MOLECULAR PHYLOGENY OF THE HAPLOSPORIDIA BASED ON TWO INDEPENDENT GENE SEQUENCES

The phylogenetic position of the Haplosporidia has confounded taxonomists for more than a century because of the unique morphology of these parasites. We collected DNA sequence data for small subunit (SSU) ribosomal RNA and actin genes from haplosporidians and other protists for conducting molecular phylogenetic analyses to help elucidate relationships of taxa within the group, as well as placement of this group among Eukaryota. Analyses were conducted using DNA sequence data from more than 100 eukaryotic taxa with various combinations of data sets including nucleotide sequence data for each gene separately and combined, as well as SSU ribosomal DNA data combined with translated actin amino acids. In almost all analyses, the Haplosporidia was sister to the Cercozoa with moderate bootstrap and jackknife support. Analysis with actin amino acid sequences alone grouped haplosporidians with the foraminiferans and cercozoans. The haplosporidians Minchinia and Urosporidium were found to be monophyletic, whereas Haplosporidium was paraphyletic. ''Microcell'' parasites, Bonamia spp. and Mikrocytos roughleyi, were sister to Minchinia, the most derived genus, with Haplosporidiumfalling between the ''microcells'' and the more basal Urosporidium. Two recently discovered parasites, one from abalone in New Zealand and another from spot prawns in British Columbia, fell at the base of the Haplosporidia with very strong support, indicating a taxonomic affinity to this group. Haplosporidia is composed of histozoic and coelozoic para- sites in a variety of freshwater and marine invertebrates; some species are significant pathogens of commercially important molluscs (Burreson et al., 2000). Since the discovery of the first species in the late 1800s, the Haplosporidia have been a trou- blesome group for taxonomists and phylogeneticists. Histori- cally, the taxon has been treated as a last resort for a diversity of spore-forming parasites that have multinucleated naked cells (plasmodia) in their life cycles and that were not readily clas- sifiable elsewhere (Sprague, 1979). There have been numerous taxonomic schemes proposed for placement of the group within the protists and for appropriate taxa within the Haplosporidia. Caullery and Mesnil (1899) es- tablished Haplosporidiumfor 2 parasites of marine annelids and placed the genus in the new order Haplosporidia in the class Sporozoa of the phylum Protozoa. Caullery (1953) recognized 6 genera in the order, and Kudo (1971) recognized 7 genera. A major change in the classification of the Haplosporidia was the separation of the Haplosporidia and the Paramyxea from other ''sporozoa'' by establishment of the new phylum Ascetospora (Sprague, 1979). This scheme proposed 2 classes: Stellatospo- rea, for the families Marteiliidae and Haplosporidiidae, and Par- amyxea, for the family Paramyxidae. The family Haplospori- diidae contained only 3 genera, i.e., Haplosporidium, Minchi- nia, and Urosporidium. Desportes and Nashed (1983) recog- nized that the family Marteiliidae belonged in the class Paramyxea, not Stellatosporea, because of its development cy- cle, and proposed 2 classes in the phylum Ascetospora, i.e., Haplosporea and Paramyxea. However, they suggested that the 2 classes probably should be raised to phylum rank because of very different developmental sequences. Recently, the phylum Ascetospora has been abandoned, and Haplosporidia and Par- amyxea have each been elevated to phylum rank (Desportes and Perkins, 1990; Perkins, 1990, 1991; Cavalier-Smith, 1993). Separate phylum rank for Haplosporidia and Paramyxea has been accepted by most researchers; however, Corliss (1994) re-