Sexual reproduction and auxospore development in the diatom Biddulphia biddulphiana.

Phylogenetic relationships among mediophycean diatoms with elliptical valve outline and elevated apices have long been a subject of interest and debate, particularly with respect to their relationship to pennates. However, results remain inconclusive, whether based on vegetative valve morphology, reproduction, or molecular phylogenetic data. Searching for phylogenetically informative features, we re-examined sexual reproduction, auxospore structure and development in the diatom Biddulphia biddulphiana. Several unique or unusual features and processes characterized its sexual reproduction. A unique spermatogenesis occurs with premeiotic separation of an anucleate protoplast containing all chloroplasts and likely other organelles. Additionally, their auxospore walls are some of the most complex documented, retaining earlier deposited layers that obscure layers formed during later stages of development. The oldest layer consists of thick, mostly organic incunabulum, underlain by outer and inner epizonia and finally transverse (TP) and longitudinal (LP) perizonia. The complexity of the fine structure of these layers is unprecedented. The orientation of some TP bands is also unique in mediophytes, with some perpendicular to the auxospore apical axis, parallel to each other, and open with aligned ends, as typically seen in pennates. The TP also contains rings slanting toward the apices, as in some other mediophytes, e.g., eupodiscaceans. However, both eupodiscaceans and biddulphiaceans show perizonial band structure derived from anastomosing radial scales, thus termed "scaly bands". Pinnate TP bands, common among pennate auxospores, were not found. Thus B. biddulphiana auxospore wall structure contains a mixture of characters specific to this clone but also known from mediophytes and araphid pennates. However, these features do not provide unequivocal evidence that this or the other Biddulphia species examined to date are the closest extant relatives of basal araphid pennates.

Haslea silbo, A Novel Cosmopolitan Species of Blue Diatoms.

Specimens of a new species of blue diatoms from the genus Haslea Simonsen were discovered in geographically distant sampling sites, first in the Canary Archipelago, then North Carolina, Gulf of Naples, the Croatian South Adriatic Sea, and Turkish coast of the Eastern Mediterranean Sea. An exhaustive characterization of these specimens, using a combined morphological and genomic approach led to the conclusion that they belong to a single new to science cosmopolitan species, Haslea silbo sp. nov. A preliminary characterization of its blue pigment shows similarities to marennine produced by Haslea ostrearia, as evidenced by UV-visible spectrophotometry and Raman spectrometry. Life cycle stages including auxosporulation were also observed, providing data on the cardinal points of this species. For the two most geographically distant populations (North Carolina and East Mediterranean), complete mitochondrial and plastid genomes were sequenced. The mitogenomes of both strains share a rare atp6 pseudogene, but the number, nature, and positions of the group II introns inside its cox1 gene differ between the two populations. There are also two pairs of genes fused in single ORFs. The plastid genomes are characterized by large regions of recombination with plasmid DNA, which are in both cases located between the ycf35 and psbA genes, but whose content differs between the strains. The two sequenced strains hosts three plasmids coding for putative serine recombinase protein whose sequences are compared, and four out of six of these plasmids were highly conserved.

Skin ionocyte remodeling in the amphibious mangrove rivulus fish (Kryptolebias marmoratus).

Amphibious fishes have evolved a variety of physiological modifications allowing them to survive in water and air. In air, the amphibious mangrove rivulus, Kryptolebias marmoratus, uses its skin as a site of ionoregulation. Skin ionocytes actively transport ions into/out of the body; however, it is unclear if there are specific morphological or functional changes occurring in skin ionocytes during air exposure. We used two microscopy techniques to describe skin ionocyte morphology and to investigate their plasticity after salinity challenges and air exposure. Immunohistochemical staining in air-exposed fish revealed ionocytes with Na + /K + ATPase (NKA), Na + /H + exchanger (NHE3b) and cystic fibrosis transmembrane conductance regulator (CFTR) immunoreactivity, whereas ionocytes from aquatic fish had only NKA (freshwater) or NKA and CFTR (brackish and hypersaline water). Following salinity challenges, we noted increases in the number and area of ionocyte apical surfaces, indicating that skin ionocyte activity increased in high salinity environments compared with control conditions. Furthermore, we show increased ionocyte area during air exposure suggesting increased ionocyte activity in all salinity conditions. Using energy dispersive X-ray spectroscopy to analyze the skin surface, we report decreases in magnesium, phosphorous, and sulfur after 7 days in air compared with fish in water, suggesting ionic movement in the skin surface during air exposure. Our study highlights morphological and functional features of skin ionocytes that are involved in ionoregulation in an air-exposed amphibious fish.

A consensus secondary structure of ITS2 for the diatom Order Cymatosirales (Mediophyceae, Bacillariophyta) and reappraisal of the order based on DNA, morphology, and reproduction.

In 1983, Hasle and colleagues removed cymatosiroid diatoms from the pennates, and erected a new centric diatom family, the Cymatosiraceae, mainly to accommodate for their newly discovered mode of sexual reproduction. The new family consisted of two subfamilies differing in frustule structure. The family was later elevated to the rank of Order Cymatosirales Round and Crawford. We revisited intra-ordinal relationships within Cymatosirales using combined genetic (DNA sequences), morphological (valve and frustule structure), and reproductive (auxospore type) characters. In total, 36 cymatosiroid strains from 19 species representing 13 genera (80% of the total number of extant genera; nine of them represented by their generitypes) were used in this study. Instead of only the commonly used loci (18S rRNA and plastidal genes) to infer diatom phylogeny, we developed a consensus secondary structure model of the Internal Transcribed Spacer 2 (ITS2) for this order and applied it to aid in sequence alignments for ITS2. This improved the alignment and thus the robustness of the phylogenetic framework. The compensatory base changes (CBCs) found in ITS2 secondary structures were mapped onto the multi-gene (18S rRNA + ITS2 + rbcL) phylogenetic tree topology. In all these trees, all species grouped into two morphologically and genetically distinct clades. Each clade was supported by multiple CBCs, as did all the clades representing genera. However, these clades did not correspond to the previously established subfamilies. Consequently, we amend the Order Cymatosirales and family Cymatosiraceae, and propose a new family, the Leyanellaceae. The structure of the auxospore was an additional synapomorphic character for Cymatosirales. Overall, we demonstrate a novel approach to study diatom phylogeny across a broader taxonomic range using ITS2 secondary structural information. Our results suggest that this approach might be useful in establishing higher taxonomic relationships in other groups of diatoms.

Structure and Development of the Auxospore in Ardissonea crystallina (C. Agardh) Grunow Demonstrates Another Way for a Centric to Look Like a Pennate.

Reproductive development in Ardissonea crystallina revealed a unique mode of enlargement involving a combination of novel and known structures. In light microscopy, auxospores of this elongated polar centric diatom were superficially similar to the auxospores of pennates. With SEM we found three different components in the auxospore wall. In the youngest, nearly spherical cell-stage, the wall consisted only of a delicate veil containing minute siliceous spherules. Incunabular elements developed underneath this layer. Second, a previously unknown form of specifically modified incunabular scales shaped the subsequent ellipsoidal-capsule auxospore stage. Third, there was a clear contribution of scales to the development of scaly transverse perizonial bands (or scaly bands, for brevity). Such bands, although noted by previous researchers, have not been fully appreciated for the evolutionary information they may convey: possibly common among polar centrics but not pennates. Finally, we propose maintaining the term transverse perizonium to refer to these bands in polar diatoms, but to introduce the differentiation of scaly bands described here from pinnate bands (currently known as typical of pennates). Further research into band types among polar centrics may provide new insights into the relationship between the groups within polar centrics that are currently unresolved by molecular methods.

Gametogenesis and Auxospore Development in the Bipolar Centric Diatom Brockmanniella brockmannii (Family Cymatosiraceae).

We examined gametogenesis and auxospore development in the cymatosiroid diatom Brockmanniella brockmannii. Our mating experiments demonstrated that the clones were homothallic, self-compatible, produced two eggs per oogonium and four hologenous uniflagellate male gametes per spermatogonium. Auxospores grew free in the culture medium and the most external layer of the auxospore wall was made up of spinescent scaly elements in their early globular stage of development. The anisodiametric expansion of the globular auxospore was rather limited, at best proceeding only to a sub-globular stage. Our data suggest that the initial epivalves, nearly pennate-like in outline, developed within such auxospores curled up against the ventral side of the auxospore wall, not across its equatorial plane, and apparently without the help of rings, hoops or bands characteristic of pennate-type transverse perizonia. Thus, B. brockmannii showed a mode of initial epivalve development thus far unknown among diatoms. We propose that progressive silicification of the initial epivalve facilitated the breakdown of the sub-globular auxospore dorsal wall to accommodate for its straightening. The structural features of the spinescent scales on the auxospore wall suggest that Cymatosiraceae could be related to the mediophycean family Eupodiscaceae, although most current molecular phylogenies do not support a direct relationship.

Sexual reproduction in plagiogrammacean diatoms: First insights into the early pennates.

The genera Plagiogramma and Dimeregramma are members of a small, but evolutionarily important group of diatoms, the "basal" araphids. They are sister to all other pennates, both araphid and raphid taxa. Thus, their phylogenetic position carries the potential for providing insights into the earliest pennates. We documented sexual reproduction, mating system and sex cell development in the first members of the "basal" araphid clade ever investigated. The mating system in all these species involved heterothally. It was, however, more complex in P. tsawwassen, where in addition to heterothallic clones, intraclonal and polysexual clones also exist. Auxospore development and wall structure was similar in all three species and demonstrated several characters also reported from "core" araphids. Of these, vigorous, pseudopodial motility of male secondary spermatocytes and gametes was most notable because it indicates that this character was likely present in the last common ancestor of all the pennates. Pseudopodial motility of the male sex cells might have afforded sufficient compensation and/or benefits to the emerging pennates for replacing flagellated sperm, present in centrics. The characters thus far uniquely present among our plagiogrammaceans but not reported from other pennates were: the "gametic" fusion between sex-compatible secondary spermatocytes, in some cases before completion of Meiosis II in males, transverse perizonial bands produced all together or in quick succession rather than being added to the auxospore apex one at a time, and expanding auxospores with 3-4 nuclei. An initial epivalve, similar in morphology to what in some diatoms had been interpreted as a "longitudinal" perizonium, may be more widespread among pennates than thus far appreciated. In addition, we discovered two species new to science (D. acutumontgo, P. tsawwassen), and refined delineation of P. staurophorum by including metric data from the original material.

Auxosporulation in Paralia guyana MacGillivary (Bacillariophyta) and Possible New Insights into the Habit of the Earliest Diatoms.

BACKGROUND: Diatoms are one of the most ecologically important aquatic micro-eukaryotes. As a group unambiguously recognized as diatoms, they seem to have appeared relatively recently with a limited record of putative remains from oldest sediments. In contrast, molecular clock estimates for the earliest possible emergence of diatoms suggest a considerably older date. Depending on the analysis, Paralia and Leptocylindrus have been recovered within the basal molecular divergences of diatoms. Thus these genera may be in the position to inform on characters that the earliest diatoms possessed. FINDINGS: Here we present auxospore development and structure of initial and post-auxospore cells in a representative of the ancient non-polar centric genus Paralia. Their initial frustules showed unusual, but not unprecedented, spore-like morphology. Similarly, initial frustules of Leptocylindrus have been long considered resting spores and a unique peculiarity of this genus. However, even though spore-like in appearance, initial cells of Paralia readily resumed mitotic divisions. In addition, Paralia post-auxospore cells underwent several rounds of mitoses in a multi-step process of building a typical, "perfect" vegetative valve. This degree of heteromorphy immediately post-auxosporulation is thus far unknown among the diatoms. IMPLICATIONS: A spore-related origin of diatoms has already been considered, most recently in the form of the "multiplate diploid cyst" hypothesis. Our discovery that the initial cells in some of the most ancient diatom lineages are structurally spore-like is consistent with that hypothesis because the earliest diatoms may be expected to look somewhat similar to their ancestors. We speculate that because the earliest diatoms may have appeared less diatom-like and more spore-like, they could have gone unrecognized as such in the Triassic/Jurassic sediments. If correct, diatoms may indeed be much older than the fossil record indicates, and possibly more in line with some molecular clock predictions.

Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis.

Freshwater fungi which can survive under metal exposure receive increasing scientific attention. Enhanced synthesis of sulphide and glutathione but no phytochelatin synthesis in response to cadmium (up to 80 µM Cd(2+) in the medium) was measured in the aquatic hyphomycete Heliscus lugdunensis. Up to 25 µmol g(-1) dry mass the fungus formed sulphide in an exponentially Cd(2+)-concentration-dependent manner. Using light microscopy, precipitates were observed outside of the hyphae which could be determined as amorphous particles by X-ray diffraction (XRD). Energy dispersive X-ray spectroscopy (EDS) analysis indicated that these particles were mainly composed of Cd and S with an atomic ratio of 1:1, but some elements of the culture medium such as P and Cl were also present. Fungal cells exposed to Cd(2+) accumulated 12-28 µmol metal g(-1) dry mass over a period of 7-28 days. The results may indicate that sulphide could sequester excess Cd(2+) under oxygen deprived conditions and thereby reduce its toxicity via an additional avoidance mechanism of this fungus.

Diversity and toxicity of the diatom Pseudo-nitzschia Peragallo in the Gulf of Maine, Northwestern Atlantic Ocean.

Multiple species in the toxic marine diatom genus Pseudo-nitzschia have been identified in the Northwestern Atlantic region encompassing the Gulf of Maine (GOM), including the Bay of Fundy (BOF). To gain further knowledge of the taxonomic composition and toxicity of species in this region, Pseudo-nitzschia isolates (n=146) were isolated from samples collected during research cruises that provided broad spatial coverage across the GOM and the southern New England shelf, herein referred to as the GOM region, during 2007-2008. Isolates, and cells in field material collected at 38 stations, were identified using electron microscopy (EM). Eight species (P. americana, P. fraudulenta, P. subpacifica, P. heimii, P. pungens, P. seriata, P. delicatissima and P. turgidula), and a novel form, Pseudo-nitzschia sp. GOM, were identified. Species identity was confirmed by sequencing the large subunit of the ribosomal rDNA (28S) and the internal transcribed spacer 2 (ITS2) for six species (36 isolates). Phylogenetic analyses (including neighbor joining, maximum parsimony, and maximum likelihood estimates and ITS2 secondary structure analysis) and morphometric data supported the placement of P. sp. GOM in a novel clade that includes morphologically and genetically similar isolates from Australia and Spain and is genetically most similar to P. pseudodelicatissima and P. cuspidata. Seven species (46 isolates) were grown in nutrient-replete batch culture and aliquots consisting of cells and growth medium were screened by Biosense ASP ELISA to measure total domoic acid (DA) produced (intracellular + extracellular); P. americana and P. heimii were excluded from all toxin analyses as they did not persist in culture long enough for testing. All 46 isolates screened produced DA in culture and total DA varied among species (e.g., 0.04 to 320 ng ml-1 for P. pungens and P. sp. GOM isolates, respectively) and among isolates of the same species (e.g., 0.24 - 320 ng ml-1 for P. sp. GOM). The 15 most toxic isolates corresponded to P. seriata, P. sp. GOM and P. pungens, and fg DA cell-1 was determined for whole cultures (cells and medium) using ELISA and liquid chromatography (LC) with fluorescence detection (FLD); for seven isolates, toxin levels were also estimated using LC - with mass spectrometry and ultraviolet absorbance detection. Pseudo-nitzschia seriata was the most toxic species (up to 3,500 fg cell-1) and was observed in the GOM region during all cruises (i.e., during the months of April, May, June and October). Pseudo-nitzschia sp. GOM, observed only during September and October 2007, was less toxic (19 - 380 fg cell-1) than P. seriata but more toxic than P. pungens var. pungens (0. 4 fg cell-1). Quantitation of DA indicated that concentrations measured by LC and ELISA were positively and significantly correlated; the lower detection limit of the ELISA permitted quantification of toxicity in isolates that were found to be nontoxic with LC methods. The confirmation of at least seven toxic species and the broad spatial and temporal distribution of toxic Pseudo-nitzschia spp. have significant implications for the regional management of nearshore and offshore shellfisheries resources.