Loss of sexual reproduction is considered an evolutionary dead end for metazoans, but bdelloid rotifers challenge this view as they appear to have persisted asexually for millions of years 1 . Neither male sex organs nor meiosis have ever been observed in these microscopic animals: oocytes are formed through mitotic divisions, with no reduction of chromosome number and no indication of chromosome pairing 2 . However, current evidence does not exclude that they may engage in sex on rare, cryptic occasions. Here we report the genome of a bdelloid rotifer, Adineta vaga (Davis, 1873) 3 , and show that its structure is incompatible with conventional meiosis. At gene scale, the genome of A. vaga is tetraploid and comprises both anciently duplicated segments and less divergent allelic regions. However, in contrast to sexual species, the allelic regions are rearranged and sometimes even found on the same chromosome. Such structure does not allow meiotic pairing; instead, we find abundant evidence of gene conversion, which may limit the accumulation of deleterious mutations in the absence of meiosis. Gene families involved in resistance to oxidation, carbohydrate metabolism and defence against transposons are significantly expanded, which may explain why transposable elements cover only 3% of the assembled sequence. Furthermore, 8% of the genes are likely to be of non-metazoan origin and were probably acquired horizontally. This apparent convergence between bdelloids and prokaryotes sheds new light on the evolutionary significance of sex.With more than 460 described species 4 , bdelloid rotifers ( Fig. 1) represent the highest metazoan taxonomic rank in which males, hermaphrodites and meiosis are unknown. Such persistence and diversification of an ameiotic clade of animals are in contradiction with the supposed long-term disadvantages of asexuality, making bdelloids an 'evolutionary scandal' 5 . Another unusual feature of bdelloid rotifers is their extreme resistance to desiccation at any stage of their life cycle 6 , enabling these microscopic animals to dwell in ephemeral freshwater habitats such as mosses, lichens and forest litter; this ability is presumably the source of their extreme resistance to ionizing radiation 7 .We assembled the genome of a clonal A. vaga lineage into separate haplotypes with a N 50 of 260 kilobases (kb) (that is, half of the assembly was composed of fragments longer than 260 kb). Assembly size was 218 megabases (Mb) but 26 Mb of the sequence had twice the average sequencing coverage, suggesting that some nearly identical regions were not resolved during assembly ( Supplementary Fig. 3); hence, the total genome size is likely to be 244 Mb, which corresponds to the estimate obtained independently using fluorometry (Supplementary Note C2). Annotation of the complete assembly (including all haplotypes) yielded 49,300 genes. Intragenomic sequence comparisons revealed numerous homologous blocks with conserved gene order (colinear regions). For each such block we computed the per-site synonymous d...

Negative selection against deleterious alleles produced by mutation influences withinpopulation variation as the most pervasive form of natural selection. However, it is not known whether deleterious alleles affect fitness independently, so that cumulative fitness loss depends exponentially on the number of deleterious alleles, or synergistically, so that each additional deleterious allele results in a larger decrease in relative fitness. Negative selection with synergistic epistasis should produce negative linkage disequilibrium between deleterious alleles and, therefore, an underdispersed distribution of the number of deleterious alleles in the genome. Indeed, we detected underdispersion of the number of rare loss-of-function alleles in eight independent data sets from human and fly populations. Thus, selection against rare protein-disrupting alleles is characterized by synergistic epistasis, which may explain how human and fly populations persist despite high genomic mutation rates.

Negative selection against deleterious alleles produced by mutation is the most common form of natural selection, which strongly influences within-population variation and interspecific divergence. However, some fundamental properties of negative selection remain obscure. In particular, it is still not known whether deleterious alleles affect fitness independently, so that cumulative fitness loss depends exponentially on the number of deleterious alleles, or synergistically, so that each additional deleterious allele results in a larger decrease in relative fitness. Negative selection with synergistic epistasis must produce negative linkage disequilibrium between deleterious alleles, and therefore, underdispersed distribution of the number of deleterious alleles in the genome. Indeed, we detected underdispersion of the number of rare loss-of-function (LoF) alleles in eight independent datasets from modern human and Drosophila melanogaster populations. Thus, ongoing selection against deleterious alleles is characterized by synergistic epistasis, which can explain how human and fly populations persist despite very high genomic deleterious mutation rates.

Sexual reproduction is almost ubiquitous among extant eukaryotes. As most asexual lineages are short-lived, abandoning sex is commonly regarded as an evolutionary dead end. Still, putative anciently asexual lineages challenge this view. One of the most striking examples are bdelloid rotifers, microscopic freshwater invertebrates believed to have completely abandoned sexual reproduction tens of Myr ago. Here, we compare whole genomes of 11 wild-caught individuals of the bdelloid rotifer Adineta vaga and present evidence that some patterns in its genetic variation are incompatible with strict clonality and lack of genetic exchange. These patterns include genotype proportions close to Hardy-Weinberg expectations within loci, lack of linkage disequilibrium between distant loci, incongruent haplotype phylogenies across the genome, and evidence for hybridization between divergent lineages. Analysis of triallelic sites independently corroborates these findings. Our results provide evidence for interindividual genetic exchange and recombination in A. vaga, a species previously thought to be anciently asexual.

Thy-1 is a cell surface glycoprotein present on normal lung fibroblasts but absent from the fibroblastic foci of idiopathic pulmonary fibrosis. Thy-1 correlates inversely with fibrogenic phenotypic characteristics and functions as a "fibrosis suppressor." Promoter region hypermethylation can silence Thy-1 expression in fibroblastic foci, suggesting that epigenetic regulation is important in programming the fibrotic phenotype. We examined whether histone modifications are important in regulating Thy-1 expression in lung fibroblasts. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) restored Thy-1 expression in Thy-1(-) cells in a time-dependent and concentration-dependent fashion and was associated with enrichment of histone acetylation. Chromatin immunoprecipitation demonstrated Thy-1 depletion of trimethylated H3K27 after 24 hours of TSA treatment, concurrent with enrichment of trimethylated H3K4 and acetylated H4. Bisulfite sequencing of the Thy-1 promoter region revealed demethylation of the previously hypermethylated CpG sites after treatment with TSA. Although Thy-1 was hypermethylated in Thy-1(-) lung fibroblasts, we observed that Thy-1(-) cells have lower global DNA methylation compared with Thy-1(+) lung fibroblasts, which was partially reversed by TSA treatment. TSA treatment up-regulates total methyltransferase activity in these cells. Our data indicate that Thy-1 silencing is regulated by histone modifications in addition to promoter hypermethylation in lung fibroblasts. Additionally, our findings indicate that alteration of histone modifications alters DNA methylation. Understanding the molecular hierarchy of events with respect to reactivation of transcription and reversal of histone modification will be critical to understand and modify the regulated expression of Thy-1, a tumor-supressor and fibrosis-suppressor gene.

15 7 8 Sexual reproduction which involves alternation of meiosis and syngamy is the 9 ancestral condition of extant eukaryotes. Transitions to asexual reproduction 10 were numerous, but most of the resulting eukaryotic lineages are rather short-11 lived. Still, there are several exceptions to this rule including darwinulid 12 ostracods 1,2 and timema stick insects 3 . The most striking of them is bdelloid 13 rotifers 4-6 , microscopic freshwater invertebrates which underwent an extensive 14 adaptive radiation after apparently losing meiosis over 10 Mya. Indeed, both the 15 lack of males in numerous bdelloid species and the lack of proper homology 16 between chromosomes 6 rule out ordinary sex. However, this does not exclude the 17 possibility of some other mode of interindividual genetic exchange and 18 recombination in their populations 7 . Recent analyses based on a few loci 19suggested genetic exchanges in this group 8,9 , although this has been 20 controversial 10 . Here, we compare complete genomes of 11 individuals from the 21 wild population of the bdelloid rotifer Adineta vaga, and show that its genetic 22 structure, which involves Hardy-Weinberg proportions of genotypes within loci 23 and lack of linkage disequilibrium between distant loci, is incompatible with 24 strictly clonal reproduction. Instead, it can emerge only under ongoing 25 recombination between different individuals within this species, possibly through 26 transformation. Such a genetic structure makes the population immune to 27

Conservation of function can be accompanied by obvious similarity of homologous sequences which may persist for billions of years (Iyer LM, Leipe DD, Koonin EV, Aravind L. 2004. Evolutionary history and higher order classification of AAA+ ATPases. J Struct Biol. 146:11–31.). However, presumably homologous segments of noncoding DNA can also retain their ancestral function even after their sequences diverge beyond recognition (Fisher S, Grice EA, Vinton RM, Bessling SL, McCallion AS. 2006. Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312:276–279.). To investigate this phenomenon at the genomic scale, we studied homologous introns in a quartet of insect species, and in a quartet of vertebrate species. Each quartet consisted of two pairs of moderately distant genomes, with a much larger evolutionary distance between the pairs. In both quartets, we found that introns that carry a regulatory segment or a conserved segment in the first pair tend to carry a conserved segment in the second pair, even though no similarity of these segments could be detected between the two pairs. Furthermore, introns from one pair that are preserved in the other pair tend to carry a conserved segment within the first pair, and be longer in the first pair, compared with the introns that were lost between pairs, even though no similarity between pairs could be detected in such preserved introns. These results indicate that selective constraint, presumably caused by conservation of the ancestral function, often persists even after the homologous DNA segments become unalignable.

Podospora anserina is a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation of P. anserina to submerged cultivation, we initiated a long-term evolution experiment. In the course of the first 4 years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, in the G-protein alpha subunit FadA, new alleles fixed in seven out of eight experimental populations, and these fixations affected just four amino acid sites, which is an unprecedented level of parallelism in experimental evolution. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed fixations were adaptive and driven by positive selection.