Parthenogenetic Offspring Research Articles

Inter-clonal competition over queen succession imposes a cost of parthenogenesis on termite colonies.

In social insect colonies, selfish behaviour due to intracolonial conflict among members can result in colony-level costs despite close relatedness. In certain termite species, queens use asexual reproduction for within-colony queen succession but rely on sexual reproduction for worker and alate production, resulting in multiple half-clones of a single primary queen competing for personal reproduction. Our study demonstrates that competition over asexual queen succession among different clone types leads to the overproduction of parthenogenetic offspring, resulting in the production of dysfunctional parthenogenetic alates. By genotyping the queens of 23 field colonies of Reticulitermes speratus, we found that clone variation in the queen population reduces as colonies develop. Field sampling of alates and primary reproductives of incipient colonies showed that overproduced parthenogenetic offspring develop into alates that have significantly smaller body sizes and much lower survivorship than sexually produced alates. Our results indicate that while the production of earlier and more parthenogenetic eggs is advantageous for winning the competition for personal reproduction, it comes at a great cost to the colony. Thus, this study highlights the evolutionary interplay between individual-level and colony-level selection on parthenogenesis by queens.

Aneuploidy during development in facultative parthenogenetic Drosophila

From concatenated chromosomes to polyploidization, large-scale genome changes are known to occur in parthenogenetic animals. Here, we report mosaic aneuploidy in larval brains of facultatively parthenogenetic Drosophila. We identified a background of aneuploidy in D. mercatorum strains and found increased levels of aneuploidy in the larval brain tissue of animals arising parthenogenetically versus those arising from sexual reproduction. There is also intra-individual variation in germline-derived aneuploidy within the same strain. To determine if this is a general feature of facultative parthenogenesis in drosophilids, we compared sexually reproduced and parthenogenetic offspring from an engineered facultative parthenogenetic strain of D. melanogaster. In addition to germline-derived aneuploidy, this revealed somatic aneuploidy that increased by up to fourfold in parthenogens compared to sexually reproduced offspring. Therefore, the genetic combination identified in D. mercatorum that causes facultative parthenogenesis in D. melanogaster results in aneuploidy, which indicates that the loss of mitotic control resulting in parthenogenesis causes subsequent genome variation within the parthenogenetic offspring. Our findings challenge the assumption that parthenogenetic offspring are near genetic replicas of their mothers.

Single hybrid population but multiple parental individuals at the origin of parthenogenetic rock lizards Darevskia sapphirina and D. bendimahiensis Schmidtler, & Eiselt Darevsky (1994) endemic to the area of Lake Van in East Turkey

Among vertebrates, obligate parthenogenesis is only found in Squamata, where it always has a hybrid origin and a few lizard genera contain most of the known hybridogenous parthenogenetic taxa. Parthenogenesis thus seems to be pre-conditioned at the genus level, but it is not clear how often the encounter between two parental sexually reproducing species can result in the parthenogenetic offspring, nor whether the success of such hybridization event requires certain conditions or the specific time frame. To address this question, we studied the rock lizards of genus Darevskia, where a pair of parental species, D. valentini and D. raddei, as well as their parthenogenetic daughter species D. bendimahiensis and D. sapphirina, are found in close proximity NE of the Lake Van in East Anatolia. Using ddRAD-seq genotyping on 19 parental and 18 hybrid individuals, we found that (i) all parthenogenetic individuals from both D. bendimahiensis and D. sapphirina have a monophyletic origin tracing back to a single initial hybrid population, but their current genetic variation is geographically structured; (ii) unlike the most probable paternal ancestor, the genetically closest extant population of the maternal ancestor is not the geographically nearest one; and (iii) in the parthenogens, about 1% of loci carry multiple haplotypes, frequently differentiated by multiple substitutions. This pattern, in addition to biases in the relative frequency of haplotypes of maternal and paternal origin, does not appear compatible with a scenario of the entire parthenogenic clonal population having descended from a single pair of parental individuals. Instead, the data suggest that multiple parental individual ancestries still persist in the parthenogenetic gene pool. This supports the notion that although hybridization leading to parthenogenesis is generally rare at the level of species, it may be more common at the individual/population level once the right conditions are met.

Costs of parthenogenesis on growth and longevity in ex situ zebra sharks Stegostoma tigrinum

The zebra shark Stegostoma tigrinum, a popular aquarium fish, is an endangered species that is known to readily reproduce both sexually and through facultative parthenogenesis while in human care. Artificial insemination trials that took place between 2011 and 2013 resulted in the hatching of 2 sexually produced (herein heterozygotes) and 10 parthenogenetic sharks that allowed for a retrospective comparison of growth, feeding and longevity between offspring produced from 2 distinct reproductive modes. Parthenogenetic offspring were generally smaller at hatch than their heterozygous counterparts and, after the first several months post-hatch, failed to increase in mass and length at the same rate as heterozygotes. Parthenogenetic offspring exhibited non-normal swimming behaviors such as spiraling, spy hopping and head standing, which may have been correlated with a gradual decline in the ability of some sharks to properly suction feed. Median lifespan for the parthenotes was 1.05 yr (range: 0.27-6.64 yr); one of the heterozygotes lived to 2.37 yr of age, and the other was alive at the time of this writing in August 2022 and had reached reproductive maturity. By contrast, the 2 longest surviving parthenotes perished just prior to reaching sexual maturity (~5.5 and ~6.5 yr). Parthenogenesis has been documented among ex situ S. tigrinum maintained in aquariums across the globe, and this study demonstrates substantial negative costs to fitness in parthenogenetic offspring compared with their heterozygous siblings. The reduced fitness of parthenotes has implications for managing populations in human care as well as for in situ conservation efforts.

Artificial Insemination and Parthenogenesis in the Zebra Shark Stegostoma tigrinum

Maintaining self-sustaining populations of zoo and aquarium collections can be challenged when natural reproduction fails within mixed-sex populations; however, reproductive success can sometimes be restored with the application of reproductive technologies. Among a population of three female and one male Zebra Sharks (Stegostoma tigrinum), production of young failed despite constant male presence with two of the females. To determine if assisted techniques could be used to rescue sexual reproduction, artificial insemination was performed in a singleton female twice over a three-year period using freshly collected semen. Hatching success for eggs laid by all three females was monitored to compare natural and artificial insemination modes. After the first insemination (December 15th, 2011), 143 yolked eggs resulted in no sexually produced offspring and four genetically-confirmed, parthenogenetic offspring. After the second insemination (September 24th, 2013), 62 yolked eggs resulted in two sexually produced offspring, 18 and 33 days after insemination, and three parthenogenetic offspring > 213 days post-insemination. For the two females housed with the male, no sexual offspring resulted. All females produced at least one hatched parthenote. This study successfully employed artificial insemination to circumvent barriers to natural reproduction in Zebra Sharks. With further development, artificial insemination represents a powerful tool that could be used for maintaining genetic diversity for animals housed in aquaria and conservation-based breeding programs for elasmobranchs.

Genome-wide data implicate terminal fusion automixis in king cobra facultative parthenogenesis

Facultative parthenogenesis (FP) is widespread in the animal kingdom. In vertebrates it was first described in poultry nearly 70 years ago, and since then reports involving other taxa have increased considerably. In the last two decades, numerous reports of FP have emerged in elasmobranch fishes and squamate reptiles (lizards and snakes), including documentation in wild populations of both clades. When considered in concert with recent evidence of reproductive competence, the accumulating data suggest that the significance of FP in vertebrate evolution has been largely underestimated. Several fundamental questions regarding developmental mechanisms, nonetheless, remain unanswered. Specifically, what is the type of automixis that underlies the production of progeny and how does this impact the genomic diversity of the resulting parthenogens? Here, we addressed these questions through the application of next-generation sequencing to investigate a suspected case of parthenogenesis in a king cobra (Ophiophagus hannah). Our results provide the first evidence of FP in this species, and provide novel evidence that rejects gametic duplication and supports terminal fusion as a mechanism underlying parthenogenesis in snakes. Moreover, we precisely estimated heterozygosity in parthenogenetic offspring and found appreciable retained genetic diversity that suggests that FP in vertebrates has underappreciated evolutionary significance.

DNA methylation differs extensively between strains of the same geographical origin and changes with age in Daphnia magna

BackgroundPatterns of methylation influence lifespan, but methylation and lifespan may also depend on diet, or differ between genotypes. Prior to this study, interactions between diet and genotype have not been explored together to determine their influence on methylation. The invertebrate Daphnia magna is an excellent choice for testing the epigenetic response to the environment: parthenogenetic offspring are identical to their siblings (making for powerful genetic comparisons), they are relatively short lived and have well-characterised inter-strain life-history trait differences. We performed a survival analysis in response to caloric restriction and then undertook a 47-replicate experiment testing the DNA methylation response to ageing and caloric restriction of two strains of D. magna.ResultsMethylated cytosines (CpGs) were most prevalent in exons two to five of gene bodies. One strain exhibited a significantly increased lifespan in response to caloric restriction, but there was no effect of food-level CpG methylation status. Inter-strain differences dominated the methylation experiment with over 15,000 differently methylated CpGs. One gene, Me31b, was hypermethylated extensively in one strain and is a key regulator of embryonic expression. Sixty-one CpGs were differentially methylated between young and old individuals, including multiple CpGs within the histone H3 gene, which were hypermethylated in old individuals. Across all age-related CpGs, we identified a set that are highly correlated with chronological age.ConclusionsMethylated cytosines are concentrated in early exons of gene sequences indicative of a directed, non-random, process despite the low overall DNA methylation percentage in this species. We identify no effect of caloric restriction on DNA methylation, contrary to our previous results, and established impacts of caloric restriction on phenotype and gene expression. We propose our approach here is more robust in invertebrates given genome-wide CpG distributions. For both strain and ageing, a single gene emerges as differentially methylated that for each factor could have widespread phenotypic effects. Our data showed the potential for an epigenetic clock at a subset of age positions, which is exciting but requires confirmation.

Acute and chronic effects of exposure to the juvenile hormone analog fenoxycarb during sexual reproduction in Daphnia magna.

Recent studies have demonstrated that insect growth regulating insecticides are able to affect reproductive endpoints in zooplankton species at very low levels. For the cyclic parthenogenetic water flea Daphnia, most of this research has focused on the asexual part of the life cycle and induction of male offspring. Even though Daphnia and many other aquatic invertebrates rely on sexual reproduction and subsequent production of dormant eggs to recover from environmentally harsh conditions, much less is known about the effects of toxicants on the sexual reproductive phase. Using fenoxycarb as a model pesticide, we exposed male and female neonate Daphnia magna, under conditions inducing a switch to sexual reproduction, and tested for effects on dormant egg (ephippia) production and sex ratio of parthenogenetic offspring. Subsequently, we assessed whether fenoxycarb exposure affected the quality of the produced dormant eggs and viability of the hatchlings. Our results showed that exposure to sub-lethal concentrations of fenoxycarb caused a sharp decrease in parthenogenetic reproduction, while inducing male offspring. Dormant egg production was marginally negatively affected, but survival and fitness of the hatched individuals were not significantly affected. This indicates that under pesticide stress, surviving adult females invested in sexual reproduction at the expense of parthenogenetic reproduction. Exposure to toxicants during the sexual reproductive phase, could affect the active aquatic phase as well as the dormant phase in natural zooplankton populations. This indicates the need for further ecotoxicological research and development of test protocols taking into account the full life cycle of zooplankton species.

First molecular genetic evidence for automictic parthenogenesis in cockroaches

Parthenogenesis is an asexual mode of reproduction that plays an important role in the evolution of sex, sociality, and reproduction strategies in insects. Some species of cockroach exhibit thelytoky, a type of parthenogenesis in which female offspring are produced without fertilization. However, the cytological and genetic mechanisms of parthenogenesis in cockroaches are not well understood. Here we provide the first molecular genetic evidence that cockroaches can reproduce through automixis. Using the American cockroach Periplaneta americana, we performed microsatellite analysis to investigate the genetic relationship between parthenogenetically produced nymphs and the parent virgin females, and found that all parthenogenetic offspring were homozygous for autosomal microsatellite markers, whereas the female parents were heterozygous. In addition, flow cytometry analysis revealed that the parthenogenetic offspring were diploid. Taken together, our results demonstrate that P. americana exhibits automixis-type thelytoky, in which diploidy is restored by gamete duplication or terminal fusion. These findings highlight the unique reproduction strategies of cockroaches, which are more varied than was previously recognized.

Exploring the relationship between tychoparthenogenesis and inbreeding depression in the Desert Locust, Schistocerca gregaria.

Tychoparthenogenesis, a form of asexual reproduction in which a small proportion of unfertilized eggs can hatch spontaneously, could be an intermediate evolutionary link in the transition from sexual to parthenogenetic reproduction. The lower fitness of tychoparthenogenetic offspring could be due to either developmental constraints or to inbreeding depression in more homozygous individuals. We tested the hypothesis that in populations where inbreeding depression has been purged, tychoparthenogenesis may be less costly. To assess this hypothesis, we compared the impact of inbreeding and parthenogenetic treatments on eight life‐history traits (five measuring inbreeding depression and three measuring inbreeding avoidance) in four laboratory populations of the desert locust, Schistocerca gregaria, with contrasted demographic histories. Overall, we found no clear relationship between the population history (illustrated by the levels of genetic diversity or inbreeding) and inbreeding depression, or between inbreeding depression and parthenogenetic capacity. First, there was a general lack of inbreeding depression in every population, except in two populations for two traits. This pattern could not be explained by the purging of inbreeding load in the studied populations. Second, we observed large differences between populations in their capacity to reproduce through tychoparthenogenesis. Only the oldest laboratory population successfully produced parthenogenetic offspring. However, the level of inbreeding depression did not explain the differences in parthenogenetic success between all studied populations. Differences in development constraints may arise driven by random and selective processes between populations.

Facultative parthenogenesis in the Ryukyu drywood termite Neotermes koshunensis.

Parthenogenesis is a relatively rare reproductive mode in nature compared to sex. In social insects, the evolution of parthenogenesis has a notable impact on their life histories. Some termites with parthenogenetic ability produce numerous non-dispersing supplementary queens asexually, whereas other castes are produced via sexual reproduction. This asexual queen succession (AQS) system is adaptive because hundreds of the asexual queens improve the reproductive potential of the colony and maintain the genetic diversity within the colony. However, the evolutionary process of the AQS system remains unclear because parthenogenetic species without this system are unknown. Here, we report facultative parthenogenesis in the drywood termite Neotermes koshunensis. Although the eggs produced by females isolated from males hatched, the hatching rate of those eggs was lower than that of the eggs produced by females kept with males. These parthenogenetic offspring inherited only the maternal alleles and showed high homozygosity, which indicates that the mechanism of ploidy restoration is terminal fusion. A previous study showed that most colonies of this species have a single queen or orphan; thus, the AQS system has not evolved despite their parthenogenetic ability. Further investigations of N. koshunensis will reveal how parthenogenesis evolved and its role in the insect societies.

Facultative parthenogenesis in the burrowing mayfly, Ephoron eophilum (Ephemeroptera: Polymitarcyidae) with an extremely short alate stage

Facultative parthenogenesis is important for mayflies with short alate stages because females are able to reproduce without mating. We studied facultative parthenogenesis in Ephoron eophilum, a mayfly with an extremely short alate stage. We examined the survival rates of embryos from unfertilized eggs, in addition to investigating the number of chromosomes in parthenogenetic offspring and the mode of inheritance by nuclear genetic analyses using Exon-Primed Intron-Crossing markers. The survival rate of thelytokous embryos was 0-70.2% (16.7 ± 26.7%, mean ± S.D.). Sixteen chromosomes were present throughout most of the mitotic metaphase in parthenogenetic offspring, which was similar to the number recorded in diploid females. All parthenogenetic offspring were homozygous in nuclear genetic analyses, despite the presence of heterozygous mothers. These results indicate that E. eophilum has the ability to reproduce via facultative parthenogenesis, producing mostly diploid thelytokous offspring. The restoration of ploidy level occurs by automixis via terminal fusion or gamete duplication, and causes rapid reduction of heterozygosity. However, despite this, significant deviation from Hardy-Weinberg equilibrium (HWE) was not observed in the studied populations. This is because facultative parthenogenesis in these circumstances normally has little influence on population genetic structuring, even though parthenogenetic embryos exhibit a high survival rate. The lack of influence of parthenogenesis on the population structure of the natural population strongly suggests that parthenogenesis rarely occurs under natural circumstances.

The persistence of facultative parthenogenesis in Drosophila albomicans.

Parthenogenesis has evolved independently in more than 10 Drosophila species. Most cases are tychoparthenogenesis, which is occasional or accidental parthenogenesis in normally bisexual species with a low hatching rate of eggs produced by virgin females; this form is presumed to be an early stage of parthenogenesis. To address how parthenogenesis and sexual reproduction coexist in Drosophila populations, we investigated several reproductive traits, including the fertility, parthenogenetic capability, diploidization mechanisms, and mating propensity of parthenogenetic D. albomicans. The fertility of mated parthenogenetic females was significantly higher than that of virgin females. The mated females could still produce parthenogenetic offspring but predominantly produced offspring by sexual reproduction. Both mated parthenogenetic females and their parthenogenetic-sexual descendants were capable of parthenogenesis. The alleles responsible for parthenogenesis can be propagated through both parthenogenesis and sexual reproduction. As diploidy is restored predominantly by gamete duplication, heterozygosity would be very low in parthenogenetic individuals. Hence, genetic variation in parthenogenetic genomes would result from sexual reproduction. The mating propensity of females after more than 20 years of isolation from males was decreased. If mutations reducing mating propensities could occur under male-limited conditions in natural populations, decreased mating propensity might accelerate tychoparthenogenesis through a positive feedback mechanism. This process provides an opportunity for the evolution of obligate parthenogenesis. Therefore, the persistence of facultative parthenogenesis may be an adaptive reproductive strategy in Drosophila when a few founders colonize a new niche or when small populations are distributed at the edge of a species' range, consistent with models of geographical parthenogenesis.

Termite queens close the sperm gates of eggs to switch from sexual to asexual reproduction

Males and females are in conflict over genetic transmission in the evolution of parthenogenesis, because it enhances female reproductive output but deprives the males' genetic contribution. For males, any trait that coerces females into sexual reproduction should increase their fitness. However, in the termite Reticulitermes speratus, queens produce their replacements (neotenic queens) parthenogenetically while using normal sexual reproduction to produce other colony members. Here, we show that termite queens produce parthenogenetic offspring in the presence of kings by closing the micropyles (sperm gates; i.e., openings for sperm entry) of their eggs. Our field survey showed that termite eggs show large variation in numbers of micropyles, with some having none. Microsatellite analysis showed that embryos of micropyleless eggs develop parthenogenetically, whereas those of eggs with micropyles are fertilized and develop sexually. Surveys of eggs among queens of different age groups showed that queens begin to lay micropyleless eggs when they are older and thus, need to produce their replacements parthenogenetically. In addition, we found clear seasonality in new neotenic queen differentiation and micropyleless egg production. This micropyle-dependent parthenogenesis is the first identification, to our knowledge, of the mechanism through which females control egg fertilization over time in diploid animals, implying a novel route of the evolution of parthenogenesis in favor of female interests without interference from males.

Laboratory generation of new parthenogenetic lineages supports contagious parthenogenesis in Artemia.

Contagious parthenogenesis—a process involving rare functional males produced by a parthenogenetic lineage which mate with coexisting sexual females resulting in fertile parthenogenetic offspring—is one of the most striking mechanisms responsible for the generation of new parthenogenetic lineages. Populations of the parthenogenetic diploid brine shrimp Artemia produce fully functional males in low proportions. The evolutionary role of these so-called Artemia rare males is, however, unknown. Here we investigate whether new parthenogenetic clones could be obtained in the laboratory by mating these rare males with sexual females. We assessed the survival and sex ratio of the hybrid ovoviviparous offspring from previous crosses between rare males and females from all Asiatic sexual species, carried out cross-mating experiments between F1 hybrid individuals to assess their fertility, and estimated the viability and the reproductive mode of the resulting F2 offspring. Molecular analysis confirmed the parentage of hybrid parthenogenetic F2. Our study documents the first laboratory synthesis of new parthenogenetic lineages in Artemia and supports a model for the contagious spread of parthenogenesis. Our results suggest recessive inheritance but further experiments are required to confirm the likelihood of the contagious parthenogenesis model.

Editorial 2012

Editorial 2012

Climate effects on life cycle variation and population genetic architecture of the black bean aphid, Aphis fabae

Aphid species may exhibit different reproductive modes ranging from cyclical to obligate parthenogenesis. The distribution of life cycle variation in aphids is generally determined by ecological forces, mainly climate, because only sexually produced diapausing eggs can survive harsh winters or periods of absence of suitable host plants. Aphids are thus interesting models to investigate intrinsic and environmental factors shaping the competition among sexual and asexual lineages. We conducted a Europe-wide sampling of black bean aphids, Aphis fabae, and combined population genetic analyses based on microsatellite data with an experimental determination of life cycle strategies. Aphids were collected from broad beans (Vicia faba) as well as some Chenopodiaceae, but we detected no genetic differentiation between aphids from different host plants. Consistent with model predictions, life cycle variation was related to climate, with aphids from areas with cold winters investing more in sexual reproduction than aphids from areas with mild winters. Accordingly, only populations from mild areas exhibited a clear genetic signature of clonal reproduction. These differences arise despite substantial gene flow over large distances, which was evident from a very low geographic population structure and a lack of isolation-by-distance among 18 sites across distances of more than 1000 km. There was virtually no genetic differentiation between aphids with different reproductive modes, suggesting that new asexual lineages are formed continuously. Indeed, a surprising number of A. fabae genotypes even from colder climates produced some parthenogenetic offspring under simulated winter conditions. From this we predict that a shift to predominantly asexual reproduction could take place rapidly under climate warming.

Ant queens adjust egg fertilization to benefit from both sexual and asexual reproduction

An enduring problem in evolutionary biology is the near ubiquity of sexual reproduction despite the inherent cost of transmitting only half the parent's genes to progeny. Queens of some ant species circumvent this cost by using selectively both sexual reproduction and parthenogenesis: workers arise from fertilized eggs, while new queens are produced by parthenogenesis. We show that queens of the ant Cataglyphis cursor maximize the transmission rate of their genes by regulating the proportion of fertilized and parthenogenetic eggs laid over time. Parthenogenetic offspring are produced in early spring, when workers raise the brood into sexuals. After the mating period, queens lay mostly fertilized eggs that will be reared as the non-reproductive caste.

A major shift in Daphnia genetic structure after the first ice‐free winter in a German reservoir

Summary1. Climate warming may cause disruption of trophic linkages in aquatic ecosystems and lead to changes in abundance and genetic structure of zooplankton populations. We monitored the community of the Daphnia galeata‐hyalina hybrid complex in the Saidenbach Reservoir (Saxony, Germany) using allozyme electrophoresis for three consecutive years (2005–07), including one (2007) following an unusually warm winter that prevented the formation of ice cover for the first time in the history of the reservoir.2. Genetic composition during the 2007 season differed substantially from the two preceding years that experienced the usual 3‐month ice period. Three abundance peaks in June, July and October 2007 were dominated by hybrids of Daphnia galeata x hyalina, whereas in the 2005 and 2006 seasons two peaks in June and September were dominated by Daphnia hyalina genotypes.3. The genetic composition of the pool of diapausing eggs produced in autumn and the rate of change of genotype abundance during the following spring indicate recruitment of the D. hyalina subpopulation from ex‐ephippial animals during the spring population increase.4. The differing potential to contribute to the overwintering animal pool or to the inoculum from diapausing eggs was confirmed by results from laboratory life‐table experiments. Daphnia galeata clones survived longer and produced parthenogenetic offspring under winter conditions, whereas D. hyalina clones showed a shorter lifespan and produced resting eggs.5. Our results indicate a profound role of recruitment strategy in the observed shift in genetic composition. Increasing winter temperatures predicted in the context of climate change may thus favour overwintering animals, leading to an increase in the contribution of these genotypes to the population. Such microevolutionary processes may dampen possible seasonal mismatches between daphnid populations and their food or predator populations.

Allelic inheritance in naturally occurring parthenogenetic offspring of the gonochoric sea star Patiria miniata

Abstract. In laboratory studies of fertilization using the gonochoric broadcast‐spawning asterinid sea star Patiria miniata, we found many cases in which some mature eggs spawned by females formed cleavage‐stage embryos and feeding bipinnaria larvae without fertilization by sperm. Segregation of maternal microsatellite alleles among the parthenogenetic offspring of known heterozygous females was consistent with several specific modes of asexual reproduction, including polar body suppression. Cryptic outcrossing by sperm contamination was ruled out by the failure to observe non‐maternal alleles. The potential for asexual reproduction by the normally outcrossing members of P. miniata may suggest a shared propensity for asexuality among asterinid species from several clades in which isolated adults can produce offspring without outcrossing.