University of British Columbia Department of Forest and Conservation Sciences

Yiyuan Zhang FRST498

B.Sc. Thesis

Phenotypic Characterization Fungal Symbionts of the Mountain Pine Beetle (MPB)

By Yiyuan (Sophia) Zhang

Supervisor Richard Hamelin Yousry El Kassaby

April 2013

Yiyuan Zhang FRST498

Abstract

The mountain pine beetle (MPB), Dendroctonus ponderosae ,causes one of the most serious diseases of pines in western North America. The three primary fungi associates carried by the beetles are Grosmannia clavigera, Leptographium longiclavatum and Ophiostoma montium. Multipartite symbioses contribute to the success of beetle survival, reproduction and outbreaks. In this study, phenotypic experiments were conducted in vitro by growing isolates of the fungal associates belonging to the three species distributed across western North America. Both the growth rate and area under the curve were measured and compared inta-specifically and inter-specifically. The results suggest that there are interspecific and intraspecific differences in growth rate at different temperatures, supporting the coexistence of fungal species in multipartite relationships.

Keywords mountain pine beetle (Dendroctonus ponderosae), Grosmannia clavigera,

Leptographium longiclavatum, Ophiostoma montium, growth rate, area under the curve.

Yiyuan Zhang FRST498

1. Introduction

1.1. MPB epidemics

The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins (Coleoptera:

Curculionidae, Scolytinae), is an indigenous species that coevolved with coniferous forest ecosystems in western North America (Bentz 2010). MPB can infest almost all species of pine, including ponderosa pine (P. ponderosa, P. Laws. Ex C. Laws), whitebark pine (Pinus albicaulis), limber pine (Pinus flexilis) and jack pine (P. banksiana Lamb.) (Gibson et al. 2009; Safranyik & Carroll 2006; Nealis &Peter

2008 ), among which lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia

Engelm) is the principal host of MPB (Shore et al. 2006). MPB causes one of the most serious diseases of pines in western North America (Cullingham et al. 2011; Kurz et al.

2008), with a widespread from northern Mexico to British Columbia (Carroll 2006). It is reported that MPB has infested 10 million ha of forests by 2006 in British

Columbia (Nealis &Peter 2008). The outbreaks and epidemics of MPB have caused massive mortality of lodgepole, resulting in enormous economic losses (Gibson et al.

2009).

1.2. Beetle - Fungal Symbiosis

The MPB is associated with a number of micro-symbiotic fauna, including mites, bacteria and fungal species (Paine et al. 1997; Six 2003; Six and Klepzig 2004).

Fungal diversity on the MPB has been extensively surveyed by Lee et al (2006a).

Yiyuan Zhang FRST498

Nine species of fungi were isolated and identified from MPB collected from infested lodgepole pines in British Columbia (Lee et al. 2006a). The nine species are

Ophiostoma montium, O. clavigerum, an O. minutum-like species, an O. nigrocarpum-like species, Leptographium longiclavatum, L. terebrantis,

Entomocorticium dendroctoni, an Entomocorticium species and an Ambrosiella species (Lee et al. 2006a). The three primary fungi associates carried by the beetles are Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer, and

Wingfield; Leptographium longiclavatum Lee, Kim, and Breuil; and Ophiostoma montium (Rumbold) von Arx (Lee et al. 2005, 2006a).

Fungi species in the family of Ophiostomataceae (Ascomycotina) play an important role in the beetle-fungal interactions (Roe et al. 2011a; Six 2003a). The multipartite symbioses contribute to the success of beetle survival, reproduction and outbreaks (Roe et al. 2011a).

1.3.Mutualistic interactions

Fungal associates of the Mountain Pine Beetle (MPB), many of which are

Ophiostomoid fungi of Ascomycota, have a beneficial mutualistic relationship with

MPB (Lee et al. 2006a; Bleiker et al. 2009). The fungi are inoculated in tree phloem when beetles build galleries and are transported by the beetles to new host trees

(Whiteney and Farris 1970; Bleiker et al. 2009). The fungi are carried on the exoskeleton or in paired sac-like mycangia on the maxillary cardines of both sexes of

Yiyuan Zhang FRST498 adults (Whiteney and Farris 1970). Mycangia are specialized integumental structures by which fungi gains access to new hosts (Batra 1963).

Fungal symbionts provide a number of benefits to the MPB, such as nutritional supplementation, overcoming tree defense and modifying environmental conditions

(Bleiker and Six 2007; Lieutier et al. 2009; Six 2003b; Six and Paine 1998).

The nutritional role of fungal associates is important for the growth of beetles

(Bleiker and Six 2007). Fungi can increase beetle size by increasing nitrogen levels in tree phloem (Six and Paine 1998; Bleiker and Six 2007). Beetles emerging with G. clavigera and/or O. montium were larger than the ones that do not carry these fungi

(Bleiker and Six 2007). Previous studies indicated that the nitrogen levels in phloem colonized by fungal associates is increased by 40% two months after attack, providing a source of nitrogen for the MPB (Bleiker and Six 2007). In addition, G. clavigera is more efficient in nitrogen concentration because beetles carrying G. clavigera have a greater size than beetles carrying O. montium (Bleiker and Six 2007). What is more, fungi symbionts/associates provide nutritional sources for both beetle adults and larvae (Adams and Six 2007; Bleiker et al. 2009). Beetle larvae feed on phloem colonized with fungal hyphae (Adams and Six 2007), and teneral adults feed on fungal conidia in pupal chambers (Leach et al.1934, Whitney 1971, Six and Paine

1998).

Previous studies have shown that the fungi may also play a role in killing trees by overwhelming tree defenses and interrupting translocation (Paine et al. 1997). Some

Yiyuan Zhang FRST498 fungi symbionts, such as Grosmannia clavigera can help beetle colonization of trees by occluding tracheid and causing water deficit (Arango-Velez et al. 2011).

1.4.Temperature

It is reported that the global land and ocean surface temperature increased 0.85 °C from 1880 to 2012 as a result of the greenhouse gases emissions (IPCC 2013).

The population of MPB is sensitive to the shifts of temperature by changing population behavior as well as the geographical distribution (Powell and Logan 2005,

Carrol 2006). Many conductive conditions under global warming contribute to the beetle expansion. The hot summers and mild winters as a consequence of climate change have favored beetle overwinter survival and reproduction (Carrol 2006). What is more, recent fire suppression has increased the number of mature pines (Taylor &

Carroll 2004; Taylor et al. 2006) and reduced the resistance of host trees (Bentz 2010).

In addition, water deficit under global warming can potentially increasing tree susceptibility to MPB and the associated fungi (McDowell et al. 2008).

Because of climate change, there are more climatically favorable habitats for mountain pine beetle (Carroll et al. 2003). The range of MPB is predicted to exceed the historic range and expand northward and eastward (Carroll et al. 2003). MPB was confined before by the Rocky Mountains historically (Carrol 2006). It is reported that MPB has spread beyond the Rocky Mountain and reached northeastern British

Columbia and north-central Alberta (Carrol 2006; Robertson et al. 2009; Bentz et al.

2010), where forest composition shifts from lodgepole pine to jack pine (Cullingham

Yiyuan Zhang FRST498

2011).

Jack pine is widely distributed across boreal forest in Canada and extends from

Alberta to Nova Scotia (Cullingham 2011). Jack pines are naïve hosts that have not been exposed to MPB and thus are susceptible (Cullingham 2011; Nealis &Peter

2008). Recent studies have demonstrated that MPB can attack pure jack pine successfully (Cullingham 2011). The MPB will potentially explore novel environments and expand eastward to Alberta where the forest is mainly composed of jack pine (Robertson et al. 2009; Bentz et al. 2010; Carroll 2006).

1.5.Adaptation of fungi associates to different environment conditions

Temperature is a critical environmental factor that influences the growth of MPB associated fungi (Rice et al. 2008), and affects fungal abundance and composition in both space and time (Six and Bentz 2007; Adams and Six 2007; Rice and Langor

2009; Rice et al. 2008). Previous studies have shown that MPB fungi symbionts have different temperature tolerances (Adams and Six 2007; Rice et al. 2008). The optimal growth temperature of the three species is different. G. clavigera is more adapted to cooler conditions, and has a lower optimal growth temperature than

O.montium (Six and Bentz 2007; Solheim and Krokene 1998). Similarly, the optimal growth temperature of L. longiclavatum is also lower than O.montium, and thus is more abundant in northern latitudes, such as northwestern Alberta (Rice and Langor

2009).

This difference determines the spatial and temporal distribution of fungi, which

Yiyuan Zhang FRST498 alter the distribution of MPB indirectly (Adams and Six 2007). Therefore, the research on temperature tolerance of fungi symbionts of MPB is important in order to predict fugal response to climate change as well as preventive actions.

To date, temperature tolerance and growth rate of these three fungal associates have been studied in limited samples sizes without previous knowledge of population structure. The influence of temperature on the growth rate of the MPB associated fungal species has only been investigated in British Columbia (BC) and Alberta (Rice et al. 2008), and inferences were limited to small sample sizes. However, population expansion associated with the shifts in temperature calls for more sampling to have a better understanding of the patterns of fungal community composition (Roe et al.

2011a).

In addition, next generation sequencing technologies have enabled the acquisition of several fungal genomes, allowing the study of population genetic structure (Li et al.

2008). Reverse ecology links genomic data with the adaptive traits (Levy and

Borenstein 2012), making feasible to find the evolved phenotypic differences by identifying genes under environmental selection (Li et al. 2008). Several genes involved in the mutualistic relationship have been identified in our laboratory.

In this study, the phenotypic experiments were conducted in vitro by growing isolates of the fungal associates belonging to 3 species (Leptographium longiclavatum,

Ophistoma montium, Grosmannia clavigera) distributed across western North

America. The isolates of these three species represent most of the entire distribution of the MPB and were selected to represent three distinctive populations identified

Yiyuan Zhang FRST498 previously with microsatellites and single-nucleotide polymorphisms (SNPs). The characteristics of the fungal isolates were compared and contrasted both intra-specifically and inter-specifically. Statistical analyses were conducted to determine if there are differences and if those differences are associated with species or genotypes. We hypothesize that there are host, vector or climate- adaptations in the

MPB associated fungi along latitudinal and altitudinal gradients.

2.Material and Methods

2.1.Population groups and fungal isolates

In total I selected 53 isolates for this experiment, representing18 isolates of L. longiclavatum, 17 isolates of O. montium and 18 isolates of G. clavigera. These isolates represent three populations across western North America according with distinctive geographic characteristics, genetic structure (Tsui et al. 2012), and MPB history.: “North BC and Alberta”, “Central BC” and “Western USA”. In each group, three locations were chosen based on the availability of samples. For each species in each group, I selected 6 individuals (Table 1). Isolates in Northern BC and

Central BC (M0024 collections) were collected during 2010, while isolates of US group (033 collections) were sampled in 2011. The fungi materials were obtained from different sources, either from the phloem of Pinus contorta or beetles found in

Pinus contorta.

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Table 1. Information for isolates of L. longiclavatum, O.montium, and G.clavigera in North BC

and Alberta, Central BC and Western USA.

L.longiclavatum O.montium G.clavigera Group Location Time phloem bettle phloem bettle phloem bettle 1 1 2 Fox creek M0024 TRIA58 TRIA37 TRIA251 TRIA292 2 2 2 TRIA47 TRIA3 TRIA1142 TRIA18 TRIA260 Grande Paririe M0024 North BC and TRIA261 Alberta 3 3 2 TRIA55 TRIA2 TRIA1090 TRIA45 Peace Area M0024 TRIA56, TRIA204 TRIA1056 TRIA268 TRIA1118 2 1 2 Canmore M0024 TRIA16 TRIA19 TRIA1106 TRIA120 TRIA210 2 3 2 TRIA61 TRIA38 TRIA1060 TRIA178 Central BC Kimberley M0024 TRIA1081 TRIA192 TRIA1086 2 1 2 Peachland M0024 TRIA63 TRIA1140 TRIA110 TRIA25 TRIA176 2 3 2 TRIA376 TRIA523 TRIA440 Colorado M033 TRIA378 TRIA377 TRIA437 TRIA442 2 0 2 Western USA California M033 TRIA893 TRIA883 TRIA891 TRIA890 2 3 2 Idaho M033 TRIA787 TRIA832 TRIA827 TRIA776 TRIA786 TRIA825 TRIA911

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2.2.Temperature tolerance experiment:

All fungi isolates were taken from 4ºC and pre-grown at room temperature to make sure they can survive and to get pure cultures. The fungi isolates were then transferred to 25ml malt extract agar (MEA) on petri dish to grow for 7 days at room temperature to make sure they were in optimal growth conditions. For each temperature, a new fresh stock of fungi was used to ensure the similar initial condition.

A plug of the same size of the media containing fungal tissue was collected at the edge of the petri dish in order to start each experiment. Plugs were inoculated on a new petri dish containing 25ml malt extract agar (MEA). The plates were placed in the incubator under four temperature conditions: 5, 15, 25, 35ºC and one at room temperature (23ºC).

2.3.Measurement

To determine growth rate, the plug was placed in the middle of the petri dish and four lines were drawn perpendicular to each other from the middle of each plug.

Growth rate was marked across these four lines on the back of each petri dish at the same time every day. The growth rate for each day was determined as the distance between two labels divided by one day. The measurements were continued until the mycelia cover the whole petri dish. Each isolate was repeated three times.

2.4.Data Analysis:

Area under the curve of fungal isolates was calculated using the growth rate

Yiyuan Zhang FRST498 according to Trapezoidal rule. Both the growth rates and area under the curve were compared both intra-specifically and inter-specifically by using Analysis of variance

(ANOVA) generated by Excel (2007).

3. Results

3.1.Optimal growth temperature

All the isolates were able to grow between 5 ºC and 25 ºC. At 5 ºC, all the species could still survive with a minimum growth rate. The growth rate at 5 ºC for G. clavigera was 0.72mm/day. L. longiclavatum and O. montium grew at a rate of

0.67mm/day and 0.71 mm/day separately at 5 ºC. At 35 ºC, none of the species were able to grow, and no growth was observed.

L. longiclavatum grew optimally at 25 ºC in North BC, Alberta and Western USA, while it grew optimally at 23 ºC in Central BC (Fig.1, Fig.2, Fig.3).

In general, the optimal growth temperature for G. clavigera is 25 ºC for all the groups (Fig.1, Fig.2, Fig.3). At optimal growth temperature (25 ºC), the population in

Central BC has the highest growth rate, and the population from North BC and

Alberta has the lowest growth rate, and USA population is intermediate (Fig.5).

The optimal temperature for O. montium is 25 ºC for all the populations. At 25 ºC, population in Central BC grows fastest, and the population in North BC and Alberta grows slowest. The population from Western USA has a medium growth rate (Fig.6).

Yiyuan Zhang FRST498

Table 2. Averaeg growth rate (mm/day) and Standard Deviation for L. longiclavatum, G. clavigera and O. montium in Western USA, Central BC and North BC and Alberta.

Species Geographic location Western USA Central BC North BC and Alberta 15C 23C 25C 15C 23C 25C 15C 23C 25C L. 3.87 4.6 5.2 5.31 6.5 6.9 3.76 5.4 5.1 longiclavatum (±1.6) (±1.5) (±1.9) (±2.9 (±3.2) (±4.1) (±2.3 (±2.7) (±2.6) ) ) G. clavigera 4.5 6.1 6.4 5.16 6.6 6.7 4.08 4.8 5.7 (±2.2) (±2.9) (±3.1) (±2.1 (±3.3) (±2.8) (±2.1 (±2.9) (±2.4) ) ) O. montium 3.2 4.5 5.2 3.5 5.3 5.6 3.2 4.8 5.0 (±1.6) (±1.4) (±2.7) (±2.2 (±2.1) (±2.5) (±1.7 (±1.6) (±2.1) ) )

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30000.00

25000.00 North BC and Alberta 20000.00

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Fig. 1. Area Under the Curve of isolates of G.clavegera, L. longiclavatum and O. montium from North BC and Alberta at temperatures of 5-35 ºC.

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Fig. 2. Area Under the Curve of isolates of G.clavegera, L. longiclavatum and O. montium from Central BC at temperatures of 5-35 ºC.

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25000.00 G. clavigera Western USA 20000.00 L. longiclavatum O. montium 15000.00

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Fig. 3. Area Under the Curve of isolates of G.clavegera, L. longiclavatum and O. montium from Western

USA at temperatures of 5-35 ºC.

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25000.00 Leptographium longiclavatum 20000.00

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0.00 0 5 10 15 20 25 30 35 40 Fig. 4. Area Under the Curve of isolates of L. longiclavatum from North BC and Alberta, Central BC and Western USA at temperatures of 5-35 ºC.

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25000.00 Grosmannia clavigera

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0.00 0 5 10 15 20 25 30 35 40 Fig. 5. Area Under the Curve of isolates of G.clavegera from North BC and Alberta, Central BC and Western USA at temperatures of 5-35 ºC.

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Fig. 5. Area Under the Curve of isolates of O. montium from North BC and Alberta, Central BC and Western USA at temperatures of 5-35 ºC.

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3.2.Interspecific variation

In general, G. clavigera outcompeted other species in North BC and Alberta and

Western USA except in Central BC where G. clavigera and L. longiclavatum have similar growth rate at all temperatures tested (Table2). In North BC and Alberta, G. clavigera grew faster than L. longiclavatum and O. montium at all treatment temperatures except at 23 ºC. At 23 ºC, G. clavigera grew more slowly than L. longiclavatum and faster than O. montium. O. montium grew most slowly at 15, 23 and 25 ºC, but the difference is not significant (P>0.05).

In Central BC, G. clavigera and L. longiclavatum had a similar growth rate, with

L. longiclavatum growing a little bit faster than G. clavigera. O. montium still had the least growth compared with G. clavigera and L. longiclavatum.

In Western USA, G. clavigera grew more quickly than either O. montium or L. longiclavatum. In contrast, L. longiclavatum other than O. montium has a minimum growth. A significant difference was found at 23 ºC among the species (F=6.705048, df=2, P=0.008311).

In North and Alberta, G. clavigera is more variable than both O. montium and L. longiclavatum except at 25 ºC (Table2). In Central BC, L. longiclavatum is the most variable species because the SD values are the highest (Table2). In Western USA, O. montium has the biggest variance than other species with higher SD values (Table2).

3.3.Intraspecific variation:

For all the species, isolates from Central BC grew fastest than ones from North

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BC and Alberta and Western USA. Among the three fungal associates of MPB, L. longiclavatum is the species with the highest intraspecific variation. In particular, isolates of L. longiclavatum from central BC grow faster than the other two locations at 15 ºC (F=8.45, df =2, P=0.003478), 23 ºC (F=7.68, df=2, P=0.005043), and 25 ºC

(F=3.98, df=2, P=0.040899). Isolates from western USA shows the slowest growth rate at 15 and 23 except at 25 ºC, where they have similar growth rate with isolates from North BC and Alberta.

In contrast to L. longiclavatum, isolates of G. clavigera have a more homogeneous growth rate. Despite the fact that isolates from Central BC population grew faster than isolates from the other locations, the difference between the populations of G. clavigera was not significant (P>0.05).

O. montium shows the least variation across all three species, and thus is the most homogeneous species. Isolates of O. montium from different locations also differed in growth rate, but the difference among the populations is not significant (P>0.05).

4. Discussion:

The beetle-fungal symbiotic system consists of three main Ophiostomatoid fungi that coexist in multipartite relationships (Six and Bentz 2007). The Mountain

Pine Beetle has a wide range of distribution in North America, so the composition and abundance of fungal symbionts are influenced by several abiotic and biotic conditions.

The difference of temperature tolerance of fungal symbionts determines the spatial and temporal distribution of fungi community (Roe et al. 2011a). The Mountain

Yiyuan Zhang FRST498

Beetle has a broad host range of pines, including almost all the native pines in western

North America (Rice et al 2007). The pine hosts produce oleoresin-based chemicals to defend beetles and fungi, including monoterpenes, diterpenes and sesquiterpenes which are fungistatic or fungicidal (Wang et al. 2013). Given the various environmental factors that the beetle-fungi complex encounters, it is an evolutionary advantage to have a multipartite fungal symbionts to secure that there is at least one fungal symbiont at all times.

There are differences among the main three fungal associates of MPB. G. clavigera is a primary fungal symbiont and has a long evolutionary history with the

MPB, while O. montium and L. longiclavatum are recent invaders (Six & Paine 1999).

G. clavigera and L. longiclavatum are carried in mycangia of beetles exclusively, and

O. montium are transported by both the mycangia and exoskeleton (Six 2003b; Lee et al. 2005; Bleiker et al. 2009 ). The virulence is also various among the three fungal species. G. clavigera is more virulent than L. longiclavatum and O. montium (Reid et al. 1967; Solheim & Krokene 1998; Lee et al. 2006b; Rice et al. 2007; Plattner et al.

2008). Previous studies indicated that the three species have significant population differentiation (Roe et al. 2011b). G. clavigera and L. longiclavatum are closely related phylogenetically, while O. montium is more distant (Zipfel et al. 2006;

Alamouti et al. 2009).

All the differences resulted in the shift in abundance of three fungi associates through the range. The abundance changes as a function of latitude. The abundance of

G. clavigera decreases when the latitude increases, while the abundance of L.

Yiyuan Zhang FRST498 longiclavatum increases with increasing latitude (Roe et al. 2011 b). O. montium also decrease in abundance as the latitude increases, but the rate of change is less than that of G. clavigera (Roe et al. 2011 b).

The genetic diversity also varies among the three species. Using microsatellite markers, Tsui et al (2012) investigated the population structure of G. clavigera. The current outbreak from North BC and Alberta is less diverse than that from Central BC

(Tsui et al 2012). L. longiclavatum has a higher genetic diversity in Central BC and a decreased diversity on the North BC and Alberta as well as in Western USA. In contrast, G. clavigera has a similar genetic diversity for the two groups identified with

SNPs.

Previous studies indicated that the three species have different temperature tolerances which determine the spatial and temporal pattern of fungal symbionts and maintain the complex multipartite symbiosis (Rice et al. 2008). In this study, we showed a more complete picture of the range of temperature tolerance of the three fungi by analyzing all three species with multiple isolates from a wide range of distribution in western North America.

4.1.Interspecific variation

It has been reported that O. montium is better adapted to higher temperatures than

G. clavigera (Six and Paine 1997; Solheim and Krokene 1998). At 30ºC, O.montium still grows at 90% of its optimal growth rate, while the growth rate of G. clavigera and L. longiclavatum is only 10% of optimal growth rate (Rice et al. 2008). Other

Yiyuan Zhang FRST498 studies also found that G. clavigera grows faster at cooler temperatures, while

O.montium grows better at warmer temperatures (Six & Bentz 2007; Rice et al. 2008),

Other more recent studies have found that L. longiclavatum has a similar environment tolerance with G. clavigera (Lee et al. 2005, 2006b; Rice et al. 2008), but is more prevalent in northern locations than G. clavigera. As the latitude increases,

G. clavigera is replaced by L. longiclavatum (Roe et al. 2011b), suggesting that L. longiclavatum should be better adapted to colder temperatures (Roe et al. 2011a,

2011b).

Our results provide evidence that these three species have differences in growth rate at different temperatures. For example, in Northern BC and Alberta, G. clavigera has a higher growth rate than the other two species. In Central BC, both G. clavigera and L. longiclavatum have a higher growth rate than O.montium. In Western USA, L. longiclavatum grows most quickly, and the difference is significant at 23 ºC. All the results support the argument that the assembly of the multipartite symbiosis is partially maintained by these differences in response to temperature. The results also indicate that O.montium is adapted to regions with higher average temperatures, while

G. clavigera and L. longiclavatum are more adaptive in colder areas. So far,

O.montium will be outcompeted by the two other species. However, more critical temperatures, such as 30 ºC, should be analyzed before a more definitive conclusion can be drawn.

Yiyuan Zhang FRST498

4.2.Intraspecific variation:

It has been found that isolates of G. clavigera from British Columbia and

California have a faster growth rate than that from Alberta at 25ºC (Rice et al.,

2008).The optimal growth temperature for isolates from Idaho/Montana and Alberta are lower than those isolates from British Columbia and California (Rice et al., 2008).

Current evidence from population genetic analyses from two species, L. longiclavatum and G. clavigera, indicate lower genetic diversity in the populations of northern Alberta (North BC and Alberta population), suggesting bottleneck due to the recent colonization (Tsui et al. 2012).

In general, our results suggest that isolates from Central BC of all three species have a higher growth rate than isolates from the other locations. It indicates that central BC could harbor more genetic diversity where the fungal symbionts have coevolved for longer time than those from the other places. In particular for L. longiclavatum, the difference of growth rate among the three locations is significant

(Fig.4). There have been more historic records of previous outbreaks from Central BC where the MPB is speculated to exist longer. Alberta has just been colonized during the recent outbreak, and genetic diversity studies have shown that Alberta has a fraction of the genetic diversity that is present in Central BC. The populations collected from this region are closer together than other populations from Central BC, reflecting a small variation. Therefore, our results suggest that there is a reduced variance in growth rates of the three species from North BC and Alberta, and that the growth rates are faster at lower temperatures from this population.

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Isolates from the Western USA of L. longiclavatum also have a lower genetic diversity. However, all the fungal samples in this study were collected from the main pine host, lodgepole pine (P. contorta), which is less abundant in Western USA. The results in this study could only reflect a decrease genetic diversity for lodgepole pine host in Western USA in comparison with Central BC samples. There more pine species where the fungal species can be found in Western USA. A more diversity of pine hosts could be investigated in future studies.

The results indicate that there are significant differences in the growth rate among the isolates from the three groups of L. longiclavatum, but not in G. clavigera and O.montium. This is in accordance with a higher population structure of L. longiclavatum (based on SSR and SNPs) than to G. clavigera. It is also in congruence with their sexual reproduction. G. clavigera is mainly reproduced asexually, and the sexual state (teleomorph) has been rarely found in the laboratory (Lee et al. 2003).

O.montium has a higher sexual reproduction than G. clavigera and L. longiclavatum.

Generally, we found that there are different levels of variation for different species in response to temperature (the SD for each species in Table2). Species with less variance have a similar response to the environment, while species that have more variance have more diverse responses. The level of variation might indicate that the population has a higher capacity to adapt to changes in the environment. Thus, a population with a higher response might indicate more genetic variations in that pool.

Previous studies have shown that O.montium has a higher degree of variation in the response to temperature than G. clavigera (Moore et al. 2013). However, this

Yiyuan Zhang FRST498 conclusion is a simplified version of a more complex response of this trait. In North

BC and Alberta, G. clavigera has the biggest variance with the highest SD values

(Table2). In Central BC, it is L. longiclavatum that has the highest level of variation

(Table2). O.montium is the most variant species only in Western USA (Table2).

Therefore, the interesting finding in this study is that the species that has the highest variation response to the temperature changes depending of the location where the fungi samples were collected.

5. Conclusion

Our studies investigated the different response to the temperature of three main fungal associates of the MPB over a large-scale geographic range. By growing isolates of the fungal associated belonging to three species at different temperatures, both the growth rate and area under the curve were measured and compared inta-specifically and inter-specifically. The results suggest that there are differences in growth rate among the three species and among the three locations within each species at different temperatures, supporting the coexistence of fungal species in multipartite relationships.

However, this experiment was conducted on Petri dishes in the laboratory, so the results may be different from natural conditions. Although previous studies showed that the fungal distribution in the field accords with the in vivo results of temperature tolerances (Adams and Six 2007), future studies should still focus on the experiments on live trees. Also, this study did not include all the temperatures. More temperatures,

Yiyuan Zhang FRST498 such as 10, 20 and 30 ºC should be investigated in future research. In addition, all the fungal materials were collected from one host species which is lodgepole pine (P. contorta). To further understand the interaction of MPB with novel host species under global warming, more pine specie should be surveyed to determine the changes of fungal community of MPB in order to predict the risk of outbreak in the future.

Acknowledgements

I wish to acknowledge the help from Dr. Richard Hamelin for the directions of the whole experiment. I also acknowledge Dario I. Ojeda Alayon for the help in the laboratory and the revision of the paper. I also thank Dr. Valerie LeMay for the help with the experiment design.

Yiyuan Zhang FRST498

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