Apomixis in flowering plants: an overview

Apomixis is a common feature of perennial plants, which occurs in ca. 60% of the British flora, but has been largely ignored by reproductive theoreticians. Successful individuals may cover huge areas, and live to great ages, favoured by 'symmetrical' selection. Apomixis is favoured by colonizing modes, for instance post-glacially. Despite its theoretical advantages, apomixis usually coexists with sexuality, suggesting 'hidden' disadvantages. Agamospermy (apomixis by seed) is relatively uncommon, but gains from the attributes of the seed. It pays agamospermy genes, which discourage recombination, to form co-adapted linkage groups, so that they become targets for disadvantageous recessive mutant accumulation. Consequently, agamospermy genes cannot succeed in diploids and agamosperms are hybrid and highly heterotic. Agamospermous endosperm may suffer from genomic imbalance, so that nutritious ovules, which can support embryos without endosperm, may be preadapted for agamospermy. When primary endosperm nucleus fertilization ('pseudogamy') continues as a requirement for many aposporous agamosperms, selfing sex becomes preadaptive and archesporial sex remains an option. Apomictic populations can be quite variable although apomictic families are much less variable than sexuals. Only in some diplosporous species does sex disappear completely, and in those species some release of variability may persist through somatic recombination. The search for an agamospermy gene suitable for genetic modification should target fertile sexuals with a single localized agamospermy (A) gene, which therefore lack a genetic load. The A gene should coexist alongside sexuality, so that it would be easy to select seedlings of sexual and asexual origins. Plants with sporophytic agamospermy provide all these attributes.     

VERY LOW GENETIC HETEROZYGOSITIES IN SEXUAL AND AGAMOSPERMOUS POPULATIONS OF EUPATORIUM ALTISSIMUM (ASTERACEAE)

An electrophoretic study revealed very low genetic heterozygosities for both sexual and agamospermous populations of Eupatorium altissimum in eastern North America. Low heterozygosity or gene diversity (H_T = 0.03) can be explained by the small sizes of the extant sexual populations, estimated to be on the order of 10⁴. There was no substantial difference in observed heterozygosity between sexual and agamospermous races. This result shows that a higher level of heterozygosity is not a prerequisite for the evolution of agamospermous races from the sexual ancestor.     

Genotypic variation in agamospermous Erigeron compositus (Asteraceae)

Few integrative analyses of the structure of agamospermous plant populations have been conducted. Erigeron compositus occurs in montane western North America and comprises both sexual and agamospermous populations. Sexual E. compositus has previously been characterized as outcrossing and predominantly diploid (2n = 18). Agamic E. compositus is usually hexaploid (2n = 54), though counts herein range from 2n = 36 to 2n = 80. Starch-gel electrophoresis, cytology, and analysis of pollen production were used to evaluate variation within and among agamospermous populations. Fifteen enzyme loci were used to identify 24 unique multilocus genotypes in seven populations, an average of 3.4 genotypes per population. Proportion of distinct genotypes per population sample size (GIN) and measures of genetic diversity (D) and evenness (E) are 0.10, 0.48, and 0.61, respectively, which indicate that E. compositus maintains levels of diversity similar to other agamospermous taxa. Most agamospermous populations are mosaics comprising groups of genetically distinct individuals that are frequently distinguished by cytotype and capacity for pollen production. The geographical and ecological separation of sexual and agamospermous populations make it unlikely that gene flow from sexual populations is a direct source of genetic variation in agamospermous populations. Instead, crossing between genetically distinct facultative agamosperms probably accounts for most variation. Genetic and morphological evidence document one such putative crossing event. Agamospermous E. compositus is very similar genetically to sexual E. compositus. Allozyme analysis further shows that genetic variation in agamospermous populations is partitioned among a few highly heterozygous genotypes, whereas sexual populations maintain numerous genotypes of relatively low heterozygosity.     

Formation of a hybrid triploid agamosperm on a sexual diploid plant: evidence from progeny tests in Taraxacum platycarpum Dahlst.

Agamospermous dandelions of hybrid origin between a native sexual diploid species (Taraxacum platycarpum Dahlst. or T. japonicum Koidz.) and an alien agamospermous triploid [T. officinale Weber and T. laevigatum (Willd.) DC.] are now widely distributed throughout mainland Japan. These hybrid dandelions are known to be genetically variable. We hypothesized that this variability is maintained by repeated ongoing hybridization, based on the fact that triploid dandelions not only produce seeds agamospermously, but also produce some functional pollen grains that are able to sire seeds of sexual dandelions. To test this hypothesis, we examined whether heads of Japanese diploid dandelions produce new hybrid seeds after fertilization by pollen from triploid agamosperms under field conditions. One of the 430 tested plants grown from sexual dandelion seeds had morphological and molecular characteristics, which are consistent with a hybrid origin. The plant formed a hybrid surrounded by many individuals having recurved involucral bracts, in which frequency of T. officinale was very low (3.5 %). Cytological data and bagging experiments demonstrate triploidy and asexual seed production of the hybrid. Taken together, these results supported that the new hybrid is probably derived from a backcross of a hybrid to the native sexual species. Our findings provide evidence for the evolution of a new agamosperm through interspecific hybridization as a contemporary population process.     

THE LEVEL OF AGAMOSPERMY IN A NEBRASKA POPULATION OF SPIRANTHES CERNUA (ORCHIDACEAE)

This study investigated reproduction in a prairie population of Spiranthes cernua in eastern Nebraska. Spiranthes cernua reproduces both sexually and asexually, through adventitious embryony. Agamospermous seeds are monoembryonic, polyembryonic, have extruded or free embryos, or lack embryos. These kinds of seeds are extremely rare in Spiranthes vernalis, a close relative that relies exclusively on sexual reproduction. Sexually derived seeds are monoembryonic or lack embryos. One objective was to quantify the amount of sexual reproduction in a natural population. In 1985 and 1986, maximum estimates of sexual seed production per capsule were 19.2% and 33.6%, respectively. Thus, every capsule examined contained a high proportion of agamospermous seeds. In addition, attempts were made to manipulate the reproductive system with experimental crosses performed in the field. The treatments examined agamospermy, autogamy, and outcrossing. In all treatments the majority of seeds in a capsule were agamospermous.     

Distribution of Sexual and Agamospermous Populations of Eupatorium (Compositae) in Asia

Abstract Examinations of chromosome number voucher specimens show that sexual diploid and agamospermous polyploid plants of Eupatorium in Asia can be distinguished by morphology, fertility, and stainability of pollen grains. Using these criteria, reproductive systems (sexual vs. agamospermous) were estimated for 558 herbarium specimens of Eupatorium in East Asia. Of 22 taxa examined, six included both sexual and agamospermous specimens; those of one taxon were all agamospermous; and those of 15 taxa were all sexual. This result shows that 21 taxa are not agamospermous microspecies but are differentiated at the diploid level. Sexual populations of most taxa had restricted geographical distributions. Eupatorium chinense var. chinense and var. oppositifolium consisted of both sexual and agamospermous populations. Sexual populations of these two varieties were allopatric and distinct in external morphology, suggesting differentiation at the species level. The specimens of E. heterophyllum s. str. were all agamospermous, while those of E. mairel , often treated as synonymous with E. heterophyllum , were sexual.
In comparing East Asian and North American Eupatorium for the distributional patterns of sexual and agamospermous populations, three differences can be pointed out: (1) Agamospermous plants of autopolyploid origin have evolved in eight species and are widespread in North America; while most agamospermous plants in East Asia may be of allopolyploid origin, have relatively restricted ranges, and are less frequent than the diploid plants with the exception of in E. chinense var. oppositifolium , (2) The number of sexual species with wide range is greater in North America than in East Asia. (3) The number of sexual species with restricted ranges is greater in East Asia than in North America. The results obtained suggest that speciation among the plants of Eupatorium has occurred more recently in East Asia than in North America.     

LOWER MITE INFESTATIONS IN AN ASEXUAL GECKO COMPARED WITH ITS SEXUAL ANCESTORS

What advantage do sexually reproducing organisms gain from their mode of reproduction that compensates for their twofold loss in reproductive rate relative to their asexual counterparts? One version of the Red Queen hypothesis suggests that selective pressure from parasites is strongest on the most common genotype in a population, and thus genetically identical clonal lineages are more vulnerable to parasitism over time than genetically diverse sexual lineages. Our surveys of the ectoparasites of an asexual gecko and its two sexual ancestral species show that the sexuals have a higher prevalence, abundance, and mean intensity of mites than asexuals sharing the same habitat. Our experimental data indicate that in one sexual/asexual pair this pattern is at least partly attributable to higher attachment rates of mites to sexuals. Such a difference may occur as a result of exceptionally high susceptibility of the sexuals to mites because of their low genetic diversity (relative to other more-outbred sexual species) and their potentially high stress levels, or as a result of exceptionally low susceptibility of the asexuals to mites because of their high levels of heterozygosity.     

The Significance of Agamospermous Triploid Pollen Donors in the Sexual Relationships between Diploids and Triploids in Taraxacum (Compositae)

Abstract We review in this article the investigations of the significance of agamospermous triploid pollen donors in the sexual relationships between diploids and triploids in Taraxacum . Crossing experiments between diploid sexual mother plants and agamospermous polyploid pollen donors and recent isozyme analyses of the progeny have exhibited the following results: 1) Pollen from Agamospermous polyploid pollen donors have the potential to give rise to the polyploid agamospermous offspring when fertilizing diploid sexual plants. Ploidy level of the progeny is usually the same or higher, but occasionally lower, compared to the pollen donor. 2) Diploid progeny also occur from diploid (♀)-polyploid (♂) crosses, however, these diploids were in our results not hybrids but the results of self-fertilization of the diploids. The self-fertilization is regarded as a cosequence of the breakdown of the self-incompatibility barrier through the sterile triploid's pollen. This breakdown is in all probability a common phenomenon in diploid (♀)-polyploid (♂) crosses. Some examples suggest that agamospermous polyploids can increase their genetic diversity through obtaining genes from coexisting diploids. The evolutionary implications of this phenomenon and the reduction mechanism of chromosome number through agamospermous pollengenesis are discussed.     

Attack of the clones: reproductive interference between sexuals and asexuals in the Crepis agamic complex

Negative reproductive interactions are likely to be strongest between close relatives and may be important in limiting local coexistence. In plants, interspecific pollen flow is common between co‐occurring close relatives and may serve as the key mechanism of reproductive interference. Agamic complexes, systems in which some populations reproduce through asexual seeds (apomixis), while others reproduce sexually, provide an opportunity to examine effects of reproductive interference in limiting coexistence. Apomictic populations experience little or no reproductive interference, because apomictic ovules cannot receive pollen from nearby sexuals. Oppositely, apomicts produce some viable pollen and can exert reproductive interference on sexuals by siring hybrids. In the Crepis agamic complex, sexuals co‐occur less often with other members of the complex, but apomicts appear to freely co‐occur with one another. We identified a mixed population and conducted a crossing experiment between sexual diploid C. atribarba and apomictic polyploid C. barbigera using pollen from sexual diploids and apomictic polyploids. Seed set was high for all treatments, and as predicted, diploid–diploid crosses produced all diploid offspring. Diploid–polyploid crosses, however, produced mainly polyploidy offspring, suggesting that non‐diploid hybrids can be formed when the two taxa meet. Furthermore, a small proportion of seeds produced in open‐pollinated flowers was also polyploid, indicating that polyploid hybrids are produced under natural conditions. Our results provide evidence for asymmetric reproductive interference, with pollen from polyploid apomicts contributing to reduce the recruitment of sexual diploids in subsequent generations. Existing models suggest that these mixed sexual–asexual populations are likely to be transient, eventually leading to eradication of sexual individuals from the population.     

Two independent loci control agamospermy (Apomixis) in the triploid flowering plant Erigeron annuus

Asexual seed production (agamospermy) via gametophytic apomixis in flowering plants typically involves the formation of an unreduced megagametophyte (via apospory or diplospory) and the parthenogenetic development of the unreduced egg cell into an embryo. Agamospermy is almost exclusively restricted to polyploids. In this study, the genetic basis of agamospermy was investigated in a segregating population of 130 F(1)'s from a cross between triploid (2n = 27) agamospermous Erigeron annuus and sexual diploid (2n = 18) E. strigosus. Correlations between markers and phenotypes and linkage analysis were performed on 387 segregating amplified fragment length polymorphisms (AFLPs). Results show that four closely linked markers with polysomic inheritance are significantly associated with parthenogenesis and that 11 cosegregating markers with univalent inheritance are completely associated with diplospory. This indicates that diplospory and parthenogenesis are unlinked and inherited independently. Further, the absence of agamospermy in diploid F(1)'s appears to be best explained by a combination of recessive-lethal gametophytic selection against the parthenogenetic locus and univalent inheritance of the region bearing diplospory. These results may have major implications for attempts to manipulate agamospermy for agricultural purposes and for interpreting the evolution of the trait.     

A comparison of within-plant karyological heterogeneity between agamospermous and sexualTaraxacum (Compositae) as assessed by the nucleolar organiser chromosome

Morphological variation for the NOR chromosome was studied for four half-siblings of a sexual outbreedingTaraxacum, for three siblings of the obligate agamospermT. pseudohamatum, and for two individuals of the agamospermT. brachyglossum. No rearrangement was detected for the 113 chromosomes of sexuals, or for 41 chromosomes of two agamospermous individuals. In the other three agamospermous individuals, 3/16, 5/50, and 5/20 chromosomes showed evidence of chromosomal rearrangement. The majority of rearrangement events (10/13) occurred to the satellite rather than to the body of the NOR-chromosome. It is considered that such high levels of somatic chromosomal rearrangement in agamospermousTaraxacum may be the result of activity by transposable genetic elements. This recombination may be of selective advantage to asexual plants which cannot generate genetic variability through the sexual process.     

Lower fecundity in parthenogenetic geckos than sexual relatives in the Australian arid zone

Theoretical models of the advantage of sexual reproduction typically assume that reproductive output is equal in sexual and parthenogenetic females. We tested this assumption by comparing fecundity between parthenogenetic and sexual races of gekkonid lizards in the Heteronotia binoei complex, collected across a 1200 km latitudinal gradient through the Australian arid zone. Under laboratory conditions, parthenogenetic geckos had approximately 30% lower fecundity when compared with their sexual progenitors, irrespective of body size. Reflecting clutch-size constraints in gekkonids, this fecundity difference was mainly because of fewer clutches over a shorter period. When parthenogens were compared more broadly with all coexisting sexual races across the latitudinal gradient, parthenogens had lower fecundity than sexuals only when corrected for body size. Differences in fecundity between parthenogens and coexisting sexual races depended on which sexual race was considered. There was no significant relationship between fecundity and parasite (mite) load, despite significantly higher mite loads in parthenogens than in sexual races.     

The complex causality of geographical parthenogenesis

Asexual organisms usually have larger and more northern distributions than their sexual relatives. This phenomenon, called geographical parthenogenesis, has been controversially attributed to predispositions in certain taxa; advantages of polyploidy and/or hybrid origin; better colonizing abilities because of uniparental reproduction; introgression of apomixis into sexuals; niche differentiation of clones; or biotic interactions. This review on apomictic plants demonstrates that each of these factors alone has not been able to explain the observed distributions. Establishment of the complex regulatory system of apomixis requires taxonomic and geographical predispositions; hybridization and/or polyploidization do create diversity, but they do not necessarily result in large distributions; colonizing abilities depend on clonal diversity and are outweighed by sexuals by self-compatibility and higher potentials for speciation; niche differentiation, ploidy levels and selfing keep sympatric sexuals and apomicts separated; and the impact of biotic interactions on distributions is uncertain. In conclusion, the distributional success of apomicts has a complex causality and depends on certain circumstances and combinations of factors. The rare establishment of apomixis may help to explain the predominance of sexuality on the large scale.     

Developmental success, stability, and plasticity in closely related parthenogenetic and sexual lizards (Heteronotia, Gekkonidae)

The developmental trajectory of an organism is influenced by the interaction between its genes and the environment in which it develops. For example, the phenotypic traits of a hatchling reptile can be influenced by the organism's genotype, by incubation temperature, and by genetically coded norms of reaction for thermally labile traits. The evolution of parthenogenesis provides a unique opportunity to explore such effects: a hybrid origin of this trait in vertebrates modifies important aspects of the genotype (e.g., heterozygosity, polyploidy) and may thus impact not only on the phenotype generally, but also on the ways in which incubation temperature affects expression of the phenotype. The scarcity of vertebrate parthenogenesis has been attributed to developmental disruptions, but previous work has rarely considered reaction norms of embryogenesis in this respect. We used closely related sexual and asexual races of the Australian gecko Heteronotia binoei, which include those with multiple origins of parthenogenesis, to explore the ways in which reproductive modes (sexual, asexual), incubation temperatures (24, 27, and 30 degrees C), and the interaction between these factors affected hatchling phenotypes. The hatchling traits we considered included incubation period, incidence of deformities, hatchling survivorship, body size and shape, scalation (including fluctuating asymmetry), locomotor performance, and growth rate. Developmental success was slightly reduced (higher proportion of abnormal offspring) in parthenogenetic lineages although there was no major difference in hatching success. Incubation temperature affected a suite of traits including incubation period, tail length, body mass relative to egg mass, labial scale counts, running speed, growth rate, and hatchling survival. Our data also reveal an interaction between reproductive modes and thermal regimes, with the phenotypic traits of parthenogenetic lizards less sensitive to incubation temperature than was the case for their sexual relatives. Thus, the evolution of asexual reproduction in this species complex has modified both mean hatchling viability and the norms of reaction linking hatchling phenotypes to incubation temperature. Discussions on the reasons why parthenogenetic organisms are scarce in nature should take into account interactive effects such as these; future work could usefully try to tease apart the roles of parthenogenesis, its hybrid origin (and thus effects on ploidy and heterozygosity, etc.), and clonal selection in generating these divergent embryonic responses.     

Comparisons of within-population genetic variation in sexual and agamospermousAmelanchier (Rosaceae) using RAPD markers

We compared genetic variation of sexualAmelanchier bartramiana and facultatively agamospermous (asexually seed-producing)A. laevis at one site where the two species are sympatric. We analyzed 77 random amplified polymorphic DNA (RAPD) markers in 29A. bartramiana individuals and 76 RAPD markers in 31A. laevis individuals. The two species do not differ significantly in mean genetic variation. However, 22.4% of genetic similarity values betweenA. laevis individuals exceed the highest value ofA. bartramiana and may represent the effect of agamospermy. Variation withinA. laevis may be the result of sexuality, hybridization, polyploidy, and other factors.     

Breeding system of diploid sexuals within the Ranunculus auricomus complex and its role in a geographical parthenogenesis scenario

The larger distribution area of asexuals compared with their sexual relatives in geographical parthenogenesis (GP) scenarios has been widely attributed to the advantages of uniparental reproduction and polyploidy. However, potential disadvantages of sexuals due to their breeding system have received little attention so far. Here, we study the breeding system of five narrowly distributed sexual lineages of Ranunculus notabilis s.l. (R. auricomus complex) and its effects on outcrossing, inbreeding, female fitness, and heterozygosity. We performed selfing and intra‐ and interlineage crossings by bagging 481 flowers (59 garden individuals) followed by germination experiments. We compared seed set and germination rates, and related them to genetic distance and genome‐wide heterozygosity (thousands of RADseq loci). Selfings (2.5%) unveiled a significantly lower seed set compared with intra‐ (69.0%) and interlineage crossings (69.5%). Seed set of intra‐ (65%) compared to interpopulation crossings (78%) was significantly lower. In contrast, all treatments showed comparable germination rates (32%–43%). Generalized linear regressions between seed set and genetic distance revealed positive relationships in general and between lineages, and a negative one within lineages. Seed set was the main decisive factor for female fitness. Germination rates were not related to genetic distance at any level, but were positively associated with heterozygosity in interlineage crossings. Experiments confirmed full crossability and predominant outcrossing among sexual R. notabilis s.l. lineages. However, up to 5% (outliers 15%–31%) of seeds were formed by selfing, probably due to semi‐self‐compatibility in a multi‐locus gametophytic SI system. Less seed set in intrapopulation crossings, and higher seed set and germination rates from crossings of genetically more distant and heterozygous lineages (interlineage) indicate negative inbreeding and positive outbreeding effects. In GP scenarios, sexual species with small and/or isolated populations can suffer from decreased female fitness due to their breeding system. This factor, among others, probably limits range expansion of sexuals.     

Evolution of clonality and polyploidy in a weevil system

The increased interest in asexual organisms calls for in-depth studies of asexual complexes that actively give rise to new clones. We present an extensive molecular study of the Otiorhynchus scaber (Coleoptera, Curculionidae) weevil system. Three forms have traditionally been recognized: diploid sexuals, triploid, and tetraploid parthenogens. All forms coexist in a small central area, but only the polyploid parthenogens have colonized marginal areas. Analyzing the phylogenetic relationship, based on three partial mitochondrial genes, of 95 individuals from 19 populations, we find that parthenogenesis and polyploidy have originated at least three times from different diploid lineages. We observe two major mitochondrial lineages, with over 2.5% sequence divergence between the most basal groups within them, and find that current distribution and phylogenetic relationships are weakly correlated. Quite unexpectedly, we also discover diploid clones that coexist with, and are morphologically indistinguishable from, the diploid sexual females. Our results support that these diploid clones are derived directly from the diploid sexuals. We also find that it is mainly an increase in ploidy level and not the benefits of asexual reproduction that confers to polyploid parthenogens the advantage over their diploid sexual relatives.     

A NEW MEASURE OF GENETIC IDENTITY BETWEEN POPULATIONS OF SEXUAL AND ASEXUAL SPECIES

We define a new genetic identity measure that is especially well suited for asexual polyploid species as it circumvents errors in the estimation of gene frequencies. It also can be applied to sexuals allowing the study of phylogenetic relationships in species complexes consisting of sexuals and asexuals of different ploidy levels. The measure groups genotypes into classes dependent on homozygosity vs heterozygosity and the number of ancestral allele types vs the number of presumed new mutations. Its value is related to evolutionary time since divergence. The application of the method is illustrated by using electrophoretic data on the species group Solenobia triquetrella (Lepidoptera: Psychidae). A high similarity of estimated relationships among the proposed as well as other genetic identity measures is shown in the case of diploid sexual and asexual races of this species group. The phylogenetic relationships within the group are reanalyzed and monophyletic vs polyphyletic origin of parthenogenesis in this species complex is discussed. The genetic identity values found by the proposed procedure are explained by a polyphyletic origin of parthenogenesis, though a monophyletic origin of parthenogenesis in a broader sense cannot be excluded. The explanation of the phylogenetic relationships is based on the assumption of hybridization between related species and the extinction of one ancestral species. Furthermore, the genetic diversity is compared among sexual and parthenogenetic races of the species.     

A population dynamic model for facultative agamosperms

Plants that can reproduce both sexually and agamically are called facultative apomicts. Some species, such as Taraxacum, contain both sexual diploids and triploid facultative apomicts. Triploids produce seeds without gamete fusion and recombination, and can also produce pollen and fertilize diploids. We present a population dynamic model that deals with gene flow and competition between diploids and triploids, with differing allocation towards reproductive investment in seeds and pollen. This paper examines whether diploids and triploids of plants with facultative agamospermy can coexist within a single population. We analyse the global behavior of such a dynamic system. Features of the system are significantly affected by the germination rates of diploids and triploids. Either diploids or triploids persist alone when the germination rate of diploids is sufficiently larger or smaller than that of triploids, respectively. Competitive exclusion occurs when both germination rates are sufficiently large. Coexistence is possible under certain specific conditions when: (I) the germination rates of both diploid sexuals and triploids are not sufficiently large, and (II) triploids produce sufficient pollen. When diploid sexuals and triploids coexist, triploids cannot exist alone, implying that the pollen of triploids is necessary to exploit diploid ovules.     

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.