Phylogeny of Amphidinium (Dinophyceae) from Guam and Okinawa, with descriptions of A. pagoense sp. nov. and A. uduigamense sp. nov.

Marine benthic dinoflagellates within the genus Amphidinium were isolated from Guam and Okinawa. Isolated strains were identified to species-level using phylogenetic analyses of 28S rRNA and ITS-5.8S rRNA genes as well as microscopy. Of the six isolated strains, two were new species: A. pagoense sp. nov. and A. uduigamense sp. nov. Other isolates included strains of A. massartii and A. operculatum from Guam, and two strains of A. operculatum from Okinawa. Both new species were described using light and electron microscopy (SEM and TEM). The combination of characteristics that make A. pagoense sp. nov. unique includes a pair of centrally-located pyrenoids, variable cell shape, absence of scales and a long, curved ventral ridge. For A. uduigamense sp. nov., a combination of several morphological features distinguishes it from other species. These include a constriction near the anterior of the hypocone, two centrally located pyrenoids, a longitudinal flagellum inserted in the posterior one-third of the cell, cell size, cell division in the motile stage and the absence of scales. Toxicity was confirmed in these two novel species by testing methanol extracts in an Artemia bioassay. Previously unrecorded ITS rRNA gene sequences from A. operculatum were also sequenced from both locations. Species identified and newly described in this study expand the taxonomic knowledge of Amphidinium in the Pacific.journal articl

Dinoflagellate nucleus contains an extensive endomembrane network, the nuclear net

Dinoflagellates are some of the most common eukaryotic cells in the ocean, but have very unusual nuclei. Many exhibit a form of closed mitosis (dinomitosis) wherein the nuclear envelope (NE) invaginates to form one or more trans-nuclear tunnels. Rather than contact spindles directly, the chromatids then bind to membrane-based kinetochores on the NE. To better understand these unique mitotic features, we reconstructed the nuclear architecture of Polykrikos kofoidii in 3D using focused ion beam scanning electron microscopy (FIB-SEM) in conjunction with high-pressure freezing, freeze-substitution, TEM, and confocal microscopy. We found that P. kofoidii possessed six nuclear tunnels, which were continuous with a reticulating network of membranes that has thus far gone unnoticed. These membranous extensions interconnect the six tunnels while ramifying throughout the nucleus to form a "nuclear net." To our knowledge, the nuclear net is the most elaborate endomembrane structure described within a nucleus. Our findings demonstrate the utility of tomographic approaches for detecting 3D membrane networks and show that nuclear complexity has been underestimated in Polykrikos kofoidii and, potentially, in other dinoflagellates

Cosmopolitan distribution of Endozoicomonas-like organisms and other intracellular microcolonies of bacteria causing infection in marine molluscs

Intracellular microcolonies of bacteria, in some cases developing large extracellular cysts, have been historically reported infecting a wide diversity of economically important mollusc species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterise those organisms, traditionally named as Rickettsia or Chlamydia -like organisms (RLO/CLO), via international collaboration, 98 samples comprising 20 mollusc species were collected over 10 countries and examined using histology and phylogenetic analysis. A 16S rRNA gene amplicon library-based sequencing showed the presence of different species of Endozoicomonas-like organisms (ELO) in all the mollusc species analysed, infecting primarily gill and digestive glands. Co-infections of ELOs with other intracellular bacteria were also observed. Subsequent phylogenetic analysis of Operational Taxonomic Units (OTU) revealed a novel microbial diversity associated with molluscan RLO/CLOs infection distributed along different taxa, including Spirochaetes phyla, Rickettsiales order, Simkaniaceae family, Mycoplasma and Francisella genera, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained. The presence of ELO sequences in the RLO/CLO infection was confirmed by standard PCR, Sanger sequencing, and by in situ hybridisation in a selection of samples. The phylogenetic analysis conducted in this study will allow for further characterization of the microbial community associated with Rickettsia and Chlamydia-like infection in marine molluscs and their correlation with severity of the lesions in order to reveal their role as endosymbionts, commensals or true pathogens.info:eu-repo/semantics/publishedVersio

Species discovery and evolutionary history of marine gregarine apicomplexans

Gregarine apicomplexans are a diverse but poorly understood group of single-celled parasites infecting a wide range of invertebrates in marine, freshwater and terrestrial environments. My thesis focuses on marine gregarines. Gregarines from marine hosts are unique because some (archigregarines) have maintained a set of pleisiomorphic characteristics from the ancestor of gregarines and apicomplexans alike. Other lineages of marine gregarines (eugregarines) are thought to have been modified from this archigregarine morphotype, and represent a wide-range of diversity with regard to general morphology, motility, and feeding strategies. My work has broadly applied molecular phylogenetics to novel species of marine gregarines from areas around British Columbia, Canada and Okinawa, Japan, with the goal of placing the evolution of gregarines in a molecular phylogenetic context. I amplified mainly SSU rDNA from a distinct life history stage (trophozoites), and coupled that with morphological data I gathered from light, confocal, as well as electron microscopy. Although my work was unable to resolve deep phylogenetic relationships among gregarines (and apicomplexans), this work did improve our understanding of evolution within gregarines. With the discovery of Veloxidium leptosynaptae from the gut of an echinoderm in Bamfield, British Columbia, and Surculinium glossobalanae from a hemichordate in Okinawa, I was able to show the paraphyly of the archigregarine morphotype, and polyphyly of other gregarine lineages, including some groups of neogregarines and eugregarines in terrestrial and freshwater environments. With the description Polyplicarium, my work uncovered and identified an ambiguous environmental sequence clade and, along with other work on Selenidium, was able to show that SSU rDNA can be reliably isolated from single cells as a method for delimiting closely related or morphologically similar species. In my final data chapter, I conducted an in-depth study on the morphology and molecular phylogenetic relationships between two sister species from the same host, Selenidium terebellae, and a newly discovered species, Selenidium melitzanae. Results from this data gave me the first opportunity to compare character evolution and niche partitioning among closely related gregarines, and provided another example of convergence of the eugregarine morphotype.Science, Faculty ofZoology, Department ofGraduat

Molecular phylogeny and ultrastructure of two novel parasitic dinoflagellates,Haplozoon gracile sp. nov. and H. pugnus sp. nov.

This study describes two novel parasitic dinoflagellates:Haplozoon gracile sp. nov. isolated from a bamboo worm (Maldanidae), 'cf.Petaloclymenesp.'sensuKobayashiet al. 2018; and,H. pugnus sp. nov. isolated fromNicomachesp. andNicomache personata(Maldanidae). Trophonts (feeding stages) were observed with light, scanning, and transmission electron microscopy. Molecular phylogenetic analyses were performed based on 18S rDNA. COI sequences were obtained for host organisms. Trophonts ofH. gracilewere linear (single longitudinal row) and relatively slender with a mean length of 190 mu m, and consisted of a long and narrow trophocyte, rectangular gonocytes (mean width = 10 mu m), and slightly rounded sporocytes. Trophonts ofH. pugnuswere pectinate (1-8 rows of sporocytes in one plane), with a mean length of 179 mu m, consisting of a bulbous trophocyte, rectangular gonocytes (mean width = 25 mu m), and rounded sporocytes. The body of both species was covered with many depressions that overlaid the amphiesmal vesicles. TEM observations of trophocytes inH. gracilerevealed a stylet with a central dense core and rich mitochondria subtending the amphiesma. Furthermore, amphiesmal vesicles appeared to contain thecal plates in both species. Phylogenetic analyses generally resolved aHaplozoonclade, andH. gracileandH. pugnuswere clearly distinguished from other species for which molecular data are available. Based on the morphological and host comparisons with all described species and their molecular phylogeny, we conclude that these two isolates are new species ofHaplozoon, H. gracile sp. nov. andH. pugnus sp. nov

Discovery of a Diverse Clade of Gregarine Apicomplexans (Apicomplexa: Eugregarinorida) from Pacific Eunicid and Onuphid Polychaetes, Including Descriptions ofParalecudinan. gen.,Trichotokara japonican. sp., andT. eunicaen. sp.

Marine gregarines are poorly understood apicomplexan parasites with large trophozoites that inhabit the body cavities of marine invertebrates. Two novel species of gregarines were discovered in polychaete hosts collected in Canada and Japan. The trophozoites of Trichotokara japonica n. sp. were oval to rhomboidal shaped, and covered with longitudinal epicytic folds with a density of six to eight folds/micron. The nucleus was situated in the middle of the cell, and the mucron was elongated and covered with hair-like projections; antler-like projections also extended from the anterior tip of the mucron. The distinctively large trophozoites of Trichotokara eunicae n. sp. lacked an elongated mucron and had a tadpole-like cell shape consisting of a bulbous anterior region and a tapered tail-like posterior region. The cell surface was covered with longitudinal epicytic folds with a density of three to five folds/micron. Small subunit (SSU) rDNA sequences of both species were very divergent and formed a strongly supported clade with the recently described species Trichotokara nothriae and an environmental sequence (AB275074). This phylogenetic context combined with the morphological features of T. eunicae n. sp. required us to amend the description for Trichotokara. The sister clade to the Trichotokara clade consisted of environmental sequences and Lecudina polymorpha, which also possesses densely packed epicyctic folds (3–5 folds/micron) and a prominently elongated mucron. This improved morphological and molecular phylogenetic context justified the establishment of Paralecudina (ex. Lecudina) polymorpha n. gen. et comb

Coral geometry and why it matters

Clonal organisms like reef building corals exhibit a wide variety of colony morphologies and geometric shapes which can have many physiological and ecological implications. Colony geometry can dictate the relationship between dimensions of volume, surface area, and length, and their associated growth parameters. For calcifying organisms, there is the added dimension of two distinct components of growth, biomass production and calcification. For reef building coral, basic geometric shapes can be used to model the inherent mathematical relationships between various growth parameters and how colony geometry determines which relationships are size-dependent or size-independent. Coral linear extension rates have traditionally been assumed to be size-independent. However, even with a constant calcification rate, extension rates can vary as a function of colony size by virtue of its geometry. Whether the ratio between mass and surface area remains constant or changes with colony size is the determining factor. For some geometric shapes, the coupling of biomass production (proportional to surface area productivity) and calcification (proportional to volume) can cause one aspect of growth to geometrically constrain the other. The nature of this relationship contributes to a species’ life history strategy and has important ecological implications. At one extreme, thin diameter branching corals can maximize growth in surface area and resource acquisition potential, but this geometry requires high biomass production to cover the fast growth in surface area. At the other extreme, growth in large, hemispheroidal corals can be constrained by calcification. These corals grow surface area relatively slowly, thereby retaining a surplus capacity for biomass production which can be allocated towards other anabolic processes. For hemispheroidal corals, the rate of surface area growth rapidly decreases as colony size increases. This ontogenetic relationship underlies the success of microfragmentation used to accelerate restoration of coral cover. However, ontogenetic changes in surface area productivity only applies to certain coral geometries where surface area to volume ratios decrease with colony size

Comparative Ultrastructure and Molecular Phylogeny of Selenidium melongena n. sp and S. terebellae Ray 1930 Demonstrate Niche Partitioning in Marine Gregarine Parasites (Apicomplexa)

Gregarine apicomplexans are a diverse group of single-celled parasites that have feeding stages (trophozoites) and gamonts that generally inhabit the extracellular spaces of invertebrate hosts living in marine, freshwater, and terrestrial environments. Inferences about the evolutionary morphology of gregarine apicomplexans are being incrementally refined by molecular phylogenetic data, which suggest that several traits associated with the feeding cells of gregarines arose by convergent evolution. The study reported here supports these inferences by showing how molecular data reveals traits that are phylogenetically misleading within the context of comparative morphology alone. We examined the ultrastructure and molecular phylogenetic positions of two gregarine species isolated from the spaghetti worm Thelepus japonicus: Selenidium terebellae Ray 1930 and S. melongena n. sp. The ultrastructural traits of S. terebellae were very similar to other species of Selenidium sensu stricto, such as having vermiform trophozoites with an apical complex, few epicytic folds, and a dense array of microtubules underlying the trilayered pellicle. By contrast, S. melongena n. sp. lacked a comparably discrete assembly of subpellicular microtubules, instead employing a system of fibrils beneath the cell surface that supported a relatively dense array of helically arranged epicytic folds. Molecular phylogenetic analyses of small subunit rDNA sequences derived from single-cell PCR unexpectedly demonstrated that these two gregarines are close sister species. The ultrastructural differences between these two species were consistent with the fact that S. terebellae infects the inner lining of the host intestines, and S. melongena n. sp. primarily inhabits the coelom, infecting the outside wall of the host intestine. Altogether, these data demonstrate a compelling case of niche partitioning and associated morphological divergence in marine gregarine apicomplexans. (C) 2014 Elsevier GmbH. All rights reserved