Phased chromosome-scale genome assembly of an asexual, allopolyploid root-knot nematode reveals complex subgenomic structure.

We present the chromosome-scale genome assembly of the allopolyploid root-knot nematode Meloidogyne javanica. We show that the M. javanica genome is predominantly allotetraploid, comprising two subgenomes, A and B, that most likely originated from hybridisation of two ancestral parental species. The assembly was annotated using full-length non-chimeric transcripts, comparison to reference databases, and ab initio prediction techniques, and the subgenomes were phased using ancestral k-mer spectral analysis. Subgenome B appears to show fission of chromosomal contigs, and while there is substantial synteny between subgenomes, we also identified regions lacking synteny that may have diverged in the ancestral genomes prior to or following hybridisation. This annotated and phased genome assembly forms a significant resource for understanding the origins and genetics of these globally important plant pathogens.

Let's talk about sex: Why reproductive systems matter for understanding algae.

Sex is a crucial process that has molecular, genetic, cellular, organismal, and population-level consequences for eukaryotic evolution. Eukaryotic life cycles are composed of alternating haploid and diploid phases but are constrained by the need to accommodate the phenotypes of these different phases. Critical gaps in our understanding of evolutionary drivers of the diversity in algae life cycles include how selection acts to stabilize and change features of the life cycle. Moreover, most eukaryotes are partially clonal, engaging in both sexual and asexual reproduction. Yet, our understanding of the variation in their reproductive systems is largely based on sexual reproduction in animals or angiosperms. The relative balance of sexual versus asexual reproduction not only controls but also is in turn controlled by standing genetic variability, thereby shaping evolutionary trajectories. Thus, we must quantitatively assess the consequences of the variation in life cycles on reproductive systems. Algae are a polyphyletic group spread across many of the major eukaryotic lineages, providing powerful models by which to resolve this knowledge gap. There is, however, an alarming lack of data about the population genetics of most algae and, therefore, the relative frequency of sexual versus asexual processes. For many algae, the occurrence of sexual reproduction is unknown, observations have been lost in overlooked papers, or data on population genetics do not yet exist. This greatly restricts our ability to forecast the consequences of climate change on algal populations inhabiting terrestrial, aquatic, and marine ecosystems. This perspective summarizes our extant knowledge and provides some future directions to pursue broadly across micro- and macroalgal species.

Histone H2B lysine 122 and lysine 130, as the putative targets of Penicillium oxalicum LaeA, play important roles in asexual development, expression of secondary metabolite gene clusters, and extracellular glycoside hydrolase synthesis.

Core histones in the nucleosome are subject to a wide variety of posttranslational modifications (PTMs), such as methylation, phosphorylation, ubiquitylation, and acetylation, all of which are crucial in shaping the structure of the chromatin and the expression of the target genes. A putative histone methyltransferase LaeA/Lae1, which is conserved in numerous filamentous fungi, functions as a global regulator of fungal growth, virulence, secondary metabolite formation, and the production of extracellular glycoside hydrolases (GHs). LaeA's direct histone targets, however, were not yet recognized. Previous research has shown that LaeA interacts with core histone H2B. Using S-adenosyl-L-methionine (SAM) as a methyl group donor and recombinant human histone H2B as the substrate, it was found that Penicillium oxalicum LaeA can transfer the methyl groups to the C-terminal lysine (K) 108 and K116 residues in vitro. The H2BK108 and H2BK116 sites on recombinant histone correspond to P. oxalicum H2BK122 and H2BK130, respectively. H2BK122A and H2BK130A, two mutants with histone H2B K122 or K130 mutation to alanine (A), were constructed in P. oxalicum. The mutants H2BK122A and H2BK130A demonstrated altered asexual development and decreased extracellular GH production, consistent with the findings of the laeA gene deletion strain (ΔlaeA). The transcriptome data showed that when compared to wild-type (WT) of P. oxalicum, 38 of the 47 differentially expressed (fold change ≥ 2, FDR ≤ 0.05) genes that encode extracellular GHs showed the same expression pattern in the three mutants ΔlaeA, H2BK122A, and H2BK130A. The four secondary metabolic gene clusters that considerably decreased expression in ΔlaeA also significantly decreased in H2BK122A or H2BK130A. The chromatin of promotor regions of the key cellulolytic genes cel7A/cbh1 and cel7B/eg1 compacted in the ΔlaeA, H2BK122A, and H2BK130A mutants, according to the results of chromatin accessibility real-time PCR (CHART-PCR). The chromatin accessibility index dropped. The histone binding pocket of the LaeA-methyltransf_23 domain is compatible with particular histone H2B peptides, providing appropriate electrostatic and steric compatibility to stabilize these peptides, according to molecular docking. The findings of the study demonstrate that H2BK122 and H2BK130, which are histone targets of P. oxalicum LaeA in vitro, are crucial for fungal conidiation, the expression of gene clusters encoding secondary metabolites, and the production of extracellular GHs.

Apomixis and the paradox of sex in plants.

BACKGROUND: The predominance of sex in eukaryotes, despite the high costs of meiosis and mating, remains an evolutionary enigma. Many theories have been proposed, none of them being conclusive on its own, and they are, in part, not well applicable to land plants. Sexual reproduction is obligate in embryophytes for the great majority of species. SCOPE: This review compares the main forms of sexual and asexual reproduction in ferns and angiosperms, based on the generation cycling of sporophyte and gametophyte (leaving vegetative propagation aside). The benefits of sexual reproduction for maintenance of genomic integrity in comparison to asexuality are discussed in the light of developmental, evolutionary, genetic and phylogenetic studies. CONCLUSIONS: Asexual reproduction represents modifications of the sexual pathway, with various forms of facultative sexuality. For sexual land plants, meiosis provides direct DNA repair mechanisms for oxidative damage in reproductive tissues. The ploidy alternations of meiosis-syngamy cycles and prolonged multicellular stages in the haploid phase in the gametophytes provide a high efficiency of purifying selection against recessive deleterious mutations. Asexual lineages might buffer effects of such mutations via polyploidy and can purge the mutational load via facultative sexuality. The role of organelle-nuclear genome compatibility for maintenance of genome integrity is not well understood. In plants in general, the costs of mating are low because of predominant hermaphroditism. Phylogenetic patterns in the archaeplastid clade suggest that high frequencies of sexuality in land plants are concomitant with a stepwise increase of intrinsic and extrinsic stress factors. Furthermore, expansion of genome size in land plants would increase the potential mutational load. Sexual reproduction appears to be essential for keeping long-term genomic integrity, and only rare combinations of extrinsic and intrinsic factors allow for shifts to asexuality.

Hybridisation has shaped a recent radiation of grass-feeding aphids.

BACKGROUND: Aphids are common crop pests. These insects reproduce by facultative parthenogenesis involving several rounds of clonal reproduction interspersed with an occasional sexual cycle. Furthermore, clonal aphids give birth to live young that are already pregnant. These qualities enable rapid population growth and have facilitated the colonisation of crops globally. In several cases, so-called "super clones" have come to dominate agricultural systems. However, the extent to which the sexual stage of the aphid life cycle has shaped global pest populations has remained unclear, as have the origins of successful lineages. Here, we used chromosome-scale genome assemblies to disentangle the evolution of two global pests of cereals-the English (Sitobion avenae) and Indian (Sitobion miscanthi) grain aphids. RESULTS: Genome-wide divergence between S. avenae and S. miscanthi is low. Moreover, comparison of haplotype-resolved assemblies revealed that the S. miscanthi isolate used for genome sequencing is likely a hybrid, with one of its diploid genome copies closely related to S. avenae (~ 0.5% divergence) and the other substantially more divergent (> 1%). Population genomics analyses of UK and China grain aphids showed that S. avenae and S. miscanthi are part of a cryptic species complex with many highly differentiated lineages that predate the origins of agriculture. The complex consists of hybrid lineages that display a tangled history of hybridisation and genetic introgression. CONCLUSIONS: Our analyses reveal that hybridisation has substantially contributed to grain aphid diversity, and hence, to the evolutionary potential of this important pest species. Furthermore, we propose that aphids are particularly well placed to exploit hybridisation events via the rapid propagation of live-born "frozen hybrids" via asexual reproduction, increasing the likelihood of hybrid lineage formation.

The Life Cycle of Aurelia aurita Depends on the Presence of a Microbiome in Polyps Prior to Onset of Strobilation.

Aurelia aurita's intricate life cycle alternates between benthic polyp and pelagic medusa stages. The strobilation process, a critical asexual reproduction mechanism in this jellyfish, is severely compromised in the absence of the natural polyp microbiome, with limited production and release of ephyrae. Yet, the recolonization of sterile polyps with a native polyp microbiome can correct this defect. Here, we investigated the precise timing necessary for recolonization as well as the host-associated molecular processes involved. We deciphered that a natural microbiota had to be present in polyps prior to the onset of strobilation to ensure normal asexual reproduction and a successful polyp-to-medusa transition. Providing the native microbiota to sterile polyps after the onset of strobilation failed to restore the normal strobilation process. The absence of a microbiome was associated with decreased transcription of developmental and strobilation genes as monitored by reverse transcription-quantitative PCR. Transcription of these genes was exclusively observed for native polyps and sterile polyps that were recolonized before the initiation of strobilation. We further propose that direct cell contact between the host and its associated bacteria is required for the normal production of offspring. Overall, our findings indicate that the presence of a native microbiome at the polyp stage prior to the onset of strobilation is essential to ensure a normal polyp-to-medusa transition. IMPORTANCE All multicellular organisms are associated with microorganisms that play fundamental roles in the health and fitness of the host. Notably, the native microbiome of the Cnidarian Aurelia aurita is crucial for the asexual reproduction by strobilation. Sterile polyps display malformed strobilae and a halt of ephyrae release, which is restored by recolonizing sterile polyps with a native microbiota. Despite that, little is known about the microbial impact on the strobilation process's timing and molecular consequences. The present study shows that A. aurita's life cycle depends on the presence of the native microbiome at the polyp stage prior to the onset of strobilation to ensure the polyp-to-medusa transition. Moreover, sterile individuals correlate with reduced transcription levels of developmental and strobilation genes, evidencing the microbiome's impact on strobilation on the molecular level. Transcription of strobilation genes was exclusively detected in native polyps and those recolonized before initiating strobilation, suggesting microbiota-dependent gene regulation.

Phase-specific transcriptional patterns of the oomycete pathogen Phytophthora sojae unravel genes essential for asexual development and pathogenic processes.

Oomycetes are filamentous microorganisms easily mistaken as fungi but vastly differ in physiology, biochemistry, and genetics. This commonly-held misconception lead to a reduced effectiveness by using conventional fungicides to control oomycetes, thus it demands the identification of novel functional genes as target for precisely design oomycetes-specific microbicide. The present study initially analyzed the available transcriptome data of the model oomycete pathogen, Phytophthora sojae, and constructed an expression matrix of 10,953 genes across the stages of asexual development and host infection. Hierarchical clustering, specificity, and diversity analyses revealed a more pronounced transcriptional plasticity during the stages of asexual development than that in host infection, which drew our attention by particularly focusing on transcripts in asexual development stage to eventually clustered them into 6 phase-specific expression modules. Three of which respectively possessing a serine/threonine phosphatase (PP2C) expressed during the mycelial and sporangium stages, a histidine kinase (HK) expressed during the zoospore and cyst stages, and a bZIP transcription factor (bZIP32) exclusive to the cyst germination stage were selected for down-stream functional validation. In this way, we demonstrated that PP2C, HK, and bZIP32 play significant roles in P. sojae asexual development and virulence. Thus, these findings provide a foundation for further gene functional annotation in oomycetes and crop disease management.

Verticillium dahliae Asp1 regulates the transition from vegetative growth to asexual reproduction by modulating microtubule dynamic organization.

Verticillium dahliae is a devastating pathogenic fungus that causes severe vascular wilts in more than 400 dicotyledonous plants. The conidiation of V. dahliae in plant vascular tissues is the key strategy for its adaptation to the nutrient-poor environment and is required for its pathogenicity. However, it remains unclear about the regulatory mechanism of conidium production of V. dahliae in vascular tissues. Here, we found that VdAsp1, encoding an inositol polyphosphate kinase, is indispensable for the pathogenicity of V. dahliae. Loss of VdAsp1 function does not affect the invasion of the host, but it impairs the colonization and proliferation in vascular tissues. The ΔVdAsp1 mutant shows defective initiation of conidiophore formation and reduced expression of genes associated with the central developmental pathway. By live-cell imaging, we observed that some of ΔVdAsp1 mutant hyphae are swollen, and microtubule arrangements at the apical region of these hyphae are disorganized. These results indicate that VdAsp1 regulates the transition from vegetative growth to asexual reproduction by modulating microtubule dynamic organization, which is essential for V. dahliae to colonize and proliferate in vascular tissues. These findings provided a potential new direction in the control of vascular wilt pathogen by targeting conidium production in vascular tissues.

Regulatory basis for reproductive flexibility in a meningitis-causing fungal pathogen.

Pathogenic fungi of the genus Cryptococcus can undergo two sexual cycles, involving either bisexual diploidization (after fusion of haploid cells of different mating type) or unisexual diploidization (by autodiploidization of a single cell). Here, we construct a gene-deletion library for 111 transcription factor genes in Cryptococcus deneoformans, and explore the roles of these regulatory networks in the two reproductive modes. We show that transcription factors crucial for bisexual syngamy induce the expression of known mating determinants as well as other conserved genes of unknown function. Deletion of one of these genes, which we term FMP1, leads to defects in bisexual reproduction in C. deneoformans, its sister species Cryptococcus neoformans, and the ascomycete Neurospora crassa. Furthermore, we show that a recently evolved regulatory cascade mediates pre-meiotic unisexual autodiploidization, supporting that this reproductive process is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern hallmark events distinguishing unisexual and bisexual reproduction in Cryptococcus.

Polyploidy as an Adaptation against Loss of Heterozygosity in Cancer.

Polyploidy is common in cancer cells and has implications for tumor progression and resistance to therapies, but it is unclear whether it is an adaptation of the tumor or the non-adaptive effect of genomic instability. I discuss the possibility that polyploidy reduces the deleterious effects of loss of heterozygosity, which arises as a consequence of mitotic recombination, and which in diploid cells leads instead to the rapid loss of complementation of recessive deleterious mutations. I use computational predictions of loss of heterozygosity to show that a population of diploid cells dividing by mitosis with recombination can be easily invaded by mutant polyploid cells or cells that divide by endomitosis, which reduces loss of complementation, or by mutant cells that occasionally fuse, which restores heterozygosity. A similar selective advantage of polyploidy has been shown for the evolution of different types of asexual reproduction in nature. This provides an adaptive explanation for cyclical ploidy, mitotic slippage and cell fusion in cancer cells.

The Origin of Asexual Brine Shrimps.

AbstractDetermining 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.

Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora litchii.

Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.

Epistatic genetic interactions govern morphogenesis during sexual reproduction and infection in a global human fungal pathogen.

Cellular development is orchestrated by evolutionarily conserved signaling pathways, which are often pleiotropic and involve intra- and interpathway epistatic interactions that form intricate, complex regulatory networks. Cryptococcus species are a group of closely related human fungal pathogens that grow as yeasts yet transition to hyphae during sexual reproduction. Additionally, during infection they can form large, polyploid titan cells that evade immunity and develop drug resistance. Multiple known signaling pathways regulate cellular development, yet how these are coordinated and interact with genetic variation is less well understood. Here, we conducted quantitative trait locus (QTL) analyses of a mapping population generated by sexual reproduction of two parents, only one of which is unisexually fertile. We observed transgressive segregation of the unisexual phenotype among progeny, as well as a large-cell phenotype under mating-inducing conditions. These large-cell progeny were found to produce titan cells both in vitro and in infected animals. Two major QTLs and corresponding quantitative trait genes (QTGs) were identified: RIC8 (encoding a guanine-exchange factor) and CNC06490 (encoding a putative Rho-GTPase activator), both involved in G protein signaling. The two QTGs interact epistatically with each other and with the mating-type locus in phenotypic determination. These findings provide insights into the complex genetics of morphogenesis during unisexual reproduction and pathogenic titan cell formation and illustrate how QTL analysis can be applied to identify epistasis between genes. This study shows that phenotypic outcomes are influenced by the genetic background upon which mutations arise, implicating dynamic, complex genotype-to-phenotype landscapes in fungal pathogens and beyond.

Effective asexual reproduction of a widespread soft coral: comparative assessment of four different fragmentation methods.

BACKGROUND: Many coral reefs worldwide are experiencing declines in hard corals, resulting in other benthic organisms, e.g., soft corals, becoming more dominant. As such, more studies on the ecophysiology of soft corals are needed. Despite many methods for asexual reproduction of hard corals, effective methods for soft corals, i.e., without a hard skeleton, are scarce. This study, thus, assessed four fragmentation methods, the glue, rubber band, tunnel mesh, and plug mesh method for the pulsating soft coral Xenia umbellata that is widely distributed in the tropical Indo-Pacific. METHODS: Methods were comparatively assessed by determining the required time and labor for the fragmentation plus the health status of the fragmented corals by measuring their oxygen fluxes and pulsation rates, i.e., a special feature of this soft coral that can be used as a proxy for its health. RESULTS: There were no significant health status differences between methods. This was indicated by similar gross photosynthesis (between 7.4 to 9.7 µg O2 polyp-1 h-1) and pulsating rates (between 35 and 44 pulses min-1) among methods. In terms of time/labor intensity and success rates, i.e., the percentage of fragments attached to the desired surface, the plug mesh method was the most efficient method with a significantly higher success rate (95 ± 5%), while the others had a success rate between 5 ± 5 and 45 ± 15%. The time needed for fragmentation, though not significant, was also the shortest (78 ± 11 s fragment-1), while other methods required between 84 ± 14 and 126 ± 8 s frag-1. The plug mesh method may thus be a valuable tool related to the reproduction of soft corals for use in subsequent experimental work.

Effects of extremely high concentrations of polystyrene microplastics on asexual reproduction and nematocyst discharge in the jellyfish Sanderia malayensis.

Numerous studies have assessed the detrimental effects of microplastics (MPs) on aquatic invertebrates due to their ubiquitous and persistent nature. In this study, the toxic effects of MPs were examined on the polyp and ephyrae of the marine hydrozoan Sanderia malayensis. The jellyfish were exposed to different sizes (1-6 µm) of non-functionalized polystyrene microbeads at a concentration of 1 × 104 particles mL-1. The MPs randomly attached to the external and internal parts of the jellyfish body, and the longest MP attachment was 52 days during the depuration after initial exposure (for 24 h). Consistent seventeen-day exposure to MPs significantly reduced the asexual reproduction of the S. malayensis polyps. To assess if the MPs can stimulate nematocyst discharge in polyp and ephyrae stages via direct contact, they were exposed to particle sizes up to 430 µm. None of the MPs or their aggregates, including the 430 µm particles, induced nematocyst discharge. These results suggest that prolonged exposure to relatively high MP concentrations affects the early stages of jellies and provides evidence for the no effect on nematocyst discharge.

Challenges and Costs of Asexuality: Variation in Premeiotic Genome Duplication in Gynogenetic Hybrids from Cobitis taenia Complex.

The transition from sexual reproduction to asexuality is often triggered by hybridization. The gametogenesis of many hybrid asexuals involves premeiotic genome endoreplication leading to bypass hybrid sterility and forming clonal gametes. However, it is still not clear when endoreplication occurs, how many gonial cells it affects and whether its rate differs among clonal lineages. Here, we investigated meiotic and premeiotic cells of diploid and triploid hybrids of spined loaches (Cypriniformes: Cobitis) that reproduce by gynogenesis. We found that in naturally and experimentally produced F1 hybrids asexuality is achieved by genome endoreplication, which occurs in gonocytes just before entering meiosis or, rarely, one or a few divisions before meiosis. However, genome endoreplication was observed only in a minor fraction of the hybrid's gonocytes, while the vast majority of gonocytes were unable to duplicate their genomes and consequently could not proceed beyond pachytene due to defects in bivalent formation. We also noted that the rate of endoreplication was significantly higher among gonocytes of hybrids from natural clones than of experimentally produced F1 hybrids. Thus, asexuality and hybrid sterility are intimately related phenomena and the transition from sexual reproduction to asexuality must overcome significant problems with genome incompatibilities with a possible impact on reproductive potential.

Rescue and vegetative propagation of Eremanthus erythropappus (DC. ) Ma ish in natural stand / Resgate e propagação vegetativa de Eremanthus erythropappus (DC. ) MacLeish em povoamentos naturais

Abstract The native stands of 'candeia' (Eremanthus erythropappus) have been explored through management plans due to the economic potential of essential oil. The rescue of adult trees, as well as the application of silvicultural techniques that favor the restoration of the stand, can contribute to the genetic conservation of this species. This study's objective was to assess the efficiency of propagation techniques for the rescue of 26 matrices of 'candeia' in a natural managed stand and discussion about the rhizogenesis. In August 2017, trees were induced to regrowth by coppice, followed by exposure and scarification of roots. The emergence of shoots and morphology were evaluated according to the origin (i.e., stump or root). After that period, 19 matrices had their sprouts collected for the preparation of apical cuttings. Indole-3-butyric acid (IBA) was applied at the base of the cuttings. Cutting survival at greenhouse exit (GE), rooting at shade house exit (SHE), morphology and root anatomy were evaluated. In 189 days, the scarification of roots promoted 76.92% of budding. The percentage of sprouted matrices, number of shoots per matrice, length, diameter, and shoot length/diameter ratio increased over time. Only 12.2% of the cuttings survived in GE, and of these, 7.9% rooted in SHE. The cutting resulted in the formation of a clonal mini-garden of 'candeia', with seven of the 19 matrices submitted to propagation. The anatomical analyses showed that bud formation occurs from cell redifferentiation in the phloem parenchyma, and presence of crystals on the walls of the vessel elements of the secondary xylem. The shoots induction from scarification of roots could be used as a silvicultural practice for the reestablishment of the native fragments handle.

Resumo Os povoamentos nativos de candeia (Eremanthus erythropappus) vêm sendo explorados por planos de manejo devido ao potencial econômico do óleo essencial. O resgate de árvores adultas, bem como a aplicação de técnicas silviculturais que favoreçam o restabelecimento do povoamento podem contribuir para a conservação genética dessa espécie. O objetivo desse trabalho foi avaliar a eficiência de técnicas de propagação para o resgate de 26 matrizes de candeia em um povoamento natural manejado e discutir sobre a rizogênese. Em agosto de 2017, as árvores foram induzidas à rebrota por meio da decepa, seguida da exposição e escarificação das raízes. A emissão brotações e morfologia foram avaliadas de acordo com a origem (toco ou raiz). Após esse período, 19 matrizes tiveram as brotações recolhidas para o preparo de estacas apicais, que foram tratadas com ácido indolbutírico (AIB). A sobrevivência das estacas na saída da casa de vegetação (SCV), o enraizamento na saída da casa de sombra (SCS), a morfologia e a anatomia da raiz foram avaliados. Aos 189 dias, a escarificação das raízes resultou em 76,92% de emissão de brotos. O percentual de matrizes brotadas, número de brotos por matriz, comprimento, diâmetro e relação comprimento/diâmetro dos brotos aumentaram ao longo do período avaliado. Somente 12,2% das estacas sobreviveram na SCV e 7,9% enraizaram na SCS. A estaquia resultou na formação de um minijardim clonal de candeia com sete das dezenove matrizes submetidas à propagação. As análises anatômicas mostraram a diferenciação das células na região do parênquima floemático e a presença de cristais de inulina nas paredes dos elementos de vaso do xilema secundário. A indução de brotos radiculares pode ser usada como prática silvicultural visando o restabelecimento de fragmentos nativos manejados.

Impact of ocean warming and ocean acidification on asexual reproduction and statolith formation of the symbiotic jellyfish Cotylorhiza tuberculata.

Ocean acidification and warming are challenging marine organisms and ecosystems around the world. The synergetic effects of these two climate change stressors on jellyfish remain still understudied. Here, we examine the independent and combined effects of these two environmental variables on polyp population dynamics of the Mediterranean jellyfish Cotylorhiza tuberculata. An experiment was conducted to examine asexual reproduction by budding and strobilation considering current and ca. 2100 winter (Trial 1, 36 days) and summer (Trial 2, 36 days) conditions under the RCP8.5 (IPCC 2013). In Trial 1, a temperature of 18°C and two pH levels (current: 7.9 and, reduced: 7.7) were tested. Trial 2 considered two temperature levels 24°C and 30°C, under current and reduced acidification conditions (8.0 and 7.7, respectively). Ephyrae size and statolith formation of released ephyrae from polyps exposed to summer temperatures under both acidification treatment was also analyzed. Zooxanthellae density inside the polyps throughout the experiment was measured. C. tuberculata polyps could cope with the conditions mimicked in all experimental treatments and no significant effect of pH, temperature, or the combination of both variables on the abundance of polyps was observed. At 18°C, strobilation was reduced under high PCO2 conditions. Under summer treatments (24°C and 30°C), percentage strobilation was very low and several released ephyrae suffered malformations and reduced size, as a consequence of reduced pH and elevated temperatures, separately. The number of statoliths was not affected by pH or temperature, however, bigger statoliths were formed at elevated temperatures (30°C). Finally, zooxanthellae density was not affected by experimental conditions, even if, the duration of the experiment significantly affected symbiont concentration. Our results show that even though polyps of C. tuberculata would thrive the future worst scenario predicted for the Mediterranean Sea, their capacity to undergo a proper strobilation and to produce healthy ephyrae will be more vulnerable to climate induced environmental conditions, thereby affecting medusae recruitment and, therefore, population dynamics of the species.

Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi.

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala-a triploid hybrid species-were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

Phenotypic plasticity rather than genotype drives reproductive choices in Hydra populations.

Facultative clonality is associated with complex life cycles where sexual and asexual forms can be exposed to contrasting selection pressures. Facultatively clonal animals often have distinct developmental capabilities that depend on reproductive mode (e.g., negligible senescence and exceptional regeneration ability in asexual individuals, which are lacking in sexual individuals). Understanding how these differences in life history strategies evolved is hampered by limited knowledge of the population structure underlying sexual and asexual forms in nature. Here we studied genetic differentiation of coexisting sexual and asexual Hydra oligactis polyps, a freshwater cnidarian where reproductive mode-dependent life history patterns are observed. We collected asexual and sexual polyps from 13 Central European water bodies and used restriction-site associated DNA sequencing to infer population structure. We detected high relatedness among populations and signs that hydras might spread with resting eggs through zoochory. We found no genetic structure with respect to mode of reproduction (asexual vs. sexual). On the other hand, clear evidence was found for phenotypic plasticity in mode of reproduction, as polyps inferred to be clones differed in reproductive mode. Moreover, we detected two cases of apparent sex change (males and females found within the same clonal lineages) in this species with supposedly stable sexes. Our study describes population genetic structure in Hydra for the first time, highlights the role of phenotypic plasticity in generating patterns of life history variation, and contributes to understanding the evolution of reproductive mode-dependent life history variation in coexisting asexual and sexual forms.