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Morphological and Molecular Characterization of Scenedesmus-like
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species from Ergene River Basin (Thrace, Turkey)
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Number of pages: 18
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Number of Tables: 4
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Number of Figures: 4
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Abstract
Scenedesmus-like species can not only been identified morphologically due to their smaller sizes and phenotypic
plasticity. Six Scenedesmus-like species had been isolated from the inland freshwater of Ergene River Basin (Thrace,
Turkey). These strains were investigated morphologically using the light microscopy (LM) and the molecular ITS1ITS4 genes. The combined morphological and phylogenetic analysis revealed that the strains studied belonged to the
genera Desmodesmus (2 strains belonging to D. communis and one strain D. armatus var. subalternas), Acutodesmus
(2 strains related to A. obliquus), and only one strain of the genus Pectinodesmus identified as P. pectinatus.
This study confirms that the morphological identifications of Scenedesmus-like species are only not enough for accurate
species delimitation, the molecular analysis has to be applied. Furthermore, more deep molecular studies using other
gene sequencing should be investigated for more easily species identification.
According to these results; only morphological observations are not enough for taxonomy of microalgae and molecular
analysis must be used for this aim.
Key words: ITS; microalgae; molecular taxonomy; Scenedesmaceae; phylogeny.
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Introduction
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The classification of the genus Scenedesmus Meyen is still unclear. To date, this genus has been
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investigated from different taxonomic standpoints and placed into different algal groups (Hegewald et al.,
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2010; Hegewald et al. 2013). Early, all autosporic coccal green algae characterized by flat and/or curved
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coenobia were described by Meyen (1829) as genus Scenedesmus (Hegewald and Wolf 2003). Turpin
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(1828) described some species of the genus Scenedesmus and transferred them into diatoms. Then later
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Ehrenberg (1834) placed them in the Family Desmidiaceae. Nevertheless, Nageli (1849) sticked the genus
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Scenedesmus, and its known species, to Order Chlorococcales and Family Hydrodictyaceae. Finally,
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Oltsmanns (1904) related the Scenedesmus-like species to Family Scenedesmaceae which belongs to Class
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Chlorophyceae (Hegewald 1997). The recent contribution of Hegewald (1978) sperated the genus
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Scenedesmus into three subgenera: Scenedesmus, Acutodesmus Hegewald and Desmodesmus R. Chodat.
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Komarek and Fott (1983) supported taxonomically this phycological revision. An et al. (1999) separated
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the genus Scenedesmus and other allied coccoidal morphospecies into two genera: Scenedesmus and
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Desmodesmus based on ITS-2 phylogenetic analysis. Therefore, Hegewald (2000) and Van Hannen et al.
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(2002) later transferred and combined all identified Desmodesmus species into the newly separated genus
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Desmodesmus.
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Both Desmodesmus and Scenedesmus taxa are characterized by a high degree of polymorphism in the
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field and in culture conditions. This distinct feature is easily recognized by the variation in number of cells
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forming the coenobium, cell dimensions and cell wall ornamentation (Hegewald 1997). Some researchers
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discussed the aforementioned characteristics in cultured strains of D. communis (e.g., Komarek and Ruzicka
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1969; Brezina et al. 1972; Steenbergen 1978; Hegewald 1989; Trainor 1992; Bica et al. 2012). Kessler et
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al. (1997) pointed out that the varied morphological characteristics and metabolic capacity are not only
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enough to distinguish clearly the different Scenedesmus and Desmodesmus taxa. Therefore, the molecular
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phylogenetic analysis was implemented to solve some this taxonomic puzzle (An et al. 1999); Van Hannen
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et al. 2002; Hegewald and Wolf 2003; Johnson et al. 2007; Vanormelingen et al. 2007; Fawley et al.2011;
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Bica et al., 2012). the recent study of Radha et al. (2013) confirmed that the combined molecular and
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morphological identification of microalgae is a very important approach for species delineation. Some
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abiotic (e.g., light intensity, nutrient concentrations, heavy metals and temperature), and biotic factors (e.g.,
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herbivores) significantly trigger the morphological plasticity in Scenedesmus, Desmodesmus and other
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related morphospecies (Jonson et al., 2007).
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In Turkey, there are a few phylogenetic studies on algae in Turkey (Tüney and Sukatar 2010; Soylu
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and Gönülol 2011; Kesici et al. 2013, Kızılkaya-Tüney et al. 2016). The main aims of the present study;
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was to investigate the molecular and morphological relationships of some Scenedesmus and Desmodesmus
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taxa isolated from the River Basin of Ergene (Turkey)using the light microscope (LM) and PCR analysis.
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Another goal is to increase our knowledge about the taxonomy and autecology of the Turkish freshwater
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microalgae.
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Material and Method
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Materials Collection, Identification and Isolation
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Water samples were collected from inland waters in River Basin of Ergene (Thrace, Turkey) in May
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2012. Coordinates and physical parameters of sampling localities are shown in Table 1. The algal samples
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were collected via phytoplankton net that has 25 µm mesh size and then put in plastic bottles and stored at
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18˚C until transferring to the laboratory by a portable cooler. The algal samples were examined
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morphologically using the light microscope (Olympus CX31). The taxonomic identification was performed
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following the classification systems adopted by (Prescott 1973; Komarek and Fott 1983; Soeder and
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Hegewald 1989). The cell shapes and arrangement, cell length and width, the outer cells, shape and length
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of spines were mainly used as the main digenetic features in species delineation. The measurements of each
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parameter was shown in Table 2.
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The specimens of Scenedesmus and Desmodesmus were isolated using the plate agar method (Bold,
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1942). Axenic cultures were grown in Bold Basal Medium (Stein, 1973), under continuous illumination of
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approximately 150 μmol photons m-2s-1, 16L:8D and 25 ± 2oC. For illumination, cold fluorescent lamps
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were used. The algal photomicrographs were taken by the digital camera, Olympus CH 51. When the culture
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reached to appropriate cell density at exponential phase, the culture was harvested and the DNA isolation
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procedure was performed.
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TABLE 1
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Molecular Analysis
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DNA isolation and PCR analysis
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Total genomic DNA from the cultured samples were isolated by the Gene JET Plant Genomic DNA
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Purification Mini Kit (Thermo Scientific, USA) according to the manufacturer’s instructions and then
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stored at -20ºC until further analysis. 25 µl PCR mix contained 1U Dream Taq DNA polymerase (Thermo
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Scientific, USA), 10× reaction buffer (Thermo Scientific, USA), 0.2 mM dNTP mix (Thermo Scientific,
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USA), 0.25 μM forward and reverse primers (Alpha DNA and IONTEK), 50 ng DNA and ultrapure water
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(HyClone). PCR analysis was performed by T100 Thermal Cycler (Biorad, USA).
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ITS1 (5'-TCC GTA GGT GAA CCT GCG G -3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC
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-3') (White et al. 1990) primers were used to amplify ITS (Internal Transcript Spaces) region of the
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identified species.
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PCR conditions for ITS1-ITS4 primer pair was started with initial denaturation at 95°C for 30 seconds
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and it was followed by 35 cycles of denaturation at 95°C for 5 s, annealing at 55°C for 5 s, extension at
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72°C for 10 s and a final extension at 72°C for 1 min.
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The PCR products were analyzed by 1% agarose gel electrophoresis in 1× Tris-Boric acid-EDTA
(TBE) buffer at 5 Vcm-1, stained with SafeView (ABM) and visualized under UV illumination.
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Sequence analysis and phylogenetic analysis
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Sequence analyses of PCR amplicons were performed at RefGen Biotechnology Research
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Laboratories (Ankara, Turkey) using the eukaryotic primers (ITS1-ITS4). The obtained sequences were
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compared to those from GenBank using the BLAST algorithm (Altschul et al. 1997). ITS gene region
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sequences were aligned using the ClustalW algorithm. Aligned data set was used for creating phylogenetic
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trees in MEGA 5 software (Tamura et al. 2011), Unweighted Pair Group Method with Arithmetic Mean
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(ML) (Michener and Sokal 1957) and Neighbor Joining (NJ) (Tamura et al. 2004) algorithms were used to
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infer phylogenetic relationships.
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Results
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Morphological Identification
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The Scenedesmus-like isolates (Table 2; Fig. 1) were morphologically identified as the following:
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Scenedesmus acutus Meyen (Sample 1), S. antennatus Brebisson (Sample 2), S. quadricauda Chodat
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(Sample 3), S. opoliensis P.G. Richter (Sample 4), S. acuminatus (Lagerheim) Chodat (Sample 5), S.
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armatus Chodat (Sample 6) (Figure 1).
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TABLE 2
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FIGURE 1
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DNA Isolation and PCR Analysis
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The presence of only one DNA band on the agarose gel indicated that there were no other
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contaminants in the sample. The quantity of isolated DNAs was measured by Nanodrop (Thermo Scientific,
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USA). The DNA samples were diluted with ultra-pure water (HyClone) to obtain 50 ng DNA for PCR
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analysis. We obtained approximately 700 base pair amplicons with ITS1-ITS2 primer pairs.
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Sequence analysis and phylogenetic analysis
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The sequenced Scenedesmus-like strains were deposited in the GenBank (see Table 3 for more
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details).
TABLE 3
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For performing the phylogenetic analyses, ITS gene sequences of both our isolates and the GenBank
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data were aligned in ClustalW tool within alignment function of MEGA 5.2. Phylogenetic trees were
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reconstructed using different methods integrated in the MEGA 5.2 software created by Tamura et al. (2007)
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such as Neighbor Joining (NJ) and Maximum Likelihood (ML) using both consensus and linearised tree
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approaches. ML and NJ phylogenetic trees gave identical results for our isolates since only the NJ tree is
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demonstrated (Figure 2) and for our isolates and GenBank data together (Figure 3 and 4).
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TABLE 4
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FIGURE 2
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FIGURE 3
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FIGURE 4
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Discussion
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The genus Scenedesmus is characterized by the following morphological features: flat coenobia of 2-
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32 cells, arranged in 1 or 2 rows, ellipsoidal cells, ovoid or crescent-shaped or tapering toward each end,
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but always elongated, with cell poles from acute to truncate/obtuse, the cell wall is smooth or ornamented
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with spines. The chloroplast is parietal and usually with a pyrenoid. Asexual reproduction occurs with
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autospores (Komarek and Fott 1983, Skaloud 2008, Sakthivel 2016).
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The genus Acutodesmus is so similar to Scenedesmus genus but, there are some differentiations such
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as acute cell poles and polar thickenings of the cell wall (Komarek and Fott, 1983; Hegewald and Hanagata,
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2000) anyway neither Scenedesmus nor Acutodesmus (sensu Tsarenko) is monophyletic in the light of
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molecular data (Hegewald and Wolf, 2003). Thus, the interpretation of the morphological evolution of the
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Scenedesmaceae is difficult owing to a high degree of plasticity or convergence (Elias et.al, 2010).
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This study confirms that Scenedesmus-like species identification is considered to be very difficult
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depending upon Light Microscopy observations. It is well-known that Scenedesmus species can be
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transformed into unicellular and small 2- or 3- celled coenobian forms under culture conditions (Soeder
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and Hegewald 1989). Therefore, the recent molecular techniques should be applied to preciously identify
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the Scenedesmus-like species.
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In this work, each strain studied has its main characteristic features. S. acutus specimens (Fig. 1) has
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elongated fusiform cells, concave middle and convex ends of outer cells, smooth cell wall. S. antennatus
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(Fig. 1) can be easily confused with S. acutus (Fig. 1). To differentiate these species, colorless mucilage
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knobs at poles of cells have to be examined (Komarek and Fott 1983).
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According to Prescott (1973), S. quadricauda (Fig. 1) is characterized by its oblong-cylindric cells
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usually in one series, outer cells with long curved spines at each pole and the inner cells without spines.
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These distinctive features are in good agreement with our specimens. The morphological similarities
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between S. opoliensis (Sample 4) and S. quadricauda (Sample 3) might lead to a mis-identification.
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However, they sharply could be separated based on the distinctive poles of outer cells. For more details,
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outer cells of S. opoliensis (Fig. 1) are fusiform-naviculoid (Prescott, 1973) and the coenobium is curved at
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the top view (Komarek and Fott, 1983). All of these features are compatible with our strain identified.
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S. acuminatus (Fig. 1) can be easily distinguished by lunate cells with sharp apices in a single row
(Prescott, 1973).
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According to Komarek and Fott (1983), S. armatus (Fig. 1) is defined by cylindric-oval cells and
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spines are up to 2 µm. These characteristics helped us to discriminate S. armatus from the other species, S.
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quadricauda (Sample 3).
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The ITS trees that we constracted according to our findings revealed that Scenedesmus-like specimens
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1 (S. acutus) and 2 (S. antennatus) could be identified as Acutodesmus obliquus; Scenedesmus-like
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specimens 3 (S. quadricauda) and 4 (S. opoliensis) were Desmodesmus communis; the Scenedesmus-like
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species sample 5 (S. acuminatus) was Pectinodesmus pectinatus and sample 6 (S. armatus) was
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Desmodesmus armatus var. subalternas (Table 3 and Table 4).
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The trees that we constructed with only our samples (Figure 2) it is seen that sample 1 and 2
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(KF470790, KF470791) are the same species that is they appeared in the same clade. It is similar for sample
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3 and 4 (KF470792 and KF470793), they appeared in the same separate clade, too. Sample 6 (KF470795)
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that revealed as Desmodesmus genus as sample 3 and 4 appeared in a sister clade.
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When we compare our samples with other sequences deposited in the GenBank we obtained similar
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results. Our samples (Sample 1 and 2; KF470790 and KF470791) that were appeared as A. obliquus are in
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the same clade with the ones from GenBank (Figure 3 and 4). As expected, samples 3 and 4 (KF470792
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and KF470793) also located in the same clade with other D. communis species from GenBank. Sample 5
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(KF470794) identified as D. armatus var. subalternas is the same clade with the other same species. Sample
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6 (KF470795) is identified as P. pectinatus by ITS region analysis while it was appeared in the same clade
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with the S. pectinatus and D. pectinatus.
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Hegewald and Wolf (2003), studied Scenedesmus and Acutodesmus species and classified them
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according to 18S rDNA and ITS-2 regions. Even S. arcuatus which is morphologically identical with S.
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hindaaakii clustered in a separate clade. While S. arcuatus showed a high bootstrap value and clustered
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together with A. regularis and A. pectinatus.
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In a similar study for revealing the Scenedesmus-like species identification (Hegewald et al., 2013),
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some Acutodesmus species (A. distendus, A. acuminatus, A. reginae and A. nyygaardii), branched with
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Scenedesmus species (S. bernardii and S. bajacalifornicus). Also these mentioned species cited as
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Scenedesmus in Lewis and Flechtner (2004)’s study.
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As we have been experienced it is hard to identify Scenedesmus and Desmodesmus species
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morphologically in LM. Molecular techniques help identification and distinguishing of problematic species.
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In our study, we identified 6 different isolates by their molecular structures and ITS regions. All isolates
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identified as Scenedesmus sp. by LM, but the results changed with the molecular techniques; three of them
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(Sample 1, 2) were found Acutodesmus species; three of them (Sample 3, 4, 6) were found Desmodesmus
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species and one of them (Sample 5) was found Pectinodesmus species.
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These studies on Scenedesmus-like species bring out a question as asked before in Hegewald et al.
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(2013)’s study; if a genetic exchange is possible in Scenedesmus-complex? Because this group usually
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reproduce vegetatively and not allow a genetic diversity in high levels. Zoospores, which allows genetic
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diversity, were rarely reported (Trainor, 1963; Trainor 1996). After Corradi et al. (1995), reported high
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zoospore production in stress conditions answered this question even if just a bit. This means zoospore
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production and so genetic fusion is not rare as its been thought before in Scenedesmus-complex species.
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To provide this sight there should be more studies done on this group.
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There is a clear need for further studies to increase the number of available sequences of genetic
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markers for different species in this complex group. We believe that establishing large-scale sequence
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databases will help to identify species easily and accurately. Also we need more studies on more strains
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from different localities to reveal the identification of this group.
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322
National d’Histoire Naturelle Paris (16): 295–344.
323
324
Tuney I., Sukatar A. 2010. DNA Extraction Protocol from Brown Algae. Biological Diversity and Conservation (3):
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325
Van Hannen E. J., Fink P. and Lurling M. A. 2002. A revised secondary structure model for the internal transcribed
326
spacer 2 of the green algae Scenedesmus and Desmodesmus and its implication for the phylogeny of these algae.
327
European Journal of Phycology (37): 203–208. doi:10.1017/S096702620200361X.
328
Vanormelingen P., Hegewald E., Braband A., Kitschke M., Friedl T., Sabbe K. and Vyverman W. 2007. The
329
systematics of a small spineless Desmodesmus species, D. costatogranulatus (Sphaeropleales, Chlorophyceae),
330
based on ITS2 rDNA sequence analyses and cell wall morphology. Journal of Phycology (43): 378–396. doi:
331
10.1111/j.1529-8817.2007.00325.x.
332
333
White T. J., Bruns T. and Lee S. 1990. PCR Protocols: A guide to methods and applications. Academic Press Inc. New
York.
334
10
335
TABLES
336
337
Table 1. Physical parameters of the sampling sites.
Algal taxa
Scenedesmus acutus
Meyen
(Sample 1)
Scenedesmus
antennatus
Brebisson (Sample
2)
Scenedesmus
quadricauda Chodat
(Sample 3)
Scenedesmus
opoliensis P.G.
Richter (Sample 4)
Scenedesmus
acuminatus
(Lagerheim) Chodat
(Sample5)
Scenedesmus
armatus Chodat
(Sample 6)
Sampling
Sites
Sazlıdere
KaşıkçıVillage
Pool
Bahçedere
Kaşıkçı Lake
Coordinates
41° 13' 22.541"
N
26° 41' 12.122"
E
40° 59' 46.499"
N
27° 11' 21.466"
E
41° 22' 5.830" N
27° 44' 45.193"
E
41° 1' 17.435" N
27° 13' 25.988"
E
Temp.
(°C)
pH
Conduct.
(µS cm-1)
Dissolved O2 (mg
l-1)
13.20
7.80
195.3
8.90
16.30
7.25
2038
5.21
20.70
7.47
760
0.98
16.30
7.25
2038
5.21
Kaşıkçı Lake
41° 1' 17.435" N
27° 13' 25.988"
E
16.30
7.25
2038
5.21
Hacıfaklı Stream
41° 42' 35.078"
N
27° 28' 43.904"
E
15.30
7.30
538
8.99
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
11
353
Table 2. Synonyms, current names and morphometric analysis of samples.
Morphological
Identification
(Komarek&Fott
1983)
Scenedesmus
acutus Meyen
(Sample 1)
Scenedesmus
antennatus
Brebisson (Sample
2)
Scenedesmus
quadricauda
Chodat
(Sample 3)
Scenedesmus
opoliensis P.G.
Richter (Sample 4)
Scenedesmus
acuminatus
(Lagerheim)
Chodat (Sample 5)
Scenedesmus
armatus Chodat
(Sample 6)
Synonyms
(Guiry&Guiry 2015)
Scenedesmus dimorphus f.
granulatus Isabella &
R.J.Patel
Arthrodesmus acutus
Ehrenberg ex Ralfs
Scenedesmus crassus Chodat
1926
Scenedesmus scenedesmoides
Chodat 1926
Desmodesmus
quadricaudatus (Turpin)
Achnanthes quadricauda
Turpin 1828
Scenedesmus carinatus f.
brevicaudatus Uherkovich
Scenedesmus opoliensis var.
setosus Dedusenko
Selenastrum acuminatum
Lagerheim
Scenedesmus obliquus var.
acuminatus (Lagerheim)
Chodat
Scenedesmus falcatus Chodat
1894
Scenedesmus acuminatus var.
minor G.M.Smith 1916
Scenedesmus acuminatus var.
elongatus G.M.Smith 1926
-
Current name
(Guiry&Guiry 2015)
Length × Width
(µm)
(Komarek & Fott
1983)
Scenedesmus obliquus
(Turpin) Kützing
(5-) 7-25 (-27)×27.5 (-14)
10×4
Scenedesmus
dimorphus (Turpin)
Kützing
9-28×1.5-5
15×4
Scenedesmus
quadricauda Chodat
(5.5)10-36×(2.1)
3-8 (-12)
11×5
Desmodesmus
opoliensis
(P.G.Richter)
E.Hegewald
8-36.5×2-9
20×7
Acutodesmus
acuminatus
(Lagerheim)
P.M.Tsarenko
9.6-48 (-50)× 1.59
22×5
Scenedesmus armatus
(Chodat) Chodat
5-13×2.3-6
11×6
354
355
356
357
358
359
360
361
362
363
12
Length × Width
(µm)
(Our samples)
364
Table 3. Morphologic and molecular identifications of the Scenedesmus-like isolates and their accession numbers in
365
the Genebank NCBI.
Samples
Morphologic Identification
Molecular Identification
Accession Numbers
1
Scenedesmus acutus
Acutodesmus obliquus
KF470790
2
Scenedesmus antennatus
Acutodesmus obliquus
KF470791
3
Scenedesmus quadricauda
Desmodesmus communis
KF470792
4
Scenedesmus opoliensis
Desmodesmus communis
KF470793
5
Scenedesmus acuminatus
Pectinodesmus pectinatus
KF470794
6
Scenedesmus armatus
Desmodesmus armatus
var. subalternas
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
13
KF470795
386
Table 4. Similarity and e-values as Blast results of ITS region of our isolates and GenBank data.
Our
Accession No
KF470790
KF470791
KF470792
KF470793
and
Our Isolates’
Molecular
Identification
Acutodesmus
obliquus
(Sample 1 and 2)
Desmodesmus
communis
(Sample 3 and 4)
KF470794
Pectinodesmus
pectinatus (Sample
5)
KF470795
Desmodesmus
armtus
var.
subalternas (Sample
6)
GenBank Data
Accession no
AB917115
AB917115
AB917117
AB917120
AB917124
KC800949
JQ082319
FR865715
FR865719
FR865726
FR865737
FR865738
JX101322
JX101323
JX101324
JX101325
JX101327
KP726233
KP726234
KP797884
JQ922412
FR865723
FR865730
FR865733
FR865735
JQ082314
JQ082321
JQ082331
JQ082332
AB917112
AB917113
AB917133
AB917138
FR865727
DQ417520
DQ417521
DQ417546
DQ417547
387
388
14
e-value
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Similarity
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
98%
98%
98%
98%
98%
98%
98%
98%
98%
98%
99%
99%
98%
98%
98%
99%
99%
99%
99%
99%
99%
99%
99%
99%
99%
389
FIGURES
390
391
392
393
394
395
Figure 1. LM photomicrographs of Scenedesmus-like species in this study 1. Scenedesmus acutus (Scale bars: 20 µm),
2. Scenedesmus antennatus (Scale bars: 20 µm), 3. Scenedesmus quadricauda (Scale bars: 20 µm), 4. Scenedesmus
opoliensis (Scale bars: 20 µm), 5. Scenedesmus acuminatus (Scale bars: 20 µm), 6. Scenedesmus armatus (Scale bars:
20 µm).
396
397
398
399
15
400
401
402
Figure 2. Neighbour Joining (NJ) tree of our isolates based on the partial sequence of the ITS region constructed by
403
Tamura-Nei method with 100 bootstrap.
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
16
421
422
423
Figure 3. Neighbour Joining (NJ) phylogenetic tree of our isolates and GeneBank data with 100 Bootstrap values
(KF536801 is outgroup).
424
17
425
426
427
428
Figure 4. Maximum Likelihood (ML) phylogenetic tree of our isolates and GeneBank data with 100 Bootstrap values
(KF536801 is outgroup).
18