Eduardo Narbona 2.4 and Rodolfo Dirzo 3


Eduardo Narbona 2.4 and Rodolfo Dirzo 3
American Journal of Botany 97(4): 672–679. 2010.
Eduardo Narbona2.4 and Rodolfo Dirzo3
de Botánica, Dpto. Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Ctra. Utrera, km 1, Sevilla
41013 Spain; and 3Department of Biology, Stanford University, Stanford, California 94305, USA
Typically, plant–pollinator interactions are recognized as mutualistic relationships. Flower visitors, however, can potentially
play multiple roles. The floral nectar in Croton suberosus has been proposed to operate as a reward for predators, especially the
wasp Polistes instabilis (Vespidae), which kills herbivorous insects, while the plant has been thought to be mainly wind-pollinated.
In this study, we reassessed the pollination mode of C. suberosus and the possible role of its flower visitors. Pollinator exclusion
experiments demonstrated that C. suberosus should be considered a strictly entomophilous species. Inflorescences of C. suberosus
were visited by a diverse entomofauna involving 28 taxa belonging to six orders; however, wasps and bees were the only visitors
that carried C. suberosus pollen. The visitation rate of wasps was approximately four times that of bees. This observation, combined with the fact that the small size of bees makes effective contact of their bodies with the stigma difficult, strongly suggests
that large wasps are responsible for most of the effective pollination of C. suberosus. Among the wasp visitors, P. instabilis seems
to be one of the most important. These findings expose an unusual plant–insect interaction, in which the plant provides nectar and
wasps pollinate and defend the plant.
Key words: bout duration; Croton suberosus; Euphorbiaceae; Mexico; neotropical dry forest; plant–animal interaction;
Polistes instabilis; pollen limitation; pollen load; pollination mode; Vespidae; visitation rate.
interaction system; the wasp Polistes instabilis Saussure
(Vespidae) defends C. suberosus foliage against herbivorous caterpillars, but the plant only produces flower nectar
(Domínguez et al., 1989). Polistes instabilis seems to be highly
effective in killing insects because levels of herbivore damage
to the leaves are extremely low, although acceptability tests indicate that leaf tissue is readily palatable to some caterpillars
and grasshoppers (Domínguez et al., 1989; R. Dirzo and E.
Narbona, unpublished data). Because this plant species has
been thought to be wind-pollinated (Domínguez et al., 1989),
nectar was regarded as a reward for predatory wasps rather than
for pollinators. However, pistillate and staminate flowers are
visited by a variety of insects, and the relative contribution of
wind pollination to overall pollination has not been assessed in
detail. Available literature (Webster, 1994; Culley et al., 2002)
has reported C. suberosus as an ambophilous species (i.e., combining both biotic and wind pollination). The pollination system of the large genus Croton is poorly known, but it is expected
to be diverse because entomophily (mainly Hymenoptera, Diptera, and Coleoptera), anemophily, and ambophily have been
documented in the genus (Bullock, 1994; Webster, 1994 and
references therein; Armbruster et al., 1999; Williams and Adam,
1999; Freitas et al., 2001).
Croton suberosus has floral traits consistent with anemophily
(sensu Culley et al., 2002), including unisexual flowers, a reduced perianth and long stigmas, but it also has traits consistent
with insect pollination, including floral nectaries and the presence of pollenkitt (E. Narbona and R. Dirzo, personal observation). However, some of the floral traits associated with
anemophily could also be attributed to wasp pollination (Faegri
and van der Pijl, 1979) or could simply represent a phylogenetic
constraint in floral design (Johnson and Steiner, 2000).
The role of P. instabilis as a defender against foliar herbivores
in C. suberosus is well established, but its role in pollination has
In plant–pollinator mutualistic interactions, nectar is produced in the flowers as a reward for pollinators, and thus plant
pollination is assured (Pellmyr, 2002). Other mutualistic interactions are well documented in plants with extrafloral nectaries,
in which extrafloral nectar is offered as a food source to predators or parasitoids that defend plants against herbivores (e.g.,
Janzen, 1966; Ness, 2006; Sugiura et al., 2006). Thus, extrafloral nectar is interpreted as a reward for defense against herbivores, whereas floral nectar is considered a reward for pollinators
(Beattie and Hughes, 2002; Pellmyr, 2002). However, exceptions to this pattern are documented in multiple interaction systems: flower nectar can be consumed by plant defenders if the
flowers have previously been damaged by nectar-robbing insects (Newman and Thomson, 2005) and flower nectar is opportunistically consumed by predatory ants that indirectly
defended the plant against herbivores (Yano, 1994).
Croton suberosus Kunth (Euphorbiaceae), a monoecious
neotropical shrub, represents another unusual plant−insect
Manuscript received 1 September 2009; revision accepted 20 January 2010.
We are much indebted to M. Arista, P. Ortíz, M. Buide, A. Shuttleworth,
S. Johnson, M. Méndez, and an anonymous reviewer for critically reading
the manuscript and providing constructive criticism. We thank T. Lambert,
J. Kuempel, B. Beverly, and S. Tram (Stanford University), and A. López
Llandes (EEZA-CSIC, Almería) for assistance in the field. K. Renton and
K. Boege provided logistical support and advice during fieldwork. Insect
determinations were provided by R. Ayala, H. Braylovsky, D. Gordon, F.
A. Noguera, E. Ruiz, S. O’Donnell, and S. Zaragoza. This work was
supported by a grant to E.N. from the Junta de Andalucía and Plan Propio
de Investigación (Univ. Pablo de Olavide) and by Stanford’s VPUE program
of summer research support. We are grateful to the Chamela Research
Station for fieldwork support.
4 Author for correspondence (e-mail: [email protected])
American Journal of Botany 97(4): 672–679, 2010; © 2010 Botanical Society of America
April 2010]
Narbona and Dirzo—Pollination of CROTON SUBEROSUS
not been quantified in spite of its importance as a floral visitor
(Domínguez et al., 1989). The main objective of this study was
to investigate the pollination system of C. suberosus and to clarify the roles of P. instabilis and other floral visitors. Specifically,
we examined the following questions: (1) What is the pollination mode of C. suberosus (wind, biotic pollination, or both)?
(2) What is the spectrum of flower-visiting insects associated
with C. suberosus, and does it differ between staminate and pistillate flowers? (3) Do floral visitors to C. suberosus transport its
pollen, and if so, which taxa transport greater pollen loads?
Species and study site—Croton suberosus is a deciduous shrub occurring in
seasonally dry tropical forests of the south Pacific coast of Mexico, typically
growing in disturbed conditions of such forests (Rzedowski, 1978; Domínguez
et al., 1989). The plant produces its leaves during the rainy season, just before
flowering time (July to August). Individuals are monoecious and usually have a
basal branch that produces successive secondary branches, and in the apex of
each branch, a dense racemose inflorescence develops. Racemes have an acropetal trend when flowering, with pistillate flowers situated at the base and staminate flowers at the apex. Each raceme produces ca. 20 pistillate flowers and
ca. 40 staminate flowers (E. Narbona and R. Dirzo, unpublished data). Inflorescence flowering duration is about 5 d for the pistillate phase and 17 d for the
staminate phase (Domínguez and Bullock, 1989). Overlapping of sexual phases
of inflorescence is extremely rare (Domínguez and Bullock, 1989) (Fig. 1), but
flowering synchrony of flowers of the same individual is not perfect; thus, geitonogamy may be possible because the species is self-compatible (Domínguez
and Bullock, 1989). Pistillate flowers are apetalous and have three large bifid
styles (Fig. 1A, B). A receptive stigmatic surface is located only at the end of
each style branch (Fig. 1A, B). Styles are straight and stigmas are sticky and
green when pistillate flowers are receptive (Fig. 1A), whereas styles become
curved and stigmas are dry and brown when flowers are not receptive (Fig. 1B,
C). Staminate flowers have five white petals (Fig. 1C, D), and their 15 stamens
produce ca. 3600 pollen grains (E. Narbona and R. Dirzo, unpublished data).
Both types of flowers produce nectar because they have floral nectaries situated
between sepals and petals in staminate flowers and between the sepals and the
base of the styles in pistillate flowers. Pistillate and staminate flowers produce
a volume of nectar of 5.1 ± 1.8 (mean ± SE) and 6.8 ± 2.3 μL/24 h, respectively
(E. Narbona and R. Dirzo, unpublished data). Daily nectar secretion occurs
between 0800 and 1600 hours (Domínguez et al., 1989). Fruits are three-seeded
capsules with explosive dispersal that mature ca. 20 d after fertilization. Ants
have been seen carrying fruits, likely performing secondary dispersal.
This study was carried out at the Chamela Biological Station, located in the
State of Jalisco, western Mexico (19°30′N, 105°03′W). Mean annual precipitation is 852 mm with most of the rains concentrated between June and October
(García-Oliva et al., 2002). The predominant vegetation is seasonally dry tropical forest (Rzedowski, 1978). We studied one population of about 400 individuals situated in a sunny area along the borders of a main road leading to the
station (150 m a.s.l.).
Pollination mode and pollen limitation—To assess the pollination mode and
the possible transfer of pollen from staminate to pistillate flowers in the same C.
suberosus inflorescence, we randomly selected 73 individuals at the beginning
of the flowering period (July) in 2006. Prior to anthesis of the pistillate flowers,
inflorescences were covered with a bag that was tied around the base of the peduncle. Due to the low number of inflorescences produced by most plants, we
used only one inflorescence per individual. Each inflorescence was randomly
assigned to one of the following treatments: “apomixis” (the production of seed
without sexual reproduction, including pseudogamy), which was tested using
pollen-proof paper bags; “anemogamy,” tested using small-mesh polyester bags
(pore size ca. 0.8 × 0.8 mm), which were permeable to airborne pollen (Neal and
Anderson, 2004); and “entomophily by small insects”, which was assessed using
large-mesh polyester bags (pore size ca. 2 × 2 mm). In all three treatments,
we removed all floral buds from staminate flowers (ca. two thirds of the upper
part of the inflorescence) to prevent transmission of pollen from staminate
to pistillate flowers in the event of some anthesis overlap. To assess this
possible transmission of pollen within inflorescences, we duplicated the three
treatments above, but left the inflorescences intact to detect (1) selfing within the
inflorescence without any dispersive agent (within inflorescence, nonmanipu-
lated geitonogamy), (2) selfing within the inflorescence in the presence of wind
(within inflorescence, anemogamy), and (3) selfing within the inflorescence in
the presence of small insects (within inflorescence, small insects). We included
a control treatment, comprising intact inflorescences open to pollination. The
influence of pollen availability on reproductive success in pistillate flowers was
assessed by supplementing open-pollinated inflorescences with extra pollen
(pollen supply treatment). When the stigmas of pistillate flowers were receptive,
we applied fresh pollen from several flowers of two remote individuals (see
Kephart, 2005) and at 2-d intervals during anthesis of the female part of the inflorescence to ensure that all flowers had been hand-pollinated.
Fruit set and seed set for all treatments were also measured. Following completion of anthesis in the pistillate flowers, we removed the bags and subsequently counted the number of developing fruit. Capsules were left on the plant
until completely mature; the inflorescences were covered with a nylon bag just
before maturation to prevent the loss of seeds.
Flower visitor activity and analysis of pollen loads—To assess quantitative
aspects of pollination, we censused flower visitors during 4 d of the flowering
peak in 2006 (2 d each for plants with either staminate-phase or pistillate-phase
inflorescences). On each census day, we selected six individuals with only one
flowering inflorescence, in the pistillate or staminate phase. The number of
flowers in anthesis per inflorescence ranged from six to eight. Random censuses
of 15-min duration were made of the selected plants from 0900 to 1800 hours,
collecting the overall activity of flower visitors (Domínguez et al., 1989; E.
Narbona, personal observation). The total number of censuses was 36 for pistillate-phase inflorescences and 32 for staminate-phase inflorescences. During
each census, we recorded (1) the insect species, (2) the number of visits of each
taxon to the inflorescence, (3) the duration of each visit, and (4) the behavior of
insects (i.e., nectar vs. pollen searching, parts of the flower contacted, and predatory behavior). We recorded “visitation rate” as the number of visits per 15
min observation period (Fishbein and Venable, 1996), and “bout duration” as
the time spent foraging on an inflorescence by an insect (Potts, 2005). We studied inflorescences rather than flowers because both pistillate and staminate
flowers generally act as a unit from the point of view of anthesis synchrony
(Domínguez and Bullock, 1989; E. Narbona, personal observation) and due to
the difficulty in differentiating isolated flowers within inflorescences (Fig. 1).
The total number of flower visitor species of a plant may be frequently underestimated as a result of poor sampling effort (Herrera, 2005). To assess control for sampling effort and gauge any possible biases regarding flower visitor
diversity, we used sample-based rarefaction curves (i.e., species accumulation
curves; Colwell et al., 2004). This method is used to estimate species richness in
an area, but can be readily applied to assess floral visitor assemblages by considering censuses instead of sampling plots and inflorescence visits instead individuals (see detailed methods in Herrera, 2005). To estimate the true number
of species of flower visitors, we used the incidence-based coverage estimator
(ICE; Colwell, 2006). Percentages of collected species were assessed by dividing the observed species by the ICE (Watkins et al., 2006). Rarefaction curves
and diversity estimators were separately computed for staminate and pistillate
inflorescences by performing 100 randomizations. We employed rarefaction
curves rescaled by inflorescence visits because we are interested in analyzing
the species richness of visitors to inflorescences (see Colwell et al., 2004).
Discrimination between true pollinators and flower visitors was assessed by
analyzing the body pollen loads (Young, 1988). Flower visitors were captured
haphazardly from C. suberosus inflorescences during the 3 d of the flowering
peak; attempts were made to include representative specimens of all taxa that
had previously been recorded as floral visitors. To avoid contamination, we
captured small and medium insects with a vial and transferred them to a freezer
(Bernhardt, 2005); butterflies and some wasps were captured individually with
a net, killed in a killing jar, and then transferred to a vial. We measured the
“total vector pollen load” (sensu Inouye et al., 1994) as the total number of pollen grains on the body of an insect, irrespective of position and without regard
to the probability of successful deposition of pollen onto a stigma. Because pollen grains are of sufficient size, pollen loads were counted directly on the insect
body using a dissecting microscope (20× magnification; Fig. 1). The vials were
checked for pollen grains that may have detached from the insects, but none
were found. Pollen grains packed in the specialized pollen-carrying organ
(metasomal sterna) of Ashmeadiella sp. (Megachilidae) were not considered
(Talavera et al., 2001). Prior to analysis of pollen loads, pollen grains of C.
suberosus and other species that flowered at the same time were determined
using an optical microscope. Pollen grain counts on the body of the insects were
based on pollen that originated only from C. suberosus. Voucher specimens of
all floral visitors are retained at the Chamela Biological Station, Universidad
Nacional Autónoma de México.
American Journal of Botany
[Vol. 97
Fig. 1. Insect visitors and aspects of (A, B) pistillate-phase and (C, D) staminate-phase inflorescences of Croton suberosus. (A) Polybia sp. sucking
nectar on a pistillate flower (white arrow indicates receptive stigma contacting the ventral surface of the insect). (B) Pistillate flowers with a brown-colored
stigmatic surface (because anthesis is finished); note that stigmatic surface only represents a small proportion of the total length of the style. (C) Polistes
instabilis searching for nectar on staminate flowers; note that several pollen grains have adhered to the ventral part of the insect body. (D) Three coleopteran
species eating pollen of staminate flowers.
April 2010]
Narbona and Dirzo—Pollination of CROTON SUBEROSUS
Data analysis—Fruit set and seed set for the pollination mode and the pollen limitation experiments were explored by generalized linear models (GLM)
with probit link function and quasi-binomial error structure (McCullagh and
Nelder, 1989). We used quasi-binomial fit instead of binomial to correct data
overdispersion (Crawley, 2005) applying an F test for analysis of deviance,
performed with software R version 2.5.0 (R Development Core Team, 2007).
Because analyzing the overall variation between all treatments is not biologically meaningful, we made the comparison by pairs of treatments biologically
relevant (e.g., anemogamy vs. open). To control for experimentwise type I error
produced by multiple comparisons, we applied the sequential Bonferroni correction for fitting the significance level (García, 2004).
Prior to analyzing data of insect visitors, we explored the link function that
was best adapted for our data, using the smaller deviance model (McCullagh
and Nelder, 1989). Differences in visitation rate and bout duration of insect
visitors were tested by means of GLMs, assuming Poisson error distribution
and the power link function. Explanatory variables tested were: groups of insects, sexual phase of the inflorescence, and their interaction. Pearson χ2 was
used to correct for overdispersion of data (StatSoft, 2001). Analyses were carried out using the GLZ module in the program STATISTICA 6.0 (StatSoft,
2001). Preliminary observations suggest that wasps and bees may play an important role in pollination process; thus we compared visitation rates and bout
durations between both groups by means of a priori contrasts using the GLM
procedure. Again, we applied sequential Bonferroni corrections (García, 2004).
Differences in pollen loads between insect groups were tested using a one-way
ANOVA with type III sum of squares due to unbalanced sample size (Shaw and
Mitchell-Olds, 1993).
We employed the EstimateS software version 8.2 (Colwell, 2006) to calculate rarefaction curves and diversity estimators. To compare diversity of flower
visitors between both types of inflorescences, we used rarefaction curves, which
were considered significantly different if and when the 95% confidence intervals did not overlap (Colwell et al., 2004).
Pollination mode and pollen limitation— Fruit set of control
inflorescences ranged from 11.1 to 100%, with an average of
66.4% (Fig. 2). In the apomixis and anemogamy treatments,
bagged flowers did not produce any fruit (Fig. 2), suggesting
that isolated pistillate flowers of C. suberosus need biotic vectors for pollination. Only 3.8% of flowers in the small insects
treatment developed fruit (Fig. 2), and this fruit set was mark-
Fig. 2. Fruit set for all treatments. The mean and 95% confidence intervals are shown. The numbers above the bars are the sample size, i.e.,
number of inflorescences and numbers of flowers (in parentheses). See
methods for explanations of treatments. inflor.: inflorescence.
edly lower than in the control treatment (F1, 19 = 25.54, P <
0.00001). Selfing within inflorescences in the absence of biotic
vectors was possible but improbable (Fig. 2), and thus the
within-inflorescence nonmanipulated geitonogamy and withininflorescence anemogamy treatments had a significantly lower
fruit set than the control treatment (F1, 24 = 59.17, P < 0.00001
and F1, 20 = 38.15, P < 0.00001, respectively). An average of
14.8% of inflorescences in the within-inflorescence small insects treatment developed fruit (Fig. 2), which was significantly
lower than in the control treatment (F1, 23 = 29.52, P < 0.0001).
Species of the Chrysomelidae, and Ashmeadiella sp. (Megachilidae) and Crematogaster sp. (Formicidae) were found inside
some bags in the small-mesh bag treatments. Seed set in this treatment was not significantly different from that of the control treatment (66.6% and 70.6%, respectively; F1, 12 = 0.758, P = 0.40).
Inflorescences in the pollen supply treatment did not develop
significantly more fruits than those in the control treatment
(F1, 28 = 2.70, P = 0.111; Fig. 2). Average seed set in the pollen
supply treatment was 74.1%, which was not significantly different from that of the control treatment (F1, 22 = 0.096, P = 0.76).
Flower visitor activity and analysis of pollen loads— Inflorescences of C. suberosus were visited by 28 insect taxa belonging to six orders (Table 1). Pistillate-phase inflorescences
received visits from 19 taxa, and staminate-phase inflorescences
were visited by 27 taxa. Our sampling effort accounted for a
considerable proportion of the total species diversity; the observed insect visitor richness was 78% and 83% of the estimated true richness, based on the ICE estimator for pistillate- and
staminate-phase inflorescences, respectively. Although pistillate-phase inflorescences were less visited than staminate-phase
inflorescences, the diversity of insect visitors in both phases
was statistically indistinguishable, as judged by the overlap of
the confidence intervals of both rarefaction curves (see Appendix S1 in Supplemental Data with online version of this article).
Reduviidae (Hemiptera) and Membracidae (Homoptera) were
casual visitors (one visit only) and were not included in the calculation of visitation rates or bout durations.
Total visitation rate for staminate-phase inflorescences was
nearly double that of pistillate-phase inflorescences (1.14 ± 0.13
vs. 0.58 ± 0.11; mean
± SE), and the difference was highly sig2
rates differed
nificant (Wald χ 1 = 14.3, P < 0.0001). Visitation
significantly among insect groups (Wald χ 5 = 65.2, P < 0.00001;
Fig. 3A), but differences among insect groups were not homogeneous when considering staminate- and pistillate-phase inflorescences (i.e., there was a signifi
cant inflorescence phase ×
insect group interaction; Wald χ 5 = 43.8, P < 0.00001; Fig. 3A).
For pistillate-phase inflorescences, ants were the most abundant
visitors, followed by lepidopterans and wasps, whereas for staminate-phase inflorescences, the most abundant visitors were
coleopterans, followed by wasps (Fig. 3A). Wasps made consistently more visits to flowers than bees in both sexual phases
of the inflorescences,
and these differences are highly signifiχ 12 = 12.7, P < 0.0001 for the pistillate phase; Wald
χ 12 = 16.4, P < 0.00001 for the staminate phase; Fig. 3A). Among
wasps, the most frequent visitors to pistillate-phase inflorescences were P. instabilis (45%) and Eumenes spp. (30%),
whereas Eumenes spp. were the most frequent visitors (51%) to
staminate-phase inflorescences, followed by P. instabilis (18%)
(Table 1). Among bees, Trigona fulviventris was the most frequent visitor to pistillate-phase inflorescences (67%), and Ashmeadiella sp. was the most frequent visitor (95%) to
staminate-phase inflorescences (Table 1).
Table 1.
[Vol. 97
American Journal of Botany
Abundance of insect visitors to the inflorescences of C. suberosus and quantitative analyses of pollen loads.
Visitation rate
(number of visits/15 min)
Order and group
Insect species (family, subfamily)
Ammophila sp. (Sphecidae)
Brachygastra sp. (Vespidae, Vespinae)
Eumenes sp. (Vespidae, Eumeninae)
Montezumia mexicana (Vespidae, Eumeninae)
Polistes instabilis (Vespidae, Polistinae)
Polybia sp. (Vespidae, Vespinae)
Zethus sp. (Vespidae, Eumeninae)
Eumeninae sp. 1 (Vespidae, Eumeninae)
Number of pollen grains per
Censuses in
pistillate phase
Censuses in
staminate phase
2.0 (1)
81.0 (2)
33.3 (4)
19.0 (2)
81.5 (4)
34.0 (2)
2.0 (1)
89.7 (3)
Ashmeadiella sp. (Megachilidae)
Trigona fulviventris (Apidae)
83.7 (3)
28.0 (2)
N, P
N, P
Camponotus sp. (Formicidae, Formicinae)
Cephalotes sp. (Formicidae, Myrmicinae)
Crematogaster sp. (Formicidae, Myrmicinae)
Pseudomyrmex sp. (Formicidae, Pseudomyrmecinae)
0 (2)
0 (1)
0 (4)
0 (1)
Rhopalophora sp. (Cerambycidae)
Stenobatyle eburata (Cerambycidae)
Trigonopeltastes burmeister (Scarabaeidae, Cetoniinae)
0 (3)
1.4 (5)
1 (2)
0 (2)
0 (4)
N, P
N, P
0 (4)
0 (3)
0 (2)
0 (1)
0 (2)
0 (1)
0 (2)
0 (1)
Euptoieta hegesia (Nymphalidae)
Microtia elva (Nymphalidae, Nymphalinae)
Nicolaea ophia (Nymphalidae, Theclinae)
Geometrinae (Geometridae)
Theclinae (Lycaenidae)
Apiomerus sp. (Reduviidae)
Note: Numbers in parentheses indicate sample size. Pollen packed in the metasomal sterna of Ashmeadiella sp. was not considered. CV, coefficient of
variation (percentage); N, nectar feeding; P, pollen feeding or collecting; D, predation.
Overall, insect taxa bout durations were not statistically different between pistillate-phase and staminate-phase
cences (204 s ± 26.4 vs. 251 s ± 22.3; Wald χ 1 = 0.42, P = 0.52).
Bout durations
differed significantly among insect groups
(Wald χ 5 = 31.1, P < 0.00001; Fig. 3B), but there was no significant interaction
between inflorescence phase and insect group
(Wald χ 5 = 5.09, P = 0.40; Fig. 3B). Coleopterans and ants had
the longest visits to both pistillate- and staminate-phase inflorescences (Fig. 3B). Wasps and bees had mean bout durations
shorter than 52 s, and bout duration was not significantly differ2
ent between these groups for either pistillate-phase (Wald χ 12 =
2.89, P = 0.09) or staminate-phase inflorescences (Wald χ 1 =
0.04, P = 0.83; Fig. 3B).
All wasps were observed feeding on nectar (Table 1; Fig. 1A,
C). Bees generally searched for nectar (Fig. 1B), but in some
cases Ashmeadiella spp. were observed collecting pollen
(packed in metasomal externa). Ants sought nectar, although
Pseudomyrmex sp. and Camponotus sp. were only observed
walking on the inflorescences. Coleopterans were active in eating pollen (Fig. 1D), although small Cantharidae and Tenebrionidae also searched for nectar. Lepidopterans and dipterans
only consumed nectar. All insect visitors that gathered nectar
took it from the nectaries at the base of sepals (Fig. 1A−D).
Members of the Reduviidae were observed predating on bees
and small coleopterans.
Only three of the eight floral visitor insect groups (wasps,
bees, and coleopterans) transported pollen, although among coleopterans the presence of pollen was trivial (<1 grain per individual, Table 1). The average number of pollen grains carried
by wasps and bees was 52.6 (coefficient of variation, CV =
121.7) and 61.4 (CV = 84.7), respectively, but high variation
was found among samples (Table 1), and consequently, differences between these insect groups were not significant (F1, 22 =
0.08, P = 0.78). Pollen was present in all taxa of wasps and
bees, but the wasps P. instabilis, Brachygastra sp., and Eumeninae sp. 1, and the bee Ashmeadiella sp. had the highest
pollen loads on their bodies (an average of 80 or more grains
per individual; Table 1).
For wasps, sticky pollen was deposited mainly on the ventral
surface of some part of their bodies when they gathered nectar
from staminate flowers and was subsequently transferred to
stigmas during nectar-seeking in pistillate flowers (Table 1;
Fig. 1A, C). The large size of wasps (body lengths of all taxa,
except Eumenes sp. > 15 mm) enabled them to touch the stigma
April 2010]
Narbona and Dirzo—Pollination of CROTON SUBEROSUS
Fig. 3. (A) Visitation rate and (B) foraging bout duration of insect
groups for pistillate-phase (black bars) and staminate-phase (gray bars) inflorescences of Croton suberosus. Means and standard error are shown.
inflor.: inflorescence, COLE: Coleoptera, LEPI: Lepidoptera, DIPT: Diptera. Insect groups included taxa shown in Table 1.
with their ventral surface, presumably resulting in pollination
(Fig. 1A). Both recorded bees are smaller than 8 mm, making
effective pollination difficult because the styles are longer than
their bodies.
Our results show that C. suberosus relies entirely on pollinators for reproduction because bagged pistillate flowers did not
produce seeds when insect pollinators were excluded. This result was expected because the presence of pollenkitts renders
difficult the dispersion of pollen by wind, and sticky pollen is a
typical feature of entomophilous taxa (Culley et al., 2002; Pacini and Hesse, 2005). Although a very improbable event, selfing within an inflorescence in the absence of biotic vectors was
found in C. suberosus. It is known that complete interfloral dichogamy (i.e., no flowering overlap between pistillate and staminate flowers) is effective in avoiding geitonogamy within
inflorescences (Bertin and Newman, 1993; Aluri et al., 1998).
Croton suberosus has a high degree of, but not complete, inter-
floral dichogamy (Domínguez and Bullock, 1989; E. Narbona,
personal observation), and thus it is possible for intrainflorescence pollination to occur if pollen grains of staminate flowers
fall onto the stigma of pistillate flowers. However, anthesis of
pistillate flowers of bagged inflorescences is much longer than
that of unbagged inflorescences (E. Narbona, personal observation) because flowers that are effectively pollinated rapidly become senescent (Primack, 1985). Thus, natural intrainflorescence
pollination of C. suberosus in the absence of biotic vectors is a
highly unlikely event.
Croton suberosus is apparently pollinated by medium-sized
and large insects because small insects (with body diameters
less than approximately 2 mm) contributed little to the overall
fruit set of the plant. However, entire inflorescences covered
with mesh bags produced a relatively higher fruit set than those
with the staminate part removed (14.8% vs. 3.8%), suggesting
that the movement of small insects throughout the inflorescences can result in some geitonogamous pollination. This experiment may have overestimated the relative pollination by
small insects because, as noted above, the probability of anthesis overlapping for pistillate and staminate flowers in unbagged
inflorescences is very low. Furthermore, the mesh bags provide
an artificial environment in which small insects can forage undisturbed by larger, aggressive ones (Keys et al., 1996), and
indeed we observed that most of the insects that entered the
mesh bags did not leave the inflorescences until the bags were
The inflorescences of C. suberosus were visited by a taxonomically diverse array of insects. The most abundant visitors
were species of the order Hymenoptera, followed by Coleoptera
and Lepidoptera. The insects were very diverse in morphology
and fed mainly on nectar, although a few fed on pollen. Such a
spectrum of insects contrasts somewhat with that observed on
other Croton species, which have been reported to be visited
mainly by dipterans and hymenopterans (Reddi and Reddi,
1985; Armbruster et al., 1999). Nectar-bearing flowers in species of this genus are typically open to insects, including those
with mouth parts unspecialized for nectar feeding. In C. suberosus and C. sarcopetalus (Freitas et al., 2001), the nectariferous
flowers are not completely open to insects because the floral
nectaries are partially covered by the sepals and the petals in
staminate flowers and by the sepals and the base of the styles in
pistillate flowers (Fig. 1). Thus, these structures must be forced
aside when insects, such as hymenopterans, insert their proboscises for nectar gathering (Faegri and van der Pijl, 1979; Aluri
et al., 1998), although some lepidopterans can insert their long,
slender proboscises between the flower parts to obtain nectar
(Faegri and van der Pijl, 1979).
Insect groups varied in their visitation rates and in the rates
of visitation between sexual phases of the inflorescence. Staminate-phase inflorescences were visited almost twice as frequently as those of the pistillate phase, a difference that can be
attributed to the greater preference of coleopterans, wasps, and
bees for staminate-phase inflorescences. Coleopterans mainly
collect pollen, and it was thus expected that they would make
more visits to staminate-phase inflorescences. Because wasps
only feed on nectar and bees search for both nectar and pollen,
the fact that staminate flowers produce almost twice as much
nectar as pistillate flowers (Dominguez et al., 1989) provides a
possible explanation for this differential preference between
flower sexual phases. In addition, staminate flowers have five
white petals, which may help to attract insects (Arista and
Ortíz, 2007).
[Vol. 97
American Journal of Botany
Pollination effectiveness can be considered in qualitative and
quantitative terms (Herrera, 1987, 1989). Assessment of pollination quality requires knowledge of the number of pollen
grains deposited on the stigma during a single visit and the preferences and movement patterns of pollinators (Herrera, 1987;
Johnson and Steiner, 2000). However, in measuring the quantity component and analyzing pollen loads in relation to insect
morphology and behavior, we were able to infer differential
pollination effectiveness (Herrera, 1987; Fishbein and Venable,
1996). In the case of C. suberosus, we were somewhat surprised
that frequent flower visitors with long residence times, including lepidopterans, ants, and coleopterans, carried little or no
pollen. We observed that coleopterans ate a large amount of
pollen, while some ant species had aggressive behavior that discouraged effective pollinators; both groups could reduce the
pollination effectiveness of other flower visitors, and consequently they may result antagonistic to C. suberosus (Ashman
and King, 2005; Larsson, 2005; Ness, 2006). Wasps and bees
were the only flower visitors that transported an appreciable
amount of pollen, and thus we conclude that C. suberosus must
be pollinated solely by these insects. Although wasps and bees
transported similar numbers of pollen grains and had visits of
the same duration, the visitation rate for wasps was approximately four times greater than that of bees for each of the two
inflorescence sexual phases, and this could have a substantial
influence on the pollination process (Gómez and Zamora, 1999;
Sahli and Conner, 2007). Furthermore, bees appeared not to be
efficient pollinators of C. suberosus because their small to medium size makes it difficult for them to touch the stigmatic surface of the pistillate flowers, as proposed for other species
(Faegri and van der Pijl, 1979; Wilson et al., 2004). Although
the role of the bees as pollinators could not definitively be excluded, all of the described factors suggest that effective pollination is, to a large extent, due to large wasps. Thus, C. suberosus
could be considered an ecological generalist in that it interacts
with a range of different insects, but a functional specialist in
terms of effective pollination (Fenster et al., 2004; Wilson et al.,
2004; Larsson, 2005). Similar specializations among the functional group of large wasps have recently been reported in other
species (Shuttleworth and Johnson, 2006, 2009; Johnson et al.,
2007). However, exclusion experiments that unequivocally permit to define the size of pollen loads delivered on stigmas by
wasps and bees are needed to elucidate their definitive role as
pollinators of plants such as C. suberosus.
The possibility that several wasp species pollinate C. suberosus may be important in ensuring its pollination success independent of fluctuations in pollinator populations in space and
time (Fishbein and Venable, 1996; Waser et al., 1996). The evolution and maintenance of a multispecies pollination system is
possible when different flower visitors have similar pollination
effectiveness (Gómez and Zamora, 1999). Similar pollination
effectiveness is expected in plants having flowers with few
ovules (Johnson et al., 1995), as occurs in C. suberosus (three
ovules per flower). Having few ovules per flower in combination with a high abundance and diversity of pollinators may decrease the probability of pollen limitation for plant reproduction
(Liu and Koptur, 2003; Ashman et al., 2004; but see Gómez et
al., 2007) and may explain why the Chamela population of C.
suberosus was not pollen-limited in the study year.
Wasps such as Eumenes sp., P. instabilis, Brachygastra sp.,
and species of the Eumeninae were the more frequent wasp visitors to inflorescences of C. suberosus. The latter three vespids
are probably the major contributors to the reproductive success
of C. suberosus because they were the wasps that transported
the largest quantities of pollen. All Polistes spp. and several
vespid wasps feed on nectar, but they also predate on lepidopteran larvae as food for their own developing larvae (Faegri
and van der Pijl, 1979; Raveret Richter, 2000). Polistes instabilis is attracted to the flowers of C. suberosus, but also preys on
foliar herbivores and defends the plant (Dominguez et al., 1989);
in so doing, these species form a double mutualistic interaction.
By providing evidence that C. suberosus produces nectar to reward pollinators, among which P. instabilis is one of the most
important, our study complements previous work showing the
role of these wasps as defenders of the plant and becomes the
first study to report a plant–insect mutualistic interaction in
which the plant provides nectar and the insect defends the plant
and takes part in its pollination.
In conclusion, large wasps seem to be specialized pollinators
of C. suberosus, although the role of bees and other mediumsized insects could not definitively be excluded. The flower
traits of C. suberosus may have evolved in response to selective
pressures exerted by functional interactions with these wasps
(Fenster et al., 2004). In addition, this specialization may be
reinforced or maintained if vespid species, including P. instabilis, affect the plant’s fitness via protection against foliar herbivores. Further studies are needed to evaluate the relative
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