Centre for the Study of Evolution, University of Sussex
Previous attempts to demonstrate, or refute, the occurrence of recombination in mitochondria have used tests designed to measure relatively frequent recombination between rather similar sequences: for example, the homoplasy test (Maynard Smith and Smith 1998
) and the regression of linkage disequilibrium between pairs of loci with distance along the chromosome (Awadalla, Eyre-Walker, and Maynard Smith 1999
). Recently, Ladoukakis and Zouros (2001
; subsequently LZ) have attempted to demonstrate rare recombination events between sequences differing at 10% or more of nucleotides. The aim of this note is, first, to evaluate the statistical support for their conclusion and, second, briefly to discuss its significance.
LZ analyze three published data sets consisting of 1,140-bp sequences of cytochrome b from eight individuals of Rana and 10 of Apodemus, and 366 bp of cytochrome oxidase I from 10 individuals of Gammarus: the Rana sequences come from different species and their origin is given in table 1 . For each set, they first examined a matrix of the variable, informative amino acids (2023 per set) and identified, by visual inspection, one potential crossover event, involving two parents (a, c) and a recombinant (b), and two break points identifying a central recombinant piece, in which b resembles c, and two flanking regions in which b resembles a (see fig. 1 ). Thus the proposed event is the transfer of a short central region from a donor, c, to a recipient, a, yielding sequence b.
|
|
However, it is not clear from their paper just how this calculation was performed. In particular, did their permutations involve all nucleotides, including those coding for amino acid variation and therefore used in formulating the hypothesis they are testing? If these nucleotides were included, then the appropriate significance test would be to randomize a matrix of all the sequences and show that no choice of three sequences and two break points would yield a value of prd as statistically significant as that observed; such a test would be difficult to perform. We have therefore repeated their permutations, accepting their identification of the parental and recombinant sequences and proposed break points, but using only sites responsible for synonymous variation in the data set and not used in formulating the hypothesis being tested. The results are shown in table 2 . There is convincing evidence of recombination in Rana and a strong suggestion of recombination in Apodemus but no reason for suspecting recombination in Gammarus.
|
|
|
|
A pattern of differences of the kind shown in figure 3 strongly suggests a recombination event affecting sequences a and b (Ra1 and Rc6). Such a pattern could not be generated by clusters of hypervariable sites, or of constrained sites, unless each sequence was subject to a unique set of constraints at synonymous sites. It would require that in sequences a and b, but not c, the sites after the break have a reduced rate of change. This seems implausible, particularly for synonymous sites.
Thus for Rana, but not the other genera, there is overwhelming evidence for regions of similarity and difference between sequences for synonymous sites. This is difficult to explain except by recombination. However, there are real difficulties with recombination as an explanation. It is not just that it requires recombination between different "species": relatively few recombination events are required to produce the observed patterns. The real difficulty is as follows. The maximum chi-square test reveals seven statistically significant crossovers, each involving two of the sequences numbered 1 to 5 in table 1 and a
similar "break point" in the range 165 to 181. An examination of the genetic distances between the eight sequences, before and after site 165, reveals that the five sequences are similar to one another after the break point (six of 10 pairwise comparisons differ at less than 20 sites) but very different before the break (nine of 10 pairwise comparisons differ at more than 75 sites). This suggests that, relatively recently, a region of DNA roughly from polymorphic site 165 (site 636) to the end of the available sequence was introduced into each of the five sequences. This seems to require five separate events (or four if one of the sequences was the donor of the DNA). This could perhaps be explained by the spread, by recombination affecting several "species," of a selectively favored region of DNA. The synonymous sites analyzed, although not themselves selected, could have hitch-hiked with the selectively favored amino acid substitutions. It is relevant that there are, in this region, 12 polymorphic amino acids present in all five sequences but that these are rare in the other three. Members of the genus Rana have a number of characteristics that may facilitate the interspecies spread of a selectively favorable region of mitochondrial DNA. These include external fertilization, weak premating isolation and hybrid amphispermy in R. esculenta (Graf and Pelaz 1989
). However, both in the laboratory and the wild, the progeny of interspecies matings are usually inviable (T. Beebee, personal communication).
To summarize, we can find no evidence for recombination in Gammarus and only weak evidence in Apodemus, but there is overwhelming evidence for a pattern of similarity and difference at synonymous sites in Rana. Although there are difficulties with recombination as an explanation, it is hard to think of any other.
Footnotes
Diethard Tautz, Reviewing Editor
Keywords: recombination
mitochondria
Rana
Apodemus
Gammarus
Address for correspondence and reprints: J. Maynard Smith, Centre for the Study of Evolution, University of Sussex, BN1 9QL, United Kingdom. E-mail: t.ellis{at}sussex.ac.uk
.
References
Awadalla P., A. Eyre-Walker, J. Maynard Smith, 1999 Linkage disequilibrium and recombination in hominid mitochondria DNA Science 286:2524-2525
Graf J.-D., M. P. Pelaz, 1989 Evolutionary genetics of the Rana esculenta complex Pp. 289301 inR. M. Dawley and J. P. Bogart, eds. Evolution and ecology of unisexual vertebrates. Bulletin 466. New York State Museum, New York
Ladoukakis E. D., E. Zouros, 2001 Recombination in animal mitochondrial DNA: evidence from published sequences Mol. Biol. Evol 18:2127-2131
Maynard Smith J., 1992 Analyzing the mosaic structure of genes J. Mol. Evol 35:126-129
Maynard Smith J., N. H. Smith, 1998 Detecting recombination from gene trees Mol. Biol. Evol 15:590-599[Abstract]
Sawyer S., 1989 Statistical tests for detecting gene conversion Mol. Biol. Evol 6:526-538[Abstract]