J. Plant Res. 114: 423-427, 2001 Journal of Plant Research 0 by The Botanical Society of Japan 2001

Pollination of Sarcandra glabra (Chloranthaceae) in Natural Populations in Japan

Yayoi Tosaki', Susanne S. Rennet and Hiroshi Takahashi3*

1 Laboratory for Plant Natural History, Faculty of Science, Kanazawa University, Kanazawa, 920-1192 Japan 2 Department of Biology, University of Missouri-St. Louis, St. Louis, MO 63121, U.S.A. 3 Department of Biology, Faculty of Education, Gifu University, Gifu, 501-1193 Japan

The pollination biology of Sarcandra glabra was inves- tiiated in its natural h a b i t in Japan. Flowers were protogynous and stigma receptivity dropped off significantly following anther dehiscence. Female-stage and bisexual- stage flowers were visited by beetles, bees, hemiptera, flies, and rarely ants that foraged for pollen and/or small droplets of liquid that occasionally were secreted by the carpels and inflorescence axes. At least the beetles, bees, and hemiptera commonly touched the stigmas and likely effect- ed pollination. Rowers were self-compatible, and auto- matic selfing sometimes occurred when pollen fell from apical flowers onto the stigmas of lower flowers.

Key words: Bees - Beetles - Chloranthaceae - Flies - Pollination - Sarcandra glabra - Self-compatibility

Chloranthaceae are a family of basal angiosperms that has received much attention from morphologists and phylogeneticists because of their unusual flowers and fossil record that goes back to the Early Cretaceous (e.g., Endress 1986,1987, Friis et a/. 1986, Crane et a/. 1989, Herendeen et a/. 1993, Brenner 1996). Less is known about their pollina- tion and breeding systems. Chloranthaceae comprise four genera of which two, Chloranthus with 15species and Sarcandra with three species, are endemic in lndomalaysia and Eastern Asia. Both appear to be insect-pollinated, although only Chloranthus has been studied in the field and greenhouse (Ma et a/. 1997, Luo and Li 1999, Wang et a/. 1999, Balthazar and Endress 1999, see Endress 2001 for a recent summary). Phylogenetically, Sarcandra and Chlor- anthus are sistergroups (Qiu et al. 1999) and derived relative to the other two genera, Hedyosmum and Ascarha, which likely are wind-pollinated (Endress 1987, 2001).

Mating systems in Chloranthaceae have been examined in the greenhouse for Sarcandra chloranthoides, S. glabra, and Chloranthus spicatus (Balthazar and Endress 1999), and in the field for C. fortunei, C. henwi, C. holostegius, and C. serratus (Ma et al. 1997, Luo and Li 1999, Wang et a/. 1999). With the exception of C. spicatus, all were found to be self-

* Corresponding author: takahash @ cc.gifu-u.ac.jp

compatible. Here we report the first field data on the pollination and mating system of S. glabra.

Materials and Methods

Field investigations were conducted at Aka0 (40 m alt.), Takatomi-cho, Yamagata-gun, Gifu Prefecture, and at Uruno (50 m alt.), Kozagawa-cho, Higashimuro-gun, Wa- kayama Prefecture. The flowering process was observed in thirty flowers at the Aka0 site in July of 1997. Insects visits were recorded at the Aka0 site for about 130 hours and at the Uruno site for about 20 hours. Each inflorescence usually consists of four spikes (cf. Balthazar and Endress 1999), and each was used for only one pollination experi- ment. Experimental crossings and selfings were performed on female-stage flowers and bisexual-stage flowers from the middle of the spikes. Pollen for cross-pollination was collected from newly opened anthers on plants more than 10 m away. Pollen for self-pollination of female-stage flowers was collected from newly opened anthers on the same inflorescence. Pollen for self-pollination of flowers in the bisexual stage came from the stamen of the same flower. Anther dehiscence could usually be predicted based on anther color (Results) and flower position because there is an almost regular progression in anther opening from the apical flowers of a spike downwards. This regular progression of spike flowering enabled us to emasculate flowers just before they shed their pollen. All flowers used in experimental crossings and apomixis tests were emasculated. With the exception of open-pollinated controls, inflorescences were bagged with I-mm or finer mesh polyethylene screens before their stigmas became receptive. Bags were removed when stigmas had dried up.

The average number of pollen grains per flower was calculated by placing mature anthers on glass slides and flushing pollen grains from the thecae with about 0.3 ml of 70% ethanol. After removal of the empty anther and evapo- ration of the ethanol, grains were immersed in a 1 : 1 mixture of glycerin and water with aniline blue and counted under a microscope. Ovules in the same flowers were counted under a binocular.

Voucher specimens of Sarcandra glabra have been de- posited in the herbarium of Gifu University, which also

424 Y. Tosaki et al.

Figs. 1-6. Sarcandm glabra flowers and their insect visitors, bar=l mm. 1. A female-stage flower, showing the single stamen to the left and the receptive stigma to the upper right. 2. Ladybird beetle (Coccinel- lidae) visiting male-stage flowers. 3. Alticinae beetle on bisexual-stage flowers. 4. Pseudomordellis- tena kaguyahime beetle on female-stage flowers. 5. Glipostenoda rosseola beetle on bisexual-stage flowers. 6. A cerambycid beetle on female-stage flowers.

Pollination of Sacandra glabra 425

I.

1

Figs. 7-10. Insects visiting Sarcandra glabra flowers, b a r 4 mm. 7 and 8. Pentatomid hemipterans visiting the flowers. 9. A syrphid fly visiting female-stage flowers. 10. A Lasiogbssum bee visiting male- stage flowers.

houses some of the insects collected. Additional insect 8 days. Flowers emit a weak fragrance just prior to anther vouchers have been deposited in the Osaka Museum of dehiscence and a stronger fragrance thereafter. Small Natural History. droplets of liquid were occasionally observed on carpels and

inflorescence axes. Results

Flowering in Sarcandra glabra Each spike of Sarcandra glabra has 6-14 flowers, and

flowering normally proceeds from the top of a spike down- wards over two to three days. The carpels are green, and the crenate stigmas become moist several days before anther dehiscence (Fig. 1). Mature but still indehisced sta- mens have a creamish white connective and cream thecae, which turn orange or reddish-brown upon opening (Figs. 2,4, 10). Anther dehiscence occurs several days after the stig- mas become receptive, mainly in the early morning (6-8 a.m.), but occasionally also throughout the day. Thecae are emptied within a few days, and the stamens fall off after 2 to

Insect visitors Table 1 lists the insects collected on the flowers of Sarcan-

dra glabra. Small, between 1.5 and 6 mm long, beetles (Figs. 2-6), hemiptera (Figs. 7,8), flies (Fig. 9), bees (Fig. lo), and rarely ants visited inflorescences and flowers just prior to anther dehiscence and during the two days after dehis- cence. The beetles probed carpels and spike axes, appar- ently to take up the droplets secreted there, and they also foraged for pollen in older flowers. They often touched the stigmas, carried numerous pollen grains on their bodies, and frequently switch between plants. The Ceratina, Lasioglos- sum, and Hylaeus bees foraged for pollen in bisexual-stage flowers and also touched the stigmas while moving on and

426 Y. Tosaki et a/.

Table 1. Insects foraging on the flowers of Sarcandra glabra Table 2. Experimental pollination in Sarcandra glabra

Insects Foraging objects" Treatment Flower Fruit set stage,, Flower Fruits

("0)

Coleoptera Cerambycidae

Chrysomllidae

Cocci nel lidae

Mordel I idae

1 sp.

1 sp.

1 sp.

Glipostenoda rosseola Marseul Pseudomordellistena kaguyahime Nomura et Kato 1 sp.

Diptera Anthomyiidae

1 sp. Syrphidae

3 SPP. Hemiptera

Pentatomidae Eysarcoris guniger Thunberg 1 sp.

Hymenoptera Formicidae

Camponotus yamaokai Terayama et Satoh Crematogaster brunnea teranishii Santschi

Ceratina japonica Cockerell

Hylaeus floralis Smith

Lasioglmsum sp.

Apidae

Colletidae

Halictidae

1) P, foraging for pollen; 0, probing carpel surface.

p, 0

P. 0

p, 0 p, 0 P. 0

P. 0

0 0

p, 0

P

P

between inflorescences. They carried large pollen loads. The hemiptera visited female- and bisexual-stage flowers and generally behaved much like the beetles except that they did not touch the stigmas as frequently. Four species of anthomyiid and syrphid flies foraged for pollen or pr6bed the carpels (apparently for the small droplets of liquid some- times secreted there), but they rarely touched the stigmas. There were no differences in the visitor spectra between the Aka0 and the Uruno sites.

Breeding experiments Results of the breeding experiments are given in Table 2.

Fruit set after experimental crossing and selfing of female- stage flowers was not significantly different (59 and 65%, respectively, chi-square=0.15, df=l, not significant). In bisexual-stage flowers, however, selfing resulted in higher fruit set than outcrossing (35 vs. 17%, chi-square=3.89, df= 1, P<0.05). Fruit set in female-stage flowers was signifi- cantly higher than fruit set in bisexual-stage flowers, both in

Cross-pollination F 22 13 59.1 B 36 6 16.7

Self-pollination F 20 13 65.0 B a 4 2 9 34.5

Bagged emasculated flowers - 50 0 0 Bagged intact flowers - 40 13 32.5 Open pollination - 86 42 48.4

l) F, female stage; 6, bisexual stage.

crossed and in selfed flowers (chi-square=11.16 for cross- pollinated flowers, df=l, P< 0.005; chi-square=6.23 for self-pollinated flowers, df=l, P<0.025). None of the emas- culated flowers produced fruit, while bagged inflorescences had a fruit set of 33%. Of the open-pollinated flowers, 48% set fruit, which was not significantly different from experi- mentally crossed female-stage flowers (chi-square=0.74, df=l) and marginally higher than the fruit set in bagged inflorescences (chi-square=2.96 df=l, P between 0.1 and 0.05).

Pollen number The average number of pollen grains in a stamen of

Sarcandra g/abra was 22.6X102 (n=lO, range 41 X102 to 11 X lo2; SD 7.35X102). Flowers contained one ovule, and the pollen/ovule ratio therefore was the same as the pollen number.

Discussion

Fruit set in female-stage selfed or outcrossed Sarcandra glabra flowers was not significantly different, indicating that S. glabra is self-compatible. A study of two plants culti- vated in a greenhouse at the University of Zurich (Balthazar and Endress 1999) found that fruit set after selfing was between 6 and 33% (crossings could not be carried out). Based on stigma receptivity tests with 5% H202 and with KMn04, Balthazar and Endress suggested that stigmas were receptive several days prior to anther dehiscence and remained receptive throughout the bisexual phase. Our results, however, indicate that stigma receptivity is signifi- cantly higher in female-stage flowers than it is in bisexual- stage flowers (59 vs. 17% in outcrossed flowers and 65 vs. 35% in selfed flowers).

Bagged inflorescences that did not receive insect visits still had 33% fruit Set, probably due to pollen from apical (older) flowers falling onto stigmas of the lower (younger) flowers of an inflorescence as also observed by Balthazar and Endress (1999). Thecae not visited by insects contain a lot of pollen, and we observed grains falling down when anthers received mechanical stimulation, for example, from strong wind.

Based on the bisexual flowers, sticky pollen, and short single stamen, Endress (1986,1987) suggested that Sarcandra

Pollination of Sarcandra glabra 427

was insect-pollinated, most likely by beetles. This first study of the natural pollination of a species of Sarcandra shows that the flowers are visited by a range of insects, including beetles, bees, pentatomid hemiptera, and flies, with at least the former three groups regularly touching the stigmas. We also found that, in addition to pollen, beetles, hemiptera, and flies apparently take up minute droplets from the carpels and inflorescence axes. In the greenhouse, Balthazar and Endress did not observe droplets on carpels or

,spike axes of S. chloranthoides. However, they found that bract apices secreted droplets through stomata (their Fig. 5a-d). In S. glabra, we did not see droplets on the bracts (at a magnification of 150 times) nor did we find stomata on the carpels that might have secreted the minute droplets.

We thank Dr. K. Yamauchi of Gifu University and Drs. S. Shiyake and S. Fujii of the Osaka Museum of Natural History for the identification of the insects, Dr. H. Takasu of Wa- kayama University for advice on the selection of study sites, and Dr. Peter Endress for comments on the manuscript. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (10640680).

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(Received June 8, 2007; accepted September IS, 2001)