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Experimental Studies on Sexual Reproduction in Diatoms
Victor A. Chepurnov,*,{ David G. Mann,{ Koen Sabbe,* and Wim Vyverman*

*Laboratory of Protistology and Aquatic Ecology, Department of Biology,
{
Ghent University, B-9000 Ghent, Belgium Royal Botanic Garden, Edinburgh, EH3 5LR Scotland, U.K.
The diatoms are the most speciose group of algae, having global ecological significance in the carbon and silicon cycles. They are almost unique among algae in being diplontic, and sexual reproduction is an obligate stage in the life cycle of most diatom species. It is unclear which are the principal factors that have fostered the evolutionary success of diatoms, but the unique life cycle (which is correlated with a curious wall structure and cell division mechanism) and size-dependent control of sexuality must have played an important part. Progress in understanding life cycle dynamics and their interrelationships with population biology and evolution will depend on how successfully sex can be initiated and manipulated experimentally, and our review provides a foundation for such work. Relevant data are scattered in time and come mostly from non-English publications, producing a false impression of diatoms as recalcitrant with respect to sexualization. Recent advances dependent on experimental cultures include the discovery of widespread heterothallism (including some complex types of behavior) in pennate diatoms, sexual diversity among clones of centric diatoms, more flexible size restitution strategies in centric diatoms than had been suspected, and use of reproductive isolation as a criterion in diatom taxonomy. We identify unsolved problems in the life history of diatoms, including aspects of sexualization, cellcell recognition, sexual reproduction, and the development of the special expanding cell (the auxospore), which is crucial to morphogenesis in this group. Some of these problems are being addressed using modern molecular genetic tools, and progress will be facilitated when whole-genome sequences are published (e.g., for Thalassiosira pseudonana). Problems of culture maintenance and methods for manipulating the life cycle are discussed.
International Review of Cytology, Vol. 237 0074-7696/04 $35.00
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Copyright 2004, Elsevier Inc. All rights reserved.
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KEY WORDS: Auxosporulation, Bacillariophyceae, Clonal culture, Diatoms, Life cycle, Mating system, Sexual reproduction. 2004 Elsevier Inc.
I. Introduction Sexual reproduction occurs in the vast majority of eukaryotic organisms and exhibits amazing constancy in its principal cytological traits (viz. meiosis, segregation, recombination, sexual fusion, karyogamy). The biological consequence of sex is the generation of new combinations of genes by mixing genomes, or portions thereof, from diVerent individuals and without the mixing produced by sex, there would not be species as we know them (Michod and Levin, 1988a). At the same time, sex remains paradoxical, because it is a costly process (Lewis, 1987), with uncertain benets in the short term. Indeed, Michod and Levin (1988b) suggest that the origin and maintenance of sex is a big (maybe the biggest) unsolved problem in evolutionary biology (see also Otto and Lenormand, 2002). As Stearns (1987) noted, most discussions concerning the evolution and maintenance of sex concentrate on higher (multicellular) animals and plants. Sexual reproduction must also be signicant for algae and protists, however, as it is very widespread among them as well (Margulis et al., 1990; van den Hoek et al., 1995). There are a few exceptions, such as the euglenids, in which sexuality and meiosis have scarcely ever been reported (Walne and Kivic, 1990), but this could as easily reect lack of study as the truth about their occurrence. Among the eukaryotic algae, the most species-rich and productive group is the diatoms (division Bacillariophyta or class Bacillariophyceae), which probably perform 20% of all photosynthetic xation of carbon (exceeding the contribution of the rainforests) and so have a crucial role in the function ing of ecosystem Earth (e.g., Field et al., 1998; Mann, 1999; Smetacek, 1999). The diatoms are a monophyletic group of unicellular or colonial eukaryotes, almost all of them autotrophic, which belong to the heterokont lineage (Bhattacharya et al., 1992; Kooistra et al., 2003b; Medlin et al., 1993; van den Hoek et al., 1995). Their hallmark is an amazingly intricate, bipartite, siliceous cell wall called the frustule (e.g., Pickett-Heaps et al., 1990; Round et al., 1990). The fossil record and molecular data indicate that the diatoms are a geologically young group that appeared only in the Mesozoic Era (<250 Ma) (Medlin et al., 1997, 2000). Since then, they have invaded almost every aquatic or semiaquatic habitat where enough light
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penetrates to allow photosynthesis, and they have diversied into hundreds of genera and perhaps 200,000 extant species (Mann, 1999). High levels of physiological and genetic variation have been detected in populations of some planktonic diatoms (e.g., Gallagher, 1980, 1982; Gallagher and Alberte, 1985; Lewis et al., 1997; Rynearson and Armbrust, 2000, 2004; Soudek and Robinson, 1983) and this has been suggested to be at least partly responsible for the success of the group, allowing rapid adaptation to changed environmental conditions (see also Wood et al., 1987). The fact that such diversity exists, however, does not explain its origin and maintenance. To understand these, data on the frequency and nature of sexual reproduction are essential, but as yet there is not a single case in which population genetics, life cycle dynamics, and sexual reproduction have been studied together for the same sets of populations of the same species. Hence, it is impossible to test the idea that the impressive diversity and abundance of the diatom algae is in some signicant degree attributable to their rened control over sexuality (Lewis, 1984). The creativity of sex in generating genetic diversity needs no explanation here, nor does the importance of sex in allowing genomes to be purged of deleterious mutations (e.g., Otto, 2003). Most diatoms are obligately sexual organisms, and clonal lineages have limited or no ability to maintain themselves for more than a few years (e.g., Drebes, 1977a; Geitler, 1973; Mann, 1988, 1993a, 1999; Round et al., 1990). No family or genus, or even a species-rich section of a genus, is known in which all the species are asexual or parthenogenetic (Mann, 1999), and so the evolutionary diversication of diatoms has taken place predominantly within sexual lineages. In addition, one more point is remarkable: Unlike other groups of algae, diatoms are highly uniform with respect to the principal traits of their life history, and especially their sexual behaviour. Sexual reproduction in diatoms has previously been reviewed by Patrick (1954), Drebes (1977a), and more briey, Round et al. (1990). There has also been a review of sexual reproduction in the pennate diatoms by Geitler (1957) and a thought-provoking article on diatom life histories by Edlund and Stoermer (1997). Recent studies, especially experimental studies of cultures, have advanced knowledge suYciently to justify a new synthesis, especially because some previous generalizations about mating systems and the auxosporulation cycle now appear to have been premature and misleading. Knowledge of diatom sex still cannot be regarded as satisfactory, however, because only a tiny minority of species have been examined, and almost nothing is known about the molecular and genetic basis of sexuality, or the dynamics of the life cycle in nature, which are clearly relevant to ecology. One reason for slow progress is that diatoms are problematic organisms to maintain in culture; ironically, this is because of sex itself and its integral place in the diatom life cycle (Section IV.A). Apt et al. (1996) noted that a
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considerable limitation on the use of diatoms as experimental organisms and commercial resources arises from the fact that diatoms grow as diploid, vegetative cells, and no one has been successful in controlling a sexual cycle in culture. Perhaps not surprisingly, therefore, those diatoms most often used as experimental organisms have highly aberrant life cycles and sexuality (if it is present at all). However, we will try to show that the situation is not so hopeless as has sometimes been thought.
II. Sexual Reproduction in the Diatom Life Cycle Typically, the diatom life cycle comprises two principal phases: a prolonged vegetative phase lasting months to years, during which the cells divide mitotically, and a comparatively short phase that includes sexual reproduction (gametogenesis and fertilization, occupying several hours) and then a complex developmental process leading to the formation of new vegetative cells (many hours to a week or more). Kobayashi et al. (2001) found that it took at least a month for auxosporulation to be completed in their rough cultures of Arachnoidiscus. In addition, most diatom life cycles conform to some other rules, as follows, though there are important exceptions, which we will highlight elsewhere in this review. Rule 1 is that the life cycle is diplontic. Diatoms are almost unique among algae in having a diplontic life cycle (Mann, 1993a). The vegetative cells are diploid, and the only haploid cells are the gametes, which have a short life. The evidence for this is extensive and is drawn from diatoms belonging to almost all of the major lineages detected by recent molecular systematic studies. Vegetative cell division has long been known to be mitotic (see, e.g., the remarkably comprehensive study by Lauterborn, 1896; reviews by Pickett-Heaps et al., 1984, 1990; Round et al., 1990). In pennate diatoms, the nuclear processes accompanying gametogenesis were shown by Klebahn (1896) and Karsten (1899, 1912) to be classical two-step meiosis (see also Geitler, 1927, 1932; Mann and Stickle, 1989, 1995; Subrahmanyan, 1947). Later (von Stosch, 1951, 1956; von Stosch et al., 1973) showed that meiosis occurs during gametogenesis in several centric diatoms. In studies of a few hundred diatom taxa, no case has been found in which meiosis is not associated with gametogenesis, and direct counts of chromosome numbers have been made in various diatom species (reviewed in Kociolek and Stoermer, 1989), conrming diploidy. Overall, diatom meiosis exhibits no special features (Geitler, 1927; Mann and Stickle, 1995). A synaptonemal complex has been observed in Lithodesmium (Manton et al., 1969b) and Gomphonema (Drum et al., 1966). During meiotic prophase, the nucleus swells considerably and becomes more
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spherical (e.g., Mann, 1989c; Mann and Stickle, 1989, 1995). Within it, during the early stages of meiotic prophase, the chromatin forms a tight knot (synizesis) that can often be seen to move around within the nuclear envelope (nuclear cyclosis; Mann and Stickle, 1989); this presumably facilitates, or is a consequence of, chromosome pairing. At this stage, the nucleoplasm surrounding the chromatin appears watery (Drum et al., 1966; Manton et al., 1969b; Figs. 6, 10). The large size of the nucleus and the ease with which it can be seen in the living gametangia of many pennates would make these organisms useful subjects for research into the mechanics of meiosis. Few studies of the chromosome complement have been made, but it can be expected that, as more species are studied, evidence will emerge in some of translocations, inversions, and so forth (a rare example is given by Mann and Stickle, 1989). Rule 2 is that vegetative multiplication is accompanied by gradual cell size reduction. This principle is also known as the MacDonaldPtzer rule (MacDonald, 1969; Ptzer, 1971; see also Crawford, 1981; Hustedt, 1967; Pickett-Heaps et al., 1990; Round, 1972; Tomaschek, 1873), from the British and German authors who rst provided a detailed explanation of cell size change during the diatom life cycle. Size change is brought about by the unique cell structure and division pattern in diatoms. The basic structure of the diatom cell wall is amazingly uniform. Almost always (the exceptions are some diatoms that live as endosymbionts and the anomalous pennate diatom Phaeodactylum), the cell wall of diatoms is silicied. The silica components together comprise the frustule, which consists of two overlapping halves called thecae. Each theca in turn consists of a large unitary end-piece, the valve, and a side wall made of a series of strips or rings called girdle bands. The whole complement of bands associated with a particular valve is called the cingulum. Within and around the silica are organic components of various kinds (Kroger et al., 1996, Round et al., 1990). The two thecae of a single cell are unequal in size, with one being slightly larger than the other, so that the frustule has a box-and-lid structure (Round et al., 1990). The reason is as follows: Mitotic cell division is followed by cytokinesis (through cleavage) at the middle of the girdle, where the two thecae overlap. After cytokinesis, therefore, the two daughter cells lie side by side within the frustule of the parent, so that each occupies one of the thecae of the mother cell. Later, after cell separation, each daughter cell inherits the parental theca that it occupies and this forms the lid (epitheca) of its new frustule. The epitheca will not be added to in the next cell cycle, nor in any subsequent cell cycle. However, to complete its frustule, each daughter cell must manufacture a new theca. This begins while the daughter cell is still contained within the two thecae of the parent cell, before cell separation. Hence, the new valvesthe hypovalves of the new cellsare at rst contained within, and are therefore smaller than, the parts of the epithecae
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that surround them. One or more girdle bands may also be produced before the parental thecae separate; others are added later to accommodate cell growth during the cell cycle. The consequence of this cell division mechanism is that one daughter cell (the cell that inherits the hypotheca of the parent cell) is smaller than the parent, whereas the other is the same size as the parent. Hence, with repeated cell division, the average size of the population decreases. If both daughter cells have more or less equal cell cycle lengths, and if both have the same chance of survival, the cell lengths within a population will approximate a binomial distribution. However, these conditions are not always met, and the variance of cell size within a population does not increase as rapidly during size reduction as would be expected from the MacDonaldPtzer mechanism (e.g., Mann, 1988). Rule 3 is that cell size is restored through development of a specialized cell called an auxospore, and formation of auxospores results from sexual reproduction. Auxosporulation involves linkage of two important processes genetic recombination and cell size restitutioninto a single chain of events. Gametes are produced and fuse to form an auxospore, which expands. After expansion is complete, a new cell (the initial cell) is formed inside the auxospore envelope, which is roughly (there are many exceptions) twice or three times as large (linear dimensions) as the auxospore mother cell(s); the initial cell then begins vegetative multiplication. During auxospore formation, the cell walls of the gamete-producing cells (gametangia) are discarded, and so the auxospore must develop the characteristic shape of the vegetative cells anew before the initial cells are formed, although further shape changes often occur after initial cell formation, as the new large vegetative cells grow and divide. Soon after plasmogamy, the auxospore begins to develop an organic wall, or more commonly, an organic wall that has siliceous components embedded within it or beneath it. For any nal shape other than an ellipsoid, morphogenesis involves local hardening of the auxospore wall during expansion, restricting growth to areas that remain plastic (Mann, 1994c). The haploid nuclei do not always fuse immediately after plasmogamy, which is why, strictly speaking, the developing auxospore cannot always be regarded as an expanding zygote (see Round et al., 1990). Rule 4 is that cells that fail to undergo sexual reproduction and auxosporulation continue dividing mitotically until they become critically small and nally die. This was rst noted by Geitler (1932) in experimental cultures of pennate diatoms. Rule 5 states that the capacity of cells to become sexualized is size dependent: Only comparatively small cells can be triggered to switch from mitotic cycles to meiosis. The rst clear demonstration of this rule was by Geitler (1932), although it had been suspected earlier (e.g., Bachmann, 1904; Ptzer, 1871), and further important studies were made by von Stosch (1965). It was
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shown that, after auxosporulation, clones cannot be induced to become sexual until the cells have declined somewhat in size. The extent of this refractory period varies between diVerent diatom species, and the upper threshold for sexual induction can range from 30% to 75% of the maximal size of the initial cells; however, for more than half of the species examined, the threshold is at 45% to 55% (reviewed by Davidovich, 2001). The lower threshold of sexual size range may coincide with the critical minimal size, in which case the sexual size range is termed open. The sexual size range is termed closed when the cells lose the capability to reproduce sexually before reaching the critical minimal size (see Drebes, 1977a). The maximal size of initial cells, together with the maximal size of cells capable of sexual reproduction and the critical minimal size, are fairly strict, species-specic characteristics; they are referred to as cardinal points in the diatom life cycle (Drebes, 1977a; Geitler, 1932; Mann and Chepurnov, 2004). Rule 6 states that the immediate products of sexual reproduction, the auxospore and initial cell, are not dormant stages. The term auxospore is misleading in that it implies that the auxospore is a resting stage or a cell specialized for dispersal. Neither is true, although a resting spore is always formed by the expanded auxospore in Leptocylindrus danicus (French and Hargraves, 1985). In contrast, sexual reproduction is often the precursor of dispersal or dormant stages in other algae (van den Hoek et al., 1995). These six rules summarize the basic plan of the diatom life cycle and the place of sex in it, and there is now overwhelming evidence that the rules operate in all of the main lineages of diatoms revealed in molecular phylogenies (e.g., Kooistra et al., 2003b). However, in some aspects of the life cycle, particularly the mechanisms of gametogenesis and sexual reproduction, diatoms exhibit immense variation. Our knowledge of relationships among diatom genera is still relatively poor, because many have yet to be incorporated into molecular systematic studies, and because the molecular phylogenies that we have, though they are based on genes (e.g., 18S rDNA and rbcL) that have proved very useful in revealing relationships in other major groups (e.g., angiosperms), often reveal a disappointingly high level of homoplasy. Nevertheless, it is already possible to see that the patterns of sexual reproduction in diatoms do correlate well in some cases with well-supported clades. At the highest levels of classication, two major groups have traditionally been recognized: centric diatoms and pennate diatoms. These two groups have been separated since the nineteenth century (Schutt, 1896), when researchers were dependent on evidence from light microscopy, particularly cell shape and the overall pattern of ornamentation of the frustule. Centric diatoms have a radial pattern of markings on the valves, and the valve outline is often circular; more rarely it is multipolar or bipolar. Careful recent
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analyses reveal that the ribs and pores of centric diatoms radiate from a ring (annulus) (Mann, 1984b; Pickett-Heaps et al., 1990; Round et al., 1990), and the presence of this structure is probably signicant in relation to pattern formation (Mann, 1984b). The valves of most pennate diatoms are bipolar and boat-shaped, with ribs and pores extending out from an elongate bar-like pattern center (sternum), as in a feather (penna/pinna in Latin). Pennate diatoms, in turn, are subdivided into two distinct groups: raphid pennates, which possess one or two slits (comprising the raphe system) that are either integrated within the sternum or associated with it, and araphid pennates, which lack a raphe. Electron microscopy (e.g., Round et al., 1990) has conrmed the reality of these three groups. However, molecular phylogenies (e.g., Kooistra et al., 2003a,b; Medlin et al., 1996, 2000) show clearly that the relationship between the three groups is hierarchical, with the centrics being paraphyletic with respect to the pennates, and the araphid pennates being paraphyletic with respect to the raphid pennates. This is, in fact, no surprise, being inherent in premolecular discussions of phylogeny (e.g., Mann, 1984b; Simonsen, 1979). Thus, the centric and araphid pennate groups are grades, with only the raphid diatoms being strictly monophyletic (holophyletic sensu; Mayr and Ashlock, 1991). However, for our discussion, the three traditional groups will be retained, because each is fairly distinctive with respect to sexual behavior.
A. Centric Diatoms: Oogamy Studies of morphology, the fossil record, and molecular data leave little doubt that the centric group is the most ancient and that it is ancestral to the pennates. Auxospores (then referred to as sporangia) were rst reported from centric diatoms in the mid-nineteenth century (Luders, 1862; Smith, 1856; Thwaites, 1847), but the nature of auxosporulation remained unclear until 1950. At rst, the preferred opinion was that the production of auxospores in Centric Diatoms is a comparatively simple process and is not dependent on any association of individuals in sexual reproduction, consisting essentially in a rejuvenescence of the protoplast (Fritsch, 1935). Essential to our understanding of the true nature of centric auxosporulation was the discovery of meiosis in the auxosporulating cells of Chaetoceros spp. (Persidsky, 1932) and in the mysterious microspores reported by early workers (Hofker, 1928; Schmidt, 1927), which are stages in male gamete formation. Geitler (1932, pp. 1112) supported Wents view (1925) that oogamous fertilization occurred in the marine planktonic diatom Chaetoceros and suggested that auxosporulation in centric diatoms might be associated with oogamous sexual reproduction. However, it took nearly 20 years before the
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WentGeitler hypothesis was conrmed by von Stosch (1950, 1951), who documented oogamous auxosporulation in Melosira varians. The next 25 years saw the conrmation of oogamy in cultures of various centrics by von Stosch and colleagues (reviewed by Drebes, 1977a). Oogamy in centric diatoms always involves fertilization of a large, nonmotile egg by a small, anteriorly uniagellate sperm (which has an unusual 9 0 internal structure: Jensen et al., 2003; Manton and von Stosch, 1966). However, auxospores can also be formed through automixis or asexually (Sections II.D and II.E). Egg formation (oogenesis) is comparatively simple. Cells triggered to become female gametangia (oogonia) can often be distinguished visually at an early stage of development by their enlarged nucleus (in prophase of meiosis I), deeper pigmentation (increased number or size of plastids), and elongation of the girdle region. Two-step meiosis follows. In most centric diatoms investigated, meiosis I proceeds without cytokinesis, and one nucleus then aborts. Furthermore, in all species studied so far, meiosis II is acytokinetic and leads to abortion of one of the haploid products. Hence, in most species, only one egg is produced. However, meiosis I is accompanied by equal cytokinesis in Odontella mobiliensis and O. granulata (Drebes, 1974; von Stosch, 1954, 1956; in all cases as Biddulphia) and Attheya decora (Drebes, 1977b), and meiosis therefore results in the production of two eggs per oogonium. In Odontella rhombus and Cerataulina smithii, there is a cytokinesis at meiosis I, but it is unequal and the small cell aborts (von Stosch, 1956); only the large cell survives to form an egg. Species in which there is no cytokinesis at meiosis I include species of Melosira (Idei and Chihara, 1992; Mizuno, 1977; von Stosch, 1951, 1958a), Cyclotella (Geitler, 1952a), Stephanopyxis (Drebes, 1966; von Stosch and Drebes, 1964), Coscinodiscus (Drebes, 1974; Schmid, 1995), Skeletonema (Migita, 1967) and Chaetoceros (von Stosch et al., 1973). Spermatogenesis is more variable and apparently more complex than oogenesis. It usually begins with a series of special diVerentiating mitotic divisions, during which the cells do not expand as they do during the mitotic cell cycle (Fig. 1AE; see also Drebes, 1977a; von Stosch and Drebes, 1964). These depauperating [Drebes, 1977b, p. 171; von Stosch and Drebes (1964) wrongly used the word depauperizing] or impoverishing divisions immediately precede the formation of the cells (spermatogonia) that undergo meiosis to produce haploid gametes. In Coscinodiscus granii, C. wailesii, and Odontella regia, A.-M. Schmid (1995) reported a formation of specic four-cell chains (tetrads) and considered these prestages of the depauperating divisions. The diVerentiating mitoses result in progressive reduction in cell size and plastid number per cell. In some diatoms, new siliceous thecae are deposited after some or all depauperating divisions, but the thecae are reduced and appear rudimentary (e.g., in
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FIG. 1 Planktonic centric diatoms from North Sea, monoclonal cultures. (AC) Coscinodiscus granii. (A) Two sibling vegetative cells, after mitosis, girdle view. (B,C) Spermatogonangium in valvar view, following the second (B) and during the fourth (C) depauperating mitoses. (D, E)
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Stephanopyxis [Drebes, 1966; von Stosch, 1954] and Chaetoceros [von Stosch et al., 1973]). No thecae are produced in some other genera (e.g., Coscinodiscus [Fig. 1B and C; see also Drebes, 1974; Schmid, 1995; von Stosch and Drebes, 1964], Rhizosolenia [Drebes, 1974], Lithodesmium, Streptotheca, and Bellerochea [von Stosch, 1954]). In Guinardia accida and Aulacodiscus argus, the depauperating mitoses are acytokinetic, and spermatogonia are therefore multinucleate (Drebes, 1977a). Finally, in Cyclotella, diVerentiating mitoses are absent (Geitler, 1952a; Schultz and Trainor, 1968). The number of depauperating mitoses varies within and among species. Formation of spermatogonia is preceded by two or three depauperating mitoses in Stephanopyxis palmeriana (Drebes, 1966), three in our clone of S. turris (Fig. 1E) and Chaetoceros didymum (Furnas, 1985; von Stosch et al., 1973), and three or four in Bacteriastrum hyalinum (Drebes, 1972). Up to six depauperating mitoses (producing 64 spermatogonia per cell) occurred in large-celled spermatogonangia of Coscinodiscus granii (Roshchin and Chepurnov, unpublished data), but in smaller spermatogonangia of the same clone, there were only three. Each spermatogonium produces four uniagellate gametes, in two-step meiosis and no nuclei abort, in contrast to oogenesis. Sperm formation is usually either merogenous or hologenous (reviews by Drebes, 1977a; Jensen et al., 2003; Round et al., 1990; there are also several records by von Stosch 1982, 1985, 1987, that are not included in the most recent review by Jensen et al.). In the merogenous type, the sperms bud oV from a residual body containing all the plastids. Examples are some species of Melosira (von Stosch, 1958a) and Stephanopyxis (Drebes, 1966; von Stosch and Drebes, 1964). Two residual bodies are produced in Odontella granulata, because meiosis I is cytokinetic (von Stosch, 1956). In hologenous sperm formation, the whole of the spermatogonium content is at rst distributed more or less equally among the four sperm. Often, therefore, the sperms possess plastids (e.g., in Melosira moniliformis var. octogona [Idei and Chihara, 1992] and species of Chaetoceros [von Stosch et al., 1973]). An intermediate type of sperm formation occurs in Coscinodiscus granii and C. concinnus (Drebes, 1974; Schmid, 1995; von Stosch and Drebes, 1964), where the plastids and vacuoles are extruded at meiosis I; after this, the spermatocyte divides hologenously. Coscinodiscus wailesii is apparently similar. In Biddulphia
Stephanopyxis turris, girdle view. (D) Two sibling vegetative cells. (E) Spermatogonangium containing eight spermatogonia. (FJ) Thalassiosira punctigera, girdle view. (E) Vegetative cell. (G) Oogonium, with the thecae partially separated and the egg surface exposed. (H) Development of auxospore (isopolar expansion). (I, J) Initial cell formation. (I) The contents of the developed auxospore have separated from the auxospore membrane at the side where the initial epivalve will be deposited. (J) The initial epitheca has formed (left side) and the hypovalve is forming (right side). Bars 50 mm (AE) and 25 mm (FJ).
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pulchella, exclusion of the plastids into a residual body occurs before meiosis (von Stosch, 1982). The ultrastructure of spermatogenesis and sperm has rarely been studied. The most detailed studies are those of Lithodesmium (Manton and von Stosch, 1966; Manton et al., 1969a,b, 1970a,b) and other available data are summarized by Jensen et al. (2003). In addition to the unusual 9 0 structure of the axoneme, diatom sperm are also unusual among the zoids of heterokont algae in their lack of discrete microtubular roots. Instead, numerous single microtubules radiate from the single basal body, or they are reduced or absent (Jensen et al., 2003). However, as in other heterokonts, transitional plates are present at the base of the axoneme. After release from the spermatogonia (if present) and spermatogonangium, sperm swim actively towards compatible eggs or oogonia. The mechanism of attraction and recognition between egg and sperm is unknown (Section III.B.2). Sperm attach to the female cell rst by the agellar tip and then via the cell body (Schmid, 1995). DiVerent mechanisms exist to allow contact between the gametes. Usually, the oogonium dehisces without releasing the egg, and this can be partial, as in Thalassiosira punctigera (Fig. 1G), Melosira (Idei and Chihara, 1992; Mizuno, 1977), and Stephanopyxis (Drebes, 1966), or complete, as in Odontella (von Stosch 1954, 1956) and Attheya (Drebes, 1977b). Complete release of the egg occurs in Lithodesmium, Streptotheca, and Bellerochea (von Stosch, 1954). After fertilization, the egg acquires an organic wall and becomes transformed into an auxospore. In the many centric diatoms with radially symmetrical, circular valves, expansion of the auxospore is isodiametric, with the auxospore becoming and remaining more or less spherical (Fig. 1H). In this case, the auxospore wall either remains unsilicied (e.g., Round, 1982) or acquires small round scales, which are embedded within the organic matrix (e.g., Crawford, 1974; Schmid, 1995; Schmid and Crawford, 2001). In centrics with bi- or multipolar valves, or asymmetrical valves, the shape of expanding auxospore is modied by unequal hardening of its wall, by the formation of siliceous bands and hoops (the properizonium, von Stosch, 1982; von Stosch et al., 1973). Several types of auxospores (i.e., intercalary [Fig. 1H], semi-intercalary, lateral, terminal, and free) can be distinguished on the basis of their position in relation to the oogonial thecae (Drebes, 1977a). Once expansion of the auxospore is complete, the initial valves are produced (Fig. 1I and J). The formation of each is preceded by an acytokinetic mitosis and the abortion of one of the daughter nuclei (Drebes, 1977a; von Stosch and Kowallik, 1969). Formation of three initial thecae occurs in Chaetoceros eibenii (von Stosch et al., 1973) and occasionally in Bacteriastrum hyalinum (Drebes, 1972); the rst formed is always discarded. In Leptocylindrus danicus (French and Hargraves, 1985), the auxospore is rst
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transformed into a resting spore, which only later germinates to produce a vegetative cell.
B. Araphid, Pennate Diatoms: Anisogamy Molecular phylogenies show that the araphid pennate diatoms evolved from a lineage of centric diatoms (Kooistra et al., 2003a,b; Medlin et al., 2000), and the earliest known araphid fossils are from the Late Cretaceous (Hajos and Stradner, 1975; Strelnikova, 1974), whereas centrics are known from the Jurassic (Rothpletz, 1896, 1900). With respect to sexual behaviour, the araphid pennates are poorly known, and only recently has there been a rst attempt to make general conclusions about the group (Chepurnov and Mann, 2004). According the most recent phylogenetic analysis (Kooistra et al., 2003b; Medlin et al., 2000), there are two major araphid clades. One contains Asterionellopsis, Asterioplanus, and Rhaphoneis, and no data on sexual behavior are available for these. All of the limited information available applies to species of the other clade, including Striatella (Chepurnov in Roshchin, 1994a), which appears to represent the sister group of the raphid diatoms (Kooistra et al., 2003b). Like the centric diatoms, the araphid diatoms are basically allogamous organisms that exhibit anisogamy: The copulating gametes diVer morphologically and behaviorally. However, the primary copulation is between gametangia, not the gametes themselves (i.e., araphid pennates exhibit gametangiogamy), no agellate gametes have been reported, and the male and female gametes do not usually diVer in size (Roshchin, 1994a; Chepurnov and Mann, 2004). Interactions between the sexual partners are required to trigger meiosis and gametogenesis, so that no gametes are produced in monoclonal cultures of heterothallic species. The mechanism of locomotion of the active gametes is unknown. von Stosch (1958b) reported amoeboid movement in male gametes of Rh. adriaticum, and male cells of other genera can be seen to move slowly over the gametangial frustules. In a pair of gametangia, one produces passive (stationary, female) gametes, which remain associated with the gametangial thecae, whereas the other produces active (migratory, male) gametes, which escape from the parental frustule and migrate toward the passive gametes to fuse. As in the oogonia of centric diatoms, only one or two gametes are produced per female gametangium, and meiosis II is acytokinetic and leads to abortion of one haploid nucleus. Two female gametes are produced in, for example, Licmophora (Chepurnov and Mann, 2004; Roshchin, 1994a; Roshchin and Chepurnov, 1994, 1999), Striatella (Chepurnov in Roshchin, 1994a), Rhabdonema arcuatum and Rh. minutum (von Stosch, 1958b, 1962), Tabularia (Synedra) tabulata, and Fragilaria delicatissima (Roshchin, 1994a). A single
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functional gamete and a residual body are produced in Grammatophora marina (Magne-Simon, 1960, 1962) and Rh. adriaticum (von Stosch, 1958b). In all the species mentioned, the male gametangia produce two gametes, except for G. marina, which produces one, plus a residual cell. The formation of the male gametes in Rhabdonema, described by von Stosch (1958b, 1962), involves depauperating mitosis, as in centric diatoms, so that the male gametes are signicantly smaller than the females. As far as is known, this is unique among pennate diatoms, and it would be consistent with an early separation of the Rhabdonema lineage. However, Rhabdonema does not consistently occupy a basal position in molecular phylogenies of pennates (e.g., Medlin et al., 2000; Kooistra et al., 2003a). von Stosch initially (1958b) referred to the sexual reproduction of Rhabdonema as oogamous, apparently because of the diVerentiating mitoses, but this prejudges homology and downplays the absence of agella. We follow von Stoschs later (1982) classication of Rhabdonema (Rh. arcuatum) as morphologically and physiologically anisogamous. Almost all of the information on auxospore development in araphid pennates comes from Rhabdonema (von Stosch, 1958b, 1962, 1982). Here, the auxospore begins by forming a composite organicsiliceous wall, like that in centrics, consisting of an organic matrix in which silica scales are embedded. This permits some expansion. However, the major phase of expansion involves formation of a new set of wall elements and is bipolar, regenerating the elongate shape of the vegetative cells by hardening the wall progressively from the center outward. The hardening occurs through the progressive formation of the transverse perizonium, which is a series of transverse silica bands; all of these are split rings, and the splits align on the same side of the auxospore to form a suture. von Stosch emphasized that, unlike the scales and properizonia of centrics, the perizonial bands are separated from the primary organic wall. After the formation of transverse perizonium is complete, a series of longitudinal bands (the longitudinal perizonium) is formed beneath the suture in Rh. arcuatum (von Stosch, 1962), but not in Rh. adriaticum (von Stosch, 1982). The function of these bands is unclear, though perhaps (here and in the raphid diatoms) they facilitate the escape of the initial cell, like the tongue of a shoe. As in centrics, two thecae are subsequently laid down within the auxospore (both preceded by an acytokinetic mitosis) to complete the initial cell. von Stosch (1982) mentioned that he had found perizonia in other, unspecied araphid pennates. In contrast, Williams (2001) found no signs that transverse or longitudinal perizonia have been detected in Fragilariforma virescens, nor do they appear to be present in Meridion or Diatoma. Williams suggested that this may be why the initial cells of these taxa are remarkably variable in shape.
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C. Raphid, Pennate Diatoms: Anisogamy and Isogamy The raphid pennates are the most recent of the major groups of diatoms they are unknown before the Tertiary (Strelnikova, 1974, 1992)and they are also the most species-rich (Round et al., 1990). Molecular data indicate that this group is monophyletic (Kooistra et al., 2003a,b; Medlin et al., 2000). Only a tiny minority of species have been studied, but sexual reproduction is nevertheless better documented here than in either of the other major groups, and the nature of the process has been understood for longer. The earliest reports revealed the essential features, that cells pair actively and that the fusion of their contents produces one or two auxospores (e.g., Luders, 1862; Smith, 1856; Thwaites, 1847), and it was in raphid diatoms that meiosis and the rules of the diatom life cycle were rst demonstrated (key works are Geitler 1927, 1932; Karsten, 1912; Klebahn, 1896). Like the centric and the araphid pennates, the raphid diatoms are predominantly allogamous. Most raphid diatoms are isogamous in the sense that the gametes produced by diVerent gametangia appear identical in shape and size (e.g., Drebes, 1977a; Geitler, 1973; Round et al., 1990). So far, only Nitzschia longissima has been found to exhibit morphological (as well as behavioral) anisogamy (Chepurnov in Roshchin, 1994a), as in the araphids. This similarity to the araphids is probably a homoplasy, because Nitzschia has an advanced type of raphe system (with silica bridges beneath the raphe slits, Round et al., 1990), which must have evolved after the many lineages with a normal type of raphe that have morphological isogamy. Furthermore, N. longissima is also atypical within Nitzschia and Pseudo-nitzschia (e.g., Davidovich and Bates, 1998; Mann, 1986, 1993b; our unpublished data, see Fig. 2FH). One or two gametes (depending on species) are produced per gametangium (Fig. 2AK), as in araphid pennates. Where one gamete is formed, meiosis I is sometimes followed by an unequal cell division, producing a residual cell (e.g., Mann, 1989a). Again as in araphids, the rst visible step in sexual reproduction is gametangiogamy. However, whereas in araphids the sexual partners are brought together by chance (Roshchin, 1986; von Stosch, 1958b), presumably through passive transport by water movements, raphid diatoms are motile and pairing is active. The details of pairing, gametogenesis, fertilization, and auxospore development are extremely diverse (e.g., Drebes, 1977a; Geitler 1957, 1969, 1973, 1979, 1984; Mann, 1993a; Round et al., 1990). One of the principal sources of variation, which needs to be taken into account when considering the mating systems of diatoms (Section III.B.1) is the behavior of the gametes. Some species are not only morphologically but also behaviorally isogamous. Recently studied examples producing two gametes per gametangium are
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FIG. 2 Patterns of allogamous sexual reproduction in raphid pennate diatoms in culture. (AE) Morphological and physiological isogamy. (A, B) Achnanthes cf. angustata from North Sea, homothallic reproduction. (A) Paired gametangia, each containing two gametes. Note the
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Rhoicosphenia (Mann, 1982b), Navicula (Mann and Stickle, 1989), Seminavis (Chepurnov et al., 2002), Haslea (Chepurnov, 1993), and Achnanthes (Fig. 2A and B, see also Chepurnov and Mann, 1997; Idei, 1991; Roshchin, 1994b). Taxa producing only one include Eunotia (Fig. 2CE, see also Geitler, 1951a, b, 1958; Mann et al., 2003), Actinella (Mayama, 1991), Nitzschia amphibia (Geitler, 1969), and some Cocconeis (Geitler, 1958, 1973, 1982). Alternatively, raphid diatoms may produce morphologically identical gametes that behave diVerently (behavioral or physiological anisogamy). Two main patterns exist: First, both gametes of one of the gametangia are active, whereas the gametes of the other gametangium are immobile. Here, therefore, allogamous fusion occurs within the connes of the passive gametangium. This pattern is termed cis-type behavioral anisogamy and occurs in Mastogloia smithii (Stickle, 1986), Amphora cf. laevissima (Mann, 1993a), Achnanthes javanica f. constricta (Mizuno, 1994), and Pseudo-nitzschia (Fig. 2FH, see also Amato et al., 2003; Davidovich and Bates, 1998). The araphid pennates also exhibit cis-type behavior. Second, each gametangium produces an active gamete and a passive one. Migration of the active gametes occurs in opposite directions, so that one auxospore develops within each gametangium. This trans-type behavioral anisogamy occurs in many genera, including Cymbella and Gomphonema (Geitler, 1973), Placoneis (Mann and Stickle, 1995), Lyrella (Mann and Stickle, 1993), Nitzschia (Mann, 1986; Roshchin 1994a), and Neidium (Mann, 1984a). Behavioral anisogamy is also exhibited by some species producing a single gamete per gametangium, for example, Sellaphora (Fig. 2IK; see also
mucilage sheath (arrows), within which the gametes will be released by separation of the gametangial thecae to allow isogamous fusion outside the parental frustules. (B) Two initial cells resulting from allogamous fusion of gametes. (CE) Eunotia cf. bilunaris from a freshwater lake in New Zealand, heterothallic reproduction. (C) A pair of gametangia of opposite mating types; in both the gametangia, the contents have divided (clearly visible in the right-hand gametangium) and produced a single functional gamete (arrows), which has begun to form a copulation papilla and a small residual cell (arrowheads). (D) The papillae have fused to produce copulation canal, through which the gametes from the two gametangia will migrate to fuse. (E) Fully developed auxospore, associated with gametangial frustules. (FK) Physiological anisogamy. (FH) Pseudo-nitzschia fraudulenta from the North Sea, heterothallic reproduction. (F) Paired cells of opposite mating type. (G) Formation of two morphologically identical gametes per gametangium. (H) Two zygotes, following physiologically anisogamous fusion: gametes from the large (lower) gametangium have migrated out of the gametangial frustule and fused with the gametes of the small gametangium, which stayed immobile and remained associated with the parental thecae. (IK) Sellaphora sp. from a freshwater lake in East Africa, homothallic reproduction. (I) Paired gametangia. (J) After fertilization, the left gametangium appears nearly empty and contains only a small residual cell (arrow), following the emigration of the active gamete, while the zygote lies within the frustule of the other cell (right). (K) Development of the zygote, through auxospore expansion, has resulted in a large initial cell, which is partially contained within the female frustule. Bars 20 mm (AH) and 10 mm (IK).
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Mann, 1989a; Mann et al., 1999) and some Cocconeis (Geitler, 1973; Mizuno and Okuda, 1985). Additional variation in sexual reproduction of the raphid species involves aspects such as pairing congurations, rearrangement of gametes within the gametangium; mucilage production; the formation of copulation apertures, canals, or tubes; and the position of the auxospores in relation to the parental frustules (Geitler 1973, 1979; Mann, 1990, 1993a). The cytological ultrastructure of sexual reproduction has been studied in only two raphid diatomsGomphonema parvulum and Neidium aYneand then only very briey (Drum et al., 1966). The results add little to what was known from light microscopical observations, except that they show that a very thin zygote wall in young Gomphonema zygotes (e.g., Drum et al., 1966) (the presence of two nuclei in the right-hand cell shows that this cannot be a gamete as Drum et al. indicate). In most of the raphid diatoms that have been studied, auxospore development and initial cell formation are like that of the araphid Rhabdonema; that is, expansion is bipolar and is accompanied by deposition of transverse and longitudinal perizonia, and the formation of each initial theca is preceded by an acytokinetic mitosis (Round et al., 1990). Often, the primary organic wall of the auxospore is ruptured at an early stage of expansion and persists during the expansion as two caplike structures, one over each pole of the auxospore (e.g., Cohn et al., 1989; Mann, 1994c; Mann and Stickle, 1989, 1991). The perizonium has the same general structure as in Rhabdonema, but whereas most transverse perizonial bands are split rings in the species studied, as in Rhabdonema, the central transverse band is a complete ring; examples studied include Rhoicosphenia (Mann, 1982b), Craticula (Cohn et al., 1989), and Caloneis (Mann, 1989c). There are some signicant variations on the main theme. Silicication of the primary wall is known in Neidium and Biremis (Mann, 1984a, 1993a), where it takes the unusual form of a hemiellipsoidal cap, and scales have been detected in Diploneis (Idei in Kaczmarska et al., 2001) and Pseudo-nitzschia (Kaczmarska et al., 2000). In Pseudo-nitzschia, Kaczmarska et al. (2000) claim to have found scales even on the gametes, which one might expect to hamper plasmogamy, but no scales are visible in the ultrathin sections of gametes and early auxospores of Gomphonema studied by Drum et al. (1966). However, scales may be more common than it currently appears, because few have looked for them using appropriate techniques (e.g., critical point drying of intact auxospores). No transverse perizonium is present in Achnanthes sensu stricto (Mizuno, 1994; Sabbe et al., 2004b; Toyoda et al., 2003; von Stosch 1962, 1982), though there is a well-developed longitudinal perizonium. A group particularly worthy of further study is the Surirellaceae, because of their unusual vegetative cell symmetry and morphogenesis. It is already known that auxospore and perizonium expansion is unipolar in some
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Surirellaceae (Mann, 1987), whereas other species lack a perizonium, and the auxospore may not even expand (e.g., Surirella angusta, Mann, 2000).
D. Automixis Infrequently, auxospore formation can result from sexual processes occurring with a single cell (automixis). Automixis has been found in various lineages, both centric (Drebes, 1977a) and pennate (Geitler, 1969, 1985; Mann 1994a). It can be facultative, the more frequent method of auxosporulation being allogamous (e.g., in Dickieia ulvacea [Mann, 1994a] and Amphora sp. [Thaler, 1972]), or it can be obligate. If obligate, however, it is often found that the closest relatives of the automictic taxa possess normal biparental sex (Geitler, 1985), and it seems likely that automixis has evolved independently in many diVerent diatom lineages. In all cases, automixis seems to represent a relatively minor modication of the allogamous pattern of reproduction found in related taxa (Geitler, 1985; Round et al., 1990). There are two main types of automixis. In both, meiosis occurs in an unpaired mother cell. Then either two normally diVerentiated gametes fuse together ( paedogamy), or a meiotic cytokinesis is suppressed and two of the four (tetrad) nuclei fuse in undivided protoplasts (autogamy) (Geitler, 1979). So far, paedogamy has never been reported to occur in the centric taxa, which is not surprising given the very diVerent pathways of male and female gametogenesis. Within the araphid pennate group, there is a single reliable report of facultative paedogamy in Synedra ulna, which normally reproduces allogamously (Geitler, 1939). All other reports of paedogamy are in the raphid group, where it has been in a few representatives of Amphora, Navicula, Gomphonema, Cymbella, Epithemia, and Nitzschia (listed by Geitler, 1985; Round et al., 1990; see also Mann, 1993a, 1994a). Paedogamy highlights the fact that, in normal allogamous pennate diatoms producing two gametes per gametangium, there must be a mechanism that prevents sister gametes from fusing. Comparisons of closely related paedogamous and allogamous species or populations may therefore help to shed light on what this mechanism is. Auxosporulation via autogamy has been found in the centrics Cyclotella meneghiniana (Iyengar and Subrahmanyan, 1944) and Melosira nummuloides (Erben, 1959; see also Fig. 3AC). Interestingly, some populations of each species have been found to be capable of spermatogenesis, but fertilization was never observed (Schultz and Trainor, 1968; von Stosch and Drebes in Drebes, 1977a; our unpublished observations). There are some other reports of auxosporulation without sperm production, in Detonula (Schoederella) schroederi (Drebes, 1977a) and Ellerbeckia arenaria forma arenaria (Schmid and Crawford, 2001) but there is insuYcient information about nuclear
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FIG. 3 Uniparental auxosporulation (AJ) and abrupt cell size reduction (KS). (AC) Centric diatom Melosira nummuloides from the North Sea. Auxosporulation is preceded by polarization of the contents in auxospore mother cell (A, upper cell) and then its unequal division (A, lower
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development to discriminate between automixis and asexual auxosporulation (Section II.E). Autogamy has never been demonstrated unambiguously in araphid pennates: Synedra vaucheriae may be autogamous or paedogamous (Geitler, 1958), whereas a report in Licmophora spp. (Kumar, 1978) is also insecure (Chepurnov and Mann, 2004). Within the raphid series, autogamous reproduction has been found in Denticula tenuis (Geitler, 1953) and a variety of Cymbella ventricosa (Geitler, 1958).
E. Apomixis and Circumvention of Sex Sexual events in diatoms always result in auxosporulation. However, sexual reproduction is not a necessary precondition for size restitution, and asexual auxosporulation has been documented in several species (Fig. 3DJ; see also Drebes, 1977a; Geitler, 1973; Nagai et al., 1995; Sabbe et al., 2004b). However, as with autogamy, in every case in which asexual auxosporulation has been found, it occurs in what are otherwise predominantly sexual diatom lineages, which indicates that asexual auxosporulation is a secondary modication of a basically sexual pathway of development. Hence, asexual auxosporulation is probably best referred to as apomixis, by analogy with higher plants, where the term generally means asexual reproduction through seeds (as opposed to purely vegetative propagation), with meiosis and fertilization being bypassed (e.g., Sadivan et al., 2001). The type of apomixis reported in the centric Actinoptychus senarius (undulatus) (Broer and von Stosch in Drebes, 1977a) and in two varieties of the raphid Cocconeis placentula (Geitler, 1927, 1973, 1982) was treated by its discoverers as diploid parthenogenesis because it appeared that meiosis is circumvented in cells already predetermined to become gametes. Another
cell). Autogamous fertilization then occurs in the larger cell (sensu Erben, 1959), which develops into an auxospore (B) and transforms into an initial cell (C) after expansion. (DJ) Asexual (apomictic) auxospore formation in Eunotia sp. from a freshwater lake, South America (E, J, I, cells stained with DAPI). In a single cell, the plastids are shifted toward the hypovalve (D, E); then mitotic division occurs, accompanied by unequal cytokinesis (F, G). The larger cell then starts to develop into auxospore, without any further nuclear transformations (H, I) and later transforms into initial cell (J). (K) Spontaneous abrupt size reduction in the Eunotia, following nonplanar cytokinesis. (LO) Pseudo-nitzschia pungens from the North Sea. (L) Cell of regular shape, with two plastids, in valvar view. (M, N) Cells with irregular valve outline that have arisen spontaneously in culture. Note the constriction that has split one of the two plastids in (M); further deepening of the constriction in subsequent cell divisions nally leads to abortion and loss of the cell part that contains half a plastid and lacks the nucleus (N, below the constriction). (O) Nonplanar cytokinesis and formation of shortened daughter cells. (PS) Nitzschia longissima from Florida Bay, Atlantic Ocean. (P) Cell of the original clone. (R, C) Cells reduced in size by shortening of one (R) and both (S) cell ends with the aid of a razor blade. Bars 20 mm (AC, LO), 10 mm (DK), and 100 mm (PS).
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type is haploid parthenogenesis (Geitler, 1979; Mann, 1994a), which is facultative in some allogamous pennate diatoms. Here, some haploid gametes that have failed to fuse can develop into auxospores and even form initial thecae (e.g., in Cymbella cesatii [Geitler, 1979], Dickeia ulvacea [Mann, 1994a], and Achnanthes longipes [Chepurnov and Roshchin, 1995]). Longer-term development is possible in Licmophora (Section III.B.4), at least in culture (Chepurnov in Roshchin, 1994a; Roshchin and Chepurnov, 1994). Purely vegetative auxosporulation (development directly from vegetative cells) occurs in the centric diatoms Melosira moniliformis var. octogona (Drebes, Broer, and von Stosch in Drebes, 1977a), Skeletonema costatum (Gallagher, 1983), and Coscinodiscus wailesii (Nagai et al., 1995). In the last two cases, the authors used the term vegetative cell enlargement to describe the process observed. However, to avoid confusion, it is important to note that the process originally described as vegetative cell enlargement by von Stosch (1965) is unlike that observed by Gallagher and Nagai et al. because the expanding cells lack the envelopes (perizonia, scaly auxospore coats, etc.) typical of auxospores. True auxosporulation, preceded not by meiosis but by a single mitotic division, was recently reported in a marine raphid diatom Achnanthes cf. subsessilis (Sabbe et al., 2004b). This further case of diploid parthenogenesis, in a genus possessing all other major types of auxosporulation, oVers an unparalleled opportunity to study the importance of sex in the diatom life cycle and how it is controlled. Some diatoms lack auxosporulation altogether, existing as permanently asexual populations. In some populations of the centric Ditylum brightwellii (e.g., in the North Sea and in Narragansett Bay; von Stosch, 1965, 1987, p. 61), cell size is restored only through vegetative cell enlargement (von Stosch, 1965). Populations of Caloneis amphisbaena and Sellaphora pupula lanceolate reveal a narrow size range that remains essentially unchanged over many years of observations, and there are no signs of sexualization in seminatural populations (Mann, 1989b; Mann et al., 2004). This agrees with, for example, Wiedlings (1943, 1948) observations of some Nitzschia species in culture, which do not reduce in size (Section IV.A).
III. Effect of Experimental Studies on Knowledge of Diatom Sexuality Experimental studies of cultures have been essential for progress in several elds, such as control of life cycle progression by size, mating systems, and the development of a species concept in diatoms. In the future, there will be increased emphasis on the molecular and genetic basis of sexual
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reproduction and auxosporulation, and the development of molecular markers for population biology studies, allowing sex to be monitored in natural populations.
A. Induction of Sexual Reproduction 1. Cell Size It was obvious from early observations (e.g., Luders, 1862; Miquel, 1892; Ptzer, 1871) that auxosporulation is a size-related process. However, the rst clear demonstration of the relationship between changes in cell size during life cycle, the capacity for sexualization, and cell size restoration via the auxospore was made by Geitler, and in this the use of monoclonal cultures was crucial (Geitler, 1932). From the changes exhibited by cultures over time, Geitler suggested that there are three cardinal points in the life history of each diatom: the maximal size of initial cells, the upper limit of the sexually inducible size range, and the critical minimal size. Subsequent studies, both in nature and culture, have conrmed the essential truth of this idea, although it has been found that there is a nongenetic dependence of initial cell size on gametangium size that complicates the Geitlerian doctrine (Section III.B.4). A further complication is that in centric diatoms, there are diVerent size thresholds for male and female gametogenesis. It is important to note that the expression of sexuality during the life cycle of a clone is truly size-dependent and not related to clone age. In most cases, of course, clone age and mean cell size are closely correlated, because of the MacDonaldPtzer rule. However, in some diatoms it is possible to change cell size abruptly, either by taking advantage of the spontaneous occurrence of unequal cell division (Fig. 3KO) or by surgical procedures, thus creating subclones of markedly diVerent sizes, but having the same age since auxosporulation (Fig. 3PS). This has been done by von Stosch (1965), Drebes (1966), Roshchin and Chepurnov (in Roshchin, 1994a), and most recently in Eunotia bilunaris by Mann et al. (2003). In all cases, cell behavior correlates with size, not age. This is also true for cells, for example, of Achnanthes longipes, that are capable of vegetative cell enlargement and are therefore able to enter the sexual size range from below the lower critical size threshold for sexualization (Roshchin and Chepurnov, 1992), or to escape above the upper limit of the sexual size range, for example, the 10-year maintenance of a clone of Stephanopyxis turris by von Stosch (1965; see also von Stosch and Drebes, 1964). The mechanism by which cells monitor size is unknown. During the mitotic cell cycle, cell volume doubles (it more than doubles in cells in which the valves are formed at a distance from each other within the girdle
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cylinder) (Crawford, 1981), and so cell volume per se is unlikely to be the measure of size used. Possibly, the cell is able to determine the ratio between the nuclear volume (or DNA content) and the cytoplasmic volume, with the former remaining more or less constant during the long diploid phase (e.g., von Stosch and Drebes, 1964; Werner, 1971a; see also Drebes, 1977a, p. 273). However, in some centric diatoms, the decrease in volume caused by diminution of the valves is partly oVset by an increase in the length of the girdle (e.g., von Stosch and Drebes, 1964, p. 220). Monoclonal culture studies of centric diatoms by von Stosch and colleagues revealed that, although many centrics are homothallic, the size ranges for oogenesis and spermatogenesis are generally diVerent, although they overlap: oogenesis starts rst, then both eggs and sperms are produced (when new clonal cultures of large cells can be established), and nally, when the cells are small, only sperms are formed. However, there are exceptions, such as Stephanopyxis (von Stosch and Drebes, 1964; Drebes, 1966). Given that the essential feature of oogamy is a diVerentiation between large female gametes and small sperms, it is not surprising that oogenesis is initiated (if environmental conditions permit) and terminated relatively early in the life cycle, while cells are still relatively large. What is unclear is why spermatogenesis is delayed and how clones and populations allocate resources appropriately to males and females, when the only way to produce males in a truly homothallic centric diatom is to prevent sexualization during the earlier, female phase. In pennate diatoms, no diVerence has yet been found between the sexual size ranges of opposite sexes (or mating types), except in Rhabdonema arcuatum, but not Rh. adriaticum (von Stosch 1958b, 1962), which correlates with the fact that the gametes are almost always more or less equal in size. However, in monoecious clones of Achnanthes longipes, the upper threshold for successful sexual reproduction depends on whether reproduction is intraclonal or interclonal (Chepurnov and Mann, 2000). Cells become capable of intercrossing when they are about 70 mm long, but they cannot reproduce intraclonally until they reach 50 mm. It is tempting to see this as an insurance strategy: Outbreeding is preferred, but if this fails, then cells are able to inbreed. Preliminary experiments indicate that the size dependence of sex may be complex in some centric species, with more than one auxosporulation size range and hence size restoration in two or more steps (Roshchin, 1994a). The ability of newly enlarged cells to undergo a further round of expansion was apparently rst reported by Schreiber (1931) in Melosira nummuloides. Three-step auxosporulation was reported by Roshchin (1994a, Fig. 18, p. 42) in Coscinodiscus granii, with short periods of size reduction occurring between auxosporulations (38 ! 127, 121 ! 198, 188 ! 248 mm). In Coscinodiscus janischii, in clonal cultures in which the cells were 150170 mm,
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oogamy resulted in initial cells of 275293 mm. After a short period of size reduction, however, cells of 230260 mm were found to be capable of transformation into oogonia, although auxospores were not observed (Roshchin, 1975, 1994a); spermatogonangia of C. janischii of 230260 mm were seen in planktonic samples. In Melosira moniliformis, Kustenko (1978) observed two-step auxosporulation in monoclonal culture: 21 ! 48 ! 79 mm, and Thalassiosira punctigera is capable of similar changes (our unpublished observations). Because Thalassiosira and Coscinodiscus (or Melosira) belong to diVerent major clades of centric diatoms (e.g., Medlin et al., 2000), complex life cycles and multistep auxosporulation may be widespread. 2. External Factors Auxosporulation is inuenced not only by intrinsic factors (e.g., cell size) but also by external conditions. Drebes (1977a) reviewed what was then known and concluded that diVerent species had diVerent requirements for sex, that few generalizations were possible, but that on the whole, there is no signicant antagonism between factors promoting vegetative growth and those eliciting gametogenesis. These comments still apply, though there are some interesting exceptions to the third one. a. Centric Diatoms Most experiments have involved centric diatoms, and light has been the main factor investigated. Gametogenesis can be induced by changes in light intensity and photoperiod, and is aVected also by temperature, for example, in Melosira nummuloides (BruckmayerBerkenbusch, 1954), Stephanopyxis (Drebes, 1966; von Stosch and Drebes, 1964), Lithodesmium undulatum (Manton and von Stosch, 1966), Coscinodiscus asteromphalus (Werner, 1971b), and C. concinnus (Holmes, 1966). Drebes (1977a) summarizes this work, but as in other Englishlanguage reviews (e.g., Round et al., 1990), Drebes did not cover the work of A. M. Roshchin and colleagues (reviewed in Russian by Roshchin, 1994a) on either centric or pennate diatoms. We will, therefore, bias our account toward this work. From his own and published studies, Roshchin (1994a) concluded that there are two major groups, distinguished according to their light responses. These were termed short-day and long-day diatoms, by analogy with higher plants. Experiments with Coscinodiscus granii and C. janischii (Roshchin, 1972, 1976; Roshchin and Lutsenko, 1972) showed that the optimal light regime for both vegetative multiplication and auxosporulation was continuous illumination, with an irradiance of 96 mmol photons m2 s1; decrease in light intensity or reduction of the photoperiod inhibited both processes. These, then, are long-day species as, apparently, (Roshchin, 1994a, p. 47) are Stephanopyxis turris (von Stosch and Drebes, 1964),
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S. palmeriana (Drebes, 1966; Steele, 1965), and Cyclotella meneghiniana (Ermolaeva, 1953). In contrast, in Chaetoceros curvisetus (Roshchin, 1976), the maximum rate of growth occurred at 50 mmol photons m2 s1 in continuous light, but this light regime completely inhibited sexualization. Induction of sex was optimal with a prolonged dark phase, and this requirement was especially noticeable in the upper part of the sexual size range. Furnas (1985) also studied induction of spermatogenesis in C. curvisetum and again found that continuous light was inhibitory, though he observed high induction of spermatogenesis with a 16:8 h lightdark cycle. A similar suppression of sexualization by continuous light occurs in Melosira nummuloides (Bruckmayer-Berkenbusch, 1954). These are short-day species, in the sense that a dark phase is essential, and M. moniliformis var. moniliforms from the Black Sea (Roshchin, 1990a) is also probably a short-day species. In a very full study of Coscinodiscus concinnus by Holmes (1966; the identication may be incorrect: Drebes, 1977a, p. 275), spermatogenesis (and oogenesis, though over a narrower range of temperature and irradiance) was promoted by a lightdark cycle of 8:16 h, in a wide range of temperatures (9 26.7 C) and irradiances (10.51745 mmol photons m2 s1; gures calculated from Holmes data using the conversions of Luning, 1981). Holmes also mentioned data of R. E. Norris recording similar promotion of auxosporulation by short days in Ditylum. Detecting daylength eVects needs care, however. In Thalassiosira weissogii, for example, Armbrust et al. (1990) found that spermatogenesis is inhib ited by continuous light at 250 mmol photons m2 s1 and 20 C but that it would occur after a dark treatment of up to 12 h, followed by transfer back to continuous light. However, the principal factor here is transfer of cells to irradiances subsaturating for growth, of <100 mmol photons m2 s1. This, not a dark period per se, is the trigger. Indeed, Vaulot and Chisholm (1987) earlier found some spermatogenesis, albeit at very low levels, during contin uous illumination at 100 mmol photons m2 s1 and 20 C. Higher irradiances are inductive in Stephanopyxis turris: von Stosch and Drebes (1964) recorded massive induction of sexual stages with transfer from about 56 to 80100 mmol photons m2 s1 and fresh culture medium, with a simultaneous increase in temperature from 15 to 21 C. Stephanopyxis palmeriana, Lithodesmium undulatum, Helicotheca tamesis, and Bellerochea malleus behave essentially similarly (Drebes, 1966, 1977a; Manton et al., 1969a). Temperature decreases are stimulatory in Chaetoceros decipiens and C. constrictus (Drebes, 1977a). Experiments with monoclonal cultures of homothallic centric diatoms that are simultaneously capable of both oogenesis and spermatogenesis have shown that the requirements for the production of eggs and sperms can diVer. In Lithodesmium undulatum, for instance, subcultures of a single
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clone placed in continuous light (though with uctuations in intensity) almost exclusively produced eggs, but with alternating light and dark, the subcultures could be induced to produce either a mixture of eggs and sperm or just sperm alone (von Stosch, 1954, p. 66). Induction of sperms and eggs by diVerent environmental conditions is counterintuitive, as both types of gamete must occur simultaneously in nature for successful auxosporulation (as in the bloom studied by Crawford, 1995). Indeed, one must suspect that any combinations of environmental factors that are inductive for one sex but not the other must be combinations that the organism does not encounter in nature. However, diVerential eVects can be exploited for experimental purposes: To induce bulk spermatogenesis of L. undulatum for electron microscopy, Manton and von Stosch (1966) used transfer from a 16:8 h lightdark cycle of 6 mmol photons m2 s1 and 15 C to a 14:10 h cycle of 400 mmol photons m2 s1 and 24 C. A whole series of other parameters, for example, monochromatic light, salinity and osmotic changes, and nutritional and organic substances (reviewed in Drebes, 1977a; see also Bruckmayer-Berkenbusch, 1954; Schmid, 1995; Schultz and Trainor, 1968, 1970; Waite and Harrison, 1992) have been tested for their eVects on sexualization of centric diatoms. No general patterns are currently detectable. Stimulation of sexual reproduction by blue and green light in Chaetoceros didymus (Baatz, 1941) hints at the involvement of cryptochrome or rhodopsin in light perception (see also Leblanc et al., 1999). The lack of a consistent link in diatoms between nutrient stress and sexual reproduction probably reects the fact that the products of reproductionauxosporesare not dormant stages, unlike, for example, the zygotes of Volvocales or chrysophytes (e.g., Sandgren, 1988). It is noteworthy that a link between low nutrient status and auxosporulation does occur in the only diatom producing a resistant stage after auxosporulation, viz. Leptocylindrus (Hargraves and French, 1983), does show nutrient dependence, reacting to low N. By contrast, the freshwater diatoms Stephanodiscus neoastraea and Cyclotella ocellata, which do not form resting stages, have been indicated as reacting to rising N or P levels, respectively (Jewson, 1992b; Perez-Martnez et al., 1992), although no experimental culture data are available to support this. Finally, an extra layer of complexities may be caused by biotic interactions, as Nagai and coworkers (Nagai and Imai, 1998, 2001; Nagai et al., 1999) have demonstrated that certain types of bacteria are necessary for spermatogenesis in the large marine centric Coscinodiscus wailesii. b. Pennate Diatoms In allogamous pennates, cellcell interactions between compatible cells seem to be the primary determinant of when and where sexual reproduction occurs (e.g., Chepurnov and Mann, 2004; Chepurnov et al., 2002; Mann et al., 1999; Roshchin, 1994a), but external factors are also
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important. We will return to cellcell interactions in Sections III.B.1 and III.B.2. Unfortunately, almost all reports of auxosporulation of pennates in nature are based on isolated observations, and long-term phenological data are entirely lacking. There are a few reports indicating that auxosporulation can be seasonal in nature. For example, Meyer (1929) recorded abundant auxosporulation of Didymosphenia in Lake Baikal in summer 1928; nearly 70 years later, one of us (D.G.M) likewise observed summer auxosporulation of this species in Lake Baikal. According to Edlund and Stoermer (1997), Cymbella and Gomphonema species often auxosporulate en masse in spring and Cocconeis placentula species seem to auxosporulate only in summer (JuneOctober) when observed in a stream in the Royal Botanic Garden Edinburgh. Given that some or most cells are usually within the sexual size range in many pennate diatom populations (Mann et al., 1999), seasonality of auxosporulation implies signicant control by factors such as irradiance, day length, or temperature. Not surprisingly, therefore, the few studies that have been undertaken reveal light and temperature eVects. Studies of the araphids Rhabdonema adriaticum (Rozumek, 1968) and Striatella unipunctata (Davidovich and Chepurnov, 1993) and the raphids Cocconeis scutellum (Mizuno and Okuda, 1985), Nitzschia lanceolata (Davidovich, 1998), N. longissima (Davidovich and Chepurnov, 1993), and Pseudonitzschia multiseries (Hiltz et al., 2000) show that there is an optimal irradiance (the parameter studied the most often) for induction of sexual reproduction and that this optimum may vary with day length and temperature (reviewed by Davidovich, 2002a). However, the conditions that favor auxosporulation, even if they are suboptimal for vegetative growth as in the case of C. scutellum (Mizuno and Okuda, 1985), nevertheless allow rapid vegetative growth, so that auxosporulating cells will incur the interruption of synthesis penalty identied by Lewis (1983). As yet, no case is known in pennate diatoms where sexual reproduction is triggered by severe nutrient (e.g., N or P) depletion. 3. Cell Cycle So far, there is only one report that unequivocally demonstrates dependence of sexualization on cell cycle stage. Armbrust et al. (1990) investigated induction of spermatogenesis in the marine centric Thalassiosira weissogii and determined, using synchronized cultures, that the inducible part of the cell cycle is in early G1; cells in the remaining portion of the cycle essentially ignore the signal and continue to divide mitotically. Experiments on the heterothallic reproduction of raphid diatom Nitzschia lanceolata suggest that here, too, induction of gametogenesis may only be possible in G1 (Davidovich, 1998), but the degree of synchrony of the cultures is unclear.
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B. Control and Mechanisms of Sexual Reproduction 1. Mating Systems Traditionally, when the mating systems of diatoms have been discussed, the terms monoecy and dioecy have been applied (e.g., Drebes, 1977a; Roshchin, 1994a; von Stosch, 1958b, 1982; Wiese, 1969), and we too have used these terms (e.g., Chepurnov and Mann, 1997, 1999, 2000), considering that they are to some extent interchangeable with nomothally and heterothally (Chepurnov et al., 2002; Mann et al., 1999). However, in many cases, a use of homothally and heterothally may be advantageous (Mann et al., 2003), because in other algal groups, for example, colonial oogamous Volvocales, monoecy and dioecy are used to characterize diVerences in mating behavior between diVerent individuals, whereas heterothally and homothally refer to diVerences at the clonal level (e.g., Starr, 1968; Wiese, 1984). In addition, homothally and heterothally are applied to the oomycete fungi, in which mating systems have been studied extensively (e.g., Elliott, 1994) and which belong to the same major evolutionary lineage (the heterokonts stramenopiles) as the diatoms. Application of a common terminology will facilitate future comparison of these two groups. Drebes (1977a) remarked that data, especially those obtained from observations on clonal cultures, indicate that the great majority of species [both centric and pennate] are monoecious. This statement has proved to be incorrect. In oogamous centric diatoms, homothallic behavior and self-compatibility of the eggs and sperms produced by a single clone have been reported from a wide variety of lineages (e.g., Drebes, 1977a,b; Roshchin and Chepurnov, 1999; Schmid, 1995; von Stosch, 1951, 1954, 1956, 1965, 1982). This indicates that homothallic behavior (and hence, phenotypic sex determination) is the predominant condition for allogamous centric diatoms. However, this conclusion needs to be treated with caution. The number of centric diatoms that have been studied in detail and shown to be capable of successful intraclonal auxosporulation in monoclonal culture is still small perhaps only a few dozen. Furthermore, when auxosporulation does occur within a clone, it cannot necessarily be assumed that this has resulted from sexual reproduction. At the very least, eggs and sperm must both have been observed, and for a more rigorous proof, fertilization must be detected, either through direct observation or via genetic analysis. For example, von Stosch (1982) recorded that his clone of Actinoptychus senarius produced sperm, but they were nonfunctional, with the auxospore developing apomictically. Some clones of C. granii isolated from the North Sea by Drebes (1968) were predominantly male, and others were predominantly female (this was termed subdioecy by Drebes), but in later years truly monoecious (homothallic)
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clones were found (Drebes, 1974, 1977a). Wholly male and predominantly female clones of C. granii have been found in the Black Sea, and also exclusively male clones of two varieties of Melosira moniliformis (Roshchin and Chepurnov, 1999, and unpublished data). Some strains of Triceratium are apparently apomictic and cannot produce viable sperm, although stages in spermatogenesis can occur (von Stosch, 1982, p. 131). Finally, there are signicant negative observations, such as von Stoschs comment concerning Biddulphiopsis membranacea (von Stosch and Simonsen, 1984), that, in spite of all eVorts, auxospores could not be obtained. Together, these data indicate that the dogma that centric diatoms are homothallic must not be accepted uncritically. By the time of Drebess 1977 review, there were only three reports of dioecious (heterothallic) reproduction in diatoms, in the araphid pennates Rhabdonema adriaticum (von Stosch, 1958b; see also Rozumek, 1968), Grammatophora marina (von Stosch and Drebes, 1964), and Subsilicea fragilarioides (von Stosch and Reimann, 1970). Overall, however, the pennates were regarded as monoecious (homothallic). In fact, by 1977 there were rather few unequivocal demonstrations of homothally, but these included well-studied cases such as Sellaphora (Navicula) seminulum, Gomphonema parvulum (Geitler, 1932), Rhabdonema arcuatum, and R. minutum (von Stosch, 1958b, 1962), which may have helped create a misleading impression. Since 1977, several further cases of homothally have been found in pennates, but the number is still not high, and out of more than 20 clones of Nitzschia species studied by Wiedling (1948), only two were shown to be capable of intraclonal auxosporulation (several were able to avoid size reduction). Recent studies have suggested that heterothally, or some other type of mating system that promotes outbreeding, is common in pennates and may be the ancestral state (e.g., Chepurnov and Mann, 2004; Chepurnov et al., 2002; Mann et al., 1999; Roshchin, 1994a). This reevaluation of the previous view that homothally is normal was prompted mainly by the research of the Russian scientist Roshchin (1994a; Roshchin and Chepurnov, 1999), who sought an explanation for the fact that most of the pennate species he cultured showed no signs of sexualization, even when information from natural populations indicated that clones were likely to be in the sexually inducible size range. Roshchin therefore inoculated pairs of clones together and obtained sexual reproduction, showing that the species were heterothallic. Application of similar methods by other authors (e.g., Chepurnov, 1993; Chepurnov and Mann, 2004; Chepurnov in Roshchin, 1994a; Chepurnov et al., 2002; Davidovich and Bates, 1998; Mann et al., 1999, 2003; Sabbe et al., 2004a; see also Fig. 2CH) has led to rapid progress. In some cases, mating types diVer in gametangium or gamete development and behavior. Thus, in the araphid group, cis anisogamy during sexual reproduction reects diVerentiation of clones into male and female
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(Chepurnov and Mann, 2004). Similarly, the anisogamy of Sellaphora pupula, in which one gametangium produces a single active gamete and the other a single passive gamete, can be associated with heterothally. For example, in interclonal crosses of the capitate deme, all the male gametangia are produced by one clone and all the females by the other (Mann et al., 1999). The same is true in Nitzschia longissima (Chepurnov in Roshchin, 1994a; Davidovich, 2002b, reported homothallic behavior in some clones of N. longissima, but the pattern of intraclonal auxosporulation was not illustrated) and Pseudo-nitzschia spp. (Amato et al., 2003; Davidovich and Bates, 1998; unpublished data, see Fig. 2FH). However, heterothally also occurs in raphid diatoms exhibiting morphological and physiological isogamy, for example, Haslea subagnita (Chepurnov, 1993), Eunotia bilunaris and E. tropica (Mann et al., 2003; see also Fig. 2CE), Seminavis cf. robusta (Chepurnov et al., 2002), and Amphora cf. proteus (Sabbe et al., 2004a). Experiments indicate that sex determination in dioecious pennate species is genotypic (e.g., Mann et al., 2003), but further research is needed to conrm this and to reveal the sex-determination mechanism. Roshchin also discovered pennates that are predominantly heterothallic, but also have some capacity for intraclonal reproduction (monoeciousdioecious species sensu Roshchin, 1994a; Roshchin and Chepurnov, 1999). For instance, in the marine araphids Tabularia tabulata and Fragilaria delicatissima, heterothallic behavior occurs (Roshchin, 1987, 1989b, 1994a) and regular and vigorous sexual reproduction takes place when clones of opposite sex are mixed. However, male clones can also produce auxospores themselves, though at a low frequency. Roshchin reported that the intraclonal reproduction of male clones diVers from heterothallic reproduction and seems to be isogamous, but more details are needed. In the raphid species Nitzschia lanceolata (Roshchin, 1990b, 1994a), each of the two mating types is capable of limited intraclonal reproduction; here, both homo and heterothallic reproduction involve trans physiological anisogamy. Mating systems have already been found that are more complex than the bipolar / types initially detected by von Stosch and Roshchin. In the marine raphid Achnanthes longipes (Chepurnov and Mann, 1997, 1999, 2000; Chepurnov and Roshchin, 1995; Roshchin, 1994b), four types of clone have been detected: clones showing relatively high levels of intraclonal reproduction that are able to mate with any other type of clone; clones of mating type 1, with low capacity for intraclonal reproduction and that are unable to mate with other clones of mating type 1, but are compatible with all other kinds of clone; clones of mating type 2, with low capacity for intraclonal reproduction and that are unable to mate with other clones of mating type 2, but are compatible with all other kinds of clone; and clones with low capacity for intraclonal reproduction that are able to copulate with cells of either mating type, or with clones capable of high levels of intraclonal
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reproduction. This diatom may be a good model for experimental study of sex determination mechanisms in pennate diatoms (Chepurnov and Mann, 2000). Some demes (biological species) within the Sellaphora pupula complex exhibit a phenomenon that, supercially at least, resembles the relative sexuality originally claimed for some other algae and oomycetes (but see Muller, 1976): In the rectangular deme (dened by Mann, 1989b), some clones are fairly consistent in their behavior, being either male or female. Others, however, behave as males when mated with female clones, but as females when mated with male clones; such clones are also capable of limited homothallic reproduction (Chepurnov and Mann, unpublished data). This complex system requires further study. Homothally has been found in some raphid diatoms in which the gametangia are clearly diVerentiated into males and females (e.g., Fig. 2IK). For example, in the elliptical deme of Sellaphora pupula, vigorous auxosporulation occurs in clonal cultures (Chepurnov and Mann, unpublished data), but within each pair of gametangia, one gametangium is male, producing an active gamete, and the other is female,) as in the heterothallic capitate deme (see above). This behavior continues after reisolation of cells to establish new subclones. At some stage during sexualization and gametogenesis, therefore, a developmental switch must operate to convert previously uncommitted cells into males and females. Similar switching may well occur in homothallic pennate species producing two gametes, but in species with trans behavioral anisogamy or isogamy, no visible diVerentiation occurs. It will be particularly instructive in such cases to study the behavior of gametes in triplets of gametangia. Here, providing the physical circumstances of sexual reproduction permit (i.e., in the absence of particularly constraining modes of plasmogamy; e.g., via copulation tubes), one might expect three auxospores to be produced in homothallic species, as each gamete would appear to have a free choice of four gametes with which to fuse. If only two auxospores are ever formed, however, this would imply that the gametangia are diVerentiated into two sexes, despite the fact that they are genetically identical, so that in a triplet of gametangia, two of the gametes (though not necessarily two belonging to the same gametangium) will be redundant. 2. Signaling and Recognition The genetic and biochemical basis of recognition and signaling mechanisms in diatoms is almost unknown. The exception is the discovery of a novel gene family (Sig) in the marine centric Thalassiosira weissogii (Armbrust, 1999), which are expressed as the cells initiate spermatogenesis. Polypeptides encoded by these genes are hypothesized to be components of the extracellular matrix and may play a role in mediating spermegg recognition.
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Sexual pheromones and their receptors probably exist, as in other algal groups (e.g., Maier, 1995; Sekimoto, 2001; Wiese, 1984), to guide gametes and gametangia during copulation. Dusenbery (2000, 2002) argues that oogamy is a likely evolutionary correlate of pheromone-mediated attraction. On the reasonable assumption that pheromone production is likely to be proportional to egg volume, the eVective size of a pheromone-producing egg as a target for sperm varies as the sixth power of the egg radius. Diatoms are known to produce chemicals like those active in sexual signaling in, for example, brown algae (Juttner, 2001; Maier, 1995; Watson, 2003), but none of these has yet been shown to play a role during the sexual reproduction of any diatom. Indeed, it has been suggested that some of these compounds may function instead to discourage grazing or parasitism (reviewed by Watson, 2003). Study of intercellular communication in diatoms should be facilitated by recent discoveries about mating systems (Section III.B.1), which will allow greater control and synchronization of sexual behavior in culture. We will summarize a few observations that hint at possible mechanisms. In dioecious pennates, the presence of compatible cells of opposite sex in a single vessel is not enough to induce gamete formation. In Licmophora ehrenbergii, Roshchin (1986) could obtain abundant sexual reproduction in well-mixed cultures of compatible clones. However, if cells of each clone were carefully inoculated on opposite sides of a 90-mm Petri dish, no auxosporulation occurred, even when the clones had grown to within 10 mm of each other. Gametogenesis began only if the distance between cells of opposite sex did not exceed the sum of their lengths (Roshchin, 1989a) (distance pairing sensu Wiese, 1969). Otherwise, cells continued dividing mitotically. In another dioecious araphid, Rhabdonema adriaticum, Rozumek (1968) attempted to induce gametogenesis in a clone of one sex by adding ltrates from cultures of the opposite sex. This failed, as did similar experiments in Licmophora abbreviata and the raphid diatom Nitzschia longissima (Chepurnov and Roshchin, unpublished data). Distance pairing between gametangia, when the partners do not normally touch, has also been reported in some other pennate diatoms, including the araphids Synedra rumpens (Geitler, 1952b), other Licmophora spp. (Chepurnov and Mann, 2004), Striatella unipunctata (Chepurnov in Roshchin, 1994a), and Fragilaria delicatissima (Roshchin, 1994a). Often, however, direct contact between partners appears to be required for the initiation of gametogenesis (contact pairing sensu Wiese, 1969), especially in raphid diatoms. During pairing of Sellaphora, cells move toward each other and then backward and forward when they are close together. Once compatible sexualized cells have come into contact, they bond rmly and move around together, sometimes in large groups of up to 10 or more, before they settle and proceed to meiosis (Mann et al., 1999, Figs. 36 and 37). In allogamous Sellaphora demes, meiosis does not usually occur in unpaired
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cells, and where a single cell is found undergoing gametogenesis, it is often possible to detect (e.g., from the pattern of bacterial settlement in nonaxenic cultures or seminatural populations) that the cell did originally have a partner. In the strictly heterothallic capitate deme of S. pupula, when large groups of cells bond to each other during the early stages of pairing, cells of the two mating types alternate within the group (Mann et al., 1999), and this occurs also in the minor deme of Neidium ampliatum (Mann and Chepurnov, unpublished data). Hence, there must be surface recognition between gametangia. The gametangia are still walled when recognition occurs, and so the reactions involved must be occurring at the surface of the frustule and its organic components. However, although contact seems necessary to stimulate gametogenesis in allogamous raphid diatoms, sexualization appears to begin earlier, as we have abundant anecdotal evidence (e.g., the reciprocal stimulation of movement by the capitate and rectangular demes of S. pupula: Mann et al., 1999) that cells do not simply rely on chance encounters between compatible cells but actively seek out a partner, apparently in response to a chemical stimulus. Pennate diatoms are diverse in their pairing congurations. One of the gametangia may attach to the other by a mucilage pad, as in the araphid Rhabdonema adriaticum (von Stosch, 1958b) and the raphid Achnanthes cf. yaquinensis (Chepurnov, Mann, and Vyrecman, unpublished data). Elsewhere, paired cells may assume strict, species-specic congurations, either side by side (e.g., in Cymbella, Sellaphora, Neidium, and Pseudo-nitzschia; Davidovich and Bates, 1998; Geitler, 1932; Mann, 1984a, 1989a; Mann et al., 1999; see also Fig. 2FK), or end to end, (e.g., in Surirellaceae; Mann, 1987, 2000). In Gomphonema, where the cells are heteropolar, the gametangia usually have opposite orientations, aligning head to tail; analogous specic orientations occur in the dorsiventral Cymbella (Geitler, 1979). All of these characteristic congurations imply a heterogeneous distribution of recognition sites over the cell surface. Elsewhere, however, the pairing conguration can be looser and more or less arbitrary, though the gametangia are often surrounded by a protective mucilage sheath [e.g., in Rhoicosphenia (Geitler, 1958; Mann, 1982b) and Dickieia (Mann, 1994a)]. 3. Plasmogamy In every diatom, plasmogamy requires the prior release of the gametes, through disengagement of the gametangial thecae. The same process occurs, of course, after vegetative mitosis, as the daughter cells separate, but it is rarely commented on (but see Kroger and Wetherbee, 2000). To some extent, the coherence of the frustule during vegetative growth and the early stages of gametogenesis can be explained by the close geometrical t between the two thecae; after all, diatom frustules often remain intact after organic material
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has been removed by oxidation, providing the treatment is gentle. However, in vivo the frustule is usually under considerable hydrostatic pressure from the turgid cell within, and when living cells are broken by excess pressure or osmotic shock, they do not usually split apart cleanly around the whole circumference of the girdle, between epitheca and hypotheca. There must, therefore, be a strong organic connection between epitheca and hypotheca, of which we know almost nothing, except for the studies of Waterkeyn and Bienfait (1987) and Kroger and Wetherbee (2000). Waterkeyn and Bienfait (1987) reported that a specic callosic b-1,3 linked glucan may be involved in maintaining the integrity of the frustule, as a gasket between the thecae, which is broken down to permit mitotic cell separation. Kroger and Wetherbee (2000) discovered that a class of proteins that they called pleuralins, which in the raphid diatom Cylindrotheca are located at the edge of the epitheca in interphase cells and bind strongly to the most abvalvar girdle bands. Pleuralins are added to the parental hypotheca after cytokinesis, before cell separation, as the hypotheca becomes the epitheca of one daughger and Wetherbee suggested (from the possibility of glycoter cell, and Kro sidic linkage between pleuralins and callose) that they may be involved in linking the girdle to the callose gasket to maintain frustule integrity, although if this is the function, one might expect the gasket to be bonded also to the hypotheca during mitotic interphase, except when the thecae are moving apart (which often occurs discontinuously during the cell cycle; Olson et al., 1986). Presumably, something similar to postmitotic separation occurs during gamete release, but the processes may not be identical, because there is often a much greater physical separation between gametes and cell wall, compared to that between mitotic daughter cells and cell wall; the gametes have often become rearranged within the gametangium before release; disengagement is sometimes only local, allowing gametes only limited access to each other; and no further development of the gametangial thecae will occur, so that maturation of the hypotheca by addition of pleuralins might be superuous. Much further research is needed, with respect to mitotic and meiotic development. Whatever the mechanism of thecal disengagement, it is likely that gamete swelling or activity also helps in many cases to separate the gametangial thecae and facilitate release. The times of gamete release and plasmogamy are of course partly dependent on how and when cells are sexually induced and on the progress of meiosis and other stages in gametogenesis. Schmid (1995) suggested that there is an internal clock controlling sperm release in Coscinodiscus granii, although it is unclear whether she meant a developmental clock, timing events in relation to when cells were sexualized (by daily alternations between two salinities and irradiances), or an endogenous clock, phasing activities relative to the solar day.
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a. Centric Diatoms In Chaetoceros didymum (von Stosch et al., 1973) and Melosira varians (von Stosch, 1951), sperm are able to penetrate the oogonia and fuse with the oocyte during prophase or anaphase of meiosis I, respectively. They then wait for the egg to mature. In contrast, meiosis is complete before sperm penetration in Stephanopyxis, Lithodesmium, Streptotheca, and Odontella (Drebes, 1966; von Stosch, 1954, 1956; von Stosch and Drebes, 1964). In Stephanopyxis palmeriana (Drebes, 1966) and Melosira moniliformis var. octogona (Idei and Chihara, 1992), in which only a small part of the egg surface is exposed for fertilization, through a crack between the oogonial thecae, the oogonium closes again almost immediately after the sperm has penetrated into the egg. In other species, it is not known how the egg protects itself from multiple penetrations of male gametes. It takes up to 56 min for the sperm (or its nucleus) to approach the female nucleus in Stephanopyxis palmeriana (Drebes, 1966) and Melosira moniliformis var. octogona (Idei and Chihara, 1992), and karyogamy occurred in 30 min in S. palmeriana. The sperm agellum is discarded almost immediately after fertilization. b. Pennate Diatoms Plasmogamy occurs only between mature gametes that have completed meiosis. The araphid Rhabdonema adriaticum is unusual in that the male gamete does not fuse with the female but only injects its nucleus into it (von Stosch, 1958b). In our observations of distance pairing in araphid diatoms, we have gained the impression that the closer the sexual partners lie to each other, the greater the chance that the male gametes will successfully fertilize the females. In Licmophora ehrenbergii (Roshchin and Chepurnov, 1994), plasmogamy is most successful when the gametangia lie opposite each other and dehisce toward each other, providing unobstructed access for the male gametes. If the gametangia lie side by side or at an angle to each other, the passage of the male gametes may be obstructed and plasmogamy can fail; the mobility of the male gametes is very restricted. Contractile vacuoles have been observed in the gametes of the freshwater araphid Synedra ulna (Geitler, 1939). In the raphid pennates, the patterns of gamete copulation and fusion are very variable. It is relatively easy to study the early stages of sexual process in natural or seminatural material, and this has allowed fertilization to be studied in many taxa (e.g., reviewed in Geitler, 1973, 1979, see also Mann, 1994a). However, with such material it is usually impossible to determine whether reproduction is intra- or interclonal, and it is not unlikely that this could aVect the mode of plasmogamy. In raphid diatoms, the gametes may or may not move bodily. If they do move, they may do so within special mucilage structures (see following), or movement and plasmogamy may occur free in the medium, provided that gametangiogamy has ensured a close association between the gametangia. This occurs, for instance, in the
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male gametes of the anisogamous, heterothallic taxa Nitzschia longissima (Chepurnov in Roshchin, 1994a) and Pseudo-nitzschia spp. (Davidovich and Bates, 1998). The gametes are also set free in some Navicula species (Mann and Stickle, 1989) and contractile vacuoles have been seen in some freshwater Navicula and Craticula species (Mann, unpublished observations Subrahmanyan, 1947). Plasmogamy can also involve pronounced contractions, rounding oV, or swelling of the gametes. Swelling alone, without movement, may be enough to bring the gametes together, as in isogamous Craticula (Mann and Stickle, 1991; Subrahmanyan, 1947), Haslea subagnita (Chepurnov, 1993), Dickieia ulvacea (Mann, 1994a), or Seminavis cf. robusta (Chepurnov et al., 2002). In many cases, after pairing but before completion of gamete formation, the gametangia surround themselves by a well-dened mucilage envelope, which presumably acts to hold the gametangia close together, protect the gametes, and restrict their migration. Among many examples are the isogamous Craticula (Mann and Stickle, 1991; Subrahmanyan, 1947), Rhoicosphenia curvata (Mann, 1982b), and the physiologically anisogamous Lyrella atlantica (Mann and Stickle, 1993) and Placoneis (Mann and Stickle, 1995). In Achnanthes sensu stricto, it is the gametes, rather than the gametangia that are surrounded by a mucilage sheath (Fig. 2A). The sheaths then unite, and plasmogamy occurs within the fused envelope (Idei, 1991; Mizuno, 1994; Roshchin, 1994b). In other raphid diatoms, plasmogamy is facilitated by the formation of discrete copulation apertures or tubes. The gametes may fuse within the copulation tube, as in isogamous Eunotia (Geitler, 1951a,b; Mann et al., 2003; see also Fig. 2CE). However, in physiologically anisogamous species, for example Neidium (Mann 1984a), some Nitzschia species (Mann, 1986) and Sellaphora spp. (Geitler, 1932; Mann et al., 1999), the gametes use the copulation tube only as a passage from one gametangium to the other. The chemical composition of the special organic structures formed to facilitate copulation and plasmogamy in raphid diatoms has not been studied, nor is it known in most cases how they are produced. The mucilage capsules around the gametangia in Lyrella, Placoneis, and related genera are produced before and during meiosis, before the gametangia dehisce (Jones et al., 2004; Mann and Stickle, 1993, 1995). Hence, because the capsules wholly surround the gametangia, they are presumably secreted over the whole surface of the cell, via the valve and girdle pores. Some of these diatoms also produce capsules during mitotic cell division, although they are usually much thinner. The copulation tubes of Eunotia (Fig. 2C, D) and some Nitzschia species grow out from one pole (Eunotia, N. amphibia) or from the center (N. recta, N. sigmoidea and related taxa) of each gametangium, and it appears that a new organic wall layer is laid down beneath the frustule beforehand. The bond between epitheca and hypotheca is then loosened locally, allowing the organic wall to be forced out between them,
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distorting the girdle greatly as it does so (Mann, 1986; Mann et al., 2003). The tubes extend by some form of tip growth until they meet and fuse, creating an open channel in which the gametes move. Some kind of chemotropism must operate to guide tube growth in Eunotia, as the tubes converge, despite great variation in the relative positions and orientations of the gametangia (Mann et al., 2003). Most pennate diatoms studied so far exhibit a single method of gamete formation and plasmogamy, which, because it is more or less constant within a species, can be a valuable source of information for diatom taxonomy (e.g., Mann, 1989a, 1993a). However, in a few cases, exible behavior of the gametes has been detected. In Dickieia ulvacea, for instance (studied in heterogeneous wild-derived material; Mann, 1994a, who also briey reviewed some other cases of reproductive plasticity), allogamous, automictic, and polyploid fusion of gametes were documented. Variation in gamete behavior has also been detected in cultures of Achnanthes longipes by Chepurnov and Roshchin (1995) and Chepurnov and Mann (1997, 1999, 2000). The typical pattern, regularly observed in natural samples and clonal cultures isolated from nature (whether reproducing intraclonally or when crossed), is isogamy: Two morphologically identical gametes are produced per gametangium in pair, which then fuse allogamously to produce two auxospores. However, during enforced inbreeding using two clones, each of them derived from one of the two initial cells produced by a single pair of gametangia, there was very diverse sexual behaviour (Chepurnov and Roshchin, 1995). The typical isogamous pattern, producing two auxospores, did occur, but it was not the most common mode of sexual reproduction. More common were variants in which only one gamete was produced per gametangium, or a 2 1 pattern. In the former type, the two gametes fused successfully to produce a single auxospore. In the latter, plasmogamy occurred between the single gamete produced by one gametangium and one of the two gametes from the other. In addition to these variants, a few gametangia were found lying alone and unpaired, containing two gametes. Unfortunately, it is not known how these became sexualized, and they may originally have been paired (as in Sellaphora; Section III.B.2), with one of the copulating cells later abandoning its partner before gametogenesis. Whatever the cause, the consequence was the production of a single auxospore through paedogamy. A new pair of sibling clones was isolated from the progeny of a single pair of gametangia in the inbred cross, in one of the few cases where a pair of gametangia produced two auxospores. Like the rst pair of sibling clones, the clones of the second inbred generation proved to be compatible (which is interesting in relation to sex determination), and again studies were made of the eVects of inbreeding on plasmogamy. Multiple pairings occurred, in which the contents of three or even four gametangia could fuse to produce polyploid zygotes (Chepurnov and Roshchin, 1995).
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4. Auxospore Development Progress in understanding auxospore development and morphogenesis in centric diatoms has been strongly associated with the development of methods to manipulate the life cycle in culture (e.g., Crawford, 1974; Hoops and Floyd, 1979; Schmid, 1995; von Stosch, 1982; von Stosch et al., 1973). In the pennates, in contrast, much has been learned from wild material (e.g., Mann, 1982a,b, 1984a, 1989c), partly because the auxospore casings are often robust and distinctive. However, greater certainty about the details of development can often be obtained from cultured material, as von Stosch showed in studies of Rhabdonema, which were the rst to reveal details of perizonium structure (von Stosch, 1958b, 1962, 1982). More recently, experimental induction of sexual reproduction in the economically important, domoic-acidproducing genus Pseudo-nitzschia (Davidovich and Bates, 1998) has allowed detailed scanning electron microscopy studies of auxospore development (Kaczmarska et al., 2000). Most studies have dealt with the formation and structure of siliceous components of the auxospore wall (silica scales, properizonium, and perizonium) and with initial cell formation. Some, however, have also provided data on nuclear processes and chloroplast behavior. In various species of allogamous centric diatoms, karyogamy of the haploid nuclei occurs before the auxospore begins to expand (e.g., Idei and Chihara, 1992; von Stosch et al., 1973). In contrast, in many allogamous raphid diatoms, karyogamy occurs during auxospore expansion or may even be delayed until expansion is more or less complete (e.g., Chepurnov et al., 2002; Round et al., 1990; Sabbe et al., 2004a), raising interesting questions about how development is regulated in the presence of two apparently independent haploid nuclei. In culture, it can be shown that there is often a correlation between the sizes of the fully expanded auxospores (and hence initial cells) and the sizes of the vegetative cells or gametangia that gave rise to them: smaller cells tend to produce smaller auxospores. This nongenetic dependence has been shown for centric species including Coscinodiscus (Nagai et al., 1995; Roshchin, 1973), Chaetoceros (Roshchin, 1976), and Skeletonema (Migita, 1967); the araphid pennates Licmophora, Striatella, and Fragilaria (Roshchin, 1994a); and the raphid diatoms Nitzschia lanceolata (Davidovich, 1994; Roshchin, 1990b, 1994a) and Sellaphora pupula (Mann et al., 1999). There is an apparently linear relationship between the lengths (diameters) of the gametangia and auxospores in some cases (e.g., Davidovich, 1994, 2001; Nagai et al., 1995; Roshchin, 1973). For N. lanceolata, Davidovich (1994, 1998) showed that the whole process of pairing, initiation of gametogenesis, plasmogamy, auxospore expansion, and initial cell formation can occur without light supply. He suggested that the successful completion of auxospore expansion (and transformation into an initial cell), driven by a swelling of the vacuoles, depends
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on the presence in the parent cells of adequate photosynthetically derived reserve material. The genetic mechanisms controlling auxospore development are unknown, but controlled breeding studies have yielded interesting observations, particularly in comparisons of outbred and inbred lineages. In the anisogamous araphid diatom Tabularia tabulata (Roshchin, 1989b, 1994a, as Synedra), two clones were crossed that were of opposite sex and had been derived from a single pair of gametangia; that is, the two clones being mated were sibling F1 clones. In the F1 crosses, as in the original parental cross, two auxospores were produced by each pair of gametangia, but only one auxospore transformed into an initial cell, whereas the other always aborted. Five new clones of the F2 generation were established from the surviving initial cells of diVerent pairs, and all ve proved to be male. One male F2 clone was back-crossed with the female F1 clone, and again only one initial cell was formed per pair of gametangia. The back-cross progeny exhibited extremely low viability. Only one of 10 cells isolated gave rise to a clone, and again, it was male. In this case, therefore, inbreeding led to selective elimination of one of the sexes during auxospore development, as well as more general loss of vigor. Inbreeding has also been shown to lead to abortion of developing auxospores and initial cells in the raphid diatoms Nitzschia lanceolata (Roshchin, 1990b, 1994a) and Achnanthes longipes (Chepurnov and Mann, 1999, 2000) with the latter showing progressive reduction in viability in successive inbred generations. Hence, the demonstration in culture that a diatom is homothallic is no guarantee that inbreeding is prevalent in the same species in nature. Jewson and Lowry (1993) reported a decline in the mean size of initial cells (from 36 mm to <25 mm) during 3 years of culturing Cymbellonitzschia diluviana. Unfortunately, no information was given on the sizes of the gametangia or the rate of size reduction, but the length of time involved indicates that the reduction may reect inbreeding depression during successive inbred generations, rather than a nongenetic cause such as the dynamics of size reduction (and hence the size spectrum of sexualizable cells). Polyploid auxospores, formed by the fusion of three or more gametes, have been reported in some raphid pennate species (e.g., Geitler, 1927, 1932; Mann, 1994a; Mann and Stickle, 1991). Their development is often abnormal but may proceed at least to the formation of complete initial cells; for example, in A. longipes (Chepurnov and Roshchin, 1995). So far, no attempts have been made to follow their further development. However, the presence within several species complexes (e.g., Sellaphora pupula, Caloneis ventricosa, Cymatopleura solea, and Neidium ampliatum: Mann, 1989b, 1999) of pairs of sympatric demes, one large-celled, the other small-celled but otherwise morphologically similar, indicates that polyploidy may often play an important part in diatom speciation.
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In natural populations of diatoms, it is often impossible to be sure whether particular auxospores have been formed sexually or not, even when opposite sexes or mating types are present. Experimental studies have shown unambiguously, however, that auxospore formation and development sometimes occur in the absence of any sexual process (e.g., Drebes, 1977a; Nagai et al., 1995; Sabbe et al., 2004b). During evolution, therefore, the link between sexual reproduction and auxosporulation has been broken in some lineages. Sexual auxosporulation must involve a tightly coordinated sequence of developmental switching and inter- and intracellular signaling, and so the evolutionary transition to asexual auxosporulation is unlikely to result from trivial genetic alteration. However, very little is known about this process. Ideally, sexual and asexual auxosporulation should be compared in a facultatively asexual species under controlled experimental conditions, but so far no such species has been identied with certainty. An alternative (which would still be valuable even if a species can be found that is facultatively asexual) is to study auxosporulation in closely related species, some of which are sexual and heterothallic, others of which are sexual and homothallic or automictic, and still others of which are asexual. One such group is Achnanthes sensu stricto (A. longipes, A. brevipes, and their relatives) and we are investigating this as a model system (Sabbe et al., 2004b). Unfused gametes are sometimes capable of transformation into auxospores (haploid parthenogenesis). Indeed, this appears to be quite common in pennate diatoms. Haploid parthenogenesis has been found in sexualized natural populations (e.g., Mann, 1994a), but the process has also been reported in culture, in the otherwise allogamous Achnanthes longipes (Chepurnov and Roshchin, 1995), Seminavis cf. robusta (Chepurnov et al., 2002), and Amphora cf. proteus (Sabbe et al., 2004a). In A. longipes, one gametic auxospore was observed to form an initial cell that divided once, and then both daughter cells died. Haploid Dickieia cells can either expand like a normal auxospore, forming a perizonium (Mann, 1994a, Figs. 5456), or they may bypass perizonium development and go on to produce an initial valve. Further development of haploid parthenogenetic cells has been obtained in two araphid species, Licmophora ehrenbergii (Roshchin and Chepurnov, 1994) and L. abbreviata (Chepurnov in Roshchin, 1994a). Both are heterothallic and anisogamous, and unfused female gametes always abort. However, unfused male gametes (see Section III.B.3) often develop into auxospores, which are smaller than their diploid equivalents. In mixed culture of clones of opposite sex in L. ehrenbergii, 20 pairs of gametangia were selected in which gametogenesis was complete but fertilization had not occurred. Female cells were removed by micromanipulation, and the development of isolated male gametangia was followed. In 10 of the 20 cells, both gametes developed into haploid auxospores, and in six cells, one gamete expanded into an auxospore
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and the other aborted; only in four gametangia did both gametes abort. Acetocarmine staining showed that one of the two haploid nuclei from meiosis II aborts quickly in both male and female gametes (this is usual in pennate diatoms, except Navicula; see Mann and Stickle, 1989), and no further nuclear divisions, transformations, or fusions occur in the male gametes during parthenogenetic development. Twenty haploid initial cells of L. ehrenbergii were isolated, but only two survived and grew in culture. Haploid cells divided more slowly than their diploid counterparts (but they otherwise appeared normal). However, because the haploid initial cells were small, the clones quickly crossed the threshold of sexually inducible size range, and attempts were made to mate them with diploid clones of known sexuality. The haploid clones were incapable of gametogenesis but could stimulate gamete production in diploid cells. Interestingly, one of the two clones stimulated gametogenesis only in female cells and the other only in male. A single haploid clone was also studied in L. abbreviata, and it initiated gamete production in diploid male clones. The presence of females and males among haploid clones derived from male gametangia indicates that, if a simple XX/XY type of sex determination mechanism is present, the males are the heterozygous sex. Licmophora may be a good genus in which to make further studies of sex determination. In addition, the capacity of haploid cells to continue dividing vegetatively, albeit slowly, indicates that the rule that diatoms are universally diplonts may not be without exceptions and that haploid parthenogenesis may be a signicant, though minor, process in diatom evolution.
IV. Applications and Interpretation A. Management of Cultures The importance of clonal cultures for microbiological research needs no explanation here, and diatoms are no exception (e.g., Mann and Chepurnov, 2004). Furthermore, everyone who tried to introduce diatoms into culture knows that the isolation and short-term maintenance in the culture of many diatom species (planktonic or benthic, marine or freshwater) do not require special techniques or unusual culture conditions, except that the medium must contain a suitable Si source (usually sodium metasilicate) (Mann and Chepurnov, 2004). Long-term maintenance of most living diatom strains is often problematic, however, because of the course of the life cycle, involving an obligatory sexual phase during auxosporulation. In the simplest case, if there is no sex, there is no size restitution and the clone will die, but the clone will also die if sexual reproduction occurs, because of genetic
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recombination, and fully heterothallic species will usually be impossible to maintain long term as clones, because no sexual reproduction can occur in the absence of compatible mating types. Thus, after a few months or years (depending on the rate of growth of the culture and the rate of size reduction per cell division), the cells will reach their minimum viable size and then die. There are ways to minimize or circumvent some of the problems. Culture maintenance strategies need to be developed for each species, based on knowledge of the principal features of its life cycle. Often, of course, these features are poorly known, but some helpful information can be obtained from the analysis of the natural sample from which a clone is isolated. Rather than throw away the sample immediately after isolation of cells, it is useful instead to inoculate a subsample of the natural assemblage into fresh culture medium and to incubate it for a few days, because sexual reproduction often occurs in these seminatural populations. It is then possible to study cytological features of auxosporulation (e.g., Mann, 1984a, 1994a,b; Mann and Stickle, 1991, 1993), and measurements can be made of gametangia and expanded auxospores, thus placing limits on the cardinal points for the species. This makes it much easier to plan when and how crosses should be attempted between clones. It may even be possible to get a preliminary indication as to whether species are likely to be homothallic or heterothallic. For example, if analysis of variance shows that paired gametangia are more similar in size than would be expected if pairs of cells were drawn at random from the whole population of sexualized cells, this could indicate that sibling cells or other closely related clonal cells are mating with each other and that the species is therefore homothallic. Over time, a clonal diatom culture is expected to reduce in size, and it is important to monitor this, so that it is known whether cells are within the sexually inducible size range, and whether they are approaching the critical minimal size (and hence the death of the culture). In many centric diatoms, auxosporulation can be expected to occur in monoclonal cultures, because the species is homothallic. At rst sight, this appears to be an advantage for the researcher, in that interclonal crosses are unnecessary, in contrast to heterothallic species. However, there are complications, because, rst, in some centric species, the size ranges for oogenesis and spermatogenesis are diVerent, though overlapping, so that the window in the life cycle during which intraclonal sexual reproduction is possible is much smaller than the whole phase during which gametogenesis can occur (e.g., Drebes, 1977a). Second, centric clones can sometimes be lost because all the cells are transformed into sperm or eggs, when there are no gametes of opposite sex present. Third, each sexual event in a clonal culture produces new recombinants (unless the parental clone was already homozygous at all loci), which means that the culture is not clonal any more. The rst problem can be mitigated by careful monitoring of cell size and provision of conditions
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appropriate for gametogenesis in that part of the sexual size range when spermatogenesis and oogenesis are both possible. The second problem can probably also be overcome because it is likely that some environmental conditions exist that suppress the induction of gametogenesis; even if gametogenesis and auxosporulation persist, it may be possible to maintain the original clone by periodic subculturing, though growth will be slow. There is, however, no remedy for the third problem and, if clonal cultures are required, new large-celled clones must be isolated from among the cells formed after auxosporulation. This has to be repeated after every instance of intraclonal sexual reproduction, although unless special mechanisms are present (e.g., reciprocal translocations; Stebbins, 1950), the inbred clones will quickly become homozygous at all loci. Unless the species is a habitual inbreeder in nature, an increase in homozygosity will probably be accompanied by inbreeding depression (Charlesworth and Charlesworth, 1987), as deleterious recessives are unmasked. It is not surprising, therefore, that some of the few experimental studies of inbred lineages give clear evidence of low viability and fertility in the F1. Examples within the centrics are given by von Stosch (1965) and von Stosch et al. (1973). Despite their homothallic behavior in monoclonal cultures, it is possible that many or most centric diatoms are habitual outbreeders in nature. Hence, to rescue a lineage, it may be necessary to test many initial cells before some are found that can successfully initiate new long-term cultures. von Stosch (1965) isolated 37 auxospores from a homothallic culture of Stephanopyxis turris, but only eight proved to be viable. Within the pennate group, experimental data reveal ever-increasing numbers of heterothallic taxa. In these, only vegetative cell multiplication can occur in monoclonal cultures, and eventually the clone will die, following critical diminution of cell size. The opportunity to maintain laboratory lineages will therefore depend on the availability of clones of opposite mating type that are within the sexually inducible cell size range. Not much is known about the sex ratios in natural populations of allogamous pennate diatoms, but there are some data indicating that it is approximately 1:1, as in most groups of organisms (e.g., Charnov, 1982). For example, in Licmophora abbreviata, Haslea subagnita, and Nitzschia longissima (Roshchin, 1994a), all of which produce two auxospores per pair of gametangia, the two sibling auxospores are of opposite mating type. So, assuming a bipolar mating system and a 1:1 ratio of mating types in natural populations, it is necessary to isolate at least seven clones of a heterothallic species before one can be reasonably certain of having both mating types (the chance that seven randomly chosen cells will be of the same mating type is 0.57 0.008) (Chepurnov et al., 2002; Mann and Chepurnov, 2004). As with centric diatoms, the possible eVects of inbreeding on naturally outbreeding taxa
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must also be taken into account; negative inuences have been reported in obligately or predominantly heterothallic pennate species (e.g., Chepurnov and Mann, 1999, 2000; Roshchin, 1989b, 1990b, 1994a; Section III.B.4). Less diYculty can be expected in managing diatoms that exhibit uniparental auxosporulation. In the centric Coscinodiscus wailesii (Nagai et al., 1995) and the pennate Achnanthes cf. subsessilis (Sabbe et al., 2004b) and Eunotia sp. from South America (Fig. 3DJ), auxosporulation is apomictic, and hence the progeny should be genetically identical to the parental cells. Thus, clones can be maintained potentially indenitely, and there is no need to reisolate new large cells after auxosporulation: Enlarged cells will simply replace the small cells over time, as the small cells auxosporulate or reach the critical minimal size. Automicts retain the potential for recombination, and so they need to be treated like homothallic diatoms, with re-isolation after auxosporulation. Unlike some homothallic species, however, habitual automicts are not expected to show inbreeding depression, because deleterious genes are purged (e.g., Barrett and Charlesworth, 1991; Crnokrak and Barrett, 2002), which is consistent with the limited experimental results available for diatoms. The cosmopolitan marine centric Melosira nummuloides exhibits uniparental auxosporulation (Fig. 3AC), and Erben (1959) demonstrated that this auxosporulation involves automixis. Roshchin isolated a clone of M. nummuloides from the Black Sea and maintained it for several years, taking it through more than 40 cycles of auxosporulation and isolating new large-celled subclones each time; no signs of inbreeding depression were observed (Roshchin and Chepurnov, 1999, as M. moniliformis var. subglobosa). Some diatoms growing in culture do not reduce in size according to the MacDonaldPtzer rule. Instead, size either remains constant or uctuates within a narrow range without reaching a critical minimum, for examples in Eunotia pectinalis var. minor (Geitler, 1932) and species of Nitzschia (von DenVer, 1949; Wiedling, 1943, 1948) and Navicula (Locker, 1950); Hendey (1945) reported loss of the silica frustule, but continued growth, in a marine Navicula. Three of the best-studied laboratory diatoms, used in a wide variety of experimental investigations, for example, Phaeodactylum tricornutum (e.g., Wilson, 1946, as Nitzschia closterium f. minutissima), Cylindrotheca fusiformis (Reimann and Lewin, 1964; Reimann et al., 1965a), and Navicula pelliculosa (Reimann et al., 1965b), also appear to lack an auxosporulation cycle, and no one has demonstrated sexual reproduction in any of them (Mann and Chepurnov, 2004). However, avoidance of cell size reduction in certain circumstances in culture does not necessarily mean that there is no sexual phase in the life cycle. In the centric Melosira nummuloides (Erben, 1959), there is a lower threshold of size below which cells divide vegetatively without cell size reduction; above the threshold, however, they can auxosporulate. In the pennate Achnanthes cf. angustata from the Black Sea (Chepurnov
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and Mann, unpublished observations), clones can persist in culture, uctuating in cell size but never reaching a critical minimum; no sex occurs in clonal cultures, but crosses have revealed that this diatom is sexual and heterothallic. In Section III.A.1, we described how experimental alteration of size has been used to show that the transition to sex within a clone is cell size dependent, not age dependent. Another important application of this is to articially shorten the life cycle. In genetic studies (e.g., laboratory experiments of speciation; Rice and Hostert, 1993), it is often desirable to minimize the generation time. In diatoms, it normally takes months (or even years) until a clone initiated from an initial cell is able to auxosporulate and give rise to the next generation. However, this time can be shortened to a few weeks or less using abrupt cell size reduction (e.g., Drebes, 1966; Mann et al., 2003; Roshchin, 1994a; von Stosch, 1965; see also Fig. 3KS). A further use of this method is to produce subclones of diVerent sizes, so that it is easier to discriminate between clones in mating experiments (Mann et al., 2003). Initiation of vegetative cell enlargement can also be useful by allowing cell size to be partially restored without genetic change. Periodic enlargement in this way can allow clones to be maintained indenitely (e.g., Roshchin, 1994a; Roshchin and Chepurnov, 1992; von Stosch, 1965). Unfortunately, not all species can be manipulated successfully.
B. Evolutionary Aspects The evolution of the diatom life cycle is still controversial (Edlund and Stoermer, 1997). The nearest relatives of the diatoms are the Bolidophyceae (Guillou et al., 1999), but it is not known whether these are sexual organisms, nor whether they are diploid or haploid. Other autotrophic heteroknotophytes exhibit a bewildering array of life histories (van den Hoek et al., 1995), and outgroup comparisons with diatoms are diYcult. The well-developed, obligate sexuality of most diatoms is consistent with Ottos (2003) analysis, that diplontic life cycles and high frequencies of sex will co-occur. One of the most striking features of sexuality in diatoms is the diVerence between the two main diatom groups, centric and pennate. There are three facets to this variation: oogamy versus anisogamyisogamy, apparently spontaneous gametogenesis versus gametangiogamy and the transfer of function (Mann, 1993a) from gametes to gametangia during the evolution of pennates, and homothallism versus true heterothallism (with apparently genetic sex determination). The diVerences are abrupt, and we have no idea how to ll the evolutionary gap between the pennates and their nearest centric relatives. Perhaps the basal pennate clade (the Asterionellopsis Rhaphoneis clade [Medlin et al., 2000]) and other taxa lying near the
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centricpennate boundary (e.g., Toxarium; Kooistra et al., 2003a,b) will provide the answers, and this is an important focus for future research. In addition, there is an urgent need to reassess the centric group in relation to sexual reproduction, now that it is clear that the centrics are paraphyletic. More evidence is required on basic aspects of sexual behavior and the life cycle from a wider spectrum of species, representing all the major centric lineages. For example, do the basal diatom lineages possess, as the primitive state, a well-regulated, classical size reductionrestitution cycle, with an obligate link between auxosporulation and sexuality, or did the classical pattern develop only later, and if so, how many times? The use of the sexual phase as a primary source of information to determine systematic relationships is less worthwhile than it was, because of the introduction of molecular systematic methods, which can provide more taxonomic characters more quickly than reproductive biology or morphology or cytology. In the past, however, knowledge of sexual behavior was sometimes crucial in solving important taxonomic problems. Perhaps the best example was the discovery that the Cymatosiraceae have agellate gametes, which prompted a reevaluation of their classication and the recognition that they are centric diatoms, despite their elongate shape (Hasle et al., 1983). A counterexample, showing how faulty interpretation can cause confusion, is the claim of oogamy in the raphid pennate Pseudo-nitzschia (Subba Rao et al., 1991), which was immediately controversial (Rosowski et al., 1992) but could not be disproved until Davidovich and Bates (1998) applied the strategies developed by Roshchin (1994a) for studying pennate mating systems. At a lower taxonomic level, molecular phylogenies can now allow us to examine how particular modes of sexual reproduction are distributed among diVerent diatom lineages and how they may have evolved. For example, rbcL data indicate that there is a large monophyletic group of raphid diatoms, containing Cymbella, Gomphonema, Lyrella, Petroneis, and Placoneis, whose sexual reproduction is characterized by the formation of robust, often multilayered mucilage capsules around the gametangia, trans physiological anisogamy, and auxospore expansion parallel to the apical axes of the gametangia (Jones et al., 2004). rbcL data also conrm expectations that the formation in Sellaphora of only one gamete per gametangium is a derived state, which prompts evaluation of what factors might select for this change, especially because of the major handicap that the number of initial cells produced per pair of gametangia is halved. In centric diatoms, there is considerable variation in spermatogenesis (Jensen et al., 2003) and oogenesis, but no convincing relationship is currently evident between the pattern of variation and phylogeny. Perhaps there is none, but more likely spermatogenesis and oogenesis require more detailed study. At the lowest level, the remarkable variation in breeding systems between closely related species in Achnanthes (e.g., Sabbe et al., 2004b) and Sellaphora
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(Mann et al., 1999, 2004) will help us, through comparative evolutionary developmental (evo-devo) studies, to understand the origin and molecular mechanisms of sexual reproduction in diatoms, and also the process of speciation. The discovery of heterothallism in many raphid pennates undermines claims that most or all diatom species are ubiquitous; that is, that they are present everywhere but often in vanishingly small, undetectable numbers, and that there are therefore no true biogeographical (as opposed to ecological) patterns of distribution in diatoms (Finlay et al., 2002). In a heterothallic diatom, because sex is obligate and must occur to allow auxosporulation, and because cells that do not undergo auxosporulation will, in most cases, die when they reach the critical minimum size, there are obvious limits to rarity and dispersal. For sex to occur, not only must compatible mating types be present in a particular water body but they must also simultaneously be at the right stage of the life cycle, and they must be in suYciently densities to allow mating. Extreme local rarity is not an option. It will be interesting to see whether there is a correlation between maximum local abundance and the ability to reproduce sexually: Are very rare species (if there are any) able to persist only because they exhibit uniparental types of auxosporulation or avoid size reduction?
C. Biological Species Concept The discovery, circumscription, and formal description of species, and the provision of ways to identify species, are perhaps the key functions of taxonomy, aVecting almost all aspects of biology. Not surprisingly, therefore, the development of a satisfactory conceptual basis for species denition has long been a very hot topic. For sexually reproducing organisms, the Biological Species Concept (BSC) is still the concept to beat, as it encapsulates a mechanismreproductive isolationthat explains the incontestable fact that there is a level (ill-dened though it may be in particular cases) below which variation is essentially reticulate and above which it is essentially hierarchically organized (Mann, 1999, pp. 438441). Mayrs (1982) formulation of the BSC is that a species is a reproductive community of populations (reproductively isolated from others) that occupies a specic niche in nature. In particular cases, there can be severe problems in translating the BSC into practical species denitions, such as where reproductive isolation is incomplete, or where it is dependent on geographical isolation (as opposed to intrinsic barriers to gene ow between populations), or where attempts are made to dene species boundaries that are valid over an evolutionarily signicant time period, or where the organisms are not sexual (e.g., prokaryotes, bdelloid rotifers). Nevertheless, reproductive
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isolation, however caused, is undoubtedly a crucial factor in the origin and maintenance of species. Current knowledge indicates that the vast majority of diatoms are biparental sexual organisms, with obligatory sexual reproduction at intervals of a few months to a few years. The BSC should therefore apply to them particularly well, and in practical terms this indicates breeding experiments as a primary test of species limits, or the indirect (but often very powerful) alternative, of detecting interbreeding via genetic analysis (e.g., using microsatellites or other suitable markers; Rynearson and Armbrust, 2000, 2004). Until recently, however (reviewed by Mann, 1999), diatom species taxonomy has developed without any such tests, and it has been correspondingly unstable and unsatisfactory. Of course, it is wholly impractical to use interbreeding as a criterion for species delimitation except in the context of hypotheses about relationships derived from other, less intensive approaches, such as inspection of morphology (with light or electron microscopy), morphometrics, or molecular ngerprinting methods. It is impossible to make every possible cross between every organism or population. However, within particular species complexes, mating tests can and should be part of the taxonomists tool kit, especially now that courier services can transport material across the globe in a few days (during which a suitably prepared diatom culture can remain perfectly healthy). We have used breeding criteria in studies of the Sellaphora pupula, Neidium ampliatum, Amphora copulata, Caloneis ventricosa, Cymatopleura solea, and Achnanthes brevipes groups. The results indicate that the pre-1990 species-level taxonomy severely underestimates diatom diversity (e.g., Behnke et al., 2004; Mann, 1989b, 1999; Mann et al., 1999, 2004; Montresor et al., 2003) have begun to apply the same approach in Pseudonitzschia). We recommend that breeding tests are applied even to apparently automictic and apomictic diatoms, to establish whether the lack of biparental sex is obligate or facultative (e.g., Sabbe et al., 2004b). Ideally, the capacity for gene exchange should be tested not only by experimental crosses between clones but also by examination of the viability and reproductive potential of the F1 and succeeding generations, and by attempts to backcross hybrids to their parents. So far, this has rarely been attempted, but the results of one longer-term study are instructive. Two morphologically distinct varieties of Achnanthes brevipes, from diVerent types of habitat, were crossed successfully to form an F1, which was then grown on and tested for its ability to produce an F2 and backcross with its parents: Neither was possible (Chepurnov and Mann, in Mann, 1999, p. 472), showing that, although there is no prezygotic barrier to mating, there is signicant reproductive isolation between the varieties. Detailed genetic analysis would be necessary to show that there is no gene ow between them, but the habitat diVerence (salt-marsh vs. rocky foreshore) must also
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reduce the likelihood of interaction; indeed, this may be why no prezygotic barrier has evolved.
D. Ecological Dynamics Because the sexual phase is an obligate stage in the life cycles of most diatom species, diatom population biology is crucially dependent on the periodicity, timing, intensity, and cost of sexual reproduction. Sexual reproduction will obviously have a profound eVect on the genetic structure of populations, and research on this is beginning to accelerate (e.g., Medlin, 2003; Rynearson and Armbrust, 2000, 2004), but it also has immediate relevance to the dynamics of population growth, because of the interruption to vegetative growth and division (Lewis, 1983), wastage of gametes or gametangia, death of gametes and zygotes because of unmasked lethal mutations, and probably increased losses to sedimentation/burial, grazers, and parasites. Few quantitative or even qualitative data are available on these costs. Hiltz et al. (2000) provided information on losses of sexualized Pseudo-nitzschia cells in culture (from all causes: Genetic, epigenetic, and environmental) during gametogenesis, plasmogamy, and auxospore development; they found a maximum of 26% conversion of gametes into initial cells, with much lower rates in unfavorable light regimes. Jewson (1992b) recorded that male stages of Stephanodiscus are preferentially grazed. Furthermore, the diVerent surface area: volume ratios of large, recently auxosporulated cells, compared to those of gametangial cells, will have eVects on a variety of physiological and physical processes (e.g., nutrient uptake; Potapova and Snoeijs, 1997), sedimentation rates, and viscous drag. Hence, if diatom populations are quantied solely by the numbers of each species present per unit volume or area, with no data on size spectra within those species, important ecological information will be missed. There have been several studies of size spectra and auxosporulation in natural populations (Mann, 1988, reviews relevant work; see Crawford, 1995; Jewson, 1992a,b; Potapova and Snoeijs, 1997; Skabichevskij, 1929). However, for a full understanding of the control and signicance of the life cycle in nature, it will be necessary to develop molecular markers for diVerent life cycle and sexual stages and occurrence and eVect of sexual reproduction in natural populations (e.g., Armbrust, 1999; Armbrust and Galindo, 2001), which will undoubtedly be facilitated by the availability of whole genome sequences, such as that of Thalassiosira pseudonana (Armbrust, 2003). Identication of suitable markers will require laboratory studies of sexualization and auxosporulation, and these will also be needed to inform interpretation of sexual reproduction in nature, for example, by providing data on mating systems, which have a major inuence on population genetics (e.g., Holsinger, 2000). Culture studies also
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have the potential to help quantify some of the costs of sex that may apply in nature. For example, it should be possible to estimate those associated with interruption of synthesis or those caused by the genetic load of lethal mutations.
V. Concluding Remarks To make signicant progress in understanding the life cycles and sexuality of diatoms, it is essential to grow a wider variety of species in culture than are currently available, and this is also true for other aspects of diatom biology and biotechnology (Lebeau and Robert, 2003a,b; Mann and Chepurnov, 2004). However, as we have shown, it is also true that success in rearing and managing diatom cultures largely depends on understanding and controlling the sexual phase. Diatom sexuality has often been regarded as mysterious, seen and studied only by a few initiates. However, the experiences of previous researchers, especially Geitler, von Stosch, and Roshchin, show how cultures can be manipulated experimentally in life cycle studies, and they provide an excellent foundation for future work. As a simple proof, most of the illustrative material included in this chapter, covering aspects of gametogenesis and auxospore development in oogamous, anisogamous, and isogamous diatoms, was obtained specially for this publication within a few months, using the instructions of previous authors and our own experience. However, it must not be assumed that there are no major surprises left in diatom reproductive biology. In many cases, we are forced by lack of information to generalize on the basis of information from just a few tens of species, representing a minority of diatom genera. If the species studied were an unbiased sample, extrapolation would be reasonable, but it is clear in many cases that the sample is not unbiased. For example, sexual reproduction is easier to study in natural populations of attached diatoms than in the epipelic or epipsammic diatoms growing associated with sediments, and reports of sexual reproduction in pennate diatoms are correspondingly dominated by attached species (e.g., Geitler, 1932, 1973, 1984); but it does not take much imagination to see that these two types of substratum must oVer very diVerent environments for diatom sex. Furthermore, it is always easier to discover homothally in pennate diatoms (because cells can pair and reproduce intraclonally) than it is to show that a species is heterothallic: If two clones do not mate, is this because they are outside the sexual size range, of the same mating type, not switched on by the environmental conditions provided, or belong to diVerent species? Overall, then, all generalizations about the diatom life cycle and sexuality, including our own, need to be viewed with caution, and intensive studies of
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the molecular biology of sexual reproduction in model species (e.g., Thalassiosira; Armbrust, 1999) need to be accompanied by a determined attempt to gain a basic knowledge of the life cycle in species drawn from all the major evolutionary groups and ecological categories of diatoms.
Acknowledgments
Financial support of this research was largely provided by FKFO projects G.0292.00 and G.0435.02, and BOF-project GOA 12050398 (Ghent University, Belgium). K. Sabbe is a Senior Research Fellow with the Fund for Scientic Research-Flanders (FWO-Flanders, Belgium). Parts of this work have been aided by the Australian Biological Resources Study and INTAS contracts 93-3605 and 93-3605-ext.
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Experimental Studies on Sexual Reproduction in Diatoms

Victor A. Chepurnov,*,{ David G. Mann,{ Koen Sabbe,* and Wim Vyverman*

International Review of Cytology, Vol. 237 0074-7696/04 $35.00

Copyright 2004, Elsevier Inc. All rights reserved.

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

SEXUAL REPRODUCTION IN DIATOMS

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Victor A. Chepurnov,,{ David G. Mann,{ Koen Sabbe, and Wim Vyverman*