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The endangered Iris atropurpurea (Iridaceae) in Israel: Honey-bees, night-sheltering male bees and female solitary bees as pollinators

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Background and AimsThe coastal plain of Israel hosts the last few remaining populations of the endemic Iris atropurpurea (Iridaceae), a Red List species of high conservation priority. The flowers offer no nectar reward. Here the role of night-sheltering male solitary bees, honey-bees and female solitary bees as pollinators of I. atropurpurea is documented.Methods Breeding system, floral longevity, stigma receptivity, visitation rates, pollen loads, pollen deposition and removal and fruit- and seed-set were investigated.Key ResultsThe main wild pollinators of this plant are male eucerine bees, and to a lesser extent, but with the potential to transfer pollen, female solitary bees. Honey-bees were found to be frequent diurnal visitors; they removed large quantities of pollen and were as effective as male sheltering bees at pollinating this species. The low density of pollen carried by male solitary bees was attributed to grooming activities, pollen displacement when bees aggregated together in flowers and pollen depletion by honey-bees. In the population free of honey-bee hives, male bees carried significantly more pollen grains on their bodies. Results from pollen analysis and pollen deposited on stigmas suggest that inadequate pollination may be an important factor limiting fruit-set. In the presence of honey-bees, eucerine bees were low removal-low deposition pollinators, whereas honey-bees were high removal-low deposition pollinators, because they removed large amounts into corbiculae and deposited relatively little onto receptive stigmas.Conclusions Even though overall, both bee taxa were equally effective pollinators, we suggest that honey-bees have the potential to reduce the amount of pollen available for plant reproduction, and to reduce the amount of resources available to solitary bee communities. The results of this study have potential implications for the conservation of this highly endangered plant species if hives are permitted inside reserves, where the bulk of Oncocyclus iris species are protected.
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The endangered Iris atropurpurea (Iridaceae) in Israel: honey-bees,
night-sheltering male bees and female solitary bees as pollinators
Stella Watts1,*, Yuval Sapir2, Bosmat Segal1and Amots Dafni1
1
Laboratory of Pollination Ecology, Institute of Evolution, University of Haifa, Haifa 31905, Israel and
2
The Botanical Garden,
Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
* For correspondence. Present address: Landscape and Biodiversity Research Group, Department of Geographical and
Environmental Sciences, University of Northampton, Avenue Campus, St George’s Avenue, Northampton NN2 6JD, UK.
E-mail hummingbird_pe@yahoo.com
Received: 14 September 2012 Returned for revision: 15 October 2012 Accepted: 20 November 2012
Background and Aims The coastal plain of Israel hosts the last few remaining populations of the endemic Iris
atropurpurea (Iridaceae), a Red List species of high conservation priority. The flowers offer no nectar reward.
Here the role of night-sheltering male solitary bees, honey-bees and female solitary bees as pollinators of
I. atropurpurea is documented.
Methods Breeding system, floral longevity, stigma receptivity, visitation rates, pollen loads, pollen deposition
and removal and fruit- and seed-set were investigated.
Key Results The main wild pollinators of this plant are male eucerine bees, and to a lesser extent, but with the
potential to transfer pollen, female solitary bees. Honey-bees were found to be frequent diurnal visitors; they
removed large quantities of pollen and were as effective as male sheltering bees at pollinating this species.
The low density of pollen carried by male solitary bees was attributed to grooming activities, pollen displacement
when bees aggregated together in flowers and pollen depletion by honey-bees. In the population free of honey-bee
hives, male bees carried significantly more pollen grains on their bodies. Results from pollen analysis and pollen
deposited on stigmas suggest that inadequate pollination may be an important factor limiting fruit-set. In the pres-
ence of honey-bees, eucerine bees were low removal– low deposition pollinators, whereas honey-bees were high
removal– low deposition pollinators, because they removed large amounts into corbiculae and deposited relative-
ly little onto receptive stigmas.
Conclusions Even though overall, both bee taxa were equally effective pollinators, we suggest that honey-bees
have the potential to reduce the amount of pollen available for plant reproduction, and to reduce the amount of
resources available to solitary bee communities. The results of this study have potential implications for the con-
servation of this highly endangered plant species if hives are permitted inside reserves, where the bulk of
Oncocyclus iris species are protected.
Key words: Endangered, Iris atropurpurea, pollination, pollinator effectiveness, Apis mellifera, night-sheltering,
eucerine bees, solitary bees, pollen removal, pollen deposition, stigma receptivity, pollen viability.
INTRODUCTION
The conservation of biodiversity has become one of the major
goals of present environmental policies, given the current pace
of global species loss (Loreau et al., 2001). Recent assess-
ments estimate that one in five of the world’s plant species
is facing extinction (Kew/Natural History Museum/IUCN,
2010). Plant –pollinator interactions play a significant role in
maintaining the functional integrity of most terrestrial ecosys-
tems (Ollerton et al., 2011b). There is mounting evidence of
large scale global declines of wild-bee populations and parallel
diversity declines of bees and insectpollinated plants across
Europe (Biesmeijer et al., 2006). Landscape modification,
habitat fragmentation and the associated decline in suitable
forage plants have been pinpointed as major global threats to
bee diversity and pollination services (Biesmeijer et al.,
2006,Tylianakis et al., 2007).
Assessing the effectiveness of different flower visitors for
pollination and subsequent seed-set is central to addressing
questions about the ecology and evolution of plant reproduc-
tion. Pollinator effectiveness is a function of multiple interact-
ing traits of both flower and flower visitors, influenced by
animal behaviour, morphology and relative sizes. Quantity
(visitation rates) and quality components (pollen removal
and deposition) of pollination service have been widely used
in the literature to rank individual pollinators and to determine
overall pollinator importance (sensu Waser and Price, 1983;
Herrera, 1987,1988;Olsen, 1997;Mayfield et al., 2001;
Ivey et al., 2003;Sahli and Conner, 2007;Ne’eman et al.,
2009;Watts et al., 2011).
Honey-bees (Apis mellifera; Apidae) vary widely in their ef-
fectiveness as pollinators of native plants (Paton, 1995;Butz
Huryn, 1997;Frietas and Paxton, 1998;Celebreeze and
Paton, 2004) and are often inferior pollinators compared
with other native flower visitors (Schaffer et al., 1983;
Taylor and Whelan, 1988;Westerkamp, 1991;Paton, 1993;
Vaughton, 1996;Gross and Mackay, 1998;Hansen et al.,
2002), and in some cases are responsible for a reduction in
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seed-set (Paton, 1993;Vaughton, 1996;Whelan et al., 2009).
However, in some systems they have comparable pollination
effectiveness with other bees (Cresswell et al., 1995;
Fishbein and Venable, 1996;Thomson and Goodell, 2001;
Ivey et al., 2003;Watts et al., 2011). In natural plant commu-
nities, honey-bees are less important as pollinators than they
are in agricultural systems, because in most regions honey-bees
are not a native species (Ollerton et al.,2011a). Although
honey-bees are native to the Mediterranean region of Israel, bee-
keepers maintain very high bee densities (90 000 beehives scat-
tered in 6300 locations, with an average area of 1.5km
2
for any
beehive location) (Shavit et al.,2009). Consequently, their es-
tablishment and spread, particularly into nature reserves, has
the potential to reduce the pool of resources available to other
native bee communities, and negatively impact on plant
fitness, including rare and endangered species (Butz Huryn,
1997;Gross and Mackay, 1998;Hansen et al., 2002;Goulson,
2003;Celebreeze and Paton, 2004;Dupont et al.,2004).
Primary floral resources utilized by bees include pollen,
nectar, oil, resin and gums, although flowers functioning as a
shelter or sleeping place could also be considered a possible
reward offered by a particular plant (Faegri and van der Pijl,
1979;Simpson and Neff, 1983). Flowers have been utilized as
night shelters by a variety of solitary male bee species (Dafni
et al., 1981;Gaglianone, 2000;Sapir et al., 2005;Monty
et al., 2006). In many species of aculeate Hymenoptera, males
form sleeping aggregations on exposed plants in the late after-
noon or early evening and disperse early the following
morning (Alcock, 1988, and references therein).
Iris atropurpurea is a Red List species endemic to the
coastal plain of Israel (Shmida and Pollak, 2007). In 2000,
the total area containing I. atropurpurea populations within
nature reserves was estimated to be 1.44 km
2
(Cohen and
Avishai, 2000). Some populations are threatened by extinction,
and about one-third have already disappeared (Cohen and
Avishai, 2000;Sapir et al., 2003). Previous studies have
reported that the main visitors to Oncocyclus irises are male
eucerine (Apidae; tribe Eucerini) (Ivri and Eisikowitch,
1988;Sapir and Shmida, 2002;Sapir et al., 2005;Monty
et al., 2006), but until now there has been no attempt to quan-
tify the pollination services of these bees directly.
Studying the reproductive biology of Iris atropurpurea is
important given its rarity and conservation status. In this
paper, we present data on observations and experiments in
which the pollination effectiveness of honey-bees was compared
with that of male solitary bees on both male and female fitness
components. We augmented data from field surveys with the ana-
lysis of pollen loads from female solitary bees. Specifically, three
questions were addressed. (1) What are the main flower visitors
of I. atropurpurea? (2) Which bee species are the most effective
pollinators in terms of visitation rate, pollen deposition and
pollen removal? (3) Are honey-bees as effective as solitary
bees as pollinators of I. atropurpurea?
MATERIALS AND METHODS
Study species
Iris atropurpurea Baker [Iris section Oncocyclus (Siemssen),
Iridaceae] is a rhizomatous clonal geophyte characterized by
a large solitary flower on the stem (Avishai and Zohary,
1980;Sapir and Shmida, 2002). The floral morphology of all
Oncocyclus irises is similar; the inner vertical tepals (stan-
dards) and outer horizontal tepals (falls) form three function-
ally separate bilabiate units (meranthium), each resembling a
single gullet flower or tunnel (Goldblatt and Bernhardt,
1999). The roof of each tunnel is formed by an expanded petal-
oid style, and the base is shaped by the outer falls which func-
tions as a landing platform for insect visitors. Each flattened
anther is pressed against the upper surface of the tunnel. The
stigma lies above the apex of the anther at the entrance of
each tunnel (Fig. 1A). Iris atropurpurea flowers have no nec-
taries and thus offer no nectar reward (Avishai, 1977). Thus,
I. atropurpurea flowers are phenotypically specialized (sensu
Ollerton et al., 2007). Flowers colour varies greatly among,
and even within, populations from deep purple, to brown or
nearly black. The fruits are a capsule (mean length ¼68.9+
18.1 mm, mean width ¼21.8+4.6 mm, n¼51) which is
divided into three parts and collectively contain many seeds
(mean ¼21.5+18.2, range 193, n¼225).
Observations and experiments were conducted at Yaqum,
Israel (coastal plain; dry Mediterranean climate, 32.258N,
34.85 8E, 20 m a.s.l.) between February and March 2009
2011. Additional observations were made at Netanya (32.20 8N,
34.51 8E, 35 m a.s.l.) during 2011. Although by law, all Iris
species in Israel are protected (Cohen and Avishai, 2000),
both populations studied are not located in official nature
reserves. These populations are isolated fragments threatened
by urban development and growth of agricultural areas. The
Iris atropurpurea population at Yaqum (area 156 650 m
2
)is
surrounded by avocado groves, Eucalyptus trees, other fruit
groves and agriculture fields. At Yaqum, honey-bee hives are
abundant throughout the site and are used for avocado pollin-
ation. The Netanya population is the largest in Israel
(370 579 m
2
) and is threatened by new housing developments.
Although no commercial bee hives are located in Netanya,
honey-bees were frequently observed.
Iris atropurpurea plants are clonal with growth via under-
ground rhizomes which creates dense patches of genotypically
identical plants (ramets). Separation of clones is clear, based
on the gap between patches. In this study, the assumption
was that ramets within a patch are closely related plants,
hence are self-incompatible if crossed (Sapir et al., 2005).
Clones were randomly selected in different patches across
entire populations. To determine visitation rates, individual
flowers were monitored for diurnal visitors during 10-min
observations periods spaced evenly throughout the day
(0800 1600 h), with a total of 193 periods. Flowers were
observed from distances of 2– 4 m. For each visit we recorded
the species of flower visitor, behavioural aspects such as
stigma contact, pollen collection, and number of flowers
visited per foraging bout and the number of tunnels entered.
Those insects which could not be identified in the field were
collected for identification in the laboratory.
Pollen viability and stigma receptivity
Pollen viability and stigma receptivity were tested at Yaqum
in 2011 using MTT (thiazolyl blue tetrazolium bromide;
M-2128, Sigma-Aldrich Inc.; Rodriguez-Rian
˜o and Dafni,
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2000). Fresh pollen grains from five flowers were collected in
the field immediately after anthesis and brought back to the la-
boratory. Flowers of five developmental stages were tested for
both pollen viability and stigma receptivity: (1) recently open
buds (0 21 d); (2) newly open flowers (12 d); (3) mature
flowers (3 d); (4) old flowers with open tunnel but wilting stan-
dards (4 d); (5) wilting standards and falls, tunnel almost
closed (5 d). Pollen grains were removed from anthers,
mounted on a microscope slide and stained with MTT. One
hundred pollen grains were counted and the proportion of
stained grains was calculated (n¼25 each with five replica-
tions per pollen sample). We used MTT to test the receptivity
of all three stigmas from five plants for each flower develop-
mental stage (100 papillae per sample were counted; n¼25
each with five replications per sample).
Breeding system
Although Oncocyclus irises are reported to be completely
self-incompatible (Avishai and Zohary, 1980;Sapir et al.,
2005), self-pollination has been found in populations of Iris
atropurpurea at Shafdan and in populations of I. haynei in
Gilboa (Y. Sapir, unpubl. res.). Therefore, we examined self-
compatibility and autogamous pollination in populations of
I. atropurpurea by bagging late-stage buds with fine mesh
bags. Flowers were treated as follows: (a) open (unbagged)
control no treatment (n¼38); (b) emasculated treatment –
flowers were carefully emasculated to remove anthers and left
unbagged throughout the duration of the observation period
(n¼26); (c) autogamy bagged unmanipulated flowers were
left covered with exclusion bags until the end of the observation
period (n¼26); (d) self-compatibility bagged flowers were
carefully emasculated and hand-pollinated with pollen from
other flowers on the same individual (n¼30); (e) xenogamy –
bagged flowers were carefully emasculated and hand-pollinated
with pollen from flowers from donor plants located at a distance
of at least 10 m (n¼22). After the autogamy and xenogamy
experiments flowers were re-covered. In addition, fruits were col-
lected to represent open-pollination treatments. Natural fruit-set
was calculated as the percentage of flowers that produced fruits
per clone, and seed-set was recorded as the number of seeds
per capsule.
The effect of pollen load on stigma on the number of seeds
per capsule was tested in Netanya in 2009. Twenty flowers, each
from a different clone, were bagged in late-bud stage and left
overnight. The following morning, bags were removed and dif-
ferent numbers of pollen grains were deposited on stigmas using
anthers from flowers .20 m away. The number of pollen grains
deposited was assigned to any of three groups: few (1– 10),
intermediate number (10– 100) and many (.100). Pollen
was deposited by brushing the anther against the receptive
side of the stigma (one stigma per flower). Immediately after de-
position, actual number of pollen grains deposited was counted
using a ×20 magnifying glass. Flowers were re-covered and left
until they completely wilted (approx. 6 d later). Five weeks
later, fruits were collected and all seeds were counted.
AB
CD
Stigma
Anther
FIG. 1 . (A) Flower structure of Iris atropurpurea: the inner vertical standards and outer horizontal falls form three functionally separate bilabiate units (mer-
anthium), each resembling a single gullet flower or tunnel. (B) Male of Synhlonia spectabilis leaving iris flower after sunrise, showing the thorax lacking pollen.
(C) Diurnal flower visitor Apis mellifera showing corbiculae loaded with iris pollen. (D) Single species aggregates of male S. spectabilis with .30 individuals
sheltering in and on top of the three tunnels. Images: (A) Elisa Mancuso, (B) Stella Watts, (C, D) David Firmage.
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Solitary-bee surveys
During three consecutive seasons (20092011 in Yaqum
and 2011 in Netanya) the flowers of Iris atropurpurea were
surveyed for sheltering male bees. We focused on night-
sheltering visits because prior observations showed that bees
rarely sheltered in flowers during the day time. Flowers were
monitored throughout entire flowering seasons from late
January to late March. The tunnels of each flower were
sampled either 1 h before sunset or 1 h before sunrise. Each
observer examined all open flowers in a designated area of
the population and each session lasted up to 60 min. In total,
72 h of observations were carried out over 73 mornings/
nights. When bees were found, flowers were tagged and we
recorded the number of individuals per species per tunnel
and the total number of flowers surveyed. Each year a
sample of bees was collected from flowers and deposited indi-
vidually into labelled vials for later identification and pollen
analyses. Voucher specimens and pollen slides are deposited
in the collection of the Laboratory of Pollination Ecology,
Institute of Evolution, University of Haifa, Israel.
Pollen deposition by solitary bees
Pollen deposition by solitary bees was examined in Netanya
and Yaqum during the peak of bee aggregation between 17 and
22 March 2011. A total of 27 stigmas (one stigma per flower)
at Netanya and 25 stigmas at Yaqum were collected from
flowers hosting male eucerine bees. Levels of natural pollin-
ation were also examined by randomly selecting 30 flowers
and collecting stigmas (one stigma per flower) at Yaqum,
and 26 flowers at Netanya. During the sampling period, no
visits by honey-bees were observed to iris flowers at
Netanya, whilst at Yaqum honey-bees had switched to
avocado flowers, and so visits to iris flowers were rare.
Fruit-set by solitary bees
We initially attempted to estimate fruit-set following single
visits to virgin flowers by male solitary bees. However, their
unpredictable behaviour when searching for a suitable night
shelter and their rapid flight, made it impossible to follow
the bees. In 2010, we bagged 60 late-stage buds in four differ-
ent areas of the Yaqum population. All the other open flowers
surrounding the target flowers (varying flower ages) were
bagged to prevent visitation, so that only the experimental
flowers remained open. Despite our efforts, no visits by male
bees were recorded. Therefore, the effectiveness of solitary
bees at facilitating fruit and seed production could only be
evaluated by tagging and monitoring flowers which hosted
solitary male bees.
Prior diurnal and early evening observations showed that
honey-bees were frequent visitors to Iris atropurpurea
throughout and across all seasons in Yaqum. Honey-bee visit-
ation was also noted in the Netanya population in 2009 and
2010, but less frequently in 2011 when bees preferred
bushes of Retama raetam (Fabaceae). Thus, due to the
strong overlap between honey-bee and solitary-bee visitation,
it was not possible to identify which taxa facilitated fruit-set.
Consequently, results are referred to as solitary bees plus
(+)Apis. It should also be noted that female solitary bees
were occasional diurnal visitors.
Pollen deposition by honey-bees
To evaluate the role of honey-bees as pollinators, pollen de-
position was investigated using single visits to 37 virgin
flowers. The flowers were treated as follows: (a) control
flowers left bagged throughout the duration of the experiment
(n¼39 stigmas), and (b) virgin flowers exposed to pollination
(n¼56 stigmas). Every flower was bagged in late-bud stage
with a fine-mesh bag and left for 24 h prior to collection.
Bags in the pollination treatment were removed the following
day at 0800 h to allow single visits by honey-bees, and flowers
were watched until all target flowers were visited. Immediately
after single visits, flowers were re-covered. On completion of
the experiment, control and visited stigmas were collected
using fine forceps and stored in separate Eppendorf tubes.
Levels of natural pollen deposition were also examined by ran-
domly selecting flowers over three seasons and collecting
stigmas (one stigma per flower).
Pollen removal by honey-bees
Pollen removal by honey-bees was investigated using mul-
tiple visits to 30 virgin flowers (one anther per flower; treat-
ment anthers, n¼45; control anthers, n¼45) on 2 March
2011. Flowers were bagged in late-bud stage and left over-
night. Treatment bags were removed from flowers the follow-
ing morning at 1000 h to allow visitation by honey-bees.
Target flowers were watched until 1230 h and control bags
were left on for the duration of the experiment. Total observa-
tion time was 100 min. When observations were concluded,
visited and control anthers were removed with fine forceps
and stored in separate Eppendorf tubes. To calculate the
number of pollen grains per anther, each anther was placed
into fresh vials containing a mixture of aniline blue and dis-
tilled water, crushed and placed in a sonic bath for 10 min.
To count pollen grains, 1 mL of solution was pipetted into a
haemocytometer and placed on a slide. A total of ten slides
per anther were counted under a microscope. The number of
pollen grains per flower was calculated by multiplying by
the number of anthers per flower (see Dafni, 1992).
Fruit- and seed-set by honey-bees
Fruit-set and seed production by honey-bees was evaluated
using 133 late-stage buds. The flowers were treated as follows:
(a) open pollinated (unbagged control no treatment (n¼
28); (b) bagged control flowers left covered with exclusion
bags until the end of the observation period (n¼49); (c)treat-
ment flowers – experimental virgin flowers (n¼56). The ex-
periment was conducted 47 March 2009. Flowers were
randomly selected and tagged over a 4-d period and assigned
to treatments and bagged controls, and left overnight. Each
morning the treatment bags were removed at 0800 h to only
allow visitation by honey-bees and at 1500 h flowers were
re-covered. The time period was based on prior honey-bee vis-
itation field data, allowing for flowers to be re-covered before
male bees started searching for night shelters. The flowers
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were subsequently monitored and, when the standards and falls
had completely wilted the bags were removed. Fruits from ex-
perimental flowers were collected and the number of seeds per
fruit counted.
Pollen loads
In 2008, 2009, 2010 and 2011, a random sample of 227
male bees found sheltering in flowers was captured and
killed using ethanol. We counted all the pollen grains depos-
ited over the entire body and pooled data from the three
body areas (thorax, occuli and frons) most likely to contact
the outer face of the stigma. Iris pollen grains are easy to rec-
ognize due to their relatively large size (100 mm), and so all
pollen grains were counted using a binocular microscope.
We also analysed pollen from the scopas of 75 female bees
(18 species) caught on other co-flowering plant species over
three seasons. To obtain pollen samples, small cubes of
fuchsin-stained gelatin were rubbed over scopas and melted
on glass slides. All iris pollen grains were counted. Given
that bees may pick up iris pollen previously deposited by
other flower visitors, we considered species with 5 pollen
grains per slide as verification of iris visitation.
Data analysis
Statistical analyses were performed using SPSS version 17.0
for Windows (2008, SPSS Inc., Chicago, IL, USA). Data were
assessed for expectations of normality and homogeneity of
variances with particular statistical tests. Data used in non-
linear regressions were square-root transformed to improve
normality. Pollen load and pollen deposition data were not nor-
mally distributed; therefore Mann– Whitney U-tests were used
with a Bonferroni correction (
a
¼0.05 divided by the number
of comparisons), yielding a critical value of 0.0034 (15 com-
parisons) and 0.0025 (20 comparisons) against which all
P-values were tested. All means in the text are presented +
s.d., and medians are indicated as required.
RESULTS
Pollen viability and stigma receptivity
Maximum pollen viability for Iris atropurpurea was 97.8+
2.3 % and 97.7+2.1 % (mean +s.d.) for recently open
buds and young flowers (1– 2 d), respectively (Fig. 2A).
There was significant variation in percentage of pollen viabil-
ity among pollen age groups (ANOVA; F¼33.43, d.f. ¼5,
P,0.0001). Pollen viability decreased gradually with flower
age. After day 5, viability was still high for old flowers with
wilting standards, and falls and tunnel almost closed.
Maximal stigmatic receptivity occurred in buds and young
flowers (1– 2 d), with an average of 97.7+2.3 % and
98.4+1.5 % papillae stained, respectively (Fig. 2B).
Stigmatic papillae remained firm for the first 3 d. A rapid
change occurred on day 3 in stigma appearance, and its papil-
lae appeared to wilt. After days 4 and 5, stigma receptivity
decreased rapidly, and on day 5 less than half of papillae
remained stained (Fig. 2B), indicating that even in old
flowers with wilting standards and falls, and when tunnels
were almost closed (4– 5 d), the stigmas remain receptive.
Thus, the end of female function is after flowers have wilted,
similar to the male function.
Experimental pollen load in 2009 in Netanya was between
16 to approx. 800 grains per stigma. The artificial pollination
resulted in fruit production in 12 out of 20 flowers pollinated.
Probability of producing a fruit was not associated with the
amount of pollen deposited (General Linear Model with bino-
mial distribution of the errors: F
1,21
¼1.16, P¼0.294). Linear
regression with Poisson distribution of the errors revealed a sig-
nificant linear slope of 0.085 (t¼3.69, P¼0.001), i.e. for a
production of one seed, 11.8 pollen grains are needed.
Breeding system
Autogamy treatments produced no fruits indicating self-
incompatibility (Table 1). Within-clone crosses (geitonogamy
treatments) in the Netanya population resulted in 10 % fruit-
100
80
60
40
20
0
Pollen viability (mean % of
stained pollen grains)
100
80
60
40
20
0
123
Sti
g
ma a
g
e (d)
Stigma receptivity (mean % of
stained papillae)
45
123
Days after anthesis
45
FIG. 2 . (A) Pollen viability (%, as tested with MTT staining) after anthesis
from flowers of five developmental stages (given as days after anthesis); n¼
25, with five replications per pollen sample, and 100 pollen grains per
sample. (B) Stigma receptivity (%, as tested with MTT staining) in relation
to stigma age; 100 papillae per sample were counted.
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set, although the mean number of seeds sired per capsule and
seed dry weight was low. Natural fruit- and seed-set measured
in Netanya at the end of the season yielded a lower value than
fruit-set following the experimental treatments (22.0%; n¼
2233 flowers in 143 clones). Supplementary hand cross-
pollination treatments revealed higher fruit-set and seeds per
fruit compared with control flowers (Table 1). Natural repro-
ductive parameters in Netanya were all higher than in
Yaqum: number of flowers per clone (General Linear Model
with Poisson distribution, P¼0.005), fruit-set (Mann
Whitney U:P,0.001), seed-set (General Linear Model
with Poisson distribution: P,0.0001) and seed weight
(Mann Whitney U:P,0.0001) (Fig. 3).
Diurnal observations of flower visitors
During the spring of 2009, 2010 and 2011, observations at
Yaqum showed that Apis mellifera was the most frequent
diurnal visitor to Iris atropurpurea. Bees from nearby hives
(in some cases 50 m) typically landed on the fall and
crawled inside the flower tunnel, rotating their bodies upside
down to collect pollen. They also burrowed into flowers
which were not fully open to harvest pollen. Honey-bees
were rarely seen to move long distances between clones;
instead they moved short distances between flowers, (usually
1 m). After gathering pollen they exited tunnels backwards,
then hovered in mid-air for a few seconds before either
re-entering the same tunnel or moving on to the next one.
This behaviour was repeated until corbiculae were loaded
with pollen (Fig. 1C), when bees returned to hives.
Other diurnal visitors were relatively rare and included
female eucerine bees of the subgenus Eucera (Synhalonia
spectabilis; Apidae), mining bees (Anthophora plumipes;
Apidae) and mason bees (Chalicadoma sicula;
Megachilidae). In total, only 30 visits were recorded during
this study.
The daily pattern of visitation by honey-bees was bimodal
with a peak during the middle of the day, followed by a
second peak in the mid-afternoon (Fig. 4). Honey-bee visit-
ation rate differed across years (
x
2
¼12.46; d.f. 2; P,
0.005); on average, honey-bees visited more iris tunnels per
minute in 2011 than in 2010 and 2009 (Mann Whitney U:
2011 vs. 2010: Z¼23.10, P,0.005; 2011 vs. 2009:
Z¼23.06, P,0.005) (Table 2). However, there was no sig-
nificant difference between visitation rates by honey-bees
during 2009 and 2010 (Z¼29.57, P.0.05).
Night-sheltering visitation
During three consecutive seasons, the tunnels of 13 717
flowers of Iris atropurpurea were sampled for sheltering
bees (Table 3). The total number of bees found in flowers
was highest in 2009, when 31 individuals were recorded in a
single flower (Fig. 1D). The number of bees found in
flowers across years ranged from 184 to 525 individuals
(Table 3).
Non-linear regressions between the number of bees found
sheltering in individual flowers versus change over time (d
number) were highly significant in all three seasons (Yaqum
2009: r
2
¼0.30; F¼25.38; P,0.001; 2010: r
2
¼0.27;
F¼25.19; P,0.001; 2011: r
2
¼0.30; F¼51.31; P,
0.0001; Netanya 2011: r
2
¼0.40; F¼167.31; P,0.0001)
(Fig. 5). In 2011, significantly fewer bees were observed in
tunnels in the Netanya population in comparison with the
Yaqum population (MannWhitney U:Z¼25.08, P,
0.0001; Z¼24.48, P,0.0001). In 2009, the peak of aggre-
gation of male bees occurred between 11 and 21 March at the
end of the flowering season, when there were significantly
fewer open flowers available in the population (t¼23.4;
d.f. ¼5; P¼0.017). In 2009, more bees were found in
flowers than in subsequent years (Table 3). Across all
seasons, the number of male bees found in iris flowers gradual-
ly increased with day number and peaked approx. 40 d after
commencement of flowering (Fig. 5). Bees were found
mostly in monospecific aggregations in all populations across
years, and some flowers hosted male bees in the same
tunnels over consecutive nights (Table 3).
Across all seasons, the assemblages of insects found in
flowers were dominated by medium-sized male bees
(mean ¼12.1mm+1.3; range ¼9.2–14.2mm, n¼35)
belonging to the Eucerini tribe. We collected a total of 227
individuals representing ten species, including three previously
undescribed species (Table 4). In 2009 and 2010, early into
each season, a few Chalicadoma sicula males were also
TABLE 1. Natural and experimental fruit and seeds set in flowers of I. atropurpurea in Yaqum 2009 and 2011 and in Netanya 2011
Year Treatment* No. of flowers No. of fruits No. of seeds per capsule (mean +s.d.) Fruit-set (%)
Yaqum
2009 1. Open pollination 1562 152 13.7+12.99
.7
2011 1. Open pollination 793 89 14.4+0.60 11.2
2009 3. Autogamy 22 0 0 0
Netanya
2011 1. Open pollination 38 13 23.5+15.834
.2
2. Emasculated 26 5 23.3+11.319
.2
3. Autogamy 26 0 0 0
4. Self-compatibility 30 3 2.0+0.010
.0
5. Xenogamy 22 20 31.1+21.090
.9
*, No treatment; 2, anthers were emasculated and left unbagged; 3, bagged unmanipulated; 4, bagged flowers emasculated and hand-pollinated from other
flowers of the same clone; 5, bagged flowers emasculated and hand-pollinated with pollen from flowers of other individuals from different clones in the same
population.
Watts et al. — Bee pollination in Iris atropurpureaPage 6 of 13
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observed in flowers at Yaqum, but these individual carried
very little pollen on the thorax (range 06 grains). The
paucity of data for these males precluded statistical
comparison. Overall, Synhalonia spectabilis was by far the
commonest flower visitor (Table 4). Other hymenoptera visi-
tors also found sheltering in tunnels of iris flowers included
wasps (Vespula germanica; Vespidae) and the bumble bee
(Bombus terrestris; Apidae) (only one record). Iris flowers
were also visited by a taxonomically wide range of insects
belonging to the orders Araneae, Coleoptera, Heteroptera
and Homoptera, of which chafer beetles (Oxythyrea spp.;
Coleoptera) were the second most abundant insects in 2009
(21.6 %). However, none of these insects carried iris pollen,
so they were not considered to be pollinators.
Pollen loads
There were significant differences among some taxa for the
number of iris pollen grains deposited on their bodies (thorax,
occuli and frons;
x
2
¼19.56, d.f. ¼5, P,0.005, entire body;
x
2
¼20.13, d.f. ¼5, P,0.0001 (Fig. 6). Overall, the density
of pollen grains carried by male solitary bees was low; 21.4%
of all bees found in flowers bore no iris pollen (range ¼02
1525), 46.4 % carried 5 pollen grains and only Synhalonia
mediterranea carried on average .200 pollen grains.
Although S. mediterranea carried more pollen grains on the
body than that of all other taxa, a Bonferroni adjustment for
the 15 comparisons rendered this finding insignificant
(Fig. 6). Sheltering bees in the Netanya population carried sig-
nificantly more iris pollen grains on their bodies than bees in
the Yaqum population (data pooled from 2008 and 2011;
TABLE 2. Visitation rates to Iris atropurpurea by Apis mellifera
Year
Mean (median) visits (+s.d.)
per minute per tunnel
Mean (median) visits (+s.d.)
per minute per flower
2009 0.41 (0.3) +0.31
a
0.28 (0.2) +0.29
a
2010 0.45 (0.3) +2.85
a
0.29 (0.2) +0.27
a
2011 0.75 (0.6) +0.58
b
0.49 (0.5) +0.34
b
Mann– Whitney U-tests, P.0.05. Medians followed by the same letters
do not differ significantly.
***
***
**
**
35 A
B
C
D
30
25
20
Number of seeds per capsuleFruit-set (%)
15
10
5
0
0·25
0·20
0·15
0·10
0·05
0
1·4
1·2
1·0
0·8
Seed weight per capsule (g)Flowers per clone
0·6
0·4
0·2
0
18
16
14
12
10
8
6
4
2
0Netanya
Stud
y
site
Yaqum
FIG. 3. Reproductive traits in populations of Iris atropurpurea at Yaqum and
Netanya: (A) number of seeds per capsule; (B) seed weight per capsule; (C)
fruit-set; (D) number of flowers per clone. Bars are mean values (+s.e.).
Asterisks denote significant differences: ** P,0.01; *** P,0.001.
0800
0
0·2
0·4
0·6
0·8
1·0
1·2
0900 1000 1100
Time of day (h)
Honey-bee visits per tunnel (min–1)
1200 1300 1400 1500
FIG. 4. Diurnal patterns of honey-bee visitation at Yaqum. Data were pooled
across three seasons and calculated as visits per minute. Error bars are standard
error of means.
Watts et al. — Bee pollination in Iris atropurpurea Page 7 of 13
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Mann Whitney U:Z¼25.60, P,0.001). The pollen data
revealed that 25 % of female bees, representing seven species
carried 5 iris pollen grains per scopa sample. The number
of iris pollen grains per sample averaged 7.58 +16.11
(range ¼0–77). The species which carried the most iris
pollen grains were Anthophora plumipes (mean ¼12.6+
22.4, n¼12 bees), Eucera cypria (mean ¼14.3+15.3, n¼
9 bees) and E. nigrilabris (mean ¼11.7+25.9, n¼8 bees).
Pollen deposition by honey-bees
The mean number of pollen grains deposited on iris stigmas
after single visits by Apis mellifera did not differ significantly
from stigmas of open-pollinated flowers across years (ANOVA:
F¼1.56, d.f. ¼3, P¼0.20; Table 5). The average number of
pollen grains found on virgin stigmas of control flowers (bagged
flowers) was significantly different from stigmas visited by
honey-bees (t¼25.5, d.f. ¼1, P,0.001). Note that due to
TABLE 3. The number and percentage of flowers hosting night-sheltering male bees in the populations of I. atropurpurea at Yaqum
(2009, 2010 and 2011) and Netanya (2011 only)
Year
Total no. of
flowers
checked
No. of
sampling
mornings/
nights
Mean (range) no. of
flowers checked each
morning/night
Mean % of
flowers hosting
bees
Total
no. of
bees
Mean no. of
bees per
flower
Maximum no.
of bees in a
single flower
% of flowers hosting bees
in the same flower tunnels
over consecutive nights
2009 3868 19 223.1 (22– 486) 22.5+37.1 525 2.9+4.231 –
2010 3915 21 217.1 (12– 419) 6.0+9.0 217 1.0+1.0 8 17 (2–4 nights)
2011 2598 18 133.5 (9– 414) 18.7+19.4 506 2.0+1.8 12 19 (2 3 nights)
2011 3336 15 219.8 (6– 613) 4.0+4.1 184 1.5+1.5 11 16 (2– 3 nights)
00 1020304050
0
0
1
2
3
4
5
6
10
Number of bees per flower Number of bees per flower
Number of bees per flower
Number of bees per flower
20 30 40 50
Time (d) Time (d)
Time (d) Time (d)
0·5
1·0
1·5
2·0
2·5
3·0
3·5
00 1020304050
0·5
1·0
1·5
2·0
2·5
3·0
3·5
00204060
0·5
1·0
1·5
2·0
2·5
3·0
3·5
4·0
A
D
B
C
FIG. 5. Non-linear regressions showing the relationship between the number of male bees per flower sheltering in tunnels of I. atropurpurea flowers as a function
of days from the start of the observations, starting (A) 6 February, 2009 (Yaqum); (B) 21 January, 2010 (Yaqum); (C) 14 February, 2011 (Yaqum); (D) 6
February, 2011 (Netanya; square-root transformed). (A, B) Lines represent polynomial best-fit models: (A) y¼0.0001x
3
–0
.0049x
2
+0.0644 +0.8491;
r
2
¼0.302; F¼25.38; P,0.001; (B) y¼0.00003x
3
–0
.0017x
2
+0.0273x+0.07314; r
2
¼0.277; F¼25.19; P,0.001. (C, D) Lines represent exponential
and power best-fit models, respectively: (C) y¼0.0902x
0·7665
;r
2
¼0.30; F¼51.31; P,0.0001; (D) y¼0.7101e
0·0177x
;r
2
¼0.40; F¼167.31; P,0.0001.
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the morphology of I. atropurpurea, removing stigmas from
flowers can result in contamination of the stigma from pollen
on the anther but, putatively, this would not create bias
among treatments, as all stigmas were removed using the
same method.
Pollen removal by honey-bees
Significant differences were found in the median number of
pollen grains per anther between visited and bagged flowers
(Mann Whitney U:Z¼222.35, P¼0.0001). The mean
(median) number of pollen grains remaining per anther after
multiple visits was 11 842 (5000) versus 43 250 (39 000) for
virgin anthers. Honey-bees removed 75 % of pollen on
virgin anthers during 100 min. The mean number of pollen
grains per flower was calculated as 129 750 for virgin
flowers, whereas the mean number of pollen grains remaining
after multiple visits was 35 526.
Pollen deposition
The median number of pollen grains deposited on iris
stigmas varied significantly among treatment groups (
x
2
¼
39.86, d.f. ¼6, P,0.0001). Open-pollinated and sheltering-
TABLE 4. List of eucerine male bee species sampled from I. atropurpurea flowers at Yaqum and Netanya 2008, Yaqum 2009 and
2010, at Yaqum and Netanya in 2011
Eucerini species
2008 2009 2010 2011
2008–2011 (%)n%n%n%n%
Eucera bidentata 131191824 6
Eucera sp.1* ––791212 4
Eucera graeca ––––12–– 1
Eucera sp. 2
12 23 6 8 – 3 6 9
Eucera sp.3* ––791224 4
Eucera nigrilabris 2434–––– 2
Eucera parnassia ––––12–– 1
Synhalonia mediterranea 1 3 15 20 1 2 7
Synhalonia rufa ––11–––– 1
Synhalonia spectabilis 37 67 36 48 35 72 41 84 65
Total 53 100 76 100 49 100 49 100 227
*S. Risch (Leverkusen, Germany, unpubl. res);
B. Tka
˚lcu (Prague, Czech Republic, unpubl. res).
Mean number of pollen grains
S. mediterranea S. spectabilis E. bidentataE. sp.3 E. sp.2 E. sp.1
Bee species
350
Entire body
Thorax/occuli/frons
33
10
11
2
7
441 11 0 0
A
a
A
a
A
aA ab A ab A ab
300
250
200
150
100
50
0
FIG. 6. Mean number of Iris atropurpurea pollen grains found on the bodies of the most abundant night-sheltering bee species. Data are pooled from populations
at Yaqum and Netanya 2008; Yaqum 2009, 2010, 2011 and Netanya 2011. Error bars are standard error of means. Numbers at the top of each bar indicate
medians. Different letters indicate significant differences (Mann– Whitney U-tests with Bonferroni correction,
a
¼0.0034). Upper- and lower-case letters are
used to distinguish between different parts of the body: A ¼entire body, a ¼thorax, occuli and frons.
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bee treatments deposited more pollen grains on iris stigmas in
Netanya than in Yaqum. However, after a Bonferroni adjust-
ment for the 20 comparisons, only bagged controls from
Yaqum (four sample medians) were significantly different
from the other six treatments (P,0.0024; Fig. 7). There
were no significant differences in the median number of
pollen grains deposited per stigma between open pollination
in Yaqum and Netanya (MannWhitney U:Z¼21.27, P¼
0.20). Similarly, open pollination and Apis mellifera, and shel-
tering bees and A. mellifera treatments were statistically indis-
tinguishable (Fig. 7).
Fruit- and seed-set by honey-bees
Fruit- and seed-set experiments following multiple visits by
Apis mellifera indicated that the mean number of seeds per capsule
between honey-bee and open-pollinated control flowers (no treat-
ment) did not differ significantly (t¼20.44, P¼0.77). Bagged
control treatments did not result in fruit-set. Naturally pollinated
flowers yielded a slightly lower percentage fruit-set than those pol-
linated by honey-bees in 2009 (Table 6).
Fruit-set by sheltering bees
Fruit-set from flowers hosting sheltering bees varied
between years and between sites. The mean number of fruits
from each treatment varied significantly among groups
(ANOVA: F¼7.48, d.f. ¼2, P,0.0001). Post-hoc tests of
multiple comparisons showed that two sample means were
highly significantly different from one another (Table 6).
Low fruit-set was observed at Yaqum in all three seasons,
despite recording the highest proportion of flowers hosting
bees in 2009 and in 2011 (Table 3). The mean number of
seeds per capsule was significantly higher in the Netanya
population than at Yaqum, in 2009 and 2011, whereas
sheltering-bee treatments were statistically indistinguishable.
DISCUSSION
The main wild pollinators of Iris atropurpurea are medium-
sized male eucerine bees. Although a wide diversity of other
insect taxa regularly sheltered in the flower tunnels, these
TABLE 5. Pollen deposition by Apis mellifera after a single visit
in 2009 season, and pollen deposition on stigmas of naturally
pollinated flowers in Iris atropurpurea in Yaqum population
during three seasons
Treatment
No. of
flowers
Mean no. of pollen grains deposited
on stigmas (+s.d.)
Open pollination
2009
49 23.1+25.0
a
Open pollination
2010
21 27.3+19.0
a
Open pollination
2011
30 36.8+42.8
a
Apis single visit 56 27.3+21.3
a
Apis single visit
control
39 9.6+13.1
b
Means with the same letter do not differ significantly ( post-hoc Tamhane
test; P.0.05)
Open-
pollinated,
Yaqum
Number of pollen grains deposited on stigmas
0
50
100
150
200
Open-
pollinated,
Netanya
Sheltering
bees,
Netanya
Apis
mellifera,
Yaqum
Bagged
control,
Yaqum
Bagged
control,
Netanya
Sheltering
bees,
Yaqum
a
(30)
a
(26)
a
(28)
a
(56)
b
(23)
ab
(39)
a
(26)
FIG. 7. Number of pollen grains deposited on iris stigmas following single visits by Apis mellifera at Yaqum, and number of pollen grains deposited on stigmas
following multiple visits by sheltering bees in Netanya and Yaqum when bees were aggregated. Box plots show the median (horizontal line) and ranges from the
25th and 75th percentiles, the black square is the mean, and the tips of the whiskers indicate the fifth and 95th percentiles. The circles and stars represent outliers.
Sample sizes are given in parenthesis. Different letters above of the error bars indicate significant differences (Mann 2Whitney U-tests with Bonferroni correc-
tion,
a
¼0.0025).
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visitors were not considered as pollinators because they did not
carry pollen. Honey-bees were found to be as effective as night-
sheltering bees at pollinating this species. Indirect evidence
of visitation to iris flowers from pollen loads revealed that
one-quarter of female solitary bees carried iris pollen, and
thus were considered as potential pollinators. Nevertheless, we
acknowledge that direct measures of effectiveness components
and more field observations and experiments are needed to
clarify the role of female solitary bees in the pollination of
I. atropurpurea.
Supplementary cross-pollination substantially increased
fruit-set (Table 1), indicating that Iris atropurpurea is pollen
limited, in agreement with other Oncocyclus iris studies
(Sapir et al., 2005;Segal et al., 2006;Shimrat, 2008),
However, no mate-limitation (sensu Campbell and Husband,
2007) exists among crosses within populations, crosses
among near populations, and crosses among far population
(Shimrat, 2008;Sapir and Mazzucco, 2012). Reproductive
parameters were all significantly higher in the Netanya popu-
lation (Fig. 3) which is not surprising given this site was
used as a refuge for plants transferred from disturbed habitats
(Y. Malihi, Israel Nature and Parks Authority, Israel, pers.
comm.), resulting in a genetic mixture derived from various
genetic sources, while Yaqum is smaller and more local, and
putatively less genetically variable.
Night-sheltering surveys of Iris atropurpurea populations
showed that the majority of bees belonged to the Eucerini
tribe, supporting earlier works by Sapir and Shmida (2002)
and Sapir et al. (2005), although this is the first detailed
study in which bee pollinators have been identified to
species level. We observed not only temporal variation in
bee assemblages, but variation in their abundance between
years. Despite such variation, however, Synhalonia spectabilis
was consistently the most frequent visitor across all seasons
(Table 4).
Honey-bees were frequent diurnal visitors across all seasons
(Table 2and Fig. 4); they were the first to visit recently opened
flowers and they removed large amounts of pollen, in agree-
ment with other studies (e.g. Paton, 1993;Vaughton, 1996;
Whelan et al., 2009). During multiple visits, honey-bees
removed approx. 90 000 times more pollen from anthers than
they deposited on stigmas. The majority of pollen was stored
in the corbiculae making it inaccessible for transfer onto
stigmas. Furthermore, honey-bees removed pollen that could
otherwise be transferred to stigmas by more effective pollina-
tors. By late afternoon when male eucerine bees started search-
ing for flowers to shelter in, we observed many anthers
stripped of pollen, possibly influencing amounts subsequently
transferred onto the bodies of sheltering bees the following
evening. Indeed, repeated pollen depletion by honey-bees
may explain why male bees in the Yaqum population carried
significantly fewer pollen grains than bees in the Netanya
population.
Pollination by male eucerine bees was facilitated at dusk by
the movement in and out of flowers, when bees visited a se-
quence of flowers in search of a suitable shelter, as observed
in a previous study (Sapir et al., 2005). Notably, we found
that the average number of pollen grains carried on the
bodies of most sheltering bees was low (Fig. 6). Low densities
of iris pollen found on the bodies of sheltering bees may also
be attributed to the extent to which pollen is groomed when
leaving flower tunnels (Rademaker et al., 1997; S. Watts,
unpubl. obs.), pollen displacement when bees aggregated to-
gether in flowers and pollen depletion by honey-bees. In the
Netanya population (although not significant), twice the
amount of pollen was deposited on stigmas for sheltering
bees and open-pollination treatments compared with those in
the Yaqum population (Fig. 7).
Some studies have found that honey-bees alter pollination
levels by reducing the amount of pollen available to native pol-
linators (Paton, 1993), reduce seed-set by depleting pollen
(Vaughton, 1996;Celebrezee and Paton, 2004;Whelan
et al.,2009) or, where active pollen-collecting bees remove
more pollen and deposit less, they may reduce total pollen
transfer (Wilson and Thompson, 1991). Here we suggest that
honey-bees can have a potentially negative effect on the pol-
lination of Iris atropurpurea for two reasons: (1) honey-bees
reduce the amount of pollen available for plant reproduction
via the male fitness component; (2) honey-bees can potentially
reduce the amount of resources available to solitary-bee popu-
lations for brood provision.
It was found that honey-bees and sheltering bees deposited
equivalent amounts of pollen on stigmas (Fig. 7), but a pos-
sible caveat important to highlight here is pollen deposition
by sheltering bees was based on unrestricted visits when
bees were aggregated, compared with single visits by honey-
bees. Therefore, if pollen deposition increased as a flower
received successive honey-bee visits (e.g. Young and
Stanton, 1990), for this component, honey-bees should be
more effective pollinators. Given that honey-bees deposited
similar amounts of pollen as naturally pollinated flowers
(Table 5), we conclude that both taxa were equally effective
at depositing pollen on the stigmas of I. atropurpurea, and
for the female component of fitness were functionally equiva-
lent (see Zamora, 2000). Our findings are in accordance with
other studies which reported similar pollen deposition efficien-
cies between honey-bees, solitary bees or bumble bees (e.g.
Cresswell et al., 1995;Freitas and Paxton, 1998;Thomson
and Goodell, 2001;Watts et al., 2011).
Similarities were also found between honey-bees and eucer-
ine bees across successively visited flowers with respect to
female fitness components. Seed production following multiple
visits by honey-bees was not only statistically indistinguishable
from that of open-pollinated treatments, it was also comparable
to that of fruit-set from flowers tagged as hosting male bees,
and to naturally pollinated flowers (Tables 1and 6). However,
even though both taxa could be considered functionally equiva-
lent in terms of their contribution to fruit-set, we were unable to
identify a single most effective pollinator, so any fruit-set was
also likely to be by female solitary bees/and or honey-bees.
Sapir et al. (2005) proposed that fruit-set might be limited
by the number or the activity of night-sheltering male solitary
bees. However, our data suggested that inadequate pollination
may be an important factor limiting fruit-set rather than bee
abundance alone, given that eucerine bees were frequently
found in flowers, and S. spectabilis was one of the most abun-
dant bee species in the plant community across all seasons
(S. Watts, unpubl. res.). In fact, even though we found a
higher proportion of flowers hosting bees, and more bees shel-
tering in flowers in the Yaqum population, fruit- and seed-set
Watts et al. — Bee pollination in Iris atropurpurea Page 11 of 13
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were still significantly lower than in the Netanya population
(Table 3and Fig. 3).
In conclusion, in the presence of honey-bees, male eucerine
bees were low removallow deposition pollinators, whereas
honey-bees were high removallow deposition pollinators.
Even though overall, both bee taxa were equally effective pol-
linators, we suggest that honey-bees not only have the potential
to reduce the amount of pollen available for plant reproduc-
tion, they also have the potential to reduce the amount of
resources available to solitary bee communities. The results
of this study have potential implications for the conservation
of this highly endangered plant species if hives are permitted
inside reserves, where the bulk of Oncocyclus iris species
are protected (Shavit et al., 2009).
In the terminology of Fenster et al. (2004),Ollerton et al.
(2007),Va
´zquez and Aizen (2006) and Watts et al. (2011),
Iris atropurpurea has high apparent generalization but low rea-
lized generalization and can be considered to be a moderate
ecological generalist (a number of species of medium-sized
bees provide pollination services) but a functional specialist
as most pollinators belong to a single functional group
(Fenster et al., 2004, although see discussion by Ollerton
et al., 2007).
ACKNOWLEDGEMENTS
We thank Haim Ashkenazi, Naomie Bensoussan, Caroline
Cawley, David Firmage, Itay Gerlitz, Richard Lewis, Elisa
Mancuso and Yuval Shimrat for field and laboratory assist-
ance; Stefan Risch and Achik Dorchin for identification of
Eucerini and Christopher O’Toole for Anthophorini.
Comments by Jeff Ollerton, Jane Stout and two referees
greatly improved the manuscript. This research was supported
by the Israeli Science Foundation (grant no. 768/08) and from
The Henk and Dorothy Schussheim Fund for Ecological
Research in Mt Carmel.
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TABLE 6. Number of fruits set in flowers of Iris atropurpurea following multiple visits by Apis mellifera compared with naturally
pollinated flowers and amount of fruit-set for each treatment in flowers tagged as hosting night-sheltering bees
Population Year Treatment No. of flowers No. of fruits Mean no. of seeds (+s.d.) Fruit-set (%)
Yaqum 2009 Open pollination 1562 152 13.7+12.9* 9.7
Apis multiple visits 47 6 13.0+9.9* 12.8
Bagged control 22 0 0 0
Yaqum 2009 Sheltering bees +Apis 102 14 9.7+6.2
a
13.7
2010 Sheltering bees +Apis 119 4 3.3
2011 Sheltering bees +Apis 187 15 11.6+13.8
a
8.0
Netanya 2011 Sheltering bees +Apis 102 30 23.0+13.4
b
29.4
* Indicates no significant difference (t-test, P.0.05). Means followed by the same letter do not differ significantly (post-hoc Tamhane test. P.0.05).
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... These new attractants may include changes in flower color and size (Gigord et al., 2001;Vereecken and Schiestl, 2009). For example, night-sheltering reward systems without a food reward are often associated with large, dark flowers (Dafni et al., 1981;Sapir et al., 2005Sapir et al., , 2006Watts et al., 2013;Lavi and Sapir, 2015). Dark petals with their highly absorptive surfaces may result in higher temperatures inside the flower, which may benefit flower visitors (Sapir et al., 2006;Watts et al., 2013). ...
... For example, night-sheltering reward systems without a food reward are often associated with large, dark flowers (Dafni et al., 1981;Sapir et al., 2005Sapir et al., , 2006Watts et al., 2013;Lavi and Sapir, 2015). Dark petals with their highly absorptive surfaces may result in higher temperatures inside the flower, which may benefit flower visitors (Sapir et al., 2006;Watts et al., 2013). As a result, in such systems flower size and color, but not food rewards, are typically under strong selection (Vereecken et al., 2013;Lavi and Sapir, 2015;Pellegrino et al., 2017). ...
... Once these irises became nectarless, the question becomes: how did they attract insect pollinators? Potentially, this is when the night-sheltering reward system, that is well-described in the Oncocyclus group, arose (Sapir et al., 2005;Monty et al., 2006;Vereecken et al., 2013;Watts et al., 2013). Oncocyclus irises are primarily pollinated by male Eucera bees (Apidae, Eucerini) that shelter in the flowers overnight (Sapir et al., 2005(Sapir et al., , 2006Watts et al., 2013). ...
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... Although irises most often reproduce vegetatively either by bulbs or rhizomes, the understanding of their reproductive biology is necessary to maintain the appropriate population size and genetic variation (Szőllősi, 2010;Szőllősi et al., 2011;Ge et al., 2013). The pollinator limitation has a substantial impact on the reproductive success of endangered cross-pollinated Iris species (Ashman et al., 2004;Watts et al., 2013;Liu et al., 2020). Genetic diversity is an effective measure of the active protection of the rare and endangered I. sibirica. ...
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... For example, seeds of I. atrofusca plants from Judea and Samaria can be used for translocation into nature reserves and national parks of the Shefela region, and seeds of I. loessicola can be used for introduction into protected areas of the western Negev, where this species has never been recorded. In search for a suitable location, presence of Eucera bees, the main pollinators of the Oncocyclus flowers (Watts et al., 2013), should be one of the criteria for location suitability. ...
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... Males of the aequata-group, among other Eucera species, are associated with irises of the section Oncocyclus (Iridaceae), which are endemic to arid parts of the Mediterranean region and the Middle East. Because entering the tunnel-like, dark flowers of these irises while looking for overnight shelter or during periods of overcast weather, they are considered as important pollinators of Iris (Watts et al. 2013). ...
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The aequata-group of the subgenus Eucera s. str. Scopoli from the Eastern Mediterranean region, Bulgaria, and Iran is described, and the three species included are revised. The little-known E. aequata Vachal 1907 known from Turkey, Cyprus, Syria and Israel, is redescribed and a Lectotype is designated. Two species are described as new: E. dafnii sp. nov. from Iran, Israel, Syria, Turkey, Bulgaria, and Greece, and E. wattsi sp. nov. from Israel and Lebanon. An identification key is provided, and natural history information including assessment of preferred pollen host plants is presented.
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The Royal Irises (section Oncocyclus) are a Middle-Eastern group of irises, characterized by extremely large flowers with a huge range of flower colors and a unique pollination system. The Royal Irises are considered to be in the course of speciation and serve as a model for evolutionary processes of speciation and pollination ecology. However, no transcriptomic and genomic data are available for these plants. Transcriptome sequencing is a valuable resource for determining the genetic basis of ecological-meaningful traits, especially in non-model organisms. Here we describe the de novo transcriptome assembly of Iris atropurpurea , an endangered species endemic to Israel’s coastal plain. We sequenced and analyzed the transcriptomes of roots, leaves, and three stages of developing flower buds . To identify genes involved in developmental processes we generated phylogenetic gene trees for two major gene families, the MADS-box and MYB transcription factors, which play an important role in plant development. In addition, we identified 1503 short sequence repeats that can be developed for molecular markers for population genetics in irises. This first reported transcriptome for the Royal Irises, and the data generated, provide a valuable resource for this non-model plant that will facilitate gene discovery, functional genomic studies, and development of molecular markers in irises, to complete the intensive eco-evolutionary studies of this group.
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Males of the bee Idiomelissodes duplocincta roost in the evening together on the stems of desert shrubs during summer months in central Arizona. The size of the aggregation can vary greatly during the few days to several months when a shrub is being used as a sleeping site. Some males return to the same plant for up to two weeks but show little site fidelity to a particular stem. When frequently-occupied stems are experimentally cut and moved to new sites in a shrub and replaced with other stems of similar dimensions, the bees rarely utilize the replacement stems but instead shift elsewhere or seek out the previously popular stems in their new locations. This result suggests that odor cues applied by sleeping bees (or some other special properties of the favored stems) attract male bees coming to specific roost sites. Sleeping aggregations are occasionally visited by a predator of the bees, the assassin bug Apiomerus flaviventris. However, the number of assassin bugs present at any one aggregation on a given evening is small (not exceeding four in the present study), and few bees are taken. Sleeping bees do not attack or harass the assassin bug. Moreover, they usually ignore approaching assassin bugs rather than flee, even when the predator vigorously attempts to subdue a neighboring bee on a sleeping stem. Thus, male bees in sleeping aggregations apparently gain anti-predator benefits largely through the dilution effect.
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Honey bees (Apis mellifera L.), native to Eurasia and Africa, have been introduced to most of the rest of the world. Many plant species are used by introduced honey bees, which suggests a high potential for disturbance of native plant/pollinatorrelationships. Few species are used intensively, however, thus decreasing the opportunity for disturbance. Pollination studies show that honey bees are effective pollinators of some native plants and less effective pollinators of others; they also reduce floral resources in some species with little or no pollination. Data are insufficient to show whether honey bee foraging on native plants significantly alters pollen and gene flow, but unusual foraging behavior by honey bees is not evident compared to many other pollinators. Honey bees do not physically damage plants; they are also unlikely to increase hybridization of native flora. Pollination by honey bees probably contributes little to the success of most weeds. Experiments have not shown competition for nesting sites between honey bees and native fauna. The presence of honey bees, however, alters the foraging behavior and abundance of some native fauna on flowers, but no studies have shown detrimental impacts of honey bees on population abundances of any native animals or plants. Anecdotal and quantitative reports of increased honey bee abundances on flowers compared with native fauna are often confounded with habitat changes induced by man.
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Abstract Bees are generally regarded as beneficial insects for their role in pollination, and in the case of the honeybee Apis mellifera, for production of honey. As a result several bee species have been introduced to countries far beyond their home range, including A. mellifera, bumblebees (Bombus sp.), the alfalfa leafcutter bee Megachile rotundata, and various other solitary species. Possible negative consequences of these introductions include: competition with native pollinators for floral resources; competition for nest sites; co-introduction of natural enemies, particularly pathogens that may infect native organisms; pollination of exotic weeds; and disruption of pollination of native plants. For most exotic bee species little or nothing is known of these possible effects. Research to date has focused mainly on A. mellifera, and has largely been concerned with detecting competition with native flower visitors. Considerable circumstantial evidence has accrued that competition does occur, but no experiment has clearly demonstrated long-term reductions in populations of native organisms. Most researchers agree that this probably reflects the difficulty of carrying out convincing studies of competition between such mobile organisms, rather than a genuine absence of competitive effects. Effects on seed set of exotic weeds are easier to demonstrate. Exotic bees often exhibit marked preferences for visiting flowers of exotic plants. For example, in Australia and New Zealand many weeds from Europe are now visited by European honeybees and bumblebees. Introduced bees are primary pollinators of a number of serious weeds. Negative impacts of exotic bees need to be carefully assessed before further introductions are carried out.
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Previous studies of introduced honey bees foraging at Agave schottii flowers suggest that Apis mellifera preferentially exploits the most productive patches of flowers and thereby reduces the standing crop of available nectar and the utilization of these sites by native bees. Results of experiments undertaken to evaluate this hypothesis are given and discussed using Apis, Bombus and Xylocopa. -Authors
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In two locations in Israel, bees were found to be sleeping in flowers of Serapias vomeracea Briq. Of these bees. Proposis spp. and Ceratina spp. were too small to be pollinators, whereas Eucera spp., Andrena spp., Osmia spp. and Tetralonia spp., mostly males, pollinated.Pollination occurs when in the afternoon hours the bees waver from flower to flower. The bees finally come to rest on a particular flower and remain there for the duration of the night. In the morning, the bees which slept in the flowers, are warmed up as a result of solar radiation which heats the flowers to 3 ***°C above ambient temperature.Since the males of many Hymenoptera sleep in holes, the hypothesis is that the flowers mimic such holes. The shortness of the flower tube can be held responsible for the observed frequent changes from flower to flower, which is so important for pollination efficiency.
Article
1. Comparisons among animal pollinators of the spatial distributions of pollen that they produce have typically been made among morphologically disparate pairs of species. In contrast, we investigated the potential extent of pollen dispersal by honey-bees (Apis mellifera) and bumble-bees (Bombus lapidarius, B. pascuorum and B. terrestris) foraging in rows of oil-seed rape (Brassica napus cv Westar). 2. We estimated the pollen carryover attributable to individual bees by using particulate fluorescent dye as a pollen analogue. Most of the dye was deposited at the first few flowers probed and smaller proportions were deposited up to the 20th successively probed flower. We found no significant interspecific differences in dye carryover mediated by individuals of A. mellifera, B. lapidarius and B. terrestris with respect to either the amount deposited or the rate of decline in deposition across successively probed flowers. We present evidence that the dye produced a reasonably good analogue of pollen transfer. 3. Bees typically flew from one plant to another nearby in the same row and were strongly directional in their movements. Bee species differed significantly in their movement patterns, with B. terrestris having the greatest mean move length and directionality. 4. We used three kinds of model (a numerical simulation and two different sets of diffusion-advection equations) to attempt to emulate bee movements. The predictions from all models were reasonably consistent with the observed bee movements, although the numerical simulation invariably made the most accurate predictions, particularly over the first few moves. 5. Predicted bee movements were combined with least-squares models of dye deposition to estimate the spatial dispersal of pollen by each bee species. All models ranked the bees in the same order of decreasing effectiveness in dye dispersal: B. terrestris, A. mellifera, B. lapidarius, B. pascuorum, although, except for long-distance dispersal, there was only minor variation among the bee species in the predicted extents of dye dispersal (e.g. the models predicted that the median dispersal distance would be approximately two intervening plants irrespective of the species of bee). Overall, the consensus of the models' predictions is that most of the pollen from a source plant is deposited on immediate neighbours, but that long-distance pollen dispersal in this system extends over approximately 20-40 intervening plants from the originating plant, depending on the identity of the pollinator.
Article
Although pollination effectiveness is a central process underlying the evolution of plant and pollinator traits, it is difficult to measure and has rarely been reported for a diverse spectrum of visitors under natural conditions. We measured the effectiveness of all common flower visitors to Asclepias tuberosa (butterfly weed) at a site in southeastern Arizona, in terms of visitation rate, per-visit rate of pollinia removal and insertion, and pollinia load. Bombus and Apis (Hymenoptera) were the most effective pollinators, counter to predictions that A. tuberosa is butterfly-pollinated. We also documented large differences between 2 yr in the pollination effectiveness of visitors, primarily due to changes in visitation rate. Bombus were the most frequent and effective pollinators in 1992. In 1993, Apis were equivalent to Bombus. Battus (Lepidoptera) were the second most effective pollinators in 1992, but were scarce in 1993. Thus, conclusions about the identity of effective pollinators based on floral traits, casual observations of visitation, or even precise measurement of effectiveness in a single season are all potentially suspect. We compare our results to those of previous studies of Asclepias pollination.