SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research. Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.     

Extensive and intimate association of the cytoskeleton with forming silica in diatoms: control over patterning on the meso- and micro-scale

BACKGROUND: The diatom cell wall, called the frustule, is predominantly made out of silica, in many cases with highly ordered nano- and micro-scale features. Frustules are built intracellularly inside a special compartment, the silica deposition vesicle, or SDV. Molecules such as proteins (silaffins and silacidins) and long chain polyamines have been isolated from the silica and shown to be involved in the control of the silica polymerization. However, we are still unable to explain or reproduce in vitro the complexity of structures formed by diatoms. METHODS/PRINCIPAL FINDING: In this study, using fluorescence microscopy, scanning electron microscopy, and atomic force microscopy, we were able to compare and correlate microtubules and microfilaments with silica structure formed in diversely structured diatom species. The high degree of correlation between silica structure and actin indicates that actin is a major element in the control of the silica morphogenesis at the meso and microscale. Microtubules appear to be involved in the spatial positioning on the mesoscale and strengthening of the SDV. CONCLUSIONS/SIGNIFICANCE: These results reveal the importance of top down control over positioning of and within the SDV during diatom wall formation and open a new perspective for the study of the mechanism of frustule patterning as well as for the understanding of the control of membrane dynamics by the cytoskeleton.     

Characterizing the silica deposition vesicle of diatoms

Summary The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.Abbreviation SDV Silica deposition vesicle     

Grazing-induced changes in cell wall silicification in a marine diatom

In aquatic environments, diatoms (Bacillariophyceae) constitute a central group of microalgae which contribute to about 40% of the oceanic primary production. Diatoms have an absolute requirement for silicon to build-up their silicified cell wall in the form of two shells (the frustule). To date, changes in diatom cell wall silicification have been only studied in response to changes in the growth environment, with consistent increase in diatom silica content when specific growth rates decrease under nutrient or light limitations. Here, we report the first evidence for grazing-induced changes in cell wall silicification in a marine diatom. Cells grown in preconditioned media that had contained both diatoms and herbivores are significantly more silicified than diatoms grown in media that have contained diatoms alone or starved herbivores. These observations suggest that grazing-induced increase in cell wall silicification can be viewed as an adaptive reaction in habitats with variable grazing pressure, and demonstrate that silicification in diatoms is not only a constitutive mechanical protection for the cell, but also a phenotypically plastic trait modulated by grazing. In turn, our results corroborate the idea that plant-herbivore interactions, beyond grazing sensu stricto, contribute to drive ecosystem structure and biogeochemical cycles in the ocean.     

Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana: insights into silica structure formation

Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme (and its gene) in the structure formation process.     

Analysis of Thalassiosira pseudonana silicon transporters indicates distinct regulatory levels and transport activity through the cell cycle

An analysis of the expression and activity of silicon transporters (SITs) was done on synchronously growing cultures of the diatom Thalassiosira pseudonana to provide insight into the role these proteins play in cellular silicon metabolism during the cell cycle. The first SIT-specific polyclonal peptide antibody was generated and used in the immunoblot analysis of whole-cell protein lysates to monitor SIT protein levels during synchronized progression through the cell cycle. Peaks in SIT protein levels correlated with active periods of silica incorporation into cell wall substructures. Quantitative real-time PCR on each of the three distinct SIT genes (TpSIT1, TpSIT2, and TpSIT3) showed that mRNA levels for the most highly expressed SIT genes peaked during the S phase of the cell cycle, a period prior to maximal silicon uptake and during which cell wall silicification does not occur. Variations in protein and mRNA levels did not correlate, suggesting that a significant regulatory step of SITs is at the translational or posttranslational level. Surge uptake rates also did not correlate with SIT protein levels, suggesting that SIT activity is internally controlled by the rate of silica incorporation. This is the first study to characterize SIT mRNA and protein expression and cellular uptake kinetics during the course of the cell cycle and cell wall synthesis, and it provides novel insight into SIT regulation.     

Characterization and Localization of Insoluble Organic Matrices Associated with Diatom Cell Walls: Insight into Their Roles during Cell Wall Formation

Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species. Results show that an insoluble organic matrix enriched in mannose, likely the diatotepum, is localized on the proximal surface of the silica cell wall. We did not identify any organic matrix embedded within the silica. We also identified a distinct material consisting of glucose polymer with variable localization depending on the species. In some species this component was directly involved in the morphogenesis of silica structure while in others it appeared to be only a structural component of the cell wall. A novel glucose-rich structure located between daughter cells during division was also identified. This work for the first time correlates the structure, composition, and localization of insoluble organic matrices associated with diatom cell walls. Additionally we identified a novel glucose polymer and characterized its role during silica structure formation.     

Multiparametric Analyses Reveal the pH-Dependence of Silicon Biomineralization in Diatoms

Diatoms, the major contributors of the global biogenic silica cycle in modern oceans, account for about 40% of global marine primary productivity. They are an important component of the biological pump in the ocean, and their assemblage can be used as useful climate proxies; it is therefore critical to better understand the changes induced by environmental pH on their physiology, silicification capability and morphology. Here, we show that external pH influences cell growth of the ubiquitous diatom Thalassiosira weissflogii, and modifies intracellular silicic acid and biogenic silica contents per cell. Measurements at the single-cell level reveal that extracellular pH modifications lead to intracellular acidosis. To further understand how variations of the acid-base balance affect silicon metabolism and theca formation, we developed novel imaging techniques to measure the dynamics of valve formation. We demonstrate that the kinetics of valve morphogenesis, at least in the early stages, depends on pH. Analytical modeling results suggest that acidic conditions alter the dynamics of the expansion of the vesicles within which silica polymerization occurs, and probably its internal pH. Morphological analysis of valve patterns reveals that acidification also reduces the dimension of the nanometric pores present on the valves, and concurrently overall valve porosity. Variations in the valve silica network seem to be more correlated to the dynamics and the regulation of the morphogenesis process than the silicon incorporation rate. These multiparametric analyses from single-cell to cell-population levels demonstrate that several higher-level processes are sensitive to the acid-base balance in diatoms, and its regulation is a key factor for the control of pattern formation and silicon metabolism.     

SYNCHRONIZED GROWTH OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) PROVIDES NOVEL INSIGHTS INTO CELL‐WALL SYNTHESIS PROCESSES IN RELATION TO THE CELL CYCLE1

Cell‐cycle effects in phytoplankton have both general and specific influences over a variety of cellular processes. Understanding these effects requires that the majority of cells in a culture are progressing through the same cell‐cycle stage, which requires synchronous growth. We report the development of a silicon starvation–recovery synchrony for the first diatom with a sequenced genome, Thalassiosira pseudonana Hasle et Heimdale, which provides several novel insights into the process of cell‐wall formation. After 24 h of silicate starvation, flow cytometry measurements indicated that 80% of the cells were arrested in the early G1 phase of the cell cycle and then upon silicate replenishment progressed synchronously through the cycle. An early G1‐arrest point was not previously documented in diatoms. After silicate replenishment, girdle‐band synthesis was confined to a particular period in G1, and cells did not lengthen in accordance with each girdle band added, which has implications related to cell growth and separation processes in diatoms. Measurements of silicic acid uptake, intracellular pools, and silica incorporation into the cell wall, coupled with fluorescence visualization of newly synthesized cell‐wall structures, provide the first direct measurements of silica amounts in individual girdle bands and valves in a diatom. Fluorescence imaging indicated why valves in T. pseudonana do not have to reduce in size with each generation and enabled visualization of intermediates in structure formation. The development of a synchrony procedure for T. pseudonana enables correlation of cellular events with the cell cycle, which should facilitate the use of genomic information.     

New insights into the complex architecture of siliceous copepod teeth

Copepods belong to the dominant marine zooplankton taxa and play an important role in particle and energy fluxes of the marine water column. Their mandibular gnathobases possess tooth-like structures, so-called teeth. In species feeding on large proportions of diatoms these teeth often contain silica, which is very probably the result of a coevolution with the siliceous diatom frustules. Detailed knowledge of the morphology and composition of the siliceous teeth is essential for understanding their functioning and their significance in the context of feeding interactions between copepods and diatoms. Based on analyses of the gnathobases of the Antarctic copepod Rhincalanus gigas, the present study clearly shows, for the first time, that the silica in the siliceous teeth features large proportions of crystalline silica that is consistent with the mineral α-cristobalite and is doped with aluminium. The siliceous structures have internal chitinous fibre networks, which are assumed to serve as scaffolds during the silicification process. The compact siliceous teeth of R. gigas are accompanied by structures with large proportions of the elastic protein resilin, likely reducing the mechanical damage of the teeth when the copepods feed on diatoms with very stable frustules. The results indicate that the coevolution with diatom frustules has resulted in gnathobases exhibiting highly sophisticated composite structures.     

Prescribing diatom morphology: toward genetic engineering of biological nanomaterials

The formation of inorganic materials with complex form is a widespread biological phenomenon (biomineralization). Among the most spectacular examples of biomineralization is the production by diatoms (a group of eukaryotic microalgae) of intricately nanopatterned to micropatterned cell walls made of silica (SiO2). Understanding the molecular mechanisms of diatom silica biomineralization is not only a fundamental biological problem, but also of great interest in materials engineering, as the biological self-assembly of three-dimensional (3D) inorganic nanomaterials has no man-made analog. Recently, insight into the molecular mechanism of diatom silica formation has been obtained by structural and functional analysis of biomolecules that are involved in this process. Furthermore, the rapid development of diatom molecular genetics has provided new tools for investigating the silica forming machinery of diatoms and for manipulating silica biogenesis. This has opened the door for the production, through genetic engineering, of unique 3D nanomaterials with designed structures and functionalities.     

Dissection of the frustules of the diatom Synedra acus under the action of picosecond impulses of submillimeter laser irradiation

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30–100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.     

Ontogenetic analysis of siliceous cell wall formation in Triparma laevis f. inornata (Parmales,Stramenopiles)

Triparma laevis f. inornata is a unicellular alga belonging to the Bolidophyceae, which is most closely related to diatoms. Like diatoms, T. laevis f. inornata has a siliceous cell wall. The cell wall of T. laevis f. inornata consists of four round plates (three shields and one ventral plate) and one dorsal and three girdle plates. But, unlike diatoms, T. laevis f. inornata cells can grow when concentrations of silica are depleted. We took advantage of this ability, using TEM to study the ontogeny of the siliceous plate, pattern center formation, and development. Two types of pattern centers (annulus and sternum) were observed in the early and middle stage of plate formation. During their formation, the annuli were initially crescent‐shaped but eventually their ends fused to make a ring. Only outward silica deposition of the branching ribs occurred on the growing annulus until it became a ring, resulting in an unfilled circle inside the annulus. The pattern center of the shield plate was always an annulus, but in ventral plates both annulus and sternum were observed. The annuli and sterna in T. laevis f. inornata round plates were very similar to the annuli and sterna in diatom valves. These results suggested that the round plates of Parmales are homologous to diatom valves. This information on the plate ontogeny of T. laevis f. inornata provides new insights into the evolution of the siliceous cell wall in the Parmales and diatoms.     

Diversity of mineral cell coverings and their formation processes: a review focused on the siliceous cell coverings

Mineral cell coverings are found in various protists. Some macroalgae accumulate calcium carbonate in the intercellular space, and some unicellular organisms use calcium carbonate or silica for the construction of loricas, scales, and frustules. Diatoms are representatives of those utilizing silica for the material of the cell covering called a frustule. The development of the frustule is initiated in a silica-deposition vesicle (SDV), which occurs just beneath the plasma membrane and, subsequently, the silicified cell covering expands its area, following the expansion of the SDV from valve face to valve mantle. Sequential valve development with whole valves is reviewed in several diatoms placed in different phylogenetic positions. Every diatom commences its valve formation from its pattern center and then develops by means of individual procedures. The results indicate that the valve development reflects the phylogeny of diatoms. In addition, recent progress in silica biomineralization is briefly reviewed, and the phylogeny of ability concerning siliceous cell covering formation is inferred. Electronic Publication     

Biogenic silica as an estimate of siliceous microfossil abundance in Great Lakes sediments

Biogenic silica concentration (BSi) in sediment cores from the Great Lakes is evaluated as an estimate of siliceous microfossil abundance. A significant linear relationship was found between measured BSi and diatom valve abundance for sediment cores from the Bay of Quinte, Lake Ontario, Lake Erie, Lake Michigan and Lake Superior and between measured BSi and diatom biovolume for Lake Erie, Lake Michigan, and Lake Superior but not for Lake Ontario. Diatom silica predicted from diatom species abundance and an estimated silica content per cell in the Lake Erie cores accounted for 117% and 103% of measured BSi, respectively. By contrast, predicted diatom silica could only account for 28% of measured BSi in the Lake Michigan core and only 25% in the Lake Superior core. A few large diatoms with a large silica content per cell comprised a major portion of predicted diatom silica in all cores. The discrepancy between chemically measured BSi and the silica predicted from diatoms in the Lake Michigan and Lake Superior cores was partially due to the inability of the regression model, used to estimate diatom silica content, to account for different degrees of silicification in the diatom asemblages from the more dissolved silica rich Lake Michigan and Lake Superior.     

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY1

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young’s modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young’s modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.     

Analytical studies on the incorporation of aluminium in the cell walls of the marine diatom Stephanopyxis turris

The eukaryotic diatoms are unicellular algae. They are well known for their filigree micro- and nanostructured cell walls which mainly consist of amorphous silica as well as various organic compounds. However, diatoms are also known to incorporate certain amounts of aluminium into their cell walls. Unexpectedly, enhanced Al concentrations in the Southern Yellow Sea were found to be correlated with a diatom spring bloom. Therefore, we have analyzed the influence of strongly enhanced Al concentrations in the culture medium upon the growth behaviour of the diatom Stephanopyxis turris (S. turris). The uptake and incorporation of Al into the cell walls was monitored. It turned out that S. turris survives aluminium concentrations up to 105.5 μM (2.85 mg/l) in the culture medium. Under the applied conditions, this corresponds to an Al/Si ratio of 1:1. These large amounts of Al had to be offered in the form of bis–tris-chelates in order to prevent uncontrolled precipitation. Under these conditions, the Al/Si ratio in the cell walls could be increased up to about 1:15 as determined by ICP-OES, the highest amount of aluminium found in diatom cell walls yet. Structural characterization of the biosilica by ATR-FTIR and solid-state ²⁷Al NMR spectroscopy revealed that an amorphous aluminosilicate phase is formed where the aluminium exists as four- and sixfold-coordinated species.     

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.     

A STUDY OF Si DEPOSITION SYNCHRONY IN RHIZOSOLENIA (BACILLARIOPHYCEAE) MATS USING A NOVEL 32Si AUTORADIOGRAPHIC METHOD

A ³²Si autoradiographic technique using a liquid photographic emulsion was developed for the study of diatom silica deposition in culture or in natural water samples. The method was used in the Central North Pacific to study silica deposition by diatoms of the genus Rhizosolenia. The species examined form centimeter-sized aggregates commonly referred to as mats. The Rhizosolenia mats examined were composed of a matrix of R. fallax Sundström chains, embedded with chains of larger cells, either R. debyana H. Peragallo or R. acuminata H. Peragallo. The autoradiographs revealed distinct rings of labeled intercalary bands and/or labeled valves. A greater proportion of the frustule of the larger species was labeled during the incubations with ³²Si, implying higher rates of silicification by R. debyana and R. accuminata compared to R. fallax. A quantitative consideration of these differences in species-specific Si production combined with abundance and surface area estimates for each species indicates that cells of the larger species carry out the majority of silica production in Rhizosolenia mats. The large cell size (pervalvar axis 240 to 3000 μm) and elongate frustule morphology of Rhizosolenia cells enabled us to localize the deposition of silica along the pervalvar axis. Positions of labeled bands along this axis indicate progress through the Si deposition cycle, and the results suggest that cell division is phased, with either a bimodal or unimodal age distribution of cells within the cell cycle for all species in a mat. Species-specific doubling times from 25 to 60 h were implied by the mean fractions of frustule that were labeled. ³²Si autoradiography revealed unique species-specific differences in diel patterns of cell division and silica deposition and has potential for studies of Si deposition by other diatom species and assemblages.