Late Miocene Speciation
Limited gene flow promotes evolutionary diversification by facilitating genetic and phenotypic divergence that may culminate in speciation (Schluter, 2001; Slatkin, 1987). The migrations between C. bifidaand C. micholitzii in both directions were very low, indicating restricted gene flow among them (Figure 5b). Since C. bifidaoccupies a lower altitude habitat in southwest China and the northern Vietnam compared to C. micholitzii in the Annam Highlands region of central Vietnam, eastern Laos and the northern Cambodia (Figure 1), it is impossible for the two species to have a second contact. Due to the separation of habitats by the RRFZ, the two taxa have obvious niche differentiation, which led to the gradual formation of two independent species. This can be proved by the two obvious clusters corresponding to the two species in genetic structure (Figure 3a) and haplotype networks (Figure 2). Based on the analyses of the three combined cpDNA and five low copy nuclear gene datasets, divergence between C. bifida andC. micholizii was estimated to have occurred approximately at c. 5.605 Mya (90% HPD interval: 2.580 - 25.407 Mya) (Figure 5c). These estimates imply that they diverged from their common ancestor in the late Miocene at a long time after India-Asia collision which formed the RRFZ. As a natural barrier, the RRFZ hindered the gene flow between the two taxa, making them more and more differentiated and finally speciation. In the haplotype networks (Figure 2), not all nuclear gene haplotypes can be separated according to the two species (e.g.AAT , SAMS ), and even some nuclear gene haplotypes can be shared by the two species (e.g. PHYP , AAT ), implying that the two species once came from the same ancestor or they were the same species, but the long-term geographical isolation resulted in high genetic differentiation between them.