Exceptional cryptic diversity and multiple origins of parthenogenesis in a freshwater ostracod
Introduction
Sex is the most common mode of reproduction in the animal kingdom (see for example, Bell, 1982). It is also the ancestral mode of all metazoa and probably all eukaryotes. Yet it is easily lost by mutation, hybridisation, or polyploidisation and it also comes with costs, such as the twofold cost of males (Maynard Smith, 1978), vulnerability to sexually transmitted infections or predators and many more (Kondrashov, 1993). The reason behind the success of sex is one of the most intriguing paradoxes of evolution still waiting to be solved. Many hypotheses have been proposed but, so far, none appears capable of providing a general and inclusive explanation for widespread sexual reproduction (West et al., 1999, Butlin, 2002). The best-supported hypotheses implicate mutation accumulation due to lack of recombination in asexuals (Kondrashov, 1988, Lynch et al., 1993) or rapidly changing selection pressures due to the ‘Red Queen’ process (Van Valen, 1973), particularly as a result of host–parasite interactions (Hamilton, 1980). It is becoming increasingly evident that several factors must be involved simultaneously (West et al., 1999) and that their contributions may differ among organisms (Burt, 2000, Schön et al., 2009).
Asexual mutants can have significant selective advantages due to superior population growth and colonising abilities. However, they have not generally replaced their sexual relatives but instead they are typically short-lived in evolutionary terms (Maynard Smith, 1978, Bell, 1982). It is difficult to estimate the ages of asexual lineages (Butlin, 2002). Therefore apparent exceptions to the rule are controversial, ironically called ‘ancient asexual scandals’ (Judson and Normark, 1996). The most well-known putative ancient asexuals are bdelloid rotifers (Welch and Meselson, 2000), darwinulid ostracods (Martens et al., 2003, Martens and Schön, 2008), and oribatid mites (Heethoff et al., 2007, Laumann et al., 2007). These are entire families that have been fully asexual for millions of years, a conclusion supported by several lines of evidence, such as genomic characteristics in the case of bdelloids (Welch and Meselson, 2001, Welch et al., 2004) and extensive fossil records without males in the case of the darwinulids (Martens et al., 2003, Martens and Schön, 2008; but see Smith et al., 2006) and oribatid mites (Maraun and Scheu, 2000).
Ancient asexual lineages are genetically diverse because of an accumulation of mutations since their origin. In other taxa, genetically diverse asexual populations may be generated by a high rate of origin resulting in a standing diversity that is dependent, at least in part, on population size (Janko et al., 2008). High rates of origin of asexuals occur in cyclical parthenogens through repeated loss of the sexual phase, as in aphids for example (Delmotte et al., 2001, Loxdale and Lushai, 2003), and where there is repeated hybridisation, as in Poeciliopsis fish (Mateos and Vrijenhoek, 2002) or Daphnia (Paland et al., 2005). In some cases, frequent origin of asexual lineages from an ancestral sexual population clearly occurs, but the mechanism remains uncertain, as in Potamopyrgus snails (Neiman et al., 2005). It is important to understand both the mechanism and rate of origin of asexual lineages because they influence models for the maintenance of co-existing sexual and asexual populations. For example, Janko et al. (2008) show that asexual lineages may be short-lived purely because of turn-over in a finite population, without the need to invoke declining fitness due to mutation accumulation or environmental change. In contrast, in Potamopyrgus, frequent origin of novel asexual lineages is necessary to maintain the asexual population in the face of co-evolving parasites (Lively et al., 2004).
The freshwater ostracod (Crustacea) Eucypris virens is a model taxon containing distinct and non-cyclical sexual and asexual lineages. The reproductive mode is the only known difference between these groups of lineages. This species belongs to the Cyprididae, a family that harbours many species with either fully asexual, fully sexual or mixed modes of reproduction (Horne et al., 1998, Martens et al., 2008). It belongs to the superfamily Cypridoidea, which is related to the superfamily Darwinuloidea whose extant representatives are the putatively ancient asexual ostracod family of the Darwinulidae, mentioned above.
Eucypris virens is characterised by high levels of variability, both at the morphological (Martens, 1998, Baltanas et al., 2002) and genetic level (Rossi et al., 1998, Schön et al., 2000, Schön, 2007). Schön et al. (2000) suggested that both multiple origins of asexual lineages and hybridisation might contribute to this high diversity. They also proposed that some asexual lineages might be old, with sequence divergence of the mitochondrial COI locus up to 21% indicating that the oldest asexual lineages might have originated 10 million years ago. However, wider sampling of lineages was needed to test these ideas and fossil evidence is scarce, as this particular species occupies only temporary pools.
Eucypris virens is also known for its distinct geographical distribution in which males are restricted to the circum-Mediterranean area and have never been recorded north of the Alps (Horne et al., 1998) whereas asexual lineages occur in Northern Europe as well as in sympatry with sexual lineages. This pattern is termed “geographical parthenogenesis” (Vandel, 1928) and has been recorded in a number of organisms (Law and Crespi, 2002, van Dijk, 2003). This phenomenon may be explained by the greater colonising ability of asexuals, their potential to adapt to novel environments because they are free of the retarding effects of gene flow (see Butlin et al., 2003, for further discussion), or the difference in environmental stability between regions (see Horne and Martens, 1999, for a review).
Here, we investigate the number of sexual and asexual lineages of E. virens in Europe and how they are related to each other phylogenetically, using partial mitochondrial cytochrome oxidase I (COI) and 16S ribosomal DNA gene sequences. This phylogenetic information provides a framework for understanding the origin of the genetic diversity within asexuals, the rate of appearance of asexual lineages and their ages, as well as their geographic distribution. Asexual organisms are thought to have originated from sexual ancestors without reversals. This assumption is supported by multiple lines of evidence (Bell, 1982) and by the argument that it requires more evolutionary steps to re-acquire sexual reproduction than it does to lose it (but see Domes et al., 2007). Although most species of the Cyprididae are asexual or include asexual lineages, it is very likely that the ancestral state of the family was sexual reproduction. Therefore, we assume that the ancestor of E. virens was sexual and test whether transitions from sexual to asexual reproduction are frequently encountered across the phylogeny or whether a single ancient event has led to diverse asexual lineages. Furthermore, we determined the number of independent sexual lineages (potential cryptic species) within the morphospecies because origin of asexuals from distinct sexual ancestors may contribute to clonal diversity.
Section snippets
Specimen collection and DNA extraction
We conducted a large-scale sampling campaign covering most of Europe and the Mediterranean including North Africa (Fig. 1). Between 50 and 100 specimens of E. virens were collected with a hand net from each of 135 temporary pools during winter and early spring of 2005/2006 and 2006/2007 and were kept alive in mineral water overnight to eliminate stomach contents. The animals were slowly killed in diluted ethanol, in order that their carapace valves stayed open, and specimens were subsequently
Phylogenetic analysis of COI data
Increasing evidence for frequent nuclear insertions of mitochondrial DNA, known as numts, has lead to the conclusion that the high diversity of COI lineages detected in DNA barcoding projects may be an overestimate of the true diversity (Song et al., 2008). This phenomenon is particularly frequent in crustaceans (Bensasson et al., 2000, Bensasson et al., 2001). However, the sequences obtained here contain neither stop codons nor indels and can be aligned unambiguously, despite a large number of
Deep phylogenetic divergence between sexual lineages and implications for intra-specific systematics
Our results reveal exceptionally high mitochondrial genetic variation within the morphospecies Eucypris virens. This is in line with the extensive allozyme diversity reported previously (Rossi et al., 1998, Rossi et al., 2008) and with an earlier analysis of mitochondrial and nuclear (ITS) sequence data (Schön et al., 2000) both of which were based on relatively restricted sampling of the species’ distribution. Not only is diversity high but it is strongly structured into distinct groups of
Acknowledgments
This work was funded by the EU Marie Curie Research Training Network ‘SexAsex’ (from Sex to Asex: a case study on interactions between sexual and asexual reproduction, contract MRTN-CT-2004-512492). K.M. and I.S. also acknowledge the financial contribution of projects 1.5.172.09 (krediet aan navorsers) and G.0118.03N (projectonderzoek) of the FWO Vlaanderen (Fund for Scientific Research, Flanders). We are very grateful to all members of the network for their practical contributions to the work
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These authors contributed equally to this work.