Supporting Information

Hussain et al. 10.1073/pnas.0803229106

ORs Zebrafish Taars 862 Neoteleost Taars AmR Shark Taars 769 944 Frog Taars 999 995 Pm Mammalian Taars AmR Chicken Taars

MP 66%-100% MP 33%-66% MP 0%-33% 503 Taar20 Supported by 3 methods: NJ, MP and ML 1000 Supported by 2 methods: NJ and ML 742 919 Taar19

545

1000 997 499 Taar18 772 954 997 Taar17 589 1000 1000 1000 1000 Taar16 1000 Taar15 1000 Taar14 985 Class III

1000 Taar28

468 1000 Taar26

783

984

381 1000

266 Taar25 524

387 456 Taar24 999

346 869 Taar23 354 522 1000 1000

855

999 Taar22 900

999 990 999 Taar9

543 1000 926 Taar8

844 998

841 Taar6 918 426 1000 954 Taar7 1000 344 Class II 1000 Taar5 1000 Taar13 238 1000 347 Taar12 773

956 Taar4 400 1000 878 1000 Taar3 1000 849 Taar2

Taar21 1000 Class I 401 1000 Taar11 813 367 Taar27

429 748 Taar10 1000 443 0.05 615 Taar1

Fig. S1. Phylogenetic tree of the taar genes. The cladogram shown here corresponds to the unrooted tree in Fig. 1. The tree is constructed by using the neighbor-joining algorithm; bootstrap support at major nodes is indicated by numbers (1,000 cycles). All subfamilies are supported by all 3 tree algorithms used (neighbor joining; maximum parsimony, 100 bootstraps; maximum likelihood), except subfamilies 23 and 24 (supported by 2 methods). Red lines represent zebrafish taar genes; orange lines, neoteleost taar genes; dark blue, cartilaginous fish taar genes; green, amphibian taar genes; light-blue, mammalian taar genes; and black represents the outgroup (OR, odorant receptors; AmR, aminergic receptors; PmAmR, Petromyzon marinus (sea lamprey) aminergic receptors). Note the segregation in 3 clades, class I to III.

Hussain et al. www.pnas.org/cgi/content/short/0803229106 1of5 Class I VM C L L YS F N FG L L T A V A I L V Y F T L C I T W S M STC T DA LS S F W NS TM2 TM3 TM6 TM7

VS A YS V F MA Y Class II F I V G L L L M LT C T F SA C I I TV G LT C C L L I ST C F C FL DE L S W VY NS TM2 TM3 TM6 TM7

L Class III L I T F SS A V A I F L L L L S I T FT YF F VY LC F V I A WVV V C GQ V VL H T L V L V I S A N S I C I I T FFL S A NS

TM2 TM3 TM6 TM7

Fig. S2. Subclass-specific amino acid sequence conservation. Conservation is displayed as a sequence logo. Four motifs are shown (end of TM2, start of TM3, preceding TM6, and start of TM7, respectively) that distinguish among the 3 classes of TAARs. TM3 and TM7 contain the 2 amino acids (filled triangle) constituting the aminergic ligand motif (1). Note the absence of the motif (open triangle) in class III genes.

Hussain et al. www.pnas.org/cgi/content/short/0803229106 2of5 30

genes 20

Number of 10

0 1 2 3 4 5 6 7 8 9 10 16-20 26 11-15 21-25

Fig. S3. Correlation of phylogenetic distance with physical distance in 2 zebrafish genomic clusters. For each gene within the clusters on chromosome 10 (dark gray bars) and chromosome 20 (light gray bars), the paralog with the highest homology was determined, and its position relative to the first gene was expressed as ordinal value, e.g., a value of 1 indicates a direct neighbor (most frequent case), and a value of 2 indicates 1 additional gene situated between the gene and its closest relative. Phylogenetic neighbors outside of the cluster occur only in 2 cases.

Hussain et al. www.pnas.org/cgi/content/short/0803229106 3of5 70

60

50

40

30 % Divergence 20

10

0

Fig. S4. Maximal divergence within rodent and pufferfish subfamilies. Bars show maximal divergence between ortholog genes in rat vs. mouse and tetraodon vs. fugu comparisons. Values are based on amino acid comparisons and ordered by size. Note that even the largest value for rodent comparisons is well below the smallest value for pufferfish comparisons.

Hussain et al. www.pnas.org/cgi/content/short/0803229106 4of5 DrTAAR20t DrTAAR19l 0,4 DrTaar10 DrTAAR12f Dr ORs 5, 6, 9

0,3

0,2 Normalized Frequency

0,1

0 0 0,2 0,4 0,6 0,8 1 relative radial distance (r/r0)

Fig. S5. Radial distribution of 4 TAAR genes. Positions of cells expressing particular TAAR genes were identified in horizontal sections of olfactory epithelia in the microscope and manually marked on printouts. Relative radial distance (r/r0) of labeled cells was measured for each lamella separately as distance from the nadir of the sensory layer, closest to the median raphe, divided by the total length of the corresponding lamella. For each section, a histogram of the radial distribution was calculated for 10 equidistant bins, frequency values obtained for each bin were normalized and averaged for several sections. Values given represent mean Ϯ SEM. Thick lines, TAAR genes; thin black lines, reference curves from left to right (peak values) for OR genes zor6, zor9, and zor5, respectively (data taken from ref. 2). Note the skewness of histogram curves for TAAR12f, 19l, 20t, similar to the skewness observed for zOR6 and zOR5. Peaks for TAAR distribution are found medially and distally, similar to the proximally, medially, and distally centered distributions described for ORs.

1. Huang ES (2003) Construction of a sequence motif characteristic of aminergic G protein-coupled receptors. Protein Sci 12:1360–1367. 2. Weth F, Nadler W, Korsching S (1996) Nested expression domains for odorant receptors in zebrafish olfactory epithelium. Proc Natl Acad Sci USA 93, 13321–13326.

Other Supporting Information Files

Table S1 (PDF) Table S2 (PDF) Table S3 (PDF) Table S4 (PDF) Table S5 (PDF) Table S6 (PDF)

Hussain et al. www.pnas.org/cgi/content/short/0803229106 5of5