A quantitative protocol for DNA metabarcoding of springtails (Collembola).
We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.
a Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
Seikoh
S
Saitoh
a Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
Hiroaki
H
Aoyama
b Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan.
Saori
S
Fujii
c Okinawa Prefectural Agricultural Research Center, 820 Makabe, Itoman 901-0336, Japan.
Haruki
H
Sunagawa
a Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
Hideki
H
Nagahama
d Department of Electrical Engineering and Computer Science, School of Industrial and Welfare Engineering, Tokai University, Toroku 9-1-1, Higashi-ku, Kumamoto 862-8652, Japan.
Masako
M
Akutsu
a Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
Naoya
N
Shinzato
b Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan.
Nobuhiro
N
Kaneko
b Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan.
Taizo
T
Nakamori
D000818:Animals; D001181:Arthropods / Q000145:classification* / Q000235:genetics*; D044822:Biodiversity; D058893:DNA Barcoding, Taxonomic; D003576:Electron Transport Complex IV / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods; D011786:Quality Control; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D012336:RNA, Ribosomal, 16S; D003576:Electron Transport Complex IV
16S; COX1; Collembola; collemboles; metabarcoding; métacodage à barres; quantification