T REPRO N DU LA C 28 The International Journal of Reproductive Biology 10(2) July, 2018, pp.133-139 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.133-139 P T 2018 Phenology and Reproductive biology of splendens G. Don 33 I F V O E

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S H Phenology and Reproductive biology of Clerodendrum splendens G. Don T Rohitash Department of Botany, B.S.A. College, Mathura-281401, India *e-mail : [email protected] Received: 21.11. 2017; Revised: 14.03.2018; Accepted and Published online: 01.06.2018 ABSTRACT Phenology and floral biology of Clerodendrum splendens () commonly known as flaming glory bower and bleeding heart vine, was studied in different parts of Mathura city, India. This beautiful climber with bunches of crimson red flowers blooms from December to April, with optimum blooming in January and February. This is highly self-incompatible. In order to avoid self-pollination, the flowers exhibit several morphological modifications in their reproductive parts (stamens and pistils). Black ants are the only floral visitors. There is no fruit formation either by self or cross-pollination, which may partly be due to cultivation by cuttings of the same clone. Only parthenocarpic fruitsdevelop in the months of March-May. The failure of fruit and seed set in C. splendens is likely to be related to its self-incompatible nature, absence of the nectar reward and the effective pollinators (butterflies, honey bees and humming birds). Keywords : Lamiaceae, flaming glory, self-incompatible, floral contrivances, parthenocarpy.

Phenology is generally described as an art of observing bicarpellary and syncarpous. The fruits blue, berry-like and the life cycle or the activities of organisms occurring parthenocarpic. throughout the year (Leith 1973). Reproductive biology of some Clerodendrum species has Reproductive biology provides information on life forms, earlier been studied (Chauhan et al. 1986, Rohitash and Jain rate of flowering, type of breeding system, plant-pollinator 2010, McMullen 2011, Rojas-Sandoval and Acevedo- interaction, breeding system, fruit and seed output, seed Rodriguez 2012, Gautam and Rohitash 2012, Raju and Kumar germination, seedling establishment, overall fitness and 2016,Rohitash 2016, 2017). However, C. splendens has survival of the species. Studies on the biology of reproduction received little attention and there is some confusion about the in endangered and threatened species are rare and useful for formation of fruits and seeds. The present paper deals with understanding the cause of their threatened status (Lavergne et phenology and floral biology, as well as changes in the floral al. 2004). Information obtained from such studies is helpful in morphology in order to prevent self- pollination in C. the in situ and ex situ management strategies for their spendens growing at different sites of Mathura city. conservation and are integral feature of all conservation projects (Moza and Bhatnagar 2007). MATERIALS AND METHODS Clerodendrum is a of subfamily Ajugoideae of Present investigation was undertaken on ten plants Lamiaceae (Harley et al. 2004), being one of the most of Clerodendrum splendens growing at five different places important and largest genera in this family, distributed in Mathura city(27.28°N 77.41°E), Uttar Pradesh, India worldwide, with over 400 species according to Harley et al. (Table 1). (2004), while Yao-Wu et al. (2010) reported only 150 species. Clerodendrum is native to tropical and warm temperate Table 1-Study sites. regions of the world, with most species occurring in tropical S. No. Site in Mathura City Africa and southern Asia, but with a few in temperate zone in 1. B.S.A. College eastern Australia and a few extending north into the temperate zone in eastern Asia (Mabberley 2008). Three species of the 2. K.R. College Phenology and Reproductive biology of Clerodendrum splendens G. Don genus Clerodendrum (C. phlomidis, C. inerme and C. 3. Birla Mandir, Mathura-Vindraban, Road Rohitash splendens) are found to grow both in wild and cultivated form 4. Krishna Janam Bhumi Department of Botany, B.S.A. College, Mathura-281401, India in different parts of north India. Clerodendrum splendens G. e-mail: [email protected] 5. Dampier Nagar Don is commonly known as flaming glory bower or bleeding Received: 21.11. 2017; Revised: 14.03.2018

heart vine. This large, sprawling, evergreen, ornamental ABSTRACT climber of about 3-4 m in height is a native of western Africa, Phenology—Phenological events on vegetative and Phenology and floral biology of Clerodendrum splendens (Lamiaceae) commonly known as flaming glory bower and bleeding heart vine, was studied in different parts from Senegal to Angola. The leaves are oval, entire, reproductive parameters were recorded on the ten marked of Mathura city, India. This beautiful climber with bunches of crimson red flowers blooms from December to April, with optimum blooming in January and February. coriaceous and arranged in opposite pairs. Flowers arranged in plants for three years (2010-13).Habit of the plant, leaf fall and This species is highly self-incompatible. In order to avoid self-pollination, the flowers exhibit several morphological modifications in their reproductive parts (stamens several corymbose spike, pentamerous, zygomorphic, leaf renewal, flowering and fruiting periods were recorded and pistils). Black ants are the only floral visitors. There is no fruit formation either by self or cross-pollination, which may partly be due to cultivation by cuttings of the hypogynous, four epipetalous stamens, gynoecium throughout the study period. same clone. Only parthenocarpic fruitsdevelop in the months of March-May. The failure of fruit and seed set in C. splendens is likely to be related to its self-incompatible nature, absence of the nectar reward and the effective pollinators (butterflies, honey bees and humming birds). Keywords: Lamiaceae, flaming glory, self-incompatible, floral contrivances, parthenocarpy.

Phenology is generally described as an art of observing the life cycle or the activities of organisms occurring throughout the year (Leith 1973). Reproductive biology provides information on life forms, rate of flowering, type of breeding system, plant-pollinator interaction, breeding system, fruit and seed output, seed germination, seedling establishment, overall fitness and survival of the species. Studies on the biology of reproduction in endangered and threatened species are rare and useful for understanding the cause of their threatened status (Lavergne et al. 2004). Information obtained from such studies is helpful in the in situ and ex situ management strategies for their conservation and are integral feature of all conservation projects (Moza and Bhatnagar 2007). Clerodendrum is a genus of subfamily Ajugoideae of Lamiaceae (Harley et al. 2004), being one of the most important and largest genera in this family, distributed worldwide, with over 400 species according to Harley et al. (2004), while Yao-Wu et al. (2010) reported only 150 species. Clerodendrum is native to tropical and warm temperate regions of the world, with most species occurring in tropical Africa and southern Asia, but with a few in temperate zone in eastern Australia and a few extending north into the temperate zone in eastern Asia (Mabberley 2008). Three species of the genus Clerodendrum (C. phlomidis, C. inerme and C. splendens) are found to grow both in wild and cultivated form in different parts of north India. Clerodendrum splendens G. Don is commonly known as flaming glory bower or bleeding heart vine. This large, sprawling, evergreen, ornamental climber of about 3-4 m in height is a native of western Africa, from Senegal to Angola. The leaves are oval, entire, coriaceous and arranged in opposite pairs. Flowers arranged in several corymbose spike, pentamerous, zygomorphic, hypogynous, four epipetalous stamens, gynoecium bicarpellary and syncarpous. The fruits blue, berry-like and parthenocarpic. Reproductive biology of some Clerodendrum species has earlier been studied (Chauhan et al. 1986, Rohitash and Jain 2010, McMullen 2011, Rojas- Sandoval and Acevedo-Rodriguez 2012, Gautam and Rohitash 2012, Raju and Kumar 2016,Rohitash 2016, 2017). However, C. splendens has received little attention and there is some confusion about the formation of fruits and seeds. The present paper deals with phenology and floral biology, as well as changes in the floral morphology in order to prevent self- pollination in C. spendens plants growing at different sites of Mathura city. MATERIALS AND METHODS Present investigation was undertaken on ten plants of Clerodendrum splendens growing at five different places in Mathura city(27.28°N 77.41°E), Uttar Pradesh, India (Table 1). Table 1-Study sites. Legend to Figures S. No. Site in Mathura City Fig. 1A. Plant in full bloom; B. Inflorescence; C. A black ant on the flowers of a mature 1. B.S.A. College inflorescence (circle); D. Parthenocarpic fruits. 2. K.R. College Fig. 2-A & B. SEM pictures showing dorsifixed anther and curved filament; B. mature pollen grain; 3. Birla Mandir, Mathura-Vindraban, Road C & D. viable pollen grains; E Pollen germinating in vitro; F. Peltate scales and unicellular 4. Krishna Janam Bhumi trichomes on corolla surface; G. bifid stigma; H. magnified view of stigmatic surface with small 5. Dampier Nagar compactly arranged papillae (an: anther, ft: filament, pg: pollen grain; pt: pollen tube, stg: stigma, ut; unicellular trochome; v: viable). Fig. 3A-I Flowers showing contrivances. Phenology- Phenological events on vegetative and reproductive parameters were recorded on the ten marked plants for three years (2010-13).Habit of the plant, leaf fall Fig. 4A & B. Viable pollen grains (v); C & D. pollen grain (pg) on stigmatic surface (fluorescence and leaf renewal, flowering and fruiting periods were recorded throughout the study period. test); E & F. Pollen (pg) germinating on stigmatic surface with small pollen tubes (pg: pollen grain; Floral Biology-Flowering period, blooming intensity (number of open flowers/inflorescence, floral longevity and flowering cycle), floral dimensions of 50 flowers, pt: pollen tube; v: viable). anthesis, anther dehiscence and stigma receptivity on twenty five flowers on five inflorescences on marked plants were observed by various methods given by Kearns and Inuoye (1993). Flower longevity-Flower longevity was determined by marking 50 buds on different branches on one plant (Gill et al. 1998). Time was recorded when new flowers opened i.e. when the petals reflexed to expose the androecium and gynoecium. The flowers were observed at regular intervals until the corolla withered. These changes were observed every day for a week. Pollen Morphology-For light microscopic (LM) studies, the pollen grains were acetolyzed following the technique after Nair (1960). The morphology of pollen grains was studied using immersion oil 100X objective in Olympus Phase contrast microscope. The size of pollen grains was measured with an ocular micrometer under light microscope by the method after McKone and Webb (1988). Scanning Electron Microscopic (SEM) studies-The pollen grains from fresh dehisced anthers and floral parts were fixed in 3% glutaraldehyde in 0.1M phosphate buffer at pH 6.8 for 8-20 hours at room temperature. Samples were washed in the same buffer by three changes. Post fixation was done in 1% osmic acid in the same buffer for four hours at 40C. Samples were passed through the graded series of ethyl alcohol and isoamyl acetate (3-methyl butyl acetate). Samples were dried with liquid

CO2 in a HCP-2 Hitachi critical point dryer and coated with gold (20 mm coating) in a sputter coating unit (Polaron equipment Ltd, Walford, England). Observation and photographs were made in LEO-EM-SEM at All India Institute of Medical Sciences (AIIMS), New Delhi. Pollen viability-Viability of pollen collected from fresh dehisced anthers was tested throughout flowering period by 0.2% TTC (2, 3, 5-tetrazolium chloride) test after Hauser and Morrison (1964) and by fluorochromatic procedure (FCR) test after Heslop-Harrison and Heslop-Harrison 1970). Pollen viability was also checked by Brewbaker and Kwack’s (1963) in vitro pollen germination test by hanging drop culture method. Pollen germination on stigma (in vivo)-In vivo pollen germination on stigmatic surface was observed by multiple staining technique after Alexander (1987). After 24 h pollinated pistils were fixed in Carnoy’s fluid for 12 h. These were washed in ethanol and brought down to distilled water through descending series and finally kept in the staining mixture containing lactic acid, malachite green, acid fuchsin, aniline blue, orange G and chloral hydrate for 12 h. The stained pistils were kept in clearing and softening mixture containing lactic acid, phenol, choral hydrate and orange G. These pistils were washed twice in lactic acid and mounted in 1:1 lactic acid and glycerol. In vivo pollen germination was also carried out by fluorescence microscopic method after (Martin 1959). After 24 h pollinated pistils were fixed in Carnoy’s fluid for 12 h, cleared in 4N NaOH and washed in distilled water. These pistils were mounted in 1:1 mixture of 0.005% decolorized aniline blue prepared in 0.05% M phosphate buffer at 11 pH and glycerin. The tissue was spread by applying gentle pressure on the cover glass. The preparation was observed under Olympus fluorescence microscope using UV filter. Stigma receptivity- To check the receptivity of the stigma, cytochemical localization of non-specific esterases was conducted by hydrolysis of the substrate α-napthyl acetate as per Mattson et al. (1974). Nectar—Volume of nectar from individual fresh open flowers at the time of flower opening (25 from each marked plant) was measured using 20 µl microcapillary tubes at 2 h intervals every day. Nectar volume was computed with the following equation after Cruden et al. (1983): mm of nectar in the capillary tube X volume of calibrated capillary tube = volume of nectar mm total length of tube Pollination Biology- Observations on type of flower foragers and their visitation rates were recorded. These were fixed in 70% alcohol and identified. Pollination efficiency of different pollinators was checked by observing the pollen load under microscope (Kearns and Inuoye 1993). Breeding behavior-Breeding system was tested by autogamy, geitonogamy and xenogamy through hand pollination tests. a. Natural/open Pollination: Five inflorescences on each marked plant were tagged and left with the buds ready for anthesis for natural pollination, while all open flowers, young buds and fruits were removed. Number of mature fruits developed in the tagged inflorescence was recorded. b. Artificial/hand Pollination: Hand pollinations were carried out at the time of maximum receptivity of the stigma, using fresh pollen from the same flower (self- pollination-autogamy), pollen from a different flower on same plant (geitonogamy), and pollen from the flowers of five plants of the same species (xenogamy). RESULTS AND DISCUSSION Phenology-Leaf fall and leaf renewal of Clerodendrum splendens takes place simultaneously, with maximum leaf fall in the months of December and January, and moderate in the months of March to September and minimum in the months of October-February. On the other hand, Raju and Kumar (2016) observed that in C. inerme leaves shed continually but leaf shedding is prominent during the dry months (March-May) and profuse leaf flushing was observed during rainy season (June- September) in Coringa mangrove ecosystem, Andhra Pradesh. C. splendens blossoms from December to April. Flowering starts in the month of December and optimum flowering takes place in the month of February and declines in March and only a limited number of flowers are left in April. Fruit formation starts in the month of March and lasts till May. Floral morphology- There are 65±15 inflorescence/ plant and there are 25±10 flowers/ inflorescence. Thus there are 2800±600 flowers /plant. Every day 14- 20 flowers open on each inflorescence and each flower lasts for 2-3 days. Flowers open between 0600-0630 h. The flowers are weakly protandrous and pollen is shed at 07:30–09:00 h within half an hour of flower opening and the stigma becomes receptive at 1100 - 1400 h. The beautiful red flowers appear in several bunches and are arranged in corymbose spike (Fig. 1A). Flowers are pedicilate, bracteates, pentamerous, complete, hermaphrodite, zygomorphic and hypogynous. The calyx consisting of five green sepals, gamosepalous with valvate aestivation (Table 2). The corolla consisting of five petals is tubular, gamopetalous, red, The androecium is epipetalous, polyandrous and didynamous, alternate with the corolla lobes, comprising four red stamens (Fig.1B). The anthers are dithecous, introrse and dorsifixed with long filaments (Fig.2A). They turn brown on maturity and dehisce longitudinally. The gynoecium is bicarpellary and syncarpous. The ovary is tetralocular, superior and four chambered with axile placentation. There are two ovules in each locule. The style is terminal and hollow throughout. The stigma is bifid like a fork (Fig. 2G) and covered with medium sized papillae (Fig. 2H). The papillate stigma is of the wet type and is receptive after anther dehiscence. The developing fruits are blue, berry-like and parthenocarpic (devoid of seeds). Only 1-3 chambers of the ovary were observed to develop into fruit (Fig.1D). They are supported by the star-shaped calyces and stay pretty for a long time.

Table 2- Floral morphology of Clerodendrum splendens (n=50) S. No. Parameters Observations 1. Flowering period December to April 2. Inflorescence Corymbose spike 3. Flower Red, 3.04±0.42 cm in diameter. 4. Calyx Five, green, gamosepalous sepals; 0.61±0.11 cm long 6. Corolla Five, red, tubular, gamopetalous petals; 2.80±0.40 cm long 7. Stamens Anther Filament Pollen Four, red, epipetalous, 2.50± 0.12 cm long0.12 ± 0.06 cm long2.38±0.2 cm5.9-55.3±0.53 μm in diameter. Colpi 53.31± 1.34 μm long. exine

3.02± 0.28 (-2.2) µm thick. 8.. Pistil Style Stigma Ovary 3.50 + 0.52 cm long Red, terminal; 2.92± 0.34 cm long 0.28± 0.13 cm broad, bifid Superior, bicarpellary, syncarpous 0.30± 0.50 cm long

± Standard deviation Floral contrivances-The flowers exhibit several interesting floral modifications (herkogamy) to avoid self-pollination (Figs.3A-I). The following different types of modifications in the position of stamens and style were observed in the flowers of the same inflorescence: 1. In nearly 20-35 % flowers/inflorescence, the stigma remains straight but closed, while two stamens bend on one side, while the other two bend to different side and remain away from the stigma (Fig. 3A). 2. In some other flowers, the stigma bends down while all the four stamens remain upright (Fig. 3B). 3. In some other flowers, two stamens remain upright, while the other two bend down and the bifid stigma remains straight, away from the stamens (Fig.3C). 4. In some other flowers, the filaments of all the four stamens bend down in one direction while the open bifid stigma is upright (Figs.3D). 5. In some other flowers, the filaments of two stamens bend in different directions, while the filaments of the other two are curled and reduced to touch the corolla surface and bend in different directions (Fig.3E). 6. In a limited number of flowers of an inflorescence, the filaments of all the four stamens are reduced and they curl and touch the corolla surface (Figs.3F, G, H). 7. In a few flowers, the filaments of all the four stamens are reduced in size, become much coiled and bend down towards the corolla tube (Fig. 3I). Raju and Kumar (2016) observed only three forms of flowers in C. inerme in respect to the position of sex organs. In the first form the stamens were elongated and the style occurred in close proximity to the stamens. In the second type, the flowers exhibited scattered position of stamens and style. In the third type of flowers, the stamens were fully extended while the style was curved away from them. According to Rohitash (2017), the flowers of C. inerme are protandrous and show four types of sex organ positions in order to avoid self-pollination. While in presently studied C. splendens seven different types of the relative position of stigma and stamens have been recorded. Trichomes-Both glandular and non-glandular trichomes are present on different floral parts viz. calyx, corolla and ovary. Peltate scales and unicellular trichomes are present on the inner surface of corolla tube (Fig. 2F). Nectar- The nectar was not available in the measurable quanitiy, it may be in traces at the time of anthesis. Pollen morphology- The pollen grains are spherical or sub-spherical, tricolporate, prolate or sub-prolate (Figs.2B,C,D). The tectum is reticulate with minute spines distributed all over the surface (Fig. 2B). Exine reticulate, thick, sexine and nexine are more or less same thickness. Rohitash (2016, 2017) observed tricolpate, sub- prolate, circular, echinate pollen grains C. phlomidis and C. inerme. Parveen and Quiser (2007) examined the pollen morphology of 13 species representing 9 genera of the family Verbenaceae now Lamiaceae from Pakistan by light and scanning electron microscope. According to them, the pollen grains were usually tricolporate, rarely tricolpate. Pollen viability-Average pollen viability as tested by FCR, and 1% TTC was 83% (Figs. 2C,D). There was 58% in vitro pollen germination with 763.65± 34 mm long pollen tubes in Brewbaker and Kwack’s medium (Fig. 2E). In vivo pollen germination-Pollen germination on the stigmatic surface as tested by Alexander’s multiple staining method and aniline blue fluorescence methods was only 5 and 8% respectively (Figs.4C,D,E,F). Pollination biology- The beautiful and attractive flowers of C. splendens are visited by black ants only (Fig.1C). These ants go deep in the corolla tubes which are devoid of nectar and they fail to contact the anthers and stigma. Other floral visitors, including honey bees and butter flies were not seen. It may be because honey bees cannot see red colour (Riddle 2016). According to the same author, honey bees see colours that human beings cannot, way up in the ultraviolet end of the spectrum and they cannot see colours in the red range too well. Red flowers would appear dark and featureless to them. Absence of butterflies may be due to absence of the reward, the nectar. Primack et al. (1989) observed bird pollination in C. inerme in Queensland. Keng (1990) reported that C. laevifolium is pollinated by bees and butterflies. According to Reddy and Reddy (1995), papilinoid butterflies (Papilio polytes, P. polymnestor and Atrophaneura hector and Atrophaneura hector) are the exclusive pollinators of C. infortunatum and effect pterogotribic pollination by striking the anthers and stigma with their wings. McMullen (2011) conducted some pollination experiments in C.molle, a widespread member of the Galαpagos flora. According to him, this species exhibited incomplete protandry and set fruit via autonomous autogamy as a result of natural selection in an environment with few faithful pollinators. Nocturnal visitors were ants, spiders, hawk moths, and roaches, whereas diurnal visitors were carpenter bees and ants. In C. inerme with creamish white flowers, Rohitash (2017) observed hawk-moth (Macroglossum sp.), bees (Apis cerana, A. dorsata, bumble bees (Bumbus lapidarius) and butterflies (Danaus genutia, Neptishylas papaja and Eurema hecabe) as floral foragers and it was observed that honey bees and bumble bees were more active as compared to butterflies. Humming birds would most probably feed on this vine’s flowers as they usually find red flowers most attractive. However, there are no humming birds in this part of the world. Breeding system- There was no fruit formation in the begged flowers indicative of self-incompatibility. Formation of fruits in open pollinated flowers in C. splendens was only 2%. However, these fruits also show some interesting features. Only 1-3 chambers and very rarely all the four chambers of the ovary swelled into fruit like structures, while the other chambers remain undeveloped (Fig.1D). The swollen structures consisted of rudimentary seeds which failed to germinate when sown on the moist filter paper. These features indicated that these fruits are parthenocarpic. Formation of parthenocarpic furits has been reported earlier in C. phlomidis (Rohitash 2016). Thus, the self-incompatible nature, absence of the important reward, the nectar and the effective pollinators (butterflies, honey bees and humming birds) are the causes of fruitlessness in C. splendens. Acknowledgements— Sincere thanks are due to Late Dr. R.K. Jain, Head, Department of Botany, B.S.A. College, Mathura for his inspiring and encouraging guidance. Thaanks are also due to the Principal, B.S.A. College, Mathura for facilties.

REFERENCES Alexander MP 1987. A method of staining pollen tube in pistil. Stain Tech. 62(2) 107-112. Brewbaker JL and Kwack BH 1963. The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50(9) 747-758. Chauhan SVS, Dhingra RK and Yadav DK 1986. Studies into causes of sterility in Clerodendrum splendens Don. J. Indian Bot. Soc. 65 185-187. Cruden RW, Hermann SM and Peterson S 1983. Patterns of nectar production and plant pollination coevolution. In: Bentley B and Eilias T (eds). Biology of Nectaries. Columbia University Press, New York. Gautam PK and Rohitash 2012. Reproductive biology of Clerodendrum splendens (Verbenaceae). J. Exp. Sci. 3(8) 14-16. Harley RM, Atkins S, Budantsev AL, Cantino PD, Conn BJ, Grayer RJ, Harley MM, de Kok RPJ, Krestovskaja TV, Morales R, Paton AJ, and Ryding PO 2004. Labiatae. Pp. 167-275. In: Kubitzki K (ed. and Kadereit JW (volume ed.). The Families and Genera of Vascular Plants volume VII. Springer-Verlag: Berlin; Heidelberg, Germany. ISBN 978-3-540-40593-1 Hauser EJP and Morrison JH 1964. The cytochemical reduction of nitro blue tetrazolium as an index of pollen viability. Am. J. Bot. 51(7) 748-752. Heslop-Harrison J and Heslop-Harrison Y 1970. Evaluation of pollen viability by enzymatically-induced fluorescence: intracellular hydrolysis of fluorescein diacetate. StainTech. 45 115-120. Kearns CA and Inouye DW 1993. Techniques for pollination Biologists. University Press of Colorado, Nivyot, Colorado. Keng H 1990. The Concise Flora of Singapore: Gymnosperms and Dicotyledons. Singapore University Press, Singapore, Pp. 222. Lavergne S, Thompson JD, Garnier E and Debussche M 2004. The biology and ecology of narrow endemic and widespread plants: a comparative study of trait variation in 20 congeneric pairs. Oikos107 505–518. Leith H 1973. Phenology in Productivity studies. In:David E Reiche (ed.). Analysis of temperate forest ecosystems. Chapman and Hall Ltd., London, Springer-Verlag, Berlin. Pp. 29-46. Mabberley DJ 2008. Mabberley’s Plant-Book third edition. Cambridge University Press: UK. ISBN 978-0-521-82071-4. Martin FW 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Tech. 34 125-128. Mattson O, Knox RB, Heslop-Harrison J and Heslop-Harrison Y 1974. Protein pellicle as a probable recognition site in incompatibility reactions. Nature 213 703-704. Mckone MJ and Webb CJ 1988. A difference in pollen size between the male and hermaphrodite flowers of two species of Apiaceae. Australian J. Bot. 36 331-337. McMullen CK 2011. Nocturnal and diurnal pollination of Clerodendrummolle (Verbenaceae) in the Galαpagos Islands. Plant Syst. Evol. 292(1) 15-23. Moza MK and Bhatnagar AK 2005. Plant reproductive biology studies crucial for conservation Curr. Sci. 89 243 244. Nair PKK 1960. A modification in method of pollen preparation. J. Sci. Indus. Res. 19 26-27. Perveen A and Qaiser M 2007. Pollen flora of Pakistan–LIII. Verbenaceae. Pak. J. Bot. 39(3) 663-669. Primack RB, Duke NC and Tomlinson PB 1981. Floral morphology in relation to pollination exocology in five Queensland costal plants. Austrobaileya 1(4) 346-355. Raju AJS and Rajendra Kumar R 2016. Pollination ecology of Clerodendruminerme (L.) Gaertn. (Lamiaceae) in Coringa mangrove ecosystem, Andhra Pradesh, India. J. Threatened Taxa. 8(5) 8777–8787 http://dx.doi.org/10.11609/jott.2276.8.5.8777-8787 Reddy TB and Reddi CS 1995. Butterfly pollination of Clerodendrum infortunatum (Verbenaceae). J. Bombay Nat. History Soc.92 166–173. Riddle S 2016. How bees see and why it that human beings cannot? Bee vision vs. Human vision. Bee Culture May, 2016. Rohitash 2016. Reproductive Biology of Clerodendrum phlomidis (Lamiaceae). Int. J. Plant Rep. Biol. 8(2) 208-210. Rohitash 2017. Floral contrivances, pollination and breeding system of Clerodendrum inerme (L.) Gaertn. (Lamiaceae). Int. J. Plant Rep. Biol. 9(2) 115-124. Rohitash and Jain RK 2010. Reproductive Biology of Clerodendrum splendens (Verbenaceae). Ad. Biores. 1(1) 84 – 86. Rojas-Sandoval J and Acevedo-Rodríguez P 2012. Clerodendrum chinense (Chinese glory bower). Invasive species compendium. www.cabi.org. Yao-Wu Y, David JM, Steane, Dorothy A and Richard GO 2010. Further disintegration and redefinition of Clerodendrum (Lamiaceae): Implications for the understanding of the evolution of an intriguing breeding strategy. Taxon 59(1) 125–133.

Legend to Figures Fig. 1A. Plant in full bloom; B. Inflorescence; C. A black ant on the flowers of a mature inflorescence (circle); D. Parthenocarpic fruits. Fig. 2-A & B. SEM pictures showing dorsifixed anther and curved filament; B. mature pollen grain; C & D. viable pollen grains; E Pollen germinating in vitro; F. Peltate scales and unicellular trichomes on corolla surface; G. bifid stigma; H. magnified view of stigmatic surface with small compactly arranged papillae (an: anther, ft: filament, pg: pollen grain; pt: pollen tube, stg: stigma, ut; unicellular trochome; v: viable). Fig. 3A-I Flowers showing contrivances. Fig. 4A & B. Viable pollen grains (v); C & D. pollen grain (pg) on stigmatic surface (fluorescence test); E & F. Pollen (pg) germinating on stigmatic surface with small pollen tubes (pg: pollen grain; pt: pollen tube; v: viable). 28 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.0-0 134 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.133-139 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 33

Floral Biology—Flowering period, blooming intensity by fluorescence microscopic method after (Martin 1959). (number of open flowers/inflorescence, floral longevity and After 24 h pollinated pistils were fixed in Carnoy’s fluid for 12 flowering cycle), floral dimensions of 50 flowers, anthesis, h, cleared in 4N NaOH and washed in distilled water. These anther dehiscence and stigma receptivity on twenty five pistils were mounted in 1:1 mixture of 0.005% decolorized flowers on five inflorescences on marked plants were aniline blue prepared in 0.05% M phosphate buffer at 11 pH observed by various methods given by Kearns and Inuoye and glycerin. The tissue was spread by applying gentle (1993). pressure on the cover glass. The preparation was observed Flower longevity—Flower longevity was determined by under Olympus fluorescence microscope using UV filter. marking 50 buds on different branches on one plant (Gill et al. Stigma receptivity— To check the receptivity of the stigma, 1998). Time was recorded when new flowers opened i.e. when cytochemical localization of non-specific esterases was the petals reflexed to expose the androecium and gynoecium. conducted by hydrolysis of the substrate α-napthyl acetate as The flowers were observed at regular intervals until the corolla per Mattson et al. (1974). withered. These changes were observed every day for a week. Nectar—Volume of nectar from individual fresh open flowers Pollen Morphology—For light microscopic (LM) studies, at the time of flower opening (25 from each marked plant) was the pollen grains were acetolyzed following the technique measured using 20 µl microcapillary tubes at 2 h intervals after Nair (1960). The morphology of pollen grains was every day. Nectar volume was computed with the following studied using immersion oil 100X objective in Olympus Phase equation after Cruden et al. (1983): contrast microscope. The size of pollen grains was measured mm of nectar in the capillary tube X volume of calibrated with an ocular micrometer under light microscope by the capillary tube = volume of nectar mm total length of tube method after McKone and Webb (1988). Pollination Biology— Observations on type of flower Scanning Electron Microscopic (SEM) studies—The foragers and their visitation rates were recorded. These were pollen grains from fresh dehisced anthers and floral parts were fixed in 70% alcohol and identified. Pollination efficiency of fixed in 3% glutaraldehyde in 0.1M phosphate buffer at pH 6.8 different pollinators was checked by observing the pollen load for 8-20 hours at room temperature. Samples were washed in under microscope (Kearns and Inuoye 1993). the same buffer by three changes. Post fixation was done in 1% Breeding behavior—Breeding system was tested by osmic acid in the same buffer for four hours at 40C. Samples autogamy, geitonogamy and xenogamy through hand were passed through the graded series of ethyl alcohol and pollination tests. isoamyl acetate (3-methyl butyl acetate). Samples were dried a. Natural/open Pollination: Five inflorescences on each

with liquid CO2 in a HCP-2 Hitachi critical point dryer and marked plant were tagged and left with the buds ready for coated with gold (20 mm coating) in a sputter coating unit anthesis for natural pollination, while all open flowers, (Polaron equipment Ltd, Walford, England). Observation and young buds and fruits were removed. Number of mature photographs were made in LEO-EM-SEM at All India fruits developed in the tagged inflorescence was recorded. Institute of Medical Sciences (AIIMS), New Delhi. b. Artificial/hand Pollination: Hand pollinations were Pollen viability—Viability of pollen collected from fresh carried out at the time of maximum receptivity of the dehisced anthers was tested throughout flowering period by stigma, using fresh pollen from the same flower (self- 0.2% TTC (2, 3, 5-tetrazolium chloride) test after Hauser and pollination-autogamy), pollen from a different flower on Morrison (1964) and by fluorochromatic procedure (FCR) test same plant (geitonogamy), and pollen from the flowers of after Heslop-Harrison and Heslop-Harrison 1970). Pollen five plants of the same species (xenogamy). viability was also checked by Brewbaker and Kwack’s (1963) RESULTS AND DISCUSSION in vitro pollen germination test by hanging drop culture method. Phenology—Leaf fall and leaf renewal of Clerodendrum Pollen germination on stigma (in vivo)—In vivo pollen splendens takes place simultaneously, with maximum leaf fall germination on stigmatic surface was observed by multiple in the months of December and January, and moderate in the staining technique after Alexander (1987). After 24 h months of March to September and minimum in the months of pollinated pistils were fixed in Carnoy’s fluid for 12 h. These October-February. On the other hand, Raju and Kumar (2016) were washed in ethanol and brought down to distilled water observed that in C. inerme leaves shed continually but leaf through descending series and finally kept in the staining shedding is prominent during the dry months (March-May) mixture containing lactic acid, malachite green, acid fuchsin, and profuse leaf flushing was observed during rainy season aniline blue, orange G and chloral hydrate for 12 h. The stained (June-September) in Coringa mangrove ecosystem, Andhra pistils were kept in clearing and softening mixture containing Pradesh. lactic acid, phenol, choral hydrate and orange G. These pistils C. splendens blossoms from December to April. were washed twice in lactic acid and mounted in 1:1 lactic acid Flowering starts in the month of December and optimum and glycerol. In vivo pollen germination was also carried out flowering takes place in the month of February and declines in 28 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.0-0 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 135 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 33

March and only a limited number of flowers are left in April. Floral contrivances—The flowers exhibit several interesting Fruit formation starts in the month of March and lasts till May. floral modifications (herkogamy) to avoid self-pollination Floral morphology— There are 65±15 inflorescence/ plant (Figs.2A-I). The following different types of modifications in and there are 25±10 flowers/ inflorescence. Thus there are the position of stamens and style were observed in the flowers 2800±600 flowers /plant. Every day 14-20 flowers open on of the same inflorescence : each inflorescence and each flower lasts for 2-3 days. Flowers 1. In nearly 20-35 % flowers/inflorescence, the stigma open between 0600-0630 h. The flowers are weakly remains straight but closed, while two stamens bend on protandrous and pollen is shed at 07:30–09:00 h within half an one side, while the other two bend to different side and hour of flower opening and the stigma becomes receptive at remain away from the stigma (Fig. 2A). 1100 - 1400 h. 2. In some other flowers, the stigma bends down while all The beautiful red flowers appear in several bunches the four stamens remain upright (Fig. 2B). and are arranged in corymbose spike (Fig. 1A). Flowers 3. In some other flowers, two stamens remain upright, while are pedicilate, bracteates, pentamerous, complete, the other two bend down and the bifid stigma remains hermaphrodite, zygomorphic and hypogynous. The calyx straight, away from the stamens (Fig.2C). consisting of five green sepals, gamosepalous with valvate 4. In some other flowers, the filaments of all the four aestivation (Table 2). The corolla consisting of five petals is stamens bend down in one direction while the open bifid tubular, gamopetalous, red, The androecium is epipetalous, stigma is upright (Figs.2D). polyandrous and didynamous, alternate with the corolla lobes, 5. In some other flowers, the filaments of two stamens bend comprising four red stamens (Fig.1B). The anthers are in different directions, while the filaments of the other dithecous, introrse and dorsifixed with long filaments (Fig.3A). two are curled and reduced to touch the corolla surface They turn brown on maturity and dehisce longitudinally. and bend in different directions (Fig.2E). The gynoecium is bicarpellary and syncarpous. The 6. In a limited number of flowers of an inflorescence, the ovary is tetralocular, superior and four chambered with axile filaments of all the four stamens are reduced and they curl placentation. There are two ovules in each locule. The style is and touch the corolla surface (Figs.2F, G, H). terminal and hollow throughout. The stigma is bifid like a fork 7. In a few flowers, the filaments of all the four stamens are (Fig. 3G) and covered with medium sized papillae (Fig. 3H). reduced in size, become much coiled and bend down The papillate stigma is of the wet type and is receptive after towards the corolla tube (Fig. 2I). anther dehiscence. The developing fruits are blue, berry-like Raju and Kumar (2016) observed only three forms of and parthenocarpic (devoid of seeds). Only 1-3 chambers of flowers in C. inerme in respect to the position of sex organs. In the ovary were observed to develop into fruit (Fig.1D). They the first form the stamens were elongated and the style are supported by the star-shaped calyces and stay pretty for a occurred in close proximity to the stamens. In the second type, long time. the flowers exhibited scattered position of stamens and style. In the third type of flowers, the stamens were fully extended Table 2- Floral morphology of Clerodendrum splendens (n=50) while the style was curved away from them. According to S. Parameters Observations Rohitash (2017), the flowers of C. inerme are protandrous and No. show four types of sex organ positions in order to avoid self- 1. Flowering period December to April pollination. While in presently studied C. splendens seven 2. Inflorescence Corymbose spike different types of the relative position of stigma and stamens 3. Flower Red, 3.04±0.42 cm in diameter. have been recorded. 4. Calyx Five, green, gamosepalous sepals; Trichomes—Both glandular and non-glandular trichomes are 0.61±0.11 cm long present on different floral parts viz. calyx, corolla and ovary. 6. Corolla Five, red, tubular, gamopetalous petals; 2.80±0.40 cm long Peltate scales and unicellular trichomes are present on the 7. Stamens Four, red, epipetalous, 2.50± 0.12 cm long inner surface of corolla tube (Fig. 3F). Anther 0.12 ± 0.06 cm long Nectar— The nectar was not available in the measurable Filament 2.38±0.2 cm quanitiy, it may be in traces at the time of anthesis. Pollen 5.9-55.3±0.53 μm in diameter. Colpi 53.31± Pollen morphology— The pollen grains are spherical or sub- 1.34 μm long. exine 3.02± 0.28 (-2.2) µm thick. spherical, tricolporate, prolate or sub-prolate (Figs.3B,C,D). 8. Pistil 3.50 + 0.52 cm long The tectum is reticulate with minute spines distributed all over Style Red, terminal; 2.92± 0.34 cm long the surface (Fig. 3B). Exine reticulate, thick, sexine and Stigma 0.28± 0.13 cm broad, bifid nexine are more or less same thickness. Rohitash (2016, 2017) Ovary Superior, bicarpellary, syncarpous 0.30± observed tricolpate, sub-prolate, circular, echinate pollen 0.50 cm long grains C. phlomidis and C. inerme. Parveen and Quiser (2007) ± Standard deviation examined the pollen morphology of 13 species representing 9 136 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.133-139

A B C D

Fig. 1A. Plant in full bloom; B. Inflorescence; C. A black ant on the flowers of a mature inflorescence (circle); D. Parthenocarpic fruits.

G H

Fig. 2-A & B. SEM pictures showing dorsifixed anther and curved filament; B. mature pollen grain; C & D. viable pollen grains; E Pollen germinating in vitro; F. Peltate scales and unicellular trichomes on corolla surface; G. bifid stigma; H. magnified view of stigmatic surface with small compactly arranged papillae (an: anther, ft: filament, pg: pollen grain; pt: pollen tube, stg: stigma, ut; unicellular trochome; v: viable). 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 137 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 33

Fig. 3A-I Flowers showing contrivances.

Fig. 4A & B. Viable pollen grains (v); C & D. pollen grain (pg) on stigmatic surface (fluorescence test); E & F. Pollen (pg) germinating on stigmatic surface with small pollen tubes (pg: pollen grain; pt: pollen tube; v: viable). 138 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.133-139

genera of the family Verbenaceae now Lamiaceae from ovary swelled into fruit like structures, while the other Pakistan by light and scanning electron microscope. chambers remain undeveloped (Fig.1D). The swollen According to them, the pollen grains were usually structures consisted of rudimentary seeds which failed to tricolporate, rarely tricolpate. germinate when sown on the moist filter paper. These features Pollen viability—Average pollen viability as tested by FCR, indicated that these fruits are parthenocarpic. Formation of and 1% TTC was 83% (Figs. 3C,D). There was 58% in vitro parthenocarpic furits has been reported earlier in C. phlomidis pollen germination with 763.65± 34 mm long pollen tubes in (Rohitash 2016). Brewbaker and Kwack’s medium (Fig. 3E). Thus, the self-incompatible nature, absence of the In vivo pollen germination—Pollen germination on the important reward, the nectar and the effective pollinators stigmatic surface as tested by Alexander’s multiple staining (butterflies, honey bees and humming birds) are the causes of method and aniline blue fluorescence methods was only 5 and fruitlessness in C. splendens. 8% respectively (Figs.4C,D,E,F). Acknowledgements— Sincere thanks are due to Late Dr. Pollination biology— The beautiful and attractive flowers of R.K. Jain, Head, Department of Botany, B.S.A. College, C. splendens are visited by black ants only (Fig.1C). These Mathura for his inspiring and encouraging guidance. Thaanks ants go deep in the corolla tubes which are devoid of nectar and are also due to the Principal, B.S.A. College, Mathura for they fail to contact the anthers and stigma. Other floral facilties. visitors, including honey bees and butter flies were not seen. It REFERENCES may be because honey bees cannot see red colour (Riddle 2016). According to the same author, honey bees see colours Alexander MP 1987. A method of staining pollen tube in pistil. that human beings cannot, way up in the ultraviolet end Stain Tech. 62(2) 107-112. of the spectrum and they cannot see colours in the red range too well. Red flowers would appear dark and featureless to Brewbaker JL and Kwack BH 1963. The essential role of them. Absence of butterflies may be due to absence of the calcium ion in pollen germination and pollen tube growth. reward, the nectar. Am. J. Bot. 50(9) 747-758. Primack et al. (1989) observed bird pollination in C. Chauhan SVS, Dhingra RK and Yadav DK 1986. Studies into inerme in Queensland. Keng (1990) reported that C. causes of sterility in Clerodendrum splendens Don. J. laevifolium is pollinated by bees and butterflies. According to Indian Bot. Soc. 65 185-187. Reddy and Reddy (1995), papilinoid butterflies (Papilio polytes, P. polymnestor and Atrophaneura hector and Cruden RW, Hermann SM and Peterson S 1983. Patterns of Atrophaneura hector) are the exclusive pollinators of C. nectar production and plant pollination coevolution. In: infortunatum and effect pterogotribic pollination by striking Bentley B and Eilias T (eds). Biology of Nectaries. the anthers and stigma with their wings. McMullen (2011) Columbia University Press, New York. conducted some pollination experiments in C.molle, a widespread member of the Galαpagos flora. According to him, Gautam PK and Rohitash 2012. Reproductive biology of this species exhibited incomplete protandry and set fruit via Clerodendrum splendens (Verbenaceae). J. Exp. Sci. 3(8) autonomous autogamy as a result of natural selection in an 14-16. environment with few faithful pollinators. Nocturnal visitors were ants, spiders, hawk moths, and roaches, whereas diurnal Harley RM, Atkins S, Budantsev AL, Cantino PD, Conn BJ, visitors were carpenter bees and ants. In C. inerme with Grayer RJ, Harley MM, de Kok RPJ, Krestovskaja TV, creamish white flowers, Rohitash (2017) observed hawk- Morales R, Paton AJ, and Ryding PO 2004. Labiatae. Pp. moth (Macroglossum sp.), bees (Apis cerana, A. dorsata, 167-275. In: Kubitzki K (ed. and Kadereit JW (volume bumble bees (Bumbus lapidarius) and butterflies (Danaus ed.). The Families and Genera of Vascular Plants volume genutia, Neptishylas papaja and Eurema hecabe) as floral VII. Springer-Verlag: Berlin; Heidelberg, Germany. foragers and it was observed that honey bees and bumble bees ISBN 978-3-540-40593-1 were more active as compared to butterflies. Humming birds would most probably feed on this vine’s flowers as they Hauser EJP and Morrison JH 1964. The cytochemical usually find red flowers most attractive. However, there are no reduction of nitro blue tetrazolium as an index of pollen humming birds in this part of the world. viability. Am. J. Bot. 51(7) 748-752. Breeding system— There was no fruit formation in the Heslop-Harrison J and Heslop-Harrison Y 1970. Evaluation of begged flowers indicative of self-incompatibility. Formation pollen viability by enzymatically-induced fluorescence: of fruits in open pollinated flowers in C. splendens was only intracellular hydrolysis of fluorescein diacetate. 2%. However, these fruits also show some interesting features. StainTech. 45 115-120. Only 1-3 chambers and very rarely all the four chambers of the 28 The International Journal of Plant Reproductive Biology 10(2) July, 2018, pp.0-0 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 139 2018 Phenology and Reproductive biology of Clerodendrum splendens G. Don 33

Kearns CA and Inouye DW 1993. Techniques for pollination Perveen A and Qaiser M 2007. Pollen flora of Pakistan–LIII. Biologists. University Press of Colorado, Nivyot, Verbenaceae. Pak. J. Bot. 39(3) 663-669. Colorado. Primack RB, Duke NC and Tomlinson PB 1981. Floral Keng H 1990. The Concise Flora of Singapore: Gymnosperms morphology in relation to pollination exocology in five and Dicotyledons. Singapore University Press, Queensland costal plants. Austrobaileya 1(4) 346-355. Singapore, Pp. 222. Raju AJS and Rajendra Kumar R 2016. Pollination ecology Lavergne S, Thompson JD, Garnier E and Debussche M 2004. of Clerodendruminerme (L.) Gaertn. (Lamiaceae) The biology and ecology of narrow endemic and in Coringa mangrove ecosystem, Andhra Pradesh, widespread plants: a comparative study of trait variation India. J. Threatened Taxa. 8(5) 8777–8787 in 20 congeneric pairs. Oikos107 505–518. http://dx.doi.org/10.11609/jott.2276.8.5.8777-8787

Leith H 1973. Phenology in Productivity studies. In:David E Reddy TB and Reddi CS 1995. Butterfly pollination of Reiche (ed.). Analysis of temperate forest ecosystems. Clerodendrum infortunatum (Verbenaceae). J. Bombay Chapman and Hall Ltd., London, Springer-Verlag, Nat. History Soc.92 166–173. Berlin. Pp. 29-46. Riddle S 2016. How bees see and why it that human beings Mabberley DJ 2008. Mabberley’s Plant-Book third edition. cannot? Bee vision vs. Human vision. Bee Culture May, Cambridge University Press: UK. ISBN 978-0-521- 2016. 82071-4. Rohitash 2016. Reproductive Biology of Clerodendrum Martin FW 1959. Staining and observing pollen tubes in the phlomidis (Lamiaceae). Int. J. Plant Rep. Biol. 8(2) 208- style by means of fluorescence. Stain Tech. 34 125-128. 210.

Mattson O, Knox RB, Heslop-Harrison J and Heslop-Harrison Rohitash 2017. Floral contrivances, pollination and breeding Y 1974. Protein pellicle as a probable recognition site in system of Clerodendrum inerme (L.) Gaertn. incompatibility reactions. Nature 213 703-704. (Lamiaceae). Int. J. Plant Rep. Biol. 9(2) 115-124. Mckone MJ and Webb CJ 1988. A difference in pollen size Rohitash and Jain RK 2010. Reproductive Biology of between the male and hermaphrodite flowers of two Clerodendrum splendens (Verbenaceae). Ad. Biores. 1(1) species of Apiaceae. Australian J. Bot. 36 331-337. 84 – 86.

McMullen CK 2011. Nocturnal and diurnal pollination of Rojas-Sandoval J and Acevedo-Rodríguez P 2012. Clerodendrummolle (Verbenaceae) in the Galαpagos Clerodendrum chinense (Chinese glory bower). Invasive Islands. Plant Syst. Evol. 292(1) 15-23. species compendium. www.cabi.org.

Moza MK and Bhatnagar AK 2005. Plant reproductive Yao-Wu Y, David JM, Steane, Dorothy A and Richard GO biology studies crucial for conservation Curr. Sci. 89 243 2010. Further disintegration and redefinition of 244. Clerodendrum (Lamiaceae): Implications for the understanding of the evolution of an intriguing breeding Nair PKK 1960. A modification in method of pollen strategy. Taxon 59(1) 125–133. preparation. J. Sci. Indus. Res. 19 26-27.