Morphology and molecular phylogeny of Pauciconfibuloides amazonica gen. n. sp. n. (Platyhelminthes,Monogenoidea) parasitizing the Amazonian croaker Plagioscion squamosissimus

An integrative study was performed to understand the phylogenetic relationships of an undescribed, freshwater species of microcotylid parasitizing Plagioscion squamosissimus from the Amazon River Basin. Based on morphological and molecular analysis (18S rDNA and partial 28S rDNA genes), a new genus is proposed to accommodate this new species, Pauciconfibuloides amazonica gen. n. sp. n. The new genus is closely related to Protastomicrocotylinae and Pauciconfibula by sharing the vagina, male copulatory organ, and genital atrium all unarmed. However, Pauciconfibuloides gen. n. can be distinguished from those taxa by the prostatic system and position of the vaginal pore. Molecular phylogenetic inference suggests a sister relationship with species of Polylabris (Prostatomicrocotylinae), but to date, there are no available 18S or 28S rDNA sequences of Pauciconfibula to be compared. This is the first report of a microcotylid parasitizing a freshwater sciaenid from South America.     

FISH mapping of the 5S and 18S-28S rDNA loci in different species of Glycine.

Wild germplasms are often the only significant sources of useful traits for crops, such as soybean, that have limited genetic variability. Before these germplasms can be effectively manipulated they must be characterized at the cytological and molecular levels. Modern soybean probably arose through an ancient allotetraploid event and subsequent diploidization of the genome. However, wild Glycine species have not been intensively investigated for this ancient polyploidy. In this article we determined the number of both the 5S and 18S-28S rDNA sequences in various members of the genus Glycine using FISH. Our results distinctly establish the loss of a 5S rDNA locus from the "diploid" (2n = 40) species and the loss of two from the (2n = 80) polyploids of GLYCINE: A similar diploidization of the 18S-28S rDNA gene family has occurred in G. canescens, G. clandestina, G. soja, and G. max (L.) Merr. (2n = 40). Although of different genome types, G. tabacina and G. tomentella (2n = 80) both showed two major 18S-28S rDNA loci per haploid genome, in contrast to the four loci that would be expected in chromosomes that have undergone two doubling events in their evolutionary history. It is evident that the evolution of the subgenus Glycine is more complex than that represented in a simple diploid-doubled to tetraploid model.     

Molecular Phylogeny of an Indian Population of Kleinstyla dorsicirrata (Foissner, 1982) Foissner et al., 2002. comb. nov. (Hypotrichia,Oxytrichidae): an Oxytrichid with Incomplete Dorsal Kinety Fragmentation

Kleinstyla dorsicirrata (Foissner, 1982) Foissner et al., 2002. comb. nov. (basionym: Gastrostyla dorsicirrata) is a slightly flexible oxytrichid, measuring about 88–115 × 27–46 μm in life and possesses cortical granules. Kleinstyla dorsicirrata is the only oxytrichid known so far with incompletely fragmented dorsal kinety. Morphological and morphogenetic data recognise K. dorsicirrata as nonstylonychine oxytrichid. Molecular phylogeny of an Indian population was inferred using 18S rRNA gene sequences and was examined with respect to oxytrichids exhibiting variation in dorsal kinety fragmentation. Kleinstyla dorsicirrata clusters with Oxytricha lanceolata; this proximity is quite significant as both show deviation from typical oxytrichid fragmentation of dorsal kinety. Molecular phylogeny of Indian population confirms its nonstylonychine oxytrichid status.     

Morphology and molecular phylogeny of the benthic dinoflagellates (Dinophyceae,Peridiniales) Amphidiniopsis crumena n. sp. and Amphidiniopsis nileribanjensis n. sp.

Amphidiniopsis is a benthic, heterotrophic and thecate dinoflagellate genus that has a smaller epitheca and larger hypotheca. The genus contains 24 described species, but is considered to be polyphyletic based on morphological characters and molecular phylogenetics. In this study, two new species were discovered from two distant sampling localities, Amphidiniopsis crumena sp. nov. from Japan, and Amphidiniopsis nileribanjensis sp. nov., from Australia. These species have a uniquely shaped, additional second postcingular plate. Both species are dorsoventrally flattened, an apical hook is present, and have six postcingular plates. The plate formula is: APC 4′ 3a 7″ ?C 4?S 6″′ 2″″. The cells of these species were examined with LM and SEM, and molecular phylogenic analyses were performed using 18S and 28S rDNA. These species are distinguished by the presence of spines on the hypotheca and touching of the sixth postcingular plate and the anterior sulcal plate. Their shape and disposition of several thecal plates also differ. Molecular phylogenetic analyses showed that the two new species formed a monophyletic clade and did not belong to any morphogroup proposed by previous studies. Considering the morphological features and the molecular phylogenetic results, a new morphogroup is proposed, Amphidiniopsis morphogroup VI (‘crumena group’).     

Chromosomal location of 18S and 5S rDNA sites in Triportheus fish species (Characiformes, Characidae)

The location of 18S and 5S rDNA sites was determined in eight species and populations of the fish genus Triportheus by using fluorescent in situ hybridization (FISH). The males and females of all species had 2n = 52 chromosomes and a ZZ/ZW sex chromosome system. A single 18S rDNA site that was roughly equivalent to an Ag-NOR was detected on the short arms of a submetacentric pair in nearly all species, and up to two additional sites were also observed in some species. In addition, another 18S rDNA cluster was identified in a distal region on the long arms of the W chromosome; this finding corroborated previous evidence that this cluster would be a shared feature amongst Triportheus species. In T. angulatus, a heterozygotic paracentric inversion involving the short arms of one homolog of a metacentric pair was associated with NORs. The 5S rDNA sites were located on the short arms of a single submetacentric chromosomal pair, close to the centromeres, except in T. auritus, which had up to ten 5S rDNA sites. The 18S and 5S rDNA sites were co-localized and adjacent on the short arms of a chromosomal pair in two populations of T. nematurus. Although all Triportheus species have a similar karyotypic macrostructure, the results of this work show that in some species ribosomal genes may serve as species-specific markers when used in conjunction with other putatively synapomorphic features.     

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes

We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata. Received: 28 August 2000 / Accepted: 27 January 2001     

Berkleasmium crunisia sp. nov. and its phylogenetic affinities to the Pleosporales based on 18S and 28S rDNA sequence analyses

Berkleasmium crunisia sp. nov. is described from a decaying rachis of Calamus sp. (Arecaceae) from Khuan Ka Long, Satun Province, Thailand. This Berkleasmium species differs morphologically from other species in possessing subtending cells and larger conidia. The phylogenetic relationship of the genus Berkleasmium among sexual ascomycetes also was examined. Sequence analyses from 18S, 28S and ITS-5.8S rDNA were analyzed phylogenetically under maximum parsimony, Bayesian and neighbor joining criteria. Phylogenies revealed that Berkleasmium is not monophyletic. Berkleasmium micronesicum and B. nigroapicale are related to Westerdykella cylindrica and Sporormia australis, which are members of the family Sporormiaceae (Pleosporales). Other species, including our new taxon, appear to share phylogenetic affinities with other anamorphic fungi, whose classification within the Pleosporales is still obscure. Analyses of 18S, 28S, ITS (+5.8S) rDNA and combined (18S+28S) gene sequences fail to give sufficient phylogenetic resolution within the Pleosporales.     

Morphology,Morphogenesis, and Molecular Phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a “Stylonychine Oxytrichidae” (Ciliophora,Hypotrichida) Without Transverse Cirri

A terrestrial oxytrichid ciliate Paraparentocirrus sibillinensis n. gen., n. sp., which was found in soil samples of a beech forest stand within the National Park of Sibillini Mountains, Italy, was investigated using live observation and protargol impregnation. The morphology of interphase, morphogenesis, and molecular phylogeny inferred from SSU rDNA sequences of this ciliate were studied. Paraparentocirrus n. gen., is mainly characterized by a semirigid body, an undulating membrane in the Oxytricha pattern, six fronto‐ventral (FV) rows, the absence of transverse cirri, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kinety 4. During morphogenesis, oral primordia develop through the proliferation of basal bodies from some cirri of FV rows 4 and 5, and FV row 6 takes part in the anlagen formation of the proter. The dorsal morphogenesis was typical of oxytrichids, with simple fragmentation of dorsal kinety 3, and the dorsomarginal rows developed from the right marginal row. Phylogenetic analyses based on the SSU rDNA sequences support the classification of this new genus in the stylonychines.     

Phylogenetic inference regarding Parergodrilidae and Hrabeiella periglandulata ('Polychaeta', Annelida) based on 18S rDNA, 28S rDNA and COI sequences

Parergodrilidae and Hrabeiella periglandulata are Annelida showing different combinations of clitellate-like and aclitellate characters. Similarities between both of these taxa and Clitellata have widely been regarded as the result of convergent evolution due to similar selection pressures. The position of the three taxa in the phylogenetic system of Annelida is still in debate. However, in analyses based on 18S rDNA sequences a close relationship of Parergodrilidae with Orbiniidae and Questidae was suggested. To infer their phylogeny the sequences of the 28S rDNA and of the cytochrome oxidase I (COI) gene of Stygocapitella subterranea , Parergodrilus heideri and H. periglandulata were determined. The data were extended by sequences of various species including species from Clitellata and Orbiniidae. Prior to tree reconstruction the dataset was analysed in detail for phylogenetic content by applying a sliding window analysis, a likelihood mapping and Modeltest V.3.04. Subsequently, generalized parsimony and maximum likelihood methods were employed. Clade robustness was estimated by bootstrapping. In addition, combined analyses of the sequences of 18S rDNA and 28S rDNA as well as of 18S rDNA, 28S rDNA and COI were performed. The combination of the data of the two structure genes and a mitochondrial gene improved the resolution obtained with the single datasets slightly. These analyses support a close relationship of Parergodrilidae and Orbiniidae but cannot resolve the position of H. periglandulata . In every analysis Clitellata cluster within 'Polychaeta', confirming previous investigations.     

Oxnerella micra sp. n. (Oxnerellidae fam. n.), a tiny naked centrohelid, and the diversity and evolution of heliozoa

We describe a new tiny naked centrohelid heliozoan, Oxnerella micra, and sequenced its 18S and 28S rRNA genes. Its extremely slender axopodia have prominent extrusomes and are normally stretched across the substratum like those of many tiny granofilosean Cercozoa. Phylogenetic analysis of 18S rDNA shows that Oxnerella does not branch within any of the six known centrohelid families but very deeply in the order Pterocystida, between Choanocystidae and Pterocystidae; therefore we place it in a new family, Oxnerellidae. Oxnerella arose from ancestors with siliceous scales by losing them; as independently did Heterophryidae and Marophryidae, which replaced them by organic spicules, and Chlamydaster that is not truly naked but retains a mucilage coat and nests extremely shallowly within Pterocystidae. 28S rDNA has a group I intron. Concatenated Bayesian 18S/28S rRNA phylogeny shows centrohelids weakly as sisters to the naked non-centrohelid heliozoan Microheliella maris (Microhelida: Heliozoa). The centrohelid Marophrys marina possesses an elongation factor α-like (EFL) protein related to that of Polyplacocystis; Microheliella also has EFL. We also analysed Hsp90 and 18S rDNA sequences from 'Pinaciophora sp.' ATCC50355; they must be from a centrohelid, probably misidentified as Pinaciophora, the rDNA sequence branching deeply within Pterocystida. We reclassify two Polyplacocystis, Luffisphaera, Phaeodaria and Rotosphaerida.     

Two new ballistoconidium-forming yeast species, Bullera melastomae and Bullera formosana, found in Taiwan

Two yeast strains, the cells of which contained xylose and Q-10 as the major ubiquinone, were isolated from a plant leaf collected in Taiwan. These yeasts were found to represent two new species of the genus Bullera in the Hymenomycetes. Identification was based on the sequence analysis of the 18S rDNA, the internal transcribed spacer (ITS) regions and the D1/D2 domain of 26S rDNA. The yeasts are named Bullera melastomae sp. nov. and Bullera formosana sp. nov. In the phylogenetic trees based on 18S rDNA and D1/D2 domain of 26S rDNA sequences, these two species constitute a cluster connected with Dioszegia cluster in the Cryptococcus luteolus lineage.     

Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.     

Molecular phylogeny of terrestrial holocarpic endoparasitic peronosporomycetes, <Emphasis Type="Italic">Haptoglossa</Emphasis> spp., inferred from 18S rDNA

Phylogenetic relationships of seven isolates of the genus Haptoglossa parasitic on terrestrial nematodes within the Peronosporomycetes were analyzed using 18S rDNA sequence data with 21 peronosporomycetes, 2 marine stramenopilous flagellates, and 2 hyphochytridiomycetes. The marine stramenopilous flagellates and hyphochytridiomycetes were used as the outgroup. All Haptoglossa isolates formed a monophyletic clade and clustered with the marine genus Eurychasma. The clade of Haptoglossa and Eurychasma formed a sister-group to the clade that consisted of all other peronosporomycetes. These results suggest that the genus Haptoglossa and other terrestrial peronosporomycetes included in the two subclasses, the Saprolegniomycetidae and the Peronosporomycetidae, might have originally adapted to the terrestrial environment individually. In the maximum-likelihood (ML) analysis, the Haptoglossa clade was divided into three subclades, one aplanosporic species clade and two zoosporic species clades. Phylogenetic analyses of combined 18S rDNA and cox2 genes among five species of Haptoglossa supported the results of the ML analysis using 18S rDNA and suggested that zoosporic species may be separated into two lineages. This topology of the analysis may suggest that aplanosporic species diverged from zoosporic species.     

Chromosomal localization of 18S + 28S and 5S ribosomal RNA genes in evolutionarily diverse anuran amphibians

The chromosomal locations of the 18S + 28S and 5S ribosomal RNA genes have been analyzed by in situ hybridization in ten anuran species of different taxonomic positions. The chosen species belong to both primitive and evolved families of the present day Anura. Each examined species has 18S + 28S rRNA genes clustered in one locus per haploid chromosome set: this locus is placed either in an intercalary position or proximal to the centromere, or close to the telomere. The 5S rRNA genes are arranged in clusters which vary in number from one to six per haploid set. The 5S rDNA sites are found in intercalary positions, at the telomeres, and at, or close to, the centromeres. Microchromosomes and small chromosomes in primitive karyotypes have been found to carry 5S rDNA sequences. The results are discussed in relation to ideas on the karyological evolution of Amphibia.     

Morphology and Molecular Phylogeny of Pseudocyrtohymena koreana n. g., n. sp. and Antarctic Neokeronopsis asiatica Foissner et al., 2010 (Ciliophora,Sporadotrichida), with a Brief Discussion of the Cyrtohymena Undulating Membranes Pattern

We discovered a new brackish water oxytrichid Pseudocyrtohymena koreana n. g., n. sp. in South Korea and investigated the new species on the basis of morphology, ontogenesis, and 18S rRNA gene sequences. The new genus has the 18 frontal‐ventral‐transverse cirri of typical oxytrichids with flexible body, cortical granules, Cyrtohymena undulating membranes (UM), and one left and one right marginal cirral row. Ontogenesis of the new species indicated that dorsal kinety anlage 3 stretches within the parental row without any fragmentations (Urosomoida pattern) and exclusively forms all caudal cirri. The new genus is morphologically similar to Cyrtohymena Foissner, 1989, but has the following distinctive features: (i) caudal cirri absent in dorsal kineties 1 and 2 (vs. present in Cyrtohymena); and (ii) dorsal kinety 3 nonfragmented (vs. fragmented in Cyrtohymena). Further, we collected an additional species Neokeronopsis asiatica Foissner et al. 2010, from King George Island, Antarctica, and the species shares the morphology of UM with Cyrtohymena. Herein, we describe the previously unidentified characteristics of N. asiatica (i.e., cortical granules, body flexibility, contractile vacuole, and 18S rRNA gene sequence). In addition, we obtained two 18S rRNA gene sequences from Cyrtohymena muscorum and Parasterkiella thompsoni to expand samples for phylogenetic analysis. Our 18S rRNA gene tree supports the hypothesis that the Cyrtohymena UM pattern might have evolved several times in hypotrichs (e.g., Neokeronopsidae, Oxytrichinae, and Stylonychinae).     

Cloning of the 18S rDNA gene, an internal transcribed spacer, and the 5' region of the 28S rDNA gene of Cope's gray treefrog, Hyla chrysoscelis

The location of rDNA genes on the chromosomes of most species is identical within that species, usually occurring on the same chromosome or chromosomes. This is not the case in Cope's gray treefrog, Hyla chrysoscelis, where the rDNA genes are polymorphic for chromosome location. The occasions leading to this polymorphism have yet to be determined. The first step in understanding the nature of the polymorphism is the characterization of the ribosomal gene array. Here we describe the cloning, sequencing, and confirmation, by fluorescence in situ hybridization, of the 18S rDNA gene, a region which includes the end of the 18S rDNA gene, an internal transcribed spacer, and a portion of the 5' end of the 28S rDNA gene in H. chrysoscelis.