Abstract: Pterocarpus angolensis is one of the most valuable timber trees in Tanzania currently extensively exploited. This study was conducted to obtain baseline information on its genetic diversity and population structure in order to formulate effective conservation plans. Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic diversity in Pterocarpus angolensis DC. collected from six natural populations in eastern part of Tanzania. A total of 42 individual plants were analysed using 11 most informative RAPD primers which amplified 73 scored DNA bands showing 75.3% polymorphism. Cluster analysis is used unweighted pair group method with arithmetic average (UPGMA) formed three major clusters which illustrated that most individuals from a given population tended to cluster together and were therefore more genetically similar than individuals from different populations. Most of the genetic variations were found to be high within population 77.13% and low among populations 22.86%. The genetic diversity information of P. angolensis populations reported in this study is very important component for efficient conservation and effective management of its genetic resources.
INTRODUCTION
Pterocarpus angolensis DC. is a leguminous tree species which belongs to the family Fabaceae. The species grows naturally in the miombo woodlands of sub-Saharan Africa and is one of the most valuable timber species within its distribution. P. angolensis is used for a range of products such as furniture, veneer, carving, general-purpose timber and traditional medicine (Monela et al., 1993; Van der Reit et al., 1998). The natural stock of large P. angolensis trees is dwindling in many areas due to commercial logging throughout their range also as a result of the valuable products derived from the species (Schwartz et al., 2002; Shackleton, 2002; Caro et al., 2005). The species has poor seed germination and collection of seed for the purpose of raising plants in a nursery is difficult because many pods are empty (Msanga, 1988; Chisha-Kasumu et al., 2007). Little also has been achieved through vegetative propagation (Magingo and Dick, 2001). High exploitation pressure to the species is threatening its genetic diversity and future existence in its natural habitats (WCMC, 1998).
Continued deforestation of tropical tree species has not only reduced size of natural populations or eliminate local populations but also may contribute to the declining genetic diversity of these species (Schwartz et al., 2002; Newton et al., 2002). The situation is particularly grave with regards to some species of economic importance, such as the P. angolensis. Efficient conservation strategies for a particular plant species can drafted if baseline information of its genetic diversity and population genetic structure is known (Newton et al., 2002). Preservation of genetic variation and evolutionary processes in viable populations has an ecological importance of preventing potential extinction for the species (Segarra-Moragues et al., 2005).
Different methodologies of assessing variability at the DNA level have been widely used, there are Restriction Fragment Length Polymorphisms (RFLP), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR) and others. Among the various used markers, Random Amplified Polymorphic DNA (RAPD) is the most popular DNA based markers (Bekessy et al., 2002; Jena et al., 2004). These markers have several advantages over other DNA marker as they are technically least demanding, relatively low cost, use small amounts of target DNA and offer a fast method for providing information from a large number of loci, particularly in species where no studies have previously been undertaken (Fontaine et al., 2004). Moreover, in terms of results, the diversity assessed with RAPDs is comparable with that obtained with Restriction Fragment Length Polymorphism (RFLP) or allozymes (Esselman et al., 2000). The use of RAPD markers in population genetic studies has been well established for tree species such as Melia volkensii (Runo et al., 2004), Vitellaria paradoxa (Bouvet et al., 2004), Rachylaena huillensis (Machua et al., 2007), Oroxylum indicum (Jayaram and Prasad, 2008) and Dalbergia melanoxylon (Amri et al., 2009).
Previous studies in assessing diversity of P. angolensis have used morphological methods (Chisha-Kasumu et al., 2009a). However, compared to molecular markers the uses of morphological markers for estimating genetic diversity in plants have limitations due to effect of stage of development or environment (Moyib et al., 2008). Morphological traits are also controlled by a relatively small number of loci (Bruschi et al., 2003). Although, Chisha-Kasumu et al. (2009b) has previously reported genetic diversity of P. angolensis populations from Zambia and Zimbabwe, there is limited knowledge available on the basic biology and genetic diversity P. angolensis populations in other areas including those from Tanzania. The information on genetic variability of P. angolensis is important for effectively formulating conservation, tree improvement and domestication plans for long term survivor of the species. This work was carried out using RAPD markers to evaluate the genetic diversity within and among P. angolensis populations in order to suggest appropriate conservation and management strategies.
MATERIALS AND METHODS
Study populations and sampling methodology: This study was conducted from August 2008 to September 2009. Leaf samples were collected from six natural populations of P. angolensis located in Morogoro region, in the eastern part of Tanzania; namely Iwonde and Nyanganje in Kilombero district, Kihiliri in Kilosa District. Other populations were Dindili, Mkindo and Kitulanghalo in Morogoro District. The information pertaining to the locations of the populations such as co-ordinates and altitudes were recorded using Global Positioning System (GPS) for each population (Table 1). Within each population, seven individual plants of P. angolensis were randomly selected at a distance of at least 100 m, in order to avoid sampling of closely genetically related individuals. Five young leaves were collected from each tree and dried rapidly in a zip locked plastic bag in the field using silica gel.
| Table 1: | Description of sampling sites of P. angolensis populations |
The collected leaf samples were stored at room temperature until DNA extraction.
DNA extraction: DNA was extracted from dried leaves, following the modified protocol as described by Dellaporta et al. (1983).
RAPD analysis and primers screening: A Polymerase Chain Reaction (PCR) for RAPD profile was carried out in a final volume of 25 μL containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1 mm mixture of dNTPs (dATP, dCTP, dGTP and dTTP), 2 μL of primer, 1 unit of Taq DNA polymerase (Sigma) and 1 μL of genomic DNA. Amplification of RAPDs was performed in Eppendorf Mastercycler (Applied Biosytems, USA) thermocycler. Optimal amplification PCR conditions used for RAPD were 1 cycle of 3 min at 94.5°C (initial denaturation), followed by 35 cycles of 1 min at 94.5°C (denaturation), 1.30 min at 40°C (annealing) and 2 min at 72°C (extension). A final step of 5 min at 72°C ensured full extension of all amplified products. Amplified PCR products were separated alongside a molecular weight marker (100-bp DNA ladder, Biolabs, USA) by electrophoresis in 1.4% agarose gel run in 0.5X TBE (Tris Borate EDTA) buffer, with subsequent staining with ethidium bromide (0.5 μg mL-1) at 100 volts for one hour. The gels were photographed under UV light and the images transferred to microcomputer for further analysis of amplified bands.
Thirty decamer primers of arbitrary sequence (‘Kit KFP’ provided by Integrated DNA Technologies Inc., USA) were initially screened to determine the suitability of each primer for the study. Eleven primers were selected for further analysis of all individuals based on their ability to amplify DNA, band intensity, number of loci amplified and reproducibility of the RAPD products (bands) over two independent runs. Only data from clear, intensely staining unambiguous bands were used for analysis.
Data analysis: The RAPD products (bands) were aligned and data were scored in a binary manner as either presence (1) or absence (0) of a DNA band at a particular locus to compile a binary matrix for cluster analysis. Only intensely stained bands were scored. The resulting data matrices were analysed using the NTSYS-pc software Version 2.10 t (Rohlf, 2000). The genetic similarity matrix of individuals was calculated using Dice coefficient (Dice, 1945) and cluster analysis run on Sequential, Agglomerative, Hierarchical and Nested (SAHN) using the Unweighted Pair Group Method with Arithmetic average (UPGMA). The binary data matrix was treated with POPGENE software Version 1.32 (Yeh et al., 1999) to calculate the genetic diversity parameters which include the gene diversity (He), Shannon’ s information indices (I), observed number of alleles (Na), effective number of alleles (Ne) and percentage of polymorphic loci. The mean Shannon’s information indices (I) was used as (Hpop) for the average diversity of the six populations and the total diversity of the whole sample population was estimated as (Hsp). Then the proportion of diversity within populations was calculated as Hpop/Hsp and the proportion of diversity among populations as (Hsp-Hpop)/Hsp.
RESULTS
Eleven most informative RAPD primers out of thirty primers screened were selected for DNA amplification reactions because they yielded many highly repeatable polymorphic bands. These primers generated a total of 73 reproducible RAPD bands with fragments ranging from 200 to 1200 bp, of which 55 (75.3%) were polymorphic (Table 2). The bands per primer produced ranged from 4 to 9 and the average band loci per primer were 6.6. Primers KFP04 gave the highest degree of polymorphic bands (87.5%) followed by 03 and KFP24 with 83.3% each, respectively. Primers 18 and KFP10 gave low polymorphism indices 62.5 and 66.6%, respectively (Table 2).
Cluster analysis: The UPGMA dendrogram based on Dice’s similarity coefficient obtained using the 73 RAPD markers exhibited three main clusters, cluster I-III, when truncated at (0.82 coefficient) 82% similarity level (Fig. 1) cluster I was the largest cluster which consisted of two sub-clusters mainly with individuals from MKD and DNL and some individuals from KTL. Genetic similarities for cluster I ranged from 85 to 94%. Cluster II was formed by individuals from population NGJ and IWD in which similarity level ranged from 83 to 97%. In cluster III, 90% all individuals were from population KHR at similarity level ranged from 82 to 92%. Based on the UPGMA dendrogram, similarity coefficients ranged from 75 to 97% among the 42 samples of P. angolensis. The dendrogram generally showed clustering reflecting both the population origin and levels of diversity, as determined by the Nei’s genetic diversity indices.
Genetic diversity estimates of the populations: Estimates of Nei’s genetic diversity, based on RAPD data, varied from 0.1142 to 0.1916 with an average of 0.1468 and the total gene diversity parameter was 0.2775. The Shannon’s indices (I) average diversity was estimated to be 0.2156 ranging from 0.1685 to 0.2769 at the population level and 0.4084 at the species level. The lowest diversity value was for Iwonde and highest value was for Kihiliri (Table 3).
At the population level, the observed number of alleles (Na) in the six populations of the P. angolensis varied from 1.3014 to 1.4521, with an average of 1.3813 (Table 3).
| Table 2: | Number of scored bands per primer and polymorphism in P. angolensis |
| Fig. 1: | UPGMA dendrogram computed from genetic similarity matrix using Dice’s similarity coefficient for six populations of P. angolensis. Populations denoted as MKD: Mkindo, DNL: Dindili, KTL: Kitulanghalo, NGJ: Nyanganje, IWD: Iwonde and KHR: Kihiliri. Numbers I-III are clusters |
| Table 3: | Genetic parameters variation in populations of P. angolensis based on RAPD analysis |
The effective number of alleles (Ne) ranged from 1.2007 to 1.3441, with an average of 1.2601 (Table 3). At species level, the observed number of alleles (Na) was 1.7397 and the effective number of alleles was 1.4891 (Table 3). At the population level, the percentage of polymorphic loci ranged from 30.14 to 45.21%, with an average of 38.13%. The Kihiliri population had the highest value of polymorphism while the Iwonde population had the lowest (Table 3).
| Table 4: | Genetic distance matrix for P. angolensis populations based on RAPD analysis |
| Populations denoted as: IWD: Iwonde, NGJ: Nyanganje, KHR: Kihiliri, MKD: Mkindo, DNL: Dindili, KTL: Kitulanghalo | |
A total of 73.97% polymorphic loci were found at the species level in the six populations indicating that P. angolensis had high genetic diversity. The mean diversity within the six populations (Hpop) of P. angolensis was 0.2156 and the total diversity of the whole sample population (Hsp) was 0.2795. The proportion of average diversity within populations (Hpop/Hsp) was 0.7713 (77.13%) while the proportion of diversity among populations (Hsp-Hpop)/Hsp was 0.2286 (22.86%) (Table 3).
The genetic distance of populations: To further evaluate the relationships among the populations, individual RAPD haplotypes were used to estimate population differentiation. The result for the genetic distances between populations of P. angolensis ranged from 0.0412 to 0.3294. The maximum value appeared between the samples from Mkindo and Iwonde, while the minimum value appeared between the samples from Kitulanghalo and Dindili (Table 4).
DISCUSSION
Analysis of the RAPD markers using cluster analysis and genetic parameters measure techniques demonstrated similar interpretations of the genetic diversity and structure of P. angolensis populations. The study revealed a high level of polymorphic loci (73.9%) at the species level in the six populations of P. angolensis. The UPGMA dendrogram illustrated that most individuals from a given population tend to cluster together and are therefore more genetically similar than individuals from different populations. Clustering of individuals from one population illustrate that there is an effective gene flow between populations. The clustering pattern found in this study is in agreement with Chisha-Kasumu et al. (2009b) and ascribed such pattern as a result of effective gene flow contributed by flower density, timely synchronisation of flowering periods and plant-pollinator interactions. The genetic distance among the populations is also important for effective gene flow. The movement of genes among populations has a significant influence on the distribution of genetic variation (Hamrick and Godt, 1990). The pattern of isolation by distance is often observed in insect-pollinated tree species when long distances within the natural range are considered (Bekessy et al., 2002; Juchum et al., 2007).
The results of the partition of variation within populations of P. angolensis by Shannon's information measure showed high genetic diversity within populations (77.13%) although slightly lower to (80.82%) reported by Chisha-Kasumu et al. (2009b) for P. angolensis populations from Zambia and Zimbabwe. The genetic diversity within populations of P. angolensis is within the range that has been estimated for other tree species reported by Newton et al. (2002) and Bouvet et al. (2004) in which Shannon’s diversity index within populations ranged from 42 to 89.7%. It has been reported that a close association exists between breeding system and the distribution of genetic diversity among population (Hamrick and Godt, 1996; Lacerda et al., 2001; Nybom, 2004). Inbreeding species are generally characterized by high levels of genetic differentiation among populations, whilst outcrossing species tend to retain considerably low variability among populations (Hamrick and Godt, 1990). According to Frankham et al. (2004), reproductive system is the most important factor in determining the genetic structure of plant populations along with the effects selection, genetic drift, mutation and migration. Studies of the biology of flowering and pollination in P. angolensis indicate that it is an outcrossing (Mbuya et al., 1994). Hamrick et al. (1991) and Nybom and Bartish (2000) reported that long-lived woody plants, outcrossing in natural ranges are likely to have high genetic variability within and low among populations. The genetic variability within populations is a very important measure of species adaptation to environmental changes and of species survival (Sofia et al., 2006).
Genetic differentiation estimates among populations for outcrossing species have been found to range between 15-38% when RAPD markers were analysed using Shannon's index (Bussell, 1999). The genetic diversity among populations of P. angolensis 22.86% is within the range and is in agreement with other outcrossing plant species that have been reported with similar range of values of diversity among populations, e.g., Cedrela odorata (37%; Gillies et al., 1997), Alkanna orientalis (32%; Wolff et al., 1997), Silene tatarica (36.9%; Tero et al., 2003). An explanation for the rather low, among population diversity for these species is the outcrossing nature of species which is the most important determinant of population genetic structure (Nybom and Bartish, 2000).
Plant species, especially the perennials like trees, rely on the available genetic diversity for stability and survival under the ever-changing environments. Populations with high level of genetic variation are valuable since they offer a diverse gene pool from which gene conservation and improvement programs can be made (Machua et al., 2007). The decrease in the genetic diversity limits species ability to keep pace with the changing selection pressure and their roles in the ecological and evolutionary development of the biosphere (Runo et al., 2004; Machua et al., 2007). Consequently, maintenance of genetic diversity is important as the diversity carries forward both ecological adaptation and microevolution. The over exploitation of P. angolensis thus threatens their genetic diversity and hence might limit their ecological and evolutionary development of the remaining populations. Therefore, knowledge of the levels and distribution of genetic diversity is important for designing conservation strategies for threatened and endangered species (Francisco-Ortega et al., 2000). Based on the information available for P. angolensis the following conservation strategies are proposed.
In situ conservation plan that would guarantee that gene flow is occurring to maintain most of the species’ genetic variation. In situ conservation is very useful for preserving inherent diversity to capture the existing local adaptation (Almeida et al., 2003). For the less diverse populations, efforts should be made for ex situ conservation based on seeds and stem cuttings collections from the population and subsequent reintroduce them into their original parental localities. Domestication of P. angolensis in field plots also proposed as means of conservation and utilization that will ensure local people needs for cash incomes from sale of tree products. Domestication will also decrease pressure on the few natural populations and protect its genetic diversity.
CONCLUSION
Genetic diversity of P. angolensis populations revealed by the use of RAPD data has indicated that there is a still high level of genetic variation in its natural populations studied. Estimates of genetic variation and structure reported herein provide a basis for both in situ and ex situ conservation strategies and effective management of its genetic resource.