(A) Maximum likelihood phylogeny of vertebrate Prg4 proteins generated using the LG model in PhyML (Guindon et al., 2009). The tree was rooted with spotted gar and human Vitronectin (Vtn) proteins. In the ray-finned lineage, after divergence of the holostean lineage (spotted gar and bowfin), the teleost genome duplication (TGD) generated Prg4a and Prg4b found in extant teleosts. GenBank/Ensembl accession numbers for sequences in the MAFFT (Katoh and Standley, 2014) alignment: zebrafish: Prg4a, NP_997918, Prg4b, XP_005160745; stickleback: Prg4a, ENSGACP00000019616, Prg4b, concatenate of ENSGACP00000009961/ENSGACP00000009951/ENSGACP00000009946; medaka: Prg4a, ENSORLP00000017289, Prg4b, ENSORLP00000011688; Spotted gar: Prg4, XP_015210531, Vtn, XP_006641129; bowfin: Prg4, AAC_TPR.1.1 [http://phylofish.sigenae.org/]; human: PRG4, NP_005798, VTN, NP_000629; mouse: Prg4, NP_067375; chicken: Prg4, ENSGALP00000008204; Anole lizard: Prg4, XP_008112908; Western clawed frog: Prg4, XP_012817383; coelacanth: Prg4, ENSLACP00000017445. (B) Orthologous pairwise synteny cluster from the Synteny Database (Catchen et al., 2009) (window size: 50 genes) shows extensive conserved synteny of Prg4 gene regions on human chromosome Hsa1 and spotted gar Loc10, supporting their orthology. (C) Composite cluster from the Synteny Database (window size: 50 genes) compares the prg4 gene regions in zebrafish to the TGD outgroup spotted gar. Double conserved synteny of prg4 on gar Loc10 and zebrafish prg4a on Dre2 and prg4b on Dre20, supports the prg4 duplication as result of the TGD.