Insect-Cycad Symbiosis and its Relation to the Pollination of Zamia furfuracea (Zamiaceae) by

Rhopalotria mollis (Curculionidae)

Author(s): Knut J. Norstog and Priscilla K. S. Fawcett
Reviewed work(s):
Source: American Journal of Botany, Vol. 76, No. 9 (Sep., 1989), pp. 1380-1394
Published by: Botanical Society of America
Stable URL: http://www.jstor.org/stable/2444562 .
Accessed: 27/03/2012 00:14

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .
http://www.jstor.org/page/info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org.

Botanical Society of America is collaborating with JSTOR to digitize, preserve and extend access to American
Journal of Botany.

http://www.jstor.org
Amer.J. Bot. 76(9): 1380-1394. 1989.

INSECT-CYCAD SYMBIOSIS AND ITS RELATION TO THE

POLLINATION OF ZAMIA FURFURACEA (ZAMIACEAE) BY
RHOPALOTRIA MOLLIS (CURCULIONIDAE)"12

KNUT J. NORSTOG AND PRISCILLA K. S. FAWCETT

FairchildTropicalGarden, 11935 Old CutlerRoad, Miami, Florida 33156

ABSTRACT
Zamiafutfuracea,a cycadof Mexicanorigin,in cultivationis pollinatedby the snout weevil,
Rhopalotriamollis, also of Mexican origin. This weevil apparentlyis host specific, swarming
upon male cones of the cycad, where mating, feeding, and oviposition occur. Sporophyllsof
male cones are rich in starch;those of female cones are poor in starch,and weevils feed upon
male cones and are visitors to but not feedersupon or within female cones. Pollen transport
to female cones occurs duringsuch visitation. All stages of metamorphosisof R. mollis occur
within male cones; larvae feed exclusivelyon parenchymaof microsporophylls,pupatewithin
stalksof microsporophylls,and emergeas adultsfromthe outerends of microsporophylls.They
do not feed on pollen and do not damage microsporangiaor pollen. Toward the end of the
breedingseason of the weevil (and the cycad),some larvaeenterdiapausein thick-walledpupal
cases within microsporangialstalks of pollen-spentcones. These remain in diapauseuntil the
next reproductiveseason of the cycad.

CYCAD pollination has been of particular in-
tetest to us because of the large number of
exotic cycad genera and species growing at
Fairchild Tropical Garden (FTG) which re-
quire artificial pollination in order to produce
viable seeds. In most cases, male and female
plants of the same species are growing and
coning at no great distance from each other;
often the sexes are separated by only a few
meters, sometimes by just a meter or two, yet
remain infertile unless hand-pollinated.
As we have related elsewhere, a fairly com-
mon assumption has been that cycads are wind-
pollinated plants, as are most other gymno-
sperms (Norstog and Stevenson, 1980; Niklas
and Norstog, 1984; Norstog, Stevenson, and
Niklas, 1986; Norstog, 1987). If so, one won-
ders why healthy and cone-producing trans-
plants to not behave in cultivation as they are
supposed to do in nature. During the past de-
cade, we have accumulated evidence, both cir-
cumstantial and experimental, that a Mexican
cycad, Zamia furfuracea, is in cultivation ef-
fectively pollinated by a small snout weevil,
Rhopalotria mollis (Fig. 1-6), which completes
at least one phase of its life cycle in pollen cones
of that cycad (Norstog and Stevenson, 1980;
Norstog et al., 1986). Field studies of polli-
nation of Z.furfuracea have not yet been made,
but R. mollis is present in wild plants in Ve-
I Receivedfor publication13 September1988;revision
accepted 10 March 1989.
2 We dedicatethis paperto Dr. ArthurMontgomeryfor
his long-time interestin and supportof cycad studies at of the relationship (Fig. 5-24). Representative
FairchildTropicalGarden.
racruz and inhabits male cones of this cycad
in much the same way as related here (A. Vo-
vides, personal communication). Now, we
present further evidence that the relationships
between this insect and cycad are much more
intimately and mutually interrelated than we,
or others, have reported earlier for any insect-
cycad interaction (see for ex. Rattray, 1913;
Baird, 1938; Norstog et al., 1986; Tang, 1987).
MATERIALS AND METHODS-Most observa-
tions reported below were made during June-
August 1988, upon a planting of 40 individuals
of Zamia furfuracea L. fil. (FTG Accession
No. 66-341) collected in Mexico and planted
at FTG (Fig. 1). Eighteen female plants are
interspersed among the male plants, and cones
of both male and female plants are sheltered
and partially hidden by foliage (Fig. 1). During
July and into August, these plants year-after-
year harbor large numbers of Rhopalotria mol-
lis Sharp, an exotic snout weevil probably in-
troduced from Mexico years ago along with Z.
furfuracea.
In order to obtain a better idea of the be-
havior of the insect (Fig. 5) during those phases
of its life in which it is involved with male and
female cones of Z. furfuracea, we collected
specimens daily and examined partially and
wholly dissected cones and their inhabitants.
Sketches, drawings, and photomicrographs
were prepared in order to record certain aspects
samples of cones and weevils were fixed in

1380
 ?Z.

^f^-#?--E~~~~~W Evv*

Fig. 1-4. Pollination of Zamia furfuraceaby Rhopalotriamollis. 1. Female (left) and male (center)cones of Z.
furfuracea.Male cones are at severalstagesof pollen developmentand weevil infestation.Note weevil swarmson two
cones in center of view. x 0.2. 2. Swarm of R. mollis on pollen cone of Z. furfuracea.Copulation,feeding, and egg
layingoccuralmost simultaneously.x 1. 3. Oldercone of Z. furfuraceaafterpollen dispersal.Note weevil holes in ends
of sporophylls(at arrows). x 2. 4. Pollen-receptivefemale cone of Z. furfuracea;note vertical cleft throughwhich
pollinatorsenter interior. x 0.8.
1382 AMERICANJOURNALOF BOTANY [Vol. 76

Fig. 5. Rhopalotriamollis Sharp.a. Female pupa. b. Adult female, ventral view. c. Adult female, dorsal view. d.
Male pupa, note its shorter snout. e. Adult male, ventral view, note inflated front femurs, short snout, large eyes,
genitaliacf. female. f. Adult male dorsal view. All are x 13.5.

FAA, CRAF, and OS04 for sectioning and
study.
Microsporophyllswere collected and fixed
in FAA just before, during, and just after egg-
layingactivity was observed;megasporophylls
were collected and fixed in FAA at the time
female cones were pollen receptive. Freehand
sections of portions of male and female cones
were stained with I2KI and sudan IV, and mi-
crotome sections of cones and weevils were
stained in PAS (periodic acid-Schiff, for car-
bohydrate),ninhydrin-Schiff(forprotein),I2KI-
erythrosin(weevils, for protein and starch)or
12KI-fastgreen(cones, for starchand cellulose),
and sudan IV (for lipids) (Jensen, 1962).
Our observations and conclusions are nec-
essarily rather subjective, since we made no
effortto do large-scalesamplings;however, in
no case do we report instances of unique be-
havior of single individuals-in most cases,
observations of particular actions were re-
peatedly observed.
RESULTSAND OBSERVATIONS-Rhopalotria
mollis is an excellent flyerand duringthe early
part of the reproductiveseason of Z. furfura-
September1989] NORSTOGAND FAWCETT-POLLINATIONOF ZAMIA 1383

4 .- ~~~~~~excavated

I U**

Fig. 6. Examplesof feedingbehaviorof R. mollis on Z. furfuracea.All but f are drawnfrom life using a dissecting
microscope.a. Three feeding(and oviposition) holes in side of microsporophyllend. This stagewhen dissectedwould
approximatelycorrespondto d. x 4.8. b. Somewhatlater than a. Some sporophyllseparationhas taken place and a
few microsporangiaare visible. This stagewould correspondapproximatelyto diagramf. x 4.8. c. Eggand earlylarva
of R. mollis. x 14.5. d. Dissection of microsporophyllto show egg chamberbeneath feeding (and oviposition) holes.
x 4.8. e. Full-sizelarva at time of pupal case formation. x 14.5. f Diagramshowing larva encapsulatedin pupal case
of its own construction.It has entirely excavated the interior of the sporophyll,but microsporangiaremain intact.
x 4.8. g. Microsporophyllafter adult weevil has emergedby chewingthroughthe tip. Althoughmicrosporangiahave
dehisced and dischargedpollen, they have not been damaged by weevils. Occasionallyweevils will emerge through
side of sporophylland then some disruptionof microsporangiaoccurs. x 4.8.

cea, males and females are attracted to male
cones where they feed upon sporophyll tissue
by making small holes in the ends of the spo-
rophylls through which they probe for food
(Fig. 2; 6a, b). Presumably,because the snouts
offemales areslightlylongerthanthose of males
(Fig. 5), they are able to probe more deeply.
Cones of both sexes of Z. furfuraceaare buff-
colored as is R. mollis, and it is noteworthy
thatanotherspeciesof Rhopalotria,R. slossoni,
1384 AMERICAN JOURNAL OF BOTANY [Vol. 76

Fig. 7. Diagramillustratingreproductivecycle of R. mollis duringearlyand middle phasesof its breedingbehavior

and of pollen productionby Z. furfuracea.a. Male plant. b. Male cone with swarmingweevils. c. Pollen-receptive
female cone and visitation by weevils. d. Cross section of male cone showing weevil feeding, oviposition, and larval
activity. e. Egg and several larvae excavatinga microsporophyll.Commonly only one larva survives in a sporophyll.
f. Larvain pupal case. g. Pupa. h. Emergenceof adult. The entire cycle is completed in about 7-9 days.

which pollinates Zamia pumila (Tang, 1987)
is black and brown and blends with the darker
cones of that cycad. Both weevils, therefore,
have coloration matching the cones of the
species they pollinate.
Mating takes place at this time and the fe-
males lay eggs in the feeding holes. This ap-
parentlyis a generalmode of oviposition among
female snout weevils which may employ their
long snouts to position the eggs in the holes
(Comstock, 1936). As a result, the eggs of R.
mollis come to lie in hollow spaces beneath
the feeding holes (Fig. 6d, 15);the diameter of
the egg is slightly less than that of the snout of

Fig. 8-13. Sections showing parenchymain distal ends of micro- and megasporophyllsof Z. furfuracea.8. Mi-
crosporophyllsection stainedwith 12KIand fast green.Cells (arrow)are filledwith starchgrains;idioblastat arrowhead.
Inset-starch (arrow)and idioblast (i). x 100; inset x 400. 9. Megasporophyllsections stainedwith I2KIand fast green
showingabsence of starch.Some idioblasts (arrow)appearempty, others (arrowhead)tend to stain with I2KI.Inset-
empty idioblast (i) containingtrabeculae. x 100; inset x 400. 10. Microsporophyllsection stained with PAS (for car-
bohydrate)showingdenselystainedstarchcells (arrow),unstainedidioblasts(arrowhead).Inset-idioblast (i) and starch
(arrow). x 100; inset x 400. 11. Megasporophyllsection stained with PAS showing absence of starch,densely stained
idioblasts (arrowhead).Inset-idioblast (i). x 100; inset x 400. 12. Microsporophyllsection stained with ninhydrin-
September 1989] NORSTOGAND FAWCETT-POLLINATION OF ZAMIA 1385

I0 '

.''''' >'4
4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

'A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A

*. 'L\ 'a

Schiff,a proteinstain, showingdensely brown-purple-staining idioblasts (arrowhead).Inset-idioblast (i). x 100; inset

x 400. 13. Megasporophyllsection stainedwith ninhydrin-Schiff.Note that few idioblasts(arrow)stain densely, others
appearempty (arrowhead).Inset-idioblast (i). x 100; inset x 400.
1386 AMERICAN JOURNAL OF BOTANY [Vol. 76

.. ~ ~ ~ ~ :

* )A

T .

~~~~~~~~~~~~j,.

ZS*cX 3 ,4 ,',

Fig. 14-19. Tissues and organsof Z. furfuraceain relationto activities of R. mollis. 14. Sectionedmale cone axis
shows relativelylight starchcontentas indicatedby isolated starchgrains(arrow),cf. Fig. 8. x 100. 15. Microsporophyll
section with egg chamberand egg (E) of R. mollis, stainedwith I2K1. Note cells filled with starch(arrow)and remnants
of what appearto have been idioblasts(largearrowhead).The lattermay indicateavoidanceof toxins by feedingadults.
x 100. 16. Cross sectionsof male cone, cut surfacesof left-handsection stainedwith 12K1.Arrowis at junction between
more heavily stainedsporophylland lightly stainedaxis. x 1.3. 17. Megasporophyllsfrom postpollinationcone (ovules
September 1989] NORSTOG AND FAWCETT-POLLINATION OF ZAMIA 1387

the female weevil. Females are gravid upon
emerging from pupation, in fact eggs are found
in late pupal stages; three large eggs per female
is the usual number. Ends of microsporophylls
often become rather hollowed out and as many
as six eggs can be present in one sporophyll.
The weevils that start the annual breeding
cycle ofR. mollis on Z.furfuraceaemerge from
pupae, which in turn develop from larvae that
"winter" in old pollen cones at the base of the
cycads they later infest. At first the breeding
cycle of the weevil involves just a few indi-
viduals, but increasingly large numbers of off-
spring in succeeding generations soon follow.
Brood-place selection - Swarming weevils
exhibit cone preferences, selecting only one or
two cones from among as many as 15-20, and
remaining upon the chosen cone 1-2 days be-
fore abandoning it (Fig. 1, 2). (Male cones of
Z. furfuracea mature sequentially; the cones
upon which swarming occurs are those in which
sporophyll separation, which gives access to
the cone's interior, will occur within about 24-
48 hr; cf. Fig. 6a, b; 18a, b.)
When a cone opens up, weevils enter, shel-
tering and feeding and, in doing so, become
covered with pollen (Fig. 7d, 24c). We have
not, however, observed weevils feeding upon
pollen. A single cone at this stage may contain
more than 100 weevils, and it is at this stage
that visitation to female cones takes place. Fe-
male cones are not fed upon, although interior
surfaces sometimes exhibit healed scars of su-
perficial, punctate wounds which may have
been made by weevils (Fig. 17).
Larval stages-Within a day or so of ovi-
position, eggs hatch, producing larvae which
eat their way inwardly from the outer end of
a microsporophyll into its stalk (Fig. 7e, 24d).
Some larvae also find their way into the cone
axis but during the period of most intense wee-
vil activity in the planting, most of the larvae
are found in the sporophylls. The larvae are
legless grubs (vermiform), as is typical of wee-
vils (Borror and DeLong, 1964), and within
the confines of the sporophylls are quite active
and respond to the slightest touch by twisting
and turning vigorously.
Although up to six eggs may be laid in one
sporophyll, and as many hatchling larvae may
be present, subsequently each sporophyll will
contain only one large larva and, later on, a
single pupa or a recently metamorphosed adult.
Possibly surplus larvae migrate to other parts
of the cone, but a more likely outcome is that
as the larvae eat away the sporophyll tissue,
they meet, and one eats the other. We have
observed larvae biting each other, have found
that when several are confined in a petri dish,
only one or two survive-the others being rep-
resented only by shrivelled bits of cuticle. We
also have observed large larvae in sporophyll
chambers accompanied by the shrivelled re-
mains of others. We have not, however, ob-
served the entire process of cannibalism.
Sexual dimorphism- Pupae and adults ofR.
mollis are sexually dimorphic. Males have rel-
atively short, broad snouts, large bulging eyes
and inflated foreleg femurs; females have lon-
ger narrower snouts, smaller eyes and less-in-
flated femurs (Fig. 5). Sex ratios based upon
samplings of pupae and emerging adults in-
cluded slightly more females (55%) than males
(79 females: 65 males), among pupae, we found
34 females and 27 males (females = 5 6%). Male
and female pupae and adults were about the
same average size but among individuals of
each sex, an appreciable variation in size was
apparent.
Feeding behavior-With the possible excep-
tion of cannibalism mentioned above, larvae
feed almost exclusively on parenchyma; the gut
is packed with material that stains lightly with
12KI but intensely with PAS. Also included are
particles that stain heavily with ninhydrin-
Schiffand these are concentrated in the hindgut
(Fig. 20). Pupae, as one might expect, have
relatively empty fore- and hindguts (Fig. 21),
and adults killed and fixed before emerging
from pupae cases also had empty fore- and
hindguts (Fig. 22). Both had extensive storage
reserves. Adults taken after emerging, feeding,
and sheltering generally had full guts and de-

removed). There are punctatescars (arrows)remainingfrom insect (weevil?)bites. x 0.75. 18. Longitudinalsections
of preelongation(a), elongated(b), and postpollination(c) cones. Cone (a) is at the weevil egg-layingstage, (b) was
largerthan (a) priorto elongationand thereforedoes not reflectespeciallygreatelongation(see text);it is at the weevil-
shelteringstage. Cone (c) has releasedmost of its early-stageweevils but retainssome larvaein diapause.Its axis has
been riddledby larvae and is filled with frass. x 0.5. 19. Longitudinal-sectionof microsporophyllstalk with encased
larva(L).The caseis composedof brittle,brown-coloredmaterial(arrow),separatedfromthe outsideonly by sporophyll
epidermis(small arrowheads).x 33.
1388 AMERICAN JOURNAL OF BOTANY [Vol. 76

. .. S -t: / .

'
4
f

Fig. 2023. Metamorphosisof R. molis. 20. Longitudinalsection of larva stained with ninhydnin-Schiff,showing
full gut (G). Gut contents include heavily stained particles(arrows),which also are found in hindgut (largearrowat
September 1989] NORSTOG AND FAWCETT-POLLINATION OF ZAMIA 1389

Fig. 24. Diagramillustratingthe late stagesof weevil infestationof Z. furfuracea.a. Male plants.b. Weevils swarm
on male cones where copulating,feeding, and egg-layingoccurs, but differsin locale from earlieractivity (cf. Fig. 7).
c. Weevils do not lay eggs in outer partsof microsporophylls(which now contain chlorophyll)but ratherat sides and
in stalksof sporophylls.d. Larvaeexcavatemuch of sporophyllbut avoid outerends. e. Some early-seasonlarvaeform
thin-walledpupalcases in which metamorphosisgoes to completion (g-h). f. Other,late-seasonlarvaeproducethick-
walledcases and enter diapause.

pleted reserves. The gut contents in emerged
adults stained faintly for starch with I2KIbut
gave an intense PAS-positive reaction. Sec-
tions stained with ninhydrin-Schiffshow par-
ticles in the gut which may be undigested re-
mains of idioblasts. These are concentratedin
the hindgut (Fig. 23).
Very little structureappeared osmiophilic,
exceptions being eyes, eggs, gut contents
(slightly osmiophilic), and in some sections

upperright). x 33. 21. Longitudinalsection of pupa stained with ninhydrin-Schiff.Note empty gut (G), abundanceof
storagematter(S). x 33. 22. Longitudinalsection of just-emergedmale adult weevil stainedwith erythrosin-I2KIand
showing an empty gut. x 33. 23. Longitudinal,ninhydrin-Schiff-stainedsection of feeding adult-gut (G) probably
containspartiallydigestedparenchymafrom microsporophylls,includingdenselystainedparticles(arrowheads)which
may be remainsof idioblasts.Note also concentrationof densely stained matterin hindgut(arrow). x 33.
1390 AMERICAN JOURNAL OF BOTANY [Vol. 76

nerve tissue. However, penetration of weevil Pupation-The larvae grow rapidly, soon
cuticle by OS04 was imperfect and the results hollowing out most of the sporophyll (Fig. 6,
therefore were inconclusive insofar as lipid 7). The epidermis of the sporophyll is tough
content is concerned. Although lipids tend to and it together with the microsporangia re-
be leached out from paraffin sections by sol- mains intact throughout the feeding period of
vents (xylene, toluene), sudan IV gave a faint the larvae. Within about 3-4 days after hatch-
reaction in some larval and pupal tissues, prin- ing, a larva constructs a chamber in the spo-
cipally those we interpret to be food reserves. rophyll stalk within which it pupates (Fig. 6f,
7g). The pupa nearly always is oriented with
Pollen foraging- Gut contents of larvae and its head toward the outer end of the micro-
adults contained no pollen grains or distin- sporophyll and its abdomen toward the base
guishable pollen fragments, hence it does not of the cone; female pupae often are found to
appear that either larval or adult weevils feed contain up to three developing eggs. The pupal
on pollen, although it is possible that some case is constructed from a clear liquid secreted
browsing on pollen by the latter may occur. by the larvae and fecal matter, which, upon
Moreover, the sporangia do not seem damaged hardening, lines the hollowed-out sporophyll
as a result of weevil activity (Fig. 6d, g). stalk with a brittle, brown-colored substance.
The case is completed by construction of end
Food content of male and female sporo- walls (Fig. 19), so the larva and later pupa is
phylls-Freehand sections of the axis of the housed within a tough, cylindrical capsule.
male cone stained less intensely with I2KI than When metamorphosis is complete, the adult
did microsporophylls, indicating that the latter emerges, usually by boring out through the tip
contain considerably more starch than does the of the sporophyll, leaving a distinctive exit hole
axis (Fig. 14, 16). The epidermis of the mi- (Fig. 3, 6g). We have not watched the entire
crosporophyll is thick and stained heavily with process of metamorphosis of any particular egg,
sudan IV. Microsporophyll parenchyma is soft but cones tagged at the beginning of swarming
and rather mushy and stained intensely with and egg laying, and observed daily, suggest the
12KI but only lightly with sudan IV. Micro- entire process is completed in 7-9 days. Thus
sporophyll sections prepared just before, dur- it is possible that over the 6-wk period, or so,
ing, and just after egg-laying activity, show of the annual coning and pollination cycle of
parenchyma cells loaded with starch grains (Fig. the cycad, at least six generations of weevils
8, 10, 15). Soon afterwards, the microsporo- may have been produced from an initial mat-
phyll parenchyma is entirely eaten by larvae. ing.
Freehand megasporophyll sections stained less
intensely with I2KI than did the microsporo- Seasonal changes-For most of the repro-
phylls and microtome sections show almost a ductive period ofboth weevils and cones, events
complete absence of starch grains (Fig. 9), but progressed pretty much as outlined above.
they did contain more lipid as evidenced by However, during the last week or so before
heavy staining with sudan IV. pollen production ceased, we observed a change
In both micro- and megasporophylls scat- in the morphology and behavior of adult and
tered storage cells (idioblasts according to Gre- larval weevils. We also noticed that the outer
guss, 1968) are present and these contain sub- ends of microsporophylls of late male cones
stance(s) that stain variously with I2KI, sudan contained quite a bit more chlorophyll than
IV, PAS and ninhydrin-Schiff (Fig. 8-13). The the earlier ones (Fig. 24). During this phase,
idioblasts are elongated in parallel with the female weevils laid 1-3 eggs either at the sides
long axis of the sporophyll but appear more- of the sporophyll ends or in the junction of the
or-less circular in cross section. In microspo- sporophyll and cone axis or in the axis itself
rophylls, they are amber-colored before stain- (Fig. 24). As a result, larvae were more fre-
ing and did not stain with sudan IV, I2KI, and quently found in the cone axes than earlier and
PAS (Fig. 8, 10) but they did stain intensely it is this "second wave" of larvae that accounts
with ninhydrin-Schiff(Fig. 12). Those ofmega- for the complete riddling and destruction of
sporophylls, stained intensely with sudan IV, the axis which becomes hollowed out and filled
and PAS (Fig. 11), variably with ninhydrin- with frass (Fig. 18).
Schiff(Fig. 13), and occasionally with I2KI (Fig. Whereas, "early-season," but late-instar lar-
9). Many idioblasts of megasporophylls ap- vae are covered with thin cuticles through which
peared to have been degraded, losing stain- internal structures are visible, "late-season,"
ability with PAS and ninhydrin-Schiff but not late-instar larvae have thick, opaque cuticles.
with sudan IV; their contents appear to consist During this latter phase, at the end of the sea-
mostly of amber-colored trabeculae (insets Fig. son, about half the eggs were laid in the corners
9, 13). of the sporophyll end, away from the green
September 1989] NORSTOG AND FAWCETT-POLLINATION
areas, and half in the sporophyll base or in the
cone axis (Fig. 24). About half of the eggs pro-
duced early-season larvae which had thin,
transparent cuticles and formed thin-walled
pupal cases from which adults soon emerged
(Fig. 24e-h); halfbecame waxy, yellowish, late-
season larvae which produced and resided in
thick-walled pupal cases (Fig. 24f). No adults
emerged from these pupal cases during Au-
gust-March; a few emerged in early April, and
many more had emerged by late May 1989, as
cones began to appear.
DISCUSSION-Rhopalotria mollis is, as far as
we know, host specific, although it also infests
an experimental hybrid of two closely related
Mexican species Z. furfuracea and Z. spartea
(Norstog et al., 1986). We have not found R.
mollis in the Florida cycad, Zamia pumila,
which has its own closely related weevil pol-
linator, Rhopalotria slossoni (Tang, 1987). But
recently, Tang (personal communication)
found several individuals of R. mollis in a fe-
male cone of Dioon spinulosum; however, at
FTG they are not regular visitors to any other
cycad. Members of the Curculionidae exhibit
host specificity or feed upon closely related
plant species; the cotton boll weevil, Anthon-
omus grandis, for example, feeds upon seven
species of Gossypium (Burke, 1976).
Brood-place selection by R. mollis-Mor-
phologically, male and female cones of Z. fur-
furacea are of similar color and form, which,
it has been suggested, may account for visit-
ations to female cones by pollen-carrying adult
weevils (Norstog et al., 1986). Because R. mol-
lis exhibits such a strong feeding preference for
male cones, we looked for differences in mor-
phology and food content of male and female
cones. Aside from size, an obvious gross dif-
ference in sporophylls, readily visible in stained
freehand and microtome sections, is the greater
amount of starch present in microsporophylls
(Fig. 8-11, 16). Because we have repeatedly
observed adult and larval weevils feeding on
microsporophylls, we assume that the feeding,
mating, and egg-laying preferences have
evolved in response to this higher starch con-
tent. There may, however, be more subtle dif-
ferences between micro- and megasporophylls
which have not been investigated or of which
we are presently unaware.
Foremost among other putative differences
between micro- and megasporophylls is the
possibility that there may be higher toxicity
levels in female tissues. Cycads are well known
for their content of toxic compounds (Vega and
Bell, 1967; Moretti, Sabato, and Siniscalco
OF ZAMIA 1391
Gigliano, 1983; Rothschild, Nash, and Bell,
1986; Spencer et al., 1987). Recently, it has
been found that micro- and megasporophylls
of Z. furfuracea contain the neurotoxin BMAA
(Mark Duncan, personal communication). Both
male and female tissues contained equivalent
concentrations of BMAA, but the analyses were
made with whole ground sporophylls so it is
not known whether toxin was localized or dis-
persed, or whether its distribution differed in
male and female tissues. Although BMAA (p3-N-
methylamino-L-alanine) has not been local-
ized within specific cells of cycads, ninhydrin-
Schiff-stained sections suggest that it might be
present in idioblasts, especially those of mi-
crosporophylls. BMAA is reported to produce
a brown-purple reaction with ninhydrin (Vega
and Bell, 1967), and all idioblasts in micro-
sporophyll sections and some in megasporo-
phyll sections stained similarly with ninhy-
drin-Schiff (Fig. 12, 13), but in the latter most
of the idioblasts appeared degraded (Fig. 13).
Idioblasts in microsporophylls, on the other
hand, do not appear to have undergone the
degree of degradation exhibited by those in
megasporophylls. In the latter, we also inter-
pret positive staining reactions with PAS and
sudan IV (cf. Fig. 10, 11) as indicating that
some substance had undergone a chemical
change, possibly with release of product (tox-
in?). We do not at this time have information
on the presence or absence of two other cycad
toxins, cycasin and macrozamin, in sporo-
phylls of Z. furfuracea, although we have been
informed that such assays are in progress (Rob-
ert Nash, personal communication).
Larvae and probably adults ingest idioblasts
of microsporophylls along with parenchyma-
tous tissue, but there also is some evidence that
adults may partially avoid ingesting idio-
blasts-see Fig. 15. Larvae, however, are in-
discriminate feeders and scattered ninhydrin-
Schiff-stained particles are present throughout
the gut contents, but are more concentrated in
the hindgut (Fig. 20). Similarly stained parti-
cles also are present in the gut of feeding adults
(Fig. 23), and in both larvae and adults these
particles might represent ingested idioblasts
which have resisted digestion. If, in fact, they
actually are undigested idioblasts, it suggests
that sequestered toxin may be eliminated in
fecal matter. Pupae cases, which are construct-
ed from fecal matter, might therefore have some
toxic properties. If idioblasts of male tissues
store toxins, equivalent toxins might not be
similarly avoidable in megasporophylls. In
them, most of the idioblasts appear to have
been modified in some way for they do not
stain with ninhydrin-Schiff and are PAS-pos-
itive. Some also appear empty and it is possible
1392 AMERICAN JOURNAL OF BOTANY [Vol. 76

that BMAA (or other toxin) has already been of chlorophyll-rich areas of microsporophylls
released into parenchyma tissue. If so, this may both by egg-laying females and by larvae (Fig.
account for avoidance of megasporophylls by 24c, d). Although the cause of diapause in-
feeding and egg-laying adult weevils. Still, nei- duction is conjectural, the fact that it occurs
ther the differential presence or absence of free when it does is further evidence of the intimacy
toxins or other compounds in micro- or mega- of this weevil/cycad symbiosis. Dormancy
sporophylls would detract from a hypothesis strategies in which an insect remains in dia-
that the reproductive behavior of R. mollis pause throughout the nonreproductive phase
exploits the high concentration of starch in of a specific host plant are well-documented
microsporophylls of Z. furfuracea. (Denlinger, 1986).
Because we have noted the metamorphosis
Exploitation of Z. furfuracea-The repro- of late larvae of R. mollis only after some 7
ductive cycle of R. mollis appears almost months in diapause, we suspect reproductive
uniquely adapted to exploitation of Z. furfur- continuity of the weevil from season to season
acea as a feeding and breeding site. Protective hinges on year-to-year survival of larvae in
coloration, mating upon male cones alone, and diapause in the remnants of pollen cones which
only those about to mature; egg deposition in are more-or-less persistent at the bases of male
food-rich microsporophylls; excavation of mi- plants.
crosporophylls, including the pupation site in
the sporophyll stalk which precisely accom- Exploitation of R. mollis-As reported by
modates the larva, pupa and pre-emergent Norstog et al. (1986) exclusion experiments
adult, all suggest an intimate and long-standing show R. mollis to be an effective pollinator of
relationship involving both behavioral and Z. furfuracea. Moreover, airborne pollen prob-
morphological adaptations on the part of the ably is unimportant in pollination of cultivated
insect. Z. furfuracea, a conclusion based on exposure
of pollen traps (greased slides) in the vicinity
Dormancy of R. mollis-If R. mollis is in- of pollen-receptive female cones. Calculations
deed host specific in its breeding cycle, then it suggested that if every trapped pollen grain had
becomes important to know what the insect instead impacted on a micropyle, fertility from
does during the 10 months, or so, when Z. that source might be 22%, whereas actual fer-
furfuracea is not coning. We have not attempt- tility of cones adjacent to slides averaged 97%
ed to follow dispersion of adult weevils after (Norstog, unpublished observations). With re-
they leave the host. They might feed on other spect to this experimental population, the ob-
plants, might even reproduce on other species, servations reinforce the conclusion that wee-
but we have no evidence of this. vils acocunt for most if not all pollinations.
Dormancy in insects is a mechanism where- We think this cycad owes its high fertility in
by some kind of adversity is circumvented, cultivation almost exclusively to pollination
whether of climate, or of availability of food by R. mollis, and see adaptations in weevil
and/or oviposition sites (Burke, 1976; Denlin- morphology and behavior that exploit the re-
ger, 1986). The dormant period, or diapause, sources of Z. furfuracea. A corollary question
may take place at any stage in the insect's life therefore is, has Z. furfuracea evolved phys-
cycle, occurring at the larval stage in about 30% iological and morphological adaptations that
of examples. Its onset can be triggered by sea- exploit parasitism by R. mollis?
sonal events such as drought, high rainfall, or It seems clear to us that a mutually profitable
changes in photo- or thermoperiod. These fac- partnership has evolved in which Z. furfuracea
tors often are less pronounced in the tropics has developed certain structural, physiological,
but diapause occurs there about as frequently and possibly other features that have resulted
as in temperate zones. Rhopalotria mollis en- in an increase in its pollination efficiency. These
ters diapause toward the end of male-cone pro- include some kind of signalling to the polli-
duction when late-season larvae develop thick, nator that certain cones have attained readi-
waxy cuticles and construct thick-walled pupal ness as brood places; interception of this signal
cases (Fig. 24). Within this brief time-frame by the pollinator then results in these cones
there probably are few climatic clues, so dia- being singled out for congregational mating,
pause most likely is induced either by endog- feeding, and brood place selection. Concentra-
enous factors in the weevil itself or is an in- tion of foodstuffs almost exclusively in ends
duction resulting from some physiological and stalks of microsporophylls and not in the
change in the tissues of the cycad. We favor cone axis can be seen as a strategy which fo-
the latter interpretation solely on the basis of cuses weevil activity at the site of pollen pro-
an increased chlorophyll content in micro- duction, and some manner of microsporangial
sporophylls, coupled with observed avoidance protection and/or isolation exists so that they
September 1989] NORSTOG AND FAWCETT-POLLINATION OF ZAMIA 1393

are not damaged by the weevils (but, equally separate the microsporophylls and allow wee-
possible, avoidance behavior has evolved in vils access to the cone's interior (Fig. 18). It
the weevil). Also, there may be differential con- may, however, not be sufficient for pollen dis-
centrations of toxins in tissues of micro- and persal by air currents. In cones of this cycad,
megasporophylls in which the former have we think the major function of starch, is that
levels sufficiently low to be attractive to R. of a nutritional reward for the cosymbiont, R.
mollis, and, importantly, provide access to pol- mollis; a secondary function may be to provide
len. Finally, we think the mode of female cone fuel for odor volatilization which probably
opening (by a vertical crack) is an adaptation serves as a signal to the pollinator. (Both male
for insect pollination which also tends to limit and female cones give off an odor we have
random visitation by other insects as well as described as "musty" [Norstog et al., 1986].)
entry of foreign pollen, mold spores, and the In pollination symbioses, a reward is pro-
like. After pollination, the cone closes up and vided animal pollinators, often in the form of
protection of ovules from predation is en- nectar, and its presence is signaled by odor or
hanced in a manner analogous to the closed color, or a combination of odor and color (Fae-
carpels of angiosperms. gri and van der Pijl, 1971; Kevan and Baker,
1984). Faegri and van der Pijl mention that
Cone expansion -While we have considered pollen may be a reward for beetles which they
the rather complex interactions between R. say might polinate some cycads, but neither
mollis and Z. furfuracea to exemplify a co- the cycad nor the pollinator are specified: "there
evolution of long standing (Norstog, 1987), it we found a possible attractant in the male flow-
is possible that structural and behavioral char- er, viz. pollen for pollen-eating beetles." Since
acteristics noted in Z. furfuracea relate to func- there is no evidence that either larvae or adults
tions other than insect/plant mutualism per se. of R. mollis feed upon pollen of Z. furfuracea,
Differential starch distribution, for example, we conclude that in this instance the reward,
may not have evolved as a pollinator reward, perhaps the sole reward, is starch-laden paren-
but relates rather to a more general mechanism chyma of microsporophylls. We have not ob-
involving thermogenesis, cone expansion, and served weevils feeding within female cones;
odor volatilization. Tang, Stemnberg,and Price possibly, they may feed upon micropylar ex-
( 1987) examined starch and lipid levels in male udates although their mode of feeding does not
cones of five cycads (Dioon edule, Encepha- seem to be consistent with lapping fluid drop-
lartos ferox, E. hildebrandtii, Macrozamia lets. We think their entry into female cones is
moorei, and Zamia pumila) during cone elon- a sheltering response to similarities of form,
gation and pollen-shedding. They report that color and, perhaps, odor of male and female
at the onset of elongation, microsporophylls of cones.
M. moorei contain high levels of starch (7.1- In addition to furnishing food for adults and
16.8% fresh wt.) and it was noted that the starch larvae, male cones function as "shelter blos-
content of the axis is relatively lower, a dif- soms." Shelter blossoms which provide food
ferential we also have noted in male cones of and a haven for adult insect pollinators and a
Z. furfuracea. In M. moorei, sporophyll starch broodplace for their young are well-known
is "significantly depleted" during cone elon- (Faegri and van der Pijl, 1972; Kevan and Ba-
gation and Tang and his colleagues conclude ker, 1984). A thrip shelters and mates in the
that cone thermogenesis and elongation is flowers of Calluna vulgaris, taking food from
fueled by starch and lipid. They further reason nectar, stamens, and petals, and laying eggs in
that thermogenesis in cycad cones is important the filaments of stamens. The eggs remain dor-
in volatilizing odors which may be attractive mant over winter in the stamen filaments of
to insect pollinators. persistent flowers before hatching into larvae.
Cone elongation, which accompanies ther- Another pollinator, the Yucca moth, Tegiticula
mogenesis in cycads, is important not only in yuccasella, does not feed at all as an adult, but
freeing pollen in those species in which wind visits and pollinates Yucca blossoms solely in
pollination is thought to occur, but it also pro- search of a place to deposit its eggs. The at-
vides access to pollen and ovules by insect tractant, in this case is odor and the reward is
pollen vectors. In some cycads, pollen cone the brood-place. Because its eggs are laid in
elongation may result in length increases ap- the pistils of Yucca blossoms and its larvae
proaching 50% or more, but is somewhat less feed upon ovules, the relationship is destruc-
in Z. furfuracea. In that species, much of the tive and some of the host's reproductive pro-
microsporophyll tissue is consumed by wee- ductivity is sacrificed in the furtherance of pol-
vils, so that the contribution of sporophyll lination. While pollination of Z. furfuracea
starch to male-cone elongation cannot be very resembles that of Yucca in some respects, it is
great; however cone elongation is sufficient to perhaps more analogous to the pollination sys-
 1394 AMERICAN JOURNAL OF BOTANY
tem of Calluna for the reason that R. mollis is
destructive neither to pollen nor ovules. The
rewardZ.furfuraceaoffersits pollinatoris food
and a brood place, and is, therefore,quite com-
parablewith the rewardsofferedpollinatorsof
angiospermflowers.
Relevance to pollination in natural popula-
tions-Can the observations reported herein
be extrapolatedto naturalpopulations?Earlier
exclusion experiments (Norstog et al., 1986)
provided strong evidence that R. mollis is an
effective pollinator of Z. furfuracea in culti-
vation, but did not clarify the role, if any, of
airbornepollen in the reproductionof Z. fur-
furacea.Among the somewhatshelteredclosely
spacedplants therewas very little airbornecy-
cad pollen, hence it probablywas not impor-
tant in the reproductionof the cycad. But be-
cause the Z. furfuraceaplanting is very dense
and is subject to massive infestations of R.
mollis, and is, moreover, surroundedby many
othergeneraand species of cycadsthatproduce
almost identical pollen, our observations of
airborne-vis a vis insect-pollination must be
viewed with some reservation in relation to
field situations.
As is often the case, exotic species, having
left competitors and predators behind, often
have rathersimplifiedecologicalrelationships.
The presentstudy, we think, should be viewed
as a laboratory situation in which the rela-
tionships between a plant and its pollinator
have been altered and perhaps simplified in
several ways. Few wild populations of Z. fur-
furacea consist of such closely spaced individ-
uals and it is also likely that both the cultivated
cycad and its pollinators are not subjectedto
many of the stressesprevailingin nature.Wild
zamias cone less frequently than they do in
cultivation (personal observation and Tang,
1985) and, of course, are subjectto many eco-
logical variables including extremes of tem-
perature and precipitation, as well as preda-
tion, fire, and other disturbing factors.
Moreover,in wild populationsof Z.furfuracea,
otherorganismsmay also in one way or another
interfere with pollinators and pollination. In
native populations of Z. pumila, for example,
there are two pollinators, Rhopalotria slossoni
and Pharaxonotha zamiae (Tang, 1987). One
of the pollinators,P. zamiae, is a pollen-eater,
a factor not encounteredin the present study
of Z. furfuracea.In addition, a moth also lives
in male cones of Z. pumila, where its larvae
probablyfeed on chitin (shed cuticle of beetle
larvaeand pupae),and a fungusthat parasitizes
the pupaeofR. slossoni is presentas well (Faw-
cett, unpublished observation). In its natural
[Vol. 76
habitat,the cycad/pollinatorrelationshipin Z.
furfuracea might be similarly complicated by
other factors, but we think the essential pol-
lination interactionswe have observedat FTG
also prevail in nature.
LITERATURE CITED
BAIRD,A. M. 1938. A contribution to the life history of
Macrozamia riedlei. J. Roy. Soc. New South Wales
25: 153-175.
BORROR,D. J., AND D. M. DELONG. 1964. An intro-
duction to the study of insects, rev. ed. Holt, Rinehart
and Winston, New York.
BURKE,H. R. 1976. Bionomics ofthe anthomine weevils.
Annual Rev. Entomol. 21: 283-303.
COMSTOCK,J. H. 1936. An introduction to entomology,
8th ed. Comstock, Ithaca, NY.
DENLINGER, D. L. 1986. Dormancy in tropical insects.
Annual Rev. Entomol. 31: 239-264.
FAEGRI,K., ANDL. VANDERPUL. 1971. The principles
of pollination biology, 2d ed. Pergamon, New York.
GREGUSS,P. 1968. Xylotomy of the living cycads. Aka-
demiai Kiado, Budapest.
JENSEN,W. A. 1962. Botanical histochemistry principles
and practices. W. H. Freeman, San Francisco.
KEVAN,P. G., ANDH. G. BAKER. 1984. Insects on flowers.
In C. B. Huffaker and R. L. Rabb [eds.], Ecological
entomology, 607-631. John Wiley and Sons, New
York.
MORETTI,A., S. SABATO,AND G. SINIsCALcoGIGLIANO.
1983. Taxonomic significance of methylazoxymeth-
anol glycosides in the cycads. Phytochemistry 22: 115-
117.
NIKLAS,K. J., AND K. NORSTOG. 1984. Aerodynamics
and pollen grain depositional pattems on cycad mega-
strobili: implications on the reproduction of three cy-
cad genera (Cycas,Dioon, and Zamia). Bot. Gaz. 145:
205-222.
NORSTOG,K. 1987. Cycads and the origin of insect pol-
lination. Amer. Sci. 75: 270-279.
- ANDD. W. STEVENSON.1980. Wind? Or insects?
The pollination of cycads. Fairchild Trop. Gard. Bull.
35: 28-30.
, AND K. J. NIKLAS. 1986. The role of
beetles in the pollination of Zamia furfuracea L. fil.
(Zamiaceae). Biotropica 18: 300-306.
RATTRAY,G. 1913. Notes on the pollination of some
South African cycads. Trans. Roy. Soc. South Africa
3: 259-270.
ROTHSCHILD, M., R. J. NASH, AND E. A. BELL. 1986.
Cycasin in the endangered butterfly Eumaeus atala
florida. Phytochemistry 25: 1853-1854.
SPENCER, P. S., P. B. NuNN, J. HUGON,A. C. LUDOLPH,
S. M. Ross, D. N. Roy, ANDR. C. ROBERTSON.1987.
Guam amyotrophic lateral sclerosis-Parkinsonism-
dementia linked to a plant excitant neurotoxin. Sci-
ence 237: 517-522.
TANG,W. 1985. Studies in cycad reproductive biology.
M.S. thesis, University of Miami, Coral Gables, FL.
. 1987. Insect pollination in the cycad Zamia
pumila (Zamiaceae). Amer. J. Bot. 74: 90-99.
, L. STERNBERG, AND D. PRICE. 1987. Metabolic
aspects of thermogenesis in male cones of five cycad
species. Amer. J. Bot. 74: 1555-1559.
VEGA,A., ANDE.A. BELL. 1967. a-Amino-B-methylami-
nopropionic acid, a new amino acid from seeds of
Cycas circinalis.Phytochemistry 6: 759-762.1.