Local diversity of heathland Cercozoa explored by in-depth sequencing

Abstract

Cercozoa are abundant free-living soil protozoa and quantitatively important in soil food webs; yet, targeted high-throughput sequencing (HTS) has not yet been applied to this group. Here we describe the development of a targeted assay to explore Cercozoa using HTS, and we apply this assay to measure Cercozoan community response to drought in a Danish climate manipulation experiment (two sites exposed to artificial drought, two unexposed). Based on a comparison of the hypervariable regions of the 18S ribosomal DNA of 193 named Cercozoa, we concluded that the V4 region is the most suitable for group-specific diversity analysis. We then designed a set of highly specific primers (encompassing ~270 bp) for 454 sequencing. The primers captured all major cercozoan groups; and >95% of the obtained sequences were from Cercozoa. From 443 350 high-quality short reads (>300 bp), we recovered 1585 operational taxonomic units defined by >95% V4 sequence similarity. Taxonomic annotation by phylogeny enabled us to assign >95% of our reads to order level and ~85% to genus level despite the presence of a large, hitherto unknown diversity. Over 40% of the annotated sequences were assigned to Glissomonad genera, whereas the most common individually named genus was the euglyphid Trinema. Cercozoan diversity was largely resilient to drought, although we observed a community composition shift towards fewer testate amoebae.

Figure 1

Linear regression of the percentage-wise DNA distance (uncorrected P-distance) of the eight hypervariable regions (V1–V9) against the entire 18S sequence from 63 complete cercozoan sequences. Dark grey shading=99% confidence intervals, light grey shading=95% confidence intervals.

Figure 2

(a) Number of OTUs in 193 named cercozoan morpho-species as a function of OTU separation threshold of the V4 region. (b) Histogram of the pairwise comparisons of the V4 region from 193 named cercozoan morpho-species. The dashed red line shows our separation threshold of 5%. The bulk of the interspecific diversity is well above dissimilarities of 3 or 5%, but it extends all the way down to zero with no separation window. (c) Number of OTUs as a function of OTU separation threshold in the cleaned 454 data set of 443 350 V4 sequences. Choosing a separation threshold of 0.5–1% consistent with the morpho-species concepts would retrieve >9000 OTUs. (d) Rarefaction curve of the observed species (OTUs at 5% threshold) with error bars from combining the two drought and control plots, resampled to 85 305 sequences per tag. The samples are reasonably saturated with no difference in raw diversity between the two treatments.

Figure 3

Heatmaps displaying the taxonomic abundance and distribution of the sequences (n=85 305) between the four sample sites, at (a) order and (b) genus level. The heatmaps are drawn using the heatmap.2 function of the gplots package, and dendrogram distances are based on Euclidean distance. Taxa constituting <1% of the total taxa have been lumped into the ‘other assigned' category. Testate taxa are marked with an asterisk. Glissomonads and Euglyphids are by far the most abundant Cercozoa in all samples. Of all taxa, the genus Trinema shows the largest relative change in response to the drought treatment.