American Journal of Botany 92(6): 931–940. 2005.
POLLINATION OF AUSTRALIAN MACROZAMIA CYCADS (ZAMIACEAE): EFFECTIVENESS AND BEHAVIOR OF SPECIALIST VECTORS IN A DEPENDENT MUTUALISM1
L. IRENE TERRY,2,7 GIMME H. WALTER,3 JOHN S. DONALDSON,4 ELIZABETH SNOW,3,6 PAUL I. FORSTER,5 AND PETER J. MACHIN5 University of Utah, Department of Biology, 257 South 1400 East, Salt Lake City, Utah 84112 USA; 3School of Integrative Biology, The University of Queensland, St. Lucia, Brisbane, 4072 Queensland, Australia; 4National Botanical Institute, Kirstenbosch Research Centre, Claremont, Republic of South Africa; 5Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Garden Mt. Coot-tha, Queensland, Australia 2
Complementary field and laboratory tests confirmed and quantified the pollination abilities of Tranes sp. weevils and Cycadothrips chadwicki thrips, specialist insects of their respective cycad hosts, Macrozamia machinii and M. lucida. No agamospermous seeds were produced when both wind and insects were excluded from female cones; and the exclusion of wind-vectored pollen alone did not eliminate seed set, because insects were able to reach the cone. Based on enclosure pollination tests, each weevil pollinates an average 26.2 ovules per cone and each thrips 2.4 ovules per cone. These pollinators visited similar numbers of ovules per cone in fluorescent dye tests that traced insect movement through cones. Fluorescent dye granules deposited by Cycadothrips were concentrated around the micropyle of each visited ovule, the site of pollen droplet release, where pollen must be deposited to achieve pollination. In contrast, Tranes weevils left dye scattered on different areas of each visited ovule, indicating that chance plays a greater role in this system. Each weevil and 25 thrips delivered 6.2 and 5.2 pollen grains, respectively, on average, to each visited ovule per cone, based on examination of dissected pollen canals. In sum, the pollination potential of 25 Cycadothrips approximates that of one Tranes weevil. Key words:
Macrozamia lucida; Macrozamia machinii; obligate pollination mutualisms; thrips; weevils; Zamiaceae.
For much of the 20th century, the generalization that conebearing plants are wind-pollinated (Chamberlain, 1919, 1935) was also applied to cycads (Chamberlain, 1919; see review by Stevenson et al., 1998), a dioecious and basal clade among the extant seed plants (Chaw et al., 2000; Hill et al., 2003). Recent cycad research has changed that perception. Now, most cycads, if not all, are considered to be involved in dependent mutualisms with specialized insect pollinators (Norstog and Nicholls, 1997; Stevenson et al., 1998). Overall, few such dependent pollinator mutualisms are known, namely those associated with yuccas, globe flowers, figs, senita cactus (Fleming and Holland, 1998; Cook and Rasplus, 2003; Pellmyr and Segraves, 2003; Cook et al., 2004; Weiblen, 2004), and the euphorbiaceous trees, Phyllanthus and Glochidion (Kato et al., 2003; Kawakita and Kato, 2004), all of which are angiosperms. Several of these angiosperm systems are better understood than are the superficially similar mutualisms of cycads, in terms of the specific roles of their pollinators and the overall structure and evolutionary history of the mutualisms. In particular, little is known about the effectiveness of individual pollinator taxa found on extant cycads. Detailed pollinator contribution studies are needed not only for plants visited
by several insect taxa, but also for plants visited by a single specialist insect, because the nature of each insect’s role cannot be assumed, especially in cases where reproductive tissues serve as substrates for the development of pollinator larvae (e.g., Donaldson, 1997; Fleming and Holland, 1998). Studies of cycad pollination have continued to challenge any simple generalizations about the origin and diversity of these systems. The combined outcome of experimental studies (Norstog et al., 1986; Tang, 1987; Donaldson, 1992, 1997; Donaldson et al., 1995; Norstog and Fawcett, 1995; Terry, 2001; Wilson, 2002; Hall et al., 2004; Terry et al., 2004b), field surveys (Vovides, 1991; Donaldson, 1993; Forster et al., 1994; Donaldson et al., 1995; Vovides et al., 1997; Chadwick, 1998; Tang et al., 1999), and insect systematics (Mound, 1991; Marullo and Mound, 1995; Oberprieler, 1995a, b; Mound and Terry, 2001; Leschen, 2003; Oberprieler, 2004) indicate that complex cycad–pollinator interactions have evolved independently on different continents in association with different cycad genera. This not only raises questions about the evolutionary dynamics and vagility of these interactions, but also about the specific relationships between the diverse insect taxa and the particular cycads with which they are associated. Within Australia, a high diversity of specialist insects is associated with cycad cones. Species of Macrozamia (Cycadales, Zamiaceae), an endemic genus with ;40 species (Jones, 2002; Forster, 2004a), are pollinated by insects in at least two unrelated taxonomic groups, although other putative pollinators have been found. Some Macrozamia species are apparently pollinated only by species of thrips in the genus Cycadothrips (Thysanoptera, Family Aeolothripidae), whereas other Macrozamia species appear to be pollinated only by species within the genus Tranes (Coleoptera, Family Curculionidae), and a few by both Cycadothrips and Tranes (see Forster et al., 1994;
Manuscript received 2 June 2004; revision accepted 17 March 2005. The authors thank the following people who contributed to this study: R. Roemer, C. Rajapakse, J. Jones, J. Beard, L. Mound, R. Oberprieler, and staff at Brisbane Forest Park, especially B. Duncombe and D. Dent, J. Cane assisted with determining pollen loads and allowed use of facilities at the USDA Bee Systematics Laboratory, Logan, UT, USA. This work was supported in part by The National Geographic Society Committee for Research and Exploration and by University of Utah Research Committee funds. 6 Present address: Alan Fletcher Research Station, Department of Natural Resources, Mines and Energy, PO Box 36, Sherwood, QLD, Australia 4075 7 Author for correspondence (e-mail:
[email protected]) phone: 801585-3139, fax: 801-581-4668 1
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Mound et al., 1998; Jones et al., 2001; Mound and Terry, 2001; Terry, 2001). All other known or putative pollinators of cycads throughout the world are beetles (Oberprieler, 1995a, b). Both Cycadothrips and Tranes weevils are obligately associated with their particular Macrozamia host species: they feed, mate, lay eggs and complete their larval development on their cycad host. Other known Australian cycads and their specialist pollinators include Lepidozamia peroffskyana (Zamiaceae) with only Tranes lyterioides (Hall et al., 2004) and both Bowenia species (Stangeriaceae, or Zamiaceae, see Treutlein and Wink, 2002; Hill et al., 2003; Rai et al., 2003) each with their own Miltotranes species (Curculionidae, Molytinae) (Wilson, 2002). Other putative pollinators need testing, including Ulomoides australis (Coleoptera, Tenebrionidae) on M. moorei (Terry et al., 2004a) and on some Cycas species (Cycadaceae) (Ornduff, 1991b; Forster et al., 1994), and Paracucujus rostratus (Coleoptera, Boganiidae) and Xenocryptus tenebroides (Coleoptera, Languriidae) on M. riedlei (Ornduff, 1991a; Connell and Ladd, 1993). Despite the advances described, little quantified testing has been conducted on the precise role of the pollinators of Australian cycads; and the vast numbers of these insects at receptive cones (see Fig. 1 of Hall et al., 2004) raise questions as to their effectiveness as pollinators. Cycads are among the most threatened groups of plants worldwide (53% are included in the IUCN Red List, Donaldson, 2003), with pollinator extinction a major threat to small populations. The vulnerability of species in dependent mutualisms to local extinction is therefore becoming an important question for conservation (Bond, 1994) and is critical for longterm population maintenance in situ for these ‘‘flagship’’ species. Several species of Macrozamia are listed as threatened (Forster, 2004b), and in situ conservation efforts will need to conserve not only the cycads but also their pollinators. Quantification of Macrozamia pollinators’ effectiveness will aid in developing specific population models to predict the vulnerability of these cycads relative to pollinator abundance, especially the minimum pollinator numbers required to obtain a full seed set. The codependence of these mutualists may render both insect and cycad especially sensitive to those properties associated with cycad population declines, including habitat fragmentation from human population encroachment, permitted and illegal collecting of adult plants, massive land clearing, and intentional killing of plants on ranch lands (Raimondo and Donaldson, 2003; Forster, 2004b). Our specific objective in this study was to test the pollinator effectiveness of Cycadothrips and Tranes on their respective Macrozamia hosts by quantifying seed set associated with different pollinator densities and by examining pollinator movement around the micropyle of ovules and pollen grain delivery into the micropyle and pollen canal within receptive female cones. Our results allow us to examine whether the dependent pollination mutualism in this gymnosperm lineage is as intricate as those of angiosperms (see previous), especially with regard to whether specific pollination-associated behavior takes place within the cycad cones. MATERIALS AND METHODS Study species and sites—Two southern Queensland Macrozamia species were studied during the 2001–2002 coning season (pollination period during October–November 2001 until seed set in April–May 2002): M. machinii P.I. Forster and D.L. Jones, which is associated only with Tranes sp. weevils; and
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M. lucida L.A.S. Johnson, which has only Cycadothrips chadwicki thrips. A follow-up experiment was conducted on M. machinii during the 2002–2003 coning season. Both cycad species are currently classified in Macrozamia section Parazamia, having belowground caudices, with leaves emerging from this underground structure. In this regard, plants of both species are relatively small in stature when compared with those of species in section Macrozamia that produce trunks with substantial crowns (Jones, 2002; Forster, 2004a). No species name is yet available for the Tranes on M. machinii (R. Oberprieler, CSIRO, personal communication). Voucher specimens of insects have been deposited in the Australian National Insect Collection (ANIC, CSIRO) in Canberra. Plant vouchers from each of the study sites that will be described have been deposited at the Queensland Herbarium (BRI) in Brisbane. Macrozamia machinii is a threatened (V, vulnerable) species restricted to a small area within the Darling Downs, west of Brisbane (Jones and Forster, 1994). Populations at two sites in the Wondul Range north of Inglewood were studied. The study site of M. lucida was in Brisbane Forest Park northwest of Brisbane, Queensland. Further descriptions of each population and study site are in Terry et al. (2004b). One M. machinii study site had ,10 coning plants of each sex, but the two other sites had at least 30 of each. Exclosure/enclosure studies—Leaves of both cycad species were clipped off at ground level so that a cone could be enclosed completely and securely inside a cage, which consisted of an inverted ‘‘bucket’’ system similar to that used by Wilson (2002). These plants experience leaf loss on an irregular basis from fire or heavy predation by butterflies and beetles when their foliage is expanding and still soft (Forster and Machin, 1994), and coning seems unaffected by such reduction in the aboveground biomass. To ensure leaf removal did not affect seed set, an additional control treatment with leaves intact was added to the test in the population with sufficient numbers of cones (i.e., the 2001 M. lucida test). A white, 20-L bucket, with 12-cm diameter windows (covered with 0.2-mm2 fine-meshed nylon cloth in Cycadothrips cages and 1-mm2 plastic mesh for Tranes cages) at the top and sides was inverted over each treatment cone and was mounted on metal stakes a few centimeters above the ground but below the sporophylls of the cone. Fine-meshed cloth skirts glued to the rim of the bucket cage were tied tightly around the cone peduncle near ground level. The peduncle was coated with Tanglefoot (The Tanglefoot Company, Grand Rapids, Michigan, USA) to prevent insects from entering at this site. This cage/window system allowed ventilation, but windblown pollen was excluded with plastic covers over screened windows. During the 2001–2002 coning season, experimental treatments consisted of: (1) exclusion of wind-vectored pollen only (by using cages that had no side windows and no mesh skirts, which blocked wind from delivering pollen but allowed insect access from ground level to ;8 cm above ground); (2) exclusion of both insects and wind-blown pollen; (3) release of pollen-coated insects into cages containing receptive female cones, using three levels of insect (sex not determined) abundance (one, two, or five pollen-coated Tranes weevils on M. machinii; and 25, 100, or 400 thrips on M. lucida); (4) open control cones (no cages over cones) that allowed access to both wind and insects; and (5) exposed control cones with all fronds still intact (M. lucida test only). The objective in treatment (3) was to use numbers of insects predicted to give low pollination, average pollination, and full pollination of all ovules and was based on numbers of insects that have been found on female cones. Those cages that enclosed insects had sleeves that could be opened to add insects when the female cone was receptive. Receptive female cones emit an odor representative of their species (Terry et al., 2004a, b) and have visible gaps between the sporophylls. Cages were placed on cones at least 1 week before any test cone became receptive. Cages were left on female cones throughout their receptive period and up to 3 weeks after receptivity. There were four and five replicates (individual caged cones) of each treatment on M. machinii and M. lucida, respectively. During the 2002–2003 coning season, we conducted an additional test to determine whether male and female Tranes vary in their effectiveness in pollinating M. machinii. Tests included treatments with one, two, or four male or female weevils inside a caged cone, which were compared with open control cones. Pollination success in these tests was determined by analyzing the proportion of ovules that set seed, which we measured when the seed began to
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Fig. 1. Macrozamia machinii female cones, ;10–14 days after period of receptivity. (a) Undamaged, pollinated cone, showing ovules beginning to expand; and (b) damaged cone, which had at least 20 weevils on it during part of its receptive period.
separate from the cone peduncle, about 5 to 8 months after the pollination period for these species. In other studied cycads, only pollinated ovules expand (Fig. 1a) and months later develop into viable seeds; they can be separated from those ovules that were not pollinated on the basis of size (Tang, 1987). Dissection of a large sample of ovules/seeds (N 5 1017) confirmed that 99% of large seeds had a starchy endosperm, an indication of fertilization and seed viability. Structural megasporophylls (at base and apex of cone) that have sterile ovules were eliminated from the analysis. The proportion of ovules that set seed (arcsin square-root transformed proportion of the ovules that developed into seeds) in each cone was used in a one-way general linear model (GLM) to test for the effects of experimental treatments on seed set. For the 2002–2003 M. machinii data, a (1) one-way and (2) two-way GLM tested for differences in seed development between (1) control cones and all other treatments; and (2) the sex and number of Tranes weevils placed inside cone cages. Specific post-hoc treatment comparisons were performed using least significant differences with Bonferronicorrected (a # 0.05/n, where n 5 number of comparisons) significance levels. Insect movement within cones—The ability of Tranes or Cycadothrips to visit and deliver pollen to the correct site in the female cone (i.e., the micropyle of ovules) was determined by observing tracks left by dye-coated insects that also carried pollen. Insects were shaken from dehiscing male cones (to ensure they carried pollen) and then placed in 25-mL cups with orange fluorescent powder dye for about 1 h. Dye-coated insects were transferred to clean cups, which were then placed inside the cage system enclosing a single receptive female cone. A single Tranes weevil or 25 Cycadothrips were placed inside a M. machinii or M. lucida cage, respectively. Cones were removed late morning of the following day and placed in a 48C refrigerator to keep insects inactive until cones were dissected (;2 weeks). Three cones of each species were used. In the laboratory, cones were dissected and separated into component tissues (sporophylls, ovules, and peduncle) and regions (top, middle, and lower sections of cones). Each ovule was examined under UV light for the presence/ absence of dye and the location of the dye (Fig. 2) with respect to the micropylar tip (where the pollen droplet is released and then retracted, pulling in deposited pollen grains through the micropyle into the pollen canal and chamber, see Fig. 2c). The percentage of ovules with dye around the micropyle was determined for each cone. ANOVA models tested for differences between species in the proportion of micropyles with dye (arcsin square-root
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Fig. 2. Macrozamia ovules (a,b, d–f, during female receptive period; c, after receptive period). Macrozamia machinii (a) under visible light with micropylar tip indicated by arrow, (b) under UV light showing the deposits of fluorescent dye left by a Tranes sp. weevil (bar 5 2 mm). (c) Section of dissected Macrozamia lucida ovule, micropylar end, under fluorescence microscopy, showing pollen grains in the pollen canal, taken from cone about 10 d after receptive period (bar 5 0.5 mm). (d–f) Macrozamia lucida ovules under UV light showing deposits of fluorescent dye left by Cycadothrips chadwicki, (d) both ovules of one sporophyll, (e) enlarged section of right ovule showing single deposits of dye granules, and (f) enlarged view of right ovule at a different angle, showing concentration of dye in the area surrounding the areole of the micropylar tip (bar 5 2 mm). transformed data) and also for position effects (top, middle, or lower section of cones) within each species. Number of pollen grains in pollen chamber—To determine whether insects delivered pollen to the micropylar canal, we dissected a subset of the ovules from cones in the dye experiments (previous section) to check for the presence and number of pollen grains in the micropylar canal and pollen chamber. All the ovules from three spirals of sporophylls (;one-third of ovules per cone) from each cone were preserved in FPA (formalin : propionic acid : 50% ethanol, 10 : 5 : 85) and then sectioned to isolate the micropylar canal and pollen chamber. Sections were cleared and prepared for fluorescence microscopy following the methods of Donaldson (1997). Pollen grains were visualized in these sections under fluorescent light on a compound microscope (Fig. 2c). ANOVA models tested for differences between cycad species in the number of grains per ovule found in the pollen tube/chamber and between ovule positions (upper, middle, lower) within cones. Pollen loads and estimates of pollen delivery rates to micropyle—Tranes weevils were collected in the field as they were leaving male cones (two males and eight females) or as they landed on female cones (two males and five females) during the pollination period. Beetles were collected from four male cones and two female cones. Each captured insect was immediately stored individually in a 2-mL Eppendorf tube containing 1 mL AGA (acetic acid : glycerine : 60% ethanol, 1 : 1 : 10). Samples were prepared for measuring pollen loads by a particle counter method (Cane et al., 1996) using a HIAC/ ROYCO (Hach Ultra Analytics, Grants Pass, Oregon, USA) particle counter, at the USDA Bee Systematics Laboratory at Logan, Utah, USA. Each collection tube containing an insect was first centrifuged for 1 min at 12 000 3 g, and the supernatant was decanted. The remaining solution and weevil were resuspended in filtered 95% ethanol and vortexed for 1 min. The solution with weevil was placed into a clean 50-mL glass vial, and the vial that originally contained the weevil and pollen was rinsed twice into the fresh glass vial. Filtered ethanol was added up to 50 mL into the fresh vial. This sample solution was sonicated for 1 min, the weevil was then removed, and 25 mL of the sample solution was passed through the particle counter. Counts of
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TABLE 1. Percentage seed set in Macrozamia machinii pollination enclosure/exclosure treatments, 2001–2002 coning season. The number of Tranes weevils added to a cage is indicated in each enclosure treatment.
Treatment (N)a d
Control (4) Exclusion of insects and wind (4) Wind exclusion (4) 1 Tranes weevil/cone (2) 2 Tranes weevils/cone (4) 5 Tranes weevils/cone (4)
Avg. no. ovules per cone (SE)
53 62 59 63 53 55
(3.4) (8.5) (5.4) (2.1) (6.3) (4.9)
Seed set (%)b
77.7 1.7 90.3 16.4 85.5 36.5
SE
4.2 1.9 12.7 16.2 1.2 7.1
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TABLE 2. Percentage seed set in Macrozamia lucida under each pollination treatment, 2001–2002 coning season. The number of Cycadothrips chadwicki added to each cage is indicated for enclosure treatments.
P valuec
a (N), total replicates per treatment; in the treatment with one weevil per cone, the ovules on two cones dried up independently of the treatment effect (as observed on an occasional uncaged female cone that was not part of the test) and were not included in the analysis. b Seed set (i.e., pollinated) 5 (enlarged seeds/potentially viable ovules) 3 100; % seed set is back-transformed. c Calculated on arcsin square-root transformed data. Model results, GLM, F5,16 5 7.73, P 5 0.0007; r2 5 70.2. For pre-planned comparisons, controls vs. each treatment, significant differences are indicated by the P values in the table. For Bonferroni-corrected post-hoc tests (P , 0.05) comparing seed set among other treatments, the treatment with one Tranes weevil per cone is significantly lower than treatments with wind exclusion and with two weevils per cone; total exclusion is significantly lower than all treatments except with one Tranes weevil per cone. d Control cones are open cones with no enclosure/exclosure cage.
particles within 15–30 mg range were included for both Macrozamia species because the grains are elliptical in shape with a width and length in this size range for both species (Deghan and Deghan, 1988; and our direct measurements). Control samples, i.e., samples of AGA used in storing insects and several ‘‘blank’’ ethanol samples were also processed through the counter to ensure the background count was insignificant. Individual thrips that were removed directly from male cones with a fine brush gave inflated pollen counts because loose pollen was always picked up simultaneously. Groups of Cycadothrips were therefore captured as they left cones through a funnel trap placed over a male cone. A clean funnel system was used for each 30–60-min interval of capture. Samples were placed on ice and later transferred to 1 mL AGA for storage. Thrips were counted and removed from the solution without sonication (to prevent their wing fringes from breaking and interfering with counts) before grains in the solution were counted. A second rinse of thrips was examined visually for additional pollen. A total of 531 thrips was obtained from six samples of two different male cones. For samples with .20 thrips, the particle counter method was used. For samples with ,20 thrips per capture, thrips were removed, the liquid was centrifuged for 30 s at 12 000 3 g, the top 600 mL was decanted, checked for pollen (no grains were found) and then discarded. Grains in the remaining solution were viewed and tallied, in 10–30 mL aliquots, through a dissecting microscope. Pollen loads were determined as the average number of grains per individual for each sample and also as averages per individual across all samples. Pollen loads of thrips arriving at female cones were determined from thrips caught on sticky trap collars, because pollen surrounding thrips bodies can be viewed through a dissecting microscope (Mound and Terry, 2001). Any remaining pollen on thrips bodies was not counted, because the only grains that can be seen are those that are pulled away from the body as they land in the Tanglefoot. To estimate the net rate of delivery of pollen grains to micropyles from the information on pollen loads (previous analysis), the number of insect visits per female receptive cone was estimated in the field. For Tranes, the number of insects visiting each cone was determined from observations on four receptive and uncaged female M. machinii cones that were excised in the early morning and covered with fine mesh. The number of weevils that emerged from each cone that evening was counted. To obtain estimates for thrips arrivals at receptive female cones, we placed a sticky trap collar (1.5 cm wide
Avg. no. ovules per cone (SE)
Treatment c
0.0007 0.379 0.016 0.745 0.057
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Control (leaves removed) Controlc (leaves not removed) Exclusion of wind and insects Wind exclusion 25 Cycadothrips per cone 100 Cycadothrips per cone 400 Cycadothrips per cone
74 72 86 57 70 77 80
(7.4) (6.2) (9.9) (8.1) (7.1) (3.7) (7.1)
Seed set (%)a
SE
P valueb
97.6 95.5 0 58.1 85.2 97.2 98.4
1.4 1.1 0 7.9 5.1 1.9 1.1
— 0.4154 0.0001 0.0001 0.0071 0.8873 0.7103
a Seed set (i.e., pollinated) 5 (enlarged seeds/potentially viable ovules) 3 100; mean % seed set is back-transformed. b Calculated on arcsin square-root transformed data. Model results of GLM, F6,38 5 64.2, P 5 0.0001, r2 5 92.1%. For pre-planned comparisons, significance of tests of differences between control without leaves and each treatment is indicated by the P value in the table. For Bonferroni-corrected post-hoc comparisons at P , 0.05, total exclusion and wind exclusion treatments had significantly lower seed set than all other treatments (and each other), and seed set in the 25 thrips per cone treatment is significantly lower than in treatments with 100 or 400 thrips per cone. c Control cones are open cones with no enclosure/exclosure cage; one set of controls had cones with no leaves removed. Plants in all other treatments had their leaves removed.
3 8 cm diameter, covering ,20% of the height of the female cone) around the middle of each of three receptive M. lucida female cones to trap arriving thrips. Counts on sticky traps were adjusted for the full cone height.
RESULTS Enclosure/exclosure experiments—Seed set differed significantly among treatments in both Macrozamia species, and insects were necessary and sufficient for seed set (Tables 1 and 2). In both Macrozamia species, the level of seed set in controls (exposed, open cones) did not differ significantly from seed set in treatments with higher densities of Tranes weevils (two or five per cone: Table 1) or Cycadothrips (100 or 400 per cone: Table 2). Removing leaves from control cones of M. lucida had no effect on seed set compared with control cones with intact leaves. Indeed, control plants with leaves removed for both species had high pollination rates (Tables 1 and 2). Statistically, treatments with the lowest densities of pollinators had a lower percentage of pollinated ovules than the control cones, although the difference for Cycadothrips was not great (85.2 vs. 97.6%: Table 2). In the M. machinii test, when both wind and insects were excluded (Table 1), no seed was set except in one replicate, where a Tranes weevil (or weevils) apparently entered the cage, and 10 of the 39 ovules set seed. (In cycad pollination studies, excluding beetles completely from cones has been consistently difficult: see review by Stevenson et al., 1998; Terry, 2001; Hall et al., 2004.) By contrast, total exclosures did apparently prevent thrips from getting to female cones on M. lucida, because no seed was set in any replicate of this treatment. Overall, no agamospermous reproduction occurs in these Macrozamia species, which agrees with other cycad studies (Norstog and Nicholls, 1997; Terry, 2001; Wilson, 2002; Hall et al., 2004), although no data are yet available for any species of Cycas. The wind exclusion treatments (which allowed insect access) returned slightly different results across the Macrozamia
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TABLE 3. Percentage seed set in Macrozamia machinii under the indicated pollination treatments, 2002–2003 season. The number of Tranes weevils added to each cage is indicated for enclosure treatments.
Tranes sex (N)a d
Open control (5) Female (6) Female (5) Female (3) Male (5) Male (6) Male (6)
No. of weevils added to cone
Open 1 2 4 1 2 4
Avg. no. ovules per cone (SE)
50.2 49.5 37.5 54.0 47.6 36.2 53.5
(3.0) (5.0) (4.0) (7.6) (7.4) (4.0) (5.7)
Seed set (%)b
SE
P valuec
82.4 63.7 49.4 68.2 58.9 66.9 51.3
6.0 9.2 17.2 22.6 15.7 9.7 8.1
0.27 0.08 0.48 0.19 0.38 0.09
a (N), number of replicates per treatment. Some additional replicates were added in some treatments because some cone replicates, already selected for treatments, were thought (mistakenly) to be postreceptive when insects were added. In the treatment with four female weevils added, two of the five replicate cones dried out, independent of the treatment effect (see Table 1 footnote). b Seed set (i.e., pollinated) 5 (enlarged seeds/potentially viable ovules) 3 100; mean % seed set is back-transformed. c Calculated on arcsin square-root transformed data. The GLM model indicated no significant effects (overall model, F5,24 5 0.75, P 5 0.614) of any of the factors, Tranes sex (P 5 0.93), number of Tranes added (P 5 0.95) and no interaction between Tranes sex 3 number of Tranes (P 5 0.93). d Control cones are open cones with no enclosure/exclosure cage.
species. In the M. machinii study, seed set did not differ significantly among the wind exclusion treatment, the open control treatment and those treatments with two or five weevils (Table 1). In the M. lucida tests, by contrast, seed set in the wind exclusion treatment was significantly reduced relative to the open controls and to the treatments with 100 or 400 thrips per cone (Table 2). However, seed set in the M. lucida wind exclusion treatment was significantly higher than in the treatment that excluded both thrips and wind and did not differ significantly from the treatment with 25 thrips per cone (Table 2). Thrips may not be as persistent, or as able, as Tranes weevils to find and get to the receptive female cone by going under the ‘‘cage.’’ In these field tests (Tables 1 and 2), relatively few individual Tranes weevils or Cycadothrips were needed to achieve levels of seed set to match the open controls. In most replicates, two Tranes weevils per M. machinii cone (with an average of 58, range 39–80, potentially viable ovules per cone) and 100 Cycadothrips per M. lucida cone (with an average of 74, range 36–120 ovules per cone) were sufficient to achieve full seed set. Variability in seed set was high in several treatments of the M. machinii test, and generally much higher than in the M. lucida test. The reduction in percentage seed set in the replicates with five weevils per cone may have resulted from damage caused by weevils because they were forced to remain on female cones for ;2–3 weeks after receptivity. Results of the 2002–2003 M. machinii test (Table 3) indicate that both sexes of Tranes weevil are capable of pollinating and that they do so to the same extent; there was no significant interaction between sex of weevil and number of weevils on cones. Both sexes of Tranes damaged cones when forced to remain on female cones, but the damage level was not clearly correlated with numbers of Tranes weevils on cones. Generally, the most damage to cones and ovules in both M. machinii tests was found in treatments with $4 weevils per cone. During the pollination period in November 2003, there were high
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numbers of Tranes weevils at one M. machinii population, and some female cones with .20 weevils during their receptive period aborted all their ovules (Fig. 1b). Also, when adult Tranes were kept on caged female cones in the laboratory for 2 weeks, the weevils laid eggs on them, and their larvae completely destroyed female sporophylls and ovule tissues. The fact that female cones may provide a larval substrate for Tranes larvae is a novel result, but it does complement other observations (Forster et al., 1994; Terry, 2001; Hall et al., 2004) that high numbers of Tranes larvae can destroy male cones. Whereas no larvae were recorded in caged female cones, some damage was detected on virtually all of them, probably from adult feeding or oviposition. Whether such damage affects pollination is not known, but there is probably some effect. In the 2002–2003 M. machinii pollination test (Table 3), 67% of those ovules from cones with medium to high damage (gum or damage on most sporophylls and ovules) had ,60% seed set, whereas 67% of those with little to no damage had .60% seed set. To estimate the number of ovules an individual insect can pollinate, we used the numbers of ovules pollinated in the treatments with the lowest densities of pollinators (i.e., one Tranes per cone and 25 thrips per cone) because they were the only ones below full seed set. Each Tranes weevil pollinated an average of 18.6 ovules (range 2, an outlier, to 23 ovules) per cone, and a single Cycadothrips pollinated an average of 2.4 ovules (range 1.1–3.8) per cone. For the 2002– 2003 M. machinii pollination test, each male or female Tranes pollinated, on average, 29.6 and 30.3 ovules, respectively. Movement of insects traced with fluorescent dye—Dye deposits left by the single Tranes weevil on each M. machinii female cone were easily detected with UV light (e.g., Fig. 2b). Dye trails were found throughout the cones and across the ovules, suggesting that a single weevil traveled extensively within a cone overnight, and dye was scattered in various areas of each contacted ovule. The amounts of dye left by Cycadothrips in M. lucida cones were much smaller and were more restricted in their distribution on ovules (Fig. 2d–f), than those left by Tranes. The dye left by Cycadothrips was mostly concentrated around the micropyles (Fig. 2f); but on a few ovules, granules of dye led toward (and/or away from) the micropyle (e.g., Fig. 2e). This suggests that Cycadothrips movement may be directed toward the micropyle, but Tranes movement is less directed, and they seem to contact the micropyle by chance. On average, 25 Cycadothrips deposited pollen on the micropyles of 36.7% of M. lucida ovules (62.8 SE, N 5 100 ovules), and a single Tranes weevil deposited pollen on the micropyles of 46.1% of M. machinii ovules (612.6 SE, from a total of 68 ovules). Based on these percentages and the total ovules per cone, each Cycadothrips pollinated an average 1.54 ovules and each Tranes weevil 32 ovules. The percentage of ovules with dye deposits was similar in all regions of the cones in both species (Table 4). Pollen grains per ovule—Pollen grains were easy to find and count within the ovule pollen chambers of both species using fluorescence microscopy (Fig. 2c). The percentage of ovules examined that had pollen in the pollen canal was similar across species (68% for M. lucida, 67% for M. machinii) (Table 5). Of those ovules that did receive pollen, no significant difference was detected in the number of pollen grains found in the pollen chambers either across Macrozamia spe-
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TABLE 4. Mean percentage (6SE) of ovules (micropyle region) with dye across the different cone regions on the Macrozamia species indicated, based on ovules from three spirals of sporophylls (one-third of each cone), three cones per species. Macrozamia machinii
Macrozamia lucida Cone region
Top Middle Bottom GLM statistics, between regions a b
Ovules (%)a
Ovules sampled (total)b
39.5 (5.4) 37.5 (10.3) 36.5 (5.6) F2,16 5 0.012, P
40 (100) 32 (100) 28 (100) . 0.98
Ovules (%)a
Ovules sampled (total)b
41.5 (14.0) 20 (68) 47.3 (14.9) 20 (70) 50.0 (17.1) 28 (70) F2,16 5 0.52 P . 0.65
Percentage of ovules (6SE) sampled with dye around the micropyle. The number of ovules sampled (total number of ovules in each region of cone).
cies or among different parts of the cone. A set of ovules was also examined from three uncaged cones (not included in any of the tests described earlier) for comparison with the results from the manipulated cones in the fluorescence tests. For both species, there was no significant difference (ztz , 1.66, df , 28, P . 0.56) between the number of pollen grains found in ovules from the earlier tests and those from uncaged cones (for M. lucida test vs. uncaged cone ovules, 5.2 [60.78 SE] vs. 5.9 [60.73 SE] grains per ovule; and for M. machinii test vs. uncaged cone ovules, 6.1 [61.07 SE] vs. 8.7 [61.25 SE]). Pollen loads—Average pollen loads of Tranes leaving male cones (see Fig. 3a of a pollen-covered Tranes weevil leaving a male cone that has ;60% dehisced) were 109 340/weevil (44 180–168 000; 13 867 SE, from 2 male, 8 female weevils); no consistent difference in loads between the sexes was discernible in these data. Weevils collected while arriving on female cones had significantly lower average pollen loads, 14 710/weevil (2278–33 840; 64815 SE, from 2 male, 5 female weevils; F test on logn-transformed values, F1,15 5 16.4, P 5 0.00031), indicating approximately an ;eight-fold average pollen-grain loss. Average pollen load on thrips leaving male cones was 107 (622.1 SE) grains per thrips, with a wide range (9.2–223 grains per thrips) among the six different samples, three from each of two cones. Pollen was found clumped around setae on the prothorax, around setae at the end of the abdomen (Fig. 3b) and on wing fringes and abdominal setae under the wings. Thrips that left male cones prior to 1100 hours had lower average loads (9 grains per thrips) compared with those of all later samples (.62 grains per thrips). This increase may correspond with increasing dehiscence of pollen later in the morning and afternoon. Thrips arriving at female cones had an average load of 29.2 (61.6 SE, range 0–145), and pollen was detected on all but 2 of the 258 thrips examined on samples from three different cones. The lowest load from a specific
sample was 13.6 (61.4 SE, range 0–31) and the highest was 44.3 (65.1 SE, range 7–123) pollen grains per thrips. Pollen delivery rates to micropyles—An average of 14 Tranes weevils emerged from the four receptive female cones collected in the field (8, 11, 14 and 22 weevils). This average is a conservative estimate of all weevils that visit a receptive cone, because it represents visitors from one night only. (If this number is reflective of the number of weevils visiting the female cone each 24 h during the cone’s receptive period of about 6 days (Norstog et al., 1986), then on average, 84 Tranes would visit one female cone.) By combining the minimum number of Tranes on a female cone during one night with the minimum pollen load data (previously given), the average potential pollen delivery is 190 grains per ovule (i.e., 8 weevils, ;2000 pollen grains per weevil, and a maximum of 84 ovules), assuming that weevils deliver all grains from their bodies and visit all ovules per cone. Even if Tranes deliver only ;2.5% of the pollen grains from their body to an ovule, each ovule would receive on average 4.8 grains—close to the average number of grains found in the pollen chamber of each ovule in our fluorescence microscopy analysis. By using average values rather than minimum ones for insect arrivals, for pollen loads and for ovules per cone, the number of grains delivered to each ovule increases by ;20 fold. (Here, 14 Tranes visit one cone of 54 ovules on a single night, and each weevil carries 14 700 grains.) Totals of 61, 110, and 72 thrips (avg. 5 81) were captured on the 1.5 cm wide sticky strip around each of three cones over 4 days. Extrapolating the sticky trap area to the complete cone surface area would give an average of 405 thrips visiting a female in 4 days (81 thrips caught 3 5, to correct for the total cone surface area). Based on minima for thrips visitors (61 thrips 3 5 5 305) and pollen loads of thrips arriving at female cones (13.2), and the maximum number of ovules recorded per cone (120), then 34 grains can be delivered to each
TABLE 5. The mean number (6SE) of pollen grains found per ovule in different cone regions of each Macrozamia species, based on random selection of three ovules from each section for each of three cones (i.e., 9 ovules per cone, or 27 total for each species). Cone region
Top Middle Bottom GLM statistics, within species Overall mean (SE) and range
Macrozamia lucida No. of grains per ovule (SE)
Macrozamia machinii No. of grains per ovule (SE)
6.6 (1.3) 3.7 (0.64) 5.6 (1.49) F2,16 5 1.7, P . 0.1, N 5 19a 5.2 (0.78) range 2–12b
4.4 (1.2) 5.3 (2.2) 8.6 (2.1) F2,15 5 1.6 P . 0.1, N 5 18a 6.1 (1.07) range 2–15b
a N is lower than 27 (9 ovules per cone 3 3 cones) because not every ovule contained pollen (i.e., all were not visited by pollinators, as indicated also by the lack of fluorescent dye on their exterior surface). Regions within a species did not differ significantly. The percentage of ovules with grains was similar between species, 70% of sampled ovules for M. lucida and 66% for M. machinii. b Overall mean of grains per ovule between species is not significantly different, F 1,35 5 0.51, P . 0.1, ns.
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Fig. 3. Insects leaving their respective male Macrozamia cone host before take-off. (a) Tranes sp. weevil, and (b) Cycadothrips chadwicki (bar 5 2 mm).
ovule, which is more than adequate for pollination of all ovules. If average values are used (405 thrips visit a cone, 29.2 grains per thrips, 74 ovules per cone), then as many as 160 grains, on average, can be delivered to each ovule. From these estimates, only 3–15% of the pollen grains from thrips bodies must be deposited to obtain five grains per ovule, the average number of grains found in the pollen chambers of dissected ovules. DISCUSSION Macrozamia pollination by insects—The results demonstrate clearly that wind-vectored pollen is not required for seed set in either M. lucida or M. machinii and that the specialist insects associated with their cones are necessary and sufficient to accomplish the task, as has been found in other cycad/insect pollination studies (see Stevenson et al., 1998; also Terry, 2001; Wilson, 2002; Hall et al., 2004). The most significant and surprising result of the current study is that the fluorescent dye tracking (Fig. 2) and seed set studies (Tables 1–3) indicate that relatively few pollen-coated Cycadothrips or Tranes (either sex) weevils are required to deliver pollen to all ovules of a cone and produce pollination rates equal to the open control cones. The visitation rates of insect vectors measured in the field suggest that many more insects visit female cones (at least they did so during this study) than are needed for full pollination on both M. lucida (avg. estimate of 405 thrips visit a cone vs. ,100 thrips for full pollination) and M. machinii (avg. 14 weevils vs. ;2 weevils for full pollination). On other Macrozamia species, female cones with 100–200 ovules per cone have attracted thousands of Cycadothrips and .100 Tranes weevils (Chadwick, 1993; Mound et al., 1998; Mound and Terry, 2001; Terry, 2001). The robustness of these results is reinforced by the fact that the complementary seed set and fluorescence experiments yielded similar values for pollinator effectiveness. Whereas the
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seed set tests were conducted under conditions that may have inflated the values (e.g., use of pollen-coated insects that remained on caged cones for many days), the dye-tracking experiments were conducted only for one night. The dye tests may even have underestimated the effectiveness of pollinators because small deposits of dye, especially from thrips, may not have been detected. The relatively low numbers of pollinator visits to female cones relative to visits to conspecific male cones observed in these and other cycads (Mound and Terry, 2001; Terry, 2001) is consistent with lower peak thermogenic temperature elevation in female cones and their lower volatile production rates (Terry et al., 2004a, b). This correlates with a lower energetic investment by the females (Seymour et al., 2004; Roemer et al., in press) indicating an energetically efficient pollinator attraction mechanism for the females, if these traits are indeed involved with attraction. Macrozamia machinii females would benefit from attracting Tranes weevils in small numbers because cones are damaged when visited by large numbers (Fig. 1b). By contrast, the larger cost of increased energetic output by male cones would be balanced by a gain in attracting pollinators, which in turn would lead to a greater potential spread of that cone’s pollen to more female cones than a cone with lower output. Even though Tranes weevils can completely destroy male cones, this generally occurs during later dehiscence stages. Most beetle cycad pollinators develop only on the male cones, and then primarily on sporophyll and peduncle tissue rather than on sporangia and pollen, and some feed on female cones at times when ovules cannot be damaged (Donaldson, 1997; Stevenson et al., 1998). With regard to damage, Cycadothrips may be a more benign partner in pollination mutualisms because adults and larvae feed only on limited amounts of pollen (Mound and Terry, 2001) and have not been observed to damage cones of either sex. Tissue damage to female M. machinii cones seems to have contributed to the much larger variation in seed set recorded on this species compared with the thrips-pollinated M. lucida (Tables 1–3). Specifically, the percentage seed set was lower on some cones that had the higher numbers of Tranes weevils. In addition, other aspects of Tranes biology may have influenced pollinator effectiveness with increasing numbers of insects per cone. For example, insects may aggregate as numbers increase, so that they move less through a cone, or mating behavior or male–male fighting may interfere with movement through a cone. Only more targeted tests will differentiate how various factors (pollen loads, damage by weevils, insect behavior) affect seed-set rates under field conditions. The variability in pollen loads among samples of insects was very high and is presumably related to the large number of factors that affect the acquisition and transport of pollen. Differences in pollen loads between insects leaving male cones and arriving at female cones was expected, because several behaviors (e.g., grooming or mating before take-off, flight, deposition of pollen on ovules) would result in pollen loss. Variability in the pollen loads of insects arriving at female cones would also naturally depend on the origins of these weevils (from female or male cones), the flight distance between cones, and the dehiscence stage of the source male cones. Lower pollen loads on Tranes lyterioides weevils than those recorded from the Tranes species used in the current study have been reported from individual weevils (directly from washes) arriving at M. communis female cones (.5050/individual) (Terry, 2001) and those on Lepidozamia peroffskyana
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male cones (,4230) (Hall et al., 2004). However, Donaldson (1997) recorded .10 000 pollen grains per individual Porthetes pearsonii weevil on the cycad Encephalartos villosus. The highest pollen loads on Tranes weevils leaving male M. machinii cones are only slightly lower than those reported for some bee species; e.g., pollen loads of 213 170–247 630 were extracted from the scopa of Megachile addenda visiting cranberry flowers (Cane et al., 1996). Pollen loads of Cycadothrips albrechti on M. macdonnellii and of C. chadwicki on M. communis (Mound and Terry, 2001; Terry, 2001) are within the low range of counts from the current studies and are also within range of those reported for other thrips species on angiosperms (Kirk, 1997). In summary, the specialist pollinators of Macrozamia take up as much pollen as do some other specialist pollinators. They also appear to have specific behaviors within the cone that ensure the successful transfer of pollen grains to the appropriate part of the ovule (Fig. 2). Rewards for visiting cones—The question often arises: why do insects visit a nonrewarding female cycad cone? Female cones have the same general pattern of volatile release and thermogenesis (but at reduced levels) as their conspecific males (Terry et al., 2004a), and thus they may lure the pollinator by deceit, if these traits are involved in attraction. However, it should first be determined whether females give any type of reward (Dufay¨ and Anstett, 2003; Renner, in press) and what cost is sustained by the pollinator for visiting a cone. Male cones provide warmth (at least during thermogenesis), food, mating sites, oviposition substrates and resources for development. Female cones, although not a normal feeding and brood site, may provide some small rewards: warmth (females of only some species); protection (Tang, 1987); and the pollen droplet, which contains amino acids and carbohydrates (Tang, 1993). Whereas the small volume of liquid in pollen droplets (0.01–0.77 mL measured from six species in six genera: Tang, 1993) may be of limited benefit to a 22 mg Tranes weevil (and much less when beetles are present in high numbers), the 0.01 mL droplet reported for M. lucida (Tang, 1993) may be a substantial reward for a ;30 mg thrips. In this study, dye granules from fluorescence experiments were concentrated around the micropyle in thrips-pollinated cones, but were scattered across the ovule and throughout the cone by weevils (Fig. 2). This indicates that thrips move toward the micropyle, possibly to feed on the pollen droplet. Dependent mutualisms in cycads—All the known insect pollinators of cycads (Oberprieler, 2004; Tang, 2004) are involved in highly specialized dependent mutualisms with their hosts, i.e., neither could survive in the long term without the other. Unlike many of the well-known obligate mutualists of angiosperms that are seed parasites as well as pollinators of their floral hosts, most, but not all, cycad pollinators tend to avoid female ovule tissue (see Stevenson et al., 1998; Mound and Terry, 2001; Hall et al., 2004). Pollination in these cycads appears to be by passive means, i.e., there are no modified or specialized morphological structures associated with acquisition or deposition of pollen that qualify as ‘‘active pollination’’ as in Yucca pollination (Pellmyr and Krenn, 2002). Both thrips and weevils seem, from the available evidence, to have specialized pollen delivery behaviors appropriate to Macrozamia pollination, including movement between cones at times when cones are thermogenic and are producing volatiles (Terry et
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al., 2004a). Whether this behavior qualifies as ‘‘active pollination’’ or whether some undetected structure is involved with pollen deposition is not clear, but the movement of insects within cones does not appear to be random, especially with regard to Cycadothrips, which concentrate their activity around the micropyle. The diversity and taxonomic breadth of specialist insects (including proven pollinators) (Oberprieler, 1995a, b, 2004) associated with the ;300 cycad species is higher than that reported in many of the highly specialized dependent angiosperm pollination systems (Cook and Rasplus, 2003; Pellmyr, 2003). Most of the pollinators of these angiosperm systems involve only one genus or family of insects. Even within the fig-wasp–fig system, with over 750 species of fig worldwide, pollinator diversity is restricted to species within the hymenopterous family Agaonidae (Chalcidoidea) (Cook and Rasplus, 2003). The diversity associated with cycads (both confirmed and putative) includes many families and subfamilies within the Curculionoidea and families within Erotylidae and Boganiidae (Tang, 2004). In part, this diversity may be related to the age of cycads, which originated by at least the Permian (Norstog and Nicholls, 1997). It is not clear whether the earliest cycads were insect-pollinated, but even species of the presumed more primitive Cycas genus (Brenner et al., 2003a, b) may be insect-pollinated (Norstog and Nicholls, 1997); and recent evidence from Triassic-aged cycad fossil cones suggests active pollinivory of cycads (Klavins et al., 2003, 2005). Many of the weevil pollinators of cycads are thought to have evolved much later, during the late Cretaceous and early Cenozoic ,100 mya (Oberprieler, 2004). In all, there may have been many gains and losses of pollinators across different regions and across geologic time periods, especially during the Cretaceous/Paleogene boundary (5 K/T boundary), such that extant cycads are the relicts of those that survived mass extinctions along with their local pollinators or that survived by acquiring new pollinators that shifted from other hosts (Oberprieler, 2004). In summary, the results of the present study contribute significant pieces to the small but growing body of knowledge regarding the pollination of cycads. Only a limited number of the known specialist beetles (members of the Languriidae [Erotylidae], Boganiidae and the more cosmopolitan superfamily Curculionoidea) on cycad cones have been tested for their pollination potential or have had their behavior examined relative to cycad pollination (see Schneider et al., 2002; Tang, 2004). Pollination studies have begun to untangle a complex set of insect–host-plant relationships associated with cycad cones, some of which are not linked to cycad pollination. For example, some insects are simply seed parasites, e.g., Antliarhinus zamiae on Encephalartos villosus (Donaldson, 1997), or phytophages feeding on other cone tissues (Donaldson et al., 1995; Oberprieler, 1995a), although diverse phytophagous insect groups are involved in cycad pollination. Many questions about the origin, diversity, and dynamics of cycad pollination systems remain unanswered, but some patterns are beginning to emerge that will be helpful in understanding the evolution of these dependent mutualisms. With regard to cycad conservation, a focus on the protection of adult plants may be most important in terms of immediate intrinsic rates of increase, according to models that test the removal of various aged/ staged plants or seeds from populations (Raimondo and Donaldson, 2003). Nevertheless, the long-term health, genetic di-
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versity, and dispersal of cycad populations ultimately depend also upon the health and viability of the pollinator population. LITERATURE CITED BOND, W. J. 1994. Do mutualisms matter? Assessing the impact of pollinator and disperser disruption on plant extinction. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 344: 83– 90. BRENNER, E. D., D. W. STEVENSON, R. W. MCCOMBIE, M. S. KATARI, S. A. RUDD, K. F. MAYER, P. M. PALENCHAR, S. J. RUNKO, R. W. TWIGG, G. DAI, R. A. MARTIENSSEN, P. N. BENFEY, AND G. M. CORUZZI. 2003a. Expressed sequence tag analysis in Cycas, the most primitive living seed plant. Genome Biology 4: R78. BRENNER, E. D., D. W. STEVENSON, AND R. W. TWIGG. 2003b. Cycads: evolutionary innovations and the role of plant-derived neurotoxins. Trends in Plant Science 8: 446–452. CANE, J. H., D. SHIFFHAUER, AND L. J. KERVIN. 1996. Pollination, foraging, and nesting ecology of the leaf-cutting bee Megachila (Delomegachile) addenda (Hymenoptera: Megachilidae) on cranberry beds. Annals of the Entomological Society of America 89: 361–367. CHADWICK, C. E. 1993. The roles of Tranes lyterioides and T. sparsus Boh (Col., Curculionidae) in the pollination of Macrozamia communis (Zamiaceae). In D. W. Stevenson and N. J. Norstog [eds.], Proceedings of CYCAD 90, second international conference on cycad biology, Townsville, 1990, QLD, Australia, 77–80. Palm and Cycad Societies of Australia Ltd., Milton, Queensland, Australia. CHADWICK, C. E. 1998. Some insects and other invertebrates associated with the Australian cycad Macrozamia communis L. Johnson (Zamiaceae). Encephalartos 54: 10–18. CHAMBERLAIN, C. J. 1919. The living cycads. University of Chicago Press, Chicago, Illinois, USA. CHAMBERLAIN, C. J. 1935. Gymnosperms: structure and evolution. University of Chicago Press, Chicago, Illinois, USA. CHAW, S.-M., C. L. PARKINSON, Y. CHENG, T. M. VINCENT, AND J. D. PALMER. 2000. Seed plant phylogeny inferred from all three plant genomes: monophyly of extant gymnosperms and origin of Gnetales from conifers. Proceedings of the National Academy of Sciences, USA 97: 4086–4096. CONNELL, S. W., AND P. G. LADD. 1993. Pollination biology of Macrozamia riedlei—the role of insects. In D. W. Stevenson and N. J. Norstog [eds.], Proceedings of CYCAD 90, second international conference on cycad biology, Townsville, 1990, QLD, Australia, 96–102. Palm and Cycad Societies of Australia Ltd., Milton, QLD, Australia. COOK, J. M., D. BEAN, S. A. POWER, AND D. J. DIXON. 2004. Evolution of a complex coevolved trait: active pollination in a genus of fig wasps. Journal of Evolutionary Biology 17: 238–246. COOK, J. M., AND J.-Y. RASPLUS. 2003. Mutualists with attitude: coevolving fig wasps and figs. Trends in Ecology and Evolution 18: 241–248. DEGHAN, B., AND N. B. DEGHAN. 1988. Comparative pollen morphology and taxonomic affinities in Cycadales. American Journal of Botany 75: 1501– 1516. DONALDSON, J. S. 1992. Adaptation for oviposition into concealed cycad ovules in the cycad weevils Antliarhinus zamiae and A. signatus (Coleoptera: Curculionidae). Biological Journal of the Linnean Society 47: 23– 35. DONALDSON, J. S. 1993. Insect predation of ovules in the South African species of Encephalartos (Cycadales: Zamiaceae). In D. W. Stevenson and N. J. Norstog [eds.], Proceedings of CYCAD 90, second international conference on cycad biology, Townsville, 1990, QLD, Australia, 103–108. Palm and Cycad Societies of Australia Ltd., Milton, QLD, Australia. DONALDSON, J. S. 1997. Is there a floral parasite mutualism in cycad pollination? The pollination biology of Encephalartos villosus (Zamiaceae). American Journal of Botany 84: 1398–1406. DONALDSON, J. S. 2003. Cycads, status survey and conservation action plan. IUCN/SSC-Cycad Specialist Group. IUCN, Gland, Switzerland. DONALDSON, J. S., I. NANNI, AND J. D. W. BO¨SENBERG. 1995. The role of insects in the pollination of Encephalartos cycadifolius. In P. Vorster [ed.], Proceedings of the third international conference of cycad biology, Pretoria, South Africa, 1993, 423–434. Cycad Society of South Africa, Stellenbosch, South Africa. DUFAY¨, M., AND M.-C. ANSTETT. 2003. Conflicts between plants and polli-
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CYCAD POLLINATORS
939
nators that reproduce within inflorescences: evolutionary variations on a theme. Oikos 100: 3–14. FLEMING, T. H., AND J. N. HOLLAND. 1998. The evolution of obligate pollination mutualisms: senita cactus and senita moth. Oecologia 114: 368– 375. FORSTER, P. I. 2004a. Classification concepts in Macrozamia (Zamiaceae) from eastern Australia. In T. Walters and R. Osborne [eds.], Cycad classification—concepts and recommendations, 85–94. CAB International, Wallingford, UK. FORSTER, P. I. 2004b. The cycads of Queensland—diversity and conservation. Palms and Cycads 82: 4–28. FORSTER, P. I., AND P. J. MACHIN. 1994. Cycad host plants for Lilioceris nigripes (Fabricius) (Coleoptera: Chrysomelidae) and Theclinesthes onycha (Hewitson) (Lepidoptera: Lycaenidae). Australian Entomologist 21: 99–102. FORSTER, P. I., P. J. MACHIN, L. A. MOUND, AND G. W. WILSON. 1994. Insects associated with reproductive structures of cycads in Queensland and north-east New South Wales, Australia. Biotropica 26: 217–222. HALL, J. A., G. H. WALTER, D. M. BERGSTROM, AND P. J. MACHIN. 2004. Pollination ecology of the Australian cycad Lepidozamia peroffskyana (Zamiaceae). Australian Journal of Botany 52: 333–343. HILL, K. D., M. W. CHASE, D. W. STEVENSON, H. G. HILLS, AND B. SCHUTZMAN. 2003. The families and genera of cycads: a molecular phylogenetic analysis of Cycadophyta based on nuclear and plastid DNA sequences. International Journal of Plant Sciences 164: 933–948. JONES, D. L. 2002. Cycads of the world. Reed New Holland, Sydney, Australia. JONES, D. L., AND P. I. FORSTER. 1994. Seven new species of Macrozamia section Parazamia (Miq.) Miq. (Zamiaceae section Parazamia) from Queensland. Austrobaileya 4: 269–288. JONES, D. L., P. I. FORSTER, AND I. K. SHARMA. 2001. Revision of the Macrozamia miquelii (F. Muell.) A.D.C. (Zamiaceae section Macrozamia) group. Austrobaileya 6: 67–94. KATO, M., A. TAKIMURA, AND A. KAWAKITA. 2003. An obligate pollination mutualism and reciprocal diversification in the tree genus Glochidion (Euphorbiaceae). Proceedings of the National Academy of Sciences, USA 100: 5264–5267. KAWAKITA, A., AND M. KATO. 2004. Evolution of obligate pollination mutualism in New Caledonian Phyllanthus (Euphorbiaceae). American Journal of Botany 91: 410–415. KIRK, W. D. J. 1997. Feeding. In T. Lewis [ed.], Thrips as crop pests, 119– 174. CAB International, Wallingford, UK. KLAVINS, S. D., D. W. KELLOGG, M. KRINGS, E. L. TAYLOR, AND T. N. TAYLOR. 2005. Coprolites in a middle Triassic cycad pollen cone: evidence for insect pollination in early cycads? Evolutionary Ecology Research 7: 479–488. KLAVINS, S. D., E. L. TAYLOR, M. KRINGS, AND T. N. TAYLOR. 2003. Gymnosperms from the middle Triassic of Antarctica: the first structurally preserved cycad pollen cone. International Journal of Plant Sciences 164: 1007–1020. LESCHEN, R. A. B. 2003. Erotylidae (Insecta: Coleoptera: Cucujoidea): phylogeny and review. Fauna of New Zealand 47: 1–108. MARULLO, R., AND L. A. MOUND. 1995. Su una classificazione sopra-generica della famiglia Aeolothripidae (Thysanoptera). Atti del XVII Congresso Nazionale Italiano di Entomologia, Udine, Italy, 1994, 87–90. Congresso Nazionale Italiano di Entomologia, Udine, Italy. MOUND, L. A. 1991. The first thrips species (Insecta, Thysanoptera) from cycad male cones, and its family level significance. Journal of Natural History 25: 647–652. MOUND, L. A., E. DEN HOLLANDER, AND L. DEN HOLLANDER. 1998. Do thrips help pollinate Macrozamia cycads? Victoria Entomologist 28: 86– 88. MOUND, L. A., AND I. TERRY. 2001. Thrips pollination of the central Australian cycad, Macrozamia macdonnellii (Cycadales). International Journal of Plant Sciences 162: 147–154. NORSTOG, K., AND P. K. S. FAWCETT. 1995. Insect pollination of cycads: evolutionary and ecological considerations. In P. Vorster [ed.], Proceedings of the third international conference of cycad biology, Pretoria, South Africa, 1993, 265–285. Cycad Society of South Africa, Stellenbosch, South Africa. NORSTOG, K. J., AND T. J. NICHOLLS. 1997. The biology of the cycads. Cornell University Press, Ithaca, New York, USA. NORSTOG, K., D. W. STEVENSON, AND K. J. NIKLAS. 1986. The role of beetles
940
AMERICAN JOURNAL
in the pollination of Zamia furfuracea L.fil. (Zamiaceae). Biotropica 18: 300–306. OBERPRIELER, R. G. 1995a. The weevils (Coleoptera: Curculionoidea) associated with cycads. 1. Classification, relationships, and biology. In P. Vorster [ed.], Proceedings of the third international conference of cycad biology, Pretoria, South Africa, 1993, 295–334. Cycad Society of South Africa, Stellenbosch, South Africa. OBERPRIELER, R. G. 1995b. The weevils (Coleoptera: Curculionoidea) associated with cycads. 2. Host specificity and implications for cycad taxonomy. In P. Vorster [ed.], Proceedings of the third international conference of cycad biology, Pretoria, South Africa, 1993, 335–365. Cycad Society of South Africa, Stellenbosch, South Africa. OBERPRIELER, R. G. 2004. ‘‘Evil weevils’’—the key to cycad survival and diversification? In A. Lindstrom [ed.], Proceedings of the 6th international cycad conference on cycad biology, Pattaya, Thailand, 2002, 170– 194. Nong Nooch Tropical Botanical Garden, Thailand. ORNDUFF, R. 1991a. Coning phenology of the cycad Macrozamia riedlei (Zamiaceae) over five-year interval. Bulletin of the Torrey Botanical Club 118: 6–11. ORNDUFF, R. 1991b. Size classes, reproductive behavior, and insect associates of Cycas media (Cycadaceae) in Australia. Botanical Gazette 152: 203– 207. PELLMYR, O. 2003. Yuccas, yucca moths, and coevolution: a review. Annals of the Missouri Botanical Garden 90: 35–55. PELLMYR, O., AND H. W. KRENN. 2002. Origin of a complex key innovation in an obligate insect–plant mutualism. Proceedings of the National Academy of Sciences, USA 99: 5498–5502. PELLMYR, O., AND K. A. SEGRAVES. 2003. Pollinator divergence within an obligate mutualism: two yucca moth species (Lepidoptera; Prodoxidae: Tegeticula) on the Joshua tree (Yucca brevifolia; Agavaceae). Annals of the Entomological Society of America 96: 716–722. RAI, H. S., H. E. O’BRIEN, P. A. REEVES, R. G. OMSTEAD, AND S. W. GRAHAM. 2003. Inference of higher-order relationships in the cycads from a large chloroplast data set. Molecular Phylogenetics and Evolution 29: 350–359. RAIMONDO, D. C., AND J. S. DONALDSON. 2003. Responses of cycads with different life histories to the impact of plant collecting: simulation models to determine important life history stages and population recovery times. Biological Conservation 111: 345–358. RENNER, S. S. In press. Rewardless flowers in the angiosperms and the role of insect cognition in their evolution. In N. M. Waser and J. Ollerton [eds.], Specialization and generalization in pollination systems. University of Chicago Press, Chicago, Illinois, USA. ROEMER, R., I. TERRY, C. CHOCKLEY, AND J. JACOBSEN. In press. Experimental evaluation and thermo-physical analysis of thermogenesis in male and female cycad cones. Oecologia.
OF
BOTANY
[Vol. 92
SCHNEIDER, D., M. WINK, F. SPORER, AND P. LOUNIBOS. 2002. Cycads: their evolution, toxins, herbivores and insect pollinators. Naturwissenchaften 89: 281–294. SEYMOUR, R. S., I. TERRY, AND R. B. ROEMER. 2004. Respiration and thermogenesis by cones of the Australian cycad, Macrozamia machinii. Functional Ecology 18: 925–930. STEVENSON, D. W., K. J. NORSTOG, AND P. K. S. FAWCETT. 1998. Pollination biology of cycads. In S. J. Owens and P. J. Rudall [eds.], Reproductive biology, 277–294. Royal Botanic Gardens, Kew, UK. TANG, W. 1987. Insect pollination in the cycad Zamia pumila (Zamiaceae). American Journal of Botany 74: 90–99. TANG, W. 1993. Nectar-like secretions in female cones of cycads. The Cycad Newsletter 16: 10–13. TANG, W. 2004. Cycad insects and pollination. In P. C. Srivastava [ed.], Vistas in paleobotany and plant morphology: evolutionary and environmental perspectives, 383–394. U.P. Offset, Lucknow, India. TANG, W., R. G. OBERPRIELER, AND S.-L. YANG. 1999. Beetles (Coleoptera) in cones of Asian Cycas: diversity, evolutionary patterns and implications for Cycas taxonomy. In C. J. Chen [ed.], Biology and conservation of cycads, proceedings of the fourth international conference on cycad biology, Panzhihua, Sichuan, China, 1996, 280–297. International Academic Publishers, Beijing, China. TERRY, L. I. 2001. Thrips and weevils as dual, specialist pollinators of the Australian cycad Macrozamia communis (Zamiaceae). International Journal of Plant Sciences 162: 1293–1305. TERRY, I., C. J. MOORE, P. I. FORSTER, G. H. WALTER, P. J. MACHIN, AND J. DONALDSON. 2004a. Pollination ecology of the genus Macrozamia: cone volatiles and pollinator specificity. In A. Lindstrom [ed.], Proceedings of the 6th international conference on cycad biology, Pattaya, Thailand. 2002, 155–169. Nong Nooch Tropical Botanical Garden, Thailand. TERRY, I., C. J. MOORE, G. H. WALTER, P. I. FORSTER, R. ROEMER, J. S. DONALDSON, AND P. J. MACHIN. 2004b. Association of cone thermogenesis and volatiles with pollinator specificity in Macrozamia cycads. Plant Systematics and Evolution 243: 233–247. TREUTLEIN, J., AND M. WINK. 2002. Molecular phylogeny of cycads inferred from rbcL sequences. Naturwissenchaften 89: 221–225. VOVIDES, A. P. 1991. Insect symbionts of some Mexican cycads in their natural habitat. Biotropica 23: 102–104. VOVIDES, A. P., N. OGATA, V. SOSA, AND E. PEN˜A-GARCI´A. 1997. Pollination of endangered Cuban cycad Microcycas calocoma (Miq.) A. DC. Botanical Journal of the Linnean Society 125: 201–210. WEIBLEN, G. D. 2004. Correlated evolution in fig pollination. Systematic Biology 53: 128–139. WILSON, G. W. 2002. Insect pollination in the cycad genus Bowenia Hook. ex Hook. f. (Stangeriaceae). Biotropica 34: 438–441.
