Pollination Biology of Jacaranda oxyphylla with an Emphasis on Staminode Function
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Annals of Botany 102: 699 –711, 2008
doi:10.1093/aob/mcn152, available online at www.aob.oxfordjournals.org
Pollination Biology of Jacaranda oxyphylla with an Emphasis
on Staminode Function
E L Z A G U I M A R Ã E S 1, * , L UI Z C L A UD IO DI S TAS I 2 and RI TA D E CA SSIA SIN DRÔ NI A
M A I M O N I - RO D E L L A 1
1
Departamento de Botânica and 2Departamento de Farmacologia, Instituto de Biociências, 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.
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† 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.
IN TROD UCT IO N seed set were found in flowers where the staminode had
been removed (Dierenger and Cabrera, 2001, 2002;
The plant family Bignoniaceae is predominantly Neotropical
Walker-Larsen and Harder, 2001).
and plays an important ecological role in the forests of these
Many species of Jacaranda grow in the Brazilian cerrado, a
regions (Lohmann, 2006), especially due to the zoophilous
savannah-like vegetation that is predominant in Central Brazil
nature of its flowers (Gentry, 1974, 1978, 1990). There are
(Mendonça et al., 1998). All species of Jacaranda are charac-
approx. 50 genera of Bignoniaceae in Brazil (Souza and
terized by the abundant glandular trichomes that are present
Lorenzi, 2005), most of them presenting flowers with
throughout the staminode (Martius et al., 1897; Gentry and
rudimentary staminodes. However, in Jacaranda and
Morawetz, 1992; Lohmann et al., 2008). These secretory
Digomphia, the staminode is well developed, conspicuous,
structures might lead to specialized interactions with anthophi-
larger than the stamens, and seems to play an important
lous animals. However, their exact role in the pollination
role in the pollination ecology of species belonging to
biology of those species is yet to be determined. In studies
these genera (Gentry, 1992; Endress, 1994).
concerning the pollination biology of some species of
The role of staminodes in pollination has been described
Jacaranda, several roles have been attributed to the
in various families of Angiosperms (Armstrong and Irvine,
staminode. Among those are a secondary pollen presentation
1990; Endress, 1994; Walker-Larsen and Harder, 2000;
(Yanagizawa and Maimoni-Rodella, 2007), a mechanism for
Decraene and Smets, 2001). Experimental studies aimed
increased bee contact with the reproductive organs through a
at ascertaining the influence of this structure on components
reduction of the space inside the floral tube (Vieira et al.,
of reproductive success have indicated its importance,
1992; Bittencourt and Semir, 2006; Yanagizawa and
especially for female reproductive success. In those
Maimoni-Rodella, 2007), visual orientation through contrast
studies, lower pollen deposition on the stigma and a lower
with the corolla (Vieira et al., 1992; Sérsic and Rando, 2004),
* For correpondence. E-mail elzaguimaraes@hotmail.com guidance through scent emission (Vieira et al., 1992; Sérsic
# The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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700 Guimarães et al. — The Pollination of Jacaranda oxyphylla
and Rando, 2004; Bittencourt and Semir, 2006) and a physical with 0.1 M phosphate buffer, pH 7.3. In addition, staminode
barrier against pollen robbers (Sérsic and Rando, 2004). samples were post-fixed with 1 % osmium tetroxide for 2 h,
Despite the ecological importance of this structure in dehydrated in a graded alcohol series, critical point dried,
species of Jacaranda, experimental studies testing how the coated with gold and examined under a Fei-Quanta 200
staminode might influence components of reproductive scanning electron microscope (Phillips, Czechoslovakia).
success are yet to be carried out. The sole study that addressed Fresh hand-cut sections were subjected to eight different
this question to any extent in Jacaranda was conducted with histochemical tests: (a) periodic acid–Schiff (PAS) reaction
Jacaranda mimosifolia (Sérsic and Rando, 2004). to detect water-insoluble polysaccharides (Jensen, 1962); (b)
In the present study, the floral biology of Jacaranda 0.02 % ruthenium red aqueous solution to detect mucilage/
oxyphylla Cham. was investigated and the role of the stami- pectin (Johansen, 1940); (c) Sudan IV to detect total lipids
node and exudates of its glandular trichomes in the inter- (Johansen, 1940); (d) naphthol þ dimethyl-paraphenylene-
actions with floral visitors was examined. In addition, the diamine (NADI) reagent to detect terpenes (David and
effect of staminode removal on the reproductive success Carde, 1964); (e) 10 % ferric trichloride aqueous solution to
of J. oxyphylla was evaluated. 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
M AT E R I A L S A N D M E T H O D S Verpoorte, 1983); and (h) Fehling’s solution to detect reducing
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Study site and study organism sugars (Purvis et al., 1964). Standard control procedures were
carried out simultaneously, following the indicated protocols.
Fieldwork was conducted from 2004 to 2006 in two frag- Temporary slides were mounted in glycerine and analysed
mented areas of cerrado located in Pratânia (228480 5200 S, under an Olympus BX 41 light microscope (Japan) equipped
488440 3500 W) and Botucatu (228570 3800 S, 488310 2200 W), in with an Olympus C7070 digital camera (Olympus, Japan).
the state of São Paulo, South-eastern Brazil. Complementary analyses were carried out with an Olympus
Jacaranda oxyphylla Cham. is widely distributed in SZ 61 (Japan) stereoscopic microscope, also equipped with
cerrado areas of South-eastern Brazil. This species is very an Olympus C7070 digital camera (Olympus, Japan).
common in open ‘campo limpo’ grasslands and the Thin-layer chromatography (TLC) was used in order to
shrubby edges of cerradão and cerrado stricto sensu investigate the presence of terpenes in staminodes. For
(Gentry and Morawetz, 1992). Jacaranda oxyphylla is a those analyses, samples from staminodes were taken from
xylopodial shrub or sub-shrub of approx. 0.5– 2.5 m tall, 102 fresh flowers and immersed in chloroform for 30 min,
with bipinnate leaves, terminal and axilar inflorescences, following Siebert (2004).
tubular – campanulate flowers above a narrow basal tube, Using a glass capillary tube, samples were spotted onto
didynamous stamens with dithecate anther and presenting a Silica gel 60 F254 (Merck) TLC plates, using toluene – ethyl
long sub-exerted staminode, a flattened – ovate ovary slightly acetate (93 : 7) as eluant. Terpenes were visualized by spray-
contracted at the base to a cylindrical – pulvinate disk, an ing the plates with AS (anisaldehyde – sulfuric acid) reagent,
elliptic fruit, thinly woody, and small-bodied seeds with heating the plates at 100 8C for 10 min, and then evaluating
hyaline – membranaceous wings (Gentry and Morawetz, the terpenes in visible light (Wagner and Bladt, 1996).
1992). The cylindrical– pulvinate disk was quoted as nectar- For the gas chromatography (GC) analysis, chloroform
iferous tissue by Yanagizawa and Maimoni-Rodella (2007). extracts of J. oxyphylla staminodes that had been ultrasonicated
Vouchers of the studied materials were collected and at room temperature for 20 min were used. The chromato-
deposited in the ‘Irina Delanova Gemtchujnicov’ graph used was a VARIAN CP-3380 coupled to an ADCB
Herbarium (BOTU) of the Biosciences Institute of the (1 V) flame ionization detector (FID), and equipped with an
Universidade Estadual Paulista, Botucatu, SP, Brazil. LM-5 capillary tube (phenyl 95 % methylpolysiloxane with
These materials are registered under numbers 24408 –24412. 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.
Staminode morphology and composition of the secretion
Operating conditions were as follows, injector ¼ 250 8C;
of staminode glandular trichomes
detector ¼ 290 8C; heating ramp-up ¼ 150–280 8C (rate of
Staminode samples were fixed with 2.5 % glutaraldehyde 10 8C/min) and 28 8C for 18 min, total time of 31 min; gas
in 0.1 M phosphate buffer, pH 7.3, for 6 – 12 h at 4 8C. In flow ¼ air at 480 mL min21, N2 at 43 mL min21 and H2 at
addition, samples were post-fixed with Karnovsky solution 2 mL min21; and gas ratios ¼ N2/H2/air 1.9 : 1.0 : 20.
(Karnovsky, 1965), dehydrated in a graded series of ethanol Authentic samples of thymol, terpineol, progesterol, tingen-
solutions and embedded in historesin (Gerrits, 1991). one, a-tocopherol, stigmasterine, campesterol, stigmasterol,
Sections of 8 mm were stained with 0.05 % toluidine blue a-espinasterol, b-sitosterol, a-amyrin, a-amyrin acetate,
(O’Brien et al., 1964). The slides were sealed with b-amyrin acetate, lupeol, lupeol acetate, friedelanol and
Entellan resin and examined under an Olympus BX 41 friedelin were injected under identical GC conditions.
light microscope (Japan) equipped with an Olympus
C7070 digital camera (Olympus, Japan).
Pollination ecology of Jacaranda oxyphylla
For morphological analyses by scanning electron
microscopy (SEM), staminode samples from five newly Flowers were monitored to check for visitors at different
opened flowers were fixed for 24 h in 2.5 % glutaraldehyde times of the day, from early in the morning at 0500 h toGuimarães et al. — The Pollination of Jacaranda oxyphylla 701
2330 h at night, a total of 180 h of field observations distrib- stereomicroscope (Japan). All pollen grains remaining per
uted over 32 non-consecutive days. Each day, plants were flower were extracted by shaking the anthers in 200 mL of
monitored for 2 – 8 consecutive hours. Visitors were cap- acetic carmine solution. Samples (20 mL) were mounted
tured to examine pollen deposition on their body, for mor- on slides and then pollen grains were counted using an
phometric analyses and for identification. Flowers from Olympus BX 41 microscope (Japan). Data from these
approx. 40 plants distributed between the two populations experiments were tested for normal distribution
were observed. Observations included the time of anthesis, (Kolmogorov – Smirnov) and compared using a two-tailed
colouring and dimensions of the floral elements, production t-test and Mann –Whitney U test, using GraphPad Instat
of aroma, presence of nectar and floral longevity. In v.3.01 software (San Diego, CA, USA).
addition, stigmatic receptivity was estimated with hydrogen
peroxide (H2O2) according to Dafni et al. (2005). Pollen
viability was estimated using acetic carmine as vital stain R E S U LT S
(Radford et al., 1974). The pollen/ovule (P/O) ratio was
Staminode morphology and composition of the secretion
estimated according to Cruden (1977) (n ¼ 6 plants, 18
of staminode glandular trichomes
flowers). Intact flowers and individual floral parts (e.g. sta-
minode, the white spot of the corolla roof ) were subjected The staminode is composed of a cylindrical filament (2.8 –
to organoleptic studies through which odour concentration 4.3 mm long) with a broader, slightly bifid tip, and a thin
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was monitored in clean glass vials (Dafni et al., 2005). base, attached to the bottom of the corolla tube. It
The presence and location of osmophores were investigated emerges obliquely and its distal end rests upon the entrance
using neutral red solution (Vogel, 1990). Nectar volume of the corolla tube (Fig. 1B). The filament of the staminode
and concentration were measured from bagged flowers is densely covered with capitate glandular trichomes over
(n ¼ 153 flowers, 50 individuals) using a glass capillary its entire length except the basal 10 mm (Fig. 1C). Its
and a Carl Zeiss (0 – 30 %) pocket-size refractometer abaxial portion shows only capitate glandular trichomes
(Jena, Germany). The presence of nectar in pre-anthesis (Fig. 1G), while the adaxial apical portion contains numer-
flowers was also investigated, and the quantity of sugar ous hyaline, simple, uniseriate, uni- to pluricellular long tri-
per mL of nectar was calculated. These data were used to chomes (Fig. 1H). The capitate glandular trichomes are
estimate the average energetic value of the nectar produced constituted by an approximately spherical multicellular
by each flower during its life span, using an exponential head, and a stalk varying in length, number of cells and
regression as suggested by Galetto and Bernardello (2005). degree of ramification. These trichomes can be divided
Pre-anthesis flowers were bagged to exclude all visitors. into three basic types according to their size. The short tri-
Subsequently, flowers in first-day anthesis were self- chomes (Fig. 2A) are distributed over the entire abaxial
pollinated ( pollen from the same flower) or cross-pollinated surface of the staminode (Fig. 1C, G). The intermediary tri-
(using pollen mix from individuals separated by at least chomes (Fig. 2C) may present ramifications, and are con-
20 m) and rebagged. Bagged flowers were tagged and left centrated at the sides of the median portion of the
intact (i.e. were not pollinated) to check for spontaneous staminode, forming a kind of channel situated 10– 35 mm
self-pollination. In addition, bagged and emasculated above the base (Fig. 1C). The third long-stalked ramificated
flowers were used to test for autonomous agamospermy. trichomes (Fig. 2D) are predominantly located on the top of
Natural fruit set was monitored by tagging unbagged the abaxial apical portion of the staminode, forming a small
flowers. Thirty individuals were used in the pollination tuft together with the simple trichomes (Fig. 1C, G).
treatments, and fruit set was recorded after 4 and 8 weeks. Trichome heads present 17– 24 cells (n ¼ 10) arranged con-
centrically (Fig. 2B) around a central cell (Fig. 2A, B).
Occasionally, trichomes with two concentric layers of
Staminode removal experiments and reproductive success
cells forming the glandular head are also present. In SEM
Staminode removal experiments were conducted to test analyses, capitate glandular heads present a marked
the effect of the staminode on female and male reproductive surface, indicating the close attachment of the cuticle to
success. Two neighbouring flower buds were bagged in the secretory upper cell walls and making the cell outlines
each plant (n ¼ 32 plants) to avoid microhabitat variation, evident (Fig. 2E). Alternatively, a small sub-cuticular space
and the bags were removed on the morning of anthesis. is formed by the detachment of the cuticle (Fig. 2F).
Staminodes from half of the flowers were removed; the In pre-anthesis, droplets were found in the style and
other half served as controls (n ¼ 32 flower per treatment, corolla. These droplets were seen in the median glandular
1 flower per plant per treatment). A control excluding visi- portion of the staminode, indicating secretory activity of
tors (bagged flowers) was not performed since pollen is this structure prior to flower opening. Under the stereomi-
released only when anthers are touched and squeezed by croscope, large droplets were observed on the head
visitors. The experiment involved a comparison of pollen surface of the capitate glandular trichomes of newly
deposition and pollen removal between flowers with and opened flowers.
without staminodes that had been exposed to open pollina- During the flower’s functional period, capitate glandular
tion during their life span. After 48 h of open-pollination, trichomes were found with different degrees of cuticle disten-
anthers and stigmas were carefully removed and fixed in sion. Some of these trichomes did not present the formation of
acetic carmine solution. Pollen grains deposited on stig- a sub-cuticular space (Fig. 2E), while others presented a
matic surfaces were counted using an Olympus SZ 61 slightly distended cuticle forming a small sub-cuticular702 Guimarães et al. — The Pollination of Jacaranda oxyphylla
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F I G . 1. Flower and staminode of Jacaranda oxyphylla. (A) Inflorescences showing flowers with the median region of the corolla curved and compressed
dorsoventrally. Scale bar ¼ 50 mm. (B) Close-up view of a flower showing the staminode (st) and white portion of the corolla roof. Scale bar ¼ 10 mm.
(C) Close-up view of a staminode. Scale bar ¼ 5 mm. (D–F) Close-up of a staminode showing its capitate glandular trichomes in variable colours. Scale
bars ¼ 500 mm. (G) Abaxial portion of the staminode with capitate glandular trichomes, (H) adaxial portion of the staminode with simple trichomes.
Scale bars ¼ 1 mm.
space filled with hyaline secretion (Fig. 2C, F), and others compounds. Treatments with ruthenium red for mucilage/
presented a wrinkled cuticle indicating previous release of pectin and with Dragendorff solution for alkaloids proved
the secretion (Fig. 2G). This variation suggested that the negative. The assays for detecting proteins, sugars, neutral
release of secretion from the capitate glandular trichomes of polysaccharides and starch showed weakly positive reac-
the staminode of J. oxyphylla is continuous. tions (Table 1) and were highly variable among neighbour-
The secretion of the capitate glandular trichomes of the ing trichomes.
staminode is composed of predominantly lipophilic TLC revealed the presence of several terpenoid com-
material. This material stained positively with Sudan IV pounds in the chloroform extract of the staminode of
and differentially with NADI reagent, indicating the pre- J. oxyphylla. With AS reagent, terpenes stained pinkish-
sence of terpenes and resinic acids. The intensity and purple zones; one of them, of Rf 0.33, was identified as
colours of the positive reaction to NADI reagent varied cineole. This compound was confirmed by comparing its
among trichomes of the same morphology located side by retention time with that of an authentic sample. The other
side. A strongly positive reaction occurred with phenolic terpenoid compounds could only be identified usingGuimarães et al. — The Pollination of Jacaranda oxyphylla 703
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F I G . 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 ¼ 30 mm. (B) Cross-section of a trichome head. Scale bar ¼ 20 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 ¼ 40 mm. (D– G) SEM. Scale bar ¼ 30 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.
Pollination ecology of Jacaranda oxyphylla
GC – FID. The gas chromatogram of J. oxyphylla stami-
nodes presented peaks that were characteristic of penta- Flowering of J. oxyphylla occurred between August and
cyclic triterpene and steroid compounds (Table 2). October. Flowers opened predominantly at around 0700 h,
The identity of these compounds was confirmed by compar- and anthesis lasted about 2 d. Each inflorescence
ing their relative retention time with those of authentic (Fig. 1A) presented one to two flowers in anthesis per
samples. day, and each plant had between one and 16 inflorescences,704 Guimarães et al. — The Pollination of Jacaranda oxyphylla
TA B L E 1. Histochemistry of mature capitate trichomes from stalks and yellow heads (Fig. 1E) or purple stalks and
the staminode of Jacaranda oxyphylla yellow heads (Fig. 1F). Flowers with staminodes carrying
trichomes of different colours presented very different
Colour Reactivity of visual patterns.
Staining procedure Target compounds observed head cells*
The median and upper portions of the corolla tube roof
Sudan IV Total lipids Red þ
present a large white spot (Fig. 1B) where the osmophores
NADI Terpenes Blue and þþ are inserted. Osmophores were revealed by intense reaction
dark red in the neutral red assay. A faint, mildly sweetish aroma was
Ruthenium red Pectin/mucilage – detected in the corolla white spot and staminode. Trichomes
Ferric trichloride Phenolic Green to þþ of the staminode presented a weak positive reaction to
compounds black
Schiff (PAS) Neutral Pink þ
neutral red that was restricted to the secretory head cells.
polysaccharides On the other hand, the corolla white spot presented a
Dragendorff Alkaloids – strong positive reaction to neutral red. This indicates that
Mercuric Proteins Dark blue þ both structures may act as osmophores.
bromophenol blue Anther dehiscence and stigmatic lobe opening occurred
Lugol Starch grains Purple þ
Fehling’s solution Sugars Reddish þ on the day prior to the onset of anthesis. At this stage, a
longitudinal dehiscence line was formed in the anthers,
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*– negative, þ slightly positive, þþ strongly positive. 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
TA B L E 2. Terpenoid composition of chloroform extract from were only released when they were lightly squeezed
the staminode of Jacaranda oxyphylla by GC –FID against the corolla tube roof by visitors. At the start of
Peak Retention time Compounds Phytochemical classes
anthesis, the corolla lobes were completely distended and
an elongated platform was formed by the lower lobe,
1 12.7 Campesterol Steroids upon which the bright tip of the staminode contrasted
2 12.9 Stigmasterol Steroids (Fig. 1B). The stigma was sensitive, and closed in a
3 16.4 b-Sitosterol Steroids matter of seconds when touched. When pollen deposition
4 16.8 a-Amyrin Pentacyclic triterpenes did not occur, the stigma remained closed for about
ursane type
5 17.7 b-Amyrin Pentacyclic triterpenes 30 min, after which it gradually opened again, completing
oleanane-type the process after 2 h.
6 18.8 Lupeol Pentacyclic triterpenes Pollen grains were white, appeared in clusters and were
lupane type covered by lipidic substances as revealed by Sudan IV assay.
7 20.5 Lupeol acetate Pentacyclic triterpenes
lupane type
The average viability of the pollen grains was 98.65 % and
8 20.9 Friedelanol Pentacyclic triterpenes the P/O ratio was 154.64 + 41.38 (mean + s.d.).
friedelane type There was no nectar production before anthesis. The
9 21.5 Friedelin Pentacyclic triterpenes accumulated nectar volume in first-day flowers was
friedelane type 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-
presenting 7.5 + 5.52 first-day flowers per day (mean + tration varied from 22 to 28 %. Not all flowers of a plant
s.d.). Three- to 6-d-old flowers remained in the inflores- produced nectar, and 43 % of the flowers analysed
cences, even though these were no longer receptive, showed no production at any time during anthesis. The esti-
perhaps acting as a visual attractor for visitors. After the mated energetic value of accumulated nectar varied from
third day, the corolla presented faded colouring, and a dar- 3.32 to 8.28 calories per flower.
kened anther and staminode, with the stigma being the last The populations studied here were preferentially xenoga-
structure to display signs of senescence. mous, with the formation of fruits occurring in only 3.85 %
The floral tube was 32– 50 mm longer (n ¼ 30) and pre- of the selfed flowers. Natural fruit set was low (7.25 %) and
sented a basal constriction (6 – 8 mm, n ¼ 30) correspond- no case of spontaneous self-pollination or autonomous aga-
ing to the nectar chamber. The median region of the mospermy was found, indicating the need for a pollen trans-
tubular – campanulate corolla was slightly curved and fer vector. The hand-cross pollination test produced greater
dorsoventrally compressed (Fig. 1A). Anthers and stigma fruit set (Table 3).
were included, and were arranged from 15 to 30 mm Small bees of the genera Ceratina, Trigona, Augochlora
above the basal constriction, remaining juxtaposed to the and Exomalopsis were observed visiting flowers of
corolla tube roof throughout the flower’s life span. In this J. oxyphylla (Table 4). Among these small bees, only
region, the staminode was aligned longitudinally, with the Exomalopsis fulvofasciata presented a legitimate visiting
reproductive structures forming a kind of lever that leaves behaviour. This bee entered deep into the floral tube,
an open space of only 2 mm. The densely glandular stami- passing over the staminode, seeking the nectar chamber
node (Fig. 1C) was recovered by capitate glandular and gathering nectar by extending its 4 mm long proboscis.
trichomes of variable colour. The trichomes may present Whenever it left the flower, its head and the dorsal portion
purple stalks and crimson heads (Fig. 1D), colourless of its thorax were covered with pollen. The narrowing ofGuimarães et al. — The Pollination of Jacaranda oxyphylla 705
TA B L E 3. Experimental pollination and fruit set of Jacaranda oxyphylla, in a cerrado patch, Botucatu, SP, Brazil
(n ¼ 30 plants)
Spontaneous Autonomous Hand Hand Natural pollination
n self-pollination agamospermy self-pollination cross-pollination (control)
Flowers 478 182 39 78 41 138
Fruits 32 0 0 3 (3.85 %) 19 (46.34 %) 10 (7.25 %)
TA B L E 4. Visitors to flowers of Jacaranda oxyphylla abdomen downwards and actively scraped the glandular
in patches of cerrado vegetation, in Botucatu and Pratânia, portion of the staminode. Then, the bees flew to other
SP, Brazil flowers of the same plant or to neighbouring plants. These
bees were never observed gathering nectar from flowers of
Visiting Foraged J. oxyphylla.
Species behaviour resource Frequency* Besides E. fulvofasciata, medium-sized bees also visited
J. oxyphylla flowers legitimately (Table 4). Eulaema
ANDRENIDAE
nigrita (Euglossini) was observed on two occasions, visiting
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Oxaea flavescens Non-legitimate Nectar High
APIDAE about ten flowers of neighbouring plants (Fig. 3E). After
Bombus Legitimate Nectar Low approaching an inflorescence, the bee landed on the anterior
(Fervidobombus) morio lobe of the corolla and then walked along the corolla tube
Ceratina (Crewella) Non-legitimate Pollen High floor, passing over the staminode, towards the nectar
maculifrons
Ceratina (Crewella) Non-legitimate Pollen High chamber. The bee touched the stigma and the anthers with
gossypii its back. After 10– 20 s, the female bee reversed out of the
Ceratina (Crewella) Non-legitimate Pollen High corolla tube, hovering for a few seconds in front of the
asuncionis visited flower, apparently cleaning its body. The anthers
Eulaema (Apeulaema) Legitimate Nectar Intermediate
nigrita
were arranged 15 – 30 mm above the nectar chamber, and
Exomalopsis Legitimate Nectar and Low the extended proboscis of E. nigrita was about 25 mm
fulvofasciata pollen long. In order to reach the nectar, individuals of E. nigrita
Trigona spinipes Non-legitimate Pollen High need to touch the reproductive structures with their head
Xylocopa sp. Non-legitimate Nectar Low and dorsal portion of their thorax, while the ventral portion
HALICTIDAE
Augochlora Non-legitimate Pollen High touches the entire median glandular region of the staminode.
(Augochlora) sp. Visits by a species of bumble-bee, Bombus morio, were
TROCHILIDAE Legitimate Nectar Intermediate also observed on two occasions (Fig. 3D). Before entering
the flowers, the bee hovered for a few seconds in front of a
*Frequency: high (30 visits d21), intermediate (1–5 visits d21), low flower and then landed on the anterior lobe of the corolla,
(,1 visit d21).
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
the tube diameter caused by dorsoventral compression and of its thorax. Individuals of B. morio were subsequently
the arrangement of the staminode in relation to the reproduc- observed visiting another flower on the same plant or
tive structures favoured the contact of E. fulvofasciata with flowers on some neighbouring plants.
those structures. Despite its small size, this bee is quite Both medium-sized bees, E. nigrita and B. morio, were
heavy and can lower the staminode upon entering the floral legitimate pollinators carrying pollen and touching the recep-
tube and treading on it. Ceratina, Augochlora and Trigona, tive surface of stigmatic lobes upon entering flowers of
on the other hand, normally entered the staminode sideways J. oxyphylla. Whenever these species presented pollen depos-
and did not present legitimate visiting behaviour. These bees ited on their dorsal region, only the sterile portions of the stig-
visited flowers from sunrise to dusk. At first, they investi- matic lobes were subsequently touched, when leaving the
gated each flower visually; if there was no other visitor, flower, restricting the possibility of self-pollination.
they entered the floral tube laterally (Fig. 3A). Upon reaching Visits by an unidentified species of hummingbird
the anthers, bees turned their ventral side toward the corolla (Trochilidae) were also observed. These hummingbirds
tube roof and collected pollen actively with their first pair of visited 5 – 15 receptive flowers per flight, between 0800 h
legs, while simultaneously cleaning their bodies and trans- and 0900 h. During flower visits, the hummingbirds thrust
ferring pollen to the subsequent pair of legs (Fig. 3B). This their heads into the floral tube for 1 or 2 s, usually remain-
process lasted from 1 to 3 min. During these movements ing in hovering flight. In some instances, the hummingbirds
various parts of the bee’s body, covered with large quantities also rested their feet briefly on the inflorescence. Given that
of pollen from this flower, established contact with the the hummingbird’s size (beak length approx. 20 mm) fit the
stigmatic surface, favouring a potential self-pollination. reproductive structures and nectar chamber of J. oxyphylla
However, stigma closure was never observed during this very well, this species may be considered a legitimate
contact. After pollen collection, the bees turned their pollinator. However, corolla abscission frequently occurred706 Guimarães et al. — The Pollination of Jacaranda oxyphylla
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F I G . 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 ¼ 10 mm.
a few seconds after the hummingbird left the flower, leaving anthers (n ¼ 32) (t-test, P ¼ 0.49) when comparing
behind only the calyx and the gynoecium and precluding flowers with and without a staminode. Flowers with stami-
further visits. nodes received pollen deposition on stigmas varying from
Lastly, Oxaea flavescens and Xylocopa sp. were observed 0 to 144 pollen grains (Fig. 4). On the other hand,
stealing nectar (Table 4). Both presented similar behaviour, flowers without staminodes only presented deposition of
landing externally on the floral tube, introducing their small quantities of pollen in all samples evaluated, with 0
mouth apparatus between the calyx and the corolla and to 15 pollen grains per stigma (Fig. 4). Small Halictidae
actively stealing nectar through longitudinal slits produced and Anthophoridae bees were observed visiting flowers
at the base of the corolla tube (Fig. 3C). 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-
Staminode removal experiments and reproductive success
quently (15 : 1) by these bees, the total pollen grain removal
Even though flowers without a staminode differed visu- was similar in flowers with and without staminodes (Fig. 5).
ally from intact flowers, no significant differences were The percentage of flowers with staminodes that received
found between treatments considering the presence and a large amount of pollen grains on the stigma (6.25 %) was
absence of staminode. No significant difference was close to the natural fructification, i.e. 7.25%. This depo-
found for pollen deposition on stigmas (n ¼ 32) (Mann – sition (.140 pollen grains per stigma, Fig. 4) was compa-
Whitney U test, P ¼ 0.89) and for pollen removal from tible with the number of ovules per flower (113.28 + 21.14,Guimarães et al. — The Pollination of Jacaranda oxyphylla 707
50 Pichersky and Gershenzon, 2002; Machado et al., 2006).
However, in the staminode of J. oxyphylla it was found
45
that the secretion was already available at flower opening
and could be scattered in the air, collected and easily trans-
40
ferred to the body of the pollinator during a visit. Sérsic and
35 Rando (2004) detected the presence of secretion com-
Flowers with staminode
ponents produced by the glandular trichomes of the stami-
30 node of J. mimosifolia in the body of B. morio, indicating
Flowers (%)
Flowers without staminode
transfer of secretion components during visits.
25 Chemical analyses of the secretion produced by capitate
glandular trichomes of the staminode of J. oxyphylla
20 allowed for a better understanding of the role of this struc-
ture in the interactions with floral visitors. Phenolic and ter-
15
penic compounds constitute the secondary compounds most
10 widely distributed among plants (Harborne, 1997). The
phenolic compounds comprise approx. 8000 molecules,
5 including flavonoids and tannins that contribute to the
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flavour, odour and colour of a variety of plants (Harborne,
0
0 1–10 11–20 21–30 131–140 141–150
1985, 1997; Nishida, 2002). Phenolic compounds in the
Number of pollen grains
capitate trichomes of the staminode of J. oxyphylla can
be of different types and may play different roles. For
F I G . 4. Number of pollen grains deposited on the stigmatic surface of example, trichomes may play a role in protection against
flowers of Jacaranda oxyphylla, with and without a staminode. ultraviolet radiation (Harborne, 1997), an important aspect
in open vegetation formations such as the cerrado.
mean + s.e.). On the other hand, the number of pollen On the other hand, terpenoids constitute a wide and
grains (,20 pollen grains, Fig. 4) deposited on stigmas complex class of secondary compounds that play essential
of flowers without staminodes was much lower than the roles in plant – animal and plant – plant interactions, acting
mean number of ovules per flower. as feeding deterrents, pheromones, defence agents and alle-
lochemicals (Harborne, 1997). The presence of monoter-
penes and sesquiterpenes in flowers has been related to
DISCUSSION the attraction of pollinators, especially bees, moths and but-
terflies, that may, in some cases, be species specific
Staminode morphology and composition of the secretion
(Harborne, 1997; Pichersky and Gershenzon, 2002). The
of staminode glandular trichomes
monoterpene cineole, identified in the capitate glandular tri-
In some species the secretion produced by glandular tri- chomes of the staminode of J. oxyphylla, comprises highly
chomes remains accumulated inside the sub-cuticular volatile fragrances associated with the localization of the
space, and is exclusively released through mechanical flowers of these plants by ‘trapliners’ and opportunistic pol-
contact. This contact is usually provided by herbivores linators, and are commonly found in Orchidaceae (Cancine
that break the cuticle and cause the release of substances and Damon, 2007).
that are normally associated with chemical defence Triterpenes make up a more diversified sub-group of ter-
(Ascensão et al., 1995, 1997; Sacchetti et al., 1999; penes and can perform ecological defence-related functions
40
Flowers with staminode
35
Flowers without staminode
30
Flowers (%)
25
20
15
10
5
0
0–10 11–20 21–30 31–40 41–50 51–60 61–70 71–80 81–90 91–100
Pollen grains (%)
F I G . 5. Percentage of pollen grains removed from anthers of flowers of Jacaranda oxyphylla, with and without a staminode.708 Guimarães et al. — The Pollination of Jacaranda oxyphylla
(Harborne, 1997; Cheng et al., 2007). In this study, pentacyc- the staminode (Gottsberger and Silberbauer-Gottsberger,
lic triterpenes and steroidal triterpenes produced by the sta- 2006). The authors reported that males, when exiting
minode of J. oxyphylla may also perform these functions. flowers, hover in front of those flowers and make typical
Pentacyclic triterpenes can be found in combinations con- movements with their legs that are associated with the trans-
taining variable quantities of terpenes mixed with other fer of liquid odour (fragrance) to the tibial capsule, similar
classes of substances (Wagner and Bladt, 1996), such as to the behaviour performed by a male of E. nigrita in
the oil – resin mixture identified in the capitate glandular tri- flowers of J. oxyphylla. Visits from Euglossini males
chomes of the J. oxyphylla staminode. Although these com- were also described in other Bignoniaceae (Roubik and
pounds are widely distributed throughout higher plants, their Hanson, 2004; Silva et al., 2007).
presence in flowers has been described for only a few species. It is also noteworthy that Euglossini bees are the main pol-
In particular, they have been found in Dalechampia linators of most species of Jacaranda studied so far, includ-
(Armbuster, 1984) and Tipuana tipu (Pereira and Aquino ing J. caroba, J. copaia, J. ulei, J. simplicifolia, J. rufa,
Neto, 2003). Floral resins, normally composed of triterpenes, J. racemosa, J. paucifoliolata, J. rugosa and J. decurrens
are collected by various genera of Neotropical Euglossini (Vieira et al., 1992; Stevens, 1994; Maués et al., 2004;
bees (Armbuster and Webster, 1979). Bittencourt and Semir, 2006; Sampaio et al., 2007;
According to Roubik and Hanson (2004), both male and Yanagizawa and Maimoni-Rodella, 2007). According to
female Euglossini bees depend greatly on non-food materials Gottsberger and Silberbauer-Gottsberger (2006), small
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in their environment. Males need to collect chemicals and bees feed on pollen, while male Euglossini bees collect
females must find nesting material. Unlike bumble-bees, fragrances from the staminode of Jacaranda. Additionally,
stingless bees and honey bees, Euglossini bees use no wax, females of Euglossini and other species of large bees feed
depending heavily on resins for nest construction. The mainly on nectar in Jacaranda, leading to what
resin from flowers remains soft and pliable for a long time, Gottsberger and Silberbauer-Gottsberger (2006) call a super-
unlike the resin or resinous sap that exudes from plant imposed pollination system. The behaviour of the females of
wounds (Roubik and Hanson, 2004). In addition to nesting E. nigrita, hovering and cleaning their bodies in front of the
material, some triterpenes, the primary compound of plant visited flower of J. oxyphylla, suggests that this bee could be
resins, provide antibiotics. Oliveira et al. (1996) tested the collecting the secretion of the staminode capitate glandular
effectiveness of resin from Clusia grandiflora flowers and trichomes transferred to its body during the visit.
found that those resins are highly effective against The presence of substances related to nest building and
Gram-positive bacteria, a deadly microbial enemy of bees. chemical defences (i.e. resins and pentacyclic triterpenes),
On the other hand, steroidal triterpenes play an important to the structural and hormonal development of bees (i.e.
role in plant – insect interactions, since many phytophagous sterols) and to the attraction of Euglossini males (cineole)
and omnivorous insects are unable to biosynthesize the suggests that the secretions of the capitate glandular tri-
steroid nucleus (Svoboda and Feldlaufer, 1991). Steroids chomes of the staminode are involved in complex chemical
such as sitosterol, campesterol and stigmasterol are essential interactions. In particular, those trichomes seem to provide
in structural and hormonal functions (Roitberg and Isman, a variety of substances that are essential to the biology of
1992). Rasmont et al. (2005) found the presence of bee pollinators of J. oxyphylla.
b-sitosterol in the pollen of a legume pollinated by
Bombus terrestris. The authors state that this compound
Pollination ecology of Jacaranda oxyphylla
has a feeding deterrent effect on Apis mellifera, which fed
on the nectar but not on the pollen of this species. This Several structural and functional features of J. oxyphylla
may be one of the reasons why no visits by A. mellifera flowers indicate pollination by bees (Faegri and Pijl, 1979;
to the flowers of J. oxyphylla were recorded. Proctor and Yeo, 1979). Flowers of J. oxyphylla are of the
Females of E. nigrita act as legitimate pollinators of Anemopaegma type described by Gentry (1974), which pre-
J. oxyphylla. Even though male visits were also observed, sents nototribic pollination carried out by medium-sized and
it is unclear whether they also act as legitimate pollinators. large bees, normally Apidae (Euglossini tribe), and are
It should be noted that males of this species collect volatile visited by nectar robbers such as Xylocopa and humming-
substances (fragrances) from floral and non-floral sources birds. Nevertheless, only legitimate hummingbird visits
and store those substances in cavities located in the pos- were recorded to J. oxyphylla flowers. This species blooms
terior tibia, where they accumulate complex, species- during the driest season of the year, when water and energetic
specific blends of fragrances (Schiestl and Roubik, 2003; resources for visitors are scarce. Considering that humming-
Eltz et al., 2006). Eltz et al. (2003) analysed the content bird visits were not observed in all studied populations, nor in
of the tibia of Euglossini males and detected mixtures of previous studies of J. oxyphylla conducted by Yanagizawa
terpenoids and aromatic compounds totalling 70 substances, and Maimoni-Rodella (2007), it is possible that humming-
including cineole. Euglossini males feed on nectar of plant bird visits may simply be opportunistic. In cerrado woody
species that are not necessarily producers of fragrances; plants opportunistic visits by hummingbirds were recorded
hence, cineole may act in the attraction of E. nigrita in .30 % of the species studied by Oliveira and Gibbs
males to flowers of J. oxyphylla, which offers nectar as a (2002).
reward, in addition to the secretions of the staminode. The intensive activity recorded for O. flavescens, a nectar
In other species of this genus, J. caroba and J. decurrens, robber, could be related to the considerable increase in
males of Euglossa were observed collecting fragrance from its population size in winter, when J. oxyphylla is inGuimarães et al. — The Pollination of Jacaranda oxyphylla 709
blossom. Oxaea flavescens is one of the most regular B. morio bees may have been due to the paucity of
and abundant nectar robbers in the Brazilian cerrado, resources available.
and is commonly observed robbing nectar in species of The low natural fruit set observed in the populations of
Bignoniaceae that occur in this biome (Gottsberger and J. oxyphylla analysed in this study could be the result of
Silberbauer-Gottsberger, 2006). the low frequency of pollinator visits recorded. Moreover,
Nectar is the main caloric resource available to pollinators the reduced number of plants flowering simultaneously in
of J. oxyphylla. Therefore, it is possible that low nectar pro- the studied populations could lead to a transfer of mostly
duction allied to intensive pillage by O. flavescens, a low incompatible pollen. Given that previous studies indicated
percentage of nectar-producing flowers (43 % of flowers selfing rates of 26 % in this species (Yanagizawa and
lacked nectar) and flower sparseness at anthesis may lead Maimoni-Rodella, 2007), the possibility that the compat-
to insufficient nectar available to pollinators. This low avail- ibility system of J. oxyphylla is flexible should not be dis-
ability of nectar may be incompatible with the energetic regarded. In the case of selfing, seed production could be
needs of pollinators and may be responsible for the low incremented through geitonogamy, since J. oxyphylla polli-
visit rate and low rate of natural fructification observed in nators visit several flowers on the same plant sequentially.
the studied populations. There is evidence that a mixed mating system combining
Considering the flexible reproductive system of high levels of allogamy with extra flexibility of permitting
J. oxyphylla, selfing could represent an alternative to propa- some selfing occurs in Bignoniaceae (Bertin and Sullivan,
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gation via seeds, especially in cerrado populations where the 1988; Bianchi et al., 2005; Bittencourt and Semir, 2006).
frequency of medium-sized and large visitors was low. The low P/O ratio observed (154.64 + 41.38) suggests
However, it was found that although Ceratina, Trigona that facultative autogamy is occurring in J. oxyphyllla.
and Augochlora removed pollen intensively from flowers, However, its nototribic flowers could represent a more
their visits resulted in a reduced deposition of pollen on precise pollination mechanism, producing a deviation of
stigmas. The findings suggest that these small bees do not the P/O ratio, as pointed out by Dafni et al. (2005) in pre-
participate substantially in the pollination of J. oxyphylla, dominantly allogamous species.
acting predominantly as pollen robbers, different from the
situation proposed by Vieira et al. (1992) for J. caroba
Staminode removal experiments and reproductive success
and by Bittencourt and Semir (2006) for J. racemosa. The
only small bee that behaved like a legitimate pollinator of Intact flowers of J. oxyphylla tended to have higher pollen
J. oxyphylla was E. fulvosfasciata, whose behaviour was deposition on the stigma, indicating the participation of the
similar to that observed by Silva et al. (2007) in Tecoma staminode in female reproductive success, as observed by
stans. Walker-Larsen and Harder (2001) and Dieringer and
Jacaranda oxyphylla presents the ‘modified steady-state’ Cabrera (2002) in Penstemon species pollinated by bees.
phenological pattern described by Gentry (1974). This Several functions have been attributed to the staminode
pattern is characterized by a scanty flower production per of Jacaranda, such as avoidance of pollen robbing, visual
day over a period of several weeks and is typical of plants orientation, visual signal of the ending of nectar production,
pollinated by bees that establish fixed daily foraging routes secondary pollen presentation, nectar guidance by odour
(e.g. Euglossini bees; Janzen, 1971). Studies related to emission and reduction of floral tube inner space
flight behaviour showed that these bees present strong orien- (Morawetz, 1982; Vieira et al., 1992; Sérsic and Rando,
tation and odour perception abilities even on extremely large 2004; Bittencourt and Semir, 2006; Yanagizawa and
areas of continuous forest (Ackerman et al., 1982; Roubik, Maimoni-Rodella, 2007).
1989; Roubik and Hanson, 2004). Even though Euglossini Overall, the staminode of J. oxyphylla does not seem to
bees are exclusively from forest habitats, E. nigrita also play any mechanical role in restricting access to pollen, as
occurs in fragmented areas (Wittmann et al., 1988; suggested for J. mimosifolia by Sérsic and Rando (2004),
Tonhasca et al., 2003; Milet-Pinheiro and Schlindwein, given that the small bees Ceratina, Augochlora and
2005). Eulaema nigrita is distributed from Costa Rica to Trigona removed similar quantities of pollen grains from
northern Argentina (Roubik and Hanson, 2004), comprising the anthers in flowers with and without a staminode. It is
the geographic distribution of J. oxyphylla (Gentry and also unlikely that the staminode of J. oxyphylla has an
Morawetz, 1992). This fact, associated with the foraging essential role in visual orientation as found in other
behaviour of E. nigrita (Roubik and Hanson, 2004), may species of Jacaranda (Vieira et al., 1992). This is due to
lead to the dispersal of pollen of J. oxyphylla over large intrapopulation variation in the colour pattern of capitate
areas. glandular trichomes encountered in J. oxyphylla, resulting
Unlike Euglossini, bees belonging to the genus Bombus in very distinctive visual patterns among flowers.
depend on the concentration of floral supplies In addition, the role of the staminode as a visual indicator
(Walther-Hellwig and Frankl, 2000) and present a beha- of flower senescence and consequent ending of nectar pro-
viour that tends to generate small-sized neighbourhoods duction was discarded for J. oxyphylla since the visual
(Schmitt, 1980). Thus, in the population of J. oxyphylla changes between fresh and old flowers were very discreet.
studied by Yanagizawa and Maimoni-Rodella (2007), the Similar results were found in J. racemosa (Bittencourt
high density of flowering individuals in a small area may and Semir, 2006). The function of secondary pollen presen-
have favoured the high frequency of visits of Bombus tation was not observed for the J. oxyphylla staminode since
atratus. In the present study, the low frequency of the pollen grains remain clustered inside the anthers.710 Guimarães et al. — The Pollination of Jacaranda oxyphylla
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