Mixed trypanosomatid infections (a simultaneous presence of two or more parasites in the same host) have long been suspected to represent an obstacle for recovering cultures that would faithfully represent original species descriptions. However, without the means to directly compare the parasites in the host and in culture, this would remain just a possibility. Here we have used PCR-based genotyping of spliced leader RNA gene repeats to analyse several novel species of insect trypanosomatids isolated from heteropteran hosts and to compare them with the parasites that had been detected in the gut smears of the same hosts. We have found that, whereas the original infections were dominated by some blastocrithidia-like parasites, most of the respective axenic cultures contained novel species of Crithidia and Leptomonas. Therefore, we concluded that, in each case, this replacement was caused by differences in cultivation properties between the original predominant blastocrithidia and the less fastidious parasite that was later recovered in culture. The properties of the new organisms, including their morphology and ultrastructure, as well as their phylogenetic affinities within the family, were investigated and used to describe five novel species.
During a survey of yeasts associated with the phylloplane of several bromeliad species in Itapuã Park in southern Brazil, we isolated four orange-coloured strains which were found to represent a novel anamorphic tremellaceous (Tremellales, Agaricomycotina, Basidiomycota) yeast species, Cryptococcus bromeliarum sp. nov. (type strain BI20T =CBS 10424T =NRRL Y-48112T). PCR-fingerprinting profiles of the four strains with primers M13 and (GTG)5 were almost identical, which suggested conspecificity among the isolates. On the basis of D1/D2 26S rDNA sequence analysis, C. bromeliarum is phylogenetically closely related to other orange-coloured Cryptococcus species, namely Cryptococcus armeniacus, C. amylolyticus, C. tibetensis and C. cistialbidi, but differed from these species by at least six nucleotide substitutions and was thus considered a separate species. Physiological differences from C. armeniacus, C. amylolyticus and C. cistialbidi included the inability of C. bromeliarum to assimilate citrate and to form starch-like compounds. Differentiation from C. tibetensis can be achieved by the ability of the latter to assimilate ethylamine.
In the course of molecular characterization of yeasts isolated from decaying leaves collected in Cambodia, anamorphic yeast strains were detected that clustered with the Microstromatales in molecular phylogenetic analyses of the chromosomal regions coding for the D1/D2 domain of the large-subunit 26S rRNA gene, the small-subunit 18S rRNA gene and the internal transcribed spacer ITS1-5.8S rRNA gene-ITS2. In the trees obtained, the isolates formed a distinct, basal branch of Microstromatales supported with high bootstrap values. As the isolates could not be identified with any known genus, we have described them as Jaminaea angkorensis gen. et sp. nov. (type strain C5bT=CBS 10918T=CCY 88-1-1T). In the chromosomal region coding for the 18S rRNA gene, the type strain of the novel species contained a group IB intron that was similar in location and sequence to introns found in certain species of Exobasidiales and Entylomatales. As no similar introns have been detected in Microstromatales, the new genus might represent a phylogenetic link connecting these three orders.