A bacterial leaf spot of Protea cynaroides (king protea)

The etiology of a new leaf spot disease on king protea was investigated. The causal organism was a Gram negative rod-shaped bacterium with between two and seven flagella. On the basis of cultural, biochemical and physiological characters it was identified as a pathovar of Pseudomonas syringae.     

Evidence for autonomous selfing in grassland Protea species (Proteaceae)

It has been assumed that species of the large African genus Protea have strong self‐incompatibility systems. However, this assumption was based largely on studies conducted on a clade of bird‐pollinated species that occur in the shrubby fynbos vegetation of the Cape region of southern Africa. To test whether self‐incompatibility occurs in a grassland/savanna Protea clade, which is largely insect‐pollinated, we performed controlled pollination experiments on four species, P. caffra, P. dracomontana, P. simplex and P. welwitschii. Although pollen–ovule ratios of all four species fall within the range for outcrossers, all four species are self‐compatible and capable of autonomous seed production. Using fluorescence microscopy, we found that self‐pollen tubes had the same probability of reaching ovules as cross‐pollen tubes. In the small tree P. caffra, selfed progeny had rates of germination and survivorship that were identical to those of crossed progeny. The grassland Protea spp. studied are likely to have mixed mating systems on account of being both visited by insects and capable of autonomous selfing. If one assumes previous reports of self‐incompatibility in Protea to be reliable, there have been at least five losses of self‐incompatibility and two gains of autonomous selfing in this genus. However, earlier studies in the genus were often methodologically flawed and a thorough re‐analysis of breeding systems in Protea is required. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 433–446.     

Species richness of arachnids associated with Protea nitida (Proteaceae) in the Cape fynbos.

Regular counts of Arachnida on Protea nitida Mill. were made in three study areas in the western Cape over a period of one year. They were also investigated in the rest of the natural distribution area of P. nitida in the southern and western Cape. Collections were made according to three standardized methods. Five arachnid orders were collected, of which the Araneae (spiders) were dominant. Thirty-two spider species (653 individuals) representing 18 families were collected, of which five families and eight genera (eight species) were recorded for the first time on the Proteaceae in the Cape fynbos. Four non-Araneae orders were collected. A microhabitat preference was observed in some families. Numbers of immature spiders peaked in autumn. Adults and immatures did not always have the same habitat distribution and did not always peak during the same season. Families also showed differences in seasonal abundance. Plant architecture and distribution influenced the number of species collected. Protea nitida hosted more spider species than less complex plants with smaller distributions. The implications of these findings are discussed in terms of biodiversity and invertebrate conservation.     

Evidence for beetle pollination in the African grassland sugarbushes (Protea: Proteaceae)

Most lineages in the African genus Protea consist of species with large unscented flowers pollinated principally by birds, and several of these lineages also show evidence of shifts to rodent pollination, associated with concealed yeasty-scented flowerheads. In this study we investigated the hypothesis that brightly coloured and fruity-scented flowerheads of four Protea species (P. caffra, P. simplex, P. dracomontana and P. welwitschii) represent a novel shift from bird to insect pollination in a grassland lineage in the genus. These species are visited by a wide range of insects, but cetoniine beetles were found to be the most important pollinators because of their abundance, size and relatively pure pollen loads. Three of the four putatively insect-pollinated Protea species have flowers presented at ground level, and experiments showed that cetoniine beetles preferred inflorescences at ground level to those artificially elevated to the height of shrubs and small trees. Relative to insects, birds were infrequent visitors to all of the study species. The nectar of all the study species contained xylose, as documented previously in bird- and rodent-pollinated Protea species, suggesting that this is a phylogenetically conserved trait. However, the very low concentration of nectar (ca. 8%), short nectar-stigma distance and the fruity scent of florets appear to be traits that are associated with specialisation for pollination by cetoniine beetles.     

The relative contributions of insect and bird pollinators to outcrossing in an African Protea (Proteaceae)

? Premise of the study: A useful, but seldom applied, measure of the effectiveness of different pollinators is their contribution to the rate of outcrossing. This measure is particularly useful in facultatively autogamous plants for which seed set cannot be used as a direct measure of pollinator effectiveness. We used selective exclusion experiments to assess the importance of insects for outcrossing in Protea caffra, a facultatively autogamous shrub with scented flowers that are visited frequently by both birds and insects (mainly beetles). ? Methods and results: Pollen loads on stigmas, pollen tube growth, seed set, seed mass, germination, and early seedling survivorship were similar for vertebrate-excluded and open-pollinated inflorescences. Pollen-supplementation mostly did not increase seed set, revealing resource limitation. Mean multilocus outcrossing rates, estimated using eight polymorphic allozyme loci, were similar for progeny from inflorescences excluded from bird visitors (0.65) and for those visited by both birds and insects (0.59). Wright's fixation indices indicated that the adult population is near Hardy-Weinberg equilibrium but differed markedly for maternal plants (F(IS) = -0.187 ± 0.065) and their early stage progeny (F(IS) = 0.258 ± 0.002). Since seed from self and cross hand-pollinations were equally viable in terms of germination, this discrepancy in F(IS) could be explained by inbreeding depression that occurs between germination and reproductive maturity. ? Conclusions: Since outcrossing rates were not reduced when birds were excluded, we infer that insects are effective agents of cross pollination in P. caffra. This helps to explain the evolution of traits associated with insect pollination, such as fruity floral scent, in this species.     

Floral scent in bird- and beetle-pollinated Protea species (Proteaceae): Chemistry,emission rates and function

Evolutionary shifts between pollination systems are often accompanied by modifications of floral traits, including olfactory cues. We investigated the implications of a shift from passerine bird to beetle pollination in Protea for floral scent chemistry, and also explored the functional significance of Protea scent for pollinator attraction. Using headspace sampling and gas chromatography–mass spectrometry, we found distinct differences in the emission rates and chemical composition of floral scents between eight bird- and four beetle-pollinated species. The amount of scent emitted from inflorescences of beetle-pollinated species was, on average, about 10-fold greater than that of bird-pollinated species. Floral scent of bird-pollinated species consists mainly of small amounts of “green-leaf volatiles” and benzenoid compounds, including benzaldehyde, anisole and benzyl alcohol. The floral scent of beetle-pollinated species is dominated by emissions of linalool, a wide variety of other monoterpenes and the benzenoid methyl benzoate, which imparts a fruity odour to the human nose. The number of compounds recorded in the scent of beetle-pollinated species was, on average, greater than in bird-pollinated species (45 versus 29 compounds, respectively). Choice experiments using a Y-maze showed that a primary pollinator of Protea species, the cetoniine beetle Atrichelaphinis tigrina, strongly preferred the scent of inflorescences of the beetle-pollinated Protea simplex over those of the bird-pollinated sympatric congener, Protea roupelliae. This study shows that a shift from passerine bird- to insect-pollination can be associated with marked up-regulation and compositional changes in floral scent emissions.     

Intraspecific trait variation influences physiological performance and fitness in the South Africa shrub genus Protea (Proteaceae)

Background and AimsGlobal plant trait datasets commonly identify trait relationships that are interpreted to reflect fundamental trade-offs associated with plant strategies, but often these trait relationships are not identified when evaluating them at smaller taxonomic and spatial scales. In this study we evaluate trait relationships measured on individual plants for five widespread Protea species in South Africa to determine whether broad-scale patterns of structural trait (e.g. leaf area) and physiological trait (e.g. photosynthetic rates) relationships can be detected within natural populations, and if these traits are themselves related to plant fitness.MethodsWe evaluated the variance structure (i.e. the proportional intraspecific trait variation relative to among-species variation) for nine structural traits and six physiological traits measured in wild populations. We used a multivariate path model to evaluate the relationships between structural traits and physiological traits, and the relationship between these traits and plant size and reproductive effort.Key ResultsWhile intraspecific trait variation is relatively low for structural traits, it accounts for between 50 and 100 % of the variation in physiological traits. Furthermore, we identified few trait associations between any one structural trait and physiological trait, but multivariate regressions revealed clear associations between combinations of structural traits and physiological performance (R² = 0.37–0.64), and almost all traits had detectable associations with plant fitness.ConclusionsIntraspecific variation in structural traits leads to predictable differences in individual-level physiological performance in a multivariate framework, even though the relationship of any particular structural trait to physiological performance may be weak or undetectable. Furthermore, intraspecific variation in both structural and physiological traits leads to differences in plant size and fitness. These results demonstrate the importance of considering measurements of multivariate phenotypes on individual plants when evaluating trait relationships and how trait variation influences predictions of ecological and evolutionary outcomes.     

The influence of pollinators and seed predation on seed production in dwarf grassland Protea “sugarbushes” (Proteaceae)

Flowers of many plant species are visited by both birds and insects, making it necessary to establish their relative contributions to seed set. In Protea, available evidence points to an overwhelming preponderance of bird-pollination systems in the genus, but the scented flowers of several dwarf grassland “sugarbush” species suggest that some Protea species may be adapted for insect pollination. In this study, we used both selective exclusion of vertebrates and complete exclusion of all visitors to investigate whether the insects that visit the scented flowerheads of three Protea species (Protea dracomontana, Protea simplex and Protea welwitschii) in KwaZulu-Natal, South Africa contribute to seed production. We also performed supplemental hand pollinations to test for pollen limitation. Seed set was generally higher in inflorescences subjected to vertebrate exclusion than in those from which all visitors were excluded, suggesting that fertile cross-pollen was deposited by insects, but these differences were slight because of high levels of self-fertilization in the study species. Pollen deposition and pollen tube growth were similar for vertebrate-excluded and open-pollinated inflorescences. Supplemental hand-pollination treatments revealed that seed set in P. simplex and P. welwitschii was not pollen-limited. Overall seed set was low, typical of the family Proteaceae, and infructescences were highly predated by lepidopteran larvae. We conclude that insects are likely to contribute to seed set of the study species, but further studies using molecular markers are required to establish the actual level of insect-mediated outcrossing.     

A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.     

Unseen fungal biodiversity and complex inter-organismal interactions in Protea flower heads

A unique microbiome occurs within the flower heads of various Protea species endemic to Africa. These include two lineages of ophiostomatoid fungi, Knoxdaviesia (Microascales) and Sporothrix (Ophiostomatales), that have members occurring exclusively in this environment and that rely on mites as their primary mode of spore dissemination. The mites, in turn, attach to the bodies of Protea-pollinating beetles and the beaks and bodies of birds for long-distance movement, establishing a hierarchical dispersal network for the ophiostomatoid fungi. This inter-organismal network is highly successful, achieving fungal dispersal over vast distances. Multiple species of fungi, mites and bacteria have been described from this unique niche over the past four decades. The intricacies of their symbiotic interactions continue to be unravelled. This review covers all current knowledge of the “distinctly African” Protea-ophiostomatoid fungus environment and illustrates the depth of a fascinating unseen fungal biodiversity niche.     

Contrasting carbon metabolism in saprotrophic and pathogenic microascalean fungi from Protea trees

Protea-associated Knoxdaviesia species grow on decaying inflorescences, yet are closely related to plant pathogens such as Ceratocystis albifundus. C. albifundus also infects Protea, but occupies a distinct niche. We investigated substrate utilization in two Knoxdaviesia saprotrophs, a generalist and a specialist, and the pathogen C. albifundus by integrating phenome and whole-genome data. On shared substrates, the generalist grew slightly better than its specialist counterpart, alluding to how it has maintained its Protea host range. C. albifundus grew on few substrates and had limited cell wall-degrading enzymes. It did not utilize sucrose, but may prefer soluble oligosaccharides. Nectar monosaccharides are likely important carbon sources for early colonizing Knoxdaviesia species. Once the inflorescence ages, they could switch to degrading cell wall components. C. albifundus likely uses its limited cell wall-degrading arsenal to gain access to plant cells and exploit internal resources. Overall, carbon metabolism and gene content in three related fungi reflected their ecological adaptations.     

Biotic and abiotic constraints that facilitate host exclusivity of Gondwanamyces and Ophiostoma on Protea

Estimations of global fungal diversity are hampered by a limited understanding of the forces that dictate host exclusivity in saprobic microfungi. To consider this problem for Gondwanamyces and Ophiostoma found in the flower heads of Protea in South Africa, we determined the role of various factors thought to influence their host exclusivity. Results showed that various biotic and abiotic factors influence the growth and survival of these fungi in vitro. Monitoring temperature and relative humidity (RH) fluctuations within infructescences in vivo revealed considerable microclimatic differences between different Protea spp. Fungal growth and survival at different RH levels experienced in the field suggested that this factor does not play a major role in host exclusivity of these fungi. Maximum temperatures within infructescences and host preferences of the vectors of Gondwanamyces and Ophiostoma appear to play a substantial part in determining colonisation of Protea in general. However, these factors did not explain host exclusivity of specific fungal species towards particular Protea hosts. In contrast, differential growth of fungal species on media containing macerated tissue of Protea showed that Gondwanamyces and Ophiostoma grow best on tissue from their natural hosts. Thus, host chemistry plays a role in host exclusivity of these fungi, although some species grew vigorously on tissue of Protea spp. with which they are not naturally associated. A combination of host chemistry and temperature partially explains host exclusivity, but the relationship for these factors on the tested saprobic microfungi and their hosts is clearly complex and most likely includes combinations of various biotic and abiotic factors including those emerging from this study.     

Energy dispersive X-ray analysis of protein bodies in Protea compacta cotyledons

Summary The elemental composition of globoids in the protein bodies of Protea compacta cotyledons was studied by means of energy dispersive X-ray analysis. The globoid crystal was rich in phosphorus and calcium with lesser amounts of magnesium and potassium suggesting the presence of phytin in these structures.Abbreviation EDX energy dispersive X-ray analysis