The Ultrastructure And Phylogenetic Analysis Of Four Oocystis Species

Oocystis is a quite common planktonic algae, belongs to phylum Chlorophyta, classTrebouxiophyceae, order Chlorellales, family Oocystaceae, widely distributed infreshwater ecosystems and predominantly in the plankton of small lakes and ponds, theyalso grow in seawater bodies. The species in the genus Oocystis are wide variety.Morphological variability in the genus is high. It is very difficult to accurately identify thespecies in Oocystis. In addition, the phylogenetic position of the genus remained unclearand the species delimitation have not been solved yet. In this paper, the ultrastructure andphylogenetic analysis of four Oocystis species were studied by transmission electronmicroscopy (TEM) and molecular biology techniques combined method. Trying todetermine the morphological characteristics and the phylogenetic relationships among theOocystis species.The TEM-investigation of4Oocystis species showed: the Oocystis lacustris CCALA396cell is enveloped by mucilage envelope, oval shape, the cell wall is composed ofseveral layers; In the cell, there is only one pyrenoid, is located in the center of thechloroplast, round in shape, the diameter of the pyrenoid body is between1and1.5μm,surrounded by a layer of starch sheath which consisted of2-5starch plates, the thickness ofthe starch sheath is between0.25and0.5μm. The Oocystis nephrocytioides CCALA397cell has no mucilage envelop, oval or round shape, the cell wall is composed of severallayers; In the cell, there are two pyrenoids which is located in the center of the chloroplast,round in shape, the diameter of the pyrenoid body is between0.75and1.25μm, the starchsheath around the pyrenoid appears like a closed ring, the thickness of the starch sheath isbetween0.15and0.75μm. The Oocystis sp. FACHB1429cell is enveloped by mucilageenvelope, round shape, the cell wall is composed of several layers; In the cell, there is onepyrenoid, is located in the center of the chloroplast, irregular shape. The pyrenoid issurrounded by some different sizes starch plates. The Oocystis sp. cell is enveloped bymucilage envelope, oval or round shape, the cell wall is composed of several layers; In thecell, there is one pyrenoid, is located in the center of the chloroplast, round or oval in shape,the diameter of the pyrenoid body is between1.25and2.5μm, surrounded by a layer ofstarch sheath which consisted of4-7starch plates. By comparison, the generalmorphological characters of the genus Oocystis are summarized as follows: presence or absence of a mucilage envelop; the cell wall is composed of several layers; the shape ofcell is oval or round; there is pyrenoid in the chloroplast, the pyrenoid is surrounded by alayer of starch sheath; the starch sheath is consisted of several starch plates.Further investigation of the pyrenoid ultrastructure showed: there are several curvedthylakoids in the pyrenoid of Oocystis lacustris CCALA396; the thylakoids is absence inthe pyrenoid of Oocystis nephrocytioides CCALA397; the pyrenoid in Oocystis sp.FACHB1429is traversed by two tubular thylakoids in parallel; in the Oocystis sp., thepyrenoid is traversed by3to5thylakoids. From the above we know that the structure ofpyrenoids and its starch sheath are diversity and species-specific. Therefore, the structureof pyrenoid matrix and its starch sheath can be used as diacritic species features inOocystis to do species identification.18S rRNA gene is the most widely used molecular markers, and the data forcomparison is the most. In this study, the18S rRNA gene differences between the speciesof the genus Oocystis is0.1%~10.8%. Indicated that the18S rRNA gene is variable, it canbe as a good molecular marker to used molecular species identification. The results ofphylogenetic tree showed that the genus Oocystis is obviously paraphyletic and has to besplit into seven independent group by Echinocoleum elegans, Lagerheimia genevensis,Terachlorella alternans, Amphikrikos sp., Oopanctella planoconvexa, Ecballocystiopsisdichotomus and makinoella tosaensis. We recommend that the genus Oocystis should begrouped and established the “small” genera for whole clusters.The28S rRNA gene differences between the species of the class Trebouxiophyceae isonly6.5%~15.4%. Indicated that28S rRNA gene is more conservative, more suitable forthe study on the genus level or above, not suitable for the genus level or below study. Thephylogenetic analysis based on28S rRNA gene is consistent with previous reports whichphylogenetic analysis based on18S rRNA gene. It further verify the reliability of18SrRNA gene.The ITS2gene differences between the species of the genus Oocystis is8.8%~45.7%.Indicated that ITS2gene is more variability, more suitable for the study on the specieslevel or below. The secondary structure of ITS2sequences is generally stable. There is asmall stem structure at the5’ end and3’ end, it is conservative. There also have a largerring structure and a longer stem structure. The ITS2secondary structure can provide areference for the future phylogenetic analysis and genetic diversity study.