Molecular analyses of protists in long-term observation programmes—current status and future perspectives

Protists (microbial eukaryotes) are diverse, major components of marine ecosystems, and are fundamental to ecosystem services. In the last 10 years, molecular studies have highlighted substantial novel diversity in marine systems including sequences with no taxonomic context. At the same time, many known protists remain without a DNA identity. Since the majority of pelagic protists are too small to identify by light microscopy, most are neither comprehensively or regularly taken into account, particularly in Long-term Ecological Research Sites. This potentially undermines the quality of research and the accuracy of predictions about biological species shifts in a changing environment. The ICES Working Group for Phytoplankton and Microbial Ecology conducted a questionnaire survey in 2013–2014 on methods and identification of protists using molecular methods plus a literature review of protist molecular diversity studies. The results revealed an increased use of high-throughput sequencing methods and a recognition that sequence data enhance the overall datasets on protist species composition. However, we found only a few long-term molecular studies and noticed a lack of integration between microscopic and molecular methods. Here, we discuss and put forward recommendations to improve and make molecular methods more accessible to Long-term Ecological Research Site investigators

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation

Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa?

We analysed the molecular and morphological features of strains of Chattonella subsalsa isolated from the western Adriatic coast (Mediterranean Sea), with the aim of confirming their classification and elucidating their phylogenetic positions within the Raphidophyceae. We sequenced parts of the ribosomal operon, including the small subunit (SSU), the internal transcribed spacer region (ITS) and the large subunit (LSU) of the rDNA. Additionally, we analysed sequences of the chloroplast-encoded subunit psaA of Photosystem I (PSI) and rbcL, encoding the large subunit of the Rubisco gene. For three phylogenetic markers (LSU, ITS, rbcL), the sequences of the strains from the Adriatic Sea were identical and for two markers (SSU, psaA) only minor differences occurred. All strains were sister to, but well separated from, sequences from isolates in culture collections and from GenBank, thus far classified as belonging to C. subsalsa. Light and electron microscopy provided evidence for morphological differences between a strain of C. subsalsa (CCMP217) from the Gulf of Mexico and the isolates from the Adriatic Sea. Differences concerned the shape and arrangement of chloroplasts and the presence of mucocysts and other surface microstructures, which were only observed in isolates from the Adriatic Sea. This is the first evidence for two different taxa classified as C. subsalsa, which are clearly separated on the basis of several genetic markers and also show morphological differences. As compared with strains assigned to C. subsalsa from the NCMA (formerly CCMP) culture collection, the Adriatic strains more closely match the original species description. This would imply that strain CCMP217 and other genetically similar strains should be described under a new species name. Nevertheless, given the high morphological plasticity of Chattonella species, the definition of the true C. subsalsa must be decided based on detailed morphological and molecular analysis of more strains from other geographical areas

Temporal changes in population structure of a marine planktonic diatom.

A prevailing question in phytoplankton research addresses changes of genetic diversity in the face of huge population sizes and apparently unlimited dispersal capabilities. We investigated population genetic structure of the pennate planktonic marine diatom Pseudo-nitzschia multistriata at the LTER station MareChiara in the Gulf of Naples (Italy) over four consecutive years and explored possible changes over seasons and from year to year. A total of 525 strains were genotyped using seven microsatellite markers, for a genotypic diversity of 75.05%, comparable to that found in other Pseudo-nitzschia species. Evidence from Bayesian clustering analysis (BA) identified two genetically distinct clusters, here interpreted as populations, and several strains that could not be assigned with ≥ 90% probability to either population, here interpreted as putative hybrids. Principal Component Analysis (PCA) recovered these two clusters in distinct clouds with most of the putative hybrids located in-between. Relative proportions of the two populations and the putative hybrids remained similar within years, but changed radically between 2008 and 2009 and between 2010 and 2011, when the 2008-population apparently became the dominant one again. Strains from the two populations are inter-fertile, and so is their offspring. Inclusion of genotypes of parental strains and their offspring shows that the majority of the latter could not be assigned to any of the two parental populations. Therefore, field strains classified by BA as the putative hybrids could be biological hybrids. We hypothesize that P. multistriata population dynamics in the Gulf of Naples follows a meta-population-like model, including establishment of populations by cell inocula at the beginning of each growth season and remixing and dispersal governed by moving and mildly turbulent water masses

Annotated 18S and 28S rDNA reference sequences of taxa in the planktonic diatom family Chaetocerotaceae.

The species-rich diatom family Chaetocerotaceae is common in the coastal marine phytoplankton worldwide where it is responsible for a substantial part of the primary production. Despite its relevance for the global cycling of carbon and silica, many species are still described only morphologically, and numerous specimens do not fit any described taxa. Nowadays, studies to assess plankton biodiversity deploy high throughput sequencing metabarcoding of the 18S rDNA V4 region, but to translate the gathered metabarcodes into biologically meaningful taxa, there is a need for reference barcodes. However, 18S reference barcodes for this important family are still relatively scarce. We provide 18S rDNA and partial 28S rDNA reference sequences of 443 morphologically characterized chaetocerotacean strains. We gathered 164 of the 216 18S sequences and 244 of the 413 28S sequences of strains from the Gulf of Naples, Atlantic France, and Chile. Inferred phylogenies showed 84 terminal taxa in seven principal clades. Two of these clades included terminal taxa whose rDNA sequences contained spliceosomal and Group IC1 introns. Regarding the commonly used metabarcode markers in planktonic diversity studies, all terminal taxa can be discriminated with the 18S V4 hypervariable region; its primers fit their targets in all but two species, and the V4-tree topology is similar to that of the 18S. Hence V4-metabarcodes of unknown Chaetocerotaceae are assignable to the family. Regarding the V9 hypervariable region, most terminal taxa can be discriminated, but several contain introns in their primer targets. Moreover, poor phylogenetic resolution of the V9 region affects placement of metabarcodes of putative but unknown chaetocerotacean taxa, and hence, uncertainty in taxonomic assignment, even of higher taxa

Placement of the individual <i>Pseudo-nitzschia multistriata</i> strains along the first two FCA axes (Genetix); specimens assigned by Structure to POP_A are indicated with grey squares, those to POP_B with black squares and those classified as putative hybrids (HYBR) with white squares.

<p>Placement of the individual <i>Pseudo-nitzschia multistriata</i> strains along the first two FCA axes (Genetix); specimens assigned by Structure to POP_A are indicated with grey squares, those to POP_B with black squares and those classified as putative hybrids (HYBR) with white squares.</p

Genotypic diversity of the inferred <i>Pseudo-nitzschia multistriata</i> populations POP_A, POP_B and the putative hybrids as identified by Structure over the four years of study.

<p>Included in the table are the number of strains (N), the number of genotypes (G) and the genotypic diversity (G/N).</p><p>Genotypic diversity of the inferred <i>Pseudo-nitzschia multistriata</i> populations POP_A, POP_B and the putative hybrids as identified by Structure over the four years of study.</p