POPULATION ASSESSMENT AND GENETIC DIVERSITY ANALYSIS …

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POPULATION ASSESSMENT AND GENETIC DIVERSITY ANALYSIS OF

THOTTEA BARBERI (GAMBLE) DING HOU - AN ENDEMIC ENDANGERED

TAXON OF SOUTHERN WESTERN GHATS, INDIA

Femy K. Haneef*1, P. M. Radhamany*2

*1,2Department Of Botany, University Of Kerala, Kariavattom, Thiruvananthapuram,

Kerala, India-695581

ABSTRACT

Floristic survey and mapping were carried out to assess the number of individuals and distribution pattern of

populations of Thottea barberi, an endemic endangered taxon from southern end of Western Ghats, India. The

development of appropriate in situ conservation measures for this endemic species is very critical because of its

high habitat specificity with few individuals. Low population density, population fragmentation and narrow

distribution range were evident from the current survey of the taxon and this reflects the critically endangered

nature of the species. Plant density showed peak value at an altitudinal limit 1200m. One way ANOVA results

proved homogeneity in distribution pattern among populations. RAPD profiling revealed genetic diversity

among and within populations of T. barberi. The phenogram generated by UPGMA showed discrete genetic

structuring in the studied populations by grouping the individuals in five clusters. Principal co-ordinate

analysis supported the observation of the phenogram. The study would provide useful information for future

conservation efforts.

Keywords: Aristolochiaceae, Thottea Barberi, Endemic Endangered Species, Distribution Pattern, ANOVA,

RAPD, Principal Co-Ordinate Analysis, UPGMA Phenogram, Conservation.

I. INTRODUCTION

Conservation of species richness assumes greater importance when the world is facing unprecedented loss of

biological diversity. For effective conservation of rare and endangered species, it is important to distinguish

between cause and consequence of rarity (Fiedler & Ahouse, 1992). Many of the world's rare plant species are

endemic due to their unique soil requirements, habitats, and restricted distribution. Western Ghats is a major

hotspot in India. Most prevailing ecosystems in Western Ghats is Tropical evergreen forests. The high degree of

endemism in the evergreen forests of the Western Ghats can be attributed to the isolation of the Ghats from

other moist formations and the prevailing drier climatic conditions in the surrounding areas (Meher, 1983).

Species richness and levels of endemism have been considered among the leading criteria for defining

conservation strategies. The study of endemic species is important for several reasons. It provides knowledge

about the species’ evolution (Keener, 1983; Major, 1991), it aids in the reconstruction of the biogeographical

history of the area occupied by the species (Humphries & Parenti, 1986), and it determines the species’

vulnerability to extinction. Risk of extinction is reported to be greater for population with a few individuals

than those having many (Pimm et al, 1988). Minimizing the extinction rate of threatened species is a major

challenge in conservation biology. A study of autecology and other biological aspects of a particular species, in

order to reveal the physical and biological reasons leading to rarity have become more relevant and prime

concern today, especially in the conservation context (Jose & Pandurangan, 1994).

The development of appropriate conservation strategies for endemic and endangered species requires its

population assessment, details on distribution pattern, occurrence and availability. To date, the assessment of

threat categories to species is mostly based on qualitative and quantitative observations (Pandey & Well, 1997).

Very few studies have used both qualitative and quantitative attributes for assessment of species status (Bhatt

et al 2006). Long-term survival and evolution of species depend on the maintenance of sufficient genetic

variation within and among populations to adapt to new selection pressures as those exerted by environmental

changes (Barrett & Kohn 1991). Understanding of the genetic variation within and between populations is

therefore required for the establishment of effective and efficient conservation strategies for endemic and

endangered plant species (Hamrick & Godt 1996, Lande 1999). Among the many kinds of available markers,

randomly amplified polymorphic DNA markers (RAPD), represent a simple and cost-effective method to

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evaluate genetic variation, regardless of any previous genomic information about the study organism (Williams

et al. 1990). Conservation of rare species requires maintenance of habitat protection, population size and

genetic diversity (Hedrick & Miller 1992; Lomolino & Channell, 1998; Neel & Cummings, 2003). Hence, the

preservation of plant genetic resources has prime importance and must be addressed adequately on a priority

basis.

The present study is focused on a single species ‘Thottea barberi’ belonging to the family Aristolochiaceae. The

plant is strictly endemic to the tropical evergreen forest of southern Western Ghats, and it is under the threat of

extinction and counted as an endangered species. The current investigation attempts to provide quantitative

details of T. barberi through assessment of distribution pattern and quantum availability of the species and

construction of its distribution map by using Global Positioning System. Random Amplified Polymorphic DNA

(RAPD) technique was employed for evaluating the population genetic structure, representing a diverse

selection of inter- and intra population samples to assess the components of genetic variation, both within and

between local populations.

II. STUDY SPECIES

Thottea barberi is a perennial shrub of height 1-1.5m with alternate lanceolate leaves and small brownish

flowers in axillary cyme. T. barberi was collected for the first time by Barber and validly published by Gamble

in Kew Bulletin in the year 1924. The floristic works made in this region have documented T. barberi as one of

the endemic species in the southern Western Ghats (Gopalan & Henry, 2000). Vajravelu & Daniel (1983)

depicted this taxon in their enumeration of threatened plants in Peninsular India. Nair & Ahmedullah (1988)

identified this plant as one of the endangered species in the Red Data Book of Indian plants published by

Botanical Survey of India, Calcutta. Mohanan & Henry (1994) described T. barberi in flora of

Thiruvananthapuram and they noted the distinguishing characters of this species as glabrous style and free

stamens. Mohanan & Sivadasan (2000) located T. barberi at higher altitudes of Western Ghats, specifically in

three regions named Athirumala, Chemmunji Hills and Pongalappara and emphasized that the plant is strictly

endemic to tropical evergreen forest of southern Western Ghats. Conventionally different species of Thottea are

used as an antidote to snake poison and its root extract has been administered internally as a remedy for

stomach ache (Anonymous, 1985; Ambasta, 1986; Binu, 1999). Mathew & Anto (2007) identified T. barberi as a

larval host plant for butterflies. There are no other scientific reports in T. barberi except some taxonomic work.

STUDY SITE

The study was conducted at the hill chain of the Western Ghats constituting the oriental realm, running parallel

to the west coast of India from 8 ° N to 21 ° N latitudes for around 1600 km, especially the regions of

Athirumala, Chemmunji Hills, Pongalappara and the foot hills of Agasthyamalai peak. These regions are the part

of tropical evergreen forest ecosystem of southern Western Ghats located between 8 o30’ and 8 o45’ North

Latitude and 77o0’ and 77 o20’ East Longitude. The altitude of these sites ranges from 600 – 1500m above MSL.

The area has typical tropical humid climate. The soil is laterite and red loamy. Its geographic position is so

unique that it has a profound effect on flora and fauna of the area.

III. METHODOLOGY

Field survey and Geo mapping

Floristic surveys were conducted to the tropical rain forest of the southern Western Ghats to gather the

information about the distribution pattern and allied aspects of Thottea barberi. The explorations were

conducted at three month intervals for three years from June 2007 to May 2010. Phytosociological parameters

were assessed using 1m2 quadrate (Kent & Cooker, 1992) in various sites where T. barberi is distributed. In

each population 20 X 20 m plots were established and 15 (1 X 1 m) quadrats placed randomly inside the plot.

The pooled quadrat information was used to analyze compositional features such as frequency, density and

abundance. The quantitative data were subjected to one way ANOVA using the SPSS software package [Version

11.5] to find out the heterogeneity / homogeneity of the distribution pattern among populations at the level of

significance p<0.05. Graphs were plotted using mean of compositional features at varying time intervals to

show the variations of different attributes at subsequent visits. Specific details of locations (altitude, latitude

and longitude) of the population were recorded using the hand held Global Positioning System (GPS) for the






construction of map. An evaluation of ecological requirements of the taxon in terms of physical and biological

elements was done during the visits. Especially, the propagational aspects were analyzed to obtain the factors

which obstruct proper establishment of T. barberi.

Random amplified polymorphic DNA (RAPD) profiling

Sampling and DNA Extraction

Individuals of Thottea barberi were randomly sampled from twelve populations of Athirumala and Chemmunji

hills, young leaves were collected from a total of 20 individual plants of each population. The material is flash

frozen in liquid nitrogen and stored at -70°C until DNA extraction. DNA was extracted from this leaf samples

using the modified CTAB method (Doyle & Doyle, 1990). Extracted DNA samples were stored in double

distilled water in required dilution. DNA yield and quality were checked by electrophoresing on 1% agarose

gel stained with ethidium bromide. DNA concentrations of purified samples ranged from 20 to 56ng/µl. Initially

twenty 10-mer primers (Integrated DNA Technologies, USA) were screened among which ten 10-mer oligo

nucleotides with maximum reproducibility and clarity were selected for RAPD analysis. The selected primers

are ONP-01: TGC CGA GCT G; ONP-2: GGT GAC GCA G;

ONP-3:GTA GAC CCG T; ONP-4:CCT TGA CGC; ONP-5: GGA GGG TGT;ONP-6:GGT GAA CGC T;

ONP-7:TGG GTC CCT C; ONP-8:GTC AGT GCG G; ONP-9:ACA GCC CCC A; ONP-10:GGT GCG GGA A

The total reaction volume was 25 µl, which consisted of 2.5µl 10X reaction buffer with 15 mM MgCl2, 4µl of

2.5mM of dNTP, 2.5µl of 0.2µM of a single 10-mer oligonucleotide, 1µl of 0.4 unit Taq polymerase and 20ng

(4µl) of template DNA and 11µl autoclaved double distilled water. All the chemicals except primers were

obtained from Genei (Bangalore, India). The polymerized chain reaction was carried out in a gradient palm

cycler (Corbet, Australia) programmed for 4 min Pre-denaturation at 95oC, followed by 40 cycles of 30 seconds

denaturation at 940C , annealing at 37 0C for 1minute and extension at 720C for 2 minutes and final extension

phase of 72°C for 5 minutes.

Agarose gel Electrophoresis and Documentation

Agarose gel was casted using Agarose, Tris Acetate EDTA (Ethylene diamine tetra acetic acid) and stained

with Ethidium Bromide. TAE buffer was used in running procedure. The dye used with sample is formed of

bromophenol blue and sucrose in double distilled water. After loading the sample in the gel, apparatus was

connected to an electric field (100 volt) of constant strength and direction. A 100bp DNA molecular weight

marker (Genei, Bangalore, India) was used as reference. Electrophoresis was performed in a horizontal gel

electrophoresis unit (Scie Plas, UK). Gel was viewed in a UV-transluminator to detect DNA bands and was

analyzed in detail by gel documentation system (Alpha Innotech, USA).

IV. DATA ANALYSIS

Genetic similarity between populations and between individuals was calculated using Euclidian distance based

on RAPD markers amplified with the ten primers. The amplification products for different samples were

screened for presence or absence of bands, presence was coded as ´1´and when it is absent it was designated as

´0´. Similarity matrix was constructed from the binary data with Euclidean coefficient. The calculation of

Euclidean distance is performed by the Pythagoras theorem (Davis, 1986), where X and Y represents vectors of

the two samples being compared:

[∑( )

]

The minimum distance possible (zero) indicate two identical samples and the maximum distance possible is

unbounded. Unweighed Pair Group Method with Arithmetic average (UPGMA) clustering and Principal

Coordinates Analysis (PCoA) were done by using the Multi Variate Statistical Package (MVSP version 3.1). A

dendrogram was constructed from the UPGMA cluster analysis based on Euclidean’s distances among the

populations and a scatter plot were generated by Principal Coordinates Analysis.






V. RESULTS

Field survey and Geo mapping

During our exploration to the hilly tracts of southern Western Ghats, we were able to locate populations of T.

barberi only from Athirumala and Chemmunji Hills. All other populations mentioned in the earlier literatures

were diminished and has gradually disappeared. Population described earlier from Pongalappara was

completely deteriorated and now the area is dominated by species of Impatiens. The results of survey

conducted in each 3 months interval from June 2007 to May 2010 is summarized in terms of mean with

standard error. These results reveal declining strength of T. barberi in different phenophases of subsequent

visits. The population strength of T. barberi varied from 0.2±0.119 individual/m2 to 0.8±0.172 individual/m2

and frequency of occurrence varied between 36% and 62%. Graphs plotted for each attributes with time

interval showed gradual decrease in the density and frequency but no such relationship is found in the case of

abundance (Figs.2&3). One way ANOVA output of phytosiociological parameters (Table. 1) showed insignificant

variation among the attributes of population, thus it can be concluded that the distribution of populations are

almost similar and it follows a homogenous pattern. In this study T. barberi exhibits highly fragmented

distribution pattern and low population density across the surveyed populations, thus indicating poor

availability of the species in study area. Poor distribution of T. barberi across the sites and localized

distribution in specific pockets (habitats) reflects its endangered status. The density and frequency of T. barberi

was found to increase up to certain altitudinal limits (1200 m) and decrease gradually thereafter, showing its

specific altitudinal preference (Figs. 4&5). Geo mapping details could be observed in the figure.1, the shaded

regions in the figure indicates the natural habitat of T. barberi where its populations are established.

Thottea barberi favour the shaded habitat with cool humid climate. It fails to acclimatize in the lower altitude in

transplantation study. Plants are grown in the sloppy terrain and small hilly pockets of southern Western

Ghats. During the field visit, plant propagational aspects were analyzed and observed that main mode of

propagation is vegetative propagation by root runners. Plants are with numerous flowers but fruit setting

percentage is extremely low. Thottea barberi displays pollen/ovule (P/O) ratio as 246.75. According to Cruden

(1977), the value between 244.7 and 2588.0 is considered as facultative xenogamous in nature. They are

partially self-compatible, but the viability of selfed seeds is lower than that of seeds produced by outcrossing.

Plants and their genes migrate through seed and pollen dispersal. Thottea barberi requires pollinators even in

the process of self pollination because flowers are dichogamous and the positions of anthers are arranged in

such a way that they never come to direct contact with receptive regions of stigma. Thottea barberi established

in the hilly pockets shows slight morphological differentiation in flowers and leaves, they are slightly larger and

flowers are randomly with four tepals. New plantlets are seldom found throughout the study period in the

Athirumala population but in the Chemmunji hills populations are devoid of seedlings. Thottea barberi favours

clonal propagation in sloppy terrain with less shaded habitat where the suitable pollinators are not available.

Plants are susceptible to various fungal diseases and insect attacks and also show stunted growth. Tribal people

of this area largely uproot this plant for medicinal purposes. The sloppy terrain is always subjected to soil

erosion during heavy rain due to deforestation. All these factors adversely affect and distress the harmonious

habitat of T. barberi.


Population of T. barberi from the sites of sloppy terrain shows negligible genetic diversity and regions of small

hilly pockets showed comparatively high level of genetic diversity. Primer 10 produced the maximum bands

(12 bands) but the least number by primer 4 (5 bands). Intra specific polymorphism is high in Athirumala

populations (12.3%) and lowest in Chemmunji hills (1.2%). The dendrogram constructed from a UPGMA

cluster analysis based on Euclidean distance among the populations of T. barberi is shown in the figure 6. Two

major clusters were observed in the dendrogram by separating the accessions of Athirumala and Chemmunji

hills. The second major cluster of Athirumala is further divided into two minor clusters. One of the minor

clusters is again divided into two; these are the populations 3 and 4 from the foot hills of Agasthyamalai with

smallest dissimilarity distance. The maximum dissimilarity distance between Chemmunji hills and Athirumala

is 2.4.






The two dimensional PCO analysis was done based on Euclidean distance to support the data produced by the

UPGMA cluster analysis (Figure.7). The first three coordinates of PCO analysis for populations describe 10.93%,

7.82% and 2.0% of the total variance. PCO reveals maximum of 79.24% variation among the accessions. The

dominant eigen value are represented by the first principal axis as 11.491.

Table 1. One way ANOVA of Phytosociological parameters

Variable N F df p

Frequency 12 0.448 7 0.869

Density 12 0.124 7 0.996

Abundance 12 0.052 7 1.000

N Sample size, F F value, df degrees of freedom, p statistical significance

Figure 1. Graphical representation of Frequency of T. barberi with time intervals in succeeding phenophases.






Figure 2. Graphical representation of Density of T. barberi with time intervals in succeeding phenophases.

Figure 3. Graphical representation of Frequency of T. barberi with increasing altitude

Figure 4. Graphical representation of Density of T. barberi with increasing altitude

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8

Fre

qu

en

cy (

%)

Time interval

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8

De

nci

ty

Time interval

0

10

20

30

40

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70

600 700 800 900 1000 1100 1200 1300

Fre

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Altitude (metres)






Figure 5. UPGMA phenogram for 12 populations of Thottea barberi based on


Figure 6. PCoA scatter plot for 12 populations of Thottea barberi based on Random amplified

polymorphic DNA (RAPD) profiling

Figure 7

0

0.2

0.4

0.6

0.8

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1.2

1.4

600 700 800 900 1000 1100 1200 1300

De

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Altitude (metres)

UPGMA

Euclidean

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12

2.4 2 1.6 1.2 0.8 0.4 0






VI. DISCUSSION

The vulnerability of species to extinction is mainly influenced by habitat specificity, limited distribution and the

small population size characteristic of most endemic species (Rabinowitz, 1981). Species distribution greatly

depends on the current environmental conditions that limit their establishment and development. Thottea

barberi is strictly demanding the climate prevailing in the higher altitudes of tropical evergreen forest. This has

a conservation implication, as the species with specific habitat requirements have greater possibilities of

extinction than the species with broad habitat range. Generally, species that are rare and have a small

population size are subject to inbreeding depression and reduced fitness resulting from homozygosity (Lacy,

1992). Monitoring seed germination of rare and endemic plant species has been also reported to be critical for

planning their ex situ conservation strategies (Kozuharova 2009). Roots of the T. barberi are uprooted

indiscriminately for the medicinal use. The removal of the entire plant before seed maturation ceases the

possibilities of development of future regeneration (Sheldon et al, 1997). A minimum population size is

required for a long term viability of T. barberi as it is a rare and endangered species. It cannot be concluded,

whether the population with distinctive morphology of flowers and leaves are genetic or plastic, that may vary

in response to edaphic, geographical and temporal factors. The need of pollinators is found to be very critical

for the sexual reproduction of T. barberi. The breeding systems, life forms and seed dispersal mechanisms of

plant species have been regarded as the main factors affecting levels of genetic diversity, genetic divergence,

genetic structure within and among plant populations and its geographical distribution pattern (Loveless &

Hamrick, 1984; Hamrick & Godt, 1996; Song et al, 2006; Mable & Adam, 2007)

Studies of within-population genetic structure are essential to the understanding of micro evolutionary

processes in plant populations. RAPD is a fingerprinting method using oligonucleotide primers to search for

variation in the entire genomic DNA and RAPD has a higher resolving power as a fingerprinting technique as it

scans the entire genome for polymorphisms (Williams et al, 1990). Due to several advantages, such as, random

sampling of the whole genome, higher levels of polymorphism than allozyme analysis, and faster and easier

analysis than microsatellites, RAPDs have been widely used in plant population studies (Nybom, 2004). RAPD

techniques are used in the population diversity analysis of T. barberi and it gave better results with high

resolution. High to moderate levels of genetic differentiation among populations is a common pattern in

endemic or narrowly distributed plant species (Hamrick & Godt, 1989; Cruse-Sanders & Hamrick 2004, Nybom

2004). This is applicable to T. barberi where the populations of Chemmunji hills accession show least diversity

but that of Athirumala shows high level of genetic diversity.

Knowledge of genetic diversity and the way it is structured in natural populations is essential in order to adopt

suitable conservation strategies (Ellstrand & Ellam 1993). A high level of population genetic diversity in a

species allows it to better adapt to environmental changes and determines its evolutionary capacity (Frankham,

1995; Hamrick & Godt, 1996). Generally, geographically restricted species exhibit lower levels of genetic

variation than widely distributed species (Hamrick & Godt, 1989). Spatial genetic structure within plant

populations is influenced by various factors such as gene flow, clonal pattern, and microenvironmental

selection( Heywood, 1991; Vekemans & Hardy, 2004). With spatially limited gene flow, populations should be

more inbred and more likely to differentiate in response to local selective force or genetic drift (Kang & Chung,

2000). The optimal balance between clonal and sexual recruitment may vary under variable growing

conditions, each strategy being favoured in different environmental circumstances (Eriksson, 1997; Eckert,

2002). Linhart & Grant (1996) showed evidence from several studies that local adaptation to different

environments (e.g.,soil parameters) can occur on the scale of a few meters (even centimeters). This may also

influence the genetic structure of T. barberi because environmental heterogeneity should promote the selection

of a number of differently adapted genotypes. The genetic diversity exhibited by T. barberi indicates that some

micro evolutionary processes are going on in the ‘Taxon’ especially at Athirumala region, for its efficient

adaptation and establishment; which may lead to the emergence of an ecotype. The ‘Plant’ is struggling hard to

acclimatize in the lower altitude and shows extremely stunted growth. It may be due to some physical or

physiological stresses that come across during its acclimatization. These may alter the production of important

metabolites which has the functional significance to protect the plant in new environment. Thus, for the

efficient conservation of T. barberi, the taxon should be protected in its natural habitat.






Information on intraspecific genetic variation among geographically isolated populations has already been

indicated as essential for the conservation of genetic resources (Gillies et al, 1997; Martin et al, 1997). This is to

ensure preservation of the levels of genetic variation in future generations (Frankel & Soule, 1981). Gillies et al.

(1997) suggested that in situ conservation strategies should seek to conserve populations that show

interpopulational genetic variation, to prevent loss of genetic diversity within a taxon. The same can be applied

to the different populations of T. barberi in the hope of conserving genetic variation. Our observations suggest

that there is a need to conserve the smaller populations of T. barberi in any attempt, in its natural habitat. The

number of populations to be conserved for a given taxon is directly related to the proportion of genetic

diversity that resides among populations (Hamrick et al, 1991). The maintenance of genetic diversity within

and among populations of plant species is a critical issue for a long term conservation program (Oyama, 1993;

Bowen, 1999). Several studies have reported that reduction in population size depresses genetic diversity

(Young et al. 1999, Rossum et al. 2004), and also may lead to decreased fitness (Lujiten et al, 2000). When

decreased fitness seriously affects the existence of T. barberi, self recovery may become impossible and it needs

immediate attention and intensive management for their survival. The study of spatial genetic structure helps

provide guidelines for selecting individuals to transplant during restoration after population destruction and

defines the meaningful within-population conservation unit.

VII. CONCLUSION

In conclusion, the study provided conservation implication for T. barberi, suggesting that, the continuous

decline in population size and recruitment rate, as well as the disappearance of populations of T. barberi is due

to its habitat specificity, stunted vegetative growth, susceptibility to fungal attacks, lack of pollinators, loss of

beneficial alleles due to inbreeding and less seed production. Thottea barberi populations appeared to

maintain high levels of genetic diversity in Athirumala region compared to Chemmunji hills. However,

destruction of their natural habitats may increase the risk of extinction. Partial destruction of this site can lead

to a loss in genetic variation and implies that different subpopulations must be conserved to maintain

differentiated genetic pools of T. barberi. On the basis of high genetic differentiation among populations and the

vulnerability of this species, it is necessary to protect existing natural populations of T. barberi in order to

preserve as much genetic variety as possible.

ACKNOWLEDGEMENTS

We do here by acknowledge our gratitude to Kerala State Council for Science, Technology and Environment,

Government of Kerala for awarding the grant for the project. We also express our sincere thanks to Head,

Department of Botany University of Kerala for providing us necessary facilities to do our work. We are also

thankful to the chief conservator of Forest (Wild Life), Government of Kerala, for giving us permission to visit

the Forest areas of Kerala. We thankfully acknowledge Mr. Sibin N. T. (Forest Ranger, Kerala Forest) and Mr.

Jyothish K. (Field Officer, Spices Board) for all help rendered by them during the field visit. We extent our

gratitude to Dr. G. Valsaladevi, Curator, Dr. E. A. Siril and Dr. Bindu R. Nair, Assistant Professors, Department of

Botany, University of Kerala and Dr. Shaiju P. N., Assistant Professor, Fathima Matha College,Kollam, for their

valuable suggestions.

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