**3. Ecological relationships: evolutionary approach**

From an evolutionary point of view, there are species that are entirely free-living, those which can live equally well both free or as symbionts, species that are almost entirely symbiotic with only occasional periods of "free" existence during their life cycles (facultative symbionts), and species which are entirely symbiotic (obligate symbionts). Most of the well documented associations between Ciliophora and Metazoa are the ones leading to a certain degree of metabolic dependence. We will use in this topic the idea of metabolic dependence to define the ecological relationships: "free-living" (no metabolic dependence), "epibiont" (facultative metabolic dependence), "mutualistic" (mutual metabolic dependence) or "parasitic" (unilateral metabolic dependence, including commensalism).

For many years the evolutionary studies for Ciliophora were based only on morphological data, mainly those related to the ultrastructural characterization of its complex infraciliature [2]. However, in recent years this scenario has been modified with the implementation of modern tools that use multidisciplinary methods to integrate morphological, phylogenetic, molecular, and ecological data [161, 172–174]. A reliably dated phylogeny is fundamental to infer a broad macroevolutionary scenario for Ciliophora [172]. The inference of diversification rates from molecular phylogenies has increasingly been used to derive macroevolutionary patterns of lineages. Understanding how the different ecological relationships evolve in Ciliophora along time is a complex task that has been developed for many years. Different hypotheses

about the origin and evolution of parasitic life have been proposed. Parasitologists suggest that the symbiotic way of life probably descended from free-living lineages that subsequently adapted to life in special habitats. Besides this, several authors suggest multiple origins of parasitism based on a comparison of morphological and ultrastructural aspects between them and their free life co-specifics [175], however, the processes that lead to its emergence are still imprecise [176–178].

Concerning the phylum Ciliophora, the vast majority of ciliates are categorized as free-living, and studies suggested that symbiosis apparently arising independently among various classes [179]. For genus *Tetrahymena* (subclass Hymenostomatia, order Hymenostomatida), all gradations of adaptations to symbiosis occur. There are species that live totally free, those that can live equally well both free and as symbionts, species that are almost entirely symbiotic with only occasional periods of "free" existence during their life cycles (optional symbionts), and species that are totally symbiotic (mandatory symbionts) [180]. Different transition routes between ecological associations have also been proposed, based on morphological and ecological characteristics. The first one proposes that free-living organisms assume habits of low metabolic dependence (mutualism, commensalism, among others), and with the strengthening of relationships, where they become parasites [176, 181]. The second hypothesis suggests that a free-living organism, when it comes into contact with a host accidentally, adapts itself to live both freely and within that host (optional parasite) [179], that is, free-living organisms adapt to live inside a host, which becomes something advantageous and increases fitness, making this a favorable way of life for the species.

Previous studies aimed to test these hypotheses based on phylogenetic analyzes of small groups within Ciliophora [174, 182, 183]. The macroevolutionary analyzes from the whole Ciliophora phylogeny presented **Figure 1** suggested that the ancestral way of life of the ciliates originated from a free-living organism and that the parasitic way of life arose numerous times and independently in Ciliophora, which was induced by two types of ancestors, free life and mutualistic (**Figure 1**). The transition to the parasitic way of life was recovered from two different origins: 1) a free-living ancestor evolved into a mutualistic organism and, later, to a parasitic organism, and 2) a free-living ancestor evolved into an organism parasite (highest number of cases). There are also cases where there has been a regression in the ciliate's way of life, where parasite clades evolved to free-living clades (**Figure 1**).

#### **Figure 1.**

*Ancestral habit reconstruction for Ciliophora showing the main routes of transitions. Blue: Free-living. Yellow: Mutualism. Red: Parasitism/commensalism.*
