Wiebe H. C. F. Kooistra

Diversity in the genus Skeletonema (Bacillariophyceae). II. An assessment of the taxonomy of S. costatum-like species with the description of four new species.

The morphology of strains of Skeletonema Greville emend Sarno et Zingone was examined in LM, TEM, and SEM and compared with sequence data from nuclear small subunit rDNA and partial large subunit rDNA. Eight distinct entities were identified, of which four were known: S. menzelii Guillard, Carpenter et Reimann; S. pseudocostatum Medlin emend. Zingone et Sarno; S. subsalsum (Cleve) Bethge; and S. tropicum Cleve. The other four species were new: S. dohrnii Sarno et Kooistra sp. nov., S. grethae Zingone et Sarno sp. nov., S. japonicum Zingone et Sarno sp. nov., and S. marinoi Sarno et Zingone sp. nov. Skeletonema species fell into four morphologically distinct groups corresponding to four lineages in the small subunit and large subunit trees. Lineage I included S. pseudocostatum, S. tropicum, S. grethae, and S. japonicum. All have external processes of the fultoportulae with narrow tips that connect with those of sibling cells via fork‐, knot‐, or knuckle‐ like junctions. Lineage II included only the solitary species S. menzelii. Lineage III comprised S. dohrnii and S. marinoi. This latter pair have flattened and flared extremities of the processes of the fultoportulae, which interdigitate with those of contiguous valves without forming knots or knuckles. Lineage IV only contained the brackish water species S. subsalsum. Some species also differ in their distribution and seasonal occurrence. These findings challenge the concept of S. costatum as a single cosmopolitan and opportunistic species and calls for reinterpretation of the vast body of research data based on this species.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Patterns of Rare and Abundant Marine Microbial Eukaryotes

BACKGROUND Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.

Intraspecific diversity in Scrippsiella trochoidea (Dinopbyceae): evidence for cryptic species

Abstract Scrippsiella trochoidea is a widely distributed neritic dinoflagellate that produces calcareous resting cysts. We assessed the level of intraspecific diversity at the molecular, morphological and physiological levels among 15 strains identified as S. trochoidea and isolated from the Gulf of Naples (Italy, Mediterranean Sea), and an additional isolate from the Faeroe Islands. We investigated the morphology of motile cells and cysts, mating modality, encystment success, and growth rates at different light irradiances. The ribosomal DNA internal transcribed spacer (ITS) region was sequenced to infer phylogenetic relationships among the S. trochoidea strains and closely related species. The molecular analysis revealed a well-supported lineage comprising strains with a Scrippsiella plate pattern. Within this clade, a number of distinct ITS haplotypes were recorded but the relationships among them were only partially resolved. The 16 S. trochoidea isolates grouped into five single-strain clades and three multi-strain clades. The grouping of haplotypes in a series of distinct clades suggests the existence of cryptic species within what has previously been considered a single species, based on the morphological features of the motile cells and cysts. Some of the ITS haplotypes were distinguishable visually, based on minor morphological features of the motile cells and cysts, but in two cases morphologically almost identical strains fell into different clades. Our results showed that the majority of the strains are homothallic; only S. trochoidea v. aciculifera from the Faeroe Islands is heterothallic. Cyst production rates were notable for their diversity, even among strains grouping with the same ITS haplotype, as were growth rates at different light irradiances. Based on phylogenetic results, two new combinations are proposed: S. operosa (Deflandre) Montresor comb. nov. and S. infula (Deflandre) Montresor comb. nov.

Phylogenetic relationships of the ‘golden algae’ (haptophytes, heterokont chromophytes) and their plastids

The phylogenetic relationships of the “golden algae”, like all algae, were rarely addressed before the advent of electron microscopy because, based upon light microscopy, each group was so distinct that shared characters were not apparent. Electron microscopy has provided many new characters that have initiated phylogenetic discussions about the relationships among the “golden algae”. Consequently, new taxa have been described or old ones revised, many of which now include non-algal protists and fungi. The haptophytes were first placed in the class Chrysophyceae but ultrastructural data have provided evidence to classify them separately. Molecular studies have greatly enhanced phylogenetic analyses based on morphology and have led to the description of additional new taxa. We took available nucleotide sequence data for the nuclear-encoded SSU rRNA, fucoxanthin/ chlorophyll photosystem I/II, and actin genes and the plastid-encoded SSU rRNA, tufA, and rbcL genes and analysed these to evaluate phylogenetic relationships among the “golden algae”, viz., the Haptophyceae (= Prymnesiophyceae) and the heterokont chromophytes (also known as chromophytes, heterokont algae, autotrophic stramenopiles). Using molecular clock calculations, we estimated the average and earliest probable time of origin of these two groups and their plastids. The origin of the haptophyte host-cell lineages appears to be more ancient than the origin of its plastid, suggesting that an endosymbiotic origin of plastids occurred late in the evolutionary history of this group. The pigmented heterokonts (heterokont chromophytes) also arose later, following an endosymbiotic event that led to the transfer of photosynthetic capacity to their heterotrophic ancestors. Photosynthetic haptophytes and heterokont chromophytes both appear to have arisen at or shortly before the Permian-Triassic boundary. Our data support the hypothesis that the haptophyte and heterokont chromophyte plastids have independent origins (i.e., two separate secondary endosymbioses) even though their plastids are similar in structure and pigmentation. Present evidence is insufficient to evaluate conclusively the possible monophyletic relationship of the haptophyte and heterokont protist host cells, even though haptophytes lack tripartite flagellar hairs. The molecular data, albeit weak, consistently fail to present the heterokont chromophytes and haptophytes as monophyletic. Phylogenetic resolution among all classes of heterokont chromophytes remains elusive even though molecular evidence has established the phylogenetic alliance of some classes (e.g., Phaeophyceae and Xanthophyceae).

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES, CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1

The genus Caulerpa consists of about 75 species of tropical to subtropical siphonous green algae. To better understand the evolutionary history of the genus, a molecular phylogeny was inferred from chloroplast tufA sequences of 23 taxa. A sequence of Caulerpella ambigua was included as a potential outgroup. Results reveal that the latter taxon is, indeed, sister to all ingroup sequences. Caulerpa itself consists of a series of relatively ancient and species‐poor lineages and a relatively modern and rapidly diversifying clade, containing most of the diversity. The molecular phylogeny conflicts with the intrageneric sectional classification based on morphological characters and an evolutionary scheme based on chloroplast ultrastructure. High bootstrap values support monophyly of C. mexicana, C. sertularioides, C. taxifolia, C. webbiana, and C. prolifera, whereas most other Caulerpa species show para‐ or polyphyly.

Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity.

The rhodophyte seaweed Asparagopsis armata Harvey is distributed in the northern and southern temperate zones, and its congener Asparagopsis taxiformis (Delile) Trevisan abounds throughout the tropics and subtropics. Here, we determine intraspecific phylogeographic patterns to compare potential causes of the disjunctions in the distributions of both species. We obtained specimens throughout their ranges and inferred phylogenies from the hypervariable domains D1‐D3 of the nuclear rDNA LSU, the plastid spacer between the large and small subunits of RuBisCo and the mitochondrial cox 2–3 intergenic spacer. The cox spacer acquired base changes the fastest and the RuBisCo spacer the slowest. Median‐joining networks inferred from the sequences revealed the absence of phylogeographic structure in the introduced range of A. armata, corroborating the species’ reported recent introduction. A. taxiformis consisted of three nuclear, three plastid and four mitochondrial genetically distinct, lineages (1–4). Mitochondrial lineage 3 is found in the western Atlantic, the Canary Islands and the eastern Mediterranean. Mitochondrial lineages 1, 2, and 4 occur in the Indo‐Pacific, but one of them (lineage 2) is also found in the central Mediterranean and southern Portugal. Phylogeographic results suggest separation of Atlantic and Indo‐Pacific lineages resulted from the emergence of the Isthmus of Panama, as well as from dispersal events postdating the closure event, such as the invasion of the Mediterranean Sea by mitochondrial lineages 2 and 3. Molecular clock estimates using the Panama closure event as a calibration for the split of lineages 3 and 4 suggest that A. taxiformis diverged into two main cryptic species (1 + 2 and 3 + 4) about 3.2–5.5 million years ago (Ma), and that the separation of the mitochondrial lineages 1 and 2 occurred 1–2.3 Ma.

PHYLOGENETIC POSITION OF TOXARIUM, A PENNATE-LIKE LINEAGE WITHIN CENTRIC DIATOMS (BACILLARIOPHYCEAE)1

The diatom genus Toxarium Bailey has been treated as a pennate because of its elongate shape and benthic lifestyle (it grows attached to solid substrata in the marine sublittoral). Yet its valve face lacks all structures that would ally it with the pennates, such as apical labiate processes, a midrib (sternum) subtending secondary ribs and rows of pores extending perpendicularly out from the midrib, or a raphe system. Instead, pores are scattered irregularly over the valve face and only form two distinct rows along the perimeter of the valve face. In our nuclear small subunit rDNA phylogenies, Toxarium groups with bi‐ and multipolar centrics, as sister to Lampriscus A. Schmidt. Thus, the genus acquired a pennate‐like shape and lifestyle independently from that of the true pennates. The two species known, T. hennedyanum Grunow and T. undulatum Bailey, differ only in a single feature: the valve perimeter of the former shows only a central expansion, whereas that of the latter possesses in addition a regular undulation. Yet both forms were observed in our monoclonal cultures, indicating that the two taxa represent extremes in a plasticity range. Toxarium resembles another elongate and supposedly araphid diatom, Ardissonea De Notaris, in being motile. Cells can move at speeds of up to 4 μm·s− 1 through secretion of mucilage from the cell poles or they remain stationary for longer periods, when they form short polysaccharide stalks. Division during longer periods of quiescence leads to the formation of small colonies of linked or radiating cells.

DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE): III. PHYLOGENETIC POSITION AND MORPHOLOGICAL VARIABILITY OF SKELETONEMA COSTATUM AND SKELETONEMA GREVILLEI, WITH THE DESCRIPTION OF SKELETONEMA ARDENS SP. NOV.†

Skeletonema costatum (Grev.) Cleve emend. Zingone et Sarno and S. grevillei Sarno et Zingone were known only from the type material collected from Hong Kong waters more than a century ago. Both species have now been collected as live material, and their morphology and phylogenetic position are investigated in this study. Eight Skeletonema strains isolated from Florida, USA; Uruguay; and Brazil are attributed to S. costatum, while one strain from Oman is ascribed to S. grevillei based on morphological similarity to the type material of these species. In addition, a new Skeletonema species, S. ardens Sarno et Zingone, is described for a strain from Singapore and two from northern Australian waters. Skeletonema ardens has terminal fultoportula processes ending in a tapered, undulate protrusion and long intercalary fultoportulae with 1:1 junctions. The rimoportula of terminal valves is located at the margin of the valve face. No major morphological variations were observed within S. grevillei and S. ardens along a salinity gradient, whereas in S. costatum, the processes shortened and the valves came into close contact at low salinities, as already described for S. subsalsum (Cleve) Bethge. Consistent with their morphology, Skeletonema costatum and Skeletonema subsalsum also had similar rDNA sequences. Skeletonema grevillei and S. ardens were distinct in the large subunit (LSU) phylogeny. Skeletonema ardens exhibited consistent intraspecific genetic differences in both the LSU and small subunit (SSU) rDNA.