* Dipartimento di Scienze Ambientali, Università degli Studi di Parma, Parma, Italy
Institute of Ecology, University of Lausanne, Lausanne, Switzerland
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Abstract |
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Key Words: ancient asexuals recombination gene conversion mosaic sequences
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Introduction |
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Unfortunately, for most taxa the very definition of their asexual status rests mainly on circumstantial evidence such as the absence of males in living populations or fossil records or the lack of observed sexual stages in their life cycles. Furthermore, many ancient asexuals are difficult to study because of small size and because they are long-lived, rendering impractical the rapid establishment of populations in the laboratory. Genetic information has the potential of providing evidence for past recombination events. Using genetic markerse.g., allozymes and DNA markershigh variability has often been reported in supposedly asexual populations. Variability alone is not informative on the issue of ancient asexuality: it could have originated by mutation accumulation after loss of sex or by repeated transitions to asexuality that captured the variability of sexual ancestors (Chaplin and Hebert 1997).
DNA sequences, although more onerous to obtain, allow the direct evaluation of recombination effects. The so-called Meselson method (see Birky 1996; Judson and Normark 1996) is a clever phylogenetic approach based on the premise that in asexual organisms, haplotype variability is only generated by mutation. Within-individual allelic divergence exceeding that observed between lineages is taken as evidence of ancient asexuality. The method was used for bdelloid rotifers (Mark Welch and Meselson 2000), the notorious and most convincing example of supposed ancient asexuals, some species of which are estimated to have been asexual between 35 and 40 Myr (Mark Welch and Meselson 2000). The finding of similar combinations of highly diverging alleles within the genome of single representatives (populations recently propagated from single individuals) of different species supports the hypothesis that bdelloids evolved without recombination. However, according to Butlin (2000), the pattern described by Mark Welch and Meselson "does not quite fit with the expectations." In some cases the observed allelic divergence is lower within than between lineages. Uncertainty was cast on the status of the few other ancient asexual taxa: no trace of a "Meselson effect" was found in the aphid tribe Tramini (Normark 1999) and in the ostracod Darwinula stevensoni, the latter estimated to have been asexual for 25 Myr (Gandolfi et al. 2001).
The lack of Meselson effect does not per se constitute evidence of outcrossing sex and does not imply the consequent rejection of the status of ancient asexual (Butlin 2002). Automixis, ploidy level variation, gene conversion, and mitotic crossing-over can account for the absence of the expected pattern (Normark 1999). Automixis and ploidy variation affect the whole genome. Gene conversion and mitotic crossing-over are within-chromosome mechanisms with effects similar to that of meiotic recombination. Gene conversion and mitotic crossing-over have the potential to both purge the genome of mutations and increase allelic richness of the species by bringing together advantageous mutations (between alleles within individuals or lineages) (Gandolfi et al. 2001).
While a population genetic approach to predictions of recombination has not been applied to data from many putative ancient asexuals because of difficulties of their culturing, it has been applied to the arbuscular mycorrhizal fungi. These fungi are thought to have been asexual for 400 Myr. Calculations of the index of association among spores of these fungi revealed a clonal population structure (Rosendahl and Taylor 1997), but this was later cast in doubt by the fact that these fungi were shown to contain genetically different nuclei (Kuhn, Hijri, and Sanders 2001). Subsequent incompatibility analysis of sequence variation in these fungi only detected a small number of recombination events in multicopy genes and no recombination events in a putative single copy gene (Kuhn, Hijri, and Sanders 2001). Unfortunately, the Meselson method could not be applied to these data sets as the method assumes that the organism is diploid and looks at allelic divergence on sister chromosomes. The ploidy of mycorrhizal fungi has not been established.
Many methods and software packages have been recently developed to test for the occurrence of recombination in DNA sequence data (see David L. Robertson, http://grinch.zoo.ox.ac.uk/RAP_links.html). These methods are based on phylogenetic, substitution, compatibility, and distance approaches. Recently, the performancei.e., power and incidence of false positivesof most of these methods was evaluated and compared on simulated and real data (Posada and Crandall 2001; Posada 2002). Most methods show a growing power of detection, as expected, at higher levels of divergence and increased rates of recombination. Some were demonstrated to be more efficient than others, with a more rapid increase of power, but with similar and constant incidence of false positives. In general, a number of methods were found to be quite reliable for detecting recombination at different frequencies of recombination, although they are not very powerful in detecting a few infrequent recombination events and may give problems if the organisms studied have particularly rapid mutation rates. Thus, the methods are rather conservative for use with data on ancient asexuals as infrequent or older recombination events are less likely to be detected.
We looked for evidence of recombination by analyzing sequence data from different loci studied in several groups of evolutionarily distant species that are considered to be ancient asexuals, using seven different types of analysis. The groups of organisms were the arbuscular mycorrhizal fungi (Glomales), D. stevensoni (Darwinuloidea crustacean ostracods), and the bdelloid rotifers (Bdelloidea), which are thought to have been asexual for the last 400, 25 (100), and 3540 Myr, respectively. The seven different analytical methods evaluated the evolutionary relationships among haplotypes, and these methods had previously been shown to be reliable for predicting the occurrence of recombination events.
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Materials and Methods |
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Most of the data sets are from rDNA regions. These multicopy gene families, highly repeated on one or several chromosomes, seem adequate material for the search of recombination evidence. Ribosomal DNA was thought to be subject to the homogenizing pressure of concerted evolution by gene conversion and mitotic crossing over within individuals and species, but this view was demonstrated in general to be incorrect (Harris and Crandall 2000), and a number of examples exist that show the absence or a slow down of homogenization processes in asexual taxa (Crease and Lynch 1991; Campbell et al. 1997; Hugall, Stanton, and Moritz 1999; Lanfranco, Delpero, and Bonfante 1999).
Of the Mark Welch and Meselson (2000) data, we only considered sequences for the hsp82 gene of two bdelloids species (individuals), Adineta vaga and Habrotrocha constricta. These were chosen because they did not show a Meselson effect and had more than two sequences per lineage, although they did not have highly divergent alleles.
An optimal opportunity to look for evidence of recombination would probably be offered by the availability of many sequences from different populations within species, as in the case of the data collected on the aphid tribe Tramini by Normark (1999). Unfortunately, almost no variation was found in the sequences: most probably an effect of strong gene conversioni.e., recombinationbut with no clear and direct evidence left. For this reason the data were excluded from the analysis.
Recombination Detection
The initial search for recombination was operated by graphic methods, because the presence of loops in networks, and not strictly bifurcating tree structure, can be evidence of recombination. Because graphical methods do not give statistical support to the recombination hypothesis, and because they cannot discriminate effective recombination from homoplasy by analogous evolution, different statistical tests of recombination were applied to the data sets. As shown by Posada and Crandall (2001) and Posada (2002), different recombination detection methods can produce different outcomes with the same data set. False negative results are a much more frequent problem than false positive outcomes, and, in general, a single-method analysis should not be considered reliable (Posada 2002).
Graphical Methods
Two different software packages were used to provide a graphic evaluation of the genealogical relationships among sequences: SplitsTree v. 3.1 (Huson 1998), based on the split decomposition method developed by Bandelt and Dress (1992), and TCS v. 1.13 (Clement, Posada, and Crandall 2000), which makes use of the parsimony method of Templeton, Crandall, and Sing (1992). Aligned sequences in NEXUS format were used as input files for both programs. Evidence for reticulate evolution is reported for the graph constructed under SplitsTree, on pairwise Hamming distances, no gaps, and using only parsimonious sites (although completely similar results were obtained with other distance measures and settings), and under TCS, with default settings. Graphical methods were not used for bdelloid data sets, because each was made up of only three sequences.
Statistical Evaluation
Statistical support to the identified network structures was assessed by different recombination detection software packages, based on different recombination algorithms, and selected on the basis of their performance according to Posada and Crandall (2001) and Posada (2002) (see Posada and Crandall 2001 for details of these methods).
Three programs were used on all the data sets. (1) Reticulate (Jakobsen and Easteal 1996) allows the construction of compatibility matrices (Sneath, Sackin, and Ambler 1975) to identify regions with different histories and the boundaries between them resulting from recombination. A no-recombination null hypothesis was tested by a randomization algorithm (1,000 runs) and a neighbor similarity score (NSS) statistic. Bdelloid data could not be analyzed by Reticulate, because the minimum required number of sequences is four. (2) GeneConv 1.81 (Stanley A. Sawyer, http://www.math.wustl.edu/sawyer/geneconv) analyzes the distribution of substitutions (Sawyer 1989) along the sequences. It detects significant clustering of polymorphic sites. Only phylogenetically informative polymorphisms, and no indels, were considered. Mismatches were allowed and set to two, and global P values were based on 10,000 permutations and were corrected for multiple comparisons (sample size). (3) RDP (Martin and Rybicki 2000) calculates, along a sliding window, the identity values for each of the three possible pairs of all possible triplets of sequences, finding potential recombinant regions; internal and external reference sequences were used and window size was set to 10 nt. Because of the limited number of sequences available, A. vaga and H. constricta were analyzed by Geneconv that considered phylogenetically uninformative sites.
Two programs based on the Maximum Chi-Squared method developed by Maynard Smith (1992), probably the most powerful method to detect recombination (Posada 2002), were used only on bdelloid rotifer data: Lard (Holmes, Worobey, and Rambaut 1999) and Maximum Chi-Squared 1.0 (Nick Ross and Brian G. Spratt, http://www.biols.susx.ac.uk/Biochem/Molbiol/maximum-chi-squared.html). The programs are computationally demanding for multiple comparisons and would have required too much time for analyzing the multiple sequence alignments of the other organisms. The Maximum Chi-Squared method identifies breakpoints that maximize a 2 x 2 2 of the difference between the proportions of polymorphic sites with equal and different bases before and after the breakpoint. Base frequencies and
parameter of the gamma distribution were estimated in DAMBE v. 4.0.75 (Xia and Xie 2001). They were used with either a single or a double breakpoint, as the parameters set in Lard (other parameters set as default). The P value of recombination was estimated by comparing the likelihood ratio (LR) of recombination (H1), calculated on real data, to that of no recombination (H0), computed for 500 sequence data sets simulated under H0 by Seq-Gen software (Rambaut and Grassly 1997) and analyzed by the same procedure as for the empirical data. The program Maximum Chi-Squared 1.0 was used to test the occurrence of multiple recombination breakpoints, following the procedure suggested in the original work by Maynard Smith (1992) for mosaic sequence structures involving more than two blocks.
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Results |
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In D. stevensoni, the recombination hypothesis suggested by the TCS graph network structure was not statistically supported. Only Reticulate gave a P value close to the 0.05 threshold (P = 0.056). Recombination events suggested by the TCS network are observed within individuals, with parental and daughter (mosaic) sequences maintained and found in the multigenic family of ITS1 rDNA.
In Bdelloids the potential recombination events were observed within single lineages recently propagated from single individuals.
Evidence of Recombination by Different Methods of Analysis
Split decomposition analysis (by SplitsTree) provided evidence of reticulate evolution in all the data sets tested except in Glomus geosporum LSU rDNA and Darwinula stevensoni ITS1 rDNA. Graphical analysis by TCS confirmed the reticulate network structure only for G. geosporum LSU rDNA, but all the data sets were completely (G. mosseae rDNA) or partially unresolved. In D. stevensoni ITS1 rDNA, however, TCS completely resolved the network and, contrary to SplitsTree, showed evidence of reticulate evolution (see Supplementary Material).
In complete agreement with the results obtained with SplitsTree, significant support for the recombination hypothesis was found by Reticulate for all data sets except G. geosporum LSU rDNA and D. stevensoni ITS1 rDNA, whose P values (0.089 and 0.056, respectively) were, however, close to the significance threshold. Geneconv confirmed these results, except in G. geosporum rDNA, but gave no support to the recombination hypothesis in A. vaga and H. constricta hsp82. Partially contrasting results emerged by RDP analysis: statistically significant evidence of recombination was observed in G. mosseae rDNA and G. intraradices BiP (table 2).
Evidence of recombination emerged in H. constricta hsp82 by the analysis with Lard, with a significant double breakpoint found between sites 370371 and either 512513 (P = 0.024) or 581582 (P = 0.020) (fig. 1B). For A. vaga and H. constricta hsp82, statistically significant support for four and two breakpoints, respectively, was obtained by Maximum Chi-Squared (fig. 1). In general, reciprocal recombinants were not observed.
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Discussion |
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The methods used are more likely to miss recombination events that occurred a long time in the past, so the analyses that we have carried out would have been more likely to miss these recombination events if they had happened before the organism became asexual, if that happened a long time ago. Recombination detection power was also found to depend on the number of sequences, the number of diverging or informative sites, and the overall level of divergence (Posada 2002). These factors are a function of sampling effort and sampling strategy. In G. geosporum and G. mosseae, each with two data sets based on different loci, a larger number of sequences and a slightly higher number of sites allowed a higher power of detection of recombination, irrespective of the increased or decreased genetic diversity observed respectively in G. mosseae and G. geosporum. Homoplasies produced by very rapid mutation rates could also potentially bias the results and give false-positive predictions of recombination. Unfortunately, the mutation rates of the organisms in these studies are not known.
The main difference in the number of cases of recombination evidence inferred by different software was observed between RDP, on the one hand, and Reticulate and Geneconv, on the other. This result is also in agreement with that reported by Posada and Crandall (2001) on simulated data, where they observed very similar performances in Reticulate and Geneconv, versus a more limited power in RDP, especially at low and intermediate values of genetic diversity.
Although a possible explanation might be the presence of PCR artifacts (Pääbo, Irwin, and Wilson 1990; Bradley and Hillis 1997; Judo, Wedel, and Wilson 1998), for some sequences we have good reasons for ruling them out. In bdelloids, Mark Welch and Meselson (2000) reported, in both A. vaga and H. constricta, three sequence variants, each found in at least five independently sequenced cloned amplicons. This is more than expected in a supposed diploid single locus, but less than expected if supernumerary variants are originated by artifactual amplification, which is likely to produce a higher number of chimeras. Moreover, Southern blot analyses performed on hsp82 in H. constricta gave the final demonstration that all three sequence variants, and no others, are present in the pool of individuals of the species. Repetitive sequencing of the same variants from cloned amplicons is also true for the BiP gene in G. intraradices and the ITS region of G. geosporum (Kuhn, Hijri, and Sanders 2001); therefore, such artifacts are also unlikely for these sequences. The evidence that the supposed recombination events are not artifactual is not as clear for some of the other sequences of Glomales and in Darwinula. The finding of the same mosaic sequences in independent amplifications from different individuals of Darwinula (Gandolfi et al. 2001), and, in general, the fact that recombination is not qualitatively (always/never) observed within homogeneous data (obtained following the same PCR protocols) also make PCR artifacts unlikely.
One possibility to explain the presence of three sequence variants in a single-copy gene in the two bdelloid species is that the mosaic sequences (AV2 and HC2 in fig. 1) originated by recombination after the clonal lineage was started from one individual and gave rise to a new sublineage, finally resulting in a pool made up of two distinct lineages. It is not clear how recently (how many generations before the analysis) the bdelloid pools had originated from a single individual. However, for this to happen, because the presence of several point mutations in the mosaic sequences compared to the parental ones, mutation rate would have to be very high. Despite the apparent functionality maintained by all the copies (Mark Welch and Meselson 2000), the presence of three variants is probably due to a duplication that could be potentially recorded within individual genome.
Detection of recombination is particularly interesting in the case of the arbuscular mycorrhizal fungi. These fungi were initially thought to be asexual because they produce spores that develop on one hypha without fusion with another individual. It was recently shown, however, that these fungi contain populations of genetically different nuclei (Kuhn, Hijri, and Sanders 2001). The original interpretation of this result was that the nuclei have diverged from each other because of accumulation of mutations in an essentially clonal lineage. Our finding that these fungi may well be recombinant would suggest that recombination can actually occur among genetically different nuclei within a spore, because several of the data sets were constructed with DNA from fungal material originating from vegetative growth, where the culture was initiated with one spore and did not have the possibility to exchange nuclei with another culture. What this means is that, because genetic variance could be generated and maintained within one fungus, the fungi can retain a recombinant population of nuclei that could negate the necessity to exchange genetic information with other mycorrhizal fungi. Furthermore, because these fungi can grow vegetatively from plant to plant, forming a very large network, the effective population size (number of different nuclei that are needed to maintain the population) could be extremely high.
Our study was not designed to investigate the mechanisms responsible for the observed recombination; however some of our results appear to shed some light on this issue. It is known that, although sexual reproduction is overwhelmingly the most common process that recombines genetic variants, recombination can occur without sex (Normark 1999; Butlin 2000, 2002). In fungi, anastomosis was suggested as the mechanism that enables exchange of different nuclei between isolates, species, or even closely related genera (Giovannetti, Azzolini, and Citernesi 1999; Clapp, Rodriguez, and Dodd 2001). Cross incompatibility is, however, most frequent (Giovannetti, Azzolini, and Citernesi 1999), and genetic exchange by fusion of hyphae could not be considered an explanation of chimera molecule formation. In all the groups analyzed chimeras and parental sequences could be observed within an individual genome (clonal isolate/spore) in multigene family markers in Glomales and Darwinula, in a single locus marker of G. intraradices, and in a triallelic system in bdelloids. This finding might be considered an indication that the recombination we observed is not necessarily due to a sexual (haploid) phase and outcrossing. Even finding parental sequences in different individuals is not necessarily evidence of outcrossing: it could simply be due to undersampling of multigene family or nuclei variants to detect both parentals.
In general, reciprocal recombinants were not observed. This could be considered a hint that gene conversion, a mechanism that leads to nonreciprocal genetic exchanges, was in action. Again, we have to stress the need for caution in interpreting this result given the sampling heterogeneity.
Ultimately, conclusive evidence that recombination events occur in ancient asexual taxa (and maybe not during a sexual phase) can only come from appropriate genealogical and well-designed population genetics studies. The difficulty in breeding and working with small individuals and the impossibility of working on single nuclei in fungi will have to be overcome. A quantitative analysis of the frequency of recombination events will indicate whether the usual justifications for the "prevalence of sex" as a reproductive mode has to be modified and attention switched to the relevance of recombination per se. However, the analyses performed in this study strongly suggest previously undetected recombination in several putative ancient asexuals.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Keith Crandall, Associate Editor
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