To analyze the evolutionary relationship of the newly discovered CEACAM genes, multiple nucleotide sequence alignments of Ig domain exons of human and dog CEACAM genes were performed and phylogenetic trees constructed. As expected, the N domain sequences of the orthologous genes (CEACAM16, CEACAM18-CEACAM20) clustered pairwise, while all new canine N domain exon sequences (CEACAM23-CEACAM30) clustered with that from dog CEACAM1 ( Figure 5A ). Similarly, the primate-specific CEACAM1-related N domain nucleotide sequences (CEACAM3-CEACAM8) clustered with the human CEACAM1 N domain exon sequence except for CEACAM21 N. The close relationship between the new dog CEACAM genes and dog CEACAM1 could also be demonstrated when the A and B domain exon nucleotide sequences were compared ( Figure 5B ). Nine of the 17 IgC-like domains of the new CEACAM1-related canine molecules are of the CEACAM1 A2 type and five each were of the CEACAM1 A1 and B type ( Figure  5B ).CEACAM23 appears to be the only CEACAM1-related gene which contains all extracellular domain exons present in CEACAM1, in particular three IgC-like domain exons (A1, A2 and B). All other members of this subgroup either lack one of these exons or contain exons corrupted by nonsense, frame shift or splice site mutations leading to smaller predicted proteins (Figure 3, 4) . Assuming that the N domain is the most relevant domain for ligand interaction, the ligand specificity of CEACAM28 seems to be most closely related to that of CEACAM1. The N domain amino acid sequence of CEACAM28 differs from that of CEACAM1 in only two positions. These amino acid changes do not affect the center of the CFG face, a topological N domain region of CEACAM members essential for ligand and pathogen interactions ( Figure 6A,B) . Interestingly, CEACAM28 lies next to CEACAM1 in the CEACAM1 gene cluster which hints to an evolutionary recent duplication event ( Figure 1B) . CEACAM25 is the most distant CEACAM1 relative within the group of CEACAM1-related genes, sharing only 66% of its N domain exon encoded amino acid sequence (Figure 5A, 6A; Table 2 ). Most of the amino acid sequence differences cluster to the CFG face of the N domains, indicating a positive selection for ligand diversification. To test this possibility, we calculated the ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitution rates on the branches of an N exon phylogenetic tree. The lowest Ka/ Ks ratio (0.1) is observed for CEACAM28. This indicates that the CEACAM28 N domain is under purifying selection. For all other canine CEACAM1-related CEACAMs, the N domain amino acid changes appear to evolve neu- (51) 56 (40) The trally or to be positively selected for during evolution since the corresponding Ka/Ks values are close to 1 or higher ( Figure 6C ).