Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Hebeloma crustuliniforme modifies root hydraulic responses of trembling aspen (Populus tremuloides) seedlings to changes in external pH

The main objective of the study was to compare the effects of short-duration pH treatments on root hydraulic properties in trembling aspen (Populus tremuloides) seedlings that were either inoculated with the ectomycorrhizal fungus Hebeloma crustuliniforme or remained non-inoculated (control). Inoculated and non-inoculated plants were exposed in solution culture to the root zone pH ranging from 4 to 9 and their root hydraulic conductivity was examined using the hydrostatic method and after subjecting the plants to treatments with 100 ??M HgCl₂ (aquaporin blocker) and 0.02% trisodium 3-hydroxy-5,8,10-pyrenetrisulfonic acid (apoplastic transport tracer). In a separate experiment, pure cultures of H. crustuliniforme were also grown on a slid medium with the pH ranging from 4 to 9 to determine their pH growth optimum and changes in medium pH over time in the presence and absence of 8 mM NH₄NO₃. When grown in pure culture, H. crustuliniforme demonstrated maximum growth at pH 7?C8 and was capable of modifying the pH of its growth media, especially in the presence of NH₄NO₃. The plants that were inoculated with H. crustuliniforme had a maximum root hydraulic conductivity at pH 7. At this pH, root hydraulic conductivity was significantly higher compared with non-inoculated plants and showed greater sensitivity of root water transport to pH changes relative to non-inoculated seedlings. Relative apoplastic flux was largely unaffected by pH in inoculated seedlings. Fungal inoculation modified the response of root hydraulic conductivity to pH. The increased root hydraulic conductivity in inoculated seedlings was likely due to an increase in aquaporin-mediated cell-to-cell water transport, particularly at the higher pH. A possible role of fungal aquaporins in the root hydraulic conductivity responses of mycorrhizzal plants should be examined.     

Microsatellite DNA somaclonal variation in micropropagated trembling aspen (Populus tremuloides)

Microsatellite DNA markers of ten simple sequence repeat (SSR) loci were used to examine somaclonal variation in randomly selected micropropagated plantlets derived from three different Populus tremuloides donor trees (genotypes). The plantlets were obtained from tissue cultures of dormant vegetative buds, and those derived from the same donor tree, grown in the greenhouse, did not exhibit any sign of visible morphological variation. No microsatellite DNA variation was observed among 13 somaclones of one tree and 4 somaclones of another tree at eight of the ten SSR loci. However, despite the small number of micropropagated progeny per tree sampled, microsatellite DNA variation was detected among the plantlets derived from the same donor trees at two SSR loci. The primer pair for the SSR locus PTR5 revealed somaclonal variation in 1 out of the 13 plantlets obtained from one genotype, while the primer pair for the PTR2 SSR locus revealed somaclonal variation in one out of the four plantlets obtained from another genotype. The variation at the PTR2 locus resulted in the appearance of a new allele of increased size, possibly due to an addition of the repeat units, while the variation at the PTR5 locus resulted in the appearance of third allele, presumably due to the presence of a single extra chromosome or duplication of a chromosomal segment. These results demonstrate that the genetic fidelity of micropropagated plants of P. tremuloides cannot always be assured and somaclonal variation can occur even when tissues of well organized vegetative buds are used for tissue cultures; that somaclonal variation cannot always be detected at the gross morphological level; and that microsatellite DNA markers provide useful and sensitive markers for determining the clonal fidelity and somaclonal variation in P. tremuloides.     

Changes in nitrogen resorption of trembling aspen (Populus tremuloides) with stand development

Variation in plant N resorption may change with stand development because plants tend to adjust their ecophysiological traits with aging. In addition, changes in soil nitrogen (N) pools associated with stand development may also affect plant N resorption. Here, we examined green- and senesced-leaf N concentrations and resorption of trembling aspen ( Populus tremuloides Michx.) in boreal forest stands of different ages (7, 25, 85 and 139 years, respectively). All sampled stands originated from wildfires and established on similar parent materials (glacial tills) and had similar climates. N concentrations in both green and senesced leaves increased between 27% and 54% along the stand age chronosequence. Resorption efficiency (percentage difference of N between green and senesced leaves) and proficiency (N concentration in senesced leaves) were higher for leaves in younger stands than in older stands. An analysis of covariance indicated that the patterns of leaf N concentration and resorption were affected significantly by stand age, but not by available soil N concentration. Our results indicate that at an intra-specific level, plants could adjust their N resorption efficiency and proficiency with stand development.     

Competition- and resource-mediated tradeoffs between growth and defensive chemistry in trembling aspen (Populus tremuloides)

Costs of defense are thought to maintain genetic variations in the expression of defense within plant populations. As with many plant species, aspen exhibits considerable variation in allocation to secondary metabolites. This study examined the independent and interactive effects of genotype, soil fertility and belowground competition on defensive chemistry and growth in trembling aspen (Populus tremuloides). Four aspen genotypes were grown with high and low soil fertility, and with and without root competition. Physiological, morphological and allocational determinants of growth were measured to identify growth-defense tradeoffs. Nutrient limitation and competition decreased growth, leaf mass ratio, leaf nitrogen concentration and photosynthesis, and increased root : shoot ratio and leaf condensed tannin concentrations. The competition treatment also resulted in increased leaf phenolic glycoside (PG) concentrations. Aspen growth was negatively correlated with PG concentrations under low fertility with competition. The relationship between growth and its major determinants was also negatively related to foliar condensed tannins expressed as a proportion of tree mass, indicating an additional indirect cost of allocation to secondary metabolites.     

Elevated growth temperatures alter hydraulic characteristics in trembling aspen (Populus tremuloides) seedlings: implications for tree drought tolerance

Although climate change will alter both soil water availability and evaporative demand, our understanding of how future climate conditions will alter tree hydraulic architecture is limited. Here, we demonstrate that growth at elevated temperatures (ambient +5 °C) affects hydraulic traits in seedlings of the deciduous boreal tree species Populus tremuloides, with the strength of the effect varying with the plant organ studied. Temperature altered the partitioning of hydraulic resistance, with greater resistance attributed to stems and less to roots in warm‐grown seedlings (P < 0.02), and a 46% (but marginally significant, P = 0.08) increase in whole plant conductance at elevated temperature. Vulnerability to cavitation was greater in leaves grown at high than at ambient temperatures, but vulnerability in stems was similar between treatments. A soil–plant–atmosphere (SPA) model suggests that these coordinated changes in hydraulic physiology would lead to more frequent drought stress and reduced water‐use efficiency in aspen that develop at warmer temperatures. Tissue‐specific trade‐offs in hydraulic traits in response to high growth temperatures would be difficult to detect when relying solely on whole plant measurements, but may have large‐scale ecological implications for plant water use, carbon cycling and, possibly, plant drought survival.     

Root water flow and leaf stomatal conductance in aspen (Populus tremuloides) seedlings treated with abscisic acid

Exogenous abscisic acid (ABA) applied to the roots and excised shoots of aspen (Populus tremuloides Michx.) inhibited stomatal conductance. However, the effect of ABA on stomatal conductance was more pronounced in the excised shoots compared with the intact seedlings. Approximately 10% of the ABA concentration applied to the roots was found in the xylem exudates of root systems exposed to a hydrostatic pressure of 0.3 MPa. A similar concentration of ABA applied to the excised shoots produced a faster and greater reduction of stomatal conductance. ABA applied to the roots had no effect on root steady-state flow rate over the 5-h experimental period. Moreover, pre-incubating root systems of intact seedlings for 12 h with 5 x 10(-5) M ABA did not significantly reduce volume flow density. Similarly, ABA had no effect on root hydraulic conductivity and the activation energy of root water flow rates.     

Growth and mortality of trembling aspen (Populus tremuloides) in response to artificial defoliation

To simulate the effects of forest tent caterpillar (FTC) defoliation on trembling aspen growth and mortality, an artificial defoliation experiment was performed over three years in young aspen stands of northwestern Quebec. Defoliation plots of 15 × 15 m were established on three sites, together with associated control stands of pure trembling aspen. In 2007, root collar diameters were measured and positions of all trees were mapped prior defoliation. Severe FTC defoliation was simulated for three successive years (2007–2009) by manually removing all leaves from all but 7–10% of the trees present in the defoliation plots. Yearly surveys of growth and mortality were conducted until 2010 to evaluate defoliation effects on defoliated as well as surrounding undefoliated trees. In absence of other factors, growth and mortality of trembling aspen decreased and increased, respectively, after defoliation. Our study further revealed that small diameter trees died after one year of artificial defoliation, while larger-diameter trees died after repeated defoliations. Distributions of tree mortality tended to be aggregated at small scales (<5 m), corroborating gap patterns observed in mature stands following FTC outbreaks. This experiment revealed that trembling aspen mortality can be directly attributed solely to defoliation. Repeated defoliations during FTC outbreaks have the potential to profoundly modify stand productivity and structure by reducing tree growth and increasing tree mortality in the absence of predisposing factors.     

Effects of genotype and nutrient availability on phytochemistry of trembling aspen (Populus tremuloides Michx.) during leaf senescence

We documented temporal patterns in phytochemical composition of Populus tremuloides Michx. during leaf senescence, and the influence of genotype and soil nutrient availability on such patterns. Levels of foliar nitrogen, carbohydrates, phenolic glycosides and condensed tannins were quantified for four aspen genotypes grown in a common garden, with low and high levels of soil nutrients. Levels of all compounds tended to decline over time, although the magnitude of change was influenced by plant genotype and nutrient availability. Genetic variation in concentrations of phytochemicals was much greater for phenolic glycosides and tannins than for nitrogen and carbohydrates, and these phenolic signatures generally persisted through leaf abscission. Our results suggest that genotypic and nutrient effects on patterns of chemical change during senescence will likely influence the performance of late-season herbivores on aspen. Moreover, nutrient and especially genotypic variation in phytochemistry of abscised leaves is likely to affect litter decomposition rates.     

Within-plant distribution of phenolic glycosides and extrafloral nectaries in trembling aspen (Populus tremuloides; Salicaceae)

Expression of foliar secondary compounds and extrafloral nectaries (EFNs) within the same leaves may be incompatible if secondary compounds repel beneficial insects that might otherwise be attracted to EFNs. This study examined the within-plant distributions of phenolic glycosides and EFNs in trembling aspen, Populus tremuloides, and their relationships to herbivore damage. Populus tremuloides expresses extrafloral nectaries (EFNs) on a subset of its leaves. We studied short and tall naturally occurring aspen ramets across multiple sites in interior Alaska. Contrary to our expectations, foliar phenolic glycoside concentrations were approximately 30% greater on leaves bearing EFNs than on leaves without EFNs. The mean concentration of foliar phenolic glycosides in short ramets was nine times that in tall ramets. Phenolic glycoside concentration was negatively related to leaf mining damage by Phyllocnistis populiella (Lepidoptera; Gracilliadae) at concentrations greater than 27 mg/g, whereas the presence of EFNs was unrelated to mining damage. The positive association of chemical defensive compounds and EFNs in leaves suggests that, for species with variation in EFN expression, negative correlations between herbivory and EFN expression may arise indirectly from associated effects of other, correlated types of defense.     

Pollen-mediated gene flow in isolated and continuous stands of bur oak, Quercus macrocarpa (Fagaceae)

? Premise of the study: Pollination patterns determine the reproductive neighborhood size of plants, the connectivity of populations, and the impacts of habitat fragmentation. We characterized pollination in three populations of Quercus macrocarpa occurring in a highly altered landscape in northeastern Illinois to determine whether isolated remnant stands were reproductively isolated. ? Methods: We used microsatellites to genotype all adults and 787 acorns from two isolated savanna remnants and a stand in an old-growth forest. One isolated remnant occurred in a highly urbanized/industrialized landscape, and one occurred in an agricultural landscape. Parentage assignment was used to assess pollen-mediated gene flow. ? Key results: Pollen donors from outside the study sites accounted for between 46% and 53% of paternities and did not differ significantly among sites, indicating that similar high levels of gene flow occurred at all three sites. Within stands, the mean pollination distance ranged from 42 to 70 meters, and when accounting for outside pollinations, mean pollination distances were well over 100 meters. Genetic diversity of incoming pollen was extremely high in all three stands. The number of effective pollen donors, N(ep), calculated from paternity assignment was higher than that estimated by an indirect correlated paternity approach. ? Conclusions: Our findings indicate that extremely isolated stands of oaks are unlikely to be genetically and reproductively isolated, and remnant stands may contribute to maintaining genetic connectivity in highly modified landscapes.     

Cycloheximide inhibits root water flow and stomatal conductance in aspen (Populus tremuloides) seedlings

Aspen (Populus tremuloides Michx.) roots were treated with cycloheximide, a protein synthesis inhibitor, to examine the role of protein synthesis in root water transport and plant water relations. Within less than 30 min following root application, cycloheximide inhibited steady‐state root water flow rates and 1 h after the application of 1 mm cycloheximide, root hydraulic conductivity had decreased by 85% compared with control roots. However, stomatal conductance showed a significant inhibition only after 2 h following cycloheximide treatment. The reduction in root hydraulic conductivity was accompanied by an almost three‐fold increase in the apoplastic water flow ratio as determined by the trisodium 3‐hydroxy‐5,8,10‐pyrenesulphonate tracer dye. Cycloheximide‐treated roots showed a decrease in the immunostaining intensity of a 32 kDa microsomal protein band that immunoreacted with the AnthPIP1; 1 antibody suggesting a decrease in the membrane aquaporin expression. These changes occurred without severe metabolic disruptions as measured by root respiration. The results point to the importance of protein‐mediated transport in roots and the rapidity of response suggests that protein synthesis may be used as a principal regulatory mechanism in root water transport in aspen.     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides)

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700‐fold higher in the hyphae within the root than in the free‐living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock‐down strains, a non‐mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H₂O₂ and CO₂ transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.     

Microsatellite analysis of seed dispersal and parentage of saplings in bur oak, Quercus macrocarpa

Microsatellite analysis was used to examine parentage and spatial distributions of 62 adult bur oaks Quercus macrocarpa, and 100 saplings in a single stand. Using genotypes scored by PCR products at four microsatellite loci, we determined that 94 saplings matched at least one parent in the stand. Saplings often occur as dense clusters of half-sibs around the presumed maternal parent, and only four adults were seed parents to a large proportion of the saplings sampled. A stump apparently was the seed parent of the largest cluster of half-sibs, which occupied a sizeable light gap opened up by the death of their maternal tree. Approximately half of the saplings appeared to have grown from seeds that had not been removed after falling from the tree, and half from seeds that were dispersed beyond the crown of their maternal parent. Long-distance seed dispersal may be more common than has been previously reported. Extremely high levels of long-distance pollination were indicated, and pollen donors within the stand were generally distributed randomly around maternal trees. More than half of the saplings had paternal parents outside of the stand. This study demonstrates the utility of microsatellite analysis for studying mating systems, seed dispersal and seedling establishment in natural plant populations.     

Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins

Main conclusion

Changes in root and leaf hydraulic properties and stimulation of transpiration rates that were initially triggered by defoliation were accompanied by corresponding changes in leaf and root aquaporin expression. Aspen (Populus tremuloides) seedlings were subjected to defoliation treatments by removing 50, 75 % or all of the leaves. Root hydraulic conductivity (L_pr) was sharply reduced in plants defoliated for 1 day and 1 week. The decrease in L _pr could not be prevented by stem girdling and it was accompanied in one-day-defoliated plants by a large decrease in the root expression of PIP1,2 aquaporin and an over twofold decrease in hydraulic conductivity of root cortical cells (L _pc). Contrary to L _pr and L _pc, 50 and 75 % defoliation treatments profoundly increased leaf lamina conductance (K _lam) after 1 day and this increase was similar in magnitude for both defoliation treatments. Transpiration rates (E) rapidly declined after the removal of 75 % of leaves. However, E increased by over twofold in defoliated plants after 1 day and the increases in E and K _lam were accompanied by five- and tenfold increases in the leaf expression of PIP2;4 in 50 and 75 % defoliation treatments, respectively. Defoliation treatments also stimulated net photosynthesis after 1 day and 3 weeks, although the increase was not as high as E. Leaf water potentials remained relatively stable following defoliation with the exception of a small decrease 1 day after defoliation which suggests that root water transport did not initially keep pace with the increased transpirational water loss. The results demonstrate the importance of root and leaf hydraulic properties in plant responses to defoliation and point to the involvement of PIP aquaporins in the early events following the loss of leaves.     

Stomatal conductance and xylem sap properties of aspen (Populus tremuloides) in response to low soil temperature

The mechanisms regulating stomatal response following exposure to low (5°C) soil temperature were investigated in aspen ( Populus tremuloides Michx.) seedlings. Low soil temperature reduced stomatal conductance within 4 h, but did not alter shoot xylem pressure potential within 24 h. The xylem sap composition was altered and its pH increased from 6.5 to 7.1 within the initial 4 h of the low temperature treatment. However, the increase in abscisic acid (ABA) concentration in xylem sap was observed later, after 8 h of treatment. These changes were accompanied by a reduction in the electrical conductivity and an increase in the osmotic potential of the xylem sap. The timing of physiological responses to low soil temperature suggests that the rapid pH change of the xylem sap and accompanying changes in ion concentration were the initial factors which triggered stomatal closure in low temperature-treated seedlings, and that the role of the more slowly accumulating ABA was likely to reinforce the stomatal closure. When leaf discs were exposed to xylem sap extracted from low soil temperature-treated plants, stomatal aperture was negatively correlated with ABA and positively correlated with K⁺ concentrations of the xylem sap. The stomatal opening in the leaf discs linearly increased in response to exogenous KCl concentrations when K⁺ concentrations were in the similar range to those detected in the xylem sap. The lowest concentration of exogenous ABA to induce stomatal closure was several-fold higher compared with the concentration present in the xylem sap.     

Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates

The biochemical anti-herbivore defense of trembling aspen ( Populus tremuloides Michx.) was investigated in a molecular analysis of polyphenol oxidase (PPO; EC 1.10.3.2). A PPO cDNA was isolated from a trembling aspen wounded leaf cDNA library and its nucleotide sequence determined. Southern analysis indicated the presence of two PPO genes in the trembling aspen genome. Expression of PPO was found to be induced after herbivory by forest tent caterpillar, by wounding, and by methyl jasmonate treatment. Wound induction was systemic, and occurred in unwounded leaves on wounded plants. This pattern of expression is consistent with a role of this enzyme in insect defense. A search for potential PPO substrates in ethanolic aspen leaf extracts using electron spin resonance (ESR) found no pre-existing diphenolic compounds. However, following a brief delay and several additions of oxygen, an ESR signal specific for catechol was detected. The source of this catechol was most likely the aspen phenolic glycosides tremulacin or salicortin which decomposed during ESR experiments. This was subsequently confirmed in experiments using pure salicortin.     

Clonal dynamics in western North American aspen (Populus tremuloides)

Clonality is a common phenomenon in plants, allowing genets to persist asexually for much longer periods of time than ramets. The relative frequency of sexual vs. asexual reproduction determines long‐term dominance and persistence of clonal plants at the landscape scale. One of the most familiar and valued clonal plants in North America is aspen (Populus tremuloides). Previous researchers have suggested that aspen in xeric landscapes of the intermountain west represent genets of great chronological age, maintained via clonal expansion in the near absence of sexual reproduction. We synthesized microsatellite data from 1371 ramets in two large sampling grids in Utah. We found a surprisingly large number of distinct genets, some covering large spatial areas, but most represented by only one to a few individual ramets at a sampling scale of 50 m. In general, multi‐ramet genets were spatially cohesive, although some genets appear to be fragmented remnants of much larger clones. We conclude that recent sexual reproduction in these landscapes is a stronger contributor to standing genetic variation at the population level than the accumulation of somatic mutations, and that even some of the spatially large clones may not be as ancient as previously supposed. Further, a striking majority of the largest genets in both study areas had three alleles at one or more loci, suggesting triploidy or aneuploidy. These genets tended to be spatially clustered but not closely related. Together, these findings substantially advance our understanding of clonal dynamics in western North American aspen, and set the stage for a broad range of future studies.     

Genetic architecture in bur oak,Quercus macrocarpa (Fagaceae), inferred by means of spatial autocorrelation analysis

Allozyme variation at seven putative polymorphic gene loci (Fle, Idh, Mdh-2, Mdh-3, 6 Pgd-2, Pgi-2, Pgm) was quantified in an isolatedQuercus macrocarpa population in northwestern Ontario, Canada. The bur oaks studied were slightly less genetically variable (H _e = 0.196,A = 3.43) than theQuercus subg.Lepidobalanus average. To assess the genetic differentiation within the topodeme,Moran's spatial autocorrelation coefficients were used for the nearest-neighbor map and correlograms. Results indicate that most alleles were randomly distributed in space. However, significant genetic patches were detected for twoFle-alleles. Patch width was inferred to be less than 10m. This spatial substructure may be due to limited gene flow and/or demographic factors. Spatially varying selection can be probably excluded as a spatial evolutionary force, since site conditions were very homogeneous.