Unequal allocation between male versus female reproduction cannot explain extreme vegetative dimorphism in Aulax species (Cape Proteaceae).

Female plants not only flower but also produce resource-rich seeds, fruits, and cones. Thus, it is generally considered that female plants allocate more resources to sexual reproduction than male plants and that this allocation difference can explain vegetative dimorphism, such as greater leaf size in females. We found significant sexual vegetative differences in the dioecious and serotinous species, Aulax umbellata and A. cancellata. Plant height, annual branch length and canopy spread were greater in males whereas leaf size, branch thickness and branch number were greater in females. Sex ratios and basal stem area were, however, equal in the sexes. Equal sex ratios imply equal allocation to sexual reproduction and equal stem areas imply equal resource use and biomass, and thus allocation to vegetative growth. Given equal allocation to reproduction and resource use, we suggest that the vegetative dimorphism is driven by intra-male-competition to be more visually conspicuous to pollinators. This implies that plant architecture is both a vegetative and a reproductive trait.

Microscale trait-environment associations in two closely-related South African shrubs.

PREMISE OF THE STUDY: Plant traits are often associated with the environments in which they occur, but these associations often differ across spatial and phylogenetic scales. Here we study the relationship between microenvironment, microgeographical location, and traits within populations using co-occurring populations of two closely related evergreen shrubs in the genus Protea. METHODS: We measured a suite of functional traits on 147 plants along a single steep mountainside where both species occur, and we used data-loggers and soil analyses to characterize the environment at 10 microsites spanning the elevational gradient. We used Bayesian path analyses to detect trait-environment relationships in the field for each species. We used complementary data from greenhouse grown seedlings derived from wild collected seed to determine whether associations detected in the field are the result of genetic differentiation. KEY RESULTS: Microenvironmental variables differed substantially across our study site. We found strong evidence for six trait-environment associations, although these differed between species. We were unable to detect similar associations in greenhouse-grown seedlings. CONCLUSIONS: Several leaf traits were associated with temperature and soil variation in the field, but the inability to detect these in the greenhouse suggests that differences in the field are not the result of genetic differentiation.

Environmental drivers and genomic architecture of trait differentiation in fire-adapted Banksia attenuata ecotypes.

Trait divergence between populations is considered an adaptive response to different environments, but to what extent this response is accompanied by genetic differentiation is less clear since it may be phenotypic plasticity. In this study, we analyzed phenotypic variation between two Banksia attenuata growth forms, lignotuberous (shrub) and epicormic resprouting (tree), in fire-prone environments to identify the environmental factors that have driven this phenotypic divergence. We linked genotype with phenotype and traced candidate genes using differential gene expression analysis. Fire intervals determined the phenotypic divergence between growth forms in B. attenuata. A genome-wide association study identified 69 single nucleotide polymorphisms, putatively associated with growth form, whereas no growth form- or phenotype-specific genotypes were identified. Genomic differentiation between the two growth forms was low (Fst = 0.024). Differential gene expression analysis identified 37 genes/transcripts that were differentially expressed in the two growth forms. A small heat-shock protein gene, associated with lignotuber presence, was differentially expressed in the two forms. We conclude that different fire regimes induce phenotypic polymorphism in B. attenuata, whereas phenotypic trait divergence involves the differential expression of a small fraction of genes that interact strongly with the disturbance regime. Thus, phenotypic plasticity among resprouters is the general strategy for surviving varying fire regimes.

Xylem adjusts to maintain efficiency across a steep precipitation gradient in two coexisting generalist species.

Background and Aims: Trees adjust the configuration of their conductive system in response to changes in water availability, maximizing efficiency in wet environments and increasing safety in dry habitats. However, evidence of this general trend is not conclusive. Generalist species growing across broad climatic gradients provide an ideal framework to assess intra-specific xylem adjustments under contrasting environmental conditions. Our aims were to compare the response of xylem traits to variations in precipitation of two co-occurring generalist tree species, and to assess climate control on xylem trait variability and co-ordination. Methods: We evaluated xylem traits of Embothrium coccineum (Proteaceae, evergreen) and Nothofagus antarctica (Nothofagaceae, deciduous) in three areas across an abrupt precipitation gradient, from 500 to 2500 mm, in southern Chile. We measured wood density, vessel lumen area and density, percentage of conductive area and vessel grouping, and estimated the hydraulic function from anatomical measurements in 60 individuals per species. Key Results: Both species shared a common pattern of response along the precipitation gradient, with an increase in vessel density with dryness, but without changes in estimated hydraulic conductivity. Xylem traits in E. coccineum were more variable and more responsive to the climate gradient, decreasing vessel lumen area and increasing wood density, whereas vessel grouping showed contrasting patterns between species. Additionally, the analysis of trait co-ordination at the individual level revealed a tighter co-ordination among xylem traits in E. coccineum. Conclusions: Estimated xylem efficiency was maintained in combination with different levels of expected xylem safety within species. Reduction in vessel lumen area was compensated through large increases in vessel density, thus breaking the trade-off between xylem efficiency and safety. Otherwise, the existence of alternative internal adjustments in coexisting species to face similar climatic constraints might increase resilience of temperate forests against unpredictable changes in climatic conditions.

Small-seeded Hakea species tolerate cotyledon loss better than large-seeded congeners.

Six Hakea species varying greatly in seed size were selected for cotyledon damage experiments. The growth of seedlings with cotyledons partially or completely removed was monitored over 90 days. All seedlings perished by the fifth week when both cotyledons were removed irrespective of seed size. Partial removal of cotyledons caused a significant delay in the emergence of the first leaf, and reduction in root and shoot growth of the large-seeded species. The growth of seedlings of small-seeded species was less impacted by cotyledon damage. The rate of survival, root and shoot lengths and dry biomass of the seedlings were determined after 90 days. When seedlings were treated with balanced nutrient solutions following removal of the cotyledons, survival was 95-98%, but 0% when supplied with nutrient solutions lacking N or P or with water only. The addition of a balanced nutrient solution failed to restore complete growth of any species, but the rate of root elongation for the small-seeded species was maintained. Cotyledons provide nutrients to support early growth of Hakea seedlings, but other physiological roles for the cotyledons are also implicated. In conclusion, small-seeded Hakea species can tolerate cotyledons loss better than large-seeded species.

Characterization of CYCLOIDEA-like genes in Proteaceae, a basal eudicot family with multiple shifts in floral symmetry.

BACKGROUND AND AIMS: The basal eudicot family Proteaceae (approx. 1700 species) shows considerable variation in floral symmetry but has received little attention in studies of evolutionary development at the genetic level. A framework for understanding the shifts in floral symmetry in Proteaceae is provided by reconstructing ancestral states on an upated phylogeny of the family, and homologues of CYCLOIDEA (CYC), a key gene for the control of floral symmetry in both monocots and eudicots, are characterized. METHODS: Perianth symmetry transitions were reconstructed on a new species-level tree using parsimony and maximum likelihood. CYC-like genes in 35 species (31 genera) of Proteaceae were sequenced and their phylogeny was reconstructed. Shifts in selection pressure following gene duplication were investigated using nested branch-site models of sequence evolution. Expression patterns of CYC homologues were characterized in three species of Grevillea with different types of floral symmetry. KEY RESULTS: Zygomorphy has evolved 10-18 times independently in Proteaceae from actinomorphic ancestors, with at least four reversals to actinomorphy. A single duplication of CYC-like genes occurred prior to the diversification of Proteaceae, with putative loss or divergence of the ProtCYC1 paralogue in more than half of the species sampled. No shifts in selection pressure were detected in the branches subtending the two ProtCYC paralogues. However, the amino acid sequence preceding the TCP domain is strongly divergent in Grevillea ProtCYC1 compared with other species. ProtCYC genes were expressed in developing flowers of both actinomorphic and zygomorphic Grevillea species, with late asymmetric expression in the perianth of the latter. CONCLUSION: Proteaceae is a remarkable family in terms of the number of transitions in floral symmetry. Furthermore, although CYC-like genes in Grevillea have unusual sequence characteristics, they display patterns of expression that make them good candidates for playing a role in the establishment of floral symmetry.

The influence of leaf size and shape on leaf thermal dynamics: does theory hold up under natural conditions?

Laboratory studies on artificial leaves suggest that leaf thermal dynamics are strongly influenced by the two-dimensional size and shape of leaves and associated boundary layer thickness. Hot environments are therefore said to favour selection for small, narrow or dissected leaves. Empirical evidence from real leaves under field conditions is scant and traditionally based on point measurements that do not capture spatial variation in heat load. We used thermal imagery under field conditions to measure the leaf thermal time constant (τ) in summer and the leaf-to-air temperature difference (∆T) and temperature range across laminae (Trange ) during winter, autumn and summer for 68 Proteaceae species. We investigated the influence of leaf area and margin complexity relative to effective leaf width (we ), the latter being a more direct indicator of boundary layer thickness. Normalized difference of margin complexity had no or weak effects on thermal dynamics, but we strongly predicted τ and ∆T, whereas leaf area influenced Trange . Unlike artificial leaves, however, spatial temperature distribution in large leaves appeared to be governed largely by structural variation. Therefore, we agree that small size, specifically we , has adaptive value in hot environments but not with the idea that thermal regulation is the primary evolutionary driver of leaf dissection.

A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy.

Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.

Environmental adaptation in stomatal size independent of the effects of genome size.

Cell sizes are linked across multiple tissues, including stomata, and this variation is closely correlated with genome size. These associations raise the question of whether generic changes in cell size cause suboptimal changes in stomata, requiring subsequent evolution under selection for stomatal size. We tested the relationships among guard cell length, genome size and vegetation type using phylogenetically independent analyses on 67 species of the ecologically and structurally diverse family, Proteaceae. We also compared how genome and stomatal sizes varied at ancient (among genera) and more recent (within genus) levels. The observed 60-fold range in genome size in Proteaceae largely reflected the mean chromosome size. Compared with variation among genera, genome size varied much less within genera (< 6% of total variance) than stomatal size, implying evolution in stomatal size subsequent to changes in genome size. Open vegetation and closed forest had significantly different relationships between stomatal and genome sizes. Ancient changes in genome size clearly influenced stomatal size in Proteaceae, but adaptation to habitat strongly modified the genome-stomatal size relationship. Direct adaptation to the environment in stomatal size argues that new proxies for past concentrations of atmospheric CO2 that incorporate stomatal size are superior to older models based solely on stomatal frequency.

Early evidence of xeromorphy in angiosperms: stomatal encryption in a new eocene species of Banksia (Proteaceae) from Western Australia.

UNLABELLED: • PREMISE OF THE STUDY: Globally, the origins of xeromorphic traits in modern angiosperm lineages are obscure but are thought to be linked to the early Neogene onset of seasonally arid climates. Stomatal encryption is a xeromorphic trait that is prominent in Banksia, an archetypal genus centered in one of the world's most diverse ecosystems, the ancient infertile landscape of Mediterranean-climate southwestern Australia.• METHODS: We describe Banksia paleocrypta, a sclerophyllous species with encrypted stomata from silcretes of the Walebing and Kojonup regions of southwestern Australia dated as Late Eocene.• KEY RESULTS: Banksia paleocrypta shows evidence of foliar xeromorphy ∼20 Ma before the widely accepted timing for the onset of aridity in Australia. Species of Banksia subgenus Banksia with very similar leaves are extant in southwestern Australia. The conditions required for silcrete formation infer fluctuating water tables and climatic seasonality in southwestern Australia in the Eocene, and seasonality is supported by the paucity of angiosperm closed-forest elements among the fossil taxa preserved with B. paleocrypta. However, climates in the region during the Eocene are unlikely to have experienced seasons as hot and dry as present-day summers.• CONCLUSIONS: The presence of B. paleocrypta within the center of diversity of subgenus Banksia in edaphically ancient southwestern Australia is consistent with the continuous presence of this lineage in the region for ≥40 Ma, a testament to the success of increasingly xeromorphic traits in Banksia over an interval in which numerous other lineages became extinct.

Maintenance of strong morphological differentiation despite ongoing natural hybridization between sympatric species of Lomatia (Proteaceae).

BACKGROUND AND AIMS: When species cohesion is maintained despite ongoing natural hybridization, many questions are raised about the evolutionary processes operating in the species complex. This study examined the extensive natural hybridization between the Australian native shrubs Lomatia myricoides and L. silaifolia (Proteaceae). These species exhibit striking differences in morphology and ecological preferences, exceeding those found in most studies of hybridization to date. METHODS: Nuclear microsatellite markers (nSSRs), genotyping methods and morphometric analyses were used to uncover patterns of hybridization and the role of gene flow in morphological differentiation between sympatric species. KEY RESULTS: The complexity of hybridization patterns differed markedly between sites, however, signals of introgression were present at all sites. One site provided evidence of a large hybrid swarm and the likely presence of multiple hybrid generations and backcrosses, another site a handful of early generational hybrids and a third site only traces of admixture from a past hybridization event. The presence of cryptic hybrids and a pattern of morphological bimodality amongst hybrids often disguised the extent of underlying genetic admixture. CONCLUSIONS: Distinct parental habitats and phenotypes are expected to form barriers that contribute to the rapid reversion of hybrid populations to their parental character state, due to limited opportunities for hybrid/intermediate advantage. Furthermore, strong genomic filters may facilitate continued gene flow between species without the danger of assimilation. Stochastic fire events facilitate temporal phenological isolation between species and may partly explain the bi-directional and site-specific patterns of hybridization observed. Furthermore, the findings suggest that F1 hybrids are rare, and backcrosses may occur rapidly following these initial hybridization events.

Climate drives vein anatomy in Proteaceae.

PREMISE OF STUDY: The mechanisms by which plants tolerate water deficit are only just becoming clear. One key factor in drought tolerance is the ability to maintain the capacity to conduct water through the leaves in conditions of water stress. Recent work has shown that a simple feature of the leaf xylem cells, the cube of the thickness of cell walls divided by the lumen width (t/b)(3), is strongly correlated with this ability. METHODS: Using ecologically, phylogenetically, and anatomically diverse members of Proteaceae, we tested the relationships between (t/b)(3) and climate, leaf mass per unit area, leaf area, and vein density. To test relationships at high phylogenetic levels (mostly genus), we used phylogenetic and nonphylogenetic single and multiple regressions based on data from 50 species. We also used 14 within-genus species pairs to test for relationships at lower phylogenetic levels. KEY RESULTS: All analyses revealed that climate, especially mean annual precipitation, was the best predictor of (t/b)(3). The variation in (t/b)(3) was driven by variation in both lumen diameter and wall thickness, implying active control of these dimensions. Total vein density was weakly related to (t/b)(3) but unrelated to either leaf area or climate. CONCLUSIONS: We conclude that xylem reinforcement is a fundamental adaptation for water stress tolerance and, among evergreen woody plants, drives a strong association between rainfall and xylem anatomy. The strong association between (t/b)(3) and climate cannot be explained by autocorrelation with other aspects of leaf form and anatomy that vary along precipitation gradients.

The impact of multiple biogeographic barriers and hybridization on species-level differentiation.

PREMISE OF THE STUDY: The glacial cycles of the Quaternary did not impact Australia in the same way as Europe and North America. Here we investigate the history of population isolation, species differentiation, and hybridization in the southeastern Australian landscape, using five species of Lomatia (Proteaceae). We use a chloroplast DNA phylogeography to assess chloroplast haplotype (chlorotype) sharing among these species and whether species with shared distributions have been affected by shared biogeographic barriers. • METHODS: We used six chloroplast DNA simple sequence repeats (cpSSR) across five species of Lomatia, sampled across their entire distributional range in southeastern Australia. Resulting size data were combined, presented as a network, and visualized on a map. Biogeographical barriers were tested using AMOVA. To explore hypotheses of chlorotype origin, we converted the network into a cladogram and reconciled with all possible species trees using parsimony-based tree mapping. • KEY RESULTS: Some chlorotypes were shared across multiple species of Lomatia in the study, including between morphologically differentiated species. Chlorotypes were either widespread in distribution or geographically restricted to specific regions. Biogeographical structure was identified across the range of Lomatia. The most parsimonious reconciled tree incorporated horizontal transfer of chlorotypes. • CONCLUSIONS: Lomatia shows evidence of both incomplete lineage sorting and extensive hybridization between co-occurring species. Although the species in the study appear to have responded to a number of biogeographic barriers to varying degrees, our findings identified the Hunter River Valley as the most important long-term biogeographic barrier for the genus in southeastern Australia.

Chemical constituents and biological studies of the leaves of Grevillea robusta.

Three new compounds: Graviquinone (1), cis-3-hydroxy-5-pentadecylcyclohexanone (2), and methyl 5-ethoxy-2-hydroxycinnamate (3), and thirty-eight known compounds were isolated and identified from the leaves of Grevillea robusta. The structures of these compounds were determined by spectroscopic and chemical transformation methods. Graviquinone (1) showed the strongest cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines. Methyl 2,5-dihydroxycinnamate (4), graviphane (13), and dehydrograviphane (14) exhibited very potent DPPH scavenging activity compared with α-tocopherol. Methyl 2,5-dihydroxycinnamate (4) and bis-norstriatol (17) demonstrated strong inhibition of L-DOPA oxidation by mushroom tyrosinase compared with kojic acid.

Uncoupled geographical variation between leaves and flowers in a South-Andean Proteaceae.

BACKGROUND AND AIMS: Geographical variation in foliar and floral traits and their degree of coupling can provide relevant information on the relative importance of abiotic, biotic and even neutral factors acting at geographical scales as generators of evolutionary novelty. Geographical variation was studied in leaves and flowers of Embothrium coccineum, a species that grows along abrupt environmental gradients and exhibits contrasting pollinator assemblages in the southern Andes. METHODS: Five foliar and eight floral morphological characters were considered from 32 populations, and their patterns of variation and covariation were analysed within and among populations, together with their relationship with environmental variables, using both univariate and multivariate methods. The relationships between foliar and floral morphological variation and geographical distance between populations were compared with Mantel permutation tests. KEY RESULTS: Leaf and flower traits were clearly uncoupled within populations and weakly associated among populations. Whereas geographical variation in foliar traits was mostly related to differences in precipitation associated with geographical longitude, variation in floral traits was not. CONCLUSIONS: These patterns suggest that leaves and flowers responded to different evolutionary forces, environmental (i.e. rainfall) in the case of leaves, and biotic (i.e. pollinators) or genetic drift in the case of flowers. This study supports the view that character divergence at a geographical scale can be moulded by different factors acting in an independent fashion.

Shallow-soil endemics: adaptive advantages and constraints of a specialized root-system morphology.

Worldwide, many rare plant species occur in shallow-soil, drought-prone environments. For most of these species, the adaptations required to be successful in their own habitats, as well as their possible consequences for establishment and persistence in others, are unknown. Here, two rare Hakea (Proteaceae) species confined to shallow-soil communities in mediterranean-climate south-western Australia were compared with four congeners commonly occurring on deeper soils. Seedlings were grown for 7 months in a glasshouse in individual 1.8 x 0.2-m containers, to allow for unconstrained root development. In addition, a reciprocal transplant experiment was carried out. The rare Hakea species differed consistently from their common congeners in their spatial root placement. They invested more in deep roots and explored the bottom of the containers much more quickly. In the reciprocal transplant experiment they showed increased survival in their own habitat, but not in others. This research suggests that shallow-soil endemics have a specialized root system that allows them to explore a large rock surface area, thereby presumably increasing their chance to locate cracks in the underlying rock. However, this root-system morphology may be maladaptive on deeper soils, providing a possible explanation for the restricted distribution of many shallow-soil endemics.

Systemic suppression of cluster-root formation and net P-uptake rates in Grevillea crithmifolia at elevated P supply: a proteacean with resistance for developing symptoms of 'P toxicity'.

Grevillea crithmifolia R. Br. is a species of Proteaceae that is resistant to developing P-toxicity symptoms at phosphorus supplies in the root environment that induce P-toxicity symptoms in the closely related Hakea prostrata (Proteaceae). It was discovered previously that development of P-toxicity symptoms in H. prostrata is related to its low capacity to down-regulate net P-uptake rates (i.e. its low plasticity). The plasticity of net P-uptake rates and whole-plant growth responses in G. crithmifolia has now been assessed in two separate experiments: (i) a range of P, from 0 to 200 micromol P d-1, was supplied to whole root systems; (ii) using a split-root design, one root half was supplied with 0, 3, 75, or 225 micromol P d-1, while the other root half invariably received 3 micromol P d-1. Fresh mass was significantly greater in G. crithmifolia plants that had received a greater daily P supply during the pretreatments, but symptoms of P toxicity were never observed. Cluster-root growth decreased from about half the total root fresh mass when the leaf [P] was lowest (c. 0.1 mg P g-1 DM) to complete suppression of cluster-root growth when leaf [P] was 1-2 mg P g-1 DM. Split-root studies revealed that cluster-root initiation and growth, and net P-uptake rates by roots were regulated systemically, possibly by shoot P concentration. It is concluded that, in response to higher P supply, G. crithmifolia does not develop symptoms of P toxicity because of (i) greater plasticity of its net P-uptake capacity, and (ii) its greater plasticity for allocating P to growth and P storage in roots. This ecologically important difference in plasticity is most probably related to a slightly higher nutrient availability in the natural habitat of G. crithmifolia when compared with that of H. prostrata.