Size, distribution, and persistence of genets in local populations of the late-stage ectomycorrhizal basidiomycete, Russula brevipes

Authors

  • Sarah E. Bergemann,

    Corresponding author
    1. University of Wyoming, Botany Department, Laramie, WY 82071–3165, USA
      Author for correspondence: Sarah Bergemann Tel: +1 (510) 643 4282 Fax: +1 (510) 643 5436 Email: sbergemann@nature.berkeley.edu
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  • Steven L. Miller

    1. University of Wyoming, Botany Department, Laramie, WY 82071–3165, USA
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Author for correspondence: Sarah Bergemann Tel: +1 (510) 643 4282 Fax: +1 (510) 643 5436 Email: sbergemann@nature.berkeley.edu

Summary

  • • The spatial mapping of genets from sporocarps allows determination of the primary modes of reproduction of ectomycorrhizal fungi. The goals of this research were to determine the relative size, density, and persistence of genets of Russula brevipes , a late successional basidiomycete, in mature stands of lodgepole pine ( Pinus contorta ) and Sitka spruce ( Picea sitchensis ).
  • • Sporocarps were mapped and genets were identified using species-specific hypervariable microsatellite markers.
  • • Most of the sporocarps sampled in clumped aggregations were the same genet and occupied areas less than 3 m. The largest distance measured between two sporocarps of the same genet was 18 m. In local populations of R. brevipes associated with Sitka spruce, genets were comprised of largely related individuals, whereas in lodgepole pine, most genets appeared unrelated.
  • • High genotypic diversity in Sitka spruce stands suggests frequent mating and recombination between local inhabitants from primary establishment of basidiospores. In lodgepole pine, fewer, unrelated genotypes of R. brevipes indicates that multiple sources of colonization arose from infrequent, long distance spore dispersal events.

Introduction

Temporal and spatial succession of ectomycorrhizal fungi (EMF) was first demonstrated in stands of Betula (Ford et al., 1980; Deacon & Fleming, 1992 for a complete review). As the trees in these studies aged, EMF that established early in succession were displaced by others and this process was thought to closely resemble seral succession in plant communities (Frankland, 1992). The persistence of early successional fungi on newly formed root systems resulted in distinct spatial patterning of EMF (Dighton & Mason, 1985; Dighton et al., 1986; Mason et al., 1987). The terms ‘early stage’ and ‘late-stage’ were adopted to differentiate between fungi found on young roots from EMF that colonized on older root systems.

The colonization and establishment of early- and late-stage EMF have been linked to physiological interactions between their hosts and environment and their reproductive strategies. Early stage EMF appear to demand small amounts of carbon from their hosts and require low concentrations of nitrogen and phosphorus, whereas late-stage fungi require greater amounts of carbon, nitrogen and phosphorus (Deacon & Fleming, 1992; Colpaert et al., 1996). Colonization by late- or early stage EMF appears to be dependent on their reproductive mode (e.g. spores or mycelia), age of the host, and substrate conditions. Early stage EMF establish readily by spores or mycelia in laboratory inoculation experiments, whereas late-stage EMF establish primarily from dikaryotic mycelia (Deacon & Fleming, 1992). Consequently, for early stage EMF, a high number of genotypes would be expected as a result of reproduction primarily by spores, whereas the formation of larger clones would be predicted if reproduction occurred primarily by mycelial expansion in late-stage EMF (Dahlberg & Stenlid, 1990, 1994; Dahlberg, 1997). Most studies of early stage EMF confirm these expectations (de la Bastide et al., 1994; Henrion et al., 1994; Gryta et al., 1997; Guidot et al., 1999).

Reports of the longevity and persistence of a genotype at a particular locale are not consistent. In independent studies of the early stage fungus, Hebeloma cylindrosporum, genets remained at a particular locale after many years (Henrion et al., 1994), in contrast to genets that were not found in successive samplings of the same host tree (Gryta et al., 1997). In early stage EMF, Laccaria bicolor and L. laccata, only a few of the genets mapped persisted over a sampling period of 3 yr (Selosse et al., 2001). Ectomycorrhizal root tips are of short duration, with a turnover time of 3 months to 3 yr (Fogel, 1983; Harley & Smith, 1983) therefore persistence over longer periods requires repeated colonization events at the root mycelium/spore interface or by persistent mycelia.

Published reports of the size and number of genets for late-stage fungi are also rare. Russula spp. are good candidates for examination of genetic structure of late-stage EMF since they often comprise the majority of sporocarps found in mature stands of temperate forests (Richardson, 1970; Harvey et al., 1976; Watling, 1984; Bills et al., 1986; Mason et al., 1987; Kernaghan et al., 1997), are generally absent or not abundant in early regeneration of stands (Keizer & Arnolds, 1994; Bradbury, 1997; Bradbury et al., 1998; Kranabetter & Wylie, 1998) and rarely form mycorrhizae from spore inoculum in laboratory experiments (Ali & Jackson, 1988).

The relative contribution of spores and vegetative growth through spread of mycelia is often studied for EMF by mapping genets. In this paper, we compare the local genetic structure of two natural populations of the late-stage ectomycorrhizal basidiomycete, Russula brevipes, to determine the size, density, and temporal persistence of genets in mature stands of lodgepole pine and Sitka spruce.

Methods and Materials

The first study site was located approximately 50 km west of Laramie, WY, USA, near the town of Fox Park in the Snowy Range of the Medicine Bow Mountains (41°04′-N, 106°10′-W) at an elevation of approximately 2775 m. The sites consisted of a large-tract even-aged (100 yr old) lodgepole pine (Pinus contorta ssp. latifolia) forest with sparse ground cover (Parsons et al., 1994a,b). The second sampling site was located in Big Lagoon County Park on the northern coast of California c. 16 km north of Trinidad, CA (41°16′-N, 124°12′-W). The sampling was conducted in Sitka spruce (Picea sitchensis) stands aged 40–60 yr with little understory at sea level.

Sporocarps of R. brevipes were collected in lodgepole pine stands during periods of fruiting in July, August, and September of 1998 and 1999. At one particular locale, voucher collections of sporocarps from 1988 were used to determine if genets were persistent over an 11-yr time frame. Sporocarps of R. brevipes associated with Sitka spruce were mapped during October and November of 2000. The positions of mapped locales were recorded with a GeoExplorer III (Trimble Navigation Ltd, Sunnyvale, CA) hand-held GPS unit. One hundred GPS positions were taken at each locale sampled. All positions were differentially corrected using GPS Pathfinder Office software (Trimble Navigation Ltd, Sunnyvale, CA) from the nearest base station and averaged to obtain one position for each locale sampled. The differentially corrected positions were imported into ArcView GIS 3.2 (ESRI Redlands, CA) and mapped. At each locale sampled, the physical distance was measured between individual sporocarps was mapped. All sites were ground-truthed for accuracy of measurements.

Approximately 100 mg of tissue were ground in liquid nitrogen and then incubated in 2X CTAB (2% CTAB, 0.1 m Tris-HCl, pH 8.0, 20 mm EDTA, pH 8.0, 1.4 m NaCl) at 65°C for 30–45 min and spun at 12 000 rpm for 15 min (Gardes & Bruns, 1993). DNA was gene cleaned following instructions with Gene Clean III kit (QBioGene, Chatsworth, CA, USA).

GIS (Genetic Identification Services, Chatsworth, CA, USA) was contracted to construct a microsatellite-enriched genomic library of di- and tri-nucleotide repeat regions and design of primers to amplify simple sequence repeats in R. brevipes. Of the 26 primer combinations designed by GIS, three primers were identified that were sufficient for identifying genets (Table 1).

Table 1.  Microsatellite primer pairs, repeat type and size range of amplified products of Russula brevipes sporocarps
LocusRepeat ArrayForward Primer 5′–3′Reverse Primer 5′–3′Size range of amplified product (bp)
RB23CAGCAG-CCG-TCT-TTC-TCT-CTC-CGCC-TTG-AAT-CAC-TAC-CTC-CA184–247
RE14GATAC-CCA-TTG-CCT-TGT-TTC-CACT-CCG-CGT-TCT-GCT-AGA-G186–204
RE102GAGGA-CTT-GTC-AGC-GTC-AAGTCA-ACC-ATC-TCA-AGG-TAT-GTC134–144

Microsatellite PCR amplifications were performed in a total volume of 10 µl containing 1.84 µl of dH2O, 1× Promega PCR buffer, 3 mm MgCl2, 2 nmol dNTP’s, 10 ng BSA, 0.9 pmol of unlabeled forward microsatellite primer, 0.1 pmol of fluorescent labeled forward primer, 1 pmol of unlabeled reverse microsatellite primer, 0.8 m betaine, 0.80 U of Taq polymerase, and 10 ng DNA. Reactions were performed for 35 cycles using the using the cycling profile: 96°C for 2 min, 35 cycles of 96°C for 20 s, 55°C for 20 s, 72°C for 30 s, and 72°C for 10 min After PCR, 1–4 µl of stop buffer (LI-COR, Lincoln, NE) was added to each reaction, centrifuged, denatured at 94°C for 3 min, and stored at 4°C until ready for use. Microsatellite PCR products were electrophoresed on 6.5% KBPlus (LI-COR, Lincoln, NE) 25 cm gel at 1500 V, 40 Watts, 40 mA and 45°C. Allele sizes were determined using SAGAGT ver. 2.0 (LI-COR, Lincoln, NE, USA). Genets were defined by identifying unique combinations of allele frequencies.

In sporocarps with identical genets, the probability of a genotype occurring by chance was computed assuming random mating. The probability of a particular multiple-locus genotype was calculated by multiplying together the frequencies of all the individual alleles and including an additional factor of 2 for each heterozygote locus. Inbreeding coefficient (FIS) was calculated for each population and the degree of genetic differentiation among populations (FST) was calculated following the methods of Weir & Cockerham (1984) using PowerSSR version 1.2 (Liu, 2001).

Results

All three microsatellite primer combinations used for analyses of genotyping were polymorphic at each locus examined (Table 1, Fig. 1). Three primer combinations yielded 28 alleles; 17 alleles were detected with RB23, five with RE14 and six alleles with RE102 (Table 1, Fig. 1).

Figure 1.

The size and frequency of alleles amplified with three primers (RB23, RE14, RE102) for sporocarps collected in lodgepole pine (LP; closed columns) stands in the Rocky Mountains and Sitka spruce (SS; open columns) stands on the northern coast of California demonstrates little overlap in allele frequencies between populations.

Analysis of simple sequence repeats (SSRs) or microsatellites is the method of choice for forensic identification of genetic individuals (Edwards et al., 1991; Urquhart et al., 1994) and has been shown to be a reliable and sensitive method for measuring intraspecific variation in EMF (Kretzer et al., 2000; Zhou et al., 2001). The results of our analyses confirmed that species-specific microsatellite primers generated diverse polymorphic patterns for measurement of intraspecific variation in R. brevipes (Table 1, Fig. 1). The expected probabilities of encountering a genotype by chance ranged from approximately 1/14–1/600 in lodgepole pine sites and 1/60–1/200 in Sitka spruce sites (Table 2).

Table 2.  The probability of a multilocus genotype occurring by chance ( POC ) for genets of Russula brevipes with multiple sporocarps. Genets A, C, D, F, G, and H were sampled in lodgepole pine stands. Genets K, M, O, P, S, T, and U were sampled in Sitka spruce sites
GenetLocusAllele sizeFreqPGenetLocusAllele sizeFreqP
ARB232170.2580.070KRB231870.4570.099
  2350.1360.070  1930.1090.099
 RE1021361.0001.000 RE1021400.5000.348
       1440.3480.348
 RE141861.0001.000 RE142020.4350.189
   POC0.070   POC0.007
CRB231990.0610.007MRB231840.3260.298
  2230.0610.007  1870.4570.298
 RE1021341.0001.000 RE1021400.5000.348
       1440.3480.348
 RE141861.0001.000 RE141980.3040.093
   POC0.007   POC0.010
DRB232080.1210.062ORB231870.4570.208
  2170.2580.062     
 RE1021361.0001.000 RE1021420.5000.348
       1440.3480.358
 RE141861.0001.000 RE142020.4350.189
   POC0.062   POC0.014
FRB231960.0450.006PRB231870.4570.099
  2470.0610.006  1900.1090.099
 RE1021340.2420.309 RE1021400.5000.348
  1360.6360.309  1440.3480.348
 RE141861.0001.000 RE141980.3040.146
       2000.2390.16
   POC0.002   POC0.005
GRB232290.0300.002SRB231840.3260.298
  2380.0300.002  1870.4570.298
 RE1021361.0001.000 RE1021400.5000.348
       1440.3480.348
 RE141861.0001.000 RE141980.3040.146
  2000.2391.000     
   POC0.002   POC0.015
HRB232170.2580.031TRB231870.4570.208
  2410.061      
 RE1021361.0001.000 RE1021420.5000.348
       1440.3480.348
 RE141861.0001.000 RE142000.2390.208
       2020.4350.435
   POC0.031   POC0.015
     URB231840.3260.071
       1900.1090.071
      RE1021400.5000.250
      RE141980.3040.265
       2020.4350.265
        POC0.005

Distribution of allele frequencies in both populations sampled indicated little overlap in allele size between sporocarps associated with lodgepole pine and Sitka spruce (Fig. 1). In the locus RB23, four alleles were amplified in Sitka spruce populations; 14 in lodgepole pine (Fig. 1). In R. brevipes associated with lodgepole pine, the amplified products were fixed at 186 bp and varied from one to four alleles ranging in size from 198 to 204 bp in the Sitka spruce populations (Fig. 1). Three alleles were amplified in the Sitka spruce populations ranging in size from 140 to 144 bp; four alleles sizes identified in lodgepole pine sporocarps ranged in size from 134 to 140 bp (Fig. 1).

At the lodgepole pine site, three of the nine genets from the 33 sampled sporocarps were represented by a single sporocarp (B, E, and I in Fig. 2). In the Sitka spruce population sampled, seven of the 23 sampled sporocarps were from a single genet (J, L, N, R, Q, V, and W in Fig. 3). The persistence of the same genet was noted over the 11-yr sampling period for genet A in the lodgepole pine site (Fig. 2). In areas where multiple sporocarps were sampled, aggregations of sporocarps were predominately the same genotype for both Sitka spruce and lodgepole pine sites (Figs 2 and 3). Generally, the distances measured between sporocarps of the same genet less than 3 m in all sites, however, distances up to 18 m were observed between sporocarps of the same genetic individual.

Figure 2.

Maps of the sporocarps sampled, the genets identified in stands, and the alleles for each locus (RB23, RE102, and RE14) of lodgepole pine. Genets of lodgepole pine are mostly unrelated. C = continuity locale where sporocarps were mapped over an 11-yr period. Arrows indicate sporocarps mapped in 1988.

Figure 3.

Maps of the sporocarps sampled, the genets identified, and the alleles for each locus (RB23, RE102, and RE14) in stands of Sitka spruce. Clusters of sporocarps typically comprise the same genet.

Estimates of high genetic differentiation were measured between populations of Russula brevipes (Table 3). This is likely a result of little overlap in allele frequencies between populations (Fig. 1). Positive inbreeding coefficients (FIS) indicated a heterozygote deficiency in Russula brevipes associated with lodgepole pine, whereas, heterozygote excess evidenced by a negative inbreeding coeffcient was identified in R. brevipes in Sitka spruce stands (Table 3).

Table 3.  Inbreeding coefficients ( FIS ) and estimate of genetic differentiation ( FST ) among populations of Russula brevipes associated with lodgepole pine and Sitka spruce
 lodgepole pineSitka spruce
FIS0.151−0.080
FST0.434 

Discussion

The results of our study indicated that mixed modes of reproduction occur in sampled populations of R. brevipes, persistence of genotypes is possible over time, and in general, identical genets were sampled in spatial aggregations of sporocarps. The results are similar to the pattern reported in Suillus bovinus, S. pungens, S. variegatus, Xerocomus chrysenteron, and X. pruinatus (Dahlberg & Stenlid, 1994; Dahlberg, 1997; Bonello et al., 1998; Fiore-Donno & Martin, 2001). In all of these species, there is evidence for temporal and spatial persistence of genets over time.

Although most genets of R. brevipes comprised areas of less than 3 m, distances up to 18 m were measured between two sporocarps of the same genet. The presence of small genets suggests recent colonization by basidiospores, whereas fewer individuals are indicative of more mature mycelial systems that have grown from point sources of individual mating events over long periods (Dahlberg & Stenlid, 1990, 1994; Dahlberg, 1997). Numerous, small genet sizes observed in R. brevipes suggests that the local population is reproducing by sexual production of basidiospores. The larger genets demonstrate that R. brevipes can colonize by mycelial expansion.

Establishment of an individual genet from mycelial systems or through colonization of basidiospores largely depends on whether the community consists of largely occupied or unoccupied territories (Dahlberg & Stenlid, 1995). If a community consists of areas with available territory, ruderal characteristics such as high spore production affords considerable advantages (Dahlberg & Stenlid, 1995). EMF studied to date that conform to pioneer species with ruderal characteristics include Hebeloma cylindrosporum (Gryta et al., 1997; Guidot et al., 1999), Laccaria amethystina (Gherbi et al., 1999; Fiore-Donno & Martin, 2001), and L. bicolor (de la Bastide et al., 1994; Henrion et al., 1994). In communities with inter- and intraspecific competition, mycelial colonization affords better competitive advantages (Dahlberg & Stenlid, 1995) unless sites are periodically disturbed (Frankland, 1992; Dahlberg & Stenlid, 1994). Under this scenario, colonization strategies involve a combination of ruderal (r), combatitive (c), and stress (s) selected strategies with primary spore reproduction, colonization, and subsequent spread of persistent mycelia (Cooke & Rayner, 1984). This is exemplified in many taxa including Suillus bovinus, S. pungens, S. granulatus, Xerocomus chrysenteron, and X. pruinatus (Dahlberg & Stenlid, 1990, 1994; Bonello et al., 1998; Fiore-Donno & Martin, 2001). In sampled populations of R. brevipes, temporally persistent mycelia conform to colonization strategies that combine r, c, and s strategies (Cooke & Rayner, 1984).

In studies of Suillus spp., the size of genets was correlated with age of host stands (Dahlberg & Stenlid, 1995). Further studies that examined the spatial extent of genets indicated that the age of stands may not adequately predict colonization strategies for all EMF. Higher frequencies of genets were recorded for Lactarius xanthogalactus and Russula cremoricolor in mature forests than predicted for late-stage EMF (Redecker et al., 2001) and were much smaller than the size of genets measured in R. brevipes. In other studies, a high frequency of genets was found in L. amethystina sporocarps compared with largely clonal genets of Xerocomus spp. mapped in the same mature forest (Gherbi et al., 1999; Fiore-Donno & Martin, 2001). It is clear from these studies that EMF colonization strategies are species-specific adaptations to the local environment, however, the autecological and biological processes that affect germination, colonization, and establishment are largely unknown.

The persistence of a genotype over an 11-yr sampling period demonstrates that the mycelium of R. brevipes is perenniating over longer direct sampling periods observed for any EMF examined to date. Large, persistent genets formed by clonal expansion were reported for some species of Suillus (Bonello et al., 1998; Dahlberg & Stenlid, 1990, 1994), and Xerocomus spp. (Fiore-Donno & Martin, 2001). These EMF possess stress-tolerant adaptive characteristics such as mycelial cords or strands that facilitate their competitive ability in mature forests (Frankland, 1992). Such adaptations are lacking in Russula spp. and growth results only from mycelia radiation in multiple directions (Read, 1992).

For EMF, little evidence exists for long distance spore dispersal (Lamb, 1979; Allen & Hipps, 1984). This is supported by high levels of genetic differentiation between populations and little overlap in allele frequencies between populations. For these populations, the Rocky Mountains may be a formidable barrier to long distance dispersal between populations. However, nonoverlapping alleles between populations indicate a strong reproductive barrier to mating rather than a simple isolation-by-distance effect. Future studies should be aimed at assessing the genetic relatedness among populations.

Genotypic variation observed in sampled populations could have originated from genetically diverse founder populations, multiple sources of colonization, and somatic mutations (Milgroom et al., 1992; Wang & Szmidt, 2000). In the Sitka spruce population, a negative inbreeding coefficient (excess of heterozygotes) is suggestive of random mating within a local genetic neighborhood. In contrast, nonrandom mating in R. brevipes associated with lodgepole pine is supported by a positive inbreeding coefficient (deficiency of heterozygotes). Nonrandom mating could have arisen due to inbreeding within the population or colonization by diverse founder individuals from multiple genetic neighborhoods. If inbreeding were common, closely spatial individuals would likely share similar allele frequencies. If the latter were true, unrelated individuals with highly divergent genetic profiles reside in the local population. In R. brevipes sampled in lodgepole pine, the bimodal distribution of allele frequencies in the lodgepole pine site and a positive inbreeding coefficient suggests that the populations are comprised largely of genetically unrelated individuals. It is unlikely that mating between unrelated individuals within the sampled area is common; rather, genotypic diversity likely arose from colonization by genetically dissimilar founders.

The sampling of sporocarps above ground may not be an adequate estimate of the size, frequency or spatial extension of genets below ground. In investigations of community structure above and below ground for EMF, the abundance of mycorrhizal morphotypes was not always indicative of the commonly occurring sporocarps (Gardes & Bruns, 1996; Dahlberg et al., 1997; Jonsson et al., 1999). Similarly, spatial extent of genotypes may not be reflected in sporocarp fruiting patterns. Future studies of the population dynamics of R. brevipes will be aimed at assessing the spatial extent and density of genets of mycorrhizae. Detailed studies employing molecular markers that estimate the relationships among individuals and that elucidate potential colonization and reproductive strategies of putative late-stage EMF such as R. brevipes are under way.

Acknowledgements

The authors would like to thank Terry McClean for technical support, R. Greg Thorn and two anonymous reviewers for critical suggestions for this manuscript. Funding was provided by Sigma Delta Epsilon Graduate Women in Science Eloise Gerry Fellowship, National Science Foundation DEB 0104976 & DEB 9974018, USDA Grant CREES 2000–02861, and a MacIntyre Stennis grant.

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