A framework phylogeny of the American oak clade based on sequenced RAD data.

Previous phylogenetic studies in oaks (Quercus, Fagaceae) have failed to resolve the backbone topology of the genus with strong support. Here, we utilize next-generation sequencing of restriction-site associated DNA (RAD-Seq) to resolve a framework phylogeny of a predominantly American clade of oaks whose crown age is estimated at 23-33 million years old. Using a recently developed analytical pipeline for RAD-Seq phylogenetics, we created a concatenated matrix of 1.40 E06 aligned nucleotides, constituting 27,727 sequence clusters. RAD-Seq data were readily combined across runs, with no difference in phylogenetic placement between technical replicates, which overlapped by only 43-64% in locus coverage. 17% (4,715) of the loci we analyzed could be mapped with high confidence to one or more expressed sequence tags in NCBI Genbank. A concatenated matrix of the loci that BLAST to at least one EST sequence provides approximately half as many variable or parsimony-informative characters as equal-sized datasets from the non-EST loci. The EST-associated matrix is more complete (fewer missing loci) and has slightly lower homoplasy than non-EST subsampled matrices of the same size, but there is no difference in phylogenetic support or relative attribution of base substitutions to internal versus terminal branches of the phylogeny. We introduce a partitioned RAD visualization method (implemented in the R package RADami; http://cran.r-project.org/web/packages/RADami) to investigate the possibility that suboptimal topologies supported by large numbers of loci--due, for example, to reticulate evolution or lineage sorting--are masked by the globally optimal tree. We find no evidence for strongly-supported alternative topologies in our study, suggesting that the phylogeny we recover is a robust estimate of large-scale phylogenetic patterns in the American oak clade. Our study is one of the first to demonstrate the utility of RAD-Seq data for inferring phylogeny in a 23-33 million year-old clade.