10Iridescent colours are colours that change depending on the angle of illumination or observation. They are 11 produced when light is reflected by multilayer structures or diffracted by gratings. While this phenomenon is well 12 understood for simple optical systems, it remains unclear how complex biological structures interact with light 13 to produce iridescence. There are very few comparative studies at interspecific level (often focusing on a single 14 colour patch for each species), resulting in an underestimation of structure diversity. Using an interdisciplinary 15 approach combining physics and biology, we here quantify the colour and structure of 36 hummingbirds species evenly 16 distributed across the phylogeny. We explore at least 2 patches per species, which are assumed to be under different 17 selective regimes. For each patch, we measure structural features (number of layers, layer width, irregularity, spacing, 18 etc.) of the feathers at different scales using both optical and electronic microscopy and we measure colour using a 19 novel approach we developed to encompass the full complexity of iridescence, including its angular dependency. We 20 discover an unsuspected diversity of structures producing iridescence in hummingbirds. We also study the effect of 21 several structural features on the colour of the resulting signal, using both an empirical and modelling approach. Our 22 findings demonstrate the need to take into account multiple patches per species and suggest possible evolutionary 23 pressures causing the evolutionary transitions from one melanosome type to another. 24 Hummingbirds are famous for their bright and shiny colours which change with the illumination or observation 26 angle: a phenomenon known as iridescence. Iridescent colours are produced by the interaction of light with periodic 27 nanometre-scale structures such as multilayers or diffraction gratings and are widespread among many taxa [1]. But few 28 taxa display colours as bright and as saturated as the hummingbirds (Trochilidae family). Most hummingbird species 29 harbour two visually distinct types of iridescent colour patches, as illustrated in fig. S1: directional patches, which are 30 only visible at a very narrow angle range [2] and are often very bright and saturated, and diffuse patches, for which 31 some colour is visible from any angle [2] and that are often not as bright as directional patches. Directional patches are 32 often located on facial or ventral patches and thought to be involved in communication while diffuse patches are often 33 located on dorsal patches and thought to be involved in camouflage [3]. Additionally, although all hummingbird species 34 display some degree of iridescence, striking differences can be noticed between the various species and body patches in 35 terms of brightness (describing how much light is reflected by the object), saturation (describing the colour "purity") 36 and directionality [4].
37Yet, the structural bases of this intra-individual and interspecific diversity...