AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.     

THE EVOLUTION OF ELONGATE SHAPE IN DIATOMS1

Diatoms have been classified historically as either centric or pennate based on a number of features, cell outline foremost among them. The consensus among nearly every estimate of the diatom phylogeny is that the traditional pennate diatoms (Pennales) constitute a well‐supported clade, whereas centric diatoms do not. The problem with the centric–pennate classification was highlighted by some recent analyses concerning the phylogenetic position of Toxarium, whereby it was concluded that this “centric” diatom independently evolved several pennate‐like characters including an elongate, pennate‐like cell outline. We performed several phylogenetic analyses to test the hypothesis that Toxarium evolved its elongate shape independently from Pennales. First, we reanalyzed the original data set used to infer the phylogenetic position of Toxarium and found that a more thorough heuristic search was necessary to find the optimal tree. Second, we aligned 181 diatom and eight outgroup SSU rDNA sequences to maximize the juxtapositioning of similar primary and secondary structure of the 18S rRNA molecule over a much broader sampling of diatoms. We then performed a number of phylogenetic analyses purposely based on disparate sets of assumptions and found that none of these analyses supported the conclusion that Toxarium acquired its pennate‐like outline independently from Pennales. Our results suggest that elongate outline is congruent with SSU rDNA data and may be synapomorphic for a larger, more inclusive clade than the traditional Pennales.     

CHARACTER DEFINITIONS AND CHARACTER STATE DELINEATION: THE BETE NOIRE OF PHYLOGENETIC INFERENCE

Abstract— Currently characters are static concepts whose definition and state delineations seldom undergo any scrutiny. Common systematic practice tends to synthesize character slates by combining or dividing observed conditions, a situation most likely due to current theoretical limitations in phylogenetic inference, which tends to ignore problems of multistate characters. This process we refer to as the “synthetic” method for character definition. Character definitions derived for the genera of North American Cochylini (Lepidoptera: Tortricidae) using “synthetic” character states postulated that the cochylines were not monophyletic. The use of cladogram characters and nearest neighbor matrices in uncovering potential flaws in character state delineation is demonstrated. The “synthetic” set of character definitions proved deficient upon such analysis, principally due to its attempt to force highly variable features into a few states. The set of character definitions produced from this analysis is referred to as “reflective” because it does not ignore observed variation. It produces characters with many states and presents problems of setting up transformation series. Three means lor deriving transformations are applied to produce transformation series for the reflective set of character definitions: the unordered outgroup method, morphocline analysis and Transformation Series Analysis (TSA). All three data sets postulated the Cochylini as monophyletic. The three sets of phylogenies were compared. Consensus trees are ambiguous when analysing changes in hierarchy. In order to summarize these results in a manner which does not destroy the phylogenetic structure, positional subtrees, a new means for summarizing multiple solution cladograms, are introduced. It was found that all three sets of transformations produced very different cladograms which in turn were very different from the tree produced by the original, synthetic definitions. The results of each of these methods were assessed for their internal consistency. TSA gave the least contradictory results.     

Frustule morphogenesis of raphid pennate diatom <Emphasis Type="Italic">Encyonema ventricosum</Emphasis> (Agardh) Grunow

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.     

A rapidly deposited pennate diatom ooze in Upper Miocene-Lower Pliocene sediment beneath the North Pacific polar front

Rapidly deposited Thalassionema-Thalassiothrix pennate diatom oozes previously have been described in Upper Miocene-Lower Pliocene sediment beneath the frontal boundary of the eastern equatorial Pacific. Here we document a new occurrence of Thalassionema-Thalassiothrix ooze in Upper Miocene-Lower Pliocene sediment beneath the frontal boundary of the subarctic North Pacific. The ooze is a 6 m interval of siliceous sediment at Ocean Drilling Program (ODP) sites 885/886 that was rapidly deposited between approximately 5.0 and 5.9 Ma. Bulk sediment in this interval may contain greater than 85% pennate diatom tests. There are also abundant laminae and pockets that are composed entirely of Thalassionema and Thalassiothrix diatoms. The presence of a rapidly deposited ooze dominated by pennate diatoms indicates unusual past conditions in the overlying surface waters. Time coincident deposition of such oozes at two distinct frontal boundary locations of the Pacific suggests that the unusual surface water conditions were causally linked to large-scale oceanographic change. This same oceanographic change most likely involved (1) addition of nutrients to the ocean, or (2) redistribution of nutrients within the ocean. The occurrence and origin of pennate diatom oozes may be a key component to an integrative understanding of late Neogene paleoceanography and biogeochemical cycling.     

High-throughput pyrosequencing of the chloroplast genome of a highly neutral-lipid-producing marine pennate diatom, Fistulifera sp. strain JPCC DA0580

The chloroplast genome of the highly neutral-lipid-producing marine pennate diatom Fistulifera sp. strain JPCC DA0580 was fully sequenced using high-throughput pyrosequencing. The general features and gene content were compared with three other complete diatom chloroplast genomes. The chloroplast genome is 134,918 bp with an inverted repeat of 13,330 bp and is slightly larger than the other diatom chloroplast genomes due to several low gene-density regions lacking similarity to the other diatom chloroplast genomes. Protein-coding genes were nearly identical to those from Phaeodactylum tricornutum. On the other hand, we found unique sequence variations in genes of photosystem II which differ from the consensus in other diatom chloroplasts. Furthermore, five functional unknown ORFs and a putative serine recombinase gene, serC2, are located in the low gene-density regions. SerC2 was also identified in the plasmids of another pennate diatom, Cylindrotheca fusiformis, and in the plastid genome of the diatom endosymbiont of Kryptoperidinium foliaceum. Exogenous plasmids might have been incorporated into the chloroplast genome of Fistulifera sp. by lateral gene transfer. Chloroplast genome sequencing analysis of this novel diatom provides many important insights into diatom evolution.     

Silicification of auxospores in the araphid diatom Tabularia fasciculata (Bacillariophyta)

We report on auxospore wall structure and development in the araphid pennate diatom Tabularia fasciculata. Similar to most other pennates, these auxospores showed a typical bidirectional elongation, but unexpectedly bore no transverse perizonium, and with no detectable silicon during much of their expansion. Energy dispersive X-ray spectroscopy (EDS) analyses segregated auxospores into two types: (1) those containing no detectable silicon and (2) those with measureable amounts. Both types were of similar size. Silica precipitation began throughout the auxospore at or near maximal length, but initially was detectable in isolated regions throughout the structure. Following this initial condition, silicon was consistently detectable throughout auxospores of comparable size and corresponded to deposition of longitudinal perizonium (visible through the thin organic outer layer of the wall in some auxospores), followed by the deposition of the initial valves. Our results raise the question as to how the tubular shape of bidirectionally expanding auxospores up to ∼90 μm long is maintained in the absence of transverse siliceous elements restricting isodiametric expansion of the cell, which are present in all other known pennate auxospores and all but one other diatom. Our study is the first to systematically examine mineral elements of the auxospore wall analytically.     

Supramolecular organization of fucoxanthin–chlorophyll proteins in centric and pennate diatoms

Fucoxanthin–chlorophyll proteins (FCP) are the major light-harvesting proteins of diatom algae, a major contributor to marine carbon fixation. FCP complexes from representatives of centric (Cyclotella meneghiniana) and pennate (Phaeodactylum tricornutum) diatoms were prepared by sucrose gradient centrifugation and studied by means of electron microscopy followed by single particle analysis. The oligomeric FCP from a centric diatom were observed to take the form of unusual chain-like or circular shapes, a very unique supramolecular assembly for such antennas. The existence of the often disputed oligomeric form of FCP in pennate diatoms has been confirmed. Contrary to the centric diatom FCP, pennate diatom FCP oligomers are very similar to oligomeric antennas from related heterokont (Stramenopila) algae. Evolutionary aspects of the presence of novel light-harvesting protein arrangement in centric diatoms are discussed.     

GENOMIC INSIGHTS INTO EVOLUTIONARY RELATIONSHIPS AMONG HETEROKONT LINEAGES EMPHASIZING THE PHAEOPHYCEAE1

Heterokonts comprise a large and diverse group of organisms unified by the heterokont biflagellate condition. Monophyly of many of these lineages is well established, but evolutionary relationships among the various lineages remain elusive. Among these lineages, the brown algae (Phaeophyceae) are a monophyletic, taxonomically diverse, and ecologically critical group common to marine environments. Despite their biological and scientific importance, consensus regarding brown algal phylogeny and taxonomic relationships is missing. Our long‐term research goal is to produce a well‐resolved taxon‐rich phylogeny of the class to assess evolutionary patterns and taxonomic relationships among brown algal lineages and their relationship to other closely related heterokont groups. To accomplish this goal and augment existing loci for phaeophycean‐wide systematic studies, we generated expressed sequence tags (ESTs) from several major brown algal lineages and from the heterokont lineage representing the closest sister group to brown algae. To date, we have successfully constructed cDNA libraries for two lineages (Choristocarpus tenellus Zanardini and Schizocladia ischiensis E. C. Henry, Okuda et H. Kawai) and in the library test phase obtained up to 1,600 ESTs per organism. Annotation results showed a gene discovery rate of 45%–50% for each library revealing 500–700 unique genes from each organism. We have identified several potential genes for phylogenetic inference and used these loci for preliminary molecular clock analyses. Our molecular clock analysis suggests that the basal divergence in brown algae occurred around the time of the pennate‐centric diatom divergence. Here we report this analysis and other uses of ESTs in brown algal phylogenomics and the utility of these data for resolving the phylogeny of this group.     

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.     

An AFLP marker approach to lower-level systematics in Eucalyptus (Myrtaceae)

Genus Eucalyptus, with over 700 species, presents a number of systematic difficulties including taxa that hybridize or intergrade across environmental gradients. To date, no DNA marker has been found capable of resolving phylogeny below the sectional level in the major subgenera. Molecular markers are needed to support taxonomic revision, assess the extent of genetic divergence at lower taxonomic levels, and inform conservation efforts. We examined the utility of 930 amplified fragment length polymorphisms (AFLPs) for analyzing relationships among Tasmanian taxa of subgenus Symphyomyrtus section Maidenaria. Phenetic and cladistic analyses resolved species into clusters demonstrating significant genetic partitioning, largely concordant with series defined in the most recent taxonomic revision of Eucalyptus. Some departures from current taxonomy were noted, indicating possible cases of morphological convergence and character reversion. Although the resolution obtained using AFLP was greatly superior to that of single sequence markers, the data demonstrated high homoplasy and incomplete resolution of closely related species. The results of this study and others are consistent with recent speciation and reticulate evolution in Maidenaria. We conclude that a combination of phylogenetic and population genetic approaches using multiple molecular markers offers the best prospects for understanding taxonomic relationships below the sectional level in Eucalyptus.     

Quantification of diatoms in biofilms: standardisation of methods

Benthic diatoms, which often dominate marine biofilms are mostly pennate along with a few centric species that have an attached mode of life. Even though the range of diatoms in biofilms is diverse, their ecology is poorly understood because of the difficulty in sampling and enumeration. Scraping or brushing are the traditional methods used for removal of diatoms from biofilms developed on solid substrata. The method of removal is the most critical step in enumerating the biofilm diatom community structure. In this study, a nylon brush and ceramic scraper were used as tools for the removal of diatoms from 1 - 4-day-old biofilms developed on fibreglass coupons and glass microscope slides. Standardisation of methods showed that the sample volume used in the analyses had the least influence on the quantification, whereas the method of removal was critical. The nylon brush was more efficient at recovering diatoms compared to a ceramic scraper. Direct microscopic enumeration of the community in the case of glass slides indicated that scraping resulted in between 30-50% underestimation. Heterogeneity in diatom community structure between replicate samples is one possible reason for such underestimation.     

Quantification of diatoms in biofilms: Standardisation of methods

Abstract

Benthic diatoms, which often dominate marine biofilms are mostly pennate along with a few centric species that have an attached mode of life. Even though the range of diatoms in biofilms is diverse, their ecology is poorly understood because of the difficulty in sampling and enumeration. Scraping or brushing are the traditional methods used for removal of diatoms from biofilms developed on solid substrata. The method of removal is the most critical step in enumerating the biofilm diatom community structure. In this study, a nylon brush and ceramic scraper were used as tools for the removal of diatoms from 1 – 4-day-old biofilms developed on fibreglass coupons and glass microscope slides. Standardisation of methods showed that the sample volume used in the analyses had the least influence on the quantification, whereas the method of removal was critical. The nylon brush was more efficient at recovering diatoms compared to a ceramic scraper. Direct microscopic enumeration of the community in the case of glass slides indicated that scraping resulted in between 30–50% underestimation. Heterogeneity in diatom community structure between replicate samples is one possible reason for such underestimation.     

Adhesive modular proteins occur in the extracellular mucilage of the motile, pennate diatom Phaeodactylum tricornutum

This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function.     

Some thoughts on taxonomic terminology

There are gaps and inconsistencies in current taxonomic vocabulary which might benefit from some attention. To address these issues, some terminological standards are proposed and explained herein. The validity of terminology derived from clear grammatical patterns is not necessarily precluded by an absence of previous use. It is still, however, a worthwhile exercise to include such potential terms in a body of text that can serve as a reference to anyone wishing to employ terminology of this nature, with the secondary benefit that it may encourage more widespread use of these terms for a more consistent use of taxonomic terminology.     

Fine structure and 18S rDNA phylogeny of a marine araphid pennate diatom Plagiostriata goreensis gen. et sp. nov. (Bacillariophyta)

A marine araphid pennate diatom Plagiostriata goreensis is described from the sand grains of Goree Island, Dakar, Republic of Senegal, based on observations of fine structure of its frustule. The most striking feature of the species is its striation, which is angled at approximately 60° across the robust sternum. The other defining features of the species are its one highly reduced rimoportula and apical pores located at both ends of the valve margin. In the 18S rDNA phylogeny, the species appears as a member of a ‘small‐celled clade’ of araphid pennate diatoms that consist of Nanofrustulum, Opephora and Staurosira. The results of the phylogenetic analyses suggest that the distinct characters of the diatom; namely, oblique striae and apical pores, may have been acquired independently. However, it remains unclear whether the rimoportula of P. goreensis is a reduced state or P. goreensis acquired its morphologically curious rimoportula independently after the loss of an ancient rimoportula at the root of the small‐celled clade.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

RpoA: a useful gene for phylogenetic analysis in diatoms

The aim of this study was to compare the usefulness of two chloroplast-encoded genes (rpoA and rbcL) and the nuclear-encoded small subunit (SSU) ribosomal RNA for reconstructing phylogenetic relationships among diatoms at lower taxonomic levels. To this end, the rpoA and rbcL genes for selected centric and pennate diatoms were sequenced. The new rpoA and rbcL sequences, and an existing nuclear-encoded SSU rRNA data set, were subjected to weighted/unweighted parsimony, maximum likelihood, minimum evolution, and Bayesian analyses. All of the tree-building methods employed showed, based on the support values, that the rpoA gene was the most useful, relative to the rbcL and SSU rRNA genes, in determining phylogenetic relationships among the sampled diatoms. The support values for the relationships among the pennate lineages were, in many instances, greater in the rpoA trees than in the SSU rRNA trees. These results suggest that rpoA might be of value in determining phylogenetic relationships among pennate lineages.     

Analytical studies of silica biomineralization: towards an understanding of silica processing by diatoms

Diatoms have continued to attract research interest over a long time. One important reason for this research interest is the amazingly beautiful microstructured and nanostructured patterning of the silica-based diatom cell walls. These materials become increasingly important from the materials science point of view. However, many aspects of diatom cell wall formation and patterning are still not fully understood. The present minireview article summarizes our recent knowledge especially with respect to two major topics related to diatom cell wall formation and patterning: (1) uptake and metabolism of silicon by living diatom cells and (2) understanding of the genetic control of cell wall formation. Analytical techniques as well as recent results concerning these two topics are highlighted in this review.