Vol. 12(48), pp. 6709-6717, 27 November, 2013 DOI: 10.5897/AJB2013.12459 ISSN 1684-5315 ©2013 Academic Journals African Journal of Biotechnology http://www.academicjournals.org/AJB Full Length Research Paper Genetic diversity and structure of potamodromous Opsaridium microlepis (Günther) populations in the in- let rivers of Lake Malawi Wisdom Changadeya1*, Hamad Stima2, Daud Kassam2, Emmanuel Kaunda2 and Wilson Jere2 1Molecular Biology and Ecology Research Unit (MBERU) DNA Laboratory, Department of Biological Sciences, Chancellor College, University of Malawi, P.O BOX 280, Zomba, Malawi. 2Aquaculture and Fisheries Science Department, Bunda College of Agriculture, Lilongwe University of Agriculture and Natural Resources, P.O. Box 219, Lilongwe, Malawi. Accepted 21 November, 2013 Studies were carried out to determine the genetic diversity and structure of endangered Opsaridium microlepis (Mpasa) populations in the affluent rivers of Lake Malawi; Linthipe, Bua, Dwangwa and North Rukuru. A total of 200 DNA samples of O. microlepis from four river populations were analyzed at 20 microsatellite loci. The primers’ discriminating power was high (mean PIC, 0.76) yielding a total of 295 alleles with a range of 10-22 and an average of 15 alleles per locus. All the populations were not in Hardy Weinberg Equilibrium probably due to outbreeding that leads to heterozygosity excess. This observation was further supported by heterozygosity excess exhibited by 100% of the population-locus combinations (mean FIS, -0.30) and lack of evidence for genetic bottleneck. The populations exhibited high genetic diversity as evidenced by high mean Shannon information Index (I=1.64) and high observed heterozygosity (Ho = 0.98). Genetic relationships among the populations appear to be less influenced by geographical distance (Mantel’s statistics Z, 0.18; p = 0.6369) implying that the populations do not fit into the isolation by distance model. Nevertheless, the populations are highly differentiated (FST = 0.17; AMOVA among populations = 16%). This is supported by inter-deme migration of less than one individual per generation (Nm=0.91) as determined by Slatkin’ private allele method. Therefore, these populations are probably still large and distinct requiring separate monitoring and management due to inferred restricted gene flow and considerable population differentiation. Key words: Mpasa, Lake Malawi, population structure, genetic diversity, microsatellites, threatened species, conservation. INTRODUCTION The genetic diversity and population structuring of native are viable (Johnson et al., 2001). Recently, molecular species populations is an important feature to be consi- markers have been commonly used in population studies. dered in conservation and management programs. The Simple sequence repeat (SSR) markers are preferable use of molecular techniques to assess patterns of genetic because they are co dominant, highly polymorphic and variation has supported conservation programs, by indi- are considered the most powerful in terms of their resol- cating which species require greater conservation efforts ving power (Creste et al., 2004; Buhariwalla et al., 2005). and also by selecting areas where natural populations In addition, microsatellites have a wide distribution in the *Corresponding author. E-mail: [email protected] or [email protected]. Tel: +265 888 877 858. 6710 Afr. J. Biotechnol. genome and can be easily identified, making them ideal extracted from individual fish and preserved in vials with 95% markers for genetic variability studies of populations ethanol. The samples were brought to Molecular Biology and (Collevatti et al., 2001). The polymorphism obtained with Ecology Research Unit DNA Laboratory, Department of Biological Sciences, Chancellor College, University of Malawi, where DNA microsatellite markers has provided useful information analysis was conducted using 20 microsatellite markers. necessary for management of fish stocks (Alam and Islam, 2005), population analysis and biodiversity conser- vation (Romana-Eguia et al., 2004; Changadeya et al., DNA extraction and amplification 2012). Total genomic DNA was extracted from O. microlepis samples The fisheries production from the wild is generally following a standard SDS- proteinase K/phenol-chloroform protocol declining and with an increase in human population, the as described by Hillis et al. (1996). The study used 20 polymorphic pressure is even greater on the resource (Government of microsatellite loci (Table 1). Amplification of DNA was done in 13.11 Malawi, 1998). Most of the fish species in Lake Malawi µL final reaction volume with 5.7 µl PCR grade water, 1 µl of 10 mM including cichlids and cyprinids are endemic. Studies by dNTP mix, 1.25 µl of 10 x PCR buffer, 1.6 µl of 25 mM magnesium Ambali et al. (2002) have also shown that there are spe- chloride (MgCl2), 0.75 µl of 15 pmol of both forward and reverse primers, 0.06 µl of 5u/µl Taq DNA Polymerase stored in buffer A cies flocks that are endemic to specific areas in the lake. (Promega, USA) and 2 µl of 25ng/ µl template DNA. The amplifi- Pollution, lakeshore development and other human acti- cations were carried out in a Master-cycler gradient 5331 Eppen- vities have degraded critical habitats for the spawning dorf Version 2.30.31-09 with the following PCR conditions: initial and early life history stages of many fishes. In addition, denaturation at 94°C for 2 min, then 10 amplification cycles of recruitment, overfishing and reduction of spawning stock denaturing at 94°C for 30 s, annealing at an optimal temperature for below a critical threshold have prevented many popula- a specific primer pair for 15 s and elongation at 72°C for 30 s. This was followed by 30 amplification cycles of denaturing at 89°C for 30 tions from rebuilding to previous levels of abundance. s, annealing at an optimal temperature for a specific primer pair for Riverine potamodromous cyprinids species are most 15 s and elongation at 72°C for 30 s. The final extension was at vulnerable because they are targeted by fishermen 72°C for 20 min followed by a soaking temperature of 4°C. during the spawning period when they swim up stream. This development has led to the fishery of some in-let Electrophoresis of PCR products on polyacrylamide gel using rivers of Lake Malawi such as North Rukuru, Bua, silver staining technique Dwangwa and Linthipe to be threatened with extinction. Though there are several commercially important riverine The amplified PCR products were resolved using 6% polyacryl- amide gel electrophoresis. The 6% polyacrylamide gel was poured cyprinids such as Mpasa (Opsaridium microlepis) that are under threat, they have been studied to a limited extent in BIORAD Sequi-Gen GT Nucleic Acid Electrophoresis Cell. A total of 6 µL of STR 3X Loading Solution (10 mM NaOH, 95% resulting in inadequate knowledge of their biogeography, formamide, 0.05% bromophenol blue, 0.05% xylene cyanol FF) was origin and genetic diversity (Snoeks, 2004). added to the PCR products in 0.2 mL PCR tubes and denatured in Mpasa, belong to the Cyprinidae family and is endemic Mastercycler gradient at 95°C for 5 min. Then 6 µL of denatured to Lake Malawi. This species was described by Günther PCR products was loaded on the 6% polyacrylamide gel and ran at in 1864, and is mostly caught while on spawning runs in 50 W. The gel plates were fixed, stained and developed following procedures in the Promega Silver SequenceTM DNA Sequencing large rivers (Lowe, 1975; Tweddle, 1981). In recent System Technical Manual. The microsatellites bands were scored years, O. microlepis catches in Lake Malawi and its in-let over a light box using pGem DNA marker (Promega, USA) and rivers have declined (Anon, 2008). The inclusion of X174 DNA/Hinf 1 (Promega, (USA) as band size standard markers. Mpasa in the 2006 IUCN Red List of Threatened species (IUCN, 2006) was because it had been heavily fished Data analysis during its spawning runs up rivers and was considered to have consequently suffered approximately 50% decline in Polymorphism information content (PIC), a variability measure of the past ten years, due to overfishing, habitat degradation each locus, was calculated as described by Zhao et al. (2007): and pollution (Kazembe et al., 2005). This is a cause for concern in the conservation of Mpasa and development n k 1 n 2 2 2 of fisheries in Malawi in general. Therefore, the objective PIC 1 pi 2Pi Pj of this study was to investigate genetic diversity and po- i1 j1 ji1 pulation structuring among populations of O. microlepis in the Lake Malawi’ river in-lets of Bua, Dwangwa, Linthipe Where, pi is the frequency of the ith allele out of the total number of alleles at a microsatellite locus, and n is the total number of and North Rukuru using microsatellite markers. different alleles for that locus. Genepop on the web (Raymond and Rousset, 1995) was used to conduct the following analyses: test for conformity to Hardy- MATERIALS AND METHODS Weinberg Equilibrium (Haldane, 1954; Weir, 1990; Guo and Thompson, 1992), test for genotyping linkage equilibrium using Sample collection Fisher’ method, tests of genic and genotypic differentiation, estima- tion of effective number of migrants using Slatikin’s private allele Muscles tissues of 200 O. microlepis specimens (50 each from method (Slatkin, 1985) and computation of Wrights statistics North Rukuru, Bua, Dwangwa and Linthipe river) (Figure 1) were (Wright, 1969). collected in February and April 2010. Tissue of about 5-10 mm2