Morphological Redescription of Opalina undulata Nie 1932 from Fejervarya limnocharis with Molecular Phylogenetic Study of Opalinids (Heterokonta, Opalinea).

The redescription of Opalina undulata Nie 1932, collected from the rectum of the frog Fejervarya limnocharis, is presented in this paper based on detailed morphological information and molecular data. Our results revealed that specimens collected from Diaocha Lake in late August were larger and had more nuclei than those collected from the same site in early May. We sequenced their SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA (5' end) and found that they were completely identical, which means that the two populations belonged to the same species. These facts gave us a hint that body dimension and number of nuclei are not reliable taxonomic parameters for opalinids during their life cycle. Therefore, we recommended that the specific identification of opalinids based on morphological features should be carried out during seasons except spring. Meanwhile, our molecular phylogenetic analysis confirmed the monophyly of Opalinata. Within Opalinata, Opalinea were monophyletic with all opalinid species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea.

Phylogenetic position of the enigmatic deep-sea nematode order Rhaptothyreida: A molecular analysis.

The placement of the rare deep-sea nematode order Rhaptothyreida remains unclear due to the unique morphology of this group, an unknown life cycle with morphologically distinct juvenile stages which may or may not be parasitic, and lack of molecular sequences. Here, we investigate the phylogenetic placement and status of the Rhaptothyreida based on SSU and D2-D3 of LSU rDNA sequences of Rhaptothyerus typicus specimens obtained from the continental slope of New Zealand. Molecular sequences of three adults and a late stage juvenile were identical, confirming that they belong to the same species despite pronounced morphological differences. We observed the presence of the rare nucleotide transition A → G and transversion G → Y in the loops of Hairpin 35 and 48 regions, which is consistent with the placement of R. typicus within the order Enoplida. Rhaptothyreus typicus was consistently recovered as a long branch clade in SSU and D2-D3 of LSU analyses, which can have a destabilising effect on tree topology. After Gblocks were used to remove sites of questionable alignment, R. typicus was placed in a clade comprising Trissonchulus, Dolicholaimus and Ironus sequences (family Ironidae, order Enoplida) in both Bayesian and Maximum Likelihood SSU topologies. Depending on which alignment algorithm was used, analyses of LSU sequences focusing on enoplid taxa either suggested a relationship between R. typicus and Halalaimus (family Oxystominidae) or did not identify any clear relationships. Overall, our results provide strong evidence for placing R. typicus and the family Rhaptothyreidae within the order Enoplida, although further work is required to clarify relationships between rhaptothyreids and other enoplid taxa. A parasitic lifestyle could explain the unique morphology of this group, their highly divergent SSU and LSU rDNA molecular sequences, and the marked morphological differences between late juveniles and adults. Further molecular investigations targeting both free-living and parasitic early juvenile life stages in potential deep-sea hosts are needed to better understand the evolution of this unusual nematode taxon.

Phylogenetic classification of Pseudomonas putida strains by MALDI-MS using ribosomal subunit proteins as biomarkers.

A new method for phylogenetic classification of bacterial strains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. This method was developed using a bioinformatics-based approach to the rapid identification of bacteria as previously proposed by Demirev and co-workers, which uses ribosomal proteins composed of approximately 50 subunit proteins as biomarkers. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations can occur at the strain level. Since ribosomal subunit proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates, sequence variation detected as mass differences by MALDI-MS may be useful for the phylogenetic classification of bacteria at strain level. In our proposed method, the first step is the selection of reliable biomarkers through characterization of the expressed ribosomal subunit proteins of a reference strain (usually a genome-sequenced strain) by MALDI-MS. The observed masses in the MALDI mass spectra of cell lysates of sample strains are then compared with the biomarker masses of the reference strain. The biomarkers for each sample strain were designated as present or absent at the reference masses, indicated by 1 or 0, respectively, which were summarized in a table. This table is processed by cluster analysis, generating a phylogenetic tree. In this study, the success of this approach was confirmed by classification of Pseudomonas putida strains because its classification is much more complicated than that of other bacterial strains. Forty-three reliable biomarkers were selected from ribosomal sub-unit proteins of a genome-sequenced strain, P. putida KT2440. The numbers and kinds of biomarkers observed for 16 strains of P. putida, including different biovars, were markedly different, reflecting the variety of the strains. The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting our proposed method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.