Panmixia defines the genetic diversity of a unique arthropod‐dispersed fungus specific to Protea flowers

Knoxdaviesia proteae, a fungus specific to the floral structures of the iconic Cape Floral Kingdom plant, Protea repens, is dispersed by mites phoretic on beetles that pollinate these flowers. Although the vectors of K. proteae have been identified, little is known regarding its patterns of distribution. Seed bearing infructescences of P. repens were sampled from current and previous flowering seasons, from which K. proteae individuals were isolated and cultured. The genotypes of K. proteae isolates were determined using 12 microsatellite markers specific to this species. Genetic diversity indices showed a high level of similarity between K. proteae isolates from the two different infructescence age classes. The heterozygosity of the population was high (0.74 ± 0.04), and exceptional genotypic diversity was encountered (Ĝ = 97.87%). Population differentiation was negligible, owing to the numerous migrants between the infructescence age classes (N_m = 47.83) and between P. repens trees (N_m = 2.96). Parsimony analysis revealed interconnected genotypes, indicative of recombination and homoplasies, and the index of linkage disequilibrium confirmed that outcrossing is prevalent in K. proteae ( = 0.0067; P = 0.132). The high diversity and panmixia in this population is likely a result of regular gene flow and an outcrossing reproductive strategy. The lack of genetic cohesion between individuals from a single P. repens tree suggests that K. proteae dispersal does not primarily occur over short distances via mites as hypothesized, but rather that long-distance dispersal by beetles plays an important part in the biology of these intriguing fungi.     

Development of polymorphic microsatellite markers for the genetic characterisation of Knoxdaviesia proteae (Ascomycota: Microascales) using ISSR-PCR and pyrosequencing

Knoxdaviesia proteae is one of the first native ophiostomatoid fungi discovered in South Africa, where it consistently occurs in the infructescences of the iconic Cape Biome plant, Protea repens. Although numerous studies have been undertaken to better understand the ecology of K. proteae, many questions remain to be answered, particularly given its unique niche and association with arthropods for dispersal. We describe the development and distribution of microsatellite markers in K. proteae through Interspersed Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) enrichment and pyrosequencing. A large proportion of the 31492 sequences obtained from sequencing the enriched genomic DNA were characterised by microsatellites consisting of short tandem repeats and di- and tri-nucleotide motifs. Seventeen percent of these microsatellites contained flanking regions sufficient for primer design. Twenty-three primer pairs were tested, of which 12 amplified and 10 generated polymorphic fragments in K. proteae. Half of these could be transferred to the sister species, K. capensis. The developed markers will be used to investigate the reproductive system, genetic diversity and dispersal strategies of K. proteae.     

Knoxdaviesia capensis: dispersal ecology and population genetics of a flower-associated fungus

Protea-associated fungi are dispersed between flower heads by mites, beetles and possibly birds. For the ophiostomatoid fungus, Knoxdaviesia proteae, these vectors offer regular dispersal between distant floral hosts. Unlike K. proteae, Knoxdaviesia capensis occupies multiple Protea host species. In this study, we aimed to determine whether the generalist K. capensis shares the long-distance dispersal pattern with specialist K. proteae and whether it moves freely between different host species. We evaluated the genetic structure of K. capensis from five populations of a wide-spread host and between sympatric hosts. Twelve K. capensis-specific microsatellite markers were developed and applied to 90 individuals. K. capensis showed high genetic diversity and almost maximal genotypic diversity. All populations were poorly differentiated, indicating the presence of long-distance dispersal. No differentiation could be detected between sympatric host populations, suggesting free dispersal between different hosts. This implies that the beetle and bird vectors that pollinate Protea species show the same non-specific movement.     

Biodiversity and ecology of flower-associated actinomycetes in different flowering stages of <Emphasis Type="Italic">Protea repens</Emphasis>

Actinomycete bacteria have previously been reported from reproductive structures (infructescences) of Protea (sugarbush/suikerbos) species, a niche dominated by fungi in the genera Knoxdaviesia and Sporothrix. It is probable that these taxa have symbiotic interactions, but a lack of knowledge regarding their diversity and general ecology precludes their study. We determined the diversity of actinomycetes within Protea repens inflorescence buds, open inflorescences, young and mature infructescences, and leaf litter surrounding these trees. Since the P. repens habitat is fire-prone, we also considered the potential of these bacteria to recolonise infructescences after fire. Actinomycetes were largely absent from flower buds and inflorescences but were consistently present in young and mature infructescences. Two Streptomyces spp. were the most consistent taxa recovered, one of which was also routinely isolated from leaf litter. Lower colonisation rates were evident in samples from a recently burnt site. One of the most consistent taxa isolated from older trees in the unburnt site was absent from this site. Our findings show that P. repens has a distinct community of actinomycetes dominated by a few species. These communities change over time and infructescence developmental stage, season and the age of the host population. Mature infructescences appear to be important sources of inoculum for some of the actinomycetes, seemingly disrupted by fire. Increased fire frequency limiting maturation of P. repens infructescences could thus impact future actinomycete colonisation in the landscape. Streptomyces spp. are likely to share this niche with the ophiostomatoid fungi, which merits further study regarding their interactions and mode of transfer.     

Early colonization of Protea flowers enable dominance of competitively weak saprobic fungi in seed cones,benefitting their hosts

Sporothrix and Knoxdaviesia fungi use pollinators to colonize Protea flowers at anthesis. These saprobes remain dominant in the nutrient-rich, fire-retardant Protea seed-cones (infructescences) for at least a year after flowering. We tested the hypothesis that they competitively exclude potentially detrimental fungi from infructescences during this time. We compared seed set and longevity of infructescences containing Sporothrix and Knoxdaviesia vs. those that contain ‘contaminant’ saprobes. Hereafter we evaluated their competitive abilities against the ‘contaminant’ saprobes. Infructescences devoid of Sporothrix and Knoxdaviesia were dominated by Penicillium cf. toxicarium, Cladosporium cf. cladosporoides and Fusarium cf. anthophilum. Sporothrix and Knoxdaviesia presence did not affect seed viability, but infructescences persisted longer than those colonised by ‘contaminant’ fungi. The ‘contaminant’ species were stronger competitors than Sporothrix and Knoxdaviesia. However, Sporothrix and Knoxdaviesia could defend captured space well against ‘contaminant’ species. This effect was enhanced when fungal taxa grew on media prepared from their usual Protea host species, clarifying their dominance and host consistency observed in the field. Sporothrix and Knoxdaviesia from Protea are therefore weak competitors against common saprobes, especially when growing on alternative hosts, and need to colonise flowers very early (before colonization by other fungi) to dominate in this environment. They may delay seed release from infructescences longer than if these are colonised by other saprobes, increasing chances of seed release to occur after fire, when conditions are more favourable for Protea recruitment.     

Pollination mutualism between a new species of the genus Colocasiomyia de Meijere (Diptera: Drosophilidae) and Steudnera colocasiifolia (Araceae) in Yunnan, China

A new species of the genus Colocasiomyia de Meijere (Diptera: Drosophilidae) was discovered from inflorescences of Steudnera colocasiifolia K. Koch (Araceae) in Yunnan, China. The new species is described as Colocasiomyia steudnerae Takenaka and Toda, sp. nov., and we investigated the reproductive ecology of both the fly and the plant species. This fly species reproduces in the inflorescences/infructescences of the plant, and depends almost throughout its entire life cycle on the host plant. The fly species is the most abundant flower visitor for S. colocasiifolia and behaves intimately with the flowering events, suggesting that it is the unique and most efficient pollinator for the host plant. Bagging (insect‐exclusion) treatment of inflorescences resulted in no fruits. These findings strongly suggest that intimate pollination mutualism has evolved between the fly and the host plant, as are known in other Colocasiomyia flies and Araceae plants. One notable feature of this system is that the new species almost monopolizes the host‐plant inflorescence as a visitor, without any cohabiting Colocasiomyia species. In comparison to other cases where two Colocasiomyia species share the same inflorescence and infructescence of Araceae host plants for reproduction by separating their breeding niches microallopatrically between the staminate (upper male‐flower) and the pistillate (lower female‐flower) regions on the spadix, C. steudnerae exhibits a mixture of stamenicolous and pistillicolous breeding habits.     

Predispersal seed predation on five Piper species in tropical rainforest

Absolute number of seeds lost to predispersal seed predators and proportion of total seeds lost per infructescence were compared among five Costa Rican Piper species of different annual fecundities. Mean seed number and mean seed size in the five species were negatively correlated. The impact of predation on these species was inversely related to the number of seeds they produced. The two early successional species had very high fecundities, a combination of many seeds per infructescence, many infructescences per plant, and, in one species, year-round reproduction. Although seed predators destroyed as many or more seeds of these early successional species than they did of the less fecund, late successional species, this loss accounted for a relatively minor proportion (9 and 12%) of the seeds of the early successional species. In contrast, late successional species produced fewer, larger seeds in a smaller number of infructescences and were not continually in fruit. One of these species, which produced intermediate numbers of intermediately sized seeds, lost 30% of the seeds in each infructescence on average. Seed predators destroyed a larger proportion (65 and 76%) of the seeds per infructescence in the two species with fewest seeds per infructescence. High levels of insect damage in these late successional species caused many of their infructescences to abort prematurely. Taken together these factors resulted in annual fecundities several orders of magnitude smaller in shade-tolerant Piper species than the annual fecundities of shade-intolerant, early successional species. Seedlings of the two early successional species were common in large gaps and other sunny clearings and seedlings of the species with 30% seed loss were occasional, whereas no seedlings were seen of the two species with the highest proportional seed loss, suggesting that seed predation on the latter species may limit seedling recruitment.     

Comparative mitogenome analysis of three species and monophyletic inference of Catantopinae (Orthoptera: Acridoidea)

To enrich the genomic database of Catantopinae (Orthoptera: Acrididae), mitogenomes of three species from different genera, Traulia nigritibialis (15,701 bp), Choroedocus capensis (16,293 bp) and Stenocatantops splendens (15,574 bp), were characterized and compared with those of other grasshoppers in the subfamily. All 13 protein-coding genes (PCGs) were initiated by ATN codons except COI with ACC (C. capensis and S.splendens) and ND6 with TTG (S. splendens). All transfer RNA (tRNA) genes had a typical clover-leaf structure, except tRNA^Ser(AGN) in which the base pairs of the dihydrouridine (DHU) arm were reduced. The phylogenetic relationships were constructed among 22 species from four subfamiles of Acrididae by classical classifications based on two datasets of their mitogenomes using both Bayesian Inference (BI) and Maximum Likelihood (ML). The phylogenetic analysis confirmed the monophyly of the three other subfamilies, but did not provided support of the monophyly of Catantopinae.     

Convergent evolution unites the population genetics of Protea-associated ophiostomatoid fungi

Knoxdaviesia and Sporothrix species occupy the flower heads of some Protea plants in southern Africa. Knoxdaviesia species display exceptional genetic diversity within the Core Cape Subregion (CCR) and are readily dispersed across large distances. This study aimed to determine whether overlapping ecologies have led to a similar population genetic structure in Sporothrix splendens. Two DNA sequence markers, β-tubulin and a microsatellite region, were amplified in 97 S. splendens strains from eight populations that span its host distribution. Genetic diversity was low in a geographically isolated population, but high elsewhere. CCR populations were closely related, showing isolation by distance with populations at the eastern edge of the sampling range. Like Knoxdaviesia species, long-distance dispersal of S. splendens spores is prevalent, although likely affected by patchy host populations. This study is the first to consider populations of a non-clinical Sporothrix species, providing insights into the population attributes of a naturally distributed species.     

Experimental evidence for pollination of Banksia spp. by non-flying mammals

Summary The importance of non-flying mammals as pollinators of Banksia integrifolia and B. spinulosa was analysed by examining the effect of pollinator exclusions on fruit-set. Visitation by potential pollinators was also measured by observation and by indirect methods. Nonflying mammals were frequent visitors to inflorescences of both Banksia species. The aluminium sleeves used to exclude non-flying mammals from B. integrifolia trees were associated with a reduction in both the number of infructescences produced and the number of fruit per infructescence, indicating that non-flying mammals were important pollinators. Bird-nets over trees also significantly reduced the number of fruit per infructescence, but had no significant effect on the number of infructescences produced. The results of exclusion experiments using single inflorescences were inconclusive due to low fruitset. No conclusions could be drawn from these experiments with B. spinulosa. However, results for B. integrifolia support the conclusions of whole-tree experiments. Analysis of the genotype frequencies in seed from B. integrifolia provided no support for the hypothesis that the relatively limited mobility of non-flying mammal pollinators would cause inbreeding.     

Intra-crown variation in leaf herbivory and seed production in striped maple,Acer pensylvanicum L. (Aceraceae)

Summary Patterns of spatial variation in leaf herbivory and the effects of this variation on seed production and twig growth were studied in striped maple, Acer pensylvanicum (Aceraceae). Experimental removal of 25% of the leaf area from each of four leaves directly subtending a developing infructescence significantly reduced seed number in that infructescence. When leaf area was removed from leaves neighboring to, but not directly subtending developing infructescences, no reduction in seed production occurred. Together, these results suggest that only photosynthate from leaves directly subtending infructescences contributes to seed production in nearby infructescences. Effects of the experimental removal of leaf area did not persist the second year, suggesting that mobilization of storage products the following spring occurred independent of prior treatment. There was probably little negative impact of leaf herbivores on this plant species during the study year due to (1) low occurrence of localized damage within the crowns of censused trees and (2) delay of leaf area loss until completion of fruit development.     

The effect of unripe fruits on ripe fruit removal by birds in Pistacia terebinthus: flag or handicap?

The shrub Pistacia terebinthus produces crowded infructescences with up to several hundred fruits, which are bright red when unripe and turn green when ripe. Most fruits contain an empty seed and never reach maturity. More ripe fruits were removed by birds from experimental bicolored fruit displays (consisting of infructescences with ten ripe fruits and stripped of unripe fruits, paired with infructescences with only unripe fruits) than from monocolored ones (single infructescences with ten ripe fruits and stripped of unripe fruits). Thus, the presence of unripe fruits seems to increase the conspicuousness or attractiveness of fruit displays to fruit-eating birds. A second experiment compared ripe fruit removal from experimental infructescences having only ripe fruits, with that from control infructescences containing both ripe fruits and natural numbers of unripe fruits, all on P. terebinthus plants. Unlike the first experiment, each bicolored display in this case consisted of a single infructescence with both unripe and ripe fruits. A higher proportion of ripe fruits was removed by birds from infructescences free of unripe fruits. This result suggests that the presence of unripe fruits reduces the accessibility of ripe fruits for fruit-eating birds. This is further supported by field observations of bird foraging behavior.     

Are Protea populations seed limited? Implications for wildflower harvesting in Cape fynbos

Abstract Little is known about the extent to which plant population growth is limited by seed production. We studied two non-sprouting Protea species, both entirely dependent on seeds for recruitment after fire, to determine how flower harvesting would affect the size of the next generation after burning. Five harvesting treatments (0, 25, 50, 75 and 100% inflorescence removal) were applied, each replicated four times in 10 × 10m plots. Seedbanks were censused before a late-summer burn for each species and each replicate. The mean proportion of seeds surviving the burn until germination the following spring was 0.46 for Protea repens and 0.57 for Protea neriifolia. Of the seedlings that emerged, 90% and 55%, respectively, survived the first summer drought. Flower production in the 9 year old stand was strongly related to population density. Optimal plant densities for maximum flower production were estimated as 150 for P. repens and 70 for P. neriifolia. These target densities were greatly exceeded by seedling populations surviving the first summer drought in unharvested stands. If future density-independent mortality is negligible, the excess represents that part of the seedbank that could have been harvested before the burn without influencing future flower production. We developed a simple static model for setting flower harvesting levels in these seed-saturated populations. The predicted harvesting levels (50% of inflorescences in P. repens and 85% in P. neriifolia) were validated against the experimental harvests. We discuss the sensitivity of harvesting levels to variation in fecundity/density relationships, seed survival through a burn and seedling survival until flowering, and the implications for flower harvesting.     

Ophiostoma gemellus and Sporothrix variecibatus from mites infesting Protea infructescences in South Africa

Ophiostoma (Ophiostomatales) represents a large genus of fungi mainly known from associations with bark beetles (Curculionidae: Scolytinae) infesting conifers in the northern hemisphere. Few southern hemisphere native species are known, and the five species that consistently occur in the infructescences of Protea spp. in South Africa are ecologically unusual. Little is known about the vectors of Ophiostoma spp. from Protea infructescences, however recent studies have considered the possible role of insects and mites in the distribution of these exceptional fungi. In this study we describe a new species of Ophiostoma and a new Sporothrix spp. with affinities to Ophiostoma, both initially isolated from mites associated with Protea spp. They are described as Ophiostoma gemellus sp. nov. and Sporothrix variecibatus sp. nov. based on their morphology and comparisons of DNA sequence data of the 28S ribosomal, beta-tubulin and internal transcribed spacer (ITS1, 5.8S, ITS2) regions. DNA sequences of S. variecibatus were identical to those of a Sporothrix isolate obtained from Eucalyptus leaf litter in the same area in which S. variecibatus occurs in Protea infructescences. Results of this study add evidence to the view that mites are the vectors of Ophiostoma spp. that colonize Protea infructescences. They also show that DNA sequence comparisons are likely to reveal additional cryptic species of Ophiostoma in this unusual niche.     

INTERFERENCE THROUGH IMPROPER POLLEN TRANSFER IN MIXED STANDS OF IMPATIENS CAPENSIS AND I. PALLIDA (BALSAMINACEAE)

Mechanisms of interference through improper pollen transfer are described for sympatric populations of the simultaneously flowering annuals Impatiens capensis and I. pallida. Stigmas of both Impatiens species are receptive to conspecific pollen from the time of androecium disarticulation until after perianth drop. Pollen of I. pallida adheres to, and germinates on, the stigmas of I. capensis. The pollen tubes reach the ovules but do not fertilize them. In contrast, pollen of I. capensis adheres poorly to I. pallida stigmas and fails to germinate. Pollen mixtures of the two Impatiens species cause a much greater reduction in fruit set in I. capensis than in I. pallida. Despite the interference of I. pallida pollen, I. capensis persists in mixed populations with I. pallida by: 1) possessing stigmas receptive to pollen throughout the female phase; 2) having the ability to produce a full seed complement via one pollination event; and 3) possessing cleistogamous flowers to ensure sexual reproduction.     

Seed, cutting and air-layering reproductive inefficiency of noxious woody vine Merremia biosiana and its implications for management strategy

Merremia boisiana (Gagnep) van Ooststr. is a noxious fast growing woody vine and is able to grow overtop other plants, causing the death of plants underneath and forming monospecies stands. To formulate management responses, we assessed its seed and vegetative reproduction efficacy through indoor and field experiments. The number of flowers counted from bagged infructescences in Guangzhou ranged from 25 to 172, with an average of 80.80. Counting the seeds of bagged infructescences had shown that there were only 1.58 hard testa seeds in each infructescence. Seed vitality tests using red ink indicated that only 68.6% of hard testa and filled seeds were viable. The emergence rate of scarified hard testa seeds in the sand bed was 31.96%. Under imitated natural conditions, 8% of hard testa seeds could germinate, and 9% still retained their germination potential in one year. Thus, seedlings should be monitored and removed in a timely fashion after any attempt of clearing. Moving soil or transplanting plant from infested patches should be strictly prohibited at least for several years. All considered, an infructescence contributed 0.3 seedlings. The investigation in the field found no seedlings either inside or at the perimeter of the patch, suggesting scant expansion by means of seed dispersal. Therefore, the elimination effort could be focused on a relatively restricted scale of patches. Of 630 cuttings of young shoots, old shoots and old lying shoots with or without growth regulators, there were only four (or 0.63%) established individuals. Air-layering shoots all died in two months. Poor cuttings and air-layering reproduction indicated that regeneration from fragments of removed stems or accidentally dropped ones was quite unlikely, and thus mechanical removal was safe.     

Infructescence size has a larger effect than light environment on the abundance of different arthropod feeding guilds dwelling on the infructescences of a terrestrial bromeliad in a xerophytic forest

Bromeliads are a conspicuous feature of many Neotropical xerophytic forests. Bromelia serra is an understory bromeliad living in xerophytic forests of the Humid Chaco, which shows high phenotypic plasticity when exposed to different environmental conditions. Its infructescences carry many fleshy fruits that are colonized by arthropods from different feeding guilds. We used structural equation modeling and redundancy analysis to evaluate the influences of light environment, plant size, and infructescence size on the abundance of five different feeding guilds of arthropods dwelling on the infructescences (‘fruit-secretion feeders,’ ‘infructescence-detritus feeders,’ ‘predators,’ ‘pulp feeders’ or ‘seed feeders’). Plant size was negatively associated with canopy openness, whereas infructescence size was positively associated with plant size. The abundance of all feeding guilds, except fruit-secretion feeders, were positively associated with infructescence size.     

Reproductive biology of the rare and endangered Banksia brownii Baxter ex R. Br. (Proteaceae)

Abstract Banksia brownii is an endangered species, now limited to ～ 15 disjunct populations in southwestern Western Australia. Data on flowering phenology, plant size, fruit set, pollination and the mating system were gathered for two of these populations between March and October 1993. Flowering for both populations followed a similar pattern, with open flowers first evident in April, and the number of inflorescences with open flowers peaking in June. At both locations, trees differed considerably with respect to their size, the total number of inflorescences produced and the length of their flowering season. Fruiting success was typically low, with approximately half of all inflorescences failing to develop into infructescences. Only 1. 8% of the flowers originally present on inflorescences developed into follicles. The distribution of follicles along each infructescence was non-random, with most forming in the middle third of the infructescence for reasons relating to nutrient supply and pollinator behaviour. More flowers opened during the day than at night, although pollen was lost from individual flowers during both periods. Honeyeaters such as Phylidonyris novaehollandiae were common at the two study sites, and often carried large loads of B. brownii pollen. Though less frequently caught, the nocturnal mammals Rattus fuscipes and Tarsipes rostratus also bore substantial amounts of pollen. Most inflorescences from which these mammals and birds were excluded remained barren. Fruiting success was further reduced when invertebrates such as Apis mellifera were also prevented from visiting inflorescences. The ability of B. brownii to set at least some fruit in the absence of biotic poli-nators indicates that the species is partially self-compatible. Honeyeaters foraged preferentially at inflorescences with one to two thirds of their flowers open, probing mainly along the ‘advancing front’ of open flowers. These animals moved more frequently between inflorescences on the same plant than between those on different plants, and were often recaptured in the same locations. Mammals also appeared to be sedentary. Both B. brownii populations had mixed mating systems, with genetically determined outcrossing rates of ～0.7. The unusually high level of selfing in each population is presumably a reflection of the species’ self-compatibility and the foraging behaviour of its pollinators.     

The effect of nutrients and inflorescence damage by insects on fruit-set by Banksia spinulosa

Summary We examined the role of added macronutrients (N, P, and K) and prevention of damage to inflorescences (ovule/seed predation and rachis damage) by insects in determining levels of fruit-set in Banksia spinulosa at two sites in southeastern Australia. Insecticide application eliminated damage to developing inflorescences (primarily by the lepidopteran, Arotrophora canthelias) and application of macronutrient fertilizer significantly increased nutrient concentrations in leaves at both sites. Only joint application of nutrients and insecticide increased fruit-set per infructescence significantly above that of the control, indicating that the effect of nutrients on fruit-set depends on the intensity of insect damage to inflorescences. The proportion of inflorescences that developed fruits differed between sites and higher levels of rachis damage at one site may partially explain the lower proportion of inflorescences that fruited. Neither nutrient addition or inflorescence damage influenced the position of fruit-set within an infructescence. Our results suggest that (1) macronutrient availability and inflorescence damage interact in determining levels of fruitset in B. spinulosa, and (2) other factors, such as pollen or micronutrient availability, may limit it at some sites. These results highlight site-specific variation and interaction in the factors that limit plant reproduction.