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Pollination biology of Canna indica (Cannaceae) with particular reference to

the functional morphology of the style

Article in Plant Systematics and Evolution · November 2010

DOI: 10.1007/s00606-010-0379-x

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Plant Syst Evol
DOI 10.1007/s00606-010-0379-x
ORIGINAL ARTICLE
Pollination biology of Canna indica (Cannaceae) with particular
reference to the functional morphology of the style
Evangelina Glinos • A. A. Cocucci
Received: 7 April 2010 / Accepted: 4 October 2010
Ó Springer-Verlag 2010
Abstract The anatomy of the bizarre style of Canna
indica is analyzed functionally and comparatively within
Zingiberales, particularly in relation to the presence of two
stigma-like areas, one apical and the other lateral and
subapical. We asked whether these areas have separate
receptive or adhesive functions and whether they are
derived from a single stigma that previously had both
functions. We expected that the mechanism of pollen
transference would be highly effective at the flower level,
i.e., that pollen limitation would affect fruit set rather than
seed set. Both areas produce a sticky carbohydrate- and
mucilage-positive exudate but only the apical one leads to
pollen tube growth into the stylar canal. The subapical and
lateral area is regarded here as being homologous to the
viscidium of Lowiaceae and Marantaceae. High pollen
limitation through fruit set is attributable to the low visi-
tation rate of the single long-billed hummingbird pollinator
(Heliomaster furcifer) and to pollen loss caused by nectar
robbers, while low limitation through seed set suggests that
the efficiency of a few visits of pollen-carrying hum-
mingbirds is high. Recordings of the pollination process
indicate that the viscidium is touched before the pollen
presenter, when hummingbirds are flying out of the
flowers.
Keywords Canna indica  Style anatomy  Pollination
biology  Hummingbirds  Stigmatic secretions  Viscidium
E. Glinos (&)  A. A. Cocucci
Laboratorio de Biologı́a Floral, Instituto Multidisciplinario
de Biologı́a Vegetal (CONICET-Universidad Nacional
de Córdoba), Casilla de Correo 495,
5000 Córdoba, Argentina
e-mail: evaglinos@gmail.com
Introduction
The flower structure of Canna (Cannaceae, Zingiberales) is
highly unusual and exhibits profound modifications in
relation to the basic pattern of monocots and to other clo-
sely related clades, such as Bromeliales and Commelinales
(Judd et al. 2008). Though a typical radially symmetric
perianth with six members in two whorls is still evident,
flower conspicuousness is due not to the perianth but rather
to the development of a colorful androecium and gynoe-
cium (Fig. 1a–c, g). The androecium, which is asymmet-
rical in development of its members, consists of three or
four flattened, unequal, petaloid staminodes in two
whorls plus one half fertile and likewise petaloid stamen
(Fig. 1b, c). One of the staminodes of the inner whorl is
turned to the ventral side of the flower and forms a label-
lum (Fig. 1c). The single anther has a lateral position on
the flattened stamen and is reduced to one theca (Fig. 1b;
Kunze 1984; Dahlgren et al. 1985; Yeo 1993; Judd et al.
2008). The style is flattened, fleshy, and also petaloid with
a marginal terminal stigma in addition to a marginal sub-
apical notch (Figs. 1e, 2). Both the terminal part and the
lateral notch have a stigma-like aspect and are wet and
sticky in fresh flowers. These two stigmatic regions, which
were recognized by early botanists (Schumann 1888;
Kränzlin 1912), are here named as apical and lateral,
respectively (Figs. 1e, 2). In a process known as ‘‘sec-
ondary pollen presentation,’’ before the flower opens,
pollen is spontaneously deposited on the flattened style as
an approximately elliptical clump below the apical and at
the side of the lateral stigmatic region (Figs. 1e, 2). After
flower opening, the style acts as the pollen-dispensing
organ or pollen presenter while the stamen folds to the back
and displaces the shrivelled anther away from the pollen
load (Bouché 1833; Yeo 1993). In Cannaceae, the flower
123

Fig. 1 Schematic representation of the flower structure of Canna
indica. a Flower diagram. b Staminode and half-fertile stamen.
c Staminode and labellum. d Cross section of flower tube. e Side view
of the style showing pollen load on the presenter at the right of the
lateral stigmatic part and below the apical stigmatic part. f Front view
of the style before (left) and after pollen delivery (right). g Calyx and
corolla. C Petal, K sepal, G style, S fertile stamen, s staminode,
L labellum, nch nectar chamber, acc section of the tube accessible to
pollinators, ap apical part of the stigma, lp lateral part of the stigma,
spp secondary pollen presenter
asymmetry evident in the unequal development of the
staminodes and in the appearance of both the fertile stamen
and the pistil do not have right- and left-handed counter-
parts as in its sister group Marantaceae in which the two
flowers of a bract are mirror images (Kirchoff 1983b).
Though the general morphology, anatomy, and devel-
opment of the Canna flower have drawn the attention of
some authors (Rao and Donde 1955; Pai 1963; Kirchoff
1983a, b; Kunze 1984; Kress 1990), the presence of the
two aforementioned stigma-like zones has apparently pas-
sed unnoticed in modern literature. Among Zingiberales,
some Canna-like features are shared by members of other
families, such as androecium reduction and secondary
pollen presentation by Marantaceae and stigma with two
distinct regions by Lowiaceae and Marantaceae. These
morphological aspects are known to be relevant in flower
functioning of Lowiaceae (Sakai and Inoue 1999) and
Marantaceae (Kunze 1984; Kress 1990; Endress 1994; Yeo
1993; Locatelli et al. 2004; Classen-Bockhoff and Heller
2008; Pischtschan and Classen-Bockhoff 2008, 2010; Ley
123
E. Glinos, A. A. Cocucci
Fig. 2 Stamen and style of Canna indica. ap Apical part of the
stigma, lp lateral part of the stigma, spp secondary pollen presenter,
S fertile stamen, t theca
and Classen-Bockhoff 2009). In addition, the morpholog-
ical distinction of Zingiberales families, which is also
supported by molecular data (Kress et al. 2001; Johansen
2005; Kress and Specht 2006), has been traditionally based
on floral characters relevant in the pollination mechanism.
Though several authors (Bouché 1833; Delpino 1873; Yeo
1993) have suggested that the particular floral organization
of Canna has a functional and morphological basis, they
have not been able to make a convincing case to explain
the bizarre flower organization from a functional view-
point. Consequently, we expected that the elucidation of
the morphological and anatomical nature of these stigmatic
structures as well as their relation to the pollination
mechanism should help us to explain some aspects of
change in the flower structure among Zingiberales from a
functional perspective.
In plants where two ‘‘stigmatic’’ regions are known
(Apocynaceae and Orchidaceae), one is receptive and the
other produces a substance that acts as an accessory pollen
adhesive, i.e., as a means that is additional to the regular
pollenkitt to glue pollen onto the pollinators (Vogel 2002;
see also Machado and Lopes 2000; Moyano et al. 2003).
For these plant families, the secretory part is interpreted as
having evolved from a single stigmatic area by special-
ization in two areas, one receptive and another secretory.
The occurrence of this division of labor in the stigma is
associated in both families with pollen-packaging strategy
in pollinia or large pollen clumps in which most seeds in a
Pollination biology of Canna indica (Cannaceae)
fruit are sired in a single or few visits (see Harder and
Johnson 2008). Consequently, our working hypothesis was
that, as in these examples, the two stigma-like structures of
Canna have receptive or adhesive functions and that they
are derived from a single stigma that previously had both
functions. In addition, we expected that the mechanism of
pollen transference would be highly effective at the flower
level, i.e., that pollen limitation would affect fruit set rather
than seed set.
Considering the above expectation, we pursued this
study to obtain a better functional explanation of the
flowers of Canna by addressing the following questions:
Do lateral and apical parts of the stigma have different
functions? Have both parts the same chemical nature and
histological characteristics? Which species visit and polli-
nate the flowers? How is pollen deposited on the pollina-
tor’s body and how is pollen retrieved by the stigma? Is this
species pollen-limited? What is the relation between fruit
set and seed set in a scenario of pollen limitation? In
addition, we traced morphological analogues of the lateral
stigmatic parts, i.e., structures that are equivalent in ana-
tomical construction and position on the style, and their
function among representatives of the Zingiberales with the
expectation that this would reveal ancestral functional
relationships in Canna.
Study species
Cannaceae (Zingiberales) are represented by a single genus
and 10 species probably native to the New World, and are
distributed in tropical and subtropical regions worldwide
(Dahlgren et al. 1985; Maas and Maas 1988). Canna indica
L. is distributed from Mexico to South America and is nat-
uralized in North America, Europe, and Southeast Asia. It
grows in temperate regions of subtropical Argentina (Novara
2001). Observations and collection of samples were carried
out in three natural populations of Canna indica located in
Cuesta Blanca 31°280 59.100 S, 64°340 33.200 W; Costa Azul
31°240 46.800 S, 64°260 51.400 W; and Agua de Oro 31°040 0400 S,
34°180 1600 W, Córdoba, Argentina. Additional morphologi-
cal and anatomical observations were made in ‘‘Canna
indica’’ garden hybrids.
Petals are basally connate and fused with the stamen and
staminodes (in this species there are three) into a
50–60 mm long tube with three entries that clearly separate
spaces accessible to pollinators and that accumulate nectar
from septal nectaries (Fig. 1d; see also Rao and Donde
1955; Pai 1963; Kunze 1984). When the flowers open, the
stamen turns to the back and the style twists axially, in such
a way that the lateral stigmatic part is placed ventrally and
the pollen presenter laterally to the flower inside. In addi-
tion, the tip of the style curves somewhat to the side during
anthesis (Fig. 1f).
For the purpose of comparisons of the style morphology
among Canna and representatives of closely related familes,
additional fresh styles of Marantaceae [Calathea cylindrica
(Roscoe) K. Schum. Ubatuba, Brazil; Maranta arundinacea
L. cultivated in Córdoba, Argentina], Strelitziaceae (Strel-
itzia reginae Aiton, cultivated in Córdoba, Argentina), and
Lowiaceae [Orchidantha maxillarioides (Ridl.) K. Schum.,
cultivated in the Botanical Gardens of Darmstadt, Germany]
were examined under the stereomicroscope.
Methods
Disposition of the glandular parts of the stigma
and anatomy of the style
Right- or left-handedness, i.e., with the lateral part of the
stigma positioned at the right or left side of the style, was
recorded in 114 flowers. For anatomical study, styles were
excised, fixed in a 1:1 glutaraldehyde-phosphate buffer pH
7 solution, and stored at 5°C for 48 h, then dehydrated in
an ethanol series and embedded in paraffin (Johansen
1940). Longitudinal and cross sections, 8 lm thick, were
made with a Leitz 1512 rotary microtome and stained with
brilliant cresyl blue (Merck). The observations were carried
out using a Leica DM LB light microscope.
Identification of the receptive part of the style
To identify the receptive area, the apical and lateral parts of
the stigma of virgin flowers were manually saturated with
cross pollen. Pollen tubes were allowed to grow for 2 days
until the style started to shrivel. Then styles were cut and
fixed in a 70% ethanol solution for at least 6 h and cleared
with 5% NaOH at 60°C for 24 h. After rinsing with water,
styles were mounted on slides with 50% glycerin and 0.1%
aniline blue in PO4HK 0.1 M buffer and, after 3 h, gently
squashed and observed with a Leica DM LB epifluores-
cence microscope (Kearns and Inouye 1993).
Chemical nature of the stigmatic secretion
Fresh flower styles were smoothly pressed onto slides to
obtain an imprint of the secretions of the two parts of the
stigma. The imprint was embedded in 0.01% ruthenium red
and left for a few minutes to reveal mucilages (Gerlach
1984). The same procedure was performed with Sudan IV
to reveal lipids (Gerlach 1984). To detect total carbohy-
drates, the imprints were treated with Schiff’s reagent
(Merck) according to Jensen (1962). The preparations
obtained were then observed using a Leica DM LB light
microscope and a Leica MS 5 stereomicroscope. Glucose
was detected by embedding a GlucostixÒ strip for a few
123

seconds with the secretion (Kearns and Inouye 1993). All
these tests were made on five flowers of three floral stages:
buds with closed thecae, buds with pollen deposited on the
presenter, and open flowers.
Breeding system and pollen attachment to the style
In the Agua de Oro population, 5–10 flowers per treatment
were used to evaluate whether flowers depend on pollina-
tors for the production of fruits and to determine repro-
ductive efficiency in this population. Three pollination
treatments were made: autonomous self-pollination, hand
cross-pollination, and open pollination. In the autonomous
self-pollination treatment, flowers were bagged before
opening to isolate them from pollinators. In the hand cross-
pollination treatment, the stigmas of virgin flowers were
saturated with pollen from other plants and then bagged.
Finally, another set of flowers was exposed to open polli-
nation. Fruit set (proportion of fruits/flowers treated 9 100)
and the number of seeds per fruit in each treatment were
recorded. The frequency of flowers setting and not setting
fruits was tested between treatments with a v2 homogeneity
test. The mean number of seeds per capsule was compared
between free- and cross-pollination treatments with a one-
way analysis of variance. A reproductive efficiency index
was calculated according to Ruiz and Arroyo (1978) as (Po/
Pc), where Po and Pc are the proportions of seeds or fruits
set under open and cross-pollination, respectively. If this
value is 1 there is no pollen limitation and therefore pollen
transfer by vectors is sufficient. Additionally, 30 flowers
were bagged to evaluate if pollen fell spontaneously when it
was not removed by pollinators. The presence of pollen on
the style presenter was recorded until the flowers shrivelled.
Floral visitors
Observations were made in the three natural populations
during a total of 15 days in January, February, and March
of 2004 and 2007. A total of 109 h of observations from
hidden posts were dedicated to identifying the animals
arriving at the flowers. During this time, posts of obser-
vations were changed approximately every 2 h. Of the total
observation time, 34 h were dedicated to determining vis-
itation frequency. To this end the number of insect and
hummingbird visits per inflorescence, as well as the visi-
tors’ behavior, was recorded at 40 min intervals. The vis-
itation rate was calculated as the number of visits/number
of inflorescences/hour of observation. Photographs and
videos of the pollination process were also taken to study
the pollination mechanism. In addition, the mechanical
interplay between flowers and pollinators was studied by
simulating visits using plastic models and embalmed
hummingbirds of the pollinating species. Exposed culmen
123
E. Glinos, A. A. Cocucci
lengths of hummingbird visitors were measured from
specimens of the Museo Argentino de Ciencias Naturales
(Buenos Aires).
Results
Disposition of the glandular parts of the stigma
and anatomy of the style
Of the 114 flowers inspected, 97% presented the lateral part
of the stigma on the left side of the style. In this position,
the pollen presenter is placed at the right side of the
secretory part (Fig. 2). Longitudinal and cross sections of
C. indica and hybrid flowers showed the stylar canal run-
ning throughout the style from the ovary to the apical part
of the stigma (Fig. 3). The epidermal cells that internally
cover this canal are flat and have a reduced lumen, a thin
cuticle, and a denser cytoplasm than the rest of the stylar
cells (Fig. 4a). These cells are presumably responsible for
the secretion of the dense material within the stylar canal
(Fig. 4a).
The lateral and the apical parts of the stigma are
similar in having abundant, long (ca. 0.1 mm), noncapi-
tate, unicellular trichomes that are swollen at the base.
The trichome cytoplasm is rich in stroma, organelles, and
small to medium vacuoles (Fig. 4b, c). The apical and
lateral parts differ in the distribution of the trichomes,
with trichomes distributed along two ridges in the apical
part or along a single ridge on the lateral part. The two
apical ridges correspond to the outer rim of the stylar
canal (Fig. 3a).
Identification of the receptive part of the style
Although pollen grains germinated on both parts of the
stigma, only pollen tubes from pollen deposited on the
stigma’s apical part developed and reached the stylar canal
(Fig. 4d–f).
Chemical nature of the stigmatic secretion
The staining of both parts of the stigma was positive for
carbohydrates (Schiff’s reagent) and mucilage (ruthenium
red). The tests for lipids and glucose were negative. Iden-
tical results were obtained for all three floral stages.
Breeding system and pollen attachment on the style
None of the flowers excluded from pollinators developed
fruits. Fruit set under free pollination and hand-crossed
pollination treatments was 20 and 86%, respectively, with
the frequency of flowers setting and not setting fruits
Pollination biology of Canna indica (Cannaceae)

Fig. 3 Canna indica.

Longitudinal and cross sections
through the style. The schematic
drawing shows the plane of the
sections. The shaded area
indicates the stylar canal.
a–c Cross sections. d, e Oblique
longitudinal sections. The stylar
canal is open throughout the
style length. The apical and the
lateral stigmatic parts have
unicellular trichomes. ap Apical
part, lp lateral part, sc stylar
canal. Scale bars 100 lm

significantly different between treatments (v2 = 5.18;
df = 1; p = 0.02). Through fruit set, the reproductive
efficiency index value obtained was 0.23, and through seed
set it was 0.84. No significant differences were found
between open pollination and hand cross-pollination in the
number of seeds produced per fruit (ANOVA; F = 0.24;
df = 1; p = 0.63).
In the flowers bagged to evaluate pollen attachment to
the secondary pollen presenter, pollen remained on the
style until flowers shrivelled even when subjected to the
effect of wind and rain. This means that pollen does not fall
spontaneously and must be removed by visitors for
pollination.
Floral visitors
Visits of two hummingbird species, Chlorostilbon aureo-
ventris (bill length: 16.64 ± 1.17 mm; n = 4) and Helio-
master furcifer (bill length: 27.86 ± 2.12 mm; n = 9),
were recorded. Visitation rate of Chlorostilbon aureoven-
tris was higher than that of Heliomaster furcifer in two of
the three populations studied (Table 1). Chlorostilbon

123
 E. Glinos, A. A. Cocucci

Fig. 4 Canna indica. a–c Detail of the stylar canal and trichomes of
the apical and lateral stigmatic parts. a Cross section of stylar canal
(arrow indicates secretion). The epidermal secretory cells that line the
canal are flat with a reduced lumen and dense cytoplasm. b,
c Longitudinal sections of the style. b Trichomes of apical part.
c Trichomes of lateral part. Trichomes of both areas are unicellular
and have a swollen base. Their cytoplasm is rich in stroma,
organelles, and small to medium vacuoles. d–f Pollen tube growth
of hand-pollinated styles, viewed with fluorescence microscopy after
clearing styles and staining with aniline blue. d View of the apical and
lateral parts of the style. Pollen tubes germinate in both parts but
reach the stylar canal only via the apical part. e Detail of the apical
part. f Detail of the lateral part. lp Lateral part, ap apical part, pt
pollen tubes. Scale bars a, b 40 lm, c 20 lm, d 200 lm, e, f 100 lm

Table 1 Percentage of
Minutes of Chlorostilbon aureoventris Heliomaster furcifer
hummingbird visits (%) and
observation
visitation rate (visits/ Percentage Visitation Percentage Visitation
inflorescence/hour) in three of visits rate (v/inf/h) of visits rate (v/inf/h)
natural populations of Canna
indica Cuesta Blanca 600 99.27 0.024 0.73 0.00018
Costa Azul 840 78.24 0.072 21.76 0.018
Agua de Oro 568 12.8 0.006 87.2 0.036
Totals 2,008 64.44 36.3

aureoventris hummingbirds showed two distinct behaviors
when taking nectar: (1) insertion of the bill into the flower
tube, and (2) nectar uptake through pre-existing perfora-
tions. These perforations were made by the carpenter bee
Xylocopa ordinaria. In neither case did the hummingbirds
of this species make contact with the fertile parts of the
flowers.
Only Heliomaster furcifer was seen carrying pollen of
Canna indica in the three study populations. The photo-
graphs and videos enabled us to evaluate some critical
aspects of the pollination mechanism. Hummingbirds of
this species do not make contact with the stigma and pollen
when entering the flowers or while taking nectar (Fig. 5a).
The simulations made with plastic models and embalmed
hummingbirds showed that their bills get tightly encased in
the floral tube. Thus, a straight backward thrust is required
for the bird to get out of the flower tube. In the videos it is
clearly evident that the hummingbirds strike a backward
wing beat when about to extract their bill from the flower
tube. It is during this backward flight that the hummingbird
touches the style. Pollen is deposited dorsally on the bill
between the apical third and the apical half of the exposed
culmen (Fig. 5b). Though the precise, very brief moment
of pollen deposition on the stigma could not be captured, it
is clear from the position of the style, protruding dorsally
and laterally, that the hummingbirds strike the style with
the bill when rearing out of the flower and touch in suc-
cession first the lateral part of the stigma and then the

123
Pollination biology of Canna indica (Cannaceae)
Fig. 5 Heliomaster furcifer hummingbird visiting a flower of Canna
indica. a The hummingbird with its beak completely inserted in the
flower tube does not make contact with the style while it is taking
nectar. b Detail of the hummingbird bill during inward movement.
The arrow indicates the pollen deposited on the pollinator’s beak
during a previous visit. Pollen is apparently deposited onto the stigma
when, during the rearing back trajectory, the beak grazes the inwardly
turned tip of the style. Style side at the back of the pollen presenter is
shown
secondary pollen presenter. Presumably, self-pollination is
prevented because the stigma is outside the trajectory of the
backward-moving hummingbird when the style is straight.
When the style curves and pollen has been removed by
pollinators, the stigma comes across this trajectory and
pollen brought by the hummingbird from other flowers can
be deposited on it.
Diurnal lepidopterans and bees were also observed vis-
iting the flowers to reach nectar but none of them made
contact with the pollen or the style (Table 2). Undeter-
mined lepidopteran species of three different families
(Hesperiidae, Nymphalidae, and Pieridae) were seen
landing on the base of the tube and inserting the proboscis
into the side of the flower through the space between the
Table 2 Percentage of insect
Minutes of
visits (%) and visitation rate
observation
(visits/inflorescence/hour) in
three natural populations of
Canna indica Cuesta Blanca 240
Costa Azul 30
Agua de Oro 308
petals and the staminodes or through pre-existing perfora-
tions. Carpenter bees always collected nectar from the
outside of the flowers and were seen making perforations at
the base of the flower. Apis mellifera also used the perfo-
rations made by the carpenter bees to take nectar. Two
bumblebee species, Bombus morio and B. opifex, got into
the flowers by climbing onto the labellum. While doing
this, they may make pollen fall down with their hindlegs,
evidently causing pollen loss. However, the stigma, which
is at a higher level, was not touched.
Discussion
Distinction between apical and lateral parts
of the stigma
The asymmetric style of Canna has two morphologically
distinct but histologically similar secretory areas, one apical
and one lateral. Both areas have the same kind of unicellular
secretory trichomes, which exude a sticky substance con-
taining mucilage and possibly other carbohydrates. When
experimentally depositing cross-pollen on both areas, pol-
len grains germinate on both. However, only tubes from
grains deposited on the apical area continue further into the
style and to the ovary. Thus, the apical part is actually
receptive, i.e., the stigma proper, in agreement with the
early suggestion by Schumann (1888) but not with that by
Kränzlin (1912), who considered both the apical and lateral
parts as receptive. The stigma proper is continuous with the
stylar canal, which is open throughout the style length and
internally covered with epidermal secretory columnar cells
as already described for other Zingiberales (Strelitzaceae,
Lowiaceae, Costaceae, Zingiberaceae, and Marantaceae;
Kronestedt and Walles 1986; Pedersen and Johansen 2004;
Winnel et al. 1992; Box and Rudall 2006; Endress 1994).
For one Orchidantha species (Lowiaceae) where secretory
and receptive parts of the style are likewise distinguishable,
Pedersen and Johansen (2004) consider the ability of the
secretory part to sustain pollen tube germination and growth
as an indication of its stigmatic origin. In Canna indica, two
histologically identical parts of the style could similarly
have a stigmatic origin, but the lateral one does not function
as receptive and is specialized in the production of adhesive
secretions. Therefore, the lateral part of the Canna indica
Visitation rate (v/h/inf)
Xylocopa sp. ? Bombus sp. Apis mellifera Lepidoptera
0.102 2.292 0
16.67 0 3.336
0.21 0.282 0.09
123

style could be called viscidium, as Pedersen and Johansen
(2004) proposed for an anatomically equivalent structure of
Orchidantha. It remains to be tested if the presence of a
viscidium is a general feature of Canna. Recently, Ciciarelli
(2007) recognized both the apical and the lateral part as
stigmatic zones in Canna ascendens, but this aspect was not
evaluated in detail.
The presence of accessory sticky substances from
flower tissues other than the tapetum becomes important
in circumstances in which pollen has to adhere to smooth
surfaces and pollenkitt is not sufficient to fulfill the
adhesive function (Vogel 2002; Moyano et al. 2003). The
differentiation of sterile areas of the stigma that produce
accessory pollen adhesives is well known in Apocynaceae
and Orchidaceae (Endress 1994). In Canna the need to
attach a few relatively large pollen grains (68 lm,
according to Zona 2001) on the smooth culmen of a
bird suggests that accessory pollen adhesive could be
advantageous.
Pollinator dependence, visitation rates, and pollination
mechanism
In at least one natural Canna indica population studied
here, plants depend on pollinators for the production of
fruits. Of two recorded hummingbird visitor species in
three populations, pollinators belonged always to one long-
billed species. Though the short-billed hummingbirds may
be locally more frequent visitors than long-billed ones, they
failed to make contact with the fertile parts of the flower
because they access the nectar either from the outside
through holes made by bees or, when accessing from the
inside, move considerably below the stigma and pollen
presenter. The observation of hummingbirds as pollinators
agrees with records from another species of Canna (Stiles
and Freeman 1993). This, however, is apparently the first
study in which the pollinator behavior and visitation rate
are recorded for a species of Canna. Long-billed hum-
mingbirds carry pollen somewhat laterally on the exposed
culmen. Pollen deposition occurs, as far as can be inter-
preted from photograph sequences, very swiftly when the
hummingbirds leave the flowers touching in a sequence the
viscidium and then the pollen clump on the pollen pre-
senter. The bending of the style tip towards the center of
the flower over the rearing trajectory of the hummingbird
apparently promotes cross-pollination. One-sided deposi-
tion of pollen on the beak of the hummingbirds and style
bending from one side to the center of the flower implies
that pollen transfer takes place by utilizing one side of the
hummingbird. This probably explains why nearly all
flowers are left-handed. A right-handed flower would have
no chance of delivering pollen to other flowers or of
receiving pollen from them.
123
E. Glinos, A. A. Cocucci
Pollen adherence to the style, pollen load loss,
and pollen limitation
It has been suggested that the presentation of the entire
pollen load on the gynoecium increases the risk of pre-
mature loss of male function by exposing unprotected
pollen to unfavorable environmental influences, such as
ultraviolet radiation, dehydration, and removal during
nonpollinating visits (Howell et al. 1993). One of the most
common ways of achieving pollen adherence to the pre-
senter is its entrapment within a brush-like structure where
trichomes are involved in the adhesion, presentation, and
staggered pollen release (Howell et al. 1993). In Canna,
pollenkitt appears to be sufficiently effective for pollen
cohesion, for adherence to the pollen presenter (pollen
remains on the style presenter of visitor-excluded flowers
even after rain and wind) and pollen delivery is not
staggered.
Bumblebees are apparently an important factor affecting
reproductive success in Canna indica because they both
steal nectar, reducing the reward available to legitimate
visitors, and favor pollen loss by rubbing the presenter and
not the stigma with their hind legs. We detected pollen
limitation through fruit set but not through seed set. The
former is presumably a result of the low visitation rate of
the pollinating hummingbird and of pollen loss caused by
illegitimate visitors. However, when pollination does take
place, pollination efficiency of one or a few visits is
actually high. If a limited lifespan of pollen exposed out-
side the anther is assumed and considering that the visita-
tion rate of pollinators is very low, the importance of an
effective pollination mechanism that ensures the transfer of
pollen to other flowers is evident.
Comparative functional morphology
Within Zingiberales, the only families in addition to
Cannaceae that have the apical part of the style divided into
stigma and viscidium are Lowiaceae and Marantaceae. The
tissues involved in the secretion of adhesive substances in
Lowiaceae are also unicellular glandular trichomes such as
those observed in Canna (Pedersen and Johansen 2004),
while in Marantaceae they are polygonal cells (Pischtschan
and Classen-Bockhoff 2010). In Orchidantha (Lowiaceae),
the viscidium is located on the ventral part of the trilobate
stigma (Pedersen and Johansen 2004), while in Maranta-
ceae, it is placed on an abaxial ridge between the stigma
and the pollen presenter (Kennedy 1978; Classen-Bockhoff
1991; Locatelli et al. 2004; Classen-Bockhoff and Heller
2008; Pischtschan and Classen-Bockhoff 2008, 2010; Ley
and Classen-Bockhoff 2009).
A comparative analysis of the style structure of the
flowers of Strelitziaceae, Lowiaceae, Cannaceae, and
Pollination biology of Canna indica (Cannaceae)
Fig. 6 Schematic representation of the styles of Strelitzia reginae,
Orchidantha maxillarioides, Canna indica, and Maranta arundinacea
all from the adaxial side. The horizontal line indicates location of the
cross sections shown above. Secretory parts of the stigma (or
viscidium in Orchidantha, Canna, and Maranta) are shaded black and
receptive parts are dark gray
Marantaceae shows transitions in the configuration and
relative position of the secretory and receptive stigma areas
(Fig. 6). In the most simple configuration, as exemplified
by Strelitzia (Kronestedt and Walles 1986), the style bran-
ches into three equally developed stylodia (one dorsal and
two ventrolateral), each having an abaxial trichome patch
anatomically equivalent to the apical and lateral stigmatic
areas of Canna. In Strelitzia, these trichomes also secrete a
viscous hyaline substance similar in appearance to that of
Canna (Cocucci, pers. obs.), but in Strelitzia a zone that
functions exclusively as viscidium is lacking, as the entire
trichome-covered surfaces of the three lobes are receptive
(Kronestedt and Walles 1986). In Lowiaceae, three stylodia
are also evident, of which only the two ventrolateral ones
are secretory, each covered with glandular trichomes along
a subapical strip. The secretory strips of both stylodia
converge and coalesce basally to form a V-shaped area
(Pedersen and Johansen 2004). The receptive apical part of
each stylodium has no trichomes, and a gradual transition
between the receptive tissue and the viscidium is evident. In
Canna, though the style is seemingly monomerous, it
reveals internally a trimerous structure in the three-pointed
star shape of the stylar canal in sections close to the ovary
(Rao and Donde 1955; Pai 1963; Kunze 1984). From a level
close to the base of the style towards the tip, the trimerous
structure becomes gradually less evident. From a compar-
ative point of view, we propose two alternative hypotheses
for the morphological and anatomical homology of the
receptive part in Canna: (1) The portion of the style bearing
the viscidium is homologous to the dorsal stylodium of
Strelitzia and of Orchidantha, while the remaining two
ventrolateral stylodia would be represented by the two flat
projections of the stigma proper. (2) The dorsal stylodium is
completely aborted and the ventrolateral stylodia are united,
forming a single ventral and subapical secretory surface.
The available evidence supports the second hypothesis,
since the ventral position of the viscidium of Canna cor-
responds to that of the secretory area in Lowiaceae. In
addition, in Canna the secretory and receptive portions are
sometimes united resulting in the aforementioned V-shaped
structure. Marantaceae have a single secretory area on a
morphologically ventral and median position (Kennedy
1978; Locatelli et al. 2004; Classen-Bockhoff and Heller
2008; Pischtschan and Classen-Bockhoff 2008, 2010; Ley
and Classen-Bockhoff 2009), which can be homologized
with the viscidium of Canna.
The tendency towards increasing dorsoventrality and
asymmetry in this group of families is associated with
different mechanisms for pollen deposition. In Strelitzia,
pollen is deposited on the feet of a bird when it sits on the
keel of the flower (Endress 1994). In Lowiaceae, where
flowers have a bilabiate architecture, pollen is deposited on
the dorsal part of an insect after it makes contact with the
viscidium. Mucilage of the viscidium is smeared over the
dorsal surface of the pollinating beetles enabling pollen to
stick to their smooth cuticle (Sakai and Inoue 1999; Pe-
dersen and Johansen 2004). In Marantaceae, flower archi-
tecture is functionally papilionate (Delpino 1873), meaning
that the pollination organs are protected by the lower lip
(Westerkamp and Weber 1999). Pollen is deposited ven-
trally on pollinators by an upward twisting of the style,
which moves the viscidium from a concealed ventral to a
dorsal position. In species of Calathea (Marantaceae),
when the style arches upward after stimulation by bees, the
pollinator’s tongue is struck in sequence by the stigma, the
viscidium, and the pollen presenter (Kennedy 1978; Clas-
sen-Bockhoff 1991; Classen-Bockhoff and Heller 2008;
Pischtschan and Classen-Bockhoff 2008, 2010; Ley and
Classen-Bockhoff 2009).
For Canna we can confirm early suggestions (Delpino
1873), that, because of its bilabiate architecture, pollen
should be deposited on the dorsal part of pollinators. It was,
however, unexpected that pollen was deposited on the
hummingbird’s culmen and not near the forehead as sug-
gested by the flower length. This indicates that pollen is
deposited on the hummingbird when it is already at a
distance from the nectar chamber. Although the mechanism
of pollen delivery and retrieval still needs further investi-
gation, our preliminary observations suggest that, in the
brief moment when hummingbirds leave the flower, first
the viscidium is touched and then the pollen presenter, thus
allowing the beak to be smeared with accessory pollen
adhesive before pollen is deposited on the same place.
123

Acknowledgments We thank Dr. Bruce Kirchoff and A. P. Wiemer
for their suggestions on earlier versions of this manuscript, and Prof.
Dr. Stefan Schneckenburger (Darmstadt) for making fresh flowers of
Orchidantha maxillarioides accessible for study. Embalmed hum-
mingbirds were kindly provided by the Zoology Museum of the
National University of Córdoba. We also thank the Biology Doctorate
Program, University of Córdoba. E. Glinos holds a fellowship and A.
A. Cocucci is a researcher in Consejo Nacional de Investigaciones
Cientı́ficas y Técnicas (Argentina). Financial support was provided by
Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (PIP
5174). We are grateful to Joss Heywood for checking English
grammar and style.
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