CHAPTER THIRTY Inferring Evolution of Fish Proteins: The Globin Case Study Agnes Dettaı ¨,* Guido di Prisco,* Guillaume Lecointre,* Elio Parisi,* and Cinzia Verde † Contents 1. Introduction 540 2. Hb Purification 542 2.1. Hemolysate preparation 542 2.2. Hb separation 542 3. Elucidation of Globin Primary Structure 542 3.1. Globin separation 542 3.2. Modification of a and b chains of fish hemoglobins 543 3.3. Protein cleavage 543 3.4. Deacylation of a-chain N terminus 544 4. Globin Characterization 545 4.1. Amino acid sequencing 545 4.2. Cloning and sequencing of globin cDNAs 545 5. Sequence Analysis 546 5.1. Homologous and paralogous copies 546 5.2. Sequence search in the databases 546 5.3. Sequence alignment 547 6. Phylogenetic Analysis 548 6.1. Overview of tree-building methods and topology interpretation 548 6.2. Approaching the species tree 551 6.3. Some reference species trees for Actinopterygians 552 6.4. Reconstruction of the history of a gene family 553 6.5. Reciprocal illumination 555 6.6. Reconstruction of character states at the nodes 557 6.7. Alternative reconstructions and interpretations 557 6.8. Adaptations and disadaptations 558 Methods in Enzymology, Volume 436 # 2008 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)36030-3 All rights reserved. * UMR, De ´partement Syste ´matique et Evolution, Muse ´um National d’Histoire Naturelle, Paris, France { Institute of Protein Biochemistry, CNR, Naples, Italy 539