A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

ABSTRACT: Mesocoelium lanfrediae sp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp nov., from Atlantic Rainforest of southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Svalbard 2010 mesocosm experiment: Protistan diversity

Hierarchical clustering. Taxonomic assignment of reads was performed using a preexisting database of SSU rDNA sequences from including XXX reference sequences generated by Sanger sequencing. Experimental amplicons (reads), sorted by abundance, were then concatenated with the reference extracted sequences sorted by decreasing length. All sequences, experimental and referential, were then clustered to 85% identity using the global alignment clustering option of the uclust module from the usearch v4.0 software (Edgar, 2010). Each 85% cluster was then reclustered at a higher stringency level (86%) and so on (87%, 88%,.) in a hierarchical manner up to 100% similarity. Each experimental sequence was then identified by the list of clusters to which it belonged at 85% to 100% levels. This information can be viewed as a matrix with the lines corresponding to different sequences and the columns corresponding to the cluster membership at each clustering level. Taxonomic assignment for a given read was performed by first looking if reference sequences clustered with the experimental sequence at the 100% clustering level. If this was the case, the last common taxonomic name of the reference sequence(s) within the cluster was used to assign the environmental read. If not, the same procedure was applied to clusters from 99% to 85% similarity if necessary, until a cluster was found containing both the experimental read and reference sequence(s), in which case sequences were taxonomically assigned as described above.

(Table 1) Statistics of size measurements of Lagenoeca antarctica sp. n. and Salpingoeca abyssalis sp. n. (floating form) sampled during POLARSTERN cruise ANT-XXII/2 and METEOR cruise M63/2

Despite their high abundance and their high importance for the oceanic matter flux, heterotrophic nanoflagellates are only poorly studied in the deep-sea regions. Studies on the choanoflagellate distribution during two deep-sea expeditions, to the South Atlantic (5038 m) and Antarctica (Weddell Sea, 2551 m), revealed the deepest records of choanoflagellates so far. A new species, (Lagenoeca antarctica) with a conspicuous spike structure on the theca is described from deep Antarctic waters. Lagenoeca antarctica sp. n. is a solitary unstalked free living salpingoecid-like choanoflagellate. The protoplast is surrounded by a typical theca with unique spikes only visible in SEM micrographs. The ovoid cell nearly fills the whole theca and ranges in size from 4 to 6 µm. The collar measures 2-3 µm and the flagellum 3-5 µm. A second species, Salpingoeca abyssalis sp. n., was isolated from the abyssal plain of the South Atlantic (5038 m depth). Floating and attached forms were observed. The protoplast ranges from to 2 to 4 µm in length and 1 to 2 µm in width. The collar is about the same length as the protoplast and the flagellum has 2 to 2.5 × the length of the protoplast. Phylogenetic analyses based on a fragment of SSU rDNA revealed Salpingoeca abyssalis to cluster together with a marine isolate of Salpingoeca infusionum while Lagenoeca antarctica clusters separately from the other codonosigid and salpingoecid taxa. Salpingoeca abyssalis and an undetermined Monosiga species seems to be the first choanoflagellate species recorded from the abyssal plain.

First report of three Kudoa species from Eastern Australia: Kudoa thyrsites from Mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus)

Fish species around the world are parasitized by myxozoans of the genus Kudoa, several of which infect and cause damage of commercial importance. In particular, Kudoa thyrsites and Kudoa amamiensis infect certain cultured fish species causing damage to muscle tissue, making the fish unmarketable. Kudoa thyrsites has a broad host and geographic range infecting over 35 different fish species worldwide, while K. amamiensis has only been reported from a few species in Japanese waters. Through morphological and molecular analyses we have confirmed the presence of both of these parasites in eastern Australian waters. In addition, a novel Kudoa species was identified, having stellate spores, with one polar capsule larger than the other three. The SSU rDNA sequence of this parasite was 1.5% different from K. thyrsites and is an outlier from K. thyrsites representatives in a phylogenetic analysis. Furthermore, the spores of this parasite are distinctly smaller than those of K. thyrsites, and thus it is described as Kudoa minithyrsites n. sp. Although the potential effects of K. minithyrsites n. sp. on its fish hosts are unknown, both K. thyrsites and K. amamiensis are associated with flesh quality problems in some cultured species and may be potential threats to an expanding aquaculture industry in Australia.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

Novel uncultivated labyrinthulomycetes revealed by 18S rDNA sequences from seawater and sediment samples

Labyrinthulomycetes (Labyrinthulea) are ubiquitous marine osmoheterotrophic protists that appear to be important in decomposition of both allochthonous and autochthonous organic matter. We used a cultivation-independent method based on the labyrinthulomycete-specific primer LABY-Y to PCR amplify, clone, and sequence 68 nearly full-length 18S rDNA amplicons from 4 sediment and 3 seawater samples collected in estuarine habitats around Long Island, New York, USA. Phylogenetic analyses revealed that all 68 amplicons belonged to the Labyrinthulea. Only 15 of the 68 amplicons belonged to the thraustochytrid phylogenetic group (Thraustochytriidae). None of these 15 were similar to cultivated strains, and 11 formed a novel group. The remaining 53 amplicons belonged either to the labyrinthulid phylogenetic group (Labyrinthulidae) or to other families of Labyrinthulea. that have not yet been described. Of these amplicons, 37 were closely related to previously cultivated Aplanochytrium spp. and Oblongichytrium spp. Members of these 2 genera were also cultivated from 1 of the sediment samples. The 16 other amplicons were not closely related to cultivated strains, and 15 belonged to 5 groups of apparently novel labyrinthulomycetes. Most of the novel groups of amplicons also contained environmental sequences from surveys of protist diversity using universal 18S rDNA primers. Because the primer LABY-Y is biased against several groups of labyrinthulomycetes, particularly among the thraustochytrids, these results may underestimate the undiscovered diversity of labyrinthulomycetes.

Assessment of prey overlap between a native (Polistes humilis) and an introduced (Vespula germanica) social wasp using morphology and phylogenetic analyses of 16S rDNA

In newly invaded communities, interspecific competition is thought to play an important role in determining the success of the invader and its impact on the native community. In southern Australia, the native Polistes humilis was the predominant social wasp prior to the arrival of the exotic Vespula germanica (Hymenoptera: Vespidae). Both species forage for similar resources (water, pulp, carbohydrate and protein prey), and concerns have arisen about potential competition between them. The aim of this study was to identify the protein foods that these wasps feed on. As many prey items are masticated by these wasps to the degree that they cannot be identified using conventional means, morphological identification was complemented by sequencing fragments of the mitochondrial 16S rRNA gene. GenBank searches using blast and phylogenetic analyses were used to identify prey items to at least order level. The results were used to construct complete prey inventories for the two species. These indicate that while P. humilis is restricted to feeding on lepidopteran larvae, V. germanica collects a variety of prey of invertebrate and vertebrate origin. Calculated values of prey overlap between the two species are used to discuss the implications of V. germanica impacting on P. humilis. Results obtained are compared to those gained by solely 'conventional' methods, and the advantages of using DNA-based taxonomy in ecological studies are emphasized.

Puccinia psidii in Queensland, Australia: disease symptoms, distribution and impact

Puccinia psidii has long been considered a significant threat to Australian plant industries and ecosystems. In April 2010, P. psidii was detected for the first time in Australia on the central coast of New South Wales (NSW). The fungus spread rapidly along the east coast and in December 2010 was found in Queensland (Qld) followed by Victoria a year later. Puccinia psidii was initially restricted to the southeastern part of Qld but spread as far north as Mossman. In Qld, 48 species of Myrtaceae are considered highly or extremely susceptible to the disease. The impact of P. psidii on individual trees and shrubs has ranged from minor leaf spots, foliage, stem and branch dieback to reduced fecundity. Tree death, as a result of repeated infection, has been recorded for Rhodomyrtus psidioides. Rust infection has also been recorded on flower buds, flowers and fruits of 28 host species. Morphological and molecular characteristics were used to confirm the identification of P. psidii from a range of Myrtaceae in Qld and compared with isolates from NSW and overseas. A reconstructed phylogeny based on the LSU and SSU regions of rDNA did not resolve the familial placement of P. psidii, but indicated that it does not belong to the Pucciniaceae. Uredo rangelii was found to be con-specific with all isolates of P. psidii in morphology, ITS and LSU sequence data, and host range.

The systematics of Endoraecium in Australia

Endoraecium (Raveneliaceae, Pucciniales) is a genus of rust that infects several species of Acacia (Fabaceae) in Australia, south-east Asia and Hawaii. Thirteen species of Endoraecium have been described, including seven species that are endemic to Australia, one species to south-east Asia and five to Hawaii. This study investigated the systematics of Endoraecium from 50 specimens in Australia and south-east Asia with a combined morphological and molecular approach. Phylogenetic analyses were conducted on combined datasets of the SSU, ITS and LSU regions of rDNA. The recovered phylogeny (i) supported a recent division of Endoraecium digitatum into five separate species based on morphology and host specificity and (ii) found lineages that did not correspond with known species.

Initiation of transcription of rDNA in rice

Three direct repeats of 320, 340 and 238 nucleotides were detected upstream to the 5′ end of the 18S rRNA gene of an rDNA unit present on a 9.8 kb EcoRT fragment of the rice DNA. The primer extension analysis showed that the site of initiation of transcription is in the 1st repeat at an A, the 623rd nucleotide upstream to the 5′ end of the 18S rRNA gene. Different stretches of the intergenic spacer DNA linked to the Chloramphenicol acetyl transferase gene were transcribed in the intact nuclei of rice embryos. The S1 nuclease protection analysis of the transcripts using [32P]-labelled Chloramphenicol acetyl transferase gene as the probe showed the presence of multiple promoters for rDNA transcription.