Melissa A. Millar

Mating system studies in jarrah, Eucalyptus marginata (Myrtaceae)

Estimates of outcrossing rate were determined for four populations of Eucalyptus marginata from the jarrah forest in south-west Western Australia. The mean multilocus outcrossing rate (t = 0.81) was high in all populations and was towards the high end of the range of outcrossing rates that have been observed in other mass-flowering eucalypt species. A significant proportion of the inbreeding detected appeared to be due to biparental inbreeding, and the levels of correlated paternity were unexpectedly high. Differences between populations were generally not significant, although trees from a disturbed site affected by disease showed lower outcrossing, higher biparental inbreeding and higher correlated paternity, suggesting an increase in population structure compared with trees from disease-free sites.

The maintenance of disparate levels of clonality, genetic diversity and genetic differentiation in disjunct subspecies of the rare Banksia ionthocarpa

The evolutionary potential of plant species that reproduce via predominantly clonal means and the conditions under which clonality is favoured are not well known. Long‐term clonal reproduction is expected to result in a number of readily detectable genetic signals not present in populations that reproduce by sexual means. We use a hierarchical sampling strategy to assess genotype probabilities and confirm that two rare sister taxa of Banksia ionthocarpa have contrasting modes of reproduction. Banksia ionthocarpa subsp. chrysophoenix reproduces clonally. Populations had low levels of genotypic diversity and were comprised of large clonal patches consisting of many ramets that covered hundreds of square metres and showed little intermixing. The taxon was genetically depauperate (mean Na = 1.97, mean P = 0.66, mean He = 0.282), had high levels of genetic differentiation between populations (θ = 0.481), and populations exhibited excess heterozygosity and linkage disequilibrium (LD) among loci, suggesting historically high levels of clonality. In contrast, the sister taxon B. ionthocarpa subsp. ionthocarpa, which occurs in an area with more than twice the annual rainfall and less extreme minimum and maximum temperatures, showed no evidence of clonality, high levels of genotypic diversity, greater genetic diversity (mean Na = 3.31, mean P = 0.81, mean He = 0.405), lower levels of genetic differentiation between populations (θ = 0.253) and no evidence of excess heterozygosity or LD among loci. We suggest that the development of clonality in subsp. chrysophoenix is associated with its more marginal environment and enhanced by response to recurrent fires.

Cultivation shapes genetic novelty in a globally important invader

Acacia saligna is a species complex that has become invasive in a number of countries worldwide where it has caused substantial environmental and economic impacts. Understanding genetic and other factors contributing to its success may allow managers to limit future invasions of closely related species. We used three molecular markers to compare the introduced range (South Africa) to the native range (Western Australia). Nuclear markers showed that invasive populations are divergent from native populations and most closely related to a cultivated population in Western Australia. We also found incongruence between nuclear and chloroplast data that, together with the long history of cultivation of the species, suggest that introgressive hybridization (coupled with chloroplast capture) may have occurred within A. saligna. While we could not definitively prove introgression, the genetic distance between cultivated and native A. saligna populations was comparable to known interspecific divergences among other Acacia species. Therefore, cultivation, multiple large‐scale introductions and possibly introgressive hybridization have rapidly given rise to the divergent genetic entity present in South Africa. This may explain the known global variation in invasiveness and inaccuracy of native bioclimatic models in predicting potential distributions.

Extensive long-distance pollen dispersal and highly outcrossed mating in historically small and disjunct populations of Acacia woodmaniorum (Fabaceae), a rare banded iron formation endemic

Background and Aims Understanding patterns of pollen dispersal and variation in mating systems provides insights into the evolutionary potential of plant species and how historically rare species with small disjunct populations persist over long time frames. This study aims to quantify the role of pollen dispersal and the mating system in maintaining contemporary levels of connectivity and facilitating persistence of small populations of the historically rare Acacia woodmaniorum. Methods Progeny arrays of A. woodmaniorum were genotyped with nine polymorphic microsatellite markers. A low number of fathers contributed to seed within single pods; therefore, sampling to remove bias of correlated paternity was implemented for further analysis. Pollen immigration and mating system parameters were then assessed in eight populations of varying size and degree of isolation. Key Results Pollen immigration into small disjunct populations was extensive (mean minimum estimate 40 % and mean maximum estimate 57 % of progeny) and dispersal occurred over large distances (≤1870m). Pollen immigration resulted in large effective population sizes and was sufficient to ensure adaptive and inbreeding connectivity in small disjunct populations. High outcrossing (mean tm = 0·975) and a lack of apparent inbreeding suggested that a self-incompatibility mechanism is operating. Population parameters, including size and degree of geographic disjunction, were not useful predictors of pollen dispersal or components of the mating system. Conclusions Extensive long-distance pollen dispersal and a highly outcrossed mating system are likely to play a key role in maintaining genetic diversity and limiting negative genetic effects of inbreeding and drift in small disjunct populations of A. woodmaniorum. It is proposed that maintenance of genetic connectivity through habitat and pollinator conservation will be a key factor in the persistence of this and other historically rare species with similar extensive long-distance pollen dispersal and highly outcrossed mating systems.

Genetic connectivity and diversity in inselberg populations of Acacia woodmaniorum , a rare endemic of the Yilgarn Craton banded iron formations

Historically rare plant species with disjunct population distributions and small population sizes might be expected to show significant genetic structure and low levels of genetic diversity because of the effects of inbreeding and genetic drift. Across the globe, terrestrial inselbergs are habitat for rich, often rare and endemic flora and are valuable systems for investigating evolutionary processes that shape patterns of genetic structure and levels of genetic diversity at the landscape scale. We assessed genetic structure and levels of genetic diversity across the range of the historically rare inselberg endemic Acacia woodmaniorum. Phylogeographic and genetic structure indicates that connectivity is not sufficient to produce a panmictic population across the limited geographic range of the species. However, historical levels of gene flow are sufficient to maintain a high degree of adaptive connectivity across the landscape. Genetic diversity indicates gene flow is sufficient to largely counteract any negative genetic effects of inbreeding and random genetic drift in even the most disjunct or smallest populations. Phylogeographic and genetic structure, a signal of isolation by distance and a lack of evidence of recent genetic bottlenecks suggest long-term stability of contemporary population distributions and population sizes. There is some evidence that genetic connectivity among disjunct outcrops may be facilitated by the occasional long distance dispersal of Acacia polyads carried by insect pollinators moved by prevailing winds.

A rapid PCR-based diagnostic test for the identification of subspecies of Acacia saligna

Subspecific taxa of species complexes can display cryptic morphological variation, and individuals and populations can often be difficult to identify with certainty. However, accurate population identification is required for comprehensive conservation and breeding strategies and for studies of invasiveness and gene flow. Using five informative microsatellite markers and a Bayesian statistical approach, we developed an efficient polymerase chain reaction-based diagnostic tool for the rapid identification of individuals and populations of the Acacia saligna species complex of Western Australia. We genotyped 189 individuals from 14 reference populations previously characterised based on morphology and used these data to investigate population structure in the species complex. High total genetic diversity (HT = 0.729) and high population differentiation (θ = 0.355) indicated strong intraspecific structuring. With the provision of prior population information, the reference data set was optimally resolved into four clusters, each corresponding to one of the four main proposed subspecies, with very high membership values (Q > 97%). The reference data set was then used to assign individuals and test populations to one of the four subspecies. Assignment was unequivocal for all test individuals from two populations of subsp. lindleyi and for all but one individual of subsp. stolonifera. Individuals from populations of subsp. saligna and subsp. pruinescens showed a degree of genetic affinity for the two subspecies in their assignments, although the majority of individuals were correctly assigned to subspecies. The diagnostic tool will assist in characterising populations of A. saligna, especially naturalised and invasive populations of unknown origin.

Contrasting patterns of clonality and fine-scale genetic structure in two rare sedges with differing geographic distributions.

For plants with mixed reproductive capabilities, asexual reproduction is more frequent in rare species and is considered a strategy for persistence when sexual recruitment is limited. We investigate whether asexual reproduction contributes to the persistence of two co-occurring, rare sedges that both experience irregular seed set and if their differing geographic distributions have a role in the relative contribution of clonality. Genotypic richness was high (R=0.889±0.02) across the clustered populations of Lepidosperma sp. Mt Caudan and, where detected, clonal patches were small, both in ramet numbers (⩽3 ramets/genet) and physical size (1.3±0.1 m). In contrast, genotypic richness was lower in the isolated L. sp. Parker Range populations, albeit more variable (R=0.437±0.13), with genets as large as 17 ramets and up to 5.8 m in size. Aggregated clonal growth generated significant fine-scale genetic structure in both species but to a greater spatial extent and with additional genet-level structure in L. sp. Parker Range that is likely due to restricted seed dispersal. Despite both species being rare, asexual reproduction clearly has a more important role in the persistence of L. sp. Parker Range than L. sp. Mt Caudan. This is consistent with our prediction that limitations to sexual reproduction, via geographic isolation to effective gene exchange, can lead to greater contributions of asexual reproduction. These results demonstrate the role of population isolation in affecting the balance of alternate reproductive modes and the contextual nature of asexual reproduction in rare species.

Characterisation of eleven polymorphic microsatellite DNA markers for Australian sandalwood (Santalum spicatum) (R.Br.) A.DC. (Santalaceae)

A genomic library was constructed and eleven novel polymorphic microsatellite markers developed for Santalum spicatum, a hemi-parasitic tree native to south Western Australia and highly valued for its sesquiterpene essential oil. Polymorphism was investigated in two populations of S. spicatum, one from the arid region (Laverton) and one from the semi-arid region (Pingrup). All loci were variable, all conformed to Hardy-Weinberg Equilibrium and none demonstrated Linkage Disequilibrium. The number of alleles per locus ranged from 9 to 10 averaging 9.909 for Laverton and from 2 to 10 averaging 7.818 for Pingrup.

Biogeographic origins and reproductive mode of naturalised populations of Acacia saligna

Acacia saligna (Labill.) H.L.Wendl. is a species complex with an extensive history of anthropogenic utilisation and distribution. The taxon is naturalised and invasive in many countries. Extensive morphological variation makes accurate taxonomic identification of populations difficult. We used population genetic analysis to determine the biogeographic origins of 12 naturalised populations sampled from throughout south-eastern South Australia and assess the mode of reproduction (seedling or root suckering) at sites with active recruitment. Ten naturalised populations were assigned to Eastern ‘saligna’, although some also showed a lesser degree of affinity with other entities. A single population was assigned to Western ‘saligna’, but showed some affinity with Eastern ‘saligna’, and one population assigned to subsp. ‘lindleyi’ showed some affinity with Northern ‘lindleyi’. These assignments suggest that although several genetic entities of A. saligna are represented in South Australia, the majority of germplasm has originated from native populations of Eastern ‘saligna’ located around Esperance on Western Australia’s southern coast. Genetic diversity is limited in naturalised A. saligna compared with that present in the native range, suggesting a restricted number of historical introductions. Reproduction is predominantly by seedling recruitment, as opposed to clonal reproduction.

Characterization of polymorphic microsatellite DNA markers in Banksia nivea, formerly Dryandra nivea

A genomic library was constructed and 10 novel polymorphic microsatellite markers were developed for Banksia nivea subsp. uliginosa, a woody shrub endemic to the southwest of Western Australia. Polymorphism was investigated for these markers in 30 individuals from one population located in restricted habitat at the base of the Whicher Range east of Busselton. Expected heterozygosity ranged from 0.124 to 0.898 and averaged 0.570. Observed heterozygosity ranged from 0.133 to 0.933 and averaged 0.544. The number of alleles per locus ranged from 2.0 to 16.0 and averaged 7.3. These markers also produced amplification products in three other Banksia species and will be used to assay genetic diversity in these species.