Historic and contemporary biogeographic perspectives on range-wide spatial genetic structure in a widespread seagrass.

Historical and contemporary processes drive spatial patterns of genetic diversity. These include climate-driven range shifts and gene flow mediated by biogeographical influences on dispersal. Assessments that integrate these drivers are uncommon, but critical for testing biogeographic hypotheses. Here, we characterize intraspecific genetic diversity and spatial structure across the entire distribution of a temperate seagrass to test marine biogeographic concepts for southern Australia. Predictive modeling was used to contrast the current Posidonia australis distribution to its historical distribution during the Last Glacial Maximum (LGM). Spatial genetic structure was estimated for 44 sampled meadows from across the geographical range of the species using nine microsatellite loci. Historical and contemporary distributions were similar, with the exception of the Bass Strait. Genetic clustering was consistent with the three currently recognized biogeographic provinces and largely consistent with the finer-scale IMCRA bioregions. Discrepancies were found within the Flindersian province and southwest IMCRA bioregion, while two regions of admixture coincided with transitional IMCRA bioregions. Clonal diversity was highly variable but positively associated with latitude. Genetic differentiation among meadows was significantly associated with oceanographic distance. Our approach suggests how shared seascape drivers have influenced the capacity of P. australis to effectively track sea level changes associated with natural climate cycles over millennia, and in particular, the recolonization of meadows across the Continental Shelf following the LGM. Genetic structure associated with IMCRA bioregions reflects the presence of stable biogeographic barriers, such as oceanic upwellings. This study highlights the importance of biogeography to infer the role of historical drivers in shaping extant diversity and structure.

Unexpectedly low paternal diversity is associated with infrequent pollinator visitation for a bird-pollinated plant.

The behaviour of pollinators has important consequences for plant mating. Nectar-feeding birds often display behaviour that results in more pollen carryover than insect pollinators, which is predicted to result in frequent outcrossing and high paternal diversity for bird-pollinated plants. We tested this prediction by quantifying mating system parameters and bird visitation in three populations of an understory bird-pollinated herb, Anigozanthos humilis (Haemodoraceae). Microsatellite markers were used to genotype 131 adult plants, and 211 seeds from 23 maternal plants, from three populations. While outcrossing rates were high, estimates of paternal diversity were surprisingly low compared with other bird-pollinated plants. Despite nectar-feeding birds being common at the study sites, visits to A. humilis flowers were infrequent (62 visits over 21,552 recording hours from motion-triggered cameras, or equivalent to one visit per flower every 10 days), and the majority (76%) were by a single species, the western spinebill Acanthorhynchus superciliosus (Meliphagidae). Pollen counts from 30 captured honeyeaters revealed that A. humilis comprised just 0.3% of the total pollen load. For 10 western spinebills, A. humilis pollen comprised only 4.1% of the pollen load, which equated to an average of 3.9 A. humilis pollen grains per bird. Taken together, our findings suggest that low visitation rates and low pollen loads of floral visitors have led to the low paternal diversity observed in this understory bird-pollinated herb. As such, we shed new light on the conditions that can lead to departures from high paternal diversity for plants competing for the pollination services of generalist nectar-feeding birds.

Optimising the conservation of genetic diversity of the last remaining population of a critically endangered shrub.

An understanding of genetic diversity and the population genetic processes that impact future population viability is vital for the management and recovery of declining populations of threatened species. Styphelia longissima (Ericaceae) is a critically endangered shrub, restricted to a single fragmented population near Eneabba, 250 km north of Perth, Western Australia. For this population, we sought to characterize population genetic variation and its spatial structure, and aspects of the mating portfolio, from which strategies that optimize the conservation of this diversity are identified. A comprehensive survey was carried out and 220 adults, and 106 seedlings from 14 maternal plants, were genotyped using 13 microsatellite markers. Levels of genetic variation and its spatial structure were assessed, and mating system parameters were estimated. Paternity was assigned to the offspring of a subsection of plants, which allowed for the calculation of realized pollen dispersal. Allelic richness and levels of expected heterozygosity were higher than predicted for a small isolated population. Spatial autocorrelation analysis identified fine-scale genetic structure at a scale of 20 m, but no genetic structure was found at larger scales. Mean outcrossing rate (t m = 0.66) reflects self-compatibility and a mixed-mating system. Multiple paternity was low, where 61 % of maternal siblings shared the same sire. Realized pollen dispersal was highly restricted, with 95 % of outcrossing events occurring at 7 m or less, and a mean pollen dispersal distance of 3.8 m. Nearest-neighbour matings were common (55 % of all outcross events), and 97 % of mating events were between the three nearest-neighbours. This study has provided critical baseline data on genetic diversity, mating system and pollen dispersal for future monitoring of S. longissima. Broadly applicable conservation strategies such as implementing a genetic monitoring plan, diluting spatial genetic structure in the natural population, genetically optimizing ex situ collections and incorporating genetic knowledge into translocations will help to manage the future erosion of the high genetic variation detected.

Variation in reproductive effort, genetic diversity and mating systems across Posidonia australis seagrass meadows in Western Australia.

Populations at the edges of their geographical range tend to have lower genetic diversity, smaller effective population sizes and limited connectivity relative to centre of range populations. Range edge populations are also likely to be better adapted to more extreme conditions for future survival and resilience in warming environments. However, they may also be most at risk of extinction from changing climate. We compare reproductive and genetic data of the temperate seagrass, Posidonia australis on the west coast of Australia. Measures of reproductive effort (flowering and fruit production and seed to ovule ratios) and estimates of genetic diversity and mating patterns (nuclear microsatellite DNA loci) were used to assess sexual reproduction in northern range edge (low latitude, elevated salinities, Shark Bay World Heritage Site) and centre of range (mid-latitude, oceanic salinity, Perth metropolitan waters) meadows in Western Australia. Flower and fruit production were highly variable among meadows and there was no significant relationship between seed to ovule ratio and clonal diversity. However, Shark Bay meadows were two orders of magnitude less fecund than those in Perth metropolitan waters. Shark Bay meadows were characterized by significantly lower levels of genetic diversity and a mixed mating system relative to meadows in Perth metropolitan waters, which had high genetic diversity and a completely outcrossed mating system. The combination of reproductive and genetic data showed overall lower sexual productivity in Shark Bay meadows relative to Perth metropolitan waters. The mixed mating system is likely driven by a combination of local environmental conditions and pollen limitation. These results indicate that seagrass restoration in Shark Bay may benefit from sourcing plant material from multiple reproductive meadows to increase outcrossed pollen availability and seed production for natural recruitment.

Seeds in motion: Genetic assignment and hydrodynamic models demonstrate concordant patterns of seagrass dispersal.

Movement is fundamental to the ecology and evolutionary dynamics within species. Understanding movement through seed dispersal in the marine environment can be difficult due to the high spatial and temporal variability of ocean currents. We employed a mutually enriching approach of population genetic assignment procedures and dispersal predictions from a hydrodynamic model to overcome this difficulty and quantify the movement of dispersing floating fruit of the temperate seagrass Posidonia australis Hook.f. across coastal waters in south-western Australia. Dispersing fruit cohorts were collected from the water surface over two consecutive years, and seeds were genotyped using microsatellite DNA markers. Likelihood-based genetic assignment tests were used to infer the meadow of origin for seed cohorts and individuals. A three-dimensional hydrodynamic model was coupled with a particle transport model to simulate the movement of fruit at the water surface. Floating fruit cohorts were mainly assigned genetically to the nearest meadow, but significant genetic differentiation between cohort and most likely meadow of origin suggested a mixed origin. This was confirmed by genetic assignment of individual seeds from the same cohort to multiple meadows. The hydrodynamic model predicted 60% of fruit dispersed within 20 km, but that fruit was physically capable of dispersing beyond the study region. Concordance between these two independent measures of dispersal provides insight into the role of physical transport for long distance dispersal of fruit and the consequences for spatial genetic structuring of seagrass meadows.

Isolation and characterization of microsatellite primers for the critically endangered shrub Styphelia longissima (Ericaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for population genetic analysis in the rare shrub Styphelia longissima (Ericaceae). METHODS AND RESULTS: We generated ca. 2.5 million sequence reads using a Personal Genome Machine semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80-480 bp. From these, 30 primer pairs were selected and screened using PCR; of these, 16 loci were found to be polymorphic, four loci were monomorphic, and 10 loci did not amplify reliably for S. longissima. For a sample of 57 plants from the only known population, the number of alleles observed for these 16 loci ranged from two to 21 and expected heterozygosity ranged from 0.49 to 0.91. These markers were also amplified in Astroloma xerophyllum, a closely related species. CONCLUSIONS: These markers will be used to characterize population genetic variation, spatial genetic structure, mating system parameters, and dispersal to aid in the management and conservation of the rare shrub S. longissima.

Characterization and transferability of microsatellites for the Kangaroo Paw, Anigozanthos manglesii (Haemodoraceae).

PREMISE OF THE STUDY: Microsatellites were developed for the future assessment of population genetic structure, mating system, and dispersal of the perennial kangaroo paw, Anigozanthos manglesii (Haemodoraceae), and related species. METHODS AND RESULTS: Using a Personal Genome Machine (PGM) semiconductor sequencer, ca. 4.03 million sequence reads were generated. QDD pipeline software was used to identify 190,000 microsatellite-containing regions and priming sites. From these, 90 were chosen and screened using PCR, and 15 polymorphic markers identified. These sites amplified di-, tri-, and pentanucleotide repeats with one to 20 alleles per locus. Primers were also amplified across congeners A. bicolor, A. flavidus, A. gabrielae, A. humilis, A. preissii, A. pulcherrimus, A. rufus, and A. viridis to assess cross-species transferability. CONCLUSIONS: These markers provide a resource for population genetic studies in A. manglesii and other species within the genus.

Isolation and characterization of 13 microsatellites for the rare endemic shrub Tetratheca erubescens (Elaeocarpaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the rare Tetratheca erubescens (Elaeocarpaceae) to assess genetic diversity and spatial structuring. METHODS AND RESULTS: We generated ca. 2.7 million sequence reads using a Personal Genome Machine (PGM) semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80-480 bp. From these, 30 primer pairs were selected and screened using PCR, from which 11 loci were found to be polymorphic and amplified reliably. For a sample of 95 plants from three populations, the number of alleles observed for these 11 loci ranged from two to seven and expected heterozygosity ranged from 0.06 to 0.72. No consistent evidence for null alleles or departure from Hardy-Weinberg equilibrium was found for any of the 11 loci. CONCLUSIONS: These markers will enable the quantification of genetic impact of proposed mining activities on the narrow endemic T. erubescens.

Reproduction at the extremes: pseudovivipary, hybridization and genetic mosaicism in Posidonia australis (Posidoniaceae).

BACKGROUND AND AIMS: Organisms occupying the edges of natural geographical ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions, often associated with climate change and exacerbated at species' geographical range edges, are known to trigger alternative responses in reproduction. This study reports the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. These novel plantlets are described and a combination of microsatellite DNA markers and flow cytometry is used to determine their origin. METHODS: Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of the adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. KEY RESULTS: All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. It was found that 87 % of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2 C value) for samples from Shark Bay and from a separate site much further south was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). CONCLUSIONS: Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. This study documents a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggests that the lower-than-usual water temperature triggered unfertilized inflorescences to 'switch' to growing plantlets that were adventitious clones of their maternal parent. This may have important long-term implications as both genetic and ecological constraints may limit the ability to adapt or range-shift; this seagrass meadow in Shark Bay already has low genetic diversity, no sexual reproduction and no seedling recruitment.

The critical role of ants in the extensive dispersal of Acacia seeds revealed by genetic parentage assignment.

Ants are prominent seed dispersal agents in many ecosystems, and dispersal distances are small in comparison with vertebrate dispersal agents. However, the distance and distribution of ant-mediated dispersal in arid/semi-arid environments remains poorly explored. We used microsatellite markers and parentage assignment to quantify the distance and distribution of dispersed seeds of Acacia karina, retrieved from the middens of Iridomyrmex agilis and Melophorus turneri perthensis. From parentage assignment, we could not distinguish the maternal from each parent pair assigned to each seed, so we applied two approaches to estimate dispersal distances, one conservative (CONS), where the parent closest to the ant midden was considered to be maternal, and the second where both parents were deemed equally likely (EL) to be maternal, and used both distances. Parentage was assigned to 124 seeds from eight middens. Maximum seed dispersal distances detected were 417 m (CONS) and 423 m (EL), more than double the estimated global maximum. Mean seed dispersal distances of 40 m (±5.8 SE) (CONS) and 79 m (±6.4 SE) (EL) exceeded the published global average of 2.24 m (±7.19 SD) by at least one order of magnitude. For both approaches and both ant species, seed dispersal was predominantly (44-84% of all seeds) within 50 m from the maternal source, with fewer dispersal events at longer distances. Ants in this semi-arid environment have demonstrated a greater capacity to disperse seeds than estimated elsewhere, which highlights their important role in this system, and suggests significant novel ecological and evolutionary consequences for myrmecochorous species in arid/semi-arid Australia.

Microsatellite markers from the Ion Torrent: a multi-species contrast to 454 shotgun sequencing.

The development and screening of microsatellite markers have been accelerated by next-generation sequencing (NGS) technology and in particular GS-FLX pyro-sequencing (454). More recent platforms such as the PGM semiconductor sequencer (Ion Torrent) offer potential benefits such as dramatic reductions in cost, but to date have not been well utilized. Here, we critically compare the advantages and disadvantages of microsatellite development using PGM semiconductor sequencing and GS-FLX pyro-sequencing for two gymnosperm (a conifer and a cycad) and one angiosperm species. We show that these NGS platforms differ in the quantity of returned sequence data, unique microsatellite data and primer design opportunities, mostly consistent with the differences in read length. The strength of the PGM lies in the large amount of data generated at a comparatively lower cost and time. The strength of GS-FLX lies in the return of longer average length sequences and therefore greater flexibility in producing markers with variable product length, due to longer flanking regions, which is ideal for capillary multiplexing. These differences need to be considered when choosing a NGS method for microsatellite discovery. However, the ongoing improvement in read lengths of the NGS platforms will reduce the disadvantage of the current short read lengths, particularly for the PGM platform, allowing greater flexibility in primer design coupled with the power of a larger number of sequences.

Characterization and cross application of novel microsatellite markers for a rare sedge, Lepidosperma gibsonii (Cyperaceae).

PREMISE OF THE STUDY: Ten polymorphic microsatellite loci for the rare sword sedge Lepidosperma gibsonii (Cyperaceae) were characterized for the future study of population structure, hybridization, and clonality. METHODS AND RESULTS: Twenty samples from each of three populations were screened with the markers to assess genetic variation. Observed population heterozygosities ranged from 0.35 to 1.00, and number of alleles observed per locus ranged from eight to 23. No departures from Hardy-Weinberg equilibrium were detected for any locus in any population. Single samples from 14 species were screened to examine the transferability of the microsatellites to other species of Lepidosperma. At least eight out of 10 loci amplified in all species tested. CONCLUSIONS: These loci will be useful for studying genetic variation, hybridization, dispersal, and breeding systems in Lepidosperma, a ubiquitous element of the flora of southern Australia.