Molecular Phylogeny Of Class Polyhymenophorea And Construction Of CDNA Library

Systematics and phylogenetics on the ciliated protozoa have been carried out for about 20 years. However, its phylogeny still remains confusing as regards the evolutionary relationships and systematic positions of many well-known groups. This is due to the high diversity of their morphology, the difficulty in recognizing which similarities are due to convergent evolution, the loss of intermediate forms during the long period of time and insufficient molecular information.Molecular marker gene were chosen e.g. SSU rRNA gene, ITS1-5.8S-ITS2 gene andα-tubulin gene as to study systemetics, phylogenetic position or relationships and evolutionary histories of some key and / or ambiguous taxa of ciliated protozoa. Based on the gene sequenceing and analyses, this work resolved some confusions of morphological taxonomy and phylogenetic positions of some key taxa within three calsses at the genetical level. We used multiple computer-assisitent algorithms inferred from combinations of molecular sequences and morphological and morphogenetic characterizations to supply important basis and parameters for molecular systematics, a reconstruction of phylogenetic positions and evolutionary relationships of key taxa ciliated protozoa.The technology of expressed tag (EST) was broadly applied in analysis of gene function and expression pattern. In this study, total rRNA of cDNA library of Marine Ciliate P. carnea was constructed using the Smart technique for the further studies, such as special nuclear dualism, complicated morphogenetic process and unique sexual reproduction (conjugation).The conclusions were confirmed:1. The order Euplotida:The typical euplotids belong to a paraphyletic group composed of three diverged clades (Uronychiidae, Euplotidae-Certesiidae-Aspidiscidae and Gastrocirrhidae); both clades formed by Euplotidae-Certesiidae-Aspidiscidae and Gastrocirrhidae were firmly supported with high bootstrap; the family Discocephalidae forms a sister group to the large clade containing Euplotidae-Certesiidae-Aspidiscidae and Gastrocirrhidae, though its position remains ambiguous due to low supports; Uronychiidae is clearly separated from the most other euplotids.2. The order Urostylida:The order Urostylida should be a paraphyletic group. (1) Pseudoamphisiella always branch firstly off the clade followed by Licnophorea which indicates that they might represent an ancestral form for urostylid species. (2) Pseudokeronopsis and Thigmokeronopsis may not share a recent common ancestor, casting doubt on the monophyly of the family Pseudokeronopsidae. (3) Our data justify the redefinition of Holosticha and confirm that Anteholosticha should be apart from Holosticha. (4) DNA sequence data can be harmonized with the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis which suggests that the urostylid midventral pattern evolved from an oxytrichine ancestor, developing a second time within the Oxytrichidae. (5) Epiclintidae clusters with Oligotrichida and Sporadotrichida, which against its establishment of the family in the order Urostylida.3. The order Sporadotrichida:(1) the oligotrichine family Halteriidae which has been transferred to the order Sporadotrichida Lynn (2008) invariably clusters within the oxytrichid clade. This contradicts the current ontogenetic findings; possibly, it is an artifact caused by taxa undersampling and/or special molecular evolutionary events. (2) The systematic position of one of the two key genera could be clarified with the SSU rRNA sequences: Gastrostyla is a stylonychine oxytrichid. (3) sequence analyses of several Oxytricha species resulted in a high molecular diversity, which does not support monophyly of this genus. (4) the molecular phylogenetic tree basically supports the establishment of the family Trachelostylidae Small & Lynn, 1985. 4. Condylostoma species and other related Heterotrich ciliates:The systematically poorly-known ciliate genus Condylostoma was erected by Vincent in 1826. (1) all the trees have similar topologies with high supports; (2) Condylostoma is mostly related to the genus Condylostentor; and (3) three Condylostoma species as well as Condylostentor auriculatus cluster together and form a sister group with other heterotrichs. This is moderately consistent with the assessment of phylogenetic relationships of Condylostoma-related heterotrichs due to morphological information. The phylogenetic relationship of some other related heterotrichs, Peritromus, Folliculina, Stentor and Blepharisma, has been also discussed.5. The cDNA library of marine ciliate, Pseudokeronopsis carnea:The cDNA library was constructed using the SMART construction kit, from which totally 237 expressed sequence tags (ESTs) were randomly selected for analyses. The capacity of the library was 1×106 pfu/mL, in which more than 90% clones are recombinant. 189 unabridged cDNA sequences lacking X or N were made homology comparison using the Blast X search; 57 of which were nearly identical to known genes in the NCBI database. These data show that the full length cDNA library of P. carnea constructed using the Smart technique is of reasonably good quality for the further studies.