The prerequisite of any program aimed at studying genetic variations is to understand distribution pattern of the target species (Bothmer and Seberg, 1995). Because the sampling procedures should recover the greatest amount of the genetic variation of the species, irrespective of the relative frequency or rarity of any genes or linked genetic combinations (Bogyo et al., 1980). In the present study, information available in floras, review articles and other related published literature was utilized to get a clear picture on the distribution of the species (Fig. 5.1). Even though A. paniculata is distributed over a broad ecogeographical range, the so called ‘native populations’ occur only in south India and Sri Lanka (Hooker, 1885; Bhat and Nanavati, 1978). Available literature also suggests that the populations found in North India, Java, Malaysia, Indonesia, West Indies, and elsewhere in Americas are ‘introduced’ (Hooker, 1885; Ridley, 1967; Backer and Brink Jr., 1967; Correll and Correll, 1982). In this study, natural populations along with a few introduced ones were included.

Apart from the wide geographical range of distribution, the habitat preferences of A. paniculata were also wide. The range of habitats (sea shore, river bed, scrub jungle, rocky exposures, fallow lands, way side, backyard of houses in villages) in which the plants were collected is indicative of the ability of the species to colonize newer areas. Besides, being a member of the family Acanthaceae, the species is well specialized in profuse setting of seeds and dispersal of the same by a rather violent bursting of the capsules. The seeds so dispersed could be easily carried away by water to distant places during the rainy season. There is no wonder therefore that this species is not likely to be endangered in the places where it occurs though genetic depletion due to indiscriminate collection might be a reality. Observations in the field also revealed luxuriant growth and seed setting in wet regions of Kerala and Kollidam and stunted growth and poor seed production in plants growing in rocky exposures of Algar Hills, probably accounting for differences in seed viability between collections recorded in the study.

Of the 56 populations collected, samples having limited number of individuals/seeds or seeds having low viability were omitted for purposes of management and planning experiments free from flaws as far as possible. Accordingly, if two populations were brought from nearby localities the one having more number of individuals/seeds was selected. After this preliminary scrutiny, nearly 40 samples were germinated and planted in an experimental plot in the TBGRI campus. Phenotypic variations of these samples were evaluated after 3-4 months and 15 samples were short listed for detailed analyses (Table 3.1). The selection included all the foreign populations except the one from Sri Lanka that showed relatively high seed dormancy.

The first set of 15 populations established as seedlings and grown in pots under uniform conditions, were mainly used to study possible among population variations. For purposes of convenience, these were treated as ex situ samples (or germplasm accessions). However, in order to find out the extent of natural genetic diversity occurring within populations and subpopulations, two metapopulations from South India, one from Sirumalai, Tamil Nadu and the other from Nallamalai, Andhra Pradesh were sampled from their natural habitats. These were designated as in situ populations and the samples were used directly without the need for establishment in pots. The knowledge of genetic diversity both at inter- and intrapopulation levels is important not only for assessing the existing variations within the species across a wide area of distribution but also for conserving populations with maximum diversity in their presumptive centers of origin and distribution (Templeton et al., 1991; Uma Shankar and Ganeshaiah, 1997).

Biology of Andrographis paniculata

Published reports on ploidy levels in A. paniculata differ. Narayanan (1951) reported the first reliable chromosome count of 2n = 28 in A. paniculata. Later in 1957, Raghavan found that 2n chromosome number in this species is 50. Some of the later investigations on the karyomorphology of south Indian Acanthaceae endorsed the latter finding of diploid number as 50 (Govindarajan and Subramanian, 1983). While studying the phylogeny of Acanthaceae, Roy (1988) found that the diploid number in this species is 30. A more recent report on the cytology of A. paniculata revealed 2n chromosome number to be 28 (Anonymous, 1996) substantiating the earlier claim of Narayanan in 1951. Roy and Datta (1988) and Roy and Paul (1991) observed the occurrence of many cytotypes in this species. The present investigation showing the occurrence of n=25 chromosomes (Fig. 4.2) in anther squashes corroborates the findings of Raghavan (1957) and Govindarajan and Subramanian (1983). The possibility of other cytotypes existing within the species, however, is not ruled out.

The present study confirms an earlier report (Anonymous, 1996) that pollen grains are dehisced at least 8-12 hrs before flower opening, thereby favouring self-pollination. A mass of pollen grains was usually seen on the surface of anthers in the bud stage itself. Stigma is also receptive in the bud stage before flower opening and remains close to anthers (Anonymous, 1996). But the present study revealed that some sort of external force is required for effective physical contact between stigma and anthers resulting in successful pollination. When the plants were kept in a closed room there occurred only 10% pollination while the rate was quite high (90%) under natural conditions. From the experiments, it was also revealed that the 'stimulating factor' could not be any biological agents. Instead the extent of this contact may be determined by environmental factors such as wind. Nesom and La Duke (1985) reported the effects of particular environmental factors for successful pollination in Trillium nivale. At night and during periods of cold and rainy weather, the flowers close into a more or less tubular form, forcing contact between the stigmas and anthers thereby favouring effective self pollination in this species.

The flowers of A. paniculata were frequently visited by the honeybee, Apis florea and one or two other unidentified Apis sp. The visitors were often found to carry pollen mass away from the flowers. However, since open pollinated (control) and netted plants gave almost similar percentage of fruits, it is assumed that the honeybees do not contribute to pollination in
A. paniculata. Occasionally certain butterflies do visit the flowers, which again could not be related to any role in pollination. Although cross pollination itself could not be entirely eliminated, it is possible that A. paniculata is essentially self pollinated even before flower opening in the morning and subsequent visits of the honeybee and butterflies during 12 h day are mostly directed towards benefiting the visitors.

Pollen analysis revealed 95% and more fertility of the pollen in line with the high percentage (96%) of pollen fertility reported earlier (Anonymous, 1996). High percentage pollen fertility may as such have a positive effect on self-pollination of the species. The exception was a population procured from a local nursery (AP51), a feral type having 100% sterile pollen grains. Genetic analysis (presence of a unique isozyme marker band in the Est3 locus in AP51, which was lacking in all the remaining populations: indicated by an arrow in Fig 4.13c,) suggested that this is possibly an interspecific hybrid, the interspecific hybridisation as such resulting in the loss of sexual reproduction and hence the plant with enhanced vegetative propagation potential.

Studies on seed germination revealed that freshly harvested seeds seldom germinated and a certain period (3 months) of dormancy / storage is needed with concomitant reduction in moisture content from 17.4 to 13.7% to obtain maximum germination. Even prolonged storage up to 9 months with the subsequent decrease in moisture level to less than 10.0% did not cause significant reduction in percentage germination (Fig 4.3). These results suggest that the seeds of A. paniculata belong to orthodox type where considerable reduction in internal moisture content without loss of viability is noticed. Apart from the observed differences in germination percentage due to moisture levels, possible genotypic effects were also evident (Fig. 5.2) as reported in wheat (Singh and Ahmad, 1997). Seeds of populations collected from Tamil Nadu gave high percentage germination (93.6%) while those from Kerala and Maharashtra gave low values (42.0% and 33.5% respectively).

Fig. 5.2 Possible genotype effects on seed germination in A. paniculata. Populations collected from different states were allowed to germinate under identical laboratory conditions.

Physiology of seed dormancy and the factors involved viz. physical and physiological are of considerable interest in recent years. The effort to breakdown the dormancy of freshly harvested seeds using organic permeation techniques particularly by infusion of seeds with plant regulators in acetone has yielded promising results for many wild and cultivated species
(Persson, 1988). Similar attempts made in the present study suggest that growth regulators break the physiological dormancy of the seeds only partially (Fig 4.4); there may be other dormancy inducing factors not linked to hormone action.

Quantitative and qualitative variations in A. paniculata

Generally phenotypic variations give valuable clue to the underlying genetic variations; however the two do not match always. It so happens because much of the variations that exist within populations or among progenies of the same individual may be selectively neutral (Baur and Schmid, 1996). The variation of phenotypic characters, especially quantitative ones, differs greatly among populations rather than within the populations (Schemske et al., 1994). The present study largely confirms this reported observation as most of the quantitative morphological attributes varied greatly among the studied 15 populations. This is evident from the polygraph presented in Fig. 4.5b. It appears that the distinct morphological characteristics of the populations may be due to certain combinations of genes which had become randomly fixed, as assumed by Stephens and Rick (1966). The representation of variations of individual plants in polygraphs or polygonal graphs (or polygon patterns) is an old (Hutchinson, 1936; Davidson, 1947) but widely appreciated method even today (Olowokudejo, 1986; Rao, 1999). In A. paniculata polygraphs offered an excellent method to compare various populations, thereby enabling the identification of the most phenotypically variable populations, AP18 and AP51. Both size and shape of the polygraphs indicated the overall phenotype of each of the population. This was further substantiated by figures 4.6 - 4.8 which actually confirmed the prevalence of morphological diversity among different populations of A. paniculata.

The existence of significant phenotypic differences in A. paniculata gave reason to assume that the same genotype may show different phenotypes in diverse habitats. The plants growing in moist shady places in the plains had comparatively broader leaves with increased surface area. They also exhibited more branching than those with narrow leaves and leaf/stem pigmentation. The plants growing in hilly tracts such as Alagar hills (Algarkoil) of Tamil Nadu were very small, their height ranged from 5-20 cm with few or no branching at all. These differences may be related to the nature of the habitat itself with the dry hard and rocky terrains supporting less growth of the plants due to non-availability of water and nutrients.

The genetic variation observed between populations was also reflected in the synthesis and accumulation of andrographolide (Fig 4.10). Since the andrographolide content was measured under uniform growing conditions, variations so observed presumably had a genetic basis. Variation analysis (Lush, 1945) was done following the equation: VP = VG + VE, where VP = total phenotypic variance, VG = genetic variance, and VE = environmental variance. Since all the plants were grown under the same growing conditions, influence of factors, if any, affecting the growth of the plants will be uniform to all populations (VE = 0) and therefore, the equation may be rewritten as VP = VG. The phytochemical diversity measured as quantitative differences in the accumulated andrographolide (ranged from 0.73 to 1.47% dry wt with an overall mean value of 0.95±0.17) and the stability of these values through three successive generations indicated genetic basis of these variations. However, a stringent correlation between genetic and phytochemical data may not be possible as the levels of variation in single locus characters do not provide insight into the levels of variation in polygenic character (Lynch, 1996; Waldmann and Anderson, 1998) such as andrographolide synthesis and accumulation.

The data presented in Table 4.2 further suggest the prevalence of intraspecific diversity among populations of A. paniculata in the concentration of the active principle they accumulate. The population AP36 with the highest andrographolide concentration (1.47%) holds the prospect of being developed as a cultivar as it is characterised by significant rates of growth and biomass production. Since the search for new germplasm as part of the present study cannot at best be considered as most extensive, an elaborate study on nation wide collection of germplasm together with the chemical scrutiny may yield even better genotypes with significantly higher levels of biomass and product synthesis that attract agricultural and industrial attention.

Plant pigments, particularly anthocyanins are considered as genetic markers in many instances (Hahlbrock, 1981). In A. paniculata under uniform conditions of growth, variation in colour of the stem, leaves and flowers was not uniform among various populations. For example, chlorophyllous leaves and stem of the populations showed variations from pale green to dark green. Plants growing under direct sun in open lands often showed reddish-brown or pink coloured leaves (Fig. 4.7) and stem mostly towards their maturity. Quantitative analysis of pigments (Table 4.3 and 4.4) clearly substantiated the phenotypic differences in colour of shoots in various populations. It was also evident that along with the increase of anthocyanin synthesis towards maturity, a proportionate decrease in total chlorophyll and carotenoid content occurred (Fig 5.3). The increase in pink-brown pigmentation of leaves and stem of mature plants as function of anthocyanin synthesis was further confirmed. It should be noted that anthocyanin biosynthesis is activated in presence of far red light irradiation (Mohr, 1972; Hahlbrock, 1981) and has protective effect against water stress and high light intensity (Howard, 1966; Veeranjaneyulu and Das, 1984).

Fig. 5.3 Chlorophyll, carotenoid and anthocyanin content of young and mature leaves of A. paniculata. The graph highlights significant inversion of chlorophyll/carotenoid vs anthocyanin concentration during different growth stages.

Genotypic variations in A. paniculata

An enormous amount of genetic diversity is characteristic of natural populations. The combined effects of random genetic drift, restricted gene flow and differential selection pressures mainly influence their genetic structure. These effects lead to low within and comparatively high among population genetic variation in species consisting of small and isolated populations (Nevo, 1983; Holderegger and Schneller, 1994). This was found to be true with A. paniculata also, the natural populations of which are in isolated patches across India and Sri Lanka.

The results of karyotypic analysis in selected populations of
A. paniculata are indicative of an identical situation existing in Plantago sp. Sen and Sharma (1990) analysed seed samples of Plantago procured from different parts of the world representing different environmental conditions. Despite the habitat differences, the populations of a particular Plantago species collected from different areas did not show any difference in karyotype, even though they differed to some extent from the earlier reports (Bocher et al., 1955; Badr and El-Kholy, 1987). In A. paniculata, even though there exist many phenotypic and genotypic variations, they may be at best considered as peripheral (affecting few nucleotide sequences) as they did not act upon the karyotype to show changes in chromosome number.

Generally, in nearby populations having better chances of gene exchange, there should be only a few differences in gene frequency, but if they are far apart, there should be strong differences (Mallet, 1996). The present study revealed moderate levels of genetic differentiation between the 15 ex situ populations (germplasm accessions) indicating possible restricted gene flow between them. None of the populations studied were from nearby localities and the minimum distance between any two of them was greater than 15 km. More over, they were brought from different habitats where they had grown under diverse climatic and edaphic conditions, which in turn might influence their life history characteristics. When brought together and grown in the same experimental plot, the 15 populations expressed differentially for both phenotypic and genotypic characters. While the experiments using the 15 ex situ populations helped to understand the extent of genetic variability and differentiation of the populations, the analysis using the in situ (natural) populations was directed at revealing (a) the extent of intrapopulation genetic diversity of A. paniculata, (b) genetic differentiation of local populations and their subpopulations and (c) theoretical gene flow between the local populations within each of the metapopulations.

Levels of genetic diversity can be better understood in a comparative context. In a comprehensive review on isozyme variation in plant species and populations, Hamrick and Godt (1989) compiled summary statistics of genetic variability in 449 plant species. Comparison of isozyme data with this and other published works indicates that overall genetic diversity in A. paniculata is moderate, in such a way that it has lower polymorphism (32%) and less number of alleles per locus (1.36) than an 'average' crop species (P = 49%; A = 2.15). Unlike isozymes, RAPD analysis will not resolve recessive alleles and hence the bands detected will be either of homozygous dominant (+/+) or heterozygous (+/-) group. As a result, the number of alleles (= bands) per locus (= primer) generated in RAPD assays will be limited. But incorporating more number of primers in the analysis the number of loci can be increased. In the present study, RAPD data was more discriminating and could contribute to an overall polymorphism of 70.5% and the effective number of alleles per locus of 1.61 that are significantly higher than that of isozyme analysis. But these values are comparatively lower than that reported for many crop species (cotton:
P = 89.1%, Iqbal et al, 1997; sweet potato: P = 77.6%, Connolly et al, 1994; Vigna: P = 78%, Sonnante et al, 1997). More over there was not a single primer (out of the 25 studied) that alone could differentiate various populations of A. paniculata. This was earlier reported in Cotton where out of the 50 primers studied, (even though number of primers were more and hence chance of locating a distinct primer was more) not a single one could differentiate clearly between all the varieties (Iqbal et al, 1997).

Since South India is presumed to be the centre of origin and distribution of A. paniculata too, areas rich in natural populations (Sirumalai and Nallamalai) of the species were selected for the studies in intrapopulation variations (Fig 5.1). These areas separated by a distance of more than 1000 km were located near the eastern spur of the Pulney hills in the Western Ghats (Sirumalai) and right in the hot spot of biodiversity that Nallamalai of the Eastern Ghats represents. The lower level of genetic diversity of the Nallamalai population revealed through isozymes and RAPDs was mainly due to reduction in heterozygocity and the lower number of alleles per locus. More over, numerous life-history factors impact levels of allozyme variations in natural populations (Hamrick and Godt, 1989). The paucity of genetic variation in the Nallamalai populations especially those from Srisailam area (N2) mimics a situation described for island populations, where many stochastic factors, particularly founder effects associated with island colonization (genetic bottlenecks) and genetic drift reduce genetic variability considerably (Wendel and Percy, 1990).

Srisailam, is a high altitude and geographically isolated pilgrim centre holding dry deciduous forest segment in the Nallamalai. The extensive collection of flowering plants including A. paniculata by the local traders in this area together with the decimation of the young plants due to recurring droughts may largely responsible for the reduction in genetic variability of natural populations in the area. Low levels of isozyme variation need not be expected in all the natural populations as evidenced from higher variability estimates in few populations from Sirumalai (S1, S2, S3 and S4), wherein the Nei's expected heterozygocity (h) ranged from 0.22 to 0.25, while the overall mean value is 0.17. Witter and Carr (1988) suggested that population size (which is higher in the S1 - S4 Sirumalai populations) and age since colonization can promote greater variability to native populations. Often it is assumed that continuous populations (stretch out to many kilometres, though individual plants may be widely scattered) would tend to retard the loss of genetic variability due to drift, thus promoting the retention of allelic diversity. However, the observed number of alleles per locus varied widely from 1.00 to 1.50 (1.25 to 1.60 in RAPD analysis) in the Nallamalai population and 1.25 to 1.50 (1.47 to 1.73) in Sirumalai population. This variation, though populations are continuous especially in the Sirumalai region, may be attributed to the loss of alleles especially due to the injudicious plant collection practices followed for years by the people in the region. On enquiry with a raw drug vender at Dindigal (Tamil Nadu), the author was told that more than 50 people regularly collect these plants from the Sirumalai forest region.

The fixation indices (mean FST = 0.34) indicated considerable genetic differentiation among the Nallamalai populations. Not only a lower estimate was recorded for Sirumalai populations (mean FST = 0.15) but also values varied widely from 0.00 to 0.36, which indicated a possible situation of differential gene flow as evidenced in the genetic variability analysis too. But the Nei's (1972) original measures of genetic distance indicated that the differentiation between the N1 and N2 (Nallamalai) was only 0.008, while neither N1 or N2 showed any affinity with S1 to S9 (Sirumalai), the least distance estimate being 0.252. The different estimates of genetic differentiation using FST values and Nei's distance estimate are quite significant. It is noted with interest that fixation indices are more appropriate than genetic distances while studying differentiation among subpopulations of the same or different varieties (Jordan and Piedrafits, 1996). Nei's distance is appropriate for long-term evolution when populations diverge because of drift and mutation. The distance is proportional to the time since divergence in the special case of the infinite allele mutation model and equilibrium in the ancestral population. The FST distance (or Coancestry Distance) is more appropriate for short-term evolution, for divergence due only to drift, and in such cases no assumptions need to be made about the ancestral population (Reynolds et al, 1983; Weir, 1990). The Sirumalai plants showed considerable differentiation ranging from 0.004 to 0.355. As expected dendrograms based on UPGMA clustering of the 11 local populations derived from isozyme and RAPD data clearly distinguished the two groups: N1 and N2 clustered into one group while the other group comprised of all the local populations from Sirumalai (S1 to S9).

An interesting point made from studying the ex situ populations, substantiated ‘spatial autocorrelation’ concept described by Sokal et al. (1989). According to this hypothesis if the population is more or less continuous, allele frequencies will fluctuate with distance due to genetic drift and selection. The correlation of allele frequencies should decline with distance in a more or less predictable way. Even though the present study did not aim at correlating the spatial scale, it was observed that among the 15 populations (possibly due to the absence of factors for gene exchange) highly variable genetic diversity parameters (Fig 5.4) were observed. This fluctuation was lowest for the populations brought from various parts of Kerala suggesting a common gene pool for the Kerala populations.

Implications of genetic data for intraspecific classification

The use of isozyme, RAPD and morphological data for intraspecific delimitation of plant species has been discussed in many studies (Wendel et al, 1992; Wendel and Percy, 1990; Maab and Klass, 1995). In A. paniculata maximum genetic identity as revealed by isozyme data was found mainly among different ex situ populations brought from various parts of Kerala. It points to a possible relation between genetic and geographical attributes. Generally the exotic and Tamil Nadu populations clustered at the bottom of the dendrogram were genetically more distant than the populations brought from different parts of Kerala (Fig. 4.14). Further, geographic distribution of various alleles from the dehydrogenase enzymes (ADH, SDH, MDH and GDH) was of considerable significance. Except AP51, AP28 and AP18 which otherwise hinder a clear interpretation of distribution patterns, all other ex situ populations were geographically classified into 3 groups: Kerala (KL), Tamil Nadu (TN) and foreign (FN) populations. The 4-dehydrogenase enzymes account for a total of 5 loci (Adh1, Sdh1, Mdh1, Gdh1 and Gdh2) which are invariant in all the KL populations. But they showed wide variations in TN and FN populations. Other genetic variability parameters such as A and P also supported categorization of these groups. The higher SD values in the TN and FN populations showed unequal distribution of alleles in these populations.

Fig 5.4 Variation in genetic variability measures of 15 populations of
A. paniculata.
Populations 6, 21, 22, 23, 24, 34, 46 and 51 were from Kerala; 28, 33 and 36 from Tamil Nadu and 48, 52 and 53 were exotic.

It was interesting to note that the dendrogram constructed using similarity measures (Gower, 1971; Adams, 1975) obtained from RAPD data ascertained the possible alliance of geographical and genetic relationships (Fig 4.18). All the 3 foreign populations were clustered into one group, i.e. group-3. AP51, showing greater differences with other populations clustered separately and appeared at the bottom of the dendrogram keeping maximum dissimilarity with other populations. However it was surprising to find AP22 clustering nearest to AP51. This may be due to the genetic make up of the population, even though it was not detectable phenotypically. Of the three Tamil Nadu populations, 2 appeared in group-2 (AP33 and AP36) while 1 (AP28) associated itself with the first group. Isozyme studies also depicted a similar situation where AP28 was clustered with Kerala populations. To an extent the RAPD dendrogram showed similarity to the dendrogram constructed using isozyme data.

The grouping of natural populations in the present study also mirrored the geographical and genetic relationships. The populations from Sirumalai and Nallamalai could be easily discriminated using the dendrograms constructed based on isozyme and RAPD data (Figs 4.16 and 4.20). It was found that in both isozyme and RAPD data analyses, the two metapopulations clustered into major arms of the dendrogram. However, there occurred differences within the Sirumalai group between both the dendrograms. The only similarity is that S6 and S8 were grouped together while all the remaining local populations cluster at varying levels.

It is widely appreciated that there could be habitat specific variations in most plant species which could be fixed and become genetic variations during the course of evolution (Briggs and Walters, 1984). Turesson (1943) found that, within collections of Euaropen Alchemilla glabra, A. monticola (A. pastoralis) and A. filicaulis, the plants from Lappland and montane areas were earlier flowering in cultivation than lowland stocks. An entirely opposite situation was also met with certain plants. For example, morphotypes recorded in Gracilaria edulis were not found to be related to habitat, as the various phenotypes could be found in the same geographic locale. Similarly the same type of morphology was found in different localities (Mal and Subramanian, 1990). In A. paniculata, plants collected from different localities exhibited mainly two classes of leaf and fruit morphology (Fig 5.5). The plants collected from northern stations of India and few locations of Tamil Nadu (e.g. Coimbatore) showed leaves having unequal base, prominent venation and ends selectively less slender (a). Fruits were much broader and seeds were of comparatively bigger size. The other group constitutes plants from Kerala and certain parts of Tamil Nadu (e.g. Alagar Hills) which showed more linear oblong leaves (b). Fruits collected from these plants were slender.

Fig. 5.5 Two types of leaf and fruit morphology in A. paniculata. (a) fruits and leaves collected from AP18, (b) fruits and leaves from AP06.

Degree of selfing and theoretical gene flow

The gene flow in higher plants is accomplished by dispersal of seeds and pollen as well as by vegetative mobility (Handel, 1985; Schmid, 1990; Parker and Hamrick, 1992). Gene flow by pollen dispersal is often low in herbaceous plants (Widen and Swenson, 1992) and in such cases a distance of 2 km is usually sufficient to keep varieties isolated in plant breeding (Levin, 1984). None of the 15 ex situ populations selected for the present study was separated by less than 15 km distance from each other. Hence the genetic structure of any of these populations is supposed to be stable provided there is no human intervention by manual gene exchange through seeds. The more similarity met within Kerala (KL) populations pointed to a likelihood of this later situation; a common gene pool of the KL family cannot be ruled out altogether.

Generally increased selfing would increase the genetic differentiation (FST) between the natural populations. Highest selfing was recorded in the N2 population (0.26) which shows highest genetic differentiation (0.37) whereas few populations (S3, S9) with theoretically minimum values of selfing (1.00) have no differentiation (0.00) at all also existed. In connection with the degree of selfing, it is appropriate to consider the rate of gene flow (Nm). Gene flow or the exchange of genes through pollen is a measure of crossing and hence, in general it reduces genetic differentiation among various populations. It was not surprising therefore that theoretically maximum value of gene flow was recorded in the S3 and S9 populations where the differentiation was nil.

Studies on the intraspecific variation of plants using ex situ populations will be successful only when utmost care is taken right at the stage of germplasm collection itself. Furthermore since characterization and maintenance of genetic profile of each collection/population is significant from the conservation point of view, maximum care was taken in this study to avoid intermixing of populations and they were maintained as progeny arrays. At the same time individuals of any one population were planted as groups (6-8 plants per ~20 cm wide pots) thus rendering enough chance for gene exchange. This is very important, as continued selfing is considered as one of the reasons for loss of vigour and productivity in many of the crop plants. As far as A. paniculata is concerned, the possibility of crossing, though at very low rate, was encountered in this study.

It is widely appreciated that genetic diversity within and among local populations is primarily due to the dynamic balance between genetic drift, which causes the local populations to lose genetic diversity but causes an increase in among-population differentiation, and gene flow, which brings new genetic diversity into the local populations and reduces genetic differentiation among populations (Baur and Schmid, 1996). Population genetic theory of finite populations predicts a loss of variation as a result of inbreeding and random genetic drift (Falconer, 1989). But as a medicinal species, the reason for the observed moderate genetic variation in A. paniculata along with high standard deviation for most of the variability parameters between populations may be probably due to unscientific plant collection from the wild including fringes of forests, particularly and even reserved forests by medicinal plant collectors across the country.

Conservation and utilization of intraspecific variations in A. paniculata

Many conservation programmes are arrived at maintaining existing levels of genetic variation in endangered and threatened species (Frankel and Soule, 1981; Simberloff, 1988; Barrett and Kohn, 1991; James and Ashburner, 1997). The moderate levels of genetic variation in an otherwise widely distributed A. paniculata as evidenced from isozyme and RAPD data indicate possible genetic bottlenecks particularly from human interference in the maintenance of genetic equilibrium. Unlike that of rare and threatened taxa, A. paniculata is not confined to specific habitat or distributional range but faces considerable threat mostly from medicinal plant collectors for use in traditional medicine or for preparing herbal extracts/products. It is a recent phenomenon that certain companies such as Natural Remedies India Pvt Ltd (India), Sabinsa Corporation (USA) and iHerb.com (USA) dealing with value addition of theis species through extraction and enchantment of andrographolide have sprung up. Therefore, over harvesting of the otherwise abundantly available medicinal species causes induced drift and resultant genetic erosion in A. paniculata. These must be reduced in order to preserve promising natural populations as part of an in situ reserve or Medicinal Plant Conservative Area (MPCA) particularly in Tamil Nadu where andrographolide rich populations (e.g. AP36 from Coimbatore) have been identified as part of this study. Rapid Assessment Surveys conducted in other parts of India also reveal that A. paniculata figures among 35 plant species showing very high quality marketing in trade and are over harvested in nature at quantity of 1000 MT and more (Anonymous, 2001).

The selection of a high yielding genotype from the wild populations with characteristic biochemical/DNA marker(s) offers an ideal option for making cultivation of A. paniculata economically viable, and also for exercising our sovereign rights over this native medicinal resource. It is particularly so since India has been legally fighting over foreign patents granted on Indian plant products/processes. The most desirable attribute should be the extraordinarily rich content of andrographolide with complementary biomass production in the farmers' field
(Dr. Rajendra Gupta, pers. commun.). Among the populations tested, AP36 with naturally higher concentration (1.47%) of andrographolide and desirable biomass production merits consideration for field-testing. As already stated countrywide scrutiny of the natural populations is a need of the hour for isolating the so-called ideal plant type. Another alternative is to isolate an improved plant type through mutation breeding. In the preliminary trials conducted on chemical mutagenesis, the author isolated a mutant (CM1) out of AP36 with somewhat improved phytochemical (1.53% andrographolide) and biomass production characteristics which were stable through three generations. These results however preliminary they are, potentially signal the feasibility of developing improved cultivars and consequently suggest the need for greater efforts in this direction. It is the well considered opinion of the author that the two way approach viz. selection of an improved natural variant for development as a cultivar and isolation of a high yielding cultivar from the selected genotype through mutagenesis should be effectively pursued not only to conserve the existing genetic diversity but also to ensure sustainable utilisation of A. paniculata.