2.3. Annotation of Mitochondrial Genome and Predictions of Secondary Structures

The MITOS2 web server (http://mitos2.bioinf.uni-leipzig.de/index.py, accessed on 18 February 2023) [24] was used to predict the protein-coding genes (PCGs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs). The annotation of the tRNAs was confirmed using ARWEN 1.2 (http://130.235.244.92/ARWEN/index.html, accessed on 18 February 2023) [25]. Protein-coding genes were manually corrected by searching for open reading frameworks (ORFs) with the NCBI tool ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 18 February 2023). Secondary structure of the RNAs was inferred using R2DT (https://rnacentral.org/r2dt, accessed on 19 February 2023) [26] and manually optimized using the 12S rRNA Eukaryote reference model [27] and the 16S rRNA Mollusca and Toxoplasma gondii (Nicolle & Manceaux, 1908) reference models [28,29]. Potential secondary structure of the control region (CR) was predicted using the RNAfold web server (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi, accessed on 19 February 2023). All secondary structures were drawn using Inkscape 1.1 (http://www.inkscape.org/, accessed on 25 February 2023). The mitochondrial genome maps of both species were drawn using the CGView tools [30] available at the web server Proksee (https://proksee.ca/, accessed on 26 February 2023). The mitochondrial genome and annotation files of P. canaliculata ({"type":"entrez-nucleotide","attrs":{"text":"KJ739609.1","term_id":"659681505","term_text":"KJ739609.1"}}KJ739609.1), P. diffusa ({"type":"entrez-nucleotide","attrs":{"text":"MF373586.1","term_id":"1565054134","term_text":"MF373586.1"}}MF373586.1), P. maculata ({"type":"entrez-nucleotide","attrs":{"text":"MF401379.1","term_id":"1624800071","term_text":"MF401379.1"}}MF401379.1) and P. occulta ({"type":"entrez-nucleotide","attrs":{"text":"KR350466.1","term_id":"854884015","term_text":"KR350466.1"}}KR350466.1) were retrieved from GenBank. We performed a re-annotation of these mitochondrial genomes using MITOS2 Web Server [24] and ORFfinder to verify current annotations.