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The evolutionary trajectory of a population both influences and is influenced by characteristics of its genome. A disjunct population, for example is likely to exhibit genomic features distinct from those of continuous populations, reflecting its specific evolutionary history and influencing future recombination outcomes. We examined genetic diversity, population differentiation and linkage disequilibrium (LD) across the highly disjunct native range of the Australian forest tree Eucalyptus globulus, using 203,337 SNPs genotyped in 136 trees spanning seven races. We found support for four broad genetic groups, with moderate FST, high allelic diversity and genome-wide LD decaying to an r2 of 0.2 within 4 kb on average. These results are broadly similar to those reported previously in Eucalyptus species and support the ‘ring’ model of migration proposed for E. globulus. However, two of the races (Otways and South-eastern Tasmania) exhibited a much slower decay of LD with physical distance than the others and were also the most differentiated and least diverse, which may reflect the effects of selective sweeps and/or genetic bottlenecks experienced in their evolutionary history. We also show that FST and rates of LD vary within and between chromosomes across all races, suggestive of recombination outcomes influenced by genomic features, hybridization or selection. The results obtained from studying this species serve to illustrate the genomic effects of population disjunction and further contribute to the characterisation of genomes of woody genera.

Eucalyptus perriniana F.Muell. ex Rodway is distributed over a wide geographic range in south-eastern Australia as a series of small and isolated populations. In Tasmania, there are only three known populations that are separated by 511 km from the closest population on mainland Australia, which is one of the largest disjunctions observed for any eucalypt species. This project utilised eight nuclear microsatellites and one chloroplast DNA marker to study the genetic diversity in E. perriniana and determine the affinities between the populations. In all, 302 individuals in nine populations across thewhole range of the specieswere sampled. The overall nuclear microsatellite diversity in E. perriniana (Ht =0.85), as well as the diversity in each population (He =0.73), was comparable to that found in widespread eucalypt species that have much larger population sizes. The microsatellites revealed that the isolated Tasmanian populations were significantly differentiated from mainland populations (FST between regions=0.08), although the mainland Baw Baw population was the most differentiated. Most populations harboured different chloroplast DNA haplotypes, but in general, there were more mutational differences among haplotypes found in Tasmania than between Tasmanian and mainland populations. The Tasmanian populations often shared chloroplast DNA haplotypes with other eucalypts from south-eastern Tasmania. In conclusion, the populations of E. perriniana are genetically variable and significantly differentiated, with geographic separation being a poor predictor of the amount of genetic divergence. The most divergent populations are those in Tasmania and on Mt Baw Baw and their conservation is important to capture the genetic diversity in the species.

F1 hybrids between exotic Eucalyptus nitens plantations and native E. ovata have previously been reported among seedlings grown from open-pollinated seed collected from E. ovata, on the island of Tasmania. Such exotic hybrid seedlings have now been found in the wild adjacent to plantations at three locations. The proportion of exotic hybrids in open-pollinated seed collected from nearby mature E. ovata was 5.5%. This level compares with only 0.4% for natural hybrids between native species at these sites (E. ovata, E. viminalis and E. rodwayi). Detection of hybrids was initially based on their deviant morphology, which was generally intermediate between parental species. This subjective classification was then successfully verified by morphometric and allozyme analyses. Pure E. nitens seedlings (wildlings) were restricted to within 30 m of these plantations, whereas established hybrids were found up to 310 m from the plantations. This pattern of establishment reflects dispersal of exotic seed and pollen respectively. It is likely that the recent expansion of the eucalypt plantation estate in Australia will cause an increase in the frequency of exotic hybrids. However, the long-term impact of such hybridisation is yet to be assessed.

The contamination of native eucalypt gene pools via exotic pollen is of concern as: (i) pollen dispersal is believed to be much more widespread than seed dispersal; (ii) reproductive barriers are often weak between closely related species; (iii) European settlement has already had a major impact on Australia’s eucalypt woodlands and mallee; (iv) there has been a rapid expansion of eucalypt plantations and restoration planting’s in Australia; and (v) Australia is the custodian of an internationally important genetic resource. The risks associated with the introduction of non-native species, provenances, and hybrids include direct effects on the gene pool through genetic pollution as well as indirect effects on dependent biodiversity. In many cases the risk of genetic pollution will be small due to strong barriers to hybridisation between distantly related species, differences in flowering time or poor fitness of hybrids. There is no risk of hybridisation between species from the different major eucalypt genera/subgenera (eg. symphyomyrts, monocalypts, eudesmids, bloodwoods and angophora). The main plantation species are symphyomyrts and within this subgenus, the probability of successful hybridisation generally decreases with increasing taxonomic distance between species. The planting of non-local provenances or improved material within the range of native populations has the potential to genetically impact on local gene pools to varying degrees, indicating the requirement for the adoption of management strategies to reduce this risk.. Pollen flow between plantation and native eucalypt species has already been reported and implementation of strategies to minimise the risk and consequences of genetic pollution is important if Australian forestry is to be certified as sustainable. Naturally small or remnant populations are at particular risk. A framework for assessment of the risk of genetic pollution is developed herein.

Tasmania is a natural laboratory for investigating the evolutionary processes of the Quaternary. It is a large island lying 40-44 degrees S, which was repeatedly glaciated and linked to southeastern continental Australia during the Quaternary. Climate change promoted both the isolation of species in glacial refugia, and an exchange between Tasmanian and mainland floras. Eucalyptus is a complex and diverse genus, which has increased in abundance in Australia over the past 100 kyr, probably in response to higher fire frequency. Morphological evidence suggests that gene flow may have occurred between many eucalypt species after changes in their distribution during the Quaternary. This paper summarizes recent genetic evidence for migration and introgressive hybridization in Tasmanian Eucalyptus. Maternally inherited chloroplast DNA reveals a long-term persistence of eucalypts in southeastern Tasmanian refugia, coupled with introgressive hybridization involving many species. Detailed analysis of the widespread species Eucalyptus globulus suggests that migration from mainland Australia was followed by introgression involving a rare Tasmanian endemic. The data support the hypothesis that changes in distribution of interfertile species during the Quaternary have promoted reticulate evolution in Eucalyptus.

Nothofagus cunninghamii (myrtle) has a widespread distribution through the temperate rainforest of southeastern Australia with some disjunct populations existing in putative glacial refugia. Polymorphic nuclear markers are required to resolve the biogeographical history of the species and will also be useful for conservation and forestry applications in this and other species of Nothofagus. Fourteen polymorphic microsatellite loci were isolated from N. cunninghamii, with four to 10 alleles amplified in 15 individuals tested. Transferability to other species of Nothofagus was successful, with six loci transferring to all eight other species tested.

With the Australian national Eucalyptus globulus breeding program entering its third generation, monitoring of genetic diversity is becoming increasingly important due to selection having the potential to reduce genetic diversity over time. To provide a baseline estimate of genetic diversity within the Australian national E. globulus breeding program, microsatellite markers were used to quantify and compare the genetic diversity of 140 first-generation selections to that observed in a sample of 340 mature individuals collected from throughout the native distribution of the species. While expected heterozygosity was high in the breeding population sample, and similar to that displayed in the native sample, allelic richness was lower, suggesting a loss of rare alleles during selection. Observed heterozygosity (individual heterozygosity), however, was consistently higher in breeding population samples than that seen in native samples, suggesting that heterozygotes have been preferentially selected within the program. The importance of maintaining genetic diversity in breeding programs and the benefit of conserving native populations as a resource of genetic diversity is discussed in relation to the long-term management of tree breeding programs.

F1 hybrids between exotic Eucalyptus nitens plantations and native E. ovata have previously been reported among seedlings grown from open-pollinated seed collected from E. ovata, on the island of Tasmania. Such exotic hybrid seedlings have now been found in the wild adjacent to plantations at three locations. The proportion of exotic hybrids in open-pollinated seed collected from nearby mature E. ovata was 5.5%. This level compares with only 0.4% for natural hybrids between native species at these sites (E. ovata, E. viminalis and E. rodwayi). Detection of hybrids was initially based on their deviant morphology, which was generally intermediate between parental species. This subjective classification was then successfully verified by morphometric and allozyme analyses. Pure E. nitens seedlings (wildlings) were restricted to within 30 m of these plantations, whereas established hybrids were found up to 310 m from the plantations. This pattern of establishment reflects dispersal of exotic seed and pollen respectively. It is likely that the recent expansion of the eucalypt plantation estate in Australia will cause an increase in the frequency of exotic hybrids. However, the long-term impact of such hybridisation is yet to be assessed.

Fungi of the genus Mycosphaerella is one of the major leaf disease of Eucalyptus globulus worldwide. The main species that infect E. globulus in southern Australia are M. cryptica and M. nubilosa. M. nubilosa is mainly confined to the juvenile foliage whereas M. cryptica may occur on both foliage types. Mycosphaerella damage to E. globulus plantations can be severe and the risk of disease damage is one of the main reasons for the shift towards planting the more resistant E. nitens in northern Tasmania. We examined the quantitative genetic variation in susceptibility to infection by M. nubilosa in a genetically diverse population of E. globulus families growing in a field trial in north-west Tasmania. The trees were two years old and still entirely in the juvenile foliage stage when a heavy epidemic of M. nubilosa occurred. Disease incidence was uniform across the trial and the mean leaf area damage (severity assessed as % of necrotic of lost leaves on whole tree basis) was very high at 34%. Significant genetic variation for susceptibility was detected with a narrow-sense heritability of disease severity of 0.6 being the highest yet reported for a Mycosphaerella disease of eucalypts. We followed the effects of this disease outbreak on growth up to age 7 years and found that M. nubilosa damage had a significantly deleterious impact on tree growth at both the phenotypic and genetic level. At age 7, the top 10% of families had a mean DBH 20.8% greater than the trial mean. Approximately half of this gain would have been achieved by early selection for disease resistance (9.1%) or height (11.0%) at age 2, with a time advantage of 5 years. This is similarly the case for selection of above average families. It is likely such gains would be reduced in homogenous plantings of resistant genotypes, or if genotype x environment interactions are significant. Nevertheless, a large component of this gain is likely to be due to disease resistance per se, and collection of seed from

Variation in the chloroplast genome of 44 accessions representing 14 Eucalyptus L'Hér. species from the series Viminales (sensu Pryor and Johnson 1971) was investigated. Southern analysis of the chloroplast genomes restricted with 12 enzymes revealed 20 restriction-site polymorphisms of which 7 were autapomorphic to individual trees. The 13 informative restriction-site polymorphisms were distributed between individuals of different species, but none was species-specific. Fourteen chloroplast haplotypes were identified for south-eastern Australian individuals. In endemic Tasmanian species, five haplotypes were identified. Chloroplast haplotypes appear to have a mosaic distribution in south-eastern Australia, more closely associated with geographical regions than with morphological species boundaries. The biogeographic distribution of chloroplast haplotypes may be explained by a combination of interspecific hybridisation and introgression, and convergent evolution. The lack of species-specificity of cpDNA variation indicates that, although cpDNA is not appropriate for species-level phylogeny analysis in the series Viminales, it may provide useful information in studies of biogeography and gene flow in Eucalyptus.

This expanded survey of ITS sequences represents the largest analysis of molecular data ever attempted on Eucalyptus. Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were included in an analysis of 90 species of Eucalyptus s.s. and 28 species representing eight other genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, Eucalyptopsis, Stockwellia, Lophostemon and Metrosideros). The results of the study indicate that Angophora and Corymbia form a well-supported clade that is highly differentiated from Eucalyptus s.s. Corymbia species are divided between two clades, one of which may be the sister to Angophora. Allosyncarpia, Arillastrum, Eucalyptopsis and 'Stockwellia' are also highly differentiated from Eucalyptus s.s. If the genus Eucalyptus is to be expanded to include Angophora and Corymbia (sensu Brooker 2000), ITS data suggest that Allosyncarpia, Eucalyptopsis, 'Stockwellia' and potentially Arillastrum should also be included in Eucalyptus s.l. The ITS data suggest that subg. Symphyomyrtus is paraphyletic and that subg. Minutifructus should be included within it. Within subg. Symphyomyrtus, only sect. Maidenaria appears to be monophyletic. Sections Adnataria and Dumaria are probably monophyletic; sections Exsertaria and Latoangulatae are very close and probably should be combined in a single section. Section Bisectae is polyphyletic and is divided into two distinct lineages. The phylogenetic groups depicted by ITS data are consistent with the frequency of natural inter-specific hybridisations as well as data from controlled crosses within subgenus Symphyomyrtus. The ITS data illustrate that subg. Idiogenes and western Australian monocalypts are early evolutionary lines relative to E. diverstfolia, E. rubiginosa (monotypic subg. Primitiva) and the eastern monocalypts and that subg. Primitiva should be sunk into subg. Eucalyptus. Subgenus Eudesmia may be monophyletic, grouping with subgenera Idiogenes and Eucalyptus. Furth

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.

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.

Eucalyptus globulus Labill. is native to southeastern Australia and is the most important temperate hardwood plantation species in the world. It consists of four subspecies that are morphologically and geographically distinct but that are linked by morphologically and geographically intermediate populations. The Jeeralang provenance, an intermediate population from southeastern Victoria, is an important source of seed for plantations and genetic material for breeding programs because of its superior growth and wood density. To determine the genetic affinities of this provenance, 154 trees from three subspecies and the Jeeralang provenance were sampled. Analysis of 12 morphological characters verified that the Jeeralang provenance is intermediate between subspecies bicostata (Maiden, Blakely, & J. Simm.) Kirkpatr., globulus and pseudoglobulus (Naudin ex Maiden) Kirkpatr., with individuals having closest affinities to either ssp. globulus or ssp. bicostata. However, eight microsatellite loci showed that the Jeeralang provenance has greater affinities to Victorian ssp. globulus to which it is geographically closest. In contrast, Tasmanian and Victorian ssp. globulus are morphologically similar yet appear to be distinct at the molecular level. This study emphasizes the importance of using traits that are unlikely to be influenced by selection when determining the origin and affinities of populations.

Eucalyptus morrisbyi is an endangered eucalypt, restricted to four populations on the island of Tasmania. The two main populations are separated by 20 km, occurring in the Risdon Hills and on Calverts Hill, and differ markedly in size and health. Although they are both in reserves, the small population at Risdon Hills has experienced a marked decline in the last two decades. The other two populations (Lumeah Point and Honeywood Drive) are very small and under threat because of urbanisation. They are close to the large Calverts Hill population and may be remnants of a once larger population in this area prior to clearing for agriculture in the 19th century. A hypervariable chloroplast marker and six nuclear microsatellites, used to quantify genetic diversity among and within populations, indicated marked genetic differences between the two main populations (Risdon and Calverts Hills), with virtually no sharing of chloroplast haplotypes and little sharing of microsatellite alleles among populations. Both of the main populations are clearly required to adequately conserve the genetic diversity in this species, whereas the Lumeah Point and Honeywood Drive populations are similar to the proximal Calverts Hill population. The two main populations showed equally high levels of genetic diversity (average HE = 0.69) in the adult trees, using microsatellites, and little difference in inbreeding levels despite the large difference in population size. Analyses of 366 offspring from 9-11 trees from each main population revealed high outcrossing rates, little biparental inbreeding and high genetic diversity (average HE = 0.65) in both seedling populations. This indicates that open-pollinated seed collections from these populations capture sufficient genetic diversity for ex situ conservation plantings. It is argued that the high genetic diversity maintained in the small Risdon Hills population is due to a combination of the longevity of its genotypes (possibly up to 1155-1523 yea

With the Australian national Eucalyptus globulus breeding program entering its third generation, monitoring of genetic diversity is becoming increasingly important due to selection having the potential to reduce genetic diversity over time. To provide a baseline estimate of genetic diversity within the Australian national E. globulus breeding program, microsatellite markers were used to quantify and compare the genetic diversity of 140 first-generation selections to that observed in a sample of 340 mature individuals collected from throughout the native distribution of the species. While expected heterozygosity was high in the breeding population sample, and similar to that displayed in the native sample, allelic richness was lower, suggesting a loss of rare alleles during selection. Observed heterozygosity (individual heterozygosity), however, was consistently higher in breeding population samples than that seen in native samples, suggesting that heterozygotes have been preferentially selected within the program. The importance of maintaining genetic diversity in breeding programs and the benefit of conserving native populations as a resource of genetic diversity is discussed in relation to the long-term management of tree breeding programs.

The mating system of the animal-pollinated forest tree species Eucalyptus globulus Labill. was investigated in a clonal seed orchard to determine the importance of self-incompatibility, floral timing, floral abundance and canopy position on explaining variation in outcrossing rates. Outcrossing rates were highly and significantly correlated with the degree of self-incompatibility of individual genotypes (R2 = 0.92). Outcrossing rates were also significantly higher in the upper than in the lower parts of tree canopies (0.78 ± 0.1 and 0.54 ± 0.1, respectively). In contrast, neither flowering time nor abundance of flowers on individual trees revealed consistent effects upon outcrossing rates, suggesting that these factors are either less important or inherently more unpredictable because their effects partly depend on complex interactions with animal pollinators. Screening seed orchards in order to identify trees with high self-incompatibility and restricting seed collection to these trees will ensure that a high proportion of the seed used for plantations is derived from outcrossing, thereby minimising the deleterious effects of inbreeding depression.

• Formylated phloroglucinols (FPCs) are key defensive compounds that influence herbivory by mammals and arthropods in eucalypts. However, the genetic architecture underlying variation in their levels remains poorly understood. • Quantitative trait loci (QTL) analysis for the concentrations of two major FPCs, sideroxylonal A and macrocarpal G, was conducted using juvenile leaves from 112 clonally duplicated progenies from an outcross F2 of Eucalyptus globulus. • Two unlinked QTL were located for macrocarpal, while another unlinked QTL was located for sideroxylonal. The sideroxylonal QTL collocated with one for total sideroxylonal previously reported using adult Eucalyptus nitens foliage, providing independent validation in a different evolutionary lineage and a different ontogenetic stage. • Given the potential widespread occurrence of these QTL, their ontogenetic stability, and their impact on a range of dependent herbivores, it is possible that they have extended phenotypic effects in the Australian forest landscape.

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were obtained for 35 species of Eucalyptus s.s. and seven taxa representing five outgroup genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, and Stockwellia). The sequences were analyzed cladistically. The data distinguished clearly between the two major subgenera of Eucalyptus s.s. (Symphyomyrtus and Monocalyptus) but indicated that subgenus Eudesmia may be paraphyletic. ITS sequence data demonstrated the potential to resolve relationships between sections within subgenus Symphyomyrtus. Within sections, however, taxa were poorly differentiated. At the generic level, Corymbia appeared to be paraphyletic due to the exclusion of Angophora. The positions of Allosyncarpia and Arillastrum relative to the ingroup remain unresolved. ITS sequence data may prove valuable for resolving other phylogenetic relationships at higher taxonomic levels within Eucalyptus.

Eucalyptus globulus Labill. is native to southeastern Australia and is the most important temperate hardwood plantation species in the world. It consists of four subspecies that are morphologically and geographically distinct but that are linked by morphologically and geographically intermediate populations. The Jeeralang provenance, an intermediate population from southeastern Victoria, is an important source of seed for plantations and genetic material for breeding programs because of its superior growth and wood density. To determine the genetic affinities of this provenance, 154 trees from three subspecies and the Jeeralang provenance were sampled. Analysis of 12 morphological characters verified that the Jeeralang provenance is intermediate between subspecies bicostata (Maiden, Blakely, & J. Simm.) Kirkpatr., globulus and pseudoglobulus (Naudin ex Maiden) Kirkpatr., with individuals having closest affinities to either ssp. globulus or ssp. bicostata. However, eight microsatellite loci showed that the Jeeralang provenance has greater affinities to Victorian ssp. globulus to which it is geographically closest. In contrast, Tasmanian and Victorian ssp. globulus are morphologically similar yet appear to be distinct at the molecular level. This study emphasizes the importance of using traits that are unlikely to be influenced by selection when determining the origin and affinities of populations.

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.

F1 hybrids between exotic Eucalyptus nitens plantations and native E. ovata have previously been reported among seedlings grown from open-pollinated seed collected from E. ovata, on the island of Tasmania. Such exotic hybrid seedlings have now been found in the wild adjacent to plantations at three locations. The proportion of exotic hybrids in open-pollinated seed collected from nearby mature E. ovata was 5.5%. This level compares with only 0.4% for natural hybrids between native species at these sites (E. ovata, E. viminalis and E. rodwayi). Detection of hybrids was initially based on their deviant morphology, which was generally intermediate between parental species. This subjective classification was then successfully verified by morphometric and allozyme analyses. Pure E. nitens seedlings (wildlings) were restricted to within 30 m of these plantations, whereas established hybrids were found up to 310 m from the plantations. This pattern of establishment reflects dispersal of exotic seed and pollen respectively. It is likely that the recent expansion of the eucalypt plantation estate in Australia will cause an increase in the frequency of exotic hybrids. However, the long-term impact of such hybridisation is yet to be assessed.

Eucalyptus morrisbyi is an endangered eucalypt, restricted to four populations on the island of Tasmania. The two main populations are separated by 20 km, occurring in the Risdon Hills and on Calverts Hill, and differ markedly in size and health. Although they are both in reserves, the small population at Risdon Hills has experienced a marked decline in the last two decades. The other two populations (Lumeah Point and Honeywood Drive) are very small and under threat because of urbanisation. They are close to the large Calverts Hill population and may be remnants of a once larger population in this area prior to clearing for agriculture in the 19th century. A hypervariable chloroplast marker and six nuclear microsatellites, used to quantify genetic diversity among and within populations, indicated marked genetic differences between the two main populations (Risdon and Calverts Hills), with virtually no sharing of chloroplast haplotypes and little sharing of microsatellite alleles among populations. Both of the main populations are clearly required to adequately conserve the genetic diversity in this species, whereas the Lumeah Point and Honeywood Drive populations are similar to the proximal Calverts Hill population. The two main populations showed equally high levels of genetic diversity (average HE = 0.69) in the adult trees, using microsatellites, and little difference in inbreeding levels despite the large difference in population size. Analyses of 366 offspring from 9-11 trees from each main population revealed high outcrossing rates, little biparental inbreeding and high genetic diversity (average HE = 0.65) in both seedling populations. This indicates that open-pollinated seed collections from these populations capture sufficient genetic diversity for ex situ conservation plantings. It is argued that the high genetic diversity maintained in the small Risdon Hills population is due to a combination of the longevity of its genotypes (possibly up to 1155-1523 yea

The mating system of the animal-pollinated forest tree species Eucalyptus globulus Labill. was investigated in a clonal seed orchard to determine the importance of self-incompatibility, floral timing, floral abundance and canopy position on explaining variation in outcrossing rates. Outcrossing rates were highly and significantly correlated with the degree of self-incompatibility of individual genotypes (R2 = 0.92). Outcrossing rates were also significantly higher in the upper than in the lower parts of tree canopies (0.78 ± 0.1 and 0.54 ± 0.1, respectively). In contrast, neither flowering time nor abundance of flowers on individual trees revealed consistent effects upon outcrossing rates, suggesting that these factors are either less important or inherently more unpredictable because their effects partly depend on complex interactions with animal pollinators. Screening seed orchards in order to identify trees with high self-incompatibility and restricting seed collection to these trees will ensure that a high proportion of the seed used for plantations is derived from outcrossing, thereby minimising the deleterious effects of inbreeding depression.

F1 hybrids between exotic Eucalyptus nitens plantations and native E. ovata have previously been reported among seedlings grown from open-pollinated seed collected from E. ovata, on the island of Tasmania. Such exotic hybrid seedlings have now been found in the wild adjacent to plantations at three locations. The proportion of exotic hybrids in open-pollinated seed collected from nearby mature E. ovata was 5.5%. This level compares with only 0.4% for natural hybrids between native species at these sites (E. ovata, E. viminalis and E. rodwayi). Detection of hybrids was initially based on their deviant morphology, which was generally intermediate between parental species. This subjective classification was then successfully verified by morphometric and allozyme analyses. Pure E. nitens seedlings (wildlings) were restricted to within 30 m of these plantations, whereas established hybrids were found up to 310 m from the plantations. This pattern of establishment reflects dispersal of exotic seed and pollen respectively. It is likely that the recent expansion of the eucalypt plantation estate in Australia will cause an increase in the frequency of exotic hybrids. However, the long-term impact of such hybridisation is yet to be assessed.

The contamination of native eucalypt gene pools via exotic pollen is of concern as: (i) pollen dispersal is believed to be much more widespread than seed dispersal; (ii) reproductive barriers are often weak between closely related species; (iii) European settlement has already had a major impact on Australia’s eucalypt woodlands and mallee; (iv) there has been a rapid expansion of eucalypt plantations and restoration planting’s in Australia; and (v) Australia is the custodian of an internationally important genetic resource. The risks associated with the introduction of non-native species, provenances, and hybrids include direct effects on the gene pool through genetic pollution as well as indirect effects on dependent biodiversity. In many cases the risk of genetic pollution will be small due to strong barriers to hybridisation between distantly related species, differences in flowering time or poor fitness of hybrids. There is no risk of hybridisation between species from the different major eucalypt genera/subgenera (eg. symphyomyrts, monocalypts, eudesmids, bloodwoods and angophora). The main plantation species are symphyomyrts and within this subgenus, the probability of successful hybridisation generally decreases with increasing taxonomic distance between species. The planting of non-local provenances or improved material within the range of native populations has the potential to genetically impact on local gene pools to varying degrees, indicating the requirement for the adoption of management strategies to reduce this risk.. Pollen flow between plantation and native eucalypt species has already been reported and implementation of strategies to minimise the risk and consequences of genetic pollution is important if Australian forestry is to be certified as sustainable. Naturally small or remnant populations are at particular risk. A framework for assessment of the risk of genetic pollution is developed herein.

Fungi of the genus Mycosphaerella is one of the major leaf disease of Eucalyptus globulus worldwide. The main species that infect E. globulus in southern Australia are M. cryptica and M. nubilosa. M. nubilosa is mainly confined to the juvenile foliage whereas M. cryptica may occur on both foliage types. Mycosphaerella damage to E. globulus plantations can be severe and the risk of disease damage is one of the main reasons for the shift towards planting the more resistant E. nitens in northern Tasmania. We examined the quantitative genetic variation in susceptibility to infection by M. nubilosa in a genetically diverse population of E. globulus families growing in a field trial in north-west Tasmania. The trees were two years old and still entirely in the juvenile foliage stage when a heavy epidemic of M. nubilosa occurred. Disease incidence was uniform across the trial and the mean leaf area damage (severity assessed as % of necrotic of lost leaves on whole tree basis) was very high at 34%. Significant genetic variation for susceptibility was detected with a narrow-sense heritability of disease severity of 0.6 being the highest yet reported for a Mycosphaerella disease of eucalypts. We followed the effects of this disease outbreak on growth up to age 7 years and found that M. nubilosa damage had a significantly deleterious impact on tree growth at both the phenotypic and genetic level. At age 7, the top 10% of families had a mean DBH 20.8% greater than the trial mean. Approximately half of this gain would have been achieved by early selection for disease resistance (9.1%) or height (11.0%) at age 2, with a time advantage of 5 years. This is similarly the case for selection of above average families. It is likely such gains would be reduced in homogenous plantings of resistant genotypes, or if genotype x environment interactions are significant. Nevertheless, a large component of this gain is likely to be due to disease resistance per se, and collection of seed from

With the Australian national Eucalyptus globulus breeding program entering its third generation, monitoring of genetic diversity is becoming increasingly important due to selection having the potential to reduce genetic diversity over time. To provide a baseline estimate of genetic diversity within the Australian national E. globulus breeding program, microsatellite markers were used to quantify and compare the genetic diversity of 140 first-generation selections to that observed in a sample of 340 mature individuals collected from throughout the native distribution of the species. While expected heterozygosity was high in the breeding population sample, and similar to that displayed in the native sample, allelic richness was lower, suggesting a loss of rare alleles during selection. Observed heterozygosity (individual heterozygosity), however, was consistently higher in breeding population samples than that seen in native samples, suggesting that heterozygotes have been preferentially selected within the program. The importance of maintaining genetic diversity in breeding programs and the benefit of conserving native populations as a resource of genetic diversity is discussed in relation to the long-term management of tree breeding programs.

Variation in the chloroplast genome of 44 accessions representing 14 Eucalyptus L'Hér. species from the series Viminales (sensu Pryor and Johnson 1971) was investigated. Southern analysis of the chloroplast genomes restricted with 12 enzymes revealed 20 restriction-site polymorphisms of which 7 were autapomorphic to individual trees. The 13 informative restriction-site polymorphisms were distributed between individuals of different species, but none was species-specific. Fourteen chloroplast haplotypes were identified for south-eastern Australian individuals. In endemic Tasmanian species, five haplotypes were identified. Chloroplast haplotypes appear to have a mosaic distribution in south-eastern Australia, more closely associated with geographical regions than with morphological species boundaries. The biogeographic distribution of chloroplast haplotypes may be explained by a combination of interspecific hybridisation and introgression, and convergent evolution. The lack of species-specificity of cpDNA variation indicates that, although cpDNA is not appropriate for species-level phylogeny analysis in the series Viminales, it may provide useful information in studies of biogeography and gene flow in Eucalyptus.

This study investigated the association between resistance of Eucalyptus globulus Labill. to autumn gum moth (Mnesempala privata Guenée) defoliation and cuticular wax compounds. In a field trial consisting of clonally replicated F2 families of E. globulus, situated in Tasmania, Australia, significant genetic variation in resistance was detected in two of three F2 families. The broad-sense heritability for defoliation within families ranged from 0.24 to 0.33. The 15 most resistant and the 15 most susceptible genotypes within each variable family were compared for their relative levels of 26 cuticular wax compounds. While no significant correlation between resistance and total wax yield estimates was found, significant differences were detected between resistant and susceptible classes in the relative quantities of several aliphatic phenylethyl and benzyl wax esters within both families. This association does not appear to be a response induced by defoliation. The broad-sense heritabilities of the variation in these compounds were high (0.82-0.94). Our findings suggest that these wax compounds are a mechanism of genetic resistance to autumn gum moth in E. globulus.

This expanded survey of ITS sequences represents the largest analysis of molecular data ever attempted on Eucalyptus. Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were included in an analysis of 90 species of Eucalyptus s.s. and 28 species representing eight other genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, Eucalyptopsis, Stockwellia, Lophostemon and Metrosideros). The results of the study indicate that Angophora and Corymbia form a well-supported clade that is highly differentiated from Eucalyptus s.s. Corymbia species are divided between two clades, one of which may be the sister to Angophora. Allosyncarpia, Arillastrum, Eucalyptopsis and 'Stockwellia' are also highly differentiated from Eucalyptus s.s. If the genus Eucalyptus is to be expanded to include Angophora and Corymbia (sensu Brooker 2000), ITS data suggest that Allosyncarpia, Eucalyptopsis, 'Stockwellia' and potentially Arillastrum should also be included in Eucalyptus s.l. The ITS data suggest that subg. Symphyomyrtus is paraphyletic and that subg. Minutifructus should be included within it. Within subg. Symphyomyrtus, only sect. Maidenaria appears to be monophyletic. Sections Adnataria and Dumaria are probably monophyletic; sections Exsertaria and Latoangulatae are very close and probably should be combined in a single section. Section Bisectae is polyphyletic and is divided into two distinct lineages. The phylogenetic groups depicted by ITS data are consistent with the frequency of natural inter-specific hybridisations as well as data from controlled crosses within subgenus Symphyomyrtus. The ITS data illustrate that subg. Idiogenes and western Australian monocalypts are early evolutionary lines relative to E. diverstfolia, E. rubiginosa (monotypic subg. Primitiva) and the eastern monocalypts and that subg. Primitiva should be sunk into subg. Eucalyptus. Subgenus Eudesmia may be monophyletic, grouping with subgenera Idiogenes and Eucalyptus. Furth

Four highly differentiated chloroplast DNA (cpDNA) lineages were identified in the forest tree species Eucalyptus globulus Labill. (Myrtaceae) in Australia using restriction site polymorphisms from Southern analysis. The cpDNA variation did not conform with ssp. boundaries, yet there was a strong geographical pattern to the distribution of the lineages. One lineage (C) was geographically central and widespread, whereas the other three lineages were found in peripheral populations: Western (W), Northern (N) and Southern (S). Thirteen haplotypes were detected in E. globulus, seven of which belonged to clade C. At least three of the cpDNA lineages (C, N and S) were shared extensively with other species. On the east coast of the island of Tasmania, there was a major north-south difference in cpDNA in the virtually continuous distribution of E. globulus. Northern populations harboured haplotypes from clade C while southeastern populations harboured a single haplotype from clade S. This difference was also reflected in several co-occurring endemic species. It is argued that the extensive cpDNA differentiation within E. globulus is likely to originate from interspecific hybridization and 'chloroplast capture' from different species in different parts of its range. Superficially, this hybridization is not evident in taxonomic traits; however, large-scale common garden experiments have revealed a steep cline in quantitative genetic variation that coincides with the haplotype transition in Tasmania. Our cpDNA results provide the strongest evidence to date that hybridization has had a widespread impact on a eucalypt species and indicate that reticulate evolution may be occurring on an unappreciated scale in Eucalyptus.

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were obtained for 35 species of Eucalyptus s.s. and seven taxa representing five outgroup genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, and Stockwellia). The sequences were analyzed cladistically. The data distinguished clearly between the two major subgenera of Eucalyptus s.s. (Symphyomyrtus and Monocalyptus) but indicated that subgenus Eudesmia may be paraphyletic. ITS sequence data demonstrated the potential to resolve relationships between sections within subgenus Symphyomyrtus. Within sections, however, taxa were poorly differentiated. At the generic level, Corymbia appeared to be paraphyletic due to the exclusion of Angophora. The positions of Allosyncarpia and Arillastrum relative to the ingroup remain unresolved. ITS sequence data may prove valuable for resolving other phylogenetic relationships at higher taxonomic levels within Eucalyptus.

Low copy number nuclear genes have been found to be useful for phylogenetic reconstruction at different taxonomic levels. This study investigated the utility of a single copy gene, cinnamoyl CoA reductase (CCR), for resolving phylogenetic relationships at the sectional level within Eucalyptus. The monophyly of sections Exsertaria and Latoangulatae was explored, using section Maidenaria as an outgroup, and the impact of intragenic recombination on phylogenetic reconstruction examined. Phylogenetic analysis did not resolve monophyletic groups. Latoangulatae and Maidenaria were polyphyletic or paraphyletic. Exsertaria species formed a clade but included a single Latoangulatae species (E. major). Recombination analysis identified two intragenic recombination events that involved species from different sections, which have probably been facilitated by inter-sectional hybridisation. One of the events most likely occurred prior to speciation, with several Latoangulatae species having the recombinant allele. The other event may have occurred after speciation, since only one of two E. globulus samples possessed the recombinant allele. This is the first detailed report of intragenic recombination in both CCR and Eucalyptus, and between species of different sections of a plant genus. The occurrence of intragenic recombination may explain the anomalous positions of some species within the phylogenetic tree, and indicates that phylogenetic analysis of Eucalyptus using nuclear genes will be problematic unless recombination is taken into account.

Low copy number nuclear genes have been found to be useful for phylogenetic reconstruction at different taxonomic levels. This study investigated the utility of a single copy gene, cinnamoyl CoA reductase (CCR), for resolving phylogenetic relationships at the sectional level within Eucalyptus. The monophyly of sections Exsertaria and Latoangulatae was explored, using section Maidenaria as an outgroup, and the impact of intragenic recombination on phylogenetic reconstruction examined. Phylogenetic analysis did not resolve monophyletic groups. Latoangulatae and Maidenaria were polyphyletic or paraphyletic. Exsertaria species formed a clade but included a single Latoangulatae species (E. major). Recombination analysis identified two intragenic recombination events that involved species from different sections, which have probably been facilitated by inter-sectional hybridisation. One of the events most likely occurred prior to speciation, with several Latoangulatae species having the recombinant allele. The other event may have occurred after speciation, since only one of two E. globulus samples possessed the recombinant allele. This is the first detailed report of intragenic recombination in both CCR and Eucalyptus, and between species of different sections of a plant genus. The occurrence of intragenic recombination may explain the anomalous positions of some species within the phylogenetic tree, and indicates that phylogenetic analysis of Eucalyptus using nuclear genes will be problematic unless recombination is taken into account.

Gauli A, Vaillancourt RE, Bailey T, Steane DA, Potts BM (2015). Evidence for local climate adaptation in early-life traits of Tasmanian populations of Eucalyptus pauciflora. Tree Genetics and Genomes (RAW DATA) Traits scored on each seedling of Eucalyptus pauciflora for quantitative genetic analysis. The table contains the description of the traits measured, codes used for each trait in the subsequent tables and text, and transformation used during analysis. Description Code Transformation Growth and developmental traits Height at 2 months (cm) HT1 Height at 6 months (cm) HT2 No. of nodes with lignotubers at 2 months LIGNO1 No. of nodes with lignotubers at 6 months LIGNO2 No. of nodes at 2 months (cotyledon = 1) NNODES1 No. of nodes at 6 months (cotyledon = 1) NNODES2 Node at which 1st petiolate leaf occurs at 6 months PET2 (NNODES2 - PET2)/NNODES2 Node at which 1st leaf alternates at 6 months ALT2 (NNODES2 - ALT2) /NNODES2 Node at which 1st leaf twists at 6 months TWIST2 (NNODES2 - TWIST2)/NNODES2 Other traits Stem oil glands (0 = absence of oil glands; 1 = presence of small oil glands 2 = protruding oil gland density) Stem OG Stem diameter at node 1 (x) (mm) at 6 months STMD Lignotuber diameter at node 1 (90° to x) Lig size √( (LIGD- STMD)/STMD) Leaf traits scored at 4th node leaf Leaf colour at node 4 at 2 months (1 = light green, 2 = intermediate, 3 = dark green) Leaf colour Leaf lamina length (cm) LL4 Lamina width LW LL4 log10(LW4/LL4) Leaf area (cm2) LA4 Lamina length to widest point from base LWP LL4 LWP 4/LL4 Petiole length (cm) PL4 log10 (PL4) Vein angle (°) VA4 Leaf traits scored at 11th node leaf Leaf lamina length (cm) LL 11 Lamina width (cm) LW LL11 log10(LW11/LL11) Leaf area (cm2) LA11 Lamina length to widest point (cm) LWP LL11 LWP11/LL11 Petiole length (cm) PL11 log10 (PL11) Vein angle (°) VA11 see also: Gauli, Archana (2014) Genetic diversity and adaptation in Eucalyptus pauciflora. PhD Thesis Univ. of Tasmania

Eucalyptus globulus is the main eucalypt species grown in Australian plantations. The focus on seedling deployment systems, coupled with exploitation of large, open-pollinated base populations for breeding purposes over the last two decades, has required a detailed understanding of the reproductive biology of this species. We review our research on the reproductive biology of E. globulus, with a focus on it’s breeding system and advances made in seed production systems. While most improved seed is still obtained from open-pollinated seedling or grafted seed orchards, the development of the one-stop/single-visit pollination procedure has revolutionised the breeding and deployment of this species. The reduced costs of controlled pollination has meant full pedigree-control can now be maintained in large advanced generation breeding populations and E. globulus is one of the few eucalypt species where large-scale production of manually pollinated seed for family forestry is being undertaken.

The first Europeans to discover Eucalyptus globulus were French explorers in 1792. Its seed was rapidly spread throughout the world in the 19th century and this was the species by which much of the world first knew the genus. However, it was in the industrial forests of the 20th century that this species, once considered the ‘Prince of Eucalypts’, achieved greatest prominence due to its fast growth and superior pulp qualities. Formal breeding first commenced in 1966 in Portugal and in the late 1980’s large base population trials from open-pollinated seed collections from native stands were established in many countries. These trials have provided unprecedented insights into the quantitative genetic control of numerous traits of economic and ecological importance and how this variation is spatially distributed in the native range of the species. However with large, fully pedigreed breeding populations becoming available for quantitative analysis and the rapidly expanding knowledge of DNA sequence variation, we are now at the threshold of a new understanding of this important eucalypt gene pool. Indications of the significance of non-additive genetic effects are becoming available. The E. globulus chloroplast genome has now been sequenced and several genome maps have been published. Studies of the variation in nuclear microsatellites and the lignin biosynthesis gene CCR confirm the complex, spatially structured nature of the native gene pool. Strong spatial structuring of the chloroplast genome has provided a tool for tracking seed migration and the geographic origin of exotic landraces. Highly divergent lineages of chloroplast DNA have been discovered and studies of the hypervariable JLA+ region argue that some components of the E. globulus gene pool have been assimilated from other species following hybridisation.

Megastigmus spp. are tiny wasps (bodies 2.5- 6.5mm in length) from the hymenopteran family Torymidae that destroy the seed of forest trees (Elliott and deLittle 1984). Many studies have examined their destructive effect on seed production in conifers, such as: Pseudotsuga; Abies; Cedrus; Cupressus; Juniperus; Larix and Picea (Roques and Skrzypczynska 2003). In Australia, they have been reported to feed on the seed of at least seven eucalypt species. However, despite reports of losses both in openpollinated seed orchard and controlled crossed seed of Eucalyptus globulus extending over many years, little is known of the biology and taxonomy of these insect species, nor the extent of the decrease in seed production they cause. The only study available suggests that the female wasps lay their eggs in eucalypt flowers (Drake 1974). After hatching, each small larva tunnel down into the developing seeds and gradually consume the content of a seed. The larva goes through several instars and the pupa develops within the seed coat. Fully-grown adult wasps emerge from the seed coat through a circular exit hole, and then exit through the top or side of the capsule usually before it opens. In this study we quantified the percentage of seed damaged by Megastigmus spp. predation in 384 seedlots of Eucalyptus globulus and examined whether there was a genetic basis to differences in the levels of seed predation observed.

While clonal propagation is used to establish some plantations of Eucalyptus globulus in countries such as Chile and Portugal, the majority of plantations worldwide are established from seed (Tibbits et al. 1997; Griffin 2001). The genetically improved seed used in industrial plantations is usually derived from open pollinated seed orchards (seedling or grafted), but with the advent of single-visit (Williams et al. 1999) and one-stop (Harbard et al. 1999) pollination procedures an increasing component is now derived from large-scale controlled pollination programs producing elite polymix or full-sib families. Regardless of the system, seed production varies markedly between trees and in most cases the choice of female can strongly influence the profitability of seed production (Leal and Cotterill 1997). In addition, as E. globulus has a mixed mating system (Griffin et al. 1987; Eldridge et al. 1993) and selfing results in severe inbreeding depression for growth traits (Hardner and Potts 1995), outcrossing rate is a key factor. Outcrossing rate will affect the genetic quality of seeds obtained from seed orchards and whether predicted additive genetic gains will be realised. One of the main drivers of the outcrossing rate is the level of self-incompatibility of the female tree (Patterson et al. 2001), which varies widely in the species (Potts and Savva 1988; Pound et al. 2002a,b, 2003). This paper reports the results of two studies aimed at determining whether there is a genetic basis to variation in traits affecting sexual reproduction in E. globulus. The first study examined the genetic basis of variation in the quantity and quality of open pollinated seed from over 800 seed orchard trees of E. globulus. The second study examined the genetic basis of the large variation in self-incompatibility observed within the species.

With the large increase in industrial eucalypt plantations in Australia over the last decade, there is growing concern that native eucalypt gene pools may be contaminated by pollen flow from locally exotic eucalypt species or provenances. We are studying the risk of hybridisation from the two most commonly planted hardwood species in industrial plantations in Australia, Eucalyptus nitens and E. globulus. While both species are native to Australia, they are frequently planted outside their natural range and in close proximity to species with which they do not co-occur naturally. While hybridisation will not occur when adjacent species belong to different subgenera to the plantation species, barriers to hybridisation within subgenera of Eucalyptus are frequently weak and exotic gene flow is then possible. A number of steps in the process of pollen-mediated gene flow have therefore been assessed. Firstly, field surveys across the entire temperate hardwood estate have confirmed that many compatible native eucalypts are found adjacent to plantations and that most older plantations are reproductively active. Secondly, open-pollinated seed collections have demonstrated that plantations can hybridise with some native species (most notably E. camaldulensis and E. ovata) and exotic F1 hybrid seedlings involving native E. ovata have been identified in the wild. Thirdly, artificial pollination studies have confirmed that numerous other hybrid combinations are possible involving species from the same subgenus as E. globulus and E. nitens. Fourthly, there is evidence to suggest that flowering time asynchrony and spatial isolation are important barriers which will reduce or prevent hybridisation in many cases. Fifthly, despite evidence that first generation hybrids are often less fit than parental species, some hybrids may survive to reproduction and backcross with native species. Full assessment of the impact of such hybridisation will depend on long-term studies of hybrid fitnes