International Journal of Marine Science, 2024, Vol.14, No.3, 172-181 http://www.aquapublisher.com/index.php/ijms 179 Figure 3 from Zhao et al. (2022) shows a flow chart of the barcoding processing steps used to assess ciliate biodiversity. It includes water sample collection, sample pretreatment, DNA extraction, PCR amplification, NGS-Illumina sequencing, reference database selection, and taxonomic assignment. The flow chart highlights potential factors that can affect biodiversity assessments, such as sampling volume, filter materials, DNA extraction methods, and PCR amplification fidelity. The figure emphasizes the importance of standardizing each step to produce reliable and reproducible results in barcoding studies, which is crucial for effective biodiversity surveys and ecological research. 7 Challenges and Future Directions 7.1 Technical and methodological challenges The study of ciliate communities faces several technical and methodological challenges. Traditional morphological methods, while valuable, often miss a significant portion of "hidden biodiversity," including rare species and resistance forms, which are more readily detected by molecular techniques (Rossi et al., 2016; Weisse, et al., 2017). The discrepancies between morphological and molecular approaches can lead to identification errors and incomplete biodiversity assessments (Rossi et al., 2016). Additionally, the reliance on single molecular markers in many studies can result in an incomplete understanding of ciliate diversity and biogeography. The integration of high-throughput DNA sequencing with traditional methods has shown promise but requires further refinement to ensure comprehensive and accurate biodiversity assessments (Fernandes et al., 2020; Kulaš et al., 2021a). 7.2 Knowledge gaps and research needs Despite significant advancements, there remain substantial knowledge gaps in our understanding of ciliate diversity and ecological functions. For instance, the ecological roles of many ciliate species, particularly those that are rare or dormant, are not well understood (Weisse et al., 2017). There is also a need for more detailed taxonomic revisions and the exploration of new habitats to uncover the full extent of ciliate diversity. Furthermore, the functional diversity of ciliates and their contributions to ecosystem processes, such as primary production and nutrient cycling, require more experimental research to link ecophysiological performance to functional genes (Weisse et al., 2017). The geographic distribution of ciliates and the factors influencing their biogeography also remain areas needing further investigation. 7.3 Emerging trends and innovations Emerging trends and innovations in the study of ciliate communities include the use of high-throughput DNA sequencing and environmental DNA (eDNA) metabarcoding, which have significantly enhanced our ability to detect and classify ciliate species (Fernandes et al., 2020; Kulaš et al., 2021a). These molecular techniques have revealed a higher number of ciliate taxa than traditional methods and have provided new insights into the community structure and ecological roles of ciliates in various environments (Kulaš et al., 2021a). Additionally, the integration of molecular data with traditional morphological approaches offers a more robust analytical framework for studying ciliate biodiversity and bioindicator potential (Kulaš et al., 2021a). Future research should focus on developing more comprehensive and integrative methodologies that combine morphological, molecular, and functional analyses to advance our understanding of ciliate communities and their ecological functions (Rossi et al., 2016; Weisse et al., 2017; Fernandes et al., 2020; Kulaš et al., 2021a). Acknowledgments The author extends sincere thanks to two anonymous peer reviewers for their feedback on the manuscript of this paper. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
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