Figure 6. Agarose gel electrophoresis, chromatogram, and alignment of sequences obtained via Sanger sequencing of the amplified DNA barcodes. The indel makers include (A) CymN1, (B) CymN2, (C) CymY, and (D) CymR. The lanes correspond to the PCR products amplified from seven individuals of C. mongolica (left) and C. daurica(right). The red squares correspond to the Indel regions. The original uncropped image is presented in Figure S5.
3.5. | Phylogenetic relationships and divergence times
Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses yielded highly consistent topologies for the three datasets (Fig. 7). The monophyly of Orobanchaceae was strongly supported. The tribes Rehmannieae, Lindenbergieae, Cymbarieae, Pedicularideae, Brandisieae, Rhinantheae, Orobancheae, and Buchnereae corresponded to well-supported clades. The hemiparasitic tribe Cymbarieae was a clade sister to the other parasitic lineages. C. mongolica and C. daurica were grouped into the monophyletic genus Cymbaria , which comprised a clade sister to the Schwalbea -Siphonostegiaclade. Divergence time analyses (Fig. 8) revealed that the common ancestor of the Orobanchaceae originated in the early Eocene (49.96 Mya), and parasites diverged from autotrophic plants in the mid-Eocene (42.95 Mya). The emergence of Cymbarieae predates the mid-Oligocene (31.44 Mya). Moreover, the diversification of Cymbaria was estimated to occur around the late Miocene (6.72 Mya).