Linear DNA-driven recombination in mammalian mitochondria.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

Global mesozooplankton communities show lower connectivity in deep oceanic layers.

Mesozooplankton is a key component of the ocean, regulating global processes such as the carbon pump, and ensuring energy transfer from lower to higher trophic levels. Yet, knowledge on mesozooplankton diversity, distribution and connectivity at global scale is still fragmented. To fill this gap, we applied DNA metabarcoding to mesozooplankton samples collected during the Malaspina-2010 circumnavigation expedition across the Atlantic, Indian and Pacific oceans from the surface to bathypelagic depths. We highlight the still scarce knowledge on global mesozooplankton diversity and identify the Indian Ocean and the deep sea as the oceanic regions with the highest proportion of hidden diversity. We report no consistent alpha-diversity patterns for mesozooplankton at a global scale, neither across vertical nor horizontal gradients. However, beta-diversity analysis suggests horizontal and vertical structuring of mesozooplankton communities mostly attributed to turnover and reveals an increase in mesozooplankton beta-diversity with depth, indicating reduced connectivity at deeper layers. Additionally, we identify a water mass type-mediated structuring of mesozooplankton bathypelagic communities instead of an oceanic basin-mediated as observed at upper layers. This suggests limited dispersal at deep ocean layers, most likely due to weaker currents and lower mixing of water mass types, thus reinforcing the importance of oceanic currents and barriers to dispersal in shaping global plankton communities.

Top3α is the replicative topoisomerase in mitochondrial DNA replication.

Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.

Genomic evidence for contrasting patterns of host-associated genetic differentiation across shared host-plant species in leaf- and bud-galling sawflies.

Resource specialization and ecological speciation arising through host-associated genetic differentiation (HAD) are frequently invoked as an explanation for the high diversity of plant-feeding insects and other organisms with a parasitic lifestyle. While genetic studies have demonstrated numerous examples of HAD in insect herbivores, the rarity of comparative studies means that we still lack an understanding of how deterministic HAD is, and whether patterns of host shifts can be predicted over evolutionary timescales. We applied genome-wide single nucleotide polymorphism and mitochondrial DNA sequence data obtained through genome resequencing to define species limits and to compare host-plant use in population samples of leaf- and bud-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae) collected from seven shared willow (Salicaceae: Salix) host species. To infer the repeatability of long-term cophylogenetic patterns, we also contrasted the phylogenies of the two galler groups with each other as well as with the phylogeny of their Salix hosts estimated based on RADseq data. We found clear evidence for host specialization and HAD in both of the focal galler groups, but also that leaf gallers are more specialized to single host species compared with most bud gallers. In contrast to bud gallers, leaf gallers also exhibited statistically significant cophylogenetic signal with their Salix hosts. The observed discordant patterns of resource specialization and host shifts in two related galler groups that have radiated in parallel across a shared resource base indicate a lack of evolutionary repeatability in the focal system, and suggest that short- and long-term host use and ecological diversification in plant-feeding insects are dominated by stochasticity and/or lineage-specific effects.

Molecular phenotyping uncovers differences in basic housekeeping functions among closely related species of hares (Lepus spp., Lagomorpha: Leporidae).

Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.

The genome of Chenopodium quinoa.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

Corrigendum: The genome of Chenopodium quinoa.

This corrects the article DOI: 10.1038/nature21370.

Microbiomes of willow-galling sawflies: effects of host plant, gall type, and phylogeny on community structure and function.

While free-living herbivorous insects are thought to harbor microbial communities composed of transient bacteria derived from their diet, recent studies indicate that insects that induce galls on plants may be involved in more intimate host-microbe relationships. We used 16S rDNA metabarcoding to survey larval microbiomes of 20 nematine sawfly species that induce bud or leaf galls on 13 Salix species. The 391 amplicon sequence variants (ASVs) detected represented 69 bacterial genera in six phyla. Multi-variate statistical analyses showed that the structure of larval microbiomes is influenced by willow host species as well as by gall type. Nevertheless, a "core" microbiome composed of 58 ASVs is shared widely across the focal galler species. Within the core community, the presence of many abundant, related ASVs representing multiple distantly related bacterial taxa is reflected as a statistically significant effect of bacterial phylogeny on galler-microbe associations. Members of the core community have a variety of inferred functions, including degradation of phenolic compounds, nutrient supplementation, and production of plant hormones. Hence, our results support suggestions of intimate and diverse interactions between galling insects and microbes and add to a growing body of evidence that microbes may play a role in the induction of insect galls on plants.

Finding flies in the mushroom soup: Host specificity of fungus-associated communities revisited with a novel molecular method.

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities.

Evidence for miRNA-mediated modulation of the host transcriptome in cnidarian-dinoflagellate symbiosis.

Reef-building corals and other cnidarians living in symbiotic relationships with intracellular, photosynthetic dinoflagellates in the genus Symbiodinium undergo transcriptomic changes during infection with the algae and maintenance of the endosymbiont population. However, the precise regulatory mechanisms modulating the host transcriptome are unknown. Here, we report apparent post-transcriptional gene regulation by miRNAs in the sea anemone Aiptasia, a model system for cnidarian-dinoflagellate endosymbiosis. Aiptasia encodes mainly species-specific miRNAs, and there appears to have been recent differentiation within the Aiptasia genome of miRNAs that are commonly conserved among anthozoan cnidarians. Analysis of miRNA expression showed that both conserved and species-specific miRNAs are differentially expressed in response to endosymbiont infection. Using cross-linking immunoprecipitation of Argonaute, the central protein of the miRNA-induced silencing complex, we identified miRNA binding sites on a transcriptome-wide scale and found that the targets of the miRNAs regulated in response to symbiosis include genes previously implicated in biological processes related to Symbiodinium infection. Our study shows that cnidarian miRNAs recognize their mRNA targets via high-complementarity target binding and suggests that miRNA-mediated modulations of genes and pathways are important during the onset and maintenance of cnidarian-dinoflagellate endosymbiosis.

The genome of Aiptasia, a sea anemone model for coral symbiosis.

The most diverse marine ecosystems, coral reefs, depend upon a functional symbiosis between a cnidarian animal host (the coral) and intracellular photosynthetic dinoflagellate algae. The molecular and cellular mechanisms underlying this endosymbiosis are not well understood, in part because of the difficulties of experimental work with corals. The small sea anemone Aiptasia provides a tractable laboratory model for investigating these mechanisms. Here we report on the assembly and analysis of the Aiptasia genome, which will provide a foundation for future studies and has revealed several features that may be key to understanding the evolution and function of the endosymbiosis. These features include genomic rearrangements and taxonomically restricted genes that may be functionally related to the symbiosis, aspects of host dependence on alga-derived nutrients, a novel and expanded cnidarian-specific family of putative pattern-recognition receptors that might be involved in the animal-algal interactions, and extensive lineage-specific horizontal gene transfer. Extensive integration of genes of prokaryotic origin, including genes for antimicrobial peptides, presumably reflects an intimate association of the animal-algal pair also with its prokaryotic microbiome.

Loss of species and genetic diversity during colonization: Insights from acanthocephalan parasites in northern European seals.

Studies on host-parasite systems that have experienced distributional shifts, range fragmentation, and population declines in the past can provide information regarding how parasite community richness and genetic diversity will change as a result of anthropogenic environmental changes in the future. Here, we studied how sequential postglacial colonization, shifts in habitat, and reduced host population sizes have influenced species richness and genetic diversity of Corynosoma (Acanthocephala: Polymorphidae) parasites in northern European marine, brackish, and freshwater seal populations. We collected Corynosoma population samples from Arctic, Baltic, Ladoga, and Saimaa ringed seal subspecies and Baltic gray seals, and then applied COI barcoding and triple-enzyme restriction-site associated DNA (3RAD) sequencing to delimit species, clarify their distributions and community structures, and elucidate patterns of intraspecific gene flow and genetic diversity. Our results showed that Corynosoma species diversity reflected host colonization histories and population sizes, with four species being present in the Arctic, three in the Baltic Sea, two in Lake Ladoga, and only one in Lake Saimaa. We found statistically significant population-genetic differentiation within all three Corynosoma species that occur in more than one seal (sub)species. Genetic diversity tended to be high in Corynosoma populations originating from Arctic ringed seals and low in the landlocked populations. Our results indicate that acanthocephalan communities in landlocked seal populations are impoverished with respect to both species and intraspecific genetic diversity. Interestingly, the loss of genetic diversity within Corynosoma species seems to have been less drastic than in their seal hosts, possibly due to their large local effective population sizes resulting from high infection intensities and effective intra-host population mixing. Our study highlights the utility of genomic methods in investigations of community composition and genetic diversity of understudied parasites.

Mountain- and brown hare genetic polymorphisms to survey local adaptations and conservation status of the heath hare (Lepus timidus sylvaticus, Nilsson 1831).

We provide the first whole genome sequences from three specimens of the mountain hare subspecies the heath hare (Lepus timidus sylvaticus), along with samples from two mountain hares (Lepus timidus timidus) and two brown hares (Lepus europaeus) from Sweden. The heath hare has a unique grey winter pelage as compared to other mountain hares (white) and brown hares (mostly brown), and face regional extinction, likely due to competitive exclusion from the non-native brown hare. Whole genome resequencing from the seven hare specimens were mapped to the Lepus timidus pseudoreference genome and used for detection of 11,363,883 polymorphic nucleotide positions. The data presented here could be useful for addressing local adaptations and conservation status of mountain hares and brown hares in Sweden, including unique subspecies.

Genomes of the willow-galling sawflies Euura lappo and Eupontania aestiva (Hymenoptera: Tenthredinidae): a resource for research on ecological speciation, adaptation, and gall induction.

Hymenoptera is a hyperdiverse insect order represented by over 153,000 different species. As many hymenopteran species perform various crucial roles for our environments, such as pollination, herbivory, and parasitism, they are of high economic and ecological importance. There are 99 hymenopteran genomes in the NCBI database, yet only five are representative of the paraphyletic suborder Symphyta (sawflies, woodwasps, and horntails), while the rest represent the suborder Apocrita (bees, wasps, and ants). Here, using a combination of 10X Genomics linked-read sequencing, Oxford Nanopore long-read technology, and Illumina short-read data, we assembled the genomes of two willow-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae: Euurina): the bud-galling species Euura lappo and the leaf-galling species Eupontania aestiva. The final assembly for E. lappo is 259.85 Mbp in size, with a contig N50 of 209.0 kbp and a BUSCO score of 93.5%. The E. aestiva genome is 222.23 Mbp in size, with a contig N50 of 49.7 kbp and a 90.2% complete BUSCO score. De novo annotation of repetitive elements showed that 27.45% of the genome was composed of repetitive elements in E. lappo and 16.89% in E. aestiva, which is a marked increase compared to previously published hymenopteran genomes. The genomes presented here provide a resource for inferring phylogenetic relationships among basal hymenopterans, comparative studies on host-related genomic adaptation in plant-feeding insects, and research on the mechanisms of plant manipulation by gall-inducing insects.

Hybridization with mountain hares increases the functional allelic repertoire in brown hares.

Brown hares (Lepus europaeus Pallas) are able to hybridize with mountain hares (L. timidus Linnaeus) and produce fertile offspring, which results in cross-species gene flow. However, not much is known about the functional significance of this genetic introgression. Using targeted sequencing of candidate loci combined with mtDNA genotyping, we found the ancestral genetic diversity in the Finnish brown hare to be small, likely due to founder effect and range expansion, while gene flow from mountain hares constitutes an important source of functional genetic variability. Some of this variability, such as the alleles of the mountain hare thermogenin (uncoupling protein 1, UCP1), might have adaptive advantage for brown hares, whereas immunity-related MHC alleles are reciprocally exchanged and maintained via balancing selection. Our study offers a rare example where an expanding species can increase its allelic variability through hybridization with a congeneric native species, offering a route to shortcut evolutionary adaptation to the local environmental conditions.

The Type and Source of Reactive Oxygen Species Influences the Outcome of Oxidative Stress in Cultured Cells.

Oxidative stress can be modeled using various different experimental approaches, such as exposing the cells or organisms to oxidative chemicals. However, the actual effects of these chemicals, outside of the immediate measured effect, have attracted relatively little attention. We show here that three commonly used oxidants, menadione, potassium bromate, and hydrogen peroxide, while known to function differently, also elicit different types of responses in HEK293T cells. Menadione and bromate exposure mainly trigger an integrated stress response, whereas hydrogen peroxide affects cellular processes more diversely. Interestingly, acute oxidative stress does not universally cause notable induction of DNA repair or antioxidant defense mechanisms. We also provide evidence that cells with previous experience of oxidative stress show adaptive changes in their responses when the stress is renewed. Our results urge caution when comparing studies where different sources of oxidative stress have been used or when generalizing the findings of these studies to other oxidant types or tissues.

DNA barcoding reveals different cestode helminth species in northern European marine and freshwater ringed seals.

Three subspecies of the ringed seal (Pusa hispida) are found in northeastern Europe: P. h. botnica in the Baltic Sea, P. h saimensis in Lake Saimaa in Finland, and P. h. ladogensis in Lake Ladoga in Russia. We investigated the poorly-known cestode helminth communities of these closely related but ecologically divergent subspecies using COI barcode data. Our results show that, while cestodes from the Baltic Sea represent Schistocephalus solidus, all worms from the two lakes are identified as Ligula intestinalis, a species that has previously not been reported from seals. The observed shift in cestode communities appears to be driven by differential availability of intermediate fish host species in marine vs. freshwater environments. Both observed cestode species normally infect fish-eating birds, so further work is required to elucidate the health and conservation implications of cestode infections in European ringed seals, whether L. intestinalis occurs also in marine ringed seals, and whether the species is able to reproduce in seal hosts. In addition, a deep barcode divergence found within S. solidus suggests the presence of cryptic diversity under this species name.

Finding Nemo's Genes: A chromosome-scale reference assembly of the genome of the orange clownfish Amphiprion percula.

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome-scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single-molecule real-time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi-C-based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein-coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.

Desert plant bacteria reveal host influence and beneficial plant growth properties.

Deserts, such as those found in Saudi Arabia, are one of the most hostile places for plant growth. However, desert plants are able to impact their surrounding microbial community and select beneficial microbes that promote their growth under these extreme conditions. In this study, we examined the soil, rhizosphere and endosphere bacterial communities of four native desert plants Tribulus terrestris, Zygophyllum simplex, Panicum turgidum and Euphorbia granulata from the Southwest (Jizan region), two of which were also found in the Midwest (Al Wahbah area) of Saudi Arabia. While the rhizosphere bacterial community mostly resembled that of the highly different surrounding soils, the endosphere composition was strongly correlated with its host plant phylogeny. In order to assess whether any of the native bacterial endophytes might have a role in plant growth under extreme conditions, we analyzed the properties of 116 cultured bacterial isolates that represent members of the phyla Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Our analysis shows that different strains have highly different biochemical properties with respect to nutrient acquisition, hormone production and growth under stress conditions. More importantly, eleven of the isolated strains could confer salinity stress tolerance to the experimental model plant Arabidopsis thaliana suggesting some of these plant-associated bacteria might be useful for improving crop desert agriculture.

DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral model Aiptasia.

The symbiotic relationship between cnidarians and dinoflagellates is the cornerstone of coral reef ecosystems. Although research has focused on the molecular mechanisms underlying this symbiosis, the role of epigenetic mechanisms, that is, the study of heritable changes that do not involve changes in the DNA sequence, is unknown. To assess the role of DNA methylation in the cnidarian-dinoflagellate symbiosis, we analyzed genome-wide CpG methylation, histone associations, and transcriptomic states of symbiotic and aposymbiotic anemones in the model system Aiptasia. We found that methylated genes are marked by histone 3 lysine 36 trimethylation (H3K36me3) and show significant reduction of spurious transcription and transcriptional noise, revealing a role of DNA methylation in the maintenance of transcriptional homeostasis. Changes in DNA methylation and expression show enrichment for symbiosis-related processes, such as immunity, apoptosis, phagocytosis recognition, and phagosome formation, and reveal intricate interactions between the underlying pathways. Our results demonstrate that DNA methylation provides an epigenetic mechanism of transcriptional homeostasis that responds to symbiosis.