School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK1
Hatherly Laboratories, School of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, UK2
Author for correspondence: Paul K. Hayes. Tel: +44 117 928 7483. e-mail: Paul.Hayes{at}Bristol.ac.uk
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ABSTRACT |
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Keywords: AS-PCR, index of association, population genetics, gene diversity
Abbreviations: AS-PCR, allele-specific polymerase chain reaction; IGS, intergenic spacer; ITS, internal transcribed spacer; PC, phycocyanin
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INTRODUCTION |
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The filamentous, diazotrophic cyanobacterium Nodularia is a convenient organism on which to perform population genetic analysis as it is reliably present during the summer months in the Baltic Sea, and sufficient template DNA for PCR can be obtained from single filaments isolated from the water column. Whilst the extraction of DNA from filaments precludes any further analysis of them, the advantages of directly analysing filaments picked from water samples, rather than clonal cultures established from them, are twofold: first, a great deal of time and effort is saved in isolating and maintaining thousands of clonal cultures; and secondly (and more importantly), culture collections may not be representative of the natural population if genetic differences between isolates have differential effects on fitness in the artificial conditions of laboratory culture.
Genetic diversity in natural populations of Nodularia has been demonstrated in the Baltic Sea (Hayes & Baker, 1997 ; Barker et al., 1999
) and elsewhere (Neilan et al., 1995
; Bolch et al., 1996
, 1999
), but these studies have either focussed on a single locus (Hayes & Barker, 1997
; Neilan et al., 1995
; Bolch et al., 1999
) or had a sample size too small for population analysis (Barker et al., 1999
). In this study we have characterized three loci from 2336 Nodularia filaments collected during research cruises in June and July 1998. The large sample size minimizes the sampling error of population genetic parameters such as gene diversity (Nei, 1987
) and index of association (IA) (Maynard Smith et al., 1993
). The three loci chosen were (1) part of the cpc operon that includes the phycocyanin intergenic spacer (PC-IGS; Neilan et al., 1995
), (2) the rDNA internal transcribed spacer (rDNA-ITS; Wilmotte, 1994
) and (3) the intergenic spacer between two adjacent copies of gvpA (the gene encoding the main structural gas vesicle protein; Tandeau de Marsac et al., 1985
), gvpA-IGS. These loci were selected for study because each is known to be polymorphic in Nodularia (Barker et al., 1999
). For the PC-IGS locus four sequence variants were found among cultured isolates. Sequence analysis of cultured isolates similarly revealed the presence of three distinct rDNA-ITS, and two gvpA-IGS genotypes (Barker et al., 1999
). The four PC-IGS sequence types fall into two main groups that can be distinguished by allele-specific PCR (AS-PCR) (Hayes & Barker, 1997
), as can the three rDNA-ITS sequence types. The AS-PCR approach uses PCR to amplify a region with broad-specificity primers and then a second, nested PCR with one broad-specificity and one or more allele-specific primers: the presence/absence and/or size of the resulting PCR products indicate which allele(s) were present in the original template DNA.
In addition to questions regarding population structure, there is currently disagreement concerning the taxonomic status of Nodularia in the Baltic Sea, with some studies suggesting that several species co-exist in this region (Komárek et al., 1993 ; Bolch et al., 1999
), whilst other data show that many phenotypic and genotypic markers are not congruent, implying that gene flow occurs between isolates (Barker et al., 1999
). The taxonomic implications of our results are discussed.
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METHODS |
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The association of alleles at different loci in Nodularia was also studied using the method of Stevens & Tibayrenc (1996 ), a modified Mantel test (Mantel, 1967
), to detect linkage disequilibrium based on correlations between independent genetic markers (the PC-IGS, rDNA-ITS and gvpA-IGS loci); non-random associations can provide evidence that gene flow is restricted in the population under study, irrespective of the reasons for this. The technique is a generalized Monte-Carlo test for linkage, independent of mating system or ploidy. In brief, the loci are shared into two groups (for example PC-IGS in one group and both rDNA-ITS and gvpA-IGS in the other). Next, a genetic distance matrix is calculated for each of these groups, showing the distance between a given individual and all other members of its group. A correlation coefficient (r) is then calculated between the two groups of distances for this, the observed arrangement of genotypes. The genotypes (the allele patterns for each locus for each Nodularia filament) are then randomly reassorted a given number of times (e.g. 100 times), each time creating a new set of Nodularia specimens of randomly mixed genetic composition. A new r-value is calculated for each random reassortment. Following the required number of genotype reassortments and r-value calculations, a frequency distribution of r-values can be constructed, allowing a probability value to be attached to the observed correlation coefficient. The process is then repeated and the loci are shared into two different groups. When the number of loci is six or less, as in this study, this is repeated until all combinations (three in this study) of loci have been evaluated. For each analysis, 100 random reassortments of each of the three combinations were used to construct each distribution of r-values. Significance levels of P<0·01 and P<0·05 were used for each combination. Computational constraints necessitated that linkage analyses were performed on 20 subsets of 100 randomly selected filaments, rather than the full 2336.
Other statistical analyses.
Gene diversity was calculated for the population as a whole, and for individual sampling stations as described by Wise et al. (1996 ). Genetic distance was calculated as described by Nei (1972
) with distance D equal to -logeI, where I, the normalized identity, is equal to Jxy/(Jxx Jyy)0·5. If there are three alleles at a given locus in population X with frequencies of p1, p2 and p3, then Jxx=
p12+p22+p32. If the equivalent allele frequencies in population Y are q1, q2 and q3, then Jyy=
q12+q22+q32, and Jxy=
p1q1+p2q2+p3q3. The arithmetic mean of each J value over all loci is used to calculate I. All data manipulations and statistical analyses were performed using Microsoft Excel 8.0 and MINITAB, except for the modified Mantel test, which was performed using DLX.FOR (Stevens & Tibayrenc, 1995
).
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RESULTS |
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The hypothesis that the genetic distance between sampling stations might correlate with the map distance was tested by calculating Neis normalized identity I and thus the genetic distance D for each pairwise combination of sampling stations. Using linear regression, we found no significant relationship between these variables (R2=1·2%), but although there is no significant trend, the scatter plot (Fig. 4) shows that at the greatest distances compared, the genetic distance between stations is always low. In order to test the hypothesis that this effect was caused by low genetic diversity at the extreme Northern and Southern limits of the sampling area, the gene diversity, averaged over all three loci, was calculated for each of the 20 stations, and compared to the distance between each station and the exact centre of the area sampled. The results (Fig. 5
) show that there is a significant relationship between gene diversity and the logarithm of the distance from the centre of the sampling locations (P= 0·037, R2=22·1%), following the equation gene diversity=0·253-(0·587 log distance).
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Modified Mantel tests for linkage disequilibrium within 20 randomly selected subsets of 100 individuals of Nodularia were performed. Analysis showed significant linkage disequilibrium between some loci combinations in 17 out of 20 subsets of data (Table 6). Having detected linkage disequilibrium in the data, we compared the observed number of filaments with each possible pairwise combination of alleles at any two loci to the number expected for a population in linkage equilibrium. The combination gvpA-IGS II+rDNA-ITS II was found to occur less often (29 individuals) than expected by chance (69 individuals): this difference is highly significant (
2=25, P=<0·01).
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DISCUSSION |
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The finding that the most distantly spaced sampling stations were genetically similar (Fig. 4) was initially surprising. A simple explanation for this observation seems to be that for the greatest distances, one is comparing stations at the extreme southwest and northeast of the sampling area. The northern limit of the population was not explored in this study, but our records show that Nodularia was not found at Litorina station 1 (54·53° N, 10·51° E: Fig. 3
), just 44 km west of station 2 (Table 1
). This indicates that station 2, the station with the lowest gene diversity, may have been close to the southwestern limit of the Nodularia population at the time of sampling. It is possible that the extremes of Nodularias range are tolerable only to a subset of genotypes, resulting in lower genetic diversity in these areas. The significant negative correlation between the level of gene diversity at each station and its distance from the centre of the study (Fig. 5
) provides some limited support for this hypothesis.
The most significant finding of this study is that genetic exchange almost certainly occurs between Nodularia filaments in the natural population. This is revealed by the fact that all 12 possible allele combinations (genotypes) were found to exist in our sample of the population. An alternative explanation for the occurrence of the 12 observed genotypes is that parallel mutation along each of several purely asexual lineages has by chance generated the same alternative alleles (but no others) many times. Suppose, for example, that the ancestor of all Nodularia in the Baltic Sea today first diverged into two clonal lineages, each of which accumulated its own unique mutations in the PC-IGS region. We must then assume that by chance, the same suite of point mutations (and no others) occurred in the rDNA-ITS of these two independent lineages, and that finally a parallel insertion/deletion event in each of the six resulting lineages gave rise to the two gvpA-IGS alleles, and thus to the 12 genotypes (see Barker et al., 1999 , for an exact description of the differences between the alternative alleles, but note that including one unpublished PC-IGS sequence, we have at least three sequences for each allele type at each locus used in this study). Given the staggering odds against the sequence of events described above actually occurring, genetic exchange clearly provides the most parsimonious explanation of the data. We have also considered the possibility that either gene conversion or gene loss could produce the observed distribution of genotypes. The gvpA and rDNA coding regions are present in multiple copies in at least some Nodularia isolates (all studied so far in the case of gvpA), and gene conversion could therefore explain changes in allele patterns without the need for gene exchange. If we take the rDNA locus first, there are three allele types: type I amplifiable with primer 9408R, type II amplifiable with 9427R and type III amplifiable with both primers. Although it might seem as though a gene conversion event could transform a type III isolate into a type I or II, sequence analysis of type III isolates has shown that the rDNA copies present are not identical to those found in either type I or type II isolates (Barker et al., 1999
). Gene conversion can not therefore account for the interconversion of any known rDNA types. Similarly, gene conversion is unlikely to explain differences in the gvpA-IGS genotype of our samples: although there are two copies of gvpA, there is just a single copy of the intergenic spacer that separates them. Of course we can not rule out the possibility that there was an ancestral Nodularia which contained all possible alleles at each locus, and that differential loss by gene conversion or deletion gave rise to the 12 types found today, but again gene exchange offers the most parsimonious explanation of the data.
This is the first population-level study suggestive of genetic exchange in a natural cyanobacterial population. The index of association is significantly different from zero, which means that whilst gene exchange could occur in Nodularia, the population is not panmictic. This conclusion is also supported by the randomization tests, which showed that whilst some combinations of filaments showed significantly more linkage disequilibrium than expected by chance, other combinations could not be distinguished from simulated random mating data (Table 6). Unfortunately it is not possible to estimate actual levels of recombination by analysing linkage disequilibrium (Maynard Smith et al., 1993
; Cohan, 1994
), as this would require other factors such as the mutation rate to be quantified. What is clear from the data is that recombination is not commonplace enough to completely randomize alleles in the population; if that were the case the index of association would not differ significantly from zero, and the randomization tests would show no significant combinations. There are three possible explanations for the non-zero index of association between loci. First, the loci studied may be closely linked (i.e. lie in close physical proximity); second, certain allelic combinations may be selectively advantageous; and third, the population may comprise a set of clones which have undergone many divisions without any genetic exchange with other genotypes.
Whilst analysis of observed and expected numbers for each pairwise combination of loci showed that rDNA-ITS II+gvpA-IGS II occurred less frequently than expected, we have no reason to suspect that this particular allele combination should have a low fitness value, especially as in the case of the rDNA region, sequence variation has only been detected in the ITS, and not in coding regions (Barker et al., 1999 ). It is possible, however, that selection could be acting on regions tightly linked to those studied. It is unlikely that the three loci chosen are closely linked, but since we do not have any information about their physical location in the Nodularia genome we can not rule out this possibility. The most likely explanation for the observed degree of linkage disequilibrium seems to be that Nodularia undergoes periods of purely asexual growth, during which time certain genotypes grow better than others, but that gene exchange, by unknown mechanisms, occurs frequently enough to ensure that no particular allele at one locus ever occurs exclusively in combination with another from a separate genomic location: such a population structure is described as epidemic by Maynard Smith et al. (1993
). There have been no other population genetic studies on cyanobacteria with which to compare the results obtained for Nodularia; therefore we have no idea whether the population structure observed for this taxon is typical of filamentous cyanobacteria or of the cyanobacteria as a whole. Given the variation in IA in the genus Bacillus (Istock et al., 1992
) it seems unwise to extrapolate widely from our findings. Other studies have addressed genetic diversity in aquatic micro-organisms; for example Wise et al. (1996
) used data from seven polymorphic allozyme loci to show that the mean gene diversity for a natural lotic population of Burkholderia cepacia was 0·52. Unfortunately a useful comparison of genetic diversity based on allozyme and DNA markers respectively is impossible. We hope that our data can form a baseline with which to compare future DNA-marker studies of other aquatic microbes.
That genetic exchange can occur between bacteria in the marine environment is not a novel finding; intra-generic conjugative transfer and transformation at frequencies of around 10-2 and 10-7 respectively have been observed in many in vitro studies with marine water and sediments (see review by Ashelford et al., 1997 ) but all of the studies to date have involved pseudomonads, Escherichia coli or Vibrio spp.
Comparison with data from a research cruise in 1996 (Hayes & Barker, 1997 ) shows that the frequency of the two PC-IGS alleles in the Baltic Sea changed dramatically over 2 years. In 1996, PC-IGS types I/II and III were present in almost equal numbers (n=91 and 88 respectively), but in 1998 type I/II was rare in comparison to type III (n=355 and 2335 respectively). Undoubtedly the abundance of filaments of genotype 9 (Table 1
) is the major contributing factor in accounting for this shift in the allelic composition of the Nodularia population. We will not be able to explain such temporal shifts in population structure until we know more about the relative fitness of different Nodularia genotypes under various environmental conditions, and what impact cyanophage and other pathogens may have on dominant genotypes.
In terms of the taxonomy of Nodularia in the Baltic Sea, this study, as with our previous study (Barker et al., 1999 ) indicates that the Baltic has only a single species, if one chooses to apply a biological species concept.
The data presented here lend support to the hypothesis of Rudi et al. (1998 ) that the occurrence of genetic exchange best explains the incongruent pattern of inheritance for the small-subunit rRNA and rbcLX regions of many cyanobacteria. What our study shows is that gene exchange occurs in some cyanobacteria at a frequency great enough not just to show up over lengthy evolutionary time scales as demonstrated already, but also to shape the genetic structure of extant populations. We are now studying temporal changes in allele frequencies in greater detail, and starting to explore the mechanisms which facilitate genetic exchange in Nodularia.
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ACKNOWLEDGEMENTS |
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Received 12 April 2000;
revised 21 July 2000;
accepted 31 July 2000.