localities in South India, mostly from Kerala. The taxa studied and their sources are provided in Appendices I and II. Verification of species

Transcription

1 3 MATERIALS AND METHODS Polleniferous materials were procured from plants growing in different localities in South India, mostly from Kerala. The taxa studied and their sources are provided in Appendices I and II. Verification of species determination was done at the Jawaharlal Nehru Tropical Botanical Garden & Research Institute, Palode, Trivandrum, Calicut University Botany department and at the Regional Centre of the Botanical Survey of India, Coimbatore. Voucher specimens were prepared and deposited at the Herbarium of the School of Environmental Sciences, Mahatma Gandhi University, Kottayam. 2.1 Techniques of pollen preparation: The morphological studies of pollen include both Light microscopy (LM) and Scanning Electron Microscopy (SEM) Light Microscopy (All the taxa). The pollen preparations have been made by the acetolysis method originally proposed by Erdtman (1952), and subsequently revised by Nair (1960c). The salient steps are as follows: 28

2 Mature flower buds were selected and fixed in 70% ethyl alcohol. Pollen preparations were made by the acetolysis method that was proposed by Erdtman (1952) as given below. Transfer the anthers with alcohol into a polyethylene centrifuge tube. Crush the anthers with a glass rod, sieve through a brass metal sieve into a glass centrifuge tube and centrifuge. Decant the supernatant, add glacial acetic acid to the sediment and centrifuge. Decant the acid and add 5 ml freshly prepared acetolysis mixture (9:1 acetic anhydride and glacial sulphuric acid) and keep the centrifuge tube in a water bath at 70⁰C and bring to boiling. The mixture is allowed to remain in the hot water till the mixture attains dark brown colour, centrifuge and decant off the supernatant. Add glacial acetic acid to the sediment, centrifuge and decant the acid. Wash this with distilled water 3-4 times, centrifuge, decant water and add a few drops of dilute glycerine and keep aside for slide preparation Scanning Electron Microscopy. A sizeable sample of taxa (62 species) was subjected to Scanning Electron Microscopy. For the (SEM) studies the acetolysed pollen grains were sputter-coated with gold in a JFC 1600 Autofine Coater, and SEM micrographs taken using JEOL-JSM 6390 LA instrument. The palynological characters were analysed by Nair s terminology (Nair, 1964). 29

3 2.2 Pollen descriptions and terminology: The exine of pollen grains embody in them the morphological characteristics which are broadly categorised into aperture, exine ornamentation, exine strata, pollen size and shape in the order of importance of its application in plant taxonomy and phylogeny (Nair, 1966). It is generally understood and acknowledged that the characters relating to apertures are primary, those of exine ornamentation secondary and the other characters like pollen size and shape tertiary in their degree of importance Apertures The apertures vary in their number, position and character (NPC), and accordingly the morphological types within a taxon are characteristic. The apertures are elongate (colpate) or circular (porate), and from these fundamental types various other aperture forms are resolved. The apertures are proximal polar, distal polar, zonal (equatorial), or global (peripheral) in distribution; and aperture classes consists of monolete, trilete, hilete (confined to monoaperturates and cryptogams), monocolpate, monoporate, zonocolpate, zonoporate (apertures ranging from 2 to many), zonocolporate, zonopororate, pantocolpate, pantoporate, spiraperturate and inaperturate. 30

4 2.2.2 Exine ornamentation. The exine surface is either smooth (psilate) or provided with excrescences or (and) depressions producing an array of architectural forms, such as spinate, spinulate, baculate, verrucate, gemmate and tuberculate among the excrescence forms; reticulate, foveolate, scrobiculate, fossulate, striate and rugulate among the depression forms (Erdtman, 1952) Exine strata. The ornamentation pattern of the exine surface is in fact the result of an organised set of elements composing the whole exine which is stratified into an internal endoexine and an external ectoexine constituted of radial rods (columella) which are arranged, fused or superimposed by wall material resulting in foot layer, the open columella layer, the tectum and the supratectum over which are elements of ornamentation Pollen Size. The measurement of pollen is an important criterion. In taking the measurements, the polar diameter and the equatorial diameter were provided with regard to radio-symmetric grains. Average pollen size was calculated based on measurements from a random sample of 100 pollen grains in each taxon. The arithmetic mean (, standard deviation (σ) and grain size ranges were given for all the species. For calculating the arithmetic mean ( and standard deviation 31

5 (σ) of pollen size, for both polar (P) and equatorial (E) views, the following formulae were used. Where, f i = frequency of pollen size x i. x i = size of pollen grain in µm for the i th size n = total number of grains = f i Shape. The shape of pollen is of least importance. But it has been shown that the percentage of shape classes within any taxon has significance in comparative morphology and taxonomy. Erdtman (1952) has recognised several shape classes on the basis of the formula P/E x 100 for radiosymmetric grains. Subsequently Walker and Doyle (1975) have proposed nine shape classes, also based on the value of P/E x 100 as given below. 32

6 Different shapes of pollen grains: P/E x 100 Shapes <50 Peroblate Oblate Suboblate Oblate-spheroidal 100 Spheroidal Prolate-spheroidal Subprolate Prolate >200 Perprolate Apart from the conventional pollen terminologies already given, a few other special terminologies are crucial in describing the myrtalean pollen. They are as follows:- 1) Subsidiary colpi (pseudocolpi): According to Faegri and Iversen (1964) a pseudocolpus (pseudopore) differs from a normal furrow (pore) in that it is not an exit for the pollen tube, while the definition of Erdtman (1971) is Colpoid streaks not functioning as apertures. Muller (1981) considers the term to be a misnomer because pseudocolpi function in the volume changes of the pollen grain during expansion and contraction in response to moisture content. Therefore he recommends the term subsidiary colpi and this is in complete accord with Patel et al., (1983). Subsidiary colpi in general have a thinner 33

7 ektexine than the surrounding mesocolpial areas but in contrast to the colpi, all exine layers are usually represented. Frequently the thinning of the ektexine is gradual and thus the subsidiary colpi are not as clearly delimited as the colpi, although the endexine is increased in thickness just as in the colpi. Subsidiary colpi are either equal to the number of colpi (isomerous) and alternating with them or there can be additional subsidiary colpi. 2) Intercolpar concavities: As originally defined by Wodehouse (1928) for pollen in the tribe Mutisieae of Compositae with unique possession of three distinct concavities appearing as impressions. Since these impressions are between the furrows, the term intercolpar-concavities is used whose position on the equator suggests further designation as equatorial concavities. These are suggested to be structurally and functionally similar to subsidiary colpi, distinguished only in that they are markedly larger. 3) Heterocolpate: This term suggests the presence of subsidiary colpi or intercolpar concavities in addition to colpi. This was originally defined by Faegri and Iversen (1950). 4) Viscin threads: These are sporopollenin containing threads, and form remarkable structural features of pollen in some angiosperms. They are themselves non-sticky, thin, long, flexible, nonelastic fibres 34

8 basically located on the surface of the pollen tetrads or single grains. One end is free and the other end is attached to the exine of the pollen. 5) Longicolpate: The colpi are long; colporate grains are longicolpate when the colpi are longer than the distance between their apices and the poles (Pike, 1956) 6) Syncolpate: In syncolpate grains, the colpi are either straight, that is meeting at the poles without becoming wider or curved where they are wider at the poles and form a triangular area. In parasyncolpate grains, the colpi bifurcate at the poles and their branches meet and outline a triangular apocolpium. 7) Brevicolpate: In the brevicolpate grains, the length of the colpi is equal to or less than the distance between their ends and the poles (Erdtman, 1971). In the brevissimicolpate grains, the colpus length is less than that of the underlying endoaperture (Erdtman, 1971). The description of the various morphological features of the pollen grains are presented in the order, the aperture character, shape, size, (range, x,σ), exine sculpturing. Photomicrographs of pollen were taken using LM and SEM and are provided in different plates for all the taxa studied. 35

9 Fig. 1. Study area