Indirect methods for conducting faunal inventories present great promise, and genomic inventories derived from environmental sources (eDNA) are improving. Invertebrate ingested DNA (iDNA) from terrestrial leeches in the family Haemadipsidae has shown potential for surveying vertebrates and biodiversity monitoring in protected areas. Here we present an initial, and critical, evaluation of the limitations and biases of current iDNA protocols for biodiversity monitoring using both standard and NGS barcoding approaches. Key findings include the need for taxon relevant multi-locus markers and reference databases. In particular, the limitations of available reference databases have profound potential to mislead and bias eDNA and iDNA results if not critically interpreted. Nevertheless, there is great potential for recovery of amplifiable DNA from gut contents of invertebrate museum specimens which may reveal both temporal patterns and cryptic diversity in protected areas with increased efficiency. Our analyses of ingested DNA (iDNA) from both freshly stored and previously collected (legacy) samples of terrestrial leeches successfully identified vertebrates from Myanmar, Australia and Madagascar and indicate the potential to characterize microbial communities, pathogen diversity and interactions at low cost.

Eusocial insects are characterized by several traits, including reproductive division of labor and caste polymorphisms, which likely modulate genome evolution. Concomitantly, evolution may act on specific genes and pathways underlying these novel, sociality-associated phenotypes. Reproductive division of labor should increase the magnitude of genetic drift and reduce the efficacy of selection by reducing effective population size. Caste polymorphism has been associated with relaxed selection and may facilitate directional selection on caste-specific genes. Here, we use comparative analyses of 22 ant genomes to test how reproductive division of labor and worker polymorphism influence positive selection and selection intensity across the genome. Our results demonstrate that worker reproductive capacity is associated with a reduction in the degree of relaxed selection but is not associated with any significant change to positive selection. We find decreases in positive selection in species with polymorphic workers, but no increase in the degree of relaxed selection. Finally, we explore evolutionary patterns in specific candidate genes associated with our focal traits in eusocial insects. Two oocyte patterning genes previously implicated in worker sterility evolve under intensified selection in species with reproductive workers. Behavioral caste genes generally experience relaxed selection associated with worker polymorphism, whereas vestigial and spalt, both associated with soldier development in Pheidole ants, experience intensified selection in worker polymorphic species. These findings expand our understanding of the genetic mechanisms underlying elaborations of sociality. The impacts of reproductive division of labor and caste polymorphisms on specific genes illuminate those genes’ roles in generating complex eusocial phenotypes.

Phenotypic plasticity is widespread and evolutionarily important, but genomic consequences of new plastic traits remain unclear. Here, we explore patterns of molecular evolution linked to the repeated evolution of Cephalotes turtle ant worker plasticity, in which smaller minor workers and distinct larger soldiers are produced from a single genomic blueprint through developmentally plastic mechanisms. We integrate developmental transcriptomics with comparative genomic approaches to test the relative relationships of selection on genes, selection on regulatory sequences, and the emergence of lineage-specific genes with the repeated evolution of the soldier morph. We find that phenotypic plasticity shields protein-coding genes from selection, whereas it imposes a strong selective constraint on the evolution of gene regulatory loci. The development of a soldier morph disproportionately involves the activity of evolutionarily ancient genes. Moreover, our data link 3 pathways—nutrition via insulin signaling, imaginal disc development, and for the first time Hippo signaling—which allow for the differential development of soldiers and workers from a single genomic background in turtle ants. Taken together, our results provide evidence that plasticity leads to relaxed selection on genes, but imposes selective constraint on regulatory elements, during the repeated evolution of the turtle ant soldier morph.

Aggression is an essential component of survival and fitness, although the expression of aggression behaviors can also carry fitness costs. As a result, aggressive behaviors vary significantly across animals and are likely acted on by natural selection to produce this variation. Aggression, and associated traits like nestmate discrimination, both complex traits, have well studied genetic components, with links to genes involved in processes like neuronal function, metabolism, and hormone and cuticular hydrocarbon (CHC) production and reception. However, whether and how natural selection acts on these genes to produce variation across species is not fully understood. Using a comparative genomics approach, we analyzed natural selection in ants (Hymenoptera: Formicidae) with candidate genes previously linked to these traits. We find that aggression is associated with shifts in selection intensity, including positive selection on neurotransmitter receptors, and that ants with low levels of nestmate discrimination experience positive or relaxed selection on several CHC genes. Interestingly, we find that most candidate genes analyzed experience positive selection across ants, regardless of aggression level or discrimination ability. Our results shed new light on the means by which natural selection may act to produce variation in aggression across the ants.

We investigated the genetic structure and diversity between populations of a rare butterfly, the Florida duskywing (Ephyriades brunnea floridensis E. Bell and W. Comstock, 1948) (Lepidoptera: Hesperiidae) across a network of South Florida pine rockland habitat fragments. Based on 81 individuals from seven populations and using multiple polymorphic microsatellite loci, our analyses support the presence of mainland Florida (peninsular) and Florida Keys (island) population groupings, with a moderate, asymmetrical gene flow connecting them, and the presence of private alleles providing unique identities to each. We additionally found that despite a prevalence in many Lepidoptera, the presence of Wolbachia was not identified in any of the samples screened. Our findings can be used to inform conservation and recovery decisions, including population monitoring, organism translocation, and priority areas for management, restoration or stepping-stone creation to help maintain the complex genetic structure of separate populations.