Supplementary Fig. 1

1. D. rerio T. rubripes T. nigroviridis T. rubripes D. rerio X. laevis X. tropicalis AncRFLC RFLC1 RFLC1 RFLC1 RFLC2 RFLC2 RFLCI RFLCI A chain B chain GHSIYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC YPSFYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC YPSFYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC DDPIYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC AGPSYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKGEISSLC RVPTFGVKLCGTEFIRAVIFTCGGSRW EAALGLSKTCCKWGCSKSQISSLC RVPTFGVKLCGREFIRAVIFTCGGSRW EAALGLSNTCCKWGCSKSQISSLC RPSTYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC RAAPYGVKLCGREFIRAVIFTCGGSRW DVLAGLSSSCCKWGCSKSEISSLC RTSPYAVKLCGREFIRAVIFTCGGSRW DVLAGVSSNCCKWGCSKTEISSLC RSPSYGMKLCGREFIRAVIFTCGGSRW DVLAGLSSNCCKWGCSKSEISSLC RVPTFGVKLCGREFIRAVIFTCGGSRW EAALGLSNTCCKWGCSKSQISSLC DDPIYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC RAPTYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC Placental RLN3 ancestor Marsupial RLN3 Monotreme RFLCI Amphibian RFLCI Teleost RFLC ancestor AncRFLC (alternative) Supplementary Fig. 1

2. AncRFLC RB12H zRFLC1 RB12H zRFLC2 RB12H AncRFLC zRFLC1 zRFLC2 INSL3 HB12A RLN3 RB12H INSL3 HB12R RLN3 RB12A RLN3 SB25P RLN3 FB20R kDa kDa kDa INSL3 RLN3 10 10 10 Non reducing 6 6 6 6 6 6 Reducing 4 4 4 AncRFLC VVA3,4LA AncRFLC NAA8,9SS RLN3 LAA3,4VV RLN3 SSA8,9NA AncRFLC pRFLCII pRFLCI oINSL3 RLN3 kDa kDa kDa INSL3 RLN3 10 10 10 Non reducing 6 6 6 6 6 6 Reducing 4 4 4 Supplementary Fig. 2

3. Human INSL3 E K L C G H H F V R A L V GAGAAGTTGTGCGGCCACCACTTCGTACGCGCGCTAGTG GAGAAGTTGTGCGGCCACCACTTCGTACGCGCGCTGGTG GCCAAGCTCTGCGGCCACCACCTGGTGCGCACGCTGGTG GCCAAGCTCTGTGGTCACCACCTGGTGCGTGCGCTGGTG GAGAAACTGTGTGGCCACCACTTCGTGCGCGCGCTCGTG GAGAAGCTGTGTGGCCACCACTTCGTGCGGGCGCTGGTG CAGAAACTCTGTGCCCACCGCTTCATCCGGGCCCTCGTC GTGAAGCTGTGTGGGCGGGAGTTCATCCGTGCTCTGATT V K L C G R E F I R A L I Human INSL3 Chimp INSL3 Mouse Insl3 Rat Insl3 Cow INSL3 Dog INSL3 Opossum INSL3 Platypus RFLCII Therian mammals Monotreme Platypus RFLCII Supplementary Fig. 3