A Study of Native Malpighiaceae in Singapore Focusing on The Pollination and Breeding Systems In Comparison with Neotropical Species Lieu Tze Ern Samuel1, Joshua Dinesh Thambiah1, Siew Jin En Ryan1, and Jeffrey Lee2 1Raffles Institution, 1 Raffles Institution Lane, Singapore 575954 2Raffles Science Institute,1 Raffles Institution Lane, Singapore 575594 Abstract We studied the methods of pollination of the native Malpighiaceae in Singapore: Aspidopterys concava, Hiptage sericea and Tristellateia australasiae. Data on pollinators were recorded by in situ observations and pollinators were analysed for identification. Breeding system experiments were conducted by isolating inflorescences in bags. Herbarium data on phenology and distribution were compiled. Flowers of T. australasiae and other Old World Malpighiaceae do not possess floral oils to attract pollinators like their counterparts in the New World. These species are not autogamous nor agamospermous. Instead, they depend on pollen-collecting stingless bees of the Apidae family for pollination. We found that A. concava is distributed mostly in the northern part of the island, whereas T. australasiae is found scattered throughout. No living or preserved specimen of H. sericea were found, but we speculate that a close species, H. benghalensis may be native rather than introduced as believed. The flowering period was determined to be April and September for A. concava, August for H. benghalensis, and January to February/July to September for T. australasiae. Introduction The Malpighiaceae, with 1200 species in 66 genera[1], is predominantly a tropical plant family, with approximately 85% of the species occurring in the New World[2]. The family exhibits a strong association between floral morphology and insect pollinator attraction[3], and it has in fact been suggested that for Malpighiaceae, specialized plant-pollinator mutualisms may account for all or part of its exceptional diversification rate[4]. The stereotypical floral morphology of the more than 1,000 New World species is very distinctive and highly conserved in terms of symmetry and pollinator reward[5]. The single upright dorsal banner petal, a critical component of this plant-pollinator mutualism, is strongly differentiated from the other petals in the corolla and helps to orient and attract only oil bee pollinators belonging to the tribes Centridini, Tetrapedini and Tapinotaspidini[6]. The petals also have very narrow bases, which allows the bees to access the calyx oil glands or elaiophores which are borne in pairs on the abaxial surfaces of the sepals[2]. The bees use the oil as larval provision and to waterproof the earthen brood-cell walls[2]. Most Malpighiaceae are outcrossing, but selfing also occurs. Some have been shown to be agamospermous[1]. In contrast to the high species diversity in the New World (~1170 species in 59 genera), lineages in the Old World are relatively species-poor (~ 140 species in 14 genera)[7]. These Old World 1 species were derived from seven independent migrations from the New World[5]. Since the specialist oil-bee pollinators are not found in the Old World, these descendent clades lost most of the stereotypical Malpighiaceae floral characteristics and evolved either radially symmetrical flowers or shifted to a different kind of zygomorphy[5]. Pollination mechanisms also shifted to having pollen or nectar becoming the principal pollinator reward, rather than oil[7]. Tristellateia, for example, maintained the zygomorphic corollas but reoriented the plane of symmetry such that two dorsal petals are displayed instead of a single banner petal. The other three petals form two lateral petals and a single ventral petal[3]. It also lost the oil gland entirely or shifted their contents to sugars, and evolved large poricidally dehiscent anthers suggesting adaptation to buzz pollination[5]. Though there have been numerous studies of the floral morphology, pollination systems and breeding systems of Malpighiaceae species, most were on Neotropical species[6]. Singapore has three native Old World species and thus offers a unique opportunity to study them, thereby contributing new knowledge to an area of which almost nothing is known. The three species of native Malpighiaceae in Singapore are the climbers Aspidopterys concava, Hiptage sericea and Tristellateia australasiae[8]. A. concava and H. sericea are endangered in Singapore and little studied so our findings would hopefully help in some ways towards their conservation and propagation. The last native stand of T. australasiae was submerged and lost as a consequence of the damming of the waterways in which they were found (W.F. Ang, pers. comm.) and all T. australasiae currently found in Singapore are cultivated individuals originating outside of the country. However, since they are widely found, we used them as a test group to trail our breeding system experiments and flower study techniques. Materials and Methods Herbarium Study Specimens of the three species in the Singapore Herbarium located at the Singapore Botanic Gardens were examined and photographed. The distribution data in Singapore and the region were analysed, and the collection locations for Singapore were plotted to get an idea of the natural distribution range of the species. When a specimen contained flowers, the collection dates were noted to construct a phenology of flowering seasons. No specimens of H. sericea were found so we collected data for the related H. benghalensis instead. Although noted as being an introduced cultivated species[9], some specimens in the Herbarium had been collected from the wild in Singapore so we decided to study it in place of H. sericea. Field Observations We observed living specimens to note the habitat, floral biology and possible pollinators associated with them. Six specimens of T. australasiae were bought from a local nursery and planted in the school garden (1o20’27”N 103o50’41”E), three in pots and three in the ground. They were observed periodically between February to May 2013. Two wild individuals of A. concava were observed from February to May 2013 at Upper Seletar Reservoir (1o23’43”N 103o47’46”E). Field observations on a stand of H. benghalensis were made from February to April 2013 at the Singapore Botanic Gardens Dell Lane (1o18’38”N 103o49’0”E). Flowering was only observed in T. australasiae during the period of observation. The behaviour of visitors on the flowers (e.g. approach, pollen/nectar collection) was noted before the visitors were collected for laboratory examination. 2 Breeding System Experiments Breeding system experiments were carried out on T. australasiae by wrapping the flowers in insect-proof cloth bags[10]. In some of the bags the anthers of the flowers were removed to test for agamospermy (reproduction that does not involve the use of the male reproductive organs). In some of the other bags, the anthers were left intact to test for autogamy (mechanical self- pollination). Laboratory examination of live flowers Live flowers of T. australasiae were dissected, examined and photographed. To determine herkogamy (spatial separation of anthers and stigma), we measured the distance between the ends of the stamens to the tip of the stigma using Vernier calipers[10]. Stigmatic receptivity was tested by observing the stigmas of the flowers under high magnification (40x objective) for a stigmatic cuticle that becomes filled with liquid when receptive, as reported for some Malpighiaceae[11]. We studied the carpels of the flower to determine the stigma morphology and dissected the individual ovaries to count the number of ovules per flower for ovule quantification. Results Description of species Aspidopterys concava We found a specimen of A. concava at Upper Seletar Reservoir Car Park A climbing on a thin palm tree (Arecaceae) and another near to Car Park C on a towering Radermachera pinnata tree (Bignoniaceae), together with a second unknown climber. We found that the pairs of opposite leaves on the vertical vine of the first specimen were about 15-20 cm apart. The leaves were also glossy, and we realised that this wild specimen has larger and rounder leaves compared to the specimens planted in school (which were rescued by the last author from land being cleared near the junction of Sembawang Road and Mandai Avenue in Nov 2012). No flowering were observed, however, a post on the Nature Society (Singapore) Nature Forum, 10 Apr 2013, showed a flowering specimen in a member’s garden which he/she grew from seeds collected from the Central Catchment Area on 27 Mar 2009. The member reported on 12 May 2013 that no seeds developed from the flowers. Hiptage benghalensis The H. benghalensis specimens we studied at the Singapore Botanic Gardens had characteristic entire moderate-sized leaves with tapered ends. It also had main branching veins on the leaves which did not extend all the way to the edge. There were wave lenticels along the length of the stem. The species forms new stalks from terminal buds on the ends of the leaflets, and these new leaves are brown and waxy. This brown colour fades to green with maturity[9]. To be sure that we were not confusing the specimens with H. sericea, we counterchecked the latter’s description in Ridley (1922)[9]. The leaves of H. sericea are described as elliptic-oblong, tapering. hairless and smooth. The branches are hairy. Each leaf has 4 pairs of nerves, each 2-6 inches long and 0.9-3 inches wide. This was the defining characteristic that allowed us to differentiate between H. sericea and H. benghalensis. A detailed note in the Singapore herbarium described H. 3 benghalensis as producing flowers with four white petals and one yellow petal. The flowers are said to be sweet scented and full of honey. Tristellateia australasiae T. australasiae, or Maiden’s Jealousy, has zygomorphic (bilaterally symmetrical) flowers with only one plane of symmetry. They have two dorsal petals, two lateral petals and one ventral petal, as can be seen from the picture below.