Phylogenetic Relationship of Birds with Crocodiles and Mammals, as ...

Letter to the Editor Phylogenetic Relationship of Birds with Crocodiles and Mammals, as Deduced from Protein Sequences’ Dan LarhammaP and Robert J. Milnee *Department of Medical Genetics, Uppsala University, Sweden; and tDepartment of Neuropharmacology, Research Institute of Scripps Clinic

The traditional fossil-based view that birds are more closely related to crocodiles than to mammals has been challenged by cladists who claim that birds share more derived characters with mammals (Gardiner 1982; Lovtrup 1985). Recently, much of the cladistic evidence has been criticized (Kemp 1988)) as reviewed in a “News and Views” commentary in Nature (Gee 1988). The protein-sequence information analyzed in the above articles included four proteins, i.e., a-crystallin A, myoglobin, hemoglobin alpha, and hemoglobin beta (see of birds with mammals fig. 1A- 1D, table 1) . Myoglobin supports a closer relationship than with crocodiles (Dene et al. 1982). a-Crystallin A (de Jong et al. 1985) and hemoglobin alpha (Perutz et al. 198 1) favor a closer chick-alligator relationship. The sequence relationships of hemoglobin beta have been more difficult to interpret [see table 1 and Goodman et al. ( 1982)]. Two additional proteins are available for sequence comparisons, namely, the peptide hormones pancreatic polypeptide (PP) and insulin. Figure 1E shows a comparison of the PP sequences of chicken (Gallus gallus) (Lance et al. 1982), American alligator (Alligator mississippiensis) ( Lance et al. 1982 ), and pig (Sus scrofa) (Chance et al. 1979). The insulin sequences are shown in figure IF. The branch lengths for the three different sequences of each protein were calculated by adding the number of positions at which a sequence has a unique amino acid (where the other two sequences are identical) to two-thirds of the number of positions where all three sequences are different. This gave for alligator, PP 1 + (2 / 3) X 3 = 3; for chicken, PP 3 + (2/3) X 3 = 5; and for pig, PP 15 + (2/3) X 3 = 17 (see table 1). The PP branch lengths suggest that the root is on the porcine-and therefore the mammalian-lineage, making birds and alligators sister taxa. To phrase the matter differently, chicken and alligator are identical at 15 positions that differ in the pig, whereas chicken and pig are identical at only one position where the alligator differs (position 36). Alligator and pig have three identical residues that differ in the chicken (positions 3, 19, and 34). In total, chicken and pig differ at 2 1 positions and alligator and pig differ at 18 positions, whereas chicken and alligator differ at only seven positions. By means of the same procedure, branch lengths were calculated for the other proteins, as summarized in table 1. The total branch lengths for the six proteins are 114.3 for the alligator sequences, 73.3 for the chick sequences, and 13 1.3 for the porcine sequences. This suggests that the root may be on the mammalian branch and that alligator and chicken are sister taxa. The previously published sequence comparisons were based on a-crystallin A, myoglobin, hemoglobin alpha, and hemoglobin beta. The latter three proteins are evolutionarily related to each other and share biochemical properties such as oxygen binding. Therefore, if in the alligator and mammalian lineages there has been con1. Key words: birds, crocodiles, mammals, phylogeny. Address for correspondence and reprints: Dr. Dan Larhammar, Department of Medical Genetics, Uppsala University, Box 589, S-751 23 Uppsala, Sweden. Mol. Biol. Evol. 6(6):693-696. 1989. 0 1989 by The University of Chicago. All rights reserved. 0737-4038/89/0606-0009$02.00

693

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FIG. I.-Alignment of protein sequences. A. = a-crystallin A; B. = hemoglobin alpha; C. = hemoglobin beta; D. = myoglobin; E. = PP; F. = insulin. Only positions that differ are shown. Dots show amino acid residues that are either unique to one of the three sequences or, on the bottom line of each panel, are different in all three sequences. The numbers of positions of each type are shown in the right-hand margin of each sequence. The pig sequence was chosen as a representative for the mammalian sequences. For PP, the pig sequence is identical to the consensus sequence of the nine mammalian sequences known. Sequences were obtained from the National Biomedical Research Foundation Protein Identification Resource data bank for protein sequences, version 20.0 ( 1989), except for the alligator sequences of PP and insulin, which are from Lance et al. (1982).

696

Letter to the Editor

Table 1 Branch Lengths of Six Proteins in Alligator, Chicken, and Pig No. OFUNIQUEPOSITIONS No. (%) TOTAL OF No. OF VARIABLE All POSITIONS POSITIONS Alligator Chick Pig Three

PROTEIN Pancreaticpolypeptide Insulin a-CrystallinA. Hemoglobinalpha Hemoglobinbeta Myoglobin . Total

36 51 173 141 146 154 iG

, . .

22 (61) 10 (20) 36 (21) 60 (43) 78 (53) *(39) 266

1 4 8 13 29 24 79

3 0 6 12 9 8 G

15

3

6

0

19 20 22 14 96

3 15 18 14 53

BRANCHLENGTH

Alligator Chick 3 4 10 23 41 33 3 ;114.3

5 0 8 22 21 17 3 ;d 73.3

Pig 17 6 21 30 34 23 3 131.3

vergent evolution influencing oxygen metabolism, it is possible that similar changes could have occurred in all three of these proteins. If so, the above values support even more strongly a closer evolutionary relationship between birds and crocodiles than between birds and mammals. Acknowledgments We thank

Dr. Ulf Gyllensten

for fruitful

discussions.

LITERATURE CITED CHANCE, R. E., M. G. JOHNSON,J. A. HOFFMANN, and T.-M. LIN. 1979. Pp. 4 19-425 in S. BABA,T. KANEKO,and N. NANAIHARA, eds. Proinsulin, insulin, C-peptide. Excerpta Medica, Amsterdam. DE JONG, W. W., A. ZWEERS, M. VERSTEEG,H. C. DESSAUER,and M. GOODMAN. 1985. aCrystallin A sequences of Alligator mississippiensis and the lizard Tupinambis teguixin: molecular evolution and reptilian phylogeny. Mol. Biol. Evol. 2:484-493. DENE, H., J. SAZY,M. GOODMAN,and A. E. ROMERO-HERRERA.1982. The amino acid sequence of alligator (Alligator mississippiensis) myoglobin. Biochim. Biophys. Acta 624:397-408. GARDINER, B. G. 1982. Tetrapod classification. Zool. J. Linnaean Sot. 74:207-232. GEE, H. 1988. Evolution: friends and relations. Nature 334: 13- 14. GOODMAN,M., A. E. ROMERO-HERRERA,H. DENE, J. CZELUSNIAK,and R. E. TASHIAN. 1982. Amino acid sequence evidence on the phylogeny of primates and other eutherians. Pp. 11519 1 in M. GOODMAN, ed. Macromolecular sequences in systematic and evolutionary biology. Plenum, New York. KEMP, T. S. 1988. Haemothermia or Archosauria? the interrelationships of mammals, birds and crocodiles. Zool. J. Linnaean Sot. 92:67- 104. LANCE, V., J. W. HAMILTON,J. B. ROUSE, J. R. KIMMEL, and H. G. POLLOCK. 1982. Isolation and characterization of reptilian insulin, glucagon, and pancreatic polypeptide: complete amino acid sequence of alligator (Alligator mississippiensis) insulin and pancreatic polypeptide. Gen. Comp. Endocrinol. 55: 112- 124. L&TRUP, S. 1985. On the classification of the taxon Tetrapoda. Syst. Zool. 34:463-470. PERUTZ, M. F., C. BAUER, G. GROS, F. LECLERCQ,C. VANDECASSERIE,A. G. SCHNEK, G. BRAUNITZER,A. E. FRIDAY, and K. A. JOYSEY . 198 1. Allosteric regulation of crocodilian haemoglobin. Nature 291:682-684. WALTER M. FITCH, reviewing Received

February

Accepted

July 19, 1989

editor

6, 1989; revision

received

July 5, 1989