Genomic Outposts Serve the Phylogenomic Pioneers: Designing Novel Nuclear Markers for Genomic DNA Extractions of Lepidoptera

Abstract

Increasing the number of characters used in phylogenetic studies is the next crucial step towards generating robust and stable phylogenetic hypotheses - i.e., strongly supported and consistent across reconstruction method. Here we describe a genomic approach to finding new protein-coding genes for systematics in nonmodel taxa, which can be PCR amplified from standard, slightly degraded genomic DNA extracts. We test this approach on Lepidoptera, searching the draft genomic sequence of the silk moth Bombyx mori, for exons > 500 bp in length, removing annotated gene families, and compared remaining exons with butterfly EST databases to identify conserved regions for primer design. These primers were tested on a set of 65 taxa primarily in the butterfly family Nymphalidae. We were able to identify and amplify six previously unused gene regions (Arginine Kinase, GAPDH, IDH, MDH, RpS2, and RpS5) and two rarely used gene regions (CAD and DDC) that when added to the three traditional gene regions (COI, EF-1alpha and wingless) gave a data set of 8114 bp. Phylogenetic robustness and stability increased with increasing numbers of genes. Smaller taxanomic subsets were also robust when using the full gene data set. The full 11-gene data set was robust and stable across reconstruction methods, recovering the major lineages and strongly supporting relationships within them. Our methods and insights should be applicable to taxonomic groups having a single genomic reference species and several EST databases from taxa that diverged less than 100 million years ago.

Diversification of the cold-adapted butterfly genus Oeneis related to Holarctic biogeography and climatic niche shifts

“…DNA was amplified by Polymerase Chain Reactions (PCR) for one mitochondrial (COI -1487 bp) and three nuclear genes (GAPDH -691 bp, RpS5 -617 bp, wingless -400 bp), using forward-reverse primer pairs described in Wahlberg and Wheat (2008). In total, the genetic information contained 3207 bp.…”

Rapid diversification and not clade age explains high diversity in neotropical Adelpha butterflies

“…For the complete set of 72 taxa (electronic supplementary material), we amplified three gene regions traditionally used in phylogenetic studies of the Lepidoptera: the cytochrome oxidase subunit I (coxI) of the mitochondrion, and two nuclear loci, elongation factor 1-a (ef1-a) and wingless (wg). Because we are also interested in examining the deeper parts of the tree and the more general patterns of diversification, we used a reduced taxon set of 29 species, and for each we amplified an additional three nuclear gene regions recently shown to provide good phylogenetic signal for among-clade relationships in other nymphalids [25]: glyceraldhyde-3-phosphate dehydrogenase (gapdh), isocitrate dehydrogenase (idh) and ribosomal protein S5 (rps5). Using both the 72 and 29 taxon datasets, we were thus able to identify within-and among-clade patterns of evolution.…”

Comprehensive gene and taxon coverage elucidates radiation patterns in moths and butterflies

“…In several cases, air-dried specimens up to 10 years old yielded DNA of good quality, indicating that also nuclear DNA of unrelaxed material kept in dry and cool conditions can successfully be amplified using standard extraction and sequencing protocols even after a relatively long time of preservation (Wahlberg & Wheat 2008). Notably, material of the ANIC and some private collections were found to be very useful, emphasizing the importance of proper preservation conditions in museum collections, and at the same time warning against water relaxation of specimens, as such an operation almost certainly fragments the nuclear DNA.…”

“…First, groups of ditrysian Lepidoptera are morphologically homogeneous, and their phylogenetic affinities are therefore especially difficult to unravel (Kristensen & Skalski 1998), and second, there has been a shortage of phylogenetically informative genetic markers suitable for routine phylogenomic analyses. Owing to recent pioneering work in designing appropriate nuclear genomic markers (Wahlberg & Wheat 2008), there is now a good set of suitable genetic markers for use in lepidopteran phylogenomics.…”