Lepidopteran Genomes Research Articles

Transposable element accumulation drives genome size increase in Hylesia metabus (Lepidoptera: Saturniidae), an urticating moth species from South America

Abstract We present the first nuclear genome assembly and a complete mitogenome for Hylesia metabus (Arthropoda; Insecta; Lepidoptera; Saturniidae). The assembled nuclear genome sequence is 1,271 Mb long, which is among the 10 largest lepidopteran genome assemblies published to date. It is scaffolded in 31 pseudo chromosomes, has a BUSCO score of 99.5%, and has a highly conserved synteny compared to phylogenetically close species. Repetitive elements make up 67% of the nuclear genome and are mainly located in intergenic regions, among which LINEs were predominant, with CR1-Zenon being the most abundant. Phylogenetic and comparative analyses of H. metabus assembly and 17 additional Saturniidae and Sphingidae assemblies suggested that an accumulation of repetitive elements likely led to the increased size of H. metabus’ genome. Gene annotation using Helixer identified 26,122 transcripts. The Z scaffold was identified using both a synteny analysis and variations of coverage for two resequenced male and female H. metabus. The H. metabus nuclear genome and mitogenome assemblies can be found and browsed on the BIPAA website and constitute useful resources for future population and comparative genomics studies.

Chromosome-Level Assembly and Annotation of the Pearly Heath Coenonympha arcania Butterfly Genome.

We present the first chromosome-level genome assembly and annotation of the pearly heath Coenonympha arcania, generated with a PacBio HiFi sequencing approach and complemented with Hi-C data. We additionally compare synteny, gene, and repeat content between C. arcania and other Lepidopteran genomes. This reference genome will enable future population genomics studies with Coenonympha butterflies, a species-rich genus that encompasses some of the most highly endangered butterfly taxa in Europe.

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.

Opsin Gene Duplication in Lepidoptera: Retrotransposition, Sex Linkage, and Gene Expression.

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.

Genome, host genome integration, and gene expression in Diadegma fenestrale ichnovirus from the perspective of coevolutionary hosts.

Polydnaviruses (PDVs) exhibit species-specific mutualistic relationships with endoparasitoid wasps. PDVs can be categorized into bracoviruses and ichnoviruses, which have independent evolutionary origins. In our previous study, we identified an ichnovirus of the endoparasitoid Diadegma fenestrale and named it DfIV. Here, DfIV virions from the ovarian calyx of gravid female wasps were characterized. DfIV virion particles were ellipsoidal (246.5 nm × 109.0 nm) with a double-layered envelope. Next-generation sequencing of the DfIV genome revealed 62 non-overlapping circular DNA segments (A1-A5, B1-B9, C1-C15, D1-D23, E1-E7, and F1-F3); the aggregate genome size was approximately 240 kb, and the GC content (43%) was similar to that of other IVs (41%-43%). A total of 123 open reading frames were predicted and included typical IV gene families such as repeat element protein (41 members), cysteine motif (10 members), vankyrin (9 members), polar residue-rich protein (7 members), vinnexin (6 members), and N gene (3 members). Neuromodulin N (2 members) was found to be unique to DfIV, along with 45 hypothetical genes. Among the 62 segments, 54 showed high (76%-98%) sequence similarities to the genome of Diadegma semiclausum ichnovirus (DsIV). Three segments, namely, D22, E3, and F2, contained lepidopteran host genome integration motifs with homologous regions of about 36-46 bp between them (Diadegma fenestrale ichnovirus, DfIV and lepidopteran host, Plutella xylostella). Most of the DfIV genes were expressed in the hymenopteran host and some in the lepidopteran host (P. xylostella), parasitized by D. fenestrale. Five segments (A4, C3, C15, D5, and E4) were differentially expressed at different developmental stages of the parasitized P. xylostella, and two segments (C15 and D14) were highly expressed in the ovaries of D. fenestrale. Comparative analysis between DfIV and DsIV revealed that the genomes differed in the number of segments, composition of sequences, and internal sequence homologies.

A Genome for Edith's Checkerspot Butterfly: An Insect with Complex Host-Adaptive Suites and Rapid Evolutionary Responses to Environmental Changes.

Insects have been key players in the assessments of biodiversity impacts of anthropogenically driven environmental change, including the evolutionary and ecological impacts of climate change. Populations of Edith’s Checkerspot Butterfly (Euphydryas editha) adapt rapidly to diverse environmental conditions, with numerous high-impact studies documenting these dynamics over several decades. However, studies of the underlying genetic bases of these responses have been hampered by missing genomic resources, limiting the ability to connect genomic responses to environmental change. Using a combination of Oxford Nanopore long reads, haplotype merging, HiC scaffolding followed by Illumina polishing, we generated a highly contiguous and complete assembly (contigs n = 142, N50 = 21.2 Mb, total length = 607.8 Mb; BUSCOs n = 5,286, single copy complete = 97.8%, duplicated = 0.9%, fragmented = 0.3%, missing = 1.0%). A total of 98% of the assembled genome was placed into 31 chromosomes, which displayed large-scale synteny with other well-characterized lepidopteran genomes. The E. editha genome, annotation, and functional descriptions now fill a missing gap for one of the leading field-based ecological model systems in North America.

Expanding the Menu: Are Polyphagy and Gene Family Expansions Linked across Lepidoptera?

Evolutionary expansions and contractions of gene families are often correlated with key innovations and/or ecological characteristics. In butterflies and moths (Lepidoptera), expansions of gene families involved in detoxification of plant specialized metabolites are hypothesized to facilitate a polyphagous feeding style. However, analyses supporting this hypothesis are mostly based on a limited number of lepidopteran species. We applied a phylogenomics approach, using 37 lepidopteran genomes, to analyze if gene family evolution (gene gain and loss) is associated with the evolution of polyphagy. Specifically, we compared gene counts and evolutionary gene gain and loss rates of gene families involved in adaptations with plant feeding. We correlated gene evolution to host plant family range (phylogenetic diversity) and specialized metabolite content of plant families (functional metabolite diversity). We found a higher rate for gene loss than gene gain in Lepidoptera, a potential consequence of genomic rearrangements and deletions after (potentially small-scale) duplication events. Gene family expansions and contractions varied across lepidopteran families, and were associated to host plant use and specialization levels. Within the family Noctuidae, a higher expansion rate for gene families involved in detoxification can be related to the large number of polyphagous species. However, gene family expansions are observed in both polyphagous and monophagous lepidopteran species and thus seem to be species-specific in the taxa sampled. Nevertheless, a significant positive correlation of gene counts of the carboxyl- and choline esterase and glutathione-S-transferase detoxification gene families with the level of polyphagy was identified across Lepidoptera.

Complete mitochondrial genome of Coleophora therinella Tengström, 1848 (Lepidoptera: Coleophoridae)

The mitochondrial genome (mitogenome) of Coleophora therinella Tengström, 1848 is the first report for the family Coleophoridae in Lepidoptera. The 15,539-bp long complete genome has an arrangement identical to that observed in most lepidopteran genomes. COI had the atypical CGA codon that is frequently found in the start region of the lepidopteran COI, and COII had the GTG codon found previously in Drosophila yakuba ND5 and Rattus norvegicus ND1. The 457-bp long A + T-rich region was the second largest, next to Blastobasis lacticolella, which belongs to Blastobasidae in the superfamily Gelechioidea. The A/T content of the whole mitogenome was 80.7%; however, it varied among the regions/genes as follows: A + T-rich region, 94.8%; srRNA, 85.0%; lrRNA, 84.3%; tRNAs, 81.5%; and PCGs, 78.9%. Phylogenetic analyses with concatenated sequences of the 13 PCGs and two RNA genes using the maximum likelihood method, placed Coleophoridae, represented only by C. therinella, as the most basal lineage of the Gelechioidea families consisted of Stathmopodidae, Scythrididae, Blastobasidae, Autostichidae, and Oecophoridae, but nodal support for this grouping was very low (27%). Currently, several families of Gelechioidea are represented by a single species. Thus, extended sampling is required for further reasonable inference for the relationships of these families.

The Dryas iulia Genome Supports Multiple Gains of a W Chromosome from a B Chromosome in Butterflies.

In butterflies and moths, which exhibit highly variable sex determination mechanisms, the homogametic Z chromosome is deeply conserved and is featured in many genome assemblies. The evolution and origin of the female W sex chromosome, however, remains mostly unknown. Previous studies have proposed that a ZZ/Z0 sex determination system is ancestral to Lepidoptera, and that W chromosomes may originate from sex-linked B chromosomes. Here, we sequence and assemble the female Dryas iulia genome into 32 highly contiguous ordered and oriented chromosomes, including the Z and W sex chromosomes. We then use sex-specific Hi-C, ATAC-seq, PRO-seq, and whole-genome DNA sequence data sets to test if features of the D. iulia W chromosome are consistent with a hypothesized B chromosome origin. We show that the putative W chromosome displays female-associated DNA sequence, gene expression, and chromatin accessibility to confirm the sex-linked function of the W sequence. In contrast with expectations from studies of homologous sex chromosomes, highly repetitive DNA content on the W chromosome, the sole presence of domesticated repetitive elements in functional DNA, and lack of sequence homology with the Z chromosome or autosomes is most consistent with a B chromosome origin for the W, although it remains challenging to rule out extensive sequence divergence. Synteny analysis of the D. iulia W chromosome with other female lepidopteran genome assemblies shows no homology between W chromosomes and suggests multiple, independent origins of the W chromosome from a B chromosome likely occurred in butterflies.

The Genome Assembly and Annotation of the Apollo Butterfly Parnassius apollo, a Flagship Species for Conservation Biology.

Conservation genomics has made dramatic improvements over the past decade, leveraging the power of genomes to infer diverse parameters central to conservation management questions. However, much of this effort has focused upon vertebrate species, despite insects providing similar flagship status with the added benefit of smaller genomes, shorter generation times and extensive historical collections in museums. Here we present the genome of the Apollo butterfly (Parnassius apollo, Papilionidae), an iconic endangered butterfly, which like many species in this genus, needs conservation genomic attention yet lacks a genome. Using 68.7 Gb of long-read data (N50 = 15.2 kb) we assembled a 1.4 Gb genome for the Apollo butterfly, making this the largest sequenced Lepidopteran genome to date. The assembly was highly contiguous (N50 = 7.1 Mb) and complete (97% of Lepidopteran BUSCOs were single-copy and complete) and consisted of 1,707 contigs. Using RNAseq data and Arthropoda proteins, we annotated 28.3K genes. Alignment with the closest-related chromosome-level assembly, Papilio bianor, reveals a highly conserved chromosomal organization, albeit genome size is highly expanded in the Apollo butterfly, due primarily to a dramatic increase in repetitive element content. Using this alignment for superscaffolding places the P. apollo genome in to 31 chromosomal scaffolds, and together with our functional annotation, provides an essential resource for advancing conservation genomics in a flagship species for insect conservation.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Complete mitochondrial genome of Scythris sinensis (Lepidoptera: Scythrididae)

The complete mitochondrial genome (mitogenome) of Scythris sinensis Felder & Rogenhofer, 1775 (Lepidoptera: Scythrididae) was 15,216 bp with a typical set of genes (13 protein-coding genes [PCGs], 2 rRNA genes, and 22 tRNA genes) and one non-coding region, with an arrangement identical to that observed in most lepidopteran genomes. Twelve PCGs had the typical ATN start codon, whereas COI had the atypical CGA codon that is frequently found in the start region of the lepidopteran COI. The 271-bp long A + T-rich region was the shortest among sequenced Gelechioidea, which ranged from 290 – 375 bp. Phylogenetic analyses with concatenated sequences of the 13 PCGs, two RNA genes, and 22 tRNA genes using the Bayesian inference (BI) method, placed S. sinensis in the Scythrididae, as a sister to the family Stathmopodidae. The nodal support for this sister relationship was the highest at Bayesian posterior probabilities = 1.

Characterization of the complete mitochondrial genome of Asia Corn Borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

In this study, the complete mitogenome sequence of Ostrinia furnacalis was described. The assembled mitogenome is 15,241 bp in length with an extreme bias of high AT content (80.9%) (GenBank accession no. MN747041). The mitochondrial genome contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (12S rRNA and 16S rRNA) genes, and a control region (D-loop region). The mitochondrial gene order was identical to that observed in most lepidopteran genomes, nine PCGs were located on the H-strand, others were located on the L-strand. 12 PCGs were initiated by typical ATN codons, except for COI with CGA instead. 21 tRNAs had the typical cloverleaf structure, while the DHU arm of the trnS1 gene did not form a stable stem-loop structure. The ‘ATAGT(A)’-like motif and a 19 bp poly-T stretch at the down-stream of the rrnS gene were observed in the A + T-rich region. The phylogenetic analysis showed that the relationship of O. furnacalis is very close to the three species in the subfamily Pyraustinae: O. nubilalis, O. penitalis and Loxostege sticticalis, and all the subfamilies of Spilomelinae, Pyraustinae, Crambinae and Nymphulinae within Crambidae formed monophyletic groups with the highest bootstrap value support.

Expansion of LINEs and species-specific DNA repeats drives genome expansion in Asian Gypsy Moths

Two subspecies of Asian gypsy moth (AGM), Lymantria dispar asiatica and L. dispar japonica, pose a serious alien invasive threat to North American forests. Despite decades of research on the ecology and biology of this pest, limited AGM-specific genomic resources are currently available. Here, we report on the genome sequences and functional content of these AGM subspecies. The genomes of L.d. asiatica and L.d. japonica are the largest lepidopteran genomes sequenced to date, totaling 921 and 999 megabases, respectively. Large genome size in these subspecies is driven by the accumulation of specific classes of repeats. Genome-wide metabolic pathway reconstructions suggest strong genomic signatures of energy-related pathways in both subspecies, dominated by metabolic functions related to thermogenesis. The genome sequences reported here will provide tools for probing the molecular mechanisms underlying phenotypic traits that are thought to enhance AGM invasiveness.

Complete mitochondrial genome of the silver stripped skipper, Leptalina unicolor (Lepidoptera: Hesperiidae)

The silver stripped skipper, Leptalina unicolor Bremer and Grey, 1853 (Lepidoptera: Hesperiidae), is listed as endangered insect in South Korea. We sequenced the whole genome (15,854 bp) of L. unicolor species using Next-Generation Sequencing method and the subsequent gap-filling method. This genome included a set of typical genes and one major non-coding A + T-rich region, with an arrangement identical to that observed in most lepidopteran genomes. Twelve protein-coding genes (PCGs) had the typical ATN start codon, whereas COI had the atypical CGA codon that is frequently found in the start region of the lepidopteran COI. The 757-bp long A + T-rich region was the second largest among completely sequenced Hesperiidae, which ranged from 234 to 793 bp. Phylogenetic reconstruction was performed by maximum-likelihood method using the concatenated sequences of 13 PCGs and two rRNAs of available species of Hesperiidae, including that of L. unicolor (a total of 28 species). The resulting phylogeny provided strong support for monophyletic Heteropterinae in which L. unicolor belongs, with the highest nodal support and a sister relationship between current L. unicolor and co-subfamilial species Carterocephalus silvicola with a bootstrap value of 91%.

Complete mitochondrial genome of Ostrinia palustralis memnialis Walker, 1859 (Lepidoptera: Crambidae)

The complete mitochondrial genome (mitogenome) of Ostrinia palustralis memnialis Walker, 1859 (Lepidoptera: Crambidae) was determined to be 15,246 bp with a typical set of genes (13 protein-coding genes [PCGs], 2 rRNA genes, and 22 tRNA genes) and one non-coding region, with an arrangement identical to that observed in most lepidopteran genomes. Twelve PCGs have the typical ATN start codon, whereas COI has an atypical CGA codon frequently found in the start region of the lepidopteran COI. At 330 bp, the A + T-rich region is well within the range found in other Pyraloidea. The A + T content of the whole genome, PCGs, srRNA, lrRNA, tRNAs, and the A + T-rich region all are well within the range found in other Pyraloidea. Phylogenetic analyses of the concatenated sequences of the 13 PCGs using Bayesian inference (BI) and maximum-likelihood (ML) methods placed O. palustralis as a sister group to Ostrinia furnacalis and Ostrinia nubilalis, with the highest nodal support. The subfamilies within Crambidae, such as Nymphulinae, Spilomelinae, and Pyraustinae, all formed monophyletic groups with the highest nodal support.

Baculovirus Kimura two-parameter species demarcation criterion is confirmed by the distances of 38 core gene nucleotide sequences.

Kimura two-parameter nucleotide distance comparisons based on polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) are a widely applied method for species demarcation for lepidopteran-specific baculoviruses. Baculoviruses are considered to belong to the same species when a pairwise distance threshold of 0.015 is not exceeded and are considered as possibly belonging to the same species with a distance of up to 0.050. In the present work this method was revised and extended for 172 entirely sequenced lepidopteran, hymenopteran and dipteran baculovirus genomes by applying the nucleotide sequences of all 38 known baculovirus core genes for pairwise distance calculations. On the basis of this large dataset, the previously established standard thresholds for baculovirus species demarcation were adjusted for pairwise nucleotide distances estimated from the alignments of all 38 core genes. With the newly applied thresholds for the 38 core-gene dataset, a more sophisticated Kimura two-parameter method was established, avoiding the possible influence of the chimerical polh gene of the Autographa californica multiple nucleopolyhedrovirus. Based on the new dataset, the present classification of baculovirus species was confirmed. Thereby the Kimura two-parameter method for baculovirus demarcation was extended to include the information from all 38 Baculoviridae core genes, which represent the established standard information for baculovirus phylogeny to date.

Complete mitochondrial genome of the female-wingless bagworm moth, Eumeta variegata Snellen, 1879 (Lepidoptera: Psychidae)

The complete mitochondrial genome of the female-wingless bagworm moth, Eumeta variegata Snellen, 1879 (Lepidoptera: Psychidae), is 15,660 base pairs (bp) and contains a typical set of genes (13 protein-coding genes [PCGs], 2 rRNA genes, and 22 tRNA genes) and one non-coding region, with an arrangement identical to that observed in most lepidopteran genomes. Twelve PCGs contained the typical ATN start codon, whereas COI had the atypical CGA codon, which is frequently detected in the start region of the lepidopteran COI. The A + T-rich region was unusually short with only 94 bp. A recent report of the same species originating from Japan revealed a lack of trnE and trnF and a 1,118 bp long A + T-rich region. Phylogenetic analyses with concatenated sequences of the 13 PCGs and two rRNA genes using the Bayesian inference method placed E. variegata in Psychidae, as a sister to a within-familial species, Mahasena colona, with the highest nodal support (Bayesian posterior probability = 1).

Conservation and lineage-specific rearrangements in the GOBP/PBP gene complex of distantly related ditrysian Lepidoptera

General odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs) form a monophyletic subfamily of insect odorant binding proteins (OBPs) specific for Lepidoptera, butterflies and moths. The GOBP/PBP genes include six subgroups (GOBP1–2, PBP-A–D) previously reported to form a complex arrayed in a conserved order in representative moths (superfamily Bombycoidea) and butterflies (Nymphalidae). Although our knowledge of lepidopteran genomes has increased greatly recently, the structure of the GOBP/PBP complex has been studied only for species that represent limited lineages of the highly diverged Ditrysia. To understand the evolution of this functionally important gene complex, we determined 69–149 kb genomic sequences that include GOBP2 and five PBP genes in three Ostrinia moths (Pyraloidea), O. nubilalis, O. furnacalis, and O. latipennis, using bacterial artificial chromosome (BAC) and fosmid clones. The structure of the GOBP2/PBP gene cluster was well conserved despite the different sex pheromone composition utilized by the three moths. Five expressed PBP genes in Ostrinia moths were the result of two duplications of PBP-A genes. Surprisingly, an allele containing a fusion gene between tandemly arrayed PBP-A genes was observed in O. nubilalis. We also revealed duplication and intra-chromosomal translocation of the GOBP1 gene in P. xylostella by fluorescence in situ hybridization (FISH) analysis. Additionally, we compared the structure of the GOBP/PBP gene complex of seventeen species covering six superfamilies and twelve families of the lepidopteran clade, Ditrysia, and found the gene order was basically conserved despite the frequent occurrence of lineage-specific gains, losses, inversions and translocations of these genes, compared with their neighboring genes. Our findings support the hypothesis that the structure of the GOBP/PBP gene complex was already established in the common ancestor of Ditrysia.

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera

BackgroundRapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology.ResultsComparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects.ConclusionsOur results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph.