Allelic diversity, genetic structure and gene flow of natural populations of Terminalia paniculata Roth. (Combretaceae) from southern parts of Western Ghats revealed by allozyme based assay.
Conservation of plant genetic diversity has recently generated a lot of interest in the tropics as a result of many years of mismanagement, adverse environment as well as socio-economic changes. Plant species, especially the perennials such as trees, rely on the available genetic diversity for stability and survival under the ever-changing environments. Understanding species population genetic structure is essential for their conservation, planning and sustainable management (Sun and Wong, 2001; Daniel et al., 2006). The level of genetic diversity in forest species is a function of both the biological characteristics of the species and the cultural breeding practices. At the species level, the conservation and the maintenance of genetic variation in forest species and their close relatives is a goal of germplasm collection and seed bank establishment (Anass et al., 2006).
Plant genetic resources not only provide basis for life on the earth but most valuable and basic raw materials to meet the current human activities, which have posed a great threat to the plant genetic resources. Therefore, there is an urgent need to conserve these genetic resources (Yu-Pin Cheng et al., 2006) Conversely the loss of genetic variability could render population more vulnerable to extinction in cases of habitat perturbation, reproductive bottlenecks etc (Duran et al., 2005; Wright, 1943).
Maintenance of genetic diversity is considered crucial for long term survival and evolutionary responses of populations to changes in the environment (Hunenke, 1991; Bohumil et al., 2006). In addition, genetic erosion would reduce the potential of the species improvement through selection. Knowledge of allozyme diversity will aid in making reasoned on conservation of forest trees. Terminalia paniculata Roth. (Combretaceae) is a tropical tree with a large natural distribution in Western Ghats. The tree extensively utilized in pharmaceutical, timber tannin, leather and tasar industries (Srivastava, 1993). Due to overexploitation and anthropogenic pressure on this species lead to habitat fragmentation and loss of the species natural population is also on the increase especially in highly settled area. In the present study, allozyme markers were used to characterize genetic diversity in the populations of T.paniculata.
Materials and Methods
Study species and Study site
Terminalia paniculata Roth. (Combretaceae) is a tropical tree species of India found on the Western Ghats between 800 to 1250 m above sea level. Samples were collected from four native populations in eastern slopes of Southern parts of Western Ghats (Courtallam--9 [degrees] 15' N, 77 [degrees] 30' E, Sasthakovil--9 [degrees] 21' N, 77 [degrees] 48' E, Kodaiyar--8 [degrees] 30' N, 77 [degrees] 18' E and Petchiparai--8 [degrees] 25' N, 77 [degrees] 13' E). Entire leaves were collected in field and stored in plastic bags with moist paper towels over ice until electrophoresis was conducted. Leaf sample were collected from randomly 50 trees along transect in each population.
Electrophoretic procedures generally followed Soltis et al., (1983). Leaf samples were prepared using the tris--HCl extraction buffer of Sun and Wong (2001) with 0.6% poly vinyl pyrrolidone, 0.04% EDTA, 0.1% of BSA, 6% Saccharaose, 0.13% Sodium diethyl carbamate and 100 [micro]l-[beta] mercaptoethanol. Small leaf sections were ground in extraction buffer, and the grindate was absorbed onto paper wicks. Wicks were stored at -80 [degrees]C until run on 10.5% starch gels. The following four gel and electrode buffer systems were used to assay the indicated enzymes. System 1 (Soltis et al., 1983): Alcohol dehydrogenase (ADH-EC.188.8.131.52), System 3: Esterase (EST--EC. 184.108.40.206) and Peroxidase (PRX--EC. 220.127.116.11), System 4: Phosphoglucomutase (PGM--18.104.22.168) and System 8 (Haufler, 1985): Aspartate aminotransferase (AAT--22.214.171.124). Staining pattern and genetic interpretation of band patterns of allozymes followed standard principles of Wendel and Weeden (1989). Putative loci and alleles were numbered or labeled sequentially from the cathode.
Using the dominant marker for diploid data analysis procedure of Yeh and Boyle (1997), implemented by POPGENE (version 1.2) software for analysis the genetic diversity parameters. Estimate the allele frequencies at each locus by number of alleles occurring within a population. The effective number of allele (ne) (Hartl and Clarke, 1989) takes into account both the number of allele and their frequencies. The mean number of alleles per locus (A) is calculated with an arithmetic mean of the allele frequency of the particular locus. The loci as polymorphic (P) considered by when the most common allele occurred at a frequency < 0.99 (Nei, 1987). From that the percentage of polymorphic loci present in each population is calculated. The estimation of Shannon's information index (I) used to measure the genetic diversity within and among the population (Shannon and Weaver, 1949).
Estimate the proportion of observed (Ho) and expected heterozygosity (He) under random mating using the algorithm of Levene (1949). Departure from Hardy-Weinberg equilibrium for each population / polymorphic loci combination was examined by Chi-square test. This test was performed to compare observed and expected genotypic frequencies and 0.05 used to probability level. Quantitative estimates of inter populational gene flow (Nm) were calculated following the "private allele model of Slatkin (1985). Nei's unbiased genetic distance (Nei, 1978) was computed for all pair wise combinations of study area. The construction dendrogram based on Nei's genetic distances using UPGMA (Unweighted Pair Group Method with Arithmetic mean).
A total of six allozymes encoded 15 putative loci (AAT-1, AAT-2, AAT-3, ADH-1, ADH-2, PRX-1, PRX-2, EST-1, PGM-1, PGM-2, PGM-3, PGM-4, PGI-1, PGI-2, PGI-3) were consistently resolved in all population of T.paniculata. Of the 15 loci surveyed, 14 were polymorphic (86.66%) in at least one of the populations sampled (Table 1). At the species level, there were 2.92 alleles per polymorphic locus (Ap), the effective no. of alleles was 1.45 and expected heterozygosity (He) was 0.21.
At the population level, on average 68.33% of the loci were polymorphic among the populations Sasthakovil and Kodaiyar having the highest values (86.66% and 73.33% repetitively). Courtallam have the lowest value (46.66%). There were 1.4515 effective alleles per locus, and 2.92 alleles at polymorphic loci. In the case of total number alleles present in the population was more in Sasthakovil (35 alleles) and low (29 alleles) Courtallam were observed. Expected heterozygosity (He) ranged from 0.2104 (Table 1). Totally 16 rare alleles were identified from the overall allele frequency. Three private alleles (ADH-2D, PGI 3D and PGI-3E) in Courtallam population and absent in other populations. The gene diversity (I) found in the populations of Sasthakovil (0.3891 [+ or -] 0.075) was higher than other populations and low at species level (0.3667 [+ or -] 0.093).
The genetic differentiation was observed in all population derived from both observed (Ho) and expected heterozygosity deviation from Hardy-Weinberg equilibrium. For every population mean observed heterozygosity (Ho) was lower than expected heterozygosity (He) at normal conditions. But in the population of Petchiparai the mean observed heterozygosity (0.2089 [+ or -] 0.0485) was higher than mean expected heterozygosity (0.1971 [+ or -] 0.0451). The genetic differentiation of He was very low in Courtallam (0.1864 [+ or -] 0.0372) and high in Sasthakovil (0.2288 [+ or -] 0.0456) (Table 1).
Interpopulational gene flow was estimated as Nm = 11.8859 indicate a high degree of gene flow among populations. The more frequency of gene flow found in ADH-1 allele and gene flow was zero in PGM-3, PGI-1. Genetic distance help to explore the genetic relationship among the populations the average distance between all pairs of populations was low (0.0086). It was lowest between populations Kodaiyar and Petchiparai; Kodaiyar and Courtallam. The genetic distance was high in between populations Sasthakovil and Kodaiyar; Sasthakovil and Petchiparai. The genetic distance values ranged from 0.0002 to 0.0123. The genetic diversity results indicate a strong differentiation of the populations based on their geographic origin. The genetic distance (Table 2) between populations increases with increase in their geographic distance, though not significantly (p > 0.05). Based on Nei's genetic distance, a dendrogram of the four populations was constructed (Fig. 1). The dendrogram indicated three clusters; the first cluster comprised population of Petchiparai and Kodaiyar, while second cluster comprised population of Courtallam. The third cluster comprised the population of Sasthakovil.
[FIGURE 1 OMITTED]
Genetic structure studies of tree species have furthered our understanding of how population genetic architecture is produced by provided insights on patterns of gametic union, gene flow and genetic sub structuring of plant populations (Hamrick, 1989). However, most of the studies to date have focused on temperate species, particularly conifers and tropical species have only recently begun to be studied in detail (Marta et al., 2008). Many temperate tree species have large population sizes, high densities and wind pollination and high out crossing rates, promote large neiborhood size and extensive gene flow. This combination of demographic and reproductive traits typically results in high genetic diversity within populations and little genetic differentiation among population (Brown et al., 1985). Tropical forest trees grow under dramatically different demographic conditions than their temperate counter parts. Tropical trees often have lower population densities than temperate species (Hubel and Foster, 1983).
Species genetic diversity can be interpreted under three criteria. One is the allelic diversity, which is measured as the total number of alleles at each locus in a population or species. A second factor that also accounts for expected heterozygosity and a third factor is the percentage of polymorphic loci present at the species level. Under the three criteria T.paniculata has higher levels of diversity than other plant species with different life history characteristics (Odasz and Savolainen, 1996; Sun and Wong, 2001; Lauren et al., 2008).
The allozyme data of T.paniculata revealed relatively higher variation at the species level (P = 87%; A = 2.67 and He 0.21) in compared with other animal- pollinated out-breeding species (P = 50%; A = 1.99 and He = 0.17) (Hamrick and Godt, 1989). Generally large population sizes and tremendous gene flow among the major factors contributing to this pattern of population structure in T.paniculata. In Spiranthes sinensis also show the higher variation at species level because of its large population size and higher rate of gene flow (Sun, 1996; Marta et al., 2008). But in the case of Goodyera procera is a good example (Wong and Sun, 1999) where despite its outbreeding system, allozyme data revealed relatively low variation at the species level (P = 33%; A = 1.33; and He = 0.15) in comparison with other animal pollinated outbreeding plant species. It shows small population sizes and lack of gene flow among the populations was the major factors contributing to low level of genetic variation pattern.
All parameters of genetic variability were high in all populations of T.paniculata, with some of them being near the maximum known for plants (Hamrick, 1989) and higher than values previously reported (Corrias et al., (1991; Felicia et al., 2006; William et al., 2008). These values are also higher than the average reported for monocots, herbs, long-lived perennials, restricted geographical distributed plants and wind-dispersed seeded plants (Hamrick and Godt, 1989; Daniel et al., 2006; Peter et al., 2008). The higher genetic variability, present in T.paniculata may be due to dependent on pollinator behaviour. The pollination biology of T.paniculata showed that the insects usually remain for a long time on the flowers and inflorescence thus being able to promote pollination between flowers of the same tree (Thangaraja and Ganesan, 2008). The similar type of trend was also observed in Pleurothalis sps. (Meve and Liede, 1994).
T.paniculata has self-incompatibility species and dichogamy it will help to prevent self-pollination. These mechanism in conjunction seen to be effective in promoting seed set by cross pollination as suggested by the high degree of genetic variability actually found. The genetic variability found in T.paniculata is similar to or higher than that usually found in cross-pollinated species, while autogamous ones tend to have much lower values of FIS value is low. Barba and Semir (1999) noted that these combined mechanisms frequently occur in fly-pollinated species in which the pollinators stay for a long time in flowers of the same individual. This, in turn, may help to maintain high genetic variability in these species.
Breeding system, seed dispersal and, most importantly, geographic range has predictive roles in shaping the genetic structure of populations. More widespread species, as well as species that outcrossed tend to have greater diversity within population (Brenan, 1983; Nicholas et al., 2008). T.paniculata occurs in gregarious groups along river systems in waterlogged conditions. During floods, the seeds of T.paniculata are often carried to long distances and deposited along the banks of rivers giving rise to gregarious stands. Thus populations of Kodaiyar and Petchiparai occur along the same river and geographically related have greater chances to be related and result in low genetic variation between these two populations.
Rare alleles (with an allelic frequency less than 0.05 within given population) were more abundant in Sasthakovil populations (12 rare alleles) than in others. The frequency of rare alleles is negatively related to gene flow (Slatkin, 1985; Lauren et al., 2008). Hence, the private alleles (alleles detected in only one population; three private alleles (PGI-3D, PGI-3E and ADH-2D) is congruent with overall high gene flow (Nm).
At the population level, the Courtallam population to be genetically depauperate compared to the rest of the populations because the size of their populations was probably seriously affected by anthropogenic pressure. The pressure was created due to tourisms and colonization--extinction episodes. However, no information is available regarding the possibility of such processes to have occurred with respect to T.paniculata of the Western Ghats. Diversity estimates obtained with allozymes indicate that these populations have lower allelic diversity and low level of observed heterozygosity. These results agree with theoretical models that indicate that these parameters are differently affected by population bottlenecks (Nicholas et al., 2008).
The levels of heterozygosity observed in all populations were lower than expected from Hardy-Weinberg equilibrium values, except Sasthakovil populations. Two possible explanations can explain the observed heterozygosity is lower than expected heterozygosity values. First, significant amounts of selfing could be occurring within T.paniculata populations. However, experimental crossings have shown that T.paniculata is strictly self-incompatible. Second, genetic diversity may be structured in neighborhoods, and mating may mainly take place among genetically related and geographically close individuals. The pollinators of T.paniculata have shown that pollinators fly nearest inflorescence while they collect the pollen and nectar.
Turner et al., (1982) was described the genetic structure of the plant species was influenced by nearest--neighbor pollination by pollinators. They concluded that it increases inbreeding, homozygosity and patchiness in the spatial distribution of genotypes. Thus, pollinator behaviour coupled with the less effective specialized seed dispersal mechanisms could be favouring the establishment of neighborhoods of related individuals.
The present investigation reveals that the estimate of allelic diversity and observed heterozygosity, T.paniculata maintains a significant level of genetic variation in the studied regions of the genome. Furthermore, a large proportion of the alleles identified (28 and 34%) occurs at frequencies below 0.05 at Sasthakovil and Kodaiyar. Therefore, T.paniculata is especially vulnerable to the loss of allelic richness due to fluctuations in population size. Based on the present investigation related to genetic variation and genetic differentiation among the populations, authors recommended that for a viable conservation programme, the genetic resources of T.paniculata are best conserved in Sasthakovil and Kodaiyar population. As a priority, the population at Sasthakovil may be targeted for conservation. This would ensure the conservation of a relatively rich proportion of genetic diversity and presence of private allele representative of that existing in other populations.
Uma Shaanker and Ganeshaiah (1997) proposed an alternate strategy involving the establishment of forest gene banks for the conservation of genetic resources of forest trees. Based on that T.paniculata, forest gene bank could be established for the conservation of genetic resources in the Southern Western Ghats. Based on the genetic diversity and gene flow parameters, Courtallam population could serve as 'sink' of the gene pool while those at the Sasthakovil and Kodaiyar can serve as 'source'.
This work was supported by a grant from the Indian Council for Forest Research and Education, DehraDun. The authors thank Er. S.Arunagiri for his help with the collection of plant material, Dr. B. Nagarajan, Scientist-E, IFGTB for technical support, and Dr. K. Jegatheesan for advice and comments.
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(1) A. Thangaraja and (2) V. Ganesan
(1) Department of Biotechnology Kamaraj College of Engineering and Technology Virudhunagar, Tamilnadu, India. 626 001 Email: email@example.com
(2) Center for Research and P.G. Studies in Botany Ayya Nadar Janaki Ammal College (Autonomous) Sivakasi, Tamilnadu, India
Table 1: Genetic variability at 15 allozyme loci in four populations and at species level in T. paniculata. Genetic Population level parameter Petchiparai Sasthakovil Polymorphic 10 13 loci % of Polymorphic 66.66 86.66 loci Mean no. of alleles / loci 2.00 2.30 Mean no. of alleles / polymorphic 2.50 2.50 loci Total no. of alleles 30 35 Rare allele 05 12 Private allele 02 02 Observed no. 2.3333 [+ or -] 1.9333 [+ or -] of alleles 0.4666 0.2559 Effective no. 1.4812 [+ or -] 1.3769 [+ or -] of alleles 0.1354 0.1192 Shannon Information 0.3891 [+ or -] 0.3115 [+ or -] Index 0.0775 0.0791 Observed 0.2222 [+ or -] 0.1797 [+ or -] heterozygosity 0.0516 0.0454 Expected 0.2288 [+ or -] 0.1864 [+ or -] heterozygosity 0.0499 0.0372 Genetic Population level parameter Kodaiyar Courtallam Polymorphic 11 7 loci % of Polymorphic 73.33 46.66 loci Mean no. of alleles / loci 2.10 1.90 Mean no. of alleles / polymorphic 2.50 2.50 loci Total no. of alleles 32 29 Rare allele 09 05 Private allele 00 03 Observed no. 2.0000 [+ or -] 2.1333 [+ or -] of alleles 0.1690 0.1831 Effective no. 1.3679 [+ or -] 1.4678 [+ or -] of alleles 0.1091 0.1371 Shannon Information 0.3302 [+ or -] 0.3614 [+ or -] Index 0.0700 0.0815 Observed 0.2089 [+ or -] 0.2095 [+ or -] heterozygosity 0.0485 0.0533 Expected 0.1971 [+ or -] 0.2163 [+ or -] heterozygosity 0.0451 0.0518 Genetic Species parameter level Polymorphic 14 loci % of Polymorphic 86.66 loci Mean no. of alleles / loci 2.66 Mean no. of alleles / polymorphic 2.92 loci Total no. of alleles 40 Rare allele 16 Private allele -- Observed no. 2.6667 [+ or -] of alleles 1.1751 Effective no. 1.4515 [+ or -] of alleles 0.6677 Shannon Information 0.3667 [+ or -] Index 0.3935 Observed 0.2042 [+ or -] heterozygosity 0.0243 Expected 0.2104 [+ or -] heterozygosity 0.0247 Table 2: Genetic distances (Nei, 1978) and geographic distances (in Kms) for all Pairs of sampled populations of T. paniculata. Population Petchiparai Sasthakovil Kodaiyar Courtallam Petchiparai -- 0.0123 (136) 0.0002 (11) 0.0085 (71) Sasthakovil -- 0.0123 (147) 0.0101 (65) Kodaiyar -- 0.0082 (82) Courtallam --