Diana Sarno

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.

Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: morphology, toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species

The genus Pseudo-nitzschia includes several species capable of producing domoic acid, the causative agent of Amnesic Shellfish Poisoning. Some of these species have been recorded frequently in the Gulf of Naples. For one of the species, P. multistriata, which has been recurrently found in our sampling area since 1995, this is the first report for European waters. Here we provide further details on the fine structure of this species. Pseudo-nitzschia multistriata was the only one found to produce domoic acid among all the Pseudo-nitzschia species from the Gulf of Naples, and this finding raises the number of potentially toxic species in this genus to nine. Phylogenetic relationships among several Pseudo-nitzschia species were assessed using the hypervariable domains (D1–D3) of the large subunit (LSU) rDNA. The match between the phylogeny obtained and important taxonomic characters used in this genus are discussed. Results show that P. multistriata clusters with wider species lacking a central larger interspace in the raphe. Close genetic relationships were determined between P. fraudulenta and P. subfraudulenta, and between P. pungens and P. multiseries. Genetic differences among these pairs of species are comparable to those among isolates of P. pseudodelicatissima from the Gulf of Naples, indicating high intraspecific genetic diversity of Pseudo-nitzschia species in the relatively conserved LSU region. This could explain the problematic results obtained when testing a match between species-specific Pseudo-nitzschia LSU probes and our sequences.

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.

Diversity in the genus Skeletonema (Bacillariophyceae). I. A reexamination of the type material of S. costatum with the description of S. grevillei sp. nov

The type material of Skeletonema costatum (Greville) Cleve was reexamined with the aims of providing an EM description of the species and clarifying which of the recently recognized Skeletonema Greville species, if any, deserves the epithet costatum. Two permanent mounts and two loose samples from the same collections from Hong Kong Bay were examined. Two distinct Skeletonema species were found in all materials. Based on the original description and on the observation of the lectotype, it was concluded that the most abundant morph in the type material is S. costatum. Cells were heavily silicified and 5–16 μm in diameter. The external processes of the fultoportulae in the terminal valves were open tubules with claw‐shaped tips. The intercalary fultoportulae processes were closed flattened tubules with a pore at their base, each attached to two processes of the sibling valve (1:2 junction). The rimoportula had a long process and was located marginally in all valves. The second morph in the type material was described as Skeletonema grevillei Sarno et Zingone sp. nov. Cells were delicate, 4–7.5 μm in diameter, and formed short colonies. The fultoportulae processes in the terminal valves were open tubes having narrow tips with truncated or spiny margins. In the intercalary valves, they formed knuckle‐like generally 1:1 junctions. A scallop‐work of silica bridges joined the bases of the processes. The rimoportula was marginal in all valves. Neither S. costatum nor S. grevillei match any Skeletonema species that have been recently analyzed by combined morphological and molecular approaches.

Comparative molecular and morphological phylogenetic analyses of taxa in the Chaetocerotaceae (Bacillariophyta)

Kooistra W.H.F.C., Sarno D., Hernández-Becerril D.U., Assmy P., Di prisco C. and Montresor M. 2010. Comparative molecular and morphological phylogenetic analyses of taxa in the Chaetocerotaceae (Bacillariophyta). Phycologia 49: 471–500. DOI: 10.2216/09-59.1 The diatom family Chaetocerotaceae (Mediophyceae) includes two exclusively phytoplanktonic genera: Chaetoceros and Bacteriastrum. Its hallmark feature constitutes setae: hollow, spine-like appendages protruding from the valves. Chaetoceros is morphologically diverse, includes c. 400 described species and is common worldwide; whereas, Bacteriastrum includes only 11, is less common and occurs mainly in temperate and tropical seas. In the present study we gathered morphological information and/or sequence data from 86 strains belonging to 17 morphologically defined species in Chaetoceros and one in Bacteriastrum (B. cf. hyalinum). The Chaetoceros species included in this study belong to 14 of the 22 sections and two of the three subgenera: Chaetoceros (Phaeoceros) and Hyalochaete. A consensus cladogram reconstructed from states associated with morphological characters gathered from strains belonging to these 18 morphological taxa in the Chaetocerotaceae and Hemiaulus hauckii as outgroup resolved Bacteriastrum inside Chaetoceros and demonstrated monophyly for the subgenus Chaetoceros; whereas, the subgenus Hyalochaete was found to be paraphyletic. Molecular phylogenies inferred from the hypervariable region (D1–D4) of the LSU rRNA gene of all strains included in this study and from a subset of these strains corroborated the findings in the cladogram and showed evidence for cryptic or pseudocryptic diversity in C. curvisetus, C. debilis, C. diadema, C. lorenzianus, C. peruvianus and C. socialis. The molecular trees differed topologically from the morphological ones, but characters exhibiting only a few state changes in the molecular tree showed also only a few changes in the morphological one; whereas, characters showing multiple changes in the molecular tree revealed many changes in the morphological tree as well.

Phytoplankton biomass and species composition in a Mediterranean coastal lagoon

Phytoplankton populations were investigated weekly at a central station in the Fusaro lagoon (Mediterranean Sea) from 27 November 1989 to 18 June 1990 to assess species composition, temporal succession and standing stock of the different species. Chlorophyll concentrations varied from 1.2 to 73.2 µg 1−1 in surface waters, and from 1.3 to 53.5 µg 1−1 at the 4.5 m depth. Phytoplankton communities were dominated by Prorocentrum micans Ehrenberg in December and January, and by small-sized diatoms in the rest of the sampling period. In surface waters, a maximum biomass of 9.5 mg C 1−1 was measured in January, in correspondence with high concentrations (8.1 × 106 cells 1−1) of P. micans, whereas an abundance peak of 159.9 × 106 cells 1−1 was registered on the last sampling date due to a massive bloom of a very small diatom, Minutocellus polymorphus (Hargraves & Guillard) Hasle, von Stosch & Syvertsen. On the whole, phytoplankton populations of the Fusaro lagoon showed distinct characters as compared to those of southern Tyrrhenian coastal waters and of other lagoons.

A new potentially toxic Azadinium species (Dinophyceae) from the Mediterranean Sea, A. dexteroporum sp. nov.

A new photosynthetic planktonic marine dinoflagellate, Azadinium dexteroporum sp. nov., is described from the Gulf of Naples (South Tyrrhenian Sea, Mediterranean Sea). The plate formula of the species, Po, cp, X, 4′, 3a, 6″, 6C, 5?S, 6‴ and 2″″, is typical for this recently described genus. Azadinium dexteroporum is the smallest rep‐resentative of the genus (8.5 μm average length, 6.2 μm average width) and shares the presence of a small antapical spine with the type species A. spinosum and with A. polongum. However, it differs from all other Azadinium species for the markedly asymmetrical Po plate and the position of the ventral pore, which is located at the right posterior end of the Po plate. Another peculiarity of A. dexteroporum is the pronounced concavity of the second intercalary plate (2a), which appears collapsed with respect to the other plates. Phylogenetic analyses based on the large subunit 28S rDNA (D1/D2) and the internal transcribed spacer (ITS rDNA) support the attribution of A. dexteroporum to the genus Azadinium and its separation from the other known species. LC/MS‐TOF analysis shows that Azadinium dex‐teroporum produces azaspiracids in low amounts. Some of them have the same molecular weight as known compounds such as azaspiracid‐3 and ‐7 and Compound 3 from Amphidoma languida, as well as similar fragmentation patterns in some cases. This is the first finding of a species producing azapiracids in the Mediterranean Sea.

Benthic protists: the under-charted majority

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBIs Short Read Archive. We suggest that the benthic realm may therefore be the worlds largest reservoir of marine protist diversity, with most taxa at present undescribed.

Strengths and weaknesses of microarray approaches to detect Pseudo-nitzschia species in the field.

The planktonic diatom genus Pseudo-nitzschia contains several genetically closely related species. Some of these can produce domoic acid, a potent neurotoxin. Thus, monitoring programs are needed to screen for the presence of these toxic species. Unfortunately, many are impossible to distinguish using light microscopy. Therefore, we assessed the applicability of microarray technology for detection of toxic and non-toxic Pseudo-nitzschia species in the Gulf of Naples (Mediterranean Sea). Here, 11 species have been detected, of which at least 5 are potentially toxic. A total of 49 genus- and species-specific DNA probes were designed in silico against the nuclear LSU and SSU rRNA of 19 species, and spotted on the microarray. The microarray was tested against total RNA of monoclonal cultures of eight species. Only three of the probes designed to be species-specific were indeed so within the limits of our experimental design. To assess the effectiveness of the microarray in detecting Pseudo-nitzschia species in environmental samples, we hybridized total RNA extracted from 11 seasonal plankton samples against microarray slides and compared the observed pattern with plankton counts in light microscopy and with expected hybridization patterns obtained with monoclonal cultures of the observed species. Presence of species in field samples generally resulted in signal patterns on the microarray as observed with RNA extracted from cultures of these species, but many a-specific signals appeared as well. Possible reasons for the numerous cross reactions are discussed. Calibration curves for Pseudo-nitzschia multistriata showed linear relationship between signal strength and cell number.

Pseudo‐nitzschia arctica sp. nov., a new cold‐water cryptic Pseudo‐nitzschia species within the P. pseudodelicatissima complex

A new nontoxic Pseudo‐nitzschia species belonging to the P. pseudodelicatissima complex, P. arctica, was isolated from different areas of the Arctic. The erection of P. arctica is mainly supported by molecular data, since the species shares identical ultrastructure with another species in the complex, P. fryxelliana, and represents a new case of crypticity within the genus. Despite their morphological similarity, the two species are not closely related in phylogenies based on LSU, ITS and rbcL. Interestingly, P. arctica is phylogenetically most closely related to P. granii and P. subcurvata, from which the species is, however, morphologically different. P. granii and P. subcurvata lack the central larger interspace which is one of the defining features of the P. pseudodelicatissima complex. The close genetic relationship between P. arctica and the two species P. granii and P. subcurvata is demonstrated by analysis of the secondary structure of ITS2 which revealed no compensatory base changes, two hemi‐compensatory base changes, and two deletions in P. arctica with respect to the other two species. These findings emphasize that rates of morphological differentiation, molecular evolution and speciation are often incongruent for Pseudo‐nitzschia species, resulting in a restricted phylogenetic value for taxonomic characters used to discriminate species. The description of a new cryptic species, widely distributed in the Arctic and potentially representing an endemic component of the Arctic diatom flora, reinforces the idea of the existence of noncosmopolitan Pseudo‐nitzschia species and highlights the need for combined morphological and molecular analyses to assess the distributional patterns of phytoplankton species.