DNA metabarcoding has become a powerful approach for analyzing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers – like the fungal ITS region, may include considerable intraspecific variation, which can lead to over-splitting of species during DNA metabarcoding analyses. Here we assess the effects of intraspecific sequence variation in DNA metabarcoding, by analyzing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS), coupled with error correction with the software DADA2. All focal species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, often occurring in low frequencies, were likely due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS-based fungal community analyses, but an extra clustering step is needed to approach species-level resolution.

Because of their steep gradients in abiotic and biotic factors, mountains offer an ideal setting to enhance our understanding of mechanisms that underlie species distributions and community assemblies. We compared the structure of taxonomically and functionally diverse soil fungal communities in soils along elevational gradients in the Neo- and Paleotropics (northern Argentina, Central America, and Borneo). We found that soil fungal community composition reflects environmental factors at both regional and pantropical scales, particularly temperature and soil pH. Elevational turnover is driven by contrasting environmental preferences among functional groups and replacement of species within functional guilds. In addition, we found that habitat preference can already be observed at the level of taxonomic orders, often irrespective of functional guild, which suggests shared physiological constraints and environmental optimum for relatively closely related taxa. Strong biogeographic structure likely reflects dispersal limitation and resulting differences in local species pools of fungi, as well as their hosts or substrates. Although the number of species shared among regions is low, remarkable similarity of functional profiles across regions suggests functional niche proportions may be driven by similar mechanisms across moist tropical forests, resulting in relatively predictable proportions of functional guilds. The pronounced compositional and functional turnover along elevation gradients driven mainly by temperature and correlated environmental factors implies that tropical montane forest fungi will likely be sensitive to climate change, resulting in variation in composition and functionality over time.