In the class Colpodea, there are many unresolved evolutionary relationships among taxa. Here, we report 30 new sequences including SSU-rRNA, ITS1-5.8S-ITS2 rRNA, and the mitochondrial small subunit ribosomal RNA (mtSSU-rRNA) genes of five colpodeans, and conduct phylogenetic analyses based on each individual gene and a two-gene concatenated dataset. For the first time, multi-genes were used to analyze phylogenetic relationships in the class Colpodea. The main findings are: (1) SSU-rRNA, ITS1-5.8S-ITS2 rRNA, and mtSSU-rRNA gene sequences of C. reniformis and C. grandis are provided for the first time, and these two species group into the clade including C. inflata, C. lucida, C. cucullus, and C. henneguyi; (2) clustering pattern and morphological similarity indicate that Bresslauides discoideus has a close relation with Colpodidae spp.; (3) Emarginatophrya genus diagnosis is improved to be 'Hausmanniellidae with sharply shortened and isometric leftmost 1-4 ciliary rows' and Colpoda elliotti is transferred to Emarginatophrya; (4) the genus Colpoda is still non-monophyletic with the addition of 10 populations from five Colpoda species sequences, but there are only two Colpoda groups left based on the present work: Group I comprises C. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis, Group II comprises C. maupasi and C. ecaudata, and the presence of diagonal grooves and the way the vestibular opens might be the two key features that differentiates Colpoda species groups;(5) a close molecular relationship, and highly similar merotelokinetal mode, somatic ciliary pattern, and basic organization of the oral apparatus with P. steinii suggests Bromeliothrix metopoides should be temporarily assigned to Colpodidae.

The application of molecular techniques to accurately identify protozoan species can correct previous misidentifications based on traditional morphological identification. Colpodea ciliates have many toxicological and cytological applications, but their subtle morphological differences and small body size hinder species delineation. Herein, we used Cox I and β-tubulin genes, alongside fluorescence in situ hybridization (FISH), to evaluate each method in delineating Colpodea species. For this analysis, Colpoda harbinensis n. sp., C. reniformis, two populations of C. inflata, Colpoda compare grandis, and five populations of Paracolpoda steinii, from the soil in northeastern China, were used. We determined that (1) the Cox I gene was more suitable than the β-tubulin gene as a molecular marker for defining intra- and interspecific level relationships of Colpoda. (2) FISH probes designed for Colpoda sp., C. inflata, Colpoda compare grandis, and Paracolpoda steinii, provided rapid interspecific differentiation of Colpodea species. (3) Colpoda harbinensis n. sp. was established and mainly characterized by its size in vivo (approximately 80 × 60   μ m ), a reniform body in outline, one macronucleus, its spherical shape, a sometimes nonexistent micronucleus, 11–15 somatic kineties, and five or six postoral kineties. In conclusion, combining oligonucleotide probes, DNA barcoding, and morphology for the first time, we have greatly improved the delineation of Colpodea and confirmed that Cox I gene was a promising DNA barcoding marker for species of Colpodea, and FISH could provide useful morphological information as complementing traditional techniques such as silver carbonate.