1. ARTICLE IN PRESS
Flora 201 (2006) 178–188
www.elsevier.de/flora
Pollination in Turnera subulata (Turneraceae): Unilateral reproductive
dependence of the narrowly oligolectic bee Protomeliturga turnerae
(Hymenoptera, Andrenidae)
Clemens Schlindweina,Ã, Petrucio C.R. Medeirosb
´
a
ˆnica, Universidade Federal de Pernambuco, (UFPE), Av. Prof. Moraes Re
ˆgo, s/n, Cidade Universita
´ria,
Departamento de Bota
50670-901 Recife, Brazil
b
´s-Graduaca em Biologia Vegetal, Universidade Federal de Pernambuco, Recife
Programa de Po
¸ ˜o
Received 15 March 2005; accepted 1 July 2005
Abstract
Turnera subulata Smith (Turneraceae) is a subshrub with distylic flowers, common as a ruderal plant in NE-Brazil.
We studied the pollination biology of a population in Joao Pessoa, Paraı´ ba, paying attention to effective pollinators
˜
and characteristics of short- and long-style morphs. The flowers attracted insects of 28 species, predominantely bees.
Several bee species were observed to be effective pollinators, including highly eusocial species, polylectic solitary
species (Centris and Xylocopa) and 1 oligolectic species, Protomeliturga turnerae (Andrenidae, Panurginae). The latter
species shows reproductive dependency on T. subulata. The plant species, on the other hand, does not depend on this
specialized bee, as reproductive success was also guaranteed by the other polylectic flower visitors. Floral
characteristics of both floral morphs are discussed with respect to pollination biology.
r 2005 Elsevier GmbH. All rights reserved.
Keywords: Turnera subulata; Heterostyly; Effective pollinators; Oligolectic bees; Northeast Brazil
Introduction
Turneraceae is 1 of at least 28 angiosperm families
showing heterostyly (Barrett, 1992, 2002; Barrett and
Richards, 1990; Ganders, 1979). Almost all species of
Turnera are distylic while homostylous populations
seem to be derived from heterostylic ancestors (Barrett,
1978; Barrett and Shore, 1987). Distyly is expressed in a
short-styled form with long stamens and a long-styled
one with short stamens and is linked to double
incompatibility, permitting fecundity only between longÃCorresponding author.
E-mail addresses: schlindw@ufpe.br (C. Schlindwein),
petruciomedeiros@ig.com.br (P.C.R. Medeiros).
0367-2530/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.flora.2005.07.002
and short-styled plants while intramorphic pollination is
incompatible (Darwin, 1877; Ganders, 1979; Richards,
1997; Vuilleumier, 1967). In general, additional features
are associated with each morph, like differences in
pollen morphology and size, number of pollen grains
and stigma morphology, which are controlled by a
supergene (Barrett, 2002; Richards, 1997; Shore and
Barrett, 1985a, b). These characters may have an
adaptive value with respect to the functioning of the
mating system (Richards, 1997).
Most studies on heterostyly have focused on the
genetic mechanism of self-incompatibility (Barrett and
Shore, 1985; Charlesworth, 1979; Ganders, 1979;
Richards, 1997; Shore and Barrett, 1985a, b, 1987,
1990) while information on interactions with pollinators

2. ARTICLE IN PRESS
C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
is scarce. Darwin (1877) suggested that visitors to
distylic flowers receive pollen at different specific parts
of the body promoting intermorphic pollination. Ganders (1979) indicated pollination especially by insects in
heterostylic plants.
Barrett (1978) studied the reproductive biology of
Turnera ulmifolia L. (vars. angustifolia (Mill.) DC.,
elegans (Otto ex Nees) Urb., intermedia Urb., surinamensis Miq.) in northern Brazil and lists insects of 13
species as flower visitors, mainly bees. None of the
flower-visiting species is oligolectic.
Ducke (1907) described Calliopsis turnerae from
´
the states of Maranhao and Ceara as one of the
˜
most common visitors of Turnera spp. flowers.
Later (Ducke 1912) he transferred the species to
the new genus Protomeliturga with P. turnerae as the
single species. Because of its mouth parts, which are
similar to those of long-tongued bees, Ruz (1991)
established the tribe Protomeliturgini (Andrenidae,
Panurginae) with P. turnerae as the monotypic
representative. The bees are oligolectic on flowers of
Turnera spp., males establish territories in flower
patches of T. subulata Smith and show multiple matings
in these flowers (Medeiros and Schlindwein, 2003,
Schlindwein, 2003).
Recently it was shown that interactions between
heterostylic plants and their pollinators can involve
highly specialized oligolectic bees like Ancyloscelis gigas
(Apidae, Emphorini) that possess extraordinarily long
mouth parts with specialized hairs to collect hidden
pollen from low-level anthers in flowers of tristylous
Eichhornia azurea (Pontederiaceae) (Alves-dos-Santos,
2003; Alves-dos-Santos and Wittmann, 1999, 2000). In
this case reproduction of bees and plants are interdependent.
In this study we asked the following questions: (1) In
which characteristics do long- and short-style flowers of
T. subulata diverge? (2) Which are the flower visitors of
both floral morphs? (3) Are bees of P. turnerae more
effective pollinators than polylectic bees? and (4) Does
reproduction of the plant and the oligolectic bee depend
on each other?
179
Field studies were carried out from March 1999 to
February 2001 in the surroundings of the agricultural
experimental research station of EMEPA (Empresa
´
Estadual de Pesquisas Agropecuarias da Paraı´ ba) near
Joao
Pessoa,
Paraı´ ba,
NE-Brazil
(71110 5800 S;
˜
0
00
34148 37 W, altitude 30–40 m, about 1 km distant from
the coast). The vegetation at the study site consists of
fruit crops and ruderal plants surrounded by seminatural vegetation of the Cerrado-like ‘‘Tabuleiro
Nordestino’’. The climate is tropical and humid (mean
annual precipitation and temperature 1600–1800 mm,
25–26 1C) (Fonseca and Azevedo, 1983; Lima and
Heckendorff, 1985).
Flower morphology and anthesis
Floral structures (diameter, length of petals, stamens,
filaments, anthers, styles) of short- and long-styled
flowers were measured in 30 flowers of different plants.
To test for significant differences in these measurements
we used test t (Zar, 1996).
Flower buds were numbered and monitored to
determine duration, time and sequence of anthesis and
number of flowers open per day and plant. The time of
stigma receptivity was determined with H2O2 (20%) and
a hand lense (Kearns and Inouye, 1993). Pollen viability
was verified by staining with acetocarmine jelly (Radford et al., 1974). The number of pollen grains per flower
was determined in 15 flowers of different plant
individuals per floral morph in non-dehisced anthers,
using Neubauer chambers.
Fresh pollen grains from 10 different flowers of each
morph were measured (30 measurements of equatorial
diameter per preparation) and the ornamentation of the
exine was analyzed under a microscope. Sugar concentration of nectar was measured with a hand refractometer (Atago, 0–90%) in previously bagged flowers.
The nectar amount was determined with micropipettes
(5 ml) every 60 min (n ¼ 10 flowers per hour).
´
Vouchers are deposed at the Herbario UPE Geraldo
Mariz, Botanical Department of the Federal University
of Pernambuco (UFP 24742, 24740, 24748).
Materials and methods
Breeding system
Study site
The breeding system was determined by a controlled
pollination experiment in both morphs: (1) spontaneous
selfing – flower buds were bagged before anthesis and
maintained closed until flower senescence; (2) hand selfpollination – flower buds were bagged before anthesis
and pollinated with pollen of the same flower; (3) interand intra-morph hand cross-pollination – bagged
flowers were opened and pollinated with pollen from
other individuals of T. subulata; (4) open, free pollination – for control, marked unbagged flowers were kept
T. subulata is a subshrub, common as a ruderal plant
in NE-Brazil. The species is part of the T. ulmifolia
complex (Solis Neffa and Fernandez, 2000; Urban,
1883). The ephemeral flowers are disc- to funnel-shaped.
The petals are cream-colored and show violaceous to
blackish nectar guides at the base that help the flower
visitors to find the food rewards (Dafni and Giurfa,
1998).

3. ARTICLE IN PRESS
180
C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
accessible to pollinators. Each treatment was performed
in 30 flowers. The flowers were monitored until fruit set.
Seeds were counted from all mature fruits.
Flower visitors and evaluation of effective pollinators
The spectrum of flower visitors was determined in
long- and short-styled morphs. Insects were captured
with entomological nets and specimens are housed in the
Entomological Collection of the Federal University of
Pernambuco. The collections were made during 5
consecutive days in January and September 2000.
The frequencies of visits by bees to the flowers of T.
subulata were determined by counting female and male
bees at the flowers during a total of 100 h of observation.
During each visit we checked whether the bees came into
contact with the stigma. The relative frequencies of
stigma contacts were calculated for males and females of
each bee species.
The relative abundance of Turnera pollen in the
scopal loads was taken to quantify the flower constancy
of the bees. Ten females of each bee species were
captured at flowers of T. subulata. Their pollen loads
were stripped off the scopa and some drops of 70%
ethanol were added. The pollen grains were mixed,
picked up on a small piece of glycerine gelatin,
transferred to a microscope slide, mounted with a cover
glass and sealed with paraffin wax. Two samples were
made of each pollen load, with pure glycerine gelatin
and with glycerine gelatin stained with alcohol–fuchsine
solution (Westrich and Schmidt, 1986). Pollen loads
were analyzed by counting at least 300 pollen grains per
sample. The number of pollen grains from short- and
long-styled morphs of T. subulata, as well as those of
other plant species was counted.
We also determined the number and type of pollen
grains attached to the stigmas during anthesis. The
stigmas of 15 long-styled and 15 short-styled flowers
were removed at 7:00, 8:00, 9:00, and 10:00 h. Each
stigma was transferred to a microscope slide containing
Table 1.
glycerin stained with fuchsine. Under the microscope,
pollen from short- and long-styled flowers of T. subulata
and of other plant species was counted. Fruit set and
deposition of pollen grains onto stigmas were determined in a period when the oligolectic bees were present
and compared to that of a period when these bees were
absent.
Results
Flower morphology and anthesis
T. subulata has erect, disc- to slightly funnel-shaped
flowers with cream-colored petals that are dark violaceous at the base (Fig. 1). Flower diameters measure
4.0–6.0 cm (x ¼ 5:1, SD ¼ 0.7, n ¼ 20). The distylic
flowers vary in 8 features (Table 1). Stamens and styles
show reciprocal hercogamy (Fig. 2). Stamen length of
short-styled flowers does not show significant difference
from style length of long-styled flowers (t ¼ 1:41,
GL ¼ 38, P40:05), as do stamens of long-styled flowers
Fig. 1. Longitudinal section of a short-styled (left) and longstyled flower of Turnera subulata.
Floral characters in the long- and short-styled morphs of Turnera subulata
Floral morph
Short-styled (x–SD–n)
Style length (mm)
Stigma length (mm)
Anther length (mm)
Filament length (mm)
Ovule number
Pollen grain number per anther
Pollen size (mm) (equatorial axis)
Ornamentation patterns of pollen
Long-styled (x–SD–n)
4.5–0.5–30
2.6–0.5–30
3.6–0.5–30
9.1–0.9–30
38.5–9.2–30
2720–54.3–15
76.8–2.38–300
Strong reticulum with 1–2 free bacula
in lumina
8.6–1.3–30
3.4–0.8–30
2.5–0.5–30
5–0.9–30
53–10.8–30
2900–52–15
64.1–1.92–300
Weak reticulum with 3–6 free bacula
in lumina

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C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
Small orifices (0.5 mm diameter) between the base of
filaments and petals allow access to the nectaries at the
base of the ovary. The distance between this aperture
and the nectary is less than 1 mm. Nectar, which is
already available at the beginning of anthesis, is
produced in small quantities (0.8–1.0 ml per flower) and
has a mean sugar concentration of 28–32% with little
variation during anthesis (Fig. 4a, b). Nectar volume
a
1.2
1
Volume [µl]
with styles of short-styled flowers (t ¼ 0:77, GL ¼ 38,
P40:05).
Pollen of both forms is sticky and orange due to
abundant pollenkitt. When pollenkitt was removed the
grains were light yellow. Pollen grains of short-styled
flowers are larger than those of long-styled flowers and
also show a more distinct reticulate ornamentation with
more free bacula in the lumina. There is no overlap in
size variation of pollen grains between both morphs
(Fig. 3). Staining with karmin acid indicated high pollen
viability in both morphs, 92% (SD ¼ 0.5, n ¼ 15) in
short-styled flowers and 94% (SD ¼ 0.6, n ¼ 15) in
long-styled flowers. Non-viable pollen grains are empty
and smaller and were not considered in the measurements of size. Short-styled flowers produce less pollen
grains (13,600) than long-styled flowers (14,500).
181
0.8
0.6
0.4
0.2
16
0
14
06:00
09:00
10:00
7:00
8:00
Hour
9:00
10:00
35
10
30
8
Concentration (%)
style length (mm)
b
08:00
Hour
6:00
12
07:00
6
4
2
25
20
15
10
5
0
0
2
4
6
8
10
12
14
16
0
stamen length (mm)
short-style
long-style
Fig. 2. Stamen and style length in short- and long-styled
morphs of Turnera subulata (n ¼ 30 for each floral morph).
Fig. 4. Nectar volume and concentration of sugars in flowers
of Turnera subulata. (a) Nectar volume, (b) sugar concentration (n ¼ 10 per hour).
long-styled morph
Number of grains
60
short-styled morph
50
40
30
20
10
0
60-62
62-64
64-66
66-68
68-70 70-72 72-74
Size classes (µm)
74-76
76-78
78-80
80-82
Fig. 3. Pollen grain size of short- and long-styled morphs of Turnera subulata (n ¼ 300).

5. ARTICLE IN PRESS
182
Table 2.
C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
Breeding system of Turnera subulata
Pollination treatment
Floral morph
n
Fruit set
(n)
Fruit set
(%)
No. of seeds
produced
Mean seed set per
capsule x (SD)
Bagged (not treated)
S
L
S
L
La  Lb
Sa  Sb
Sa  Lb
La  Sb
S
30
30
30
30
30
32
33
32
30
0
0
0
0
0
0
29
31
26
0
0
0
0
0
0
88
97
87
0
0
0
0
0
0
915
713
389
0
0
0
0
0
0
27.7 (14.75)
23 (10.35)
19.5 (9.4)
L
30
29
97
397
19.9 (11.2)
Hand self-pollinated
Hand cross-pollinated
Open pollinated flowers
(controls)
Compatibility and seed set following controlled self-pollination, and intra- and inter-morph cross-pollination (S ¼ short-styled morph, L ¼ longstyled morph, x ¼ average, SD ¼ standard deviation).
a
Pollen receptor.
b
Pollen donor.
and concentration of sugars in short-styled flowers did
not differ from that of long-styled flowers.
T. subulata flowered and set fruit during the whole
year. Per individual plant, 3–15 flowers opened daily at
6:00 h synchronically closing at about 11:00 h. The fruits
took 8–10 days to reach maturity.
Breeding system
The flowers of T. subulata are self-incompatible
(Table 2). Hand self-pollinated as well as intra-morph
hand cross-pollinated flowers did not set fruit. Fruit set
of open pollinated short- and long-styled flowers was
similar. Seed set of these flowers, on the other hand, was
reduced when compared with inter-morph hand crosspollinated flowers (Table 2). Short-styled flowers accessible to flower visitors produced on average only 30%
and long-styled flowers 36% of the number of seeds as
the hand cross-pollinated flowers, and the number of
seeds per fruit that resulted from pollination by flower
visitors was almost the same in the 2 morphs. Shortstyled flowers visited by bees showed similar seed set
(x ¼ 19:5; SD ¼ 16.6; n ¼ 30) as long-styled flowers
(x ¼ 19:9; SD ¼ 9.1; n ¼ 30). Pollen–ovule ratio (P/O
ratio) was 353.2 in short-styled flowers and 273.6 in
long-styled flowers. Considering the mean number of
ovules, short-styled flowers produced 43.8% and longstyled flowers 56% of the potential number of seeds in
the hand cross-pollinated treatments.
Flower visitors, their pollen loads and deposition of
pollen grains on stigmas
The flowers of T. subulata were visited by insects
of 28 species of the orders Hymenoptera, Lepidoptera
and Coleoptera (Table 3). Bees were predominant
(24 spp.), especially those in the family Apidae. The
bees did not differentiate between short- and long-styled
flowers. We counted 1408 (48%) flower visits in
short-styled flowers and 1534 (52%) in long-styled
flowers.
In January the most frequent species to visit both
flower morphs were highly eusocial species (Apis
mellifera 31.5% of the visits, Trigona spinipes 23.9%,
Frieseomelitta doederleinii 16.2%) while in September
bees of the solitary panurgine Protomeliturga turnerae
were the most frequent (27%) followed by social A.
mellifera (24%), Trigona spinipes (17%), F. doederleinii
(16%), and Plebeia flavocinta (9%) (Fig. 5). In January
P. turnerae did not occur. Flower visits were most
frequent between 6:30 and 8:00 h, soon after beginning
of anthesis of T. subulata (Fig. 6).
With the exception of F. doederleinii, bees of all
species touched the style frequently (Fig. 7). Large bees,
like those of Centris and Xylocopa showed a higher rate
of stigma contact than medium- or small-sized species,
but showed low frequencies of flower visits. The
frequency of stigma contacts during flower visits in
long-styled flowers was slightly higher than those in
short-styled flowers.
Styles of long-styled flowers received more pollen
grains than short-styled flowers. In long-styled flowers
55.2% of the pollen grains were legitimate, while shortstyled received only 44.5% of legitimate pollen grains. A
higher amount of pollen of short-styled flowers reached
the stigmas of both flower morphs. Only 4% of the
pollen grains deposited on the stigmas came from other
plant species. After only 1 h after the beginning of
anthesis 55.1% of the final mean number of pollen
grains was deposited on short-styled stigmas and 93%
on stigmas of long-styled flowers (Fig. 8).

6. ARTICLE IN PRESS
C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
Table 3.
183
Flower visitors of Turnera subulata in Joao Pessoa, Paraı´ ba
˜
Flower visitors
Males
Females
Hymenoptera
Andrenidae
Protomeliturga turnerae (Ducke, 1907)
Psaenythia variabilis Ducke, 1910
+
À
+
+
Apidae
Apis mellifera Linnaeus, 1758
Centris (Centris) aenea (Lepeletier, 1841)
Centris (Centris) flavifrons (Fabricius, 1775)
Centris (Centris) leprieuri (Spinola, 1841)
Centris (Hemisiella) tarsata Smith, 1874
Centris (Xanthemisia) lutea Friese, 1899
Ceratina (Crewella) maculifrons Smith, 1844
Ceratinula muelleri Moure, 1941
Epicharis (Xanthepicharis) bicolor (Smith, 1854)
Eulaema nigrita Lepeletier, 1841
Frieseomelitta doederleinii (Friese, 1900)
Plebeia flavocincta (Cockerell, 1912)
Trigona spinipes (Fabricius, 1793)
Xylocopa (Megaxylocopa) frontalis (Olivier, 1789)
Xylocopa (Neoxylocopa) cearensis Ducke, 1910
Xylocopa (Neoxylocopa) suspecta Moure & Camargo, 1988
Xylocopa (Schonnherria) muscaria (Fabricius, 1775)
À
+
À
+
+
À
À
À
À
+
À
À
À
À
À
À
À
+
+
+
+
+
+
+
+
+
À
+
+
+
+
+
+
+
Halictidae
Augochloropsis sp.
Augochlorella sp.
Pereirapis semiaurata (Spinola, 1851)
Pseudoaugochlora sp.
À
+
+
+
+
+
Megachilidae
Dicranthidium arenarium (Ducke, 1907)
+
À
Lepidoptera
Hesperiidae
Nisoniades macarius (Herrich-Schaffer, 1870)
¨
Urbanus dorantes dorantes (Stoll, 1790)
Urbanus proteus proteus (Linnaeus, 1758)
À
À
+
+
+
+
Coleoptera
Curculionidae
Pristimerus calcaratus (Boheman, 1843)
+
+
A mean of 5.2 pollen grains of long-styled flowers
were deposited on stigmas of short-styled flowers to
fertilize 1 ovule and 6 grains of short-styled flowes to
fertilize 1 ovule of long-styled flowers.
Analysis of the pollen loads of females of the 6 most
frequent flower-visiting bee species showed that P.
turnerae and Plebeia flavocinta exclusively collected
pollen of T. subulata (Fig. 9). The loads of the highly
eusocial A. mellifera, Trigona spinipes and F. doederleinii
contained only small quantities (1–2%) of pollen grains
of other plant species. Females of Augochloropsis sp.
always had mixed pollen loads containing predominantely pollen of Borreria verticillata (Rubiaceae) besides
those of T. subulata. The pollen loads of all bee species
contained more pollen grains of short-styled flowers
than of long-styled flowers of T. subulata. Thus, the
pollen ratio short- to long-styled to foreign flowers of all
visitors was similar to that deposited on the stigmas of
both floral morphs, indicating that pollen is not directed
to the legitimate stigma.
The behavior during flower visits of P. turnerae, A.
mellifera, Augochloropsis sp., F. doederleinii, and Trigona spinipes was similar. When visiting flowers of shortstyle-morphs, these bees alighted on the anthers, and
collected the pollen grains with the forelegs while
maintaining the head directed to the center of the
flower. At the same time they generally touched the
stigma. Pollen grains adhered mainly to the ventral

7. ARTICLE IN PRESS
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C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
300
Flower visits
250
200
150
100
50
Centris lutea
Xylocopa
frontalis
Centris hyptidis
Centris aenea
Xylocopa
muscaria
Centris
flavifrons
Ceratina
maculifrons
Augochloropsis
sp.
Plebeia
flavocinta
Frieseomelitta
doederleinii
Trigona
spinipes
Protomeliturga
turnerae
Apis mellifera
0
Fig. 5. Frequency of flower visitors of Turnera subulata in January and September 2000; January 2000 white bars, September 2000
black bars (50 h of observation in both months).
Apis mellifera
Trigona spinipes
Frieseomelitta doederleinii
Plebeia flavocincta
Ceratina maculifrons
0
11
0:3
10
0:0
10
30
9:
:0
0
10
0:
-1
-9
00
9:
:3
00
0
:3
0
8:
30
-9
:0
0
8:
00
-8
:3
0
7:
30
-8
:0
0
:3
7:
00
-7
:0
-7
30
6:
6:
00
-6
:3
0
40
35
30
25
20
15
10
5
0
0
Number of flower visits
Protomeliturga turnerae
Hour (30-min intervalls)
Fig. 6. Frequency of flower visits of the most abundant species to Turnera subulata during anthesis (observations during 5
subsequent days in September 2000).
region of the meso- and metasoma of the bees. In longstyled flowers, the bees landed on the stigmas, touching
them with the ventral region of their meso- and
metasoma. Then, they penetrated the flowers with their
heads directed downwards to collect nectar. Pollen was
collected with the forelegs. Pollen grains adhered mainly
to the head and anterior portion of the mesosoma.
Females of P. turnerae collected pollen grains of longstyled flowers with rigid hairs of the labrum.
Besides the bees, beetles of 3 species were recorded in
the flowers of T. subulata: Pristimerus calcaratus
(Curculionidae), a meloid and a chrysomelid beetle.
The beetles started flower visits at about 8:00 h,
collecting nectar, eating stigmas and pollen. Further-
more, they used the flowers as mating sites. Ocassionally, we observed that the Pristimerus beetles perforated
the petals with their proboscides and closed the flowers
with their claws.
Discussion
Effective pollinators of T. subulata
The flowers of T. subulata attract insects of numerous
species, especially bees. At the study site, bees of 24
species visited the flowers. When other localities in

8. ARTICLE IN PRESS
185
100
80
60
40
20
Urbanus
dorantes
Xylocopa
muscaria
Centris
flavifrons
Ceratina
maculifrons
Augochloropsis
sp.
Friesiomelitta
doederleini
Trigona
spinipes
Apis mellifera
0
Protomeliturga
turnerae
Frequency of stigma contacts (%)
C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
Fig. 7. Relative frequency of stigma contacts by flower visitors to short-styled (black bars) and long-styled flowers (white bars) of
Turnera subulata.
a
Pollen of short-styled flowers
Pollen of long-styled flowers
Pollen of other spp.
Number of pollen grains
350
300
250
200
150
100
50
0
7:00 h
8:00 h
9:00 h
10:00 h
Hour
b
Number of pollen grains
350
300
250
200
150
100
50
0
7:00 h
8:00 h
9:00 h
10:00 h
Hour
Fig. 8. Number (mean, SD) of pollen grains deposited by flower visitors on the stigmas of short-styled (a) and long-styled flowers (b)
of Turnera subulata during anthesis. Legitimate pollen in long-styled flowers comes from the long stamens of short-styled morphs,
and in short-styled flowers from the short stamens of long-styled morphs.
NE-Brazil are included, this number rises to 46 species
(Schlindwein and Medeiros, not published). This shows
the importance of the Turnera flowers as pollen and
nectar sources for bees, and suggests that they are
generalized melittophilous blossoms. As the plants set
flowers throughout the year, they are reliable food

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Pollen of short-styled flowers
Pollen of long-style flowers
Pollen of other spp.
70
Pollen amount [%]
60
50
40
30
20
10
0
Apis mellifera
Trigona spinipes Protomeliturga Augochloropsis
turnerae
sp.
Plebeia
flavocincta
Frieseomelita
doederleini
Fig. 9. Pollen amount (mean, SD) of short-styled flowers, long-styled flowers and of other plant species in the pollen loads of the
most frequent flower-visiting bees of Turnera subulata (n ¼ 10 bees per species).
sources, especially for perennial bee species. The easy
access to pollen and nectar allows flower visits of large
and small bees and of bees with short or long tongues.
No morphological adaptations seem to be necessary to
collect floral resources from T. subulata.
By comparing several measurements related to
pollinator effectivity like frequency of stigma contacts,
relative frequency of flower visits and flower constancy,
several species have to be considered effective pollinators: the solitary P. turnerae, the very common, highly
eusocial A. mellifera and Trigona spinipes, as well as the
less frequent, large, solitary Centris and Xylocopa
species.
P. turnerae is the only oligolectic species among all
flower visitors. Reproduction of this species depends
completely on the presence of T. subulata flowers. These
flowers are not only the unique pollen source of this
species at the study site, but also the sites of male
territories and mating (Medeiros and Schlindwein,
2003). Because of this close relation of P. turnerae bees
to Turnera flowers, it was to be expected that these bees
are more effective pollinators than polylectic species. In
fact, in most cases oligolectic bees are the most effective
pollinators of their specific food plants (Schlindwein,
2004; Schlindwein and Wittmann, 1995, 1997a, b;
Schlindwein and Martins, 2000; Schlindwein et al.,
2005). Because of morphological adaptations of the
oligolectic bees combined with a highly efficient pollen
collection behavior, their specific food plants generally
quickly become unattractive for polylectic species in the
presence of the specialized bees.
In the studied case, P. turnerae indeed is an effective
pollinator of T. subulata, but we found no evidence that
indicates a competitive advantage of this species over,
for instance, the polylectic workers of A. melifera or
Trigona spinipes. The dependency of the Protomeliturga
bees on the flowers of Turnera is not accompanied by
any dependency of the plant species on the oligolectic
bee. In the absence of Protomeliturga bees at the study
site (January) and at the campus of the Federal
University of Pernambuco, Recife, where bees of P.
turnerae do not occur, fruit set and pollen deposition on
the stigmas of T. subulata is high as well (Schlindwein
and Medeiros, not published).
For the tristylous flowers of Eichhornia azurea
(Swartz) Kunth (Pontederiaceae), Alves-dos-Santos
and Wittmann (1999, 2000) showed that the oligolectic
bees of Ancyloscelis gigas (Apidae, Emphorini) were the
most important pollinators of the short-styled morph,
the most hidden level of stigmas and anthers. In our
study, the bees of P. turnerae did not show preference to
the low-level morph. Stigma contacts to the short-styled
morph were not more frequent than those of competitors, and pollen grains from low-level anthers (those of
the long-styled morph) were less frequent in their
scopae, just as in the other bee species.
Thus, bees of P. turnerae are no better or more
important pollinators than most of the other bee species
found in flowers of T. subulata and dependency of the
oligolectic species is unilateral.
Surprisingly, the flower-closing curculionid beetle,
Pristimerus calcaratus, which competes for floral resources of Pavonia cancellata Cav. (Malvaceae) with the
olgolectic bee Ptilothrix plumata (Apidae, Emphorini)
(Schlindwein and Martins, 2000), also visited the
flowers of sympatric T. subulata. However, their
frequency in T. subulata flowers was low and, therefore,
an impact on other flower visitors is not to be expected.

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C. Schlindwein, P.C.R. Medeiros / Flora 201 (2006) 178–188
The questions of whether the beetles use the T. subulata
flowers as well as the flowers of P. cancellata, or if they
mistake Turnera for Pavonia flowers, which are similar
in shape, color and time of anthesis, need further
investigation.
Differences between short- and long-styled morphs
Long- and short-styled morphs of T. subulata differ in
at least 8 characteristics which are similar to the ones
detected in other species of the genus (Barrett, 1978;
Barrett and Shore, 1985, 1987; Belaoussoff and Shore,
1995; Shore and Barrett, 1984). Short-styled flowers
produce less ovules (38.5) and pollen grains (13,600)
than long-styled flowers (53, 14,500). P/O ratio is 353.2
for short-styled flowers and 273.6 for long-styled
flowers. This P/O ratio, however, is related to illegitimate pollination. The corrected relationship (legitimate
pollination), ovules of short-styled flowers compared to
pollen of long-styled flowers and vice versa, results in a
bigger difference of P/O ratios among both morphs: P/O
ratio in short-styled flowers is 376.6 and in long-styled
flowers 256.6. Following Cruden (1977), the more
difficult legitimate pollen deposition in a given breeding
system is, the higher the P/O ratio is, then the shortstyled flowers of T. subulata would receive less pollen
grains than the long-styled ones. In fact, in our study the
morph which received less pollen, reflecting a hindered
pollen deposition, showed the higher P/O ratio (see
Fig. 8). In addition, more pollen from short-styled
flowers – in other words from long stamens – was
deposited on stigmas of both morphs. Despite of the
difference of ovule number in both morphs, mean seed
set was the same in short- and long-styled flowers of T.
subulata.
Different as predicted in species with reciprocal
hercogamy by Darwin (1877), in the case with T.
subulata legitimate pollen transfer is not favored by the
flower visitors. The higher P/O ratio in short-styled
flowers might be interpreted as a compensation of
hampered legitimate pollen flow in this floral morph. It
is surprising that heterostyly in T. subulata seems not to
have a function in directing pollen in the expected way.
Acknowledgements
We thank the EMEPA for the permission to work at
the Agricultural Experimental Research Station in Joao
˜
Pessoa, Celso Feitosa Martins (UFPB) and Isabel
Cristina Machado (UFPE) for suggestions, Evelise
Locatelli, Reisla Oliveira Darrault and Celso F. Martins
for their help in the field, Scott Vinson Heald (Cornell
University) for revising the English and the Brazilian
Research Council (CNPq) for financal support.
187
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