Elizabeth A. James

Assessing the benefits and risks of translocations in changing environments: a genetic perspective

Translocations are being increasingly proposed as a way of conserving biodiversity, particularly in the management of threatened and keystone species, with the aims of maintaining biodiversity and ecosystem function under the combined pressures of habitat fragmentation and climate change. Evolutionary genetic considerations should be an important part of translocation strategies, but there is often confusion about concepts and goals. Here, we provide a classification of translocations based on specific genetic goals for both threatened species and ecological restoration, separating targets based on ‘genetic rescue’ of current population fitness from those focused on maintaining adaptive potential. We then provide a framework for assessing the genetic benefits and risks associated with translocations and provide guidelines for managers focused on conserving biodiversity and evolutionary processes. Case studies are developed to illustrate the framework.

Clonality and sexual reproductive failure in remnant populations of Santalum lanceolatum (Santalaceae).

Habitat fragmentation can have important conservation consequences for clonal plant species that possess self-incompatibility mechanisms, as lack of genetic variability within remnant populations may result in sexual reproductive failure. Allozymes and RAPDs were used in this study to determine the extent of clonality in remnant Victorian populations of the northern sandalwood, Santalum lanceolatum (Santalaceae), a species that has been heavily wild-harvested. S. lanceolatum can reproduce asexually by root suckers, and each population was identified as a unique single clone composed of numerous ramets of a single genet. Examination of pollination and fruit set indicated that little or no sexual reproduction was occurring in the remnants, due to pollen sterility in one population and self-incompatibility or pistil dysfunction in others. Clonality, genetic isolation and sexual reproductive failure indicate that preservation of each population, and possibly the establishment of new ones, should be objectives of the conservation strategy for the S. lanceolatum remnants.

Limitations to Reproductive Output and Genetic Rescue in Populations of the Rare Shrub Grevillea repens (Proteaceae)

BACKGROUND AND AIMS When conserving rare plant species, managers are often faced with small and/or isolated populations displaying low levels of sexual reproduction and genetic variation. One option for reinvigorating these populations is the introduction of genetic material from other sites, but in some cases fitness may be reduced as a result of outbreeding depression. Here the pollination biology of the rare shrub Grevillea repens is studied across its natural range and reproductive responses following cross-pollination among populations are examined to determine factors that may be limiting sexual reproduction and the potential for genetic rescue. METHODS Pollen manipulation treatments (self-, autogamous self-, cross- and open pollination) were applied to flowers to examine the breeding system and fruit and seed production in five populations of G. repens. Pollen production, presentation and viability were investigated and interpopulation crosses of increasing genetic distance performed among the populations. KEY RESULTS The study species is self-incompatible and displayed very low natural seed set over two seasons, due partly to low pollen viability in one of the populations. Within-population crossing increased fruit and seed production at some sites, indicating pollinator limitation. Interpopulation crosses further increased reproductive output in one population, suggesting mate limitation, and for this site there was a positive relationship between genetic distance among populations and the size of genetic rescue benefits. However, in other populations there was a decrease in fruit and seed set with increasing genetic distance. CONCLUSIONS The results highlight that management strategies involving interpopulation crosses can improve reproductive output in small, isolated populations of rare plants, but guidelines need to be developed on a population by population basis.

Reproductive biology and genetic marker diversity in Grevillea infecunda (Proteaceae), a rare plant with no known seed production

Grevillea infecunda D.J.McGillivray is a narrow endemic that reproduces vegetatively via root suckering. The reproductive biology of five Grevillea infecunda populations was investigated by scanning electron microscopy and light microscopy. Stigma maturation follows normal Grevillea development. However, a large number of pollen grains with aberrant forms were documented. A fluorochromatic reaction (FCR) test revealed 0.04% pollen viability. All pollen samples collected from two populations were completely sterile. All viable grains were >100 µm in diameter and included aberrant forms. Amplified fragment length polymorphism (AFLP) technique was used to investigate genotypic diversity in G. infecunda. Three primer pairs revealed sufficient variation in 109 loci to assign a unique phenotype to every individual (N = 40) sampled. This suggests that the populations were established from founder seedlings. The species has apparently lost the ability to reproduce sexually but genotypic variation is maintained through asexual reproduction via root suckers.

Divergent levels of genetic variation and ploidy among populations of the rare shrub, Grevillea repens (Proteaceae)

Rare plant species are often restricted to small and/or isolated populations that can have reduced reproductive output and adaptive potential, resulting in an increased probability of extinction. Nevertheless, evolutionary changes might occur in such populations that increase their likelihood of persisting. In Australia, many threatened species from the ecologically important genus Grevillea (Proteaceae) are found in disjunct populations and these often display varied modes of reproduction from sexual to exclusively clonal. Here we use microsatellite markers to show that isolated populations across the entire range of G. repens have developed diverse patterns of genetic variation. The largest population has a relatively low level of genetic variation, one small population displays inbreeding, two populations show evidence of clonal reproduction and two contain both triploids and diploids. The global estimate of FST was moderately high (0.272) suggesting limited gene flow between populations and historical isolation. These findings indicate that the genetically distinct G. repens populations exhibit very different patterns of genetic variation and we propose that the development of clonality and polyploidy in small or isolated populations may allow persistence but also reduces the effective size of the sexual population. Grevillea repens populations from its eastern and western/western central centres of distribution should be viewed as separate units for conservation management.

Development of microsatellite loci in Triglochin procera (Juncaginaceae), a polyploid wetland plant

Eleven polymorphic microsatellite loci were developed from the polyploid wetland plant Triglochin procera (Juncaginaceae). Loci were screened for variability among 20 individuals from each of two populations in Victoria, Australia. The number of alleles amplified per locus ranged from 5 to 17, with a mean of 9.5. Nei’s genetic diversity (HE) ranged from 0.463 to 0.898 with a mean of 0.725. These primers provide the opportunity to use polymorphic DNA markers to study the population genetic structure, breeding system and dispersal in T. procera and related species.

Spatial genetic structure reflects extensive clonality, low genotypic diversity and habitat fragmentation in Grevillea renwickiana (Proteaceae), a rare, sterile shrub from south-eastern Australia

BACKGROUND AND AIMS The association of clonality, polyploidy and reduced fecundity has been identified as an extinction risk for clonal plants. Compromised sexual reproduction limits both their ability to adapt to new conditions and their capacity to disperse to more favourable environments. Grevillea renwickiana is a prostrate, putatively sterile shrub reliant on asexual reproduction. Dispersal is most likely limited by the rate of clonal expansion via rhizomes. The nine localized populations constituting this species provide an opportunity to examine the extent of clonality and spatial genotypic diversity to evaluate its evolutionary prospects. METHODS Ten microsatellite loci were used to compare genetic and genotypic diversity across all sites with more intensive sampling at four locations (n = 185). The spatial distribution of genotypes and chloroplast DNA haplotypes based on the trnQ-rps16 intergenic spacer region were compared. Chromosome counts provided a basis for examining genetic profiles inconsistent with diploidy. KEY RESULTS Microsatellite analysis identified 46 multilocus genotypes (MLGs) in eight multilocus clonal lineages (MLLs). MLLs are not shared among sites, with two exceptions. Spatial autocorrelation was significant to 1·6 km. Genotypic richness ranged from 0 to 0·33. Somatic mutation is likely to contribute to minor variation between MLGs within clonal lineages. The eight chloroplast haplotypes identified were correlated with eight MLLs defined by ordination and generally restricted to single populations. Triploidy is the most likely reason for tri-allelic patterns. CONCLUSIONS Grevillea renwickiana comprises few genetic individuals. Sterility has most likely been induced by triploidy. Extensive lateral suckering in long-lived sterile clones facilitates the accumulation of somatic mutations, which contribute to the measured genetic diversity. Genetic conservation value may not be a function of population size. Despite facing evolutionary stagnation, sterile clonal species can play a vital role in mitigating ecological instability as floras respond to rapid environmental change.

Mycorrhizal specificity of Diuris fragrantissima (Orchidaceae) and persistence in a reintroduced population

This study investigated the diversity and specificity of mycorrhizal fungi associated with five Diuris (Orchidaceae) taxa in south-eastern Australia, as part of a reintroduction program for the endangered species Diuris fragrantissima. We compared fungi isolated from D. fragrantissima occurring naturally in the only remaining population with those from artificially cultivated plants and reintroduced plants 18 months after planting in a new field site west of Melbourne. Genetic similarity of nuclear internal transcribed spacer and nuclear large subunit DNA sequences showed that Diuris taxa associate with a narrow taxonomic range of fungi within the cosmopolitan family Tulasnellaceae in the Rhizoctonia alliance. All fungal isolates induced host seed germination and hence were considered mycorrhizal. Fungal isolates from naturally occurring D. fragrantissima plants showed a higher level of genetic similarity than fungi isolated from cultivated plants. This observation suggests that, historically, the species may have associated with a more genetically variable range of Tulasnella fungi. Artificially cultivated D. fragrantissima were propagated aseptically from seed and spontaneously formed mycorrhizal associations within 6 months of transfer to potting media. Wild collected D. fragrantissima plants maintained in cultivation for over 30 years were found to contain mycorrhizal fungi similar to those isolated from naturally occurring plants in 2004–2006. Mycorrhizal associations in artificially cultivated D. fragrantissima were present in 18 randomly sampled plants 18 months after reintroduction. Further, associations formed between several reintroduced plants and a fungus concurrently inoculated into site soil. We propose that future orchid reintroductions may benefit from the concurrent addition of suitable mycorrhizal fungi to site soil. Maintenance of orchid mycorrhizal relationships after reintroduction is essential to improve long-term viability of reintroduced populations.

Effects of environmental conditions on the production of hypocotyl hairs in seedlings of Melaleuca ericifolia (swamp paperbark)

The production of hypocotyl hairs in the early stages of seedling development can strongly influence the success with which plants recruit sexually in harsh environments. Although wetlands are one type of environment in which seedlings might be expected to develop hypocotyl hairs, there have been few studies of these structures in the woody aquatic plants. We investigated the production of hypocotyl hairs in Melaleuca ericifolia Sm., a small wetland tree widely distributed across swampy coastal areas of south-eastern Australia, in relation to water availability, salinity, temperature and light regime. Hypocotyl hairs were ~20 mm long × 30 μm wide; in contrast, root hairs were generally less than 5 mm long and 15 μm wide. Hypocotyl hairs were produced only under a narrow range of environmental conditions—low salinity, low water availability, moderate temperature, and darkness—and seedlings that failed to produce hypocotyl hairs did not survive. Since the conditions under which hypocotyl hairs were produced were at least as, and possibly even more, restricted than those required for successful germination of seeds, it is likely that the successful sexual recruitment of M. ericifolia would be rare and episodic under conditions existing in most coastal wetlands in south-eastern Australia.

Phylogeny of the holly grevilleas (Proteaceae) based on nuclear ribosomal and chloroplast DNA

Abstract. The holly grevilleas are an informal grouping of 15 species (19 taxa) of woody shrubs from south-eastern Australia, with a centre of distribution in central to western Victoria. Many of the species are narrowly endemic. The present study is the first molecular-phylogenetic analysis of the group, with the aim of providing an evolutionary framework for assessing species-level taxonomy and conservation priorities. Analyses using the nrDNA internal transcribed spacer (ITS) regions were complicated by the presence of divergent paralogues, including inferred pseudogenes; analyses restricted to presumed orthologous, functional ITS sequences were uninformative. Combined analyses of three chloroplast intergenic spacers (trnQ–5′rps16, trnL–trnF and rpoB–trnC) strongly support the monophyly of a core group of 16 taxa (the ‘southern holly grevilleas’) from Victoria and South Australia. However, nodes outside this group are poorly resolved and poorly supported, and the relationships of taxa from New South Wales and eastern Victoria (the ‘northern holly grevilleas’) are unclear. Among the southern holly grevilleas, the following four distinct and partly sympatric cpDNA clades are identified: the ‘Grevillea ilicifolia’, ‘G. aquifolium’, ‘G. dryophylla’ and ‘G. repens’ clades, among which the earliest and most strongly supported divergence is that of the western-most ‘G. ilicifolia’ clade. Variation in cpDNA is incongruent with current species-level taxonomy, especially for G. aquifolium (polyphyletic), G. montis-cole (polyphyletic, but the two subspecies each monophyletic) and G. microstegia (nested in G. aquifolium). The effects of incomplete chloroplast lineage sorting, gene flow through hybridisation or introgression, and inappropriate taxonomy are possible explanations for this incongruence. The formal conservation listing for some species within the holly grevillea group requires re-evaluation.