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General Introduction General Introduction As angiosperms diversified and flourished in terrestrial habitats, some species were able to colonize freshwater or marine environments and became aquatic (Philbrick and Les, 1996). The transition to an aquatic life has been achieved by only 2% of the approximately 350,000 species of angiosperms (Cook, 1990). Although aquatic plants are typically discussed as a unified biological group, the ways these plants have evolved in the aquatic milieu are as diverse as their different evolutionary lineages that became aquatic (Hutchinson, 1975; Sculthorpe, 1967). Podostemaceae represents one such diverse assemblage of fresh water flowering plants with several unusual vegetative and reproductive features. Podostemaceae, the largest family of fresh water angiosperms, has caught the interest of botanists for decades because of many striking attributes that truly make them the conundrum for being an absolute deviation from the usual flowering plants. These plants (Podostemads) are often addressed as evolutionary misfits because of a set of unique morphological, anatomical, embryological and evolutionary features (Mohan Ram and Sehgal, 1992). There are nearly 50 genera and 280 species in Podostemaceae that dwell in the tropical and sub-tropical regions of the world (Cook and Rutishauser, 2007). The family is subdivided into three subfamilies: Podostemoideae, Tristichoideae, and Weddellinoideae (Engler, 1930; Kita and Kato, 2001; Moline et al., 2007; Koi et al., 2012b). These plants grow in fast-flowing waters of rivers, rapids or cataracts, clinging tightly to the rocks with the help of small haptera or holdfasts and adhesive hairs. The haptera and adhesive hairs secrete mucilage, which is often termed as ‘super-glue’ because it helps the plant body to withstand the force of the mighty currents (Mohan Ram and Sehgal, 1992). Podostemads are so tenaciously attached to the rocks on which they grow, that it is difficult to remove them unless a sharp instrument is used. A great majority of Podostemaceae are annual, strongly seasonal and their life cycle is correlated with rainy season (Willis, 1902b; Mohan Ram and Sehgal, 1992; Philbrick and Novelo, 1995). The plants remain vegetative all through the rainy season under 1 water and with the retreat of monsoon, the water level in the rivers drops which elicits flowering in the plants. Thus, the periodic occurrence of dry and wet periods clearly underlines the life history of these plants. The podostemads are not only unique in their habitat but also in their habit. They are often mistaken for liverworts, lichens or algae, because their plant body is thalloid which lacks a typical root-shoot bipolar organization. Most interestingly, the podostemads show a marked structural reduction in their adaptive features, which they have evolved to survive in gushing fresh water streams. The prostate axis occurs in various forms and has been the storm-centre of various interpretations by the botanists. In addition to the main plant body, other organs like shoots, leaves, roots in do not a show complete homology with the organs of typical classical root-shoot model (CRS model; Rutishauser and Huber, 1991). Arber, as early as in 1950, proposed the concept of “partial shoot theory of leaf” for such structural misfits in angiosperms and gave the concept of ‘Fuzzy Arberian Morphology’. The concept was later referred by Rutishauser in 1995 to propose the term ‘fuzzy morphology’ to describe the plant body and organs of the podostemads. According to him the terms ‘root’, ‘shoot/stem’, and ‘leaf’ are taken as fuzzy sets in this family, as structural categories with fuzzy border- lines that allow the recognition of intermediates or mosaic organs (Rutishauser, 1995). The structural category ‘shoot’ is defined as a ‘stem’ that carries’ leaves’. Thus, the terms ‘shoot’ and ‘stem’ have, by definition, partially identical connotations (Rutishauser and Sattler, 1985). The other structural novelties of Podostemaceae, not found in the other angiosperms, are double sheathed leaves i.e., leaves with two boatshaped stipular sheaths each having a flower bud, resulting in unique branching pattern (Rutishauser et al., 2003), syndesmy (syndesmy refers to the portions of shoots in which all organs are fused to each other, Jäger-Zürn, 2005), endogenous origin of shoots and leaf-shoot mosaic (Rutishauser, 1997; Rutishauser and Huber, 1991; Jäger-Zürn, 2005; Sehgal et al., 2002). The plants of Podostemaceae are exceptional, as they lack aerenchyma, one of the characteristic anatomical attributes of aquatic plants. Instead abundant silica bodies are found in the epidermal cells which may be an adaptation by plants to remain rigid in the fast flowing water. Vegetative reproduction, a frequent feature and a dominant 2 mode of reproduction among the aquatic plants, is also absent in the family. Therefore, these plants are dependent only on sexual reproduction for their perpetuation. Profuse flowering and high percentage of seed production in a fruit, as observed in Podostemaceae, are also unusual among the aquatic plants. The Podostemaceae have a set of embryological features which do not occur in any other family. Interestingly, double fertilization and endosperm formation, the two defining features of the angiosperms, are invariably absent. The lack of endosperm is redressed by a ‘pseudo-embryo sac’, which plays the surrogate and provides nutrition to the developing embryo. The mature embryo is extremely simplified as it lacks an identifiable radicle or plumule. The plant body develops laterally from the hypocotyl, a feature which has not been reported from any other angiosperm. Podostemads are difficult to study due to hitches involved in their collection and the preservation. As seed germination coincides with the rainy season, it is almost impossible to collect the seedlings from the swollen rivers (Khanduri et al, 2009). For several weeks, the plants remain completely submerged. For many decades, the details of seed germination and establishment of the plants had not been clearly understood. In earlier attempts of studies, researchers at the University of Delhi, developed an in vitro technique to successfully germinate the seeds of Podostemaceae (Vidyashankari and Mohan Ram, 1987) thereby enabling a detailed histological investigation of the origin and ontogeny of the thallus and even induction of flowering for the first time (Sehgal et al., 1993). Within the family, there is a marked diversity with regard to size and the number of various plant parts. Generally, the New World podostemads are larger and more conspicuous than their Old World counterparts which is reflected not only in the vegetative structures but also in the number of floral organs. In the Asian Podostemoideae, shoots are unbranched and short (1 cm long or less) (Cusset, 1992), whereas many African and American members of this subfamily form elongate and branched shoots, exceeding over 80 cm in length (van Royen, 1951, 1953, 1954; Taylor, 1953; Cusset, 1987). Similarly, leaves may be simple and thread like as in the Asian Podostemoideae or compound with coiled pinnae as in the American Podostemoideae. The New World podostemads bear numerous, large, polysymmetric and multistemanous 3 flowers arranged in an inflorescence whereas those of the Old World bear solitary, small, monosymmetric flowers with only two stamens. Evolutionarily, there is a progressive dorsiventrality shown by the members of Podostemaceae (Willis, 1902a, 1902b). No other family above the liverworts show such a marked dorsiventrality. Flowers of the New World genera are generally erect, whereas those of the Old World genera are dorsiventral. The structural dorsiventrality of the flowers is accompanied by two other evolutionary trends- (a) reduction in the size and number of perianth, stamens, carpels, ovules; and (b) fusion of the filaments in the stamens. Although these morphological manifestations may not appear significant, they tend to increase dorsiventrality as well as autogamy. A great majority of podostemaceous species exhibit a narrow distribution range (van Royen, 1951; Taylor, 1953; Sculthorpe, 1967; Graham and Wood, 1975; Cook, 1996a, 1996b). Most of them are endemics, restricted to a country or even a single river or waterfall (Mohan Ram and Sehgal, 2001; Novelo and Philbrick, 1993a, 1993b). The only exception is Tristicha trifaria (Bory ex Willd.) Spreng. which has a pantropical distribution (Cusset and Cusset, 1989). Members of Tristichoideae are less specialized and more widespread and those of Podostemoideae are highly distinctive and occupy a narrow distribution range (Willis, 1902b). However, reasons for such a high degree of endemism are still not clear. The Indian sub-continent is home to approximately half of the world’s known aquatic flowering plants comprising 470 species (Lavania et al., 1990). India harbours 10 genera and 28 species of Podostemaceae, of which 23 are endemic and are largely confined to Kerala and Karnataka. Besides these two states, the occurrence of Podostemaceae has been also reported from Arunachal Pradesh, Meghalaya, Assam, Orissa, Madhya Pradesh, Maharashtra, Goa and Tamil Nadu (Cook, 1996b). A perusal of literature indicates that there are several gaps in our knowledge of the biology of these enigmatic plants. Moreover, the extremely fragile riverine ecosystems in which these plants inhabit are under a constant threat due to anthropogenic activities. Thus, it becomes important to strengthen our knowledge of these little known plants which defy not a few but many rules of the biology of flowering plants. 4 We owe our knowledge of Podostemaceae in the Indian subcontinent to the pioneering work of late Sir J. C. Willis (Former Director, Botanic Gardens, Rio de Janeiro, Brazil) and to C.R. Nagendran (Professor, Department of Post-graduate Studies and Research in Botany, University of Mysore). Studies on the morphology and ecology of Indian Podostemaceae were initiated by J. C. Willis way back in 1902. He studied several members of the Podostemaceae from India and Ceylon over a span of nearly 26 years. Since then, the Indian and European workers have carried out detailed taxonomical and embryological studies. The family Podostemaceae has attracted the Indian plant scientists for long because of its unique embryological features. The female gametophyte development and embryo development is the most studied aspect (Mukkada, 1962; Nagendran, 1975; Nagendran and Arekal, 1976; Arekal and Nagendran, 1974, 1975a, 1975b, 1977a, 1977b; Nagendran et al., 1981). However, several issues pertaining to the absence of double fertilization and reproductive ecology have not been addressed in the Indian Podostemaceae. The discovery of dicotyledonous embryo in 1920’s recognized the dicotyledonous status of Podostemaceae and also underlined the fact that embryo in this family develops without an endosperm, as double fertilization is absent. While the developmental biology of Podostemads is still a riddle, understanding the molecular biology of embryo formation and seed development in general has been understood to a certain extent but whether or not a similar regulatory network is present in podostemads, still needs an answer. After Mohan Ram and Vidyashankari developed a simple and reproducible technique of raising Podostemads in aseptic conditions (Vidyashankari, 1988 a, 1988b; Vidyashankari and Mohan Ram, 1987), interesting features of the life cycle of podostemads such as early germination events, origin and development of plant body came to light and paved way for the developmental studies of various genera of Podostemaceae. All the studies reported variable germination patterns which is not only surprising for an angiospermic family but also indicates that the data from seedling culture are useful in investigating the evolution of body plan in Podostemaceae. 5 Recent molecular data have provided an insight into phylogenetic relationships among the podostemads which clearly suggests their possible biogeographic relationships (Tippery et al., 2011; Moline et al., 2007; Koi et al., 2012b). Using a chloroplast mapping gene (matK) and internal transcribed spacer (ITS) regions of nuclear ribosomal RNA gene, the workers have found a common ancestor for a few genera (Kita and Kato, 2004a, 2004b). So far, similar information on the Indian podostemads is lacking, in spite of the availability of sufficient number of species in India. In this work, the main objectives and results have been presented in the form of five chapters. I have focused on four species viz. Zeylanidium olivaceum (Gardn.), Engler, Polypleurum munnarense (Nagendran and Arekal), Podostemum subulatum Gardn., Willisia arekaliana Shivam. and Sadanand for sections on reproductive ecology and developmental biology. The species were selected primarily on the basis of their availability and the fact that little information is available for them. The present work was attempted to study the following aspects: 1. Reproductive ecology of selected species of Podostemaceae. 2. Female gametophyte development and reasons for absence of double fertilization in the selected species of Podostemoideae; Podostemaceae. 3. Developmental morphology and anatomy of seedling development in the selected species of Podostemaceae. 4. Molecular characterization of FERTILIZATION INDEPENDENT ENDOSPERM (FIE) gene from Podostemaceae. 5. Phylogenetic relationships among the Indian podostemads. I performed a detailed study of the floral structure, the temporal and spatial arrangement of floral parts leading to pollination in the selected species. Additionally, the path of pollen tubes was traced from its initiation until the entry into ovules. Besides the details of self- and cross-pollination studies, quantitative and qualitative data on ovule, pollen and seed set has also been provided to establish the breeding systems. The results of above mentioned study are provided in Chapter 1. To determine the reasons for the absence of double fertilization in Podostemaceae, female gametophyte development was observed from the onset of 6 differentiation of megaspore mother cell till fertilization in the species. I have employed resin-embedded sectioning to trace the development of female gametophyte. The details are provided in Chapter 2. Work done by the previous workers became the premise of my work on seedling development. By combining information from the previous studies and with the data generated from my work, I have provided a possible line of evolution of seedlings in the Indian Podostemaceae in Chapter 3. By using fresh material collected from the field, I isolated an ortholog of polycomb group gene FIE from two species of Podostemaceae (Zeylanidium olivaceum, Polypleurum stylosum var. stylosum) and examined its expression patterns through various stages of development of plant. The work forms Chapter 4 of the present study. I generated DNA sequence data from the herbarium specimens and fresh material collected during fieldwork and used molecular phylogenetic methods as well as traditional morphological data to infer the phylogenetic relationships among the Indian podostemads. My work has led to inferring of phylogenetic relationships among Indian species. The phylogenetic study constitutes Chapter 5 of the thesis. 7