Broadly, we are interested in the evolution of novel adaptations, the occurrence of such traits, the physiological mechanisms that produce them, the ecological factors that drive the evolution of these adaptations, and the impact of novel adaptations on the biodiversity of lineages that possess them. Below we outline current projects that are ongoing in the lab to address these questions with regards to cyclopeptide tolerance in mushroom-feeding Drosophila.
Vinegar flies (genus Drosophila) utilize a wide variety of food resources that include sap, fruits, flowers, cacti, and mushrooms. Mushroom-feeding species that occur in the immigrans-tripunctata radiation are generalists on fleshy Basidomycota mushrooms, including edible and toxic species. These flies are among a very limited number of eukaryotes that can tolerate cyclopeptide toxins found in some species of Amanita mushrooms (e.g., destroying angels). While the toxic mushrooms make up only a small portion of their possible diet, all mushroom-feeding Drosophila that have been assayed can tolerate high concentrations of the toxins.
Cyclopeptide toxins act by binding to RNA polymerase II, which inhibits the production of messenger RNA and leads to cell death in most multi-cellular organisms. The physiological mechanism of toxin tolerance within mushroom-feeding Drosophila is poorly understood, but we do know the flies do not have mutations that would prevent the toxin from acting. To better characterize the physiological mechanism, we are using metabolomic and transcriptomic analyses of larvae after they are reared on diets with and without the toxin. We are analyzing the spectra generated by global NMR of two tolerant species (D. guttifera and D. recens) and one susceptible species (D. deflecta) to identify both the impact of the toxin on the larval metabolome and the manner in which the larvae metabolize it. In addition, we are comparing gene expression within D. guttifera and D. recens larvae after feeding on diets with and without toxin to identify genes that may contribute to toxin tolerance. We are expanding these analyses to include transcriptomic data for 12 tolerant and 6 susceptible species and metabolomic data for eight tolerant species and two susceptible species with our Dimensions of Biodiversity Grant (DEB-1737869).
Expanding on our transcriptomic and metabolomic analyses of toxin tolerance, we are sequencing the genomes of tolerant and susceptible species in the immigrans-tripunctata radiation. The evolution of novel adaptations can produce changes, such as gene duplications and/or changes in the rate of molecular evolution, in the genomes of species in which they occur. We are sequencing the genomes of these species using a combination of long reads (Oxford Nanopore), short reads (Illumina HiSeq), and structural data. The construction of a hybrid, de novo assembly for each species is worked on in lab and in BIO3545-Genomics: DNA to Gene ID. We are annotating these assemblies using other Drosophila genomes and the RNAseq reads generated for each species. Once the genomes are annotated, we will examine them in a phylogenetic context to identify potential genomic signals of toxin tolerance, such as gene duplication events in detoxification gene families.
Within species, the genetic basis of novel adaptations can be a single gene or multiple genes. When intraspecific variation is observed within a population for a trait of interest this indicates a polygenic basis for the adaptation. We have examined variation of cyclopeptide tolerance in a population of D. tripunctata that occurs on the campus of the University of Alabama. We found significant genetic variation for toxin tolerance and also demonstrated two families of detoxification genes did not contribute significantly to tolerance. Examining differential gene expression in a line with higher fitness on a diet with a natural toxin concentration has produced a very exciting hypothesis of how the larvae detoxify the cyclopeptide alpha-amanitin (Stay tuned for the upcoming publication!). We will shortly begin 'evolve-and-resequence' experiments in four species to identify alleles that contribute to toxin tolerance.
To examine the evolution of mushroom-feeding and cyclopeptide tolerance in Drosophila, a well resolved phylogeny is needed. Our current understanding of the evolutionary relationships is a phylogenetic analysis based on 40 protein-coding loci sequenced for 27 species (most quinaria group spp. and all testacea group spp.; Scott Chialvo et al. 2019). The resulting tree supported the monophyly of the testacea, but not the quinaria species group. Neither cyclopeptide tolerance nor responses to gene inhibition assays predicted the relationship recovered. With our recent award from NSF, we have begun expanding our taxon sampling to include 16 additional species from the already sampled groups and other closely related species groups. For this upcoming analysis, we will use transcriptomic data to reconstruct a phylogenetic hypothesis of the group and examine the evolution of cyclopeptide tolerance.
