International Journal of Genetics and Molecular Biology Vol. 2(7), pp. 130-139, July 2010 Available online at http://www.academicjournals.org/IJGMB ISSN2006-9863 ©2010 Academic Journals Full Length Research Paper Molecular phylogeny of Bulinus (Gastropoda: Planorbidae) reveals the presence of three species complexes in the Albertine Rift freshwater bodies A. Nalugwa 1*, A. Jørgensen 3, S. Nyakaana 1 and T. K. Kristensen 2 1Makerere University Institute of Environment and Natural Resources: Molecular Biology laboratory P. O. Box 7298, Kampala, Uganda. 2The Mandahl-Barth Research Centre for Biodiversity and Health, DBL-Parasitology, Health and Development, Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 57, DK-1871 Frederiksberg C, Denmark. 3The Molecular Systematic Laboratory, the Natural History Museum of Denmark, Sølvgade 83, DK-1307 Copenhagen K, Denmark. Accepted 19 July, 2010 In this study, partial mitochondrial DNA cytochrome oxidase subunit I (mtCOI) sequences (612 bp) of Bulinus snails sampled from 31 freshwater bodies in the Albertine Rift were analyzed to investigate the extent of genetic variation and phylogenetic relationships. Bayesian phylogenetic inferences clustered the samples into three species groups; Bulinus truncatus/tropicus , Bulinus forskalii and Bulinus africanus . Twenty-two haplotypes were identified within the B. truncatus/tropicus species group which clustered into two well-differentiated lineages; with 2.7% sequence divergence between them. Significant genetic variation was also observed within the B. forskalii group, with the Maramagambo forest haplotype being separated by 55 mutational changes from the rest of the haplotypes. The B. truncatus/tropicus species group showed early divergence from the two B. forskalii and B. africanus species groups which were more closely related. A single species B. globosus in the B. africanus species group was identified in the Albertine Rift. We report the presence of five Bulinus species in the Albertine Rift; two in the B. truncatus/tropicus group, two in the B. forskalii group (one species yet to be identified) and one species in the B. africanus group. The findings of this study highlight the limitations of relying solely on shell characteristics to delineate snail species within the genus Bulinus . Key words: Bulinus species, cytochrome oxidase c subunit I, mitochondrial DNA, phylogenetic relationships, Albertine Rift. INTRODUCTION Freshwater snails of the genus Bulinus are widely distri- of Bulinus are on the increase due to the taxon’s role as buted in Africa, the East African islands, the Middle East intermediate hosts in the transmission of schistosomiasis and some Mediterranean countries. Bulinus is comprised in humans, domestic and wild animals. The Albertine Rift of 37 recognized species (Brown, 1994) categorized into is the western arm of the Great Rift Valley and it is four species groups; the Bulinus forskalii group (11 occupied for more than half of its length by water, forming species), Bulinus truncatus/tropicus complex (14 the five great Lakes Albert, Edward, George, Kivu and species), Bulinus africanus group (10 species) and Tanganyika (Kaufman et al., 1996). The Albertine Rift Bulinus reticulatus group (2 species). Taxonomic studies also harbors a number of volcanic crater lakes found along the eastern side of the Rift valley in western Uganda between Kabarole and Kasese districts. The Albertine Rift harbors more endemic mammals, *Corresponding author. E-mail: [email protected], birds and amphibians than any other region in Africa and [email protected]. Tel: +246 -414-532789. Fax: +256- has consequently been declared a biodiversity hotspot 414-530134 (Myers et al., 2000). The geological events that created Nalugwa et al. 131 the mountains of this hotspot have also yielded some of phylogenetic affinities between Bulinus species groups the world’s most extraordinary lakes that harbor a from different localities within the Albertine Rift water number of Bulinus species on which very little information bodies. New data on the geographical distribution and exists regarding their identity, molecular genetic diversity genetic diversity gathered on the potential Bulinus inter- and phylogenetic relationships. Earlier morphological mediate host snails will contribute towards the identify- work to characterize the genus Bulinus at the species cation of target areas for focal schistosomiasis control in level proved problematic due to high levels of variation the Albertine Rift. especially in shell form within and among populations (Mandahl-Barth, 1965; Brown, 1994). Techniques such MATERIALS AND METHODS as morphometrics (Kristensen and Christensen, 1989) and biochemical studies (Rollinson and Southgate, 1979; Sample collection and DNA extraction Rollinson and Wright, 1984; Jelnes, 1986; Mimpfoundi and Greer, 1990), cytogenetic studies (Burch, 1960; Snail samples were collected from 26 localities across the five great Goldman et al., 1980; Brown and Shaw, 1989) and lakes (Albert, Edward, George, Kivu, Tanganyika) and ten crater molecular genetic studies (Stothard and Rollinson, 1997; lakes as well as permanent and temporary ponds found in the Albertine Rift as shown in Table 1 and Figure 1. The taxonomic Raahauge and Kristensen, 2000; Stothard et al., 2002; status of the sampled individuals was assessed by shell Jorgensen et al., 2007a; Kane et al., 2008) have all en- morphology using the field identification key of Kristensen (1987) abled researchers to search for genetic differences that (depicted in Figure 2). may help to solve the taxonomic questions in Bulinus . The sorted Bulinus snails were later preserved in 80 % ethanol Over the past two decades, molecular approaches and stored in the laboratory at -80°C. The frozen samples were thawed at room temperature prior to DNA extraction. Genomic DNA have increasingly proven to be valuable not only in was extracted from individual snails using the DNeasy Tissue Kit resolving phylogenetic uncertainties, but also in providing (Qiagen, Inc. Valencia, CA), following the manufacturer’s an insight into the time scales of evolutionary divergence. instructions. Unlike the relatively slow evolving nuclear rRNA genes that have been widely used in studies attempting to resolve relationships among groups that have a long Amplification of mtDNA COI and sequencing history of evolutionary divergence, the more rapidly The mitochondrial cytochrome oxidase subunit I (mtDNA COI) evolving mitochondrial coding genes are increasingly fragment was amplified using universal primers LCO1490 (5´-GGT being employed to infer relationships among groups with CAA CAA ATC ATA AAG ATA TTG G-3´) HCO2198 (5´-TAA ACT a more recent ancestry. TCA GGG TGA CCA AAA AAT CA-3´) (Folmer et al., 1994). Mitochondrial cytochrome oxidase subunit I (mtCOI) Amplification was performed with Taq DNA polymerase (Roche) in and cytochrome b (cyt b) have so far been the favorite 50 µL total reaction volume using the following PCR profiles: initial denaturation (5 min at 95°C), followed by 35 - 40 cycles of candidate genes for this purpose. Although both genes denaturation (2 min at 94°C), annealing (2 min at 56 - 58°C), show a high incidence of base substitutions at third posi- extension (2 min 30 s at 72°C) and final extension (5 min at 72°C). tion nucleotides thereby allowing the discrimination of PCR products were purified using the QIAquick PCR purification Kit closely related species, mtCOI possesses two important (QIAGEN) and sequenced in both directions using Big Dye advantages over cyt b, both associated with its slower Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an automated ABI PRISM ® 3700 DNA sequencer (Applied rate of molecular evolution. Firstly, the universal primers Biosystems). for this gene are very robust, enabling the recovery of its 5' end from most animal species and secondly, mtCOI has a greater taxonomic signal range than cyt b. These Genetic variation and phylogenetic analyses characteristics have therefore made mtCOI a popular molecular marker for resolving both recent and deeper Nucleotide sequences were multiple aligned using ClustalW v1.4 (Thompson et al., 1994) and edited using BioEdit v7.0.5.3 (Hall, taxonomic affinities between taxa (Remigio and Hebert, 1999). Sequence similarity with other gastropod sequences in the 2003). GenBank was determined using BLAST (Basic Local Aligned Mitochondrial DNA COI sequence variation has Search Tool, hptt://www.ncbi/BLAST /index/html) to exclude the increasingly been widely employed in phylogenetic possibility of amplification of the nuclear pseudogene copies of studies of the genera Bulinus , Biomphalaria and other mtDNA COI region. freshwater gastropods (Stothard and Rollinson, 1997; The COI sequences were also translated into amino acid sequences using the invertebrate mitochondrial genetic code and Davis et al., 1998; Campbell et al., 2000; Remigio and inspected for stop codons using BioEdit v7.0.5.3 (Hall, 1999) to Hebert, 2003; Sørensen et al., 2005; Jørgensen et al., further eliminate the possibility of amplification of numts. Nucleotide 2007a, b, 2008; Plam et al., 2008; Sengupta et al., 2009). variations in the mtDNA COI region sequences were estimated Different other studies have also used sequence diversity using the program POPSTR version 1.2 (H. R. Siegismund, of the COI as a DNA barcode for the identification of unpublished). Phylogenetic analyses were performed using PAUP software