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Population Biology |
2Department of Biology and Mountain Lake Biological Station, University of Virginia, P.O. Box 400327, Charlottesville, Virginia 22904-4327 USA; 3Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409 USA
Received for publication June 27, 2002. Accepted for publication September 17, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: allozyme variation • Castanea dentata • Cryphonectria parasitica • Fagaceae • genetic diversity • heterozygosity • population structure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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). The accidental introduction of the fungal pathogen Cryphonectria parasitica (Murr.) Barr from eastern Asia approximately 100 yr ago resulted in a sweeping pandemic destroying more than 3.5 billion trees (Anagnostakis, 1982
). Many rootstocks persist today, with small shoots being continually pruned by fungal infection. As a result, Castanea dentata has been transformed from a majestic canopy tree to a predominantly clonal, understory shrub. This paper explores the possibility that this change in life history may have influenced the population genetics of C. dentata.
Natural history of Castanea dentata
The American chestnut was a regionally distributed species, very common in the Appalachian Mountains of the eastern United States and extreme southern reaches of Ontario, Canada (Little, 1980
). In the northernmost latitudes it is limited to elevations below 130 m a.s.l. in New Hampshire (Russell, 1987
). The American chestnut has historically been, and continues to be, more abundant at high elevations in the southern Appalachians. The American chestnut is a calciphobe and consequently exhibits poor performance on limestone soils, with its native range evidently limited to some extent by limestone-based soils (Russell, 1987
). It is markedly more abundant in areas having a sandstone substrate. In Virginia, currently the highest densities of American chestnut are found at the higher elevations (750–1200 m a.s.l.) (Griffin, 2000
). In Giles County, Virginia, USA, where this study was conducted, the ridges are capped by a thin layer of Clinch or Tuscarora sandstone (Butts, 1933
; Braun, 1950
; Woodward and Hoffman, 1991
), and historically, the American chestnut has been a dominant part of the local forest community (Braun, 1950
; Stephenson, Adams, and Lipford, 1991
). The dominance of chestnut along the ridges of the southern Appalachians is not unusual for the species; it often occurred in almost pure stands (Brooks, 1937
) and was present in the mixed northern hardwood type forests where it comprised about 25% of the canopy (Cochran, 1990
; Griffin, 2000
).
Chestnut blight
Cryphonectria parasitica was introduced into North America on imported Asian Castanea species and was first observed in New York in 1904. Recent population genetic analyses provide evidence the North American strains of the fungus originated in Japan (Milgroom et al., 1996
). Within the genus, Castanea dentata is the species that is most susceptible to Cryphonectria parasitica and its destructive effects. Allozyme studies have found evidence the Chinese chestnut (Castanea mollissima) is the progenitor of C. dentata, and it has been suggested that C. dentata lost its blight resistance as it evolved in America in the absence of Cryphonectria parasitica (Huang, Dane, and Norton, 1994
). The combination of sexual ascospores dispersed by wind and asexual conidia dispersed by water, insects, and birds (Hepting, 1974
; Anagnostakis, 1987
) enabled the fungus to spread rapidly and within 40–50 yr of its introduction into North America. This disease was found in all areas of the natural range of the American chestnut, from its southern limit in Alabama to its northern limit in Maine (Griffin, 2000
).
Infection by Cryphonectria parasitica does not affect Castanea dentata root systems, only the aboveground stems. Cryphonectria parasitica invades cracks in the bark of Castanea dentata, forms a canker and develops a mycelium, eventually girdling the stem and killing the vascular cambium (Griffin, 2000
). Sexual reproduction of chestnut in the forest understory is exceedingly rare because few individuals ever escape infection long enough to reach the level of maturity to flower and bear fully developed fruit (Stephenson, Adams, and Lipford, 1991
; Paillet, 1993
). It is only by producing sprouts from the root collar that C. dentata has escaped extirpation from the Appalachian forest ecosystem. Following the infection and death of the main stem, the release of dormant meristems at the root collar allows the growth of clonal stems from the original root collar. Moreover, it might be possible the larger root collars may fragment into separate clumps of stems creating an incremental clonal expansion of genotypes.
Genetic diversity and population structure
The ecological changes and the diminished importance of sexual reproduction in C. dentata, consequences of the C. parasitica pandemic, could have several effects on the amount and distribution of genetic diversity. First, the overall level of genetic diversity may have been reduced. The life history and ecological characteristics of woody plants tending to have a combination of widespread geographical distribution, outcrossing, and wide seed and pollen dispersal suggest C. dentata should have had high levels of within-population diversity and relatively little population structure (Hamrick, Godt, and Sherman-Broyles, 1992
). The chestnut blight pandemic significantly altered the ecology of the species. The virtual elimination of sexual reproduction and the gradual attrition of the chestnut root stocks may have resulted in the loss of genetic diversity.
The second effect that the blight pandemic could have on the chestnut population is on the distribution of genetic variance within and among populations. This could occur by genetic drift from the reduced population sizes or from the vegetative expansion of root collars, both of which would tend to diminish genetic variance within patches. Studies examining the genetic diversity of Castanea species have found a relatively small amount of genetic variation distributed among populations: about 11.0% in C. dentata (Huang, Dane, and Kubisiak, 1998
) and 10% in C. sativa (Pigliucci, Benedettelli, and Villani, 1990
). These values are also consistent with the overall levels of population structure in other members of the Fagaceae (Hamrick, Godt, and Sherman-Broyles, 1992
). In this paper, we examine the population structure at a finer scale and look for evidence of possible clonal expansion by multiple occurrences of identical unique genotypes in stems originating from separate root collars.
Selection among clones may also be influencing the population genetics of C. dentata. The vigorous growth of hybrid individuals and the effects of "inbreeding depression" in highly inbred or homozygous individuals have suggested a connection between allozyme heterozygosity and fitness or performance. Levels of single and multilocus heterozygosity have been associated with performance and fitness characters of some species. This is not a universal observation; other studies of plants and animals have found no association, so this topic continues to be a subject of debate. A number of studies found no association between heterozygosity and fitness parameters (Booth, Woodruff, and Gould, 1990
; Savolainen and Hedrick, 1995
; Cullum, 1997
; Aravanopoulos, 2000
). Associations of heterozygosity and increased overall growth or growth rate have been observed in a variety of organisms (Mitton and Grant, 1980
; Ledig, Guries, and Bonefeld, 1983
; Garton, Koehn, and Scott, 1984
; Zouros, Romero-Dorey, and Mallet, 1988
; Quattro and Vrijenhoek, 1989
). One study, for example, found a positive association between heterozygosity and winter survival rates in populations of western toads in the Cascade Range of central Oregon (Samollow and Soule, 1983
). The general perception is that more heterozygous individuals are less variable and are better adapted in fluctuating environments. Some studies support this theorized association. However, other studies have found a positive association between heterozygosity and variability; both were of long-lived tree species, the ponderosa pine, Pinus ponderosa (Knowles and Grant, 1981
), and quaking aspen, Populus tremuloides (Mitton and Grant, 1980
).
The fact that many long-lived tree species show an excess of heterozygosity suggests that selection favoring heterozygotes is relatively subtle and hence is more likely to have an effect over the course of a long life span. The American chestnut provides a unique opportunity to observe this type of selection. With the chestnut having become predominantly clonal, there are virtually no recruits to restore populations toward Hardy-Weinberg equilibrium. Moreover, even subtle differences in the performance of genotypes may have been magnified in importance as the clones have aged. Over the last half-century, even relatively small fitness effects may accumulate to have conspicuous effects on the genetics of the populations.
The objectives of this study were to examine how the changes in life history of C. dentata have influenced its population genetics. First, we estimated the amount and spatial distribution of genetic diversity of American chestnut to look for evidence of clonal expansion beyond the root collar. Second, we looked for evidence of selection favoring heterozygotes by testing for an association between heterozygosity, growth rate, and growth rate variability in the American chestnut.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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). The three Salt Pond Mountain sites are near the summit. The MLBS site is located at the University of Virginia's Mountain Lake Biological Station (elevation about 1190 m a.s.l.), War Spur (Wspur) is on the east side of state route 613 (elevation about 1130 m a.s.l.) in the Jefferson National Forest, near the War Spur Loop Trail 2.6 km northeast of MLBS; the Old Golf Course (OGC) site (elevation about 1210 m a.s.l.) is along a road to an abandoned golf course on land belonging to the Wilderness Conservancy at Mountain Lake, 2.9 km southwest of MLBS. The fourth site is on nearby Butt Mountain (elevation about 1210 m a.s.l.) on the west side of state route 714 in the Jefferson National Forest, 8.2 km west of MLBS. These sites were chosen because of the relative abundance of American chestnut in the understory of the predominantly oak (Quercus spp.) forest along the mountainous ridges (Stephenson, 1974
).
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Leaf tissue for horizontal starch-gel electrophoresis was collected in July and August 2000. Eleven enzyme systems on three buffer media were screened for variation (Table 1). LAP, EST, HK, and GOT were resolved on a lithium-hydroxide system (Werth, 1985
). IDH, MDH, PGM, 6PGDH, and SKDH were resolved on a morpholine-citrate buffer system (Werth, 1991
). PGI and TPI were resolved on buffer system 6 (Soltis et al., 1983
). Electrophoretic settings were as follows: LiOH, 400 V and <100 mA; System 6, 250 V and <100 mA; and Morpholine-citrate, 250 V and approximately 50 mA for 5–5.5 h. Gels were immediately sliced and stained for their respective enzyme systems using the protocols established by Werth (1990)
except LAP, EST, and GOT, which were stained as baths. Genotypes were scored directly from the gel shortly after staining.
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) using the methods of Weir and Cockerham (1984)
to estimate F statistics: the inbreeding coefficient, FIS; the total fixation index, FIT; and the proportion of differentiation among the subpopulations and populations, FST (Wright, 1951
). The allele frequencies (Table 2) and observed heterozygosity (Ho) (Table 3) at each locus were calculated directly from the data set. Assuming Hardy-Weinberg equilibrium, the expected heterozygosity (He) was calculated by the following equation: He = 1 – 
xi2. Where xi is the mean frequency of the ith allele. Differentiation among the subpopulations leads to a decreased level of overall expected heterozygosity. The expected heterozygosity, when corrected for population structure, was calculated as He = (1 – xi2)(1 – FST). A chi-square test was used to determine if the expected and observed heterozygosities at each locus and over all polymorphic loci were significantly different than expected (Table 3).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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Repeated samples of stems from the same root collar always had the same multi-locus genotype, and the spatial patterning of genotypes gave no evidence of clonal expansion beyond what was obviously an existing root collar (i.e., genotypes were restricted to well-defined groups of stems; data not shown).
Several factors were used to evaluate the vegetative performance of homozygotes relative to heterozygotes at the MLBS site. The size-specific growth rate, variability of growth rate, health, number of sprouts produced at the root collar, and conspecific stem density were examined. The size-specific growth rate was not normally distributed and differed in variance, with the heterozygous growth rate significantly more variable than the homozygous growth rate (F34,54 = 2.203, P = 0.010). Due to the nonnormal distribution and unequal variances, a nonparametric Mann-Whitney test of the median size-specific growth rate was used to test the suggested superior performance of the individuals having at least one heterozygous locus. Two levels of heterozygosity were used as only five samples exhibited multiple (two) heterozygous loci. The median size-specific growth rate was higher for the heterozygous individuals (Mann-Whitney W = 2239.5, P = 0.081). In a posteriori testing, the size-specific growth rates were assigned to slow (N = 26), medium (N = 38), and fast (N = 24) categories based upon the discontinuous distribution of growth rates (Fig. 2). Chi-square analysis revealed a statistically significant difference (Table 5) in the distribution of growth rates for homozygotes and heterozygotes. The largest contributor to the chi-square statistic was the excess of heterozygotes in the fast growth rate category (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONLITERATURE CITED |
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; Bush and Smouse, 1992
). This is probably why an excess of heterozygotes is often seen in long-lived species (Mitton and Grant, 1980
). In studies of different age classes (seeds, seedlings, and stands of differing age) an increase in heterozygosity with increasing age has been reported in other tree species (Ledig, Guries, and Bonefeld, 1983
; Hokanson et al., 1993
). The difference between those studies and the present situation is that practically all extant chestnut genotypes are more than 70 yr old, and many that succumbed to the blight as mature canopy trees will be much older. In the chestnut, therefore, as selection enriches the population for heterozygotes, there are no new recruits to restore the population toward Hardy-Weinberg equilibrium. We would therefore expect the excess of heterozygotes to continue to accumulate, as the age structure of the chestnut clones grows older and older. Our results also show that the mean size-specific growth rate of chestnut stems has a positive but weak association with allozyme heterozygosity. Although the growth rate differences were subtle, the 3-yr time scale in which the data were obtained may not have been sufficient time to observe a more marked variation in growth rate. It is also important to point out that we did not observe enough mortality of chestnut clones to detect a relationship between clone mortality and stem growth or heterozygosity, so the causal connection between higher stem growth of heterozygotes and the excess of heterozygous clones in the population is not entirely clear. Taken together, however, the data suggest there is selection favoring heterozygous individuals, and that the prolonged absence of sexual reproduction in C. dentata has allowed these fitness differences to accumulate to the extent that they have had significant effects on the genetics of chestnut populations.
It is not clear why selection may favor heterozygotes in C. dentata. One possibility is that higher heterozygosity at allozyme loci reflects higher heterozygosity throughout the genome and reflects a masking of deleterious recessive alleles. However, we cannot reject the possibility of direct selection on the allozyme loci. The enzymes used in most allozyme studies are known to be important in carbohydrate metabolism, and studies have shown that different proteins at a single locus perform differently (Fincham, 1972
; Mitton and Grant, 1984
). For example, some have suggested heterozygosity may confer developmental homeostasis in fluctuating environments (Mitton, 1978
; Knowles and Mitton, 1980
; Vrijenhoek and Lerman, 1982
; Mitton and Grant, 1984
; Quattro and Vrijenhoek, 1989
).
Although our data are consistent with the chestnut blight pandemic having altered the patterns of genetic diversity within populations, its effect, if any, on overall levels of genetic diversity is less clear. Our Virginia population of C. dentata had a lower percentage of polymorphic loci (41.7%) compared with 77.8% across its natural range (Huang, Dane, and Kubisiak, 1998
) and with a mean of 49.3% for long-lived woody plants (Hamrick, Godt, and Sherman-Broyles, 1992
). However, the number of alleles per locus (A = 2.0) is slightly higher than reported for C. dentata by Huang, Dane, and Kubisiak (1998
; A = 1.69) and for long-lived woody plants (A = 1.76; Hamrick, Godt, and Sherman-Broyles, 1992
). The overall diversity measures in this study and Huang, Dane, and Kubisiak (1998)
were less than that observed in many closely related taxa in the Fagaceae family, such as Quercus spp. (Hamrick, Godt, and Sherman-Broyles, 1992
; Hokanson et al., 1993
; Berg and Hamrick, 1994
; Montalvo et al., 1997
), Fagus grandifolia (Houston and Houston, 1994
), and other species of Castanea (Pigliucci, Benedettelli, and Villani, 1990
; Villani et al., 1991
).
The high mortality of C. dentata stems in conjunction with a near total elimination of sexual reproduction could have resulted in the loss of some (mostly rare) alleles (Loveless and Hamrick, 1984
; Leberg, 1992
). It is not clear, however, whether this slightly lower genetic diversity is a result of the chestnut blight epidemic. For example, our study sampled alleles over a relatively small spatial scale, so we would expect to see lower diversity. Moreover, Huang, Dane, and Kubisiak (1998)
suggested that the low genetic diversity of the American chestnut resulted in the high susceptibility of C. dentata to attack by Cryphonectria parasitica, rather than that the low genetic diversity was a direct consequence of the C. parasitica pandemic, and that other Castanea species with more diverse allozyme variation are less susceptible to epidemics. In any case, without any knowledge of pre-blight population structure it is difficult to make any definitive statement on changes in genetic diversity due to the pandemic.
The F statistics averaged across the four sites indicate no differentiation among populations (FST = –0.0156). These results were expected given the close proximity of the four populations and considering the life-history characteristics of the American chestnut. Historical levels of gene flow were probably very high among sites within a 10-km area, and the American chestnut has historically been abundant in the study area. The F statistics do indicate a substantial amount of differentiation among the subpopulations (FST = 0.1273). We found no evidence for clonal expansion of genotypes beyond the root collar. Therefore, this fine-scale clustering in the face of gene flow, coupled with a lack of differentiation at larger spatial scales, is due perhaps to limited seed movement and local founder effects leading to the clustering of families (McCauley et al., 1996
).
One unexpected result was the significant positive association between homozygosity and conspecific stem density. While the homozygotes are more commonly found in the smaller size classes and slower growth rate classes, these individuals may be in more dense aggregations because the smaller trees are experiencing less competition in the limited canopy openings than the larger, more heterozygous individuals; thus, the lower density of heterozygotes could be due to a competitive advantage such as the aforementioned higher growth rate. There was no association, however, between stem density and overall growth rate, so the growth differences between heterozygous and homozygous individuals does not appear to be an artifact of the two kinds of genotypes experiencing different density environments.
Infection rate and root sprout production were not associated with heterozygosity class; however, the root sprout production was significantly higher after infection by Cryphonectria parasitica. The release of meristems at the root collar coincides with infection and imminent death of the main stem. The infection rate at the MLBS site (approximately 11% per year) is much higher than observed (approximately 5% per year) by Paillet (1993)
at a lower elevation site in Loudon County, Virginia. The harsh winters at high elevations have been coupled with the change of superficial blight cankers to more lethal cankers in the vascular cambium, likely due to a stress-induced weakening of the already vulnerable host defense mechanism (Griffin, 2000
).
The American chestnut was driven to clonal reproduction by the chestnut blight pandemic. The lack of sexual reproduction over the past 70 yr appears to have produced a significant change in the population genetics of C. dentata. Specifically, without new recruits to return the population toward Hardy-Weinberg equilibrium, a slight growth advantage for heterozygous genotypes appears to have significantly enriched the population for heterozygous genotypes over time since the pandemic. Our results also suggest there is relatively fine-scale population structure in chestnut populations, but that this is more likely due to factors such as limited seed movement before the epidemic rather than clonal expansion of root stocks since the epidemic.
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FOOTNOTES |
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4 Author for reprint requests, current address: Rt. 4, Box 648, North Tazewell, Virginia 24630 USA (kstilwel{at}pen.k12.va.us
)
5 Charles R. Werth passed away during the preparation of this manuscript. The rest of the authors are indebted to him for his contribution to this work and for his long-standing contributions to the intellectual community at Mountain Lake Biological Station
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2 = 0.000 + 0.000 + 0.940 + 1.493 + 1.517 + 2.410 = 6.361, df = 2, P = 0.042