Evolution of Dimorphisms of the Proteasome Subunit Beta Type 8 Gene (PSMB8) in Basal Ray-Finned

Evolution of Dimorphisms of the Proteasome Subunit Beta Type 8 Gene (PSMB8) in Basal Ray-Finned Fish

Original paper

Megumi Noro, Masaru Nonaka

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan

Corresponding author: Masaru Nonaka Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: 81-3-5841-7589 Fax: 81-3-5841-4990 Email: [email protected]

Supplementary Table 1 Nucleotide sequences of the gene specific primers for RACE 5' RACE primer 3' RACE primer P. buccholzi F 5'-GACATGGTTCCCAGCAG-3' (LLGTM) 5'-TCATCTGGATCCTATGTCAG-3' (SSGSYV) P. buccholzi A 5'-CAGCAGCGCTGCCTGACATG-3' (MSGSAA) 5'-TGTGGACTCCAGAGCCTCTG-3' (VDSRAS) G. kidako F 5'-CTGCACTCTTTAGCCAGTAG-3' (LLAKEC) 5'-TGTGGACTCCAGAGCCTCTG-3' (VDSRAS) G. kidako A 5'-CTGCACTCTTTAGCCAGTAG-3' (LLAKEC) 5'-TATATTAGCACCCAATTTGC-3' (YISTQF) Thalassenchelys sp. F 5'-TCTCCTTGTTCCTCAGCTTG-3' (KLRNKE) 5'-TGAGCGGGAACATGTTCTCC-3' (SGNMFS) N. parviceps A 5'-ATCCTGCATTCCTTGGC-3' (AKECR) 5'-TGAGCGGGAACATGTTCTCC-3' (SGNMFS) C. myriaster (A/V) 5'-CGGACATTGTGCCCAGTAAG-3'(LLGTMS) 5'-GAAGATCAGGCTGAGCCATG-3' (KIRLSH) C. japonicus A 5'-CGGACATTGTGCCCAGTAAG-3'(LLGTMS) 5'-GAAGATCAGGCTGAGCCATG-3' (KIRLSH) O. macrorhynchus F 5'-ATCCTGCATTCCTTGGC-3' (AKECR) 5'-TGAGCGGGAACATGTTCTCC-3' (SGNMFS) A. japonica (Y/A) 5'-GTAAGTAGGGGTTTATCTCG-3' (EINPYL) 5'-GTTTACAAGCTGAGGAAC-3' (VYKRN) H. nipponensis F 5'-TGGCGTGAAAGATGGCT-3' (AIFHA) 5'-ATCTACAAGCTGAGGAAC-3' (IYKLRN) H. nipponensis A 5'-GGGTGGCGTGGACGATG-3' (IVHAT) 5'-CTGTACAAGCTGAGGAAC-3' (LYKLRN)

Supplementary figure legends Supplementary Figure 1 Phylogenetic trees of the PSMB8 The nucleotide sequences encoding the mature protein were aligned by Clustal X and evolutionary analysis was conducted in MEGA5.1. The phylogenetic trees were constructed by the neighbor joining (NJ) method. Bootstrap percentages were determined with 1000 bootstrap replications. * indicates the sequences newly determined in this study.

Supplementary Figure 2 Multiple alignment of the nucleotide sequences of G. kidako The nucleotide sequences of G. kidako (n = 4) were aligned separately by their types in Supplementary Figure (A) and (B). The alphabet and the numbers after the names represent their types (A- or F-) and identificationindividual numbers, respectively. Each Genotypes identifiedof each individual elucidated by cDNA typing weare shown by the right side of the namesin parentheses. The codons at the polymorphic sites weare shown by the underlines and letters withthe shading represents the codons correspond to the 31 st residue of the mature protein. Boxed letters represent the variations found by direct sequenceings. PCR amplifications were performed using type-specific primer sets. (A) Nucleotide alignment of the A-types. 96 Y represents a double peak of C and T. (B) Nucleotide alignment of the F-types. 225 R represents a double peak of A and G.

Supplementary Figure 3 Multiple alignment of the nucleotide sequences of H. nipponensis The nucleotide sequences of H. nipponensis (n = 9) caught in Lake Suwa were aligned separately by their types as described in the legend to Supplementary Figure 2. in Supplementary Figure (A) and (B). (A) Nucleotide alignment of the A-types. 386 K represents a double peak of G and T. The variations found in C 380 T and G 386 T replacements are accompanied with amino acid substitutions, Ser to Phe (S 127 F) and Gly to Val (G 129 V), respectively. (B) Nucleotide alignment of the F-types. 255 S represents a double peak of G and C. The variation found in G 2 5 5C represents a double peak of C and Gis a nonsynonymous substitution, and G 255 C replacement is accompanied with amino acid substitution , Lys to Asn (K 85 N).

Supplementary Figure 4 The evolutionary history of PSMB8 in teleosts The phylogenetic tree of ray-finned fish discussed here based on Miya et al. (2003) and Inoue et al. (2001) is schematically shown at left, and A type (31A or 31V) and F type (31F or 31Y) alleles identified in each species are shown separately for the A and F lineages at right. Symbols on the phylogenetic tree represent the evolutionary events of PSMB8 dimorphism as follows. Open circle, interallelic homogenization replacing most part of the A lineage sequences with the F lineage sequences; closed circle, interallelic homogenization replacing most part of the F lineage sequences with the A lineage sequences; open triangle, intermission of continuous interallelic homogenization; closed rectangle, loss of F type; black heavy line, continuous interallelic homogenization. The timing of intermission of interallelic homogenization was inferred from the nucleotide sequence diversity between the A- and F- type alleles. The recent interallelic homogenization inat exon 6 and intron 5 of A. japonica was shown by the vertical line inat the end of the blanch. 100 Gymnothorax kidako F* Gymnothorax kidako A* Nettastoma parviceps A* 100 69 Conger japonicus A* 59 94 Conger myriaster V* 100 Conger myriaster A* 57 Anguilla japonica Y* 99 100 Anguilla japonica A* PSMB8 F lineage Thalassenchelys sp. F* 100 99 Ophisurus macrorhynchus F* Hypomesus nipponensis F* 100 Salmo salar F 73 Oncorhynchus mykiss F 83 Pantodon buchholzi F* Danio rerio F 71 Polypterus senegals F 100 Tetraodon nigroviridis F 100 Tetraodon nigroviridis A 92 Oryzias latipes Y 95 Oryzias latipes V 57 100 Hypomesus nipponensis A* Salmo salar A PSMB8 A lineage 100 57 Oncorhynchus mykiss A 77 50 Danio rerio A Pantodon buchholzi A* Polypterus senegals A 100 Takifugu rubripes PSMB5 100 Danio rerio PSMB5 Homo sapiens PSMB5 Petromyzon marinus PSMB5

0.05 Supplementary Figure 1 Supplementary Figure1 Gymnothorax kidakoA-1 (A/F) : AC T AC A AC T C TGGCC T T C A A A T TCC AGC ACGGAGT GA TGGT GGCCGTGGAC T CC AGAGCC T C TGCCGGGT : 70 Gymnothorax kidakoA-3 (A/F) : ...... : 70 Gymnothorax kidakoA-4 (A/A) : ...... : 70

31Ala Gymnothorax kidakoA-1 (A/F) : CC T A T A T T AGC ACCC A A T T TGCC A AC A AGGTGA ACGAGA T A A A T CCGT ACC T GC TGGGC AC A A TGT C T GG: 140 Gymnothorax kidakoA-3 (A/F) : ...... : 140 Gymnothorax kidakoA-4 (A/A) : ...... Y ...... : 140

Gymnothorax kidakoA-1 (A/F) : A AGCGC T GC T GAC TGC A T C T T T TGGGA A AGGC T AC T GGC T A A AGAGTGC AGGA T A T A T GAGC TGAGA A AC : 210 Gymnothorax kidakoA-3 (A/F) : ...... : 210 Gymnothorax kidakoA-4 (A/A) : ...... : 210

Gymnothorax kidakoA-1 (A/F) : A AGGA A AGGA TC TC T GT GT CGGCCGCC TC T A AGC T GC TGGCC A AC A TGGT GGT TGGT T A T AGAGGGA T GG: 280 Gymnothorax kidakoA-3 (A/F) : ...... : 280 Gymnothorax kidakoA-4 (A/A) : ...... : 280

Gymnothorax kidakoA-1 (A/F) : GCC T T TC A A T GGGC ACC A T GGTC T GT GGC TGGGAC A AGA AGGGT CC TGGCC T GT AC T A T GT TGA TGA T T C : 350 Gymnothorax kidakoA-3 (A/F) : ...... : 350 Gymnothorax kidakoA-4 (A/A) : ...... : 350

Gymnothorax kidakoA-1 (A/F) : T GGTC TGCGGA TGTCCGGGA AC A T GT T C TCC AC T GGT TCGGGT A AC TCC T ACGCC T A T GGCGT T A T GGAC : 420 Gymnothorax kidakoA-3 (A/F) : ...... : 420 Gymnothorax kidakoA-4 (A/A) : ...... : 420

Gymnothorax kidakoA-1 (A/F) : AGTGGC T A T CGGT ACGA T C T A ACGGT CCCGGAGGC T T A TGAGC T GGCCC A A AGAGCC A T C T TCC ACGCC A : 490 Gymnothorax kidakoA-3 (A/F) : ...... : 490 Gymnothorax kidakoA-4 (A/A) : ...... : 490

Gymnothorax kidakoA-1 (A/F) : CC TGCCGAGA TGC T T AC T CCGGGGGA AC TGTC A AC A TGT ACC AC A TGCGAGA AGA TGGC T GGA TC A A AGT : 560 Gymnothorax kidakoA-3 (A/F) : ...... : 560 Gymnothorax kidakoA-4 (A/A) : ...... : 560

Gymnothorax kidakoA-1 (A/F) : GT C AC AGGA AGA TGT GGGGGA TC T T T AC A ACC A A T T CGCC A A AGA A A AGA A A : 612 Gymnothorax kidakoA-3 (A/F) : ...... : 612 Gymnothorax kidakoA-4 (A/A) : ...... : 612 Supplementary Figure 2 (A) Gymnothorax kidakoF-1 (A/F) : AC T AC A AC TC TGGCC T TC A AA T TCC AGC ACGGAGTGA TGGTGGCCGTGGAC TCC AGAGCC TCC TC AGGGT : 70 Gymnothorax kidakoF-2 (F/F) : ...... : 70 Gymnothorax kidakoF-3 (A/F) : ...... : 70

31Phe Gymnothorax kidakoF-1 (A/F) : CC T A T A T TGGC ACCC AGT T AT TC AAC A AGGTGA T TGAGA T AA ATCC AT ACC TGC TGGGC AC AA TGTC TGG : 140 Gymnothorax kidakoF-2 (F/F) : ...... : 140 Gymnothorax kidakoF-3 (A/F) : ...... : 140

Gymnothorax kidakoF-1 (A/F) : AAGTGC TGC TGAC TGTGTC T A T TGGGA AAGGC T AC TGGC T AA AGAGTGC AGGAT AT AC A AGC TGAGA AAC : 210 Gymnothorax kidakoF-2 (F/F) : ...... : 210 Gymnothorax kidakoF-3 (A/F) : ...... : 210

Gymnothorax kidakoF-1 (A/F) : AAGGAA AGGA TC TCGGTGTCGGCCGCC TC T A AGC TGC TGGCC AAC A TGGTGGT TGGT T A T AGAGGGA TGG : 280 Gymnothorax kidakoF-2 (F/F) : ...... R ...... Y ...... Y ...... : 280 Gymnothorax kidakoF-3 (A/F) : ...... : 280

Gymnothorax kidakoF-1 (A/F) : GCC T T TC A ATGGGC ACC A TGGTC TGTGGC TGGGAC AAGA AGGGTCC TGGCC TGT AC T ATGT TGATGAGTC : 350 Gymnothorax kidakoF-2 (F/F) : ...... : 350 Gymnothorax kidakoF-3 (A/F) : ...... : 350

Gymnothorax kidakoF-1 (A/F) : TGGCC AGCGGT TGACCGGGAAC A TGT TC TCC AC TGGT TCGGGT A AC TCC T ACGCC T A TGGCGT T A TGGAC : 420 Gymnothorax kidakoF-2 (F/F) : ...... : 420 Gymnothorax kidakoF-3 (A/F) : ...... : 420

Gymnothorax kidakoF-1 (A/F) : AGTGGC T A TCGGT ACGATC TGACGGTGCCGGAGGC T T ATGAGC TGGCCC A AAGAGCC ATC T TCC ACGCC A : 490 Gymnothorax kidakoF-2 (F/F) : ...... : 490 Gymnothorax kidakoF-3 (A/F) : ...... : 490

Gymnothorax kidakoF-1 (A/F) : CC TGCCGAGA TGC T T AC TCCGGGGGAAC TGTC A AC ATGT ACC AC ATGCGAGA AGATGGC TGGA T T AA AGT : 560 Gymnothorax kidakoF-2 (F/F) : ...... : 560 Gymnothorax kidakoF-3 (A/F) : ...... : 560

Gymnothorax kidakoF-1 (A/F) : C TC AC AGGAAGA TGTGGGGGACC T T T AC T ACC A AT TCGCC AA TGAA AAGA AG : 612 Gymnothorax kidakoF-2 (F/F) : ...... : 612 Gymnothorax kidakoF-3 (A/F) : ...... : 612 Supplementary Figure 2 (B) Hypomesus nipponensisA-1 (A/F) : ACC AC C ACGC T GGC T T T C A A A T T CCGCC A T GGGGT C A T C GT GGCGGT GG AC T CC AG AGCC T C AGC T GGC A : 70 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

31Ala Hypomesus nipponensisA-1 (A/F) : G T T AC A T CGCG T CC A AGGAGGC T A AC A AGG T G A T AG AG A T C A ACC C T T ACC T GC T GGGG ACC A T G T C T GG : 140 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

Hypomesus nipponensisA-1 (A/F) : C AG T GC T GC AG AC T G T C AG T AC T GGG AG AGGC T GC T GGC C A AGG AGT GC AGGC T G T AC A AGC T GAGG A AC : 210 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

Hypomesus nipponensisA-1 (A/F) : A A T C AGAGG A T C T CGG T GT C T GC T GCC T CC A AGC T GC T G T CC A AC A T G A T GC AGGGG T AC AGGGGC A T GG : 280 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

Hypomesus nipponensisA-1 (A/F) : GGC T G T CC A T GGGC AGC A T G A T C A T T GGC T GGG AC A AC A AGGGCC C T GG T C T G T AC T ACG T GGA T G AC A A : 350 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

S127F G129V Hypomesus nipponensisA-1 (A/F) : T GGG ACCCG T C T G T C T GGT CGC ACG T T C T CC AC T GGC T G T GG T AGC AGC T A T GCG T ACGGCG T GG T GG AC : 420 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... Y ...... : Hypomesus nipponensisA-6 (A/A) : ...... Y ...... : Hypomesus nipponensisA-7 (A/F) : ...... T ...... : Hypomesus nipponensisA-8 (A/A) : ...... Y . . . . . K ...... :

Hypomesus nipponensisA-1 (A/F) : AG T GG T T ACC ACG AGG AC A T G ACGG T GG AGG AGGCC T AC GAGC T GGG ACGC CGCGGC A T CG T CC ACGC C A : 490 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

Hypomesus nipponensisA-1 (A/F) : CCC AC CGGG AC GCC T AC T C T GG AGG AG T GG T C A ACC T G T ACC AC A T GC AGG AGG ACGGC T GG A T A A A AGT : 560 Hypomesus nipponensisA-3 (A/F) : ...... : Hypomesus nipponensisA-5 (A/A) : ...... : Hypomesus nipponensisA-6 (A/A) : ...... : Hypomesus nipponensisA-7 (A/F) : ...... : Hypomesus nipponensisA-8 (A/A) : ...... :

Hypomesus nipponensisA-1 (A/F) : G T G T A AGG AGG ACG T G T C AG AGC T G A T C C A T CGC T AC AGGA AGGGC A T G T T C : 612 Hypomesus nipponensisA-3 (A/F) : ...... : 612 Hypomesus nipponensisA-5 (A/A) : ...... : 612 Hypomesus nipponensisA-6 (A/A) : ...... : 612 Hypomesus nipponensisA-7 (A/F) : ...... : 612 Hypomesus nipponensisA-8 (A/A) : C ...... : 612 Supplementary Figure 3 (A) Hypomesus nipponensisF-1 (A/F) : ACC ACC AC GT T T GCC T T T A AG T T CC A AC A T GG AG T G A T GG T GGC AG T GG AC T CC AGGGCC T CC T C T GGG T : 70 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... :

31Phe Hypomesus nipponensisF-1 (A/F) : CC T A T G T C AGC AC T C AG A T G T T C A AG A A AG T G A T AG AG A T T A ACCC C T ACC T GC T T GGG ACC A T G T C AGG : 140 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... :

Hypomesus nipponensisF-1 (A/F) : A AG T GC T GC T G A T T GC T GC T AC T GGG AGAGGG T CC T GGCC A A AG AG T GC AGG A T C T AC A AGC T G AGG A AC : 210 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... :

K85N Hypomesus nipponensisF-1 (A/F) : A AGGAGCG A A T C T C T G T GT C T GC T GCC T CC A AGC T GC T GGC T A AG A T GG T GG T GGGC T AC AGGGGC A T GG : 280 Hypomesus nipponensisF-2 (F/F) : ...... S ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... S ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... S ...... :

Hypomesus nipponensisF-1 (A/F) : GGC T G T CC A T GGGC ACC A T GG T C T G T GGC T GGG AC A AG A AGGGCCC T GGGC T G T AC T A T G T AG A T GA A A A : 350 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... :

Hypomesus nipponensisF-1 (A/F) : CGG A ACG AGAC T G AG T GGG A AC A T G T T C T CC ACCGGGT CGGGC AG T T C T T ACGCC T ACGG AG T G A T GG AC : 420 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... :

Hypomesus nipponensisF-1 (A/F) : AGC GGC T ACC AC T ACG ACC T G AGT G T C CCCG AGGC T T ACG ACC T GGCGC AG AG AGCG A T C T T T C ACGCC A : 490 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... S ...... :

Hypomesus nipponensisF-1 (A/F) : C AC A T AG AGACGC C T A T T CCGG T GGC AC AG T C A AC A T G T AC C AC A T GAG AG AG AG T GGC T GG A T C A AGG T : 560 Hypomesus nipponensisF-2 (F/F) : ...... : Hypomesus nipponensisF-3 (A/F) : ...... : Hypomesus nipponensisF-4 (F/F) : ...... G ...... : Hypomesus nipponensisF-7 (A/F) : ...... : Hypomesus nipponensisF-9 (F/F) : ...... :

Hypomesus nipponensisF-1 (A/F) : G T C T C AGG AGG A T G T GGGAG AC C T G T A T T A T C GC T T CGCC A A T G A A A AG A AG : 612 Hypomesus nipponensisF-2 (F/F) : ...... : 612 Hypomesus nipponensisF-3 (A/F) : ...... : 612 Hypomesus nipponensisF-4 (F/F) : ...... : 612 Hypomesus nipponensisF-7 (A/F) : ...... : 612 Hypomesus nipponensisF-9 (F/F) : ...... : 612 Supplementary Figure 3 (B) A lineage F lineage Polypterus senegalus* Polypteriformes 31A 31F

Pantodon buchholzi* Osteoglossiformes 31A 31F

Gymnothorax kidako* - 31F, 31A

Conger myriaster* - 31A, 31V Anguilliformes Conger japonicus* - 31A

Anguilla japonica* - 31Y, 31A

Danio rerio Cypriniformes 31A 31F Oncorhynchus mykiss 31A 31F Salmoniformes Salmo salar 31A 31F

Hypomesus nipponensis* Osmeriformes 31A 31F Zenopsis nebulosa* Zeiformes 31A - Myripristis kuntee* Beryciformes 31A - Oryzias latipes Beloniformes 31V, 31Y -

Tetraodon nigroviridis Tetraodontiformes 31A, 31F -

Supplementary Figure 4