Bdelloid Genome Structure.

We find that the sequences around the four copies of hsp82 in P. roseola occur as two colinear pairs, a quartet in which the two pairs share a limited number of genes in the same order and orientation but with genes present in each pair that are absent from the other. That the quartet structure of the hsp82 region of P. roseola is conserved in Bdelloidea is supported by the finding of an hsp82-containing quartet with a gene content and structure closely similar to that depicted in Fig. 1 in the bdelloid Adineta vaga (23), a species belonging to a family that separated from that of P. roseola ≈60 million years ago (12, 19, 23). Moreover, the existence of such quartet structures along entire chromosomes is suggested by the finding in P. roseola of a quartet organization of the regions surrounding a Hox gene and by the observation by FISH that the Hox and hsp82 quartets appear to be far apart on the same four mid-size chromosomes (J.L.M.W., unpublished observations).

That bdelloid genomes are generally organized as quartets is also consistent with the karyotypes of P. roseola and A. vaga. The karyotype of A. vaga consists of 12 mid-size chromosomes, probably comprising three quartets, whereas that of P. roseola comprises 10 mid-size chromosomes, a single chromosome of approximately twice that length, and two dots of unequal size (18, 24). The large chromosome appears to be an isochromosome, because probes that hybridize to it do so at pairs of sites, each approximately equidistant from its center (ref. 25 and unpublished data). The 13 chromosomes of P. roseola may therefore consist of two quartets of mid-size chromosomes, with the two remaining mid-size chromosomes and the two arms of the isochromosome making up the third quartet. The two dots may be B chromosomes, because one or the other is often missing in karyotypes (J.L.M.W., unpublished observations).

Thus, the evidence suggests that bdelloids are degenerate tetraploids. The lack of any discernable homology between pairs outside of protein coding regions and the similarity of the hsp82-containing quartet of P. roseola to that of A. vaga indicate that this tetraploidy originated long ago, probably before the radiation of modern bdelloids. Tetraploidization could have resulted from whole-genome duplication (26, 27) followed by extensive gene loss. Although it cannot be said whether any particular segment present in one colinear pair and not in the other has simply been lost through decay and deletion or is instead the result of translocation to or from a site elsewhere in the genome, rampant translocation of small segments would have destroyed evidence of initial linkage. In that case, except for the hsp82-containing segment on which our selection of cosmids for sequencing was based, we would not expect to find segments common to both colinear pairs. In fact, as may be seen in Fig. 1, there are three such additional segments, in the same order and orientation on all four contigs. In addition, the evidence from FISH that hsp82 and a Hox gene are on the same four chromosomes suggests that there are few translocations.

Comparison with other degenerate tetraploids such as Saccharomyces cerevisiae, Arabidopsis thaliana, and the teleost Tetraodon nigroviridis reveals both similarities and apparent differences from what we find in P. roseola (28⇓⇓–31). In all of these cases, consistent with the pattern we see in bdelloids, most genes have been lost from one or the other of the genomes of the initial tetraploid, mainly as the result of deletions covering one or a few genes. Although deletions greatly predominate over rearrangements in S. cerevisiae, A. thaliana, and T. nigroviridis, many rearrangements have nevertheless occurred. Individual chromosomes in these species are mosaics of numerous blocks, each containing a minority of its genes common to a block on a different chromosome, most of them in the same order and orientation in both blocks, so that each such pair of blocks resembles the A and B contigs depicted in Fig. 1.

Colinear Chromosome Pairs and DNA Repair.

As described in the companion article (32), bdelloids are extraordinarily resistant to ionizing radiation, being able to continue reproducing after exposure to a dose causing hundreds of DNA double-strand breaks (DSBs) per genome. As discussed there, the extraordinary resistance of bdelloid rotifers to radiation and desiccation, like that of the bacterium Deinococcus radiodurans (33), is almost certainly the result of evolutionary adaptation to repair DNA breaks and to prevent or repair other damage caused by the episodes of desiccation to which bdelloids are exposed in their ephemerally aquatic habitats.

The ability of bdelloid rotifers to remain fertile after experiencing high levels of radiation-induced DSBs and the implication that frequent DNA breakage and repair is a characteristic of the bdelloid lifestyle offer an explanation for why bdelloid genomes are made up of colinear chromosome pairs. Bdelloid primary oocyte nuclei are in G₁ and are therefore without sister chromatids (34⇓–36). The colinear chromosomes are therefore the only templates available for the accurate repair of DSBs. Sequence homology between the members of a pair could be maintained by conversion and mitotic crossing-over associated with DSB repair, particularly that occurring in association with desiccation and rehydration, and by selection against clones in which sequence difference reaches levels that substantially reduce the efficiency or accuracy of repair.

The occurrence of homogenizing events is indicated by the sizeable tracts of identity or near-identity between colinear contigs depicted in Fig. 1. Such tracts cannot be chance occurrences. The probability of their chance occurrence may be estimated by using Stephens's runs test (37) under the assumption that nucleotide differences are distributed at random within a region of given length and overall sequence divergence. For example, the probability of the 470-bp tract of identity in the A lineage contigs occurring anywhere in the region of their overlap is 3.0 × 10⁻⁶, and that of the 400-bp tract in the B lineage is 2.8 × 10⁻⁷. Inspection of the colinear pairs reveals additional long tracts that are nearly identical. Such tracts of identity and near-identity appear to have arisen recently, possibly associated with DSB repair, which may also produce tracts too short to be recognized as such. The correlation of divergence values between adjacent windows is very strong (r ≈ 0.7) for windows up to ≈2 kb and becomes insignificant by about twice that length (data not shown). This pattern, seen over a wide range of divergence values, would be expected for gene conversion tracts usually of no more than a few kilobases occurring over millions of years. The contigs depicted in Fig. 1 exhibit no evidence of recent conversion between A and B lineages, and these are now probably too divergent to function as templates for interlineage homologous repair.

In addition to satisfying the requirement for templates for accurate DNA repair in G₁, colinear chromosome pairs may be of additional benefit to bdelloid rotifers by allowing the accumulation of heterotic interactions. Because the average synonymous divergence we find for gene pairs on colinear chromosomes is ≈7%, such interactions could accumulate and persist for appreciable times between homogenization events. Also, the presence of colinear chromosome pairs and the occurrence of gene conversion may produce clones with combinations of mutations within a gene that are beneficial only in cis more rapidly than could be achieved if each chromosome were represented only once. Furthermore, given the requirement for colinear chromosome pairs, conversion and mitotic crossing-over in clones heterozygous for recessive beneficial mutations may hasten the appearance of better-adapted clones that otherwise could arise only by further mutation in the clone in which they occur, analogous to the effect of allelic segregation and syngamy in sexual species (38, 39).

Implications of Colinear Pairs for Bdelloid Asexuality.

Our findings call for a reevaluation of the significance of the highly diverged pairs of genes we have found in individual bdelloid genomes (19). Previously, although not rejecting the possibility that bdelloids descend from an ancient polyploid ancestor and engage in some rare or elusive form of sexual reproduction, we thought it more plausible, consistent with other indications of bdelloid asexuality and the presumed rareness of polyploidy in animals (40), to interpret the highly diverged copies of hsp82 we found in diverse bdelloid species as descendants of alleles that had ceased segregation and had not undergone homogenization by conversion or mitotic crossing-over since the separation of bdelloid families and therefore as evidence for ancient asexuality (19). The present evidence that bdelloids are degenerate tetraploids suggests that the highly diverged gene pairs are the result of ancient whole-genome duplication, perhaps involving a hybridization between closely related species, rather than descendants of former alleles and raises the question of whether the colinear chromosomes are meiotic homologs. Although it is not entirely excluded that bdelloids engage in rare sexual reproduction, as explained above an entirely different explanation for the maintenance of colinear chromosome pairs follows from the discovery of the extraordinary resistance of bdelloid rotifers to ionizing radiation (32) and the implication that colinear chromosome pairs are required in order for bdelloids to repair DNA breakage associated with their ephemerally aquatic habitats.