Annals of Botany 103: 1065– 1075, 2009

doi:10.1093/aob/mcp049, available online at www.aob.oxfordjournals.org

The ant-pollination system of Cytinus hypocistis (Cytinaceae), a Mediterranean

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root holoparasite
Clara de Vega1,*, Montserrat Arista2, Pedro L. Ortiz2, Carlos M. Herrera1 and Salvador Talavera2
1
Estación Biológica de Doñana, Consejo Superior de Investigaciones Cientı́ficas (CSIC), Avenida de Marı́a Luisa, E-41013
Sevilla, Spain and 2Departamento de Biologı́a Vegetal y Ecologı́a, Facultad de Biologı́a, Universidad de Sevilla, Apdo-1095,
41080 Sevilla, Spain

Received: 22 November 2008 Returned for revision: 6 January 2009 Accepted: 20 January 2009 Published electronically: 3 March 2009

† Background and Aims The genus Cytinus is composed of rootless, stemless and leafless parasites whose flowers
are only visible during the reproductive period when they arise from the host tissues. Most of the taxa occur in
Madagascar and South Africa, where mammal pollination has been suggested for one species. There is only one
species in the Mediterranean region, and its pollination system has been unknown. Here, a long-term field obser-
vation study is combined with experimental pollination treatments in order to assess the pollination biology and
reproductive system in the Mediterranean species Cytinus hypocistis.
† Methods Field studies were carried out in six populations in southern Spain over 4 years. Temporal and spatial
patterns of variation in the composition and behaviour of floral visitors were characterized. Pollen loads and
pollen viability were observed, and exclusion and controlled-pollination treatments were also conducted.
† Key Results Cytinus hypocistis is a self-compatible monoecious species that relies on insects for seed pro-
duction. Ants were the main visitors, accounting for 97.4 % of total floral visits, and exclusion experiments
showed that they act as true pollinators. They consistently touched reproductive organs, carried large pollen
loads and transported viable pollen, although the different ant species observed in the flowers differed in their
pollination effectiveness. The abundance of flying visitors was surprisingly low, and only the fly Oplisa aterrima
contributed to fruit production and cross-pollination.
† Conclusions Mutualistic services by ant are essential for the pollination of Cytinus hypocistis. Although this
parasite does not exhibit typical features of the ‘ant-pollination syndrome’, many other characteristics indicate
that it is evolving to a more specialized ant-pollination system. The striking interspecific differences in the polli-
nation systems of Mediterranean Cytinus (ant-pollinated) and some South African Cytinus (mammal-pollinated)
make this genus an excellent model to investigate the divergent evolution of pollination systems in broadly
disjunct areas.

Key words: Ant, breeding system, Cytinus hypocistis, Cytinaceae, insects, flies, Mediterranean Basin, parasitic
plant, pollination, Rafflesiaceae.

IN T RO DU C T IO N roots or stems of the host (Kuijt, 1969; Meijer, 1993; de

Vega et al., 2007). The flowers are only visible during the
Biological oddities have traditionally attracted the attention of
reproductive period when they arise from the host tissues
naturalists, either for understanding their particular way of life
(Kuijt, 1969; de Vega, 2007). Because of the close similarity
or for the study of their evolutionary history. In the plant
of their lifestyles, these endophytic parasites were long con-
kingdom one of the most intriguing groups of plants are the
sidered to constitute a monophyletic group, the family
parasites, which depend partially or completely on their host
Rafflesiaceae s.l. However, data from recent molecular phylo-
for the supply of carbon, nutrients and water (Stewart and
genetic studies together with differences in the morphology of
Press, 1990; Nickrent, 2002a; Shen et al., 2006). The parasitic
flowers, ovaries and seeds indicate that these endophytes are
mode of life in angiosperms has evolved independently at least
composed of four independent families, even belonging to
11 times (Barkman et al., 2007), and is found in approx. 4000
different orders: (1) Apodanthaceae, with minute, solitary
species in 18– 22 dicot families (Nickrent et al., 1998;
flowers (genera Apodanthes, Berlinianche and Pilostyles);
Nickrent, 2002b). Among them, the Rafflesiaceae s.l. has
(2) Cytinaceae, with flowers arranged in an inflorescence
been considered as one of ‘the most extraordinary products
(Bdallophyton and Cytinus); (3) Mitrastemonaceae, with hypo-
of the plant kingdom’ (Kuijt, 1969, p. 104) due to the
gynous, solitary flowers (Mitrastema); and (4) Rafflesiaceae
extreme parasitism found in the species and the unique floral
s.s., with large, solitary flowers (Rafflesia, Rhizanthes and
features of some of their taxa.
Sapria) (Bouman and Meijer, 1994; Barkman et al., 2004;
All the species in Rafflesiaceae s.l. lack chlorophyll, and
Nickrent et al., 2004; Davis et al., 2007).
show extreme reduction in their morphological characters,
Despite the wide morphological variation exhibited by their
being rootless, stemless and leafless parasites with a vegetative
flowers (e.g. size, shape, colour, floral scent), Rafflesiaceae s.l.
body reduced to an endophytic system that lives within the
has been traditionally associated with the pollination syndrome
* For correspondence. E-mail cvega@ebd.csic.es of sapromyiophily (i.e. pollination by carrion flies; van der
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
1066 de Vega et al. — Pollination in Mediterranean Cytinus
Pijl, 1961; Molau, 1995). However, it is noteworthy that nearly
all studies on the pollination biology of these plants have
focused on species of the Rafflesiaceae s.s. (e.g. Beaman
et al., 1988; Bänzinger, 1996; Hidayati et al., 2000;
Bänzinger and Pape, 2004; but see Garcı́a-Franco and
Rico-Gray, 1997). Field observations on the pollination of
species of the other newly recognized families suggest that,
besides flies, flowers are visited by a great diversity of
pollen vectors including bees (in Apodanthaceae; de
Vattimo, 1971; Gómez, 1983) and birds (in
Mitrastemonaceae; Matuda, 1947; Beehler, 1994), although
it remains uncertain as to which the true pollinators are.
Only recently, a distinct and novel pollination system carried
out by two ground-dwelling mammal species has been con-
firmed in a South African species of Cytinus (Johnson et al.,
2008; S.D. Johnson, University of Pietermaritzburg, South
Africa, unpubl. res.), a genus whose reproductive biology
and pollination ecology remain poorly known.
The genus Cytinus comprises some eight species
(Mabberley, 1997) that occur as holoparasites on the roots of
diverse plant families. Most of the species occur in South
Africa and Madagascar, where the genus presents one centre
of diversification. In this area the species are dioecious
and parasitize roots of Asteraceae, Hamamelidaceae,
Rhamnaceae, Rosaceae and Rutaceae (Visser, 1981;
Burgoyne, 2006). The other centre of diversification in this
genus occurs in the Mediterranean Basin, where the taxa are
monoecious and parasitize exclusively roots of Cistaceae
(Webb, 1964; de Vega et al., 2007). Recent molecular analyses
indicate that only one species occurs in the Mediterranean,
Cytinus hypocistis, which comprises several genetic races
that infect different Cistaceae species (de Vega et al., 2008).
Regarding the pollination biology in Cytinus, besides the
small mammals previously mentioned, birds and ants have
been proposed as possible pollinating agents of the South
African species (Visser, 1981), while bees have been
suggested as pollination vectors in the Mediterranean
(Harms, 1935). Pollination by flies has not been documented
for Cytinus, in contrast with the sapromyiophilous pollination
described in Bdallophyton, the other genus of Cytinaceae,
endemic to the American tropics (Garcı́a-Franco and
Rico-Gray, 1997). It is noteworthy that reliable studies on
the pollination of Cytinaceae species have been restricted to
tropical or subtropical areas, with Mediterranean taxa con-
spicuously absent.
In the present study, we combined a long-term field obser-
vation study conducted over 4 years with experimental pollina-
tion treatments at six study sites in order to assess the
pollination biology and reproductive systems of three genetic
races of the Mediterranean Cytinus. The goals were to charac-
terize temporal and spatial patterns of variation in the compo-
sition and behaviour of their floral visitors, to evaluate the
relative importance of different insect species in the reproduc-
tive success of the taxa, and to characterize the breeding
systems of Mediterranean Cytinus. This information enables
us to address whether the pollination systems of the
Mediterranean taxa resembles those of Cytinaceae species
from tropical regions, and represents a first step towards a com-
parative study of the pollination biology of the intercontinental
disjunct genus Cytinus.
MAT E RI AL A ND M E T HO DS
Study area
The study was carried out over 4 years (2002– 2005) in six
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populations of Cytinus hypocistis L. on three Cistaceae host
species: two populations parasitizing Cistus ladanifer (here-
after referred to as Cl1 and Cl2), two populations on Cistus
salviifolius (Cs1 and Cs2) and two populations on Halimium
halimifolium (Hh1 and Hh2). The populations were separated
by 0.5 – 2.5 km and were located at 80 – 90 m a.s.l in Huelva
province, south-west Spain. The climate is typically
Mediterranean, and climatic conditions were similar in the
six populations. During the study period, annual mean temp-
eratures ranged from 16.6 to 17.6 8C and annual precipitation
was 910.5 mm in 2001 – 2002 (September– August),
661.5 mm in 2002 – 2003 and 970.3 mm in 2003 – 2004; in
2004 – 2005 total precipitation was markedly lower, being
only 226 mm.
Study species
Cytinus hypocistis is a monoecious perennial endophyte that
shows a marked reduction of morphological characters, with a
vegetative body reduced to an endophytic system that grows
exclusively inside its host (de Vega et al., 2007). Only in
the blooming period (March – May) are the plants visible,
with inflorescences bursting through the host root tissues.
Inflorescences appear at ground level in clusters of 1 – 22 on
the same host root, and belong to the same or different, geneti-
cally distinct individuals (de Vega, 2007). The inflorescence is
a simple short spike with 5.6 + 0.1 (mean + s.e.) female
flowers (5.3 – 14.9 mm corolla width) at basal positions and
6.2 + 0.1 male flowers (4.1 – 16.1 mm corolla width) at distal
ones (de Vega, 2007). Female and male flowers produce
similar amounts of nectar, approx. 1 mL d21, which is accumu-
lated in four nectariferous cavities, with higher nectar sugar
concentration in female than in male flowers (32 % and 29 %
sucrose equivalents, respectively; de Vega, 2007). Each
flower lasts approx. 6 d and within inflorescences female
flowers open 1– 2 d earlier than males. The fruit is a berry
that ripens from May to July and contains thousands of dust-
like seeds (de Vega and de Oliveira, 2007).
Pollen vectors
In order to check the possibility of anemophily in
C. hypocistis, airborne pollen was sampled in populations
Cl1, Cs1 and Hh1. In each population three pollen traps
were placed at ground level at 0.5 – 1 m around each of ten ran-
domly chosen C. hypocistis plants. Each pollen trap comprised
a plastic tube surrounded by a cigarette paper coated with a
thin layer of petroleum jelly (see Arista and Talavera, 1994).
Pollen traps were collected after a 48-h exposure time and
the pollen content was determined under a microscope.
The composition and abundance of floral visitors was deter-
mined in the six populations over 4 years (2002– 2005). In
each population, clusters of C. hypocistis inflorescences on
15– 22 different host individuals were studied. Diurnal cen-
suses were always made by observers, situated 1.5 m from
focal flower clusters, who recorded the number and activity
 de Vega et al. — Pollination in Mediterranean Cytinus
of insects foraging in flowers during 15-min-long watching
periods (‘censuses’ hereafter) separated by 15-min intervals
throughout daytime (0800– 2200). Given that many floral visi-
tors were small ants, we made four additional censuses of
5 min each hour at 0.5 m from the plants in order to register
their activity more accurately. Data for insect composition
and abundance are shown pooled across years in the Results
section. We recorded insect identity, number of visitors per
flower (n ¼ 4764 visits; throughout the text insect abundance
are expressed as individuals per 5 min ), time spent in
flowers from arrival to departure (n ¼ 2248 foraging times)
and number of flowers visited. The number of inter- and intra-
plant movements (n ¼ 656 foraging sequences) and type of
reward collected were also recorded in every instance. A
total of 262.65 h of diurnal censuses were made, distributed
as follows: 64.5 h in Cl1, 29.4 h in Cl2, 45.75 h in Cs1,
28.25 h in Cs2, 44.5 h in Hh1 and 50.25 h in Hh2. In addition,
a total of 16.5 h of nocturnal censuses (2200– 0300) were
made in populations Cl1, Cs1, Hh1 and Hh2 using red light
to minimize the effect of illumination on flower visitors
(Peitsch et al., 1992). Voucher specimens of all species
recorded visiting flowers were collected, identified and
measured under a binocular microscope.
Ant pollen loads
The size of pollen loads carried by individual ants was esti-
mated for 15 individuals of each of the three most abundant
species. Pollen was removed from all parts of the insects by
dabbing them with small cubes of stained glycerine jelly
under a binocular microscope. The jelly was then transferred
to a slide, melted, and all pollen grains were counted. Ants
were always collected while on C. hypocistis female flowers,
to avoid the likely pollen contamination that would have
ensued from collections in male flowers.
Pollen viability after contacting ant bodies
Most authors assume that ants are ineffective pollinators,
mainly due to the ‘antibiotic hypothesis’ according to which
ants produce antibiotic secretions, mainly from metapleural
glands, that reduced pollen viability and germination percen-
tage (Beattie et al., 1984, 1985; Hull and Beattie, 1988). To
determine the possible effect of ant secretions on pollen ger-
minability, experiments were carried out using the three
most abundant diurnal ant species and the nocturnal one (see
Results). Male flowers of C. hypocistis were collected, and
in the laboratory their pollen was mixed and distributed
between vials. One ant was placed in each vial and left in
contact with the pollen for 10 or 30 min. Ten individual ants
per species were used for each exposure period. In other
vials, control pollen was left for the same time but with no
ant present. Pollen from the vials was then transferred to
culture slides with Brewbaker– Kwack medium (200 g L21
sacarose, 0.1 g L21 boric acid, 0.3 g L21 calcium nitrate,
0.2 g L21 magnesium sulphate and 0.1 g L21 potassium
nitrate) for an incubation period of 48 h, and germination
rate was then assessed under a microscope (n ¼ 11 476 ant-
treated pollen grains and 11 270 control pollen grains
counted).
1067
Breeding system and reproductive success
The breeding system of C. hypocistis was studied by hand-
pollinations in plants from five populations (Cl1, Cs1, Cs2,
Hh1 and Hh2) using within-plant self-pollination (n ¼ 212
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flowers pollinated), cross-pollination (n ¼ 173), total exclu-
sion (to test for possible apomixis, n ¼ 65), and controls
(open pollination, n ¼ 6071; fruit set after open pollination
was recorded in all populations from 2002 to 2005). Before
pollination, each female bud was covered using a plastic
tube with cotton on the top to exclude pollinators. A sub-
sample of the hand-pollinated female flowers was collected 1
week after the pollinations, softened with KOH, stained with
aniline blue, and observed under a fluorescence microscope
to detect pollen tubes in the style and ovules (Martin, 1959).
The remaining pollinations were left in the field until fruit
maturation, and the percentage of flowers that set fruit was
recorded. The number of seeds per fruit was counted in 15
fruit per treatment by means of a particle counter (Coulter
Multisizer III, Beckman Coulter, UK). Germination tests are
critical for assessing seed viability, but unfortunately for
Cytinus in particular, and for all species traditionally included
in Rafflesiaceae s.l. in general, such studies are extremely dif-
ficult due to the special requirements for germination. So, we
used an alternative technique described by de Vega and de
Oliveira (2007) to examine the viability of seeds. Briefly,
seeds were treated successively with Franklin’s and with
Jeffrey’s softening fluids, and then with Herr’s clearing fluid.
The combined action of the two softening fluids caused suffi-
cient seed coat rupture to allow Herr’s fluid to clear the seed
tissues, permitting effective observation of the embryo and
hence assessment of viability.
Exclusion experiments
In order to assess the contribution of flying visitors to polli-
nation we tried to exclude ants from C. hypocistis plants apply-
ing Tanglefootw around the base of 35 inflorescences.
However, ant exclusion was ineffective since inflorescences
arise at ground level and ants forage primarily on plants
under which they nest. To asses the effect of ants as pollina-
tors, flying visitors were excluded from flowers. In populations
Cl1, Cs1 and Hh2, 10 – 14 plants were randomly selected and
were covered prior to blooming with a small net tent (15 cm
tall, 0.25 mm2 mesh) so that only small, crawling insects
could visit the enclosed inflorescences. In each population
we used inflorescences freely exposed to all insects as ‘con-
trols’. Pollen tubes in the style and ovules were examined in
a subsample of flowers, as described above, and seed
number was counted in 16 fruit in each population.
Statistical analysis
To analyse whether flower choice made by ants was statisti-
cally independent of the sex Wilcoxon’s tests were used, and
when comparing the time spent in female and male flowers
we used generalized linear models (GLZs) with a Poisson dis-
tribution and a log link-function (for statistical analysis the
GLZs were used when the response variables followed distri-
butions other than the normal). The number of flowers
1068 de Vega et al. — Pollination in Mediterranean Cytinus

TA B L E 1. Characteristics and behaviour of all Cytinus hypocistis pollinators that accounted for at least 0.2 % of total floral visits
(pooled across all populations and years).

Body length Metapleural Percent of total floral

Taxa (mm) glands1 Pollen load2 visits Reward3 Populations

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HYMENOPTERA
Formicidae
Aphaenogaster senilis 6.89 + 0.25 Yes – 1.53 N Cs1, Cs2, Hh2
Camponotus pilicornis 7.76 + 0.24 No – 0.29 N Cl1, Cs1
Crematogaster auberti 3.69 + 0.09 Yes 123.33 + 15.25 (53–237) 13.43 N Cl1, Cs1 Cs2, Hh2
Crematogaster 4.79 + 0.23 Yes – 2.69 N Cl1, Cs1, Hh2
scutellaris
Pheidole pallidula 2.11 + 0.10 Yes 122.53 + 17.64 (40–268) 35.26 N Cl1, Cl2, Cs1, Hh1, Hh2
Plagiolepis pygmaea 1.63 + 0.06 278.33 + 47.47 (66–669) 19.37 N Cl1, Cl2, Cs1, Cs2
Plagiolepis schmitzii 2.16 + 0.19 Yes – 5.71 N Hh1, Hh2
Tapinoma nigerrimum 3.82 + 0.17 Yes – 1.36 N Cs1, Hh2
Tetramorium ruginode 3.05 + 0.07 Yes – 9.05 N Hh2
Tetramorium semilaeve 2.32 + 0.10 Yes – 8.67 N Cl1, Cs2, Hh2
Halictidae
Lasioglossum sp. 6.95 + 0.21 – – 1.09 P, N All
DIPTERA: Rinophoridae
Oplisa aterrima 3.25 + 0.38 – – 1.34 N, TS All

1
Presence or absence of metapleural glands.
2
Pollen loads are given for the three most abundant species. Values are means + s.e. (range in parentheses).
3
Reward collected: P, pollen; N, nectar; TS, tepal secretions.
Insects that accounted for less than 0.2 % of total floral visits were Apis mellifera (Apidae, 0.02 % of total floral visits), Bombus terrestris (Apidae, 0.04 %),
an unidentified Braconidae (0.04 %), an unidentified Camilidae (0.02 %), and an unidentified Rinophoridae (0.06 %).

visited was compared among insect species and between type
of visitors (flying and ants) by means of a GLZ with a Poisson
distribution and a log link-function.
Pollen loads on insects were compared among species by
means of a one-way ANOVA, and differences in pollen ger-
minability between control and ant-treated pollen were com-
pared using a GLZ with a Poisson distribution and a log
link-function.
Differences in fruit set after hand-pollination treatments were
compared using a GLZ with a binomial distribution and a logit
link-function, and the same method was used to compare fruit
set after exclusion experiments vs. control. Mean numbers of
seeds per fruit between pollination treatments and populations
were compared by means of a factorial ANOVA, and differences
among seeds produced after exclusion and control experiments
were compared by a one-way ANOVA. All statistical analyses
were performed in Statistica 6.0 (StatSoft, 2001). Throughout
the text, all means are shown + s.e.
RES ULT S
Floral visitor assemblage
Pollen traps showed a low presence of C. hypocistis pollen
grains (0.9 + 0.23 grains per trap; n ¼ 90) indicating that
wind-pollination is unlikely in this species.
The flowers of C. hypocistis were visited by 17 insect species
belonging to six families, with ants being much more abundant
than flying visitors and accounting for 97.4 % of total floral
visits (n ¼ 4638; Table 1, Fig. 1). The most abundant daytime
ant species were Pheidole pallidula (35.3 % of total floral
visits), Plagiolepis pygmaea (19.4 %) and Crematogaster
auberti (13.4 %). During the night only the ant Camponotus
pilicornis visited the flowers of C. hypocistis, appearing in 50
% of the nocturnal censuses, but only accounting for 0.3 % of
total visits. These four ant species and Plagiolepis schmitzii
foraged on C. hypocistis flowers in all the years of the study,
their abundance being consistent across years, but none
of them appeared in all the populations studied (Table 1).
The other ant species recorded only appeared in some
years (Aphaenogaster senilis and Crematogaster scutellaris:
2002, 2003 and 2005; Tapinoma nigerrimum: 2002, 2004;
Tetramorium ruginode: 2002; and Tetramorium semilaeve:
2002 – 2004).
The number of individuals foraging on female and male
flowers of C. hypocistis varied greatly among ant species,
ranging from 0.18 (C. pilicornis) to 12.05 individuals per
5 min (T. ruginode, Table 2). It is noteworthy that, although
the two Tetramorium species appeared in a low number of
censuses, when they did appeared they were very abundant
(Fig. 2, Table 2).
Flying visitors mainly foraged in C. hypocistis inflorescences
without ants; however, aggressive interactions between ants and
flying insects were not seen. The fly Oplisa aterrima (1.3 % of
overall floral visits, and 51.2 % of visits of flying insects;
Fig. 1F) and solitary female bees of Lasioglossum sp. (1.1 %
and 41.6 %, respectively) were the most abundant flying
species (Table 1). Oplisa aterrima was observed in all the popu-
lations and years studied, with its abundance being consistent
across years, whereas Lasioglossum appeared in all populations
but only during 2005 (Table 1).
Floral visitor foraging behaviour
Ants and flying insects visited both male and female flowers
and made contact with the plant reproductive organs when
foraging for floral resources, acting as potential pollinators
 de Vega et al. — Pollination in Mediterranean Cytinus 1069

A B C

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D E F

F I G . 1. Flowers and pollinators of Cytinus hypocistis: (A) Cytinus hypocistis, (B) Aphaenogaster senilis, (C) Crematogaster auberti, (D) Pheidole pallidula,
(E) Tetramorium semilaeve and (F) Oplisa aterrima. Scale bars ¼ 5 mm.

(Fig. 1). Ants foraged for nectar, Lasioglossum foraged both
for pollen and nectar, and Oplisa aterrima for nectar and
secretions from tepal glandular trichomes; none of them
destroyed floral organs. Some of the visitors foraged more fre-
quently and spent more time in flowers of a given sex (Figs 2
and 3; Table 2). For each species, the number of visits and
mean time spent in a flower was highly variable throughout
the day (Fig. 3).
The mean number of flowers foraged per plant by each indi-
vidual insect ranged from 2.62 to 3.65, the differences being
significant among species (x2 ¼ 20.574, d.f. ¼ 9, P ¼ 0.015).
The mean number of flowers per plant foraged by ants
(2.83 + 0.05; all species combined) was similar to that for
winged pollinating insects (2.68 + 0.20, x2 ¼ 1.075, d.f. ¼ 1,
P ¼ 0.299). Between 76.19– 96.97 % of movements made by
ants in the daytime were between flowers of the same plant;
however, such movements were much less for the nocturnal
C. pilicornis (40 %; Table 2). Oplisa aterrima and
Lasioglossum made approx. 60 % of flights within plants
(Table 2).
Pollen loads and germination ability
All ants consistently touched the anthers with their bodies
and carried pollen loads that were significantly different
among species (x2 ¼ 20.79, d.f. ¼ 2, P , 0.0001). Pollen
loads were not related to body size, since the smallest ant,
P. pygmaea, carried the highest pollen loads (Table 1). In all
ant species pollen grains were attached to the head, thorax
and gaster. There was no detrimental effect to pollen germina-
tion after contact with the integument of C. auberti and
P. pallidula, either in the treatment of 10 min or 30 min

TA B L E 2. Behaviour of visitors while foraging on Cytinus hypocistis flowers. All species are ants except those marked with an
asterisk

No. of individuals per 5 min Time spent in flowers (s)

Species Female flowers Male flowers Female flowers Male flowers No flowers visited Within-plant movements (%)

Aphaenogaster senilis 0.48 + 0.12 0.55 + 0.14 20.90 + 3.91 74.85 + 24.84 3.65 + 0.50 76.19
Camponotus pilicornis 0.18 + 0.07 0.24 + 0.08 9.00 + 3.76 8.67 + 5.17 3.00 + 0.52 40.00
Crematogaster auberti 2.88 + 0.34 1.51 + 0.166 70.05 + 11.70 60.71 + 8.59 2.97 + 0.11 95.87
Crematogaster scutellaris 1.03 + 0.18 1.03 + 0.17 69.00 + 35.13 24.33 + 17.84 2.64 + 0.17 92.13
Lasioglossum sp.* 0.15 + 0.11 1.18 + 0.18 15.48 + 4.86 13.71 + 2.11 2.62 + 0.29 58.62
Oplisa aterrima* 0.69 + 0.09 0.49 + 0.09 27.09 + 6.15 19.41 + 8.43 2.77 + 0.22 61.29
Pheidole pallidula 4.94 + 0.65 4.94 + 0.53 69.81 + 6.24 80.80 + 8.39 2.62 + 0.07 87.57
Plagiolepis pygmaea 0.60 + 0.06 1.00 + 0.07 41.01 + 4.38 43.43 + 3.99 2.83 + 0.09 98.61
Plagiolepis schmitzii 0.93 + 0.12 1.17 + 0.12 145.96 + 23.13 61.18 + 15.73 2.65 + 0.14 92.50
Tapinoma nigerrimum 0.76 + 0.18 0.78 + 0.17 37.05 + 17.52 16.34 + 3.86 2.82 + 0.18 96.97
Tetramorium ruginode 10.74 + 1.19 12.05 + 1.73 75.14 + 26.74 86.11 + 26.71 3.01 + 0.52 91.94
Tetramorium semilaeve 5.26 + 0.74 5.78 + 0.88 27.40 + 8.13 38.47 + 7.38 3.32 + 0.31 89.47

Values are means + s.e.

1070 de Vega et al. — Pollination in Mediterranean Cytinus

Aphaenogaster senilis Crematogaster auberti Camponotus pilicornis

On flowers
1·4 6 1·2
No. individuals

1·2 On flowers 5 1·0

per 5 min

1·0 4 0·8
0·8 3 0·6

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0·6
0·4 2 0·4
0·2 1 0·2
0·0 0 0·0

00

00

00

00

00

00

00

00
20

21

22

23

00

01

02

03
2·5 Crematogaster scutellaris 2·0 Lasioglossum sp.* 1·2 Oplisa aterrima*
No. individuals

2·0 1·0
per 5 min

1·5
1·5 0·8
1·0 0·6
1·0
0·4
0·5 0·5
0·2
0·0 0·0 0·0

10 Pheidole pallidula Plagiolepis pygmaea 2·0 Plagiolepis schmitzii

2·0
No. individuals

8 1·6 1·6
per 5 min

6 1·2 1·2
4 0·8 0·8
2 0·4 0·4
0 0·0 0·0
30 Tetramorium ruginodis 20 Tetramorium semilaeve
2·5 Tapinoma nigerrimum
No. individuals

2·0 25 16
per 5 min

20 12
1·5
15
1·0 10 8
0·5 5 4
0·0 0 0
00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00
07

09

11

13

15

17

19

21

07

09

11

13

15

17

19

21

07

09

11

13

15

17

19

21
Time of day Time of day Time of day

F I G . 2. Insects visiting the flowers of Cytinus hypocistis during the day. Note that for each species the y-axis differs, and the x-axis is also different for the
nocturnal Camponotus pilicornis. Differences in the number of male and female flowers visited were observed for C. auberti (Z ¼ 3.96; P , 0.0001),
O. aterrima (Z ¼ 1.91; P ¼ 0.055) Lasioglossum (Z ¼ 3.69; P ¼ 0.0002), P. pygmaea (Z ¼ 7.71; P ¼ ,0.0001) and P. schmitzii (Z ¼ 1.97; P ¼ 0.049). All
species are ants except those marked with an asterisk.

(P . 0.25 in all cases; Table 3). In contrast, P. pygmaea and
C. pilicornis significantly reduced pollen germination
(Table 3); however, it is noteworthy that, even in the latter
two species, a considerable fraction of pollen remained
viable after contact.
Pollen loads were not measured on winged insects.
However, during the censuses it was observed that the fly
Oplisa aterrima and the bee Lasioglossum carried pollen
loads on the legs and body.
Breeding system
None of the permanently covered female flowers produced
fruit, indicating that pollinators are required to set seeds.
Microscopic observations revealed many pollen tubes on the
style and also penetrating the ovules of all flowers examined
in both geitonogamous and cross-pollination, giving evidence
of self-compatibility in this species. Cytinus hypocistis showed
high values of fruit set after self-pollination (89.85– 98.11 %),
similar to those observed after cross-pollination and open-
pollination (Table 4). Significant differences were not found
either among treatments (x2 ¼ 2.59, d.f. ¼ 2, P ¼ 0.274) or
populations (x2 ¼ 9.35, d.f. ¼ 4, P ¼ 0.063). Differences
among years for each population in the percentage of fruit
set after open-pollination were not significant (in all cases
P . 0.6).
The mean number of seeds per fruit was 19 305 + 2171 in
self-pollinated flowers, 19 200 + 2333 in cross-pollinated
flowers, and 24 163 + 3847 in open-pollinated flowers.
Mean number of seeds per fruit did not vary among treatments
(F ¼ 0.96, d.f. ¼ 2, P ¼ 0.394) or among populations
(F ¼ 2.68, d.f. ¼ 4, P ¼ 0.083).
Exclusion experiments
Flying-visitor exclusion experiments showed that ants act as
true pollinators. In the three populations studied, plants
exposed only to ants showed a fruit set of 80 – 87 %, and in
all flowers analysed pollen tubes were observed on the styles
and ovules.
The fruit set of plants of the ‘ant-only’ treatment was stat-
istically lower than that of control flowers (x2 ¼ 43.07,
d.f. ¼ 1, P , 0.0001), suggesting that flying insects also con-
tribute significantly to fruit production in C. hypocistis
despite the low quantitative importance of their visitation
rates. Although differences among populations were statisti-
cally significant (x2 ¼ 7.53, d.f. ¼ 2, P ¼ 0.023), fruit set
after the ant-only treatment was similarly reduced relative to
control plants in all populations ( population-by-treatment
interaction not significant: x2 ¼ 2.33, d.f. ¼ 2, P ¼ 0.311).
However, the mean number of seeds per fruit in plants
exposed only to ants (19 418 + 2776) was not significantly
 de Vega et al. — Pollination in Mediterranean Cytinus 1071

Aphaenogaster senilis Crematogaster auberti Camponotus pilicornis

Time in flowers (s)
150 450 25
125 On flowers 375 20
100 On flowers 300 15
75 225
50 150 10

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25 75 5
0 0 0
00 00 00 00 00 00
21 22 23 00 01 02

Crematogaster scutellaris Lasioglossum sp.* Oplisa aterrima*

Time in flowers (s)

250 75 90
200 60 75
150 45 60
45
100 30
30
50 15 15
0 0 0
Pheidole pallidula Plagiolepis pygmaea Plagiolepis schmitzii
Time in flowers (s)

250 120 400

125 100
300
100 80
75 60 200
50 40
100
25 20
0 0 0

Tapinoma nigerrimum Tetramorium ruginodis Tetramorium semilaeve

Time in flowers (s)

200 150 100

160 125 80
120 100 60
75
80 50 40
40 25 20
0 0 0
00 00 00 00 600 800 000 200 00 00 00 00 600 800 000 200 00 00 00 00 600 800 000 200
08 10 12 14 1 1 2 2 08 10 12 14 1 1 2 2 08 10 12 14 1 1 2 2
Time of day Time of day Time of day

F I G . 3. Time spent by different pollinators on female and male flowers of Cytinus hypocistis during the day. Note that for each species the y-axis differs, and the
x-axis is also different for the nocturnal Camponotus pilicornis. Aphaenogaster senilis spent more time on male flowers (x2 ¼ 11.83; P ¼ 0.0006), whereas
P. schmitzii (x2 ¼ 22.01; P , 0.0001) and T. nigerrima (x2 ¼ 22.01; P , 0.0001) spent more time on female flowers. In the other species no differences
were found. All species are ants except those marked with an asterisk.

TA B L E 3. Percentage germination of Cytinus hypocistis pollen
without contacting ants (control) or after contact with ants of
different species (ant-treated pollen) for either 10 or 30 min.
Sample sizes (total number of pollen grains examined) are
shown in parentheses
Germination (%)
Contact time and
ant species Control
different to those produced after open-pollination (F ¼ 1.02,
d.f. ¼ 1, P ¼ 0.321).
Ripe seeds from ant-, cross-, open- and self-pollination
seemed to be viable. All of them presented a small embryo
composed of about ten cells surrounded by an endosperm com-
posed of larger cells, as is typical of C. hypocistis seeds (see de
Vega and de Oliveira, 2007).
Ant-treated F P

10 min DISCUSSION
Crematogaster 64.05 + 7.94 (2305) 58.45 + 5.75 (2358) 0.327 0.574
auberti
This study has shown that Cytinus hypocistis is a self-
Pheidole 65.52 + 7.65 (2207) 53.56 + 7.19 (2226) 1.298 0.269 compatible species consistently exhibiting a high fruit set
pallidula and extensive seed production in natural conditions (i.e. open-
30 min pollination). The species relies on insects for seed production
Crematogaster 57.92 + 6.50 (2203) 47.85 + 3.98 (2128) 1.744 0.203 since neither wind pollination nor agamospermy were
auberti
Pheidole 60.75 + 7.19 (2204) 51.15 + 6.37 (2181) 0.999 0.331 recorded. The long-term field observations and exclusion
pallidula experiments strongly suggest that ants are the main pollinators
Plagiolepis 63.39 + 9.94 (1088) 26.71 + 5.29 (1319) 11.725 0.008 in C. hypocistis, accounting for most flower visits in all
pygmaea populations and years, and yielding a fruit set close to 80 %
Camponotus 63.28 + 4.48 (1263) 14.76 + 9.27 (1264) 22.192 0.001
pilicornis
when other potential visitors were excluded. Accordingly,
this study joins a growing body of evidence highlighting the
1072 de Vega et al. — Pollination in Mediterranean Cytinus
TA B L E 4. Percentage fruit set of Cytinus hypocistis after self-pollination, cross-pollination, open-pollination, ant-only pollination or
total exclusion of flower visitors (sample sizes are shown in parentheses)
Cl1 Cs1
Self-pollination 97.9 (53) 95.4 (41)
Cross-pollination 94.1 (36) 96.1 (23)
Open-pollination 97.3 (1555) 98.9 (871)
Ant-only pollination 80.2 (55) 86.3 (42)
Total exclusion 0 (15) 0 (10)
prominent role that ants can play in some plant – pollinator
systems (Wyatt, 1981; Peakall, 1989; Peakall and Beattie,
1989; Gómez and Zamora, 1992, 1999; Garcı́a et al., 1995;
Ramsey, 1995; Gómez et al., 1996; Puterbaugh, 1998;
Gómez, 2000; Schürch et al., 2000; Ashman and King,
2005; Sugiura et al., 2006). However, contrary to most pre-
vious studies in which single ant species were involved in pol-
lination (but see Gómez et al., 1996; Ashman and King, 2005),
in C. hypocistis as many as ten species can be considered as
true pollinators.
The effectiveness of a given pollinator not only depends on
their abundance and frequency of flower visitation but also on
the efficiency with which they remove and deposit pollen
(quantity and quality components of the plant – pollinator inter-
action, respectively; Herrera, 1987, 1989). Ants were the most
abundant pollinators during both the day and night, accounting
for 97.36 % of floral visits, and while foraging for nectar they
frequently touched the anthers (each male flower may produce
more than one million pollen grains; de Vega, 2007) and the
wide stigmatic surface (4.2  2.5 mm in size). In addition,
we have demonstrated that ants carried large pollen loads
and that they spent a long time foraging at each flower,
which increases the probability for them to contact the repro-
ductive organs of C. hypocistis, and thus promoting pollina-
tion. Furthermore, pollen germination experiments showed
that the ants’ metapleural secretions did not play a major
role in inhibiting pollen viability, in contrast to the hypothesis
stating that these antimicrobial secretions mostly prevent the
transfer of viable pollen (Beattie et al., 1984, 1985; Hull and
Beattie, 1988; but see Peakall and Beattie, 1989; Gómez and
Zamora, 1992; Garcı́a et al., 1995; Gómez et al., 1996). It
may be possible that the high pollen production could ‘neutral-
ize’ the effects of ant metapleural secretions, as has been
suggested by Hull and Beattie (1988). Two of the most abun-
dant ant species, P. pallidula and C. auberti, have metapleural
glands, but they did not significantly reduce pollen viability. It
is noteworthy that although C. pilicornis reduced pollen ger-
minability (the same trend has been observed for other
Camponotus species; Beattie et al., 1985; Hull and Beattie,
1988), this ant species has no metapleural glands, a common
feature in the genus (Hölldobler and Engel-Siegel, 1984).
Perhaps antibiotic substances are distributed throughout the
cuticle or may be secreted from a different gland (Beattie
et al., 1985; Hull and Beattie, 1988). Our findings have
shown that when different ant species are pollinating a given
plant species they can differ in their pollination effectiveness,
as is well known to occur in other pollinator guilds (Herrera,
1987; Larsson, 2005; Fumero-Cabán and Meléndez-
Ackerman, 2007). Consequently, an ant can be either an
Cs2 Hh1 Hh2
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90.4 (69) 100 (15) 100 (34)
90.4 (68) 98.0 (11) 99.1 (35)
96.2 (707) 96.9 (1258) 98.5 (1680)
– – 88 (31)
0 (15) 0 (10) 0 (15)
excellent or a poor pollinator, the same as for a bee or a but-
terfly, and so generalizations about the ability and quality of
ants as pollinators should be carefully considered.
Even though C. hypocistis flowers offer abundant nectar and
pollen, the diversity and abundance of flying insects foraging
in this parasite is surprisingly low, in spite of many bees,
flies, butterflies, moths and beetles being observed foraging
on the flowers of other plant species nearby. Similar obser-
vations were done in more than 50 populations elsewhere in
Spain and Morocco (C. de Vega, CSIC, Sevilla, Spain,
unpubl. res.). Since winged-visitors were only observed in
inflorescences with few or no ants, it is likely that the presence
of ants deterred flying pollinators from visiting the flowers, as
has been suggested in other species (McDade and Kinsman,
1980; Schürch et al., 2000; Philpott et al., 2005). Among the
flying insects, only the fly O. aterrima was a predictable
visitor of C. hypocistis, appearing in all populations and
years, whereas other winged insects only appeared during
the drought year of 2005, when the dramatically low availability
of flowers of other species in the populations probably forced
them to search for alternative feeding resources. Temporal
variation in the identity and importance of pollinators has
been observed in other plant species (Herrera, 1988; Gómez
and Zamora, 1999; Thompson, 2001; Ivey et al., 2003);
nonetheless, for C. hypocistis, despite the differences found in
pollinator composition between years, the pollinator assemblage
that accounted for most visits remained remarkably constant.
When pollinator assemblages are taxonomically hetero-
geneous, determination of the behaviour of each species is
important for understanding their differential contribution to
plant reproduction (Herrera, 1987; Giménez-Benavides et al.,
2007; Li et al., 2008). Our findings suggest that despite their
low proportional frequency, the fly O. aterrima might play a
key role in producing outcrossed progeny in C. hypocistis,
since it tends to visit few flowers per individual and move
more often among different plants. In contrast, ants exhibited
a restricted foraging area and repeatedly visited individual
flowers and inflorescences, thus potentially enhancing the
occurrence of geitonogamous selfing, as has been observed
in other ant-pollination systems (Svensson, 1985; Peakall and
Beattie, 1991; Gómez and Zamora, 1992). Although selfing
often negatively affects plant reproductive success, self-
compatibility may be advantageous in species with small
population sizes, subjected to strong stochastic demographic
fluctuations (Stebbins, 1957; Schemske and Lande, 1985;
Pannell and Barrett, 1998) as it occurs in C. hypocistis
(de Vega, 2007; de Vega et al., 2008). In C. hypocistis pollina-
tion exclusively by ants resulted in lower fruit set than open-,
cross- and geitonogamous pollinations; however, it is unlike
 de Vega et al. — Pollination in Mediterranean Cytinus
that this decrease is due to the self-pollen carried by ants,
because geitonogamous hand-pollinations result in similar
levels of fruit set to cross-pollinations. Our field observations
suggest that ant behaviour could account for this reduction in
fruit set, given that ants frequently avoided some particular
flowers without contacting them. However, it could also be
plausible that some female flowers did not receive enough
visits (and enough pollen) to set fruit. Since there was no
decrease in the seed production of ant-pollinated flowers com-
pared to that obtained after hand- and open-pollinations, our
findings indicate that when ants continuously visit flowers
they act as effective pollinators.
Selfing and different types of crosses can affect the pro-
portion of viable seeds (Wallace, 2003; Jersáková and
Johnson, 2006), such that self-pollinated plants sometimes
produce fruits with empty seeds (Ferdy et al., 2001;
Smithson, 2006). However, in C. hypocistis seeds produced
after open-, cross-, geitonogamous and ant-pollination shared
similar features, containing a small embryo and endosperm
Although it has been not possible so far to experimentally
induce the germination of C. hypocistis, a recent genetic
study based on AFLP markers has suggested that geitonoga-
mous seeds produced naturally do germinate and the resulting
plants become established in the host populations (de Vega,
2007). Moreover, the low levels of genetic diversity recorded
in all populations (de Vega, 2007; de Vega et al., 2008)
were similar, or even lower, than those characteristically
found in species with high selfing rates (Nybom, 2004).
To summarize, our results indicate that mutualistic services
by ants are essential for the pollination of the Mediterranean
root holoparasite C. hypocistis. As in other reported cases of
ant pollination (Peakall et al., 1987; Peakall and Beattie,
1991), C. hypocistis does not exhibit some features of the pur-
ported ‘ant-pollination syndrome’ (Hickman, 1974), such as
small, open flowers with a small amount of pollen, readily
accessible nectaries with very little nectar production, and a
low number of ovules. However, many other features exhibited
by this parasite – self-compatibility, the large amount of pollen
produced per flower, the big size of reproductive organs, the
long floral life span and the low stature of plants – seem to
play a crucial role in enhancing the effectiveness of ants as
efficacious pollen carriers. Although it seems unlikely that
these features of C. hypocistis have evolved to facilitate polli-
nation by ants, the fact that they contribute to successful pol-
lination, together with our recent findings suggesting that
floral scents attract ants (C. de Vega, CSIC, Sevilla, Spain,
unpubl. res.), may indicate that this species could be evolving
to a more specialized pollination system. The sweet scent
emitted by C. hypocistis flowers is clearly different to the
yeasty and unpleasant scent produced by the dark flowers of
Bdallophyton (also Cytinaceae) to attract their pollinators
(Sarcophagidae flies; Garcı́a-Franco and Rico-Gray, 1997).
Perhaps some minor floral morphological changes and
altered scent production could be playing key roles in a shift
between those pollination systems. The fact that striking inter-
specific differences exist between the pollination systems of
Mediterranean Cytinus (ant-pollinated) and South African
Cytinus (mammal-pollinated; Johnson et al., 2008;
S. D. Johnson, University of Pietermaritzburg, South Africa,
unpubl. res.) make this genus an excellent model to investigate
1073
the divergent evolution of pollination systems in broadly dis-
junct areas. Floral morphology, nectar characteristics and
floral scents could be playing crucial roles in establishing
mutualistic pollination interactions in Cytinus, and may be
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critical for understanding the evolution of pollination
systems in this poorly known genus.
ACK NOW L E D G ME NT S
We thank R. Vega-Durán, E. Durán, and J. L. Garcı́a-Castaño
for help with the field work, R. G. Albaladejo for taking some
of the pictures of ants, and A. Tinaut (ants), F. J. Ortiz (bees)
and A. Sánchez (flies) for identification of insects. Special
thanks to R. G. Albaladejo and S. D. Johnson for helpful com-
ments on the manuscript. This work was supported by a Ph.D.
grant from the Spanish Ministerio de Educación y Ciencia to
C. de V. and by funds from the Ministerio de Educación y
Ciencia and Fondo Europeo de Desarrollo Regional
(REN2002 – 04354-C02-02, CGL 2005 – 01951 to M.A.) and
Consejerı́a de Innovación, Ciencia y Empresa, Junta de
Andalucı́a (EXC/2005/RNM-204 to S.T., P06-RNM-01627
to C.M.H.).
L I T E R AT U R E CI T E D
Arista M, Talavera S. 1994. Pollen dispersal capacity and pollen viability of
Abies pinsapo Boiss. Silvae Genetica 43: 155– 158.
Ashman TL, King EA. 2005. Are flower-visiting ants mutualists or antagon-
ists? A study in a gynodioecious wild strawberry. American Journal of
Botany 92: 891– 895.
Bänzinger H. 1996. Pollination of a flowering oddity: Rhizanthes zippelii
(Blume) Spach (Rafflesiaceae). Natural History Bulletin of the Siam
Society 44: 113– 142.
Bänzinger H, Pape T. 2004. Flowers, faeces and cadavers: natural feeding and
laying habits of flesh flies in Thailand (Diptera: Sarcophagidae,
Sarcophaga spp.). Journal of Natural History 38: 1677– 1694.
Barkman TJ, Lim SH, Salleh M, Nais J. 2004. Mitochondrial DNA
sequences reveal the photosynthetic relatives of Rafflesia, the world’s
largest flower. Proceedings of the National Academic of Sciences of the
USA 101: 787 –792.
Barkman TJ, McNeal JR, Lim SH, et al. 2007. Mitochondrial DNA suggests
at least 11 origins of parasitism in angiosperms and reveals genomic chi-
merism in parasitic plants. BMC Evolutionary Biology 7: 248.
doi:10.1186/1471-2148-7-248
Beaman RS, Decker PJ, Beaman JH. 1988. Pollination of Rafflesia
(Rafflesiaceae). American Journal of Botany 75: 1148–1162.
Beattie AJ, Turnbull C, Knox RB, Williams EG. 1984. Ant inhibition of
pollen function: a possible reason why ant pollination is rare. American
Journal of Botany 71: 421– 426.
Beattie AJ, Turnbull C, Hough T, Jobson S, Knox RB. 1985. The vulner-
ability of pollen and fungal spores to ant secretions: evidence and some
evolutionary implications. American Journal of Botany 72: 606– 614.
Beehler BM. 1994. Canopy-dwelling honeyeater aggressively defends terres-
trial nectar resource. Biotropica 26: 459 –461.
Bouman F, Meijer W. 1994. Comparative structure of ovules and seeds in
Rafflesiaceae. Plant Systematics and Evolution 193: 187– 212.
Burgoyne PM. 2006. A new species of Cytinus (Cytinaceae) from South
Africa and Swaziland, with a key to the Southern African species.
Novon 16: 315– 319.
Davis CC, Latvis M, Nickrent DL, Wurdack KJ, Baum DA. 2007. Floral
gigantism in Rafflesiaceae. Science 315: 1812.
Ferdy JB, Loriot S, Sandmeier M, Lefranc M, Raquin C. 2001. Inbreeding
depression in a rare deceptive orchid. Canadian Journal of Botany 79:
1181– 1188.
Fumero Cabán J, Meléndez-Ackerman E. 2007. Relative pollination effec-
tiveness of floral visitors of Pitcairnia angustifolia (Bromeliaceae).
American Journal of Botany 94: 419– 424.
1074 de Vega et al. — Pollination in Mediterranean Cytinus
Garcı́a MB, Antor RJ, Espadaler X. 1995. Ant pollination of the paleoen-
demic dioecious Borderea pyrenaica (Dioscoreaceae). Plant Systematics
and Evolution 198: 17–27.
Garcı́a-Franco JG, Rico-Gray V. 1997. Reproductive biology of the holopar-
asitic endophyte Bdallophyton bambusarum (Rafflesiaceae). Botanical
Journal of the Linnean Society 123: 237–247.
Giménez-Benavides L, Dötterl S, Jürgens A, Escudero A, Iriondo JM.
2007. Generalist diurnal pollination provides greater fitness in a plant
with nocturnal pollination syndrome: assessing the effects of a Silene–
Hadena interaction. Oikos 116: 1461–1472.
Gómez JM. 2000. Effectiveness of ants as pollinators of Lobularia maritima:
effects on main sequential fitness components of the host plant.
Oecologia 122: 90–97.
Gómez JM, Zamora R. 1992. Pollination by ants: consequences of the
quantitative effects on a mutualistic system. Oecologia 91: 410 –418.
Gómez JM, Zamora R. 1999. Generalization vs. specialization in the pollina-
tion system of Hermantophylla spinosa (Crucifera). Ecology 80:
796–805.
Gómez JM, Zamora R, Hódar JA, Garcı́a D. 1996. Experimental study of
pollination by ants in Mediterranean high mountain and arid habitats.
Oecologia 105: 236–242.
Gómez LD. 1983. Flora Costaricensis: Rafflesiaceae. Fieldiana Botany 13:
89– 93.
Harms H. 1935. Rafflesiaceae, Tribe IV. Cytineae. In: Engler A, Harms H eds.
Die Natürlichen Planzenfamilien. Leipzig: Wilhelm Engelman,
276–281.
Herrera CM. 1987. Components of pollinator ‘quality’: comparative analysis
of a diverse insect assemblage. Oikos 50: 79– 90.
Herrera CM. 1988. Variation in mutualism: the spatiotemporal mosaic of a
pollinator assemblage. Biological Journal of the Linnean Society 35:
95– 125.
Herrera CM. 1989. Pollinator abundance, morphology, and flower visitation
rate: analysis of the ‘quantity’ component in a plant–pollinator system.
Oecologia 80: 241 –248.
Hickman JC. 1974. Pollination by ants: a low-energy system. Science 184:
1290–1292.
Hidayati SN, Meijer W, Baskin JM, Walck JL. 2000. A contribution to the
life history of the rare Indonesian holoparasite Rafflesia patma
(Rafflesiaceae). Biotropica 32: 408–414.
Hölldobler B, Engel-Siegel H. 1984. On the metapleural gland of ants.
Psyche 91: 201–224.
Hull DA, Beattie AJ. 1988. Adverse effects on pollen exposed to Atta texana
and other North American ants: implications for ant pollination.
Oecologia 75: 153 –155.
Ivey CT, Martinez P, Wyatt R. 2003. Variation in pollinator effectiveness in
swamp milkweed (Asclepias incarnata). American Journal of Botany 90:
212–223.
Jersáková J, Johnson SD. 2006. Lack of floral nectar reduces self-pollination
in a fly-pollinated orchid. Oecologia 147: 60–68.
Johnson SD, Burgoyne PM, Harder LD, Dötterl S. 2008. Evidence for pol-
lination by small mammals in the parasitic plant Cytinus visseri
(Cytinaceae). South African Journal of Botany 74: 369 [SAAB Annual
Meeting Abstracts].
Kuijt J. 1969. The biology of parasitic flowering plants. Berkeley, CA:
University of California Press.
Larsson M. 2005. Higher pollinator effectiveness by specialist than generalist
flower-visitors of unspecialized Knautia arvensis (Dipsacaceae).
Oecologia 146: 394–403.
Li P, Luo Y, Bernhardt P, Kou Y, Perner H. 2008. Pollination of
Cypripedium plectrochilum (Orchidaceae) by Lasioglossum spp.
(Halictidae): the roles of generalist attractants versus restrictive floral
architecture. Plant Biology 10: 220– 230.
Mabberley D. 1997. The plant book, 2nd edn. Cambridge: Cambridge
University Press.
Martin FW. 1959. Staining and observing pollen tubes in the styles by means
of fluorescence. Stain Technology 34: 125– 128.
Matuda E. 1947. On the genus Mitrastemon. Bulletin of the Torrey Botanical
Club 74: 133–141.
McDade LA, Kinsman S. 1980. The impact of floral parasitism in two neo-
tropical hummingbird-pollinated plant species. Evolution 34: 944 –958.
Meijer W. 1993. Rafflesiaceae. In: Kubitzki K, Rohwer JG, Bittrich V eds.
The families and genera of vascular plants, vol. II. Berlin:
Sringer-Verlag, 557– 563.
Molau U. 1995. Reproductive ecology and biology. In: Press MC, Graves JD
eds. Parasitic plants. London: Chapman and Hall, 141 –177.
Nickrent DL. 2002a. Plantas parásitas en el mundo. In: López-Sáez JA,
Catalán P, Sáez L eds. Plantas Parásitas de la Penı́nsula Ibérica e
Islas Baleares. Madrid: Mundi-Prensa Libros S.A., 7 –28.
Downloaded from https://academic.oup.com/aob/article-abstract/103/7/1065/104685 by UNIVERSITAT DE BARCELONA. Biblioteca user on 01 April 2020
Nickrent DL. 2002b. Origen filogenético de las plantas parásitas. In:
López-Sáez JA, Catalán P, Sáez L eds. Plantas Parásitas de la
Penı́nsula Ibérica e Islas Baleares. Madrid: Mundi-Prensa Libros S.A.,
29–56.
Nickrent DL, Duff RJ, Colwell AE, et al. 1998. Molecular phylogenetic and
evolutionary studies of parasitic plants. In: Soltis DE, Soltis PS, Doyle JJ
eds. Plant molecular systematics II. Boston, MA: Kluwer, 211– 241.
Nickrent DL, Blarer A, Qiu YL, Vidal-Russell R, Anderson FE. 2004.
Phylogenetic inference in Rafflesiales: the influence of rate heterogeneity
and horizontal gene transfer. BMC Evolutionary Biology 4: 40.
doi:10.1186/1471-2148-4-40
Nybom H. 2004. Comparison of different nuclear DNA markers for estimating
intraspecific genetic diversity in plants. Molecular Ecology 13:
1143–1155.
Pannell JR, Barrett SCH. 1998. Baker’s law revised: reproductive assurance
in a metapopulation. Evolution 52: 657 –668.
Peakall R. 1989. The unique pollination of Leporella fimbriata (Orchidaceae):
Pollination by pseudocopulating male ants (Myrmecia urens,
Formicidae). Plant Systematics and Evolution 167: 137– 148.
Peakall R, Beattie AJ. 1989. Pollination of the orchid Microtis parviflora
R. Br. by flightless worker ants. Functional Ecology 3: 515– 522.
Peakall R, Beattie AJ. 1991. The genetic consequences of worker ant pollina-
tion in a self-compatible, clonal orchid. Evolution 45: 1837– 1848.
Peakall R, Beattie AJ, James SH. 1987. Pseudocopulation of an orchid by
male ants: a test of two hypotheses accounting for the rarity of ant pollina-
tion. Oecologia 73: 522– 524.
Peitsch D, Fietz A, Hertel H, Desouza J, Ventura DF, Menzel R. 1992. The
spectral input systems of hymenopteran insects and their receptor-based
color-vision. Journal of Comparative Physiology A 170: 23–40.
Philpott SM, Greenberg R, Bichier P. 2005. The Influence of ants on the
foraging behavior of birds in an agroforest. Biotropica 37: 468–471.
van der Pijl L. 1961. Ecological aspects of flower evolution. II. Zoophilous
flower classes. Evolution 15: 44– 59.
Puterbaugh MN. 1998. The roles of ants as flowers visitors: experimental
analysis in three alpine plant species. Oikos 83: 36–46.
Ramsey M. 1995. Ant pollination of the perennial herb Blandfordia grandi-
flora (Liliaceae). Oikos 74: 265– 272.
Schürch S, Pfunder M, Roy BA. 2000. Effects of ant on the reproductive
success of Euphorbia cyparissias and associated pathogenic rust fungi.
Oikos 88: 6– 12.
Schemske DW, Lande R. 1985. The evolution of self-fertilization and
inbreeding depression in plants. II. Empirical observations. Evolution
39: 41– 52.
Shen H, Ye W, Hong L, et al. 2006. Progress in parasitic plant biology: host
selection and nutrient transfer. Plant Biology 8: 175– 185.
Sugiura N, Miyazaki S, Nagaishi S. 2006. A supplementary contribution of
ants in the pollination of an orchid, Epipactis thunbergii, usually polli-
nated by hover flies. Plant Systematics and Evolution 258: 17– 26.
Smithson A. 2006. Pollinator limitation and inbreeding depression in orchid
species with and without nectar rewards. New Phytologist 169: 419– 430.
StatSoft Inc. 2001. STATISTICA (data analysis software), version 6. http://
www.statsoft.com
Stebbins GL. 1957. Self-fertilization and population variability in the higher
plants. American Naturalist 91: 337 –354.
Stewart GR, Press MC. 1990. The physiology and biochemistry of parasitic
angiosperms. Annual Review of Plant Physiology and Plant Molecular
Biology 41: 127– 151.
Svensson L. 1985. An estimate of pollen carryover by ants in a natural popu-
lation of Scleranthus perennis L. (Caryophyllaceae). Oecologia 66:
373– 377.
Thompson JD. 2001. How do visitation patterns vary among pollinators in
relation to floral display and floral design in a generalist pollination
system? Oecologia 126: 386–394.
de Vattimo I. 1971. Contribuição ao conhecimento da tribu Apodantheae
R. Br. Parte 1 Conspecto das espécies (Rafflesiaceae). Rodriguésia 26:
37–62.
de Vega C. 2007. Reproductive biology of Cytinus hypocistis (L.) L.:
host-parasite interactions. Ph.D. Thesis, University of Seville, Spain.
 de Vega et al. — Pollination in Mediterranean Cytinus
de Vega C, de Oliveira RC. 2007. A new procedure for making observations
of embryo morphology in dust-like seeds with rigid coats. Seed Science
Research 17: 63–67.
de Vega C, Ortiz PL, Arista M, Talavera S. 2007. The endophytic system of
Mediterranean Cytinus (Cytinaceae) developing on five host Cistaceae
species. Annals of Botany 100: 1209–1217.
de Vega C, Berjano R, Arista M, Ortiz PL, Talavera S, Stuessy TF. 2008.
Genetic races associated with the genera and sections of host species in
the holoparasitic plant Cytinus (Cytinaceae) in the Western
Mediterranean basin. New Phytologist 178: 875– 887.
1075
Visser J. 1981. South African parasitic flowering plants. Capetown: Juta &
Co.
Wallace LE. 2003. The cost of inbreeding in Platanthera leucophaea
(Orchidaceae). American Journal of Botany 90: 235–242.
Webb DA. 1964. Cytinus. In: Tutin TG, Heywood VH, Burges NA, Valentine
Downloaded from https://academic.oup.com/aob/article-abstract/103/7/1065/104685 by UNIVERSITAT DE BARCELONA. Biblioteca user on 01 April 2020
DH, Walters SM, Webb DA eds. Flora Europaea. Cambridge: Cambridge
University Press, 75.
Wyatt R. 1981. Ant-pollination of the granite outcrop endemic Diamorpha
smallii (Crassulaceae). American Journal of Botany 68:
1212– 1217.