Untitled

Experimental Studies on SexualReproduction 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, cell–cell 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 91 Copyright 2004, Elsevier Inc.0074-7696/04 $35.00 All rights reserved.

92 CHEPURNOV ET AL.

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 con-

sequence 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 benefits 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 significant 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 reflect 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 fixation 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

SEXUAL REPRODUCTION IN DIATOMS 93

penetrates to allow photosynthesis, and they have diversified 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 adap-

tation 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 significant degree attributable

to their refined 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 them-

selves 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 diversification 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 briefly, 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 auxo-

sporulation 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

94 CHEPURNOV ET AL.

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 reproduc-

tion (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), confirming diploidy.

Overall, diatom meiosis exhibits no special features (Geitler, 1927; Mann

and Stickle, 1995). A synaptonemal complex has been observed in Lithodes-

mium (Manton et al., 1969b) and Gomphonema (Drum et al., 1966). During

meiotic prophase, the nucleus swells considerably and becomes more


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 MacDonald–Pfitzer rule

(MacDonald, 1969; Pfitzer, 1971; see also Crawford, 1981; Hustedt, 1967;

Pickett-Heaps et al., 1990; Round, 1972; Tomaschek, 1873), from the British

and German authors who first 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 silicified. 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 valves—the hypovalves of the new cells—are at first

contained within, and are therefore smaller than, the parts of the epithecae

96 CHEPURNOV ET AL.

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 MacDonald–Pfitzer 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 repro-

duction. Auxosporulation involves linkage of two important processes—

genetic recombination and cell size restitution—into 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 forma-

tion, 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 compo-

nents embedded within it or beneath it. For any final 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 plasmoga-

my, 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 auxospor-

ulation continue dividing mitotically until they become critically small and

finally die. This was first noted by Geitler (1932) in experimental cultures of

pennate diatoms.

Rule 5 states that the capacity of cells to become sexualized is size depen-

dent: Only comparatively small cells can be triggered to switch from mitotic

cycles to meiosis. The first clear demonstration of this rule was by Geitler

(1932), although it had been suspected earlier (e.g., Bachmann, 1904; Pfitzer,

1871), and further important studies were made by von Stosch (1965). It was


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-specific 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 phylo-

genies (e.g., Kooistra et al., 2003b). However, in some aspects of the life

cycle, particularly the mechanisms of gametogenesis and sexual reproduc-

tion, 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 classification, 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

98 CHEPURNOV ET AL.

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 significant 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

confirmed 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 monophy-

letic (holophyletic sensu; Mayr and Ashlock, 1991). However, for our dis-

cussion, 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 first 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

first, 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 under-

standing 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. 11–12) supported Went’s 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


Went–Geitler hypothesis was confirmed by von Stosch (1950, 1951), who

documented oogamous auxosporulation in Melosira varians. The next 25

years saw the confirmation 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 uniflagellate sperm (which has an unusual 9 þ 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 nucle-

us 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. 1A–E; 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 ‘‘impoverish-

ing’’ divisions immediately precede the formation of the cells (spermatogo-

nia) that undergo meiosis to produce haploid gametes. In Coscinodiscus

granii, C. wailesii, and Odontella regia, A.-M. Schmid (1995) reported a

formation of specific four-cell chains (‘‘tetrads’’) and considered these pre-

stages 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

FIG. 1 Planktonic centric diatoms from North Sea, monoclonal cultures. (A–C) 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)

100 CHEPURNOV ET AL.


Stephanopyxis [Drebes, 1966; von Stosch, 1954] and Chaetoceros [von Stosch

et al., 1973]). No thecae are produced in some other genera (e.g., Coscino-

discus [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 flaccida 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 uniflagellate 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 first 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. (F–J) 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 ¼ 50 mm (A–E) and 25 mm (F–J).


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 9þ 0 structure

of the axoneme, diatom sperm are also unusual among the zoids of hetero-

kont 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 spermatogonan-

gium, sperm swim actively towards compatible eggs or oogonia. The mecha-

nism of attraction and recognition between egg and sperm is unknown

(Section III.B.2). Sperm attach to the female cell first by the flagellar 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 Stephano-

pyxis (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 trans-

formed into an auxospore. In the many centric diatoms with radially sym-

metrical, 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 unsilicified (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 cen-

trics with bi- or multipolar valves, or asymmetrical valves, the shape of

expanding auxospore is modified 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 pro-

duced (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 Bacterias-

trum hyalinum (Drebes, 1972); the first formed is always discarded. In Lepto-

cylindrus danicus (French and Hargraves, 1985), the auxospore is first


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; Strel’nikova, 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 first

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 beha-

vior 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 morpho-

logically and behaviorally. However, the primary copulation is between

gametangia, not the gametes themselves (i.e., araphid pennates exhibit game-

tangiogamy), no flagellate 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

(¼Synedra) tabulata, and Fragilaria delicatissima (Roshchin, 1994a). A single


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 significantly 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 ooga-

mous, apparently because of the diVerentiating mitoses, but this prejudges

homology and downplays the absence of flagella. We follow von Stosch’s

later (1982) classification 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 organic–siliceous wall, like that

in centrics, consisting of an organic matrix in which silica scales are embed-

ded. 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, unspe-

cified 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.


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 (Strel’nikova, 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 first demonstrated (key works are

Geitler 1927, 1932; Karsten, 1912; Klebahn, 1896).

Like the centric and the araphid pennates, the raphid diatoms are predom-

inantly 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 simi-

larity 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. 2F–H).

One or two gametes (depending on species) are produced per gametangium

(Fig. 2A–K), 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 first visible step in sexual

reproduction is gametangiogamy. However, whereas in araphids the sex-

ual 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 devel-

opment 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

FIG. 2 Patterns of allogamous sexual reproduction in raphid pennate diatoms in culture. (A–E)

Morphological and physiological isogamy. (A, B) Achnanthes cf. angustata from North Sea,

homothallic reproduction. (A) Paired gametangia, each containing two gametes. Note the



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. 2C–E, 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’’ anisoga-

my). 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 confines of the ‘‘pas-

sive’’ 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. 2F–H, 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). Be-

havioral anisogamy is also exhibited by some species producing a single

gamete per gametangium, for example, Sellaphora (Fig. 2I–K; 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. (C–E) 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. (F–K) Physiological

anisogamy. (F–H) 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: ga-

metes 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. (I–K) 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 ¼ 20 mm (A–H) and 10 mm (I–K).


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 configurations, 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 diatoms—Gomphonema parvulum and Neidium aYne—and

then only very briefly (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 develop-

ment 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 stud-

ied, 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 significant variations on the main theme. Silicification 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 perizo-

nium. 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


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 occur-

ring 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 auxospor-

ulation 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 modification 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 (¼tetrad) 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. 3A–C). 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 (¼Schoederella)

schroederi (Drebes, 1977a) and Ellerbeckia arenaria forma arenaria (Schmid

and Crawford, 2001) but there is insuYcient information about nuclear

FIG. 3 Uniparental auxosporulation (A–J) and abrupt cell size reduction (K–S). (A–C) 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



development to discriminate between automixis and asexual auxosporula-

tion (Section II.E). Autogamy has never been demonstrated unambiguously

in araphid pennates: Synedra vaucheriae may be autogamous or paedoga-

mous (Geitler, 1958), whereas a report in Licmophora spp. (Kumar, 1978) is

also insecure (Chepurnov and Mann, 2004). Within the raphid series, autog-

amous 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. 3D–J; see also

Drebes, 1977a; Geitler, 1973; Nagai et al., 1995; Sabbe et al., 2004b). How-

ever, 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 mod-

ification 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

(¼undulatus) (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. (D–J) 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. (L–O) 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 finally 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. (P–S)

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 ¼ 20 mm (A–C, L–O), 10 mm (D–K), and 100mm (P–S).


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 de-

scribe 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, Wiedling’s (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 ofDiatom Sexuality

Experimental studies of cultures have been essential for progress in several

fields, 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


reproduction and auxosporulation, and the development of molecular mar-

kers 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;

Pfitzer, 1871) that auxosporulation is a ‘‘size-related’’ process. However, the

first 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 confirmed 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

MacDonald–Pfitzer 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. 3K–O) or by surgical procedures, thus creating

subclones of markedly diVerent sizes, but having the same age since auxo-

sporulation (Fig. 3P–S). 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


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 collea-

gues revealed that, although many centrics are homothallic, the size ranges

for oogenesis and spermatogenesis are generally diVerent, although they

overlap: oogenesis starts first, then both eggs and sperms are produced

(when new clonal cultures of large cells can be established), and finally,

when the cells are small, only sperms are formed. However, there are excep-

tions, 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 arcua-

tum, 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 intra-

clonal 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 first 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 Cosci-

nodiscus janischii, in clonal cultures in which the cells were 150–170 mm,


oogamy resulted in initial cells of 275–293 mm. After a short period of size

reduction, however, cells of 230–260 mm were found to be capable of trans-

formation into oogonia, although auxospores were not observed (Roshchin,

1975, 1994a); spermatogonangia of C. janischii of 230–260 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 influenced 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 sign-

ificant 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 (Bruckmayer-

Berkenbusch, 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 English-

language 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 m�2 s�1;

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),


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 m�2 s�1 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 light–dark 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 identification may be incorrect: Drebes,

1977a, p. 275), spermatogenesis (and oogenesis, though over a narrower

range of temperature and irradiance) was promoted by a light–dark cycle

of 8:16 h, in a wide range of temperatures (9�–26.7

�C) and irradiances

(10.5–1745 mmol photons m�2 s�1; figures 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 weissflo-

gii, for example, Armbrust et al. (1990) found that spermatogenesis is inhib-

ited by continuous light at 250 mmol photons m�2 s�1 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 m�2 s�1. 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 m�2 s�1 and 20�C. Higher irra-

diances are inductive in Stephanopyxis turris: von Stosch and Drebes (1964)

recorded massive induction of sexual stages with transfer from about 5–6 to

80–100 mmol photons m�2 s�1 and fresh culture medium, with a simulta-

neous increase in temperature from 15�

to 21�C. Stephanopyxis palmeriana,

Lithodesmium undulatum, Helicotheca tamesis, and Bellerochea malleus be-

have essentially similarly (Drebes, 1966, 1977a; Manton et al., 1969a). Tem-

perature 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


clone placed in continuous light (though with fluctuations 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 pur-

poses: To induce bulk spermatogenesis of L. undulatum for electron micros-

copy, Manton and von Stosch (1966) used transfer from a 16:8 h light–dark

cycle of 6 mmol photons m�2 s�1 and 15�C to a 14:10 h cycle of 400 mmol

photons m�2 s�1 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 reflects the fact that the

products of reproduction—auxospores—are 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 auxosporula-

tion, viz. Leptocylindrus (Hargraves and French, 1983), does show nutrient

dependence, reacting to low N. By contrast, the freshwater diatoms Stepha-

nodiscus 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-Martınez et al., 1992), although no experimental culture data

are available to support this.

Finally, an extra layer of complexities may be caused by biotic interac-

tions, 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, cell–cell 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


important. We will return to cell–cell 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

(June–October) 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 significant 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 uni-

punctata (Davidovich and Chepurnov, 1993) and the raphids Cocconeis

scutellum (Mizuno and Okuda, 1985), Nitzschia lanceolata (Davidovich,

1998), N. longissima (Davidovich and Chepurnov, 1993), and Pseudo-

nitzschia 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 tempera-

ture (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 identified 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 weissflogii

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.


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 ‘‘het-

erothally’’ (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 diVer-

ences 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 ¼ 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 obser-

vations 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-compatibil-

ity’’ 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 deter-

mination) 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 (¼homothallic)


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 significant ‘‘negative’’ observations, such as von Stosch’s 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 Drebes’s 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 pen-

nates 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 (¼Navicula) 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 heterothal-

lic. 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. 2C–H) 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 reflects diVerentiation of clones into ‘‘male’’ and ‘‘female’’


(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 pro-

duced 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. 2F–H). However, heterothally also occurs in

raphid diatoms exhibiting morphological and physiological isogamy, for

example, Haslea subagnita (Chepurnov, 1993), Eunotia bilunaris and E. tro-

pica (Mann et al., 2003; see also Fig. 2C–E), 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 confirm this and

to reveal the sex-determination mechanism.

Roshchin also discovered pennates that are predominantly heterothallic,

but also have some capacity for intraclonal reproduction (‘‘monoecious-

dioecious’’ 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 intraclo-

nal 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 mat-

ing 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 þ/� 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 intraclo-

nal 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


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, superficially at least, resembles the ‘‘relative

sexuality’’ originally claimed for some other algae and oomycetes (but see

Muller, 1976): In the ‘‘rectangular’’ deme (defined 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 game-

tangia are clearly diVerentiated into males and females (e.g., Fig. 2I–K). For

example, in the ‘‘elliptical’’ deme of Sellaphora pupula, vigorous auxospor-

ulation 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 ‘‘capi-

tate’’ deme (see above). This behavior continues after reisolation of cells to

establish new subclones. At some stage during sexualization and gametogen-

esis, 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 circum-

stances 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 weissflogii (Armbrust,

1999), which are expressed as the cells initiate spermatogenesis. Poly-

peptides encoded by these genes are hypothesized to be components of the

extracellular matrix and may play a role in mediating sperm–egg recognition.


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 auxospor-

ulation 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 anoth-

er dioecious araphid, Rhabdonema adriaticum, Rozumek (1968) attempted to

induce gametogenesis in a clone of one sex by adding filtrates 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 firmly 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


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 recognit-

ion 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, sexua-

lization appears to begin earlier, as we have abundant anecdotal evidence

(e.g., the reciprocal stimulation of movement by the ‘‘capitate’’ and ‘‘rectan-

gular’’ 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 configurations. 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). Else-

where, paired cells may assume strict, species-specific configurations, 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. 2F–K), 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 specific

orientations occur in the dorsiventral Cymbella (Geitler, 1979). All of these

characteristic configurations imply a heterogeneous distribution of recogni-

tion sites over the cell surface. Elsewhere, however, the pairing configuration

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 fit between the two

thecae; after all, diatom frustules often remain intact after organic material


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 specific 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 ‘‘pleur-

alins,’’ 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 daugh-

ter cell, and Kroger and Wetherbee suggested (from the possibility of glyco-

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 dur-

ing 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; disen-

gagement 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

superfluous. 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 depen-

dent 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.


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, respec-

tively. They then ‘‘wait’’ for the egg to mature. In contrast, meiosis is

complete before sperm penetration in Stephanopyxis, Lithodesmium, Strep-

totheca, 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

5–6 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 flagellum 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


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 contrac-

tions, 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-defined 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 cur-

vata (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. 2C–E). 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 stud-

ied, 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 gametangi-

um, 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,


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 chemo-

tropism 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, flexible behavior of

the gametes has been detected. In Dickieia ulvacea, for instance (studied

in heterogeneous wild-derived material; Mann, 1994a, who also briefly

reviewed some other cases of reproductive plasticity), allogamous, automic-

tic, 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 auxo-

spores, did occur, but it was not the most common mode of sexual reproduc-

tion. More common were variants in which only one gamete was produced

per gametangium, or a ‘‘2 þ 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 first 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 oc-

curred, in which the contents of three or even four gametangia could fuse

to produce polyploid zygotes (Chepurnov and Roshchin, 1995).


4. Auxospore Development

Progress in understanding auxospore development and morphogenesis in

centric diatoms has been strongly associated with the development of meth-

ods 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 first to reveal details of perizonium

structure (von Stosch, 1958b, 1962, 1982). More recently, experimental in-

duction of sexual reproduction in the economically important, domoic-acid-

producing 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 perizo-

nium) 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, auxo-

spore expansion, and initial cell formation can occur without light supply. He

suggested that the successful completion of auxospore expansion (and trans-

formation into an initial cell), driven by a swelling of the vacuoles, depends


on the presence in the parent cells of adequate photosynthetically derived

reserve material.

The genetic mechanisms controlling auxospore development are un-

known, but controlled breeding studies have yielded interesting observations,

particularly in comparisons of outbred and inbred lineages. In the anisoga-

mous 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 auxo-

spore 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 five 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 auxo-

spores 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 homothal-

lic 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 diluvi-

ana. 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 reflect inbreeding depression during successive inbred genera-

tions, 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 mor-

phologically similar, indicates that polyploidy may often play an important

part in diatom speciation.


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 unam-

biguously, 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 facul-

tatively asexual species under controlled experimental conditions, but so far

no such species has been identified 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 Ach-

nanthes 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 auxo-

spores (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. 54–56),

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 hetero-

thallic and anisogamous, and unfused female gametes always abort. Howev-

er, 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


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 significant, 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


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 first 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, first, 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 trans-

formed into sperm or eggs, when there are no gametes of opposite sex

present. Third, each sexual event in a clonal culture produces new recombi-

nants (unless the parental clone was already homozygous at all loci), which

means that the culture is not clonal any more. The first problem can be

mitigated by careful monitoring of cell size and provision of conditions


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 game-

togenesis 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 num-

bers 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 ¼ 0.008)

(Chepurnov et al., 2002; Mann and Chepurnov, 2004). As with centric

diatoms, the possible eVects of inbreeding on naturally outbreeding taxa


must also be taken into account; negative influences 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 ‘‘unipa-

rental’’ 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. 3D–J), auxosporulation is apomictic,

and hence the progeny should be genetically identical to the parental cells.

Thus, clones can be maintained potentially indefinitely, 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 auto-

micts 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 avail-

able for diatoms. The cosmopolitan marine centric Melosira nummuloides

exhibits uniparental auxosporulation (Fig. 3A–C), and Erben (1959) demon-

strated 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

MacDonald–Pfitzer rule. Instead, size either remains constant or fluctuates

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 tricornu-

tum (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 auxospor-

ulate. In the pennate Achnanthes cf. angustata from the Black Sea (Chepurnov


and Mann, unpublished observations), clones can persist in culture, fluctuat-

ing 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

artificially shorten the life cycle. In genetic studies (e.g., laboratory experi-

ments 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. 3K–S). 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 indefinitely (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 Bolidophy-

ceae (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

Otto’s (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 anisogamy–isogamy, 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 fill 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


centric–pennate 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 reduction–restitution 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 intro-

duction of molecular systematic methods, which can provide more taxonomic

characters more quickly than reproductive biology or morphology or cytolo-

gy. 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 flagellate gametes, which prompted a

reevaluation of their classification 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 confirm 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


(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 ecologi-

cal) 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, there-

fore, the development of a satisfactory conceptual basis for species definition

has long been a very ‘‘hot’’ topic. For sexually reproducing organisms, the

Biological Species Concept (BSC) is still the concept to beat, as it encapsu-

lates a mechanism—reproductive isolation—that explains the incontestable

fact that there is a level (ill-defined though it may be in particular cases)

below which variation is essentially reticulate and above which it is essential-

ly hierarchically organized (Mann, 1999, pp. 438–441). Mayr’s (1982) formu-

lation of the BSC is that a species is ‘‘a reproductive community of

populations (reproductively isolated from others) that occupies a specific

niche in nature.’’ In particular cases, there can be severe problems in trans-

lating the BSC into practical species definitions, such as where reproductive

isolation is incomplete, or where it is dependent on geographical isolation

(as opposed to intrinsic barriers to gene flow between populations), or

where attempts are made to define species boundaries that are valid over

an evolutionarily significant time period, or where the organisms are not

sexual (e.g., prokaryotes, bdelloid rotifers). Nevertheless, reproductive


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 bipa-

rental sexual organisms, with obligatory sexual reproduction at intervals of a

few months to a few years. The BSC should therefore apply to them particu-

larly 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 micro-

satellites 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 morphol-

ogy (with light or electron microscopy), morphometrics, or molecular finger-

printing methods. It is impossible to make every possible cross between every

organism or population. However, within particular species complexes, mat-

ing tests can and should be part of the taxonomist’s 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, Cymato-

pleura 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 Pseudo-

nitzschia). 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

significant reproductive isolation between the varieties. Detailed genetic

analysis would be necessary to show that there is no gene flow between

them, but the habitat diVerence (salt-marsh vs. rocky foreshore) must also


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, plas-

mogamy, 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 quantified 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 significance 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). Identification 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

influence on population genetics (e.g., Holsinger, 2000). Culture studies also


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 significant 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 illustra-

tive material included in this chapter, covering aspects of gametogenesis and

auxospore development in oogamous, anisogamous, and isogamous dia-

toms, was obtained specially for this publication within a few months,

using the ‘‘instructions’’ of previous authors and our own experience. How-

ever, 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 repro-

duce 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


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 Scientific 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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