Interactions between multiple fungi isolated from two bark beetles, Dendroctonus brevicomis and D. frontalis (Coleoptera: Curculionidae)

Full text

Journal of Yeast and Fungal Research Vol. 1(7). pp. 118 - 126, September, 2010
Available online http://www.academicjournals.org/JYFR
ISSN 2141-2413 © 2010 Academic Journals
Full Length Research Paper
Interactions between multiple fungi isolated from two
bark beetles, Dendroctonus brevicomis and
Dendroctonus frontalis (Coleoptera: Curculionidae)
Thomas S. Davis
1
, Richard W. Hofstetter
1
, Kier D. Klepzig
2
, Jeffrey T. Foster
3
and Paul Keim
3
1
Northern Arizona University, School of Forestry, P. O. BOX 15018, Flagstaff, Arizona, USA, 86011.
2
USDA Forest Service, Southern Research Station, Pineville, Louisiana, USA.
3
Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, USA.
Accepted 29 June, 2010
Antagonism between the fungal symbionts of bark beetles may represent a biologically significant
interaction when multiple beetle species co-occur in a host tree. Since high density bark beetle
populations rapidly and dramatically shift forest characteristics, patterns of competition between the
obligate fungal associates of sympatric bark beetle species may have broad ecological effects. Primary
and competitive resource acquisition between allopatric and sympatric isolates of mutualist fungi
associated with the bark beetles Dendroctonus frontalis and Dendroctonus brevicomis were
investigated. Growth assays at multiple temperatures suggest that primary resource acquisition by
fungi growing in the absence of competitors varies regionally, and that optimal growth rate is likely to
correspond to average summertime maximum temperatures. In competition assays, interactions were
asymmetric between fungi isolated from sympatric beetle populations and fungi isolated from allopatric
beetle populations: sympatric isolates out-competed allopatric isolates. However, competition between
fungi from beetle populations in sympatry was found to be equal. These studies are the first to
investigate interactions between the mycangial fungi of multiple Dendroctonus species, and the results
suggest that competition is likely to occur when the mycangial fungi of multiple beetle species occur
together.
Key words: Allopatric, competition, coexistence, mutualism, mutualist, mycangial fungi, sympatric.
INTRODUCTION
In many ecosystems, symbiotic associations are
ubiquitous (Bronstein, 1994). For species that co-evolve
with symbionts, interaction with symbionts is strongly
correlated with population performance (e.g. fig/fig wasp,
yucca/ yucca moth, ants/myrmecophytes, and
beetle/fungus mutualisms; Bronstein, 1992; Huth and
Pellmyr, 1997; Klepzig et al., 2001; Palmer et al., 2003);
however, little research has investigated competitive
symmetry among species with multiple symbionts
(Palmer et al., 2003). Dendroctonus beetles (Coleoptera:
Curculionidae) represent a useful system for studying
*Corresponding author. E-mail: tsd3@nau.edu. Tel: 928-523-
6452. Fax: 928-523-1080.
interactions between symbiotic species (Six and Klepzig,
2004). Dendroctonus beetles associate with an extensive
community of microorganisms, including mites, nema-
todes, fungi, yeasts, and bacteria (Whitney, 1982; Klepzig
et al., 2001; Kenis et al., 2004; Kirisits, 2004; Scott et al.,
2008). The composition and abundance of these micro-
bial communities considerably impact beetle population
dynamics (Bridges, 1983; Paine et al., 1997; Hofstetter et
al., 2006), and previous studies have cited a need for
investigating interactions among the microbial associates
of Dendroctonus beetles (Klepzig and Wilkens, 1997;
Harrington, 2005).
Dendroctonus species construct tunnels in the vascular
tissue of host conifers in order to lay eggs (Wood, 1982).
During excavation, tunnels are inoculated with fungal
symbionts that grow throughout host tissues and deve-

loping beetle larvae feed upon them (Harrington, 2005).
In most cases Dendroctonus species are allopatric or
colonize different tree species (Wood, 1982; Lieutier et
al., 2004), so the opportunity for antagonism among
fungal symbionts are avoided during larval development
(Schlyter and Anderbrandt, 1993). However, in the
ponderosa pine (Pinus ponderosa var brachyptera)
forests of Southwestern North America, Dendroctonus
brevicomis LeConte and Dendroctonus frontalis
Zimmerman have been reported to co-colonize tissues of
ponderosa pine (Pinus ponderosa) and with no apparent
negative impacts on the fitness or fecundity of either
beetle species (Davis and Hofstetter, 2009).
For D. brevicomis and D. frontalis, fungal symbionts in
the genera Ceratocystiopsis and Entomocorticium confer
important benefits to developing larvae (Klepzig and
Wilkens, 1997; Hsiau and Harrington, 1997). For exam-
le, the presence of these filamentous fungi in feeding
chambers is correlated with adult beetle size, fecundity,
and nitrogen content (Bridges, 1983; Coppedge et al.,
1995; Ayres et al., 2000). Also, beetles have evolved
glandular structures (termed ‘mycangia’) that are used for
transporting these fungi between host trees (Barras and
Perry, 1971; Hsiau and Harrington, 1997; Yuceer et al.,
2010). Due to the prevalence of association and inter-
dependence of mycangial fungi and multiple Dendroc-
tonus species, the beetle-fungal relationship is often
considered a mutualism (Six, 2003). However, the sign
(+, −) of interaction between multiple fungal symbionts of
sympatric Dendroctonus species is unknown. But,
interactions between fungal species that co-inhabit a
niche have been shown to be antagonistic in many
natural systems (Klepzig and Wilkens, 1997; Yuen et al.,
1999; Murphy and Mitchell, 2001; Klepzig, 2006; Boddy,
2007; Licyayo et al., 2007). Uncolonized pine vascular
tissue is a limiting resource for mycangial fungi; this
should create an interface for antagonism between fungal
mutualists when multiple beetle species co-colonize a
host. Among sympatric Dendroctonus species that co-
inhabit host tissue, competition between fungal symbionts
of beetles may represent an interaction that limits beetle
fitness or population growth.
Here, the authors report on patterns of primary and
competitive resource acquisition by mycangial fungi of
sympatric and allopatric Dendroctonus beetles. Through-
out this report, “primary resource acquisition” is defined
as the rate at which fungal isolates acquired resource
area in the absence of competitors. “Competitive
resource acquisition” is defined as the average proportion
of trials in which individual fungal isolates acquired
resource space that was occupied by another fungal
isolate. The authors experimentally investigated the
effects of temperature and biotic interactions on growth
patterns of 33 isolates of mutualistic mycangial fungi
associated with D. brevicomis and D. frontalis, from both
sympatric and allopatric beetle populations. We ask two
questions: (1) Does primary resource acquisition by
mycangial isolates vary by fungal species, beetle species, or
Davis et al. 119
beetle populations? (2) Does the symmetry of competitive
interactions by mycangial isolates vary with sympatry or
allopatry of beetle populations?
MATERIALS AND METHODS
Beetle collection and acquisition of fungal isolates
Bark beetles used for fungal isolation were collected from three
locations: Coconino National Forest in Arizona, U.S.A., Homochitto
Ranger District National Forests in Mississippi, U.S.A., and Plumas
National Forest in California, U.S.A. (Figure 1). In Arizona,
populations of D. frontalis and D. brevicomis occur in sympatry and
co-colonize ponderosa pine (Pinus ponderosa var brachyptera). In
Mississippi only D. frontalis occurs and colonizes multiple pine
species. In California only D. brevicomis occurs and colonizes P.
ponderosa var benthamiana. There is moderate climatic variability
between the three forests in terms of both annual mean
precipitation and maximum temperature. On the Coconino National
Forest annual precipitation averages 44.7 cm per year and mean
summer maximum temperature is 25°C (Hereford, 2007), on the
Homochitto National Forest annual precipitation averages 162.5 cm
per year and mean summer maximum temperature is 30°C
(Southern Regional Climate Center), and on the Plumas National
Forest annual precipitation averages 38.8 cm per year and mean
summer maximum temperature is 29.6°C (Western Regional
Climate Center).
To obtain mycangial fungi, live beetles were trapped in the field
using Lindgren funnel traps baited with pine beetle pheromone
lures containing frontalin, exo-brevicomin, and -pinene (Synergy
Semiochemicals Corp, Lot No. WPP10416). Beetles were placed
individually into clear, size 0 gelatin capsules (Torpac, Lot No.
1100049271), and stored in the lab in dark environmental chambers
at 5°C until used for the isolation of fungi. All insects specimens
used for microbial isolations mentioned in this study were collected
between May 18, 2007 and July 18, 2007. Healthy female beetles
were dissected and the thorax removed. Each thorax was surface
sterilized using HgCl
2
and de-ionized water described by (Kopper et
al., 2004) and then split dorsoventrally and placed in 2% malt
extract media (Malt extract – MP Biomedicals LLC, Lot No. 6753J;
Agar – BioServ, Lot No. 1740.01). The pH of the media was 4.7 ±
0.2 according to manufacturer specifications. Malt extract media
(2%) and 95 × 15 mm Petri dishes (Fisherbrand) were used for all
isolations and assays. Dishes containing isolates were sealed using
Parafilm and incubated in dark environmental chambers at 15°C
until used in assays.
Fungal identification
Fungal colonies were determined to be one of two fungal genera,
Entomocorticium and Ceratocystiopsis, and were putatively
identified based on microscopic observations of hyphal morphology
and degree of melanization (Klepzig et al., 2004). Twenty strains of
mutualist mycangial fungi from D. brevicomis and D. frontalis in
sympatry (9 D. frontalis and 11 D. brevicomis), and thirteen fungal
strains from allopatric beetle populations (6 from D. frontalis in
Mississippi and 7 from D. brevicomis in California) were isolated.
Fungi from D. brevicomis and D. frontalis are Ceratocystiopsis
brevicomi and Entomocorticium sp. B, and Ceratocystiopsis
ranaculosus (J.R. Bridges and T.J. Perry) Hausner and
Entomocorticium sp. A, respectively (Hsiau and Harrington, 1997).
The identity of fungal strains were confirmed by sequencing of
internal transcribed spacer (ITS) regions 1 and 2 between the
ribosomal RNA genes 18S and 28S. DNA was extracted using a
Qiagen DNeasy plant kit (Valencia, CA) with the modified protocol

120 J. Yeast Fungal Res.
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Figure 1. The locations of beetle populations where fungal isolates were collected. D. brevicomis and D.
frontalis occur in sympatry in northern Arizona (Coconino National Forest), D. brevicomis in allopatry in
California (Plumas National Forest), and D. frontalis in allopatry in Mississippi (Homochitto National
Forest).
for yeasts, which includes a sorbitol buffer and lyticase enzyme to
digest cell walls. The author used primers 5.8SF (5’-
CGCTGCGTTCTTCATCG-3’) and 5.8SR (5’-
TCGATGAAGAACGCAGCG-3’) from White et al. (1990) and paired
them with newly developed primers ITS-18S (5’-
CTTSAACGAGGAATNCCTAGTA-3’) and ITS-28S
(CATWCCCAAACWACYCGACTC) for ITS regions 1 and 2,
respectively (Cindy Liu et al. unpublished manuscript).
The authors used the following parameters for a touchdown
PCR: hot start 95°C for 4 min; then 20 cycles at 95°C for 30 s, 60°C
for 1 min decreasing 0.5°C each subsequent cycle, 72°C for 1 min;
12 cycles at 95°C for 30 s, 45°C for 30 s, 72°C for 30 s; finishing
with 72°C for 7 min. PCR reagents were used in the following final
concentrations: Invitrogen PCR buffer 1 x (Carlsbad, CA), primers
0.2 uM each, MgCl
2
2.5 mM, dNTPs 0.8 uM, and Invitrogen
Platinum taq polymerase 1.4 U. PCR amplicons were cleaned up
using ExoSAP-IT (USB, Cleveland, OH), cycle sequenced with ABI
PRISM BigDye Terminator 3.1 (Applied Biosystems, Foster City,
CA), and run on an ABI 3130 x l Genetic Analyzer. Sequences for
both reads were edited and compiled in Sequencher 4.9 (Gene
Codes, Ann Arbor, MI) and BLASTed against all GenBank
accessions. Identifications were based on the highest identity value
(complete match) and read length. Representative voucher
specimens of fungi were preserved in 80% glycerol/20% malt
extract broth (MEB; Difco; Lot No. 7306921) and placed in storage
freezers held at -80°C in the Microbial Genetics and Genomics
Center in Flagstaff, Arizona, U.S.A.
Primary resource acquisition by mycangial fungi
Radial growth is a primary mode of resource acquisition for fila-
mentous fungi, and primary resource acquisition was defined as the
rate at which fungal colonies occupied media area in the absence
of competitors. All fungal strains were incubated in dark environ-
mental chambers at six temperatures of 5, 10, 15, 20, 25 and 28°C.
Fungi were transferred from original isolates to sterile 2% malt
extract agar by extracting a 1 x 1 mm section of growth media from
hyphal tips of isolations during the linear growth phase using a
flame-sterilized spatula. Hyphal growth was traced every 48 h
beginning at day zero (initial transfer of colony) for 15 d. The growth
rate for each fungal colony at each temperature was determined by
dividing the distance between tracings by the number of days of
growth. This study was replicated twice for each strain and growth
rate was quantified in mm growth/day to 1 x 10
-1
mm.
Competitive resource acquisition by mycangial fungi
Combative interaction between organisms is a secondary means of
resource acquisition when limited resources are occupied by
competitors (Tilman, 1982). The authors divided competitive
resource acquisition into two parts: (1) Resource
acquisition/capture: the mean frequency with which a fungal isolate
colonized media resources occupied by a competing fungal isolate,
and (2) Resource defense: the mean frequency with which a fungal
strain resisted colonization by a competing fungal colony.
Competitive interactions between mycangial fungi were tested
using a pairwise approach. All fungi were paired in Petri dishes by
placing 1 x 1 mm media sections containing fungal hyphae at
opposing ends of the dish, and fungal strains were transferred from
original isolate colonies as described above. Each isolate was also
tested against itself. This full factorial design (33 x 33) was
replicated twice (n = 2178 assays).
Variation in the growth patterns of these fungi on 2% malt extract

Davis et al. 121
Table 1. Fungal growth rates (mm/day
-1
) at multiple temperatures and passive / competitive resource acquisition patterns by fungal species and beetle host
isolated from sympatric beetle populations. ANOVA results also shown. Bold values indicate significance differences in means at  = 0.05.
Beetle species Fungal genera
D. brevicomis D. frontalis Ceratocystiopsis Entomocorticium
Variable Mean ± SE Mean ± SE
F
df
P
Mean ± SE Mean ± SE
F
df
P
5°C 0.091 ± 0.033 0.040 ± 0.029 1.312 1.16 0.268 0.074 ± 0.027 0.057 ± 0.036 0.141 1.16 0.711
10°C 0.425 ± 0.117 0.368 ± 0.103 0.133 1.16 0.720 0.446 ± 0.094 0.347 ± 0.127 0.372 1.16 0.550
15°C 0.704 ± 0.150 0.665 ± 0.131 0.040 1.16 0.843 0.592 ± 0.121 0.777 ± 0.162 0.808 1.16 0.381
20°C 1.086 ± 0.254 1.361± 0.222 0.660 1.16 0.428 1.016 ± 0.205 1.432 ± 0.276 1.410 1.16 0.252
25°C 1.518 ± 0.299 1.999 ± 0.262 1.452 1.16 0.245 1.584 ± 0.242 1.933 ± 0.325 0.715 1.16 0.409
28°C 1.250 ± 0.228 0.95 ± 0.152 1.193 1.16 0.306 1.320 ± 0.216 a 0.880 ± 0.125 b 3.100 1.16 0.116
Resource defense 0.488 ± 0.047 0.497 ± 0.041 0.016 1.16 0.900 0.594 ± 0.038 a 0.391 ± 0.051 b 9.707 1.16 0.006
Resource capture 0.386 ± 0.076 0.425 ± 0.067 0.141 1.16 0.712 0.445 ± 0.061 0.366 ± 0.183 0.571 1.16 0.460
*Letters indicate differences in means (Tukey's HSD test) by row.
media is consistent with that found in tree phloem (Rayner and
Webber, 1984; Klepzig and Wilkens, 1997; Hofstetter et al., 2005).
Following establishment of strains on dishes (1 - 2 d); hyphal
growth was traced every 48 h for 30 d. Assays were done at 25°C
in the dark. The outcomes of paired competition assays are
reported in terms of the mean frequency of resource acquisition and
the mean frequency of resource defense by each fungal strain.
These two observational metrics yielded four basic outcome
categories for each colony in each pairing: (1) Fungal isolate A
grew over media colonized by its paired competitor fungal isolate B;
(2) Media colonized by fungal isolate A was grown over by its
paired competitor fungal isolate B, (3) Both fungal isolates A and B
successfully grew into others colonized area, or (4) Both fungal
isolates A and B resisted overgrowth or formed a partition
(Tuininga, 2005). Thus, each isolate in each trial received both a
resource capture score and a defense score. Scoring for every
pairing was verified microscopically (10 - 100 x magnification) by
examining hyphal interactions.
The outcomes (mean frequency of resource capture and
resource defense) of paired competition assays were converted “a
posteriori” to binary values (0 = failure to defend/capture; 1 =
successful defense/capture) for each isolate and averaged over all
assays to yield an index of each fungal isolates’ competitive
performance on a continuous scale. Thus, each isolate received a
relative frequency score (ranging from 0 - 1) that described the
proportion of competitive resource acquisitions and defensive
responses. For example, an isolate with a resource defense score
of 0.87 indicates that the fungi successfully resisted colonization in
87% of competition trials.
Statistical analyses
All statistics were computed using JMP 7.0 software (SAS Institute).
Statistical tests were prefaced by checking statistical assumptions.
Assumptions of normality were verified using a Shapiro Wilk Test,
and no transformations were required. In comparisons among fungi
in sympatry (n = 20 sympatric fungal isolates), a two-way ANOVA
was performed to analyze the fixed effects of beetle species, fungal
species, and beetle species x fungal species interaction on
response variables of radial growth rate, mean resource capture
and mean resource defense. The beetle species x fungal species
interaction effect did not contribute significant variation to the
statistical model, so the interaction effect is not reported in the
ANOVA summary for ease of display.
In comparisons between isolates across beetle populations (n =
33 fungal strains [20 sympatric/13 allopatric]), a one-way ANOVA
was performed to analyze the fixed effect of sympatry or allopatry
on response variables of radial growth rate, mean resource
acquisition and mean resource defense. For this analysis sympatric
isolates were considered as a single (Arizonan) population. The
effects of temperature on fungal growth were analyzed as a fixed
effect nested by location and differences between means were
tested using contrasts. Statistical significance was established at 
= 0.05 for statistical tests and ANOVA models for growth and
competition assays were analyzed using F-tests to establish the
significance of effects. Where differences in mean growth and
competitive responses were detected in ANOVA models,
directionality was established using Tukey’s HSD Test.
RESULTS
Primary resource acquisition by mycangial fungi
Sympatric fungi: Growth rates of fungi from Arizona did
not vary by beetle species, fungal species, or beetle species
x fungal species interaction at 5, 10°C, 15, 20, 25 or 28°C
(Table 1, Figure 2). Thus, fungi from sympatric beetle
populations behaved statistically identically in terms of
growth rates across temperatures by both fungal species
and beetle species. However, fungal growth rates did
consistently increase as temperature increased then
declined once ambient temperature surpassed 28°C.
Entomocorticium species exhibited greater variability in
growth rates across temperatures than Ceratocystiopsis
species.
Sympatric and allopatric fungi: Growth rates did not
vary among the three populations at 5 or 10°C (Table 2,
Figure 2). Primary resource acquisition from the
sympatric fungi was significantly higher than the rate of
primary resource acquisition by fungi from D. brevicomis
in California at 15 and 20°C, and fungi from D. frontalis in
Mississippi were intermediate. There was no difference in
primary resource acquisition by population at 25°C, but at
28°C primary resource acquisition by sympatric isolates

122 J. Yeast Fungal Res.
Table 2. Fungal growth rates (mm/day
-1
) at multiple temperatures and passive / competitive resource acquisition patterns by fungal species and beetle host
isolated from sympatric and allopatric beetle populations. ANOVA results also shown. Bold values indicate significance differences in means at  = 0.05.
Population
Sympatric (D. brevicomis and D. frontalis) Allopatric (D. brevicomis) Allopatric (D. frontalis)
Variable Mean ± SE Mean ± SE Mean ± SE
F
df
P
5°C 0.064 ± 0.034 0.034 ± 0.054 0.187 ± 0.060 2.199 2.30 0.137
10°C 0.408 ± 0.065 0.237 ± 0.079 0.356 ± 0.086 1.367 2.30 0.277
15°C 0.799 ± 0.084 a 0.428 ± 0.100 b 0.588 ± 0.108 ab 4.093 2.30 0.032
20°C 1.341 ± 0.180 a 0.577 ± 0.216 b 1.136 ± 0.233 ab 3.752 2.30 0.041
25°C 2.016 ± 0.174 a 0.841 ± 0.259 b 1.810 ± 0.280 a 6.386 2.30 0.007
28°C 1.100 ± 0.201 b 2.362 ± 0.241 a 2.987 ± 0.260 a 18.233 2.30 <0.001
Resource defense 0.800 ± 0.031 a 0.707 ± 0.038 b 0.616 ± 0.041 b 3.593 2.30 0.046
Resource capture 0.496 ± 0.078 a 0.185 ± 0.101 b 0.210 ± 0.109 b 3.533 2.30 0.050
*Letters indicate differences in means (Tukey's HSD test) by row.
was significantly lower than for allopatric isolates
(Figure 2). Growth rates consistently increased
with temperature for all fungal isolates until 25°C,
where sympatric isolates showed a substantial
decline but allopatric fungi achieved optimal
growth.
Competitive resource acquisition by
mycangial fungi
Sympatric fungi: Patterns of competitive resource
acquisition varied significantly with fungal species
but were not variable with beetle species (Table
1). Specifically, Ceratocystiopsis species had
higher mean frequencies of resource defense
than Entomocorticium species (Table 1). Thus,
neither beetle species was associated with a
consistently more competitive fungal symbiont.
Sympatric and allopatric fungi; Competitive
resource acquisition by fungal isolates varied
significantly with sympatry and allopatry of source
beetle populations (Table 2). Isolates from the
sympatric beetle populations exhibited signifi-
cantly higher mean frequencies of competitive
resource acquisition and resource defense of
growth media than fungi isolated from either
allopatric beetle population (Table 2). Fungi from
opposing allopatric populations were not
significantly different from each other.
DISCUSSION
Primary resource acquisition
The radial growth rates of fungi were found to vary
by population at multiple temperatures (Table 2).
This is in agreement with the findings of Six and
Bentz (2007), which showed that ambient tempe-
rature was a mediator of fungal abundances, and
that this variation was related to both site and
seasonality in a Dendroctonus ponderosae sys-
tem. Here, they show that the growth rates of
mycangial fungi vary across beetle populations,
which in the current study are separated by large
geographic regions. However, growth rates did
not vary among fungal isolates for fungal species
or beetle species within sympatric populations.
The studies did not sample fungi from multiple
sites within sympatric populations, so they might
have detected greater variation in growth rates by
assessing multiple sites within each region.
In contrast to the present study, Hofstetter et al.
(2007) showed that Entomocorticium sp. A,
exhibited optimal growth at a lower ambient
temperature than C. ranaculosus in a D. frontalis
system in Mississippi. In the present study, no
differences in response to temperature were
detected for growth by fungal species from sym-
patric beetle populations in Arizona or an
allopatric beetle population in California (Figure
2). However, the authors did support their findings
that Entomocorticium sp. A, and C. ranaculosus
had different optimal growth rates in a Mississippi
population of D. frontalis (Figure 2). One expla-
nation for this pattern is overall variability in daily
temperature: daily temperature range is greater in
Arizona and California than in Mississippi

Davis et al 123
Entomocorticium spp.
Radial growth rate (mm/day
-1
)
0
1
2
3
4
0
1
2
3
4
Arizona, D. frontalis, E.a.
Arizona, D. brevicomis, E.b.
Mississippi, D. frontalis, E.a.
California, D. brevicomis, E.b.
Ceratocystiopsis spp.
Temperature (C)
5 10 15 20 25 30
Radial growth rate (mm/day
-1
)
0
1
2
3
4
0
1
2
3
4
Arizona, D.frontalis, C.r.
Arizona, D. brevicomis, C.b.
Mississippi, D. frontalis, C.r.
California, D. brevicomis, C. b.
Figure 2. Growth rates of Ceratocystiopsis spp. and Entomocorticium spp. isolated from sympatric
bark beetle populations in Arizona, an allopatric population of D. brevicomis in California, and an
allopatric population of D. frontalis in Mississippi. Sympatric populations were pooled in this figure
since there were no significant differences between radial growth rates of sympatric fungi. Error bars
represent one standard error.
(Hereford, 2007; Southern Regional Climate Center,
2008; Western Regional Climate Center, 2008). Thus,
optimal growth rate and species abundances of
mycangial fungi may be influenced by daily temperature
range, in addition to regional and seasonally mediated
temperature differences. The rates of primary resource
acquisition by sympatric fungi corresponded to the
average maximum summer temperature in the region. In
northern Arizona, climate records show that maximum
summer (June-September) temperatures average 25°C
(Hereford, 2007), which is the range where the greatest
average growth rates were observed for sympatric fungi
(Figure 2). In the experiments, an increase in ambient
temperature of only 3°C above the average summer
maximum correlated with a dramatic decrease in growth
rates of the sympatric fungi. In contrast, mycangial
isolates from allopatric populations showed no apparent
decrease in growth rates as ambient temperatures

124 J. Yeast Fungal Res.
increased, and isolates from both populations grew
optimally at 28°C. Unfortunately, no inferences can be
made about primary resource acquisition by fungi beyond
ambient temperatures of 28°C. However, previous work
shows that the growth rates of mycangial fungi (both
Entomocorticium and Ceratocystiopsis) isolated from D.
frontalis in Mississippi declines once ambient
temperatures exceeds 28°C (Hofstetter et al., 2007). If it
is true that fungal growth rates correspond to regionally
defined average maximum temperatures during summer
months, then they would predict that isolates from
allopatric D. brevicomis will grow optimally at 29.6°C, and
isolates from allopatric D. frontalis will grow optimally at
30°C. Future studies with these organisms could
benefit from assaying growth rates of mycangial fungi
from both within and between beetle populations across a
broader temperature range and at smaller intervals, since
isolates appear to be highly sensitive to relatively small
incremental variations in temperature.
Competitive resource acquisition
Fungi isolated from the sympatric beetle populations
exhibited significantly greater frequencies of competitive
acquisition and defense of media in a resource-limited
environment. In sympatric beetle populations, Cerato-
cystiopsis species defended media resources from
colonization by opposing isolates with significantly higher
frequency than Entomocorticium species (Table 1). Data
regarding competitive resource acquisition and resource
defense by allopatric fungi were strongly asymmetric: in
almost, no case (< 5%) was a fungal isolate from an
allopatric beetle population able to colonize media
occupied by a fungal isolate from a sympatric beetle
population. Thus, competitive resource acquisition by
allopatric isolates occurred almost exclusively in pairings
with other allopatric isolates. The opposite was not true
for sympatric fungi, which were able to competitively
acquire media resources colonized by isolates from all
beetle populations. However, all competition assays were
performed at 25°C, where sympatric fungi exhibited their
optimal growth. Thus, it is possible that the observed
differences in competitive performance between sym-
patric and allopatric isolates were due to localized
adaptations to temperature or the seasonality of collec-
tion (Six and Bentz, 2007). However, optimal growth rate
of mycangial fungi from D. frontalis collected in
Mississippi and Alabama is reported between 25 - 28
˚
C
(Klepzig et al., 2001; Hofstetter et al., 2007). The authors
suggest that future studies related to competition
between multiple mycangial species focus on testing
interactions across a broader range of temperatures. In
nature, many factors may contribute to the outcomes of
competitive interactions between multiple fungi. For
example, Licyayo et al. (2007) found that ammonia con-
centrations and pH had strong effects on interspecific
interactions between fungal species. Similarly, melanin
and other pigments have been shown to strongly mediate
competition between fungal species (Yuen et al., 1999;
Klepzig, 2006). In a D. frontalis system, water availability
was determined to play an important role in fungal com-
petition (Klepzig et al., 2004). The studies only account
for differences in competitive ability among fungi between
regions of sympatry and allopatry, and by beetle species
and fungal species within sympatric populations. How-
ever, the present study represents a first assessment of
combative interactions between the mutualists of two or
more bark beetle species, and suggests that fungi are
likely to adapt to a competitive environment when
multiple mycophagous beetle species inhabit a single
plant host. Future studies of competitive interactions
could benefit by testing interspecific fungal interactions
across a gradient of host plant Dendroctonus beetles are
frequently exposed to a terpenoid-saturated environment
during the colonization of host tissues, and exposure to
terpenoid compounds strongly impacts the growth
performance of beetle – associated fungi (Paine and
Hanlon, 1994; Hofstetter et al., 2005).
In conclusion, the mycangial fungi associated with D.
brevicomis and D. frontalis were variable with respect to
primary resource acquisition and competitive interactions.
Growth rates of fungi did not vary by beetle species or
fungal species when beetle populations were sympatric,
however fungal growth rates did show substantial
variation across regions. In sympatric populations,
Ceratocystiopsis species were more likely to resist colo-
nization by a competitor than Entomocorticium species.
Mycangial isolates from sympatric beetle populations
were more likely to competitively acquire resources.
Interactions between sympatric isolates and allopatric
isolates were asymmetric: sympatric isolates were better
competitors and frequently colonized media resources
inhabited by an allopatric isolate. Interestingly, com-
petition also appeared to be symmetric among allopatric
isolates.
These data reported here support the hypothesis that
interactions between mycangial fungi of multiple Den-
droctonus species are antagonistic (−, −), since fungi that
occurred in sympatry were stronger competitors. Further-
more, these findings may be extendable to other systems
where multiple insects with fungal associates colonize the
same plant host and insect-fungal associations have been
increasingly recognized as a central theme in arthropod
ecology (Blackwell and Vega, 2005). The importance of
these interactions for beetle larval performance and
fungal-beetle relationships are still unknown and further
experiments are needed to determine how competition
between multiple fungal associates of Dendroctonus
species affect beetle fitness.
ACKNOWLDEGEMENTS
The author thanked Amanda Garcia, Sherri Smith, and

Danny Cluck for capturing and sending bark beetles to
their lab. The author appreciated the efforts of the USDA
Forest Service, Southern Research Station laboratory
technicians and the USDA Forest Service, Rocky
Mountain Research Station for providing us with
laboratory space. The author thanked Brandy Francis for
assistance with sequencing efforts, Cindy Liu for access
to unpublished fungal sequencing primers, and Laine
Smith for laboratory assistance. The author also thanked
one anonymous reviewer for comments that improved the
quality of the manuscript. The author acknowledged the
USDA Forest Service funding sources: Southern
Research Station Cooperative Agreement 06-CA-
11330129-046 and Rocky Mountain Research Station
Joint-Venture Agreement 05-PA-11221615-104.
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