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Background and Aims In plants, the spatial and genetic distance between mates can influence reproductive success and offspring fitness. Negative fitness consequences associated with the extremes of inbreeding and outbreeding suggest that there will be an intermediate optimal outcrossing distance (OOD), the scale and drivers of which remain poorly understood. In the bird-pollinated Anigozanthos manglesii (Haemodoraceae) we tested (1) for the presence of within-population OOD, (2) over what scale it occurs, and (3) for OOD under biologically realistic scenarios of multi-donor deposition associated with pollination by nectar-feeding birds. Methods We measured the impact of mate distance (spatial and genetic) on seed set, fruit size, seed mass, seed viability and germination success following hand pollination from (1) single donors across 0 m (self), Key results Inter-mate distance had a significant impact on single-donor reproductive success, with selfed and nearest-neighbour ( Conclusions Collectively, single- and multi-donor pollinations indicated evidence for a near-neighbour OOD within A. manglesii. A survey of the literature suggests that within-population OOD may be more characteristic of plants pollinated by birds than those pollinated by insects.

Background: Metallic silver nanoparticles with antibacterial properties were biosynthesised for the first time using an indigenous Australian plant Anigozanthos manglesii. Methods: A practical, straight-forward and eco-friendly technique used the Anigozanthos manglesii leaf extract, which acted as both reducing and capping agents to create stable silver nanoparticles. The antibacterial activities of the nanoparticles were investigated using the Kirby-Bauer sensitivity method. Results: Characterisation revealed the nanoparticles ranged in size from 50 nm up to 150 nm, and their morphologies included cubes, triangular plates and hexagonal plates. Antibacterial studies revealed Deinococcus was sensitive and susceptible to the biosynthesised nanoparticles. Escherichia coli and Staphylococcus Epidermis strains were also found to be less susceptible to the silver nanoparticles. Conclusions: The present study has shown that silver nanoparticles biosynthesised using Anigozanthos manglesii leaf extracts have antibacterial activity against Deinococcus , Escherichia coli and Staphylococcus Epidermis bacterial strains.

Plants pollinated by vertebrates are often visited by native and exotic insects foraging for pollen and nectar. We compared flower visitation rates, foraging behaviour, and the contribution to reproduction of nectar-feeding birds and the introduced honeybee Apis mellifera in four populations of the bird-pollinated Anigozanthos manglesii (Haemodoraceae). The behaviour of floral visitors was quantified with direct observations and motion-triggered and hand-held cameras. Pollinator access to flowers was manipulated by enclosure in netting to either exclude all visitors or to exclude vertebrate visitors only. Apis mellifera was the only insect observed visiting flowers, and the most frequent flower visitor, but primarily acted as a pollen thief. Although birds visited A. manglesii plants only once per week on average, they were 3.5 times more likely to contact the anther or stigma as foraging honeybees. Exclusion of birds resulted in 67% fewer fruits and 81% fewer seeds than flowers left open and unmanipulated. Unnetted flowers that were open to bird and insect pollinators showed pollen-limitation and a large variation in reproductive output within and between sites. Although honeybees have been shown to pollinate other Australian plants, compared to birds, they are highly inefficient pollinators of A. manglesii.

A root rot and wilt disease of Anigozanthos manglesii (Kangaroo Paw) grown in greenhouses in Israel, for exporting as cut flowers to Europe, was characterized. Pythium myriotylum (Drechs.) and Rhizoctonia solani (Kuhn) were the prevalent pathogens in diseased plants collected from commercial greenhouses. Fusarium oxysporum, Fusarium spp. and Myrothecium sp. were also isolated, but P. myriotylum or R. solani were not detected in samples from symptomless plants in tissue cultures (Australian origin) or plants at different stages in the nursery; non-pathogenic F. oxysporum and Fusarium spp. were detected in several samples. In pathogenicity tests carried out in pots, plant mortality occurred 7 days after inoculation with P. myriotylum. In a field experiment carried out in methyl bromide-fumigated soil, the incidence of dead plants following inoculation with P. myriotylum alone was 22% 10 days after inoculation, increasing to 78% after an additional 25 days. The incidence of dead plants following inoculation with R. solani alone was only 5% and in plants inoculated simultaneously with both pathogens, disease incidence was 88% 35 days after inoculation. Mortality reached 90-100% in plants inoculated with P. myriotylum, either singly or combined with R. solani 60 days after inoculation, whereas in plants inoculated with R. solani it was 5%. The maximum mortality in plants inoculated with R. solani was 25%, 76 days after inoculation. These results clearly demonstrate that P. myriotylum was the dominant pathogen in the root rot and wilt of A. manglesii.

The influence of the storage environment on seed viability and antioxidant potential was examined for four species native to Western Australia: Acacia bivenosa DC., Anigozanthos manglesii D. Don, Banksia ashbyi E.G. Baker, and Mesomelaena tetragona (R. Br.) Benth. Seeds were stored at four water contents (at c. 5%, 11–15%, 20–23% and 50% relative humidity) at each of five temperatures (–196, –18, 5, 23 and 50°C), and seed germination and seedling vigour monitored over an 18-month period. Deterioration was apparent in all species (except A. bivenosa) stored at 50°C, with 11% RH maximizing longevity for B. ashbyi and M. tetragona seeds, and 5% or 11% RH preventing deterioration for A. manglesii seeds. Seed viability generally remained high for all species stored at 23°C or less. Notably, however, germination and seedling vigour of A. manglesii and M. tetragona seeds gradually declined when stored at –18°C, suggesting that storage at this temperature was detrimental. The antioxidant activity of lipid extracts of seeds after 18 months storage at 5, 23 and 50°C was also examined to determine whether the seed viability decline was associated with a loss of antioxidants. Antioxidant activity varied between storage treatments and was not related to seed viability.

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.

Exposure to high temperatures (40–60 °C) over several days to months (simulating summer soil conditions) or to extremely high temperatures (80–120 °C) for periods ranging from minutes to several days (simulating the heat of an extreme fire) promoted germination in five (including one legume species) of the seven species tested from southwestern Australia. Heat, a collective term for high temperature (HT) and extremely high temperature (EHT), has previously been used to promote germination in species with impermeable seed coats (many of which are legumes). If heat is found to promote germination in species with permeable seed coats, it could be of use in the release of dormancy for commercial development of native plants. Here we report that elevated storage temperatures (50 °C for 45 and 90 d or 60 °C for 45 d) and EHT (100 °C for 3 h and 120 °C for 30 min) alone, and in addition to smoke, directly promote germination of the refractory species Actinotus leucocephalus, Anigozanthos manglesii, Gompholobium knightianum, Loxocarya striatus and Stylidium affine. Germination of Sowerbaea laxiflora seed was not improved with heat alone but was improved by the combination of heat and smoke. Seed of Schoenus unispiculatus did not germinate in response to any of the treatments. Interactions between the effect of temperature, the duration of heat exposure or smoke were detected in all species. Whilst most species demonstrated an interaction between heat and time, not all species were affected by smoke alone. This was illustrated by an interaction between smoke and HT found only in Actinotus leucocephalus, Anigozanthos manglesii and Stylidium affine or smoke and EHT in A. manglesii andS. affine . Smoke and exposure time to heat influenced the germination of Actinotus leucocephalus,Anigozanthos manglesii and L. striatus in response to HT and only S. affine appeared to demonstrate an interaction between smoke and EHT. The variations found in the interactions between treatments suggest that germination events occur over a range of conditions both spatially and temporally which may facilitate seedling survival and limit competition between emergents.

Anigozanthos manglesii (Haemodoraceae) is a colourful, herbaceous perennial exhibiting intra- and inter-populational variation in germination in response to smoke and heat. This study investigated the extent and nature of this variation in A. manglesii populations collected along a 550 km latitudinal cline from kwongan scrub (30°S) to more mesic jarrah forest habitats (34°S) in southwestern Australia. Variation in seed germination along a maturing inflorescence was also investigated. Germination of this species is known to be prolific following fire, and two germination treatments, aerosol smoke and heat, were used as germination cues. There was a trend of increasing responsiveness of A. manglesii to smoke with increasing latitude along a clinal range, but there were no differences in germination of seeds along the cline in response to control or heat treatments. There was no significant difference in intra-population seed germination in response to any treatment. Lastly, a significant and increasing response to smoke for seeds from the apex to base of the inflorescence was detected. This latter trend may be attributed to higher resource allocation and an accelerated after-ripening of basipetal compared to acropetal seeds. Possible reasons for the clinal variation are discussed.

Patterns of dormancy and smoke responsiveness in artificially buried seeds were investigated in eight native plant species from the species-rich mediterranean-type climate zone of south-western Australia. A comparison was made between germination and viability behaviour of shelf- and field-soil-stored seed, with and without smoke treatment, at least every 3 months. These comparisons corresponded with each of the four seasons. The species chosen germinated with the aid of smoke under field or glasshouse conditions (termed ‘dormant’; n = 4) or produced low and variable germination under glasshouse conditions with smoke (termed ‘deeply dormant’; n = 4). Three trends were observed in viability of soil-stored seeds over 450 d: no decline, gradual decline or late-onset decline. In addition, various germination responses to soil burial and aerosol smoke were observed. Burial was not required for optimal germination in Anigozanthos manglesii. However, for all other species tested, maximum germination was observed only following a period of burial. This was manifested in a germination response without smoke after a short period of burial ( Stylidium affine and Conospermum triplinervium ) or a longer period of burial ( Conostylis neocymosa, Hibbertia commutata, Leucopogon conostephioides, Stirlingia latifolia and Stylidium crossocephalum ). Smoke treatment led to high germination in buried seed of S. affine , S. crossocephalum and H. commutata . The patterns of germination detected in this limited number of species indicate that a variety of mechanisms may exist, both temporally and spatially, in south-western Australian species, and support further research of this type for horticulture and land restoration.

The viability of kangaroo paw pollen (Anigozanthos manglesii) was estimated by germination in vitro and by fluorescence and the more reliable method was then used to assess the effect of storage conditions on pollen longevity. Pollen germination was more accurate than staining for pollen of low and intermediate viability levels. Effective storage could be achieved in a number of ways depending on the duration required. Dehydrated pollen retained viability longer in storage than did hydrated pollen. Pollen from intact anthers could be stored for up to 5 weeks at room temperature with 50% or better germination provided it was stored on silica gel. Pollen isolated from anthers and stored at 2°C had reduced viability in comparison with that stored at room temperature or at ‐80°C. Pollen stored at ‐196°C lost only 5% viability after 20 weeks and presumably this would be the most suitable method for long term storage for breeding or conservation purposes.

Gamma irradiation doses above 0.05 kGy for Geraldton wax, 0.1 kGy for banksia and 1.0 kGy for kangaroo paw caused significant (P

Many species in fire-prone environments germinate after fire including most taxa in the genus Anigozanthos Labill. Following preliminary studies with Anigozanthos manglesii D.Don subsp. manglesii, the response of several Anigozanthos taxa to germination stimulants relating to the post-fire environment including smoke, karrikinolide (KAR1, 3-methyl-2H-furo[2,3-c]pyran-2-one) and nitrate were compared in seed when freshly collected and after a period of burial. Following after-ripening, the response of seed to water, smoke-water, KAR1 and glyceronitrile (2,3-dihydroxypropanenitrile) was examined. Seed of 16 Anigozanthos taxa, and the related monotypic genus Blancoa Lindl., were highly dormant at maturity. Investigations of eight Anigozanthos taxa and B. canescens indicated that these taxa had morphophysiological dormancy. Following a period of either 3- to 4 months or 1 year of burial and exhumation in autumn, many taxa remained dormant. However, dormancy was alleviated and smoke-water stimulated some germination of A. manglesii subsp. manglesii, A. flavidus DC., A. viridis Endl. subsp. viridis, and A. viridis Endl. subsp. Cataby (S.D. Hopper 1786). Nitrate also stimulated germination of A. flavidus in the light. Following 3–3.5 years of laboratory after-ripening, 13 of the 17 Anigozanthos taxa examined were smoke-responsive. When testing individual smoke-derived compounds these taxa germinated in response to glyceronitrile and not KAR1. In contrast, smoke-responsive B. canescens seed responded to KAR1 and not glyceronitrile. These findings suggest a phylogenetic link between responsiveness to different chemicals in smoke in this family that may shed light on the evolutionary development of these smoke responses.

Exposure of dormant seed to cold smoke derived from burnt native vegetation had a positive influence on germination in one or more seed provenances in 45 out of 94 species of native Western Australian plants that are normally hard to germinate. When tested under controlled conditions some species showed earlier germination in smoke treatments than controls; in others smoke-treated seeds continued to germinate for several weeks after controls had achieved full germination. In the remainder, treated and control seeds germinated to similar time schedules. A group of 23 species which responded positively had previously been recorded as extremely difficult or impossible to germinate using conventional techniques. These included members of the genera Geleznowia (Rutaceae), Hibbertia (Dilleniaceae), Stirlingia (Proteaceae), Verticordia (Myrtaceae), Actinostrobus (Cupressaceae) and Pimelea (Thymelaeaceae). Both large- and small-seeded species were encountered amongst the positively responding taxa, which encompassed representatives of 15 families and 26 genera of dicotyledons, 5 families and 8 genera of monocotyledons and the gymnosperm Actinostrobus acuminatus. Sowing seeds on smoke-fumigated filter papers or watering with aqueous eluates of smoke elicited similar degrees of stimulation of germination, as did exposure to gaseous smoke in a readily germinating species Anigozanthos manglesii (Haemodoraceae) and the normally intractable species Lysinema ciliatum (Epacridaceae). Exposing recently burnt and unburnt natural bushland sites to smoke, smoked water or smoked dry sand elicited a significant germination response in 15 species. Over one third of the species sampled in the burnt site exhibited germination additional to that caused by the fire. Data are discussed in relation to previous germination studies on Australian and other taxa.

The relationship between storage temperature, relative humidity and seed water content was investigated for four species native to Western Australia: Acacia bivenosa DC., Anigozanthos manglesii D.Don., Banksia ashbyi E.G.Baker and Mesomelaena tetragona (R.Br.) Benth. Water sorption isotherms were constructed at 5, 23 and 50�C and the enthalpy of water sorption was calculated by van�t Hoff analysis. Seeds of three species, A. manglesii, B.�ashbyi and M. tetragona, showed a sigmoidal relationship between seed water content and relative humidity. Intact seeds of Acacia bivenosa maintained a constant water content at temperatures of 23�C or less due to the impermeable seed coat; however, isotherms of scarified seeds were similar in shape to those of the other species at all temperatures. The enthalpy of water sorption ranged from –19 kJ mol–1 for M. tetragona seeds to –29 kJ mol–1 for B. ashbyi seeds and was dependent on water content. However, all species had a maximum sorption strength at 2–6% water content and three regions of water-binding strength were evident. Each of these species has water sorption characteristics consistent with orthodox storage behaviour and the results of this study provide a framework for improving seed storage methods for the highly diverse Western Australian flora.

Germination of seed of Anigozanthos (Haemodoraceae) species is usually low and unreliable which hinders the propagation of sexually generated progeny, impedes plant breeding activities and their use in horticulture. This study was undertaken to investigate the basis of these germination problems. Seeds of cultivated Anigozanthos manglesii D. Don. were shown to possess two mechanisms for regulating seed germination, both of which were external to the embryo. Embryos germinated freely on excision and culture on nutrient agar. This first barrier is a physico-chemical barrier and can be eliminated by a brief treatment with 5 M KOH and less effectively by physical abrasion with 50% H2SO4 or 0.7% NaOCl. Release of this barrier was correlated with permeability to fluorescein diacetate (FDA) and this proved to be a more useful indicator of seed viability than 2,3,5-triphenyl tetrazolium chloride (TTC). The other barrier was physiological and could be eliminated by treatment with GA3 or KNO3. Treatment of A. viridis Endl., A. humilis Lindley and A. flavidus DC. with KOH or H2SO4 with or without GA(3) elicited a small, a nil or negative response showing that mechanisms of control of germination may differ between taxa. Observations on the effects of temperature and duration of storage suggest mechanisms which may be significant in natural systems: moist stratification at 5°C caused reduced germination but sustained viability (as assessed by FDA) while dry seed stored at room temperature, showed increased germination with increasing duration of storage. The chilling response may indicate the capacity to initiate secondary dormancy under unfavourable germination conditions.

The origin and development of the inflorescence of two species of kangaroo paw, Anigozanthos manglesii (D. Don) and A. flavidus (DC) have been described using scanning electron microscopy. In both species the first signs of transition to flowering are swelling of the apical dome, a change in leaf phyllotaxy from alternate to radial and commencement of rachis elongation. In branched inflorescences, pedicels are initiated spirally and acropetally. The spike of kangaroo paws is composed of flowers in a sequence of diminishing size, formed because one flower primordium continues to enlarge and differentiate organs whilst the other divides again to form the next flower until the specific number have been formed. Floral organs are initiated in alternate fashion in whorls on a radially symmetric disc from the outer edge towards the centre. Elongation of the floral tube and zygomorphy appear late in the floral process.

The flower production, fruit set, seed set and seed germination of parental and hybrid Anigozanthos individuals from the Gingin cemetery, Western Australia, were investigated to establish whether differences in reproductive capacity could have been responsible for observed short-term numerical changes in the population. A. manglesii had a six- to ninefold reproductive advantage over A. humilis due to greater seed set and better germination. F1 hybrids and backcrosses had reproductive capacities equivalent to or slightly better than A. humilis. The agreement between calculated reproductive capacity values and the trend of numerical changes in the cemetery supports the conclusion that differences in reproductive performance have played a significant role in the short-term dynamics of the population.

The specificity and spatial pattern of foraging movements of red wattlebirds (Anthochaera carunculata) were studied in a hybrid population of Anigozanthos manglesii D. Don and A. humilis Lindl in the shire cemetery at Gingin, W.A. Differences in phenology, nectar production, stem height and floral dimensions between F1 hybrids and the two parental Anigozanthos species were also documented to assess their influence on foraging behaviour and interspecific pollen flow. The red wattlebirds showed a 97 % fidelity for A. humilis at its seasonal peak in August 1976 and, a month later, an equally strong fidelity (97 %) for A. manglesii at its seasonal peak. Interspecific foraging movements constituted only 1.2% of the total of 925 observed during these two study periods. The spatial distribution of flowering individuals and plant stature appeared to be principal factors determining assortative pollination, presumably because of their influence on the energetics of foraging. It is suggested that the observed nearest-neighbour foraging pattern results in spatial restrictions on pollen flow and may be responsible for the confinement of hybrids to ecotonal zones where the parental species grow intermixed. Gene exchange between species is also restricted by differences in pollen deposition/retrieval sites arising from the divergent floral structures of A. hurnilis and A. rnanglesii. It is proposed that honeyeaters have generated strong selective pressures influencing the stature, floral structure and phenology of these kangaroo paws.

Poor competitive ability has been invoked often to explain the confinement of hybrids to disturbed ecotones at the junction of parental habitats. This hypothesis has not as yet been verified in an experimental study. Two-month-old seedlings of Anigozanthos manglesii, A, humilis and synthesized F1 hybrids were grown singly and in various paired combinations in small pots for 172 days to test for competitive interactions. F1 hybrids and A. manglesii performed comparably in most competition trials, while A. humilis suffered a considerable competitive disadvantage. It is proposed that competitive interactions may play a minor role in confining naturally occurring F1 hybrids to ecotonal areas between habitats preferentially occupied by A. humilis and A. manglesii. Spatial limitations on pollen and seed flow appear to be the major factors controlling hybrid distribution in this case.

The present-day vegetation of a cemetery at Gingin, Western Australia, is described by means of normal and inverse information analysis. In spite of severe disturbance during the past 10 years through annual burning and slashing, a well-defined floristic pattern is identifiable, even in the absence from the analysis of the two most characteristic species, Anigozanthos manglesii and A. humilis. It is suggested that whereas, in the past, parts of the cemetery were ecologically distinct and characterized by different groups of species, regular disturbance is altering these ecological conditions and causing changes in the distribution and behaviour of the species present. This changing ecology appears to be most sensitively demonstrated by the behaviour of A. manglesii and A. humilis.

The structure of a hybrid kangaroo paw population (Anigozanthos Labill.) in the shire cemetery at Gingin, Western Australia, was analysed from multivariate morphometric, pollen fertility and floral colour data. A. manglesii D. Don. comprised 87% of the population of 9547 flowering individuals in 1976, and occupied the lower, wetter areas of the cemetery to the exclusion of A. humilis Lindl. A. humilis (11 % of the population) occurred at the greatest density on the higher, drier areas. The intervening ecotonal regions were occupied by both species, and by the majority of F1 hybrids (0.5 % of the population) and backcrosses (c. 2%). Census data documented a threefold increase in the number of A. manglesii individuals, a slight increase in the number of F1 hybrids, and a decrease in the number of A. humilis by a third over a 3-year study period. These data suggest the occurrence of a limited amount of introgressive gene exchange in the face of potent barriers to interspecific hybridization.