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Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation—both in terms of length and repeat motif order—between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 Mya: the insect clade containing caddisflies and butterflies and spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests common mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation.

Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation—both in terms of length and repeat motif order—between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 million years ago—(1) the insect clade containing caddisflies and butterflies and (2) spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation.

Whole genome sequencing for generating SNP data is increasingly used in population genetic studies. However, obtaining genomes for massive numbers of samples is still not within the budgets of many researchers. It is thus imperative to select an appropriate reference genome and sequencing depth to ensure the accuracy of the results for a specific research question, while balancing cost and feasibility. To evaluate the effect of the choice of the reference genome and sequencing depth on downstream analyses, we used five confamilial reference genomes of variable relatedness and three levels of sequencing depth (3.5×, 7.5× and 12×) in a population genomic study on two caddisfly species

BackgroundLarvae of Trichoptera produce silk to build various underwater structures allowing them to exploit a wide range of aquatic environments. Theheavy chain fibroin(h-fibroin) gene encodes the primary protein component of their silk. Studies on this long (>20 kbp) and highly repetitive gene have been limited by difficulties in its sequence assembly. Recently, high-quality long-read sequencing techniques have been successfully applied to obtain the full-lengthh-fibroinsequence.ResultsWe used three new and five previously published genomes to identify eight full-length h-fibroin gene and protein sequences of h-fibroin across the order Trichoptera covering various endpoints of the diversity of ecological silk use in caddisflies. We analyze these together with four existing high-quality h-fibroin sequences. Across the order, we observed conserved patterns in h-fibroin (high similarity of amino(n)-/carboxyl(c)-termini, presence of characteristic repeating structural modules). However, the sequence, number, and arrangement of these repeating modules varied across clades with increasing structural complexity of h-fibroin in fixed retreat and tube-case builders compared to cocoon-builders. We also found a higher percentage of proline in fixed-retreat makers.ConclusionThis study provides characterizations of the primary structure of h-fibroin from a diverse set of caddisflies. The interplay of conserved termini and basic motif structure with high variation in repeating modules as well as the variation in the percentage of proline might be linked to differences in mechanical properties (i.e., tensile strength, toughness) related to the different silk usage. This sets a starting point for future studies to screen and correlate amino acid motifs and other sequence features with quantifiable silk properties.

The divergence of sister orders, Trichoptera (caddisflies) and Lepidoptera (moths and butterflies), from a silk-spinning ancestor occurred around 290 million years ago. Trichoptera larvae are mainly aquatic and Lepidoptera larvae are almost entirely terrestrial, distinct habitats that required molecular adaptation of their silk for deployment in water versus air. The major protein components of their silks are Heavy chain and Light chain fibroins. In an effort to identify molecular changes in L-fibroins that may have contributed to the divergent use of silk in water versus air, we used the ColabFold implementation of AlphaFold2 to predict three-dimensional structures of L-fibroins from both orders. Comparison of the structures revealed that despite the ancient divergence, profoundly different habitats, and low sequence conservation, a 10-helix core structure was strongly conserved in L-fibroins from both orders. Previously known intra- and intermolecular disulfide linkages were accurately predicted. Structure variations observed outside of the core may represent molecular changes that contributed to the evolution of silks adapted to water or air. The distributions of electrostatic potential, for example, are not conserved and present distinct order-specific surfaces for potential interactions with or modulation by external factors. Also, the interactions of L-fibroins with the H-fibroin C-termini are different between the orders; lepidopteran L-fibroins have N-terminal insertions, not present in trichopteran L-fibroins, which form an unstructured ribbon in isolation but become part of an intermolecular β-sheet when folded with their corresponding H-fibroin C-termini. The results may serve as another example of protein structure prediction from deep sequence data of understudied proteins made possible by AlphaFold2.

Whole-genome sequencing for generating SNP data is increasingly used in population genetic studies. However, obtaining genomes for massive numbers of samples is still not within the budgets of many researchers. It is thus imperative to select an appropriate reference genome and sequencing coverage to ensure the accuracy of the results for a specific research question, while balancing cost and feasibility. To evaluate the effect of the choice of the reference genome and sequencing coverage on downstream analyses, we used five confamilial reference genomes of variable relatedness and three levels of sequencing coverage (3.5x, 7.5x and 12x) in a population genomic study on two caddisfly species: Himalopsyche digitata and H. tibetana. Using these 30 datasets (five reference genomes × three coverages × two target species), we estimated population genetic indices (inbreeding coefficient, nucleotide diversity, pairwise and genome-wide FST) based on variants and population structure (PCA and admixture) based on genotype likelihood estimates. The results showed that both distantly related reference genomes and lower sequencing coverage lead to degradation of resolution. In addition, choosing a more closely related reference genome may significantly remedy the defects caused by low coverage. Therefore, we conclude that population genetic studies would benefit from closely related reference genomes, especially as the costs of obtaining a high-quality reference genome continue to decrease. However, to determine a cost-efficient strategy for a specific population genomic study, a trade-off between reference genome relatedness and sequencing depth can be considered.

Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes. Yet, RE dynamics remain understudied in many taxonomic groups, preventing holistic understanding of how genomes and species evolve. Here, we investigated REs across 601 insect species (20 orders) to better understand the RE landscape of insects and to evaluate automated RE annotation methods in the era of biodiversity genomics. We identified wide variation in the types and frequency of REs across insect groups. We quantified associations between REs and protein-coding genes and found an elevated frequency of associations in insects with abundant long interspersed nuclear elements (LINEs). Sequencing technology impacts RE detection; ∼36% more REs could be identified in long-read versus short-read assemblies. Long terminal repeats (LTRs) showed markedly improved detection in long-read assemblies (162% more), while DNA transposons and LINEs showed less respective technology-related bias. We illustrate fundamental challenges to efficient study of REs in diverse groups, showing that in most insect lineages, 25–85% of repetitive sequences were “unclassified” compared to only ∼13% of unclassified repeats in Drosophila species. Our findings suggest this RE-annotation bottleneck, driven largely by uneven taxonomic representation in RE reference databases, is worsening. Although the diversity of available insect genomes has rapidly expanded, the rate of community contributions to RE databases (essential for RE annotation) has not kept pace, preventing high resolution study of REs in most groups. We highlight the tremendous opportunity and need for the field of biodiversity genomics to embrace REs and suggest collective steps for making progress towards this goal.

We sequence, assemble, and annotate the genome of Atopsyche davidsoni Sykora, 1991, the first whole-genome assembly for the caddisfly family Hydrobiosidae. This free-living and predatory caddisfly inhabits streams in the high-elevation Andes and is separated by more than 200 Myr of evolutionary history from the most closely related caddisfly species with genome assemblies available. We demonstrate the promise of PacBio HiFi reads by assembling the most contiguous caddisfly genome assembly to date with a contig N50 of 14 Mb, which is more than 6× more contiguous than the current most contiguous assembly for a caddisfly (Hydropsyche tenuis). We recover 98.8% of insect BUSCO genes indicating a high level of gene completeness. We also provide a genome annotation of 12,232 annotated proteins. This new genome assembly provides an important new resource for studying genomic adaptation of aquatic insects to harsh, high-altitude environments.

Members of the speciose insect order Trichoptera (caddisflies) provide important ecosystem services, for example, nutrient cycling through breaking down of organic matter. They are also of industrial interest due to their larval silk secretions. These form the basis for their diverse case-making behavior that allows them to exploit a wide range of ecological niches. Only five genomes of this order have been published thus far, with variable qualities regarding contiguity and completeness. A low-cost sequencing strategy, that is, using a single Oxford Nanopore flow cell per individual along with Illumina sequence reads was successfully used to generate high-quality genomes of two Trichoptera species, Plectrocnemia conspersa and Hydropsyche tenuis. Of the de novo assembly methods compared, assembly of low coverage Nanopore reads (∼18×) and subsequent polishing with long reads followed by Illumina short reads (∼80–170× coverage) yielded the highest genome quality both in terms of contiguity and BUSCO completeness. The presented genomes are the shortest to date and extend our knowledge of genome size across caddisfly families. The genomic region that encodes for light (L)-chain fibroin, a protein component of larval caddisfly silk was identified and compared with existing L-fibroin gene clusters. The new genomic resources presented in this paper are among the highest quality Trichoptera genomes and will increase the knowledge of this important insect order by serving as the basis for phylogenomic and comparative genomic studies.

We provide a new, annotated genome assembly of Neomicropteryx cornuta, a species of the so-called mandibulate archaic moths (Lepidoptera: Micropterigidae). These moths belong to a lineage that is thought to have split from all other Lepidoptera more than 300 Ma and are consequently vital to understanding the early evolution of superorder Amphiesmenoptera, which contains the order Lepidoptera (butterflies and moths) and its sister order Trichoptera (caddisflies). Using PacBio HiFi sequencing reads, we assembled a highly contiguous genome with a contig N50 of nearly 17 Mb. The assembled genome length of 541,115,538 bp is about half the length of the largest published Amphiesmenoptera genome (Limnephilus lunatus, Trichoptera) and double the length of the smallest (Papilio polytes, Lepidoptera). We find high recovery of universal single copy orthologs with 98.1% of BUSCO genes present and provide a genome annotation of 15,643 genes aided by resolved isoforms from PacBio IsoSeq data. This high-quality genome assembly provides an important resource for studying ecological and evolutionary transitions in the early evolution of Amphiesmenoptera.

Background Genome size is implicated in the form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: polyploidy (i.e., whole-genome duplication) or smaller duplication events and bursts in the activity of repetitive elements. Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages. Results We detect a ∼14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single-copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments). Conclusion Our findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

The first insect genome assembly (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 insect species representing 20 orders. In this study, we analyzed the most-contiguous assembly for each species and provide a “state-of-the-field” perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technologies. Relative to species richness, genomic efforts have been biased toward four orders (Diptera, Hymenoptera, Collembola, and Phasmatodea), Coleoptera are underrepresented, and 11 orders still lack a publicly available genome assembly. The average insect genome assembly is 439.2 Mb in length with 87.5% of single-copy benchmarking genes intact. Most notable has been the impact of long-read sequencing; assemblies that incorporate long reads are ∼48× more contiguous than those that do not. We offer four recommendations as we collectively continue building insect genome resources: 1) seek better integration between independent research groups and consortia, 2) balance future sampling between filling taxonomic gaps and generating data for targeted questions, 3) take advantage of long-read sequencing technologies, and 4) expand and improve gene annotations.

BackgroundGenome size is implicated in form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: Polyploidy (i.e., whole genome duplication: WGD) or smaller duplication events and bursts in the activity of repetitive elements (RE). Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages.ResultsWe detect a ~14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element (RE) expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments).ConclusionOur findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

Trichoptera (caddisflies) play an essential role in freshwater ecosystems; for instance, larvae process organic material from the water and are food for a variety of predators. Knowledge on the genomic diversity of caddisflies can facilitate comparative and phylogenetic studies thereby allowing scientists to better understand the evolutionary history of caddisflies. While Trichoptera are the most diverse aquatic insect order, they remain poorly represented in terms of genomic resources. To date, all long-read based genomes have been sequenced from individuals in the retreat-making suborder, Annulipalpia, leaving ∼275 Ma of evolution without high-quality genomic resources. Here, we report the first long-read based de novo genome assemblies of two tube case-making Trichoptera from the suborder Integripalpia, Agrypnia vestita Walker and Hesperophylax magnus Banks. We find that these tube case-making caddisflies have genome sizes that are at least three-fold larger than those of currently sequenced annulipalpian genomes and that this pattern is at least partly driven by major expansion of repetitive elements. In H. magnus, long interspersed nuclear elements (LINEs) alone exceed the entire genome size of some annulipalpian counterparts suggesting that caddisflies have high potential as a model for understanding genome size evolution in diverse insect lineages.SignificanceThere is a lack of genomic resources for aquatic insects. So far, only three high-quality genomes have been assembled, all from individuals in the retreat-making suborder Annulipalpia. In this article, we report the first high-quality genomes of two case-making species from the suborder Integripalpia, which are essential for studying genomic diversity across this ecologically diverse insect order. Our research reveals larger genome sizes in the tube case-makers (suborder Integripalpia, infraorder Phryganides), accompanied by a disproportionate increase of repetitive DNA. This suggests that genome size is at least partly driven by a major expansion of repetitive elements. Our work shows that caddisflies have high potential as a model for understanding how genomic diversity might be linked to functional diversification and forms the basis for detailed studies on genome size evolution in caddisflies.Data depositionThis project has been deposited at NCBI under the Bioproject ID: PRJNA668166

A supra-annual, community-level synchronous flowering prevails in several parts of the tropical forests of Southeast Asia and its evolution has been hypothesized to be linked to pollinator shifts. The aseasonal Southeast Asian lowland rainforests are dominated by Dipterocarpaceae, which exhibit great floral diversity, a range of pollination syndromes and include species with annual and supra-annual gregarious flowering. Phylogenetic relationships within this family are still unclear, especially in the tribe Shoreeae. Here, we develop a pipeline to maximize recovery of genome-wide SNPs from restriction-site associated DNA sequencing (RADseq) in non-model organisms across wide phylogenetic scales. We then infer phylogenomic relationships in the tribe Shoreeae using both traditional and coalescent analyses. The phylogenetic trees obtained with these methods are congruent to each other and highly resolved. They allow reconstructing the evolutionary patterns of floral traits (number of stamens, anther structure and anther/appendage size) in the group. Our inferences indicate that species with many stamens, but smaller, globose anthers and longer appendages and have evolved multiple times from species with fewer stamens, but larger, oblong anthers and shorter appendages. This could have happened in parallel to iterative shifts in pollinators across the uncovered phylogeny from larger, longer generation to smaller, shorter-generation insects that can quickly build up the necessary population sizes during mass flowering episodes.

Milleporidae are of high ecological and economic importance, as, together with the scleractinian corals, they belong to the main reef builders of tropical coral reefs. Coral reefs face severe threats mainly due to anthropogenic disturbance. Understanding their population structure and dynamics is crucial for any conservation effort. Here we report the first microsatellite loci for the Milleporidae. Eleven polymorphic markers were developed for the hydrozoan corals Millepora dichotoma from the Great Barrier Reef (Australia) and tested for amplification in M. dichotoma from the Red Sea (Egypt), as well as for Millepora platyphylla from the Pacific Ocean (Moorea, French Polynesia). All loci were variable with 4–15 alleles per locus. Nine loci were transferable between geographic regions and species. These are the first microsatellites for hydrozoan corals. They will provide valuable tools for characterizing the population structure and genetic diversity of the group thereby benefitting coral reef conservation.

Premise of the study: PCR amplification of the matK barcoding region is often difficult when dealing with multiple angiosperm families. We developed a primer cocktail to amplify this region efficiently across angiosperm diversity. Methods and Results: We developed 14 matK primers (seven forward, seven reverse) for multiplex PCR, using sequences available in GenBank for 178 taxa belonging to 123 genera in 41 families and 18 orders. Universality of these new multiplexed primers was tested with 53 specimens from 44 representative angiosperm families in 23 different orders. Our primers showed high PCR amplification and sequencing success. Conclusions: These results show that our newly developed primers are highly effective for multiplex PCR and can be employed in future barcode projects involving taxonomically diverse samples across angiosperms. Using multiplex primers for barcoding will reduce the cost and time needed for PCR amplification.

Plectranthus hadiensis is an important medicinal plant in Sri Lanka. It was considered a separate species, P. zeylanicus, endemic to the island until its inclusion, as P. hadiensis var. tomentosus, together with morphs from southern Africa in the revised species concept of P. hadiensis. However, there are morphological, chemical, and therapeutic differences between the African and Sri Lankan morphs. We used eight molecular markers in a phylogenetic study to clarify the species concept of P. hadiensis and to investigate whether it should include the Sri Lankan morph. We examined the position of the two P. hadiensis morphs in relation to eight other Plectranthus species. The maximum likelihood tree revealed three clades: a weakly supported clade including P. calycinus, P. glabratus, P. fruticosus, and P. malabaricus; a highly supported clade including P. amboinicus and African and Sri Lankan specimens of P. hadiensis; and a highly supported clade formed by P. barbatus, P. caninus, and P. hadiensis var. tomentosus. The African P. hadiensis specimens form a highly supported subclade sister to a subclade containing the Sri Lankan P. hadiensis, suggesting that the subclades correspond to either two sister species or two subspecies. We propose that they are more likely to be sister species given the differences in morphology, chemistry, and chromosome number.