The phylogenetic utility of the nuclear protein-coding gene EF-1 alpha for resolving recent divergences in Opiliones, emphasizing intron evolution

Our focus was to design harvestmen-specific PCR primers to target both introns and exons of the nuclear protein-coding gene Elongation Factor 4 alpha (EF-1 alpha). We tested this primer set on ten genera representing all primary lineages of Opiliones, with sets of close phylogenetic relatives (i.e., sets of several congeners) included to specifically assess utility at shallow phylogenetic levels. Our research also included the collection of parallel mitochondrial protein-coding DNA sequence datasets for the congeneric sets to compare relative rates of evolution and gene tree congruence for EF-1 alpha versus mitochondrial data. The harvestmen primers resulted in successful amplification for nine of ten tested genera. Exon sequences for these nine genera appear orthologous to previously-reported EF-1 alpha Opiliones sequences, which were generated using RT-PCR methods. Newly-generated exon sequences are interrupted by three separate spliceosomal introns; two introns are restricted to one or two genera, but a third intron is conserved in position across all surveyed genera. Phylogenetic analyses of EF-1 alpha nucleotide data for congeneric sets result in gene trees that are generally congruent with mitochondrial gene trees, with EF-1 alpha phylogenetic signal coming from both intron and exon sites, and resolving apparently recent divergences (e.g., as recent as one million years ago). Overall, the combination of gene orthology, conserved intron position, and gene tree congruence at shallow levels suggest that this gene region will prove generally useful for both phylogeographic and species-level phylogenetic analyses in Opiliones, complementing already-documented utility at higher taxonomic levels.