Rhizopogon spore bank communities within and among California pine forests

Transcription

1 Mycologia, 95(4), 2003, pp by The Mycological Society of America, Lawrence, KS Rhizopogon spore bank communities within and among pine forests Rasmus Kjøller 1 Botanical Institute, University of Copenhagen, Øster Farimagsgade 2D, DK-1353 Copenhagen, Denmark Thomas D. Bruns Department of Plant and Microbial Biology, 311 Koshland Hall, University of at Berkeley, Berkeley, Abstract: In this study we examine the distribution of Rhizopogon species in spore banks from five pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences. Key words: bioassay, community structure, ectomycorrhiza, ITS, Pinus muricata, RFLP, sequencing INTRODUCTION Seed banks are well known in plant communities, where they function as a reservoir of plant diversity that often is expressed after disturbances (Harper 1977, Thompson and Grime 1979). Miller et al (1994) similarly suggested that fungal spores are Accepted for publication November 21, Corresponding author. rasmusk@bot.ku.dk long-lived and that the spore bank plays an important function in postfire settings. Recent work at Point Reyes National Seashore (, U.S.A.) supports this view for the fungal ectomycorrhizal community associated with bishop pine (Pinus muricata). In this community, the spore bank survives fire and its component species are among the most common postfire colonists of pine seedlings (Horton et al 1998, Baar et al 1999, Grogan et al 2000). One of the most common taxa in the spore bank in western North American pinaceous ecosystems is Rhizopogon (Amaranthus and Perry 1989, Baar et al 1999, Taylor and Bruns 1999). Rhizopogon contains more than described species (Smith and Zeller 1966, Martín 1996) and, with minor exceptions, all species are restricted to hosts within the Pinaceae. Furthermore, the majority of Rhizopogon species are host specific, being limited to a single genus, or subgenus within the Pinaceae (Molina et al 1999). Most are specialized on Pinus or Pseudotsuga, although species in subgenus Amylopogon exhibit broader host ranges within the Pinaceae and the Ericaceae (Massicotte et al 1994, Bidartondo et al 2000). In mature pine forests, Rhizopogon species are found as ectomycorrhizal root tips in low abundance (Gardes and Bruns 1996, Horton and Bruns 1998, Stendell et al 1999, Taylor and Bruns 1999) but, in postfire pine seedling communities, Rhizopogon species often are dominant (Horton et al 1998, Baar et al 1999, Grogan et al 2000). Spores from Rhizopogon sporocarps either remain in situ after sporocarp decomposition (Miller et al 1994) or are dispersed locally by vertebrates (Maser et al 1978, Johnson 1996). Both processes might be expected to result in a patchy distribution of spores within a forest site. However, a study by Horton et al (1998) revealed an unexpected uniformity of Rhizopogon inoculum at a previously nonforested site adjacent to burned forest. In the current study, we wanted to extend the sampling both to additional sites, to see if these too were similarly homogeneous as the one described by Horton et al (1998) but also to examine the variation in spore bank structure at a regional scale. The latter has not been examined previously, but we predicted that there would be differences among sites due to the combination of host specificity and the limited 603

2 604 MYCOLOGIA FIG. 1. Map of showing the five sample sites. dispersal among noncontiguous coast pine forests. Four of the sites were natural discontinuous populations of bishop pine (Pinus muricata D. Don) along the n coast, while the fifth was a ponderosa pine (Pinus ponderosa Laws.) forest in Sierra National Forest, separated from the others by the San Joaquín Valley (FIG. 1). The fungi were brought into axenic culture by sampling Rhizopogon ectomycorrhizal morphotypes from bioassay pine seedling roots. This strategy was adopted, instead of direct PCR identification, because an additional goal was to obtain cultures for ongoing population genetic and microcosm studies. MATERIALS AND METHODS General sampling strategy. Four plots (A D) were sampled at each of the five sites (FIG. 1). The four plots within a site were separated by 10 m between A and B, m between AB and C, and 0 m between ABC and D to provide a broad range of distances. The distance between A, B and C were measured precisely when possible. The distances between ABC and D were measured by a combination of GPS, detailed maps and car odometer and are estimated to be within m. There was no linear orientation from A through D. Site descriptions. Point Reyes National Seashore. Samples were taken on Mount Vision in Point Reyes National Seashore, Marin County, September 1999 at elevation 260 m. All plots were sampled in or adjacent to mature bishop pine forest. A, B ( N, W) and D were taken in closed forest with some shrub vegetation and a thick needle layer. Plot C was over a ridge top from A and B in an opening in the forest with grass, about 7 m from the nearest trees. Plot D was downhill from A C. The distance to the ocean was 6.5 km (2.5 to Tomales Bay). Salt Point State Park. Samples were taken September 1999 in mature bishop pine forest in Salt Point State Park, Sonoma County, at elevation 110 m, 0.6 km from the ocean. The forest floor at Plots A C ( N, W) were covered with grass; some shrub, especially Rhododendron, and a few small deer-grazed Douglas fir seedlings also were present. Plot D was sampled farther north in the park in a closed-canopy bishop pine forest with no herbaceous understory and a thick needle layer. Monterey Peninsula. Samples were taken November 1999 at the Huckleberry Hill area on Monterey Peninsula, Monterey County, at elevation 125 m. Plots A C ( N, W) were located in two small isolated mature bishop pine stands within a mature Monterey pine forest. These plots contained no herbaceous understory but some shrubs were present. There was 130 m between AB and C. Plot D was in a 12-year-old, dense, postfire stand. The sampling area was 2.5 km from the ocean. Santa Barbara County. Samples were taken November 6, 1999, along the Harris Grade Road in Purisima Hills just north of Lompoc, Santa Barbara County. ( N, W). All plots were in a 5-year-old postfire stand. The terrain was quite steep and the forest dense. The distance to plot C and D, therefore, could not be accurately measured but was estimated to be m and 0 and 1300 m respectively. The plots were at elevation 320 meters, 16 km from the ocean. Sierra National Forest. Samples were taken October 1999 along U.S. Forest Service Road 10S67 (Ross Crossing Road) in Sierra National Forest, Fresno County, at elevation 1450 m, 250 km from the ocean. Plots were sampled under ponderosa pine. Plots A C ( N, W) were taken next to plots 1 5 described by Stendell et al (1999). Plots A and B were south of the road in a unburned stand with some understory. Plot C was north of the road at a prescribed fire site established in No understory was present, but some incense cedars were mixed with the pines. Plot D was taken on a steep slope under a few ponderosa pines. Sampling and processing of soils. At each plot, four soil samples were taken around a 1 m diameter ring. Previous studies showed that most Rhizopogon propagules are found in mineral soil (Miller et al 1994, Taylor and Bruns 1999). Therefore, the organic layer was removed and the upper cm of mineral soil retained. The four soil samples were pooled for a total volume of approximately 5 l. In the laboratory the soil was sifted through a 1.0-mm sieve and air dried in paper bags at room temperature 2 wk. One l of the dried soil was retained for inoculation and the rest was mixed with an equal volume of coarse sand and autoclaved 1 h for diluting the inoculum soil and for use in controls. Bioassay. Pinus muricata seeds, collected at Point Reyes National Seashore, were surface sterilized 20 min in 30%

3 KJøLLER AND BRUNS: RHIZOPOGON SPORE BANK COMMUNITIES 605 hydrogen peroxide, including one drop of Tween-20. The seeds were rinsed and soaked overnight in distilled water and pregerminated 1 week in moist vermiculite. Germinated seeds were transplanted to pots containing 80 ml of growth medium. The growth medium consisted of inoculum soil diluted 1:50 with the autoclaved soil/sand mixture. The dilution was chosen to optimize Rhizopogon colonization with respect to other ectomycorrhizal fungi (Baar et al 1999, Taylor and Bruns 1999). From each plot, 60 pots (RLC-4 Super Stubby Cell Cone-tainer from Stuewe & Sons Inc., Corvallis, ) were sown with two seeds in each pot, which were covered by 2 cm of sterilized sand. As a control for contamination, seedlings were planted in pots containing only the autoclaved soil/sand mixtures. Pots were maintained in a greenhouse and watered 3 times a wk with tap water. Extra inoculum soil from the plots is archived at the Deptartment of Plant and Microbial Biology, University of at Berkeley. Isolation and pure culturing of Rhizopogon sp. from root tips. Pots were harvested 4 5 months after planting. Rhizopogon ectomycorrhizal root tips were identified based on their typical features: densely coralloid branching pattern, felty surface, whitish color often with rusty deposits or red or blue staining and abundant rhizomorphs in similar colors. From successful pots, 3 10 healthy-looking Rhizopogon colonized branches were transferred to fresh distilled water in a Petri dish. In a laminar-flow hood, tips were placed in 30% hydrogen peroxide for 30 s and then back to fresh distilled water. Finally, tips were transferred to the bottom of a sterile Petri dish lid to drain and then placed on dilute MMN medium [1 g Glucose, 2.5 g Malt extract, 50 mg CaCl 2 2(H 2 O), 25 mg NaCl, 150 mg MgSO 4 7(H 2 O), 25 mg (NH 4 ) 2 HPO 4,50mgKH 2 PO 4, 12 mg ferro-citrate, 1 mg thiamin-hcl, 20 g agar, 50 mg chloramphenicol, streptomycin, ampicillin, 1 mg benomyl to 1 L distilled water, ph 5.5]. This version of MMN was relatively low in glucose, malt extract, nitrogen and phosphorus and included the antibiotics ampicillin, streptomycin, chloramphenicol, as well as the fungicide benomyl. The margin of successful cultures were transferred to full strength MMN media without benomyl and antibiotics [2.5 g Glucose, 10 g Malt extract, 50 mg CaCl 2 2(H 2 O), 25 mg NaCl, 150 mg MgSO 4 7(H 2 O), 250 mg (NH 4 ) 2 HPO 4, 500 mg KH 2 PO 4, 12 mg ferro-citrate, 1 mg thiamin-hcl, 20 g agar to 1 L distilled water, ph 5.5] and grown 2 4 weeks. From these cultures 0.5 cm plugs were transferred to a 5 5 cm sterilized cellophane disk (gel drying film, Promega, Madison, Wisconsin) placed on full strength MMN media. These cultures were grown 4 wk at which time most cultures had reached the margin of the cellophane disk. All cultures were maintained at room temperature. At harvest, cultures gently were peeled from the cellophane and placed in 1.5 ml screwcap tubes with two 2.5 mm glass beads. The tubes were snap frozen in liquid nitrogen and freeze dried. Freeze-dried mycelia were stored at room temperature until DNA extraction. Molecular identification of isolates. Freeze-dried mycelia were crushed in a bead beater after which DNA was extracted with the DNeasy Tissue Kit, supplied by QIAGEN (QIA- GEN Inc., Valencia, ). The protocol supplied with the kit was followed, except that the lysed material was centrifuged twice at 7000 rpm to pellet cell debris and, in addition, a chloroform:isoamyl (24:1 vol.) extraction step was included before the filtering steps. PCR amplification of the internal transcribed spacer (ITS) and subsequent restriction digests were performed as described by (Gardes and Bruns 1996). The ITS region was amplified with the primer combination ITS1F-ITS4B (Gardes and Bruns 1993) and restricted with the enzymes Hinf-I and Hha-I (purchased from New England Biolabs. Inc., Beverly, Massachusetts). RFLP patterns were compared within individual RFLP gels, and all unique patterns were sequenced. Both strands were sequenced with ITS1F and ITS4 (White et al 1990) as sequence primers following the procedures provided with the ABI Prism BigDye Cycle Sequencing ready reaction Kit (PE Biosystems, Foster City, ). Electrophoresis and data collection was performed with an ABI 377 sequencer (PE Biosystems). Sequences were compiled with Sequence Navigator software (version 1.01) and visually aligned in the BioEdit Sequence Alignment Editor version 5 (Hall 1999) with sequences of known Rhizopogon species (TABLE I). Phylogenetic analysis was performed with a PAUP*4.0 beta version (Svofford 1998). Data analysis. The similarity of taxa composition within and among sites was analyzed using the complement Bray- Curtis measure (Krebs 1999). The index range is 0 1, where 0 means that two communities do not share any species and 1 means that the communities B 1 n ij i 1 n ij i 1 X X ik (X X ik ) contain all the same species at identical frequency. The index is dominated by the most-frequent species and is calculated as follows: X ij and X ik are the frequency of individuals of taxa i in each site j and k, n is the number of taxa at each site. Rarefaction (Krebs 1999) was used to get an estimate of how well each site was sampled with respect to the total species. Using the observed data, this method allows the calculation of the expected number of species as a function of a random sample of individuals taken from a collection. The frequencies of the taxa were compared for the complete site/taxa table and for each pair of individual sites, using conventional X 2 statistics with the Null Hypothesis that the frequency of taxa was evenly distributed among sites. RESULTS Cultures. Of 619 pots harvested, 92% were colonized with ectomycorrhizal fungi and 366 pots (59%) contained ectomycorrhizae with Rhizopogon-like morphology (TABLE II). From the pots with Rhizopogonlike ectomycorrhiza, 172 root tips were successfully brought into culture, DNA extracted, PCR-ITS amplified and typed by RFLP. This number excludes cul-

4 606 MYCOLOGIA TABLE I. Origin of ITS sequences used for comparison with the spore bank isolates Species Voucher a Location Accession Reference R. arctostaphyli (HOLOTY PE) R. burlinghamii R. ellenae (HOLOTY PE) R. ellenae R. evadens (HOLOTY PE) R. evadens R. evadens R. fuscorubens R. occidentalis R. occidentalis R. ochraceorubens (HOLOTY PE) R. ochraceorubens R. olivaceotinctus R. roseolus R. salebrosus (HOLOTY PE) R. vulgaris R. vulgaris Unidentified Rhizopogon sp. Sarcodes symbiont Pterospora symbiont Pterospora symbiont JPT5705 JMT AHS JMT AHS JMT JMT c JMT JMT LCG 211 AHS JMT HDT JMT 8227 AHS69273 JMT JMT OSC58561 SaHarT2290 Cres2380 Cres2398 Idaho South Carolina Idaho Idaho Idaho AF AF AF AF AF AF AF AF AF AF AF AF AJ AF AF AF AF AF AF AF AF (Bidartondo and Bruns 2002) Direct GenBank Submission This study b (Bidartondo and Bruns 2001) (Bidartondo and Bruns 2002) (Bidartondo and Bruns 2001) (Bidartondo and Bruns 2001) (Bidartondo and Bruns 2001) a In case of the three monotropoid symbiont sequences collection IDs are given. b This former unpublished R. olivaceotinctus sequence was kindly provided by Lisa C. Grubisha. c This collection was originally identified as R. smithii by James M. Trappe. J. Trappe later reexamined the sporocarp and identified it as R. evadens. tures originating from the same pot with identical RFLP fingerprints (TABLE II). From individual pots, cultures from replicate root tips were mostly RFLP identical (70 pots). Cultures with two different RFLP fingerprints were found in three pots and one pot contained cultures of three different types. Generally, between 85 % of the pots from individual plots were ectomycorrhizal. Molecular identification of cultures. We followed the nomenclature of subgeneric groups proposed by and assigned clade names to sequence-defined groups. First, we tested the spore bank-derived sequences against a full range of ITS sequences of known Rhizopogon species ( Johannesson and Martin 1999, Taylor and Bruns 1999, Kretzer et al 2000, Bidartondo and Bruns 2001, Bidartondo and Bruns 2002, Grubisha et al 2002). No sequences recovered belonged within subgenus Villosuli, a group in which all species are host-specific associates of Pseudotsuga spp. (Massicotte et al 1994, Molina and Trappe 1994). Sixty percent of the sequences fell within subgenus Amylopogon, 34% within subgenus Rhizopogon and 6% within subgenus Roseoli (FIG. 2 and TABLE III). Sequences then were assigned clades by the following criteria. A group of sequences had to be a monophyletic group with short branch lengths and supported by bootstrap analysis. Of the 12 clades defined, nine had internal sequence similarity 99% and three 98% (TABLE III). For convenience, we named each group by a voucher species contained within it. Voucher collections assigned different species names fall into some of these clades. In these cases, we used names associated with holotype collections, when such data were available. In lieu of type sequences, we named clades by the mostcommon named species contained in it and, in lieu of identified collections within a clade, we name it by its subgeneric group followed by a roman numeral. A few of the isolated cultures turned out not to be Rhizopogon species. Isolates from PtR-C were 99% sequence identical with Suillus pungens, and four isolates from Salt Point -C were 99% identical to Suillus tomentosus (Kretzer et al 1996, Kretzer and Bruns 1997). Both Suillus species also are specific associates of Pinus, and their mycorrhizae superficially are similar to those of Rhizopogon. Variation within and among sites. We used rarefaction curves (Krebs 1999) of expected number of species versus number of cultures to examine how thoroughly we had sampled species. The method does not extrapolate beyond the total number of individuals in the collection, but the shape of the curve in-

5 KJøLLER AND BRUNS: RHIZOPOGON SPORE BANK COMMUNITIES 607 TABLE II. Pots harvested and success in obtaining Rhizopogon cultures Sites and plots No. of pots used % pots ecto mycorhizal (EM) % pots with Rhizopogon EM morphotypes % isolation success from pots with Rhizopogon a No. of Rhizopogon cultures a PtR-A PtR-B PtR-C PtR-D PtR total Salt Pt-A Salt Pt-B Salt Pt-C Salt Pt-D SPt total SNF-A SNF-B SNF-C SNF-D SNF total Monterey-A Monterey-B Monterey-C Monterey-D Mont. Total St. Barb-A St. Barb-B St. Barb-C St. Barb-D St. B total Grand total a Cultures from the same pot are not included unless they proved to belong to different sequence groups. dicates how well the community has been sampled (if new species are found in each new sample the result will be a positively sloped straight line). Inspection of FIG. 3 indicates that increased sampling effort would result in more species, although the main proportion of the species seems to have been isolated at all sites. It should be noted that these calculations only apply to the specific bioassay and isolation conditions used in this experiment. Changing these conditions, e.g., working with a range of soil dilutions in the bioassay, might increase the total number of species encountered (Baar et al 1999, Taylor and Bruns 1999). The frequency of isolates in each clade was used to compare the Rhizopogon community within and among sites. Overall, the null hypothesis that the frequency of sequence groups was evenly distributed among sites could be rejected (X 2 test P 0.001). Likewise, for all pairwise comparisons the null hypothesis that the frequency of sequence groups was evenly distributed between two sites could be rejected (X 2 test, P 0.005). In Santa Barbara County and Sierra National Forest, the dominating sequence groups were distributed homogeneously among plots (FIG. 4 and TABLE IV). Monterey, Point Reyes and Salt Point contained more heterogeneity and, therefore, lower within-site Bray-Curtis values (TABLE IV). Salt Point showed the most complicated pattern with distinct Rhizopogon communities at each plot (FIG. 4); this resulted in a lower similarity index value within Salt Point than among Salt Point and three of the other sites (TABLE III). These trends are summarized visually with detrending correspondence analysis (DCA), which shows species composition of plots within a site often tend to be more similar than the composition among sites (FIG. 5). This pattern is obvious for Monterey, Sierra National Forest, Santa Barbara and three of the Point Reyes plots. The outlying nature of plot D at Point Reyes and wide variation at Salt Point also stand out. DISCUSSION Perspectives on the methodology. Our results are clearly a biased view of the Rhizopogon species at these sites, but this view is useful for several reasons. Although the bioassay likely recovers species with abun-

6 608 MYCOLOGIA FIG. 2. Phylogenetic comparison of 72 ITS sequences from spore bank derived cultures, 18 Rhizopogon basidiocarp sequences and three sequences from mycorrhizal monotropoid plants. Phylogeny was calculated using PAUP*4.0 [Swofford, 1998#433]. Gaps were treated as missing and trees were midpoint rooted. MaxTrees was set to 0, and 0 trees were retained. Bootstrap values following bootstrap replicates 80% are given near branches. Branches present in the strictconsensus tree are bold. Subgenera as proposed by are shown. mon Monterey, stb Santa Barbara, snf Sierra National Forest, ptr Point Reyes and spt Salt Point. See Fig. 4 for distribution of each sequence type within plots and sites.

7 KJøLLER AND BRUNS: RHIZOPOGON SPORE BANK COMMUNITIES 609 TABLE III. Sequence identity, number of cultures assigned to each clade and presence of voucher sequences in each clade Sequence groups Sequence identity (%) Sequences obtained Number of cultures Voucher sequences salebrosus a clade b 99% R. salebrosus (HT) Pterospora symbiont Cres2380 amylopogon clade I 99% 4 7 none amylopogon clade II 99% 8 33 Basidiocarp OSC58561 arctostaphyli a clade 99% 7 23 R. arctostaphyli (HT) Pterospora symbiont Cres2398 ellenae a clade 98% 2 4 R. ellenae (HT) R. ellenae JMT Sarcodes symbiont roseolus clade 99% 3 7 R. roseolus R. burlinghamii vulgaris clade 98% 1 3 R. vulgaris JMT and JMT occidentalis a clade 98% R. occidentalis JMT and LCG 211 R. ochraceorubens (HT) AHS and JMT fuscorubens clade 99% 4 8 R. fuscorubens evadens a clade 99% 3 12 R. evadens (HT) AHS versicolores clade I 99% 1 2 R. evadens JMT olivaceotinctus clade 99% 3 6 R. olivaceotinctus a Name based on ITS sequence from holotypes of Rhizopogon species. b Many herbarium collections assignable to this clade are identified as R. subgelatinosus, R. semireticulatus, R. subcaerulescens or R. ellenae, but the holotypes for the two latter and more common used names fall into the ellenae clade (Bidartondo and Bruns 2002). Spore bank derived sequences have been deposited in the EMBL database with accession numbers AJ AJ and AJ AJ Voucher codes are shown here only when more than one collection of a species are included. For further information on the voucher sequences inspect TABLE I. dant responsive spores, this is a relevant group to examine because species identified from bioassays were among the most common colonizers found in postfire seedling communities (Horton et al 1998, Baar et al 1999, Grogan et al 2000). Culturing introduces a second potential bias, and we might have lost unique species in this step. Although, because Rhizopogon species are known to grow well in culture (Molina et al 1999), we do not believe that we have introduced a strong bias. In addition, our primary screening was based on ectomycorrhizal morphotypes; this is an advantage because only one ectomycorrhizal individual typically is present on each root tip. Thus culturing from them does not tend to select for the fastest-growing species, as would be the case with soil dilution plate techniques. Furthermore, we saw no evidence for differential success in isolating cultures from different Rhizopogon morphotypes, and the 12 sequence groups we isolated covered most of the known pine associated Rhizopogon diversity as well as one previously unknown clade (the amylopogon clade I from Monterey). Isolation success from colonized pots was fairly similar, with an average of 50% (TABLE II). In addition, neither the bioassay nor the culture biases discussed above are likely to vary among sites. Thus among-site and plot comparisons should be valid. If species interactions result in replacement or exclusion within bioassay pots, then this would have the potential to cause site-specific biases. However, the high dilution of soil that we used (50-fold), and the growth period ( 6 months) should have minimized species interactions within individual pots. In any case, we required isolates for our long-term goal of examining the population genetics of some of the common species, so culturing was a necessary source of potential bias. One major advantage of the approach is that it provided a very efficient way to recover multiple genotypes of particular Rhizopogon species from a spatially defined sample with minimal disturbance to the community. Essentially we could recover any size population that we desired for the more common species by simply planting sufficient bioassay seedlings. In comparison, one normally must rake extensively to find hypogeous sporocarps, and because fruiting phenology varies, single collection days also produce a bias. Sim-

8 610 MYCOLOGIA TABLE IV. Inverse Bray-Curtis similarity indices comparing frequencies of clades within and among the five sites FIG. 3. Theoretical species-sampling curve calculated with the rarefaction method (Krebs 1999). The method uses the recorded total number of individuals, total number of species and the frequency of each species to calculate the expected number of species in a random sample of individuals taken from a collection. Note that in this experiment approximately three seedlings were needed to obtain one culture (TABLE I). Bray-Curtis Index Salt Point Point Reyes Monterey St. Barbara Sierra National Forest Salt Point Point Reyes 0.18 a 0.51 b 0.34 Monterey St. Barbara S.N.F a Values within sites (in bold) are means of the six pairwise comparisons among plot A-D. b Index was calculated using standardized frequencies to normalize the differences among the total number of cultures obtained from each plot/site. Using the absolute numbers produced very similar results to the ones in this table (data not shown). ilarly, sampling roots directly from nature is not a very efficient way to recover most pine-associated Rhizopogon species because they tend to be low abundance components of a complex community in these forests. The species concept within Rhizopogon is not resolved, nor are we attempting to resolve it in this paper. Evidence from prior studies has shown that species names have been applied in a very inconsistent way to sporocarps collections and even many paratype collections (Bidartondo and Bruns 2002; A. Kretzer pers comm). We have taken the pragmatic approach of treating tight ITS-defined groups as useful taxonomic units, and when possible we have used existing sequences from holotype material and other collections to limit the possible species names ap- FIG. 4. For each site, frequencies of isolates of each clade in the individual plots are shown. For all sites, frequencies of isolates of each clade are compared among sites. Bars represent percentages of each type, but the actual numbers of isolates are shown in parenthesis after each plot and clade name respectively.

9 KJøLLER AND BRUNS: RHIZOPOGON SPORE BANK COMMUNITIES 611 FIG. 5. Detrending correspondence analysis (DCA) of species abundances in 19 plots from five sites. DCA was calculated with the CANOCO 4.0 software packet. Data were standardized to give relative abundance of species in plots. Axis 1 and 2 accounted for 58 and 29% of the variation respectively. plied to these. However, we are aware that ITS sequences are unlikely to differentiate all closely related species. Thus, many of these sequence-defined clades might be species groups rather than species. Data from multilocus approaches will be necessary to further differentiate species in this taxonomically challenging genus, but in the meantime these ITSdefined groups provide a useful tool for exploring the biology and ecology of Rhizopogon. Distribution of Rhizopogon inoculum and patterns of within site variation. During the past 10 years, the ectomycorrhizal community associated with bishop pine at Point Reyes National Seashore in has been analyzed extensively (Horton et al 1998, Baar et al 1999, Taylor and Bruns 1999, Grogan et al 2000). In this system, Rhizopogon species are only lowabundance members of the mature forest but are highly abundant on seedlings established after wildfire and abundant in seedling bioassays, both with mature as well as with fire-treated soil. Rhizopogon spores either stay where basidiomes decompose or rely on animals, especially rodents, for dispersal ( Johnson 1996, Molina et al 1999). Although rodents act to smooth out the initial pointsource pattern of the fruiting bodies, the dispersal biology of these fungi still would predict a clumped distribution of inoculum within a forest. However, the nearly uniform occurrence of Rhizopogon on postfire seedlings within formerly non-forested shrub communities was at odds with this prediction (Horton et al 1998). Similar uniformity was found in the current study. Not only did 19 of 20 plots produce seedlings with Rhizopogon colonized root tips, but at all sites, except Salt Point, the dominating clade were found in three or four of the plots within a site. This pattern includes the D plots that were spaced 1 km from the other three plots (FIGS. 4 and 5). These results show that at the scale of 10 0 m, distance is not very predictive of spore-bank similarity. This is especially impressive because the soils were diluted 50-fold before bioassaying. Thus, Rhizopogon spores were not just present, they were abundantly present at each site. A high longevity of Rhizopogon spores would explain how inoculum becomes fairly homogenized within a site because time would smooth out the patchy spatial dispersal. In addition, high longevity coupled with high abundance would enable other means of dispersal, because any process that moves soil would move Rhizopogon. Patterns of among-site variation. In coastal, pines are found in rather small discontinuous populations (Vogl et al 1990). Because of the discontinuity of their hosts and their hypogeous nature, the potential for Rhizopogon species to spread among sites might be limited. The difficulty of spore dispersal beyond local forests could lead to communities differing among sites. Some evidence for both geographic structure and the lack of it are seen in these data. The similarity indexes (TABLE III) indicate that Monterey, Sierra National Forest and Santa Barbara are more homogenous within each site than among each of these and other sites, while Salt Point and Point Reyes, the sites that are the closest pair (75 km), shared most of their sequence groups, giving a high similarity index. On the other hand, the DCA plot (and the Bray-Curtis values) shows that Sierra National Forest communities do not appear to be strikingly different from those on the coast. This is somewhat unexpected because both the host pine species and the climate at this site differ from all other sites, and the unforested San Joaquín Valley separates Sierra National Forest from all other sites. A striking case of geographic diversification is seen in subgenus Amylopogon. Amylopogon clades I and II, sister groups, were found only in Monterey and Santa Barbara, respectively. These are the two southernmost coastal sites. The sister group of these is the arctostaphyli clade, which was retrieved only from Sierra National Forest. Salt Point and Point Reyes, the two northern coastal sites, lacked all three of these clades but did have the related salebrosus clade, which was lacking at the southern coastal sites but present in the Sierra. In sites where the salebrosus clade was present, no significant ITS variation correlated with geography. Similarly, the occidentalis clade, from subgenus Rhizopogon, was found at all

10 612 MYCOLOGIA sites except Santa Barbara and there was no significant ITS variation among sites. If there is genetic differentiation among geographic regions within clades shared among sites, ITS sequences are not sensitive enough to reveal it. A preliminary AFLP (amplified fragment-length polymorphism) screening of the isolates in the salebrosus clade from Salt Point, Point Reyes and Sierra National Forest indicated that the populations from these sites indeed could be separated into site-specific groups and that the populations from Salt Point and Point Reyes were more similar to each other than to the population in Sierra National Forest (R. Kjøller unpubl). This suggests that the isolation may be a driving force in the Rhizopogon diversification among these forests. On the other hand, differences in local conditions (e.g., soil characteristics, climate, etc.) among sites also could lead to the greater similarity, and this effect cannot be ruled out from this experiment, because Point Reyes and Salt Point have the most-similar climatic conditions. Studies comparing isolates from clades shared among sites under various physical and chemical conditions are needed to clarify this further. The Santa Barbara community was strikingly different from the four others because it was dominated almost totally by one sequence group. The reasons for this limited diversity are not clear. The conditions in Purisima Hills, where Santa Barbara samples were taken, are harsher than other coastal sites with less rainfall and warmer temperatures. These plots also are more isolated in terms of distance to other closed-cone pine stands. Both factors might have reduced the diversity at this site. Ongoing work on Rhizopogon spore banks from the nearby Channel Islands, which are even more isolated and provide a similar environment, also have revealed limited Rhizopogon species diversity but with different species from those found in Santa Barbara (L. Grubisha pers comm). Unlike our other sites, all Santa Barbara plots recently were burned by a stand-replacing fire. However, reduced Rhizopogon diversity resulting from a stand-replacing fire is not consistent with the studies at Point Reyes (Horton et al 1998, Baar et al 1999, Taylor and Bruns 1999, Grogan et al 2000). These demonstrated that a rich Rhizopogon spore-bank community survived that fire intact and dominated the roots of pine seedlings in the first postfire years. In addition, plots -C in Sierra National Forest and -D in Monterey also had been burned recently from either a prescribed ground fire (Sierra) or a small standreplacing fire (Monterey). Yet these plots were not noticeably different from the unburned plots at the same sites. Conclusions. Much remains unknown about the biology of pine-associated Rhizopogon species. However, it is now clear that they are important components of the spore bank in many widely dispersed n settings. We suspect that nearly uniform occurrence of Rhizopogon partially is due to the longevity of the spores, but this conjecture still needs to be demonstrated. Within local forest areas spores of Rhizopogon species are fairly homogeneously distributed. High spore longevity and mixing by soil and water movement might explain this. At the geographic scale, it is clear that there is spatial structure in these communities. This observation fits with an expectation of limited dispersal and leads to the prediction of limited gene flow among populations. To test this we will need a more refined delimitation of the species and more variable genetic markers. ACKNOWLEDGMENTS We wish to thank Martin Bidartondo and Lisa Grubisha for providing access to their Rhizopogon sequences before publication and for comments on drafts of this manuscript, Jenny Tan and Sermed Naaman for help in the laboratory and Lee Taylor for collecting the Santa Barbara samples. The Danish Natural Science Research Council to R. Kjøller and NSF grant DEB to T.D. Bruns provided partial financing of this study. LITERATURE CITED Amaranthus MP, Perry DA Interaction effects of vegetation type and Pacific madrone soil inocula on survival, growth, and mycorhiza formation of Douglas fir. Canadian Journal of Forest Research 19: Baar J, Horton TR, Kretzer AM, Bruns TD Mycorrhizal colonization of Pinus muricata from resistant propagules after a stand-replacing wildfire. New Phytologist 143: Bidartond MI, Kretzer AM, Pine EM, Bruns TD High root concentration and uneven ectomycorrhizal diversity near Sarcodes sanguinea (Ericaceae): a cheater that stimulates its victims? 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