First trial of rapid detection of macroalgal seed bank on cobbles: application of DNA metabarcoding using next-generation sequencing

Spores or microscopic stages of macroalgae are known on rocky substrata in urchin barren as well as in macroalgal beds. The components of such macroalgal seed bank can affect restoration of macroalgal beds in urchin barrens. Previously, macroalgal seed bank on a barren bottom was detected by incubation in herbivore-free enriched seawater, which took a few months. In the present study, a rapid detection was challenged using DNA metabarcoding approach with the plastid rbcL partial gene (688 bp) and next-generation sequencing for the first time. A total of twenty-four cobbles were collected from three different depth (2, 4 and 6 m in depth) of macroalgal beds and urchin barrens in northeastern Kyushu, Japan. The organisms attached on the cobbles were scraped off and suspended in sterilized seawater, from which DNA was extracted. After 454 pyrosequencing, program and manual based chimera-check were used for OTUs-picking. Combining both methods, 133 OTUs were detected.Composition of seed bankdetected from macroalgal beds and urchin barrens were significantly different (p = 0.0053).The method can shorten the time for detecting seed banks and contribute to accurate identification of the component algal species by enabling the comparison with molecular database.