Molecular investigation of genet distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests

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1 New Phytol. (1999), 142, Molecular investigation of genet distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests N. A. SAWYER, S. M. CHAMBERS AND J. W. G. CAIRNEY* Mycorrhiza Research Group, School of Science, University of Western Sydney (Nepean), PO Box 10, Kingswood, NSW 2747, Australia Received 15 September 1998; accepted 12 March 1999 SUMMARY The size and distribution of Cortinarius rotundisporus genets at three sclerophyll forest field sites in New South Wales, Australia, were estimated by using microsatellite-primed PCR (MS-PCR) of DNA extracted from sporocarp tissue. MS-PCR fingerprints generated with the primers (GTG) and (GACA) indicated that two to five genets were present at each site, with each site being characterized by a single large genet (9 30 m in diameter). Analysis of internal transcribed spacer (ITS)-RFLP patterns from individual sporocarps used in the study suggested that three distinct RFLP types were present in the sampled C. rotundisporus population. ITS sequence data indicate that the three RFLP types had less than 88.4% sequence identity to each other, strongly suggesting that C. rotundisporus is a complex of three species. Key words: ectomycorrhizal fungi, microsatellite-primed PCR, internal transcribed spacer (ITS) sequences, ITS-RFLP. INTRODUCTION Cortinarius is a widespread basidiomycete genus, of which most species are thought to be ectomycorrhizal (Liu et al., 1997). Members of this genus have been recorded in both Northern and Southern Hemisphere forests, associated with coniferous and eucalypt hosts respectively (Allen et al., 1995). On the basis of sporocarp records, Cortinarius is among the most frequently recorded ectomycorrhizal fungal genera in many European and North American coniferous forests (Villeneuve et al., 1989; Bradbury et al., 1998; Comandini et al., 1998; Kranabetter & Wylie, 1998), Australian eucalypt forests (Malajczuk et al., 1987) and arctic-alpine tundra (Gardes & Dahlberg, 1996). Carpophore records further suggest that the genus is an important late-stage colonizer in such forests (Bowen, 1994; Visser, 1995). In a study of ectomycorrhiza morphotype diversity, Visser (1995) found that Cortinarius-type ectomycorrhizas constituted approx. 13% of the total ectomycorrhizal root tip community in a Pinus *Author for correspondence ( j.cairney nepean.uws.edu.au). banksiana stand after wildfire. More recent molecular studies also indicate that Cortinarius species can account for 5 14% of total infected root tips in North American Pinus muricata forests (Gardes & Bruns, 1996) and Swedish Picea abies forests (Ka re n et al., 1997), respectively. Despite such information, we still have a poor understanding of the functional roles played by Cortinarius species in natural and man-managed forest ecosystems. This can be explained partly by the acknowledged difficulties associated with isolating and maintaining axenic cultures of Cortinarius species (Brundrett et al., 1996) and the concomitant lack of physiological investigation that the genus has received. It also reflects the current paucity of information regarding the population biology and spatial structure of Cortinarius mycelia in soil. Thus, although some Cortinarius species are known to produce rhizomorphs (sensu Cairney et al., 1991) during growth (e.g. Agerer, 1988), the extent of spread of individual mycelia in forest soils is at present unknown. The apparent late-stage status of the genus suggests, however, that Cortinarius species are likely to be combative (C) or stress-tolerant (S) strategists (sensu Cooke & Rayner, 1984) and therefore might produce extensive, long-lived mycelia in soil.

2 562 N. A. Sawyer et al. Somatic incompatibility testing has revealed that individual mycelia (genets) of some ectomycorrhizal fungal taxa might be extensive, presumably reflecting S or C life-history strategies. In Suillus bovinus and Suillus variegatus, for example, genet diameter has been estimated at up to 20 m, whereas genets of Laccaria bicolor can reach a diameter of 12.5 m (Dahlberg & Stenlid, 1990, 1994; Baar et al., 1994; Dahlberg, 1997)). Depending on the variationgenerating mechanisms operating in a particular population, somatic incompatibility testing might not be reliable in all cases; however, there is some evidence that molecular methods can provide more reliable markers of genetic diversity in fungal populations (e.g. Jacobson et al., 1993). Indeed, in the specific case of Cortinarius, difficulties associated with establishment in axenic culture preclude the use of somatic compatibility testing for most species. A range of molecular methods have so far been used to estimate genet size in ectomycorrhizal fungi. Anderson et al. (1998b) used fingerprinting with a combination of random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MS- PCR) to show that genets of an Australian Pisolithus species extended for up to 30 m along a roadside verge. Bonello et al. (1998) adopted a two-step PCR and single-strand conformational polymorphism (SSCP) analysis to show that genets of Suillus pungens associated with P. muricata can be as large as 40 m in diameter. By contrast, genets of Hebeloma cylindrosporum in a Pinus pinaster plantation in France, as revealed by combined data from four separate DNA typing methods, did not exceed 5 m (and were generally only a few centimetres) in diameter (Gryta et al., 1997). Although populations of ectomycorrhizal fungal taxa that display C or S strategies typically comprise a low density of extensive genets, in disturbed forests a high density of smaller genets might prevail for the same taxa (Dahlberg & Stenlid, 1995). In such circumstances, disturbance is seen as creating opportunities for the establishment of new mycelia by sexual spore dispersal, which would otherwise be unable to compete with the high inoculum potential of established mycelia. Small-scale gaps resulting from windthrow of trees or larger gaps created by clear-felling or intense fire thus increase genetic diversity in local mycelial populations by promoting the establishment of new mycelia from spores. Cortinarius rotundisporus is a common and widely distributed ectomycorrhizal basidiomycete in native mixed sclerophyll forests in southern Australia (Horak & Wood, 1990; Bougher & Syme, 1998). Despite its widespread occurrence, we know nothing about the autecology of this taxon. We have employed MS-PCR to discriminate between genets of C. rotundisporus at three native forest sites within the greater Sydney region of New South Wales, Australia, and report here estimates of genet size and distribution at the field sites on the basis of these data. The taxonomy of C. rotundisporus is currently somewhat confused. Horak & Wood (1990) considered three previously held species (C. rotundisporus Clel. & Cheel, C. austro-evernius Clel. & Cheel and C. oleaginus Clel. & Harris) as syntaxic, placing all three under C. rotundisporus comb. nov. More recently, however, Grgurinovic (1997) reverted to the earlier classification of three separate species. A secondary aim of our study was thus to use molecular methods to clarify whether C. rotundisporus is a single species or a complex of several cryptic species. MATERIALS AND METHODS Sporocarp collection Sporocarps of Cortinarius rotundisporus (Clel. & Cheel) Horak & Wood (sensu Horak & Wood, 1990) were collected from three field sites in the Sydney region of New South Wales, Australia, during the period April-June 1997; their location was mapped to scale (see Figs 2 4). Thirty-eight sporocarps were thus collected from a 480 m site at Warrimoo Trail, Ku-ring-gai Chase National Park, 32 from a 160 m site at Lovers Jump Creek Reserve, Turramurra, and 15 from a 100 m site at Girrahween Park, Bardwell Park. The site at Warrimoo Trail is native sclerophyll forest that has been disturbed only by occasional low-intensity fire activity. The other sites, also native sclerophyll forest, both display signs of disturbance in the form of invasion by alien weed species (largely Ligustrum lucidum, Ligustrum sinense and Lantana camara) as a result of nutrient addition via storm water run-off (Fox & Adamson, 1979; Buchanan, 1988). All sporocarps were frozen on collection and stored at 20 C until DNA extraction. DNA extraction and MS-PCR Genomic DNA was extracted from sporocarp stipe material by using the method of Gardes & Bruns (1993). In cases where PCR amplification was unsuccessful, genomic DNA from sporocarps was further purified with the WizardTM DNA Clean-Up System (Promega, Madison, WI, USA). MS-PCR reactions were conducted with the primers (GTG) and (GACA) (Beckman, Fullerton, CA, USA). Each 50 µl PCR reaction contained: approx. 100 ng of DNA, 200 pmol of primer, 50 mm KCl, 10 mm Tris-HCl, 0.1% (v v) Triton X-100, 1.5 mm MgCl, 200 pmol each of datp, dctp, dgtp and dttp, and 2.5 units of Taq DNA polymerase (Promega). All amplifications were performed in duplicate in a PTC-100 thermocycler (MJ Research, Watertown, MA, USA) to confirm the reproducibility of amplifications, and the reactions were

3 Genets and genetics of Cortinarius rotundisporus in Australia 563 Table 1. Summary of restriction fragment sizes for selected Cortinarius rotundisporus sporocarps and their groupings as ITS RFLP types Restriction fragment sizes (base pairs) ITS RFLP Sporocarps HinfI MboI HaeIII type, W06, Lr06 380, , 250, , 220 I 370, 150, , , 200 II 360, , , 195 III cycled by a method modified from Groppe et al. (1995). Cycling conditions (30 cycles) were: for 1 min at 94 C, 2 min at 60 C ((GTG) ) or 48 C ((GACA) ) and 3 min at 72 C, followed by a final extension at 72 C for 10 min. A negative control, containing no fungal DNA, was included in each PCR reaction run. Amplification products were subjected to electrophoresis in 1.5% (m v) agarose gels and viewed under UV after being stained with ethidium bromide. Sporocarps that had different fingerprints were regarded as belonging to different genets, whereas sporocarps that had identical fingerprints with (GTG) and identical fingerprints with (GACA) were regarded as belonging to the same genet (Anderson et al., 1998b; Liu et al., 1998). Internal transcribed spacer (ITS) amplification and ITS-RFLP Sporocarps from genets L2 and L3 at the Lovers Jump Creek Reserve site exhibited a high degree of MS-PCR polymorphism compared with all other sporocarps in the study. To investigate this further, the ITS region of rdna was amplified for sporocarps (genet L2, from Girrahween Park) and (genet L3, from Lovers Jump Creek Reserve), along with, Lr06 and W06 (from Warrimoo Trail). Amplifications were performed with the primers ITS1 and ITS4 (White et al., 1990), by using a method modified from White et al. (1990). Amplifications (in duplicate) were thus performed in a 50 µl reaction volume containing c. 100 ng of genomic DNA, 25 pmol of each primer, 50 mm KCl, 10 mm Tris-HCl, 0.1% Triton X-100, 2.5 mm MgCl, 200 pmol each of datp, dctp, dgtp and dttp, and 1.5 units of Taq DNA polymerase. All amplifications were performed in a PTC-100 thermocycler (MJ Research) with 35 cycles of 94 C for 1 min, 50 C for 1 min and 72 C for 1 min, followed by 72 C for 10 min. A negative control, containing no fungal DNA, was included in each PCR reaction run to test for the presence of contaminants. Amplification products were subjected to electrophoresis and detected as outlined for MS-PCR. Each ITS product was then digested with the restriction endonucleases HinfI, MboI and HaeIII (Promega) by incubating c. 2 µg of PCR product with each enzyme at 37 C for 3 h. Restriction fragments were separated by electrophoresis in 3% (m v) agarose gels and detected as described for MS-PCR. Sequencing of amplified ITS regions Combined data from the ITS-RFLP analysis indicated that three RFLP groups existed within the collected sporocarp population (Table 1). The ITS regions from single sporocarps (, and ) representing each RFLP type were sequenced to permit a more detailed comparison. Before sequencing, PCR products were cloned with the pgem-t easy vector system (Promega). Two or three clones for each sporocarp were sequenced with the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an automated fluorescent DNA sequencer ABI model 373-A (Applied Biosystems). Sequencing reactions were performed with the primers T7 and SP6 (Promega). Sequences were aligned by using the PILEUP and PRETTY programs and the percentage similarity was calculated by using the HOMOLOGY program (all programs are in the EGCG extensions to the Wisconsin Package, Version (Rice, 1996)). Sequences were screened for closest matches in the GenBank and EMBL nucleotide databases by using the program FASTA 3.0 (Pearson & Lipman, 1988). RESULTS MS-PCR Reproducible fingerprints were obtained with both primers for all sporocarps collected; fragments ( base pairs (bp)) were consistently amplified for each sporocarp with (GTG), whereas fragments ( bp) were produced with (GACA) (Fig. 1). Sporocarps that produced different fingerprints with either primer were considered to belong to genetically distinct individual mycelia (genets). Where sporocarps displayed identical fingerprints with (GTG) and identical fingerprints with (GACA), they were regarded as belonging to the same genet and grouped accordingly. Thus five genets were identified at the Lovers Jump Creek site, with four and two at the Warrimoo Trail

4 564 N. A. Sawyer et al. (a) 2645 bp 1198 bp and Girrahween Park sites, respectively (Figs 2 4). Each site was characterized by a single large genet (c m in diameter) that extended across most of the site area, along with one to four smaller genets (6 m in diameter). 676 bp 460 bp 350 bp (b) 2645 bp 1198 bp 676 bp 460 bp 350 bp Fig. 1. MS-PCR fingerprints of selected Cortinarius rotundisporus sporocarps from the Lovers Jump Creek site using the primers (a) (GTG) or (b) (GACA). Isolates Lr01, Lr03, Lr05, Lr06, Lr08, Lr09, Lr11 and Lr12 had identical fingerprints with (GTG) and identical fingerprints with (GACA) and were thus assigned to the same genet (L1). Isolates and Lr10 were similarly grouped as genet L3, whereas Lr07 and Lr02 had distinctive fingerprints with both primers and were regarded as belonging to separate genets (L4 and L5 respectively). Isolate codes are listed above each lane. M, pgem molecular size markers (Promega). ITS-RFLP and ITS sequence analyses Although most of the sporocarps from which DNA was amplified with the use of MS-PCR yielded similar fingerprints, the sporocarps that constituted two of the smaller genets (L2 and L3; Fig. 2) at the Lovers Jump Creek site exhibited a high degree of polymorphism compared with sporocarps from all other genets identified at the three study sites. The ITS regions of rdna from sporocarps and (representing genets L2 and L3 respectively), along with Lr06, W06 and (representing the remaining sporocarp population at each field site) were therefore amplified. Single ITS products (c. 690 bp) were amplified for each of the selected sporocarps (results not shown) and the restriction endonucleases HinfI, MboI and HaeIII produced clear RFLP patterns for each sporocarp. Restriction digests with HinfI, MboI and HaeIII yielded two or three variable fragments for each C. rotundisporus sporocarp (Fig. 5). Taken together, the ITS-RFLP data indicated that three different RFLP types were present within the sporocarp population collected from the three sites (Table 1). Genets L2 and L3 (isolates and ) comprised RFLP types II and III, respectively, whereas all other genets contained sporocarps of type I. L4 L L L L5 1 m Fig. 2. Schematic map of the distribution of sampled Cortinarius rotundisporus sporocarps (filled circles) at the Lovers Jump Creek site. Sporocarps enclosed by shaded areas were considered to belong to the same genet (L1-L5). Genets comprising ITS-RFLP types I, II and III are indicated by grey shading, light stippling and diagonal hatching, respectively.

5 Genets and genetics of Cortinarius rotundisporus in Australia W (a) 553 bp 427 bp 311 bp 200 bp bp (b) W2 553 bp 427 bp 311 bp 249 bp 200 bp W1 151 bp 118 bp (c) bp 427 bp W3 1 m Fig. 3. Schematic map of the distribution of sampled Cortinarius rotundisporus sporocarps (filled circles) at the Warrimoo Trail site. Sporocarps enclosed by shaded areas were considered to belong to the same genet (W1-W4). All genets at this site were of ITS-RFLP type I bp 200 bp Fig. 5. ITS-RFLP patterns for selected Cortinarius rotundisporus sporocarps after digestion with (a) HinfI, (b) MboI or (c) HaeIII. Sporocarp codes are listed above each lane. M, φx174 DNA-HinfI molecular size markers (Promega) G m Fig. 4. Schematic map of the distribution of sampled Cortinarius rotundisporus sporocarps (filled circles) at the Girrahween Park site. Sporocarps enclosed by shaded areas were considered to belong to the same genet (G1 or G2). All genets at this site were of ITS-RFLP type I. Full-length ITS sequences (excluding the primer sequences) of c. 650 bp were obtained for sporocarps, and (RFLP types I-III, respectively) (Fig. 6). The aligned sequences indicated that the three sporocarps had % identity to each other. A number of small insertions and deletions were found in the sequences, especially within the ITS1 and ITS2 regions from sporocarps and. Sporocarp had a four-base insertion at nucleotide 144, whereas had a seven-base insertion at nucleotide 480. Single-base and double-base insertions or deletions, along with base substitutions, were common throughout the ITS1 and ITS2 regions (Fig. 6). The full-length sequences were also screened for closest matches to other fungal taxa for which ITS sequences are available in the GenBank and EMBL databases by using the program FASTA. Both Lr06 and displayed the highest degree of identity to the ITS sequence of Cortinarius delibutus (GenBank accession code CDU56025) (91.7% and 92.4%, respectively). Sporocarp displayed the highest identity to Dermocybe olivaceopicta (GenBank accession code DOU56049) (93.9%).

6 566 N. A. Sawyer et al. Fig. 6. Sequence alignment of the ITS region from selected Cortinarius rotundisporus sporocarps of each of the three ITS-RFLP types (, type II;, type I;, type III;, consensus sequence). GenBank accession codes:, AF136739;, AF136738;, AF *, bases identical across all sequences;, deletions or substitutions. DISCUSSION The size and distribution of genets of C. rotundisporus at three field sites were estimated by using combined data from MS-PCR analysis with the primers (GTG) and (GACA). The data suggest that, at all sites, the below-ground C. rotundisporus populations were dominated by single large genets (9 30 m in diameter). However, these estimates of genet size must be regarded as conservative. Data were derived on the basis of DNA extracted from sporocarps; there is overwhelming evidence that, although the presence of a sporocarp indicates the presence of the parent mycelium in soil, the absence of sporocarps does not necessarily mean that mycelia are absent from soil (Taylor & Alexander, 1989; Gardes & Bruns, 1996; Dahlberg et al., 1997). Furthermore, the sizes of the field sites at the three locations were established according to the area within which C. rotundisporus fruited during a single fruiting season; it is conceivable that sampling during successive seasons would have yielded sporocarps associated with the outer edges of the identified genets. Thus genets at the three sites might be more extensive than the current data suggest. The above notwithstanding, it is clear that genets of C. rotundisporus in native Australian sclerophyll forests can be at least as extensive as those of a Pisolithus species in a patch of similar forest (Anderson et al., 1998b) and Suillus species in Northern Hemisphere conifer forests (Dahlberg & Stenlid, 1990, 1994; Dahlberg, 1997; Bonello et al., 1998). Genets of such proportions must have arisen by mycelial extension through soil. Although data on growth rates of Cortinarius spp. mycelia through soil have not been reported, known rates of growth of other ectomycorrhizal basidiomycetes ( 0.5 m yr ; Read, 1992) suggest that the largest genets at our field sites will have taken many years to reach their current dimensions. C. rotundisporus thus seems to be a long-lived C or S strategist, of the type that would be expected to prevail in mature forest systems (Dighton & Mason, 1985). Indeed, our unpublished observations indicate that C. rotundisporus mycelium forms apically diffuse simple rhizomorphs (see Cairney et al., 1991) during growth through soil, which would facilitate the long-distance transport of nutrients required for the establishment of such large mycelial systems (Cairney, 1992). Rhizomorph production is also likely to be important for the maintenance of such long-lived mycelial complexes in soil, by providing protection against edaphic stresses (Thompson, 1984) and by facilitating temporal shifts in patterns of translocation according to changing spatial patterns of physiological activity and source-sink demands in the heterogeneous soil environment (Cairney & Burke, 1996). Ecological disturbance is thought to disrupt large genets of ectomycorrhizal fungi and permit the establishment of new mycelia by the establishment

7 Genets and genetics of Cortinarius rotundisporus in Australia 567 and germination of basidiospores (Dahlberg & Stenlid, 1995). Sites subject to a major disturbance such as clear-felling are thus characterized by highdensity populations of small genets (Dahlberg & Stenlid, 1990, 1994; Dahlberg, 1997). The Warrimoo Trail site, situated in Ku-ring-gai Chase National Park, comprises pristine native sclerophyll forest that seems to be undisturbed other than by occasional fire activity. By contrast, the Girrahween Park and Lovers Jump Creek sites are situated in patches of native sclerophyll forest around which urban development has encroached, and both receive sporadic nutrient-rich anthropogenic storm-water run-off (Fox & Adamson, 1979; Buchanan, 1988). Although the mature Eucalyptus spp. and Angophora costata (presumed to be the host species for C. rotundisporus) at these sites remain, much of the native understorey (largely Acacia, Banksia, Grevillea and Hakea spp. in addition to juvenile eucalypts) has been replaced by the exotic weed species Ligustrum spp. and L. camara, along with the invasive native species Pittosporum undulatum. This seems to have had no obvious effect on C. rotundisporus populations at the sites, with a single large genet dominating at each site. However, it might be that the degree of fragmentation of the large genets into ramets differs between the three sites, although this is impossible to ascertain with the techniques used. On the basis of similar microscopic characters, Horak & Wood (1990) placed the three species C. rotundisporus, C. austro-evernius and C. oleaginus into a single taxon, C. rotundisporus. More recently, Grgurinovic (1997) separated the three species in her treatment of the fungal flora of South Australia, whereas Bougher & Syme (1998) preferred to retain the amalgamation of the species into the single C. rotundisporus taxon. In the present study, three RFLP groups were clearly identified within the C. rotundisporus sporocarps collected. RFLP groups II and III (represented by sporocarps and, respectively), were present in two genets at the Lovers Jump Creek site. The remaining genets at this site, and the genets at the other two sites, were all of RFLP group I (represented by sporocarp ). The ITS sequence data obtained for the three representative sporocarps showed a divergence of up to 16%. In fact the three RFLP groups had higher sequence identity ( %) to other Cortinarius or Dermocybe species than they had to each other, strongly supporting the likelihood that they represent three separate taxa. This suggests that C. rotundisporus (sensu Horak & Wood, 1990) represents a complex of three species, probably representing the species C. rotundisporus, C. austro-evernius and C. oleaginus. More extensive collections and detailed studies of morphological characteristics, combined with molecular analysis, will be required to test this hypothesis. These data, however, emphasize the value of molecular approaches for discriminating morphologically difficult taxa, as already used successfully for the Pisolithus tinctorius complex (Anderson et al., 1998a; Martin et al., 1998) and for other non-mycorrhizal fungal taxa (see, e.g. Roy et al., 1998). ACKNOWLEDGEMENTS We thank the New South Wales National Parks and Wildlife Service and Ku-ring-gai Municipal Council for permission to conduct field work and collect sporocarps from the Warrimoo Trail and Lovers Jump Creek sites respectively. 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