Phylogenetic tree of bitter taste receptor (T2R) genes based on amino acid sequences. Genes in humans and mice are shown as solid and shaded, respectively. The tree was reconstructed by the NJ method (Saitou and Nei 1987) on the basis of the p distances, and only >50% bootstrap values are shown at each node (1000 replications). Pseudogenes in this study are defined by the presence of ORF disruptive mutations and are indicated by asterisks (*). The codon frames in pseudogenes are inferred from those of closely related functional genes. The nomenclature of human T2R genes is the same as that in Conte et al. (2002) and Shi et al. (2003). However, because of the different designation systems of the mouse genes used by two research groups (Conte et al. 2002, 2003; Shi et al. 2003), we have renamed the mouse genes. Detailed nomenclatures in humans and mice are in Table 1. The numbers in parentheses stand for those of nonsense and indel mutations, respectively. The definitions of categories A, B, and C are given in the text.

Phylogenetic tree of 305 T2R genes based on amino acid sequences in primates and mice. Genes used are from hominoids (blue), OWMs (red), NWMs (green), prosimians (yellow), tupais (gray), and mice (black). The NJ tree is reconstructed by using the p distances. The numbers in boldface type correspond to those in the locus designation system for human T2R genes. Pseudogenes are indicated by asterisks (*). Nonsense (red crosses) and indel (blue crosses) mutations responsible for pseudogenization are placed along branches. The definitions of categories A, B, and C are the same as in Figure 1.

Partial alignments of the nucleotide sequences of primate T2R genes. Dots show the same nucleotides as those in the top sequence, which were retrieved from GenBank. Both asterisks and shaded boxes show the location of mutations that impair intact ORFs. (A) T2R62 is pseudogenized by two nonsense mutations (CAG → TAG at codon position 235 and GAA → TAA at 292). (B) T2R2 is polymorphic in terms of a two-base deletion at codon position 160 in human populations. Only Adygei (from Eastern Europe), Biaka, and Mbuti Pygmies (both from Africa) possess the intact genes, while the deletion is found in all sequences collected from 10 other human populations (Karitiana, Surui, Waorani Indians from South America, Russians from Eastern Europe, Druze from the Middle East, Atayal, Chinese, Japanese from Eastern Asia, and Khmers and Melanesians from Southeast Asia) and from GenBank resources. (C) T2R64 is a human-specific pseudogene (TGG → TGA at 280). This gene also became a pseudogene in the orangutan through an independent nonsense mutation (TGG → TGA at 49). (D) Independent mutations disrupt T2R55 in the four apes: chimpanzees (five-base deletion at 172–173), gorillas (one-base insertion at 271), orangutans (one-base deletion at 303), and gibbons (two-base deletion at 103); in the prosimians: ring-tailed lemurs (two one-base deletions at 43 and 227, two-base deletion at 116, one-base insertion at 271, and two nonsense mutations at 90 and 236); and in the tupai: common tree shrews (one-base insertion at 66).

The rate of pseudogenization in humans and nonhuman anthropoidea. Each cross on a terminal branch shows species-specific pseudogenization. The divergence times (MY) represented above each branch are from Goodman et al. (1998). While three human-specific pseudogenes have arisen within 6 MY, 15 (1 + 2 + 2 + 2 + 2 + 3 + 2 + 1) pseudogenes in nonhuman anthropoidea have accumulated within 117 MY (6 + 7 + 14 + 18 + 14 + 14 + 22 + 22). Pseudogenization that occurred along internodal branches (dashed lines) is excluded from consideration.

The extent of positive and negative selection on individual codons. Schematic positions of three functional domains of the T2R gene (ECs, TMs, and ICs) are shown on the x-axis. The value above and below the x-axis, respectively, indicates an excess and deficiency in the number of per-site nonsynonymous substitutions (d_N) over per-site synonymous ones (d_S) in an individual codon. The zero value means d_N = d_S. Asterisks (*) show significant positive selection (P < 0.05) based on the extended binominal distribution (http://www.datamonkey.org/).

The b_S and b_N values of category A genes (▵) and category C genes (•) in ECs (top) and TMs + ICs (bottom). The x- and y-axis stand for b_S and b_N, respectively. The primate genes used for category A are T2R15, T2R43-44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R55, and those for category C are T2R1, T2R2, T2R3, T2R4, T2R7, T2R16, T2R38, T2R39, T2R40, T2R41, T2R56, and T2R62.