A Comprehensive Mitochondrial Cytochrome c Oxidase Subunit II Gene (cox2)-Based Framework for Genotyping and Phylogenetic Analysis of Pythium insidiosum and Related Taxa

Pythium insidiosum is an important oomycete pathogen that causes pythiosis, a severe infectious disease affecting humans and animals worldwide. Understanding its genetic diversity is essential for accurate species identification, genotyping, and molecular characterization. Although the ribosomal DNA internal transcribed spacer (ITS) region is widely used as a DNA barcode, its limited variability can restrict fine-scale genotyping, highlighting the need for alternative genetic markers. Previous studies demonstrated the utility of the mitochondrial cytochrome c oxidase subunit II gene (cox2) for molecular identification and phylogenetic analysis of P. insidiosum. However, a comprehensive global assessment of cox2 sequence diversity has not been conducted. In this study, we established a comprehensive cox2 sequence dataset representing diverse P. insidiosum isolates from multiple hosts and geographic regions. A total of 249 unique cox2 sequences were analyzed, including 171 retrieved from the NCBI database and 78 newly generated from our culture collection. Using a standardized 387-bp cox2 fragment, phylogenetic analysis resolved four major phylogenetic clades (I–IV), with additional subclade resolution. Sequence alignment identified an 18-nucleotide signature motif for genotype differentiation. A 98.5% sequence identity threshold was used as a practical guideline for sequence screening and species-level identification. Clade II was the most prevalent lineage, accounting for 120 of 249 isolates (48.2%), and was further resolved into two distinct subclades (IIa, n = 82; IIb, n = 38), indicating substantial genetic diversification within the clade. Collectively, these findings establish cox2 as a robust molecular marker and provide a comprehensive reference dataset for P. insidiosum genotyping. This resource supports species identification, genotyping, and molecular characterization.