Appendix 2 Alignment of Nucleotide Sequences of a Portion of the Α-Amylase Gene (1188 Bp)

Appendix 2 Alignment of nucleotide sequences of a portion of the α-amylase gene (1188 bp) from the four prickleback species Xiphistermucosus,Xiphisteratropurpureus,Cebidichthysviolaceus, and Anoplarchuspurpurescens, and from Pseudopleuronectesamericanus, which was used as a probe for the in situ hybridization assays (see “Materials and Methods”). Sequences were aligned using ClustalW multiple alignment software and ranked in descending pairwise alignment scores. Asterisks indicate identical residues among all species.

Xiphistermucosus ---AGATCTCCCCTCCTAATGAACACATTGTGCTGAATGATCCCTGGAGG 47

Xiphisteratropurpureus ---AGATCTCCCCTCCTAATGAACACATTGTGCTGAATGATCCCTGGAGG 47

Cebidichthysviolaceus ---AGATCTCCCCTCCAAATGAGCACATTGTGCTGGATAATCCCTGGAGG 47

Anoplarchuspurpurescens ---AGATCTCCCCTCCAAATGAGCACATTGTGCTGGACAATCCCTGGAGG 47

PseudopleuronectesamericanusTTCAGATCTCCCCCCCGAACGAGCACATCATGCTGGACAACCCCTGGAGG 50

********** ** ** ** ***** ***** * * *********

Xiphistermucosus CCCTGGTGGCAGAGATACCAACCTGTCGGCTACAACCTTTGCTCCAGATC 97

Xiphisteratropurpureus CCCTGGTGGCAGAGATATCAACCAGTCGGCTACAACCTTTGCTCCAGATC 97

Cebidichthysviolaceus CCCTGGTGGCAGAGATACCAACCAGTCGGCTACAACCTGTGCTCCAGATC 97

Anoplarchuspurpurescens CCCTGGTGGCAGAGATACCAACCAGTCGGCTACAACCTGTGCTCCAGATC 97

PseudopleuronectesamericanusCCCTGGTGGATGAGGTACCAGCCAATCAGCTACAACCTGTGCTCCCGATC 100

********* *** ** ** ** ** ********** ****** ****

Xiphistermucosus AGGCAATGAGAACGAGCTGAGAGACATGATCACCAGATGCAACAACGTTG 147

Xiphisteratropurpureus AGGCAATGAGAACGAGCTGAGAGACATGATCACCAGATGCAACAACGTTG 147

Cebidichthysviolaceus AGGCAATGAGAACGAGCTGAGAGACATGATCACCAGATGCAACAACGTTG 147

Anoplarchuspurpurescens AGGCACTGAGAACGAGCTGAGAGACATGATCACCAGATGCAACAACGTTG 147

PseudopleuronectesamericanusCGGCAGCGAGGACGAGCTGAGAGACATGATCACCAGATGCAACAACGTTG 150

**** *** ***************************************

Xiphistermucosus GGGTCAATATCTATGTGGACGCTATCATCAACCACATGTGCGGCGCCGGC 197

Xiphisteratropurpureus GGGTCAATATCTATGTGGACGCTATCATCAACCACATGTGCGGCGCCGGC 197

Cebidichthysviolaceus GGGTCAATATTTACGTGGACGCTATCATTAACCATATGTGCGGCTCCGGC 197

Anoplarchuspurpurescens GGGTCAATATCTATGTGGACGCTATCATCAACCACATGTGCGGCGCCGGC 197

PseudopleuronectesamericanusGGGTCAACATCTACGTGGACGCGGTCATCAACCACATGTGTGGAGCCAGC 200

******* ** ** ******** **** ***** ***** ** ** **

Xiphistermucosus GGTGGAGAGGGAACCCACTCTTCATGTGGGAGCTGGTTCAGTGCTGGCAG 247

Xiphisteratropurpureus GGTGGAGAGGGAACCCACTCTTCATGTGGGAGCTGGTTCAGTGCTGGCAG 247

Cebidichthysviolaceus GGTGGAGAGGGAACCCATTCTTCATGTGGAAGCTGGTTCAGTGCTGGCAG 247

Anoplarchuspurpurescens GGTGGAGAGGGAACCCACTCTTCATGTGGAAGCTGGTTCAGTGCTGGCAG 247

PseudopleuronectesamericanusGGTGGAGAAGGAACACACTCCTCATGTGGAAGCTGGTTCGACGCCGGCAA 250

******** ***** ** ** ******** ********* ** ****

Xiphistermucosus GAAGGACTTCCCCAGTATCCCGTTTACCCATTTGGACTTTAATGACCACA 297

Xiphisteratropurpureus GAAGGACTTCCCCAGTATCCCGTTTACCCACTTGGACTTTAATGACCACA 297

Cebidichthysviolaceus GAAGGACTTCCCCAGTATCCCCTTTACCCAGTCGGACTTCAATGACCACA 297

Anoplarchuspurpurescens GAAGGACTTCCCCAGTATCCCCTTTACCCAGTTGGACTTCAATGACCACA 297

PseudopleuronectesamericanusGAAGGACTTTCCCAGCGTCCCCTTCTCCAACTGCGACTTCAACGACAACA 300

********* ***** **** ** ** * * ***** ** *** ***

Xiphistermucosus AATGCAGGACTGGCAGTGGCAATATTGAGAACTATGGTGATGCCAATCAG 347

Xiphisteratropurpureus AATGCAGGACTGGCAGTGGCAATATTGAGAACTATGGTGATGCCAATCAG 347

Cebidichthysviolaceus AATGCAGGACTGGCAGTGGCAATATTGAGAACTATGGTGATGCCAGTCAG 347

Anoplarchuspurpurescens AATGCAGAACTGCCAGTGGCAATATTGAGAGCTATAGTGATGCCAATCAG 347

Pseudopleuronectesamericanus AATGCAGGACAGGCAGTGGAAACATTGAGAACTACGGTGATGCTAATCAG 350

******* ** * ****** ** ******* *** ******* * ****

Xiphistermucosus GTGCGTGACTGTCGTCTGGTGGGTCTGTTGGACCTCGCCTTGGAGAAAGA 397

Xiphisteratropurpureus GTGCGTGACTGTCGTCTGGTGGGTCTGTTGGACCTCGCCTTGGAGAAAGA 397

Cebidichthysviolaceus GTGCGTGACTGTCGTCTGGTGGGTCTGTTGGACCTCGCCTTGGAGAAAGA 397

Anoplarchuspurpurescens GTGCGTGACTGTCGTCTGGTGAGTCTGTTGGACCTCAACTTGGAGAAAGA 397

Pseudopleuronectesamericanus GTGCGTGACTGTCGTCTGGTCAGCCTGCTGGACCTCGCCCTGGAGAAGGA 400

******************** * *** ******** * ******* **

Xiphistermucosus CTATGTCAGGGGCAAGGTGGCCGACTTCATGAACAAGCTGATTGACATGG 447

Xiphisteratropurpureus TTATGTCAGGGGCAAGGTGGCCGACTTCATGAACAAGCTGATTGACATGG 447

Cebidichthysviolaceus TTATGTCAGGGGCAAGGTGGCCAACTTCATGAACAAGCTGATTGACATGG 447

Anoplarchuspurpurescens TTACGTCAGGGGCAAGGTGGCCGACTTCCTGAACGAGCTGGTTGACATGG 447

Pseudopleuronectesamericanus TTACGTCCGGGGGAAGGTGACCGACTTCCTGAACGTCCTGATCGACATGG 450

** *** **** ****** ** ***** ***** *** * *******

Xiphistermucosus GTGTGGCTGGATTCAGAGTGGATGCCTGCAAGCACATGTGGCCAGGTGAC 497

Xiphisteratropurpureus GTGTGGCTGGATTCAGAGTGGATGCCTGCAAGCACATGTGGCCAGGTGAC 497

Cebidichthysviolaceus GTGTGGCTGGATTCAGAGTGGATGCCTGCAAGCACATGTGGCCCGGTGAC 497

Anoplarchuspurpurescens GTGTGGCTGGATTCAGAGTGGATGCCTGCAAGCACATGTGGCCAGGTGAC 497

Pseudopleuronectesamericanus GTGTGGCTGGATTCCGAGTGGACGCCTGCAAGCACATGTGGCCCGGCGAC 500

************** ******* ******************** ** ***

Xiphistermucosus CTGTCTGCTATCTACGGTCGTCTGCACAACCTCAACACCAAGTGGTTCAA 547

Xiphisteratropurpureus CTGTCTGCTATCTACGGTCGTCTGCACAACCTCAACACCAAGTGGTTCAA 547

Cebidichthysviolaceus CTGTCTGCTATCTACGGTCGTCTGCACAACCTCAACACCAAGTGGTTCAA 547

Anoplarchuspurpurescens CTGTCTGCTATCTACGGTCGTCTGCACAACCTCAACACCAAGTGGTTCAA 547

Pseudopleuronectesamericanus CTGGCTGCCGTGTACGGGGGTCTGCACAACCTCAACACCAAGTGGTGGTC 550

*** **** * ***** ***************************

Xiphistermucosus TGGTGCCTCCAGACCCTTCATCTTCCAGGAGGTTATTGATTTGGGAGGTG 597

Xiphisteratropurpureus TGGTGGCTCCAGACCCTTCATCTTCCAGGAGGTTATTGATTTGGGAGGTG 597

Cebidichthysviolaceus TGGTGGCTCCAGACCCTTTATCTTCCAGGAGGTTATTGATTTGGGAGGTG 597

Anoplarchuspurpurescens TGGTGGCTCCAGACCCTTCATCTTCCAGGAGGTTATTGATTTGGGAGGTG 597

Pseudopleuronectesamericanus CGCCGGCTCCAGACCCTTCATCTTCCAGGAGGTTATTGATCTGGGAGGTG 600

* * ************ ********************* *********

Xiphistermucosus AGCCCATCACATCTAGAGAGTACTCCCATCTGGGAAGGGTGACTGAGTTC 647

Xiphisteratropurpureus AGCCCATCACATCTAGAGAGTACTCCCATCTGGGAAGGGTGACTGAGTTC 647

Cebidichthysviolaceus AGCCCATCACATCTAAAGAGTACACACATCTGGGAAGGGTGACTGAGTTC 647

Anoplarchuspurpurescens AGGGCATCACATCTGGAGAGTACTCCCATCTGGGAAGGGTGACTGAGTTC 647

Pseudopleuronectesamericanus AGTCCATCACAACTAAGGAGTACGTCAATCTGGGAAGAGTGACCGAGTTC 650

** ******* ** ****** ********** ***** ******

Xiphistermucosus AAATATGGTGCCAAACTGGGAGGTATCTTCAGAAAGTGGAATGGCGAGAA 697

Xiphisteratropurpureus AAATATGGTGCCAAACTGGGAGGTATCTTCAGAAAGTGGAATGGCGAGAA 697

Cebidichthysviolaceus AAATATGGTGCCAAACTGGGAACTATCTTCAGAAAGTGGAATGGCGAGAA 697

Anoplarchuspurpurescens AAATATGGTGCCAAACTGGGAACTGTCTTCAGAAAGTGGAATGGCGAGAA 697

Pseudopleuronectesamericanus AAATATGGAGCCAAACTGGGAACTGTCTTCAGAAAGTGGAACGGCGAGAA 700

******** ************ * **************** ********

Xiphistermucosus GCTGTCTTACACCAAAAACTGGGGAGAGGGATGGGGTTTCATGCCCAACG 747

Xiphisteratropurpureus GCTGTCTTACACCAAAAACTGGGGAGAGGGATGGGGTTTCATGCCCAACG 747

Cebidichthysviolaceus GCTGTCTTACACCAAGAACTGGGGAGAGGGATGGGGTTTCATGCCCAACG 747

Anoplarchuspurpurescens GCTGTCTTACACCAAGAACTGGGGAGAGGGATGGGGATTCATGCCCAACG 747

Pseudopleuronectesamericanus GCTGTGCTACACCAGGAACTGGGGAGAGGGCTGGGGTTTCATGTCCGATG 750

***** ******* ************** ***** ****** ** * *

Xiphistermucosus GCGATGCTCTCGTCTTCGTTGACAACCACGACAACCAGAGAGGCCATGGT 797

Xiphisteratropurpureus GCGATGCTCTCGTCTTCGTTGACAACCACGACAACCAGAGAGGCCATGGT 797

Cebidichthysviolaceus GCAATGCTCTCGTCTTCGTTGACAACCACGACAACCAGAGAGGCCATGGT 797

Anoplarchuspurpurescens GCAATGCTCTCGTCTTCGTTGACAACCACGACAACCAGAGAGGCCATGGT 797

Pseudopleuronectesamericanus GAAACGCTGTCGTCTTCGTTGACAACCACGACAACCAGAGGGGCCACGGT 800

* * *** ******************************* ***** ***

Xiphistermucosus GCTGGTGGTGCATCCATCGTTTCCTTCTGGGACTCCAAGCTGCACAAGAT 847

Xiphisteratropurpureus GCTGGTGGTGCATCCATCGTTTCCTTCTGGGACTCCAAGCTGCACAAGAT 847

Cebidichthysviolaceus GCTGGTGGTGCCTCCATCGTTTCCTTCTGGGACTCCAAGCTGCACAAGAT 847

Anoplarchuspurpurescens GCTGGTGGTGCATCCATCGTTTCCTTCTGGGACTCCAAGCTGCACAAGAT 847

Pseudopleuronectesamericanus GCCGGGGGCGCTGCCATCGTCACCTTCTGGGACCCCAGGCCACACAAGAT 850

** ** ** ** ******* *********** *** ** ********

Xiphistermucosus GGCTGTCAGCTACATGCTGGCCCACCCTTACGGAGTAACCAGGGTGATGT 897

Xiphisteratropurpureus GGCTGTCAGCTACATGCTGGCCCACCCTTACGGAGTAACCAGGGTGATGT 897

Cebidichthysviolaceus GGCTGTCAGCTACATGCTGGCCCACCCTTACGGAGTAACCAGGGTGATGT 897

Anoplarchuspurpurescens GGCTGTCGGCTACATGCTGGCCCACCCTTACGGAGTAACCAGGGTGATGT 897

Pseudopleuronectesamericanus GGCCGTAGCCTACATGCTGGCTCATCCATATGGAGTGACCAGAGTGATGT 900

*** ** ************ ** ** ** ***** ***** *******

Xiphistermucosus CTAGCTTCCGCTGGAATCGCCAAATTGTCAATGGAAAGGATCAGAATGAC 947

Xiphisteratropurpureus CTAGCTTCAGCTGGAATCGCCAAATTGTCAATGGAAAGGATCAGAATGAC 947

Cebidichthysviolaceus CTAGCTTCCGCTGGAATCGCAACATTGTCAATGGAAAGGATCAGAATGAC 947

Anoplarchuspurpurescens CTAGCTTCCGCTGGAATCGCTACATTGTCGATGGAAAGGATCAGAATGAC 947

Pseudopleuronectesamericanus CGAGCTTCCGCTGGAACCGACACATTGTGAACGGAAAGGATCAGAATGAC 950

* ****** ******* ** * ***** * ******************

Xiphistermucosus TGGATGGGCCCTCCCAGCCATGGTGATGGATCCACTAAGTCTGTTCCCAT 997

Xiphisteratropurpureus TGGATGGGCCCTCCCAGCCATGGGGATGGATCCACTAAGTCTGTTCCTAT 997

Cebidichthysviolaceus TGGATGGGCCCTCCCAGCCATGGTGATGGATCCACTAAGTCCGTTCCCAT 997

Anoplarchuspurpurescens TGGATGGGCCCTCCAAGCCATGGTGATGGATCCACTAAGTCTGTTCCCAT 997

Pseudopleuronectesamericanus TGGATGGGACCTCCCAGCCACAGTGATGGATCCACCAAGTCGGTTCCCAT 1000

******** ***** ***** * *********** ***** ***** **

Xiphistermucosus CAACGCCGACCAGACTTGTGGAGATGGATGGGTGTGTGAGCACAGATGGC 1047

Xiphisteratropurpureus CAACGCCGACCAGACTTGTGGAGATGGATGGGTGTGTGAGCACAGATGGC 1047

Cebidichthysviolaceus CAAAGCTGACCAGACTTGTGGAGATGAATGGGTGTGTGAGCACAGATGGC 1047

Anoplarchuspurpurescens CAACGCTGACCAGACTTGTGGAGATGGATGGGTGTGTGAGCACAGATGGC 1047

Pseudopleuronectesamericanus CAACCCCGATCAGACCTGTGGAGACGGATGGGTGTGTGAGCACAGATGGC 1050

*** * ** ***** ******** * ***********************

Xiphistermucosus ATCAGATCAAGAACATGATTATTTTCCGCAATGTGGTCAATGGACAGCCT 1097

Xiphisteratropurpureus GTCAGATCAAGAACATGGTTATTTTCCGCAATGTGGTCAATGGACAGCCT 1097

Cebidichthysviolaceus GTCAGATCACGAACATGGTTATTTTCCGCAATGTGGTCAATGGACAGCCT 1097

Anoplarchuspurpurescens GTCAGATCACGAACATGGTTATTTTCCGCAATGTGGTCAGTGGACAGCCT 1097

Pseudopleuronectesamericanus GTCAGATCACCAACATGGTGAAGTTCCGTAACGTGGTCGACGGACAGCCT 1100

******** ****** * * ***** ** ****** *********

Xiphistermucosus CATTCCAACTGGTGGGACAACAAGAGCAACCAGGTTGCTTTTGGACGTGG 1147

Xiphisteratropurpureus CATTCCAACTGGTGGGACAACCAGAGCAACCAGGTTGCCTTTGGACGTGG 1147

Cebidichthysviolaceus CATTCCAACTGGTGGGACAACCAGAGCAACCAGGTTGCCTTTGGACGTGG 1147

Anoplarchuspurpurescens CATTCCAACTGGTGGGACAACCAGAGCAACCAGGTTGCCTTTGGACGTGG 1147

Pseudopleuronectesamericanus CACTCCAACTGGTGGGACAACCAGAGCAACCAGGTCGCCTTCGGACGTGG 1150

** ****************** ************* ** ** ********

Xiphistermucosus TAATCGTGGTTTCATCGTCTTCAACAATGATGACTGGAACT--- 1188

Xiphisteratropurpureus TAATCGTGGTTTCATCGTCTTCAACAATGATGACTGGAACT--- 1188

Cebidichthysviolaceus TAATCGTGGTTTCATCGTCTTCAACAATGATGACTGGAACC--- 1188

Anoplarchuspurpurescens TAATCGTGGTTTCATCGTCTTCAACAATGATGACTGGAACC--- 1188

Pseudopleuronectesamericanus AAACCGTGGATTCATCGTCTTTAACAATGACGACTGGGACCTGG 1194

** ***** *********** ******** ****** **