T REPRO N DU The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.38-50 LA C P T I F V O E

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S H Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) T A Mangrove-Associate species Sourav Singh Deo*, Prashant Pant8** and A. K. Bhatnagar*** *Department of Botany, Deshbandhu College, University of Delhi, Delhi-110014; **Department of Botany Government College, Daman, UT of Daman and Diu, ***Department of Botany, University of Delhi, Delhi-110014, e-mail : *[email protected] Received : 30.07.2018; Revised: 06.11.2018; Accepted and Published online: 15.11.2018 ABSTRACT Excoecaria agallocha is an important mangrove-associate medicinal plant. Owing to the enormous demand for the drug, the plant has been subjected to crude and destructive utilization leading to its depletion from the natural habitats. The present work has been undertaken to investigate reproductive biology of this species such as vegetative and reproductive phenology, floral biology, pollination mechanism and breeding behavior subsidized projects for the management and conservation of this important mangrove associate species. The study was undertaken at various Mangrove regions of the country with major sampling done at Odisha (Orissa, India) from 2004-2010. The reproductive was long (ca. seven months). Excoecaria agallocha, was an obligate cross-pollinated species. It is non-apomictic and required pollen vectors (wind and ) for sexual reproduction. Main biotic biotic pollinators were Xylocopa sp., Apis dorsata, stollii, Eristalinus arvorum and Eristalis solitus. Butterflies such as Euploea core, Anosia melanippus, Pieris brassicae, Tirumala limnaceae, and Hypolimnas bolina were also potential pollinators. Pollen tubes took 18 to 24 h for fertilization. The fruit is a three-lobed schizocarp, containing single seed in each of its three locules. At maturity the fruit dehisced with an explosion liberating the seeds while it still was attached to the parent tree Keywords : Mangrove Associate species, Pollination Ecology of Mangroves

Mangroves are the forest ecosystems inhabiting the inter- management of this species and its associated biotic factors, it tidal coastal and deltaic regions formed by major rivers, where is crucial to understand its breeding behavior, seed biology most angiosperms cannot survive due to hyper salinity (Kim et and pollination ecology. Considering these factors, a al. 2016). The alarming rate at which these ecosystems are comprehensive study on pollination ecology and reproductive being destroyed for human need is an issue of grave concern. Biology of E. agallocha was taken up. The fury of tsunami in 2004 made us realize how important MATERIAL AND METHODS these forests are for the sustenance of human life along the coast. The mangroves act as a natural barrier against sea water The study material of Excoecaria agallocha were surges, and minimize the impact on human settlements collected from three different sites in the state of Orissa. (Spalding et al. 2014). In view of their socio-economic and Battighar Island was the core site located at 200 19.8’N latitude ecological significance, the National Biodiversity Action Plan and 860 44.5’E longitude at an elevation of 38m above mean approved by the Union Cabinet of the Government of India on seal level at Kendrapara district, whereas Hukitola Island 6th November, 2008 has accorded high priority to conservation situated at 200 40’N latitude and 860 80’E longitude, and and sustainable use of the mangrove ecosystems. Therefore, Bhitarkanika situated at 200 40’N latitude and 860 52’E knowing the reproductive biology and ecophysiology of longitude were taken for comparison. Sampling and collection mangrove species are essential for their conservation (Singh of Excoecaria agallocha was carried in mangrove areas in Deo and Pant 2018). In the present study, one of the mangrove eastern and western coast of India (Fig. 1 A, B). However, for associate species Excoecaria agallocha (Euphorbiaceae) has reproductive biology and pollination ecology was studied in been selected because this tree species is quiet common and limited populations of Odisha (Orissa), India (Fig.1). In spite forms an important element of mangrove plant cover (Singh of the fact that Excoecaria agallocha occupies landward Deo and Pant 2018). This species is listed under IUCN Red fringe of mangrove ecosystems, the plants are not easily Data List and has been included under vulnerable category and accessible because they grow in dense clusters and have it is being cleared for purposes, largely for cultivation of compact growth pattern. Branches are interwoven with each aquaculture purposes. E. agallocha plays an important role in other making them impenetrable (Fig. 2). The trees were maintaining mangrove forests ecology, but little information approached through narrow creeks by small, lightweight, is available on its reproduction and propagation (Suvarnaraju manually driven boats in order to gain access during both low et al. 2012, Upadhyay and Mishra 2014, Karamsetty and and high tides without disturbing the pollinators. Hand rowing Aluri 2018) or its ethnobotanical and commercial importance is preferred as the pollinators are very sensitive to any (Singh Deo and Pant 2018). For effective ecological unnatural movement and sound other than wind. The branches 2019 Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) 39

sometime loom down on to the creeks and become more easily electron microscopic studies floral buds were fixed were fixed approachable. in 3% glutaraldehyde. These were dehydrated through

aqueous acetone series, dried with CO2 in Jumbo Critical Point Dryer (Polaron). The samples were coated with gold (20 mm) in a Balzer Union SCPO 020 sputter coater and observed in a EVO-18 Zeiss, UK with 30 kV Acc. voltage; and EDS Image analyzer system. Flowering and fruiting intensity was observed recorded according to the method given by Zakaria (1991). Pollen grains at various stages of development of flowers were A B collected and tested with fluorochromatic reaction (FCR) test as given by Heslop-Harrison and Heslop-Harrison (1970). To Fig.1— Physical maps of the study site. Peninsular part of Indian sub- check whether the pollen grains are shed at two or three continent showing study sites in Orissa nucleate stage, ethidium bromide test adapted by Shivanna The periodicity of various vegetative and reproductive and Rangaswamy (1992) was followed. Mature pollens at the phenoevents that occurred was carefully documented and a time of anther dehiscence were collected and their size was phenological chart was prepared by evaluating various determined by the methodology of McKone and Webb (1988). events as suggested by Sparks et al. (2000). Environmental A small amount of pollen grains was placed in gelatin capsules parameters such as temperature, relative humidity and and stored over a drying agent (calcium chloride) in a rainfall were taken into consideration for the study of above- desiccator and kept in deep freezer (Chanon and Jourdan mentioned phenophase and to assess their influence on plant’s 1998). To determine pollen viability for stored pollen in vitro vegetative and reproductive behaviour. Reproductive period pollen germination for freshly harvested pollen grains were starts from the month of June and continues till November. conducted by hanging drop culture method using Brewbaker Significant changes in the floral morphoplogy and adaptations and Kwack’s (1963) medium supplemented with varying were visually observed and recorded. Measurements were concentration of sucrose ranging from 5 to 30 per cent. Stigma taken with the help of Vernier-calliper or by taking the receptivity (extent and duration analyses) in the field was impression of plant parts on graph sheets and then counting the determined visually by using hand lens to see any secretion on number of squares. Progressive stages of flowering and seed the stigmatic surface. Apart from the above method, four other development were fixed in FAA for sectioning. For scanning methods used are as follows: Non-specific esterases as

A B C

D E F

Fig.2. Morphological and adaptive features. A. Habit: Tidal water influx. B. Exposed system of roots. C. Lenticels at base of tree trunk (Scale bar = 30 mm). D. Lenticels in sectional view showing cellular details (Scale bar = 40 µm). E. Crenate margin of leaf bearing spiny projections (Scale bar = 90 mm). F. Scanning electron micrograph of leaf margin showing spines (Scale bar = 100 µm) 40 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.38-50

described by Mattsson et al. (1974), aniline blue fluorescence dioecious systems tend to flower earlier than the females in a microscopy method by Shivanna and Rangaswamy (1992), season (Stephenson and Bertin 1983). Both male and female hydrogen-peroxide test by Dafni (1992), and fruit set trees look similar and have no distinguishing features till they experiment method. The pollen-ovule ratios were determined start flowering. There was no difference in vegetative parts of by the method proposed by Cruden (1976). Experiments to either type except that the male inflorescence in monoecious determine the mechanism of pollination, nature and the trees bears some female flowers at the base (Fig.5). The trees activities of the pollinator, and the interaction between the have a month-long bud-stage period. It is believed that plant and the pollinators were conducted according to Faegri reduction in water stress and a sharp fall in temperature during and Pijl (1979). Controlled pollination experiments on wet season release bud dormancy and promotes flower selected trees at the time of maximum stigma receptivity using development (Opler et al. 1976). The longevity of both male pollen grains from freshly dehisced anthers to check for fruit and female inflorescences varied. Male inflorescence set following natural and artificial pollination. The manually persisted for 10 days after anthesis and anther dehiscence pollinated flowers were bagged again to prevent any occurred in flowers in acropetal succession, flowers at the top contamination or experimental error. Since Excoecaria of the rachis being the last to contribute to this process. agallocha is a dioecious species are closely placed against Flowering occurs once in a year from end of May and each other, therefore, possibility of existence of apomixis was continues till early September, with peak period in early July. A prominent peak in the flowering occurred during rainy also examined by withholding pollination (Hörandl 2010). season that lasts from early July to early September. The Occurrences of one or two monoecious individuals were also number of flowers per inflorescence varied from 56±4 for observed where in the male inflorescence a few female male inflorescence and 27±3 in female inflorescence. flowers were observed at the base. These were tested for cross- Flowering in male initiates from end of May while in female it pollination. . starts from first week of June. During this phase, pale yellow RESULTS AND DISCUSSION or green, mildly fragrant flowers borne in erect inflorescence were conspicuous (Figs. 3D-E). Inflorescence is catkin-like Phenology—Excoecaria agallocha is a medium sized, structure arising within the leaf bearing portion of the shoot. It dioecious mangrove-associate tree with 5-7 m. Branching may be erect or sometime drooping. Male inflorescence is starts from base, giving the plant a bushy appearance, with 7.9±0.7 cm long, two or three together in an axil with a series snake-like roots, superficially running over the substratum and of spirally arranged bracts, each bract subtending a male exposed during low tides (Figs. 2A-B). The surface of the roots flower. A bracteole is present on either side of each flower. shows lenticels that help the plant to survive in waterlogged Male buds take 35-40 days to develop into a mature flower. condition. Lenticels are present throughout the plant but these Male flowers are pale yellow, almost sessile, tepals are three, are in high density near the base of the main trunk (Figs. 2C- stamens usually three but sometimes 4 or 5, filaments are free, D). Leaf fall begins in mid-May before the arrival of monsoon, anthers are basifixed or versatile, bilobed, dehiscing due to the development of an abscission zone near the petiole. longitudinally (Figs. 5A-C). There is steady increase in the Before shedding, the leaves turn red, the tree remaining length of anther filaments after anthesis attaining a maximum leafless for 10-15 days, with bare trunk and silver-grey bark, length of 2.5±0.5 mm in seven days. After this there is only resuming leaf and floral setting by the end of May (Figs. 3A- increase in the length of inflorescence. Longevity of a male C). The entire phenophase are shown in Figure 4. blossomed inflorescence was 10 days of the beginning of the Floral Biology—The flowers of E. agallocha are unisexual, anthesis. axillary, sometime 2-3 originate from the same axil and they Female inflorescence is usually shorter than the male are pale green when covered by bract, which in fact a feature inflorescence, 2.8±0.6 cm long. Female buds take 35-40 days regarded as characteristic of anemophilous plants (Reddi and for anthesis. Flowers are shortly pedicellate, each flower with Reddi 1984). E. agallocha possesses mass flowering pattern. a pair of basal bracteoles. Tepals are three, closely adpressed to Mass flowering has been frequently reported in tropical trees the ovary. Staminodes are absent (Fig. 5C). Pistil is usually in dry season, while steady state flowering is observed in tricarpellary but sometime it may be bi-, tetra-, or understory species (Frankie et al. 1974, Gentry 1974, pentacarpellary (Fig. 5D), syncarpous, glabrous, ovary Augspurger 1980, Opler et al. 1980, Bawa 1983). This superior, trilocular with one pendulous ovule in each locule. prolonged duration of flowering allows the plant to Increase in the length of style is observed for initial seven days accumulate and channelize the resources for subsequent seed after anthesis attaining a maximum length of 2.5±0.5 mm. maturation in sites where the resource supply is unpredictable (Figs. 6A-D). The ratio between male and female trees is 2:1 or sparse (Bawa 1983). and male and female flowers in a tree are 2:1. The pollinated The date of first flowering and flowering duration were female inflorescences are retained on the tree and later almost similar for both male and female trees. However, male produce fruits and seeds. Fruiting starts from end of June and precedes female trees by few days. Male plants in most of the 2019 Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) 41

Fig.3—Phenological events showing both vegetative and reproductive Fig.4—Excoecaria agallocha: Phenological calendar showing timing phases. A. Leaves turn red prior to shedding from the tree. B. Branches of leaf fall, leaf flushing, flowering and fruiting of Excoecaria agallocha of same tree devoid of leaves. C. Simultaneous flushing of young foliar at Battighar Island, Orissa. and reproductive buds. D. A male tree at peak period of flowering. E. A female tree at peak period of flowering. F. A tree at the time of maximum fruiting. continues till November, with peak in August (Fig. 3F). The from the pollinating agents. The anthers have a longitudinal rich bloom, though short lasting, serves to attract a number of line of dehiscence facing outwards (Fig. 8A). Yellow colored pollinators. At the same time, it ensures that maximum seeds pollen dust was seen scattered around the inflorescence. are shed during the monsoon when the tidal water is least Pollen is released into the atmosphere or carried away by saline. insects. During anther dehiscence, the temperature and Anthesis occurred after 35-40 days in both male and relative humidity recorded were 30 ± 20C and 87 ± 2% female flowers from the date of first appearance. The respectively. The anther dehiscence and subsequent pollen emergence of one of the three anthers or the stigma from the release is often correlated with relative humidity and bract that covered both male and female flowers in the temperature conditions (Yates and Sparks 1993). The trees of inflorescence indicated the initiation of anthesis. Excoecaria agallocha occur in close stands where each In male inflorescences anthesis started at 00:00h, but individual tree canopy intercepts the adjacent canopy. Such maximum opening, i.e. about 62%, was observed between closely-spaced individual trees present their pollen efficiently 02:00 and 04:00 h. Dehiscence of anthers takes place between to wind under turbulent as well as calm weather condition. 08:00 and 10:00h in an asynchronous manner. The anthers Hence, the pollen grains are released gradually and slowly. emerged out of the bract one by one at an interval of about 2 h. Wind is not a common pollen vector for species-rich tropical Thus, spatial distribution and duration of pollen exposure and trees, where conspecific individuals are widely spaced and availability was extended to gain some reproductive benefits have one or more plants of other species growing 42 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.38-50

Fig. 5—Excoecaria agallocha: Morphological variation in reproductive Fig. 6—Development of flowers and fruits. A. Male spike showing three structures. A. A branch with many spikes, each having 1-2 female anthers, nectaries (arrow), bracts (arrow head) and bracteoles (Scale flowers at base and many male flowers. B. Developmental stages of bar = 15 mm).B. Different stages of development in male inflorescence androgynous inflorescences after 5, 10, 15, 20, 25, 30, 35, and 40 days after 7, 14, 21, 28, 35, and 42 days of emergence (Scale bar = 5 mm). C. of their emergence (Scale bar = 5 mm). C. Variation in number of Female inflorescence showing trifid stigma, globular floral base, bracts stamens, showing tri-, tetra-, and penta-staemenous (Scale bar = 40 and bracteoles. Nectaries are present near pedicel (arrow) (Scale bar = mm). D. Variation in number of carpels, showing bi-, tri-, tetra-, and 20 mm). D. Different stages of development of female inflorescence penta-carpellary conditions as displayed by number of stigmas (Scale bar = 15 mm). E. Developmental stages of tetra-locular fruits at 10, 20, after 5, 10, 15, 20, 25, 30, 35, and 40 days of emergence (Scale bar = 4 30, 40, 50 days of maturity. Also present is 30 days old penta-locular mm). E. Developmental stages of 10, 20, 30, 40, 50, and 60 days old fruit, with one abortive locule (arrow) (Scale bar = 16 mm). fruit (Scale bar = 13 mm). between them (Janzen 1975). Mangrove forests are species- frequently shed at three celled stage (Brewbaker 1967, poor and thus wind plays an important role in pollen Webster and Rupert 1973). According to Heslop-Harrison and dispersal. Shivanna (1977), binucleate pollen grains are observed in Copious amount of pollen was produced by each male most species with wet stigma. Pollen grains are shed at two flower and it was found that each anther produces on an celled stage in almost 70% of plant species, whereas the average approximately 1475±75 pollen grains. Scanning remaining 30% species have pollen dehiscence at three celled electron microscopic study revealed that the pollen grains stage (Brewbaker 1957). It is observed that two celled pollen were spherical and tricolpate with reticulate exine (Fig. 8B). grains retain viability for longer period than three celled pollen Average diameter of pollen grain was 40±0.05 µm. Viability due to their higher respiratory activity (Hoekstra and of pollen grains was observed to be maximum on the day of Bruinsma 1975). The prolonged viability of pollen establishes anther dehiscence (Figs. 8D-E). Viability of pollen grains the potential for cross-pollination (xenogamy) and thus results decreased with the increase in the number of days and ceases in genetic variability. In vitro pollen germination showed that completely in seven days from the day of dehiscence. at the time of anther dehiscence, 98.4% pollen germination Viability of pollen grains was maximum on the day anther was observed in Brewbaker and Kwack’s (1963) medium dehisce (Fig. 8D, E). Pollen was shed at two nucleate stage supplemented with 15% sucrose concentration, which was (Fig. 8C). However, in Euphorbiaceae pollen grains optimal for maximum pollen germination. The pollen tubes 2019 Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) 43

grew as long as four times or more the diameter of pollen grain affects seed setting, followed by propagules development and within two hours (Fig. 8E). The pollen grains are dry and did seedling establishment along the seashore. According to not adhere to each other but were spread separately or occured Faegri and Pijl (1979), in majority of the flowering plants in small groups. This is an adaptation for both wind and insect pollen grains are rich in proteins but for those plants where pollination (Wragg and Johnson 2011). Pollen grains are pollen grains are the major floral reward for the pollinator, endowed with rich deposition of polysaccharides, protein, and they tend to be predominantly lipidic. lipids and serve as nutritional rewards for the pollinators (Figs. In female inflorescence, anthesis starts at 00:00h but 8F-H). According to Elisens and Freeman (1988), pollen maximum opening, i.e. about 59% occurred between 04:00 grains are considered to be the original attractant for and 06:00 h. Between 10:00 and 12:00 h all the three anthers pollinators, nectar being the secondary adaptation. But emerged out of the bract representing a fully opened male unfortunately knowledge about pollen fertility, pollen flower. The female flower was considered open when the trifid dispersal, and most importantly pollination control of stigma was well exposed to the agents of pollination between mangroves is meager (Tyagi and Singh 1998). Pollen fertility 08:00 and 10:00 h. Although flowering was asynchronous,

Fig. 8—Anther dehiscence and pollen grains. A. Scanning electron micrograph showing longitudinal line of anther dehiscence (Scale bar = 20 µm). B. Scanning electron micrograph of pollen grain showing wall reticulation and tricolporate condition (Scale bar = 3 µm).C. Pollen grain having two nuclei at the time of dehiscence when stained with 0.1 per Fig. 7—Excoecaria agallocha: Extra-floral nectaries. A. Female cent ethidium bromide (Scale bar = 10 µm). D. Pollen viability test using inflorescence showing the presence of conspicuous nectaries near the fluorescein diacetate. The viable pollen grains fluoresce bright (Scale pedicel (arrow) (Scale bar = 30 mm). B. Longitudinal section of female bar = 80 µm).E. In vitro pollen germination in modified Brewbaker and inflorescence showing linear arrangement of the floral structures such Kwack’s medium supplemented with 15 per cent sucrose showing as nectary, followed by bracteole, bract and the ovary (Scale bar = 270 maximum pollen germination (Scale bar = 80 µm).F. Pollen grains µm). C. Longitudinal section of nectary showing its cellular details showing the presence of lipid droplets when stained with Auramin O (Scale bar = 80 µm). D. Magnified view of the nectary showing (Scale bar = 80 µm). G. Pollen grains stained with iodine-potassium epithelium-like secretory layer covered by cuticle (Scale bar = 40 µm). iodide (I2KI), showing the presence of starch grains (Scale bar = 80 E. Scanning electron micrograph (SEM) of a pair of extra-floral µm). H. Pollen grains stained with aniline blue black, dark blue staining nectaries showing their location. Each nectary has four lobes and a shows presence of protein (Scale bar = 80 µm).I. Pollen grains stained central pore (arrow) (Scale bar = 300 µm). F. Magnified view of a with periodic acid Schiff’s (PAS) reagent. Magenta red staining nectary showing surface contour. Lobes are separated by grove with indicates abundant insoluble polysaccharides (Scale bar = 200 µm). J. Pollen grains stained with Sudan black B, dark colour indicates the central pore (Scale bar = 30 µm). presence of lipids (Scale bar = 80 µm). 44 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.38-50

many male and female flowers opened at same time and were, ascertained by the appearance of surface esterase in certain therefore, available for pollination. Temperature and relative plants (Knox et al. 1986), a more appropriate method may be humidity was recorded at midnight when floral anthesis used to assess it through studies on pollen germination and occurred was in the range of 23±20C and 68±2% respectively. pollen tube growth in manually pollinated pistils in order to The female inflorescence remained intact in the tree after check the per cent fruit set. pollination and developed into bunches of fruits. Nearly 84% flowers developed into fruits if pollinated on The pistil is composed of a globular, superior ovary and a the day of anthesis. With delayed pollination, the percentage well spread trilobed stigma at its top. Sometime the number of declined to 68, 40, 24, 14 and 4 with each day. On the sixth day radiating stigmatic lobes varied from four to five. Stigma is of the stigma entirely lost its receptivity. Fruit initiation was a wet type, sticky and non-papillate but having only slightly recorded in the last week of June and it reached its peak by raised cells. The style is solid, with a central core of 6-8 August. By this time every tree was loaded with trilocular layered thick transmitting tissue surrounded by schizocarpic fruits. Fruit maturation is a long process taking parenchymatous cells, terminating just distal to where the almost five months, from July to November. Seeds are obturator originates (Figs. 9A-D). The cells of the transmitting released from the fruit with an explosion at maturity. tissue are compactly arranged and do not show any Nectaries: Excoecaria shows entomophilous features intercellular spaces. Epidermal cells of the style have thick e.g. presence of large, 4-lobed extra floral nectaries near the cuticle on the exterior wall which is folded. Continuous base of the pedicel. This seems to have considerable stretches of prominent vascular supply are observed towards ecological significance (Davis et al. 1988, Nepi et al. 1996). The presence of extra-floral nectaries is common in the lower surface of the style (Fig. 9E). Scanning electron Euphorbiaceae. In this family conspicuous extra-floral microscopy revealed the presence of stomata at base of style nectaries may be present which are: (i) Patelliform, (ii) near ovary and rectangular blocks covered with hair-like Glandular, on leaf margin, or (iii) Morulose (Metcalfe and projections (Figs. 9B-F). Permanently open stomata are Chalk 1950). These glands are also reported particularly in present on stigmatic surface as observed under scanning species of Croton, Hevea, Macaranga, Micrandra and electron micrograph (Fig. 10F). Each ovary has one ovule in Ricinus (Dave and Patel 1975). The position of the nectary each locule. Ovule is anatropous, with a prominent raphe, within the flower determines the nature of plant-pollinator occupying the ovarian cavity fully. interactions (Nepi and Pacini 1993). The adaptive significance Stigma is trifid, wet and non-papillate type, during of extra-floral nectaries has been of interest to evolutionary anthesis it is green, and the colour remained same as long as it biologists (Keeler 1989, Huxley and Cutler 1991). Currently, was receptive. Pollinated stigmas changed their colour from the most well established hypothesis for the evolution and green to light yellow to complete orange. Finally, the stigma maintenance of extra-floral nectaries is that ants kill or turned brown and persisted till the end of the reproductive dislodge herbivores from plant tissue, thereby reducing the phase of the propagule (Figs. 10A-B). Nonspecific esterases, loss of plant tissue to herbivores (Becerra and Venable 1989). localized on the surface of stigma, play an important role in The extra-floral nectaries in Excoecaria agallocha more pollen adhesion and pollen tube growth. Maximum receptivity likely serve the function of distraction of crawling insects such of stigma was observed on the day of anthesis as confirmed by as ants, and preventing them from reaching the flowers. The hydrogen peroxide test and bright red colour of stigma floral nectar is conserved for the flying insect pollinators that surface. In Aniline blue fluorescence method, using manually promote cross pollination. Nectaries are large, tetralobed pollinated stigmas, pollen grains start germinating after four present on either side near the base of pedicel of both male and hours from landing on the stigmatic surface (Figs. 11A-B). female flowers (Fig. 7A). In sections these extra-floral The pollen tube takes 18-24 h to reach ovule. The pollen tubes nectaries, were seen as situated below the bracteoles in a linear sometime penetrated the stylar tissue through the opening of arrangement (Fig. 7B). The nectariferous tissue was stomata (Fig. 11C). Stigma receptivity is maximum on the day composed of small cells with thin walls, relatively large of anthesis and remains so for five days. Success rate of in vivo nuclei, dense granular cytoplasm, and small vacuoles. The pollen germination and pollen tube growth suggests that the glands receive well developed vascular bundles with a high availability of pollen grains is not the limiting factor for fruit proportion of phloem elements. The secretion is brought about set in this mangrove-associate species. At the time of by parenchymatous epidermal cells to the outer environment maximum receptivity, surface of stigma became saturated directly through a pore. The cuticle is disrupted near the pore with exudates, which glisten against sunlight, as observed in or the opening of the nectary facilitating smooth passage for curcas (Euphorbiaceae) (Solomon Raju and nectar secretion (Figs. 7C-D). Secretion of nectar continues for two consecutive days from the day of anthesis. Secretion Ezradanam 2002). It is observed that the volume of exudates begins at 04:00h and continues till 16:00h with maximum secreted by stigma varies greatly from species to species nectar secretion (about 3µl) between 09:00 and 11:00h in within a family. Although stigma receptivity can be 2019 Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) 45

Fig. 9—Morphology and anatomical details of style (tt: transmitting Fig.10—Morphology and physiology of stigma. A. An excised female tissue, vs: vascular supply). A. Longitudinal section of female flower flower having trifid stigma with coiled stigmatic lobes (Scale bar = 20 showing one of three stylar lobes. Style is solid type (Scale bar = 240 mm).B. Scanning electron micrograph of female inflorescence µm). B. Scanning electron micrograph showing the presence of showing surface contour of stigma (Scale bar = 100 µm). C. Scanning projecting stomata at base of style near ovary (Scale bar = 30 µm). C. electron micrograph of stigmatic surface showing numerous pollen Magnified portion of style showing 6-8 layeres thick transmitting tissue grains on stigma (Scale bar = 30 µm). D. Magnified view of C, showing and prominent vascular supply (Scale bar = 120 µm). D. Longitudinal pollen grains adhering to stigmatic surface (Scale bar = 10 µm). E. section of style showing variation in thickness. Stigmatic surface is Hydrogen per oxide test for maximum stigma receptivity as observed uneven and becomes thicker in middle (Scale bar = 240 µm). E. by the liberation of air bubbles (arrow) close to stigmatic surface (Scale Vascular tissue with spiral thickenings. F. SEM of surface of style bar = 12 mm). F. Scanning electron micrograph of stomata present on showing rectangular blocks covered with hair-like projections (Scale the surface of stigma (Scale bar = 10 µm). bar = 10 µm).

Fig. 11— Aniline blue fluorescence method for determining pollen tube growth. A. Cleared preparation of the trifid stigma stained observed under fluorescence microscope, showing large number of germinated pollen grains and pollen tubes entering the style (Scale bar = 500 µm). B. One of the lobes of trifid stigma with germinated pollen grains on stigma surface and many pollen tubes in style (Scale bar = 400 µm). C. Tricolpate pollen grains with a pollen tube from each that has penetrated stigma (Scale bar = 4 µm). 46 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.38-50

male, and (1.8µl) between 10:00 and 11:00h in female on first recruitment process and ultimately towards the maintenance day. On second day the secretion was (1.2µl) between 08:00 of the population. Though a wide variety of potential and 09:00h in male, and (0.3µl) between 08:00 and 09:00h in pollinators visit the flowers, yet there is a low fruit set female. During the day time when temperature was high, observed in open pollination, which suggests that either concentration of nectar sugar increased, perhaps due to pollinators are not in sufficient number, or the flowers are less evaporation (35-38%). Environmental condition affected efficient in tapping their services. Tomlinson et al. (1979) concentration of nectar sugar (Kenoyer 1917). Flowers reported a wide variety of pollination mechanisms including continued secretion of nectar for two days from the day of wind pollination in Rhizophoraceae species, bird pollination anthesis. Scanning electron microscopic studies revealed a in large-flowered species and butterfly pollination in small minute central pore or opening through which the nectar is flowered Bruguiera sp. and moth pollination in some of secreted (Figs. 7E-F). Nectar is an important source of Ceriops sp. Sun et al. (1998) observed that in Kandelia candel, carbohydrate needed for the growth and development of a member of Rhizophoraceae, non-specialized pollination honey bees, whereas pollen is the main protein source (Lin et mechanism involves several generalist pollinators, such as al. 1993). Generally, mangrove plants are regarded as good bees and butterfly. Sunbirds and carpenter bees are equally honey plants (Jana and Bera 2004, Naskar 2004). important as Anisomeles pollinators, and they effectively Pollination biology— Pollination is accomplished by both pollinate Anisomeles (Lamiaceae) flowers and blue Acanthus wind and insects. Floral morphology is well adapted for cross ilicifolius (Acanthaceae). In Avicennia officinalis pollination. Features such as inflorescence being a dense (Acanthaceae) pollination is accomplished by the flies spike, yellow to pale yellow flowers with mild fragrance, and (Solomon Raju 1990). presence of extra-floral nectaries facilitate insect pollination, Among the pollinating insects were Xylocopa sp., Apis whereas wet and well exposed trifid stigma helps in adhesion dorsata, Chrysocoris stollii, Eristalinus arvorum and Eristalis of wind borne pollen. Nectar production was copious, and solitus. Butterflies (Euploea core, Anosia melanippus, Pieris continuous, and it was found to be concurrent with the brassicae, Tirumala limnaceae and Hypolimnas bolina) were temporal activities of floral visitor. Bees are attracted by pale also prominent pollinators (Fig. 12). yellow colour of the flower. It was interesting to note that the Activity of insects began at 05:00 h and reached its peak frequency of insect visitation is directly proportional to nectar between 08:00 and 10:00 h. Decline in foraging activity was secretion during the day. Nectar as reward was not concealed; observed between 10:00 and 14:00 h because of rise in rather the nectary surface glitters in sunlight and directs the temperature. However, between 14:00 and 16:00 h there was insects to probe the flowers and acts as a substitute for nectar again a rise in insect activity and thereafter it ceased guides. By offering pollen as a reward to the unspecialized completely. There is hardly any difference in the foraging opportunistic bees, E. agallocha derives reproductive benefits. timing between the male and female inflorescences except for In the part of the season when E. agallocha flowers no other the fact that the number of visits received by female mangrove species is seen flowering. Therefore, this species inflorescences was less as compared to their male serves as the only source of nectar for the insects for nearly a counterparts. The nectar production was not concealed and is month. Other mangrove species have either completed their accessible to almost all insects, both pollinating and non- flowering phase or will do so in recent future. Thus, the insects pollinating insects that exhibit differential foraging behaviour are very sensitive to variations in nectar rewards. (Table 1). The pollinators in E. agallocha are diverse and include Bees were the most frequent, active and effective insects with short mouth parts (allophylics) and dystrophic foragers. They foraged at almost all levels in the canopy of the visitors (not having adaptations to specific floral trees as compared to other insects. Giant Asian honey bee Apis morphologies). By offering pollen and nectar as a food dorsata was the most prominent, since its individuals carried resource, this mangrove-associate species exploits many the maximum number of pollen grains (average 2833 per flower visitors for pollen transport. Therefore, E. agallocha individual, Table 1) on the thorax (coxa) and in their pollen has characteristics related to both anemophily and basket (corbiculum) (Fig. 12B). The bees generally landed entomophily indicating a mixed system of pollination known directly on stamens of the youngest flower at the top of as ambophily (Karamsetty and Aluri 2918). Similar breeding inflorescence and followed a circular path while foraging for mechanism is also found in many members of the family nectar and pollen. A significant accumulation of pollen grains Euphorbiaceae, e.g. Croton bonplandianum and Phyllanthus on the pollen basket situated on their hind legs and the pinnatus (Reddi and Reddi 1984). P. pinnatus is a dioecious abdomen was observed (Fig. 12C). Xylocopa sp. is another species and exhibits synchronous flowering. Such important pollinator and it was observed foraging at the synchronization brings appropriate genetic exchange and seed highest level of the canopy (Fig. 12A), flying very swiftly set. The potential of E. agallocha, to convert floral visitation to from one tree to another (Table 1) collecting mainly pollen flower fertilization and fruit set is an important step in the grains. 2019 Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) 47

Amongst the syrphidae, Eristalinus arvorum, Eristalis Average fruit set after open pollination was about 35% solitus, and Chrysocoris stollii were the most common (Figs. (Table 2). As is expected the number of pollen grains deposited 12D-F) catching both pollen and nectar. Species of both cumulatively increases with increasing time, but the rate of Eristalinus and Eristalis used the inflorescence rachis as the deposition decreased considerably once the stigma started landing platform and started foraging first from the older loosing its stickiness and receptivity. Artificial/hand flowers situated at the base of each inflorescence and proceeded pollination revealed that fruit set was 65% which is much upward to the younger flowers. Chrysocoris stollii was the higher as compared to open pollination. Efficacy of wind as a most sluggish amongst the tree species. It was interesting to pollinating agent was assessed by fruit set experiment to be note that pollen grains were found adhering on the leg bristles 24.5% when wind was the sole agent of pollination. The and especially within the claws. Several species of butterflies species is non apomictic. Ovaries of these unpollinated (Euploea core, Anosia melanippus, Pieris brassicae, Tirumala flowers aborted within seven days after emergence of the limnaceae and Hypolimnas bolina) prefered to search for nectar at the apex of the canopy (Figs. 12 I-L). They tend to visit only stigma from surrounding bracts. one flower of E. agallocha at a time before flying away to visit Ants are common flower visitors, and their interaction another plant. They carry considerable amount of pollen grains with the plants can take a variety of forms. They are in their body. These feed exclusively on the nectar. Their long opportunists and respond to locally available nectar proboscis facilitates easy access to nectar. It seems that this resources, which are modified by the abundance and floral mechanism is intended for multiple visits so that performances of other nectar foragers. In other situations, pollination is affected by these legitimate pollinators which are ants can act as antagonists. These may negatively affect plant capable of manipulating the pollen receptacle to reach the reproductive success by removing nectar without pollination nectar present at the base of pedicel in each flower. There were (acting as nectar thieves), by disrupting the visitation of the several other insects such as dipteran flies, Musca domestica, effective pollinators and thus reducing pollination (Wyatt Camponotus sericeus, Nupserha sp., Tabanidae fly, Acraea 1980, Fritz and Morse 1981), or by damaging reproductive violae and some species of butterflies which were observed structures such as pistils, ovaries (Puterbaugh 1998, Galen foraging the inflorescence (Figs. 13A-F). However, their 1999) or pollen (Wagner 2000, Galen and Butchart 2003). movements were restricted to only a few trees or within a few Recent researches have shown that ants are aggressive and inflorescences in a single tree. These carried a relatively low impose a cost to the host tree by driving away useful number of pollen grains on their bodies. Another interesting pollinators (Willmer and Stone 1997). In E. agallocha, ants thing to note was that while foraging the female inflorescence negatively affect plant reproductive success by removing none of their body parts carrying the pollen grains came in nectar without pollinating (acting as nectar thieves), by contact with the stigma surface.

Table 1— Excoecaria agallocha: Efficacy of insects during their visitation

Name of insect Pollen load Foraging duration/ No. of Reward (No. of pollen inflorescence inflorescence Pollen/Nectar grains/ insect) (in seconds) visited/min Xylocopa sp. 2.4 25 P Apis dorsata 2833 5.1 12 P+N Eristalis solitus 700 8.3 7 P+N Chrysocoris stollii 330 200 0.3 P Euploea core 733 120 0.5 N Anosia melanippus 883 8.9 7 N Pieris brassicae 683 4.9 12 N Tirumala limniace 750 5.04 12 N Papilio nikobarus 633 7.5 8 N Camponotus sericeus 162 40.7 1 N Ropalidia ferruginea 295 7.0 8 P Muscidae domestica 152 16.3 3 N Nupserha sp. 152 40.9 1 N Rodolia cardinalis 13 13.1 4 - Pachliopta hector 112 5.4 11 N 48 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.38-50

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Table 2— Excoecaria agallocha : Fruit set following natural and manual pollination

Mode of Total No. 2010 2011 2012 2013 pollination of flowers No. of Per cent No. of Per cent No. of Per cent No. of Per cent pollinated fruit set fruit set fruit set fruit set fruit set fruit set fruit set fruit set Open 2800 583 29.15 688 34.4 811 40.55 718 35.90 Hand Pollination 520 139 69.5 160 80 97 48.5 124 62 Apomixis 200 0 0 0 0 0 0 0 0 Wind 208 41 20.5 64 32 36 18 55 27.5

Fig. 12 — Excoecaria agallocha: Pollinators in action. A. Xylocopa sp. on male flowers (Apidae). B. Apis dorsata with pollen basket (Apidae). C. Scanning electron micrograph of ventral surface of Apis dorsata, showing clumps of pollen grains on its body surface (Scale bar = 10 µm). D. Eristalinus arvorum (Syrphidae). E. Eristalis solitus (Syrphidae). F. Chrysocoris stollii (Syrphidae).G. Euploea core (Nymphalidae). H. Anosia melanippus (Nymphalidae). I. Scanning electron micrograph of Fig. 13—Excoecaria agallocha: Non-pollinating visitors. A. claw of Chrysocoris stollii having pollen grains trapped in claw (Scale Dipteran fly on male inflorescence. B. Musca domestica (Muscidae). C. bar = 30 µm). J. Pieris brassicae (Pieridae). K. Tirumala limnaceae Camponotus sericeus (Formidiae). D. Nupserha sp. (Cerambycidae). (Nymphalidae). L. Hypolimnas bolina (Nymphalidae). E. Tabanidae fly (Diptera). F. Acraea violae (Acraeidae). 2019 Reproductive Biology of Excoecaria agallocha L. (Euphorbiaceae) 49

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