bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1

1 Equality between the sexes in plants for costs of reproduction; evidence from the dioecious 2 Cape genus Leucadendron (Proteaceae).

3 Jeremy J. Midgley*1, Adam G. West1, Michael D. Cramer1

4 1Dept Biological Sciences, University of Cape Town, P bag Rondebosch 7701, South Africa

5 [email protected]; [email protected]; [email protected].

6 Keywords Life history evolution, plants, sexual allocation, dioecy, Leucadendron, 7 dimorphism

8 Running title: Equality between the sexes in plants

9 bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2

10 Abstract

11 It has been argued that sexual allocation is greater for female function than male function in 12 plants in general and specifically for the large dioecious Cape genus Leucadendron. Here, 13 we use new interpretations of published information to support the hypothesis of equality 14 between sexes in this genus. The explanations are based on the fire ecology of the Cape that 15 results in reproductive synchrony, reproductive doubling and competitive symmetry. Firstly, 16 strict post-fire seedling establishment of the reseeder life-history in the Cape results in single- 17 aged populations. Consequently, the reproductive and vegetative schedules of males must 18 synchronously track that of females. This implies equal allocation to sex. Secondly, after 19 fires, dioecious females have double the seedling to adult ratio of co-occurring 20 hermaphrodites. This indicates that being liberated from male function allows females access 21 to resources that double their fitness compared to hermaphrodites. Therefore, male and 22 female costs of reproduction are equal in hermaphrodites. Thirdly, competitive symmetry 23 must occur because males and female plants will frequently encounter each other as close 24 near neighbours. Competitive asymmetry would both reduce mating opportunities and skew 25 local sex ratios. The evidence to date is for 1:1 sex ratios in small plots and this indicates 26 competitive symmetry and a lack of dimorphic niches. Finally, vegetatively dimorphic 27 species must also allocate equally to sex, or else sexual asynchrony, lack of reproductive 28 doubling or competitive asymmetry will occur.

29

30 bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 3

31 Introduction

32 Understanding allocation by the different sexes is a key focus in evolutionary studies and in 33 organisms where both parents contribute genes equally to off-spring, sexual allocation (e.g. 34 sex ratios, parental care of off-spring of different sexes, costs of reproduction) is expected to 35 be equal (Fischerian) (Hamilton 1967; Charnov 1982). It is difficult to investigate sexual 36 allocation in plants because most plants (>90%) are hermaphrodites, nevertheless, it is 37 generally assumed that the resource costs of reproduction are greater for females (Barrett and 38 Hough 2013). Although only female plants bear the costs of producing large seeds, fruits and 39 cones after flowering, it is nevertheless controversial whether total female costs are higher 40 than male costs. For example, Harris and Pannell (2010) argued that serotiny (storage of 41 seeds in closed cones that open after plant death in fire) is a form of maternal care that 42 imposes extra selection pressure on females of the Cape Proteaceae genus Leucadendron for 43 greater water efficiency. They also argue that vegetative dimorphism, such as the larger 44 leaves and lower rates of branching in females, results from this extra ecophysiological 45 selection on females. Cones on females need to maintain a water supply to prevent them from 46 drying out during the dry, hot Mediterranean summers and opening prematurely and thus 47 releasing seeds into the unfavourable inter-fire environment. The proposed mechanism for 48 greater water efficiency in females is the hydraulic benefit of their less frequent branching 49 than occurs in males. Barrett et al. (2010) noted that males in serotinous L. gandogeri 50 matured first and argued that the delayed maturation of females reflects their higher costs of 51 reproduction.

52 In contrast to these suggestions of higher female costs, Midgley (2010) showed that δ 13 C (an 53 integrated measure of an individual’s water use efficiency) did not differ between males and 54 females of eight Leucadendron species, including a co-occurring vegetatively monomorphic 55 and very dimorphic species, as well as serotinous and non-serotinous species. Midgley (2010) 56 also argued that vegetative dimorphism in leaf size and branching is not explained by 57 ecophysiological differences between the sexes, but differences in the process of pollen 58 transfer and capture. Thus, whether the costs of sex are higher for females and whether this 59 could lead to vegetative dimorphism is unresolved for Leucadendron. Here we use new 60 interpretations of published data to further support the hypothesis that the costs of sex are 61 equal amongst the sexes. Our hypothesis for sexual equality should apply generally to all 62 plants and could be tested using some of the many hundreds of Cape dioecious species (such bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4

63 as > 300 Restionaceae species) and using dioecious species from other similarly fire-prone 64 environments such as in Western Australia.

65 The hypothesis of higher allocation to reproduction in female plants, besides being of 66 fundamental evolutionary interest, may also have a conservation implication for dioecious 67 taxa such as Leucadendron. It has been argued that global change will lead to a decline in 68 dioecious species (Hultine et al. 2016). A more stressed future environment (e.g. greater 69 aridity) is argued to favour male survival if males allocate relatively more to vegetative 70 growth and less to reproduction than females. Female biased mortality will then have 71 negative population consequences such as male biased sex ratios.

72 The fire-prone vegetation of the Cape (fynbos), South Africa, is an excellent place to 73 test the hypothesis of greater female allocation to reproduction; allocation to sexual 74 reproduction rather than vegetative reproduction is critical for post-fire seedling recruitment, 75 dioecy is common and the fitness of co-occurring dioecious and hermaphroditic taxa can be 76 compared. Also, sexual dimorphism in the genus Leucadendron (Proteaceae) can be extreme 77 at a global scale.

78 The fynbos environment

79 Leucadendron contains about 80 species and occurs in fire-prone Cape (South Africa) 80 vegetation known as fynbos (sensu Rebelo et al. 2006). The Proteaceae dominate the fynbos 81 canopy layer which is about 2 m tall, mainly through the genera Protea and Leucadendron. 82 The family (about 360 fynbos species) includes both dioecious (25%) and hermaphroditic 83 taxa and which frequently co-exist Figure 1). Most Proteaceae species are reseeders (i.e. non- 84 resprouters) (Le Maitre and Midgley 1992). Since reseeders cannot resprout, all plants die in 85 fires. As they are relatively short-lived (1-3m tall, 5-30 years), plants also die in senescent 86 vegetation which occurs in the prolonged absence of fire (Bond 1980). Successful 87 recruitment of reseeders only takes place after a fire (Bond et al. 1987; Le Maitre and 88 Midgley 1992). About 60% of fynbos Proteaceae species are serotinous (Le Maitre and 89 Midgley 1990). When serotinous cones dry-out and open after death of adults in fire, seeds 90 are released en masse into the optimum post-fire environment where seedling recruitment 91 takes place in situ Figure 2). The most common soil seed-bank (35% of fynbos Proteaceae 92 species) is due to ant burial of seeds (Slingsby and Bond 1985; Le Maitre and Midgley 1992) 93 and most of remaining 5% of species have seeds that are scatter-hoarded by small mammals bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 5

94 (Midgley and Anderson 2005). Species with soil seed-banks also germinate after fire as do 95 serotinous species; except in this case typically fire is needed to crack their relatively thick 96 seed coats. All three of these types of seed-stores occur in Leucadendron.

97 Given that only fires create suitable opportunities for regeneration, there is no strong 98 selection in fynbos for long distance seed dispersal such as by birds (Le Maitre and Midgley, 99 1992). Plant seeds merely wait in situ in soil or canopy seed-banks for recruitment 100 opportunities post-fire. Dispersing long distances could result in hazardous movement away 101 from recently burned sites to unsuitable unburned sites.

102 Short distance seed dispersal thus predominates in the fynbos Proteaceae and is due to 103 both the dispersal agents and short plant size. Scatterhoarding by small mammals (Midgley 104 and Anderson 2005) and ant-dispersal (Slingsby and Bond 1985) results in dispersal at the 105 scale of metres. Dispersal of winged serotinous Leucadendron seeds has not been studied but 106 is also expected to be very local, given that they are released from very short plants (0.5-3 m 107 tall) and that release height dominates wind dispersal distances (Augsberger et al. 2016). 108 Wind dispersed Leucadendron species have small wings (Williams, 1972) and relatively high 109 fall-rates (Midgley et al. in prep.). Genetic studies indicate median dispersal distance in 110 relatively large winged Banksia (Proteaceae) seeds is only 5 m (Krauss et al. 2009). Some 111 species from both hermaphroditic and dioecious taxa produce seeds that are hairy and can be 112 wind-rolled along the soil but are rapidly captured by obstacles and depressions (Bond 1988, 113 95 % of seedlings < 15 m from parent plant in Manders 1986). The features of the Proteaceae 114 in the fynbos environment that are relevant to considering sexual costs of reproduction are; (i) 115 co-occurring dioecious and hermaphrodite species, (ii) single-aged populations of the 116 reseeder life history and (iii) short gene (see pollination aspect later) dispersal distances, 117 which render post-fire recruitment patterns as a measure of local dead adult fitness. Below we 118 argue why these characteristics should result in equal reproductive allocation between the 119 sexes of dioecious plants.

120 Reproductive synchrony; Leucadendron populations are single-aged and this forces 121 males to track females

122 Fynbos reseeder populations are single-aged, dating back to winter germination after the most 123 recent fire. Individual female reproductive schedules will evolve in concert with the 124 historical fire regimes such that maximum output coincides with the most likely fire 125 frequency. The historical variance around the mean fire frequencies would constrain age to bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 6

126 maturity and senescence, because reseeders need to be reproductive if fire occurs in young 127 vegetation and serotinous females need to be alive in old vegetation. Since fynbos Proteaceae 128 grow and mature within a few years (Midgley and Rebelo 2010) and because serotiny is 129 generally weak (cones are typically only retained for a year or two, seed viability declines 130 with time), the cumulative seed crop is dominated by the contribution of the most recent few 131 years (Midgley, 2000). Therefore, given no immigration by long distance dispersal, the level 132 of post-fire seedling recruitment is largely dominated by the reproductive condition of the 133 females at the time of the fire, Figure 2). For example, fire in very young or senescent 134 vegetation results in very low dead adult:seedling ratios (e.g. Bond 1980; Kraaij et al. 2013).

135 The scheduling of male reproductive allocation in single-aged reseeders must 136 synchronously match that of females, whatever the factors are that constrain the scheduling 137 female reproductive allocation. Males that allocate more to sexual function and less to 138 vegetative function at an earlier age than females would experience limited mating 139 opportunities due to being asynchronous with ovule numbers for their whole lifetime. 140 Similarly, a male that allocated more to vegetative growth than females to achieve larger size 141 at maturity would also be asynchronous with females. Also, large relatively immature males 142 forego future breeding opportunities when a fire takes place because fires kill all individuals. 143 Synchronous allocation to reproduction also implies equal allocation to vegetative growth as 144 there are no advantages of delayed maturity to achieve relatively larger size or longevity, 145 given that fire kills all plants.

146 Reproductive doubling; post-fire recruitment levels of dioecious females is double that 147 of hermaphrodites

148 Because adults die in fires and seed dispersal distances are short, local extinction will result 149 after fire in a stand of immature or senescent plants. Post-fire recruitment levels (i.e. dead 150 adults:seedling ratios) of the Proteaceae have therefore been widely measured in fynbos, to 151 determine responses of the reseeders to fires in stands of different age and in different 152 seasons (Bond 1984; Bond et al. 1984; Midgley 1987; Kraaij et al. 2013). These surveys are 153 typically determined 1-4 years after fires. However, since most fynbos Proteaceae species 154 grow and mature rapidly (generally within 1-5 years, Midgley and Rebelo 2008, Kraaij et al. 155 2013) these recruits are not seedlings (i.e. dependent on seed reserves) but can be advanced 156 juveniles that have already survived summer drought and competitive interactions. Given the 157 predominance of short distance dispersal, we argue that post-fire dead adult:seedling ratios of bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 7

158 reseeders are therefore a direct measure of mean lifetime fitness and that this then allows 159 fitness comparisons between co-occurring dioecious and hermaphroditic taxa. Since 160 dioecious and hermaphrodites co-occur, mean fitness must be similar.

161 Species from fynbos dioecious genera frequently co-occur in the same landscape, 162 even the same plots with those from similar sized species from hermaphroditic genera; 163 Leucadendron and Aulax co-occur with hermaphroditic genera Protea, Leucospermum and 164 Mimetes (Supplementary Figure 1 and see also community descriptions in Rebelo et al. 165 2006). Because dioecy is rare amongst plant species (< 5%), it is often considered to be 166 disadvantageous, although this is questioned by Sabath et al. (2016). In fynbos, the 167 widespread co-occurrence of dioecious and hermaphroditic taxa indicates there is no nett 168 benefit, or disadvantage, of either breeding system. Thus, proteas and leucadendrons are 169 ecological equivalents given their fine-scale co-occurrence as well as other forms of 170 equivalence such as in plant size, reseeding life-history, serotiny and seedling growth rates 171 (Laurie et al. 1997). This ecological equivalence and stable co-occurrence means that they 172 must both have the same mean post-fire fitness (i.e. dead adult:seedling ratio). For example, 173 if the female dioecious female adult:seedling ratio is double the hermaphrodite adult:seedling 174 ratio, this implies a 1:1 sex ratio in the dioecious taxa.

175 Post-fire recruitment data indicates dioecious female individuals do produce double 176 the number of seedlings as do co-occurring hermaphrodites (Midgley 1987), such that the 177 standard measurement of dioecious recruitment levels is female adults*2/seedlings (e.g. 178 Kraaij et al. 2013). For a recent example, our analysis of the post-fire recruitment data in 179 Kraaij et al. (Fig. 4, 2013) indicates there is no difference between in seedling:adult ratios in 180 Protea versus seedling:2*female ratios in Leucadendron (M-W p >0.2). Dioecious females 181 achieve higher seedling recruitment in part by having higher seed set and thus seed 182 production than hermaphrodites. For example, seed set (from Mustart et al. 1994) in co- 183 occurring Protea obtusifolia (19.5%) is lower than in female L. meridianum (54.7%) as is P. 184 susannae (12.7%) compared to co-occurring L. coniferum (87.1%).

185 The doubling of female seedling production in Leucadendron is not because low seed 186 production in hermaphrodites is limited by out-crossed pollen. Most of the hermaphroditic 187 Proteaceae are also out-crossers (Collins and Rebelo 1987), mainly through strong protandry 188 and self-incompatibility. Despite the out-bred genetic structure of most hermaphroditic 189 Proteaceae (e.g. Goldingay and Carthew 1998), the widely observed low seed-set (<25%) is bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 8

190 not strongly improved by artificially adding cross pollen (e.g. Whelan and Denham 2009; 191 Steenhuisen and Johnson 2012). This generally low seed in the Proteaceae is not maladaptive, 192 it reflects functional andromonoecy (Rebelo and Collins 1987); most flowers/florets have 193 evolved to achieve fitness by male function and the distribution of pollen. Support for the 194 functional andromonoecy hypothesis is the higher seed set (> 50%) in female florets in 195 dioecious taxa such as in Aulax and Leucadendron (Rebelo and Collins 1987; Mustart et 196 al.1997; Hemborg and Bond 2005), but that this is normalised when lack of seed-set in the 197 many male florets on male plants is considered. Also, seed-set is very high in the 198 hermaphrodite flowers in morphologically andromonoecious reseeding proteoids, but it too is 199 very low when lack of seed-set in the numerous male flowers per plant is considered (Ladd 200 and Connell, 1994). In the few somewhat self-compatible Proteaceae species that do occur, 201 seed-set per floret is still very low (< 1-3 % in several stands of Banksia baxteri) (Wooller 202 and Wooller 2001). In this species, seed-set and seed quality is much higher in open controls 203 than manually-selfed flowers (Wooller and Wooller 2004), indicating that most seed-set in 204 open flowers is due to out-crossed pollen. Furthermore, Hemborg and Bond (2005) noted that 205 95% of female L. xanthoconus flowers were pollinated, but only about 50% developed into 206 seeds. In summary, nett seed-set in both dioecious and hermaphroditic taxa is similar and in 207 neither, is specifically out-cross pollen limited.

208 Double Leucadendron female fitness (dead adult:seedlings) compared to co-occurring 209 hermaphrodites indicates that male and female costs of reproduction in hermaphrodites are 210 equal; a dioecious female liberated from male function can produce twice as many recruits as 211 a hermaphrodite. Double female fitness also indicates that the adult sex ratio in dioecious 212 plants is 1:1; a dioecious male and female have the same fitness as two hermaphrodites. The 213 above reproductive argument applies to male components of fitness, but it is more difficult to 214 measure than is the seedling ratios of females. For example, burned males do not have cones 215 which thus prevents them being identified and counted.

216 Our argument for equal costs of male and female function appears to run counter to 217 arguments of differential resource utilisation by the sexes. For example, it has been argued 218 that males and females use different resources for reproduction, such as nitrogen for pollen 219 and carbon for seeds (Harris and Pannell, 2008). However, the doubling of seedling 220 production of dioecious females relative to that of hermaphrodites indicates that the release 221 from the cost of male function must be equal in magnitude to that of the female function. bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 9

222 Thus, even if males and females use resources at different stoichiometries, the nett cost of 223 these functions to the plant must be equal.

224 Competitive symmetry is needed to maintain 1:1 sex ratios

225 Short distance seed dispersal from large seed-banks will cause intense post-fire competitive 226 interactions. Numbers of seeds stored in individual serotinous adult Leucadendron canopies 227 can be large (e.g. > 24 000 per female in L. coniferum and > 2500 in L. meridianum in 228 Mustart et al. 1994). Since fynbos Proteaceae often occur in mesic areas, dense stands of 229 seedlings typically arise after fire in mature stands. The number of 1-4-year-old seedlings 230 within the space occupied by an adult is up to 50 in Bond et al. (1987) and 40 in Kraaij et al. 231 (2013), with densities up to > 200 m-2 in L. uliginosum (Midgley 1987). In many animal 232 species, competition between sexes can be avoided, but not in dense stands of plants. Thus, 233 many potential competitive intra-specific near neighbour interactions will occur in 234 Leucadendron. In dioecious fynbos Proteaceae, seed dispersal attributes do not differ 235 between the sexes because the dispersal attributes (elaiosome, wings or hairs) are maternal 236 (i.e. they are fruits, Midgley 1987), not off-spring tissue. Given equal dispersal of an equal 237 ratio of male and female seeds, 50% of seedling interactions would randomly be mixed-sex 238 encounters and 50% same-sex encounters. If males allocated less to reproduction than 239 females, they would be vegetatively superior and out-compete females, thus causing local 240 non 1:1 sex ratios and local mate competition.

241 Many entomophilous Leucadendron species are pollinated by very small (< 2mm in 242 length, Williams, 1972) largely sedentary nitidulid insects (Hemborg and Bond 2005; 243 Welsford et al. 2014). Thus, biased sex ratios may complicate pollination by increasing 244 between sex distances that very small insects must travel. A male that allocates less to sexual 245 reproduction and more to vegetative growth than conspecifics could outcompete near 246 neighbour males and females. However, for this large male mutant that is capable of out- 247 competing near neighbour females to be successful, it still faces the hurdle of dispersing 248 pollen to other more distant females which will likely have smaller, but closer, near- 249 neighbour males. This is unlikely because to be vegetatively competitive this mutant male 250 must be allocating less to reproduction. Also, the spread of such an aggressive male 251 genotype would in any event eventually be terminated by the strong decline in females 252 (leading to 1:3 sex ratio) and the necessary evolution of compensatory female biased sex 253 ratios of seeds (> 2:1), in contradiction of the evolutionary stable 1:1 sex ratio strategy for bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 10

254 most dioecious organisms and the ratio expected and found in Leucadendron. Barrett et al. 255 (2010) documented 1:1 sex ratios in mature mesic Leucadendron stands and analysis of the 256 data of Mustart et al. (1994) indicates no difference in sex ratio from 1:1. Given the argument 257 above that competitive interactions between the sexes are highly likely in fynbos, this sex 258 ratio equality implies competitive symmetry across the sexes. This implies resource use is 259 equal between the sexes.

260 Serotiny, higher female costs and vegetative dimorphism

261 Besides the above arguments for equality between the sexes, there are theoretical and 262 mechanistic reasons why the argument of Harris and Pannell (2010) based on extra maternal 263 costs of serotiny cannot explain leaf dimorphism. Firstly, maternal care is not an extra cost 264 compared to male sexual allocation, but a cost that must be integrated into female 265 reproductive allocation. Storing seeds in canopies is merely an alternative adaptation to soil- 266 stored seeds as a mechanism for recruiting after fynbos fires. For example, many 267 scatterhoarded Leucadendron species have large seeds with a thicker seed coat, than is the 268 case with serotinous species (Williams 1972; Midgley et al. 2015). The thick seed coat of 269 these species is an alternative allocation to a cone, and cone maintenance, not an extra cost.

270 Cones remain closed for many years in the serotinous dioecious Proteaceae genus 271 Aulax, but according to the leaf size data in Rourke (1987), there is no sexual leaf 272 dimorphism. Intense serotiny is therefore not a general correlate of dimorphism. Leaf 273 dimorphism in Leucadendron matches floral dimorphism via Corner’s Rules (Bond and 274 Midgley 1988); few, large inflorescences on fewer larger branches in females versus many, 275 smaller inflorescences and branches in males. Explaining dimorphism based on water 276 relations cannot explain why then males have gone the apparently expensive “many-small” 277 route rather than also evolving this more effective habit (Midgley 2010). Midgley (2010) 278 argued that the “many-small versus few-big” is due to differences in dispersing and receiving 279 pollen.

280 Regarding ecophysiological mechanisms for the evolution of dimorphism, the 281 hydraulic advantages of larger leaf size and lower branching are controversial. Despite the 282 allometric correlations summarised in Corner’s Rules being important in plants (e.g. Westoby 283 et al., 2002), there has been no argument that lower rates of branching have a positive 284 hydraulic effect. Lower branching associated with larger leaf size per se is not predicted to 285 make a substantial positive impact on carbon and water economy, because larger leaf size has bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 11

286 other balancing positive and negative correlates. Larger leaf (and associated branch) size is 287 correlated with lower specific leaf area (e.g. Midgley 2010 for Leucadendron), more self- 288 shading and a longer path-length, but a larger xylem diameter and smaller total leaf area 289 (West et al. 1999; Westoby et al. 2002; Smith et al. 2017). Within Leucadendron mean leaf 290 size is smaller in the more arid areas (Thuiller et al. 2004), in contradiction of the expectation 291 of Harris and Pannell (2010). Although cones transpire and respire at a total rate equivalent to 292 only a few leaves (Cramer and Midgley 2000), transpiring cones must nevertheless compete 293 with transpiring leaves on the same plant for water (Midgley and Enright 2000). Even if 294 reduced branching rates was positively correlated with hydraulic efficiency, it would equally 295 influence all leaves and cones on the same plant and thus not improve relative access to water 296 of cones of different ages. If lower branching patterns positively influenced plant water 297 status, then this should also apply to non-serotinous species as well. Finally, Bond and 298 Midgley (1988) noted leaves on Leucadendron seedlings resemble those of females, 299 suggesting that leaf dimorphism is primarily due to selection on males, not females. In 300 conclusion, there is no expectation to the contention that lower branching/larger leaves is 301 positively associated with water use efficiency and measurements showed no sexual 302 differences in water use efficiency (Midgley 2010).

303 Male costs of reproduction in Leucadendron

304 The large investment in cones and seeds by females tends to dominate the view of higher 305 female’s costs of reproduction. However, if male and female reproductive costs are the same, 306 where do the high male’s costs come from? There are features of male reproduction in 307 Leucadendron, besides the cost of pollen, that incur higher costs than in females. During 308 flowering in Leucadendron the bracts and leaves surrounding male inflorescences of insect 309 pollinated species are far more yellow and less green than females; in many species, the 310 whole male plant may become yellow/red, presumably to attract pollinators. This sexual 311 dimorphism in attractiveness is due to a total break-down of thylakoids in leaves and bracts, 312 which then exposes hidden yellow carotenoids (Schmeisser et al. 2010). This break-down of 313 chlorophyll and the accompanying loss of photosynthesis, must be far more expensive to 314 males than females because it occurs in many more leaves and floral bracts in male plants 315 Figure 3). Males produce more scent and with a greater diversity of volatiles than females 316 (Welsford et al. 2015). Also, males in wind-pollinated Leucadendron species produce many 317 more florets than do their females (Midgley 2010). Thus, male allocation to reproduction is as 318 high as in females; through pollen, greater attractiveness and high male:female floret ratios. bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 12

319 Sexual niche segregation is unlikely

320 Females could sustain higher reproductive allocation if they out-competed males in more 321 resource rich sites. This is often seen in riparian ecosystems where steep and stable 322 environmental gradients occur over short spatial scales, allowing sexual niche-segregation 323 and specialisation (e.g. Hultine et al. 2008). However, this is unlikely to be the case in the 324 fynbos for several reasons. We have already argued that in fynbos dioecious males will 325 always track female allocation, so this competitive elimination of one sex by the other in 326 some habitats is unlikely. Furthermore, we suggest that frequent fire, short lifespans and 327 limited gene dispersal in space and time by either seeds or pollen reduces the opportunities 328 for the sexes to specialise in different microsites. Firstly, such microsites would have to be at 329 a very fine geographic scale (a few metres) in the fynbos to match the limited gene dispersal 330 patterns. Secondly, frequent fire resets sexual spatial patterns in this landscape because all 331 adults die in fire, but seed dispersal of both sexes only occurs from female plants. For a male 332 mutant genotype that is advantageous in an arid microsite to succeed, this genotype must 333 repeatedly disperse in seeds away from maternal plants in the mesic sites, out-compete 334 females in the arid sites and then disperse in pollen back from the arid sites to females in the 335 mesic sites, whilst competing with the mesic adapted males for mating opportunities. 336 Furthermore, these sites must be burned in the same fires or else populations of each sex will 337 be at different stages of reproductive maturity. This back-and-forward scenario from females 338 after every fire is very unlikely. Finally, the data available suggest no habitat specialisation; 339 males and females co-occur equally and at a very fine scale (such as in 5 x 5 m plots in 340 Barrett et al. 2010 and 10 x 10 m plots in Mustart et al. 1994).

341 Even if there were ecophysiological consequences of vegetative dimorphism, say of 342 differences in leaf size or hydraulic architecture, this would not change how present 343 dimorphic females experience the past benefit of avoiding allocation to male function. This 344 would have been set when dioecy first evolved and per the above arguments appears to have 345 been a 50% reproductive benefit. Therefore, dimorphism in Leucadendron cannot be due to 346 differences in sexual allocation because reproductive asynchrony, lack of reproductive 347 doubling or competitive asymmetry would then occur. We have argued above that sexual 348 niche dimorphism is unlikely and therefore sexual vegetative dimorphism cannot be a result 349 of sexual niche dimorphism. Vegetative dimorphism is therefore not due to ecophysiological 350 differences, habitat differences nor to allocation differences but could be due to differences in 351 sexual efficiency related to pollination (Midgley 2010). bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 13

352 Early maturation of males does not necessarily reflect higher costs of reproduction

353 We interpret the earlier maturation of Leucadendron males (Barret et al., 2010) not to 354 represent sexual differences in total reproductive allocation but to differences in costs of 355 producing individual inflorescences. Given that Cape Proteaceae are single-aged there would 356 be strong selection preventing males from flowering if no females were flowering. Male 357 inflorescences are far smaller than females (e.g. 0.6 % female mass in L. rubrum and 2.5% in 358 L. ericifolium and L. teretifolium from Bond and Midgley 1988). Given that only one 359 inflorescence needs to be produced to determine the sex of a plant and that size thresholds for 360 maturity are widespread in plants, it is to be expected that the sex producing the cheaper 361 single inflorescence will mature first. Although about double the number of males than 362 females have inflorescences in 6-year-old stands of L. gandogeri (Barret et al. 2010), because 363 females nevertheless have many more florets per single inflorescence than males, precocious 364 male flowering is not maladaptive nor evidence of lower male total allocation to 365 reproduction.

366

367 Conclusion

368 Three separate arguments support the hypothesis that the costs of reproduction are equal in 369 Leucadendron. Single-agedness forces males to allocate to reproduction in synchrony with 370 females. Dioecious females achieve double the female fitness of hermaphrodites by being 371 relieved of male function. Competitive interactions are likely in fynbos and since the data 372 suggests 1:1 sexual ratios this indicates competitive symmetry. We conclude also that the 373 costs of reproduction must be equal whether the taxa are dimorphic or not. Our argument 374 applies in the Cape, where allocation to sexual reproduction is critical and where dimorphism 375 can be extreme, but should be tested globally.

376 We predict that global change will impact dioecious Proteaceae to the same extent as 377 hermaphrodites, because the latter being bi-parental and largely out-crossed, also allocate 378 equally to the sexes. Plants from the different sexes in dimorphic species in Leucadendron 379 have very different shapes and sizes. We nevertheless predict that whole plant resource use, 380 sex ratios and near neighbour associations will be equivalent between the sexes within both 381 monomorphic and dimorphic species and will not be differentially impacted by global 382 change. bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 14

383 Authors Contribution

384 JJM, AGM and MDC were all involved in all stages of this project.

385 Funding

386 We thank the University Research Council for funding.

387 Conflict of Interest Statement

388 We have no conflict of interest.

389 Acknowledgements

390 We thank Bruce Anderson, William Bond, Richard Cowling, Todd Dawson, Allan Ellis, 391 Steve Johnson, Phil Ladd, Peter Linder, Laurence Midgley, John Pannell, Tony Verboom and 392 Joe White for critical opinions.

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502 Figure 1.

503 504 505 Figure 1. Distribution of hermaphrodite (Brabejum, Diastella, Faurea, Leucospermum, 506 Mimetes, Orothamnus, Paranomus, Protea, Serruria, Sorocephalus, Spatalla and 507 Vexatorella) and dioecious genera (Leucadendron and Aulax) obtained from the location 508 records for Proteaceae in Protea Atlas dataset (www.proteaatlas.org.za). The coastline and 509 provincial borders are shown for reference. 510

511

512 bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20

513 Figure 2.

514

515 Figure 2. a.Stylised life-history of a serotinous reseeding proteoid. Phases 1-3 depict rapid 516 increase of seeds over post-fire time. Cones remain closed for about two years and most 517 cones open simultaneously after fire (Phase 4) and release seeds. Single-aged cohorts of 518 seedlings establish in situ post-fire (Phase 5). b. Rapid increase in number of ovules over 519 post-fire phases for males to compete for, and numbers of seeds available for post-fire 520 recruitment.

521 bioRxiv preprint doi: https://doi.org/10.1101/212555; this version posted November 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 21

522 Figure 3.

523

524

525

526 Figure 3. Representative male and female flowering shoots of Leucadendron sessile in colour 527 (left) and Normalised Difference Vegetation Index (right). NDVI image created using a 528 Parrot Sequoia multispectral camera. Note low NDVI in more male terminal leaves and 529 bracts than females.

530

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