Chloroplast divergence in C. americana appears to be driven by accelerated rates of evolution in a subset of chloroplast genes. In particular, the small ribosomal proteins, clpP, ycf1, and ycf2, exhibit accelerated rates of nucleotide substitution and elevated dN/dS ratios. The small ribosomal proteins, ycf1, and ycf2, also exhibit levels of elevated pN/pS when looking at polymorphism within C. americana. These rapidly evolving chloroplast genes may select for complimentary mutations in nuclear-encoded, chloroplast-targeted genes to maintain chloroplast function, leading to cytonuclear co-evolution within chlorolast clades, but cytonuclear mismatch between.

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The cytonuclear incompatibility in C. americana can be very strong, leading to seedlings that are completely white and survive only a few days. However, when doing our between-clade crosses, we observed not only white and green seedlings but variegated seedlings (seedlings that are part green, part white). Genotyping of green and white leaf tissue punches from variegated plants found that green leaf tissue always contains Appalachian clade chloroplasts, and white leaf tissue always contains Western clade chloroplast. Thus, Western chloroplasts are incompatible with Appalachian nuclear genes, confirming the genetic incompatibility is cytonuclear, and also demonstrating that in C. americana chloroplasts can be inherited from both mom and dad. Further work demonstrated that while chloroplast inheritance is predominantly maternal in C. americana, up to 50% of offspring can have biparental (maternal and paternal) inheritance, depending upon the cross.

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