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Page 1: Supplementary Fig. 1

Supplementary Fig. 1

Placental RLN3 ancestorMarsupial RLN3Monotreme RFLCIAmphibian RFLCITeleost RFLC ancestor

AncRFLC (alternative)

GHSIYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLCYPSFYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLCYPSFYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLCDDPIYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLCAGPSYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKGEISSLCRVPTFGVKLCGTEFIRAVIFTCGGSRW EAALGLSKTCCKWGCSKSQISSLCRVPTFGVKLCGREFIRAVIFTCGGSRW EAALGLSNTCCKWGCSKSQISSLC

RPSTYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC

RAAPYGVKLCGREFIRAVIFTCGGSRW DVLAGLSSSCCKWGCSKSEISSLCRTSPYAVKLCGREFIRAVIFTCGGSRW DVLAGVSSNCCKWGCSKTEISSLCRSPSYGMKLCGREFIRAVIFTCGGSRW DVLAGLSSNCCKWGCSKSEISSLCRVPTFGVKLCGREFIRAVIFTCGGSRW EAALGLSNTCCKWGCSKSQISSLCDDPIYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC

RAPTYGVKLCGREFIRAVIFTCGGSRW DVVVGLSNACCKWGCSKSEISSLC

A chainB chainD. rerio T. rubripes T. nigroviridisT. rubripes D. rerio X. laevis X. tropicalis

AncRFLC

RFLC1RFLC1RFLC1RFLC2RFLC2RFLCIRFLCI

Page 2: Supplementary Fig. 1

zRFLC

1zR

FLC1

RB12HzR

FLC

2zR

FLC2

RB12HAncR

FL

CAncR

FLC

RB12H

RLN3 RB12 H

RLN3

RLN3 RB12 A

RLN3 FB20 R

RLN3 SB25 P

INSL3

INSL3 H

B12 R

INSL3 H

B12 A

AncRFLC

AncRFLC V

VA3,4 LA

AncRFLC N

AA8,9 SS

RLN3 LA

A3,4 VV

RLN3 SSA8,9 NA

RLN3

RLN3

oINSL3

INSL3

pRFLCI

pRFLCII

4

66

10

4

66

10

kDa kDa kDa

kDa kDa kDa

Nonreducing

4

66

10

Nonreducing

Reducing

4

66

10

Reducing4

66

10

4

66

10

Supplementary Fig. 2

Page 3: Supplementary Fig. 1

Human INSL3

Platypus RFLCII

Supplementary Fig. 3

Therian mammals

Monotreme

Human INSL3Chimp INSL3Mouse Insl3Rat Insl3Cow INSL3Dog INSL3Opossum INSL3

Platypus RFLCII

E K L C G H H F V R A L VGAGAAGTTGTGCGGCCACCACTTCGTACGCGCGCTAGTGGAGAAGTTGTGCGGCCACCACTTCGTACGCGCGCTGGTGGCCAAGCTCTGCGGCCACCACCTGGTGCGCACGCTGGTGGCCAAGCTCTGTGGTCACCACCTGGTGCGTGCGCTGGTGGAGAAACTGTGTGGCCACCACTTCGTGCGCGCGCTCGTGGAGAAGCTGTGTGGCCACCACTTCGTGCGGGCGCTGGTGCAGAAACTCTGTGCCCACCGCTTCATCCGGGCCCTCGTC

GTGAAGCTGTGTGGGCGGGAGTTCATCCGTGCTCTGATTV K L C G R E F I R A L I


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