Experiment 1: Quantitative genetics study

Genetic-based variation in wax chemistry at the sub-race and family within sub-race levels was studied by growing families with known female pedigrees in a replicated common environment field trial (Hamilton et al. 2013; O'Reilly-Wapstra et al. 2013b); located at Salmon River (41° 01' S, 144° 52' E) in northwest Tasmania, Australia. The trial was established in 2006 using progeny derived from open-pollinated seed (families) from 140 trees sampled across the natural range of

E. globulus. Full trial details are given in Hamilton et al. (2013). In brief, families were planted as single-tree plots in a randomized incomplete block design consisting of 25 replicates and 13 incomplete blocks per replicate. A total of 246 trees from 13 geographic sub-races were sampled, with 7-13 families per sub-race and 2 trees per family. One sub-race was chosen to represent each of the 13 geographic races of E. globulus. Ten fully expanded adult leaves were randomly collected from the mid-canopy on the north side of each tree for cuticular wax extraction and analysis in

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April 2012. Leaves were sealed in plastic bags in the field and stored at -20°C until extraction (July-August 2012).

Cuticular wax extraction and analysis was done using a method originally designed for essential oils as this allowed for both oils and waxes to be assessed simultaneously. Cuticular waxes were extracted by immersing 1 gram of wet leaf cut into 6 mm diameter disks from a pool of the 10 leaves in 10 mL of dichloromethane containing 100 mg heptadecane (C17) per litre as an internal standard. Samples were left to extract at room temperature overnight followed by ultrasonication for 30 minutes in a Unisonics FXP-12 M. Extracts were decanted and stored at 4°C. The extraction process was repeated two more times on the same leaf disks, yielding a pooled 30 mL of extract per sample. Surplus leaf material was oven-dried for 7 days at 60°C to determine percentage dry matter.

Extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Varian 3800 GC coupled to a Bruker-300 triple quadrupole MS. Helium was used as the carrier gas, with a pressure program of 9.7 psi to 18.l8 psi at 0.46 psi/min, then to 38 psi at 15psi/min, then to 45 psi at 1.12 psi/min with a final hold time of 12 mins. Waxes were separated with 30 m x 0.25 mm (internal diameter) VF5-ms column, with a 0.25 μm film thickness. One microliter injections were made with an injection port temperature of 280°C and a 3:1 split, 3 minute solvent delay. Initial column temperature was 60°C for 1 min, raised to 210°C at 8°C/min, then raised to 300°C at 12°C/min with a final hold time of 12 mins. Mass spectra was collected from m/z 35 to 650, scanned in 0.2s, plus Selected Ion Monitoring of the following characteristic ions for 30ms per ion; m/z 58, 82, 91, 100, 104,108. Individual compounds were identified bases on an ‘in-house’ MS database of wax compounds, in tandem with the Kovats’ retention index of the analyses. Concentrations of individual compounds are expressed as equivalents of heptadecane, on a milligram per gram of leaf dry matter basis. Overall, nine aliphatic ester, two aliphatic β-diketones and two flavonoid cuticular wax compounds were quantified from these adult leaf samples (Table 4.1), all of which had been previously reported in juvenile leaves (Jones et al. 2002; Li et al. 1997) and a few in adult leaves of E. globulus (Li et al. 1997).

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Table 4.1. Trial means and quantitative genetic parameters describing the variation in leaf cuticular wax compounds in a range wide study of Eucalyptus globulus (Experiment 1).

Mean (mg/gD M)

Sub-race

Cuticular Wax Compound Code F P Qst H2

Aliphatic esters

Benzyl n-tetracosanoate Benzyl C24 0.085 61.5 0.000* 0.39 0.40±0.237

Benzyl n-hexacosanoate Benzyl C26 0.201 91.7 0.000* 0.65

*

0.45±0.266

Benzyl n-octacosanoate Benzyl C28 0.127 55.7 0.000* 0.28 0.47±0.233

Phenylethyl n-eicosanoate Phenylethyl C20 0.014 18.9 0.090 0.04 0.45±0.221 Phenylethyl n-docosanoate Phenylethyl C22 0.022 15.4 0.221 0.03 0.35±0.225 Phenylethyl n-tetracosanoate Phenylethyl C24 0.066 18.5 0.100 0.05 0.30±0.226 Phenylethyl n-pentacosanoate Phenylethyl C25 0.008 23.0 0.028* 0.34 0.09±0.227 Phenylethyl n-hexacosanoate Phenylethyl C26 0.096 21.5 0.043* 0.06 0.38±0.223 Phenylethyl n-octacosanoate Phenylethyl C28 0.071 29.7 0.003* 0.08 0.60±0.218 Aliphatic β-diketones

n-Hentriacontane-14, 16-dione β-diket C31 0.155 74.2 0.000* 0.28 0.72±0.260

n-Tritriacontane-16, 18-dione β-diket C33 4.639 12.3 0.419 0.01 0.47±0.223

Total Wax Yield 5.48 16.4 0.174 0.03 0.38±0.225

Flavonoids

Desmethyl eucalyptin Desmethyl 0.089 41.9 0.000* 0.13 0.73±0.223

Eucalyptin Eucalyptin 0.213 46.8 0.000* 0.16 0.62±0.227

Mixed- model univariate tests of significance of the variation in cuticular wax concentrations between sub- races, the associated quantitative inbreeding coefficient (Qst), and broad-sense heritability estimates (H2)

are shown. Asterisks indicate significance (p<0.05), with boldface indicating significance after a Bonferroni adjustment to p=0.0035. Qst values significantly different from the reported average Fst (0.09) between sub-

races for microsatellite markers are shown in boldface, with asterisks indicating Qst values significantly

different from the recorded maximum Fst (0.201) (see O'Reilly-Wapstra et al. 2013b). Statistical

significance was tested using a Wald-F test for the fixed sub-race effect.

Phenotypic variation in the composition of all 13 cuticular compounds was summarized with a principal component analysis (PCA) using prcomp from the base stats package in R version 3.1.2 (R Core Team, 2013). Prior to PCA, compounds were standardized by total yield per sample using

decostand from the package vegan in R. Correlations between cuticular wax compounds were summarized through ordination of the principal components (PCs) with the compound loadings on the components (i.e. correlations between PCs and individual cuticular wax compounds) displayed as vectors.

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A linear discriminant analysis maximizing the differences between sub-races was conducted on the raw data of all thirteen cuticular wax compounds using lda from the package MASS in R. Differences among sub-races were summarized through ordination of sub-race means on the main discriminant axes. The significance of discriminant axes in differentiating between geographic sub-races were tested using manova from the base stats package in R. Minimum spanning Mahalanobis distances between geographic sub-races were determined based on the Euclidian distances among sub-races in the discriminant space using spantree from the package vegan in R. Nine climatic variables obtained using home-site data from the natural sub-race populations (sub- races; where seed was originally collected) in ANUCLIM v6.1 (Xu 2011), as well as latitude, longitude, altitude and a drought susceptibility score (Dutkowski and Potts 2012), were fit as vectors into the discriminant space using envfit from the package vegan in R. This was also done for individual cuticular wax compounds, using sub-race means to provide insight into their influence on sub-race differentiation.

To examine the genetic variation in individual foliar wax compounds, we used the following mixed model fitted with ASReml in R (asreml-r) version 3 (Gilmour et al. 2009):

y = μ + Sub-race + Family(Sub-race) + Replicate + Residuals

where Sub-race is a fixed effect, and Family(Sub-race) and Replicate are the random effects corresponding to the variation of families within sub-races and amongst field trial replicates. All compounds, as well as total wax yield (sum of aliphatic esters and diketones), were square root transformed to improve normality and homoscedasticity. Significance of the sub-race fixed effect in the univariate analysis was determined using a Wald-F test. Random effects were tested using a one-tailed log-likelihood ratio test of the full model against a model with the random effect of interest removed.

Variance components for estimating trait heritability and quantitative inbreeding coefficient (Qst) were obtained by fitting sub-race as a random effect in asreml-r. Within sub-race narrow-sense heritability estimates (h2_{op) and among sub-race quantitative inbreeding coefficients (Qst) were} calculated from the variance components obtained from univariate analyses. These analyses

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assume base parents from which the families are derived are unrelated and an average coefficient of relationship of 0.4 to account for an assumed selfing rate of 30% in the open-pollinated families. Assumptions, calculations and tests followed those outlined in Dutkowski and Potts (2012) and Hamilton et al. (2013). Qst estimates among sub-races were tested to determine if they were greater than the published estimate of the mean microsatellite Fst (0.09) as well as the maximum-recorded Fst (0.201 in Yeoh et al., 2012) using a one-tailed likelihood test. These Fst values are from microsatellite studies of similar range-wide populations of E. globulus but different individuals. These Fst values are relatively stable despite the different studies using different microsatellite loci (e.g. mean Fst values of 0.09 have been reported by both Steane et al. 2006; Yeoh et al. 2012). If Qst is significantly greater than Fst, this is a line of evidence suggesting that diversifying selection has influenced the sub-race divergence (Whitlock 2008).

For individual wax compounds found to vary significantly at the family within sub-race level, pair- wise additive genetic correlations (family within sub-race level correlations) were estimated with a bivariate analysis using asreml-r and the same model as above. Inter-trait genetic correlations were tested from zero using a two-tailed likelihood ratio test (Costa e Silva et al. 2009). Wax compounds found to vary significantly at the sub-race level were tested for correlations among sub-race least-squares means derived from univariate analyses using Pearson correlations calculated with cor.test from the base stats package in R. Overall phenotypic correlations for all compounds were tested using bivariate analyses in asreml-r with the no model terms fitted. For wax compounds shown to have a significant Qst. Pearson correlations were also used to test for associations of sub-race least-squares means with home-site spatial and climate variables using

cor.test from the base stats package in R. Only climate variables shown to have a significant fit to sub-race variation in the discriminant space were tested.

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