Can RNA-Seq resolve the rapid radiation of advanced moths and butterflies (Hexapoda: Lepidoptera: Apoditrysia)? An exploratory study

Abstract

Recent molecular phylogenetic studies of the insect order Lepidoptera have robustly resolved family-level divergences within most superfamilies, and most divergences among the relatively species-poor early-arising superfamilies. In sharp contrast, relationships among the superfamilies of more advanced moths and butterflies that comprise the mega-diverse clade Apoditrysia (ca. 145,000 spp.) remain mostly poorly supported. This uncertainty, in turn, limits our ability to discern the origins, ages and evolutionary consequences of traits hypothesized to promote the spectacular diversification of Apoditrysia. Low support along the apoditrysian "backbone" probably reflects rapid diversification. If so, it may be feasible to strengthen resolution by radically increasing the gene sample, but case studies have been few. We explored the potential of next-generation sequencing to conclusively resolve apoditrysian relationships. We used transcriptome RNA-Seq to generate 1579 putatively orthologous gene sequences across a broad sample of 40 apoditrysians plus four outgroups, to which we added two taxa from previously published data. Phylogenetic analysis of a 46-taxon, 741-gene matrix, resulting from a strict filter that eliminated ortholog groups containing any apparent paralogs, yielded dramatic overall increase in bootstrap support for deeper nodes within Apoditrysia as compared to results from previous and concurrent 19-gene analyses. High support was restricted mainly to the huge subclade Obtectomera broadly defined, in which 11 of 12 nodes subtending multiple superfamilies had bootstrap support of 100%. The strongly supported nodes showed little conflict with groupings from previous studies, and were little affected by changes in taxon sampling, suggesting that they reflect true signal rather than artifacts of massive gene sampling. In contrast, strong support was seen at only 2 of 11 deeper nodes among the "lower", non-obtectomeran apoditrysians. These represent a much harder phylogenetic problem, for which one path to resolution might include further increase in gene sampling, together with improved orthology assignments.

Figure 1. Summary of previous “backbone” phylogeny results (483 taxa/19 genes), modified from Regier et al.[22]

ML topology shown for degen1 (non-synonymous change only) is based on 100 GARLI searches. Bootstrap percentages are degen1 followed by nt123 (all nucleotides), based on 1000 bootstrap replicates with 15 search replicates each. Only values greater than 50% are shown. Branch lengths are arbitrary. ‘-’ = node not found in ML tree for nt123. Numbers in parentheses after taxon names indicate number of families/number of exemplars studied. Names in bold denote clades in which larvae are not typically phytophagous. Names in serif font denote clades in which adults typically bear ultrasound-detecting tympanic organs on the thorax and/or abdomen. Classification follows van Nieukerken et al. [1].

Figure 2. ML tree for 46 taxa, 741 paralogy-filtered genes, degen-1 (non-synonymous change only).

Bootstrap percentages: 741 genes consensus method, followed by 741 genes Rrepresentative method in parentheses but only when these two differ, followed by 19 genes, each based on 1000 bootstrap replicates with 5 search replicates each. ‘-’ = node not found in ML tree for 19 genes.