High heteroplasmy is associated with low mitochondrial copy number and selection against non-synonymous mutations in the snail Cepaea nemoralis.

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Temporal genomics in Hawaiian crickets reveals compensatory intragenomic coadaptation during adaptive evolution.

Theory predicts that compensatory genetic changes reduce negative indirect effects of selected variants during adaptive evolution, but evidence is scarce. Here, we test this in a wild population of Hawaiian crickets using temporal genomics and a high-quality chromosome-level cricket genome. In this population, a mutation, flatwing, silences males and rapidly spread due to an acoustically-orienting parasitoid. Our sampling spanned a social transition during which flatwing fixed and the population went silent. We find long-range linkage disequilibrium around the putative flatwing locus was maintained over time, and hitchhiking genes had functions related to negative flatwing-associated effects. We develop a combinatorial enrichment approach using transcriptome data to test for compensatory, intragenomic coevolution. Temporal changes in genomic selection were distributed genome-wide and functionally associated with the population's transition to silence, particularly behavioural responses to silent environments. Our results demonstrate how 'adaptation begets adaptation'; changes to the sociogenetic environment accompanying rapid trait evolution can generate selection provoking further, compensatory adaptation.

Comparative mitochondrial genomics in Nematoda reveal astonishing variation in compositional biases and substitution rates indicative of multi-level selection.

BACKGROUND: Nematodes are the most abundant and diverse metazoans on Earth, and are known to significantly affect ecosystem functioning. A better understanding of their biology and ecology, including potential adaptations to diverse habitats and lifestyles, is key to understanding their response to global change scenarios. Mitochondrial genomes offer high species level characterization, low cost of sequencing, and an ease of data handling that can provide insights into nematode evolutionary pressures. RESULTS: Generally, nematode mitochondrial genomes exhibited similar structural characteristics (e.g., gene size and GC content), but displayed remarkable variability around these general patterns. Compositional strand biases showed strong codon position specific G skews and relationships with nematode life traits (especially parasitic feeding habits) equal to or greater than with predicted phylogeny. On average, nematode mitochondrial genomes showed low non-synonymous substitution rates, but also high clade specific deviations from these means. Despite the presence of significant mutational saturation, non-synonymous (dN) and synonymous (dS) substitution rates could still be significantly explained by feeding habit and/or habitat. Low ratios of dN:dS rates, particularly associated with the parasitic lifestyles, suggested the presence of strong purifying selection. CONCLUSIONS: Nematode mitochondrial genomes demonstrated a capacity to accumulate diversity in composition, structure, and content while still maintaining functional genes. Moreover, they demonstrated a capacity for rapid evolutionary change pointing to a potential interaction between multi-level selection pressures and rapid evolution. In conclusion, this study helps establish a background for our understanding of the potential evolutionary pressures shaping nematode mitochondrial genomes, while outlining likely routes of future inquiry.

Genome evolution in intracellular parasites: Microsporidia and Apicomplexa.

Microsporidia and Apicomplexa are eukaryotic, single-celled, intracellular parasites with huge public health and economic importance. Typically, these parasites are studied separately, emphasizing their uniqueness and diversity. In this review, we explore the huge amount of genomic data that has recently become available for the two groups. We compare and contrast their genome evolution and discuss how their transitions to intracellular life may have shaped it. In particular, we explore genome reduction and compaction, genome expansion and ploidy, gene shuffling and rearrangements, and the evolution of centromeres and telomeres.

The genome sequence of the Tipped Oak Case-bearer, Coleophora flavipennella (Duponchel 1843).

We present a genome assembly from an individual female Coleophora flavipennella (the Tipped Oak Case-bearer; Arthropoda; Insecta; Lepidoptera; Coleophoridae). The genome sequence is 989.3 megabases in span. Most of the assembly is scaffolded into 57 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.77 kilobases in length.

MarkerScan: Separation and assembly of cobionts sequenced alongside target species in biodiversity genomics projects.

Contamination of public databases by mislabelled sequences has been highlighted for many years and the avalanche of novel sequencing data now being deposited has the potential to make databases difficult to use effectively. It is therefore crucial that sequencing projects and database curators perform pre-submission checks to remove obvious contamination and avoid propagating erroneous taxonomic relationships. However, it is important also to recognise that biological contamination of a target sample with unexpected species' DNA can also lead to the discovery of fascinating biological phenomena through the identification of environmental organisms or endosymbionts. Here, we present a novel, integrated method for detection and generation of high-quality genomes of all non-target genomes co-sequenced in eukaryotic genome sequencing projects. After performing taxonomic profiling of an assembly from the raw data, and leveraging the identity of small rRNA sequences discovered therein as markers, a targeted classification approach retrieves and assembles high-quality genomes. The genomes of these cobionts are then not only removed from the target species' genome but also available for further interrogation. Source code is available from https://github.com/CobiontID/MarkerScan. MarkerScan is written in Python and is deployed as a Docker container.

This article addresses a common issue in genetic research: the accidental mixing of genetic information from different species in public databases, often due to mislabelling or contamination. Interestingly, this 'contamination' can sometimes lead to exciting discoveries, like identifying DNA from unexpected species in a sample, revealing insights about organisms that live in the environment of the target organism. In our study, we developed a tool called MarkerScan for identifying these additional species found alongside the target species in eukaryotic genome sequencing projects. The method includes a way to sequence the whole genomes of the additional species. Our method involves sorting through the genetic data to identify certain small RNA sequences, which we then use as markers. These markers help to classify and assemble high-quality genomes from these additional species. This not only cleans up the main target species' genome data but also provides new, valuable genomes for further exploration.

Comparative genomics reveals the dynamics of chromosome evolution in Lepidoptera.

Chromosomes are a central unit of genome organization. One-tenth of all described species on Earth are butterflies and moths, the Lepidoptera, which generally possess 31 chromosomes. However, some species display dramatic variation in chromosome number. Here we analyse 210 chromosomally complete lepidopteran genomes and show that the chromosomes of extant lepidopterans are derived from 32 ancestral linkage groups, which we term Merian elements. Merian elements have remained largely intact through 250 million years of evolution and diversification. Against this stable background, eight lineages have undergone extensive reorganization either through numerous fissions or a combination of fusion and fission events. Outside these lineages, fusions are rare and fissions are rarer still. Fusions often involve small, repeat-rich Merian elements and the sex-linked element. Our results reveal the constraints on genome architecture in Lepidoptera and provide a deeper understanding of chromosomal rearrangements in eukaryotic genome evolution.

Polyploidy is widespread in Microsporidia.

Microsporidia are obligate intracellular eukaryotic parasites with an extremely broad host range. They have both economic and public health importance. Ploidy in microsporidia is variable, with a few species formally identified as diploid and one as polyploid. Given the increase in the number of studies sequencing microsporidian genomes, it is now possible to assess ploidy levels across all currently explored microsporidian diversity. We estimate ploidy for all microsporidian data sets available on the Sequence Read Archive using k-mer-based analyses, indicating that polyploidy is widespread in Microsporidia and that ploidy change is dynamic in the group. Using genome-wide heterozygosity estimates, we also show that polyploid microsporidian genomes are relatively homozygous, and we discuss the implications of these findings on the timing of polyploidization events and their origin.IMPORTANCEMicrosporidia are single-celled intracellular parasites, distantly related to fungi, that can infect a broad range of hosts, from humans all the way to protozoans. Exploiting the wealth of microsporidian genomic data available, we use k-mer-based analyses to assess ploidy status across the group. Understanding a genome's ploidy is crucial in order to assemble it effectively and may also be relevant for better understanding a parasite's behavior and life cycle. We show that tetraploidy is present in at least six species in Microsporidia and that these polyploidization events are likely to have occurred independently. We discuss why these findings may be paradoxical, given that Microsporidia, like other intracellular parasites, have extremely small, reduced genomes.

The genome of Litomosoides sigmodontis illuminates the origins of Y chromosomes in filarial nematodes.

Heteromorphic sex chromosomes are usually thought to have originated from a pair of autosomes that acquired a sex-determining locus and subsequently stopped recombining, leading to degeneration of the sex-limited chromosome. The majority of nematode species lack heteromorphic sex chromosomes and determine sex using an X-chromosome counting mechanism, with males being hemizygous for one or more X chromosomes (XX/X0). Some filarial nematode species, including important parasites of humans, have heteromorphic XX/XY karyotypes. It has been assumed that sex is determined by a Y-linked locus in these species. However, karyotypic analyses suggested that filarial Y chromosomes are derived from the unfused homologue of an autosome involved in an X-autosome fusion event. Here, we generated a chromosome-level reference genome for Litomosoides sigmodontis, a filarial nematode with the ancestral filarial karyotype and sex determination mechanism (XX/X0). By mapping the assembled chromosomes to the rhabditid nematode ancestral linkage (or Nigon) elements, we infer that the ancestral filarial X chromosome was the product of a fusion between NigonX (the ancestrally X-linked element) and NigonD (ancestrally autosomal). In the two filarial lineages with XY systems, there have been two independent X-autosome chromosome fusion events involving different autosomal Nigon elements. In both lineages, the region shared by the neo-X and neo-Y chromosomes is within the ancestrally autosomal portion of the X, confirming that the filarial Y chromosomes are derived from the unfused homologue of the autosome. Sex determination in XY filarial nematodes therefore likely continues to operate via the ancestral X-chromosome counting mechanism, rather than via a Y-linked sex-determining locus.

Ancient diversity in host-parasite interaction genes in a model parasitic nematode.

Host-parasite interactions exert strong selection pressures on the genomes of both host and parasite. These interactions can lead to negative frequency-dependent selection, a form of balancing selection that is hypothesised to explain the high levels of polymorphism seen in many host immune and parasite antigen loci. Here, we sequence the genomes of several individuals of Heligmosomoides bakeri, a model parasite of house mice, and Heligmosomoides polygyrus, a closely related parasite of wood mice. Although H. bakeri is commonly referred to as H. polygyrus in the literature, their genomes show levels of divergence that are consistent with at least a million years of independent evolution. The genomes of both species contain hyper-divergent haplotypes that are enriched for proteins that interact with the host immune response. Many of these haplotypes originated prior to the divergence between H. bakeri and H. polygyrus, suggesting that they have been maintained by long-term balancing selection. Together, our results suggest that the selection pressures exerted by the host immune response have played a key role in shaping patterns of genetic diversity in the genomes of parasitic nematodes.

Fine mapping of the Cepaea nemoralis shell colour and mid-banded loci using a high-density linkage map.

Molluscs are a highly speciose phylum that exhibits an astonishing array of colours and patterns, yet relatively little progress has been made in identifying the underlying genes that determine phenotypic variation. One prominent example is the land snail Cepaea nemoralis for which classical genetic studies have shown that around nine loci, several physically linked and inherited together as a 'supergene', control the shell colour and banding polymorphism. As a first step towards identifying the genes involved, we used whole-genome resequencing of individuals from a laboratory cross to construct a high-density linkage map, and then trait mapping to identify 95% confidence intervals for the chromosomal region that contains the supergene, specifically the colour locus (C), and the unlinked mid-banded locus (U). The linkage map is made up of 215,593 markers, ordered into 22 linkage groups, with one large group making up ~27% of the genome. The C locus was mapped to a ~1.3 cM region on linkage group 11, and the U locus was mapped to a ~0.7 cM region on linkage group 15. The linkage map will serve as an important resource for further evolutionary and population genomic studies of C. nemoralis and related species, as well as the identification of candidate genes within the supergene and for the mid-banding phenotype.

Genomics Reveals Complex Population History and Unexpected Diversity of Eurasian Otters (Lutra lutra) in Britain Relative to Genetic Methods.

Conservation genetic analyses of many endangered species have been based on genotyping of microsatellite loci and sequencing of short fragments of mtDNA. The increase in power and resolution afforded by whole genome approaches may challenge conclusions made on limited numbers of loci and maternally inherited haploid markers. Here, we provide a matched comparison of whole genome sequencing versus microsatellite and control region (CR) genotyping for Eurasian otters (Lutra lutra). Previous work identified four genetically differentiated "stronghold" populations of otter in Britain, derived from regional populations that survived the population crash of the 1950s-1980s. Using whole genome resequencing data from 45 samples from across the British stronghold populations, we confirmed some aspects of population structure derived from previous marker-driven studies. Importantly, we showed that genomic signals of the population crash bottlenecks matched evidence from otter population surveys. Unexpectedly, two strongly divergent mitochondrial lineages were identified that were undetectable using CR fragments, and otters in the east of England were genetically distinct and surprisingly variable. We hypothesize that this previously unsuspected variability may derive from past releases of Eurasian otters from other, non-British source populations in England around the time of the population bottleneck. Our work highlights that even reasonably well-studied species may harbor genetic surprises, if studied using modern high-throughput sequencing methods.

MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio high fidelity reads.

BACKGROUND:  PacBio high fidelity (HiFi) sequencing reads are both long (15-20 kb) and highly accurate (> Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing. RESULTS:  MitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (368 Metazoa and 6 Fungi species) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats. CONCLUSIONS:  MitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on GitHub ( https://github.com/marcelauliano/MitoHiFi ). MitoHiFi is available with its dependencies as a Docker container on GitHub (ghcr.io/marcelauliano/mitohifi:master).

Genomes on a Tree (GoaT): A versatile, scalable search engine for genomic and sequencing project metadata across the eukaryotic tree of life.

As genomic data transform our understanding of biodiversity, the Earth BioGenome Project (EBP) has set a goal of generating reference quality genome assemblies for all ~1.9 million described eukaryotic taxa. Meeting this goal requires coordination among many individual regional and taxon-focussed projects working under the EBP umbrella. Large-scale sequencing projects require ready access to validated genome-relevant metadata, such as genome sizes and karyotypes, but these data are dispersed across the literature, and directly measured values are lacking for most taxa. To meet these needs, we have developed Genomes on a Tree (GoaT), an Elasticsearch-powered datastore and search index for genome-relevant metadata and sequencing project plans and statuses. GoaT indexes publicly available metadata for all eukaryotic species and interpolates missing values through phylogenetic comparison. GoaT also holds target priority and sequencing status information for many projects affiliated to the EBP to aid project coordination. Metadata and status attributes in GoaT can be queried through a mature API, a web front end, and a command line interface. The web front end additionally provides summary visualisations for data exploration and reporting (see https://goat.genomehubs.org). GoaT currently holds direct or estimated values for over 70 taxon attributes and over 30 assembly attributes across 1.5 million eukaryotic species. The depth and breadth of curated data, frequent updates, and a versatile query interface make GoaT a powerful data aggregator and portal to explore and report underlying data for the eukaryotic tree of life. We illustrate this utility through a series of use cases from planning through to completion of a genome-sequencing project.

The genome sequence of the spotted kaleidoscope jellyfish, Haliclystus octoradiatus (James-Clark, 1863).

We present a genome assembly from an individual Haliclystus octoradiatus (the spotted kaleidoscope jellyfish; Cnidaria; Staurozoa; Stauromedusae; Haliclystidae). The genome sequence is 262 megabases in span. Most of the assembly (98.3%) is scaffolded into nine (9) chromosomal pseudomolecules. The mitochondrial genome was also assembled and is 18.3 kilobases in length.

Phylogenomic analysis of Wolbachia genomes from the Darwin Tree of Life biodiversity genomics project.

The Darwin Tree of Life (DToL) project aims to sequence all described terrestrial and aquatic eukaryotic species found in Britain and Ireland. Reference genome sequences are generated from single individuals for each target species. In addition to the target genome, sequenced samples often contain genetic material from microbiomes, endosymbionts, parasites, and other cobionts. Wolbachia endosymbiotic bacteria are found in a diversity of terrestrial arthropods and nematodes, with supergroups A and B the most common in insects. We identified and assembled 110 complete Wolbachia genomes from 93 host species spanning 92 families by filtering data from 368 insect species generated by the DToL project. From 15 infected species, we assembled more than one Wolbachia genome, including cases where individuals carried simultaneous supergroup A and B infections. Different insect orders had distinct patterns of infection, with Lepidopteran hosts mostly infected with supergroup B, while infections in Diptera and Hymenoptera were dominated by A-type Wolbachia. Other than these large-scale order-level associations, host and Wolbachia phylogenies revealed no (or very limited) cophylogeny. This points to the occurrence of frequent host switching events, including between insect orders, in the evolutionary history of the Wolbachia pandemic. While supergroup A and B genomes had distinct GC% and GC skew, and B genomes had a larger core gene set and tended to be longer, it was the abundance of copies of bacteriophage WO who was a strong determinant of Wolbachia genome size. Mining raw genome data generated for reference genome assemblies is a robust way of identifying and analysing cobiont genomes and giving greater ecological context for their hosts.

The genome sequence of the common earthworm, Lumbricus terrestris (Linnaeus, 1758).

We present a genome assembly from an individual Lumbricus terrestris (the common earthworm; Annelida; Clitellata; Haplotaxida; Lumbricidae). The genome sequence is 1,056.5 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.93 kilobases in length.

Caenorhabditis nematodes colonize ephemeral resource patches in neotropical forests.

Factors shaping the distribution and abundance of species include life-history traits, population structure, and stochastic colonization-extinction dynamics. Field studies of model species groups help reveal the roles of these factors. Species of Caenorhabditis nematodes are highly divergent at the sequence level but exhibit highly conserved morphology, and many of these species live in sympatry on microbe-rich patches of rotten material. Here, we use field experiments and large-scale opportunistic collections to investigate species composition, abundance, and colonization efficiency of Caenorhabditis species in two of the world's best-studied lowland tropical field sites: Barro Colorado Island in Panamá and La Selva in Sarapiquí, Costa Rica. We observed seven species of Caenorhabditis, four of them known only from these collections. We formally describe two species and place them within the Caenorhabditis phylogeny. While these localities contain species from many parts of the phylogeny, both localities were dominated by globally distributed androdiecious species. We found that Caenorhabditis individuals were able to colonize baits accessible only through phoresy and preferentially colonized baits that were in direct contact with the ground. We estimate the number of colonization events per patch to be low.

Genome-enabled discovery of candidate virulence loci in Striga hermonthica, a devastating parasite of African cereal crops.

Parasites have evolved proteins, virulence factors (VFs), that facilitate plant colonisation, however VFs mediating parasitic plant-host interactions are poorly understood. Striga hermonthica is an obligate, root-parasitic plant of cereal hosts in sub-Saharan Africa, causing devastating yield losses. Understanding the molecular nature and allelic variation of VFs in S. hermonthica is essential for breeding resistance and delaying the evolution of parasite virulence. We assembled the S. hermonthica genome and identified secreted proteins using in silico prediction. Pooled sequencing of parasites growing on a susceptible and a strongly resistant rice host allowed us to scan for loci where selection imposed by the resistant host had elevated the frequency of alleles contributing to successful colonisation. Thirty-eight putatively secreted VFs had very different allele frequencies with functions including host cell wall modification, protease or protease inhibitor and kinase activities. These candidate loci had significantly higher Tajima's D than the genomic background, consistent with balancing selection. Our results reveal diverse strategies used by S. hermonthica to overcome different layers of host resistance. Understanding the maintenance of variation at virulence loci by balancing selection will be critical to managing the evolution of virulence as part of a sustainable control strategy.