Lost in parameter space: a road map for stacks

Although population genomic studies using next generation sequencing (NGS) data are becoming increasingly common, studies focusing on phylogenetic inference using these data are in their infancy. Here, we use NGS data generated from reduced representation genomic libraries of restriction-site-associated DNA (RAD) markers to infer phylogenetic relationships among 16 species of cichlid fishes from a single rocky island community within Lake Victoria's cichlid adaptive radiation. Previous attempts at sequence-based phylogenetic analyses in Victoria cichlids have shown extensive sharing of genetic variation among species and no resolution of species or higher-level relationships. These patterns have generally been attributed to the very recent origin ( 5.8 million base pairs in width), species are reciprocally monophyletic with high bootstrap support, and the majority of internal branches on the tree have high support. Given the difficulty of the phylogenetic problem that the Lake Victoria cichlid adaptive radiation represents, these results are striking. The strict interpretation of the topologies we present here warrants caution because many questions remain about phylogenetic inference with very large genomic data set and because we can with the current analysis not distinguish between effects of shared ancestry and post-speciation gene flow. However, these results provide the first conclusive evidence for the monophyly of species in the Lake Victoria cichlid radiation and demonstrate the power that NGS data sets hold to resolve even the most difficult of phylogenetic challenges. © 2012 Blackwell Publishing Ltd.

The distinction between model and nonmodel organisms is becoming increasingly blurred. High-throughput, second-generation sequencing approaches are being applied to organisms based on their interesting ecological, physiological, developmental, or evolutionary properties and not on the depth of genetic information available for them. Here, we illustrate this point using a low-cost, efficient technique to determine the fine-scale phylogenetic relationships among recently diverged populations in a species. This application of restriction site-associated DNA tags (RAD tags) reveals previously unresolved genetic structure and direction of evolution in the pitcher plant mosquito, Wyeomyia smithii, from a southern Appalachian Mountain refugium following recession of the Laurentide Ice Sheet at 22,000-19,000 B.P. The RAD tag method can be used to identify detailed patterns of phylogeography in any organism regardless of existing genomic data, and, more broadly, to identify incipient speciation and genome-wide variation in natural populations in general.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico. © 2014 John Wiley & Sons Ltd.