Genetic tests of ancient asexuality in Root Knot Nematodes reveal recent hybrid origins

Background  

The existence of "ancient asexuals", taxa that have persisted for long periods of evolutionary history without sexual recombination, is both controversial and important for our understanding of the evolution and maintenance of sexual reproduction. A lack of sex has consequences not only for the ecology of the asexual organism but also for its genome. Several genetic signatures are predicted from long-term asexual (apomictic) reproduction including (i) large "allelic" sequence divergence (ii) lack of phylogenetic clustering of "alleles" within morphological species and (iii) decay and loss of genes specific to meiosis and sexual reproduction. These genetic signatures can be hard to assess since it is difficult to demonstrate the allelic nature of very divergent sequences, divergence levels may be complicated by processes such as inter-specific hybridization, and genes may have secondary roles unrelated to sexual reproduction. Apomictic species of Meloidogyne root knot nematodes have been suggested previously to be ancient asexuals. Their relatives reproduce by meiotic parthenogenesis or facultative sexuality, which in combination with the abundance of nematode genomic sequence data, makes them a powerful system in which to study the consequences of reproductive mode on genomic divergence.     

Sex drives intracellular conflict in yeast

Theory predicts that sex can drive the evolution of conflict within the cell. During asexual reproduction, genetic material within the cell is inherited as a single unit, selecting for cooperation both within the genome as well as between the extra‐genomic elements within the cell (e.g. plasmids and endosymbionts). Under sexual reproduction, this unity is broken down as parental genomes are distributed between meiotic progeny. Genetic elements able to transmit to more than 50% of meiotic progeny have a transmission advantage over the rest of the genome and are able to spread, even where they reduce the fitness of the individual as a whole. Sexual reproduction is therefore expected to drive the evolution of selfish genetic elements (SGEs). Here, we directly test this hypothesis by studying the evolution of two independent SGEs, the 2‐μm plasmid and selfish mitochondria, in populations of Saccharomyces cerevisiae. Following 22 rounds of sexual reproduction, 2‐μm copy number increased by approximately 13.2 (± 5.6) copies per cell, whereas in asexual populations copy number decreased by approximately 5.1 (± 1.5) copies per cell. Given that the burden imposed by this parasite increases with copy number, these results support the idea that sex drives the evolution of increased SGE virulence. Moreover, we found that mitochondria that are respiratory‐deficient rapidly invaded sexual but not asexual populations, demonstrating that frequent outcrossed sex can drive the de novo evolution of genetic parasites. Our study highlights the genomic perils of sex and suggests that SGEs may play a key role in driving major evolutionary transitions, such as uniparental inheritance.     

Using a meiosis detection toolkit to investigate ancient asexual "scandals" and the evolution of sex

Sexual reproduction is the dominant reproductive mode in eukaryotes but, in many taxa, it has never been observed. Molecular methods that detect evidence of sex are largely based on the genetic consequences of sexual reproduction. Here we describe a powerful new approach to directly search genomes for genes that function in meiosis. We describe a "meiosis detection toolkit", a set of meiotic genes that represent the best markers for the presence of meiosis. These genes are widely present in eukaryotes, function only in meiosis and can be isolated by degenerate PCR. The presence of most, or all, of these genes in a genome would suggest they have been maintained for meiosis and, implicitly, sexual reproduction. In contrast, their absence would be consistent with the loss of meiosis and asexuality. This approach will help to understand both meiotic gene evolution and the capacity for meiosis and sex in putative obligate asexuals.     

Genetic signature of amphimixis allows for the detection and fine scale localization of sexual reproduction events in a mainly parthenogenetic nematode

Asexuality is an important mode of reproduction in eukaryotic taxa and has a theoretical advantage over sexual reproduction because of the increased ability to propagate genes. Despite this advantage, hidden signs of cryptic sex have been discovered in the genomes of asexual organisms. This has provided an interesting way to address the evolutionary impact of sex in plant and animal populations. However, the identification of rare sexual reproduction events in mainly asexual species has remained a challenging task. We examined the reproductive history in populations of the plant parasitic nematode Xiphinema index by genotyping individuals collected from six grapevine fields using seven microsatellite markers. A high level of linkage disequilibrium and heterozygous excess suggested a clonality rate of 95–100%. However, we also detected rare sexual reproduction events within these highly clonal populations. By combining highly polymorphic markers with an appropriate hierarchical sampling, and using both Bayesian and multivariate analysis with phylogenetic reconstructions, we were able to identify a small number of sexually produced individuals at the overlapping zones between different genetic clusters. This suggested that sexual reproduction was favoured when and where two nematode patches came into contact. Among fields, a high degree of genetic differentiation indicated a low level of gene flow between populations. Rare genotypes that were shared by several populations suggested passive dispersal by human activities, possibly through the introduction of infected plants from nurseries. We conclude that our method can be used to detect and locate sexual events in various predominantly asexual species.     

Radical amino acid mutations persist longer in the absence of sex

Harmful mutations are ubiquitous and inevitable, and the rate at which these mutations are removed from populations is a critical determinant of evolutionary fate. Closely related sexual and asexual taxa provide a particularly powerful setting to study deleterious mutation elimination because sexual reproduction should facilitate mutational clearance by reducing selective interference between sites and by allowing the production of offspring with different mutational complements than their parents. Here, we compared the rate of removal of conservative (i.e., similar biochemical properties) and radical (i.e., distinct biochemical properties) nonsynonymous mutations from mitochondrial genomes of sexual versus asexual Potamopyrgus antipodarum, a New Zealand freshwater snail characterized by coexisting and ecologically similar sexual and asexual lineages. Our analyses revealed that radical nonsynonymous mutations are cleared at higher rates than conservative changes and that sexual lineages eliminate radical changes more rapidly than asexual counterparts. These results are consistent with reduced efficacy of purifying selection in asexual lineages allowing harmful mutations to remain polymorphic longer than in sexual lineages. Together, these data illuminate some of the population‐level processes contributing to mitochondrial mutation accumulation and suggest that mutation accumulation could influence the outcome of competition between sexual and asexual lineages.     

Genetic variation and asexual reproduction in the facultatively parthenogenetic cockroach Nauphoeta cinerea: implications for the evolution of sex

Asexual reproduction could offer up to a two‐fold fitness advantage over sexual reproduction, yet higher organisms usually reproduce sexually. Even in facultatively parthenogenetic species, where both sexual and asexual reproduction is sometimes possible, asexual reproduction is rare. Thus, the debate over the evolution of sex has focused on ecological and mutation‐elimination advantages of sex. An alternative explanation for the predominance of sex is that it is difficult for an organism to accomplish asexual reproduction once sexual reproduction has evolved. Difficulty in returning to asexuality could reflect developmental or genetic constraints. Here, we investigate the role of genetic factors in limiting asexual reproduction in Nauphoeta cinerea, an African cockroach with facultative parthenogenesis that nearly always reproduces sexually. We show that when N. cinerea females do reproduce asexually, offspring are genetically identical to their mothers. However, asexual reproduction is limited to a nonrandom subset of the genotypes in the population. Only females that have a high level of heterozygosity are capable of parthenogenetic reproduction and there is a strong familial influence on the ability to reproduce parthenogenetically. Although the mechanism by which genetic variation facilitates asexual reproduction is unknown, we suggest that heterosis may facilitate the switch from producing haploid meiotic eggs to diploid, essentially mitotic, eggs.     

Fine-scale adaptation in a clonal sea anemone

Local adaptation in response to fine-scale spatial heterogeneity is well documented in terrestrial ecosystems. In contrast, in marine environments local adaptation has rarely been documented or rigorously explored. This may reflect real or anticipated effects of genetic homogenization, resulting from widespread dispersal in the sea. However, evolutionary theory predicts that for the many benthic species with complex life histories that include both sexual and asexual phases, each parental habitat patch should become dominated by the fittest and most competitive clones. In this study we used genotypic mapping to show that within headlands, clones of the sea anemone Actinia tenebrosa show restricted distributions to specific habitats despite the potential for more widespread dispersal. On these same shores we used reciprocal transplant experiments that revealed strikingly better performance of clones within their natal rather than foreign habitats as judged by survivorship, asexual fecundity, and growth. These findings highlight the importance of selection for fine-scale environmental adaptation in marine taxa and imply that the genotypic structure of populations reflects extensive periods of interclonal competition and site-specific selection.     

Sexual reproduction as a response to H2O2 damage in Schizosaccharomyces pombe.

Although sexual reproduction is widespread, its adaptive advantage over asexual reproduction is unclear. One major advantage of sex may be its promotion of recombinational repair of DNA damage during meiosis. This idea predicts that treatment of the asexual form of a facultatively sexual-asexual eucaryote with a DNA-damaging agent may cause it to enter the sexual cycle more frequently. Endogenous hydrogen peroxide is a major natural source of DNA damage. Thus, we treated vegetative cells of Schizosaccharomyces pombe with hydrogen peroxide to test if sexual reproduction increases. Among untreated stationary-phase S. pombe populations the sexual spores produced by meiosis represented about 1% of the total cells. However, treatment of late-exponential-phase vegetative cells with hydrogen peroxide increased the percentage of meiotic spores in the stationary phase by 4- to 18-fold. Oxidative damage therefore induces sexual reproduction in a facultatively sexual organism, a result expected by the hypothesis that sex promotes DNA repair.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis

Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.     

Asexuality Associated with Marked Genomic Expansion of Tandemly Repeated rRNA and Histone Genes

How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits—mode of genomic inheritance—influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S–5.8S–28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA–histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction—with potential functional consequences related to the loss of sexual reproduction.     

Food quality triggers the reproductive mode in the cyclical parthenogen <Emphasis Type="Italic">Daphnia</Emphasis> (Cladocera)

Cyclic parthenogenesis (heterogony) is a widespread reproductive mode found in diverse taxa such as digenean trematodes, gall wasps, gall midges, aphids, cladocerans and rotifers. It is of particular interest as it combines the advantages of asexual reproduction (rapid population growth) and sexual reproduction (recombination). Usually sexual reproduction is initiated when, or slightly before, environmental conditions deteriorate, and often results in the production of resting stages. The optimal timing of diapause induction must thus be under strong natural selection. Using the cladoceran Daphnia as a model system, we show here for the first time that the switch from parthenogenetic to sexual reproduction in a cyclical parthenogenetic organism can be influenced by the chemical composition of food. Under crowding conditions Daphnia reproduced parthenogenetically with subitaneous eggs when fed the algal species Cryptomonas sp., but started the production of resting eggs when fed with the green algal species Scenedesmus obliquus. Supplementation experiments with lipids and especially proteins showed that the induction of resting egg production in two clones of different Daphnia species was due to a dietary deficiency in the green alga. Hence, the low food quality induced a switch in the reproductive mode that may contribute to optimal timing of the sexual reproduction of Daphnia in nature. Furthermore, our results have two other major implications: first, they suggest that protein compounds should be added to the list of diet constituents potentially limiting or influencing Daphnia reproduction. Second, we show that the role of food quality goes far beyond the up to now documented effects of food quality on somatic growth and trophic transfer efficiency of herbivores: due to its effects on sexual reproduction and the production of resting eggs, food quality might influence genetic diversity and long-term persistence of Daphnia in lakes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Retrotransposon Sequence Variation in Four Asexual Plant Species

Transposable elements (TEs) can be viewed as genetic parasites that persist in populations due to their capacity for increase in copy number and the inefficacy of selection against them. A corollary of this hypothesis is that TEs are more likely to spread within sexual populations and be eliminated or inactivated within asexual populations. While previous work with animals has shown that asexual taxa may contain less TE diversity than sexual taxa, comparable work with plants has been lacking. Here we report the results of a study of Ty1/copia, Ty3/gypsy, and LINE-like retroelement diversity in four asexual plant species. Retroelement-like sequences, with a high degree of conservation both within and between species, were isolated from all four species. The sequences correspond to several previously annotated retroelement subfamilies. They also exhibit a pattern of nucleotide substitution characterized by an excess of synonymous substitutions, suggestive of a history of purifying selection. These findings were compared with retroelement sequence evolution in sexual plant taxa. One likely explanation for the discovery of conserved TE sequences in the genomes of these asexual taxa is simply that asexuality within these taxa evolved relatively recently, such that the loss and breakdown of TEs is not yet detectable through analysis of sequence diversity. This explanation is examined by conducting stochastic simulation of TE evolution and by using published information to infer rough estimates of the ages of asexual taxa. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov     

Extreme genetic diversity in asexual grass thrips populations

The continuous generation of genetic variation has been proposed as one of the main factors explaining the maintenance of sexual reproduction in nature. However, populations of asexual individuals may attain high levels of genetic diversity through within‐lineage diversification, replicate transitions to asexuality from sexual ancestors and migration. How these mechanisms affect genetic variation in populations of closely related sexual and asexual taxa can therefore provide insights into the role of genetic diversity for the maintenance of sexual reproduction. Here, we evaluate patterns of intra‐ and interpopulation genetic diversity in sexual and asexual populations of Aptinothrips rufus grass thrips. Asexual A. rufus populations are found throughout the world, whereas sexual populations appear to be confined to few locations in the Mediterranean region. We found that asexual A. rufus populations are characterized by extremely high levels of genetic diversity, both in comparison with their sexual relatives and in comparison with other asexual species. Migration is extensive among asexual populations over large geographic distances, whereas close sexual populations are strongly isolated from each other. The combination of extensive migration with replicate evolution of asexual lineages, and a past demographic expansion in at least one of them, generated high local clone diversities in A. rufus. These high clone diversities in asexual populations may mimic certain benefits conferred by sex via genetic diversity and could help explain the extreme success of asexual A. rufus populations.     

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.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Cytonuclear evidence for hybridogenetic reproduction in natural populations of the Australian carp gudgeon (Hypseleotris: Eleotridae)

Although most vertebrates reproduce sexually, a small number of fishes, amphibians and reptiles are known in which reproduction is asexual, i.e. without meiotic recombination. In fishes, these so-called unisexual lineages usually comprise only females and utilize co-occurring males of a related sexual species to reproduce via gynogenesis or hybridogenesis. Here, we examine patterns of microsatellite and mitochondrial DNA (mtDNA) variation in a widespread group of freshwater fishes (carp gudgeons; Hypseleotris spp.) to investigate a long-standing proposal that this group includes unisexual forms. We show that the mtDNA genome of most carp gudgeons in tributaries of the Goulburn River belongs to one of two deeply divided clades (～10% cyt b divergence) and that nuclear variation divides the same individuals into four distinct groups. Group 1 exhibits the genotypic proportions of a random mating population and has a 1:1 sex ratio. Two other groups are extremely sex-biased (98% male, 96% female), exhibit excess heterozygosity at most loci and share at least one allele per locus with group 1. We propose that these two groups represent 'unisexual' hybridogenetic lineages and that both utilize co-occurring group 1 as sexual host. Interestingly, the fourth distinct group appears to represent hybrid offspring of the two putative hybridogenetic lineages. The propagation of clonal haploid genomes by both males and females and the ability of these clones to unite and form sexually mature diploid hybrid offspring may represent a novel mechanism that contributes to the dynamics of coexistence between hybridogenetic lineages and their sexual hosts.     

Exceptional cryptic diversity and multiple origins of parthenogenesis in a freshwater ostracod

The persistence of asexual reproduction in many taxa depends on a balance between the origin of new asexual lineages and the extinction of old ones. This turnover determines the diversity of extant asexual populations and so influences the interaction between sexual and asexual modes of reproduction. Species with mixed reproduction, like the freshwater ostracod (Crustacea) morphospecies Eucypris virens, are a good model to examine these dynamics. This species is also a geographic parthenogen, in which sexual females and males co-exist with asexual females in the circum-Mediterranean area only, whereas asexual females occur all over Europe. A molecular phylogeny of E. virens based on the mitochondrial COI and 16S fragments is presented. It is characterised by many distinct clusters of haplotypes which are either exclusively sexual or asexual, with only one exception, and are often separated by deep branches. Analysis of the phylogeny reveals an astonishing cryptic diversity, which indicates the existence of a species complex with more than 40 cryptic taxa. We therefore suggest a revision of the single species status of E. virens. The phylogeny indicates multiple transitions from diverse sexual ancestor populations to asexuality. Although many transitions appear to be ancient, we argue that this may be an artefact of the existence of unsampled or extinct sexual lineages.     

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.