Microb Ecol DOI 10.1007/s00248-015-0593-8

FUNGAL MICROBIOLOGY

Effects of Temperature on Growth, Sporulation, and Competition of Mountain Pine Beetle Fungal Symbionts Melissa L. Moore & Diana L. Six

Received: 30 December 2014 / Accepted: 26 February 2015 # Springer Science+Business Media New York 2015

Abstract The mountain pine beetle, Dendroctonus ponderosae, depends on two fungi, Grosmannia clavigera and Ophiostoma montium, to augment a nutrient-poor woody food resource. Because the two fungi exert differential effects on the host beetle, temperature-driven differences in fungal growth and competition outcomes have a strong potential to influence host population dynamics. Weisolated fungi from beetles and wood from three locations in Montana and Utah, USA, and measured their growth rates and sporulation between 5 and 35 °C on artificial media. We also measured growth rates and percent resource capture for each fungus at 10, 15, 21, and 25 °C during inter- and intra-specific competition. G. clavigera excelled at resource capture at most temperatures. Its optimal growth temperature occurs around 20 °C while that of O. montium occurs near 30 °C. There was no effect of collection site on growth or sporulation; however, O. montium exhibited greater variability in response to temperature than did G. clavigera. Sporulation of G. clavigera was greatest at 30 °C while O. montium sporulated at low levels across all temperatures. During competition experiments, G. clavigera captured more resources than O. montium at most temperatures and captured a greater percentage of resources at a greater rate during inter-specific competition than during intra-specific competition. In contrast, O. montium captured a greater percentage of resources during intraspecific competition. These results demonstrate that temperature can differentially affect growth, sporulation, and resource capture of the two symbionts, indicating that it may be an important factor influencing the composition and dynamics of the symbiosis.

M. L. Moore : D. L. Six (*) Department of Ecosystem and Conservation Sciences, The University of Montana, Missoula, MT 59812, USA e-mail: [email protected]

Keywords Bark beetle . Symbiosis . Dendroctonus ponderosae . Grosmannia clavigera . Ophiostoma montium

Introduction The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) (Coleoptera: Curculionidae, Scolytinae) is a bark beetle indigenous to western North America. This insect is capable of killing a wide range of hosts in Pinus [38] and can cause the mortality of millions of trees during outbreaks. In recent years, damage caused by MPB has increased dramatically due to extensive outbreaks exacerbated by climate change, fire suppression, and logging practices [24, 28]. The beetle excels in the forest environment due, in part, to its participation in symbioses with two fungi, Grosmannia clavigera (Rob. & Jeff.) Zipfel, Z.W. de Beer & M.J. Wingfield and Ophiostoma montium (Rumbold) Arx, which are consistently associated with the beetle across its geographic range [18, 26, 34, 37]. MPB feeds on phloem, which is a poor nutrient resource relative to the needs of the insect [6, 32]. G. clavigera and O. montium provide nutritional supplementation by concentrating nitrogen [4, 6, 11, 15] and producing sterols [4]. Beetle larvae feed on the fungi along with phloem throughout their development [1, 6]. After completing development, the larvae create chambers in the phloem where they pupate and eclose to the adult form [32]. New adults feed on fungal spore layers that form on the walls of the pupal chamber [32]. As the beetles feed, they pack mycangia, specialized fungus transport structures in the exoskeleton, with spores that are then carried to the next tree and the next generation of beetles [9, 37]. Feeding on fungi has significant benefits for the beetles. Larvae that feed on phloem containing symbiotic fungi attain

M. L. Moore, D. L. Six

a larger adult body size [6, 32] which is positively correlated with fecundity [19]. They also have higher survival rates; larvae that feed on phloem alone seldom survive [37]. In addition, it appears that feeding on spores by new adults is required for reproduction [32]. While both fungi are able to support beetle growth and development in trees, they differ in the degree of benefit they confer [6, 11, 32]. G. clavigera concentrates nitrogen better than O. montium [11] which may account for observations that beetles developing with G. clavigera are bigger and have higher productivity and survival rates than those developing with O. montium [6, 15, 32]. Because differential nutrient supplementation by the two fungi translates directly to fitness effects on beetle productivity, the relative prevalence of the two fungi is predicted to influence MPB population dynamics. MPB must overwhelm tree defenses to colonize a tree and for the mycangial fungi to establish. To accomplish this, the beetles initiate a mass attack using a complex attractantantiattractant pheromone system that results in an even spacing of attacks on the tree bole [23]. This also produces a regular pattern of inoculation of the fungal associates within the tree. As the fungi grow out from their respective inoculation points, they eventually encounter other fungal individuals, either hetero- or con-specifics, growing from nearby beetle entry points. The relative prevalence of each species of fungus inoculated into the tree depends upon the proportion of the two symbionts being carried by attacking beetles that, in turn, is determined by temperature [34]. Once in the tree, rates of MPB development and fungal growth are directly controlled by temperature [2, 3, 5, 16]. Because both fungi are introduced into the tree at the same time, are limited to colonizing a finite phloem and sapwood resource, and ultimately must gain access to dispersing beetles for their spores to be transported to a new host tree, the two are expected to be in strong competition. Observational and experimental evidence in vitro, and in vivo in standing naturally attacked trees, indicates that the two fungi associated with MPB exhibit exploitation competition [6–8] where the outcome is determined solely by how efficiently or rapidly each species captures resources. Competitive interactions are thought to be destabilizing to multi-partite mutualisms and are predicted to result in the loss of inferior competitors over time [13]. However, the three-way symbiosis between MPB and its two fungi has remained remarkably stable over long periods of evolutionary time [30, 33]. Coexistence of competing organisms can occur when competitors respond differently to environmental conditions such that no one species is favored at all times [10]. Only two studies have directly examined how environmental factors may affect competition between the two fungi associated with MPB. In one study, conducted in vitro with the fungi grown at one temperature but at various water potentials mimicking those that occur in dying trees over time, both species

generally exhibited reduced growth rates and percent resource capture when grown with the other [8]. However, the growth rate of G. clavigera increased when it was grown in close proximity to O. montium at low water potentials. This indicates that under certain conditions, some fungal partners may be able to use volatiles, by-products, or enzymes diffusing from other partners to their advantage [8]. In the other study, the relative ability of the two fungi to capture space in naturally attacked trees was found to vary seasonally and depended upon the timing of their introduction to the host tree [7]. Together, these studies suggest that the fungi compete and capture resources in a manner highly dependent upon environmental conditions. Several studies have examined the growth rate responses of the two fungi at different temperatures. These studies found that G. clavigera is able to grow at lower temperatures than O. montium, while O. montium can grow at warmer temperatures than G. clavigera [25, 31, 36]. This may explain the shifting prevalence of the two fungi that has been observed over time in naturally attacked trees [1, 8]. Adams and Six [1] observed that G. clavigera was more often isolated from phloem adjacent to third and fourth instar larvae developing during spring and early summer when conditions were relatively cool, while O. montium was isolated more often from phloem adjacent to pupae, teneral adults, and eggs which develop in mid-to-late summer when temperatures are warmer. Temperature has also been correlated with the relative prevalence of the two fungi carried by dispersing beetles [34]. These studies collectively indicate that temperature determines the relative proportion of each fungus in a population and that this proportion shifts constantly over time. The effects of temperature on symbiont prevalence have important implications for both fungal and beetle fitness. For example, at sites that are generally cool, or at times of the year when conditions are overall cooler, G. clavigera would be expected to be at an advantage and capture the most resources within the tree. In contrast, at sites where conditions are generally warm, or during times of the year when conditions are warmer, O. montium would be expected to dominate. At sites where conditions fluctuate greatly, the two fungi would be expected to capture resources at different rates at different times of the year, with highly variable outcomes in total resources captured over time. Such variability in resource capture by the fungi should translate directly to effects on beetle fitness through differential nutrient availability during larval development. For example, an increase in the prevalence of O. montium during larval development should result in the production of more small adult female beetles [7] and a subsequent reduction in fecundity. While this initially may appear detrimental to MPB, a redundancy of nutritional symbionts, even if one is inferior to the other, may be critical to MPB persistence under fluctuating environmental conditions. By having more than one symbiont, each with different

Effects of Temperature on Mountain Pine Beetle Fungal Symbionts

temperature tolerances, MPB larvae may minimize the likelihood of being caught without a symbiont as conditions vary through time and space [34]. Differential responses to temperature may also support coexistence of the fungi over time by never allowing any one fungus to move to fixation with the host [2]. Our aim was to better understand the effects of temperature on the fungi associated with MPB. Past studies described temperature effects on growth rates of these fungi using very small numbers of isolates [25, 31, 36] and no studies have quantified how temperature affects sporulation or competition. In this study, we assessed the effects of temperature on the primary fitness parameters of the two fungi (growth rate, sporulation, and ability to compete with hetero- and con-specifics) in vitro. We included a large number of isolates from several locations in order to more accurately estimate growth rates and to capture the range of variability in responses to temperature within each species.

Methods Fungal Isolates A total of 88 isolates (50G. clavigera, 38 O. montium) were isolated from beetles and wood from two locations in MT and one location in UT, USA. Sites in MT were Vipond Park (45° 42.31′ N, 112° 55.54′ W, elevation 2438 m) located in the Beaverhead National Forest, and Lubrecht Experimental Forest (46° 53.30′ N, 113° 26.03′ W, elevation 1263 m) located in Missoula County. The UT site, Stump Hollow (41° 57.34′ N, 111° 31.47′ W, elevation 2136 m), was located in the UintaWasatch-Cache National Forest. Multiple sites were included to allow us to better describe the variability within each species, and also to detect if population sub-structuring occurred among fungi from geographically distant locations. All isolates were obtained either from dissections of MPB mycangia using methods described by Six and Paine [31], or from phloem removed from MPB galleries in trees using a sterile cork borer. Pure fungal isolates were obtained using single spore isolations and then incubated at room temperature on 2 % malt extract agar (MEA) enriched with sterile pine twig cuttings to encourage sporulation. Isolates were identified using cultural characteristics and morphology. Effects of Temperature on Growth Rates of G. Clavigera and O. Montium The 88 isolates were used to determine the growth rates of the fungi at various temperatures and to determine their upper and lower limits for growth. The isolates were grown for 5 days on 2 % MEA prior to use in each experiment to ensure that the cultures were in an active growth phase. On the fifth day, a 13-

mm2 circular plug of actively growing mycelium was extracted from the leading edge of the culture and placed mycelium side down onto the center of a 100-mm diameter Petri dish containing 2 % MEA. The inoculated plates were incubated for 14 days at 5, 10, 15, 21, 25, 30, and 35 °C. Each treatment was replicated three times for each isolate. At the same time each day, beginning on the second day, the area colonized by each fungus was traced on the bottom of the Petri dish. On the tenth day, the bottoms of the Petri dishes were photographed with a digital camera and photos uploaded onto a computer where the area colonized by each fungus per day was measured using ImageJ v.1.43u (ImageJ, Bethesda, MD). The cultures were held for an additional 4 days to assess sporulation (see below). Data Analyses Area measurements were regressed by time since inoculation and slopes fit by regression analysis. The slopes of the regression lines for each replicate were averaged for each isolate at each temperature, resulting in an average absolute growth rate for each. The mean, range, and standard error of the growth rate for each species at each temperature were determined. A logarithmic transformation was used to obtain relative growth rate estimates [21]. Mixed model analysis of variance (ANOVA, maximum likelihood method) with isolate as a random variable was used to test for the fixed effects of temperature, species, and site, and their interactions on relative growth rate. Significant F-tests for all interactions were followed by Tukey-Kramer’s honestly significant different (HSD) tests. For all tests, here and elsewhere, α was set at P<0.05. Growth rates for each isolate and the mean growth rate for each species were plotted against temperature to produce growth curves. A lack of overlap in 95 % confidence intervals was used to detect temperatures at which the fungi grew at significantly different rates [17]. All statistical analyses here and throughout were conducted using R [22]. The Effects of Temperature on Sporulation of G. Clavigera and O. Montium Cultures used in the growth rate study were also used to assess temperature effects on sporulation. After 14 days, two replicates of each isolate from each temperature treatment were removed from incubation. A known amount of sterile deionized water was added to the surface of each culture. A sterile bent glass rod was used to dislodge spores from the surface of the agar into the sterile water. The resulting spore mixture was extracted from the surface of the plate, placed into a centrifuge tube, and vortexed for 30 s. A 10-μm aliquot of suspension was then injected into a hemocytometer, and the number of spores in a subset of randomly chosen grids of the hemocytometer was counted. Data analyses The hemocytometer spore counts were converted to spores/ml using the dilution factor. Spores/ml was

M. L. Moore, D. L. Six

then scaled by the area of colonization at day 14 to produce an estimate of spores/mm2. Replicates were averaged for each isolate at each temperature. The mean, range, and standard error in spore production for each species at each temperature were determined. A logarithmic transformation was used to obtain estimates of the number of spores/mm2 [21]. A mixed model ANOVA (maximum likelihood method) with isolate as a random variable was used to test for fixed effects of temperature, species, and site, and their interactions on relative growth rate. Observations from 35 °C were not included in the model because no growth was observed for either fungus at this temperature. Significant F-tests for all interactions were followed by Tukey-Kramer’s HSD tests. Comparisons between species were not conducted because the fungi respond differently to growth on artificial media [6, 7]. The Effects of Temperature on Resource Capture by G. Clavigera and O. Montium During Intraand Inter-Specific Competition A total of 30 isolates (15G. clavigera, 15 O. montium) were used in this experiment. To investigate the rate and percent of resource capture when isolates of the same species or isolates of the two species were grown together, we created an arena that would allow estimations of linear growth of the fungi in vitro. To create the arena, we inserted a 55-mm-diameter Petri dish inside a 100-mm-diameter Petri dish. The space between the smaller dish and the larger dish was filled with 2 % MEA forming a ring of growth medium (Fig. 1). This design allowed us to measure how temperature affects resource capture in the context of a natural pattern of beetle attack and fungal inoculation, where the growth of an

Fig. 1 Arena used to test for effects of temperature on resource capture by Grosmannia clavigera and Ophiostoma montium during intra- and inter-specific competition. Arena consists of a small Petri dish placed inside a larger Petri dish with 2 % malt extract agar poured into the space between the edges of the two dishes. The black dots indicate the locations of inoculations

individual colony approaches a competitor primarily on a two-dimensional plane. The isolates used in this experiment were grown for 5 days on 2 % MEA prior to use. On the fifth day, a 13-mm2 circular plug of mycelia was extracted from the leading edge of the culture. Four plugs were placed mycelium side down, equidistant from one another, onto the surface of the ring of agar. For the intra-specific competition treatment (G. clavigera growing toward G. clavigera, O. montium growing toward O. montium), four plugs of one isolate were used per plate. For the inter-specific competition treatment, one isolate of each species from the same site were inoculated equidistant from each other and in an alternating pattern on the surface of the ring of agar in the dish. Three replicates of each treatment were incubated at 10, 15, 21, and 25 ° C. Measurements were taken daily for fungal growth from each plug by marking the bottom of the dish parallel to hyphal extension and perpendicular to the edge of the Petri dish. A tape measure wrapped around the circumference of a 55-mm-diameter Petri dish was used to measure the distance from the center of the inoculation plug to the leading edge of growth extending from each plug each day. The total distance captured by each isolate was recorded once the cultures met. Data Analysis Two metrics were analyzed: growth rate approaching a competitor and the percent resource capture by each competitor once the arena was entirely colonized. Changes in rate of resource capture as an isolate approaches a competitor indicate that the fungus is responding to the presence of the competitor. If rates increase, facilitation is indicated, while if rates decrease, interference competition is occurring. No change in rate is indicative of exploitation competition. The percent resource captured indicates the total amount of resource captured over the trial by each fungus at each temperature. The daily growth rates of G. clavigera and O. montium were used in a regression analysis. The slopes of the regression lines formed using observations within individual plates were averaged to obtain an estimate of linear growth rate (mm/day). A logarithmic transformation was used to obtain relative mm/day estimates [21]. Comparisons were made between species as well as within species using mixed model ANOVA (maximum likelihood) with isolate as a random effect. The percent resource capture by each individual was measured by dividing the distance captured by the distance between opposing individuals and multiplying by 100 and then averaged within each plate for each competition treatment. Comparisons were made between species as well as within species using mixed model ANOVA (maximum likelihood) with isolate as a random effect. For the growth rate model, as well as the percent resource capture model comparing competition treatments within each

Effects of Temperature on Mountain Pine Beetle Fungal Symbionts

species, significant F-tests for interactions were followed by Tukey-Kramer’s HSD tests. In addition, mean growth rates and percent resource capture were compared between species at each temperature.

Results Growth Rates

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Growth curves for the two fungi are shown in Fig. 2. Individual isolates exhibited considerable variability in response to temperature (Fig. 3), but in general, G. clavigera grew better at cooler temperatures while O. montium grew better at warmer temperatures. The highest mean growth rates occurred at 21 °C for G. clavigera (457.4 mm2/day) and 30 °C for O. montium (436.2 mm2/day) (Table 1). The majority of isolates of both species grew between 5 and 30 °C. At 5 °C, four isolates of G. clavigera, and six isolates of O. montium did not grow but were able to grow when moved to room temperature (~25 °C). At 30 °C, four isolates of G. clavigera failed to grow, while 15 grew initially but stopped growing after day 4; however, all were able to grow when moved to room temperature. No isolate of either species was able to grow when held at 35 °C; however, all were able to grow when moved to room temperature. The effect of temperature on the growth of the two species of fungi was significant (F=442.9; df 5, 344; P<0.0001). There was also a significant interaction between temperature and species (F=28.4; df 5, 341; P<0.0000). Mean growth of G. clavigera isolates was significantly greater than that of

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O. montium isolates at 10 °C (Tukey’s HSD; P<0.0000) and 15 °C (Tukey’s HSD; P=0.0003), while at 30 °C, the mean growth of O. montium isolates was significantly greater than that of G. clavigera (Tukey’s HSD; P<0.0000) (Table 1). Average growth rates for G. clavigera were not significantly different between 15, 21 and 25 °C, or between 10 and 30 °C. Average growth rates for O. montium were not significantly different between temperatures 21, 25, and 30 °C (Table 1). A significant interaction was found between temperature and site (F=5.34; df 10, 344; P<0.0001). Post hoc HSD tests did not detect significant differences between sites for the growth of either fungus at any temperature. In contrast, when the two species were modeled separately, drop in deviance t tests found a significant difference for growth of O. montium by site, but not for G. clavigera. This was due to a lower mean growth rate for isolates of O. montium collected from Vipond Park compared to isolates from Lubrecht Experimental Forest (t=−2.55; df 34; P=0.0156). Sporulation The greatest mean number of spores/mm2 was observed at 30 °C for both G. clavigera (4190.4 spores/mm2, standard error (SE)=1173.0) and O. montium (2401.0 spores/mm2, SE=1599.6) (Table 2). The majority of isolates from both species produced spores between 5 and 30 °C. Two isolates of G. clavigera and two isolates of O. montium failed to produce spores at 5 °C. The same was true for one isolate of O. montium at 10 °C, one isolate of O. montium at 25 °C, and four isolates of G. clavigera at 30 °C. While sporulation for G. clavigera peaked at 30 °C, O. montium sporulated at approximately equal rates across all temperatures (Fig. 4). ANOVA detected a significant effect of temperature (F= 26.89, df 5, 210, P<0.0001) as well as a significant effect of species (F=30.14, df 1, 210, P<0.0001) on sporulation. The interaction between temperature and species was also significant (F=13.16, df 5, 210, P<0.0001). There were significant differences in sporulation by G. clavigera between 5 and 10 °C (Tukey’s HSD; P=0.0210), and 15 and 21 °C (Tukey’s HSD; P<0.0001); however, no significant differences in sporulation were found for O. montium among temperatures (Table 2). The interaction between temperature and site had a significant effect on mean sporulation (F=2.29, df 10, 210, P=0.0146).

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Fig. 2 Mean growth rate (mm2/day) for Grosmannia clavigera (black) and Ophiostoma montium (gray) (combined data for isolates collected at two sites in Montana and one site in Utah). Bars represent 95 % confidence intervals

Growth and Resource Capture During Intraand Inter-Specific Competition In all replicates, growth of the fungi halted when the growing edges of the isolates met, although, in many cases, a slight intermingling of hyphae occurred. However, in the intraspecific competition treatment for G. clavigera, a zone lacking

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Fig. 3 Mean growth rates of individual Grosmannia clavigera (top) and Ophiostoma montium (bottom) isolates from study sites in Montana (Lubrecht, Vipond Park) and Utah (Stump Hollow)

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M. L. Moore, D. L. Six

Temperature (°C)

melanization (both G. clavigera and O. montium typically produce abundant melanin in wood and in culture) developed where the hyphae of isolates met. This did not occur in the intra-specific competition treatment involving O. montium, or any inter-specific competition treatments. No barrage or necrosis zones were observed as a result of any interactions between any isolate combinations. Temperature significantly affected mean growth rates of the fungi during competition for both O. montium (F = 530.41; df=3, 279; P<0.0001) and G. clavigera (F=196.13, df 3, 294, P<0.0001). In addition, mean growth rates of O. montium were significantly greater during intra-specific competition than during inter-specific competition (F = 10.91; df 1, 279; P=0.0011) (Table 3). No differences occurred in growth rates for G. clavigera in either competition treatment (Table 3). A significant effect was found for the interaction between competition treatment and site for O. montium (F = 5.85, df 2, 279, P = 0.0033). Isolates of O. montium collected from Vipond Park grew more slowly overall than isolates from Stump Hollow (Tukey’s HSD; P= 0.0001) and Lubrecht Experimental Forest (Tukey’s HSD; P= 0.0002) (Table 3). The interaction between temperature and site had a significant effect on mean growth rates during competition for O. montium (F=3.58, df 6, 285, P=0.0020) and G. clavigera (F=4.28, df 6, 300, P=0.0004) (Table 3). O. montium isolates collected from Vipond Park grew significantly more slowly at 15 °C than isolates collect from Lubrecht Experimental Forest

(Tukey’s HSD; P=0.0025) and Stump Hollow (Tukey’s HSD; P=0.0001). In addition, G. clavigera isolates collected from Stump Hollow grew more slowly at 10 °C than isolates from Lubrecht Experimental Forest (Tukey’s HSD; P<0.0001). Of the two fungi, only O. montium was affected by the interaction between temperature, competition treatment, and site (F= 2.73, df 6, 279, P 0.0137). O. montium isolates from Vipond Park grew slower than isolates from Lubrecht Experimental Forest (Tukey’s HSD; P = 0.0437) and Stump Hollow (Tukey’s HSD; P=0.0200). During intra-specific competition at 15 °C, O. montium isolates from Vipond Park grew slower than isolates from Stump Hollow (Tukey’s HSD; P=0.0452) (Table 3). Temperature had a significant effect on percent substrate captured by O. montium (F=7.98, df 3, 278, P<0.0001), but not by G. clavigera (F = 0.86, df 3, 293, P = 0.4621). G. clavigera captured significantly more resources during inter-specific competition than during intra-specific competition (F=50.52, df 1, 293, P<0.0001) (Table 4). The reverse was true for O. montium which captured significantly more resources during intra-specific competition (F=36.04, df 1, 278, P<0.0001). The interaction between temperature and competition treatment had a significant effect on percent resource capture by G. clavigera (F=2.78, df 3, 293, P=0.0413) as well as by O. montium (F=3.00, df 3, 278, P=0.0311); G. clavigera captured significantly more resources during inter-specific competition than during intra-specific competition at 10 °C (Tukey’s HSD; P<0.0001) and 15 °C (Tukey’s

Effects of Temperature on Mountain Pine Beetle Fungal Symbionts Table 1 Mean (SE) and range of growth rate (mm2/day) of Grosmannia clavigera and Ophiostoma montium grown on artificial medium (malt extract agar) at six temperatures Temperature (°C) 5

10

15

21

25

30

167.54 (16.89) b1 31.04–608.50 47

399.94 (24.08)c1 69.87–821.03 48

457.35 (14.96)c1 208.23–787.80 48

433.80 (18.32)c1 142.81–751.33 49

162.47 (20.59)b1 7.16–624.20 47

191.48 (41.40) 31.92–608.50 14

365.50 (49.68) 69.87–821.03 15

501.51 (17.33) 409.20–625.35 16

482.79 (32.56) 142.81–751.33 17

172.01 (38.73) 41.61–624.20 15

6.11 (1.31) 0.39–21.25 21

186.57 (21.1`0) 40.56–447.40 22

468.90 (28.66) 291.07–649.50 21

424.96 (19.84) 272.50–634.50 20

398.67 (20.45) 264.28–602.53 22

141.69 (29.61) 7.16–494.90 21

11.65 (3.67) 1.86–33.46 10

99.015 (16.98) 31.04–251.90 11

322.33 (43.95) 134.20–651.28 12

452.47 (41.81) 208.23–787.80 12

427.781 (51.55) 228.15–720.45 10

189.13 (44.22) 52.98–466.17 11

60.40 (7.27)b2 1.33–149.85 36

186.42 (18.22)c2 50.72–387.90 27

343.75 (27.46)d1 70.74–648.00 33

424.24 (30.46)d1 108.00–850.27 29

436.16 (32.52)d2 39.28–775.90 29

42.23 (9.38) 2.92–91.07 9

166.68 (36.55) 61.85–298.30 6

423.73 (62.84) 70.74–648.00 9

466.79 (82.21) 108.00–850.27 8

462.55 (59.05) 322.03–755.90 7

83.24 (9.31) 12.30–149.85 20

228.20 (25.08) 94.45–387.90 14

299.90 (32.83) 85.74–535.20 17

427.90 (34.43) 248.73–723.45 16

450.63 (41.70) 39.28–744.83 18

18.49 (6.36) 1.33–45.19 7

119.78 (22.14) 50.72–225.80 7

347.38 (55.28) 150.14–524.40 7

344.47 (46.87) 171.50–454.83 5

324.85 (100.80) 54.96–524.77 4

G. clavigera All sites combined Mean (SE) 7.48 (1.40)a1 Range 0.39–39.03 N 42 Lubrecht Experimental Forest Mean (SE) 6.29 (3.32) Range 1.14–39.03 N 11 Stump Hollow Mean (SE) Range N Vipond Park Mean (SE) Range N O. montium

All sites combined Mean (SE) 8.96 (1.78)a1 Range 0.30–32.46 N 26 Lubrecht Experimental Forest Mean (SE) 11.23 (.2.14) Range 5.19–20.91 N 7 Stump Hollow Mean (SE) 7.83 (2.69) Range 0.30–32.46 N 16 Vipond Park Mean (SE) 9.69 (3.69) Range 3.86–16.52 N 3

Same letters within rows indicate that means are not significantly different between temperatures. Same numbers within columns indicate that means are not significantly different between species within temperatures (all sites combined only). Comparisons between sites within species and temperatures were not performed because ANOVA found no temperature * species * site interaction SE standard error

HSD; P=0.0003) (Table 4). O. montium, in contrast, captured more resources during intra-specific competition than during inter-specific competition at 10 °C (Tukey’s HSD; P=0.0005) and at 15 °C (Tukey’s HSD; P=0.0026) (Table 4), and less resource during inter-specific competition at 10 °C than at 20 (Tukey’s HSD; P=0.0005) and 25 °C (Tukey’s HSD; P=0.0099) (Table 4). In addition, the interaction between

temperature and site had a significant effect on percent resource capture by O. montium (F = 2.59, df 6, 278, P=0.0187). During inter-specific competition, temperature had a significant effect on the rate of resource capture (F=344.86, df 3, 258, P<0.0001). G. clavigera captured a greater percentage of resources overall (F=27.24, df 1, 24, P<0.0001) as well as

M. L. Moore, D. L. Six Table 2 Mean (SE) and range of sporulation (spores/mm2) of Grosmannia clavigera and Ophiostoma montium grown on artificial medium (malt extract agar) at six temperatures Temperature (C) 5

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21

25

30

G. clavigera All sites combined 262.77 (67.03)a

Mean (SE) Range

0.00–1307.47

N

22

74.87 (23.60)b 1.39–525.96

74.36 (17.84)b

1589.60 (430.96)c

0.92–462.74

28

35

42.33 (19.08)b1

74.64 (35.30)b1

47.36–15,803.58 45

2112.47 (254.52)c 114.61–5291.53 30

4190.39 (1172.99)c 9.30–34,460.14 35

Lubrecht Experimental Forest 386.08 (128.71)a1

Mean (SE) Range

0–1307.47

N

10

2.84–289.84

0.92–462.74

15

14

46.07 (20.96)b1

46.70 (18.95)b1

1169.28 (366.52)c1

2136.27 (333.95)c1

4470.81 (1341.34)c1

56.38–5326.01

353.49–5291.53

518.63–15,684.59

15

17

14

Stump Hollow 164.39 (164.39)a1

Mean (SE) Range

0–328.77

N

2

2486.20 (949.47)c1

4.15–67.11

1.85–189.97

52.68–15,803.58

3

9

19

1110.18 (732.93)c1

5324.43 (3288.97)d1

114.61–2539.73

175.06–34,460.14

13

10

Vipond Park 159.13 (52.89)1a

Mean (SE) Range

7.14–536.22

N

10

132.32 (56.82)1a 1.39–525.96 10

94.79 (29.42)a1

614.09 (160.12)1b

2372.70 (460.71)1c

12.33–391.28

47.36–1805.11

161.75–4274.82

12

11

10

2802.55 (1654.65)1c 9.30–18,624.69 10

O. montium All sites combined 136.22 (36.74) a

Mean (SE) Range

0.00–360.59

N

13

2099.81 (1971.26)a 0.00–23,775.25 12

107.81 (35.38)a

1980.75 (839.40)a

2.97–843.37

2.48–19,233.28

26

34

182.41 (109.73)a 0.00–2326.89 21

2400.96 (1599.61)a 0.00–26,391.33 19

Lubrecht Experimental Forest 81.38 (37.04)a1

Mean (SE) Range

0–279.59

N

7

162.39 (80.97)a1 0–701.11 8

30.97 (10.81)a1

2575.94 (2326.54)a1

380.71 (324.96)a1 5624 (3614.19)a1

4.93–88.67

8.01–18,841.47

6.78–2326.89

7.39–26,391.33

7

8

7

8

Stump Hollow Mean (SE) Range N

248.06(85.30)a1

NA

80.76–360.59

NA

3

0

100.35 (36.18)a1

2132.07 (1144.44)a1

2.97–388.80

4.62–19,233.28

12

19

77.18 (55.19)a1 0–457.94 8

57.82 (24.43)a1 0–161.96 7

Vipond Park Mean (SE) Range N

152.35 (91.21)a1 19.06–326.84 3

5974.67 (5933.54)a1 197.46 (113.15)a1 24.26–23,775.25 4

17.87–843.37 7

889.82 (814.97)a1

91.34 (35.33)a1

54.76 (8.70)a1

2.48–5776.78

12.02–244.86

38.82–79.49

7

6

4

Same letters within rows indicate that means are not significantly different between temperatures. Comparisons between species at the same temperature were not performed due to differential sporulation responses to artificial media by the two fungi. Comparisons between sites at the same temperature were not performed for either species as ANOVA detected no interaction between temperature * species * site. There was no sporulation by O. montium from Stump Hollow at 10 °C SE standard error

capturing resources at a greater rate overall (F=9.67, df 1, 24, P<0.0048) than O. montium (Table 4). The interaction between temperature and site had a significant effect on the rate of resource capture during inter-specific competition (F=5.70, df 6, 258, P<0.0001). HSD tests detected no differences in the rate of resource capture between sites within temperatures

(Table 4). The interaction between temperature and species had a significant effect on the percentage of resource captured (F=7.84, df 3, 258, P<0.0001) as well as the rate of resource capture (F =4.92, df 3, 258, P=0.0024). G. clavigera captured a greater percentage of resources than O. montium at 10 °C (Tukey’s HSD; P<0.0001), 15 °C (Tukey’s HSD; P<0.0001),

Effects of Temperature on Mountain Pine Beetle Fungal Symbionts

Discussion

Fig. 4 Mean sporulation (spores/mm2) of Grosmannia clavigera (black line) and Ophiostoma montium (gray line) isolates (all sites combined). Points are mean sporulation values for individual isolates

and 25 °C (Tukey’s HSD; P<0.0231) (Table 4), as well as at a greater rate than O. montium at 10 (Tukey’s HSD; P<0.0001) and 15 °C (Tukey’s HSD; P=0.0024).

Temperature plays a major role in the life history, vital rates, and distribution of poikilotherms. It is not surprising, therefore, that it is also a major factor determining the formation, dynamics, and stability of their symbioses. For example, temperature influences the acquisition and loss of Xoozanthellae with corals, endosymbiotic bacteria with ants, and secondary bacterial symbionts with sap-sucking insects [12, 14, 20]. Despite the importance of temperature in maintaining (or disrupting) the stability of symbioses and, subsequently, in influencing the fitness of the interacting partners, its effects remain unstudied except in a handful of systems. In this study, we investigated how temperature may affect important metrics of fitness (growth and reproduction) of the two fungi associated with the MPB, and how it may mediate competition between the symbionts. We found that the mean growth rates of the two fungi are differentially affected by temperature. Growth rates of G. clavigera are significantly greater than those of O. montium at the lower temperatures tested while the converse was true for the warmest temperatures (Fig. 2). These differences are likely to result in differential rates of resource capture within trees over time which, in turn, would affect host dynamics. In the field, low temperatures are most likely to occur during late

Table 3 Mean (SE) growth rate (mm/day) of G. clavigera and O. montium when growing toward individuals of the same species (intra-specific competition) or toward individuals of the other species (inter-specific competition) Temperature

10 °C

Inter-specific competition: G. clavigera All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park Intra-specific competition: G. clavigera

1.38 (0.06)a1 1.69 (0.09)a 1.11 (0.07)a 1.39 (0.112)a

N 42 13 15 14

1.91 (0.09)b1 2.13 (0.10)ab 1.98 (0.13)b 1.64 (0.18)a

N 30 10 9 11

3.10 (0.11)c1 3.10 (0.23)b 3.08 (0.18)bc 3.13 (0.18)b

N 41 12 13 16

3.35 (0.11)c1 3.26 (0.19)b 3.47 (0.20)c 3.30 (0.17)b

N 42 13 15 14

All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park Inter-specific competition: O. montium

1.49 (0.11)a1 1.88 (0.25)a 1.11 (0.09)a 1.40 (0.07)a

46 18 15 13

2.19 (0.15)b1 2.70 (0.39)ab 1.91 (0.15)b 2.03 (0.15)ab

45 14 18 13

2.91 (0.09)c1 3.10 (0.13)bc 2.58 (0.22)bc 2.96 (0.13)b

42 12 10 20

3.23 (0.20)c1 4.07 (0.45)c 2.91 (0.11)c 2.58 (0.22)b

47 17 15 15

All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park Intra-specific competition: O. montium

0.99 (0.04)a1 1.01 (0.05)a 0.96 (0.06)a 1.00 (0.10)a

42 13 15 4

1.45 (0.09)b1 1.61 (0.11)a 1.67 (0.11)b 1.15 (0.16)a

29 9 9 11

2.88 (0.12)c1 2.69 (0.16)b 3.26 (0.17)c 2.73 (0.22)b

41 12 13 16

2.97 (0.12)c1 3.03 (0.17)b 3.04 (0.23)c 2.83 (0.24)b

42 14 15 13

0.97 (0.05)a1 1.05 (0.05)a 1.09 (0.07)a 0.71 (0.12)a

39 15 14 10

1.48 (0.06)b1 1.50 (0.05)a 1.70 (0.10)b 1.23 (0.16)b

46 20 13 13

2.62 (0.11)c1 2.70 (0.12)b 2.70 (0.18)c 2.38 (0.31)c

39 16 14 9

2.85 (0.09)c1 2.91 (0.10)b 2.95 (0.18)c 2.71 (0.19)c

42 14 12 16

All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park

15 °C

21 °C

25 °C

Same letters within rows indicate no difference in rate of resource capture between temperatures. Same numbers within columns indicate no difference in rate of resource capture between competition treatments within species (all sites combined only)

M. L. Moore, D. L. Six Table 4 Mean (SE) percent resource capture for G. clavigera and O. montium when growing toward individuals of the same species (intra-specific competition) or toward individuals of the other species (inter-specific competition) Temperature

10 °C

Inter-specific competition: G. clavigera All sites combined Lubrecht Experimental Forest Stump Hollow

56.35 (1.49)a1 59.15 (1.57)a 52.68 (2.06)a

N 42 13 15

57.01 (1.43)a1 56.41 (2.77)a 53.75 (2.03)a

N 30 10 9

52.73 (1.80)a1 54.62 (3.59)a 50.68 (2.19)a

N 41 12 13

53.10 (1.33)a1 52.24 (2.73)a 51.98 (2.07)a

N 42 13 15

Vipond Park Intra-specific competition: G. clavigera

57.68 (3.49)a

14

60.24 (2.31)a

11

52.97 (3.41)a

16

55.10 (2.24)a

14

All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park Inter-specific competition: O. montium

48.47 (0.95)a2 50.01 (0.22)a 46.29 (2.36)a 48.86 (1.92)a

46 18 15 13

49.42 (0.17)a2 48.92 (0.32)a 49.55 (0.26)a 49.77 (0.24)a

45 14 18 13

49.72 (0.41)a1 50.15 (0.85)a 48.46 (1.14)a 50.10 (0.38)a

42 12 10 20

49.71 (0.33)a1 50.16 (0.26)a 48.34 (0.60)a 50.56 (0.71)a

47 17 15 15

All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park Intra-specific competition: O. montium

40.74 (1.65)a1 36.65 (1.54)a 44.13 (2.32)a 40.91 (3.92)a

42 13 15 14

42.35 (1.29)a1 42.81 (2.01)ab 45.50 (2.19)a 39.39 (2.17)a

29 9 9 11

47.11 (1.43)b1 46.64 (2.65)ab 50.71 (1.92)a 44.55 (2.55)a

41 12 13 16

46.21 (1.53)b1 48.95 (2.82)b 44.83 (2.57)a 44.85 (2.58)a

42 14 15 13

47.34 (0.93)a2 45.16 (2.23)a 49.50 (0.34)a 47.58 (0.94)a

39 15 14 10

48.81 (0.38)a2 49.54 (0.35)a 48.93 (0.40)a 47.57 (1.14)a

46 20 13 13

49.69 (0.32)a1 49.59 (0.19)a 49.31 (0.44)a 50.45 (1.20)a

39 16 14 9

49.31 (0.34)a1 49.12 (0.31)a 49.95 (0.65)a 49.00 (0.72)a

42 14 12 16

All sites combined Lubrecht Experimental Forest Stump Hollow Vipond Park

15 °C

21 °C

25 °C

Same letters within rows indicate no difference in percent resource capture between temperatures. Same numbers within columns indicate no difference in percent resource capture between competition treatments within species (all sites combined only)

autumn, early winter, and early spring and would support greater resource capture and a greater prevalence of G. clavigera with the host at those times of year, as was observed in a field-based study by Adams and Six [1]. On the other hand, in late spring and summer, when temperatures often reach 30 °C or more, O. montium should excel at resource capture and increase in both its prevalence and in its effects on the host beetle. Resource capture is also likely to be affected by the general thermal conditions at a site resulting in greater numbers of MPB developing with and dispersing G. clavigera at sites that are overall cooler and O. montium at sites that are on average warmer. This prediction is supported by field observations that G. clavigera dominates at cool sites while O. montium dominates at hot sites [34]. While the temperature ranges supporting growth of G. clavigera and O. montium observed in this study are in close agreement with those reported in previous studies using smaller samples [8, 25, 31, 36], we observed substantially more variability within each species. Such variability may be critically important in maintaining the symbiosis, particularly in light of climate change (discussed further below). Both the amount of resource (area) captured in a tree as well as the timing of sporulation are important factors affecting fungal and beetle fitness and the maintenance of the symbiosis. The greater the area within the tree that is captured by a

fungus, the greater the potential number of hosts that can disperse it. However, to be dispersed, a fungus must also sporulate at the time the beetles eclose so that their spores are available to the beetles for feeding and packing in mycangia prior to emergence and dispersal. For efficient dispersal, the optimal temperature for sporulation is likely to be different than the optimal temperature for resource capture. In this study, the greatest mean sporulation of G. clavigera occurred at 30 °C (Fig. 4 and Table 2). A temperature of 30 °C is suboptimal for growth of this fungus; however, if it has captured substantial territory prior to the onset of warm conditions that often accompany beetle eclosion and dispersal, its ability to sporulate well at this temperature increases its odds of dissemination. MPB tends to disperse at temperatures between 22 and 32 °C [27]. Both fungi are capable of producing spores at these temperatures (Fig. 4 and Table 2). It is likely that both the degree of resource capture and the amount of spores produced at the time of beetle eclosion interact with temperature to determine the relative proportion of the two fungi dispersed by the beetles at a given time. The occasional co-occurrence of both fungi in the mycangia of individual beetles [34] is likely due to the coincidence of temperatures that support sporulation and the pupation of beetles in locations where the fungi interface within the tree. That the two fungi can occur together in single

Effects of Temperature on Mountain Pine Beetle Fungal Symbionts

pupal chambers further supports observations that the fungi do not exhibit strong antagonism toward one another. Results of the competition study confirmed the findings of previous studies indicating that interference competition does not occur between the two fungi [6–8]. G. clavigera and O. montium did not grow differently in response to the presence of con- and hetero-specifics than they did alone indicating that antagonism or facilitation does not occur regardless of temperature. Both fungi increased in rate of growth from 10 to 15 °C and held steady from 21 to 25 °C. However, temperature did affect competition outcomes and these were different for the two fungi. O. montium captured more resources at the lower temperatures during intra- than it did during interspecific competition (Table 4). In contrast, G. clavigera captured more resources at lower temperatures during inter- than it did during intra-specific competition (Table 4). Growth rates during competition (Table 3) differed from growth rates for the two fungi when grown without competitors (Table 1). These differences were likely due to differences in the type of arenas used in the two experiments. In the competition experiment, fungal expansion was constrained primarily to linear growth extending in two directions from the initial inoculation point. In the growth experiment, the fungi were able to grow out in all directions from inoculations made in the center of agar-filled Petri dishes. However, within experiments, outcomes were consistent for both studies; G. clavigera grew more quickly than O. montium at lower temperatures, and the growth rates of the two species were similar at intermediate temperatures. Our results indicate that G. clavigera and O. montium exhibit exploitation competition (where the outcome of competition is determined by the rate of resource capture). During inter-specific competition at the lower temperatures tested, G. clavigera captured resources at a greater rate than O. montium (Table 3) and was also able to capture more resources than O. montium at most temperatures (Table 4). Our results also suggest that, as temperatures increase due to climate change, O. montium may come to increasingly dominate this symbiosis. Temperature-driven models predict that G. clavigera will be lost in areas that experience even moderate warming [2]. While redundancy of nutritional symbionts in this system likely has supported a broad ecological amplitude for the host beetle, the loss or severe decline of one symbiont may act to reduce the range of the beetle in the future as warming increases. Because the superior symbiont is the partner that is expected to be marginalized and finally lost due to warming, this means that MPB populations will increasingly depend upon O. montium. This may result in lower overall fitness which may translate to an overall dampening of MPB population dynamics. Therefore, while warming is currently acting to exacerbate MPB impacts on forests by reducing tree defenses and enhancing beetle survival and development [29],

in the long term, it may actually begin to reduce the capacity of the beetle to develop widespread outbreaks. A caveat to the work presented here is that it was conducted in the lab on artificial media. Neither O. montium nor G. clavigera sporulate as readily on artificial media as they do on natural substrates [6, 8]. Likewise, growth on artificial media is likely different than that which occurs in trees due to differences in substrate structure, nutrients, secondary chemistry, and differences in virulence [35] of the two species. However, this study was necessarily confined to artificial media to control variability in the system and due to the difficulty in tracking fungal growth within trees or logs. Observational studies investigating effects of temperature on fungal growth and sporulation under field conditions, however difficult, will still be needed to fully describe how temperature influences the function and stability of this important symbiosis. Acknowledgments We thank David Affleck and Brian Steele for assistance with data analysis and Audrey Addison, Barbara Bentz, Jim Powell, Cory Cleveland, John McCutcheon, Caleb Craft, Joseph Caleb Dysthe, Pamela Erm, Brenna Hannapel, Dara McDevitt, Emily Newman, Austin Stewart, and Aspen Ward for general assistance. This work was supported by National Science Foundation grant DEB 0918756 to Diana L. Six. The authors declare that they have no conflict of interest.

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