Botanical Journal of the Linnean Society, 2009, 160, 357–368. With 14 figures
Floral biology and mechanisms of spontaneous
self-pollination in five neotropical species of
Gentianaceae
LEANDRO FREITAS1* and MARLIES SAZIMA2
1
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915, 22460-030, Rio
de Janeiro – RJ, Brazil
2
Universidade Estadual de Campinas, Departamento de Botânica, Caixa Postal 6109, 13083-970,
Campinas – SP, Brazil
Received 16 March 2009; accepted for publication 11 June 2009
Cross- and self-fertilization in angiosperms are regulated by several factors, and a knowledge of the mechanism
and time of spontaneous self-pollination offers opportunities for a better understanding of the evolution of mating
systems and floral traits. The floral biology of five species of Gentianaceae found in high-altitude neotropical
grassland is presented, with emphasis on the mechanisms that promote spontaneous self-pollination. A presumed
floral Batesian mimicry system is suggested between the rare and rewardless Zygostigma australe and Calydorea
campestris, a species of Iridaceae with pollen-flowers, pollinated by syrphids and bees. The floral morphology of the
other four gentian species points to three different pollination syndromes: melittophily, phalaenophily and
ornithophily. However, with the exception of the nocturnal Helia oblongifolia, flowers are nectarless and appear to
exhibit non-model deceptive mechanisms, providing similar floral cues to some sympatric rewarding species with
the same syndrome. The similar mechanism of spontaneous self-pollination in Calolisianthus pedunculatus,
Calolisianthus pendulus and H. oblongifolia (Helieae) is based on the stigmatic movements towards the anthers.
Selfing is promoted by movements of the style/stigma and of the corolla in Deianira nervosa and Z. australe
(Chironieae), respectively. The movements of stamens, style and stigma during anthesis seem to be the most
common method of spontaneous self-pollination in angiosperms. It is suggested that the evolution of delayed
spontaneous self-pollination would be more expected in those taxa with dichogamous flowers associated with
herkogamy. Such a characteristic is frequent in long-lived flowers of certain groups of Asteridae, which comprise
most documented cases of autonomous selfing. Thus, the presence of dichogamy associated with herkogamy (which
supposedly evolved as a result of selection to promote both separation of male and female functions and the efficient
transfer of cross pollen) may be the first step in the adaptive evolution of delayed selfing to provide reproductive
assurance. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368.
ADDITIONAL KEYWORDS: autonomous self-pollination – exaptation traits – flowering phenology – nectary
– pollination by deceit – sexual reproduction – south-eastern Brazil.
INTRODUCTION
Cross- and self-fertilization in angiosperms are regulated by the genetic, physiological and morphological
factors of plants and by the interaction between
plants and both their pollinators and neighbours
(Lloyd, 1992; Armbruster et al., 2002; Goodwillie,
*Corresponding author. E-mail: [email protected]
Kalisz & Eckert, 2005). This generates a high level of
variation in the degree of cross-fertilization among
flowering species, from seed production only by
outcrossing in dioecious species to complete selffertilization in cleistogamous flowers. Crossfertilization is advantageous, for example, in
generating genetic diversity, mainly by higher heterozygosity in polymorphic loci; in contrast, selffertilization may promote higher reproductive
efficiency in harsh environments, where pollinators
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
357
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L. FREITAS and M. SAZIMA
are scarce (Richards, 1997). Such questions have
made the comparison between cross- and selffertilization a central subject in reproductive biology
since the founder studies on inbreeding depression
were carried out by T. A. Knight and C. Darwin in
the 18th and 19th centuries (Lloyd, 1992; Lloyd &
Schoen, 1992; Armbruster et al., 2002). Two main
approaches to the study of self- and cross-fertilization
can be distinguished as: (1) functional studies emphasizing the operation of pollination mechanisms and
aspects of the natural history of flowers; and (2)
genetic studies dealing with frequencies of selffertilization, expression of inbreeding depression
and genetic structure of populations. The genetic
approach has been predominant in the last few
decades (Lloyd & Schoen, 1992; Herlihy & Eckert,
2002), with increasing interest in the understanding
of the molecular and genetic basis of pollen/pistil
interaction in the past 10 years (Wheeler, FranklinTong & Franklin, 2001; Lord & Russell, 2002; Ge,
Tian & Russell, 2007).
Despite many studies carried out on the subject,
the functional approach of self-fertilization has been
overlooked, even though it offers opportunities to
increase our knowledge of the evolution of mating
systems and floral traits (Lloyd & Schoen, 1992; Armbruster et al., 2002; Sato, 2002; Qu et al., 2007; Zhang
& Li, 2008). In theory, spontaneous selfing is evolutionarily favoured in certain scenarios, such as environments with a low frequency of pollinator visits to
flowers and in plants with specialized pollination
systems, and in monocarpic species, which have only
one opportunity to contribute to the next generation
(see Lloyd, 1992; Richards, 1997; Sato, 2002; Fenster
& Martén-Rodríguez, 2007). Functional aspects that
remain poorly studied include the modes of autonomous selfing, i.e. without the intervention of a pollinating agent.
Gentianaceae (c. 1600 species) are mostly found in
boreal temperate areas, but with an extensive tropical
and subtropical diversity of genera (for example, 54%
of the genera occur in the neotropics and 77% are
endemics of this region) (Albert & Struwe, 2002).
Floral morphology is highly diversified in the family,
which suggests the adaptive radiation of species to
pollination by different animal groups, such as bees,
moths, flies, bats and hummingbirds. However,
studies on pollination biology in the family are scarce
and have concentrated on a few genera, such as
Gentiana L. and Gentianella Moench (for example,
Beattie, Breedlove & Ehrlich, 1973; Webb & Littleton,
1987; Fischer & Matthies, 1997; Machado, Sazima &
Sazima 1998; Petanidou et al., 1998; Kozuharova,
1999; Albert & Struwe, 2002). Spontaneous selfpollination has been recorded in some species of
Gentianaceae (Weaver, 1972; Webb, 1984; Fischer &
Matthies, 1997; Petanidou et al., 1998; Struwe et al.,
2002; Kozuharova & Anchev, 2006), and has also been
found in five species that occur in high-altitude grasslands in south-eastern Brazil: Calolisianthus pedunculatus (Cham. & Schltdl.) Gilg, Calolisianthus
pendulus (Mart.) Gilg, Helia oblongifolia Mart.,
Deianira nervosa Cham. & Schltdl. and Zygostigma
australe (Cham. & Schltdl.) Griseb. (Freitas &
Sazima, 2006). These plants belong to two of the six
tribes of Gentianaceae (after Struwe et al., 2002) and
their floral traits fit three different pollination syndromes. In this article, the floral biology of these five
species is addressed, emphasizing the mechanisms
that promote spontaneous self-pollination. In addition, a possible floral Batesian mimicry system
between the rewardless gentian Z. australe and the
irid Calydorea campestris Baker is described.
MATERIAL AND METHODS
STUDY SITE AND SPECIES
Our study was conducted in the montane area of the
Parque Nacional da Serra da Bocaina in the Serra
do Mar Range, south-eastern Brazil (c. 22 °44′S,
44 °36′W, 1500–1800 m a.s.l.). This area is covered
mainly by Araucaria Juss. forest and high-altitude
grasslands, a subtype of the Atlantic Forest ecosystem. The annual rainfall is up to 2100 mm, with a
rainy season mostly from October to March and a dry
season (with a monthly rainfall lower than 50 mm)
from June to August. The average annual temperature is c. 15 °C; temperatures may fall below 0 °C
during the dry season and frost may occur (for details,
see Segadas-Vianna & Dau, 1965; Freitas & Sazima,
2006). The five species of Gentianaceae (Table 1) were
studied in these grasslands, which are a mosaic of
shrubs and herbs of many families within a matrix of
Cyperaceae and Poaceae (for details, see Freitas &
Sazima, 2006).
The studied species are small, sun-loving, shortliving perennials (D. nervosa and H. oblongifolia) or
annual herbs (Z. australe, C. pedunculatus and C.
pendulus; Fig. 1). They occur in well-drained areas,
except for H. oblongifolia, which is typical of temporary swamps that are flooded in the summer months.
The populations of the species are small at Serra da
Bocaina (less than 50 individuals were found). Only
four individuals of D. nervosa and eight of Z. australe
were found during 25 field trips made between
December 1997 and February 2000. The plants are
generally distributed in small clusters in areas up to
20 m2. The only exception is C. pendulus, which forms
groups of several individuals, in a wide range from a
few plants to large clusters of up to dozens of plants,
spread across Bocaina grasslands. Voucher specimens
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
359
were deposited at the herbarium of the Universidade
Estadual de Campinas (UEC).
Syrphid (?)
Small bee,
syrphid
PROCEDURES
Actino, actinomorphic; Zygo, zygomorphic; Pa, protandry; Pg, protogyny; N, nectar; P, pollen; Undet, undetermined; (?), doubtful.
Melittophily
Deceit/melittophily
Feb–Apr, June (Feb–Mar)
Feb–Apr (Feb–Mar)
P
P (?)
Pg
Pg
Pink
Violet
Dish
Dish
CHIRONIEAE
Deianira nervosa
Zygostigma australe
Actino
Actino
Undet
Undet
Undet
Phalaenophily
Ornithophily
Melittophily
Jan–Mar (Jan–Feb)
Dec–Apr (Jan–Feb)
Jan–May, July (Feb)
N
N (?)
N (?)
Pa
Pa
Pa, Pg
Green
Red
Violet
Tube
Tube
Tube
HELIEAE
Helia oblongifolia
Calolisianthus pedunculatus
Calolisianthus pendulus
Zygo
Zygo
Zygo
Flowering (peak)
Resource
Dichogamy
Colour
Symmetry
Shape
TRIBE and species
Table 1. Floral traits of Gentianaceae species in high-altitude grassland at Serra da Bocaina, south-eastern Brazil
Pollination syndrome
Pollinators
SPONTANEOUS SELF-POLLINATION IN GENTIANACEAE
The flowering time (months in which each species
was in flower) and the blooming peak (months in
which more than 50% of the individuals of each
species were in flower) were recorded on a monthly
basis from December 1998 to February 2000. The
following floral attributes were registered in the
field: colour, shape, symmetry, dimensions, time
and phases of anthesis, occurrence of herkogamy and
dichogamy, longevity and presence of odour and
rewards (Kearns & Inouye, 1993). Pollen viability
was estimated using the acetocarmine technique
(Radford et al., 1974), and the stigma receptivity was
verified with the H2O2 catalase activity method
(Zeisler, 1938). Freshly picked flowers, both in the
morning and at night, were enclosed in a glass jar
for 30–60 min; the lid was then removed and the
inside of the jar was smelt to detect the presence of
odour (Kearns & Inouye, 1993). Twelve flowers of
H. oblongifolia were tagged and bagged in the bud
stage, and during the night nectar was extracted
with a graduated microlitre syringe (Hamilton©,
USA). The nectar volume was registered immediately, and the nectar sugar concentration was measured with a handheld refractometer (Atago©).
For scanning electron microscopy, the corolla of
three flowers of C. pendulus, which were observed
secreting nectar droplets in the field, were fixed in
2.5% glutaraldehyde in 0.05 M sodium cacodylate
buffer, pH 7.0, and post-fixed in 1.0% osmium tetroxide. They were then dehydrated though a graded
ethanol–acetone series. Flowers were critical point
dried in a Balzers CPD 030 instrument using CO2
as the replacement fluid. Dried specimens were
mounted on stubs and coated with gold in a Balzers
SCD 050 sputter coater. The material was examined
with a Phillips 505 scanning electron microscope at
25 kV.
Buds of the five species were bagged with nylon
mesh to verify fruit set by autonomous selfpollination. Previously bagged flowers of H. oblongifolia and C. pendulus were manually pollinated
(cross- and self-pollinated), and the pistils were fixed
in formalin–acetic acid–alcohol (FAA) at 12 and 24 h
after pollination, and analysed via fluorescence
microscopy to observe pollen tube growth (Martin,
1959). A total of 100 h were spent watching floral
visitors during 1999 and 2000 in order to record the
pollinators and their frequency and behaviour during
the visits. The insects visiting the flowers were collected as far as possible by means of an insect net and
were identified by specialists.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
360
L. FREITAS and M. SAZIMA
Figures 1–6. Species of Helieae (Gentianaceae) from Serra da Bocaina grasslands, south-eastern Brazil. Fig. 1. Habit
and habitat of Calolisianthus pendulus. Fig. 2. Nocturnal phalaenophilous flower of Helia oblongifolia. Fig. 3. Nectarless
ornithophilous-like flower of C. pedunculatus. Fig. 4. Melittophilous-like flower of C. pendulus. Fig. 5. Flower of C.
pendulus at the onset of anthesis. Note the closed anthers and the anther–stigma separation (protandry associated with
herkogamy). Fig. 6. Spontaneous self-pollination of C. pendulus after stigmatic movements towards the anthers.
RESULTS
FLORAL
TRAITS
The flowering time of the five species was concentrated
in the rainy season (Table 1). Flowers of the three
species belonging to tribe Helieae Gilg in Engler &
Prantl are bisexual, pentamerous and zygomorphic.
The stamens are epipetalous with dorsifixed and
introrse anthers and the pollen is in tetrads. The
dimerous gynoecium has a bilocular ovary, a long style
and a bilobed stigma, with papillae on the ventral
surface of each lobe. In general, a single flower per
inflorescence is open at a time. Flowers last for at least
5 days and show different degrees of herkogamy and
dichogamy. The three species appear to have three
different pollination syndromes (Table 1).
Helia oblongifolia has horizontally placed, hypocrateriform, yellowish-green flowers (corolla tube
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
SPONTANEOUS SELF-POLLINATION IN GENTIANACEAE
c. 20 ¥ 3 mm) (Fig. 2). The anthers are positioned side
by side at the same height in the lower portion of the
corolla tube, and pollen grains face the tube lumen.
The stigma lobes are placed above the anthers in the
upper portion of the tube. Such a position of female
and male organs characterizes approach herkogamy.
Anthesis is nocturnal, when a sweet odour can be
detected. An ovarian-based disc secretes between 4
and 8 mL of nectar at night (sugar concentration,
14–21%, N = 12). The nectar is completely reabsorbed
by the morning.
Calolisianthus pedunculatus has horizontally
directed or pendant red flowers (corolla tube
c. 32 ¥ 12 mm) (Fig. 3), without any perceptible odour.
The stamens and style/stigma are placed in a parallel
position in the lower portion of the corolla tube. The
anthers are positioned side by side (3 + 2) and separated by the style. The flowers are slightly herkogamous, because the anthers are positioned one to a few
millimetres below the stigma. The pollen grains face
the tube lumen and many are deposited on the dorsal,
non-receptive, portions of the stigma lobes. The floral
morphology suggests ornithophily, but floral nectaries
were not observed.
The lilac to violet flowers (corolla tube c. 25 ¥ 9 mm)
of C. pendulus are horizontal (Fig. 4), without any
perceptible odour. The stamens are aligned in two or
three parallel lines in the lower portion of the corolla
tube. The anthers are clumped together and face the
corolla tube lumen or apex. The flowers are herkogamous because the style is longer than the anthers
(Figs 5, 6). There is no nectariferous disc but, in a few
plants, minute glands (nectarioles sensu Vogel, 1998)
(Figs 7, 8), placed at the mid-length of the corolla,
secrete small nectar droplets. The floral morphology
suggests melittophily, and the absence of the nectar
in most flowers suggests pollination by deceit.
The two species of Chironieae Dumort. are erect
herbs with flowers arranged in dense inflorescences
in D. nervosa and solitary in Z. australe. The flowers
are erect and either tetramerous (D. nervosa) or pentamerous (Z. australe). The corolla is hypocrateriform
with large lobes, but the flowers are functionally
dish-shaped, because the bicarpellate, unilocular
ovary almost fills the tube and the anthers are placed
above the ovary (Figs 9, 11, 13–14). Moreover, there is
no nectariferous disc and the basal outer part of the
corolla is green and constricted by the sepals.
Deianira nervosa flowers are white in bud and
reddish-lilac to pink at anthesis. In the last 5–6 days,
it emits a sweet odour. The stamens are epipetalous,
but two are erect and the others are slightly curved;
thus, the four anthers are placed at the same height
on the posterior side of the corolla (Fig. 9). The style
is long and curved towards the ventral face of the
corolla, such that the stigma is placed at a higher
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Figures 7, 8. Scanning electron micrographs of the
abaxial corolla surface of Calolisianthus pendulus. Fig. 7.
Medial region of the corolla with some nectarioles
(arrows). Fig. 8. Detail of the nectariole. Scale bars: Fig. 7,
100 mm; Fig. 8, 25 mm.
position and opposite side of the anthers (Fig. 9).
Thus, the flowers are herkogamous and slightly zygomorphic. The style ends in a bifid, large, papillate
stigma. The dehiscence of the anthers and, consequently, pollen availability is progressive. They first
open with an apical pore, which then opens into a slit
towards the base (Figs 9, 10). The floral morphology
suggests pollination by bees in search of pollen.
Zygostigma australe has actinomorphic (Fig. 11),
purple, slightly odorous flowers. The stamens are
epipetalous; the anthers are small, sagittate and
introrse, and the pollen grains are in monads. The
short style (c. 1 mm) ends in a c. 3 mm long papillate
stigma with two oblong lobes. They are slightly
herkogamous, as the stigma is a few millimetres
apart from the anthers (Fig. 11). The flowers last for
3–4 days. During anthesis, they close through corolla
movements in the early afternoon and open again the
following morning. Between 2% and 19% of the pollen
grains germinate inside the anthers (N = 4 flowers).
The general flower aspect fits melittophily, but the
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362
L. FREITAS and M. SAZIMA
Figures 9–14. Species of Chironieae (Gentianaceae) (except Fig. 12) from Serra da Bocaina grasslands, south-eastern
Brazil. Fig. 9. Nectarless melittophilous-like flower of Deianira nervosa at the onset of anthesis. Note the closed anthers
and globose stigma in its receptive state (protogyny), and the anther–stigma separation caused by anther and style/stigma
displacement. Fig. 10. Spontaneous self-pollination of D. nervosa after style movement towards the posterior side of the
flower and the progressive recurvature of the stigmatic lobes, which acquire the shape of a roll. Fig. 11. Nectarless
melittophilous-like flower of Zygostigma australe on the second day of anthesis. Note the wide stigmatic surface and
opened anthers. Fig. 12. Flower of Calydorea campestris (Iridaceae) during a visit by an unidentified species of syrphid
in search of pollen. This is the probable model of Z. australe in a possible Batesian mimetic relationship. Fig. 13.
Spontaneous self-pollination of Z. australe after corolla closing, which results in the stimulation of anther movement
towards the stigma. Fig. 14. Allograpta exotica visiting a flower of Z. australe. Note that this syrphid fly is touching the
stigma with its head.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
SPONTANEOUS SELF-POLLINATION IN GENTIANACEAE
low plant density and some floral traits, such as
solitary flowers, lack of nectar, wide stigmatic area
and well-exposed pollen (Fig. 11), suggest pollination
by deceit.
DICHOGAMY
AND SPONTANEOUS SELF-POLLINATION
MECHANISMS
The flowers of the three species belonging to Helieae
have a similar spontaneous self-pollination mechanism, which is based on stigmatic movements
towards the anthers. Most flowers of C. pendulus are
protandrous; the style is not fully elongated and the
stigmatic lobes are not yet exposed on the first day of
anthesis, but pollen grains are already exposed on the
anthers. The style elongates and reaches a higher
position than the anthers 2 or 3 days after the onset
of anthesis. At this stage, the stigmatic lobes recurve,
increasing the receptive area, and face the corolla
mouth (Fig. 5). At this stage, male and female functions overlap if the visitors do not remove the pollen
first. On the seventh or eighth day after anthesis, one
of the stigmatic lobes is more recurved so that it
touches the anthers and receives pollen (Fig. 6). Some
flowers of this species are protogynous as the anthers
are closed and the stigma is deflected and receptive at
the beginning of anthesis (Fig. 3). Pollen is exposed
after the second or third day of anthesis, and selfpollination occurs at the end of anthesis by the same
mechanism as observed in the protandrous flowers.
Pollinator visits in several flowers were simulated by
removing a large portion of the pollen grains from the
anthers with a toothpick. After this, the anthers wilt,
such that, at the end of anthesis, the stigma cannot
touch the anthers.
The dynamics of anthesis and the self-pollination
mechanism in H. oblongifolia are similar to those
found in the protandrous flowers of C. pendulus.
However, the contact between the stigma and the
anthers in the flowers of H. oblongifolia takes place
around the third day of anthesis, at the beginning of
the stigmatic lobe inflection, as the diameter of the
tube is smaller and the anther–stigma distance is
shorter in this species. The protandrous flowers of C.
pedunculatus are spontaneously self-pollinated at the
beginning of the female phase, as herkogamy is slight
or absent. Pollen grains are exposed and the stigma is
not receptive on the first day of anthesis. On the
second day, the inflection of the stigmatic lobes begins
and contact with the anthers takes place when the
inflection angle reaches around 45 °.
Self-pollination mechanisms differ between the two
species of Chironieae. The movements of the gynoecium and corolla in D. nervosa and Z. australe,
respectively, promote self-pollination. In the newly
opened flowers of D. nervosa, the anthers are closed
363
and the stigmatic lobes expand at angles of 30–45 °
(Fig. 9). Anther opening begins on the second or third
day of anthesis. Male and female functions overlap
because the stigma remains receptive until the end of
anthesis. The style bends towards the posterior side
of the flower and the stigmatic lobes recurve, attaining the form of a roll on the fourth or fifth day of
anthesis (Fig. 10). As a consequence, the stigma,
which is separated from the stamens and has a receptive area facing upwards at the beginning of anthesis,
touches the anthers and receives a pollen charge at
the end of anthesis (Figs 9, 10).
The opening of Z. australe flowers takes place
around 08.00–09.00 h and is characterized by the
deflection of petals and stigma bending, such that
its receptive surface is enhanced (Figs 11, 13). The
anthers in most flowers remain closed, but opening
begins on the first day in some flowers. Petal inflection begins around 11.00 h and flowers are completely
closed between 12.00 and 14.00 h. Flowers reopen and
anther dehiscence is completed on the following day.
Flowers close and reopen on the third day and on the
fourth day in a few cases. As stamens are epipetalous,
the closing of the corolla results in anther movement
towards the stigma (Fig. 13). As a consequence, selfpollen is deposited on the stigma from the second day
onwards, but, in some flowers, self-pollination occurs
on the first day of anthesis.
As the flowers of H. oblongifolia and C. pedunculatus last for at least 5 days, cross-pollination can
occur simultaneously with or after self-pollination,
that is ‘competing and prior spontaneous selfpollinations’ (sensu Lloyd, 1992). In the ‘late spontaneous self-pollination’ mechanisms (sensu Lloyd,
1992) of C. pendulus and D. nervosa, self-pollination
occurs after opportunities for cross-pollination. The
mechanism of Z. australe varies between competing
and delayed selfing.
Pollen tube growth does not differ among self- and
cross-pollinated flowers of H. oblongifolia and C. pendulus. Most grains germinate and penetrate within
the style, and ovule fertilization occurs within 24 h of
pollination in both treatments. Pollen viability was
high (85–97%) when recorded at any time during
anthesis in all five species. All unmanipulated bagged
buds set fruits with well-developed seeds (number of
flowers: H. oblongifolia, 6; C. pendulus, 18; C. pedunculatus, 5; D. nervosa, 13; Z. australe, 6).
FLORAL
VISITORS
Only one pollination event was recorded in D. nervosa
during 6 h of observation, when two flowers were
visited by an unidentified species of Syrphidae. It
approached the flower at its centre and landed with
the head pointed towards the anthers. Both the
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L. FREITAS and M. SAZIMA
stigma and anthers touched the ventral part of its
body during the visit. However, the two visits lasted
less than 1 s each and apparently the visitor did not
ingest pollen, because syrphids typically stay on the
flowers for many seconds when feeding on pollen (see
Freitas & Sazima, 2003). Thus, this visitor may be
only a casual pollinator of this species.
Zygostigma australe was visited four times during
8.5 h of observation, twice by Allograpta exotica
(Syrphidae), once by Ceratalictus sp. (AugochloriniHalictidae) and once by an unidentified syrphid
species. The three species landed on the lobes of the
corolla and directed their head towards the centre of
the flower (Fig. 14). Visits were fast (less than 2 s),
and the insects did not ingest (flies) or collect (bee)
pollen. Visitors touched the anthers and the stigma
with the head (Fig. 14), and thus may act as pollinators. No visits were observed on the flowers of the
three Helieae species (total observation time: 20 h at
night and 8 h during the day on H. oblongifolia;
19.5 h on C. pedunculatus and 38.5 h on C. pendulus).
PRESUMED
MIMICRY BETWEEN
CALYDOREA
Z.
AUSTRALE AND
CAMPESTRIS
Calydorea campestris (Iridaceae) is a herb with one
to three rhipidia that bear dish-shaped, trimerous,
actinomorphic (c. 25 mm diameter) and nectarless
flowers. The tepals are purple, and pollen is the single
floral resource. Anthers open longitudinally and
pollen is easily accessible to pollinators, syrphids and
small bees, which ingest or actively collect pollen,
respectively (see Freitas & Sazima, 2006). From a
human perspective, the flower architecture, colour,
size and display of C. campestris resemble that of Z.
australe (Figs 11, 12).
DISCUSSION
Although the flowers of the studied species of Gentianaceae at Serra da Bocaina have characteristics
attractive to different pollinator groups, indicating
different syndromes, the absence of nectar or other
floral resources in four of these species suggests pollination by deceit (for example, Lin & Bernardello,
1999; Pansarin, Pansarin & Sazima, 2008). Floral
visitors were absent or scarce, and their behaviour
during the visits was also typical of visitors attracted
to flowers without resources and pollinated by deceit
(see Agren & Schemske, 1991). Moreover, different
forms of pollen aggregation, as pollen tetrads, seem to
evolve most commonly when the pollinator visits are
infrequent, and may also be associated with pollination by deceit (Harder & Johnson, 2008). Thus, with
the exception of Z. australe and H. oblongifolia, the
gentians studied here seem to be engaged in non-
model deceptive mechanisms, providing similar floral
cues to some sympatric rewarding species that share
the same groups of pollinators (for the functional
groups of plants in this community, see Freitas &
Sazima, 2006). The low frequency of visits associated
with deceit mechanisms suggests that selfpollination, although predominant, is facultative in
these gentians, and some cross-pollination can occasionally occur, possibly by visits of naive individuals
of anthophilous animals.
The evolution of spontaneous self-pollination to
ensure sexual reproduction under conditions of scarce
opportunities for cross-pollination (i.e. ‘reproductive
assurance’) was first proposed by Darwin (1878,
1885). Ever since, this idea has been the central
element of the hypothesis for self-pollination evolution in angiosperms, in spite of the deleterious effects
of self-fertilization (see Lloyd, 1992; Herrera et al.,
2001). A wealth of indirect evidence (Dole, 1990; Lloyd
& Schoen, 1992; Qu et al., 2007) and experimental
tests in a few species (for example, Piper, Charlesworth & Charlesworth, 1986; Elle & Carney, 2003)
indicate the validity of reproductive assurance to
explain the evolution of spontaneous self-pollination
mechanisms. However, the results of a few studies do
not match the expectations of this idea (Eckert &
Schaefer, 1998; Herrera et al., 2001), and other
factors, such as founder effects and small population
size, may favour self-pollination evolution (Lloyd,
1992). Although it is still not possible to indicate
reproductive assurance as the ultimate cause of the
origin of self-pollination in the gentians from Bocaina,
the full fruit set achieved by spontaneous selfing is
the possible proximate reason for its maintenance.
The mechanisms of autonomous self-pollination are
diverse and, as also observed in H. oblongifolia, C.
pedunculatus, C. pendulus and D. nervosa, include
style or stigma movements in species of Lamiales,
previously placed in Scrophulariaceae (Fetscher &
Kohn, 1999; Kalisz et al., 1999; Armbruster et al.,
2002), Bignoniaceae (Yang et al., 2004), Campanulaceae and Asteraceae (Faegri & van der Pijl, 1979),
Malvaceae s.l. (Buttrose, Grant & Lott, 1977; Klips &
Snow, 1997; Ruan, Qin & Xi, 2005), Violaceae (Culley,
2002), Ranunculaceae (Yu & Huang, 2006) and Zingiberaceae (Zhang & Li, 2008). In addition, the movement of stamens promotes selfing in representatives
of Ericaceae (Rathcke & Real, 1993), Rutaceae (Faegri
& van der Pijl, 1979), Fabaceae (Etcheverry, Protomastro & Westerkamp, 2003), Papaveraceae s.l.
(Lyon, 1992), Ranunculaceae (Eckert & Schaefer,
1998) and Orchidaceae (Liu et al., 2006). Although it
is still not possible to identify consistent patterns of
spontaneous self-pollination mechanisms among
angiosperms because detailed descriptions are insufficient and concentrated in just a few families, move-
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
SPONTANEOUS SELF-POLLINATION IN GENTIANACEAE
ments of the reproductive organs seem to be involved
in most cases. Other mechanisms are based on other
factors, including incomplete dichogamy (sensu Lloyd
& Webb, 1986) in Fuchsia (Onagraceae) and Helleborus (Ranunculaceae; Traveset, Willson & Sabag,
1998; Herrera et al., 2001), pollen/pollinia sliding in
Caulokaempferia (Zingiberaceae) and Cyrtopodium
(Orchidaceae; Wang et al., 2004; Pansarin et al.,
2008), and corolla abscission in Incarvillea (Bignoniaceae), Mimulus (Phrymaceae), Pedicularis (Orobanchaceae) and Verbascum (Scrophulariaceae; Dole,
1990; Donnelly, Lortie & Aarssen, 1998; Qu et al.,
2007; Vickery, 2008). In Fumana juniperina (Cistaceae) and Bulbine vagans (Xanthorrhoeaceae),
delayed self-pollination occurs when the perianth
closes and pushes the anthers onto the stigma (Carrio
et al., 2008; Vaughton, Ramsey & Simpson, 2008),
resembling but distinct from the mechanism of Z.
australe. Among gentians, it is possible that the
selfing mechanisms in Gentiana lineata Kirk and
Gentianella uliginosa (Willd.) Börner (Webb, 1984;
Petanidou et al., 1998) and some Bulgarian Gentiana
spp. (Kozuharova & Anchev, 2006) are equivalent to
that in Z. australe.
Among the many possible mating strategies, one of
the most advantageous is to primarily let cross pollen
reach stigmas, but also to ensure or increase seed
production in cases of either failure by or inefficiency
of pollinator visits (Lloyd & Schoen, 1992). This gives
the potential for physical mechanisms for the promotion of both cross-pollination at the onset of anthesis
and self-pollination at the end of anthesis, and thus to
combine the positive results of reproductive assurance with the reduced pollen and ovule discounting
and low inbreeding depression (Barrett, 2002). In this
sense, the evolution of delayed spontaneous selfpollination would be more likely in taxa with flowers
that show dichogamy associated with herkogamy, by
means of movements of stamens and style/stigma
during anthesis. This is frequent in long-lived flowers
of certain groups, such as families of Lamiales (Acanthaceae, Bignoniaceae, Gesneriaceae, Lamiaceae,
Plantaginaceae, Scrophulariaceae and Verbenaceae;
see Endress, 1994) and other families of Asteridae (for
example, Asteraceae, Boraginaceae, Campanulaceae,
Escalloniaceae and Gentianaceae), which comprise
most documented cases of autonomous selfing.
Thus, the presence of dichogamy associated with
herkogamy, which supposedly evolved as a result of
selection to promote both the separation of male and
female functions and the efficient transfer of cross
pollen from anthers to stigma by pollinators (Lloyd &
Webb, 1986; Barrett, 2002), may be a first step in the
adaptive evolution of delayed selfing mechanisms
selected by reproductive assurance. At least the
results of Armbruster et al. (2002) concerning evolu-
365
tion in the tribe Collinsieae Meisn. appear to support
this idea. In addition to the families of Asteridae, the
occurrence of spontaneous self-pollination is frequent
in two other unrelated groups: Malvaceae s.l. and
Ranunculales. Finally, as found for four of the gentians studied here, there is an apparent relationship
between spontaneous selfing and pollination by deceit
for Orchidaceae (see Liu et al., 2006; Pansarin et al.,
2008).
Flowers of C. pedunculatus found at Serra da
Bocaina have no nectar. In a population found in the
Serra do Cipó, Minas Gerais, S. Vogel (Faculty of Life
Sciences, University Vienna, pers. comm.) observed
disc nectaries in the flowers. Likewise, flowers of this
species in São Tomé das Letras, Minas Gerais, secreted
up to 15 mL of nectar (M. Sazima and I. SanMartinGajardo, unpubl. data). Thus, floral nectaries seem to
be a plastic trait in C. pedunculatus. These three
populations are separated from each other by hundreds of kilometres, and the presence/absence of nectar
could be related to pollinator activity and autonomous
self-pollination frequency within each population.
Strong evidence to support the reproductive assurance
hypothesis can be provided by patterns of variation of
self-pollination at regional scales and their relationship to floral traits and pollinator efficiency (see
Herrera et al., 2001). There are no data for these
parameters for C. pedunculatus populations, but the
results mentioned above indicate that this species is a
good model to test reproductive assurance in plants.
The daily blooming pattern of Z. australe, i.e.
repeated flower opening and closure at regular intervals, is apparently not widespread in angiosperms.
In the few species that have been studied in detail,
the endogenous rhythms are highly dependent on
changes from light to darkness (and/or vice versa);
rarely, a temperature-dependent endogenous rhythm
may exist (reviewed in van Doorn & van Meeteren,
2003). A similar blooming pattern to that seen in Z.
australe in several other species of Gentianaceae was
considered to be dependent on weather changes
(Webb, 1984; Petanidou et al., 1998; Bynum & Smith,
2001; Kozuharova & Anchev, 2006). Factors, such as
protection of the reproductive parts against pathogens and harsh weather and reduction of costs for
scent production, were proposed as possible selective
forces driving floral opening/closure mechanisms (van
Doorn & van Meeteren, 2003). As observed in Z.
australe, the nastic movements of the corolla in five
Bulgarian Gentiana spp. result in different degrees of
spontaneous selfing, which suggests that repeated
flower opening and closure are related to reproductive
assurance.
The floral appearance of Z. australe and Calydorea
campestris is similar from a human perspective, and
both species share their uncommon daily blooming
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
366
L. FREITAS and M. SAZIMA
pattern over 3–4 days and with petals closing in the
afternoon and reopening the next morning (Freitas &
Sazima, 2003, 2006). In addition, this irid is common
in the Bocaina grasslands (hundreds of plants), and
its pollen-flowers are pollinated by the same animal
groups as Z. australe (Freitas & Sazima, 2006). Such
a set of characteristics, in addition to the higher
frequency (c. 1.5 visits per hour) and behaviour of
pollinators in C. campestris flowers, suggest that this
species may be the model for Z. australe in a Batesian
mimetic relationship. Although it is well accepted
that several systems marked by floral similarity are
probable cases of Batesian mimicry (for example,
Dafni, 1984; Johnson, 1994), clear demonstrations
remain scarce. It is difficult to test whether such a
similarity is important to mimic fitness, and hence a
result of an adaptive process (Roy & Widmer, 1999),
especially because the number of individuals of nonreward mimics is usually not sufficient for extensive
manipulative experiments (as an example, Z. australe
is so rare that it was considered to be extinct in São
Paulo State before our collection in Serra da Bocaina).
Moreover, floral mimicry may have characteristics
beyond the binary conceptual boundaries of protective
Batesian and Müllerian mimicry in animal systems
(see Dafni, 1984). For instance, although Roy &
Widmer (1999) have pointed out as a condition for
floral Batesian mimicry that mimics must be nonrewarding and both species must require pollinators
for seed set, a low rewarding and autonomous delayed
self-pollination mimic could have increased fitness in
a non-Müllerian, Batesian-like system.
In spite of such practical limitations and theoretical
caveats, two additional traits suggest a possible
mimetic interaction between Z. australe and C.
campestris: first, the broad distribution range of both
species overlaps, as they occur in open and cold/mesic
areas, near latitudes 20 and 30 °S in south-eastern
South America (Struwe et al., 2002; Chukr, 2003; Cordeiro & Hoch, 2005); second, the flowering peak of
Z. australe is in February and March at Serra da
Bocaina, overlapping with the final flowering time of
C. campestris (Freitas & Sazima, 2006). Although we
have suggested the relationship between Z. australe
and C. campestris as a possible case of Batesian
mimicry, no robust conclusion can be drawn before a
deeper investigation of this system. Measurements
of the flower appearance of both species from the
perspective of floral visitors (for example, Gumbert,
Kunze & Chittka, 1999) and data on the mimic fitness
in the absence and presence of the model (for
example, Johnson, 2000) may provide robust support
for our suggestion of mimicry (see Roy & Widmer,
1999).
In conclusion, the spontaneous self-pollination
mechanisms of the five gentian species reinforces the
suggestion that the adaptive evolution of delayed
selfing mechanisms selected by reproductive assurance is linked to the presence of dichogamy associated
with herkogamy.
ACKNOWLEDGEMENTS
We are grateful to E. F. Guimarães, I. SanMartinGajardo and S. Vogel for unpublished data and fruitful discussions, I. Cordeiro and B. W. Coelho for plant
and bee species identification, respectively, and two
anonymous reviewers for their useful comments
and linguistic improvement. Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) and Fundo de Apoio ao Ensino e à
Pesquisa (FAEP-UNICAMP) and Petróleo Brasileiro
S.A. (PETROBRAS-PMA), provided financial and
logistic support, respectively.
REFERENCES
Agren J, Schemske DW. 1991. Pollination by deceit in a
Neotropical monoecious herb, Begonia involucrata. Biotropica 23: 235–241.
Albert VA, Struwe L. 2002. Gentianaceae in context. In:
Struwe L, Albert VA, eds. Gentianaceae: systematics and
natural history. Cambridge: Cambridge University Press,
1–20.
Armbruster WS, Mulder CPH, Baldwin BG, Kalisz S,
Wessa B, Nute H. 2002. Comparative analysis of late floral
development and mating-system evolution in tribe Collinsieae (Scrophulariaceae s.l.). American Journal of Botany
89: 37–49.
Barrett SCH. 2002. Sexual interference of the floral kind.
Heredity 88: 154–159.
Beattie AJ, Breedlove DE, Ehrlich PR. 1973. The ecology
of the pollinators and predators of Frasera speciosa. Ecology
54: 81–91.
Buttrose MS, Grant WJR, Lott JNA. 1977. Reversible
curvature of style branches of Hibiscus trionum L., a pollination mechanism. Australian Journal of Botany 25: 567–
570.
Bynum MR, Smith WK. 2001. Floral movements in response
to thunderstorms improve reproductive effort in the alpine
Gentiana algida (Gentianaceae). American Journal of
Botany 88: 1088–1095.
Carrio E, Herreros R, Bacchetta G, Guemes J. 2008.
Evidence of delayed selfing in Fumana juniperina (Cistaceae). International Journal of Plant Science 169: 761–767.
Chukr NS. 2003. Calydorea Herb. (Iridaceae). In: Wanderley
MGL, Shepherd GJ, Giulietti AM, Melhem TS, orgs. Flora
fanerogâmica do Estado de São Paulo, 1a edn, vol. 3. São
Paulo: Fapesp/Hucitec, 129–130.
Cordeiro I, Hoch AM. 2005. Gentianaceae. In: Wanderley
MGL, Shepherd GJ, Melhem TS, Giulietti AM, orgs. Flora
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
SPONTANEOUS SELF-POLLINATION IN GENTIANACEAE
fanerogâmica do Estado de São Paulo, 1a edn, vol. 4. São
Paulo: Fapesp/Rima, 211–222.
Culley TM. 2002. Reproductive biology and delayed selfing in
Viola pubescens (Violaceae), an understory herb with chasmogamous and cleistogamous flowers. International
Journal of Plant Science 163: 113–122.
Dafni A. 1984. Mimicry and deception in pollination. Annual
Review of Ecology and Systematics 15: 259–278.
Darwin C. 1878. The effects of cross and self fertilisation in
the vegetable kingdom, 2nd edn. London: John Murray
(facsimile reprint by Kessinger Publ.).
Darwin C. 1885. On the various contrivances by which
orchids are fertilised by insects, 2nd edn. London: John
Murray (facsimile reprint by University Press Pacific
Honolulu).
Dole JA. 1990. Role of corolla abscission in delayed selfpollination of Mimulus guttatus (Scrophulariaceae). American Journal of Botany 77: 1505–1507.
Donnelly SE, Lortie CJ, Aarssen LW. 1998. Pollination in
Verbascum thapsus (Scrophulariaceae): the advantage of
being tall. American Journal of Botany 85: 1618–1625.
van Doorn WG, van Meeteren U. 2003. Flower opening and
closure: a review. Journal of Experimental Botany 54: 1801–
1812.
Eckert CG, Schaefer A. 1998. Does self-pollination provide
reproductive assurance in Aquilegia canadensis (Ranunculaceae)? American Journal of Botany 85: 919–924.
Elle E, Carney R. 2003. Reproductive assurance varies
with flower size in Collinsia parviflora (Scrophulariaceae).
American Journal of Botany 90: 888–896.
Endress PK. 1994. Diversity and evolutionary biology of
tropical flowers. Cambridge: Cambridge University Press.
Etcheverry AV, Protomastro JJ, Westerkamp C. 2003.
Delayed autonomous self-pollination in the colonizer Crotalaria micans (Fabaceae: Papilionoideae): structural and
functional aspects. Plant Systematics and Evolution 239:
15–28.
Faegri K, van der Pijl L. 1979. The principles of pollination
ecology. New York: Pergamon Press.
Fenster CB, Martén-Rodríguez S. 2007. Reproductive
assurance and the evolution of pollination specialization.
International Journal of Plant Science 168: 215–228.
Fetscher AE, Kohn JR. 1999. Stigma behavior in Mimulus
aurantiacus (Scrophulariaceae). American Journal of
Botany 86: 1130–1135.
Fischer M, Matthies D. 1997. Mating structure and inbreeding and outbreeding depression in the rare plant Gentianella germanica (Gentianaceae). American Journal of
Botany 84: 1685–1692.
Freitas L, Sazima M. 2003. Daily blooming pattern and
pollination by syrphids in Sisyrinchium vaginatum (Iridaceae) in southeastern Brazil. Journal of the Torrey
Botanical Society 130: 55–61.
Freitas L, Sazima M. 2006. Pollination biology in a tropical
high-altitude grassland in Brazil: interactions at the community level. Annals of the Missouri Botanical Garden 93:
465–516.
Ge LL, Tian HQ, Russell SD. 2007. Calcium function and
367
distribution during fertilization in angiosperms. American
Journal of Botany 94: 1046–1060.
Goodwillie C, Kalisz S, Eckert CG. 2005. The evolutionary
enigma of mixed mating systems in plants: occurrence,
theoretical explanations, and empirical evidence. Annual
Review of Ecology, Evolution and Systematics 36: 46–79.
Gumbert A, Kunze J, Chittka L. 1999. Floral color diversity
in plant communities, bee colour space, and a null model.
Proceedings of the Royal Society London B 266: 1711–1716.
Harder LD, Johnson SD. 2008. Function and evolution of
aggregated pollen in angiosperms. International Journal of
Plant Science 169: 59–78.
Herlihy CR, Eckert CG. 2002. Genetic cost of reproductive
assurance in a self-fertilizing plant. Nature 416: 320–323.
Herrera CM, Sánchez-Lafuente AM, Medrano M,
Guitián J, Cerdá X, Rey P. 2001. Geographical variation
in autonomous self-pollination levels unrelated to pollinator
service in Helleborus foetidus (Ranunculaceae). American
Journal of Botany 88: 1025–1032.
Johnson SD. 1994. Evidence for Batesian mimicry in a
butterfly-pollinated orchid. Biological Journal of the
Linnean Society 53: 91–104.
Johnson SD. 2000. Batesian mimicry in the non-rewarding
orchid Disa pulchra, and its consequences for pollinator
behaviour. Biological Journal of the Linnean Society 71:
119–132.
Kalisz S, Vogler D, Fails B, Finer M, Shepard E, Herman
T, Gonzales R. 1999. The mechanism of delayed selfing in
Collinsia verna (Scrophulariaceae). American Journal of
Botany 86: 1239–1247.
Kearns CA, Inouye DW. 1993. Techniques for pollination
biologists. Niwot, CO: University Press of Colorado.
Klips RA, Snow AA. 1997. Delayed autonomous selfpollination in Hibiscus laevis (Malvaceae). American
Journal of Botany 84: 48–53.
Kozuharova EK. 1999. Pollination ecology of Gentiana
species present in Bulgarian Flora. Comptes Rendus de
l’Académie Bulgare des Sciences 52: 73–76.
Kozuharova EK, Anchev ME. 2006. Nastic corolla movements of nine Gentiana species (Gentianaceae), presented in
the Bulgarian flora. Phytologia Balcanica 12: 255–265.
Lin S, Bernardello G. 1999. Flower structure and reproductive biology in Aspidosperma quebracho-blanco (Apocynaceae), a tree pollinated by deceit. International Journal of
Plant Science 160: 869–878.
Liu KW, Liu ZJ, Huang LQ, Li LQ, Chen LJ, Tang GD.
2006. Self-fertilization strategy in an orchid. Nature 441:
945–946.
Lloyd DG. 1992. Self- and cross-fertilization in plants. II. The
selection of self-fertilization. International Journal of Plant
Science 153: 370–380.
Lloyd DG, Schoen DJ. 1992. Self- and cross-fertilization in
plants. I. Functional dimensions. International Journal of
Plant Science 153: 358–369.
Lloyd DG, Webb CJ. 1986. The avoidance of interference
between the presentation of pollen and stigmas in
angiosperms. I. Dichogamy. New Zealand Journal of Botany
24: 135–162.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368
368
L. FREITAS and M. SAZIMA
Lord EM, Russell SD. 2002. The mechanisms of pollination
and fertilization in plants. Annual Review of Cell and Development Biology 18: 81–105.
Lyon DL. 1992. Bee pollination of facultatively xenogamous
Sanguinaria canadensis L. Bulletin of the Torrey Botanical
Club 119: 368–375.
Machado ICS, Sazima I, Sazima M. 1998. Bat pollination of
the terrestrial herb Irlbachia alata (Gentianaceae) in northeastern Brazil. Plant Systematics and Evolution 209: 231–
237.
Martin FN. 1959. Staining and observing pollen tubes in the
style by means of fluorescence. Stain Technology 34: 125–
128.
Pansarin LM, Pansarin ER, Sazima M. 2008. Reproductive
biology of Cyrtopodium polyphyllum (Orchidaceae): a Cyrtopodiinae pollinated by deceit. Plant Biology 10: 650–659.
Petanidou T, Ellis-Adam AC, den Nijs JCM, Oostermeijer JGB. 1998. Pollination ecology of Gentianella uliginosa, a rare annual of the Dutch coastal dunes. Nordic
Journal of Botany 18: 537–548.
Piper JG, Charlesworth B, Charlesworth D. 1986. Breeding system evolution in Primula vulgaris and the role of
reproductive assurance. Heredity 56: 207–217.
Qu R, Li X, Luo Y, Dong M, Xu H, Chen X, Dafni A. 2007.
Wind-dragged corolla enhances self-pollination: a new
mechanism of delayed self-pollination. Annals of Botany
100: 1155–1164.
Radford AE, Dickinson WC, Massey JR, Bell CR. 1974.
Vascular plant systematics. New York: Harper & Row.
Rathcke B, Real L. 1993. Autogamy and inbreeding depression in mountain laurel, Kalmia latifolia (Ericaceae). American Journal of Botany 80: 143–146.
Richards AJ. 1997. Plant breeding systems, 2nd edn. London:
Chapman and Hall.
Roy B, Widmer A. 1999. Floral mimicry. A fascinating yet
poorly understood phenomenon. Trends in Plant Science
418: 325–330.
Ruan CJ, Qin P, Xi YG. 2005. Floral traits and pollination
modes in Kosteletzkya virginica (Malvaceae). Belgian
Journal of Botany 138: 39–46.
Sato H. 2002. The role of autonomous self-pollination in floral
longevity in varieties of Impatiens hypophylla (Balsaminaceae). American Journal of Botany 89: 263–269.
Segadas-Vianna F, Dau L. 1965. Ecology of the Itatiaia
range, southeastern Brazil. II. Climates and altitudinal
climatic zonation. Arquivos do Museu Nacional 53: 31–53.
Struwe L, Kadereit J, Klackenberg J, Nilsson S, Thiv M,
von Hagen KB, Albert VA. 2002. Systematics, character
evolution, and biogeography of Gentianaceae, including a
new tribal and subtribal classification. In: Struwe L, Albert
VA, eds. Gentianaceae: systematics and natural history.
Cambridge: Cambridge University Press, 21–309.
Traveset A, Willson MF, Sabag C. 1998. Effect of nectarrobbing birds on fruit set of Fuchsia magellanica in Tierra
del Fuego: a disrupted mutualism. Functional Ecology 12:
459–464.
Vaughton G, Ramsey M, Simpson I. 2008. Does selfing
provide reproductive assurance in the perennial herb
Bulbine vagans (Asphodelaceae)? Oikos 117: 390–398.
Vickery RK Jr. 2008. How does Mimulus verbenaceus (Phrymaceae) set seed in the absence of pollinators? Evolutionary
Biology 35: 199–207.
Vogel S. 1998. Remarkable nectaries: structure, ecology, organophyletic perspectives. II. Nectarioles. Flora 193: 1–29.
Wang Y, Zhang D, Renner SS, Chen Z. 2004. A new
self-pollination mechanism. Nature 431: 39–40.
Weaver RE Jr. 1972. A revision of the Neotropical genus
Lisianthus (Gentianaceae). Journal of the Arnold Arboretum 53: 76–100.
Webb CJ. 1984. Constraints on the evolution of plant breeding systems and their relevance to systematics. In: Grant
WF, ed. Plant biosystematics. Toronto: Academic Press, 249–
270.
Webb CJ, Littleton J. 1987. Flower longevity and protandry
in two species of Gentiana (Gentianaceae). Annals of the
Missouri Botanical Garden 74: 51–57.
Wheeler MJ, Franklin-Tong VE, Franklin FCH. 2001.
The molecular and genetic basis of pollen–pistil interactions. New Phytologist 151: 565–584.
Yang SX, Yang CF, Zhang T, Wang QF. 2004. A mechanism
facilitates pollination due to stigma behavior in Campsis
radicans (Bignoniaceae). Acta Botanica Sinica 46: 1071–
1074.
Yu Q, Huang SQ. 2006. Flexible stigma presentation assists
context-dependent pollination in a wild columbine. New
Phytologist 169: 237–242.
Zeisler M. 1938. Über die Abgrenzung der eigentlichen Narbenfläche mit Hilfe von Reaktionen. Beihefte zum Botanischen Centralblatt 58: 308–318.
Zhang ZQ, Li QJ. 2008. Autonomous selfing provides reproductive assurance in an alpine ginger Roscoea schneideriana (Zingiberaceae). Annals of Botany 102: 531–538.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 357–368