Revisiting the free-living <i>Trebouxia</i> strains of the Coimbra Collection of Algae (ACOI): <i>Trebouxia valentina</i> sp. nov. (Trebouxiophyceae, Chlorophyta)

<i>Trebouxia</i> microalgae have previously been mostly reported as lichen photobionts. However, in recent years, reports of their occurrence outside the lichen symbiosis has been more common. This study provides evidence of free-living <i>Trebouxia</i> species, as well as the re-evaluation of six strains from the Coimbra Collection of Algae (ACOI 1141, ACOI 1821, ACOI 2621, ACOI 2622, ACOI 2623 and ACOI 3426) obtained via micropipette single-cell isolation. The nuclear Internal Transcribed Spacer rDNA was sequenced to infer its phylogenetic position within <i>Trebouxia</i> genus. Strains ACOI 1141, ACOI 1821, ACOI 2621 and ACOI 3426 clustered in a large clade with a species-level lineage previously named as <i>Trebouxia</i> sp. A13. This lineage comprises sequences belonging to three previously described <i>Trebouxia</i> species: <i>Trebouxia crenulata, Trebouxia arboricola</i> and <i>Trebouxia aggregata</i>. Strain ACOI 2623 clustered with a species-level lineage not yet formally described, but previously detected and coded as <i>Trebouxia</i> sp. A19. ACOI 2622 separated into an independent clade distinct from all other sequences included in the dataset. This strain has not been previously identified as a species-level lineage in other studies, thus we code it as A60 and describe it as <i>Trebouxia valentina</i> sp. nov. based on morphological, ultrastructural, molecular and phylogenetic analyses. Our findings highlight the ability of these microalgae, typically associated with lichens, to thrive independently and expand our understanding of their biology and ecology. Free-living <i>Trebouxia</i> species were isolated and added to the ACOI.ACOI 2623 clustered with a species-level lineage not yet described.ACOI 2622 is here described as <i>Trebouxia valentina</i> sp. nov. Free-living <i>Trebouxia</i> species were isolated and added to the ACOI. ACOI 2623 clustered with a species-level lineage not yet described. ACOI 2622 is here described as <i>Trebouxia valentina</i> sp. nov.