Insight into the morphology and genetic diversity of the Chaetoceros tenuissimus (Bacillariophyta) species complex

ABSTRACT

Among the marine planktonic diatoms, Chaetoceros is among the most species-rich genera, and many Chaetoceros species are considered important primary producers. However, little is known about the ecology and distribution of the few small solitary species within this genus, including Chaetoceros tenuissimus. This article describes a minute Chaetoceros strain, identified as C. tenuissimus (named CT16ED) that was isolated at a coastal lagoon in Corsica Island, Western Mediterranean. The strain was characterized by light microscopy and scanning and transmission electron microscopy, with a specific focus on the fine structure and construction of setae and its behaviour in culture. The CT16ED strain was compared with other strains we isolated from the species type locality (Ostend Harbour, North Sea) by sequencing a fragment of the nuclear ribosomal DNA (rDNA) spanning from the 18S rDNA to the D3 region of the 28S rDNA, and the plastid rbcL gene that codes the large RuBisCO subunit. Based on the literature and the available sequence data, the analysed strains were similar to C. tenuissimus but the phylogenetic analysis indicated a C. tenuissimus species complex that contained several clades, therefore the current taxonomic status of C. tenuissimus is discussed. The comparison with the available rDNA and rbcL sequencing data of strains assigned to species considered as synonyms of C. tenuissimus, including Chaetoceros simplex var. calcitrans, Chaetoceros calcitrans and Chaetoceros calcitrans f. pumilus, suggested that these taxa are paraphyletic within the genus Chaetoceros.

KEYWORDS:

• Cell division
• Chaetoceros tenuissimus
• internal transcribed spacer (ITS)
• morphology
• phylogeny
• rDNA
• ultrastructure

Acknowledgements

Thanks to the Vlaams Institute of Marine Sciences (VLIZ) for its welcome and support at the Marine Station of Ostend (MSO) facility; thanks to MSO staff members for their help, especially to D. Cattrijsse and M. De Rijcke. Thanks to S. Pantani for assistance in the genetic analysis of the Ostend strains during a technician internship. The authors gratefully acknowledge S. Van Pé, M. Soleil and C. André (University of Corsica, UMS Stella Mare) and R. Pradelles (Microphyt) for assistance with the isolation and culture of microalgae. We acknowledge the anonymous reviewers for their helpful comments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary information

The following supplementary material is accessible via the Supplementary Content tab on the article’s online page at https://doi.org/10.1080/09670262.2022.2029949

Supplementary table S1. PCR and sequencing primers used in this study.

Supplementary fig. S1. Photography of the Chaetoceros tenuissimus description drawings in Figure 55 from the article by Meunier (1913). The coin (diameter of 23 mm) was used as a size scale of the published drawing. Photograph obtained by courtesy of the botanical library of the Muséum National d’Histoire Naturelle, Paris, France.

Supplementary fig. S2. Phylogenetic tree of the genus Chaetoceros inferred by maximum likelihood analysis, based on the sequences of the D1–D2 region in the 28S rDNA gene, with Leptocylindrus aporus and Leptocylindrus convexus (Chaetocerotophycidae, Leptocylindrales) as outgroups. The branch length is proportional to the number of substitutions per site (see scale bar at the bottom). Branch support (if posterior probability value > 0.5) is provided at the main nodes.

Supplementary fig. S3. Alignment of the Chaetoceros tenuissimus 18S rDNA sequences and from Chaetoceros sp. strains. All sequence fragments begin after the forward PCR primer 18S-F used in this study and end at the binding site of the reverse PCR primer 18S-R (3ʹ end of 18S rDNA, see Supplementary table S1). The shown alignment is a subset of the one used for the 18S rDNA phylogenetic analysis (Fig. 33). All sequences, but one (Chaetoceros sp. CHMS01), are identical, but for some insertions or deletions located in homopolymer repeats that might be sequencing errors. The Chaetoceros sp. CHMS01 sequence (accession number AF145226) differs only by one T substitution at position 543 in the alignment. The last sequence (accession AB246746), which was attributed to the diatom endosymbiont of the dinoflagellate Blixaea quinquecornis (formerly Peridinium quinquecorne), is identical to the C. tenuissimus sequences, except at the 3ʹ end where the A substitution (circled in red) corresponds to a mismatch in the reverse PCR primer SR12b (rectangular black box) used by Horiguchi & Takano (2006).

Supplementary fig. S4: Alignment of the Chaetoceros tenuissimus 28S (D1-D2) rDNA sequences. The alignment spans from the binding site of the forward primer D1R (5ʹ end of 28S rDNA) to that of the reverse primer D2C (3ʹ end of the D2 domain of 28S rDNA), used for sequencing in this study (see Supplementary table S1). The red arrows and circles at positions 109 and 476 indicate two unique substitutions in the CT16ED sequence. The alignment is a subset of the one used for the 28S rDNA phylogenetic analysis (Fig. 34).

Supplementary fig. S5: Alignment of the Chaetoceros tenuissimus ITS region sequences and from Chaetoceros spp. strains. The alignment spans from the binding site of the reverse PCR primer 18S-R (3ʹ end of 18S rDNA) to that of the forward primer D1R (5ʹ end of 28S rDNA) used in this study (Supplementary table S1). The alignment is a subset of the one used for the ITS-based phylogenetic analysis (Fig. 35).

Supplementary fig. S6: RuBisCO large subunit (rbcL) gene. Alignments of the nucleotide sequences (sequence included between the used PCR primers), and the corresponding amino acid sequences. The sequences including the 1311 nucleotide long fragment (positions 59-1369) were used for the phylogenetic analysis (Fig. 36). The sequence with accession number AB246745, attributed to the diatom endosymbiont of the dinoflagellate B. quinquecornis (formerly P. quinquecorne), is also included. Most sequences code identical proteins, with the exception of two strains from Ostend Harbour in Clade I (accession numbers: MZ189415 and MZ198421). The red arrows and circles indicate the A/T nucleotide substitution at position 1065 in the nucleotide alignment of these two strains and the corresponding phenylalanine (F) to tyrosine (Y) substitution at position 355 of the amino acid alignment.

Author contributions

V. Pasqualini conceptualized the project, isolated the Corsican strain and performed cultures. D. Grzebyk isolated the Ostend strains in Belgium, performed the molecular work and the phylogenetic analyses, and contributed to the morphological description. M. Garrido microscopically determined the Corsican strain and described its morphology. Y. Quilichini performed scanning electron and transmission electron microscopy studies. C. Pereto performed growth experiments. P. Cecchi organized and supervised the activities. All authors equally contributed to the draft preparation and to its redaction.

Additional information

Funding

This research was supported by the European Funds for Economic and Regional Development, the State-region program with the Corsican regional council ‘Collectivité de Corse’ and the University of Corsica (Gerhyco programme). The genetic work was carried out with the support of LabEx CeMEB, an ANR ‘Investissements d’Avenir’ programme (ANR-10-LABX-04-01), through the use of the GenSeq sequencing facility. The research leading to some of these results received partial funding from the European Union Horizon 2020 research and innovation programme under grant agreement number 730984, for the European consortium ASSEMBLE Plus project: specifically, the sampling in Ostend (Belgium) was supported by the Transnational Access programme through the project CHAETOTYPE.