Light is a key factor in triggering sexual reproduction in the pennate diatom Haslea ostrearia

Sexual reproduction is an obligatory phase in the life cycle of most diatoms, as cell size decreases with successive vegetative divisions and the maximal cell size is only restored by a specialized cell, the auxospore, which follows zygote formation as a result of sexual reproduction. While in pennate diatoms the induction of sexual reproduction depends primarily on cell–cell interactions, the importance of different external factors for the induction of sexual reproduction is less well known. Here, we investigated the effects of light on sexualization in the marine benthic pennate diatom Haslea ostrearia (Gaillon) R. Simonsen. Compatible clones were crossed and exposed to different combinations of light levels, qualities, and photoperiods. Light was found to be a key factor for sexualization, and to a certain extent, to control auxosporulation in H. ostrearia . The light conditions most favorable for sexual reproduction were low irradiances (<50 μmolphotons m⁻² s⁻¹) and short photoperiods (6–10 h), conditions that prevail during winter, and to a lesser extent, the higher irradiances and longer photoperiods that correspond to the spring and fall, when blooms of this organism form in the natural environment. Auxospore formation was very rare in continuous light, and maximum in presence of red radiation, while it was never observed in darkness or in radiation other than red.     

Linkage Mapping Identifies the Sex Determining Region as a Single Locus in the Pennate Diatom Seminavis robusta

The pennate diatom Seminavis robusta, characterized by an archetypical diatom life cycle including a heterothallic mating system, is emerging as a model system for studying the molecular regulation of the diatom cell and life cycle. One of its main advantages compared with other diatom model systems is that sexual crosses can be made routinely, offering unprecedented possibilities for forward genetics. To date, nothing is known about the genetic basis of sex determination in diatoms. Here, we report on the construction of mating type-specific linkage maps for S. robusta, and use them to identify a single locus sex determination system in this diatom. We identified 13 mating type plus and 15 mating type minus linkage groups obtained from the analysis of 463 AFLP markers segregating in a full-sib family, covering 963.7 and 972.2 cM, respectively. Five linkage group pairs could be identified as putative homologues. The mating type phenotype mapped as a monogenic trait, disclosing the mating type plus as the heterogametic sex. This study provides the first evidence for a genetic sex determining mechanism in a diatom.     

The sexual phase of the diatom <Emphasis Type="Italic">Pseudo-nitzschia multistriata</Emphasis>: cytological and time-lapse cinematography characterization

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.     

Mechanism of male gamete motility in araphid pennate diatoms from the genus Tabularia (Bacillariophyta)

During sexual reproduction, araphid pennate diatoms of the genus Tabularia (Kützing) D. M. Williams and Round released male gametes directly into the medium, sometimes at a considerable distance from the female gametes. This raised the question of how male gametes, suspended in water, manage to reach female ones, given that no locomotive organelles have been described in gametes of pennate diatoms. Optical microscopic investigation revealed cytoplasmic projections produced by male gametes of Tabularia tabulata (C. A. Agardh) Snoeijs and T. fasciculata (C. A. Agardh) D. M. Williams and Round. Morphology and behavior of these projections is consistent with pseudopodia, however, which specific type of pseudopodia they may be, remains inconclusive. The growth and retraction of the pseudopodia coincided with gamete motility and so we postulate that it explains the otherwise apparent random movement of male gametes. Spinning, shuffling and chaotic patterns of motility were documented. In theory, gamete mobility increases the probability of gamete encounter thus enhancing the probability of syngamy. This is the first known case where cytoplasmic projections have been described in diatom gametes, and possibly in mature gametes in general.     

MATING SYSTEM,SEXUAL REPRODUCTION,AND AUXOSPORULATION IN THE ANOMALOUS RAPHID DIATOM EUNOTIA (BACILLARIOPHYTA)1

The diatom genus Eunotia is unusual among raphid diatoms in having a raphe system consisting of two short slits that are not integrated into the primary pattern center. This and other characteristics, particularly the presence of rimoportulae, are consistent with the hypothesis that Eunotia is a basal lineage within the raphid group. We studied auxosporulation in E. bilunaris (Ehrenberg) Mills and E. tropica Hustedt for comparison with other raphid pennate diatoms and with araphid pennates; E. bilunaris was studied in parental and F1 generations. Like araphid pennates, E. bilunaris and E. tropica are heterothallic. Clones of the same mating type did not interact sexually, and intraclonal sexual reproduction was absent or very rare. Clones retained the same sex throughout the life cycle, as shown by experiments using abrupt size reduction to produce clones of similar age but different size and using subclones derived from a single initial cell within six mitotic generations. Unlike in araphid pennate diatoms, in the Eunotia species the gametes are not visibly or behaviorally differentiated. Gametogenesis is merogenous, because the gametangium formed a supernumerary cell as well as a single gametic cell, both undergoing meiosis II to form a surviving functional nucleus and a nucleus that quickly degenerated. Plasmogamy is via papillae that grew out toward each other from the ends of the gametangia to create a copulation canal. After plasmogamy, the gametes moves bodily into the copulation canal, producing an elongate zygote, which expands to form a curved sausage‐like auxospore.     

AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.     

Supramolecular organization of fucoxanthin–chlorophyll proteins in centric and pennate diatoms

Fucoxanthin–chlorophyll proteins (FCP) are the major light-harvesting proteins of diatom algae, a major contributor to marine carbon fixation. FCP complexes from representatives of centric (Cyclotella meneghiniana) and pennate (Phaeodactylum tricornutum) diatoms were prepared by sucrose gradient centrifugation and studied by means of electron microscopy followed by single particle analysis. The oligomeric FCP from a centric diatom were observed to take the form of unusual chain-like or circular shapes, a very unique supramolecular assembly for such antennas. The existence of the often disputed oligomeric form of FCP in pennate diatoms has been confirmed. Contrary to the centric diatom FCP, pennate diatom FCP oligomers are very similar to oligomeric antennas from related heterokont (Stramenopila) algae. Evolutionary aspects of the presence of novel light-harvesting protein arrangement in centric diatoms are discussed.     

SEXUALITY, INCOMPATIBILITY, SIZE VARIATION, AND PREFERENTIAL POLYANDRY IN NATURAL POPULATIONS AND CLONES OF SELLAPHORA PUPULA (BACILLARIOPHYCEAE)

The capitate and rectangular demes of the freshwater epipelic diatom Sellaphora pupula (Kütz.) Mereschk. are dioecious, the first such report for any freshwater diatom. Sexual differentiation, which is probably determined genetically, involves recognition at the cell surface as well as differences in gamete behavior (one gametangium produces an active "male" gamete, the other a passive "female" gamete). In culture, successful sexual reproduction occurs only when compatible clones are mixed. All cells of a clone behave identically in interclonal crosses, being either male or female, regardless of the stage of the life cycle, in contrast to the sequential hermaphroditism of centric diatoms. Males and females have identical frustule morphology. As in other diatoms, there is an upper size threshold for sexual reproduction, below which cells become progressively easier to sexualize. In culture, sexual interactions occur in cells much smaller than those ever seen in natural populations, so that in nature the sexual size range is effectively open. Natural populations almost always contain sexualizable cells; often, most of the cells are below the upper sexual size threshold. Male gametangia are, on average, slightly larger than females in the capitate deme, which may be produced by preferential polyandry, depleting the population of males and making them younger at mating. Rarely, selfing occurs producing zygotes, but these abort before producing initial cells. The sizes of the gametangia and initial cells are correlated but this does not invalidate the use of "cardinal points" of the life cycle in taxonomy. No interbreeding occurs between the rectangular and capitate demes. However, when males of one deme are mixed with females of the other, there is a stimulation of activity, as during the early stages of pairing in compatible intrademic crosses.     

Diatom elemental and morphological changes in response to iron limitation: a brief review with potential paleoceanographic applications

Diatoms are a major group of phytoplankton that account for approximately 40% of the ocean carbon fixation and the vast majority of biogenic silica production through the construction of their cell walls (termed frustules). These frustules accumulate and are partially preserved in the ocean sediments. Diatom growth and nutrient utilization in high‐nitrate, low‐chlorophyll regions of the world’s oceans are mostly regulated by iron availability. Diatoms acclimate to iron limitation by decreasing cell size. The associated increase in surface area‐to‐volume ratio and decrease in diffusive boundary layer thickness may improve nutrient uptake kinetics. In parallel, cellular silicon (Si) contents are elevated in iron‐limited diatoms relative to nitrogen (N) and carbon (C). Variations in degree of silicification and nutritional requirements of iron‐limited diatoms have been hypothesized to account for higher cellular Si and/or lower cellular N and C, respectively. However, in some diatoms, frustule silicification does not significantly change when cells are iron‐limited. Instead, changes in the Si‐containing valve surface area relative to volume within these diatoms is hypothesized to be responsible for the variations in the cellular Si : N and Si : C ratios. In particular, some examined iron‐limited pennate diatoms have reduced widths relative to their lengths (i.e. lower length‐normalized widths, LNW) compared to iron‐replete cells. In the pennate diatom Fragilariopsis kerguelensis, the mean LNWs of valves preserved in sediments throughout the Southern Ocean (a well‐characterized iron‐limited region) is positively correlated with satellite‐derived, climatological net primary productivity in the overlying waters. Because of the specific morphological changes in pennate diatom frustules in response to iron availability, the valve morphometerics (e.g. LNWs) can potentially be used as a diagnostic tool for iron‐limited diatom growth and relative changes in the Si : N (and Si : C) ratios in extant diatom assemblages as well as those preserved in the sediments.     

Effect of culture density on sexual reproduction of <Emphasis Type="Italic">Ardissonea crystallina</Emphasis> (Bacillariophyta) inhabiting the Black Sea

One of the key factors that affect the sexual reproduction of diatoms is the cell concentration in the mating experiments. The concentration of pheromones, which probably initiate gametogenesis in the mixture of cells of opposite sexes, depends on the culture density. The influence of the cell concentration and inoculation pattern on the sexual reproduction has been studied in the experiments with the taxonomically important marine diatom Ardissonea crystallina (C. Agardh) Grunow. Several clones have been isolated from samples collected near Sevastopol (Black Sea). The cell concentration that is most favorable for the species reproduction has been estimated. A low initial density may also increase the time required to start heterothallic sexual reproduction. The optimum cell concentration that is most favorable for the species reproduction has been estimated. Larger volume of the medium allowed reproduction at higher cell concentration. If the initial concentration of cells was greater than the optimal density, reproduction often did not occur, probably due to the cell metabolism products accumulated in the culture.     

Seasonal changes on planktonic diatom communities along an inshore-offshore gradient in the Gulf of Gabes (Tunisia)

We investigated the composition, biomass and cell size dynamics of the marine planktonic diatom species along a coastal-open sea gradient in relation to the hydrological characteristics, during four oceanographic cruises in July 2005, May–June 2006, September 2006 and March 2007 in the Gulf of Gabes. The study of the marine planktonic diatoms throughout the sampling period showed the presence of 40 different species belonging to 22 pennate and 18 centric diatom species. Centric diatoms were more abundant than the pennate ones; 56% and 44% of total diatom abundance, respectively. Diatoms were very abundant, representing about 60% of the total phytoplankton abundance, with an exception in July 2005 (19%) during which Dictyochophyceae were the most dominant group (41% of the total phytoplankton abundance). Diatoms, which were dominant in the coastal samples, mainly proliferate in the semi-mixing conditions (May–June 2006), whereas they declined in the offshore area, most likely due to silicate shortage. In this period, in spite of the high abundance of diatom planktonic cells, only diatom biomass was correlated with silica amount, proving that biomass was a better ecological bio-indicator than abundance. The results suggest that the marine planktonic diatoms taxa were generally adapted to specific hydrological structure. In fact, the dominance index of diatoms showed that the biodiversity of diatoms increased gradually along the coastal-open sea gradient except in May–June 2006 during which a slight decrease along the inshore towards offshore areas was observed. This index increase depended on a coastal-open sea distance during thermal stratification (July 2005 and September 2006) and mixing periods (March 2007).     

The dynamics of sexual phase in the marine diatom Pseudo‐nitzschia multistriata (Bacillariophyceae)

Sexual reproduction represents a fundamental phase in the life cycle of diatoms, linked to both the production of genotypic diversity and the formation of large‐sized initial cells. Only cells below a certain size threshold can be sexualized, but various environmental factors can modulate the success of sexual reproduction. We investigated the role of cell density and physiological conditions of parental strains in affecting the success and timing of sexual reproduction in the marine heterothallic diatom Pseudo‐nitzschia multistriata. We also studied the dynamics of the sexual phase in still conditions allowing cell sedimentation and in gently mixed conditions that keep cells in suspension. Our results showed that successful sexual reproduction can only be achieved when crossing parental strains in the exponential growth phase. Evidence was provided for the fact that sexual reproduction is a density‐dependent event and requires a threshold cell concentration to start, although this might vary considerably amongst strains. Moreover, the onset of the sexual phase was coupled to a marked reduction in growth of the vegetative parental cells. The crosses carried out in physically mixed conditions produced a significantly reduced number of sexual stages as compared to crosses in still conditions, showing that mixing impairs sexualization. The results of our experiments suggest that the signaling that triggers the sexual phase is favored when cells can accumulate, reducing the distance between them and facilitating contacts and/or the perception of chemical cues. Information on the progression of the sexual phase in laboratory conditions help understanding the conditions at which sex occurs in the natural environment.     

THE EVOLUTION OF ELONGATE SHAPE IN DIATOMS1

Diatoms have been classified historically as either centric or pennate based on a number of features, cell outline foremost among them. The consensus among nearly every estimate of the diatom phylogeny is that the traditional pennate diatoms (Pennales) constitute a well‐supported clade, whereas centric diatoms do not. The problem with the centric–pennate classification was highlighted by some recent analyses concerning the phylogenetic position of Toxarium, whereby it was concluded that this “centric” diatom independently evolved several pennate‐like characters including an elongate, pennate‐like cell outline. We performed several phylogenetic analyses to test the hypothesis that Toxarium evolved its elongate shape independently from Pennales. First, we reanalyzed the original data set used to infer the phylogenetic position of Toxarium and found that a more thorough heuristic search was necessary to find the optimal tree. Second, we aligned 181 diatom and eight outgroup SSU rDNA sequences to maximize the juxtapositioning of similar primary and secondary structure of the 18S rRNA molecule over a much broader sampling of diatoms. We then performed a number of phylogenetic analyses purposely based on disparate sets of assumptions and found that none of these analyses supported the conclusion that Toxarium acquired its pennate‐like outline independently from Pennales. Our results suggest that elongate outline is congruent with SSU rDNA data and may be synapomorphic for a larger, more inclusive clade than the traditional Pennales.     

Apomixis in Achnanthes (Bacillariophyceae); development of a model system for diatom reproductive biology

The availability of extensive experimental data and remarkable intra- and interspecific variation in breeding behaviour make Achnanthes Bory sensu stricto an especially good model for studying the reproductive and population biology of pennate diatoms. In most Achnanthes species studied, auxospore formation is accompanied by biparental sexual reproduction, but we found uniparental auxosporulation in Achnanthes cf. subsessilis. Auxosporulation appears to be apomictic and follows contraction of the contents of unpaired cells and then a mitotic division, which is normally acytokinetic: one nucleus aborts before the cell develops into an auxospore. Rarely, both daughter nuclei survive and cytokinesis produces two auxospores (two auxospores per mother cell is highly unusual in pennate diatoms); one may abort. Expansion of auxospores is not accompanied by deposition of a transverse perizonium, but a longitudinal perizonium is produced and consists of a wide central strip (structurally similar to the araphid valve) and at least one narrow lateral strip. This newly discovered asexual lineage in Achnanthes is discussed in relation to other reproductive systems found in the genus, and also in relation to the ‘sex clock’ hypothesis concerning the adaptive significance of the diatom life cycle. Brief information on chloroplast division and nuclear dynamics over the cell cycle is also presented.     

A rapidly deposited pennate diatom ooze in Upper Miocene-Lower Pliocene sediment beneath the North Pacific polar front

Rapidly deposited Thalassionema-Thalassiothrix pennate diatom oozes previously have been described in Upper Miocene-Lower Pliocene sediment beneath the frontal boundary of the eastern equatorial Pacific. Here we document a new occurrence of Thalassionema-Thalassiothrix ooze in Upper Miocene-Lower Pliocene sediment beneath the frontal boundary of the subarctic North Pacific. The ooze is a 6 m interval of siliceous sediment at Ocean Drilling Program (ODP) sites 885/886 that was rapidly deposited between approximately 5.0 and 5.9 Ma. Bulk sediment in this interval may contain greater than 85% pennate diatom tests. There are also abundant laminae and pockets that are composed entirely of Thalassionema and Thalassiothrix diatoms. The presence of a rapidly deposited ooze dominated by pennate diatoms indicates unusual past conditions in the overlying surface waters. Time coincident deposition of such oozes at two distinct frontal boundary locations of the Pacific suggests that the unusual surface water conditions were causally linked to large-scale oceanographic change. This same oceanographic change most likely involved (1) addition of nutrients to the ocean, or (2) redistribution of nutrients within the ocean. The occurrence and origin of pennate diatom oozes may be a key component to an integrative understanding of late Neogene paleoceanography and biogeochemical cycling.     

White Cells Facilitate Opposite- and Same-Sex Mating of Opaque Cells in Candida albicans

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTL a) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.