this paper does not constitute endorsement by the USDA Forest Service.
Re
´sume
´
Caracte
´risation des espe`ces dÕArmillaria d’Ame
´rique du Nord: relations ge
´ne
´tiques de
´termine
´es a` partir
des se
´quences de l’ADN ribosomal et de marqueurs AFLP
Les relations phyloge´ne´tiques et ge´ne´tiques entre 10 espe`ces nord-ame´ ricaines dÕArmillaria ont e´te´
analyse´es a` partir d’une part de donne´es de se´quences de l’ADN ribosomal (ADNr), incluant
l’espaceur inter-ge´nique (IGS-1), les espaceurs internes transcrits et le 5.8S associe´ (ITS + 5.8S), et
160 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
l’ADNr nucle´aire de la grande sous-unite´ (nLSU), d’autre part de marqueurs AFLP. D’apre`s les
donne´es de se´quence de l’ADNr, la re´ gion nLSU est moins variable entre espe` ces dÕArmillaria que les
re´gions ITS + 5.8S et IGS-1 (nLSU < ITS + 5.8S < IGS-1). Les analyses phyloge´ne´ tiques base´ es sur
les se´quences de l’ADNr sugge`rent que A. mellea,A. tabescens,etA. nabsnona sont bien se´ pare´ es des
autres espe`ces dÕArmillaria (A. ostoyae,A. gemina,A. calvescens,A. sinapina,A. gallica, NABS X, et
A. cepistipes). Plusieurs espe`ces dÕArmillaria (A. calvescens,A. sinapina,A. gallica, NABS X, et
A. cepistipes) se trouvent regroupe´es avec l’utilisation de ces se´ quences. En se basant sur les isolats
utilise´s dans cette e´tude, il apparaıˆt donc que les techniques base´ es sur l’IGS-1, ITS + 5.8S, et/ou le
domaine D/extre´mite´3¢de nLSU sont insuffisantes pour distinguer A. calvescens,A. sinapina,
A. gallica,etA. cepistipes. Toutefois, les donne´es d’AFLP ont permis de diffe´ rencier ces espe` ces et
l’analyse AFLP est cohe´rente avec la classification taxonomique e´tablie a` partir des me´ thodes
conventionnelles (morphologie et tests d’interfertilite´). Nos re´sultats montrent l’inte´reˆ t des marqueurs
AFLP pour distinguer les diffe´rentes espe`ces biologiques nord-ame´ricaines d’Armilllaria pour des
e´tudes de biologie, dÕe´cologie et de taxonomie.
Zusammenfassung
Charakterisierung von nordamerikanischen Armillaria-Arten: Bestimmung der genetischen
Beziehungen anhand von ribosomalen rDNA-Sequenzen und AFLP-Markern
Die phylogenetischen und genetischen Beziehungen zwischen zehn nordamerikanischen Armillaria-
Arten wurden mit Hilfe von Sequenzdaten der ribosomalen DNA (rDNA), welche die Regionen
IGS-1, ITS + 5,8 S und nLSU einschliesst, sowie mit AFLP-Markern analysiert. Die Sequenzdaten
ergaben, dass die nLSU-Region bei Armillaria-Arten weniger stark variiert als die ITS + 5,8S sowie
die IGS-1 Regionen (nLSU < ITS + 5,8 S <IGS-1). Aus den phylogenetischen Analysen der rDNA-
Sequenzen ergab sich eine klare Trennung von A. mellea,A. tabescens und A. nabsnona von den
u
¨brigen Arten (A. ostoyae,A. gemina,A. calvescens,A. sinapina,A. gallica, NABS X und A.
cepistipes). Verschiedene Arten (A. calvescens,A. sinapina,A. gallica, NABS X und A. cepistipes)
bildeten aufgrund ihrer rDNA-Sequenzen eine Gruppe. Am untersuchten Material zeigte sich, dass
Techniken, die auf IGS1, ITS + 5,8 S und/oder D-Domain/3¢-Enden der nLSU basieren, nicht
zuverla
¨ssig zwischen A. calvescens,A. sinapina,A. gallica und A. cepistipes unterscheiden ko
¨nnen. Im
Gegensatz dazu konnten diese Arten mit AFLP-Markern voneinander abgegrenzt werden und die
Analyse der AFLP-Daten besta
¨tigte die taxonomische Klassifikation aufgrund konventioneller
Methoden (Morphologie und Interfertilita
¨tstests). Die Befunde zeigen, dass AFLP-Marker zur
Unterscheidung der derzeit akzeptierten nordamerikanischen biologischen Arten von Armillaria
sowie fu
¨r zuku
¨nftige biologische, o
¨kologische und taxonomische Untersuchungen eingesetzt werden
ko
¨nnen.
References
Anderson, J. B.;Kohn, L. M., 1995: Clonality in soilborne, plant-pathogenic fungi. Annu. Rev.
Phytopathol. 33, 369–391.
Anderson, J. B.;Stasovski, E., 1992: Molecular phylogeny of Northern Hemisphere species of
Armillaria. Mycologia 84, 506–516.
Anderson, J. B.;Ullrich, R. C., 1979: Biological species of Armillaria mellea in North America.
Mycologia 71, 402–414.
Anderson, J. B.;Petsche, D. M.;Smith, M. L., 1987: Restriction fragment polymorphisms in
biological species of Armillaria mellea. Mycologia 79, 69–76.
Anderson, J. B.;Bailey, S. S.;Pukkila, P. J., 1989: Variation in ribosomal DNA of Armillaria,a
genus of root-infecting fungi. Evolution 43, 1652–1662.
Appel, D. J.;Gordon, T. R., 1996: Relationships among pathogenic and nonpathogenic isolates of
Fusarium oxysporum based on the partial sequence of the intergenic spacer region of the ribosomal
DNA. Mol. Plant Microbe Interact. 9, 125–138.
Banik, M. T.;Volk, T. J.;Burdsall, H. H., Jr, 1996: Armillaria species of the Olympic Peninsula of
Washington state, including confirmation of North American biological species XI. Mycologia 88,
492–496.
Be
´rube
´,J.A.;Dessureault, M., 1988: Morphological characterization of Armillaria sinapina sp. nov.
Can. J. Bot. 66, 2027–2034.
Brasier, C. M., 2000: The rise of hybrid fungi. Nature 405, 134–135.
Bruns, T. D.;Shefferson, R. P., 2004: Evolutionary studies of ectomycorrhizal fungi: recent advances
and future directions. Can. J. Bot. 82, 1122–1132.
161Characterization of North American Armillaria spp.
Chillali, M.;Wipf, D.;Guillaumin, J.-J.;Mohammed, C.;Botton, B., 1998: Delineation of the
European Armillaria species based on the sequences of the internal transcribed spacer (ITS) of
ribosomal DNA. New Phytol. 138, 553–561.
Coetzee, M. P. A.;Wingfield, B. D.;Harrington, T. C.;Dalevi, D.;Coutinho, T. A.;
Wingfield, M. J., 2000: Geographic diversity of Armillaria mellea s.s. based on phylogenetic
analysis. Mycologia 92, 105–113.
Coetzee, M. P. A.;Wingfield, B. D.;Bloomer, P.;Ridley, G. S.;Kile, G. A.;Wingfield, M. J.,
2001: Phylogenetic relationships of Australian and New Zealand Armillaria species. Mycologia 93,
887–896.
Coetzee, M. P. A.;Wingfield, B. D.;Bloomer, P.;Ridley, G. S.;Wingfield, M. J., 2003:
Molecular identification and phylogeny of Armillaria isolates from South America and Indo-
Malaysia. Mycologia 95, 285–293.
Crease, T. J.;Lynch, M., 1991: Ribosomal DNA variation in Daphnia pulex. Mol. Biol. Evol. 8, 620–
640.
Duchesne, L. C.;Anderson, J. B., 1990: Location and direction of transcription of the 5S rRNA gene
in Armillaria. Mycol. Res. 94, 266–269.
Elder, J. F., Jr; Turner, B. J., 1995: Concerted evolution of repetitive DNA sequences in eukaryotes.
Q. Rev. Biol. 70, 297–320.
Eriksson, T.;Hibbs, M. S.;Yoder, A. D.;Delwiche, C. F.;Donoghue, M. J., 2003: The phylogeny
of Rosoideae (Rosaceae) based on sequences of the internal transcribed spacers (ITS) of the nuclear
ribosomal DNA and the TRNL/F region of chloroplast DNA. Int. J. Plant Sci. 164, 197–211.
Fell, J. W.;Boekhout, T.;Fonseca, A.;Scorzetti, G.;Tallman, A., 2000: Biodiversity and
systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain
sequence analysis. Int. J. Syst. Evol. Microbiol. 50, 1351–1371.
Felsenstein, J., 1985: Confidence limits on phylogenies: an approach using the bootstrap. Evolution
39, 783–791.
Gardes, M.;Bruns, T. D., 1993: ITS primers with enhanced specificity for basidiomycetes-application
to the identification of mycorrhizae and rust. Mol. Ecol. 2, 113–118.
Gezahgne, A.;Coetzee, M. P. A.;Wingfield, B. D.;Wingfield, M. J.;Roux, J., 2004:
Identification of the Armillaria root rot pathogen in Ethiopian plantations. For. Pathol. 34, 133–
145.
Hall, T. A., 1999: BioEdit: a user-friendly biological sequence alignment editor and analysis program
for Windows 95/98/NT. Nucleic Acids. Symp. Ser. 41, 95–98.
Hall, G. H., 2001: Phylogenetic Trees Made Easy: a How-to Manual for Molecular Biologists.
Sunderland, MA: Sinauer Associates, Inc., 164 pp.
Hanna, J. W., 2005: Armillaria ostoyae: genetic characterization and distribution in the western
United States. MS Thesis, University of Idaho, Moscow, ID, 116 pp.
Harrington, T. C.;Wingfield, B. D., 1995: A PCR-based identification method for species of
Armillaria. Mycologia 87, 280–288.
Hasegawa, M.;Kishino, H.;Yano, T., 1985: Dating of the human-ape splitting by a molecular clock
of mitochondrial DNA. J. Mol. Evol. 22, 160–174.
Hintikka, V., 1973: A note on the polarity of Armillariella mellea. Karstenia 13, 32–39.
Hopple, J. S.,Jr;Vilgalys, R., 1999: Phylogenetic relationships in the mushroom genus Coprinus and
dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal
subunit RNA: divergent domains, outgroups and monophyly. Mol. Phylogenet. Evol. 13, 1–19.
Huelsenbeck, J. P.;Ronquist, F., 2001: MrBayes: Bayesian inference of phylogenetic trees.
Biometrics 17, 754–755.
Jahnke, K. D.;Bahnweg, G.;Worrall, J. J., 1987: Species delimitation in the Armillaria mellea
complex by analysis of nuclear and mitochondrial DNAs. Trans. Br. Mycol. Soc. 88, 572–575.
Kauserud, H.;Schumacher, T., 2003: Genetic structure of Fennoscandian populations of the
threatened wood-decay fungus Fomitopsis rosea. Mycol. Res. 107, 155–163.
Kim, M.-S.;Klopfenstein, N. B.;McDonald, G. I.;Arumuganathan, K.;Vidaver, A. K., 2000:
Characterization of North American Armillaria species by nuclear DNA content and RFLP
analysis. Mycologia 92, 874–883.
Kim, M.-S.;Klopfenstein, N. B.;McDonald, G. I.;Arumuganathan, K.;Vidaver, A. K., 2001:
Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific
and interspecific matings of Armillaria species. Mycol. Res. 105, 153–163.
Korhonen, K., 1978: Infertility and clonal size in the Armillaria complex. Karstenia 18, 31–42.
Korhonen, K., 1995: Armillaria since Elias Fries. Symb. Bot. Upsal. 30, 153–161.
162 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
Kuzoff, R. K.;Sweere, J. A.;Soltis, D. E.;Soltis, P. S.;Zimmer, E. A., 1998: The phylogenetic
potential of entire 26S rDNA sequences in plants. Mol. Biol. Evol. 15, 251–263.
Martin, F. N., 1990: Variation in the ribosomal DNA repeat unit within single-oospore isolates of the
genus Pythium. Genome 33, 585–591.
Michot, B.;Hassouna, N.;Bachellerie, J., 1984: Secondary structure of mouse 28S rRNA and a
general model for the folding of the large rRNA in eukaryotes. Nucleic Acids Res. 12, 4259–4279.
Michot, B.;Qu, H.-L.;Bachellerie, J. P., 1990: Evolution of large-subunit ribosomal RNA
structure: the diversification of divergent D3 domain among major phylogenetic groups. Eur. J.
Biochem. 188, 219–230.
Miller, O. K.;Johnson, J. L.;Burdsall, H. H.;Flynn, T., 1994: Species delimitation in North
American species of Armillaria as measured by DNA reassociation. Mycol. Res. 98, 1005–1011.
Moncalvo, J.-M.;Lutzoni, F. M.;Rehner, S. A.;Johnson, J.;Vilgalys, R., 2000: Phylogenetic
relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Syst. Biol.
49, 278–305.
Moncalvo, J.-M.;Vilgalys, R.;Redhead, S. A.;Johnson, J. E.;James, T. Y.;Aime, M. C.;
Hofstetter, V.;Verduin, S. J. W.;Larsson, E.;Baroni, T. J.;Thorn, R. G.;Jacobsson, S.;
Clemencon, H.;Miller, O. K.,Jr, 2002: One hundred and seventeen clades of euagarics. Mol.
Phylogenet. Evol. 23, 357–400.
Motta, J. J.;Korhonen, K., 1986: A note on Armillaria mellea and Armillaria bulbosa. Mycologia 78,
963–965.
Nei, M.;Li, W. H., 1979: Mathematical model for studying genetic variation in terms of restriction
endonucleases. Proc. Natl. Acad. Sci. U.S.A. 76, 5269–5273.
Nylander, J. A. A., 2003: MrModeltest 1.0b. A Simplified Version of David Posada’s ÔÔModeltest
3.06‘‘. Uppsala, Sweden: Department of Systematic Zoology, http://www.ebc.uu.se/systzoo/staff/
nylander.html.
O’Donnell, K., 1992: Ribosomal DNA internal transcribed spacers are highly divergent in the
phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris). Curr. Genet. 22, 213–
220.
O’Donnell, K.;Cigelnik, E., 1997: Two divergent intragenomic rDNA ITS2 types within a
monophyletic lineage of the fungus Fusarium are nonorthologous. Mol. Phylogenet. Evol. 7, 103–
116.
Otieno, W.;Pe
´rez-Sierra, A.;Termorshuizen, A., 2003: Characterization of Armillaria isolates
from tea (Camellia sinensis) in Kenya. Mycologia 95, 160–175.
Pe
´rez-Sierra, A.;Guillaumin, J.-J.;Spooner, B. M.;Bridge, P. D., 2004: Characterization of
Armillaria heimii from Africa. Plant Pathol. 53, 220–230.
Piercey-Normore, M. D.;Egger, K. N.;Be
´rube
´,J.A., 1998: Molecular phylogeny and evolutionary
divergence of North American Biological Species of Armillaria. Mol. Phylogenet. Evol. 10, 49–66.
Rauscher, J. T.;Doyle, J. J.;Brown, H. D., 2002: Internal transcribed spacer repeat-specific primers
and the analysis of hybridization in the Glycine tomentella (Leguminosae) polyploid complex. Mol.
Ecol. 11, 2691–2702.
Saitou, N.;Nei, M., 1987: The neighbor-joining method: a new method for reconstructing
phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
Schulze, S.;Bahnweg, G.;Tesche, M.;Sandermann, H.,Jr, 1995: Identification of European
Armillaria species by restriction-fragment-length polymorphisms of ribosomal DNA. Eur. J. For.
Pathol. 25, 214–223.
Schulze, S.;Bahnweg, G.;Mo¨ ller, E. M.;Sandermann, H.,Jr, 1997: Identification of genus
Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation
within A. ostoyae by rDNA-RFLP and RAPD analysis. Eur. J. For. Pathol. 27, 225–239.
Shaw, C. G., III; Kile, G. A., 1991: Armillaria Root Disease. USDA Forest Service, Agriculture
Handbook No. 691, 233 pp.
Simmons, M. P.;Ochoterena, H., 2000: Gaps as characters in sequence-based phylogenetic analyses.
Syst. Biol. 49, 369–381.
Smith, M. L.;Anderson, J. B., 1989: Restriction fragment length polymorphisms in mitochondrial
DNA’s of Armillaria: identification of North American biological species. Mycol. Res. 93, 247–
256.
Smith, M. L.;Duchesne, L. C.;Bruhn, J. N.;Anderson, J. B., 1990: Mitochondrial genetics in a
natural population of the plant pathogen Armillaria. Genetics 126, 575–582.
Swofford, D. L., 2001: PAUP*: Phylogenetic Analysis Using Parsimony and Other Methods
(PAUP* version 4.0 beta 10). Sunderland, MA: Sinauer.
163Characterization of North American Armillaria spp.
Terashima, K.;Cha, J. Y.;Yajima, T.;Igarashi, T.;Miura, K., 1998: Phylogenetic analysis of
Japanese Armillaria based on the intergenic spacer (IGS) sequences of their ribosomal DNA. Eur. J.
For. Pathol. 28, 11–19.
Termorshuizen, A. J.;Arnolds, E. J. M., 1987: On the nomenclature of the European species of
Armillaria mellea group. Mycotaxon 30, 101–116.
Termorshuizen, A. J.;Arnolds, E. J. M., 1997: Compatibility groups, species concepts and
nomenclature in European Armillaria species. Mycotaxon 65, 263–272.
Veldman, G. M.;Klootwijk, J.;de Regt, V. C. H. F.;Rudi, J., 1981: The primary and secondary
structure of yeast 26S rRNA. Nucleic Acids Res. 9, 6935–6952.
Volk, T. J.;Burdsall, H. H.,Jr, 1995: A Nomenclatural Study of Armillaria and Armillariella
Species (Basidiomycotina, Tricholomataceae). Synopsis Fungorum 8, Oslo, Norway, 121 pp.
Volk, T. J.;Burdsall, H. H.,Jr;Banik, M. T., 1996: Armillaria nabsnona, a new species from
western North America. Mycologia 88, 484–491.
Vos, P.;Hogers, R.;Bleeker, M.;Reijans, M.;de Lee, T.;Hornes, M.;Frijters, A.;Pot, J.;
Peleman, J.;Kuiper, M.;Zabeau, M., 1995: AFLP: a new technique for DNA fingerprinting.
Nucleic Acids Res. 23, 4407–4414.
Weiß, M.;Oberwinkler, F., 2001: Phylogenetic relationships in Auriculariales and related groups –
hypotheses derived from nuclear ribosomal DNA sequences. Mycol. Res. 105, 403–415.
White, T. J.;Bruns, T. D.;Lee, S. B.;Taylor, J. W., 1990: Analysis of phylogenic relationships by
amplification and direct sequencing of ribosomal DNA genes. In: PCR Protocols: a Guide to
Methods and Applications. Ed. by Innis, M. A.;Gelfand, D. H.;Sninsky, J. J.;White, T. J. New
York: Academic Press, pp. 315–322.
White, E. E.;Dubetz, C. P.;Cruickshank, M. G.;Morrison, D. J., 1998: DNA diagnostic for
Armillaria species in British Columbia: within and between species variation in the IGS-1 and IGS-
2 regions. Mycologia 90, 125–131.
Yang, Z., 1993: Maximum likelihood estimation of phylogeny from DNA sequences when
substitution rates differ over sites. Mol. Biol. Evol. 10, 1396–1402.
164 M.-S. Kim, N. B. Klopfenstein, J. W. Hanna and G. I. McDonald
