Water-filled tree holes present a unique ecosystem in forests that has long fascinated ecologists. Water-filled tree holes provide habitats for a diver array of aquatic and semiaquatic species. Insect larvae, mesozoa, and other organisms inhabit the water bodies, feeding on the deposited detritus and forming food webs. The vertical stratification of tropical forests results in significant differences in environmental conditions between the canopy and understory. Several studies have been conducted on differences in community formation in water-filled tree holes across vertical strata. However, some species are too small to be detected visually. Recent development of eDNA metabarcoding provides unique opportunity to study aquatic biodiversity by detecting the community members of tree holes. In addition, studies using water-filled artificial tree holes have shown that almost all species found in natural tree holes are present. Therefore, in this study, the eDNA metabarcoding method was applied to investigate the community assembly of species associated with water-filled tree holes in tropical rainforests.
Artificial water-filled holes were attached to 10 trees at 1.5 m (understory) and over 60 m (canopy) above the ground from July to September 2019. Each water sample was analyzed with a high-throughput sequencer. A total of 6408 operational taxonomic units (OTUs) were found and 53 arthropod families were assigned. Of these, dipteran families were the most dominant throughout the study period. The results showed that water-filled tree holes in the canopy and the understory were the main habitats of the dipteran species. The eDNA metabarcoding results were largely consistent with studies based on visual observations, although some groups (e.g., Acarina) were not consistent. This suggest that the eDNA method can detect small organisms that were not detected by visual observations. No differences in arthropod species composition were found between the canopy and understory. Result of NMDS analysis showed that community structure of artificial water-filled tree holes can be explained by height, water temperature, pH, electrical conductivity, and mass of litter falling. This study demonstrates that the eDNA metabarcoding method is useful for studying community structure in water-filled tree holes.