title: Molecular detection and species identification of Alexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline.
abstract: OBJECTIVE
  On the basis of morphological evidence, the species involved in South American Pacific coast harmful algal blooms (HABs) has been traditionally recognized as Alexandrium catenella (Dinophyceae). However, these observations have not been confirmed using evidence based on genomic sequence variability. Our principal objective was to accurately determine the species of Alexandrium involved in local HABs in order to implement a real-time polymerase chain reaction (PCR) assay for its rapid and easy detection on filter-feeding shellfish, such as mussels.
  
  
  METHODS
  For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1-D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences. Species-specific primers were used to amplify signature sequences within the genomic DNA of the studied species by conventional and real-time PCR.
  
  
  RESULTS
  Phylogenetic analysis determined that the Chilean strain falls into Group I of the tamarensis complex. Our results support the allocation of the Chilean Alexandrium species as a toxic Alexandrium tamarense rather than A. catenella, as currently defined. Once local species were determined to belong to Group I of the tamarensis complex, a highly sensitive and accurate real-time PCR procedure was developed to detect dinoflagellate presence in Mytilus spp. (Bivalvia) samples after being fed (challenged) in vitro with the Chilean Alexandrium strain. The results show that real-time PCR is useful to detect Alexandrium intake in filter-feeding molluscs.
  
  
  CONCLUSIONS
  It has been shown that the classification of local Alexandrium using morphological evidence is not very accurate. Molecular methods enabled the HAB dinoflagellate species of the Chilean coast to be assigned as A. tamarense rather than A. catenella. Real-time PCR analysis based on A. tamarense primers allowed the detection of dinoflagellate DNA in Mytilus spp. samples exposed to this alga. Through the specific assignment of dinoflagellate species involved in HABs, more reliable preventive policies can be implemented.
authors.affiliation: FAVET-INBIOGEN, Faculty of Veterinary Sciences ,  University of Chile ,  Santa Rosa No 11.735 (PO 8820808), Santiago ,  Chile.
authors.forename: Ana
authors.initials: A
authors.lastname: Jedlicki
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Fernández
authors.forename: Marcela
authors.initials: M
authors.lastname: Astorga
authors.forename: Pablo
authors.initials: P
authors.lastname: Oyarzún
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Toro
authors.forename: Jorge M
authors.initials: JM
authors.lastname: Navarro
authors.forename: Víctor
authors.initials: V
authors.lastname: Martínez