title: Culture-Independent Study of the Late-Stage of a Bloom of the Toxic Dinoflagellate Ostreopsis cf. ovata: Preliminary Findings Suggest Genetic Differences at the Sub-Species Level and Allow ITS2 Structure Characterization.
abstract: Available genomic data for the toxic, bloom-forming, benthic Ostreopsis spp. are traditionally obtained from isolates rather than from individuals originally present in environmental samples. Samples from the final phase of the first reported Ostreopsis bloom in European North Atlantic waters (Algarve, south coast of Portugal) were studied and characterized, using a culture-independent approach. In the first instance, a microscopy-based analysis revealed the intricate complexity of the samples. Then, we evaluated the adequacy of commonly used molecular tools (i.e., primers and nuclear ribosomal markers) for the study of Ostreopsis diversity in natural samples. A PCR-based methodology previously developed to identify/detect common Ostreopsis species was tested, including one new combination of existing PCR primers. Two sets of environmental rRNA sequences were obtained, one of them (1052 bp) with the newly tested primer set. These latter sequences encompass both the ITS1-5.8S-ITS2 region and the D1/D2 domain of the LSU rRNA gene, leading us to an accurate identification of ITS2. In turn, this allowed us to predict and show for the first time the ITS2 secondary structure of Ostreopsis. With 92 bp in length and a two-helix structure, the ITS2 of this genus revealed to be unique among the dinoflagellates. Both the PCR approach as the phylogenetic analyses allowed to place the Ostreopsis cells observed in the samples within the O. cf. ovata phylospecies' complex, discarding the presence of O. cf. siamensis. The (phylo)genetic results point out a certain level of nucleotide sequence divergence, but were inconclusive in relation to a possible geographic origin of the O. cf. ovata population from the Algarve's bloom.
authors.affiliation: CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. vtr.rms@gmail.com.
authors.forename: Vitor
authors.initials: V
authors.lastname: Ramos
authors.affiliation: CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO, Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal. danielesalvi.bio@gmail.com.
authors.forename: Daniele
authors.initials: D
authors.lastname: Salvi
authors.affiliation: CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. jprmachado@gmail.com.
authors.forename: João Paulo
authors.initials: JP
authors.lastname: Machado
authors.affiliation: CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. mica.vale@gmail.com.
authors.forename: Micaela
authors.initials: M
authors.lastname: Vale
authors.affiliation: CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. joana_passo@hotmail.com.
authors.forename: Joana
authors.initials: J
authors.lastname: Azevedo
authors.affiliation: CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. vmvascon@fc.up.pt.|Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal. vmvascon@fc.up.pt.
authors.forename: Vitor
authors.initials: V
authors.lastname: Vasconcelos
mesh_terms: D001483:Base Sequence; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D004141:Dinoflagellida / Q000235:genetics* / Q000648:ultrastructure; D004784:Environmental Monitoring; D020459:Genes, rRNA / Q000235:genetics*; D057097:Harmful Algal Bloom; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009792:Oceans and Seas; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011174:Portugal; D012339:RNA, Ribosomal, 28S / Q000235:genetics*; D012623:Seawater; D013045:Species Specificity
chemical_list: D021903:DNA, Ribosomal Spacer; D012339:RNA, Ribosomal, 28S
keywords: HAB; ITS2 secondary structure; NE Atlantic; Ostreopsis; Portugal; culture-independent approach; environmental samples; phylogeny