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.1

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). II. AN ASSESSMENT OF THE TAXONOMY OF S. COSTATUM‐LIKE SPECIES WITH THE DESCRIPTION OF FOUR NEW SPECIES1

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.     

EFFECTS OF ARSENIC SPECIATION AND PHOSPHATE CONCENTRATION ON ARSENIC INHIBITION OF SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Arsenate is taken up readily by Skeletonema costatum (Greville) Cleve due to its chemical similarity to phosphate, and it inhibits primary productivity at concentrations as low as 67 nM when the phosphate concentration is low. A phosphate enrichment of greater than 0.3 μM alleviates this inhibition; however, the arsenate stress causes an increase in the cell's requirement for phosphorus. Arsenite is also toxic to Skeletonema at similar concentrations. Methylated species, such as dimethylarsinic acid, did not affect cell productivity at the levels examined. Thus, the reduction and methylation of arsenate to dimethylarsinic acid by the cell produces a stable, non-toxic compound.     

EFFECT OF POLYCARBONATE CONTAINERS ON THE GROWTH OF TWO SPECIES OF MARINE DIATOMS1

Two species of marine diatoms [Skeletonema costatum (Greville) Cleve and Thalassiosira pseudonana (Hustedt) Hasle and Heimdal] were grown in glass and polyarbonate containers. S. costatum exhibited a signzJicantly lower exponential growth rate and maximal yield and a signajcantly longer lag phase when grown in polycarbonate. Exponential growth rate and maximal yield of T. pseudonana was significantly reduced (P < 0.05 in all cases). This study suggests that a difference in diatom growth between glass and polyarbonate containers might arise in certain cases. However, such a difference may not be detectable with all biomass measurement techniques or with low within-treatment replication.     

The amino acid and gross composition of marine diatoms potentially useful for mariculture

The amino acid and gross compositions of three benthic diatoms (Cylindrotheca fusiformis Reimann and Lewin, Navicula jeffreyi Hallegraeff et Burford and Nitzschia closterium (Ehr. W. Smith) and three chain-forming diatoms (Lauderia annulata Cleve, Skeletonema costatum (Greville) Cleve and a tropical Skeletonema sp. (CS-252)), were determined during late-logarithmic growth phase.Four of the six species were rich in protein, ranging from 31% (S. costatum) to 38% (N. closterium) of the dry weight, and contained 4.9 to 6.5% carbohydrate and 9.4 to 18% ash. The other two species, C. fusiformis and L. annulata contained only 16% protein but had nearly twice the total carbohydrate (11 to 12%) and two to three times the ash (29 to 35%) of the other species. All species contained a similar percentage of lipid (18 to 20%), and a high protein quality based on their total amino acid composition.The usefulness of these diatoms for mariculture will be determined by growth rates, gross composition and acceptability to the animal. N. closterium had the fastest growth rates of the benthic mat-forming diatoms, it was rich in protein, and it may be a good candidate for abalone culture. The two Skeletonema spp. had the fastest growth rates of the chain-forming diatoms, and are already widely used for prawn larval culture in Australia. Feeding trials are now necessary to confirm the high nutritional value of these diatom species for specific animals.     

Nutrient-induced competition between two species of marine diatoms

Competition experiments betweenPhaeodactylum tricornutum andSkeletonema costatum showed that even at temperatures higher than 10°C (i.c. 14°C), the development ofSkeletonema can be favoured by adjusting nutrient levels and nutrient ratios. Low NSi ratios were found to favourSkeletonema. Additionally, high NP ratios further enhanced the ability ofSkeletonema to dominate the cuftures. Contrary to some statements in literature, it seems that high concentrations of silicates are more important for the dominance ofSkeletonema costatum in large-scale cultures than just low temperatures. This finding is important with regard to stimulating the blooming ofSkeletonema costatum in natural phytoplankton populations as food for bivalve molluscs.     

MORPHOLOGY OF THE DIATOM GENUS CAMPYLONEIS (BACILLARIOPHYCEAE,BACILLARIOPHYTA), WITH A DESCRIPTION OF CAMPYLONEIS JULIAE SP. NOV. AND AN EVALUATION OF THE FUNCTION OF THE VALVOCOPULAE1

A revision of the monoraphid pennate diatom genus Campyloneis Grunow was carried out based on LM and EM observations. The material examined originated from various herbarium collections and from extant epiphytic diatom communities on leaves of Posidonia spp. We also examined the generitype C. grevillei (Smith) Grunow and the fossil material of C. gheyselinchi Reinhold from which the author extracted the type. Our results clarified the fine structure of C. grevillei and C. gheyselinchi. Of the various varieties of C. grevillei, only the variety argus (Grunow) Cleve was retained. This differs from the nominate variety in the arrangement and shape of the areolae adjacent to the sternum of the araphid valve. The newly described taxon Campyloneis juliae De Stefano differs from all Campyloneis species in areolae ultrastructure and morphology of the valvocopulae. As for the fossil species C. gheyselinchi, the sternum valve areolae are similar to those of C. grevillei, but scarcity of frustules in the type material prohibited evaluation of its variability. For this reason we provisionally maintained its rank of species. The elaborate linking systems among the valvocopulae and valves in Campyloneis species appear to provide structural reinforcement against pressure from neighboring epiphytic diatoms and scouring of seagrass leaves.     

BRACKISH WATER AND FRESHWATER SPECIES OF THE DIATOM GENUS SKELETONEMA. II. SKELETONEMA POTAMOS COMB. NOV.1

Electron microscope investigations of the siliceous frustule show that the diatom described by Hustedt as Stephanodiscus subsalsus (A. Cleve) Hust. is not Skeletonema subsalsum (A. Cleve) Bethge (Melosira subsalsa A. Cleve) but is Microsiphona potamos Weber. This species is so similar to Skeletonema costatum (Grev.) Cleve and Skeletonema subsalsum that the combination Skeletonema potamos (Weber) Hasle is suggested. Present records classify Skeletonema potamos as a freshwater species of lakes and rivers. In Sandusky Bay, Lake Erie (U.S.A.) and in River Wümme, a tributary of the River Weser (Germany) it grows with Skeletonema subsalsum. In nature, and when grown in cultures at a salinity of 0%, the processes are extremely short; when grown at salinities of 2% or more, the processes are much longer.     

Morphology and molecular phylogeny of Thalassiosira sinica sp. nov. (Bacillariophyta) with delicate areolae and fultoportulae pattern

Thalassiosira Cleve is one of the most species-rich marine diatom genera. Previous studies have mainly focused on polar and temperate areas, but recent studies on material from Asian waters suggested that a high and undescribed species diversity of Thalassiosira occurs in Asia. On the basis of plankton samples collected from the South China Sea, a new species, T. sinica sp. nov. Y. Li & Y. Q. Guo is described. The morphology of the cells was examined by light and scanning electron microscopy. The hypervariable region of the nuclear large-subunit ribosomal DNA and the relatively conserved region of the nuclear small-subunit ribosomal DNA were sequenced for information on phylogenetic relationships. The living cells are usually solitary and drum-shaped. The areolae on the valve are delicate, small and arranged in fascicles. In addition to a regular ring of marginal fultoportulae on the valve edge, T. sinica possesses one central fultoportula and a number of fultoportulae arranged into 2–3 irregular rings on the valve face. A rimoportula located inside the ring of marginal fultoportulae possesses a long and strong external tube. The valvocopula and the copulae have rows of pores, but the pores on the valvocopula are larger than those on the copulae. Thalassiosira sinica appears to be included in subgroup C sensu Gedde because of a rimoportula with a distinct external tube located on the valve face. The molecular phylogeny, inferred from both SSU and LSU sequences, does, however, not support the validity of subgroup C, as the closest allies of T. sinica here turned out to be T. diporocyclus and T. lundiana, species in which the rimoportulae are located on the valve margin.     

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Two new species of the diatom genusChaetoceros (Bacillariophyta)

Two new planktonic diatom species of the genusChaetoceros are described herein:Ch. octagonus andCh. rectus, from material of Baja California coasts and the Gulf of California, respectively;Ch. rectus was also found in a sample from Australian waters.Ch. octagonus is a robust species, with a characteristic pyramidal valve shape, heavily silicified valves and setae, and one central rimoportula on every valve in the chain; it is compared with related species, which show a similar morphology, especially by electron microscopy. It belongs into subg.Chaetoceros, sect.Borealia. Ch. rectus is placed in subg.Hyalochaete, and provisionally within sect.Anastomosantia. Its major characters are: chains, cells, and setae are delicate and fragile, cells are joined in the chain by a bridge which connects sibling intercalary setae, valves and setae are weakly silicified, and one rimoportula is present on terminal valves only. Comparison is made with another species of the same section.Ch. rectus shows a different manner of chain-forming within the genus than previously described in the literature.     

INFLUENCE OF ULTRAVIOLET RADIATION ON THE EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN AND DNA POLYMERASE α IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

To evaluate the effects of UV radiation on the expression of DNA replication‐related genes in phytoplankton, the mRNA levels of DNA polymerase α and proliferating cell nuclear antigen (PCNA) in a marine diatom, Skeletonema costatum (Greville) Cleve, were studied using the methods of real‐time quantitative PCR. Treating the algal cultures with UVC radiation for 15 min caused severe mortality during the 24‐h period after treatment. A significant amount of thymine dimers was detected in the treated cultures, and the mRNA levels of DNA polymerase α and PCNA increased by as much as 140 and 23 pmol·(g total RNA)^?1, respectively, compared with the control experiments. In contrast, massive cell deaths did not occur in cultures receiving UVA/B radiation, and the formation of thymine dimers was inconspicuous. Also, UVA/B did not enhance the expression levels of DNA polymerase α or PCNA. Based on the calculation of biologically effective UV doses, daily exposure to sunlight may increase the expression of DNA polymerase α or PCNA genes in S. costatum by 12% at sea surface. This level of increase does not seriously affect the value of using these genes as growth indicators, but caution is needed in the extrapolation of this conclusion to all phytoplankton species.     

COMPARATIVE SEQUENCE ANALYSIS OF DIATOM SILICON TRANSPORTERS: TOWARD A MECHANISTIC MODEL OF SILICON TRANSPORT1

Silicon is an important element in biology, for organisms ranging from unicellular algae to humans. It acts as a structural material for both plants and animals, but can also function as a metabolite or regulator of gene expression, affecting a wide range of cellular processes. Molecular details of biological interaction with silicon are poorly understood. Diatoms, the largest group of silicifying organisms, are a good model system for studying this interaction. The first proteins shown to directly interact with silicon were diatom silicon transporters (SITs). Because the basis for substrate recognition lies within the primary sequence of a protein, identification of conserved amino acid residues would provide insight into the mechanism of SIT function. Lack of SIT sequences from a diversity of diatoms and high sequence conservation in known SITs has precluded identification of such residues. In this study, PCR was used to amplify partial SIT sequences from eight diverse diatom species. Multiple gene copies were prevalent in each species, and phylogenetic analysis showed that SITs generally group according to species. In addition to partial SIT sequences, full‐length SIT genes were identified from the pennate diatom, Nitzschia alba (Lewin and Lewin), and the centric diatom Skeletonema costatum (Greville) Cleve. Comparing these SITs with previously identified SITs showed structural differences between SITs of centrics and pennates, suggesting differences in transport mechanism or regulation. Comparative amino acid analysis identified conserved regions that may be important for silicon transport, including repeats of the motif GXQ. A model for silicon uptake and efflux is presented that is consistent with known aspects of transport.     

The capacity for change and the unpredictability of the phytoplankton of the east coast of the United States

Differences in the change of numerical abundance from time totime through the year and place to place along the U.S. eastcoast show Skeletonema costatum to have a greater capacity forchange than Thalassionema nitzschioides. These differences emergefrom the unpredictable occurrence and absence of Skeletonemafrom time to time through the year and from year to year atWoods Hole and from place to place along the U.S. east coast.Skeletonema's greater capacity for change than Thatassionema'stransmit an inherently variable capacity for change to the wholephytoplankton, and the unpredictability of these species transmitsthis characteristic to the whole population too.     

Description of Conticribra tricircularis,a new genus and species of Thalassiosirales,with a discussion on its relationship to other continuous cribra species of Thalassiosira Cleve (Bacillariophyta) and its freshwater origin

A new diatom genus Conticribra is erected to accommodate C. tricircularis, described from a freshwater Pliocene deposit in Trout Creek, Oregon (USA). The genus accommodates species possessing: (i) loculate areolae with (semi-) continuous cribra; (ii) non-plicated valve face; (iii) rimoportula located on the valve mantle, replacing a fultoportula. Conticribra tricircularis has no valve face fultoportulae and can easily be distinguished by its marginal fultoportulae with four satellite pores arranged in three rings. Three species are transferred to the new genus from Thalassiosira sensu lato. Using evidence from the fossil record and recent molecular data, a hypothesis concerning the freshwater origin of Conticribra is discussed.     

Utility of mitochondrial‐encoded cytochrome c oxidase I gene for phylogenetic analysis and species identification of the planktonic diatom genus Skeletonema

Small subunit (SSU) and large subunit (LSU) rDNA sequences have been commonly used to delineate the taxonomy and biogeography of the planktonic diatom genus Skeletonema, but the genes occur as multiple copies and are therefore not suitable for barcoding purposes. Here, we analyzed phylogenetic relationships of Skeletonema using the mitochondrial‐encoded cytochrome c oxidase I gene (cox1), as well as partial LSU rDNA (D1–D3) and SSU rDNA, to identify the factors that define species and to evaluate the utility of these three markers for this taxon. Twelve Skeletonema species were divided into six clades, I–VI, each of which comprised the same species by the three markers: clades I (S. japonicum, S. grethae, S. pseudocostatum, and S. tropicum), II (S. menzelii), III (S. dohrnii and S. marinoi), IV (S. costatum, S. potamos, and S. subsalsum), V (S. grevillei), and VI (S. ardens). However, the branching order among these clades was incongruent among the markers. In clade III, six S. marinoi strains had identical cox1 sequences. These S. marinoi strains branched along with S. dohrnii, except for strains from the Gulf of Naples, with high support in cox1. Species delimitation between S. dohrnii and S. marinoi was therefore not supported. In clade IV, S. costatum and S. subsalsum were robustly clustered, with S. potamos as a sister clade in the cox1 tree, not in the LSU and SSU trees. In clade II, cox1 also confirmed that S. menzelii includes three subclades potentially distinguishable from each other by morphological features. Cox1 proved to be the most useful marker for the identification of Skeletonema species because it gave a tree with highly supported clades, has sufficient variation within and among species, encodes a protein in a single copy, and requires relatively few primers.     

Molecular Phylogeny and Taxonomy of the Genus Minidiscus (Bacillariophyceae), with Description of Mediolabrus gen. nov

The genus Minidiscus comprises a group of ecologically-important and globally-distributed planktonic diatoms that are characterized by their small cell size, high mantle and processes more or less concentrated in the valve center. Monoclonal strains were established from collections along the Chinese coast. In the phylogenetic analyses inferred from a LSU and SSU dataset, six Minidiscus species clustered into two well-supported clades. The first clade was located within a larger clade formed mainly by Thalassiosira taxa, and the second clade appeared as sister to a clade comprising the genus Skeletonema. Hence, presently known Minidiscus do not form a monophyletic clade, but rather make up a phenotypic grouping. Based on the morphology of the type species, M. trioculatus, as well as morphological characters of all taxa in the clade with M. trioculatus, Minidiscus is characterized by having fultoportula(e) in the valve center or sub-centered close to a single rimoportula, and the cells are usually cylindrical. Mediolabrus gen. nov. is proposed to accommodate species in the second clade. The main difference between Minidiscus and Mediolabrus is the type of process found in the valve (sub-)center, with fultoportula(e) close to a rimoportula in the former, and only a single rimoportula in the latter. According to the above criteria, previously described Minidiscus taxa were re-examined, and either retained in Minidiscus or transferred to Mediolabrus.     

GENERAL FEATURES OF PHOTOPROTECTION BY ENERGY DISSIPATION IN PLANKTONIC DIATOMS (BACILLARIOPHYCEAE)1

Planktonic diatoms (Bacillariophyceae) have to cope with large fluctuations of light intensity and periodic exposure to high light. After a shift to high light, photoprotective dissipation of excess energy characterized by the nonphotochemical quenching of fluorescence (NPQ) and the concomitant deepoxidation of diadinoxanthin to diatoxanthin (DT) were measured in four different planktonic marine diatoms (Bacillariophyceae): Skeletonema costatum (Greville) Cleve, Cylindrotheca fusiformis Reimann et Lewin, Thalassiosira weissflogii (Grunow) Fryxell et Hasle, and Ditylum brightwellii (West) Grunow in comparison to the model organism Phaeodactylum tricornutum Böhlin. Upon a sudden increase of light intensity, deepoxidation was rapid and de novo synthesis of DT also occurred. In all species, NPQ was linearly related to the amount of DT formed during high light. In this report, we focused on the role of DT in the dissipation of energy that takes place in the light‐harvesting complex. In S. costatum for the same amount of DT, less NPQ was formed than in P. tricornutum and as a consequence the photoprotection of PSII was less efficient. The general features of photoprotection by harmless dissipation of excess energy in planktonic diatoms described here partly explain why diatoms are well adapted to light intensity fluctuations.     

Triphenyltin inhibits photosynthesis and respiration in marine microalgae

Summary The effects of diphenyltin and triphenyltin (TPhT) on gross photosynthesis and respiration by the diatomSkeletonema costatum (Greville) Cleve and the chlorophyteDunaliella tertiolecta (Butscher) were investigated by measuring the rates of change of oxygen concentration in samples which were alternately illuminated unilluminated. Measurements were carried out for 90 min after organotin addition. Triphyltin at concentrations in the nM to M range inhibited photosynthesis and respiration in both ogranisms. Levels of TPhT inhibiting these processes were two to three orders of magnitude higher forD. tertiolecta than forS. costatum. Photosynthesis and respiration byD. tertiolecta were resistant to diphenyltin at concentrations up to its limit of solubility (0.84 mM). WithS. costatum, inhibitory levels of diphenyltin were one to two orders of magnitude higher than those for triphenyltin. Inhibition was often progressive over the period after organotin addition. This effect varied in intensity and was more noticeale with the more resistantD. tertiolecta. Comparison of our results with levels of organotins which have been obeserved by others in Mediterranean coastal waters indicate that environmental levels of TPhT could influence phytoplankton composition and dynamics.