Species are often treated as evolutionarily significant units of diversity that reflect patterns of gene flow and selection. In contrast, higher taxa are mostly regarded as convenient labels for levels in the tree of life, which reflect evolutionary history if defined cladistically but are assumed to have no real significance for ongoing evolution. We test the alternative hypothesis that some higher taxa are evolutionarily significant units with coherent patterns of selection on their constituent species. Specifically, we ask whether the big 4 orders of holometabolous insects, namely Coleoptera, Diptera, Hymenoptera, and Lepidoptera, display divergent, but internally conserved patterns of selection acting on protein-coding genes. Analyzing orthologous genes from whole genome sequence data for multiple species per order, we find that, in most genes, selection on roughly one fifth of codons is conserved within each order but differs significantly among orders. The shift is associated with variation in GC content among orders, but primarily at codon 2nd positions hence due to selection rather than mutational or repair bias. Comparison of alternative models assigning different taxonomic levels (either more lumped or divided than orders) shows that best models always specify Hymenoptera and Lepidoptera as coherent units, whereas patterns of selection on protein-coding genes within Coleoptera and especially Diptera are better explained by subdividing them further. We hypothesise that some aspect of the general lifestyle, body plan or genetic makeup of orders (or of nested clades within Coleoptera and Diptera) leads to conserved patterns of selection across protein-coding genes within them, whereas constraints differ among them. The emergence of whole-genome data for broad and deep phylogenetic samples will allow this hypothesis of evolutionarily significant higher taxa versus more evenly dispersed shifts in selection across genes to be tested further.

The distinction between sexual and asexual reproduction is fundamental to eukaryotic evolution. Testing theories about the evolution of reproductive modes first requires knowing whether sex is present or absent in a population. While this seems straightforward, the literature on asexuality reflects a history of shifting claims and uncertainty regarding reproductive mode, especially where sex is potentially rare or cryptic. Here, we develop a new framework to explore the challenges in detecting and quantifying sexual reproduction from population genomic data, based on genetic simulations. We first show that commonly calculated population genetic statistics do not reliably distinguish sexual and obligate asexual scenarios if asexuality is accompanied by sex-independent homologous recombination, as emerging evidence suggests is often the case. We then present a new method to quantify the relationship between evolutionary trees and mode of reproduction by exhaustively classifying local trees for pairs of diploid individuals using ancestral recombination graphs (ARGs). This approach accurately distinguishes signatures of genetic exchange and homologous recombination, although uncertainty remains due to unavoidable biases in the steps needed to reconstruct trees from genome data. We introduce a new statistic and simulation models to account for common reconstruction biases. Our approach offers the potential for improved quantitative inference of reproductive modes that is extendable and applicable to a broad range of eukaryotes. Significance Statement Determining how often, if at all, organisms have sex has implications across biology. Studies often use population genomic data to interrogate the private life of putative asexuals, but the answers prove surprisingly inconclusive. We develop a framework to explore the challenges in detecting and quantifying rates of sex. New simulation models show how sex-independent recombination, which occurs widely across a range of asexual eukaryotes, causes genetic patterns to resemble sexual populations, even when sex is absent. An approach based on ancestral recombination graphs (ARGs) and classification of local trees accounts for these problems and quantifies remaining uncertainty due to inevitable reconstruction biases. Our framework will enable improved inferences of reproductive mode across a wide range of eukaryotes. ### Competing Interest Statement The authors have declared no competing interest.

Understanding the structural changes that enable enzymes to remain active in extreme thermal conditions is of broad scientific interest for both fundamental and applied biological research. Three key mechanisms that underlie the thermal adaptation of enzymes are modifications in structural flexibility, compactness, and the contacts formed among amino acids. However, most previous studies on these topics have been limited to small sample sizes or a narrow taxonomic focus, and the importance of these factors to thermal adaptation remains poorly understood. In this study, we combined molecular dynamics simulations and phylogenetic comparative analyses to thoroughly analyze the structural factors underlying thermal adaptation in adenylate kinase—a key enzyme involved in cellular energy balance and homeostasis—across 70 prokaryotic species. We detect systematic increases in the flexibility of the enzyme with temperature, both across and within species. In contrast, structural compactness appears to be almost completely independent of temperature. Finally, we uncover a remarkable diversity in the number and types of amino acid contacts observed in different adenylate kinases that cannot be explained solely by temperature. Our results suggest that there are multiple paths toward the adaptation of prokaryotic adenylate kinases to extreme thermal environments and that these paths are generally accessible through changes in flexibility.

Most knowledge about bacterial evolution and ecological interactions comes from laboratory studies. One difference between the wild and most laboratory experiments is the diversity of bacterial taxa present. Understanding how wild bacteria respond to perturbation therefore requires consideration of how ecological sorting, colonization, and genetic changes of constituent species interact. Ecological sorting of species might reduce evolutionary rates and make communities robust to disturbance, or it could amplify selection pressures and lead to unstable co-evolutionary cascades. Even estimates of basic rates of ecological sorting, dispersal, and genetic change are rare. Here, we addressed these knowledge gaps by liming wild decomposer communities living in beech tree holes and tracking ecological and evolutionary responses for 12 weeks. Overall, tree hole communities were extremely robust to liming involving short-term pulses up to 4 pH units and long-term increases up to 2 pH units. Species diversity and composition displayed significant but small changes in treatment tree holes compared to control ones. New bacterial taxa colonized at a low rate that did not vary with liming. Genetic changes in the frequency of single nucleotide polymorphisms in metagenome assembled genomes occurred at rates that were both comparable to and correlated with ecological changes in the same metagenome assembled genomes, but the rate of genetic changes did not vary between limed and control tree holes. Analysis of rates of genetic change estimated low effective population size (~104) and generation times of roughly 1 day. Our study provides estimates of rates of ecological and evolutionary processes in wild bacterial communities, which displayed remarkable robustness to our experimental perturbation.

Coevolutionary antagonism generates relentless selection that can favour genetic exchange, including transfer of antibiotic synthesis and resistance genes among bacteria, and sexual recombination of disease resistance alleles in eukaryotes. We report an unusual link between biological conflict and DNA transfer in bdelloid rotifers, microscopic animals whose genomes show elevated levels of horizontal gene transfer from non-metazoan taxa. When rotifers were challenged with a fungal pathogen, horizontally acquired genes were over twice as likely to be upregulated as other genes - a stronger enrichment than observed for abiotic stressors. Among hundreds of upregulated genes, the most markedly overrepresented were clusters resembling bacterial polyketide and nonribosomal peptide synthetases that produce antibiotics. Upregulation of these clusters in a pathogen-resistant rotifer species was nearly ten times stronger than in a susceptible species. By acquiring, domesticating, and expressing non-metazoan biosynthetic pathways, bdelloids may have evolved to resist natural enemies using antimicrobial mechanisms absent from other animals.

The endpoint of speciation has been viewed as complete isolation and the absence of gene flow between species. If the influx of genes from another species is maladaptive because species have different adaptations and genetic backgrounds, selection should favour the closing of species boundaries and zero gene flow, a process known as reinforcement. Recently, numerous cases of gene flow between species have been identified, many of which involved adaptive introgression of beneficial alleles. These cases could reflect transient states on the way to closed species boundaries or the result of declining strength or efficacy of selection for reinforcement as the level of gene flow approaches zero. An alternative hypothesis, however, is that selection favours porous species boundaries that allow beneficial alleles to cross, especially in changing environments. This perspective evaluates the conditions that would be needed for selection to favour porous species boundaries and the evidence for them. A contrast is made between hybridization in sexual eukaryotes and gene transfer via homologous recombination in bacteria. Current evidence is inconclusive on whether non-zero gene flow is favoured by selection. Studies are needed that quantify selection gradients on rates of gene flow and test for evolution towards intermediate values, especially experiments that manipulate conditions and track evolution for multiple generations. Estimation of gene flow networks for more clades and regional assemblages using emerging genome data will also allow the evolutionary determinants of interspecific gene flow to be better understood.

Bdelloid rotifers constitute a class of microscopic animals living in freshwater habitats worldwide. Several strange features of bdelloids have drawn attention: their ability to tolerate desiccation and other stresses, a lack of reported males across the clade despite centuries of study, and unusually high numbers of horizontally acquired, non-metazoan genes. Genome sequencing is transforming our understanding of their lifestyle and its consequences, while in turn providing wider insights about recombination and genome organisation in animals. Many questions remain, not least how to reconcile apparent genomic signatures of sex with the continued absence of reported males, why bdelloids have so many horizontally acquired genes, and how their remarkable ability to survive stress interacts with recombination and other genomic processes.

Outbreaks of fungal diseases have devastated plants and animals throughout history. Over the past century, the repeated emergence of coffee wilt disease caused by the fungal pathogen Fusarium xylarioides severely impacted coffee production across sub-Saharan Africa. To improve the disease management of such pathogens, it is crucial to understand their genetic structure and evolutionary potential. We compared the genomes of 13 historic strains spanning 6 decades and multiple disease outbreaks to investigate population structure and host specialisation. We found that F. xylarioides comprised at least 4 distinct lineages: 1 host-specific to Coffea arabica, 1 to C. canephora var. robusta, and 2 historic lineages isolated from various Coffea species. The presence/absence of large genomic regions across populations, the higher genetic similarities of these regions between species than expected based on genome-wide divergence and their locations in different loci in genomes across populations showed that horizontal transfers of effector genes from members of the F. oxysporum species complex contributed to host specificity. Multiple transfers into F. xylarioides populations matched different parts of the F. oxysporum mobile pathogenicity chromosome and were enriched in effector genes and transposons. Effector genes in this region and other carbohydrate-active enzymes important in the breakdown of plant cell walls were shown by transcriptomics to be highly expressed during infection of C. arabica by the fungal arabica strains. Widespread sharing of specific transposons between F. xylarioides and F. oxysporum, and the correspondence of a putative horizontally transferred regions to a Starship (large mobile element involved in horizontal gene transfers in fungi), reinforce the inference of horizontal transfers and suggest that mobile elements were involved. Our results support the hypothesis that horizontal gene transfers contributed to the repeated emergence of coffee wilt disease.

The clade Syndermata includes the endoparasitic Acanthocephala, the epibiotic Seisonidea, and the free-living Bdelloidea and Monogononta. The phylogeny of Syndermata is highly debated, hindering the understanding of the evolution of morphological features, reproductive modes, and lifestyles within the group. Here, we use publicly available whole-genome data to re-evaluate syndermatan phylogeny and assess the credibility of alternative hypotheses, using a new combination of phylogenomic methods. We found that the Hemirotifera and Pararotatoria hypotheses were recovered under combinations of datasets and methods with reduced possibility of systematic error in concatenation-based analyses. In contrast, the Seisonidea-sister and Lemniscea hypotheses were recovered under dataset combinations with increased possibility of systematic error. Hemirotifera was further supported by whole-genome microsynteny analyses and species-tree methods that use multi-copy orthogroups after removing distantly related outgroups. Pararotatoria was only partially supported by microsynteny-based phylogenomic reconstructions. Hence, Hemirotifera and partially Pararotatoria were supported by independent phylogenetic methods and data-evaluation approaches. These two hypotheses have important implications for the evolution of syndermatan morphological features, such as the gradual reduction of locomotory ciliation from the common ancestor of Syndermata in the stem lineage of Pararotatoria. Our study illustrates the importance of combining various types of evidence to resolve difficult phylogenetic questions.

Fungal plant pathogens cause major crop losses worldwide, with many featuring compartmentalised genomes that include both core and accessory regions, which are believed to drive adaptation. The highly host-specific fungus Colletotrichum lupini greatly impacts lupin (Lupinus spp.) cultivation. This pathogen is part of clade 1 of the C. acutatum species complex and comprises four genetically uniform, presumably clonal, lineages (I-IV). Despite this, variation in virulence and morphology has been observed within these lineages. To investigate the potential sources of genetic variability in this asexual fungus, we compared the genomes of 16 C. lupini strains and 17 related Colletotrichum species. Phylogenomics confirmed the presence of four distinct lineages, but further examination based on genome size, gene content, transposable elements (TEs), and deletions revealed that lineage II could be split into two groups, II-A and II-B. TE content varied between lineages and correlated strongly with genome size variation, supporting a role for TEs in genome expansion in this species. Pangenome analysis revealed a highly variable accessory genome, including a minichromosome present in lineages II, III, and IV, but absent in lineage I. Accessory genes and effectors appeared to cluster in proximity to TEs. Presence/absence variation of putative effectors was lineage-specific, suggesting that these genes play a crucial role in determining host range. Notably, no effectors were found on the TE-rich minichromosome. Our findings shed light on the potential mechanisms generating genetic diversity in this asexual fungal pathogen that could aid future disease management.

Fungi produce a vast number of secondary metabolites that shape their interactions with other organisms and the environment. Characterizing the genes underpinning metabolite synthesis is therefore key to understanding fungal evolution and adaptation. Lichenized fungi represent almost one-third of Ascomycota diversity and boast impressive secondary metabolites repertoires. However, most lichen biosynthetic genes have not been linked to their metabolite products. Here we used metagenomic sequencing to survey gene families associated with production of anthraquinones, UV-protectant secondary metabolites present in various fungi, but especially abundant in a diverse order of lichens, the Teloschistales (class Lecanoromycetes, phylum Ascomycota). We successfully assembled 24 new, high-quality lichenized-fungal genomes de novo and combined them with publicly available Lecanoromycetes genomes from taxa with diverse secondary chemistry to produce a whole-genome tree. Secondary metabolite biosynthetic gene cluster (BGC) analysis showed that whilst lichen BGCs are numerous and highly dissimilar, core enzyme genes are generally conserved across taxa. This suggests metabolite diversification occurs via re-shuffling existing enzyme genes with novel accessory genes rather than BGC gains/losses or de novo gene evolution. We identified putative anthraquinone BGCs in our lichen dataset that appear homologous to anthraquinone clusters from non-lichenized fungi, suggesting these genes were present in the common ancestor of the subphylum Pezizomycotina. Finally, we identified unique transporter genes in Teloschistales anthraquinone BGCs that may explain why these metabolites are so abundant and ubiquitous in these lichens. Our results support the importance of metagenomics for understanding the secondary metabolism of non-model fungi such as lichens.

BACKGROUND:Intraspecific public goods are commonly shared within microbial populations, where the benefits of public goods are largely limited to closely related conspecifics. One example is the production of iron-scavenging siderophores that deliver iron to cells via specific cell envelope receptor and transport systems. Intraspecific social exploitation of siderophore producers is common, since non-producers avoid the costs of production but retain the cell envelope machinery for siderophore uptake. However, little is known about how interactions between species (i.e., interspecific interactions) can shape intraspecific public goods exploitation. Here, we predicted that strong competition for iron between species in diverse communities will increase costs of siderophore cooperation, and hence drive intraspecific exploitation. We examined how increasing microbial community species diversity shapes intraspecific social dynamics by monitoring the growth of siderophore producers and non-producers of the plant-growth promoting bacterium Pseudomonas fluorescens, embedded within tree-hole microbial communities ranging from 2 to 15 species.RESULTS:We find, contrary to our prediction, that siderophore production is favoured at higher levels of community species richness, driven by increased likelihood of encountering key species that reduce the growth of siderophore non-producing (but not producing) strains of P. fluorescens.CONCLUSIONS:Our results suggest that maintaining a diverse soil microbiota could partly contribute to the maintenance of siderophore production in natural communities.

Coffee wilt disease, caused by the fungus Fusarium xylarioides , is a vascular wilt disease that has affected coffee production in sub-Saharan Africa over the past century. Today, the disease has two host-specific populations specialising on arabica and robusta coffee crops, which grow at high and low altitude, respectively. Here we test whether adaptation to different temperatures contributes to specialisation of the fungi on each crop. Firstly, climate models show that the severity of the arabica and robusta populations of coffee wilt disease correlates with temperature. The robusta population shows higher peak severity than the arabica population overall, but the latter has greater cold tolerance. Secondly, growth assays of thermal performance of fungal strains in vitro show that, while robusta strains grow faster than arabicas at intermediate temperatures, the arabica strains have higher sporulation and spore germination rates at temperatures below 15ºC. The match between environmental patterns of severity in nature with thermal performance of fungal cultures in the laboratory supports a role for temperature adaptation in specialisation on arabica and robusta coffee. Extrapolating our temperature-models to future climate change predicts that disease severity could decline on average due to increased temperature but could increase in some coffee-growing regions.

Gut microbiomes in infancy have a profound impact on health in adulthood. CRISPRs play an essential role in the interaction between bacteria and phages. However, the dynamics of CRISPRs in gut microbiomes during early life are poorly understood. In this study, using shotgun metagenomic sequencing data from 82 Swedish infants' gut microbiomes, 1882 candidate CRISPRs were identified, and their dynamics were analysed. We found large- scale turnover of CRISPRs and their spacers during the first year of life. As well as changes in relative abundance of the bacteria containing CRISPR, acquisition, loss and mutation of spacers were observed within the same CRISPR array sampled over time. Accordingly, the inferred interaction network of bacteria and phage was distinct at different times. This research underpins CRISPR dynamics and their potential role in the interaction between bacteria and phage in early life.

The first microbial culture collection was established in 1890, and subsequently, over the course of the twentieth century, the number of culture collections grew substantially. One such collection was the CABI-IMI Genetic Resources Collection, informally initiated at the origins of the Commonwealth Mycological Institute in 1920 and established as a UK National Collection in 1947. Its holdings reflect the activities of CABI in agriculture and the environment. Like many collections, it was originally established as a taxonomic reference resource. Over the years, deposits have included strains isolated from disease outbreaks in major crops from all parts of the world. Recent developments in genome sequencing technology and bioinformatic analysis have opened up the potential to characterise historic strains to shed more insights on their biology and evolution. In this paper we describe how the resources held within the CABI-IMI culture collection have helped track the emergence and divergence of Fusarium xylarioides, the coffee wilt pathogen, with a specific focus on the evolution of pathogenicity. Such studies illustrate the value held within the world’s culture collections—their importance in underpinning science and developing our understanding and evolution of plant diseases as well as how the impact of disease can be mitigated in response to climate change, in order to increase yields and feed the world’s burgeoning population.

Intraspecific public goods are commonly shared within microbial populations, where the benefits of public goods are largely limited to closely related conspecifics. One example is the production of iron-scavenging siderophores that deliver iron to cells via specific cell envelope receptor and transport systems. Intraspecific social exploitation of siderophore producers is common, since non-producers avoid the costs of production but retain the cell envelope machinery for siderophore uptake. However, little is known about how interactions between species (i.e. interspecific interactions) can shape intraspecific public goods exploitation. Here, we predicted that strong competition for iron between species in diverse communities will increase costs of siderophore cooperation, and hence select for increased intraspecific exploitation. We examined how increasing microbial community species diversity shapes intraspecific social dynamics by monitoring the growth of siderophore producers and non-producers of the plant-growth promoting bacterium Pseudomonas fluorescens , embedded within tree-hole microbial communities ranging from 2-15 species. We find, contrary to our prediction, that siderophore exploitation is reduced in high diversity communities, driven by increased likelihood of encountering key species that reduce the growth of siderophore non-producing (but not producing) strains of P. fluorescens . Our results suggest that maintaining a healthy soil microbiota could contribute to the maintenance of siderophore production in natural communities.

PREMISE:Plants have evolved different ecological strategies in response to environmental challenges, and a higher lability of such strategies is more common in plant groups that adapt to various niches. Crassula (Crassulaceae), occurring in varied mesic to xeric habitats, exhibits a remarkable diversity of life-forms. However, whether any particular life-form trait has shaped species diversification in Crassula has remained unexplored. This study aims to investigate diversification patterns within Crassula and identify potential links to its life-form evolution.METHODS:A phylogenetic tree of 140 Crassula taxa was reconstructed using plastid and nuclear loci and dated based on the nuclear DNA information only. We reconstructed ancestral life-form characters to estimate the evolutionary trends of ecophysiological change, and subsequently estimated net diversification rates. Multiple diversification models were applied to examine the association between certain life-forms and net diversification rates.RESULTS:Our findings confirm a radiation within Crassula in the last 10 million years. A configuration of net diversification rate shifts was detected, which coincides with the emergence of a speciose lineage during the late Miocene. The results of ancestral state reconstruction demonstrate a high lability of life-forms in Crassula, and the trait-dependent diversification analyses revealed that the increased diversification is strongly associated with a compact growth form.CONCLUSIONS:Transitions between life-forms in Crassula seem to have driven adaptation and shaped diversification of this genus across various habitats. The diversification patterns we inferred are similar to those observed in other major succulent lineages, with the most-speciose clades originating in the late Miocene.

The dynamics of the gut microbiome in infancy have a profound impact on health in adulthood, but the ecological mechanism underlying the dynamics between bacteria and bacteriophages remains poorly understood. CRISPR is a bacterial adaptive immune system to resist bacteriophages; however, the role they play in the dynamics in infants’ gut microbiota is unknown. In this work, using large-scale metagenomic sequencing data from 82 Sweden infants’ gut microbiomes, 1882 candidate CRISPRs were identified and their dynamics were analyzed. The results showed CRISPRs were distributed in dominant bacteria and could target distinct bacteriophages at different time points with largely alternated spacers. In the putative identical CRISPRs, we found the CRISPRs could acquire new spacers and loss old spacers during the first year. In addition, it is the first time to report that gender was a major factor to determine the bacterial richness and the number of CRISPRs and host range of bacteriophages was narrow in silico . Therefore, we concluded that CRISPRs were associated with bacterial expansion. This work improves the understanding of the ecological mechanism behind dynamics in the early life of the human gut and substantially expands the repertoire of predicted CRISPRs providing a resource to study the function of bacterial unknown genes and to enhance the performance of these beneficial bacteria by CRISPR gene-editing technology. ### Competing Interest Statement The authors have declared no competing interest.

Plasmids are extrachromosomal segments of DNA that can transfer genes between bacterial cells. Many plasmid genes benefit bacteria but cause harm to human health by granting antibiotic resistance to pathogens. Transfer rate is a key parameter for predicting plasmid dynamics, but observed rates are highly variable, and the effects of selective forces on their evolution are unclear. We apply evolutionary analysis to plasmid conjugation models to investigate selective pressures affecting plasmid transfer rate, emphasizing host versus plasmid control, the costs of plasmid transfer, and the role of recipient cells. Our analyses show that plasmid-determined transfer rates can be predicted with three parameters (host growth rate, plasmid loss rate, and the cost of plasmid transfer on growth) under some conditions. We also show that low-frequency genetic variation in transfer rate can accumulate, facilitating rapid adaptation to changing conditions. Furthermore, reduced transfer rates due to host control have limited effects on plasmid prevalence until low enough to prevent plasmid persistence. These results provide a framework to predict plasmid transfer rate evolution in different environments and demonstrate the limited impact of host mechanisms to control the costs incurred when plasmids are present.