Nodes represent genotypes and edges represent mutations. Only pathways that increase the antibiotic resistance at each step are shown. Parallelograms formed by four connected genotypes indicate additivity among mutations. Asymmetrical quadrilaterals indicate the presence of epistasis.

a | To understand the evolution of new spectral sensitivities in the short-wave opsins (SWO), Yokoyama and colleagues inferred their phylogeny. The wavelength to which each pigment is most sensitive is shown in parentheses; UV-sensitive pigments are shown in purple and blue-sensitive pigments in blue. Sequences of ancestral SWOs at each node were inferred, and the optimal wavelength for each ancestral pigment is shown in circles. The shift from the UV-sensitive pigment in the bird-reptile ancestor (represented as a purple circle) to the blue-sensitive pigment in the ancestor of birds (represented as a blue circle) is indicated by an arrow. b | The inferred sequences of ancenstral SWOs at each node were ‘resurrected’ by mutagenesis, expressed in mammalian cells (COS1), reconstituted with the chromophore 11-cis-retinal and tested in vitro. c | Introducing four historical substitutions from this branch into the bird-reptile ancestral SWO is sufficient to recapitulate the evolution of the bird ancestor’s new spectral phenotype. Parts a and c reproduced with permission from REF. 69 © (2003) National Academy of Sciences (USA). Part b reproduced with permission from REF. 126 © (2002) Elsevier Sciences.

Aa | The catalytic cycle of isopropylmalate dehydrogenase (IMDH) to produce ATP in the Krebs cycle; ancestral use of NAD⁺ as a coenzyme has been conserved in all known IMDHs. Ab | The catalytic cycle of isocitrate dehydrogenase (IDH) to produce NADPH for biosynthesis; use of NADP⁺ as a coenzyme has evolved in some but not all IDH lineages. Binding of the reduced coenzyme — NADH to IMDH or NADPH to IDH — inhibits catalysis by tying up the enzyme in abortive complexes. Cellular concentrations of NADH are low, so inhibition of IMDH is insignificant. Although abundant, NADPH has a relatively low affinity for IDH. B | The adaptive landscape controlling the evolution of coenzyme use by IMDH. For 90 IMDH mutants, fitness is plotted against coenzyme performances. The fitted phenotype-fitness map is shown in blue. C | Structural basis for reduced NADPH inhibition in IDH. In both IDH and IMDH, the nicotinamide ring of the coenzyme lies atop the γ-moiety of the bound substrate (isopropylmalate or isocitrate). During catalysis, hydrid transfer to the C4 neutralizes the positive charge on the ring. In IMDH, the resulting hydrophobic attraction between the reduced ring and the γ-isopropyl increases affinity. In IDH, the loss of the charge on the ring breaks the salt bridge to the γ-carboxylate and lowers affinity. Hence, cellular NADPH is a potent inhibitor of IMDH but a relatively weak inhibitor of IDH, allowing IDHs to evolve NADP⁺ use. Parts B and C modified with permission from REF. 88 © (2006) American Association for the Advancement of Science.

a | Diazinon, like other organophosphates, binds to acetylcholinesterase (AChE) and blocks neurotransmitter turnover, causing acetylcholine accumulation and neurotoxicity. b | Diazinon also binds to and blocks E3, another esterase. Newcomb et al. found that, in populations of diazinon-resistant blowflies, the esterase isozyme E3 hydrolyses diazinon, rendering it non-toxic57. Compared with sensitive blowfly populations, E3 from resistant populations contains five amino-acid replacements (represented as grey circles). c | Newcomb et al. prepared chimeric E3s to test the role of Gly137Asp replacement, the only replacement that is located in the enzyme’s active site. Chimaera 1 contains all the residues from sensitive E3 (represented as green circles) except Asp137 (from the resistant allele, represented as a grey circle). Chimaera 2 contains all the residues from the resistant allele except Gly137. The graph shows the loss of carboxyesterase activity and the gain of diazinon-hydrolysis activity in the resistant allele and Chimaera 1, but not Chimaera 2. d | A model of the active-site region of a resistant esterase with the Gly137Asp replacement (Asp119 in the model). Asp119 is positioned to act as a general base, activating a water molecule (WAT) for nucleophylic attack on the insecticide’s phosphorous atom. The P-O bond to Ser200 breaks, diethyl phosphate is released and the active E3 enzyme is restored. Panel d reproduced with permission from REF. 57 © (1997) National Academy of Sciences (USA).

a | Timeline for evolution of receptors for three structurally similar steroid hormones. Deoxycorticosterone (DOC) is an ancient vertebrate hormone, whereas aldosterone evolved much later in the tetrapod lineage (as indicated by a black arrow). Modern mineralocorticoid receptors (MRs) can be activated by aldosterone, DOC, and to a lesser extent cortisol. The glucocorticoid receptor (GR) is activated only by cortisol in bony vertebrates. The resurrected ancestral corticoid receptor (AncCR) has MR-like sensitivity to all three hormones. Resurrection of GRs from the ancestral jawed vertebrate (AncGR1) and the ancestral bony vertebrate (AncGR2) show that GR’s cortisol specificity evolved in the interval between AncGR1 and AncGR2 (represented as a blue box). Dates from the fossil record are indicated in million of years ago (mya). b | Evolution of GR’s cortisol specificity. When two historical replacements from the AncGR1-AncGR2 interval were introduced into the ancestral background, they switched receptor preference from aldosterone to cortisol. Structures of the ancestral proteins show that replacement Ser106Pro causes a kink in the protein backbone, destabilizing the ligand-receptor complex and reducing activation by all ligands; Ser106Pro also repositions site 111, so that replacement Leu111Gln forms hydrogen bonds with the C17-hydroxyl that is unique to cortisol. c | Optimization of the GR phenotype. Three other strict replacements from the same interval abolish function when introduced into AncGR1 with Ser106Pro and Leu111Gln — an unlikely evolutionary path under selection (as indicated by the inhibitory arrow). The ancestral structures identified two other replacements from this interval (out of 37 total) that in isolation have little effect on function, but when combined with the conserved substitutions yield a complete GR-like phenotype (as indicated by the black arrow). Part b reproduced with permission from REF. 93 © (2007) American Association for the Advancment of Science.