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Ingestion of the cycad toxins β-methylamino-L-alanine (BMAA) and azoxyglycosides is harmful to diverse organisms. However, some insects are specialized to feed on toxin-rich cycads with apparent immunity. Some cycad-feeding insects possess a common set of gut bacteria, which might play a role in detoxifying cycad toxins. Here, we investigated the composition of gut microbiota from a worldwide sample of cycadivorous insects and characterized the biosynthetic potential of selected bacteria. Cycadivorous insects shared a core gut microbiome consisting of six bacterial taxa, mainly belonging to the Proteobacteria, which we were able to isolate. To further investigate selected taxa from diverging lineages, we performed shotgun metagenomic sequencing of co-cultured bacterial sub-communities. We characterized the biosynthetic potential of four bacteria from Serratia, Pantoea, and two different Stenotrophomonas lineages, and discovered a suite of biosynthetic gene clusters notably rich in siderophores. Siderophore semi-untargeted metabolomics revealed a broad range of chemically related yet diverse iron-chelating metabolites, including desferrioxamine B, suggesting the occurrence of an unprecedented desferrioxamine-like biosynthetic pathway that remains to be identified. These results provide a foundation for future investigations into how cycadivorous insects tolerate diets rich in azoxyglycosides, BMAA, and other cycad toxins, including a possible role for bacterial siderophores.

 

An insect’s living systems—circulation, respiration, and a branching nervous system—extend from the body into the wing. Wing hemolymph circulation is critical for hydrating tissues and supplying nutrients to living systems such as sensory organs across the wing. Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered “lifeless” cuticle, and flows remain largely unquantified. High-speed fluorescent microscopy and particle tracking of hemolymph in the wings and body of the grasshopperSchistocerca americanarevealed dynamic flow in every vein of the fore- and hindwings. The global system forms a circuit, but local flow behavior is complex, exhibiting three distinct types: pulsatile, aperiodic, and “leaky” flow. Thoracic wing hearts pull hemolymph from the wing at slower frequencies than the dorsal vessel; however, the velocity of returning hemolymph (in the hindwing) is faster than in that of the dorsal vessel. To characterize the wing’s internal flow mechanics, we mapped dimensionless flow parameters across the wings, revealing viscous flow regimes. Wings sustain ecologically important insect behaviors such as pollination and migration. Analysis of the wing circulatory system provides a template for future studies investigating the critical hemodynamics necessary to sustaining wing health and insect flight.

 

Next-generation sequencing (NGS) technologies have revolutionized phylogenomics by decreasing the cost and time required to generate sequence data from multiple markers or whole genomes. Further, the fragmented DNA of biological specimens collected decades ago can be sequenced with NGS, reducing the need for collecting fresh specimens. Sequence capture, also known as anchored hybrid enrichment, is a method to produce reduced representation libraries for NGS sequencing. The technique uses single-stranded oligonucleotide probes that hybridize with pre-selected regions of the genome that are sequenced via NGS, culminating in a dataset of numerous orthologous loci from multiple taxa. Phylogenetic analyses using these sequences have the potential to resolve deep and shallow phylogenetic relationships. Identifying the factors that affect sequence capture success could save time, money, and valuable specimens that might be destructively sampled despite low likelihood of sequencing success. We investigated the impacts of specimen age, preservation method, and DNA concentration on sequence capture (number of captured sequences and sequence quality) while accounting for taxonomy and extracted tissue type in a large-scale butterfly phylogenomics project. This project used two probe sets to extract 391 loci or a subset of 13 loci from over 6,000 butterfly specimens. We found that sequence capture is a resilient method capable of amplifying loci in samples of varying age (0–111 years), preservation method (alcohol, papered, pinned), and DNA concentration (0.020 ng/μl - 316 ng/ul). Regression analyses demonstrate that sequence capture is positively correlated with DNA concentration. However, sequence capture and DNA concentration are negatively correlated with sample age and preservation method. Our findings suggest that sequence capture projects should prioritize the use of alcohol-preserved samples younger than 20 years old when available. In the absence of such specimens, dried samples of any age can yield sequence data, albeit with returns that diminish with increasing age. 

The Albany pitcher plant, Cephalotus follicularis , has evolved cup-shaped leaves and a carnivorous habit completely independently from other lineages of pitcher plants. It is the only species in the family Cephalotaceae and is restricted to a small region of Western Australia. Here, we used metabarcoding to characterize the bacterial and eukaryotic communities living in C. follicularis pitchers at two different sites. Bacterial and eukaryotic communities were correlated in both richness and composition; however, the factors associated with richness were not the same across bacteria and eukaryotes, with bacterial richness differing with fluid color, and eukaryotic richness differing with the concentration of DNA extracted from the fluid, a measure roughly related to biomass. For turnover in composition, the variation in both bacterial and eukaryotic communities primarily differed with fluid acidity, fluid color, and sampling site. We compared C. follicularis -associated community diversity with that of Australian Nepenthes mirabilis , as well as a global comparison of Southeast Asian Nepenthes and North American Sarracenia . Our results showed similarity in richness with communities from other pitcher plants, and specific bacterial taxa shared among all three independent lineages of pitcher plants. Overall, we saw convergence in richness and particular clades colonizing pitcher plants around the world, suggesting that these highly specialized habitats select for certain numbers and types of inhabitants. 

We present an economical imaging system with integrated hardware and software to capture multispectral images of Lepidoptera with high efficiency. This method facilitates the comparison of colors and shapes among species at fine and broad taxonomic scales and may be adapted for other insect orders with greater three-dimensionality. Our system can image both the dorsal and ventral sides of pinned specimens. Together with our processing pipeline, the descriptive data can be used to systematically investigate multispectral colors and shapes based on full-wing reconstruction and a universally applicable ground plan that objectively quantifies wing patterns for species with different wing shapes (including tails) and venation systems. Basic morphological measurements, such as body length, thorax width, and antenna size are automatically generated. This system can increase exponentially the amount and quality of trait data extracted from museum specimens.

 

The last Xerces blue butterfly was seen in the early 1940s, and its extinction is credited to human urban development. This butterfly has become a North American icon for insect conservation, but some have questioned whether it was truly a distinct species, or simply an isolated population of another living species. To address this question, we leveraged next-generation sequencing using a 93-year-old museum specimen. We applied a genome skimming strategy that aimed for the organellar genome and high-copy fractions of the nuclear genome by a shallow sequencing approach. From these data, we were able to recover over 200 million nucleotides, which assembled into several phylogenetically informative markers and the near-complete mitochondrial genome. From our phylogenetic analyses and haplotype network analysis we conclude that the Xerces blue butterfly was a distinct species driven to extinction. 

The Hengduan Mountains region is a biodiversity hotspot known for its topologically complex, deep valleys and high mountains. While landscape and glacial refugia have been evoked to explain patterns of interspecies divergence, the accumulation of intra‐species (i.e., population level) genetic divergence across the mountain‐valley landscape in this region has received less attention. We used genome‐wide restriction site‐associated DNA sequencing (RADseq) to reveal signatures of Pleistocene glaciation in populations ofThitarodes shambalaensis(Lepidoptera: Hepialidae), the host moth of parasiticOphiocordyceps sinensis(Hypocreales: Ophiocordycipitaceae) or caterpillar fungus” endemic to the glacier of eastern Mt. Gongga. We used moraine history along the glacier valleys to model the distribution and environmental barriers to gene flow across populations ofT.shambalaensis. We found that moth populations separated by less than 10 km exhibited valley‐based population genetic clustering and isolation‐by‐distance (IBD), while gene flow among populations was best explained by models using information about their distributions at the local last glacial maximum (LGM_L, 58 kya), not their contemporary distribution. Maximum likelihood lineage history among populations, and among subpopulations as little as 500 m apart, recapitulated glaciation history across the landscape. We also found signals of isolated population expansion following the retreat of LGM_Lglaciers. These results reveal the fine‐scale, long‐term historical influence of landscape and glaciation on the genetic structuring of populations of an endangered and economically important insect species. Similar mechanisms, given enough time and continued isolation, could explain the contribution of glacier refugia to the generation of species diversity among the Hengduan Mountains.

 

Coevolution between plants and insects is thought to be responsible for generating biodiversity. Extensive research has focused largely on antagonistic herbivorous relationships, but mutualistic pollination systems also likely contribute to diversification. Here we describe an example of chemically-mediated mutualistic species interactions affecting trait evolution and lineage diversification. We show that volatile compounds produced by closely related species of Zamia cycads are more strikingly different from each other than are other phenotypic characters, and that two distantly related pollinating weevil species have specialized responses only to volatiles from their specific host Zamia species. Plant transcriptomes show that approximately a fifth of genes related to volatile production are evolving under positive selection, but we find no differences in the relative proportion of genes under positive selection in different categories. The importance of phenotypic divergence coupled with chemical communication for the maintenance of this obligate mutualism highlights chemical signaling as a key mechanism of coevolution between cycads and their weevil pollinators. 

Here, we present the largest, global dataset of Lepidopteran traits, focusing initially on butterflies (ca. 12,500 species records). These traits are derived from field guides, taxonomic treatments, and other literature resources. We present traits on wing size, phenology,voltinism, diapause/overwintering stage, hostplant associations, and habitat affinities (canopy, edge, moisture, and disturbance). This dataset will facilitate comparative research on butterfly ecology and evolution and our goal is to inspire future research collaboration and the continued development of this dataset.