Microsatellite variation for phylogenetic, phylogeographic and population-genetic studies in Lomatia (Proteaceae)

Lomatia R.Br. is a genus of 12 species in South America and eastern Australia. Hybridisation is extensive in the Australian species and molecular work is required to understand phylogenetic relationships and examine potential gene flow among species. We developed a library of microsatellite markers for Lomatia silaifolia (Sm.) R.Br. These markers were tested across population samples of L. silaifolia and L. myricoides (C.F.Gaertn.) Domin, assessed for cross amplification across all 12 species of Lomatia, sequenced and inspected for variation in the microsatellite flanking region (MFR), and utilised for phylogeographic and phylogenetic analysis. Nineteen microsatellite markers were tested, 13 of which were polymorphic in size analysis. Four of the markers amplified consistently within Lomatia and the MFR had equivalent to or more sequence variation than the three universal markers (psbA-trnH intergenic spacer, PHYA, ITS). Variation within five individuals of L. silaifolia indicated that MFR could be phylogeographically informative. Combined phylogenetic analysis of Lomatia using universal markers and MFR resulted in a well supported tree; however, phylogenetic analysis of only MFR suggested non-monophyly of the species. Phylogenetic trees supported South American species as being diverged from the Australian species but paraphyletic with respect to the Australian lineage. Relationships among Australian species of Lomatia are correlated with geography rather than morphology. We found that microsatellite markers designed for Lomatia and the flanking regions can be informative at population, phylogenetic and phylogeographic levels.