Re Vaillancourt

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Genetic pollution of native eucalypt gene pools—identifying the risks

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 Australias eucalypt woodlands and mallee, (iv) there has been a rapid expansion of eucalypt plantations and restoration plantings in Australia and (v) Australia is the custodian of an internationally important genetic resource. 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 considered sustainable. 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 and/or subgenera (e.g. 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 have an impact on local gene pools to varying degrees, indicating the requirement for the adoption of management strategies to reduce this risk. Naturally small or remnant populations are at particular risk. A framework for assessment of the risk of genetic pollution is developed herein.

Partitioning and distribution of RAPD variation in a forest tree species, Eucalyptus globulus(Myrtaceae).

Eucalyptus globulus is an important species for pulpwood production in many countries. The pattern and partitioning of variation is important baseline knowledge for tree breeding. Currently the species is divided into four subspecies: globulus, bicostata, pseudoglobulus and maidenii. Random Amplified Polymorphic DNA (RAPD) markers were used to analyse variation in 173 representatives of 37 natural populations of E. globulus: 31 localities of ssp. globulus (148 individuals), two localities each of ssp. bicostata (nine individuals), ssp. maidenii (ten individuals) and ssp. pseudoglobulus (six individuals). Ten 10-mer primers amplified a total of 162 scorable bands, of which 149 (91.9 per cent) were polymorphic, AMOVA analysis of a Euclidean distance matrix based on presence/absence of polymorphic bands found most variation within localities, but significant differences between localities and regions. Principal components analysis (PCA) identified a major latitudinal cline in RAPD phenotype that differentiated southern Tasmanian localities from other ssp. globulus localities on mainland Australia. Many localities previously identified as intermediate between subspecies globulus and other subspecies in morphology were not intermediate in RAPD phenotype. In some cases regions which showed marked differentiation between localities in capsule and juvenile leaf morphology showed Utile RAPD differentiation between localities. RAPDs also provided new insights into the affinities of outlying localities. Although RAPD technology has not yet been applied to many forest tree species, patterns of variation were similar to those found in other outcrossing species studied using both RAPDs and other molecular markers.

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

The status of genomics and genetics research in the Myrtaceae, a large family of dicotyledonous woody plants, is reviewed with Eucalyptus as the focal genus. The family contains over 5,650 species in 130 to 150 genera, predominantly of neo-tropical and Southern Hemisphere distribution. Several genera are well known for their economic importance worldwide. Myrtaceae are typically diploids with small to intermediate genome size. Microsatellites have been developed for several genera while higher throughput marker systems such as diversity arrays technology and single nucleotide polymorphism are available for Eucalyptus. Molecular data have been fundamental to current perspectives on the phylogeny, phylogeography and taxonomy of the Myrtaceae, while numerous studies of genetic diversity have been carried out particularly as it relates to endangered, rare, fragmented, overharvested or economically important species. Large expressed sequence tag collections for species of Eucalyptus have recently become public to support the annotation of the Eucalyptus grandis genome. Transcriptomics in Eucalyptus has advanced by microarrays and next-generation sequencing focusing on wood development. Linkage maps for Eucalyptus display high synteny across species and have been extensively used to map quantitative trait loci for a number of traits including growth, wood quality, disease and insect resistance. Candidate gene-based association genetics have successfully found marker–trait associations for wood and fiber traits. Genomic selection experiments have demonstrated clear potential to improve the efficiency of breeding programs while freeze-tolerant transgenic Eucalyptus trials have recently been initiated. The recently released E. grandis genome, sequenced to an average coverage of 8×, will open up exceptional opportunities to advance Myrtaceae genetics and genomics research.

Chloroplast sharing in the Tasmanian Eucalypts

Abstract The biogeographic pattern of chloroplast DNA (cpDNA) haplotypes in Eucalyptus on the island of Tasmania is consistent with reticulate evolution, involving at least 12 Tasmanian species from the subgenus Symphyomyrtus. Intraspecific cpDNA polymorphism in 14 of 17 species is coupled with extensive sharing of identical haplotypes across populations of different species in the same geographic area. Haplotype diversity is lowest in central regions of Tasmania formerly occupied by alpine vegetation during glacial intervals and in northern regions that were periodically linked to continental Australia by land bridges. The observed distribution of several cpDNA haplotypes unique to Tasmania coincides with modeled locations of glacial refugia in coastal areas of Tasmania and shows the power of cpDNA in unraveling the complex history of past distributions of Eucalyptus. The results suggest that the model of evolution of the eucalypts should be reassessed to allow for the anastomosing effects of interspecific hybridization and introgression. Corresponding Editor: O. Savolainen

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

Eucalyptus globulus (Myrtaceae) is a forest tree native to southeastern Australia, but is grown globally for pulpwood and timber. Eight microsatellite loci were used to determine the degree of selectively neutral differentiation between native populations of the geographic races of E. globulus that are used in a national breeding programme. Spatial differentiation was detected among 340 samples from across the species range (FST=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 (QST) and molecular (FST) 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.

Incongruence between chloroplast and species phylogenies in Eucalyptus subgenus Monocalyptus (Myrtaceae).

Seventy-eight polymorphic cpDNA (chloroplast DNA) characters were found in 13 closely related taxa from Eucalyptus series Amygdalinae (subgenus Monocalyptus) and seven potential outgroup taxa. The strict consensus of six cladograms generated from cpDNA data confirmed monophyly of Monocalyptus. However, cpDNA phylogeny within Monocalyptus was incongruent with taxonomic classification, being more related to geography, even when accessions were from divergent series. Monocalyptus cpDNA formed two major clades. On the island of Tasmania cpDNA was restricted to a single clade, exhibited very little variation, and was phylogenetically related to cpDNA found in central and western Victoria. In contrast, cpDNA of mainland monocalypt taxa was more variable, even within the Amygdalinae. Four out of six Tasmanian Amygdalinae species were polymorphic. The difference between cpDNA of replicates was often greater than differences between species from different series. The low level of cpDNA variation and extensive morphological intergradation between the Tasmanian endemics suggest recent speciation. However, the transfer of cpDNA through hybridization between lineages is the most likely explanation for the observed sharing of cpDNA across series. This study highlights that the geographical pattern to cpDNA variation in Eucalyptus may be an important source of information on past plant distributions in Australia.

Chloroplast DNA phylogeography of Eucalyptus globulus

The pattern of variability in chloroplast DNA (cpDNA) of Eucalyptus globulus Labill. (Myrtaceae) was studied using 270 samples from southern Australia. Forty variable sequence characters were found, defining 105 haplotypes. Haplotypes were assigned to three major cpDNA clades based on their phylogeny. The pattern of cpDNA variation did not conform to subspecies boundaries; however, there was a strong geographic structure to the distribution of clades and haplotypes. One clade (JC) was geographically central and widespread and was found in 163 samples from Tasmania and continental Australia. Less-common clades occurred in more localised regions— southern (Js, 77 samples) and eastern (Jet, 12 samples). The distribution of the Jet and Js clades coincides with hypothesised glacial refugia in Tasmania, suggesting limited seed-mediated dispersal since deglaciation.

Population genetic analysis and phylogeny reconstruction in Eucalyptus (Myrtaceae) using high-throughput, genome-wide genotyping

A set of over 8000 Diversity Arrays Technology (DArT) markers was tested for its utility in high-resolution population and phylogenetic studies across a range of Eucalyptus taxa. Small-scale population studies of Eucalyptus camaldulensis, Eucalyptus cladocalyx, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus nitens, Eucalyptus pilularis and Eucalyptus urophylla demonstrated the potential of genome-wide genotyping with DArT markers to differentiate species, to identify interspecific hybrids and to resolve biogeographic disjunctions within species. The population genetic studies resolved geographically partitioned clusters in E. camaldulensis, E. cladocalyx, E. globulus and E. urophylla that were congruent with previous molecular studies. A phylogenetic study of 94 eucalypt species provided results that were largely congruent with traditional taxonomy and ITS-based phylogenies, but provided more resolution within major clades than had been obtained previously. Ascertainment bias (the bias introduced in a phylogeny from using markers developed in a small sample of the taxa that are being studied) was not detected. DArT offers an unprecedented level of resolution for population genetic, phylogenetic and evolutionary studies across the full range of Eucalyptus species.

Chloroplast DNA evidence for reticulate evolution in Eucalyptus (Myrtaceae)

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.