Slow molecular evolution in an ancient asexual ostracod

Genetic variability of the non-marine ostracod species Darwinula stevensoni was estimated by sequencing part of the nuclear and the mitochondrial genome. As Darwinulidae are believed to be ancient asexuals, accumulation of mutations should have occurred, both between alleles within lineages and between lineages, during the millions of years of parthenogenetic reproduction. However, our sequence data show the opposite: no variability in the nuclear ITS1 region was observed within or among individuals of D. stevensoni, despite sampling a geographical range from Finland to South Africa. Lack of allelic divergence might be explained by concerted evolution of rDNA repeats. Homogeneity among individuals may be caused either by slow molecular evolution in ITS1 or by a recent selective sweep. Variability of mitochondrial cytochrome oxidase (COI) was similar to intraspecific levels in other invertebrates, thus weakening the latter hypothesis. Calibrating interspecific, genetic divergences among D. stevensoni and other Darwinulidae using their fossil record enabled us to estimate rates of molecular evolution. Both COI and ITS1 evolve half as fast, at most, in darwinulids as in other invertebrates, and molecular evolution has significantly slowed down in ITS1 of D. stevensoni relative to other darwinulids. A reduced ITS1 mutation rate might explain this inconsistency between nuclear and mitochondrial evolution in D. stevensoni.     

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.     

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.     

The potential role of chromosome telomere resetting consequent upon sex in the population dynamics of aphids: an hypothesis

Models of population structure have emphasized the importance of sex in maintaining lineages. This is because, despite the well known ‘two‐fold cost of sex’ compared with asex, it is considered that recombination rids the genome of accumulated mutations and increases its potential for adaptive variation. However, asexual lineages of eukaryotic organisms can also rapidly gain genetic variance directly by various mutational processes, thereby proving that so‐called ‘clones’ do not have strict genetic fidelity ( Lushai & Loxdale, 2002 ; Loxdale & Lushai, 2003a ), whereas the variation so produced may well have adaptive advantage during the evolutionary process. This being so, obligated asexuals or cyclical parthenogens that occasionally indulge in sexual recombination (‘rare sex’) cannot be deemed as ‘evolutionary dead‐ends’( Lushai, Loxdale & Allen, 2003a ). In addition, the persistence of asexual lineages (i.e. lineage longevity) may also involve the integrity of the telomere region, the physical end of the chromosomes ( Loxdale & Lushai, 2003b ). In this earlier study on this topic, we argued that the persistence and ultimate senescence of eukaryotic cell lineages (based upon the frequency of ‘capped’ and ‘uncapped’ chromosomes related to telomere functionality; Blackburn, 2000 ) may directly relate to the survival and persistence of lineages of whole asexual organisms. Aphids are a good model system to test this hypothesis because they show a variety of sexual/asexual reproductive strategies, whereas their mode of asexual reproduction is of the mitotic (= apomictic) type. We also suggested that many aphid lineages require occasional or even rare sexual recombination to re‐set telomere length to allow lineages to persist. Ample empirical evidence from diverse taxa, lineages, and different developmental stages now reveals that the telomere states are indeed re‐set by recombination (homologous or meiotic), thereby rejuvenating the lineage in question. The generational clock element of telomeric functionality has also been successfully described in artificially‐induced mammalian clonal systems. It thus appears that telomere function is a central molecular mechanism instigating and promoting lineage continuity per se. By contrast, we hypothesized that other long‐lived asexuals, or the rare category of ancient asexuals such as bdelloid rotifers, have compensatory mechanisms for maintaining chromosome functional integrity, which are somewhat different from conventional telomeric repeats. In the present study, we carry the analogy between eukaryotic cell functionality and aphid lineages a stage further. Here, we hypothesize that the changing frequency of capped and uncapped telomeres, progressing to senescence in a stochastic manner, may be an underlying factor that significantly contributes to population dynamics in asexual lineage evolution. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 90 , 719–728.     

Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda)

Background

Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.

Methodology/Principal Findings

We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.

Conclusions/Significance

Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.     

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.     

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.     

Ancient asexual scandals

Asexual organisms that are descended from ancient asexual lineages defy current thinking on the evolution of sexual reproduction; theoreticians have been anxious to explain away their existence. However, a number of groups of organisms, from ferns to rotifers, have been suggested to be anciently asexual, and favourable evidence is being accumulated. Furthermore, new techniques for assessing claims of ancient asexuality have been proposed. Although ancient asexuals challenge current theories of sex, understanding how they manage to persist will help to explain why most organisms are sexual.     

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.     

Integrative taxonomy in two free-living nematode species complexes

Integrative taxonomy considers species boundaries from multiple, complementary perspectives, with the main objective being to compare the observed data against the predictions of the methodologies used. In the present study we used three methods for delineating species boundaries within the cosmopolitan nematode species Rhabditis ( Pellioditis ) marina and Halomonhystera disjuncta . First, phylogenetic relationships among molecular sequences from the mitochondrial cytochrome oxidase c subunit 1 gene (COI), and from two nuclear regions, internal transcribed spacer (ITS) and D2D3, were analysed. Subsequently, multivariate morphometric analysis was used to investigate whether concordant molecular lineages were also morphologically distinct. When morphological differences were found, typological taxonomy was performed to identify fixed or non-overlapping characters between lineages. Interbreeding experiments were conducted between the two closest related lineages of R . ( P. ) marina to investigate potential reproductive isolation. This integrative approach confirmed the presence of several species within each nominal species: molecular lineages were concordant across two independent loci (COI and ITS), and were characterized by significant morphological divergence. Most lineages were also detectable in the D2D3 region, but were less resolved. The two lineages investigated in our study did not produce offspring. Our results highlight that classical taxonomy grossly underestimates species diversity within the phylum Nematoda.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 737–753.     

Recent and ancient asexuality in Timema walkingsticks

Determining the evolutionary age of asexual lineages should help in inferring the temporal scale under which asexuality and sex evolve and assessing selective factors involved in the evolution of asexuality. We used 416 bp of the mitochondrial COI gene to infer phylogenetic relationships of virtually all known Timema walkingstick species, including extensive intraspecific sampling for all five of the asexuals and their close sexual relatives. The asexuals T. douglasi and T. shepardii were very closely related to each other and evolutionarily young (less than 0.5 million years old). For the asexuals T. monikensis and T. tahoe, evidence for antiquity was weak since only one population of each was sampled, intraspecific divergences were low, and genetic distances to related sexuals were high: maximum-likelihood molecular-clock age estimates ranged from 0.26 to 2.39 million years in T. monikensis and from 0.29-1.06 million years in T. tahoe. By contrast, T. genevieve was inferred to be an ancient asexual, with an age of 0.81 to 1.42 million years. The main correlate of the age of asexual lineages was their geographic position, with younger asexuals being found further north.     

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.     

No slave to sex

Fully asexual lineages cannot purge accumulating mutations from their genome through recombination. In ancient asexuals that have persisted without sex for millions of years, this should lead to high allelic divergences (the 'Meselson effect') as has been shown for bdelloid rotifers. Homogenizing mechanisms can counter this effect, resulting in low genetic diversity within and between individuals. Here, we show that the ancient asexual ostracod species Darwinula stevensoni has very low nucleotide sequence divergence in three nuclear regions. Differences in genetic diversity between embryos and adults furthermore indicate that up to half of the observed genetic changes in adults can be caused by somatic mutations. Likelihood permutation tests confirm the presence of gene conversion in the multi-copy internal transcribed spacer sequence, but reject rare or cryptic forms of sex as a general explanation for the low genetic diversity in D. stevensoni. Other special mechanisms (such as highly efficient DNA repair) might have been selected for in this ancient asexual to overcome the mutational load and Muller's ratchet. In this case, our data support these hypotheses on the prevalence of sex, even if the two extant ancient asexual groups (bdelloids and darwinulids) seem to follow opposite evolutionary strategies.     

High levels of genetic diversity in populations of Iris aphylla L. (Iridaceae), an endangered species in Poland

We used RAPDs (Random Amplified Polymorphic DNAs) to investigate genetic diversity and its partition within and between three populations of Iris aphylla in Poland. Analysis of Molecular Variance (AMOVA) of 84 distinct RAPD multiband genotypes revealed higher variation within populations (77.2%) than genetic differentiation between them (22.8%, P  < 0.002). Values of genetic diversity indices ( H ) were similar in all three sites (0.21–0.24). The differentiation of the populations corresponded to low average gene flow ( Nm  = 0.81). Our results indicated that genetic diversity was independent of population size. We concluded that although sexual reproduction and gene flow between populations of I. aphylla were very limited, they preserved high levels of genetic diversity. Relatively large number of seeds, which migrated in the past to populations, as well as patterns of reproduction and life history of I. aphylla may explain this situation.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 65–72.     

Did Pleistocene glaciations shape genetic patterns of European ostracods? A phylogeographic analysis of two species with asexual reproduction

Nested clade analyses (NCA) and likelihood mapping were applied to DNA sequence data of the ribosomal ITS1 and mitochondrial COI region from two non-marine ostracod species. The aim was to test whether Pleistocene glaciations may have shaped genetic and geographic patterns. According to the results from both types of analyses, evidence was lacking for any kind of geographic grouping of European (and one African) population from the putatively ancient asexual ostracod, Darwinula stevensoni. This counters the possibility that a recent selective sweep could have caused the low intraspecific, genetic diversity observed in this species. One of the most cited hypotheses to explain geographic parthenogenesis invokes faster, postglacial colonization by asexual lineages. However, no evidence for northern ‘range expansion’ of asexual haplotypes was found for Eucypris virens, a species with geographic parthenogenesis. Rather, the outcome of the NCA reveals that phylogeographic relationships are characterized by ‘restricted dispersal with isolation by (geographic) distance’. This result suggests that either no faster, postglacial range expansion of asexuals occurred in E. virens or, that patterns of subsequent colonization became ‘overwritten’ by more recent dispersal events. Likelihood mapping provides evidence for the second scenario because genetic instead of geographic clustering was statistically supported. Handling editor: K. Martens     

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.     

Distribution, phenology and demography of sympatric sexual and asexual dandelions (Taraxacum officinale s.l.): geographic parthenogenesis on a small scale

In many plant and animal species, sexual and asexual forms have different geographical distributions ('geographic parthenogenesis'). The common dandelion Taraxacum officinale s.l. provides a particularly clear example of differing distributions: diploid sexuals are restricted to southern and central Europe, while triploid asexuals occur across Europe. To get a better understanding of the factors underlying this pattern, we studied the distribution and demography of sexuals and asexuals in a mixed population that was located at the northern distribution limit of the sexuals. In this population three adjacent, contrasting microhabitats were found: a foreland and south and north slopes of a river dike. Comparative analyses of the distribution, phenology and demography indicated that sexuals had a stronger preference for the south slope than did asexuals. We therefore propose that the large-scale geographic parthenogenesis in T. officinale is shaped by an environmental gradient which acts upon the sexuals.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 205–218.     

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.     

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.     

Phylogenetic evidence for hybrid origins of asexual lineages in an aphid species

Understanding the mode of origin of asexuality is central to ongoing debates concerning the evolution and maintenance of sexual reproduction in eukaryotes. This is because it has profound consequences for patterns of genetic diversity and ecological adaptability of asexual lineages, hence on the outcome of competition with sexual relatives both in short and longer terms. Among the possible routes to asexuality, hybridization is a very common mechanism in animals and plants. Aphids present frequent transitions from their ancestral reproductive mode (cyclical parthenogenesis) to permanent asexuality, but the mode of origin of asexual lineages is generally not known because it has never been thoroughly investigated with appropriate molecular tools. Rhopalosiphum padi is an aphid species with coexisting sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) lineages that are genetically distinct. Previous studies have shown that asexual lineages of R. padi are heterozygous at most nuclear loci, suggesting either that they have undergone long-term asexuality (under which heterozygosity tends to increase) or that they have hybrid origins. To discriminate between these alternatives, we conducted an extensive molecular survey combining the sequence analysis of alleles of two nuclear DNA markers and mitochondrial DNA haplotypes in sexual and asexual lineages of R. padi. Both nuclear and cytoplasmic markers clearly showed that many asexual lineages have hybrid origins, the first such demonstration in aphids. Our results also indicated that asexuals result from multiple events of hybridization between R. padi and an unknown sibling species, and are of recent origin (contradicting previous estimates that asexual R. padi lineages were of moderate longevity). This study constitutes another example that putatively ancient asexual lineages are actually of much more recent origin than previously thought. It also presents a robust approach for testing whether hybrid origin of asexuality is also a common phenomenon in aphids.