16TH INTERNATIONAL SYMPOSIUM, 25 AUG.-l SEPT. 2000, ATHENS & AEGEAN ISLANDS PROCEEDINGS 2001 (A. ECONOMOU - AMILLI, ED.), 601 PP., UNIVERSITY OF ATHENS, GREECE

A review ofauxospore structure, ontogeny and diatom phylogeny

I Kaczmarska, I., I Ehrman, J.M. and S.S. Bates 2

1. Department ofBio1ogy, Mount Allison University, Sack:ville, NB, E4L IG7, Canada

2. Fisheries and Oceans Canada, Gu1fFisheries Centre, P.O. Box 5030, Moncton, NB;EIC 9B6, Canada

Abstract Interest in phylogenetic relationships within the has been recently rejuvenated by a number of reports based on the molecular approach that do not agree with the current phenetic phylogenies. Ontogeny has been successfully applied in studies of the evolutionary history of many animal and seed plants but has not been extensively tested with respect to diatoms. Here we propose such an approach. The literature indicates a considerable diversity in auxospore ontogeny and structure. Three basic types of auxospores occur: isometric (usually with siliceous scales in an organic wall), anisometric (with scales in the primary wall and a properizonium) and bilateral (with an organic· primary wall and a siliceous perizonium). These three types ofauxospores corroborate the recently developed molecular phylogeny of diatoms (Medlin et ai. 1997) where two major clades are apparent in this tree: Diatom Clade I (DC I) contains centric species with tubular processes located in most cases at the periphery of the valve. These diatoms produce isometric auxospores covered with scales. Clade 2 (DC2) consists of a diverse assemblage of diatoms, usually defined as multipolar centrics and pennates, and is divided into two sub-clades. The first (DsCzJ consists of mainly multipolar centrics with tubular processes located approximately at the center of the valve, and these diatoms produce anisometric auxospores with properizonia. The other (DsCZb) clusters pennate genera that produce bilateral auxospores with perizonia. Thisjntriguing correlation between the clades and auxospore types warrants further studies of auxospore fme structure. We suggest that auxospore ontogeny and structure may be useful characters in defining high level taxa such as classes or orders in a natural system ofdiatoms.

Background Many studies have investigated the ongm of diatoms and the phylogenetic relationships among major lineages within the diatoms (e.g., Simonsen 1979, Round & Crawford 1981, Mann & Marchant 1989, Round et aI. 1990, Harwood & Gersonde 1990, Edlund & Stoermer 1997). In spite of this, neither aspect has yet been conclusively resolved, although considerable advances have been recently made in some areas using molecular techniques (Bhattacharya et aI. 1992, Medlin et aI. 1997b, Guillou et aI. 1999). Interest in phylogenetic relationships within the diatoms has been rejuvenated by several reports based on molecular approaches (Medlin et aI. 1993, Philippe et aI.

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1994, Kooistra & Medlin 1996, Medlin et al. 1996a,b, Medlin 1997a), partially because these findings differ significantly from phenetic phylogenies that have prevailed in diatom literature for decades. Modem thinking about diatom evolution was initiated by Round & Crawford (1981, 1984), who hypothesized a monophyletic origin of diatoms and their phylogenetic connection to scale-bearing progenitors. This hypothesis is supported by molecular work where diatom monophyly is consistently shown by trees based on a variety of sequences (Bhattacharya et af. 1992, Medlin et aJ. 1997b, Daugbjerg & Anderson 1997, Van de Peer & De Wachter 1997, Guillou et aJ. 1999). Round et af. (1990, p. 122) pointed out how significant it would be to find scales in the gametes, and these have been recently found in the pennate diatom Pseudo-nitzschia multiseries (Hasle) Hasle (Kaczmarska et af. 2000) and possibly in oocytes ofActinocyclus sp. (Idei, pers. comm. 2000). Identifying the protoctistan ancestors of diatoms seems to be even more elusive. A close ancestry between diatoms and a number of taxa has been speculated over the last several decades: e.g., xanthophytes (Pascher 1921), Synurophyceae (Korshikov 1930, Round & Crawford 1989), parmalean algae (Mann & Marchant 1989), and lately, oomycetes (Schmid 1988, Leipe et af. 1994, Medlin et ai. 1997b, Sogin & Silberman 1998). Silica metabolism is present in all these groups (Bhattacharya et af. 1992), and some are diplobiontic and possess life-forms with scales. Recently, a molecular sister group of diatoms was serendipitously discovered among planktonic picobiflagellates (Guillou et af. 1999), clearly suggesting that microbial diversity has not yet been adequately researched foc a putative relative of diatoms. Phylogenetic relationships within the diatoms are equally difficult to unravel. Traditionally, valve symmetry, morphology and a few other characters (e.g., cytological, reproductive) were used to infer evolutionary rebdedness among the taxa. Most broadly accepted modem systems were developed by Hustedt (1930-1966), Simonsen (1979) and Round et ai. (1990). All these systems adopted basically a view of diatom diversity that supported Schutt's distinction between radially symmetric Centrics and bilateral Pennates. Different systematic ranks have been given to these two groups, commonly either classes or orders, e.g., Centra1es and . This distinction was substantiated by two modes of , oogamy in centric and isogamy in pennate diatoms. However, this system was not universally accepted (Hendey 1964, Patrick & Reimer 1960). Somewhat earlier, pa1aeodiatomists working in the former Soviet Union collectively erected a third taxon, Mediales, to accommodate many fossil diatoms whose valve symmetry and morphology could not be reconciled with the systematic division ofdiatoms into the two orders Centrales and Pennales (Proshkina-Lavrenko 1949-1950). Many ofthe taxa assigned to the Mediales were species with a non-circular valve outline but with a radial pattern of valve ornamentation. Ontogeny has been successfully applied in studies ofthe evolutionary history ofmany animals and seed plants. The feasibility of this approach has not been extensively tested with diatoms, although it has already been postulated by Kociolek & Williams

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(1987). Here, we theorize on the significance of auxospore ontogeny in reconstructing phylogenetic relationships within diatoms and suggest such an approach to identitY their majorJineages. We fir~t summarize the observations on auxospore structure and development (based on a review of the literature and on our own research), and then place this information within the context of classical diatom systematics, contrasting this with what is currently known about the molecular phylogeny ofdiatoms.

Definition ofterms Although some ofthe following terms are defined in several publications (Anonymous 1975, Ross et al. 1979, Stosch 1982, Round et al. 1990, Kaczmarska et al. 2000), they may not be obvious to all readers. Here, we define these terms as they are used in this paper. 1. : a with a diploid nucleus resulting from fusion oftwo gametes. 2. Auxospore: a cell destined to restore large size individuals in a diatom population. Depending on species and the stage of development, the auxospore may contain one diploid nucleus (zygote) or two haploid nuclei (dikaryon state). In addition, in some auxospores a varying number ofpycnotic nuclei may also be present. 3. : protective layer external to the plasma membrane. Here, cell wall is used interchangeably with the term cell envelope. 4. Properizonium: a secondary auxospore wall, built of siliceous rings produced inside and coalescent with the primary, scaly wall (Stosch 1982). These bands, rings, loops or hoops are laid at various orientation relative to the direction of the initial valve expansion. 5. Perizonium: secondary wall of the bilateral auxospore consisting of closed or open rings laid at fixed orientation relative to the direction ofinitial valve expansion. 6. Isometric auxospores: auxospores with a dilatable cell wall, normally containing scales of various size and shape (sometimes even elongated); covering the entire surface area ofa usually spherical cell. 7. Anisometric auxospores: auxospores with partitioned walls, one part (primary) containing scales, the other (secondary) properizonium; the scaly wall and properizo­ nium each cover different areas ofthe protoplast. 8. Bilateral auxospores: auxospores with perizonia and sometimes with scales. 9. Initial valves: valves produced within the auxospore, may number 2-5, depending on species. . 10. Initial : first two valves produced within the auxospore. 11. Initial cell: cell liberated from the auxospore and capable of at least one mitotic ---\. division outside the auxospore. Initial cell mayor may not contain an initial frustule. 12. Auxospore ontogeny: a development of auxospore from fertilization (plasmogamy ofgametes) to maturity (immediately prior to liberation ofinitial cell).

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Auxospore structure and development The auxospore is unique to the diatoms. Most commonly the cell results from allogamOlis fusion, and its major function in diatom life history is to restore the larger sizes in a vegetatively propagating population. Typically the auxospore cell wall consists oftwo biogenic constituents: organic matter and siliceous elements, though the relationships between the two differ from those in vegetative cells (Stosch 1982, Round et ai. 1990). The sequence of deposition oforganic and siliceous elements into the auxospore wall and the shape and spatial orientation ofthe siliceous components is different in each ofthe three types of auxospores: isometric, anisometric and bilateral. During growth, an auxospore undergoes a sequence of developmental stages (ontogeny) that results in one ofthese three auxospore types, depending on the diatom species. The review presented in this paper is based on sources that could provide: a) comparable level of structural detail and b) unambiguous reconstruction of walles) development in a sexual auxospore. Occasional, anecdotal accounts on auxospores based exclusively on light microscopy (LM) observations were excluded here due to their lack of information on the fine structure and sequence of development of auxospore wall components. LM documentation is too cursory for structures that are minute, lightly silicified, often superimposed on each other or parental , and frequently all together enveloped in a gelatinous matrix. The necessity of resolving such structures is of the utmost importance in the case of centric auxospores where there is a great deal ofdiversity in structural elements which are not resolvable even in the best light optics. For example, compare LM images of the auxospore wall in diatoms as large as Coscinodiscus centralis Ehrenberg (Drebes 1974, p. 36.) to SEM images from Schmid (1994, p. 147) for C. granii Gough, to observe scales that are not detectable using LM. Not all stages of auxospore development are equally well understood. Thus, by necessity this summary is limited to auxospore wall structure and development and to the form of initial valves. The patterns of diatom sexual reproduction (sometimes also referred to as auxosporulation but in fact representing patterns in gametogenesis and gamete behavior) have been recently reviewed by Mann (1993) who concluded that 'although Hustedt's four main categories do reflect important evolutionary changes, the variation pattern indicates that these changes have occurred several times quite independently, and that some changes are probably easier than others'. As such they are not reliable predictors ofevolutionary relatedness ofthe high rank taxa. The behavior of the auxospore nucleus/nuclei is well documented for many pennates through the work ofMann (summarized in Round et ai. 1990). This work demonstrates that karyogamy may be postponed (in raphid and araphid diatoms) until later stages of auxospore development, and that no obvious evolutionary pattern emerges from .. -.'---. considering this character. In centric diatoms, the·minute size of the sperm nucleus makes observations more difficult, and presumably because ofthis there are only a few studies that documented the whole process with necessary scrutiny, e.g., Drebes (1969), Stosch et ai. (1973). Nonetheless, Drebes (1977b) was able to conclude that as

-156- AUXOSPORE STRUCTURE, ONTOGENY AND DIATOM PHYLOGENY in pennates, karyogamy in centrics does not need to immediately follow plasmogamy. Moreover, the very limited number of species studied would not inspire confidence in any pattern that may arise. Below we summarize what is known about auxospore ontogeny in each of the three types ofauxospores (isometric, anisometric and bilateral), with the focus on auxospore wall and form ofinitial valves.

Isometric auxospores (sensu Drebes 1977b), Fig. 1. These are globular or ellipsoidal cells throughout the existence of the auxospore. The auxospore wall is dilatable, contains organic matter and embedded siliceous scales. Their shape results from even growth of the cell in most or all directions (when not constrained by parental theca). Growth is accomplished by deposition of new scales and organic matter as the auxospore expands. These auxospores are found in representatives ofthe following genera: Melosira (Crawford 1974, 1975; Stosch 1982), Cyclotella (Hoops & Floyd 1979), Coscinodiscus (Schmid 1994), Stephanopyxis, Actinoptychus (Stosch 1982), Ellerbeckia (Schmid, pers. comm. 2000), Orthoseira (Crawford 1975, Roemer & Rosowski 1980), Actinocyclus (Idei et al. 2000) and Thalassiosira (Schmid 1984). A few species, e.g., Stephanodiscus niagarae Ehrenberg (Edlund & Stoermer 1991) and Aulacoseira herzogii (Lemmermann) Simonsen (Jewson et al. 1993), were reported lacking siliceous scales. However, some of this work dealt with auxospores at late stages of development (already containing initial cells) and so some ofthe earlier components ofthe wall might have been lost. The youngest auxospore walls are delicate, easy to distort and exclusively organic (Fig. lA; Crawford 1975, Hoops & Floyd 1979). They later become more rigid when siliceous scales are added (Fig. IB). Auxospore scales may differ in number, size, shape and surface morphology (Round et al. 1990), but most often they are small, somewhat rounded, radial and siliceous (Crawford 1975, Hoops & Floyd 1979, Stosch 1982). Recently, elongated band-scales were found in Ellerbeckia arenaria (Ralfs ex Moore) Crawford (Schmid, pers. comm. 2000), but they were integrated into the scaly layer rather than separated into a properizonium. When illustrated, scales are located close to the plasma membrane (Crawford 1974) and thus called secondary walls by Stosch (1982). At maturity (Fig. 1c), the isometric auxospore contains two hemispherical initial valves. These valves will become the initial cell frustule. Future vegetative divisions of this cell will restore species specific form and ornamentation to the valves. Spherical initial frustules are produced even by species with cylindrical vegetative frustules. In some species, one or both oogonial theca remain attached to the mature auxospore, resulting in a small elevation on the otherwise hemispherical initial valves. It is not clear what becomes of the female gamete wall in this type of auxospore. Possibly, it becomes the outermost part ofthe auxospore envelope. The ontogeny of isometric auxospores has not been well documented (particularly the earliest stages), so we are not in a position to determine precisely when scales are produced during auxospore development and to what extent some gamete wall

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components are retained by the auxospore. Recent work ofIdei et al. (2000) shows that siliceous scales in Actinocyclus sp. are produced immediately after fertilization and perhaps even in oocytes (ldei, pers. comm. 2000). Because ofthis, Fig. lA-C illustrates a simplified representation of the auxospore ontogeny inferred from Crawford (1974, 1975), Stosch (1982) and our own observations, but we do not attribute it to any given species.

A

B

eecondary auxospore wall with scalee

Fig. 1. Schematic representation of the stages in development of an isometric auxospore inferred from Crawford (1974, 1975) and von Stosch (1982). A young auxospore with organic walls (A) expands to surpass the size of the parental theca (B). At this stage the wall may contain siliceous scales. A mature auxospore contains an initial frustule with form and morphology that is very different from the vegetative pattern (C).

Anisometric auxospores (sensu von Stosch 1982), Fig. 2. When young, these auxospores are globular but change shape later in development. This change of form is facilitated by construction of a specialized secondary wall, the properizonium. Available documentation indicates that in many species, the young auxospore exits the oogonial theca soon after fertilization, but remains attached to its external wall. The mechanism of attachment is unknown. As soon as it is free of the parental theca, but still in the globular stage, the auxospore wall consists of organic matter containing small, round siliceous scales, and is called the primary auxospore wall (Fig. 2A). Several distinct types ofscales were reported to concentrate either in the innermost or outer parts of the wall (Stosch 1982), depending on species. Further enlargement of anisometric auxospores is facilitated by formation of a secondary wall that complements, but does not replace, the primary scaly wall.

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B secondary auxospore wall ¥"(properizonium)

initial --- cell

Fig. 2. Schematic representation of the stages in development of an anisometric auxospore. The genus is used as an example, based on work by von Stosch et at. (1973) and von Stosch (1982). A young auxospore free of the parental theca, at the globular stage of enlargement (A) has a primary wall that may contain organic matter and siliceous scales. Older auxospores at the secondary wall stage (B) have added the properizonium to accommodate cell enlargement. A mature auxospore (C) contains an initial frustule whose general shape is similar to that ofvegetative cells.

The secondary wall contains elongated siliceous structures (cups, hoops, loops or rings) which compose the properizonium. In Chaetoceros species, the properizonium bands are arranged in a convex fan-shape (Fig. 2B). In other genera, however, these bands may be organized into concentric rings or a variety of loops covering the part of the auxospore protoplasm that protrudes from the confines ofthe primary wall. In some species, these loops and hoops may be very long and elaborately wrapped around a multipolar protoplast (Lithodesmium). In other species, long and well developed bands may be accompanied by shorter ones. Although this type of auxospore possesses the greatest variety of scale types, types of bands, and arrangement of these elements relative to the plane of auxospore expansion, all anisometric auxospores examined using EM share the same basic design. In all anisometric auxospores, the primary wall is ruptured by the properizonium, which complements, but does not replace it (Stosch ~ .., 1982). The relative proportion of the surface area of the auxospore protoplast covered by either primary or secondary wall varies between taxa (Stosch 1982). As with isometric auxospores, the fate of the gametal walls after fertilization is unclear in the ontogeny ofthis auxospore. When mature, anisometric auxospores produce two, or in some species three, initial

-159- KACZMARSKA, I., EHRMAN, J.M. AND S.S. BATES valves. When three initial valves are produced, the first initial valve does not contribute to the initial cell wall. The general shape of initial cell frustule is similar to that of vegetative cells (Fig. 2c). In addition to species ofthe genus Chaetoceros, auxospores with properizonium are known in representatives of the genera Attheya, Bellerochea, Biddulphia, Lithodesmium, Odontella and Amphitetras (Drebes 1977a, Stosch 1982). Cerataulus laevis (Ehrenberg) Ralfs auxospores likely belong here as well (Ehrlich et al. 1982). A unique fonn of properizonium is known from hyalinum Lauder, where one half of the bipartite wall is comprised of many convex concentric rings, while the other half is cup-shaped. In addition, the entire properizonium is enclosed in the persisting primary auxospore wall (Drebes 1972). It is not known if this primary wall contains any scales. The fine structure and composition ofthe cup-shaped part of perizonium is also unknown. Fig. 2 illustrates a simplified representation ofthe ontogeny of an auxospore seen in a species of the genus Chaetoceros and is inferred from the work of von Stosch et af. (1973) and von Stosch (1982).

Bilateral auxospores (sensu von Stosch 1962), Fig. 3. As in the two previous types, bilateral auxospores are initially spherical and possess an organic primary wall. In contrast to the two previous types, however, the primary wall ofthe typical bilateral auxospore is often reported to be free of siliceous scales (Stosch 1982, Round et al. 1990). Although the presence of silica could not be confirmed, scales were recently found in primary wall of auxospores in Pseudo-nitzschia multiseries (Fig. 3A-C; Kaczmarska et al. 2000) and Diploneis papula (Schmidt) Cleve (Idei, pers. comm. 2000). In another raphid pennate, the presence of silica in these scales has been confirmed by EDS analysis (Kaczmarska, unpublished). The primary wall ofthe auxospore is ruptured by a secondary wall, the siliceous perizonium, in later stages of development, when the expanding protoplast outgrows the primary wall (Fig. 3A). The ruptured remnants may later dissolve (Stosch 1982) or be retained at the perizonium apices as caps (Fig. 3B), presumably for protection (e.g., Mann & Stickle 1989) or, as in the case of Neidium affine (Ehrenberg) Pfitzer, to control perizonium development (Mann 1984). The perizonium is seen as a highly specialized (secondary) wall of the pennate auxospore that arose later in the evolution of diatoms and may be homologous to properizonium in anisometric auxospores (Stosch 1962, 1982). It is constructed ofa set oflongitudina1 and/or transverse bands ofvarying width which may fonn a closed ring, but most commonly are open. The rings usually grow in a bipolar, telescoping fashion which leads to the production of a rod-shaped mature auxospore (Fig. 3B,C). The auxospores of species of Cymatopleura and Surirella are exceptions; their growth is unidirectional, but both nevertheless produce bilateral auxospores (Thaler 1972, Stosch 1982, Mann 1987). Mature auxospores may contain 2-5 initial valves, though two are most common. The youngest initial valves become the initial cell frustule with a shape generally similar to that of vegetative cells. The initial cell frustule ornamentation is

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often imperfect (Fig. 3c). All bilateral auxospores follow this general sequence of development (Kaczmarska et al. 2000). A simplified and schematic representation is given in Fig. 3A-C, for which the auxospore of Pseudo-nitzschia multiseries serves as an example.

A shed gamete v" wall

passive gamete anchoring scale-'"

parental theca ~

B

Fig. 3. Schematic representation of the stages in development of a bilateral auxospore, based on Kaczmarska et al. (2000). Pseudo-nitzschia multiseries is used as an example. Young auxospores are globular and rupture along the equator ofthe primary wall (A). The gametal scale facilitates attachment to the parental theca. Older auxospores with a developing perizonium (B) have the primary auxospore wall retained as apical caps. Note scales lining the inner primary wall surface. The largest auxospores display a fully mature perizonium and two initial frustules (C).

There is considerable variation in the fine structure and orientation of siliceous elements in the perizonium and, to a certain extent, in the primary auxospore walls. A combination of these elements may be used to classify perizonia into several categories. Much more work is needed before these specific categories may be considered in an evolutionary context. First, we will discuss those bilateral auxospores with scales in the primary walls, then we will address variations in perizonia. i Unlike other bilateral auxospores, the primary walls of two species of Rhabdonema persist throughout the life span of the auxospore and seem to be thickened by the addition of both organic matter and siliceous scales as the auxospore expands. The retention of the primary wall of the auxospore is thought to approximate an ancestral (transitional) characteristic among evolutionarily less advanced araphid pennate diatoms such as R. arcuatum (Lyngbye? Agardh) Kiitzing (Stosch 1962, 1982). How­ ever, the bilateral auxospore of Neidium affine was also reported to contain fused siliceous scales in the organic primary wall (Mann 1984), which controlled expansion

-161- KACZMARSKA, I., EHRMAN, J.M. AND S.S. BATES of the perizonium. Mann later speculated that these scales may be homologous to the scales of centric auxospores (in Round et al. 1990), similar to Rhabdonema species. Thus, the auxospores ofRhabdonema species may be said to be bilateral with a lasting primary wall in contrast to all other bilateral auxospores where the primary wall is split about the cell circumference. The fact that scales are now known from the auxospores ofdiatoms as diverse as those discussed above (Rhabdonema, Diploneis, Pseudo-nitzschia), indicate that scales may be more commonly present in the primary walls ofbilateral auxospores than previously believed. Furthermore, the ability to produce scales is known in raphid pennate diatoms under some unusual circumstances (Schmid 1980, Lee & Xenophanthes 1989). All bilateral auxospores form a perizonium composed of a variety of siliceous bands. Depending on the type of bands and their orientation with respect to the elongation axis of the auxospore, we distinguish three basic types of perizonium architecture: transverse, longitudinal and converging (Kaczmarska et al. 2000). Transverse and longitudinal perizonia may occur alone or together in the same auxospore. Transverse and longitudinal perizonia, the most common type, occur together in e.g., Rhabdonema arcuatum (Stosch 1962), Rhoicosphenia (Mann 1982a) and Neidium (Mann 1984). Transverse perizonia alone occur in e.g., Rhabdonema adriaticum Kiitzing (Stosch 1962), Licmophora (Mann 1982b), Craticula (Cohn et al. 1989), Caloneis (Mann 1989), (Mann & Stickle 1989), and Pseudo-nitzschia (Kaczmarska et al. 2000). Longitudinal perizonia alone are present in Achnanthes longipes Agardh and A. javanica f. subconstricta (Meister) Hustedt (Stosch 1982, Mizuno 1994). Converging perizonia are found only in Cocconeis pellucida Grunow (Mizuno 1998, figs. 15, 16). Auxospore wall characteristics of these and many other taxa are summarized in Kaczmarska et al. 2000. The bilateral auxospore develops outside the parental theca. Many benthic pennate diatoms, such as most of the species discussed above, confine the developing auxospores in a mucilaginous envelope (Mann 1993). In general, the pennate auxospore shares more similarities with the anisometric auxospore of multipolar centric diatoms than with the isometric auxospore of radial centric diatoms (Fig. 4). Evolutionary significance of these similarities was also discussed by von Stosch (1982).

Auxospore structure and diatom phylogeny This review shows that the general structure of auxospores in diatoms is strongly consef¥ed within major lineages of diatom cell architecture. Globular (isometric) auxospores with dilatable, scaly walls are known among species whose valves are mostly circular and possess processes at the valve periphery. These species are called radial centric by Medlin and coworkers (Medlin et al. 1996a, Kooistra & Medlin 1996). Anisometric auxospores with properizonia are most common among centric diatoms whose valves are bi(multi)polar and most have processes located at the valve center. These are designated bi(multi)polar centrics (Medlin et al. 1996a, Kooistra & Medlin 1996). Bipolar (bilateral) auxospores are typical ofpennate species.

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Classical Phylogeny Valves Centrics Pennates Radial Multipolar Araphid Raphid '-----~II' radial symmetry bilateral symmetryID flagellated male gametes non-flagellated male gametesI Molecular Phylogeny (ssu rRNA) Clade 1 Clade 2a Clade 2b Auxospores ~ ~

auxospores auxospores WITh with scales bands (rings, hoops)

Fig. 4. Diagrammatic summary of the relationship between auxospore structure and classical versus molecular phylogenies (only selected diagnostic characteristics are included, see text for further discussion). Only auxospore walls are represented in the bottom panel.

These auxospore types do not correlate well with classical diatom systematics based mainly on phenetic characters ofvegetative cells and type of sexual reproduction. The centrics sensu Simonsen (1979) and Hustedt (1930-1966) produce two distinctly different types of auxospores: isometric and anisometric. The division of diatoms into one centric () and two pennate classes ( and Bacillariophyceae; Round et al. 1990) requires that one class of diatoms produces two different types ofauxospores, while two different classes of diatoms produce basically the same type ofauxospore (Fig. 4). In many plants and animals, reproductive structures are highly conserved because of the potential cost of diminished reproductive success for those organisms that produce mutated ~(and usually inferior) reproductive structures. Owing to this property, reproductive characters are often used to circumscribe taxa of all levels; e.g., details of conjugation in desmids and zygnemales, pre- and post-fertilization structure of carpogonia in floridean red algae, and the structure of flowers in flowering plants. The usefulness of post fertilization reproductive structures in diatom systematics has been largely unexplored until now. The three types of diatom auxospores correlate best with the recently advanced molecular phylogeny of diatoms (Fig. 4), which established a paraphyletic origin of centric diatoms. This is inconsistent with several classical higher order diatom systematics where centric diatoms are consistently regarded as a coherent evolutionary

-163- KACZMARSKA, I., EHRMAN, J.M. AND 8.8. BATES unit. However, this view has. been debated repeatedly (e.g., Proshkina-Lavrenko 1949-1950, Patrick & Reimer 1960, Hendey 1964). Based on comparison of small subunit (ssu) rRNA sequences of 30 diatom species, Medlin et al. (1997b) divides diatom genera into two major clades: a small Diatom Clade 1 (DC I) and large Diatom Clade 2 (DC2). A similar molecular tree was generated based on mitochondrial gene sequences (Ehara et al. 2000). The most significant difference between molecular and classical systematics is the segregation of diatoms with the centric pattern of valve ornamentation into two separate clades. This segregation is also the focus ofour review. Clade' 1 consists of the centric species with radial valve symmetry and tubular processes located mostly on the periphery of the valve (Medlin et al. 1996a, Kooistra & Medlin 1996). We note that it also groups the diatoms that reproduce oogamously and produce isometric auxospores with relatively simple, dilatable walls carrying siliceous scales integrated into one envelope (e.g., Melosira, Aulacoseira, Coscinodiscus and Actinocyclus). It is unfortunate that there is no report on the fine structure and development of auxospores in Rhizosolenia and Corethron, the sister group to two other branches in this clade. However, for one of the members of this group, Corethron hystrix Hensen, Cupp (1943, fig. 34b) reported an LM based image of an auxospore stage that suggests the presence ofa spherical initial frustule, one with characteristic spines. Thus, further enlargement of the initial cell should be facilitated by elongation of the pervalvar axis by addition of scale-like girdle bands. Properizonium would not be needed in this auxospore. Clade 2 contains a more diverse assemblage of diatoms usually defined as bi(multi)polar centrics by Medlin et al. (l996a) and pennates. The topology of this branch is relatively well resolved only in the case ofpennates. In this large clade, two sub-groups. emerge: the first (DC2a) contains mainly multipolar centrics with tubular processes located approximately at the center of the valve and which reproduce oogam.ously. These diatoms produce anisometric auxospores with a complex secondary wall (properizonium) containing a great variety of siliceous bands and scales (e.g., Chaetoceros, Ditylum, Odontella and Cymatosira). Only the globular auxospores with scaly walls seen in thalassiosiroid diatoms fit poorly into DC2.. as do their radial valves and ornamentation. However, this difficulty may be reconciled, for example, by convergent evolution or retention ofancestral characters.

The other sub-group (DC2b) tightly clusters pennate genera, all of which possess non-flagellated gametes, produce bilateral auxospores with simpler primary walls than in the previous sub-group and have a simple arrangement of siliceous bands in the perizonium (e.g., Nitzschia). The molecular phylogeny of a variety of microorganisms indicates that many characters previously used to define higher level taxa may be convergent and are therefore not helpful in reconstructing natural phylogenetic lineages; e.g., the pigment composition and chloroplast structure in broadly defined chrysophytes or green algae and the land plants. In these and many other groups, a new set of informative characters was identified that allowed construction ofa biologically coherent assembly

-164- AUXOSPORE STRUCTURE, ONTOGENY AND DIATOM PHYLOGENY among these morphologically diverse organisms; e.g., a mitotic phragmoplast unifies charophycean algae and terrestrial plants into viridaeplantae, while mastigonemate flagella defines heterokontae that include diatoms, chrysophytes, brown kelps and oomycetes. Taylor (1999) argued convincingly that such characters serve as a 'control for protistan molecular phylogeny'. We suggest that ontogeny and structure of the diatom auxospore may allow this because auxospores are remarkably similar in diatom taxa that are not immediately related (genera, families), yet they provide enough diversity to allow separation of higher level taxa (classes or orders). With further studies, including a greater range ofspecies, particularly those producing isometric and anisometric auxospores, the structure and development of the auxospore may become useful even in elucidating evolutionary relationships between taxa within each of the three clades. Diatom progenitors, the Dr-diatoms, are hypothesized to be small, spherical or hemispherical cells covered with numerous rounded scales containing silica (Round & Crawford 1981). Some of these scales were thought to have developed into two large structures (valves) and several smaller elements (cingulae) over the course of evolution, during the differentiation of these organisms into true diatoms. Isometric auxospores with small rounded scales, such as those seen in species of the genus Stephanopyxis, may be ontogenetically reminiscent of such an evolutionary stage (Round & Crawford 1981, Round et aI. 1990). This stage seems to be still present in anisometric auxospores and in some bilateral auxospores, but is restricted to the earliest steps of auxospore development. Diatoms using anisometric or bilateral auxospores to restore the larger sizes of cells, evolved a system of bands (properizonium and perizonium) to facilitate enlargement oftheir valves. The evolutionary trend in auxospore development among centric diatoms seems to be that of increasing complexity: from isometric forms with a variety of scales dispersed through the primary and secondary walls, to the anisometric auxospore of multipolar centrics with a secondary wall containing a properizonium with loops and rings (Stosch 1982). The trend seems to be opposite in pennates. Assuming that the Rhabdonema-type of auxospore illustrates a possible evolutionary transition between species producing anisometric and bilateral auxospores (Stosch 1982), bilateral auxospores with perizonia evolved into progressively simpler forms, i.e., in raphid diatoms there are fewer scales and fewer types ofelongated elements. The evolutionary trend from spherical Dr-diatoms to diatoms with isometric auxospores, to those with anisometric, and then to those with bilateral auxospores, is thus one logical scenario that can-be drawn from existing data.

Acknowledgments We thank Claude Leger for his assistance in growing Pseudo-nitzschia .sexual cells. We thank one anonymous reviewer for careful reading of this manuscript. This research was supported by an NSERC Research Grant awarded to I.K.

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References ANONYMOUS (1975). Proposal for standarization of diatom terminology and diagnoses. Nova Hedwigia, Beiheft 53, 323-354. BHATTACHARYA, D., MEDLIN, L., WAINRIGHT, P.O., ARIZTIA, E. V., BIBEAU, C., STICKEL, S. K. & SOGIN, M. L. (1992). Algae containing chlorophyll a+c are paraphyletic: molecular evolution analysis ofthe Chromophyta. Evolution, 46, 1801-1817. COHN S. A., SPURCK, T. P., PICKETT-HEAPS, J. D. & EDGAR, L. W. (1989).Perizonium and initial valve formation in the diatom Navicula cuspidata (Bacillariophyceae). Journal ofPhycology, 25, 15-26. CRAWFORD, R. M. (1974). The auxospore wall ofthe marine diatom Melosira nummuloides (Dilw.) C. Ag. and related species. British phycological Journal, 9, 9-20. CRAWFORD, R. M. (1975). The frustule ofthe initial cells of some species ofthe diatom genus Melosira C. Agardh. Nova Hedwigia,Beiheft 53,37-50. DAUGBJERG, N. & ANDERSEN, R. A. (1997). A molecular phylogeny of the heterokont algae based on analyses ofchloroplast-encoded rbcL sequence data. Journal ofPhycology, 33, 1031-1041. DREBES, G. (1969). Stephanopyxis turris (Centrales) Geschiechtliche Fortpflanzung Differenzierung der Oogonien Auxosporenbildung. Institutfiir Wissenschlaftlichen Film, E 1344: 3-10. Gottingen. DREBES, G. (1972). The life history of the centric diatom Bacteriastrum hyalinum Lauder. Nova Hedwigia. Beiheft 39, 95-11 O. DREBES, G. (1974). Marine . 186 pp. Thieme, Stuttgard. DREBES, G. (I977a). Cell structure, , and sexual reproduction of Attheya decora West (Bacillariophyceae, Biddulphiineae). Nova Hedwigia, Beihefte 54, 167-178. DREBES, G. (1977b). Sexuality. In: The Biology ofDiatoms.(D. Werner, ed.). Botanical Monographs 13: 250-283, University ofCalifornia Press, Berkeley. EHRLICH, A., CRAWFORD, R. M. & ROUND, F. E. (1982). A study ofthe diatom Cerataulus laevis­ the structure ofthe auxospore and the initial cell. British phycological Journal, 17, 205-214. EDLUND, M. B. & STOERMER, E. F. (1991). Sexual reproduction in Stephanodiscus niagarae (Bacillariophyta). Journal ofPhycology, 27, 780-793. EDLUND, M. B. & STOERMER, E. F. (1997). Ecological, evolutionary, and systematic significance of diatom life histories. Journal ofPhycology, 33, 897-918. EHARA, M., INAGAKI, Y., WATANABE, K. I., & OHAMA, T. (2000). Phylogenetic analysis of diatom coxl genes and implications of a fluctuating GC content on mitochondrial genetic code evolution. Current Genetics, 37, 29-33. GUILLOU, L., CHRETIENNOT-DINET, M.-J., MEDLIN, L. K., CLAUSTRE, H., LOISEAUX-DE GOER, S. & VAULOT, D. (1999). Bolidomonas: a new genus with two species belonging to a new algal class, the Bolidophyceae (Heterokonta). Journal ofPhycology, 35, 368-381. HENDEY, N. I. (1964). An introductory account ofthe smaller algae ofBritish coastal waters. Part V. Bacillariophyceae (Diatoms). Ministry ofAgriculture, Fisheries and Food, Fisheries Investigations, Series IV. 317 pp. HMSO, London. HOOPS, H. J. & FLOYD, G. L. (1979). Ultrastructure of the centric diatom, Cyclotella meneghiniana: vegetative cell and auxospore development. Phycologia, 18, 424-435. HUSTEDT, F. (1930-1966). Die Kieselalgen Deutschland, Osterreichs und der Schweiz. In: Kryptogamen-Flora von Deutschland, 6sterreich und del' Schweiz. VII. -Akademishe ~erlagsgesellschaft, (L. Rabenhorst, ed.), 3 vols. Leipzig. IDEI, M., OSADA, K. & NAGUMO, T. (2000). Specifying the initial stage ofscale formation in a centric diatom Actinocyclus sp. Abstracts ofthe 16th International Diatom Symposium, p. 48, Athens, Greece. JEWSON, D. H., KHONDKER, M., RAHMAN, M. H. & LOWRY, S. (1993). Auxosporulation the .-. freshwater diatom Aulacoseira herzogii in Lake Banam, Bangladesh. Diatom Research, 8, 403-418. KACZMARSKA, I., BATES, S. S., EHRMAN, J. M. & LEGER, C. (2000). Fine structure of the gamete, auxospore and initial cell in the peunate diatom Pseudo-nitzschia multiseries (Bacillariophyta). Nova Hedwigia, 71, 337-357. KOCIOLEK, J. P. & WILLIAMS, D. M. (1987). Unicell ontogeny and phylogeny: examples from the diatoms. Cladistics, 3, 274-284.

-166- AUXOSPORE STRUCTURE, ONTOGENY AND DIATOM PHYLOGENY

KORSHIKOV, A. A. (1930). On the origin of diatoms. Beihefte zum Botanischen Centralblatt, 46, 460-469. KOOISTRA, W. H. C. F. & MEDLIN, L. K. (1996). Evolution of the diatoms (Bacillariophyta). IV. A reconstruction of their age from small subunit rRNA coding regions and the fossil record. Molecular Phylogenetics and Evolution, 6, 391-407. LEE, J. l & XENOPHANTHES, X. (1989). The unusual life cycle of Navicula muscatineii. Diatom Research, 4, 69-77. LEIPE, D. D., WAINRIGHT, P.O., GUNDERSON, J. H., PORTER, D., PATTERSON, D. J., VALOIS, F., HIMMERICH, S. & SOGIN, M. L. (1994). The stramenopiles from a molecular perspective: 16S-like rRNA sequences from Labyrinthuloides minuta and Cafeteria roenburgensis. Phycologia, 33, 369-377. MANN, D. G. (1982a). Structure, life history and systematics of Rhoicosphenia (Bacillariophyta). II. Auxospore formation and perizonium structure ofRh. curvata. Journal ofPhycology, 18,264-274. MANN, D. G. (1982b). Auxospore formation in Licmophora (Bacillariophyta). Plant Systematics and Evolution, 139, 289-294. MANN, D. G. (1984). Auxospore formation and development in Neidium (Bacillariophyta). British phycologicalJournal, 19, 319-331. MANN, D. G. (1987). Sexual reproduction in Cymatopleura. Diatom Research, 2, 97-112. MANN, D. G. (1989). On auxospore formation in Caloneis and the nature of Amphiraphia (Bacillariophyceae). Plant Systematics and Evolution, 163,43-52. MANN, D. G. (1993). Patterns ofsexual reproduction in diatoms. Hydrobiologia, 269/270,11-20. MANN, D. G. & MARCHANT, H. l (1989). The origin of diatoms and their life cycle. In: The Chromophyte Algae, problems and perspectives. Systematic Association Special Volume 38 (lC. Green, B.S.C. Leadbeater & W.L. Diver, eds), 307-323. Clarendon Press, Oxford. MANN, D. G. & STICKLE, A. J. (1989). Meiosis, nuclear cyclosis and auxospore formation in Navicula sensu stricto (Bacillariophyta). British phycologicalJournal, 24, 167-181. MEDLIN, L. K., KOOISTRA, W. H. C. F., GERSONDE, R. & WELLBROCK, D. (1996a). Evolution of diatoms (Bacillariophyta). II. Nuclear-encoded small subunit rRNA sequence comparison confirm a paraphyletic origin for the centric diatoms. Molecular Biology andEvolution, 13, 67-75. MEDLIN, L. K., KOOISTRA, W. H. C. F., GERSONDE, R. & WELLBROCK, D. (1996b). Evolution of diatoms (Bacillariophyta). III. Molecular evidence for the origin of the . Nova Hedwigia, Beiheft 112, 221-234. MEDLIN, L. K., KOOISTRA, W. H. C. F., GERSONDE, R., SIMS, P. & WELLBROCK, D. (1997a). Is the origin ofthe diatoms related to the end-Permian mass extinction? Nova Hedwigia, 65, 1-11. MEDLIN, L. K., KOOISTRA, W. H. C. F, POTTER, D., SAUNDERS, G. W. & ANDERSON, R. A. (1997b). Phylogenetic relationships of the 'golden algae' (hepatophytes, heterokont chrysophytes) and their plastids. Plant Systematics and Evolution (Supp!.), 11, 187-210. MEDLIN, L. K., WILLIAMS, D. M. & SIMS, P. (1'993). The evolution of diatoms (Bacillariophyta). I. Origin of the group and assessment of the monophyly of its major divisions. European Journal of Phycology, 28, 261-275. MIZUNO, M. (1994). Sexual reproduction and auxospore formation in Achnanthes javanica f. subconstricta. Diatom Research, 9, 133-141. MIZUNO, M. (1998). Sexual reproduction and auxospore formation of the marine monoraphid diatom Cocconeis pellucida. Diatom Research, 13, 103-112. PASCHER, A. (1921). Uber die Ubereinstimmung zwischen den Diatomeen, Heterokonten und Chrysomonaden. Berichte der deutschen botanischen Gesellschaft, 39, 236-248. PATRICK, R. & REIMER, C. W. (1966). The Diatoms of the United States, exclusive ofAlaska and Hawaii. Academy of Natural Sciences of Philadelphia, Monograph No. 13, 688 pp. Livingston Pub!., Philadelphia, Pennsylvania PHILIPPE, H., SORHANNUS, D., BAROIN, A., PARASSO, R., GASSE, F. & ADODTTE, A. (1994). Comparison of molecular and paleontological data in diatoms-suggest a major gap in the fossil record. Journal ofEvolutionary Biology, 7, 247-265. PROSHKINA-LAVRENKO, A. I. (1949-1950). Dyatomovy Analyz. Kniga 2-3. Opredelytel iskopayemyh i sovremennyh dyatomovyh vodorosley. [Diatom analysis. A guide to fossil and contemporary diatoms.),

-167- KACZMARSKA, 1., EHRMAN, J.M. AND 8.8. BATES

2-3, 398 pp. Izdatelsvo Geologitscheskoy Literatury Akademi Nauk SSSR, Moskva. ROEMER, S. C. & ROSOWSKI, J. R. (1980). Valve and band morphology of some freshwater diatoms. Ill. Pre- and post-auxospore frustules and the initial cell of Melosira rosea/la. Journal ofPhycology, 16, 399-411. ROSS, R., COX, E. J., KARAYEVA, N. L, MANN, D. G., PADDOCK, T. B. B., SIMONSEN, R. & SIMS, P. A. (1975). An amended terminology for the siliceous coponents of the diatom cell. Nova Hedwigia, Beihefte 64,513-533. ROUND, F. E. & CRAWFORD, R. M. (1981). The lines of evolution of the Bacillariophyta. I. Origin. Proceedings ofthe Royal Society ofLondon Series B- Biological Sciences, 211, 237-260. ROUND, F. E. & CRAWFORD, R. M. (1984). The lines of evolution of the Bacillariophyta. II. The centric series. Proceedings ofthe Royal Society ofLondon Series B- Biological Sciences, 221, 169-188. ROUND, F. E. & CRAWFORD, R. M. (1989). Corethron and Mallomonas - some striking morphological similarities. In: The Chromophyte Algae, problems and perspectives. Systematic Association Special Volume 38 (J.e. Green, B.S.C. Leadbeater & W.L. Diver, eds), 295-305. Clarendon Press, Oxford. ROUND, F. E., CRAWFORD, R. M. & MANN, D. G. (1990). The Diatoms. Biology & morphology ofthe genera. 747 pp. Cambridge University Press, Cambridge. SCHMID, A-M. M. (1980). Valve morphogenesis in diatoms: A pattern-related filamentous system in pennates and the effect ofAPM, colchicine and osmotic pressure. Nova Hedwigia, 33, 811-846. I SCHMID, A-M. M. (1984). Wall morphogenesis in Thalassiosira eccentrica a comparison of auxospore formation and the effect of MT - inhibitors. In: Proceedings ofthe 7th International Diatom Symposium (D.G. Mann ed.), 47-70, Koeltz; Koeningstein. SCHMID, A.-M. M. (1988). The special Golgi-ER-mitochondrium unit in the diatom genus I Coscinodiscus. Plant Systematics and Evolution, 158,211-223. SCHMID, A.-M. M. (1994). Sexual reproduction in Coscinodiscus granii Gough in culture: preliminary report. In: Proceedings ofthe l3th International Diatom Symposium (D. Marino, ed.), 139-159. Biopress, Bristol. SIMONSEN, R. (1979). The diatom system: ideas on phylogeny. Bacillaria, 2, 9-71. SOGIN, M. L. & SILBERMAN, J. D. (1998). Evolution of the protists and protistan parasites from the perspective ofmolecular systematics. International Journal ofParasitology, 28, 11-20. TAYLOR, F. 1. R. (1999). Ultrastructure as a control for protistan molecular phylogeny. The American Naturalist (Suppl.), 154, 125-136. THALER, F. (1972). Beitrag zur Entwicklungsgeschichte und zum Zellbau einiger Diatomeen. 6sterreichische Botanische Zeitschrift, 120,313-347. STOSCH, H. A. VON (1962). Uber das Perizonium der Diatomeen. Vortriige aus dem Gesamtgebiet der Botanik. N.R. 1, 43-52. STOSCH, H. A. VON (1982). On auxospore envelopes in diatoms. Bacillaria, 5, 127-156. STOSCH, H. A. VON, THEIL, G. & KOWALLIK, K. V. (1973). Entwicklungsgeschichtliche Unter­ suchungen an zentrischen Diatomeen. V. Bau und Lebenszyklus von Chaetoceros didymum, mit Beobachtungen iiber einige andere Arten der Gattung. Helgoliinder Meeresuntersuchungen, 25, 384-445. VAN DE PEER, Y. & DE WACHTER, R. (1997). Evolutionary relationships among the eukaryotic crown taxa taking into account site-to-site rate variation in 18S rRNA. Journal ofMolecular Evolution, 45, 619­ 630.

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