Key questions on the evolution and biogeography of New Zealand alpine insects.

New Zealand alpine environments host a diverse assemblage of insect lineages, with virtually every major insect group represented. The modern mountain ranges of New Zealand are relatively young and large areas of habitat above the tree line have only been in continual existence for the past one million years. We discuss the geological history and physical characteristics of New Zealand alpine environments and the resulting selective pressures placed on insect species. Some notable alpine taxa and previous faunistic research is highlighted. We discuss examples where single lineages have colonised the alpine zone and contrast these with larger radiations of alpine species which in some cases are the result of multiple colonisation events. The age of most alpine lineages is consistent with the young geological age of the mountains, nevertheless there are some much older alpine lineages of uncertain evolutionary history. We show that alpine species have employed a very broad range of morphological, physiological, and behavioural adaptations to survive in the alpine zone, and new studies are starting to unpick their genomic basis. Finally, we look to the future and assess threats to the unique New Zealand alpine insect fauna.

A population genomics analysis of the Aotearoa New Zealand endemic rewarewa tree (Knightia excelsa).

Rewarewa (Knightia excelsa, Proteaceae) is a tree species endemic to Aotearoa New Zealand, with a natural distribution spanning Te Ika-a-Maui (North Island) and the top of Te Waipounamu (South Island). We used the pseudo-chromosome genome assembly of rewarewa as a reference and whole genome pooled sequencing from 35 populations sampled across Aotearoa New Zealand, including trees growing on Maori-owned land, to identify 1,443,255 single nucleotide polymorphisms (SNPs). Four genetic clusters located in the northern North Island (NNI), eastern North Island (NIE), western and southern North Island (NIWS), and the South Island (SI) were identified. Gene flow was revealed between the SI and NIE genetic clusters, plus bottleneck and contraction events within the genetic clusters since the mid-late Pleistocene, with divergence between North and South Island clusters estimated to have occurred ~115,000-230,000 years ago. Genotype environment analysis (GEA) was used to identify loci and genes linked with altitude, soil pH, soil carbon, slope, soil size, annual mean temperature, mean diurnal range, isothermality, annual precipitation, and precipitation seasonality. The location of the SNPs associated with these environmental variables was compared with the position of 52,192 gene-coding sequences that were predicted in the rewarewa genome using RNA sequencing. This new understanding of the genetic variation present in rewarewa and insights into the genetic control of adaptive traits will inform efforts to incorporate the species in restoration plantings and for marketing rewarewa honey based on provenance.

Multiple hybridization events and repeated evolution of homoeologue expression bias in parthenogenetic, polyploid New Zealand stick insects.

During hybrid speciation, homoeologues combine in a single genome. Homoeologue expression bias (HEB) occurs when one homoeologue has higher gene expression than another. HEB has been well characterized in plants but rarely investigated in animals, especially invertebrates. Consequently, we have little idea as to the role that HEB plays in allopolyploid invertebrate genomes. If HEB is constrained by features of the parental genomes, then we predict repeated evolution of similar HEB patterns among hybrid genomes formed from the same parental lineages. To address this, we reconstructed the history of hybridization between the New Zealand stick insect genera Acanthoxyla and Clitarchus using a high-quality genome assembly from Clitarchus hookeri to call variants and phase alleles. These analyses revealed the formation of three independent diploid and triploid hybrid lineages between these genera. RNA sequencing revealed a similar magnitude and direction of HEB among these hybrid lineages, and we observed that many enriched functions and pathways were also shared among lineages, consistent with repeated evolution due to parental genome constraints. In most hybrid lineages, a slight majority of the genes involved in mitochondrial function showed HEB towards the maternal homoeologues, consistent with only weak effects of mitonuclear incompatibility. We also observed a proteasome functional enrichment in most lineages and hypothesize this may result from the need to maintain proteostasis in hybrid genomes. Reference bias was a pervasive problem, and we caution against relying on HEB estimates from a single parental reference genome.

Wing pattern variation and DNA barcodes defy taxonomic splitting in the New Zealand Pimelea Looper Notoreas perornata (Walker) (Lepidoptera: Geometridae: Larentiinae): the importance of populations as conservation units.

The endemic Notoreas perornata (Walker, 1863) complex (Lepidoptera: Geometridae: Larentiinae) from the North Island and northern South Island of New Zealand is reviewed. Larvae feed on Pimelea spp. (Thymelaeaceae), frequently in highly fragmented and threatened shrubland habitats. Allopatric populations tend to differ in size and wing pattern characteristics, but not in genitalia; moreover extensive variation renders recognition of subspecies / allopatric species based on any species concept problematic. A mitochondrial DNA gene tree is not congruent with morphology and indicates rapid recent divergence that has not settled into diagnosable lineages. Based on our results, we synonymise Notoreas simplex Hudson, 1898 with N. perornata (Walker, 1863), and retain N. perornata as a single, highly diverse but monotypic species. All known populations are illustrated to display variation. For conservation purposes, we recommend the continued recognition within the species of 10 populations or groups of populations that appear to be on the way to diverging at subspecific level based on morphological and/or DNA data. The conservation status of all these populations is reviewed. One conservation unit, comprising the populations from Westland, has not been seen since 1998 and is feared possibly extinct.

Fast-tracking bespoke DNA reference database generation from museum collections for biomonitoring and conservation.

Despite recent advances in high-throughput DNA sequencing technologies, a lack of locally relevant DNA reference databases limits the potential for DNA-based monitoring of biodiversity for conservation and biosecurity applications. Museums and national collections represent a compelling source of authoritatively identified genetic material for DNA database development, yet obtaining DNA barcodes from long-stored specimens may be difficult due to sample degradation. Here we demonstrate a sensitive and efficient laboratory and bioinformatic process for generating DNA barcodes from hundreds of invertebrate specimens simultaneously via the Illumina MiSeq system. Using this process, we recovered full-length (334) or partial (105) COI barcodes from 439 of 450 (98%) national collection-held invertebrate specimens. This included full-length barcodes from 146 specimens which produced low-yield DNA and no visible PCR bands, and which produced as little as a single sequence per specimen, demonstrating high sensitivity of the process. In many cases, the identity of the most abundant sequences per specimen were not the correct barcodes, necessitating the development of a taxonomy-informed process for identifying correct sequences among the sequencing output. The recovery of only partial barcodes for some taxa indicates a need to refine certain PCR primers. Nonetheless, our approach represents a highly sensitive, accurate and efficient method for targeted reference database generation, providing a foundation for DNA-based assessments and monitoring of biodiversity.

An exploration of assembly strategies and quality metrics on the accuracy of the rewarewa (Knightia excelsa) genome.

We used long read sequencing data generated from Knightia excelsa, a nectar-producing Proteaceae tree endemic to Aotearoa (New Zealand), to explore how sequencing data type, volume and workflows can impact final assembly accuracy and chromosome reconstruction. Establishing a high-quality genome for this species has specific cultural importance to Maori and commercial importance to honey producers in Aotearoa. Assemblies were produced by five long read assemblers using data subsampled based on read lengths, two polishing strategies and two Hi-C mapping methods. Our results from subsampling the data by read length showed that each assembler tested performed differently depending on the coverage and the read length of the data. Subsampling highlighted that input data with longer read lengths but perhaps lower coverage constructed more contiguous, kmers and gene-complete assemblies than short read length input data with higher coverage. The final genome assembly was constructed into 14 pseudochromosomes using an initial flye long read assembly, a racon/medaka/pilon combined polishing strategy, salsa2 and allhic scaffolding, juicebox curation, and Macadamia linkage map validation. We highlighted the importance of developing assembly workflows based on the volume and read length of sequencing data and established a robust set of quality metrics for generating high-quality assemblies. Scaffolding analyses highlighted that problems found in the initial assemblies could not be resolved accurately by Hi-C data and that assembly scaffolding was more successful when the underlying contig assembly was of higher accuracy. These findings provide insight into how quality assessment tools can be implemented throughout genome assembly pipelines to inform the de novo reconstruction of a high-quality genome assembly for nonmodel organisms.

Divergent Gene Expression Following Duplication of Meiotic Genes in the Stick Insect Clitarchus hookeri.

Some animal groups, such as stick insects (Phasmatodea), have repeatedly evolved alternative reproductive strategies, including parthenogenesis. Genomic studies have found modification of the genes underlying meiosis exists in some of these animals. Here we examine the evolution of copy number, evolutionary rate, and gene expression in candidate meiotic genes of the New Zealand geographic parthenogenetic stick insect Clitarchus hookeri. We characterized 101 genes from a de novo transcriptome assembly from female and male gonads that have homology with meiotic genes from other arthropods. For each gene we determined copy number, the pattern of gene duplication relative to other arthropod orthologs, and the potential for meiosis-specific expression. There are five genes duplicated in C. hookeri, including one also duplicated in the stick insect Timema cristinae, that are not or are uncommonly duplicated in other arthropods. These included two sister chromatid cohesion associated genes (SA2 and SCC2), a recombination gene (HOP1), an RNA-silencing gene (AGO2) and a cell-cycle regulation gene (WEE1). Interestingly, WEE1 and SA2 are also duplicated in the cyclical parthenogenetic aphid Acyrthosiphon pisum and Daphnia duplex, respectively, indicating possible roles in the evolution of reproductive mode. Three of these genes (SA2, SCC2, and WEE1) have one copy displaying gonad-specific expression. All genes, with the exception of WEE1, have significantly different nonsynonymous/synonymous ratios between the gene duplicates, indicative of a shift in evolutionary constraints following duplication. These results suggest that stick insects may have evolved genes with novel functions in gamete production by gene duplication.

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The genus Mecodema Blanchard 1853 (Coleoptera: Carabidae: Broscini) from the North Island, New Zealand.

Mecodema (Coleoptera: Carabidae: Broscini) is a hyperdiverse endemic New Zealand genus of ground beetles with only a few geographically widespread species found throughout the two main islands, as well as many offshore islands. Using specimens from a number of private and institutional collections, in addition new specimens were acquired by extensive pitfall trapping, we describe or redescribe all of the known North Island Mecodema species. Additionally, we redescribe three South Island species from the former genus Metaglymma, as morphological evidence shows that these species are nested within Mecodema. Species descriptions are formed by using 128 morphological characters, which include external characters, as well as both male and female internal structures. There are four new combinations: Mecodema antarctica comb. n., M. aberrans comb. n., M. moniliferum comb. n. and M. tibiale comb. n. We synonymise M. occiputale under Mecodema curvidens, and M. sulcatum under Mecodema oblongum, and reinstate M. scitulum Broun (northwest Hunua Range, Auckland). Twenty four new species are described: Mecodema argentum sp. n., M. atuanui sp. n., M. dunnorum sp. n., M. genesispotini sp. n., M. godzilla sp. n., M. jacinda sp. n., M. kipjac sp. n., M. kokoroiho sp. n., M. mohi sp. n., M. ngaiatonga sp. n., M. ngaitahuhu sp. n., M. papake sp. n., M. perexiguus sp. n., M. rusticulus sp. n., M. temata sp. n., M. teparawhau sp. n., M. teroroa sp. n., M. tewhara sp. n., M. tuhoe sp. n., M. undecimus sp. n., M. wharekahika sp. n., M. xylanthrax sp. n., M. yconomus sp. n., M. zonula sp. n. North Island regional species endemism is very high in Northland (15/16 endemic species), with species becoming more widespread in the southern regions, e.g. Wellington only has two endemic species from a total of eight species. This research increases the total number of described Mecodema species to 102, and will allow a modern taxonomic framework for completion of the revision of the South Island species.

A new leaf-mining moth from New Zealand, Sabulopteryxbotanica sp. nov. (Lepidoptera, Gracillariidae, Gracillariinae), feeding on the rare endemic shrub Teucriumparvifolium (Lamiaceae), with a revised checklist of New Zealand Gracillariidae.

Sabulopteryxbotanica Hoare & Patrick, sp. nov. (Lepidoptera, Gracillariidae, Gracillariinae) is described as a new species from New Zealand. It is regarded as endemic, and represents the first record of its genus from the southern hemisphere. Though diverging in some morphological features from previously described species, it is placed in genus Sabulopteryx Triberti, based on wing venation, abdominal characters, male and female genitalia and hostplant choice; this placement is supported by phylogenetic analysis based on the COI mitochondrial gene. The life history is described: the larva is an underside leaf-miner on the endemic divaricating shrub Teucriumparvifolium (Lamiaceae), and exits the mine to pupate in a cocoon in a folded leaf of the host plant. The remarkable history of the discovery and rediscovery of this moth is discussed: for many years it was only known from a single sap-feeding larva found in a leaf-mine in a pressed herbarium specimen of the host. The adult was discovered by BHP in Christchurch Botanic Gardens in 2013. Most distribution records of the moth come from a recent search for mines and cocoons on herbarium specimens of T.parvifolium. Sabulopteryxbotanica has high conservation status, and is regarded as 'Nationally Vulnerable' according to the New Zealand Department of Conservation threat classification system, based on the rarity and declining status of its host plant. However, the presence of apparently thriving populations of S.botanica on cultivated plants of T.parvifolium, especially at the type locality, Christchurch Botanic Gardens, suggests that encouraging cultivation of the plant could greatly improve the conservation status of the moth. A revised checklist of New Zealand Gracillariidae is presented, assigning all species to the currently recognised subfamilies. The Australian Macarostolaida (Meyrick, 1880) is newly recorded from New Zealand (Auckland), where it is established on Eucalyptus.

New Zealand Tree and Giant Weta (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci.

Exposure to low temperatures requires an organism to overcome physiological challenges. New Zealand weta belonging to the genera Hemideina and Deinacrida are found across a wide range of thermal environments and therefore subject to varying selective pressures. Here we assess the selection pressures across the weta phylogeny, with a particular emphasis on identifying genes under positive or diversifying selection. We used RNA-seq to generate transcriptomes for all 18 Deinacrida and Hemideina species. A total of 755 orthologous genes were identified using a bidirectional best-hit approach, with the resulting gene set encompassing a diverse range of functional classes. Analysis of ortholog ratios of synonymous to nonsynonymous amino acid changes found 83 genes that are under positive selection for at least one codon. A wide variety of Gene Ontology terms, enzymes, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways are represented among these genes. In particular, enzymes involved in oxidative phosphorylation, melanin synthesis, and free-radical scavenging are represented, consistent with physiological and metabolic changes that are associated with adaptation to alpine environments. Structural alignment of the transcripts with the most codons under positive selection revealed that the majority of sites are surface residues, and therefore have the potential to influence the thermostability of the enzyme, with the exception of prophenoloxidase where two residues near the active site are under selection. These proteins provide interesting candidates for further analysis of protein evolution.

Weak premating isolation between Clitarchus stick insect species despite divergent male and female genital morphology.

Documenting natural hybrid systems builds our understanding of mate choice, reproductive isolation and speciation. The stick insect species Clitarchus hookeri and C. tepaki differ in their genital morphology and hybridize along a narrow peninsula in northern New Zealand. We utilize three lines of evidence to understand the role of premating isolation and species boundaries: (a) genetic differentiation using microsatellites and mitochondrial DNA; (b) variation in 3D surface topology of male claspers and 2D morphometrics of female opercular organs; and (c) behavioural reproductive isolation among parental and hybrid populations through mating crosses. The genetic data show introgression between the parental species and formation of a genetically variable hybrid swarm. Similarly, the male and female morphometric data show genital divergence between the parental species as well as increased variation within the hybrid populations. This genital divergence has not resulted in reproductive isolation between species, instead weak perimating isolation has enabled the formation of a hybrid swarm. Behavioural analysis demonstrates that the entire mating process influences the degree of reproductive isolation between species undergoing secondary contact. Mechanical isolation may appear strong, whereas perimating isolation is weak.

Estimating the biodiversity of terrestrial invertebrates on a forested island using DNA barcodes and metabarcoding data.

Invertebrates are a major component of terrestrial ecosystems, however, estimating their biodiversity is challenging. We compiled an inventory of invertebrate biodiversity along an elevation gradient on the temperate forested island of Hauturu, New Zealand, by DNA barcoding of specimens obtained from leaf litter samples and pitfall traps. We compared the barcodes and biodiversity estimates from this data set with those from a parallel DNA metabarcoding analysis of soil from the same locations, and with pre-existing sequences in reference databases, before exploring the use of combined data sets as a basis for estimating total invertebrate biodiversity. We obtained 1,282 28S and 1,610 COI barcodes from a total of 1,947 invertebrate specimens, which were clustered into 247 (28S) and 366 (COI) OTUs, of which ≤ 10% were represented in GenBank. Coleoptera were most abundant (730 sequenced specimens), followed by Hymenoptera, Diptera, Lepidoptera, and Amphipoda. The most abundant OTU from both the 28S (153 sequences) and COI (140 sequences) data sets was an undescribed beetle from the family Salpingidae. Based on the occurrences of COI OTUs along the elevation gradient, we estimated there are ~1,000 arthropod species (excluding mites) on Hauturu, including 770 insects, of which 344 are beetles. A DNA metabarcoding analysis of soil DNA from the same sites resulted in the identification of similar numbers of OTUs in most invertebrate groups compared with the DNA barcoding, but less than 10% of the DNA barcoding COI OTUs were also detected by the metabarcoding analysis of soil DNA. A mark-recapture analysis based on the overlap between these data sets estimated the presence of approximately 6,800 arthropod species (excluding mites) on the island, including ~3,900 insects. Estimates of New Zealand-wide biodiversity for selected arthropod groups based on matching of the COI DNA barcodes with pre-existing reference sequences suggested over 13,200 insect species are present, including 4,000 Coleoptera, 2,200 Diptera, and 2,700 Hymenoptera species, and 1,000 arachnid species (excluding mites). These results confirm that metabarcoding analyses of soil DNA tends to recover different components of terrestrial invertebrate biodiversity compared to traditional invertebrate sampling, but the combined methods provide a novel basis for estimating invertebrate biodiversity.

Systematics of the New Zealand Weevil Etheophanus Broun (Curculionidae: Molytinae).

Etheophanus Broun is considered a molytine based on the form of the pharyngeal plate, presence of a small spiculum relictum in the male, and presence of a pair of small internal apodemes on the antero-lateral corners of the 5th abdominal ventrite of the female. Examination of primary type specimens and newer material confirm one new species Etheophanus kuscheli sp. n. and two synonomies (Etheophanus nitidellus Broun, 1923 [= Etheophanus obscurus Broun, 1923] and Etheophanus striatus Broun, 1910 [=Etheophanus punctiventris Broun, 1914]). Generic and species diagnoses, a key to the species, and lectotype designations for three species are included. Phylogenetic reconstructions based on a combined analysis of the nuclear 28S rRNA and mitochondrial cytochrome c oxidase subunit I genes confirmed the status of E. kuscheli and a species complex, the E. nitidellus/E. optandus clade distributed in the southern portion of the South Island. The relationship E. pinguis [northern North Island] (E. striatus [southern North Island, northern South Island] (E. kuscheli [northwestern South Island] (E. nitidellus, E. optandus [southwestern North Island]) corresponds to geographic patterns found in other beetle lineages. Etheophanus striatus is composed of three lineages, one widespread in the north and south islands and two allopatric populations in the northwest South Island. The E. nitidellus/E. optandus complex includes four distinct lineages, one restricted to Fiordland, the other three sympatric in the region affected by the Haast Corridor.

Evolutionary history of Polyneoptera and its implications for our understanding of early winged insects.

Polyneoptera represents one of the major lineages of winged insects, comprising around 40,000 extant species in 10 traditional orders, including grasshoppers, roaches, and stoneflies. Many important aspects of polyneopteran evolution, such as their phylogenetic relationships, changes in their external appearance, their habitat preferences, and social behavior, are unresolved and are a major enigma in entomology. These ambiguities also have direct consequences for our understanding of the evolution of winged insects in general; for example, with respect to the ancestral habitats of adults and juveniles. We addressed these issues with a large-scale phylogenomic analysis and used the reconstructed phylogenetic relationships to trace the evolution of 112 characters associated with the external appearance and the lifestyle of winged insects. Our inferences suggest that the last common ancestors of Polyneoptera and of the winged insects were terrestrial throughout their lives, implying that wings did not evolve in an aquatic environment. The appearance of the first polyneopteran insect was mainly characterized by ancestral traits such as long segmented abdominal appendages and biting mouthparts held below the head capsule. This ancestor lived in association with the ground, which led to various specializations including hardened forewings and unique tarsal attachment structures. However, within Polyneoptera, several groups switched separately to a life on plants. In contrast to a previous hypothesis, we found that social behavior was not part of the polyneopteran ground plan. In other traits, such as the biting mouthparts, Polyneoptera shows a high degree of evolutionary conservatism unique among the major lineages of winged insects.

Positive selection and comparative molecular evolution of reproductive proteins from New Zealand tree weta (Orthoptera, Hemideina).

Animal reproductive proteins, especially those in the seminal fluid, have been shown to have higher levels of divergence than non-reproductive proteins and are often evolving adaptively. Seminal fluid proteins have been implicated in the formation of reproductive barriers between diverging lineages, and hence represent interesting candidates underlying speciation. RNA-seq was used to generate the first male reproductive transcriptome for the New Zealand tree weta species Hemideina thoracica and H. crassidens. We identified 865 putative reproductive associated proteins across both species, encompassing a diverse range of functional classes. Candidate gene sequencing of nine genes across three Hemideina, and two Deinacrida species suggests that H. thoracica has the highest levels of intraspecific genetic diversity. Non-monophyly was observed in the majority of sequenced genes indicating that either gene flow may be occurring between the species, or that reciprocal monophyly at these loci has yet to be attained. Evidence for positive selection was found for one lectin-related reproductive protein, with an overall omega of 7.65 and one site in particular being under strong positive selection. This candidate gene represents the first step in the identification of proteins underlying the evolutionary basis of weta reproduction and speciation.

Assembling large genomes: analysis of the stick insect (Clitarchus hookeri) genome reveals a high repeat content and sex-biased genes associated with reproduction.

BACKGROUND: Stick insects (Phasmatodea) have a high incidence of parthenogenesis and other alternative reproductive strategies, yet the genetic basis of reproduction is poorly understood. Phasmatodea includes nearly 3000 species, yet only the genome of Timema cristinae has been published to date. Clitarchus hookeri is a geographical parthenogenetic stick insect distributed across New Zealand. Sexual reproduction dominates in northern habitats but is replaced by parthenogenesis in the south. Here, we present a de novo genome assembly of a female C. hookeri and use it to detect candidate genes associated with gamete production and development in females and males. We also explore the factors underlying large genome size in stick insects. RESULTS: The C. hookeri genome assembly was 4.2 Gb, similar to the flow cytometry estimate, making it the second largest insect genome sequenced and assembled to date. Like the large genome of Locusta migratoria, the genome of C. hookeri is also highly repetitive and the predicted gene models are much longer than those from most other sequenced insect genomes, largely due to longer introns. Miniature inverted repeat transposable elements (MITEs), absent in the much smaller T. cristinae genome, is the most abundant repeat type in the C. hookeri genome assembly. Mapping RNA-Seq reads from female and male gonadal transcriptomes onto the genome assembly resulted in the identification of 39,940 gene loci, 15.8% and 37.6% of which showed female-biased and male-biased expression, respectively. The genes that were over-expressed in females were mostly associated with molecular transportation, developmental process, oocyte growth and reproductive process; whereas, the male-biased genes were enriched in rhythmic process, molecular transducer activity and synapse. Several genes involved in the juvenile hormone synthesis pathway were also identified. CONCLUSIONS: The evolution of large insect genomes such as L. migratoria and C. hookeri genomes is most likely due to the accumulation of repetitive regions and intron elongation. MITEs contributed significantly to the growth of C. hookeri genome size yet are surprisingly absent from the T. cristinae genome. Sex-biased genes identified from gonadal tissues, including genes involved in juvenile hormone synthesis, provide interesting candidates for the further study of flexible reproduction in stick insects.

Analysis of the genome of the New Zealand giant collembolan (Holacanthella duospinosa) sheds light on hexapod evolution.

BACKGROUND: The New Zealand collembolan genus Holacanthella contains the largest species of springtails (Collembola) in the world. Using Illumina technology we have sequenced and assembled a draft genome and transcriptome from Holacanthella duospinosa (Salmon). We have used this annotated assembly to investigate the genetic basis of a range of traits critical to the evolution of the Hexapoda, the phylogenetic position of H. duospinosa and potential horizontal gene transfer events. RESULTS: Our genome assembly was ~375 Mbp in size with a scaffold N50 of ~230 Kbp and sequencing coverage of ~180×. DNA elements, LTRs and simple repeats and LINEs formed the largest components and SINEs were very rare. Phylogenomics (370,877 amino acids) placed H. duospinosa within the Neanuridae. We recovered orthologs of the conserved sex determination genes thought to play a role in sex determination. Analysis of CpG content suggested the absence of DNA methylation, and consistent with this we were unable to detect orthologs of the DNA methyltransferase enzymes. The small subunit rRNA gene contained a possible retrotransposon. The Hox gene complex was broken over two scaffolds. For chemosensory ability, at least 15 and 18 ionotropic glutamate and gustatory receptors were identified, respectively. However, we were unable to identify any odorant receptors or their obligate co-receptor Orco. Twenty-three chitinase-like genes were identified from the assembly. Members of this multigene family may play roles in the digestion of fungal cell walls, a common food source for these saproxylic organisms. We also detected 59 and 96 genes that blasted to bacteria and fungi, respectively, but were located on scaffolds that otherwise contained arthropod genes. CONCLUSIONS: The genome of H. duospinosa contains some unusual features including a Hox complex broken over two scaffolds, in a different manner to other arthropod species, a lack of odorant receptor genes and an apparent lack of environmentally responsive DNA methylation, unlike many other arthropods. Our detection of candidate horizontal gene transfer candidates confirms that this phenomenon is occurring across Collembola. These findings allow us to narrow down the regions of the arthropod phylogeny where key innovations have occurred that have facilitated the evolutionary success of Hexapoda.