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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
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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
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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
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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.
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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.
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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
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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.
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