Considerations for fatty acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

Fatty acid accumulation was studied in the parthenogenetic all-female marbled crayfish Procambarus virginalis using six arbitrarily designed experimental feeds and related to individuals with glair glands (sexual maturity) after 100 days of ad libitum feeding at 21 °C, including gravid females from the wild as a reference. Fatty acids 16:0 and 18:1n-9 comprised 40% of the total amount of fatty acids and tended to up-concentrate in bodies. Shorter chain 14:0 depleted from feed to body. Across diets, there was a concomitant decrease in precursor fatty acid and increase in product fatty acid, such as reinforcements in monounsaturated fatty acid (18:1n-9), eicosanoid precursors 20:4n-6 (arachidonic acid, ARA) and 20:5n-3 (eicosapentaenoic acid, EPA) in-vivo, but not 22:6n-3 (docosahexaenoic acid, DHA) except when deficient in CHI or CHI + SPI diets. Saturation kinetics modeling (R2 0.7-0.9, p < 0.05) showed that when the ARA share is ~ 1%, the EPA share is ~ 8%, and the DHA share is ~ 2% in the food lipids, the accumulation of fatty acids in body lipids levels off. The lowest DHA in the CHI (0% glair glands) or CHI + SPI (0-3.9% glair glands) diets, and the lowest ARA in SER (0% glair glands) or SER + SPI (0-3% glair glands) diets, were synchronous with negligible sexual maturity despite a wide range of observed specific growth rates (2.77-3.60% per day), body size (0.44-0.84 g), ≤ 5% crude lipid and 40-46% crude protein feed. The FISH and SHRIMP diets (56% protein, 11-14% lipid) with the highest ARA, EPA, and DHA together seem to be the most conducive diets for sexual maturity (up to 20% of individuals with glair glands). We propose a fatty acid profile mimicking the FISH or SHRIMP diets as a starting point for designing the lipid content required in the marbled crayfish standardized reference diet.

Maternal inheritance of functional centrioles in two parthenogenetic nematodes.

Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species.

Considerations for protein and amino acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

The concept of a standardized reference diet (SRD) is used in laboratory model organisms to ensure nutritional control between studies and laboratories. Although models using the genetically identical, all female parthenogenetic marbled crayfish (Procambarus virginalis) are growing in popularity, research into nutrition in this species still has many knowledge gaps. To fast track the development of a SRD in terms of protein and amino acids (SRDprotein) for this species, we first analyzed the composition of its body amino acids to determine the ideal protein concept (IPC) of indispensable amino acids in wild-caught P. virginalis (which had an unusually high preponderance of leucine and arginine). Then, we strategically evaluated three common clusters of types of fish feed: (1) ornamental fish feed (SER) fortified with a naturally occurring alga (Spirulina). This type of feed was protein-high in arginine and leucine (SER + SPI) that fulfils the species' IPC for iso-protein (~ 40%), iso-phosphorus (~ 0.8%) and near iso-energetic (~ 475 kcal 100 g-1); (2) freeze-dried live feed consisting of chironomid larvae (CHI) fortified with Spirulina (CHI + SPI) that fulfils the IPC for iso-protein (~ 46%), iso-phosphorus (~ 0.7%) and near iso-energetic (~ 405 kcal 100 g-1); and (3) a commercially standardized 'starter diet' for carnivorous fish larvae (FISH) and post-larval shrimps (SHRIMP) with iso-protein (~ 56%) and iso-phosphorus (~ 1.6%). A total of six diets, embracing a diverse range of proteinaceous feeds, were used in a 100-day ad libitum feeding and growth trial. The FISH group outperformed all the other groups (p < 0.05) and our exploratory multivariate analysis revealed an ideal demand of > 44% protein (tailored to deliver high arginine 3% and leucine 4%, followed by the usual lysine > 3.5% and methionine 1.2%) but also the lowest carbohydrate level (21%). For SRDprotein, our findings show that the FISH diet is ideal and suggest the possibilities of using a CHI + SPI diet for further optimization (more economic use of protein and phosphorus).

Effects of bisphenol A and tauroursodeoxycholic acid on maturation of porcine oocytes and parthenogenetic development of embryos.

Prolonged exposure of bisphenol A (BPA) has adverse effects on in vitro maturation (IVM) of oocytes, but treatment with tauroursodeoxycholic acid (TUDCA) can improve the IVM and development of embryos. The purpose of this study was to investigate the effects of BPA and both BPA and TUDCA on IVM and parthenogenetic development of embryos. The results showed that BPA treatment adverse effects on the cumulus expansion index, survival rate, polar body rate, mitochondrial distribution of the oocytes after maturation culture, and that it also decreased the cleavage rate and blastocyst rate of embryos after parthenogenetic develpoment. In addition, BPA treatment upregulated expression of genes related to endoplasmic reticulum stress and apoptosis and increased the intracellular reactive oxygen species (ROS) level, while it decreased expression of genes related to cumulus expansion. However, the supplementation of TUDCA relieved these adverse effects of BPA except polar body rate, blastocyst rate, and expression of BCL2 and PTGS1. In conclusion, the supplementation of TUDCA can partly attenuate the negative effects of BPA on IVM and parthenogenetic development of embryos, possibly by modification of the expression of genes related to endoplasmic reticulum stress, apoptosis and cumulus expansion, intracellular ROS level, and mitochondrial distribution.

Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR.

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.

Post-meiotic mechanism of facultative parthenogenesis in gonochoristic whiptail lizard species.

Facultative parthenogenesis (FP) has historically been regarded as rare in vertebrates, but in recent years incidences have been reported in a growing list of fish, reptile, and bird species. Despite the increasing interest in the phenomenon, the underlying mechanism and evolutionary implications have remained unclear. A common finding across many incidences of FP is either a high degree of homozygosity at microsatellite loci or low levels of heterozygosity detected in next-generation sequencing data. This has led to the proposal that second polar body fusion following the meiotic divisions restores diploidy and thereby mimics fertilization. Here, we show that FP occurring in the gonochoristic Aspidoscelis species A. marmoratus and A. arizonae results in genome-wide homozygosity, an observation inconsistent with polar body fusion as the underlying mechanism of restoration. Instead, a high-quality reference genome for A. marmoratus and analysis of whole-genome sequencing from multiple FP and control animals reveals that a post-meiotic mechanism gives rise to homozygous animals from haploid, unfertilized oocytes. Contrary to the widely held belief that females need to be isolated from males to undergo FP, females housed with conspecific and heterospecific males produced unfertilized eggs that underwent spontaneous development. In addition, offspring arising from both fertilized eggs and parthenogenetic development were observed to arise from a single clutch. Strikingly, our data support a mechanism for facultative parthenogenesis that removes all heterozygosity in a single generation. Complete homozygosity exposes the genetic load and explains the high rate of congenital malformations and embryonic mortality associated with FP in many species. Conversely, for animals that develop normally, FP could potentially exert strong purifying selection as all lethal recessive alleles are purged in a single generation.

Reproductive manipulation: Wolbachia induce host parthenogenesis using a stolen transformer.

The Wolbachia strain that infects the parasitoid wasp Encarsia formosa induces female-producing parthenogenesis. A new study shows that a Wolbachia-encoded gene has replaced the use of the ancestral wasp homologue that normally controls sexual reproduction, resulting in parthenogenesis.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes.

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Wolbachia symbionts control sex in a parasitoid wasp using a horizontally acquired gene.

Host reproduction can be manipulated by bacterial symbionts in various ways. Parthenogenesis induction is the most effective type of reproduction manipulation by symbionts for their transmission. Insect sex is determined by regulation of doublesex (dsx) splicing through transformer2 (tra2) and transformer (tra) interaction. Although parthenogenesis induction by symbionts has been studied since the 1970s, its underlying molecular mechanism is unknown. Here we identify a Wolbachia parthenogenesis-induction feminization factor gene (piff) that targets sex-determining genes and causes female-producing parthenogenesis in the haplodiploid parasitoid Encarsia formosa. We found that Wolbachia elimination repressed expression of female-specific dsx and enhanced expression of male-specific dsx, which led to the production of wasp haploid male offspring. Furthermore, we found that E. formosa tra is truncated and non-functional, and Wolbachia has a functional tra homolog, termed piff, with an insect origin. Wolbachia PIFF can colocalize and interact with wasp TRA2. Moreover, Wolbachia piff has coordinated expression with tra2 and dsx of E. formosa. Our results demonstrate the bacterial symbiont Wolbachia has acquired an insect gene to manipulate the host sex determination cascade and induce parthenogenesis in wasps. This study reveals insect-to-bacteria horizontal gene transfer drives the evolution of animal sex determination systems, elucidating a striking mechanism of insect-microbe symbiosis.

AtRKD5 inhibits the parthenogenic potential mediated by AtBBM.

Parthenogenesis, the development of unfertilized egg cells into embryos, is a key component of apomixis. AtBBM (BABY BOOM), a crucial regulator of embryogenesis in Arabidopsis, possesses the capacity to shift nutritional growth toward reproductive growth. However, the mechanisms underlying AtBBM-induced parthenogenesis remain largely unexplored in dicot plants. Our findings revealed that in order to uphold the order of sexual reproduction, the embryo-specific promoter activity of AtBBM as well as repressors that inhibit its expression in egg cells combine to limiting its ability to induce parthenogenesis. Notably, AtRKD5, a RWP-RK domain-containing (RKD) transcription factor, binds to the 3' end of AtBBM and is identified as one of the inhibitory factors for AtBBM expression in the egg cell. In the atrkd5 mutant, we successfully achieved enhanced ectopic expression of AtBBM in egg cells, resulting in the generation of haploid offspring via parthenogenesis at a rate of 0.28%. Furthermore, by introducing chimeric Arabidopsis and rice BBM genes into the egg cell, we achieved a significant 4.6-fold enhancement in haploid induction through the atdmp8/9 mutant. These findings lay a strong foundation for further exploration of the BBM-mediated parthenogenesis mechanism and the improvement of haploid breeding efficiency mediated by the dmp8/9 mutant.

Chlorogenic acid improves the development of porcine parthenogenetic embryos by regulating oxidative stress and ameliorating mitochondrial function.

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA-induced porcine parthenogenetic activation (PA) in early-stage embryos were investigated. Our results showed that 50 µM CGA treatment during the in vitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early-stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early-stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early-stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis-related gene NRF-1 in porcine PA early-stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early-stage embryos by regulating oxidative stress and improving mitochondrial function.

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.

Parthenogenetic Stick Insects Exhibit Signatures of Preservation in the Molecular Architecture of Male Reproduction.

After the loss of a trait, theory predicts that the molecular machinery underlying its phenotypic expression should decay. Yet, empirical evidence is contrasting. Here, we test the hypotheses that (i) the molecular ground plan of a lost trait could persist due to pleiotropic effects on other traits and (ii) that gene co-expression network architecture could constrain individual gene expression. Our testing ground has been the Bacillus stick insect species complex, which contains close relatives that are either bisexual or parthenogenetic. After the identification of genes expressed in male reproductive tissues in a bisexual species, we investigated their gene co-expression network structure in two parthenogenetic species. We found that gene co-expression within the male gonads was partially preserved in parthenogens. Furthermore, parthenogens did not show relaxed selection on genes upregulated in male gonads in the bisexual species. As these genes were mostly expressed in female gonads, this preservation could be driven by pleiotropic interactions and an ongoing role in female reproduction. Connectivity within the network also played a key role, with highly connected-and more pleiotropic-genes within male gonad also having a gonad-biased expression in parthenogens. Our findings provide novel insight into the mechanisms which could underlie the production of rare males in parthenogenetic lineages; more generally, they provide an example of the cryptic persistence of a lost trait molecular architecture, driven by gene pleiotropy on other traits and within their co-expression network.

Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Parthenogenesis, identity, and value.

Parthenogenesis is a form of asexual reproduction in which a gamete (ovum or sperm) develops without being fertilized. Tomer Jordi Chaffer uses parthenogenesis to challenge Don Marquis' future-like-ours (FLO) argument against abortion. According to Marquis, (1) what makes it morally wrong to kill us is that it would deprive us of a possible future that we might come to value-a future "like ours" (FLO) and (2) human fetuses are numerically identical to any adult human organism they may develop into, and thus have a FLO. Chaffer contends that if human ova are capable of parthenogenesis, then they would have a FLO, which contraception may deprive them of, but contends this is absurd. Bruce P. Blackshaw challenges Chaffer, contending sexually fertilized embryos are not identical to unfertilized ovum, but this would yield a more absurd implication, that fertilization deprives an ovum of a FLO! Here I show Marquis' account of identity rules out both Chaffer's and Blackshaw's accounts.

Parvatrema spp. (Digenea, Gymnophallidae) with parthenogenetic metacercariae: diversity, distribution and host specificity in the palaearctic.

There are several species of gymnophallid digeneans in the genus Parvatrema that are unique in developing metacercariae that reproduce by parthenogenesis in the second intermediate host. Transmission of these digeneans takes place in coastal ecosystems of the North Pacific and North Atlantic seas. The first intermediate hosts are bivalves, the second ones are gastropods, and the definitive hosts are migratory birds. We integrated data accumulated over 25 years of research and differentiated a complex of five closely related species. They differ in the molluscan second intermediate hosts, distribution ranges, and life cycles patterns. The type I life cycle includes two generations of parthenogenetic metacercariae, followed by development of metacercariae which are invasive for the definitive host. In the type II life cycle, the number of generations of parthenogenetic metacercariae is unlimited, and they can also produce cercariae. These cercariae emerge into the environment and can infect new individuals of the second intermediate host. We conclude that the type I life cycle is a derived option that has evolved as a better fit to transmission in the unstable conditions in the intertidal zone. Another evolutionary trend in Parvatrema is transition from inhabiting the extrapallial space of the gastropod second intermediate host to endoparasitism in its mantle and internal organs. rDNA sequence analysis highlighted that Parvatrema spp. with parthenogenetic metacercariae form a monophyletic clade and suggested the Pacific origin of the group, with two transfers to the North Atlantic and colonisation of new second intermediate host species. Apparently the group formed in the late Pliocene-Pleistocene and diversified as a result of recurrent isolation in inshore refugia during glacial periods. We argue that parthenogenetic metacercariae in Parvatrema may serve as a model for early digenean evolution, demonstrating the first steps of adopting the molluscan first intermediate host and becoming tissue parasites.

Plant kleptomaniacs: geographical genetic patterns in the amphi-apomictic Rubus ser. Glandulosi (Rosaceae) reveal complex reticulate evolution of Eurasian brambles.

BACKGROUND AND AIMS: Rubus ser. Glandulosi provides a unique model of geographical parthenogenesis on a homoploid (2n = 4x) level. We aim to characterize evolutionary and phylogeographical patterns in this taxon and shed light on the geographical differentiation of apomicts and sexuals. Ultimately, we aim to evaluate the importance of phylogeography in the formation of geographical parthenogenesis. METHODS: Rubus ser. Glandulosi was sampled across its Eurasian range together with other co-occurring Rubus taxa (587 individuals in total). Double-digest restriction site-associated DNA sequencing (ddRADseq) and modelling of suitable climate were used for evolutionary inferences. KEY RESULTS: Six ancestral species were identified that contributed to the contemporary gene pool of R. ser. Glandulosi. Sexuals were introgressed from Rubus dolichocarpus and Rubus moschus in West Asia and from Rubus ulmifolius agg., Rubus canescens and Rubus incanescens in Europe, whereas apomicts were characterized by alleles of Rubus subsect. Rubus. Gene flow between sexuals and apomicts was also detected, as was occasional hybridization with other taxa. CONCLUSIONS: We hypothesize that sexuals survived the last glacial period in several large southern refugia, whereas apomicts were mostly restricted to southern France, whence they quickly recolonized Central and Western Europe. The secondary contact of sexuals and apomicts was probably the principal factor that established geographical parthenogenesis in R. ser. Glandulosi. Sexual populations are not impoverished in genetic diversity along their borderline with apomicts, and maladaptive population genetic processes probably did not shape the geographical patterns.

Identification of Parthenogenesis-Inducing Effector Proteins in Wolbachia.

Bacteria in the genus Wolbachia have evolved numerous strategies to manipulate arthropod sex, including the conversion of would-be male offspring to asexually reproducing females. This so-called "parthenogenesis induction" phenotype can be found in a number of Wolbachia strains that infect arthropods with haplodiploid sex determination systems, including parasitoid wasps. Despite the discovery of microbe-mediated parthenogenesis more than 30 yr ago, the underlying genetic mechanisms have remained elusive. We used a suite of genomic, computational, and molecular tools to identify and characterize two proteins that are uniquely found in parthenogenesis-inducing Wolbachia and have strong signatures of host-associated bacterial effector proteins. These putative parthenogenesis-inducing proteins have structural homology to eukaryotic protein domains including nucleoporins, the key insect sex determining factor Transformer, and a eukaryotic-like serine-threonine kinase with leucine-rich repeats. Furthermore, these proteins significantly impact eukaryotic cell biology in the model Saccharomyces cerevisiae. We suggest that these proteins are parthenogenesis-inducing factors and our results indicate that this would be made possible by a novel mechanism of bacterial-host interaction.