Floral Morpho-Anatomy and the Pollination Mechanism of Atalantia Monophylla DC

T REPRO N DU LA C The International Journal of Plant Reproductive Biology 10(1) Jan., 2018, pp.90-95 P T I F V O E

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S H Floral morpho-anatomy and the pollination mechanism of T Atalantia monophylla DC. S. Krishna Veni and P. Selva Singh Richard* Department of Botany, Madras Christian College, Tambaram, Chennai-600059, India *e-mail: [email protected] Received: 28.09.2017; Revised: 13.12.2017; Accepted and Published online: 01.01.2018 ABSTRACT Morphological and anatomical characters of Atalantia monophylla DC. (Rutaceae) flowers were studied using both light microscopic and scanning electron microscopic observations in order to understand its pollination mechanism. The study revealed that the flowers of A. monophylla are usually fragrant and nectariferous. High P/O ratio indicate that the outbreeding nature of this species. The olfaction and rewarding nature of the flower attracts a wide range of floral visitors particularly bees, butterflies and diurnal moths. Insect's behavior on the blossom showed that Apis dorsata and A. indica were the potential pollinators over butterflies and other floral visitors. Keywords: Atalantia monophylla, floral characters, pollination mechanisms, floral visitors

The genus Atalantia (Rutaceae) is closely similar to and the behaviour were also photographically recorded in Citrus; the members are usually spinous small to medium- order to understand its pollination mechanism. sized trees with white, fragrant flowers and small globose Floral morphology—Fresh flowers and buds samples (n=15 greenish-yellow fruits (Ranade et al. 2009). There are 11 each) were fixed and stored in 2ml sample vials containing closely associated species of the genus Atalantia commonly Formalin Acetic Acid reagent (Formalin 5ml + Glacial Acetic distributed in most of the countries of Oriental region, of acid 5 ml + 70% Ethanol 90 ml) for floral morphometric which A. monophylla, A. racemosa, A. simplicifolia and A. studies. 10 measurements were made on each floral part wightii are occurring in Indian subcontinent (Swingle and (sepals, petals, stamens and pistils) using both stereoscopic Reece 1967, Ranade et al. 2009). A. monophylla is one of the light microscope and Scanning Electron Microscope (SEM). dry evergreen tree species of tropical dry evergreen forests Scanning Electron Microscopy—Fresh floral samples were (TDEF), confined to the east coast region of southern India dehydrated by passing through acetone series, and were (Blasco and Legris 1973, Venkateswaran and Parthasarathy sputter-coated with gold for SEM studies. The gold coated 2003, Parthasarathy et al. 2008). In recent years, this species specimens were mounted and the floral morphological gaining importance due to its application in traditional characters were observed using FEI QUANTA 450 FEG medicine for treating various ailments (Sankaranarayanan et Scanning Electron Microscope. al. 2010); its phytochemicals and volatile aromatic oil with Localisation of osmophores—The presence of osmophores significant larvicidal activity against mosquito larvae (Baskar on the floral parts was determined by immersing fresh flowers et al. 2009) and its antiviral property (Chansakaow et al. in Neutral Red stain (Effmert et al. 2005). 1996). However, there are no studies on the floral biology or Floral anatomy—Open flowers and buds were fixed in FAA pollination ecology as well as the breeding system of this reagent for a period of 24 h and then dehydrated with graded species was ever undertaken. Therefore, the present series of Tertiary-Butyl Alcohol (TBA). Wax infiltration of the investigation was carried out to focus on the floral biology of specimens was carried out by gradual addition of pure paraffin Atalantia monophylla DC. one of the important taxon of wax with a melting point of about 58 – 60oC. Later, the tropical dry evergreen forest. The study was conducted using specimens were casted into paraffin blocks using paper boats. light and scanning electron microscopic observations in order The specimens were sectioned both longitudinally and to understand the pollination requirements of this species. transversely at 8 or 9 or 10 µm thickness using a rotary microtome followed by a standard dewaxing protocol MATERIALS AND METHODS (Johansen 1940; Sass, 1940). The sections were stained in This study was carried out in an area of tropical dry toluidine blue O´ (O´ Brien et al. 1964) and the mounted on evergreen forest around Tambaram, Chennai from September 1mm thick microslides using DPX. to December, 2016. Field observations on the blossoms of A. Pollen morphology—Pollen grains of A. monophylla were monophylla were made during this peak flowering season and subjected to standard acetolysis procedure for permanent the floral characters such as colour, scent, symmetry, pollen slide preparation (Erdtman 1960). The acetolysed architecture and shape were noted. In addition, preliminary pollen grains were mounted using Glycerine jelly (Kisser observations on the floral visitors particularly their diversity 1935) and sealed with paraffin wax. Pollen measurements 2018 Floral morpho-anatomy and the pollination mechanism of Atalantia monophylla DC. 91

(n=50) were taken in order to record the pollen size, shape and Piedade and Ranga 1993, Cervancia and Manila 2000, Lopes wall thickness. Pollen characters like pollen wall 2002, Chauhan 2015, El Ottra et al. 2016). The flower with a ornamentation and aperture type were also recorded. tubular androecium formed by monadelphous stamens can Pollen–ovule ratio—Pollen production and its ratio between acts as a floral tube and hence stops nectar robbers (Faegri and ovule was analyzed by following the methods of Subba and van der Pijl 1966); their accumulation at the depth also aids in Reddi (1986). Pollen count was carried out using binocular reducing nectar evapouration (Endress 1994). research microscope under 40x and 10x magnifications, Pollen Morphology—The anthers of A. monophylla dehisce respectively. The mean pollen count of the five flower buds longitudinally along the line of dehiscence and the pollen were considered to be the actual pollen count. Similarly, grains were usually released in monads. Size of the pollen number of ovule/ovary was also counted from five flowers and grains ranges from 31 – 40 (35.5) µm in polar axis (P) and 16 – finally, the pollen ovule ratio was computed (Cruden 1977). 25 (21.9) µm in equatorial axis (E) with 1.62 P/E ratio (prolate shape). Each pollen grain had 4 colporate (compound – colpus RESULTS AND DISCUSSION and Os) apertures. Colpus (boat-shaped) was linear with The flowering in A. monophylla has commenced from 2nd granulate colpal membrane, acute apex and costate. Os or 3rd week of October and continued till 2nd week of (circular/ oval) was lalongate with acute apex. The pollen wall December. Young fruits started appearing in 3rd week of (exine) was 2 µm thick with micro-reticulate or micro- December. The mature fruits of A. monophylla were green, perforates exine ornamentation (Fig. 2 B). Outer sexine and small-globose berry, predominantly consumed by fruit bats inner nexine wall layers were almost equal in thickness with during night. columellate infratectum. In Psydrax dicoccos, Subha and Floral morphology—Flowers were dull-coloured, usually Richard (2016) reported the accumulation of lipid bodies in borne on axillary raceme or panicle inflorescence (Fig. 1 A). inter-columellar space of the pollen wall promoting the Dull-coloured blossoms were observed in other members of stickiness of pollen. According to Fenster et al. (2004) pollen Rutaceae such as Aegle marmelos (Singhal et al. 2011), aperture types and pollen wall ornamentations are the Feronia limonia (Chauhan 2015) and in Conchocarpus important pollen characteristics features for determining the macrophyllus (El Ottra et al. 2016). Flowers were trimerous or pollination syndrome. Colporate aperture type and micro- pentamerous, hermaphrodite, actinomorphic and measured perforate or micro-reticulate pollen wall ornamentation was about7-10 mm x 5 - 6 mm. Calyx was light green, fused, 2.5 – 3 observed to be associated with butterfly pollination (Perveen mm in length, terminated with 3 – 5 lobes and producing weak 1993, Richard 2012). pleasant scent. Glandular trichomes were usually present on Gynoecium of A. monophylla composed of a superior the outer surface of the sepals. Corolla was white or greenish- ovary, stout style and a capitate stigma with many papillae- white composed of 3 – 5 free petals. Petals were 8 – 10 mm in like structures (Fig. 2 C & D). Nectar was the major floral length, usually glabrous with profuse oil glands and recurved reward of A. monophylla secreted from an annular nectar disc, in mature flowers. Neutral Red stained flowers of A. situated exactly at the base of the ovary (Fig. 1 D). This monophylla indicate the presence of osmophores on the petals concentric nectar disc (ND) was about 30µm thick and 60µm (particularly on the adaxial side) and the stigma. When in height, consisting of 5 hemispherical masses. The anatomy compared to stigma, petals were deeply stained due to the of ND showed single epidermal layer of rectangular cells and a copious amount of osmophores. Therefore, the fragrance of homogenous and parenchymatous Ground Tissue (GT) (Figs. the flower was mostly from its petals. Rbeiro et al. (2016) 1 B, C, D & E). Similarly, the flowers of Citrus sinensis also reported that the osmosphores on the flowers of Citrus had a concentric nectar disc of about 6.5mm in diameter sinensis var. Pera Rio were responsible for the release of (Ribeiro et al. 2016). The nectar exuded from the gynoecial fragrance. According to Esau (2006), the volatile aromatic nectary was the most important reward which the flower can compounds were one of the important factors for attracting give (Kulloli et al. 2011). pollinators. Protandry had been noticed in A. monophylla, Floral anatomy—The anatomical investigation of A. flowers with 6 – 8 fertile stamens bloom between 5.30 am and monophylla flower showed that sepals with single-layered 11.00 am. Pomball and Morellato (2000); Souza et al. (2004) epidermis on both adaxial and abaxial sides. The adaxial noted protrandrous condition and usual self-incompatiblity in surface epidermal cells were short, thick and slightly projected Metrodorea nigra, another member of Rutaceae.The above the surface, whereas the abaxial surface epidermal cells filaments of A. monophylla were fused together were short with swollen Epidermal Trichomes (ETr) (Fig.1C). (monadelphous) to form a narrow staminal tube. Anthers were These epidermal trichomes were non-glandular, measured dithecous, each 7 – 9 mm in length, cordate-shaped, sub- about 50µm in length and 30µm in thickness. The ground basifixed on the terminal lobes of the staminal tube. Flowers tissue was parenchymatous with a single vascular trace with similar short staminal tube were also recorded in several forming a midrib. The CS of petal showed a thick midrib other members of the family Rutaceae (Amstrong 1979, (200µm) and gradually tapering lateral wings (15µm). The 92 The International Journal of Plant Reproductive Biology 10(1) Jan. 2018, pp.90-95

Fig. 2. Atalantia monophylla, A. C.S. of young ovary; B. L.S. of ovule Fig. 1- Atalantia monophylla, A. An axillary inflorescence; B. Toluidine (LM); C. L.S. of stigma; D. Stigma (SEM); E. Pollen grains (SEM) (SC - Blue O stained L.S. flower bud (LM); C. C.S. sepal showing Epidermal Secretory Cavity, MSP - Megasporogenous tissue, GT - Ground Trichome (ETr); D. C.S. petal showing Fibrillar Trichome (Ftr); E. Nectar Tissue, VS - Vascular strand, Ep - Epidermis, MP - Micropyle, OI - Disc (ND); F. Scanning Electron Micrograph of L.S. flower bud. (Sl - Outer Integument, II - Inner Integument, ES - Embryo Sac, St. sur. - sepal, Pl - petal, ND - Nectary Disc, ETr – Epidermal Trichome, Ftr – Stigmatic surface, St - Stigma, GT - Ground Tissue, Stl - Style, Po – FibrillarTrichome, Tr - Trichome, SC - Secretory Cavity, Ory - Ovary, Pollen). Ovl - Ovule, Cpl - Carpel, Sep - Septa, St – Stamen). cells in the adaxial epidermal layer were prominently globular, pyriform with dense inclusions whereas the abaxial epidermal cells were smooth, produces long cylindrical non-glandular Fibrillar Trichomes (FTr) (Fig. 1D). Ground tissue usually parenchymatous, enclosing a prominent midrib with single vascular trace and many small vascular strands along the lateral wings. In CS of mature anther, the disintegration of middle layer and tapetum as well as the line of dehiscence along the length of the anther were distinctly noticed. The CS of young ovary was circular in outline (2mm wide) with single-layered epidermis and parenchymatous ground tissue (GT). Numerous circular secretory cavities (SC) were distributed along the periphery of the GT. The central portion of the young ovary had four spindle-shaped elliptical cavities, possessing young primordial ovules with Megasporogenous tissue (MSP) (Fig. 2 A). As the ovary matures, there were 4 Fig. 3-Some floral visitors of Atalantia monophylla, A.Giant honey bee wide carpellary chambers, each with a single orthotropous (Apis dorsata), B.Yellow carpenter bee (Xylocopa aestuans), C. ovule on an axile placentation (Fig. 1B). Ovule was elliptical Greater Egg fly (Hypolominas bolina), D. Bevan's Swift (Borbo bevani) 2018 Floral morpho-anatomy and the pollination mechanism of Atalantia monophylla DC. 93

in outline, enclosing a central Embryo Sac (ES) which was observed that the medium-sized bees with short tongue visited covered by a continuous Inner Integument (II) and a only to C. macrophyllus flowers due to its short corolla tube discontinuous Outer Integument (OI), led to a micropyle (MP) when compared to A. bracteata where the corolla tube was (Fig. 2B). relatively long. In Citrus sinensis, bees such as Apis mellifera Pollen-Ovule ratio—The pollen production per anther of A. and Melipona scutellaris were the potential pollinators monophylla ranges from 1763 – 2244 (1849) and the total (Ribeiro et al. 2016). Singhal et al. (2011) reported that honey pollen production of a single flower was calculated to 14792 bee (A. dorsata) was the major and legitimate pollinator of pollen grains. The pollen ovule ratio (P/O) ratio was Aegle marmelos while the rest of the insects were either minor considerably high (3698:1). Similarly, Ribeiro et al. (2016) pollinator or mere visitors. The family Rutaceae shows a wide reported high P/O ratio (2832:1) in Citrus sinensis and Singhal variety of pollination mechanisms such as pollination in et al. (2011) in Aegle marmelos. The high P/O ratio determines Feronia limonia by honey bees, halicitid bees, yellow that A. monophylla as an obligatory outbreeding species. This butterflies and hoverflies (Chauhan 2015). Records on corresponds to the findings of Cruden (1977, 2000). pollination by bees, butterflies, moths, sometimes According to Kale et al. (2017) the cross-pollinated plant taxa hummingbirds were found in Angostura bracteata, usually have high pollen: ovule ratio. Conchocarpus macrophyllus and Erythrochiton brasiliensis Floral visitors—The present observation revealed that the (Lopes 2002, El Ottra et al. 2016). floral structure plays a significant role in attracting the Table 1. Floral characters of Atalantia monophylla DC. pollinators or visitors (Table 1). Short corolla, hidden nectary and closed staminal tube made the visitors to struggle in order Floral Characters access the floral reward. Therefore, limited group of insects Flower type : Hermaphrodite like bees, butterflies, moths and wasps were visiting the Flower symmetry : Actinomorphic blossoms of A. monophylla. Bee species such as Apis dorsata Flower shape : Tubular (Giant honey bee), Apis indica (Asian honey bee), Flower colour : Calyx green and Tetragonulair idipennis (Indian stingless bee) and Xylocopa Corolla greenish white aestuans (Yellow carpenter bee); butterflies such as Barbo Flower scent : Weak pleasant bevani (Bevan's swift), Hasora chromus (Common banded No. of sepals : 3 – 5 awl), (Hypolimnas bolina) Great egg fly, Phalanta phantha No. of petals : 3 – 5 (Common leopard), Junonia lemonias (Lemon pansy) and No. of anthers : 6 – 8 diurnal moth such as Cephonodes spp. (Pellucid hawk moth) No. of carpels : 4 were some of the common visitors of A. monophylla. However, the frequency of the bees is less when compared to Number of ovule/flower : 4 the butterflies due to the availability of nectar as floral reward. Number of pollen per anther : 1763 - 2244 (1849) Several studies highlight that the flowers with light greenish or Pollen production per flower : 14104 - 17952 (14792) white corolla and shorter staminal tube with concealed nectar Pollen ovule ratio : 3526:1 - 4488:1 (3698:1) were reported to attract visitors mostly having small proboscis or mouthparts reaching into the short, wide staminal tube; The floral biology of A. monophylla highlighting the visitors like butterflies (Armstrong 1979; Corbet 2000; floral morphology, floral anatomy, pollen ovule ratio, nectar Malleshappa and Richard 2011, Subha and Richard 2016), and also the floral visitors indicate its cross-pollinating nature. bees (Faegri and van der Pijil 1979), few day flying moths The hidden nectar source limits the group of visitors yet the (Wilmer 2011), also beetles and moths during night (Kevan less frequently visiting bee species were the potential 1983, Scogin 1983). pollinators. This species also served many insect communities as well as frugivores by providing essential floral rewards and It was observed that bees with short tongue had to come fruits in the tropical dry evergreen forest ecosystem. much closer to the blossom in order to access the nectar source Therefore, it is very imperative to conserve and protect this (Fig. 3 A& B). Due to this behaviour their entire body had to species by studying extensively on other propagation constantly contact with all the reproductive parts of the flower. methods. The butterflies with long proboscis reached the nectar resource without having much contact with the vital parts of Acknowledgements—We thank Dr. P. Jayaraman, Plant the flower (Fig. 3 C & D). This indicated that though the Anatomy Research Centre (PARC), Chennai for his valuable butterflies make frequent visit they can't be the effective suggestions in processing the floral sample for anatomical pollinator when compared to the bees. El Ottra et al. (2016) on studies; The Head, Department of Chemistry, Indian Institute comparing the two co-flowering Rutaceae members, of Technology Madras (IIT), Chennai for providing SEM Angostura bracteata and Conchocarpus macrophyllus, they facilities. 94 The International Journal of Plant Reproductive Biology 10(1) Jan. 2018, pp.90-95

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