María Susana Rossi

Phylogenetic divergence, morphological and physiological differences distinguish a new Neotyphodium endophyte species in the grass Bromus auleticus from South America

The fungi of genus Neotyphodium are systemic, constitutive, symbionts of grasses of subfamily Pooideae. In the southern hemisphere most of these asexual endophytes are the result of the hybridization between two sexual species, Epichloë festucae and E. typhina, from the northern hemisphere. However the ancestral sexual species have not been detected in this region. Several grasses from Argentina are infected by Neotyphodium species. These endophytes are in general very similar macro-and micromorphologically and phylogenetically conform to species N. tembladerae. However the Neotyphodium spp. endophytes of some hosts, Bromus auleticus and Poa spicifomis var. spiciformis, have not been included in this species. In this work we studied the incidence and characterized the diversity of Neotyphodium species in populations of the native grass Bromus auleticus from Argentina. The incidence of endophytes was 100% in all populations investigated. Two groups of endophytes were differentiated by their morphologies, growth rates, conidial ontogenies and by relative resistance to the fungicide benomyl. Phylogenetic trees inferred from tefA and tubB intron sequences indicated that both N. tembladerae and the novel morphotype were hybrids of E. festucae and E. typhina, but the ancestral E. typhina genotype distinguished them. Isolates from plants that inhabit coastal dunes, xerophytic forests, savannahs and hills were similar morphologically and phylogenetically to N. tembladerae, whereas the endophytes from the humid pampa plains conformed to the novel group. We propose the endophyte of Bromus auleticus from humid pampas as a new species, Neotyphodium pampeanum.

Phylogeny of Euglenophyceae based on small subunit rDNA sequences: Taxonomic implications

Small subunit rDNA sequences of 42 taxa belonging to 10 genera were used to infer phylogenetic relationships among euglenoids. Members of the phototrophic genera Euglena, Phacus, Lepocinclis, Colacium, Trachelomonas, and Strombomonas plus the osmotrophs Astasia longa, Khawkinea quartana, and Hyalophacus ocellatus were included. Six major clades were found in most trees using multiple methods. The utility of Bayesian analyses in resolving these clades is demonstrated. The genus Phacus was polyphyletic with taxa sorting into two main clades. The two clades correlated with overall morphology and corresponded in large part to the previously defined sections, Pleur‐ aspis Pochmann and Proterophacus Pochmann. Euglena was also polyphyletic and split into two clades. In Bayesian analyses species with less plastic pellicles and small disk‐like chloroplasts diverged at the base of the tree. They grouped into a single clade which included the two Lepocinclis spp., which also are rigid and bear similar chloroplasts. The metabolic Euglena species with larger plastids bearing pyrenoids and paramylon caps arose near the top of the tree. The loricates Strombomonas and Trachelomonas formed two well‐ supported, but paraphyletic, clades. The strong support for the individual clades confirmed the value of using lorica features as taxonomic criteria. The separation of the osmotrophic species A. longa, K. quartana, and H. ocellatus into different clades suggested that the loss of the photosynthetic ability has occurred multiple times.

Novel processing in a mammalian nuclear 28S pre-rRNA: tissue-specific elimination of an 'intron' bearing a hidden break site.

Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp ‘intron’ located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis‐specific processing eliminates the intron and concomitantly the break site, producing non‐split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.

Chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys

The chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys was analyzed in eight species by in situ hybridization, using a probe isolated from C. porteousi. The hybridization patterns showed different numbers of chromosomes with positive pericentromeric regions and/or complete short arms. In some species, a positive signal was scarce (or not detectable, as in C. opimus), and was usually located in the pericentromeric areas (C. occultus and C. latro). In those species where the satellite was highly amplified, its chromosomal localization tended to encompass the entire length of the short arms. These patterns were compared with C-band distribution patterns in the same species. We discuss the putative evolutionary trend of this satellite DNA in the genus Ctenomys and suggest that it evolved from a strictly pericentromeric localization to comprising the whole short arms of some chromosomes.

Retroviral-like features in the monomer of the major satellite DNA from the South American rodents of the genus Ctenomys.

It is well known that uninfected mammalian cells contain DNA sequences which are closely related to retroviral genomic segments. However, these sequences seldom (if ever) have been found associated to highly repetitive (satellite) DNA. RPCS is a 348 bp monomer of a major satellite DNA from the South American rodents of the genus Ctenomys. It was found that RPCS contains several elements which are typical of the U3 region of retroviral LTRs. These elements are: a) a polypurine tract; b) two enhancer core sequences; c) two NF1 binding sites; d) two C/EBP binding sites; e) two CCAAT-motifs; f) a TATA box, and g) two putative polyadenylation motifs. Furthermore, the relative positions of these elements are as in the U3 retroviral regions.

Species diversity of Epichloë symbiotic with two grasses from southern Argentinean Patagonia

In this work we performed morphological and molecular phylogenetic analyses (based on sequences of calmodulin M [calM], translation-elongation factor 1-α [tefA] and β-tubulin [tubB] genes) to characterize the diversity of Epichloë endophytes in Bromus setifolius and Phleum alpinum. The phylogenies obtained from the three genes were congruent and allowed differentiation of three lineages of endophytes that also presented morphological differences. One lineage corresponds to the previously described species Epichloë tembladerae, which is present in a wide range of native grasses from Argentina including B. setifolius and P. alpinum. Another genotype isolated only from B. setifolius is a non-hybrid endophyte, a rare condition for the South American Epichloë endophytes. Isolates of this genotype, described herein as a new variety, Epichloë typhina var. aonikenkana, presented waxy colonies at maturity and a low production of conidia. The third lineage, exclusively found in isolates from P. alpinum, is a hybrid between E. typhina and a common ancestor of E. amarillans and E. baconii. Isolates of this lineage produce abundant conidia that are variable in shape and size. Based on its unique phylogenetic position and morphology, we propose the new species, Epichloë cabralii for this lineage. The new combinations Epichloë tembladerae and E. pampeana also are proposed for the previously described Neotyphodium tembladerae and Neotyphodium pampeanum species.

mRNA Encoding a Putative RNA Helicase of the DEAD‐Box Gene Family is Up‐Regulated in Trypomastigotes of Trypanosoma cruzi

Abstract Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.

Diversity of tuco-tucos (Ctenomys, Rodentia) in the Northeastern wetlands from Argentina: mitochondrial phylogeny and chromosomal evolution

Tuco-tucos (small subterranean rodents of the genus Ctenomys) that inhabit sandy soils of the area under the influence of the second largest wetland of South America, in Northeastern Argentina (Corrientes province), are a complex of species and forms whose taxonomic status were not defined, nor are the evolutionary relationships among them. The tuco-tuco populations of this area exhibit one of the most ample grades of chromosomal variability within the genus. In order to analyze evolutionary relationships within the Corrientes group and its chromosomal variability, we completed the missing karyotypic information and performed a phylogenetic analysis. We obtained partial sequences of three mitochondrial markers: D-loop, cytochrome b and cytochrome oxidase I. The Corrientes group was monophyletic and split into three main clades that grouped related karyomorphs. The phylogeny suggested an ancestral condition of the karyomorph with diploid number (2n) 70 and fundamental number (FN) 84 that has evolved mainly via reductions of the FN although amplifications occurred in certain lineages. We discuss the relationship between patterns of chromosomal variability and species and groups boundaries. From the three main clades the one named iberá exhibited a remarkable karyotypic homogeneity, and could be considered as an independent and cohesive evolutionary lineage. On the contrary, the former recognized species C. dorbignyi is a polyphyletic lineage and hence its systematic classification should be reviewed.

The library model for satellite DNA evolution: a case study with the rodents of the genus Ctenomys (Octodontidae) from the Iberá marsh, Argentina

On the basement of the library model of satellite DNA evolution is the differential amplification of subfamilies through lineages diversification. However, this idea has rarely been explored from an experimental point of view. In the present work, we analyzed copy number and sequence variability of RPCS (repetitive PvuII Ctenomys sequence), the major satellite DNA present in the genomes of the rodents of the genus Ctenomys, in a closely related group of species and forms inhabiting the Iberá marsh in Argentina. We studied the dependence of these two parameters at the intrapopulation level because in the case of interbreeding genomes, differences in RPCS copy number are due to recent amplification/contraction events. We found an inverse relationship among RPCS copy number and sequence variability: amplifications lead to a decrease in sequence variability, by means of biased homogenization of the overall satellite DNA, prevailing few variants. On the contrary, the contraction events that involve tandems of homogeneous monomers contribute—by default—minor variants to become “evident”, which otherwise were undetectable. On the other hand, all the RPCS sequence variants are totally or partially shared by all the studied populations. As a whole, these results are comprehensible if these RPCS variants preexisted in the common ancestor of this Ctenomys group.

Integrative lineage delimitation in rodents of the Ctenomys Corrientes group

Abstract The tuco-tucos rodents (genus Ctenomys) of the Corrientes group comprise several populations that inhabit the vast area under the influence of the Iberá wetland. Lineage delimitation within the recently diverged Corrientes group is a challenging task as morphological differentiation is not conspicuous between populations. However, delimitation is crucial for evolutionary studies and conservation issues. In this study, we performed a phylogenetic analysis including cytochrome b (cyt-b) sequences from taxa that had never been studied in a comprehensive context. We integrated previously published chromosomal studies, mitochondrial phylogenies and simple sequence repeat (SSR) variability analyses, and applied a delimitation criterion over the basis of chromosomal incompatibilities and genetic exclusivity. Under this integrative approach seven independently evolving lineages were delimited in the Corrientes group: Ctenomys roigi, which conserves its former definition, Ctenomys dorbignyi and Ctenomys perrensi complex which were redefined, Sarandicito which includes the population of Paraje Sarandicito and probably a group of nearby poorly studied populations, and Iberá i, ii and iii distributed at both sides of the Iberá wetland. We discuss future perspectives to evaluate the proposed lineages and conservation issues concerning these tuco-tucos.