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Contents lists available at ScienceDirect
Flora
j ournal homepage: www.elsevier.com/locate/flora
Low legitimate pollen flow in distylic Turnera hermannioides
(Passifloraceae) and its consequences on fruit and seed set
a,∗ b c
Paulo Milet-Pinheiro , Diana Corrêa de Andrade Penante , Clemens Schlindwein
a
Institute of Experimental Ecology, University of Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany
b
Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901, Brazil
c
Departamento de Botânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil
a r t i c l e i n f o a b s t r a c t
Article history: Turnera hermannioides is a ruderal distylic subshrub, native to NE-Brazil. In the Catimbau National
Received 16 May 2013
Park, situated within the semi-arid Caatinga region, we studied the pollination ecology of this species,
Accepted 23 September 2013
emphasizing (1) effective pollinators; (2) characteristics of short- and long-styled flowers; (3) intra-
Available online xxx
and intermorph pollen flow; and (4) fruit and seed set. Short and long-styled morphs differ in pollen
size and ornamentation, stigmatic surface, style and stamen length and nectar production. The flowers
Keywords:
are obligate intermorphic outcrossers and depend on animals for pollination. The flowers of T. herman-
Heterostyly
nioides attracted insect visitors of 25 species, among them butterflies, beetles, but mainly bees. Polylectic
Oligolectic bees
bees, such as Apis mellifera, stingless bees, and solitary Callonychium brasiliense were the most frequent
Polylectic bees
visitors and the principal pollinators. The frequency of visits, however, was very low (on average 1.9
Pollen flow
Pollination visits/flower/day), resulting in a low and unbalanced legitimate pollen flow, i.e. a much lower number
Turneraceae-clade of pollen grains from short-styled flowers reaching stigmas of long-styles than vice versa, which in turn
compromised fruit and seed set. Surprisingly, Protomeliturga catimbaui and P. turnerae, both oligolectic
on flowers of the Turneraceae-clade of the Passifloraceae s.l., were very rare flower visitors and did not
contribute significantly to the pollination of Turnera hermannioides. We discuss how the low visitation
rate and the inefficiency of pollinators (i.e. inefficiency in promoting legitimate pollination) compromise
pollen flow and, consequently, fruit and seed set in this species.
© 2013 Elsevier GmbH. All rights reserved.
Introduction fruit set (Ganders, 1979). In some cases, associations involve
oligolectic bees, which restrict pollen collection to a few plant
Heterostyly is a heteromorphic self-incompatibility system in species of a given genus or family to rear their offspring (Cane
which individuals of the same species present a floral polymor- and Sipes, 2006; Robertson, 1925). Recent studies documented
phism that is expressed in reciprocal differences in anther and very tight relationships of oligolectic bees to their heterosty-
stigma length, and often by additional features, such as number, lous host plants, showing mutual reproductive dependence. In
size and ornamentation of pollen grains, and length of stigmatic these cases, the pollinating bees are provided with morphologi-
lobes and papillae (Barrett, 1990; Darwin, 1877; Ganders, 1979). In cal and/or behavioral adaptations to optimize resource gathering
the two (short-and long-styled; distyly) or three (short-, mid- and in those plants (Alves-dos-Santos and Wittmann, 2000; Carvalho
long-styled; tristyly) flower morphs of heterostylic plants, fecun- and Schlindwein, 2011; Milet-Pinheiro and Schlindwein, 2010;
dity is only possible (or often seed set is maximized) if legitimate Schlindwein and Wittmann, 1995, 1997a, 1997b). The absence of
pollination occurs, i.e. if intermorphic pollen grains of the appropri- the specialized pollinators generally results in diminished fruit set
ate height level are deposited on the stigmas. This heteromorphic (Carvalho and Schlindwein, 2011; Milet-Pinheiro and Schlindwein,
self-incompatibility system has evolved independently in several 2010) and in di- or tristylic species even in breakdown of het-
angiosperm lineages and has been recorded in at least 28 families erostyly (Alves-dos-Santos, 2002; Barrett, 1988). In other cases,
(Vuilleumier, 1967). oligolectic bees depend on their specific host plants as exclu-
Heterostylous plants are often obligate outcrossers and, accord- sive pollen sources, but the plants, in general, do not depend on
ing to the pollination system, pollinators are required to guarantee the bees as exclusive pollinators (Minckley and Roulston, 2006;
Schlindwein, 2004).
Turnera Smith (Passifloraceae) is the largest genus of the Turn-
∗ eraceae clade of Passifloraceae s.l. (formerly Turneraceae; APG III,
Corresponding author.
E-mail address: [email protected] (P. Milet-Pinheiro). 2009; Thulin et al., 2012), distributed mainly in the neotropics, and
0367-2530/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.flora.2013.09.005
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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most of the about 130 species are distylous (Shore et al., 2006). flower opening and wilting, anther dehiscence, stigma receptivity
Flowers in this genus are pollinated mainly by bees, including both (with H2O2 at 20%, Kearns and Inouye, 1993), and nectar availabil-
polylectic and oligolectic species (Barrett, 1978; Schlindwein and ity. Nectar amount was measured at 12:00 h in bagged flowers of
Medeiros, 2006). Protomeliturgini is a monogeneric bee tribe rep- 20 plants for each morph (n = 1 flower per individual) with micro-
®
resented by two species, Protomeliturga turnerae (Ducke, 1912) and capillaries (0.5 l, Duran ).
P. catimbaui Schlindwein and Moure, 2005. Protomeliturga turnerae The proportion of short and long-styled individuals in the pop-
is an oligolectic species that collects pollen exclusively in flow- ulation was determined for 65 plants.
ers of Turnera. In addition to the use as a food source, this bee
uses flowers of T. subulata as mating site; the males establish ter- Pollen
ritories in flowers for several days, where they patrol in search of
females (Medeiros and Schlindwein, 2003). In spite of this food and To describe exine ornamentation and measure the size of the
reproductive dependence by P. turnerae, its absence does not com- pollen grains, we prepared microscope slides from short- and long-
promise the fruit set of T. subulata, since polylectic flower visitors styled flowers (n = 10 for each morph) of different individuals.
are also effective pollinators (Schlindwein and Medeiros, 2006). Grains were removed from anthers with a small piece of glyc-
Protomeliturga catimbaui is a rare species endemic to the Brazilian erin gelatine, mounted with a cover glass and sealed with paraffin
Caatinga (a tropical dry forest, characterized by succulent plants wax (Louveaux et al., 1978). Under a Zeiss Axiolab microscope (at
×
and thorn-shrubs) and so far it was recorded only in the Catimbau 1000 ), we measured the pollen grains in equatorial view (50 per
National Park (Northeastern Brazil) in flowers of Piriqueta sidifolia flower, n = 10 flowers per morph) and analyzed the ornamentation
(Cambess) Urban/Passifloraceae, Turneraceae clade (Schlindwein of the exine. Unviable empty pollen grains were not considered for
and Moure, 2005). the measurements of size.
We studied the pollination ecology of Turnera hermannioides, a Pollen viability was determined using the methods of Radford
distylic subshrub (Barrett and Shore, 1985), which is common to et al. (1974). Grains from short- and long-styled flowers (n = 10
the Catimbau National Park, and asked: (1) which features diverge for each morph) were removed from anthers fixed in FAA, and
between short- and long-styled flowers? (2) What is the spectrum transferred to microscope slides with acetocarmine solution. Three
and frequency of flower visitors? (3) How does pollen flow between hundred pollen grains per flower were counted under the micro-
and within morphs? (4) What is the fruit and seed set of short- and scope to determine the proportion of stained viable vs. non-stained
long-styled morphs? unviable grains.
The number of pollen grains per flower was determined in
Materials and methods flower buds of 15 plants for each morph (n = 1 flower per indi-
vidual). The grains were removed from all anthers available in a
Study site flower, placed in a Petri dish, and counted under a stereomicro-
scope (Zeiss, Stemi SV8). We placed a grid paper below the Petri dish
The study was performed between August 2007 and November to avoid counting grains twice or more. The ovaries of short- and
◦ ◦
2008 in the Catimbau National Park (S 08 32 54,2 ; W 37 14 49,6 ), long-styled flowers (n = 15 for each morph) were dissected and the
situated in the municipalities of Buíque, Tupanatinga, and Ibimirim, ovules counted under a Zeiss Stemi SV8 stereomicroscope (Dafni
State of Pernambuco, Northeastern Brazil. The Park covers about et al., 2005).
62,000 hectares with altitudes varying from 700 to 1100 m (IBAMA,
2008). The climate is tropical semi-arid and the average annual Breeding system
◦
temperature and precipitation is 21.5 C and 1100 mm, respectively
(ITEP, 2008). The vegetation of the Park is composed by ever- To determine the breeding system of T. hermannioides, the fol-
green shrubs and small trees among which typical species of the lowing pollination experiments were performed for both morphs:
surrounding Caatinga intermingle, the common vegetation of semi- (a) spontaneous self-pollination – flowers were bagged and left
arid NE-Brazil (Andrade et al., 2004; Gomes et al., 2006; Rodal et al., unmanipulated; (b) hand self-pollination – bagged flowers were
1998). pollinated with self-pollen; (c) inter- and intramorph hand cross-
pollination – flowers were pollinated with pollen donors of either
Studied species the same (intra) or the alternate morph (inter), from distances of at
least 1 km; and (d) pollination of open flowers accessible to flower
Turnera hermannioides Cambess is a distylic perennial shrub, visitors under natural conditions (control). For each treatment in
native to Brazilian Northeast, recorded in the States of Sergipe, each morph, 40 flowers bagged before anthesis were used, except
Bahia, and Pernambuco (Barrett and Shore, 1985). This ruderal the 45 marked flowers per morph used as control. Mature fruits
species occurs mainly in places where original vegetation was were counted and collected to count the seeds. For each treatment,
removed for pasture or agriculture. we measured fruit and seed set as the ratio of fruits to flowers and
as the mean number of seeds per flower, respectively.
Flower biology
Flower visitors
Flower morphology is described using fresh flowers from both
morphs (n = 30 flowers per morph). We measured the corolla diam- The frequency of flower visitors was determined in March and
eter and the length of stamens and styles using a digital caliper April 2008. In each month, observations were made on three con-
(Stainless Digimess). Additionally, floral parts were analyzed by a secutive days, between 07:00 and 13:00 h, summing up 36 h of
scanning electron microscope Zeiss LEO VP 438 (SEM). Material was observation. Ten short- and ten long-styled flowers were observed
dehydrated in a graded ethanol series, critical point dried follow- per day; each group of 10 flowers was monitored by one person.
ing the standard procedure, and sputter coated with gold (Robards, During observations, we described the behavior of each visitor
1978). species and recorded the resources sought. Effective pollinators
Thirty-six flower buds from each morph (n = 10 individuals per were determined based on their frequency, contacts to stigmas and
morph) were bagged, marked, and monitored from their opening anthers, and flights performed among con-specific plants. Addi-
to abscission in order to describe anthesis. We recorded the time of tionally, we screened the body of flower-visiting insects (bees,
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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Fig. 1. Turnera hermannioides and insect visitors in the Catimbau National Park. (A) Upper view and (B) longitudinal section of short- (left) and long-styled flowers (right).
Note the similar height of style and stamens in flowers of different morphs. Flower visitors: (C) Apis mellifera and (D) Trigona spinipes.
beetles, and butterflies) for the presence of pollen using a stereo- seed set among treatments and pollen flow (legitimate vs. illegiti-
microscope. mate, legitimate vs. legitimate and illegitimate vs. illegitimate) in
The relative frequency of pollen from short- and long-styled short- vs. long-styled flowers (emasculated and non-emasculated
morphs of T. hermannioides in the scopae of flower visiting female flowers were analyzed separately). The Tukey–Kramer test for non-
bees was taken to determine their flower fidelity. Uniform scopa parametric data was used as post hoc test (Siegel and Castellan,
loads were considered indicative of flower constancy. Samples of 1988). The normality of the samples for all tests was verified by
pollen from the scopa of the more frequent flower visitors were Kolmogorov–Smirnov tests. All statistical analyses were performed
removed from five individuals per species with small pieces of glyc- using Statistica 7 (StatSoft, 2004), except the G-tests and Fisher’s
erin gelatine, transferred to a microscope slide, mounted with a exact tests, which were performed using the spreadsheets provided
cover glass and sealed with paraffin wax. Pollen loads were ana- by http://udel.edu/∼mcdonald.html (accessed 26.8.13, McDonald,
lyzed by counting the first 300 pollen grains per sample. The ratio 2009).
of pollen grains from short- and long-styled morphs of T. herman-
nioides, as well as those of other plant species adhering to the scopae
of the visitors, was counted under the microscope. Voucher specimens
The flower-visiting insects are deposited in the Entomologi-
Pollen flow
cal Collection of the Federal University of Pernambuco (UFPE) and
the Federal University of Minas Gerais (UFMG). Plant vouchers are
For qualitative and quantitative assessment of pollen flow
stored in the Herbarium Geraldo Mariz UPE, Recife.
between flowers, we measured pollen deposition on stigmas,
simultaneously with the frequency counts of flower visitors. To
determine inter- and intramorph pollen flow (hereafter ‘legitimate’
Results
and ‘illegitimate’ pollen flow, respectively), we emasculated 10
flowers of each morph on different individuals at the beginning
Flower biology
of anthesis. At the end of anthesis, the stigmas were removed and
mounted on microscope slides as described above for pollen count.
Turnera hermannioides bears open disk- to funnel-shaped flow-
Simultaneously to the measurements for emasculated flowers, we
ers. The corolla is cream-colored becoming gradually yellowish
measured the legitimate and illegitimate pollen flow at the end of
toward the center of the flowers (Fig. 1A). Nectar and pollen are
anthesis for non-emasculated flowers (n = 10 flowers per morph).
accessible to flower visitors; small channels between the bases of
filaments and petals lead to the nectar disk at the ovary base, where
Statistical analyses pollinators inserted their mouthparts to collect nectar. Corolla
diameter of short- and long-styled flowers was on average 30.3 mm
Student’s t-test (independent samples) was used to test for (±2.7, SD) and 32.6 mm (±3.3), respectively. The flowers show
differences between short- and long-styled flowers with regard reciprocal herkogamy (Fig. 1B) with respect to style and stamen
to nectar amount, number and mean size of pollen grains, and length, i.e. anthers and stigmas of different morphs are disposed
number of ovules (Zar, 1999). Differences between both the fre- at the same height. Style length was, on average, 6.8 mm (±0.6 SD,
quency of short- and long-styled individuals in the population n = 30) for short- and 11.5 mm (±0.6, n = 30) for long-styled flowers.
and the frequency of visits to short- and long-styled flowers, was Stamen length was, on average, 11.2 mm (±0.8 SD, n = 30) and 7 mm
2 ±
assessed by goodness-of-fit tests. A G-test was performed to ( 0.6, n = 30) for short- and long-styled morphs, respectively. Stig-
assess differences in fruit set among pollination treatments. Fisher’s matic lobes of short-styled flowers were inserted at a single level,
exact tests were used for post hoc comparisons between each while in long-styled ones they ramified within an area of about
treatment pair. Kruskal–Wallis tests were performed to compare 0.7 mm of the stylar apex.
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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Table 1
Controlled pollination experiments in Turnera hermannioides. S: short-styled; L: long-styled; r: pollen receptor; d: pollen donor. Distinct capital letters (A–C) indicate
significant differences among treatments in terms of the proportion of flowers that set fruits and flowers that did not set fruits, assessed using a G-test (G = 198.7, df = 9, p = 0)
and Fisher’s exact test as post hoc comparisons. Distinct low letters (a–d) indicate significant differences in mean seed set among treatments, assessed using a Kruskal–Wallis
test (H(9,398) = 203.9, p < 0.001) and Tukey–Kramer test as post hoc comparisons.
Treatment Morph Flowers (n) Fruit set (%) Seed set (mean ± SD) Comparisons (fruit/seed)
Spontaneous self-pollination S 39 2.6 0.05 ± 0.3 A/a
L 40 2.5 0.3 ± 1.7 A/a
Hand self-pollination S 39 5.5 0.5 ± 3.2 A/a
L 39 2.6 0.05 ± 0.3 A/a
r d
Hand cross-pollination L × L 39 5.5 0.05 ± 0.2 A/a
r d
S × S 39 15.4 0.4 ± 1.1 A/ab
r d
× ± S L 40 77.5 14.8 11.1 B/d
r d
L × S 36 83.3 18.3 ± 10.5 B/d
Open-pollinated flowers (control) S 43 51.2 5.9 ± 8.6 C/bc
L 44 47.7 6.1 ± 7.6 C/bc
Anthesis was synchronous among plants and started between Flower visitors
06:00 h and 07:30 h, depending on weather conditions; flowers
closed between 12:00 h and 13:00 h. Tests with hydrogen perox- We recorded 25 species of insects in flowers of T. hermannioides,
ide indicated that stigmas are receptive throughout the anthesis. predominantly bees (15 spp.) and butterflies (6 spp.) (Table 2
Anthers have already dehisced as flowers start to open. Short- and Fig. 1C and D). Apis mellifera was the most frequent flower
styled flowers produce, on average, more nectar (0.18 l ± 0.06 SD, visitor (17.7%), followed by Euptoieta hegesia (13%), Callonychium
n = 20) than long-styled ones (0.12 l ± 0.05, n = 20; T = 2.8, df = 38, brasiliense (10.8%), Plebeia flavocincta (6.4%), Perditomorpha sp.
p < 0.01). (5.5%), and Trigona spinipes (4%) (Fig. 3 and Table 3). Further species
Frequency of short- and long-styled individuals in the popu- were sporadic visitors. Frequency of bee visits to short- (on average
2
lation was similar (33 vs. 32 individuals, respectively; = 0.15; only 2.1 visits a day) and long-styled flowers (1.7) was similar and
df = 1; p = 0.9). low (Table 3). However, the most frequent visits of species were
recorded predominantly in short-styled flowers (52 vs. 28 for Apis
mellifera and 33 vs. 16 for Callonychium brasiliense).
Pollen
Pollen grains from short-styled flowers were significantly larger
Table 2
(54.4 m ± 2.9 SD, n = 10 flowers; pollen size for each flower is
Flower visitors of Turnera hermannioides. P: pollen and N: nectar.
the mean size of 50 grains) than those from long-styled flowers
Flower visitors Sex Resource
(46.7 m ± 2.2, n = 10; T = 12.47, df = 18, p < 0.001). Ornamentation
of the exine also varies between morphs. While pollen grains Hymenoptera
from short-styled flowers have a reticulum with larger brochi, and Andrenidae
Callonychium brasiliense (Ducke, 1907) ♀ P/N
lumina with 2–13 free baculae, those of long-styled flowers have
Protomeliturga catimbaui Schlindwein and Moure, 2005 ♀/♂ P/N
smaller brochi and lumina with 1–6 free baculae (Fig. 2A and B).
Protomeliturga turnerae (Ducke, 1907) ♀/♂ P/N
Pollen viability was similar in long- (84% ± 11.9 SD, n = 15) and
Apidae
short-styled flowers (86% ± 12.7, n = 15; T = −0.6, df = 28, p = 0.5).
Apis mellifera Linnaeus, 1758 ♀ P/N
Long- (10,627 ± 2052 SD, n = 15) and short- (9672 ± 1865, n = 15)
Ceratina (Ceratinula) sp. ♀ P/N
styled flowers produced similar numbers of pollen grains (T = 1.33, Ceratina (Crewella) maculifrons Smith, 1844 ♀ P/N
♀
df = 28, p = 0.19). The mean number of ovules per flower was sim- Plebeia flavocincta (Cockerell, 1912) P
Ptilothrix plumata Smith, 1853 ♂ N
ilar in long- (28.8 ± 4.4 SD, n = 15) and short-styled ones (28.7 ± 5,
Trigona sp. ♀ P/N
n = 15; T = -0.4, df = 28, p = 0.96).
Trigona spinipes (Fabricius, 1793) ♀ P/N
Xylocopa (Neoxylocopa) grisescens Lepeletier, 1841 ♀ P/N
Xylocopa (Neoxylocopa) ordinaria Smith, 1874 ♀ P/N
Breeding system Colletidae
Hylaeus sp. ♀ ?
♀ ♂
Controlled pollination experiments showed that the flow- Perditomorpha sp. / N
ers of T. hermannioides are self-incompatible (Table 1). In the Halictidae
self-pollination treatments, as well as under intramorph hand Augochlora (Oxystoglossella) sp. ♀ ?
cross-pollination, flowers set only a few fruits (2.5–15%) and seeds Lepidoptera
♀
(on average 0.05–0.5 seeds per flower). Furthermore, these fruits Agraulis vanillae maculosa (Stichel, 1908) /♂ N
♀ ♂
were deformed and differed morphologically from those fruits Euptoieta hegesia (Cramer, 1779) / N
Eurema elathea (Cramer, 1775) ♀/♂ N
formed in the treatments of legitimate hand cross-pollination and
Heliopyrgus domicella willi (Plötz, 1884) ♀/♂ N
in open flowers (control). Only open-pollinated and intermorph
Phoebis sennae (Linnaeus, 1758) ♀/♂ N
hand cross-pollinated flowers showed substantial fruit (47–83%)
Pyrisitia nise tenella (Boisduval, 1836) ♀/♂ N
and seed set (on average 6–18 seeds per flower). Natural fruit (51.2%
Coleoptera
short- and 47.7% long-styled flowers) and seed set (about 6 seeds
Cantharidae sp. – N
per flower) were similar in both morphs, but about 30% lower than Chrysomelidae sp. ♀/♂ N
♀ ♂
that of intermorph hand cross-pollinated flowers (Table 1). Fruits Nitidulidae sp. 1 / P
Nitidulidae sp.2 ♀/♂ P
matured in eight to ten days.
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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Fig. 2. Pollen grains from short- (A) and long-styled flowers (B) of Turnera hermannioides (SEM).
Fig. 3. Number of flower visits per flower and day (mean ± SE) by bees and butterflies to flowers of Turnera hermannioides (n = 60 flowers for each morph).
Table 3
Number of visits to short- and long-styled flowers of Turnera hermannioides recorded during the observation of frequency of flower visitors. Differences between the numbers
of visits to short- and long-styled flowers were assessed by chi-square goodness-of-fit tests (df = 1 for all tests). N.T – not tested (given the small number of events observed).
Flower visitor species Number of visits Observed vs. expected frequency
2
Short-styled (n = 60) Long-styled (n = 60) ; p
2
Bees + butterflies + beetles 248 204 = 4.28; p < 0.05
2
Butterflies 58 30 = 8.9; p < 0.01
2
Euptoieta hegesia 36 23 = 2.9; p > 0.05
2
Phoebis sennae 12 3 = 5.4; p < 0.05
2
Others 10 4 = 2.6; p > 0.05
2
Bees 128 104 = 2.5; p > 0.05
2
Apis mellifera 52 28 = 7.2; p < 0.01
2
Callonychium brasiliense 33 16 = 5.9; p < 0.05
Ceratina sp. 2 2 n.t.
Ceratina (Ceratinula) sp. 1 4 n.t.
2
Perditomorpha sp. 11 14 = 0.3; p > 0.05
2
Plebeia flavocincta 17 30 = 3.6; p > 0.05
Protomeliturga catimbaui 0 4 n.t.
2
Trigona sp. 12 6 = 2; p > 0.05
2
Beetles 62 70 = 0.5; p > 0.05
2
Nitidulidae sp 1/2 55 63 = 0.5; p > 0.05
2
Cantharidae/chrysomelidae sp. 7 7 = 0; p > 0.05
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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Fig. 4. Percentage of pollen grains (mean ± 0.95 × SE) from short- and long-styled flowers of Turnera hermannioides and from other species in the pollen loads of flower-visiting
bees.
Females of Apis mellifera, Callonychium brasiliense, Ceratina mac- but those of short-styled flowers predominantly on short styles
ulifrons, Ceratina sp., Protomeliturga catimbaui, P. turnerae, and (Fig. 5B). Legitimate pollen flow to short-styled flowers was much
Trigona spinipes gathered pollen and nectar in flowers of T. herman- higher (on average 233 pollen grains per flower) compared to long-
nioides and flew among con-specific individuals. During pollen or styled flowers (42). Illegitimate pollen flow to short- (on average
nectar collection, females contacted stigmas of both floral morphs. 238 grains per flower) and long-styled (191) flowers was similar.
Males of Perditomorpha sp. and Protomeliturga catimbaui patrolled Non-emasculated short- and long-styled flowers received, on aver-
the flowers of T. hermannioides, alighted and waited for females. age, 8 (47.3% of the total deposited on the stigmas) and 1.4 (17.5%
While patrolling, males of these two species frequently search for of the total deposited on the stigmas) legitimate pollen grains per
nectar in flowers, contacting stigmas and anthers. ovule. Legitimate pollen flow in short-styled flowers represented
Butterflies were frequent visitors seeking nectar in flowers of 2.2% of the total pollen produced by long-styled flowers and 0.4%
T. hermannioides. They flew among con-specific individuals, but of those produced by short-styled ones.
did not contact stigmas while taking up nectar nor did they carry Deposition of pollen grains (legitimate + illegitimate) on stigmas
Turnera-pollen grains on the body surface or mouthparts. of non-emasculated flowers was about three times higher in short-
Nitidulid beetles were frequent flower visitors and always styled flowers (471 vs. 125) and twice in long-styled flowers (233
remained for a long time (frequently more than an hour) in a single vs. 109), when compared to emasculated flowers (Fig. 5A and B).
flower, where they ate pollen and mated. They rarely flew among Deposition of pollen grains from alien species was low in all anal-
con-specific individuals of T. hermannioides. yses (14.8 or 2% in non-emasculated flowers and 20.4 or 8% in
Analyses of the scopa pollen loads of female bees showed that emasculated flowers).
the ratio of pollen grains from short- and long-styled flowers of T.
hermannioides varied among the different pollinator species (Fig. 4).
While grains from long-styled flowers were predominant in the
pollen loads of A. mellifera and Trigona spinipes (between 60% and
70%), those of short-styled flowers were predominant in Plebeia
flavocincta and Protomeliturga catimbaui (between 66 and 77%).
Females of C. brasiliense carried almost exclusively pollen from
long-styled flowers. Worker bees of P. flavocincta were the only
visitors carrying a substantial number of pollen grains from other
species (20%).
Pollen flow
For emasculated flowers, pollen grains of long- and short-styled
plants were deposited in similar numbers on stigmas of both
morphs (Fig. 5A). Legitimate pollen flow to short-styled flowers was
much higher (96 pollen grains per flower on average) compared to
long-styled flowers (only 8 grains per flower on average). Illegit-
imate pollen flow especially occurred to long-styled flowers and
to a lesser extent to short-styled flowers (100 vs. 30 pollen grains,
on average). Emasculated short- and long-styled flowers received,
on average, 3.3 (or 70% of the total deposited on stigmas) and 0.3
(6.7% of the total deposited on stigmas) legitimate pollen grains per
Fig. 5. Mean number of pollen grains of Turnera hermannioides deposited by
ovule, respectively. The mean number of pollen grains deposited in flower visitors on the stigma of emasculated (A) and non-emasculated (B) long-
and short-styled flowers (n = 10 for each morph and treatment) at the end of
short-styled flowers corresponds to 0.9% of the grains produced by
the anthesis. Continuous and dotted lines represent legitimate and illegitimate
long-styled flowers (10,627), while that in long-styled ones to only
pollen flow, respectively. Distinct low letters indicate significant differences in
0.08% of the grains produced by short-styled flowers (9672).
pollen flow within emasculated (a, b) and non-emasculated (x, y) flowers, assessed
For non-emasculated flowers, pollen grains of long-styled plants using a Kruskal–Wallis test (emasculated: H(3,40) = 14.5, P < 0.01; non-emasculated:
were deposited in similar numbers on stigmas of both morphs, H(3,40) = 15.45069, P < 0.01) and Tukey–Kramer tests as post hoc comparisons.
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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P. Milet-Pinheiro et al. / Flora xxx (2013) xxx–xxx 7
Discussion of this level, in general, are attached more frequently on body
parts of any nectar foraging male or female bee. Such pattern has
Breeding system and morph-related features been frequently observed in heterostylous species (see, among
others, Alves-dos-Santos and Wittmann, 2000; Milet-Pinheiro and
Turnera hermannioides shows a heteromorphic self- Schlindwein, 2010; Schlindwein and Medeiros, 2006).
incompatibility system, similar to that found in other distylic The comparison of the pollen flow in emasculated vs.
species, i.e. pollen from short-styled flowers is compatible almost non-emasculated flowers reveals that within-flower illegitimate
exclusively with long-styled flowers and vice versa (Barrett and (intramorph) pollen flow is responsible for about twice of the pollen
Shore, 1985; Darwin, 1877; Ganders, 1979; Milet-Pinheiro and deposition in long-style morphs; in short-styled flowers, however,
Schlindwein, 2010; Schlindwein and Medeiros, 2006), while fruit this self-pollen contributes to an even much higher percentage of
and seed set after self- and intramorph cross-pollination is very the stigmatic pollen loads (almost ten times higher). This indicates
low. Besides differences in anther height and style length, the that spatial disposition of anthers and stigma in long-styled flowers
two morphs of T. hermanniodes also diverge in ornamentation and helps to reduce intra-floral self-pollen deposition, but not in short-
size of pollen grains, length of stigmatic lobes and nectar amount. styled flowers. This might be due to vertical top-down transport of
Except the difference in nectar amount (Cawoy et al., 2006; pollen, partially by grains falling down from the anthers to the low-
Milet-Pinheiro and Schlindwein, 2010; Ornelas et al., 2004), these level stigmas. The much lower legitimate pollen flow in both levels
other variations have been frequently recorded in distylic plants of emasculated flowers, when compared to non-emasculated flow-
(Ganders, 1979). In heterostylic plants, differences in size and ers, might reflect a lower visitation rate experienced by antherless
ornamentation of pollen grains from different morphs provide an flowers. Removing anthers changes the visual display and possibly
excellent means to investigate pollen flow within a population (see the scent bouquet of a flower, and this might have led to a decreased
Barrett and Glover, 1985; Milet-Pinheiro and Schlindwein, 2010; flower visitation. Given that pollen flow in emasculated and non-
Rama Swamy and Bahadur, 1984; Schlindwein and Medeiros, emasculated flowers was measured simultaneously, fluctuation in
2006). Given that these peculiarities were also observed in T. pollinator availability does not explain this difference. Although we
hermannioides, we were able to determine the origin of pollen have not measured the frequency of visits in emasculated and non-
grains in stigmas of short- and long-styled flowers and in the emasculated flowers separately, accumulating evidences show that
pollen load of flower visitors. bees are able to evaluate pollen availability using olfactory (Dobson
and Bergström, 2000; Dobson et al., 1999) and visual cues (Lunau,
Flower visitors and pollen flow 2000) of pollen, so that this might have led to the differences found.
Analyses of the scopa pollen loads showed that females/worker
Turnera hermannioides is a xenogamous species that depends on bees of A. mellifera and C. brasiliense, the two most frequent pollina-
pollinators to set fruits and requires crossing between morphs. In tors, as well as those of Trigona spinipes, collected significantly more
spite of its large and conspicuous flowers, where pollen and nectar pollen grains from low-level anthers, even if they visited short-
are easily accessible to visitors, the number of flower visits (at least styled flowers more frequently. This could explain the encountered
during the time periods explored in this study) was remarkably mismatch of the stigmas’ pollen loads. The unbalanced pollen flow,
low. A single flower was visited only 1.9 times a day, a visitation however, did not result in unbalanced fruit and seed set of long
frequency that is much lower than those observed for other bee- and short-styled morphs in the studied population. It is interesting,
pollinated species occurring in the Caatinga region, such as Cordia that the scopa loads of the rare Protomeliturga catimbaui contained
leucocephala (Cordiaceae, Milet-Pinheiro and Schlindwein, 2010), conspicuously more pollen grains from high-level anthers (short-
Jacaranda rugosa (Bignoniaceae, Milet-Pinheiro and Schlindwein, styled flowers) than from low-level anthers, contrary to the three
2009), Hydrocleys martii (Alismataceae, Carvalho and Schlindwein, above-mentioned species. In the Catimbau National Park, thus, we
2011). Bees were the main pollinators of T. hermannioides; they interpret that the low and unbalanced pollen flow in distylic T.
contacted stigmas and anthers during visits, carried pollen grains hermannioides is caused by both a general low frequency and prob-
on their body surface, showed flower fidelity and flew among ably an inefficiency of pollinators. Given that natural fruit set is
con-specific individuals of different morphs. This complex set of not under pressure of limited resources (legitimate hand cross-
behaviors, necessary to pollinate flowers, was not shown by butter- pollinated flowers set more fruits than open flowers), we speculate
flies and beetles. The low frequency of flower visits by pollinators that higher general rates of flower visitors and especially higher
must compromise pollen flow and, consequently, fruit and seed abundance of oligolectic P. catimbaui would lead to a higher natural
set in T. hermannioides. In fact, analyses of pollen grains deposited fruit and seed set of both floral morphs.
on stigmas at the end of anthesis showed an extraordinary low In T. subulata, a species sharing similar habit and floral traits with
legitimate pollen flow in both non-emasculated and emasculated T. hermannioides, natural fruit set was almost 100% (Schlindwein
flowers. and Medeiros, 2006). The number of legitimate pollen grains
In the studied population, the number of pollen grains per flower deposited on the stigmas of T. subulata was six and five for each
of short- and long-styled morphs was similar, which fact diverges ovule in short- and long-styled flowers, respectively. This corre-
from what is the case with most heterostylic plants (Ganders, 1979) sponds to a somewhat lower number of pollen grains deposited
and even from a population of the same species from Sergipe on stigmas of short-styled flowers (8 grains per ovule in non-
(Barrett and Shore, 1985). Furthermore, the ratio of short- and emasculated flowers), but 3.5 times more pollen grains on stigmas
long-styled plant individuals was also similar. Consequently, we of long-styled flowers (1.4 grains per ovule), as compared to
would expect a 1:1 ratio of pollen from short- and long-styled T. hermannioides. Unlike T. hermannioides, flowers of T. subu-
flowers in stigmas of both morphs. The analysis of pollen loads lata experienced a high frequency of visits by the oligolectic
deposited at the stigmas, however, evidenced a strong mismatch bee Protomeliturga turnerae and several polylectic bee species
in pollen flow: pollen grains from long-styled flowers (low-level (Schlindwein and Medeiros, 2006). This might explain the higher
anthers) were much more frequent on stigmas of short- and long- pollen flow and natural fruit set in this plant.
styled morphs in both non-emasculated and emasculated flowers. The higher number of pollen grains deposited on stigmas of
This indicates that pollen of the low-level anthers are preferen- short-styled flowers indicates that pollination in this morph is
tially (or more easily) collected by female bees and then dispersed more efficient. We measured that the general frequency of flower
among other flowers on subsequent visits, or that pollen grains visits was significantly higher in short-styled flowers, which could
Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae)
and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005
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8 P. Milet-Pinheiro et al. / Flora xxx (2013) xxx–xxx
reflect the higher amount of nectar produced by flowers of this Acknowledgements
morph. Cawoy et al. (2006), investigating distylic Fagopyrum escu-
lentum (Polygonaceae), also found a positive correlation between We thank Gerhard Gottsberger for using the SEM, Carlos
nectar amount and the number of flower visits by A. mellifera to the Eduardo Nobre and Artur Maia for identifying butterflies and bee-
more nectar-rich morph. This corroborates the findings that flowers tles, respectively, João Silva, Dona Maria, and the association of field
providing more resources (e.g. nectar and pollen) are more attrac- guides for their support in the nature reserve, IBAMA for the per-
tive to flower visitors than those with less resources (Dobson and mission to work in the National Park of Catimbau and for logistic
Bergström, 2000; Dobson et al., 1999; Howell and Alarcón, 2007; support, and Stefan Dötterl and an anonymous referee for com-
Thomson, 1988). If certain foraging bees really prefer one of the two ments on an earlier version of the manuscript. This study was
floral morphs due to differences in nectar availability, than they supported by the Coordenac¸ ão de Aperfeic¸ oamento de Pessoal de
must be able to differentiate short-style from long-style flowers. It Nível Superior (CAPES), Conselho Nacional de Desenvolvimento
would be interesting to confirm these probably learned preferences Científico e Tecnológico (CNPq), Fundac¸ ão de Amparo à Ciência
in an experimental setup. de Pernambuco (FACEPE), and Fundac¸ ão o Boticário de Protec¸ ão
The evaluation of pollen flow in T. hermannioides also revealed à Natureza. CNPq also provided an undergraduate student grant
that the pollination service is expensive for the plants. More than (PIBIC) to D.C.A.P. and a research grant (PQ) to C.S.
99% of the pollen grains produced in the population were gathered
by bees for brood provisioning, while the rest was either lost in
the flowers, eaten by flower visitors or remained on the visitors’
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