Detection and visualization of spatial genetic structure in continuous Eucalyptus globulus forest

Visualizing the pattern of variation using microsatellites within a Eucalyptus globulus forest on the island of Tasmania provided surprising insights into the complex nature of the fine-scale spatial genetic structure that resides in these forests. We used spatial autocorrelation and principal coordinate analysis to compare fine-scale genetic structure between juvenile and mature cohorts in a study area, 140 m in diameter, located within a typical, continuous E. globulus forest. In total, 115 juvenile and 168 mature individuals were genotyped with eight highly polymorphic microsatellite loci. There was no significant difference in the level of genetic diversity between cohorts. However, there were differences in the spatial distribution of the genetic variation. Autocorrelation analysis provided clear evidence for significant spatial genetic structure in the mature cohort and significant, but weaker, structure in the juvenile cohort. The spatial interpolation of principal coordinate axes, derived from ordination of the genetic distance matrix between individuals, revealed a spatially coherent family group which was evident in both cohorts. Direct comparison of the genetic structure within each cohort allowed visualization of a shift in the spatial distribution of genetic variation within the population of approximately 10 m. As the shift coincided with the direction of prevailing winds, it is hypothesized that this phenomenon is due to downwind dispersal of seeds and is indicative of the important role of prevailing winds in forcing eastward gene flow in these high-latitude forests.     

Controlling genetic variability by mathematical programming in a selection scheme on an open-pollinated population in Eucalyptus globulus

A model using integer quadratic mathematical programming has been developed to control the inbreeding level (or genetic diversity) through group coancestry in a selection programme for a forestry population structured in terms of maternal families coming from different locations. A method to calculate the average group coancestry between- and within-families for these open-pollinated populations is also proposed. This model has been applied to data from a breeding programme of Australian Eucalyptus globulus. The strategy proved to be effective as reductions of up to 50% for the group coancestry of the selected individuals were reached with a loss of only 5% of the maximum attainable selection differential (corresponding to truncation selection). Received: 14 October 1999 / Accepted: 26 July 2000     

Identification of spatial genetic boundaries using a multifractal model in human population genetics

There are two purposes in displaying spatial genetic structure. One is that a visual representation of the variation of the genetic variable should be provided in the contour map. The other is that spatial genetic structure should be reflected by the patterns or the gradients with genetic boundaries in the map. Nevertheless, most conventional interpolation methods, such as Cavalli-Sforza's method in genography, inverse distance-weighted methods, and the Kriging technique, focus only on the first primary purpose because of their arbitrary thresholds marked on the maps. In this paper we present an application of the contour area multifractal model (CAMM) to human population genetics. The method enables the analysis of the geographic distribution of a genetic marker and provides an insight into the spatial and geometric properties of obtained patterns. Furthermore, the CAMM may overcome some of the limitations of other interpolation techniques because no arbitrary thresholds are necessary in the computation of genetic boundaries. The CAMM is built by establishing power law relationships between the area A (> or =rho) in the contour map and the value p itself after plotting these values on a log-log graph. A series of straight-line segments can be fitted to the points on the log-log graph, each representing a power law relationship between the area A (> or =rho) and the cutoff genetic variable value for rho in a particular range. These straight-line segments can yield a group of cutoff values, which can be identified as the genetic boundaries that can classify the map of genetic variable into discrete genetic zones. These genetic zones usually correspond to spatial genetic structure on the landscape. To provide a better understanding of the interest in the CAMM approach, we analyze the spatial genetic structures of three loci (ABO, HLA-A, and TPOX) in China using the CAMM. Each synthetic principal component (SPC) contour map of the three loci is created by using both Han and minority groups data together. These contour maps all present an obvious geographic diversity, which gradually increases from north to south, and show that the genetic differences among populations in different districts of the same nationality are greater than those among different nationalities of the same district. It is surprising to find that both the value of p and the fractal dimension alpha have a clear north to south gradient for each locus, and the same clear boundary between southern and northern Asians in each contour map is still seen in the zone of the Yangtze River, although substantial population migrations have occurred because of war or famine in the last 2,000 or 3,000 years. A clear genetic boundary between Europeans and Asians in each contour map is still seen in northwestern China with a small value of alpha, although the genetic gradient caused by gene flow between Europeans and Asians has tended to show expansion from northwestern China. From the three contour maps another interesting result can be found: The values of alpha north of the Yangtze River are generally less than those south of the Yangtze River. This indicates that the genetic differences among the populations north of the Yangtze River are generally smaller than those in populations south of the Yangtze River.     

Origin of spatial genetic structure in an expanding oak population

Spatial genetic structure (SGS) results from the interplay of several demographical processes that are difficult to tease apart. In this study, we explore the specific effects of seed and pollen dispersal and of early postdispersal mortality on the SGS of a seedling cohort (N = 786) recruiting within and around an expanding pedunculate oak (Quercus robur) stand. Using data on dispersal (derived from parentage analysis) and mortality (monitored in the field through two growing seasons), we decompose the overall SGS of the cohort into its components by contrasting the SGS of dispersed (i.e. growing away from their mother tree) vs. nondispersed (i.e. growing beneath their mother tree) and initial vs. surviving seedlings. Patterns differ strongly between nondispersed and dispersed seedlings. Nondispersed seedlings are largely responsible for the positive kinship values observed at short distances in the studied population, whereas dispersed seedlings determine the overall SGS at distances beyond c. 30 m. The paternal alleles of nondispersed seedlings show weak yet significantly positive kinships up to c. 15 m, indicating some limitations in pollen flow that should further promote pedigree structures at short distances. Seedling mortality does not alter SGS, except for a slight increase in the nondispersed group. Field data reveal that mortality in this group is negatively density‐dependent, probably because of small‐scale variation in light conditions. Finally, we observe a remarkable similarity between the SGS of the dispersed seedlings and that of the adults, which probably reflects dispersal processes during the initial expansion of the population. Overall, this study demonstrates that incorporating individual‐level complementary information into analyses can greatly improve the detail and confidence of ecological inferences drawn from SGS.     

AFLP genetic maps of Eucalyptus globulus and E. tereticornis

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient technique for detecting large numbers of DNA markers in eucalypts. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of two clones of different Eucalyptus species (E. tereticornis and E. globulus). Of 606 polymorphic fragments scored, 487 segregated in a 1 : 1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. In the maternal E. tereticornis map, 268 markers were ordered in 14 linkage groups (919 cM); the paternal E. globulus map had 200 markers in 16 linkage groups (967 cM). Results from PGRI software were compared with MAPMAKER. The average density of markers was approximately 1 per 3.9 cM. Framework markers were ordered with an average confidence level of 90%, covering 80–100% of the estimated Eucalyptus genome size. In order to investigate the homologies between the E. tereticornis and the E. globulus genetic linkage maps, we included 19 markers segregating 3 : 1 in the analysis. Some homeologous linkage groups were recognized. The linkage data developed in these maps will be used to detect loci controlling commercially important traits. Received: 17 July 1997 / Accepted: 13 October 1997     

Predicted genetic gain and realised gain in stand volume of Eucalyptus globulus

Realised gains in stand volume of Eucalyptus globulus Labill. families at 7–9 years were compared with gains predicted at 3.5 years. Gain predictions were based on height, diameter at breast height (DBH), sectional area, and stem volume for 153 full-sib families and 18 commercial checks in five-tree line plots on three West Australian sites. Single-site narrow-sense heritability estimates were 0.12–0.24 for height, 0.08–0.12 for DBH, 0.09–0.13 for sectional area, and 0.14–0.19 for stem volume. Genetic dominance effects were significant (p?<?0.05) in most cases, and the estimated dominance/additive genetic variance ratio was heterogeneous for height and DBH. Stand volume was measured for 93 of the same families and checks in 40-tree block plots on four sites. Heritability of stand volume was 0.25–0.76, with an across-sites estimate of 0.41. Dominance effects were statistically absent at two sites. Estimated region-wide additive genetic correlations between selection traits (in line plots) and stand volume (in block plots) ranged from 0.86 to 0.90. Estimated stand volume gain was 23 % of the mean for the best 12 % of families and 14 % of the mean for the best 24 % of families. Realised gain was under-estimated by predictions based on height, DBH, and sectional area, which had smaller coefficients of additive variation than did stem volume. It is concluded that although BLUP analysis of early-age height and DBH can provide for indirect selection on E. globulus stand volume, analysis of stem volume is required to predict genetic gain at an appropriate scale.     

Lignin composition and structure in young versus adult Eucalyptus globulus plants

Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied in situ by a new procedure enabling the acquisition of two-dimensional nuclear magnetic resonance (2D-NMR) spectra on wood gels formed in the NMR tube as well as by analytical pyrolysis-gas chromatography-mass spectrometry. In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, pyrolysis-gas chromatography-mass spectrometry, and thioacidolysis. The data indicated that p-hydroxyphenyl and guaiacyl units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that β-O-4' and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open β-1' structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end groups were depleted with age, spirodienone abundance increased, and the main substructures (β-O-4' and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important nonether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed guaiacyl-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl, and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are approximately 50% acetylated) and 4-O-methylglucuronosyl signals.     

A comparative analysis of population structure of a forest tree, Eucalyptus globulus (Myrtaceae), using microsatellite markers and quantitative traits

Eucalyptus globulus (Myrtaceae) is a forest tree native to southeastern Australia, but is grown globally for pulpwood and timber. Eight microsatellite loci were used to determine the degree of selectively neutral differentiation between native populations of the geographic races of E. globulus that are used in a national breeding programme. Spatial differentiation was detected among 340 samples from across the species range (F _ST=0.09±0.02). Analysis of molecular variance showed that there was significant variation between the races, and an unweighted pair group method with arithmetic mean analysis of Nei’s genetic distance between races showed that geographically proximal races tended to be more closely related than geographically distant races. This contrasted markedly with analyses based on quantitative genetic data, where some races appeared to be highly divergent from their geographically closest neighbours. Comparison of racial differentiation based on quantitative (Q _ST) and molecular (F _ST) data suggested that at least five of the quantitative traits used for defining races of E. globulus have been influenced by natural selection, resulting in cases of both phenotypic divergence of parapatric races and phenotypic convergence of allopatric races. We conclude that selectively neutral molecular markers are more useful than quantitative genetic data for identifying the evolutionary affinities and lineages within E. globulus. However, both sources of information should be used in defining evolutionarily important units for conservation. The population structure observed in E. globulus has important consequences for future association studies and may also affect breeding strategies if significant genome co-adaptation has occurred.     

Evidence of spatial genetic structure in a California bunchgrass population

We investigated the scale of genetic variation of purple needlegrass (Nassella pulchra), a species commonly used in California for grassland restoration. Common garden and field data revealed evidence of genetic differentiation between two intermixed microhabitats characterized by differences in soil depth and community composition. We assessed the genetic variation within a single population using randomly amplified polymorphic DNA (RAPD) data collected from clusters of five individuals in 40 locations. We found no evidence for genetic structure at the whole population level. At smaller spatial scales, however, we found strong evidence that genetic subdivision of the population occurs at the level of the maternal neighborhood. We suggest that the interaction between widespread pollen dispersal and restricted seed dispersal may be the primary factor generating these results; panmictic pollen dispersal will make detection of genetic patterning difficult at larger spatial scales while limited seed dispersal will generate local genetic structure. As a result, the detection of population genetic structure will depend on the spatial scale of analysis. Local selection gradients related to topography and soil depth are also likely to play a role in structuring local genetic variation. Since N. pulchra is widely used in California in grassland and woodland habitat restoration, we suggest that, as a general rule, care should be exercised in transferring germplasm for the purposes of conservation when little is known about the within-population genetic subdivision of a plant species. Received: 23 December 1996 / Accepted: 20 May 1997     

Species discrimination, population structure and linkage disequilibrium in Eucalyptus camaldulensis and Eucalyptus tereticornis using SSR markers

Eucalyptus camaldulensis and E. tereticornis are closely related species commonly cultivated for pulp wood in many tropical countries including India. Understanding the genetic structure and linkage disequilibrium (LD) existing in these species is essential for the improvement of industrially important traits. Our goal was to evaluate the use of simple sequence repeat (SSR) loci for species discrimination, population structure and LD analysis in these species. Investigations were carried out with the most common alleles in 93 accessions belonging to these two species using 62 SSR markers through cross amplification. The polymorphic information content (PIC) ranged from 0.44 to 0.93 and 0.36 to 0.93 in E. camaldulensis and E. tereticornis respectively. A clear delineation between the two species was evident based on the analysis of population structure and species-specific alleles. Significant genotypic LD was found in E. camaldulensis, wherein out of 135 significant pairs, 17 pairs showed r(2)≥0.1. Similarly, in E. tereticornis, out of 136 significant pairs, 18 pairs showed r(2)≥0.1. The extent of LD decayed rapidly showing the significance of association analyses in eucalypts with higher resolution markers. The availability of whole genome sequence for E. grandis and the synteny and co-linearity in the genome of eucalypts, will allow genome-wide genotyping using microsatellites or single nucleotide polymorphims.     

Control of transpiration in an irrigated Eucalyptus globulus Labill. plantation

Stomatal conductance and transpiration were measured concurrently in an irrigated Eucalyptus globulus Labill. plantation. Canopy stomatal conductance, canopy boundary layer conductance and the dimensionless decoupling coefficient (Ω) were calculated (a) summing the conductance of three canopy layers (g_c) and (b) weighting the contribution of foliage according to the amount of radiation received (g_c′). Canopy transpiration was then calculated from g_c and g_c′ for Ω = 1 (E_eq), Ω = 0 (E_imp) and by weighting E_eq and E_imp using Ω (E_Ω). E_eq, E_imp and E_Ω were compared to transpiration estimated from measurements of heat pulse velocity. The mean value of Ω was 0·63. Transpiration calculated using g_c and assuming perfect coupling (12·5 ± 0·9 mmol m^?2 s^?1) significantly overestimated measured values (8·7 ± 0·8 mmol m^?2 s^?1). Good estimates of canopy transpiration were obtained either (a) calculating E_Ω separately for the individual canopy layers or (b) treating the canopy as a single layer and using g_c′ in a calculation of E_imp (Ω = 0). The latter approach only required measurement of stomatal conductance at a single canopy position but would be unsuitable for use in combined models of canopy transpiration and assimilation. It should however, be suitable for estimating transpiration in forests regardless of the degree of coupling.     

Analysis of spatial genetic structure in an expanding Pinus halepensis population reveals development of fine-scale genetic clustering over time

We analysed the change of spatial genetic structure (SGS) of reproductive individuals over time in an expanding Pinus halepensis population. To our knowledge, this is the first empirical study to analyse the temporal component of SGS by following the dynamics of successive cohorts of the same population over time, rather than analysing different age cohorts at a single time. SGS is influenced by various factors including restricted gene dispersal, microenvironmental selection, mating patterns and the spatial pattern of reproductive individuals. Several factors that affect SGS are expected to vary over time and as adult density increases. Using air photo analysis, tree-ring dating and molecular marker analysis we reconstructed the spread of reproductive individuals over 30 years beginning from five initial individuals. In the early stages, genotypes were distributed randomly in space. Over time and with increasing density, fine-scale (< 20 m) SGS developed and the magnitude of genetic clustering increased. The SGS was strongly affected by the initial spatial distribution and genetic variation of the founding individuals. The development of SGS may be explained by fine-scale environmental heterogeneity and possibly microenvironmental selection. Inbreeding and variation in reproductive success may have enhanced SGS magnitude over time.     

Optimization model of spatial population structure: example of poplar moth laying eggs on leaves

The authors analyze spatial distribution and survival of populations of poplar moth Litchcolletis populifoliella Tr. on its feeding plant--balsam poplar Populus balsamifera. Imago of the moth glue its eggs on the leaves thus determining the future location of their offspring on the host plant. Spatial distribution of eggs on leaf surface and distribution of leaves according egg numbers are not random. On the short distance from each egg the average number of eggs is less, than it should be in case of random distribution. While this distance increases up to some particular value the occurrence of eggs is higher than random. Thus, the eggs of moth are located by groups on the leaf surface. Within each group eggs are situated not very close to each other, this allowing larvae to lower competition for common resource. It is suggested that on the same feeding plant individuals have different interactions: competition, caused by limited quantity of resource and cooperation that is necessary to resist leaf defensive (antibiosis) reaction.     

Hierarchical spatial genetic structure in a distinct population segment of greater sage-grouse

Greater sage-grouse (Centrocercus urophasianus) within the Bi-State Management Zone (area along the border between Nevada and California) are geographically isolated on the southwestern edge of the species’ range. Previous research demonstrated that this population is genetically unique, with a high proportion of unique mitochondrial DNA (mtDNA) haplotypes and with significant differences in microsatellite allele frequencies compared to populations across the species’ range. As a result, this population was considered a distinct population segment (DPS) and was recently proposed for listing as threatened under the U.S. Endangered Species Act. A more comprehensive understanding of the boundaries of this genetically unique population (where the Bi-State population begins) and an examination of genetic structure within the Bi-State is needed to help guide effective management decisions. We collected DNA from eight sampling locales within the Bi-State (N = 181) and compared those samples to previously collected DNA from the two most proximal populations outside of the Bi-State DPS, generating mtDNA sequence data and amplifying 15 nuclear microsatellites. Both mtDNA and microsatellite analyses support the idea that the Bi-State DPS represents a genetically unique population, which has likely been separated for thousands of years. Seven mtDNA haplotypes were found exclusively in the Bi-State population and represented 73 % of individuals, while three haplotypes were shared with neighboring populations. In the microsatellite analyses both STRUCTURE and FCA separate the Bi-State from the neighboring populations. We also found genetic structure within the Bi-State as both types of data revealed differences between the northern and southern part of the Bi-State and there was evidence of isolation-by-distance. STRUCTURE revealed three subpopulations within the Bi-State consisting of the northern Pine Nut Mountains (PNa), mid Bi-State, and White Mountains (WM) following a north–south gradient. This genetic subdivision within the Bi-State is likely the result of habitat loss and fragmentation that has been exacerbated by recent human activities and the encroachment of singleleaf pinyon (Pinus monophylla) and juniper (Juniperus spp.) trees. While genetic concerns may be only one of many priorities for the conservation and management of the Bi-State greater sage-grouse, we believe that they warrant attention along with other issues (e.g., quality of sagebrush habitat, preventing future loss of habitat). Management actions that promote genetic connectivity, especially with respect to WM and PNa, may be critical to the long-term viability of the Bi-State DPS.     

Thinning strategies for Eucalyptus dunnii population: balance between breeding and conservation using spatial variation and competition model

Tree Genetics & Genomes - Malus baccata (L.) Borkh. and Malus toringo (Siebold) Siebold ex de Vriese of the genus Malus Mill. (Rosaceae) are wild crabapples occurring in temperate East Asia....     

Bayesian estimation of genetic parameters for growth,stem straightness,and survival in Eucalyptus globulus on an Andean Foothill site

Knowledge of the genetic variation of key economic traits in Eucalyptus globulus under cold conditions is crucial to the genetic improvement of environmental tolerances and other economic traits. A Bayesian analysis of genetic parameters for quantitative traits was carried out in 37 E. globulus open-pollinated families under cold conditions in southern Chile. The trial is located in the Andean foothills, in the Province of Bío-Bío. The Bayesian approach was performed using Gibbs sampling algorithm. Multi-trait linear and threshold models were fitted to phenotypic data (growth traits, survival, and stem straightness). Fifteen years after planting, height, diameter at breast height, and stem volume were found to be weakly to moderately heritable with Bayesian credible intervals (probability of 90 %): $ {\widehat{h}}^2 $ ?=?0.009–0.102, $ {\widehat{h}}^2 $ ?=?0.031–0.185, and $ {\widehat{h}}^2 $ ?=?0.045–0.205, respectively. Stem straightness was found to be weakly to moderately heritable ranging from 0.032 to 0.208 (Bayesian 90 % credible interval); posterior mode $ {\widehat{h}}^2 $ ?=?0.091. Tree survival at age of 15 years was high in the trial (84.8 %) with such heritability values ranging from 0.072 to 0.157. Survival was non-significantly genetically correlated to growth and stem straightness. Stem volume had the highest predicted genetic gains ranging from 17.9 to 23.7 % (selection rate of 15.8 and 8.3 %, respectively). The results of this study confirm the potential for selective breeding of this eucalypt in areas of southern Chile where cold is a significant constraint.     

QTL influencing growth and wood properties in Eucalyptus globulus

Regions of the genome affecting physical and chemical wood properties (quantitative trait loci (QTL)), as well as growth, were identified using a clonally replicated, outbred F₂ family (112 genotypes, each with two ramets) of Eucalyptus globulus, planted in a field trial in north-west Tasmania. Traits studied were growth (assessed by stem diameter), wood density, cellulose content, pulp yield and lignin content. These traits are important in breeding for pulpwood, and will be important in breeding for carbon sequestration and biofuel production. Between one and four QTL were located for each trait, with each QTL explaining between 4% and 12% of the phenotypic variation. Several QTL for chemical wood properties were co-located, consistent with their high phenotypic correlations, and may reflect pleiotropic effects of the same genes. In contrast, QTL for density and lignin content with overlapping confidence intervals were considered to be due to independent genes, since the QTL effects were inherited from different parents. The inclusion of fully informative microsatellites on the linkage map allowed the determination of homology at the linkage group level between QTL and candidate genes in different pedigrees of E. globulus and different eucalypt species. None of the candidate genes mapped in comparable studies co-located with our major QTL for wood chemical properties, arguing that there are important candidate genes yet to be discovered.     

Nuclear and chloroplast genetic structure indicate fine-scale spatial dynamics in a neotropical tree population

Dicorynia guianensis is a canopy tree, endemic to the tropical rain forest of French Guiana. We compared generational and spatial genetic structure for maternally and biparentally inherited markers in two cohorts (adult and seedling) in order to infer processes shaping the distribution of genetic diversity. The study was conducted on a 40 ha study plot located at Paracou near Kourou, where 172 adults trees and 375 saplings were sampled. Aggregation of trees was therefore suggested at different distances, ranging from 100 to 400 m. There was a strong link between demographic and genetic spatial structures at small distances (less than 100 m) that is likely to be the consequence of restricted seed dispersal. Genetic differentiation was more pronounced between spatial aggregates than between cohorts. Despite the spatial differentiation, the species was able to maintain high levels of diversity for maternal genomes, suggesting rapid turnover of aggregates. Spatial autocorrelation was larger for chloroplast than nuclear markers indicating a strong asymmetry between pollen and seed flow. Fixation indices indicated a lower heterozygote deficiency for the adults, maybe because of gradual elimination of selfed trees. Genetic relatedness at lower distances was higher in adult trees than in saplings, as a result of generation overlapping in the adult cohort. Overall, our results confirm earlier biological knowledge about the dispersion mechanisms of the species, and lead to an enhanced role of spatial processes in the dynamics of genetic diversity of D. guianensis.     

Early ovule development following self- and cross-pollinations in Eucalyptus globulus Labill. ssp. globulus

The study was conducted to identify the self-incompatibility mechanism in Eucalyptus globulus ssp. globulus. Controlled self- and cross-pollinations were conducted on individual flowers from three mature trees that had self-incompatibility levels of 76, 99.6 and 100%. Flowers were harvested at 4, 6 and 8 weeks after pollination. Embryology was investigated by bright field microscopy on material harvested at 4 and 6 weeks after pollination. Fertilization had taken place at 4 weeks after pollination with zygotes and free nuclear endosperm visible. There was a greater proportion of healthy, fertilized ovules in the cross- compared with the self-pollination treatment, and approx. half the ovules examined from both pollen treatments were not fertilized or were degenerating. By 6 weeks after pollination a few zygotes were starting to divide. The number of healthy, fertilized ovules was still greater in the cross-pollination treatment, but the number of healthy fertilized ovules was lower in both treatments compared with 4 weeks after pollination, and many ovules were degenerating. Fertilized ovules were significantly larger than non-fertilized or degenerating ovules and this difference was detectable by eye at 6 and 8 weeks after pollination. The mechanism of self-incompatibility appears to have both late pre- and post-zygotic components.