Table 1. Melt-MAMA primers targeting canonical SNPs for 8 new phylogenetic branches discovered in this study^a.

Branch	AmesAnc positionb	Genome SNP state (D/A)c	Melt-MAMA primer sequenced	Conc, µMe	Annealing temp °C
A.Br.026	3,640,599	T/C	A:CTTCTTTTAATACATCTAAGTAAGTAAGCGTTgC D:cggggcggggcggggcggggCTTCTTTTAATACATCTAAGTAAGTAAGCGTTcTC:ATTGACCCAACAGCTACGAAATAC	0.45 0.15 0.15	60
A.Br.027	4,355,524	A/G	A:CCCATTCCAAGTGACACACTcG D:cggggcggggcggggcggggCCCATTCCAAGTGACACACTgA C:AGCACTTGCTTATCTTGGAGCTT	0.60 0.15 0.15	60
A.Br.028	791,256	A/G	A:ACAGAGAAGGTTATAAGTCCAGAcGG D:cggggcggggcggggcggggACAGAGAAGGTTATAAGTCCAGAaGA C:CTCGCTTTTCCTGTTCTTTTATTCAC	0.15 0.15 0.15	60
A.Br.029	3,960,657	A/G	A:AGTATTCCAACCATTACTATAGTCACTaG D:ggggcggggcggggcggggcggggcAGTATTCCAACCATTACTATAGTCACTcAC:GTACTTATTGGTGGTACTGCCAAATT	0.15 0.15 0.15	60
A.Br.030	3,528,668	A/G	A:CAATCCCTCGATTTACATATAAATATAAaG D:ggggcggggcggggcggggcggggcCAATCCCTCGATTTACATATAAATATAAcAC:AGGTATGTATGAATTAGAAGGGAAGAA	0.15 0.15 0.15	60
A.Br.031	3,018,054	C/T	A:ACTATCGCCAAAAGCAATTGaAT D:cggggcggggcggggcggggACTATCGCCAAAAGCAATTGtAC C:TATTTTAGACAAGTACGAACTAGATAAATCAA	0.15 0.15 0.15	55
A.Br.032	3,520,170	G/A	A:CCACCAACAACGAATGGAAGtA D:cggggcggggcggggcggggCCACCAACAACGAATGGAAGaG C:AGCATTTAATGAACGGCGTAAGTAATA	0.45 0.15 0.15	60
A.Br.033	3,610,151	C/T	A:TAAATAACCAAGGCGTCTTGCCAT D:ggggcggggcggggcggggcggggcCTAAATAACCAAGGCGTCTTGCtAC C:TGTAGGACGTAGTATGGTGAAAGTAGTAGAT	0.60 0.15 0.15	60

^aMelt-MAMA, melt-mismatch amplification mutation assay; SNP, single nucleotide polymorphism; con, concentration.

^bAmes Ancestor reference genome (NC_006570).

^cSNP states are presented according to the top strand in the Ames ancestor AE017334 D: Derived SNP state; A: Ancestral SNP state.

^dMelt-mismatch amplification mutation assay (MAMA), A: Ancestral; D: Derived; C: Common. Primer tails and antepenultimate or penultimate mismatch bases are in lower case.

^eFinal concentratinon of each primer in Melt-MAMA genotyping assays.