The taxonomy of yeasts found in exudates of certain trees and other natural breeding sites of some species ofDrosophila

Summary A survey was made of the yeasts occurring in slime fluxes ofQuercus kellogii (black oak),Abies concolor (red fir) and in certain mushrooms and dead logs in the mountains of the Yosemite region of California. Most of the 134 isolates identified were found to be non-fermentative or poorly fermentative (fermenting glucose only and usually weakly). The isolates were placed in the following genera:Pichia (34),Debaryomyces (35), imperfect forms ofHansenula (39),Endomyces (3),Saccharomyces (2),Candida (11),Trichosporon (7),Torulopsis (1),Cryptococcus (1) andSporobolomyces (1). Four new species have been described,Pichia silvestris, Pichia quercibus, Pichia carsonii andDebaryomyces fluxorum. P. quercibus andP. carsonii are unusual representatives of the genusPichia in that they form a very primitive pseudomycelium and lack pellicles on malt extract. To accommodate such species an amended diagnosis of the genusPichia has been proposed byPhaff in an accompanying paper.Saccharomyces pastori, of which 12 isolates were obtained, has been transferred to the amended genusPichia asP. pastori (Guilliermond) nov. comb.     

DNA relatedness among saturn-spored yeasts assigned to the generaWilliopsis andPichia

Saturn-spored species assigned to the generaWilliopsis andPichia were compared from extent of nuclear DNA complementarity. Of thePichia spp., four were recognized as distinct taxa:P. dispora, P. saitoi, P. zaruensis andPichia sp. nov. AmongWilliopsis spp., the following were accepted:W. californica, W. mucosa comb. nov.,W. pratensis, W. saturnus var.saturnus, W. saturnus var.mrakii comb. nov.,W. saturnus var.sargentensis comb. nov.,W. saturnus var.subsufficiens comb. nov. andWilliopsis sp. nov. The newPichia andWilliopsis species are described elsewhere. Moderate (36–68%) DNA relatedness was detected between the formerPichia sargentensis and varieties ofW. saturnus again demonstrating that nitrate assimilation is not a reliable criterion for separating yeast species.     

Differential toxinogenesis in the genusPichia detected by an anti-yeast killer toxin monoclonal antibody

The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genusPichia that were previously classified in the genusHansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain ofPichia anomala (UCSC 25F). The killer phenotypes of thePichia isolates were evaluated by their ability to kill each other. The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genusPichia as well as the presumptive dual killer/sensitive identity for each single isolate.     

Two new species ofPichia isolated from slime fluxes of deciduous trees

Two new homothallic species ofPichia, associated with exudates ofPopulus trichocarpa andSalix sp. have been described.Pichia trehalophila sp.n. was isolated from slime exudates in two widely separatedPopulus trichocarpa trees andPichia salictaria sp.n. was isolated from two exudates ofSalix sp. The latter species was previously present as an unidentified strain ofPichia in the culture collection of the Centraalbureau voor Schimmelcultures and had been isolated from a patient in Germany. The natural habitat ofP. salictaria is considered to be in association with willow trees.     

A proposal for amendment of the diagnosis of the genusPichia hansen

Summary An amended diagnosis has been given for the genusPichia Hansen to allow inclusion of species characterized by a lack of pellicle formation, by the absence or a strongly reduced pseudomycelium or by a combination of both properties. The following species fit the amended diagnosis ofPichia: P haplophila Shifrine et Phaff,P. carsonii Phaff et Knapp,P. quercibus Phaff et Knapp andP. xylosa Phaff, Miller et Shifrine. Furthermore, it has been proposed to transferSaccharomyces pastori Guilliermond,Saccharomyces pini Holst andDebaryomyces vini Zimmermann to the amended genusPichia, where they fit well rather than occupying and exceptional position in their present genera.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

The utilization ofL-amino acids as carbon source by yeasts of the generaHansenula andTrichosporon

The assimilation of singleL-amino acids as carbon source in the absence of added nitrogen has been studied for all the described species in the generaHansenula andTrichosporon. The range in the number of amino acids used by individual species varied in the two genera, inHansenula from 0 to 6, inTrichosporon from 0 to 16. These characters were considered in relation to the phylogenetic scheme and ecological origin ofHansenula.     

Systématique du genre Kluyveromyces: Hybridation ADN-ADN

Deoxyribonucleic acids of 8 species ofKluyveromyces and one round-spored species ofPichia have been compared with P³² labelled DNA ofK. marxianus in view of a systematic study by a method of hybridization in liquid media.The species related toK. marxianus by currently employed systematic characters show generally a good nucleotide sequence homology (> 70%), exceptK. wickerhamii.On the contrary,K. africanus, K. phaffii andP. abadiae show a very low percentage of hybridization withK. marxianus.This molecular approach yields useful information to test the value of usual criteria of yeast systematic.

---

---

Collaboration technique: Mme Geneviéve Billon-Grand.     

The taxonomy of yeasts isolated fromDrosophila in the Yosemite region of California

Summary A survey was made of the yeasts occurring in the intestinal tract of wild species ofDrosophila occurring in the Yosemite Region of California. Two hundred and forty one yeasts, representing 42 species and varieties, were identified. Each isolate was obtained from a different fly. Almost half of the isolates belong toSaccharomyces. The most common species in this genus wereS. montanus (36 isolates),S. veronae (30 isolates),S. cerevisiae var.tetrasporus (22 isolates) andS. drosophilarum (13 isolates). Further species are listed in Table 1.Zygosaccharomyces fermentati Naganishi was shown to be a distinct species and not a synonym ofS. cerevisiae. In order to avoid confusion with another yeast of the same name, it has been proposed to change the nameZ. fermentati toS. montanus Naganishi. Two new species ofSaccharomyces were described,S. wickerhamii andS. kluyveri. S. mangini var.tetrasporus has been renamedS. cerevisiae var.tetrasporus. A non-cellobiose attacking strain ofS. drosophilarum has been designated tentativelyS. drosophilarum var.acellobiosa. A new species of the genusPichia was described asP. xylosa. Saccharomyces pastori andSaccharomyces pini were transferred to the genusPichia on the basis of arguments given in the preceding paper. A new species ofTrichosporon was described asTr. aculeatum on the basis of the presence of characteristic needlelike cells. Common species besides those mentioned inSaccharomyces wereHansenula angusta (19),Kloeckera apiculata (15),Kl. magna (13), andTorulopsis stellata (10). Other genera represented wereHanseniaspora, Cryptococus, Rhodotorula, Candida andOospora. Evidence was obtained that many species of imperfect genera consist of distinctly different physiological types.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Serratia ficaria sp. nov., a bacterial species associated with Smyrna figs and the fig waspBlastophaga psenes

Fourteen strains isolated from figs, caprifigs, and fig wasps collected in California and Tunisia, and from a small black ant in France, constitute a new DNA hybridization group that is 25–56% related toSerratia species, and 6–17% related to other species of Enterobacteriaceae. This homogeneous group (90% relatedness within the group) constitutes a new species that is namedSerratia ficaria sp. nov. (type strain, ICPB 4050, ATCC 33105). Strains of this species have a characteristic odor, similar to that ofS: odorifera andPseudomonas perolens. No strain ofS. ficaria has yet been recovered from clinical specimens.     

Systematics of the generaDebaryomyces andMetschnikowia: Proton magnetic resonance spectra of their mannans as an aid in classification

The proton magnetic resonance spectra of the mannans of a number ofDebaryomyces andMetschnikowia species (Endomycetales) were determined. The spectra of all of the mannans had several similar characteristics, regardless of the species from which they originated. Pichia vini, a species originally classified asDebaryomyces vini, formed a mannan with a spectrum almost identical with those ofMetschnikowia (Candida) reukaufii andPichia haplophila. Debaryomyces vanrijii, originally placed in the genusPichia, formed a mannan with a spectrum identical with those ofPichia robertsii andCandida (Pichia) guilliermondii mannans.The authors wish to thank Mr. M. Mazurek for the determination of the p.m.r. spectra, and Mr. N. R. Gardner and Mr. R. J. Magus for valuable technical assistance.     

Species of hypocrella

Summary Additional information is given from studies of types and authentic specimens for 25 species of Hypocrella.Stereocrea aurantiaca is transferred to Hypocrella asH. aurantiaca (Petch) comb. nov.,H. caulium is considered an invalid name andH. brasiliana (P. Henn.) comb. nov. is proposed. It is concluded thatH. sloaneae, H. nectroides, H. citrina, H. melaena, H. disjuncta andH. amazonica all are valid species.H. verruculosa is considered conspecific withH. phyllogena. H. fluminensis is a synonym ofH. epiphylla andH. guaranitica, H. spegazzini, H. orbicularis andH. cornuta are synonyms ofH. palmae. Similarities between several species of the eastern and western hemispheres are noted.Paper from the Herbarium and the Department of Botany, University of Michigan, Ann Arbor, Michigan.     

Phylogenetic relationships among species ofWilliopsis andSaturnospora gen. nov. as determined from partial rRNA sequences

Phylogenetic relationships among those yeast species that form saturn-shaped ascospores and which are assigned to the generaWilliopsis andPichia were estimated from their extent of nucleotide sequence divergence in three regions of ribosomal RNA. ThePichia species (P. dispora, P. saitoi, P. zaruensis andP. sp. nov.) are a closely clustered group only distantly related toWilliopsis, and it is proposed that they be reassigned toSaturnospora gen. nov. The extent of divergence amongWilliopsis species (W. californica, W. mucosa, W. pratensis, W. saturnus andW. sp. nov.) is greater than that previously observed within other ascomycetous yeast genera.     

Studies on DNA sequences in the Osmundaceae

Summary Phylogenetic relationships ofOsmunda cinnamomea, O. claytoniana, andO. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences ofO. claytoniana are nearly equally diverged from those ofO. cinnamomea andO. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the threeOsmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hybridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.     

Evolution of single-copy DNA and the ADH gene in seven drosophilids

Summary Single-copy DNA was isolated fromDrosophila melanogaster and hybridized with total genomic DNA ofD. melanogaster, D. mauritiana, D. simulans, D. pseudoobscura, D. willistoni, D. hydei andD. virilis. The duplexes were thermally eluted from hydroxyapatite and the data used to assess the relatedness of each species toD. melanogaster. The general pattern of relatedness was similar to that predicted by morphological methods but with some notable exceptions. The rate of nucleotide substitution was estimated to be greater than 0.66% of bases per million years. An unexpected, rapidly evolving component ofD. melanogaster single-copy DNA was identified. The relatedness of these species was also studied with respect to the gene coding for alcohol dehydrogenase (ADH). The ADH gene, previously cloned fromD. melanogaster (Goldberg 1980), was hybridized with Southern blots of genomic digests of the seven species. The intensity and position of the hybridizing bands suggest the amount of divergence of the gene. Divergence was quantitated by reassociation of a fragment of the cloned ADH gene with total DNA of the seven drosophilids and thermal elution of the resultant duplexes from hydroxyapatite. The ADH gene was isolated from genomic clone libraries ofD. melanogaster, D. simulans andD. mauritiana and further studied by comparison of position of restriction sites. Species relationships deduced from comparison of total single-copy DNA and the ADH gene were consistent, demonstrating that a single gene can reflect divergence of the entire genome.     

Species-specific banding patterns of restriction endonuclease-digested mitochondrial DNA from the genusPythium

Restriction endonuclease-digested mitochondrial DNA from 29Pythium spp. showed distinctly different species-specific electrophoretic banding patterns. Numerical comparisons among species were conducted by calculating the percentage of restriction fragments having the same apparent molecular size. The greatest interspecific similarity in banding patterns (67%) was observed betweenHindIII digests ofPythium heterothallicum andP. sylvaticum. However, comparisons among other species generally revealed similarities of less than 50%, and often less than 30%. The lack of similarity of restriction banding patterns was observed even with several species that share many common morphological features:P. arrhenomanes vsP. graminicola (20%),P. myriotylum vsP. aristosporum (28%), andP. torulosum vsP. vanterpoolii (32%). In contrast to the fragment size heterogeneity among different species, isolates of the same species have highly conserved restriction patterns. Ten isolates ofP. oligandrum, collected from the United States, South Africa, and Czechoslovakia, had a minimum of 86% similarity inHindIII banding patterns. Similar results were observed with eight isolates ofP. ultimum, five ofP. acanthicum, six ofP. spinosum, five ofP. sylvaticum, and eight ofP. irregulare. However, two isolates ofP. irregulare exhibited a higher degree of heterogeneity and shared only 64 to 76% comigrating bands with the eight other isolates of this species.     

Hybridation ADN-ADN chez quelques espèces du genre Hansenula

The genus Hansenula was considered a long time ago as a good pattern for phylogenetic research. In 1969, Wickerham proposed an evolutive scheme based upon morphological, physiological and ecological criteria. Recently, relatedness among yeasts were analysed by DNA-DNA hybridization in liquid medium. H. anomala var. anomala (G + C content: 37,1%) was compared with H. anomala var. schneggii (37.6%), H. subpelliculosa (33,8%), H. ciferrii (33,1%), H. holstii (37%) belonging to the same line 2, and also with H. beckii (38,3%) line 3, H. sydowiorum (40,1%) and H. muscicola (37,1%).These results showed little relatedness between H. anomala var. anomala /H. ciferrii and H. anomala var. anomala/ H. subpelliculosa. On the other hand, H. anomala var. schneggii shared 89,5% of its nucleotide sequences with H. anomala var. anomala. These 2 strains were considered to represent the same species. H. holstii showed 67.1% complementarity with H. anomala var. anomala: this strain is considered to represent valid species, different from H. anomala var. anomala, but H. muscicola with 72.5% relatedness to H. anomala var. anomala could be considered as a limit species. An unexpected finding was that H. beckii was closely related to H. anomala var. anomala (84.8%). These data suggested the inadequacy of current criteria used to establish the phylogenetic lines in genus Hansenula.     

Legionella bozemanii sp. nov. andLegionella dumoffii sp. nov.: Classification of two additional species ofLegionella associated with human pneumonia

Deoxyribonucleic acid (DNA) relatedness was used to distinguish strains ofLegionella-like organisms (LLO) fromLegionella pneumophila. Two of these LLO strains, WIGA and MI 15, showed sufficient DNA relatedness to one another to be classified in the same species. The nameLegionella bozemanii species nova is proposed for this new species. The type strain ofL. bozemanii is WIGA (=ATCC 33217) Two other LLO strains, NY 23 and Tex-KL, were shown to represent a new species. The nameLegionella dumoffii species nova is proposed for this species. The type strain ofL. dumoffii is NY 23 (=ATCC 33279). These two species joinL. pneumophila andL. micdadei in the genusLegionella.