1 2 DR. ALICIA TOON (Orcid ID : 0000-0002-1517-2601) 3 4 5 Article type : Invited Review 6 7 8 Insect pollination of cycads 9 10 Alicia Toon1, L. Irene Terry2, William Tang3, Gimme H. Walter1, and Lyn G. Cook1 11 12 1The University of Queensland, School of Biological Sciences, Brisbane, Qld, 4072, 13 Australia

2 14 University of Utah, School of Biological Sciences, Salt Lake City, UT 84112, USA 15 3 USDA APHIS PPQ South Florida, P.O.Box 660520, Miami, FL 33266, USA 16 17 Corresponding author: Alicia Toon 18 [email protected] Ph: +61 (0) 411954179 19 Goddard Building, The University of Queensland, School of Biological Sciences, Brisbane, 20 Qld, 4072, Australia. 21 22 23 24 25 26 27 28 29

30 Manuscript Author 31

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/AEC.12925

This article is protected by copyright. All rights reserved 32 33 Acknowledgements 34 We would like to thank Dean Brookes for discussions about genetic structure in cycad 35 pollinating thrips populations. Also, thanks to Mike Crisp for discussions about plant 36 diversification and Paul Forster for information on Australian cycads. This work was funded 37 by ARC Discovery Grant DP160102806. 38 39 Abstract 40 Most cycads have intimate associations with their insect pollinators that parallel those of 41 well-known flowering plants, such as sexually-deceptive orchids and the male wasps and 42 bees they deceive. Despite this, the mistaken belief that cycads are mostly wind-pollinated is 43 still commonly expressed. Perhaps as a consequence, cycad-pollinator systems are rarely 44 exemplified in studies of the role of pollinators in plant evolution and diversification. 45 Although first recognized more than a century ago, specialised associations between cycads 46 and their insect pollinators have been elucidated experimentally only in the past few decades. 47 This review covers the history of understanding pollination in cycads, the advances that have 48 been made since the 1980s using field observations and experiments, and analyses of 49 molecular data from the population to phylum level. We outline areas for future research to 50 address how such interactions might have affected speciation and extinctions. We stress that 51 inclusion of cycads in broader considerations of the role of pollinators in plant diversification 52 is important because they are phylogenetically distant from flowering plants and their 53 pollination systems might have evolved independently of one another. This review is timely 54 because cycads are a globally threatened group that might be vulnerable to co-extinction with 55 pollinator loss. 56 57 58 59 60 61 Author Manuscript Author 62 63 Key words: diversification, cycads, insect pollination, push-pull 64 Terms:

This article is protected by copyright. All rights reserved 65 Cospeciation: parallel speciation among interacting taxa. 66 Phylogenetic tracking: pattern of cospeciation where speciation in one taxon is a consequence 67 of the dependence on another taxon. 68 Coevolution: the process of parallel speciation among multiple taxa, as a consequence of 69 reciprocal selection on each other. 70 Brood-site pollination: the offspring of pollinators complete part of their life cycle within 71 reproductive tissues of their host that developed as a consequence of host fertilisation 72 facilitated by that pollinator. 73 74 Introduction 75 Until recently, it was generally taught that all gymnosperms are wind pollinated. This 76 is not the case, with most species of cycads (Terry et al. 2012) and gnetales (Ickert-Bond and 77 Renner 2016) being pollinated by insects. Thus, two of the four extant phyla of gymnosperms 78 are insect, rather than wind, pollinated. 79 Despite almost all cycads being pollinated by insects, they are rarely mentioned in 80 reviews of plant-pollinator diversification and cospeciation (but see Dufaÿ and Anstett 2003). 81 This lack of recognition might be because cycads are not as speciose as their angiosperm 82 cousins, their reproductive organs are not as showy as those of many flowering plants, and 83 their dependence on insects has only recently been accepted among the broader scientific 84 community (Terry et al. 2012). Given that cycads form a divergent lineage within the 85 gymnosperms, they represent a distant and independent case of association with insect 86 pollinators, and could add substantial breadth to our understanding of the effects on plant 87 populations of pollination interactions. 88 In this review, we outline the history of discovery of insects as pollinators of cycads 89 and speculate as to why it took so long before early reports were verified. We then explain 90 the pollination system in cycads and its variations, and what is not yet understood. We cover 91 the potential ecological consequences of cycads being reliant on insects as pollinators, and in 92 particular how insects might affect gene flow, population structure and long-term resilience 93 of cycads, and propose future directions for cycad-pollinator research. 94 Author Manuscript Author 95 Pollination of seed plants

96 Interactions with animals are central to understanding the diversification of seed plants, 97 i.e., angiosperms and gymnosperms. For many seed plants, animals are involved in

This article is protected by copyright. All rights reserved 98 pollination and the dispersal of seeds, as well as being herbivores and the vectors of disease. 99 Some of these types of interactions likely date to the very origin of seed plants, around 300 100 million years ago (Linkies et al. 2010) when early insects were diversifying (Grimaldi and 101 Engel 2005), including the beetles (Zhang et al. 2018), hemipterans (Johnson et al. 2018) and 102 flies (Wiegmann et al. 2011). While many seed plants are wind-pollinated, such as the 103 species-rich grasses and pines, the vast majority of seed plants are pollinated by animals, 104 mostly insects (Ollerton et al. 2011).

105 The astounding diversity of flowering plants and insects is often attributed to their biotic 106 interactions (Ehrlich and Raven 1964; Grant and Grant 1965; Suchan and Alvarez 2015). 107 Interactions with animals have greatly affected the ecology and diversification of plants, 108 especially by seed dispersal and pollen transport that might lead to plant isolation and or 109 affect gene flow (Ballesteros-Mejia et al. 2016; Ghazoul 2005; Krauss et al. 2017). For 110 example, fleshy fruits that are dispersed by animals have evolved repeatedly in the 111 Myrtaceae, and the switch to fleshy fruit is generally accompanied by dramatic increases in 112 the diversification rate of the plant lineage compared with lineages without fleshy fruit 113 (Biffin et al. 2010). In contrast, evolutionary shifts among animal pollinators (e.g., from 114 using insects to using birds, or between insects with different feeding styles (guilds)) are 115 correlated with increased diversification rates or, in some cases, with decreases (Kay and 116 Sargent 2009; Serrano-Serrano et al. 2017; Smith 2010; Toon et al. 2014). While other 117 factors probably play a more important role in the huge species richness of angiosperms 118 compared with other land plants (e.g., Brodribb and Feild 2010; Amborella Genome Project 119 2013), the situation in gymnosperms still needs to be clarified (e.g., Bolinder et al. 2016).

120 Only a minority of animal-pollinated plant species are pollinated by a single species of 121 animal: instead, pollination typically involves guilds of relatively generalist pollinators (e.g., 122 Myrtaceae and flies, beetles and butterflies) (Waser et al. 1996) or there has been diffuse 123 coevolution with a particular guild of pollinator (Lunau 2004), e.g, buzz-pollination bees and 124 Solanum (Buchmann and Cane 1989). Of the more specialised pollination systems, the 125 obligate mutualisms Yucca—yucca moth (Pellmyr 2003), fig—fig wasp (Herre et al. 2008) 126 and Glochidion—leafflower moth are the most well studied. In Yucca (Pellmyr et al. 1996) Author Manuscript Author 127 and Glochidion (Kato et al. 2003), pollination is achieved by the female moth actively 128 collecting pollen and placing it on the style of another flower, then laying her eggs within the 129 flower of the plant where the larvae will feed on a subset of the developing seeds. Even in

This article is protected by copyright. All rights reserved 130 such ecologically specialised mutualisms, co-pollinators are known and one-to-one 131 relationships are rare (Hembry and Althoff 2016; Herre et al. 2008).

132 There has been considerable research investigating co-diversification of host and 133 pollinator in obligate pollination systems. Phylogeographic concordance among host and 134 pollinators, e.g., European globeflower, Trollius europaeus, and their pollinating flies, 135 Chiastocheta spp. (Espíndola et al. 2014), and Ficus and their pollinating wasps (Rodriguez 136 et al. 2017; Tian et al. 2015), supports the idea that strong ecological associations might 137 affect co-genetic structure, at least within species. Although evidence for coevolution is 138 weak and phylogenetic congruence at the species level is rare (Hembry and Althoff 2016), 139 Yucca—yucca moth (Althoff et al. 2012), fig—fig wasp (Yang et al. 2015) and some clades 140 of Glochidion—leafflower moth (Hembry and Althoff 2016) show significant cophyletic

141 structure across phylogenies. This pattern of phylogenetic congruence at the clade level, 142 might in part be explained by conservation of the strong ecological association between host 143 and pollinator, combined with host-switching (Hembry and Althoff 2016). Although there 144 are obligate pollination systems within gymnosperms, little is currently known of the extent 145 to which pollinators and hosts affect each other’s ecology or evolution.

146

147 Evolution of pollination by insects

148 Currently we know little about the origins of biotic pollination, how it evolved or how 149 many times it has originated. Direct evidence for ancient insects as pollinators is scant, 150 although insects with pollinator-like phenotypes, including beetles (Coleoptera), scorpion 151 flies (Mecoptera), lacewings (Neuroptera) and true flies (Diptera), date to the early-mid 152 Mesozoic (Labandeira et al. 2007; Labandeira et al. 2016; Peñalver et al. 2015; Peris et al. 153 2017; Ren et al. 2009). Coprolites (fossilised excrement) within fossil cycad pollen cones 154 from the middle Triassic of Antarctica are thought to be from beetles, based on their 155 morphology (Klavins et al. 2005), and represent some of the earliest evidence of herbivory 156 on cycad pollen by insects. The earliest direct evidence of insects interacting with pollen is

157 from 100 million year Manuscript Author old thrips (melanthripids) bearing Cycadopites pollen in amber from 158 Spain (Peñalver et al. 2012), and a 100 million year old wasp feeding on eudicot pollen in 159 Burmese amber (Grimaldi et al. 2019). Another amber fossil, a boganiid beetle, 160 Cretoparacucujus cycadophilus, from mid-Cretaceous amber from Myanmar, has been found

This article is protected by copyright. All rights reserved 161 associated with cycad pollen, but it does not bear pollen on the body (Cai et al. 2018). Thus, 162 by the mid-Cretaceous, insect-plant interactions, and potentially pollination, had evolved 163 among gymnosperms and flowering plants.

164 One trait of gymnosperms, pollination droplets or micropylar droplets, has been proposed 165 as a potential mechanism involved in the origin of insect pollination (Labandeira et al. 2007; 166 Nepi et al. 2009). Pollination droplets, produced and secreted by the ovule of most 167 gymnosperms, are used for pollen capture (Labandeira et al. 2007; von Aderkas et al. 2018). 168 Most species secrete a pollination droplet from the ovule through a narrow canal (the 169 micropylar tube): the droplet captures pollen and is then withdrawn, transporting the pollen to 170 the nucellus where the pollen produces pollen tubes (Nepi et al. 2009). The presence of 171 sugars and proteins in pollination droplets might have attracted insect visitors and could 172 have been involved in the evolution of insect associations (Nepi et al. 2009). Pollination 173 droplets have been proposed to be a reward for pollinators of cycads and gnetophytes (two 174 groups of gymnosperms that are pollinated by insects) (Ickert-Bond and Renner 2016; Tang 175 1987b).. Consistent with a reward hypothesis is that gymnosperm species that are insect 176 pollinated have higher concentrations of sugars and/or amino acids (in their micropylar 177 droplets) required by insects than those of wind-pollinated gymnosperms (Nepi et al. 2017). 178 Pollen capture using pollination droplets appears to be an ancestral trait of gymnosperms 179 (von Aderkas et al. 2018), but it is not known whether pollination droplets were present in 180 the common ancestor of all seed plants or are a synapomorphy for gymnosperms.

181

182 Cycad distribution, evolution and pollination

183 Cycads are seed plants with a woody trunk, a crown of evergreen, usually pinnate, 184 leaves, and produce reproductive cones on separate male and female plants. They have a 185 global distribution, found throughout tropical, subtropical and warmer temperate regions of 186 Africa, Asia, Australia and America. Currently 10 genera are recognised, comprising 353 187 species (World List of Cycads accessed 10 October2018). While species diversity is highest

188 in Mexico, central and Manuscript Author south America, Australia has the highest phylogenetic diversity of 189 cycads, with four genera (three endemic) from across the breadth of the cycad phylogeny 190 (Fig. 1).

This article is protected by copyright. All rights reserved 191 The placement of cycads within gymnosperms is uncertain: recent molecular evidence 192 suggests they are most likely sister to the rest of the gymnosperms (Crisp and Cook 2011; Lu 193 et al. 2014; Mathews 2009), or sister to ginkgo (Li et al. 2017; Ruhfel et al. 2014; Wickett et 194 al. 2014; Xi et al. 2013). In either case, they have a long history, with fossils dating from the 195 Palaeozoic (Hermsen et al. 2006), with high abundance and diversity in the Mesozoic, and 196 they have a conserved general morphology with extant plants similar to those of the fossil 197 record (Taylor et al. 2009). Rather than representing “living fossils”, though, the extant cycad 198 species derive from a much more recent (Miocene) diversification (Crisp and Cook 2011; 199 Nagalingum et al. 2011)(Fig. 1) that followed several periods of mass extinction (Crepet and 200 Niklas 2009). Cycads are also now under threat, with more than half of all species listed "at 201 risk" (IUCN red list, accessed 17th June 2019), with major risks being loss of habitat, over- 202 harvesting of wild plants, climate change, and reproduction failure from loss of pollinating 203 insects (Mankga and Yessoufou 2017).

204 Most cycads are pollinated by insects, with wind pollination far less important for 205 most species, but this idea has been widely accepted since the 1980s. Pollination of cycads by 206 insects was first suggested by Sir Joseph Hooker, based on the observation of the large 207 quantity of pollen-grains within ovules of cycads, “so many more than the wind is likely to 208 have brought” (Oliver and Scott 1905). Weevils of the genus Porthetes (originally identified 209 as Phlaeophagus and later synonymised by Oberprieler (1996)) were first reported as 210 potential pollinators of the cycad, Encephalartos villosus, in the early 1900’s (Pearson 1906), 211 based on observations that these beetles are associated only with cycads and that the rostrum 212 of each specimen examined was heavily laden with cycad pollen. The seed parasite, 213 Antliarhinus zamiae, was also proposed as an important pollinator of Encephalartos cycads 214 (Rattray and Pearson 1913). The idea that a seed parasite was a pollinator of cycads, led to a 215 hypothesis of a floral parasite mutualism (Marloth 1914). Donaldson (1997) has since 216 rejected this hypothesis, because A. zamiae account for only a small proportion of pollination, 217 but he did find support for the earlier observation of a mutualism between Porthetes sp. and 218 species of Encephalartos.

219 Observations and experiments on Zamia furfuracea (Norstog 1987; Norstog et al. Author Manuscript Author 220 1986) and on Zamia pumila (Tang 1987b) in the 1980s contributed some of the first widely 221 acknowledged direct evidence that beetles are the principal pollinators of cycads. This lag 222 between the first hypotheses reported early in the 20th century and tests of entomophily in

This article is protected by copyright. All rights reserved 223 cycads was at least in part because of the prevailing idea that cycads were primarily 224 pollinated by wind (Chamberlain 1934), although Baird (1939) and Faegri (1979) reported 225 evidence of insect pollination. Research on pollination of cycads has shown that most cycads 226 are pollinated by insects (Hall et al. 2004; Norstog 1987; Norstog et al. 1986; Suinyuy et al. 227 2009; Tang et al. 2018b; Terry 2001; Wilson 2002) and that many of these interactions are 228 highly specific and specialised (Norstog and Fawcett 1989; Terryet al. 2005b; Wilson 2002) 229 (See Appendix S1). Wind pollination might play an important role for some species of Cycas 230 which have more exposed ovules, especially in the absence of insect pollinators (Hall and 231 Walter 2018; Hamada et al. 2015b; Kono and Tobe 2007). Wind might serve a role 232 sporadically among other cycads that are in close proximity.

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234

235 Insect pollinators of cycads

236 Pollinating insects of extant cycads are mostly beetles, including weevils (Curculionidae 237 and Belidae) and non-weevils (Erotylidae, Nitidulidae Boganiidae, Tenebrionidae) 238 (Oberprieler 2004; Terry et al. 2012) (Table 1). There are some exceptions, though, with 239 about a quarter of species of Macrozamia pollinated by thrips (Aeolothripidae, Cycadothrips) 240 (Forster et al. 1994; Mound and Terry 2001; Terry 2001) and the other species pollinated by 241 members of several beetle families. Moths of the genus Anatrachyntis (Cosmopterigidae) are 242 pollinators of Cycas micronesica (Marler 2010; Terry et al. 2009), with possible 243 contributions by more generalist insects as pollen vectors of this island cycad (Terry et al. 244 2009), and wind as vector in more open habitats (Hamada et al. 2015b). Many other cycad 245 species have not been surveyed for pollinators nor studied empirically to demonstrate 246 pollination by specific insects and, in some species that have been surveyed, no insects have 247 ever been found associated with cones (Appendix S1), which might indicate pollinator loss, 248 wind pollination or that the survey was conducted at the wrong time.

249 The diversity of independent pollinator lineages associated with cycads across Author Manuscript Author 250 continents (Fig. 1) has led to the idea of multiple origins of pollinators from among extant 251 insects rather than a long-term co-diversification of pollinators since the origin of cycads 252 (Oberprieler 1995a). There are at least seven unrelated beetle families, as well as thrips and 253 moths that are now known to pollinate cycads (Table 1). Host-shifts of insect pollinators

This article is protected by copyright. All rights reserved 254 among cycads might also be important processes contributing to the extant diversity of cycad 255 pollinators. For example, host-shifts have been proposed to account for discordance between 256 the phylogenetic relationships of Encephalartos and their pollinators (Downie et al. 2008; 257 Tang et al. 2018b) and taxonomy of Macrozamia and their thrips pollinators (Brookes et al. 258 2015). Although further molecular work to clarify phylogenetic relationships in both cycads 259 and their pollinators is needed before testing whether host-shifts might affect diversity or 260 evolution of cycads and their pollinators.

261

262 Dating the origins of any individual cycad-pollinator relationship is complicated 263 because extant species of cycads represent recent diversifications (mostly Miocene, 264 (Condamine et al. 2015)). Without independent evidence (i.e., relationships between fossils 265 and extant taxa), we are currently limited in how far back we can extrapolate. For example, 266 thrips pollinate different species of Macrozamia on either side of Australia, and it has 267 therefore been suggested that this thrips-cycad relationship might be ancient (Terry 2002). 268 The oldest it can be currently dated, based on molecular data, is that of the crown of 269 Macrozamia, which is between 1 and 17 Ma (95% highest posterior density) (Ingham et al. 270 2013). It is possible that some extant interactions are ancient. For instance, some fossil 271 beetles (e.g. Boganiidae ca. 99 million years old) found associated with cycad pollen (Cai et 272 al. 2018) are from a family also represented among extant species (Table 1). Also, 273 Pharaxonothinae (Erotylidae) beetle pollinators of the Asian genus Cycas and the New World 274 genera Ceratozamia, Dioon and Zamia might suggest an ancient origin of pollinator-cycad 275 relationships prior to the breakup of Laurasia (Skelley et al. 2017; Tang et al. 2018a; Tang et 276 al. 2018b; Xu et al. 2015). There is however, currently too little data to test whether any 277 fossils represent ancestors of extant pollinators or to perform any robust phylogenetic testing 278 of biogeographic relationships.

279 Biogeographic histories of cycad-insect relationships within regions are just starting 280 to be explored based on distributions and molecular data. The distributions of the 281 allocorynine weevils and pollinating erotylid beetles on New World cycads have enabled 282 Tang (2018b) to propose Manuscript Author hypotheses about the evolutionary relationships between cycads and 283 their pollinators in this region. Overall, the pattern observed from phylogenetic relationships 284 of the beetles and their host patterns (Tang et al. 2018b) suggest that there has been some 285 level of pollinator host switching with no evidence for strict co-speciation between the

This article is protected by copyright. All rights reserved 286 species of the four genera of New World cycads and their pollinators. For example, the 287 presence of allocorynine weevils in all Dioon species but only on some Zamia species, and 288 not on species at the periphery of their geographic distribution, has led to the hypothesis that 289 Dioon is their earliest cycad host with subsequent host shifts onto some Zamia species (Tang 290 et al. 2018b). However, the base of the allocorynine phylogeny remains unresolved 291 (polytomy among three clades on either Dioon or Zamia), and thus the direction from Dioon 292 to Zamia, or vice versa, cannot be tested yet based on molecular data.

293 In most cycad genera, at least some of the species associate with more than one 294 specialist pollinator (Appendix. S1). Such co-pollinators are recorded for species of 295 Stangeria and Encephalartos, not all of which are obligate, and they probably differ in 296 effectiveness (Procheş and Johnson 2009; Suinyuy et al. 2009). Most species of Macrozamia 297 are pollinated by a single obligate pollinator species, either weevils (Tranes) or thrips 298 (Cycadothrips), but at least four species are pollinated by both beetles and thrips (Terry 299 2001), or multiple species of beetle (Ornduff 1991). Only a single species of obligate 300 pollinator is known for each of the two species of Bowenia (Wilson 2002) and for 301 Lepidozamia peroffskyana (Hall et al. 2004). Most species of cycads in the Americas 302 (Zamia, Dioon, Ceratozamia, Microcycas) host more than one species of pollinator, some up 303 to three species (Tang et al. 2018b), and pollinators are obligatorily associated with their 304 local host plant (Terry et al. 2012). For most cycads, there are not enough data yet to say 305 how tightly linked the pollinators are to a host species, but when molecular data have been 306 used (e.g., Brookes et al. 2015; Downie and Williams 2009), the insects are more diverse 307 than previously thought, with a high level of specialization on local host cycads.

308

309 Pollination process and mechanisms in cycads

310 Cycads are strictly dioecious: male plants produce pollen cones and female plants 311 produce ovulate cones (also known as seed cones). Cones are spiral arrangements of leaf 312 homologs (microsporophylls in males and megasporophylls in females) around a cone axis

313 (Norstog 1987). Pollen Manuscript Author cones of all genera form many narrow crevices between 314 microsporophylls during dehiscence and these provide shelter for small insects. Ovulate 315 cones are more rigid than pollen cones, but narrow cracks, usually only 3-4 mm wide, form 316 between megasporophylls during receptivity that provide access into an interior with many

This article is protected by copyright. All rights reserved 317 narrow passageways. These cracks may be limited to particular regions of the cone (Calonje 318 et al. 2011), and cracks close soon after receptivity has ended. In all species, except those in 319 the genus Cycas, each megasporophyll has two ovules that are oriented inward and the 320 micropylar tips face the cone axis. Species within the genus Cycas do not produce true 321 ovulate cones, but have a whorl of megasporophylls at the stem apex without an axis to 322 connect them. Most Cycas megasporophylls have eight ovules (Jones 2002) but some species 323 have up to 12 ovules (Osborne et al. 2019), and their megasporophylls overlap tightly, but 324 loosen during receptivity. In some island Cycas species, such as C. micronesica, a large 325 proportion of ovules is exposed when receptive, allowing for wind vectored pollen to access 326 the micropylar tip directly (Hamada et al. 2015b).

327 Although there are similarities across cycad and angiosperm pollination organs, such as 328 between floral nectars and cycad micropylar drops, there are significant differences. For 329 example, most angiosperms have bisexual flowers (Renner 2014), and therefore their 330 reproductive structures (flowers) all possess the same rewards (small amounts of nectar and 331 pollen), present identical signals (flower shape, color pattern, scents) and exert the same 332 motivations for pollinators to depart (usually depletion of nectar or pollen reward). The 333 architecture of cycad cones, as with some dioecious and monoecious insect pollinated 334 angiosperms, present much greater asymmetry across the sexes in rewards, signals and 335 motivations to pollinators. For instance, pollen cones can offer pollen as a reward and 336 ovulate cones can offer micropylar droplets or ovule tissue as rewards, but not vice versa. 337 The pollen cones of most species are thermogenic in a daily pattern with concomitant peak 338 emission of volatile compounds at peak thermogenesis, while ovulate cones are generally 339 weakly thermogenic (or non-thermogenic in some species) with corresponding lower volatile 340 emission rates (Suinyuy et al. 2010; Tang 1987a; Terry et al. 2016).

341 The micropylar drops of cycads can be compared with floral nectaries of angiosperms and 342 play a potential role as rewards. In tests where suspected pollinators were coated with 343 fluorescent dyes, then released near ovulate cones and their paths traced, beetles as well as 344 Cycadothrips were found to crawl on and near micropyles, suggesting effective delivery of 345 pollen to micropyles by these pollinators (Donaldson 1997; Suinyuy et al. 2009; Terry et al. Author Manuscript Author 346 2005a). In the case of Cycadothrips, dye trails were concentrated around micropyles rather 347 than other structures within the cone indicating that their movement was directed toward 348 them, possibly to feed on micropylar droplets (Terry et al. 2005a). Tang (1987b; 1993)

This article is protected by copyright. All rights reserved 349 found amino acids in droplets, however, sugar levels appear to be lower than in nectars of 350 animal pollinated flowers (Nepi et al. 2009). Nepi et al. (2009) also found significant levels 351 of β-alanine in micropylar drops of Zamia furfuracea. This is a non-protein amino acid 352 usually found in higher levels in nectars of insect pollinated plants and appears to be a 353 nutritional attractant for insects.

354 Over a dozen cycad pollination systems have been studied in detail in the past 35 years. 355 Donaldson (2007) proposed 16 pollination models for cycads based on attraction or 356 repellency of their cone odors and thermogenesis. Here we categorize cycad pollination 357 systems into four general models (Table 2A-D) based on (1) types of rewards offered to 358 pollinators, (2) signals (odors, thermogenesis, shapes) that allow pollinators to find cones, 359 and (3) motivators for pollinators to depart cones. These factors more completely encompass 360 the critical components or mechanisms involved in these pollination systems. In most 361 species that have been surveyed, pollinating insects feed and reproduce within pollen cones 362 (Mound and Terry 2001; Norstog et al. 1986; Suinyuy et al. 2009; Wilson 2002) and this 363 appears to be a critical reward and component of the pollination systems of these species. 364 These pollination systems may fit into either models A or B (Table 2).

365 In model A (pollen cone brood-site), the pollen cone provides either pollen, nutritious 366 sporophyll or axis tissue, or a combination of these, and mating adults and developing larvae 367 feed on these resources. Examples of model A are Zamia integrifolia and its weevil 368 pollinator Rhopalotria slossoni and Z. furfuracea and its weevil pollinator R. furfuracea (see 369 Tables 1, 2A). In both these examples, the weevil oviposits into microsporophylls of pollen 370 cones, within which the developing larvae feed on starch-rich tissue (Norstog and Fawcett 371 1989; Tang 1987b). Zamia integrifolia and its beetle pollinator, Pharaxonotha floridana, 372 also fit model A. The adults and early instar larvae of this beetle feed on pollen released by 373 microsporangia (Norstog et al. 1992). Adults of all three of these beetle species, having 374 finished feeding on the deteriorating cone, as well as adults that have emerged from their 375 pupal cases, will leave the vicinity of the depleted pollen cone, usually with their bodies 376 laden with pollen, in search of another pollen cone brood site. In the process, they may visit 377 an ovulate cone, possibly by mistake, since ovulate cones possess similar signals (scent, Author Manuscript Author 378 color, texture). The ovulate cone offers shelter and possibly some ephemeral nutrition in the 379 form of a sugary micropylar drop. The beetle departs to find a more suitable cone in which to 380 mate and reproduce. This system has elements of the “mistake pollination” model proposed

This article is protected by copyright. All rights reserved 381 by Baker (1976) for papaya, in which female flowers, unlike male flowers, provide no nectar 382 or other reward, but lure in pollinators by mimicking the male flower.

383 Model B (pollen cone brood-site with push-pull) of cycad pollination (see Table 2B) is 384 similar to model A except that the adult pollinators that feed, mate and oviposit in the pollen 385 cone are driven out temporarily during a daily physiological cycle of the cone in which heat 386 production, high relative humidities, and the release of volatiles reach repellent levels. When 387 temperature and chemical levels fall later in the day the pollen cones become attractive again 388 to pollinators. This pollinator mechanism has been called “push-pull” and may be unique to 389 certain species of cycads, e.g. Macrozamia lucida and its pollinating thrips Cycadothrips 390 chadwicki (Terry et al. 2007; Terry et al. 2014; see Box 1). As in model A, the ovulate cone 391 may provide shelter and a temporary food source in the form of micropylar drops, but thrips 392 depart from ovulate cones when they undergo thermogenesis similar to that of pollen cones, 393 with high relative humidity and volatile emissions. 394 In pollination model C (ovulate cone brood-site), ovulate cones provide a brood site 395 reward for pollinators while no pollinator reproduction occurs in pollen cones. This 396 pollination mechanism was first demonstrated by Donaldson (1997) for Antliarhinus zamiae, 397 a weevil that spends time in the pollen cones of the South African cycad Encephalartos 398 villosus, but oviposits its eggs only into ovules of the ovulate cone. Despite destruction of a 399 portion of developing seeds by its larvae, which may reach levels above 80%, this weevil 400 appears to contribute 10% of the pollination of this cycad under natural conditions. Other 401 beetles also pollinate this cycad (see Appendix S1) and may pollinate a higher percent of 402 ovules, so there is the possibility that the efficiency of A. zamiae as a pollinator may be 403 higher in the absence of other pollinators. Another example of a female brood site system in 404 cycads is Cycas revoluta and its nitidulid beetle pollinators on Yonaguni Islands of Okinawa, 405 Japan (Kono and Tobe 2007, see also Table 2, Appendix S1). In this species beetles were 406 observed in pollen cones where they appear to consume pollen, but did not reproduce. 407 During and after the pollination period, hundreds of beetle adults and larvae were observed 408 sheltering in megasporophyll clumps, which appear to offer mating and brood sites. Beetles 409 may consume micropylar droplets, but the primary food appeared to be megasporophyll

410 tissue. Up to 10% Manuscript Author of megasporophylls and their ovules were consumed by these beetles, but 411 most were undamaged. Even the most damaged ovulate cones achieved a seed set of over 412 30%. When male plants were situated within 2 m of a female, wind played an important role 413 in pollination, so this species is ambophilous, being both insect and wind pollinated, under

This article is protected by copyright. All rights reserved 414 these conditions (Kono and Tobe 2007). Both the Encephalartos villosus and Cycas revoluta 415 pollination systems display some parallels to ovule parasite mutualisms found in yuccas, figs 416 and other specialized angiosperm pollination systems (Bain et al. 2016; Dufaÿ and Anstett 417 2003; Herre et al. 2008; Pellmyr 2003).

418 A subset of pollination model C is one in which insect pollinators breed within both 419 pollen and ovulate cones during the cones’ pollination phase. Some authors mention the 420 presence of adults and larvae of known or potential pollinators in both pollen and ovulate 421 cones (Connell and Ladd 1993; Donaldson 1997; Donaldson et al. 1995). Details of such a 422 system, if it exists, need verification. For example, it appears that in many of these 423 observations beetles are feeding in older ovulate cones that had been pollinated months 424 before and their presence may merely be predatory without involving any pollination of the 425 female cycad.

426 A fourth model of cycad pollination, model D (food-deceiving model), is found in 427 Stangeria eriopus and its nitidulid beetle pollinators (Procheş and Johnson 2009). In this 428 system neither pollen nor ovulate cones serve as brood sites, although they offer some 429 potential reward in the form of shelter and pollen in the case of pollen cones, and shelter and 430 micropylar drops in the case of females. The cone chemical volatiles emitted are the same as 431 those of fermenting fruit, suggesting that both pollen and ovulate cones mimic the fruits of 432 angiosperms (Procheş and Johnson 2009). The authors conclude that the system is a case of 433 deceit (or mistake) pollination in which the cones mimic decaying fruit, which is the usual 434 food for the nitidulid beetles, but offer little reward.

435 Niklas & Norstog (1984) proposed one other model of cycad pollination that involves 436 insects. Based on wind tunnel studies they suggested that wind may carry pollen to female 437 megasporopyhylls of Cycas, which are oriented vertically during receptivity. The pollen thus 438 delivered, is subsequently carried by rain or insects from various surfaces of the 439 megaporophylls to the micropyles of the ovules. The authors suggest that this mode of 440 transport may be more prevalent in species that produce large pollen cones with copious 441 amounts of pollen. The apparent wind pollination in Cycas revoluta described by Kono & 442 Tobe (2007) for females Manuscript Author within 2 m of a pollen cone may, in part be due to this mode of 443 transport, as the nitdulid beetles inhabiting those cones may have carried initially wind- 444 transported pollen the final step to the ovules. Tests have not been proposed or carried out to 445 separate or measure the contribution of this mode of pollination, but see Hamada et al.

This article is protected by copyright. All rights reserved 446 (2015a; 2015b) for details of potential wind contributions toward pollination in Cycas 447 micronesica on Guam.

448 Besides, micropylar droplets and shelter, another feature that all four proposed cycad 449 pollination models share is the role that cone scent plays as an informative signal to 450 pollinating insects. All cycad species that have been well-studied release cone scents that are 451 particularly strong during the daily periods of thermogenesis, primarily during pollen 452 shedding phase in pollen cones or during receptivity in ovulate cones (Pellmyr et al. 1991; 453 Suinyuy et al. 2010; Suinyuy et al. 2012; Suinyuy et al. 2013a; b; Tang 1987a; Terry et al. 454 2004a; Terry et al. 2016). The ovulate cones of most of these cycads have the same or fewer 455 compounds than that of their conspecific pollen cones (Azuma and Kono 2006; Pellmyr et al. 456 1991; Terry et al. 2004a; Terry et al. 2009; Terry et al. 2004b). Although only a small 457 fraction of all cycad species have been examined, over 150 different compounds across many 458 classes of chemistry have been detected, including terpenoids (both monoterpenes and 459 sesquiterpenes), benzenoids, aldehydes, alkenes and N-containing methoxy pyrazines. This 460 diversity is comparable to that of angiosperm floral and leaf volatiles (Knudsen and Tollsten 461 1993). Some species produce only a few compounds, such as some beetle-pollinated species 462 of Zamia and Macrozamia, whereas some species of Macrozamia and Cycas produce over 20 463 compounds (Terry et al. 2004a; Terry et al. 2004b), and some Encephalartos produce over 60 464 compounds (Suinyuy et al. 2012; Suinyuy et al. 2013a; b). Many of the same monoterpenes 465 are found across many species within several genera, while other compounds seem to be 466 unique to a few species. Notably, the N-containing methoxy pyrazines (found only in some 467 species of Cycas and Encephalartos (Kaiser 2006; Suinyuy et al. 2013a; Terry et al. 2012)) 468 and 1,3-octadiene (found only in some species of Zamia and Encephalartos (Pellmyr et al. 469 1991; Suinyuy et al. 2013a)) are major components in the species that produce them.

470 Choice tests with cones’ scent components on some cycad pollinators have demonstrated 471 their effectiveness as attractants (Suinyuy et al. 2015; Terry et al. 2008b; Terry et al. 2014). 472 Electroantennograms (EAGs) or gas chromatography- linked to electroantennogram detection 473 (GC-EADs) have been used to determine what odors that pollinators’ antennae can detect 474 physiologically (Suinyuy et al. 2015; Terry et al. 2008b; Terry et al. 2007). For example, Author Manuscript Author 475 Cycadothrips physiologically respond to the major cone volatile emission component, the 476 monoterpene β-myrcene, of their host Macrozamia lucida , at concentrations over several 477 orders of magnitude, suggesting a high sensitivity to this compound (Terry et al. 2008b).

This article is protected by copyright. All rights reserved 478 However, they are ‘blind’ to the linalool that is the major component in the volatile emissions 479 of Macrozamia machinii, pollinated by the weevil Tranes. Tranes responds to both linalool 480 and β-myrcene, the major components of its host cone odors. Suinyuy et al. (2015) 481 demonstrated with GC-EADs that pollinator beetles of Encephalartos respond to their own 482 local host cone’s volatile emission components.

483 Other aspects of the four pollination models involving insects presented here require 484 more study. In systems where “push-pull” does not appear to be active, other motivators for 485 pollinators to depart their brood cones, such as depletion of brood sites, depletion of food 486 resources and overcrowding need to be tested. The role of toxin levels may be important for 487 the choice of brood sites. For example, the presence of idioblasts that contain toxins, in the 488 cone tissue of species of Zamia (Norstog and Fawcett 1989; Vovides et al. 1993) and some 489 other cycads (Vovides 1991a) might restrict feeding to pollen cones that keep the idioblasts 490 intact, and not ovulate cones where idioblasts break down (Terry et al. 2012). Larvae of 491 Rhopalotria weevils feeding on pollen sporophylls that have intact idioblasts may sequester 492 the toxin and may be able to use it as defense against predators, whereas idioblasts in ovulate 493 cones appear to mobilize the toxin and break down the idioblasts and release the toxin to 494 avoid predation and protect their ovules (Norstog and Fawcett 1989; Vovides et al. 1993). 495 Another factor that is largely unresolved is the role of thermogenesis in cycad pollination. It 496 has been difficult to separate the possible attractant or repellent effects of thermogenesis from 497 those of volatile chemicals that are released concomitantly. For example, in some species of 498 Encephalartos, volatiles have been shown to play a role in attracting pollinating beetles to 499 cones and encouraging movement between cones, but pollinators do not appear to respond 500 strongly to peak temperatures, which the authors interpret as a possible active attraction 501 model (attraction to cone odors and possibly to warm cones) with no "push" (Suinyuy et al. 502 2013a). Terry et al. (2014) demonstrated several possible roles for cone thermogenesis in 503 Macrozamia lucida and M. macleayi. Specifically, experiments showed that cone 504 thermogenesis was a result of the intense increase in cone respiration, and that increased 505 respiration and cone heating was not only essential for the production of the high levels of β- 506 myrcene that become repellant, but warm cone temperatures alone caused Cycadothrips to

507 become more active, Manuscript Author and move toward bright light away from the cone. Thus, thermogenesis 508 could play a direct role by increasing pollinator activity (feeding or mating).

509

This article is protected by copyright. All rights reserved 510

511

512 Box: Push-pull pollination 513 The push-pull pollination system of cycads is exemplified by interactions between cones of 514 some Macrozamia species and their pollinator, Cycadothrips, but may also occur in some 515 Macrozamia pollinated by Tranes beetles. The Cycadothrips adults feed on pollen and 516 produce offspring that develop by feeding only on pollen among the sporophylls of the pollen 517 cones. During their pollination phase, pollen and ovulate cones have daily thermogenic 518 events that last for several hours (Terry et al. 2016). Cones ramp up their respiratory 519 metabolism for a few hours each day, resulting in increased cone temperatures 520 (thermogenesis), increased relative humidity, and increased volatile emission rates (mainly 521 the monoterpene β-myrcene in Cycadothrips-pollinated M. lucida) (Terry et al. 2012). High 522 levels of β-myrcene are repellent to thrips but at lower levels slightly attract thrips. At peak 523 temperatures (sometimes up to 12 °C above ambient), high relative humidity, and maximum 524 volatile release, throngs of pollen-laden thrips rapidly exit pollen cones over about 30 min. 525 Some of the multitudes of flying thrips are attracted to ovulate cones (Fig. 2) (Roemer et al. 526 2017; Terry et al. 2007; Terry et al. 2014) as cone thermogenesis subsides along with lower 527 emission rates of volatiles. Once within the ovulate cones, pollen from the insects is 528 deposited at the tip of the micropyle (Terry et al. 2005b). Whether thrips feed on the 529 pollination droplet is not known. Ovulate cones also undergo thermogenic events, when 530 thrips rapidly exit cones. The identity of specific attractive chemicals are under further study 531 in thrips-pollinated species.

532 Push-pull pollination occurs on a daily cycle, over a period of up to two weeks within a cone, 533 and this occurs when a cone is actively dehiscing pollen (pollen cones) or are receptive to 534 pollen (ovulate cones) (Roemer et al. 2017).

535 Future directions

536 Studies are needed to characterise species interactions among most cycads and their Author Manuscript Author 537 pollinators. There are still many species where the pollinators are unknown, not described, or 538 where there is taxonomic uncertainty. This basic science is necessary to be able to answer 539 more complex questions addressing the diversification and evolution of cycads. Detailed 540 knowledge will also allow for better comparisons with the pollination systems of flowering

This article is protected by copyright. All rights reserved 541 plants and will make cycad pollination systems more suitable as platforms for addressing 542 more general questions in ecology and evolutionary biology. Below are some questions that 543 need to be answered before cycad-pollinator associations are fully understood.

544 1. What are the pollinators of cycads and what is their level of specificity (i.e, is each 545 cycad species associated with one or more pollinator species)? There are still many 546 species of cycads whose pollinator is unknown. In Australia for example, many 547 species of Cycas have not been examined or surveyed for pollinators. In Macrozamia, 548 some species have been surveyed but either no insects have been found or found only 549 in one survey (e.g., M. moorei, M. fearnsidei) but not in later surveys. In the New 550 World, more than 80% of species of Dioon have been surveyed (O'Brien and Tang 551 2015), yet more than half the species of Zamia and Ceratozamia have no published 552 pollinator surveys. 553 2. What is the pollination mechanism of each species of cycad? Of the many cycads that 554 have been surveyed, very few (some species of Macrozamia, Zamia, Bowenia, 555 Encephalartos and Cycas) have been examined in enough detail to know how the 556 pollination system functions. 557 3. Are pollination-related traits of cycads, such as cone volatiles and thermogenesis, 558 correlated with species of pollinator? Encephalartos exhibit variation in composition 559 of cone volatiles across the landscape (Suinyuy et al. 2012; Suinyuy et al. 2018) and 560 between species (Suinyuy et al. 2013b), and differences correlate with different 561 pollinators, indicating that pollination traits might be important in speciation 562 processes. However, studies of the importance and role of pollination-related traits 563 have only been accomplished in a few species of cycads. 564 4. Do pollinators affect population structure and diversity of populations (via pollen 565 movement)? Do close associations result in pollinators and cycads being 566 phylogeographically/phylogenetically congruent? There is some evidence that thrips 567 pollinators of Macrozamia disperse long distances among isolated populations 568 (Brookes et al. 2015), although it is unknown whether this involves movement of 569 pollen. Few genera of cycads have complete surveys of pollinators or an adequate

570 understanding Manuscript Author of host genus systematics to answer questions regarding congruency or 571 co-diversification at present. There is evidence that host switching is common among 572 species within genera of Macrozamia (Brookes et al. 2015) and Encephalartos 573 (Downie et al. 2008; Tang et al. 2018b), and among the genera Dioon, Zamia and

This article is protected by copyright. All rights reserved 574 Ceratozamia (Tang et al. 2018b), but how host shifts affect the evolution of host or 575 pollinator has not been examined. Comparative phylogeographic studies among 576 pollinators and their cycad hosts are needed to address these questions at the 577 species/landscape level. 578 5. Does pollinator loss lead to the localised extinction of hosts or is wind pollination 579 sufficient? Insect exclusion experiments have demonstrated that the loss of insects 580 would result in reduced fertility for most genera of cycads (Donaldson et al. 1995; 581 Hall et al. 2004; Kono and Tobe 2007; Norstog et al. 1986; Suinyuy et al. 2009; Terry 582 2001; Terry et al. 2005b). Wind pollination is most likely only important for a few 583 species that have light pollen and are found in exposed conditions (Hall and Walter 584 2018; Hamada et al. 2015b; Terry et al. 2009). Cycads are long-lived plants and it’s 585 possible that localised temporary loss of pollinators, might not be detrimental to a 586 population in the short term. Currently, little is known about population dynamics 587 and dispersal of pollinators for most species and this area of research is in need of 588 attention for long-term conservation and management of cycads. 589 590 Conclusions

591 Research on cycads since the 1980s has established that most cycads are pollinated by 592 host-specialist insects, most are beetles from several different families but also thrips and 593 moths, and that there have been multiple shifts in the associations. It is clear that some 594 cycads and their pollinators have evolved an intricate mutualism that rivals the most 595 specialised mechanisms present among flowering plants, yet they are largely ignored in 596 reviews of pollination systems. 597 There is still much we do not know about cycad pollination and, as with angiosperms, 598 there is little understanding of the importance of the role of pollinators in plant 599 diversification. 600 601 602

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This article is protected by copyright. All rights reserved 1050 Table 1: List of insect pollinators of cycads, based on detailed study, presence of large numbers of adults and/or larvae in pollen cones, and/or 1051 close taxonomic affinity with well-studied pollinators. Included are the likely pollination model that they employ (see Table 2) and whether the 1052 insect has been studied as a pollinator. Insects that occur sporadically in cones, in small numbers, have clear alternate hosts, or have life 1053 histories that make them unlikely pollinators (e.g., are generalists pollen feeders), are excluded from this list. Species names of insects and 1054 cycads are updated to their most currently accepted binomials. Known Pollination Studied Pollinator taxon range Found in cones or stem apex of model indepth** Remarks References COLEOPTERA: CLEROIDEA: BIPHYLLIDAE Genus Biphyllus Cycas edentata, C. elongata, C. nongnoochii, C. pachypoda, C. Apparently one species is commonly pectinata, C. siamensis, C. associated with pollen cones of many Biphyllus sp. Asia zeylanica ?D Cycas species in Asia; Tang et al. 1999 COLEOPTERA: CUCUJOIDEA: BOGANIIDAE Genus Metacucujus May occur with Amorphocerus sp., Endrody-Younga & Erotylidae sp., Phacecorynus Crowson 1986, variegatus on E. cycadifolius; A. Donaldson et al. rufipes, Erotylidae sp., Platymerus 1995, Downie et al. Metacucujus Encephalartos cycadifolius, E. sp., Porthetes hispidus on E. friderici- 2008, Suinyuy et al. encephalarti South Africa friderici-guilielmi, E. lanatus A Y guilielmi; larvae feed on pollen 2009 May occur with Amorphocerus sp., Donaldson et al. Antliarhinus zamiae, Erotylidse sp., 1995, Downie et al. Author Manuscript Author M. goodei South Africa E. ghellinckii, E. villosus A Y Xenoscelinae sp. 1 & 2, Lobariidae, 2008, Suinyuy &

This article is protected by copyright. All rights reserved Platymerus sp., Porthetes sp. n. 6; Johnson 2018 larvae only in pollen cones Genus Paracucujus Ornduff 1991, Ladd Australia May occur with Cycadothrips & Connell 1995, (W. emmaliami, Tranes vigorsii, Oberprieler 1995, Paracucujus rostratus Australia) Macrozamia riedlei ?A Xenocryptus tenebroides Mound et al. 1998 COLEOPTERA: CUCUJOIDEA: EROTYLIDAE Genus Ceratophila Only known from Ceratozamia CERATOPHILA (subgenus CERATOPHILA) Ceratophila (C.) May occur with Pharxonotha sp. chemnicki Mexico Ceratozamia euryphilidia ?A D0221 Tang et al. 2018a C. (C.) gregoryi Mexico C. mixeorum A May occur with C. (V.) mixeorum Tang et al. 2018a C. alvarezii, C. mirandae, C. May occurs with C. (V.) chiapensis C. (C.) picipennis Mexico norstogi, C.vovidesii A and Pharaxonotha sp. Tang et al. 2018a Tang et al. 2018a, May occurs with C. (V.) vazquezi and Santiago-Jimenez et C. (C.) sanchezae Mexico C. tenuis A Pharaxonotha tenuis al. 2019 CERATOPHILA (subgenus VOVIDESA) Ceratophila (V.) C. alvarezii, C. mirandae, C. May occur with C. (C.) picipennis and chiapensis Mexico norstogi, C.vovidesii A Pharaxonotha sp. Tang et al. 2018a C. (V.) mixeorum Mexico C. mixeorum A May occur with C. (C.) gregoryi Tang et al. 2018a Tang et al. 2018a, May occur with C. (C.) sanchezae and Santiago-Jimenez et Author Manuscript Author C. (V.) vazquezi Mexico C. tenuis A Pharaxonotha tenuis al. 2019

This article is protected by copyright. All rights reserved Genus Cycadophila Skelley et al. 2017 CYCADOPHILA (subgenus CYCADOPHILA) LATA SPECIES GROUP China, Laos, Cycadophila (C.) collina Vietnam Cycas collina, C. tanqingii A May occur with Cycadophila nigra Skelley et al. 2017 China, May occur with C. (C.) fupingensis, C. (C.) debaonica Vietnam C. debaoensis, C. haobinhensis A C. (C.) nigra and C. (C.) yunnanensis Xu et al. 2015 C. (C.) lata Vietnam ? C. ferruginea ?A Cycas ferruginea is a probable host Skelley et al. 2017 CYCADOPHILA (subgenus CYCADOPHILA) FUPINGENSIS SPECIES GROUP Cycas debaoensis, C. Cycadophila (C.) China, diananensis, C. dolichophylla, May occur with C. (C.) debaonica, C. Xu et al. 2015, fupingensis Vietnam C. haobinhensis A (C.) nigra and C. (C.) yunnanensis Skelley et al. 2017 Some members of this species group (not listed here) are not known to CYCADOPHILA (CYCADOPHILA) NIGRA SPECIES GROUP coccur on cycads Cycadophila (C.) abyssa China Cycas hainanensis ?A or D Skelley et al. 2017 China, India, C. collina, C. debaoensis, C. Xu et al. 2015, C. (C.) nigra Vietnam diannanensis, C. haobinhensis ?A or D Skelley et al. 2017 This beetle is composed of 3 cryptic C. (C.) yunnanensis China, Laos C. collina, C. debaoensis D species or subspecies Xu et al. 2015 CYCADOPHILA (subgenus CYCADOPHILA) PAPUA SPECIES GROUP Papua New ? C. rumphii or C. Possible hosts include Cycas Cycadophila (C.) pupua Guinea schumanniana ?A schumanniana and C. rumphii Skelley et al. 2017 May occur with species of Skelley et al. 2017, C. (C.) samara Philippines Cycas edentata, C. nitida A Nanoplaxes type B4 Tang et al. (in press) Author Manuscript Author CYCADOPHILA (subgenus STROBILOPHILA)

This article is protected by copyright. All rights reserved Cycadophila (Strobilophila) May occur with species of Skelley et al. 2017, assamensis India Cycas pectinata A Nanoplaxes type A & B3 Tang et al. (in press) May occur with species of Skelley et al. 2017, C. (S.) hiepi Vietnam C. elongata, C. pachypoda A Nanoplaxes types A & C Tang et al. (in press) Host is currently recognized as Cycas C. (S.) kwaiensis Thailand C. sp. A siamenis (A. Lindstrom pers. Comm. Skelley et al. 2017 C. elephantipes, C. pectinata, C. clivicola ssp. Lutea, C. May occur with species of Skelley et al. 2017, C. (S.) tansachai Thailand tansachana A Nanoplaxes types A, B3 & C Tang et al. (in press) May occur with Cycadophila (C.) tansachai and species of Nanoplaxes Skelley et al. 2017, C. (S.) yangi Thailand C. pranburiensis, C. pectinata A types A, B3 & C Tang et al. (in press) Genus Hapalips Cycas cairnsiana, C. Australia megacarpa, C. media, C. May occur with Ulomoides australis , Hapalips sp. (Queensland) ophiolitica, C. platyphylla ? indet. Cossoninae Forster et al. 1994 Genus Pharaxonotha PHARAXONOTHA EARLY-DIVERGING LINEAGES Central America, Pharaxonotha kirschii Mexico Zamia sp. and other hosts ?D or not a pollinator Also a stored products pest Tang et al. 2018b May occur with Ceratophila Santiago-Jimenez et P. tenuis Mexico Ceratozamia tenuis A sanchezae and C. vazquezi al. 2019 Author Manuscript Author P. sp. D0057 Mexico Z. inermis ?D or not a pollinator Forms a cryptic species complex with Tang et al. 2018b

This article is protected by copyright. All rights reserved P. kirschii P. sp. D0021 Colombia Z. pyrophylla A Tang et al. 2018b *Attracted to bait cones of closely related Z. ipetiensis placed in Z. Tang et al. 2018b, A. P. sp. D0063 Panama in habitat of Z. cunaria* ?A cunaria habitat Taylor (perS. com.) May occur with Parallocorynus Tang et al. 2018b, P. sp. D0177 Mexico Dioon holmgrenii, D. merolae A salasae & P. schiblii or with P. jonesi Tang (pers. obs.) May occur with Ceratophila (C.) P. sp. D0221 Mexico Ceratozamia euryphilidia A chemnicki Tang et al. 2018a,b May occur with Parallocorynus Tang et al. 2018b, P. sp. D0250 Mexico D. edule (Farallon population) A perez-farrerai & P. iglesiasi Tang (pers. obs.) D. edule (Palma Sola May occur with Parallocorynus Tang et al. 2018b, P. sp. D0324 Mexico population) A perez-farrerai & P. iglesiasi Tang (pers. obs.) D. edule (Mt. Oscuro May occur with Parallocorynus Tang et al. 2018b, P. sp. D0253 Mexico population) A perez-farrerai & P. iglesiasi Tang (pers. obs.) May occur with Rhopalotria sp. Tang et al. 2018b, P. sp. D0288 Mexico Z. soconucensis A D0289 Tang (pers. obs.) D. angustifolium (San Luis May occur with Parallocorynus Tang et al. 2018b, P. sp. D0322 Mexico Potosi population) A norstogi & P. inexpectatus Tang (pers. obs.) D. angustifolium (Tamaulipas May occur with Parallocorynus Tang et al. 2018b, P. sp. D0323 Mexico population) A norstogi & P. inexpectatus Tang (pers. obs.) May occur with Parallocorynus Navarrete-Heredia P. sp. Mexico D. tomasellii A andrewsi 2018 C. alvarezii, C. mirandae, C. May occur with C. (V.) chiapensis and Author Manuscript Author P. sp. Mexico norstogi, C.vovidesii A C. (C.) picipennis Tang et al. 2018a

This article is protected by copyright. All rights reserved PHARAXONOTHA CARIBBEAN RADIATIONS P. zamiae is a junior synomym of this USA species; may occur with Rhopalotria Tang 1987, Tang et Pharaxonotha floridana (Florida) Zamia integrifolia A Y slossoni and Anatrachyntis badia al. 2018 Puerto Rico, Z. erosa, portoricensis, Z. Franz and Skelley Dominican pumila ngustifolia, Z. lucayana, A cryptic species complex likely to be 2008, Tang et al. P. portophylla Republic Z. sp. A divided into three species 2018b O'Brien & Tang 2015, Tang et al. P. sp. D0007 Jamaica Z. erosa A May occur with R. meerowi 2018b O'Brien & Tang Z. angustifolia, Z. integrifolia, 2015, Tang et al. P. sp. D0036 Bahamas Z. lucayana A May occur with R. dimidiata 2018b O'Brien & Tang Cayman 2015, Tang et al. P. sp. D0067 Islands Z. integrifolia A May occur with R. dimidiata 2018b PHARAXONOTHA RECENT RADIATION Pharaxonotha Bolivia, Skelley & Segalla cerradensis Brazil Z. boliviana A Sole pollinating insect 2019 Probably extends to Atlantic drainage Pakaluk 1988, Tang P. clarkorum Costa Rica Zamia neurophyllidia A of Panama on Z. skinneri complex et al. 2018b Pakaluk 1988, O'Brien & Tang Costa Rica, Z. fairchildiana, Z. May occur with Notorhopalotria 2015, Tang et al. Author Manuscript Author P. confusa Panama pseudomonticola A montgomeryensis 2018b

This article is protected by copyright. All rights reserved Costa Rica, Panama, May occur with N. panamensiis (in Colombia Z. dressleri, Z. elegantissima, Z. Panama) and N. platysoma (in P. sp. D0022 (Choco) nana, Z. obliqua, Z. stevensonii A Colombia) Tang et al. 2018b P. sp. D0072 Colombia Z. tolimensis A Tang et al. 2018b O'Brien & Tang 2015, Tang et al. P. sp. D0009 Belize Z. decumbens A May occur with Rhopalotria calonjei 2018b Host likely to include other Zamia of O'Brien & Tang the Mexico Altantic drainage, occurs 2015, Tang et al. P. sp. D0025 Mexico Z. loddigesii A with R. furfuracea 2018b Range likely confined to the Pacific O'Brien & Tang drainage of Mexico, occurs with R. 2015, Tang et al. P. sp. D0176 Mexico Z. paucijuga A mollis 2018b Vovides 1991, O'Brien & Tang Dioon califanoi, D. caputoi, D. May occur with Parallocorynus 2015, Tang et al. P. sp. D0235 Mexico purpusii A gregoryi and P. chemnicki 2018b Valencia-Montoya P. sp. Colombia Z. incognita A Y Sole pollinating insect et al. 2017 Genus Xenocryptus Ornduff 1991, Ladd Australia May occur with Cycadothrips & Connell 1995, (W. emmaliami, Paracucujus rostratus, Oberprieler 1995, Author Manuscript Author Xenocryptus tenebroides Australia) Macrozamia riedlei ?A Tranes vigorsii Mound et al. 1998

This article is protected by copyright. All rights reserved Genus undescribed May occur with Amorphocerus sp. ?A&C (=Porthetes sp. N. 6?), Metacucujus Donaldson et simultaneo encephalarti, Phacecorynus al.1995, Downie et Languriidae sp. South Africa Encephalartos cycadifolius usly Y variegatus al. 2008 May occur with Amorphocerus rufipes, M. encephalarti, Platymerus sp., Porthetes hispidus; larvae feed on Erotylidae sp. South Africa E. friderici-guilielmi A Y pollen Suinyuy et al. 2009 May occur with Antliarhinus zamiae, M. goodei, Porthetes sp. n. 6, Xenoscelinae sp. 2; larvae in femalecone but only several months Donaldson 1997, Xenoscelinae sp. 1 South Africa E. villosus A Y after pollination Downie et al. 2008 May occur with A. zamiae, M. goodei, Donaldson 1997, Xenoscelinae sp. 2 South Africa E. villosus A Y Porthetes sp. n. 6, Xenoscelinae sp. 1 Downie et al. 2008 May occur with Metacucujus goodei, Platymerus sp., Amorphocerus sp., Suinyuy & Johnson Erotylidae sp. South Africa E. ghellinckii ?A Lobariidae sp. 2018 COLEOPTERA: CUCUJOIDEA: NITIDULIDAE Typically feed on decaying fruit; Genus Carpophilus cycads are not obligate hosts May occur with Epuraea Carpophilus chalybeus Japan Cycas revoluta C Y mandibularis Kono & Tobe 2007 Author Manuscript Author C. dimidiatus Guam, Rota C. micronesica ?D Y May occur with Anatrachyntis sp. Terry et al. 2009,

This article is protected by copyright. All rights reserved 2012 May occur with Carpophilus fumatus Procheş & Johnson C. hemipterus South Africa Stangeria eriopus D Y and Urophorus picinus 2009 May occur with Carpophilus Procheş & Johnson C. fumatus South Africa Stangeria eriopus D Y hemipterus and Urophorus picinus 2009 Terry et al. 2009, C. mutilatus Guam, Rota C. micronesica ?D Y May occur with Anatrachyntis sp. 2012 Genus Epuraea May occur with Carpophilus Epuraea mandibularis Japan Cycas revoluta C Y chalybeus Kono & Tobe 2007 Genus Urophorus May occur with Carpophilus Procheş & Johnson Urophorus picinus South Africa Stangeria eriopus D Y hemipterus and C. fumatus 2009 COLEOPTERA: CURCULIONOIDEA: BELIDAE Genus Notorhopalotria Pakaluk 1988, O'Brien & Tang Notorhopalotria Costa Rica, Zamia fairchildiana, Z. May occur with Pharaxonotha 2015, Tang et al. montgomeryesnsis Panama pseudomonticola A confusa 2018b O'Brien & Tang Z. dressleri, Z. elegantissima, Z. May occur with Pharaxonotha sp. 2015, Tang et al. N. panamensis Panama nana, Z. obliqua, Z. stevensonii A D0022 2018b O'Brien & Tang May occur with Pharaxonotha sp. 2015, Tang et al. Author Manuscript Author N. platysoma Colombia Z. obliqua A D0022 2018b

This article is protected by copyright. All rights reserved O'Brien & Tang 2015, Tang (pers. N. taylori Panama Z. pseudoparasitica A May occur with Pharaxonotha sp. obs.) Genus Parallocorynus PARALLOCORYNUS (subgenus PARALLOCORYNUS) O'Brien & Tang Parallocorynus (P.) May occur with Pharaxonotha sp. 2015, Tang et al. bicolor Mexico Dioon caputoi, D. planifolium A D0235 on D. caputoi 2018b May occur with Parallocorynus chemnicki on D. califanoi and with O'Brien & Tang D. argenteum, D. califanoi, D. Pharaxonotha sp. D0235 on D. 2015, Tang et al. P. (P.) gregoryi Mexico purpusii A califanoi & D. purpusii 2018b O'Brien & Tang May occur with Pharaxonotha sp. 2015, Tang et al. P. (P.) jonesi Mexico D. merolae A D0177 2018b May occur with Parallocorynus O'Brien & Tang inexpectatus and Pharaxonotha sp. 2015, Tang et al. P. (P.) norstogi Mexico D. angustifolium A D00322 & D0323 2018b May occur with Parallocorynus O'Brien & Tang iglesiasi and Pharaxonotha sp. D0250 2015, Tang et al. P. (P.) perezfarrerai Mexico D. edule A & D0324 2018b O'Brien & Tang May occur with Parallocorynus 2015, Tang et al. P. (P.) salasae Mexico D. holmgrenii A schiblii and Pharaxonotha sp. D0177 2018b Author Manuscript Author PARALLOCORYNUS (subgenus EOCORYNUS)

This article is protected by copyright. All rights reserved May occur with Parallocorynus O'Brien & Tang gregoryi and Pharaxonotha sp. 2015, Tang et al. P. (E.) chemnicki Mexico D. califanoi A D0235 2018b O'Brien & Tang Occurs with Parallocorynus salasae 2015, Tang et al. P. (E.) schiblii Mexico D. holmgrenii A and Pharaxonotha sp. D0177 2018b PARALLOCORYNUS (subgenus NEOCORYNUS) May occur with Parallocorynus O'Brien & Tang perezfarrerai and Pharaxonotha sp. 2015, Tang et al. P. (N.) iglesiasi Mexico D. edule A D0250 & D0324 2018b May occur with Parallocorynus O'Brien & Tang norstogii and Pharaxonotha sp. 2015, Tang et al. P. (N.) inexpectatus Mexico D. angustifolium A D00322 & D0323 2018b PARALLOCORYNUS (subgenus DYSICORYNUS) O'Brien & Tang 2015, Navarrete- P. (D.) andrewsi Mexico D. stevensonii, D. tomasellii A May occur with Pharaxontha sp. Heredia 2018 O'Brien & Tang P. (D.) sonorensis Mexico D. vovidesii A 2015 Genus Protocorynus O'Brien & Tang Protocorynus bontai Honduras Dioon mejiae A 2015 Genus Rhopalotria RHOPALOTRIA (subgenus RHOPALOTRIA) Author Manuscript Author Rhopalotria (R.) Zamia angustifloia, Z. erosa, Z. A May occur with Pharaxonotha sp. O'Brien & Tang

This article is protected by copyright. All rights reserved dimidiata integrifolia, Z. lucayana, Z. D0007, D0036 & D0067 2015, Tang et al. pygmaea, Z. stricta 2018b, Tang (pers. obs.) Cayman Islands, Cuba, O'Brien & Tang Bahamas, May occur with Pharaxonotha sp. 2015, Tang et al. R. (R.) meerowi Jamaica Z. erosa A D0007 2018b O'Brien & Tang May occur with Pharaxonotha 2015, Tang et al. R. (R.) slossoni Jamaia Z. integrifolia A Y floridana and Anatrachyntis badia 2018b RHOPALOTRIA (subgenus ALLOCORYNUS) O'Brien & Tang USA May occur with Pharaxonotha sp. 2015, Tang et al. Rhopalotria (A.) calonjei (Florida) Z. decumbens A D0009 2018b May occur with Pharaxonotha sp. Norstog et al. 1996, D0025 on Z. loddigesii; pollination O'Brien & Tang Z. furfuracea, Z. loddigesii, Z. confirmed by exclusion experiment on 2015, Tang et al. R. (A.) furfuracea Belize spartea, Z. splendens A Y Z. furfuracea 2018b O'Brien & Tang May occur with Pharaxonotha sp. 2015, Tang et al. R. (A.) mollis Mexico Z. paucijuga A D0176 2018b O'Brien & Tang 2015, Tang (pers. Author Manuscript Author R. (A.) vovidesi Mexico Dioon spinulosum A May occur with Pharaxonotha sp. obs.)

This article is protected by copyright. All rights reserved COLEOPTERA: CURCULIONOIDEA: BRENTIDAE Genus Antliarhinus Encephalartos altensteinii, E. Larvae feed on female sporophyll lehmannii, E. longifolius, tissue; importance in pollination, if Donaldson 1993, Antliarhinus peglereae South Africa E.natalensis, E. trispinosus ?C any, not studied Oberprieler 1995 E. altensteinii, E.arenarius, E. horridus, E. lehmannii, E. Larvae grow in developing seeds; longifolius, E. natalensis, E. importance in pollination, if any, not Donaldson 1993, A. signatus South Africa trispinosus ?C studied Oberprieler 1995 E. altensteinii, E. arenarius, E. caffer, E. ferox, E. horridus, E. On E. villosus it is a seed predator lehmanni, E. lebomboensis, E. which plays a minor role in Donaldson 1993, longifolius, E. natalensis, E. pollination and occurs with 1997, Oberprieler princeps, E. trispinosus, E. Metacucujus goodei, Porthetes sp. n. 1995, Downie et al. A. zamiae South Africa umbeluziensis, E. villosus C Y 6, Xenoscelinae sp. 1 & 2; 2008 A. verdcourti Kenya E. tegulaneus ?C Ovulate cone Oberprieler 1995 Larvae feed on the ovulate cone axis; importance in pollination, if any, not A. sp. nr. verdcourti South Africa E. altensteinii ?C studied Donaldson 1993 A. sp. 2 Zimbabwe E. concinnus ?C Oberprieler 1995 Genus Platymerus Encephalartos friderici- Ovulate cone axis; importance in Platymerus eckloni South Africa guilielmi ?C pollination, if any, not studied Oberprieler 1995 Ovulate cone sporophylls; importance Author Manuscript Author P. zeyheri South Africa E. friderici-guilielmi ?C in pollination, if any, not studied Oberprieler 1995

This article is protected by copyright. All rights reserved Ovulate cone sporophylls; importance P. ? winthemi South Africa E. friderici-guilielmi ?C in pollination, if any, not studied Oberprieler 1995 Ovulate cone sporophylls; importance P. sp. n. South Africa E. cycadifolius ?C in pollination, if any, not studied Oberprieler 1995 Found on pollen cones, but importance in pollination, if any, not P. sp. South Africa E. friderici-guilielmi ?C Y studied Suinyuy et al. 2009 May occur with Metacucujus goodei, Amorphocerus sp., Erotylidae sp., Suinyuy & Johnson P. sp. South Africa E. ghellinckii ?C Lobariidae sp. 2018 COLEOPTERA: CURCULIONOIDEA: CURCULIONIDAE: AMORPHOCERINI Genus Amorphocerus May occur with Erotylidae sp., Metacucujus encephalarti, Encephalartos friderici- Platymerus sp., Porthetes hispidus on Downie et al. 2008, Amorphocerus rufipes South Africa guilielmi, E. ghellinckii A E. friderici-guilielmi Suinyuy et al. 2009 May occur with Porthetes dissimilis, P. sp. n. 14 on E. altensteinii; P. sp. n. E. altensteinii, E. arenarius, E. 3 on E. lehmanii; P. zamiae, P. sp. n. latifrons, E. lehmanii, 2, P. sp. n. 13 on E. longifolius; P. sp. A. talpa South Africa longifolius, E. trispinosus A n. 4 on E. trispinous Downie et al. 2008 May occur with Porthetes sp. n. 9 on A. sp. n. 1 South Africa E. altensteinii A E. altensteinii Downie et al. 2008 A. sp. n. 2 South Africa E. cycadifolius A May occur with A. setosus Downie et al. 2008 Author Manuscript Author A. sp. South Africa E. ghellinckii ?A Y May occur with Metacucujus goodei, Suinyuy & Johnson

This article is protected by copyright. All rights reserved Platymerus sp., Erotylidae sp., 2018 Lobariidae sp. Genus Porthetes May occur with Amorphocerus talpa on E. latifrons and E. altensteinii, A. Porthetes dissimilis South Africa E. altensteinii, E. latifrons A sp. 1 on E. altensteinii Downie et al. 2008 Also placed under the genus Oberprieler 1995, P. gedyei Kenya E. hildebrandtii A Peltostethus Downie et al. 2008 May occur with Amorphocerus rufipes, Metacucujus encephalarti, Platymerus sp., Erotylidae sp.; larvae feed within tissues of Downie et al. 2008, P. hispidus South Africa E. friderici-guilielmi A Y microsporophylls Suinyuy et al. 2009 P. zamiae South Africa E. longifolius A May occur with A. talpa Downie et al. 2008 P. sp. n. 1 South Africa E. senticosus A Downie et al. 2008 P. sp. n. 2 South Africa E. longifolius A May occur with A. talpa Downie et al. 2008 May occur with A. talpa on E. P. sp. n. 3 South Africa E. lehmanii, E. horridus A lehmanii Downie et al. 2008 P. sp. n. 4 South Africa E. trispinosus A Downie et al. 2008 P. sp. n. 5 South Africa E. horridus A Downie et al. 2008 May occur with A. zamiae, M. goodei, Xenoscelinae sp. 1 & 2; larvae feed Donaldson et al. within microsporophylls but not 1995, Downie et al. P. pearsoni (sp. n. 6) South Africa E. villosus A Y pollen 2008 Author Manuscript Author P. sp. n. 7 South Africa E. ferox A Downie et al. 2008

This article is protected by copyright. All rights reserved P. sp. n. 8 South Africa E. laevifolius A Downie et al. 2008 P. sp. n. 9 South Africa E. altensteinii A May occur with A. sp. n. 1 Downie et al. 2008 P. sp. n. 11 South Africa E. caffer A Downie et al. 2008 P. sp. n. 12 South Africa E. aplanatus, E. umbeluziensis A Downie et al. 2008 P. sp. n. 13 South Africa E. longifolius A May occur with A. talpa Downie et al. 2008 E. altensteinii, E. lebomboensis, May occur with A. talpa on E. P. sp. n. 14 South Africa E. natalensis A altensteinii Downie et al. 2008 COLEOPTERA: CURCULIONOIDEA: CURCULIONIDAE: COSSONINAE Genus undescribed nr. Cossonideus Australia (N. Territory, new sp. 1 Queensland) Cycas armstrongii, C. media ?A Oberprieler 1995 Undetermined genus Australia sp. 1 (Queensland) Cycas sp. ?A Oberprieler 1995 Australia sp. 2 (Queensland) Cycas sp. Oberprieler 1995 COLEOPTERA: CURCULIONOIDEA: CURCULIONIDAE: MOLYTINI Genus Melanotranes May occur with Cycadothrips Forster 1994, Ladd Australia emmaliami, Paracucujus rostratus, & Connell 1995, (W. Tranes luteroides, T. vigorsii, Oberprieler 1995, Melanotranes roei Australia) M. riedlei ?A Xenocryptus tenebroides Mound et al. 1998

Genus Miltotranes Manuscript Author Miltotranes prosternalis Australia Bowenia spectabilis A Y Wilson 2002

This article is protected by copyright. All rights reserved (Queensland) Australia M. subopaca (Queensland) Bowenia serrulata A Y Wilson 2002 Genus Tranes Australia Tranes insignipes (Queensland) ? Lepidozamia hopei ?A Oberprieler 1995 Australia May occur with Cycadothrips Chadwick 1993, (New South L. peroffskyanna, Macrozamia chadwicki 5, and/or Tranes sparsus on Forster 1994, Terry T. lyterioides Wales) communis ?A Y M. communis 2001 May occur with Cycadothrips Australia chadwicki 5 (on M. communis), (New South Tranes internatus, T. lyterioides; Chadwick 1993, T. sparsus Wales) M. communis ?A Y found on pollen but not ovulate cones Oberprieler 1995 Australia May occur with Cycadothrips Forster 1994, Ladd (W. emmaliami, Paracucujus rostratus, & Connell 1995, T. vigorsi Australia) M. riedlei ?A Xenocryptus tenebroides Mound et al. 1998 Insect pollination confirm by Australia exclusion experiment; no other insect T. sp. (Queensland) Lepidozamia peroffskyana ?A Y consistently associated with cones Hall et al. 2004 Insect pollination confirm by Australia exclusion experiment; no other insect T. sp. (Queensland) M. machinii ?A,B Y consistently associated with cones Terry et al. 2005 Australia (New South Author Manuscript Author T. sp. 1 Wales) Macrozamia fawcettii ?A Forster 1994

This article is protected by copyright. All rights reserved M. crassifolia, M. douglasii, M. lomandroides, M. pauli- Australia guilielmi, M. parcifolia, M. sp. Forster 1994, Terry T. sp. 2 (Queensland) aff. plurivernia ?A 2001 Australia (New South May occur with Cycadothrips Forster 1994, Terry T. sp. 2 Wales) M. johnsonii, M. montana ?A,B chadwicki in M. montana (pers. obs.) COLEOPTERA: CURCULIONOIDEA: CURCULIONIDAE: TRYPETIDINI Genus Nanoplaxes India, Myanmar, May occur with Tychiodes B3 Heller 1913, Tang Nanoplaxes ferruginea Thailand Cycas pectinata ?A (D0138) & C (D0139) (in press, pers. obs.) N. merrilli Philippines ? Cycas ?A Heller 1913 May occur with Cycadophila Tang et al. 1999, in N. sp. D0051 India C. pectinata ?A assamensis, Tychiodes B3 (D0148) press Tang et al. 1999, in N. sp. D0079 Vietnam C. pachypoda ?A May occur with Tychiodes C (D0078) press May occur with Tychiodes B2 Tang et al. 1999, in N. sp. D0113 Thailand C. nongnoochii, C. siamensis ?A (D0114) & C (D0121) press N. sp. D0145 Thailand C. macrocarpa ?A May occur with Tychiodes B3 Tang (in press) N. sp. D0338 India C. sp. aff. siamensis ?A Tang (in press) Genus Tychiodes Type locality is 270 km from nearest Wollaston 1873, Tychiodes adamsii Japan Unlikely to have cycad host ?A cycad population Tang (in press) Author Manuscript Author T. jansoni Philippines ? Cycas sp. ?A Oberprieler 1995

This article is protected by copyright. All rights reserved Solomon T. rennelli Islands ? Cycas seemannii ?A Oberprieler 1995 India (Andaman & T. sp. Nicobar Isl) C. zeylanica ?A Oberprieler 1995 Tychiodes B1 Philippines C. edentata ?A May occur with Cycadophila samara Tang, in press May occur with Nanoplaxes sp. Tang et al. 1999, in Tychiodes B2 (D0114) Thailand C. nongnoochii, C. siamensis ?A D0113 press Tang et al. 1999, in Tychiodes B2 (D0141) Thailand C. clivicola, C. edentata ?A press India, C. elephantipes, C. macrocarpa, May occur with Nanoplaxes Tang et al. 1999, in Tychiodes B3 Thailand C. pectinata ?A ferruginea, Tychiodes C (D0078) press C. nitida, sancti-lasellii, C. Tychiodes B4 Philippines vespertillo ?A Tang (in press) India, Thailand, C. elephantipes, C. pachypoda, May occur with Nanoplaxes spp., Tang et al. 1999, in Tychiodes C (D0111) Vietnam C. pectinata, C. petraea ?A Tychiodes B3 press May occur with Nanoplaxes sp. Tang et al. 1999, in Tychiodes C (D0121) Thailand C. siamensis ?A D0113, Tychiodes B2 (D00114) press May occur with N. sp. D0113, Tang et al. 1999, in Tychiodes C (D0133) Thailand C. nongnoochii ?A Tychiodes B2 (D0114) press Tychiodes C (D0150) Philippines C. nitida ?A May occur with Cycadophila samara Tang, in press Genus Tychiosoma Tychiosoma Author Manuscript Author gracilirostre Philippines ? Cycas sp. ?A Wollaston 1873

This article is protected by copyright. All rights reserved COLEOPTERA: TENEBRIONOIDEA: TENEBRIONIDAE Genus Ulomoides Cycas cairnsiana, C. megacarpa, C. media, C. Australia ophiolitica, C. platyphylla, May occur with Hapalips sp. , indet. Forster et al. 1994, Ulomoides australis (Queensland) Macrozamia moorei ?A Cossoninae on Cycas spp. Terry et al. 2004 Australia May occur with Cossoninae nr. U. xamiaphila (Queensland) C. armstrongii ?A Cossonideus Ornduff 1992 LEPIDOPTERA: COSMOPTERIGIDAE Genus Anatrachyntis Terry et al. 2009, Anatrachyntis sp. Guam, Rota Cycas micronesica ?A Y May occur with Carpophilus spp. 2012 May occur with Pharaxonotha USA floridana & Rhopalotria slossoni; A. badia (Florida) Zamia integrifolia ?A pollen found on adults Hua et al. 2019 THYSANOPTERA: AELOTHRIPIDAE Genus Cycadothrips Australia (N. Mound and Terry Cycadothrips albrechti Territory) Macrozamia macdonnellii B Y 2001 Brookes et al. C. chadwicki 1 (northern Australia M. macleayi, M. miquelii, M. C. chadwicki is a cryptic species 2015;Forster et al. clade) (Queensland) serpentina B Y complex consisting of 5 species 1994 C. chadwicki 2 (central Australia C. chadwicki is a cryptic species Brookes et al. 2015; clade) (Queensland) M. platyrhachis ?B Y complex consisting of 5 species Terry et al. 2008 Author Manuscript Author C. chadwicki 3 (central Australia M. longispina, M. macleayi, M. B Y C. chadwicki is a cryptic species Brookes et al.

This article is protected by copyright. All rights reserved clade) (Queensland) mountperriensis complex consisting of 5 species 2015;Forster et al. 1994 Brookes et al. 2015, C. chadwicki 4 (central Australia C. chadwicki is a cryptic species Terry et al. 2005; clade) (Queensland) M. lucida, M. macleayi B Y complex consisting of 5 species Forster et al. 1994 C. chadwicki (central Australia Jones 2001; Brookes clade?) (Queensland) M. cardiacensis ?B May occur with Tranes sp. & Terry (pers. obs.) Australia C. chadwicki is a cryptic species C. chadwicki 5 (southern (New South complex consisting of 5 species, may Brookes et al. 2015, clade) Wales) M. communis, M. montana ?B Y occur with Tranes lyterioides Terry 2001 Connell & Ladd Australia May occur with Paracucujus 1993, Oberprieler (W. rostratus, Tranes vigorsi, Xenocryptus 1995, Mound et al. C. emmaliami Australia) M. riedlei ?B tenebroides 1998 *Model A,B,C, or D (see text) is proposed for the pollination system based on experimental work or behavioral observations. A question mark is included where information is limited and model is presumed. Models may not be exclusive of other models.

** Studied in depth with at least detailed observations or including enclosure/exclosure tests with pollinators versus wind; in some cases, author’s conclusions of pollination by the insect were used. 1055

1056 Manuscript Author 1057

This article is protected by copyright. All rights reserved 1058 1059 1060 1061 1062 1063 1064 1065 Author Manuscript Author

This article is protected by copyright. All rights reserved 1066 Table 2: Models of cycad pollination systems focusing on rewards, signals and motivators 1067 for insect pollinators to depart, for pollen and ovulate cones. 1068 1069 MODEL A (Pollen Cone Brood-Site): pollen cone tissues serve as larval food, ovulate & pollen cones 1070 pull the insect in, ovulate cone mimics pollen cone. Pollen and ovulate cones are asymmetric in 1071 rewards and motivators for pollinator to move, but similar in signals. Based on Zamia integrifolia & 1072 Pharaxonotha floridana, Rhopalotria slossoni systems and Z. furfuracea & R. furfuracea system (see 1073 text). 1074 POLLEN CONE OVULATE CONE

REWARDS  Food for adult pollinators  Micropylar drops (other tissue  Toxins in idioblasts if present possibly toxic) in sporophylls-may be  Shelter sequestered for defense

 Mating & brood site  Shelter SIGNALS  Cone shape  Mimic pollen cone shape  Cone color  Mimic pollen cone color  Cone scent  Mimic pollen cone scent MOTIVATORS  Depletion of food and brood  Food is ephemeral TO DEPART sites 1075 1076 1077 MODEL B (Pollen Cone Brood-Site With Push-Pull): Similar to Model A except pollen cones exert a 1078 push-pull effect, ovulate cones mimic pollen cone. Pollen and ovulate cones are asymmetric in their 1079 rewards. Based on Macrozamia lucida & Cycadothrips chadwicki system (see text). 1080 POLLEN CONE OVULATE CONE

REWARDS  Food for adult pollinators:  Micropylar drops pollen  Shelter  Manuscript Author Mating & brood site (pollen)  Shelter SIGNALS  Cone shape/texture  Mimic pollen cone shape/texture

This article is protected by copyright. All rights reserved  Cone color  Mimic pollen cone color  Cone scent  Mimic pollen cone scent MOTIVATORS  High cone temperatures and  Food is limited and/or ephemeral TO DEPART scent during thermogenic event  High cone temperatures & scent MOTIVATOR  Lowering cone temperature & TO RETURN scent

 Cone scent 1081 1082 1083 MODEL C (Ovulate Cone Brood-Site): Larvae develop in ovules or megasporophylls, ovulate cone 1084 pull- pollen cone pull, pollen cones mimic ovulate cones. Pollen and ovulate cones are asymmetric in 1085 rewards and motivators to depart, but similar in signals. Based on the Encephalartos villosus & 1086 Antliarhinus zamiae system and Cycas revoluta & Nitidulidae system (see text). 1087 POLLEN CONE OVULATE CONE

REWARDS  Food for adult pollinators:  Mating and brood sites pollen  Micropylar drops & sporophyll  Shelter tissue  Shelter SIGNALS  Mimic ovulate cone  Cone shape/texture shape/texture  Cone color  Mimic ovulate cone color  Cone scent  Mimic ovulate cone scent MOTIVATORS  No brood sites  None TO DEPART  Insufficient food 1088 1089 MODEL D (Food-Deceiving): Larvae do not develop in cones, ovulate cone pull- pollen cone pull, 1090 ovulate & pollen cone mimic angiosperm fruit, attraction mainly by deceit. Pollen and ovulate cones 1091 asymmetric in reward, but similar in motivators to depart and signals. Based on Stangeria eriopus & 1092 Nitidulidae system (see text).

1093 Manuscript Author POLLEN CONE OVULATE CONE

REWARDS  Food for adult pollinators:  Micropylar drops pollen  Shelter

This article is protected by copyright. All rights reserved  Shelter SIGNALS  Mimic scent of angiosperm  Mimic scent of angiosperm fruit fruit (usual food of pollinator)

MOTIVATORS  Insufficient food  Food is limited and/or ephemeral TO DEPART 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 Figure Headings 1109 Figure 1: Phylogeny of cycads (modified from Condamine et al. 2015) showing the 10 1110 extant cycad genera and their primary pollinators: weevils (Coleoptera: Curculionoidea), 1111 other beetles (Coleoptera: Cleroidea, Cucujoidea and Tenebrionoidea), thrips (Thysanoptera: 1112 Aelothripidae), moths (Lepidoptera: Cosmopterigidae) and wind. In some cycad phylogenies, 1113 Bowenia is sister to all cycads excluding Dioon and Cycas (Salas-Leiva et al. 2013). The 1114 clade size is relative to the number of species and number of species are in brackets (World 1115 List of Cycads accessed 10 October2018). Refer to Table 1 for genera of pollinators 1116 associated with each cycad genera. 1117

1118 Figure 2: Push-pull Manuscript Author pollination in Macrozamia cycads and Cycadothrips. During their 1119 pollination period when pollen is dehiscing and ovulate cones are receptive, reproductive 1120 tissues undergo a daily thermogenic cycle over many days with cones heating up for several 1121 hours releasing high levels of volatiles (Terry et al. 2016). Thrips respond to peak

This article is protected by copyright. All rights reserved 1122 temperature and volatile expression (mainly the monoterpene β-myrcene in Cycadothrips- 1123 pollinated M. lucida) (Terry et al. 2012) by leaving cones and return to cones as they cool. 1124 Pollination is accomplished by some thrips moving pollen from pollen cones to ovulate 1125 cones. 1126 1127 Appendix S1. List of all cycad species where potential insect pollinators have been collected, 1128 locations where pollinators (see Table 1) were found or studied, and the level of study that 1129 each plant-pollinator pair has received. Species names of cycads and insects are updated to 1130 their most currently accepted binomials. Author Manuscript Author

This article is protected by copyright. All rights reserved aec_12925_f1.pdf Key to taxa and geography Macrozamia (41) weevils Australia Lepidozamia (2) other Africa beetles America Encephalartos (65) thrips Madagascar, moths Africa, Asia, Ceratozamia (30) Pacific Islands, wind Australia Stangeria (1) Microcycas (1)

Zamia (81)

Bowenia (2) Dioon (16)

Cycas (117)

Ginkgo

CPTKJ P N 300 200 100 0 Millions of years Author Manuscript Author

This article is protected by copyright. All rights reserved aec_12925_f2.pdf

pollen ovulate cone cone ii. During mid-day: thermogenic pollen and ovulate cone tissues peak in temperature and expression of volatiles, and thrips move out of cones, covered in pollen from pollen cones.

iii. Later in the day: pollen and ovulate i. In the morning: cones are cool, cone tissues cool and volatile expression volatile expression is low, thrips is lower, thrips return to cones, some to are relatively sedentary within pollen cones and some to ovulate cones. the sporophylls of the cones. Author Manuscript Author

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