Pollen adaptation to ant pollination: a case study from the Proteaceae.

BACKGROUND AND AIMS: Ant-plant associations are widely diverse and distributed throughout the world, leading to antagonistic and/or mutualistic interactions. Ant pollination is a rare mutualistic association and reports of ants as effective pollinators are limited to a few studies. Conospermum (Proteaceae) is an insect-pollinated genus well represented in the south-western Australia biodiversity hotspot, and here we aimed to evaluate the role of ants as pollinators of C. undulatum. METHODS: Pollen germination after contact with several species of ants and bees was tested for C. undulatum and five co-flowering species for comparison. We then sampled the pollen load of floral visitors of C. undulatum to assess whether ants carried a pollen load sufficient to enable pollination. Lastly, we performed exclusion treatments to assess the relative effect of flying- and non-flying-invertebrate floral visitors on the reproduction of C. undulatum. For this, we measured the seed set under different conditions: ants exclusion, flying-insects exclusion and control. KEY RESULTS: Pollen of C. undulatum, along with the other Conospermum species, had a germination rate after contact with ants of ~80 % which did not differ from the effect of bees; in contrast, the other plant species tested showed a drop in the germination rate to ~10 % following ant treatments. Although ants were generalist visitors, they carried a pollen load with 68-86 % of suitable grains. Moreover, ants significantly contributed to the seed set of C. undulatum. CONCLUSIONS: Our study highlights the complexity of ant-flower interactions and suggests that generalizations neglecting the importance of ants as pollinators cannot be made. Conospermum undulatum has evolved pollen with resistance to the negative effect of ant secretions on pollen grains, with ants providing effective pollination services to this threatened species.

Specialization to Extremely Low-Nutrient Soils Limits the Nutritional Adaptability of Plant Lineages.

Specialization to extreme selective situations promotes the acquisition of traits whose coadaptive integration may compromise evolutionary flexibility and adaptability. We test this idea in the context of the foliar stoichiometry of plants native to the South African Cape. Whereas foliar concentrations of nitrogen, phosphorus (P), potassium (K), calcium, magnesium, and sodium showed strong phylogenetic signal, as did the foliar ratios of these nutrients to P, the same was not true of the corresponding soil values. In addition, although foliar traits were often related to soil values, the coefficients of determination were consistently low. These results identify foliar stoichiometry as having a strong genetic component, with variation in foliar nutrient concentrations, especially [P] and [K], being identified as potentially adaptive. Comparison of stoichiometric variation across 11 similarly aged clades revealed consistently low foliar nutrient concentrations in lineages showing specialization to extremely low-nutrient fynbos heathlands. These lineages also display lower rates of evolution of these traits as well as a reduced tendency for foliar [P] to track soil [P]. Reduced evolutionary lability and adaptability in the nutritional traits of fynbos-specialist lineages may explain the floristic distinctness of the fynbos flora and implies a reduced scope for edaphically driven ecological speciation.

Plant functional traits of dominant native and invasive species in mediterranean-climate ecosystems.

The idea that dominant invasive plant species outperform neighboring native species through higher rates of carbon assimilation and growth is supported by several analyses of global data sets. However, theory suggests that native and invasive species occurring in low-resource environments will be functionally similar, as environmental factors restrict the range of observed physiological and morphological trait values. We measured resource-use traits in native and invasive plant species across eight diverse vegetation communities distributed throughout the five mediterranean-climate regions, which are drought prone and increasingly threatened by human activities, including the introduction of exotic species. Traits differed strongly across the five regions. In regions with functional differences between native and invasive species groups, invasive species displayed traits consistent with high resource acquisition; however, these patterns were largely attributable to differences in life form. We found that species invading mediterranean-climate regions were more likely to be annual than perennial: three of the five regions were dominated by native woody species and invasive annuals. These results suggest that trait differences between native and invasive species are context dependent and will vary across vegetation communities. Native and invasive species within annual and perennial groups had similar patterns of carbon assimilation and resource use, which contradicts the widespread idea that invasive species optimize resource acquisition rather than resource conservation. .

Genome-wide scans detect adaptation to aridity in a widespread forest tree species.

Patterns of adaptive variation within plant species are best studied through common garden experiments, but these are costly and time-consuming, especially for trees that have long generation times. We explored whether genome-wide scanning technology combined with outlier marker detection could be used to detect adaptation to climate and provide an alternative to common garden experiments. As a case study, we sampled nine provenances of the widespread forest tree species, Eucalyptus tricarpa, across an aridity gradient in southeastern Australia. Using a Bayesian analysis, we identified a suite of 94 putatively adaptive (outlying) sequence-tagged markers across the genome. Population-level allele frequencies of these outlier markers were strongly correlated with temperature and moisture availability at the site of origin, and with population differences in functional traits measured in two common gardens. Using the output from a canonical analysis of principal coordinates, we devised a metric that provides a holistic measure of genomic adaptation to aridity that could be used to guide assisted migration or genetic augmentation.

Plasticity of functional traits varies clinally along a rainfall gradient in Eucalyptus tricarpa.

Widespread species often occur across a range of climatic conditions, through a combination of local genetic adaptations and phenotypic plasticity. Species with greater phenotypic plasticity are likely to be better positioned to cope with rapid anthropogenic climate changes, while those displaying strong local adaptations might benefit from translocations to assist the movement of adaptive genes as the climate changes. Eucalyptus tricarpa occurs across a climatic gradient in south-eastern Australia, a region of increasing aridity, and we hypothesized that this species would display local adaptation to climate. We measured morphological and physiological traits reflecting climate responses in nine provenances from sites of 460 to 1040 mm annual rainfall, in their natural habitat and in common gardens near each end of the gradient. Local adaptation was evident in functional traits and differential growth rates in the common gardens. Some traits displayed complex combinations of plasticity and genetic divergence among provenances, including clinal variation in plasticity itself. Provenances from drier locations were more plastic in leaf thickness, whereas leaf size was more plastic in provenances from higher rainfall locations. Leaf density and stomatal physiology (as indicated by δ(13)C and δ(18)O) were highly and uniformly plastic. In addition to variation in mean trait values, genetic variation in trait plasticity may play a role in climate adaptation.

Dynamics of phreatophyte root growth relative to a seasonally fluctuating water table in a Mediterranean-type environment.

While seasonal redistribution of fine root biomass in response to fluctuations in groundwater level is often inferred in phreatophytic plants, few studies have observed the in situ growth dynamics of deep roots relative to those near the surface. We investigated the root growth dynamics of two Banksia species accessing a seasonally dynamic water table and hypothesized that root growth phenology varied with depth, i.e. root growth closest to the water table would be influenced by water table dynamics rather than surface micro-climate. Root in-growth bags were used to observe the dynamics of root growth at different soil depths and above-ground growth was also assessed to identify whole-plant growth phenology. Root growth at shallow depths was found to be in synchrony with above-ground growth phenophases, following increases in ambient temperature and soil water content. In contrast, root growth at depth was either constant or suppressed by saturation. Root growth above the water table and within the capillary fringe occurred in all seasons, corresponding with consistent water availability and aerobic conditions. However, at the water table, a seasonal cycle of root elongation with drawdown in summer followed by trimming in response to water table rise and saturation in winter, was observed. The ability to grow roots year-round at the capillary fringe and redistribute fine root biomass in response to groundwater drawdown is considered critical in allowing phreatophytes, in seasonally water-limited environments, to maintain access to groundwater throughout the year.

Emission factors and composition of PM2.5 from laboratory combustion of five Western Australian vegetation types.

This study investigated the emission of PM10 and PM2.5 (particulates with diameters of less than 10 µm and 2.5 µm, respectively) and the chemical composition of PM2.5 from laboratory combustion of five Australian vegetation types (three grasslands, a woodland and a forest). A mix of plants representative of Banksia (woodland) and Jarrah (forest) and three types of grasses (Spinifex - Triodia basedowii; Kimberley grass - Sehima nervosum and Heteropogon contortus; and an invasive grass (Veldt) - Ehrharta calycina) were burnt in 9 combustion conditions comprised of 3 fuel moisture levels (dry, moist, wet) and 3 air flow rates (no, low and high flow). PM (particulate matter) samples were collected onto filters and measured using gravimetric analysis. PM2.5 was then extracted and analyzed for water-soluble metals and polycyclic aromatic hydrocarbons (PAH) concentrations. The largest proportion of PM10 (98%) from vegetation fires was PM2.5. Banksia yielded the highest PM2.5 emission factor (EF), followed by Jarrah and Spinifex. Veldt grass combustion generated significantly higher emissions of PM2.5 compared with the other two grass types. High moisture contents and flow rates resulted in larger emissions of PM2.5. A strong correlation (R2 = 0.84) was observed between the EF for PM2.5 and combustion efficiency, suggesting higher PM emission with lower combustion efficiencies. Potassium and sodium were the most abundant PM2.5-bound water soluble metals, accounting for more than 97% of the total mass of metals analyzed. PAHs were found in significant concentrations, including the carcinogenic benzo(a)pyrene. Pyrene and fluoranthene were the most abundant PAHs detected, accounting for nearly 40% mass of the total PAHs. Indeno(1,2,3-cd)pyrene and benzo(g,h,i)perylene ratio (IND/IND + BghiP) appeared to be produced in a diagnostic ratio that indicated that the PAHs were derived from vegetation fires rather than other sources of emissions. The EF for PM2.5 and its chemical composition (water-soluble metals and PAHs) were strongly influenced by the type of vegetation burned. The results presented in this study could be useful in predicting the risks of human health effects on firefighters and the public who may be exposed to regular bushfires in Australia.

Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability.

Ratios of nitrogen (N) isotopes in leaves could elucidate underlying patterns of N cycling across ecological gradients. To better understand global-scale patterns of N cycling, we compiled data on foliar N isotope ratios (delta(15)N), foliar N concentrations, mycorrhizal type and climate for over 11,000 plants worldwide. Arbuscular mycorrhizal, ectomycorrhizal, and ericoid mycorrhizal plants were depleted in foliar delta(15)N by 2 per thousand, 3.2 per thousand, 5.9 per thousand, respectively, relative to nonmycorrhizal plants. Foliar delta(15)N increased with decreasing mean annual precipitation and with increasing mean annual temperature (MAT) across sites with MAT >or= -0.5 degrees C, but was invariant with MAT across sites with MAT < -0.5 degrees C. In independent landscape-level to regional-level studies, foliar delta(15)N increased with increasing N availability; at the global scale, foliar delta(15)N increased with increasing foliar N concentrations and decreasing foliar phosphorus (P) concentrations. Together, these results suggest that warm, dry ecosystems have the highest N availability, while plants with high N concentrations, on average, occupy sites with higher N availability than plants with low N concentrations. Global-scale comparisons of other components of the N cycle are still required for better mechanistic understanding of the determinants of variation in foliar delta(15)N and ultimately global patterns in N cycling.

Water stress vulnerability of four Banksia species in contrasting ecohydrological habitats on the Gnangara Mound, Western Australia.

This study investigated the interspecific differences in vulnerability to xylem embolism of four phreatophytes - two facultative phreatophytes (Banksia attenuata and B. menziesii) and two obligate phreatophytes (B. ilicifolia and B. littoralis). Species differences at the same position along an ecohydrological gradient on the Gnangara Groundwater Mound, Western Australia were determined in addition to intraspecific differences to water stress between populations in contrasting ecohydrological habitats. Stem- and leaf-specific hydraulic conductivity, as well as Huber values (ratio of stem to leaf area), were also determined to support these findings. We found that where water is readily accessible, there were no interspecific differences in vulnerability to water stress. In contrast both facultative phreatophyte species were more resistant to xylem embolism at the more xeric dune crest site than at the wetter bottom slope site. B. ilicifolia did not differ in vulnerability to embolism, supporting its classification as an obligate phreatophyte. Other measured hydraulic traits (K(S), K(L) and Huber value) showed no adaptive responses, although there was a tendency for plants at the wetter site to have higher K(S) and K(L). This study highlights the influence site hydrological attributes can have on plant hydraulic architecture across species and environmental gradients.

Browsing and fire interact to suppress tree density in an African savanna.

Disturbances from fire and herbivory strongly affect savanna vegetation dynamics. In some savannas, fire especially may be instrumental in preserving the coexistence of trees and grasses. The role of herbivory by large mammals is less clear; herbivory has been shown variously to promote and to suppress tree establishment. Here we ask how interactions between herbivory and fire act to shape savanna vegetation dynamics via their effects on tree populations in Hluhluwe iMfolozi Park in KwaZulu Natal, South Africa, a savanna with a full complement of native large mammals. We examined the effects of herbivore exclusion on tree growth, mortality, and seedling establishment from 2000 to 2007 at 10 sites located in areas of low and high herbivore pressure throughout the park. Results were analyzed statistically and using Leslie matrix models of population dynamics. Herbivory and fire acted primarily to suppress sapling growth rather than on sapling mortality or seedling establishment. This indicates that browsing, like fire, suppresses tree density by imposing a demographic bottleneck on the maturation of saplings to adults. Model results suggest that, while browsing and fire each alone impacted growth, a combination of browsing and fire had much greater effects on tree density. Only fire and browsing together were able to prevent increases in tree density. These results suggest that, while soil resources, including nutrients and moisture, are probably instrumental in determining tree growth rates, disturbances from fire and herbivory may be instrumental in limiting tree cover and facilitating the coexistence of trees and grasses in savannas.

Floral display and habitat fragmentation: Effects on the reproductive success of the threatened mass-flowering Conospermum undulatum (Proteaceae).

Fragmentation of natural vegetation is currently one of the largest threats to plant populations and their interactions with pollinators. Plant reproductive susceptibility to habitat fragmentation has been investigated in many species; however, the response of wild mass-flowering species is poorly known, with research limited to mainly boreal plant species.Here, we studied twelve remnant populations of the threatened mass-flowering shrub Conospermum undulatum in the southwest Australian biodiversity hotspot, each presenting different population size, level of isolation, and floral display. We assessed the impact of fragmentation on (a) fruit and seed production; and (b) seed germination. To gain a deeper understanding of factors influencing the reproductive success of C. undulatum, we performed pollinator exclusion and self-pollination treatments to experimentally assess the mating system of this threatened shrub.We found C. undulatum to be strictly self-incompatible and totally reliant on pollinators visiting with an outcrossed pollen load to complete the reproductive cycle. Further, we found that fruit production dropped from 35% to <20% as a result of decreasing floral display. A reduction in population size from 880 to 5 plants and from ~700 to 0.21 in the floral display index led to a decrease in seed output, while a similar reduction in seed output, from 6% to 3%, was observed as a result of increasing isolation index from -21.41 to -0.04. Overall, seed germination was positively related to population size, and a negative relationship was found between germination and isolation. Synthesis and applications. Our results demonstrate the important relationship between pollinators and floral morphology in plants of southwest Australia that have coevolved with native pollinators and developed characteristic flower morphologies over long time frames. Indeed, due to its characteristic pollination mechanism, the self-incompatible C. undulatum can only rely on specialized native pollinators for pollen flow and cannot rely on its mass-flowering trait to attract generalist pollinators from coflowering species; neither can it compensate for the lack of visitors by promoting geitonogamy. Consequently, fragmentation has a significant effect on the reproductive output of C. undulatum, and size, isolation, and floral display of populations are important factors to be considered when planning conservation actions for the species.

Isolation, characterization, and cross-amplification of 20 microsatellite markers for Conospermum undulatum (Proteaceae).

PREMISE: Recent habitat fragmentation is posing a risk to the wavy-leaved smokebush, Conospermum undulatum (Proteaceae), a rare plant species endemic to southwestern Western Australia. Microsatellite markers are required to characterize the genetic diversity and structure of the species for conservation purposes and to facilitate ecological studies. METHODS AND RESULTS: Illumina MiSeq high-throughput sequencing was used to develop 20 novel microsatellite markers for C. undulatum. Polymorphism at each locus was assessed using 72 individuals from three natural populations. Nineteen markers were polymorphic, with the number of alleles per locus ranging from two to 21, and observed and expected heterozygosity ranging from 0.000 to 1.000 and 0.117 to 0.919, respectively. All markers successfully amplified in three congeneric species (C. stoechadis, C. canaliculatum and C. triplinervium). CONCLUSIONS: The microsatellite markers will be useful for revealing patterns of genetic diversity, dispersal dynamics, and hybridization events for C. undulatum to inform future conservation efforts.

Nutrient concentration ratios and co-limitation in South African grasslands.

*Assessing plant nutrient limitation is a fundamental part of understanding grassland dynamics. The ratio of concentrations of nitrogen (N) and phosphorus (P) in vegetation has been proposed as an index of the relative limitation of biomass production by N and P, but its utility has not been tested well in grasslands. *At five sites in Kruger National Park, South Africa, across soil and precipitation contrasts, N and P were added in a factorial design to grass-dominated plots. *Although the N:P ratio of unfertilized vegetation across all sites (5.8) would have indicated that production was N-limited, aboveground production was consistently co-limited by N and P. Aboveground production was still greater in plots fertilized with N and P than in those fertilized with just N, but the N:P ratio did not exceed standard thresholds for P limitation in N-fertilized vegetation. Comparisons among sites showed little pattern between site N:P ratio and relative responses to N and P. *When combined with results from other grassland fertilization studies, these data suggest that the N:P ratio of grasses has little ability to predict limitation in upland grasslands. Co-limitation between N and P appears to be much more widespread than would be predicted from simple assumptions of vegetative N:P ratios.

Assessing nitrogen fixation in mixed- and single-species plantations of Eucalyptus globulus and Acacia mearnsii.

Mixtures of Eucalyptus globulus Labill. and Acacia mearnsii de Wildeman are twice as productive as E. globulus monocultures growing on the same site in East Gippsland, Victoria, Australia, possibly because of increased nitrogen (N) availability owing to N(2) fixation by A. mearnsii. To investigate whether N(2) fixation by A. mearnsii could account for the mixed-species growth responses, we assessed N(2) fixation by the accretion method and the (15)N natural abundance method. Nitrogen gained by E. globulus and A. mearnsii mixtures and monocultures was calculated by the accretion method with plant and soil samples collected 10 years after plantation establishment. Nitrogen in biomass and soil confirmed that A. mearnsii influenced N dynamics. Assuming that the differences in soil, forest floor litter and biomass N of plots containing A. mearnsii compared with E. globulus monocultures were due to N(2) fixation, the 10-year annual mean rates of N(2) fixation were 38 and 86 kg ha(-1) year(-1) in 1:1 mixtures and A. mearnsii monocultures, respectively. Nitrogen fixation by A. mearnsii could not be quantified on the basis of the natural abundance of (15)N because such factors as mycorrhization type and fractionation of N isotopes during N cycling within the plant confounded the effect of the N source on the N isotopic signature of plants. This study shows that A. mearnsii fixed significant quantities of N(2) when mixed with E. globulus. A decline in delta(15)N values of E. globulus and A. mearnsii with time, from 2 to 10 years, is further evidence that N(2) was fixed and cycled through the stands. The increased aboveground biomass production of E. globulus trees in mixtures when compared with monocultures can be attributed to increases in N availability.

In vitro assessment of the toxicity of bushfire emissions: A review.

Bushfires produce many toxic pollutants and the smoke has been shown to have negative effects on human health, especially to the respiratory system. Bushfires are predicted to increase in size and frequency, leading to a greater incidence of smoke and impacts. While there are many epidemiological studies of the potential impact on populations, there are few studies using in vitro methods to investigate the biological effects of bushfire emissions to better understand its toxicity and significance. This review focused on the literature pertaining to in vitro toxicity testing to determine the state of knowledge on current methods and findings on the impacts of bushfire smoke. There was a considerable variation in the experimental conditions, outcomes and test concentrations used by researchers using in vitro methods. Of the studies reviewed, most reported adverse impacts of particulate matter (PM) on cytotoxic and genotoxic responses. Studies on whole smoke were rare. Finer primary particulates from bushfire smoke were generally found to be more toxic than the coarse particulates and the toxicological endpoints of bushfire PM different to ambient PM. However the variation in study designs and experimental conditions made comparisons difficult. This review highlights the need for standard protocols to enable appropriate comparisons between studies to be undertaken including the assessment of physiologically relevant outcomes. Further work is essential to establish the effect of burning different vegetation types and combustion conditions on the toxicity of bushfire emissions to better inform both health and response agencies on the significance of smoke from bushfires.

Genomic Scans across Three Eucalypts Suggest that Adaptation to Aridity is a Genome-Wide Phenomenon.

Widespread species spanning strong environmental (e.g., climatic) gradients frequently display morphological and physiological adaptations to local conditions. Some adaptations are common to different species that occupy similar environments. However, the genomic architecture underlying such convergent traits may not be the same between species. Using genomic data from previous studies of three widespread eucalypt species that grow along rainfall gradients in southern Australia, our probabilistic approach provides evidence that adaptation to aridity is a genome-wide phenomenon, likely to involve multiple and diverse genes, gene families and regulatory regions that affect a multitude of complex genetic and biochemical processes.

Phylogenetics of the grass genus Ehrharta: evidence for radiation in the summer-arid zone of the South African Cape.

Climatic and geological change may play a key role in stimulating biological radiations. Here, we use phylogenetic data to test whether the comparatively high diversity of ehrharteoid grasses in the Cape region of South Africa is the result of rapid radiation associated with the onset of a seasonally arid climate during the late Miocene. A phylogenetic hypothesis based on morphological and nucleotide sequence (nuclear ITS1 and plastid trnL-F) data confirms the monophyly of the African Ehrharta species and shows that the diversification of this lineage was centered in the Cape region. Sequence divergence data (ITS1 + trnL-F) indicate a pulse of rapid speciation, which may explain poor phylogenetic resolution within the African Ehrharta clade. Alternative calibrations yield a broad range of time estimates for the start and end of this radiation, most of which indicate a radiation inside the last 11 million years. A calibration based on the age of Ehrhartoideae suggests that radiation started 9.82 +/- 0.20 million years ago and ended 8.74 +/- 0.21 million years ago. Under alternative calibrations, estimated speciation rates during the period of radiation range between 0.87 and 4.18 species per million years. Parsimony optimization of habitat parameters reveals that radiation was correlated with the occupation of seasonally arid succulent karoo environments, wet heathy (fynbos) environments being ancestral. These data support earlier suggestions that late Miocene climatic change stimulated floristic radiation at the Cape, and highlight the potential importance of environmental change in powering diversification in continental floras.

Pine as fast food: foraging ecology of an endangered cockatoo in a forestry landscape.

Pine plantations near Perth, Western Australia have provided an important food source for endangered Carnaby's Cockatoos (Calyptorhynchus latirostris) since the 1940s. Plans to harvest these plantations without re-planting will remove this food source by 2031 or earlier. To assess the impact of pine removal, we studied the ecological association between Carnaby's Cockatoos and pine using behavioural, nutritional, and phenological data. Pine plantations provided high densities of seed (158,025 seeds ha(-1)) over a large area (c. 15,000 ha). Carnaby's Cockatoos fed throughout these plantations and removed almost the entire annual crop of pine cones. Peak cockatoo abundance coincided with pine seed maturation. Pine seed had energy and protein contents equivalent to native food sources and, critically, is available in summer when breeding pairs have young offspring to feed. This strong and enduring ecological association clearly suggests that removing pine will have a significant impact on this endangered species unless restoration strategies, to establish alternative food sources, are implemented.

Nitrogen deposition effects on Mediterranean-type ecosystems: an ecological assessment.

We review the ecological consequences of N deposition on the five Mediterranean regions of the world. Seasonality of precipitation and fires regulate the N cycle in these water-limited ecosystems, where dry N deposition dominates. Nitrogen accumulation in soils and on plant surfaces results in peaks of availability with the first winter rains. Decoupling between N flushes and plant demand promotes losses via leaching and gas emissions. Differences in P availability may control the response to N inputs and susceptibility to exotic plant invasion. Invasive grasses accumulate as fuel during the dry season, altering fire regimes. California and the Mediterranean Basin are the most threatened by N deposition; however, there is limited evidence for N deposition impacts outside of California. Consequently, more research is needed to determine critical loads for each region and vegetation type based on the most sensitive elements, such as changes in lichen species composition and N cycling.