title: Inclusion of chloroplast genes that have undergone expansion misleads phylogenetic reconstruction in the Chlorophyta.
abstract: OBJECTIVE
  Chlorophytes comprise a substantial proportion of green plant diversity. However, sister-group relationships and circumscription of the classes Chlorophyceae, Trebouxiophyceae, and Ulvophyceae have been problematic to resolve. Some analyses support a sister relationship between the trebouxiophycean Leptosira and chlorophyceans, potentially altering the circumscription of two classes, also supported by a shared fragmentation in the chloroplast gene rpoB. We sought to determine whether the latter is a synapomorphy or whether the supporting analyses are vulnerable to systematic bias.
  
  
  METHODS
  We sequenced a portion of rpoB spanning the fragmented region in strains for which it had not previously been sampled: four Chlorophyceae, six counterclockwise (CCW) group (ulvophyceans and trebouxiophyceans) and one streptophyte. We then explored the effect of subsampling proteins and taxa on phylogenetic reconstruction from a data set of 41 chloroplast proteins.
  
  
  RESULTS
  None of the CCW or streptophyte strains possessed the split in rpoB, including inferred near relatives of Leptosira, but it was found in all chlorophycean strains. We reconstructed alternative phylogenies (Leptosira + Chlorophyceae and Leptosira + Chlorellales) using two different protein groups (Rpo and Rps), both subject to coding-region expansion. A conserved region of RpoB remained suitable for analysis of more recent divergences.
  
  
  CONCLUSIONS
  The Rps sequences can explain earlier findings linking Leptosira with the Chlorophyceae and should be excluded from phylogenetic analyses attempting to resolve deep nodes because their expansion violates the assumptions of substitution models. We reaffirm that Leptosira is a trebouxiophycean and that fragmentation of rpoB has occurred at least twice in chlorophyte evolution.
authors.affiliation: Allan Herbarium, Landcare Research, P.O. Box 69040, Lincoln 7640, New Zealand.
authors.forename: Phil M
authors.initials: PM
authors.lastname: Novis
authors.forename: Rob
authors.initials: R
authors.lastname: Smissen
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Buckley
authors.forename: Kishore
authors.initials: K
authors.lastname: Gopalakrishnan
authors.forename: Gabriel
authors.initials: G
authors.lastname: Visnovsky
mesh_terms: D020418:Algal Proteins / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000460:Chlorophyta / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D060365:Chloroplast Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D020418:Algal Proteins; D060365:Chloroplast Proteins
keywords: Chlorophyta; chloroplast genes; genome evolution; green algae; rpoB; topological incongruence