Supporting Information for "Twenty-five new microsporidian genomes: Insights into ploidy, reproduction, and the group's pangenome"

Supporting Information for "Twenty-five new microsporidian genomes: Insights into ploidy, reproduction, and the group’s pangenome". This dataset contains: the 25 genomes generated in this study. Please note the genomes will eventually be released on ENA/NCBI, the names they will be released under will be added here when applicable. the Prokka annotations for all the genomes included in this study (both novel and publicly available). the OrthoFinder file (Orthogroups.tsv) which lists what genes constitute each orthogroup, and which genomes they are found in. the OrthoFinder file (Orthogroups.GeneCount.tsv) which lists the copy number of each orthogroup in every genome. Table S1: Microsporidian genome assembly statistics and host metadata. Full list of recovered microsporidian genome assemblies, their associated metadata, and host metadata. Table S2: List of genomes used in this study. Genome accessions are listed where applicable. Text S1: Code snippet underlying species delineation. In the phylogeny, genomes are named “Genus_species_GCA_XXXX” where applicable. Fig. S1 Example of spurious Stringtie assembly. IGV (Robinson et al. 2011) snapshot of microsporidian genome from Noctua janthina, with PacBio reads and RNA-seq reads aligned to it. RNA-seq coverage islands represent genes undergoing expression. Some transcription at the intergenic regions is observed, with no evidence of split reads, indicating this is a case of polycistronic transcription and not multiple exons belonging to one long gene. Stringtie (version 3.0.1) (Pertea et al. 2015) spuriously assembles through many of the neighbouring genes, generating long polycistronic transcripts despite most transcription being monocistronic. Fig. S2 Example of spurious intron inferred by Stringtie. IGV (Robinson et al. 2011) snapshot of microsporidian genome from Noctua janthina, with RNA-seq reads aligned to it. Stringtie (version 3.0.1) (Pertea et al. 2015) spuriously infers an intron due to HISAT2 (version 2.2.1) (Kim et al. 2019) splitting some reads over repetitive regions, instead of extending the alignment. Note the beginning of the intron and the beginning of the following exon. Table S3: Summary of RNA-seq-based annotations for the three microsporidian genomes explored. Table S4: Performance of ab initio annotation tools on the three explored microsporidian genomes. The number of coding sequences identified from TransDecoder’s (GitHub: https://github.com/TransDecoder/TransDecoder) output, for each ab initio annotation, was calculated using MMSeqs2 (Steinegger and Söding 2017). Percentages represent the proportion of TransDecoder coding sequences retrieved. BUSCO scores were calculated using microsporidia_ob10, version 5.4.6 (Simão et al. 2015). CDS: coding sequences. Fig. S3: The topology of the microsporidian phylogeny depends on the gene alignment and phylogenetic inference methods employed. Four phylogenetic topologies, labelled by the methods used to generate them, are shown in the figure. The positions of Glugeida and Neopoereziida shift according the methods employed. Trimming, where used, was carried out using TrimAl (-gt 0.8 -st 0.001 -resoverlap 0.75 -seqoverlap 80, version 1.4.1) (Capella-Gutiérrez et al. 2009). Alignments were generated using MAFFT (--globalpair --maxiterate 1000, version 7.25) (Katoh et al. 2002), and the phylogenies were inferred using IQ-TREE (version 3.0.1) (Wong et al. 2026), or ASTRAL (version 5.7.8) (Zhang et al. 2018) in combination with IQ-TREE. All topologies show high support values, including the ASTRAL trees, despite gene concordance factors and site concordance factors varying dramatically. The genes underlying these trees are the BUSCO set (microsporidia_obd10, version 5.4.6) (Simão et al. 2015). The figure was generated using iTOL (Letunic and Bork 2024), and manually annotated with InkScape (version 1.4.4, inkscape.org). The genomes included are listed in Supporting Information Table S2. Table S5: List of orthogroups unique and conserved in each large microsporidian taxonomic group explored. ***The code underlying the analyses carried out in this study can be found on GitHub (https://github.com/Amjad-Khalaf/MicrosporidiaPangenome).