John Boyette

Emerging fungal diseases are contributing to population declines and species extinctions across vertebrates. In amphibians, the fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, an infectious disease responsible for mass amphibian die-offs worldwide. After initial declines, many species in Brazil have bounced back and populations are stabilizing. However, the mechanisms contributing to the development of this observed resilience are poorly understood. Fortunately, a rich trove of historical data is available in museum collections. This project will leverage emerging historical DNA extraction and sequencing techniques to quantify immunogenomic responses to Bd through time. Such insights at the genomic level will aid in understanding broader population and community-level processes such as species decline and recovery.

Bd has been introduced at least twice in Brazil, with the latest introduction occurring in the late 1970s. This introduction led to species declines which were documented by Ronald Heyer between 1979 and 1982 in the Boraceia municipality of São Paulo. Ronald Heyer was Curator of Herpetology at the Smithsonian’s National Museum of Natural History, and his personal collections at the museum include over 1,300 frogs from Boraceia and the surrounding region, including species sampled in the 70s (before the decline) as well as the 80s (after the decline). This collection provides an unparalleled opportunity to characterize historical Bd prevalence and immunogenomic profiles of frog populations in the decade proceeding and following observed frog declines in Boraceia.

Given the emerging feasibility of historical DNA extraction and sequencing techniques, we are presented with a groundbreaking opportunity to characterize immunogenomic profiles of amphibian populations before, during, and after Bd outbreaks. By focusing our sequencing efforts on candidate Bd resistance or susceptibility genes, we will gain transformative insight into Bd-mediated selection occurring over the course of Bd outbreaks, test for genomic evidence of population bottlenecks, and further identify candidate polymorphisms associated with Bd resistance within and potentially across species. Collectively, these data will provide novel and potentially transformative insight into the development of amphibian resilience through time.

As a NSF REU intern with the Bell Lab at the Smithsonian’s National Museum of Natural History, I contributed to a project characterizing physiological and genomic underpinnings of visual evolution in frogs. Frogs are an excellent model for studies of visual adaptation because they exhibit a vast diversity of ecologies and lifestyles, each with unique spectral environments and visual challenges. My summer project focused on the evolution of a diverse group of light-sensitive proteins known as non-visual opsins. These proteins are expressed in the eyes, skin, and brain of many animals and are involved in various non-visual light detection functions, including circadian rhythm regulation and melatonin release.

Previous research has primarily explored the function of non-visual opsins in model vertebrates, with few studies investigating their molecular evolution or diversity across non-model species. To address this gap, I set out to: 1) identify which non-visual opsin genes are expressed in the eyes of frogs; 2) compare selective pressure (dN/dS) among non-visual opsin genes; and 3) test for potential adaptive evolution within each gene by comparing selective pressure between discrete ecological classes of frogs living in different light environments (e.g. nocturnal vs. diurnal).

Using annotated frog genomes for BLAST reference, I extracted 18 non-visual opsin genes from de novo eye transcriptomes of 48 frog species. I created phylogenetic trees and used PAML selection analyses to test for variation in dN/dS between discrete ecological classes of frogs. These analyses revealed variation in selective constraint across genes and species which may reflect functional adaptation to different ecologies. A poster summarizing my initial findings can be found below:

In June 2020, I continued my investigation of frog visual evolution as an intern with the California Academy of Sciences. I extracted non-visual opsin genes from 46 additional species, bringing the project’s total frog sampling to 94 species. This sampling spans frog ecological diversity and provides a powerful framework for further investigating patterns detected in the preliminary dataset.

In January 2021, I presented my findings at the Society of Integrative and Comparative Biology conference and was honored to be awarded the Marvalee and David Wake Award for Best Student Talk!

MHC class II genes code for molecules found on the surface of dedicated antigen-presenting cells. These molecules present pathogen-derived peptides to T cells to initiate acquired immune responses. Due to strong, pathogen-mediated selection, MHC genes display naturally high rates of allelic variation, a feature critical to their function. However, diversity of MHC genes has shifted in domestic animals as a consequence of artificial breeding. I wanted to quantify this shift and explore its potential consequences. As student supervisor for the Peters Lab, I developed three objectives: 1) identify patterns of MHC class II polymorphism between breeds and species of domestic ruminants; 2) investigate if these patterns correspond to artificial selection for production traits (e.g. milk production); and 3) explore how these patterns compare to a wild ruminant species (the white-tailed deer, Odocoileus virginianus).

To meet these objectives, the lab and I collected blood samples from 242 sheep (Ovis aries, representing four breeds) and 142 cattle (Bos taurus, representing three breeds), as well as tissue samples from 129 white-tailed deer (Od. virginianus). By early 2020, I had overseen preparation of 355 samples for sequencing. Unfortunately, the COVID-19 pandemic prematurely halted lab efforts. When it became apparent that the MHC project would be delayed, Dr. Peters invited me to collaborate on a project analyzing a genetic dataset he had previously collected. In spring 2020 we coauthored a manuscript exploring differential expression of growth-related genes across age classes in quarter horses that is now published in the Journal of Equine Veterinary Science. This paper is available below: