The authors have declared that no competing interests exist.
Benthic diatoms isolated from tidal flats in the west coast of Korea were identified through both traditional morphological method and molecular phylogenetic method for methodological comparison. For the molecular phylogenetic analyses, we sequenced the 18S rRNA and the ribulose bisphosphate carboxylase large subunit coding gene,
Diatoms are the most dominant taxa among the various microalgae and are known to account for ca. 40% of the total primary production in the ocean [
Since molecular techniques were applied to diatom research for the first time in the 1980s [
Specific marker genes are used for molecular phylogenetic analyses. Different DNA regions within the nuclear rRNA gene, as well as mitochondrial and chloroplast genes, have been used for the phylogenetic analysis of diatoms [
In this study, morphological and molecular taxonomic characteristics of benthic diatoms isolated from tidal flats were investigated to evaluate the applicability of molecular phylogenetic approaches using 18S rRNA and
Benthic diatoms were collected mainly from tidal flats of Geunso Bay in Taean (36° 44' 12.06" N 126° 10' 47.52" E), Eulwang-ri (37° 26' 43.67" N 126° 22' 18.07" E), and saline Sihwa (37° 18' 46.73" N 126° 36' 32.64" E) along the west coast of Korea (
(TA: Taean, SH: Sihwa, EW: Eulwang-ri).
To obtain sediment samples containing diatoms, the surface of the tidal flat was scratched to a depth of ca. 2 mm and the sediment collected in a conical tube. Samples were transported to the laboratory under refrigerated conditions and then incubated at ± 2°C of the
Monoclonal cultures of benthic diatom strains were identified to the genus or species level by morphological features based on observations under light and scanning electron microscopy. For the light microscopy examination, diatom cultures were treated with acid to prepare cleaned frustules [
For DNA extraction, the cultured strain (100 μl) was harvested by centrifugation at 14,000 × g for 1 min and the cell pellet was resuspended in 1 ml of sterilized STE (sodium chloride-Tris-EDTA, pH 7.8) buffer solution. Two cycles of freezing (–80°C) and thawing (95°C) were followed by vigorous vortexing with sterilized silica/zirconium beads to break the cells. To remove cell debris, the lysate was centrifuged at 8,000 × g for 1 min. The supernatant was dispensed into a clean tube and used as template DNA for PCR.
PCR amplification was performed using two primer sets: Diatom9F (
Species names were determined by morphological analyses.
Species name by morphological characteristics | Strain |
Collection information |
Accession number | |
---|---|---|---|---|
18S | ||||
EW234 | Eulwang-ri, Incheon, Korea / 20 Apr 2012 | KY320376 | KY320315 | |
SH349 | Sihwa, Siheung, Korea / 8 Mar 2013 | KY320377 | KY320316 | |
TA256 | Geunso Bay, Taean, Korea / 11 Apr 2011 | KY320373 | KY320312 | |
TA46 | Geunso Bay, Taean, Korea / 21 Jan 2011 | KY320374 | KY320313 | |
TA198 | Geunso Bay, Taean, Korea / 23 Mar 2011 | KY320375 | KY320314 | |
Dilu38 | Geunso Bay, Taean, Korea / 22 Mar 2012 | KY320391 | KY320330 | |
TA139 | Geunso Bay, Taean, Korea / 23 Mar 2011 | KY320379 | KY320318 | |
TA44 |
Geunso Bay, Taean, Korea / 21 Jan 2011 |
KY320393 |
KY320332 |
|
TA37 | Geunso Bay, Taean, Korea / 21 Jan 2011 | KY320381 | KY320321 | |
SH366 | Sihwa, Siheung, Korea / 8 Mar 2013 | KY320382 | KY320320 | |
TA353 | Geunso Bay, Taean, Korea / 23 Feb 2012 | KY320378 | KY320331 | |
TA426 | Geunso Bay, Taean, Korea / 5 Dec 2013 | KY320392 | KY320317 | |
TA394 | Geunso Bay, Taean, Korea / 22 Mar 2012 | KY320383 | KY320322 | |
TA406 | Geunso Bay, Taean, Korea / 22 Jan 2014 | KY320380 | KY320319 | |
EW229 | Eulwang-ri, Incheon, Korea / 20 Apr 2012 | KY320389 | KY320328 | |
TA-45 |
Geunso Bay, Taean, Korea / 17 Apr 2014 |
KY320384 |
KY320323 |
|
TA341 |
Geunso Bay, Taean, Korea / 23 Feb 2012 |
KY320395 |
KY320337 |
|
TA311 | Geunso Bay, Taean, Korea / 27 Jan 2012 | KY320386 | KY320325 | |
Dilu16 | Geunso Bay, Taean, Korea / 22 Mar 2012 | KY320387 | KY320326 | |
TA61 | Geunso Bay, Taean, Korea / 21 Jan 2011 | KY320388 | KY320327 | |
TA409 | Geunso Bay, Taean, Korea / 22 Jan 2014 | KY320396 | KY320338 | |
TA-85 | Geunso Bay, Taean, Korea / 17 Apr 2014 | KY320397 | KY320334 | |
TA424 | Geunso Bay, Taean, Korea / 5 Dec 2013 | KY320346 | KY320285 | |
TA440 | Geunso Bay, Taean, Korea / 5 Dec 2013 | KY320345 | KY320284 | |
TA387 | Geunso Bay, Taean, Korea / 22 Mar 2012 | KY320352 | KY320291 | |
SH381 | Sihwa, Siheung, Korea / 8 Mar 2013 | KY320351 | KY320290 | |
TA280 | Geunso Bay, Taean, Korea / 11 Apr 2011 | KY320350 | KY320289 | |
TA291 | Geunso Bay, Taean, Korea / 11 Apr 2011 | KY320353 | KY320292 | |
TA105 | Geunso Bay, Taean, Korea / 10 Feb 2011 | KY320357 | KY320296 | |
TA289 | Geunso Bay, Taean, Korea / 11 Apr 2011 | KY320358 | KY320297 | |
TA414 | Geunso Bay, Taean, Korea / 5 Dec 2013 | KY320359 | KY320298 | |
TA413 TA441 | Geunso Bay, Taean, Korea / 5 Dec 2013 |
KY320360 |
KY320299 |
|
TA316 TA439 | Geunso Bay, Taean, Korea / 27 Jan 2012 |
KY320362 |
KY320301 |
|
TA402 | Geunso Bay, Taean, Korea / 22 Jan 2014 | KY320364 | KY320303 | |
TA416 | Geunso Bay, Taean, Korea / 22 Jan 2014 | KY320365 | KY320304 | |
TA407 | Geunso Bay, Taean, Korea / 22 Jan 2014 | KY320354 | KY320293 | |
TA204 | Geunso Bay, Taean, Korea / 23 Mar 2011 | KY320366 | KY320305 | |
TA83 | Geunso Bay, Taean, Korea / 21 Jan 2011 | KY320372 | KY320311 | |
TA298 | Geunso Bay, Taean, Korea / 11 Apr 2011 | KY320367 | KY320306 | |
TA64 | Geunso Bay, Taean, Korea / 21 Jan 2011 | KY320368 | KY320307 | |
EW220 | Eulwang-ri, Incheon, Korea / 20 Apr 2012 | KY320370 | KY320309 | |
TA323 | Geunso Bay, Taean, Korea / 27 Jan 2012 | KY320369 | KY320308 | |
TU3 | Geunso Bay, Taean, Korea / 5 Dec 2013 | KY320371 | KY320310 | |
TA308 |
Geunso Bay, Taean, Korea / 27 Jan 2012 |
KY320355 |
KY320294 |
|
TA305 | Geunso Bay, Taean, Korea / 27 Jan 2012 | KY320398 | KY320335 | |
TA152 |
Geunso Bay, Taean, Korea / 23 Mar 2011 |
KY320347 |
KY320347 |
|
TA34 | Geunso Bay, Taean, Korea / 21 Jan 2011 | KY320349 | KY320288 | |
TA208 |
Geunso Bay, Taean, Korea / 23 Mar 2011 |
KY320339 |
KY320278 |
|
TA410 | Geunso Bay, Taean, Korea / 22 Jan 2014 | KY320341 | KY320280 | |
TA350 |
Geunso Bay, Taean, Korea / 23 Feb 2012 |
KY320343 |
KY320282 |
|
EW239 | Eulwang-ri, Incheon, Korea / 20 Apr 2012 | KY320344 | KY320283 | |
TA201 | Geunso Bay, Taean, Korea / 23 Mar 2011 | KY320399 | KY320336 |
For phylogenetic analysis, 18S rRNA and
The 61 diatom isolates were identified by morphometric characteristics using light and scanning electron microscopy and their detailed information is shown in
(a)
(a)
(a)
(a, b)
Species name and sequence identity of the closest relative found in GenBank using BLASTn.
Species name | Strain |
Morphometrics | Ref. | BLASTn | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
L |
W |
Striae in 10 μm | L |
F |
18S rDNA | ||||||||
T |
L |
O |
Species name | Ident |
Species name | Ident. |
|||||||
EW234 | 55.7 | 6.5 | 23 | 20 | 7 | [ |
99.9 | 94.3 | |||||
Unidentified |
SH349 | 115.7 | 10.0 | 20 | 24 | 10 | 98.5 | 94.3 | |||||
TA256 | 159.4 | 3.8 | 15 | [ |
99.6 | 95.6 | |||||||
TA46 | 156.3 | 4.0 | 20 | [ |
98.7 | 95.1 | |||||||
Unidentified |
TA198 | 45.6 | 2.8 | 20 | 98.7 | 95.0 | |||||||
Dilu38 | 7.4 | 3.3 | 55 | 22 | [ |
99.6 | 95.1 | ||||||
TA139 | 25.7 | 5.3 | 40 | 22 | 16 | [ |
99.6 | 95.3 | |||||
TA44 |
37.8 |
5.8 |
48 | 6 | [ |
97.6 |
95.6 |
||||||
TA37 | 55.2 | 12.0 | 23 | 10 | [ |
99.8 | 94.6 | ||||||
SH366 | 48.3 | 5.0 | 38 | 16 | [ |
98.7 | 93.4 | ||||||
TA353 | 21.1 | 2.7 | 21 | 11 | [ |
96.7 | 94.5 | ||||||
TA426 | 22.0 | 7.6 | 26 | 11 | [ |
98.9 | 95.3 | ||||||
TA394 | 50.0 | 4.5 | 36 | 9 | [ |
98.8 | 94.1 | ||||||
TA406 | 19.1 | 5.0 | 48 | 8 | [ |
98.9 | 94.1 | ||||||
EW229 | 73.7 | 7.5 | 33 | 14 | [ |
99.2 | 92.9 | ||||||
TA-45 |
20.0 |
4.4 |
51 |
18 |
[ |
99.3 |
95.3 |
||||||
TA341 |
277.1 |
8.2 |
28 |
6 | [ |
96.0 |
94.8 |
||||||
TA311 | 84.8 | 5.2 | 27 | 10 | [ |
97.5 | 91.2 | ||||||
Unidentified |
Dilu16 | 11.3 | 3.7 | 52 | 18 | 99.2 | 95.2 | ||||||
Unidentified |
TA61 | 9.8 | 3.0 | 56 | 20 | 99.5 | 95.0 | ||||||
Unidentified |
TA409 | 26.9 | 7.8 | 40 | 12 | 99.7 | 94.3 | ||||||
TA-85 | 30.8 | 6.7 | 16 | [ |
99.6 | 96.5 | |||||||
TA424 | 11.7 | 3.9 | 36 |
[ |
99.4 | 94.7 | |||||||
TA440 | 15.9 | 4.1 | 28 |
[ |
99.8 | 99.5 | |||||||
TA387 | 36.7 | 12.7 | 19 | [ |
98.92 | 94.2 | |||||||
SH381 | 130.5 | 17.8 | 26 | 28 | [ |
99.7 | 95.9 | ||||||
TA280 | 48.4 | 6.0 | 41 | 35 | [ |
100.0 | 96.7 | ||||||
TA291 | 18.7 | 4.7 | 18 | 15 | [ |
99.5 | 96.0 | ||||||
TA105 | 33.6 | 6.4 | 14 | 40 | [ |
99.9 | 99.2 | ||||||
TA289 | 25.5 | 5.2 | 18 | 29 | [ |
99.9 | 95.4 | ||||||
TA414 | 19.2 | 3.5 | 16 | 48 | [ |
99.6 | 95.6 | ||||||
TA413 TA441 | 4.3 |
2.0 |
26 | 40 | [ |
100.0 | 94.0 |
||||||
TA316 TA439 | 25.1 |
5.7 |
12 |
38 |
[ |
99.8 |
97.7 |
||||||
TA402 | 37.0 | 11.7 | 15 | 33 | [ |
99.5 | 96.0 | ||||||
TA416 | 20.3 | 6.9 | 18 | 42 | [ |
99.6 | 95.4 | ||||||
TA407 | 36.3 | 13.8 | 14 | 31 | 99.5 | 95.6 | |||||||
TA204 | 12.3 | 3.5 | 20 | 40 | [ |
99.5 | 95.8 | ||||||
TA83 | 49.0 | 12.5 | 14 | 30 | [ |
99.5 | 95.2 | ||||||
Unidentified |
TA298 | 24.0 | 6.3 | 12 | 99.6 | 95.5 | |||||||
Unidentified |
TA64 | 36.8 | 10.4 | 9 | 98.6 | 95.7 | |||||||
Unidentified |
EW220 | 29.5 | 7.1 | 12 | 20 | 99.6 | 95.6 | ||||||
Unidentified |
TA323 | 11.6 | 5.0 | 18 | 36 | 99.6 | 96.9 | ||||||
Unidentified |
TU3 | 10.6 | 5.0 | 16 | 39 | 99.9 | 99.6 | ||||||
Unidentified |
TA308 |
11.8 | 5.6 | 20 | 45 |
99.7 |
95.7 |
||||||
Unidentified |
TA305 | 15.9 | 6.4 | 18 | 45 | 98.9 | 95.6 | ||||||
TA152 |
61.6 |
10.7 |
22 |
2628 | [ |
99.7 | 96.9 | ||||||
Unidentified |
TA34 | 91.4 | 20.0 | 24 | 20 | 98.7 | 93.9 | ||||||
Surirellales D.G.Mann | |||||||||||||
TA208 |
41.4 |
35.9 |
22 |
[ |
86.5 |
96.9 |
|||||||
Unidentified |
TA410 | 30.2 | 15.7 |
25 |
95.6 | 96.7 | |||||||
Unidentified |
TA350 |
50.3 |
29.9 |
15 |
98.7 |
97.7 |
|||||||
Unidentified |
EW239 | 53.2 | 41.0 |
16 |
94.6 | 96.5 | |||||||
TA201 | 55.8 | 25.8 | [ |
83.8 | 96.9 |
1 L, length;
2 W, width;
3 T, transverse;
4 L, longitudinal;
5 O, oblique;
6 L, lineolae;
7 F, fibulae;
8 In girdle view;
9 In the middle of frustule;
10 Striae composed of doubly-punctate striae
Both 18S rRNA and
From the phylogenetic trees, phylogenetic relationships among the isolates can be determined (Figs
Using the sequences obtained in this study, we analyzed divergence levels of the 18S rRNA and
Order | Genus | No. of strains | Genetic distance | |
---|---|---|---|---|
18S rDNA | ||||
21 | 0.015 | 0.050 | ||
2 | 0.037 | 0.047 | ||
2 | 0.003 | 0.049 | ||
2 | 0.003 | 0.021 | ||
2 | 0.022 | 0.056 | ||
20 | 0.036 | 0.078 | ||
3 | 0.010 | 0.065 | ||
6 | 0.074 | 0.048 | ||
Average | 0.060 | 0.089 |
In this study, we attempted to identify and classify benthic diatoms by the polyphasic approach using both morphological characteristics and molecular markers and suggested that molecular approach using
The quality of a database has a direct and absolute influence on the applicability and efficiency of DNA barcoding techniques [
In this study, six groups of diatoms, namely, Bacillariaceae, Naviculaceae, Pleurosigmataceae, Berkeleyaceae, Entomoneidaceae, and Surirellaceae, were clearly distinguished and formed monophyletic groups in the phylogenetic trees of
Conversely, the
Despite the ecological importance of benthic diatom community, their identification and classification systems still need to be improved. In this study, we showed that a large proportion of diatoms could not be identified by morphological characteristics and that genetic information should be expanded for molecular phylogenetic analyses. Furthermore,
We thank anonymous reviewers for providing constructive comments and Hwa Young Lee and Seong Jun Chun for help with sampling and algal culturing. We also thank Dr. Eun Chan Yang for his helpful comments on a previous version of this manuscript.