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

Spores or microscopic stages of macroalgae are present on rocky substrata in shallow waters and even on barrens. The components of such a “macroalgal seed bank” can influence the restoration of macroalgal beds from urchin barrens. Previously, we characterized “macroalgal seed banks” on cobbles collected from the barren bottom by incubation in herbivore-free enriched seawater, which required a few months. Herein, we attempted rapid detection of the macroalgal seed banks by employing DNA metabarcoding and next-generation sequencing of the plastid rbcL partial gene (688 bp). A total of 24 cobbles were collected from three depths (2, 4, and 6 m) of macroalgal beds and urchin barrens in northeastern Kyushu, Japan. Organisms attached on the cobbles were scraped off and suspended in sterilized seawater, after which DNA was extracted. Samples were sequenced by 454 pyrosequencing, and chimera identification and operational taxonomic unit (OTU) picking were conducted following program and manual based protocols. The combined approach identified 133 OTUs; of these, 102 and 31 OTUs were assigned to 14 orders in Rhodophyta and 5 orders in Phaeophyceae, respectively. Macroalgal beds and urchin barrens showed significant differences in seed bank composition based on PERMANOVA (F = 3.011, p = 0.0059). Seed banks of macroalgal beds predominantly comprised Diplura spp. (Ishigeales familia incertae sedis) (36.6%), Halymeniaceae (17.2%), Gigartinaceae (13.5%), and Peyssonneliaceae (13.2%), while urchin barrens predominantly comprised Halymeniaceae (46.6%) and Diplura spp. (23.6%). Our proposed method significantly reduced the time and facilitated comprehensive taxonomic identification for characterizing “macroalgal seed banks.”