Diatom of the Month January 2019 - Chaetocerotaceae

In this first 2019 Diatom of the Month post, Chetan Chandrakant Gaonkar* present us the marine diatom planktonic family Chaetocerotaceae and how to unravel its complex diversity by using up-to-date molecular techniques

The family Chaetocerotaceae is arguably among the most diverse among planktonic diatoms. Species are particularly common in coastal waters and upwelling zones where they are responsible for a substantial part of the primary productivity^1-3. The hallmark of this family constitutes hollow siliceous tubular structures, called the setae, emerging from the corner of the valves. The family includes two extant genera, Bacteriastrum Shadbolt and Chaetoceros Ehrenberg⁴, the latter being way more diverse. Within the genus Chaetoceros two subgenera are commonly recognized, Chaetoceros (a.k.a. Phaeoceros),which is characterized by exhibiting plastids in the setae, and Hyalochaete, whose setae lack plastids. Chaetoceroscells exhibits bipolar/bilateral symmetry and possess two setae per valve (Fig. 1 A-B), whereas Bacteriastrumcells have radial symmetry and more than two setae per valve (Fig. 1 C-D). Most of the species in the family form colonies early on in the vegetative part of their life cycle, in the form of chains of cells held together by brief fusion of setae emerging from sibling valves. As in most other diatoms, cell size diminishes when progressing through the vegetative life cycle and cells tend to become solitary. Species are homothallic, meaning that micro- and macrogametes are formed by cells belonging to one and the same strain and they can conjugate. Last but not least, most if not all species in the subgenus Hyalochaete, and a few in Bacteriastrum and in subgenus Chaetoceros form resting spores that tend to form when conditions become less favorable for growth. These spores sink from the water column onto the underlaying sediment where they can remain dormant for long periods of time. The recovery of these dormant cells from sedimentary archives is a promising tool in ‘resurrection ecology’ to unravel questions of past environmental and evolutionary conditions.

Figure 1 (A-D). Illustrations of the two representatives extant genera Chaetoceros and Bacteriastrumof the family Chaetocerotaceae. A-B represents the members of the genus Chaetoceros; and C-D represents the members of the genus Bacteriastrum. 

The diversity of the family has been traditionally characterized and assessed using light microscope (LM). Since the 1960s, electronic microscope (EM) revealed a wealth of ultrastructural details of the frustule useful for species identification. Molecular phylogenetic approaches making use of polymerase chain reaction (PCR) amplification and Sanger sequencing of selected DNA markers have uncovered a wealth of cryptic and pseudo-cryptic diversity (i.e., highly variable species morphologically similar and thus difficult to separate seem even more complex at the molecular level) and several species new to science^5-11. One of the key outcomes of all these molecular-morphological taxonomic explorations is the amassing of reference barcodes¹², i.e., marker sequences generated from taxonomically identified strains of which the morphological and ultrastructural details are available in public databases. These reference barcodes are useful for various research applications such as high throughput sequencing (HTS) metabarcoding. 

During my PhD thesis research, I assessed the Chaetocerotacean species diversity at the Long Term Ecological Research station MareChiara (LTER-MC) in the Gulf of Naples, Italy (GoN). This station is located ca. two miles offshore from Naples and has been monitored regularly since 1984 with the aim of assessing the structure and functioning of planktonic communities¹³. Chaetocerotaceae, and especially Chaetoceros, comprise a substantial part of the plankton at this site, with marked seasonal turnover in the composition of its species¹⁴. As many of the specimens belonging to Chaetocerosencountered at the site could not be identified, I isolated 433 Chaetocerotacean specimens from the GoN, brought them into monoclonal cultures and characterized morphologically, ultrastructurally using scanning electron microscope (SEM) and transmission electron microscopy (TEM) and genetically (18S and partial 28S rDNA)¹². Recently, HTS metabarcoding is been viewed as a proxy for the regular classical LM monitoring. The HTS approach inlcudes, DNA extraction, PCR amplification of a short marger region of a gene, then seqencing using a high throughut sequence. The reason for sequencing the 18S rDNA region was that in most of the plankton diversity studies using a HTS metabarcoding approach, the targeted markers are the hypervariable V4 or the V9 regions within the 18S rDNA^15-19. Most of the previous molecular taxonomic studies of Chaetocerotaceae included as genetic marker only ca. 700 bp at the 5’-end of the 28S rDNA^5-8. The obtained 18S and 28S rDNA sequences were used also to infer the phylogenetic relationship among the studied species, and to evaluate the universality of the potential metabarcode primers across the family. 

Phylogenies inferred from the rDNA genes revealed seven major clades in the family Chaetocerotaceae, and are reported in Gaonkar et al. 2018. Some of the major outcomes of my research were (i) the presence of several bits of nucleotides fragments in the rDNA gene that did not belong/correspond to the rDNA gene region. Upon evaluation, these nucleotide fragments turned out to be “introns”. Introns, in general, are nucleotide fragments that are present in the functional gene, but are eliminated at the final stages. Further characterization based on the primary and secondary structures revealed that these introns are in turn characterized as spliceosomal and groupI C1 introns, and are restricted among two of the principal clades¹².  Furthermore, (ii) all species analyzed in the phylogenetic study could be differentiated also with the V4 region alone and the primers were mostly universal to the family, except in few cases. This means that each of these species is detectable also with HTS metabarcoding environmental samples using the V4 region as marker. Moreover, (iii) even when using only the V4 marker of numerous diatom strains in a phylogenetic analysis, the Chaetocerotacean references resolve in a clade, suggesting that V4 metabarcodes can be assigned reliably to Chaetocerotaceae. Unfortunately, (iv) this does not account for the much shorter V9 region (see Gaonkar et al. 2018). The present study (v) provides a wealth of reference barcodes needed for the scientific community to study, and assess the species diversity of Chaetocerotaceae in their native locality. My collaborators and I are working on a publication assessing the species diversity in the family Chaetocerotaceae using a HTS metabarcoding approach.

This is collaborative work in which I acknowledge the contribution, training and mentoring from Wiebe HCF Kooistra, Carina Lange, David G Mann, Marina Montresor, Diana Sarno, Adriana Zingone, Roberta Piredda, Nina Lundholm, Yang Li and the staff of SZN (Italy).

*Chetan Chandrakant Gaonkar is a post-doctoral associate at the Department of Oceanography, Texas A&M University, College Station, Texas, USA.

References

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5.    Kooistra W.H.C.F, Sarno D., Hernández-Becerril D.U., Assmy P., Di Prisco C., & Montresor M. (2010) Comparative molecular and morphological phylogenetic analyses of taxa in the Chaetocerotaceae (Bacillariophyta). Phycologia, 5:471–500.

6.    Chamnansinp A., Li Y., Lundholm N., & Moestrup Ø. (2013) Global diversity of two widespread, colony-forming diatoms of the marine plankton, Chaetoceros socialis(syn. C. radians) and Chaetoceros gelidussp. nov. Journal of Phycology, 49:1128–41. https://doi.org/10.1111/jpy.12121

7.    Li Y., Lundholm N., & Moestrup Ø. (2013) Chaetoceros rotosporussp. nov. (Bacillariophyceae), a species with unusual resting spore formation. Phycologia, 52:600–08. https://doi.org/10.2216/13-168.1.

8.    Li Y., Zhu S., Lundholm N., & Lu S. (2015) Morphology and molecular phylogeny of Chaetocerosdayaensissp. nov. (Bacillariophyceae), characterized by two 90˚ rotations of the resting spore during maturation. Journal of Phycology, 51:469–79. https://doi.org/10.1111/jpy.12290.

9.    Balzano S., Percopo I., Siano R., Gourvil P., Chanoine M, Marie D., et al. (2017) Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetocerosneogracilisspecies complex. Journal of Phycology, 53:161–87. https://doi.org/10.1111/jpy.12489.

10.  Gaonkar C.C., Kooistra W.H.C.F., Lange C.B., Montresor M., & Sarno D. (2017) Two new species in the Chaetoceros socialiscomplex (Bacillariophyta): C. sporotruncatusand C. dichatoensis, and characterization of its relatives, C. radicansand C. cinctus. Journal of Phycology, 53:889–907. https://doi.org/10.1111/jpy.12554.

11.  Li Y., Boonprakob A., Gaonkar C.C., Kooistra W., Lange C.B., Hernandez-Becerrill D., et al. (2017) Diversity in the globally distributed diatom genus Chaetoceros(Bacillariophyceae): Three new species s from warm-temperate waters. PLoS ONE, 12(1):e168887. https://doi.org/10.1371/journal.pone.0168887.

12.  Gaonkar C.C., Piredda R., Minucci C., Mann D.G., Montresor M., Sarno D., et al. (2018) Annotated 18S and 28S rDNA reference sequences of taxa in the planktonic diatom family Chaetocerotaceae. PLoS ONE 13(12): e0208929.  https://doi.org/10.1371/journal.pone.0208929.

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16.  Hu S.K., Liu Z., Lie A.A., Countway P.D., Kim D.Y., Jones A.C., et al. (2015) Estimating Protistan Diversity Using High-Throughput Sequencing. Journal of Eukaryotic Microbiology, 62:688–93. https://doi.org/10.1111/jeu.12217.

17.  Massana R., Gobet A., Audic S., Bass D., Bittner L., Boutte C., et al. (2015) Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing. EnvironmentalMicrobiology, 17:4035–49. https://doi.org/10.1111/1462-2920.12955. 

18.  Piredda R., Tomasino M., D’Erchia A., Manzari C., Pesole G., Montresor M., et al. (2017) Diversity and temporal patterns of planktonic protist assemblages at a Mediterranean Long Term Ecological Research site. FEMS Microbiology and Ecology,  93:fiw200.
19.  Piredda R., Claverie J., Decelle J, de Vargas C. Dunthorn M., et al. (2018) Diatom diversity through HTS-metabarcoding in coastal European seas. Scientific Reports, 8:18059