Comparisons of protocols for decontamination of environmental ice samples for biological and molecular examinations.

Drilling and laboratory manipulations of glacial ice cores introduce contemporary microbes and biomolecules onto the cores. We report herein a systematic comparative study of several decontamination protocols. Only treatment with 5% sodium hypochlorite eliminated all external contaminating microbes and nucleic acids while maintaining the integrity of those within the cores.

Quantitative genetics of seminal receptacle length in Drosophila melanogaster.

The length of the female's primary sperm-storage organ, the seminal receptacle, has undergone rapid divergence within the Drosophila genus. Quantitative genetic analysis of seminal receptacle length was carried out on two laboratory strains of Drosophila melanogaster that had undergone artificial selection for both increased and decreased organ length. Realized heritabilities were 0.176 and 0.270 for the two experiments. Parental strains, F1, F1r (reciprocal), F2, backcross, and backcross reciprocal generations were used in a line-cross (generation means) analysis. This analysis revealed that additive, dominance, and additive-by-dominance epistasis contributed significantly to the means. No significant maternal effects were found. Variance analysis indicated that a completely additive model was adequate to explain the variances observed in these lines. Castle-Wright minimal estimates of 5.25 and 1.91, segregating loci responsible for mean differences, were found for the two respective experiments. There were significant positive correlations between additive effects of seminal receptacle length and thorax length in both experiments. The correlated evolution of sperm and seminal receptacle length is discussed.

The quantitative genetics of fluctuating asymmetry.

Fluctuating asymmetry (subtle departures from identical expression of a trait across an axis of symmetry) in many taxa is under stabilizing selection for reduced asymmetry. However, lack of reliable estimates of genetic parameters for asymmetry variation hampers our ability to predict the evolutionary outcome of this selection. Here we report on a study, based on analysis of variation within and between isofemale lines and of generation means (line-cross analysis), designed to dissect in detail the quantitative genetics of positional fluctuating asymmetry (PFA) in bristle number in natural populations of Drosophila falleni. PFA is defined as the difference between the two sides of the body in the placement or position of components of a meristic trait. Heritability (measured at 25 degrees C) of two related measures of PFA were 13% and 21%, both of which differed significantly from zero. In contrast, heritability estimates for fluctuating asymmetry in the total number of anterior (0.7%) and transverse (2.4%) sternopleural bristles were smaller, not significant, and in quantitative agreement with previously published estimates. Heritabilities for bristle number (trait size) were considerably greater than that for any asymmetry measure. The experimental design controlled for the potentially confounding effects of common familial environment, and repeated testing revealed that PFA differences between lines were genetically stable for up to 16 generations in the laboratory at 25 degrees C. We performed line cross analysis between strains at the extremes of the PFA distribution (highest and lowest values); parental strains, F1, F1r (reciprocal), F2, backcross, and backcross reciprocal generations were represented. The inheritance of PFA was described best by additive and dominance effects localized to the X-chromosomes, whereas autosomal dominance effects were also detected. Epistatic, maternal, and cytoplasmic effects were not detected. The inheritance of trait size was notably more complex and involved significant autosomal additive, dominance, and epistatic effects; maternal dominance effects; and additive and dominance effects localized to the X-chromosomes. The additive genetic correlation between PFA and its associated measure of trait size was negative (-0.049), but not statistically significant, indicating that the loci contributing additive genetic effects to these traits are probably different. It is suggested that PFA may be a sensitive measure of developmental instability because PFA taps the ability of an organism to integrate interconnected developmental pathways.

Candida orba sp. nov., a new cactus-specific yeast species from Queensland, Australia.

A new species of yeast from decaying cladodes of Opuntia cactus, Candida orba, is described. This species is a member of a four-species clade of cactophilic yeasts. The new species has only been found in one region of Queensland, Australia, where it was presumably introduced during attempts to eradicate prickly pear cactus. DNA-DNA relatedness, phylogenetic analysis, physiological differences, killer-sensitivity profiles and mating reactions establish the distinctness of the taxon as a new species. C. orba is most closely related to Phaffomyces thermotolerans, a species found associated with columnar cacti in the North American Sonoran Desert. The type strain of C. orba, isolated from rotting cladodes of Opuntia stricta in the State of Queensland, Australia, is strain UCD-FST 84-833.1T (= CBS 8782T = NRRL Y-27336T = ATCC MYA-341). Only the h- mating type of the species has been recovered. The lack of the opposite mating type could be the result of a bottleneck during its introduction to Australia. The original geographic/host distribution of this species in the Americas is unknown.

Metschnikowia lochheadii and Metschnikowia drosophilae, two new yeast species isolated from insects associated with flowers.

Two new haplontic heterothallic species of Metschnikowia were isolated from floricolous insects and flowers. Metschnikowia lochheadii was recovered from insects found in various flowers on the Hawaiian Islands of Kauai and Maui, and from Conotelus sp. (Coleoptera: Nitidulidae) in northwestern Guanacaste Province, Costa Rica. The morphology, physiology, and sexual cycle are typical of the large-spored Metschnikowia species, and the partial ribosomal DNA large subunit (D1D2) sequences suggest that the new species is most closely related to Candida ipomoeae. Metschnikowia lochheadii is nearly indistinguishable from its ascogenous relatives and conjugates freely with Metschnikowia continentalis, forming sterile asci. It also exhibits asymmetric mating with Metschnikowia hawaiiensis. Metschnikowia drosophilae was found in morning glory (Ipomoea sp.) flowers and associated Drosophila bromeliae on Grand Cayman Island. Its nutritional profile is atypical of the genus, being the only species that does not utilize sucrose or maltose as carbon sources, and one of the few that does not utilize melezitose. D1D2 sequences show that Metschnikowia drosophilae is a sister species to Candida torresii, to which it bears considerable similarity in nutritional profile. The type cultures are: Metschnikowia lochheadii, strains UWO(PS)00-133.2 = CBS 8807 (h+, holotype) UWO(PS)99-661.1 = CBS 8808 (h-, isotype); and Metschnikowia drosophilae, strains UWO(PS)83-1135.3 = CBS 8809 (h+, holotype) and UWO(PS)83-1143.1 = CBS 8810 (h-, isotype).

Phylogenetic structure of the Sporopachydermia cereana species complex.

A large number of isolates previously referred to as members of the 'Sporopachydermia cereana species complex' were examined by various DNA characterization methods, leading to the conclusion that the complex is in fact made up of 10 species, one of which contains three varieties. The sequences of the internal transcribed spacer (ITS) region and the D1/D2 divergent domains of the large subunit rDNA were determined for representatives of each taxon and specific primers based on differences in the ITS were designed for rapid identification of five of the taxa. Whereas the data provide additional elements for the calibration of the ITS as a criterion for species delineation, the emerging pattern is that the ITS region does not function as well as the D1/D2 domains as an evolutionary clock. Some taxa appear to be specific for the geographical regions where they were isolated, and the distribution of many taxa is mutually exclusive.

Yeast-Yeast Interactions in Guava and Tomato Fruits.

The host specificity of yeast-yeast interactions was investigated for two yeast types, represented by six pairs of Pichia membranifaciens clade yeasts (Pichia membranifaciens or Issatchenkia occidentalis) with apiculate yeasts (Kloeckera apis, Kloeckera africana, or Saccharomycodes ludwigii), commonly found in fruits. Competitive interactions between the two types were detected in both ripe tomato and guava fruit pulp. The differences in growth rates and carrying capacities depended on fruit type (host) and culture conditions (monocultures versus bicultures). These differences were probably due to nutrient composition of each fruit. Pichia membranifaciens did not show host dependent responses, but the apiculate yeasts and Issatchenkia occidentalis did. Depending on yeast strain and culture conditions (i.e., monoculture or biculture), carbon, nitrogen, and vitamins were investigated as potential limiting growth factors in guava fruit. Both singular and multiple limiting nutrients were implicated.

Biogeography of the yeasts of ephemeral flowers and their insects.

We studied specific yeast communities vectored by beetles, drosophilids, and bees that visit ephemeral flowers, mostly in the genus Hibiscus and in the families Convolvulaceae and Cactaceae, in the Neotropical, Nearctic, and Australian biogeographic regions. The communities consist mostly of yeasts in four clades centered around the genera Metschnikowia, Kodamaea, Wickerhamiella, and Starmerella. The largest geographic discontinuity occurs as a function of the nitidulid beetle species that dominate the non-pollinator insect visitors of the flowers. This partitions the New World, where the dominant beetle is in the genus Conotelus, from the Australian biogeographic region, dominated by species of Aethina. Distinct but sympatric insects may also carry radically different yeast communities.

Pichia hawaiiensis sp. nov., occurring in decaying bark of Charpentiera trees in the Hawaiian archipelago.

A description is given for Pichia hawaiiensis sp. nov., a nitrate-utilizing member of the genus Pichia E. C. Hansen emend. Kurtzman. Seven strains of the new species were isolated during the years 1972, 1973 and 1978 from rotting bark of the Hawaiian tree genera Charpentiera, Pisonia and Cheirodendron. P. hawaiiensis is heterothallic but appears to occur in nature mainly in the diploid state. Asci are deliquescent and produce up to four hat-shaped spores per ascus. Phylogenetic analysis of the 600 nucleotide D1/D2 domain of the 26S rDNA showed that P. hawaiiensis is most closely related to Pichia populi and Williopsis californica (syn. Hansenula californica). The type strain of P. hawaiiensis, isolated on the island of Hawaii from the rotting bark of Charpentiera sp. containing insect larvae, is strain UCD-FST 72-181T (= ATCC MYA-137T = CBS 8760T = NRRL Y-27270T).

Ribosomal DNA, species structure, and biogeography of the cactophilic yeast Clavispora opuntiae.

The ribosomal DNA of the cactophilic yeast species Clavispora opuntiae was studied in order to clarify the global distribution of the yeast. Over 500 strains, including isolates from several new localities worldwide, were characterized by rDNA restriction mapping. An unusual restriction pattern previously encountered only in one strain, from Conception Island in the Bahamas, was found in several Brazilian isolates. Sequences of the D1/D2 and D7/D8 divergent domains of the large subunit (LSU) and of the intergenic spacers (IGS) confirmed that these strains represent a genetically distinct variety of Clavispora opuntiae. This divergence had previously been hypothesized on the basis of reduced genetic recombination in inter-varietal crosses and the presence of a polymorphic ApaI restriction site located in the LSU. The exact position of the ApaI site in the D8 divergent domain and the nature of the variation that it reveals were determined. The complete sequences of 12 intergenic spacers clarified the significance of the species-wide variation uncovered by restriction mapping. Most of the polymorphic sites occur in the IGS1 and IGS2 regions, on either side of the 5S gene, and the variation is largely due to differences in the numbers and the sequences of internal repeats. Two other polymorphic sites are located in the external transcribed spacer (ETS) region. The reliability of various sites as indicators of overall spacer sequence divergence differed from one case to another. Variety-specific probes were devised and used to screen 120 strains for the presence of recombinant rDNA spacers. Three strains gave ambiguous results, but these did not constitute evidence that inter-varietal recombination has taken place in nature. The hypothesis that the global movement of Clavispora opuntiae has been influenced by the worldwide biological control of prickly pear with Cactoblastis cactorum, a moth of Argentinian origin, has received additional support from the demonstration that Argentinian strains have rDNAs similar to those found where the moth has been introduced. A dramatic founder effect was identified in a yeast population collected in cacti (Maui, Hawaii) in a site where the moth had been recently introduced.

Reproductive characteristics of the flower-breeding Drosophila hibisci Bock (Drosophilidae) in eastern Australia: within-population genetic determinants of ovariole number.

Genetic variation for ovariole number in the flower-breeding Drosophila hibisci was studied within populations obtained from three separate sites in the centre of the species distribution along the east coast of Australia. Heritability for ovariole number (adjusted for body size), derived from isofemale lines from each site, was estimated to be h2+/-SE = 0.564+/-0.160. The variance of ovariole number within sites (sigma2(within) = 2.039) was comparable to the variance between sites (sigma2(between) = 2.048) obtained from an earlier study of populations sampled over 14 degrees of latitude. Two isofemale lines (from within one site) that differed by an average of 4.6 ovarioles were used to generate F1, F2 and backcross generations. Analysis of mean ovariole number for these generations showed that only additive gene effects were important and that dominance, digenic epistasis and maternal effects were not significant. This within-population result contrasted with earlier results between populations that revealed additive and digenic epistasis for the same trait. High heritability within populations and the relatively large within-population variation for ovariole number suggest that substantial microhabitat variation is influencing this fitness-related trait.

On the biogeography of yeasts in the Wickerhamiella clade and description of Wickerhamiella lipophila sp. nov., the teleomorph of Candida lipophila.

We describe the new yeast species Wickerhamiella lipophila, the teleomorph of Candida lipophila, a haploid heterothallic yeast previously isolated from insects associated with morning glories in Hawaii. Both mating types were recovered in the eastern region of Maui, and a single strain was found in the Waimea region of Kauai. We reexamined the mating compatibility of the several strains of Candida lipophila previously collected on the island of Hawaii and found them to be fertile mating types that had been overlooked because of the unpredictability of mating and ascus formation. The type culture of Candida lipophila [UWO(PS)91-681.3 = CBS 8458, h+] is transferred to the genus Wickerhamiella, and strain UWO(PS)00-340.1 (CBS 8812, h-) is designated as isotype. Also found on Maui and Kauai were strains of Candida drosophilae that produced a strong extracellular protease. An update on the global distribution of members of the Wickerhamiella clade is given.

Genetic architecture of a wing size measure in Drosophila hibisci from two populations in eastern Australia.

Two models of evolutionary change invoke either additive genetic contributions to phenotypic traits (Fisher) or epistatic as well as additive effects (Wright). An earlier study of the flower-breeding Drosophila hibisci from two sites in eastern Australia reported additive and epistatic genetic effects as well as environmental effects on ovariole number. The present study of the same flies examines the genetic architecture of wing width, a trait that is correlated phenotypically with ovariole number and body size. A generation means analysis of flies reared at 25 degrees C indicated additive and epistatic genetic effects, but no consistent maternal effects, whereas for flies reared at three temperatures (18 degrees C, 21.5 degrees C, and 25 degrees C) linear and nonlinear environmental effects interacted with additive genetic effects. The genetic correlation matrix for ovariole number and wing width suggested negative genetic correlations between additive effects on one trait and epistatic effects on the other. Both traits provide evidence of genetic effects consistent with assumptions of Wright's shifting balance theory of evolution.

Pichia lachancei sp. nov., associated with several Hawaiian plant species.

A description is given of Pichia lachancei sp. nov., a new species of yeast that occurs in association with several Hawaiian plant species of the genera Tetraplasandra, Cheirodendron and Clermontia. The new species is heterothallic and occurs in nature in the haploid as well as the diploid state. Upon conjugation of complementary mating types, zygotes are formed that reproduce by budding as diploid cells. When placed on sporulation medium, four hat-shaped spores are produced which are rapidly released from the ascus. Phylogenetic analysis showed that P. lachancei is most closely related to Pichia rhodanensis and Pichia jadinii. The diploid type strain of P. lachancei, isolated from rotting bark of Tetraplasandra hawaiiensis on the island of Hawaii, is strain UCD-FST 79-9T (= ATCC 201914T = CBS 8557T = NRRL Y-27008T).

Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers.

Two new yeast species were isolated from flowers of Hibiscus species in Eastern and Northern Australia. Kodamaea kakaduensis is heterothallic, haploid, and similar to other Kodamaea species and to Candida restingae. Buds are often produced on short protuberances, and a true mycelium is formed. The new species differs from others by the assimilation of trehalose, melezitose, and xylitol, and is reproductively isolated. The cells of Candida tolerans are small and a pseudomycelium is formed. The carbon and nitrogen assimilation pattern is reminiscent of that of Zygosaccharomyces rouxii but the two are not closely related. Sequences of the D1/D2 domain of large subunit ribosomal DNA confirm the membership of K. kakaduensis in the genus Kodamaea and indicate that C. tolerans belongs to the Clavispora-Metschnikowia clade, with a moderate relatedness to Candida mogii. The type strains are: K. kakaduensis, UWO(PS)98-119.2 (h+, holotype, CBS 8611) and UWO(PS)98-117.1 (h-, isotype, CBS 8612); and C. tolerans, UWO(PS)98-115.5 (CBS 8613).

Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL) proteins.

Delta/Serrate/LAG-2 (DSL) proteins are putative transmembrane signaling molecules that regulate cell differentiation in metazoans. DSL proteins are characterized by the presence of a motif unique to these proteins, the DSL motif, and a variable number of tandemly repeated copies of an epidermal growth factor-like (EGF) motif. We have completed a phylogenetic analysis of 15 DSL proteins from eight species. Our findings reveal that at least one gene duplication occurred prior to the divergence of the Drosophila melanogaster and vertebrate lineages, with subsequent duplications in vertebrates. The three known Caenorhabditis elegans proteins likely arose by two independent duplications in the nematode lineage. Analysis of EGF repeats suggests that EGF 2 has been conserved among DSL proteins in vertebrates and D. melanogaster. The sequences of two EGF repeats have been perfectly conserved in vertebrate orthologs: EGF 2 in Delta and EGF 15 in Jagged/Serrate. Finally, the linear order of EGF repeats has been conserved in the vertebrate Jagged/Serrate orthologs and vertebrate Delta orthologs.

Kodamaea nitidulidarum, Candida restingae and Kodamaea anthophila, three new related yeast species from ephemeral flowers.

Three new yeast species were discovered during studies of yeasts associated with ephemeral flowers in Brazil, Australia and Hawaii. Their physiological and morphological similarity to Kodamaea (Pichia) ohmeri suggested a possible relationship to that species, which was confirmed by rDNA sequencing. Kodamaea nitidulidarum and Candida restingae were found in cactus flowers and associated nitidulid beetles in sand dune ecosystems (restinga) of South-eastern Brazil. Over 350 strains of Kodamaea anthophila were isolated from Hibiscus and morning glory flowers (Ipomoea spp.) in Australia, and from associated nitidulid beetles and Drosophila hibisci. A single isolate came from a beach morning glory in Hawaii. Expansion of the genus Kodamaea to three species modified the existing definition of the genus only slightly. The type and isotype strains are as follows: K. nitidulidarum strains UFMG96-272T (h+; CBS 8491T) and UFMG96-394I (h-; CBS 8492I); Candida restingae UFMG96-276T (CBS 8493T); K. anthophila strains UWO(PS)95-602.1T (h+; CBS 8494T), UWO(PS)91-893.2I (h-; CBS 8495I) and UWO(PS)95-725.1I (h-; CBS 8496I).

Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, five new related yeast species from flowers and associated insects.

Five new yeast species, Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, are described to accommodate isolates recovered from flowers and floricolous insects of Australian Hibiscus trees, cosmopolitan morning glories (Ipomoea spp.) and Brazilian cereoid cacti. The new Wickerhamiella species are heterothallic, occur in the haploid condition and are clearly separated reproductively from one another. Although they exhibit little physiological variation, they are easily delineated from Wickerhamiella domercqiae, the only species known previously, by their resistance to cycloheximide and the production of strong extracellular lipases. C. drosophilae and C. lipophila share the latter property, but unlike the Wickerhamiella species, they fail to utilize nitrate as sole nitrogen source. PFGE indicates that these yeasts have an unusually low number of chromosomes. The large-subunit rDNA (D1/D2) sequences demonstrate a close relationship between the five species and Candida vanderwaltii and Candida azyma. Their relationship with W. domercqiae is more distant, but all share, with some other Candida species, a single monophyletic clade. The type and isotype strains are as follows: W. australiensis strains UWO(PS)95-604.3T (h+; CBS 8456T) and UWO(PS)95-631.3I (h-; CBS 8457I); W. cacticola strains UFMG96-267T (h+; CBS 8454T) and UFMG96-381I (h-; CBS 8455I); W. occidentalis strains UWO(PS)91-698.4T (h+; CBS 8452T) and UFMG96-212I (h-; CBS 8453I); C. drosophilae UWO(PS)91-716.3T (CBS 8459T); and C. lipophila UWO(PS)91-681.3T (CBS 8458T).

Candida ipomoeae, a new yeast species related to large-spored Metschnikowia species.

Numerous strains of an unusual asexual yeast species were isolated from flowers of morning glory (Ipomoea spp., Convolvulaceae) and associated drosophilids and sap beetles of the genus Conotelus sampled in Hawaii and in Brazil. The nutritional profile of this yeast is similar to those of Metschnikowia hawaiiensis and Metschnikowia continentalis, which share the same habitats. The cells are large, hydrophobic, and tend to remain attached after budding, causing the colonies on agar media to have a convoluted appearance, reminiscent of popcorn. The sequences of the D1/D2 domain of large subunit rDNAs of strains from three different localities confirmed that a single species is involved, and that it is related to large-spored Metschnikowia species. The type strain is UWO(PS)91-672.1 (CBS 8466).

Parasite-induced risk of mortality elevates reproductive effort in male Drosophila.

A trade-off between sex and somatic maintenance is fundamental to life-history theory. Tests of this trade-off usually emphasize deleterious consequences of increased reproduction on life span. Here we show the reverse effect, that reductions in the expected life span elevate sexual activity. Experimentally parasitized male Drosophila nigrospiracula lived shorter lives, but before dying, they courted females significantly more than unparasitized controls. This greater courtship resulted in increased mating speed, and potentially greater reproductive success than parasitized males would have achieved otherwise. The results show that an environmental reduction in life span increases reproductive effort, and support the hypothesis of a trade-off between these key life-history traits.