Over the last decades, lakes have been exposed to environmental changes (anthropogenic stressors and natural changes) with important implications on the biological communities. Recent paleolimnlogical studies have provided new insights on the long-term responses of microbial assemblages by using DNA-based methods15. Although previous paleolimnological investigations of overall microeukaryotes communities reported a strong response of the ciliates to nutrients inputs13,33, specific long-term changes in the ciliates communities structure and functional ecology had not yet been investigated. The present study represents the first paleolimnological reconstruction of ciliate communities and demonstrate the potential of using heterotrophic protists as indicators of environmental change at the community and functional levels.
Analyses of the ciliate communities indicate an overall decline in the β-diversity in recent times following the same trends as the overall micro-eukaryotes diversity18. Through the use of primers specifically targeting ciliates, our study brings an innovative perspective and provides additional information by highlighting changes in some heterotrophic and mixotrophic communities that were not previously revealed in the analysis of the overall microeukaryote communities using generalist primers. Interestingly, the ciliates did not undergo a large turnover but rather our results indicate a spatial homogenization of the diversity with a reorganisation of the community structure (i.e. switch in dominance) (Figs. 2 and 3). This spatial homogenization of the ciliate communities was marked by the replacement of three clusters by one homogeneous community dominated by Haptoria across all lakes. Biotic homogenization of communities is a well documented phenomenon that has been observed in terrestrial and aquatic ecosystems largely influenced by a reduction of environmental heterogeneity and the availability of diverse ecological niches34–36. In aquatic ecosystems, change in climate and productivity, and anthropogenic alteration of the watershed are the most prevalent causes of biotic homogenization37. Several of our studied lakes are exposed to similar stressors which includes nutrient enrichment, agricultural and urban development of watershed and climate change38–41. As such, our results support that these factors acted as deterministic filters selecting for a more homogeneous group of species dominated by generalist ciliates that display more flexible life strategies.
A stronger restructuration of the ciliate communities was observed in the low elevation lakes demonstrating that environmental changes in lowland lakes impacted several trophic levels, including non-photosynthetic protist communities. The geographical variation in the amplitude of changes in diversity and community turnover of microorganisms associated with an elevation gradient have been previously demonstrated in terrestrial42 and aquatic ecosystems43. These patterns are explained by the more pronounced human footprint in lowlands42,44, including nutrient-enrichment in freshwater ecosystems6. Supporting this trend, the present day trophic status of our studied lakes was significantly higher for lowland lakes than for high elevation lakes (Supplemental Fig. S3). As such, human-induced nutrient increase might have influenced the observed changes in the ciliate community diversity of lowland lakes.
Changes in the dominant functional groups of ciliates in the 48 studied lakes highlighted modifications of the physico-chemical conditions and biotic interactions of the pelagic and benthic zones.
The recent increase in the ciliates that can display mixotrophic life strategies whereby ciliates harbor algal endosymbionts or sequester plastid from their prey45, suggests that mixotrophic ciliates are directly or indirectly responding to new environmental conditions45. Interestingly, mixotrophic organisms tends to benefit from a more flexible nutrition strategy and have been found to prone during transition phases between autotrophy-dominated and heterotrophy-dominated systems46,47. As such, they can easily adapt to the more frequent exposure to extreme events or highly-variable environmental conditions that have occurred in lakes over the last decades48. Moreover, Limnostrombidium (synonym of Strombidium, from marine waters) is a common freshwater genus regrouping specialists mixotrophic species that preferentially feed and use the chloroplast of picophytoplankton31,45,49. Their significant increase in recent time thus indicates that recent changes in autotrophic picoplankton dynamic and structure, that have been previously recorded in some of our studied lakes16, might have provided them with a competitive advantage. Importantly, however, more empirical studies are still needed in order to solidify inference made between the relative increase in mixotrophic life strategies and associated ecological conditions.
Mixotrophic ciliates play a major role in foodweb structure of freshwater lakes24,50. The mixotrophic ciliate can contribute to the enhancement of the primary production51, while as prey, they represent a more direct transfer of the solar energy to the zooplankton24. Through this process, mixotrophic ciliates can enhance the efficiency of carbon transfer and energy flow in the food web24. Their recent increase thus suggests that lakes might have undergone important trophodynamic changes as mixotrophy is becoming an increasingly important pathway in aquatic food webs. These results also highlight the importance of integrating the mixotrophic component in food web modelling and carbon flow studies52.
Change in the benthic ciliate communities indicates that the benthic environment has also been impacted by recent environmental changes. The significant increase in the facultative or obligate anaerobic ciliate associated with the benthos, such as Metopus, suggests that the ciliate communities have been directly influenced by the widespread deoxygenation of temperate lakes5 (Fig. 5 and Supplemental Fig. S2). The significant decline in the benthic and hypolimnitic ciliates associated with well-oxygenated conditions further support that the habitability of the sediment-water interface has been declining for this particular group of ciliates. The depletion of oxygen concentrations in the profundal zone of freshwater lakes is a well recorded global phenomenon that can have a pervasive impact on the overall ecosystem functioning53. These changes have been associated with stronger and longer thermal stratification, as well as a loss of water clarity, in part due to the increases in pelagic production5. Supporting this hypothesis, some of our studied lakes, where the strict anaerobe bacteriophage Metopus have been recently thriving, have experienced unprecedented episodes of eutrophication or cyanobacterial bloom over the last decades and subsequent periods of deep water hypoxia54–56.
Significant changes in several other functional groups of ciliate provide additional evidence of recent changes in aquatic food web structures and habitats. For instance, the significant increase in pelagic ciliates that preferentially lives in the stratified epilimniun further support that lakes have been exposed to longer and stronger periods of stratification in recent time. Furthermore, periphytic species, such as the sessile or sedentary forms Peritrichia and Suctoria, is consistent with more frequent pelagic blooms or macrophytes under higher nutrient and warmer conditions57,58. However, the lack of knowledge about the ciliate ecology, their biotic interactions and regulatory factors limit our capacity to decipher the relative importance of each potential stressors and the underlying mechanisms remain elusive.
Altogether, the diagnosis of the changes in the ciliate communities across the 48 studied lakes supports the use of ciliates as indicators of environmental changes59 and provides evidence that the ciliate communities are strongly responding to the environmental changes that have occurred over the last century which includes widespread deoxygenation of deep waters, changes in thermal stratification and nutrient-enrichment. Playing a key role in the metabolic pathways of aquatic ecosystems51,60, they can provide valuable insight into the functional ecology of lakes, and their strong response recorded in the sedimentary archives suggests important changes in the main pathways for the transfer of energy within the microbial food webs61. Although, molecular studies of protist communities and working with ancient DNA is quite challenging with several aspects that need to be taken into consideration for future studies (as summarized in Methods section of the Supplemental Material), the present study as well as several previous studies27,51,62 have shown the great success of using such approaches to assess environmental changes in aquatic ecosystems. Their integration to environmental assessment using high-throughput sequencing and metabarcoding technologies is thus promising, providing a more holistic overview of the response of aquatic ecosystems to environmental changes. This is even more relevant as the science is moving toward ecosystem-wide food web modelling20,51,63, and protists, as key players of the microbial food-web, serve an important function of recycling carbon and energy in lakes.