Implementing ITS1 metabarcoding for the analysis of soil microeukariotic diversity in the Mountain Cloud Forest

Abstract

Purpose In threatened diversity hotspots, such as mountain cloud forests, microbiome studies have focused essentially on bacteria. Unlike prokaryotic microbiomes, the study of the microeukaryotes has largely been restricted to the visual identification of specific groups. Herein, microeukaryotic edaphic diversity from a pristine mountain cloud forest (MCF) of Mexico was analyzed via the metabarcoding of the ITS1 region of ribosomal DNA. Materials and methods An exploratory triangular sampling was conducted in the mountain cloud forest located in El Relámpago Mount, Santiago Comaltepec, Oaxaca, Mexico. Each vertex was located adjacent to a dominant plant species in the ecosystem (Oreomunnea mexicana and Alsophila salvinii). After DNA extraction the ITS1 region (rDNA) was amplified. Microeukaryotic sequences were filtered by computational subtraction against the ITS2 Database. Next, alpha and beta diversity indexes were calculated, and the relationship between abiotic variables and diversity patterns were inferred by means of a Canonical Correspondence Analysis. Results Overall, 138 inferred sequence variants were identified, including 87 protists, 35 animals (microfauna), and 16 algae. Within the animals, the nematodes were the dominant group, chlorophytes dominated algae, and in Protista, no dominance patterns were observed given the high diversity and equitability of this group. Soil available carbon, carbon degrading enzymes and the pH play a key role in modeling the community structure. Remarkably, high beta diversity levels were obtained, evidencing a strong spatial heterogeneity at the small scale. Conclusions The ITS metabarcoding proved to be a useful tool to conduct multi-taxa diversity assessments for microeukaryotes, allowing the identification of alpha and beta diversity patterns and overcoming limitations of sampling and the direct observation of individuals. The results presented in this work evidenced high microeukaryotic diversity levels in the soil of MCF and encourage future studies aiming to explore the taxonomic diversity of individual taxa.