Sexual auxosporulation of the marine diatom Navicula directa var. directa

Sexual auxosporulation was observed in a mixed culture of two strains of Navicula directa var. directa. Two gametes did not re‐arrange in their gametangium and each adhered to the inner surfaces of the gametangial theca. Each of the two gametes of one gametangium fused with a gamete of the other gametangium iso‐gamously. As a result, two zygotes and hence two auxo‐spores were produced per paired gametangia. As the gametangial thecae kept close to the gametes during fusion, the zygote became associated with two different thecae. The presence of type IB2a of Geitler's (1973) system was confirmed by the present observations.     

Patterns of sexual reproduction in diatoms

Sexual reproduction takes many forms within the diatoms. The variation has been classified by several authors, but in most cases the distinctions between their main categories have depended on the number of gametes produced per gametangium (and thus on how many zygotes per pair of copulating cells), and upon whether fusion is oogamous, anisogamous or isogamous. These classifications are not themselves an adequate basis for taxonomic comparison, which should be based on individual characteristics of the sexual process. Diatoms seem to be primitively oogamous. In araphid pennate diatoms and some raphid diatoms the gametes and gametangia are morphologically alike but physiologically distinct; one gametangium produces active gametes and the other passive ones. This may be the primitive condition in pennate diatoms, providing a link to the oogamy of centrics via the morphological anisogamy of Rhabdonema Kütz.     

SEXUAL REPRODUCTION IN NAVICULA CRYPTOCEPHALA (BACILLARIOPHYCEAE)1

Homothallic sexual reproduction and auxosporulation were studied in monoclonal cultures and seminatural populations of the freshwater epipelic diatom Navicula cryptocephala Kütz. Gametangia paired via the girdle, one gamete was formed per gametangium (and hence one zygote per pair of gametangia), and gamete fusion took place without the formation of any copulation envelope or copulation canal. Superfluous nuclei from meiosis survived unusually long, so that gametes and young zygotes were probably functionally polyploid; later, all but two haploid nuclei degenerated. Expanded auxospores had a swollen center, but during formation of the initial valves, the auxospore contracted away from the perizonium to produce linear‐lanceolate valves. The pattern of reproductive behavior found in N. cryptocephala can be classified as type IIA2a auxosporulation in Geitler's system. The same type of zygote and auxospore formation seen in clonal cultures was observed in seminatural material from four lakes in Scotland and the Czech Republic. Variation in nuclear structure and auxosporulation in the N. cryptocephala species complex is discussed, as is the evolution of type II auxosporulation (one zygote per pair of gametangia) from type I auxosporulation (two zygotes per pair). The penalty of smaller numbers of zygote produced in type II may be outweighed by formation of larger auxospores (prolonging the vegetative phase) or more vigorous auxospores. The variation present among members of the N. cryptocephala complex indicates that biogeographical analyses based on use of the name N. cryptocephala, as performed recently to support the ubiquity hypothesis of protist distributions, are almost meaningless.     

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.     

MASS SEXUAL REPRODUCTION IN THE TOXIGENIC DIATOMS PSEUDO‐NITZSCHIA AUSTRALIS AND P. PUNGENS (BACILLARIOPHYCEAE) ON THE WASHINGTON COAST,USA1

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)₄:NO₃ ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L^?1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g^?1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.     

The Life History of Acetabularia mobii Solms-Laubach

Acetabularia mobii Solms-Laubach is a perennial and littoralalga along the coast of Karachi. Plants bear 15–18 gametangiawhich are strongly calcined. The contents of gametangia aretransformed into 64–85 thick-walled multinucleate sphericalcysts which produce anisogamous biflagellate gametes. Micro-and macro-gametes are produced in two different cysts of thesame gametangium. The germinating zygotes produce directly erectcylindrical tube-like structures which ultimately form adultplants.     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole     

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

 The reproductive strategy of a marine alga with a heteromorphic biphasic life cycle was studied by analyzing various sexual reproductive characters in light of the evolution of anisogamy. Gametophytes of Monostroma angicava were dioecious and their gametes were slightly anisogamous. Volume of gametangium, density of gametangia and area of mature gametangial parts on each gametophyte did not differ from male to female. Therefore, the reproductive biomass investment for gamete production was considered to be the same for each sex. Anisogamy in this alga appeared to be derived from the difference in the number of cell divisions during gametogenesis, because the majority of male gametangia each produced 64 (2⁶) gametes and the female produced 32 (2⁵) gametes. This corresponded with measurements of cell size in male and female gametes. Further, the sex ratio was 1:1 for sexually mature plants sampled at Charatsunai. Therefore, it was suggested that in the field twice as many male gametes are released as female gametes. Liberated gametes of both sexes showed positive phototaxis. The swimming velocity of freshly liberated male gametes was a little higher than that of female gametes. Male gametes had the potential to swim for ca. 72 h and female gametes for ca. 84 h. The difference in gamete motility between the two sexes seemed to be related to cell size. Planozygotes were negatively phototactic and swam more rapidly than gametes of either sex. Received: 5 March 1997 / Revision accepted: 18 July 1997     

SEXUAL REPRODUCTIO IN CODIUM FRAGILE SSP. TOMENTOSOIDES (CHLOROPHYCEAE) FROM THE NORTHEAST COAST OF NORTH AMERICA1

Male and female gametes were formed within the same gametangiurn on reproductive plants of Codium fragile (Sur.) Hariot ssp. tomentosoides (Van Goor) Silva growing off Appledore Island in the Gulf of Maine, USA. The female gametes were approximately twice the diameter of male gametes and outnumbered male gametes in the same gametangium by approximately six times. Fusion appears to require gametes from different gametangia if not different plants.     

A Virus Infection in the Marine Brown Alga Ectocarpus siliculosus (Phaeophyceae)

Laboratory cultures of Ectocarpus siliculosus originating from New Zealand showed a defect in gametangium formation. Nuclear divisions in gametangium initials are not followed by cell wall formation. In the resulting multinucleate cells nuclear DNA increases dramatically, and nuclear membranes disintegrate. Eventually, the entire structure is filled with hexagonal particles of approximately 130 nm diameter. These were isolated and shown by EM to consist of a dense core surrounded by a 3-layered shell. They are released into the culture medium when the host cells burst. Ectocarpus gametes from healthy cultures could be infected by these particles. The resulting partheno-sporophytes developed pathological symptoms, suggesting that the particles are viruses. The expression of the defect is temperature dependent. At 10°C all gametangia are abnormal, while between 15 and 20 °C defective and normal gametangia and gametes are formed on the same plant. Partheno-sporophytes developing from such gametes carry the viral particles expressed in deformed unilocular and plurilocular sporangia.     

GAMETOGENESIS AND GAMETE RELEASE OF ULVA MUTABILIS AND ULVA LACTUCA (CHLOROPHYTA): REGULATORY EFFECTS AND CHEMICAL CHARACTERIZATION OF THE “SWARMING INHIBITOR”1

Gametophytes of Ulva mutabilis Føyn and Ulva lactuca L. were artificially induced to form gametangia by removal of sporulation inhibitors. After this treatment, U. mutabilis gametes were ready for swarming on the third morning after induction, while U. lactuca gametangia needed 1–2 d longer for maturation. Release of gametes of U. lactuca was dependent solely upon exposure to the first light in the morning. Gametangia of U. mutabilis, however, also required sufficient dilution of the swarming inhibitor (SWI). SWI was excreted transiently by both Ulva species early during gametogenesis. While the SWI concentration in U. mutabilis medium remained above the inhibitory concentration until the gametangia were mature, the concentration of U. lactuca‐SWI dropped rapidly below this level. In the presence of sufficient SWI, mature gametes of U. mutabilis remained motionless within the gametangia despite light and open exit pores. However, using SEM, an additional seal was detected within these pores, which probably prevented premature swarming until dilution of SWI and exposure to light. Observations by time lapse microscopy and experiments with the myosin kinase inhibitor BDM suggest that the gametes may be either extruded by the gametangium or leave the exit pore by active gliding motion, driven by a myosin‐like motor protein. The SWIs were purified from both Ulva species, and mass spectral analysis showed their molecular masses (292 Da) were identical.     

Preferential digestion of chloroplast DNA in male gametangia during the late stage of gametogenesis in the anisogamous algaBryopsis maxima

Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).     

THE ULTRASTRUCTURE OF DERBESIA TENUISSIMA (DE NOTARIS) CROUAN. I. ORGANIZATION OF THE GAMETOPHYTE PROTOPLAST,GAMETANGIUM, AND GAMETANGIAL PORE1,2,3

The fine structure of vegetative and reproductive gametophytes of Derbesia tenuissima is described. Development of the gametangium and release of the gametes progress as follows: (1) In initial stages of gametangium formation, prior to 24 hr before gamete release, there is an accumulation and proliferation of nuclei, chloroplasts, and other organelles. (2) This is followed by separation of the gametangium from the rest of the plant by a gametangial membrane; segregation of organelles into gametes has begun by 12 hr before release and the process is completed by 2.5 hr before release. (3) Enzymatic wall dissolution of the pore area occurs between 2.5 and, 12 hr before normal lights-on time. (4) The release mechanism appears to be an instantaneous light-induced increase in lurgor pressure rupturing the weakened pore area, of the wall and causing a forcible expulsion of the gametes. (5) Following release, the pore is sealed by organellar debris and the gametangial membrane. Additional wall layers are presumed to be laid down internal to the plugged pore by the vegetative protoplasm which migrates into the area.     

Sexual reproduction in British Ectocarpus Siliculosus (Phaeophyta)

One male and one female gametophyte of Ectocarpus siliculosus have been detected in a sample of 19 plants taken from the Isle of Man. Fertilization takes place between gametes formed in plurilocular gametangia. Clear cross-fertility exists with Mediterranean strains. This result confirms the notion that the concept of the gametic character of meiospores in Ectocarpus should be abandoned.     

SEXUAL REPRODUCTION,MATING SYSTEM,AND PROTOPLAST DYNAMICS OF SEMINAVIS (BACILLARIOPHYCEAE)1

Cell division, the mating system, and auxosporulation were studied in the marine epipelic diatom Seminavis cf. robusta Danielidis & D. G. Mann. The interphase protoplast contains two girdle‐appressed chloroplasts, each with an elongate bar‐like pyrenoid, and also a central nucleus, located in a bridge between two vacuoles. Before cell division, the chloroplasts divide transversely and translocate onto the valves. The nucleus relocates to the ventral side for mitosis. After cytokinesis and valve formation, the chloroplasts move back to the girdle, showing a constant clockwise movement relative to the epitheca of the daughter cell. Seminavis cf. robusta is dioecious, and sexual reproduction is possible once cells are less than 50 μm. In crosses of compatible clones, gametangia pair laterally, without the formation of a copulation envelope, and produce two gametes apiece. The intensity of sexualization increases as cells reduce further in size below the 50‐μm threshold. At plasmogamy, the gametangia dehisce fully and the gametes, which were morphologically and behaviorally isogamous, fuse in the space between the gametangial thecae. The auxospore forms a transverse and longitudinal perizonium. After expansion is complete, there is an unequal contraction of the protoplast within the perizonium, creating the asymmetrical shape of the vegetative cell. Apart from this last feature, almost all characteristics exhibited by the live cell and auxospores of Seminavis agree with what is found in Navicula sensu stricto, supporting the classification of both in the Naviculaceae. Haploid parthenogenesis and polyploid auxospores were found, lending support to the view that change in ploidy may be a significant mechanism in diatom evolution.     

Variation in the sexual behaviour of Achnanthes longipes (Bacillariophyta). III. Progeny of crosses between monoecious and unisexual clones

The allogamous raphid diatom Achnanthes longipes C. A. Agardh possesses a complex breeding system involving interactions between three types of clone: monoecious, unisexual and bisexual. Previous studies showed that these three types can be crossed with each other, with a tendency for sexual characteristics to be inherited: inbred monoecious lineages gave rise to monoecious or, very rarely, to bisexual clones, while inbred unisexual lineages yielded unisexual and bisexual clones. The current paper reports on the progeny of crosses between monoecious and unisexual clones and their inbred offspring. All three types of clone appeared in the F₁ and F₂, although unisexual clones of opposite sex to the parental clone were not found. Inbreeding depression was observed and also a tendency for ‘normal’ auxosporulation (producing two auxospores per pair of gametangia) to be replaced by ‘reduced’ or ‘intermediate’ auxosporulation (producing one auxospore per pair). In addition, patterns of incompatibility were observed that were not seen during earlier studies of clones isolated directly from nature. These included the inability of some F₁ clones to mate with each other, in spite of compatibility with all other clones examined (unisexual, bisexual and monoecious).     

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.     

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.     

Gamete discharge by Bryopsis plumosa (Codiales, Chlorophyta) induced by blue and UV-A light

Gamete discharge by the coenocytic green alga Bryopsis plumosa (Hudson) C. Agardh is induced by light. The mature male gametangia consist of a mass of quiescent male gametes and a large central vacuole. Within a few minutes after the onset of irradiation, breakdown of the tonoplast of the central vacuole and initiation of gamete motility occur simultaneously. This is followed by a forced discharge of moving gametes through a hole ruptured at the subapical region of the gametangium. The action spectrum for the light-induced gamete discharge was determined from a series of fluence-response curves. This action spectrum, having two major maxima at 370 and 450 nm, indicates the involvement of a blue light/UV-A-absorbing photoreceptor previously described as ‘cryptochrome’.