Pollination
biology of Impatiens cuspidata Wight
and Arn. (Balsaminaceae), a rare and endemic balsam of the Western Ghats, India
A.K. Sreekala 1, A.G.
Pandurangan 2, R. Ramasubbu 3 & Shrishail K. Kulloli4
1,2 Scientist,
Plant Systematics and Evolutionary Science Division, Tropical Botanic Garden
and Research Institute
Pacha-Palode,
Thiruvananthapuram, Kerala 695562, India
3 In charge,
Horticulture Section, CSIR Madras Complex, Tarmani, Chennai, Tamil Nadu 600113,
India
4 Field
Officer, Spice Board, Developmental wing, Somawarpet, Karnataka, India
Email: 1 ak.sreekala@gmail.com (corresponding
author), 2 agpandurangan@mail.com, 3 racprabha@yahoo.com,4 sreekulloli@gmail.com
Date of
publication (online): 26 June 2011
Date of
publication (print): 26 June 2011
ISSN 0974-7907
(online) | 0974-7893 (print)
Editor: K.R. Sasidharan
Manuscript details:
Ms # o2385
Received 12
January 2010
Final revised
received 11 May 2011
Finally
accepted 28 May 2011
Citation: Sreekala, A.K., A.G. Pandurangan, R.
Ramasubbu & S.K. Kulloli (2011). Pollination biology of Impatiens cuspidata Wight and Arn. (Balsaminaceae), a rare and endemic balsam of the
Western Ghats, India. Journal of
Threatened Taxa 3(6): 1818–1825.
Copyright: © A.K.
Sreekala, A.G. Pandurangan, R. Ramasubbu & Shrishail K. Kulloli 2011.
Creative Commons Attribution 3.0 Unported License. JoTT allows unrestricted use
of this article in any medium for non-profit purposes, reproduction and
distribution by providing adequate credit to the authors and the source of
publication.
Author Details: Dr. A.K. Sreekala is currently holding a scientist position
in TBGRI. She is recipient of several awards and published as many research
papers in National and International journals. She presently working on
reproductive biology of RET plant species from the Western Ghats. Dr. A.G. Pandurangan is scientist F & Head, Plant
Systematics and Evolutionary Science Division, Tropical Botanic Garden and
Research Institute. He is the recipient of several national and international
awards. He is selected as a member on expert committee of many organizations.
He has more than 200 research papers in national and international Journals. He
is presently working on systematics of grasses, sedges, balsams,
Asclepiadaceae, floristic study of Kerala, reproductive biology of RET Balsams
and. Dr. R. Ramasubbu was working
as senior research fellow in the DST funded projects and currently holding a
position of In-charge, Horticulture section, CSIR-CMC, Chennai. He has
published several research papers in national and international journals. Mr. Shrishail K. Kulloli was also worked
as senior research fellow for the same project. Now he is field officer, Spice
Board, Somawarpet, Karnataka. He has published several research papers and has
contributed articles on Western Ghats.
Author contributions: AKS designed
the work, corrected the manuscript and guided during the study. AGP coordinated
the work, raised the funds, provided necessary comments and guidance for the
field work and manuscript preparation. RR carried out the field and laboratory
work and consolidated the results and prepared the manuscript. SKK assisted
during the filed work and manuscript preparation
Acknowledgement: The financial
support received through a Major Research Project [No SP/SO/PS-09/2003 dated
17-03-2005] from the Department of Science Technology, New Delhi is gratefully
acknowledged. We thank Director TBGRI for extending a necessary logistic
support during the project. Thanks
are due to R. Thulashidharan for help during field collection.
Abstract: The
pollination biology of Impatiens
cuspidata, a rare and endemic balsam from the Western Ghats, has been
studied with special reference to phenology, pollination, pollen-pistil
interactions, breeding experiments and stigma receptivity. It flowers at night between 2330 and
0430 hr; flowering days extend up to 160 days in a year. The anther dehisced one day before
anthesis, which confirmed the protandrous condition of the flower. Pollen-ovule ratio was calculated as
1729:1. Hawk moths, honeybees,
flies and butterflies are the major pollinators of Impatiens cuspidata. Pollen grains are oval, having an average diameter of 28.24μm. Pollen viability by FCR test confirmed
that 82% pollen grains are viable on the day of anthesis. Best pollen germination along with 1636μm
tube development was achieved in Brewbakers medium. Stigma was more receptive (up to 80%) on the first day of
flower opening. It chiefly
reproduced by means of cross pollination, where the fruit set was only 40%, but
artificial cross-pollination through xenogamy enhanced fruit set up to 80%. The plant is an obligate out-crosser
and self incompatible, as confirmed by various hand pollination
experiments. Seed germination in
natural and controlled conditions was only 20%. Its dependence on a specialized
habitat, bottlenecks in sexual reproduction, low percentage of seed germination
and other abiotic factors could be reasons for its limited distribution and
endemism.
Keywords: Impatiens
cuspidata, phenology, pollination biology, stigma receptivity, xenogamy.
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INTRODUCTION
Pollination
biology provides a framework to test a diverse array of paradigms in several
subdisciplines of biology (Bawa et al. 1993). It also plays a critical role in the
formation of seed and fruits. There are very few attempts made on pollination biology of Impatiensexcept by Rust (1977, 1979), Tian et al. (2004), Sreekala et al. (2007,
2008). A comprehensive study on
pollination biology of Impatiens was fundamental to investigations into
its reproductive syndrome, systematics and evolutionary biology.
The
family Balsaminaceae comprises two genera; HydroceraBlume and Impatiens Linn., which are commonly known as
balsams, jewel weed, snap weed or touch-me-not (Willis 1973). The genus Impatiens is supposed
to be one of the largest group among the flowering plants (Sreekala et al.
2007), comprising more than 1000 species (Grey-Wilson 1980; Clifton 2000;
Pieter et al. 2006). It is
essentially sub-cosmopolitan species and mainly distributed in montane areas in
the Old World tropics and subtropics (Grey-Wilson 1980; Yuan et al. 2004), but
several species occur in temperate Eurasia and North America (Pieter et al.
2006). Native species are absent
from the South America and Australia. Impatiens is phytogeographically a unique genus,
which has its greatest development in the Indian region and is found chiefly in
moist sub-temperate areas.
In
India, the concentration of Impatiens species is remarkably local and occurs in
three major centers of diversity including the Himalaya in the north, Western
Ghats in the south and parts of northeastern states. Although the altitude in southern India is lower compared
with the Himalaya, conditions are favourable for the growth of Impatienssince the region gets rainfall from both southwest and northeast monsoons. There is no doubt that in respect of Impatiens,
the Western Ghats are the second richest area in the Indian subcontinent and
perhaps in the World (Bhaskar 1981). The genus contains over 206 species in India (Vivekananthan et al. 1997;
Vishwanathan & Manikandan 2003; Bhasker 2006), half of which occur in
southern India and more than 86 are endemic to the Western Ghats (Nair
1991). Due to their restricted
distribution, nearly 30 species of Impatiens are already threatened with uncertain
future (Vajravelu & Daniel 1983; Pandurangan & Pushpangadan 1997;
Sreekala et al. 2008).
Though
the ideal climatic conditions prevailing in the Western Ghats region provide
suitable habitat for the balsams, their populations are rapidly declining due
to various biotic and abiotic factors. A comprehensive study on pollination biology of endemic balsams has not
been made so far due to their habitat specificity, island biogeography, delicate
nature and explosive fruits. Against this background, one such wild ornamental balsam namely Impatiens
cuspidata has been selected for the present
investigation on pollination biology encompassing phenology, pollination,
pollinator behaviour, pollen viability, stigma receptivity and breeding behaviour
to find out possible reasons for its limited distribution in the Western Ghats.
MATERIALS AND METHODS
Impatiens
cuspidata naturally occurs in evergreen, sholas and
montane grasslands of southern Western Ghats (Fig. 1). The study was conducted on Impatiens
cuspidata in natural conditions, which was located
from Neymakkad gap of Munnar, Idukki District, Kerala, India. The area is located between 9015’–10021’N and 76015’–77025’E. Impatiens cuspidata is a shrub and may attain
a height of 1.5m or even more in dense shola forests at an altitude of
1500–2000 m (Image 1a). This
species is found to be associated with Impatiens leschenaultii, I. coelotropis,
I. henslowiana, Rubussp. Alternanthera sp. and Rhodomytrussp. The area experiences rainfall from both southwest and northeast monsoons
and receives an average rainfall of 3000mm except in Anaimudi where some times
rainfall exceeds 7000mm. The
region harbours vegetation types such as moist deciduous, semievergreen,
evergreen, shola and montane grasslands. The study was conducted during March 2005 and December 2007. Five populations were selected for the
present investigation in the natural condition. Twenty healthy plants were selected from each population and
observations were made on a day-to-day basis in natural habitats on flowering
phenology, which include season, habit, development, anthesis etc. Floral morphology was also studied with
the help of hand lens and dissection microscope. Fifty flower buds were selected from different populations
and observations were made between 2330 to 0830 hrs to study the time of flower
opening (anthesis) and anther dehiscence. The number of pollen grains contained in each anther was determined by
the method suggested by Cruden (1977) to determine P/O ratio of the candidate
species.
Pollen
fertility was assessed by acetocarmine and glycerin staining technique. The stained pollen grains were treated
as fertile and unstained pollens were counted as sterile. Pollen viability was checked by FCR
(fluorochromatic reaction) test using fluorescein diacetate (FDA). To study the pollen germination in
vitro, pollen grains were incubated in sucrose medium of different
concentrations (2, 5, 10, 15, & 20 %) and Brewbakers medium (Brewbaker
& Kwack 1963) containing 2% sucrose for two hours. After two hours the percentage of
pollen germination and tube elongation was noticed. Stigma receptivity was studied visually with the help of
hand lens and by hydrogen peroxide (H2O2) test according
to the method of Scribailo & Posluszny (1984). In vivo pollen germination
was checked by using aniline blue (Aldrich chemical 86.102-2) florescence
microscopic method as designed by Shivanna & Rangaswamy (1992). The preparations were observed under
the fluorescent microscope (Lieca DME Germany). Percentages of pollen
germination in the stigmatic surface and average tube length were calculated.
Continuous
observations during July 2005 to March 2006 were made on behaviour of different
pollinators. The pollinators were
collected and identified with the help of experts from Kerala Agricultural
University and KFRI (Kerala Forest Research Institute). The foraging period and the type of
food collected by different visitors on daily basis were recorded by close
observations. Different pollination systems such as autogamous self pollination
(B), emasculation and hand crossing (S), emasculation and hand out crossing (O)
and natural pollination (N) were tested in the field. Treatment ‘S’ was conducted to examine geitonogamy through
artificial pollination using pollens from different flowers of the same
plant. Treatment ‘O’ was conducted
to examine xenogamy through artificial cross pollination by using pollens from
flowers of different plants but within the populations. Twenty- five healthy flowers for each
treatment from each population were chosen randomly and observed for fruit
set. The developed fruits and
seeds were collected and their numbers were recorded. In addition, the weight of each seed was also measured and
recorded. Twenty mature capsules
were selected randomly from five populations in the field for this
experiment. The distance to which
seeds ejected from the capsules were measured. In the laboratory 240 seeds, from capsules of each plant
were taken and soaked overnight to soften the seed coat. The soaked seeds were then placed on
germination paper and incubated at 250C and germination percentage
was recorded.
RESULTS
The
flowers of I. cuspidata are light pink in colour (Image 1b) born
at apical cyme in pair or solitary and spur is straight, oblong and
glabrous. Capsules are ellipsoid
and cuspidate in nature; contains 14–16 seeds in each capsule. The plant starts
flowering in the month of August and it extends up to December with peak
flowering during October. The
flower buds take 6–11 days from initiation to full bloom. The flowering period is extended up to
160 days in a year and the average life span of the individual flower is
2–3 days. The flowers
bloom in the night between 2330 and 0430 hr, confirming their nocturnal
nature. Anther dehisced one day
before flower opening which confirmed the protandrous condition of the flowers
(Table 1). The mean number of
pollen grains per flower was found to be 34,571 and the mean number of ovules
was twenty. Therefore the pollen ovule ratio was calculated as 1729: 1 (Table
1).
Pollen
grains are oval and have an average size of 28.24µm in diameter. The acetocarmine staining technique
revealed that 80% of the pollen grains were fertile. Pollen viability by FCR test confirmed
that 82% pollen grains were viable on the day of anthesis (Image 1c) and
gradually reduced after second day of anthesis. In vitro pollen germination studies by using different
concentration of sucrose and Brewbakers medium revealed that pollen grains were
viable only up to the minimal period. Effect of sucrose on in vitro pollen germination studies revealed that
68% of pollen grains germinated and produced 1003µm tube in 5% sucrose medium. Best pollen germination (96%) along
with 1636µm tube development was achieved in Brewbakers medium after four hours
of incubation (Image 1d).
Receptivity of stigma is a critical factor for successful
completion of post pollination events. The stigma is wet and non-papillate type (Heslop-Harrison & Shivanna 1977). Stigmas were more receptive up to 80%
on the first day of flower opening (Table 2 & Image 1e); by showing 62% in
vivo germinating pollens along with 917µm long pollen tube on the stigmatic surface
(Table 2 & Image 1g). Subsequently, the receptivity percentages and in vivo germinating
pollens were decreased on successive days after blooming. In I. cuspidata, pollen grains were well adhered on the stigmatic
surface due to sticky nature and presence of pollen threads. Pollen tubes penetrate the stigmatic
region and reached up to the ovary and fertilized the ovules. The fertilized ovules developed into
seeds with only 20% germinability.
When I. cuspidata flowers bloom in the night between 2330 and 0430 hr,
two species of hawk moths: Macroglossum corythus and M. variegatum were found to forage actively. They helped in pollination by their
rapid and frequent visit to flowers for nectar collection. Pollen loads were found on the head and
long proboscis of hawk moths, which transferred pollen grains from one flower
to another from the same plant or another plant and thus favoring geitonogamy
or xenogamy. The foraging periods
of honeybees were mainly during daytime from 0700 to 1600 hr. Honey Bees visited many flowers and
spent an average of 2–4 seconds in each flower, whereas hawk moths spent
slightly long duration from 3–6 seconds in each flower. Bees (Apis cerana indica &Trigona sp.) were found to
be the most abundant visitors and they visited more flowers than any other
pollinators. Butterflies were
active during daytime and in fine weather, they actively visited the flowers,
spending an average of 3–5 seconds on individual flower for nectar. However, when the weather was cloudy,
butterflies were less active and in rainy days, they were completely
inactive. Flies were intermittent
visitors and found to be poor pollinators (Table 3). In general, hawk moths in night and honey bees in day time
served as better pollinators, but they were not sufficient to pollinate all the
flowers in the selected populations.
In I. cuspidata, different breeding experiments were carried out to
find the reproductive capacity of the plant. In natural condition, 40% fruit set was observed (Image 1f).
The fruit set was not observed in autogamous self pollination. However, 56% fruit set was observed in
geitonogamy and 76% in xenogamy (Table 4; Fig. 2). Breeding experiments like geitonogamy and xenogamy produced
more fruits and seeds than the natural pollination. The average weight of the individual seeds produced by the
treatments of natural, geitonogamy and xenogamy were 1.5, 1.7, 2.0 mg
respectively which indicated that the seeds produced by artificial pollination
were more healthy and viable than natural system because of combination of
gametes from different flowers or plants. The fruit development took 25–30 days for attaining maturity after
fertilization. As capsules mature,
the fruit wall ruptured and the seeds were ejected up to 0.60–1.25 m away
from the mother plant and this is the only way of dissemination of seeds in the
present taxa. Seeds germinate
after dehiscing from the capsule in a favorable place but very few of them
established into seedlings in the natural condition. But seeds developed through xenogamy produced more
seedlings. In the laboratory
condition, the germination of seeds obtained through natural pollination was
20%.
DISCUSSION
The
members of Balsaminaceae have their greatest development in the Indian region
and are remarkably endemic. Endemism gives us clue that they are in restricted distribution, either
due to their reproductive syndrome or by anthropogenic pressures. Knowledge on phenology and floral
morphology are essential for conducting studies on breeding systems
particularly on pollination syndrome if any. Impatiens cuspidata starts flowering in the
month of August and continued up to December and reached a peak during October
and anthesis commenced between 2330 and 0430 hr on the next day. About 62% of Impatiens species
in the Western Ghats flower during July–December, 16% during
April–June and 15% during January–March. Interestingly 18% of the balsams flower throughout the year
if conditions are favorable (Rajalal et al. 1996). Bhaskar & Razi (1974)
had reported that majority of the wild balsams grown in the high altitude areas
are night blooming and have a wide range of timing with regard to pollen
germination. The anther dehisced
one day before anthesis, which in turn confirmed their protandrous condition. This observation was similar to that ofI. platypetala, I. korthalsii, and I. eubotrya in
Sumatra (Kato et al. 1991).
In I. cuspidata, pollen
viability is highest on the day of anthesis and then gradually decreased on
successive days after anthesis. This observation is similar to that of I. reptans in
China (Tian et al. 2004). In vitro pollen germination test indicated that highest
percentage of pollen germination and tube elongation was observed in Brewbakers
medium. Sucrose acts as a
nutritive material for pollen germination (Johri & Vasil 1961) and it helps
in maintaining osmotic balance between the germination media and pollen
cytoplasm (Mukerjee & Das 1964). Germination percentages were significantly low in higher concentration
of sucrose medium. According to
Shivanna & Johri (1985), the optimum concentration of sucrose varies from
species to species. In the present
investigation, Brewbakers medium is the most suitable for pollen germination inI. cuspidata. Besides the medium contain
carbohydrates, boron and calcium which are other important substances required
for pollen germination and tube growth (Brewbaker & Kwack 1963). Pollen germination and subsequent post pollination events depend upon the
receptivity of the stigma, its nature and compatibility.
It
is well known that the flowers of Impatiens have enormous diversity and different
pollinators. Impatiens
cuspidata is pollinated by honeybees, hawk moths
and butterflies. In different
climatic regions, species of pollinators vary. In sub tropical regions of Africa the Impatiensspecies are pollinated by humming birds as well as by insects. In temperate zones, pollinators are
bumblebees and humming birds (Rust 1977, 1979; Heinrich 1979; Kato et al.
1989). In I. cuspidata, bees
(Apis cerana indica and Trigona sp.)
are the most important pollinators and visited more flowers than any other
pollinators during day time for nectar and pollen gathering. There is a strong relationship between
the weather and foraging activity of pollinators. When the weather is fine, butterflies are more active and
spend on an average 2–6 seconds on a flower at each visit. But when the weather is cloudy and
rainy, the butterflies and hawk moths are less active. The present investigation agrees with
the findings on I. coelotropis (Sreekala et al. 2008).
In I. cuspidata,40% fruit set was observed in natural pollination. However, artificial cross pollination (geitonogamy and
xenogamy) enhanced the fruit set rate up to 76%. Pollination experiments demonstrated that, artificial
cross-pollination enhanced the rate of fruit and seed set in I. cuspidata. The balsams are highly evolved members
among the order Geranials as evident from their marked zygomorphic flowers and
nectariferous spur. The
arrangement of stamens, pistil and spur are markedly adapted for cross
pollination in Impatiens (Bhaskar & Razi 1974) and hence most
of the species of Impatiens reproduce by cross pollination (Schmitt
& Gamble 1990; Lu 2000, 2002).
Stigma
receptivity is a critical factor for successful completion of post pollination
events. Usually it is highest soon
after anthesis but it varies from species to species, depending upon the
temperature and humidity (Shivanna & Johri 1989). But in I. cuspidata, stigmas remained receptive only after
the shedding of androecium and gradually increased for 8–14 hr. The receptivity ends after 14 hours but
at the same time its pollen viability reduced drastically. The adhesion of pollens on the stigma
is a primary requirement for successful pollination. After landing on the stigmatic surface, pollen grains are
subjected to hydration and then pollen wall proteins are released on to the
stigmatic surface (Heslop-Harrison et al. 1975). In I. cuspidata,pollen grains are well adhered on the stigmatic surface. Pollen tubes penetrate the stigmatic
surface and reached up to the ovary and successfully fertilize the ovules. The fertilized ovules developed into
seeds. The percentage of seed set was only 60%.
Seeds
are not dormant and they germinate immediately. Experimental results also
substantiate the same. Very few
germinated seeds were established into seedlings and remaining perished. This may be due to insufficient flow of
nutrients into seeds. Therefore
the study suggests that, absence of dormancy, protandry, self-incompatibility,
pollinator limitation and perishing of considerable percentage of seedlings
prior to establishment in combination with other abiotic traits are
contributing factors for regulation of population size of I. cuspidata in
its natural condition.
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