Indian Journal of Experimental Biology Vol. 57, July 2019, pp. 516-525 Reproductive biology and pollinators of a steno-endemic and critically endangered tree, Monoon tirunelveliense (Annonaceae), from Kalakkad-Mundanthurai Tiger Reserve (KMTR), India MB Viswanathan*, C Rajasekar & P Sathish Kumar Centre for Research and Development of Siddha-Ayurveda Medicines (CRDSAM), Department of Botany, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India Received 06 June 2014; revised 27 June 2015 Reproductive biological studies on the endemic and threatened plants are vital to understand pollinators and their role in seed setting and their dispersal, and thereby identify appropriate initiatives for conservation. In this study, we investigated Monoon tirunelveliense (M.B. Viswan. & Manik.) B. Xue & R.M.K. Saunders (Annonaceae), a steno-endemic and critically endangered tree species from the Kalakkad-Mundanthurai Tiger Reserve of India for its phenology, pollen morphology and viability, pollinators and conditions required to increase individuals and populations. We used Global Positioning System mapping to collect required data. Recording of mere 171 individuals in 7 populations justify its inclusion in IUCN Red List Category of critically endangered. Though flowering occurs throughout the year, it is at peak in July. Flowers are protogynous and cantharophilous and bear 215+10 anthers/flower, 750+60 pollen grains/anther, 1,65,000+100 pollen grains/flower, 25+12 ovules/flower and 6,600:1 pollen/ovule. Predominant pollinators are beetles belonging to Carpophilus plagiatipennis and Cerambycid species. Other pollinators include species of Aphis, Azteca, Endaeus, Pseudococcus and Psylla. Species of Halyzia and Scolopendra have also been noticed. Pollinators left behind black markings after feeding. Pollen germination was high (73.44% to MTT assay; 73.10% at 20% sucrose concentration). While humus-free soil significantly promotes seed germination, it requires human intervention for successful conservation. Keywords: Aphids, Beetles, Black ants, Conservation, Mealy bugs, Humus-free soil, Weevils Reproductive biological studies provide better in Annonaceae. Reproductive biology of Annonaceae understanding of not only the pollinators and their role has been reported in neotropical species, viz., Asimina in seed setting and their dispersal; identify initiatives obovata and Asimina pygmaea4, Asimina parviflora5, required for conservation through human intervention Anaxagorea crassipetala6 and in paleotropical species, but also to understand their influence in the process of viz., Polyalthia littoralis7, Uvaria elmeri8, Popowia evolution1,2. Some investigators claimed that frequent pisocarpa9, Annona cherimola10 and Polyalthia occurrence of inbreeding caused by low population coffeoides and Polyalthia korinti11,12. density in tropical rain forests accelerates species Annonaceae has more specialized pollination diversification. Population density, phenology, sexual systems than the primitive families in Magnoliaceae systems, and physiological self-incompatibility and Eupomatiaceae13 as the flowers of Annonaceae are mechanisms determine and influence the breeding visited by a diverse array of insects which is considered patterns of plants3. Floral morphology and biology, to be a prototype of the beetle pollination. Such critical such as size of the flowers and floral chamber, amount studies, fundamental to understand evolutionary of pollen, corrugated inner side of petals having rich in mechanisms in Angiosperms, still remain obscure in carbohydrates and minute quantity of vitamins and Annonaceae14,15. Major pollinators in Annonaceae are lipids, colour, thickness and odour of petals and beetles16,17. Other insect guilds of Annonaceae include stigmatic secretions, pollination patterns and thrips9,16-19, flies5,20-21, bees17,20-25 and cockroaches8. quantitative studies of genetic variations are important Mode of pollination has been reported in new and old for understanding and characterizing breeding systems world Annonaceae26,27. Interactions among pollinator behaviour, pollen carryover, flowering phenology or —————— their effects on pollen dispersal and plant mating *Correspondence: 28 Phone: +91 431 2407061; 2407045; (Mob.): +91 94432 29677 systems in palaeotropical species were elucidated . E-mail: [email protected] Pollination of Annonaceae has been reported from VISWANATHAN et al.: REPRODUCTIVE BIOLOGY OF MONOON TIRUNELVELIENSE (ANNONACEAE) IN INDIA 517 Brazil, Asian tropics, and Lambir hills in Sarawak29,30. average of 38ºC. But in the forests, annual average A new species called Polyalthia tirunelveliensis was temperature ranges from 13.5°C in the southern published from the Kalakkad-Mundanthurai Tiger Tropical wet evergreen forests to 23°C in the Reserve (KMTR) in the Southern Western Ghats of Tirunelveli semi-evergreen and dry, moist or mixed Peninsular India31. It is a steno-endemic and critically deciduous forests. Rainfall varies from a minimum of endangered species. The genus Monoon with 56 900 mm in the plains to a maximum of 5,000 mm per species was resurrected wherein 37 species were year depending upon the altitude and forest type (Fig. 1). transferred from Polyalthia, including Polyalthia Study species tirunelveliensis, 18 species from Enicosanthum and one Monoon tirunelveliense is a lofty tree growing up to species of Woodiellantha32. 25 m high. Leaves are elliptic-lanceolate, oblong- Considering the pivotal role played by the elliptic or oblong-oblanceolate, margin undulate, dark reproductive biological studies in the conservation of greenish brown above. Flowers (Fig. 2) are bisexual, endemic and threatened species, here, we investigated in aggregated clusters on trunk at scars of the branches Monoon tirunelveliense (M.B. Viswan. & Manik.) B. and at leaf axils, 8-25 per cluster. Sepals are 3, green. Xue & R.M.K. Saunders, a steno-endemic and critically Petals are 6, arranged in 2 rows, 3 in each row, green to endangered tree from the Kalakkad-Mundanthurai yellow through greenish yellow, rarely 4, arranged in Tiger Reserve, southern Western Ghats, to increase the 2 + 2 manner. Stamens are 215 + 10, green to yellow individuals and populations of the species. through greenish yellow, arranged spirally. Carpels are 25 + 12, green to yellow through greenish yellow, Materials and Methods arranged spirally. Ripe carpels/fruits are in clusters Study area (Fig. 3), 12-25 per cluster, pale to dark green when The Kalakkad-Mundanthurai Tiger Reserve mature, fallen ones turn to dark black from dark brown, (KMTR) is geographically located at 8°25′–8°53′N ellipsoid, 6.4-7.6 × 1.8-3.0 cm. Seeds are creamish white, ellipsoid, 2.8-3.5 × 1.2-2.4 cm. and 77°10′–77°35′E in the Southern Western Ghats of Peninsular India and covers an area of 895 km². The Distribution of populations foothills up to 350 m MSL record 44ºC during March All the individuals of 7 populations were recorded to May, 24ºC during December and January and an with their respective latitude, longitude and altitude using Garmin eTrex Vista® HCx Global Positioning System (GPS), transferred the data to the Garmin Map Source, and calculated area of occupancy (AOO), extent of occurrence (EOO) and total area of occupancy (TAOO). Light intensity Light readings were recorded before sunrise between 0500 and 0530 h at ground level at the intersection of each location. During this period, no direct light entered the forest and shadow had no influence. Light reading in an open area was recorded for control. Standard value was obtained by comparing the open area and forest reading by measuring incidence of light on the canopy cover and the forest floor. Phenology Growth and formation of leaves, origin and development of inflorescences, anthesis in flowers, pollinators, feeding mechanism of predators, quantity of pollen output, stigmatic secretions, change in coloring pattern of petals, formation of black spots from the cut edges by the predation of beetles, weevils and lice, fruit set and maturity were recorded Fig. 1 — Distribution of seven populations in the KMTR. and analyzed for 35 individuals, 5 per population 518 INDIAN J EXP BIOL, JULY 2019 from 7 populations, every month from January 2009 with 5% sucrose. The pollen grain was considered to May 2012. viable if it turned into deep pink or colourless but showed irregular black lines over its surface34. Observation and identification of floral visitors and pollen grains Fluoresceine diacetate (FDA) test Pollinators were studied right from the bud phase Pollen grains were added with a 0.5 mol sucrose to fertilization phase as the flowers are protogynous solution and FDA (2 mg dissolved with 1 mL and cantharophilous-pollinated ones. The peak of acetone) solution35 and incubated for 30 min at room flowering season has helped to record visit duration temperature (28°C). After incubation, water drops and visit frequency in a particular flower. Activities of were added with the sample to dilute the solution floral visitors both inside and outside of the floral mixture. This process was repeated until the solution chambers and their arrival patterns were recorded at has become clear and colorless. Thereafter, the 1-2 h intervals in a day. Pollen grains of Monoon solution was decanted and removed excess water with tirunelveliense were photographed under Motic the help of filter paper. Stained and unstained pollen Stereo-Zoom Microscope, Italy, and compared with grains were observed under the microscope. the photographs of the