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 .

Hypotrichs are the most complex and highly differentiated ciliate lineages and have great potential for evolutionary novelties. Problems in hypotrich systematics are mainly due to discordance between the morphological and genetic data (mainly small subunit rRNA gene). Species with morphologies that are characteristic of two or more higher rank taxa are probably a major contributing factor to these conflicts. The present study describes a Chinese population of a poorly known organism with numerous endosymbiotic zoochlorellae, the morphology of which corresponds well with the type population of Limnoholosticha viridis (Kahl, 1932) Li et al., 2017. Newly obtained information shows this species has a zigzag midventral cirral pattern that is diagnostic of urostylids, whereas the dorsal ciliature shares features (presence of dorsomarginal kinety and dorsal kinety 3 fragmentation) that are typical of oxytrichids. Molecular phylogenetic analyses reveal a close relationship with oxytrichids. An integrative approach combining morphological, morphogenetic, phylogenetic and ecological analyses indicates that L. viridis represents a new genus and new family which might be an intermediate form between uorstylids and dorsomarginalians. Thus, Bourlandellidae fam. nov. and Bourlandella gen. nov. are here established. Lastly, we speculate that phenotypic convergence and mixtrophy might confer on the new combination, Bourlandella viridis (Kahl, 1932) comb. nov., the ability to adapt to a wide range of environmental conditions.

ATP-binding cassette subfamily B (ABCB) has been implicated in various essential functions such as multidrug resistance, auxin transport and heavy metal tolerance in animals and plants. However, the functions, the genomic distribution and the evolutionary history have not been characterized systematically in lower eukaryotes. As a lineage of highly specialized unicellular eukaryotes, ciliates have extremely diverse genomic features including nuclear dimorphism. To further understand the genomic structure and evolutionary history of this gene family, we investigated the ABCB gene subfamily in 11 ciliates. The results demonstrate that there is evidence of substantial gene duplication, which has occurred by different mechanisms in different species. These gene duplicates show consistent purifying selection, suggesting functional constraint, in all but one species, where positive selection may be acting to generate novel function. We also compare the gene structures in the micronuclear and macronuclear genomes and find no gene scrambling during genome rearrangement, despite the abundance of such scrambling in two of our focal species. These results lay the foundation for future analyses of the function of these genes and the mechanisms responsible for their evolution across diverse eukaryotic lineages.