Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho-species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.