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The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120 bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10 pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

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The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20-25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

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Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences.

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Ganoderma includes species of great economic and ecological importance, but taxonomists judge the current nomenclatural situation as chaotic and poorly studied in the neotropics. From this perspective, phylogenetic analyses inferred from ribosomal DNA sequences have aided the clarification of the genus status. In this study, 14 specimens of Ganoderma and two of Tomophagus collected in Brazil were used for DNA extraction, amplification and sequencing of the ITS and LSU regions (rDNA). The phylogenetic delimitation of six neotropical taxa (G. chalceum, G. multiplicatum, G. orbiforme, G. parvulum, G. aff. oerstedtii and Tomophagus colossus) was determined based on these Brazilian specimens and found to be distinct from the laccate Ganoderma from Asia, Europe, North America and from some specimens from Argentina. Phylogenetic reconstructions confirmed that the laccate Ganoderma is distinct from Tomophagus, although they belong to the same group. The use of taxonomic synonyms Ganoderma subamboinense for G. multiplicatum, G. boninense for G. orbiforme and G. chalceum for G. cupreum was not confirmed. However, Ganoderma parvulum was confirmed as the correct name for specimens called G. stipitatum. Furthermore, the name G. lucidum should be used only for European species. The use of valid published names is proposed according to the specimen geographical distribution, their morphological characteristics and rDNA analysis. Rev. Biol. Trop. 62 (3): 1197-1208. Epub 2014 September 01.

Ganoderma incluye especies de gran importancia económica y ecológica, sin embargo, su nomenclatura actual es caótica y poco estudiada en el neotrópico. En este estudio se utilizaron 14 muestras de Ganoderma y dos de Tomophagus recolectados en Brasil para la extracción de ADN, amplificación y secuenciación de las regiones ITS y LSU. La delimitación filogenética de seis táxones neotropicales fue discutida con base en especímenes brasileños y secuencias del GenBank. Estas especies mostraron ser distintas de los Ganoderma lacados de Asia, Europa, América del Norte y de algunos ejemplares de Argentina. Las reconstrucciones filogenéticas confirman que los Ganoderma lacados son distintos de Tomophagus, aunque pertenecen al mismo grupo. No se confirman los sinónimos de G. subamboinense a G. multiplicatum, de G. boninense a G. orbiforme y G. chalceum a G. cupreum. G. parvulum se confirma como el nombre correcto para G. stipitatum. G. lucidum sólo se debe utilizar para especies europeas. Por lo tanto, se propone el uso de nombres publicados válidamente de acuerdo con la distribución geográfica de las muestras, características morfológicas y análisis de ADNr.

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Based on morphological investigations and a phylogeny constructed with partial sequences of the SSU rDNA gene, we revalidate the genus Alwisia and propose the combination Alwisia bombarda Berk. & Broome to be used against Tubifera bombarda (Berk. & Broome) G.W. Martin. Two new species, Alwisia morula and A. repens, are described based on material collected respectively in Costa Rica and Australia. Both new species lack a capillitium and possess individually stalked subspherical sporothecae. Alwisia repens differs from A. morula by its procumbent stalks and iridescent peridium. A comparison of 83 sequences of species in the genera Lycogala, Reticularia and Tubifera with a recent two-gene phylogeny of the bright-spored myxomycetes resulted in a similar topology of both Bayesian and maximum likelihood trees and placed A. bombarda, A. morula and A. repens in one well delimited clade within Reticulariaceae.

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