T REPRO N DU LA C The International Journal of Reproductive Biology 9(2) Jul., 2017, pp.129-138 P T I F V O E

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S H Phenology, pollination mechanism, breeding system, seed dispersal and germination T in Aquilaria malaccensis Lam. a threatened tropical evergreen forest tree of North East N. Venugopal* and Ester Jones Marbaniang** Department of Botany, Centre for Advanced Studies in Botany, North Eastern Hill University, Shillong-793 022, India e-mail : *[email protected]; **[email protected] Received: 01. 04. 2017; Revised: 28.05.2017; Accepted and Published online : 01.06.2017 ABSTRACT Aquilaria malaccensis Lam. of is an economically important tree for the production of agar oil. The species has been placed on Appendix II of CITES and belongs to the “Threatened” category of the IUCN Red List. Flowers are small and yellowish green and flowering takes place from March end to April first week followed by fruiting in May. Honey bees, beetles and thrips are the pollinators. The breeding system is xenogamous. In A. malaccensis wind and insects (ants) are the major dispersal agents of seeds. Seeds of A. malaccensis are recalcitrant and therefore, the percentage of viability, moisture content and germination of the seeds decreases with increasing the storage time, hence sowing of seeds soon after harvest is essential for better and higher percentage of germination. A. malaccensis is an evergreen tree but remain leafless from December to February due to caterpillars. The attack of caterpillars on the leaves and tender branches acts as a triggering mechanism for the formation of new leaves and buds. Keywords : Aquilaria malaccensis Lam.; Thymelaeaceae; Threatened tree; Pollination; Breeding behaviour; Myrmecochorous dispersal; Meghalaya; North East India.

Knowledge on pollination ecology and detailed studies Most of the species of Aquilaria as well as the genera on reproductive biology of Indian plant species, especially and of the family Thymelaeaceae are also trees, are very meager suggesting the need to investigate this pollinated by various Lepidoptera, Hymenoptera Coleoptera field of biology (Aluri 1990, Tandon et al. 2003). and Diptera insects, and both butterflies and settling moths Angiosperm tree species have a low reproductive efficiency; (Whitehead et al. 1987, Johnson 1992). The number of therefore, it is important to study the pollination mechanism flowers borne by a mature tree is very high in most of the and reproductive biology for developing strategies that will species of Aquilaria, but the proportion of flowers developing help to preserve the genetic potential of rare and threatened into a fruit was very low (Soehartono and Newton 2000). In species (Holsinger 1991, Bernardello et al. 1999, Moza and Daphne gnidium more than half of the flowers fell down Bhatnagar 2007). A. malaccensis Lam. commonly known as immediately after anthesis, and a very limited number (only agar tree belongs to family Thymelaeaceae. It is economically 8%) reached maturity (Jordano 1987, Herrera 1988, Guitian important tree, for the production of good-quality agar wood. and Guitian 1990, Roccotiello et al. 2009). Seeds of Aquilaria The resin is valuable and used in medicine, perfumes and other are considered to be recalcitrant and germinate rapidly within aromatic products. Because of its immense monetary value for 3 weeks after sowing with 86% germination (Beniwal 1989, the communities who exploit these trees and hence the Soehartono and Newton 2001). Keeping in view of the population of A. malaccensis has decreased in the natural importance of A. malaccensis, drastic decline of population in the natural habitat, a study was conducted on its reproductive forests(Barden et al. 2000). biology for its conservation and restoration. India is home to two Aquilaria species, A. khasiana and A. malaccensis whereas the third species Aquilaria macrophylla MATERIAL AND METHODS Miq. is reported from Nicobar islands (Giri 2003). A. Study area—Aquilaria malaccensis Lam. (syn A. khasiana is found mainly in the Khasi Hills of Meghalaya and agallocha Roxb.) trees are growing in the wet sub-tropical A. malaccensis is native to eight north-eastern states of India forest in Mawlyngroh, South West Khasi Hills District of and is also widely distributed in south and south-east Asia o Meghalaya, India. This area lies between 25 14`48.6`` North (Barden et al. 2000). In Meghalaya, A. malaccensis is o latitudes and 91 24`57.90`` East longitudes at an altitude of confined to the dense tropical evergreen forests located in about 487m amsl. The flower buds, from the initiation to the between 800-1000 m amsl (Haridasan and Rao 1987). The mature flowers and different stages of fruits were collected to species was placed on Appendix II of the Convention on the study the developmental aspects of anther, embryo and International Trade in Endangered Species (CITES) and endosperm. belongs to the “Threatened” category according to the IUCN Phenology and floral biology—Phenological events

Red List (Oldfield et al. 1998, CITES 2005, Saikia and Khan from bud initiation to seed dispersal were recorded over a 2012a) and considered 'critically endangered' in India (Tabin period of four flowering seasons (2011-2014). The average and Shrivastava 2014). numbers of flower borne on an were counted 130 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.129-138

from a set of randomly selected 500 flowering branches of 10 4. Apomixis: possibility of occurrence of apomixes was trees. The time of anther dehiscence and period of stigma also assessed by preventing pollination and subsequent receptivity were also observed. fertilization. The receptivity of stigmatic surface was confirmed by 5. Open pollination: Flowers were marked and tagged one localizing the non-specific esterase and phosphatase activity day before anthesis to check for pollination and fruit set with α-naphthyl acetate (Mattsson et al. 1974) and α-naphthyl and as they occur in nature. acid phosphate (Scandlios 1969) as respective substrates. For The above treatments were conducted on 50 flowers from estimating the average number of pollen grains produced per randomly selected 10 trees. The total sample sizes are given in flower, mature but undehisced anthers (n = 20) randomly Table 2 according to the methods followed by Dafni (1992) selected from 10 different trees were squashed in two to three Tandon et al. (2003) Kaul and Koul (2009). drops of 25 % glycerol + 1 drop of 1 % acetocarmine. Pollen Fruit-set and seed-set—In A. malaccensis, fruit and seed to ovule ratio (P/O ratio) was calculated following the method given by Cruden (1977). Pollen viability of fresh pollen grains set were estimated by counting the total number of flowers as was tested by using Fluorochromatic reaction (FCR) Test well as the total number of fruits formed per inflorescence (Heslop-Harrison and Heslop-Harrison 1970) and confirmed from 10 branches selected randomly from 10 trees. The fallen by pollen germination using the method described by flowers from each selected inflorescence were also taken care Brewbaker and Kwack (1963) and Dashek (2000). of. Mature fruits were harvested before dehiscence and the Morphological characters of flowers, fruits and seeds were numbers of seeds in the respective fruit locule were counted observed with the help of a Magnus MSZ-TR stereo and the percentage of one seeded and two seeded fruits were microscope. Different stages of development of pistil, anther, calculated. fruit and seed were studied through microtome sectioning of Fruit and seed dispersal—Five isolated trees at different paraffin embedded materials (Johansen 1940, Sass 1958, locations within the population were marked to check for the Berlyn and Miksche 1976). Scanning electron microscope dispersal of fruits and seeds. The distance travelled by fruits as (SEM) was also used during the study after Dey et al. (1989) well as seeds after their dispersal was measured from the base and Dashek (2000) to confirm the pollen grains of A. of the trunk of respective trees (Tandon et al. 2003). The malaccensis on the pollinators bodies as well to study the dispersal agents were also observed. pollen sculpture. Seed viability, germination and moisture content— Pollination and breeding system— The floral visitors of The viability of seeds was determined by staining them with A. malaccensis were observed continuously during the peak Tetrazolium salt solution (Henry 1962). Seed germination was period of flowering (March end to April first week), flower opening time from 8.00 am to 5.00 pm for 6 days from March tried in laboratory and net house conditions and compared 30th to April 1st to 4th during 2011- 2014 at Mawlyngroh with those in the field. location. The floral visitor observations were conducted in A comparison was made in respect to the seed parallel by five observers. During these observations the germination percentage in the laboratory and net house behaviour of the flower visitors, such as movement on the conditions and seed viability in relation to time, For this, seeds flower and contact with floral organs, was recorded and it was were washed thoroughly in tap water for 10 minutes, noted whether flower visitors were carrying pollen on their disinfected with 70% ethanol for 45 seconds and rinsed 3 proboscises or bodies parts. Some flower visitors were caught times with double distilled water, followed by soaking the and preserved for later identification by insect taxonomists at seeds in 0.1% (w/v) aqueous mercuric chloride for 5 minutes Zoological Survey of India, Shillong. and rinsed 3 times again with double distilled water. The seeds To find out the breeding behaviour in Aquilaria were then kept on the moistened seed germinating paper and malaccensis, following natural and artificial pollination kept inside the seed germinator at 28-300C. Seeds were also manipulations were carried out. sowed in the net house, Department of Botany, NEHU- 1. Autogamy : Flowers were bagged prior to anthesis and Shillong by mixing cow dung, soil and sand in the mixture of left undistributed to find out spontaneous self- 1:2:2. Seed moisture content was estimated by weighing 100 pollination. fresh seeds and dried in an oven at 1030C for 17 hours and then 2. Geitonogamy : Flowers were emasculated prior to anthesis and pollinated with pollen grains dusted on the weighed again and the present moisture content calculated on receptive stigma with fine and clean brush from the freshly wet basis (Roberts and Roberts 1972). Statistical analysis of dehisced anthers of other flowers of the same plant. mean data (one – way ANOVA) was done to interpret 3. Xenogamy : Flowers were emasculated and pollinated significant difference between mean values of seed with pollen grain collected from of a different germination in laboratory conditions and natural habitat as population and bagged. well as seed moisture content. 2017 Phenology, pollination mechanism, breeding system, seed dispersal and germination ...... 131

RESULTS pubescent and reduced to scale like appendages (Fig.1D). The colour of the changes from pale to yellowish green upon Phenology—Aquilaria malaccensis is a relatively slow- maturation. Although flowers are small but its petaloid growing, medium-sized tree, on average of 15-25 m tall with a are prominent. The androecium consists of 10 epipetalous moderately straight stem. The bark is smooth, thin, pale gray stamens bearing dorsifixed anthers. On average 3,840 pollen with dense, dark foliage of shiny elliptical leaves. Flowering st grains were found per anther and a total of an average 38,400 took place in the from the end of March to the 1 week of April, pollen grains were recorded from a single flower. The pollen which lasted only for 10-15 days and mature fruits were grains are pentaporate with crotonoid exine (Fig.1E and F). observed in May followed by the emergence of seedlings in Anthers are yellowish green at anthesis and turn brown after June and July. The entire reproductive phase is completed dehiscence and release pollen grains. within four months and these phenophases are correlated with the increase in temperature and rainfall. A. malaccensis is evergreen but most of the trees remain leafless during December, January and February. This is because as the leaves are eaten up by caterpillars which eventually give rise to the Aquilaria moth. Those trees which are not attacked by caterpillars remain green throughout the year. The caterpillars clustered on the leaves and eat up the leaves and young tender branches. They can completely defoliate young and mature trees as well (Fig.1A). On receiving the first rains, the caterpillars begin to disappear and new leaves started sprouting along with the initiation of flower buds and a fully green tree can be seen in March.

Fig. 2- Aquilaria malaccensis. A. Dehisced mature anthers (A) and a single pistil (arrow) (Bar = 0.5cm); B. L.S. of bilocular gynoecium showing a single ovule (O) in each locule. The style is inconspicuous; stigma (S) is capitate and yellowish(Bar = 5cm); C. An enlarged view of the stigma showing ridges (R) and intensely stained non specific esterases (Bar = 0.3cm); D. Pollen viability tested by FCR test, viable pollen grains showing bright greenish fluorescence (Bar = 100µm); E. In vitro pollen germination (Bar = 100µm); F. Stereo microscopic photograph of a bee showing the pollen grains on its body (Red circle) (Bar = 2.75cm); G. An enlarged view of part of the bee in (F) with yellow pollen grains on its body parts (Bar = 6.5cm). Pistil is monocarpellary with superior, ovoid, massive and highly pubescent ovary, inconspicuous style and a prominent stigma. Ovary is biloculate with a single bitegmic ovule in each locule but sometimes owing to ovule abortion; only one ovule is formed (Fig.2A and B). Stigma is capitate Fig. 1- Aquilaria malaccensis—A. Enlarged view of a caterpillar eating leaf (Bar = 1.3cm); B. An enlarged view of an inflorescence consisting with 5 ridges alternating with 5 furrows and covered with of 10-13 flowers (Bar = 0.5cm); C. Five sepals (S) larger than the unicellular papillate outgrowths. The stigmatic surface is filiform petals, gamosepalous, persistent and densely pubescent (Bar = 0.3cm); D. Highly pubescent petals (P) reduced into scale like 1mm in diameter and the ovary is 2.5-3 x 1.5-2 mm. The ovule appendages and yellowish anthers (Bar = 0.2cm); E. Scanning is anatropous and the embryo sac is polygonum type. At electron micrograph (SEM) of a mature laterally dehisced anther lobe receptivity stigma is yellowish. It remains receptive for 2-3 releasing pollen grains; F. SEM of a single pollen grain showing the cratonoid ornamentation of the exine. days and later loses receptivity, turns brown and dries off. On Floral morphology— Each inflorescence in A. the ridges of the stigmatic surfaces, the exudates stain malaccensis was composed of 10-13 flowers, their maturation intensely for non-specific esterases and phosphatases is centripetal, younger flowers are towards the centre and the (Fig.2C). older ones at the periphery (Fig.1B). Flowers of A. The percentages of pollen viability (Fig.2D) and pollen malaccensis are small, about 8-10 mm in diameter, germination in vitro recorded was 88.32 ± 2.41% and 78.00 ± protogynous, bisexual, and ebracteate. Sepals are 5, 3.5% respectively with the average pollen tubes length of 595 gamosepalous, persistent, petaloid, pubescent and larger than ± 10.12 µm. Pollen grains remain viable for 2-3 days under the filiform petals (Fig.1C). Petals are 10, free, highly laboratory conditions. Pollen grains germinated at a wide 132 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.129-138

range of pH (i.e. from pH 5 to pH 7.5), but the best result was obtained at a pH of 7.3 at 280C for 4-5 hours (Fig.2E). Increasing or decreasing the temperature lead to decrease in the percentage pollen germination.

Fig. 4–Aquilaria malaccensis – A. Dehisced fruit exposing the two seeds hanging upside down by an intact vasculature with persistent aril (arrow)(Bar = 1.5cm); B. Single and double seeded fruits (Bar =1.5cm); C. Mature and dry fruits colonizing ants inside (black arrow). Note in one of the seed the aril is completely consumed by the ants (red arrow) (Bar = 1.5cm); D & E. Showing the viable and nonviable seeds tested by tetrazolium chloride (Bar = 0.2cm); F. Germinating seed inside the seed germinator at 28-300 C (Bar = 0.6cm).

Fig. 3–Aquilaria malaccensis. A. An enlarged view of part flower with thrip on the anthers (Bar = 0.1cm); B. An enlarged view of flower with a beetle (brown) on top of the anthers. Bar = 0.2cm); C. An inflorescence and a beetle (black) on one of the flowers (Bar = 1cm); D. SEM of a thrip showing the pollen grains adhered on the lateral side of its abdomen. E. Brown beetle with pollen grains near its mouth parts; F. A black beetle with pollen grains on its middle leg part and proboscis (red and Fig. 5–Aquilaria malaccensis – A. Epigeal seed germination. Note two black circles); G. An enlarged view of the red circle in (F) with pollen cotyledons are raised above the ground (Bar = 2.5cm); B. Seedlings grains on the proboscis; H. An Enlarged view of circle in (F) with pollen maintained in the net house (Bar = 2.0 cm); C. Seed germination in the grains on their leg parts. natural condition (Bar = 4cm). Table 1—Aquilaria malaccensis – Percentage of insect visitation/hour, foraging time per visit and peak period for the pollinators Pollinators(insects) Percentage of insect’s Foraging time per visit Peak period for the pollinators visitation per hour (%) Hymenoptera(Honey Bees) 41.66 ± 0.92 10-15 (sec.) 10:30 amto12:30 pm Thysanoptera(Thrips) 25.00 ± 0.92 02-08 (min.) Coleoptera(Beetles) 16.66 ± 2.01 02-03 (min.) Values are mean ± standard deviation

Table 2—Aquilaria malaccensis - Percentage of fruit set per inflorescence through manual pollination and open pollination Breeding No. of trees No. of flowers bagged Fruit set/in florescence Percentage of fruit experiments used set/inflorescence

Autogamy 10 50 Nil Nil Geitonogamy 10 50 Nil Nil Xenogamy 10 50 20-25 (22.50 ± 3.5) 45 Apomixis 10 50 Nil Nil Open pollination 10 50 39-40 (39.50 ± 0.7) 79 Values in the parenthesis are mean ± standard deviation. One way analysis of variance (ANOVA) shows significant difference at P = 0.05 2017 Phenology, pollination mechanism, breeding system, seed dispersal and germination ...... 133

Table 3— Aquilaria malaccensis – Percentage of seeds viability, germination (seed germinator & net house) and seed moisture content. No. of weeks Percentage of Percentage of seed Percentage of seed Percentage of seed seed viability (%) germination in seed germination in net moisture content (%) germinator (%) house (%)

1st week 87.00-92.00 80.00-85.00 76.00-82.00 70.30-71.26 (89.75 ± 2.50) (81.66 ± 2.99) (79 ± 3.08) (70.78 ± 0.64) 2nd week 75.00-80.00 70.00-75.00 65.00-68.00 55.00-60.00 (78.50 ± 2.63) (73.33 ± 2.99) (67 ± 1.79) (57.50 ± 3.55) 3rd week 50.00-55.00 45.00-48.00 34.00-40.00 40.50-47.78 (53.75 ± 2.89) (46.66 ± 1.53) (37.66 ± 3.24) (44.14 ± 5.13) 4th week 32.00-35.00 25.00-28.00 04.00-05.00 25.30-29.39 (31.75 ± 2.55) (26.25 ± 1.53) (03 ± 2.62) (27.34 ± 2.80) 5th week 7.00-10.00 09.00-12.00 (08.25 ± 0.50) Nil Nil (10.50 ± 2.10) Values in the parenthesis are mean ± standard deviation. One way analysis of variance (ANOVA) shows significant difference at P = 0.05 for percentage of seed viability, germination & moisture content.

Pollination— The pollinators of A. malaccensis belong fertilization does not take place the development of aril is to three insect orders namely order Hymenoptera (honey bees arrested and area remains unchanged, and after some days its - 41.66 ± 0.92%), Thysanoptera (thrips – 25.00 ± 0.92%) and cells begin to degrade and die together with the ovule. An Coleoptera (beetles – 16.66± 2.01%) (Figs.2F, G & 3 A-H). increase in the size of aril involves a functional change in its Maximum visitation of pollinators was recorded between cells, from meristematic to storage. 10.30 am to 12.30 pm. and the time spent by a single insect on Fruit-set and seed-set— It has been observed that the the flower was varied from 10 seconds to 15 minutes per visit number of flowers in a single inflorescence varies from 10-13 (Table 1). Beetles and thrips enter first, followed by bees at but the number of fruits formed varies from 4-5. This shows anthesis. that most of the flowers in an inflorescence will not lead to the Breeding system — The pollen to ovule ratio is 19,200:1 development of fruits. Most of the flowers get infected by and the breeding system is obligate xenogamy. Open larvae of Aquilaria moth which are usually found inside pollinated flowers produced maximum number of fruits and unopened as well as opened flowers, and these larvae eat up seeds than those of artificially cross-pollinated ones. Manual the anthers and the gynoecium reducing the fruit set. cross pollination gives 45% fruit set while open pollination Fruit and seed dispersal— Fruits of A. malaccensis gives 79%. Flowers bagged to check for autogamous, remain hanging until their maturation, the pericarp of the geitonogamous and apomixis gives a negative result i.e. fruits matured fruit splits open naturally and the seeds are dispersed are not formed in these experiments, because flowers withered to 4-5 metres away from the mother tree with the help of the and fall off after three-four days of bagging (Table 2). mechanical force exerted during splitting. Seeds are light Fruit and seed morphology — The fruits are capsules, weight varying from 0.06 to 0.11g and are frequently green, oval in shape, bi-valved consisting of one seed per locus dispersed by wind. When the spilt fruit fall down on the forest in A. Malaccensis. Exocarp is leathery with fine white hairs. floor, ants begin to colonize it and within one or two weeks Although there are two ovules in each ovary, unsuccessful they carry these seeds to their nest. Ants are often seen to carry fertilization of one of the ovule leads to the formation of a the seeds to some distances aiding their dispersal. The entire single seeded fruit (Fig.4B). Even then majority are 2 seeded, aril is consumed by caterpillars and ants leaving the seed. For out of 100 fruits examined, 79% of the fruits have two seeded ants, arils are edible enticement and serve as bait for and 21% are one seeded. myrmecochorous dispersal (Fig. 4C). The pericarp of the fruit splits open at the time of Viability, germination and moisture content of dehiscence and remains hanging upside down by a thread like seeds— Seeds of A. malaccensis are recalcitrant and are not structure or the vasculature which is usually intact with the able to withstand dehydration. Viable seeds stained red but persistent and fleshly funicle at the base of the seed. This non-viable seeds did not when soaked in tetrazolium solution appendage is called the true aril because it develops from the (Figs. 4D, E). The viability of seeds of A. malaccensis funicular outgrowths (Fig. 4A). Seeds are white when young decreases with the increase in the number of days from but turn brown on maturity. They are connoid and covered 89.75% ± 2.50 in the first week to 08.00% ± 0.50 in the fifth with short and stiff brown hairs. The development of the aril is week. In A. malaccensis seed germination is epigeal and stimulated by fertilization and completed with the started to germinate 5-7 days inside the seed germinator (Fig. development of embryo and endosperm. In A. malaccensis, if 4F). The viability of seeds is observed up to 30 days after that 134 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.129-138

there is no germination (Table 3). The percentage of petals of A. malaccensis which are shorter than the stamens, germination also decreases with storage time from 81.66 ± makes easier for the insects to enter into the flowers but once 2.99% in the first week to 26.25± 1.53% in the fourth week in the insects entered inside the flower, the insects will not be the seed germinator and from 76.00% ± 3.08 during the first able to move out easily since the petals are filiform and week to 03.00% ±2.62 during the fourth week in the net house incurved, there is greater chances to get entangle with these respectively. The moisture content of the seeds declined filiform petals. Therefore, filiform petals may provide a drastically with storage time from 70.78% ± 0.64 during the suitable platform for the pollinators to pollinate the flowers. first week to 10.50 %± 2.10 during the fifth week. The In A. malaccensis, the stigma is dry and according to percentage of seed viability, germination and moisture content Heslop-Harrison and Shivanna (1977), dry stigma is classified are given in (Table 3). into two categories: the first group includes those stigmas with Seedling survival rate— In the net house of the the plumose form where there are discontinuities along the Department of Botany, NEHU, Shillong, the survival of receptive surface and the second groups includes those seedling varies from 11-13% in the first year and gradually stigmas where the receptive surface is concentrated into reduces to 08% in the fourth year (Figs.5A, B). While only 08- distinct ridges, zones or heads. The stigma of A. malaccensis 10% seedlings are survive in the natural habitat. The reduced belongs to the second group in which the stigma has a limited seedling survival is due to anthropogenic activities and surface secretion and the receptive cells are concentrated in Aquilaria caterpillars (Fig. 5C). distinct ridges and as a result, pollen grains landing on these DISCUSSION AND CONCLUSION ridges will be able to bring about pollen tube germination and subsequently fertilization. A. malaccensis is an evergreen tree and the phenological Heslop-Harrison and Shivanna (1977) also stated that the patterns show variations in the different regions of the world as well as north east India and appear to flower and fruit structural and physiological features of pollen and stigmatic sporadically at different times of the year (Gunn et al. 2004, surfaces differ considerably between families and sometimes Saikia and Khan 2012a, Soehartono and Newton 2001). In the within families and these characteristics are often related to present study as well as Beniwal (1989) found that in A. the operation of the breeding system malaccensis flowering started in March to June. These In A. malaccensis, the percentage of pollen viability was differences in the flowering and fruiting season in the different found to be 88.32±2.40% and the percentage of pollen parts of the world, particularly southeast Asian countries, is germination in vitro was found to be 78.00±3.56% with the largely due to the effect of climatic condition. Borchert et al. average pollen tube length of 595µm ± 7.90 maintaining a (2004) stated that the variation in flowering time is induced by a temperature at 280C. Pollen viability and pollen tube growth variety of factors like precipitation, photoperiod or drought and are important characteristics that are responsible for the has resulted in a number of flowering patterns in tropical trees. quality and quantity of seed production, regeneration of forest Most of the trees remain leafless during the month of stands and survival of certain populations of forest trees December, January and February as the leaves are being eaten (Hedhly et al. 2005, Varela et al. 2008). up by caterpillars. Soon after receiving the first rainfall, the The pollination mechanism in most members of the caterpillars began to leave the trees following with the Thymelaeaceae family is entomophilous (Whitehead et al. initiation of new leaves and flower buds. Therefore, the attack 1987). In A. malaccensis the mode of pollination is of these caterpillars acts as a triggering mechanism for the entomophilous and all pollinators are pollen carriers and their formation of new leaves and buds. Precipitation together with inter plant movement facilitates cross pollination. A. temperature changes associated with it seems the best malaccensis is self-incompatible and depends completely on candidate to stimulate the initiation of leaf and bud insects for the production of seeds. development leading to the sequence of flowering. A similar Honey bees, thrips and beetles belonging to three orders view was also expressed by Kramer and Kolzoeski (1979) that viz. Hymenoptera, Thysanoptera and Coleoptera respectively temperature was a significant factor for bud break following are the only pollinators recorded in A. malaccensis which are reactivation and subsequent shoot growth. very less when compared to A. crassna with a total of 103 Flowering in A. malaccensis lasted for 10-15 days and the insect species belonging to four orders. Climatic conditions longevity of a flower determines the time to be visited by which include temperature, humidity, rainfall, light intensity pollinators. Floral longevity is also an important floral trait that influences the reproduction of plant (Primack 1985, etc. has affected the flowering behaviour of A. malaccensis Ashman and Schoen 1994, Steinacher and Wagner 2010). and also the foraging activity of the pollinators. However, The petals in A. malaccensis are small and filiform, a Tasen et al. (2009) reported that the diversity and abundance similar condition is reported in Epiranthes and Oncidium of of pollinators depends on several factors, including the family Orchidaceae (Pandey and Misra 2009). The small geographic and climatic conditions. 2017 Phenology, pollination mechanism, breeding system, seed dispersal and germination ...... 135

A. malaccensis is protogynous and this nature of the plant induce a divergent fruit production among the plants. Low promotes out crossing and prevents selfing. Protogyny is fruit and seed set were also reported in A. crassna (Guitian commonly associated with wind, beetle, fly and thrips- 1993, Sutherland 1986, Stephenson 1981) and in Dirca pollination but generally not bee-pollination (Endress 2010). mexicana of the family Thymelaeaceae (Graves 2008). Breakdown of protogyny was observed in A. malaccensis as In A. malaccensis, ovule and seed abortion was also bees also play a role in pollination mechanism a similar view observed in which a single seeded fruits were also encountered was observed by Borg-Karlson (1996) in some species of instead of double seeded fruits. However, production of a Aquilaria. The same phenomena has also been reported in chemical of indole nature, such as 4-chloroindoleacetic acid in Uvaria concave and Unonopsis guatterioides that the bees were Dalbergia sissoo (Mohan Raju et al. 1996), and in Syzigium observed in collecting both stigmatic exudates and pollen grains cumini (Krishnamurthy et al. 1997) has lead to the production inspite of the protogynous nature of these plants (Carvalho and of a single seed and the abortion of the other seed. But this Webber 2000, Silberbauer and Gottsberger et al. 2003). aspect has not been investigated in A. malaccensis. In A. malaccensis, there is a change in the colour of petals Venugopal and Marbaniang (2015) reported that the aril from pale to yellowish green when matured. Beetles and in the matured seed of A. malaccensis has abundant starch, thrips are the insects to enter the flower at the initial stage of carbohydrates, lipids and total proteins as reserve foods. anthesis when the petals of A. malaccensis are pale colour. It Hence, arils are edible enticement, encouraging transport by has been suggested that petals of beetle and thrips pollinated animals and thereby assisting in seed dispersal. Like in the species are generally pale-coloured, often greenish or whitish case of A. malaccensis (Venugopal and Marbaniang 2015), in (Saunders 2010, Kirk 1998) and Kirk (1998) also stated that Cytisus multiflorus and C. striatus (Rodri-Guez et al. 2006) small to medium-sized beetles are sometimes co-pollinators the arils are eaten by ants but in Myristicaceae, arils are eaten along with thrips. by birds (Endress 1973) and human (Uphof 1959). When the petals of A. malaccensis turn yellowish, bees The fruits and seeds of A. malaccensis are dispersed only are then seem to visit and pollinate the flowers. The yellowish to a few meters away from the mother tree. Seed dispersal is petals act both as a guiding and landing pad for the bees, considered to be an important process to determine the spatial attracting them to the flowers. The change in colour of petals and dynamic structure of plant population and is influenced by and foraging by a variety of insects indicate the possibility of a number of factors including the height of the tree, entomophily in Crateva adansonii (Mangla and Tandon dispersibility of the seeds, activity of the dispersal agent, etc. 2011). Even though the foraging time of the bee is lesser when (Harper 1977, Nathan and Muller-Landau 2000). Seeds compared to thrips and beetles, but the percentage of visitation dispersed only to a few metres distance would consist of per hour was higher, indicating that the visitation of bees is genetically related neighbouring individuals and as a result of more and as a result bees may be considered to play a relatively which, homozygosity of the population would further major role in the pollination of A. malaccensis. increases as in Butea monosperma (Tandon et al. 2003). The breeding system according to Cruden (1977) as well In A. malaccensis wind and insects (ants) are the major as bagging experiment indicates that the breeding system in A. dispersal agents which can also known as myrmecochorous malaccensis is obligate xenogamous, the same breeding dispersal (Venugopal and Marbaniang 2015). In this type of system reported in most of the Aquilaria species (Sohartono dispersal the seed will be dispersed first by wind and after the and Newton 2000). Thus, the chances of pollination can be seed fall on to the ground, the ants will be the next dispersal improved by pollinators and could be an alternative way to agent which carry the seed to some distances and help in the increase fruit production. However, an investigation of tropical mechanism of dispersal. The hanging posture of the seed of A. tree breeding systems has revealed that obligate out crossing is malaccensis plays a significant role in wind dispersal the main mode of reproduction in tropical trees (Bawa 1974). mechanism. Apart from wind and ants, wasps (Manohara Flowers of A. malaccensis left for open pollination produced 2013) are also the major agents for the dispersal of seeds of A. maximum number of fruits and seeds whereas those artificially malaccensis. cross-pollinated give minimum number of fruits, a similar Seeds of A. malaccensis are recalcitrant and are not able situation has been reported in A. crassna (Tasen et al. 2009). to withstand dehydration. The recalcitrant nature of seeds of Most of the flowers withered and fall down after manual Aquilaria species which germinate rapidly within 3 weeks pollination and as such great increase of fruit set would not be after sowing is also reported by Beniwal (1989) and expected if manual pollination is preferred. Soehartono and Newton (2000). The seed moisture content, Comparing to the number of flower per inflorescence in the viability and germination percentage declined drastically A. malaccensis, the rate of fruit setting was very low, a with storage time. Therefore, the seeds should be sown soon phenomenon usually observed in hermaphroditic plants after harvesting for better and higher percentage of exhibiting self-incompatibility. The large number of fallen germination. Seeds of tropical trees and some temperate flowers due to larvae of caterpillars recorded in A. species are recalcitrant and cannot be dried without losing malaccensis during anthesis might be interpreted as a viability, and cannot be stored for longer period of time reduction of the functionality of female organs, and frequently (Roberts 1975). 136 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.129-138

In A. Malaccensis, the moisture content of the seed is Berjak P and Pammenter NW 2004. Recalcitrant seeds. In: correlated with viability and as the moisture content of the Benech-Arnold RL and Sanchez RA (eds.) Handbook of seed decreases, viability also decreases and once the moisture Seed Physiology: Applications to Agriculture: Haworth content dries off, the endosperm began to shrink and Press, New York. Pp. 305-345. eventually both the embryo and endosperm lose their water Berlyn GP and Miksche JP 1976. Botanical Micro-technique content and becomes non viable. This finding holds true for and Cytochemistry. Iowa State University Press, Iowa, recalcitrant species where even slight water loss brings about USA. death of embryonic cells (Berjak and Pammenter 2004). This Bernardello G, Gregory J, Lopez PS, Maryke A, Cleland phenomenon is accompanied by the dehydration of the seeds Stuessy TF and Crawford DJ 1999. Reproductive biology as in the case of Quercus robur, Castanea sativa, Aesculus of Lactoris fernandeziana (Lactoridaceae). Am. J. Bot. hippocastanum (Finch-Savage et al. 1994) and tropical 86 829-840. species Shorea robusta (Chaitanya and Naithani 1994) and as a result of which the seeds failed to germinate. Borchert R, Meyer SA, Felger RS and Porter-Bolland L Insects especially caterpillars are also a major problem for 2004. Environmental control of flowering periodicity in the survival of the seedlings in the natural condition. These Costa Rican and Mexican tropical dry forests Global Eco. caterpillars mostly attack the seedling and sometimes Biogeo. 13 409-425. completely defoliate and kill the seedlings, a common problem Borg-Karlson AK, Unelius CR, Valterová I and Nilsson LA also encountered by Saikia and Khan (2012a, b) in A. 1996. Floral fragrance chemistry in the early flowering malaccensis growing in Assam and Arunachal Pradesh of Daphne mezereum. Phytochemistry 41 1477-1483. North-East India and this has affected the growth and survival of Brewbaker JL and Kwack BH 1963. The essential role of seedlings. In the net house although no insects were found to calcium ion in pollen germination and pollen tube attack the seedlings but climate and type of soil has play a growth. Am. J. Bot. 50 859-865. crucial role for the low percentage of the seedlings to survive. Hau and Corlett (2003) reported that, seasonal drought, below Carvalho R and Webber AC 2000. Biologia floral de ground competition and low soil nutrients significantly affect Unonopsis guatterioides (ADC) RE Fr uma Annonaceae the growth of the seedlings in Schima superb, Castanopsis fissa, polinizada por Euglossini. Brazilian J. Bot. 23 421-425. Schefflera heptaphylla and Sapium discolor in Hong Kong. Chaitanya KSK and Naithani SC 1994. Role of superoxide, Acknowledgements—We acknowledge the financial lipid peroxidation and superoxide dismutase in support of the Ministry of Environment and Forest (MoEF) membrane perturbation during loss of viability in seeds of project No. 22/3/2010-RE, New Delhi, Govt. of India. Shorea robusta Gaertn. f. New Phytologist 126 623-627. Thanks are due to the Department of Botany, North Eastern CITES 2005. Convention on International Trade in Hill University, Shillong for providing facilities. We also Endangered Species of Wild Fauna and Flora. thank DFO and staff of Mawsynram forest division for their Appendices I, II and III of CITES. UNEP 48. help in locating the plant and allowing us to collect the plant for the study. Cruden RW 1977. Pollen-Ovule Ratios: a conservative indicator of breeding system in flowering plants. REFERENCES Evolution 31 32-46. Aluri RJS 1990. Studies on pollination in India: A review. Dafni A 1992. Pollination ecology: a practical approach. Proc. Acad. Nat. Sci. 56 375-388. Oxford University Press, New York. Ashman TL and Schoen DJ 1994. How long should flowers Dashek WV 2000. Methods in plant electron microscopy and live? Nature 371 788-791. cytochemistry. Humana Press, New Jersey, USA. Barden A, Anak NA, Mulliken T and Song M 2000. Heart of Dey S, Basu BTS, Roy B and Dey D 1989. A new rapid method of air drying for scanning electron microscopy the Matter: Agarwood use and trade and Convention on using tetra methyl silane. J. Microscopy 156 259-261. International Trade in Endangered Species of wild flora and fauna (CITES) Implementation for Aquilaria Endress PK 1973. Arils and aril-like structures in woody malaccensis. ranales. New Phytologist 72 1159-1171. Bawa KS 1974. Breeding systems of tree species of a lowland Endress PK 2010. The evolution of floral biology in basal tropical community. Evolution 28 85-95. angiosperms. Philo. Tran. Royal Soc. Biol. Sci. 365 411-421. Beniwal BS 1989. Silvical characteristics of Aquilaria Finch-Savage WE, Pramanik SK and Bewley JD 1994. The agallocha Roxb. Indian Forester 115 17-21 expression of dehydrin proteins in desiccation- 2017 Phenology, pollination mechanism, breeding system, seed dispersal and germination ...... 137

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