Causes et conséquences évolutives de l’asexualité non-clonale chez Artemia

The majority of parthenogenetic species are often thought to be clonal. Clonality is costly in the long term, as it can result in accumulation of deleterious mutations and lower adaptability. However, cases reporting non-clonal asexuals are accumulating. Non-clonal asexuality has very different genomic and fitness consequences compared to clonality, and may be a key intermediate step in the transition from sex to asexuality. Additionally, asexuality may be often non-obligate, with events of cryptic sex. These events may also shape the genome and evolution of asexual lineages. In this PhD, I investigated the reproductive mode of Artemia parthenogenetica and its role in the transition from sex to asexuality and the evolution of asexual lineages. Specifically, I used the capacity of asexually produced males (“rare males”) to cross with sexual females and transmit asexuality to their offspring (contagious asexuality), to experimentally generate new lineages. I showed that diploid asexual Artemia have a non-clonal reproductive mode, in which recombination results in loss of heterozygosity (LOH) in the offspring. LOH is costly as it can reveal recessive deleterious mutations. Perhaps due to selection caused by the deleterious consequences of LOH, the recombination rate in these asexuals was lower than in a closely related sexual species. I also found that sex-asex hybrids had a mixed sexual and asexual reproduction, and that asexual females from natural populations were capable of rare sex. This means that rare events of sex in asexual Artemia could occur between a rare male and an asexual female reproducing sexually. In a review of how asexual reproductive modes were identified in the literature, I found that there was a bias in the identification and general perception of asexuals toward clonality, as an important part of the asexual species reviewed were in fact non-clonal, and evidence for clonality was often missing. Furthermore, the maj ority of non-clonal asexuals had reproductive modes that resulted in low LOH. This suggests that non-clonal asexuals often evolve secondarily toward a more clonal-like reproduction, so that even clonal species may not have been clonal throughout their evolutionary history. Finally, using genomics on contagion-generated lineages, I found that in Artemia, rare males are produced asexually through recombination and thus LOH on the ZW sex chromosomes. We know that contagious asexuality, and possibly between-lineages crosses, occurred in the evolutionary history of A. parthenogenetica. Perhaps, contagious asexuality and/or within asexual sex events provide opportunities for the gene(s) controlling asexuality to escape declining lineages into new ones. In this case, contagious asexuality through rare males may be the reason why recombination persists in asexual Artemia. Whether non-clonal asexuality and sex events occur in many parthenogenetic species is still unclear, and requires thorou gh investigation. Theoretically, there is a strong need for models taking into account the genomic consequences of non-clonal and non-obligate asexuality, and their role in the transition from sex to asexuality and the maintenance of sex.La majorité des espèces parthénogétiques sont souvent perçues comme clonales. La clonalité est coûteuse à long terme, car elle peut entraîner l'accumulation de mutations délétères et une moins bonne capacité d’adaptation. Cependant, les cas d’espèces asexuées non clonales s'accumulent. L’asexualité non-clonale génère des conséquences génomiques et de fitness très différentes de la clonalité, et pourraient représenter une étape-clé dans la transition du sexe vers l’asexualité. De plus, l’asexualité peut être souvent non-obligatoire, avec des événements de sexe cryptiques. Ces évènements peuvent aussi façonner le génome et l'évolution des lignées asexuées. Dans cette thèse, j'ai étudié le mode de reproduction d'Artemia parthenogenetica, et son rôle dans la transition du sexe vers l'asexualité et l'évolution des lignées asexuées. En particulier, j'ai utilisé la capacité des mâles produits par voie asexuée (“mâles rares”) à se croiser avec des femelles sexuées et à transmettre l’asexualité à leurs descendants (asexualité contagieuse), pour générer expérimentalement de nouvelles lignées. J’ai montré que les Artemia asexués diploïdes ont un mode de reproduction non-clonal, dans lequel la recombinaison entraîne une perte d'hétérozygotie (LOH, pour “loss of heterozygosity”) chez les descendants. Le LOH est coûteux car il peut révéler des mutations délétères récessives. Peut-être en raison de la sélection causée par les conséquences délétères du LOH, le taux de recombinaison chez les Artemia asexués était plus faible que chez une espèce sexuée apparentée. J'ai également constaté que les hybrides sexués avaient une reproduction mixte sexuée et asexuée, et que les femelles asexuées issues de populations naturelles étaient capables de sexe rare. Cela signifie que des événements rares de sexe chez les Artemia asexués pourraient se produire entre un mâle rare et une femelle asexuée se reproduisant sexuellement. En effectuant une revue de la façon don t les modes de reproduction asexués sont identifiés dans la littérature, j'ai constaté que l'identification et la perception générale des asexués étaient biaisées en faveur de la clonalité, car une grande partie des espèces asexuées examinées étaient en fait non-clonales, et les preuves de la clonalité étaient souvent insuffisantes. En outre, la majorité des asexués non-clonaux avaient des modes de reproduction qui entraînaient de faibles taux de LOH. Cela suggère que les asexués non-clonaux évoluent souvent secondairement vers une reproduction plus clonale. Ainsi, même les espèces clonales pourraient ne pas avoir été clonales au cours de leur histoire évolutive. Enfin, avec une analyse génomique sur de nouvelles lignées générées par contagion, j'ai démontré que chez Artemia, les mâles rares sont produits asexuellement par recombinaison et donc LOH sur les chromosomes sexuels ZW. Nous savons que l'asexualité contagieuse, et peut-être des croisements entre lignées, ont eu lieu au cou rs de l'histoire évolutive d'A. parthenogenetica. L'asexualité contagieuse et/ou des événements sexuels chez les asexués constituent peut-être des opportunités pour que le(s) gène(s) contrôlant l'asexualité s'échappe(nt) des lignées en déclin vers de nouvelles lignées. Dans ce cas, l'asexualité contagieuse par le biais de mâles rares pourrait être la raison pour laquelle la recombinaison persiste chez les Artemia asexués. Chez de nombreuses espèces, l’identification de l’asexualité non clonale et des événements de sexe n'est toujours pas claire et nécessite une étude approfondie. Théoriquement, il y a un fort besoin de modèles prenant en compte les conséquences génomiques de l'asexualité non-clonale et non-obligatoire, et leur rôle dans la transition du sexe vers l'asexualité et la maintenance du sexe

Causes et conséquences évolutives de l’asexualité non-clonale chez Artemia

The majority of parthenogenetic species are often thought to be clonal. Clonality is costly in the long term, as it can result in accumulation of deleterious mutations and lower adaptability. However, cases reporting non-clonal asexuals are accumulating. Non-clonal asexuality has very different genomic and fitness consequences compared to clonality, and may be a key intermediate step in the transition from sex to asexuality. Additionally, asexuality may be often non-obligate, with events of cryptic sex. These events may also shape the genome and evolution of asexual lineages. In this PhD, I investigated the reproductive mode of Artemia parthenogenetica and its role in the transition from sex to asexuality and the evolution of asexual lineages. Specifically, I used the capacity of asexually produced males (“rare males”) to cross with sexual females and transmit asexuality to their offspring (contagious asexuality), to experimentally generate new lineages. I showed that diploid asexual Artemia have a non-clonal reproductive mode, in which recombination results in loss of heterozygosity (LOH) in the offspring. LOH is costly as it can reveal recessive deleterious mutations. Perhaps due to selection caused by the deleterious consequences of LOH, the recombination rate in these asexuals was lower than in a closely related sexual species. I also found that sex-asex hybrids had a mixed sexual and asexual reproduction, and that asexual females from natural populations were capable of rare sex. This means that rare events of sex in asexual Artemia could occur between a rare male and an asexual female reproducing sexually. In a review of how asexual reproductive modes were identified in the literature, I found that there was a bias in the identification and general perception of asexuals toward clonality, as an important part of the asexual species reviewed were in fact non-clonal, and evidence for clonality was often missing. Furthermore, the maj ority of non-clonal asexuals had reproductive modes that resulted in low LOH. This suggests that non-clonal asexuals often evolve secondarily toward a more clonal-like reproduction, so that even clonal species may not have been clonal throughout their evolutionary history. Finally, using genomics on contagion-generated lineages, I found that in Artemia, rare males are produced asexually through recombination and thus LOH on the ZW sex chromosomes. We know that contagious asexuality, and possibly between-lineages crosses, occurred in the evolutionary history of A. parthenogenetica. Perhaps, contagious asexuality and/or within asexual sex events provide opportunities for the gene(s) controlling asexuality to escape declining lineages into new ones. In this case, contagious asexuality through rare males may be the reason why recombination persists in asexual Artemia. Whether non-clonal asexuality and sex events occur in many parthenogenetic species is still unclear, and requires thorou gh investigation. Theoretically, there is a strong need for models taking into account the genomic consequences of non-clonal and non-obligate asexuality, and their role in the transition from sex to asexuality and the maintenance of sex.La majorité des espèces parthénogétiques sont souvent perçues comme clonales. La clonalité est coûteuse à long terme, car elle peut entraîner l'accumulation de mutations délétères et une moins bonne capacité d’adaptation. Cependant, les cas d’espèces asexuées non clonales s'accumulent. L’asexualité non-clonale génère des conséquences génomiques et de fitness très différentes de la clonalité, et pourraient représenter une étape-clé dans la transition du sexe vers l’asexualité. De plus, l’asexualité peut être souvent non-obligatoire, avec des événements de sexe cryptiques. Ces évènements peuvent aussi façonner le génome et l'évolution des lignées asexuées. Dans cette thèse, j'ai étudié le mode de reproduction d'Artemia parthenogenetica, et son rôle dans la transition du sexe vers l'asexualité et l'évolution des lignées asexuées. En particulier, j'ai utilisé la capacité des mâles produits par voie asexuée (“mâles rares”) à se croiser avec des femelles sexuées et à transmettre l’asexualité à leurs descendants (asexualité contagieuse), pour générer expérimentalement de nouvelles lignées. J’ai montré que les Artemia asexués diploïdes ont un mode de reproduction non-clonal, dans lequel la recombinaison entraîne une perte d'hétérozygotie (LOH, pour “loss of heterozygosity”) chez les descendants. Le LOH est coûteux car il peut révéler des mutations délétères récessives. Peut-être en raison de la sélection causée par les conséquences délétères du LOH, le taux de recombinaison chez les Artemia asexués était plus faible que chez une espèce sexuée apparentée. J'ai également constaté que les hybrides sexués avaient une reproduction mixte sexuée et asexuée, et que les femelles asexuées issues de populations naturelles étaient capables de sexe rare. Cela signifie que des événements rares de sexe chez les Artemia asexués pourraient se produire entre un mâle rare et une femelle asexuée se reproduisant sexuellement. En effectuant une revue de la façon don t les modes de reproduction asexués sont identifiés dans la littérature, j'ai constaté que l'identification et la perception générale des asexués étaient biaisées en faveur de la clonalité, car une grande partie des espèces asexuées examinées étaient en fait non-clonales, et les preuves de la clonalité étaient souvent insuffisantes. En outre, la majorité des asexués non-clonaux avaient des modes de reproduction qui entraînaient de faibles taux de LOH. Cela suggère que les asexués non-clonaux évoluent souvent secondairement vers une reproduction plus clonale. Ainsi, même les espèces clonales pourraient ne pas avoir été clonales au cours de leur histoire évolutive. Enfin, avec une analyse génomique sur de nouvelles lignées générées par contagion, j'ai démontré que chez Artemia, les mâles rares sont produits asexuellement par recombinaison et donc LOH sur les chromosomes sexuels ZW. Nous savons que l'asexualité contagieuse, et peut-être des croisements entre lignées, ont eu lieu au cou rs de l'histoire évolutive d'A. parthenogenetica. L'asexualité contagieuse et/ou des événements sexuels chez les asexués constituent peut-être des opportunités pour que le(s) gène(s) contrôlant l'asexualité s'échappe(nt) des lignées en déclin vers de nouvelles lignées. Dans ce cas, l'asexualité contagieuse par le biais de mâles rares pourrait être la raison pour laquelle la recombinaison persiste chez les Artemia asexués. Chez de nombreuses espèces, l’identification de l’asexualité non clonale et des événements de sexe n'est toujours pas claire et nécessite une étude approfondie. Théoriquement, il y a un fort besoin de modèles prenant en compte les conséquences génomiques de l'asexualité non-clonale et non-obligatoire, et leur rôle dans la transition du sexe vers l'asexualité et la maintenance du sexe

Not so clonal asexuals: Unraveling the secret sex life of Artemia parthenogenetica

International audienceThe maintenance of sex is paradoxical as sexual species pay the “twofold cost of males” and should thus quickly be replaced by asexual mutants reproducing clonally. However, asexuals may not be strictly clonal and engage in “cryptic sex,” challenging this simple scenario. We study the cryptic sex life of the brine shrimp Artemia parthenogenetica, which has once been termed an “ancient asexual” and where no genetic differences have ever been observed between parents and offspring. This asexual species rarely produces males, which can hybridize with sexual females of closely related species and transmit asexuality to their offspring. Using such hybrids, we show that recombination occurs in asexual lineages, causing loss-of-heterozygosity and parent-offspring differences. These differences cannot generally be observed in field-sampled asexuals because once heterozygosity is lost, subsequent recombination leaves no footprint. Furthermore, using extensive paternity tests, we show that hybrid females can reproduce both sexually and asexually, and transmit asexuality to both sexually and asexually produced offspring in a dominant fashion. Finally, we show that, contrary to previous reports, field-sampled asexual females also rarely reproduce sexually (rate ∼2‰). Overall, most previously known facts about Artemia asexuality turned out to be erroneous. More generally, our findings suggest that the evidence for strictly clonal reproduction of asexual species needs to be reconsidered, and that rare sex and consequences of nonclonal asexuality, such as gene flow within asexuals, need to be more widely taken into account in more realistic models for the maintenance of sex and the persistence of asexual lineages

Asexual male production by ZW recombination in Artemia parthenogenetica

In some asexual species, parthenogenetic females occasionally produce males, which may strongly affect the evolution and maintenance of asexuality if they cross with related sexuals and transmit genes causing asexuality to their offspring (“contagious parthenogenesis”). How these males arise in the first place has remained enigmatic, especially in species with sex chromosomes. Here, we test the hypothesis that rare, asexually produced males of the crustacean Artemia parthenogenetica are produced by recombination between the Z and W sex chromosomes during non-clonal parthenogenesis, resulting in ZZ males through loss of heterozygosity at the sex determination locus. We used RAD-sequencing to compare asexual mothers with their male and female offspring. Markers on several sex-chromosome scaffolds indeed lost heterozygosity in all male but no female offspring, suggesting that they correspond to the sex-determining region. Other sex-chromosome scaffolds lost heterozygosity in only a part of the male offspring, consistent with recombination occurring at a variable location. Alternative hypotheses for the production of these males (such as partial or total hemizygosity of the Z) could be excluded. Rare males are thus produced because recombination is not entirely suppressed during parthenogenesis in A. parthenogenetica . This finding may contribute to explaining the maintenance of recombination in these asexuals

The origin of asexual brine shrimps

Determining how and how often asexual lineages emerge within sexual species is central to our understanding of sex-asex transitions and the long-term maintenance of sex. Asexuality can arise “by transmission” from an existing asexual lineage to a new one, through different types of crosses. The occurrence of these crosses, cryptic sex, variation in ploidy and recombination within asexuals greatly complicates the study of sex-asex transitions, as they preclude the use of standard phylogenetic methods and genetic distance metrics. In this study we show how to overcome these challenges by developing new approaches to investigate the origin of the various asexual lineages of the brine shrimp Artemia parthenogenetica. We use a large sample of asexuals, including all known polyploids, and their sexual relatives. We combine flow cytometry with mitochondrial and nuclear DNA data. We develop new genetic distance measures and methods to compare various scenarios describing the origin of the different lineages. We find that all diploid and polyploid A. parthenogenetica likely arose within the last 80,000 years through successive and nested hybridization events that involved backcrosses with different sexual species. All A. parthenogenetica have the same common ancestor and therefore likely carry the same asexuality gene(s) and reproduce by automixis. These findings radically change our view of sex-asex transitions in this group, and show the importance of considering asexuality “by transmission” scenarios. The methods developed are applicable to many other asexual taxa.N

The origins of asexual brine shrimps

[Methods] Flow cytometry of 147 individuals (+59 individuals from Nougué et al. 2015) COI sequencing of 336 individuals using primers 1/2COI_Fol-F and 1/2COI_Fol-R following the protocol of Muñoz et al. (2010). COI sequencing of 23 individuals using primers Co1APAR-F(5’-259 TTTGGAGCTTGAGCAGGAAT-3’) and Co1APAR-R(5’-260 TGCGGGATCAAAGAAAGAAG-3’). Genotyping of 432 individuals with a panel of 12 microsatellite markers (see Muñoz et al. 2008; Nougué et al. 2015 for details regarding markers and amplification protocol) [Usage Notes] See README files.Determining how and how often asexual lineages emerge within sexual species is central to our understanding of sex-asex transitions and the long-term maintenance of sex. Asexuality can arise “by transmission” from an existing asexual lineage to a new one, through different types of crosses. The occurrence of these crosses, cryptic sex, variation in ploidy and recombination within asexuals greatly complicates the study of sex-asex transitions, as they preclude the use of standard phylogenetic methods and genetic distance metrics. In this study we show how to overcome these challenges by developing new approaches to investigate the origin of the various asexual lineages of the brine shrimp Artemia parthenogenetica. We use a large sample of asexuals, including all known polyploids, and their sexual relatives. We combine flow cytometry with mitochondrial and nuclear DNA data. We develop new genetic distance measures and methods to compare various scenarios describing the origin of the different lineages. We find that all diploid and polyploid A. parthenogenetica likely arose within the last 80,000 years through successive and nested hybridization events that involved backcrosses with different sexual species. All A. parthenogenetica have the same common ancestor and therefore likely carry the same asexuality gene(s) and reproduce by automixis. These findings radically change our view of sex-asex transitions in this group, and show the importance of considering asexuality “by transmission” scenarios. The methods developed are applicable to many other asexual taxa.Peer reviewe

The origins of asexual brine shrimps

Determining how and how often asexual lineages emerge within sexual species is central to our understanding of sex-asex transitions and the long-term maintenance of sex. Asexuality can arise "by transmission" from an existing asexual lineage to a new one, through different types of crosses. The occurrence of these crosses, cryptic sex, variation in ploidy and recombination within asexuals greatly complicates the study of sex-asex transitions, as they preclude the use of standard phylogenetic methods and genetic distance metrics. In this study we show how to overcome these challenges by developing new approaches to investigate the origin of the various asexual lineages of the brine shrimp Artemia parthenogenetica. We use a large sample of asexuals, including all known polyploids, and their sexual relatives. We combine flow cytometry with mitochondrial and nuclear DNA data. We develop new genetic distance measures and methods to compare various scenarios describing the origin of the different lineages. We find that all diploid and polyploid A. parthenogenetica likely arose within the last 80,000 years through successive and nested hybridization events that involved backcrosses with different sexual species. All A. parthenogenetica have the same common ancestor and therefore likely carry the same asexuality gene(s) and reproduce by automixis. These findings radically change our view of sex-asex transitions in this group, and show the importance of considering asexuality "by transmission" scenarios. The methods developed are applicable to many other asexual taxa.Peer reviewe

The Origin of Asexual Brine Shrimps

Determining how and how often asexual lineages emerge within sexual species is central to our understanding of sex-asex transitions and the long-term maintenance of sex. Asexuality can arise “by transmission” from an existing asexual lineage to a new one through different types of crosses. The occurrence of these crosses, cryptic sex, variations in ploidy, and recombination within asexuals greatly complicates the study of sex-asex transitions, as they preclude the use of standard phylogenetic methods and genetic distance metrics. In this study we show how to overcome these challenges by developing new approaches to investigate the origin of the various asexual lineages of the brine shrimp Artemia parthenogenetica. We use a large sample of asexuals, including all known polyploids, and their sexual relatives. We combine flow cytometry with mitochondrial and nuclear DNA data. We develop new genetic distance measures and methods to compare various scenarios describing the origin of the different lineages. We find that all diploid and polyploid A. parthenogenetica likely arose within the past 80,000 years through successive and nested hybridization events that involved backcrosses with different sexual species. All A. parthenogenetica have the same common ancestor and therefore likely carry the same asexuality gene(s) and reproduce by automixis. These findings radically change our view of sex-asex transitions in this group and show the importance of considering scenarios of asexuality by transmission. The methods developed are applicable to many other asexual taxa.Peer reviewe