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AOBPreview originally published online on June 30, 2004
Annals of Botany 2004 94(2):229-232; doi:10.1093/aob/mch142
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Annals of Botany 94/2, © Annals of Botany Company 2004; all rights reserved

Density Affects Gametophyte Growth and Sexual Expression of Osmunda cinnamomea (Osmundaceae: Pteridophyta)

YAO-MOAN HUANG1, HSUEH-MEI CHOU1 and WEN-LIANG CHIOU2,*

1 Department of Biology, National Taiwan Normal University, Taipei 106, Taiwan and 2 Division of Forest Biology, Taiwan Forestry Research Institute, Taipei 100, Taiwan

* For correspondence. E-mail chiou{at}serv.tfri.gov.tw

Received: 14 January 2004    Returned for revision: 18 March 2004    Accepted: 20 April 2004    Published electronically: 30 June 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

• Background and aims To understand how gametophyte densities affect the sexual expression and sizes of Osmunda cinnamomea and to provide information on the density of growth needed to favour successful reproduction, fresh spores were sown at various densities and subsequent gametophyte growth was studied.

• Methods Spores were sown and cultured in the laboratory. Subsequent gameophytes at different population densities were sampled and their sexual expression and sizes were recorded.

• Key results One-year-old multispore cultures of the fern O. cinnamomea demonstrated that population density affected gametophyte growth and sexual expression. As density increased, gametophytes became significantly smaller and more slender. Female and asexual gametophytes dominated in populations of low and high densities, respectively. At intermediate population densities, hermaphroditic and male gametophytes were dominant. Female gametophytes were larger than gametophytes of all other types. Hermaphroditic gametophytes were larger than male gametophytes, which were larger than asexual gametophytes. Large gametophytes were wide-cordate, whereas smaller ones tended to be narrow-spathulate.

• Conclusions Gametophyte size of O. cinnamomea is negatively related to the population density, which significantly affects gametophytes' sexual expression. The presence of unisexual and bisexual gametophytes at intermediate densities indicates that both intergametophytic and intragametophytic selfing may occur.

Key words: Gametophyte density, Osmunda cinnamomea, sexual expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
To understand fern reproduction, it is necessary to understand sexual expression and the factors that affect it. Although homosporous ferns have the potential to be hermaphroditic, many factors may affect their growth and sexual expression (Korpelainen, 1994Go; Eberle and Banks, 1996Go; Banks, 1997Go; Verma and Selvan, 2001Go). Population density is one of the factors affecting sexual expression and growth in gametophyte communities. In overcrowded populations resources are limited by competition, and gametophytes are often asexual or male, and narrow (usually spathulate). Female and hermaphroditic gametophytes, on the other hand, often occur in sparse populations (Klekowski and Lloyd, 1968Go; Lloyd and Gregg, 1975Go; Cousens, 1979Go).

Osmunda cinnamomea is found in Siberia, Manchuria, Korea, Japan and China. It is rare in north-eastern Taiwan (Shieh, 1994Go). Artificial propagation ex situ, for example in botanical gardens, is one of the ways to conserve this rare fern. Its gametophyte morphology has been described (Momose, 1967Go; Nayar and Kaur, 1971Go). However, whether its sexual expression and gametophyte sizes are influenced by population density is unclear. In this study, the effect of gametophyte density on the sexual expression and growth of this fern was examined to provide information on the density of growth needed to favour successful reproduction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Fresh spores of Osmunda cinnamomea L. were obtained from a plant growing in a marsh at Tsao-Pi (24°45'N, 121°33'E), Ilan County, in north-eastern Taiwan. Spores were sown on eight sheets of membrane filter (pore 0·45 µm, 47 mm in diameter; Gelman Laboratory). Each sheet lay on 2–4 cm deep moist Sphagnum moss in a plastic box (114 x 86 x 65 mm). All cultures were maintained under white fluorescent illumination at about 24 µmol m–2 s–1, 12 h d–1 and the temperature was between 20 and 28 °C. Because spores disperse randomly in nature, instead of being controlled at specific densities as in most experiments, spores in this study were sown randomly and gametophytes were sampled randomly in the culture. One year later, gametophytes growing at different population densities (1, 2, 3, 6, 10, 12, 14, 32, 37, 41, 66, 92, 131 and 253 individuals cm–2) were sampled. The length and width of each gametophyte was measured (Fig. 1), and their genders were recorded. A stereomicroscope (Leica, Wild M8) and a compound light microscope (Leitz, Dialux 20) were used to work out the density and to examine the morphology, sizes and genders of the gametophytes.



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  		FIG. 1. Measurement of gametophyte width and length in this study.

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The chloroplast-bearing spores germinated within 1 week of sowing. A typical, mature Osmunda cinnamomea gametophyte was cordate and without trichomes. Initiation of antheridia and archegonia did not occur until 6 and 9 months after sowing, respectively. Antheridia formed on the dorsal and ventral surfaces of the wing, while archegonia formed on the ventral surface of the anterior cushion. Sporophytes were produced 1 year after the spores were sown.

One year after spores were sown, hermaphroditic, male, female and asexual gametophytes appeared in the culture synchronously. Of 17 hermaphroditic gametophytes examined, nine had antheridia containing antherozoids and eight had empty antheridia. At the density of 1, 2 and 3 individuals cm–2 all gametophytes were female, but at 41 individuals cm–2 there were no female gametophytes (Fig. 2). In contrast, the frequency of asexual gametophytes increased with density. At densities over 66 individuals cm–2, only asexual gametophytes were produced. Male gametophytes occurred at densities between 12 and 41 individuals cm–2, and hermaphroditic gametophytes occurred at densities between 6 and 37 individuals cm–2 (Fig. 2).



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  		FIG. 2. Gender frequencies of Osmunda cinnamomea grown at different gametophyte densities.

 
Female gametophytes were much larger, in all respects, than other types of gametophytes (Table 1). Hermaphroditic gametophytes were larger than male gametophytes, which were larger than asexual gametophytes. Gametophyte size could be estimated by the regression equation:

where A = width x length (mm2) and D = individuals cm–2. This equation clearly shows that gametophyte size decreased significantly as density increased. When the population density was more than 16 individuals per cm2, the size of all gametophytes did not exceed 3·38 mm2 (Fig. 3). Large, usually wide, cordate gametophytes occurred in low-density populations and were female or hermaphroditic. Small, usually slender-spathulate gametophytes occurred in high-density populations and were asexual (Fig. 4). The mid-sized male gametophytes appeared in populations of intermediate density.


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  		TABLE 1. Growth characteristics of Osmunda cinnamomea gametophytes of different genders (average ± s.d.)

 


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  		FIG. 3. The relationship between Osmunda cinnamomea population density and average gametophyte size after 1 year in culture. A, size; D, density.

 


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  		FIG. 4. Two types of Osmunda cinnamomea gametophytes: (A) female gametophytes are wide-cordate; (B) asexual gametophytes are spathulate.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Gametophyte growth and sexual expression are affected by many factors, including spore size, germination time, nutrition source, growth density and antheridiogen (Schedlbauer, 1976Go; Nester and Schedlbauer, 1981Go; Korpelainen, 1994Go). The density of cultured gametophytes has been shown to modify sexual expression in several species of homosporous ferns (Cousens and Horner, 1970Go; Rashid, 1970Go; Lloyd and Gregg, 1975Go; Cousens, 1979Go; Rubin et al., 1985Go; Warne and Lloyd, 1987Go; Greer, 1993Go). In this study, it was found that population density affected the sexual expression of Osmunda cinnamomea. Gametophyte densities above a specific level yield relatively constant sex ratios (Warne and Lloyd, 1981Go). In this study, solely asexual gametophytes were produced when the population density exceeded 66 individuals cm–2. The high asexual/sexual percentage (74/26) of the laboratory-cultured gametophytes and the low asexual/sexual percentage (20/80) of field gametophytes of Sadleria cyatheoides and S. pallida (Ranker and Houston, 2002Go) may partly be explained by the influence of density as demonstrated in this study.

Female and hermaphroditic gametophytes are usually larger than male and asexual gametophytes (Korpelainen, 1994Go). This was true of O. cinnamomea gametophytes. In this observation, density affected gametophyte size and shape, and size was closely related to gametophyte gender. Miller (1968)Go stated that weak vegetative growth favours the formation of male and asexual gametophytes, whereas vigorous growth favours the formation of female and hermaphroditic gametophytes. When gametophytes are sparse, each gametophyte can obtain more resources than those gametophytes in dense populations. This may explain, in part, why the gametophyte size was significantly negatively correlated with population density.

Antheridium formation may consume energy and reduce gametophyte growth (Miller, 1968Go; Näf, 1979Go). Antheridiogen, a hormone promoting antheridium formation, affects size and size-influenced sexual expression in Dryopteris filix-mas (Korpelainen, 1994Go). Potential vegetative growth is diverted to antheridium formation by antheridiogen in polypodiaceous ferns (Näf, 1956Go). However, further studies are needed to determine whether antheridiogen is present in O. cinnamomea. Chemicals other than antheridiogen may also affect gametophyte growth. For example, the gametophytes of some ferns in the Polypodiaceae may produce growth inhibitors (Chiou and Farrar, 1997Go). Reciprocal allelopathy has been demonstrated between O. cinnamomea and Dryopteris intermedia, but gametophyte growth was not retarded by spraying with the supernatant of the same species (Petersen and Fairbrothers, 1980Go). However, in this study, it is unclear whether the growth of gametophytes in high-density populations was inhibited by allelochemicals.

Different patterns of sexual expression may provide evidence for different mating systems (Klekowski, 1972Go; Pajarón et al., 1999Go). Unisexual gametophytes favour outcrossing, whereas bisexual ones provide the opportunity for intragametophytic selfing (Schneller et al., 1990Go; Haufler, 2002Go). The occurrence of unisexual (female and male) and bisexual gametophytes of O. cinnamomea indicates that both intergametophytic and intragametophytic selfing may produce sporophytes, although possible limitations of genetic load on the latter have not been tested. In nature, most fern spores are dispersed only a limited distance, with very few or even only single spores travelling longer distances (Peck et al., 1990Go). The habitat in this study is fully covered by a Sphagnum mat. Dispersed spores of O. cinnamomea may either gather on the Sphagnum mat or float further and separate to various densities. Gametophytes growing in different densities will express different sexualities and may produce sporophytes through different mating systems accordingly.

These results were derived from a single plant and thus may not represent the species as a whole. However, this study does indicate the potential that ex situ conservation of this species may be achievable by sowing spores sparsely, as it will lead to subsequent development of gametophytes of both sexes and promote successful sporophyte production.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Drs Donald R. Farrar, Tom A. Ranker and Liz Sheffield for their valuable comments. The assistance of Dr Alan Warneke in editing the manuscript is gratefully acknowledged.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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    Chiou WL, Farrar DR. 1997. Antheridiogen production and response in Polypodiaceae species. American Journal of Botany 84: 633–640.[Abstract]

    Cousens MI. 1979. Gametophyte ontogeny, sex expression, and genetic load as measures of population divergence in Blechnum spicant. American Journal of Botany 66: 116–132.[CrossRef][ISI]

    Cousens MI, Horner Jr HT. 1970. Gametophyte ontogeny and sex expression in Dryopteris ludoviciana. American Fern Journal 60: 13–27.[CrossRef]

    Eberle JR, Banks JA. 1996. Genetic interactions among sex-determining genes in the fern Ceratopteris richardii. Genetics 142: 973–985.[Abstract]

    Greer GK. 1993. The influencce of soil topography and spore-rain density on gender expression in gametophyte populations of the homosporous fern Aspidotis densa. American Fern Journal 83: 54–59.[CrossRef]

    Haufler CH. 2002. Homospory 2002: an odyssey of progress in pteridophyte genetics and evolutionary biology. BioScience 52: 1081–1093.[CrossRef]

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    Lloyd RM, Gregg TL. 1975. Reproductive biology and gametophyte morphology of Acrostichum danaeifolium from Mexico. American Fern Journal 65: 105–120.[CrossRef]

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    Pajarón S, Pangua E, García-Álvarez L. 1999. Sexual expression and genetic diversity in populations of Cryptogramma crispa (Pteridaceae). American Journal of Botany 86: 964–973.[Abstract/Free Full Text]

    Peck JH, Peck CJ, Farrar DR. 1990. Influences of the history attributes on formation of local and distant fern population. Amercian Fern Journal 80: 126–142.[CrossRef]

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