Botany
Induced cleistogamy: A strategy for reproductive assurance in Murdannia nudiflora (Commelinaceae)
Journal: Botany
Manuscript ID cjb-2019-0007.R3
Manuscript Type: Article
Date Submitted by the 25-Jun-2019 Author:
Complete List of Authors: V, VEENA; University of Calicut, BOTANY Nampy, Santhosh; University of Calicut
anthesis, breeding systems, chasmogamous, entomophilous, Keyword: spontaneousDraft autogamy Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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Induced cleistogamy: A strategy for reproductive assurance in Murdannia
nudiflora (Commelinaceae)
Veena V. & *Santhosh Nampy
Department of Botany, University of Calicut, Malappuram District, Kerala, India- 673635
*[email protected], [email protected]
*Corresponding Author Draft
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Abstract
Murdannia nudiflora is a day flower that is dependent on entomophilous pollination. Despite the lack of pollinator attractants, and its short flower longevity, M. nudiflora shows high fecundity rates, which lead to their quick dispersal. In addition to monitoring vegetative reproductive methods and the effect of select environmental parameters (atmospheric temperature, relative humidity, and precipitation rates), we evaluated mating systems in this species via artificial crosses. This species has evolved to undergo autogamy to ensure reproductive success for when pollinators are insufficient; cleistogamous flowers are produced for optimal pollinator activity when environmental conditions are unfavorable. The number of cleistogamous flowers produced was foundDraft to be positively correlated with humidity and precipitation rates, and negatively correlated with temperature. In addition, it was found that high humidity and precipitation always hinder insect pollinators. Therefore, the combination of vegetative reproduction, entomophily, and cleistogamous self-pollination helps this weedy species to propagate to its maximum efficiency. M. nudiflora employs pre-anthesis cleistogamy as a mating strategy for reproductive assurance while retaining mechanisms that promote out-crossing.
Key words: anthesis; breeding systems; chasmogamous; entomophilous; pollen viability; spontaneous autogamy.
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Introduction
Murdannia Royle is a genus within the dayflower family Commelinaceae, which includes 56 species
worldwide. The genus is primarily Asiatic, with species inhabiting diverse habitats. Murdannia nudiflora
(L.) Brenan, one of the most widespread species of this genus (Ancy and Nampy 2015), was reported to
be invasive (Faden 1982, 2000; GCW 2007) in most parts of the United States, Central and South
Americas, and western Australia (Keighery 1982; Waterhouse 1993; Faden 2000). It is classified as one
of the world's worst weeds, infesting no less than 16 crops in 23 countries (Holm et al. 1977; Moody 1989). Draft Murdannia nudiflora are usually erect to decumbent herbs with rarely branched tufted shoots and roots at
the nodes. Its flowers are bisexual (rarely unisexual) and are compactly arranged on the inflorescence axis
(Ancy and Nampy 2015). Despite the short blooming period of its flowers, M. nudiflora shows high
reproductive success and propagation from one growing season to the next. Like most members of the
Commelinaceae family, M. nudiflora uses entomophily as its chief mode of pollination. Generally, short
duration of flower opening and a lack of nutritional rewards other than pollen (Faden 1992) for pollinators
limit population increase by sexual methods in entomophilous species. Arguably, this species may be
relying on vegetative propagation to ensure continuous population increase. Though their ability to root
readily at nodes and propagate clonally through stems attests to their potential for vegetative propagation,
these ‘propagules’ are unable to survive the harsh summer and perennate to the next season.
Contrary to expectations, the Invasive Species Compendium (Centre for Agriculture and Biosciences
International, https://www.cabi.org/isc/datasheet/35180) reported that M. nudiflora produces 500-2200
seeds per plant, which is a relatively high number for a plant with a short flowering time, lacks nectar, and
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is dependent on insect pollinators. In this study, we aimed to unravel the strategies for reproductive assurance in M. nudiflora across seasons.
Materials and methods
Study area
Natural populations of M. nudiflora growing in the Calicut University Botanical Garden (CUBG,
11⁰7.9850'N 75⁰53.3710'E), Malappuram District, Kerala, and Vellimadukunnu (VK, 11⁰18.1070'N
75⁰48.9250E), Kozhikode District, Kerala, separated by a distance of 30 km, were studied over the course of two years (2016-2017). The first populationDraft was comprised of 73 plants spread over a 3 × 2 m plot, and the second population contained 30 plants spread over a 1 × 1 m plot. Data on humidity, temperature, and precipitation were recorded and pooled.
Floral morphology and phenology
Inflorescence of M. nudiflora is terminal and axillary, consisting of single or sometimes paired pedunculate cincinni. The flowers are usually bisexual and are very rarely male. They are compactly arranged in the cincinnus, and are trimerous with free, ovate-elliptic, pale green, glabrous sepals, and free, obovate, and lilac to lavender colored petals. There are two antesepalous stamens, which are dorsifixed with dense hairs at the basal half of the filament. Anther lobes are deep bluish purple with purplish-white connectives, and dehisce longitudinally to release ellipsoid, creamy-yellowish white pollen. There are four staminodes, of which three are antepetalous and one is antesepalous. They are either glabrous or possess a few hairs at the base with trilobed, white antherodes; the antherode of the antisepalous staminode is reduced to a knob. The ovary is greenish and glabrous, while the style is white, with papillate stigma. Capsules are ellipsoid-triangular, with two uniseriate seeds in each locule (Fig. 1. A-F).
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We randomly chose 10 chasmogamous and 10 cleistogamous flowers from each of the populations. Floral
traits, including lengths of sepals, petals, stamens, and staminodes, were compared using a Zeiss Stemi
508 stereomicroscope.
Floral phenoevents were recorded for one hour between anthesis and flower closure. A total of 35
inflorescences (20 at the first site and 15 at the second) were observed on 20 random days in each season.
A total of 234 flowers were studied.
Pollen production
Pollen production for 10 flowers from the CalicutDraft University Botanical Garden (CUBG) was studied with
a hemocytometer (Shore and Barrett 1984; Barrett 1985; Dudash 1991); mean value was estimated.
Pollen dispersal
To study pollen dispersal, pollen traps were set by fixing microscope cover-slides on wooden sticks to the
ground at the level of the inflorescence, facing the direct of the prevailing wind (Mulugata et al. 1994).
Ten traps were set each day for 25 days every season, before anthesis. Following flower closure, traps
were removed and compared with a known sample under the microscope.
Observations were made on floral visitors for 15 days each season, from anthesis to flower closure. The
number of floral visitors, visiting time, foraging nature, foraging hour, time spent in each flower, stigma
touch by insects, and frequency of their visits were recorded; the presence of pollen on pollinators was
confirmed. Insects were identified with assistance from the Division of Entomology, Department of
Zoology, University of Calicut.
Pollen viability
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Pollen viability tests were carried out in flowers taken from the CUBG. To determine pollen viability, the
1,2,3-triphenyl tetrazolium chloride (TTC) method (Shivanna and Rangaswamy 1992) was used. Tests were carried out at one-hour intervals on freshly collected pollen for two days each season.
Pistil receptivity
The stigma receptivity period was tested (at CUBG) at one-hour intervals with the α-naphthyl acetate test
(Pearse 1985); results were confirmed via in vivo germination tests. The pollinated stigmas were fixed at various periods, and only those that supported pollen germination were considered to be receptive.
Breeding system and fruit set Draft
Twenty-five randomly selected flowers from each of the two populations were subjected to seven pollination treatments, as outlined below (Kaul and Koul 2008):
1. Flowers were tagged before anthesis and left for natural pollination to provide estimates of fruit
set under natural conditions.
2. Flowers were emasculated before anthesis and were left for natural pollination.
3. Flowers were left for natural pollination with anthers and staminodes removed; filaments were
retained.
4. Manual cross-pollination of emasculated flowers followed by bagging to evaluate cross-
compatibility.
5. Manual self-pollination of emasculated flowers followed by bagging to test for self-compatibility.
6. Bagging flower buds to evaluate autonomous selfing.
7. Cleistogamous flowers were tagged to estimate fruit set.
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For manual self-pollination, pollen grains were collected either from the same flower or from another
flower of the same plant. For cross-pollination, pollen grains were collected from flowers of different
plants and different populations. Emasculation (including staminodes) was performed 5 h before anthesis.
All treatments were carried out for two seasons, with the exception of the third treatment, which was only
tested in the second season. The percentage of fruit set from various treatments was compared to evaluate
the efficiency of different breeding systems.
Seed production per plant and seed germinationDraft
Twenty-five plants (at CUBG) were monitored throughout their life cycles, and the average number of
seeds produced by each plant through natural pollination was estimated.
The seeds were washed and thoroughly cleaned before being placed in petri-dishes containing water-
soaked cotton to assess the extent of seed germinability. Petri-dishes were subjected to normal
temperature and light conditions. Two sets of 50 seeds, (i) 10 days after collection from plants and (ii) 6
months of storage post-collection from plants, were treated in this manner.
Perennation of vegetative propagules
Two experimental plots were used at CUBG; each plot was planted with 10 stem cuttings of M. nudiflora.
Seeds were periodically removed from plants to ensure that no regeneration occurred through sexual
means. The plots were monitored for vegetative reproduction in the following season.
Statistical Analysis
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Mean, correlation analysis, and t-tests were carried out for all measurements using Microsoft Excel 2010.
T-test at P < 0.05 was used to compare the means, and to determine the significance of differences between the variables.
The formula given below was used for correlation analysis
푛 ― 2 푡 = 푟 × 1 ― 푟2 , where r = correlation coefficient, and n = number of observations. The resulting value was compared to the table ‘t’ value to determine the statisticalDraft significance.
Results
Floral morphology and phenology
We found that M. nudiflora produced both chasmogamous (CH) and two types of cleistogamous (CL) flowers. A comparison of their floral traits revealed no significant morphological differences (P > 0.05 for all cases) between the CH and CL flowers of the same population. CH and CL flowers of one population were also comparable to those of the other population. Mean ± SE of the floral traits and the results of their t-tests are shown in Tables 1 and 2, respectively. Of the two types of CL flowers, one remains completely closed and lacks anthesis; the other exhibits preanthesis cleistogamy, where anther dehiscence and self-pollination occur in the bud, followed by anthesis.
The number of CL flowers produced was found to be significantly positively correlated with humidity
(77.26%) and precipitation (58.13%), while being negatively correlated with atmospheric temperature
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(63.67%). Correlation analysis of the environmental parameters listed in Table 3 was performed, and the
results are shown in Table 4. Maximum CL flower production occurred in June (36%), July (32%), and
August (34%), when humidity (99%, 95%, and 97%, respectively), and precipitation rates (440 mm, 400
mm, and 390 mm, respectively) were at their highest, and temperature (26.1⁰C, 26⁰C, and 25.5⁰C,
respectively) was at its lowest. In October, when temperature was at its highest, and humidity and
precipitation rates were at their lowest, almost no CL flowers were produced (Table 3). The
environmental parameters and the percentage of cleistogamous flowers were plotted against observational
days in Fig. 2. Seed germination and appearance of seedlings occurred between late May and the beginning of June, coinciding with onset Draft of the monsoon. Flowering began by the end of June to the beginning of July. Anthesis usually took place between 10:00 to 11:30 am; flowers in shaded areas were
10-15 minutes late in anthesis. By approximately 2:00 to 2:30 pm, all flowers were closed and further
deliquesced.
Pollen production
An anther produced 799.11 ± 41.57 (mean ± S.E.) pollen on average. As each flower bears two such
anthers, the pollen to ovule ratio of a flower was 1599.10:6.
Pollen dispersal
Anther dehiscence began five to ten minutes post-anthesis, except in preanthesis CL flowers, where
anthesis was delayed and anther dehiscence preceded anthesis.
When the anthers dehisced prior to anthesis, a small portion of the pollen was transferred to the stigma.
On days when anther dehiscence followed anthesis, insect visitors carried flower pollen (Fig. 1 G) on
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their various body parts, especially their hind legs, lower abdomen, head, corbiculae (pollen basket), and hairs.
It was observed that insects started to hover above these flowers prior to anthesis. The peak pollination period was found to be between 11:00 am and 1:00 pm. The flowers were visited by Osmia sp., Nomia sp., Halictus sp., Amegilla zonata, and an unidentified sp. (Fig. 3). Of these, Osmia sp., Nomia sp.,
Halictus sp., and the unidentified sp. were found to be effective pollinators. Amegilla zonata made quick occasional visits to the flowers, where it spent less than a second on the flowers, and did not result in pollination. Osmia sp. was the most frequent and effective pollinator; three to eight bees of this species were found around the flowers on each day.Draft During each visit, they would spend approximately 8-30 seconds on a flower, the duration shortening with repeated visits. They would land on the stamens in a flower and work their way around, eating pollen and inevitably being covered with pollen.
Halictus sp. was observed for six days in 2016, and two days in 2017; the unidentified sp. was found on five of the 15 observation days in 2017. Nomia sp. was also seen foraging on three days in 2016 and five days in 2017 (Table 5).
The pollen load in the anthers was exhausted nearly an hour before the flower closed on days when 5-8 repeated visits to a flower were made by any of its legitimate pollinators, each lasting 3-5 seconds.
The vaseline-coated slides did not show any pollen, clearly indicating the absence of wind mediated during pollen dispersal.
Pollen viability and stigma receptivity
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Pollen became viable approximately 30 minutes before anthesis, while stigma became receptive only 10-
15 minutes before anthesis. Pollen viability during the peak pollination period (11:30 am to 12:30 pm)
was 89 ± 01% (mean ± SE), after which it declined (Fig. 4). The stigma attained maximum receptivity
between 12:00–12:30 pm and remained receptive for up to two hours after the flower wilted (Fig. 4).
Pollen tubes were found to be longer and closer to the ovary in flowers which were pollinated
immediately after anthesis.
Breeding systems and fruit set
When open pollination was allowed in intactDraft flowers, 73.5 ± 0.83% fruit set was observed; emasculated flowers only showed 7.75 ± 0.74% fruit set. However, when filaments of stamens were retained in
flowers, the fruit set percentage increased to 22.5 ± 0.35%.
Manual cross-pollination and self-pollination resulted in 100 ± 0% and 98.25 ± 0.74% fruit set,
respectively. On the other hand, 45.5 ± 0.90% fruit set was observed when flowers were bagged without
emasculation, and 96 ± 0.61% fruit set was observed in cleistogamous flowers (Table 6).
Seed production per plant and seed germination
A plant produced 282.72 ± 32.8 (mean ± SE) seeds during the three and a half months of its life cycle.
When seeds were allowed to germinate immediately after collection, 14% seed germination was observed
in the laboratory under normal temperature and light conditions; 49% of the seeds germinated after six
months of storage.
Vegetative propagation
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While it was observed that the plants were able to spread through rooted stem nodes to a larger area within the plot, none were able to survive to the next season; barren plots were observed in the following season.
Discussion
Even though M. nudiflora is a very short-lived day flower and lacks nectar, it shows high fecundity rates.
Pollen dispersal studies demonstrated that similar to other members of the Commelinaceae family, wind pollination does not occur in M. nudiflora (Faden 1992). The major pollinators of this species are insects, which should have resulted in reproductiveDraft disadvantage, as the flower lack any nectar to offer as a reward. Having only pollen to attract visitors will exclude entire classes of pollinators, which may lead to severe consequences. However, floral morphology studies revealed an array of adaptations that attract pollinators. The closely-placed purple flowers in inflorescence, hairy, staminal filaments, the purple anther lobes, the creamy yellowish-white pollen, and the staminodes with trilobed antherodes seem to be designed, so as to deceive pollinators by seeming to offer more reward than it actually does. An increased percentage of fruit set in flowers where staminal filaments are retained, as compared to completely emasculated flowers, is indicative of their deceptive potential. The retention of bearded staminal filaments can lead to increased insect visits, thereby increasing the fruit set (Fig. 5). Observations of insect visitors and evaluation of breeding system in these plants confirmed entomophily-mediated pollen dispersal.
The peak periods of pollen viability and stigma receptivity coincides with peak pollination periods. On days with high humidity, cloud cover, or rain, a decline in insect visits was observed (Primack 1983;
Arroyo et al. 1985). On such days, the plant must rely on self-pollination mechanisms.
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The CL flowers produced in this species are examples of induced cleistogamy, as explained by Culley
(2007), where environmental changes induce the production of CL flowers. In contrast to dimorphic
cleistogamy, there is no morphological difference between the CL and CH flowers (Lord 1981). In such
species, unfavorable conditions such as drought and low temperatures often encourage the production of
CL flowers (Uphof 1938). We also found that in M. nudiflora, high humidity, low temperatures, and high
precipitations are correlated with production of CL flowers. Of the two types of CL flowers observed in
this species, preanthesis CL flowers may also offer an opportunity for outcrossing (Culley 2007), rather
than solely acting as a fail-safe mechanism for seed production. As in vivo pollen germination experiments suggest, the pollen tube requiresDraft a long time to reach the ovary, and by allowing the flowers to pollinate themselves prior to opening, a large number of them are able to be fertilized.
The presence of CL flowers in this species is a mechanism for reproductive assurance. The high
percentage of fruit set in bagged flowers confirms the occurrence of spontaneous autogamy and self-
compatibility. As Darwin (1876) has suggested, self-pollination may have been adapted to provide
reproductive assurance in case cross-pollination fails. Plants self-pollinate when pollinators and/or
potential mates are scarce to ensure seed production it is considered as reproductive assurance (Herlihy
and Eckert 2002). According to Lloyd and Schoen (1992), reproductive assurance may be promoted
through autonomous autogamy or facilitated autogamy (caused by animal pollinators). In M. nudiflora
environmental parameters seem to be playing a more crucial role in the need for an assurance mechanism
rather than the lack of pollinators. Keighery (1982) reported the occurrence of facultative cleistogamy
during rainy seasons, and CH flowers on appear on sunny days in this species. We found that both CH
and CL flowers are present during the rainy season and almost only CH flowers on sunny days, with
extremely rare occurrences of cleistogamy. According to Culley (2007), when environments vary across a
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season, the fitness of a phenotype is optimized by the production of a different flower type within each environment to maximize its reproductive success.
The peak flowering season for the species in India falls right during the extreme monsoon conditions, which not only limits the chances of pollinator visits, but also reduces fructifications, as open flowers are usually ruined by heavy rains (Hagerup 1950). Weather is an important factor in determining insect visitation rates (McCall and Primack 1992). Insects are found to be less active when temperatures are exceptionally high (Hagerup 1932; Kevan and Baker 1983; Arroyo et al. 1985), and when conditions are cool, cloudy, or windy (Muller 1881; HagerupDraft 1951; Mani 1962; Downes 1965; Beattie 1971; Cruden 1972; Primack 1983; Arroyo et al. 1985). Consequently, the number of CL flowers produced during rainy, cloudy, and humid days was significantly higher as compared with that on warm sunny days. The inconsistencies in pollen availability across the years may also have prompted adaptive modifications
(Schoen 1991) that resulted in a mixed mating system that produces both self and cross seeds. The almost simultaneous maturation of both male and female reproductive structures, evident from the viable pollen and fertile stigma, may also play a role in facilitating self-reproduction to ensure reproductive success. It is known that selfing mechanisms elevate seed production in natural populations (Elle and Carney 2003;
Kaliz and Vogler 2003). Though our studies revealed lower seed production as compared with that from a previous report by the invasive species compendium (Centre for Agriculture and Biosciences
International, https://www.cabi.org/isc/datasheet/35180), it is significantly more than that reported (100 seeds per plant in 3.5 months of growth cycle) in the unpublished study referred to in the same database.
It is possible that the variation in number is due to differences in the environment and area of study.
In this species, vegetative reproduction ensures that local spread of the population is relatively fast, as opposed to dispersal of propagules to new sites and perennation. Vegetative propagation techniques
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enhance their chances of survival by increasing the population size within shorter time frame, and thereby
adding to its troublesome weedy nature.
In the case of M. nudiflora, the combination of entomophilous pollination and induced cleistogamy helps
this delicate weed to propagate through harsh environments and across seasons. This species seems to
have evolved a mechanism that allows it to produce an abundance of CL flowers in response to various
environmental conditions to promote sexual reproduction. The occurrence of spontaneous cleistogamy in
the species appears to be a preadaptation, which allows induced cleistogamy to be effective in assuring
seed set. They tend to be highly successful, in part, because of their ability to produce an abundance of
seeds, which have relatively long life spansDraft in the soil due to a handful of adaptive reproductive
mechanisms.
Acknowledgements
This work was supported by the Kerala State Council for Science Technology and Environment. We also
thank Dr. K.V. Mohanan, Retired Professor, Department of Botany, University of Calicut, for all statistical
interpretations.
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References
Ancy, A.A., and Nampy, S. 2015. Murdannia Royle (Commelinaceae) of India, Centre for Research in
Indigenous Knowledge, Science & Culture, Kerala, India.
Arroyo, M.T.K., Armesto, J., and Primack, R. 1985. Community studies in pollination ecology in the high
Andes of central Chile. II. Effect of temperature on visitation rates and pollination possibilities. Plant
Syst. Evol. 149: 187–203.
Barrett, S.C.H. 1985. Floral trimorphism and monomorphism in continental and island populations of
Eichhornia paniculata (Spreng.) Solms. (Pontederiaceae).Draft Biol. J. Linn. Soc. 25(1): 41–60.
Beattie, A. J. 1971. Itinerant pollinators in a forest. Madrono, 21: 120–124.
Cruden, R. W. 1972. Pollinators in high-elevation ecosystems: relative effectiveness of birds and bees.
Science, 176: 1439–1440.
Culley, T.M., and M.R. Klooster. 2007. The cleistogamous breeding system: a review of its frequency, evolution, and ecology in angiosperms. Bot. Rev. 73(1): 1.
Darwin, C. 1876. The effects of cross and self fertilisation in the vegetable kingdom. J. Murray, London,
UK.
Downes, J. A. 1965. Adaptations of insects in the Arctic. Annu. Rev. Entomol. 10: 257–274.
Dudash, M.R. 1991. Plant size effects on female and male function in hermaphroditic Sabatia angularis
(Gentianaceae). Ecology, 72(3): 1004–1012.
16 https://mc06.manuscriptcentral.com/botany-pubs Page 17 of 34 Botany
Elle, E., and Carney, R. 2003. Reproductive assurance varies with flower size in Collinsia parviflora
(Scrophulariaceae). Am. J. Bot. 90(6): 888–896.
Faden, R. 1982. Commelinaceae. In Monocot weeds 3: monocot weeds excluding grasses. Edited by B.E.
Hafliger. CIBA-GEIGY Ltd. Basle, Switzerland. pp. 100–111.
Faden, R.B. 2000. Floral Biology of Commelinaceae. In Monocots: systematics and evolution. Edited by
K.L. Wilson and D.A. Morrison. CSIRO, Melbourne. pp. 309–317.
GCW (Global Compendium of Weeds) .2007. Available from
http://www.hear.org/gcw/species/murdannia_nudiflora/.Draft [accessed 19 December 2018].
Herlihy, C.R., and Eckert, C.G. 2002. Genetic cost of reproductive assurance in a self-fertilizing plant.
Nature, 416(6878): 320.
Hagerup, O. 1932. On pollination in the extremely hot air in Tim- buctu. Danske Botaniske Archiv, 8: 1–
20.
Hagerup, O. 1950. Rain-pollination.I kommission hos E. Munksgaard.
Hagerup, O. 1951. Pollination in the Faroes-in spite of rain and poverty of insects. Det KongeligeDanske
Videnskabernes Selskab Biologiske Meddelelser, 18: 1–16.
Holm, L.R., Plucknett, D.L., Panco, J.V., and Herberger J.P. 1977. The World’s Worst Weeds:
Distribution and Biology. Honolulu, USA: University Press of Hawaii.
Kalisz, S., and Vogler, D.W. 2003. Benefits of autonomous selfing under unpredictable pollinator
environments. Ecology, 84(11): 2928–2942.
17 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 18 of 34
Kaul, V., and Koul, A.K. 2008. Floral phenology in relation to pollination and reproductive output in
Commelina caroliniana (Commelinaceae). Aust. J. Bot 56(1): 59–66.
Keighery, G.J. 1982. Facultative cleistogamy in Murdannia nudiflora (Commelinaceae). Flora, 172: 327–
328.
Kevan, P., and Baker. H. 1983. Insects as flower visitors and pollinators. Annu. Rev. Entomol. 28: 407–
453.
Lloyd, D.G., and Schoen, D.J. 1992. Self-and cross-fertilization in plants. I. Functional dimensions. Int. J.
Plant Sci. 153(3, Part 1): 358–369. Draft
Lord, E.M. 1981. Cleistogamy: a tool for the study of floral morphogenesis, function and evolution. Bot.
Rev. 47(4): 421–449.
Mani, M.S. 1962. Introduction to high altitude entomology: Insect life above the timber-line en the North-
West Himalaya No. 5957. Methuen & Co Ltd London.
McCall, C., and Primack, R.B. 1992. Influence of flower characteristics, weather, time of day, and season on insect visitation rates in three plant communities. Am. J. Bot. 79(4): 434–442.
Moody, K. 1989. Weeds reported in rice in South and Southeast Asia. International Rice Research
Institute, Los Banos, Philippines.
Muller, H. 1881. Die alpenblumen, ihrebefruchtundurchinsekten und ihreanpassungandieselbe. Verlag von Wilhelm Elgelmann, Leipzig, Germany.
18 https://mc06.manuscriptcentral.com/botany-pubs Page 19 of 34 Botany
Mulugeta, D., Maxwell, B.D., Fay P.K., and Dyer W.E. 1994. Kochia (Kochia scoparia) pollen
dispersion, viability and germination. Weed Sci. 42(4): 548–552.
Pearse, A.G.E. 1985. Analytical Technology. Histochemistry, Theoretical and Applied vol. 2. Churchill
Livingstone.
Primack, R. 1983. Insect pollination in the New Zealand mountain flora. N. Z. J. Bot. 21: 317–333.
Schoen, D.J., and Brown, A.H. 1991. Whole‐and part‐flower self‐pollination in Glycine clandestina and
G. argyrea and the evolution of autogamy. Evolution, 45(7): 1651–1664. Draft Shore, J.S., and Barrett, S.C.H. 1984. The effect of pollination intensity and incompatible pollen on seed
set in Turnera ulmifolia (Turneraceae). Can. J. Bot. 62(6): 1298–1303.
Shivanna K.R., and Rangaswamy, N.S. 1992. Pollen biology: a laboratory manual. New Delhi: Narosa
Publishing House.
Uphof, J.T. 1938. Cleistogamic flowers. Bot. Rev. 4(1): 21–49.
Waterhouse, D.F. 1993. The Major Arthropod Pests and Weeds of Agriculture in Southeast Asia. ACIAR
Monograph No. 21. Canberra, Australia: Australian Centre for International Agricultural Research.
19 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 20 of 34
Table 1. Floral trait comparison of CH and CL flowers at the two selected localities
Organ Site Mean CH CL SL 3.10±0.60 3.05±0.05 SB 1.60±0.90 1.50±0.07 PL CUBG 5.20±0.18 4.90±0.15 PB 5.00±0.14 4.80±0.13 St 4.83±0.15 4.85±0.14 Sd 1.41±0.07 1.45±0.05
SL 3.10±0.90 3.00±0 SB 1.60±0.60 1.50±0.07 PL VK 5.25±0.10 5.05±0.15 PB 5.05±0.04 4.90±0.09 St 5.08±0.07 4.90±0.09 Sd Draft 1.55±0.05 1.45±0.05 Note: SL- sepal length, SB- sepal breadth, PL-petal length, PB- petal breadth, St- stamen, Sd- staminode
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Table 2. Result of t-test for floral traits
Organ P value
CH flowers of population 1 and SL 0.50* population 2 SB 0.50* PL 0.41* PB 0.38* St 0.09* Sd 0.06*
CH flowers of population 1 and SL 0.17* population 2 SDraftB 0.50* PL 0.26* PB 0.28* St 0.39* Sd 0.50*
CH and CL flowers of population SL 0.28* 1 SB 0.22* PL 0.12* PB 0.17* St 0.46* Sd 0.32*
CH and CL flowers of population SL 0.17* 1 SB 0.16* PL 0.16* PB 0.10* St 0.08* Sd 0.09* *no significant difference at 5% level of significance
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Table 3. Temperature, Humidity and precipitation data with the percentage of cleistogamous flowers produced.
Percentage of Temperature Humidity Precipitation cleistogamous Month (⁰C) (%) (mm) flowers Day 1- June 25.75 94 70 30 Day 2- June 28 88 90 16 Day 3- June Draft 25.5 98 550 28 Day 4- June 25.5 98 150 28 Day 5- June 30 82 0 16 Day 6- June 28.5 83 30 18 Day 7- June 30 80 50 12 Day 8- June 25.6 82 170 18 Day 9- June 26.1 99 440 36 Day 10- June 27.9 90 220 24 Day 1- July 25.5 97 175 20 Day 2- July 25 97 25 26 Day 3- July 27 90 300 28 Day 4- July 26 89 320 22
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Day 5- July 26 95 400 32 Day 6- July 24.7 93 250 28 Day 7- July 25.3 95 180 31 Day 8- July 26.8 94 140 26 Day 9- July 27.4 88 120 15 Day 10- July 27.8 87 200 17 Day 1- August 25.5 Draft98 800 32 Day 2- August 28 86 0 12 Day 3- August 26.9 97 210 29 Day 4- August 28.6 87 250 12 Day 5- August 25.8 97 140 24 Day 6- August 25.5 97 390 34 Day 7- August 29.4 85 300 19 Day 8- August 28.1 90 450 26 Day 9- August 25.1 98 310 28 Day 10- August 24.1 97 0 17 Day 1- September 27.8 82 10 0 Day 2- September 29.5 84 12 0
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Day 3- September 27.05 86 130 0 Day 4- September 29.5 90 260 0 Day 5- September 26.8 94 1000 28 Day 6- September 25.65 98 510 26 Day 7- September 28.25 88 40 0 Day 8- September 28.6 78 0 0 Day 9- September 27.5 Draft96 480 24 Day 10-September 28 88 340 0 Day 1- October 29.3 80 0 0 Day 2- October 27.4 84 50 0 Day 3- October 27.9 83 0 0 Day 4- October 28.1 86 30 0 Day 5- October 26.2 91 320 1 Day 6- October 27.8 85 0 0 Day 7- October 29.1 84 0 0 Day 8- October 28.4 81 0 0 Day 9- October 28.6 81 0 0 Day 10- October 28 85 0 0
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Draft
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Table 4. Correlation analysis of temperature, Humidity, Precipitation and the percentage of cleistogamous flowers produced
Temp Humidity Precipitation % of CL Temp 1 Humidity -0.78272 1 Precipitation -0.36359 0.579498 1 % of CL -0.63674* 0.772608* 0.58136* 1 *no significant difference at 5% level of significance
Draft
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Table 5. Insect visitors
Sl Name of taxa Location Foraging Foraging Time Stigma No and Family for hours spent on touch each visit 1. Osmia sp. CUBG and VK pollen 11.00 am to 8-30 + Megachilidae 01.30 pm seconds
2. Halictus sp. CUBG and VK Pollen 11.15 am to 3-5 + Halictidae 02.00 pm seconds 3. Unidentified CUBG Pollen 11.00 am to 3-8 + sp. 2 02.00pm seconds Syrphidae 4. Nomia sp. CUBG Pollen 11.30 am to 2-5 + Halictidae .1.30 pm seconds 5. Amegilla CUBG and VK Draft- 12.00 pm to 1-2 _ zonata 01.00 pm seconds Apidae
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Table 6. Fruit set percentages for different breeding systems over the two seasons
Season 1 Season 2 Mean±SE CUBG VK CUBG VK Open pollination 72 76 74 72 73.5±0.83
Manual cross-pollination 100 100 100 100 100±0
Manual self-pollination 100 98 96 99 98.25±0.74
Emasculated open 7 6 8 10 7.75±0.74 pollination Emasculation without - - 23 22 22.5±0.35 removing filaments and open pollination Emasculated and bagged 46 Draft48 43 45 45.5±0.90 Cleistogamous flowers 98 96 95 95 96±0.61
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LEGEND TO FIGURES
Fig. 1. Murdannia nudiflora: A. Habit; B. Flower; C. Stamen; D. Staminode; E.Gynoecium; F. Seeds; G.
Osmia sp. with pollen on body.
Fig. 2. Studied environmental parameters and the percentage of cleistogamous flowers plotted against
days of observation. A. Temperature (in ⁰C) against days observed; B. Relative humidity (in percentage)
against days observed; C. Rate of precipitation (in mm) against days observed; D. Percentage of
cleistogamous flowers against days observed.Draft
Fig.3. Insect visitors: A. Osmia sp.; B. Halictus sp.; C. Nomia sp.; D. Unidentified sp.
Fig.4. A. Pollen grains stained by TTC. Red staining (red arrow) indicates viable pollen. Lack of staining
(yellow arrow) indicates non-viable pollen; B. Pollen grains germinated on the stigma; C. Stigma
receptivity as indicated by α-naphthyl acetate test.
Fig.5. A. Insect visit on a completely emasculated flower; B. Insect visit on emasculated flower retaining
stamen.
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Draft
Fig. 1. Murdannia nudiflora: A. Habit; B. Flower; C. Stamen; D. Staminode; E.Gynoecium; F. Seeds; G. Osmia sp. with pollen on body.
209x296mm (300 x 300 DPI)
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Draft
Fig. 2. Studied environmental parameters and the percentage of cleistogamous flowers plotted against days of observation. A. Temperature (in ⁰C) against days observed; B. Relative humidity (in percentage) against days observed; C. Rate of precipitation (in mm) against days observed; D. Percentage of cleistogamous flowers against days observed.
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Draft
Fig.3. Insect visitors: A. Osmia sp.; B. Halictus sp.; C. Nomia sp.; D. Unidentified sp.
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Draft
Fig.4. A. Pollen grains stained by TTC. Red staining (red arrow) indicates viable pollen. Lack of staining (yellow arrow) indicates non-viable pollen; B. Pollen grains germinated on the stigma; C. Stigma receptivity as indicated by α-naphthyl acetate test.
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Draft
Fig.5. A. Insect visit on completely emasculated flower; B. Insect visit on emasculated flower retaining stamen
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