| Dictyostelid cellular slime molds(Dictyostelids),social amoebae,live primarily in forest soils that is the ancient microorganism which feed primarily on bacteria and play an important role in maintaining the balance between bacteria and other microorganisms in soils and the recycling of soil nutrient.They are also ideal materials for biological experments because of their special feeding way.On account of these characteristics,they are also a kind of ideal objects suitable for molecular biology,cytology and genetics.In the study,2 new species of Dictyostelium were found,and 2 identification keys and 3 groups of Dictyostelium and Polysphondylium were given based on morphology.Based on the morphology of dictyostelid cellular slime molds,two genera and thirty-three species of dictyostelids were analyzed by multigene,phospholipid fatty acids of biochemistry method and the combination of three methods.Four genetic sequences were used to classify the dictyostelids.The fatty acids library about dictyostelids has been established.And Four pairs of primers have been screened,primers of eEF-1? gene are EF51,ER960;primers of CO?gene are COXF,COXR;primers of ITS sequence are ITS1,ITS4;primers of 18 S rRNA gene are NS1,NS2.There are 113 sequences amplified in the study,including 43 sequences of ITS sequence,20 sequences is new,fragment length is about 780-1000 bp;23 sequences of CO?sequence,22 sequences is new,fragment length is about 700-1000 bp;19 sequences of eEF-1?sequence,15 sequences is new,fragment length is about 700-900 bp;28 sequences of 18 S rRNA gene sequence,15 sequences is new,fragment length is about 1650-1804 bp.Pairwise K2 P distances for the entire ITS region,CO?,eEf-1? and 18 S rRNA gene were calculated using MEGA.Phylogenetic analyses were conducted using MrBays based on ITS sequences,CO?sequences,EF-? sequences and 18 S rRNA gene sequences.Besides,a multigene phylogenetic tree was been tried to constructed.Phylogenetic trees conform to the taxonomy system of Paulin Schaap and Maria Romeralo which based on morphology.Twenty species of dictyostelids were selected(including 19 strains of Dictyostelium,1 species of Polysphondylium),D.multi-stipes,D.recurvusbasicus,D.semiprostratum,D.homostipes,D.sphaerocephalum,D.mucoroides,D.crassicaule,D.membranosum,D.cespitosum,D.roridum,D.multicellularium,D.magnum,D.giganteum,D.culliculosum,D.clavatum,D.implicatum,D.globisporum,D.tenue,D.discoideum,P.candidum and extracted fatty acids.The PLFAs biomarkers of the 20 species were 75.There were 10 of PLFAs biomarkers that the content was much more than other PLFAs biomarkers.They are 12:00,14:00,16:00,18:00,15:0 ANTEISO,17:0 cyclo,19:0 cyclo w8 c,Feature 2 | 14:0 3OH/16:1 iso I,Feature 3 | 16:1 w6c/16:1 w7 c,Feature 8 | 18:1 w6 c | 18:1 w7 c,content 1.84~14.22%,2.58~10.77%,10.08~37.46%,0.92~16.0%,0.05~4.9%,2.23~24.75%,0.41~5.80%,1.23~15.56%,3.15~11.09%,2.77~8.38%?Based on the type and content of fatty acid,we got some special fatty acids biomarkers of the 20 dictyostelids species.The special fatty acid can be used as the biomarker in the taxonomy of dictyostelid cellular slime molds.And the ratio of some fatty acids also can be used as the biomarker.The cluster analysis of 20 dictyostelids species based on 75 kinds of fatty acids,there were two class group when ??14.They can be separated primely and comform to the taxonomy of dictyostelids.There are another two phylogenetic trees that contain 6 species of dictyostelids that were extracted fatty acids and got ITS sequences and 18 S rRNA gene sequences.It shows that the cluster analysis,morphology and phylogenetic analysis are coincident.Finally,in the classifition of dictyostelid cellular slime molds,we should choose the multigene analysis,fatty acid analysis and morphology together,to get a more natural classification and thorough method. |
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