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 000). 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 especially 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 15or20mMMgCl2,10mMTrispH84, 50 mM KCl, 001% gelatine, 00625 mg ml1 bovine serum albumin, 04 mM of each primer, 200 mM of each dNTP, and 025– 05 units of Taq polymerase (Roche Diagnostics, Indianapolis, IN, U.S.A.). The temper- ature profile consisted of 30 s denaturation at 95° C, 30 s annealing at 50° C and 60 s extension at 72° C (35 cycles). Following electrophoresis through 2% agarose gels, amplified fragments were purified using Qiaquick Gel Extraction columns (Qiagen) and sequenced using Thermosequenase Radiolabeled Terminator Cycle Sequencing Kits (Amersham Biosciences, Montreal, Quebec, Canada).

DATA ANALYSIS Estimates of genetic diversity (nucleotide diversity p and haplotype diversity h) and analysis of molecular variance (AMOVA) were made using Arlequin (Schneider et al.,

# 2006 The Authors Journal compilation # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 69 (Supplement B), 189–199 192 S. GARNER ET AL.

FIG. 1. Map of Lake Victoria showing sampling locations in Kenya and Uganda. Inset shows location of Lake Baringo, Kenya.

2000), and variation was tested for deviations from neutrality using Ewens–Watterson’s and Chakraborty’s tests (Ewens, 1972; Watterson, 1978; Chakraborty, 1990), also im- plemented in Arlequin. A network of the substitutional relationships between haplotypes was reconstructed using statistical parsimony (Templeton, 1998, 2004) with TCS version 1.13 software (Clement et al., 2000). Phylogenetically informative inser- tions and deletions (indels) were treated as a fifth character state since sequence align- ment was unambiguous. A nested clade analysis (Templeton, 2004) was performed to test both for phylogeographic structure and for a historic population bottleneck and expansion: the haplotype network was nested following the rules of Templeton (1998), clades with non-random distributions were identified using GEODIS (Posada et al., 2000) with 10 000 randomizations of the data, and the inference key available at http://darwin.uvigo.es/software/geodis.html was used to identify evidence of phylogeographic structure and population expansion. FLUCTUATE software (version 1.4; Kuhner et al., 1998) was used to estimate female effective population size (Nf) at various times in the past. This software takes advantage of the effects of demographic patterns on coalescence times. Gene trees for growing populations will have shorter internal branches than those for stationary populations, while declining populations are expected to have relatively long internal branches (Kuhner et al., 1998). The following input parameters were used. The mutation rate used was estimated for cichlids, i.e. 28% per site per 106 years (Nagl et al., 2000). Because estimates for control region mutation rate vary across species and because there is no specific information about mutation rate in marbled lungfish, simulations were run using a wide range of values. Marbled lungfish generation time was estimated to be 7 years using the rationale that the age of first reproduction in females is 3–4 years (Dunbrack et al., 2006). If the reproductive life span is assumed to be twice this value, the average female reproduces to age 9–12 years. Females are therefore c 7 years old at the midpoint of their reproductive lives, which was the value used for generation time. The transition:transversion ratio and initial value for Y were set at 20 and 10, respectively. Change in population size was permitted with initial rate of change (g) set at 10. Results were robust even to large changes in these initial values. To ensure the results of FLUCTUATE were converging on the true values of Y and g,

# 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 193 simulations were run using a range of random seeds and with sequences entered in different orders. Simulations were also run with a range of values for short and long chain- lengths and chain numbers. Regardless of the initial parameters the simulations provided similar estimates of Y and g. Because the results were only weakly affected by these parameters, results are presented using the default settings, i.e. with the transition:transversion ratio and initial value for Y set at 20 and 10, respectively. Ten short chains with 200 steps each were followed by two long chains of 10 000 steps.

RESULTS The 1107 base pair (bp) P. aethiopicus control region sequence (Genbank accession numbers DQ320659–DQ320739) showed 75% identity with the P. dolloi control region. Thirty-five haplotypes were defined by 39 polymorphic sites including 30 transitions, one transversion, two sites containing a transition and a transversion in different individuals and six indels. Polymorphic sites were found throughout most of the control region, but were absent in the 154 bp region containing conserved sequence blocks II and III. No evidence of deviation from neutrality was found from the Ewens–Watterson or Chakraborty tests (all P > 010). Control region variation was high in marbled lungfish from Lakes Victoria (21 haplotypes), Kanyaboli (16 haplotypes) and Nabugabo (five haplotypes). In contrast, only one haplotype (Pae01) was found in Lake Baringo (Table I). Haplotype Pae01 was observed in one individual from Lake Kanyaboli but not in Lakes Victoria or Nabugabo. Estimates of haplotype diversity were high in Lakes Victoria, Nabugabo and Kanyaboli, while estimates of nucleotide diversity were relatively low (Table II). Haplotype diversity and nucleotide diversity were zero in Lake Baringo. The global estimate of population genetic structure across the region sampled reveals significant structure (FST ¼ 039, P < 0001). If Lake Baringo is removed, FST is essentially zero, as are pair-wise estimates of FST and d (Table II). Pair-wise estimates involving Lake Baringo are high and significantly greater than zero. Any population genetic structure is clearly due to the effect of the Lake Baringo sample. Results of the nested clade analysis are consistent with AMOVA results. The TCS tree reveals three principal clades (Fig. 2). No phylogeographic structure is evident outside Lake Baringo. For clade 1-2, the nested clade and nesting clade distances were both significantly small for haplotype Pae01 (DC ¼ 348, P < 0001; DN ¼ 113, P < 0001); the nested clade distance was significantly large for haplotype 19 (DN ¼ 359, P < 001), and the interior-tip distance was significantly small (DC ¼ 629, P < 0001), suggesting restricted gene flow with some long-distance colonization within this clade, reflecting the introduction to Lake Baringo. In clade 2-1, the nested clade distance for clade 1-2 was significantly large (DN ¼ 159, P < 0001), suggesting restricted gene flow with isolation by distance. In the analysis of the total cladogram, the nested clade distance was significantly large for clade 1-4 (DC ¼ 161, P < 005) but the result from the inference key was inconclusive. No evidence was detected for population expansion. The variation in mtDNA observed in the Lake Victoria sample is not consistent with a population bottleneck during the recent past. As there is no

# 2006 The Authors Journal compilation # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 69 (Supplement B), 189–199 194 S. GARNER ET AL.

TABLE I. Marbled lungﬁsh control region haplotype frequencies for the four lakes sampled

Sampling location

Haplotype Baringo Victoria Nabugabo Kanyaboli

01 20 1 02 1 03 5 4 6 04 1 05 2 06 1 07 3 1 08 1 09 1 10 1 11 1 12 1 13 1 1 14 1 2 15 2 1 16 1 17 1 18 1 19 1 2 20 1 21 1 22 1 23 1 24 1 25 1 26 2 27 1 28 1 29 1 30 1 31 1 32 1 33 1 34 1 35 1

apparent population structure outside Lake Baringo, the samples from the three other lakes were pooled and treated as a single population in estimates of past Nf. FLUCTUATE indicated a period of slow but statistically signiﬁcant population growth [growth parameter (g) ¼ 362, P < 005)] and Nf remained large across a wide range of control region mutation rates (Table III). For 8 mutation rate on the order of 10 , Nf is estimated to be on the order of 5 4 10 . A 10-fold higher mutation rate yields Nf estimates on the order of 10 . Despite large-scale variation in water levels of Lake Victoria since the late

# 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 195

TABLE II. Pair-wise estimates of FST (above diagonal) and d (below diagonal) for the four lakes sampled. Also shown are estimates S.D. of haplotype diversity (h) and nucleotide diversity (p)

Baringo Victoria Nabugabo Kanyaboli

Baringo — 058* 069* 070* Victoria 078* — 000 000 Nabugabo 060* 000 — 000 Kanyaboli 098* 000 000 — h 000 000 096 003 081 012 093 004 p 000 000 087 046 088 051 072 039

*P < 0001.

Pleistocene, marbled lungﬁsh appear to have maintained a large population. This result is not consistent with a situation in which a prolonged population bottleneck resulted in low levels of genetic variation.

DISCUSSION

LAKE VICTORIA Abundant genetic variation is present in marbled lungﬁsh populations now occupying Lakes Victoria, Nabugabo and Kanyaboli. Neither AMOVA nor

Haplotype frequency key

3–5 2–8 23 5 32 1–18 1–17 1 06 34 14 17 2–9 1–19 2–1 13 22 04 1–16 09 4–3 3–1 1–1 31 2–7 4–1 1–15 07

3–4 2–6 16 19 1–2 1–15 2–2 12 15 3–2 20 1–12 30 2–4 4–2 1–6 05 21 1–14 2–3 02 1–3 1–4 1–7 28 01 24 29 10 33 03 26 25 1–5 1–11 3–3 27 2–5 18 1–10 1–9 1–8 08 11 35

FIG. 2. Haplotype network generated with TCS. The area of each ellipse is proportional to the frequency of that haplotype. Each line represents one mutational step, with d; indicating intermediate haplotypes that were not observed.

# 2006 The Authors Journal compilation # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 69 (Supplement B), 189–199 196 S. GARNER ET AL.

TABLE III. The Nf estimates generated by FLUCTUATE for the pooled samples from Lakes Victoria, Nabugabo and Kanyaboli. Separate estimates are shown for three different mutation rates for the present-day population, and populations of 100, 1000, 2000 and 4000 generations past

Generations past

Mutation rate 4000 2000 1000 100 Present

109 549 106 550 106 550 106 550 106 550 106 108 542 105 546 105 548 105 549 105 550 105 107 473 104 510 104 529 104 547 104 550 104 nested clade analysis indicate genetic heterogeneity among these populations. This result is not surprising since Lake Victoria was much larger during the early Holocene humid phase than it is today (Stager et al., 1997). Elevated lake levels during this time would have subsumed the three lakes into a single large lake, effectively creating a single marbled lungfish population from which the present-day populations would be derived when levels subsequently dropped. Periodic gene flow between these populations may also be occurring during modern times. Given the abundant control region variation and large estimates of historic Nf, the Lake Victoria marbled lungfish population does not appear to have been bottlenecked during the late Pleistocene dry period. How can this finding be reconciled with the view of Johnson et al. (1996) that Lake Victoria was dry at that time? Two hypotheses are suggested, however, in order to test them additional sampling outside the Lake Victoria basin will be necessary. One hypothesis is that marbled lungfish, along with the entire fish fauna of Lake Victoria, were exterminated during the arid period and subsequently recolon- ized the lake from unknown (and potentially several) sources after it refilled. Under this situation the abundant genetic variation present today represents variation imported recently from outside the Lake Victoria basin. An alterna- tive hypothesis is that Lake Victoria did not dry out completely during the late Pleistocene and marbled lungfish survived through this period in significant numbers within the basin. The present results are consistent with this hypothesis in as much as the nested clade analysis revealed no evidence for an extensive population bottleneck and the FLUCTUATE simulations revealed evidence for neither population bottleneck nor expansion. While several authors (Nagl et al., 2000; Seehausen et al., 2003) have assumed that complete lake desiccation occurred, Fryer (1997, 2001, 2004) has argued against the widely held assumption of complete desiccation of the Lake Victoria basin on biological and geophysical grounds. Fryer (2001) suggested that extensive swampy areas including numerous lakelets capable of supporting large and diverse communities of fishes (and other aquatic organisms) could have per- sisted throughout the arid period. Such conditions would certainly have been suitable for marbled lungfish, which are characteristically associated with shallow, weedy, inshore habitats (Greenwood, 1986). Furthermore, the well-known abilities of marbled lungfish to breathe air and survive seasonal dry periods are consistent with the idea that the species survived in large numbers in the Lake

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Victoria basin throughout the late Pleistocene arid period. Abundant genetic variation has been documented in other Lake Victoria fish populations including numerous species of cichlids (Nagl et al., 1998; Seehausen et al., 2003) and the African catfish Clarias gariepinus (Burchell) (Giddelo et al., 2002). Such variation, especially the presence of old mtDNA haplotypes in Lake Victoria haplochromines, led Verheyen et al. (2003) to the conclusion that the lake did not dry out completely during the late Pleistocene. The presence of long internal branches in the TCS tree (Fig. 2) can result from either historic fragmentation or long-term demographic stability in a single large population. The first situation is consistent with extirpation of marbled lungfish in a completely desiccated Lake Victoria during the late Pleistocene dry period followed by recolonization from separate refugia. The second situation is in keeping with a viable population surviving through the dry period in a lake of reduced, but sufficient, size to sustain a large population. Unfortu- nately there is insufficient power in the nested clade analysis to distinguish between these alternatives.

INTRODUCTION OF MARBLED LUNGFISH TO LAKE BARINGO The introduction of marbled lungfish into Lake Baringo, as described by Mlewa & Green (2006), is consistent with the present observation of the absence of control region variation there. Given the lack of variation, the population could have been derived from a single pair of founders, which may have originated from Lake Victoria even though the Lake Baringo haplotype (Pae01) was not observed in the Lake Victoria sample. While the sample from Lake Victoria is reasonably large (n ¼ 29), it cannot be claimed that haplotype Pae01 is absent from that population. The large number of haplotypes recorded (21) and the low frequency of most haplotypes (17 of 21 occurred in single individuals), clearly indicate that additional variants would be detected in Lake Victoria with additional sampling, and that haplotype Pae01, if present, occurs at low frequency. The presence of Pae01 in Lake Kanyaboli suggests this haplotype probably occurs in Lake Victoria because, as previously noted, the lakes were part of the same larger water body during the early Holo- cene and probably more recently. The recent nature of the introduction to Lake Baringo is evident from the apparent lack of new haplotypes generated by mutation. Because mtDNA is inherited maternally, the data are not informative about the number of male founders. If the Lake Baringo population is descended from only three founders, as described by Mlewa & Green (2006), the maximum number of males could have been two. The present data, however, would not detect a greater number of male founders. Population genetic structure has been studied in the African catfish, a species also present in East Africa (Giddelo et al., 2002). As in marbled lungfish, mtDNA variation in African catfish, as determined by restriction fragment length polymorphism analysis of a 25 kb segment containing the ND5 and ND6 genes, was high in Lake Victoria. Less diversity was found in Lake Bar- ingo but that African catfish population was not fixed for any one haplotype.

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A population bottleneck was cited as the most likely reason for the low variation at this location. African catfish populations appear to be partitioned into three major regional groups: an eastern group (present on the plateau supporting Lake Victoria), a northern group (present in the middle and lower Nile, Sudd Marshes, Lake Tchad, Niger and Senegal Rivers) and a south-central group (present south of the Great Ruaha drainage). Although the results of the African catfish and marbled lungfish studies are not directly comparable due to different sampling regimes and analytical methods, within Lake Victoria the marbled lungfish mtDNA lineages appear to be considerably older than those in African catfish. This suggests that the species have very different his- tories within the Lake Victoria basin. Additional sampling of marbled lungfish populations outside the basin will be required before the history of the species can be compared with that of African catfish and other species in East Africa. The apparent lack of genetic variation in Lake Baringo marbled lungfish could pose problems for the long-term viability of this population relating to inbreeding depression (Hedrick & Kalinowski, 2000). Fishery managers might consider augmenting the existing variation by introducing additional founder stock from surrounding populations although such action needs to be consid- ered carefully given the problems caused by alien fish introductions in East Africa, especially Lake Victoria.

We thank H. Tarnowski for laboratory assistance, R. Ficken for preparing Fig. 1, and two anonymous reviewers for thoughtful suggestions. This work was supported by the National Sciences and Engineering Research Council of Canada (summer under- graduate scholarship to S. G. and a discovery grant to V. L. F.), Canadian Interna- tional Development Agency (graduate fellowship to C. M. M.), Lake Victoria Environment Program (to C. M. M.) and the Canadian International Development Research Centre (sabbatical grant to J. M. G.). We thank Moi University for logistical support and transportation.

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