In many butterflies, the ancestral trichromatic insect colour vision, based on UV-, blue- and green-sensitive photoreceptors, is extended with red-sensitive cells. Physiological evidence for red receptors has been missing in Nymphalid butterflies, although some species can discriminate red hues well. In eight species from genera Archaeoprepona, Argynnis, Charaxes, Danaus, Melitaea, Morpho, Heliconius and Speyeria we found a novel class of green-sensitive photoreceptors that have hyperpolarising responses to stimulation with red light. These green-positive, red-negative (G+R–) cells are allocated to positions R1/2, normally occupied by UV and blue-sensitive cells. Spectral sensitivity, polarisation sensitivity and temporal dynamics suggest that the red opponent units (R–) are the basal photoreceptors R9, interacting with R1/2 in the same ommatidia via direct inhibitory synapses. We found the G+R– cells exclusively in butterflies with red-shining ommatidia containing longitudinal screening pigments. The implementation of the red colour channel with R9 is different from Pierid and Papilionid butterflies, where cells R5-8 are the red receptors. The Nymphalid red-green opponent channel and the potential for tetrachromacy seem to have been switched on several times during evolution, balancing between the cost of neural processing and the value of extended colour information.

Open files ending with .___ using software, download link: .wcp WINWCP http://spider.science.strath.ac.uk/sipbs/software_ses.htm .pzfx Prism https://www.graphpad.com/scientific-software/prism/ .m GNU Octave Funding provided by: Air Force Office of Scientific ResearchCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000181Award Number: FA9550-15-1-0068Funding provided by: Ministrstvo za Izobraževanje, Znanost in ŠportCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100005989Award Number: 5442-1/2018/434Funding provided by: Javna Agencija za Raziskovalno Dejavnost RSCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004329Award Number: P3-0333

Electrophysiological data (.wcp files) were collected using WINWCP software.