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Supplementary material Note S1 Ancestral Reconstructed sequences and topology. The three reconstructed sequences are output following Ancetral sequence reconstruction (ASR) analysis in HyPhy (joint, marginal and sampled). As few differences occurred between the three different reconstructions we estimated number of different non-synonymous and synonymous substitutions between the mitogenome sequences of living freshwater eels and the ancestral reconstructions by averaging over all three reconstructed sequences. The ATP6 part of the sequences did not show any differences in estimated amino acid changes. The analysis was based on a GTR substitution model (the best fitted model estimated in HyPhy) and a topology identical to the one found in Jacobsen et al. (1) using a posterior threshold of 0.90 to determine pylogenetic relationships (see newick tree below). Stemonidium hypomelas was used as outgroup. Site-to-site variation was modeled using beta-gamma variation and three rate classes were used. Topology: (((((NC_006537,NC_006541),(((NC_006543,NC_006544),((NC_006545,(NC_006534,NC_006535)),NC_006539)),NC _006540)),NC_002707,NC_006546),((NC_006531,NC_006547),((NC_006532,NC_006533),NC_006538)),NC_00653 6,NC_006542),((NC_013436,NC_013627),NC_013628)); Reconstructed sequences: > ATP6_reconstructed_Ancestral sequence_joint ATGAACACAATCCTTACTCACATCATTAATCCTCTAGCATATATTGTCCCAGTACTTCTCGCCGTCGCATTCCTAACACTTCTAGAACG AAAAGTATTAGGATATATACAACTACGAAAAGGGCCAAACATCGTTGGACCATACGGCCTCCTTCAACCAATCGCCGACGGAGTAAA ACTATTTATTAAAGAACCAGTACGCCCTTCAACCGCATCCCCATTCCTATTTTTAGCCACCCCAACTCTTGCACTAACCCTCGCCTTAA CACTATGAGCCCCCATACCAATACCTTATCCAGTAGTAGAACTAAACCTAGGAATTCTATTCGTACTAGCCCTATCAAGCCTCGCAGT TTACTCAATCCTGGGCTCAGGCTGGCCTCAAACTCAAAATATGCTCTAATTGGGGCCCTACGTGCCGTAGCCCAAACAATTTCATATGAAGTAAGCCTAGGCCTAATCCTTC TTTCAATTATTATTATCGTAGGAGGATTCAACCTAAAAACATTCAACATCGCACAAGAAGCAACATGATTAATAGCCCCAGCCTGACC ACTAGCAGCAATATGATAATCTCAACACTAGCTGAAACAAACCGAGCCCCATTTGACCTTACAGAAGGAGAATCAGAATTAGTATCAGGCTTTAACGTAGAATAT GCAGGAGGACCATTTGCCCTATTCTTCCTAGCCGAATACTCAAACATCCTACTAATAAATACCTTATCAACAATCCTATTTCTAGGAGC AATACACACCCCACTAATTCCAGAACTAACAACAATAAACCTAATAATAAAAGCCACTATACTTTCCATTATATTCTTATGAGTACGA GCCTCCTACCCACGATTTCGGTACGACCAGCTAATACATCTAATATGAAAAAACTTCCTACCACTAACCCTAGCCCTACTTATCTGAA ACCTAGCCTTGCCCATTACCATGGCAGGCCTCCCCCCAAACAACATGAACCCATATGTAACCTTCATCATACTAACAAGCTTGGGACT AGGCACCACAATCACATTCGCTAGCTCACACTGACTGCTCGCCTGAATAGGATTGGAAATTAATACACTAGCCATTATTCCCCTTATA GCCCAACACCACCACCCACGAGCCGTAGAAGCAACAACAAAATACTTCCTCACACAAGCCACAGCAGCAGCACTAATACTATTTACA ACCACATCAAATGCATGAATTACAGGACAATGAGAAATTCAACAACTATCTCACCCAATAATCACCACAATTACAATCCTTGCCCTAG GACTTAAAGTAGGACTAGCCCCAATACATTTTTGACTACCAGAAGTCCTACAAGGCCTAGACCTAACTACAGGACTAATCCTGTCAAC ATGACAAAAACTAGCGCCCATAGCCCTAATTTACCAACTCTCACCAGGAGTAGAACAACCACTAATAATTATACTAGGAATGATATCT GCCCTCGTAGGAGGATGAGGTGGATTAAACCAAACACAACTACGAAAAATCCTAGCATACTCCTCAATCGCACACATAGGATGAATA ATAATTGTAATAAAATACTTACCAAATTTAATAATCATAAACCTAATAATCTATATCATCATGACATCATCAGCTTTCATAGCACTAAA AATAACCACCGCTACAAAAATTAATACACTAGCAACAGGTGAACAAAAGCCCCAATCCTTACAGCACTAACTATGGCCACCATACTATCATTAGGAGGTTTACCCCCACTAACCGGATTCATACCAA AATGAATAATTCTACAAGAACTAACCAAACAAGACCTCCCACTAATCGCTACGCTAATAGCAATAACAGCCCTACTAAGTCTATTCTT CTACCTACGACTATGCTACGCCATAACACTAACAATTTCACCAAACACAAACAATGCTAAAACACCATGACGATTAAAATCAAAACA AATAACAATGCCCCTCTCAATTACAATAATCATAACAGCTATAATACTCCCGGTAACCCCAGCAGTAATAGCAATAACAACAGTGGC AATCACCCGTTGATTCTTTTCTACTAATCACAAAGACATTGGTACCCTATATCTAGTATTTGGTGCCTGAGCCGGAATAGTGGGCACCG CACTAAGCCTTCTAATCCGTGCCGAATTAAGTCAACCAGGCGCCCTTCTTGGAGATGACCAAATTTACAATGTCATCGTCACAGCGCA TGCCTTTGTAATGATTTTCTTTATAGTAATACCAGTAATAATTGGAGGATTTGGCAACTGACTTGTGCCATTAATAATCGGCGCCCCAG ACATAGCATTCCCCCGAATAAACAACATAAGCTTCTGACTTTTACCCCCATCATTTCTTCTACTACTAGCCTCCTCAGGAGTAGAGGCT GGGGCTGGTACAGGTTGAACTGTATATCCACCTCTAGCTGGAAACTTAGCCCACGCCGGAGCATCTGTTGACCTGACAATTTTCTCAC TTCACCTTGCAGGAATTTCATCAATTCTAGGGGCCATTAATTTTATTACTACAATTATTAACATGAAACCGCCTGCCATTACACAGTAC CAAACCCCTCTGTTTGTATGAGCTGTTTTAGTAACCGCTGTTCTGCTACTCCTATCCCTGCCAGTCCTAGCCGCAGGTATTACAATACTT CTAACTGACCGAAATCTAAATACAACCTTCTTTGACCCTGCAGGGGGTGGAGACCCAATCCTCTACCAACACCTATTCTGATTCTTCG GCCACCCAGAAGTATACATTTTAATCTTACCAGGATTTGGAATAATCTCACACATTGTTGCTTATTATTCCGGTAAGAAAGAACCATTT GGGTATATAGGAATGGTCTGAGCAATGATGGCTATCGGACTACTAGGATTCATTGTATGAGCACACCATATGTTTACAGTAGGAATAG ACGTAGACACCCGTGCTTACTTCACTTCCGCCACAATAATCATCGCAATTCCAACTGGGGTAAAAGTATTCAGCTGACTAGCCACATT ACACGGAGGGGTCATCAAATGAGAAACCCCACTTCTTTGAGCTTTAGGTTTTATTTTCCTATTTACAGTTGGGGGCCTAACAGGTATCG TACTAGCAAACTCATCAATCGATATTGTATTACATGACACATACTATGTAGTAGCCCATTTCCATTATGTTCTGTCCATAGGAGCAGTA TTTGCTATTATAGGAGGCTTCGTACACTGATTCCCCCTATTCTCAGGCTATACACTACACGACACATGAACCAAAGTACACTTTGGAAT TATGTTCGTAGGAGTAAACCTAACCTTCTTCCCACAACATTTCCTAGGCTTAGCAGGAATACCACGACGTTACTCAGACTACCCAGAT GCCTACACCCTATGAAATACAATCTCCTCTATTGGATCCCTAATTTCTCTCACAGCCGTAGTCCTGTTCCTATTTATCCTCTGAGAAGC ATTTACTGCCAAACGAGAAGTAAAATGAGTAGAACTTACAGAAACAAATGTTGAATGACTACACGGATGTCCTCCACCATATCACAC ATTCGAAGAACCAGCGTACGTCCGAGTTCAAATGGCACATCCCTCACAGCTAGGTTTCCAAGACGCAGCCTCACCCCTGATAGAAGA ACTACTTCATTTCCACGACCATGCGCTAATAATTGTTTTCCTAATTAGCGTCCTAGTACTTTATATTATTGTGGCAATGGTAACTGCCA AAGTTACTAACATGTTCATCCTAGATTCACAAGAGATTGAAATGTGTGAACCGTATTGCCAGCAGCAATTCTAATTCTAATCGCACTCCCCTCTCTACGGATCCTTTATCTAATAGACGAAATCAATGACCC ACATTTAACAATTAAAGCAATCGGACATCAATGATACTGAAGCTACGAGTATACTGACTATGAAGACCTTGGATTTGACTCGTACATA ATCCCAACACAAGACTTAACCCCAGGACAATTCCGACTGCTAGAAACAGACCATCGAATAGTAGTACCAATAGAATCACCTGTACGA GTATTAGTTACAGCAGAAGACGTCTTACACTCATGAGCAGTCCCATCCTTAGGGGTGAAAATGGACGCAGTCCCAGGACGCCTAAAC CAAACAGCATTTATTGCCGCCCGACCGGGAGTATATTATGGACAATGCTCTGAAATCTGCGGCGCAAACCACAGCTTTATACCAATCG TAGTTGAAGCAGTTCCTCTACAACACTTCGAAAACTGATCCTCAATAATGCTAGAAGACGCCATGATACTAAGTTTTTTCGACCAATT CATAAGCCCCACATATATAGGAATTTCTTTAATTACTTTAGCCTTAACCCTACCATGAATTCTTTATCCTACCCCAACATCCCGATGAC TAAATAACCGAATCCTAACCCTACAAAGCTGTTCGTTAACCGATTCACACAACAACTCCTTCTACCACTAAATGTTGGAGGACATAAATGAGCAGTTATACTAACATCTCTAATACTATT CCTATTAACAATAAACCTGCTAGGACTGCTCCCATACACATTTACACCAACAACCCAATTATCCCTAAATATGGGATTTGCAGTCCCA CTATGACTAGCCACCGTAATCATCGGTATACGAAATCAACCAACTGTAGCACTAGGACACCTACTGCCAGAAGGAACACCAGTCCCT CTAATCCCTGTACTTATTATCATCGAAACAATTAGCTTATTTATCCGTCCACTAGCCCTAGGTGTACGACTTACAGCAAACCTGACAGC AGGCCATCTCTTAATTCAACTCATTGCCACTGCAGTCTTTGTACTTTTACCAATAATGCCTACAGTAGCTATTCTAACAGCAACAGTAC TATTTCTTTTAACATTACTAGAAGTAGCAGTTGCTATAATCCAAGCTTACGTATTTGTACTTCTACTAAGCCTATATCTTCAAGAAAAC GTAATGCCACAGTTAAACCCCGCCCCCTGATTCACGATCCTAGTATTCTCATGGGCCGTGTTCTTAGCTATTCTCCCAACAAAAGTAAT AGCACACACGTTTAATAATGAGCCCAACCTACAAACTGCAAAAAAACCAAAACTAGACTCTTGAAACTGACCATGATACATGGCACA CCAAGCACACGCATATCACATAGTTGACCCAAGCCCATGACCCCTAACAGGCGCAGTAGCCGCCCTACTAATAACATCAGGAACAGC CATATGATTCCACTTCCAAACAACCACCCTAATAACATTAGGAATAATTCTACTTTTACTTACGATATACCAATGATGACGAGACATT GTACGAGAAGGGACTTTCCAAGGACACCATACACCGCCAGTACAAAAAGGACTACGATACGGAATAATCCTATTTATTACCTCAGAA GTATTCTTTTTCCTAGGGTTCTTCTGGCTTTCTACCATTCAAGCCTAGCTCCAACCCCAGAACTAGGGGGATGCTGACCTCCAACTGGTATTATTACTCTAGACCCATTTGAAGT ACCCCTACTAAACACAGCTGTCCTACTTGCCTCAGGCGTTACAGTCACATGAGCACACCACAGCATTATAGAAGGAGAACGAAAACA AGCCATCCAATCCCTAACCCTCACAATCATTTTAGGTTTTTATTTTACATTCCTACAAGCCATAGAATATTATGAAGCCCCATTTACCA TTGCAGATGGAGTTTATGGCTCAACATTCTTCGTGGCCACCGGATTCCACGGCCTACATGTCATTATTGGCTCTACCTTCTTAGCAGTG TGCCTCCTACGCCAAATCAAATATCACTTTACTTCAGAACATCACTTTGGATTCGAAGCCGCCGCATGATACTGACACTTCGTTGACGT AGTATGACTATTCCTATATGTCTCAATTTATTGATGAGGCTCAATGAATCCAATTATTTCAATCCTAATTATCACCACTACCCTTTCCTG CGTACTGATTACAGTCTCATTTTGACTTCCACAGATAAACCCTGACTCAGAAAAACTTTCCCCCTATGAATGCGGCTTTGACCCACTCG GATCCGCCCGACTCCCATTCTCAATACGCTTCTTTCTAGTGGCAATCCTATTCCTGCTATTTGACCTAGAAATTGCACTTCTACTCCCCC TTCCATGAGGGGACCAACTGCCTGACACTACACACGCATTCTTCTGAGCTATATCAATTATTATTCTACTAACACTAGGACTGGTATAC GAATGAATTCAAGGAGGACTAGAATGAGCTGAAATGACTCCCGTACACTTCAGCTTCACATTGGCATTTACCCTAGGGTTCTCAGGCC TAGCCTTCCACCGAAAACATTTATTATCCGCCCTCCTTTGTCTAGAAGCAATAATATTATCACTGTACATTGCTATAGCCTTATGTCTTTCCAAACAGAATCCACCGTATTCTCCTCAGCACCAATAATACTACTAGCCTTTTCCGCCTGTGAAGCCAGTGCAGGCCTAGCCCT CCTAGTAGCTACCTCACGTACACACGGCACAGATCACCTAATAAACCTTAACCTACTACAATGCATGCTAAAAGTACTAATTCCTACC ATCATGCTCATCCCCACCACTTGTTAGTAAACAAAAAATGACTATGAACCACAACTACCTACCAAAGTTTCATCATCGCCTCCATCAGCCTA--ATGATTTAA-TGGACTCAGAAATAGGATGATCTACCACAAACACTTATCTAGCAACAGACCCCCTATCAACGCCACTGCTAGTTCTATCCTGCTGCTTCTCCCATTAATAATTCTAGCAAGCCAAAACCACATGCGCCTAGAACCAATTAACCGCCAACGATCCTACATTACACTACTAATTT CCCTACAAATATTCTTAATCATAGCATTCGGAGCAACAGAAATCATTATATTCTATGTAATATTTGAAGCCACATTAATCCCAACCTTA ATTATTATTACCCGATGAGGAAACCAAACAGAACGACTCAACGCAGGAACTTACTTTCTATTCTATACACTAGCAGGCTCACTCCCAC TGCTTGTTGCACTACTTGCACTACAAAAAGACCTCGGCACTCTATCAATACTCACAATTCAATATACAAAGCCCCTCATTTTATCTTCA TGAGGTGATAAACTATGATGAGCAGGCTGCTTAATAGCATTCCTAGTAAAAATACCACTTTATGGGGTCCACTTATGACTACCAAAAG CTCATGTAGAGGCCCCAGTAGCAGGGTCCATAGTCCTAGCCGCCGTACTACTAAAACTAGGTGGATATGGAATAATACGAATAATTAT TATTTTAACCCCACTAACCAAAGAACTAGCCTATCCATTCATCATCCTCGCCCTCTGAGGAATCATTATAACCGGCTCTATCTGCTTAC GACAAACAGACCTAAAATCCATAATTGCATACTCATCAGTCAGCCACATAGGCCTAGTGGCAGGAGGAATTCTCATCCAAACCCCAT GAGGATTCACAGGAGCAATTATCCTAATAATTGCCCACGGACTAGTATCATCAGCATTATTTTGTCTAGCCAACACCAACTACGAACG AACCCATAGCCGCACCCTACTACTAGCCCGAGGCCTACAAATAATCCTTCCTTTAATAGCCGCCTGATGATTTATTGCCAACCTAGCC AACTTGGCACTCCCACCACTACCAAATCTAATAGGAGAACTGATAATCATCACATCCATGTTTAACTGTCATACTGATCTATTGCATTAACAGGCTTAGGAACCCTAATCACAGCCGGATATTCACTATACATATTCCTAATAACACAACGGGGCC CAACCCCAAATCACATCATTGGTTTAGAACCATCACATACCCGAGAACACCTACTAATTGCTATACACCTCATTCCAGTACTCCTTCTA GTACTAAAACCTGAACTAATATGAGGATGATGCTTAATGCCCTTAACAACTTTAACACTAAATTCGAGCCTTCTAATCATTCTCACGCT ACTAATTTACCCTATTATAATGACATTAAACCCAAATCCAATAAAAAAAGACTGGCTGTAACACATGTTAAAACTGCTGTTCAAATAGCATTTTTCGTAAGCCTGATCCCACTATTCCTGTTCTTAGACCAAGGAATAGAAAC AGTACTAACAAACTGCAATGAGCTAATACAATAACATTTGATTTAAATACAAGCTTCAAATTTGACCATTACTCAATTATCTTCACCCCAGTTGCCCTATACGT TACATGATCAATCCTAGAGTTCGCCTCATGATACATGCACGCAGACCCAAACATGAACCGATTCTTTAAATATTTACTTATATTCCTAG TAGCAATAATCATTCTAGTAACAGCCAACAACCTATTTCAACTATTTATTGGCTGAGAAGGTGTAGGAATTATATCATTCCTCCTAATT GGCTGATGTACGGACGGGCAGATGCAAACACCGCAGCACTCCAAGCCGTCATCTACAACCGAGTTGGTGATATTGGCCTCATCCTAGCTATAGCCT GAATAGCAATAAACCTAAACAGCTGAGAAATCCAACAAGTATTTATTATCTCAAAAGAAATAGACCTCACACTTCCTCTTATAGGACT AGTCATTGCTGCAACAGGAAAATCCGCCCAATTCGGACTACACCCATGACTACCATCAGCAATAGAAGGCCCCACACCAGTCTCTGCC CTACTGCATTCAAGCACTATGGTCGTAGCAGGAATCTTCCTATTAATCCGACTGCACCCAATAATAGAAAACAACCAAACAGTTCTAT CAACCTGCCTATGTTTAGGAGCACTAACCACGCTATTCACAGCCACTTGTGCACTAACACAAAATGATATCAAAAAAATTGTTGCATT TTCAACATCCAGCCAACTCGGCCTTATAATAGTAACTATTGGACTAAACCAACCACAACTTGCATTCATACATATTTGTACACACGCA TTCTTTAAAGCAATACTATTCCTCTGTTCAGGGTCTATCATTCACAGCCTAAATGACGAACAAGATATTCGAAAAATAGGAGGACTAC ACAAACTGCTTCCATTTACTTCATCTTGCATAACAATTGGCAGCTTAGCCCTCACAGGCACCCCATTCCTAGCAGGATTCTTCTCCAAA GACGCAATCATCGAAGCCATAAACACATCCTACCTTAACGCCTGAGCCCTTACCTTAACCTTGATCGCCACCTCATTTACAGCCGTAT ATAGCTTCCGAATTATCTTCTTTGCTTCAATAGGACAACCACGGTTTCTTCCACTCTCTCCAATTAACGAAAACAACCCTGCAGTTTTA AACCCAATCAAACGACTCGCTTGAGGAAGCATCATTGCAGGTCTAATCATTACATCAAATTTCCTACCAATAAAAACACCAATTATAA CAATACACCCCACATTAAAACTAAGCGCTCTAATAGTTACCGTTATTGGACTCTTTACCGCCATAGAGCTAGCAAACCTAACAAACAA GCAATACAAAACTAAACCATACACTAAAACACATAACTTTTCAAATATACTAGGCTACTTCCCAGCCGTAATCCACCGAATGGCCCCA AAACTAACTCTAGTACTAGGACAAAAAGTAGCCACCCAACTAGTAGATCAAACATGATTTGAAAAACTAGGACCAAAAGGAATTGTA AACATTCAACTACCAATAATCAAAATTATTAACAACCCACAACAAGGACTTATTAAAGTATACCTAGCAACATTCTTCCTAACAAATG CCCTAATTATCCTCATAATAATAATATTCATGGCAAACCTACGAAAAACCCACCCACTTCTAAAAATTGCTAACGATGCCCTAGTGGA TCTACCAACCCCATCCAATATTTCAGCATGATGAAATTTTGGCTCTCTCCTAGGATTATGCCTTATCTCACAAATCCTTACAGGACTAT TCCTAGCCATACACTATACATCAGACATCTCAACTGCCTTTTCCTCAGTAGCCCACATCTGCCGAGACGTCAACTATGGATGATTAATC CGTAACCTACATGCAAACGGAGCCTCCTTCTTCTTTATTTGCCTCTACCTCCACATTGCCCGAGGACTTTACTACGGCTCATACCTTTAC AAAGAGACATGAAACATTGGAGTCGTATTATTCCTATTAGTAATAATAACAGCATTCGTAGGATATGTACTTCCATGAGGACAAATAT CATTCTGAGGTGCTACAGTAATTACCAACCTACTATCTGCCGTCCCATACGTAGGAGACTCCCTAGTCCAATGAATCTGAGGAGGCTT CTCAGTTGACAACGCCACATTAACCCGATTCTTCGCATTCCACTTCCTATTCCCATTTGTAGTTGCCGGAGCTACAATACTTCACCTCCT ATTCCTCCATGAAACAGGATCAAACAACCCAGTAGGACTGAACTCTGACGCAGACAAAATCCCATTCCACCCATACTTCTCCTACAAA GACCTACTAGGATTCATTATTATACTCACCGCCCTAACAATACTTGCCCTATTCTACCCAAACCTCCTTGGAGACCCAGACAACTTCAC CCCTGCAAACCCAATAGTTACCCCACCACACATCAAGCCAGAATGATATTTCCTATTTGCCTACGCCATTCTACGATCAATTCCTAACA AACTCGGCGGGGTATTAGCCCTACTATCCTCTATCCTAGTCCTAATAGTAGTACCAATTCTTCACACCTCAAAACAACGAGGACTTAC CTTCCGACCAGCTTCACAACTACTATTCTGAATTTTAGTAGCAGACATACTAGTACTAACATGAATCGGAGGAATACCAGTAGAACAC CCATACATTATCATTGGCCAAGTAGCATCAGTACTTTATTTTTCCCTATTTCTGGTGCTAAACCCATTAGTAGGTGCCTAGAAAACAAAGTAATAAATGCATGAGTTATTTTATTTTTCTTTTTTTGGTTATATTGGTGTTGGGGTTTTTGGGTGTGGCTTCTAATCCTGCTCCTTATTTTGCTGCTTT AGGGTTGGTGTTGGCAGCTGCTGGGGGTTGTGGGGTTTTAGCAGGGTATGGTGGGTCGTTTATTTCTTTAGTACTGTTTCTTATTTATTT GGGTGGCATGTTAGTGGTATTTGCATATTCTGCTGCTCTTGCTGCTGAGCCATACCCGGAGTCATG-GGGGATGGTCTGTTTTGGGGTATGTAGTTGGTTATGTTTTTCTTTGTGTTTTAGGTATTGGTGTATTTTATGTGGAGTGTTTGATTGTTATTGTGGAATTGTTGATGAATATCGGGATTTTTCAGTGTTGCGTGGGGATTTTAGTGGGGTTTCTTTTATTTATTATTTG GGTGGTGGGATGTTAATTATTTGTGGGTGGCTTTATTGCTTACTCTTTTTGTTGTTCTTGAGTTAACTCGTGGTCGTAGTCGTGGGGCTTTGCGAGCAATT > ATP6_reconstructed_Ancestral sequence_marginal ATGAACACAATCCTTACTCACATCATTAATCCTCTAGCATATATTGTCCCAGTACTTCTCGCCGTCGCATTCCTAACACTTCTAGAACG AAAAGTATTAGGATATATACAACTACGAAAAGGGCCAAACATCGTTGGACCATACGGCCTCCTTCAACCAATCGCCGACGGAGTAAA ACTATTTATTAAAGAACCAGTACGCCCTTCAACCGCATCCCCATTCCTATTTTTAGCCACCCCAACTCTTGCACTAACCCTCGCCTTAA CACTATGAGCCCCCATACCAATACCTTACCCAGTAGTAGAACTAAACCTAGGAATTCTATTCGTACTAGCCCTATCAAGCCTCGCAGT TTACTCAATCCTGGGCTCAGGCTGGCCTCAAACTCAAAATATGCTCTAATTGGGGCCCTACGTGCCGTAGCCCAAACAATTTCATATGAAGTAAGCCTAGGCCTAATCCTTC TTTCAATTATTATTATCGTAGGAGGATTCAACCTAAAAACATTCAACATCGCACAAGAAGCAACATGATTAATAGCCCCAGCCTGACC ACTAGCAGCAATATGATAATCTCAACACTAGCTGAAACAAACCGAGCCCCATTTGACCTTACAGAAGGAGAATCAGAATTAGTATCAGGCTTTAACGTAGAATAT GCAGGAGGACCATTTGCCCTATTCTTCCTAGCCGAATACTCAAACATCCTACTAATAAATACCTTATCAACAATCCTATTTCTAGGAGC AATACACACCCCACTAATTCCAGAACTAACAACAATAAACCTAATAATAAAAGCCACTATACTTTCCATTATATTCTTATGAGTACGA GCCTCCTACCCACGATTTCGGTACGACCAGCTAATACATCTAATATGAAAAAACTTCCTACCACTAACCCTAGCCCTACTTATCTGAA ACCTAGCCTTGCCCATTACCATGGCAGGCCTCCCCCCAAACAACATGAACCCATATGTAACCTTCATCATACTAACAAGCTTGGGACT AGGCACCACAATCACATTCGCTAGCTCACACTGACTGCTCGCCTGAATAGGATTGGAAATTAATACACTAGCCATTATTCCCCTTATA GCCCAACACCACCACCCACGAGCCGTAGAAGCAACAACAAAATACTTCCTCACACAAGCCACAGCAGCAGCACTAATACTATTTACA ACCACATCAAATGCATGAATTACAGGACAATGAGAAATTCAACAACTATCTCACCCAATAATCACCACAATTACAATCCTTGCCCTAG GACTTAAAGTAGGACTAGCCCCAATACATTTTTGACTACCAGAAGTCCTACAAGGCCTAGACCTAACTACAGGACTAATCCTGTCAAC ATGACAAAAACTAGCGCCCATAGCCCTAATTTACCAACTCTCACCAGGAGTAGAACAACCACTAATAATTATACTAGGAATGATATCT GCCCTCGTAGGAGGATGAGGTGGATTAAACCAAACACAACTACGAAAAATCCTAGCATACTCCTCAATCGCACACATAGGATGAATA ATAATTGTAATAAAATACTTACCAAATTTAATAATCATAAACCTAATAATCTATATCATCATGACATCATCAGCTTTCATAGCACTAAA AATAACCACCGCTACAAAAATTAATACACTAGCAACAGGTGAACAAAAGCCCCAATCCTTACAGCACTAACTATGGCCACCATACTATCATTAGGAGGTTTACCCCCACTAACCGGATTCATACCAA AATGAATAATTCTACAAGAACTAACCAAACAAGACCTCCCACTAATCGCTACGCTAATAGCAATAACAGCCCTACTAAGTCTATTCTT CTACCTACGACTATGCTACGCCATAACACTAACAATTTCACCAAACACAAACAATGCTAAAACACCATGACGATTAAAATCAAAACA AATAACAATGCCCCTCTCAATTACAATAATCATAACAGCTATAATACTCCCGGTAACCCCAGCAGTAATAGCAATAACAACAGTGGC AATCACCCGTTGATTCTTTTCTACTAATCACAAAGACATTGGTACCCTATATCTAGTATTTGGTGCCTGAGCCGGAATAGTGGGTACCG CACTAAGCCTTCTAATCCGTGCCGAATTAAGTCAACCAGGCGCCCTTCTTGGAGATGACCAAATTTACAATGTCATCGTCACAGCGCA TGCCTTTGTAATGATTTTCTTTATAGTAATACCAGTAATAATTGGAGGATTTGGCAACTGACTTGTGCCATTAATAATCGGCGCCCCAG ACATAGCATTCCCCCGAATAAACAACATAAGCTTCTGACTTTTACCCCCATCATTTCTTCTACTACTAGCCTCCTCAGGAGTAGAGGCT GGGGCTGGTACAGGTTGAACTGTATATCCACCTCTAGCTGGAAACTTAGCCCACGCCGGAGCATCTGTTGACCTGACAATTTTCTCAC TTCACCTTGCAGGAATTTCATCAATTCTAGGGGCCATTAATTTTATTACTACAATTATTAACATGAAACCGCCTGCCATTACACAGTAC CAAACCCCTCTGTTTGTATGAGCTGTTTTAGTAACCGCTGTTCTGCTACTCCTATCCCTGCCAGTCCTAGCCGCAGGTATTACAATACTT CTAACTGACCGAAATCTAAATACAACCTTCTTTGACCCTGCAGGGGGTGGAGACCCAATCCTCTACCAACACCTATTCTGATTCTTCG GCCACCCAGAAGTATACATTTTAATCTTACCAGGATTTGGAATAATCTCACACATTGTTGCTTATTATTCCGGTAAGAAAGAACCATTT GGGTATATAGGAATGGTCTGAGCAATGATGGCTATCGGACTACTAGGATTCATTGTATGAGCACACCATATGTTTACAGTAGGAATAG ACGTAGACACCCGTGCTTACTTCACTTCCGCCACAATAATCATCGCAATTCCAACTGGGGTAAAAGTATTCAGCTGACTAGCCACATT ACACGGAGGGGTCATCAAATGAGAAACCCCACTTCTTTGAGCTTTAGGTTTTATTTTCCTATTTACAGTTGGGGGCCTAACAGGTATCG TACTAGCAAACTCATCAATCGATATTGTATTACATGACACATACTATGTAGTAGCCCATTTCCATTATGTTCTGTCCATAGGAGCAGTA TTTGCTATTATAGGAGGCTTCGTACACTGATTCCCCCTATTCTCAGGCTATACACTACACGACACATGAACCAAAGTACACTTTGGAAT TATGTTCGTAGGAGTAAACCTAACCTTCTTCCCACAACATTTCCTAGGCTTAGCAGGAATACCACGACGTTACTCAGACTACCCAGAT GCCTACACCCTATGAAATACAATCTCCTCTATTGGATCCCTAATTTCTCTCACAGCCGTAGTCCTGTTCCTATTTATCCTCTGAGAAGC ATTTACTGCCAAACGAGAAGTAAAATGAGTAGAACTTACAGAAACAAATGTTGAATGACTACACGGATGTCCTCCACCATATCACAC ATTCGAAGAACCAGCGTACGTCCGAGTTCAAATGGCACATCCCTCACAGCTAGGTTTCCAAGACGCAGCCTCACCCCTGATAGAAGA ACTACTTCATTTCCACGACCATGCGCTAATAATTGTTTTCCTAATTAGCGTCCTAGTACTTTATATTATTGTGGCAATGGTAACTGCCA AAGTTACTAACATGTTTATCCTAGATTCACAAGAGATTGAAATGTGTGAACCGTATTGCCAGCAGCAATTCTAATTCTAATCGCACTCCCCTCTCTACGGATCCTTTATCTAATAGACGAAATCAATGACCC ACATTTAACAATTAAAGCAATCGGACATCAATGATACTGAAGCTACGAGTATACTGACTATGAAGACCTTGGATTTGACTCGTACATA ATCCCAACACAAGACTTAACCCCAGGACAATTCCGACTGCTAGAAACAGACCATCGAATAGTAGTACCAATAGAATCACCTGTACGA GTATTAGTTACAGCAGAAGACGTCTTACACTCATGAGCAGTCCCATCCTTAGGGGTGAAAATGGACGCAGTCCCAGGACGCCTAAAC CAAACAGCATTTATTGCCGCCCGACCGGGAGTATATTATGGACAATGCTCTGAAATCTGCGGCGCAAACCACAGCTTTATACCAATCG TAGTTGAAGCAGTTCCTCTACAACACTTCGAAAACTGATCCTCAATAATGCTAGAAGACGCCATGATACTAAGTTTTTTCGACCAATT CATAAGCCCCACATATATAGGAATTTCTTTAATTACTTTAGCCTTAACCCTACCATGAATTCTTTATCCTACCCCAACATCCCGATGAC TAAATAACCGAATCCTAACCCTACAAAGCTGTTCGTTAACCGATTCACACAACAACTCCTTCTACCACTAAATGTTGGAGGACATAAATGAGCAGTTATACTAACATCTCTAATACTATT CCTATTAACAATAAACCTGCTAGGACTGCTCCCATACACATTTACACCAACAACCCAATTATCCCTAAATATGGGATTTGCAGTCCCA CTATGACTAGCCACCGTAATCATCGGTATACGAAATCAACCAACTGTAGCACTAGGACACCTACTGCCAGAAGGAACACCAGTCCCT CTAATCCCTGTACTTATTATCATCGAAACAATTAGCTTATTTATCCGTCCACTAGCCCTAGGTGTACGACTTACAGCAAACCTGACAGC AGGCCATCTCTTAATTCAACTCATTGCCACTGCAGTCTTTGTACTTTTACCAATAATGCCTACAGTAGCTATTCTAACAGCAACAGTAC TATTTCTTTTAACATTACTAGAAGTAGCAGTTGCTATAATCCAAGCTTACGTATTTGTACTTCTACTAAGCCTATATCTTCAAGAAAAC GTAATGCCACAGTTAAACCCCGCCCCCTGATTCACGATCCTAGTATTCTCATGGGCCGTGTTCTTAGCTATTCTCCCAACAAAAGTAAT AGCACACACGTTTAATAATGAGCCCAACCTACAAACTGCAAAAAAACCAAAACTAGACTCTTGAAACTGACCATGATACATGGCACA CCAAGCACACGCATATCACATAGTTGACCCAAGCCCATGACCCCTAACAGGCGCAGTAGCCGCCCTACTAATAACATCAGGAACAGC CATATGATTCCACTTCCAAACAACCACCCTAATAACATTAGGAATAATTCTACTTTTACTTACGATATACCAATGATGACGAGACATT GTACGAGAAGGGACTTTCCAAGGACACCATACACCGCCAGTACAAAAAGGACTACGATACGGAATAATCCTATTTATTACCTCAGAA GTATTCTTTTTCCTAGGGTTCTTCTGGCTTTTTACCATTCAAGCCTAGCTCCAACCCCAGAACTAGGGGGATGCTGACCTCCAACTGGTATTATTACTCTAGACCCATTTGAAGT ACCCCTACTAAACACAGCTGTCCTACTTGCCTCAGGCGTTACAGTCACATGAGCACACCACAGCATTATAGAAGGAGAACGAAAACA AGCCATCCAATCCCTAACCCTCACAATCATTTTAGGTTTTTATTTTACATTCCTACAAGCCATAGAATATTATGAAGCCCCATTTACCA TTGCAGATGGAGTTTATGGCTCAACATTCTTCGTGGCCACCGGATTCCACGGCCTACATGTCATTATTGGCTCTACCTTCTTAGCAGTG TGCCTCCTACGCCAAATCAAATATCACTTTACTTCAGAACATCACTTTGGATTCGAAGCCGCCGCATGATACTGACACTTCGTTGACGT AGTATGACTATTCCTATATGTCTCAATTTATTGATGAGGCTCAATGAATCCAATTATTTCAATCCTAATTATCACCACTACCCTTTCCTG CGTACTGATTACAGTCTCATTTTGACTTCCACAGATAAACCCTGACTCAGAAAAACTTTCCCCCTATGAATGCGGCTTTGACCCACTCG GATCCGCCCGACTCCCATTCTCAATACGCTTCTTTCTAGTGGCAATCCTATTCCTGCTATTTGACCTAGAAATTGCACTTCTACTCCCCC TTCCATGAGGGGACCAACTGCCTGACACTACACACGCATTCTTCTGAGCTATATCAATTATTATTCTACTAACACTAGGACTGGTATAC GAATGAATTCAAGGAGGACTAGAATGAGCTGAAATGACTCCCGTACACTTCAGCTTCACATTGGCATTTACCCTAGGGTTCTCAGGCC TAGCCTTCCACCGAAAACATTTATTATCCGCCCTCCTTTGTCTAGAAGCAATAATATTATCACTGTACATTGCTATAGCCTTATGTCTTTCCAAACAGAATCCACCGTATTCTCCTCAGCACCAATAATACTACTAGCCTTTTCCGCCTGTGAAGCCAGTGCAGGCCTAGCCCT CCTAGTAGCTACCTCACGTACACACGGCACAGATCACCTAATAAACCTTAACCTACTACAATGCATGCTAAAAGTACTAATTCCTACC ATCATGCTCATCCCCACCACTTGTTAGTAAACAAAAAATGACTATGAACCACAACTACCTACCAAAGTTTCATCATCGCCTCCATCAGCCTA--ATGATTTAA-TGGACTCAGAAATAGGATGATCTACCACAAACACTTATCTAGCAACAGACCCCCTATCAACGCCACTGCTAGTTCTATCCTGCTGCTTCTCCCATTAATAATTCTAGCAAGCCAAAACCACATGCGCCTAGAACCAATTAACCGCCAACGATCCTACATTACACTACTAATTT CCCTACAAATATTCTTAATCATAGCATTCGGAGCAACAGAAATCATTATATTCTATGTAATATTTGAAGCCACATTAATCCCAACCTTA ATTATTATTACCCGATGAGGAAACCAAACAGAACGACTCAACGCAGGAACTTACTTTCTATTCTATACACTAGCAGGCTCACTCCCAC TGCTTGTTGCACTACTTGCACTACAAAAAGACCTCGGCACTCTATCAATACTCACAATTCAATATACAAAGCCCCTCATTTTATCTTCA TGAGGTGATAAACTATGATGAGCAGGCTGCTTAATAGCATTCCTAGTAAAAATACCACTTTATGGGGTCCACTTATGACTACCAAAAG CTCATGTAGAGGCCCCAGTAGCAGGGTCCATAGTCCTAGCCGCCGTACTACTAAAACTAGGTGGATATGGAATAATACGAATAATTAT TATTTTAACCCCACTAACCAAAGAACTAGCCTATCCATTCATCATCCTCGCCCTCTGAGGAATCATTATAACCGGCTCTATCTGCTTAC GACAAACAGACCTAAAATCCATAATTGCATACTCATCAGTCAGCCACATAGGCCTAGTGGCAGGAGGAATTCTTATCCAAACCCCAT GAGGATTCACAGGAGCAATTATCCTAATAATTGCCCACGGACTAGTATCATCAGCATTATTTTGTCTAGCCAACACCAACTACGAACG AACCCATAGCCGCACCCTACTACTAGCCCGAGGCCTACAAATAATCCTTCCTTTAATAGCCGCCTGATGATTTATTGCCAACCTAGCC AACTTGGCACTCCCACCACTACCAAATCTAATAGGAGAACTGATAATCATCACATCCATGTTTAACTGTCATACTGATCTATTGCATTAACAGGCTTAGGAACCCTAATCACAGCCGGATATTCACTATACATATTCCTAATAACACAACGGGGCC CAACCCCAAATCACATCATTGGTTTAGAACCATCACATACCCGAGAACACCTACTAATTGCTATACACCTCATTCCAGTACTCCTTCTA GTACTAAAACCTGAACTAATATGAGGATGATGCTTAATGCCCTTAACAACTTTAACACTAAATTCGAGCCTTCTAATCATTCTCACGCT ACTAATTTACCCTATTATAATGACATTAAACCCAAATCCAATAAAAAAAGACTGGCTGTAACACATGTTAAAACTGCTGTTCAAATAGCATTTTTCGTAAGCCTGATCCCACTATTCCTGTTCTTAGACCAAGGAATAGAAAC AGTACTAACAAACTGCAATGAGCTAATACAATAACATTTGATTTAAATACAAGCTTCAAATTTGACCATTACTCAATTATCTTCACCCCAGTTGCCCTATACGT TACATGATCAATCCTAGAGTTCGCCTCATGATACATGCACGCAGACCCAAACATGAACCGATTCTTTAAATATTTACTTATATTCCTAG TAGCAATAATCATTCTAGTAACAGCCAACAACCTATTTCAACTATTTATTGGCTGAGAAGGTGTAGGAATTATATCATTCCTCCTAATT GGCTGATGTACGGACGGGCAGATGCAAACACCGCAGCACTCCAAGCCGTCATCTACAACCGAGTTGGTGATATTGGCCTCATCCTAGCTATAGCCT GAATAGCAATAAACCTAAACAGCTGAGAAATTCAACAAGTATTTATTATCTCAAAAGAAATAGACCTCACACTTCCTCTTATAGGACT AGTCATTGCTGCAACAGGAAAATCCGCCCAATTCGGACTACACCCATGACTACCATCAGCAATAGAAGGCCCCACACCAGTCTCTGCC CTACTGCATTCAAGCACTATGGTCGTAGCAGGAATCTTCCTATTAATCCGACTGCACCCAATAATAGAAAACAACCAAACAGTTCTAT CAACCTGCCTATGTTTAGGAGCACTAACCACGCTATTCACAGCCACTTGTGCACTAACACAAAATGATATCAAAAAAATTGTTGCATT TTCAACATCCAGCCAACTCGGCCTTATAATAGTAACTATTGGACTAAACCAACCACAACTTGCATTCATACATATTTGTACACACGCA TTCTTTAAAGCAATACTATTCCTCTGTTCAGGGTCTATCATTCACAGCCTAAATGACGAACAAGATATTCGAAAAATAGGAGGACTAC ACAAACTGCTTCCATTTACTTCATCTTGCATAACAATTGGCAGCCTAGCCCTCACAGGCACCCCATTCCTAGCAGGATTCTTCTCCAAA GACGCAATCATCGAAGCCATAAACACATCCTACCTTAACGCCTGAGCCCTTACCTTAACCTTGATCGCCACCTCATTTACAGCCGTAT ATAGCTTCCGAATTATCTTCTTTGCTTCAATAGGACAACCACGGTTTCTTCCACTCTCTCCAATTAACGAAAACAACCCTGCAGTTTTA AACCCAATCAAACGACTCGCTTGAGGAAGCATCATTGCAGGTCTAATCATTACATCAAATTTCCTACCAATAAAAACACCAATTATAA CAATACACCCCACATTAAAACTAAGCGCTCTAATAGTTACCGCTATTGGACTCTTTACCGCCATAGAGCTAGCAAACCTAACAAATAA GCAATACAAAACTAAACCATACACTAAAACACATAACTTTTCAAACATACTAGGCTACTTCCCAGCCGTAATCCACCGAATGGCCCCA AAACTAACTCTAGTACTAGGACAAAAAGTAGCCACCCAACTAGTAGATCAAACATGATTTGAAAAACTAGGACCAAAAGGAATTGTA AACATTCAACTACCAATAATCAAAATTATTAACAACCCACAACAAGGACTTATTAAAGTATACCTAGCAACATTCTTCCTAACAAATG CCCTAATTATCCTCATAATAATAATATTCATGGCAAACCTACGAAAAACCCACCCACTTCTAAAAATTGCTAACGATGCCCTAGTGGA TCTACCAACCCCATCCAATATTTCAGCATGATGAAATTTTGGCTCTCTCCTAGGATTATGCCTTATCTCACAAATCCTTACAGGACTAT TCCTAGCCATACACTATACATCAGACATCTCAACTGCCTTTTCCTCAGTAGCCCACATCTGCCGAGACGTCAACTATGGATGATTAATC CGTAACCTACATGCAAACGGAGCCTCCTTCTTCTTTATTTGCCTCTACCTCCACATTGCCCGAGGACTTTACTACGGCTCATATCTTTAC AAAGAGACATGAAACATTGGAGTCGTATTATTCCTATTAGTAATAATAACAGCATTCGTAGGATATGTACTTCCATGAGGACAAATAT CATTCTGAGGTGCTACAGTAATTACCAACCTACTATCTGCCGTCCCATACGTAGGAGACTCCCTAGTCCAATGAATCTGAGGGGGCTT CTCAGTTGACAACGCCACATTAACCCGATTCTTCGCATTCCACTTCCTATTCCCATTTGTAGTTGCCGGAGCTACAATACTTCACCTCCT ATTCCTCCATGAAACAGGATCAAACAACCCAGTAGGACTGAACTCTGACGCAGACAAAATCCCATTCCACCCATACTTCTCCTACAAA GACCTACTAGGATTCATTATTATACTCACCGCCCTAACAATACTTGCCCTATTCTACCCAAACCTCCTTGGAGACCCAGACAACTTCAC CCCTGCAAACCCAATAGTTACCCCACCACACATCAAGCCAGAATGATATTTCCTATTTGCCTACGCCATTCTACGATCAATTCCTAACA AACTCGGCGGGGTATTAGCCCTACTATCCTCTATCCTAGTCCTAATAGTAGTACCAATTCTTCACACCTCAAAACAACGAGGACTTAC CTTCCGACCAGCTTCACAACTACTATTCTGAATTTTAGTAGCAGACATACTAGTACTAACATGAATCGGAGGAATACCAGTAGAACAC CCATACATTATCATTGGCCAAGTAGCATCAGTACTTTATTTTTCCCTATTTCTGGTGCTAAACCCATTAGTAGGTGCCTAGAAAACAAAGTAATAAATGCATGAGTTATTTTATTTTTCTTTTTTTGGTTATATTGGTGTTGGGGTTTTTGGGTGTGGCTTCTAATCCTGCTCCTTATTTTGCTGCTTT AGGGTTGGTGTTGGCAGCTGCTGGGGGTTGTGGGGTTTTAGCAGGGTATGGTGGGTCGTTTATTTCTTTAGTACTGTTTCTTATTTATTT GGGTGGCATGTTAGTGGTATTTGCATATTCTGCTGCTCTTGCTGCTGAGCCATACCCGGAGTCATG-GGGGATGGTCTGTTTTGGGGTATGTGGTTGGTTATGTTTTTCTTTGTGTTTTAGGTATTGGTGTATTTTATGTGGAGTGTTTGATTGTTATTGTGGAATTGTTGATGAATATCGGGATTTTTCAGTGTTGCGTGGGGATTTTAGTGGGGTTTCTTTTATTTATTATTTG GGTGGTGGGATGTTAATTATTTGTGGGTGGCTTTATTGCTTACTCTTTTTGTTGTTCTTGAGTTAACTCGTGGTCGTAGTCGTGGGGCTTTGCGAGCAATT > ATP6_reconstructed_Ancestral sequence _sampled ATGAACACAATCCTTACTCACATCATTAATCCTCTAGCATATATTGTCCCAGTACTTCTCGCCGTCGCATTCCTAACACTTCTAGAACG AAAAGTATTAGGATATATACAACTACGAAAAGGGCCAAACATCGTTGGACCATACGGCCTCCTTCAACCAATCGCCGACGGAGTAAA ACTATTTATTAAAGAACCAGTACGCCCTTCAACCGCATCCCCATTCCTATTTTTAGCCACCCCAACTCTTGCACTAACCCTCGCCTTAA CACTATGAGCCCCCATACCAATACCTTACCCAGTAGTAGAACTAAACCTAGGAATTCTATTCGTACTAGCCCTATCAAGCCTCGCAGT TTACTCAATCCTGGGCTCAGGCTGGCCTCAAACTCAAAATATGCTCTAATTGGGGCCCTACGTGCCGTAGCCCAAACAATTTCATATGAAGTAAGCCTAGGCCTAATCCTTC TTTCAATTATTATTATCGTAGGAGGATTCAACCTAAAAACATTCAACATCGCACAAGAAGCAACATGATTAATAGCCCCAGCCTGACC ACTAGCAGCAATATGATAATCTCAACACTAGCTGAAACAAACCGAGCCCCATTTGACCTTACAGAAGGAGAATCAGAATTAGTATCAGGCTTTAACGTAGAATAT GCAGGAGGACCATTTGCCCTATTCTTCCTAGCCGAATACTCAAACATCCTACTAATAAATACCTTATCAACAATCCTATTTCTAGGAGC AATACACACCCCACTAATTCCAGAACTAACAACAATAAACCTAATAATAAAAGCCACTATACTTTCCATTATATTCTTATGAGTACGA GCCTCCTACCCACGATTTCGGTACGACCAGCTAATACATCTAATATGAAAAAACTTCCTACCACTAACCCTAGCCCTACTTATCTGAA ACCTAGCCTTGCCCATTACCATGGCAGGCCTCCCCCCAAACAACATGAACCCATATGTAACCTTCATCATACTAACAAGCTTGGGACT AGGCACCACAATCACATTCGCTAGCTCACACTGACTGCTCGCCTGAATAGGATTGGAAATTAATACACTAGCCATTATTCCCCTTATA GCCCAACACCACCACCCACGAGCCGTAGAAGCAACAACAAAATACTTCCTCACACAAGCCACAGCAGCAGCACTAATACTATTTACA ACCACATCAAATGCATGAATTACAGGACAATGAGAAATTCAACAACTATCTCACCCAATAATCACCACAATTACAATCCTTGCCCTAG GACTTAAAGTAGGACTAGCCCCAATACATTTTTGACTACCAGAAGTCCTACAAGGCCTAGACCTAACTACAGGACTAATCCTGTCAAC ATGACAAAAACTAGCGCCCATAGCCCTAATTTACCAACTCTCACCAGGAGTAGAACAACCACTAATAATTATACTAGGAATGATATCT GCCCTCGTAGGAGGATGAGGTGGATTAAACCAAACACAACTACGAAAAATCCTAGCATACTCCTCAATCGCACACATAGGATGAATA ATAATTGTAATAAAATACTTACCAAATTTAATAATCATAAACCTAATAATCTATATCATCATGACATCATCAGCTTTCATAGCACTAAA AATAACCACCGCTACAAAAATTAATACACTAGCAACAGGTGAACAAAAGCCCCAATCCTTACAGCACTAACTATGGCCACCATACTATCATTAGGAGGTTTACCCCCACTAACCGGATTCATACCAA AATGAATAATTCTACAAGAACTAACCAAACAAGACCTCCCACTAATCGCTACGCTAATAGCAATAACAGCCCTACTAAGTCTATTCTT CTACCTACGACTATGCTACGCCATAACACTAACAATTTCACCAAACACAAACAATGCTAAAACACCATGACGATTAAAATCAAAACA AATAACAATGCCCCTCTCAATTACAATAATCATAACAGCTATAATACTCCCGGTAACCCCAGCAGTAATAGCAATAACAACAGTGGC AATCACCCGTTGATTCTTTTCTACTAATCACAAAGACATTGGTACCCTATATCTAGTATTTGGTGCCTGAGCCGGAATAGTGGGTACCG CACTAAGCCTTCTAATCCGTGCCGAATTAAGTCAACCAGGCGCCCTTCTTGGAGATGACCAAATTTACAATGTCATCGTCACAGCGCA TGCCTTTGTAATGATTTTCTTTATAGTAATACCAGTAATAATTGGAGGATTTGGCAACTGACTTGTGCCATTAATAATCGGCGCCCCAG ACATAGCATTCCCCCGAATAAACAACATAAGCTTCTGACTTTTACCCCCATCATTTCTTCTACTACTAGCCTCCTCAGGAGTAGAGGCT GGGGCTGGTACAGGTTGAACTGTATATCCACCTCTAGCTGGAAACTTAGCCCACGCCGGAGCATCTGTTGACCTGACAATTTTCTCAC TTCACCTTGCAGGAATTTCATCAATTCTAGGGGCCATTAATTTTATTACTACAATTATTAACATGAAACCGCCTGCCATTACACAGTAC CAAACCCCTCTGTTTGTATGAGCTGTTTTAGTAACCGCTGTTCTGCTACTCCTATCCCTGCCAGTCCTAGCCGCAGGTATTACAATACTT CTAACTGACCGAAATCTAAATACAACCTTCTTTGACCCTGCAGGGGGTGGAGACCCAATCCTCTACCAACACCTATTCTGATTCTTCG GCCACCCAGAAGTATACATTTTAATCTTACCAGGATTTGGAATAATCTCACACATTGTTGCTTATTATTCCGGTAAGAAAGAACCATTT GGGTATATAGGAATGGTCTGAGCAATGATGGCTATCGGACTACTAGGATTCATTGTATGAGCACACCATATGTTTACAGTAGGAATAG ACGTAGACACCCGTGCTTACTTCACTTCCGCCACAATAATCATCGCAATTCCAACTGGGGTAAAAGTATTCAGCTGACTAGCCACATT ACACGGAGGGGTCATCAAATGAGAAACCCCACTTCTTTGAGCTTTAGGTTTTATTTTCCTATTTACAGTTGGGGGCCTAACAGGTATCG TACTAGCAAACTCATCAATCGATATTGTATTACATGACACATACTATGTAGTAGCCCATTTCCATTATGTTCTGTCCATAGGAGCAGTA TTTGCTATTATAGGAGGCTTCGTACACTGATTCCCCCTATTCTCAGGCTATACACTACACGACACATGAACCAAAGTACACTTTGGAAT TATGTTCGTAGGAGTAAACCTAACCTTCTTCCCACAACATTTCCTAGGCTTAGCAGGAATACCACGACGTTACTCAGACTACCCAGAT GCCTACACCCTATGAAATACAATCTCCTCTATTGGATCCCTAATTTCTCTCACAGCCGTAGTCCTGTTCCTATTTATCCTCTGAGAAGC ATTTACTGCCAAACGAGAAGTAAAATGAGTAGAACTTACAGAAACAAATGTTGAATGACTACACGGATGTCCTCCACCATATCACAC ATTCGAAGAACCAGCGTACGTCCGAGTTCAAATGGCACATCCCTCACAGCTAGGTTTCCAAGACGCAGCCTCACCCCTGATAGAAGA ACTACTTCATTTCCACGACCATGCGCTAATAATTGTTTTCCTAATTAGCGTCCTAGTACTTTATATTATTGTGGCAATGGTAACTGCCA AAGTTACTAACATGTTCATCCTAGATTCACAAGAGATTGAAATGTGTGAACCGTATTGCCAGCAGCAATTCTAATTCTAATCGCACTCCCCTCTCTACGGATCCTTTATCTAATAGACGAAATCAATGACCC ACATTTAACAATTAAAGCAATCGGACATCAATGATACTGAAGCTACGAGTATACTGACTATGAAGACCTTGGATTTGACTCGTACATA ATCCCAACACAAGACTTAACCCCAGGACAATTCCGACTGCTAGAAACAGACCATCGAATAGTAGTACCAATAGAATCACCTGTACGA GTATTAGTTACAGCAGAAGACGTCTTACACTCATGAGCAGTCCCATCCTTAGGGGTGAAAATGGACGCAGTCCCAGGACGCCTAAAC CAAACAGCATTTATTGCCGCCCGACCGGGAGTATATTATGGACAATGCTCTGAAATCTGCGGCGCAAACCACAGCTTTATACCAATCG TAGTTGAAGCAGTTCCTCTACAACACTTCGAAAACTGATCCTCAATAATGCTAGAAGACGCCATGATACTAAGTTTTTTCGACCAATT CATAAGCCCCACATATATAGGAATTTCTTTAATTACTTTAGCCTTAACCCTACCATGAATTCTTTATCCTACCCCAACATCCCGATGAC TAAATAACCGAATCCTAACCCTACAAAGCTGTTCGTTAACCGATTCACACAACAACTCCTTCTACCACTAAATGTTGGAGGACATAAATGAGCAGTTATACTAACATCTCTAATACTATT CCTATTAACAATAAACCTGCTAGGACTGCTCCCATACACATTTACACCAACAACCCAATTATCCCTAAATATGGGATTTGCAGTCCCA CTATGACTAGCCACCGTAATCATCGGTATACGAAATCAACCAACTGTAGCACTAGGACACCTACTGCCAGAAGGAACACCAGTCCCT CTAATCCCTGTACTTATTATCATCGAAACAATTAGCTTATTTATCCGTCCACTAGCCCTAGGTGTACGACTTACAGCAAACCTGACAGC AGGCCATCTCTTAATTCAACTCATTGCCACTGCAGTCTTTGTACTTTTACCAATAATGCCTACAGTAGCTATTCTAACAGCAACAGTAC TATTTCTTTTAACATTACTAGAAGTAGCAGTTGCTATAATCCAAGCTTACGTATTTGTACTTCTACTAAGCCTATATCTTCAAGAAAAC GTAATGCCACAGTTAAACCCCGCCCCCTGATTCACGATCCTAGTATTCTCATGGGCCGTGTTCTTAGCTATTCTCCCAACAAAAGTAAT AGCACACACGTTTAATAATGAGCCCAACCTACAAACTGCAAAAAAACCAAAACTAGACTCTTGAAACTGACCATGATACATGGCACA CCAAGCACACGCATATCACATAGTTGACCCAAGCCCATGACCCCTAACAGGCGCAGTAGCCGCCCTACTAATAACATCAGGAACAGC CATATGATTCCACTTCCAAACAACCACCCTAATAACATTAGGAATAATTCTACTTTTACTTACGATATACCAATGATGACGAGACATT GTACGAGAAGGGACTTTCCAAGGACACCATACACCGCCAGTACAAAAAGGACTACGATACGGAATAATCCTATTTATTACCTCAGAA GTATTCTTTTTCCTAGGGTTCTTCTGGCTTTTTACCATTCAAGCCTAGCTCCAACCCCAGAACTAGGGGGATGCTGACCTCCAACTGGTATTATTACTCTAGACCCATTTGAAGT ACCCCTACTAAACACAGCTGTCCTACTTGCCTCAGGCGTTACAGTCACATGAGCACACCACAGCATTATAGAAGGAGAACGAAAACA AGCCATCCAATCCCTAACCCTCACAATCATTTTAGGTTTTTATTTTACATTCCTACAAGCCATAGAATATTATGAAGCCCCATTTACCA TTGCAGATGGAGTTTATGGCTCAACATTCTTCGTGGCCACCGGATTCCACGGCCTACATGTCATTATTGGCTCTACCTTCTTAGCAGTG TGCCTCCTACGCCAAATCAAATATCACTTTACTTCAGAACATCACTTTGGATTCGAAGCCGCCGCATGATACTGACACTTCGTTGACGT AGTATGACTATTCCTATATGTCTCAATTTATTGATGAGGCTCAATGAATCCAATTATTTCAATCCTAATTATCACCACTACCCTTTCCTG CGTACTGATTACAGTCTCATTTTGACTTCCACAGATAAACCCTGACTCAGAAAAACTTTCCCCCTATGAATGCGGCTTTGACCCACTCG GATCCGCCCGACTCCCATTCTCAATACGCTTCTTTCTAGTGGCAATCCTATTCCTGCTATTTGACCTAGAAATTGCACTTCTACTCCCCC TTCCATGAGGGGACCAACTGCCTGACACTACACACGCATTCTTCTGAGCTATATCAATTATTATTCTACTAACACTAGGACTGGTATAC GAATGAATTCAAGGAGGACTAGAATGAGCTGAAATGACTCCCGTACACTTCAGCTTCACATTGGCATTTACCCTAGGGTTCTCAGGCC TAGCCTTCCACCGAAAACATTTATTATCCGCCCTCCTTTGTCTAGAAGCAATAATATTATCACTGTACATTGCTATAGCCTTATGTCTTTCCAAACAGAATCCACCGTATTCTCCTCAGCACCAATAATACTACTAGCCTTTTCCGCCTGTGAAGCCAGTGCAGGCCTAGCCCT CCTAGTAGCTACCTCACGTACACACGGCACAGATCACCTAATAAACCTTAACCTACTACAATGCATGCTAAAAGTACTAATTCCTACC ATCATGCTCATCCCCACCACTTGTTAGTAAACAAAAAATGACTATGAACCACAACTACCTACCAAAGTTTCATCATCGCCTCCATCAGCCTA--ATGATTTAA-TGGACTCAGAAATAGGATGATCTACCACAAACACTTATCTAGCAACAGACCCCCTATCAACGCCACTGCTAGTTCTATCCTGCTGCTTCTCCCATTAATAATTCTAGCAAGCCAAAACCACATGCGCCTAGAACCAATTAACCGCCAACGATCCTACATTACACTACTAATTT CCCTACAAATATTCTTAATCATAGCATTCGGAGCAACAGAAATCATTATATTCTATGTAATATTTGAAGCCACATTAATCCCAACCTTA ATTATTATTACCCGATGAGGAAACCAAACAGAACGACTCAACGCAGGAACTTACTTTCTATTCTATACACTAGCAGGCTCACTCCCAC TGCTTGTTGCACTACTTGCACTACAAAAAGACCTCGGCACTCTATCAATACTCACAATTCAATATACAAAGCCCCTCATTTTATCTTCA TGAGGTGATAAACTATGATGAGCAGGCTGCTTAATAGCATTCCTAGTAAAAATACCACTTTATGGGGTCCACTTATGACTACCAAAAG CTCATGTAGAGGCCCCAGTAGCAGGGTCCATAGTCCTAGCCGCCGTACTACTAAAACTAGGTGGATATGGAATAATACGAATAATTAT TATTTTAACCCCACTAACCAAAGAACTAGCCTATCCATTCATCATCCTCGCCCTCTGAGGAATCATTATAACCGGCTCTATCTGCTTAC GACAAACAGACCTAAAATCCATAATTGCATACTCATCAGTCAGCCACATAGGCCTAGTGGCAGGAGGAATTCTTATCCAAACCCCAT GAGGATTCACAGGAGCAATTATCCTAATAATTGCCCACGGACTAGTATCATCAGCATTATTTTGTCTAGCCAACACCAACTACGAACG AACCCATAGCCGCACCCTACTACTAGCCCGAGGCCTACAAATAATCCTTCCTTTAATAGCCGCCTGATGATTTATTGCCAACCTAGCC AACTTGGCACTCCCACCACTACCAAATCTAATAGGAGAACTGATAATCATCACATCCATGTTTAACTGTCATACTGATCTATTGCATTAACAGGCTTAGGAACCCTAATCACAGCCGGATATTCACTATACATATTCCTAATAACACAACGGGGCC CAACCCCAAATCACATCATTGGTTTAGAACCATCACATACCCGAGAACACCTACTAATTGCTATACACCTCATTCCAGTACTCCTTCTA GTACTAAAACCTGAACTAATATGAGGATGATGCTTAATGCCCTTAACAACTTTAACACTAAATTCGAGCCTTCTAATCATTCTCACGCT ACTAATTTACCCTATTATAATGACATTAAACCCAAATCCAATAAAAAAAGACTGGCTGTAACACATGTTAAAACTGCTGTTCAAATAGCATTTTTCGTAAGCCTGATCCCACTATTCCTGTTCTTAGACCAAGGAATAGAAAC AGTACTAACAAACTGCAATGAGCTAATACAATAACATTTGATTTAAATACAAGCTTCAAATTTGACCATTACTCAATTATCTTCACCCCAGTTGCCCTATACGT TACATGATCAATCCTAGAGTTCGCCTCATGATACATGCACGCAGACCCAAACATGAACCGATTCTTTAAATATTTACTTATATTCCTAG TAGCAATAATCATTCTAGTAACAGCCAACAACCTATTTCAACTATTTATTGGCTGAGAAGGTGTAGGAATTATATCATTCCTCCTAATT GGCTGATGTACGGACGGGCAGATGCAAACACCGCAGCACTCCAAGCCGTCATCTACAACCGAGTTGGTGATATTGGCCTCATCCTAGCTATAGCCT GAATAGCAATAAACCTAAACAGCTGAGAAATTCAACAAGTATTTATTATCTCAAAAGAAATAGACCTCACACTTCCTCTTATAGGACT AGTCATTGCTGCAACAGGAAAATCCGCCCAATTCGGACTACACCCATGACTACCATCAGCAATAGAAGGCCCCACACCAGTCTCTGCC CTACTGCATTCAAGCACTATGGTCGTAGCAGGAATCTTCCTATTAATCCGACTGCACCCAATAATAGAAAACAACCAAACAGTTCTAT CAACCTGCCTATGTTTAGGAGCACTAACCACGCTATTCACAGCCACTTGTGCACTAACACAAAATGATATCAAAAAAATTGTTGCATT TTCAACATCCAGCCAACTCGGCCTTATAATAGTAACTATTGGACTAAACCAACCACAACTTGCATTCATACATATTTGTACACACGCA TTCTTTAAAGCAATACTATTCCTCTGTTCAGGGTCTATCATTCACAGCCTAAATGACGAACAAGATATTCGAAAAATAGGAGGACTAC ACAAACTGCTTCCATTTACTTCATCTTGCATAACAATTGGCAGCTTAGCCCTCACAGGCACCCCATTCCTAGCAGGATTCTTCTCCAAA GACGCAATCATCGAAGCCATAAACACATCCTACCTTAACGCCTGAGCCCTTACCTTAACCTTGATCGCCACCTCATTTACAGCCGTAT ATAGCTTCCGAATTATCTTCTTTGCTTCAATAGGACAACCACGGTTTCTTCCACTCTCTCCAATTAACGAAAACAACCCTGCAGTTTTA AACCCAATCAAACGACTCGCTTGAGGAAGCATCATTGCAGGTCTAATCATTACATCAAATTTCCTACCAATAAAAACACCAATTATAA CAATACACCCCACATTAAAACTAAGCGCTCTAATAGTTACCGCTATTGGACTCTTTACCGCCATAGAGCTAGCAAACCTAACAAATAA GCAATACAAAACTAAACCATACACTAAAACACATAACTTTTCAAACATACTAGGCTACTTCCCAGCCGTAATCCACCGAATGGCCCCA AAACTAACTCTAGTACTAGGACAAAAAGTAGCCACCCAACTAGTAGATCAAACATGATTTGAAAAACTAGGACCAAAAGGAATTGTA AACATTCAACTACCAATAATCAAAATTATTAACAACCCACAACAAGGACTTATTAAAGTATACCTAGCAACATTCTTCCTAACAAATG CCCTAATTATCCTCATAATAATAATATTCATGGCAAACCTACGAAAAACCCACCCACTTCTAAAAATTGCTAACGATGCCCTAGTGGA TCTACCAACCCCATCCAATATTTCAGCATGATGAAATTTTGGCTCTCTCCTAGGATTATGCCTTATCTCACAAATCCTTACAGGACTAT TCCTAGCCATACACTATACATCAGACATCTCAACTGCCTTTTCCTCAGTAGCCCACATCTGCCGAGACGTCAACTATGGATGATTAATC CGTAACCTACATGCAAACGGAGCCTCCTTCTTCTTTATTTGCCTCTACCTCCACATTGCCCGAGGACTTTACTACGGCTCATATCTTTAC AAAGAGACATGAAACATCGGAGTCGTATTATTCCTATTAGTAATAATAACAGCATTCGTAGGATATGTACTTCCATGAGGACAAATAT CATTCTGAGGTGCTACAGTAATTACCAACCTACTATCTGCCGTCCCATACGTAGGAGACTCCCTAGTCCAATGAATCTGAGGGGGCTT CTCAGTTGACAACGCCACATTAACCCGATTCTTCGCATTCCACTTCCTATTCCCATTTGTAGTTGCCGGAGCTACAATACTTCACCTCCT ATTCCTCCATGAAACAGGATCAAACAACCCAGTAGGACTGAACTCCGACGCAGACAAAATCCCATTCCACCCATACTTCTCCTACAAA GACCTACTAGGATTCATTATTATACTCACCGCCCTAACAATACTTGCCCTATTCTACCCAAACCTCCTTGGAGACCCAGACAACTTCAC CCCTGCAAACCCAATAGTTACCCCACCACACATCAAGCCAGAATGATATTTCCTATTTGCCTACGCCATTCTACGATCAATTCCTAACA AACTCGGCGGGGTATTAGCCCTACTATCCTCTATCCTAGTCCTAATAGTAGTACCAATTCTTCACACCTCAAAACAACGAGGACTTAC CTTCCGACCAGCTTCACAACTACTATTCTGAATTTTAGTAGCAGACATACTAGTACTAACATGAATCGGAGGAATACCAGTAGAACAC CCATACATTATCATTGGCCAAGTAGCATCAGTACTTTATTTTTCCCTATTTCTGGTGCTAAACCCATTAGTAGGTGCCTAGAAAACAAAGTAATAAA- TGCATGAGTTATTTTATTTTTCTTTTTTTGGTTATATTGGTGTTGGGGTTTTTGGGTGTGGCTTCTAATCCTGCTCCTTATTTTGCTGCTTT AGGGTTGGTGTTGGCAGCTGCTGGGGGTTGTGGGGTTTTAGCAGGGTATGGTGGGTCGTTTATTTCTTTAGTACTGTTTCTTATTTATTT GGGTGGCATGTTAGTGGTATTTGCATATTCTGCTGCTCTTGCTGCTGAGCCATACCCGGAGTCATG-GGGGATGGTCTGTTTTGGGGTATGTGGTTGGTTATGTTTTTCTTTGTGTTTTAGGTATTGGTGTATTTTATGTGGAGTGTTTGATTGTTATTGTGGAATTGTTGATGAATATCGGGATTTTTCAGTGTTGCGTGGGGATTTTAGTGGGGTTTCTTTTATTTATTATTTG GGTGGTGGGATGTTAATTATTTGTGGGTGGCTTTATTGCTTACTCTTTTTGTTGTTCTTGAGTTAACTCGTGGTCGTAGTCGTGGGGCTTTGCGAGCAATT Note S2 For the endemic New Zealand eel (A. differenbachii) the spawning migration was estimated to average 3000 km. This was based on the assumption of a spawning ground situated between New Caledonia and Fiji, as proposed by larval migration route modeling (2) and further confirmed by studies employing popup tags for tracking migrating adult eels (3, 4). Estimates of age at metamorphosis were based on studies investigating increments in the Sr/Ca ratio in the otoliths. When several estimates were available the average among all studies were used. However, for the giant mottled eel (A. marmorata) that shows possible population structure (5), only estimates from the North Pacific population (including Japan, Taiwan, Philippines, Sulawesi) were used in order to match the sampling locality of the mitogenome sequence (6). Further analyses were conducted in order to assess the robustness of the results considering 1) earlier estimates of migratory distances (7) and 2) the possibility that age of metamorphosis in temperate (A. japonica, A. Anguilla, A. rostra, A. dieffenbachia and A. australis ssp.) freshwater eels, that all show large migratory loops, is underestimated. The latter could be a result of colder and less productive environments that potentially can lead to decreased growth and thereby decreased rate of otolith increments (8, 9). In total, we conducted correlation analyses (linear correlation or spearman rank; Table 3) between the observed genotype changes and four other datasets (Table 4). The first two considered earlier estimates of migratory distances by Aoyama et al. (7). They provided minimum and maximum estimates for five different species of freshwater eels; A. celebesensis, A. japonica, A. borneensis, A. anguilla and A. rostrata. Thus we replaced the average estimates presented in the paper (Table 1) for these species and reassessed the correlations. Furthermore, we calculated the average between both studies (7, 10) and used that for analyses. To assess the robustness of the results in case of an underestimation of the age of metamorphoses in temperate species, we increased the estimated ages for these species by either 20 or 40%. Correlation analyses were conducted for all above datasets and the results were overall identical with the result in the paper. The only major difference was that distance shows a significant positive correlation with number of amino acid changes at the five candidate positions when using the average distances from Aoyama et al. and Aoyama (7, 10). Overview of these datasets and results can be found in Table S3 and S4. Finally it should be mentioned that information was available for larval phase duration in thirteen species, whereas information on migratory distance was available for only eight species (Table 1, paper). Thus the higher statistical power associated with the former trait may be correlated to the larger sample size. Note 3 To evaluate whether the two examined life-history related traits also are correlated when correcting for the phylogenetic relationship, we conducted a comparative analysis of independent contrasts (CAIC) (11). This test calculates the phylogenetic independent contrast of two continues traits like the ones examined in the paper. In this way phylogenetic relatedness will not bias the result, making it possible to assess whether the two traits are truly correlated in freshwater eels. We conducted the CAIC test as implemented in the R-package ‘CAPER’ (12). We used the ‘comparative.data’ function to calculate contrasts that are depicted in Figure 1B (main paper). Subsequently these contrasts were further evaluated using the ‘crunch’ function. This function is similar to the CAIC test described in Purvis and Rambaut (11). We used the estimated trait-values from Table 1 (main paper) and used a phylogenetic tree similar to Jacobsen et al. (1) using a posterior threshold of 0.90 to determine phylogenetic relationships (Tree A below). We also analyzed the data using the original tree without taken the posterior into account (Tree B below). Both analyses showed significant linear relationships as evaluated using the ‘crunch’ function; A) adjusted R2 = 0.8802 (P=0.0003526), B) adjusted R2 = 0.9016 (P=0.0001939). Tree A with associated branch length (see text above). Names are Genbank accession numbers of the analyzed species: TreeA=(((((NC_006537:6.710798380105512,NC_006541:6.710798380105512):2.9497974363657 926,(((NC_006543:0.46856817025425235,NC_006544:0.46856817025425235):5.4583334242142 26,((NC_006545:4.5317981719257805,(NC_006534:1.45681856351162,NC_006535:1.456818563 51162):3.0749796084141607):1.0228622013721393,NC_006539:5.55466037329792):0.37224122 117055813):0.7525228432524482,NC_006540:6.679424437720926):2.9811713787503784):0.969 68494923635000,NC_002707:10.630280765707656,NC_006546:10.630280765707656):3.089719 2342924000,(((NC_006547:3.367422269745780,NC_006531:3.367422269745780):7.4421166896 416056,((NC_006532:0.3237748459895941,NC_006533:0.3237748459895941):8.8509863769305 76,NC_006538:9.174761222920171):1.6347777364672265):2.9104610406126100,NC_006536:13. 7200000000000000,NC_006542:13.7200000000000000)):38.493967059563104,((NC_013436:16. 425946884535055,NC_013627:16.425946884535055):3.793732144489759,NC_013628:20.21967 9029024814):31.994042005710387); Tree B with associated branch length (see text above). Names are Genbank accession numbers of the analyzed species: TreeB=((((((NC_006537:6.710798380105512,NC_006541:6.710798380105512):2.9497974363657 926,(((NC_006543:0.46856817025425235,NC_006544:0.46856817025425235):5.4583334242142 26,((NC_006545:4.5317981719257805,(NC_006534:1.45681856351162,NC_006535:1.456818563 51162):3.0749796084141607):1.0228622013721393,NC_006539:5.55466037329792):0.37224122 117055813):0.7525228432524482,NC_006540:6.679424437720926):2.9811713787503784):0.419 57271622032444,NC_002707:10.08016853269163):0.5501122330160264,NC_006546:10.630280 765707656):3.089473209464444,((((NC_006547:3.367422269745780,NC_006531:3.36742226974 5780):7.4421166896416056,((NC_006532:0.3237748459895941,NC_006533:0.323774845989594 1):8.850986376930576,NC_006538:9.174761222920171):1.6347777364672265):0.579456488121 5649,NC_006536:11.388995447508963):0.8425037842219041,NC_006542:12.231499231730867) :1.4882547434412334):38.493967059563104,((NC_013436:16.425946884535055,NC_013627:16. 425946884535055):3.793732144489759,NC_013628:20.219679029024814):31.994042005710387 ); Tables Table S1. Overview of the studies used for estimation of distance to spawning ground and length of larval stage. Estimates for the individual studies are also shown. Species Distance to Length of larval Individual estimates for the individual studies spawning (leptocephali) stage (days) (mean and sample size) ground Mean (km) NA Ref. - Mean (days) NA Ref. - NA - NA - A. bengalensis labiata /A. nebulosa labiata NA - 117.50 13 A. obscura NA - NA - A. interioris A. bengalensis bengalensis /A. nebulosa nebulosa 1 117.5 (N=2) A. bicolor bicolor NA - 92.60 13,14 46.20 (N=11) 139.00 (N=12) A. bicolor pacifica 1000 10 136.25 15,16,17,18 160.00 (N=15) 109.00 (N=2) 141.00 (N=15) 135.00 (N=25) A. marmorata 1000 10 130.77 14,15,16,17,18,22 143.00 (N=16) 159.00 (N=2) 128.00 (N=68) 121.50 (N=32) 122.20 (83) 112.40 (41) A. celebesensis 50 10 101.50 A. megastoma NA - NA - A. reinhardtii NA - 144.50 20 A. japonica 2500 10 134.56 21,22 A. Malgumora /A. Borneensis 100 10 133.00 16 133.00 (N=1) A. mossambica NA - 102.10 13 102.10 (N=12) A. anguilla 6000 10 350.20 23 350.20 (N=56) A. rostrata 2500 10 200.00 23 200.00 (N=125) 14,15,16,19 90.00 (N=14) 88.00 (189) 124.00 (13) 104.00 (N=43) 144.50 (N=176) 128.42 (N=131) 140.70 (N=131) A. dieffenbachii 3000 2,3,4 248.00 15 248 (N=22) A. australis australis NA - 181.59 15,16,24,25 204.00 (N=10) A. australis schmidtii NA - NA - 173.70 (N=150) 170.00 (N=45) 178.66 (N=30) (13),(14), (15),(16),(17),(18),(19),(20),(21),(22),(23),(24),(25) Table S2. Normality test results for all datasets. For datasets showing significant differences from a normal distribution, logarithmic transformations were applied and the data tested for normal distribution. Distance to Duration of Test spawning larval stage ground Mean (km) Contrasts distance Contrasts Log(larval phase) Mean (days) Mean (km) Mean (days) Synonymous Nonsynonymous dN/dS All sites 5 sites1 All codons Five codons1 Codon 522 Overall number of changes (all genes) Number of nonsynonymous changes in ATP6 Overall physio-chemical change in ATP6 Initial values N 8 13 7 7 18 18 18 18 18 18 18 18 Shapiro-Wilk W 0.9154 0.8001 0,9354 0,8832 0.8825 0.9729 0,9692 0.8969 0.8642 0.9409 0.8646 0.6617 p(normal) 0.3937 0.006834 0,5979 0,2409 0.02875 0.85 0,7822 0.05071 0.01432 0.2999 0.01451 2.958E-5 Log(10) values N 13 18 18 18 18 18 Shapiro-Wilk W 0.9118 0.8229 0.956 0.8087 0.8087 0.6294 p(normal) 0.1938 0.003262 0.5259 0.00268 0.00268 2.035E-5 1: Codon positions 43, 52, 83, 105 and 195, candidates for positive selection between Atlantic eels (1, 26) 2: Candidate for differential selection across all species of freshwater eels (1) Table S3. Linear regression analyses with associated r-corefficients and P-values. Datasets are explained under the table and further shown in Table S4. Bold values represent Spearman rank test results. Trait correlated to genotype 1 Km to spawning ground Overall number of changes (all genes) Number of nonsynonymous changes in ATP6 Overall physiochemical and structural change in ATP6 Synonymous Nonsynonymous All sites 5 sites All codons Five codons Codon 52 Distance to spawning ground (km)1 r=0.920, P=0.001 r=-0.479, P=0.233 r=-0.524, P=0.182 r=-0.476, P=0.424 r=0.752, P=0.043 r=-0.491, P=0.217 0.825 P=0.015 r=0.387, P=0.363 Distance to spawning ground (km)2 r=0.922, P=0.001 r=-0.503, P=0.0.209 r=-0.477, P=0.232 r=-0.455, P=0.258 r=0.690, P=0.072 r=-0.483, P=0.225 r=0.777, P=0.028 r=0.484, P=0.244 Length of larval (leptocephali) stage (days)3 r=0.945, P<0.001 r=-0.703, P=0.007 r=-0.381, P=0.200 r=-0.025, P=0.934 r=0.783, P=0.002 r=-0.213, P=0.484 r=0.772, P=0.002 r=0.609, P=0.027 Length of larval (leptocephali) stage (days)4 r=0.954, P<0.001 r=-0.703, P=0.007 r=-0.403, P=0.172 r=-0.052, P=0.867 r=0.783, P=0.002 r=-0.242, P=0.425 r=0.772, P=0.002 r=0.609, P=0.027 Distance to spawning ground estimated by calculating the average distances based on minimum and maximum distances provided in Aoyama et al. (7). Distances of A. biocolor and A. marmorata are provided by Aoyama (10). 2 Distance to spawning ground estimated by calculating the average distances between estimates provided by Aoyama et al. (7) and Aoyama (10). 3 Age at metamorphosis for the five temperate species A. japonica, A. anguilla, A. rostrata, A. dieffenbachii and A. australis australis are increased 20% to account for the possibility of decreased rate of otolith increments in these species, due to colder and less productive environment (8, 9). 4 Age at metamorphosis for the five temperate species A. japonica, A. anguilla, A. rostrata, A. dieffenbachii and A. australis australis are increased 40% to account for the possibility of decreased rate of otolith increments in these species, due to colder and less productive environment (8, 9). Table S4. Estimated values of either distance to the spawning ground or age of metamorphosis used for correlation analyses shown in Table S4. Numbers correspond to analyses in Table S3. Species Distance to Distance to Duration of Duration of spawning ground1 spawning ground2 larval stage3 larval stage4 Mean (km) Mean (km) Mean (days) Mean (days) A. interioris NA NA NA NA A. bengalensis bengalensis /A. nebulosa nebulosa NA NA NA NA A. bengalensis labiata /A. nebulosa labiata NA NA 117.50 117.50 A. obscura NA NA NA NA A. bicolor bicolor NA NA 92.60 92.60 A. bicolor pacifica 1000 1000 136.25 136.25 A. marmorata 1000 1000 130.77 130.77 A. celebesensis 190 120 101.50 101.50 A. megastoma NA NA NA NA A. reinhardtii NA NA 144.50 144.50 A. japonica 2750 2625 161.47 188.38 A. Malgumora /A. Borneensis 565 332.5 133.00 133.00 A. mossambica NA NA 102.10 102.10 A. anguilla 6000 5500 420.24 490.28 A. rostrata 3200 2850 240.00 280.00 A. dieffenbachii 3000 3000 297.90 347.20 A. australis australis NA NA 217.91 254.23 A. australis schmidtii NA NA NA NA T T T T T T T T T T T T T T T I I T T T T T T T T T T 29 I I I I I I I I I I I I V V I I V I I 31 Y F F F F F F F F F F Y Y Y Y Y Y Y Y 40 N N N N N N N N N S N N N N N N N N N 43 I I I I I I I I I I T V V I I T I I I 48 51 S V G I G I G I G I G I G I G V G I S V S V G V S V S V S V S V S I S V S V 52 N N N N N N N N N G N N S N G N S S S 64 V L L L L L L V V V V V V V V V I V V 71 I I I I I I I V I I I I V V V V V V V 72 M M M M M M M I L M M M M M M M M M M 81 L L I I L L L I L L L L L L L L L L L 82 83 T M T M S M S M T M T T T T T M T T T M T M T T T M T M T M T T T M T M T M 85 L L L L L L L M L L L L L L L L L L L 101 L L L L L L L M L L L L L L L L L L L 105 F F F F F F F F F F F F F F L F F F F 107 V V V V V V V V V V I V I V V V V V V 109 L L L L L L L M L M L L L L L L L L L 111 A A A A A A A A A S A A A A A A A A A 116 I I I I I I I V I I I I I I I I I I I 136 V A V V V V V V V V V V V V V V V V V 176 A A A A A A A A A A A A S A A A A A A 179 V V V V V V V A V V V V V V V V V V V 180 F F F F F F F F F F F F L F F F F F F 183 L L L L L L L L L L L L L L M M M M M 185 M M I I M M M M M I M M M M M M M M M 194 A A A A A A A S A A A A A A A A A A A 195 T T T T T T T T T T T T T T I V A M M 198 F F F F F F F L F F F F F F F F F F F HMMTOP T T MEMSAT2 T T TM1 A T TM2 A T TM3 T T TM4 T 25 TM5 21 TM6 Y M TMHMM v2 Y M TM1 Y L TM1 Y M TM2 A. australis schmidtii Y M TM3 A. dieffenbachii A. australis australis Y M TM2 A. rostrata F M TM4 A. anguilla F M TM3 A. mossambica F M TM5 A. malgumora/ A. borneensis L M TM4 A. japonica F M TM5 A. reinhardtii L M TM6 A. megastoma F M TM6 A. marmorata A. celebesensis F M GENEIOUS PRO ver 5.4.6 A. bicolor pacifica L M TM1 A. obscura A. bicolor bicolor L M TM2 A. b. labiata/A. n. labiata L M TM3 A. b. bengalensis/A. n. nebulosa L M TM5 A. interioris Y M TM6 Codon position Reconstructed Ancestral sequence 14 15 Figure S1: Figure showing the amino acid changes in ATP6 of the 18 examined species of freshwater eel compared to the reconstructed ancestral sequence shown to the left. 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