ORI GI NAL ARTI CLE

Factors affecting pollinator movement and plant tness

in a specialized pollination system
Julieta Nattero

Romina Malerba

Rodrigo Medel

Andrea Cocucci
Received: 4 January 2011 / Accepted: 16 May 2011 / Published online: 9 June 2011
Springer-Verlag 2011
Abstract The rate of pollen exchange within and among
owers may depend on pollinator attraction traits such as
oral display size and owering plant density. Variations in
these traits may inuence pollinator movements, pollen
receipt, and seed number. To assess how oral display size
and owering plant density affect parameters of pollinator
visitation rate, pollen receipt per ower, seed number per
fruit and the between-plant pollinator movements, we
studied the self-incompatible plant, Nierembergia linarii-
folia. Per-ower pollinator visitation rate and bout length
increased linearly with increasing oral display size. Pollen
receipt per ower increased linearly with increasing ow-
ering plant density. For seed number per fruit, a polynomial
model describing an increased seed number per fruit at low
density and a decreased seed number per fruit at high
density provided a signicant t. Per-ower pollinator
visitation rate was not associated with pollen receipt per
ower and seed number per fruit. Bees visited plants
located near to the center of the population more frequently
than plants located at the periphery. Increases in both oral
display size and owering plant density led to an increased
chance of a plant being chosen as the center of the polli-
nator foraging area. These results suggest that even though
large oral displays and high owering plant density are
traits that attract more pollinators, they may also reduce
potential mate diversity by restricting pollen movement to
conspecic mates that are closely located.
Keywords Centris tricolor Floral display size
Flowering plant density Nierembergia linariifolia
Pollen receipt Pollinator visitation rate Seed number
Introduction
In animal-pollinated plants, the number of open owers per
plant (oral display size) may inuence the attraction of
pollinators, their movement within and among plants, and
hence the rate of pollen exchange and seed production (e.g.,
Makino et al. 2007; Ohashi and Yahara 1999; Willson and
Price 1977). A large oral display increases the attractive-
ness to pollinators since it is often associated with a high rate
of reward and reduced traveling time (Kacelnik et al. 1986;
Makino et al. 2007). Variations in oral display size may
affect pollinator response in two ways. First, several studies
have shown a positive relationship between oral display
size and the frequency of pollinator visits at the individual
plant level (i.e., plant visitation rate; e.g., Klinkhamer and de
Jong 1990; 15 out of 17 studies reviewed by Ohashi and
Yahara 1999, 2002). This pollinator response may promote
outcrossing through enhanced pollen receipt but may also be
associated with important costs in terms of lost mating
opportunities through the male function and pollen dis-
counting (Harder and Barrett 1995, 1996). Second, because
the number of owers that pollinators probe per plant within
J. Nattero R. Malerba A. Cocucci
Instituto Multidisciplinario de Biolog a Vegetal,
CONICET-Universidad Nacional de Cordoba,
Casilla 495, X5000JJC Cordoba, Argentina
R. Medel
Departamento de Ciencias Ecologicas, Facultad de Ciencias,
Universidad de Chile, Casilla 653, Santiago, Chile
Present Address:
J. Nattero (&)
Catedra de Introduccion a la Biolog a,
Facultad de Ciencias Exactas, F sicas y Naturales,
Universidad Nacional de Cordoba, Avda. Velez Sarseld 299,
piso 5, X5000JJC Cordoba, Argentina
e-mail: jnattero@efn.uncor.edu
1 3
Plant Syst Evol (2011) 296:7785
DOI 10.1007/s00606-011-0477-4
a foraging bout (bout length) tends to increase with
increasing oral display size (Grindeland et al. 2005; studies
reviewed by Ohashi and Yahara 1999; Pyke 1979), large
oral displays would be expected to promote within-plant
pollinator movements and longer bouts. Although the
importance of oral display size for pollen dispersal may
depend on the pollinator species involved, in general, oral
display size may affect selng rate and the evolution of the
breeding system through the expression of pollen dis-
counting and stigma clogging in self-incompatible species
(Ritland 1991; Robertson and Macnair 1995).
The number of neighboring plants (owering plant den-
sity) may also inuence pollinator movement and hence
plant reproductive success (e.g., Elliot and Irwin 2009;
Ghazoul and Shaanker 2004; Holland et al., 2002; Maron
and Crone, 2006; Mitchell et al. 2004). Like oral display,
increasing owering plant density may increase pollinator
attraction and pollinator service per unit time (Dreisig 1995;
Grindeland et al. 2005; Klinkhamer and de Jong 1990).
Dense patches may be more attractive to pollinators because
they represent clumped resources that permit a reduction in
travel time among multiple sparse patches (Kacelnik et al.
1986). Plants in turn may benet frombeing in dense patches
if abundant oral resources attract more pollinators and/or
provide an ample supply of compatible pollen donors
(Hegland and Boeke 2006; Kunin 1993; Waites and A

gren
2004). In dense patches, pollinators may visit owers from
different plants favoring pollen exchange among plants and
reducing bout length and visitation rate per plant. Thus,
depending on bout length and visitation rate per plant,
owering plant density may affect pollination either posi-
tively (e.g., Ehlers et al. 2002; Klinkhamer and de Jong
1990; Kunin 1997; Nielsen and Ims 2000) or negatively
(Mustajarvi et al. 2001; Stout et al. 1998). Plants in dense
patches may experience increased levels of intraspecic
competition for pollinator visits (Steven et al. 2003). If seed
set is pollinator-limited and pollinators visit a smaller pro-
portion of owers per plant in dense rather than low-density
patches, then seed set per ower may decline in dense pat-
ches (Garc a-Robledo et al. 2005). Notwithstanding, seed set
may decline as a result of the action of other ecological
forces such as abiotic factors (Galen et al. 1999; Haig and
Westoby 1988). For instance, it has been proposed that
natural selection may bring female reproductive effort to a
point where seed production is limited by both pollen supply
and provisioning resources (Haig and Westoby 1988).
Pollinator movement may have important implications
for pollen ow among plants. Most evidence on pollinator
movement has been quantied as total visitation rate (total
visits to a plant made by unidentied individual pollina-
tors). However, visitation rate consists of (a) returns made
by one or a few individuals to owers, and (b) occasional
visits made by different individuals to owers (Makino
et al. 2007). These components may have different con-
sequences for the way pollen is dispersed across plants. For
example, while potential mate diversity may increase with
an increase in the number of individual pollinators that visit
the plant per unit of time, geitonogamous pollination,
pollen discounting and stigma clogging in self-incompati-
ble species may increase with an increase in the number of
foraging bouts (de Jong et al. 1993; Richards 1997; Ritland
1991; Robertson and Macnair 1995).
One way to understand the way display size and ow-
ering plant density inuence potential mate diversity and
pollen movement within and among plants is to study the
total visitation rate and patterns of individual pollinator
movements within and between plants and their inuence
on plant tness. In this work we examined the effect of
display size and plant density on multiple steps of the
pollination and seed production process in Nierembergia
linariifolia Graham, a self-incompatible plant with a spe-
cialized pollination system. More specically, we sought to
determine whether per-ower pollinator visitation rate, the
pattern of between-plant pollinator movement, pollen
receipt per ower, and seed number per fruit are variables
that depend on display size and plant density.
Nierembergia linariifolia is a plant species with oil-
secreting owers that receives the pollination service of
solitary oil-collecting bees. Previous studies of this system
have shown that this species depends exclusively on poll-
inators for reproduction, and is pollen-limited for fruit and
seed production through quantity (i.e., via pollinator visits
and number of pollen grains deposited on stigmas) and
quality components (i.e., via compatibility of the pollen
delivered) (Cosacov et al. 2008). The aims of this work
were to evaluate whether oral display size and owering
plant density in this species affect: (1) the movement of the
specialized insects involved in the pollination process (per-
ower pollinator visitation rate, bout length, proportion of
visited owers), (2) the amount of pollen receipt per ower
and/or seed number per fruit, and (3) the pattern of polli-
nator movements within and among plants.
Materials and methods
Study system
Nierembergia linariifolia var. pinilioides (Millan) Andr.
Cocucci et Hunziker (Solanaceae) is a perennial shrub
endemic to Sierras Chicas in Central Argentina. Populations
occur in open elds, and can also be found on road-sides and
disturbed areas where soil had been recently exposed. Plants
are distributed in isolated patches, with usually fewer than
50 plants and exceptionally more than 200 in each popula-
tion. Flowers are violet, with a thin nectarless tube and a
78 J. Nattero et al.
1 3
somewhat horizontal well-developed corolla limb. One petal
lobe of the corolla limb, which is called the labellum, is
usually larger than the remaining lower ones. The fertile
parts, consisting of ve stamens and the stigmatic portion of
the pistil, are exserted, and emerge from the center of the
almost at limb (Cocucci 1991). The ower reward consists
of fatty oils distributed on a restricted area of the corolla
limb, a purple ring where glandular hairs (elaiophores) are
located. In the studied population plants are strictly self-
incompatible and pollen-limited by quality and quantity,
depending exclusively on pollinators for reproduction
(Cosacov et al. 2008). Daily oral display size varies from
one to more than 100 owers per plant. Flower longevity is
about 3 days (Nattero et al. 2010).
This study was conducted during the austral spring and
summer of 2008 in a population near Capilla del Monte
(30852
0
31.0
00
S, 64832
0
12.1
00
W, Cordoba province,
Argentina). This population is located in the Chaco Serrano
Forest, a montane variant of the Chaco Forest, the most
extensive dry forest area in South America (Cabrera,
1971). Altitude ranges between 400 and 1,300 m, mean
January and July temperatures are 23C and 11C,
respectively, and the mean annual rainfall is 550 mm. The
vegetation consists of low and open woodland dominated
by Prosopis species and Aspidosperma quebracho-blanco
(Cabrera 1971). In this site, N. linariifolia is distributed in
multispecic patches and coincides in owering with
Sphaeralcea cordobensis, Oenothera afnis, Turnera
sidoides, Glandularia dissecta, Glandularia chiloensis,
Gaillardia megapotamica and Petunia axillaris. among
others. All these plants offer nectar as reward, whereas
only N. linariifolia offers oils as oral reward.
We selected a population of 50 plants that covered an
area of 77 9 132 m. All owering plants in the population
were mapped (Fig. 2a) using the polar coordinates method
with respect to a xed reference point. The population map
allowed the estimation of the owering plant density per
meter squared around each plant. Since we were interested
in understanding the way display size and plant density
determines pollinator visits and returns to plants, we
counted the number of plants within a circle of 0.5-m
radius around each plant, and used these data to calculate
owering plant density. We chose circles of 0.5-m radius
around plants because studies on the spatial memory and
movement patterns of bumblebees indicate that insects
memorize spatial congurations of 10-cm distance between
owers in near- and far-searching strategies and reward
detection (Burns and Thomson 2006).
Pollinators
Pollinators were observed over 5 days distributed
throughout the owering period (2 months). Pollinators
were observed for a total of 57 h spread evenly during the
observation days by at least two observers. Because this
species is not pollinated at night, observations were done
from 10 a.m. to 6 p.m., the time interval when the two bee
species are active in the study site. In each sampling day, a
variable number of 30-min focal observations per plant
were performed, and pollinator visits and the identity of the
pollinator involved were recorded. A visit was recorded if
the bee contacted the fertile part of the ower. Specically,
for each observation day we recorded: the number of open
owers per plant, the number of times each plant was
visited, and the number of owers probed per visit. From
these data we estimated: the average oral display size per
plant across observation days, per-ower pollinator visita-
tion rate (owers visited per plant per hour standardized by
the number of owers in the plant), bout length (the
number of owers probed during a single plant visit) and
the proportion of owers visited (bout length divided by
oral display size).
To study the pattern of bee movement among plants, we
followed a visiting bee and recorded the sequence of
individual plants visited and the number of owers probed
per plant until it left the population. We recorded a total of
70 bee paths during the ve observation days. The most
frequent pollinator was the bee Centris tricolor (Apidae,
Centridini) (Fig. 1) which accounted for 99% of the total
visits recorded. The bee Tapinotaspis chalybaea (Apidae,
Tapinotaspidini) was responsible for the remaining 1% of
total pollinator visits (Nattero et al. 2010). Because the bee
T. chalybaea was too rare and sporadic in this population,
we only considered the pollination activity of C. tricolor to
describe the pattern of pollinator movement.
Fig. 1 Visual description of the study system. Centris tricolor bee
collecting oil from a Nierembergia linariifolia ower
Factors affecting pollinator movement and plant tness 79
1 3
Pollen receipt per ower and seed number per fruit
To estimate pollen receipt per ower we collected stigmas
from three randomly chosen senescent owers per plant at
the end of each observation day. Stigmas were mounted
on microscope slides, and the pollen was stained with
basic fuchsine dye (Kearns and Inouye 1993). We counted
the stained grains of N. linariifolia under a microscope.
Heterospecic pollen on stigmas was very rare. The
number of pollen grains deposited on the entire stigma
surface was averaged over three owers per plant. For
each plant in the population, we estimated the seed
number per fruit as the mean seed production from ve
randomly collected fruits shortly before seed dispersal.
Seeds were counted in the laboratory under a binocular
microscope.
Data analysis
Since we were interested in assessing the joint effect and
relative importance of oral display size and owering
plant density on per-ower pollinator visitation rate, bout
length, and proportion of owers visited, a multiple
regression was used for each dependent variable. Floral
display size and owering plant density showed a positive
but nonsignicant correlation (r = 0.126; p = 0.396),
which permits us to rule out a potential multicollinearity of
independent variables.
To analyze the relationship between oral display size,
owering plant density, per-ower visitation rate and bout
length on pollen receipt per ower and seed number per
fruit we used independent models of univariate linear
regression. We did not include a multiple regression
because our interest was to understand the effect of each
variable on female tness by separate. Since preliminary
inspection of data suggested a unimodal rather than linear
relationship between owering plant density and seed
number per fruit we included a quadratic term in all these
univariate regressions.
To analyze the pattern of pollinator movements among
plants we followed the methodology proposed by Makino
et al. (2007). To quantify the shape of the distribution
among the bee paths we calculated the Simpsons measure
of evenness (E) as follows:
E
NN 1
P
n
i
n
i
1

1
s
;
where n
i
is the number of visits to plant i by a bee in a path,
N is the total number of plant visits made by a bee in a path
(equal to Rn
i
), and s is the total number of individual plants
visited by a bee in a path (Krebs 1999; Makino et al. 2007).
According to this measure, E equals 1 when bee visits are
distributed randomly among plants in a path and
approaches 0 when bees visit an increasingly restricted set
of plants in a path.
To examine whether the distribution of visits to plants
differed among bee paths, we used a chi-squared test for
homogeneity with rows representing the observed bee
paths and columns the number of visits received per plant
in decreasing order. If the expected frequencies were fewer
than ve in more than 20% of the cells, we summed the
adjacent columns to correct for bias in the chi-squared
calculations (Sokal and Rohlf 1995). To evaluate whether
bees visited more frequently those plants located at the
central part of the population, we performed a Kendalls
rank correlation analysis between the distance of each plant
to the centroid of the population and the number of visits
received. The centroid of the population was estimated by
averaging the geographic coordinates (latitude and longi-
tude) of every plant in the population. A similar procedure
was used for bee paths, this time calculating the centroid of
the foraging area of the bee paths (i.e., by averaging the
geographic coordinates of every plant visited in each bee
path). We then calculated the distance from all the plants to
the centroid of the foraging area of each bee path and the
number of ower visits to the plant by the bee and per-
formed a Kendalls rank correlation analysis considering
individual bees as blocking factor.
The effects of oral display size and owering plant
density on the center location of a bees foraging area was
tested by ANCOVA, considering the distance from the
centroid as the dependent variable, oral display size and
owering plant density as covariates, and individual bees
as the main factor. For all the results, mean values are
presented with SD values.
Results
Effects of oral display and plant density on pollination
and reproductive success
The average oral display size was 12.43 9.53 open
owers per plant per day. Mean owering plant density
around each plant was 4.1 2.56 plants/m
2
. For the 57
observation hours, we recorded 804 visits to plants and
2,903 visits to owers. Per-ower visitation rate was 5.05
owers visited per plant/hour. The number of visits each
plant received per day varied greatly among plants
(Fig. 2b). While some plants attracted only 3 visitors in the
57 h observation period, others received more than 200
visits (maximum number of visits 257). The average bout
length per plant was 3.56 3.54, and the maximum
number of owers probed per plant during a single plant
visit was 28. Of the 804 bouts, 23% were just one ower
long.
80 J. Nattero et al.
1 3
Per-ower pollinator visitation rate and bout length
increased signicantly with increasing oral display size
(Table 1; Fig. 3). Plant density negatively and signicantly
affected the bout length, indicating that intraplant (geito-
nogamous) movements tend to decrease with increasing
plant density (Table 1). Plant density did not affect per-
ower pollinator visitation rate. The average proportion of
owers visited was not affected either by oral display size
or owering plant density (Table 1).
Pollen receipt per ower and seed number per fruit were
not signicantly associated (r = 0.08, p = 0.564). Floral
display size did not affect pollen receipt per ower or seed
number per fruit (Table 2). However, the amount of pollen
received per ower increased linearly with increasing plant
density (Fig. 4a). A polynomial model described an
increased seed number per fruit at low plant densities and a
reduction in seed production at high plant densities. The
maximum seed number per fruit was observed at a density
near 12 plants/m
2
(Table 2; Fig. 4b).
The per-ower pollinator visitation rate was not related
to pollen receipt, but was related with borderline signi-
cance to seed number per fruit (Table 2). Bout length was
not related to pollen receipt per ower or seed number per
fruit (Table 2).
Patterns of bee movement across plants
The number of plants visited in bee paths ranged from 5 to
29 with an average of 10.38 6.32 plants per path. The
mean number of visited owers per bee path was
38.19 24.93. In 35 out of the 70 bee paths observed, the
visitors returned to at least one of the previously visited
A
B
10 m
10 m
E W
N
S
E W
N
S
Fig. 2 Plant location in the study population of Nierembergia
linariifolia. a The size of each circle indicates the average oral
display size per sampling day. b The size of the circle indicates the
average number of visits per day each plant received
Table 1 Multiple regression models that test the effect of oral
display size and owering plant density on per-ower pollinator
visitation rate (owers visited per hour per plant), bout length
(number of owers probed during a single plant visit) and proportion
of owers visited (bout length per oral display size)
Response variable Per-ower pollinator visitation rate Bout length Proportion of owers visited
b SE b SE b SE
Floral display size 0.21 0.09* 0.07 0.01** -0.01 0.03
Flowering plant density 0.01 0.01 -0.07 0.02** -0.04 0.05
Model R
2
= 0.10; F = 2.72; p = 0.076 R
2
= 0.54; F = 29.02; p = 0.001 R
2
= 0.02; F = 0.02; p = 0.997
* p \0.01; ** p \0.001
Display size
0 10 20 30 40 50 60
V
i
s
i
t
a
t
i
o
n

r
a
t
e

(
f
l
o
w
e
r
s

v
i
s
i
t
e
d
/
p
l
a
n
t
/
h
o
u
r
)
0
5
10
15
20
25
Fig. 3 Relationship between Nierembergia linariifolia oral display
size and per-ower pollinator visitation rate (owers visited per plant
per hour)
Factors affecting pollinator movement and plant tness 81
1 3
plants. The distribution of visits differed among bee paths
(v
2
= 125.02; df 483; p \0.0001). Mean Simpsons
evenness (E) values was 0.68 ranging between 0.32 and
0.86, indicating that while some bees in paths allocated
their visits evenly among plants (e.g., E = 0.86), others
concentrated their activities on a small proportion of plants
(e.g., E = 0.32).
The results of the Kendalls rank correlation analysis
between the centroid of the population and the number of
visits showed a signicant and negative relationship
(Kendalls blocked s = -0.181; p \0.05), indicating that
bees more frequently visited plants located near to the
center of the population than plants located in the periph-
ery. Similar results were found for bee paths (Kendalls
blocked s = -0.089, p \0.05). The distance from the
plant to the centroid of a bees foraging area tended to
increase with increasing oral display size and plant den-
sity (Table 3), implying that these two variables increased
the chance of a plant of being chosen by a bee as the center
of the foraging area path.
Discussion
Several studies have evaluated the inuence of oral dis-
play size on total visitation rate. Results from these studies
indicate that per-ower visitation rate may increase (e.g.,
Grindeland et al. 2005; Klinkhamer et al. 1989; Makino
et al. 2007; Mitchell 1994), decrease (e.g., Andersson
1988; Klinkhamer and de Jong 1990) or remain unchanged
with increasing oral display size (e.g., Ohashi and Yahara
2002; Robertson and Macnair 1995; Vaughton and Ramsey
1998). Our data in N. linariifolia indicate that an increase
in oral display size led to an important increase in per-
ower bee visitation rate (Fig. 3). Likewise, empirical
evidence indicates that bout lengths tend to increase with
increasing oral display size, but this increase is often
Table 2 Results of the linear and polynomial (quadratic term) regression (b SE) between oral display size, owering plant density, per-
ower pollinator visitation rate and bout length, and pollen receipt per ower and seed number per fruit
Pollen receipt per ower Seed number per fruit
Linear t Polynomial t Linear t Polynomial t
Floral display size 1.46 2.17 0.00 0.00 0.73 0.76 0.21 0.11
Flowering plant density 6.34 2.35** -0.41 0.42 -0.11 0.91 -0.47 0.15**
Per-ower pollinator visitation rate 2.13 3.27 -0.22 0.47 1.89 0.33* 0.15 0.16
Bout length 2.99 9.39 2.12 2.70 3.83 3.29 0.05 0.95
* p \0.10; ** p \0.01
Fig. 4 Relationship between Nierembergia linariifolia plant density
(owering plants/m
2
) and (a) pollen receipt per ower and (b) seed
number per fruit
Table 3 ANCOVA results for effects of oral display size, plant
density, and bee paths on the distance from the plant to centroid of the
foraging area in Nierembergia linariifolia
Source of variation Degrees of
freedom
Mean
square
F b
a
Floral display size 1 18.26 7.62*** -0.03
Plant density (plants/m
2
) 1 128.77 53.78*** -0.16
Bee path 69 723.05 28.17***
Error 382 598.23
*** p \0.001.
a
Regression coefcient in a general linear model.
82 J. Nattero et al.
1 3
accompanied by a decrease in the proportion of owers
visited (references in Mitchell et al. 2004). In this study, we
found that bout lengths of bee pollinators increased with
increasing display size in N. linariifolia, but this increase
was not accompanied by a signicant decrease in the
proportion of owers visited (Table 1). However, bout
length decreased with increasing owering plant density
(Table 1), suggesting that ight costs associated with plant
movement tend to decrease when plants are more densely
distributed (see also Grindeland et al. 2005).
We found that oral display size did not show a sig-
nicant association with seed number per fruit (Table 2),
indicating that the strong and positive association found
between display size and per-ower pollinator visitation
rate did not translate into a benet in the reception of
compatible pollen. There are at least two potential
explanations that may account for this pattern. First,
because an increase in plant attractiveness often results in
a concomitant increase in the rate of geitonogamy (polli-
nation between owers on the same plant) (e.g., Harder
and Barrett 1995; Karron et al. 2004), the chance of cross-
pollination for an individual ower will be a decreasing
function of display size or plant attractiveness to pollina-
tors. Large oral displays will therefore be penalized, as
selection through the female function is particularly strong
under conditions of pollen-limitation (Ashman and
Morgan 2004; Totland 2001; Vaughton and Ramsey
2010). In such circumstances, traits that increase pollinator
attraction without conveying a reproductive cost in
geitonogamy should be selected for, especially in self-
incompatible species. Previous information on N. linarii-
folia populations indicates pervasive pollen limitation in
terms of quality and quantity (Cosacov et al. 2008), and
pollinator-mediated selection coefcients show consistent
selection on oral area instead of total oral display size
(Nattero et al. 2010). Second, because plants need to divert
more resources to ower maintenance in larger than in
smaller displays, seed production may be severely curtailed
by resource availability and provisioning (e.g., Andersson
2005, 2006; Haig and Westoby 1988), hence diluting any
effect of the display size/visitation rate relationship on plant
fecundity.
Pollen receipt per ower increased linearly with
increasing owering plant density (Table 2). This result
might be explained by an increased per-ower pollinator
visitation rate and pollen receipt at higher plant densities
(Engel and Irwin 2003). However, per-ower pollinator
visitation rate did not inuence pollen receipt per ower
(Table 2). This result is intriguing since pollen receipt and/
or removal often increase with increasing pollinator visi-
tation rate (e.g., Duncan et al. 2004; Elliot and Irwin 2009,
2003; Johnson et al. 2003; Karron et al. 2006). It is likely
that our estimation of per-ower pollinator visitation rate
was too coarse to detect a relationship between per-ower
visitation rate and pollen receipt per ower, or pollen
receipt was saturated with surplus pollinator visits.
A polynomial model described the relationship between
owering plant density with seed number per fruit
(Fig. 4b). Seed number increased with increasing plant
density up to a maximum value near 12 plants/m
2
, after
which it decreased with increasing plant density. Plant
density affected pollen receipt and seed number in different
ways (Fig. 4a, b). While pollen receipt per ower depends
on the quantity of pollen deposited onto the stigma (either
self- or outcross pollen), seed number per fruit probably
relates to the quality of the pollen that is deposited (only
cross-compatible outcross pollen will produce seeds).
Several studies have described signicant associations
between plant density and tness-related variables such as
seed number per fruit (e.g., Pettersson 1997; Sabat and
Ackerman 1996; Somanathan and Borges 2004; Spigler
and Chang 2008), and pollen receipt and/or removal (e.g.,
Duncan et al. 2004; Elliot and Irwin 2009). To our
knowledge, only one study has found a hump-backed curve
for the relationship between conspecic density and
reproductive success (Elliot and Irwin 2009)
Half of the foraging paths made by C. tricolor bees
showed that they returned to at least one of the visited
plants. The memory hypothesis states that when foragers
reuse patches, they can potentially benet from remem-
bering the most protable patches, and return preferentially
to these. There is good evidence that bumblebees have a
strong long-term spatial memory, in that they repeatedly
return to the same foraging areas (Osborne et al. 1999,
2001), remember and return to highly rewarding plants
(Cartar, 2004), and sometimes repeat the same sequence of
plant visits on each foraging trip over several days (Man-
ning, 1956; Williams and Thomson, 1998). Bees may use
their past experience to preferentially exploit richer patches
to increase foraging gain (Cartar 2004). If plants with large
oral displays offer a high rate of reward due to a reduced
travelling time, returning to plants with large displays may
increase the pay-off for pollinators. In Cirsium purpura-
tum, Makino et al. (2007) found that large oral displays
increased pollinator return to owers therefore partially
verifying the memory hypothesis. In addition to oral
display, other oral traits may also serve as clues for
pollinators to revisit plants. In the study system, the bee
C. tricolor often uses the labellum of owers as a landing
platform to collect oil from elaiophores (70% of landings)
(Nattero et al. 2010). In this population, the labellum width
is a trait that correlates positively with the number of visits
and the per-ower pollinator visitation rate. Similarly, the
number of visits is related in a positive way to the elaio-
phore area and the oral reward advertisement (Nattero
et al., unpublished data). These observations suggest that
Factors affecting pollinator movement and plant tness 83
1 3
oral shape may be an important character involved in
oral attraction and adaptation to specialized pollinators.
Bees make more frequent visits to plants near to the
center than the periphery of the population. Similar results
were found when individual bee paths were considered.
This pattern of visits was signicantly associated with
oral display size and owering plant density. It is likely
that a bee species the location of every plant within its
foraging area and concentrates its visits around the center
of the foraging area to plants having large oral displays
and zones with high plant density (Table 3). These results
suggest that the way a bee structures its visitation pattern
depends on ower display size, owering plant densities,
and the location of plants within its foraging area.
In this study, we sought to determine whether oral dis-
play size and owering plant density inuence pollinator
movements within and among plants (per-ower pollinator
visitation rate, bout length, proportion of visited owers)
and tness-related variables in a highly specialized polli-
nation system. Our data provide an insight into the way a bee
may structure its plant visitation pattern. In general, we
found that bees responded strongly to oral display size and
owering plant density, two variables that in addition to
plant location within foraging areas, determine the overall
pattern of bee visitation to plants. While display size posi-
tively affected per-ower visitation rate and bout length, a
decrease in plant density led to a decrease in the number of
consecutive intraplant (geitonogamous) movements, sug-
gesting that the pattern of pollinator visitation rate in this
specialized pollination system results from the balance
between the number of owers per plant and the number of
plants in the neighborhood. Furthermore, our results stress
that large oral display and high owering plant density are
variables that besides attracting pollinators, increase the
chance of a plant being chosen by a bee as the center of its
foraging area path, ensuring pollen movements with con-
specic mates that are closely located.
Acknowledgments We are grateful to M. Calumi and C. Lazarte
who assisted us during the eld work. J.N. and A.A.C. are fellow
researchers from CONICET. We would also thank two anonymous
reviewers for their useful comments. This study was funded by
Consejo Nacional de Investigaciones Cient cas y Tecnicas (PIP
5174) and Secretar a de Ciencia y Tecnica Universidad Nacional de
Cordoba (197/05).
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