James Leavitt and Dr. Michael Whiting, Department of Biology

Goal/Purpose of the Project

The orthopteran superfamily Acridoidea (Orthoptera, Caelifera), which includes grasshoppers and their nearest relatives, is the largest of the orthopteran superfamilies and provides an excellent model for studying the evolution of mitochondrial genomes. Ultimately I was able to reconstruct a phylogeny for Caelifera based on entire mitochondrial genomes (mtgenomes) in order to (1) evaluate the relative effectiveness of various data partitioning strategies within a Maximum Likelihood (ML) framework; (2) evaluate the conservation of anti-codons across the group and elucidate patterns among the start and stop codons of the mitochondrial protein coding genes (MPCGs); and (3) specifically test the monophyly of Acridoidea and hypothesize phylogenetic relationships among the major lineages.

Importance of Project

Despite previous attempts to address acridoidean phylogeny (1,2), most evolutionary relationships within Acridoidea are still poorly understood. On a molecular level, the start and stop codons of the MPCGs of Acridoidea exhibit varying degrees of conservation, but it is unclear whether or not this variation represents any meaningful phylogenetic patterns. Using Acridoidea as a model group, Mitochondrial genome (mtgenome) data have proven effective in resolving deep evolutionary relationships in other insect groups, but they have yet to be broadly applied to Acridoidea (2-4). My project investigated the optimal treatment of mtgenome data within an ML framework by reconstructing a robust phylogeny for Acridoidea by analyzing 34 complete mtgenomes representing 9 of the 11 acridoidean families and 4 other closely related families under various partition schemes. Given this phylogeny I found the following: 1) That over-partitioning can be detrimental in a phylogenetic analysis; 2) That anti-codons show varying levels of conservation across the group and that patterns in start codon conservation may provide meaningful molecular synapomorphies; and 3) That the superfamily Acridoidea is monophyletic with the group Ommexechidae+Romaleidae as sister to Acrididae.

Previous research based on morphological and limited molecular characters has returned a poorly resolved Acridoidea (1). Recent innovations in DNA sequence generation and annotation have made phylogenetic analyses using entire mtgenomes more feasible (5). However, there is still some debate regarding the partitioning of data sets with multiple loci. I evaluated partitioning strategies and the monophyly of Acridoidea by reconstructing trees based on the mtgenomes of key taxa representing the diversity of Acridoidea using a battery of partitioning strategies under a ML framework. My research showed that mtgenome data contains sufficient information to resolve some ambiguous relationships among the acridoideans, demonstrated that basal caeliferans do not exhibit the same gene rearrangement found in more apical caeliferans, and in the published paper discussed using PartitionFinder to identify optimal partitioning strategies.

Materials and Methods

Four complete mtgenomes were generated for this project. Genomes generated in the Whiting Lab were uploaded to GenBank. A total of 34 complete mtgenomes representing 9 of the 11 acridoidean families and 4 other closely related families were used in the analysis. Sequence files were uploaded and annotated in MOSAS (http://mosas.byu.edu). Protein-coding genes were translated into amino acid sequences MacClade 4.0 and aligned using the G-INS-i strategy in MAFFT and were then back-translated into DNA sequences (MacClade 4.0). Ribosomal RNA and transfer RNA sequences were aligned using the E-INS-i strategy in MAFFT. These data were then partitioned by unique combinations of gene, gene family, codon position, and as many relevant interactions as possible. Phylogenies were reconstructed in a maximum likelihood (RAxML) framework and compared across partition strategies using LRT, hLRT, and other pertinent metrics to identify the optimal partition strategy. Anti-codons, start and stop codons, and tRNA structure were mapped onto the topologies to determine their relative positions within the phylogeny.

Academic Outcome

I presented the preliminary analyses of this data at the national meeting of the Entomological Society of America (ESA) on Nov. 14, 2011 as a competitor for the President’s Prize. The project was also presented at the National Conference for Undergraduate Research (NCUR) March 2012. As senior author I submitted a manuscript that was published in Molecular Phylogenetics and Evolution in Spring 2013. This project provided me with direct experience in molecular biology, DNA sequencing and analysis, and provided the experiences that helped me better understand data manipulation and analysis for complex datasets. Additionally, presentation at scientific meetings and the publication contributed to me being a more competitive graduate in the job market.

References

1. Flook, P. K., Klee, S. & Rowell C.H.F. Combined Molecular Phylogenetic Analysis of the Orthoptera (Arthropoda, Insecta) and Implications for Their Higher Systematics. Syst. Biol. 48, 233-253 (1999).
2. Fenn, J. D., Song, H., Cameron, S. L. & Whiting, M. F. A preliminary mitochondrial genome phylogeny of Orthoptera (Insecta) and approaches to maximizing phylogenetic signal found within mitochondrial genome data. Mol. Phylogenet. Evol. 49, 59-68 (2008).
3. Cameron, S. L., Lambkin, C. L., Barker, S. C. & Whiting, M. F. A mitochondrial genome phylogeny of Diptera: whole genome sequence data accurately resolve relationships over broad timescales with high precision. Syst. Entomol. 32, 40-59 (2007).
4. Sheffield, N. C., Song, H., Cameron, S. L. & Whiting, M. F. A Comparative Analysis of Mitochondrial Genomes in Coleoptera (Arthropoda: Insecta) and Genome Descriptions of Six New Beetles. Mol. Biol. Evol. 25, 2499-2509 (2008).
5. Hwang, U.W., Park, C. J., Yong, T. S. & Kim, W. One-step PCR amplification of complete arthropod mitochondrial genomes. Mol. Phylogenet. Evol. 19, 345-352 (2001).
6. Boore, J. L. & Brown, W. M. Big trees from little genomes: mitochondrial gene order as a phylogenetic tool. Current Opinion in Genetics & Development. 8, 668-674 (1998).