Genetic barcoding of Ecuadorian epilithic diatom species suitable as water quality bioindicators.

Diatom identification is a key step in using these microorganisms as water quality bioindicators. Morphological diagnosis is a difficult task due to the enormous number of species and their microscopic size. This can be overcome using molecular tools to complement the diagnosis. The main goal of this work was to obtain the DNA barcode of Ecuadorian epilithic diatoms with a wide geographical distribution, a well-defined ecological range and characteristics that allow them to be reliable indicator species. Unialgal diatom cultures were obtained from environmental samples of Ecuadorian Andean streams. Morphological characterization of cultures was carried out under SEM microscopy. For molecular characterization, 18SV4 and rbcL barcodes were sequenced from each strain and blasted against a GenBank database. A phylogenetic tree for each barcode was constructed using the ML method including sequences of strains of the studied species from different geographical locations. The results showed the following five species to be suitable as bioindicators and these were isolated. Sellaphora seminulum (strain JA01b, c), Nitzschia fonticola (strain SP02a) and N. palea (strain CA01a) are tolerant to eutrophication; Eolimna minima (strain CH02a) is a mesotrophic water bioindicator, and Achnanthidium minutissimum (strain JA01a) is an oligotrophic water bioindicator. The comparison with the GenBank database of the barcoding regions supported the morphological identification. The barcoding sequences of the strains showed a high percentage of identity with the sequences reported in INSDC databases for the same species. The topology of the phylogenetic trees demonstrates that epilithic diatoms from Ecuador are closely related to those of same species isolated from other geographical regions. This study is a first attempt to establish a morphological and molecular taxonomic reference library for neotropical diatoms. This study demonstrates that it would be feasible to use the existing barcoding data for diatoms to develop molecular tools for the bioassessment of aquatic ecosystems in the Ecuadorian Andean region.

L'identification des diatomées est une étape clé dans l'utilisation de ces microorganismes comme bioindicateurs de la qualité de l'eau. Le diagnostic morphologique est une tâche difficile en raison du nombre considérable d'espèces et de leur dimension microscopique. Il est possible de surmonter cette difficulté en utilisant des techniques moléculaires pour compléter le diagnostic. L'objectif principal de ce travail était d'obtenir le code-barre de l'ADN des diatomées épilithiques équatoriennes ayant une large distribution géographique, une niche écologique bien définie et des caractéristiques leur permettant d'être des espèces indicatrices fiables. Des cultures de diatomées unialgales ont été obtenues à partir d'échantillons environnementaux de cours d'eau des Andes équatoriennes. La caractérisation morphologique des cultures a été réalisée sous microscopie MEB. Pour la caractérisation moléculaire, les codes-barres 18SV4 et rbcL ont été séquencés à partir de chaque souche et comparés à la base de données GenBank. Pour chaque code-barres, un arbre phylogénétique a été construit à partir de la méthode ML comprenant des séquences de souches des espèces étudiées, provenant de différents lieux géographiques. Les résultats ayant montré que les cinq espèces suivantes étaient appropriées comme bioindicateurs, elles ont été isolées. Sellaphora seminulum (souche JA01b, c), Nitzschia fonticola (souche SP02a) et N. palea (souche CA01a) sont tolérantes à l'eutrophisation ; Eolimna minima (souche CH02a) est un bioindicateur d'eau mésotrophe, et Achnanthidium minutissimum (souche JA01a) est un bioindicateur d'eau oligotrophe. La comparaison avec la base de données GenBank des régions de code-barres a supporté leurs identifications morphologiques. Les séquences de code-barres des souches ont montré un pourcentage élevé d'identité génétique avec les séquences signalées dans les bases de données de l'INSDC pour la même espèce. La topologie des arbres phylogénétiques démontre que les diatomées épilithiques de l'Équateur sont étroitement liées à celles des mêmes espèces isolées d'autres régions géographiques. Cette étude est une première tentative d'établir une bibliothèque de référence morphologique et taxonomique moléculaire pour les diatomées néotropicales. Cette étude démontre qu'il serait possible d'utiliser les données de code-barres existantes pour les diatomées afin de développer des instruments moléculaires pour la bioévaluation des écosystèmes aquatiques dans la région andine équatorienne.

DNA Sequencing of Type Material Reveals Pneophyllum marlothii comb. nov. from South Africa and P. discoideum comb. nov. (Chamberlainoideae, Corallinales, Rhodophyta) from Argentina.

A partial rbcL sequence from the type material of Spongites discoideus from southern Argentina showed that it was distinct from rbcL sequences of South African specimens to which that name had been applied based on morpho-anatomy. A partial rbcL sequence from an original syntype specimen, herein designated the lectotype, of Lithophyllum marlothii, type locality Camps Bay, Western Cape Province, South Africa, was identical to rbcL sequences of South African field-collected specimens assigned to S. discoideus. Based on phylogenetic analyses of rbcL and/or psbA sequences, both of these species belong in Pneophyllum and are transferred there as P. discoideum comb. nov. and P. marlothii comb. nov. The two species exhibit a distinct type of development where thick, secondary, monomerous disks are produced from thin, primary, dimerous crusts. Whether this type of development represents an example of convergent evolution or is characteristic of a clade of species within Pneophyllum remains to be resolved.

A segment of rbcL gene as a potential tool for forensic discrimination of Cannabis sativa seized at Rio de Janeiro, Brazil.

Cannabis sativa, known by the common name marijuana, is the psychoactive drug most widely distributed in the world. Identification of Cannabis cultivars may be useful for association to illegal crops, which may reveal trafficking routes and related criminal groups. This study provides evidence for the performance of a segment of the rbcL gene, through genetic signature, as a tool for identification for C. sativa samples apprehended by the Rio de Janeiro Police, Brazil. The PCR amplified and further sequenced the fragment of approximately 561 bp of 24 samples of C. sativa rbcL gene and showed the same nucleotide sequences, suggesting a possible genetic similarity or identical varieties. Comparing with other Cannabaceae family sequences, we have found 99% of similarity between the Rio de Janeiro sequence and three other C. sativa rbcL genes. These findings suggest that the fragment utilized at this study is efficient in identifying C. sativa samples, therefore, useful in genetic discrimination of samples seized in forensic cases.

PHYLOGEOGRAPHY OF BATRACHOSPERMUM MACROSPORUM (BATRACHOSPERMALES, RHODOPHYTA) FROM NORTH AND SOUTH AMERICA(1).

Phylogeographic trends in Batrachospermum macrosporum Mont. were investigated using the mitochondrial intergenic spacer between the cytochrome oxidase subunit 2 and 3 genes (cox2-3). A total of 11 stream segments were sampled with seven in the coastal plain of North America and four in tropical areas of South America. Fifteen thalli were sampled from seven streams, 14 thalli from two streams, and eight thalli from two streams. There were 16 haplotypes detected using 149 individuals. Of the eight haplotypes from locations in North America, all were 334 base pairs (bp) in length, and of those from South America, five were 344 bp, and three were 348 bp. Two individual networks were produced: one for the haplotypes from North America and another for those from South America, and these could not be joined due to the large number of base pair differences. This split between haplotypes from North and South America was confirmed with sequence data of the rbcL gene. There was very little genetic variation among the haplotypes from the North American locations, leading us to hypothesize that these are fairly recent colonization events along the coastal plain. In contrast, there was high variation among haplotypes from South America, and it would appear that the Amazon serves as a center of diversity. We detected considerable variation in haplotypes among streams, but frequently, a single haplotype in an individual stream segment, which is consistent with data from previous studies of other batrachospermalean taxa, may suggest a single colonization event per stream.