Protists are the most diverse and widespread eukaryotes (de Vargas et al., 2015; Adl et al., 2019; Sieber et al., 2020). They perform important ecosystem functions and play a fundamental part in aquatic food webs (Weisse et al., 2016). Phagotrophic protists (those that utilize food through phagocytosis) have recently been shown to be even more diverse than their phototrophic counterparts (Singer et al., 2021). As the most important microbial predators in aquatic and soil ecosystems, phagotrophic protists transfer carbon and energy between micro- and macrofauna, significantly influencing ecosystem-level processes (Sherr and Sherr, 1984, 2002; Risse-Buhl et al., 2015; Geisen et al., 2017).

Due to their ubiquity, abundance and sensitivity, phagotrophic protists have been recognized as excellent bioindicators in a variety of environments (Payne, 2013). Ciliates (Ciliophora) and testate amoebae (Amoebozoa and Rhizaria) are very common groups of phagotrophic protists in freshwater and soil ecosystems (Singer et al., 2021). They are also the best-studied protists, partly because they are easy to isolate and have a distinctive morphology (Foissner, 1999; Todorov and Bankov, 2019). Naked amoebae, on the other hand, are understudied due to their smaller size and a lack of knowledge on their morphological features, although they dominate many protist communities, particularly in periphyton and benthos, as well as in soil (Coleman and Wall, 2015; Geisen, 2016; Singer et al., 2021).

Different protist groups may play opposing or complementary roles in ecosystem functioning. Thus, analyzing their functional diversity (FD) may enhance our understanding of various protist traits and their role in the ecosystem (Dumack et al., 2020). Functional diversity analysis is based on linking ecosystem processes to species’ functional traits, i.e., organisms’ characteristics that influence not only their fitness, but also their interactions with the environment (Nock et al., 2016). Trait-based approaches offer an opportunity to understand the relationship between microbial diversity and ecosystem functioning better than taxonomy-based approaches, especially since taxonomic and functional diversity are not always linearly correlated (Laliberté and Legendre, 2010). However, the trait-based approach has rarely been applied to protists (Weisse, 2017; Fiore-Donno et al., 2019).

Phagotrophic protists have already been recognized as good bioindicators of the karst environments’ (e.g., tufa-depositing lakes, rivers, and streams) quality (Primc-Habdija et al., 2001; Matoničkin Kepčija et al., 2011; Kulaš et al., 2021). A recent study by Gulin et al. (2021) showed that periphyton responds to revitalization of dry tufa-depositing streams, opening new possibilities for the application of periphytic organisms as proxies of the stream revitalization success. The study focused on evaluating the response of periphyton based on sampling natural substrates, i.e., stream substrate containing tufa particles, which allowed analysis not only of the abundance and diversity of periphytic taxa, but also of the granulometric and mineral constituents of the substrate itself. However, the method of sampling with a corer proved too aggressive for periphytic organisms, resulting in low abundance and diversity of periphyton. In contrast, the present study is an improvement, because it is based on evaluating the response by monitoring periphyton development on artificial substrates over time so that the periphyton can be studied directly without damaging it. It focuses on phagotrophic protists (ciliates and amoeboid protists) and analyses both their taxonomic and functional diversity. Since periphyton exhibits complex spatio-temporal colonization trends (Franco et al., 1998; Risse-Buhl and Küsel, 2009), 1 and 2-months immersion periods were covered.

Our first objective was to assess whether taxonomic (abundance, species richness and diversity) and functional (i.e., functional diversity) metrics of ciliates and amoeboid protists differed between control (C) sites (located within permanent streams) and revitalized (N) sites (located within reactivated streams). We expected higher abundance and diversity (taxonomic and functional) of ciliates and amoeboid protists at revitalized sites than at control sites. This was based on the fact that conditions that prevail at reactivated streams due to newly created waterways and soil drainage, represent a positive disturbance, which can improve the diversity of habitats, species and their survival strategies (Timoner et al., 2014). A better understanding of the effects of disturbance on functional diversity is critical because functional diversity is likely related to ecosystem resilience, i.e., the capacity of a system to absorb shocks, reorganize, and maintain the same structure and function (Walker, 1995).

The second objective was to determine the influence of several environmental factors (flow velocity, temperature, dissolved oxygen concentration, pH, conductivity, alkalinity, total water hardness, concentrations of nitrites, nitrates and orthophosphates, and chemical oxygen demand) on the taxonomic and functional metrics of ciliates and amoeboid protists. Some of these factors have already shown a significant effect on the assemblage metrics of periphytic taxa in revitalized tufa streams (Gulin et al., 2021). Based on previous findings, we expected several organic matter associated factors (e.g., chemical oxygen demand, orthophosphates) to increase the abundance and taxonomic diversity of phagotrophic protists while others (e.g., nitrites) were expected to yield the opposite (decreasing) effect (Gulin et al., 2021).

Our third objective was to estimate how periphyton-associated factors (organic and inorganic matter (tufa) content, chlorophyll a concentration) affect the taxonomic and functional metrics of ciliates and amoeboid protists. Based on the findings of Gulin et al. (2021), we expected that increased organic matter content (and subsequently chlorophyll a concentration) would lead to increased abundance of protists. On the other hand, we expected that increased inorganic matter (i.e., tufa) content would decrease the taxonomic and functional diversity of protists as intense tufa deposition can lead to detachment and sloughing (Pitois et al., 2001, 2003).

Finally, our fourth objective was to examine the extent to which duration of immersion in water influences changes in taxonomic and functional metrics of ciliates and amoeboid protists at revitalized sites. We expected to observe a trend of increasing values of taxonomic and functional metrics with longer immersion duration, for both ciliates and amoeboid protists from revitalized sites.

The links between ecosystem functioning and the taxonomic and functional diversity of phagotrophic protists could be fundamental to resource management and conservation planning, particularly in protected and sensitive areas such as national parks. The results of this research would make an important contribution to understanding fragile and complex tufa-depositing ecosystems.

Our results revealed that ciliates and amoeboid protists respond differently to stream revitalization at the assemblage level. While ciliate assemblages at N sites had higher abundance and taxonomic diversity compared to those at C sites for both immersion periods, taxonomic metrics of amoeboid assemblages differed little between sites, even with longer immersion. The two protist assemblages also differed in their functional response: while the functional diversity of ciliates was lower at N sites compared to C sites for both immersion periods, that of amoeboid protists was higher at N sites for the 1-month period but lower for the 2-months period, in comparison to C sites.

The lower functional diversity of ciliates at N sites compared to C sites for both immersion periods suggests that the level of disturbance in reactivated streams, in the form of occasionally changing hydrologic conditions (high flow or drought), was either too high or too low to promote functional diversity, judging by the intermediate disturbance hypothesis (IDH). IDH rests on the assumption that a maximum level of diversity is achieved when the assemblage is exposed to an intermediate level of disturbance (Connell, 1978). It is likely that in our case, disturbance filtered out incompatible suites of traits, so that only a subset of species with disturbance-resistant traits could colonize a disturbed habitat (Lavorel et al., 2008; Biswas and Mallik, 2010). Thus, compared to C sites, ciliate assemblages at N sites could be expected to consist mainly of prospective colonizers, with disturbance-intolerant species (r-strategists) absent or their share in the assemblage minimized, leading to increased similarity and functional redundancy within the assemblage (Segovia et al., 2016). On the other hand, it appears that amoeboid assemblages at N sites during the 1-month period consisted mainly of highly tolerant or even drought-resistant species (K-strategists) that would thrive under hydrologically extreme conditions (Carballeira and Pontevedra-Pombal, 2021), in contrast to the r-strategists that probably dominated the 2-months period, leading to differences in functional diversity between the two periods.

Our results suggested that the reasons for these different responses to stream revitalization in terms of taxonomic and functional metrics could also lie in the effects of environmental and periphyton-associated factors. While some factors had a similar effect on both assemblages, others affected the assemblages quite differently. The overall observed positive effect of COD, pH, and organic matter content on taxonomic metrics of both ciliates and amoeboid protists is consistent with previous findings by numerous authors who have worked with these protists (Cowling, 1994; Smith, 1996; Verhoeven, 2002; Bates et al., 2013; Bradford, 2016; Geisen, 2016; Fiore-Donno et al., 2019). The likely reason for this is the increased diversity and quantity of bacteria that are their main food source, as the deposited organic material supports attachment of bacteria that decompose both dissolved and particulate organic matter (Foissner, 2014). However, the dominance of bacteria as a food source could have led to functional homogeneity and explain why functional diversity of both ciliates and amoeboid protists was negatively affected by COD and/or organic matter.

Inorganic material, i.e., tufa, on the other hand, had a different effect. While it positively affected the taxonomic and functional metrics of both protist groups for the 1-month period, the same was not observed for the 2-months period. It appears that the higher inorganic matter content during the 2-months period, whose deposition was facilitated by slightly higher temperature, conductivity, and flow velocity at N sites (Supplementary Table 3), negatively affected the ciliate taxonomic and functional metrics, but did not cause any negative effects on those of the amoeboid assemblages. Although the presence of inorganic matter can increase microhabitat heterogeneity (Beisel et al., 2000; Bednar et al., 2017; Singer et al., 2021) and thus serve as an “inoculum” for periphyton supporting high abundance and diversity (Primc and Habdija, 1987; Wörner et al., 2000; Zimmermann-Timm, 2002), there appears to be a critical point at which inorganic matter content no longer promotes high abundance and taxonomic diversity of ciliates. It could be that ciliates are much more sensitive to burial and sloughing than amoeboid protists because they do not predominantly glide along the substrate as amoebae do but exhibit various modes of locomotion: many of them are free-swimmers or even move by jumping and rotating (Foissner and Berger, 1996; Esteban and Fenchel, 2021). Amoeboid protists (especially naked amoebae) are also smaller in size compared to ciliates, which allows them to savor the microhabitats and be very closely associated with the substrate (Preston, 2003).

The two assemblages also responded differently with respect to immersion duration: while ciliate taxonomic and functional metrics at N sites increased with longer immersion, the same was observed only for some taxonomic metrics of amoeboid protists (species richness and True diversity values), while the abundance decreased, and functional metrics remained more or less the same. Since chloropyhll a concentration showed significant correlations with most taxonomic and functional metrics of both assemblages and reached extreme values at times during the 2-months period at N sites, it is reasonable to assume that the different responses to immersion duration could be due to this. Although the observed orthophosphate levels during the 2-months period were still quite low and within the range characteristic of tufa-depositing streams (Primc-Habdija et al., 2005), they may have caused algal proliferation and subsequently higher chlorophyll a concentration. The dense algal coating of the substrate may have contributed to heterogeneity of food sources and microhabitats (Algarte et al., 2017) which together led to niche diversification, an increase in the share of disturbance-resistant ciliate species and subsequently an increase in taxonomic and functional diversity. Another possible explanation could be that changing hydrological conditions at N sites activate the dormant part of the microbial assemblages, leading to increased functional diversity over time (Velasco-González et al., 2020). On the other hand, the alginate covering the bacteria likely triggered a different response in the amoeboid assemblages, leading to reduced or even absent uptake of bacteria by amoeboid protists. This is likely due to the alginate coating increasing the overall size of the bacterial particles or altering their “flavor” (Heaton et al., 2001; Parry, 2004), resulting in reduced abundance of amoeboid protists during the 2-months immersion period.

The taxonomic and functional diversity of protists is a valuable indication of environmental conditions and ecosystem stability. Understanding the factors that structure protist assemblages is a prerequisite for using these organisms to predict environmental quality and future changes in ecosystem functioning. Protists have been used in environmental monitoring, but only to a limited extent because of the difficulties associated with sophisticated morphological identification methods. However, comprehensive studies that also consider their functional traits (Fournier et al., 2012) could make them more recognizable and increase the frequency of using protists as indicators of ecosystem functioning.

Linking taxonomic and functional metrics to environmental conditions can improve our understanding of biological processes, especially in ecosystems undergoing extensive changes, such as revitalized streams. Our results showed that taxonomic and functional metrics of ciliates and amoeboid protists responded to the prevailing conditions characteristic of revitalized tufa-depositing streams: changing hydrology (occasional high flow or drought), soil drainage, and extensive inorganic matter, i.e., tufa deposition, although their responses were somewhat different. While both protist assemblages benefited from COD and the presence of organic matter supported by favorable pH, as this provided their main food source–bacteria, ciliates were more sensitive to the presence of inorganic matter content than amoeboid protists. However, in terms of chlorophyll a concentration, ciliates appeared to benefit more from the algal coating as it resulted in heterogeneity of food sources and microhabitats, which increased their taxonomic and functional diversity, while it decreased the uptake of bacteria for amoeboid protists, negatively affecting their abundance. The two assemblages also showed different responses of taxonomic and functional metrics with respect to immersion duration: while the taxonomic and functional diversity of ciliates at N sites increased with longer immersion, indicating niche diversification, those of amoeboid protists hardly changed with time. The results presented suggest that a comprehensive analysis of taxonomic and functional metrics of ciliates and amoeboid protists could be a good proxy for assessing revitalization of tufa-depositing streams. However, the temporal component should always be considered when conducting such studies, as the colonization processes of ciliates and amoeboid protists are quite complex, especially in tufa-depositing streams.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

VG, MSP, and RMK contributed to conception and design of the study. VG and BV organized the trait database. VG and FR performed the statistical analysis. VG wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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