Genome Sequence of the Wheat Stem Sawfly, Cephus cinctus, Representing an Early-Branching Lineage of the Hymenoptera, Illuminates Evolution of Hymenopteran Chemoreceptors

Abstract

The wheat stem sawfly, Cephus cinctus, is a major pest of wheat and key ecological player in the grasslands of western North America. It also represents the distinctive Cephoidea superfamily of sawflies (Symphyta) that appeared early during the hymenopteran radiation, but after three early-branching eusymphytan superfamilies that form the base of the order Hymenoptera. We present a high-quality draft genome assembly of 162 Mb in 1,976 scaffolds with a scaffold N50 of 622 kb. Automated gene annotation identified 11,210 protein-coding gene models and 1,307 noncoding RNA models. Thirteen percent of the assembly consists of ∼58,000 transposable elements partitioned equally between Class-I and Class-II elements. Orthology analysis reveals that 86% of Cephus proteins have identifiable orthologs in other insects. Phylogenomic analysis of conserved subsets of these proteins supports the placement of the Cephoidea between the Eusymphyta and the parasitic woodwasp superfamily Orussoidea. Manual annotation and phylogenetic analysis of families of odorant, gustatory, and ionotropic receptors, plus odorant-binding proteins, shows that Cephus has representatives for most conserved and expanded gene lineages in the Apocrita (wasps, ants, and bees). Cephus has also maintained several insect gene lineages that have been lost from the Apocrita, most prominently the carbon dioxide receptor subfamily. Furthermore, Cephus encodes a few small lineage-specific chemoreceptor gene family expansions that might be involved in adaptations to new grasses including wheat. These comparative analyses identify gene family members likely to have been present in the hymenopteran ancestor and provide a new perspective on the evolution of the chemosensory gene repertoire.

Fig. 1.

—Life cycle of the wheat stem sawfly, Cephus cinctus Norton. (A) After overwintering in diapause in a senesced wheat stem, a prepupa metamorphoses in late spring or early summer. The pupal stage lasts 10–12 days, after which the newly eclosed adult chews a hole in a frass plug left by the larva to emerge from the stem. (B) The adults can mate immediately after emergence, either on the wheat stubble or on new host stems. The short-lived females oviposit 30–40 eggs before dying in 7–10 days. (C) Newly deposited eggs in the stem lumen, usually from multiple females, hatch in 5–10 days. The neonate larvae feed on the parenchyma lining the stem interior. As the larvae grow through four to five larval instars they forage on the stem lining throughout the stem by boring through the nodes. The duration of larval feeding can range from 3 to 8 weeks. (D) Within the stem, cannibalism leads to a single surviving larva that migrates to the base of the stem at plant senescence and ripening. The larva girdles the interior of the ripened stem wall, cutting a groove that encircles the entire stem. Wind or gravity causes the weakened stem to break and lodge, leaving the unharvested wheat head on the soil. Below this groove, the larva makes a frass plug before migrating to the base of the stem near the crown. Within a thin hibernaculum, the prepupa remains protected in obligate diapause from late summer through to warming the following spring. This period of inactivity can last >9 months. Image created by Megan L. Hofland and Norma J. Irish, Department of Land Resources and Environmental Sciences, Montana State University.

Fig. 2.

—Phylogenomics, orthology, sequence identity, and functional annotation of Cephus cinctus genes. (A) Maximum likelihood molecular phylogeny estimated from the aligned protein sequences of 852 single-copy orthologs from 17 selected insect species, including three sawflies, Athalia rosae, C. cinctus, and Orussus abietinus, with the banded demoiselle, Calopteryx splendens (Odonata), as the outgroup. Branch lengths represent substitutions per site, all nodes except for the placement of O. abietinus (labeled) achieved 100% bootstrap support. (B) Total gene counts per species partitioned into categories from single-copy orthologs in all 17 species, present (i.e., allowing gene duplications) in all or most (>12) or with patchy species distributions (≤12), to lineage-restricted orthologs (sawflies and outgroups, non-hymenopteran Holometabola, Blattodea, Hemiptera, or Apocrita), genes with orthologs from other sequenced insect genomes, and those with currently no identifiable orthologs. (C) Distributions of percent amino acid identities of 1,035 single-copy orthologs from each of the three sawflies with Apis mellifera, Pogonomyrmex barbatus, and Nasonia vitripennis. Violin plots show the smoothed densities of each distribution, with boxplots indicating medians and upper and lower quartiles. Asterisks indicate statistically significant differences with P values <1e-108 (**) and <1e-39 (*). (D) Functional inferences for C. cinctus genes from Gene Ontology (GO) annotations of orthologs from A. mellifera, Anopheles gambiae, Drosophila melanogaster, or Tribolium castaneum. Of the 7,595 C. cinctus genes with orthologs from any of these four species, 57% were in orthologous groups with GO terms shared among at least two of the four species (common annotations), 22% with GO terms from only one species (unique annotations), and 21% where no GO terms were associated with any of the orthologs (no annotations).

Fig. 3.

—Phylogenetic relationships of the gustatory receptor family in Hymenoptera. The tree was rooted by declaring the sugar and carbon dioxide receptor lineages as the outgroup, based on their basal location within insect GRs in an analysis of the entire GR family in insects and other animals (Robertson 2015). GR names and the branches leading to them are colored by species, purple for Cephus cinctus, cyan for Nasonia vitripennis, brown for Pogonomyrmex barbatus, green for Bombus terrestris, blue for Apis mellifera, and black for other species. The CcinGr lineages are indicated outside the circle of names, and major lineages with CcinGr representatives are highlighted in colored segments. The scale bar indicates substitutions per site, and the filled circle indicates approximate likelihood ratio test (aLRT) support of 1.

Fig. 4.

—Expression levels of selected ORs. Expression levels are shown as Reads Per Kilobase transcript per Million reads (RPKM) in each library for 8 of the 73 OR family genes and the 17 RNAseq libraries (detailed in supplementary table S1, Supplementary Material online) that sampled different life stages and tissues of male and female sawflies collected from 2011 to 2016. Abbreviations: abd, abdomen; ant, antennae; F, female; M, male.

Fig. 5.

—Expression levels of selected GRs. Expression levels in RPKM for 8 of the 36 GR family genes. Other details as for figure 4.

Fig. 6.

—Expression levels of selected IRs. Expression levels in RPKM for 8 of the 49 IR family genes. Other details as for figure 4.

Fig. 7.

—Expression levels of selected OBPs. Expression levels RPKM for 8 of the 15 Obp family genes. Other details as for figure 4.