Species boundaries in tintinnid ciliates: a case study--morphometric variability, molecular characterization, and temporal distribution of Helicostomella species (Ciliophora, Tintinnina).

Tintinnids are a large group of planktonic ciliates with diverse morphologies. The range of variability in lorica shapes and sizes can be very high even within a single species depending on life stages and environmental conditions, which makes the delimitation of different species based on morphological criteria alone very difficult. Accordingly, comparisons of morphological and molecular variability in tintinnids are necessary to provide a pragmatic approach for establishing species boundaries within this diverse and poorly understood group. We investigated the temporal distribution of species of the hyaline tintinnid Helicostomella (Ciliophora, Tintinnina), which were collected daily from September 2008 to August 2009 in Jangmok Bay of Geoje Island on the south coast of Korea. Two forms - a long form and a short form, were discovered. The long form was found in cold waters in February and March whereas the short form occurred in warm waters from June to October. Thus, these two forms were seasonally isolated. However, all the morphological characteristics for these two forms overlap to some degree. A comparison of the small subunit (SSU) rDNA, ITS1-5.8S-ITS2, and ITS2 sequences from two forms revealed 0.5%, 3.8%, and 5.6% divergences, respectively. Morever, one compensatory base change (CBC) and three hemi-CBCs were identified from ITS2 secondary structures of these two forms. All these data suggest that these two forms represent two distinct species despite their highly similar lorica morphology. The phylogenetic position of the genus Helicostomella was also examined using SSU rDNA sequences.