3.2 GB-RFLP marker analysis for the assignment of sympatric crater lake species
Next, we designed markers to distinguish the sympatric species of crater lakes Apoyo (A. astorquii , A. chancho , A. flaveolus , A. globosus , A. supercilius and A. zaliosus ) and Xiloá (A. amarillo , A. sagittae , A. viridis and A. xiloaensis ). Although all sympatric species clearly form separate genetic clusters, the number of shared alleles is extensive due to ongoing gene flow and/or recent divergence (Kautt et al., 2016, 2018). Indeed, there are no alternatively fixed differences, but we show that there are some nearly fixed ones based on which we should reach 90 to 100% accuracy — however it is possible that these predictions might be limited because our genomic samples do not accurately enough reflect the actual population frequencies.
Overall, the quality of the species-specific RFLP markers (Fig. S4) was much lower than the lake specific markers (Fig. S3). The percentage of correctly assigned species ranged from 50% (equal to a random assignment) to 100%. Only two markers achieved values above 90% (A. chancho , Fig. S4F and A. viridis , Fig. S4O). In contrast to the lake-specific markers, species specific-markers showed a much lower accuracy in the GB-RFLP assay compared to the bootstrapping dataset with 12/16 samples having >20% less correctly assigned samples than in the bootstrapping dataset (Fig. 3D–F), which is substantially different from the lake-specific markers (1/18). The combination of markers did not improve the accuracy.