**4. Future perspectives**

The analytical improvement for morphological, ultrastructural, molecular, and evolutionary characterizations in Ciliophora culminated in an "Age of Integration", which several disciplines interact to infer patterns of biodiversity [184]. Although it is an age in full expansion, several gaps often prevent a study of diversity in its diverse areas in a complete way.

We are in a period of the paradigm shift, where Next Generation Sequencing (NGS) techniques have been applied exponentially, and, therefore, it is expected that new discoveries will emerge and new panoramas will be drawn on the diversity of the strains, as well as their respective ecological interactions. The transition from phylogenetic studies to phylogenomics is based on technological progress combined with exponential sequencing of molecular sequences (DNA, RNA), reduced associated costs, increased computational capacity, and improved analytical protocols. It is important to make efforts in studies to expand such technologies to lineages with little sampling in databases. For example, the classes Prostomatea, Oligohymenophorea, Litostomatea, and Phyllopharyngea, which present several examples of symbiosis, do not have available molecular sequences which prevents the evolutionary inferences of these lineages, requiring in the future more studies to refine the evolutionary hypotheses about the phylum. Efforts to expand metataxonomy using metagenomics and metatranscriptome methods have fed the databases exponentially in several lineages, revolutionized the analysis of environmental microbial diversity [175, 185, 186]. In fact, the generation of data for the target sequencing of phylogenetic, metagenomic, and metatranscriptomic markers is now reasonably well established, and several DNA sequencing platforms based on different technologies are currently available as well as different bioinformatics programs for each level of data extraction. However, due to the limited size of the molecular sequences produced by the platforms (~ 500 bp), phylogenetic estimates may be inadequate. With longer readings comes an improved phylogenetic signal, and we show that it is possible to employ a complete phylogenetic signal approach to taxonomically classify sequences and obtain a robust evolutionary structure of environmental diversity. New sequencing technologies such as nanopore sequencing, which offer long reads, improved the phylogenetic signal and more robust taxonomic patterns, can be an alternative in future studies [187].

With the significant increase in the number of available sequences from NGS sequencing, more effective and less subjective methodologies have been proposed to define the limits and number of independent evolutionary entities, to accelerate the biodiversity assessment process. In the last two decades, the field of species delimitation has intensified in relation to the number of methods available. For this, several methodologies have been proposed, based on biological [188], ecological [189], and molecular data [190], in addition to combining phylogenetic theory and population genetics [191–193]. The use of these methodologies in ciliates performed very recently to delimit organisms of free life, as species of the genus *Frontonia*, using the mitochondrial gene COX1 [194], species of the genus *Spirostomum*, using the ITS spacer region genes [195], and COI and 18S markers of the *Paramecium* genus.

Finally, several authors have emphasized the lack of studies on the distribution and occurrence of ciliates associated with Metazoa in natural conditions and the the lack of information on the ecology and interactions between epibionts and hosts. Few studies are exploring the natural history and complexity of life cycles, which makes it difficult to characterize optional and mandatory relationships. The absence of the characterization of the ciliate at the stage it is in the host, most studies, only in the environment, making it difficult to characterize the life cycle. Relevant information about habitat, life cycle, infection site is rare for Ciliophora [160, 196, 197].
