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The repeated evolutionary specialization of distantly related insects to cardenolide-containing host plants provides a stunning example of parallel adaptation. Hundreds of herbivorous insect species have independently evolved insensitivity to cardenolides, which are potent inhibitors of the alpha-subunit of Na + ,K + -ATPase (ATPα). Previous studies investigating ATPα-mediated cardenolide insensitivity in five insect orders have revealed remarkably high levels of parallelism in the evolution of this trait, including the frequent occurrence of parallel amino acid substitutions at two sites and recurrent episodes of duplication followed by neo-functionalization. Here we add data for a sixth insect order, Orthoptera, which includes an ancient group of highly aposematic cardenolide-sequestering grasshoppers in the family Pyrgomorphidae. We find that Orthopterans exhibit largely predictable patterns of evolution of insensitivity established by sampling other insect orders. Taken together the data lend further support to the proposal that negative pleiotropic constraints are a key determinant in the evolution of cardenolide insensitivity in insects. Furthermore, analysis of our expanded taxonomic survey implicates positive selection acting on site 111 of cardenolide-sequestering species with a single-copy of ATPα, and sites 115, 118 and 122 in lineages with neo-functionalized duplicate copies, all of which are sites of frequent parallel amino acid substitution. This article is part of the theme issue ‘Convergent evolution in the genomics era: new insights and directions’.

One of the most enduring mysteries in biology concerns the evolution of complex adaptations made up of interacting component traits. When these component traits do not enhance fitness independently of one another, their origin requires that they evolve sequentially through intermediate steps that do not produce their full adaptive value as a combined trait, or alternatively, that they arise via simultaneous, synergistic evolution. We tested these alternatives using the powerful but accessible example of leaf masquerade in katydids, where in some species, highly modified wings strikingly mimic vegetation to avoid predator recognition. Combining a field predation experiment with a phylogenetic comparative analysis of wing morphology in 58 Neotropical katydid species, we show that color and shape synergistically interact to enhance survival in the wild, and modifications in both traits evolved concurrently during diversification of this clade. Our findings identify the adaptive value of masquerade camouflage in the wild and show how concordant evolutionary change in separate traits—evolutionary synergy—can generate extraordinarily specialized, multi-component adaptations.

Phylogenomic data are revolutionizing the field of insect phylogenetics. One of the most tenable and cost-effective methods of generating phylogenomic data is target enrichment, which has resulted in novel phylogenetic hypotheses and revealed new insights into insect evolution. Orthoptera is the most diverse insect order within polyneoptera and includes many evolutionarily and ecologically interesting species. Still, the order as a whole has lagged behind other major insect orders in terms of transitioning to phylogenomics. In this study, we developed an Orthoptera-specific target enrichment (OR-TE) probe set from 80 transcriptomes across Orthoptera. The probe set targets 1828 loci from genes exhibiting a wide range of evolutionary rates. The utility of this new probe set was validated by generating phylogenomic data from 36 orthopteran species that had not previously been subjected to phylogenomic studies. The OR-TE probe set captured an average of 1037 loci across the tested taxa, resolving relationships across broad phylogenetic scales. Our detailed documentation of the probe design and bioinformatics process is intended to facilitate the widespread adoption of this tool.

Curated DNA barcode reference libraries are crucial for advancing environmental DNA (eDNA) studies, monitoring biological invasions, reliable biodiversity assessments, accurate species identification, etc. However, DNA barcode databases remain highly incomplete for most invertebrate taxa. In this study, we present the most comprehensive reference library to date for the family Tettigoniidae (Orthoptera) from the Iberian Peninsula—the most species-rich orthopteran family globally, with over 8,000 valid species. We generated 402 new DNA barcodes from at least 121 tettigoniid species from the Iberian Peninsula and integrated these with 169 previously published sequences. The resulting dataset comprises 571 barcoded specimens, representing 49 genera and 123 species, including many recently described taxa. Notably, we provide DNA barcodes for at least 68 described species that previously lacked them. Our dataset covers 85% of the tettigoniid species in the Iberian Peninsula and approximately 25% of European bush-cricket species. Furthermore, our analyses show that most tettigoniid species (95%) can be reliably identified using DNA barcoding. However, mitochondrial introgression events were detected in several species of the subfamilies Bradyporinae and Tettigoniinae, highlighting the need for cautious application of this molecular identification tool.

Background In evolutionary biology, identifying and quantifying inter-lineage genome size variation and elucidating the underlying causes of that variation have long been goals. Repetitive elements (REs) have been proposed and confirmed as being among the most important contributors to genome size variation. However, the evolutionary implications of genome size variation and RE dynamics are not well understood. Results A total of 35 Ensifera insects were collected from different areas in China, including nine species of crickets and 26 species of katydids. The genome sizes of seven species were then determined using flow cytometry. The RepeatExplorer2 pipeline was employed to retrieve the repeated sequences for each species, based on low-coverage (0.1 X) high-throughput Illumina unassembled short reads. The genome sizes of the 35 Ensifera insects exhibited a considerable degree of variation, ranging from 1.00 to 18.34 pg. This variation was more than 18-fold. Similarly, the RE abundances exhibited considerable variation, ranging from 13.66 to 61.16%. In addition, the Tettigonioidea had larger genomes and contained significantly more REs than did the Grylloidea genomes. Analysis of the correlation between RE abundance and the genome size of 35 Ensifera insects revealed that the abundance of REs, transposable elements (TEs), long terminal repeats (LTRs), and long interspersed nuclear elements (LINEs) are significantly correlated with genome size. Notably, there is an inflection point in this correlation, where species with increasingly large genomes (e.g., > 5–10 pg) have repeats that contribute less to genome expansion than expected. Furthermore, this study revealed contrasting evolutionary directions between the Tettigonioidea and Grylloidea clades in terms of the expansion of REs. Tettigonioidea species exhibit a gradual increase in ancestral genome size and RE abundance as they diverge, while Grylloidea species experience sustained genome contraction. Conclusions This study reveals extensive variation in genome size and RE abundance in Ensifera insects, with distinct evolutionary patterns across two major groups, Tettigonioidea and Grylloidea. This provides valuable insights into the variation in genome size and RE abundance in Ensifera insects, offering a comprehensive understanding of their evolutionary history.

A high portion of the earliest known insect fauna is composed of the so-called ‘lobeattid insects’, whose systematic affinities and role as foliage feeders remain debated. We investigated hundreds of samples of a new lobeattid species from the Xiaheyan locality using a combination of photographic techniques, including reflectance transforming imaging, geometric morphometrics, and biomechanics to document its morphology, and infer its phylogenetic position and ecological role. Ctenoptilus frequens sp. nov. possessed a sword-shaped ovipositor with valves interlocked by two ball-and-socket mechanisms, lacked jumping hind-legs, and certain wing venation features. This combination of characters unambiguously supports lobeattids as stem relatives of all living Orthoptera (crickets, grasshoppers, katydids). Given the herein presented and other remains, it follows that this group experienced an early diversification and, additionally, occurred in high individual numbers. The ovipositor shape indicates that ground was the preferred substrate for eggs. Visible mouthparts made it possible to assess the efficiency of the mandibular food uptake system in comparison to a wide array of extant species. The new species was likely omnivorous which explains the paucity of external damage on contemporaneous plant foliage.

Background DNA barcoding has been developed as a useful tool for species discrimination. Several sequence-based species delimitation methods, such as Barcode Index Number (BIN), REfined Single Linkage (RESL), Automatic Barcode Gap Discovery (ABGD), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP), were used. Our aim was to estimate Chinese katydid biodiversity using standard DNA barcode cytochrome c oxidase subunit I (COI-5P) sequences. Results Detection of a barcoding gap by similarity-based analyses and clustering-base analyses indicated that 131 identified morphological species (morphospecies) were assigned to 196 BINs and were divided into four categories: (i) MATCH (83/131 = 64.89%), morphospecies were a perfect match between morphospecies and BINs (including 61 concordant BINs and 22 singleton BINs); (ii) MERGE (14/131 = 10.69%), morphospecies shared its unique BIN with other species; (iii) SPLIT (33/131 = 25.19%, when 22 singleton species were excluded, it rose to 33/109 = 30.28%), morphospecies were placed in more than one BIN; (iv) MIXTURE (4/131 = 5.34%), morphospecies showed a more complex partition involving both a merge and a split. Neighbor-joining (NJ) analyses showed that nearly all BINs and most morphospecies formed monophyletic cluster with little variation. The molecular operational taxonomic units (MOTUs) were defined considering only the more inclusive clades found by at least four of seven species delimitation methods. Our results robustly supported 61 of 109 (55.96%) morphospecies represented by more than one specimen, 159 of 213 (74.65%) concordant BINs, and 3 of 8 (37.5%) discordant BINs. Conclusions Molecular species delimitation analyses generated a larger number of MOTUs compared with morphospecies. If these MOTU splits are proven to be true, Chinese katydids probably contain a seemingly large proportion of cryptic/undescribed taxa. Future amplification of additional molecular markers, particularly from the nuclear DNA, may be especially useful for specimens that were identified here as problematic taxa.

The mitochondrial genome (mitogenome) has been widely used to infer the phylogeny, origin and evolution of Orthopteran insects. Although several mitogenomic data have been used to study the phylogenetic relationships of Tetrigoidea (Orthoptera), the phylogenetic status of several subfamilies and tribes was still unclear due to the limited sampling of taxon. To further analyze the mitogenomic features and phylogeny of Tetrigoidea, five mitogenomes ( Zhengitettix curvispinus , Z. hainanensis , Scelimena melli , Eucriotettix oculatus and Thoradonta yunnana ) were sequenced and analyzed in this study, with Z. hainanensis being the newly published mitogenome and Z. curvispinus and S. melli being the complete mitogenomes. Nucleotide composition showed that more A and T bases than C and G bases were found in the sampled mitogenomes, with A- and C-skew. A large intergenic region containing tandem repeats was identified between trnS(ucn) and nad1 in the Z. curvispinus mitogenome. The protein-coding genes (PCGs) used ATG and TAA as the most common initiation and termination codons, respectively. The tRNAs showed a typical clover secondary structure in the Z. curvispinus . The A+T-rich region contained tandem repeats in Z. curvispinus . Phylogenetic analyses of Tetrigoidea based on the maximum likelihood (ML) and Bayesian inference (BI) method supported several non-monophyly subfamilies and tribes, such as Scelimeninae and Thoradontini. Divergence time results showed that Tetrigoidea is one of the basal branch of Orthoptera, with Batrachideinae splitting first, followed by Tripetalocerinae. The genera diverged over a relatively long period, expanding from the Jurassic to the Neogene. These results provide useful data for the study of the mitogenome characteristic of the Tetrigoidea and even the whole Orthoptera, and were basic resources for their phylogeny and evolution study.

Myrmecophilus acervorum , previously considered a parthenogenetic species widely-distributed in Europe, has been observed to have both sexes in populations inhabiting the central part of the distribution range. Specimens from those heterosexual populations have been found being infected with Wolbachia . New mitochondrial data (COI and 16S markers) revealed the well-supported differentiation of M. acervorum populations inhabiting western Polesie (Poland) and southern Europe. In turn, analyses of EF1α marker support the hypothesis on the unfinished lineage sorting at the nuclear DNA level. Interestingly, we found that parthenogenetic populations inhabiting western Polesie are infected with Wolbachia belonging to supergroup A, while endosymbionts occurring in sexual populations of M. acervorum observed in Romania belong to supergroup B. Furthermore, new and potentially diagnostic characteristics in the external structures of the eyes of M. acervorum were identified. The surface of ommatidia in specimens occurring in southern Europe was smooth. In contrast, the ommatidia surface of individuals collected in Poland was visibly sculptured. To sum up, the significant genetic variability found in the present case, and the differentiating morphological character, are almost certainly effects of cryptic species being present within M. acervorum. This is indicative of ongoing speciation within the populations of this insect, and of simultaneous unfinished lineage sorting at the nuclear DNA level.

Recently, the heteroxenous eyeworm, Oxyspirura petrowi, has gained attention due to its prevalence in the declining game bird, Northern bobwhite (Colinus virginianus), but the intermediate hosts of many nematodes remain unknown. However, identifying the intermediate host of O. petrowi with traditional techniques would be difficult and time-consuming, especially considering there are more than 80 potential orthopteran hosts just in Texas. To screen a large number of samples quickly and effectively, primers for nested PCR (nPCR) were developed using the internal transcribed spacer 1 (ITS1) region. Then the nPCR was used to identify which of the 35 species collected from the Order Orthoptera were potential intermediate hosts of O. petrowi. With this technique, 18 potential intermediate hosts were identified. Later, we collected live specimens of species that tested positive to confirm the presence of larvae, but larvae were not found in the live specimens, nor in the extra tissue of the species that had tested positive for O. petrowi DNA. Despite this, this study demonstrated that nPCR is more sensitive than traditional techniques and can be a valuable tool in determining the intermediate hosts of parasites.