| Cyclotella(Kützing) Brébisson is a quite common planktonic algae, belongs to phylum Bacillariophyta, class Centriae, order Cosconodiscales, family Coscinodiscaceae,some are frequently found in freshwater and some are tolerant of brackish or marine environments. The species in the genus Cyclotella are wide variety, the morphological differentiation among species are limited, it is very difficult to accurately identify the species in Cyclotella. In addition, the phylogenetic position of the genus remained unclear and the species delimitation had not been solved yet. In this paper, the ultrastucture and phylogenetic analysis of three Cyclotella species were studied by scanning electron microscope(SEM) and molecular biology techniques combined method, trying to determine the morphological characteristics and phylogenetic relationships among Cyclotella species, and furthermore, we explore the application of DNA barcodes in identification and classification of Cyclotella species, for helping solve the identification difficulties.The SEM-investigation of three Cyclotella species showed: C. meneghiniana range from 5–45 μm in diameter. The central area of the valve face is more or less tangentially undulated. Small silica granules are irregularly distributed on the central area of the valve,striae 5–8 are 5 μm. Spines are present at the junction of the valve face and mantle at the interstriae. There is one central fultoportula, 6–13 marginal fultoportulae in per valve. The valves of C. cryptica are circular, nearly flat and 5–10 μm in diameter. Spines can be found at the junction of the valve face and mantle at every interstria. There is one central fultoportula and 11–18 marginal fultoportulae per valve. The C. pseudostelligera ranging from 4-9 μm in diameter, the central area of the valve face is more or less tangentially undulated, small silica granules are irregularly distributed on the central area of the valve.Striae 8-10 in 3 μm, the striaes consisted of two rows of areolae. There is none central fultoportula on the valve of central area. But on the marginal area, there are 6-10 marginal fultoportula. The single rimoportula was formed with a labium open in the direction of the upward of both above Cyclotella species. By comparison, the general morphological characters of the Cyclotella are summarized as follows: the shape of valve are circular,there is a single rimoportula on marginal area. The shape and density of striaes on the valve are different; presence or absence of central fultoportula and small silica granules, the position and quantity of marginal fultoportula, the ratio of central area and marginal areaare also different.Compared with 28 S and ITS, 18 S and rbc L owned higher conserved sites and lower variable sites. Higher parsimony-informative(PI) sites were recorded for the 28S(15.27%)and ITS(16.97%) than for the 18S(5.41%) and rbc L(6.69%). The genetic distance(p-distance) of 18 S, 28 S, rbc L and ITS genes are 0.037, 0.134, 0.063 and 0.168,respectively. 18 S and rbc L own lower p-distance score. Phylogenetic and comparative data analyses suggested that the D1-D2 region of 28 S and rbc L are more suitable than the 18 S and ITS r DNA as molecular markers for the classification and identification of the Cyclotella species.Phylogenetic analyses indicates that C. cryptica always forms a single clade sister with the C. meneghiniana clade, which implies that C. cryptica are genetically homogeneous, but share a close relationship with C. meneghiniana. On the other hand, C.pseudostelligera had no central fultoportulae and small number of marginal fultoportula.Phylogenetic analyses show that C. pseudostelligera had a far relationship with other Cyclotella species, but have a close relationship with Discostella species.The strains C. meneghiniana which live in marine are range from 12–30 μm in diameter. The central area is nearly flat and shows the opening of the central fultoportula.The valve has marginal striae of equal length, and the striae 4–6 at 5 μm, spines are presented. The valve is smooth and shows 3–14 central fultoportulae and 16–23 marginal fultoportulae per valve, each single fultoprtula is surrounded by three satellite pores.Detailed morphological studies showed that they demonstrate rather distinct ultrastructure features, such that C. meneghiniana from saline waters were considerably larger in diameter and contained many more central and marginal fultoportulae than the C.meneghiniana from freshwater. Phylogenetic analyses of DNA sequences(18S, 28 S, ITS r DNA and rbc L) indicated that C. meneghiniana from entirely distinct salinity environments were clustered within the same clade, which suggested that C. meneghiniana from different habitats, possess quite different ultrastructural characteristics but share a nearly identical genotype, thus may represent different a ecophoentypes of the same species.Eight strains of Cyclotella meneghiniana were isolated from widely distributed locations globally and compared to evaluate intraspecific genetic diversity and boundaries.Phylogenetic analyses identified all strains as C. meneghiniana. In the phylogenetic analyses, C. meneghiniana clade in the 28 S r DNA tree had three small ramifications and,in the rbc L tree, there were four small ramifications. These paraphyletic clades indicate that C. meneghiniana collected from different localities are homologous, but independentevolution occurred subsequently likely due to an extensive period of geographical isolation.The genetic distance in the C. meneghiniana calculated based on 28 S r DNA and rbc L genes ranged from approximately 0 to 5.2% and 0 to 0.7%, respectively. This suggests that C. meneghiniana from the same localities may have little or no genetic variation, but there is significant biogeographic genetic diversity among C. meneghiniana from various wide-reaching areas. |
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