Floral Variation and Breeding System in Distylous and Homostylous Species of Clonal Aquatic 'Nymphoides' (Menyanthaceae)

This study focuses on floral variation and the breeding system of two species of the clonal aquatic 'Nymphoides' (Menyanthaceae), in particular, the phenomena of distyly, loss of sex and homostyly. Distyly is a floral design that promotes efficient pollen transfer while reducing self-interference and self-pollination. Populations of distylous species contain two floral morphs that differ in the reciprocal positioning of the sex organs; i.e. reciprocal herkogamy. Distylous species usually possess a dimorphic incompatibility system in which only pollination between flowers of different morphs produce seed. There are, however, atypical features of distyly, which provide excellent examples to study the maintenance or evolutionary transition of this breeding system. 'Nymphoides montana' is a perennial clonal aquatic species, native to Australia, found in shallow water on the edges of wetlands. Selective factors that maintain distyly were sought by examining floral morphologies, compatibility relationships, morph frequencies and reproductive success in three 'N. montana' populations

The expansion of sterile 'Arundo donax' (Poaceae) in southeastern Australia is accompanied by genotypic variation

Reproductive modes and dispersal mechanisms shape the genotypic structure of populations which in turn influences a species' capacity for successful invasion. Understanding the limitations in a species' reproductive capacity may assist with finding the best approach to control the expansion of an invasive species. To understand the founder history against the spread of 'Arundo donax', we examined genotypic variation, using Inter Simple Sequence Repeats (ISSR) markers, and we determined ploidy and fertility in 10 single (one patch) and seven multiple (spatially distinct patches) stands scattered along three river systems in southeastern Australia. No seed was detected in any plant and shriveled anthers did not produce pollen. Somatic chromosome counts identified uneven ploidy levels (2n = 7x = 84), which may be the cause of sterility in 'A. donax'. Of the 58 plants sampled we detected 38 genotypes, and genotypic variation was moderate to high within each river system (G/N = 0.485-1.000 and Simpson's D = 0.881-1.000). Three genotypes were found in more than one stand, suggesting that the invasive spread of 'A. donax' by vegetative propagules is not a rare event. Genetically distinct genotypes were detected in all single stands. Sections of the river systems that contained multiple stands of 'A. donax' revealed that each stand was a unique genotype; this may be attributable to multiple founders, somatic mutations, and/or polyploidy. Our results indicate that sterility has had little effect on retarding the dispersal of heptaploid 'A. donax' in southeastern Australia, and reproduction through vegetative fragments is an effective dispersal mechanism

The discovery of microsatellite markers for 'Hardenbergia violacea' (Fabaceae), using next-generation sequencing

'Hardenbergia violacea' is a climbing to prostrate shrub endemic to woodlands in the south east of Australia. We used next generation sequencing to develop a species-specific primer set. We obtained 106,529 reads in FASTA format with an average read length of 548 bp. DNA was successfully amplified for 20 markers and eight markers were characterized. The degree of polymorphism of these loci was tested on 95 samples from five populations. The total number of alleles ranged from two to 11 and observed and expected heterozygosities ranged from 0.0 to 0.807 and 0.0 to 0.933, respectively. Inbreeding coefficients were highly variable among populations, suggesting a mixture of selfing and outcrossing in nature. The characterized loci will be useful for studies of population structure, gene flow, mating systems, and the conservation and selection of provenances for restoration projects in degraded habitats

Somatic mutations contribute to genotypic diversity in sterile and fertile populations of the threatened shrub, 'Grevillea rhizomatosa' (Proteaceae)

Background and Aims: 'Grevillea rhizomatosa' is a spreading shrub which exhibits multiple breeding strategies within a narrow area in the fire-prone heathlands of eastern Australia. Reproductive strategies include self-compatibility, self-incompatibility and clonality (with and without sterility). The close proximity of contrasting breeding systems provides an opportunity to explore the evolution of sterility and to compare and contrast the origins of genotypic diversity (recombinant or somatic) against degrees of sexual expression. Methods: ISSR markers for 120 band positions (putative loci) were used to compare genetic diversity among five populations at a macro-scale of 5 m between samples (n = 244 shrubs), and at a micro-scale of nearest neighbours for all plants in five 25-m² quadrats with contrasting fertilities (n = 162 shrubs). Nearest-neighbour sampling included several clusters of connected ramets. Matrix incompatibility (MIC) analyses were used to evaluate the relative contribution of recombination and somatic mutation to genotype diversity. Key Results: High levels of genotypic diversity were found in all populations regardless of fertilities (fertile populations, G/N ≥ 0.94; sterile populations, G/N ≥ 0.97) and most sterile populations had a unique genetic profile. Somatic mutations were detected along connected ramets in ten out of 42 ramet clusters. MIC analyses showed that somatic mutations have contributed to diversity in all populations and particularly so in sterile populations. Conclusions: Somatic mutations contribute significantly to gene diversity in sterile populations of 'Grevillea rhizomatosa', the accumulation of which is the likely cause of male and female sterility. High levels of genetic diversity therefore may not always be synonymous with sexual fitness and genetic health. We hypothesize that frequent fires drive selection for clonal reproduction, at the cost of flowering such that sexual functions are not maintained through selection, and the build-up of somatic mutations in meristems results in high genotype diversity at the cost of pollen and ovule fertilities

Cost-Effective Microsatellite Markers for <i>Banksia integrifolia</i> (Proteaceae)

Premise of the study: Microsatellite markers were developed to assess the level of genetic variation and population structure in <i>Banksia integrifolia</i>, a widespread species endemic to eastern Australia. Methods and Results: We used next-generation sequencing approaches to identify and develop 11 polymorphic microsatellite markers with perfect tri- and tetranucleotide repeats. We tested these markers with 71 specimens from three populations. Observed and expected heterozygosities ranged from 0.0 to 0.875 and 0.0 to 0.763, respectively. Conclusions: The developed markers will be valuable for studies of the population structure, mating system, and selection of provenances for restoration projects involving <i>B. integrifolia</i>

Clonal growth is enhanced in the absence of a mating morph: a comparative study of fertile stylar polymorphic and sterile monomorphic populations of Nymphoides montana (Menyanthaceae)

BACKGROUND AND AIMS: Many aquatic species with stylar polymorphisms have the capacity for clonal and sexual reproduction and are sensitive to the balance of the two reproductive modes when there are a limited number of mating morphs within a population. This study asked how the clonal and sexual reproductive modes perform in populations that contain only a single morph and where fitness gain through sexual reproduction is rare. In clonal aquatic Nymphoides montana, polymorphic populations normally contain two mating morphs in equal frequencies. Populations are sexually fertile and appear to be maintained by pollen transfer between the two partners. However, in a monomorphic population of N. montana where mating opportunities are unavailable, female and male function is impaired and clonality maintains the population. Here, the consequences of intraspecific variation in sexuality were explored between monomorphic and polymorphic N. montana populations in eastern Australia. METHODS: Comparative measurements of male and female fertility, total dry mass and genotypic diversity using ISSR markers were made between populations with variable sexuality. KEY RESULTS AND CONCLUSIONS: Very few seeds were produced in the monomorphic population under natural and glasshouse conditions due to dysfunctional pollen and ovules. Stigma–anther separation was minimal in the monomorphic population, which may be a consequence of the relaxed selective pressures that regulate the maintenance of sexual function. However, clonal reproduction was favoured at the expense of sexual reproduction in the monomorphic population; this may facilitate the establishment of sterility throughout the population via resource reallocation or pleiotropic effects. The ISSR results showed that the monomorphic population was one large, single genotype, unlike the multi-genotypic fertile polymorphic populations. Evolutionary loss of sex in a clonal population in which a mating morph is absent was evident; under these conditions clonal growth may assure reproduction and expand the population via spreading stolons