Spatial sorting and range shifts: consequences for evolutionary potential and genetic signature of a dispersal trait.

Species are shifting their ranges under climate change, with genetic and evolutionary consequences. As a result, the spatial distribution of genetic diversity in a species' range can show a signature of range expansion. This genetic signature takes time to decay after the range stops expanding and it is important to take that lag time into account when interpreting contemporary spatial patterns of genetic diversity. In addition, the return to spatial equilibrium on an ecologically relevant timescale will depend on migration of genetic diversity across the species' range. However, during a range shift alleles may go extinct at the retracting range margin due to spatial sorting. Here we studied the spatial pattern of genotypes that differ in dispersal rate across the species range before, during and after a range shift, assessed the effect of range retraction on this pattern, and quantified the duration of the ephemeral genetic signature of range expansion for this trait. We performed simulation experiments with an individual-based metapopulation model under several contemporary climate change scenarios. The results show an increase of the number of individuals with high dispersal rate. If the temperature increased long enough the allele coding for low dispersal rate would go extinct. The duration of the genetic signature of range expansion after stabilisation of the species' distribution lasted up to 1200 generations after a temperature increase for 60 years at the contemporary rate. This depended on the total displacement of the climate optimum, as the product of the rate of temperature increase and its duration. So genetic data collected in the field do not necessarily reflect current selection pressures but can be affected by historic changes in species distribution, long after the establishment of the current species' range. Return to equilibrium patterns may be hampered by loss of evolutionary potential during range shift.

Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora.

The replication and segregation of multi-copy mitochondrial DNA (mtDNA) are not under strict control of the nuclear DNA. Within-cell selection may thus favour variants with an intracellular selective advantage but a detrimental effect on cell fitness. High relatedness among the mtDNA variants of an individual is predicted to disfavour such deleterious selfish genetic elements, but experimental evidence for this hypothesis is scarce. We studied the effect of mtDNA relatedness on the opportunities for suppressive mtDNA variants in the fungus Neurospora carrying the mitochondrial mutator plasmid pKALILO. During growth, this plasmid integrates into the mitochondrial genome, generating suppressive mtDNA variants. These mtDNA variants gradually replace the wild-type mtDNA, ultimately culminating in growth arrest and death. We show that regular sequestration of mtDNA variation is required for effective selection against suppressive mtDNA variants. First, bottlenecks in the number of mtDNA copies from which a 'Kalilo' culture started significantly increased the maximum lifespan and variation in lifespan among cultures. Second, restrictions to somatic fusion among fungal individuals, either by using anastomosis-deficient mutants or by generating allotype diversity, prevented the accumulation of suppressive mtDNA variants. We discuss the implications of these results for the somatic accumulation of mitochondrial defects during ageing.

Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina.

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the 'plasmid-based' senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5' UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species.

Sexual and vegetative compatibility genes in the aspergilli.

Gene flow within populations can occur by sexual and/or parasexual means. Analyses of experimental and in silico work are presented relevant to possible gene flow within the aspergilli. First, the discovery of mating-type (MAT) genes within certain species of Aspergillus is described. The implications for self-fertility, sexuality in supposedly asexual species and possible uses as phylogenetic markers are discussed. Second, the results of data mining for heterokaryon incompatibility (het) and programmed cell death (PCD) related genes in the genomes of two heterokaryon incompatible isolates of the asexual species Aspergillus niger are reported. Het-genes regulate the formation of anastomoses and heterokaryons, may protect resources and prevent the spread of infectious genetic elements. Depending on the het locus involved, hetero-allelism is not tolerated and fusion of genetically different individuals leads to growth inhibition or cell death. The high natural level of heterokaryon incompatibility in A. niger blocks parasexual analysis of the het-genes involved, but in silico experiments in the sequenced genomes allow us to identify putative het-genes. Homologous sequences to known het- and PCD-genes were compared between different sexual and asexual species including different Aspergillus species, Sordariales and the yeast Saccharomyces cerevisiae. Both het- and PCD-genes were well conserved in A. niger. However some point mutations and other small differences between the het-genes in the two A. niger isolates examined may hint to functions in heterokaryon incompatibility reactions.

Reducing the cost of resistance; experimental evolution in the filamentous fungus Aspergillus nidulans.

We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans.

Comparing artificial and natural selection in rate of adaptation to genetic stress in Aspergillus nidulans.

In an experimental study of adaptation to negative pleiotropic effects of a major fungicide resistance mutation in the filamentous fungus Aspergillus nidulans we have investigated the relative effectiveness of artificial selection vs. natural selection on the rate of compensatory evolution. Using mycelial growth rate as a fitness measure, artificial selection involved the weekly transfer of the fastest growing sector onto a fresh plate. Natural selection was approximated by transferring random samples of all the spores produced by the mycelium. Fungicide resistant and fungicide sensitive haploid and diploid strains were used in an evolution experiment over 10 weekly transfers, which is equivalent to 1200 cell cycles. Two different environmental conditions were applied: a constant fungicide-free environment and a weekly alternation between presence and absence of fungicide. Results show that for all strains and conditions used the transfer of a random sample of all spores leads to more rapid adaptation than the transfer of the visually 'fittest' sector. The rates of compensatory evolution in the constant and the alternating environment did not differ. Moreover, haploid strains tend to have a higher rate of adaptation than isogenic diploid strains.

Genetic analysis in the asexual fungus Aspergillus niger.

The genetics of A. niger has been developed since 1980. An overview is presented of the advances in developing methods and collecting data. Important tools have been a) the application of essentially different methods to isolate mutants, b) the adaptation to A. niger ofA. nidulans methodology for analysis of the parasexual cycle, c) the choice of marker genes, and in some cases the artificial introduction of such genes, to select homozygous segregants arising from mitotic recombination. With the use of parasexual recombination, a genetic linkage map of A. niger has been established. In total, 110 nuclear and 1 cytoplasmic (mitochondrial) markers are available. The application of A. niger genetics in applied research is illustrated by examples.

The 'rare allele phenomenon' in a ribosomal spacer.

We describe the increased frequency of a particular length variant of the internal transcribed spacer 1 (ITS-1) of the ribosomal DNA in a hybrid zone of the land snail Albinaria hippolyti. The phenomenon that normally rare alleles or other markers can increase in frequency in the centre of hybrid zones is not new. Under the term 'hybrizyme' or 'rare allele' phenomenon it has been recorded in many organisms and different genetic markers. However, this is the first time that it has been found in a multicopy locus. On the one hand, the pattern fits well with the view that purifying selection in hybrid populations works on many loci across the genome and should thus have its effect on many independent molecular markers. On the other hand, the results are puzzling, given that the multiple copies of rDNA are not expected to respond in unison. We suggest two possible explanations for these conflicting observations.

Meiotic recombination in sexual diploid and apomictic triploid dandelions (Taraxacum officinale L.).

Taraxacum officinale L. (dandelion) is a vigorous weed in Europe with diploid sexual populations in the southern regions and partially overlapping populations of diploid sexuals and triploid or tetraploid apomicts in the central and northern regions. Previous studies have demonstrated unexpectedly high levels of genetic variation in the apomictic populations, suggesting the occurrence of genetic segregation in the apomicts and (or) hybridization between sexual and apomictic individuals. In this study we analysed meiosis in both sexual diploid and apomictic triploid plants to find mechanisms that could account for the high levels of genetic variation in the apomicts. Microscopic study of microsporocytes in the triploid apomicts revealed that the levels of chromosome pairing and chiasma formation at meiotic prophase I were lower than in that of the sexual diploids, but still sufficient to assume recombination between the homologues. Nomarski DIC (differential interference contrast) microscopy of optically cleared megasporocytes in the apomicts demonstrated incidental formation of tetrads, which suggests that hybridization can occur in triploid apomicts.

Spore-killing meiotic drive factors in a natural population of the fungus Podospora anserina.

In fungi, meiotic drive is observed as spore killing. In the secondarily homothallic ascomycete Podospora anserina it is characterized by the abortion of two of the four spores in the ascus. We have identified seven different types of meiotic drive elements (Spore killers). Among 99 isolates from nature, six of these meiotic drive elements occurred in a local population. Spore killers comprise 23% of the natural population of P. anserina in Wageningen, The Netherlands, sampled from 1991 to 1997. One Spore-killer type was also found in a French strain dating from 1937. All other isolates found so far are sensitive to spore killing. All seven Spore killer types differ in the percentage of asci that show killing and in their mutual interactions. Interactions among Spore killer types showed either mutual resistance or dominant epistasis. Most killer elements could be assigned to linkage group III but are not tightly linked to the centromere.

Evolutionary origin and consequences of uniparental mitochondrial inheritance.

In the great majority of sexual organisms, cytoplasmic genomes such as the mitochondrial genome are inherited (almost) exclusively through only one, usually the maternal, parent. This rule probably evolved to minimize the potential spread of selfish cytoplasmic genomic mutations through a species. Maternal inheritance creates an asymmetry between the sexes from which several evolutionary consequences follow. Because natural selection on mitochondria operates only in females, mitochondrial mutations may have more deleterious effects in males than in females. Strictly uniparental inheritance creates asexual mitochondrial lineages that are vulnerable to mutation accumulation (Muller's ratchet). There is evidence that over evolutionary time mitochondrial genomes have indeed accumulated slightly deleterious mutations. Mutation accumulation in animal mitochondrial genomes is probably slowed down mainly by two processes: a severe reduction in germline mitochondrial genome copy number at some point in the life cycle, enabling more effective elimination of mutations by natural selection, and occasional recombination between maternal and paternal mitochondrial genomes following paternal leakage.

The evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes) in northwestern Europe: a phylogenetic approach.

To reconstruct the evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes), phylogenetic relationships were determined between strains that belong to a clade consisting of nine intercompatibility groups (ICGs, biological species). Four of these nine ICGs are partially compatible and belong to the H. crustuliniforme aggregate. Different levels of partial compatibility have been found between these four ICGs. Between ICGs 3 and 4, 15% of the combinations were compatible. One strain was compatible with all isolates of both ICGs 3 and 4 and also with one isolate of ICG 2. Both a nuclear phylogeny, based on ribosomal IGS sequence data, and a mitochondrial phylogeny, based on a group-I intron located in the large subunit ribosomal RNA gene (LrRNA), were reconstructed. The level of incompatibility was compared with the phylogenetic history of individuals belonging to this clade. Different relationships were found between the level of compatibility and the relative age of the most recent common ancestor (MRCA) for different ICGs. On the one hand, the evolution of incompatibility between ICGs 2 and 3/4 is most consistent with the class of "divergence- first" models because a positive correlation was found between the relative age of the MRCA and the level of incompatibility for ICG 2 versus 3/4. On the other hand, the lack of such a correlation for ICGs 3 and 4 shows that (partial) incompatibility between these ICGs has arisen without strong divergence. The ecological (and to a lesser extent geographical) differences found between ICGs 3 and 4 suggest that selection for incompatibility, associated with host tree preference, has been important in the evolution of incompatibility between these two ICGs. The incongruence between the nuclear and mitochondrial trees for ICG 1 could be explained by a hybrid origin of this ICG, with different donors of the mitochondrial and nuclear sequences.

Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions and the 5.8 S rRNA gene.

Phenotypic features and sequences of the internal transcribed spacer (ITS) regions and the 5.8 S rRNA gene of type or neotype strains and other isolates of the 17 species currently assigned to Aspergillus section Circumdati and some potentially related species were analyzed. Parsimony analysis of sequence data indicated that Aspergillus section Circumdati is paraphyletic. Aspergillus campestris, A. lanosus, and A. dimorphicus with A. sepultus were found to be more closely related to Aspergillus sections Candidi, Flavi, and Cremei, respectively. These results were also supported by phenotypic data. A. robustus and A. ochraceoroseus were found not to be related to any of the species examined. Species of the proposed revised Aspergillus section Circumdati formed two main clades, which could also be distinguished based on phenotypic methods. Phylogenetic analysis of sequence data of other isolates assigned to species of the revised section indicates that either some of these isolates were misidentified or species concepts of A. ochraceus, A. melleus, and A. petrakii should be reconsidered.

Molecular analysis of variability within the toxigenic Aspergillus ochraceus species.

Genetic variability of Aspergillus ochraceus was examined at the DNA level. Based on the HaeIII-Bg/II generated mitochondrial DNA restriction profiles, most isolates could be classified into two distinct groups. These two groups could also be distinguished by the random amplified polymorphic DNA technique, and with telomeric PCR amplifications. Phylogenetic analysis of sequences of the intergenic transcribed spacer region of some of the strains resulted in a dendrogram with the same topology as that based on mitochondrial DNA and amplified DNA data. None of the isolates with type 2 mtDNA profiles produce ochratoxins. Some strains (e.g., A. ochraceus ICMP 939) displayed strain-specific mitochondrial DNA patterns, and their amplified DNA profiles were also different from all other A. ochraceus strains examined.

A statistical analysis of the effect of warfare on the human secondary sex ratio.

Many factors have been hypothesized to affect the human secondary sex ratio (the annual percentage of males among all live births), among them race, parental ages, and birth order. Some authors have even proposed warfare as a factor influencing live birth sex ratios. The hypothesis that during and shortly after periods of war the human secondary sex ratio is higher has received little statistical treatment. In this paper we evaluate the war hypothesis using 3 statistical methods: linear regression, randomization, and time-series analysis. Live birth data from 10 different countries were included. Although we cannot speak of a general phenomenon, statistical evidence for an association between warfare and live birth sex ratio was found for several countries. Regression and randomization test results were in agreement. Time-series analysis showed that most human sex-ratio time series can be described by a common model. The results obtained using intervention models differed somewhat from results obtained by regression methods.

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina.

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids.

Intra- and interspecies virus transfer in Aspergilli via protoplast fusion.

Intra- and interspecies transfer of dsRNA viruses between black Aspergilli and Aspergillus nidulans strains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of black Aspergillus and A. nidulans strains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found among A. nidulans strains, transfer among black Aspergilli was frequently unsuccessful. The lack of virus transfer between black Aspergillus isolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible black Aspergilli. The inefficient transfer of dsRNA viruses between black Aspergilli is not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of black Aspergilli are discussed.

Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli.

The extent of heterokaryon (also termed somatic or vegetative) incompatibility among black Aspergillus strains was examined using nitrate non-utilising mutants selected on chlorate medium. Pairings of complementary mutants showed that somatic compatibility between different strains is exceptional in natural populations of the asexual black Aspergilli. Mycoviruses are present in a considerable fraction of the sampled natural population, but surprisingly, horizontal transfer of mycoviruses only occurs-at least under laboratory conditions-between the (very rare) compatible combinations of strains. Thus, unlike other fungal species, somatic incompatibility in black Aspergilli efficiently blocks virus transfer. Viruses present in black Aspergillus isolates are highly efficiently transmitted to asexual progeny.

An experimental test for synergistic epistasis and its application in Chlamydomonas.

Theoretically, one of the most general benefits of sex is given by its function in facilitating selection against deleterious mutations. This advantage of sex may be deterministic if deleterious mutations affect the fitness of an individual in a synergistic way, i.e., if mutations increase each others' negative fitness effect. We present a new test for synergistic epistasis that considers the skewness of the log fitness distribution of offspring from a cross. We applied this test to data of the unicellular alga Chlamydomonas moewussii. For this purpose, two crosses were made: one between two strains that are presumed to have accumulated slightly deleterious mutations, the other between two strains without a history of mutation accumulation. Fitness was measured by estimating the two parameters of logistic growth in batch culture, the maximum growth rate (r) and the carrying capacity (K). The finding of a negatively skewed distribution for K in the accumulation cross suggests synergism between mutations affecting the carrying capacity, while the absence of skewness for tau in both crosses is consistent with independent effects of mutations affecting this parameter. The results suggest a possible alternative explanation for the general observation that sex is related to constant environments, where selection on K predominates, while asexual reproduction is found in more variable environments, where selection on r is more important.

Factors affecting the spread of double-stranded RNA viruses in Aspergillus nidulans.

Viruses are common in asexual Aspergilli but not in sexual Aspergilli. We found no viruses in 112 isolates of the sexual Aspergillus nidulans. We have investigated factors that could play a role in preventing the spread of mycoviruses through populations of A. nidulans. Experiments were performed with A. nidulans strains infected with viruses originating from A. niger. Horizontal virus transmission was restricted but not prevented by somatic incompatibility. Viruses were transmitted vertically via conidiospores but not via ascospores. Competition experiments revealed no effect of virus infection on host fitness. Outcrossing was found to limit the spread of viruses significantly more than selfing. It is concluded that the exclusion of viruses from sexual Aspergilli could be due to the formation of new somatic incompatibility groups by sexual recombination.