Isolating Nuclear Genes and Identifying Lineages withoutMonophyly: An Example of Closely Related Species from Southern Madagascar

Megistostegium, a small genus endemic to southern Madagascar, comprises three morphologically distinct species that live in sympatry, lack obvious prezygotic barriers to gene flow, and occasionally hybridize. The genus is one among many plant groups that contain distinct morphological species despite genetic permeability. Such systems are difficult to study because of the need to utilize rapidly evolving nuclear genes, which may be difficult and expensive to isolate. Additionally, analytical methods investigating genealogical patterns at the boundary between the phylogenetic level and the population genetic level are in their nascent stages. As a solution to the first problem, we collected nuclear genotypic data for four loci with the aid of single‐stranded conformational polymorphism as a means to isolate alleles from heterozygotes without cloning. As a solution to the second, we used the genealogical sorting index (GSI) and developed a new taxonomic autocorrelation test, both of which can be used to determine how closely predefined species (or geographic areas) approach monophyly. We applied these methods to generate and analyze sequence data for four nuclear loci and plastid DNA from Megistostegium. None of the three traditionally delimited species was monophyletic for any gene. Nonetheless, the GSI and autocorrelation tests detected situations in which the taxonomic species were significantly closer to monophyly than expected under the null hypothesis of no taxonomic structure. These analyses suggest that while phylogenetic signal is confounded by hybridization and perhaps incomplete lineage sorting, the three Megistostegium species have meaningful, if incomplete, genetic distinctiveness.