The phylum Ciliophora plays important roles in a wide range of biological studies. However, the evolutionary relationships of many groups remain unclear due to a lack of sufficient molecular data. In this study, molecular dataset was expanded with representatives from 55 orders and all major lineages. The main findings are: (1) 14 classes were recovered including one new class, Protocruziea n. cl.; (2) in addition to the two main branches, Postciliodesmatophora and Intramacronucleata, a third branch, the Mesodiniea, is identified as being basal to the other two subphyla; (3) the newly defined order Discocephalida is revealed to be a sister clade to the euplotids, strongly suggesting the separation of discocephalids from the hypotrichs; (4) the separation of mobilids from the peritrichs is not supported; (5) Loxocephalida is basal to the main scuticociliate assemblage, whereas the thigmotrichs are placed within the order Pleuronematida; (6) the monophyly of classes Phyllopharyngea, Karyorelictea, Armophorea, Prostomatea, Plagiopylea, Colpodea and Heterotrichea are confirmed; (7) ambiguous generaAskenasia,CyclotrichiumParaspathidiumandPlagiocampashow close affiliation to the well known plagiopyleans; (8) validity of the subclass Rhynchostomatia is supported and (9) the systematic positions of Halteriida and Linconophoria remain unresolved and are thus regarded asincertae sediswithin Spirotrichea.

Recent advances in high-throughput sequencing and metabarcoding technologies are revolutionizing our understanding of the diversity and ecology of microbial eukaryotes (protists). The interpretation of protist diversity and the elucidation of their ecosystem function are, however, impeded by problems with species delimitation, especially as it applies to molecular taxonomy. Here, using the ciliate Euplotes as an example, we describe approaches for species delimitation based on integrative taxonomy by using evolutionary and ecological perspectives and selecting the most appropriate metabarcoding gene markers as proxies for species units. Our analyses show that: Euplotes ( sensu lato ) comprises six distinct clades, mainly as result of ecological speciation; the validity of the genera Euplotes ( sensu stricto ), Euplotoides , Euplotopsis and Moneuplotes are not supported; the vannus -type group, which includes species without distinct morphological differences, seems to be undergoing incipient speciation and contains cryptic species; the hypervariable V4 region of the small subunit rDNA and D1–D2 region of the large subunit rDNA are the promising candidates for general species delimitation in Euplotes .

The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.

Pathogenic scuticociliates, which are common in the haemolymph or tissues of maricultured animals and often cause serious diseases, are a species-rich assemblage with mostly unresolved systematic relationships, especially in some less-studied groups. In the present study, we sequenced the small-subunit rRNA gene of six species of scuticociliates, Uronemella parafilificum, Metanophrys sinensis, Parauronema longum, Cohnilembus verminus, Porpostoma notata and Ancistrum crassum, the last two of which have not been studied previously using molecular analyses. Phylogenetic trees were constructed using Bayesian inference, maximum-likelihood and maximum-parsimony methods to assess the inter- and intra-generic relationships of scuticociliates. Results revealed the following: 1) Porpostoma did not cluster with the Philasteridae, Cohnilembidae or any other family of the order Philasterida; 2) sequences of Uronemella parafilificum and Uronemella filificum showed a difference of 1.02 % (15 nt sites), revealing a close relationship between them; 3) the approximately unbiased test rejected monophyly of both Metanophrys and Parauronema, indicating that the terminal position of the anterior end of the paroral membrane and the structure of membranelle 1 are unreliable characters for distinction of genera in this group of scuticociliates; 4) Ancistrum crassum grouped with Boveria subcylindrica, showing a close phylogenetic relationship between the orders Thigmotrichida and Pleuronematida; and 5) Parauronema longum, Cyclidium plouneouri and Cyclidium porcatum should be removed from their currently assigned genera.