Holometabolous Orders Research Articles

Male is the default sex: functional significance of the sex determination cascade in horned dung beetles.

Sex-specific trait expression represents a striking dimension of morphological variation within and across species. The mechanisms instructing sex-specific organ development have been well studied in a small number of insect model systems, suggesting striking conservation in some parts of the somatic sex determination pathway while hinting at possible evolutionary lability in others. However, further resolution of this phenomenon necessitates additional taxon sampling, particularly in groups in which sexual dimorphisms have undergone significant elaboration and diversification. Here, we functionally investigate the somatic sex determination pathway in the gazelle dung beetle Digitonthophagus gazella , an emerging model system in the study of the development and evolution of sexual dimorphisms. We find that RNA interference (RNAi) targeting transformer1 (tra1) caused chromosomal females to develop morphological traits largely indistinguishable from those normally only observed in males, and that tra1 RNAi is sufficient to induce splicing of the normally male-specific isoform of doublesex in chromosomal females, while leaving males unaffected. Further, intersex RNAi was found to phenocopy previously described RNAi phenotypes of doublesex in female but not male beetles. These findings match predictions derived from models of the sex determination cascade as developed largely through studies in Drosophila melanogaster . In contrast, transformer2 RNAi resulted in larval mortality and was not sufficient to affect doublesex splicing, whereas RNAi targeting Sex-lethal and two putative orthologs of hermaphrodite yielded no obvious phenotypic modifications in either males or females, raising the possibility that the function of a subset of sex determination genes may be derived in Diptera and thus non-representative of their roles in other holometabolous orders. Our results help illuminate how the differential evolutionary lability of the somatic sex determination pathway has contributed to the extraordinary morphological diversification of sex-specific trait expression found in nature.

A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

GC Content Across Insect Genomes: Phylogenetic Patterns, Causes and Consequences

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.

Cover Caption

AbstractThe pleasing lacewings (Dilaridae) are a distinctive family of the holometabolous order Neuroptera, mainly characterized by the sexually dimorphic antennae and long tubular ovipositor, which are widely distributed in most zoogeographic regions except for Australia. The female egg‐laying behaviors, and the anatomy of the larval head are reported in detail for understanding their phylogeny and larval adaptation to subterranean habitat (see pages 1445–1463). Photo provided by Yuezheng Tu.

The earliest known species of Permeca (Insecta, Mecoptera, Permochoristidae) from the late Guadalupian Yinping Formation of China

Mecoptera are commonly known as scorpionflies, as some mecopterans have bulbous genitals that are upturned, resembling the stinger of scorpions. As one of the most ancient holometabolous orders (Kristensen, 1981; Dunford & Somma, 2008), the stem group of mecopterans can be dated back to the early Permian (Rasnitsyn, 2004). Mecopterans play an important role in our understanding of the origin of fleas. Some Mesozoic giant fleas have been suggested to be derived from extinct long-proboscid mecopterans (Huang et al., 2012), and recent phylogenomic evidence has clearly demonstrated that fleas are nested within Mecoptera (Tihelka et al., 2020). Mecoptera are very abundant in the geological past, with more than 700 species and 210 genera placed in 39 families (Novokshonov et al., 2016; Ren et al., 2019; Wang, 2020). Extant faunas are represented by ca. 700 species and 40 genera assigned to 9 families (Bicha, 2018; Wang, 2020).

Establishing the phenology of the Bombyliidae Family (Insecta: Diptera)

Despite the high number of species of this family, the biology of juveniles of most species is poorly understood. The postembryonic development is of the type hypermetamorphic, with parasitoid or hyperparasitoid larvae. Exceptions are the larvae of Heterotropinae, whose biology is similar to that of other Asiloidea, with predatory larvae that do not undergo hypermetamorphosis. Hosts of bee flies belong to different orders of insects, but mostly are among the holometabolous orders. The objective of this work is to research the biogeography, bioecology, habitat, geographic distribution, taxonomy, life cycle, phenology, parasitoidism (parasitism) and predatism of the Bombyliidae family (Insecta: Diptera). The research was carried out in studies related to quantitative aspects taxonomic and conceptual aspects. A literature search was carried out containing articles published from 1937 to 2021. The mini review was prepared in Goiânia, Goiás, from September to October 2021. The mini-review was prepared in Goiânia, Goiás, from September to October 2021, through, SEEK education, Periodicals CAPES, Google Academic, Bioline International, VADLO, Scopus, Web of Science, LILACS, Medline, LIS and Portal of Scientific Journals in Health Sciences.

The earliest beetle with mouthparts specialized for feeding on nectar is a parasitoid of mid-Cretaceous Hymenoptera

BackgroundDuring the Mesozoic, there were many insects in several holometabolous orders (Neuroptera, Mecoptera and Diptera) with elongated mouthparts adapted for feeding on nectar. The evolutionary history of the megadiverse order Coleptera, which has a great diversity of mouthparts and feeding strategies, is well documented since early Permian with a significant peak in diversity in the Triassic. Currently, however, there is no evidence that in the Mesozoic these beetles fed on nectar despite the recorded specializations for pollination of flowering plants in several families since the mid-Cretaceous.ResultsHere we describe a new wedge-shaped beetle Melanosiagon serraticornis gen. et sp. nov. from mid-Cretaceous Burmese amber attributed to Macrosiagonini (Ripiphoridae: Ripiphorinae), which has elongated galea comparable to that in the extant parasitoid genus Macrosiagon, and a well known example of adaptation for nectar feeding in Coleoptera. Furthermore, Salignacicola gen. nov. is established for Macrosiagon ebboi Perrichot, Nel et Néraudeau, 2004, based on the holotype found in mid-Cretaceous amber from France. Systematic positions of both newly established genera are discussed. A list of potential wasp and bee hosts of Ripiphorinae from the Mesozoic is provided.ConclusionsThis study presents evidence of the earliest occurrence of specialized nectar feeding mouthparts in Coleoptera. Melanosiagon serraticornis is closely related to extant Macrosiagonini. In all genera belonging to subfamily Ripiphorinae the primary larvae are adapted for parasitism on aculeate Hymenoptera (bees and wasps) and adults are associated with blossoms of flowering plants, in terms of their specialized morphology. Adults of Macrosiagon visit blossoms of flowering plants to obtain nectar and lay eggs from which the hatching larvae attack visiting wasps and bees. An association with flowers of some tropical trees is already corroborated in some extant species. Interestingly the larvae of Ripiphorinae are also found in Burmese amber. Thus, both life stages of the mid-Cretaceous Ripiphorinae indicate a close association of this lineage with flowering trees.

Mining the Species Diversity of Lacewings: New Species of the Pleasing Lacewing Genus Dilar Rambur, 1838 (Neuroptera, Dilaridae) from the Oriental Region.

Simple SummaryThe pleasing lacewing (Dilaridae) is a little known family of the holometabolous order Neuroptera, and our understanding of their species diversity has long remained poor. Here, we present descriptions of 12 new species of the pleasing lacewing genus Dilar Rambur, which is widely distributed in the Palaearctic and Oriental regions. We found disparate wing marking patterns as well as several unique characters of the male genitalia of the new species, which highlight the diverse morphologies of Dilar. Based on a faunal analysis, eight areas of endemism of Dilar were distinguished, and the state of their species diversity and endemism were summarized. The Oriental part of China was revealed as the region with the highest species diversity of this genus, and Yunnan within this region stood out as the most species-rich subregion.The species diversity of insects is extraordinarily rich, but still has been insufficiently explored or underestimated particularly for uncommon groups. The pleasing lacewings (Dilaridae) are a little known family of Neuroptera with distinct sexually dimorphic antennae. The species diversity of pleasing lacewings was recently found to be severely underestimated and requires a comprehensive investigation, as well as systematic reviews. Here, we report on 12 new species of the pleasing lacewing genus Dilar Rambur, 1838, from the Oriental region, namely D. forcipatus sp. nov. and D. laoticus sp. nov. from Laos (new country record of Dilar); D. malickyi sp. nov., D. phraenus sp. nov. and D. rauschorum sp. nov. from northern Thailand; D. striatus sp. nov. from northern Vietnam; D. cangyuanensis sp. nov., D. daweishanensis sp. nov., D. nujianganus sp. nov., D. weibaoshanensis sp. nov., D. yucheni sp. nov., and D. zhangweiae sp. nov. from Yunnan and Tibet, both in southwestern China. The new species of Dilar display several types of wing marking patterns, and the morphology of the male genitalia is highly diverse. A comprehensive examination of the species diversity and distribution of Dilar concluded that Yunnan (southwestern China) represents a biogeographic region with high endemism and the richest species diversity. The potential correlation between vertical distribution and geographical latitude in Dilar was also analyzed.

Discovery of Raphidioptera (Insecta: Neuropterida) in Xizang, China, with description of a new species of Inocellia Schneider.

The holometabolous order Raphidioptera is recorded from Xizang Autonomous Region for the first time. A new species of the family Inocelliidae, Inocellia tibetana sp. nov., from southeastern Xizang is described and its two sexes illustrated. Based on the male gonocoxite 9 that is longer than width of its base, the new species belongs to the I. fulvostigmata species-group, and it appears to be closely related to I. fulvostigmata U. Aspöck, Rausch H. Aspöck, 1968. Both species are distributed near the southern edge of the Himalayas. The male of the new species is characterized in the genitalia by the presence of a membranous, short and digitiform gonostylus 9, the gonarcus (fused gonocoxites 11) subtriangular in caudal view dorsally with a pair of short tubercular processes, and the reduction of bristle tuft on the endophallus.

New antlions (Insecta: Neuroptera: Myrmeleontidae) from the mid-Cretaceous of Myanmar and their phylogenetic implications

Myrmeleontidae (antlions) is the most species-rich family of the holometabolous order Neuroptera. Evolutionary history of this diverse lacewing family remains largely unexplored. Here we report on three new genera and four new species of antlions from the mid-Cretaceous amber of Myanmar, namely Allopteroneura burmana Lu, Zhang & Liu gen. et sp. nov., Phylloleon elegans Lu, Wang & Liu gen. et sp. nov., Phylloleon stangei Lu, Ohl & Liu gen. et sp. nov. and Nanoleon wangae Hu, Lu & Liu gen. et sp. nov. A phylogenetic analysis of the Burmese amber antlions is performed based on morphological characters. The result shows that the antlion species presently described belong to different major clades across the phylogenetic tree, suggesting a diverse palaeofauna of this family from the mid-Cretaceous of Myanmar. The phylogenetic status of the extinct subfamily Araripeneurinae is also discussed.http://zoobank.org/urn:lsid:zoobank.org:pub:90D26B8B-1A59-465B-A530-0C0662D85E72

Genomic Mining of Phylogenetically Informative Nuclear Markers in Bark and Ambrosia Beetles.

Deep level insect relationships are generally difficult to resolve, especially within taxa of the most diverse and species rich holometabolous orders. In beetles, the major diversity occurs in the Phytophaga, including charismatic groups such as leaf beetles, longhorn beetles and weevils. Bark and ambrosia beetles are wood boring weevils that contribute 12 percent of the diversity encountered in Curculionidae, one of the largest families of beetles with more than 50000 described species. Phylogenetic resolution in groups of Cretaceous age has proven particularly difficult and requires large quantity of data. In this study, we investigated 100 nuclear genes in order to select a number of markers with low evolutionary rates and high phylogenetic signal. A PCR screening using degenerate primers was applied to 26 different weevil species. We obtained sequences from 57 of the 100 targeted genes. Sequences from each nuclear marker were aligned and examined for detecting multiple copies, pseudogenes and introns. Phylogenetic informativeness (PI) and the capacity for reconstruction of previously established phylogenetic relationships were used as proxies for selecting a subset of the 57 amplified genes. Finally, we selected 16 markers suitable for large-scale phylogenetics of Scolytinae and related weevil taxa.

Global patterns of insect diversification: towards a reconciliation of fossil and molecular evidence?

Macroevolutionary studies of insects at diverse taxonomic scales often reveal dynamic evolutionary patterns, with multiple inferred diversification rate shifts. Responses to major past environmental changes, such as the Cretaceous Terrestrial Revolution, or the development of major key innovations, such as wings or complete metamorphosis are usually invoked as potential evolutionary triggers. However this view is partially contradicted by studies on the family-level fossil record showing that insect diversification was relatively constant through time. In an attempt to reconcile both views, we investigate large-scale insect diversification dynamics at family level using two distinct types of diversification analyses on a molecular timetree representing ca. 82% of the extant families, and reassess the insect fossil diversity using up-to-date records. Analyses focusing on the fossil record recovered an early burst of diversification, declining to low and steady rates through time, interrupted by extinction events. Phylogenetic analyses showed that major shifts of diversification rates only occurred in the four richest holometabolous orders. Both suggest that neither the development of flight or complete metamorphosis nor the Cretaceous Terrestrial Revolution environmental changes induced immediate changes in diversification regimes; instead clade-specific innovations likely promoted the diversification of major insect orders.

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.

New transitional fossil snakeflies from China illuminate the early evolution of Raphidioptera.

BackgroundRaphidioptera (snakeflies) is a holometabolous order of the superorder Neuropterida characterized by the narrowly elongate adult prothorax and the long female ovipositor. Mesozoic snakeflies were markedly more diverse than the modern ones are. However, the evolutionary history of Raphidioptera is largely unexplored, as a result of the poorly studied phylogeny among fossil and extant lineages within the order.ResultsIn this paper, we report a new snakefly family, Juroraphidiidae fam. nov., based on exquisitely preserved fossils, attributed to a new species Juroraphidia longicollumgen. et sp. nov., from the Jiulongshan Formation (Middle Jurassic) in Inner Mongolia, China. The new family is characterized by an unexpected combination of plesiomorphic and apomorphic characters of Raphidioptera. Based on our phylogenetic analysis, Juroraphidiidae fam. nov. together with Raphidiomorpha form a monophyletic clade, which is the sister to Priscaenigmatomorpha. The snakefly affinity of Priscaenigmatomorpha is confirmed and another new family, Chrysoraphidiidae fam. nov., is erected in this suborder.ConclusionsJuroraphidiidae fam. nov. is determined to be a transitional lineage between Priscaenigmatomorpha and Raphidiomorpha. Diversification of higher snakefly taxa had occurred by the Early Jurassic, suggesting that these insects had already had a long but undocumented history by this time.

The first mitochondrial genome for caddisfly (insecta: Trichoptera) with phylogenetic implications.

The Trichoptera (caddisflies) is a holometabolous insect order with 14,300 described species forming the second most species-rich monophyletic group of animals in freshwater. Hitherto, there is no mitochondrial genome reported of this order. Herein, we describe the complete mitochondrial (mt) genome of a caddisfly species, Eubasilissa regina (McLachlan, 1871). A phylogenomic analysis was carried out based on the mt genomic sequences of 13 mt protein coding genes (PCGs) and two rRNA genes of 24 species belonging to eight holometabolous orders. Both maximum likelihood and Bayesian inference analyses highly support the sister relationship between Trichoptera and Lepidoptera.

Discovery of Raphidioptera (Insecta: Neuropterida) in Hainan Island, China, with description of a new species of the genus Inocellia Schneider

Based on the discovery of a new species of Inocelliidae, Inocellia hainanica sp. n., described in both sexes, the holometabolous order Raphidioptera is recorded from Hainan, the southernmost island of China, for the first time. I. hainanica can be regarded as a relic species from colder periods, which has survived in higher altitudes of this island, which is attributed to the Oriental realm, and is characterized by tropical rainforests.

A mitochondrial genome phylogeny of the Neuropterida (lace‐wings, alderflies and snakeflies) and their relationship to the other holometabolous insect orders

We present a mitochondrial (mt) genome phylogeny inferring relationships within Neuropterida (lacewings, alderflies and camel flies) and between Neuropterida and other holometabolous insect orders. Whole mt genomes were sequenced for Sialis hamata (Megaloptera: Sialidae), Ditaxis latistyla (Neuroptera: Mantispidae), Mongoloraphidia harmandi (Raphidioptera: Raphidiidae), Macrogyrus oblongus (Coleoptera: Gyrinidae), Rhopaea magnicornis (Coleoptera: Scarabaeidae), and Mordella atrata (Coleoptera: Mordellidae) and compared against representatives of other holometabolous orders in phylogenetic analyses. Additionally, we test the sensitivity of phylogenetic inferences to four analytical approaches: inclusion vs. exclusion of RNA genes, manual vs. algorithmic alignments, arbitrary vs. algorithmic approaches to excluding variable gene regions and how each approach interacts with phylogenetic inference methods (parsimony vs. Bayesian inference). Of these factors, phylogenetic inference method had the most influence on interordinal relationships. Bayesian analyses inferred topologies largely congruent with morphologically‐based hypotheses of neuropterid relationships, a monophyletic Neuropterida whose sister group is Coleoptera. In contrast, parsimony analyses failed to support a monophyletic Neuropterida as Raphidioptera was the sister group of the entire Holometabola excluding Hymenoptera, and Neuroptera + Megaloptera is the sister group of Diptera, a relationship which has not previously been proposed based on either molecular or morphological data sets. These differences between analytical methods are due to the high among site rate heterogeneity found in insect mt genomes which is properly modelled by Bayesian methods but results in artifactual relationships under parsimony. Properly analysed, the mt genomic data set presented here is among the first molecular data to support traditional, morphology‐based interpretations of relationships between the three neuropterid orders and their grouping with Coleoptera.

Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis

Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC). Highly conserved orthologs of BTBBRC and all four ZBRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrupt, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, which revealed that the ZBRC clade includes Zabrupt. All four ZBRC domains, including Z4BRC, which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated ZBRC exons even when they contribute to common developmental processes.

Neuropteran (Insecta) palaeodiversity with predictions for the Cretaceous fauna of the Wealden

The Wealden Group of southern England is a succession of non-marine sediments from the lower Cretaceous ( c. 141–119 Ma), which also outcrop in Belgium and northwest Germany. Most insect orders are present as fossils, including Neuroptera, one of the most basal holometabolous orders. The fossil record of Neuroptera is diverse, spanning Permian–Recent, with all major family radiations having occurred prior to the Wealden. However, Wealden Neuroptera have received little attention to date, with only three families reported and one species described. The fossil record in general is notoriously incomplete, and palaeoenvironmental reconstructions can facilitate predictions of what other organisms were present in the habitat, but which were either not preserved or only occasionally preserved and yet to be found as fossils. We examine the Wealden palaeohabitat and global neuropteran palaeodiversity and consider these in relation to palaeo/biogeography of fossil and extant taxa (including habitat preferences). We predict that the following families will form components of the Cretaceous Wealden fauna: Nymphidae, Polystoechotidae, Chrysopidae, Hemerobiidae and Coniopterygidae.

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects.

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.