Mating-System Variation,Demographic History and Patterns of Nucleotide Diversity in the Tristylous Plant Eichhornia paniculata

Inbreeding in highly selfing populations reduces effective size and, combined with demographic conditions associated with selfing, this can erode genetic diversity and increase population differentiation. Here we investigate the role that variation in mating patterns and demographic history play in shaping the distribution of nucleotide variation within and among populations of the annual neotropical colonizing plant Eichhornia paniculata, a species with wide variation in selfing rates. We sequenced 10 EST-derived nuclear loci in 225 individuals from 25 populations sampled from much of the geographic range and used coalescent simulations to investigate demographic history. Highly selfing populations exhibited moderate reductions in diversity but there was no significant difference in variation between outcrossing and mixed mating populations. Population size interacted strongly with mating system and explained more of the variation in diversity within populations. Bayesian structure analysis revealed strong regional clustering and selfing populations were highly differentiated on the basis of an analysis of F_st. There was no evidence for a significant loss of within-locus linkage disequilibrium within populations, but regional samples revealed greater breakdown in Brazil than in selfing populations from the Caribbean. Coalescent simulations indicate a moderate bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years before the present. Our results suggest that the recent multiple origins of selfing in E. paniculata from diverse outcrossing populations result in higher diversity than expected under long-term equilibrium.THE rate of self-fertilization in hermaphrodite organisms is expected to affect a number of important features of population genetic structure and diversity. Most directly, homozygosity increases as a function of the selfing rate and thus reduces the effective population size (N_e), up to twofold with complete selfing (Pollak 1987; Charlesworth et al. 1993; Nordborg 2000). Further, because of increased homozygosity, crossing over rarely occurs between heterozygous sites, thus increasing linkage disequilibrium (LD). Higher LD causes stronger hitchhiking effects such as selective sweeps, background selection, and Hill–Robertson interference, all of which are expected to further reduce the amount of neutral genetic variation within populations (reviewed in Charlesworth and Wright 2001).Population genetic processes resulting from inbreeding may be further augmented by demographic and life-history characteristics associated with the selfing habit. In particular, selfing populations can be founded by single individuals, resulting in striking reductions in diversity as a result of genetic bottlenecks and reproductive isolation. The capacity for uniparental reproduction gives many selfers prolific colonizing ability and the capacity to establish after long-distance dispersal, especially in comparison with obligate outcrossers (Baker 1955; Pannell and Barrett 1998). The colonization–extinction dynamics typical of many selfing species and limited pollen-mediated gene flow also increase differentiation among populations, resulting in considerable population subdivision (Hamrick and Godt 1990, 1996; Schoen and Brown 1991). Although the total amounts of among-population variation may be less affected by these processes (Pannell and Charlesworth 1999; Ingvarsson 2002), the demographic and life-history characteristics of many selfing species are likely to result in nonequilibrium conditions occurring in selfing populations.In many taxa where selfing has evolved it may be of relatively recent origin (Schoen et al. 1997; Takebayashi and Morrell 2001; Foxe et al. 2009; Guo et al. 2009). Where selfing has recently established, demographic forces associated with colonization may be as important as the mating system per se in structuring patterns of diversity. For example, if selfing originates through the establishment of a small number of founders, we would expect a sharp reduction in diversity relative to the outcrossing progenitor and a strong signature of a genetic bottleneck. In contrast, if selfing has evolved recently through the spread of genetic modifiers of small effect, newly established populations may retain significant amounts of ancestral polymorphism from their outcrossing progenitors. In this latter case populations may retain considerably more variation than expected under long-term equilibrium predictions.Molecular evidence for reduced nucleotide diversity and greater differentiation among populations of selfing taxa compared to populations of related outcrossing taxa has been reported from Leavenworthia (Liu et al. 1998, 1999), Arabidopsis (Savolainen et al. 2000; Wright et al. 2002), Solanum (Baudry et al. 2001), Mimulus (Sweigart and Willis 2003), Amsinckia (Perusse and Schoen 2004), and Caenorhabditis (Graustein et al. 2002; Cutter et al. 2006; Cutter 2008). In each case the reduction in diversity was more severe than the twofold reduction predicted for selfing populations at equilibrium. This indicates that factors in addition to the mating system are reducing diversity, but it has been difficult to uncouple the relative importance of genetic hitchhiking from the ecology and demographic history of selfing taxa. This challenge parallels similar difficulties in efforts to distinguish selective from demographic explanations in population genetic studies of Drosophila (Haddrill et al. 2005; Ometto et al. 2005; Thornton and Andolfatto 2006; Jensen et al. 2008). However, in many plant populations, especially those with annual life histories and small structured populations, demographic processes may play a more prominent role in causing reduced diversity than increased hitchhiking associated with selfing.Molecular population genetic studies of selfing in plants have generally focused on either small samples from a large number of populations (e.g., Sweigart and Willis 2003; Nordborg et al. 2005) or relatively large within-population samples from a small number of populations (e.g., Baudry et al. 2001). Ideally, a deeper sampling both within and among populations combined with independent ecological and historical information is required to improve understanding of the interplay of demographic and selective factors. Here we address these issues by examining patterns of nucleotide diversity within a large sample of populations of Eichhornia paniculata (Pontederiaceae), an annual species for which there is considerable ecological and demographic information (reviewed in Barrett and Husband 1997).E. paniculata occurs primarily in northeastern (N.E.) Brazil and the Caribbean islands of Cuba and Jamaica. Various lines of evidence suggest that Brazil is the original source region for Caribbean populations (reviewed in Barrett et al. 2009). Populations of E. paniculata exhibit striking mating-system diversity, ranging from predominantly outcrossing to those that are highly selfing (outcrossing rate, t = 0.002–0.96; n = 54 populations) (Barrett and Husband 1990; Barrett et al. 1992). Variation in mating system is associated with the evolutionary breakdown of the species'' tristylous genetic polymorphism and the spread and fixation of selfing variants capable of autonomous self-pollination (Barrett et al. 1989). Populations of E. paniculata are characterized by three morph structures: trimorphic with long-, mid-, and short-styled morphs (hereafter L-, M-, and S-morphs); dimorphic, with two floral morphs, most commonly the L- and M-morphs; and monomorphic, primarily composed of selfing variants of the M-morph. The morph structure and presence of selfing variants within populations explain ∼60% of the variation in outcrossing rates among populations (Barrett and Husband 1990). Trimorphic populations are largely outcrossing, dimorphic populations display mixed mating, and monomorphic populations are highly selfing. Patterns of allozyme variation indicate a reduction in diversity with increased selfing rates and greater among-population differentiation (Glover and Barrett 1987; Barrett and Husband 1990; Husband and Barrett 1993). Finally, studies of the inheritance of mating-system modifiers (Fenster and Barrett 1994; Vallejo-Marín and Barrett 2009) in combination with allozyme (Husband and Barrett 1993) and molecular evidence (Barrett et al. 2009) indicate that the transition from outcrossing to selfing in E. paniculata has occurred on multiple occasions.The goal of our study was to investigate the relation between mating-system variation and neutral molecular diversity for a large sample of E. paniculata populations encompassing most of the geographical range. This was accomplished by collecting multilocus nucleotide sequence data from 225 individuals sampled from 25 populations including trimorphic, dimorphic, and monomorphic populations. Because it has been previously demonstrated that this sequence of morph structures is strongly associated with increasing rates of self-fertilization (see Barrett and Husband 1990), we predicted a decrease in neutral diversity and increases in F_st and linkage disequilibrium from floral trimorphism to monomorphism. This extensive population-level sampling across a wide range of selfing rates allowed us to investigate the relative importance of mating system, geography, and current population size in structuring genetic variation. We also applied the approaches of Bayesian clustering (Pritchard et al. 2000; Falush et al. 2003; Gao et al. 2007) and divergence population genetics (Wakeley and Hey 1997; Hey and Nielsen 2004; Becquet and Przeworski 2007) to investigate the demographic history of E. paniculata and to provide a framework for understanding island colonization and the transition from outcrossing to selfing.     

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI⁺], [URE3], and [RNQ/PIN⁺] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI⁺] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI⁺] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.     

Transgenic studies on the involvement of cytokinin and gibberellin in male development

Numerous plant hormones interact during plant growth and development. Elucidating the role of these various hormones on particular tissue types or developmental stages has been difficult with exogenous applications or constitutive expression studies. Therefore, we used tissue-specific promoters expressing CKX1 and gai, genes involved in oxidative cytokinin degradation and gibberellin (GA) signal transduction, respectively, to study the roles of cytokinin and GA in male organ development. Accumulation of CKX1 in reproductive tissues of transgenic maize (Zea mays) resulted in male-sterile plants. The male development of these plants was restored by applications of kinetin and thidiazuron. Similarly, expression of gai specifically in anthers and pollen of tobacco (Nicotiana tabacum) and Arabidopsis resulted in the abortion of these respective tissues. The gai-induced male-sterile phenotype exhibited by the transgenic plants was reversible by exogenous applications of kinetin. Our results provide molecular evidence of the involvement of cytokinin and GA in male development and support the hypothesis that the male development is controlled in concert by multiple hormones. These studies also suggest a potential method for generating maintainable male sterility in plants by using existing agrochemicals that would reduce the expense of seed production for existing hybrid crops and provide a method to produce hybrid varieties of traditionally non-hybrid crops.     

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.