Concordance between DNA-based species boundaries and reproductive isolating barriers in the Scytosiphon lomentaria species complex (Ectocarpales, Phaeophyceae)

DNA-based species delimitation methods often predict many putative cryptic species. Interpretation of these putative species (i.e. do they represent distinct species, or is this an erroneous estimate based on DNA variability?) is challenging because estimates of DNA-based delimitation are often difficult to verify using nongenetic data such as morphology or geographical distribution. In the present study, estimates of DNA-based delimitation methods were verified based on the biological species concept in Japanese populations of Scytosiphon lomentaria. Three DNA-based species delimitation methods (Generalised Mixed Yule Coalescent, Poisson Tree Processes and Automatic Barcode Gap Discovery) were conducted using mitochondrial (cox1) and nuclear (second intron of centrin gene) DNA sequence datasets. In the S. lomentaria species complex, five putative cryptic species (Ia-Va) were well-supported by DNA-based species delimitation, and these putative species were often found in the same locality. To verify the estimates of DNAbased delimitation, crossing experiments were conducted among the putative species, using cultured isolates. Gametic incompatibility, which prevented hybrid zygote formation, was observed among them, except in the crosses between species IIa male and species IIIa female, in which hybrid zygotes successfully formed. These hybrid zygotes grew up to sporophytes and matured with unilocular sporangia in culture; however, the survival rate of their meiospores (zoospores) was significantly lower than that of meiospores from sporophytes derived from intraspecific crosses. In the S. lomentaria species complex in Japan, well-supported DNA-based species boundaries were concordant with biological species boundaries (intrinsic barriers to gene exchange). The five cryptic species are reproductively isolated from each other by gametic incompatibility (pre-zygotic barrier) and low survival rate of hybrid meiospores (post-zygotic barrier).