Genetic Variation in the Marbled Lungfish Protopterus Aethiopicus In

Journal of Fish Biology (2006) 69 (Supplement B), 189–199 doi:10.1111/j.1095-8649.2006.01224.x, available online at http://www.blackwell-synergy.com Genetic variation in the marbled lungfish Protopterus aethiopicus in Lake Victoria and introduction to Lake Baringo, Kenya S. GARNER*k,T.P.BIRT*†,C.M.MLEWA‡, J. M. GREEN§, A. SEIFERT{ AND V. L. FRIESEN* *Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6, Canada, ‡Department of Fisheries, Moi University, P. O. Box 3900, Eldoret, Kenya, §Department of Biology, Memorial University, St John’s, Newfoundland, A1B 3X9, Canada and {Department of Zoology, University of Florida, Gainesville, FL 32611, U.S.A. (Received 27 May 2005, Accepted 23 May 2006) Marbled lungfish Protopterus aethiopicus in Lake Victoria and two nearby smaller lakes were found to have high levels of DNA sequence variation in their mitochondrial control regions (35 haplotypes in 61 fish) but no population genetic structure (FST ¼ 0Á00). In contrast, marbled lungfish in Lake Baringo, Kenya, appeared to be fixed for a single control region haplotype, which occurred at low frequency in the other lakes. Using FLUCTUATE software, the female effective population size in Lake Victoria during the late Pleistocene was estimated to be c. 500 000, similar to the value estimated for the present-day population. These observations suggest that, during the late Pleistocene dry period, a large marbled lungfish population survived either in wet refugial areas within the lake basin or in surrounding areas. Marbled lungfish were reported to have been introduced into Lake Baringo 30 years ago with a founding population of only three individuals. The lack of control region variation in the Lake Baringo population is consistent with that situation. # 2006 The Authors Journal compilation # 2006 The Fisheries Society of the British Isles Key words: control region; Lake Victoria; marbled lungfish; mtDNA. INTRODUCTION Lake Victoria, Africa’s largest lake (69 000 km2), is situated on a plateau sep- arating the eastern and western rift valleys. Unlike the rift lakes of East Africa, Lake Victoria is shallow, with a maximum depth of c. 80 m (Stager et al., 1997). The hydrologic history of the lake is controversial. Seismic reflection profiles and sediment cores taken in Lake Victoria have been interpreted as evidence that the lake dried out completely for several thousand years at the end †Author to whom correspondence should be addressed. Tel.: þ1 613 533 6000 ext. 77530; fax: þ1 613 533 6617; email: [email protected] kPresent address: Department of Biology, University of Western Ontario, London, Ontario, N6A 5B8, Canada. 189 # 2006 The Authors Journal compilation # 2006 The Fisheries Society of the British Isles 190 S. GARNER ET AL. of the Pleistocene before filling again 12 400 14C years before present (Johnson et al., 1996). If correct, this situation implies that the fish community inhabiting the lake during this period must have been exterminated and that the endemic haplochromine cichlid species flock present today (comprising several hundred species) evolved in situ since the late Pleistocene (Seehausen, 2002). Some workers have argued that the Lake Victoria basin did not dry out completely during the late Pleistocene. Fryer (2004) criticized the interpretation of the geophysical evidence used to infer complete desiccation and further argued that biological evidence is not consistent with such a hydrologic history. Likewise, Verheyen et al. (2003) concluded, from an analysis of mtDNA control region variation in cichlids from Lake Victoria and surrounding areas, that these fishes were not eliminated from the lake basin during the late Pleistocene. Whether or not Lake Victoria was completely dry during the period in ques- tion, there is general agreement that the adaptive radiation of the cichlid species flock was rapid. Rapid diversification, however, was not universal in Lake Victoria fishes, and investigation of the impact of the reduced lake level on other taxa is of interest. The marbled lungfish Protopterus aethiopicus Heckel occurs in waters of central and eastern Africa including the Congo and Nile drainages (Greenwood, 1986) and is present in many lakes of the Great Lakes region including Victoria, Albert, Edward, George, Kyoga and Tanganyika (Greenwood, 1986; Mlewa & Green, 2004). It is usually found in shallow swampy habitat in lakes or rivers but has been recorded in deeper offshore waters. Lungfishes are obligate air breathing fishes and are adapted to survive seasonal bouts of habitat desiccation. This species probably could have survived the late Pleistocene dry period in the Lake Victoria basin and surrounding areas if refugia such as remnant streams or ponds were available. The size and number of such refugia would have influenced the population size of marbled lungfish and hence the degree to which population genetic variation could be maintained through the dry period. If habitat was limited, marbled lungfish populations would have been bottlenecked for an extended period leading to loss of genetic diversity through genetic drift. Alternatively, if suitable habitat was extensive, population bottlenecking and loss of genetic diversity would have been unlikely. One objective of the present study was to assess the level of genetic diversity in the present-day Lake Victoria marbled lungfish population. A finding of limited genetic diversity would be consistent with an extended period of reduced effective population size resulting from extensive habitat loss followed by little or no gene flow from other areas. Alternatively, a finding of abundant genetic diversity would be evidence against a strong population bottleneck, or alternatively, recolonization of the lake following the dry period from surrounding areas. Such recolonization could have occurred as an isolated event or as multiple invasion episodes from one or more source populations. Records of marbled lungfish in Lake Baringo, a small Kenyan lake (137 km2) situated in the East African Rift Valley, do not exist prior to 1984 when the species began to appear in commercial fish catches. Since then, a substantial fishery has developed. The sudden and recent appearance of marbled lungfish in Lake Baringo is consistent with local information indicating that the species was intentionally introduced in 1975 using three founders from Lake Victoria (Mlewa & Green, 2006). If this introduction situation is correct, the Lake Baringo # 2006 The Authors Journal compilation # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 69 (Supplement B), 189–199 LUNGFISH POPULATION STRUCTURE 191 marbled lungfish represent an apparently viable population that has recently passed through a severe, but brief, bottleneck. As there are relatively few exam- ples of such severe bottlenecks (Miller & Lambert, 2004) the Lake Baringo marbled lungfish are of interest from a variety of perspectives including fishery management, conservation and evolution. Marbled lungfish is a protein source for many east Africans, hence effective management of the resource is espe- cially important. Effective fishery management relies upon information about the species’ basic biology, little of which is available for P. aethiopicus. Mlewa & Green (2004) described aspects of the biology of this species in Lake Baringo and they documented maximum daily movements in excess of 5 km in individuals tagged with sonic transmitters (Mlewa et al., 2005). Genetic information that could be informative about dispersal on a larger scale and gene flow among marbled lungfish populations is completely lacking. The second objective of this study therefore, was to determine if genetic variation in Lake Baringo marbled lungfish was consistent with the introduction situation reported by Mlewa & Green (2006). If the apparently self-sustaining population was derived from only three founders, a maximum of two mitochondrial haplotypes can be present assuming no additional haplotypes have arisen through mutation or arrived by dispersal since the introduction. A finding of more than two haplotypes would not be consistent with the introduction situation or could be the result of gene flow from other populations. MATERIALS AND METHODS SAMPLES AND LABORATORY METHODS Samples of liver or finclips were collected from marbled lungfish at three Kenyan sites (Fig. 1) including Lakes Victoria (Kissumu area; n ¼ 29), Baringo (n ¼ 20) and Kanyaboli (n ¼ 23). One lake was sampled in Uganda (Nabugabo; n ¼ 9). The DNA was prepared using either standard phenol–chloroform extraction after digestion with Proteinase K (Sambrook & Russell, 2001) or the DNeasy DNA extraction method (Qiagen, Mississauga, Ontario, Canada) The mtDNA control region was amplified using polymerase chain reaction primers with binding sites in phenylalanine (Pae00031H, 59-TTAACTCCCACCGCCGGCTCCCA-39) and proline (Pae15443L, 59-GGCTCCCAAAGCTGATGTTC-39) tRNAs. These primers were developed using the complete mtDNA sequence of Protopterus dolloi Boulenger (Zardoya & Meyer, 1996) as a model. Two additional primers (Pae15825L, 59-GCTGATTCTTTGGTTAA- TACTC-39 and Pae16401H, 59-GTGCTTCAAAAACCGTCATTAG-39) were used for sequencing. Control region amplifications were performed in 15 ul volumes containing either 1Á5or2Á0mMMgCl2,10mMTrispH8Á4, 50 mM KCl, 0Á01% gelatine, 0Á0625 mg mlÀ1 bovine serum albumin, 0Á4 mM of each primer, 200 mM of each dNTP, and 0Á25– 0Á5 units of Taq polymerase (Roche Diagnostics, Indianapolis, IN, U.S.A.). The temper- ature profile consisted of 30

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