Karyotype Analysis And Molecular Systematics Of Clematis

The genus Clematis L. belongs to the family Ranunculaceae and comprises 230-355 species in the world. In this study, based on existing achievement of Clematis system research, modern cytology and molecular biology techniques were used to launch cytology and molecular systematics research in the method of karyotype sequence analysis. Target of the research was to accumulate some molecular evidence for solving system issues of Clematis and perfecting classification system of Clematis. The main results were as follow:1.7 species of the genus Clematis were studied. Among them, C. glauca, C. sibirica, C. cadmia, C. chinensis, C. viorna were reported for the first time. The main results were as follow:(1) All the analyzed species showed the same stable chromosome number (2n=2x=16) and basic chromosome number (8).(2) The 7 species of Clematis chromosomes were mainly composed by median region (m), submedinal region (st) and terminal region (t) chromosome. All of them belong to Type 2A, an original type.(3) 32 species of the genus Clematis, including upper 7 species and some reported species were divided into five groups by cluster analysis. The first group comprised of C. apiifolia, C. patens and other 17species, the second group comprised of C. fusca var. violacea Maxin., C crispa and other 3 species, the third group comprised of C. cadmiaå’ŒC. viorna, C. crispa, the fourth group comprised of C. macropetala, C. japonica and other 4 species, and the fifth group comprised of C. huchouensis and C. terniflora.2. The internal transcribed spacer (ITS) regions of 18 species of the genus Clematis (including ITS1,5.8S and ITS2) were sequenced. The results showed:(1) The ITS region ranged from 547 to 566bp and average G+C content was 61.8%. The ITS1 region ranged from 170-189bp, slightly less than ITS2 (210-214bp). With Pulsatilla chinensis, P. turczaninovii, Anemone rivularis, Naravelia laurifolia and Knowltonia sp. as out-groups, the ITS region was 601 sites after alignment and sequencing, including 184 variant sites (30.6%) and 137 informative sites (21.1%).(2) Based on the measured ITS region of 18 species of the genus Clematis together with out-groups, MP tree was constructed by the Maximum Parsimony method. As a result,18 species were clustered to 5 groups. C. fusca, C hexapetala, C. japonaca, C. terniflora were clustered to a group, and the support level reached 96%.(3) Sequences of measured 18 species were compared with sequences of other 71 species of 4 subgenus,13 groups downloaded from GenBank. The ITS region was 601 sites after alignment and sequencing, including 230 variant sites (37.6%) and 180 informative sites (29.4%).99 sequences were divided to 9 main classes by constructing MP tree.3. The matK,rpoB-trnC,psbA-trnQ united regions of 18 species of the genus Clematis were sequenced. The results showed:(1) The length of complete matk region was 1403 sites after alignment and sequencing, and length variation ranged from 1397-1403bp with a difference of 6bp. The average G+C content of matk region was 32.2%. With Naravelia and Knowltonia as out-groups,85 variant sites occupied 6.1 percent of the complete ITS region and 18 informative sites occupied 1.3 percent. The length of complete rpoB-trnC region was 1147 sites after alignment and sequencing, and length variation ranged from 1135-1146bp with a difference of llbp. The G+C content of rpoB-trnC region was 30.3%-31.2%while the average value was 30.7%. The length of complete psbA-trnQ region was 582 sites after alignment and sequencing, and length variation ranged from 540-562bp with a difference of 22bp. The average G+C content of psbA-trnQ region was 34.7%.(2) Based on the measured matK, rpoB-trnC and psbA-trnQ united regions of 15 species of the genus Clematis together with out-groups, MP tree was constructed by the Maximum Parsimony method. As a result,15 species were clustered to 3 groups, and C. acerifolia was clustered as a group separately.(3) Sequences of measured 15 species were split joint with matK, rpoB-trnC and psbA-trnQ united regions of Clematis downloaded from GenBank. Together with out-groups, MP tree was constructed by the Maximum Parsimony method. As a result,45 samples were clustered to 4 groups, and C. acerifolia was clustered as a group separately. System classification by united matK, rpoB-trnC and psbA-trnQ united regions was different from classification by separate sequence.(4) Sequences of measured 15 species were split joint with matK, rpoB-trnC, psbA-trnQ and ITS united regions of Clematis downloaded from GenBank. Together with out-groups, MP tree was constructed by the Maximum Parsimony method. As a result,45 samples were clustered to 8 groups, and MP tree constructed by ITS region were not the same as that constructed by matK, rpoB-trnC and psbA-trnQ united regions. Difference between nuclear and cytoplasmic evolutionary rate of Clematis may explain that.4. The genus Clematis was found as a paraphyly including Naravelia and Knowltonia by karyotype and sequence analysis. For subordinate classification, there was no support for system Wang and any other morphology system in this research. Researched 18 species of 4 subgenus,7 groups and downloaded sequences (4 subgenus,7 groups) were polyphyly or paraphyly, and relationship between all the groups were complicated.