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The brown algal genus Padina (Dictyotales, Phaeophyceae) is distributed worldwide in tropical and temperate seas. Global species diversity and distribution ranges, however, remain largely unknown. Species-level diversity was reassessed using DNA-based, algorithmic species delineation techniques based on cox3 and rbcL sequence data from 221 specimens collected worldwide. This resulted in estimates ranging from 39 to 61 putative species (ESUs), depending on the technique as well as the locus. We discuss the merits, potential pitfalls, and evolutionary and biogeographic significance of algorithmic species delineation. We unveil patterns whereby ESUs are in all but one case restricted to either the Atlantic or Indo-Pacific Ocean. Within ocean basins we find evidence for the vast majority of ESUs to be confined to a single marine realm. Exceptions, whereby ESUs span up to three realms, are located in the Indo-Pacific Ocean. Patterns of range-restricted species likely arise by repeated founder events and subsequent peripatric speciation, hypothesized to dominate speciation mechanisms for coastal marine organisms in the Indo-Pacific. Using a three-gene (cox3, psaA and rbcL), relaxed molecular clock phylogenetic analysis we estimated divergence times, providing a historical framework to interpret biogeographic patterns.

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In brown algae (Phaeophyceae), only a few taxa are reported to have plastids containing pyrenoids and this character has often been used for systematic delineation. Members of the Ectocarpales display large, stalked and exserted pyrenoids in their plastids, whereas the Scytothamnales, as well as the genera Asterocladon, Asteronema and Bachelotia, exhibit plastids arranged in a stellate configuration with pyrenoids embedded in the plastid stroma. The evolution of plastid characters has remained difficult to establish, especially because of a persistent lack of resolution in brown algal molecular phylogenies. Here, we generated a seven-locus dataset for a species-rich taxon sampling, to reassess phylogenetic relationships of pyrenoid-bearing brown algal taxa. Within the Ectocarpales, the relationships among families are clarified. The minute chordariacean genus Herponema is assigned to the family Acinetosporaceae. The systematic positions of two enigmatic genera are revised: Chordariopsis is assigned to the Adenocystaceae, and Spongonema is transferred back to the Ectocarpaceae. The new order Asterocladales, which is sister to the Ectocarpales, is proposed to accommodate species of the genus Asterocladon. The order Scytothamnales is shown to include the genus Bachelotia in a new family Bachelotiaceae, as well as another new family Asteronemataceae, accommodating the genus Asteronema. These new taxonomic insights also shed light on the evolution of plastid morphology and arrangement in brown algae. Stellate plastid configurations have evolved independently in Asterocladales and Scytothamnales. Evolutionary scenarios underlying the diversity of stellate configurations in the Scytothamnales and the Asterocladales-Ectocarpales lineage are discussed.

Large-scale DNA molecular studies require reliable and efficient tools for DNA extractions. However, for some plant species and brown algae, isolation of high-quality DNA is difficult. We developed a novel method for isolating high-quality DNA from the polysaccharide-rich and polyphenol-rich brown algae based on a commercial kit and protocol (Qiagen) by optimizing the lysis step and including a chloroform ⁄ isoamyl alcohol supplementary purification step. DNAs from 24 brown algal species extracted using the original and the modified Qiagen protocol were compared for yield, quality, and effectiveness in PCR amplification. There was no significant difference in the yields between protocols. However, a statistically significant increase in DNA purity was obtained with the modified protocol, for which the A 260 ⁄ A 280 and A 260 ⁄ A 230 absorbance ratios averaged 1.66 ± 0.05 and 1.31 ± 0.01, respectively, compared to 1.37 ± 0.04 and 0.52 ± 0.04 with the original protocol. DNAs extracted by the modified procedure were more successfully amplified by PCR (nuclear, mitochondrial, and chloroplastic regions) than DNAs extracted using the original commercial kit and protocol. Importantly, the modified protocol can be applied in a high-throughput (e.g., 96-well plate) format, allowing a higher efficiency for downstream molecular analysis. In addition, improved DNA quality could increase its stability for long-term storage.