Every refuge has its price: Ostreobium as a model for understanding how algae can live in rock and stay in business.

Ostreobium is a siphonous green alga in the Bryopsidales (Chlorophyta) that burrows into calcium carbonate (CaCO3) substrates. In this habitat, it lives under environmental conditions unusual for an alga (i.e., low light and low oxygen) and it is a major agent of carbonate reef bioerosion. In coral skeletons, Ostreobium can form conspicuous green bands recognizable by the naked eye and it is thought to contribute to the coral's nutritional needs. With coral reefs in global decline, there is a renewed focus on understanding Ostreobium biology and its roles in the coral holobiont. This review summarizes knowledge on Ostreobium's morphological structure, biodiversity and evolution, photosynthesis, mechanism of bioerosion and its role as a member of the coral holobiont. We discuss the resources available to study Ostreobium biology, lay out some of the uncharted territories in Ostreobium biology and offer perspectives for future research.

Molecular phylogeny and taxonomic revision of the genus Wittrockiella (Pithophoraceae, Cladophorales), including the descriptions of W. australis sp. nov. and W. zosterae sp. nov.

Wittrockiella is a small genus of filamentous green algae that occurs in habitats with reduced or fluctuating salinities. Many aspects of the basic biology of these algae are still unknown and the phylogenetic relationships within the genus have not been fully explored. We provide a phylogeny based on three ribosomal markers (ITS, LSU, and SSU rDNA) of the genus, including broad intraspecific sampling for W. lyallii and W. salina, recommendations for the use of existing names are made, and highlight aspects of their physiology and life cycle. Molecular data indicate that there are five species of Wittrockiella. Two new species, W. australis and W. zosterae, are described, both are endophytes. Although W. lyallii and W. salina can be identified morphologically, there are no diagnostic morphological characters to distinguish between W. amphibia, W. australis, and W. zosterae. A range of low molecular weight carbohydrates were analyzed but proved to not be taxonomically informative. The distribution range of W. salina is extended to the Northern Hemisphere as this species has been found in brackish lakes in Japan. Furthermore, it is shown that there are no grounds to recognize W. salina var. kraftii, which was described as an endemic variety from a freshwater habitat on Lord Howe Island, Australia. Culture experiments indicate that W. australis has a preference for growth in lower salinities over full seawater. For W. amphibia and W. zosterae, sexual reproduction is documented, and the split of these species is possibly attributable to polyploidization.

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts.

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single-celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra-isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co-evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.

New Ulvaceae (Ulvophyceae, Chlorophyta) from mesophotic ecosystems across the Hawaiian Archipelago.

Ulvalean algae (Chlorophyta) are most commonly described from intertidal and shallow subtidal marine environments worldwide, but are less well known from mesophotic environments. Their morphological simplicity and phenotypic plasticity make accurate species determinations difficult, even at the generic level. Here, we describe the mesophotic Ulvales species composition from 13 locations across 2,300 km of the Hawaiian Archipelago. Twenty-eight representative Ulvales specimens from 64 to 125 m depths were collected using technical diving, submersibles, and remotely operated vehicles. Morphological and molecular characters suggest that mesophotic Ulvales in Hawaiian waters form unique communities comprising four species within the genera Ulva and Umbraulva, each with discrete geographic and/or depth-related distributional patterns. Three genetically distinct taxa are supported by both plastid (rbcL and tufA) and nuclear (ITS1) markers, and are presented here as new species: Umbraulva kaloakulau, Ulva ohiohilulu, and Ulva iliohaha. We also propose a new Umbraulva species (Umbraulva kuaweuweu), which is closely related to subtidal records from New Zealand and Australia, but not formally described. To our knowledge, these are the first marine species descriptions from Hawai'i resulting from the collaboration of traditional Hawaiian nomenclature specialists, cultural practitioners and scientists. The difficulty of finding reliable diagnostic morphological characters for these species reflects a common problem worldwide of achieving accurate identification of ulvalean taxa using solely morphological criteria. Mesophotic Ulvales appear to be distinct from shallow-water populations in Hawai'i, but their degree of similarity to mesophotic floras in other locations in the Pacific remains unknown.

Small doses, big troubles: modeling growth dynamics of organisms affecting microalgal production cultures in closed photobioreactors.

The destruction of mass cultures of microalgae by biological contamination of culture medium is a pervasive and expensive problem, in industry and research. A mathematical model has been formulated that attempts to explain contaminant growth dynamics in closed photobioreactors (PBRs). The model simulates an initial growth phase without PBR dilution, followed by a production phase in which culture is intermittently removed. Contaminants can be introduced at any of these stages. The model shows how exponential growth from low initial inocula can lead to "explosive" growth in the population of contaminants, appearing days to weeks after inoculation. Principal influences are contaminant growth rate, PBR dilution rate, and the size of initial contaminant inoculum. Predictions corresponded closely with observed behavior of two contaminants, Uronema sp. and Neoparamoeba sp., found in operating PBRs. A simple, cheap and effective protocol was developed for short-term prediction of contamination in PBRs, using microscopy and archived samples.

Molecular and morphological diversity of Narragansett Bay (RI, USA) Ulva (Ulvales, Chlorophyta) populations.

Macroalgal bloom-forming species occur in coastal systems worldwide. However, due to overlapping morphologies in some taxa, accurate taxonomic assessment and classification of these species can be quite challenging. We investigated the molecular and morphological characteristics of 153 specimens of bloom-forming Ulva located in and around Narragansett Bay, RI, USA. We analyzed sequences of the nuclear internal transcribed spacer 1 region (ITS1) and the chloroplast-encoded rbcL; based on the ITS1 data, we grouped the specimens into nine operational taxonomic units (OTUs). Eight of these OTUs have been previously reported to exist, while one is novel. Of the eight OTUs, all shared sequence identity with previously published sequences or differed by less than 1.5% sequence divergence for two molecular markers. Previously, 10 species names were reported for Ulva in Rhode Island (one blade and nine tube-forming species) based upon morphological classification alone. Of our nine OTUs, three contained blade-forming specimens (U. lactuca, U. compressa, U. rigida), one OTU had a blade with a tubular stipe, and six contained unbranched and/or branched tubular morphologies (one of these six, U. compressa, had both a blade and a tube morphology). While the three blade-forming OTUs in Narragansett Bay can frequently be distinguished by careful observations of morphological characteristics, and spatial/temporal distribution, it is much more difficult to distinguish among the tube-forming specimens based upon morphology or distribution alone. Our data support the molecular species concept for Ulva, and indicate that molecular-based classifications of Ulva species are critical for proper species identification, and subsequent ecological assessment or mitigation of Ulva blooms.

MOLECULAR PHYLOGENY AND TAXONOMY OF THE AEGAGROPILA CLADE (CLADOPHORALES, ULVOPHYCEAE), INCLUDING THE DESCRIPTION OF AEGAGROPILOPSIS GEN. NOV. AND PSEUDOCLADOPHORA GEN. NOV.(1).

The Aegagropila clade represents a unique group of cladophoralean green algae occurring mainly in brackish and freshwater environments. The clade is sister to the species-rich and primarily marine Cladophora and Siphonocladus lineages. Phylogenetic analyses of partial LSU and SSU nrDNA sequences reveal four main lineages within the Aegagropila clade, and allow a taxonomic reassessment. One lineage consists of two marine 'Cladophora' species, for which the new genus Pseudocladophora and the new family Pseudocladophoraceae are proposed. For the other lineages, the family name Pithophoraceae is reinstated. Within the Pithophoraceae, the earliest diverging lineage includes Wittrockiella and Cladophorella calcicola, occurring mainly in brackish and subaerial habitats. The two other lineages are restricted to freshwater. One of them shows a strong tendency for epizoism, and consists of Basicladia species and Arnoldiella conchophila. The other lineage includes Aegagropila, Pithophora and a small number of tropical 'Cladophora' species. The latter are transferred to the new genus Aegagropilopsis. Previously, polypyramidal pyrenoids had been suggested to be apomorphous for this clade, but we report the finding of both polypyramidal and bilenticular pyrenoids in members of the Pithophoraceae, and thus show that this character has no diagnostic value.

Subulatomonas tetraspora nov. gen. nov. sp. is a member of a previously unrecognized major clade of eukaryotes.

While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and 'slightly brackish' environments, respectively. Phylogenetic analyses of these two taxa plus closely related sequences from environmental surveys provide support for a novel clade of eukaryotes that is distinct from the major clades including the Opisthokonta, Excavata, Amoebozoa and 'SAR' (Stramenopile, Alveolate, Rhizaria).

THE GREEN ALGAL GENUS CLONIOPHORA REPRESENTS A NOVEL LINEAGE IN THE ULVALES: A PROPOSAL FOR CLONIOPHORACEAE FAM. NOV.(1).

The green algal genus Cloniophora has been classified in the Chaetophorales (Chlorophyceae) based on morphological characters. This study uses DNA sequence data from the nucleus (SSU) and the chloroplast (tufA) from collections in the Hawaiian Islands and a culture originating from Portugal to test this classification. Taxonomic identities of contemporary collections were confirmed by sequencing small fragments of DNA (rbcL and SSU) from type specimens, including the generitype, Cloniophora willei L. H. Tiffany. These molecular data show that Cloniophora does not have close affinities to the Chaetophorales and belongs instead to the Ulvales (Ulvophyceae). The morphological features of eight or more reproductive products per cell and a pyrenoid with a traversing thylakoid membrane support the molecular data and confirm the placement of this group in the Ulvales. As this genus does not belong to any recognized family in the Ulvales, the family Cloniophoraceae, containing the single genus Cloniophora, is proposed.

Environmental controls, oceanography and population dynamics of pathogens and harmful algal blooms: connecting sources to human exposure.

Coupled physical-biological models are capable of linking the complex interactions between environmental factors and physical hydrodynamics to simulate the growth, toxicity and transport of infectious pathogens and harmful algal blooms (HABs). Such simulations can be used to assess and predict the impact of pathogens and HABs on human health. Given the widespread and increasing reliance of coastal communities on aquatic systems for drinking water, seafood and recreation, such predictions are critical for making informed resource management decisions. Here we identify three challenges to making this connection between pathogens/HABs and human health: predicting concentrations and toxicity; identifying the spatial and temporal scales of population and ecosystem interactions; and applying the understanding of population dynamics of pathogens/HABs to management strategies. We elaborate on the need to meet each of these challenges, describe how modeling approaches can be used and discuss strategies for moving forward in addressing these challenges.

Microheterogeneity and coevolution: an examination of rDNA sequence characteristics in Neoparamoeba pemaquidensis and its prokinetoplastid endosymbiont.

Neoparamoeba pemaquidensis, the etiological agent of amoebic gill disease, has shown surprising sequence variability among different copies of the 18S ribosomal RNA gene within an isolate. This intra-genomic microheterogeneity was confirmed and extended to an analysis of the internal transcribed spacer (ITS) region. High levels of intra-genomic nucleotide diversity (Pi=0.0201-0.0313) were found among sequenced ITS regions from individual host amoeba isolates. In contrast, the ITS region of its endosymbiont revealed significantly lower levels of intra-genomic nucleotide diversity (Pi=0.0028-0.0056) compared with the host N. pemaquidensis. Phylogenetic and ParaFit coevolution analyses involving N. pemaquidensis isolates and their respective endosymbionts confirmed a significant coevolutionary relationship between the two protists. The observation of non-shared microheterogeneity and coevolution emphasizes the complexity of the interactions between N. pemaquidensis and its obligate endosymbiont.

Characterization of Neoparamoeba pemaquidensis strains: PCR-RFLP of the internal transcribed spacer region from the amoeba and endosymbiont.

Neoparamoeba pemaquidensis continues to be an ongoing problem for commercial finfish aquaculture and has also sporadically been associated with mass mortalities of commercially relevant marine invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of the biology as it applies to host range, pathogenicity, tissue tropism, and geographic distribution is severely lacking. This may stem from the inability of current diagnostic tests based on morphology, immunology, and molecular biology to differentiate strains at the subspecies level. In the present study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the internal transcribed spacer (ITS) region that can accurately differentiate amoeba strains of N. pemaquidensis. The investigation focused on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont, Ichthyobodo necator related organism (IRO), ITS region as an alternative marker. The combination of host amoeba and endosymbiont ITS PCR-RFLP analyses was successfully used to correctly identify and characterize an N. pemaquidensis isolate from an outbreak of amoebic gill disease in Atlantic salmon Salmo salar from the west coast of North America (Washington State, USA).

Protistan grazing analysis by flow cytometry using prey labeled by in vivo expression of fluorescent proteins.

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: two new amitochondrial heterolobosean amoebae from anoxic environments.

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Two new small-subunit ribosomal RNA gene lineages within the subclass gymnamoebia.

Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.

How oxymonads lost their groove: an ultrastructural comparison of Monocercomonoides and excavate taxa.

Despite being amongst the more familiar groups of heterotrophic flagellates, the evolutionary affinities of oxymonads remain poorly understood. A re-interpretation of the cytoskeleton of the oxymonad Monocercomonoides hausmanni suggests that this organism has a similar ultrastructural organisation to members of the informal assemblage 'excavate taxa'. The preaxostyle, 'R1' root, and 'R2' root of M. hausmanni are proposed to be homologous to the right, left, and anterior roots respectively of excavate taxa. The 'paracrystalline' portion of the preaxostyle, previously treated as unique to oxymonads, is proposed to be homologous to the I fibre of excavate taxa. Other non-microtubular fibres are identified that have both positional and substructural similarity to the distinctive B and C fibres of excavate taxa. A homologue to the 'singlet root', otherwise distinctive for excavate taxa, is also proposed. The preaxostyle and C fibre homologue in Monocercomonoides are most similar to the homologous structures in Trimastix. suggesting a particularly close relationship. This supports and extends recent molecular phylogenetic findings that Trimastix and oxymonads form a clade. We conclude that oxymonads have an excavate ancestry, and that the 'excavate taxa' sensu stricto form a paraphyletic assemblage.

The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution.

The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.