Microsatellites are the genetic markers of choice for many population genetic studies, but must be isolated de novo using recombinant approaches where prior genetic data are lacking. Here we utilized high-throughput genomic sequencing technology to produce millions of base pairs of short fragment reads, which were screened with bioinformatics toolsets to identify primers that amplify polymorphic microsatellite loci. Using this approach we isolated 13 polymorphic microsatellites for the blue duck (Hymenolaimus malacorhynchos), a species for which limited genetic data were available. Our genomic approach eliminates recombinant genetic steps, significantly reducing the time and cost requirements of marker development compared with traditional approaches. While this application of genomic sequencing may seem obvious to many, this study is, to the best of our knowledge, the first attempt to describe the use of genomic sequencing for the development of microsatellite markers in a non-model organism or indeed any organism.

RAD-tag sequencing is a promising method for conducting genome-wide evolutionary studies. However, to date, only a handful of studies empirically tested its applicability above the species level. In this communication, we use RAD tags to contribute to the delimitation of species within a diverse genus of deep-sea octocorals, Chrysogorgia, for which few classical genetic markers have proved informative. Previous studies have hypothesized that single mitochondrial haplotypes can be used to delimit Chrysogorgia species. On the basis of two lanes of Illumina sequencing, we inferred phylogenetic relationships among 12 putative species that were delimited using mitochondrial data, comparing two RAD analysis pipelines (Stacks and PyRAD). The number of homologous RAD loci decreased dramatically with increasing divergence, as 470% of loci are lost when comparing specimens separated by two mutations on the 700-nt long mitochondrial phylogeny. Species delimitation hypotheses based on the mitochondrial mtMutS gene are largely supported, as six out of nine putative species represented by more than one colony were recovered as discrete, well-supported clades. Significant genetic structure (correlating with geography) was detected within one putative species, suggesting that individuals characterized by the same mtMutS haplotype may belong to distinct species. Conversely, three mtMutS haplotypes formed one well-supported clade within which no population structure was detected, also suggesting that intraspecific variation exists at mtMutS in Chrysogorgia. Despite an impressive decrease in the number of homologous loci across clades, RAD data helped us to fine-tune our interpretations of classical mitochondrial markers used in octocoral species delimitation, and discover previously undetected diversity.