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A quantitative protocol for DNA metabarcoding of springtails (Collembola)
Seikoh Saitoh1, Hiroaki Aoyama1, Saori Fujii2
1a Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
We developed a new DNA metabarcoding protocol for analyzing soil Collembola communities. This method provides superior quantitative results for soil microarthropod analysis, enabling accurate comparisons of Collembola populations.
Area of Science:
- Ecology
- Molecular Biology
- Soil Science
Background:
- Collembola are crucial soil microarthropods, but their community analysis is challenging.
- DNA metabarcoding offers a powerful tool for biodiversity assessment.
- Existing methods for Collembola metabarcoding require optimization for quantitative accuracy.
Purpose of the Study:
- To develop and validate a novel DNA metabarcoding protocol for quantitative analysis of Collembola communities.
- To identify optimal PCR primers targeting mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes for Collembola.
- To assess the quantitative performance of the protocol using natural and mock soil communities.
Main Methods:
- Designed degenerate PCR primers for conserved regions of mtCOI and mt16S genes in Collembola.
- Selected the best primer pair based on broad-spectrum amplification across Collembola species.
- Extracted DNA from natural temperate forest soil samples and cultured mock communities.
- Amplified target genes, prepared libraries for 454 sequencing, and analyzed sequence data.
- Defined operational taxonomic units (OTUs) at 90% sequence similarity and compared with microscopic identification.
Main Results:
- Recovered 32 (mtCOI) and 36 (mt16S) OTUs from natural communities, comparable to 25 morphospecies identified microscopically.
- Demonstrated strong correlation (R = 0.91-0.99) between normalized sequence abundances and actual individual counts in mock communities.
- Observed quantitative accuracy in species abundance estimation, despite some skewing in relative abundances based on sequence data.
Conclusions:
- The developed DNA metabarcoding protocol provides a robust and quantitative method for assessing Collembola community composition.
- The protocol enables reliable comparisons of Collembola populations across different soil environments.
- This advancement facilitates deeper ecological understanding of soil microarthropod communities.
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