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Background Almost 10% of all known animal species belong to Lepidoptera: moths and butterflies. To understand how this incredible diversity evolved we assess the role of gene gain in driving early lepidopteran evolution. Here, we compared the complete genomes of 115 insect species, including 99 Lepidoptera, to search for novel genes coincident with the emergence of Lepidoptera. Results We find 217 orthogroups or gene families which emerged on the branch leading to Lepidoptera; of these 177 likely arose by gene duplication followed by extensive sequence divergence, 2 are candidates for origin by horizontal gene transfer, and 38 have no known homology outside of Lepidoptera and possibly arose via de novo gene genesis. We focus on two new gene families that are conserved across all lepidopteran species and underwent extensive duplication, suggesting important roles in lepidopteran biology. One encodes a family of sugar and ion transporter molecules, potentially involved in the evolution of diverse feeding behaviours in early Lepidoptera. The second encodes a family of unusual propeller-shaped proteins that likely originated by horizontal gene transfer from Spiroplasma bacteria; we name these the Lepidoptera propellin genes. Conclusion We provide the first insights into the role of genetic novelty in the early evolution of Lepidoptera. This gives new insight into the rate of gene gain during the evolution of the order as well as providing context on the likely mechanisms of origin. We describe examples of new genes which were retained and duplicated further in all lepidopteran species, suggesting their importance in Lepidoptera evolution.

The proportions of A:T and G:C nucleotide pairs are often unequal and can vary greatly between animal species and along chromosomes. The causes and consequences of this variation are incompletely understood. The recent release of high-quality genome sequences from the Darwin Tree of Life and other large-scale genome projects provides an opportunity for GC heterogeneity to be compared across a large number of insect species. Here we analyse GC content along chromosomes, and within protein-coding genes and codons, of 150 insect species from four holometabolous orders: Coleoptera, Diptera, Hymenoptera, and Lepidoptera. We find that protein-coding sequences have higher GC content than the genome average, and that Lepidoptera generally have higher GC content than the other three insect orders examined. GC content is higher in small chromosomes in most Lepidoptera species, but this pattern is less consistent in other orders. GC content also increases towards subtelomeric regions within protein-coding genes in Diptera, Coleoptera and Lepidoptera. Two species of Diptera, Bombylius major and B. discolor, have very atypical genomes with ubiquitous increase in AT content, especially at third codon positions. Despite dramatic AT-biased codon usage, we find no evidence that this has driven divergent protein evolution. We argue that the GC landscape of Lepidoptera, Diptera and Coleoptera genomes is influenced by GC-biased gene conversion, strongest in Lepidoptera, with some outlier taxa affected drastically by counteracting processes.

Abstract Color vision in insects is determined by signaling cascades, central to which are opsin proteins, resulting in sensitivity to light at different wavelengths. In certain insect groups, lineage-specific evolution of opsin genes, in terms of copy number, shifts in expression patterns, and functional amino acid substitutions, has resulted in changes in color vision with subsequent behavioral and niche adaptations. Lepidoptera are a fascinating model to address whether evolutionary change in opsin content and sequence evolution are associated with changes in vision phenotype. Until recently, the lack of high-quality genome data representing broad sampling across the lepidopteran phylogeny has greatly limited our ability to accurately address this question. Here, we annotate opsin genes in 219 lepidopteran genomes representing 33 families, reconstruct their evolutionary history, and analyze shifts in selective pressures and expression between genes and species. We discover 44 duplication events in opsin genes across ∼300 million years of lepidopteran evolution. While many duplication events are species or family specific, we find retention of an ancient long-wavelength-sensitive (LW) opsin duplication derived by retrotransposition within the speciose superfamily Noctuoidea (in the families Nolidae, Erebidae, and Noctuidae). This conserved LW retrogene shows life stage–specific expression suggesting visual sensitivities or other sensory functions specific to the early larval stage. This study provides a comprehensive order-wide view of opsin evolution across Lepidoptera, showcasing high rates of opsin duplications and changes in expression patterns.

Abstract There is conflicting evidence as to whether Porifera (sponges) or Ctenophora (comb jellies) comprise the root of the animal phylogeny. Support for either a Porifera-sister or Ctenophore-sister tree has been extensively examined in the context of model selection, taxon sampling, and outgroup selection. The influence of dataset construction is comparatively understudied. We re-examine five animal phylogeny datasets that have supported either root hypothesis using an approach designed to enrich orthologous signal in phylogenomic datasets. We find that many component orthogroups in animal datasets fail to recover major lineages as monophyletic with the exception of Ctenophora, regardless of the supported root. Enriching these datasets to retain orthogroups recovering ≥3 major lineages reduces dataset size by up to 50% while retaining underlying phylogenetic information and taxon sampling. Site-heterogeneous phylogenomic analysis of these enriched datasets recovers both Porifera-sister and Ctenophora-sister positions, even with additional constraints on outgroup sampling. Two datasets which previously supported Ctenophora-sister support Porifera-sister upon enrichment. All enriched datasets display improved model fitness under posterior predictive analysis. While not conclusively rooting animals at either Porifera or Ctenophora, we do see an increase in signal for Porifera-sister and a decrease in signal for Ctenophore-sister when data are filtered for orthologous signal. Our results indicate that dataset size and construction as well as model fit influence animal root inference.

Conflicting studies place a group of bilaterian invertebrates containing xenoturbellids and acoelomorphs, the Xenacoelomorpha, as either the primary emerging bilaterian phylum, or within Deuterostomia, sister to Ambulacraria. While their placement as sister to the rest of Bilateria supports relatively simple morphology in the ancestral bilaterian, their alternative placement within Deuterostomia suggests a morphologically complex ancestral Bilaterian along with extensive loss of major phenotypic traits in the Xenacoelomorpha. More recently, further studies have brought into question whether Deuterostomia should be considered monophyletic at all. Hidden paralogy presents a major challenge for reconstructing species phylogenies. Here we assess whether hidden paralogy has contributed to the conflict over the placement of Xenacoelomorpha. Our approach assesses previously published datasets, enriching for orthogroups whose gene trees support well resolved clans elsewhere in the animal tree of life. We find that the majority of constituent genes in previously published datasets violate incontestable clans, suggesting that hidden paralogy is rife at this depth. We demonstrate that enrichment for genes with orthologous signal alters the final topology that is inferred, whilst simultaneously improving fit of the model to the data. We discover increased, but ultimately not conclusive, support for the existence of Xenambulacraria in our orthology enriched set of genes. At a time when we are steadily progressing towards sequencing all of life on the planet, we argue that long-standing contentious issues in the tree of life will be resolved using smaller amounts of better quality data that can be modelled adequately. Competing Interest Statement The authors have declared no competing interest.

The specification of the anterior-posterior axis in the embryo is a crucial step in early insect development. Despite its importance, the underlying genetic and regulatory architecture controlling this process varies significantly between species. In cyclorrhaphan flies, such as Drosophila melanogaster, anterior determination is controlled by the transcription factor bicoid, which emerged through duplication of the ancestral Hox3 gene (called zen in insects). With new, high-quality genomic data we mine the genomes of 186 dipteran species, and find presence of bicoid in non-cyclorrhaphan flies, including in the bee flies (family Bombyliidae) and the stiletto flies (Therevidae). We confirm maternal expression and localisation of the non-cyclorrhaphan bicoid mRNA to the anterior region of the unfertilised oocyte in the dark edged bee fly, Bombylius major. To determine the timing and origin of bicoid, we address uncertainty in the dipteran phylogeny, uncovering a ladder-like topology in the branching orders of the early Brachycera lineages. This new species phylogeny suggests that bicoid emerged at the common ancestor of Bombyliidae, Asiloidea, and Eremoneura (collectively Heterodactyla), and was subsequently lost at least 16 times. These findings expand our understanding of the early developmental processes in flies and provide new insights into the backbone phylogeny of Diptera and the evolution of bicoid.

The proportions of A:T and G:C nucleotide pairs are often unequal and can vary greatly between animal species and along chromosomes. The causes and consequences of this variation are incompletely understood. The recent release of high-quality genome sequences from the Darwin Tree of Life and other large-scale genome projects provides an opportunity for GC heterogeneity to be compared across a large number of insect species. Here we analyse GC content along chromosomes, and within protein-coding genes and codons, of 150 insect species from four holometabolous orders: Coleoptera, Diptera, Hymenoptera, and Lepidoptera. We find that protein-coding sequences have higher GC content than the genome average, and that Lepidoptera generally have higher GC content than the other three insect orders examined. GC content is higher in small chromosomes in most Lepidoptera species, but this pattern is less consistent in other orders. GC content also increases towards subtelomeric regions within protein-coding genes in Hymenoptera, Coleoptera and, most strikingly, Lepidoptera. Two species of Diptera, Bombylius major and B. discolor, have very atypical genomes with ubiquitous increase in AT content, especially at third codon positions. Despite dramatic AT-biased codon usage, we find no evidence that this has driven divergent protein evolution. We argue that the GC landscape of Lepidoptera, Hymenoptera and Coleoptera genomes is influenced by GC-biased gene conversion, strongest in Lepidoptera, with some outlier taxa affected drastically by counteracting processes.

Colour vision in insects is determined by signalling cascades, central to which are opsin proteins, resulting in sensitivity to light at different wavelengths. In certain insect groups, lineage specific evolution of opsin genes, in terms of copy number, shifts in expression patterns, and functional amino acid substitutions, has resulted in changes in colour vision with subsequent behavioural and niche adaptations. Lepidoptera are a fascinating model to address whether evolutionary change in opsin content and sequence evolution are associated with changes in vision phenotype. Until recently, the lack of high quality genome data representing broad sampling across the lepidopteran phylogeny has greatly limited our ability to accurately address this question. Here, we annotate opsin genes in 219 lepidopteran genomes representing 33 families, reconstruct their evolutionary history, and analyse shifts in selective pressures and expression between genes and species. We discover 44 duplication events in opsin genes across ∼300 million years of lepidopteran evolution. While many duplication events are species or family specific, we find retention of an ancient long-wavelength sensitive (LW) opsin duplication derived by retrotransposition within the speciose superfamily Noctuoidea (in the families Nolidae, Erebidae, and Noctuidae). This conserved LW retrogene shows life stage specific expression suggesting visual sensitivities or other sensory functions specific to the early larval stage. This study provides a comprehensive order-wide view of opsin evolution across Lepidoptera, showcasing high rates of opsin duplications and changes in expression patterns.

A molecular level perspective on how novel phenotypes evolve is contingent on our understanding of how genomes evolve through time, and of particular interest is how novel elements emerge or are lost. Mechanisms of protein evolution such as gene duplication have been well established. Studies of gene fusion events show they often generate novel functions and adaptive benefits. Identifying gene fusion and fission events on a genome scale allows us to establish the mode and tempo of emergence of composite genes across the animal tree of life, and allows us to test the repeatability of evolution in terms of determining how often composite genes can arise independently. Here we show that ∼5% of all animal gene families are composite, and their phylogenetic distribution suggests an abrupt, rather than gradual, emergence during animal evolution. We find that gene fusion occurs at a higher rate than fission (73.3% vs 25.4%) in animal composite genes, but many gene fusions (79% of the 73.3%) have more complex patterns including subsequent fission or loss. We demonstrate that nodes such as Bilateria, Euteleostomi, and Eutheria, have significantly higher rates of accumulation of composite genes. We observe that in general deuterostomes have a greater amount of composite genes as compared to protostomes. Intriguingly, up to 41% of composite gene families have evolved independently in different clades showing that the same solutions to protein innovation have evolved time and again in animals. New genes emerge and are lost from genomes over time. Mechanisms that can produce new genes include, but are not limited to, gene duplication, retrotransposition, de novo gene genesis, and gene fusion/fission. In this work, we show that new genes formed by fusing distinct homologous gene families together comprise a significant portion of the animal proteome. Their pattern of emergence through time is not gradual throughout the animal phylogeny - it is intensified on nodes of major transition in animal phylogeny. Interestingly, we see that evolution replays the tape frequently in these genes with 41% of gene fusion/fission events occurring independently throughout animal evolution.