Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio-temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next-generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub-Antarctic seabird, the thin-billed prion (Pachyptila belcheri), that we tested for cross-species amplification in other Pachyptila and related sub-Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific FST provided better approximations of mtDNA relationships among the studied species than those estimated using DC, even though FST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross-species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.

NGS shotgun data from Pachytila belcheriThese are the 454 sequencing reads generated from two Pachyptila belcheri individuals (Kerguelen Archepelago) from which the 26 microsatellite loci used in this study were isolated.Prions_Msat_genotypes_by_Species_FinalThese are the genotypes for the 25 unlinked microsatellite loci analysed in this paper. The Excel file is in "Covert" format, divided into species populations for easy conversion into any other data format.Tree files and pairwise distance matricesThese are the MEGA input distance matrices for Fst and DC distances calculated from 25 microsatellite loci. Also included are the newick format tree files for each matric. These are the trees presented in Figure 2A and B.Cytochrome B ALignment and Tree