Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.

The work was supported by HHMI International Early Career Scientist Program (55007424), the MINECO (BFU2012-31329, BFU2012-37168, BFU2015-68351-P and BFU2015-68723-P), Spanish Ministry of Economy and Competitiveness Centro de Excelencia Severo Ochoa 2013-2017 grant (SEV-2012-0208), the Unidad de Excelencia María de Maeztu funded by the MINECO (MDM-2014-0370), Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat AGAUR program (2014 SGR 0974), the CERCA Programme of the Generalitat de Catalunya, Russian Foundation for Basic Research grant (18-04-01173), the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie programme (665385) and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013, ERC grant agreement 335980_EinME and Synergy Grant 609989). KSS was supported by EMBO long-term fellowship (ALTF 107-2016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.