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 (), 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 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., in seed setting and their dispersal; identify initiatives obovata and Asimina pygmaea4, Asimina parviflora5, required for conservation through human intervention crassipetala6 and in paleotropical species, but also to understand their influence in the process of viz., 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 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 pollen grains attached on the head, mouth parts and body surface of the pollinators. In vitro germination of pollen grains (PG-test) Pollen grains from flower buds (fixed in FAA- A water solution with various sucrose solution formalin:alcohol:acetic acid 90:5:5) were dissected concentrations (0, 5, 10, 15 and 25%) and 50 ppm out, mounted either with glycerin or water, sealed H3BO4 served as the test medium. A meagre quantity with DPX mount, and observed under the Optika of pollen grains was incubated for 16 h under dark at Trinocular Microscope (Inverted) with EPI room temperature (20-24°C). After incubation, it was Fluorescence XDS 2, Italy. Pollen grains were smeared on a microscopic slide, removed excessive acetolyzed, directly attached by adhesive carbon tabs moisture using filter paper, observed under microscope and calculated the percentage of in stubs, coated with gold ion sputter (JEOL) at 25 36 to 30 nm thickness for two min and exposed to germination . take Scanning Electron Microscope (SEM model: In vivo germination of pollen grains (PG-test) S-3000H/Hitachi, Japan) photographs at an After pollination, the pistils were preserved in FAA accelerating voltage of 20 kV, with magnifications of immediately, fixed for 24 h, transferred to 70, 50, 30 33 3000X, 5000X and 10000X . and 10 alcohol solutions and 2 mol/L NaOH solution

Pollen viability for 12 h, washed twice in distilled water, then stained The following methods were used to estimate with 0.05% aniline blue solution for 12 h, placed on pollen viability: Carmine acetic acid (CAA-test): the clean glass slides for observation under the Fluorescent Microscope and captured photographs. Pollen samples were stained with one drop of 4% CAA on a microscopic slide. After 1 or 2 min, viable Pollen:ovule ratio and proportion of fruit setting pollen grains stained with red were observed and Pollen/ovule ratio was determined and randomly, recorded. flowers were collected during full bloom (70%) and fruit-setting period of June and July in the early TTC (2,3,5-triphenyl tetrazolium chloride) test morning before anthesis, preserved in FAA, Pollen grains were incubated for 2-6 h under the hydrolyzed androecium from each flower in cover of darkness at 37°C in a solution of 0.2-1.0% hydrochloric acid (HCl) to make one mL of pollen TTC in 1/15 mol phosphate buffer (pH 7.2). grain suspension, observed a sample of 5 µL of each Aniline blue (AB-test) pollen grain suspension under Optika Trinocular Pollen grains were incubated with 0.2 mL of Microscope, counted the number of pollen grains 0.001 mg/mL aniline blue with 0.1 M K2HPO4 pH10 using haemocytometer and calibrated the mean buffer for 5 min. Then stained pollen grains were number of pollen grains per flower. Young pistils observed under the microscope. were dissected out from flowers and observed

MTT (2,5-diphenyl tetrazolium bromide) test under Motic Stereo-Zoom Microscope to determine Pollen grains were fixed with the test solution the number of ovules per flower and the mean made up of a 1% concentration of the substrate 2,5- number of seeds per flower produced by natural diphenyltetrazolium bromide (MTT or thiazolyl blue) pollination. VISWANATHAN et al.: REPRODUCTIVE BIOLOGY OF MONOON TIRUNELVELIENSE (ANNONACEAE) IN INDIA 519

Results and carpels (Fig. 2E-L). This enclosure increases the Intensive and in-depth field investigation of the chamber temperature more than the ambient entire KMTR made possible to record 171 individuals temperature but outer row apertures certainly (23 seedlings, 55 saplings, and 93 mature individuals) dissipate chamber temperature. Their presence was by Garmin GPS in seven populations in the northern often noticed during the pistillate and staminate part of KMTR between 8°46′–8°49′N and 77°15′– phases (Fig. 5). Pseudococcus species and mealy bugs 77°16′E (Fig. 1) from 898 and 1504 m in the southern (Fig. 6A) were present on peduncle, sepals, petals, Tropical wet evergreen forests. Area of occupancy and fruits, right from bud to fruit. Psylla species, (AOO) was calculated with respect to location with jumping plant lice (Fig. 6B), Aphis species – aphids number of individuals in parentheses, points, length, (Fig. 6 C-E) were present on the outer and inner side and area of each population: Nondimangadu-1 (28), of the sepals and petals, and on stamens and carpels. 2 30, 1.44840 km, 0.091559 km ; Nondimangadu-2 (50), 30, 0.643737 km, 0.014861 km2; Nondimangadu-3 (22), 33, 0.482803 km, 0.008027 km2; Nondimangadu-4 (23), 19, 0.110337 km, 0.000906 km2; Nondimangadu-5 (12), 17, 0.09387 km, 0.000586 km2; Thalamalai boundary-1 (16), 38, 1.28747 km, 0.060318 km2, and Thalamalai boundary-2 (20), 30, 0.80467 km, 0.027444 km2. Red List Category of Critically Endangered of the International Union for Conservation of Nature and Natural Resources37 was assigned on the basis of B1: extent of occurrence (EOO), 1.123757 km2 (<100 km2); B2: total area of occupancy (TAOO), 0.374937 km2 (<10 km2); and C: small population size and decline, 93 mature individuals (<250).

Light intensity Light intensity measurements were recorded as 65, 60, 55, 48, 45, 40 and 25% for Thalamalai boundary-1, Nondimangadu-3, Thalamalai boundary-2, Nondi- mangadu-1, Nondimangadu-2, Nondimangadu-4 and

Nondimangadu-5, respectively during the flowering and fruiting periods. Fig. 2 — Developmental stages from bud to flower in Monoon tirunelveliense. (A) A pair of green floral buds emerges from Phenology, pollinators and visitors trunk; (B) Slightly upright arcuate greenish pale yellow floral bud Flowering occurs throughout the year (Figs. 2-4). at leaf axil; (C) Upright greenish yellow floral bud starts open Flowers were recorded every month from 35 selected from closed condition; (D) Opened immature flower at leaf axil individuals, calculated average and mentioned here. with recurved outward sepals; (E) Opened flower showing Flowers were bisexual and their numbers/tree were greenish pale yellow petals, stamens and carpels, outer 3 petals highly variable every month viz. 28 in February, 47 in start for outward folding, inner 3 petals ready to form a chamber; (F) Greenish yellow petals, outer ones folded outward, inner ones March, a slump of 32 in April, 42 in May, surged to folded outward and starting to geniculate; (G) Greenish yellow 70 in June, peaked to 90 in July and again a slump of petals, folded and fully spread petals, inner outward folded 28 in August (Fig. 4). Duration from bud to flowers is slightly geniculate petals, greenish yellow stamens and carpels; 18 days. Peduncles are accrescent, erect to slightly (H) Yellow petals, outer outward folded petals, inner outward folded and geniculate petals; (I) Yellow petals and inner arcuate towards maturity. Sepals are recurved geniculate petals; (J) Yellow petals, outer outward folded petals, 3 outwards (Fig. 2). Petals are green to yellow through lateral apertures, inner outward folded with geniculation; (K) yellowish green (Fig. 2). Outer 3 petals are folded Yellow petals, 3 outward folded petals, 3 lateral apertures, 3 outwards, slightly geniculate, and leave apertures to geniculate inner petals, pollination chamber; and (L) Pistillate the pollination chamber to facilitate smooth entry of phase: dark yellow folded petals, with pollination chamber, 3 lateral apertures. [Scale bars: (A) = 0.5 cm; (B) = 1 cm; (C) = 1.5 pollinators (Fig. 2E-L). Whereas inner 3 petals are cm; (D and E) = 2 cm; (F and G) = 4 cm; (H and I) = 5 cm; (J, K geniculate, form a chamber, and enclose both stamens and L) = 5.5 cm] 520 INDIAN J EXP BIOL, JULY 2019

Fig. 4 — Phenology of Monoon tirunelveliense.

Fig. 5 — Stamens and carpels towards maturity. (A) Greenish yellow stamens and carpels; (B) Pistillate phase: stigmatic exudates and formation of compitum; (C) Yellowish green indehiscent anthers; and (D) Staminate phase: dehiscing anthers and released pollen grains. [Magnification: (A-D) = 15X]

staminate and pistillate phases and were mainly active from 08.00-10.00 a.m. The aphids entered and left the floral chamber during functionally active phases of

the flower, visited several flowers, fed on greenish

Fig. 3 — Fertilization to fruit in Monoon tirunelveliense. yellow or yellow petals, left black markings on the (A) Downward arcuate flowers after fertilization, sepals partially petals and released honeydew from the terminations withered, petals fully withered; (B) Yellowish immature of their alimentary canals. tomentose fruits without sepals and petals; (C) Yellow fruits with Black ants (Azteca species; Fig. 6A) visited the accrescent fruit stipe; (D) Green fruits with appressed tomentose hairy; (E) Enlarged stipes and fruits with disappearing tomentose flowers more actively from 08.00 a.m.-12.00 noon, hairs; (F) Ellipsoid matured fruits on ground with accrescent stipe; built their nests with periderm fragments in between and. (G) Fruits on trunk at different stages of growth. [Scale bars: folds on the dorsal surface of petals, fed the (A) = 0.5 cm; (B) = 0.7 cm; (C) = 1 cm; (D) = 1.5 cm; (E) = 10 honeydew of the mealy bugs (Fig. 6A), jumping plant cm; (F) = 2.5 cm; (G) = 20 cm] lice (Fig. 6B), and aphids (Fig. 6 C-E) showing Pollination chamber served as shelter for parasitic mutual relationship (symbiosis), decreased in number aphids which are present in larger groups often towards sunset and finally no ant visited after sunset. observed in copulation, although neither eggs nor Often, Halyzia species, lady beetle, could be seen. larvae were present in pollination chamber during Ants (Fig. 6A), mealy bugs (Fig. 6A), jumping plant VISWANATHAN et al.: REPRODUCTIVE BIOLOGY OF MONOON TIRUNELVELIENSE (ANNONACEAE) IN INDIA 521

indicate dead tissues resulted out of the feeding by beetles and weevils which were seen in pairs or larger groups and often in copulation that concomitantly pollinated flowers and left behind black markings of dead stamens and carpels.

But weevils remained in the floral chamber for most of the time and mated. Pollen grains were more abundant on the surface of the weevils than aphids. Body parts of the weevils and black ants bore hairs that attach pollen grains on to the body surface during the course of dusting while feeding. The pendent orientation of the flowers facilitates application of gravity which ultimately assists in attaching pollen grains on to the body surface of the pollinators with much ease. Pollen connecting threads may increase the efficiency of the pollination by attaching more pollen grains on to the pollinator during a single visit. The SEM studies revealed the presence of pollen grains on the body surface, mainly on hairs, legs and mouth parts of the insects (Fig. 6B, G and H). At the end of the pistillate phase, compitum (sticky mass of stigmas; Fig. 5) fell off collectively from the flower as th dried black exudate. Staminate phase started on 18

Fig. 6 — Pollinators. (A) Black ants, Azteca species, feeding on day at 05.30 p.m. and lasted up to 09.30 p.m. Anthers honeydew of mealy bugs, Pseudococcus species; (B) SEM of dehisce longitudinally (Fig. 5 C and D). Ratios of body surface of Psylla species, jumping plant lice, insert showing different parameters were 1,65,000 + 100 pollen an individual; (C and D) Aphis species – aphids on sepals, petals, stamens and carpels; (E) An individual Aphis species; (F) Dorsal grains/flower, 750 + 60 pollen grains/anther, 25 + 12 view of sap-feeding beetle, Carpophilus plagiatipennis; (G) SEM ovules/flower and 6,600:1 of pollen/ovule ratio. of mouth parts of Carpophilus plagiatipennis; (H) Pollen grains Pollen grains on the body surface of ants, mealy bugs, on the hairs of Carpophilus plagiatipennis, arrow-marked. (I) A jumping plant lice (Fig. 6B), and aphids were part of longhorn beetle, Cerambycid species; (J) Weevil — Endaeus species; (K) Beetle and weevil-fed black markings on recorded. During that phase, beetles and weevil feed petals, stamens and carpels; and (L) Dead black tissues in stigmatic exudates, adaxial side of the petals at the immature fruits due to feeding. [Scale bars: (A, C, F and I) = 4 base and pollen which probably contained starch, mm; (B and G) = 500 µm; (D) = 5 mm; (E) = 2 mm; (H) = 200 lipids, mucilages and tannins. Black markings on the µm; (I) = 500 µm; (J) = 8 mm; (K) = 1.5 mm; (L) = 5 cm] petals indicate the feeding by beetles and weevils. lice (Fig. 6B), aphids (Fig. 6C-E), and lady beetle Cockroaches visit the flowers at all times. Centipedes, could be seen till the end of 2nd week of flowering. Scolopendra species, which fed on them, were present Flowers became fully pendent before the start of on the inner side of the floral chamber. At the end of pistillate phase which started on 17th day at 05.30 p.m. the staminate phase, petals and sepals fell off the and lasted up to 12.00 noon on 18th day. During that flowers. Remnants and markings were conspicuous at phase, petals turned into deep yellow, emitted the early stages. When fertilized carpels developed into fragrance that attracted sap-feeding beetle, fruits, tender ones were tomentose in nature (Fig. 3). Carpophilus plagiatipennis (Coleoptera: Nitidulidae; When they mature, the fruits become glabrous with Fig. 6F), and longhorn beetle, Cerambycid species accrescent stipe. Ripe carpels/fruits are 6.48-7.67 g (Coleoptera: Cerambycidae; Fig. 6I) and Weevil when fresh and 5.25-6.40 g when dry (Fig. 3F). Endaeus species (Coleoptera: Curculionidae; Fig. 6J). Moisture content of the ripe carpels/fruits ranged from Beetles fed on stigmatic exudates (Fig. 5B). Four to 4.60-23.50%. Seed coat thickness was 3-5 mm. five beetles could be seen at a time inside the floral SEM structure of pollen chamber that fed and mated. Often, the petals and The pollen grains (Fig. 7) were loosely fruits bear black markings (Fig. 6 K and L) that arranged in tetrads, later found dispersed in 522 INDIAN J EXP BIOL, JULY 2019

Fig. 7 — SEM of pollen. (A) Loosely arranged tetrads; (B) Boat- shaped pollen grains; (C) Distant equatorial view of a pollen Fig. 8 — Pollen viability analysis. (A) Unstained pollen grains; grain; (D) Closer equatorial view of a pollen grain; (E) Closer (B) CAA; (C) TTC; (D) AB; (E) MTT; (F) FDA; (G) In vitro view of germination zone; and (F) Rugulate surface of exine. pollen tube germination; and (H) In vivo germination of pollen [Scale bars: (A) = 100 µm; (B) = 40 µm; (C) = 30 µm; (D) = 20 tubes. [Magnifications: (A-G) = 6000X; (H) = 1500X; (A-E and µm; (E) = 10 µm; (F) = 5 µm] G) Brightfield; (F, H) Epifluorescence]

Table 1—Pollen viability test (%) of Monoon tirunelveliense estimated by different staining techniques Pollen viability test at different staining techniques Population AB TTC FDA CAA MTT Means for a No. Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Populations Pop. 1 34.8b±1.03 60.3b±0.40 62.2c±1.27 70.2c±1.44 72.0cd±1.15 59.90 AB Pop. 2 36.2b±0.69 62.6b±1.50 74.6b±2.65 72.5c±0.98 75.3bc±0.40 64.24 AB Pop. 3 50.0a±1.15 71.1a±0.51 61.7c±1.90 79.3b±1.61 78.1b±0.51 68.04 A Pop. 4 32.7bc±1.55 58.0b±1.15 58.5c±2.59 60.2d±2.02 69.5d±2.02 55.78 AB Pop. 5 30.3c±1.32 42.0c±1.27 54.2d±2.42 55.5e±1.67 54.3e±1.55 47.26 B Pop. 6 52.2a±1.27 74.0a±2.59 80.6a±0.80 86.1a±0.92 85.4a±0.92 75.66 A Pop. 7 49.4a±0.34 72.9a±2.94 78.2ab±1.27 78.4b±1.03 79.5b±1.44 71.68 A Means 40.80B 62.98A 67.14A 71.74A 73.44A [aMeans followed by the same letter are not significantly different (P = 0.05), AB, aniline blue; CAA, carmino acetic acid; FDA, fluoresceine diacetate; MTT, 2,5-diphenyl tetrazolium bromide; SD, standard deviation; TTC, 2,3,5-triphenyl tetrazolium chloride] monads, granular/boat-shaped, c. 45 × 50 µm, with respectively (Table 1; Fig. 8). The highest percentage rugulate surface bearing one furrow and one of pollen germination was 73.10 recorded at 20% germination zone and monosulcate aperture. Exine sucrose solution for in vitro PG-test (Table 2). Pollen reduction was towards the sulcus, lack of grains which bear remarkable endoaperture-shed endexine, otherwise uniform. Pollen connecting exine before pollen tube germination. threads bore short exinal connections which link and form tetrads. Discussion

Pollen viability Light intensity Pollen viability varied such as 40.80, 62.98, 67.14, Light intensity is one of the most important factors 71.74 and 73.44 to AB, TTC, FDA, CAA, and MTT, in floral development, pollinator activity and seedling VISWANATHAN et al.: REPRODUCTIVE BIOLOGY OF MONOON TIRUNELVELIENSE (ANNONACEAE) IN INDIA 523

Table 2—Effect of in vitro pollen tube growth (%) of Monoon tirunelveliense in different sucrose concentrations In vitro PG - test at different sucrose concentrations Population 0% 5% 10% 15% 20% 25% Means for No. Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Populationsa Pop. 1 2.4ab±0.55 24.1c±0.63 43.3c±1.90 52.5b±1.46 73.1c±1.80 65.4b±0.82 43.46C Pop. 2 2.0bc±0.90 21.3d±0.75 40.4c±0.81 50.8b±1.66 68.3cd±2.49 49.2d±2.44 38.60C Pop. 3 3.5a±0.57 32.4b±1.38 63.6a±0.96 75.7a±2.15 82.2b±1.28 71.4a±1.39 54.80A Pop. 4 1.8b±0.57 18.6de±0.34 34.3d±2.09 43.6c±2.10 63.6d±1.52 43.7e±2.15 34.26C Pop. 5 1.2c±1.21 16.8e±1.03 31.0d±4.60 35.5d±2.03 54.4e±2.55 36.6f±0.36 29.25C Pop. 6 3.4a±0.63 38.4a±1.38 66.5a±1.99 76.9a±1.69 89.6a±3.27 61.6bc±0.96 56.06A Pop. 7 2.6ab±0.75 24.3c±2.13 48.3b±0.96 72.8a±1.84 80.7b±1.57 59.4c±0.31 48.01B Means 2.41D 25.12C 46.77B 58.25B 73.10A 55.32B [aMeans followed by the same letter are not significantly different (P = 0.05). PG, pollen germination; SD, standard deviation] growth. It is evident from the fact that tree branches anthesis (Nitidulidae, Curculionidae, Chrysomelidae) wherever exposed to sunlight recorded more flowers and the third type of beetles are nocturnally active, and fruits. large and voracious dynastid scarab beetles which

Phenology feed on large floral chambers enclosed by thick petals 16 Most of the species in Polyalthia are that emit strong odors . Beetles which were attracted protogynous7,10,17. In Monoon tirunelveliense also, by the odour of the petals and stigmatic exudates, flowers are protogynous and are present throughout entered the floral chamber during the pistillate phase, the year. Greyish brown petals at bud condition turn used it as a mating site, fed on stigmatic exudates and into greyish green, pale green and pale greenish petals during the pistillate phase and pollen during the yellow when mature in Polyalthia littoralis9, staminate phase. In order to protect from feeding by Polyalthia coffeoides and Polyalthia korinti11,12. In voracious beetles, the stamens possess peltate Monoon tirunelveliense also, flowers turned into connective shields which were also present in yellow when mature through yellowish green from Monoon tirunelveliense also (Fig. 5C and D). green at bud condition (Fig. 2). Compitum was detached at the end of the pistillate

Pollinators and visitors phase in Monoon tirunelveliense (Fig. 3A). Beetles and weevil feed petals of Polyalthia species in the Insect pollination of trees appears to be more 16,17 common in the tropics, particularly the humid tropics, Neotropics . than in the temperate regions where wind pollination Beetle and weevil feeding were reported in Polyalthia species from Lambir hills, Sarawak and is ineffective. Even among the insects, beetles are 17 attracted by amount of food available in the floral Sumatra; Xylopia aromatica from Brazil ; and chamber, thickness of the petal tissue and dimension Polyalthia coffeoides and Polyalthia korinti from 11,12 of the flowers increasing with size, number and Sri Lanka . In Monoon tirunelveliense, black voraciousness. Because of that, Annonaceae flowers markings of the petals are indicators of beetle and are pollinated predominantly by beetles weevil feeding (Fig. 6K). Jumping plant lice (Fig. 6B) (Coleoptera)16,17. But other insects which effect and beetles (Fig. 6H) had pollen grains on their pollination either by one group or more than one surface in Monoon tirunelveliense. Floral evolution, group include weevils (Coleoptera)16,17, thrips pest feeding of pollen and petals and pollination shifts (Thysanoptera)9,16-19, flies (Diptera)5,20,21, and rarely by simplification are reported in Monanthotaxis 38 bees (Hymenoptera)17,22,25 and cockroaches (Annonaceae) . Similar observation was recorded (Dictyoptera)8, in Annonaceae. Interestingly in here in Monoon tirunelveliense (Fig. 6 K and L). Monoon tirunelveliense, flowers are predominantly Flowers pollinated by small beetles open fully pollinated by beetles and weevils. But other pollinators between the staminate and pistillate phases, e.g., such as ants (Fig. 6A), mealy bugs (Fig. 6A), jumping Polyalthia species11 and Xylopia species39. Partially plant lice (Fig. 6B), aphids (Fig. 6 C-E), lady beetle enclosed pollination chamber in beetle-pollinated and centipedes were also recorded. These pollinators flowers of Annonaceae was reported40,41. Here, in were recorded possibly for first time in the flowers of Monoon tirunelveliense also, 3 outer slightly Annonaceae. geniculate petals provide access to pollinators through Two types of beetles feed relatively small floral their three lateral apertures to the pollination chamber chambers which exhibit diurnal and/or nocturnal (Fig. 2 D-I). Small- to large-beetle pollination, overall 524 INDIAN J EXP BIOL, JULY 2019

increased size of flowers, and thick fleshy petals as termites which decay the fruits of Monoon food source are an evolutionary change in tirunelveliense relatively faster. Therefore, the matured Annonaceae40,41. Small and large beetles pollinate fruits should be collected and sown in humus-free soil flowers of Monoon tirunelveliense because of its or humus should be cleared before the fruits fall off large-sized flowers and food source of thick fleshy from branches on the ground in its habitats. Higher petals. Majority of the beetle-pollinated flowers in amount of germination and establishment of seedlings Annonaceae never open their internal petals during in humus-free soil in natural habitats is indicative of staminate phase and form a pollination chamber16. In this fact. This simple intervention by the KMTR Forest M. tirunelveliense, the petals become dark yellow, department may ensure better survival of this tree and inner petals geniculate more than the outer ones, thereby increase its population. contagious with each other, form a pollination chamber and leave three apertures at the base of the Conflict of Interest three outer petals (Fig. 2). Beetles and weevil which The authors declare that there is no conflict of feed stigmatic exudates, adaxial side of the petals at interest regarding the publication of this paper. the base and pollen probably contain starch, lipids, mucilages and tannins. Pollination by large beetles Acknowledgement (cantharophily) is considered as special condition The authors thank the Department of acquired most probably late in evolution in Biotechnology, Government of India, New Delhi, for 17 Annonaceae . Cockroach pollination has been fund assistance vide Sanction Order ref. BT/PR- 8 reported previously only in Uvaria elmeri . 9368/BCE/08/563/2007 dt.25.06.2008. The authors

Pollen structure also thank the Chief Wildlife Warden, Principal Chief Tetrads and loose pollen grains (Fig. 7) are common Conservator of Forests, Chennai, and Chief in Annonaceae. On the basis of pollen character, Conservator of Forests and Field Director, Project phylogenetic relationship of c. 40 genera was prepared42 Tiger, Tirunelveli, for necessary permission to carry . Rugulate exine bearing pollen in west Malesian out research in the KMTR. Pseuduvaria species is reported already21. Similar type of pollen was observed in the present study (Fig. 7F). References 1 Gottsberger G, Evolutionary steps in the reproductive Exine reduction is towards the sulcus, otherwise biology of Annonaceae. Rev Bras Frutic, 36 (2014) 32. uniform. Aperture is monosulcate and lack of endexine 2 Guo X, Thomas DC & Saunders RM, Organ homologies and indicates its primitive form among the angiosperms. The perianth evolution in the Dasymaschalon Alliance presence of short exinal connections (Fig. 7B) linking (Annonaceae): inner petal loss and its functional pollen grains in tetrads enhanced pollination efficiency consequences. Front Plant Sci, 9 (2018) 174. 3 Kress WJ & Beach JH, reproductive in this species. Pollen connecting threads help to transfer systems. In: Ecology and Natural History of a Neotropical large aggregates of pollen grains and facilitate Rain Forest, (Ed. Mcdade LA, Bawa KS, Hespenheide HA entanglement on hairs and legs of the pollinators43. The & Hartshorn GS; The University of Chicago Press, London), SEM studies showed pollen grains on the body surface, 1994, 161. 4 Norman EM & Clayton D, Reproductive biology of two hairs, legs, mouth parts and head of the beetles and Florida pawpaws: Asimina obovata and A. pygmaea jumping plant lice (Fig. 6 B, G and H). (Annonaceae). Bull Torrey Bot Club, 113 (1986) 16.

Pollen viability 5 Norman EM, Rice K & Cochran S, Reproductive biology of Asimina parviflora (Annonaceae). Bull Torrey Bot Club, 119 The positive results of staining reactions to all the (1992) 1. samples reveal high variability (Table 1; Fig. 8). 6 Armstrong JE & Marsh D, Floral herbivory, floral Enzyme assays make it possible to detect the actual phenology, visitation rate, and fruit set in Anaxagorea viability and fertility of pollen samples35. The highest crassipetala (Annonaceae), a lowland rain forest tree of Costa Rica. J Torrey Bot Soc, 124 (1997) 228. pollen germinations of 73.44% in MTT assay (Table 1; 7 Okada H, Reproductive biology of Polyalthia littoralis Fig. 8E) and 73.10% in 20% sucrose solution (Table 2) (Annonaceae). Plant Syst Evol, 170 (1990) 237. were recorded in the present study. 8 Nagamitsu T & Inoue T, Cockroach pollination and breeding system of Uvaria elmeri (Annonaceae) in a lowland mixed Conclusion dipterocarp forest in Sarawak. Am J Bot, 84 (1997) 208. 9 Momose K, Nagamitsu T & Inoue T, Thrips cross-pollination Humus on forest floor in the southern tropical wet of Popowia pisocarpa (Annonaceae) in a lowland evergreen forests is enriched with microbes and dipterocarp forest in Sarawak. Biotropica, 30 (1998) 444. VISWANATHAN et al.: REPRODUCTIVE BIOLOGY OF MONOON TIRUNELVELIENSE (ANNONACEAE) IN INDIA 525

10 Tsukada M, Higuchi H, Furukawa T & Yoshida A, Flower biology, pollinator trapping and plant breeding system. Sci visitors to cherimoya, Annona cherimola (: Rep, 6 (2016) 14. Annonaceae) in Japan. Appl Entomol Zool, 40 (2005) 317. 27 Xicohténcatl-Lara L, Figueroa-Castro DM, Andrés- 11 Ratnayake RMCS, Gunatilleke IAUN, Wijesundra DSA & Hernández AR & Campos-Villanueva Á, Aspects of the Saunders RMK, Reproductive biology of two sympatric reproductive biology of Stenanona flagelliflora species of Polyalthia (Annonaceae) in Sri Lanka. I. (Annonaceae). Pak J Bot, 48 (2016) 211. Pollination by Curculionid beetles. Int J Plant Sci, 167 28 Gottsberger G, Silberbauer-Gottsberger I, Webber AC & (2006a) 483. Dötterl S, Populations of Unonopsis guatterioides 12 Ratnayake RMCS, Su YCF, Gunatilleke IAUN, Wijesundara DSA (Annonaceae) in Amazonas and Minas Gerais, Brazil, & Saunders RMK, Reproductive biology of two sympatric potentially represent different species: Floral scent, flower species of Polyalthia (Annonaceae) in Sri Lanka. II. characters and pollinators revisited. Biochem Syst Ecol, 78 Breeding systems and population genetic structure. Int J (2018) 17. Plant Sci, 167 (2006b) 495. 29 Gottsberger G, Beetle pollination and flowering rhythm of 13 Irvine AK & Armstrong JE, Beetle pollination in tropical Annona spp. (Annonaceae) in Brazil. Plant Syst Evol, 167 forests in Australia. In: Reproductive Ecology of Tropical (1989) 165. Forest Plants, (Ed. KS Bawa & M Hadley M; UNESCO, 30 Gottsberger G, Comments on flower evolution and beetle Paris) 1990, 135. pollination in the genera Annona and Rollinia (Annonaceae). 14 Lamoureux CH, Phenology and floral biology of Monodora Plant Syst Evol, 167 (1989) 189. myristica (Annonaceae) in Bogor, Indonesia. Ann Bogor, 6 31 Viswanathan MB & Manikandan U, Polyalthia (1975) 1. tirunelveliensis (Annonaceae), a new species from Peninsular 15 Willson MF & Schemske DW, Pollinator limitation, fruit India. Kew Bull, 56 (2001) 217. production and floral display in Pawpaw (). 32 Xue B, Su YCF, Thomas DC & Saunders RMK, Pruning the Bull Torrey Bot Club, 107 (1980) 401. polypheletic genus Polyalthia (Annonaceae) and resurrecting 16 Gottsberger G, Pollination and evolution in neotropical the genus Monoon. Taxon, 61 (2012) 1021. Annonaceae. Plant Spec Biol, 14 (1999) 143. 33 Erdtman G, The acetolysis method, a revised description. 17 Silberbauer-Gottsberger I, Gottsberger G & Webber AC, Svensk Bot Tidskr, 54 (1960) 561. Morphological and functional flower characteristics of New 34 Khatum S & Flowers T J, The estimation of pollen viability and Old World Annonaceae with respect to their mode of in rice. J Exp Bot, 46 (1995) 151. pollination. Taxon, 52 (2003) 701. 35 Kison HU & Franke R, La détermination de la viabilité du 18 Webber AC & Gottsberger G, Floral biology and pollination pollen. Arch Zuchtungs-forch, Berlin, 10 (1980) 171. of Bocageopsis multiflora and Oxandra euneura in central 36 Shivanna KR & Rangaswamy NS, Pollen biology: A Amazonia, with remarks on the evolution of stamens in laboratory manual. Springer-Verlag, Berlin (1992) 119. Annonaceae. Feddes Repert, 106 (1995) 515. 37 IUCN Standards and Petitions Subcommittee, Guidelines for 19 Kuchmeister H, Webber AC, Silberbauer-Gottsberger I & Using the IUCN Red List Categories and Criteria. Version Gottsberger G, A polinizacao e sua relacao com a 9.0. Prepared by the Standards and Petitions Subcommittee termogênese em especies de Arecaceae e Annonaceae da (2011). Downloadable from http://www.iucnredlist.org/ Amazônia Central. Acta Amaz, 28 (1998) 217. documents/ Red List Guide lines.pdf. Accessed 23 20 Gottsberger G, Pollination and dispersal in the Annonaceae. November 2012. Annonaceae Newsletter, 1 (1985) 6. 38 Hoekstra PH, Wieringa JJ, Smets E & Chatrou LW, Floral 21 Su YCF, Mols JB, Takeuchi W, Kebler PJA & Saunders RMK, evolution by simplification in Monanthotaxis (Annonaceae) Reassessing the generic status of Petalolophus (Annonaceae): and hypotheses for pollination system shifts. Sci Rep, 8 evidence for the evolution of a distinct sapromyophilous lineage (2018) 12. within Pseuduvaria. Syst Bot 30 (2005) 494. 39 Ratnayake RMCS, Gunatilleke IAUN, Wijesundara DSA & 22 Olesen JM, Flower mining by moth larvae vs. pollination by Saunders RMK, Pollination ecology and breeding system of beetles and bees in the cauliflorous palanga Xylopia championii (Annonaceae) curculionid beetle (Annonaceae) in Costa Rica. Flora, 187 (1992) 9. pollination, promoted by floral scents and elevated floral 23 Carvalho R & Webber AC, Biologia floral de Unonopsis temperatures. Int J Plant Sci, 168 (2007) 1255. guatterioides (A.D.C.) R.E. Fr., uma Annonaceae polinizada 40 Saunders RMK, Floral evolution in the Annonaceae: por Euglossini. Rev Bras Bot, 23 (2000) 421. hypotheses of homeotic mutations and functional 24 Teichert H, Pollination and floral biology in five species of convergence. Biol Rev, 85 (2010) 571. the family Annonaceae in French Guiana. Phyton, 46 41 Saunders RMK, The diversity and evolution of pollination (2007) 219. systems in Annonaceae. Bot J Linn Soc, 169 (2012) 222. 25 Teichert H, Dotterl S, Zimma B, Ayasse M & Gottsberger G, 42 Doyle JA & Le Thomas A, Cladistic analysis and Perfume-collecting male euglossine bees as pollinators of a pollen evolution in Annonaceae. Acta Bot Gallica, 141 basal angiosperm: The case of Unonopsis stipitata (1994) 149. (Annonaceae). Plant Biol, 11 (2009) 29. 43 Hesse M, Halbritter H & Weber M, Beschorneria 26 Lau JYY, Pang CC, Ramsden L & Saunders RMK, yuccoides and Asimina triloba (L.) Dun: Examples for Reproductive resource partitioning in two sympatric proximal polar germinating pollen in Angiosperms. Grana, Goniothalamus species (Annonaceae) from Borneo: Floral 48 (2009) 151.