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).