Genomic signatures of ancient asexual lineages

Ancient asexuals – organisms that have lived without sex for millions of years – offer unique opportunities for discriminating among the various theories of the maintenance of sex. The last few years have seen molecular studies of a number of putative ancient asexual lineages, including bdelloid rotifers, Darwinulid ostracods, and mycorrhizal fungi. To help make sense of the diverse findings of such studies, we present a review and classification of the predicted effects of loss of sex on the eukaryotic genome. These include: (1) direct effects on the genetic structure of individuals and populations; (2) direct effects on the mutation rate due to the loss of the sexual phase; (3) decay of genes specific to sex and recombination; (4) effects of the cessation of sexual selection; (5) dis-adaptation due to the reduced efficiency of selection; and (6) adaptations to asexuality. We discuss the utility of the various predictions for detecting ancient asexuality, for testing hypotheses of the reversibility of a transition to asexuality, and for discriminating between theories of sex. In addition, we review the current status of putative ancient asexuals.  © 2003 The Linnean Society of London. Biological Journal of the Linnean Society 2003, 79 , 69–84.     

Evolution in the slow lane: molecular rates of evolution in sexual and asexual ostracods (Crustacea: Ostracoda)

Parthenogenetic lineages within non-marine ostracods can occur either in mixed (with sexual and asexual females) or exclusively asexual taxa. The former mode of reproduction is associated with a high intraspecific diversity at all levels (genetic, morphological, ecological) and, at least in the Cypridoidea, with geographical parthenogenesis. Obligate asexuality is restricted to the Darwinuloidea, the strongest candidate for an ancient asexual animal group after the bdelloid rotifers, and is characterized by low diversity. We have compared rates of molecular evolution for the nuclear ITS1 region and the mitochondrial COI gene amongst the three major lineages of non-marine ostracods with sexual, mixed and asexual reproduction. Absolute rates of molecular evolution are low for both regions in the darwinulids. The slow-down of evolution in ITS1 that has been observed for Darwinula stevensoni (Brady & Robertson) apparently does not occur in other darwinulid species. ITS1 evolves more slowly than COI within non-marine ostracod families, including the darwinulids, but not between superfamilies. The ancient asexuals might have a higher relative substitution rate in ITS1, as would be expected from hypotheses that predict the accumulation of mutations in asexuals. However, the speed-up of ITS could also be ancient, for example through the stochastic loss of most lineages within the superfamily after the Permian–Triassic mass extinction. In this case, the difference in rate would have occurred independently from any effects of asexual reproduction.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 93–100.     

Discordance between nuclear and mitochondrial genomes in sexual and asexual lineages of the freshwater snail Potamopyrgus antipodarum

The presence and extent of mitonuclear discordance in coexisting sexual and asexual lineages provides insight into 1) how and when asexual lineages emerged, and 2) the spatial and temporal scales at which the ecological and evolutionary processes influencing the evolution of sexual and asexual reproduction occur. Here, we used nuclear single‐nucleotide polymorphism (SNP) markers and a mitochondrial gene to characterize phylogeographic structure and the extent of mitonuclear discordance in Potamopyrgus antipodarum. This New Zealand freshwater snail is often used to study the evolution and maintenance of sex because obligately sexual and obligately asexual individuals often coexist. While our data indicate that sexual and asexual P. antipodarum sampled from the same lake population are often genetically similar, suggesting recent origin of these asexuals from sympatric sexual P. antipodarum, we also found significantly more population structure in sexuals vs. asexuals. This latter result suggests that some asexual lineages originated in other lakes and/or in the relatively distant past. When comparing mitochondrial and nuclear population genetic structure, we discovered that one mitochondrial haplotype (‘1A’) was rare in sexuals, but common and widespread in asexuals. Haplotype 1A frequency and nuclear genetic diversity were not associated, suggesting that the commonness of this haplotype cannot be attributed entirely to genetic drift and pointing instead to a role for selection.     

Deleterious mutation accumulation in asexual Timema stick insects

Sexual reproduction is extremely widespread in spite of its presumed costs relative to asexual reproduction, indicating that it must provide significant advantages. One postulated benefit of sex and recombination is that they facilitate the purging of mildly deleterious mutations, which would accumulate in asexual lineages and contribute to their short evolutionary life span. To test this prediction, we estimated the accumulation rate of coding (nonsynonymous) mutations, which are expected to be deleterious, in parts of one mitochondrial (COI) and two nuclear (Actin and Hsp70) genes in six independently derived asexual lineages and related sexual species of Timema stick insects. We found signatures of increased coding mutation accumulation in all six asexual Timema and for each of the three analyzed genes, with 3.6- to 13.4-fold higher rates in the asexuals as compared with the sexuals. In addition, because coding mutations in the asexuals often resulted in considerable hydrophobicity changes at the concerned amino acid positions, coding mutations in the asexuals are likely associated with more strongly deleterious effects than in the sexuals. Our results demonstrate that deleterious mutation accumulation can differentially affect sexual and asexual lineages and support the idea that deleterious mutation accumulation plays an important role in limiting the long-term persistence of all-female lineages.     

Transposon proliferation in an asexual parasitoid

The widespread occurrence of sex is one of the most elusive problems in evolutionary biology. Theory predicts that asexual lineages can be driven to extinction by uncontrolled proliferation of vertically transmitted transposable elements (TEs), which accumulate because of the inefficiency of purifying selection in the absence of sex and recombination. To test this prediction, we compared genome-wide TE load between a sexual lineage of the parasitoid wasp Leptopilina clavipes and a lineage of the same species that is rendered asexual by Wolbachia-induced parthenogenesis. We obtained draft genome sequences at 15-20× coverage of both the sexual and the asexual lineages using next-generation sequencing. We identified transposons of most major classes in both lineages. Quantification of TE abundance using coverage depth showed that copy numbers in the asexual lineage exceeded those in the sexual lineage for DNA transposons, but not LTR and LINE-like elements. However, one or a small number of gypsy-like LTR elements exhibited a fourfold higher coverage in the asexual lineage. Quantitative PCR showed that high loads of this gypsy-like TE were characteristic for 11 genetically distinct asexual wasp lineages when compared to sexual lineages. We found no evidence for an overall increase in copy number for all TE types in asexuals as predicted by theory. Instead, we suggest that the expansions of specific TEs are best explained as side effects of (epi)genetic manipulations of the host genome by Wolbachia. Asexuality is achieved in a myriad of ways in nature, many of which could similarly result in TE proliferation.     

Transposable elements in clonal lineages: lethal hangover from sex

Long-term coevolution of transposable elements (TEs) in sexual hosts leads to evolution of extremely active and dangerous mutagens kept in tenuous check by host-derived mechanisms and via natural selection against TE-rich genomes. To the extent that sexual reproduction and recombination are important in maintaining a stable TE copy number and a tolerable mutation load, the switch to clonality from sexual reproduction can be extremely damaging and, generally, should lead to clonal lineage extinction. Surprisingly however, the loss of powerful selective mechanisms constraining TEs can be beneficial in the short-term by immediately eliminating selective load and possibly promoting the early success of clonal lineages. The clonal lineages that do survive in the long-term must find a way to eliminate or domesticate TEs. Indeed bdelloid rotifers, which are ancient asexuals, do appear to have lost most of the otherwise wide-spread TEs and might have domesticated others. The path to this TE-free haven is anything but clear at the moment. We have considered a novel scenario of instantaneous inactivation of TEs by starting off with a genome carrying repressive host alleles for all TEs in the genome. We show that such a scenario appears plausible and provide some limited empirical evidence in its support.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 33–41.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Male offspring production by asexual Potamopyrgus antipodarum, a New Zealand snail

As only females contribute directly to population growth, sexual females investing equally in sons and daughters experience a two-fold cost relative to asexuals producing only daughters. Typically, researchers have focused on benefits of sex that can counter this 'cost of males' and thus explain its predominance. Here, we instead ask whether asexuals might also pay a cost of males by quantifying the rate of son production in 45 experimental populations ('lineages') founded by obligately asexual female Potamopyrgus antipodarum. This New Zealand snail is a powerful model for studying sex because phenotypically similar sexual and asexual forms often coexist, allowing direct comparisons between sexuals and asexuals. After 2 years of culture, 23 of the 45 lineages had produced males, demonstrating that asexual P. antipodarum can make sons. We used maximum-likelihood analysis of a model of male production in which only some lineages can produce males to estimate that ~50% of lineages have the ability to produce males and that ~5% of the offspring of male-producing lineages are male. Lineages producing males in the first year of the experiment were more likely to make males in the second, suggesting that some asexual lineages might pay a cost of males relative to other asexual lineages. Finally, we used a simple deterministic model of population dynamics to evaluate how male production affects the rate of invasion of an asexual lineage into a sexual population, and found that the estimated rate of male production by asexual P. antipodarum is too low to influence invasion dynamics.     

Role of parasite load and differential habitat preferences in maintaining the coexistence of sexual and asexual competitors in fish of the Cobitis taenia hybrid complex

In the context of the paradoxical ubiquity of sex, we tested whether stable coexistence of sexual and asexual fish of the genus Cobitis is mediated by parasites, as asexual fish suffer more from parasitic infections because of their lower genetic variability [the Red Queen hypothesis (RQH)], or by partial niche shift of the two strains differing in mode of reproduction. We did not find a clear correlation between infection risk with a helminth parasite and the proportion of sexuals, and we found similar infection rates among sexual females and co‐occurring asexuals in general, including the most frequent clone in particular. These results suggest that the mechanisms of the RQH are not directly engaged in stabilizing this asexual complex. On the other hand, the temporally stable gradient in sexual/asexual proportions along the river correlated with gradients in environmental parameters (physicochemical water parameters, velocity, and shading of the habitat) and turnover in the fish assemblage structure. Sexual and asexual forms thus appear to prefer different habitats. The Cobitis teania asexual complex thus contributes to the view that persistence of sex may, as in many taxa, be driven by case‐specific processes. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 220–235.     

Poor male function favours the coexistence of sexual and asexual relatives

Classical models of the evolution of sex typically assume that an asexual lineage, once derived, is reproductively separate from the sexual lineage from which it was derived. However, many asexuals, including hermaphrodite plants, produce male gametes capable of fertilising the eggs of co-existing sexuals, giving rise to sexual and asexual progeny. This male function of asexuals may be poor, and it has been proposed that this could favour sexuality and adversely affect the successful establishment of asexual lineages. We show that things are more complicated than this; the effect is frequency-dependent and poor male function may sometimes favour asexuality. In a spatially distributed population of flowering plants, it can prevent the successful invasion of either reproductive mode by the other via long-range dispersal. Consequently invasions must be driven by short-range dispersal, and are therefore extremely slow. Thus poor male function favours long-term co-existence of sexuals and asexuals. When coupled with the superior ability of asexuals to colonise virgin territory after an Ice Age, it may explain current ecological distribution patterns.     

Can resource costs of polyploidy provide an advantage to sex?

The predominance of sexual reproduction despite its costs indicates that sex provides substantial benefits, which are usually thought to derive from the direct genetic consequences of recombination and syngamy. While genetic benefits of sex are certainly important, sexual and asexual individuals, lineages, or populations may also differ in physiological and life history traits that could influence outcomes of competition between sexuals and asexuals across environmental gradients. Here, we address possible phenotypic costs of a very common correlate of asexuality, polyploidy. We suggest that polyploidy could confer resource costs related to the dietary phosphorus demands of nucleic acid production; such costs could facilitate the persistence of sex in situations where asexual taxa are of higher ploidy level and phosphorus availability limits important traits like growth and reproduction. We outline predictions regarding the distribution of diploid sexual and polyploid asexual taxa across biogeochemical gradients and provide suggestions for study systems and empirical approaches for testing elements of our hypothesis.     

Heritable genetic variation and potential for adaptive evolution in asexual aphids (Aphidoidea)

Aphid life cycles can encompass cyclical parthenogenesis, obligate parthenogenesis, obligate parthenogenesis with male production and an intermediate 'bet-hedging' strategy where an aphid genotype will over-winter by continuing to reproduce by parthenogenesis and by investment in sexually produced eggs. In this paper, we focus on aphid lineages that reproduce entirely parthenogenetically (asexual aphids), in contrast to those that have any sexual forms in the annual cycle. Using modern molecular techniques, aphid biologists have made many empirical observations showing that asexual lineages are widespread both geographically and temporally. Indeed, we are collectively beginning to gather data on the evolution and persistence of these lineages through time. Here we review aphid karyology and parthenogenesis, both essential for interpretation of the molecular and ecological evolution of aphid asexual lineages. We describe the growing list of studies that have identified aphid genotypes that are both temporally and geographically widespread. We then collate examples of molecular and chromosomal evolution in asexual aphids and review the literature pertaining to phenotypic evolution and ecological diversification of asexual aphid lineages. In addition, we briefly discuss the potential of bacterial endosymbionts and epigenetic effects to influence the evolution of asexual aphid lineages. Lastly we provide a list of aphid taxa believed to be obligately asexual. This will be a useful resource for those seeking parthenogenetic animals as study systems. In conclusion, we present guidelines for the use of the term clone in aphid biology and stress the need for well-designed and well-executed studies examining the potential of asexual aphid lineages for adaptive evolution.  © 2003 The Linnean Society of London. Biological Journal of the Linnean Society , 2003, 79 , 115–135.     

Twigs on the tree of life? Neutral and selective models for integrating macroevolutionary patterns with microevolutionary processes in the analysis of asexuality

Neutral models characterize evolutionary or ecological patterns expected in the absence of specific causal processes, such as natural selection or ecological interactions. In this study, we describe and evaluate three neutral models that can, in principle, help to explain the apparent 'twigginess' of asexual lineages on phylogenetic trees without involving the negative consequences predicted for the absence of recombination and genetic exchange between individuals. Previously, such phylogenetic twiggyness of asexual lineages has been uncritically interpreted as evidence that asexuality is associated with elevated extinction rates and thus represents an evolutionary dead end. Our first model uses simple phylogenetic simulations to illustrate that, with sexual reproduction as the ancestral state, low transition rates to stable asexuality, or low rates of ascertained 'speciation' in asexuals, can generate twiggy distributions of asexuality, in the absence of high extinction rates for asexual lineages. The second model, developed by Janko et   al . (2008 ), shows that a dynamic equilibrium between origins and neutral losses of asexuals can, under some conditions, generate a relatively low mean age of asexual lineages. The third model posits that the risk of extinction for asexual lineages may be higher than that of sexuals simply because asexuals inhabit higher latitudes or altitudes, and not due to effects of their reproductive systems. Such neutral models are useful in that they allow quantitative evaluation of whether empirical data, such as phylogenetic and phylogeographic patterns of sex and asexuality, indeed support the idea that asexually reproducing lineages persist over shorter evolutionary periods than sexual lineages, due to such processes as mutation accumulation, slower rates of adaptive evolution, or relatively lower levels of genetic variability.     

Escape from the Red Queen: an overlooked scenario in coevolutionary studies

Almost all eukaryotic organisms undergo sexual recombination at some stage of their life history. However, strictly asexual organisms should have higher per capita rate of reproduction compared with those that have sex, so the latter must convey some advantage which overrides the reproductive benefit of asexuality. For example, sexual reproduction and recombination may play an important role in allowing organisms to evolutionarily ‘keep up’ with parasites. Host–parasite coevolution can operate via negative frequency‐dependent selection whereby parasite genotypes adapt to infect host genotypes as they become locally common. By producing more genetically diverse offspring with unique genotypes, sexual organisms have an advantage over asexual counterparts. Essentially, sexual hosts are more difficult for coevolving parasites to ‘track’ over time. This scenario has been named the “Red Queen hypothesis”. It refers to a passage in Lewis Carroll's ‘Through the Looking Glass’ in which the Red Queen tells Alice: ‘it takes all the running you can do, to keep in the same place’; this statement resembles the negative frequency‐dependent dynamics of host–parasite coevolution.     

Molecular evidence for ancient asexuality in timema stick insects

Asexuality is rare in animals in spite of its apparent advantage relative to sexual reproduction, indicating that it must be associated with profound costs [1-9]. One expectation is that reproductive advantages gained by new asexual lineages will be quickly eroded over time [3, 5-7]. Ancient asexual taxa that have evolved and adapted without sex would be "scandalous" exceptions to this rule, but it is often difficult to exclude the possibility that putative asexuals deploy some form of "cryptic" sex, or have abandoned sex more recently than estimated from divergence times to sexual relatives [10]. Here we provide evidence, from high intraspecific divergence of mitochondrial sequence and nuclear allele divergence patterns, that several independently derived Timema stick-insect lineages have persisted without recombination for more than a million generations. Nuclear alleles in the asexual lineages displayed significantly higher intraindividual divergences than in related sexual species. In addition, within two asexuals, nuclear allele phylogenies suggested the presence of two clades, with sequences from the same individual appearing in both clades. These data strongly support ancient asexuality in Timema and validate the genus as an exceptional opportunity to attack the question of how asexual reproduction can be maintained over long periods of evolutionary time.     

The cost of males in Daphnia pulex

Sex is paradoxical, because asexuals should replace their sexual ancestors by avoiding the demographic cost of producing males (hereafter referred to as the cost‐of‐males). Despite the large body of theoretical and empirical work dealing with the paradox of sex, the cost‐of‐males assumption has been rarely tested. In the present study, we tested the cost‐of‐males assumption in the cladoceran Daphnia pulex. Populations of this species consist of both cyclically parthenogenetic (i.e. sexuals) and obligately parthenogenetic (i.e. asexuals) lineages. In addition, some of the asexual lineages produce only female offspring, whereas others produce functional males, which can mate with sexual females. We compared the reproductive investment of sexuals, male‐producing asexuals, and non‐male‐producing asexuals when raised separately under various environmental conditions. We also determined the outcome of competition between pair‐wise combinations of these reproductive modes. The cost of males was evident when sexual and asexual females were raised separately: sexuals produced fewer female offspring. However, there was no cost of males when reproductive modes were raised in pairs, as sexuals won the competition with asexuals. Our results directly relate to the field conditions experienced by D. pulex. Sexuals might suffer the cost of males at the beginning of the season, when resource competition is low; but when conditions deteriorate as the population approaches carrying capacity, sexuals seem to be better competitors in spite of male production.     

Pollen profile,spatial structure,and access to sex in asexual hermaphrodites

Classical cost‐of‐sex models predict the rapid fixation of asexual reproduction. Coexistence of sexuals and asexuals is common among hermaphrodite plants, however, providing asexuals with access to sex via their male function; some of the sexually reproduced progeny they sire will be asexual. The ability of asexuals to sire progeny is often hindered by the production of poor quality pollen. Using cellular automata, it is shown that decreases in pollen quality in asexuals can greatly increase the period of coexistence of sexuals and asexuals and, consequently, the cumulative contribution of sex to asex. Extensive periods of coexistence are only likely, however, if pollen and seed are dispersed locally, in which case coexistence over thousands of generations can be achieved. It is argued that, with local dispersal, the negative relationship between pollen quality and the period of coexistence of sex and asex will result in patterns of geographic parthenogenesis in which asexuals that coexist with sexuals will exhibit a poor male function, whereas asexuals with a very efficient male function will occur in exclusively asexual populations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 954–966.     

Contrasting patterns of synonymous and nonsynonymous sequence evolution in asexual and sexual freshwater snail lineages

In asexual lineages, both synonymous and nonsynonymous sequence polymorphism may be reduced due to severe founder effects when asexual lineages originate. However, mildly deleterious (nonsynonymous) mutations may accumulate after asexual lineages are formed, because the efficiency of purifying selection is reduced even in the nonrecombining mitochondrial genome. Here we examine patterns of synonymous and nonsynonymous mitochondrial sequence polymorphism in asexual and sexual lineages of the freshwater snail Campeloma. Using clade-specific estimates, we found that synonymous sequence polymorphism was significantly reduced by 75% in asexuals relative to sexuals, whereas nonsynonymous sequence polymorphism did not differ significantly between sexuals and asexuals. Two asexual clades had high negative values for Tajima's D statistic. Coalescent simulations confirmed that various bottleneck scenarios can account for this result. We also used branch-specific estimates of the ratio of amino acid to silent substitutions, K(a)/K(s). Our study revealed that K(a)/K(s) ratios are six times higher in terminal branches of independent asexual lineages compared to sexuals. Coalescent-based reconstruction of gene networks for all sexual and asexual clades indicated that nonsynonymous mutations occurred at a higher frequency in recently derived asexual haplotypes. These findings suggest that patterns of synonymous and nonsynonymous nucleotide polymorphism in asexual snail lineages may be shaped by both severe founder effect and relaxed purifying selection.     

Loss of sexual recombination and segregation is associated with increased diversification in evening primroses

The loss of sexual recombination and segregation in asexual organisms has been portrayed as an irreversible process that commits asexually reproducing lineages to reduced diversification. We test this hypothesis by estimating rates of speciation, extinction, and transition between sexuality and functional asexuality in the evening primroses. Specifically, we estimate these rates using the recently developed BiSSE (Binary State Speciation and Extinction) phylogenetic comparative method, which employs maximum likelihood and Bayesian techniques. We infer that net diversification rates (speciation minus extinction) in functionally asexual evening primrose lineages are roughly eight times faster than diversification rates in sexual lineages, largely due to higher speciation rates in asexual lineages. We further reject the hypothesis that a loss of recombination and segregation is irreversible because the transition rate from functional asexuality to sexuality is significantly greater than zero and in fact exceeded the reverse rate. These results provide the first empirical evidence in support of the alternative theoretical prediction that asexual populations should instead diversify more rapidly than sexual populations because they are free from the homogenizing effects of sexual recombination and segregation. Although asexual reproduction may often constrain adaptive evolution, our results show that the loss of recombination and segregation need not be an evolutionary dead end in terms of diversification of lineages.