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.
INTRODUCTION
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 (Ha
et 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 (Oliva
et al
. 2008). However, morphological discrimination of these species is very difficult, because of small size (less than 15 μm) and a number of recorded different
Stephanodiscus
species (approximately 124 taxa) according to Guiry and Guiry (2009). In addition, morphologies of
Stephanodiscus
are similar to those of other freshwater centric diatoms, e.g.
Cyclotella
and
Discostella
(formerly, these represented the stelligeroid group of
Cyclotella
[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; Ki
et 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
, and
Hsp90
). The rDNA sequences have been used for the discrimination of centric diatoms and for the phylogenetic analyses (Alverson
et al
. 2007; Kaczmarska
et al
. 2007). Most studies on the freshwater centric diatoms have been biased to
Cyclotella
, particularly for the ystematics and phylogenetic relationships of cyclotelloid diatoms (Beszteri
et al
. 2005, 2007; Alverson
et al
. 2007). More recently, genetic divergence between
Cyclotella
and
Discostella
has been studied, by comparisons of a wide range of rDNA sequences (Jung
et al
. 2009). With regard to molecular analyses of
Stephanodiscus
, Kaczmarska
et al
. (2005) showed for the first time that
Cyclotella
and
Stephanodiscus
were not belonging to the same phylogenetic clade, making the family Stephanodiscaceae paraphyletic. Recently, Alverson
et al
. (2007) reported phylogenetic relationships of thalassiosiroid diatoms, representing the separations of the freshwater centric diatoms, e.g.
Cyclotella, Discostella
and
Stephanodiscus
. Recently, the author reported high molecular genetic divergences between
Cyclotella
and
Discostella
, suggesting that rDNA may be a suitable molecular marker for the discrimination of the two genera and species (Jung
et al
. 2009). Excluding these works, little attention has been paid on the molecular analyses of
Stephanodiscus
.
In the present study, the author sequenced nuclear rDNA, spanning the 18S to the 28S rDNA, of
S. hantzschii
and
Stephanodiscus
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 selected
Stephanodiscus
to reveal the rDNA relationships of the freshwater centric diatoms. In addition, molecular divergences between
Stephanodiscus
with other centric diatoms
Cyclotella
and
Discostella
were compared to evaluate their usefulness for the discrimination of freshwater centric diatoms. The studied
S. hantzschii
is one of the planktonic, cosmopolitan species and the
Stephanodiscus
blooms were recorded annually in Korean waters, particularly in the Paldang Reservoir and Nakdong River (Kim 1998; Ha
et al.
2002; Han
et al
. 2002; Kim
et al
. 2008).
MATERIALS & METHODS
- Cultures ofStephanodiscus
Water samples were collected from Paldang Reservoir (a reservoir in Han River) of Korea, when
Stephanodiscus
blooms occurred. The author isolated single cells of
Stephanodiscus
from field samples using the capillary method (Ki and Han 2005), and established a clonal culture (KHR001) of
Stephanodiscus
. An additional strain (UTCC 267) of
S. 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, (Beakes
et 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.
- DNA sequencing
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® BigDye
TM
? 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) and
Discostella
sp. (GQ148713). DNA sequences determined here have been deposited to GenBank as accession numbers GQ844873 and GQ844874.
- Comparisons ofStephanodiscusrDNA
BLAST (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 Korean
Stephanodiscus
were compared with those of other
Stephanodiscus
(see
Table 1
). DNA similarity scores of individual rDNA molecules were calculated by using pairwise sequences among nine selected species of
Stephanodiscus
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 (Tamura
et al
. 2007). Statistical analyses on the nucleotide comparisons were performed using SPSS 10.0.7 (SPSS Inc., Chicago, IL).
Origins of the centric diatoms,Stephanodiscus, CyclotellaandDiscostella, and their DNA sequence GenBank accession numbers
Origins of the centric diatoms, Stephanodiscus, Cyclotella and Discostella, and their DNA sequence GenBank accession numbers
- Phylogenetic relationships ofStephanodiscusspecies
Phylogenetic 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 ten
Stephanodiscus
, four selected
Discostella
, and six selected
Cyclotella
rDNAs (see
Table 1
). A total of 21 sequences, including the outgroup (
Thalassiosira gessneri
#AN02-08), were aligned with the Clustal W 1.8 (Thompson
et 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 (Tamura
et al
. 2007). For the 28S rDNA tree, Bayesian and NJ analyses were performed the same way as in the 18S analysis.
Sequence length and G+C content (%) measured from the Stephanodiscus rDNA determined in the present study
Sequence length and G+C content (%) measured from the Stephanodiscus rDNA determined in the present study
RESULTS AND DISCUSSION
- Nuclear rDNA ofS. hantzschiiand 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 Alverson
et al
. (2007). The rDNA ITS regions were only revealed from a few species, including
S. hantzschii
(U03078),
S. niagarae
(U03074-6, AF455267-9), and
S. yellowstonensis
(U03077). By using the ITS data, Wolf
et al
. (2002) demonstrated the same species of
S. neoastraea
and
S. 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 from
Stephanodiscus
(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 Korean
Stephanodiscus
sp. #KHR001 was highest matched with
S. hantzschii
#CCAP 1079/4 (GenBank DQ093370) with 99.5% similarity by 18S rDNA comparison, and was also matched to
S. 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 with
S. niagarae
(U03076) of 89.9% DNA similarity. BLAST searches of individual rDNA of
S. hantzschii
#UTCC 267 were similar to those of Korean
Stephanodiscus
spp. In the latter, the rDNA ITS was highly matched at 94.1% similarity with
S. yellowstonensis
(U03077) and at 96.9% similarity with
S. hantzschii
(U03078), respectively. Overall similari-ties of the coding rDNAs, e.g. 18S and 28S, were high within the genus
Stephanodiscus
.
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 ofStephanodiscus
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
- Molecular similarities and genetic divergence of rDNA
Molecular comparisons showed that a Korean isolate of
Stephanodiscus
had a different genotype compared with other
Stephanodiscus
(Table 3)
, including
S. hantzschii
. The present Korean isolate was presumably identified as
S. hantzschii
based on routine morphological observations and previous studies (Han
et al
. 2002). As noted previously, morphological characteristics of
Stephanodiscus
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 discriminated
Stephanodiscus
sp. #KHR001 (or named as
Stephanodiscus
sp. cf.
S. hantzschii
). Considering these molecular and morphological characteristics of
Stephanodiscus
, the author selected nine species of
Stephanodiscus
, 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 nine
Stephanodiscus
(>;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 with
S. agassizensis, S. binderanus, S. hantzschii
and
S. minutulus
, respectively. By 28S comparisons, DNA similarities were highly recorded among the
Stephanodiscus
, 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 the
Stephanodiscus
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
and
Discostella
(Jung
et 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
and
Stephanodiscus
, 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 of
Cyclotella
and
Discostella
, in which phylogenetic trees were inferred with Bayesian method, using 18S and 28S rDNA data (Jung
et al
. 2009). In the present study, the author focused on
Stephanodiscus
relationships against the two other genera, as well as inter-species relationships within the genus
Stephanodiscus
. Considering our previous work (Jung
et al
. 2009), the author constructed new data matrices, including certain members of
Cyclotella
and
Discostella
. In the present analyses, a total of 20 species, including six
Cyclotella
, four
Discostella
and ten
Stephanodiscus
, with the outgroup of
Thalassiosira
, 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 of
Stephanodiscus
formed a cluster (1.00 PP, 100% BP), of which clade was separated from a
Discostella
cluster.
Stephanodiscus
and
Discostella
are a sister relationship, separating a clade of
Cyclotella
, showing that these patterns were in agreement with Alverson
et al
. (2007). In the
Stephanodiscus
linage, most species, excluding a cluster of
S. niagarae, S. reimerri
and
S. 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 Korean
Stephanodiscus
isolate was positioned at an early divergent place, clearly being separated from other
Stephanodiscus
(1.00 PP, 100% BP).
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.
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 with
Discostella
, of which clade was clustered with
Cyclotella
(1.00 PP, 100% BP). Within theses analyses,
Stephanodiscus
formed a polytomy, excluding a cluster of
S. niagarae, S. reimerii
and
S. yellowstonensis
(Fig. 2)
. The 28S rDNA phylogeny showed that the Korean isolate was not separated from other
Stephanodiscus
. Overall 28S phylogeny was in good accordance with the 18S phylogeny described above.
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.
- Molecular divergences of 18S, ITS, 28S rDNAs
The present rDNA sequences of
Stephanodiscus
were graphically compared with those of the
Cyclotella
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 (Jung
et 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).
Comparisons of 18S and 28S rDNA nucleotide divergences based on correctedp-distances ofStephanodiscus(St),StephanodiscusversusCyclotella(St vs. Cy) andStephanodiscusversusDiscostella(St vs. Di). Genetic distances between each paired sequence from 20 species listed inTable 1were calculated with Kimura two-parameter model.
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.
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.
- DNA identity of Stephanodiscus from Paldang Reservoir
The centric diatoms, including
Cyclotella, Discostella
, and
Stephanodiscus
, commonly occur in freshwaters, including the Han River (Han
et al.
2002; Jung
et al
. 2009). According to the previous studies (Kim 1998; Han
et 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 by
Cyclotella
and
Discostella
(e.g. Jung
et al.
2009), and sometimes
Stephanodiscus
blooms occurred in Paldang Reservoir (Kim 1998; Han
et al
. 2002). The blooming
Stephanodiscus
in Paldang Reservoir were morphologically considered as
S. hantzschii
(Han
et al
. 2002). In addition, the author isolated a Korean
Stephanodiscus
cell (KHR001) from a water sample of Paldang Reservoir when
Stephanodiscus
cells were present predominantly, and identified them as
S. hantzschii
, judging by routine morphological observa-tions and previous reports (Kim 1998; Han
et 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 between
Stephanodiscus
sp. #KHR001 with other
S. hantzschii
(the present UTCC 267, WTC21, ATN2), the present Korean isolate should be a different species, than
S. hantzschii
, judging from the present phylogenetic analyses and molecular similarities
(Table 3
;
Figs. 1
,
2)
. Previously, Kim (1998) discriminated three species of
Stephanodiscus
, 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, while
S. 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 blooming
Stephanodiscus
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 Korean
Stephanodiscus
sp. #KHR001. Molecular comparisons showed high genetic similarities (or low genetic divergence) within the genus
Stephanodiscus
compared with those of
Cyclotella
and
Discostella
. From these facts, the author concludes that nuclear rDNA sequences of
Stephanodiscus
are considerably similar to each other, but they are significantly different (
p
< 0.01) from other freshwater centric diatoms (e.g.
Cyclotella
and
Discostella
).
Acknowledgements
The author thanks Dr. H.-U. Dahms for English editing and critical comments on the manuscript. Also the author thanks two anonymous reviewers for their constructive comments. This work was supported by a research grant of the Sangmyung University funded to J.-S. Ki (2009).
View Fulltext
Alverson A.J
,
Jansen R.K
,
Theriot E.C
2007
Bridging the Rubicon: Phylogenetic analysis reveals repeated colonizations of marine and fresh waters by thalassiosiroid diatoms
Molecular Phylogenetics and Evolution
45
(1)
193 -
210
DOI : 10.1016/j.ympev.2007.03.024
Beakes G
1988
Zoospore ultrastructure of Zygorhizidium affluens Canter and Z planktonicum Canter two chytrids parasitizing the diatom Asterionella formosa Hassall
Botany
66
(6)
1054 -
1067
DOI : 10.1139/b88-151
Beszteri B
,
Acs E
,
Medlin L.K
2005
Conventional and geometric morphometric studies of valve ultrastructural variation in two closely related Cyclotella species (Bacillariophyta)
European Journal of Phycology
40
(1)
89 -
103
DOI : 10.1080/09670260500050026
Beszteri B
,
John U
,
Medlin L.K
2007
An assessment of cryptic genetic diversity within the Cyclotella meneghiniana species complex (Bacillariophyta) based on nuclear and plastid genes and amplified fragment length polymorphisms
European Journal of Phycology
42
(1)
47 -
60
DOI : 10.1080/09670260601044068
Bruder K
,
Medlin L.K
2007
Molecular assessment of phylogenetic relationships in selected species/genera in the naviculoid diatoms (Bacillariophyta) I The genus Placoneis
Nova Hedwigia
85
(3)
331 -
352
DOI : 10.1127/0029-5035/2007/0085-0331
Guiry G.M
,
Guiry M.D
,
Guiry G.M
2009
"Stephanodiscus” AlgaeBase World-wide electronic publication National University of Ireland Galway
http://wwwalgaebaseorg/
Ha K
,
Jang M.H
,
Joo G.J
2002
Spatial and temporal dynamics of phytoplankton communities along a regulated river system the Nakdong River Korea
Hydrobiologia
470
235 -
245
DOI : 10.1023/A:1015610900467
Hall Hall
1999
BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT
Nucl Acids Symp Ser
41
95 -
98
Han M.S
,
Lee H.R
,
Hong S.S
,
Kim Y.O
,
Lee K
,
Choi Y.K
,
Kim S
,
Yoo K.I
2002
Ecological studies on Togyo reservoir system in Chulwon Korea V Seasonal changes of size-fractionated standing crops and chlorophyll a of phytoplankton in Kyungan Stream of Pal’tang Reservoir Korea
Korean J Environ Biol
20
91 -
99
Hassouna Hassouna
,
Michot B
,
Bachellerie J.P
1984
The complete nucleotide sequence of mouse 28S rRNA gene Implications for the process of size increase of the large subunit rRNA in higher eukaryotes
Nucleic Acids Research
12
(8)
3563 -
3583
DOI : 10.1093/nar/12.8.3563
Houk V
,
Klee R
2004
The stelligeroid taxa of the genus Cyclotella (Kutzing) Brebisson (Bacillariophyceae) and their transfer into the new genus Discostella gen nov
Diatom Res
19
203 -
231
Han R
,
Jung S.W
,
Han M.S
,
Ki J.S
2009
Molecular genetic divergence of the centric diatom Cyclotella and Discostella (Bacillariophyceae) revealed by nuclear ribosomal DNA comparisons
J Appl Phycol
Kaczmarska I
,
Beaton M
,
Benoit A.C
,
Lee J
,
Park B.S
,
Kang S.H
,
Han M.S
2005
Comprehensive comparisons of three pennate diatoms Diatoma tenuae Fragilaria vaucheriae and Navicula pelliculosa isolated from summer Arctic reservoirs (Svalbard 79oN) by fine-scale morpholog
Journal of Phycology
42
(1)
121 -
138
DOI : 10.1111/j.1529-8817.2006161.x
Karsten U
,
Schumann R
,
Rothe S
,
Jung I
,
Medlin L
2006
Temperature and light requirements for growth of two diatom species (Bacillariophyceae) isolated from an Arctic macroalga
Polar Biology
29
(6)
476 -
486
DOI : 10.1007/s00300-005-0078-1
Ki Ki
,
Cho S.Y
,
Katano T
,
Jung S.W
,
Lee J
,
Park B.S
,
Kang S.H
,
Han M.S
2009
Comprehensive comparisons of three pennate diatoms Diatoma tenuae Fragilaria vaucheriae and Navicula pelliculosa isolated from summer Arctic reservoirs (Svalbard 79℃N) by fine-scale morpholog
Polar Biology
32
(2)
147 -
159
DOI : 10.1007/s00300-008-0514-0
Ki J.S
,
Han M.S
2005
Sequence-based diagnostics and phylogenetic approach of uncultured freshwater dinoflagellate Peridinium (Dinophyceae) species based on single-cell sequencing of rDNA
Journal of Applied Phycology
17
(2)
147 -
153
DOI : 10.1007/s10811-005-7211-y
Ki J.S
,
Han M.S
2007
Informative characteristics of 12 divergent domains in complete large subunit rDNA sequences from the harmful dinoflagellate genus Alexandrium (Dinophyceae)
J Eukaryotic Microbiol
54
210 -
219
DOI : 10.1111/j.1550-7408.2007251.x
Kim M.C
,
La G.H
,
Kim H.W
,
Jeong K.S
,
Kim D.K
,
Joo G.J
2008
The Effect of Water Temperature on Proliferation of Stephanodiscus sp in vitro from the Nakdong River South Korea
Korean Journal of Limnlolgy
41
(1)
26 -
33
Kim Y.J
1998
Ecological characteristics of phytoplankton community in lake Paltang Dam
Korean J Limnol
31
225 -
234
Lenaers G
,
Maroteaux L
,
Michot B
,
Herzog M
1989
Dinoflagellates in evolution A molecular phylogenetic analysis of large subunit ribosomal RNA
Journal of Molecular Evolution
29
(1)
40 -
51
DOI : 10.1007/BF02106180
Nylander K
2004
MrModeltest v2
Uppsala Univ
Uppsala Sweden
Oliva M.G
,
Lugo A
,
Alcocer J
,
Cantoral-Uriza E.A
2008
Morphological study of Cyclotella choctawhatcheeana Prasad (Stephanodiscaceae) from a saline Mexican lake
Saline Systems
4
(1)
17 -
DOI : 10.1186/1746-1448-4-17
Page R.D.M
1996
TREEVIEW: an application to display of phylogenetic trees on personal computers
Comput Appl Biosci
12
357 -
358
Tamura K
,
Dudley J
,
Nei M
,
Kumar S
2007
MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 40
Molecular Biology and Evolution
24
(8)
1596 -
1599
DOI : 10.1093/molbev/msm092
Thompson J.D
,
Higgins D.G
,
Gibson T.J
1997
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice
Nucleic Acids Research
22
(22)
4673 -
DOI : 10.1093/nar/22.22.4673
Wolf M
,
Scheffler W
,
Nicklisch A
2002
Stephanodiscusneoastraea and Stephanodiscus heterostylus (Bacillariophyta)are one and the same species
Diatom Res
17
445 -
451