Max-Planck-Institut für Biologie, Abteilung Immungenetik, Tübingen, Germany
Correspondence: E-mail: akie.sato{at}tuebingen.mpg.de.
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Abstract |
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Key Words: cichlids haplochromine fishes Mhc mtDNA control region SINE crater lakes
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Introduction |
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Genetic characterization of a fish population can provide information about the probable size and composition of the founding population, the time of the founding event, and other related questions. This information can then be used to make inferences not only about the circumstances of the colonization but also about the process of speciation and species divergence. Here we use three genetic systems to probe the origin, population dynamics, and speciation of lacustrine fishesmitochondrial (mt) DNA, genes of the major histocompatibility complex (Mhc), and short interspersed repetitive elements (SINEs). The mtDNA control region with its high substitution rate and maternal transmission is the tool of choice in efforts to identify the most recent common ancestor (MRCA) of a population (Avise et al. 1987). Our survey of mtDNA control region sequences in lacustrine and fluviatile haplochromine cichlids of the Lake Victoria basin revealed the existence of seven major haplogroups, designated I through VII (Nagl et al. 2000). Each haplogroup is characterized by a set of diagnostic substitutions and by the clustering of its members in a single clade on phylogenetic trees. The haplogroups are distributed differentially over the basin, the most widespread being haplogroup V centered on Lake Victoria, the Lake Edward region (including Lake George and Lake Albert), and the rivers around these lakes. A number of additional haplogroups apparently exist among the fishes of Lake Tanganyika and Lake Malawi, which were involved in the survey only tangentially. Haplogroup V is subdivided further into subgroups VA through VD, which are distinguished by a more restrictive set of diagnostic substitutions. Each of the subgroups, however, may be subdivisible even further on typing additional specimens. In particular, subgroups VB and VC show a heterogeneity indicative of future splitting.
Mhc genes with their high polymorphism and long persistence of allelic lineages are well suited for exploring the population dynamics in the history of a species (Klein et al. 1998). Fish Mhc genes, like those of other jawed vertebrates, fall into two classes, I and II, and each class into two subclasses, A and B, encoding the and ß chains of the
ß heterodimeric protein (Klein et al. 1997; Shand and Dixon 2001). Since most of the work on cichlid Mhc genes was done on the class II B genes (Klein et al. 1993; Ono et al. 1993a, 1993b; Sato et al. 1997; Málaga-Trillo et al. 1998; Figueroa et al. 2000), we focused on the most variable part of these genes, exon 2 encoding the ß1 domain of the class II ß chain.
Fish SINEs are retroposons that are several hundred base pairs long and contain a region homologous to a transfer RNA gene in their 5' part and frequently also contain a region corresponding to a segment of a long interspersed repetitive element (LINE) in their 3' part (Okada 1991; Schmid and Maraia 1992; Okada et al. 1997). In a recent study, Terai and colleagues (2003) have shown that haplochromines of the Lake Victoria basin contain two types of SINEs, fixed and polymorphic. The former are found in all individuals of a given species or species assemblage, whereas the latter are present in only some individuals of a population. As there is no indication for selection influencing the evolution of SINEs, these loci provide a nuclear counterpart to the neutrally evolving control region segment of the mtDNA on the one hand and the selection driven evolution of the Mhc genes on the other hand. Furthermore, the presumed random distribution of SINEs among the chromosomes enables sampling of different segments of the nuclear genome.
The two main aims of the present study were (1) to determine the origin, structure, and effective size of the haplochromine populations in Lake Lutoto and Lake Nshere and to ascertain the genetic relationship between them and (2) to provide a basis for comparing the genetic variability existing in these two isolated populations with that present in the endemic Lake Victoria species and thus pave a way toward a better understanding of the speciation process in these fishes.
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Materials and Methods |
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Phylogenetic Analysis
The multiple alignments of sequence data were made by using ClustalW 1.82 (Thompson, Higgins, and Gibson 1994) and checked by eye. The phylogenetic trees were drawn by the neighbor-joining (NJ) method (Saitou and Nei 1987) and evaluated by 1,000 bootstrap replications.
In the analyses of mtDNA control region sequences, the sites 518 to 535 of the alignment in Nagl et al. (2000) were eliminated because they contain many single nucleotide repeats and the sequences are not reliable. Sites with indels were also deleted. NJ trees were drawn using p-distances. Unless the substitution rate varies extensively in different lineages, p-distance is better in obtaining a correct tree topology than distances based on a complex substitution model, even when the substitution pattern of the nucleotide changes follows a complex model, particularly for closely related sequences (Nei and Kumar 2000). The alignment of group V sequences (fig. 4 [only variable sites are shown]) includes sequences from Nagl et al. (2000). The NJ tree for the group V sequences (fig. 5) was drawn based on the alignment in figure 4 with 810 shared sites.
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Since in the mtDNA control region the transition-transversion ratio is relatively high and the substitution rate varies extensively across sites (e.g., Tamura and Nei 1993), Kimura's two-parameter distance with gamma correction was used for time estimation. The gamma parameter ( = 0.12) was estimated by the maximum-likelihood method using the tree topology of the NJ tree and the baseml in PAML3.12 program (Yang 2002). In the maximum-likelihood analysis, the transition-transversion rate ratio was estimated as 7.1. Observed base frequencies were 0.31, 0.31, 0.16, and 0.23 for A, T, G, and C, respectively.
The average distance (h) between the two descendant clusters of interior nodes of the mtDNA control region NJ tree was computed as
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The average distance between the mtDNA control region sequences of Lake Malawi and Lake Victoria fishes was 0.0445 ± 0.0073. Assuming that the separation of these two groups occurred 1 to 2 MYA (see also Sturmbauer et al. 2001), the mutation rate for the mtDNA control region was estimated as 2.2 - 4.5 x 10-8 per site per year. This rate is similar to the mutation rate (1.8 x 10-8) estimated for other fish (snook [Donaldson and Wilson 1999]), but much lower than the rate for human mtDNA control region (0.75 x 10-7 [Tamura and Nei 1993]).
An NJ tree of the cichlid Mhc class II B exon 2 sequences was drawn using all the available cichlid and closely related sequences in the GenBank database. In the NJ tree shown in figure 5, only representative sequences were included because of space limitations, and medaka fish (Oryzias latipes) sequences were used as an outgroup. In the alignment, there were 160 shared nucleotide sites after sites with missing nucleotides or indels were excluded. Jukes-Cantor distances were used.
Detailed information regarding the fish samples and the sequence data obtained for mtDNA control region, Mhc class II B exon 2, and SINE 1357 are available as Supplementary Material online.
Computer Simulation of Coalescence Process
The coalescence theory (Hudson 1983; Tajima 1983) and the polymorphism found at the SINE loci were used to obtain the lower limit of the effective population size. According to the theory, allelic lineages that exist in a current population have a genealogy that can be traced back to an MRCA. For a neutral nuclear locus of a population with an effective size N, the time (tj) during which exactly j allelic lineages exist in the population corresponds to a random number generated according to the probability density of the exponential distribution with a mean of 2/[ j( j - 1)] in units of 2N generations (Simonsen, Churchill, and Aquadro 1995). In the computer simulation starting with n lineages, the coalescence process was repeated until na - 1 lineages remained in the population, where n is the number of genes sampled in either the H. "Nshere" or H. "Lutoto" populations, and na is the number of alleles shared between H. "Nshere" or H. "Lutoto" and other lake populations. The time
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Computer Simulation for Founding Population Size and the Number of Alleles Retained
To examine the relationship between the minimum number of Mhc alleles retained in the population, the founding population size, and the population growth rate after the founding event, another computer simulation was conducted. The simulation was a slight modification of that used by Vincek et al. (1997) who assumed overdominant selection for Mhc loci with the selection coefficient s = 0.01. The alleles for the founding population of Nb individuals were drawn at random from the parental population. After the founding event, population growth started immediately with rate r and continued until the number of individuals in the population reached 10,000. Then, the number of alleles retained in the population was recorded. This process was repeated 1,000 times for each combination of the values of Nb and r. The ranges of the Nb and r values varied from 5 to 500 and from 0.01 to 0.5, respectively. The allele frequencies in the ancestral population were assumed to be those reported for the HLA-DRB1 locus in Caucasians (Imanishi, Wakisaka, and Gojobori 1991). In a preliminary study, we also assumed that the frequencies of alleles in the ancestral population are equal. The results for these two situations were essentially the same.
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Results |
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mtDNA Control Region
Control regions of mtDNA were sequenced from 28 fish caught in Lake Nshere and 20 specimens from Lake Lutoto. Ten distinct H. "Nshere" and seven H. "Lutoto" haplotypes were found (fig. 4) among the 48 sequences obtained. The H. "Nshere" sequences showed more variability (one to seven differences) than those of H. "Lutoto" (one to three differences). Phylogenetic analysis revealed all 48 sequences to belong to haplogroup V characterized by the presence of T, C, C, A, and A at sites 27, 87, 96, 348, and 825, respectively (see Nagl et al. 2000 and fig. 4). Each of the two sets of sequences forms a separate cluster (a clade on a phylogenetic tree [fig. 5]) and each is identified by distinct diagnostic substitutions. All Lake Nshere sequences have A and T at sites 107 and 830, respectively, whereas all Lake Lutoto sequences share T, T, and G at sites 100, 347, and 830, respectively (fig. 4). We denote the control region subgroup of the VB haplogroup present in H. "Lutoto" as VE and that of H. "Nshere" as VF. The H. "Lutoto" sequences appear to be most closely related to a particular subset of VB sequences with which they share an A at site 635 (fig. 4). Similarly, the H. "Nshere" sequences are most closely related to another subset of VB sequences that have a G at site 635. Some of the VF sequences also share an A at site 167 with some members of this VB subset, but this nucleotide is also present in some members of the VC subgroup (figs. 4 and 5). These observations lead us to three conclusions. First, the haplochromine fishes inhabiting Lake Lutoto and Lake Nshere originated in an area in which the haplogroup VB is common. Second, the fishes of these two lakes originated from two distinct but related populations, probably occupying different parts of the area of wide VB-haplogroup distribution (see Discussion). Third, the fish populations of the two lakes have been isolated from other populations long enough to attain fixation of diagnostic substitutions. A network presentation of mtDNA haplotypes of the VB group and the crater lake fishes is available as Supplementary Material online.
Using the mutation rate of 2.2 - 4.5 x 10-8 per site per year (see Material and Methods), we estimate the age of the nodes that separate the crater lake fishes from fishes of other localities (nodes 3 and 4 in fig. 5) as 0.18 ± 0.06 to 0.09 ± 0.03 MYA for H. "Nshere" and 0.12 ± 0.05 to 0.06 ± 0.03 MYA for H. "Lutoto" (table 2). These estimates give the upper limit for the time when the isolation of the crater lake fish occurred. The common ancestral nodes of the Lake Nshere and the Lake Lutoto fishes (nodes 5 and 6 in fig. 5) are estimated to date to 0.13 ± 0.04 to 0.06 ± 0.02 and 0.080 ± 0.04 to 0.04 ± 0.02 MYA, respectively. In contrast, the common ancestral node (node 7 in fig. 5) of the Lake Victoria fishes is dated to 0.23 ± 0.08 to 0.12 ± 0.04 MYA (table 2). These estimates suggest that the isolation of Lake Victoria fishes from other fishes occurred somewhat earlier than that of the crater lake fishes.
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One unique sequence of H. "Nshere" is included in lineage II; all other sequences of fishes from the two crater lakes are included in lineage I and are approximately equally distributed between the sublineages IA and IB (fig. 7). Thus, a minimum of three class II B loci must exist in fishes from crater lakes (IA, IB, and II). Additional loci are defined by distinct clades within lineage I. Altogether, seven possible class II B loci are defined in the haplochromines of the two crater lakes (fig. 7). The predicted number of loci is also supported by the results of the Southern blot analysis (fig. 8).
Distribution of SINEs
Terai and his coworkers (2003) identified a set of SINEs that have been inserted into the genomes of endemic Lake Victoria and Lake Edward region haplochromines after the divergence of the Lake Victoria from the Lake Malawi flock and persist in most of the species and populations as presence or absence polymorphisms. To test for the presence or absence of these SINEs in the individual H. "Nshere" and H. "Lutoto" specimens, we used primers specific for the flanking regions of SINEs 1350, 1840, 1424, 1801, 1807del, 1909, and 1919 in PCR. Of the seven SINEs tested, two were found to be polymorphic in H. "Nshere" (1918 and 1801), whereas only one (1918) was polymorphic in H. "Lutoto"; all other SINEs were fixed in the two populations (table 3 [see also Terai et al. 2003]). Comparison of observed and expected frequencies of the genotypes at the polymorphic SINEs revealed both populations to be in Hardy-Weinberg equilibrium (table 3). This result suggests that neither of the two populations is structured.
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The Lower Limit for the Effective Population Size of Crater Lake Fishes
Since polymorphism at some of the SINE loci is shared by the crater lake fishes and fishes of the neighboring region, we used the number of shared alleles and the coalescence theory to obtain the lower limit of the effective population size for H. "Nshere" and H. "Lutoto." As described in Materials and Methods, we computed the probability of na allelic lineages persisting for 50,000 years by the coalescence algorithm (Hudson 1983; Tajima 1983) for various population sizes (N) and searched for the value of N for which na lineages persist for more than 50,000 years in 5% of the iterations of the computer simulation.
We can assume na = 2 in the case of the presence/absence polymorphism of SINEs 1918 and 1801 in H. "Nshere" and 1918 in H. "Lutoto"; and na = 4 for the alleles determined by sequencing SINE 1357 and found to be shared between H. "Nshere" and other lake populations. Using the na = 2 value, we can reject the hypothesis that N was smaller than 6,000 individuals at the 5% significance level for the past 50,000 years of existence of the H. "Nshere" and H. "Lutoto" populations. The 6,000 individuals thus represent the lower limit for the effective sizes of these two populations. Using the na = 4 value, the results of the computer simulation indicate that N = 24,000 is the lower limit of the effective H. "Nshere" population size that assures the persistence of at least four allelic lineages for the last 50,000 years.
Population History
Tajima (1989) designed a test that examines
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In a computer simulation similar to that described by Vincek et al. (1997), we examined the number of alleles retained for various founding population sizes (Nb= 5 to 500) and growth rates (r = 0.01 to 0.5) (see Materials and Methods). Only representative values (r = 0.01, 0.05, and 0.5) are shown in figure 10. In figure 10A, the average numbers of alleles (nave) retained when the number of individuals reached 10,000 in 1,000 replications of computer simulation are shown in relation to the founding population size (Nb). In figure 10B, instead of the average number of alleles (nave), the 95% upper limit of the number of alleles (nm) retained in the population is shown. That is, in at least 95% of the replications, the number of alleles retained in the population was smaller than nm. For each Nb value, we can reject that the number of alleles retained in the population is smaller than nm at the 5% significance level. Thus, Nb values in figure 10B can give a lower limit of the founding population size for the observed number of alleles nm. The result of the simulation suggests that four or five alleles can be retained even in the case of a small founding population (5 to 10 individuals) if the growth rate of the population after the founding event is not small (< 0.1 to 0.2). The founding population must contain at least 20 breeding individuals to retain 12 alleles, even for a growth rate as high as 0.5. For a relatively low growth rate (0.01), the founding population must be at least 50 to 70 to retain four to five alleles and at least 300 individuals to retain 12 alleles.
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Discussion |
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Geologists date the earliest volcanic activity in the Lake Lutoto and Lake Nshere region to approximately 50,000 years ago (Boven et al. 1998). This date places an upper limit on the origin of the two lakes and hence also on the time of their colonization by fishes. The date is younger than the estimated time required for the mtDNA control region haplotypes now present in H. "Nshere" and H. "Lutoto" to coalesce to the MRCA. The difficulty with coalescence time estimates, however, is that the calibration of the mtDNA molecular clock is unreliable. For the East African cichlid fishes, the clock is commonly calibrated by the geological age of Lake Malawi (Meyer et al. 1990; Nagl et al. 2000; Sturmbauer et al. 2001), which, however, is uncertain (Schlüter 1997). The estimates range from 0.5 to 4 Myr, depending on the part of the lake examined and the dating method. In an earlier publication (Nagl et al. 2000), we calibrated the clock on both the 2 Myr and 4 Myr age of Lake Malawi. Here, we also take the younger age estimate (1 Myr) into account. Thus calibrated, the molecular clock dates the MRCAs of the mtDNA control region of H. "Nshere" to 60,000 or 130,000 years ago and that of H. "Lutoto" to 40,000 or 80,000 years ago (the two alternatives represent datings based on the 1 or 2 Myr age of Lake Malawi). Both estimates for H. "Nshere" exceed the upper limit set by the geological dates, but the H. "Lutoto" estimates come reasonably close to it. Of course, large errors are associated with these time estimates (table 2), and the geological dating of the crater lakes is subject to uncertainty (Boven et al. 1998). It is therefore difficult to tell whether or not the age of the MRCAs of the crater lake fishes really exceeds the geological date of the lakes. In principle, there can be two reasons for the discrepancy between molecularly and geologically estimated dates. First, the coalescence time to the MRCA need not coincide with the time of lake colonization and emergence of a species because polymorphism could have passed through the speciation phase. In the case of H. "Nshere" and H. "Lutoto," however, there is no indication that any of the mtDNA control region haplotypes now present in the population existed before the emergence of these species. Second, Lake Malawi could in fact be younger than 1 Myr or the divergence of the Lake Victoria basin cichlids from the Lake Malawi fishes might have significantly postdated the emergence of the lake (Sturmbauer et al. 2001). Be this as it may, a large standard error of the molecular clock estimate leaves the door open for a younger age of the MRCA. In relative terms, the ages of H. "Nshere" and H. "Lutoto" may be comparable to those of the endemic Lake Victoria species.
Both the SINE and the mtDNA data indicate that the populations of H. "Nshere" and H. "Lutoto" are quite large. The lower limit of N (effective size of a whole population) from the SINE data is 6,000 for H. "Lutoto" and 24,000 for H. "Nshere," and the Nf (female effective population size) estimate from the mtDNA control region data is 38,000 to 77,000 for H. "Nshere" and 8,000 to 17,000 for H. "Lutoto." For lakes that have each a surface area of less than 1 km2, these are indeed large effective population sizes. In a large population, selectionwhere applicablegains the upper hand over random genetic drift. It can lead to the rapid fixation of mutations introducing adaptive modifications. This potential does not seem to have been exploited by fishes of Lake Nshere and Lake Lutoto, but has apparently been capitalized on by fishes in some other crater lakes and by haplochromines of Lake Victoria. In West Africa, the volcanic crater lakes Barombi Mbo and Bermin in Cameroon harbor 11 and nine tilapiine cichlid species, respectively (Trewavas, Green, and Corbet 1972; Stiassny, Schliewen, and Dominey 1992), which apparently arose in situ from a single ancestor species by sympatric speciation (Schliewen, Tautz, and Pääbo 1994). Similarly, Lake Tana in Ethiopia provides a home for 14 morphotypes of large barbels (Barbus intermedius complex), all adapted to different ecological niches, exploiting different food resources, and derived from a common ancestral species within the lake (Nagelkerke, Sibbing, and Osse 1995; Dixon et al. 1996). The tremendous morphological diversity of the endemic Lake Victoria haplochromines is well documented (Greenwood 1981; Seehausen 1996). The difference between Lake Victoria on the one hand and Lake Nshere and Lake Lutoto on the other is, of course, that Lake Victoria, being the third largest lake in the world, offers many different ecological niches for adaptive radiation that the crater lakes do not. By contrast, Lake Barombi Mbo and Lake Bermin on the one hand and Lake Nshere and Lake Lutoto on the other are comparable in size. Whether they are also comparable in the number of ecological niches they potentially provide for fishes is not known to us. The reasons why cichlids adaptively radiated in the West African but not in the two East African crater lakes are unclear.
The lower limits of N (6,000 for H. "Lutoto" and 24,000 for H. "Nshere") obtained in this study were based on the assumption that the size remained constant over 50,000 years. If this was the case, the founding populations of these two species must have been quite large. Although a bottleneck phase is not excluded by these considerations, if it did occur it must have been of short duration and followed by very rapid expansion of the population to its present size. The lower limit of the bottleneck is indicated by the Mhc data. It is 10 to approximately 300 breeding individuals, depending on how the data are interpreted. These numbers make the hypothesis involving the "seeding" of the lakes by fish eggs or fry transmitted by animals an unlikely proposition. Even if birds were able to deliver up to 300 founders into each of the two lakes, one would not expect the eggs or fry to be of the same origin in terms of species and locality, whereas all the available evidence suggests a monophyletic origin of both H. "Nshere" and H. "Lutoto" in a single founding event. Most likely, the founders of H. "Nshere" and H. "Lutoto" reached the lakes by way of rivers, and perhaps the way to Lake Lutoto was longer or more difficult than the way to Lake Nshere, so fewer fish reached the former than the latter body of water. In both cases, however, the genetic diversity of the founding populations might have been greater than was ultimately retained. This conclusion is indicated by a comparison of the genetic variability currently found in the crater lake populations and that present among the Kazinga Channel fishes. Some loss of diversity apparently occurred by random genetic drift during the expansion phase after the founding event. The isolation of the crater lake population might have been another factor contributing to the loss of variability.
All the genetic systems we tested consistently indicate that compared with H. "Nshere," H. "Lutoto" is genetically a more homogeneous population. Whereas in H. "Nshere" we detected 10 different mtDNA control region haplotypes, found four of the six Mhc class II B loci to be polymorphic with two to four alleles per locus, could establish the presence or absence of polymorphism at two of the five SINE loci, and found four alleles at the SINE 1357 locus, in H. "Lutoto" we found seven mtDNA alleles, all the Mhc class II B loci to be monomorphic, one of the SINE loci to be polymorphic, and only one SINE 1357 allele. The reason for this difference between H. "Nshere" and H. "Lutoto" populations is not immediately apparent. The founding population of H. "Lutoto" might have been smaller than that of H. "Nshere," as mentioned earlier, or, as the Tajima test indicates, the H. "Lutoto" population might have experienced a recent bottleneck and currently be in a recovering phase as a consequence of some event such as a change in water level.
In contrast to molecular diversification, morphological diversification reveals just the opposite trend when the populations of the crater lakes and Lake Victoria are compared. The two crater lake species are rather similar in appearance, the differences between them in body shape and coloration being inextensive. Similarly, the morphological variation within each crater lake population is relatively minor (H. Tichy and E. Schraml, unpublished data). By contrast, the endemic Lake Victoria species have diverged much more extensively from each other morphologically and each shows considerable intraspecies variation (Greenwood 1981; Seehausen 1996). These disparate trends can be attributed to the differences in opportunities for adaptation to distinct environmental niches on the one hand and to opportunities to mix diverging populations in the large and small lakes on the other hand.
At the molecular level, the most striking difference between speciation of haplochromines in the crater lakes and in Lake Victoria is in the fixation of substitutions. Although, as stated earlier, the endemic haplochromines in the crater lakes and in Lake Victoria had approximately the same length of time to evolve, the crater lakes species each accumulated two or three diagnostic substitutions in the control region of their mtDNAs since the time of their origin from the founding populations, but the endemic Lake Victoria species tested thus far have not accrued any such fixed changes (Nagl et al. 1998, 2000). This difference is most likely attributable to the different degrees of isolation of the populations. Whereas the crater lake populations apparently evolve in complete physical isolation with only one species per lake, in Lake Victoria the reproductive barriers between the emerging species may be leaky. The barriers may be sufficiently strong to segregate characters under selection, differentiating the populations morphologically and behaviorally for longer periods of time, but not strong enough to assure fixation of neutral mutations by random genetic drift. Occasional breaches of the barrier may keep these mutations in a polymorphic state. An important implication of these deductions is the tentativeness of the early phases of speciation in the endemic Lake Victoria haplochromines. We suggest that as long as the environmental conditions in the lake remain stable, the different species may evolve in isolation from one another. Dramatic changes in the conditions may, however, usher in a phase of a limited gene flow between some of the species and so prevent fixation of neutral mutations. The ease with which the Lake Victoria haplochromines produce interspecies hybrids in aquaria attests to the weakness of the reproductive barriers between the species.
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Acknowledgements |
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Footnotes |
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Naruya Saitou, Associate Editor
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