Austin Burt

The Evolutionary Biology and Population Genetics Underlying Fungal Strain Typing

SUMMARY Strain typing of medically important fungi and fungal population genetics have been stimulated by new methods of tapping DNA variation. The aim of this contribution is to show how awareness of fungal population genetics can increase the utility of strain typing to better serve the interests of medical mycology. Knowing two basic features of fungal population biology, the mode of reproduction and genetic differentiation or isolation, can give medical mycologists information about the intraspecific groups that are worth identifying and the number and type of markers that would be needed to do so. The same evolutionary information can be just as valuable for the selection of fungi for development and testing of pharmaceuticals or vaccines. The many methods of analyzing DNA variation are evaluated in light of the need for polymorphic loci that are well characterized, simple, independent, and stable. Traditional population genetic and new phylogenetic methods for analyzing mode of reproduction, genetic differentiation, and isolation are reviewed. Strain typing and population genetic reports are examined for six medically important species: Coccidioides immitis, Histoplasma capsulatum, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and A. flavus. Research opportunities in the areas of genomics, correlation of clinical variation with genetic variation, amount of recombination, and standardization of approach are suggested.

Sex increases the efficacy of natural selection in experimental yeast populations

Why sex evolved and persists is a problem for evolutionary biology, because sex disrupts favourable gene combinations and requires an expenditure of time and energy. Further, in organisms with unequal-sized gametes, the female transmits her genes at only half the rate of an asexual equivalent (the twofold cost of sex). Many modern theories that provide an explanation for the advantage of sex incorporate an idea originally proposed by Weismann more than 100 years ago: sex allows natural selection to proceed more effectively because it increases genetic variation. Here we test this hypothesis, which still lacks robust empirical support, with the use of experiments on yeast populations. Capitalizing on recent advances in the molecular biology of recombination in yeast, we produced by genetic manipulation strains that differed only in their capacity for sexual reproduction. We show that, as predicted by the theory, sex increases the rate of adaptation to a new harsh environment but has no measurable effect on fitness in a new benign environment where there is little selection.

Perspective: sex, recombination, and the efficacy of selection--was Weismann right?

Abstract The idea that sex functions to provide variation for natural selection to act upon was first advocated by August Weismann and it has dominated much discussion on the evolution of sex and recombination since then. The goal of this paper is to further extend this hypothesis and to assess its place in a larger body of theory on the evolution of sex and recombination. A simple generic model is developed to show how fitness variation and covariation interact with selection for recombination and illustrate some important implications of the hypothesis: (1) the advantage of sex and recombination can accrue both to reproductively isolated populations and to modifiers segregating within populations, but the former will be much larger than the latter; (2) forces of degradation that are correlated across loci within an individual can reduce or reverse selection for increased recombination; and (3) crossing-over (which can occur at different places in different meioses) will create more variability than having multiple chromosomes and so will have more influence on the efficacy of selection. Several long-term selection experiments support Weismanns hypothesis, including those showing a greater response to selection in populations with higher rates of recombination and higher rates of recombination evolving as a correlated response to selection for some other character. Weismanns hypothesis is also consistent with the sporadic distribution of obligate asexuality, which indicates that clones have a higher rate of extinction than sexuals. Weismanns hypothesis is then discussed in light of other patterns in the distribution of sexuality versus asexuality. To account for variation in the frequency of obligate asexuality in different taxa, a simple model is developed in which this frequency is a function of three parameters: the rate of clonal origin, the initial fitness of clones when they arise, and the rate at which that fitness declines over time. Variation in all three parameters is likely to be important in explaining the distribution of obligate asexuality. Facultative asexuality also exists, and for this to be stable it seems there must be ecological differences between the sexual and asexual propagules as well as genetic differences. Finally, the timing of sex in cyclical parthenogens is most likely set to minimize the opportunity costs of sex. None of these patterns contradict Weismanns hypothesis, but they do show that many additional principles unrelated to the function of sex are required to fully explain its distribution. Weismanns hypothesis is also consistent with what we know about the mechanics and molecular genetics of recombination, in particular the tendency for chromatids to recombine with a homolog rather than a sister chromatid at meiosis, which is opposite to what they do during mitosis. However, molecular genetic studies have shown that cis-acting sites at which recombination is initiated are lost by gene conversion as a result, a factor that can be expected to affect many fine details in the evolution of recombination. In summary, although Weismanns hypothesis must be considered the leading candidate for the function of sex and recombination, nevertheless, many additional principles are needed to fully account for their evolution. Corresponding Editor: C. Lively

A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae

Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.

Site-specific selfish genes as tools for the control and genetic engineering of natural populations

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

Population genomics of the wild yeast Saccharomyces paradoxus: Quantifying the life cycle

Most microbes have complex life cycles with multiple modes of reproduction that differ in their effects on DNA sequence variation. Population genomic analyses can therefore be used to estimate the relative frequencies of these different modes in nature. The life cycle of the wild yeast Saccharomyces paradoxus is complex, including clonal reproduction, outcrossing, and two different modes of inbreeding. To quantify these different aspects we analyzed DNA sequence variation in the third chromosome among 20 isolates from two populations. Measures of mutational and recombinational diversity were used to make two independent estimates of the population size. In an obligately sexual population these values should be approximately equal. Instead there is a discrepancy of about three orders of magnitude between our two estimates of population size, indicating that S. paradoxus goes through a sexual cycle approximately once in every 1,000 asexual generations. Chromosome III also contains the mating type locus (MAT), which is the most outbred part in the entire genome, and by comparing recombinational diversity as a function of distance from MAT we estimate the frequency of matings to be ≈94% from within the same tetrad, 5% with a clonemate after switching the mating type, and 1% outcrossed. Our study illustrates the utility of population genomic data in quantifying life cycles.

A synthetic homing endonuclease-based gene drive system in the human malaria mosquito

Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito’s ability to serve as a disease vector. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations. We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions and the homing endonuclease gene I-SceI, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

Genomewide Screening Reveals High Levels of Insertional Polymorphism in the Human Endogenous Retrovirus Family HERV-K(HML2): Implications for Present-Day Activity

ABSTRACT The published human genome sequence contains many thousands of endogenous retroviruses (HERVs) but all are defective, containing nonsense mutations or major deletions. Only the HERV-K(HML2) family has been active since the divergence of humans and chimpanzees; it contains many members that are human specific, as well as several that are insertionally polymorphic (an inserted element present only in some human individuals). Here we perform a genomewide survey of insertional polymorphism levels in this family by using the published human genome sequence and a diverse sample of 19 humans. We find that there are 113 human-specific HERV-K(HML2) elements in the human genome sequence, 8 of which are insertionally polymorphic (11 if we extrapolate to those within regions of the genome that were not suitable for amplification). The average rate of accumulation since the divergence with chimpanzees is thus approximately 3.8 × 10−4 per haploid genome per generation. Furthermore, we find that the number of polymorphic elements is not significantly different from that predicted by a standard population genetic model that assumes constant activity of the family until the present. This suggests to us that the HERV-K(HML2) family may be active in present-day humans. Active (replication-competent) elements are likely to have inserted very recently and to be present at low allele frequencies, and they may be causing disease in the individuals carrying them. This view of the family from a population perspective rather than a genome perspective will inform the current debate about a possible role of HERV-K(HML2) in human disease.

Population Genetics of the Wild Yeast Saccharomyces paradoxus

Saccharomyces paradoxus is the closest known relative of the well-known S. cerevisiae and an attractive model organism for population genetic and genomic studies. Here we characterize a set of 28 wild isolates from a 10-km2 sampling area in southern England. All 28 isolates are homothallic (capable of mating-type switching) and wild type with respect to nutrient requirements. Nine wild isolates and two lab strains of S. paradoxus were surveyed for sequence variation at six loci totaling 7 kb, and all 28 wild isolates were then genotyped at seven polymorphic loci. These data were used to calculate nucleotide diversity and number of segregating sites in S. paradoxus and to investigate geographic differentiation, population structure, and linkage disequilibrium. Synonymous site diversity is ∼0.3%. Extensive incompatibilities between gene genealogies indicate frequent recombination between unlinked loci, but there is no evidence of recombination within genes. Some localized clonal growth is apparent. The frequency of outcrossing relative to inbreeding is estimated at 1.1% on the basis of heterozygosity. Thus, all three modes of reproduction known in the lab (clonal replication, inbreeding, and outcrossing) have been important in molding genetic variation in this species.

DIVERSIFICATION IN SEXUAL AND ASEXUAL ORGANISMS

Abstract Sexual reproduction has long been proposed as a major factor explaining the existence of species and species diversity. Yet, the importance of sex for diversification remains obscure because of a lack of critical theory, difficulties of applying universal concepts of species and speciation, and above all the scarcity of empirical tests. Here, we use genealogical theory to compare the relative tendency of strictly sexual and asexual organisms to diversify into discrete genotypic and morphological clusters. We conclude that asexuals are expected to display discrete clusters similar to those found in sexual organisms. Whether sexuals or asexuals display stronger clustering depends on a number of factors, but in at least some scenarios asexuals should display a stronger pattern. Confounding factors aside, the only explanation we identify for stronger patterns of diversification in sexuals than asexuals is if the faster rates of adaptive change conferred by sexual reproduction promote greater clustering. Quantitative comparisons of diversification in related sexual and asexual taxa are needed to resolve this issue. The answer should shed light not only on the importance of the different stages leading to diversification, but also on the adaptive consequences of sex, still largely unexplored from a macroevolutionary perspective.