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the Third International Conference on Cycad Biology. The Cycad Society of South Africa, Stellenbosch, South Africa. O’Brien, C. W. & G. J. Wibmer. 1982. Annotated checklist of the weevils (Curculionidae sensu lato) of North America, Central America, and the West Indies (Coleoptera: Curculionoi- dea). Mem. Amer. Entomol. Inst. 34. Stevenson, D. W. 1993. The Zamiaceae in Panama with comments on phytogeography and species relationships. Brittonia 45: 1– 16. Tang, W. 1987. Insect pollination in the cycad Zamia pumila (Zamiaceae). Amer. J. Bot. 74: 90– 99. ———. 2004. Cycad insects and pollination. Pp. 383– 394 in P. C. Srivastava (ed.), Vistas in Palaeobotany and Plant Morphology: Evolutionary and Environmental Perspectives, Professor D. D. Pant Memorial Volume. U. P. Off set, Lucknow, India. Terry, I., G. Walter, C. Moore, R. Roemer & C. Hull. 2007. Odor- mediated push-pull pollination in cycads. Science 318(5847): 70. Van Bael, S., P. Bichier, I. Ochoa & R. Greenberg. 2007. Bird diversity in cacao farms and forest fragments of western Panama. Biodivers. & Conservation 16: 2245– 2256. Vovides, A. 1991. Insect symbionts of some Mexican cycads in their natural habitat. Biotropica 23:102– 104. Zherikhen, V. V. & V. G. Gratshev. 1995. A comparative study of the hind wing venation of the superfamily Curculionoidea with phyloge ne tic implications. Pp. 634– 777 in J. Pakaluk & S. A. Slipinski (eds.), Biology, Phylogeny, and Classifi cation of Coleoptera. Papers Celebrating the 80th Birthday of Roy A. Crowson. Vol. 2. Muzeum i Instytut Zoologii PAN, Warsaw, Poland.

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An Overview of Cycad Pollination Studies Irene Terry, William Tang, Alberto S. Taylor Blake, John S. Donaldson, Rita Singh, Andrew P. Vovides, and Angélica Cibrián Jaramillo

Abstract • 353

Resumen • 353

Introduction • 353

Results and Discussion • 357 Cycad Cone Traits • 357 Cone Insects of Cycas in Southeast Asia, India, and Guam • 358 India • 359 Guam • 360 Pollination of Australian Cycads • 363 Pollination of African Cycads • 366 Pollination of American Cycads • 368 Baiting Cycad Pollinators on the Isthmus of Panama • 369 Idioblasts and Cycad Cones • 375 Population- genetic Techniques in the Study of Cycad Pollination • 381

Conclusions • 384

Ac know ledg ments • 386

Literature Cited • 386 -1— 0— +1—

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Abstract

Interest in cycad pollination biology has steadily increased since the 1980s, when the fi rst defi nitive studies demonstrated that specialist insects were the pollen vectors of their cycad host. In this review several aspects of cycad pollination are discussed briefl y, in- cluding the known and putative insect pollinators of cycads from diff erent regions, sam- pling techniques to determine putative pollinator- adult habitats during coning or nonconing season, experiments on cones excluding/including insects or wind to deter- mine their roles in pollination, cone thermogenesis and volatile emission studies, and behavioral studies of insect pollinators. In addition, understudied areas and new research questions and techniques are identifi ed.

Resumen

El interés en la biología de la polinización de cícadas ha aumentado constantemente desde la década de 1980, cuando los primeros estudios defi nitivos demostraron que in- sectos especialistas eran los vectores de polen de sus hospederos de cícadas. En esta re- visión se discuten brevemente varios aspectos de la polinización de cícadas, con la inclusión de polinizadores de cícadas conocidos y putativos de diferentes regiones, técni- cas de muestreo para determinar polinizadores adultos putativos en hábitats durante la época de formación o no de conos de polinización , experimentos sobre conos que ex- cluyen/incluyen insectos o viento para determinar su papels en la polinización, estudios de la termogénesis y emisiones volátiles del cono, y estudios del comportamiento de los insectos polinizadores. Además, se identifi can áreas poco estudiadas y preguntas nove- dosas sobre investigación y técnica.

Introduction

Reports of insect associations with cycad cones began as early as 1772 with Carl Thun- berg’s discovery of a long-snouted weevil infesting seeds of a South African cycad (see Oberprieler, 1989). During the early 20th century there were several more accounts from diff erent regions reporting that cycads have beetles closely associated with their cones —-1 (see reviews by Stevenson et al., 1998; Tang, 2004). Pronouncements by Chamberlain —0 —+1

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Figure 24-1. A. Receptive ovulate cone of Dioon sp. showing tightly overlapping sporophylls except at the base of the cone, where beetles gain entry. B. Part of a cross section of ovulate cone showing a pollen droplet at tip of micropyle.

(1935), a leading authority on gymnosperm biology, that these fi ndings had little to do with cycad pollination, as “gymnosperms” are wind- pollinated, probably suppressed study of this topic for many decades. During the 1980s and early 1990s several studies laid the foundation for a reassessment of cycad pollination, a pro cess that is currently ongoing. The closed nature of the ovulate cones (Fig. 24- 1A) of most cycads, except possibly in the family Cycadaceae, suggested that wind- blown pollen cannot penetrate through the small gaps between sporophylls and reach the inwardly facing micropylar tip, where a pollen droplet is released. Wind- tunnel studies with cones of Dioon edule Lindl., Zamia furfuracea L.f, Z. integrifolia L.f., and Cycas rumphii Miq. (Niklas & Norstog, 1984) confi rmed that wind alone was ineff ec tive as a pollen vector for cycads. The pro- duction of heat and odors was shown to be widespread in cycad cones (Tang, 1987a; Pellmyr et al., 1991), suggesting that cycad pollination systems have similar traits as those used by insect-pollinated fl owering plants. Wind and insect exclusion experiments on ovulate cones and detailed observations of the life cycle and behavior of the cone beetles in two Zamia species (Norstog et al., 1986, 1992; Tang, 1987b; Norstog & Fawcett, -1— 1989) demonstrated the primary role of specialist insects as pollen vectors. These and 0— later studies demonstrated that some of the pollination interactions entail intricate be- +1—

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haviors between the cones and their insect inhabitants, with partic ular cone thermo- genic and volatile cues eliciting specifi c responses in the insects, i.e., movement between pollen and ovulate cones (e.g., Norstog & Fawcett, 1989; Donaldson, 1997; Terry et al., 2007). How these cone characters and cone cues may choreograph pollinator activity is an important focus of current research. Surveys of cycads in Australia (Forster et al., 1994), Africa (Oberprieler, 1995a), Asia (Tang et al., 1999), North and South America (Vovides, 1991a; Tang & O’Brien, 2012) have revealed the widespread presence of certain insect groups in cycad cones as well as more specialized insects that are restricted to cycads on certain landmasses. The most common insects are beetles (Coleoptera)— mainly Curculionoidea and Erotylidae beetles. Erotylidae beetles have been found on cycad cones from all of these continents; however, the erotylid genera on cycads of three of the major regions outlined above are not closely related and appear to have colonized cycad cones in de pen dently (Leschen, 2003; Oberprieler, 2004). Weevils (Curculionoidea), again in diverse lineages, appear to have colonized cycad cones in de pen dently in Africa, Asia, Australia, and Central Amer- ica (Oberprieler, 2004). Boganiidae is another beetle family that has members that are specialists inhabiting cycad cones and these insects are found on cycads in Africa and western Australia. Members of the genus Cycadothrips of the insect order Thysanoptera are sole pollinators of some species of Macrozamia cycads found in Australia. A summary of the important cycad- cone insects and their hosts is displayed in Table 24-1. In the following we present an overview of pollination studies of cycads based on individual pre senta tions during a pollinator workshop at the Eighth International Cycad Conference in January 2008. These summaries focus on par tic ular aspects of pollination biology whereas more comprehensive examinations of cycad cone insects and their cy- cad associations and interactions can be found in one of several reviews (Norstog et al., 1995; Oberprieler, 1995a, 1995b, 2004; Norstog & Nicholls, 1997; Stevenson et al., 1998; Tang, 2004). The following presen ta tion summarizes what is known about polli- nators of cycads in the major geographic regions and then discusses more specifi c topics of cone thermogenesis and volatile emissions, idioblast chemistry and structure of sporo- phyll tissue, manipulation of pollen cones as baits for sampling insects to determine their presence in natural habitats, and the use of population ge netics to analyze wind versus insect pollination. We highlight perplexing questions concerning pollinator-cycad in- —-1 teractions that require focused investigation of various cone chemical and physical traits —0 —+1

529-50504_ch02_2P.indd 355 7/26/12 5:52 PM s & Johnson (2009), ons to be likely candidates (Lepidoptera: (Aeolothripidae) (Nitidulidae) (Nitidulidae) (Nitidulidae) (Nitidulidae) (Boganiidae) (Boganiidae) (Tenebrionidae) (Tenebrionidae) (Biphyllidae) — Carpophilus Carpophilus Cycadothrips Paracucujus Ulomoides Metacucujus Ulomoides Carpophilus Anatrachyntis Cosmopterigidae) Carpophilus Other insects and family in parentheses) (order Biphyllus (Brentidae: (Belidae: Oxycoryninae) — (Belidae: Oxycoryninae) — (Anthribidae: Anthribinae) (Curculionidae: Molytinae) — (Curculionidae: Cossoninae) (Curculionidae: Molytinae) (Curculionidae: Molytinae) — — —— Antliarhinus, Platymerus Antliarhinus, Antliarhinae) Apinotropis Amorphocerus, Porthetes (Curculionidae: Molytinae) Miltotranes Tranes Undet. genus (Curculionidae: Cossoninae) Beetles (family and subfamily in parentheses) Tychiodes Undet. genus — — —— Genus near Pharaxonotha —— cant distances; list is based on Oberprieler Tang (2004), (1995a), (2007), Kono & Tobe Proche¸ . a CeratozamiaDioonMicrocycas Pharaxonotha Pharaxonotha Pharaxonotha Rhopalotria Zamia Pharaxonotha Rhopalotria Macrozamia Xenocryptus Tranes Encephalartos Bowenia Lepidozamia Cycas Hapalips Cycas Cycas Cycas Terry et al. (2009). -1— as pollinators; not included in this table are insect species that appear to be occasionaltor ability visitors, to disperse detritivores pollen for signifi visiting spent cones, or not having the locomo- These are insects that have been detected in high frequency and numbers or have been shown in experiments or detailed observati N. & C. America Africa Australia N. AustraliaN. Guam/Rota Table 24- 1 Beetle and thrips genera found on cycad cones in various regions of the world, that are likely likely that are 1 Beetle and thrips genera found on cycad cones in various regions of the world, 24- Table candidates as pollinators Geographic region AsiaS. Cycad genus Erotylidae a 0— Japan +1—

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and insect responses. One goal of this review is to stimulate more interest in cycad pollination-related research by presenting how cycads serve as interesting models for testing basic biological questions.

Results and Discussion Cycad Cone Traits

Cycad cones exhibit partic ular traits such as odor, heat, and color that occur during the pollination period and are associated with the appearance of cone insect visitors. In most species studied, pollen cones emit odor bouquets that may be species- specifi c (or some- times pollinator specifi c) in their composition (Pellmyr et al., 1991; Terry et al., 2004a, 2004b; Azuma & Kono, 2006; Proche¸s & Johnson, 2009; Suinyuy et al., 2012), and ovu- late cones emit odors that are nearly identical to those of the pollen cones but may vary in the levels and ratio of the volatile components. Ovulate cones therefore are generally thought to mimic pollen cone odors to attract the pollinators, though emitting a weaker signal. In some species, these odor components change or increase dramatically through- out the day or possibly as cones mature (Terry et al., 2004b) and both heat and odor coincide with pollinator movement between cones. Receptive ovulate cones discharge pollen droplets at the tip of the micropyle that trap any insect-deposited pollen (Fig. 24- 1B). These droplets contain nutrients, sugars and amino acids (Tang, 1993b), that may provide a reward to some pollinators (albeit in very small volumes), although such a function has not been demonstrated. Dye-covered pollinators released onto caged cones showed that the insects crawl around inside the ovulate cone and reach the micropylar tips, where some of their load of dye or pollen is trapped by the pollen droplets (Donaldson, 1997; Terry et al., 2005; Proche¸s & Johnson, 2009). Cones of many studied cycad species have a daily thermogenic event that lasts for several hours and occurs daily for a period of up to two weeks (Tang, 1987a, 1993a; Tang et al., 1987; Seymour et al., 2004; Terry et al., 2004b; Roemer et al., 2005, 2008), usu- ally with one or two peaks each day, but this pattern may be moderated by environmen- tal temperature (Tang, 1987a; Roemer et al., 2008). In some species, volatile emissions —-1 greatly increase during thermogenesis. This thermogenic activity results from increased —0 —+1

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aerobic respiration (metabolism) within sporophylls with additional heat energy from the futile dissipation of redox potential via alternative oxidase pathways of the electron transport system rather than the cytochrome pathway (Tang et al., 1987; Skubatz et al., 1993). Although upregulation of uncoupling proteins (UCPs) has been associated with thermogenesis in some plant tissues (Ito & Seymour, 2005), a similar association has not been tested in cycad sporophylls to date. In pollen cones of Zamia furfuracea and Cerato- zamia miqueliana H. Wendl., individual sporophylls display oscillatory heat production (mea sured in μW), with periods of 12– 14 min mea sured during ca. 90 min test times (Skubatz et al., 1993) suggesting that what we mea sure in terms of temperature increase in cones is due to a summation of the individual sporophyll metabolic oscillations. For most cycad species how or whether these traits contribute to the overall cone attractive- ness (or possibly repellency) awaits further research. Raskin et al. (1990) found high levels of salicylic acid in male cones during the elongation and pollen dehiscence phases in two species of Dioon and three species of Encephalartos. This suggests that salicylic acid may be an inducer of thermogenic events in cycads; however, more research is required before the enzymatic or gene tic regulation of this process is understood.

Cone Insects of Cycas in Southeast Asia, India, and Guam

In Asia and islands associated with its continental shelf, insect pollination studies of the one indigenous cycad genus, Cycas, are still in the survey stage for possible insect polli- nators. Of the ca. 100 species of Cycas, just over half are found in mainland Asia and its nearshore islands, and the rest occur in northern Australia, Indonesia, the Philippine Islands, and some of the western Pacifi c Islands, and a single species in Africa and Mada- gascar (Hill, 1994, 1996). Tang et al. (1999) surveyed China, Thailand, and Vietnam and found that erotylid beetles, currently placed in a genus near Pharaxonotha (Leschen, 2003) are widespread in pollen cones of the Cycas species in the taxonomic sections Stangerioides, Indosinenses, and Cycas (Table 24- 1). Beetles of this genus are known polli- nators of Zamia, Dioon, and Ceratozamia in the Americas (Table 24- 1). Weevils (Curcu- lionoidea) in the genus Tychiodes were found only in the more-derived sections -1— Indosinenses and Cycas, and a beetle species in the genus Biphyllus (Biphyllidae) was also 0— found in a number of Cycas species in these two sections (Table 24-1). Cycas revoluta +1—

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Thunb. (section Asiorientales), on the Japanese Island of Yonaguni, has been thoroughly surveyed for cone insects and pollination studies have demonstrated that ovulate cones in close proximity to pollen cones can be pollinated by wind- blown pollen but those farther away require insect vectors, such as Carpophilus chalybeus Murray (Kono & Tobe, 2007).

India

On the Indian subcontinent and its nearshore islands there are eight recognized species of Cycas (in sections Cycas and Indosinenses). Surveys for cone-associated insects have been conducted in several populations of section Cycas species, including C. circinalis L. and C. annaikalensis Rita Singh & P. Radha in the Western Ghats, and C. zeylanica (J. Schust.) A. Lindst. & K. D. Hill on the Andaman and Nicobar Islands. The beetles in the cones of C. zeylanica have been identifi ed as belonging to the genera Tychiodes and Biphyllus (Oberprieler 1995a) (Table 24- 1; Fig. 24-2) and those in the Western Ghats tentatively to a genus near Pharaxonotha, Carcinops troglodytes (Paykull), Tribolium, and a

Figure 24-2. Beetles species collected from pollen cones of Cycas zeylanica. A. Lateral view and B. dorsal anterior view of Tychiodes sp. C. Dorsal and D. dorsal —-1 anterior view of Biphyllus sp. (Scale bar, A, C = 1 mm; B, D = 0.1 mm). —0 —+1

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Figure 24-3. A. Longitudinal section of predehiscent pollen cone of Cycas circina- lis with insect larvae visitors. B. Cycas pectinata pollen sporophylls with Tychiodes weevils. C. Cycas pectinata pollen sporophylls hollowed by beetle feeding.

genus near Tychiodes (Table 24- 1). In the Western Ghats, larvae and adult weevils of the latter genus were seen feeding in large numbers in pollen sporophyll tissues (Fig. 24- 3). These Pharaxonotha-like beetles, coated with pollen, have also been found in ovulate cones.

Guam

The only native cycad species in Guam, Cycas micronesica K. D. Hill, now critically en- dangered due to high mortality of trees and seedlings by the combined eff ects of invasive scale and lepidopteran pests, has also been surveyed for insects. This species belongs in section Cycas, in the rumphii species complex (Hill, 1994, 1996), which is widespread from Africa and Madagascar to Southeast Asia and some western Pacifi c Islands. Several -1— species of this complex, living in Asia and nearshore islands, harbor Tychiodes weevils in 0— their pollen cones (Tang et al., 1999). Very little is known about insects from species in +1—

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the Pacifi c Islands. In Guam, numerous insect species were collected from the cones of C. micronesica, but none of the mainland Asian or nearshore island beetles was found. However, at least two species are regarded as putative pollinators based on their consis- tent presence on pollen cones and ovulate sporophylls: the microlepidopteran Anatra- chyntis sp. (Cosmopterigidae) and several species of Carpophilus (Coleoptera: Nitidulidae) (Terry et al., 2009, 2012). The genus Anatrachyntis is cosmopolitan, and its larvae are known to infest agricultural crops (see Terry et al., 2009). The moths do use pollen cones as nurseries and larvae complete their development on the microsporophyll tissue; how- ever, it is not known whether the nitidulid beetles use either ovulate or pollen cones for larval development (Marler & Muniappan, 2006). None of the Carpophilus species is native to Guam (Ewing & Cline, 2005), but they do exist on the fruits of several crop species, many of which have been introduced to Guam agriculture, all of which suggests that they only very recently became associated with these cycad cones. Nitidulid beetles, including members of Carpophilus, were demonstrated to be pollinators of C. revoluta (Kono & Tobe, 2007), Stangeria eriopus (Kunze) Baill., as well as other angiosperm species (Proche¸s & Johnson, 2009). Insect surveys on cones of C. micronesica of Guam and the adjacent island of Rota in the Mariana Islands have revealed similar insects on these two islands (Terry et al., 2009) and detailed pollination exclusion/inclusion studies are underway. Of other Cycas species in the C. rumphii complex, only a few have been surveyed, includ- ing some Asian C. rumphii species (see above; Tang et al., 1999) with the weevil Tychiodes being found on some species. Cycas seemannii A. Braun in the southwestern Pacifi c Islands has been reported to be wind pollinated but this is based on circumstantial evidence (few insects were found during a limited survey and more seed set occurred on the windward side of the cones) (Keppel, 2001). Of C. rumphii species found in Indonesia, Papua New Guinea, or Solomon Islands, little is known except that Tychiodes rennelli Marshall is de- scribed from the Solomon Islands (Oberprieler, 1995a, 1995b), but the specifi c host is not known. Two other species of Tychiodes with unknown hosts have also been described, one from Japan (T. adamsii Wollaston) and another from the Philippines (T. jansoni Wollaston) (Oberprieler, 1995a, 1995b). Many more surveys and pollination studies (treatments ex- cluding wind, insect or wind and insect and including insects) are needed on both island and mainland Cycas species before we can attempt to piece together the evolution of the pollination systems related to C. rumphii species complex. Based on morphological evi- —-1 dence and Cycas species distributions, Hill (1996) proposed that the fl otation layer of the —0 —+1

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C. rumphii species complex is recent and that Cycas species arrival on the southern Pacifi c Islands is relatively recent. If C. seemannii has recently arrived in this area, it could be that no specifi c pollinators have established yet on these plants. Volatile emissions of four Cycas species have been analyzed, but at diff erent levels of detail; namely C. micronesica from Guam and Rota and from a specimen purchased as “C. micronesica” and grown in a private garden in Queensland (Terry, unpublished ob- servations), C. thouarsii R. Br. ex Gaudich. from Madagascar (Kaiser, 2006), C. revoluta from Japan (Azuma & Kono, 2006), and C. seemannii from New Caledonia (Pellmyr et al., 1991). All these species are members of the C. rumphii complex except for C. revoluta, whose volatiles are diff erent from those of the other three species (Table 24- 2). While both pollen and ovulate cones of C. micronesica from Guam and Rota share several odor components with C. seemannii (Table 24- 2), their odor descriptions are very diff erent, and the two species diff er substantially from the others in both major components and organoleptics. The “C. micronesica” specimen from a private garden is unusual in that it closely resembles C. thouarsii in major components (sampled only once for C. thouarsii but several times for “C. micronesica” in the garden) and organoleptics, suggesting that it may have been mislabeled or its origin confused. Because volatile chemistry can change with cone stage and time of day, the results are only tentative and require more sampling of cones over time. However, the organoleptics of the “C. micronesica” cone from the gar- den was noted to have the same “capsicum” (methoxypyrazine) fragrance with no sweet methyl isovalerate fragrance during afternoons and evenings over several days (Terry, unpublished observations). By contrast, numerous volatile samples of several pollen and ovulate cones of C. micronesica on Guam and Rota did not contain any methoxypyr- azines and the capsicum fragrance was never noted during diff erent times of day or as cones matured. Clearly, more studies of cycad species cone volatiles are needed, with collections from both ovulate and pollen cones over diff erent times of day and during diff erent stages of cone phenology to determine both qualitative and quantitative changes that might refl ect on interaction with cone insects as has been done for some cycad species (Terry et al., 2007; Suinyuy et al., 2012). In summary, more surveys are needed on island and mainland Cycas species as well as Cycas in Asia, Africa, and Australia. In addition, pollination insect/wind exclusion -1— experiments and insect/cone interaction studies are needed for cones of Cycas from the 0— diff erent taxonomic sections of the genus. +1—

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Table 24- 2 Major volatile components reported from pollen and ovulate Cycas cones.

Cycas species Locality Organoleptics Major components Reference

C. micronesica Guam, Rota bubble gum, methyl isovalerate, Terry & Marler, unpubl. pineapple linalool C. ‘micronesica’ Garden in QLD green capsicum methoxypyrazines Terry & Moore, unpubl. C. seemanniii New Caledonia stale, fermented, 2-pentanol, 2- pentyl Pellmyr et al., 1991 pollen cone pollen acetate, 2-pentanone C. seemannii New Caledonia — methyl isovalerate, Pellmyr et al., 1991 ovulate cone 2-pentanol C. thouarsii Madagascar green capsicum methoxypyrazines Kaiser, 2006 C. revoluta Okinawa, Japan unpleasant, estragole Azuma & Kono, 2006 pine

Pollination of Australian Cycads

Australia has a high diversity of cycads, spanning four of the 11 genera and ca. 75 of the 327 species currently known worldwide and representing the families Cycadaceae (genus Cycas) and Zamiaceae (Lepidozamia and Macrozamia). The taxonomic position of the fourth genus, Bowenia, is not clear; some studies have placed Bowenia in the family Stan- geriaceae as sister taxon to Stangeria (e.g., Stevenson, 1990, among others; see Chaw et al., 2005), but most molecular analyses suggest that Stangeria and Bowenia are not sister taxa and are not closely related (Hill et al., 2003; Rai et al., 2003; Chaw et al., 2005; Zagurski et al., 2008). Insects have been found associated with cones of all these genera (Table 24- 1), and most of them are Coleoptera (Ornduff , 1991; Forster et al., 1994; Oberprieler, 1995a, 1995b; Hall et al., 2004; Tang, 2004). Thrips (Thysanoptera) species in the genus Cycado- thrips is the exception, which occurs on some Macrozamia species only (Mound, 1991; Forster et al., 1994; Mound & Terry, 2001). Most of the pollination studies in Australia have focused on Macrozamia (comprising ca. 40 species), but many species have not even been surveyed for insects. Some Macrozamia species are pollinated solely by Tranes weevils (Curculionidae: Molytini), others solely by Cycadothrips, and a few species by both insects (Terry et al., 2005, 2008). To date, there are three species of Cycadothrips, C. chadwicki —-1 Mound on east-coast Macrozamia (Mound, 1991; Forster et al., 1994), C. albrechti Mound & —0 —+1

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Ter r y on M. macdonnellii (F. Muell.ex Miq.) A. DC. near Alice Springs (Mound & Terry, 2001), and C. emmaliami Mound & Marullo on M. riedlei (Gaudich.) C. A. Gardner in southwestern Australia (Mound et al., 1998). In the other Australian cycad genera, Hall et al. (2004) conducted wind and insect exclusion experiments on Lepidozamia peroff skyana and showed that it is pollinated by a host- specifi c Tranes weevil. Wilson (2002) reported that both species of Bowenia, B. spectabilis Hook. ex Hook.f. and B. serrulata (W. Bull) Chamb., are pollinated exclusively by the Molytini weevils, Miltotranes prosternalis (Lea) and M. subopaca (Lea), respectively. On Cycas species, several beetles have been found as- sociated with pollen cones, Hapalips sp. (Languriidae), Ulomoides australis (Carter) (Tene- brionidae), an undetermined Cossoninae, and a Nitidulidae (Ornduff , 1991; Forster et al. 1994). To date no pollinator exclusion/inclusion studies have been reported for any the Australian Cycas sp. and most Australian Cycas species (ca. 30) need to be surveyed for insects or have their insect visitors identifi ed. For some Macrozamia species, the identity of the pollinator remains a mystery and much more investigation is needed. In south- central Queensland, M. moorei F. Muell. was surveyed for pollinators in three diff erent years. In 2001 and 2002, the beetle Ulo- moides australis (Tenebrionidae) was found on both pollen and ovulate cones in three distinct M. moorei populations (Terry et al., 2004a), but in 2006, during a mass- coning year, no insects were found or trapped on cones (Terry, unpublished observations). An- other species, M. fearnsidei D. L. Jones, was also surveyed in three diff erent years, yield- ing no beetles or thrips, but in 2008, small Lepidoptera, Conobrosis sp. (Oecophoridae) moths were caught fl ying around the pollen cones (Terry, unpublished observations). Conobrosis larvae are associated with pollen cones of many Macrozamia species (Chad- wick, 1993; Mound & Terry, 2001; Terry, 2001) and are considered detritivores but not pollinators. In M. platyrhachis F. M. Bailey, only a few Cycadothrips specimens were trapped on cones in one year but none in the following year at some of the same sites (Terry et al., 2008). In addition to these Macrozamia species, only about one third of the ca. 40 Macrozamia species have been surveyed for insects, or tested for pollinator eff ec- tiveness with exclusion/inclusion tests, or sampled for volatile emissions, including any qualitative or quantitative changes during cone maturation. Thermogenesis has been well characterized and analyzed for a few Macrozamia spe- -1— cies (Tang, 1987a; Terry et al., 2004b; Roemer et al., 2005, 2008). Respirometry studies 0— of cones in the fi eld (Seymour et al., 2004) (Fig. 24-4A, 24- 4B) and the laboratory (Roe- +1—

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Figure 24-4. A. Preparation of Macrozamia machinii cones for respirometry mea sure ments. B. Macrozamia machinii cones enclosed in respiratory chamber to measure diel oxygen consump- tion during thermogensis in situ.

mer et al., unpublished observations) show that thermogenic cones increase their me- tabolism. Basic energy balance models can accurately predict the metabolism from mea sured ambient and cone temperatures (Roemer et al., 2005, 2008, 2012). Because it is diffi cult to take respiratory measure ments in closed chambers and observe normal in- sect behavior during this period, the model predictions are useful to examine the timing of metabolic activity, volatile release, and thermogenesis relative to insect movement. Insect movement between cones appears to be correlated with the increase in both cone temperature and volatile emissions (Terry et al., 2004b). In M. lucida L. A. S. Johnson and M. macleayi Miq., cone volatiles attract the pollinating thrips, Cycadothrips chadwicki Mound, at low concentrations, but emissions of some volatiles, particularly the acyclic monoter- pene β– myrcene, can reach very high and repellent levels such that the pollinators leave the pollen cones en masse (Terry et al., 2007). Light and temperature also play a role in this exodus (Terry, unpublished observations). This syndrome has been characterized as a “push- pull” pollination system (Terry et al., 2007). Whether or not this phenome- —-1 non functions in other cycad pollinator interactions requires more testing. Behavioral —0 —+1

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observations of Tranes sp. on M. machinii P. I. Forst. & D. J. Jones suggest that the adult insects will move out of cones after dark if cones and ambient conditions are warm, but not under cold ambient or cone temperatures (Terry, unpublished observations). For Porthetes sp. on Encephalartos villosus Lem. the odor emissions are higher in the evening, but the beetles are attracted to the odor at all levels tested (see Suinyuy et al., 2012).

Pollination of African Cycads

Three genera of cycads, Cycas, Stangeria, and Encephalartos, belonging to the families, Cycadaceae, Stangeriaceae, and Zamiaceae, respectively, are found in Africa with a total of ca. 70 species. There is only one species of Cycas in Africa, and Stangeria is monotypic with the majority of species in the genus Encephalartos. Studies by Pearson (1906) and Rattray (1913) provided early evidence of insects associated with cones of the South Af- rican Encephalartos villosus and that these insects likely acted as pollinators of this cycad species. Only much later were defi nitive tests conducted that demonstrated that a diver- sity of beetles is involved in the pollination of Encephalartos. Metacucujus encephalarti En- drody Younga (Boganiidae) and an undescribed erotylid beetle pollinate E. cycadifolius (Jacq.) Lehm. (Donaldson et al., 1995). Suinyuy et al.(2009) demonstrated with pollina- tion tests that one of the same beetles, M. encephalarti, as well as Erotylidae sp. nov. (Cu- cujoideae) and Porthetes hispidus (Boheman) (Curculionidae) can vector pollen and pollinate E. friderici-guilielmi Lehm. Several beetles are associated with E. villosus cones and were tested for their pollination potential (Donaldson, 1997). The most effi cient pollinator was found to be a species of Porthetes, while Antliarhinus zamiae (Thunberg) and an undescribed species of Erotylidae may play a minor role (Table 24- 1). Antliarhinus zamiae is also an obligate ovule predator (Oberprieler, 1995a; Donaldson, 1997), sug- gesting that any benefi t from its pollination will be off set by some potential seed loss due to ovule damage. Proche¸s & Johnson (2009) demonstrated through pollinator exclusion tests and release of dye- coated insects onto caged receptive ovulate cones that several species of sap beetles (Nitidulidae) are capable of pollinating S. eriopus. Two of the pol- linating sap beetles are in the genus Carpophilus, and members of this genus are known to pollinate C. revoluta and are associated with C. micronesica cones (see above), but they -1— also pollinate many species of angiosperms and are associated with the fruits of many 0— angiosperm crops (Proche¸s & Johnson, 2009). +1—

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Many unanswered questions remain in relation to cycad pollination in general and of African cycads in partic ular. For one, there are no defi nitive studies showing whether pollinators cospeciated with their hosts. Further, among African cycads no pollination studies have been undertaken in countries outside of South Africa, which harbors about half of the African cycad species, and almost no studies have been conducted on savan- nah cycads, which have the highest species richness. Some African cycads share genera of cone beetles, specifi cally Amorphocerus, Porthetes, Platymerus, Antliarhinus, and Xe- nocryptus-like erotylids. Co- evolutionary patterns between cycads and their pollinators can be detected only by comparing the phyloge netic relationships of plants and insects and by identifying the cycad cone traits that mediate pollinator behavior. A recent molecular- systematic study of Amorphocerini weevils (genera Porthetes and Amorpho- cerus) associated with Encephalartos cones (Downie et al., 2008) largely confi rmed earlier phylogene tic results based on morphology (Oberprieler, 1996) that these weevils have likely colonized Encephalartos relatively recently and that co-evolution between them and their host species has probably occurred both by cospeciation (among closely related cycad species) and by host shifting onto more distantly related but geograph i cally close Encephalartos species. Downie et al. (2008) concluded that much more research on both Encephalartos species and their associated cone insects is needed to thoroughly investigate these questions. Very little is also known about insect responses to cone cues of African cycads, so as to determine how the pollinators are guided between cones, and very little has been published on relevant cone traits except for studies of thermogenesis and volatile emis- sion in Encephalartos villosus (Donaldson, 1997; Suinyuy et al., 2012) and of odor compo- nents of E. altensteinii Lehm. pollen cones (Pellmyr et al., 1991). In E. villosus, pollen and ovulate cones emit similar odors, the major components being eucalyptol and a me- thoxypyrazine, and numerous minor volatiles (Suinyuy et al., 2012). The movement activity of the Porthetes species associated E. villosus coincides with peak cone tempera- tures and volatile emissions. Cone volatiles do vary throughout the day, with lower emissions during the morning and higher emissions in the eve ning, but the levels emit- ted always attract the pollinator, suggesting that other cues are responsible for motivat- ing the pollinating beetles to leave their pollen cones. More research is needed to analyze the role of individual components in attraction. In addition, nothing is yet —-1 known about the function of the antennal pits found in Porthetes (Oberprieler, 1996), —0 —+1

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which may play a role in the detection of specifi c volatiles. In E. altensteinii, the major volatile of pollen and ovulate cones was found to be 1,3- octadiene, comprising ca. 88% of total emissions, with all other components accounting for ca. 4% or less of the total emissions (Pellmyr et al., 1991). Much more information is needed on cone fragrance chemistry of other Encephalartos species and how volatile emissions and chemistries vary during diff erent cone stages and time of day.

Pollination of American Cycads

There are four genera of cycads in the New World, all endemic, with well over 100 rec- ognized species. This is a level of diversity equivalent to or exceeding that found in Asia, Australia, or Africa, yet in comparison to these other regions, insect diversity at the level of genus and family in cones of New World cycads, is relatively low. Surveys have re- vealed only two beetle genera, Pharaxonotha (Cucujoidea: Erotylidae) and Rhopalotria (Curculionoidea: Belidae). Pharaxonotha is found in the cones of all four genera, Cerato- zamia, Dioon, Microcycas, and Zamia (Vovides, 1991a; Chaves & Genaro, 2005). The pres- ence of a close relative of Pharaxonotha in Asian Cycas (Leschen, 2003) suggests that the cone fauna of the New World may once have been part of a broader Laurasian cycad cone fauna. Rhopalotria is found in Dioon and in part of the range of Zamia (Tang & O’Brien, 2011). Its apparent absence in the peripheral range of Zamia, in the more distant islands of the Greater Antilles and portions of Panama and South America, coupled with a mor- phological analysis by Oberprieler (2004), suggests that Rhopalotria has evolved and colo- nized New World cycads relatively recently. Both Pharaxonotha and Rhopalotria have been demonstrated to pollinate two species of Zamia, Z. pumila L. and Z. furfuracea, in wind/insect exclusion experiments and detailed observations of the life cycles of the beetles (Norstog et al., 1986, 1992; Tang, 1987b; Norstog & Fawcett, 1989; ). Lepidoptera larvae, in the families Blastobasidae, Cosmopterigidae, and Oecophoridae, have been collected on cones of a few Zamia species (Terry et al., 2011); however, their role in pol- lination, if any, awaits further research. What is the cause of this relatively low diversity pattern in New World cycad cone insects? Part of the reason may lie in restricted geographic distributions of some New -1— World cycad genera, now or in the past. Ceratozamia and Dioon are restricted to Meso- 0— america and have patchy distributions ranging from Mexico to Honduras. In the Pleisto- +1—

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cene this distribution was even more limited and the current ranges of these genera may be the result of recent expansion from refugia (Gregory & Chemnick, 2004; Vovides et al., 2004). Similarly, Microcycas is currently confi ned to a small region in western Cuba. Contraction of geographic ranges and concomitant reductions in population size may have lead to extinction among cycad cone insects at the genus and family levels. The recent expansion of these ranges may have allowed speciation in surviving cone insect genera, but there may not have been enough time to allow for recolonization of other insect families or genera. Unlike other New World cycad genera, Zamia is very diverse, with some 70 species, and a distribution ranging from Mexico and Florida to Bolivia. Why does this genus not have a greater variety of cone insects? Perhaps part of the reason lies in the small size of Zamia male cones compared to other cycad genera. Typically less than 10 cm long, excluding their peduncles, and often forming singly or in small numbers, male Zamia cones are a small food source compared with those in other cycads. Also, most Zamia inhabit forest or other mesic habitats where spent cones decay rapidly. These two factors make them a relatively inconspicuous and ephemeral food source and thus a less likely target for colonization by insects compared to cycad cones in regions such as Africa and Australia, where habitats tend to be drier and cycad cones much larger. Small size may also limit the number of available niches within a cone and limit the number of insects that can coexist on this resource. Due to the numerous po liti cal boundaries that cross the geo graphi cal range of New World cycads, surveys of cone insects has been slow in the New World and most research in pollination biology has been conducted in Florida. More fi eldwork and analysis of cone biology need to be carried out throughout the region for a more-complete assessment of pollinator diversity and pollination mechanisms in New World cycads. Such surveys will determine whether the diversity patterns currently seen are real or merely a sampling artifact.

Baiting Cycad Pollinators on the Isthmus of Panama

Pollinators, either confi rmed by exclusion experiments or assumed from sampling de- hiscing pollen cones in natural populations, have been collected from individuals of the 16 described Zamia species, and one pending description of Zamia, known to occur in —-1 Panama (Taylor B., 2002, 2007, unpublished observations). The insects found on cones —0 —+1

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are Pharaxonotha sp. clavicorn beetles (found on all species) and Rhopalotria sp. weevils (found on seven species). An eff ective technique to sample putative pollinators has been to take dehiscing pollen cones (with no insects present) to natural habitats as baits during the coning season or in the “off - season” (no pollen or ovulate cones present in the popu- lation). Either small potted plants with dehiscing pollen cones or freshly excised pollen cones from natural populations or nursery-/garden- grown plants (Fig. 24-5A, 24- 5B) in natural habitats (Fig. 24- 5C) have been used. Kept in a water- absorbent material, ex- cised cones continued to dehisce (Fig. 24-5A) on the plant as has been shown by many other cycad species (Tang, 1987a; Roemer et al., 2008). Cones were retrieved and exam- ined for insects after only two days in the fi eld because cones of Panamanian Zamia are viable with adult insects for only two to three days. This technique has been tested in many populations and species of Zamia on the Isthmus of Panama (Table 24-3). This bait- sampling technique has revealed intriguing aspects about putative polli- nators in Panamanian Zamia species. For one, these “surrogate cones” have successfully attracted adult Pharaxonotha beetles and Rhopalotria weevils (Fig. 24- 5D) in almost every Zamia population tested (Table 24-3). Rhopalotria has not been found on cones of Z. acuminata Oers. ex Dyer in its native habitat. Another striking result is that bait cones have attracted adult pollinators of both genera throughout the year even in the off - season in several Zamia species. Bait traps of an excised Z. obliqua A. Braun cone from a nursery in Panama City attracted beetles in a population of Z. obliqua in the Darién area in the far- eastern part of Panama (Fig. 24-5C; Table 24- 3). In another experiment, Z. aff . elegantissima was sampled throughout the year with pollen-cone baits taken from garden-grown Z. acuminata, Z. elegantissima Schutzman, Vovides & Adams, Z. aff . elegantissima, Z. obliqua, and Z. cunaria Dressler & D. W. Stev. (Table 24- 3). The species used as bait during a partic ular sample period depended on which Zamia species had pollen cones. Except for Z. cunaria, bait cones of these species collectively attracted many individuals of Pharaxonotha during every month, including ten off - season months. Rhopalotria, however, was trapped only during seven months, including fi ve off - season months. During three of these off - season months, only fi ve Rhopalotria adults were collected. Trapping adults during the off - season suggests one of several possibilities: these putative pollinators are present as adults, possibly in a repro- -1— ductive diapause in the off - season “waiting” for a cone; they are diapausing as pupae 0— ready to eclose in the presence of a cone; or they are active adults on an alternate host. +1—

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Figure 24-5. A. Cut dehiscing pollen cone from nursery- germinated Zamia acuminata placed in water- absorbing material and used as bait to attract pollinators. B. Selection of dehiscing pollen cone of nursery-grown Z. acuminata to be used as bait or this potted plant with multiple cones can be used as bait plant. C. Bait cone set up at Z. obliqua site in the Darién area of Pan- —-1 ama. D. Beetles trapped on bait cones: Pharaxonotha (two beetles in upper panels), and Rho- —0 palotria (lower panel). (Scale bars = 1 mm). —+1

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Table 24- 3 Zamia speciesa cones tested for attracting putative pollinators in diff erent Zamia species habitats.

Insects found on bait cone Zamia species Zamia species bait b Sourced habitat Pharaxonotha Rhopalotria OSe CSe

Z. obliqua nursery Z. obliqua Yes Yes Yes Z. obliqua nursery Z. dressleri Yes Yes Yes Z. obliqua nursery Z. aff . elegantissima Yes Yes Yes Z. acuminatac garden Z. aff . elegantissima Yes Yes Yes Yes Z. acuminatac habitat Z. dressleri Yes Yes Yes Z. acuminatac habitat Z. pseudoparasitica No No Yes Z. acuminata habitat Z. aff . elegantissima Yes Yes Yes Yes Z. ipetiensisc habitat Z. cunaria Yes No Yes Z. aff . elegantissima garden Z. aff . elegantissima Yes Yes Yes Yes Z. aff . elegantissimac garden Z. dressleri Yes No Yes Z. elegantissima garden Z. aff . elegantissima Yes Yes Yes Z. dressleri habitat Z. elegantissima Yes Yes Yes Z. dressleri habitat Z. aff . elegantissima Yes Yes Yes Z. cunaria habitat Z. aff . elegantissima No No Yes Z. pseudomonticola habitat Z. dressleri Yes Yes Yes

aSpecies authority of species not mentioned in text: Z. pseudoparasitica Yates in Seem. bCones are excised from plant unless otherwise noted. cA few cones were on potted plant rather than excised cone. dSource of bait cone refers to where the plant with the cone was located; however, some plants in gardens were obtained from the native habitat or are of unknown origin. eCS = Coning Season, OS = Off -Season.

Although it has been suggested that these beetles might pollinate palms (D. Stevenson, pers. comm.), this is unlikely because pollinators of neotropical palms are known to be species of derelomine weevils (Schatz, 1990; Listabarth, 2001; Berry & Gorchov, 2004; Franz, 2005; Franz & Valente, 2006). At present, we have no data on the cycad beetles pollinating or utilizing other plants. It is possible that pollinators are ready to eclose from pupae on or in the ground (Figs. 24- 6A, 24-6B). Rhopalotria diapause in pupa cases in old dehisced pollen cones (Fig. 24- 6B) in natural populations, but not Pharaxonotha. Although Pharaxonotha larvae (Fig. 24-6C) are only found in dehiscing cones, the larvae can pupate in the ground (Fawcett & Norstog, 1993). Further research is needed on the -1— basic biology and life cycle of these pollinators to determine whether adults are already 0— present or they eclose in the presence of bait cones during the off - season. +1—

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In natural populations of Zamia that have both pollinators, more individuals of Pharaxonotha than Rhopalotria were collected from cones during the pollination season. This was also true for bait cones. Pharaxonotha was also collected for a longer period dur- ing the year than the weevil. This indicates that Pharaxonotha is more abundant in natu- ral populations (or perhaps it is more attracted to these cones) and it may have a shorter diapause time (or the pupae take less time to eclose than the weevils when cones are present). If both insects are equally eff ective as pollinators, then Pharaxonotha is likely the principal pollinator of Zamia in the populations studied. The bait technique has attracted insects to cones from a diff erent species than that of the population in which pollinators are sought (Table 24-3). This indicates that these cycad species share some similar cone traits that attract the same pollinators. Whether or not the insects caught on bait cones are the same species of beetle as found on natural cones is currently being examined. However, other data suggest that cone traits across some Zamia species may be similar enough to attract the same species of beetle. A cycad nursery in central Panama, ca. 2 km from a Z. aff . elegantissima population, has many individuals of several other Zamia species that are far removed (more than 25 km for Z. cunaria, the closest, and more than 120 km for Z. ipetiensis D. W. Stev.) from their natural populations. Ovulate cones are produced throughout the year and attract polli- nators that are assumed to be from the nearby natural Z. aff . elegantissima cycad popula- tion. (Alternatively, there may be beetles that are now resident in the nursery so studies are underway to compare beetles from the nursery and from the nearby natural Z. aff . elegantissima cycad population.) Here, individuals of Z. obliqua, Z. cunaria, and Z. elegan- tissima produce viable seeds (Figs. 24-6D, 24- 6E). The beetles that arrive on the ovulate cones could be carrying pollen from any of the species in the nursery, or a mixture of species pollen, and pollinating the same or diff erent species ovulate cones. However, successful pollination appears to have been between cones of the same species of Zamia, with the exception of Z. elegantissima, the progeny of which could be a possible hybrid between this species and Z. aff . elegantissima. These results suggest that cone cues be- tween these Panamanian Zamia species are similar, beetle host specifi city is low, and that there is the possibility of cross pollination of some species. One other experiment also suggests that pollen-cone attractants are similar among some species: bait cones from one of the western groups of Zamia (Z. pseudomonticola L. D. Gómez) also attract beetles —-1 (both Rhopalotria and Pharaxonotha) in a population of the eastern groups (Z. dressleri —0 —+1

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Figure 24-6. A. Old Zamia aff . elegantissima pollen cone with beetle exit holes. B. Rhopatotria weevil larva in hard opened pupa case. C. Pharaxonotha beetle larvae from natural population. D, E. Viable seeds respectively with embryos of Z. obliqua and Z. elegantissima plants in a nursery in central Panama.

D. W. Stev.). This is despite the fact that Rhopalotria groups of the western Panamanian cycad species are taxonomically distinct from those of the central and eastern popula- tions (Tang & O’Brien, 2011). Another indication of a lack of host- specifi c cone traits among some species is found with dehiscing cones of Z. acuminata, which attract only a species of Pharaxonotha (the only pollinator known for Z. acuminata) in its natural popu- -1— lation in north- central Panama, but attract large numbers of Pharaxonotha plus Rhopalo- 0— tria on bait cones from this population in other natural Zamia, Z. dressleri, and Z. aff . +1—

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elegantissima populations in which both pollinators have been extracted from native de- hiscing cones (Taylor, unpublished observations). In summary, the bait technique has been a useful sampling tool for pollinators of Panamanian cycads, it has revealed interesting questions about cycad cone attractions, and it has raised many new questions about the biology and lifecycle of Pharaxonotha and Rhopalotria pollinators. Another application of this baiting method is to search for and collect pollinators in cycad species where the pollinator is not known. The method also can be used to seek out the presence of cycads in places where natural populations are likely but have not been found (e.g., in parts of the Darién area of eastern Panama) or in areas that are diffi cult to explore. The absence of pollinators during the off - season with some experiments (Table 24-3) may be due to the lack of pollinator adults during that period, so care is needed in interpreting the data. Based on these results, bait cones should be set up during every month of one year to ensure getting all possible pollinators. In addition, we fi nd that small potted plants grown in the nursery off er advantages to excis- ing cones from fi eld populations: potted plants usually have many cones that mature se- quentially that can be used to obtain samples over multiple periods, they help preserve natural cones in habitats, and natural cones are not available during much of the year.

Idioblasts and Cycad Cones

The cycad Zamia furfuracea is pollinated by the weevil Rhopalotria mollis (Sharp), which completes its life cycle in the pollen cones of the cycad and eff ectively pollinates its ovu- late cones (Norstog et al., 1986). Norstog & Fawcett (1989) characterized the internal structure and chemistry of this weevil and cone parenchyma tissue using stains specifi c for starch, lipids, carbohydrate, and protein. They discovered idioblasts (gold-cells sensu D. Stevenson), specialized storage cells diff ering from surrounding cells, in sporophyll tissue that remain intact in the pollen cone but in the ovulate cone appear to release their content a few days before receptivity (Fig. 24- 7). The idioblasts stained for high levels of protein in pollen cones, but in ovulate cones the remaining few intact idioblasts stained for lower levels of protein and higher ones of carbohydrates and lipids. Based on staining and starch- grain content, the microsporophyll parenchyma contains high levels of starch, whereas megasporophyll tissue has little starch but more lipids. From these results, Nor- —-1 stog & Fawcett (1989) proposed that the diff erences in pollen and ovulate cone tissues —0 —+1

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Figure 24-7. Sporophyll sections of Zamia furfuracea. A. Dehiscing pollen cone, showing ex-

tensive starch grains in parenchyma (I2KI [Lugol’s] test) and intact idioblasts (gold cells) with no stain. B. Dehiscing pollen cone, showing idioblasts stained for protein with Ninhydrin- Schiff (NIN) +ve. C. Prereceptive ovulate cone, showing intact idioblasts stained with NIN

+ve. D. Receptive ovulate cone, showing no starch (I2KI test) and idioblasts breaking down (unstained). E. Receptive ovulate cone, showing breakdown of idioblasts, stained for protein with NIN +ve (from Norstog & Fawcett, 1989).

could be related to a complex pollination syndrome such that toxins are sequestered in pollen tissues within these idioblasts but are dispersed in ovulate sporophyll tissue. This phenomenon could be responsible for restricting pollinator feeding to pollen- cone tissue and complete avoidance of ovulate-cone tissue. The weevil larvae may deal with toxins in pollen cones by either feeding on sporophyll tissue around the idioblasts and/or in- gesting these intact (as found in the hindgut of larvae after feeding on pollen tissue). Expanding on these studies, Vovides et al. (1993) tracked changes in idioblasts in the parenchyma of Z. furfuracea cones throughout cone maturation and examined the chem- istry of the idioblasts. Throughout pollen-cone development to pollen maturation, idio- blasts appeared structurally intact, and feeding weevils consumed much of the starch- rich -1— parenchyma tissue, including idioblasts. During this activity no appreciable change in 0— structure or staining reactions of pollen- cone idioblasts was detected. Prior to receptiv- +1—

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ity, ovulate cone tissue contained idioblasts that resembled those of pollen cones, but thereafter many ovulate- cone idioblasts showed marked changes in structure and break- down of idioblasts, which was not induced by weevil feeding as it occurred in the absence of weevils (Table 24-4). In addition, idioblasts in both pollen and ovulate cones appear to contain toxic compounds, including at least one neurotoxin, 2-amino- 3-(methylamino) propanoic acid (BMAA), a non- protein amino acid (Table 24-5). The results of the stud- ies by Vovides et al. (1993) therefore corroborate the suggestions of Norstog & Fawcett (1989) that idioblast changes in ovulate cones may function as a defense of ovulate- cone resources against predation by animals, including pollinating weevils, and that this may be aff ected by the mobilization of toxins from idioblasts. This mechanism would also protect the ovules and developing seeds from predation. Since the pollen- cone tissue maintains the integrity of these idioblasts and the hindgut of weevil larvae contains similarly intact idioblasts after feeding on such tissue, these toxins are likely stored in sequestered form in idioblasts in the pollen cones. Further, since pupa cases of the wee- vils, formed largely from larval excreta, contained three to six times the amount of BMAA as found in pollen or ovulate cone tissues (Vovides et al., 1993; see Table 24- 5), the cycad toxins would also aff ord protection from predation to the weevil during this vulnerable stage in the pollen- cone bagasse. Vovides (1991b) examined other cycad species to determine whether these show a similar idioblast structure as their cones mature, as exhibited by Zamia furfuracea prior to

Table 24- 4 Mea sure ments (μm) of idioblast diameter at diff erent developmental stages of pollen and ovulate cones of Zamia furfuracea (N = 25) (from Vovides et al., 1993, with permission, Wiley- Blackwell Publishing).

Developmental stage Early Dehiscence Receptive Post- pollination

Pollen 47.0 ± 2.6 69.5 ± 3.7 —a —a Ovulate 37.0 ± 2.4 —b 58.7 ± 2.2 120.7 ± 6.7

aFollowing dehiscence, parenchyma is consumed by weevil larvae or becomes senescent. —-1 bOvulate cones are receptive at the time pollen cones are dehiscent. —0 —+1

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Table 24- 5 2- Amino- 3-(methylamino)propanoic acid (BMAA) content of tissues of Zamia furfuracea and of larvae and pupa cases of Rhopalotria mollis, a faithful pollinator of Z. furfuracea (from Vovides et al., 1993, with permission, Wiley- Blackwell Publishing).

Sample Name BMAA (μg g−1) (wet wt.) BMAA (μg g−1) (dry wt.)

Pollen cone, 1 9.2 — Pollen cone, 2 5.7 — Ovulate cone 11.2 — R. mollis, larvae 9.0 ± 1.4 (N = 5) — R. mollis, pupa cases — 34.2 ± 21.7 (N = 3) Idioblasts Pellet BMAA (μg g- 1) Supernatant BMAA (μg g- 1) Pollen 1a 252.0 186.0 Pollen 2b 207.0 189.0 Ovulatec 362.0 193.0

aPollen 1, predehiscent stage. bPollen 2, late predehiscent cone. cOvulate receptive cone.

and during the dehiscence phase. Of the 16 species studied, most showed a similar devel- opmental and cone- type related pattern as discovered in Z. furfuracea (Table 24- 6; Fig. 24- 8A). In contrast, Cycas rumphii and Stangeria eriopus did not contain idioblasts in their sporophyll parenchyma tissue, while in Macrozamia lucida idioblasts occurred in the sporo- phylls of both cone types, and released their content during the pollination phase (Table 24-6; Fig. 24- 8B–D). These latter species appear to have pollination syndromes diff erent from that of Z. furfuracea. For M. lucida, it is pollinated by the thrips Cycadothrips chadwicki, which feeds only on pollen and not on sporophyll tissue (Terry et al., 2005), so there is no need for the cycad to restrict it from feeding on sporophyll tissue but it may restrict other insects that are cone predators. Within the C. rumphii complex, more species should be examined for their cone idioblasts to determine whether species that have Tychiodes beetles associated with the pollen cones diff er from species that have nonweevil insects feeding on pollen cones (see section on C. micronesica from Guam) or no insects associated with cones. Experimental studies demonstrated that nitidulid beetles are capable of pollinating S. erio- pus but the beetles appear to feed only slightly on pollen cones and not sporophyll tissue, -1— and the cones show less volatile emissions and heat production (Proche¸s & Johnson, 2009). 0— This is in agreement with cone idioblast data of Vovides (1991b) who suggested a diff erent +1—

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pollination system in Stangeria to that of Z. furfuracea owing to the lack of idioblasts in both pollen and ovulate sporophyll parenchyma in S. eriopus but not so in the rest of the Zamia- ceae (see Vovides, 1991b, fi gs. 16, 17). Further investigation of what traits may inhibit feeding on sporophyll tissues and the role of idioblasts is needed. This work raises the prospect that such changes in idioblast structure and chemistry may be of pivotal importance in regulating pollinator activity in at least those cycad species pollinated by weevils and other beetles that feed on tissues of the pollen cone. Volatiles of pollen and ovulate cones are likely to attract pollinators, but in cycad species in which the pollinator is dependent on the pollen cone for its life cycle, it may only visit the ovulate

Table 24- 6 Sporophyll idioblast data from 11 species in ten cycad genera (modifi ed from Vovides, 1991b, 1991©, University of Chicago Press).

Idioblast Species Cone sex breakdown Starch Ninhydrin- Schiffd

Bowenia spectabilis Mnondc +ve Ceratozamia mexicanaa Mnoyes +ve C. mexicanaa Fnoyes +ve Cycas rumphii Mnoyes − ve C. rumphii Fnoyes +ve Dioon spinulosumab Mnoyes +ve D. spinulosumab Fyesno +ve Encephalartos Mnoyes +ve hildebrandtiiab E. hildebrandtiiab Fyesno +ve Lepidozamia hopeia Fyesno +ve Macrozamia lucida Myesyes +ve M. lucidab Fyesno +ve Microcycas calocomaa Myesndc − ve M. calocomaa Fyesndc +ve Stangeria eriopus Mnoyes − ve S. eriopus Fyesno +ve Zamia furfuraceab Mnoyes +ve Z. furfuraceab Fyesno +ve Z. restrepoia Fyesyes − ve

aSpecies authorities of species not mentioned in text: Ceratozamia mexicana Brongn.; Dioon spinulosum Dyer ex Eichler; Encephalartos hildebrandtii A. Braun & C. D. Bouché; Lepidozamia hopei (W. Hill) Regel; Microcycas calocoma (Miq.) A. DC.; Zamia restrepoi (D. W. Stev.) A. Lindstr. bBold = cones have Zamia furfuracea idioblast breakdown pattern. cnd = starch not detected. —-1 dNinhydrin- Schiff , test for protein. —0 —+1

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cone long enough to deliver the pollen it carries on its body and may be repelled by the presence of dispersed toxins if it attempts to feed on sporophyll tissue. Par tic ular pollina- tion syndromes thus may be associated with diff erences in idioblast presence in pollen and ovulate cone tissue, idioblast chemistry, or idioblast changes throughout cone maturity. Unfortunately too few details are known about the pollination systems of most cycads

Figure 24-8. Sporophyll sections of diff erent cycad species. A. Lepidozamia hopei (ovulate), NIN +ve, protein positive, secretory idioblast (large arrow) and tannin- fi lled idioblast near epidermis (ferrous sulfate localization); insert a. detail of protein- stained Ninhydrin- Schiff (NIN) +ve idioblast. B. Macrozamia lucida (ovulate), NIN +ve, secretory idioblasts (arrows); insert a. pollen, NIN +ve idioblast, insert b. tannin-fi lled idioblast (ferrous sulfate localiza- tion). C. Stangeria eriopus (pollen), tannin- fi lled epidermal idioblast (ferrous sulfate localiza-

tion); insert a. starch-fi lled parenchyma (arrowed), I2KI stain; insert b. pollen, tannin- fi lled epidermal idioblasts (FS stain), note lack of parenchyma idioblasts. D. Stangeria eriopus (ovu- late), mostly NIN - ve epidermal idioblasts; insert a. detail; insert b. NIN +ve, epidermal glan- -1— dular hair, note lack of parenchyma idioblasts (modifi ed from Vovides, 1991b). Scale bar = 50 μ. 0— (From Vovides, 1991b, fi gs. 13, 14, 16, and 17, © 1991 by University of Chicago Press.) +1—

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relative to this hypothesis. For example, in some Encephalartos species, such as E. villosus, the weevil pollinator (a species of Porthetes) does occasionally develop in ovulate cone tis- sues (Donaldson, 1997), and in Macrozamia machinii adult Tranes weevils sometimes feed on and damage megasporophyll tissue (Terry et al., 2005). However, the occurrence of idio- blasts and toxins has not been investigated in these cycad species. Clearly more studies of both idioblast structure of many more cycads and the specifi c feeding patterns of insect visitors and pollinators on both pollen and ovulate cones are needed to determine how these insects deal with, by sequestering, tolerating, or detoxifying, these idioblast toxins.

Population- genetic Techniques in the Study of Cycad Pollination

The role that wind may play in cycad pollination has not been thoroughly investigated in the genus Cycas. In some species, the megasporophylls separate enough to expose re- ceptive ovules to wind- blown pollen, so that wind could play a primary (if not exclusive) role in the pollination of such species, in contrast to cycad genera whose ovules are en- closed inside cones. Keppel (2001) reported little evidence for insect pollination in C. seemannii as based on limited circumstantial evidence (lack of insects on cones, higher seed set on leeward side of cones), but he also stressed the need for more extensive sur- veys of cone insects in this species. In contrast, many Asian species of Cycas have insects associated with their cones (Tang et al., 1999; Tang, 2004). For C. revoluta on the Japa- nese island of Yanaguni, Kono & Tobe (2007) demonstrated that wind can transport pollen over a distance of only a few meters from the pollen cone, whereas the nitidulid beetle Carpophilus chalybeus pollinates ovules of megasporophylls farther away. Results of wind-tunnel tests conducted on Cycas rumphii showed that pollen blown around cones lands on the blades of the sporophylls but not on ovules (Niklas & Norstog, 1984). In this case, insects or rain may carry pollen to the ovules. In some cycad species, such as C. micronesica on the island of Guam, the role of wind may vary depending on habitat and aspect, as plants of some cliff - dwelling and high- elevation populations are directly buff eted by consistent trade winds that blow across the Mariana Islands, but plants in populations deep in the understory are protected from these winds. Thus, wind is less available to transport pollen away from dehiscing pollen cones in the understory, but —-1 may play a larger role where plants are more exposed to trade winds. —0 —+1

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Population- genetic techniques can provide invaluable evidence to explore the links between pollination syndromes, pollinators, and gene fl ow in natural plant populations. Among and within populations, the availability and source of pollen do- nors is critical for determining ge netic structure (Sork & Smouse, 2006; Fortuna et al., 2008). Mating system involves patterns of relatedness within a plant population and establishes the genotypic composition of the progeny cohort and pollen dispersal dis- tance, and the shape of the dispersal kernel determines the spatial scale at which prog- eny infl uence ge netic structure (Nason et al., 1997; Fortuna et al., 2008). Pollinators can also shape the spatial assortment of mating events among conspecifi cs, often by distributing pollen unevenly among a subset of available mother plants (Fortuna et al., 2008). Traditionally, gene fl ow has been estimated indirectly from the eff ective num- ber of immigrants received by a subpopulation of each generation with F-statistics (Wright, 1969). Highly variable molecular markers such as microsatellites— simple tandem repeats present in all eukaryote genomes— have facilitated a direct ge netic ap- proach for measur ing pollen gene fl ow, mainly through parentage analysis where genotype frequencies from a series of individuals are characterized and compared to the genotype frequencies present in the target population (Dick et al., 2003; Latouche- Halle et al., 2004). Other methods include Bayesian approaches (Rannala & Moun- tain, 1997; Pritchard et al., 2000) and two- generation analysis (two-gener), in which the observed gene tic variation among sampled pollen pools is due to pollen dispersal distance (Sork et al., 2002). Two- gener models can fi t several families of dispersal curves, refl ecting diverse dispersal behaviors, rendering this approach yet more pow- erful for estimating the role of pollen dispersal in plant population ge netic structure (Austerlitz et al., 2004; Sork & Smouse, 2006). Cycads are an excellent system for exploring the contribution of seed and pollen- mediated gene fl ow: pollen and ovulate trees are easily identifi ed in the fi eld; they have generally restricted gene fl ow, which increases the chances of sampling putative parents; and seeds contain both progeny and maternal tissue that can be used for maternity analy- ses, which reduces collection eff orts of putative mother adult plants (e.g., Abe et al., 2006). A set of 14 microsatellites from an EST (Expressed Sequence Tag) library of Cycas rumphii have been developed and adapted to C. micronesica (Cibrian- Jaramillo et al., 2008). -1— These microsatellites were used to examine the ge netic variation and structure among 0— +1—

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and within populations of these species across their range in the South Pacifi c. They were specifi cally used to estimate the contribution of pollen-mediated gene fl ow in popula- tions of C. micronesica on Guam and to identify populations that are critical for maintain- ing population ge netic structure across the island and thus are fi rst targets for conservation initiatives. Initial fi ndings show that gene tic connectivity in C. micronesica across the is- land is low, with most of the variation concentrated within populations and a gene tic di- vision between contiguous northern and fragmented southern populations. Patterns of gene fl ow throughout Guam were associated to smaller seed size and the presence of riv- ers, but also to restricted pollen movement by an obligate mutualist and generalist insects. Overall, factors such as seed dispersal, movement of plants by humans or insect dispersal of pollen, and possibly fi ne- scale demographic factors related to the dioecious mating system of this species may shape the ge netic structure of this species on Guam (Cibrian et al., unpublished observations). We are now exploring the correlations between gene fl ow and gradients of wind and insect pollination to establish the impact of either mech- anism on gene tic structure. This will help determine which pollination mechanism is predominant (i.e., insects vs. wind, short- range insects vs. long- range) how far pollen is moving, and which pollen-cone plants are contributing within and among populations. Understanding parental contribution enlightens fi ne- scale demographic factors related to mating system and provides a very precise mea sure of the extent of gene fl ow through the landscape, which in turn infl uences conservation decisions (Dick, 2001; Lidicker, 2002). A gene tic profi le of seeds and mother plants of Cycas micronesica in three or four Guam populations will be used to determine the exact contribution of pollen- mediated gene fl ow (methods reviewed in Sork & Smouse, 2006). This approach can be applied to other cycads, and in partic ular to Cycas species. Preliminary results on C. zambalensis Madulid & Agoo, C. edentata de Laub., and C. wadei Merrill suggest that patterns of gene fl ow in cycads are more complex than previously suspected and worth exploring based on pollen contributions (Cibrian- Jaramillo et al., unpublished observations). A subset of the pub- lished EST-microsatellites (Cibrian- Jaramillo et al., 2008) have also been optimized for C. chamberlainii W. H. Brown & Kienholz (= C. riuminiana Porte ex Regel), C. nitida K. D. Hill & A. Lindst., C. circinalis, and C. bougainvilleana K. D. Hill, expanding the use of ge netic tools to understand the role of pollen movement in the evolutionary history of this group (Cibrian- Jaramillo et al., unpublished observations). —-1 —0 —+1

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Conclusions

In this overview of cycad pollination research, we have reviewed some of the history of pollination biology and ge netic studies, outlined some current fi ndings, stressed in some sections par tic ular shortcomings in our knowledge, and proposed research needs. We also have presented methods for using pollen cones as sampling devices in areas with no known cycads or for determining presence of pollinators in off - season, and the potential application of population-genetic techniques using molecular markers for the parentage analysis across and within populations to determine the potential role of wind to vector pollen. Finally, we have reviewed earlier studies of the association of idioblasts within developing ovulate and pollen cones that might regulate pollinator behavior and feeding on diff erent tissues. While we have learned much about pollination systems in terms of their insect visitors and putative pollinators in some species since the 1980s, most of the cycad species (more than 300) have not been even surveyed for insects and only a small number of species have been tested for the eff ectiveness of pollinators or for interactions between cone cues and pollinator responses. Still relatively unstudied areas are the use of gene tic and molecular tools for understanding the role of pollinators within and among wild populations and the relationships and potential co- evolution between insect polli- nators and their host species; however, since most cycads have not had their pollinators identifi ed, we need basic work on the identity of the pollinators. We encourage more survey work, life history studies of pollinators in natural habitats, behavioral studies of the insects in response to host cues, and trait analysis of pollen and ovulate cones, as well as pollinator effi ciency tests. We hope that this review of studies and techniques will promote more research in understudied areas of cycad pollination biology. We propose these specifi c research needs:

(1) Surveys of cone- associated insects and cone traits: most of the species of the ca. 105 species of Cycas have not been sampled for insects. This is especially true for species in Australia, Africa, most of the Pacifi c Islands, and Indonesia. The most extensive surveys have been in parts of Asia (Tang et al., 1999) and some species in India. For Macrozamia, found only in Australia, only about one-third -1— of the ca. 40 Macrozamia species have had their pollinators identifi ed. In Africa, 0— very few of the ca. 65 species of Encephalartos have had their pollinators identi- +1—

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fi ed, although ca. 30 species have been surveyed for cone insects. Only a few of the Mexican and Central and South American species have been surveyed for insects. This includes species of Dioon, Ceratozamia, and Zamia. (2) Pollination inclusion/exclusion experiments: there is an almost universal need for pollination tests of cycads on all cycad- bearing continents. Most important are tests to determine the possible role of insects that have not yet been been proven as pollinators on any cycad species. For example, within the genus Cy- cas, only C. revoluta has been thoroughly examined (Kono & Tobe, 2007), and Carpophilus (Nitidulidae) is the pollinator. The cones have specifi c volatiles not found in other Cycas species that have been examined. The role of wind should also be tested more thoroughly, especially for Cycas species, by having some treatments as “wind-only” excluding insects but allowing wind to deposit pol- len as part of the experimental design in pollination tests. Including more studies on wind pollination may be more important on island cycads, espe- cially those Cycas more distant from mainland cycads. Population- genetic techniques using molecular markers to identify pollen parents of seeds may be invaluable in determining how much of a roll wind plays in the pollination process of par tic ular cycads. Once pollen vectors are determined for a species, then the role of cone traits can be tested to examine how the pollination system functions. (3) Testing specifi c cone traits and insect interactions: we know very little about pollination syndromes in cycads or what specifi c cone/plant traits are respon- sible for mediating pollinator behavior in most cycad species. For example, cone thermogenesis and volatile emissions (qualitative and quantitative analysis of emissions over time of day and cone stage) are the primary traits associated with or are directly involved in mediating behavior of some pollinating in- sects. These studies will help identify patterns of cone traits associated with specifi c pollinator- cycad associations. Specifi c questions related to understand- ing how the system functions are the following. What are the major determi- nants of attraction? Are volatile components only attractants or are any of them repellents, or are some volatile chemistries attractive and repellent depending upon their concentration (i.e., test of the push- pull pollination hypothesis)? —-1 Do components work synergistically? What role do cone idioblasts play in —0 —+1

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thermogenesis and volatile emissions, if any? Can cone baits (as in Panama studies) be used to attract potential pollinators and help determine when cones attract pollinators or whether pollinators are present during the off - season in other cycad species populations? Do cone volatiles induce diapausing pupae to eclose? Can we use results of volatile analyses to test diff erent components and blends in the fi eld as attractants (although cones often outcompete artifi cial baits)? Does thermogenesis play a direct role to induce the pollinator to leave by warming wing muscles for fl ight or by making the pollinator more active or does thermogenesis indirectly aff ect the pollinator by increasing volatile release or production? Physiological tests, electroantennograms (EAGs), or gas chromatography-electroantennographic detection (GC- EADs), can be used to identify specifi c components of cone volatiles for further testing insect behav- ioral responses in bioassays in the laboratory or fi eld. Finally, what is the role of cone toxin chemistry, including idioblast changes and toxicology, in pollinator interactions or in anti- herbivore defense? (4). Taxonomy, phylogeny and life histories of pollinators: there are numerous groups of insects needing revisions and species descriptions. In addition, more phylogene tic information is needed based on both morphological and molecu- lar traits to determine any co- evolutionary or co-speciation patterns.

Ac know ledgments

We thank all those researchers who have contributed to our knowledge of cycad pollination systems, especially insect taxonomists and those who provided some of the fi rst experimen- tal evidence for insect involvement in the pollination of cycads, Knut Norstog, Priscilla Fawcett, Dennis Stevenson, and Karl Niklas. We also acknowledge the kind permission of Gaspar Silvera, Panama, to use his cycad nursery for long-term pollination studies.

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