AbstractLichen-forming fungi are a diverse and ecologically important group of obligate mutualistic symbionts. Due to difficulties with maintaining them in culture and their extremely slow growth, lichenologists are increasingly opting for metagenomic sequencing followed by symbiont genome separation using bioinformatic pipelines. However, without knowing the true genome size of the lichen-forming fungus, we cannot quantify the completeness of the genome assembly and the efficacy of the bioinformatic filtering. To address this issue, we report here the first whole-genome assembly for the lichen-forming fungus Ramalina farinacea (L.) Ach. sequenced with Oxford Nanopore long-read technology alongside direct measurements of its genome size using flow cytometry. The assembly showed high contiguity (N50 = 1.55 Mb) and gene set completeness (BUSCO = 95.8%). The highly robust genome size of 33.61 Mb/1C (coefficients of variation = 2.98) that was obtained showed our assembly covered 97% of the entire genome. Our results demonstrate that accurate genome size measurements can be obtained directly from lichen thalli and used to provide a benchmark for assessing true cytometric completeness of metagenome-derived assemblies.