Species delimitation is ever more critical for assessing biodiversity in threatened regions of the world, especially when undescribed lineages may be at risk from habitat loss. Mouse lemurs (Microcebus) are an example of a rapid radiation of morphologically cryptic species that are distributed throughout Madagascar in its rapidly vanishing forested habitats. Here, we focus on two pairs of sister lineages that occur in a region in northeastern Madagascar that shows high levels of microendemism. We revisit previous hypotheses of species diversity by filling geographic sampling gaps and by generating new genomic data for three named species, as well as an undescribed lineage previously identified to be of interest due to its highly divergent mtDNA. We analyzed RADseq data with multiple species delimitation methods based on the multispecies coalescent (MSC) model while accounting for introgression. Non-sister lineages occur sympatrically in two instances, despite an estimated divergence time of less than 1 Ma, thus suggesting rapid evolution of reproductive isolation in the mouse lemur clade. We note, however, that the divergence time estimates reported here are based on the MSC and calibrated with pedigree-based primate mutation rates. These dates are considerably more recent than previous analyses that used traditional relaxed clock methods and distant fossil calibrations. One pair of sister lineages passed all species delimitation tests while the other pair failed most, largely due to differences in Ne between the two pairs of lineages. Nevertheless, delimitation results were also supported by differences in levels of gene flow and patterns of isolation-by-distance between the two pairs. We conclude that MSC-based species delimitation methods are valuable tools for evaluating cryptic species, even though these methods can be strongly affected by variable Ne. We suggest that this result has general implications for species delimitation studies of other recently diverged lineages.

In the tarball, there are four folders describing analyses, metadata, scripts, and the sequence data. Each folder and their subdirectories have readme files describing the contents.