Comparative Molecular Analysis of Freshwater Centric Diatoms with Particular Emphasis on the Nuclear Ribosomal DNA of Stephanodiscus (Bacillariophyceae)
- Author: Ki Jang-Seu
- Organization: Ki Jang-Seu
- Publish: ALGAE Volume 24, Issue3, p129~138, 01 Sep 2009
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ABSTRACT
DNA-based discrimination of species is a powerful way for morphologically otherwise similar species, like centric diatoms. Here, the author sequenced long-range nuclear ribosomal DNAs, spanning from the 18S to the D5 region of the 28S rDNA, of Stephanodiscus, particularly including a Korean isolate. By comparisons, high DNA similarities were detected from the rDNAs of nine Stephanodiscus (>99.4% in 18S rDNA, >98.0% in 28S rDNA). Their genetic distances, however, were significantly different (Kruskal-Wallis test, p < 0.01) compared to two related genera, namely
Cyclotella and Discostella. In addition, genetic distances of 18S rDNAs were significantly different (Student’s t-test, p = 0.000) against those of the 28S rDNAs according to individual genera (Cyclotella , Discostella, and Stephanodiscus). Phylogenetic analyses showed that Stephanodiscus and Discostella showed a sister taxon relationship, and their clade was separated from a cluster of Cyclotella (1.00 PP, 100% BP). This suggests that Stephanodiscus has highly conserved sequences of both 18S and 28S rDNA; however, Stephanodiscus is well-separated from other freshwater centric diatoms, such as Cyclotella and Discostella, at the generic level.
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KEYWORD
freshwater diatom , nucleotide divergence , phylogeny , ribosomal DNA , Stephanodiscus
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The centric diatom
Stephanodiscus Ehrenberg 1846 is commonly present in freshwater environments, and several species are important bio-indicators of water quality, particularly for eutrophicated waters (Haet al . 2002). Conventionally, their taxonomic identities are determined by microscopic observations of certain morphological characters, such as the pattern of the central area of the exoskeleton, and density and branching of the striae (Olivaet al . 2008). However, morphological discrimination of these species is very difficult, because of small size (less than 15 μm) and a number of recorded differentStephanodiscus species (approximately 124 taxa) according to Guiry and Guiry (2009). In addition, morphologies ofStephanodiscus are similar to those of other freshwater centric diatoms, e.g.Cyclotella andDiscostella (formerly, these represented the stelligeroid group ofCyclotella [Houk and Klee 2004]). Moreover, several centric diatoms of different species sometimes are co-occurring. Many uncertainties about the proper identities of the centric diatoms are, therefore, remaining.Recently, DNA-based taxonomy is widely used for the discrimination of small-size organisms, including diatoms and dinoflagellates (Karsten
et al . 2005; Kiet al . 2009). Indeed, molecular analyses (e.g. immunoassays, PCR assay, DNAchip), including phylogenetic inferences, are very effective to discriminate morphologically similar, microscopic-size organisms. In most cases, these molecular approaches are based on the DNA sequences of the nuclear ribosomal DNA (rDNA), because it occurs in all living organisms, and many rDNA sequences are available, compared to other genes (e.g.actin , α-, β?-tubulin , andHsp90 ). The rDNA sequences have been used for the discrimination of centric diatoms and for the phylogenetic analyses (Alversonet al . 2007; Kaczmarskaet al . 2007). Most studies on the freshwater centric diatoms have been biased toCyclotella , particularly for the ystematics and phylogenetic relationships of cyclotelloid diatoms (Beszteriet al . 2005, 2007; Alversonet al . 2007). More recently, genetic divergence betweenCyclotella andDiscostella has been studied, by comparisons of a wide range of rDNA sequences (Junget al . 2009). With regard to molecular analyses ofStephanodiscus , Kaczmarskaet al . (2005) showed for the first time thatCyclotella andStephanodiscus were not belonging to the same phylogenetic clade, making the family Stephanodiscaceae paraphyletic. Recently, Alversonet al . (2007) reported phylogenetic relationships of thalassiosiroid diatoms, representing the separations of the freshwater centric diatoms, e.g.Cyclotella, Discostella andStephanodiscus . Recently, the author reported high molecular genetic divergences betweenCyclotella andDiscostella , suggesting that rDNA may be a suitable molecular marker for the discrimination of the two genera and species (Junget al . 2009). Excluding these works, little attention has been paid on the molecular analyses ofStephanodiscus .In the present study, the author sequenced nuclear rDNA, spanning the 18S to the 28S rDNA, of
S. hantzschii andStephanodiscus sp., including a Korean isolate, and characterized molecular features of various rDNA regions according to each rDNA. Comparative analyses of individual 18S, 28S rDNAs were performed with some rDNAs of selectedStephanodiscus to reveal the rDNA relationships of the freshwater centric diatoms. In addition, molecular divergences betweenStephanodiscus with other centric diatomsCyclotella andDiscostella were compared to evaluate their usefulness for the discrimination of freshwater centric diatoms. The studiedS. hantzschii is one of the planktonic, cosmopolitan species and theStephanodiscus blooms were recorded annually in Korean waters, particularly in the Paldang Reservoir and Nakdong River (Kim 1998; Haet al. 2002; Hanet al . 2002; Kimet al . 2008).Water samples were collected from Paldang Reservoir (a reservoir in Han River) of Korea, when
Stephanodiscus blooms occurred. The author isolated single cells ofStephanodiscus from field samples using the capillary method (Ki and Han 2005), and established a clonal culture (KHR001) ofStephanodiscus . An additional strain (UTCC 267) ofS. hantzschii was commercially obtained from the University of Toronto Culture Collection of Algae and Cyanobacteria (UTCC). All the cultures were routinely maintained in Diatom Medium, DM, (Beakeset al . 1988), and were grown at 15℃, 12:12 h light:dark cycle, with a photon flux density of about 65 μmol photons m?2 s?1.> DNA extraction and PCR amplification
A total of 50 ml clonal cultures were harvested by centrifugation centrifugation at 8,000 rpm for 15 min. The concentrated cells were transferred to 1.5 ml micro tubes, 100 μl of TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) was added and the tubes were stored at ?20℃ until DNA extraction. Genomic DNA was isolated from the stored cells using the DNeasy Plant mini kit (Qiagen, Valencia, CA).
Polymerase chain reaction (PCR) was subject to amplifythe 18S-28S rDNA of Stephanodiscus genomic DNA. Inthis case, the author used a set of PCR primers that targetedto bind nuclear 18S rDNA (a forward AT18F01, 5’-ACC TGG TTG ATC CTG CCA GTA G-3’) and 28SrDNA (a reverse PM28-R1318, 5’-TCG GCA GGT GAGTTG TTA CAC AC-3’), which are specific for diatoms(Jung et al. 2009). PCR was performed with 50 μl reactionmixtures containing 30.5 μl sterile distilled water, 5 μl 10x LA PCR buffer II (TaKaRa, Kyoto, Japan), 8 μl dNTPmix (4 mM), 5 μl of each primer (5 M), 0.5 μl LA Taqpolymerase (2.5 U), and 1 μl of template. PCR cyclingwas performed in a Bio-Rad iCycler (Bio-Rad, Hercules,CA) with 94°C for 2 min, following 35 cycles of 94°C for20 sec, 55°C for 30 sec, and 72°C for 2 min, and a finalextension at 72°C for 10 min. Resulting PCR productswere electrophoresed in a 1.0% agarose gel (Promega,Madison, WI), stained with ethidium bromide, and visualizedby ultraviolet transillumination.
For DNA sequencing, desired PCR products were purified with a QIAquick PCR purification Kit (Qiagen GmbH, Germany). DNA sequencing reactions were performed in a ABI PRISM® BigDyeTM? Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster City, CA) using the PCR products (2 μl) as the template and 10 picomoles of the above PCR and internal walking primers. Labeled DNA fragments were analyzed on an automated DNA sequencer (Model 3700, Applied Biosystems, Foster City, CA).
Editing and contig assembly of DNA sequences were performed using Sequencher 4.1.4 (Gene Codes, Ann Arbor, MI). The coding rDNAs were identified by comparison with those of other diatoms, including
Cyclotella meneghiniana (GenBank No. GQ148712) andDiscostella sp. (GQ148713). DNA sequences determined here have been deposited to GenBank as accession numbers GQ844873 and GQ844874.> Comparisons of
Stephanodiscus rDNABLAST (The Basic Local Alignment Search Tool) searches were performed with the present rDNA sequence data and the available DNA sequences in the National Center for Biotechnology Information (NCBI) database. In addition, DNA sequences of
S. hantzschii and a KoreanStephanodiscus were compared with those of otherStephanodiscus (see Table 1). DNA similarity scores of individual rDNA molecules were calculated by using pairwise sequences among nine selected species ofStephanodiscus in BioEdit 5.0.6 (Hall 1999). In addition, dot-plot analysis was carried out using the MegAlign 5.01 (DNAstar Inc., Madison, WI). Molecular genetic divergences of the nine species were measured with the Kimura two-parameter model in MEGA 4.0 (Tamuraet al . 2007). Statistical analyses on the nucleotide comparisons were performed using SPSS 10.0.7 (SPSS Inc., Chicago, IL).> Phylogenetic relationships of
Stephanodiscus speciesPhylogenetic analyses of the freshwater centric diatoms were carried out, following our previous work (Jung
et al . 2009). In the present case, the author constructed two new data matrixes of individual 18S and 28S rDNAs, including tenStephanodiscus , four selectedDiscostella , and six selectedCyclotella rDNAs (see Table 1). A total of 21 sequences, including the outgroup (Thalassiosira gessneri #AN02-08), were aligned with the Clustal W 1.8 (Thompsonet al . 1997). The aligned sequences were trimmed each end to the same length. In addition, various regions and uncertain sequences were further corrected manually. Finally, only unambiguous positions of the aligned sequences were used in the subsequent analyses: 1,689 out of 1,813 alignment positions for 18S, 506 out of 1,264 for 28S). MrModeltest2 (Nylander 2004) was used to find the optimal model of DNA substitution for the Bayesian tree construction. As best-fit models for the present 18S rDNA dataset, the author selected the General Time Reversible plus Invariant sites plus Gamma distributed model (GTR+I+G) for 18S (-lnL = 3859.3) and for 28S (-lnL = 2260.4) from the Akaike Information Criterion (AIC), respectively. A Bayesian tree of the 18S was implemented with the selected GTR+I+G substitution model in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). The Markov chain Monte Carlo process was set to two chains (MCMC), and 1,000,000 generations were conducted. The sampling frequency was assigned as every 100 generations. After analysis, the first 2,000 trees were deleted as burn-in and a consensus tree was constructed. The phylogenetic tree was visualized with TreeView ver.1.6.6 (Page 1996). Bayesian posterior probabilities (PP) of more than 0.50 were indicated at each branch node. An additional Neighbor-Joining (NJ) tree was constructed with the same data matrix of 18S rDNA using the Maximum Composite Likelihood model in MEGA 4.0 (Tamuraet al . 2007). For the 28S rDNA tree, Bayesian and NJ analyses were performed the same way as in the 18S analysis.> Nuclear rDNA of
S. hantzschii and KoreanStephanodiscus In the present study, DNA sequences of nuclearrDNAs, spanning the 18S to the D5 domain of the 28SrDNA, were determined from S. hantzschii #UTCC 267(3,768 bp; 47.9% GC) and a Korean Stephanodiscus sp.#KHR001 (3,682 bp; 48.3% GC), as shown in Table 2.Their gene structures were organized in the typicaleukaryotic fashion of rDNA (i.e. 18S-ITS1-5.8S-ITS2-28S).In general, the 28S rDNA, the largest rDNA codingregion, contains twelve hyper-variable domains(Hassouna et al. 1984; Lenaers et al. 1989), often designatedas divergent (D) domains. Of them, the presentsequences included D1 to D5 of the 28S rDNA. Uponcomparisons, most sequences available in public databases(e.g. DDBJ, EMBL, NCBI) were revealed fromD1/D2 domains of the 28S rDNA, while the others containmuch genetic information (Ki and Han 2007). Thepresent data included wider range of the 28S rDNA fromthe genus Stephanodiscus. With these data, the authorevaluated their molecular characteristics and compared 2representatives of Stephanodiscus with freshwater centricdiatom data available in the NCBI. Particularly, completelengths of the 18S rDNA sequences of S. hantzschii#UTCC 267 and Stepahnodiscus sp. #KHR001 were estimatedto be 1,805 bp, after incorporating undeterminednucleotides of the 18S rDNA 5’end into the present 18Ssequences, taking into account of available data (e.g.AM712618, DQ093370) recorded in GenBank (e.g.AM712618, DQ093370). These were nearly identical tothose of other relatives, including Cyclotella andDiscostella (Jung et al. 2009).
By database searches, the author found many partial 18S, 28S rDNA sequences revealed from
Stephanodiscus , particularly by the work of Alversonet al . (2007). The rDNA ITS regions were only revealed from a few species, includingS. hantzschii (U03078),S. niagarae (U03074-6, AF455267-9), andS. yellowstonensis (U03077). By using the ITS data, Wolfet al . (2002) demonstrated the same species ofS. neoastraea andS. heterostylus . All the DNA sequence data (e.g. 18S, ITS, 28S) available in databases have been partially sequenced at a given locus. Here the author compared the present data with available partial sequences reported fromStephanodiscus (Tables 3, 4). Firstly, these sequences were compared with those of the NCBI database using the BLAST search algorithm. BLAST searches of individual rDNA sequences showed that a KoreanStephanodiscus sp. #KHR001 was highest matched withS. hantzschii #CCAP 1079/4 (GenBank DQ093370) with 99.5% similarity by 18S rDNA comparison, and was also matched toS. hantzschii #AT-N2 (AJ878502) with 98.5% similarity by 28S comparison. On the other hand, the ITS rDNA comparison showed that the top hit was recorded withS. niagarae (U03076) of 89.9% DNA similarity. BLAST searches of individual rDNA ofS. hantzschii #UTCC 267 were similar to those of KoreanStephanodiscus spp. In the latter, the rDNA ITS was highly matched at 94.1% similarity withS. yellowstonensis (U03077) and at 96.9% similarity withS. hantzschii (U03078), respectively. Overall similari-ties of the coding rDNAs, e.g. 18S and 28S, were high within the genusStephanodiscus .> Molecular similarities and genetic divergence of rDNA
Molecular comparisons showed that a Korean isolate of
Stephanodiscus had a different genotype compared with otherStephanodiscus (Table 3), includingS. hantzschii . The present Korean isolate was presumably identified asS. hantzschii based on routine morphological observations and previous studies (Hanet al . 2002). As noted previously, morphological characteristics ofStephanodiscus are similar to each other, and a number of species (at least 124 species) have been described so far. n the present study, the author tentatively discriminatedStephanodiscus sp. #KHR001 (or named asStephanodiscus sp. cf.S. hantzschii ). Considering these molecular and morphological characteristics ofStephanodiscus , the author selected nine species ofStephanodiscus , including Korean one, and measured DNA similarities of both 18S and partial 28S rDNA sequences (Table 3). High DNA similarities were recorded among rDNA pairs of nineStephanodiscus (>;99.4% in 18S rDNA, >;98.0% in 28S rDNA). Strikingly, DNA similarities of 18S rDNAs were considerably similar to one another. The present Korean isolate (KHR001) showed more than 99.5% similarity withS. agassizensis, S. binderanus, S. hantzschii andS. minutulus , respectively. By 28S comparisons, DNA similarities were highly recorded among theStephanodiscus , while these data included the most variable domain D1/D2 within the 28S rDNA (Ki and Han 2007). These suggest that molecular genetic divergences within theStephanodiscus are considerably low with approximately 1% in 18S rDNA and 2% in 28S rDNA, respectively. However, we detected high genetic divergences of other freshwater centric diatoms, e.g.Cyclotella andDiscostella (Junget al . 2009). Genetic divergences of freshwater centric diatoms may, therefore, be taxon-dependent rather than general molecular characteristics in the three diatom groups.> Phylogenetic relationships of freshwater centric diatoms
Molecular relationships of three major freshwater centric diatoms, namely
Cyclotella, Discostella andStephanodiscus , were inferred from Bayesian, Neighbor-Joining analyses, using their available 18S and partial 28S rDNA sequences, respectively (Figs 2, 3). Recently, we reported the phylogenetic relationships ofCyclotella andDiscostella , in which phylogenetic trees were inferred with Bayesian method, using 18S and 28S rDNA data (Junget al . 2009). In the present study, the author focused onStephanodiscus relationships against the two other genera, as well as inter-species relationships within the genusStephanodiscus . Considering our previous work (Junget al . 2009), the author constructed new data matrices, including certain members ofCyclotella andDiscostella . In the present analyses, a total of 20 species, including sixCyclotella , fourDiscostella and tenStephanodiscus , with the outgroup ofThalassiosira , were subjected to phylogenetic analyses with Bayesian and NJ methods (Fig. 1). Phylogenetic analyses showed that the three genera included here were well separated (1.00 PP, 100% BP). Overall topologies of the Bayesian tree were compatible with those of the NJ tree. All the species ofStephanodiscus formed a cluster (1.00 PP, 100% BP), of which clade was separated from aDiscostella cluster.Stephanodiscus andDiscostella are a sister relationship, separating a clade ofCyclotella , showing that these patterns were in agreement with Alversonet al . (2007). In theStephanodiscus linage, most species, excluding a cluster ofS. niagarae, S. reimerri andS. yellowstonensis , formed a polytomy including six species. These were caused by low genetic divergences and high DNA similarities, detected in Table 3. Within this linage, a KoreanStephanodiscus isolate was positioned at an early divergent place, clearly being separated from otherStephanodiscus (1.00 PP, 100% BP).In addition to this, phylogenetic analyses of partial 28S rDNA of the three centric diatom groups showed similar branch patterns, when compared with those of 18S rDNA phylogenies.
Stephanodiscus was a sister relationship withDiscostella , of which clade was clustered withCyclotella (1.00 PP, 100% BP). Within theses analyses,Stephanodiscus formed a polytomy, excluding a cluster ofS. niagarae, S. reimerii andS. yellowstonensis (Fig. 2). The 28S rDNA phylogeny showed that the Korean isolate was not separated from otherStephanodiscus . Overall 28S phylogeny was in good accordance with the 18S phylogeny described above.> Molecular divergences of 18S, ITS, 28S rDNAs
The present rDNA sequences of
Stephanodiscus were graphically compared with those of theCyclotella sensu lato, by using dot-matrix and entropy-plot analyses (Fig. 3). Here the dot-plot was obtained using sliding windows of 60 nucleotides along the compared rDNAs. The plot showed a clear distribution of both variable and conserved positions along the rDNA sequences: the coding regions were conserved, the other non-coding regions were highly variable (Fig. 3). This was in good accordance with our previous study (Junget al . 2009).Nucleotide divergences of the 18S and 28S rDNA sequences were compared, using pairwise genetic distances calculated with the Kimura two-parameter model (Table 4). In most cases, DNA divergences within nine Stephanodiscus (listed in Table 3) were considerably low both in 18S (less than 0.2%) and in 28S rDNA (less than 1.0%). By comparisons, divergences of the 28S rDNA were significantly different compared to the 18S rDNA (Student’s t-test, p = 0.000). In addition, divergences of individual 18S, 28S rDNA among the three groups, Cyclotella, Discostella, and Stephanodiscus, were significantly different according to the Kruskal-Wallis Test (p < 0.01). By comparisons of Stephanodiscus with Cyclotella and Discostella, high genetic divergences were calculated from 18S (Stephanodiscus versus Cyclotella, 5.4%, SD = 0.45) and 28S rDNA (Stephanodiscus versus Cyclotella, 15.6%, SD = 2.9). These support that Stephanodiscus has high similarities of both 18S and 28S rDNA (Table 3), but Cyclotella and Discostella shows low similarities in both genes (Jung et al. 2009).
> DNA identity of Stephanodiscus from Paldang Reservoir
The centric diatoms, including
Cyclotella, Discostella , andStephanodiscus , commonly occur in freshwaters, including the Han River (Hanet al. 2002; Junget al . 2009). According to the previous studies (Kim 1998; Hanet al . 2002), high abundance of the centric diatoms were frequently observed in water samples collected from Paldang Reservoir and Han River during early spring. Some blooms were caused byCyclotella andDiscostella (e.g. Junget al. 2009), and sometimesStephanodiscus blooms occurred in Paldang Reservoir (Kim 1998; Hanet al . 2002). The bloomingStephanodiscus in Paldang Reservoir were morphologically considered asS. hantzschii (Hanet al . 2002). In addition, the author isolated a KoreanStephanodiscus cell (KHR001) from a water sample of Paldang Reservoir whenStephanodiscus cells were present predominantly, and identified them asS. hantzschii , judging by routine morphological observa-tions and previous reports (Kim 1998; Hanet al . 2002). However, comparative molecular data done with BLAST searches and similarity scores (Table 3) were not in accordance with morphological identity. Upon rDNA comparisons betweenStephanodiscus sp. #KHR001 with otherS. hantzschii (the present UTCC 267, WTC21, ATN2), the present Korean isolate should be a different species, thanS. hantzschii , judging from the present phylogenetic analyses and molecular similarities (Table 3; Figs. 1, 2). Previously, Kim (1998) discriminated three species ofStephanodiscus , e.g.S. hantzschi f.tenuis ,S. parvus ,S. invistatus , from spring water samples of Paldang Reservoir. These suggest that the blooming species may be some of these recorded species (e.g.S. hantzschii ,S. hantzschi f.tenuis ,S. parvus, S. invistatus ) possibly including unrecorded species, whileS. hantzschii have been considered only to be the blooming species in Paldang Reservoir for a long time. Thus, existing ecological and morphological discrimination of the bloomingStephanodiscus may be reinvestigated, considering the present molecular data available.In conclusion, the present study determined longrange sequences of rDNA from
S. hantzschii #UTCC 267 and a KoreanStephanodiscus sp. #KHR001. Molecular comparisons showed high genetic similarities (or low genetic divergence) within the genusStephanodiscus compared with those ofCyclotella andDiscostella . From these facts, the author concludes that nuclear rDNA sequences ofStephanodiscus are considerably similar to each other, but they are significantly different (p < 0.01) from other freshwater centric diatoms (e.g.Cyclotella andDiscostella ).-
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[Table 1.] Origins of the centric diatoms, Stephanodiscus, Cyclotella and Discostella, and their DNA sequence GenBank accession numbers
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[Table 2.] Sequence length and G+C content (%) measured from the Stephanodiscus rDNA determined in the present study
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[Table 3.] Similarity scores between 9 pairs of the aligned sequence data of the nearly complete 18S rDNA (above diagonal) and partial 28S rDNA (below diagonal) from nine selected species of Stephanodiscus
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[Fig. 1.] Phylogenetic relationships of three centric diatom genera, Cyclotella, Discostella, and Stephanodiscus, inferred by nearly complete18S rDNA sequences with (a) Bayesian and (b) NJ algorithms, respectively. Both analyses were used as the same datamatrix, with different nucleotide substitution models (e.g. GTR + I + G in Bayesian, and Maximum Composite Likelihood in NJalgorithm). Likelihood scores as the Bayesian tree were calculated at ?lnL = 3,898.6. The centric diatom, Thalassiosira gessneri#AN02-08 (GenBank no. DQ514864), was used as the outgroup. Bayesian posterior probabilities less than 0.50 and bootstrap proportionless then 50% were not shown.
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[Fig. 2.] Phylogenetic relationships of three centric diatom genera, Cyclotella, Discostella, and Stephanodiscus, inferred by partial 28SrDNA sequences with (a) Bayesian and (b) NJ algorithms, respectively. Both analyses were used as the same data matrix, withdifferent nucleotide substitution models (e.g. GTR + I + G in Bayesian, and Maximum Composite Likelihood in NJ algorithm).Likelihood scores of Bayesian tree was calculated at ?lnL = 2,296.2. The centric diatom, Thalassiosira gessneri #AN02-08 (GenBankno. DQ512413), was used as the outgroup. Bayesian posterior probabilities less than 0.50 and bootstrap proportion less then 50%are not shown.
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[Table 4.] Comparisons of 18S and 28S rDNA nucleotide divergences based on corrected p-distances of Stephanodiscus (St), Stephanodiscus versus Cyclotella (St vs. Cy) and Stephanodiscus versus Discostella (St vs. Di). Genetic distances between each paired sequence from 20 species listed in Table 1 were calculated with Kimura two-parameter model.
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[Fig. 3.] A dot-matrix plot and an entropy-plot of the nuclearrDNA of Stephanodiscus and other close relatives. The dotmatrixplot was drawn with the rDNA sequence comparisonof S. hantzschii (UTCC 267) and C. meneghiniana(HYK0210-A1). In addition, the entropy plot was drawn bycalculating the amount of nucleotide variability amongfour rDNAs, including C. meneghiniana, Discostella sp., S.hantzschii, and Stephanodiscus sp. (see Table 1). Color scalebar represents consecutive sequence length of some regionsdetected similarly between the two sequence pair. A lineon the entropy plot displays the normalized curve on eachhistogram.