The Diatoms: a Primer

P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

2 The diatoms: A primer

MATTHEW L. JULIUS AND EDWARD C. THERIOT Texas Natural Science Center, The University of Texas at Austin, Austin, TX 78705, USA

2.1 Introduction than the specialized teeth found in mammals, including those Diatoms have long been lauded for their use as powerful in the reader’s mouth (Shubin, 2008). Dates for the origin and reliable environmental indicators (Cholnoky, 1968; Lowe, of the diatom common ancestor are bracketed by molecular 1974). This utility can be attributed to their high abundance clock estimates (Sorhannus, 2007) and the oldest stratigraphic and species diversity, which is distributed among most aquatic observation (Rothpletz, 1896, 1900). Both of these estimates environments. Additionally, their remains are highly durable are inherently biased. The temporal proximity of each esti- and well preserved in accumulated sediments. Often, scientists mate to one another does, however, suggest a certain degree of exploiting the group simply as environmental proxies give little accuracy, given the complimentary nature of the biases. Molec- thought as to how and why the species diversity exists in these ular estimates represent an attempt to identify the absolute environments. This may be a by-product of how diatoms are moment two populations diverged from one another. The old- collected and identified. Diatoms are most often recognized by est stratigraphic observation represents a period where fossil the presence of a siliceous cell wall, the frustule. This structure remains were sufficiently abundant to allow discovery. Given varies considerably in shape and architecture among species the expected disparity between the moment two populations (Figure 2.1) and virtually all taxonomic diagnosis of taxa is diverged, and the time it would take divergent populations to based upon frustular morphology. To properly observe diatom develop sufficient numbers allowing paleontological discov- frustules for taxonomic identification, living and sedimentary ery, the 10 million year gap between the two estimates does not collections are typically subjected to various “cleaning” tech- appear to be overly large in context of other estimates in this niques designed to remove all organic materials (e.g. Battarbee temporal range. et al., 2001; Blanco et al., 2008), allowing unobstructed observa- Discussion of when diatoms originated begs the question: tionofthefrustuleinthemicroscope.Thisfrequentobservation what did they originate from? Diatoms share ancestry with of inorganic components of the cell without reference to the heterokonts. Heterokonts (or stramenopiles) are a group of organicfeaturesallowsobserversto“forget”thatthespecimens protists with unequal flagella (Leedale, 1974; Hoek, 1978) seen in the microscope represent individual organisms com- that includes both chloroplast-bearing and non-chloroplast- peting in the selective environments driven by biotic and abiotic bearing representatives (Patterson, 1989) whose common ecological pressures. The abundance and taxonomic diversity ancestor is thought to have arisen ∼725 million years before can be attributed to the extraordinary success of diatoms in the present (Bhattacharya & Medlin, 2004). The group contains competitive ecological arena. an array of morphologically diverse groups including giant The casual observer frequently regards diatoms, like most kelps (>60 m) at the large end of the size spectrum and protists, as primitive ancestral lineages to multicellular organ- the Bolidomonads and Pelagomonads (1–2 μm) at the small isms. While some protists may fit this description, diatoms end of the size spectrum (North, 1994; Andersen et al., 1993; do not. Diatoms are a relatively recent evolutionary group with Guillou et al., 1999). The heterokonts may be part of the larger the common ancestor’s origin considered to be between 200 “chromalveolate” evolutionary group, which includes cryp- and 190 million years before present (Rothpletz, 1896, 1900; tophytes, dinoflagellates, ciliates, apicomplexans, and hap- Medlin et al., 1997). As a point of reference, the origin of this tophytes (Yoon et al., 2002; Cavalier-Smith, 2003; Harper & “first diatom” is approximately 60 to 70 million years younger Keeling, 2003, Ryall et al. 2003, Bachvaroff et al. 2005, Harper

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The diatoms: A primer 9

abcThis statement about heterokonts easily extends to diatoms. Once an understanding of origin is achieved, an apprecia- tion should be given to the speed of diversification. Currently, d >24,000 diatom species have valid scientific names (Fourtanier & Kociolek, 2009a, b). Many of these have only been illustrated in the literature with light microscopy, and few have yet been the subject of any other genetic, ecological, or physiological study. Mann and Droop (1996) conservatively estimated that there are 200,000 diatom species. If these numbers are taken at face value, 12% of the diatom flora is currently described. This means the modern diatom taxonomic community has a major- ity of the 24,000 described species to observe in the electron microscope, and an additional 176,000 species to describe. In e f addition, completing a phylogeny for these 200,000 species should also be an objective. Julius (2007a) demonstrated that the rate of species description in diatoms is approximately 183– 185 per year and that this rate has remained constant for nearly a century. At this pace it will take approximately 951 years to describe diatom species completely! With this in mind, it is easy to understand why the diatom systematics community is still grappling with the collection of g detailed ultrastuctural information for most species, description of species, and the proper way to analyze these data. Many modern diatom systematic studies deal with taxa at the generic or higher level, avoiding unresolved issues concerning ultra- h structure and species concepts. Several recent studies suggest Figure 2.1 Variations in frustule morphology within diatoms species. diatom diversity is much greater than previously imagined (a) Amphicampa eruca,(b)Navicula cryptocephala,(c)Cymbella inaequalis, (Theriot & Stoermer, 1984; Bourne et al., 1992), causing (d)Hydroserawhampoensis,(e)Acanthocerasmagdeburgensis,(f)Cyclotella researchers to suggest continued emphasis on species descrip- striata,(g)Cymatopleura solea,(h)Gyrosigma acuminatum.Scalebars tion is most essential in developing phylogenetic hypotheses equal 5 μm. (Kociolek, 1997; Lange-Bertalot, 1997; Mann, 1997; Round, 1997). In many instances, researchers also continue to argue et al. 2005). This proposed relationship is controversial and about what classes of data should be emphasized, valve highly debated (e.g., Falkowski et al., 2004; Grzebyk et al., 2004; morphology or cytological features, in classification systems Keelingetal.,2004;Bachvaroffetal.,2005;Bodyl,2005).Within (Round, 1996) without regard to any sense of evolution. heterokonts, individual groups are well established and easily Systematic studies in the twenty-first century must incor- diagnosable, but the relationship between these groups has porate all types of character information in some sort of an yet to be definitively identified (Saunders et al., 1995; Sorhan- analysis emphasizing the similarity between evolved features nus, 2001; Goertzen & Theriot, 2003). Molecular techniques (cladistic analysis is currently the most prominent system). utilizing multiple data sets have identified the bolidophytes This character information must be presented for individual (Goertzen & Theriot, 2003) as the heterokont most closely species, not broad generic groups. This requires considerable related to diatoms. The bolidophytes are a group of marine uni- additional descriptive work. Gradually, a classification more cellular flagellates that were unknown to science prior to the reflective of evolutionary history will develop. Simply put, there late 1990s (Guillou et al., 1999). This relatively recent discovery is a great deal of work to be done. We are gradually develop- of the diatoms sister group reflects how much discovery and ing a more structured way of handling the problem and, with description-level science remains uncompleted in heterokont luck, progress will be made. One indication of this activity biology. is the distinctly non-linear trend seen in the rate of generic P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

10 Matthew L. Julius and Edward C. Theriot

description over the last two decades (Fourtanier & Kociolek, distributed along a gradient moving from basal radially sym- 1999). Generic descriptions increased at an exponential rate metric groups to more recently diverged bilaterally symmetric duringthistime,contrastingwiththelinearrateseeninspecies. groups. This may indicate that existing taxa are being placed into newly Simonsen (1979) was the first to discuss a phylogeny for all created higher taxonomic categories and a greater interest diatoms in the context of a taxonomic system. While Simonsen is being taken in the relationship between one species and presentedanevolutionarytreefordiatomfamilies,hewasreluc- another. tant to deconstruct class and order designations in a manner reflecting monophyly. Most notable is the presentation of centric diatoms as distinctly non-monophyletic while maintaining 2.2 Classification the traditional taxonomic category for the group. Round et al. Modernsystematicsstrivestoachievenatural,ormonophyletic, (1990) presented a taxonomic system for genera and higher- groups when designating categories above the species level. level groups. This work treated the diatoms as a division with These natural groups contain an ancestral lineage and all of three classes consisting of the radially symmetric taxa, the ara- its descendants (monophyly). Many taxonomic groups were phid pennate taxa, and raphid pennate taxa, suggesting that established prior to the acceptance of monophyly as a goal. evolution was along this line and that the centric diatoms pre- Taxonomic designations for diatoms are no exception, and ceded pennates. This text remains the most recent comprehen- researchers have only recently begun attempts to test and adjust sive coverage for diatoms, but the classification system was diatom taxonomic schemes to reflect monophyletic groupings. not developed in an evolutionary context and many of the tax- Not all individuals establishing genera and other categories onomic designations are being reconsidered and modified. for diatoms view monophyly as a goal, despite its widespread A comprehensive evolutionary tree for the diatoms is cur- acceptance elsewhere in biology, and proceed in their endeav- rently a popular research topic (summarized in Alverson et al., ors in an evolutionary free context (Williams & Kociolek, 2007). 2006 and Theriot et al., 2009). While molecular systematics Individuals utilizing taxonomic schemes for diatoms should has advanced rapidly in other areas of biology, only the small be aware of the unstable status of many higher taxonomic subunit of the nuclearly encoded ribosomal rDNA gene (SSU) categories (genus and above). Species are frequently moved has been used for comprehensive analyses of diatom phy- in and out of categories and new categories are continually logeny (other genes and morphology have generally just been being established. This process, hopefully, reflects the gradual employed selectively at the ordinal level or below). Trees pro- transition to a monophyletic taxonomic system and the over- duced using the SSU molecule have uniformly obtained a grade whelming level of species description remaining incomplete. of multiple lineages of centric diatoms generally with radial Individualsutilizing diatomsasindicatorspeciesoftenfind this symmetry of valve elements, then a series of lineages of centrics fluctuation in higher taxonomic categories frustrating. To cir- with generally bipolar or multipolar symmetry, then a series of cumvent this taxonomic instability, identifications should be araphid taxa and, finally, the clade of raphe-bearing pennates. made to the species level whenever possible, because species The only exceptions to this are when only a few diatoms were names can always be referenced back to the original popula- included in the analysis or the analytical techniques used were tion described no matter how many times a name is modified improperly applied (Theriot et al., 2009). nomenclaturally. Goertzen and Theriot (2003) noted the effect of taxon sam- Diatoms are traditionally classified as one of two biological pling on topologies generated in phylogenetic analyses of het- orders, the Centrales (informally referred to as centrics) and the erokont taxa. Diatom diversity presents a challenge, in this Pennales (or pennates). Diagnostic features cited supporting context, to attempts at reconstructing phylogenies for the the two classes typically include (1) valve formation developing group using molecular data. All attempts to date have sampled radially around a “point” in centrics, contrasted by deposition <0.01% of the described diatom flora (and obviously have not originating along a “plane” in pennates and (2) oogamous sex included extinct taxa). A very small amount of morphologi- with relatively small motile flagella bearing sperm and a large cal data has been formally analyzed and it supports the trees non-motile egg in the centrics, contrasted by isogamous sex derived from the SSU gene (Theriot et al., 2009). Again, how- with ameboid gametes in the pennates. These features are not ever, results are weakly supported and the analyses did not distinctly distributed among bilaterally and radially symmetric include extinct taxa. What can be conservatively concluded is morphologies on the diatom evolutionary tree, but are instead that no investigation has produced a comprehensive and robust P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

The diatoms: A primer 11

phylogenetic hypothesis for the group. Nevertheless, there seems to be mounting evidence that only raphid pennates are monophyletic among the major diatom taxa. Readers should understand that, like the terms “invertebrate” or “fish”, the words“centric”and“araphidpennate”aresimplytermsofcon- venience and do not reflect independent phylogenetic groups. While a comprehensive diatom phylogeny is desirable, it is not a requirement for utilizing diatom species for environmen- talreconstructionandotherapplications.Reliablespeciesiden- tification is, however, essential. Two text collections represent thestandardsforfreshwaterspeciesidentificationwiththelight microscope.PatrickandReimer(1966,1975)areEnglishguides to the North American flora. Taxa are illustrated with line draw- ings. Unfortunately, the work was never completed and contains only a partial coverage of pennate diatoms. Some major pennate groups (e.g. keel-bearing genera such as Nitzschia and Figure 2.2 Complete Stephanodiscus minutulus frustule. Both valves theorderSurirellales)andcentricdiatomswerenottreated.Vol- and girdle bands comprising the “pill box” architecture are visible. ume 1 (Patrick & Reimer, 1966) is still available for purchase, Scale bar equals 5 μm. but volume 2 (Patrick & Reimer, 1975) has become difficult membrane can be altered quickly in response to environmen- to acquire. Krammer and Lange-Bertalot (1986, 1988, 1991a, tal challenges (Higgins et al., 2003). Adhesion responses have 1991b) are German language guides to the central European been tied to specific plasma membrane receptors (Almqvist flora. Taxa are illustrated with photographs. The texts have et al., 2004). been used to identify species from around the world and repre- The cell interior (Figure 2.3) typically has a central cytoplas- sent a reasonably comprehensive coverage of species. All four mic bridge containing the nucleus. This is bounded by a large volumes are currently available for purchase. vacuole,whichcanaccountforupto70%ofthetotalcellbiovol- ume (Sicko-Goad et al., 1984). The vacuole is frequently associ- 2.3 The diatom cell ated with laterally positioned chloroplasts, which may deposit The diatom cell has the same general pattern as many plastid- photosynthetic products in the vacuole. The silica deposition bearing protists. It has an outer cell wall lined internally with vesicles (SDVs) are closely associated with the golgi, and in a plasma membrane containing the cytoplasm and a collection some species mitochondria may be found in close proximity of organelles. The cell wall, as previously mentioned, is distinct to both the SDVs and golgi. Little is understood about diatom and is composed of silicon dioxide (SiO2). Generally, the cell physiology and most efforts focus on frustule formation or wall consists of two halves, termed valves. The two valves have plastid function. slightly different sizes. The larger valve is termed the epitheca and the smaller the hypotheca. The two valves fit together 2.3.1 Reproduction to form a box. Between the valves are a series of siliceous Diatoms, like many other unicells, reproduce primarily via bands or belts termed girdle bands. The valves plus all girdle asexual mitotic divisions with relatively rare instances of sex- bands comprise a complete cell wall that is termed a frustule ual reproduction. Cells are typically diploid with chromosome (Figure 2.2). numbers varying considerably across species, having as few The frustule has many openings allowing the cell’s organic as 4 and as many as 68 (Kociolek & Stoermer, 1989). Some component to make contact with its surrounding environ- species exhibit high growth rates and may divide once per day in ment. The openings can range from simple pores to specialized optimal environments (Rivkin, 1986). The frustule is involved structures of extremely complex micro-architecture. The cell’s in the divisional process to varying degrees with some taxa, plasma membrane, like other eukaryotes, is rich with proteina- specifically Thalassiosiraceae and Coscinodiscaceae, possess- ceous receptors (Scherer et al., 2007). Little work has been done ing dedicated structures for positioning and manipulating the concerning these receptors (Julius et al., 2007), but basic phys- nucleus (Schmid & Volcani, 1983; Chepurnov et al., 2006). In ical properties of extra cellular material emanating from the most species, the arrangement of the epitheca and hypotheca P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

12 Matthew L. Julius and Edward C. Theriot

involved in sperm and egg recognition. Sexual events are typically observed in cells approximately one third of the maximum cell size. A single cell cycle, the process of cell size reduction and restoration via sex, can take place over decades (Julius et al., 1998). These factors must be accounted for when making deci- sions about the morphological variability of a population over time. Size ranges contained within a species category should have the largest individuals being at least three times the size abof the smallest individuals. It may only be possible to observe this range in size over several seasons or numerous sediment intervals, depending upon where the population is in the size reduction/sexual cycle when observed. Two basic forms of sexual reproduction are presented in reviews dealing with the subject (Mann, 1993; Edlund & Stoermer, 1997): oogamy in centric diatoms and isogamy in pennate diatoms. As mentioned in the introduction, these features are not distributed between radially and bilaterally symmetric diatoms in a strictly categorical fashion. Variety also exists and numerous modifications in sexual reproduction are known in the gradation ancestral to more recently diverged diatom lineages. Mann (1993) illustrates a transitional form of c oogamy in an early pennate lineage in which flagella are absent Figure 2.3 Transmission electron micrographs of Aulacoseira ambigua in “sperm” but the gamete migrates toward a large egg to com- and Nitzschia sp. (a) A. ambigua, transmission electron micrograph plete syngamy. All sexual events in diatoms result in auxospore (TEM) cross section through region of central cytoplasmic bridge. production. The auxospore is a covering composed of siliceous (b) A. ambigua, TEM longitudinal section showing fine structure of scales or bands, which surrounds a newly formed zygote. frustule and arrangements of cytoplasmic components. (c) Nitzschia sp. cross section: the hypothecal valve can be seen resting inside the The zygote and the auxospore expand together, ranging in epithecal valve. Scale bars equal 10 μm. Photographs courtesy Linda shape from spheres to cigar-shaped structures. Within the aux- Goad. ospore a maximally sized frustule is formed, called the initial valve. Initial valves can appear slightly irregular in form as influence mitotic products. Both the epitheca and hypotheca of they are sometimes formed directly against the auxospore and the mother cells will become epitheca in the next generation, resemble the inflated shape of this structure. These irregular- one for each daughter cell. The mitotic products result in one ities diminish as mitotic products are produced. Some taxa daughter cell equal in size to the mother cell and one daughter are capable of producing auxospores without sexual repro- cell smaller than the mother cell. Repeated division results in duction and will undergo automixis in which gametes form a gradual decrease in population mean cell size over time. within a single individual and fuse with one another giving rise MacDonald (1869) and Pfitzer (1869, 1871) described this to an auxospore (Geitler, 1973). Parthenogenesis has also been process over a century ago. described in some taxa (Geitler, 1982). These non-sexual mech- Cell size is restored via sexual reproduction, with a syngamy anisms for regenerating size appear to be adaptations existing event producing a single maximally sized cell. Triggers for sex- in addition to standard sexual processes producing auxospores ual reproduction are not well understood, but are generally and initial cells. thought to include both environmental factors and size cues (Mann, 1993). A collection of genes that are expressed at the 2.3.2 Resting cells onset of sexual reproduction has been identified (Armbrust, The ability to produce resting cells and spores is thought to 1999). The three genes identified in this collection were named have arisen early in diatoms (Round & Sims, 1981; Harwood Sig 1, Sig 2, and Sig 3 or sexually induced genes 1–3. Armbrust & Nikolaev, 1995). This speculation is unconfirmed, but rest- (1999) found that the genes encoded for a protein are similar ing cell and spore production is a known component of many in structure to an animal epidermal growth protein potentially diatoms since the Cretaceous (Harwood & Nikolaev, 1995). P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

The diatoms: A primer 13

Resting spores are regarded as an ancestral structure (Har- et al., 2004; Kidder & Erwin, 2001). Proponents of this hypoth- graves & French, 1983), and they are given credit as one rea- esis argue that this process makes silica readily available to son the diatoms were able to survive the Cretaceous extinction diatoms, providing a competitive advantage allowing displace- (Kitchell et al., 1986; Harwood, 1988). Resting spores produced ment of other photosynthetic protists. This hypothesis is not through asexual division generally differ morphologically from corroborated by the current dataset available for fossil diatoms vegetative valves and generally possess two distinctive, heavily (Rabosky & Sorhannus, 2009), and development and domi- silicified valves. The spores are rich in storage products and nance of diatoms over the last 65 millions years is a more have faster sinking rates and higher chlorophyll content than complex story than previously postulated. vegetativevalves(Hargraves&French,1983).Restingsporesare While the mechanisms and extent of diatom dominance viable for a relatively short time (<3 years); in contrast, resting since the Cenozoic remains an open question, the immediate cells are viable for long periods when in anoxic sediments (at availability of silica in aquatic systems is an important factor in least 20–30 years; Sicko-Goad et al., 1986). Resting cells are determining the locally dominant primary producer (Schelske morphologically indistinguishable from vegetative valves, dif- & Stoermer, 1971; Conley, 1988). Excess silica in combination fering only in the dark, highly condensed cytoplasm of the cell. with excess nitrogen frequently results in diatom-dominated Resting cell formation has largely replaced resting spore pro- environments. Generalizations can also be made about silica ductioninfreshwaterdiatoms(Sicko-Goadetal.,1986).Resting utilization in marine and freshwater species. Marine taxa typi- cells are physiologically similar to resting spores, having high cally contain ten times less silica than freshwater taxa (Conley quantities of storage products. et al., 1989). This reflects a dichotomy in the availability of Resting cells, however, do not undergo a mitotic division silica in freshwater and marine systems, in that silica is far during formation. Instead, they form from a vegetative cell by less abundant in marine environments (Conley, 2002). Despite condensing the chloroplast and accumulating storage prod- this, silica availability does not appear to be a major factor driv- ucts (Sicko-Goad et al., 1986). This fact suggests that although ing selection of silica transport genes in modern marine and both resting spores and resting cells are very effective survival freshwater species (Alverson, 2007; Alverson et al., 2007). strategies, the resting cells are more physiologically efficient, Diatom cell wall formation is the subject of considerable bypassing the cellular expense (time and energy) of an addi- interest within the nanotechnology community (see Gordon, tional mitotic division. this volume). The structure is unique to diatoms and is com-

Both of the above resting structures are triggered into forma- posed of opaline silica (SiO2) deposited via a specialized tion or encystment by very similar environmental cues. These organelle, the silica deposition vesicle. Within this organelle, include the availability of various nutrients (N, P, Si), tempera- silica is sequestered and deposited along a proteinaceous ture, light intensity, and photoperiod (Round et al., 1990). Once template during cytokinesis following the initiation of mito- formation has occurred, both resting spores and resting cells sis. In older lineages valve formation is initiated during sink, to the pycnocline in many marine species (Hargraves & anaphase/telophase; and in more recent pennate diatom lin- French, 1983) and into the sediments for many freshwater eages the process begins after cleavage (Schmid, 1994). Frus- taxa. Eventually the resting structures will be resuspended into tule formation involves an unusual interaction between organic the water column during favorable environmental conditions, and inorganic molecules. Wetherbee (2002) provides a sum- when the above environmental triggers will cause vegetative mary of these interactions. Organic molecules regulating silica cell production. deposition in diatoms are proteins called silaﬁns (Kroger¨ et al., 1999), and a small collection of silica transporter genes (SITs) 2.3.3 Silica and frustule formation is responsible for silica uptake in the cell (Hildebrand et al., Diatomsaretheprimaryorganismsinvolvedinthemediationof 1998). Silica available for valve formation in aquatic systems is

silica in modern oceans (Conley, 2002) and are thought to have in the form of silicic acid (Si(OH)4). Acquisition of this mate- occupied this role since the Cenozoic (Martin-Jezequel et al., rial is synchronized with the cell cycle, providing adequate raw 2000; Katz et al., 2004). They also achieved dominance over material for valve formation during cytokinesis, with peak sili- other phytoplankton during this same period. Diatom domi- cic acid intake from the environment occurring during the G2 nanceinthemarineandfreshwaterfossilrecordsincetheCeno- and M phases of the cell cycle (Sullivan, 1977; Brzezinski et al., zoic is often attributed to geochemical coupling between ter- 1990; Brzezinski, 1992). restrial grasslands and aquatic ecosystems as part of the global Anunderstandingofthevalveformationprocessisstilldevel- silicon cycle (Kidder & Gierlowski-Kordesch, 2005; Falkowski oping and is best understood from microscopic observations P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

14 Matthew L. Julius and Edward C. Theriot

of the process. Molecular-level dynamics of the process are the subject of intense study, and information concerning this process should develop rapidly. Gordon and Drum (1994) present a preliminary review of the chemical and physical parameters accounting for certain siliceous structures, and Schmid (1994) performed the most extensive studies of valve morphogenesis via light microscopic observation and provides a summary of nearly two decades of her work on the subject. Following nuclear division, the valves of the mother cell become the new epithecal valves of daughter cells. Between these mother-cell valves each hypothecal valve is formed inside an individual vesicle, called a silica deposition vesicle. The membrane of this vesicle is called the silicalemma. The silicalemma controls the release (flow and position) of silica from the SDVs. In all species of diatoms investigated, a structure known as the cleavage plasmalemma adheres to the silicalemma during expansion and shaping of the new valve. The pattern of siliceous structures (pore fields, chambers, the raphe, or strutted processes) is produced as a result of the cell’s ability to localize silica and molding organelles to particular regions of the cleavage plasmalemma. The morphogenic infor- Figure 2.4 Chloroplast acquisition by photosynthetic protists. mation for a new valve is carried from the plasmalemma to the Endosymbiotic events from acquisition of original cyanobacterial silicalemma. The mechanics and regulation of this information endosymbiont through events leading to modern photosynthetic pro- transfer remain an open question. tist lineages. As with all proteins, silafins are directly coded by nuclear DNA, but function and shape can be modified by environ- over an environmental gradient and suggested that the forms mental conditions. This undoubtedly causes a certain degree represent a single highly plastic species. This variation should of environmentally regulated plasticity, but evolutionary pro- encourage caution when making taxonomic diagnoses based cesses at the genetic level inherently regulate expression in upon valve morphology. The feature is absolutely the product valve morphology. Prior work has documented environmen- of evolutionary processes, but is variable like morphological tally induced variation in valve morphology. Julius et al. (1997) features in all organisms. Conclusions should be reached via found that specific features commonly used in identifying radi- observation of multiple valves from repeated collections of a ally symmetric diatoms may be highly prone to environmental specific locality. variation and encouraged the use of features passing tests for evolutionary homology when diagnosing species. It is com- 2.3.4 Plastids monly suggested (Round et al., 1990) that researchers look The diatom plastid, like that of all other photosynthetic eukary- for “janus valves” (cells in which the frustule has two dis- otes, originated as the result of an endosymbiotic event involv- tinctly different valve types in species for which the two valves ing an as-yet unidentified eukaryotic protist or a photosynthetic normally appear identical (McBride & Edgar, 1998)) if they bacterium (Sanchez-Puerta & Delwiche, 2008). The engulfed are concerned about differing environmental forms. Stoermer bacterium likely shared an ancestry with the cyanobacteria. In (1967) identified this phenomenon in field collections of the addition to this original endosymbiotic event, additional klep- pennate diatom Mastogloia. Modifications in levels of total dis- toplastic events involving only eukaryotes have also occurred. solved solids apparently caused two distinct morphotypes to One of these events involving a non-plastid bearing eukary- appear. Tuchman et al. (1984) noted similar modifications in ote and a red alga presumably resulted in the plastid-bearing the valve structure of the centric diatom Cyclotella meneghiniana cell giving rise to organisms with plastids containing chloro- with increased salinity under experimental conditions. Other phyll a and c (Keeling, 2004) (Figure 2.4). The diatom plastid researchers have observed similar taxonomic forms occurring arose from this chlorophyll a-andc-bearing cell through an P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

The diatoms: A primer 15

Green algae Cyanobacteria Red algae Alveolates Cryptomonads Haptophytes Heterokonts* land plants

Loss of one plastid membrane A

Floridean starch as Chlorophyll b photosynthetic product B Three plastid membranes Tertiary Fucoxanthin endosymbiotic event Chrysolaminarin as photosynthetic product Chlorophyll c Large sub-unit RUBISCO Four plastid Small sub-unit RUBISCO Two plastid nuclear encoded membranes nuclear encoded membranes

Chlorophyll a Tertiary endosymbiotic event Chlorophyll Starch as at A or B binding proteins photosynthetic product Non-cyclic photophosphorolation Primary Secondary Cyanophycean endosymbiotic event endosymbiotic event starch

Figure 2.5 Chloroplast phylogeny, adapted from Sanchez-Puerta and 1994). Specific proteins found in diatoms are termed the fucox- Delwiche (2008), as proposed by the chromalveolate hypothesis. Solid anthin, chlorophyll a/c binding proteins (FCPs). These proteins lines in the tree represent phylogenetic relation at the chloroplast and function on the thylakoid membranes and channel light energy host level. Dashed lines represent phylogenetic relation for only the to chlorophyll a (Owens, 1986). At least two FCP gene clus- chloroplast. Hash marks on the tree represent acquisition of specific features. ters exist with multiple FCP gene copies contained within each cluster (Bhaya & Grossman, 1993). These proteins are nuclear encoded, and protein transport to the plastids differs from that additional endosymbiotic event (Sanchez-Puerta & Delwiche, in other photosynthetic eukaryotes with less than four plastid 2008). membranes (Bhaya & Grossman, 1991). Plastid acquisition history in diatoms encourages caution Plastid number and morphology varies among different when making generalizations about diatoms and other photo- diatom evolutionary lineages. Non-pennate diatoms and basal synthetic organisms. Consideration must be given to the plas- pennate lineages generally have numerous discoid chloro- tid’s history as a cyanobacterial cell, the time it spent within plasts. Chloroplast number in these diatom lineages varies the red algal lineage, and events since its removal from that with environmental cues. Modifications in chloroplast number lineage. To do this, it is best to think of the plastid’s evolution- associated with such cues can be tied to variations in FCP gene ary history separately from the “host” organism (Figure 2.5). activity (Leblanc et al., 1999). More recently diverged diatom Photosynthesis in diatoms mechanistically resembles the pro- lineages can have fixed numbers of morphologically distinct cess in cyanobacteria and other photosynthetic eukaryotes, in plastids. These attributes have been used as an identification that it is chlorophyll a centered, non-cyclic, and involves the tool for distinguishing living specimens from one another same number of proteins in the reactions. Diatoms can be (Cox, 1986). distinguished from other non-heterokont algae through the Chloroplasts directly produce chrysolaminarin, a biological possession of (a) chlorophyll a, c,fucoxanthin,andβ-carotene polymer composed of β 1–3 glucose molecules. This is used as pigments, (b) four plastid membranes with the outer mem- directly as a food source by the diatoms, but is transformed brane functionally bound to the golgi apparatus, and (c) the and stored as lipid in the cell’s vacuole. Diatoms typically have utilization of chrysolaminarin as a photosynthetic product. one or two large vacuoles which can account for >60% of the Pigments in diatom plastids are bound together by nuclear cells volume (Sicko-Goad et al., 1977). Stored lipid can occupy encodedproteinshomologoustothoseinothereukaryoticpho- a major portion of the vacuole (Figure 2.6) with lipid account- totrophs (Owens, 1986; Bhaya & Grossman, 1993; Apt et al., ing for a large percentage of cell dry weight. Modifications in P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

16 Matthew L. Julius and Edward C. Theriot

(Arts et al., 2009). While animals are predominately associated with these heterotrophic consumers, members of other evolutionary groups also graze on diatom taxa. Canter and Lund (1948, 1951, 1953), for example, detail the relationship between fungal chytrids and diatom populations. This description is frequently used as a model for describing predator–prey dynamics in ecology texts. While discussions of microalgae as a food source for animals frequently focus on members of the invertebrate community (Bott, 1996), spectacular examples exist in among vertebrate grazers. Julius et al. (2005), for example, detail the behavior and feeding mechanics of a gobid ﬁsh adapted for grazing on planktonic diatoms as a larvae and stalk-forming benthic diatoms as an adult. The abundance and diversity of diatom grazers directly Figure 2.6 Transmisssion electron micrograph of Cyclotella meneghiniana in cross section. LB denotes lipid bodies in the vacuole, which are reﬂects diatom species abundance and diversity. Diatom often located near chloroplasts. Scale bar equals 5 μm. species are found in a wide array of nutrient, pH, salinity, and temperature regimes. The many combinations of these chemical and physical parameters provide the diversity of niches chloroplast number may be made in times of environmental required to sustain the thousands of known species. Function- stress to maintain lipid reserves, and chlorophyll to lipid ratios ally the diatoms can be described by growth habit, with the have been successfully used as a metric for cell health (Julius understanding that certain strategies likely evolved multiple et al., 2007). times in distantly related diatom species. Species can be easily found with adaptations for plank- 2.4 Ecology and growth habits tonic, tychoplanktonic, benthic, and subaerial existences. Taxa The production of high-energy lipid reserves make diatoms found in each of these environments can occur as unicells or a highly desirable food source for many aquatic heterotrophs colonially. The widest diversity in growth habits is found in (Volkman et al., 1989; Ahlgren et al., 1990). Lipids, especially benthic taxa. Adaptations can be found for growth on a vari- those from diatoms, play a critical role in the aquatic food web ety of substrates including sediments, rocks, photosynthetic providing essential fatty acids (EFAs) to higher trophic levels macrophytes, and animals (Figure 2.7). Other species remain

(a) (b)

Figure 2.7 Some growth variations in attached benthic diatoms: (a) the tuft-forming diatom Synedra acus; (b) the stalk-forming diatom Rhoicosphenia curvata. Scale bars equal 200 μmforAand10μm for B. P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

The diatoms: A primer 17

motile and rarely adhere to substrates. Substrate availability 2.5 Sample preparation and (Townsend & Gell, 2005) and flow (Tuji, 2000) can dramat- material preservation ically restructure diatom communities. Substrate disturbance frequency and magnitude is also a major consideration (Tuji, Diatom studies are somewhat of an enigma. The diversity and 2000), as many taxa dominate in disturbed rather than stable ecological dominance in aquatic systems demonstrate their systems or vice versa (see Stevenson et al., this volume). Varia- importance and utility in ecological investigations. At the tions in growth forms directly impact the availability of cells to same time, the lack of understanding in virtually all aspects grazers. Alterations in physical parameters can alter dominant of the group’s biology is, at times, overwhelming. This con- diatom growth types making taxa unavailable to grazers (Julius, trast should not intimidate investigators interested in utilizing 2007b) with subtle changes in physical parameters. Variations diatoms in research pursuits. It need only encourage investiga- in growth habit can also cause selective collection of some tors to make the best estimates possible and voucher samples species over others when sampling benthos. Factors influenc- used to make those estimates. Diatoms are inherently suited for ing growth habit can also dramatically impact the quantity of vouchering. Appropriate preparation and curation of samples biomass in a benthic system. Peak biomass typically occurs will allow vouchered material to be available to future gener- in low to moderate flow settings with rock substrates where ations interested in reexamination in light of new discoveries diatom taxa can readily grow on attached green algae (Biggs, and an expanded knowledge base. 1996). When a sample is collected, two considerations must be Growth habit often may or may not reflect shared evolu- given to the collection in context of archival preservation. First, tionary history. Barriers encountered by taxa requiring adap- some portion of the material has to be processed in a manner tations for growth may, in some instances, have provided resultingintheremovaloforganicmaterials,withonlysiliceous an advantage to species allowing them to radiate and colo- components remaining (Battarbee et al., 2001). Second, the nize many similar habitats. In other cases, chemical barri- expanding techniques and power of molecular data sets also ers may have presented a greater challenge, and adaptation encourage the preservation of organic components. Proto- to nutrient regimes many have provided selective advan- cols exist for each sample preparation method (Blanco et al., tage, allowing variations in growth forms to evolve multiple 2008). times. Monoraphid diatoms, for example, are now known A variety of techniques exist for removal of organic mate- to have multiple evolutionary origins (Kingston, 2003) and rial allowing observation of the frustule (Battarbee et al., 2001). taxa adapted for disparate nutrient conditions achieve sim- These methods vary in time, expense, and the generation of ilar growth solutions for attachment to substrates. Conver- dangerous by-products. Investigators must select a method gent evolution has also been described for stalk-forming producing the best product within constraints of specific labo- clavate-shaped diatoms (Kociolek & Stoermer, 1988). Caution ratorysettings.Untilrecently,frustulesweremostoftencleaned

should be taken when taxonomic classiﬁcation is based upon with concentrated nitric acid (HNO3) augmented with potas-

growth habit without reference to other features useful for sium dichromate (KCr2O7) as a catalyst. The chromium disas- diagnosis. sociated in this process has made the technique less popular. Variations in habitat provide opportunity for speciation and Similar results can be achieved via extended exposure (multi- divergence of diatom populations. This can occur rapidly ple hours) to heated nitric acid. Diatom acid slurries must be through seemingly minor environmental alterations. Theriot repeatedly rinsed with distilled water. In this process, water is et al. (2006) documented species divergence of two Stephanodis- added to a sample being cleaned and diatoms are allowed to cus species in Yellowstone Lake (Wyoming, USA) occurring in settle. Liquids are decanted and distilled water is again added fewer than 10,000 years. The number of possible habitat combi- and diatoms are allowed to settle. This process continues until nations and abundance of diatoms mean new species discovery a neutral pH is achieved. Forcing diatom sinking via centrifu- is possible in any ecological endeavor involving diatoms. Inves- gation can accelerate the process. Caution must be taken prior

tigators should not hesitate to label unknown taxa as unknown, to application of nitric acid. If calcium carbonate (CaCO3)or

rather than forcing a speciﬁc entity into an existing species cat- ethanol (C2H6O) is present in samples nitric acid addition may egory (see Stoermer, 2001). Materials used in these investiga- cause a violent reaction. Calcium carbonate can be eliminated tions should be archived and made available for investigation via pretreatment with hydrochloric acid (HCl). In the case of and classiﬁcation. ethanol, nitric acid must be avoided completely. P1: SJT Trim: 276mm × 219mm Top: 10mm Gutter: 21.089mm CUUK1067-02 cuuk1067/Smol ISBN: 978 0 521 50996 1 May 20, 2010 11:46

18 Matthew L. Julius and Edward C. Theriot

Once samples are cleaned, a portion of the siliceous rem- Diatoms are the most abundant group of heterokonts contain- nants should be archived for electron microscope and other ing these lipid storage products, making them the preferred types of observation. Cleaned material should be dried before food source of many animals and aquatic fungi. The high archiving to avoid individual frustules dissolving and reprecip- species diversity and critical role in the aquatic food web make itating due to Ostwald ripening (a phenomenon in which larger diatoms an ideal choice for environmental investigations; addi- particles are thermodynamically favored over many small parti- tionally the diatom frustule preserves well in sediments provid- cles). To prepare a light microscope slide, materials should be ing a data set ideal for paleoecological-oriented studies. Histor- mountedinahighlyrefractivematerial,ideallyonewitharefrac- ically diatom investigations have focused on descriptive aspects tive index of ∼1.7. Two products are currently commonly used of biology, but recent technological developments, specifically for these preparations, NaphraxTM and MeltmountTM. Cleaned molecular tools, have allowed process-oriented questions to material is dried onto cover slips and then adhered to a micro- be explored. These activities are rapidly expanding the diatom scope slide with one of these high-refractive-index materials. knowledgebase and prior descriptive efforts are beginning to Observation of slides prepared is typically conducted at 1000× be understood at a genetic and metabolic level. This under- with a high-resolution objective. standing should continue to expand at an unprecedented rate When preserving organic materials for future molecular during the next few decades. investigations formalin-based preservatives should be avoided in favor of ethanol. While pigments will be extracted into the preservationmedia,nucleicacidswillnotbedamaged.Iforgan- References ics need to be preserved in a manner leaving pigments intact, a Ahlgren, G., Lundstedt, L., Brett, M., and Forsberg, C. (1990). Lipid separate preparation should be made. composition and food quality of some freshwater phytoplankton The degree to which samples should be processed and for cladoceran zooplankters. Journal of Plankton Research, 12, 809– archived is at the discretion of the investigator. At a mini- 18. Almqvist, N., Bhatia, R., Primbs, G., et al. (2004). Elasticity and adhe- mum, materials should be prepared to accomplish the project sion force mapping reveals real-time clustering of growth factor for which collections were made. Maximally, materials should receptors and associated changes in local cellular rheological be archived in light of potential future investigations. Materials properties. Biophysical Journal, 86, 1753–62. archived in a manner allowing access to future investigators Alverson, A. J. (2007). Strong purifying selection in the silicon trans- lend themselves to a more rapid advancement in an under- porters of marine and freshwater diatoms, Limnology and Oceanog- standing of diatom biology. raphy, 52, 1420–9 Alverson, A. J., Cannone, J. J., Gutell, R. R., and Theriot, E. C. (2006). The evolution of elongate cell shape in diatoms. Journal of Phycol- 2.6 Summary ogy, 42, 655–668. Alverson, A. J., Jansen, R. K., and Theriot, E. C. (2007). Bridging the Diatoms diverged from other “golden” algae relatively recently Rubicon: phylogenetic analysis reveals repeated colonizations of and represent a unique evolutionary lineage less than 200 mil- marine and fresh waters by thalassiosiroid diatoms. Molecular lionyearsold.Duringthistime,thegroupdiversifiedtoadegree Phylogenetics and Evolution, 45, 193–210. rivaled only by the insects and contains at least 200,000 species. Andersen, R. A., Saunders, G. W., Paskind, M. P., and Sexton, Diatom researchers have examined less than 15% of this esti- J. P. (1993). Ultrastructure and 18S rRNA gene sequence for mated diversity, and many gaps exist in the current understand- Pelagomonas calceolata gen. et sp. nov. and the description of a new algal class, the Pelagophyceae classis nov. Journal of Phycol- ingofdiatombiology.Diatomscanbedistinguishedfromother ogy, 29, 701–715. unicellular organisms by the presence of a siliceous cell wall, Apt, K. K., Bhaya, D., & Grossman, A. R. (1994) Characterization of which typically consists of two halves fitting together to form the genes encoding the light-harvesting proteins in diatoms: the a box containing the organic components of the cell. Cellular biogenesis of the fucocanthin chlorophyll a/c protein complex, material within the box, known as a frustule, is similar in com- Journal of Applied Phycology 6, 225–30. position to other photosynthetic protists. Diatoms are mem- Armbrust, E. V. (1999). Identification of a new gene family expressed during the onset of sexual reproduction in the centric diatom bers of a larger evolutionary group known as the heterokonts. Thalassiosira weissflogii. Applied Environmental Microbiology, 65, Like many heterokonts, diatoms possess chlorophyll a and c 121–8. and convert photosynthetic products into high-energy lipids. Arts, M. T., Brett, M. T., and Kainz, M. J. (2009). Lipids in Aquatic Theselipidscontainfattyacidsrequiredforanimalmetabolism. Ecosystems. New York: Springer. 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22 Matthew L. Julius and Edward C. Theriot

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