Of an Evergreen Broad-Leaf Understorey Species, Eurya Japonica Thunb

Bull. Kitakyushu Mus. Nat. Hist., 15: 137-144, pis. 13-15. March 28, 1996

Variations in Sex Expression and the Reproductive Function of an Evergreen Broad-leaf Understorey Species, Eurya japonica Thunb. (Theaceae)

Tohru Manabe

Kitakyushu Museum and Institute of Natural History, Nishihonmachi 3, Yahatahigashiku, Kitakyushu 805, Japan Received November 28, 1995)

Abstract Variations in sex expression and the reproductive function of Eurya japonica Thunb., evergreen broad-leaf understorey species, were investigated at a Quercus serrata secondary forest in Okayama City and at a burnt pine forest in Kurc City, southwestern Japan. A few individuals (1.5- 16.4% of flowering individuals) produced flowers similar to hermaphrodite flowers in shape (hcrmaphroditc-typc flowers), whereas most of individuals did either male or female flowers. Trends in the data for germination test of seeds and pollen grains and observed state of pollen production in hermaphrodite-type flowers indicated that hermaphrodite-type flowers were divided into two types according to their reproductive function: hermaphrodite flowers which produced both fertile seeds and pollen grains; and 'pseudo-hermaphrodite' flowers, functionally female ones, which produced fertile seeds and no pollen grains. Individuals having hermaphrodite flowers always had male flowers. Individuals having pseudo-hermaphrodite flowers, on the other hand, always had female flowers. Thus, sex expression of individuals was classified into three types: male individuals which produced only male flowers (44.8- 53.6% of flowering individuals); female individuals which produces only female flowers (32.4-44.8%) and those which produced both female and pseudo-hermaphrodite flowers (2.3- 19.6%); and andromonoccious individuals which produced both male and her maphrodite flowers (1.8-6.5%). Therefore, sex types of E. japonica was classified as polygamodioecy from a functional view point.

Introduction

In flowering plants, flowers having reproductive function have been classified three types such as two unisexual flowers (male and female flowers) and hermaphro dite flowers. Sex expression of individuals can be determined by the type of flowers produced: male or female individuals which produce only male or female flowers, respectively; monoecious ones which produce both male and female flowers; her maphrodite ones which produce only hermaphrodite flowers; and individuals which produce both unisexual flowers and hermaphrodite flowers in various combination. Therefore, sex type of plant species is complicated, for some species are consisted of individuals, which indicate various sex expression, in various combination. 138 Tohru Manabe

There are species having individuals which change sex expression in response to changes in size or age and/or to changes in ambient environment (Freeman el al., 1980). In some species of Acer, for example, sex expression of individuals change from male to female (Hibbs and Fischer, 1979; Primack and McCall,1986; Matsui, 1995). There are also species having individuals which are not known their sex expression exactly from a morphological view point of the flowers. Callicarpa subpubescens Hook, et Arn. produces hermaphrodite-like flowers in shape. The sex type of the species was, therefore, recognized as hermaphrodity as well as other congeneric species. Flowers of the species were, however, divided into two types from a functional view point: male flowers which produce only fertile pollen grains; and female flowers which produce fertile seeds and pollen grains having no germination ability (Kawakubo, 1990). Further, sex type of the species was dioecy, because the individuals never produce both male and female flowers simultaneously. Thus, it is need for understanding sex types of flowering plants to evaluate the reproductive functions in addition to morphology of flowers. To understand sex types from a functional view point is also important to consider evolution of sex expression and sex type (e.g. Charlesworth and Charles- worth, 1978; Lewis, 1942: Ross, 1978). Sex expression and sex type are not always known exactly, although those are one of important characters of plants. Eurya japonica Thunb. is an evergreen broad-leaf understorey tree, in the family Theaceae. Congeneric species are 6 and ca. 130 in Japan (Kitamura and Murata, 1980) and in the world (Shaw, 1973), respectively. E. japonica occurs in southeast Asia, China, Taiwan, south part of Korea and Japan (Ohwi and Kitagawa, 1992). A few individuals of E. japonica produce flowers similar to hermaphrodite flowers in shape (Hermaphrodite-type flowers: Makino, 1940; Furuike, 1989; Manabe et al., 1991; Murata et al., 1991; Higo, 1994), although most flowering individuals are either male or female. Sex type of the species has not been known exactly because of lack of information on the reproductive function of hermaphrodite-type flowers. In this paper, I describe the reproductive function of hermaphrodite-type flowers. Sex expression of individuals and sex type of E. japonica are also described from the functional view point.

Study sites The study sites are located at Mt. Handa (34°41'N, 133°55'E) in Okayama City and at a burnt secondary pine forest (31°14'N, 132°43'E) burnt in early in May, 1984 in Kure City, southwestern Japan. Both sites locate in warm-temperate zone. The study stand at Mt. Handa is a mature secondary forest with a canopy dominated by deciduous broad-leaf trees such as Quercus serrata Thunb., Platycarya strobilacea Sieb. et Zucc. and Prunus jamasakura Sieb. ex Koidz., and with an understorey dominated by evergreen broad-leaf trees such as Dendropanax trifidus Sex Expression and the Reproductive Functionof Eurya japonica 139

(Thunb.) Makino, Ilex chinensis Sims, Cinnamomum japonicum Sieb. ex Nakai and Eurya japonica (Nishimura et al., 1990). The stand is on a gentle slope (average ca. 20°). Soils are brown forest soils derived from sandstone. The study stand on the burnt site is located at area where was left alone after forest fire. Dominant trees of the stand are the species invaded after fire such as Pinus densiflora Sieb et Zucc, the species sprouted after fire such as Quercus serrata, Eurya japonica and Lyonia ovalifolia (Wall.) Drude var. elliptica (Sieb. et Zucc.) Hand.- Mazz. and the species recruited from the soil seed bank such as Lespedeza cyrtobolorya Miq. and Mallotus japonicus (Thunb.) Muell (Manabe et al., 1988). The stand is on a steep slope (average inclination = 27°). Soils do not develop well and bed mother rocks are granite.

Methods

Field survey Two 10 mXlO m plots (HI and H2) and a 20-mX20 m plot (H3) were established at Mt. Handa at the end of March, 1988 and 1991, respectively. A 10 mX 10 m plot (Bl) was established at a burnt site at the end of September, 1988. A 20 mX20 m plot (B2) containing Bl was also established at the end of November, 1990. All individuals of Eurya japonica more than 5 cm in height were tagged when the plots were established. All flowering individuals were scored their sex expression based on morphology of their flowers at all plots during the end of March to early April from the years when the plots were established to 1991 except B2 which was scored in only 1991. All fruiting individuals were recorded at all plots during the end of November to early December in every year when the sex expression was scored. Fruiting individuals were defined as individuals with more than one fruit.

Germination tests of seeds and pollen grains At the survey on the sex expression of individuals in 1989, two types of hermaphrodite-type flowers were recognized such as flowers which produced pollen grains and no pollen grains. Twenty fruits each were collected from two types of hermaphrodite-like flowers and female flowers derived from each one individual at Mt. Handa at the end of October, 1989. Collected fruits were kept hermetically in a refrigerator at 4°C separately until use in germination test following fruits collection. Early April, 1990, each 50 seeds, which were sampled at random from each sampled fruits, were placed in the Petri dishes layered a filter paper separately, and were kept in a incubator controlled at 27.5°C (± 1.0°C) during 120 days. Germinated seeds were counted almost everyday and filter papers were kept suitable moisture for germination throughout the test. Pollen grains were collected from hermaphrodite-type flowers and male flowers separately at Mt. Handa at the end of March. Each pollen grains were sown 140 Tohru Manabe separately soon after pollen collection on cellophane papers which were dipped into 10% of sucrose solution. Those cellophane papers were placed into the Petri dishes layered a filter paper dipped same sucrose solution, and were kept in a incubator at 22.0°C (± 1.0°C) during 6 hours. State of pollen grains were observed using a light microscope. Pollen grain having elongated pollen tube was defined as fertile one.

Results

Flowering condition There were individuals having flowers similar to hermaphrodite flowers in shape (hermaphrodite-type flowers) as well as male ones having only male flowers (Fig. 1) and female ones having only female flowers (Fig. 2) at all plots in all years investigated (Tables 1 and 2). Individuals having hermaphrodite-type flowers also bore male or female flowers. No individuals had only hermaphrodite-type flowers. Hermaphrodite-type flowers had a pistil-like organ and stamen-like organs. Stamen like organs of the flowers were also recognized different types in shape: organs which were not similar to stamens of male flowers (Fig. 3) and those which were similar to stamens of male flowers (Figs. 4 and 5). Two types of hermaphrodite-type flowers were recognized until investigation in 1990: flowers which produced no pollen grains (Figs. 3 and 4) and did pollen grains (Fig. 5). Thus, flowers having no male function were contained hermaphrodite-type flowers, although hermaphrodite-type flowers were supposed to have both male and

Table 1. The number of Eurya japonica individuals in each sex at the plots at Mt. Handa (HI and H2) in 1988, 1989 and 1990, and at the plot in a burnt site (Bl) in 1898and 1990. The number of fruiting individuals are given in parenth-

•i) Plot Year Density" Sex expression (100m-2) Ma Fe He

HI 1988 115 35 24 (23) 3( 2)

1989 115 33 26 (25) 5( 4)

1990 115 32 26 (25) 6( 4)

H2 1988 66 15 16 (13) 1 ( 1) 1989 66 18 16 (12) 2( 1) 1990 66 14 17(15) 2( 1)

Bl 1989 73 26 31 (31) 12 (12) 1990 73 26 30 (30) 12 (12) 1) Density of all individuals taller than 5 cm 2) Ma: individuals having only male flowers, Fe: those having only female flowers, He: those having flowers which are similar to hermaphrodite flowers in shape (hermaphrodite-type flowers) Sex Expression and the Reproductive Function of Eurya japonica 141

Table 2. The number of Eurya japonica individuals in each sex at the plotsat Mt. Handa (HI, H2 and H3) and at the plot in a burnt site (B2) in 1991. The number of fruiting individuals are given in parentheses.

Sex cxpressi on Plot Individual Flower1' HI H2 H3 B2

Male M 31 13 119 93 Female F 24 (22) 13 (10) 94 (88) 66 (66) F+p-H 2( 2) 1 ( 1) 4( 3) 27 (27) F+ p-H + N 1 ( 1) 0 1 ( 0) 13 (13) Andromonoccious M + H 4( 2) 2( 1) 4( 3) 5( 5) No flowers 50 36 247 8 1) M: male flowers, F: female flowers, p-H (pseudo-hermaphrodite): functionally female flowers having organs which are similar to stamen (produce no pollen grains), N: functionally neuter flowers having organs which are similar to stamen (produce no pollen grains), H: functionally hermaphrodite flowers which produce both fertile seedsand pollen grains. female functions. Hermaphrodite-type flowers were, therefore, divided into two types: flowers having no male function (pseudo-hermaphrodite flowers) and those producing pollen grains (hermaphrodite-like flowers). There were also neuter flowers, which produced no pollen grains, having only stamen-like organs. In 1991, sex expression was investigated more detail (Table 2). Individuals having hermaphrodite-like flowers always bore male flowers. Individuals having pseudo-hermaphrodite and/or neuter flowers, on the other hand, always bore female flowers. No individuals had both female flowers and hermaphrodite-like flowers, and both male flowers and pseudo-hermaphrodite and/or neuter flowers. The ratio of individuals having hermaphrodite-like flowers to all flowering individuals were 2- 7% and ca. 2.5% at Mt. Handa and at burnt site, respectively. The ratio of individuals having pseudo-hermaphrodite flowers to all flowering individuals were significantly higher (P<0.001) at burnt site (ca. 20%) than at Mt. Handa (2-5%). There were no individuals which changed their sex expression from male to female or from female to male during the periods investigated. Most of female individuals bore fruits at all plots in all years investigated (Tables 1 and 2). Individuals having hermaphrodite-like flowers and having pseudo-hermaphrodite flowers also bore fruits.

Germination tests of seeds and pollen grains Germination ratios of seeds collected from female, hermaphrodite-like and pseudo-hermaphrodite flowers of Eurya japonica were about 30-40%, and there were no significant difference (P>0.05) among the flower types (Table 3). Pollen grains collected from hermaphrodite-like flowers (Fig. 6) as well as male flowers (Fig. 7) in 142 Tohru Manabe

Table 3. The number of germinated seeds collected from female, pseudo-hermaphrodite and hermaphrodite flowers of Eurya japonica.

Source flower" Number of Number of seeds germinated seeds

Female 50 15 Pseudo-hermaphrodite"' 50 20 Hermaphrodite" 50 15

1) Flowers from which seeds were collected 2) Pseudo-hermaphrodite: functionally female flowers having organs which are similar to stamen (produce no pollen grains) 3) Hermaphrodite: functionally hermaphrodite (lowers which produce both fertile seeds and pollen grains the species elongated their pollen tubes. Thus, pseudo-hermaphrodite flowers had only female function, whereas hermaphrodite-like flowers, which had both female and male functions, were hermaphrodite flowers in reproductive function.

Discussion

Some reports have pointed out that Eurya japonica produces hermaphrodite-type flowers (e.g. Makino, 1940; Furuike, 1989; Manabe et al., 1991; Murata et al., 1991; Higo, 1994). In detail study on the morphological variations in flowers of E. japonica (Murata et al., 1991), some characters such as the number of stamens per flower, the number of ramifications in a style, flower color and shape, and so on, were compared among male, female and hermaphrodite-type flowers. But, Murata et al. (1991) did not clarify the reproductive function of hermaphrodite-type flowers, although they pointed out some hermaphrodite-type flowers produced pollen grains and some ones had an ovule. In this study, hermaphrodite-type flowers of Eurya japonica, which had been supposed to have both male and female functions, were classified into two types by the reproductive function: hermaphrodite flowers which produced both fertile seeds and pollen grains, and pseudo-hermaphrodite flowers, functionally female ones, which produced fertile seeds and no pollen grains. Thus, four types of flowers such as male, female, hermaphrodite and neuter flowers were recognized in the species. Further, the sex expression of individuals was classified into three types: male individuals having only male flowers; female individuals having only female flowers and having female flowers, pseudo-hermaphrodite flowers and/or neuter flowers; and andromonoecious individuals having male and hermaphrodite flowers. Sex type of the species was, therefore, classified as polygamodioecy from the functional view point. Sex Expression and the Reproductive Function of Eurya japonica 143

Murata etal. (1991) have suggested that a population of Eurya japonica contains individuals having female and hermaphrodite flowers and having male, female and hermaphrodite flowers in addition to those having male and hermaphrodite flowers. Furuike (1989) has also pointed out that some female individuals of the species have hermaphrodite-type flowers. Pseudo-hermaphrodite flowers might be regard as hermaphrodite ones, as seen in the figure (Fig. 1) of Murata et al. (1991). Some species showed varying population sex ratios owing to their population density (Onyekwelu and Harper, 1979). The ratio of individuals having her maphrodite flowers to all flowering individuals of Eurya japonica was similar among the plots, suggesting that hermaphrodite flowers might always appear in low frequency. On the other hand, the ratio of individuals having pseudo-hermaphro dite flowers to all flowering individuals varied among the plots. The ratio of individuals having pseudo-hermaphrodite flowers of the species might not be affected by population density, because population densities of H2 and B2 did not differ largely. This might suggest that appearance of pseudo-hermaphrodite flowers is affected by environmental factors and the developmental process in flowers is easily deviated from the ordinal process under unusual ambient physical conditions. Dioecy and other dicliny in flowering plants might have evolved from an hermaphrodite ancestral condition (e.g. Lewis, 1942; Charlesworth and Charles- worth, 1978: Bawa, 1980). Polygamodioecy in Eurya japonica may evolve from hermaphrodity, for most of species in the family Theaceae are hermaphrodity in their sex type. Male-sterile mutant might spread more easy in the hermaphrodite population than female-sterile mutant (e.g. Bawa, 1980). In hermaphrodite populations, genetic variation which brings male-sterility might spread easily in the populations, if the amount of seed production was more abundant at individuals which lost male function (that is, female individuals) than at hermaphrodite individuals (Lloyd, 1974). Further, in the hermaphrodite populations occurring inbreeding depression, male-sterile gene may be able to invade the population, even if female individuals have same amount of seed production as hermaphrodite ones (Charlesworth and Charlesworth, 1978). In Eurya japonica populations surveyed, no individuals produced both female and hermaphrodite flowers, although some individuals did both male and hermaphrodite ones. This fact might suggest that male function was lost previously and female function was hardly lost in the processes from an ancestral hermaphrodite condition to a polygamodioecious condition in E. japonica.

Acknowledgments

I express my appreciation to Kyozo Chiba, Okayama University, and Hideaki Ohba, University of Tokyo for their encouragement. I am also grateful to Makoto Amano, Natural History Museum and Institute, Chiba, Kiyoshi Matsui, Kyoto 144 Tohru Manabe

University, Shin-Ichi Yamamoto, Okayama University and Kunito Nehira, Hiroshi ma University for their kind suggestions improving the manuscript.

References

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Tohru Manabe

Plates 13-15 Explanation of Plate 13

Figs. 1-2. Flowers of Eurya japonica

1. Male flower 2. Female flower Manabe, T. Sex Expression and the Reproductive Function ofEurya japonica Plate 13 Explanation of Plate 14

Figs. 3-5. Flowers of Eurya japonica

3- 4. Hcrmaphrodite-likc flowers (functionally female flowers), which have organs similar to stamen, produced fertile seeds and no pollen grains 5. Hermaphrodite flower produced both fertile seeds and pollen grains. Manabe. T. Sex Expression and the Reproductive Function of Eurya japonica Plate 14 Explanation of Plate 15

Figs. 6-7. Germinated pollen grains of Eurya japonica

6. Pollen grains collected from male flowers 7. Pollen grains collected from hermaphrodite flowers Manabe, T. Sex Expression and the Reproductive Funclion of Eurya japonica Plate 15