Do bark beetles facilitate the establishment of rot fungi in Norway spruce?

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Do bark beetles facilitate the establishment of rot fungi in Norway spruce? Ylva PERSSON*, Katarina IHRMARK, Jan STENLID Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, SE-75007 Uppsala, Sweden

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abstract

Article history:

Bark beetles, mycelia and wood were sampled from the vicinity of insect galleries in the

Received 20 August 2010

bark of Picea abies high stumps of four different age classes in southeastern Sweden.

Revision received 23 December 2010

Molecular methods were used for fungal species identification. From 203 samples a total of

Accepted 20 January 2011

21 fungal taxa were found, including 12 ascomycetes and 9 basidiomycetes. Of the fila-

Available online 24 March 2011

mentous fungal species, 50 % were found both in bark and bark beetles, and 37 % were

Corresponding editor:

found in bark, wood and bark beetles. Yeasts dominated in stumps that were 1-yr old and

Lynne Boddy

in control samples without insect activity. In 2- and 3-yr-old stumps, filamentous ascomycetes were present but also common wood decay basidiomycetes such as Stereum

Keywords:

sanguinolentum, Phlebiopsis gigantea, Trichaptum abietinum and Fomitopsis pinicola were found

Bark beetle

as mycelia associated with insect galleries and on bark beetles. The results indicate that

DNA sequencing

insect facilitation of the establishment of wood decay fungi cannot be neglected.

Fungal colonization

ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved.

High stumps PCR Picea abies T-RLFP Wood decay basidiomycetes

Introduction About 30 % of the 25 000e30 000 Fennoscandian multicellular forest species depend on dead or dying wood for some part of their life cycle, and it is thus a key component for biodiversity in these forests (de Jong et al. 2004). The most species-rich groups of saproxylic organisms in Fennoscandia are fungi and insects, represented by more than 2 500 and 3 000 taxa, respectively (Petersen 2003; de Jong et al. 2004). Due to the introduction of intensive forestry practices at the beginning of the 20th century, the amount of dead wood in forests has decreased (Fridman & Walheim 2000). In accordance with forest certification schemes such as the Programme for the Endorsement of Forest Certification (PEFC) and the Forest Stewardship Council (FSC), one way of increasing the amount

of dead or dying wood in boreal forest is to leave high stumps after clear felling (Jonsell et al. 2004). Dead wood is a suitable habitat for both insect and fungal species and the interactions range from fungal pathogens of insects to insect grazing of fungi, and from ephemeral connections to truly mutualistic relationships (Carlile & Watkinson 1994). The symbiotic relationship between ambrosia fungi and ambrosia beetles is well known (Muller et al. 2002). Ambrosia beetles make up around 3 400 of the 7 500 species in the weevil subfamily Scolytinae (Mueller et al. 2005), and can significantly increase the fungal decomposition of bolts of spruce trees by boring into the wood (Muller et al. 2002). Ambrosia beetles feed exclusively, or near exclusively, on fungi (Mueller et al. 2005), including basidiomycetes, ascomycetes, yeasts and mitosporic fungi (Carlile & Watkinson 1994). The

* Corresponding author. Tel.: þ46 18 672376; fax: þ46 18 673599. E-mail address: [email protected] (Y. Persson). 1754-5048/$ e see front matter ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2011.01.005

Do bark beetles facilitate the establishment of rot fungi?

female beetle carries the ambrosia fungi in glandular invaginations on the surface of their body called mycangia (Carlile & Watkinson 1994; Paine et al. 1997). The wood is inoculated when the female beetle bores tunnels into the sapwood and exudes yeast-like fungal cells from the mycangia before laying eggs. This ensures that there is a ready supply of food available when the larvae hatch (Carlile & Watkinson 1994). Some of the primary saproxylic bark beetles, including members of Pityogenes sp. and Crypturgus sp. that colonize living conifer trees, also carry fungi in unspecialized structures on their body surface. Primary scolytid species can sometimes kill a considerable number of living trees if there is an explosion in the population (Paine et al. 1997). Attacks by some species, for example, Dendroctonus ponderosae and Ips typographus, are thought to be aided by aggressive blue-stain fungi vectored by the beetles (Paine et al. 1997; Krokene & Solheim 1998; Rice & Langor 2009). Important factors that affect the fungal community involved in wood decomposition are the physical and chemical properties of the host tree, the micro climate of the forest site and how the tree died (Sippola & Renvall 1999). Wood is made up of lignin, cellulose and hemicelluloses, which are attractive to different fungal species depending on their ability to decompose the different wood components (Petersen 2003). Brown rot fungi, such as Fomitopsis pinicola, preferentially decompose the wood cellulose and hemicellulose, whereas white rot fungi, such as Stereum sanguinolentum, are able to utilize lignin as well as cellulose and hemicellulose (Rayner & Boddy 1988). Most basidiomycetes have small (4e24 mm), often asymmetric, spores that are actively discharged when mature. The most common method of spore dispersal is thought to be passively by wind (Rayner & Boddy 1988; Edman et al. 2004). However, insects may facilitate the spread by carrying spores on their body and transporting them to a suitable site. Insects may also help fungal spores to penetrate the bark of trees. With parental tracking, Guidot et al. (2003) have shown that the sexual cycle of the fire-dependent fungus Daldinia loculata can only be completed with the aid of pyrophilous insects. The insects acted as vectors for the male and female fungal gametes within a localized burned forest site by grazing on the conidia and flying between nearby trees, enabling the male gametes to fertilize the female gametes (Guidot et al. 2003). The beetle Dendroctonus pseudotsugae has also been shown to act as a vector in the transfer of the brown rot fungus F. pinicola to trees and may thereby significantly advance the initiation of wood decay (Harrington et al. 1981). Our aim was to study the fungal flora and its possible association with bark- and wood-boring insects in mechanically created high stumps of Norway spruce (Picea abies). The main questions were whether bark beetles have the potential to facilitate the establishment of wood decay fungi and whether the fungal flora differs between 1-, 2- and 3-yr-old high stumps.

Materials and methods Collecting mycelia, wood and insect material Samples were collected from mechanically created high stumps (approx. 3 m in height, and 20e40 cm in diameter at

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1.3 m above ground) of Norway spruce of four different ages. The high stumps were created in the springtime of consecutive years at four clear cut sites in southeast Sweden: the stumps of newly cut trees (i.e. year zero) were located at Kungstomt (N60 70 56.73" E17 480 59.80"); 1-yr-old stumps (i.e. the trees were cut 1 yr before samples were collected) were located at Skyttorp (N60 50 27.00" E17 440 32.11"); 2-yr-old stumps were located at Gimo (N60 110 31.65" E18 70 20.53"); and € 3-yr-old stumps were located at Osterbybruk (N60 90 48.65" E18 20 38.30). In total, 66 mycelia samples, 87 wood samples, 18 wood control samples (collected where no insect activity was noted) and 32 insect samples were collected from 16 high stumps, divided equally between the four clear cut sites plus two control trees. Bark at breast height (approx. 1.3 m above the ground) with signs of bark beetle activity was lifted off using a knife, and samples of mycelia were scraped off the interface of the collected bark sample and placed in a microcentrifuge tube. Bark samples (approx. 15  15 cm) were collected where mycelia and bark beetle galleries were visible. The bark was carefully examined and any bark beetles in the bark were collected for identification and further examination. The totally dominating species of bark beetles were Pityogenes chalcographus and Crypturgus sp., whereas e.g. I. typographus was absent. The beetles were examined molecularly to verify whether they were carrying fungal propagules. Wood samples were collected in association with the mycelial area in the bark and the bark beetle marks by drilling three holes 2e4 cm into the high stump. Care was taken to remove all remnants of bark before sampling the wood. The drill chips from the three holes were pooled and collected in plastic bags attached to the stump below the drill holes before being moved to 2-ml screw cap tubes. Each sample was collected in a separate plastic bag. Wood control samples were collected from high stumps in a newly felled area (year zero) that were without any trace of bark beetle activity or mycelial outgrowth. To estimate the natural fungal flora that was not directly associated with bark beetle activity in 0-, 1-, 2and 3-yr-old high stumps, samples were taken from areas of the stump that had no sign of bark beetle activity or mycelial outgrowth. The wood control samples were collected by drilling three holes into each high stump as described above. To sterilize the instruments before each sample was collected the instruments were sprayed with alcohol and then left to dry. All the samples were stored in a cooling bag in the field for transportation back to the laboratory where they were stored at 20  C before further analysis.

Molecular identification of fungal species DNA was extracted according to the CTAB protocol (Gardes & Bruns 1993) with some modifications. Each wood sample was prepared by filling approximately half a 2-ml screw cap tube with drill chips. A screw and a nut were added and the wood was homogenized by shaking for 30 sec in a bead beater (Fast prep FP 120, Savant Instrument Inc., NY, USA). Next, 1.2 ml of CTAB-buffer (3 % cetyltrimethylammonium bromide, 2 mM EDTA, 150 mM TriseHCl, 2.6 M NaCl, pH 8) was added to each tube and the sample was heated at 65  C for 1 hr. After centrifugation the supernatant was sequentially extracted with an equal volume of chloroform, centrifuged for 7 min at

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16 200 g, precipitated with 1.5 volumes of 2-propanol, washed with 70 % ethanol, dried and then redissolved in 50 ml of double deionized water (ddwater). The mycelia samples were prepared as above but without the screw in the homogenization step. The insect samples were homogenized using a pestle and mortar before being subjected to the same treatment as the mycelia and wood samples. No attempt was made to separate the external and internal part of insect bodies. Insects of the same species that were collected from the same bark sample were pooled and ground together. To verify if the DNA extracted was of a sufficiently high quality, a PCR was run with unmarked primers, internal transcribed spacer (ITS) 1-F (CTT GGT CAT TTA GAG GAA GTA A) (Gardes & Bruns 1993) and ITS4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990), as described by Lindahl et al. (2007). PCRproducts were verified electrophoretically on a 1 % D-1 low electroendosmosis agarose gel stained with ethidium bromide and visualized in UV-light.

Restriction enzymes To visualize the restriction fragment patterns, WellRED (SigmaAldrich, St. Louis, MO, USA) fluorescence labeled primers ITS1-F with D3 and ITS4 with D4 (Sigma-Aldrich, St. Louis, MO, USA) were used. Following PCR, samples were digested using two different restriction enzymes (AluI and CfoI). These enzymes were chosen because they have been extensively tested with fungal species in ITS studies and give a sufficient amount of variation. From each sample, 3 ml of PCR-product was mixed with 0.2 ml of AluI enzyme, 0.6 ml of AluI buffer and 2.2 ml of ddwater, according to the manufacturers’ description (Roche Diagnostics GmbH, Mannheim, Germany). A similar mix was also made for each sample using CfoI (Promega, Madison, WI, USA) and CfoI-buffer as active ingredients. The two mixes were placed in a 37  C water bath for 2 hr. After incubation 2 ml of each sample was desalted according to Beckman Coulter DNA Size Standard Kit-600. Three ml of each sample was placed in a new well and 30 ml of sample loding solution (Beckman Coulter) with a 0.2 ml size marker was added. The samples were overlaid with one drop of light mineral oil and the T-RFLP (terminal restriction fragment length polymorphism) patterns were then analyzed with program Frag-4 (Beckman Coulter Seq 2000 Genetic Analysis System).

Cloning fungal DNA To identify unknown fungal species and their specific T-RFLP pattern, the ITS region of some of the samples was cloned. A PCR with unmarked primers was conducted to select the species that appeared to be the most different on visualization. Cloning was then performed using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). For each sample, 16 clones were collected with a pipette from the selective plates and mixed with 150 ml of ddwater followed by a PCR with primers M13F (GTA AAA CGA CGG CCA G) and M13R (CAG GAA ACA GCT ATG ACC), as described by Lindahl et al. (2007). A T-RFLP analysis was performed on the selected clones. The cloned PCR-products generated with primers M13F and M13R were cleaned with Viogene DNA Extraction PCR-M Kit (ViogeneBiotek Corp.) to obtain samples that were as clean as possible.

Y. Persson et al.

Sequencing was performed on a CEQ 2000 (Beckman Coulter) using a DTCS Quick Start Kit. We detected up to 14 different fungal species from DNA clones derived from a single insect sample. All cloned and sequenced material was used for building the reference T-RFLP database.

Insect samples Because ITS4 is also able to bind to insect DNA, a nested PCR was performed with primers NLC2 (GAG CTG ATT CCC AAA CAA CTC) and NSA3 (AAA CTC TGT CGT GCT GGG GAT A) (Martin & Rygiewicz 2005) before the insect samples underwent the same treatment as the mycelia and wood samples. NLC2 and NSA3 primers only bind to fungal species and therefore the chimaeras of insect and fungal DNA that could have been generated if primer ITS4 had been used were avoided.

Sequence analyses Sequences were aligned and manually edited using the Lasergene software package (version 5.07, DNASTAR, Madison, WI, USA) and all ambiguous data were removed before the sequences were assembled into similar groups (95e100 % similarity). The sequences were identified by blasting them against NCBI’s sequence database GenBank (Altschul et al. 1990). The criterion for species identification was minimum 98 % similarity with a known ITS sequence. Given that the cloned samples had been sequenced and identified, the peak patterns from the fragment analysis of the clones were used as a reference library in TRAMP (Terminal Restriction Analysis and Matching Program) (Dickie et al. 2002). This enabled the identification of previously unknown patterns that are frequently found in mycelia, wood and insect samples. The results for all the samples from the fragment analysis were processed in TRAMP. Each pattern was compared against the reference library that had been developed from the samples that had been sequenced. The threshold for fragment identity was set at three base pairs.

Statistics Chi-squared tests were performed on dominant species to determine if the identified species were equally distributed among the different sample categories (mycelia, wood and insects). Species were considered dominant if Pi > 1/S, where Pi is the proportion of total sample represented by species i and S (species richness) is the number of competing species present in the community (Camargo 1993).

Results In total, 21 fungal species were found at the four test sites (Fig 1). Of these, eight were Basidiomycota and twelve were Ascomycota. In total, 203 samples were collected, of which 66 were mycelia samples, 87 wood samples, 18 wood control samples and 32 insect samples. The most frequently sampled species were two unidentified Cryptococcus sp. followed by a Saccharomycetales sp. The most frequently recorded wood decay fungus was Phlebiopsis gigantea, which was found in 28 of the 203

Do bark beetles facilitate the establishment of rot fungi?

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Fig 1 e Pooled data of fungal species detected in samples of wood, mycelia in bark and insects from newly cut, 1-, 2- and 3-yr-old high stumps of Norway spruce.

samples (Table 1). There were 23 samples that were unidentified and did not have a match in the fungal clone library. The collected material, except the samples collected immediately after clear cut (year zero), showed signs of activity by bark- and wood-boring insects. Approximately 45 % of the collected bark samples contained living bark beetles of two different species: P. chalcographus and Crypturgus sp. No beetles were found immediately after clear cut. Forty Crypturgus sp. and 67 P. chalcographus beetles were found in 1-yr-old stumps. However, Crypturgus sp. was the dominant beetle species in 2and 3-yr-old stumps with 77 and 409 beetles recorded, respectively. P. chalcographus was not found in 2-yr-old stumps and only 11 beetles were found in 3-yr-old stumps. In total, 604 beetles were collected. From 30 samples of insects, mycelia and wood, 17 fungal species were detected from the insect samples, 11 fungal species from the mycelia samples and 6 fungal species from the wood samples. The species accumulation curves clearly indicated that more fungal species are likely to be detected if more than 30 wood and mycelia samples were analyzed, whereas 30 samples appeared to be sufficient to detect all the fungal species that were likely to be present in the beetle samples (Fig 2). Stump age had an impact on the fungal communities that were detected. Year zero stumps were part of the control samples and only two fungal species were detected: Cryptococcus sp. 1 and 2. In older stumps the fungal community was more diverse: Cladosporium sp., Pezizomycotina sp. and Lecythophora sp. were detected with similar frequency from all the analyzed samples taken from 1-, 2- and 3-yr-old stumps. Saccharomycetales sp. and Cryptococcus sp. 1 and 2 were detected with decreasing frequency in older stumps, whereas S. sanguinolentum and P. gigantea were detected with increasing frequency

(Fig 3). Based on mycelia samples, the similarity between the species composition of 1- and 2-yr-old stumps was greater (70 % similarity) than that between 1- and 3-yr-old stumps (25 %) or between 2- and 3-yr-old stumps (44 %). This trend was reversed in samples of wood or insects (data not shown). The sampling method also had an impact on the fungal species that were detected: 38.1 % of the species were detected using all three sampling methods, 19 % were exclusively detected in insect samples, 23.8 % were detected in both insect and mycelia samples, 4.7 % were exclusively detected in mycelia or wood samples and 4.7 % occurred in both mycelia and wood samples and in insect and wood samples. Several known wood decay fungi, for example, F. pinicola and Trichaptum abietinum, occurred in samples of mycelia in bark and in bark beetle samples (Table 1). P. gigantea was also frequently detected in wood samples. The ascomycete species Capronia, Dothioraceae and Cadophora and the basidiomycete in the Tremellales were exclusively recorded with bark beetles. Of the dominating species, the two basidiomycete yeasts Cryptococcus sp. 1 and 2 were detected significantly more often in control tree samples than in the other samples; Cryptococcus sp. 2 was not detected in any insect samples (Table 1). By contrast, the four dominating ascomycete species (in Cladosporium, Lecythophora, Pezizomycotina and Saccharomycetales) were detected significantly more often in insect samples than in other samples and were absent or underrepresented in samples from undisturbed control trees (Table 1). P. gigantea, the only filamentous basidiomycete among the dominant species, did not show a significant bias for the various sample types although it could not be detected in undisturbed wood.

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Y. Persson et al.

Table 1 e Fungal taxa detected from various substrata (mycelia, wood, beetles) Fungal taxa

Gene Bank accession no.

Total no. of detections in mycelia, wood and beetle samples

Basidiomycetes Bjerkandera adusta Cryptococcus sp. Cryptococcus sp. 2 Fomitopsis pinicola Phlebiopsis gigantea Pleurotus sp. Stereum sanguinolentum Tremellales sp. Trichaptum sp.

AY854083.1 AF087487.1 AY540327.1 EU673084.1 EF060790.1 AY781273.1

3 44 85 13 28 2 7 2 3

Ascomycetes Cadophora sp. Capronia sp. Chaetothyriales sp. Cladosporium sp. Cosmospora vilior Dothioraceae sp. Herpotrichiellaceae sp. Lecyhophora sp. Pezizomycotina sp. Pichia sp. Saccharomycetales sp. Trichoderma citrinoviride

AY781242.1 AF397136.1 AF050242 AJ279487.1 U57673.1 AJ875381.1 AY156968.1 AY781228.1 DQ317330.1 AY790541 AY770414.1 AM498489.1

3 8 1 16 1 7 8 21 26 5 30 3

EF441742.1 AY761170.1

No. of samples analyzed No. of fungal taxa detected No. of samples analyzed with no match with clonal library

Sample Mycelia

1 9 28 9 11 1 3

Wood

25 57 13 1

2

Beetle

2 10

Control

7 13

4 4 1 3 2 1

3 8 1 1

15

1 3 2 4 1 5 1

1 1 4 1 4

7 4 18 18 3 21 2

66 15 9

87 11 8

32 17 4

1 1

18 4 2

Taxon frequency (%) detected in all samples (%)

Chisquared testb

0.9 13.9a 26.9a 4.1 8.9a 0.6 2.2 0.6 0.9

n.t. 0.05 0 n.t. 0.86 n.t. n.t. n.t. n.t.

0.9 2.5 0.3 5.1a 0.3 2.2 2.5 6.6a 8.2a 1.6 9.5a 0.9

n.t. n.t. n.t. 0 n.t. n.t. n.t. 0 0 n.t. 0 n.t.

203 18 23

0.67

Abbreviation: n.t. not tested. a Dominant species. Species is considered dominant if Pi > 1/S, where Pi is the proportion of total sample represented by species i and S (species richness) is the number of competing species present in the community (Camargo 1993). b Chi-squared tests were performed on dominant species in order to analyze if the identified species were equally distributed among the different sample categories (mycelia, wood and insects).

Fig 2 e Species accumulation curve of fungal species detected in samples of wood, mycelia in bark and bark beetles from Norway spruce high stumps. Fungal species were detected from amplicons of the ribosomal internal transcribed spacer (ITS). DNA was extracted from wood, mycelia and bark beetles.

Fig 3 e Temporal sequence of fungal species detected in the various samples taken from high stumps of Norway spruce.

Do bark beetles facilitate the establishment of rot fungi?

Discussion This study demonstrated a succession of fungal species in the bark of high stumps of Norway spruce in the first 3 yr after tree harvest. Initially, two Cryptococcus spp. dominated the barkinhabiting flora. A fungal flora associated with the bark beetles Crypturgus sp. and P. chalcographus built up in the bark and wood. Earlier studies have reported ophiostomoid fungi commonly associated with these insects together with a range of other ascomycetes in low frequencies (Kirschner 2001; Kirisits 2004). Not only did we detect ascomycete yeasts and filamentous fungi directly from the bark beetle samples, but we were also able to detect a range of basidiomycete decay fungi that commonly form fruit bodies on Norway spruce high stumps (Jonsell et al. 2005) in a reproducible manner. Several of the fungal species detected on bark beetles were also detected from samples of mycelial growth in or closely associated with insect breeding galleries in the bark. Most of the known decay fungi that were recorded in this study were detected in association with insects but not from the wood underneath the galleries. This is strong circumstantial evidence that bark beetles have a role as facilitators of the spread of certain decay fungi and that they might even function as unspecific vectors. According to the species accumulation curve 30 samples were satisfactory for the insect samples but were not sufficient for bark and mycelia samples. T-RLFP of a greater number of mycelia and wood samples would probably have identified a greater number of fungal species, and hence a more accurate understanding of which species exist within the high stumps and the frequency of these species would have been obtained. Fungal species were found in all three sample types, indicating that most of the fungi found as mycelia in bark and in wood have some connection with insects. The species composition of the wood and mycelia samples was similar, which is probably because the mycelia grow into the wood and therefore the fungal species appear in both sampling types. To determine if the insects actively spread the fungi requires more investigation, but it is clear from this study that fungi can be present on the body surface of insects or in their stomachs. The wood control samples collected from each stump were similar to the control samples from newly cut stumps (year zero), and were dominated by the two Cryptococcus species. Insect activity and the vegetative growth of other fungal species might have stimulated competing wood decay fungi within the high stumps during the 3 yr following cutting. The decline of Cryptococcus spp. in the oldest stumps was probably due to microhabitat change and competition with the other fungal species. The living cells of the high stumps immediately after cutting (year zero) might prevent insect attack or the newly created clear cut area might simply not have been located by the bark beetles yet. The basidiomycete yeasts Cryptococcus spp. are commonly found in soil, and two species of this genus dominate the year zero samples. The human pathogen Cryptococcus neoformans has been isolated from environmental samples such as tree bark and from pigeon droppings (Lin & Heitman 2006). These niches are not strikingly different from the bark habitat of Norway spruce trees and high stumps reported here.

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Both species of bark beetle found in the high stumps are usually found in conifers such as Norway spruce. The bark beetle genus Crypturgus is more common in trees that have been dead for longer time periods than trees that are colonized by P. chalcographus, because Crypturgus species can reuse primary insect galleries under the bark. The bark beetle P. chalcographus is most common in newly dead or dying trees € m & Axelsson 2002). The inner bark of trees from 3-yr(Ehnstro old stumps was structurally disintegrated to a large extent. This was probably caused by primary bark beetle or boring insect species that were not detected in this study. Studies of beetle diversity on dead spruce wood in the region reported that the beetle assemblage was disproportionately dominated by the genus Crypturgus while P. chalcographus was the third € m et al. 2008). That study also most common species (Djupstro reported high species diversity although no species apart from € m et al. Crypturgus sp. was present at all studied sites (Djupstro 2008). It is also possible that some of the other bark beetles may contribute in facilitating colonization by fungi. Most of the fungal species found with the bark beetles were also detected using the other sampling methods (Table 1). Of 18 fungal species identified from bark beetles, 14 were found on both beetle genera and four were only associated with Crypturgus sp. It seems likely that the insect-associated fungal species were picked up by emerging beetles and transported to the new substratum during the spring migration from the hibernation tree or picked up from debris on the ground. The hibernation habitat has been shown to play a significant role for the fungal biota carried by I. typographus (Persson et al. 2009). Similarly to earlier reports, ophiostomatoid fungi did not dominate the fungal biota of the investigated insect species habitat. This is in agreement with the colonizing strategy of Crypturgus sp. and P. chalcographus, which do not depend on specific fungi for establishing in the bark. More primary beetles, such as I. typographus, have more close association with ophiostomoid fungi. The species detected in the control samples have a shifting resemblance to the species detected in the mycelia, wood and insect samples. There was low similarity between species detected in the control samples and the insect samples, probably because the control samples were taken where there was no evidence of insect activity. The species detected in the control samples taken from the two types of woody tissuefresh high stumps with no colonization of insects, and wood samples from colonized high stumps but away from insect activity, were not surprisingly the ones that most resembled each other since they were collected from the same type of tissue. The fungal species recorded for the mycelia and insect samples were similar to each other but dissimilar to the control samples, probably because the sampling methods used for the mycelia and insect samples were different to that used for the wood and control samples. Depending on their ability to compete, fungal species have different abilities to colonize high stumps. Often the fungus that arrives first has an advantage over the other species until the substratum loses its nutritional value or the microhabitat changes. The dominating fungal species increased over time, except for Saccharomycetales sp. and Cryptococcus sp. 1 and 2, which decreased. The explanation for changes in species composition over time is probably connected not only to changes in the habitat, such as humidity, sun exposure and

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bark, but also to what species were brought to the tree by insects and the death of tree cells. Heterobasidion annosum is one of the most economically important root rots of conifers (Asiegbu et al. 2005). The local forest company (Bergvik AB, Sweden) has since 1995, used P. gigantea as an inhibitor of H. annosum on stumps, but it is usually only applied when the last thinning occurs. This practice would make P. gigantea spores more common in the air fungal flora and could in theory have contributed to the pronounced increase in P. gigantea detected in samples from 3-yr-old stumps compared with that found in 2-yr-old stumps. However, Samils et al. (2009) did not find that introducing P. gigantea had a strong influence on the local population structure of the fungus away from the local forest stands where it had been used. The prevalence of the saprotrophic and parasitic fungus Stereum sanguinolentum normally increases over time in wounded trees and stumps (Vasiliauskas & Stenlid 1998). The fungus could theoretically enter the stump via bark beetle entry holes, which would explain the greater frequency of S. sanguinolentum in 3-yr-old stumps compared with that found in 2-yr-old stumps. F. pinicola is typically found on dead standing conifer wood or on fallen logs, which are rapidly decayed (Hogberg et al. 1999; Jonsell et al. 2005). The fungus occurred in association with insects and was found more frequently in 2-yr-old stumps than in 1-yr-old stumps; however, it was not present in 3-yr-old stumps. This indicates that insects might facilitate the spread of the fungus although the spatiotemporal stochastic variations make it hard to detect in all samples. The bark beetles that were found at the test sites are not connected with F. pinicola fruit bodies but are likely to transport mycelia on their body when moving from an infested tree. F. pinicola has previously been detected on beetles in flight (Pettey & Shaw 1986). In Swedish ecosystems, F. pinicola (and also T. abietinum) commonly fruit on high stumps of Norway spruce and are important determinants of the insect fauna on older high stumps (Jonsell et al. 2005; Abrahamsson et al. 2008). For example, a positive correlation between the occurrence of F. pinicola fruit bodies and the beetle Hadreule elongatula has been found in several studies (Jonsell et al. 2005; Schroeder et al. 2006; Abrahamsson et al. 2008). Approximately 25 insect species are known to lay eggs and develop their larval stage in the F. pinicola fruit body (Jonsell et al. 2001). The frequency with which decay fungi were detected in the high stumps might be enough to account for the frequency with which they are found to colonize recently dead trees or logs. We detected F. pinicola and T. abietinum in about 2 % of examined insect galleries. This might seem a low frequency but, given that hundreds or thousands of insect galleries that can be found in the bark of a dead spruce tree, the likelihood is relatively high for insect facilitation of decay fungi in dead trees or logs. In theory insect facilitation could easily account for the inoculation of up to 19 different genotypes of these species found in a single standing or fallen tree (Norden 1997; Kauserud & Schumacher 2003). The aggressive primary Norway spruce bark beetle I. typographus carries the pathogenic fungi Ceratocystis polonica and Grosmannia europhioides. In addition to the pathogenic ascomycetes a range of non-pathogenic ophiostomatoid and yeast fungi are also associated with I. typographus (Solheim 1992; Kirschner & Oberwinkler 1998; Kirisits 2010). In

Y. Persson et al.

a recent investigation of fungi vectored by I. typographus following hibernation, Persson et al. (2009) detected four of the wood decay basidiomycetes also found in the present study, which suggests that insect facilitation of fungal colonization is more common than currently recognized in forest ecology. In the present study it was apparent that bark beetles acted as facilitators of wood decay by wood decay fungi. Of the 21 fungal species detected in the high stumps, 18 were associated with insects. Either these fungi could have been directly introduced by bark beetles or they could have gained entry via the bark beetle entry hole in the bark when dispersed by air or rain. Even if the bark beetle was in theory clean of spores when arriving at the high stump, the fungal flora present in the high stump could be transferred by the second generation of bark beetles when immigrating to a new habitat. The bark beetles role as a vector of root rot fungi cannot be neglected, but further investigation is needed to confirm which insect species are the most effective vector.

Acknowledgments This study was financially supported by the Swedish Energy Agency (STEM) and FORMAS. We are grateful to Martin Schroeder for entomological expert comments. We thank Petra Fransson, Katarina Ihrmark for reading and commenting on the manuscript and Caroline Woods for constructive linguistic comments.

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