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It has been hypothesized that in ancient apomictic, nonrecombining lineages the two alleles of a single copy gene will become highly divergent as a result of the independent accumulation of mutations (Meselson effect). We used a partial sequence of the elongation factor-1α (ef-1α) and the heat shock protein 82 (hsp82) genes to test this hypothesis for putative ancient parthenogenetic oribatid mite lineages. In addition, we tested if the hsp82 gene is fully transcribed by sequencing the cDNA and we also tested if there is evidence for recombination and gene conversion in sexual and parthenogenetic oribatid mite species. The average maximum intra-specific divergence in the ef-1α was 2.7% in three parthenogenetic species and 8.6% in three sexual species; the average maximum intra-individual genetic divergence was 0.9% in the parthenogenetic and 6.0% in the sexual species. In the hsp82 gene the average maximum intra-individual genetic divergence in the sexual species Steganacarus magnus and in the parthenogenetic species Platynothrus peltifer was 1.1% and 1.2%, respectively. None of the differences were statistically significant. The cDNA data indicated that the hsp82 sequence is transcribed and intron-free. Likelihood permutation tests indicate that ef-1α has undergone recombination in all three studied sexual species and gene conversion in two of the sexual species, but neither process has occurred in any of the parthenogenetic species. No evidence for recombination or gene conversion was found for sexual or parthenogenetic oribatid mite species in the hsp 82 gene. There appears to be no Meselson effect in parthenogenetic oribatid mite species. Presumably, their low genetic divergence is due to automixis, other homogenizing mechanisms or strong selection to keep both the ef-1α and the hsp82 gene functioning.
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- Materials and methods
Putative ancient parthenogenetic taxa, such as bdelloid rotifers, darwinulid ostracods and some clusters within oribatid mites (Mark Welch & Meselson, 2000; Schön & Martens, 2003; Maraun et al., 2004), are challenging theories of evolutionary biology. They are species rich (darwinulid ostracods: 26 species; bdelloid rotifers: 363 species; oribatid mites: between 54 and 156 species in different clusters) and may have radiated while being parthenogenetic. These taxa have been termed ‘evolutionary scandals’ (Maynard Smith, 1978) because theoretical considerations suggest that long-term advantages of sexual reproduction should eventually overcome the short-term advantages of parthenogenesis. Consequently, parthenogenetic lineages are doomed to extinction in the long term. Understanding how species managed to persist over long periods of time without sex and recombination will contribute significantly to our understanding of the prevalence of sex in the living world.
No single evolutionary theory is able to explain why sex is the prevalent reproductive mode in eukaryotes (Bell, 1982; Kondrashov, 1988,1993; Roughgarden, 1991; Browne, 1992; Lynch et al., 1993; Crow, 1994; West et al., 1999; Butlin, 2002). From the selfish DNA point of view parthenogenesis is much easier to explain than sexual reproduction, yet only a minority – about 1%– of the known eukaryotic species reproduce via parthenogenesis (White, 1978). It is still not clear if the main function of sex is the homogenisation of genomes (Roughgarden, 1991; Otto & Nuismer, 2004) or the production of different genotypes that allow adaptation to changing environments (Ghiselin, 1974; Maynard Smith, 1978; Hamilton, 1980; Perlman et al., 2003). Ancient asexual taxa provide the opportunity to test the hypothesis that sexual reproduction evolved not to produce genetic variation in the short term but to homogenize genomes over long periods of time. Contradicting the latter hypothesis, ancient asexual taxa have more genetic variation than sexual taxa of similar age (Barraclough & Herniou, 2003; Barraclough et al., 2003).
A precondition for understanding the survival of ancient asexuals is to know (a) whether these taxa really are ancient and (b) whether they are apomicts or automicts.
Of the three putative ancient asexual animal taxa, bdelloid rotifers are apomicts (Mark Welch & Meselson, 2000; Birky, 2004). In asexual, nonmarine ostracods, only apomixis has been reported so far (Butlin et al., 1998) although the Darwinulidae have not yet been investigated extensively. The exceptionally low genetic divergence of ITS1 in the ancient asexual species of the family, Darwinula stevensoni, could be attributed to gene conversion (Schön & Martens, 2003) whereas homogenising mechanisms such as highly efficient DNA repair (Schön & Martens, 1998) could keep other genomic regions intact (Schön & Martens, 2003). In contrast, there is evidence for automixis in parthenogenetic taxa of oribatid mites (e.g. Platynothrus peltifer) (Taberly, 1987a,b; Wrensch et al., 1994; Butlin et al., 1998; Gorelick, 2003).
Definitions of sexual and parthenogenetic reproduction are sometimes contradicting, which is why we provide our definition below. We use the term sex for the fusion of meiotically and independently produced gametes of two individuals. Parthenogenetic reproduction as used here relates specifically to thelytoky, the production of females from unfertilised eggs. Further, we distinguish between apomictic and automictic thelytoky. Apomixis describes a process in which oocytes are produced by mitotic cell division in the germ line. Daughters inherit a complete unrecombined maternal genome, which results in increased heterozygosity at any given locus over time as mutations accumulate. Automixis refers to the reconstitution of the diploid state from meiotically reduced oocytes within one organism. Several mechanisms for reconstituting the numbers of chromosomes in the presumptive egg are known, each either resulting in increased heterozygosity or homozygosity among the offspring. Doubling the chromosomes before meiosis and pairing of homologous chromosomes increases homozygosity as does post-meiotic fusion of meiotically produced ova (eggs with polar bodies), if nonhomologous chromosomes pair, crossing over can generate genetically diverse offspring. The degree of heterozygosity at a given locus depends on whether the first or the second meiotic division is suppressed. The same holds for the fusion of the first (central fusion) or the second (terminal fusion) polar body with the oocyte. In general, central fusion automixis results in heterozygosity (except in cross-over regions) whereas terminal fusion automixis increases homozygosity (except in cross-over regions) (Maynard Smith, 1978; Bell, 1982; Suomalainen et al., 1987). The patterns of these hypothesized mechanisms can be overwritten by nonmeiotic homogenising processes, such as gene conversion, mitotic recombination, or DNA repair.
One way to test for long-term absence of recombination is the so-called ‘Meselson effect’, as proposed by Birky (1996) and Mark Welch & Meselson (2000). The Meselson effect assumes that the two allelic copies at a given locus (in a diploid) become divergent over time as a result of the independent accumulation of mutations. Over millions of years of evolution the intra-individual allelic divergence of ancient apomicts should be higher than that of sexual species. However, the effect should not exist in ancient automicts, in which homogenising mechanisms such as recombination, gene conversion (Butlin, 2000), or DNA repair (Schön & Martens, 2000) should lower allelic diversity (Gorelick, 2003; Gandolfi et al., 2003). Therefore, the power of testing the Meselson effect is asymmetrical. Its presence indicates long-term lack of recombination but its absence does not distinguish sexual reproduction from any other sort of homogenising mechanism (Butlin, 2000). So far, the Meselson effect has been found in bdelloid rotifers (Mark Welch & Meselson, 2000) but not in D.stevensoni (Schön & Martens, 2003) or any other putative ancient parthenogenetic group (Birky, 2004). Even the presence of the Meselson effect does not prove long-term absence of recombination; Ceplitis (2003) showed that the effect is also compatible with low rates of sexual reproduction.
In contrast to darwinulid ostracods and bdelloid rotifers, the various putative ancient asexual taxa of oribatid mites have so far hardly been studied in an evolutionary context with molecular techniques. These mites are decomposer animals that reach high densities in virtually all soils of the world, ranging from a few hundreds in agricultural sites up to 500 000 m−2 in northern boreal forests (Maraun & Scheu, 2000). Fossils are known from Devonian sediments indicating an age of at least 380 My (Shear et al., 1984, Norton et al., 1988). There is increasing evidence that several species-rich clusters of oribatid mites represent ancient asexual lineages that radiated while being parthenogenetic (Norton & Palmer, 1991; Norton et al., 1993; Maraun et al., 2003,2004).
This study tests for the presence of the Meselson effect in parthenogenetic species of oribatid mites. The intra-individual genetic divergence in parthenogenetic taxa is compared with that of sexually reproducing oribatid mite species. We used the elongation factor-1α (ef-1α) and the heat shock protein 82 (hsp82) genes; both are conserved and most likely single copy genes (Klompen, 2000; Mark Welch & Meselson, 2000). The hsp82 gene has been used for the analysis of the Meselson effect in bdelloid rotifers (Mark Welch & Meselson, 2000) and darwinulid ostracods (Schön & Martens, 2003) facilitating the comparison of results from the different groups of ancient asexuals. The ef-1α gene has been successfully used for phylogenetic studies (e.g. Klompen, 2000). Additionally, the functionality of hsp82 was verified in the current study by sequencing its cDNA. We also applied likelihood permutation tests (McVean et al., 2002) to test for recombination and gene conversion in ef-1α and hsp82 from both sexual and parthenogenetic taxa.