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South African Journal of Botany 79 (2012) 77–83 www.elsevier.com/locate/sajb

The influence of pollinators and seed predation on seed production in dwarf “sugarbushes” () ⁎ S.-L. Steenhuisen , S.D. Johnson

School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209,

Received 8 November 2011; received in revised form 25 November 2011; accepted 5 December 2011

Abstract

Flowers of many are visited by both birds and , making it necessary to establish their relative contributions to seed set. In Protea, available evidence points to an overwhelming preponderance of bird- systems in the , but the scented flowers of several dwarf grassland “sugarbush” species suggest that some Protea species may be adapted for pollination. In this study, we used both selective exclusion of 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 ) in KwaZulu-Natal, South Africa contribute to seed production. We also performed supplemental hand to test for limitation. Seed set was generally higher in inflorescences subjected to 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 estab- lish the actual level of insect-mediated outcrossing. © 2011 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Pollination; Protea; Resource limitation; Seed predation;

1. Introduction in , and Protea (Faegri, 1965; Hargreaves et al., 2004; Mostert et al., 1980; Rebelo, 2001) The large Gondwanan plant family Proteaceae shows con- although rodent pollination has been recorded for a few geo- siderable diversity in reproductive systems (Ayre and Whelan, florous Protea species (e.g. Protea amplexicaulis and Protea 1989; Collins and Rebelo, 1987; Goldingay and Carthew, humiflora)(e.g.Wiens and Rourke, 1978), and insect and 1998). Among Australian genera, bird- and mammal- wind pollination is inferred for most species pollination dominates in and (Collins and (Collins and Rebelo, 1987) and other genera. In Protea,the Rebelo, 1987), while insect pollination has been recorded in distribution and guild composition of insects inhabiting inflo- Banksia, Dryandra, Grevillea, , and rescences have been documented over many years, mainly in (Blanche et al., 2006; Carolin, 1961; Lamont, 1982; the context of the marketability of cut Protea flowers Lamont and Collins, 1988; Lamont et al., 1998; Wallace et al., (Coetzee and Latsky, 1986; Gess, 1968; Myburgh and Rust, 1996). Among the African genera, bird pollination dominates 1975; Myburgh et al., 1973; Wright and Giliomee, 1990; Wright and Samways, 2000). For most Protea species, there is still considerable uncertainty whether the insects that fre- ⁎ Corresponding author. Tel.: +27 33 2605657; fax: +27 33 2605105. quent contribute to seed production (Coetzee E-mail address: [email protected] (S.-L. Steenhuisen). and Giliomee, 1985; Collins and Rebelo, 1987).

0254-6299/$ -see front matter © 2011 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2011.12.004 78 S.-L. Steenhuisen, S.D. Johnson / South African Journal of Botany 79 (2012) 77–83

Exclusion experiments, in which avian visitors were prevented ex J.M.Wood , located on the grassland slopes of the sum- from visiting inflorescences covered with wire mesh cages, have mit of Mount Gilboa (29.29°S, 30.29°E, 1770 m), KwaZulu- been conducted to ascertain the contribution of bird versus insect Natal, was used for experiments conducted in January 2002 and pollinators in a few Protea species. Caged inflorescences of 2005. A population of about 300 plants of P. welwitschii Engl., , , and Protea located on steep grassland slopes of a residential area in Winston roupelliae set significantly less seed than open-pollinated treat- Park (SE-facing slope in 2003, NW-facing slope (Giba Gorge) in ments, supporting the idea that these species are predominantly 2004) (28.75°S, 30.75°E, 550 m), was used in 2003 and 2005. A bird-pollinated (Hargreaves et al., 2004; Wright et al., 1991). In large population (approximately 500 plants) of P. dracomontana contrast, caged and open-pollinated inflorescences of Protea Beard on the lower slopes of Garden Castle (29.74°S, 29.20°E, cynaroides, and set similar amount 1900 m) in the Drakensberg mountains was used for this study of seed (Coetzee and Giliomee, 1985; Wright et al., 1991). For in 2007. All three species have a flowering period ranging from five of these seven species, a treatment excluding all visitors December to March with a January peak. These sites receive was not included and it is thus unclear whether high seed set in summer rainfall. Voucher specimens have been deposited in the vertebrate-excluded inflorescences was due to insect pollination University of KwaZulu-Natal (voucher numbers: or to autonomous self-fertilization. It is thus essential to establish Steenhuisen 55, 57, 59, 60, 62). The species were identified the breeding system of each species in order to determine self- according to Rourke (1980). compatibility and, if so, whether they can set seed autonomously (without the use of pollen vectors). At least some Protea species 2.2. Experimental design are self-compatible, viz. P. repens (Van der Walt, 1995)and P. roupelliae (Hargreaves et al., 2004), and we have recently To investigate the pollination effectiveness of beetles and documented self-compatibility and autogamy in four grassland other invertebrates, exclusion experiments were performed on Protea species (unpublished results). Nevertheless, autogamy is the three Protea study species. Wire mesh cages painted most often facultative rather than obligate, and pollinators may green to reduce their conspicuousness were erected over single therefore contribute to seed production in these Protea species. inflorescences to exclude vertebrate visitors to the flowers. While insects have been shown to affect pollination in Treatments applied to inflorescences consisted of (1) enclosure ornithophilous Protea species, their contribution to pollination in a small diameter (15 mm) mesh cage that excludes verte- of Protea species with floral traits suggestive of insect- brates and larger insects, (2) enclosure in a larger diameter pollination has not been investigated. We excluded vertebrate (30 mm) mesh cage that excludes vertebrates while allowing visitors from three putatively insect-pollinated grassland Protea most insects to pass through, (3) enclosure in a fine mesh bag species (Protea dracomontana, Protea simplex, and Protea that excludes all visitors and thereby tests for autogamous welwitschii) to investigate the contribution of insect visitors to seed production, (4) open inflorescences as a control to assess seed set. These three species have floral traits similar to bird- natural seed set, and (5) supplemental hand-pollination to test pollinated Protea species, such as colorful involucral for pollen limitation. A wire support in (3) kept the bag from and abundant rewards, but they also have floral traits extensively touching the and its pollen presenters. conforming to a beetle-pollination syndrome, notably a strong, Inflorescences in (4) were cross-pollinated at least twice by fruity floral scent (Steenhuisen et al., 2010), smaller and more brushing five or more freshly exposed pollen presenters from bowl-shaped inflorescences, immense pollen rewards and low another inflorescence of a different plant over the stigmatic plant growth form (Steenhuisen and Johnson, in press). The in- grooves of all florets of each inflorescence during its recep- florescences of these species are visited by a variety of general- tive stage, determined by previous observations (unpublished ist insects, but most frequently by cetoniine beetles foraging on results). Bags and cages were applied at the bud stage before both nectar and pollen (Steenhuisen and Johnson, in press). De- the inflorescences opened. From occasional observations of spite being self-compatible and partially autogamous (unpub- insects visiting caged inflorescences, although this was not for- lished results) these species are characterized by low seed set mally quantified, it became apparent that similar-sized insects (b40% florets set seed). We used supplemental hand- werefoundtovisitinflorescencescoveredbydifferentcage pollination (Bierzychudek, 1981) to test whether seed set in mesh sizes (e.g. Cetoniinae ranging from 10 to 25 mm in the study species was limited by either pollen or resource avail- length; honeybees; flies; various ). We thus pooled ability (Ayre and Whelan, 1989). We also quantified the rate of data for all caged inflorescences. All treatments were applied to seed predation and identified insect seed predators (e.g. inflorescences on the same plant whenever possible or on adja- Myburgh et al., 1973). cent plants in order to control for local habitat effects. In all ex- periments, 20–24 inflorescences were used for each treatment 2. Materials and methods and corresponding open-pollinated controls. No pollen supple- mentation treatment was performed on P. dracomontana. 2.1. Study species To investigate pollen deposition by insects on stigmas of P. welwitschii¸ five stigmas from each open, caged and pollen- The role of insect pollinators in seed production in three grass- supplemented inflorescence were collected from experimental land Protea species was investigated in KwaZulu-Natal, South plants in 2004 once the inflorescence bracts had withered and Africa. A population of approximately 550 P. simplex E.Phillips the anthers senesced. We did not collect stigmas from bagged S.-L. Steenhuisen, S.D. Johnson / South African Journal of Botany 79 (2012) 77–83 79 plants for this experiment as self-pollen coats pollen presenters appropriate, and compared treatments using pairwise contrasts and stigmas of these species and would not have been removed with sequential sidak adjustment for multiple comparisons by pollinators or wind/rain. The tips were squashed (Hosmer and Lemeshow, 2000). I models were used to ac- fresh in fuschin gel, which was melted to form permanent slides count for the effect of year before treatment on the proportion of (Beattie, 1971) and the number of foreign (distinguishable from styles with pollen tubes, the number of pollen tubes per style Protea pollen in shape and texture) and Protea pollen grains (2003 and 2004), and seed set (2003 and 2005) for P. welwitschii. (using the unique smooth-edged triangular shape of Protea pollen Treatment effects on the number of pollen grains deposited on as a reference) deposited on each stigma determined. A further stigmas and pollen tubes growing in styles were tested using five stigmas from each experimental inflorescence (including means per inflorescence rounded to the nearest integer and fitted bagged ones) in P. simplex (2002) and P. welwitschii (2003/ to models with a Poisson error distribution and log link functions. 2004) were collected to determine pollen tube growth in the style. Each stigma was fixed in 2 ml 3:1 70% ethanol: acetic 3. Results acid for 1 h, washed with distilled water, and stored in 2 ml 70% ethanol. Fixed stigmas were prepared for pollen tube analy- Very pure pollen loads (N90% Protea pollen) were deposited sis using a softening and staining procedure modified from on all stigmas of unbagged P. welwitschii experimental inflores- Martin (1959), allowing for the examination of pollen tubes in cences (Table 1). Stigmas of caged inflorescences received the style through aniline blue UV-induced fluorescence of callose slightly lower pollen loads than open controls, while pollen associated with the pollen tube wall. The stigmas were rinsed in supplementation increased pollen loads by about 60% distilled water for 10 min, softened and cleared by suspending (Table 1). Over 80% of P. welwitschii stigmas received pollen them in 4 N NaOH for 48 h, rinsed in tap water for 1 h, and in all treatments. A small percentage (0–7%) of pollen loads stained with aniline blue-0.1 N K2HPO4 for 4 h. The stained found on P. welwitschii stigmas was comprised of pollen from stigmas were stored in glycerin for no longer than 3 days before up to eight other species (see pollen purity examination. Stigmas were mounted on slides in a drop of stain measures in Table 1). and glycerin, and flattened with a coverslip. The proportion of Seed set for all species was very low, never reaching above styles with pollen tubes in the upper first centimeter and the num- 40%, despite evidence of prolific pollen tube growth on most ber of pollen tubes per stigma for each treatment were determined stigmas in pollen supplemented inflorescences in P. simplex by examining the stigmas with an Olympus Provis, AX-70 and P. welwitschii (Figs. 1 and 2). For P. simplex, we observed equipped with a UV filter system consisting of a dichroic mirror lower proportions of stigmas with pollen tubes (2002, treatment: – 2 (400 nm), an ultraviolet excitation filter (330 385 nm) and a bar- χ (3) = 23.302, Pb0.001) and lower numbers of pollen tubes per 2 rier filter (420 nm). In all analyses, insect-damaged stigmas and stigma (2002, treatment: χ (3) =139.631, Pb0.001) for bagged, the rare case of pollen/pollen tube loads of over 1000 grains caged and open-pollinated inflorescences compared to pollen were excluded from analyses. supplemented inflorescences (Fig. 1a,c). This pattern was not Infructescences were collected 4 months after each flowering reflected by seed set for which we recorded higher seed set for season, and the proportion of florets that set seed was determined caged and pollen supplemented inflorescences compared to 2 for each treatment. Plump ovaries were treated as containing open-pollinated and bagged inflorescences (year: χ (1) =15.167, 2 fertile seeds. Damaged infructescences were assessed for evi- Pb0.001; treatment: χ (3) = 17.579, P=0.001; Fig. 2a). dence of predation (dried frass, emergence holes in the base, Pollen supplementation increased the proportion of stig- damaged styles, eaten seeds) and excluded from analyses. The mas with pollen tubes in P. welwitschii (2003–2004, year: percentage of infructescences damaged by insect predators (partially or completely), lost to uncontrolled fires through the study sites or premature release of seeds was determined. Lepi- Table 1 dopteran larvae found in damaged infructescences were collected The effect of natural pollination, caging and pollen supplementation on into glass vials, and allowed to pupate and metamorphose. stigmatic pollen loads for Protea welwitschii in 2004. Emerged adults were identified by Dr M. Krüger (Transvaal 2 Treatment (sample size) χ (2) Museum), and voucher specimens stored in the entomological Open Cage Pollen collection at the University of KwaZulu-Natal. control (40) supplemented (57) (20) 2.3. Statistical analyses Pollen load per stigma Mean 12.1a 10.5a 78.6b 184.914* Lower SE 1.5 1.7 6.8 We analyzed the effects of treatment on the number of pollen Upper SE 1.8 2.0 7.4 grains per stigma, proportion of stigmas that received pollen, pro- Proportion of sampled Mean 0.86a 0.83a 0.99b 22.258* portion of deposited pollen that was Protea pollen, proportion of stigmas with Protea Lower SE 0.02 0.03 0.02 pollen Upper SE 0.02 0.02 0.01 stigmas with pollen tubes in the upper style, the number of pollen Protea proportion of Mean 0.93a 0.96a 1.00b 45.082* tubes per style, and the proportion of florets that set seed using pollen load Lower SE 0.01 0.02 0.003 generalized linear models (GZLMs). Unless otherwise stated Upper SE 0.01 0.01 0.002 we used likelihood ratio Chi-square statistics, logit link functions, Significant differences between treatments are indicated by different letters next binomial error distributions corrected for overdispersion where to the means (significance level: *Pb0.001). 80 S.-L. Steenhuisen, S.D. Johnson / South African Journal of Botany 79 (2012) 77–83

(a) (b) Seed set in P. dracomontana did not differ significantly be- 2 tween bagged, caged and open-pollinated treatments (χ (2) = 0.908, P=0.635; Fig. 2c). Insect predator species typically laid their eggs on the base of the inflorescence buds and the hatched larvae bored through the involucral bracts and devoured the , ovaries, or maturing seeds in the inflorescence. Lepidopteran larvae (, ) were solely responsible for seed and receptacle predation, while other lepidoteran larvae (Psychidae) (c) (d) and very large Coleoptera (Dynastinae, Cetoniinae, Scarabaeinae) predated on floral parts above the involucral bracts, damaging inflorescences and preventing them from setting seed. From pupa- tion and subsequent emergence of lepidopteran predators from larvae found in experimental inflorescences, we determined that the majority of predation was due to the Cydia sp. cf. ocnogramma (Meyrick, 1910) (Tortricidae: Olethreutinae: Grapholitini). Minor predators were the orange banded protea , alpheus (Cramer, 1777) (Lycaenidae: : ), and the small moth, Epichorista sp. cf. galeata Meyrick, 1921 (Tortricidae: : ). Of all the Fig. 1. The effect of “full” exclusion of pollinators by bagging, “partial” exclusion larvae collected from experimental inflorescences (48 individ- of pollinators by caging (allowing access to invertebrates only), natural (“none”) uals) from Mt Gilboa, 50.0% were determined to be C. sp. cf. “ ” – and supplemental hand-pollination ( supp. )onthe(a b) proportion of stigmas ocnogramma,4.2%E. sp. cf. galeata, and the remaining 45.8% with pollen tubes growing in the style, and (c–d) the pollen tube loads on stigmas for P. simplex (Mount Gilboa 2002) and P. welwitschii (Winston Park 2003 and failed to metamorphose and were not identified. Very few infruc- 2004). Different letters indicate statistically significant differences between treat- tescences opened prematurely or released dry, withered and ments (Pb0.05). aborted fruits without visible evidence of predation (Table 2). Missing infructescences were usually from the exclusion experi- ments and could be attributed to damage from caging and wires supporting them. Those missing from experimental plants of 2 2 χ (1) =9.389, P =0.002; treatment: χ (3) =20.207, P b0.001), P. dracomontana were mostly due to removal by baboons. and along with bagging, more than doubled the number of pollen tubes found on open or caged stigmas (2003–2004, year: 2 2 χ (1) =32.891, Pb0.001; treatment: χ (3) =104.239, Pb0.001); 4. Discussion Fig. 1b,d). Seed set in P. welwitschii was very low (b15%), and with the This study provides limited support for the hypothesis of insect exception of slightly higher seed set after pollen supplementation pollination in P. dracomontana, P. simplex and P. welwitschii. compared to caged inflorescences, seed set was similar for all Exclusion of vertebrates had little effect on the number of pollen 2 other pairwise comparisons of treatments (year: χ (1) =14.621, tubes or seed set in all three species (Table 1; Figs. 1 and 2). 2 Pb0.001; treatment: χ (3) = 12.221, P=0.007; Fig. 2b). With the exception of P. dracomontana, inflorescences from

(a) (b) (c)

Fig. 2. The effect of “full” exclusion of pollinators by bagging, “partial” exclusion of pollinators by caging (allowing access to invertebrates only), natural (“none”) and supplemental hand-pollination (“supp.”) on the adjusted mean proportion of florets to set seed per inflorescence for (a) P. simplex (Mount Gilboa 2002 and 2005 combined), (b) P. welwitschii (Winston Park 2003 and Giba Gorge 2005 combined), and (c) P. dracomontana (Garden Castle 2007). Different letters indicate statistically significant differences between treatments (Pb0.05). S.-L. Steenhuisen, S.D. Johnson / South African Journal of Botany 79 (2012) 77–83 81

Table 2 The proportion of experimental inflorescences lost to predation and other environmental factors for three Protea species spanning various years and sites. Species Undamaged Partially Completely Dry Burnt Missing Released seeds Total no. of (%) eaten (%) eaten (%) (%) (%) (%) prematurely (%) inflorescences sampled Protea simplex Gilboa 2002 58.33 27.27 12.12 2.27 0.00 0.00 0.00 132 Protea simplex Gilboa 2005 50.31 20.13 22.64 0.00 0.00 6.92 0.00 159 Protea welwitschii Winston Park 2003 25.42 41.53 32.20 0.85 0.00 0.00 0.00 118 Protea welwitschii Winston Park 2005 42.68 8.54 0.00 0.00 44.51 0.00 4.27 164 Protea dracomontana Garden Castle 2007 15.05 23.66 51.61 0.00 0.00 9.68 0.00 93 which vertebrates but not insects were excluded generally had similar lack of response in seed set to pollen supplementation slightly higher seed set than inflorescences bagged to exclude was reported in the bird-pollinated grassland species P. roupelliae insects as well, indicating that insects transfer viable cross- (Hargreaves et al., 2004). In contrast, the effect of pollinator limi- pollen. These results do not, however, provide unambiguous tation was clearly demonstrated by extremely low natural fruit set evidence for the importance of insect pollination because there in Protea subvestita studied by Carlson and Holsinger (only 18% was also substantial autogamous seed production in bagged inflo- of naturally pollinated infructescences investigated contained seed, rescences. Supplemental pollination had little effect on natural 2010). Seed set may also vary from year to year (Vaughton, 1991) seed set in two species, suggesting that seed production in the and pollen supplementation may affect seed set in subsequent study species was not pollen-limited. flowering seasons by draining the plant's resources (Janzen et A more precise method for investigating the contribution of al., 1980). However, pollen supplementation failed to significant- different pollinators in autogamous species is to use emasculated ly increase seed set in P. welwitschii in two separate seasons flowers, so that seed set reflects only transfer of cross-pollen. This (2003 and 2005). was not feasible in inflorescences of these Protea species due to The high levels of seed predation recorded in this study are the gradual maturity of florets within an inflorescence and the typical for Proteaceae (Mustart et al., 1995; Wright, 1994; immense amount of self-pollen covering pollen presenters and Wright and Samways, 2000). Insect predators halved seed set stigmas. Whelan et al. (2009) successfully washed self-pollen per plant in var. neoanglica (Vaughton, off pollen presenters of to measure pollen 1990), and reduced seed set to zero for some Cape Protea species deposition by pollinators. They were able to show that despite the (Carlson and Holsinger, 2010). Coetzee and Giliomee (1987) high frequency of visits by honeybees to this species, that they found that more than 80% seeds of P. repens were destroyed by deposited fewer pollen grains than birds. The presence of foreign insect predators within 2 years after flowering. Like Wright and pollen on stigmas of caged P. welwitschii inflorescences indicat- Samways (2000), we found that the major predators of seeds in ed that insects were transferring pollen from other flowering Protea infructescences in KwaZulu-Natal were Cydia species in the community. and other Olethreutinae species (Tortricidae), which are econom- Seed set in autogamous plants is less likely to be pollen- ically important pest species in South Africa (Timm et al., 2010). limited than in allogamous plants but pollen-limitation is Other predators that we recorded included C. alpheus (Lycaeni- known to occur in some species in which autogamy is not effi- dae) and species of Curculionidae, and Psyllidae. cient enough to result in all setting seed (Rodger et al., Seed predators are undoubtedly a factor limiting seed production 2010). In such plants, which are typically facultatively autoga- but their effect is hard to quantify. If we had applied insecticide mous, pollinators can make significant contributions to seed (as done in Vaughton (1990) and Zammit and Hood's (1986) production. In our study, inflorescences of P. simplex that were Banksia studies), then cross pollination by insects would have supplemented with pollen produced more seeds than those that been compromised. We did attempt application of insecticide were bagged but this was not evident in P. welwitschii,suggest- after pollination in some trials, but most lepidopteran predators ing that autogamy in P. simplex does not reach the threshold had already laid eggs at the bud stage (mesh bags did not exclude that is physiologically possible. predators from laying eggs on experimental inflorescences), Previous studies in the Proteaceae have identified a range of and there was little effect on subsequent predation. Similarly, factors limiting seed production (Ayre and Whelan, 1989; weevils laid eggs before experiments could commence on Vaughton, 1991; Whelan and Denham, 2009). In some species, , leading to predation of 79% of infructescences seed set can be increased with the addition of nutrients (e.g. Bank- (Abbott, 1985). Wallace and O'Dowd (1989) were also able to sia cunninghamii; Vaughton, 1991), and Vaughton and Ramsey increase seed set in B. spinulosa after applying insecticide to (1998) increased seed mass, but not seed set, from a plants, but this increase was significant only with the addition redistribution of plant resources by removing inflorescences of nutrients. However, as pointed out by Ayre and Whelan from . Resource limitation was proposed as (1989), none of these manipulations (pollen-supplementation, re- the major constraint on fruit and seed set in several Grevillea spe- source addition, removal of seed predators) result in fruit:flower cies (Hermanutz et al., 1998; Holmes et al., 2008). The lack of a ratios near unity, indicating that there are other factors limiting significant increase in seed set following pollen supplementation seed set in this plant family. in P. simplex and P. welwitschii suggests that seed set in these spe- Due to confounding factors of low seed set, self-compatibility cies is also limited by resources rather than pollen availability. A and high seed predation, our exclusion experiments provided 82 S.-L. Steenhuisen, S.D. Johnson / South African Journal of Botany 79 (2012) 77–83 limited evidence for the contribution of insect pollinators to seed Goldingay, R.L., Carthew, S.M., 1998. Breeding and mating systems of Australian – production. One solution to this problem would be to compare out- Proteaceae. Australian Journal of Botany 46, 421 437. Hargreaves, A.L., Johnson, S.D., Nol, E., 2004. Do floral syndromes predict crossing rates for seeds derived from bagged, vertebrate-excluded specialization in plant pollination systems? An experimental test in an and open-pollinated inflorescences. Preliminary results from stud- “ornithophilous” African Protea. Oecologia 140, 295–301. ies using multilocus outcrossing rates in ,agrassland Hermanutz, L., Innes, D., Denham, A., Whelan, R., 1998. Very low fruit: flower species with scented flowers, indicate that there is substantial out- ratios in Grevillea (Proteaceae) are independent of breeding system. Australian – crossing in seed derived from inflorescences from which verte- Journal of Botany 46, 465 478. Holmes, G.D., James, E.A., Hoffmann, A.A., 2008. 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We are grateful to Mondi forestry company for permission to Lamont, B.B., Collins, B.G., 1988. Flower colour change in :a work in the Mt Gilboa Estate; KZN Wildlife for permitting us to signal for pollinators. Australian Journal of Ecology 13, 129–135. work in protected areas in KZN (project number NA/20094/02); Lamont, B.B., Olesen, J.M., Briffa, P.J., 1998. Seed production, pollinator — Greg Anderson for guidance with fluorescence microscopy; attractants and breeding system in relation to fire response are there re- productive syndromes among co-occurring Proteaceous ? Australian Anna Hargreaves, Dave Thompson, Paulo Massinga, Craig Journal of Botany 46, 377–385. Peter, Peter Mutabazi, Natalie Swanepoel, James Rodger, Martin, F.W., 1959. Staining and observing pollen tubes in the style by means Mariaan Ponsie, Katherine Johnston and the Hunter and of fluorescence. Stain Technology 34, 125–128. 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Edited by JC Manning