Irena Kaczmarska

Evolution of the Diatoms: V. Morphological and Cytological Support for the Major Clades and a Taxonomic Revision.

Abstract Phylogenetic reconstructions of the diatoms have been inferred with the 18S and 16S ribosomal RNA genes. Previous studies have shown that the group is divided into two major clades, with support coming initially from the arrangement of the Golgi bodies inside the cells in extant taxa. Features of extinct taxa that also support these clades can be found in the earliest fossil record of the diatoms and include the presence or absence of a central structure in the valve wall and the type of peripheral linking mechanisms between cells. Here we demonstrate that the general pattern of the auxospore expansion and the structure of their walls, the structure of the pyrenoid and the ultrastructure of the spermatozoid further support the molecular clades. Given the combined molecular and morphological support, we propose two new subdivisions (Coscinodiscophytina and Bacillariophytina), emend the classes Coscinodiscophyceae and Bacillariophyceae and propose a new class, the Mediophyceae for the bipolar centrics and the Thalassiosirales.

Barcoding diatoms: Is there a good marker?

The promise of DNA barcoding is based on a small DNA fragment divergence coinciding with biological species separation. Here we evaluated the performance of three markers as diatom barcodes, the small ribosomal subunit (1600 bp), a 5′ end fragment of cytochrome c oxidase subunit 1 (430 bp), and the second internal transcribed spacer region combined with the 5.8S gene (5.8S + ITS‐2, 300–400 bp). Forty‐four sequences per marker representing 28 species from all diatom classes were analysed. Sequence alignment of the three genetic markers and uncorrected genetic distances (P) were calculated at the intra‐ and heterospecific level. All three markers correctly separated the species examined and had advantages which contribute to their feasibility as a DNA barcode. Small ribosomal subunit had the largest GenBank data set, its success rate in amplification and sequencing was assumed to be the highest of all three and was readily aligned. However, it required a long fragment to recover divergence sufficient for species separation and small genetic distances increased the potential for misidentifications. Cytochrome c oxidase subunit 1 demonstrated a substantial heterospecific divergence level and was also readily alignable, but it showed very low amplification and sequencing success rates with currently existing primers. 5.8S + ITS‐2 was amplified and sequenced with high success rate and was the most variable of the three markers, but its secondary structure was needed to aid in alignment. However, since it has been recently suggested that ITS‐2 may provide insight into sexual compatibility, this marker offers an additional advantage. We therefore propose that the 5.8S + ITS‐2 fragment is the best candidate as a diatom DNA barcode.

MOLECULAR PHYLOGENY OF SELECTED MEMBERS OF THE ORDER THALASSIOSIRALES (BACILLARIOPHYTA) AND EVOLUTION OF THE FULTOPORTULA1

Recent phylogenetic studies of the diatoms indicate that members of the order Thalassiosirales occupy an interesting position in the diatom evolutionary tree. Despite their radial morphology and scaly auxospores, they are consistently recovered in molecular analyses as a member of subdivision Bacillariophytina and a sister clade to non‐fultoportulate and non‐radial lithodesmioids. This study included 46 species from nine traditionally accepted extant genera, and analyzed 43 nuclear small subunit (SSU) rRNA sequences in parallel with a survey of the variation in fultoportula structure. Three possible scenarios leading to the evolution of the fultoportula are discussed in the context of molecular and morphological similarities between the examined Thalassiosirales and their SSU rRNA sister clade Lithodesmiales. We speculate that the fultoportula might be derived by a modification of either a cribrum in an areola (fultoportula within an areola), or structures similar to marginal ridges now seen in lithodesmioids around a cluster of poroids (fultoportula in a tube), or finally, that the central fultoportula may have an origin different from the marginal fultoportulae. Our data confirm that fultoportula‐bearing diatoms constitute a natural phylogenetic group. The families Thalassiosiraceae, Skeletonemaceae, and Stephanodiscaceae and the genus Thalassiosira Cleve were unexpectedly found to be paraphyletic. Further, Cyclotella Kutz. and Stephanodiscus Ehr. may not be closely related and some species of these genera are more closely allied to other species of Thalassiosira. The generitype, T. nordenskioeldii, is embedded within a large poorly structured cluster of species that includes several members of Thalassiosira, Planktoniella sol, Minidiscus trioculatus, and two members of Stephanodiscus. An emendment of the order Lithodesmiales and the family Lauderiaceae are proposed.

Survey of the Efficacy of a Short Fragment of the rbcL Gene as a Supplemental DNA Barcode for Diatoms

DNA barcoding is a tool that uses a short, standard segment of DNA to identify organisms. In diatoms, a consensus on an appropriate DNA barcode has not been reached, but several markers show promise. These include the 5.8S gene plus a fragment of the internal transcribed spacer 2 (ITS‐2) of nuclear‐encoded ribosomal RNA, a 420‐bp segment of the 18S rRNA gene, and a 748‐bp fragment at the 3′‐end of the ribulose bisophosphate carboxylase large subunit (rbcL) gene. Here, we tested a 540‐bp fragment 417‐bp downstream of the start codon of the rbcL gene for its efficacy in distinguishing diatom species in a wide range of taxa. Overall, 381 sequences representing 66 genera and 245 species from the classes Mediophyceae and Bacillariophyceae were examined. Intra/interspecific thresholds were set at p = 0.01 differences per site (diff./site) for Mediophyceae and p = 0.02 diff./site for Bacillariophyceae and correctly segregated 96% and 93% of morphological congeners, respectively. When testing reproductively isolated or biological species, which are only available from Bacillariophyceae, 80% of species were discriminated. Therefore, we concluded that, alone, the rbcL region tested herein as potential a DNA barcode was not a sufficient discriminator of all diatoms. We suggest that this fragment could be used in a dual‐locus barcode with the more variable 5.8S+ITS‐2 to discriminate species without sufficient interspecific divergences in the tested rbcL region and to provide insight into species identity from a separately evolved genome.

Relationship between propagule pressure and colonization pressure in invasion ecology: a test with ships' ballast

Increasing empirical evidence indicates the number of released individuals (i.e. propagule pressure) and number of released species (i.e. colonization pressure) are key determinants of the number of species that successfully invade new habitats. In view of these relationships, and the possibility that ships transport whole communities of organisms, we collected 333 ballast water and sediment samples to investigate the relationship between propagule and colonization pressure for a variety of diverse taxonomic groups (diatoms, dinoflagellates and invertebrates). We also reviewed the scientific literature to compare the number of species transported by ships to those reported in nature. Here, we show that even though ships transport nearly entire local communities, a strong relationship between propagule and colonization pressure exists only for dinoflagellates. Our study provides evidence that colonization pressure of invertebrates and diatoms may fluctuate widely irrespective of propagule pressure. We suggest that the lack of correspondence is explained by reduced uptake of invertebrates into the transport vector and the sensitivity of invertebrates and diatoms to selective pressures during transportation. Selection during transportation is initially evident through decreases in propagule pressure, followed by decreased colonization pressure in the most sensitive taxa.

Distance and Character-Based Evaluation of the V4 Region of the 18S rRNA Gene for the Identification of Diatoms (Bacillariophyceae)

DNA barcoding is a molecular tool that exploits a unique DNA sequence of a standardized gene or non-coding region for the species identification of unknown individuals. The investigation into a suitable barcode for diatoms is ongoing and there are several promising candidates including mitochondrial, plastidial and nuclear markers. We analyzed 272 sequences from 76 diatoms species in the orders Thalassiosirales, Lithodesmiales and Cymatosirales, using distance and character based approaches, to assess the applicability of a DNA barcode based on the hypervariable V4 region of the nuclear 18S rRNA gene. We show that the proposed V4 barcode separated ca. 97% of all centric diatom taxa tested using a threshold p-distance of 0.02 and that many problem pairs were further separated using a character based approach. The reliability of amplification, extensive reference library and variability seen in the V4 region make it the most promising candidate to date for a barcode marker for diatoms particularly when combined with DNA character analysis.

Effect of light: dark cycles and cell apical length on the sexual reproduction of the pennate diatom Pseudo-nitzschia multiseries (Bacillariophyceae) in culture

Abstract Sexual reproduction in the domoic acid–producing pennate diatom Pseudo-nitzschia multiseries (Hasle) Hasle is dioecious and involves pairing of two morphologically isogamous parent cells (gametangia) and the production of two gametes per gametangium. Gamete fusion leads to the formation of zygotes, which elongate into auxospores. Within each auxospore, a long initial cell is formed, thereby restoring the longer cell sizes in the population. Mating experiments were performed by mixing active “male” and passive “female” cells from pairs of clones and growing them under different light : dark (L : D) cycle regimes. Two pairs of clones were studied: one pair was ontogenetically older and therefore shorter than the other. After mating, the relative abundance of gametes, zygotes, auxospores, and initial cells was determined for a minimum of four consecutive days. Cell size, which reflects population age, influences sexualization. The short-celled clones had a lower maximum proportion of gametes per vegetative cell (4%) than the long-celled clones (49%), suggesting that the former were nearing the minimal cell length capable of sexualization. The size range within which P. multiseries is able to reproduce sexually is 23–70% of the maximum cell length. A positive correlation was found between hours of light exposure per 24 h at approximately 100 μmol photons m−2 s−1 and sexual cell production of the ontogenetically younger clones. Greatest production of initial cells per female gamete (26%) occurred in 10 h of light. Results from this study indicate that the most biologically successful light regimen in culture corresponds in nature to the day length of the fall bloom from which these clones were isolated.

Proposals for a terminology for diatom sexual reproduction, auxospores and resting stages

The past few decades have brought about a significant expansion in our understanding of the diatom life cycle, particularly its sexual part. Presented here is a set of proposals for the terminology of processes and structures associated with sexual reproduction and for the resting stages of diatoms, some of which have at times been confused with each other. The proposals fill the void present in widely used publications offering standardized terminology related to diatom frustule micro-architecture.

Eucampia Index as an indicator of the Late Pleistocene oscillations of the winter sea-ice extent at the ODP Leg 119 Site 745B at the Kerguelen Plateau

A new paleoenvironmental proxy, Eucampia Index, was used to trace the Late Pleistocene oscillations of winter ice extent at ODP Leg 119, Site 745B (59° 35.71′ S and 85° 51.60′ E) on the Kerguelen Plateau. The index is calculated as the ratio of winter terminal to intercalary valves of the diatom Eucampia antarctica sensu lato. During the early Brunhes the winter sea-ice edge was positioned south from Site 745. It started expanding northward, closer to the site location soon after 0.4 Ma and progressed in a manner of a several wide oscillations. For approximately the last 0.1 Ma the winter sea-ice edge oscillated less and retained similar range of oscillations. The ice edge oscillated in periods which correspond closely to that of Milankovitch oscillations of Earth obliquity, although the significance of individual periods appears to vary in time.

Cryptic diversity in a cosmopolitan diatom known as Asterionellopsis glacialis (Fragilariaceae): Implications for ecology, biogeography, and taxonomy

PREMISE OF THE STUDY Diatoms have long been known as the most species-rich of algal groups, with a wide range of estimates for species number (20-200 k) due to differing species concepts. The fine valve structure in Asterionellopsis glacialis, a diatom believed cosmopolitan and eurytopic, has never been systematically examined using modern microscopy and is an excellent candidate to genetically test morphology-based conspecificity among its geographically distant culture isolates. METHODS Isolates from the Atlantic and Pacific Oceans that were morphologically delineated as A. glacialis were genetically characterized using three nuclear DNA regions (two 18S rDNA fragments and ITS region) and one plastidal (rbcL) and one mitochondrial gene (cox1) and related to SEM-based morphometrics. KEY RESULTS Five genetically distinct groupings were found, four of which are new to science. ITS2 RNA transcript secondary structure was species specific as were plastidal and mitochondrial genes, while the 18S gene fragments did not diverge sufficiently to segregate new species efficiently. We genetically circumscribed the A. glacialis epitype. CONCLUSIONS The morphological diversification of the species examined in this study lags behind their genetic divergence. The currently accepted 2% cutoff level of operational taxonomic unit (OTU) clustering in 18S rDNA environmental sequencing is too high to recognize genetic diversity in Asterionellopsis and very likely in many other species. Our results support the notion that a considerable number of species and diversity remain to be discovered among diatoms and that species number may be more in line with higher estimates. Molecular signatures of the species discovered here will aid in their globally consistent identification and ultimate understanding of their ecology.