Alexandra M. Gottlieb

Molecular analyses of the genus Ilex (Aquifoliaceae) in southern South America, evidence from AFLP and ITS sequence data.

In order to clarify the relationships among southern South American (sSA) representatives of the genus Ilex, an amplified fragment length polymorphism (AFLP) analysis was accomplished. In addition, the phylogenetic relationships of the species were studied using ribosomal internal transcribed spacer (ITS) sequence data alone and in combination with AFLP data, taking into account the possible existence of paralogous sequences and the influence of alignment parameters. To explore stability of phylogenetic hypotheses, a sensitivity analysis was performed using 15 indel-substitution models. Within each species assayed, the AFLPs allowed the recognition of several diagnostic bands. Furthermore, the AFLP analysis revealed that individuals belonging to the same morpho-species formed coherent clades. In addition, some cases of geographical association were noted. Studies on ITS sequences revealed divergence between data obtained herein and sequence data downloaded from GenBank. The sensitivity analyses yielded different interspecific hypotheses of relationships. Notwithstanding, analyses of the ITS data alone and in combination with AFLPs, rendered clades stable to variation in the analytical parameters. Topologies obtained for the AFLPs, the ITS data alone and the combined analyses, demonstrated the existence of a group formed by I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, and I. theezans, and that I. dumosa and I. paraguariensis were distantly related to the former. Incongruence with traditional taxonomical treatments was found.

Isoenzymes of Ganoderma species from southern South America

One hundred and thirty four dikaryotic isolates of Ganoderma, morphologically determined as G. adspersum, G. annulare, G. applanatum, G. brownii, G. lobatoideum, G. lobatum, G. lucidum, G. oerstedii, G. resinaceum, and G. tornatum, were examined by horizontal PAGE for eight enzymatic activities. A total of 94 bands were analysed. The objective was to evaluate the relatedness among the specific taxa by analysing numerically the isoenzymic patterns. To discern fungal individuals somatic incompatibility tests were used. Within each enzyme system several allozyme phenotypes could be observed. The enzymic systems analysed failed to yield diagnostic bands for each morphologically defined group, except the AKP system for G. resinaceum. Some bands were shared by all the isolates studied. On the other hand, diagnostic bands at the species complex level were encountered.

Inonotus s. l. in Argentina - morphology, cultural characters and molecular analyses

A survey of the hymenochaetaceous Inonotus in Argentina was conducted. At least seven Inonotus sensu stricto species were recorded for the region. These are I. ochroporus, I. patouillardii, I. pertenuis, I. quercustris, I. rickii, I. serranus and I. texanus. A detailed description of each species is given, and a key for their identification is provided. Each morphospecies could also be distinguished from both RFLPs and sequence data obtained from PCR products of the internal transcribed spacer region of the nuclear ribosomal RNA gene (ITS). A high level of nucleotide sequence variation was found among Inonotus. Molecular data also indicate that one morphospecies, I. patouillardii, may in fact represent two distinct species. Both cultural and molecular data support the view that Ptychogaster cubensis represents an anamorphic state of I. rickii. Two new combinations are proposed, namely Phellinus crustosus and Inocutis jamaicensis. I. pertenuis is reported for the first time from Argentina.

Taxonomy of Ganoderma from southern South America: subgenus Ganoderma

The macro- and micromorphology of 45 specimens and 19 holotypes of the subgenus Ganoderma (= G. lucidum complex) were examined, together with isoenzymic data. The objective was to evaluate relationships by analysing numerically morphological and isoenzymic data to discern the number of species occurring in southern South America, and their delimitation. A total of 73 isozymic bands and 26 morphological characters were considered. Spore ornamentation was studied under SEM. Nine taxa appear to be represented in the region (G. lucidum var. lucidum, G. multiplicatum, G. sessiliforme, G. praelongum, G. subincrustatum, G. platense, G. zonatum, G. sessile and G. tuberculosum) and the presence of another eight is doubtful (G. argillaceum, G. bibadiostraum, G. chaffangeonii, G. lucidum var. capense, G. lucidum var. dorsale, G. subamboinense var. laevisporum, G. resinaceum and G. tropicum). Three major cutis types were defined and illustrated. Although congruence between dendrograms derived from both data sets was low, some similar groupings were formed. Species descriptions and a key for identification are provided.

Taxonomy of Ganoderma from southern South America: subgenus Elfvingia

Macro- and micromorphology of 124 specimens and 11 holotypes of South American taxa of subgenus Elfvingia (= G. applanatum complex) were examined, and were compared with isoenzymic data previously obtained. Two major dermis types were defined and illustrated. Spore ornamentation was inspected under SEM. Sixty-four per cent of the specimens had an anamixodermis, 80% had spores with longitudinal furrows under SEM and 83% had a context layer without streaks of melanoid deposits. Some of our South American collections could be ascribed to G. lipsiense, G. tornatum, G. testaceum and G. lobatum . Other taxa are critically discussed. The position adopted in this work was to join under synonymy closely related taxa on the grounds of morphology and geographical distribution. In general, correlation between morphological features and isoenzymic patterns could not be established. In this group, phenotypic plasticity appears to be great.

Integral Phylogenomic Approach over Ilex L. Species from Southern South America

The use of molecular markers with inadequate variation levels has resulted in poorly resolved phylogenetic relationships within Ilex. Focusing on southern South American and Asian species, we aimed at contributing informative plastid markers. Also, we intended to gain insights into the nature of morphological and physiological characters used to identify species. We obtained the chloroplast genomes of I. paraguariensis and I. dumosa, and combined these with all the congeneric plastomes currently available to accomplish interspecific comparisons and multilocus analyses. We selected seven introns and nine IGSs as variable non-coding markers that were used in phylogenomic analyses. Eight extra IGSs were proposed as candidate markers. Southern South American species formed one lineage, except for I. paraguariensis, I. dumosa and I. argentina, which occupied intermediate positions among sampled taxa; Euroasiatic species formed two lineages. Some concordant relationships were retrieved from nuclear sequence data. We also conducted integral analyses, involving a supernetwork of molecular data, and a simultaneous analysis of quantitative and qualitative morphological and phytochemical characters, together with molecular data. The total evidence tree was used to study the evolution of non-molecular data, evidencing fifteen non-ambiguous synapomorphic character states and consolidating the relationships among southern South American species. More South American representatives should be incorporated to elucidate their origin.

Multiple isoforms for the catalytic subunit of PKA in the basal fungal lineage Mucor circinelloides.

Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal.