Mapping species differences for adventitious rooting in a Corymbia torelliana x Corymbia citriodora subspecies variegata hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana x Corymbia citriodora subspecies variegata hybrid family (n=186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.

Genetic control of propagation traits in a single Corymbia torelliana x Corymbia variegata family

Genetic control of vegetative propagation traits was described for a second-generation, outbred, intersectional hybrid family (N = 208) derived from two species, Corymbia torelliana (F. Muell.) K.D. Hill & L.A.S. Johnson and Corymbia variegata (F. Muell.) K.D. Hill & L.A.S. Johnson, which contrast for propagation characteristics and in their capacity to develop lignotubers. Large phenotypic variances were evident for rooting and most other propagation traits, with significant proportions attributable to differences between clones (broad-sense heritabilities 0.2-0.5). Bare root assessment of rooting rate and root quality parameters tended to have the highest heritabilities, whereas rooting percentage based on root emergence from pots and shoot production were intermediate. Root biomass and root initiation had the lowest heritabilities. Strong favourable genetic correlations were found between rooting percentage and root quality traits such as root biomass, volume, and length. Lignotuber development on a seedling was associated with low rooting and a tendency to poor root quality in cuttings and was in accord with the persistence of species parent types due to gametic phase disequilibrium. On average, nodal cuttings rooted more frequently and with higher quality root systems, but significant cutting type x genotype interaction indicated that for some clones, higher rooting rates were obtained from tips. Low germination, survival of seedlings, and rooting rates suggested strong hybrid breakdown in this family.

Local dispersion and damage of Corymbia citriodora subsp. variegata (Myrtaceae) regrowth by eriophyid mites (Acari: Eriophyoidea).

Eriophyid mites (Acari: Eriophyoidea: Eriophyidae: Rhombacus sp. and Acalox ptychocarpi Keifer) are recently-emerged pests of commercial eucalypt plantations in subtropical Australia. They cause severe blistering, necrosis and leaf loss to Corymbia citriodora subsp. variegata (F. Muell.) K.D. Hill & L.A.S. Johnson, one of the region's most important hardwood plantation species. In this study we examine the progression, incidence and severity of these damage symptoms. We also measure within-branch colonisation by mites to identify dispersive stages, and estimate the relative abundance of the two co-occurring species. Rhombacus sp., an undescribed species, was numerically dominant, accounting for over 90% of all adult mites. Adults were the dispersive stage, moving mostly within branches, but 12% of recruitment onto new leaves occurred on previously uninfested branches. Damage incidence and severity were correlated, while older leaves had more damage than younger leaves. "Patch-type" damage was less frequent but was associated with higher mite numbers and damage scores than "spot-type" damage, while leaf discoloration symptoms related mostly to leaf age.

Wood properties and knot occlusion of plantation grown Eucalyptus dunnii and Corymbia citriodora subsp. variegata in a pruning and thinning experiment.

A 2 × 2 factorial combination of thinned or unthinned, and pruned or unpruned 11-year-old Eucalyptus dunnii (DWG) and 12-year-old Corymbia citriodora subsp. variegata (CCV) was destructively sampled to provide 60 trees in total per species. Two 1.4 m long billets were cut from each tree and were rotary veneered in a spindleless lathe down to a 45 mm diameter core to expose knots which were classified as either alive, partially occluded or fully occluded. Non-destructive evaluation of a wider range of thinning treatments available in these trials was undertaken with Pilodyn and Fakopp tools. Disc samples were also taken for basic density and modulus of elasticity. Differences between treatments for all wood property assessments were generally small and not significantly different.Thinning and pruning had little effect on the stem diameter growth required to achieve occlusion, therefore occlusion would be more rapid after thinning due to more rapid stem diameter growth. The difference between the treatments of greatest management interest, thinned and pruned (T&P) and unthinned and unpruned (UT&UP) were small. The production of higher value clear wood produced after all knots had occluded, measured as the average stem diameter growth over occlusion of the three outermost knots, was approximately 2 centimetres diameter. Two of the treatments can be ruled out as viable management alternatives: (i) the effect of thinning without pruning (T&UP) is clear, leading to a large inner core of stem wood containing knots (large knotty core diameter) and (ii) pruning without thinning (UT&P) results in a small knotty core diameter, however the tree and therefore log diameters are also small.

Reproductive Biology of Corymbia citriodora subsp. variegata and Effective Pollination across its Native Range.

The spotted gum species complex represents a group of four eucalypt hardwood taxa that have a native range that spans the east coast of Australia, with a morphological cline from Victoria to northern Queensland. Of this group, Corymbia citriodora subsp. variegata (CCV) is widespread in south-eastern Queensland and northern New South Wales. It is currently the most commonly harvested native hardwood in Queensland. However, little basic knowledge of the reproductive biology of the species is available to inform genetic improvement and resource management programmes. Here we take an integrative approach, using both field and molecular data, to identify ecological factors important to mating patterns in native populations of CCV. Field observation of pollinator visitation and flowering phenology of 20 trees showed that foraging behaviour of pollinator guilds varies depending on flowering phenology and canopy structure. A positive effect of tree mean flowering effort was found on insect visitation, while bat visitation was predicted by tree height and by the number of trees simultaneously bearing flowers. Moreover, introduced honeybees were observed frequently, performing 73% of detected flower visits. Conversely, nectar-feeding birds and mammals were observed sporadically with lorikeets and honeyeaters each contributing to 11% of visits. Fruit bats, represented solely by the grey-headed flying fox, performed less than 2% of visits. Genotyping at six microsatellite markers in 301 seeds from 17 families sampled from four of Queensland's native forests showed that CCV displays a mixed-mating system that is mostly outcrossing (tm = 0.899 ± 0.021). Preferential effective pollination from near-neighbours was detected by means of maximum-likelihood paternity analysis with up to 16% of reproduction events resulting from selfing. Forty to 48% of fertilising pollen was also carried from longer distance (>60 m). Marked differences in foraging behaviour and visitation frequency between observed pollinator guilds suggests that the observed dichotomy of effective pollen movement in spotted gums may be due to frequent visit from introduced honeybees favouring geitonogamy and sporadic visits from honeyeaters and fruit bats resulting in potential long-distance pollinations.

Rotary veneering of plantation-grown spotted gum (Corymbia citriodora subsp. variegata) and Dunn's white gum (Eucalyptus dunnii)

This report evaluates the wood and veneer properties of plantation-grown spotted gum (Corymbia citriodora subsp. variegata, or CCV) and Dunn's white gum (Eucalyptus dunnii), grown at different stockings, in thinning trials near Ellangowan in north-east New South Wales (mean annual rainfall 1050 mm) and Kingaroy in south-east Queensland (mean annual rainfall 873 mm). Thinning trials were established at age seven years. Both species showed a significant increase in stem diameter growth of the dominant trees in response to thinning. At age 10 years, trees from the unthinned (950–1270 stems ha-1) and 300 stems ha-1 treatments were selected for veneering. Five dominant trees were felled from each combination of species x sites x thinning treatment. Diameter at breast height over bark of the selected trees ranged from 20 cm to 27 cm at Ellangowan, and 19 cm to 26 cm at Kingaroy. From each tree, 1.5 m long billets were removed at two positions: a butt billet from 0.3–1.8 m above ground and a top billet from approximately 5.5–7.0 m. Log end splitting was assessed 24 hours after harvesting and again after steaming, approximately four days after harvesting. Disks from just above both billets were collected for assessment of wood properties. Billets were peeled on a spindleless veneer lathe to produce a full veneer ribbon with a target green thickness of 2.8 to 3.0 mm. The 1.55 m wide (tangential dimension) veneer sheets were dried and graded according to AS/NZ Standard 2269:2008, which describes four veneer grades. Veneer samples taken along the length of the veneer ribbon, at regular intervals of 1.55 m, were tested for stiffness, shrinkage and density. Veneer length measurements were used to calculate the radial distance of each sample from the central axis of the billet. Overall veneer gross recoveries ranged from 50% to 70%. They were significantly lower at the Kingaroy site, for both species. The veneer recoveries achieved were 2–3 times higher than typical green off saw recoveries from small plantation hardwood logs of similar diameter. Most of the veneer recovered was classified as D-grade. CCV trees from the Ellangowan site yielded up to 38% of the better C-grade and higher grade veneers. The main limiting factors that prevented veneer from meeting higher grades were the presence of kino defects and encased knots. Splits in E. dunnii veneer also contributed to reduced grade quality. Log end splits were higher for E. dunnii than for CCV, and logs from Ellangowan exhibited more severe splitting. Split index was generally higher for top than for butt billets. Split index was strongly correlated with the average veneer grade from corresponding billets. The Ellangowan site, where rainfall was higher and trees grew faster, yielded significantly denser and stiffer veneers than did the drier sites near Kingaroy, where tree growth was slower. The difference was more pronounced for E. dunnii than for CCV. Differences in measured wood properties between thinned and unthinned treatments were generally small and not significant. On average, 10% of billet volume was lost during the peeling rounding-up process. Much of the wood laid down following thinning was removed during rounding-up, meaning the effect of thinning on veneer properties could not be effectively assessed. CCV was confirmed as having high veneer density and very good veneer stiffness, exceeding 15 GPa, making it very suitable for structural products. E. dunnii also demonstrated good potential as a useful structural plywood resource, achieving stiffness above 10 GPa. Veneer stiffness and density in CCV increased from pith to bark at both sites, while for E. dunnii there was a radial increase in these properties at the Ellangowan site only. At the drier Kingaroy site, veneer stiffness and density declined from mid-radius to the log periphery. This may be associated with prolonged drought from 2005 to 2009, corresponding to the later years of tree growth at the Kingaroy site. CCV appeared to be less sensitive to drought conditions. Standing tree acoustic velocity, determined by the Fakopp time-of-flight method, provided a reliable prediction of average veneer stiffness for both species (R2=0.78 for CCV and R2=0.90 for E. dunnii) suggesting that the Fakopp method may be a useful indicator of tree and stand quality, in terms of veneer stiffness in standing trees.

Association genetics in Corymbia citriodora subsp variegata identifies single nucleotide polymorphisms affecting wood growth and cellulosic pulp yield

Wood is an important biological resource which contributes to nutrient and hydrology cycles through ecosystems, and provides structural support at the plant level. Thousands of genes are involved in wood development, yet their effects on phenotype are not well understood. We have exploited the low genomic linkage disequilibrium (LD) and abundant phenotypic variation of forest trees to explore allelic diversity underlying wood traits in an association study. Candidate gene allelic diversity was modelled against quantitative variation to identify SNPs influencing wood properties, growth and disease resistance across three populations of Corymbia citriodora subsp. variegata, a forest tree of eastern Australia. Nine single nucleotide polymorphism (SNP) associations from six genes were identified in a discovery population (833 individuals). Associations were subsequently tested in two smaller populations (130160 individuals), validating our findings in three cases for actin 7 (ACT7) and COP1 interacting protein 7 (CIP7). The results imply a functional role for these genes in mediating wood chemical composition and growth, respectively. A flip in the effect of ACT7 on pulp yield between populations suggests gene by environment interactions are at play. Existing evidence of gene function lends strength to the observed associations, and in the case of CIP7 supports a role in cortical photosynthesis.

Genetic control of flowering in spotted gum, Corymbia citriodora subsp. variegata and C. maculata

Genetically controlled asynchrony in anthesis is an effective barrier to gene flow between planted and native forests. We investigated the degree of genetically controlled variation in the timing of key floral developmental stages in a major plantation species in subtropical Australia, Corymbia citriodora subsp. variegata K.D. Hill and L.A.S Johnson, and its relative C. maculata K.D. Hill and L.A.S. Johnson. Flowering observations were made in a common garden planting at Bonalbo in northern New South Wales in spring on 1855 trees from eight regions over three consecutive years, and monthly on a subset of 208 trees for 12 months. Peak anthesis time was stable over years and observations from translocated trees tended to be congruent with the observations in native stands, suggesting strong genetic control of anthesis time. A cluster of early flowering provenances was identified from the north-east of the Great Dividing Range. The recognition of a distinct flowering race from this region accorded well with earlier evidence of adaptive differentiation of populations from this region and geographically-structured genetic groupings in C. citriodora subsp. variegata. The early flowering northern race was more fecund, probably associated with its disease tolerance and greater vigour. Bud abundance fluctuated extensively at the regional level across 3 years suggesting bud abundance was more environmentally labile than timing of anthesis. Overall the level of flowering in the planted stand (age 12 years) was low (8–12% of assessed trees with open flowers), and was far lower than in nearby native stands. Low levels of flowering and asynchrony in peak anthesis between flowering races of C. citriodora subsp. variegata may partially mitigate a high likelihood of gene flow where the northern race is planted in the south of the species range neighbouring native stands

Genetic variation in growth and wood-quality traits of Corymbia citriodora subsp. variegata across three sites in south-east Queensland, Australia

Ten growth or wood-quality traits were assessed in three nearby Corymbia citriodora subsp. variegata (CCV) open-pollinated family-within-provenance trials (18 provenances represented by a total of 374 families) to provide information for the development of a breeding program targeting both pulp and solid-wood products. Growth traits (diameter at breast high over bark [DBH], height and conical volume) were assessed at 3 and 7 years of age. Wood-quality traits (density [DEN], Kraft pulp yield [KPY], modulus of elasticity [MoE] and microfibril angle [MfA]) were predicted using near-infrared spectroscopy on wood samples collected from these trials when aged between 10 and 12 years. The high average KPY, DEN and MoE, and low average MfA observed indicates CCV is very suitable for both pulp and timber products. All traits were under moderate to strong genetic control. In across- trials analyses, high (>0.4) heritability estimates were observed for height, DEN, MoE and MfA, while moderate heritability estimates (0.24 to 0.34) were observed for DBH, volume and KPY. Most traits showed very low levels of genotype × site interaction. Estimated age–age genetic correlations for growth traits were strong at both the family (0.97) and provenance (0.99) levels. Relationships among traits (additive genetic correlation estimates) were favourable, with strong and positive estimates between growth traits (0.84 to 0.98), moderate and positive values between growth and wood-quality traits (0.32 to 0.68), moderate and positive between KPY and MoE (0.64), and high and positive between DEN and MoE (0.82). However, negative (but favourable) correlations were detected between MfA and all other evaluated traits (−0.31 to −0.96). The genetic correlation between the same trait expressed on two different sites, at family level, ranged from 0.24 to 0.42 for growth traits, and from 0.29 to 0.53 for wood traits. Therefore simultaneous genetic improvement of growth and wood property traits in CCV for the target environment in south-east Queensland should be possible, given the moderate to high estimates of heritability and favourable correlations amongst all traits studied, unless genotype × site interactions are greater than was evident. © 2016 NISC (Pty) Ltd

The risk of pollen-mediated gene flow from exotic Corymbia plantations into native Corymbia populations in Australia

Sectors of the forest plantation industry in Australia are set to expand in the near future using species or hybrids of the spotted gums (Corymbia, Section Politaria). Plantations of these taxa have already been introduced across temperate and subtropical Australia, representing locally exotic introductions from native stands in Queensland and New South Wales. A literature review was undertaken to provide insights into the potential for pollen-mediated gene flow from these plantations into native populations. Three factors suggest that such gene flow is likely; (1) interspecific hybridisation within the genus has frequently been recorded, including between distantly related species from different sections, (2) apparent high levels of vertebrate pollinator activity may result in plantation pollen being moved over hundreds of kilometres, (3) much of the plantation estate is being established among closely related taxa and therefore few barriers to gene flow are expected. Across Australia, 20 of the 100 native Corymbia taxa were found to have regional level co-occurrence with plantations. These were located most notably within regions of north-east New South Wales and south-east Queensland, however, co-occurrence was also found in south-west Western Australia and eastern Victoria. The native species found to have co-occurrence were then assessed for the presence of reproductive barriers at each step in the process of gene flow that may reduce the number of species at risk even further. The available data suggest three risk categories exist for Corymbia. The highest risk was for gene flow from plantations of spotted gums to native populations of spotted gums. This was based on the expected limited existence of pre- and post-zygotic barriers, substantial long-distance pollen dispersal and an apparent broad period of flowering in Corymbia citriodora subsp. variegata plantations. The following risk category focussed on gene flow from Corymbia torelliana × C. c. variegata hybrid plantations into native C. c. variegata, as the barriers associated with the production and establishment of F1 hybrids have been circumvented. For the lowest risk category, Corymbia plantations may present a risk to other non-spotted gum species, however, further investigation of the particular cross-combinations is required. A list of research directions is provided to better quantify these risks. Empirical data will need to be combined within a risk assessment framework that will not only estimate the likelihood of exotic gene flow, but also consider the conservation status/value of the native populations. In addition, the potential impacts of pollen flow from plantations will need to be weighed up against their various economic and environmental benefits.

Construction of microsatellite linkage maps for Corymbia

The genus Corymbia is closely related to the genus Eucalyptus, and like Eucalyptus contains tree species that are important for sub-tropical forestry. Corymbia's close relationship with Eucalyptus suggests genetic studies in Corymbia should benefit from transfer of genetic information from its more intensively studied relatives. Here we report a genetic map for Corymbia spp. based on microsatellite markers identified de novo in Corymbia sp or transferred from Eucalyptus. A framework consensus map was generated from an outbred F 2 population (n = 90) created by crossing two unrelated Corymbia torelliana x C. citriodora subsp. variegata F1 trees. The map had a total length of 367 cM (Kosambi) and was composed of 46 microsatellite markers distributed across 13 linkage groups (LOD 3). A high proportion of Eucalyptus microsatellites (90%) transferred to Corymbia. Comparative analysis between the Corymbia map and a published Eucalyptus map identified eight homeologous linkage groups in Corymbia with 13 markers mapping on one or both maps. Further comparative analysis was limited by low power to detect linkage due to low genome coverage in Corymbia, however, there was no convincing evidence for chromosomal structural differences because instances of non-synteny were associated with large distances on the Eucalyptus map. Segregation distortion was primarily restricted to a single linkage group and due to a deficit of hybrid genotypes, suggesting that hybrid inviability was one factor shaping the genetic composition of the F2 population in this inter-subgeneric hybrid. The conservation of microsatellite loci and synteny between Corymbia and Eucalyptus suggests there will be substantial value in exchanging information between the two groups.

Genetic variation within two sympatric spotted gum eucalypts exceeds between taxa variation

Population substructure and hybridization, among other factors, have the potential to cause erroneous associations in linkage disequilibrium (LD) mapping. Two closely related spotted gum eucalypts, Corymbia variegata and C. henryi (Myrtaceae) occur in sympatry in the east coast of Australia and potentially interbreed. They are morphologically similar but are distinguished as separate species based on capsule and foliage size. To determine whether they hybridize in nature and its implications for LD mapping, we investigated the level of molecular divergence between the two species at two sympatric locations separated by 300 kilometres. Very few individuals of intermediate morphology were identified, despite the two species occurring only metres apart. Analysis of genetic structure using 12 microsatellite loci showed that genetic differentiation between populations of the same species at different locations (FST = 0.07 for both species; p = 0.0001) was significantly higher than that observed between species at each location (mean FST = 0.02 and 0.04 for Cherry tree and Bunyaville respectively; p = 0.0001; all Mann-Whitney U-test p ≤ 0.01). No species-specific alleles or significant allele frequency differences were detected within a site, suggesting recurrent local gene flow between the two species. The lack of significant allele frequency differences implies no population stratification along taxonomic lines. This suggested that there is little concern for cryptic hybridization when sampling from sites of sympatry for LD mapping.

Determination of specific leaf area of some commercially useful sub-tropical hardwood species

Variability of specific leaf area (SLA) across taxa, sites and crown zones was determined for four sub-tropical hardwood species, Eucalyptus grandis, E. cloeziana, E. argophloia and Corymbia citriodora ssp. variegata, growing in south-eastern Queensland. Mean SLA values were stable amongst those taxa sampled on dry sites but varied markedly between provenances of E. grandis on a moist site. Mean SLA did not vary significantly with crown zone in any of these four sub-tropical eucalypts, which is in contrast to that observed in temperate species, both in Australia and overseas. A provenance of E. cloeziana from a moist coastal site exhibited the largest SLA of all taxa studied.

Paropsine beetles (Coleoptera: Chrysomelidae) in south-eastern Queensland hardwood plantations: identifying potential pest species

Paropsine chrysomelid beetles are significant defoliators of Australian eucalypts. In Queensland, the relatively recent expansion of hardwood plantations has resulted in the emergence of new pest species. Here I identify paropsine beetles collected from Eucalyptus cloeziana Muell. and E. dunnii Maiden, two of the major Eucalyptus species grown in plantations in south-eastern Queensland, and estimate the relative abundance of each paropsine species. Although I was unable to identify all taxa to species level, at least 17 paropsine species were collected, about one-third of which have not been previously associated with hardwood plantations. Paropsis atomaria Olivier and P. charybdis Stål were the most abundant species collected in E. cloeziana plantations, while Chrysophtharta cloelia (Stål) and P. atomaria were most commonly collected from E. dunnii. Occasional collections from Corymbia citriodora (Hook.) Hill and Johns, ssp. variegata revealed an additional four species implicated in plantation damage. Abundance and voltinism varied between species and sites. I predict which paropsine species are likely to threaten plantation productivity.

In vitro propagation of Corymbia torelliana x C. citriodora (Myrtaceae) via cytokinin-free node culture

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively to establish forestry plantations in subtropical Australia. Methods were developed for in vitro seed germination, shoot multiplication and plantlet formation that could be used to establish in vitro and ex vitro clone banks of juvenile Corymbia hybrids. Effects of sodium hypochlorite concentration and exposure time on seed contamination and germination, and effects of cytokinin and auxin concentrations on shoot multiplication and subsequent rooting, were assessed. A two-step surface sterilisation procedure, involving 70% ethanol followed by 1% sodium hypochlorite, provided almost no contamination and at least 88% germination. A novel method of cytokinin-free node culture proved most effective for in vitro propagation. Lateral bud break of primary shoots was difficult to induce by using cytokinin, but primary shoots rooted prolifically, elongated rapidly and produced multiple nodes in the absence of exogenous cytokinin. Further multiplication was obtained either by elongating lateral shoots of nodal explants in cytokinin-free medium or by inducing organogenic callus and axillary shoot proliferation with 2.2 µm benzyladenine. Plantlets were produced using an in vitro soil-less method that provided extensive rooting in sterile propagation mixture. These methods provide a means for simultaneous laboratory storage and field-testing of clones before selection and multiplication of desired genotypes.