Barcoding of cryptic stages of marine brown algae isolated from incubated substratum reveals high diversity in Acinetosporaceae (Ectocarpales, Phaeophyceae)

To identify cryptic stages of marine brown macroalgae present in the "bank of microscopic forms", we incubated natural substrata of different geographical origins and isolated emerging Phaeophyceae into clonal cultures. A total of 431 clones were subsequently identified by barcoding using 5'-COI. A proportion of 98% of the isolates belonged to the Ectocarpales. The distribution of pairwise genetic distances revealed a K2P divergence of 1.8% as species-level cut-off. Using this threshold, the samples were ascribed to 83 different species, 39 (47%) of which were identified through reference sequences or morphology. In the Ectocarpaceae, 16 lineages of Ectocarpus fulfilled the barcode criterion for different species, while three putative new species were detected. In the Chordariaceae, numerous microthalli were microstages of known macroscopic taxa. A separate cluster contained Hecatonema maculans and other microscopic species. Taxa traditionally classified in Acinetosporaceae were split in two species-rich groups containing Pylaiella and Hincksia in one and Acinetospora in the other. Feldmannia species were present in both clusters. The present study shows that the germling emergence method is suited to reveal the diversity of hidden life-history stages, albeit with a bias towards early successional species.