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Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPR-Cas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterialconjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both singlegene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPR-Cas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.

Aim To infer species identity, population isolation, and geographical variation in inter‐specific hybridization among corals of the genus Porites from the central and eastern tropical Pacific, with a focus on the timing of separation between populations of P. evermanni and P. lobata divided by the Eastern Pacific Barrier. Location Hawaii, American Samoa, Panama and the Galapagos Islands of Ecuador. Methods Maximum likelihood gene trees were obtained for mitochondrial DNA (COI), the internal transcribed spacer (ITS), and 5 single‐copy nuclear (scn) gene regions. Allelic networks were used to group multi‐locus scn data into species clusters despite some allele sharing. Coalescent analyses (IMa2) of the 5 scn markers were used to estimate the time of population divergence and test for introgression between P. evermanni and P. lobata. Results Allelic networks based on scn gene sequences agreed with mtCOI and ITS designations. Divergence times between Hawaiian and eastern Pacific populations are consistent with an early Pleistocene recolonization of the eastern Pacific by P. evermanni followed by a more recent arrival of P. lobata. The two species were fully isolated in Hawaii/American Samoa populations, but introgression from P. evermanni into P. lobata was evident in the eastern Pacific. Main conclusions These results are consistent with a scenario where a bout of introgression with P. evermanni, an early‐arriving colonizer of the eastern Pacific suited to marginal environmental conditions, facilitated the later colonization of the more sensitive P. lobata.