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The relationship between cross success and spatial proximity of Eucalyptus globulus ssp. globulus parents.

The spatial distribution of genetic variation has important implications for the evolution of populations (Wright 1943, 1978; Levin and Kerster 1974; Turner et al. 1982; Uyenoyama 1986; Epperson 1992). Within plant populations, clusters of relatives that share at least a half-sib relationship may develop if seed dispersal is limited (Fenster 1991a; Epperson 1992). Inbreeding may then occur through mating among related neighbors, particularly when pollination is via insects and other animals that tend to follow a localized foraging pattern (Loveless and Hamrick 1984; Ellstrand 1992). If inbred progeny survive to reproductive maturity (cf. Hardner and Potts 1997), then several generations of related matings may lead to the development of genetic neighborhoods (Wright 1943; Levin and Kerster 1974) and a localized deme structure (Turner et al. 1982). Alternatively, spatially structured genetic variation may arise from geographical heterogeneity in natural selection (Endler 1977).

In this study, we examine the spatial genetic structure of natural populations of Eucalyptus globulus ssp. globulus (Labill.), a long-lived, temperate forest tree. This species is a common codominant of the native forests of Tasmania and southeastern Australia; however, little is known about the genetic structure and evolutionary dynamics of its natural populations. Seed dispersal in eucalypts is mainly by wind or gravity and is virtually limited to twice the tree height (Potts and Wiltshire 1997), leading several authors to suggest that family group clusters exist in native forests (Griffin 1989; Eldridge et al. 1993). In addition, as eucalypts are mainly pollinated by nonspecific insect and other animal vectors (Pryor 1976; Griffin 1980), pollen dispersal is also expected to be relatively restricted (Hopper and Moran 1981; Savva et al. 1988; Griffin 1989).

Spatial clustering of genotypes has been observed in other animal-pollinated forest trees with limited seed dispersal (Acer saccharum, Perry and Knowles 1991; Quercus laevis, Berg and Hamrick 1995; and Quercus rubra, Sork et al. 1993) and in conifers that also have limited seed dispersal but are wind pollinated (Pinus ponderosa, Linhart et al. 1981; P. strobus, Beaulieu and Simon 1995; Picea abies, Brunel and Rodolphe 1985 and Linhart et al.'s 1981 reanalysis of Tigerstedt 1973). However, spatial clustering has not been observed in another P. abies study (Leonardi et al. 1996) nor in Pinus contorta (Epperson and Allard 1989).

The poorer vigor of progenies from nearest neighbor crosses, relative to longer distances crosses, has been interpreted as inbreeding depression and an indirect measure of relatedness in Picea glauca (Coles and Fowler 1976; Park and Fowler 1984), Larix laricina (Park and Fowler 1982), and a number of small perennial species (e.g., Waser and Price 1989; Hauser and Loeschcke 1994; Waser and Price 1994), but has not been detected in some populations of short-lived plants (McCall et al. 1989; Newport 1989). Conversely, outbreeding depression, that is, the poorer performance of long-distance crosses relative to crosses involving intermediate distances, has also been suggested in some herbaceous species (e.g., Price and Waser 1979, Waser and Price 1989, 1994). However, again this observation is not universal (McCall et al. 1989; Newport 1989; Hauser and Loeschcke 1994) and has not been reported in any forest tree study (Coles and Fowler 1976; Park and Fowler 1982, 1984). Here, we examine the genetic structure of E. globulus forests by studying the vigor of progenies from crosses among parents separated by varying distances. Severe inbreeding depression in seed set and field growth following self-pollination has been reported in this species (Hardner and Potts 1995), suggesting that a reduction in the vigor of progenies from crosses between proximate parents may indicate a spatial structure to relatedness.


Experimental Design

Controlled pollinations were undertaken on seven mature Eucalyptus globulus ssp. globulus individuals (females) growing in the Tinderbox locality (43 [degrees] 3 [minutes] S, 147 [degrees] 19 [minutes] E), approximately 20 km south of Hobart, Tasmania, Australia. These trees were remnants of the mature native forest, grew in clumps of between 50 to 80 individuals, and were between 0.5 and 1 km from the nearest continuous native forest. The average canopy height was approximately 25 m. Females were only selected on the basis of accessibility of flowers for controlled pollination.

For each female, pollen was sampled from individuals (males) at seven different distances classes. These classes were: (1) the same tree (self); (2) nearest flowering neighbor (average 21 m); (3) 250 m; (4) 500 m; (5) 1 km; (6) 10 km; and (7) 100 km. Except for self-pollination, two males were sampled within each distance class for each female. Males used for the 1 km and less distance classes were from the Tinderbox locality. Males for the 10 km class were sampled from South Arm (43 [degrees] 2 [minutes] S, 147 [degrees] 25 [minutes] E), east of Hobart, and those for the 100 km class were from the Mayfield (42 [degrees] 15 [minutes] S, 148 [degrees] 1 [minutes] E) locality on the east coast of Tasmania. Controlled pollinations followed Hardner and Potts (1995), with selfs undertaken in exactly the same manner as the cross pollinations. Mature capsules were collected in late 1991. Open-pollinated (OP) families were also collected from each female and male used in the crossing scheme.

Seeds were extracted from dried capsules and seed set quantified as the number of viable seed per capsule following Hardner and Potts (1995). Seedlings raised from the viable seed were used to establish a field trial 4 km west of Geeveston (43 [degrees] 8 [minutes] S, 146 [degrees] 52 [minutes] E), 70 km south of Hobart on an ex-native E. obliqua forest site. There were 14 replications. Within each replicate, each family of female OPs, self and controlled outcross progenies was represented by single tree plots. These were grouped by female parent (blocks) in a split-plot design to optimize comparisons of offspring of the same female. Adjacent to each replicate, an additional planting was established containing randomized single tree plots of the OP families of all parents used in the crossing scheme. Distances between all plants were 3 m x 4 m. Height and diameter at breast height (DBH) were measured four years after planting and used to calculate conic volume.


The relationship between spatial proximity of parents and cross success (seed set and field growth) was tested with a mixed-model analysis using restricted maximum likelihood (REML) in the procedure PROC MIXED in SAS (SAS Institute 1992). The statistical model included terms for the fixed replicate effect (only for field growth), the fixed effect of the distance between parents, the random block within replicate effect (only for field growth), the random effect of female, and the random interaction between female and distance between parents. Type III tests were undertaken for the fixed effects. Variance components were estimated for random effects and their significance tested by dividing the estimate by its standard error, which is asymptotically equivalent to an F-test with infinite denominator degrees of freedom (SAS Institute 1992). Least squares means were estimated for each distance class, and standard errors and confidence intervals at P = 0.05 were calculated by SAS. The significance between the distance classes was tested by a simple comparison of the confidence intervals and the means. For the initial analysis of conic volume, the OPs of the females were included as an additional distance class.

However, under this analysis, differences in growth amongst distance classes could be confounded by variation in the additive effect of males from different localities (i.e., Tinderbox, South Arm, Mayfield). In an attempt to account for this effect, an independent mixed-model analysis was undertaken on the growth of the OP families of all parents used in the crossing design with a model that included the fixed effect of replicate and male parent locality and the random effect of OP families within locality. The estimated effect of male parent locality from this analysis was then used to adjust each observation from the controlled crosses progenies. Subsequently, the analysis for the relationship between spatial proximity of parents and growth was repeated using the adjusted data without inclusion of the female OPs.

Inbreeding depression for each distance class ([[Delta].sub.dist]) was calculated by assuming the level of inbreeding of the 1 km cross was f = 0 and using the formula:

[[Delta].sub.dist] = 100([X.sub.1km] - [X.sub.dist])/[X.sub.1km],

where [X.sub.1km] was the least squares mean of the 1 km cross and [X.sub.dist] was the least squares mean for the different distance classes.


There was a highly significant (P [less than] 0.01) effect of distance between mates on seed set and volume at four years after planting, but no significant effect of female parent (Table 1). For volume at four years, the interaction between female parent and distance between mates was slightly significant. Seed set was severely depressed by selfing (84%), however, there was no significant difference between, or systematic trend in, mean seed set for the cross pollination treatments [ILLUSTRATION FOR FIGURE 1a OMITTED]. Selfs were also significantly smaller (P [less than] 0.05) than outcrosses after four years in the field [ILLUSTRATION FOR FIGURE 1b OMITTED]. Progenies from OP and nearest neighbor crosses (21 m) were intermediate to, and significantly different from, selfs and the progenies from crosses between parents separated by 250 m or more for growth to four years. Growth of the 10-km cross was superior to all but that of the 1-km and 100-km outcrosses. However, virtually all differences in the growth among distance classes of 250 m or greater were removed after adjustment was made for the average effect of male parent locality determined from an independent analysis of the OP families collected from all males used in the cross design [ILLUSTRATION FOR FIGURE 1c OMITTED]. Relative to the 1-km cross, inbreeding depression due to selfing, OP, and nearest neighbor crossing was, 48%, 22%, and 18%, respectively.


Selfing has a severe detrimental effect on seed set and growth in E. globulus. Levels of inbreeding depression in viable seed per capsule and conic volume at four years due to selfing are of similar magnitude to those in studies of this and other eucalypt species (Hardner and Potts 1995). Inbreeding depression in field growth has been shown to be strongly correlated with inbreeding depression in later age survival, and hence fitness, in eucalypts (Hardner and Potts 1997).

The growth data in this study provides strong evidence that nearest neighbors sampled from the mature stratum of the E. globulus forest are related. Progenies from crosses between nearest neighbors were significantly less vigorous than crosses between mates separated by greater distances. On average, nearest neighbors appear to share a relationship equivalent to at least that of half-sibs. Relative to the 1-km cross, the reduction in growth of progenies from crossing between nearest neighbors is one-third of that due to selfing (18% cf. 48%). In comparison, inbreeding depression in progenies from crossing between half-sibs is expected to be one-fourth that of selfing, assuming a linear relationship between inbreeding depression and the inbreeding coefficient, f. However, seed set from crossing nearest neighbors did not appear to exhibit inbreeding depression [ILLUSTRATION FOR FIGURE 1a OMITTED]. Effects other than relatedness may be more important for this trait (e.g., maternal effects, Fenster 1991b). Other life-cycle traits such as seed germination or early height growth (when competition was presumably absent, cf. Hardner and Potts 1997) were also examined; however, even selfing was not significant for these traits until three years in the field (results not presented).

There appears little relatedness among parents separated by at least 250 m, as seed set and growth for the 250-m cross was as good as, or better than, that for longer distance crosses. Adjusting for the average effect of male parent locality, independently estimated from the OP families of each male, removed differences among the long distance crosses, possibly [TABULAR DATA FOR TABLE 1 OMITTED] indicating the extent of variation in adaptation among localities. However, reciprocal transplant experiments would be required to investigate this further. It is therefore unlikely that outbreeding depression due to disruption of coadapted gene complexes (Templeton 1986; Potts et al. 1987; Lynch 1991) exists in this species even when parents are separated by 100 km.

The reduction in growth of the OP progenies of E. globulus (present study; Hardner and Potts 1995) suggests that OP families contain both inbred and outcross progenies. Multilocus estimates of selfing rates in native E. globulus stands vary between 0.00 and 0.52 (Hardner et al. 1996). However, these are estimates of effective selfing rates and the actual level of selfs may be over estimated if biparental inbreeding occurs (Ritland 1984). In native eucalypt forests, biparental inbreeding may be frequent as most eucalypt pollen appears to be distributed close to a source (e.g., Pryor 1976) and, as the present study suggests, neighbors appear to be closely related. Mean foraging movements of birds are only marginally greater than the distance between neighbors in eucalypt species with avian pollination (e.g., E. stoatei, Hopper and Moran 1981; E. urnigera, Savva et al. 1988). In addition, insect pollen vectors in eucalypts appear to follow a localized foraging pattern, working whole inflorescences (Griffin 1980), although more work is needed to confirm the extent of between neighbor insect pollinations. Biparental inbreeding is further favored in E. globulus as flowering is under strong genetic control (Gore and Potts 1995), suggesting related neighbors tend to flower at the same time. In E. regnans, biparental inbreeding arising from mating among related neighbors is believed to explain the lower outcrossing rate in a native forest (0.75) compared to a nearby seed orchard (0.91) where a family structure was absent (Moran et al. 1989). In this context, Griffin (1980) reports that in native populations of E. regnans there is a tendency for nearest neighbors to flower synchronously.

The most likely explanation for our observation that nearest neighbors in native E. globulus populations appear to share a relationship equivalent to at least that of half-sibs is that family clusters develop each generation due to limited seed dispersal. Under this scenario, half-sibs would be expected to be grow around the site of their maternal parent (Griffin 1989; Eldridge et al. 1993). As seed dispersal in eucalypts is generally limited to twice the tree height (Potts and Wiltshire 1997), the radius of each cluster may be strongly determined by the height of the maternal parents. In this context, the detection of relatives within 50 m of each other, but not 250 m, fits with the height of the seed trees in this study being on average 25 m.

An alternative explanation is that genetic neighborhoods (Wright 1943) have developed within eucalypt forests due to an accumulation of inbreeding over generations because of isolation by distance from limited pollen and seed dispersal. However, the development of genetic neighborhoods will depend on the degree to which inbred progeny from one generation contribute to the next generation. The poorer fitness of inbred eucalypt progenies suggest they will be selected against as intense competition develops in native forests (Hardner and Potts 1997). In this context, our results suggest that the cumulative impact of inbreeding in eucalypt forests may be relatively small, as mates separated by only 250 m appear to be as related as parents separated by distances up to 100 km. Thus, while the mating system, the population structure and the pollen and seed distribution patterns suggest gene dispersal in E. globulus is limited, selection against the products of inbreeding would be expected to increase realized gene flow by affecting patterns of gene establishment (sensu Fenster 1991c). Such selection would result in genetic neighborhoods that are larger than predicted from gene dispersal patters alone, but which are composed of tight family group clusters that are renewed each generation through limited seed dispersal.


This research was undertaken with the assistance of the Tasmanian Forest Research Council and Forestry Tasmania. We would also like to thank R. Vaillancourt for his comments on the manuscript.


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Author:Hardner, Craig M.; Potts, Bradley M.; Gore, Peter L.
Date:Apr 1, 1998
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