Protein accession number Gene accession number ENSGALP00000034837 ENSGALG00000022066 ENSGALP00000034929 ENSGALG00000022152 ENSGALP00000035229 ENSGALG00000022422 ENSGALP00000034919 ENSGALG00000022147 ENSGALP00000028102 ENSGALG00000022627 ENSGALP00000034729 ENSGALG00000021959 ENSGALP00000034962 ENSGALG00000022179 ENSGALP00000034780 ENSGALG00000022004 ENSGALP00000028108 ENSGALG00000022444 ENSGALP00000034818 ENSGALG00000022040 ENSGALP00000034941 ENSGALG00000022164 ENSGALP00000035408 ENSGALG00000022608 ENSGALP00000019806 ENSGALG00000012141 ENSGALP00000028197 ENSGALG00000021958 ENSGALP00000035315 ENSGALG00000022513 ENSGALP00000035394 ENSGALG00000022593 ENSGALP00000035207 ENSGALG00000022399 ENSGALP00000035396 ENSGALG00000022595 ENSGALP00000015212 ENSGALG00000009351 ENSGALP00000028377 ENSGALG00000022560 ENSGALP00000035368 ENSGALG00000022568 ENSGALP00000034731 ENSGALG00000021961 ENSGALP00000019013 ENSGALG00000011647 ENSGALP00000034808 ENSGALG00000022027 ENSGALP00000035169 ENSGALG00000022360 ENSGALP00000035053 ENSGALG00000022260

Supplemental data 1. Correspondence between Ensembl genes and proteins accession numbers

Supplemental Data 2. Chicken FFAR2 expression in embryonic tissue, liver and adipose tissue using RNA-Seq data.

Ensembl gene ID Ensembl transcript ID Embryos Liver Adipose tissue

ENSGALG00000009351 ENSGALT00000015228 718 1 2

ENSGALG00000011647 ENSGALT00000019036 1 8 29

ENSGALG00000012141 ENSGALT00000019833 1 3 16

ENSGALG00000022027 ENSGALT00000035577 13 4 34

ENSGALG00000022066 ENSGALT00000035607 63 7 29

ENSGALG00000022260 ENSGALT00000035824 30 5 3

ENSGALG00000022360 ENSGALT00000035941 388 0 6

ENSGALG00000022422 ENSGALT00000036001 1 15 30

ENSGALG00000022513 ENSGALT00000036089 174 1 2

ENSGALG00000022560 ENSGALT00000028448 0 2 13

ENSGALG00000022595 ENSGALT00000036173 86 21 63

Total reads   1475 166 132

Values are the read sum of either liver and adipose of eight 15 weeks old birds or twenty 4.5-day embryos. Filtering criterion of 10 reads was used to identify expressed genes (in pink).

Supplemental Data 3. Percentage of identity between nucleic acids between chicken FFAR-2 paralogs

After multiple sequences alignment using CLUSTAL O(1.2.1), the Percent Identity Matrix was created by Clustal2.1. A high degree of sequence identity (>80% in green and >90% in pink) is observed between the FFAR2 paralogs (annotated as “Novel Ensembl prediction” in the Ensembl database (Genome assembly WASHUC2 (Ensembl release 70)). Red boxes correspond to the highest scores (> 98.5%).

Ensembl Gene name Ensembl Transcrpt ID 35549 35824 19833 28253 35500 35689 35498 28448 36171 19036 36001 35607 15228 35577 36173 36144 28161 36185 35699 35711 35587 28155 35979 35732 35941 36089 26926 24170 38562 24171 35006 04056Novel Ensembl prediction ENSGALT00000035549 100Novel Ensembl prediction ENSGALT00000035824 93.01 100Novel Ensembl prediction ENSGALT00000019833 97.58 94.44 100Novel Ensembl prediction ENSGALT00000028253 97.7 93.72 98.1 100Novel Ensembl prediction ENSGALT00000035500 95.39 95.11 96.86 95.89 100Novel Ensembl prediction ENSGALT00000035689 93.54 89.94 96.28 97 94.54 100Novel Ensembl prediction ENSGALT00000035498 94.35 89.94 97.12 97.84 95.12 92.57 100Novel Ensembl prediction ENSGALT00000028448 96.63 94.71 97.99 97.43 97.06 97.92 98.28 100Novel Ensembl prediction ENSGALT00000036171 96.63 93.97 97.99 97.18 96.81 98.16 98.28 99.02 100Novel Ensembl prediction ENSGALT00000019036 92.78 89.15 93.86 94.49 92.52 91.15 90.38 95.79 96.04 100Novel Ensembl prediction ENSGALT00000036001 90.55 89.79 90.53 90.37 90.28 91.82 91.45 92.55 92.55 92.59 100Novel Ensembl prediction ENSGALT00000035607 93.6 94.63 94.57 93.72 95.24 92.32 91.67 94.46 94.96 93.79 90.41 100Novel Ensembl prediction ENSGALT00000015228 93.97 96.72 95.13 94.4 96.47 92.95 91.92 95.59 95.3 93.45 89.03 96.68 100Novel Ensembl prediction ENSGALT00000035577 93 96.2 93.95 93.6 94.89 92.32 91.34 95.08 94.1 93.11 90.53 95.96 97.7 100Novel Ensembl prediction ENSGALT00000036173 94.76 92.64 95.05 94.57 93.83 91.19 87.23 95.57 95.57 92.68 93.23 89.95 88.76 89.18 100Novel Ensembl prediction ENSGALT00000036144 92.16 89.15 94.36 95.32 93.03 94.56 90.94 96.08 96.32 91.26 91.94 92 90.64 90.9 90.78 100Novel Ensembl prediction ENSGALT00000028161 95.77 93.83 96.79 96.14 95.72 93.86 94.08 96.68 96.43 93.2 91.64 92.77 93.73 93.43 91.48 94.09 100Novel Ensembl prediction ENSGALT00000036185 95.56 92.38 95.47 95.08 94.31 94.12 93.36 95.83 95.83 93.54 93.53 91.24 91.68 91.79 92.68 93.7 96.72 100Novel Ensembl prediction ENSGALT00000035699 95.41 92.68 95.31 94.69 94.53 94.41 93.31 96.07 96.07 93.52 94.22 91.75 92.02 92.08 92.43 93.44 96.71 98.91 100Novel Ensembl prediction ENSGALT00000035711 95.29 92.34 95.19 94.81 94.41 93.86 93.2 95.95 95.95 93.4 94.1 91.42 91.51 91.97 92.54 93.33 96.6 98.8 99.02 100Novel Ensembl prediction ENSGALT00000035587 94.8 93.2 96.28 95.65 94.94 93.07 93.59 96.53 96.53 93.06 91.28 94.56 93.53 92.79 95.69 95.03 96.33 95.1 95.21 95.21 100Novel Ensembl prediction ENSGALT00000028155 95.65 92.49 96.42 96.01 95.24 94.08 94.08 97.05 97.05 93.32 92.25 92.88 91.93 92 92.46 95.3 96.39 96.17 96.82 96.71 97.96 100Novel Ensembl prediction ENSGALT00000035979 95.77 91.93 95.68 95.29 94.77 94.85 94.52 96.56 96.06 95.49 92 92.84 93.04 93.06 94.52 96.31 96.42 96.53 97.19 97.08 96.6 96.87 100Novel Ensembl prediction ENSGALT00000035732 93.5 90.67 96.01 97.1 94.53 95.85 91.6 97.66 97.17 91.05 90.9 92.11 92.7 92.33 91.8 95.45 95.08 93.77 94.07 93.96 94.25 95.52 97.54 100Novel Ensembl prediction ENSGALT00000035941 94.99 92.08 96.38 95.89 95.11 93.7 89.23 96.92 96.92 90.9 91.14 91.45 91.54 91.45 94.02 94.32 94.31 93.44 93.74 93.63 96.73 95.19 97.65 95.64 100Novel Ensembl prediction ENSGALT00000036089 94.71 93.48 96.05 95.65 94.55 96.74 96.38 96.68 96.43 93.98 91.54 93.72 93.39 93.6 94.32 96.62 96.38 95.77 96.24 95.87 96.73 96.98 97.46 97.58 98.31 100P2RY8 ENSGALT00000026926 39.85 40.55 39.81 39.49 39.55 38.29 38.29 39.07 39.33 38.66 40.66 39.47 40.05 40.43 38.34 37.25 39.05 39.43 39.23 39.47 39.8 39.29 40.55 38.61 37.69 39.05 100F2R ENSGALT00000024170 45.26 45.69 46.98 46.55 45.26 46.12 46.12 45.45 45 46.53 46.12 45.26 45.7 45.69 45.69 45.26 46.12 46.55 45.26 46.12 46.12 46.12 44.4 46.12 44.83 44.83 47.32 100F2RL2 ENSGALT00000038562 47.84 49.57 48.71 49.14 49.14 48.71 48.71 47.73 46.82 49.5 48.28 49.14 47.51 49.57 48.71 47.84 49.14 49.14 48.71 49.57 49.14 49.57 48.71 49.57 47.84 48.28 46.29 45.35 100F2RL1 ENSGALT00000024171 42.99 41.79 43.23 43.51 43.48 43 41.68 43.49 43.37 43.73 41.53 43.31 43.34 42.98 42.96 42.29 41.88 41.93 41.44 41.44 43.14 42.32 42.87 43.13 42.78 43.35 47.03 46.58 46.88 100F2RL3 ENSGALT00000035006 50.86 50.86 51.29 51.29 50.43 51.72 51.72 50.45 50.45 50 50.86 51.29 49.77 51.72 51.29 50.43 51.72 51.29 51.72 51.72 50.86 51.72 50.86 51.72 50.43 50.43 45.05 45.12 49.28 48.16 100Novel Ensembl prediction ENSGALT00000004056 53.02 53.02 53.45 53.45 53.02 53.88 53.88 53.18 52.73 50.99 53.02 53.02 52.04 53.88 52.59 53.02 54.31 53.45 53.02 52.59 53.02 53.02 52.16 53.02 52.59 52.59 45.03 43.8 45.49 47.65 54.89 100

Supplemental Data 4. Free fatty acid receptors accession number in human, mouse, chicken and pig species

Human Mouse Chicken Pig Gene name Gene

descriptionEnsembl Gene ID

Ensembl Transcript ID Ensembl Gene ID Ensembl

Transcript ID Ensembl Gene ID Ensembl Transcript ID

Ensembl Gene ID

Ensembl Transcript ID

FFAR2 (GPR43) free fatty acid receptor 2

ENSG00000126262ENST00000599180

ENSMUSG00000051314

ENSMUST00000053156 26 genes (See Supplemental data 1)   ENSSSCG00000002881 ENSSSCT00000003183

ENST00000246549 ENSMUST00000168528        

    ENSMUST00000186339        

    ENSMUST00000186534        

    ENSMUST00000186059        

    ENSMUST00000163504        

FFAR3 (GPR41) free fatty acid receptor 3

ENSG00000185897ENST00000327809 ENSMUSG00000019429 ENSMUST00000094583     ENSSSCG00000002891 ENSSSCT00000003195

ENST00000594310 ENSMUST00000185748

FFAR1 (GPR40) free fatty acid receptor 1

ENSG00000126266 ENST00000246553 ENSMUSG00000044453 ENSMUST00000052700     ENSSSCG00000002892 ENSSSCT00000003196

FFAR4 (GPR120, O3FAR1)

free fatty acid receptor 4

ENSG00000186188 ENST00000371481 ENSMUSG00000054200 ENSMUST00000067098 ENSGALG00000026733 ENSGALT00000045426 ENSSSCG00000010478 ENSSSCT00000011466

ENSG00000186188 ENST00000371483

Supplemental Data 5. Percentage of identity between nucleic acids FFAR sequences of human (A), mouse (B) and pig species (C).

A

B

Gene name Ensembl gene ID Transcript ID 1 2 3 4 5 6 7 8 9 10 111

FFAR2 ENSG00000126262ENST00000599180 100.0

2 ENST00000246549 100.0 100.0 3

FFAR3 ENSG00000185897ENST00000327809 53.0 53.6 100.0

4 ENST00000594310 55.0 56.4 96.8 100.0 5

GPR42 ENSG00000126251ENST00000454971 52.9 53.5 99.3 96.2 100.0

6 ENST00000597214 55.0 56.4 96.2 99.4 96.8 100.0 7 FFAR1 ENSG00000126266 ENST00000246553 43.7 43.6 44.2 47.5 44.1 47.3 100.0 8

FFAR4 ENSG00000186188ENST00000371481 33.5 33.3 33.6 35.1 33.7 35.2 35.4 100.0

9 ENST00000371483 33.9 33.7 33.5 34.9 33.6 34.9 35.6 100.0 100.0 10

GPR84 ENSG00000139572ENST00000267015 34.12 33.8 36 35.8 36 35.8 35.4 35.9 36.1 100

11 ENST00000551809 34 34 35 35 35 35 35 36 36 96 100

Gene name Ensembl gene ID Transcript ID 1 2 3 4 5 6 7 8 9 10 111

FFAR2 ENSMUSG00000051314

ENSMUST00000186534 100.0 2 ENSMUST00000186059 89.5 100.0 3 ENSMUST00000163504 90.0 100.0 100.0 4 ENSMUST00000053156 76.6 84.9 95.7 100.0 5 ENSMUST00000168528 84.1 88.0 94.8 90.6 100.0 6 ENSMUST00000186339 93.6 92.5 94.2 90.8 96.6 100.0 7

FFAR3 ENSMUSG00000019429ENSMUST00000094583 54.0 54.3 50.5 50.3 53.8 54.0 100.0

8 ENSMUST00000185748 48.9 51.9 53.0 52.3 52.0 52.6 96.5 100.0 9 FFAR1 ENSMUSG00000044453 ENSMUST00000052700 45.1 45.8 42.5 42.5 41.4 43.6 43.2 42.7 100.0 10 FFAR4 ENSMUSG00000054200 ENSMUST00000067098 42.0 40.7 38.4 38.8 37.8 40.9 40.9 40.5 35.5 100.0 11 GPR84 ENSMUSG00000063234 ENSMUST00000079824 41.6 42.3 37.5 36.6 38.5 40.7 37.0 37.3 39.4 36.6 100.0

Gene name Ensembl gene ID Transcript ID 1 2 3 4 51 FFAR2 ENSSSCG00000002881 ENSSSCT00000003183 100.0 2 ENSSSCG00000024282 ENSSSCT00000030352 78.2 100.0 3 FFAR3 ENSSSCG00000002891 ENSSSCT00000003195 56.1 57.7 100.0 4 FFAR1 ENSSSCG00000002892 ENSSSCT00000003196 47.8 45.9 49.5 100.0 5 FFAR4 ENSSSCG00000010478 ENSSSCT00000011466 44.9 43.0 45.2 41.2 100.0

C

The percentage of identity was determined using Clustal Omega version 2.1. High degree of sequences identity (>80%) is colored in pink. Red boxes correspond to the highest scores (> 98.5%). Other FFAR2 paralogs ((human F2R,F2RL1,F2RL2,F2RL3,GPR132,GPR4,GPR68,and P2RY8) and (mouse F2R,F2RL1,F2RL2,F2RL3,GPR132,GPR65 and GPR84) and (pig F2R,F2RL1,F2RL2,F2RL3,GPR132,GPR4 and GPR68)) shared <50% identity with FFAR2 sequences.

Supplemental Data 6. Percentage of identity of amino acids FFAR sequences of human (A), mouse (B) and pig species (C).

Gene name Ensembl gene ID Transcript ID 1 2 3 4 5 6 7 8 9 10 111

FFAR2 ENSG00000126262ENST00000599180 100

2 ENST00000246549 100 100 3

FFAR3 ENSG00000185897ENST00000327809 41 41 100

4 ENST00000594310 41 41 100 100 5

GPR42 ENSG00000126251ENST00000454971 41 41 98 98 100

6 ENST00000597214 41 41 98 98 100 100 7 FFAR1 ENSG00000126266 ENST00000246553 28 28 31 31 31 31 100 8

FFAR4 ENSG00000186188ENST00000371481 17 17 19 19 19 19 15 100

9 ENST00000371483 17 17 19 19 19 19 15 100 100 10

GPR84 ENSG00000139572ENST00000267015 19 19 21 21 21 21 18 20 19 100

11 ENST00000551809 19 19 21 21 21 21 18 20 19 100 100

A

Gene name Ensembl gene ID Transcript ID 1 2 3 4 5 6 7 8 9 10 111

FFAR2 ENSMUSG00000051314

ENSMUST00000186534 100.0 2 ENSMUST00000186059 100.0 100.0 3 ENSMUST00000163504 100.0 100.0 100.0 4 ENSMUST00000053156 100.0 100.0 100.0 100.0 5 ENSMUST00000168528 100.0 100.0 100.0 100.0 100.0 6 ENSMUST00000186339 100.0 100.0 100.0 100.0 100.0 100.0 7

FFAR3 ENSMUSG00000019429ENSMUST00000094583 43.1 43.1 44.6 46.4 47.3 43.1 100.0

8 ENSMUST00000185748 43.1 43.1 44.6 46.4 47.3 43.1 100.0 100.0 9 FFAR1 ENSMUSG00000044453 ENSMUST00000052700 29.3 29.3 30.1 32.4 33.1 29.3 30.2 30.2 100.0 10 FFAR4 ENSMUSG00000054200 ENSMUST00000067098 22.8 22.8 21.5 24.8 25.0 22.8 23.0 23.0 16.3 100.0 11 GPR84 ENSMUSG00000063234 ENSMUST00000079824 22.6 22.6 24.2 26.1 27.1 22.6 21.9 21.9 18.6 22.1 100.0

B

Gene name Ensembl gene ID Transcript ID 1 2 3 4 51 FFAR2 ENSSSCG00000002881 ENSSSCT00000003183 100.0 2 ENSSSCG00000024282 ENSSSCT00000030352 73.7 100.0 3 FFAR3 ENSSSCG00000002891 ENSSSCT00000003195 41.0 44.7 100.0 4 FFAR1 ENSSSCG00000002892 ENSSSCT00000003196 30.3 28.3 31.7 100.0 5 FFAR4 ENSSSCG00000010478 ENSSSCT00000011466 19.2 17.2 19.1 15.5 100.0

C

The percentage of identity was determined using Clustal Omega version 2.1. The percentages of amino acid identity >67% is colored in pink. Red boxes correspond to the highest scores (100%). Other FFAR2 paralogs ((human F2R,F2RL1,F2RL2,F2RL3,GPR132,GPR4,GPR68,and P2RY8) and (mouse F2R,F2RL1,F2RL2,F2RL3,GPR132,GPR65 and GPR84) and (pig F2R,F2RL1,F2RL2,F2RL3,GPR132,GPR4 and GPR68)) shared <50% identity with FFAR2 sequences.

Supplemental Data 7. Multiple amino-acid sequence alignment of the 26 FFAR2 paralogs

Supplemental Data 8. FFAR2 fragment amplified with universal primers.

100pb

50pb

63pb

3483

7

3492

9

3522

9

3491

9

2810

2

3472

9

3539

4

3496

2

1980

6

3478

0

3520

7

2810

8

3481

8

3494

1

3540

8

2837

7

1521

2

3536

8

3473

1

1901

3

2819

7

3480

8

3539

6

3516

9

3505

3

3531

5

ENSGALP00000034837 439-504 439-504 439-591

ENSGALP00000034929 543-1068 422-548 439-548 460-601 422-548 422-548

ENSGALP00000035229 306-425 306-419 306-425 357-425 306-425 276-438 543-1068 306-419 607-793 205-409 607-721

ENSGALP00000034919 276-540 364-540

ENSGALP00000028102ENSGALP00000034729ENSGALP00000035394 421-540 426-540 421-540

ENSGALP00000034962 422-591 422-548 422-548 439-680 399-777 399-613

ENSGALP00000019806 421-504 426-504 421-504

ENSGALP00000034780 421-504 426-504 421-504

ENSGALP00000035207 422-548 422-587 422-587 460-587 251-548 306-548

ENSGALP00000028108 417-548 257-548 439-591 460-548 422-591 422-591

ENSGALP00000034818 460-548 411-548 460-680

ENSGALP00000034941 439-548 460-601 422-548 422-548

ENSGALP00000035408 439-548 460-601 422-548 422-548

ENSGALP00000028377 379-540 379-540

ENSGALP00000015212 475-591 421-504

ENSGALP00000035368 475-591 439-680 475-591 439-613

ENSGALP00000034731ENSGALP00000019013 463-547 463-547

ENSGALP00000028197 426-504 421-504

ENSGALP00000034808ENSGALP00000035396 460-548 460-548

ENSGALP00000035169ENSGALP00000035053ENSGALP00000035315

Supplemental Data 9. Gene conversion results

The colors correspond to the results of the three different p-values calculated by Geneconv. Yellow: one significant p-value out to three, orange: two significant p-values, and red: the three p-values are significant. The coordinates of the fragments are indicated for each event and correspond to the codon multiple sequences alignment coordinates.

Supplemental Data 10.Alignment of the sequences of the human proteinase-activated receptor 1 (PAR1; pdb 3vw7; Zhang et al. 2012) and of chicken FFAR2(ENSGALP00000034780).Secondary structures, as observed in the experimental 3D structure of PAR1, are reported above the alignment. Amino acid critical for the FFAR2 function, as depicted in Supplemental Data 4 and in Figure 3, are reported in grey, whereas amino acids under positive selection are reported in pink and orange, respectively. The position of T4 lysozyme (T4L), inserted within intracellular loop 3 to allow crystallization, is boxed in yellow.