Cryptic Dioecy of Cryptic Dioecy of Symplocos wikstroemiifoliaSymplocos wikstroemiifolia Hayata Hayata (Symplocaceae) and (Symplocaceae) and Ternstroemia gymnantheraTernstroemia gymnanthera (Wight & Arn.) (Wight & Arn.)

Sprague (Pentaphylacaceae) in TaiwanSprague (Pentaphylacaceae) in Taiwan

Yu-Chen Wang (Yu-Chen Wang ( 王有禎王有禎 ) and Jer-Ming Hu () and Jer-Ming Hu ( 胡哲明胡哲明 ))

IntroductionIntroductionInstitute of Ecology and Evolutionary Biology, College of Life Science, National Taiwan University Institute of Ecology and Evolutionary Biology, College of Life Science, National Taiwan University

Androdioecy is a rare breeding system in angiosperms. Theoretical models predicted that the condition for the evolution of Androdioecy is a rare breeding system in angiosperms. Theoretical models predicted that the condition for the evolution of androdioecy is stringent, and most morphologically androdioecious plants were proved to be cryptically dioecious. In the 2androdioecy is stringent, and most morphologically androdioecious plants were proved to be cryptically dioecious. In the 2ndnd edition of edition of the Flora of Taiwan, only two species, the Flora of Taiwan, only two species, Symplocos wikstroemiifoliaSymplocos wikstroemiifolia and and Ternstroemia gymnantheraTernstroemia gymnanthera, were documented as , were documented as morphologically androdioecious. morphologically androdioecious. Symplocos wikstroemiifoliaSymplocos wikstroemiifolia had been already proposed to be cryptically dioecious, but for had been already proposed to be cryptically dioecious, but for T. T. gymnantheragymnanthera the sexual characteristics of its breeding system is unclear. This thesis is aimed to investigate the possibility of the the sexual characteristics of its breeding system is unclear. This thesis is aimed to investigate the possibility of the existence of androdioecy in Taiwan and to enhance the knowledge on cryptic dioecy.existence of androdioecy in Taiwan and to enhance the knowledge on cryptic dioecy.

Reproductive Biology of Reproductive Biology of Symplocos wikstroemiifolia Symplocos wikstroemiifolia Study SpeciesStudy Species Flower MorphologyFlower Morphology Sex RatiosSex Ratios

Unusual Natural MutantsUnusual Natural Mutants

Reproductive Biology of Reproductive Biology of Ternstroemia gymnanthera Ternstroemia gymnanthera

ConclusionsConclusions

Study SpeciesStudy Species Pollen MorphologyPollen Morphology

Pollen VolumePollen VolumePollen Number per FlowerPollen Number per Flower

In vitroIn vitro Pollen Germination Pollen Germination

Artificial PollinationArtificial Pollination

Symplocos wikstroemiifolia Symplocos wikstroemiifolia (Symplocaceae)(Symplocaceae) is an is an evergreen shrub or tree species. The male flowers evergreen shrub or tree species. The male flowers usually have 15~20 stamens, but the morphologically usually have 15~20 stamens, but the morphologically bisexual flowers only have about five stamens. bisexual flowers only have about five stamens.

Figure 2Figure 2 Anthers of Anthers of S. wikstroemiifolia.S. wikstroemiifolia.

Based on these evidences, Based on these evidences, S. wikstroemiifoliaS. wikstroemiifolia and and T. gymnanthera T. gymnanthera are cryptically dioecious species. The morphologically bisexual are cryptically dioecious species. The morphologically bisexual flowers are actually functionally female. The discovery of mutant plants in flowers are actually functionally female. The discovery of mutant plants in S. wikstroemiifoliaS. wikstroemiifolia may imply that some female plants could may imply that some female plants could restore the male reproductive function in some circumstances. restore the male reproductive function in some circumstances. Ternstroemia gymnanthera Ternstroemia gymnanthera exhibits floral and pollen dimorphism, and exhibits floral and pollen dimorphism, and pollen grains from female flowers are inaperturate and sterile.pollen grains from female flowers are inaperturate and sterile. In conclusion, no plant species in Taiwan are discovered to be In conclusion, no plant species in Taiwan are discovered to be functionally androdioecious so far. functionally androdioecious so far.

Figure 8 Figure 8 Mean pollen volume Mean pollen volume (± SE) of (± SE) of T. gymnanthera.T. gymnanthera. B, B, pollen grains from pollen grains from morphologically bisexual morphologically bisexual flowers. M, pollen grains from flowers. M, pollen grains from male flowers. N, the number male flowers. N, the number of pollen grains was used to of pollen grains was used to estimate pollen volume. estimate pollen volume.

Figure 6Figure 6 Mean pollen Mean pollen grain per flower (± SE) of grain per flower (± SE) of T. gymnanthera.T. gymnanthera. B, B, morphologically bisexual morphologically bisexual flowers. M, male flowers. flowers. M, male flowers. N, the number of flowers N, the number of flowers was used to estimate was used to estimate pollen grains. pollen grains.

FlowersFlowers FruitsFruits Fruit-sets (%)Fruit-sets (%)

BB 66 00 00

SS 66 00 00

MM 77 55 71.471.4

Table 2Table 2 Fruit-setsFruit-sets (%) of artificial (%) of artificial pollination experiments. B, Pollen grains pollination experiments. B, Pollen grains from the morphologically bisexual plants; from the morphologically bisexual plants; S, pollen grains from the same plant S, pollen grains from the same plant (selfing); M, pollen grains from the male (selfing); M, pollen grains from the male plant. plant.

Figure 1Figure 1 Inflorescences and flowers of Inflorescences and flowers of S. S. wikstroemiifoliawikstroemiifolia..

Ternstroemia gymnantheraTernstroemia gymnanthera (Pentaphylacaceae) is (Pentaphylacaceae) is an evergreen shrub or tree species. Both male and an evergreen shrub or tree species. Both male and morphologically bisexual flowers have five sepals, morphologically bisexual flowers have five sepals, five petals and numerous stamens. Carpels are five petals and numerous stamens. Carpels are rudimentary in male but well-developed in rudimentary in male but well-developed in morphologically bisexual flowers. morphologically bisexual flowers.

Figure 5 Figure 5 Flowers of Flowers of T. gymnanthera.T. gymnanthera.Figure 7 Figure 7 Scanning electron micrographs of pollen Scanning electron micrographs of pollen grains from grains from T. gymnanthera. T. gymnanthera.

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Male plantsMale plantsMorphologically Morphologically bisexual plantsbisexual plants

Male flowersMale flowersMorphologically Morphologically bisexual flowersbisexual flowers

Male flowersMale flowersMorphologically Morphologically bisexual flowersbisexual flowers

Male flowersMale flowers Morphologically bisexual flowersMorphologically bisexual flowers

The sex ratios in the population of Peichatienshan were near 1:1 The sex ratios in the population of Peichatienshan were near 1:1 in two consecutive years (2007~2008), and no plants altered their in two consecutive years (2007~2008), and no plants altered their sexual expressions.sexual expressions.

YearYear MM BB Sex RatioSex Ratio Male FrequencyMale Frequency χχ22

20072007 3535 3030 1:0.861:0.86 0.540.54 0.24 < 0.24 < χχ22 0.05, 10.05, 1

20082008 2424 1818 1:0.751:0.75 0.570.57 0.59 < 0.59 < χχ22 0.05, 10.05, 1

Plant no. 189Plant no. 189Plant no. 29Plant no. 29

Figure 3 Figure 3 Flowers of two natural mutants of Flowers of two natural mutants of S. wikstroemiifoliaS. wikstroemiifolia. Note that the styles of . Note that the styles of plant no. 189 are shorter than normal ones plant no. 189 are shorter than normal ones (see figure 1).(see figure 1).

Two natural mutants, plant no. 29 and Two natural mutants, plant no. 29 and plant no. 189, were found in the plant no. 189, were found in the population. They produced morphologically population. They produced morphologically bisexual flowers and viable pollen grains, bisexual flowers and viable pollen grains, but it is not known whether they have the but it is not known whether they have the female reproductive function because the female reproductive function because the stigmas are morphologically different from stigmas are morphologically different from normal morphologically bisexual flowers. normal morphologically bisexual flowers.

Figure 9Figure 9 Pollen grains that were Pollen grains that were cultured in 5% sucrose solution after cultured in 5% sucrose solution after 24 hours. The pollen and pollen 24 hours. The pollen and pollen tubes were stained with methylene tubes were stained with methylene blue. Bar = 50μm.blue. Bar = 50μm.

Male flowersMale flowersMorphologically Morphologically bisexual flowersbisexual flowers

Table 1Table 1 Sex ratios and male frequencies of the population in 2007 Sex ratios and male frequencies of the population in 2007 and 2008. M, male plants. B, morphologically bisexual plants. The and 2008. M, male plants. B, morphologically bisexual plants. The sex ratios aresex ratios are not significantly different from 1:1 (not significantly different from 1:1 (χχ22 0.05, 1 0.05, 1 = 3.84= 3.84). ).

Plant no. 29Plant no. 29Morphologically Morphologically bisexual flowersbisexual flowers

Figure 4 Figure 4 The stigmas of The stigmas of S. wikstroemiifoliaS. wikstroemiifolia. . Note that the stigmas of plant no. 29 are Note that the stigmas of plant no. 29 are smaller than normal ones. smaller than normal ones.

The male flowers The male flowers contained significantly more contained significantly more pollen grains than the pollen grains than the morphologically bisexual morphologically bisexual flowers. flowers. (P < 0.0001(P < 0.0001).).

Means of pollen volume Means of pollen volume were significantly larger in were significantly larger in morphologically bisexual morphologically bisexual flowers than in male flowers flowers than in male flowers ((P < 0.0001P < 0.0001).).

Pollen grains from male flowers were prolate to prolate-Pollen grains from male flowers were prolate to prolate-spheroidal and tricolporate, whereas spheroidal and tricolporate, whereas pollen grains from pollen grains from morphologically bisexual flowers were spheroidal and morphologically bisexual flowers were spheroidal and inaperturate. inaperturate.

There were no pollen grains in the anthers of the There were no pollen grains in the anthers of the morphologically bisexual flowers in anthesis. morphologically bisexual flowers in anthesis.

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Pollen grains from morphologically bisexual and male flowers of Pollen grains from morphologically bisexual and male flowers of T. T. gymnantheragymnanthera were cultured in 5% sucrose solution. Only the pollen were cultured in 5% sucrose solution. Only the pollen grains from male flowers could produce pollen tubes. Some of the grains from male flowers could produce pollen tubes. Some of the pollen grains from morphologically bisexual flowers released some pollen grains from morphologically bisexual flowers released some substances during the cultured periods, but they never formed pollen substances during the cultured periods, but they never formed pollen tubes. Meantubes. Mean pollen germination rate (%) of the pollen grains from pollen germination rate (%) of the pollen grains from morphologically bisexual flowersmorphologically bisexual flowers was significantly different from male was significantly different from male flowers (flowers (P < 0.0001P < 0.0001).).

Figure 10Figure 10 Mean pollen Mean pollen germination rate (± SE) of germination rate (± SE) of T. T. gymnantheragymnanthera. B, morphologically . B, morphologically bisexual flowers. M, male flowers. bisexual flowers. M, male flowers. N, the number of flowers was N, the number of flowers was used to conduct pollen used to conduct pollen germination experiments. germination experiments.

Artificial pollination experiments revealed that only the pollen Artificial pollination experiments revealed that only the pollen grains from male flowers could set fruits. Treatments were grains from male flowers could set fruits. Treatments were significantly different (significantly different (χχ22 = 11.6, P < 0.005 = 11.6, P < 0.005).).

台灣產蕘花葉灰木與厚皮香之隱性雌雄異株之研究台灣產蕘花葉灰木與厚皮香之隱性雌雄異株之研究