DEVELOPMENTAL PATTERNS OF CLEISTOGAMY AND CHASMOGAMY IN
TRIODANIS PERFOLIATA. by
Elizabeth A. Bernetski
A Senior Honors Project Presented to the
Honors College
East Carolina University
In Partial Fulfillment of the
Requirements for
Graduation with Honors by
Elizabeth A. Bernetski
Greenville, NC
May 2015
Approved by:
Carol Goodwillie, Ph.D.
Thomas Harriot College of Arts and Science
Department of Biology
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I hereby declare I am the sole author of this thesis. It is the result of my own work and is not the outcome of work done in collaboration, nor has it been submitted elsewhere as coursework for this or another degree.
Signed: Date:
Elizabeth A. Bernetski
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Developmental Patterns of Cleistogamy and Chasmogamy in Triodanis perfoliata
Elizabeth A. Bernetski
Department of Biology, East Carolina University, Greenville N.C. 27858
ABSTRACT - Some plants exhibit mixed mating, in which individual plants utilize both cross and self-fertilization. Dimorphic cleistogamy is one form of mixed mating. In plants that display dimorphic cleistogamy, both chasmogamous (CH) and cleistogamous (CL) flowers may occur on the same individual. CH flowers are typical open flowers that are predominantly cross-fertilized.
CL flowers are closed, lack petals, and are obligate self-fertilizing. Benefits of cross-fertilizing
CH flowers are that the resulting offspring may have a high level of fitness; however, they have a high energetic cost, and the flowers rely on pollinators. In CL flowers, the ability to self-fertilize may benefit plants in that reproduction can occur without the need to find a separate mating partner. However, self-fertilization generates populations that have little genetic variation and results in higher levels of inbreeding depression. Although the fitness advantages of both flower types have been hypothesized, the factors that maintain this mixed strategy have not been fully explained. Triodanis perfoliata is a local annual plant that exhibits dimorphic cleistogamy.
Observing the patterns of CH and CL flowers in Triodanis perfoliata may provide clarity to how a mixed mating strategy can be maintained. In a growth room study, individual flowers were observed to compare rates of development of the two flower types. Individual plants were observed weekly to determine the spatial and temporal development of CH and CL flowers throughout the lifespan of a plant. By sequentially numbering the nodes on the main spike of the plants, the type and developmental stage of each flower at the individual node was observed.
Characterizing the spatial and temporal patterns of development of CH and CL flowers in
Triodanis perfoliata may provide insight to the adaptive value of dimorphic cleistogamy.
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Acknowledgments
I would sincerely like to thank the people and organizations that made this study possible: Dr.
Tom Fink, who taught and allowed me to use the microscopy equipment, my Parents, Joseph and
Sandra Bernetski, the Department of Biology, East Carolina University, and the Honors College.
I am especially grateful to my project advisor, Dr. Carol Goodwillie, for her knowledge and guidance.
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Title Page
Abstract
Acknowledgements
Table of contents
List of Tables
List of Figures
Introduction
Materials and Methods
Study species
Plant cultivation
Observational study
Microscopy
Results
Observational study
Microscopy
Discussion
Literature cited
Table of Contents
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List of Tables
Table 1 : Survey of the occurrence of cleistogamy and chasmogamy in eight plant species. 23
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List of Figures
Figure 1: The spatial occurrence of CL and CH flowers along the main flowering spike
Figure 2: The temporal occurrence of CL and CH flowers
Figure 3: The timing of developmental events of both CL and CH flowers
Figure 4: Bright field microscopy of CL flowers at various ages
Figure 5: Pollen tubes of CL and CH flowers viewed under DAPI filter
Figure 6: Anthers from CL and CH flowers viewed under a FITC filter
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Introduction
Plant mating systems can be divided into two basic types, dioecy and monoecy, which influence the mode of fertilization. In a dioecious species, individual plants have either all male or all female flowers. With the flowers all being of one sex, dioecious plants require crosspollination for fertilization. The monoecious plant species has either separate male and female flowers on the same plant or has hermaphroditic flowers containing both male and female organs
(Charlesworth 2006). The hermaphroditic plant can fertilize through cross-fertilization, selffertilization, or mixed mating, which is a combination of self and cross fertilization (Barrett
2002).
Cross fertilization occurs between individuals with the exchange of gametes of the opposite sex. For the monoecious plant, male and female structures used for fertilization can be separated spatially in different flowers, on different areas of a single flower, or can be separated by the timing of reproductive functioning, all of which reduce the likelihood of self-fertilization
(Barrett 2002). The advantage of cross fertilization is that outbred offspring often have a higher fitness (Darwin 1877). New generations produced by cross pollination between genetically diverse parent plants tend to display heterosis or hybrid vigor resulting in benefits such as increased size, fecundity, and disease resistance (Schmitt & Gamble 1990). As a result of heterosis, offspring with genetic diversity may be better suited to thrive in a variety of heterogeneous habitats (Mitchell-Olds & Waller 1985). A disadvantage of cross fertilization is that large flowers required for pollinator attraction can have a high energetic cost and fertilization mainly depends on pollinators (Barrett 2002).
Self-fertilization can occur in individual plants when both female and male gametes are produced by the same plant (Barrett 2002). A benefit of self-fertilization is the
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increase in the transmission of mature sex cells that form the next generation (Fisher 1941) because plants act as both mother and father of each seed (Pannell & Barrett 1998). The disadvantages of self-fertilization include increased inbreeding depression and populations that are genetically static (Barrett 2002).
Inbreeding depression occurs when selfed offspring display lower fitness in comparison to outbred offspring (Barrett 2002). This change in fitness is due to the increased expression of harmful recessive alleles in the inbred offspring (Charlesworth & Charlesworth 1995). When individuals are carriers of a harmful recessive trait, the trait is not expressed, but when they inbreed, one quarter of the offspring produced are expected to express the harmful recessive allele. Inbreeding depression usually affects the rate of growth, resistance to disease, survival of the offspring, and often has substantial effects on fertility (Charlesworth & Charlesworth 1987).
Inbreeding depression is a major factor in the evolutionary selection of cross pollination
(Charlesworth & Charlesworth 1995). While a large amount of inbreeding generally results in lower fitness, there are indications that some plants that are naturally inclined to self-fertilize, show less significant inbreeding depression than those plants that are naturally cross-fertilized
(Wright 1977). Successful selfing species may be eliminating the deleterious recessive alleles in their populations through natural selection (Schemske & Lande 1985).
Some plant species have evolved to take advantage of both cross and self-fertilization methods, which is referred to as mixed mating. One form of mixed mating is dimorphic cleistogamy, in which both open, chasmogamous (CH) flowers, and closed, cleistogamous (CL) flowers, may occur on the same individual. Chasmogamous flowers predominantly participate in cross-fertilization, but may occasionally utilize self-fertilization (Culley 2000, 2002).
Cleistogamous flowers solely use self-fertilization.
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Cleistogamy occurs in various forms. Dimorphic cleistogamy is one such variation.
Dimorphic cleistogamy is also referred to as true cleistogamy in Lord’s classification (1981).
The CH and CL flowers of dimorphic cleistogamy have noticeable differences in their morphology. The CL flowers are significantly reduced in size compared to CH flowers and do not produce petals (Lord 1981). The differences in the floral morphology are a result of the CL and CH forms developing from alternate routes (Culley & Klooster 2007). The type of flower produced may be influenced by environmental factors such as the amount of light, nutrients, pollinators, and herbivore activity (Culley & Klooster 2007). Once the individual primordial bud has gone down one of the developmental pathways, the bud is only able to produce one type of flower and is unable to change to produce the other. The change in flower type within the growing season is gradual because the change has to originate from new bud growth since the developmental pathway between CH or CL is not reversible (Culley & Klooster 2007).
Induced cleistogamy is another form of this reproductive system, which is classified as pseudocleistogamy in Lord’s (1981) classification. In induced cleistogamy, certain environmental conditions such as drought (Uphof 1938) cause buds that are initially developing as CH flowers to halt petal development prior to anthesis, preventing the flower from opening, which results in the formation of a CL flower (Schoen & Lloyd 1984). Because of this aspect in development of the CL flower there are relatively few morphological differences between the CL and CH flowers beside floral expansion and anthesis (Lord 1981). Another result of this development pattern is that seasonal shifts between flower types occur relatively quickly (Culley
& Klooster 2007).
Complete cleistogamy is a third variation in which the individual plant produces only CL flowers (Culley & Klooster 2007). Some orchids (Uphof 1938) and grasses (Connor 1979) have
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been observed to have complete cleistogamy, although most of these observations have been based on a few individuals in an artificial environment (Culley & Klooster 2007). Darwin
(1877) was unconvinced of reports of complete cleistogamy. It is hard to confirm complete cleistogamy in nature because of the level of observation needed to completely deny the possibility of any cross-fertilization occurring (Culley & Klooster 2007).
The spatial occurrence of CH and CL flowers varies among different plant species.
There is a trend of CH flowers being produced near the apex of the plant, while the CL flowers are formed more towards the base, as in Centaurea melitensis (Porras and Munoz 2000) and
Oxalis acetosella (Berg 2000). In another spatial orientation, the CL flowers are subterranean while the CH flowers are aerial or above ground, as seen in Vigna minima (Gopinathan & Babu
1987). This may occur because being above ground makes CH flowers more accessible to the pollinators necessary for cross-fertilization, while the CL flowers have no need for pollinators and therefore can produce fruits underground. The subterranean growth of CL flowers allows the plant to produce additional flowers without using more resources for the plant to grow taller.
In other words, by moving the CL flowers below ground, the plant can allocate a greater portion of the existing plant for flower production.
In addition to spatial patterns, temporal differences in the occurrence of CH and CL flowers have also been observed. The floral type may change during the season by altering the production of primordial buds which develop into either CL or CH flowers (Culley & Klooster
2007).
Impatiens noli-tangere is an example of a plant in which the CH and CL flowers can have a large overlap (Masuda & Yahara 1994). The CL flowers have been seen to develop first and then CH flowers are produced alongside the CL flowers. In Scutellaria indica (Sun 1999) and
O xalis acetosella (Berg & Redbo-Torstensson 2000), the CH flowers appear first and then the
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CL flowers appear with very little overlap between the two flower types. However, in
Centaurea melitensis , while the two flower types also have a small period in which they overlap, the CL flowers are produced first (Porras & Munoz 2000). CH flower production may be timed to coincide with optimal pollinator activity, with pollinators being most active when CH flowers are produced and least active when CL flowers are produced (Masuda et al. 2004). Not all plants produce both flower types during the same period. Viola pubescens is an example of one that produces each type of flower with no overlap (Culley 2002). The CH flowers are produced first for 2 to 3 weeks and then after which, CL flowers are produced. The timing of flower type development is hypothesized to be associated with changes in light availability in this case
(Culley 2002), as opposed to pollinator activity. A sunny environment provides the high energy required for CH flowers to be produced. When the environment becomes shady due to the leafing out of trees, the CL flowers have the advantage of requiring less energy to be produced.
The various types of fertilization have different costs and benefits. In a cross-fertilized plant that produces only CH flowers the costs of self-fertilization, which are inbreeding depression and the expression of harmful recessive traits, is minimal due to the constant outcrossing (Charlesworth & Charlesworth 1995). For plants that only have CL flowers, the detrimental recessive traits can be purged from the population, which would raise the fitness of the plants and lower inbreeding depression (Schemske & Lande 1985). Questions remain as to why a plant would maintain both CH and CL flowers. If plants producing CL flowers have purged harmful alleles, why continue to produce the costly CH flowers? Alternatively, if a plant produces CH flowers, why also produce CL flowers that come with the risk of inbreeding depression? A definitive reasoning for why mixed mating strategy using both CH and CL flowers is maintained in many plant species has not been established. By investigating the
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developmental patterns of where and when each type of flower form in relation to each other may shed light on the benefits of CH and CL flowers and why both types of flowers have continued to be maintained.
This study is an exploratory study to characterize the occurrence of CL and CH flowers in Triodanis perfoliata and to document their spatial and temporal patterns. Data were collected to clarify where on the plant the CH and CL flowers form, the order of appearance and maturation of CH and CL flowers and fruits, the length of the developmental period from flower appearance to fruit dehiscence of the two flower types, taking note of any overlap in production of the two flower types.
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Materials and Methods
Study species
Triodanis perfoliata is an annual herb in the family Campanulaceae that exhibits dimorphic cleistogamy.
This weedy plant is widely distributed across the United States and
Canada and flowers during the summer season (USDA 2015). The CL flowers produced typically have only three or four sepals, and petals are absent. The CH flowers produced have five violet blue petals, are 1-1.5 cm in diameter, and commonly have five sepals. The CH flowers demonstrate protandry. Before anthesis, dehiscence takes place in the anthers resulting in the male phase in which the pollen is released, followed two days later by the female phase when stigmas opens and become receptive. With the separation of when the male and female reproduction organs function, the chance of self-fertilization in the CH flowers is reduced.
Pollinators to these flowers include small bees, wasps, and beetles (Gara & Muenchow 1990;
Goodwillie & Stewart 2013). Multiple CH and CL flowers are produced along the spike of
Triodanis perfoliata .
Plant cultivation
In a greenhouse that is provided with both ambient light and artificial lighting in a 14 hour daylight period (6 am to 8 pm), Triodanis perfoliata seeds from a local population in Pitt
County were planted in a seed tray with a mixture of half greenhouse potting mix and half
Miracle Gro potting soil. Once the seeds germinated, the seedlings were transplanted into individual conetainers (Stuewe and Sons, Corvallis, OR) containing the same soil mixture and were suspended in water. The plants were then fertilized every other week.
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Observational Study
Six Triodanis perfoliata plants were observed to identify the spatial and temporal development of CH and CL flowers throughout the plant’s lifespan. Upward growth of the flowering stem began eight weeks after the seedlings were transplanted. At this point the nodes and bracts could clearly be identified. The nodes along the length of the main spike of each plant were numbered on the bracts sequentially from the lowermost to the uppermost, after which weekly observations were made on each of the nodes, taking note of bud appearance, flower type, and stage of development. The plants continued to be observed until the end of plant senescence.
To get more precise data on the duration of the flower’s development period, individual nodes were labeled on some plants for more frequent observation of individual flowers of both
CL and CH types. These nodes were observed every other day for five weeks to gather more detailed data on how long it took for each flower to progress through each developmental stage.
For the CL flowers observations noted were first appearance of CL flowers as well as timing of dehiscence. With the CH flowers, timing of first appearance and dehiscence were noted as well as when the male and female form developed.
Microscopy
Throughout the study, CL flowers were removed from the plant at varying ages and CH flowers were removed from the plant before and after pollination. The flowers were placed in specimen dishes and were fixed with a solution of 70% ethanol and 30% acetic acid. During the period of fixation the dishes were wrapped in plastic wrap to prevent evaporation. The flowers were then treated with a 10 M NaOH solution to degrade the external tissue, which results in the internal structures becoming more visible. After two days in the NaOH solution, the flowers
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were treated with 1% aniline blue in 33mM potassium phosphate solution to stain the flowers and allow the pollen tubes to be seen (Martin 1959). The flowers were then observed under both bright field and epifluorescence microscopy using DAPI and FITC filters.
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Results
Observational Study
One out of the six Triodanis perfoliata plants numbered for observation died before data could be collected. The remaining five plants were observed until plant senescence, for a total period of 21 weeks. The height of the plants varied, with the number of nodes ranging from 64 to 37. In all of the plants, the CH flowers occurred in the uppermost nodes of the plant. The CL flowers were found along the length of the spike, and were concentrated in the middle and the top of the spike. While the CH flowers occurred only as primary flowers, the CL flowers occurred as both primary and secondary, so multiple CL flowers were found in a single node, and secondary CL flowers also were produced later in the same nodes as primary CH flowers.
Both flower types were absent in the lowermost nodes of the plant.
Both CH and CL flowers were visible by the 12th week after germination. A peak in CH flower production occurred at week 16 with plants having on average 12 pre-dehiscence CH flowers. The CL flowers had a sharp increase in production around week 15, and production peaked at week 18 with an average of 42 pre-dehiscence CL flowers on each plant.
CL flowers observed (n=57) had a mean of 14 days from first appearance to fruit dehiscence with a standard deviation of 0.625783. CH flowers observed (n=47) had an average of 25.9 days from first appearance until fruit dehiscence with a standard deviation of 0.649486.
CH flowers exhibit protandry, and the length of each developmental period was noted. The interval between first appearance to when the CH flower initially opened in the male phase was approximately nine days. From male phase to female phase took approximately two days. From female to petal senescence took approximately two days, and from petal senescence to fruit dehiscence took approximately eleven days.
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Microscopy
In CL flowers, the pollen tubes were not visible in the specimen collected five days after initial appearance, but in the specimen collected seven days after initial appearance the pollen tubes were visible. By day seven the CL ovules had been fertilized and had greatly increased in size from 63 µm at day one to 471 µm. The pollen tubes in the CH flower were visible after flower senescence. Anthers seen in the CL flower were found located below the sepals, were
145 µm in length and contained approximately 14 pollen grains per anther. The anthers in the
CH flower were located above the sepals, were 1489 µm in length and contained approximately
160,000 grains of pollen per anther.
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Discussion
Triodanis perfoliata produces both CL and CH flowers, which display differences in appearance, timing, and maturation rates. The CL flowers mature rapidly and are predominantly created at the end of the plant’s life, while the CH flowers typically take an additional two weeks to mature and are produced more towards the beginning of the flowering season. On average the ovules of CL flowers are fertilized and seeds mature before the CH flower fertilizes its ovules.
The CL fruits take an average of 14 days to dehisce from first appearance on the plant. In contrast, the CH fruits on average take 25.9 days to dehisce. Similar patterns have been observed in other plants such as Impatiens noli-tangere (Masuda, et al. 2004) , Viola odorata (Mayers &
Lord 1983) , Oxalis acetosella (Berg & Redbo-Torstensson 1998) , and Lamium amplexicaule
(Lord 1982) in regards to CL flower buds maturing at a faster rate than CH buds. While all of these plants demonstrate more rapidly maturing CL fruits, they do not all show the same degree of difference in maturation rate. For example Viola odorata has fruits that have relatively long maturation periods, with CL maturing around 45 days and with CH around 60 days (Mayer’s &
Lord 1983). CH and CL flowers had overlapping growing periods on Triodanis perfoliata. CH flower production started to decline after week 16. CL flowers peaked in production in week 18 and did not show a decline until closer to plant senescence in week 21. Centaurea melitensis exhibits a similar pattern in flower development with the CH flowers developing first, and then switching to mainly producing CL flowers (Porras & Muñoz 2000). This study on Triodanis perfoliata provides more insight into the process in the process of a mixed mating system and the data may be used to better understand why the species maintains both self and cross fertilization methods.
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Mixed mating systems continue to be a viable method of reproduction for some plant species, and there are many questions as to why a plant would maintain both cleistogamy and chasmogamy as forms of reproduction. There are costs and benefits for utilizing either form, and any number of factors may be involved in the underlying reasons as to why either form does not become the sole method of fertilization. Factors such as the length of the growing season, fruit maturation rates, and pollen production, all may play a role in why mixed mating continues to be maintained in many plant species.
Cleistogamous flower production near the end of the flowering period is more effective in producing a higher output of seeds with the limited resources of a plant nearing senescence
(Porras & Muñoz 2000). The higher effectiveness of CL flowers towards the end of the plants life may explain why Triodanis perfoliata produces CL flowers as both primary and secondary flowers but only produces CH flowers as primary flowers. The plant is limited in its resources and its growing season. Reproductive success is increased by producing CL flowers in nodes already occupied with either flower type as the primary flower, instead of using its limited resources less effectively to make more nodes by growing taller or by branching. The observed timing of development of CH and CL flowers on Triodanis perfoliata was similar to a study on
Viola pubescens. In this study CH flower production was observed to peak first, followed by a peak in CL flower production with continued CL flower production until the end of the season
(Culley 2002). In Viola pubescens, this system promotes reproductive success by producing the different flower types at the most cost effective times. CH flowers are produced early in the flowering season, when pollinators are active and resources are abundant, followed by production of CL flower later in the flowering season when pollinators are less available and resources and time are limited therefore maximizing the plant’s seed output (Culley 2002).
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Triodanis perfoliata has a finite growing season; therefore by not solely relying on the longer maturing CH flowers and producing more of the faster maturing CL flowers towards the end of the plant’s life, it allows
Triodanis perfoliata to successfully utilize more of the growing season and maximizes mature seed output. This may be a contributing factor as to why dimorphic cleistogamy has continued to be maintained. The plant may produce each flower type according to the amount of resources, and time available: CH flowers, which have the advantage of producing more seeds and higher fitness offspring, are produced earlier in the growing season when there are ample resources and time, and more CL flowers are produced later in the season, which optimizes the total viable seed output (Schoen & Lloyd 1984).
Pollen production observed in Triodanis perfoliata CH flowers was significantly higher than in CL flowers. Both CH and CL flowers generally develop five anthers but, CH exhibited approximately 160,000 pollen grains per anther, while CL contained approximately 14 pollen grains per anther. This higher pollen production in CH flowers was also observed in a study on
Scutellaria indica , although the difference was not as great (Sun 1999). This difference in pollen production can be attributed to the different flower structures and pollination methods. In the CL flower, the pollen is produced right next to the ovules it needs to fertilize, so CL flowers only need to produce enough pollen so that each ovule inside the enclosed flower can be successfully fertilized. Meanwhile, the CH flowers, being open flowers, rely on external factors such as wind and pollinators to successfully transport the pollen to the receiving stigma allowing the ovules to be fertilized, which is much less reliable and therefore the plant must produce an excess of pollen to increase the chances of successful fertilization. CH flowers need to produce showy petals to attract pollinators and must also produce a large number of pollen in order to be successful, which costs the plant more in resources and time than the production of CL flowers.
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This study gave interesting data on the timing of production of the CL and CH flowers, the maturation time of each flower type, and the pollen abundance in both types of flowers, but to more fully understand the selective factors responsible for the maintenance of dimorphic cleistogamy, further studies must be completed. An observational study examining changes through time in pollinator activity, and CH and CL flower production rates could determine whether there is a correlation between the two. A controlled fertilization study examining the fitness of the offspring of cross and self-fertilized plants would provide data on the level at which
Triodanis perfoliata is affected by inbreeding depression. A quantitative study on the energetic cost per seed of CH and CL flowers of Triodanis perfoliata, by measuring the dry biomass of the pre-dehiscence fruit and counting the number of seeds, would provide insight to the cost/benefit ratio of each flower type.
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Table 1. Survey of the occurrence of cleistogamy and chasmogamy in eight plant species.
Species Temporal Occurrence Citation
Centaurea melitensis
Impatiens noli-tangere
Monochoria vaginalis
Oxalis acetosella
Type of
CL
Spatial
Occurrence
Dimorphic -CL occur near base
-CH occur near apex
Dimorphic -CL occur near apex
-CH occur near apex
Induced -induced CL occur where CH flowers are
-CH occur at the apex
Dimorphic -CL occur near base
-CH occur near apex
Little Overlap
-CL produced first
-CH produced in middle of flowering season
-CL produced at the end of flowering season
Overlap
-CL produced first
-CH produced in middle of flowering season
Overlap
-CL and CH are produced at same time
(Porras & Munoz
2000)
(Masuda, et al. 2004)
(Imaizumi, et al.
2008)
(Berg 2000; Berg and Redbo-
Torstensson 2000)
Ruellia nudiflora
Scutellaria indica
Dimorphic -CL occur more basal than CH
Little Overlap
-CH produced first
-CL produced at the end of flowering season
Little Overlap
-CL produced first
-CH produced in middle of flowering season
-CL produced at the end of flowering season
Little overlap
-CH produced first
-CL produced at the end of flowering season
(Munguia-Rosas, et al. 2012)
(Sun 1999)
Vigna minima
Viola pubescens
Dimorphic -CH occur on terminal inflorescence
-CL occur on separate inflorescence
Dimorphic -CH occur near apex
-CL occur subterranean
Dimorphic -CH occur on longer peduncles than CL
Overlap
-CH produced first
-CL produced near the end of flowering season
No overlap
-CH produced first
-After CH production is over
CL are produced until the end of the flowering season
(Gopinathan & Babu
1987)
(Culley 2002)
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Node Number
Figure 1. The spatial occurrence of CL (blue) and CH flowers (red) along the main flowering spike of five different Triodanis perfoliata plants . The nodes were numbered from the bottom of the plant to the top.
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Figure 2. The mean temporal occurrence of CL and CH flowers in five Triodanis perfoliata plants starting from germination. The blue and red lines represent CL and CH flowers predehiscence respectfully. The green and the purple lines represent CL and CH flowers postdehiscence respectfully.
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Dehiscence
First
Appearance
Male
Phase
Female
Phase
Flower
Senescence
Dehiscence
Figure 3. The mean timing of developmental events of both CL (n=57) and CH (n=47) flowers on Triodanis perfoliata.
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Figure 4. Bright field microscopy of CL flowers at various ages from Triodanis perfoliata . CL flowers are at ages of 1,3,5,7, and 9 days (starting from upper left). From day one, the sepals, anthers, vascular tissue, and immature ovules are visible. By day seven, maturing seeds are visible along with unfertilized ovules.
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Figure 5. Pollen tubes of CL (top) and CH (bottom) flowers from Triodanis perfoliata viewed with a DAPI filter and epiflourescence microscope. The pollen in the CL flower was visible seven days after first appearance. The pollen in the CH flower was visible after fertilization in the female phase of the flower.
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Figure 6. Anthers from CL (top) and CH (bottom) flowers of Triodanis perfoliata viewed with a
FITC filter and epifluorescence microscope.
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Literature cited
Barrett, S.C.H. (2002). The Evolution of Plant Sexual Diversity. Nature Reviews Genetics, 3 (4),
274-284.
Berg, H. (2000). Differential seed dispersal in Oxalis acetosella, a cleistogamous perennial herb.
Acta Oecologica, 21, 109-118.
Berg, H. & Redbo-Torstensson, P. (1998). Cleistogamy as a bet-hedging strategy in Oxalis acetosella , a perennial herb. Journal of Ecology, 86, 491-500.
Berg, H. & Redbo-Torstensson, P. (2000). Offspring Performance in Oxalis acetosella, a
Cleistogamous Perennial Herb. Plant Biology, 2, 638-645
Charlesworth, D. (2006). Evolution of Plant Breeding Systems. Current Biology, 16, R726-
R735 .
Charlesworth, D. & Charlesworth B. (1987). Inbreeding Depression and Its Evolutionary
Consequences. Ann. Rev. Ecol. Syst., 18, 237-268 .
Charlesworth, D. & Charlesworth, B. (1995). Quantitative Genetics in Plants: The Effects of
Breeding Systems on Genetic Variability. Evolution , 53, 911-920.
Connor, H. E. (1979). Breeding Systems in the Grasses: A Survey. New Zealand J. Bot., 17 , 547-
574.
Culley, T. M. 2000. Inbreeding Depression and Floral Type Differences in Viola canadensis
(Violaceae), a Perennial Herb with Chasmogamous and Cleistogamous Flowers. Canad.
J. Bot., 78 (11), 1420-1429.
Culley, T. M. 2002. Reproductive Biology and Delayed Selfing in Viola pubescens (Violaceae), an Understory Herb with Chasmogamous and Cleistogamous Flowers. Int. J. Pl. Sci., 163 ,
113-122.
Culley, T.M. & Klooster, M.R. (2007). The Cleistogamous Breeding System: A Review of Its
Frequency, Evolution, and Ecology in Angiosperms. Botanical Review, 73(1), 1-30 .
Darwin, C. (1877). The Different Forms of Flowers on Plants of the Same Species. John Murray,
London. Reprint, 1986. University of Chicago Press, Chicago, Illinois.
Fisher, R.A. (1941). Average Excess and Average Effect of a Gene Substitution. Ann. Eugen., 11,
53-63.
Gara, B. & Muenchow, G. (1990). Chasmogamy/cleistogamy in Triodanis perfoliata
(Campanulaceae): some CH/CL comparisons in fitness parameters. American Journal of
Botany, 77, 1-6.
30

Goodwillie, C. & Stewart, E. (2013). Cleistogamy and hybridization in two subspecies of
Triodanis perfoliata (Campanulaceae). Rhodora, 115, 42-60.
Gopinathan, M.C. & Babu, C.R. (1987). Breeding systems and pollination in Vigna minima
( Leguminosae, Papilionoideae).
Plant System Evolution, 156, 117-126.
Imaizumi, T., Wang, G., & Tominaga, T. (2008). Pollination of chasmogamous flowers and the effects of light and emergence time on chasmogamy and cleistogamy in Monochoria vaginalis . Weed Biology and Management, 8, 260-266.
Lord, E.M. (1981). Cleistogamy: A Tool for the Study of Floral Morphogenesis, Function and
Evolution. Botanical Review, 47(4), 421-449 .
Lord, E.M. (1982). Floral Morphogenesis in Lamium amplexicaule L. (Labiatae) with a Model for the Evolution of the Cleistogamous Flower. Botanical Gazette, 143, 63-72.
Martin, F.W. (1959). Staining and Observing Pollen Tubes in the Style by Means of
Fluorescence. Biotechnic & Histochemistry, 34, 125-128.
Masuda, M., Yahara, T. (1994). Reproductive ecology of a cleistogamous annual, Impatiens noli-tangere L., occurring under different environmental conditions. Ecological Research ,
9, 67-75.
Masuda, M., Yahara, T., & Maki, M. (2004). Evolution of floral dimorphism in a cleistogamous annual, Impatiens noli-tangere L. occurring under different environmental conditions.
Ecological Research , 19, 571-580.
Mayers, A.M., & Lord, E.M. (1983). Comparative Flower Development in the Cleistogamous
Species Viola odorata. I. A Growth Rate Study. American Journal of Botany, 70, 1548-
1555.
Mitchell-Olds, T. & Waller, D.M. (1985). Relative Performance of Selfed and Outcrossed
Progeny in Impatiens capensis. Evolution, 39 , 533-544.
Munguia-Rosas, M.A., Parra-Tabla, V., Ollerton, J., & Cervera, J.C. (2012). Environmental control of reproductive phenology and the effect of pollen supplementation on resource allocation in the cleistogamous weed, Ruellia nudiflora (Acanthaceae). Annals of Botany ,
109, 343-350.
Pannell, J.R. & Barrett, S.C.H. (1998). Baker’s Law Revistited: Reproductive Assurance in
Metapopulation. Evolution, 52 (3), 657-668.
Porras, R. & Munoz, J.M. (2000). Cleistogamy in Centaurea melitensis (Asteraceae): capitulum variability and spatio-temporal development patterns. Plant System Evolution, 223, 251-
262
31

Schemske, D.W., Lande, R. (1985). The evolution of self-fertilization and inbreeding depression in plants. II. Empirical observations. Evolution, 39, 41-52.
Schmitt, J. & Gamble, S.E. (1990). The Effect of Distance from the Parental Site on Offspring
Performance and Inbreeding Depression in Impatiens capenisis: A Test of the Local
Adaptation Hypothesis. Evolution, 44 (8), 2022-2030.
Schoesn, D.J. & Lloyd, D. G. (1984). The Selection of Cleistogamy and Heteromorphic
Diaspores. Biol. J. Linn. Soc., 23 , 303-322.
Sun, M. (1999). Cleistogamy in Scutellaria indica (Labiatae): effective mating system and population genetic structure. Molecular Ecology, 8, 1285-1295.
Uphof, J. C. (1938). Cleistogamic flowers . Bot. Rev., 4 , 21-49.
USDA, NRCS. (2015). The PLANTS Database National Plant Data Team, Greensboro, NC.
Website (http://plants.usda.gov) Accessed April 2015
Wright, S. (1977). Evolution and the Genetics of Populations. Experimental Results and
Evolutionary Deductions. Chicago: Univ. of Chicago Press.
32