Acacia crassicarpa is a tropical forestry species that is increasingly being planted in South-East Asia.Using next-generation sequencing, we developed 12 nuclear microsatellite markers for A. crassicarpa and optimised them for assay in three multiplex sets.We tested the 12 loci on 34 A. crassicarpa samples and found that polymorphism ranged from 4 to 8 alleles per locus (average = 5.8).The loci were easy to score in terms of binning and their reproducibility and polymerase chain reaction success rate were high.Given their usability and polymorphism we believe that these 12 loci will be useful for DNA profiling and mating system analysis with direct application in breeding programmes and the conservation of wild populations.Six of the markers also amplified products in related A. mangium and A. auriculiformis, with four being polymorphic in three samples of each species.Therefore, these markers added more broadly to the genomic resources available in Acacia.

AimThe distribution of genetic variation in the Australian dry sclerophyll plant Hardenbergia violacea (Fabaceae) is examined in the context of Pleistocene climate change in order to identify likely refugia. Particular consideration is given to the origin of range disjunctions in South Australia and Tasmania, and to determining whether the Tasmanian population is indigenous or recently introduced from mainland Australia.LocationSoutheastern Australian mainland and Tasmania.MethodsA combination of chloroplast polymerase chain reaction-restriction fragment length polymorphism and genomic amplified fragment length polymorphism (AFLP) marker systems was used to examine the genetic structure of 292 individuals from 13 populations across the range of H. violacea in southeastern Australia.ResultsHardenbergia violacea populations in Tasmania and southern Victoria were characterized by low, almost monotypic chloroplast diversity. New South Wales showed higher haplotype diversity and haplotype sharing among widely distributed populations. Principal coordinates analysis (PCoA) of the AFLP data found a strong latitudinal cline in AFLP variation from northern New South Wales south to Tasmania. The Tasmanian population formed an isolated and somewhat disjunct genetic cluster at one end of this cline. However, the South Australian population was an exception to the clinal variation shown by all other populations, forming a highly disjunct cluster in the PCoA. Within-population genetic diversity was low in both disjunct populations.Main conclusionsThe genetic evidence indicates that the Tasmanian population is likely to be indigenous and probably the product of vicariance, which was followed by range contraction at the Last Glacial Maximum or an earlier glacial event. The deep phylogenetic disjunction in South Australia is evidence of a much earlier separation on mainland Australia. The chloroplast structure indicates that, during the Pleistocene, H. violacea underwent broad-scale recolonization in southern Victoria and Tasmania, possibly from a large continental refugium in eastern New South Wales. We conclude that H. violacea, and presumably the sclerophyll communities in which it occurs, have undergone multiple range contractions to large continental refugia during different Pleistocene glaciations in southeastern Australia.

Few chloroplast-based genetic studies have been undertaken for plants of mesic temperate forests in the southern hemisphere and fossil-based models have provided evidence of vegetation history only at the broadest scales in this region. This study investigates the chloroplast DNA phylogeography of Tasmannia lanceolata (Winteraceae), a fleshy-fruited, bird-dispersed shrub that is widespread in the mountains of southeastern Australia and Tasmania. Thirty haplotypes were identified after sequencing 3206 bp of chloroplast DNA in each of 244 individuals collected across the species' range. These haplotypes showed unexpectedly strong phylogeographic structuring, including a phylogeographic break within a continuous part of the species' range, with the distribution of four major clades mostly not overlapping, and geographic structuring of haplotypes within these clades. This strong geographic patterning of chloroplast DNA provided evidence for the survival of T. lanceolata in multiple putative wet forest refugia as well as evidence for additional wet forest species refugia in southeastern Australia. In western Tasmania lower haplotype diversity below the LGM tree line compared to above the LGM tree line suggests that glacial refugia at high altitudes may have been important for T. lanceolata. The level of geographic structuring in T. lanceolata is similar to gravity dispersed southern hemisphere plants such as Nothofagus and Eucalyptus. Behavioural traits of the birds transporting seed may have had a strong bearing on the limited transport of T. lanceolata seed, although factors limiting establishment, possibly including selection, may also have been important.

Numerous studies within plant genera have found geographically structured sharing of chloroplast (cp) DNA among sympatric species, consistent with introgressive hybridization. Current research is aimed at understanding the extent, direction and significance of nuclear (nr) DNA exchange that accompanies putative cpDNA exchange. Eucalyptus is a complex tree genus for which cpDNA sharing has been established between multiple species. Prior phylogeographic analysis has indicated cpDNA introgression into the widespread forest species Eucalyptus globulus from its rare congener E. cordata. In this study, we use AFLP markers to characterize corresponding nrDNA introgression, on both a broad and fine spatial scale. Using 388 samples we examine (i) the fine-scale spatial structure of cp and nrDNA introgression from E. cordata into E. globulus at a site in natural forest and (ii) broad-scale patterns of AFLP marker introgression at six additional mixed populations. We show that while E. globulus and E. cordata retain strongly differentiated nuclear gene pools overall, leakage of nrDNA occurs at mixed populations, with some AFLP markers being transferred to E. globulus recurrently at different sites. On the fine scale, different AFLP fragments show varying distances of introgression into E. globulus, while introgression of cpDNA is extensive. The frequency of E. cordata markers in E. globulus is correlated with spatial proximity to E. cordata, but departs from expectations based on AFLP marker frequency in E. cordata, indicating that selection may be governing the persistence of introgressed fragments in E. globulus.

Hybridisation through pollen dispersal from exotic plants is increasingly recognised as a threat to the genetic integrity of native plant populations. Its genetic impact can be greater in rare taxa, due to their vulnerability to pollen swamping by more abundant congeners. We assessed the likelihood of pollen dispersal from exotic eucalypt plantations into all of Australia's rare native eucalypts, and conducted a case study of Eucalyptus perriniana, which is rare in Tasmania. The Australia-wide study involved spatial analyses of the locations for each rare species superimposed on distributions of eucalypt plantations, which were combined with known taxonomically based reproductive barriers. Of the 74 nationally listed rare eucalypt taxa, 22 had locations within 10 km of plantations of the same genus, and eight were within 1 km. These eight proximal taxa are considered priorities for monitoring. In the most extreme case, 30% of point locations originating from herbarium records and field surveys for Eucalyptus conglomerata were within 1 km of exotic plantations. In the case study, E. perriniana revealed considerable reproduci.ive compatibility with adjacent recently established Eucalyptus nitens plantations. However, F-1 hybridisation between these species was limited, with 0.2% of the 18,625 seedlings grown from 100 single-tree open-pollinated seedlots being hybrids. For now, the probability of exotic gene flow into E. perriniana appears to be low, however this probability is likely to increase as more E. nitens flowers in the surrounding landscape. These studies suggest that understanding the breeding system and biology of these populations may reveal surprising resistance to such exotic hybridisation as well as identifying high risk situations to focus conservation management. (C) 2010 Elsevier B.V. All rights reserved.

Glacial aridity of the Pleistocene was inhospitable for the cool temperate rainforest tree Nothofagus cunninghamii over most of its current range in southeastern Australia, particularly in eastern Tasmania. A chloroplast DNA phylogeographic study was undertaken to investigate whether this species was likely to have survived in situ or conforms to a dispersal model of postglacial recovery.Twenty-three chloroplast haplotypes were identified by PCR-RFLP and direct sequencing of 2164 base pairs from 213 N. cunninghamii individuals collected in a range-wide survey. Fine-scale haplotype distribution was investigated using PCR-RFLP in eastern Tasmania.Deep chloroplast divergence occurred in N. cunninghamii. The single haplotype of the sister species, N. moorei, was nested among N. cunninghamii haplotypes. The distribution of N. cunninghamii haplotypes supports: multiple glacial refugia in coastal and inland western Tasmania, the centre of haplotype diversity; glacial survival in the central highlands of Victoria, corroborating pollen data; and the long-term occupation of eastern Tasmania because of the presence of a unique deeply diverged chloroplast lineage.Nothofagus cunninghamii withstood glacial aridity within multiple regions in apparently nonequable climates. This finding contributes to a growing understanding of how the resilience of temperate species during glacial periods has shaped modern biota.

Sustainable management of native forest gene pools requires an understanding of the levels and spatial patterns of genetic diversity in tree species. This diversity impacts on numerous management issues including seed transfer guidelines and in situ genetic resource conservation. Our long-term studies of Eucalyptus globulus (Tasmanian Blue Gum) have provided unprecedented insights into the complexity of the genetic variation that can exist in natural populations. We demonstrate that E. globulus comprises a fine-scale mosaic of family groups superimposed on a complex spatial pattern of local and broad-scale adaptive and non-adaptive genetic variation. The high level of genetic diversity observed within forest tree species such as E. globulus, coupled with their flexible breeding systems, provides the fundamental elements for adaptation to futureenvironmental challenges.

Genus Eucalyptus , with over 700 species, presents a number of systematic difficulties including taxa that hybridize or intergrade across environmental gradients. To date, no DNA marker has been found capable of resolving phylogeny below the sectional level in the major subgenera. Molecular markers are needed to support taxonomic revision, assess the extent of genetic divergence at lower taxonomic levels, and inform conservation efforts. We examined the utility of 930 amplified fragment length polymorphisms (AFLPs) for analyzing relationships among Tasmanian taxa of subgenus Symphyomyrtus section Maidenaria . Phenetic and cladistic analyses resolved species into clusters demonstrating significant genetic partitioning, largely concordant with series defined in the most recent taxonomic revision of Eucalyptus . Some departures from current taxonomy were noted, indicating possible cases of morphological convergence and character reversion. Although the resolution obtained using AFLP was greatly superior to that of single sequence markers, the data demonstrated high homoplasy and incomplete resolution of closely related species. The results of this study and others are consistent with recent speciation and reticulate evolution in Maidenaria . We conclude that a combination of phylogenetic and population genetic approaches using multiple molecular markers offers the best prospects for understanding taxonomic relationships below the sectional level in Eucalyptus .

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 the whole range of the species were 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.

Three small populations of a dwarf ecotype of the forest tree Eucalyptus globulus are found on exposed granite headlands in south-eastern Australia. These populations are separated by at least 100 km. Here, we used 12 nuclear microsatellites and a chloroplast DNA marker to investigate the genetic affinities of the dwarf populations to one another and to their nearest populations of tall E. globulus. Cape Tourville was studied in greater detail to assess the processes enabling the maintenance of distinct ecotypes in close geographical proximity. The three dwarf populations were not related to one another and were more closely related to adjacent tall trees than to one another. At Cape Tourville the dwarf and tall ecotypes were significantly differentiated in microsatellites and in chloroplast DNA. The dwarf and tall populations differed in flowering time and no evidence of pollen dispersal from the more extensive tall to the dwarf population was found. The three dwarf populations have evolved in parallel from the local tall ecotypes. This study shows that small marginal populations of eucalypts are capable of developing reproductive isolation from nearby larger populations through differences in flowering time and/or minor spatial separation, making parapatric speciation possible.