Dictyostelid species classification has traditionally relied on morphology, a time-intensive method requiring expert knowledge. This study evaluated the potential and limitations of using the 18S rDNA sequence for species-level classification. 18S rDNA sequences of 16 samples from the Dicty stock center, including 14 samples found in Thailand, were analyzed. Signature sequence analyses confirmed genus-level identification with high accuracy. These sequences were analyzed alongside 309 database entries retrieved from the GenBank database. The analyses confirmed genus-level identification accuracy but highlighted challenges in distinguishing species due to overlapping intraspecific and interspecific variations, negative barcoding gaps, and incorrectly grouped samples to putative taxa by species delimitation analyses. Species delimitation methods, including maximum likelihood (ML) phylogenetic analysis, achieved limited success, with ML showing the highest accuracy but not exceeding 50%. However, species with high barcoding gaps, such as Raperostelium and Rostrostelium, demonstrated potential for accurate classification. These findings support using 18S rDNA for genus-level identification and suggest its possible application for certain species. Expanded sampling is needed to improve species-level classification and to identify more robust DNA markers for dictyostelid diversity studies.

Dictyostelium intermedium is a member of dictyostelids, the unicellular eukaryotes with a unique life cycle, including a social cycle. Despite the high diversity of dictyostelids, only five species' complete mitochondrial genome sequences were reported. This study aimed to add the D. intermedium mitochondrial genome sequence to the list. The size of this genome is 58,627 bp, with 73.99% A/T, containing 62 genes located on one strand: 41 protein-coding genes, three ribosomal RNA genes, and 18 transfer RNA genes. The 41 protein-coding genes comprised 18 oxidative phosphorylation-related, 16 ribosomal, and seven hypothetical protein-coding genes. The cox1/2 and rnl gene contained introns, similar to other species of Dictyostelium. The phylogenetic tree built based on 34 protein sequences supported the monophyletic clade of Dictyostelium and the dictyostelids' ancestor's position between the two dictyostelids orders: Dictyosteliales and Acytosteliales.