Calculating Nucleotide Diversity in O. colligata

For calculating the average number of nucleotide differences (π) between O. colligata genomes, we used pixy v.0.95 (Korunes and Samuk 2021 (link)). Beforehand, we filtered out alleles with less than half or more than double the average sample coverage. Finally, we averaged the estimated π-values over one Kilobase pair (kb) windows. Similarly, we calculated the non-/synonymous per-site nucleotide diversity (π_N and π_S) for the coding sequences of O. colligata using the script selectionStats.py (https://github.com/tatumdmortimer/popgen-stats). Both methods are consistent with theoretical expectations and comparable among species as they take invariant sites into account for their calculations (Korunes and Samuk 2021 (link)). The ratio of π_N and π_S was separately calculated for BUSCO genes (Benchmarking Universal Single-Copy Orthologs) identified using BUSCO v.4.0.1 (Seppey et al. 2019 ) and its microsporidia_odb10 database (Creation date: August 05, 2020) and compared with published π_N/π_S values of H. tvaerminnensis (Angst et al. 2022 (link)).