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Pollination Biology of Jacaranda oxyphylla with an Emphasis
on Staminode Function
ELZA GUIMARA
˜ES1,*, LUIZ CLAUDIO DI STASI2and RI TA D E CA SSIA S I N D R O
ˆNIA
MAIMONI-RODELLA1
1
Departamento de Bota
ˆnica and
2
Departamento de Farmacologia, Instituto de Biocie
ˆncias, Universidade Estadual
Paulista (UNESP), Campus de Botucatu, PO Box 510, SP, 18618-000, Brazil
Received: 18 April 2008 Returned for revision: 30 June 2008 Accepted: 22 July 2008 Published electronically: 2 September 2008
†Background and Aims Bignoniaceae is a Neotropical family with .100 genera, only two of which, Jacaranda and
Digomphia, have a developed staminode. Jacaranda oxyphylla, whose flowers possess a conspicuous glandular sta-
minode, is a zoophilous cerrado species. Here, the composition of the secretion of the glandular trichome and the
influence of the staminode on the pollination biology and reproductive success of J. oxyphylla were studied.
†Methods The floral morphology, pollen viability, stigma receptivity, nectar volume and nectar concentration were
studied. Compatibility system experiments were performed and floral visitors were observed and identified.
Experiments comparing the effect of staminode presence and absence on pollen removal and pollen deposition effi-
ciency were conducted in open-pollinated flowers. Histochemistry, thin-layer chromatography (TLC) and gas chrom-
atography coupled to flame ionization detection (GC– FID) analyses were performed to determine the main chemical
components of the staminode’s glandular trichome secretion.
†Key Results Flower anthesis lasted 2 d and, despite the low frequency of flower visitation, pollination seemed to
be effected mainly by medium-sized Eulaema nigrita and Bombus morio bees, by the small bee Exomalopsis
fulvofasciata and occasionally by hummingbirds. Small bees belonging to the genera Ceratina,Augochlora and
Trigona were frequent visitors, collecting pollen. Jacaranda oxyphylla is predominantly allogamous. Staminode
removal resulted in fewer pollen grains deposited on stigmas but did not affect total pollen removal. The secretion
of capitate glandular trichome occurs continually; the main chemical compounds detected histochemically were
phenolic and terpenoid (essential oils and resins). Monoterpene cineole, pentacyclic triterpenes and steroids were
identified by TLC and GC–FID.
†Conclusions The staminode of J. oxyphyllla is multifunctional and its importance for female reproductive success
was attributed mainly to the secretion produced by capitate glandular trichomes. This secretion is involved in
complex chemical interactions with pollinating bees, including the solitary bees Euglossini. These bees are
common pollinators of various species of Jacaranda.
Key words: Bignoniaceae, Jacaranda oxyphylla, pollination, bee, staminode, glandular trichomes, reproductive success,
terpenes, steroids, phenolics.
INTRODUCTION
The plant family Bignoniaceae is predominantly Neotropical
and plays an important ecological role in the forests of these
regions (Lohmann, 2006), especially due to the zoophilous
nature of its flowers (Gentry, 1974, 1978, 1990). There are
approx. 50 genera of Bignoniaceae in Brazil (Souza and
Lorenzi, 2005), most of them presenting flowers with
rudimentary staminodes. However, in Jacaranda and
Digomphia, the staminode is well developed, conspicuous,
larger than the stamens, and seems to play an important
role in the pollination ecology of species belonging to
these genera (Gentry, 1992; Endress, 1994).
The role of staminodes in pollination has been described
in various families of Angiosperms (Armstrong and Irvine,
1990; Endress, 1994; Walker-Larsen and Harder, 2000;
Decraene and Smets, 2001). Experimental studies aimed
at ascertaining the influence of this structure on components
of reproductive success have indicated its importance,
especially for female reproductive success. In those
studies, lower pollen deposition on the stigma and a lower
seed set were found in flowers where the staminode had
been removed (Dierenger and Cabrera, 2001, 2002;
Walker-Larsen and Harder, 2001).
Many species of Jacaranda grow in the Brazilian cerrado, a
savannah-like vegetation that is predominant in Central Brazil
(Mendonc¸a et al., 1998). All species of Jacaranda are charac-
terized by the abundant glandular trichomes that are present
throughout the staminode (Martius et al., 1897; Gentry and
Morawetz, 1992; Lohmann et al., 2008). These secretory
structures might lead to specialized interactions with anthophi-
lous animals. However, their exact role in the pollination
biology of those species is yet to be determined. In studies
concerning the pollination biology of some species of
Jacaranda, several roles have been attributed to the
staminode. Among those are a secondary pollen presentation
(Yanagizawa and Maimoni-Rodella, 2007), a mechanism for
increased bee contact with the reproductive organs through a
reduction of the space inside the floral tube (Vieira et al.,
1992; Bittencourt and Semir, 2006; Yanagizawa and
Maimoni-Rodella, 2007), visual orientation through contrast
with the corolla (Vieira et al., 1992; Se
´rsic and Rando, 2004),
guidance through scent emission (Vieira et al.,1992;Se
´rsic
* For correpondence. E-mail elzaguimaraes@hotmail.com
#The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Annals of Botany 102: 699–711, 2008
doi:10.1093/aob/mcn152, available online at www.aob.oxfordjournals.org
and Rando, 2004; Bittencourt and Semir, 2006) and a physical
barrier against pollen robbers (Se
´rsic and Rando, 2004).
Despite the ecological importance of this structure in
species of Jacaranda, experimental studies testing how the
staminode might influence components of reproductive
success are yet to be carried out. The sole study that addressed
this question to any extent in Jacaranda was conducted with
Jacaranda mimosifolia (Se
´rsic and Rando, 2004).
In the present study, the floral biology of Jacaranda
oxyphylla Cham. was investigated and the role of the stami-
node and exudates of its glandular trichomes in the inter-
actions with floral visitors was examined. In addition, the
effect of staminode removal on the reproductive success
of J. oxyphylla was evaluated.
MATERIALS AND METHODS
Study site and study organism
Fieldwork was conducted from 2004 to 2006 in two frag-
mented areas of cerrado located in Prata
ˆnia (2284805200S,
4884403500W) and Botucatu (2285703800 S, 4883102200W), in
the state of Sa
˜o Paulo, South-eastern Brazil.
Jacaranda oxyphylla Cham. is widely distributed in
cerrado areas of South-eastern Brazil. This species is very
common in open ‘campo limpo’ grasslands and the
shrubby edges of cerrada
˜o and cerrado stricto sensu
(Gentry and Morawetz, 1992). Jacaranda oxyphylla is a
xylopodial shrub or sub-shrub of approx. 0.5–2.5 m tall,
with bipinnate leaves, terminal and axilar inflorescences,
tubular–campanulate flowers above a narrow basal tube,
didynamous stamens with dithecate anther and presenting a
long sub-exerted staminode, a flattened –ovate ovary slightly
contracted at the base to a cylindrical – pulvinate disk, an
elliptic fruit, thinly woody, and small-bodied seeds with
hyaline–membranaceous wings (Gentry and Morawetz,
1992). The cylindrical– pulvinate disk was quoted as nectar-
iferous tissue by Yanagizawa and Maimoni-Rodella (2007).
Vouchers of the studied materials were collected and
deposited in the ‘Irina Delanova Gemtchujnicov’
Herbarium (BOTU) of the Biosciences Institute of the
Universidade Estadual Paulista, Botucatu, SP, Brazil.
These materials are registered under numbers 24408 –24412.
Staminode morphology and composition of the secretion
of staminode glandular trichomes
Staminode samples were fixed with 2.5 % glutaraldehyde
in 0.1Mphosphate buffer, pH 7.3, for 6– 12 h at 4 8C. In
addition, samples were post-fixed with Karnovsky solution
(Karnovsky, 1965), dehydrated in a graded series of ethanol
solutions and embedded in historesin (Gerrits, 1991).
Sections of 8 mm were stained with 0.05 % toluidine blue
(O’Brien et al., 1964). The slides were sealed with
Entellan resin and examined under an Olympus BX 41
light microscope (Japan) equipped with an Olympus
C7070 digital camera (Olympus, Japan).
For morphological analyses by scanning electron
microscopy (SEM), staminode samples from five newly
opened flowers were fixed for 24 h in 2.5 % glutaraldehyde
with 0.1Mphosphate buffer, pH 7.3. In addition, staminode
samples were post-fixed with 1 % osmium tetroxide for 2 h,
dehydrated in a graded alcohol series, critical point dried,
coated with gold and examined under a Fei-Quanta 200
scanning electron microscope (Phillips, Czechoslovakia).
Fresh hand-cut sections were subjected to eight different
histochemical tests: (a) periodic acid– Schiff (PAS) reaction
to detect water-insoluble polysaccharides (Jensen, 1962); (b)
0.02 % ruthenium red aqueous solution to detect mucilage/
pectin (Johansen, 1940); (c) Sudan IV to detect total lipids
(Johansen, 1940); (d)naphtholþdimethyl-paraphenylene-
diamine (NADI) reagent to detect terpenes (David and
Carde, 1964); (e) 10 % ferric trichloride aqueous solution to
label phenolic compounds (Johansen, 1940); ( f) mercuric
bromophenol blue to detect total proteins (Mazia et al., 1953);
(g) Dragendorff reagent to detect alkaloids (Svendsen and
Verpoorte, 1983); and (h) Fehling’s solution to detect reducing
sugars (Purvis et al., 1964). Standard control procedures were
carried out simultaneously, following the indicated protocols.
Temporary slides were mounted in glycerine and analysed
under an Olympus BX 41 light microscope (Japan) equipped
with an Olympus C7070 digital camera (Olympus, Japan).
Complementary analyses were carried out with an Olympus
SZ 61 (Japan) stereoscopic microscope, also equipped with
an Olympus C7070 digital camera (Olympus, Japan).
Thin-layer chromatography (TLC) was used in order to
investigate the presence of terpenes in staminodes. For
those analyses, samples from staminodes were taken from
102 fresh flowers and immersed in chloroform for 30 min,
following Siebert (2004).
Using a glass capillary tube, samples were spotted onto
Silica gel 60 F
254
(Merck) TLC plates, using toluene–ethyl
acetate (93 : 7) as eluant. Terpenes were visualized by spray-
ing the plates with AS (anisaldehyde– sulfuric acid) reagent,
heating the plates at 100 8C for 10 min, and then evaluating
the terpenes in visible light (Wagner and Bladt, 1996).
For the gas chromatography (GC) analysis, chloroform
extracts of J. oxyphylla staminodes that had been ultrasonicated
at room temperature for 20 min were used. The chromato-
graph used was a VARIAN CP-3380 coupled to an ADCB
(1 V) flame ionization detector (FID), and equipped with an
LM-5 capillary tube (phenyl 95 % methylpolysiloxane with
a length of 15 m, internal diameter of 0.33 m and film thick-
ness of 0.5 mm). Results were recorded on a computer
equipped with VARIAN GW-V509NO Workstation software.
Operating conditions were as follows, injector ¼250 8C;
detector ¼290 8C; heating ramp-up ¼150– 280 8C(rateof
10 8C/min) and 28 8C for 18 min, total time of 31 min; gas
flow ¼air at 480 mL min
21
,N
2
at 43 mL min
21
and H
2
at
2mLmin
21
; and gas ratios ¼N
2
/H
2
/air 1.9:1
.0 : 20.
Authentic samples of thymol, terpineol, progesterol, tingen-
one, a-tocopherol, stigmasterine, campesterol, stigmasterol,
a-espinasterol, b-sitosterol, a-amyrin, a-amyrin acetate,
b-amyrin acetate, lupeol, lupeol acetate, friedelanol and
friedelin were injected under identical GC conditions.
Pollination ecology of Jacaranda oxyphylla
Flowers were monitored to check for visitors at different
times of the day, from early in the morning at 0500 h to
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla700
2330 h at night, a total of 180 h of field observations distrib-
uted over 32 non-consecutive days. Each day, plants were
monitored for 2–8 consecutive hours. Visitors were cap-
tured to examine pollen deposition on their body, for mor-
phometric analyses and for identification. Flowers from
approx. 40 plants distributed between the two populations
were observed. Observations included the time of anthesis,
colouring and dimensions of the floral elements, production
of aroma, presence of nectar and floral longevity. In
addition, stigmatic receptivity was estimated with hydrogen
peroxide (H
2
O
2
) according to Dafni et al. (2005). Pollen
viability was estimated using acetic carmine as vital stain
(Radford et al., 1974). The pollen/ovule (P/O) ratio was
estimated according to Cruden (1977) (n¼6 plants, 18
flowers). Intact flowers and individual floral parts (e.g. sta-
minode, the white spot of the corolla roof) were subjected
to organoleptic studies through which odour concentration
was monitored in clean glass vials (Dafni et al., 2005).
The presence and location of osmophores were investigated
using neutral red solution (Vogel, 1990). Nectar volume
and concentration were measured from bagged flowers
(n¼153 flowers, 50 individuals) using a glass capillary
and a Carl Zeiss (0 –30 %) pocket-size refractometer
(Jena, Germany). The presence of nectar in pre-anthesis
flowers was also investigated, and the quantity of sugar
per mL of nectar was calculated. These data were used to
estimate the average energetic value of the nectar produced
by each flower during its life span, using an exponential
regression as suggested by Galetto and Bernardello (2005).
Pre-anthesis flowers were bagged to exclude all visitors.
Subsequently, flowers in first-day anthesis were self-
pollinated (pollen from the same flower) or cross-pollinated
(using pollen mix from individuals separated by at least
20 m) and rebagged. Bagged flowers were tagged and left
intact (i.e. were not pollinated) to check for spontaneous
self-pollination. In addition, bagged and emasculated
flowers were used to test for autonomous agamospermy.
Natural fruit set was monitored by tagging unbagged
flowers. Thirty individuals were used in the pollination
treatments, and fruit set was recorded after 4 and 8 weeks.
Staminode removal experiments and reproductive success
Staminode removal experiments were conducted to test
the effect of the staminode on female and male reproductive
success. Two neighbouring flower buds were bagged in
each plant (n¼32 plants) to avoid microhabitat variation,
and the bags were removed on the morning of anthesis.
Staminodes from half of the flowers were removed; the
other half served as controls (n¼32 flower per treatment,
1 flower per plant per treatment). A control excluding visi-
tors (bagged flowers) was not performed since pollen is
released only when anthers are touched and squeezed by
visitors. The experiment involved a comparison of pollen
deposition and pollen removal between flowers with and
without staminodes that had been exposed to open pollina-
tion during their life span. After 48 h of open-pollination,
anthers and stigmas were carefully removed and fixed in
acetic carmine solution. Pollen grains deposited on stig-
matic surfaces were counted using an Olympus SZ 61
stereomicroscope (Japan). All pollen grains remaining per
flower were extracted by shaking the anthers in 200 mLof
acetic carmine solution. Samples (20 mL) were mounted
on slides and then pollen grains were counted using an
Olympus BX 41 microscope (Japan). Data from these
experiments were tested for normal distribution
(Kolmogorov– Smirnov) and compared using a two-tailed
t-test and Mann–Whitney Utest, using GraphPad Instat
v.3.01 software (San Diego, CA, USA).
RESULTS
Staminode morphology and composition of the secretion
of staminode glandular trichomes
The staminode is composed of a cylindrical filament (2.8–
4.3 mm long) with a broader, slightly bifid tip, and a thin
base, attached to the bottom of the corolla tube. It
emerges obliquely and its distal end rests upon the entrance
of the corolla tube (Fig. 1B). The filament of the staminode
is densely covered with capitate glandular trichomes over
its entire length except the basal 10 mm (Fig. 1C). Its
abaxial portion shows only capitate glandular trichomes
(Fig. 1G), while the adaxial apical portion contains numer-
ous hyaline, simple, uniseriate, uni- to pluricellular long tri-
chomes (Fig. 1H). The capitate glandular trichomes are
constituted by an approximately spherical multicellular
head, and a stalk varying in length, number of cells and
degree of ramification. These trichomes can be divided
into three basic types according to their size. The short tri-
chomes (Fig. 2A) are distributed over the entire abaxial
surface of the staminode (Fig. 1C, G). The intermediary tri-
chomes (Fig. 2C) may present ramifications, and are con-
centrated at the sides of the median portion of the
staminode, forming a kind of channel situated 10– 35 mm
above the base (Fig. 1C). The third long-stalked ramificated
trichomes (Fig. 2D) are predominantly located on the top of
the abaxial apical portion of the staminode, forming a small
tuft together with the simple trichomes (Fig. 1C, G).
Trichome heads present 17– 24 cells (n¼10) arranged con-
centrically (Fig. 2B) around a central cell (Fig. 2A, B).
Occasionally, trichomes with two concentric layers of
cells forming the glandular head are also present. In SEM
analyses, capitate glandular heads present a marked
surface, indicating the close attachment of the cuticle to
the secretory upper cell walls and making the cell outlines
evident (Fig. 2E). Alternatively, a small sub-cuticular space
is formed by the detachment of the cuticle (Fig. 2F).
In pre-anthesis, droplets were found in the style and
corolla. These droplets were seen in the median glandular
portion of the staminode, indicating secretory activity of
this structure prior to flower opening. Under the stereomi-
croscope, large droplets were observed on the head
surface of the capitate glandular trichomes of newly
opened flowers.
During the flower’s functional period, capitate glandular
trichomes were found with different degrees of cuticle disten-
sion. Some of these trichomes did not present the formation of
a sub-cuticular space (Fig. 2E), while others presented a
slightly distended cuticle forming a small sub-cuticular
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla 701
space filled with hyaline secretion (Fig. 2C, F), and others
presented a wrinkled cuticle indicating previous release of
the secretion (Fig. 2G). This variation suggested that the
release of secretion from the capitate glandular trichomes of
the staminode of J. oxyphylla is continuous.
The secretion of the capitate glandular trichomes of the
staminode is composed of predominantly lipophilic
material. This material stained positively with Sudan IV
and differentially with NADI reagent, indicating the pre-
sence of terpenes and resinic acids. The intensity and
colours of the positive reaction to NADI reagent varied
among trichomes of the same morphology located side by
side. A strongly positive reaction occurred with phenolic
compounds. Treatments with ruthenium red for mucilage/
pectin and with Dragendorff solution for alkaloids proved
negative. The assays for detecting proteins, sugars, neutral
polysaccharides and starch showed weakly positive reac-
tions (Table 1) and were highly variable among neighbour-
ing trichomes.
TLC revealed the presence of several terpenoid com-
pounds in the chloroform extract of the staminode of
J. oxyphylla. With AS reagent, terpenes stained pinkish-
purple zones; one of them, of R
f
0.33, was identified as
cineole. This compound was confirmed by comparing its
retention time with that of an authentic sample. The other
terpenoid compounds could only be identified using
FIG. 1. Flower and staminode of Jacaranda oxyphylla. (A) Inflorescences showing flowers with the median region of the corolla curved and compressed
dorsoventrally. Scale bar ¼50 mm. (B) Close-up view of a flower showing the staminode (st) and white portion of the corolla roof. Scale bar ¼10 mm.
(C) Close-up view of a staminode. Scale bar ¼5 mm. (D– F) Close-up of a staminode showing its capitate glandular trichomes in variable colours. Scale
bars ¼500 mm. (G) Abaxial portion of the staminode with capitate glandular trichomes, (H) adaxial portion of the staminode with simple trichomes.
Scale bars ¼1 mm.
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla702
GC–FID. The gas chromatogram of J. oxyphylla stami-
nodes presented peaks that were characteristic of penta-
cyclic triterpene and steroid compounds (Table 2).
The identity of these compounds was confirmed by compar-
ing their relative retention time with those of authentic
samples.
Pollination ecology of Jacaranda oxyphylla
Flowering of J. oxyphylla occurred between August and
October. Flowers opened predominantly at around 0700 h,
and anthesis lasted about 2 d. Each inflorescence
(Fig. 1A) presented one to two flowers in anthesis per
day, and each plant had between one and 16 inflorescences,
FIG. 2 . Morphology of the capitate glandular trichomes from the staminode of Jacaranda oxyphylla. (A, B) Light microscopy. (A) Section through a
short-stalked capitate glandular trichome showing one basal cell, one stalk cell and a large head formed by secretory cells disposed in a single layer around
a central cell. Scale bar ¼30 mm. (B) Cross-section of a trichome head. Scale bar ¼20 mm. (C) Stereomicroscopic view of the capitate glandular tri-
chome showing the small sub-cuticular space filled with hyaline secretion on the head cell. Scale bar ¼40 mm. (D–G) SEM. Scale bar ¼30 mm. (D)
Long-stalked ramified capitate glandular trichome. (E, F) Successive stages of sub-cuticular space development. (E) Glandular head with the cuticle
attached to the secretory upper cell walls. (F) Detachment of the cuticle forming a small sub-cuticular space. (G) Wrinkled cuticle indicating the post-
secretory stage on the right.
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla 703
presenting 7.5+5.52 first-day flowers per day (mean +
s.d.). Three- to 6-d-old flowers remained in the inflores-
cences, even though these were no longer receptive,
perhaps acting as a visual attractor for visitors. After the
third day, the corolla presented faded colouring, and a dar-
kened anther and staminode, with the stigma being the last
structure to display signs of senescence.
The floral tube was 32 – 50 mm longer (n¼30) and pre-
sented a basal constriction (6 –8 mm, n¼30) correspond-
ing to the nectar chamber. The median region of the
tubular–campanulate corolla was slightly curved and
dorsoventrally compressed (Fig. 1A). Anthers and stigma
were included, and were arranged from 15 to 30 mm
above the basal constriction, remaining juxtaposed to the
corolla tube roof throughout the flower’s life span. In this
region, the staminode was aligned longitudinally, with the
reproductive structures forming a kind of lever that leaves
an open space of only 2 mm. The densely glandular stami-
node (Fig. 1C) was recovered by capitate glandular
trichomes of variable colour. The trichomes may present
purple stalks and crimson heads (Fig. 1D), colourless
stalks and yellow heads (Fig. 1E) or purple stalks and
yellow heads (Fig. 1F). Flowers with staminodes carrying
trichomes of different colours presented very different
visual patterns.
The median and upper portions of the corolla tube roof
present a large white spot (Fig. 1B) where the osmophores
are inserted. Osmophores were revealed by intense reaction
in the neutral red assay. A faint, mildly sweetish aroma was
detected in the corolla white spot and staminode. Trichomes
of the staminode presented a weak positive reaction to
neutral red that was restricted to the secretory head cells.
On the other hand, the corolla white spot presented a
strong positive reaction to neutral red. This indicates that
both structures may act as osmophores.
Anther dehiscence and stigmatic lobe opening occurred
on the day prior to the onset of anthesis. At this stage, a
longitudinal dehiscence line was formed in the anthers,
but no separation of the edges was encountered. The
thecal valves did not open completely and the pollen
grains remained clustered inside the anthers. Pollen grains
were only released when they were lightly squeezed
against the corolla tube roof by visitors. At the start of
anthesis, the corolla lobes were completely distended and
an elongated platform was formed by the lower lobe,
upon which the bright tip of the staminode contrasted
(Fig. 1B). The stigma was sensitive, and closed in a
matter of seconds when touched. When pollen deposition
did not occur, the stigma remained closed for about
30 min, after which it gradually opened again, completing
the process after 2 h.
Pollen grains were white, appeared in clusters and were
covered by lipidic substances as revealed by Sudan IV assay.
The average viability of the pollen grains was 98.65 % and
the P/O ratio was 154.64 +41.38 (mean +s.d.).
There was no nectar production before anthesis. The
accumulated nectar volume in first-day flowers was
3.45 +2.03 mL (mean +s.d.) and in second-day flowers
was 6.62 +3.22 mL (mean +s.d.), suggesting that nectar
production follows a continuous pattern. Nectar concen-
tration varied from 22 to 28 %. Not all flowers of a plant
produced nectar, and 43 % of the flowers analysed
showed no production at any time during anthesis. The esti-
mated energetic value of accumulated nectar varied from
3.32 to 8.28 calories per flower.
The populations studied here were preferentially xenoga-
mous, with the formation of fruits occurring in only 3.85 %
of the selfed flowers. Natural fruit set was low (7.25 %) and
no case of spontaneous self-pollination or autonomous aga-
mospermy was found, indicating the need for a pollen trans-
fer vector. The hand-cross pollination test produced greater
fruit set (Table 3).
Small bees of the genera Ceratina,Trigona,Augochlora
and Exomalopsis were observed visiting flowers of
J. oxyphylla (Table 4). Among these small bees, only
Exomalopsis fulvofasciata presented a legitimate visiting
behaviour. This bee entered deep into the floral tube,
passing over the staminode, seeking the nectar chamber
and gathering nectar by extending its 4 mm long proboscis.
Whenever it left the flower, its head and the dorsal portion
of its thorax were covered with pollen. The narrowing of
TABLE 1. Histochemistry of mature capitate trichomes from
the staminode of Jacaranda oxyphylla
Staining procedure Target compounds
Colour
observed
Reactivity of
head cells*
Sudan IV Total lipids Red þ
NADI Terpenes Blue and
dark red
þþ
Ruthenium red Pectin/mucilage –
Ferric trichloride Phenolic
compounds
Green to
black
þþ
Schiff (PAS) Neutral
polysaccharides
Pink þ
Dragendorff Alkaloids –
Mercuric
bromophenol blue
Proteins Dark blue þ
Lugol Starch grains Purple þ
Fehling’s solution Sugars Reddish þ
*– negative, þslightly positive, þþ strongly positive.
TABLE 2. Terpenoid composition of chloroform extract from
the staminode of Jacaranda oxyphylla by GC– FID
Peak Retention time Compounds Phytochemical classes
112
.7 Campesterol Steroids
212
.9 Stigmasterol Steroids
316
.4b-Sitosterol Steroids
416
.8a-Amyrin Pentacyclic triterpenes
ursane type
517
.7b-Amyrin Pentacyclic triterpenes
oleanane-type
618
.8 Lupeol Pentacyclic triterpenes
lupane type
720
.5 Lupeol acetate Pentacyclic triterpenes
lupane type
820
.9 Friedelanol Pentacyclic triterpenes
friedelane type
921
.5 Friedelin Pentacyclic triterpenes
friedelane type
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla704
the tube diameter caused by dorsoventral compression and
the arrangement of the staminode in relation to the reproduc-
tive structures favoured the contact of E. fulvofasciata with
those structures. Despite its small size, this bee is quite
heavy and can lower the staminode upon entering the floral
tube and treading on it. Ceratina,Augochlora and Trigona,
on the other hand, normally entered the staminode sideways
and did not present legitimate visiting behaviour. These bees
visited flowers from sunrise to dusk. At first, they investi-
gated each flower visually; if there was no other visitor,
they entered the floral tube laterally (Fig. 3A). Upon reaching
the anthers, bees turned their ventral side toward the corolla
tube roof and collected pollen actively with their first pair of
legs, while simultaneously cleaning their bodies and trans-
ferring pollen to the subsequent pair of legs (Fig. 3B). This
process lasted from 1 to 3 min. During these movements
various parts of the bee’s body, covered with large quantities
of pollen from this flower, established contact with the
stigmatic surface, favouring a potential self-pollination.
However, stigma closure was never observed during this
contact. After pollen collection, the bees turned their
abdomen downwards and actively scraped the glandular
portion of the staminode. Then, the bees flew to other
flowers of the same plant or to neighbouring plants. These
bees were never observed gathering nectar from flowers of
J. oxyphylla.
Besides E. fulvofasciata, medium-sized bees also visited
J. oxyphylla flowers legitimately (Table 4). Eulaema
nigrita (Euglossini) was observed on two occasions, visiting
about ten flowers of neighbouring plants (Fig. 3E). After
approaching an inflorescence, the bee landed on the anterior
lobe of the corolla and then walked along the corolla tube
floor, passing over the staminode, towards the nectar
chamber. The bee touched the stigma and the anthers with
its back. After 10–20 s, the female bee reversed out of the
corolla tube, hovering for a few seconds in front of the
visited flower, apparently cleaning its body. The anthers
were arranged 15– 30 mm above the nectar chamber, and
the extended proboscis of E. nigrita was about 25 mm
long. In order to reach the nectar, individuals of E. nigrita
need to touch the reproductive structures with their head
and dorsal portion of their thorax, while the ventral portion
touches the entire median glandular region of the staminode.
Visits by a species of bumble-bee, Bombus morio, were
also observed on two occasions (Fig. 3D). Before entering
the flowers, the bee hovered for a few seconds in front of a
flower and then landed on the anterior lobe of the corolla,
walking on the tube floor towards the flower base. When
B. morio left the flower, it displayed a large quantity of
white pollen deposited on its head and the dorsal region
of its thorax. Individuals of B. morio were subsequently
observed visiting another flower on the same plant or
flowers on some neighbouring plants.
Both medium-sized bees, E. nigrita and B. morio, were
legitimate pollinators carrying pollen and touching the recep-
tive surface of stigmatic lobes upon entering flowers of
J. oxyphylla. Whenever these species presented pollen depos-
ited on their dorsal region, only the sterile portions of the stig-
matic lobes were subsequently touched, when leaving the
flower, restricting the possibility of self-pollination.
Visits by an unidentified species of hummingbird
(Trochilidae) were also observed. These hummingbirds
visited 5 – 15 receptive flowers per flight, between 0800 h
and 0900 h. During flower visits, the hummingbirds thrust
their heads into the floral tube for 1 or 2 s, usually remain-
ing in hovering flight. In some instances, the hummingbirds
also rested their feet briefly on the inflorescence. Given that
the hummingbird’s size (beak length approx. 20 mm) fit the
reproductive structures and nectar chamber of J. oxyphylla
very well, this species may be considered a legitimate
pollinator. However, corolla abscission frequently occurred
TABLE 4. Visitors to flowers of Jacaranda oxyphylla
in patches of cerrado vegetation, in Botucatu and Prata
ˆnia,
SP, Brazil
Species
Visiting
behaviour
Foraged
resource Frequency*
ANDRENIDAE
Oxaea flavescens Non-legitimate Nectar High
APIDAE
Bombus
(Fervidobombus)morio
Legitimate Nectar Low
Ceratina (Crewella)
maculifrons
Non-legitimate Pollen High
Ceratina (Crewella)
gossypii
Non-legitimate Pollen High
Ceratina (Crewella)
asuncionis
Non-legitimate Pollen High
Eulaema (Apeulaema)
nigrita
Legitimate Nectar Intermediate
Exomalopsis
fulvofasciata
Legitimate Nectar and
pollen
Low
Trigona spinipes Non-legitimate Pollen High
Xylocopa sp. Non-legitimate Nectar Low
HALICTIDAE
Augochlora
(Augochlora) sp.
Non-legitimate Pollen High
TROCHILIDAE Legitimate Nectar Intermediate
*Frequency: high (30 visits d
21
), intermediate (1– 5 visits d
21
), low
(,1 visit d
21
).
TABLE 3. Experimental pollination and fruit set of Jacaranda oxyphylla, in a cerrado patch, Botucatu, SP, Brazil
(n¼30 plants)
n
Spontaneous
self-pollination
Autonomous
agamospermy
Hand
self-pollination
Hand
cross-pollination
Natural pollination
(control)
Flowers 478 182 39 78 41 138
Fruits 32 0 0 3 (3.85 %) 19 (46.34 %) 10 (7.25 %)
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla 705
a few seconds after the hummingbird left the flower, leaving
behind only the calyx and the gynoecium and precluding
further visits.
Lastly, Oxaea flavescens and Xylocopa sp. were observed
stealing nectar (Table 4). Both presented similar behaviour,
landing externally on the floral tube, introducing their
mouth apparatus between the calyx and the corolla and
actively stealing nectar through longitudinal slits produced
at the base of the corolla tube (Fig. 3C).
Staminode removal experiments and reproductive success
Even though flowers without a staminode differed visu-
ally from intact flowers, no significant differences were
found between treatments considering the presence and
absence of staminode. No significant difference was
found for pollen deposition on stigmas (n¼32) (Mann–
Whitney Utest, P¼0.89) and for pollen removal from
anthers (n¼32) (t-test, P¼0.49) when comparing
flowers with and without a staminode. Flowers with stami-
nodes received pollen deposition on stigmas varying from
0 to 144 pollen grains (Fig. 4). On the other hand,
flowers without staminodes only presented deposition of
small quantities of pollen in all samples evaluated, with 0
to 15 pollen grains per stigma (Fig. 4). Small Halictidae
and Anthophoridae bees were observed visiting flowers
with and without a staminode, displaying the same beha-
viour (Fig. 3A, B) in both treatments. Although flowers
whose staminodes had been removed were visited more fre-
quently (15 : 1) by these bees, the total pollen grain removal
was similar in flowers with and without staminodes (Fig. 5).
The percentage of flowers with staminodes that received
a large amount of pollen grains on the stigma (6.25 %) was
close to the natural fructification, i.e. 7.25%. This depo-
sition (.140 pollen grains per stigma, Fig. 4) was compa-
tible with the number of ovules per flower (113.28 +21.14,
FIG. 3. Interactions between flower and bee in Jacaranda oxyphylla. (A) Ceratina sp. entering the floral tube. (B) Ceratina sp. collecting pollen; note the
white cluster of pollen on the bee’s legs; the pollen was collected and stored exclusively from this opening flower. (C) Oxaea flavescens stealing nectar.
(D) Bombus morio visiting a flower. (E) Eulaema nigrita visiting a flower. Scale bars ¼10 mm.
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla706
mean +s.e.). On the other hand, the number of pollen
grains (,20 pollen grains, Fig. 4) deposited on stigmas
of flowers without staminodes was much lower than the
mean number of ovules per flower.
DISCUSSION
Staminode morphology and composition of the secretion
of staminode glandular trichomes
In some species the secretion produced by glandular tri-
chomes remains accumulated inside the sub-cuticular
space, and is exclusively released through mechanical
contact. This contact is usually provided by herbivores
that break the cuticle and cause the release of substances
that are normally associated with chemical defence
(Ascensa
˜oet al., 1995, 1997; Sacchetti et al., 1999;
Pichersky and Gershenzon, 2002; Machado et al., 2006).
However, in the staminode of J. oxyphylla it was found
that the secretion was already available at flower opening
and could be scattered in the air, collected and easily trans-
ferred to the body of the pollinator during a visit. Se
´rsic and
Rando (2004) detected the presence of secretion com-
ponents produced by the glandular trichomes of the stami-
node of J. mimosifolia in the body of B. morio, indicating
transfer of secretion components during visits.
Chemical analyses of the secretion produced by capitate
glandular trichomes of the staminode of J. oxyphylla
allowed for a better understanding of the role of this struc-
ture in the interactions with floral visitors. Phenolic and ter-
penic compounds constitute the secondary compounds most
widely distributed among plants (Harborne, 1997). The
phenolic compounds comprise approx. 8000 molecules,
including flavonoids and tannins that contribute to the
flavour, odour and colour of a variety of plants (Harborne,
1985, 1997; Nishida, 2002). Phenolic compounds in the
capitate trichomes of the staminode of J. oxyphylla can
be of different types and may play different roles. For
example, trichomes may play a role in protection against
ultraviolet radiation (Harborne, 1997), an important aspect
in open vegetation formations such as the cerrado.
On the other hand, terpenoids constitute a wide and
complex class of secondary compounds that play essential
roles in plant–animal and plant– plant interactions, acting
as feeding deterrents, pheromones, defence agents and alle-
lochemicals (Harborne, 1997). The presence of monoter-
penes and sesquiterpenes in flowers has been related to
the attraction of pollinators, especially bees, moths and but-
terflies, that may, in some cases, be species specific
(Harborne, 1997; Pichersky and Gershenzon, 2002). The
monoterpene cineole, identified in the capitate glandular tri-
chomes of the staminode of J. oxyphylla, comprises highly
volatile fragrances associated with the localization of the
flowers of these plants by ‘trapliners’ and opportunistic pol-
linators, and are commonly found in Orchidaceae (Cancine
and Damon, 2007).
Triterpenes make up a more diversified sub-group of ter-
penes and can perform ecological defence-related functions
50
45
40
35
30
25
20
Flowers (%)
15
10
5
00 1–10 11–20 21–30 131–140 141–150
Flowers with staminode
Flowers without staminode
Number of pollen grains
FIG. 4 . Number of pollen grains deposited on the stigmatic surface of
flowers of Jacaranda oxyphylla, with and without a staminode.
40
35
30
25
20
Flowers (%)
15
10
5
0
Pollen grains (%)
0–10 11–20 21–30 31–40 41–50 51–60 61–70 71–80 81–90 91–100
Flowers with staminode
Flowers without staminode
FIG. 5 . Percentage of pollen grains removed from anthers of flowers of Jacaranda oxyphylla, with and without a staminode.
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla 707
(Harborne, 1997; Cheng et al., 2007). In this study, pentacyc-
lic triterpenes and steroidal triterpenes produced by the sta-
minode of J. oxyphylla may also perform these functions.
Pentacyclic triterpenes can be found in combinations con-
taining variable quantities of terpenes mixed with other
classes of substances (Wagner and Bladt, 1996), such as
the oil–resin mixture identified in the capitate glandular tri-
chomes of the J. oxyphylla staminode. Although these com-
pounds are widely distributed throughout higher plants, their
presence in flowers has been described for only a few species.
In particular, they have been found in Dalechampia
(Armbuster, 1984) and Tipuana tipu (Pereira and Aquino
Neto, 2003). Floral resins, normally composed of triterpenes,
are collected by various genera of Neotropical Euglossini
bees (Armbuster and Webster, 1979).
According to Roubik and Hanson (2004), both male and
female Euglossini bees depend greatly on non-food materials
in their environment. Males need to collect chemicals and
females must find nesting material. Unlike bumble-bees,
stingless bees and honey bees, Euglossini bees use no wax,
depending heavily on resins for nest construction. The
resin from flowers remains soft and pliable for a long time,
unlike the resin or resinous sap that exudes from plant
wounds (Roubik and Hanson, 2004). In addition to nesting
material, some triterpenes, the primary compound of plant
resins, provide antibiotics. Oliveira et al. (1996) tested the
effectiveness of resin from Clusia grandiflora flowers and
found that those resins are highly effective against
Gram-positive bacteria, a deadly microbial enemy of bees.
On the other hand, steroidal triterpenes play an important
role in plant–insect interactions, since many phytophagous
and omnivorous insects are unable to biosynthesize the
steroid nucleus (Svoboda and Feldlaufer, 1991). Steroids
such as sitosterol, campesterol and stigmasterol are essential
in structural and hormonal functions (Roitberg and Isman,
1992). Rasmont et al. (2005) found the presence of
b-sitosterol in the pollen of a legume pollinated by
Bombus terrestris. The authors state that this compound
has a feeding deterrent effect on Apis mellifera, which fed
on the nectar but not on the pollen of this species. This
may be one of the reasons why no visits by A. mellifera
to the flowers of J. oxyphylla were recorded.
Females of E. nigrita act as legitimate pollinators of
J. oxyphylla. Even though male visits were also observed,
it is unclear whether they also act as legitimate pollinators.
It should be noted that males of this species collect volatile
substances (fragrances) from floral and non-floral sources
and store those substances in cavities located in the pos-
terior tibia, where they accumulate complex, species-
specific blends of fragrances (Schiestl and Roubik, 2003;
Eltz et al., 2006). Eltz et al. (2003) analysed the content
of the tibia of Euglossini males and detected mixtures of
terpenoids and aromatic compounds totalling 70 substances,
including cineole. Euglossini males feed on nectar of plant
species that are not necessarily producers of fragrances;
hence, cineole may act in the attraction of E. nigrita
males to flowers of J. oxyphylla, which offers nectar as a
reward, in addition to the secretions of the staminode.
In other species of this genus, J. caroba and J. decurrens,
males of Euglossa were observed collecting fragrance from
the staminode (Gottsberger and Silberbauer-Gottsberger,
2006). The authors reported that males, when exiting
flowers, hover in front of those flowers and make typical
movements with their legs that are associated with the trans-
fer of liquid odour ( fragrance) to the tibial capsule, similar
to the behaviour performed by a male of E. nigrita in
flowers of J. oxyphylla. Visits from Euglossini males
were also described in other Bignoniaceae (Roubik and
Hanson, 2004; Silva et al., 2007).
It is also noteworthy that Euglossini bees are the main pol-
linators of most species of Jacaranda studied so far, includ-
ing J. caroba,J. copaia,J. ulei,J. simplicifolia,J. rufa,
J. racemosa,J. paucifoliolata,J. rugosa and J. decurrens
(Vieira et al., 1992; Stevens, 1994; Maue
´set al., 2004;
Bittencourt and Semir, 2006; Sampaio et al., 2007;
Yanagizawa and Maimoni-Rodella, 2007). According to
Gottsberger and Silberbauer-Gottsberger (2006), small
bees feed on pollen, while male Euglossini bees collect
fragrances from the staminode of Jacaranda. Additionally,
females of Euglossini and other species of large bees feed
mainly on nectar in Jacaranda, leading to what
Gottsberger and Silberbauer-Gottsberger (2006) call a super-
imposed pollination system. The behaviour of the females of
E. nigrita, hovering and cleaning their bodies in front of the
visited flower of J. oxyphylla, suggests that this bee could be
collecting the secretion of the staminode capitate glandular
trichomes transferred to its body during the visit.
The presence of substances related to nest building and
chemical defences (i.e. resins and pentacyclic triterpenes),
to the structural and hormonal development of bees (i.e.
sterols) and to the attraction of Euglossini males (cineole)
suggests that the secretions of the capitate glandular tri-
chomes of the staminode are involved in complex chemical
interactions. In particular, those trichomes seem to provide
a variety of substances that are essential to the biology of
bee pollinators of J. oxyphylla.
Pollination ecology of Jacaranda oxyphylla
Several structural and functional features of J. oxyphylla
flowers indicate pollination by bees (Faegri and Pijl, 1979;
Proctor and Yeo, 1979). Flowers of J. oxyphylla are of the
Anemopaegma type described by Gentry (1974), which pre-
sents nototribic pollination carried out by medium-sized and
large bees, normally Apidae (Euglossini tribe), and are
visited by nectar robbers such as Xylocopa and humming-
birds. Nevertheless, only legitimate hummingbird visits
were recorded to J. oxyphylla flowers. This species blooms
during the driest season of the year, when water and energetic
resources for visitors are scarce. Considering that humming-
bird visits were not observed in all studied populations, nor in
previous studies of J. oxyphylla conducted by Yanagizawa
and Maimoni-Rodella (2007), it is possible that humming-
bird visits may simply be opportunistic. In cerrado woody
plants opportunistic visits by hummingbirds were recorded
in .30 % of the species studied by Oliveira and Gibbs
(2002).
The intensive activity recorded for O. flavescens, a nectar
robber, could be related to the considerable increase in
its population size in winter, when J. oxyphylla is in
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla708
blossom. Oxaea flavescens is one of the most regular
and abundant nectar robbers in the Brazilian cerrado,
and is commonly observed robbing nectar in species of
Bignoniaceae that occur in this biome (Gottsberger and
Silberbauer-Gottsberger, 2006).
Nectar is the main caloric resource available to pollinators
of J. oxyphylla. Therefore, it is possible that low nectar pro-
duction allied to intensive pillage by O. flavescens, a low
percentage of nectar-producing flowers (43 % of flowers
lacked nectar) and flower sparseness at anthesis may lead
to insufficient nectar available to pollinators. This low avail-
ability of nectar may be incompatible with the energetic
needs of pollinators and may be responsible for the low
visit rate and low rate of natural fructification observed in
the studied populations.
Considering the flexible reproductive system of
J. oxyphylla, selfing could represent an alternative to propa-
gation via seeds, especially in cerrado populations where the
frequency of medium-sized and large visitors was low.
However, it was found that although Ceratina,Trigona
and Augochlora removed pollen intensively from flowers,
their visits resulted in a reduced deposition of pollen on
stigmas. The findings suggest that these small bees do not
participate substantially in the pollination of J. oxyphylla,
acting predominantly as pollen robbers, different from the
situation proposed by Vieira et al. (1992) for J. caroba
and by Bittencourt and Semir (2006) for J. racemosa. The
only small bee that behaved like a legitimate pollinator of
J. oxyphylla was E. fulvosfasciata, whose behaviour was
similar to that observed by Silva et al. (2007) in Tecoma
stans.
Jacaranda oxyphylla presents the ‘modified steady-state’
phenological pattern described by Gentry (1974). This
pattern is characterized by a scanty flower production per
day over a period of several weeks and is typical of plants
pollinated by bees that establish fixed daily foraging routes
(e.g. Euglossini bees; Janzen, 1971). Studies related to
flight behaviour showed that these bees present strong orien-
tation and odour perception abilities even on extremely large
areas of continuous forest (Ackerman et al., 1982; Roubik,
1989; Roubik and Hanson, 2004). Even though Euglossini
bees are exclusively from forest habitats, E. nigrita also
occurs in fragmented areas (Wittmann et al., 1988;
Tonhasca et al., 2003; Milet-Pinheiro and Schlindwein,
2005). Eulaema nigrita is distributed from Costa Rica to
northern Argentina (Roubik and Hanson, 2004), comprising
the geographic distribution of J. oxyphylla (Gentry and
Morawetz, 1992). This fact, associated with the foraging
behaviour of E. nigrita (Roubik and Hanson, 2004), may
lead to the dispersal of pollen of J. oxyphylla over large
areas.
Unlike Euglossini, bees belonging to the genus Bombus
depend on the concentration of floral supplies
(Walther-Hellwig and Frankl, 2000) and present a beha-
viour that tends to generate small-sized neighbourhoods
(Schmitt, 1980). Thus, in the population of J. oxyphylla
studied by Yanagizawa and Maimoni-Rodella (2007), the
high density of flowering individuals in a small area may
have favoured the high frequency of visits of Bombus
atratus. In the present study, the low frequency of
B. morio bees may have been due to the paucity of
resources available.
The low natural fruit set observed in the populations of
J. oxyphylla analysed in this study could be the result of
the low frequency of pollinator visits recorded. Moreover,
the reduced number of plants flowering simultaneously in
the studied populations could lead to a transfer of mostly
incompatible pollen. Given that previous studies indicated
selfing rates of 26 % in this species (Yanagizawa and
Maimoni-Rodella, 2007), the possibility that the compat-
ibility system of J. oxyphylla is flexible should not be dis-
regarded. In the case of selfing, seed production could be
incremented through geitonogamy, since J. oxyphylla polli-
nators visit several flowers on the same plant sequentially.
There is evidence that a mixed mating system combining
high levels of allogamy with extra flexibility of permitting
some selfing occurs in Bignoniaceae (Bertin and Sullivan,
1988; Bianchi et al., 2005; Bittencourt and Semir, 2006).
The low P/O ratio observed (154.64 +41.38) suggests
that facultative autogamy is occurring in J. oxyphyllla.
However, its nototribic flowers could represent a more
precise pollination mechanism, producing a deviation of
the P/O ratio, as pointed out by Dafni et al. (2005) in pre-
dominantly allogamous species.
Staminode removal experiments and reproductive success
Intact flowers of J. oxyphylla tended to have higher pollen
deposition on the stigma, indicating the participation of the
staminode in female reproductive success, as observed by
Walker-Larsen and Harder (2001) and Dieringer and
Cabrera (2002) in Penstemon species pollinated by bees.
Several functions have been attributed to the staminode
of Jacaranda, such as avoidance of pollen robbing, visual
orientation, visual signal of the ending of nectar production,
secondary pollen presentation, nectar guidance by odour
emission and reduction of floral tube inner space
(Morawetz, 1982; Vieira et al., 1992; Se
´rsic and Rando,
2004; Bittencourt and Semir, 2006; Yanagizawa and
Maimoni-Rodella, 2007).
Overall, the staminode of J. oxyphylla does not seem to
play any mechanical role in restricting access to pollen, as
suggested for J. mimosifolia by Se
´rsic and Rando (2004),
given that the small bees Ceratina,Augochlora and
Trigona removed similar quantities of pollen grains from
the anthers in flowers with and without a staminode. It is
also unlikely that the staminode of J. oxyphylla has an
essential role in visual orientation as found in other
species of Jacaranda (Vieira et al., 1992). This is due to
intrapopulation variation in the colour pattern of capitate
glandular trichomes encountered in J. oxyphylla, resulting
in very distinctive visual patterns among flowers.
In addition, the role of the staminode as a visual indicator
of flower senescence and consequent ending of nectar pro-
duction was discarded for J. oxyphylla since the visual
changes between fresh and old flowers were very discreet.
Similar results were found in J. racemosa (Bittencourt
and Semir, 2006). The function of secondary pollen presen-
tation was not observed for the J. oxyphylla staminode since
the pollen grains remain clustered inside the anthers.
Guimara
˜es et al. — The Pollination of Jacaranda oxyphylla 709
An additional function attributed to the staminodes is the
role of guidance, through odour emission. This role has
been attributed to the staminodes of other species of
Jacaranda (Vieira et al., 1992; Se
´rsic and Rando, 2004;
Bittencourt and Semir, 2006). However, it was shown
here that the white spot of the corolla tube may also carry
out this function, given that it produces a similar mild
and sweetish aroma as well as the fact that it reacts posi-
tively to neutral red. This emission might also be comple-
mented by the capitate glandular trichomes of the
staminode, which, together, form a tunnel of aroma emis-
sion that converges towards the reproductive structures
and nectar chamber.
From a structural viewpoint, the spatial arrangement of
the staminode in the floral tube of J. oxyphylla may cause
the reduction of floral tube inner space, favouring the
contact of some small bees with reproductive organs, as
observed for E. fulvofasciata.
The staminode may be involved in complex chemical
interactions considering the presence of cineole, resins,
steroids, pentacyclic triterpenes and phenolic compounds
in the secretions of the capitate glandular trichomes.
Some of these substances, such as cineole and other terpe-
noids, may act as secondary attractants for pollinating bees.
On the other hand, considering the behaviour of females of
E. nigrita in flowers of J. oxyphylla and the functions that
have been ascribed to other substances found in the
secretion of the capitate glandular trichomes, the possibility
that the staminode may act as the primary attractant should
not be disregarded.
In conclusion, it is suggested that the staminode of
J. oxyphylla is multifunctional and positively influences
female reproductive success, acting physically as a lever
and chemically as an attractant for pollinating bees.
ACKNOWLEDGEMENTS
This research was supported by CAPES (Coordenac¸a
˜ode
Aperfeic¸oamento de Pessoal de Nı
´vel Superior). We
thank the referees for valuable suggestions on the manu-
script, Dr Joa
˜o Semir for botanical identification, Dr Joa
˜o
Camargo for bee identification, Dr Wagner Villegas and
Dr Luis F. Rolim for support with gas chromatography,
Dr Sı
´lvia Rodrigues Machado and Lu
´cia M. G. Santos
for helpful comments on the manuscript, Clı
´via
C. F. Possobom for lab assistance, and the technicians
from the Electron Microcopy Center, UNESP, Botucatu,
for SEM assistance.
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