High-throughput sequencing covers greater nematode diversity than conventional morphotyping on natural cedar forests in Yakushima Island, Japan

Abstract

Massive sequence reads obtained via high-throughput sequencing (HTS) methods have provided opportunities to extensively identify nematodes in certain habitats, thereby enabling the generation of assemblage patterns depicting nematode communities in natural ecosystems. However, we do not know whether HTS analysis yields assemblage patterns for nematode communities that are similar to those generated by conventional morphological analyses. We compared the community structures of soil nematodes determined by HTS and morphological analyses in natural Japanese cedar (Cryptomeria japonica) forests more than 1,000 years old on Yakushima Island, South Japan. Soil nematodes were collected at two sites, namely, Shiratani (SR) and Arakawa (AR), and identified to the genus level under a compound light microscope. Nematodes were also subjected to DNA sequence analysis, focusing on partial small subunit rRNA. Morphological analysis divided the nematodes into 9 orders, 34 families, and 42 genera. The 509 amplicon sequence variants were assigned to 11 orders, 45 families, and 76 genera. Based on the phylogenetic analysis of Aphelenchoides, our sequences were divided into 12 independent clades with bootstrap values greater than 70%. From both morphological and HTS analyses, 77 and 72 genera were identified for SR and AR, respectively, and HTS analysis covered 88% and 85% of the total nematode genera in samples from SR and AR, respectively. The nematode abundance obtained via HTS analysis was significantly correlated with the morphological abundance of Acrobeloides, Aphelenchoides, Helicotylenchus, Rhabdolaimus, and Wilsonema. The non-metric multidimensional scaling scatter plot indices showed that nematode community structures were significantly different between SR and AR, regardless of the methodology. Thus, HTS analysis is more useful than morphological analysis for identifying the nematode communities of natural ecosystems to uncover hidden diversity.