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.     

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.     

SEXUAL REPRODUCTION IN STEPHANODISCUS NIAGARAE (BACILLARIOPHYTA)1

We observed sexual reproduction in a clonal culture of Stephanodiscus niagarae Ehrenb. and used light and scanning electron microscopy to absent flagellated male cells, auxospore growth, initial valve structure and production, and subsequent daughter cell division. Free auxospores were spherical and nonsiliceous throughout growth, producing hemispherical initial valves devoid of spines and with nonfasciculate striae. Pregametangial cells averaged 43% of the diameter of the daughter cell population and were 1/9 the biovolume of initial, cells. This paper is the first confirmed report of sexual reproduction in S. niagarae, although it appears that specimens of Actinocyclus niagarae H. L. Smith, described from Lake Erie in 1878, are actually initial valves of S. niagarae.     

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.     

OOGAMOUS REPRODUCTION,WITH TWO‐STEP AUXOSPORULATION,IN THE CENTRIC DIATOM THALASSIOSIRA PUNCTIGERA (BACILLARIOPHYTA)1

Thalassiosira species are common components of marine planktonic communities worldwide and are used intensively as model experimental organisms. However, data on life cycles and sexuality within the genus are fragmentary. A clone of the cosmopolitan marine diatom Thalassiosira punctigera Cleve emend. Hasle was isolated from the North Sea and oogamous sexual reproduction was observed in culture. Cells approximately 45 μm and smaller became sexualized. Oogonia were produced preferentially and spermatogenesis was infrequent. Unfertilized oogonia always aborted and their development was apparently arrested at prophase of meiosis I. Further progression through meiosis and auxospore formation occurred only after a sperm had penetrated into the oocyte. Many cells of the new large‐celled generation (approximately 90–120 μm in size) immediately became sexualized again but only oogonia were produced. A few of the large oogonia became auxospores and produced initial cells 132–153 μm in diameter. The second step of auxosporulation probably involved fertilization of large‐celled oocytes by the sperm of the small‐celled spermatogonangia that were still present in the culture. An F₁ clone obtained after selfing within the small‐celled auxosporulation size range was investigated. Like the parent clone, the F₁ clone was homothallic but no auxosporulation was observed: spermatogonangia were unable to produce viable sperm, apparently because of inbreeding depression. Aggregation and interaction of oogonia were documented, and may be relevant for understanding the mechanisms of signaling and recognition between sexualized cells and the evolution of sexuality in pennate diatoms.     

AUXOSPORE FORMATION BY THE SILICA‐SINKING,OCEANIC DIATOM FRAGILARIOPSIS KERGUELENSIS (BACILLARIOPHYCEAE)1

Size restoration by the auxospore that develops from the zygote is a crucial stage in diatom life cycles. However, information on sexual events in pelagic diatom species is very limited. We report for the first time auxospore formation by the pennate diatom Fragilariopsis kerguelensis (O'Hara) Hustedt during an iron‐induced bloom in the Southern Ocean (EIFEX, European Iron Fertilization EXperiment). Auxospores of F. kerguelensis resembled those described for Pseudo‐nitzschia species. The auxospore was characterized by an outer coating, the perizonium; two caps, one at each distal end; and four chloroplasts, one at each end and two in the central part. Different stages of auxospore elongation were recorded, with a length of 24–91 μm, but only the largest auxospores contained the initial cell, whose apical axis ranged between 76 and 90 μm. Gametangial cell walls were often attached to the auxospores and ranged from 10 to 31 μm in length. Auxospore abundances were consistently higher in the fertilized patch, where an increase in the F. kerguelensis population was observed, as compared with surrounding waters.     

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.     

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.     

PERIZONIUM AND INITIAL VALVE FORMATION IN THE DIATOM NAVICULA CUSPIDATA (BACILLARIOPHYCEAE)1

This paper describes the perizonium and initial valve formation in Navicula cuspidata Kütz., based on light microscope (LM) and scanning electron microscope (SEM) observations. The perizonium consists of concentric over-lapping bands, laid down sequentially at the tips of the expanding biconical auxospore during its elongation. The central perizonial band has fimbriate edges and is considerably more rigid than the more distal bands. During auxospore elongation and the band secretion, the chloroplasts continuously oscillate between the two ends of the cell; this oscillation ceases once the elongation is complete. The initial valves, formed within the perizonium, are molded into the basically biconical shape of the perizonium except for a central flattening of each valve face. In contrast to the raphes in gametangial and vegetative valves which are surrounded by a smooth axial area, the raphes in initial valves lie within a raised ridge running along the apical axis of the valve. The regular pattern of apically oriented ridges on the outer surface of vegetative valves is also lacking on initial valves. Comparison of pore–pore spacing within striae of gametangial valves, initial values and post-initial valves (first division and vegetative cells) reveals that the pore–pore distance within striae is conserved at all sexual stages. However, the distance between striae is considerably larger in initial valves than in gametangial and post-initial valves. Vegetative interstriae spacing as well as the planar morphology of the valve face is regained at the first division of the initial cell. This suggests that the spacing between striae is dependent on the sexual stage of the cell during valve formation (i.e. not directly dependent on the cell size) and can be altered independently of the pore–pore spacing.     

THE NATURAL LIFE CYCLE IN WILD POPULATIONS OF DIATOMA MONILIFORMIS (BACILLARIOPHYCEAE) AND ITS DISRUPTION IN AN ABERRANT ENVIRONMENT1

We studied how size variation in populations of Diatoma moniliformis Kütz. was influenced by environmental effects on the diatom life cycle. One of the two populations sampled monthly in the northern Baltic Sea grew under natural conditions; the other population was in a cooling water discharge channel of a nuclear power plant, where the temperature and flow rate of the water were artificially higher. The life cycle was synchronous at the natural site, with sexual reproduction occurring in the winter; most of the initial cells were found in March-April. After this, a reduction in cell size occurred, and the vegetative life cycle consisted of two parts. During the first part, cell volume decreased, whereas the surface area to volume ratio increased, and during the second part of the cycle, both of these parameters decreased. No direct evidence was found for the existence of a supra-annual life cycle in D. moniliformis, as convincing modes fm large cells were lacking in the size-frequency distributions. It was concluded from extrapolations of the data that the natural life cycle of D. moniliformis probably lasts 2 or 3 years. The changes in cell proportions during the life cycle fit well with annual growth cycles of D. moniliformis at the natural site (i.e. the cells had high surface area to volume ratios during the period of optimal growth in late spring [May-June]). At the site affected by cooling water discharge, the synchronization of the natural life cycle was disrupted, but some seasonal size variation did occur. Under natural conditions, auxosporulation is probably triggered by a combination of small cell size, low water temperature (0–3° C), and rapidly increasing light intensity or daylength in late winter to early spring. When these conditions were not met (e.g. at the heated site, the required low temperature was absent), auxosporulation did not occur simultaneously. This paper also presents scanning electron photomicrographs showing the typical shape and fine structure of the initial cell of D. moniliformis. These cells are semispherical in cross section, possess a pronounced curvature along the longitudinal axis, and are bent in the perualvar plane.     

Natural populations of the Closterium ehrenbergii (Desmidiales, Chlorophyta) species complex in Nepal

Several natural populations of the Closterium ehrenbergii Meneghini ex Ralfs species complex were collected in Nepal, in October–December 1982. Water temperature and pH were also recorded. Clonal isolates from these populations were identified to one of four mating groups (H, I, J and M) by test crossing with standard mating-type strains of known mating groups. Groups H and M have smooth walled zygospores, while Groups I and J have scrobiculated zygospore walls. Several undetermined isolates were found in some population samples. In contrast to the previously reported population samples from Nepal, especially from dried soil samples, some of these populations appeared to be rather heavily loaded with mutations that are deleterious to the sexual cycle (i.e. sexual compatibility, zygospore formation and germination). By genetic analysis, a zygote maturation-defective mutation (zym) was detected. One reason for such a heavy genetic load was suggested to be that most population samples had been maintained exclusively by asexual reproduction for a long period in large lakes and nearby ponds, or left-over vegetative populations in paddy fields after other members entered into dormancy through sexual reproduction. The significance of studying such mutations at sexual gene loci is discussed in the light of speciation problems in microalgae.     

Repeated evolution of uniparental reproduction in Sellaphora (Bacillariophyceae)

Diatoms possess a remarkable life cycle in which cell size decreases slowly during vegetative cell division and then increases rapidly via special expanding cells called ‘auxospores’, which are usually formed as a result of biparental sexual reproduction. However, auxospores are sometimes produced by single unpaired cells, i.e. uniparentally. We examined the nature of uniparental auxosporulation in Sellaphora and used a two-gene dataset to study phylogenetic relationships between uniparental and biparental Sellaphora demes and species; we tested whether uniparental reproduction has evolved once or repeatedly in the genus. In at least two of the uniparental demes auxosporulation occurred through autogamy (i.e. intra-tetrad mating within an undivided cell). Maximum likelihood phylogenies indicated four lineages of uniparental Sellaphora and significance tests of alternative topologies, in which combinations of uniparental Sellaphora were constrained to be monophyletic, coupled with likelihood reconstruction of ancestral character states, led to rejection of the hypothesis that uniparental auxosporulation evolved only once in the genus. Uniparentally reproducing lineages appear to arise not infrequently in diatoms but do not persist. Two small extranuclear bodies, apparently containing DNA and lying outside the chloroplast (one close to each pole of the cell), were revealed by DAPI staining.     

THE SEXUAL AND ASEXUAL LIFE CYCLE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The sexual and asexual portions of the life cycle of Glenodiniopsis steinii were examined at both the light and scanning electron microscopic levels. Asexual reproduction by cell division occurs every 2 to 3 d under optimal conditions. Gamete formation and sexual reproduction can be induced by either nitrogen deprivation or aging of cultures. Fusion of gametes peaks about 10 hr into the light cycle and requires about 10 hr for completion. Cells remain motile during fusion but the zygote loses motility. The cell then undergoes changes that give it a thick-walled, warty appearance.     

夜光藻有性繁殖研究进展

夜光藻是全球最主要的赤潮生物之一,也是我国近海常见的浮游甲藻。根据营养方式分为异养的红色夜光藻和混合营养的绿色夜光藻,前者广泛分布于温带和亚热带近岸水域,后者仅分布于热带西太平洋、阿拉伯海、阿曼湾和红海。夜光藻的生活史包括无性繁殖和有性繁殖过程。少部分营养细胞自发转变为配子母细胞,启动了有性繁殖。每个配子母细胞可形成大量配子,具有横沟、纵沟和2根鞭毛,形态与裸甲藻接近。配子两两融合形成合子,合子不经过休眠孢囊阶段直接发育成新的营养细胞。目前,对配子母细胞形成的调控机制、合子发育的影响因素的认识还存在分歧。研究发现,营养细胞经过一定次数的二分裂后都会转变为配子母细胞,而配子的存在能够中止此过程,使营养细胞继续进行二分裂。因此,有性繁殖可能通过产生新个体对种群增长做出贡献,还可能通过释放配子维持无性繁殖,进而促进种群增长。配子在相模湾水域全年都有分布,其丰度峰值与营养细胞丰度峰值同步或提前出现,配子的大量出现可能是赤潮形成的必要条件。对有性繁殖的研究佐证了夜光藻在甲藻的系统进化中处于较为古老的地位。此外,还简单介绍了研究夜光藻有性繁殖的主要方法,回顾了国内的夜光藻研究,并对相关研究进行了展望。     

SEXUAL REPRODUCTION OF PERIDINIUM WILLEI (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium willei Huitfeld-Kass when exponentially growing cells were inoculated into nitrogen deficient medium. Small, naked vegetative cells produced by division of thecate cells acted as gametes. The zygote remained motile 13–14 days, during which time it enlarged and the theca formed became warty. Fourteen to 15 days following plasmogamy the zygote was nonmotile with the protoplast contracted. A large red oil droplet appeared and the wall thickened, becoming chitinized. Hypnozygotes with 4 nuclei were observed 7–8 wk following formation. Meiosis was inferred. The hypnozygote germinated, within 8 wk producing one post-zygotic cell retaining the red oil droplet. This cell divided within 24 h into 2 daughter cells each with a prominent red oil droplet. These daughter cells divided after 2 to 3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

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.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

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).     

Aplanogamous sexual reproduction in Carteria eugametos (Volvocales,Chlorophyta)

Morphological details of sexual reproduction in Carteria eugametos (Volvocales, Chlorophyta) were studied under controlled laboratory conditions. Protoplasts of the two pairing, flagellate cells were released from cell walls to become isogametes. Such gametes were nonmotile and soon fused to form a completely immobile zygote. The zygote then secreted a cell wall to enter the dormant period. After dark treatment, the zygote produced four, eight or 16 quadriflagellate germ cells, and a transparent vesicle enclosing all the germ cells was released from the zygote wall. This type of zygote germination and aplanogamy in C. eugametos is unique and may be related to its peculiar phylogenetic position within the Volvocales (Chlamydomonadales).