4.1. Survival and Hybrid Performance in Arauco and Valdivia
Although the annual temperature profiles of Arauco and Valdivia (
Figure 2) are reasonably similar, Valdivia is cooler, with a minimum mean monthly temperature (MMT) around 2.6 °C lower across the year and a difference near 3 °C in the coldest month of the year, July. Moreover, Valdivia is a more frost-prone region with 10.2 frost days in winter per year, compared to 3.1 in Arauco (see
Table 1). The temperature difference between zones is probably a primary factor in the differences in survival observed in the two zones. Age 8-year survival of both species and the hybrid were lower in Valdivia than Arauco; however, the decrease was larger for the hybrid than for the pure species: 8.1% lower survival for
E. globulus and 2.4% lower for
E. nitens, compared to 11.9% lower for GloNi (
Figure 3). Tibbits et al. [
37] reviewed and studied the performance of a number of
Eucalyptus hybrids and concluded that, on average, F1 hybrids tend to be intermediate to the parent species for frost tolerance with a slight tendency toward the more frost-susceptible species. These authors also observed this to be true for the
E. nitens x
E. globulus hybrid in particular, with sporadic statistically significant deviation toward the less tolerant
E. globulus, depending on the trait and the time of year. Potts et al. [
11] comment on high levels of abnormal phenotypes (dwarfs) and mortality in
E. nitens ×
E. globulus nursery and field experiments in Australia. In the current clonal trials in Chile, it seems likely that many abnormal phenotypes would be culled during the rooting phase of the selection process. However, the lower survival at 8 years could still be the result of some level of incompatibility for this hybrid that becomes more apparent with age and under stress from competition, frost, and other factors. Costa e Silva et al. [
38] and Larcombe et al. [
39] reported outbreeding depression in both survival and growth in long-term studies (14 and 20 years, respectively) of hybrids between
E. globulus and
E. nitens. Nevertheless, in the current study, the best 20% clones in volume gain (BLUP) had an average survival of 89% and 90% in Arauco and Valdivia, respectively; this is higher than the overall average survival of the hybrid observed at 8 years in
Figure 3, especially in Valdivia, where the difference is around 16% higher survival of the best 20% of the clones in the zone.
This climatic difference also has an impact on the individual tree volume of the pure species and the hybrid in the two zones. At 8 years of age, all three varieties have larger individual tree volume in the Arauco zone than in Valdivia; however, the magnitude of the difference varies considerably. Comparing individual tree volume at 8 years between Arauco and Valdivia (
Figure 3),
E. nitens grows some 22.2% less in Valdivia than Arauco. The
E. globulus grows 29.6% less volume in Valdivia and shows much more variation in growth rate, perhaps indicating a slightly lower degree of adaptation to the zone. However, the GloNi hybrid also grows less in Valdivia but has a decrease of only 7.7% compared to the growth in Arauco. This phenomenon is partly due to the higher mortality (lower survival) observed in Valdivia. However, even accounting for this, it seems that the hybrid tree volume is less affected by the environment of Valdivia compared to Arauco in general.
In this study, the GloNi hybrid had wood properties intermediate to the parent species in both zones, being consistent with the conclusions of various authors indicating that, on average,
Eucalyptus hybrids tend to have intermediate values for wood density and other wood property traits [
11,
40,
41]
4.2. Genetic Parameters
The genetic variances in these clonal populations of GloNi were, in general, high for volume, pulp yield, specific consumption, and moderate for basic density. Broad sense heritabilities ranged from H
2 = 0.22 to 0.60 for wood traits (basic density, pulp yield, and specific consumption) and were above 0.50 for volume (
Table 4). All traits in the Arauco breeding zone had higher H
2 estimates than in Valdivia, and this was also reflected in a lower level of G×E interaction observed in the Arauco zone. At the clonal level, rB
G values for all traits ranged from rB
G = 0.92 to 0.99 in Arauco and were typically lower in Valdivia, ranging from rB
G = 0.86 to 0.94 in Valdivia (
Table 4). In general, the low levels of G × E interaction suggest that in the future, more clones can be tested for growth across fewer sites, and for wood properties, samples could also be taken in fewer tests.
Volker et al. [
10], working with
E. nitens ×
E. globulus trials in Australia, reported narrow-sense heritability for the growth trait DBH, and the wood property trait Pilodyn penetration. Pilodyn is a useful indirect measurement of wood basic density [
42,
43]. In the mentioned research, Volker et al. [
10] reported a narrow-sense heritability (h
2) of h
2 = 0.42 for DBH and h
2 = 0.20 for Pilodyn, roughly comparable to the estimates of H
2 ≈ 0.50 for growth and H
2 ranging from 0.22 to 0.36 for basic density observed in this study. The estimated H
2 for basic density in the hybrid population in this study was lower than might have been expected, based on published heritability estimates for the parental species. Raymond [
44] reported a summary of wood genetic parameters for the species
E. globulus and
E. nitens based on a decade of publications, and reported a mean h
2 estimate for basic density around 0.60 and 0.70 for
E. nitens (based on 12 publications) and
E. globulus (based on 7 publications), respectively. For pulp yield, the mean h
2 estimates for both
E. nitens and
E. globulus were around 0.40 (based on 7 and 5 publications, respectively), and these values correspond more closely to the H
2 = 0.55 and 0.60 in the two zones in this study.
For volume, there was a strong effect of the E. nitens parent on the growth performance of the hybrid in both breeding zones. In addition, there was a substantial clonal variation found within full-sib GloNi families, and the data suggests that it should be possible to find clones ranging from ±80% in volume gain, relative to the mean of each family. There was no SHA variance found for these interspecific crosses for volume, and there was no GHA variance found for volume among E. globulus parents.
It is important to remember that there were only 8
E. globulus fathers evaluated in this study. Simulation results for hybrid populations suggest a low (but non-zero) chance of estimating a zero variance for a random effect when the number of parents is low and the true underlying genetic variance is small [
45]. So even if the true GHA
GLO variance (
) is not zero, it is likely that
is relatively low and much less important than the other effects for hybrid tree volume. Other authors have estimated zero GHA variance for one of the parent species in
Eucalyptus hybrids. In a study with a large hybrid population of
Eucalyptus grandis x
E. urophylla with seedling progeny, van den Berg et al. [
20] reported a broad-sense heritability of H
2 = 0.37, but estimated the GHA variance for the 30
E. grandis parents to be zero. There was substantial GHA variance for the 27
E. urophylla parents, but there was also a considerable non-additive SHA variance, indicating a high amount of dominance genetic variance. In the current study, there was no SHA variance detected for volume in either zone. However, there appears to be a substantial amount of non-additive epistasis variation observed in both zones.
The estimates of epistasis (
) for volume was large in both zones, making up 42% to 50% of the total genetic variation. In contrast, there was no evidence of epistasis for any of the wood traits in either zone. These results are consistent with results reported by Tan et al. [
46] in an extensive study of the progeny of 476 full-sib hybrid families of
E. urophylla ×
E. grandis. The families were derived from 86
E. urophylla and 95
E. grandis parents and represented by 35 individuals each. In one trial, the hybrids were tested in a randomized complete block design in single-tree plots and 35 replications. Using 41,304 SNP markers, genomic models were evaluated that accounted for additive, dominance, and first-order epistatic interactions for two growth traits (Circumference at Breast Height (CBH) and Height) and two wood traits (basic density and pulp yield) evaluated at 3 and 6 years-old. The study results showed significant epistasis variation in height and CBH at 3 years, with the epistasis variance comprising 91% and 65% of the total genetic variance, respectively. In the measurement at 6 years, the epistasis variance was zero for height but still accounted for 36% of the total genetic variance in CBH. Similar to the current GloNi study results, Tan et al. [
46] found no epistasis variation for any of the wood traits at either age.
By applying Foster and Shaw’s [
31] epistasis variance estimation procedure on the variances components reported by van den Berg et al. [
20] for a sizeable clonal population of
Eucalyptus grandis x
E. urophylla, discussed above, the epistasis variance for tree volume was estimated to be 40% of the total genetic variation, a value roughly similar to the estimates found in this study for the same trait. It seems that the direction of the cross for
E. urophylla x
E. grandis does not affect the amount of epistatic variation; comparing the result of van den Berg et al. [
20] with Tan et al. [
46], both show high epistasis variation for growth in these hybrid populations.
At the pure species level, Costa e Silva et al. [
33] estimated the effect of epistasis in a study with full-sib families and clonally replicated progeny of
E. globulus in Portugal. Epistasis variation was estimated for DBH growth and Pilodyn penetration measured at 4 years of age. That study reported a very low amount of epistasis variance for DBH, accounting for only 3% of the total genetic variation. In contrast, for Pilodyn penetration, a substantial epistasis effect was reported, corresponding to 23% of the total genetic variance.
In another study of
E. globulus in Portugal, Araújo et al. [
32] partitioned additive and non-additive variation for DBH growth in a clonal population, testing more than 4200 genotypes in 40 sites. These authors reported a small amount of epistasis variance
= −0.02, not significantly different from zero (using the expectations of genetic variances derived by Costa e Silva et al. [
33]). They did report some important non-additive genetic variation in this
Eucalyptus clonal population, but this was dominance variation (
= 0.096) of a similar size to the additive variance (
= 0.096), followed for the clonal within family variation (
= 0.055).
Overall, the studies of Costa e Silva et al. [
33] and Araújo et al. [
32] are in accord with a low to zero epistasis variance for the DBH growth trait in
E. globulus. For
E. nitens, there are no studies that characterize epistasis.
Isik et al. [
47], studying clonally replicated progeny tests with loblolly pine (
Pinus taeda), composed of 9-full sib families, partitioned genetic variance into additive, dominance, and epistatic components, and found a negative epistasis variance for growth traits (Total height, DBH, and volume), which was interpreted as zero variance.
Costa e Silva [
33] and Isik et al. [
47] both mention that the epistasis variance estimates obtained using the Foster & Shaw method [
31] could be underestimates, since that model assumes that epistasis comes mostly from high-level loci interaction. Instead, if low-level loci interaction occurs, the additive variance could be slightly overestimated, and similarly, dominance variance could be slightly overestimated. However, in general, the Foster and Shaw [
31] method should give a good and straightforward approximation of the epistasis variation.
4.3. Pure Species GCA—Hybrid GHA Correlations
Positive correlations from small to moderate size were found for
E. nitens pure species GCA and hybrid GHA (r
HP) for volume in both the Arauco and Valdivia zones, with correlation values of r
HP = 0.44 and 0.55, respectively. These correlations (
Figure 5) suggest that the pure species genetic value for growth traits could be used as an indicator of genetic worth as a hybrid parent. Since there was zero GHA variance found for
E. globulus, r
HP for this species could not be defined.
There was a relationship between E. nitens GCA and GHA in both the Arauco and Valdivia zones for wood property traits, but with much higher correlations found in Valdivia (rHP = 0.65 to 0.71) than in Arauco (rHP = 0.4 to 0.5). For E. globulus, there were quite strong correlations between GCA and GHA for all three wood traits in the Arauco zone, ranging from rHP = 0.81 to 0.99. However, in Valdivia, the correlations were near zero for BD and SC, and were non-estimable for PY since the E. globulus GHA was zero for this trait.
Volker et al. [
10], working with
E. nitens, reported a correlation for GCA-GHA of r
HP = 0.67 for 6-year-old DBH and r
HP = 0.65 for 10-year-old DBH. In contrast, for
E. globulus, they reported a GCA-GHA correlation of r
HP = 0.16 for 6-year-old DBH and a negative correlation for 10-year-old DBH. For Pilodyn penetration (an indirect measure of basic density (BD) in the current study), GCA-GHA correlations of r
HP = 0.60 and 0.65 were found for
E. globulus and
E. nitens, respectively. In all cases, the standard errors of the correlation estimates were high, but in general, those results correspond well to the current study results. Thus, it appears that
E. nitens GCA values are moderate predictors of the hybrid GHA for both growth and wood properties, while for
E. globulus, only for wood traits are GCA values related to GHA values.
For a different hybrid,
E. grandis ×
E. urophylla, van den Berg et al. [
20] found a statistically significant correlation of r
HP = 0.58 between GCA and GHA for
E. urophylla for DBH. As there was very little GHA variance for
E. grandis, the GCA-GHA correlation for
E. grandis was not reported. These authors concluded that individual tree breeding values for growth traits would be relatively good indicators of GHA. However, they also noted a large amount of non-additive genetic variation for DBH in the hybrid. Nevertheless, there would be some value in selecting the best pure species
E. urophylla parents for growth to test as hybrid parents in a hybrid breeding program, similar to the case for
E. nitens parents and the GloNi hybrid variety in Chile.
Finally, it is important to note that the current population of GloNi does not have many NIT and GLO parents to date, and relatively few crosses per parent (approximately 2 per E. nitens, and 4 per E. globulus), so the GCA-GHA correlations have to be viewed with some caution. Nevertheless, for tree volume, the data suggest a clear tendency with E. nitens for high GCA values to be associated with high GHA in both breeding zones (Arauco and Valdivia). For wood properties in Arauco, E. globulus GCA is an excellent predictor of the GHA, and E. nitens GCA is a moderate GHA predictor. For wood properties in Valdivia, E. nitens GCA is an excellent GHA predictor.
4.4. Impact of Environment on Hybrid Genetic Architecture
An interesting pattern emerged in this study where there was a clear relationship between the environment (i.e., the Arauco and Valdivia zones) and the expression of genetic variances related to the E. nitens or E. globulus parentage of the GloNi hybrid. Comparing the two parental pure species, it is clear that E. nitens should be better adapted to the Valdivia zone than Arauco, and the reverse is true for E. globulus. The magnitude of the E. nitens-related genetic parameters was greater in Valdivia than in Arauco, while the opposite was true for the E. globulus-related parameters.
Regarding E. nitens, the GHA variance was higher in Valdivia than in Arauco for volume and the wood traits SC and BD. The correlation between E. nitens GCA and GHA for volume was low in Arauco and moderate Valdivia (rHP = 0.44 and 0.55, respectively). However, for the three wood traits, this correlation was much higher in Valdivia than in Arauco (rHP = 0.65 to 0.71 in Valdivia, and rHP = 0.42 to 0.5 in Arauco). Concerning E. globulus, GHA variance was higher in Arauco than in Valdivia for all three wood traits. The correlation between E. globulus GCA and GHA for wood traits was very high in Arauco (rHP = 0.81 to 0.99) and low to near-zero in Valdivia (rHP = 0.29 to 0.19).
It is conceivable that in a hybrid tree variety where one species brings adaptability to specific environmental conditions and stresses, more of the genetic variation in hybrid performance could derive from that species relative to the other parental species. He et al. [
48] examined genetic variation in
E. urophylla ×
E. tereticornis in a cool frost-prone environment where
E. tereticornis would be expected to bring frost tolerance to the hybrid. These authors found higher (and statistically significant) GHA variance for 4-year volume in the
E. tereticornis parents than the
E. urophylla parents. Additionally, they found that genetic variation in “cold hardiness” (i.e., field assessed cold and frost damage) derived only from the
E. tereticornis parents.
Trials of
E. grandis ×
E. tereticornis and
E. grandis ×
E. camaldulensis hybrids were planted on four sites in Zimbabwe that were considered marginal for
E. grandis due to low rainfall [
1], and where the
E. tereticornis and
E. camaldulensis parents were intended to bring drought tolerance to the hybrid. There was no GHA variance for 43-month height and DBH among the
E. grandis parents in either hybrid combination in these four tests. In contrast, there was significant GHA variance among the
E. tereticornis parents for both height and DBH, and among the
E. camaldulensis parents for DBH.
The above examples involve growth traits and seem consistent with the pattern observed in this study for volume, where E. nitens (GHA) contribute more genetic variation for volume in the cooler zone Valdivia than in the warmer zone of Arauco. However, the current results appear to be the first observation of this kind of pattern with wood traits, where E. globulus parents explain more of the hybrid performance in the warmer zone (Arauco), and E. nitens parents explain more of the hybrid performance in the cooler zone (Valdivia).
This last observation is somewhat surprising, as generally, there is a low level of G×E interaction reported for wood properties in forest trees, and specifically, this has been found to be true for pure species
E. globulus and
E. nitens. For
E. globulus, no G×E interaction was found for basic density and pulp yield in a study conducted by Raymond et al. [
49] in Tasmania, Australia, or for basic density and Kraft pulp yield in another study by Nickolas et al. [
50], also in Tasmania. In
E. nitens, there was no significant G×E interaction in wood properties in multiple studies in different environments in Victoria and Tasmania, Australia. In those studies, Greaves et al. [
42] evaluated the Pilodyn for indirect measurement of wood density, Blackburn et al. [
51] used acoustic wave velocity for indirect selection of trees related to MOE, and Hamilton et al. [
51] evaluated wood density and cellulose content, among other traits. As pure species, both
E. nitens and
E. globulus present very stable behavior across sites in wood properties evaluations.
4.5. Implications for Crossing Strategy for F1 GloNi Clone Production
This study provides some guidance for the formulation of crossing strategies to identify new GloNi clones. All traits examined show considerable genetic variation for clones within hybrid family, and volume appears to have a large amount of epistatic variation. These results first suggest that it is essential to test large numbers of clones, as this will be the only way to capture these potential genetic gains. However, the selection of specific E. nitens and E. globulus parents can also provide some genetic gains.
First, an important strategy to improve volume in both breeding zones would be to increase the number of E. nitens parents used in the crossing design, as there is important GHA variation due to E. nitens in both zones. Thus, a sizable increment of gain could be achieved by testing more E. nitens females in hybrid crosses, and E. nitens parents could be selected based on their performance as pure species due to the moderately high correlation observed between GCANIT and GHANIT in both breeding zones. Moreover, it appears that increments of GCANIT might result in larger increments of GHANIT in the hybrid, as the regression coefficient suggests a multiplier of 1.26 and 1.1 for Arauco and Valdivia, respectively.
GHA variation for wood properties was also due to E. nitens in both zones, and GCA-GHA correlations were moderate (Arauco) to high (Valdivia), so some emphasis could also be placed on wood properties when selecting E. nitens parents.
There was substantial GHA variation for wood properties due to
E. globulus in both zones and a very high GCA-GHA correlation in the Arauco zone. Similarly, to the discussion above, there may be some scale effect where increments of GCA
GLO for wood traits might result in larger increments of GHA
GLO in the hybrid: the regression coefficients suggest a multiplier of 1.36 for BD, 1.72 for SC and 1.87 for PY in Arauco (
Figure 6 D, E and F, respectively). Since
E. globulus does not contribute GHA variation to hybrid performance in volume, it seems likely that breeders can ignore pure species volume and focus only on wood properties when selecting
E. globulus parents for hybrid crosses. Possibly a breeder would want to be cautious with the interpretation of these results since a very small number of parents were tested, and therefore would not want to ignore pure species volume GCA completely. Nevertheless, even selecting the top half of the population for volume would allow substantial selection intensity for wood traits.
Finally, the low amount of G x E interaction observed within hybrid zones (almost all type-B genetic correlations ranging from 0.80 to 1.00) indicates that clones should perform in essentially the same way across all sites within the Arauco and Valdivia breeding zones. In other words, the top 10 clones selected on one or a small number of sites should be excellent performers on any other site within the zone. Therefore, clones could be tested on relatively few sites within zones, allowing more hybrid families and clones to be included in the testing program.
In summary, to obtain gain in growth and wood properties for the Arauco zone, it is proposed to select the best E. nitens females with high performance in growth and the best E. globulus parents with good performance for wood properties, using the results of the parent-tested as pure species or hybrid crosses. In Valdivia, parental selection should focus more on the performance of E. nitens females for volume and wood properties.