Establishment of zygomorphy on an ontogenic spiral and evolution of perianth in the tribe Delphinieae (Ranunculaceae)

Background and Aims

Ranunculaceae presents both ancestral and derived floral traits for eudicots, and as such is of potential interest to understand key steps involved in the evolution of zygomorphy in eudicots. Zygomorphy evolved once in Ranunculaceae, in the speciose and derived tribe Delphinieae. This tribe consists of two genera (Aconitum and Delphinium s.l.) comprising more than one-quarter of the species of the family. In this paper, the establishment of zygomorphy during development was investigated to cast light on the origin and evolution of this morphological novelty.

Methods

The floral developmental sequence of six species of Ranunculaceae, three actinomorphic (Nigella damascena, Aquilegia alpina and Clematis recta) and three zygomorphic (Aconitum napellus, Delphinium staphisagria and D. grandiflorum), was compared. A developmental model was elaborated to break down the successive acquisitions of floral organ identities on the ontogenic spiral (all the species studied except Aquilegia have a spiral phyllotaxis), giving clues to understanding this complex morphogenesis from an evo-devo point of view. In addition, the evolution of symmetry in Ranunculaceae was examined in conjunction with other traits of flowers and with ecological factors.

Key Results

In the species studied, zygomorphy is established after organogenesis is completed, and is late, compared with other zygomorphic eudicot species. Zygomorphy occurs in flowers characterized by a fixed merism and a partially reduced and transformed corolla.

Conclusions

It is suggested that shifts in expression of genes controlling the merism, as well as floral symmetry and organ identity, have played a critical role in the evolution of zygomorphy in Delphinieae, while the presence of pollinators able to exploit the peculiar morphology of the flower has been a key factor for the maintenance and diversification of this trait.Key words: Delphinieae, development, evolution, evo-devo, nectar spurs, ontogenic spiral, Ranunculaceae, zygomorphy     

Floral development and evolution of capitulum structure in Anacyclus (Anthemideae,Asteraceae)

Background and Aims

Most of the diversity in the pseudanthia of Asteraceae is based on the differential symmetry and sexuality of its flowers. In Anacyclus, where there are (1) homogamous capitula, with bisexual, mainly actinomorphic and pentamerous flowers; and (2) heterogamous capitula, with peripheral zygomorphic, trimerous and long-/short-rayed female flowers, the floral ontogeny was investigated to infer their origin.

Methods

Floral morphology and ontogeny were studied using scanning electron microscope and light microscope techniques

Key Results

Disc flowers, subtended by paleae, initiate acropetally. Perianth and androecium initiation is unidirectional/simultaneous. Late zygomorphy occurs by enlargement of the adaxial perianth lobes. In contrast, ray flowers, subtended by involucral bracts, initiate after the proximal disc buds, breaking the inflorescence acropetal pattern. Early zygomorphy is manifested through the fusion of the lateral and abaxial perianth lobes and the arrest of the adaxials. We report atypical phenotypes with peripheral ‘trumpet’ flowers from natural populations. The peripheral ‘trumpet’ buds initiate after disc flowers, but maintain an actinomorphic perianth. All phenotypes are compared and interpreted in the context of alternative scenarios for the origin of the capitulum and the perianth identity.

Conclusions

Homogamous inflorescences display a uniform floral morphology and development, whereas the peripheral buds in heterogamous capitula display remarkable plasticity. Disc and ray flowers follow different floral developmental pathways. Peripheral zygomorphic flowers initiate after the proximal actinomorphic disc flowers, behaving as lateral independent units of the pseudanthial disc from inception. The perianth and the androecium are the most variable whorls across the different types of flowers, but their changes are not correlated. Lack of homology between hypanthial appendages and a calyx, and the perianth double-sided structure are discussed for Anacyclus together with potential causes of its ray flower plasticity.     

Evolutionary Trends in the Flowers of Asteridae: Is Polyandry an Alternative to Zygomorphy?

Background and Aims

Floral symmetry presents two main states in angiosperms, actinomorphy (polysymmetry or radial symmetry) and zygomorphy (monosymmetry or bilateral symmetry). Transitions from actinomorphy to zygomorphy have occurred repeatedly among flowering plants, possibly in coadaptation with specialized pollinators. In this paper, the rules controlling the evolution of floral symmetry were investigated to determine in which architectural context zygomorphy can evolve.

Methods

Floral traits potentially associated with perianth symmetry shifts in Asteridae, one of the major clades of the core eudicots, were selected: namely the perianth merism, the presence and number of spurs, and the androecium organ number. The evolution of these characters was optimized on a composite tree. Correlations between symmetry and the other morphological traits were then examined using a phylogenetic comparative method.

Key Results

The analyses reveal that the evolution of floral symmetry in Asteridae is conditioned by both androecium organ number and perianth merism and that zygomorphy is a prerequisite to the emergence of spurs.

Conclusions

The statistically significant correlation between perianth zygomorphy and oligandry suggests that the evolution of floral symmetry could be canalized by developmental or spatial constraint. Interestingly, the evolution of polyandry in an actinomorphic context appears as an alternative evolutionary pathway to zygomorphy in Asteridae. These results may be interpreted either in terms of plant–pollinator adaptation or in terms of developmental or physical constraints. The results are discussed in relation to current knowledge about the molecular bases underlying floral symmetry.Key words: Floral symmetry, architectural constraints, Asteridae, comparative analysis, composite tree, correlated evolution, evolutionary scenario     

Identification of Selection Signatures in Cattle Breeds Selected for Dairy Production

Genetic correlations are expected to be high among functionally related traits and lower between groups of traits with distinct functions (e.g., reproductive vs. resource-acquisition traits). Here, we explore the quantitative-genetic and QTL architecture of floral organ sizes, vegetative traits, and life history in a set of Brassica rapa recombinant inbred lines within and across field and greenhouse environments. Floral organ lengths were strongly positively correlated within both environments, and analysis of standardized G-matrices indicates that the structure of genetic correlations is ∼80% conserved across environments. Consistent with these correlations, we detected a total of 19 and 21 additive-effect floral QTL in the field and the greenhouse, respectively, and individual QTL typically affected multiple organ types. Interestingly, QTL × QTL epistasis also appeared to contribute to observed genetic correlations; i.e., interactions between two QTL had similar effects on filament length and two estimates of petal size. Although floral and nonfloral traits are hypothesized to be genetically decoupled, correlations between floral organ size and both vegetative and life-history traits were highly significant in the greenhouse; G-matrices of floral and vegetative traits as well as floral and life-history traits differed across environments. Correspondingly, many QTL (45% of those mapped in the greenhouse) showed environmental interactions, including approximately even numbers of floral and nonfloral QTL. Most instances of QTL × QTL epistasis for floral traits were environment dependent.EVOLUTIONARY responses to selection are dependent on genetic architecture. The proportion of phenotypic variation with a heritable genetic basis affects the response to selection, as does the structure of genetic correlations among selected traits. For example, an evolutionary response will be constrained if selection favors an increase in the value of two traits that are negatively correlated; i.e., a negative correlation is antagonistic to the joint vector of selection. Alternatively, if the vector of selection is parallel to the genetic correlation, then trait covariation is reinforcing and the population mean may more rapidly approach favored trait values (Etterson and Shaw 2001; Merilä and Björklund 2004). One measure of genetic architecture is the G-matrix (Lynch and Walsh 1998), which is composed of genetic variances (diagonal matrix elements) and genetic covariances among traits (off-diagonal matrix elements). G-matrices have been shown to vary across environments (Donohue et al. 2000; Conner et al. 2003; Brock and Weinig 2007), indicating that the molecular-genetic underpinnings of matrix elements (e.g., identity and/or relative effect of additive and epistatic loci, degree of pleiotropy, etc.) and the traits'' evolutionary potential vary across environments. Few studies, however, have related matrix and QTL architectures; and, therefore, the molecular-genetic underpinnings of quantitative-genetic estimates remain unclear (but see Gardner and Latta 2007; Kelly 2009).In angiosperms, covariances between floral whorls (e.g., petal and stamen length) are frequently positive among functionally related traits. These positive correlations can arise from pollinator-mediated (or pollination-mediated) selection for specific allometric relationships among floral traits and ensuing linkage disequilibrium (LD) among causal loci (Berg 1959, 1960; also referred to as phenotypic integration, see Pigliucci 2003; Klingenberg 2008). For example, in outcrossing species, male fitness may be more dependent on the frequency and efficiency of pollinator visitation than female fitness (Bell 1985; but see Hodgins and Barrett 2008). Anther placement relative to the corolla opening can affect the efficiency of pollen dissemination (Conner and Via 1993; Morgan and Conner 2001); in addition, comparative work indicates that petal–stamen length correlations are stronger than stamen–pistil length correlations in outcrossers, whereas species that reproduce via autogamous selfing show the opposite pattern (Ushimaru and Nakata 2002). Alternatively, strong floral integration could be attributed to the developmental hypothesis that genetic correlations arise due to pleiotropic genes coregulating floral whorls (Herrera 2001; Herrera et al. 2002). Strong correlations resulting from linkage disequilibrium or from developmentally based pleiotropy may constrain the evolution of novel reproductive morphologies when biotic or abiotic factors (and selection) change (Cheverud 1984; Clark 1987; Smith and Rausher 2008; Agrawal and Stinchcombe 2009).Similar to genetic covariances among floral traits, covariances between floral and nonfloral traits could also alter the evolutionary response of reproductive traits. In contrast to hypotheses regarding the adaptive significance of floral-trait integration, genetic correlations between floral and nonfloral traits (e.g., vegetative or phenological traits) are hypothesized to be disadvantageous (Berg 1960). More specifically, floral allometry may be shaped by selection for reproductive success, as described above, whereas vegetative morphology is shaped primarily by selection to optimize other functions, such as light capture. If floral and nonfloral traits have a common genetic basis, then selection on phenological or morphological traits may result in maladaptive expression of floral organ size. As a result, functionally integrated floral traits are predicted to be genetically decoupled from vegetative and phenological traits (Berg 1960).QTL mapping provides a powerful tool to explore the genetic architecture of evolutionarily important traits. The QTL architecture of interspecific floral traits has been explored in diverse systems (Bradshaw et al. 1995; Fishman et al. 2002; Goodwillie et al. 2006; Bouck et al. 2007; Moyle 2007); however, insight into the molecular genetic basis of intraspecific floral variation comes almost exclusively from Arabidopsis thaliana (Juenger et al. 2000, 2005) and Mimulus guttatus (Hall et al. 2006). Floral traits in these intraspecific crosses are polygenic with a majority of detected QTL being of small to moderate effect size. Consistent with other quantitative-genetic studies (reviewed in Ashman and Majetic 2006), floral traits in A. thaliana and M. guttatus mapping populations exhibited moderate to high genetic correlations. In both systems, mapped QTL often affected multiple floral traits. In the few cases where QTL underlying intraspecific floral morphology have been evaluated, only a single growth environment was used; estimation of floral quantitative genetics across environments and subsequent comparison with the QTL architecture underlying observed across-environment patterns are lacking.Using a segregating progeny of Brassica rapa (recombinant inbred lines, RILs) and a small sample of crop and wild accessions, we examine the quantitative-genetic and QTL architecture of floral traits under field and greenhouse environments. Specifically, we address the following questions: (1) Does this RIL population express significant genetic (co)variation for floral traits when growing in the field or greenhouse? (2) Is there significant genetic variation for vegetative traits and days to flowering in field and greenhouse environments, and is there evidence for genetic correlations between floral and nonfloral traits? (3) Does the genetic architecture of floral and nonfloral traits, as measured by the G-matrix, differ across environments? (4) What is the number and effect size of additive and epistatic QTL in field and greenhouse environments? (5) What is the relationship between mapped QTL and quantitative genetic estimates of trait (co)variation within and between floral and nonfloral traits? And (6) what is the relationship between the quantitative-genetic architecture of floral traits in the RILs vs. in the accessions?     

Ontogenetic origins of floral bilateral symmetry in Moringaceae (Brassicales)

Floral morphology of the 13 species of Moringa ranges from actinomorphic flowers with little hypanthium to highly zygomorphic flowers with well-developed hypanthia. Scanning electron and light microscopy were used to identify ontogenetic differences among two actinomorphic and eight zygomorphic species. All species show traces of zygomorphy between petal organogenesis and anther differentiation. At late organogenesis, zygomorphy is manifest by one petal being larger than the others, slight unidirectional maturation of the anthers, and in many species, some staminodes may be missing. At organ differentiation and beyond, the actinomorphic species show a trend toward increasing actinomorphy, whereas the zygomorphic features of early ontogeny are progressively accentuated throughout the ontogeny of the zygomorphic species. Because of the early traces of zygomorphy throughout the family, ontogeny in Moringa does not resemble that known from the sister taxon Caricaceae, which has flowers that are actinomorphic throughout ontogeny. Great intraspecific variation was found in floral plan in the actinomorphic-flowered species in contrast to the zygomorphic species. Each of the main clades in the family is distinguished by at least one feature of floral ontogeny. In general, ontogenetic differences that are congruent with deeper phylogenetic splits tend to occur earlier in ontogeny than those congruent with more recent divergences.     

Identification of the Major Sex-Determining Region of Turbot (Scophthalmus maximus)

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.SEX ratio is a central demographic parameter directly related to the reproductive potential of individuals and populations (Penman and Piferrer 2008). The phenotypic sex depends on the processes of both sex determination and sex differentiation. Exogenous factors, such as temperature, hormones, or social behavior, can modify the gonad development pathway in fish (Baroiller and D''Cotta 2001; Piferrer and Guiguen 2008). Both genetic (GSD) and environmental sex determination has been reported in this group (Devlin and Nagahama 2002; Penman and Piferrer 2008), although primary sex determination is genetic in most species (Valenzuela et al. 2003). Among GSD, single, multiple, or polygenic sex-determining (SD) gene systems have been documented (Kallman 1984; Matsuda et al. 2002; Lee et al. 2004; Vandeputte et al. 2007).Sex determination in fish can evolve very rapidly (Woram et al. 2003; Peichel et al. 2004; Ross et al. 2009). Different sex determination mechanisms have been reported between congeneric species and even between populations of the same species (Almeida-Toledo and Foresti 2001; Lee et al. 2004; Mank et al. 2006). The evolution of sex chromosomes involves the suppression of recombination between homologous chromosomes probably to maintain sex-related coadapted gene blocks (Charlesworth et al. 2005; Tripathi et al. 2009). The sex determination pathway appears to be less conserved than other developmental processes (Penman and Piferrer 2008). However, differences are more related to the top of the hierarchy in the developmental pathway, while downstream genes are more conserved (Wilkins 1995; Marín and Baker 1998). As a consequence, the SD master gene in fish can vary among related species (Kondo et al. 2003; Tanaka et al. 2007; Alfaqih et al. 2009). In this sense, fish represent an attractive model for studying the evolution of SD mechanisms and sex chromosomes (Peichel et al. 2004; Kikuchi et al. 2007).A low proportion of fish species have demonstrated sex-associated chromosome heteromorphisms (Almeida-Toledo and Foresti 2001; Devlin and Nagahama 2002; Penman and Piferrer 2008). This is congruent with the rapid evolution of the SD region in fish, and thus in most species the male and female version of this chromosome region appears largely undifferentiated. In spite of this, indirect clues related to progenies of sex/chromosome-manipulated individuals or to segregation of morphologic/molecular sex-associated markers indicate that mechanisms of sex determination in fish are similar to other vertebrates (Penman and Piferrer 2008). With the arrival of genomics, large amounts of different genetic markers and genomic information are available for scanning genomes to look for their association with sex determination. Quantitative trait loci (QTL) (Cnaani et al. 2004; Peichel et al. 2004) or marker association (Felip et al. 2005; Chen et al. 2007) approaches have been used to identify the SD regions in some fish species. Also, microarrays constructed from gonadal ESTs have been applied to detect differentially expressed genes in the process of gonadal differentiation (Baron et al. 2005). Further, the increased genomic resources in model and aquaculture species have allowed the development of both comparative genomics (Woram et al. 2003; Kikuchi et al. 2007; Tripathi et al. 2009) and candidate gene (Shirak et al. 2006; Alfaqih et al. 2009) strategies to identify and characterize the SD region in fish. This has permitted the identification of the SD region in eight fish, including both model and aquaculture species (reviewed in Penman and Piferrer 2008).The turbot is a highly appreciated European aquaculture species, whose harvest is expected to increase from the current 9000 tons to >15,000 tons in 2012 (S. Cabaleiro, personal communication). Females of this species reach commercial size 4–6 months before males do, explaining the interest of the industry in obtaining all-female populations. Although some differences between families can be observed in the production process at farms, sex ratio is usually balanced at ∼1:1. Neither mitotic nor meiotic chromosomes have shown sex-associated heteromorphisms in turbot (Bouza et al. 1994; Cuñado et al. 2001). The proportion of sexes observed in triploid and especially gynogenetic progenies moved Cal et al. (2006a,b) to suggest an XX/XY mechanism in turbot with some additional, either environmental or genetic, factor involved. However, Haffray et al. (2009) have recently claimed a ZZ/ZW mechanism on the basis of the analysis of a large number of progenies from steroid-treated parents. These authors also suggested some (albeit low) influence of temperature in distorting sex proportions after the larval period. Finally, hybridizations between brill (Scophthalmus rhombus) and turbot render monosex progenies, depending on the direction of the cross performed, which suggests different SD mechanisms in these congeneric species (Purdom and ThaCker 1980).In this study, we used the turbot genetic map (Bouza et al. 2007, 2008; Martínez et al. 2008) to look for sex-associated QTL in this species. The identification of a major QTL in a specific linkage group (LG) in the five families analyzed prompted us to refine the genetic map at this LG and to perform a comparative genomics approach against model fish species for a precise location and characterization of the putative SD region. Also, sex-associated QTL markers were screened in a large natural population to provide additional support to our findings and to obtain population parameters at sex-related markers that could aid in interpreting the evolution of this genomic region.     

Estimating the Parameters of Selection on Nonsynonymous Mutations in Drosophila pseudoobscura and D. miranda

We present the results of surveys of diversity in sets of >40 X-linked and autosomal loci in samples from natural populations of Drosophila miranda and D. pseudoobscura, together with their sequence divergence from D. affinis. Mean silent site diversity in D. miranda is approximately one-quarter of that in D. pseudoobscura; mean X-linked silent diversity is about three-quarters of that for the autosomes in both species. Estimates of the distribution of selection coefficients against heterozygous, deleterious nonsynonymous mutations from two different methods suggest a wide distribution, with coefficients of variation greater than one, and with the average segregating amino acid mutation being subject to only very weak selection. Only a small fraction of new amino acid mutations behave as effectively neutral, however. A large fraction of amino acid differences between D. pseudoobscura and D. affinis appear to have been fixed by positive natural selection, using three different methods of estimation; estimates between D. miranda and D. affinis are more equivocal. Sources of bias in the estimates, especially those arising from selection on synonymous mutations and from the choice of genes, are discussed and corrections for these applied. Overall, the results show that both purifying selection and positive selection on nonsynonymous mutations are pervasive.SURVEYS of DNA sequence diversity and divergence are shedding light on a number of questions in evolutionary genetics (for recent reviews, see Akey 2009; Sella et al. 2009). Two of the most important questions of this kind concern the distribution of selection coefficients against deleterious mutations affecting protein sequences and the proportion of amino acid sequence differences between related species that have been fixed by positive selection. Several different methods have been proposed for studying each of these questions, using different features of data on polymorphism and divergence at nonsynonymous and silent sites.For example, the parameters of the distribution of selection coefficients against deleterious amino acid mutations have been estimated by contrasting the numbers of nonsynonymous and silent within-species polymorphisms and fixed differences between species (Sawyer and Hartl 1992; Bustamante et al. 2002; Piganeau and Eyre-Walker 2003; Sawyer et al. 2007); by fitting the frequency spectra of nonsynonymous and silent variants to models of selection, mutation, and drift (Akashi 1999; Eyre-Walker et al. 2006; Keightley and Eyre-Walker 2007; Kryukov et al. 2007; Boyko et al. 2008; Eyre-Walker and Keightley 2009); or by comparing levels of nonsynonymous and silent diversities between species with different population sizes (Loewe and Charlesworth 2006; Loewe et al. 2006). The results of these different approaches generally agree in suggesting that there is a wide distribution of selection coefficients against nonsynonymous mutations and that the mean selection coefficient against heterozygous carriers of such mutations is very small. The results imply that a typical individual from a human population carries several hundred weakly deleterious mutations (Eyre-Walker et al. 2006; Kryukov et al. 2007; Boyko et al. 2008); for a typical Drosophila population, with its much higher level of variability, the number is probably an order of magnitude greater (Loewe et al. 2006; Keightley and Eyre-Walker 2007).The presence of this large load of slightly deleterious mutations in human and natural populations, most of which are held at low frequencies by natural selection, has many implications. From the point of view of understanding human genetic disease, it means that we have to face the likelihood that susceptibility to a disease can be influenced by variants at many loci, each with small effects (Kryukov et al. 2007). The pervasive presence of deleterious mutations throughout the genome contributes to inbreeding depression (Charlesworth and Willis 2009) and may mean that the effective population size is reduced by background selection effects, even in regions of the genome with normal levels of genetic recombination (Loewe and Charlesworth 2007). Their presence may contribute so strongly to Hill–Robertson effects (Hill and Robertson 1966; Felsenstein 1974) that they cause severely reduced levels of diversity and adaptation in low-recombination regions of the genome (Charlesworth et al. 2010) and create a selective advantage to maintaining nonzero levels of recombination (Keightley and Otto 2006; Charlesworth et al. 2010). In addition, having an estimate of the distribution of selection coefficients against deleterious nonsynonymous mutations allows their contribution to between-species divergence to be predicted, providing a way of estimating the fraction of fixed nonsynonymous differences caused by positive selection (Loewe et al. 2006; Boyko et al. 2008; Eyre-Walker and Keightley 2009).It is thus important to collect data that shed light on the properties of selection against nonsynonymous mutations in a wide range of systems and also to compare the results from different methods of estimation, since they are subject to different sources of difficulty and biases. In a previous study, we proposed the use of a comparison between two related species with different effective population sizes for this purpose (Loewe and Charlesworth 2006; Loewe et al. 2006), using Drosophila miranda and D. pseudoobscura as material. These are well suited for this type of study, as they are closely related, live together in similar habitats, and yet have very different levels of silent nucleotide diversity, indicating different effective population sizes (N_e). This study was hampered by our inability to compare the same set of loci across the two species and by the small number of loci that could be used. We here present the results of a much larger study of DNA variation at X-linked and autosomal loci for these two species, using D. affinis as a basis for estimating divergence. We compare the results, applying the method of Loewe et al. (2006) with that of Eyre-Walker and Keightley (2009) for estimating the distribution of deleterious selection coefficients and with McDonald–Kreitman test-based methods for estimating the proportion of nonsynonymous differences fixed by positive selection. While broadly confirming the conclusions from earlier studies, we note some possible sources of bias and describe methods for minimizing their effects.     

Simple Y-Autosomal Incompatibilities Cause Hybrid Male Sterility in Reciprocal Crosses Between Drosophila virilis and D. americana

Postzygotic reproductive isolation evolves when hybrid incompatibilities accumulate between diverging populations. Here, I examine the genetic basis of hybrid male sterility between two species of Drosophila, Drosophila virilis and D. americana. From these analyses, I reach several conclusions. First, neither species carries any autosomal dominant hybrid male sterility alleles: reciprocal F₁ hybrid males are perfectly fertile. Second, later generation (backcross and F₂) hybrid male sterility between D. virilis and D. americana is not polygenic. In fact, I identified only three genetically independent incompatibilities that cause hybrid male sterility. Remarkably, each of these incompatibilities involves the Y chromosome. In one direction of the cross, the D. americana Y is incompatible with recessive D. virilis alleles at loci on chromosomes 2 and 5. In the other direction, the D. virilis Y chromosome causes hybrid male sterility in combination with recessive D. americana alleles at a single QTL on chromosome 5. Finally, in contrast with findings from other Drosophila species pairs, the X chromosome has only a modest effect on hybrid male sterility between D. virilis and D. americana.SPECIATION occurs when populations evolve one or more barriers to interbreeding (Dobzhansky 1937; Mayr 1963). One such barrier is intrinsic postzygotic isolation, which typically evolves when diverging populations accumulate different alleles at two or more loci that are incompatible when brought together in hybrid genomes; negative epistasis between these alleles renders hybrids inviable or sterile (Bateson 1909; Dobzhansky 1937; Muller 1942). Classical and recent studies in diverse animal taxa have provided support for two evolutionary patterns that often characterize the genetics of postzygotic isolation (Coyne and Orr 1989a). The first, Haldane''s rule, observes that when there is F₁ hybrid inviability or sterility that affects only one sex, it is almost always the heterogametic sex (Haldane 1922). Over the years, many researchers have tried to account for this pattern, but only two ideas are now thought to provide a general explanation: the “dominance theory,” which posits that incompatibility alleles are generally recessive in hybrids, and the “faster-male theory,” which posits that genes causing hybrid male sterility diverge more rapidly than those causing hybrid female sterility (Muller 1942; Wu and Davis 1993; Turelli and Orr 1995; reviewed in Coyne and Orr 2004). In some cases, however, additional factors might contribute to Haldane''s rule, including meiotic drive, a faster-evolving X chromosome, dosage compensation, and Y chromosome incompatibilities (reviewed in Laurie 1997; Turelli and Orr 2000; Coyne and Orr 2004).The second broad pattern affecting the evolution of postzygotic isolation is the disproportionately large effect of the X chromosome on heterogametic F₁ hybrid sterility (Coyne 1992). This “large X effect” has been documented in genetic analyses of backcross hybrid sterility (e.g., Dobzhansky 1936; Grula and Taylor 1980; Orr 1987; Masly and Presgraves 2007) and inferred from patterns of introgression across natural hybrid zones (e.g., Machado et al. 2002; Saetre et al. 2003; Payseur et al. 2004). However, in only one case has the cause of the large X effect been unambiguously determined: incompatibilities causing hybrid male sterility between Drosophila mauritiana and D. sechellia occur at a higher density on the X than on the autosomes (Masly and Presgraves 2007). Testing the generality of this pattern will require additional high-resolution genetic analyses in diverse taxa (Presgraves 2008). But whatever its causes, there is now general consensus that the X chromosome often plays a special role in the evolution of postzygotic isolation (Coyne and Orr 2004).The contribution of the Y chromosome to animal speciation is less clear. Y chromosomes have far fewer genes than the X or autosomes, and most of these genes are male specific (Lahn and Page 1997; Carvalho et al. 2009). In Drosophila species, the Y chromosome is typically required for male fertility, but not for viability (Voelker and Kojima 1971). How often, then, does the Y chromosome play a role in reproductive isolation? In crosses between Drosophila species, hybrid male sterility is frequently caused by incompatibilities between the X and Y chromosomes (Schafer 1978; Heikkinen and Lumme 1998; Mishra and Singh 2007) or between the Y and heterospecific autosomal alleles (Patterson and Stone 1952; Vigneault and Zouros 1986; Lamnissou et al. 1996). In crosses between D. yakuba and D. santomea, the Y chromosome causes F₁ hybrid male sterility, and accordingly, shows no evidence for recent introgression across a species hybrid zone (Coyne et al. 2004; Llopart et al. 2005). In mammals, reduced introgression of Y-linked loci (relative to autosomal loci) has been shown across natural hybrid zones of mice (Tucker et al. 1992) and rabbits (Geraldes et al. 2008), suggesting that the Y chromosome contributes to reproductive barriers.Here I examine the genetic basis of hybrid male sterility between two species of Drosophila, D. virilis and D. americana. These species show considerable genetic divergence (K_s ∼0.11, Morales-Hojas et al. 2008) and are currently allopatric: D. virilis is a human commensal worldwide with natural populations in Asia, and D. americana is found in riparian habitats throughout much of North America (Throckmorton 1982; McAllister 2002). Nearly 70 years ago, Patterson et al. (1942) showed that incompatibilities between the D. americana Y chromosome and the second and fifth chromosomes from D. virilis cause hybrid male sterility, a result that was confirmed in a more recent study (Lamnissou et al. 1996). Another study suggested that the X chromosome might play the predominant role in causing hybrid male sterility between D. virilis and D. americana (Orr and Coyne 1989). But because previous genetic analyses had to rely on only a few visible markers to map hybrid male sterility, they lacked the resolution to examine the genomic distribution of incompatibility loci.Using the D. virilis genome sequence, I have developed a dense set of molecular markers to investigate the genetic architecture of hybrid male sterility between D. virilis and D. americana. In this study, I perform a comprehensive set of crosses to address several key questions: What is the effect of the X chromosome on hybrid male sterility between D. virilis and D. americana? What is the effect of the Y chromosome? Approximately how many loci contribute to hybrid male sterility between these Drosophila species? Perhaps surprisingly, the answers to these questions differ dramatically from what has been found for other Drosophila species, including the well-studied D. melanogaster group.     

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C⁴- and C⁷-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C³-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.     

Genetic Control of Photoperiod Sensitivity in Maize Revealed by Joint Multiple Population Analysis

Variation in maize for response to photoperiod is related to geographical adaptation in the species. Maize possesses homologs of many genes identified as regulators of flowering time in other species, but their relation to the natural variation for photoperiod response in maize is unknown. Candidate gene sequences were mapped in four populations created by crossing two temperate inbred lines to two photoperiod-sensitive tropical inbreds. Whole-genome scans were conducted by high-density genotyping of the populations, which were phenotyped over 3 years in both short- and long-day environments. Joint multiple population analysis identified genomic regions controlling photoperiod responses in flowering time, plant height, and total leaf number. Four key genome regions controlling photoperiod response across populations were identified, referred to as ZmPR1–4. Functional allelic differences within these regions among phenotypically similar founders suggest distinct evolutionary trajectories for photoperiod adaptation in maize. These regions encompass candidate genes CCA/LHY, CONZ1, CRY2, ELF4, GHD7, VGT1, HY1/SE5, TOC1/PRR7/PPD-1, PIF3, ZCN8, and ZCN19.MAIZE (Zea mays L. subsp. mays) was domesticated in southern Mexico and its center of diversity is in tropical Latin America (Goodman 1999; Matsuoka et al. 2002), where precipitation rates and day lengths cycle annually. The presumed ancestor of maize, teosinte (Zea mays L. subsp. parviglumis), likely evolved photoperiod sensitivity to synchronize its reproductive phases to the wetter, short-day growing season (Ribaut et al. 1996; Campos et al. 2006). A critical event in the postdomestication evolution of maize was its spread from tropical to temperate regions of the Americas (Goodman 1988), requiring adaptation to longer day lengths. The result of this adaptation process is manifested today as a major genetic differentiation between temperate and tropical maize (Liu et al. 2003) and substantially reduced photoperiod sensitivity of temperate maize (Gouesnard et al. 2002). Tropical maize exhibits delayed flowering time, increased plant height, and a greater total leaf number when grown in temperate latitudes with daily dark periods <11 hr (Allison and Daynard 1979; Warrington and Kanemasu 1983a,b). Identifying the genes underlying maize photoperiod sensitivity will provide insight into the postdomestication evolution of maize and may reduce barriers to the use of diverse tropical germplasm resources for improving temperate maize production (Holland and Goodman 1995; Liu et al. 2003; Ducrocq et al. 2009).Natural variation at key genes in flowering time pathways is related to adaptation and evolution of diverse plant species (Caicedo et al. 2004; Shindo et al. 2005; Turner et al. 2005; Cockram et al. 2007; Izawa 2007; Slotte et al. 2007). Identification of some of the genes controlling adaptation in numerous plant species relied on regulatory pathways elucidated in Arabidopsis (Simpson and Dean 2002). Many key genes in the Arabidopsis flowering time regulatory pathways are conserved across diverse plant species (Kojima et al. 2002; Hecht et al. 2007; Kwak et al. 2008), but their functions have diverged, resulting in unique regulatory pathways in some phylogenetic groups (Colasanti and Coneva 2009). For example, FRI and FLC control most natural variation for vernalization response in Arabidopsis (Caicedo et al. 2004; Shindo et al. 2005), but wheat and barley appear to lack homologs of these genes and regulate vernalization response with different genes (Yan et al. 2004).Maize exhibits tremendous natural variation for flowering time (Gouesnard et al. 2002; Camus-Kulandaivelu et al. 2006), for which numerous QTL have been identified (Chardon et al. 2004). In contrast, only a few flowering time mutants are known and only a handful of flowering time genes, including DWARF8 (D8), DELAYED FLOWERING1 (DLF1), VEGETATIVE TO GENERATIVE TRANSITION1 (VGT1), and INDETERMINATE GROWTH1 (ID1), have been cloned in maize (Thornsberry et al. 2001; Colasanti et al. 2006; Muszynski et al. 2006; Salvi et al. 2007; Colasanti and Coneva 2009). Variation at or near D8 and VGT1 is related to latitudinal adaptation, but these genes do not appear to regulate photoperiod responses and account for only a limited proportion of the standing flowering time variation in maize (Camus-Kulandaivelu et al. 2006, 2008; Ducrocq et al. 2008; Buckler et al. 2009).Quantitative trait loci (QTL) mapping was a key first step to identifying the genes underlying natural variation for flowering time in Arabidopsis (Koornneef et al. 2004). Photoperiodic QTL have been mapped previously in individual biparental maize mapping populations (Koester et al. 1993; Moutiq et al. 2002; Wang et al. 2008; Ducrocq et al. 2009). Such studies are informative with respect to the parents from which the populations were derived, but often do not reflect the genetic heterogeneity of broader genetic reference populations (Holland 2007).Association mapping (Thornsberry et al. 2001; Ersoz et al. 2007) and combined analysis of multiple biparental crosses (Rebaï et al. 1997; Rebaï and Goffinet 2000; Blanc et al. 2006; Verhoeven et al. 2006; Yu et al. 2008) represent alternative approaches to understanding the variation in genetic control for complex traits among diverse germplasm. Association mapping has limited power to identify genes that affect traits closely associated with population structure, such as flowering time in maize (Camus-Kulandaivelu et al. 2006; Ersoz et al. 2007). In contrast, joint QTL analysis of multiple populations is not hindered by the associations between causal genes and population structure. Combined QTL analysis of multiple mapping populations provides improved power to detect QTL, more precise estimation of their effects and positions, and better understanding of their functional allelic variation and distribution across more diverse germplasm compared to single-population mapping (Rebaï et al. 1997; Wu and Jannink 2004; Jourjon et al. 2005; Blanc et al. 2006; Verhoeven et al. 2006; Yu et al. 2008; Buckler et al. 2009). Joint analysis also provides a direct test of the importance of higher-order epistatic interactions between founder alleles at individual loci with genetic backgrounds (Jannink and Jansen 2001; Blanc et al. 2006). In this study, joint analysis of multiple populations was used to test directly the hypothesis that diverse tropical maize lines carry functionally similar alleles at key photoperiod loci, which would imply genetic homogeneity for a common set of mutations and a shared evolutionary pathway for photoperiod insensitivity.The objective of this study was to integrate candidate gene analyses with photoperiod QTL mapping across multiple maize populations. We tested candidate floral regulators known from other species for associations with natural variation for photoperiod response in maize. We analyzed flowering time in four interrelated recombinant inbred line (RIL) populations, each derived from crosses between temperate and tropical maize parents (Figure 1), in both long- and short-day environments to characterize their responses to distinct photoperiods. Joint population analysis provided high resolution of many QTL positions, permitting robust testing of underlying candidate genes. We directly and indirectly mapped homologs of flowering time candidates genes from Arabidopsis, rice, and barley on a dense consensus genetic map of these four populations, permitting identification of homologs that colocalize with genome regions associated with variation for photoperiod response. These mapping families are being integrated into the maize nested association mapping (NAM) population (Buckler et al. 2009; McMullen et al. 2009) because they were genotyped with the maize NAM map SNP markers, they involve the common parent B73, and their seed and genotypic information (File S1 cont.) are publicly available. Their availability further expands the genetic diversity represented by the maize NAM population and enhances this valuable public community resource.Open in a separate windowFigure 1.—Factorial mating of two temperate (B73 and B97) and two tropical (CML254 and Ki14) inbred maize lines to create four related recombinant inbred line mapping populations.     

Anecdotal,Historical and Critical Commentaries on Genetics: Personal Reflections on the Origins and Emergence of Recombinant DNA Technology

The ability to identify genetic markers in nonmodel systems has allowed geneticists to construct linkage maps for a diversity of species, and the sex-determining locus is often among the first to be mapped. Sex determination is an important area of study in developmental and evolutionary biology, as well as ecology. Its importance for organisms might suggest that sex determination is highly conserved. However, genetic studies have shown that sex determination mechanisms, and the genes involved, are surprisingly labile. We review studies using genetic mapping and phylogenetic inferences, which can help reveal evolutionary pattern within this lability and potentially identify the changes that have occurred among different sex determination systems. We define some of the terminology, particularly where confusion arises in writing about such a diverse range of organisms, and highlight some major differences between plants and animals, and some important similarities. We stress the importance of studying taxa suitable for testing hypotheses, and the need for phylogenetic studies directed to taxa where the patterns of changes can be most reliably inferred, if the ultimate goal of testing hypotheses regarding the selective forces that have led to changes in such an essential trait is to become feasible.THE ever-increasing accessibility of genetic markers is allowing sex-determining regions to be genetically mapped in a growing number of nonmodel organisms. There are several reasons for studying sex determination. In animals, gonadal differences are often accompanied by striking somatic secondary sexual dimorphisms, which are interesting in an evolutionary context (Shine 1989; Badyaev 2002). In plants, females and males often differ in flower morphology and abundance (Dawson and Geber 1998), and, although sex differences are often minor outside the flowers (or inflorescences), they do exist (Dawson and Geber 1998; Eppley and Wenk 2001). The genetic control of these phenotypes is a fundamental biological process, and studying sex determination pathways is important in animal developmental biology (Adams and McLaren 2002; Pinyopich et al. 2003), including genetic pathway evolution (Wilkins 1995; Williams and Carroll 2009).Until recently, sex determination was generally studied by testing for genetic control vs. partial or complete environmental influences. Genetic systems were examined cytologically to determine the level of heteromorphism between the sex chromosomes and to identify whether females or males are heterogametic (see the comprehensive review in Bull 1983). Male heterogametic systems, referred to as XY, were also tested to identify whether the Y chromosome carries a male-determining gene, as in almost all therian mammals and most dioecious plants so far studied, or whether sex is determined through X–autosome balance, as in Drosophila and Caenorhabditis elegans (Haag 2005). For female heterogametic (ZW) species, analogous tests were used to identify how femaleness is determined.Until recently, sex-determining genes and regions could be genetically mapped in only a few model species, but now that molecular genetic markers can be developed in nonmodel species, new information is becoming available about how genetic sex determination (GSD) mechanisms have changed during evolutionary history. It has long been known from genetic mapping in model systems, including mammals, and (more recently) birds, that sex chromosomes often have large nonrecombining regions (Bull 1983; Charlesworth 1991; Charlesworth et al. 2005). However, in other organisms, nonrecombining regions are not always large and may sometimes be absent. The evolution of sexual reproduction and recombination have been the focus of many years of discussion in evolutionary biology (Otto 2009), and studies of sex chromosomes are important for understanding why recombination is often lost, and elucidating the evolutionary consequences of recombination suppression (Charlesworth 1996; Otto and Barton 1997; Barton and Charlesworth 1998). The adaptation of genes on the sex chromosomes is also interesting, because this location affects the outcome of sex-specific selection pressures (Rice 1984; Charlesworth et al. 1987; Vicoso and Charlesworth 2006; Mank 2009a). Finally, the mechanism of sex determination can affect sex ratios (West and Sheldon 2002; Dorken and Pannell 2008; West 2009) and is therefore significant in evolutionary ecology.Sex determination is also relevant in applied biology. In many domesticated animals, one sex may be of greatest economic interest to farmers and breeders. Modern meat production is largely based on males, including industrial production of chicken, cattle, and many fish, whereas females are the sex required for milk (cattle) and egg (chicken) production. Similarly, a few crop plants are dioecious, and, in some of these, the crop is produced by females (e.g., grapes, dates, and papaya), while in other species the sexes differ in characteristics such as fiber or chemical content. Because immature birds, fish, and plants have no obvious phenotypic sex differences, maximizing agricultural returns often requires genetically sexing juveniles. Mapping sex determination is an important first step toward identifying the sex-determining genes or finding other sex-specific markers to develop molecular sexing methods.In this review, we first summarize recent developments in genetic mapping of sex determination, concentrating on nonmodel plants and animals with genetic sex determination. We show how this information can be useful for understanding the evolution of sex determination and sex chromosomes and identify some important unanswered questions.     

Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to ∼10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.HORIZONTAL gene transfer (HGT) contributes to bacterial evolution by providing access to DNA evolved and retained in separate species or strains (Cohan 1994a,b; Bergstrom et al. 2000; Ochman et al. 2000; Feil et al. 2001; Koonin 2003; Lawrence and Hendrickson 2003; Fraser et al. 2007). Multilocus sequence typing (MLST) has provided strong evidence for frequent transfer and recombination of chromosomal DNA between related bacterial strains within the same species (Maiden et al. 1998; Enright et al. 2002). HGT occurring by natural transformation allows bacteria to exploit the presence of nucleic acids in their environment for the purposes of nutrition, DNA repair, reacquisition of lost genes, and/or acquisition of novel genetic diversity (Redfield 1993; Mehr and Seifert 1998; Dubnau 1999; Claverys et al. 2000; Szöllösi et al. 2006; Johnsen et al. 2009). It can be inferred from observations of the presence of extracellular DNA in most environments that bacteria are constantly exposed to DNA from a variety of sources, without such exposure necessarily producing observable changes in the genetic compositions of bacterial populations over evolutionary time (Thomas and Nielsen 2005; Nielsen et al. 2007a,b).The absence of sequence similarity between the donor DNA and the DNA of the recipient bacterium is the strongest barrier to the horizontal acquisition of chromosomal genes in bacteria (Matic et al. 1996; Vulic et al. 1997; Majewski 2001; Townsend et al. 2003) as illegitimate recombination occurs only at extremely low frequencies in bacteria (Hülter and Wackernagel 2008a). Single-locus transfer models have been extensively applied and have demonstrated a log-linear decrease in recombination frequencies with increasing sequence divergence for Bacillus subtilis (Roberts and Cohan, 1993; Zawadzki et al. 1995), Acinetobacter baylyi (Young and Ornston 2001), Escherichia coli (Shen and Huang 1986; Vulic et al. 1997), and Streptococcus pneumoniae (Majewski et al. 2000). For instance, heterogamic transformation between nonmutator isolates at the rpoB locus of B. mojavensis is undetectable at sequence divergences >16.7% (Zawadzki et al. 1995) and between S. pneumoniae isolates with sequence divergences >18% (Majewski et al. 2000). In A. baylyi, the nonmutator sequence divergence limit for detectable transformation at the pcaH locus of strain ADP1 was found to be 20% (Young and Ornston 2001), and up to 24% overall divergence yielded transformants at 16S rRNA loci in strain DSM587 (Strätz et al. 1996).Several recent studies also show that short stretches (<200 bp) of DNA sequence identity can facilitate additive or substitutive integration of longer stretches (>1000 bp) of heterologous DNA in bacteria (Prudhomme et al. 1991, 2002; de Vries and Wackernagel 2002; Hülter and Wackernagel 2008a). Thus, the uptake of DNA in bacteria can facilitate larger substitutions within gene sequences and the integration of additional DNA material on the basis of recombination initiated in flanking DNA stretches (either at one or both ends) with high sequence similarity (Nielsen et al. 2000). On the other hand, segments of heterologous DNA interrupting the synteny of homologous DNA have also been shown to be a barrier in intraspecies transformation in S. pneumoniae (Pasta and Sicard 1996, 1999).The various studies of the interspecies transfer potential of single genes demonstrate that the immediate local sequence divergence of the transferred locus is of high importance in determining recombination frequencies in hosts up to 20% divergent (at the housekeeping gene level). However, it can be hypothesized that the broader structural, organizational, and biochemical properties of the genome region surrounding a particular locus will determine its transfer potential to more divergent host species (Cohan 2001; Lawrence 2002). The interspecies transfer potential of various genome regions/loci between more diverged species (>20% at the housekeeping gene level) may therefore differ substantially from a log-linear model (determined experimentally for more closely related species) as local gene organization becomes less conserved with evolutionary time. The barriers to gene exchange between divergent bacterial species is likely a combination of inefficient recombination due to both mismatched base pairs (the main determinator in the log-linear model) and conflicting gene order and organization across the local recombining DNA regions. In addition, selective barriers due to negative effects on host fitness of the transferred DNA regions may become increasingly important for the removal of recombination events from the bacterial population. Recent bioinformatics-based genome analysis of E. coli and Salmonella genomes suggests various parts of the bacterial genome may have different suceptibilities to undergo evolutionarily successful recombination leading to temporal fragmentation of speciation (Lawrence 2002; Retchless and Lawrence 2007). Nevertheless, few studies have experimentally tested the effect of variable species and chromosome locations of genes on their transfer potential between bacteria (Ravin and Chen 1967; Ravin and Chakrabarti 1975; Siddiqui and Goldberg 1975; Cohan et al. 1991; Huang et al. 1991; Fall et al. 2007).Here, we determine to what extent genome location contributes to sexual isolation between the recipient A. baylyi strain ADP1 and 19 sequence divergent (24–27% divergent at the mutS/trpE loci) donor Acinetobacter strains and species (carrying a selectable nptI gene in a total of 95 random genome locations).     

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.     

Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.POLYPLOIDY is common among plants (Masterson 1994; Levin 2002) and is an important aspect of plant evolution. Widespread paleopolyploidy in flowering plant lineages suggests that ancient polyploidization events have contributed to the radiation of angiosperms (Soltis et al. 2009; Van de Peer et al. 2009a). Whole genome duplications are thought to be the sources of evolutionary novelty (Osborn et al. 2003; Freeling and Thomas 2006; Chen 2007; Hegarty and Hiscock 2008; Flagel and Wendel 2009; Leitch and Leitch 2008). Other attributes of polyploids considered to promote evolutionary success include increased vigor, masking of recessive alleles, and reproductive barriers arising from loss of one of the duplicate genes (Soltis and Soltis 2000; Comai 2005; Otto 2007; Van de Peer et al. 2009b). Among crop species, polyploidy likely contributed to trait improvement under artificial selection (Paterson 2005; Udall and Wendell 2006; Dubcovsky and Dvorak 2007; Hovav et al. 2008).Disomic inheritance in polyploids, in contrast to polysomic inheritance, presents opportunities for duplicated genes to diverge and evolve new functions. The relative age of whole genome duplications and the extent of homology between subgenomes greatly influence chromosomal pairing at meiosis (Soltis and Soltis 1995; Wolfe 2001; Ramsey and Schemske 2002). Polysomic inheritance resulting from random chromosome pairing is associated with doubling of a single set of chromosomes. Disomic inheritance resulting from preferential pairing is often associated with polyploidy arising from combinations of divergent genomes. The evolutionary process of diploidization leads to a shift from random to preferential pairing that is not well understood but is genetically defined in systems such as Ph1 of wheat (Triticum aestivum) and PrBn of Brassica napus (Riley and Chapman 1958; Vega and Feldman 1998; Jenczewski et al. 2003). The degree of preferential pairing also affects allelic diversity and the ability to detect linkage. Accurate information about chromosome pairing and whole or partial genome duplications is thus important for both evolutionary studies and in linkage analysis.Such information is extremely limited in the C4 panicoid species Panicum virgatum (switchgrass), which is now viewed as a promising energy crop in the United States and Europe (Lewandowski et al. 2003; McLaughlin and Kszos 2005) and is planted extensively for forage and soil conservation (Vogel and Jung 2001). Little is known about either its genome structure or inheritance. Much current bioenergy feedstock development is focused on tetraploid cytotypes (2n = 4x = 36) due to their higher yield potentials, and an initial segregation study indicated a high degree of preferential pairing in a single F1 mapping population (Missaoui et al. 2005). A once-dominant component of the tallgrass prairie in North America, switchgrass is largely self-incompatible (Martinez-Reyna and Vogel 2002) with predominantly tetraploid or octoploid cytotypes (Hultquist et al. 1997; Lu et al. 1998). Limited gene flow appears possible between different cytotypes suggested by DNA content variation within collection sites and seed lots (Nielsen 1944; Hultquist et al. 1997; Narasimhamoorthy et al. 2008). True diploids appear to be rare (Nielsen 1944; Young et al. 2010). Multivalents in meiosis have not been observed in tetraploids or F1 hybrids between upland and lowland tetraploids, although rare univalents occurred (Barnett and Carver 1967; Martinez-Reyna et al. 2001). However, polysomic inheritance may occur with random bivalent pairing (Howard and Swaminathan 1953).Sustainable production of switchgrass for bioenergy to meet the goal of reducing greenhouse gas emissions will require advances in feedstock production that include improvements in yield (Carroll and Somerville 2009). Switchgrass has extensive genetic diversity and potential for genetic improvements, but each cycle of phenotypic selection can take several years (McLaughlin and Kszos 2005; Parrish and Fike 2005; Bouton 2007). Detailed understanding of genome structure to enable efficient marker-assisted selection (MAS) can speed this process considerably. Complete linkage maps are therefore required to both understand chromosome pairing and allow MAS.We report the construction of the first complete linkage maps of two switchgrass genotypes. The linkage maps provide genetic evidence for disomic inheritance in lowland, tetraploid switchgrass. Gene-derived markers enabled a comparative analysis to sorghum, revealing syntenic relationships between the diploid sorghum genome and the tetraploid switchgrass subgenomes. Transmission ratio distortion and multilocus interactions were analyzed in detail to document their potential influence on map accuracy and map-based studies in switchgrass.     

l-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation

l-Serine is required to synthesize membrane lipids such as phosphatidylserine and sphingolipids. Nevertheless, it remains largely unknown how a diminished capacity to synthesize l-serine affects lipid homeostasis in cells and tissues. Here, we show that deprivation of external l-serine leads to the generation of 1-deoxysphingolipids (doxSLs), including 1-deoxysphinganine, in mouse embryonic fibroblasts (KO-MEFs) lacking d-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step in the de novo synthesis of l-serine. A novel mass spectrometry-based lipidomic approach demonstrated that 1-deoxydihydroceramide was the most abundant species of doxSLs accumulated in l-serine-deprived KO-MEFs. Among normal sphingolipid species in KO-MEFs, levels of sphinganine, dihydroceramide, ceramide, and hexosylceramide were significantly reduced after deprivation of external l-serine, whereas those of sphingomyelin, sphingosine, and sphingosine 1-phosphate were retained. The synthesis of doxSLs was suppressed by supplementing the culture medium with l-serine but was potentiated by increasing the ratio of l-alanine to l-serine in the medium. Unlike with l-serine, depriving cells of external l-leucine did not promote the occurrence of doxSLs. Consistent with results obtained from KO-MEFs, brain-specific deletion of Phgdh in mice also resulted in accumulation of doxSLs in the brain. Furthermore, l-serine-deprived KO-MEFs exhibited increased formation of cytosolic lipid bodies containing doxSLs and other sphingolipids. These in vitro and in vivo studies indicate that doxSLs are generated in the presence of a high ratio of l-alanine to l-serine in cells and tissues lacking Phgdh, and de novo synthesis of l-serine is necessary to maintain normal sphingolipid homeostasis when the external supply of this amino acid is limited.