Table I. Amino acid residues under selection pressure in the CslF6 ancestral lineage.

Alignment Coordinatea	Pb	Position in Enzymec	Variation in Grasses	Notes
Ile-295	0.99		Ile in all CslF6s, mostly Trp in other CslFs
Ala-422	0.98		Ala in all CslF6s, except Ser in B. distachyon, Cys in most other CslFs
Ile-453	0.98		Ile in all CslF6s, Gly/Arg/Lys in other CslFs
Arg-459	0.91		Arg in all CslF6s, Met in most other CslFs
Ile-468	0.90	Substrate-binding cleft	Ile in all CslF6s, divergent in others, mostly Arg/Cys	Adjacent to the G660D mutantd
Thr-506	0.95		Thr in all CslF6s, divergent in other CslFs, mostly Arg
Tyr-507	0.92		Tyr in barley/B. distachyon CslF6s, Phe in other CslF6, Arg in all other CslFs
Tyr-550	0.88	Transmembrane pore	Tyr in all F6s, divergent in other CslFs, mostly Val/Ile
Ser-588	0.87	Transmembrane pore	Ser in all CslF6s, also CslF3 and CslF10, others mostly Arg
Cys-589	0.98	Transmembrane pore	Cys in all CslF6s, Thr in all other CslFs, except divergent in CslF10
Ser-590	0.90	Transmembrane pore	Ser in all CslF6s, also CslF3 and CslF10, others mostly Gly
Cys-597	0.92	Near the gating loop	Cys in all CslF6s except rice (Leu), mostly Leu in other CslFs
Ser-651	0.87	Transmembrane pore	Ser in all CslF6s, Ile in most other CslFs, sometimes Val	Adjacent to mutantse
Lys-681	0.96		Lys in all CslF6s, Leu in all other CslFs
His-687	0.98		His in all CslF6s, Trp in all other CslFs

^aAlignments are shown in Supplemental Figure S2.  ^bPosterior probability.  ^cOnly indicated if the amino acid residue is near the active site or in the transmembrane pore through which the nascent polysaccharide is extruded across the membrane.  ^dβ-Glucan-less mutant (Taketa et al., 2012).  ^eWong et al. (2015) and Hu et al. (2014) describe mutations in this position that result in reduced (1,3;1,4)-β-glucan levels in barley grain.