Genes of interest were identified from previously published studies of anaerobic mitochondria, the published mitochondrial proteome of Tetrahymena thermophila [121], and recent publications on mitochondrial metabolism of Apicomplexa [15, 51, 61, 65, 75, 85, 122124]. Candidate sequences from our gregarine data and other publicly available genome-scale datasets for apicomplexans and their close relatives (Additional file 1: Table S1) were recovered by BLAST using representative seed sequences and collecting hits with e-value <1e−5 were extracted and added to the initial datasets for each gene. When available, datasets published by Rotterova et al. [61, 124] were used as starting datasets. Alternatively, datasets for phylogenetic analysis were constructed by using BLAST searches of the seed sequences against nr (max target seqs = 100) to thoroughly sample apicomplexans and other close relatives. These BLAST results were supplemented with sequences selected from gene name search results from the NCBI protein and RefSeq databases to ensure broad eukaryotic and prokaryotic taxonomic sampling and were then clustered using CD-Hit [125] at 75%. Each gene dataset was filtered for sequencing errors and non-homologous sites using PREQUAL, aligned using MAFFT (--localpair --maxiterate 1000), and alignment uncertainties and errors were filtered using DIVVIER (-partial -mincol 4 -divvygap) and single gene trees were constructed using IQtree (LG+C20+F+G) with 100 RAxML rapid bootstraps using constructed under the PROTGAMMALG4X model. Each alignment/tree has been repeatedly inspected by the eye, and sequences were added/removed as necessary. Ultimately, each tree was manually inspected, enabling precise inference of the presence/absence of proteins involved in mitochondrial metabolism in the studied lineages. Prediction of subcellular localization for all sequences identified as apicomplexan homologs in this study was conducted in TargetP v2 [126] and DeepLoc [127]. Files containing all initially considered sequences, final datasets, final alignments, sequences of all selected Apicomplexan homologs, the resulting trees (raw and colored to show selected apicomplexan homologs), and the subcellular localization prediction results for all mitochondrial genes discussed in this study are available on figshare [128].

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