Phylogeny of dermatophytes with genomic character evaluation of clinically distinct Trichophyton rubrum and T. violaceum.

Trichophyton rubrum and T. violaceum are prevalent agents of human dermatophyte infections, the former being found on glabrous skin and nail, while the latter is confined to the scalp. The two species are phenotypically different but are highly similar phylogenetically. The taxonomy of dermatophytes is currently being reconsidered on the basis of molecular phylogeny. Molecular species definitions do not always coincide with existing concepts which are guided by ecological and clinical principles. In this article, we aim to bring phylogenetic and ecological data together in an attempt to develop new species concepts for anthropophilic dermatophytes. Focus is on the T. rubrum complex with analysis of rDNA ITS supplemented with LSU, TUB2, TEF3 and ribosomal protein L10 gene sequences. In order to explore genomic differences between T. rubrum and T. violaceum, one representative for both species was whole genome sequenced. Draft sequences were compared with currently available dermatophyte genomes. Potential virulence factors of adhesins and secreted proteases were predicted and compared phylogenetically. General phylogeny showed clear gaps between geophilic species of Arthroderma, but multilocus distances between species were often very small in the derived anthropophilic and zoophilic genus Trichophyton. Significant genome conservation between T. rubrum and T. violaceum was observed, with a high similarity at the nucleic acid level of 99.38 % identity. Trichophyton violaceum contains more paralogs than T. rubrum. About 30 adhesion genes were predicted among dermatophytes. Seventeen adhesins were common between T. rubrum and T. violaceum, while four were specific for the former and eight for the latter. Phylogenetic analysis of secreted proteases reveals considerable expansion and conservation among the analyzed species. Multilocus phylogeny and genome comparison of T. rubrum and T. violaceum underlined their close affinity. The possibility that they represent a single species exhibiting different phenotypes due to different localizations on the human body is discussed.

Three New Species of the Genus Ochroconis.

Ochroconis bacilliformis, O. phaeophora and O. robusta, three novel species of the melanized genus Ochroconis (Sympoventuriaceae, Venturiales), are described, illustrated and distinguished phenotypically and molecularly from previously described species in the genus Ochroconis. Their potential significance for infection of cold-blooded vertebrates is discussed.

Effects of immune challenge on the oviposition strategy of a noctuid moth.

Infections can have detrimental effects on the fitness of an animal. Reproducing females may therefore be sensitive to cues of infection and be able to adaptively change their oviposition strategy in the face of infection. As one possibility, females could make a terminal investment and shift reproductive effort from future to current reproduction as life expectancy decreases. We hypothesized that females of the noctuid moth Heliothis virescens make a terminal investment and adapt their oviposition timing as well as their oviposition site selectivity in response to an immune challenge. We indeed found that females that were challenged with the bacterial entomopathogen Serratia entomophila laid more eggs than control females one night after the challenge. Additionally, bacteria-challenged females were less discriminating between oviposition sites than control females. Whereas control females preferred undamaged over damaged plants, immune-challenged females did not differentiate between the two. These results indicate that terminal investment is part of the life history of H. virescens females. Moreover, our results suggest that the strategy of terminal investment in H. virescens oviposition represents a fitness trade-off for females: in the face of infection, an increase in oviposition rate enhances female fitness, whereas low oviposition site selectivity reduces female fitness.

Revision of agents of black-grain eumycetoma in the order Pleosporales.

Eumycetoma is a chronic fungal infection characterised by large subcutaneous masses and the presence of sinuses discharging coloured grains. The causative agents of black-grain eumycetoma mostly belong to the orders Sordariales and Pleosporales. The aim of the present study was to clarify the phylogeny and taxonomy of pleosporalean agents, viz. Madurella grisea, Medicopsis romeroi (syn.: Pyrenochaeta romeroi), Nigrograna mackinnonii (syn. Pyrenochaeta mackinnonii), Leptosphaeria senegalensis, L. tompkinsii, and Pseudochaetosphaeronema larense. A phylogenetic analysis based on five loci was performed: the Internal Transcribed Spacer (ITS), large (LSU) and small (SSU) subunit ribosomal RNA, the second largest RNA polymerase subunit (RPB2), and translation elongation factor 1-alpha (TEF1) gene. In addition, the morphological and physiological characteristics were determined. Three species were well-resolved at the family and genus level. Madurella grisea, L. senegalensis, and L. tompkinsii were found to belong to the family Trematospheriaceae and are reclassified as Trematosphaeria grisea comb. nov., Falciformispora senegalensis comb. nov., and F. tompkinsii comb. nov. Medicopsis romeroi and Pseudochaetosphaeronema larense were phylogenetically distant and both names are accepted. The genus Nigrograna is reduced to synonymy of Biatriospora and therefore N. mackinnonii is reclassified as B. mackinnonii comb. Nov. Mycetoma agents in Pleosporales were phylogenetically quite diverse despite their morphological similarity in the formation of pycnidia, except for the ascosporulating genus Falciformispora (formerly in Leptosphaeria). Most of the species diagnosed from human mycetoma were found to be related to waterborne or marine fungi, suggesting an association of the virulence factors with oligotrophism or halotolerance.

Elucidation of distribution patterns and possible infection routes of the neurotropic black yeast Exophiala dermatitidis using AFLP.

Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-dominated environment, where it occasionally causes human pulmonary or fatal disseminated and neurotropic disease. The hypothesis of its natural niche was investigated by comparing a set of 178 strains from natural and human-dominated environments in Thailand with a worldwide selection of 107 strains from the reference collection of the CBS Fungal Biodiversity Centre, comprising 75.7% clinical isolates. Many isolates had unique AFLP patterns and were too remote for confident comparison. Eight populations containing multiple isolates could be distinguished, enabling determination of geographic distributions of these populations. Some of the populations were confined to Thailand, while others occurred worldwide. The local populations from Thailand contained strains from natural and urban environments, suggesting an environmental jump of the fungus. Strains from human brain belonged to widely dispersed populations. In some cases cerebral isolates were identical to isolates from the human intestinal tract. The possibility of cerebral infection through intestinal translocation was thus not excluded.

Rapid screening for genotypes as possible markers of virulence in the neurotropic black yeast Exophiala dermatitidis using PCR-RFLP.

A simple method for fungal genotype screening was developed for the black yeast Exophiala dermatitidis based on RFLP of ribosomal ITS regions currently used as potential virulence markers. In a study set of 502 strains of the species, two main genotypes were recognized. Only 0.97% of lanes were difficult to interpret as they did not clearly present one of the expected genotypes. Twenty strains were deviating and proved to be E. spinifera after sequencing. Eight common, related species (based on SSU data) with clinical significance yielded different patterns with TaqI digestion, and thus the method is also usable for routine diagnostics.

The neurotropic black yeast Exophiala dermatitidis has a possible origin in the tropical rain forest.

The black yeast Exophiala dermatitidis is known as a rare etiologic agent of neurotropic infections in humans, occurring particularly in East and Southeast Asia. In search of its natural habitat, a large sampling was undertaken in temperate as well as in tropical climates. Sampling sites were selected on the basis of the origins of previously isolated strains, and on the basis of physiological properties of the species, which also determined a selective isolation protocol. The species was absent from outdoor environments in the temperate climate, but present at low abundance in comparable habitats in the tropics. Positive outdoor sites particularly included faeces of frugivorous birds and bats, in urban as well as in natural areas. Tropical fruits were found E. dermatitidis positive at low incidence. Of the human-made environments sampled, railway ties contaminated by human faeces and oily debris in the tropics were massively positive, while the known abundance of the fungus in steam baths was confirmed. On the basis of the species' oligotrophy, thermotolerance, acidotolerance, moderate osmotolerance, melanization and capsular yeast cells a natural life cycle in association with frugivorous animals in foci in the tropical rain forest, involving passage of living cells through the intestinal tract was hypothesized. The human-dominated environment may have become contaminated by ingestion of wild berries carrying fungal propagules.

Genetic differentiation and natural hybridization between the Sardinian endemic Maniola nurag and the European Maniola jurtina.

The Mediterranean island of Sardinia is known for its multitude of unique genetic lineages. We view one of them in a larger phylogeographic context. The endemic Sardinian Meadow Brown butterfly, Maniola nurag, is restricted to the mountainous areas of the island, whereas its widespread close relative, Maniola jurtina, also occurs on the coast. At intermediate altitudes the species' distributions overlap. There, a number of individuals exhibit phenotypic characteristics intermediate between the two species. We examined patterns of intra- and interpopulation variation in 10 M. nurag populations from Sardinia and 16 M. jurtina populations from Sardinia and continental Europe, as well as 17 intermediate individuals, sampled in 1999-2002, by means of allozyme markers, combining it with a morphometric analysis based on 18 wing-characters of 52 males. At the 15 loci studied (aldolase, aat-1, aat-2, g6pdh, gpd, idh-1, idh-2, mdh-1, mdh-2, mpi, me, leu-ala, pgi, pgm, and 6pgdh), 76 different alleles were detected, 63 of which were shared by M. nurag and M. jurtina. None of the loci was found to be alternatively fixed between the two species. In that respect, this study testifies to the difficulties that may arise when trying to identify hybrids from genotypic data. Levels of genetic variation in island populations (M. jurtina: H(O) = 0.137-0.189; M. nurag: H(O) = 0.141-0.270) were comparable to those of mainland M. jurtina (H(O) = 0.141-0.236). A Bayesian admixture analysis supported the hypothesis of mixed (hybrid) ancestry of individuals occurring at intermediate altitudes. Similarly, neighbour-joining and unweighted pair-group method with arithmetic averaging (UPGMA) analyses, as well as morphometrics hinted at the existence of a Maniola-hybrid zone in Sardinia at intermediate altitudes. We discuss the results in the light of the phylogeography of other Sardinian taxa with the aim to reach a general understanding of the biogeographic history of this island's endemic species.

Spatial and temporal genetic structure of the planktonic Sagitta setosa (Chaetognatha) in European seas as revealed by mitochondrial and nuclear DNA markers.

Little is known about the spatial and temporal scales at which planktonic organisms are genetically structured. A previous study of mitochondrial DNA (mtDNA) in the holoplanktonic chaetognath Sagitta setosa revealed strong phylogeographic structuring suggesting that Northeast (NE) Atlantic, Mediterranean and Black Sea populations are genetically disjunct. The present study used a higher sampling intensity and a combination of mitochondrial and four microsatellite markers to reveal population structuring between and within basins. Between basins, both marker sets indicated significant differentiation confirming earlier results that gene flow is probably absent between the respective S. setosa populations. At the within-basin scale, we found no evidence of spatial or temporal structuring within the NE Atlantic. In the Mediterranean basin, both marker sets indicated significant structuring, but only the mtDNA data indicated a sharp genetic division between Adriatic and all other Mediterranean populations. Data were inconclusive about population structuring in the Black Sea. The levels of differentiation indicated by the two marker sets differed substantially, with far less pronounced structure detected by microsatellite than mtDNA data. This study also uncovered the presence of highly divergent mitochondrial lineages that were discordant with morphology, geography and nuclear DNA. We thus propose the hypothesis that highly divergent mitochondrial lineages may be present within interbreeding S. setosa populations.

Short-term impact of disturbance on genetic diversity and structure of Indonesian populations of the butterfly Drupadia theda in East Kalimantan.

We investigated the short-term impact of disturbance on genetic diversity and structure of the tropical butterfly Drupadia theda Felder (Lepidoptera: Lycaenidae). Populations were sampled from five landscapes in East Kalimantan (Borneo, Indonesia) which were differentially disturbed by selective logging and the 1997/1998 El Niño Southern Oscillation (ENSO)-induced drought and fires. Sampling occurred before (in 1997) and after the forest fires (in 1998, 1999, 2000, and 2004). Drupadia theda populations underwent serious population size reductions following the 1997/1998 ENSO event. For a total of 208 individuals, we sequenced a 509-bp segment of mtDNA containing the control region plus the 5' end of the 12S rDNA gene. Haplotype diversity in D. theda populations ranged from 0.468 to 0.953. Just after the 1997/1998 ENSO event, number of recorded individuals and genetic diversity were very low in D. theda populations sampled in the two severely burned areas and in a small pristine forest fragment that was surrounded by burned forest and thereby affected by drought. Interestingly, higher levels of genetic diversity were observed in logged forest compared to proximate pristine forest. After 1998, the genetic composition within the three ENSO-disturbed areas diverged. In the twice-burned forest, the genetic diversity in 1999 already approached pre-fire levels, while it remained nearly unchanged in proximate once-burned forest. Our data suggest that the 1997/1998 ENSO-induced drought and fires caused massive reductions in the genetic diversity of D. theda and that population recoveries were linked to their geographical position relative to patches of unburned forest (and thus to source populations).

Spatial ecological and genetic structure of a mixed population of sexual diploid and apomictic triploid dandelions.

Ecological differentiation is widely seen as an important factor enabling the stable coexistence of closely related plants of different ploidy levels. We studied ecological and genetic differentiation between co-occurring sexual diploid and apomictic triploid Taraxacum section Ruderalia by analysing spatial patterns both in the distribution of cytotypes and in the distribution of genetic variation within and between the cytotypes. A significant relationship between ploidy level and elevation was found. This mode of ecological differentiation however, was not sufficient to explain the significant spatial structure in the distribution of diploids and triploids within the population. Strong congruence was found between the spatial genetic patterns within the diploids and within the triploids. We argue that this congruence is an indication of gene flow between neighbouring plants of different ploidy levels.

Multiple paternity and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea).

Multiple paternity in the olive ridley sea turtle (Lepidochelys olivacea) population nesting in Suriname was demonstrated using two microsatellite loci, viz., Ei8 and Cm84. The large number of offspring sampled per clutch (70 on average, ranging from 15 to 103) and the number of alleles found at the two loci (18 and eight alleles, respectively) enabled unambiguous assessment of the occurrence of multiple paternity. In two out of 10 clutches analysed, the offspring had been sired by at least two males, which was confirmed at both loci. In both clutches, unequal paternity occurred: 73% and 92% of the offspring had been sired by the primary male. The probability of detecting multiple paternity was 0.903, and therefore there is a small chance that multiple paternity occurred but remained undetected in some of the eight clutches that appeared to be singly sired. Analysis of 703 offspring revealed a high mutation rate for locus Ei8 (micro = 2.3 x 10(-2)) with all 33 mutations occurring in maternal alleles. In particular, one allele of 274 bp mutated at a high frequency in a clutch to which the mother contributed the allele, but in another clutch where the father contributed the same allele, no such mutations were observed. Inferred allele-specific mutation rates for Ei8 and expected numbers of mutations per clutch confirmed that maternal alleles for Ei8 are more likely to mutate in the olive ridley sea turtle than paternal alleles. Possible explanations are discussed.

Heterologous amplification of microsatellite markers from colubroid snakes in European natricines (serpentes: natricinae).

Eighteen microsatellite loci developed for a range of snake species (New World natricines, elapids, crotalids) were tested against European natricines (Natrix natrix, N. maura, and N. tessellata) in cross-species amplification experiments. Five loci were polymorphic (average expected heterozygosity 0.749 for a population of N. natrix in Amsterdam, mean sample size 47.8) and three loci were monomorphic. The remainder could not be consistently scored or failed to amplify. Further tests on single individuals of a diverse set of eight species of colubroid snakes showed that 15 of the 18 loci could be cross-amplified in at least one of these species. We conclude that our results show promise for the utilization of these markers for experimental assessments of genetic variation in the phylogenetically closely related group of European natricine snakes with emphasis on N. natrix. The full suite of microsatellite markers now available for snakes may show additional potential for subsequent investigation across a broader range of colubroid snakes.

SEX-INVESTMENT RATIOS AND RELATEDNESS IN THE MONOGYNOUS ANT LASIUS NIGER (L.).

The genetic variation at two marker loci in three populations of the monogynous ant Lasius niger was used to analyze the importance of relatedness structure to sex-investment ratios in sexuals produced by colonies living in different resource conditions. From a resource-rich monoculture to a population in a resource-limited environment, dry weight investment in queens decreased from female-biased (0.76) to equality with male investment (Boomsma et al., 1982). The investment ratios in sexuals expected from the estimated relatedness ratios, resulting from kin-selection theory (Trivers and Hare, 1976), were in good agreement with the observed ratios in all populations. This indicated that the workers can capitalize on the asymmetry in relatedness, opposing the queen's interest, despite the contrast in environmental conditions in the different populations. This asymmetry, however, almost disappeared in the marginal population, due to a high frequency of double mating and worker production of males, resulting in a much reduced queen-worker conflict. We suggest that different levels of polyandry might be favored at different points of the resource gradient, with the sex ratio secondarily depending on these polyandry levels. As there was no population subdivision or spatial structure within the populations, group-selection and local-mate-competition models cannot account for the observed female-biased ratios, whereas they were rather accurately predicted by kin-selection theory.