Soil Micro-eukaryotic Diversity Patterns along Elevation Gradient Are Best Estimated by Increasing the Number of Elevation Steps Rather Than Within Elevation Band Replication.

The development of high-throughput sequencing (HTS) of environmental DNA (eDNA) has stimulated the study of soil microbial diversity patterns and drivers at all scales. However, given the heterogeneity of soils, a challenge is to define effective and efficient sampling protocols that allow sound comparison with other records, especially vegetation. In studies of elevational diversity pattern, a trade-off is choosing between replication within elevation bands vs. sampling more elevation bands. We addressed this question for soil protists along an elevation gradient on Mt. Asahi, Hokkaido, Japan. We compared two sampling approaches: (1) the replicate strategy (five replicates at six elevational bands, total = 30) and (2) the transect strategy (one sample in each of 16 different elevational bands). Despite a nearly twofold lower sampling effort, the transect strategy yielded congruent results compared to the replicate strategy for the estimation of elevational alpha diversity pattern: the regression coefficients between diversity indices and elevation did not differ between the two options. Furthermore, for a given total number of samples, gamma diversity estimated across the entire transect was higher when sampling more elevational bands as compared to replication from fewer elevational bands. Beta diversity (community composition turnover) was lower within a given elevational band than between adjacent bands and increased with elevation distance. In redundancy analyses, soil organic matter-related variable (the first principal component of soil organic matter, water content, total organic carbon, and nitrogen by whom were highly correlated) and elevation best explained elevational beta diversity pattern for both sampling approaches. Taken together, our results suggest that sampling a single plot per elevation band will be sufficient to obtain a good estimate of soil micro-eukaryotic diversity patterns along elevation gradients. This study demonstrated the effectiveness of the transect strategy in estimating diversity patterns along elevation gradients which is instructive for future environmental or even experimental studies. While not advocating for completely replacing replication-based sampling practices, it is important to note that both replicate and transect strategies have their merits and can be employed based on specific research goals and resource limitations.