Spontaneous mutation rate estimation in the large-genome unicellular eukaryote Euglena gracilis

The spontaneous mutation rate per nucleotide (µ) is a fundamental parameter shaping genome evolution, yet it remains unknown for many eukaryotic groups. Here, we estimated µ in the freshwater unicellular eukaryote Euglena gracilis, which possesses a 2.4 Gb genome, among the largest reported for unicellular species. We conducted a mutation accumulation experiment using 10 independent MA lines propagated through repeated single-cell bottlenecks and then identified 153 de novo nucleotide mutations. After correction for false negatives, the spontaneous mutation rate was estimated at µ=6.60 × 10⁻¹⁰ mutations per nucleotide per generation. E. gracilis exhibits a mutation rate within the range reported for other unicellular eukaryotes and bacteria, supporting the predominant role of effective population size rather than genome size in mutation-rate evolution. Most detected mutations occurred in intergenic regions, and the mutation spectrum was strongly biased toward GC → AT mutations. These results provide a first estimate of the spontaneous mutation rate in Euglenophyte group and contribute to documenting mutation-rate evolution in large-genome unicellular eukaryotes.