Comparing the efficiency of pollination mechanisms in Papilionoideae

Abstract

The pump pollination mechanism is typical of basal clades within Papilionoideae and might be associated with less efficient pollen transfer systems, while the explosive tripping mechanism is considered more advanced and might represent the highest expression of the trend in pollen economy. Crotalaria pumila, C. stipularia, Desmodium incanum and D. subsericeum present secondary pollen presentation with pump and explosive pollination mechanisms, respectively. In the present study, we evaluate and compare (1) pollen removal, (2) pollen deposition and (3) pollen transfer efficiency of both mechanisms, considering single visits by Megachile spp., common pollinators of the four plant species in Salta Province, Argentina. Comparisons of visit durations are made in relation to the type of mechanism and rewards offered. We detected significant differences between both mechanisms in the proportion of pollen grains removed and deposited in a flower after a single visit of Megachile. We found that efficiency in pollen transfer was significantly higher for explosive mechanism (2.13 ± 0.42 pollen grains deposited per 100 removed) than for pump mechanism (0.33 ± 0.17 pollen grains deposited per 100 removed). This is the first study that compares efficiency between pollination mechanisms in this group of plants.

Full text

Vol.:(0123456789)
1 3
Arthropod-Plant Interactions (2017) 11:273–283
DOI 10.1007/s11829-017-9515-7
ORIGINAL PAPER
Comparing theefficiency ofpollination mechanisms
inPapilionoideae
TrinidadFigueroaFleming1· ÁngelaVirginiaEtcheverry1
Received: 1 October 2016 / Accepted: 17 March 2017 / Published online: 11 April 2017
© Springer Science+Business Media Dordrecht 2017
Introduction
Floral evolution in Leguminosae is characterized by a
clear-cut trend toward maximizing pollen and nectar econ-
omy. This trend, in turn, has resulted in a gradual selective
development of increasingly sophisticated pollinators and
the elimination of non-specialized vectors (Arroyo 1981).
Each of the three subfamilies has achieved a characteristic
grade of advancement in this subject. Leppik (1966) and
Arroyo (1981) affirmed that more efficient pollen transfer
systems appear to be a major trend in the evolution of the
subfamily Papilionoideae. In papilionaceous flowers, pol-
len deposition and removal can occur through four types
of specific pollination mechanisms: valvular, pump, explo-
sive and brush (Arroyo 1981; Yeo 1993; Westerkamp 1997;
Westerkamp and Weber 1999; López etal. 1999; Galloni
etal. 2007; Etcheverry etal. 2012). The valvular mecha-
nism is considered to be a type of primary pollen presen-
tation, while the remaining three are types of secondary
pollen presentation (Yeo 1993). The probable functions of
secondary pollen presentation appear to be (1) harmoni-
zation of presentation sites and reception of pollen in the
flower, (2) protection of pollen against robbery, (3) place-
ment of the pollen on the vector so that the latter cannot
misuse it and (4) the issue of pollen in separate doses.
Besides, these mechanisms favour cross-pollen transfer
(Arroyo 1981; Suzuki 2003; Alemán etal. 2014).
As stated by Arroyo (1981), the valvular and pump
mechanisms are typical of basal clades within Papilio-
noideae and may be associated with less efficient pol-
len transfer systems. Explosive tripping mechanism is, in
contrast, the most advanced form representing the highest
expression of pollen economy, where pollinator attraction is
potentially based on intoxicating effects. It is hypothesized
Abstract The pump pollination mechanism is typical of
basal clades within Papilionoideae and might be associated
with less efficient pollen transfer systems, while the explo-
sive tripping mechanism is considered more advanced and
might represent the highest expression of the trend in pol-
len economy. Crotalaria pumila, C. stipularia, Desmodium
incanum and D. subsericeum present secondary pollen
presentation with pump and explosive pollination mecha-
nisms, respectively. In the present study, we evaluate and
compare (1) pollen removal, (2) pollen deposition and (3)
pollen transfer efficiency of both mechanisms, considering
single visits by Megachile spp., common pollinators of the
four plant species in Salta Province, Argentina. Compari-
sons of visit durations are made in relation to the type of
mechanism and rewards offered. We detected significant
differences between both mechanisms in the proportion of
pollen grains removed and deposited in a flower after a sin-
gle visit of Megachile. We found that efficiency in pollen
transfer was significantly higher for explosive mechanism
(2.13 ± 0.42 pollen grains deposited per 100 removed) than
for pump mechanism (0.33 ± 0.17 pollen grains deposited
per 100 removed). This is the first study that compares effi-
ciency between pollination mechanisms in this group of
plants.
Keywords Pollination efficiency· Pollination
mechanisms· Papilionoideae· Megachile· Crotalaria·
Desmodium
Handling Editor: Isabel Alves dos Santosand Isabel Machado.
* Trinidad Figueroa Fleming
tricafig@yahoo.com.ar
1 Facultad de Ciencias Naturales, Universidad Nacional de
Salta, Avenida Bolivia 5150, 4400Salta, Argentina

274 T.Figueroa Fleming, Á.V.Etcheverry
1 3
that pump, explosive and brush mechanisms have evolved
from the valvular mechanism.
Flowers with pump mechanism can receive more than
one visit during anthesis. This mechanism is often associ-
ated with two whorls of five stamens each, with differential
growth and dimorphic anthers, as in flowers of Crotalaria
micans (Etcheverry 2001a). The longer anthers of the outer
stamens dehisce and deposit pollen around the style. Fila-
ments of the inner and smaller stamens develop after the
flower opens, acting as pistons that force the mass of pollen
progressively into the tip of the keel. When a bee visits a
flower, the stylar brush is pushed through the pollen mass
and moves it out of the keel (Etcheverry 2001a). The total
pollen production of a flower is divided into separate units,
acting as a dispensing mechanism that maximizes pollen
dispersal, by limiting the pollen delivery to visitors. Dis-
pensation of pollen can be flexible and plants may execute
adjustments based on the frequency of visits (Harder and
Thomson 1989). For instance, in C. micans, long intervals
between visits led to greater pollen removal than did short
intervals (Etcheverry 2001a). The same was observed for
Lupinus sericeus (Harder and Wilson 1994).
In explosive pollination, the staminal column (formed
by ten fused stamens) and stigma erupt from the keel when
the latter is depressed, and pollen is sprayed over a large
part of the visitor’s body. Arroyo (1981) hypothesized that
the explosive mechanism could only have evolved in flow-
ers where nectar—not pollen—is the primary floral reward.
However, explosive pollen release was also observed in the
genus Desmodium, which completely lacks nectar secretion
(Willmer etal. 2009, Etcheverry etal. 2012).This pollina-
tion system was interpreted as an adaptation geared toward
the efficient removal and deposition of pollen during single
flower visits of pollinators, because a large amount of pol-
len is likely to be lost from a flower tripped open by a pol-
linator (Suzuki 2003). Further studies are needed to fully
understand the evolutionary aspects to pollination.
According to Arroyo (1981), pollination mechanisms
evolve by convergence in different tribes, without implying
phylogenetic relatedness among them. Pump pollination is
present in the Crotalarieae and Dalbergieae tribes, while
explosive pollination occurs in Desmodieae and Indigofer-
eae (following the classification of Lavin etal. 2005).
Comparisons of pollination efficiency across pollination
mechanisms are required. The spatial and temporal patterns
of pollen release are still a hot spot of evolution in papil-
ionaceous plants and are related to flower morphology, pol-
lination biology and mating systems (Huang etal. 2013).
Crotalaria species growing in Salta Province, Argentina,
exhibit secondary pollen presentation and pump-type pol-
lination mechanism (Etcheverry 2001a, b; Alemán 2014).
In a global study of the genus, Le Roux and Van Wyk
(2012) observed that Crotalaria presented a modified pump
mechanism, showing an improved pumping action, through
trichomes on the apical part of the style, brushing the pol-
len out of the keel tip. Within Crotalarieae, this mechanism
is unique to Crotalaria and is present in all but one of the
700 species in the genus (Le Roux and Van Wyk 2012).
Pollen is presented secondarily when legitimate pollina-
tors, typically bees strong enough to depress the keel (Etch-
everry et al. 2003; Jacobi et al. 2005; Brito et al. 2010),
expose the stigma and push out a mass of pollen grains
(Endress 1996; Westerkamp 1997). The flowers of Cro-
talaria species have a long floral cycle (3–4 days) (Etch-
everry 2001b; Alemán 2014). Native solitary bees such as
Megachile, Epanthidium, Melissodes, Coelioxys and honey
bees are pollinators (Figueroa Fleming 2014). Only a few
species in the genus were reported to be bird-pollinated
(Du Puy and Labat 2002; Polhill 1976, 1982). Crotalaria
species are self-compatible, with a mixed mating system
and also produce fruits and seeds through autonomous self-
pollination (Etcheverry 2001b; Alemán 2014).
Desmodium species growing in Salta Province, Argen-
tina, posses secondary pollen presentation and their flow-
ers have an explosive pollination mechanism (Alemán etal.
2014). The reproductive column (stamens and stigma) is
enclosed inside the flower keel, and is exposed when the
pollinator presses against the wing and the keel petals. Con-
sequently, once the reproductive column comes into con-
tact with the insect body for the first time, the wing–keel
complex cannot return to its original position and the
explosive mechanism gets deactivated (Arroyo 1981; Yeo
1993; Westerkamp 1997; López et al. 1999; Galloni and
Cristofolini 2003; Galloni etal. 2007; Alemán etal. 2014).
However, it was observed in flowers of D. setigerum that
floral parts partially return to their original positions after
the first visit (Willmer etal. 2009; Stanley etal. 2016), and
the same situation was reported in other legume species
with this mechanism (López etal. 1999; Galloni and Cris-
tofolini 2003; Solomon Raju and Purchandra Rao 2006).
The mechanism is activated if a given threshold strength is
reached due to the action of an animal visiting the flower
(Suzuki 2003; Solomon Raju and Purchandra Rao 2006;
Córdoba and Cocucci 2011). Thus, only a portion of all of
the potential pollinators could efficiently activate the mech-
anism. It is commonly assumed that flowers possessing this
mechanism release almost all the pollen in the first and only
visit; however, at present, only two studies have evaluated
this finding in explosive flowers, the first in Cytisus sco-
parius, outside its natural geographic range (Suzuki 2003),
and the second in Desmodium incanum and D. subsericeum
(Alemán etal. 2014). The flowers have a short floral cycle
(<4h) (Alemán etal. 2014). Although Desmodium species
depend on pollinators because the mechanism cannot self-
activate, they are self-compatible, and there is evidence that
D. incanum and D. subsericeum produce fruits and seeds

275Comparing theefficiency ofpollination mechanisms inPapilionoideae
1 3
also through autonomous self-pollination (Alemán et al.
2014). Native solitary bees such as Megachile, Epanthid-
ium, Melissodes, Psaenythia, the eusocial Bombus and Apis
mellifera honey bees form the list of pollinators (Figueroa
Fleming 2014).
Knowledge of the pollen–ovule ratio (P/O) is useful for
detecting possible evolutionary trends in pollen economy
(Arroyo 1981). For instance, explosive pollen delivery
is expected to be associated with low P/O ratios (Arroyo
1981). This hypothesis was confirmed for the genus Med-
icago L., with explosive pollination and lower P/O ratios
than related genera from the tribe Trifolieae with different
pollination mechanisms (Small 1988). Rodrıíguez-Riaño
et al. (1999) analysed P/O ratios associated with several
pollination mechanisms across 168 taxa from nine tribes
and reported that the highest P/O ratio was presented by
the pump type, while explosive type was associated with
a considerably lower ratios. Another study using 32 Medi-
terranean legume species showed that those with brush
mechanism had the lowest P/O ratio; the explosive mecha-
nism yielded intermediate values, and pump and valvular
mechanisms accounted for the highest results (Galloni etal.
2007). In accordance with this finding, a study of all mech-
anism types in the Papilionoideae concluded that the pump
mechanism had higher P/O than explosive mechanism
(Etcheverry etal. 2012). To gain a better understanding of
the selective advantages and disadvantages of the different
pollination mechanisms, more comparative studies on the
efficiency of pollen transfer are needed.
Ne’eman etal. (2010) reviewed pollinator performance.
Their scheme to measuring pollinator performance based
on pollen deposition per single visit is divided into two
main assessment concepts: pollination success and plant
reproductive success. Not every floral visitor that is effec-
tive (‘good’ in terms of pollen deposition) is also efficient
(‘good’ in terms of seed production), but any visitor that
is efficient also has to be effective (Ne’eman etal. 2010).
Single visit deposition of pollen has been put forward as
the most practical measure of pollinator effectiveness in a
range of tropical and temperate plant species (King etal.
2013). Pollinator efficiency takes into account variables
involved in the plant’s female reproductive success such as
ovule production, and reflect wether a pollinator deposits
enough pollen to achieve full seed set per flower. To assess
male reproductive success, it is necessary to include pol-
len removal and pollen loss as suggested by Thomson etal.
(2000). This evaluation of pollen transfer efficiency is con-
sidered an appropriate derived index by Ne’eman et al.
(2010).
In the present study, we evaluated and compared (1) the
effectiveness in pollen deposition, (2) pollen removal and
(3) pollen transfer efficiency in two pollination mechanisms
characteristic of the subfamily Papilionoideae: the pump
type in two species of Crotalaria and the explosive type
in two Desmodium species. We considered single visits by
Megachile spp., common pollinators of the four plant spe-
cies. Comparisons of visits’ durations are made in relation
to the type of mechanism and rewards offered. We also
reported the major visitors at Crotalaria and Desmodium
flowers in the studied area.
Materials andmethods
Plant species
Crotalaria and Desmodium species (Table 1) belong to
the Leguminosae family (subfamily Papilionoideae). All
plant species occur in the same area and they grow form-
ing monospecific or heterospecific patches. Natural popu-
lations of them are distributed north of the Lerma Valley,
Salta Province, NW Argentina (24.34.53°–25.31.38°S and
65.22.30°–65.39.70°W), in a transition region between
Yungas rain forest and Chaco Serrano (Olson et al. 2001)
with a maximum altitude of 1360 m. The climate is strongly
seasonal with summer rains concentrated between Novem-
ber and May. The average annual rainfall is 662.58 mm
(Bianchi and Yañez 1992), while the average annual tem-
perature is 17 °C (Bianchi 1996).
Crotalaria pumila and C. stipularia blooms from late
summer to early winter, with yellow flowers that offer nec-
tar and pollen as reward, arranged in racemes with acro-
petal maturation. The flowers have a pump pollination
mechanism and pollen is presented secondarily by a stylar
brush (Etcheverry etal. 2012). C. pumila flowers measure
8.2 ± 0.5mm (n = 10) and C. stipularia 8.1 ± 2.1 (n = 10).
Desmodium incanum and D. subsericeum have pink-
lilac flowers, with post pollinated changes in both the
corolla colour and the position of the floral pieces (Willmer
etal. 2009; Alemán etal. 2014). They offer only pollen as
reward. Flowers have an explosive pollination mechanism
(Etcheverry et al. 2012). Floral length of D. incanum is
8.9 ± 5.5mm (n = 10) while D. subsericeum is 9.1 ± 0.7mm
(n = 10). The flowering period of D. incanum is the long-
est, and last between November and May, while D. subseri-
ceum coincides with that of the Crotalaria species.
Voucher specimens were deposited in the Herbarium of
the Museum of Natural Sciences from Salta—MCNS (No.
10408, 10410, 10416, 10420, 12067).
Floral visitors
For each plant species, the identity of floral visitors, their
relative importance (visit proportion) and duration of their
visits were determined trough focal observations dur-
ing the 2011 blooming season. The relative abundance of

276 T.Figueroa Fleming, Á.V.Etcheverry
1 3
pollinators (proportion of individuals of each insect spe-
cies in respect to the total) was estimated (Dafni 1992).
The observations were conducted in a total of 11 quadrants
(except for C. stipularia with four quadrants) of 1m2 in
monospecific patches, during two or three daily periods of
15min each, from 9:00 to 14:00h. Samples of insects visit-
ing flowers were taken for identification. All six species of
Megachile were considered as a group, because their simi-
larities in size and behaviour.
Pollen removal anddeposition
In our experimental work, we analysed the effectiveness
in deposition (D) and removal (R) of pollen after a single
visit by native solitary bees of the genus Megachile. With
the obtained values of pollen removal and deposition, the
proportion of deposited pollen from the total removed pol-
len was calculated (D/R) as an estimate of the pollen trans-
fer efficiency. We completed field observations in summer
2011, studying 3–6 days of the flowering period of each
plant species. Pollen and ovule numbers were estimated
from 10 randomly selected flower buds of each species
(1 flower×10 individuals). All 10 anthers from each sin-
gle flower were softened in a 70% ethanol solution; then
anthers were transferred to 0.5ml ethanol/detergent solu-
tion, and macerated with a glass rod. In order to homog-
enize the mixture, the macerated samples were vortexed for
60s. Immediately after vortexing, a sample was placed in a
Table 1 Principal visitors at Crotalaria flowers with pump pollination mechanism and Desmodium flowers with explosive pollination mecha-
nism, from Northwestern Argentina
Legend for behaviour P pollinator; PO occasional pollinator; NT nectar thief; PT pollen thief
Plant species Visitors
Family Tribe Bee species Cast or sex Behaviour
Crotalaria pumila Ortega Apidae Apini Apis mellifera L. Worker P/NT
Meliponini Paratrigona glabella (Camargo & Moure 1994) Worker PT
Megachilidae Anthidiini Epanthidium nigrescens (Friese 1906) P
Epanthidium erythrocephalum (Schrottky 1902) P
Megachilini Megachile (Dactylomegachile) sp. 1 ♀P
Megachile (Chrysosarus) sp. 2 ♀P
Megachile (Leptorachis) sp. 5 ♀P
Megachile (Leptorachis) sp. 6 ♀P
Coelioxys sp ♀PO
Crotalaria stipularia Desv Apidae Apini Apis mellifera L Worker P/NT
Eucerini Melissodes (Ecplectica) tintinnans (Holmberg
1884)
P/NT
Megachilidae Anthidiini Epanthidium nigrescens (Friese 1906) P
Megachilini Megachile (Leptorachis) sp. 3 ♀P
Megachile (Leptorachis) sp. 5 ♀P
Desmodium incanum DC Apidae Apini Apis mellifera L Worker P
Meliponini Paratrigona sp Worker PT
Tapinotaspidini Arhysoceble picta (Friese 1899) ♀PO
Andrenidae Protandrenini Psaenythia sp ♀P
Megachilidae Megachilini Megachile (Leptorachis) sp. 5 ♀P
Megachile (Leptorachis) sp. 6 ♀P
Desmodium subsericeum Malme Apidae Apini Apis mellifera L Worker P/PT
Bombini Bombus (Thoracobombus) atratus (Franklin 1913) Worker P
Bombus (Thoracobombus) morio (Swederus 1787) Worker P
Eucerini Melissodes (Ecplectica) tintinnans (Holmberg
1884)
♀P
Megachilidae Anthidiini Epanthidium sp. PO
Megachilini Megachile (Chrysosarus) sp. 2 ♀P
Megachile (Leptorachis) sp. 4 ♀P
Megachile (Leptorachis) sp. 6 ♀P

277Comparing theefficiency ofpollination mechanisms inPapilionoideae
1 3
haemocytometer and the pollen grains were counted. This
value was then used to estimate the total number of grains
per flower, following Dafni (1992) and Kearns and Inouye
(1993). Ovule number for each plant species was directly
determined from dissections of ovaries from the same 10
randomly selected flower buds (1 flower × 10 individuals)
under a stereoscopic microscope.
In order to evaluate pollen removal (R), 10–15 flower
buds per species were marked daily. Once flowers had fully
opened, we observed until a single visitor foraged. After a
single visit, the flower was collected, and its anthers were
removed and stored separately in Eppendorf tubes con-
taining 0.5 ml of 70% ethanol solution. The hemocytom-
eter method of dilution and counting was used to calcu-
late the remaining pollen grains per flower (Dafni 1992).
As the control for pollen removal, we used pollen num-
ber per flower obtained previously. The amount of pollen
removed was calculated as the difference between the total
number of pollen grains per flower and pollen remaining.
The proportion of pollen removed for each plant species
was calculated and arcsine-transformed for further statisti-
cal analysis. We compared proportions of removed pollen
among species and among pollination mechanism, using
Kruskal–Wallis non-parametric tests. Post hoc pairwise
comparisons were made between the means of treatment
ranges. The procedure used to judge the significance of
multiple comparisons is described in Conover (1999).
To measure pollen deposition on stigmas (D), 10–15
flower buds per species were marked daily and covered
with paper bags to exclude flower visitors. At approxi-
mately 9:00h., when flowers had fully opened, they were
uncovered. After a single visit of Megachile to an experi-
mental flower (i.e. an insect contacted floral reproductive
parts activating the pollination mechanism), the flower
was collected, the stigma was removed using fine for-
ceps, placed on a microscopic slide in a drop of warmed
glycerine jelly and covered with cover slips. The number
of pollen grains deposited at stigmas was counted under a
light microscope (400X). Because stigmas of the studied
species received pollen from more than the four species
used in our research, pollen grains were only counted if
morphologically conspecific. Also, 15–25 stigmas per spe-
cies without visits were mounted as controls for self-pollen
transfer (one taken from each individual plant) and pollen
grain numbers deposited by automatic self-pollination were
counted with light microscope. A value of mean single visit
deposition (D) for each plant species was determined, sub-
stracting mean pollen grains deposited by automatic self-
pollination from the number of pollen grains counting at
stigmas visited. We compared D values among pollination
mechanisms and plant species using Kruskal–Wallis non-
parametric tests, followed by post hoc pairwise tests (Cono-
ver 1999). The level of significance was set at α = 0.05.
To estimate the pollen transfer efficiency (D/R) (Thom-
son et al. 2000; Castellanos et al. 2006), for pollination
mechanism and plant species, we compared mean pollen
deposited relative to pollen removed using Kruskal–Wallis
non-parametric tests. Post hoc pairwise comparisons were
made between the means of treatment ranges (Conover
1999).
INFOSTAT (Di Rienzo et al. 2010) was used for all
statistical analyses and figures. Mean values and standard
error, median, 25th and 75th percentiles are detailed in
Table2.
Results
Floral visitors
Flowers of the four plant species studied were visited by
161 specimens of Apoidea species from Apidae, Megachi-
lidae and Andrenidae families (Table 1). Solitary native
bees of the genus Megachile were the most abundant (44%
Table 2 Ovule number, proportion of pollen removed from anthers
(R), pollen deposition in stigmas (D) and pollen transfer (D/R) by sin-
gle visits of Megachile spp. in Crotalaria species with pump polli-
nation mechanism and Desmodium species with explosive pollination
mechanism, from Northwestern Argentina
Mean ± standard error, (sample size), and median values (Me), 25th and 75th percentiles (Q1 and Q3) are informed. Different letters indicate significant
differences (P < 0.05)
Plant species Ovule number Pollen removed (R) Pollen deposition (D) Pollen transfer (D/R)
(Mean ± S.E.)
(n = 10)
(Mean ± S.E.) (n) Me (Q1; Q3) (Mean ± S.E.) (n) Me (Q1; Q3) (Mean ± S.E.) (n) Me (Q1; Q3)
Crotalaria pumila 8.10 ± 0.18 0.18 ± 0.05 (23)a0.06 (0;0.37) 8.23 ± 4.87 (26)a0 (0; 0) 0.57 ± 0.30 (23)a0 (0; 0)
Crotalaria stipularia 35.40 ± 2.22 0.19 ± 0.07 (18)a0 (0; 0.49) 20.93 ± 6.62 (27)ab 0 (0; 44) 0.03 ± 0.02 (18)a0 (0; 0)
Desmodium incanum 6.82 ± 0.33 0.81 ± 0.02 (41)b0.84 (0.68; 1) 15.54 ± 2.09 (39)b13 (0; 26) 0.53 ± 0.08 (39)b0.39 (0; 0.79)
Desmodium subsericeum 7.82 ± 0.33 0.72 ± 0.06 (31)b0.73 (0.73; 1) 78.03 ± 12.66 (32)c48.5 (31; 92) 4.22 ± 0.83 (30)c2.24 (0.7; 8.14)

278 T.Figueroa Fleming, Á.V.Etcheverry
1 3
of relative abundance), followed by eusocial bumblebees of
the genus Bombus (22%), social non-native bee Apis mel-
lifera (13%) and solitary native bees of Epanthidium genera
(12%), Melissodes (5%), Psaenythia (2%) and Coelioxys
(<1%). Arhysoceble picta occasionally visited D. incanum
flowers (Table1). All these bees were able to activate the
pollination mechanisms making “legitimate” visits. Apis
mellifera behaved in some “illegitimate” visits to C. pumila
and C. stipularia as nectar thief accessing the flowers lat-
erally, without activating the pump mechanism. Melissodes
tintinnans also had a dual behaviour, activating the pump
mechanism in approximately 50% of visits to flowers of C.
stipularia and behaving like a thief of nectar in the rest of
the “illegitimate” visits to flowers of this plant. Paratrigona
glabella was not able to activate the pollination mecha-
nisms and was observed collecting pollen in D. incanum
flowers with the tripped mechanism and collecting pollen
from the end of the keel in flowers of C. pumila previously
visited by other insects (Table1). Megachile species have
the highest relative importance in the bee assemblage, and
they accounted for a visit proportion of 52%.
Pollen removal
We detected significant differences between both mecha-
nisms in the proportion of pollen grains removed (R)
from a flower after a single visit of Megachile (H = 53.73;
P < 0.0001). During a single visit, pollinators removed,
on average, 77 ± 3% (n = 72) and 18 ± 4% (n = 41) pollen
grains of the available pollen at the flower, respectively,
for explosive and pump mechanisms (Fig. 1). Also we
observed statistical differences between plant species in
pollen removal (H = 54.03; P < 0.0001) (Table2).
Pollen deposition
A single visit of Megachile resulted, on average, in a
deposit of 43 ± 7 (n = 71) pollen grains for flowers with
explosive mechanism and 15 ± 4 (n = 53) pollen grains for
flowers with pump mechanism. This difference between
mechanisms was statistically significant (H = 25.33;
P < 0.0001) (Fig.2).
There were significant differences in the average number
of pollen grains deposited on stigmas among plant species
(H = 45.42; P < 0.0001) (Table 2), with the highest value
recorded in D. subsericeum stigmas.
Megachile spp. were effective pollinators, because they
deposited after a single visit the same amount of pollen
grains per stigma (H = 0.13, P = 0.7152, pump mechanism)
or more pollen grains per stigma (H = 51.68, P < 0.0001,
explosive mechanism) than those deposited by autonomous
self-pollination.
Transfer efficiency: D/R
Efficiency of pollen transfer was significantly higher for
explosive mechanism (2.13 ± 0.42 pollen grains deposited
per 100 removed) than for pump mechanism (0.33 ± 0.17
Explosive (72)
Pump (41)
0.11
0.29
0.47
0.65
0.84
Removed pollen (R)
a
b
Fig. 1 Proportions of pollen grains removed from anthers (R)
(mean ± SE) by single visits of Megachile from flowers with explo-
sive and pump pollination mechanism. Numbers in brackets indicate
sample size for each mechanism. Means with different letters are dif-
ferent at P < 0.05
Explosive (71) Pump (53)
8.51
19.52
30.54
41.56
52.57
Pollen deposition (D)
a
b
Fig. 2 Numbers of pollen grains deposited on stigmas (D)
(mean ± SE) by single visits of Megachile in flowers with explosive
and pump pollination mechanism. Numbers in brackets indicate sam-
ple size for each mechanism. Means with different letters are different
at P < 0.05

279Comparing theefficiency ofpollination mechanisms inPapilionoideae
1 3
pollen grains deposited per 100 removed) (H = 31.65;
P < 0.0001) (Fig. 3). Average pollen grains deposited per
100 removed for Desmodium species was significantly
higher than for Crotalaria species. Among C. pumila and
C. stipularia, bees deposited per single visit less than one
pollen grain in a stigma per 100 pollen grains removed from
the anthers (0.57 ± 0.30; Me = 0; n = 23 and 0.03 ± 0.02;
Me = 0; n = 18, respectively), differing statistically from D.
incanum (0.53 ± 0.08; Me = 0.39; n = 39) and D. subseri-
ceum (4.22 ± 0.83; Me = 2.24; n = 30) (Table2). Pollination
equivalences showed that Megachile would have to visit a
flower with pump mechanism about four times to deposit
the same amount of pollen in a single visit in flowers with
explosive mechanism.
Visit duration
Time spent on flowers varied greatly between the two
mechanisms (H = 871.04; P < 0.0001), and Megachile
bees performed, on average, significantly shorter vis-
its in flowers with explosive than with pump mechanism
(1.86 ± 0.04 s; Me = 2; n = 853 vs. 3.95 ± 0.05 s; Me = 4;
n = 1450) (Fig. 4). Visit duration varied between plant
species (H = 967.41; P < 0.0001). C. stipularia received
the longest visits (5.01 ± 0.26 s; n = 104), followed by
C. pumila (3.87 ± 0.05 s; n = 1346) and D. subsericeum
(2.33 ± 0.06s; n = 399), while D. incanum had the shortest
visits (1.45 ± 0.04s; n = 454).
Conclusion anddiscussion
The results of our study on two Desmodium and Crota-
laria species confirm that explosive mechanism is more
efficient than pump mechanism in terms of pollen transfer.
This finding is in accordance with the prediction made by
Arroyo (1981).
Many studies have examined the amount of removed
and deposited pollen grains (Snow and Roubik 1987; Galen
and Stanton 1989; Young and Stanton 1990; Wilson and
Thomson 1991; Rademaker etal. 1997; King etal. 2013;
Zych et al. 2013). However, none of these studies have
investigated removal and deposition of pollen in Papil-
ionoideae species. The only records available are studies
made in Lupinus sericeus focusing only on pollen dispersal
(Harder 1990; Harder and Wilson 1994). To the authors’
knowledge the present study is the first to compare the effi-
ciency of pump versus explosive pollination mechanisms in
Papilionoideae.
The two genera considered in this study differed in some
of the traits involved in pollination, such as the tripping
mechanism, floral reward offered (nectar and/or pollen)
and colours of flowers. Also the bee assemblage visiting
Desmodium and Crotalaria flowers is taxonomically and
functionally diverse. It included native and exotic species
differing in size (small, such as Psaenythia, and large, such
as Bombus) and social behaviour (eusocial and solitary).
Bumblebee workers visited only Desmodium subsericeum
flowers. Apis mellifera and Megachile species were com-
mon visitors of all the four species. The native solitary bee
Explosive (69) Pump (41)
0.04
0.70
1.36
2.02
2.68
D/R
a
b
Fig. 3 Ratios of pollen grains deposited / pollen grains removed
(D/R) (mean ± SE) by single visits of Megachile in flowers with
explosive and pump pollination mechanism. Numbers in brackets
indicate sample size for each mechanism. Means with different letters
are different at P < 0.05
Pump (1450)Explosive (853)
0.15
4.83
9.50
14.18
18.85
Visit duration (s)
a
b
Fig. 4 Visit duration (mean ± SE) of Megachile in flowers with
explosive and pump pollination mechanism. Numbers in brackets
indicate sample size. Different letters over the data indicate that medi-
ans are significantly different at P < 0.05

280 T.Figueroa Fleming, Á.V.Etcheverry
1 3
species of Epanthidium, Melissodes, Psaenythia and Coe-
lioxys were able to activate both mechanisms, but had a
lower abundance and visit proportions, which places them
second in importance. Further research is needed to analyse
their effectiveness and efficiency in pollination.
Not all the floral visits were legitimate. Honeybees had
a dual behaviour, sometimes activated pollination mecha-
nisms and other times accessed laterally to the flowers
extracting nectar, without triggering the pump mechanism
of Crotalaria flowers. Paratrigona glabella (Meliponini)
was recorded, sporadically, robbing pollen in flowers of
C. pumila and D. incanum. This species was previously
reported stealing pollen in Vigna caracalla flowers (Etch-
everry etal. 2008).
Taking into account the visits of Megachile, the results
suggest that the four plant species are not pollen-limited at
the study area. Megachile bees are abundant, and can be
considered of great importance due to the proportion of
visits they made. They are effective in terms of pollen dep-
osition, pollen removal and efficient in relation of their con-
tribution for the female reproductive success of the studied
species. However, they deposited a smaller percentage of
pollen in species with pump mechanism. Recently it was
found that one species of Megachile deposited more pollen
per single visit (>250) than most other visitors in explosive
flowers of D. setigerum (Stanley et al. 2016). The reasons
for their success in pollen deposition are related to pollen-
carrying hairs on the underside of the abdomen, potentially
making visitors more likely to deposit pollen when posi-
tioning themselves on the keel of flowers. The other rea-
son is a morphological “fit” (or size matching) for achiev-
ing contact with the reproductive parts of flowers (Stanley
etal. 2016). These results, together with those obtained in
the present study, reinforce the idea that papilionate flowers
with an explosive mechanism are more specialized in terms
of its flower visitors than usually assumed for papilionate
flowers (Stanley etal. 2016).
In the studied species of Desmodium, with explosive
mechanism, approximately 77% of pollen was removed
from the flower after a single visit by Megachile spp. This
proportion is close to that reported for Cytisus scoparius,
with the same mechanism, in which Apis mellifera removed
ca. 75% (Suzuki 2003). Even lower values were reported
for non-papilionaceous or non-leguminous species with dif-
ferent pollination mechanisms and a similar duration of flo-
ral visit in Harder etal. (1985), Snow and Roubik (1987),
Galen and Stanton (1989), Young and Stanton (1990), Wil-
son and Thomson (1991), Harder and Barrett (1993) and
Rademaker etal. (1997). The removal of such a high per-
centage of pollen from Desmodium flowers means that lit-
tle pollen remains after a single flower visit by Megachile
spp. This may be the reason why bees rarely visits already
exploded flowers. Our results support the hypothesis that
species with explosive pollination mechanisms are visited
just once (Arroyo 1981; López etal. 1999; Galloni etal.
2007; Suzuki 2003; Alemán et al. 2014), and that bees
avoid those flowers in which the mechanism has already
been activated. As explained by Alemán et al. (2014),
Desmodium flowers have a unique opportunity to obtain an
effective cross-pollination during a short floral cycle, with
post-pollination changes (in position of the floral parts and
in corolla colour) which function as cues that allow polli-
nators to avoid them. Previous studies have shown that the
decrease in the amount of available pollen in the already
foraged flowers reduces their attractiveness to pollinators
(Harder and Thomson 1989; Young and Stanton 1990).
In Crotalaria species studied here, only 18% of pollen
was removed after a single flower visit by Megachile spp.
This value is very close to that obtained in Lupinus seri-
ceus (19%) having the same mechanism, but in the latter
case the removal was performed by hand (Harder 1990).
Flowers with pump mechanism can receive multiple vis-
its, depending on the length of total floral cycle, amounts
of rewards, and also on the pollinators’ abundance and
visit frequency. Many legume species, such as Lupinus,
Vicia, Astragalus (Faegri and van der Pijl 1971; Haynes
and Mesler 1984; Harder and Thomson 1989; Juncosa and
Webster 1989; Harder 1990; Dafni 1992; Proctor et al.
1996; Kittelson and Maron 2000) and Crotalaria micans
(Etcheverry 2001a), possess mechanisms that delimit the
amount of pollen removed during single flower visits of
pollinators. In flowers that have pollen dispensing, pol-
len deposition is a decelerating function of pollen removal
(Harder and Thomson 1989). The effect of inter-visit inter-
val on pollen removal in L. sericeus (Harder and Thomson
1989) and C. micans (Etcheverry 2001a) was studied per-
forming, by hand, manipulations at three different intervals.
In L. sericeus, it was reported a value close to 9% during
the first removal, while in C. micans this value was ca. 20%
of the total available per flower. These values are in accord-
ance with the results we obtained for the proportion of pol-
len removed by Megachile spp. during the first visit to C.
pumila and C. stipularia flowers. Our findings suggest that
both species limit the amount of pollen exposed to indi-
vidual pollinators. Generally speaking, plants that receive
many visits can maximize the amount of pollen donated
to other stigmas by presenting their pollen in many small
doses rather than all at once (Thomson et al. 2000). The
more pollen a plant puts on a bee, the more likely the bee
is to remove that pollen from circulation (Thomson 1983;
Harder 1990). Concerning their natural history, bees are
probably better than other visitors at removing whatever
pollen is presented, because of their branched body hairs
that facilitate pollen removal (Thomson etal. 2000). How-
ever, in stereotyped grooming movements, they drag spe-
cialized rakes or combs through their pile, gathering the

281Comparing theefficiency ofpollination mechanisms inPapilionoideae
1 3
loose pollen into their scopae or corbiculae (Michener etal.
1978; Thorp 1979). Therefore, if a plant doses a bee with a
large load, most of that pollen will be packed away immedi-
ately, whereas grains applied in small doses tend to remain
longer on active sites on the animal (Thomson etal. 2000).
This seems to be true for the pump mechanism of the Cro-
talaria species, exhibiting pollen dosage and scopal loads
in Megachile may in fact form active pools of pollen for
donation.
Some studies have shown that the number of pollen
grains deposited on the stigma should exceed a minimum
threshold for the formation of a fruit. Threshold values
reported, expressed as the ratio between the number of pol-
len grains deposited and the number of ovules, are about
1:1–12:1 (Herrera 1987), i.e. fruit formation can occur
even with the minimum intensity in pollination and the
minimum number of pollen grains required is generally the
same as the total ovules. Desmodium species with explo-
sive mechanism have surpassed the minimum threshold
required for the formation of fruit. The highest D value was
recorded in D. subsericeum stigmas, with pollen deposition
ten times higher than the average of ovules in their flowers.
In D. incanum, the deposited pollen was twice the mean
number of ovules per flower (Table2). The observed values
of pollen deposition in both species correspond to what we
might expect in flowers that usually receive a single visit
throughout anthesis (Arroyo 1981).
In C. pumila, the amount of pollen grains deposited by
Megachile spp. during single visits was enough to ferti-
lize all ovules (1:1) but in C. stipularia was less than mean
ovules number (0.6:1) (Table 2). In species with pump
mechanism, it is necessary to take into account the positive
effect that multiple visits can have to increase the amount
of pollen deposited, which allows reaching the minimum
threshold required for fruit formation.
Regarding pollen transfer efficiency, Megachile spp.
removed and deposited a higher proportion of pollen grains
in flowers with explosive mechanism than in those with
pump mechanism. Consequently, transfer efficiency of pol-
len was higher for explosive mechanism (two pollen grains
deposited per 100 removed) than for pump mechanism (less
than one pollen grain deposited per 100 removed). Galen
and Stanton (1989) reported a higher pollination efficiency
of individual bees in comparison with our study, because
nearly 3% of the pollen removed by bumblebees was
deposited on compatible stigmas of Polemonium viscosum.
In the case of Fritillaria meleagris, the fraction of pollen
produced in flowers reaching conspecific stigma during a
single visit ranged 1.3–2.2%, depending on the insect taxon
(Zych etal. 2013).
The expectation that high removal leads to high depo-
sition was met in Desmodium species receiving a single
visit, but not in Crotalaria species, where both variables
were low. However, Crotalaria species could have hetero-
geneity among floral visitors in subsequent visits. If flow-
ers were visited many times, the situation could be fur-
ther complicated by the sequence of visitors. Single visits
by nectar collecting bees and by pollen collecting bees
have significantly different consequences for pollen trans-
fer, because pollen collectors remove lots of pollen but
deposit very little of it, while nectar collecting removes
less but deposits more (Wilson and Thomson 1991).
The shorter visits were observed in the explosive flow-
ers of Desmodium. However, the relatively short manip-
ulation time involves a greater operative force than the
flowers with other mechanisms of pollination (Córdoba
and Cocucci 2011). In the flowers with pump mecha-
nism, bees must perform a series of piston movements
to remove the pollen from the flowers which extends the
duration of visits a few seconds more with respect to the
previous mechanism. Additionally, flowers with pump
mechanism also offer nectar as reward; bees may collect
a single or both rewards. In theory, longer visits could
increase visitor contact with, and/or transfer of pollen
to, a stigma (Harder 1990); but they could also indicate
‘ineffective’ feeding (excessive grooming, eating pol-
len or floral tissues, avoiding anther or stigma contacts)
(King et al. 2013). In our study, the assumption that a
longer residence time in the flower leads to greater pol-
len removal and deposition was not met. Megachile visits
were significantly longer in the flowers that had lower D.
This observation could be explained by King etal. (2013)
who concluded that it is problematic to use visit duration
as a proxy for pollination, because no particular ‘kind’ of
relation between visit duration and single visit deposition
can be assumed for a visitor group or for a single visitor
species.
It can be assumed that in Papilionoideae, the numer-
ous groups with secondary pollen presentation arose from
groups with primary pollen presentation and that the devel-
opment of pollination mechanisms would appear by con-
vergence in different tribes. But there is still not enough
evidence on the systematic distribution and the evolution-
ary sequence in the occurrence of pollination mechanisms
within the subfamily. Future questions are posed at vari-
ous levels on related topics ranging from structure to the
mechanics of pollen delivery, from pollinator behaviour to
pollen dispensing, pollen transport and pollination success
to delineate the overall picture of floral evolution in the
Papilionoideae.
Acknowledgements Insects were identified by Dr. Alberto Abra-
hamovich (Museo de la Plata). Carolina Noemí Yañez and Andrea
Florencia Romero helped with field work. The authors thank María
Schulze, Francisco Sylvester and Hugo Lesser for their assistance
with the English version of the manuscript. This research was sup-
ported by grants from Consejo Nacional de Investigaciones Científicas

282 T.Figueroa Fleming, Á.V.Etcheverry
1 3
y Técnicas and Consejo de Investigación de la Universidad Nacional
de Salta.
References
Alemán M (2014) Biología floral, mecanismos de polinización y sis-
tema reproductivo de Papilionoideas nativas del Valle de Lerma
(Salta). Tesis de Doctorado en Ciencias Biológicas. Universidad
Nacional de Salta
Alemán M, Figueroa-Fleming T, Etcheverry A, Sühring S, Ortega-
Baes P (2014) The explosive pollination mechanism in Papilio-
noideae (Leguminosae): an analysis with three Desmodium spe-
cies. Plant Syst Evol 300:177–186
Arroyo MTK (1981) Breeding systems and pollination biology
in Leguminosae. In: Polhill RM, Raven PH (eds) Advances
in legume systematics, Part 2. Royal Botanic Garden, Kew,
pp723–769
Bianchi AR (1996) Temperaturas medias estimadas para la región
noroeste de Argentina. INTA, Salta
Bianchi AR, Yáñez CE (1992) Las precipitaciones en el noroeste
argentino. INTA, Salta
Brito VLG, PinheiroM, Sazima M (2010) Sophora tomentosa and
Crotalaria vitellina (Fabaceae): reproductive biology and inter-
actions with bees in the restinga of Ubatuba, São Paulo. Biota
Neotrop 10: 185–192
Castellanos MC, Wilson P, Keller SJ, Wolfe AD, Thomson JD (2006)
Anther evolution: pollen presentation strategies when pollinators
differ. Am Nat 167:288–296
Conover WJ (1999) Practical nonparametric statistics. Wiley, New
York
Córdoba SA, Cocucci AA (2011) Flower power: its association with
bee power and floral functional morphology in papilionate leg-
umes. Ann Bot (Lond) 108:919–931
Dafni A (1992) The principles of pollination ecology. Oxford Univer-
sity Press, New York
Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M,
Robledo CW (2010) InfoStat versión 2010. Grupo InfoStat,
FCA, Universidad Nacional de Córdoba, Argentina
Du Puy DJ, Labat JN (2002) Crotalaria. In: Du Puy DJ (ed) The
Leguminosae of Madagascar. Royal Botanic Gardens, Kew,
pp.672–708
Endress PK (1996) Diversity and evolutionary biology of tropical
flowers. Cambridge University Press, Cambridge
Etcheverry AV (2001a) Dynamic dispensing of pollen: an empirical
study. In: Proceedings of the 8th international pollination sympo-
sium on acta horticulture, vol 561, pp 67–70
Etcheverry AV (2001b) Floral biology and pollination in Crotalaria
stipularia (Fabaceae: Papilionoideae). In: Proceedings of the 8th
international pollination symposium on acta horticulture, vol
561, pp 339–342
Etcheverry AV, Protomastro JJ, Westerkamp C (2003) Delayed
autonomous self-pollination in the colonizer Crotalaria micans
(Fabaceae: Papilionoideae): structural and functional aspects.
Plant Syst Evol 239:15–28
Etcheverry AV, Alemán MM, Figueroa Fleming T (2008) Flower
morphology, pollination biology and mating system of the com-
plex flower of Vigna caracalla (Fabaceae: Papilionoideae). Ann
Bot (Lond) 102:305–316
Etcheverry AV, Alemán MM, Figueroa-Fleming T, López-Spahr
D, Gómez CA, Yáñez C, Figueroa-Castro DM, Ortega-Baes P
(2012) Pollen:ovule ratio and its relationship with other flo-
ral traits in Papilionoideae (Leguminosae): an evaluation with
Argentine species. Plant Biol 14:271–278
Faegri K, van der Pijl L (1979) The principles of pollination ecol-
ogy. 2nd Ed. Pergamon, Oxford
Figueroa Fleming T (2014) Interacciones planta-polinizador en
Papilionoideas (Leguminosae) simpátricas nativas de Salta,
Argentina. Tesis de Doctorado en Ciencias Biológicas. Univer-
sidad Nacional de Salta
Galen C, Stanton ML (1989) Bumble bee pollination and floral
morphology: factors influencing pollen dispersal in the alpine
sky pilot, Polemonium viscosum (Polemoniaceae). Am J Bot
76:419–426
Galloni M, Cristofolini G (2003) Floral rewards and pollination in
Cytiseae (Fabaceae). Plant Syst Evol 238:127–137
Galloni M, Podda L, Vivarelli D, Cristofolini G (2007) Pollen pres-
entation, pollen–ovule ratios, and other reproductive traits in
Mediterranean legumes (Fam. Fabaceae-Subfam. Faboideae).
Plant Syst Evol 266:147–164
Harder LD (1990) Pollen removal by bumble bees and its implica-
tions for pollen dispersal. Ecology 71:1110–1125
Harder LD, Barret SCH (1993) Pollen removal from tristylous Pon-
tederia cordata: effects of anther position and pollinator spe-
cialization. Ecology 74:1059–1072
Harder LD, Thomson JD (1989) Evolutionary options for maxi-
mizing pollen dispersal of animal-pollinated plants. Am Nat
133:323–344
Harder LD, Wilson WG (1994) Floral evolution and male reproduc-
tive success: optimal dispensing schedules for pollen dispersal
by animal-pollinated plants. Evol Ecol 8:542–559
Harder LD, Thomson JD, Cruzan MB, Unnasch RS (1985) Sex-
ual reproduction and variation in floral morphology in an
ephemeral vernal lily, Erythronium americanum. Oecologia
67:286–291
Haynes J, Mesler M (1984) Pollen foraging by bumblebees: Forag-
ing patterns and efficiency on Lupinus polyphyllus. Oecologia
61:249–253
Herrera CM (1987) Components of pollinator ‘‘quality’’: compara-
tive analysis of a diverse insect assemblage. Oikos 50:79–90
Huang L, Jin L, Zhang S, Li J, Yang Y, Zhang X, Wang X
(2013) Pollen release mechanisms of papilionaceous plants
(Faboideae). Acta Pratacult Sin 22: 305–314
Jacobi CM, Ramalho M, Silva M (2005) Pollination biology of the
exotic rattleweed Crotalaria retusa L. (Fabaceae) in NE Bra-
zil. Biotropica 37:357–363
Juncosa AM, Webster BD (1989) Pollination in Lupinus nanus
subsp. latifolius (Leguminosae). Am J Bot 76: 59–66
Kearns CA, Inouye DW (1993) Techniques for pollination biolo-
gists. University Press of Colorado, Niwot
King C, Ballantyne G, Willmer PG (2013) Why flower visitation
is a poor proxy for pollination: measuring single-visit pollen
deposition, with implications for pollination networks and con-
servation. Methods Ecol Evol 4:811–818
Kittelson PM, Maron JL (2000) Outcrossing rate and inbreed-
ing depression in the perennial yellow bush lupine, Lupinus
arboreus (Fabaceae). Am J Bot 87: 652–660
Lavin M, Herendeen PS, Wojciechowski MF (2005) Evolutionary
rates analysis of Leguminosae implicates a rapid diversifica-
tion of lineages during the Tertiary. Syst Biol 54: 575–594
Le Roux MM, Van Wyk BE (2012) The systematic value of flower
structure in Crotalaria and related genera of the tribe Crotalar-
ieae (Fabaceae). Flora 207:414–426
Leppik EE (1966) Floral evolution and evolution in Leguminosae.
Ann Bot Fennici 3: 99–308
López J, Rodríguez-Riaño T, Ortega-Olivencia A, Devesa JA, Ruiz
T (1999) Pollination mechanisms and pollen-ovule ratios in
some Genisteae (Fabaceae) from Southwestern Europe. Plant
Syst Evol 216:23–47

283Comparing theefficiency ofpollination mechanisms inPapilionoideae
1 3
Michener CD, Winston ML, Jander R (1978) Pollen manipulation
and related activities and structures in bees of the family Apidae.
Univ Kansas Sci Bull 51:575–601
Ne’eman G, Jurgens A, Newstrom-Lloyd LE, Potts SG, Dafni A
(2010) A framework for comparing pollinator performance:
effectiveness and efficiency. Biol Rev 85:435–451
Olson DM, Dinerstein E, Wikramanayake ED, Burgess ND, Powell
GVN, Underwood EC, D’Amico JA, Itoua I, Strand HE, Mor-
rison JC, Loucks CJ, Allnutt TF, Ricketts TH, Kura Y, Lamor-
eux JF, Wettengel WW, Hedao P, Kassem KR (2001) Terrestrial
ecoregions of the worlds: a new map of life on earth. Bioscience
51:933–938
Polhill RM (1976) Genisteae (Adans.) Benth. and related tribes
(Leguminosae). Syst Bot 1: 143–368
Polhill RM (1982) Crotalaria in Africa and Madagascar. A. A.
Balkema, Rotterdam
Proctor M, Yeo P, Lack A (1996) The natural history of pollination.
Timber Press, Portland
Rademaker MCJ, De Jong TJ, Klinkhamer PGL (1997) Pollen
dynamics of bumble-bee visitation on Echium vulgare. Funct
Ecol 11:554–563
Rodríguez-Riaño T, Ortega-Olivencia A, Devesa AJ (1999) Repro-
ductive biology in two Genisteae (Papilionoideae) endemic of
the Western Mediterranean Region: Cytisus striatus and Retama
sphaerocarpa. Can J Bot 77:809–820
Small E (1988) Pollen–ovule patterns in tribe Trifoliae (Legumi-
nosae). Plant Syst Evol 160:195–205
Snow AA, Roubik DW (1987) Pollen deposition and removal by bees
visiting two tree species in Panama. Biotropica 19:57–63
Solomon Raju AJ, Purnachandra Rao S (2006) Explosive pollen
release and pollination as a function of nectar-feeding activity of
certain bees in the biodiesel plant, Pongamia pinnata (L.) Pierre
(Fabaceae). Curr Sci 90: 7–10
Stanley DA, Otieno M, Steijven J, Sandler Berlin E, Piiroinen T,
Willmer P, Nuttman C (2016) Pollination ecology of Desmodium
setigerum (Fabaceae) in Uganda: do big bees do it better? J Pol-
linat Ecol 19:43–49
Suzuki N (2003) Significance of flower exploding pollination on the
reproduction of the Scotch broom, Cytisus scoparius (Legumi-
nosae). Ecol Res 18:523–532
Thomson JD (1983) Component analysis of community-level interac-
tions in pollination systems. In: Jones CE, Little RJ (eds) Hand-
book of experimental pollination biology. Van Nostrand Rein-
hold, New York, pp451–460
Thomson JD, Wilson P, Valenzuela M, Malzone M (2000) Pollen
presentation and pollination syndromes, with special reference to
Penstemon. Plant Species Biol 15:11–29
Thorp RW (1979) Structural, behavioral, and physiological adapta-
tions of bees (Apoidea) for collecting pollen. Ann Mo Bot Gard
66:788–812
Westerkamp C (1997) Keel blossoms: bee flowers with adaptations
against bees. Flora 192:125–132
Westerkamp C, Weber A (1999) Keel flowers of the Polygalaceae and
Fabaceae: a functional comparison. Bot J Linn Soc 129:207–221
Willmer P, Stanley DA, Steijven K, Matthews IM, Nuttman CV
(2009) Bidirectional flower color and shape changes allow a sec-
ond opportunity for pollination. Curr Biol 19:919–923
Wilson P, Thomson JD (1991) Heterogeneity among floral visitors
leads to discordance between removal and deposition of pollen.
Ecology 72:1503–1507
Yeo PF (1993) Secondary pollen presentation. Form, function and
evolution. Springer, New York
Young HJ, Stanton ML (1990) Influences of floral variation on pollen
removal and seed production in wild radish. Ecology 71:536–547
Zych M, Gold J, Stpiczynska M (2013) The most effective pollinator
revisited: pollen dynamics in a spring-flowering herb. Arthro-
pod-Plant Interactions 7:315–322