a, MHC genes in platypus and echidna are located on two different chromosomes, but the classical class I and II genes involved in antigen presentation are located within a single cluster in each genome. b, Phylogenetic relationship of class I genes in representative mammals and chicken. Classical class I genes (red) in monotremes exhibit high similarity, which is rarely observed in other species. Only bootstrap values with >50% support are shown. c, d, Phylogenetic relationship of MHC class II alpha (c) and beta (d) genes. Genes with prefix ‘HLA’, ‘Modo’, ‘Phci’, ‘Oran’, ‘Taac’ and ‘Gaga’ indicate genes in human, opossum, koala, platypus, echidna and chicken, respectively. Only bootstrap values with >50% support are shown. e, Phylogenetic relationship among putative functional Vγ sequences from platypus (yellow), echidna (purple), koala (green), mouse (orange), human (red), sheep (grey), cow (dark red) and chicken (dark yellow). Groups according to a previous study90 are displayed around the outside of the tree, with the putative marsupial–monotreme-specific group denoted by a ‘?’. Only bootstrap values with greater than 50% support are shown. f, Synteny conservation of beta-defensin genes in monotremes and loss of functional venom defensins in echidna. Venom defensins (OavDLP genes) and venom-like defensin (DEFB-VL genes) are shown in red. Only putative functional defensins are shown. g, Putative OavDLP loss in echidna. OavDLP genes and DEFB-VL each contain two exons (indicated by a box and triangle) in platypus. Both exons of platypus DEFB-VL can be mapped to echidna chromosome X2. A single platypus OavDLP exon can be mapped to echidna chromosome X2 while the second exons cannot. Grey links indicate platypus–echidna LASTZ alignment. h, Phylogenetic relationship of DEFB-VL and OavDLP genes suggested that ancestral monotremes had all three OavDLP genes but that echidna has lost the two of them (OavDLP-B and OavDLP-C). Branch length is not shown. ta, echidna; oa, platypus. Silhouettes of the human, opossum, koala and frog are from https://www.flaticon.com/. The silhouette of the platypus is created by S. Werning and is reproduced under a Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).