Floral Structure and Pollination in Relation to Fruit Set in Cynanchum otophyllum Schneid *Ding-Kang Wang, Shu-Hua Zhai, Bin Wang Gui-Fang Sun Biology Department, Kunming University, Clinical Skill Center, Kunming Medical College, Kunming 650214, China. Kunming 650031, China *[email protected] Abstract—We studied the floral morphology and floral biology floral structure and function of C. otophyllum in Yunnan, of Cynanchum otophyllum Schneid in experimental plots and China. We addressed the following three points: (1) What field sites. Some observations were augmented by laboratory kind of pollination system exists in C. otophyllum, (2) What studies of floral traits, including scanning electron microscopy floral characters of the flowers are correlated to pollination in and light microscopy. The flower was characterized by a C. otophyllum, and (3) What causes the formation of low fruit staminal corona. The pollinia were lodged in sacs on each side of set in C. otophyllum. the stigma and needed pollen vector for fruit production. C. otophyllum has characteristics similar to bee-pollinated plants. Honeybees (Apis cerana Fabricius) were the main pollinators. 1 MATERIALS AND METHODS Pollinaria removal and pollinia insertion rates were low at 5.4% and 0.45%, respectively. The fruit set was only 2.2% in natural 1.1 Study area population. The flowering span of C. otophyllum was about 3 We established and monitored two experimental areas: A months, and the functional longevity of individual flowers was and B. Area A was located on a hill with yellow soil near the 6–8 days. The extended period may be related to the relatively town of Longchang, Xuanwei County, Yunnan Province low levels of effective pollinator activity. The flowers were self- (104009’ E, 26011’ N). The climate is subtropical with an incompatible. Umbels displayed open flowers for 9–10 days, and annual average temperature of 14.1 °C. During the study there was a large overlap in flowering time within and among period, rainfall was 45–65 mm, mean temperature was inflorescences in a single plant. Therefore, a high level of self- 21.6 °C (18.5–28.5 °C), and RH was 61.6% (59.8%–64.3%) pollination is possible. From the significant increases in fruit set at 0700 h. C. otophyllum plants grew in a dry area, covering in cross-pollinated flowers (12.6%) compared with self- about 3000 m2 and usually thriving in patches. The pollinated flowers (1.52%), the low fruit set in C. otophyllum could be partially explained by pollen limitation. surrounding landscape was a mixture of small woodlots and agricultural crops. There were about 500 plants in the whole Keywords-Cynanchum otophyllum; floral biology; honeybees; area. Area B included plants grown from seeds collected in pollination; fruit set) Area A and transplanted to the Botany Experimental Field of Kunming University in early March 2006. A voucher specimen from the population of C. otophyllum studied was 0 INTRODUCTION housed in Kunming University. Voucher specimens of insect The complex floral morphologies found in Asclepiadaceae species observed were also deposited in Kunming University. have intrigued botanists for two centuries [1]. Most data for Asclepiadaceae have been principally accumulated from the 1.2 Floral morphology and anatomy observations of Asclepias in North America [2]; Thus, We picked some mature flowers and immediately fixed Ollerton and Liede [3] considered that overdependence on them in FAA for morphological and anatomical examinations. information about Asclepias could bias our conclusions about Some flowers were sectioned in paraplast and stained with Asclepiadaceae as a whole. either hematoxyline or safranine and observed under an Asclepiadaceae are characterized by low fruit set, typically Olympus BX60 microscope. We also compared self- and averaging 1%–5% [2]. Cynanchum is a large genus of some cross-pollen tube growth in hand-pollinated flowers using 400 described species with a tropical and subtropical fluorescence microscope (Martin 1959). These flowers were distribution in Africa, Madagascar, Australia, North and South fixed 72 hr after pollination in a solution of 3:1 ethanol: acetic America, and some parts of Asia [4]. Cynanchum is acid for at least 24 hr and sectioned. All microtome sections characterized by a staminal corona originating from a ring- stained with decolorized aniline blue for 30 min. The stained shaped meristem [5] that can be extremely variable in shape pollen tubes fluoresced brightly when viewed with and relationship to the gynostegium. Observations in epifluorescence optics on an Olympus BX60 microscope. Cynanchum are still scarce[3] . Critical point dried samples were examined through scanning electron spectroscopy (SEM). C. otophyllum is a medicinal plant endemic to China which produces bountiful flowers but not all of them mature into fruits [6]. This is the bottleneck restricting their 1.3 Flowering phenology cultivation. We studied the species with regard to flowering We determined the flowering patterns from weekly counts phenology, pollination system, and the relationship between of the number of umbels with open flowers in field 2011 IEEE International Conference on Systems Biology (ISB) 179 Zhuhai, China, September 2–4, 2011 978-1-4577-1666-9/11/$26.00 ©2011 IEEE populations from July to October 2006 and 2007. In Areas A B). The flower consists of five parts (Fig. 1: B), and each part and B, 86 and 74 plants, respectively, were individually has a stigma and a pair of pollinia lodged in sacs on each side tagged with thin, red thread. Opening and wilting dates for (Fig. C, E). The brown corpuscular of five pollinaria are easy each were recorded during the flowering season (2 July to 6 to see against the yellow background of the central October 2006). In these two areas, the flowering dynamics of gynostegium (Fig. 1: B). Nectar is often visible at the base of C. otophyllum population were recorded. More detailed the petals. The flowers have a sweet, fruity odor. observations were made in Area B from 23 inflorescences tagged while in bud, each part consisting of 2–5 flowers The perianth consists of five sepals surrounding five petals depending on inflorescence size. Every 3 h, we observed and (Fig. 1: B). The corona is inserted at the base of the recorded floral traits including the timing and duration of gynostegium (Fig. 1: B), which is membranous and deeply flowering. divided into five sections. The corona encircles the gynostegium, the union of the stamens with the fleshy stigma head at the flower's center (Fig. 1: B). The gynostegium 1.4 Hand pollination consists of two pistils attached to the gynostegium at the point On 17–19 July, 23–25 August, and 4–6 September 2006, of connection between the style and the stigma head (Fig. 1: 3–5 flowers at a single node on each of the 60 plants were C). The stigma head is probably stylar in origin because the randomly assigned to the following treatment: (a) bagged true stigmatic surfaces are enclosed within the stigmatic without pollination; (b) emasculation, i.e., removal of all five chambers between adjacent anthers (Fig. 1: D). pollinarium; (c) self-pollen, i.e., emasculation and insertion of pollinaria from another flower of the same plant; (d) cross- The androecium consists of five stamens whose filaments pollen, i.e., emasculation and insertion of pollinaria from are fused to form the column (Fig. 1: C). Each anther is another plant, and all the flowers were bagged before bilocular (Fig. 1: E) and tipped with a white outgrowth of the flowering and bagged again after treatment except the bagged connective that partially covers the stigma head. Each anther without pollination; and (e) unmanipulated control. In the self- sac encloses a pollinium (Fig. 1: E), where approximately and cross-pollination treatments, the pollinarium was removed 170–190 pollen grains are held together by a waxy coating. by inserting an entomological pin beneath the corpuscular. The pollinia of each pair are connected by a thin band called The removed pollinaria were then inserted in one of the five translator arms to a brown, tubular structure called nectar-filled cavities lying directly beneath the corpuscular. corpusculum on top of the stigmatic slit (Fig. 1: C, E). The All other flower buds were removed from the plants as they entire pollination unit (i.e., two pollinia, two translator arms, formed so that resources would be allocated to the and a corpusculum) has the general shape of a wishbone and experimental flowers. We recorded the state of follicle is called a pollinarium (Fig. 1: C). The corneous margins of initiation and maturation every 3 days. All initiated follicles two adjacent anthers form the anther slit (guide rails), which were characterized by visibly enlarged ovaries. Fully provides the narrow entrance to the stigmatic chamber (Fig. 1: developed follicles were considered mature when no F). The corpusculum joining adjacent pollinia of different additional enlargement occurred during three consecutive anthers is directly seated above the alar slit (Fig. 1: C, F). weeks. Nectar serves as the pollinator attractant and is secreted by the basal glands at the base of the sepals (Fig. 1: J). The 1.5 Pollinator and natural pollination efficiency nectar accumulates in the opening of flower as well as in the Pollinator observations were made for 5 days in Area A. stigmatic chambers where the nectar is removed by pollinators Observations were made between 0800 and 0630 h. The (Fig.1: H). Often part of the tarsus and mouthparts of the behaviors of visitors were recorded, and some insects were pollinators, enters the opening between the corneous anther collected for later identification or examination upon receipt slit. Because the upper edges of the anther slit overlap the of the pollen. A total of 400 flowers were checked for the corpusculum (Fig.1: C, F), with the upward pull of the insect, number of removed pollinaria and inserted pollinia under a the tarsus or mouthparts become wedged in the groove of the dissecting microscope.