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Concerted evolution refers to the pattern in which copies of multigene families show high intraspecific sequence homogeneity but high interspecific sequence diversity. Sequence homogeneity of these copies depends on relative rates of mutation and recombination, including gene conversion and unequal crossing over, between misaligned copies. The internally repetitive intergenic spacer (IGS) is located between the genes for the 28S and 18S ribosomal RNAs. To identify patterns of recombination and/or homogenization within IGS repeat arrays, and to identify regions of the IGS that are under functional constraint, we analyzed 13 complete IGS sequences from 10 individuals representing four species in the Daphnia pulex complex.
Gene conversion and unequal crossing over between misaligned IGS repeats generates variation in copy number between arrays, as has been observed in previous studies. Moreover, terminal repeats are rarely involved in these events. Despite the occurrence of recombination, orthologous repeats in different species are more similar to one another than are paralogous repeats within species that diverged less than 4 million years ago. Patterns consistent with concerted evolution of these repeats were observed between species that diverged 8-10 million years ago. Sequence homogeneity varies along the IGS; the most homogeneous regions are downstream of the 28S rRNA gene and in the region containing the core promoter. The inadvertent inclusion of interspecific hybrids in our analysis uncovered evidence of both inter- and intrachromosomal recombination in the nonrepetitive regions of the IGS.
Our analysis of variation in ribosomal IGS from Daphnia shows that levels of homogeneity within and between species result from the interaction between rates of recombination and selective constraint. Consequently, different regions of the IGS are on substantially different evolutionary trajectories.
We expect duplicated gene copies to accumulate mutations independently of one another, which results in greater sequence diversity among paralogs than among orthologs. However, in some multigene families (MGF), including ribosomal DNA (rDNA), tandemly arrayed paralogs are more similar to each other than they are to orthologs in closely related species. This pattern is referred to as concerted evolution [1], and Arnheim [2] invoked gene conversion and unequal crossing over between misaligned members of the gene family to explain it. Thus, the degree of sequence homogeneity within a MGF will depend on the relative rate of mutation and recombination between misaligned copies on homologous and nonhomologous chromosomes.
The ubiquity and high degree of interspecific sequence conservation of the genes encoding ribosomal RNA (rRNA) makes them a valuable system for studying MGF evolution. Tandem copies of these coding sequences alternate with the less-conserved intergenic spacer (IGS) and internal transcribed spacer (ITS) to form a complete ribosomal DNA (rDNA) unit. In many species, the IGS is internally repetitive, and contains one or more arrays of repeats with elements that may be involved in transcription regulation (Drosophila [3], Xenopus [4], Arabidopsis [5], rat [6], mouse [7], Acanthamoeba [8]). Furthermore, these elements are involved in chromosomal pairing in Drosophila [9]. The iterative nature of rDNA, the homogeneity of its copies and the regulatory functions played by the IGS suggest that recombination in the form of gene conversion and unequal crossover is frequent, and may be the result of DNA repair mechanisms influenced by protein/DNA interactions within it [10,11].
In a study of IGS repeat array variation in three populations of Daphnia pulex, Crease [12] reported greater similarity between orthologous copies of IGS repeats than between paralogous copies in the same repeat array. Hayworth [13] described similar results in a study of IGS variation in six species of Arabidopsis, although patterns typical of concerted evolution emerged as divergence times between species increased. In a previous study, we [14] reported few differences in IGS array organization and repeat sequences between the closely related species, Daphnia pulicaria and North American Daphnia pulex (D. pulexNA), but we observed clear differences between Daphnia parvula and Daphnia obtusa, which are in a different species complex than D. pulex or D. pulicaria and diverged from them on the order of 50-90 million years ago [15]. However, we also observed differences between an IGS array in European D. pulex (D. pulexE) and an array from D. pulicaria and D. pulexNA, all three of which are members of the D. pulex species complex.
In this study, we focus on evolutionary changes across the IGS, including the repeat arrays, by analyzing complete IGS sequences from representatives of four species in the D. pulex complex: D. pulexE, D. pulexNA, D. pulicaria, and D. tenebrosa (Table 1). Daphnia pulexNA is the dominant Daphnia species in ephemeral ponds that lack fish across North America while D. pulicaria has approximately the same geographic distribution but has invaded permanent lakes that contain fish. Daphnia tenebrosa is an Arctic endemic that lives in permanent ponds and lakes [16], and D. pulexE inhabits ponds in the temperate regions of Europe [17]. The divergence time between D. tenebrosa and D. pulexNA or D. pulicaria (~4-5 million years) is about half the divergence time between D. pulexE and the latter two species (~8-10 million years, [17]). Our objectives are to 1) identify patterns of recombination and/or homogenisation within rDNA repeat arrays, 2) estimate the divergence time at which repeats become more similar within species than between, and 3) identify regions of the IGS that may be experiencing functional constraint.
Table 1. Daphnia individuals included in this study.
IGS sequence variation
We sequenced 13 complete IGS including one from each of three D. pulexNA (DpxNA1, DpxNA2, DpxNA3), three D. pulicaria (Dpc1, Dpc2, Dpc3) and one D. tenebrosa (Dten) individual. We sequenced two complete IGS from each of three D. pulexE individuals (DpxE1a, DpxE1b, DpxE2a, DpxE2b, DpxE3a and DpxE3b). Each individual was sampled from a different population (Table 1). We partitioned each IGS sequence into three regions: N1, the nonrepetitive segment located immediately downstream of the 28S rRNA coding region; R, the repetitive midsection; and N2, the nonrepetitive segment located downstream of the repeat arrays and 5' to the 18S rRNA coding region (Figure 1). The tandem arrays in the R-region were further dissected into repeat types A, B, and C.
Figure 1. Typical rDNA IGS from species in the Daphnia pulex complex. 28S = 28S rRNA gene. 18S = 18S rRNA gene. N1 = nonrepetitive region 1. N2 = nonrepetitive region 2. R = repetitive mid section composed of A (blue), B (green), and C (red) repeats. N2 contains the external transcribed spacer (ETS).
Length variation among the IGS sequences is as high as 20% and can be attributed to the presence of indels in the nonrepetitive regions (N1 and N2), and variable numbers of the three repeat types (A, B, and C) in the repetitive region, which begins between nt 659 (DpxNA3) and nt 797 (Dpc1), downstream of the 28S rRNA coding region (Figure 2, Additional file 1: Structure of complete IGS sequences). The three DpxNA IGS sequences have a 107 nt deletion beginning at nt 493 in the multiple alignment that is not present in any of the other species, and is not limited to these three populations (Crease TJ, unpublished data). Region N2 ranges from 2492 nt (DpxE1b) to 2843 nt (DpxE3a) in length.
Figure 2. Diagram of complete IGS sequences from representatives of four species in the Daphnia pulex complex. Dpc = D. pulicaria, DpxE = European D. pulex, DpxNA = North American D. pulex, Dten = D. tenebrosa. A repeats are represented by rectangles, B repeats by ovals, and C repeats by pentagons. Repeats that cluster with one another in Neighbor-joining dendrograms are indicated by the same color. Open shapes bounded by dashed lines indicate putative deletions. Nonrepetitive regions upstream and downstream of the repeats are shown with thick black lines.
Additional file 1. Structure of complete IGS sequences. PDF file showing features of 13 IGS sequences from 4 species in the Daphnia pulex complex.
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Neighbor-joining (NJ) trees based on the complete condensed (see methods) IGS, and the N1 and N2 regions (Figures 3, 4 and 5 ) show that the sequences from DpxE3 do form the sister group to the remaining sequences, as expected from phylogenies of the D. pulex complex based on mtDNA [17]. However, the DpxE1a/b and DpxE2a/b sequences do not cluster with them in any of these trees. In addition, Dten is not the sister group to a DpxNA+Dpc cluster, as expected based on the mtDNA phylogeny [17]. Indeed, the only consistent features of the three trees is the sister group relationship of the DpxE3a/b sequences to all the others, and the occurrence of two groups consisting of [Dpc1+DpxE2a/b] and [Dten+DpxE1a/b].
Figure 3. Unrooted Neighbour-joining dendrogram of complete IGS sequences from representatives of four species in the Daphnia pulex complex. The inset shows a phylogeny of these species based on the mitochondrial ND5 gene [17]. Dpc = D. pulicaria, DpxE = European D. pulex, DpxNA = North American D. pulex, Dten = D. tenebrosa.
Figure 4. Unrooted Neighbour-joining dendrogram of the IGS N1 region from representatives of four species in the Daphnia pulex complex. Dpc = D. pulicaria, DpxE = European D. pulex, DpxNA = North American D. pulex, Dten = D. tenebrosa.
Figure 5. Unrooted Neighbour-joining dendrogram of the IGS N2 region from representatives of four species in the Daphnia pulex complex. Dpc = D. pulicaria, DpxE = European D. pulex, DpxNA = North American D. pulex, Dten = D. tenebrosa.
Mean sequence divergence (p-distance) in the complete condensed IGS and nonrepetitive regions is slightly higher between species than within them, with the highest values occurring in the N1 region (Table 2). Mean values of intra- and interspecific sequence divergence in the N1 and N2 regions are similar to those obtained for the complete IGS. The AMOVA results show that over half of the variation in complete IGS and N1 sequences occurs among species, but all the variation in N2 sequences occurs within species, although this value is not significant (Table 3). This result is consistent with the fact that N2 sequences show less clustering by species (Figure 5) than do the full IGS or N1 sequences (Figure 3, 4). For example, intraspecific sequence divergence is always lowest in DpxNA (Table 2), but all three sequences from this species do not cluster together in the NJ tree based on N2 sequences (Figure 5).
Table 2. Pairwise sequence divergence (PSD) between IGS sequences from ten individuals representing four species in the Daphnia pulex complex.
Table 3. Analysis of molecular variance in the rDNA IGS from ten individuals representing four species in the Daphnia pulex complex.
In the repetitive region of the IGS, one to five copies of the A repeat, ranging from 184 to 222 nt in length, are interleaved with B repeats (Additional file 1, Figure 2). All sequence variation among A repeats is within individuals (Table 3). With the exception of two A repeats from DpxE3b and an anomalous Dpc2 A repeat, there is a tendency for repeats to cluster according to their position in the array (Figure 6). Mean sequence divergence between A repeats within clusters based on the NJ tree is 0.04 while that between sequences from different clusters is substantially higher at 0.125 (Table 4).
Figure 6. Unrooted Neighbour-joining dendrogram of IGS A repeats from representatives of four species in the Daphnia pulex complex. Dpc = D. pulicaria, DpxE = European D. pulex, DpxNA = North American D. pulex, Dten = D. tenebrosa.
Table 4. Sequence divergence (p-distance) among IGS repeats in ten individuals representing four species in the Daphnia pulex species complex.
The number of B repeats per IGS ranges from two to six (Figure 2, Additional file 1). As with A repeats, all of the sequence variation is found within individuals (Table 3). Mean sequence divergence between sequences within the clusters based on the NJ tree (Figure 7) is 0.027 while that between sequences from different clusters is 0.168 (Table 4).
Figure 7. Unrooted Neighbor-joining dendrogram of IGS B repeats from representatives of four species in the Daphnia pulex complex. Dpc = D. pulicaria, DpxE = European D. pulex, DpxNA = North American D. pulex, Dten = D. tenebrosa.
C repeats occur as two tandem copies in all but the two DpxE3 IGS sequences, which each contain a single C repeat (Figure 2). All sequence variation among C repeats is within individuals (Table 3) and repeats cluster by position (Figure 8). The single C repeat in DpxE3a groups with repeats in the second position, while the single C repeat in DpxE3b groups with those in the first position (Figure 8). Mean divergence between sequences within the clusters based on the NJ tree is 0.013 while that between sequences from different clusters is 0.057 (Table 4).
Figure 8. Unrooted Neighbour-joining dendrogram of IGS C repeats from representatives of four species in the Daphnia pulex complex. Dpc = D. pulicaria, DpxE = D. pulex Europe, DpxNA = D. pulex North America, Dten = D. tenebrosa.
Although analysis of the complete condensed IGS using GARD partitioned it into five hypothetical nonrecombinant sections, the server's execution time limit per job was reached before the analysis was completed. Further analysis of N1 identified five hypothetical nonrecombinant sections, and although four such sections were identified in N2 (Table 5), the server once again timed out before the analysis of the alignment was complete. In the complete IGS, the first putative recombination breakpoint falls immediately upstream of the repeat array and the second is located within the A repeat consensus sequence, just upstream from the spacer promoter [18,19]. The position of the last two breakpoints coincides with two of the three breakpoints identified in the separate analysis of the N2 region. The third IGS breakpoint is about 150 nt downstream of the core promoter, and the fourth IGS breakpoint is located in the vicinity of a (GT)n microsatellite, a few hundred nt upstream of the 18S rRNA coding region.
Table 5. Pairwise sequence divergence (p-dist) among fragments within each section of the IGS from ten individuals representing four species in the Daphnia pulex complex.
Mean inter- and intraspecific p-distances differ substantially among the regions identified by the GARD analysis (Table 5). The regions with the least variation are located just downstream of the 28S rRNA coding region (N1-1 and N1-2) while the regions with the highest variation are just downstream of that (N1-3) and in the A repeat consensus (IGS-2) (Table 5, Figure 9).
Figure 9. Mean pairwise p-distance between IGS sequences from representatives of four species in the Daphnia pulex complex. (a) The complete IGS. (b) Nonrepetitive regions, N1 and N2. Values are plotted for segments defined by recombination analysis of multiple sequence alignments using the program, GARD [44]. Segment 2 in the IGS corresponds to the R region, which contains the repeat arrays. Vertical black lines are standard errors of the overall mean p-distance (intraspecific and interspecific).
Analysis of complete IGS sequences using GENECONV identified 12 significant putative gene conversion tracts between ancestors of two sequences in the alignment. All 12 fragments are located within the N2 region, and range in length between 106 and 1620 nt (Additional file 2: Gene conversion analysis of complete IGS sequences). Three of these conversion tracts, including the longest one, are between IGS sequences from the same individual (DpxE1a × DpxE1b and DpxE3a × DpxE3b). Three other tracts are between sequences from different individuals of the same species (DpxE1b × DpxE2a; DpxE1b × DpxE2b; DpxE1a × DpxE3a). The six remaining exchanges involve interspecific gene conversion events; four tracts involve Dpc2: two with Dten and one each with DpxE1b and DpxE1a. Two tracts involve DpxE2b; one with Dten and one with DpxNA3 (Additional file 2). A separate analysis of the N1 region identified only two gene conversion tracts, both of which cover the same region, between DpxE3a and each of two DpxNA individuals (Additional file 3: Gene conversion analysis of the IGS N1 region.). In a similar analysis of N2 (Additional file 4: Gene conversion analysis of the IGS N2 region), four of the seven gene conversion tracts are interspecific, involving Dten with Dpc2 (2 tracts), DpxE3a and DpxE2b.
Additional file 2. Gene conversion analysis of complete IGS sequences. PDF file showing results of a gene conversion analysis of complete IGS sequences from 4 species in the Daphnia pulex complex using GENECONV.
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Additional file 3. Gene conversion analysis of the IGS N1 region. PDF file showing results of a gene conversion analysis of IGS N1 sequences from 4 species in the Daphnia pulex complex using GENECONV.
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Additional file 4. Gene conversion analysis of the IGS N2 region. PDF file showing results of a gene conversion analysis of IGS N2 sequences from 4 species in the Daphnia pulex complex using GENECONV.
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There is little statistical support for putative recombination breakpoints within the aligned consensus repeat sequences using the GARD algorithm. However, the GENECONV algorithm identified seven putative gene conversion tracts in the A repeat alignment and nine in the B repeat alignment (Additional files 5: Gene conversion analysis of the IGS A repeat region, Additional file 6: Gene conversion analysis of the IGS B repeat region). All putative gene conversion tracts in the B repeat alignment were between the fourth copy of the B repeat from DpxE3b and the second or third copies of B repeats from all other species. Two putative gene conversion tracts were identified between the C repeat from DpxE3a and the second C repeat copies from Dpc2 and DpxNA3 (Additional file 7: Gene conversion analysis of the IGS C repeat region).
Additional file 5. Gene conversion analysis of the IGS A repeat region. PDF file showing results of a gene conversion analysis of IGS A repeat region from 4 species in the Daphnia pulex complex using GENECONV.
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Additional file 6. Gene conversion analysis of the IGS B repeat region. PDF file showing results of a gene conversion analysis of IGS B repeat region from 4 species in the Daphnia pulex complex using GENECONV.
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Additional file 7. Gene conversion analysis of the IGS C repeat region. PDF file showing results of a gene conversion analysis of IGS C repeat region from 4 species in the Daphnia pulex complex using GENECONV.
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We found inconsistencies between the topology of NJ trees based on IGS sequences and the species phylogenies based on mtDNA sequences. The most plausible explanation for these differences is the introduction of allospecific nuclear DNA through hybridization between D. pulexE and both D. tenebrosa and D. pulicaria. The NJ tree of complete IGS sequences reveals that only the two DpxE3 sequences form a separate branch as predicted by the divergent mtDNA sequence of this individual. The IGS sequences of the other two D. pulexE individuals cluster with those from D. pulicaria or D. tenebrosa. While hybridization between D. pulexE and D. pulicaria or D. tenebrosa has not been documented in the literature, the divergence among their mitochondrial 12S rDNA sequences falls well below the 14% threshold for species which are known to hybridize [[15] and references within]. Colbourne and Hebert [15] note that the lack of evidence for hybridization between species with low levels of 12S rDNA sequence divergence involves taxa with allopatric distributions, which is generally thought to be the case for D. pulexE relative to the other species. However, D. pulexNA and D. pulicaria have been found in Europe [16,20] so opportunities for hybridization do exist.
The phylogenetic relationship among the four Daphnia species in this study, based on mtDNA sequences, is most closely reflected in the relationship among N1 sequences. We observed tight clustering of the three N1 sequences from D. pulexNA, while the N1 sequences of the three D. pulicaria individuals form a looser grouping. However, evidence for introgression is seen in four of the six DpxE N1 sequences. N1 sequences from DpxE1 cluster with the Dten N1 sequence, and N1 sequences from DpxE2 cluster with the Dpc N1 sequences.
The tree topologies of A and B repeats, which are interleaved with one another, are similar. Aside from the repeat sequences from DpxE3, major clusters are formed by orthologous rather than paralogous repeats for both A and B types. The occurrence of this structure in all but the most divergent species suggests that it has persisted for several million years, despite the occurrence of recombination between repeats (discussed below, [12]). Unfortunately, because all but one of the IGS arrays from D. pulexE appears to have been impacted by hybridization, it is not entirely clear if this position-specific pattern also occurs in this species. However, two observations suggest that it may. First, the only A repeats from different DpxE IGS sequences (DpxE3a-A1 and DpxE3b-A1) that cluster with one another in the NJ tree (Figure 6) are both in the same (first) position. Second, branch lengths between the A repeats in the DpxE3a array are more similar to branch lengths between array positions than within them in the other species (Figure 6).
All but the last of the six B repeats in the DpxE3a array cluster with one another, which is consistent with the pattern observed for A repeats. However, orthologous clustering of terminal F repeats was observed in the IGS of Drosophila melanogaster and Dr. orena [14]. Others have also reported the apparent escape from homogenization experienced by terminal repeats relative to interior paralogs [21-23].
With the exception of the three D. pulexE individuals, for which two complete IGS were sequenced, our data are limited to a single IGS sequence for each individual, and three IGS sequences per species in D. pulexNA and D. pulicaria. This, in combination with the introgression mentioned above, limits the confidence with which we are able to estimate the divergence time necessary for IGS sequences to appear more similar within than between species. However, divergence times between the species in this study, based on the mitochondrial genes [17], suggest that the threshold for detecting patterns consistent with concerted evolution for the complete IGS must be greater than 4 million years.
Recombination in the IGS
We expect the hierarchically iterative nature of rDNA to facilitate recombination and homogenization at this locus. Indeed, our GARD and GENECONV analyses confirm that recombination occurs at multiple locations across the Daphnia IGS, including the repeats although these analyses do not identify recombination hotspots. The GARD algorithm identifies nonrecombinant segments rather than precise recombination break points and adopts the convention that breakpoints coincide with variable sites because breakpoints can only be resolved to the nearest variable site [24]. In fact, actual breakpoints may be located at invariant sites [25].
Although it is possible that some of the intraindividual recombination that we observed is due to template switching during PCR amplification, we used a long extension time and a total of 30 cycles. Thus, it seems unlikely that recombination during the PCR reaction is a substantial source of the variation we observed. Indeed, recombination among IGS repeats has been observed in sequences from D. pulex obtained by cloning directly from genomic DNA [12]. However, the frequency with which such artefacts occur could also be tested empirically by combining cloned divergent IGS sequences and amplifying them under our PCR conditions.
The copy number of A repeats, which contain a putative enhancer motif [18], ranges between one (Dpc1) and five (DpxNA2). Crease [12] reported that 18 of 21 arrays from seven D. pulexNA individuals contained four repeats, while the remaining three contained either five or six. This length variation is strong evidence that unequal crossing over is occurring between misaligned IGS repeats. Despite this, A and B repeats cluster by position in the array rather than species. This pattern was also observed by Luchetti et al. [26] in the IGS arrays of Triops cancriformis, which contain three copies of a ~200 nt repeat. In a previous study, we [14] found that the homogeneity of tandem and interleaved repeats increases as their number increases in arthropod IGS sequences. Thus, the rate of recombination in short arrays may be too low to fully homogenize the repeats. We also observed that duplication and deletion events rarely involve terminal repeats, which is consistent with the results of earlier work in plants. For example, Markos and Baldwin [27] found that interior repeats evolve in concert in Lessingia spp. (Compositae, Astereae), and Baldwin and Markos [28] found that sequence similarity of flanking repeats is higher between orthologs than paralogs in Calycadenia (Asteraceae).
Previous studies have suggested that intrachromosomal exchange (between sister chromatids) is more frequent than interchromosomal exchange (between homologues) in rDNA. For example, Crease [12] showed that intrachromosomal recombination is most likely responsible for patterns of sequence diversity within the IGS repeat arrays of D. pulexNA. Similarly, Schlötterer and Tautz [29] suggested that intrachromosomal exchange mechanisms are the most parsimonious explanation for the homogenization process in the ITS of Drosophila melanogaster. In contrast, our results suggest that many of the putative gene conversion tracts in the nonrepetitive regions of the IGS occurred between, rather than within, species (i.e. between homologous chromosomes in hybrids). This is consistent with the results of Polanco et al. [30] who showed that homogenization of the Drosophila IGS is the result of interchromosomal recombination. Our results do not exclude the possibility that intrachromosomal exchange occurs at an equal or even higher frequency than interchromosomal exchange. However, they do suggest that recombination within the IGS occurs during a phase in the cell cycle when homologous chromosomes are in close proximity, either following S phase during meiosis or when actively transcribed rRNA genes come together to form the nucleolus. Recombination can also occur between rDNA arrays on nonhomologous chromosomes, but D. pulex has only a single rDNA array per haploid genome (D. Tsuchiya, unpublished data). The number of rDNA arrays has not been determined for the other species, but they have similar genome sizes [31] and the same number of chromosomes (n = 12) as D. pulex [32]. Taken together, the above studies corroborate Polanco et al.'s [33] assertion that different regions within the rDNA unit follow different evolutionary trajectories.
Conserved regions within the IGS
The exceptionally low sequence diversity in the first ~350 nt of N1 suggests that it undergoes homogenization along with the 28S rRNA gene. Liao [34] also reported that the homogenization of flanking regions in bacterial rRNA genes was the result of hitchhiking, or co-conversion with genic sequences. Moreover, the mean sequence divergence and the topology of NJ trees differs between N1 and N2, and from the repetitive region that connects them. This may be due to differences in the strength of natural selection acting on regulatory regions within the IGS, as well as the frequency with which recombination occurs between paralogous repeat copies whose sequences predispose them to frequent breakage and repair.
Because concerted evolution reduces mean intraspecific p-distance among the members of a MGF despite interspecific divergence, we would expect the ratio of mean intra- and interspecific p-distance (p-distance ratio) to be less than one and decrease with divergence time. On the other hand, if natural selection is constraining sequence divergence, then mean intra- and interspecific p-distance should be low and similar, especially among closely related taxa such as those included in this study. In this case, the p-distance ratio would remain close to one regardless of divergence time.
Although hybridization has blurred the species boundaries between individuals in this study, a comparison of mean p-distances within and between species does suggest that some of regions of the IGS may be under functional constraint. For example, the most conserved of the four N2 segments delimited by GARD breakpoints (N2-3), with a p-distance ratio of 1.0, is located between the putative core promoter and the breakpoint at nt 3900 in the full IGS, which may be the location of an rRNA processing site [35-37]. In contrast, the region that appears to be under the least functional constraint (N2-4, Table 5) is just downstream of this region and upstream of the 18S rRNA coding region, which is highly conserved both within and between species. This increase in both mean intra- and interspecific p-distance is also evident when mean p-distance is calculated after dividing the IGS into sequential 500 nt sections (data not shown).
As previously noted, the lowest overall sequence diversity occurs at the 3' end of the 28S rRNA coding region (N1-1 and N1-2). In contrast, the highest sequence diversity occurs just downstream of this region, in the middle section of N1 (N1-3), which includes a GAn dinucleotide repeat. The p-distance ratio is relatively low in this region (0.76, Table 5), but the lowest ratio (0.5) occurs in region N1-5, which is separated from N1-3 by the only region in the IGS (N1-4) where mean intraspecific divergence actually exceeds mean interspecific divergence (ratio = 1.27). The explanation for this pattern is unclear, but it should be noted that all of the regions in N1 are relatively short (151 - 181 nt). Further examination of this pattern will require analysis of species that diverged from a common ancestor at least 4 million years ago, and between which hybridization does not occur.
The region of the IGS with the highest mean intraspecific sequence divergence is the repeat region, although the p-distance ratio is also high at 0.94 (Table 5). This high level of diversity is primarily driven by differences between repeats in different positions in the array (Figure 3 and Figure 4). As suggested above, one explanation for this is low rates of recombination. However, it has also been suggested that this pattern may be maintained by natural selection despite the occurrence of recombination [12]. Indeed, the A repeats contain an ~27-nt putative TATA motif, which is highly conserved among all A repeats in this and the previous study [12]. This motif is also be found in the IGS repeats of other arthropods [14] and those containing the motif were found to be significantly more homogeneous than those without it in these taxa. These results suggest that selection is able to maintain homogeneity or diversity among functionally important repeat types regardless of the level of recombination among them [12].
The occurrence of length variation in the IGS repeat array suggests that unequal crossing over occurs in this region, which is consistent with previous work. However, we also found evidence of interchromosomal gene conversion in the nonrepetitive regions of the IGS. Levels of sequence homogeneity vary across the IGS, due to the interaction between rates of recombination and selective constraint. The pattern of position-specific variation in Daphnia IGS repeat arrays likely predates speciation in the D. pulex species complex, and has persisted for millions of years. Although this pattern may be a function of low rates of recombination in short repeat arrays, the occurrence of a highly conserved TATA motif in the A repeats suggests that natural selection may also play a role in the maintenance of this pattern. The unintentional inclusion of hybrids in this study provided insight into evolutionary patterns within the IGS, but analysis of D. pulexE individuals that are not hybrids, as well as other sister species pairs in the genus Daphnia, is required to more precisely estimate the divergence time at which paralogous IGS repeats show evidence of concerted evolution.
Cloning and sequencing the IGS
Complete copies of the IGS were amplified from genomic DNA of Daphnia individuals using the Expand Long Template PCR System (Roche Diagnostics) and primers complimentary to highly conserved regions at the 3' end of the 28S rRNA gene (5' GTTTAGACCGTCGTGAGACAGGTTAG) and the 5' end of the 18S rRNA gene (5' TCAGGCTCCCTCTCCGG). The PCR conditions were as follows: 95°C for 5 min, followed by 10 cycles of 92°C for 30s, 60°C for 30s, and 68°C for 8 minutes. The next 20 cycles were 92°C for 30s, 60°C for 30s, and 68°C for 8 min plus 2s/cycle, ending with a final extension at 68°C for 7 minutes. Depending on the species, the major fragment generated in these amplifications ranged between ~4000 and 5500 nt, in agreement with earlier characterizations of the D. pulex IGS [12].
The PCR fragments were gel purified and cloned using the TOPO XL PCR cloning Kit (Invitrogen) according to manufacturer's recommendations. After screening plasmid clones for inserts of the expected size using gel electrophoresis, the presence of the 5' 18S and 3' 28S rRNA gene termini were confirmed by sequencing plasmid DNA with the M13 Forward and Reverse primers and the ABI Prism TaqFS dye terminator kit (Applied Biosystems). Sequences were resolved on an ABI 377 automated sequencer (Applied Biosystems). The Erase-a-base system (Promega) was used to generate nested deletion subclones from a single plasmid clone from each individual. Overlapping deletion subclones were sequenced and a contig sequence of each insert from each individual was created in Sequencher (Gene Codes). Additional clones from each species, and regions that were not well covered by deletion subclones were sequenced by primer-walking.
IGS sequence analysis
We located the 5' end of the 18S rRNA gene and the 3' end of the 28S rRNA gene in each IGS sequence by consensus alignment with the homologous sequence from D. pulex [GenBank:AF014011] and D. longicephala [GenBank:AF346516] [38]. Repetitive regions in individual IGS sequences were located visually in Dotmatcher [39] using a window size of 25 and a threshold of 40. We estimated IGS repeat boundaries by performing 25 local self-alignments using the default settings in the local similarity program, SIM [40,41]. Finally, we refined putative IGS repeat boundaries by visual inspection of the aligned IGS repeat sequences in Multiple Sequence Alignment Program (MAP) [42] with default settings.
To facilitate alignments between individuals with different numbers and/or organization of A, B and C repeats, we aligned all copies of each repeat type for each IGS sequence and created a consensus repeat sequence. We then condensed the repeat array in each full IGS sequence by replacing it with one copy of the consensus for each repeat type and aligned the condensed IGS sequences using ClustalW [43]. The resulting alignment was adjusted by eye.
To incorporate a conservative estimate of the effects of indels on sequence divergence, we replaced the first position in each gap with a nucleotide that was unique to that position. We used the nucleotide p-distance algorithm in MEGA v3.1 [44] to calculate pairwise sequence divergence between entire condensed IGS sequences, the nonrepetitive regions (N1 and N2) and the repeat sequences. We used pairwise deletion of indels and assumed homogeneous patterns of nucleotide substitution among lineages and uniform mutation rates among sites for these analyses. MEGA was also used to construct Neighbor-joining dendrograms from the nucleotide p-distance matrices.
Statistical analyses
We performed an Analysis of Molecular Variance (AMOVA) using Arlequin 3.0, [45] to partition the genetic variance in condensed IGS sequences within and between species. In addition, variation in each repeat type was partitioned within and between species. Levels of significance were based on 1023 random permutation replicates.
We used the program Genetic Algorithm Recombination Detection (GARD) [46] to look for evidence of recombination within each condensed IGS. In addition, we analyzed sequence alignments of the nonrepetitive N1 and N2 regions separately because the entire condensed IGS sequences caused the program to "time out" before all breakpoints had been identified. Goodness of fit was assessed by small sample Akaike Information Criterion (AIC) derived from a maximum likelihood model fit to each segment. Nucleotide substitution bias models were defined for each alignment using the model selection tool in GARD (general discrete rate variation with four rate classes, and the GARD detection method, which looks for multiple rather than a single breakpoint).
We also used the GENECONV v1.81 computer program [47] to find statistical evidence of putative gene conversion events between the ancestors of two sequences in an alignment. GENECONV identifies global inner fragments that contain evidence of a possible gene conversion event between ancestors of two sequences in the alignment. It also identifies outer fragments, which contain evidence of putative gene conversion events that may have originated from outside of the alignment, or from within the alignment, but with evidence of the source destroyed by later mutation or gene conversion [48].
bp: base pair; ETS: external transcribed spacer; IGS: intergenic spacer; ITS: internal transcribe spacer; MGF: multigene family; N1: nonrepetitive region 1; N2: nonrepetitive region 2; NJ: Neighbor-joining; nt: nucleotide; R: repetitive region; rDNA: ribosomal DNA; rRNA: ribosomal RNA
Authors' contributions
CDA collected the sequence data, performed the statistical analyses and drafted the manuscript. TJC conceived of the study, participated in its design and coordination and helped to draft the manuscript. Both authors read and approved the final manuscript.
Funding for this work was provided by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to TJC. CDA was partially funded by Ontario Graduate Scholarships. We thank staff in the Genomics Facility at the University of Guelph for sequencing the Daphnia IGS clones. D. Irwin, R. Danzmann, E. Clare, R. Floyd and 2 anonymous reviewers provided helpful comments on the manuscript.
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Chapter 2 : Teddy, Tears, and Treacle Tart
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Teddy, Tears, and Treacle Tart
I have the worst luck on the face of the planet.
Nanny made treacle tart for dessert.
I know that I really didn't have to go to live with Great Aunt Petunia if I didn't want to, since only Stan knew about my little promise to myself, but I had given my word. And I've heard it said that anyone is only ever as good as their word- and I don't want to be a bad person! I want very much to be a good person. Because good people go to heaven and get to swim in giant chocolate fountains and jump on giant clouds made out of marshmallows.
So, in order to be a better person, I needed to keep my promise. I had thought that it wouldn't be so bad if I had to go, but as soon as I saw the delicious treat that usually made my whole insides light up with happiness, I started to cry.
I didn't want to go. I wanted to live with my mummy and my daddy and Al- even Jamesie! I wanted to snuggle with Padfoot the crup and walk through Godric's Hollow, singing at the top of my lungs. I liked my life!
But Jamesie's words rang throughout my head over and over again, as if they'd been recorded and put onto one of those muggle gadgets, set to constantly repeat. I was a squib- no better than a muggle. Mummy and daddy were always sad and frowning now, and they hadn't been paying much attention to Al or James. Not that Jamesie needed more attention, really. He was just bitter because he wasn't always the centre of the universe. But I would've gladly given up the spotlight if it meant I could be normal.
But I wasn't. I didn't fit in, and I never would. If I didn't leave now, I'd always be a failure in my parents' eyes. I would always be the Potter girl that never lived up to her parents. There was no way I could ever live with myself, because everyone would just expect me to fail at everything. I would be nothing but one, big disappointment.
But I didn't want to be that. I wanted to be just like everybody else. If I lived like a muggle, no one would know I came from a family of witches and wizards. No one would expect anything great of me- I could be just Lily, instead of the Squib. As much as I hated the idea of leaving home to live with a mean, old lady, that idea sounded very appealing. Maybe I'd finally find someplace where I belong.
"What's wrong, love?" Nanny asked me as I sobbed, looking shocked and a tad disturbed. The tart was in her hands and she looked a little hurt- as if I was rejecting what was usually my favourite treat. This only made me feel guilty, which added to the volume of my tears. I heard James groan from across the table.
"Can't we just eat withou- owwww!"
I didn't look to see who it was that had kicked him under the table, but I guessed it was probably Al.
Big, warm arms wrapped me in a bear hug and I sniffed into Nanny's shoulder as she held me, just like mummy had earlier today.
"I can make you anything you like, dear." She said kindly, planting a kiss on my cheek. "You don't have to have treacle tart if you don't want any."
"What's wrong with her?" Hugo piped up, and I looked at him with a frown. He didn't seem to notice that he was prying into other people's personal business; he sat there, licking the last of the gravy off his spoon, kicking his legs impatiently as he waited for dessert.
"Nothing's wrong with her!" Jamesie said, sounding annoyed. "She's just being a baby!"
"James Sirius!" My mother snapped, her eyes practically spitting fire at my brother. I could tell by her expression that he was sitting on the Naughty Step again when we got home. I stuck my tongue out at him.
"Put that back in your mouth, young lady, or I'll hex it there!" Mummy scolded me, and I sank in my chair. You would've thought she'd be nice to me, since I'm going through such a tough time! But no, she still told me off, and now Jamesie was looking all smug.
"You can have my chocolate frog, Lils." Al jumped into the short silence that followed my reprimand, the rustling of a wrapper audible as he dug in the pocket of his trousers. He pulled out a slightly crushed frog and stretched out over the table. I smiled at him and took it, but not before shooting a glare at Jamesie, whom I could tell had seriously considered snatching it out of Al's hand and eating it first.
"She's had a rough day." My mum explained to the rest of the family, and I could feel my face warming up as they all stared at me. I couldn't blame them, really. After all- what kind of freak just randomly burst into tears at the dinner table?
A squib. That's what kind of freak.
Suddenly, as I chewed the half-melted chocolate, I remembered why it was I'd begun to cry in the first place. But I couldn't cry again, 'cause I'd cried all the tears out of my eyes for that night, so I'd be all out until it rained- because that's when everyone got their tears back, or so Jamesie tells me. Instead, I just felt very tired. But I couldn't let myself be sleepy, because I had to run away, and that would take energy.
"Liiiiily," a whiny voice caught my attention, and I turned my head to see Hugo looking at me imploringly. His mum gave him a warning look, but he ignored it, holding out his hand eagerly. I let out a very long, drawn-out sigh and gave him the rest of my chocolate frog. He grinned at me and shoved it into his mouth all at once- probably worried his mum would tell him to give it back.
The rest of the dinner passed without much excitement. After we ate, everyone had to sit around in the living room, like every Sunday. It was boring, like always, and most of the people just slept on the floor. Hugo and Al were catching fireflies, and I wanted to go, too, but Jamesie was out there. I didn't want to talk to him just yet. I followed Rose around for a little while and asked her to braid my hair for me, but she told me to go away. She was reading a boring book about Hogwarts that was bigger than my whole body, practically, and she didn't want to talk, except to ask her mum for help on the big words (there were lots of them). Dejected, I sat by myself on the floor.
I looked to my left and saw that Teddy was one of those members of the family that had decided to take a nap on the carpet. I had to tell him about my plan- he wouldn't tattle, and he could tell me what to do. I crawled over the floor and climbed up onto his tummy and he let out a small grunt of pain.
"Lils, you're not four anymore!" He said without opening his eyes, and I smiled as I leaned over and pulled a lid up. A bright green eye looked back at me, and I grinned. That was my favourite colour.
"I'm not that heavy." I argued, still not budging. Teddy grinned up at me and raised a heavy hand to pat my head.
"No, but you're getting pretty big!" He said cheerfully. "How old are you, anyway- twenty?"
I giggled. Teddy was always silly like that.
"No, you goose! I'm seven!" I looked at him sternly, as if I was upset he'd forgotten. But, of course, no one could ever be put-out with Teddy. He was just like his name- cuddly and meant for hugging.
"Ahhh, that's right." He said with a nod, voice still strained because my entire body-weight was pushing down on his diaphragm. "So that means you're going to be eight this August, eh? One month, Lil' Lily!"
I nodded, but it wasn't enthusiastic. It was a miserable sort of nod; an acceptance of my fate. Teddy frowned, and I saw his hair change from brown to black.
"What's wrong, silly?" He asked, brushing some of my ginger bangs out of my eyes. "You're not supposed to hate birthdays until you get old like your mum-"
"I heard that." Mummy's voice sounded from across the room and I giggled, unable to help myself. Teddy was smiling, too, but I could tell he was still worried about me. That's why I liked him so much. Even when mummy and daddy didn't seem to give an owl's tail about whether or not I was happy, Teddy always seemed to care. It was sad that he had to be at Hogwarts all the time, and I never got to see him in summer. What was worse, I wouldn't even get to spend a year with him at the castle. He was going to be graduating at the end of next term, and I wasn't going to Hogwarts for two more years after that.
No, wait. I wasn't going at all.
"Lily, are you okay?" Teddy had lowered his voice and was brushing a tear off my cheek, his eyes turning to grey. I sniffed, shaking my head. No, I was definitely not okay.
With a groan, Teddy pushed himself off the floor and into a sitting position, so that I was curled up in his lap. He hugged me- one of his famous teddy bear hugs- and patted my hair.
"Do you wanna talk about it?" He asked me quietly, so none of the other nosy relatives could hear. Sniffing into his shirt, I nodded. He picked me up- with a little bit of difficulty, since I'd gotten so big- and carried me out to the kitchen. All the food had been put away and the table scrubbed nice and neat, and it was on this that Teddy sat, still holding me protectively.
"I wish you were my big brother." I said wistfully, my eyes travelling to the window, where I could see the figures of the others jumping for bugs. "Jamesie is a meanie-head, and he hates me."
"He doesn't hate you." Teddy said gently.
"But he does." I protested stubbornly, twisting where I sat so I could frown at him. He just smiled and shook his head. But he was smart enough not to argue, and instead changed the subject.
"What's wrong, Lils?" The question was a simple one, but it seemed as if he was prepared for a very long answer. I leaned back and pulled his arms tighter around me. My lip stuck out in a pout.
"I'm a squib."
It was awful, admitting it out loud like that, without any doubt in my voice. I didn't quite know what to expect from Teddy- perhaps tears, or a dramatic scream of 'Noooo! Not Lily! Anyone but my favourite not-really-cousin-but-close-enough-to-be-a-cousin in the world!' But what I had not expected was for him to laugh.
"It's not funny!" I whined, tearing up again.
"I'm sorry." Teddy said, attempting to sober up. He failed, however, because his eyes were a twinkling blue and the corner of his mouth kept twitching. But one look at the hurt expression on my face and he sighed, resigned to take me seriously. Thank Merlin. "You're not a squib, Lils-"
"I am!" I interrupted, but he put his hand over my mouth. I pouted and let him finish.
"You're not a squib- you just haven't had anything to stimulate your powers yet. Uncle Neville didn't do anything magical until he was eight, and he's one of the greatest wizards of the century. You just have to be patient." I went to push his hand away, but he wasn't done. "And even if you were a squib- which you're not- then no one would care. We're your family, and we're going to love you, no matter what."
Finally, he let me breathe again.
His words hadn't had any affect on me whatsoever.
"I don't care what you say." I said, crossing my arms tightly across my chest. I thrust my nose into the air. "Jamesie told me I should go live with people like me, and so I'm going. I'm running away to stay with muggles."
Now there was no laughing.
"You're- you... what?" Teddy stuttered, seemingly unable to fathom what I had just said.
Well, at least he was taking me seriously this time.
"Lils, you're crazy!" He finally managed, looking at me with widened eyes. "You can't run-"
"Shhhh!" I shushed him, giving him a stern look. I glanced at the door that separated the kitchen from the living area. "They'll hear you!" I whispered, glaring at him. But then I felt bad, because glaring isn't nice. I sighed and hugged him again, and I could tell that he wasn't mad. He never got mad.
"Where are you going to go, Lily?" He asked after a short silence, and he sounded very worried still. "You can't just wander off and live with strangers- it's not safe!"
I turned and frowned at him, my nose wrinkled.
"You're not going to tell, are you?" I asked, watching him carefully. He shifted uncomfortably, and I saw him glance out the window, where my dad was standing and watching the boys.
"Lily, I can't let you put yourself in danger… I love you. We all love you-"
I could feel my heart sinking. I had thought, out of everyone, that maybe Teddy would understand. But he didn't. No one could. Because nobody had this squirming in their tummies every time anyone looked at them funny, and nobody else felt sad instead of cheery when they saw those scary-looking owls bring Hogwarts letters to the kids. Nobody knew how gloomy it was, being a squib.
"I should've never told you nothing." I choked, tears in my eyes again. "Now you're gonna tattle and they're never gonna leave me alone. I can't tell anybody anything." I let another little sob escape me as I tried to slide off his lap. "I'm all alone. But so is she! So I thought we'd help each other…"
Teddy's strong arms pulled me back, holding me in place. I didn't struggle long, and just leaned back, and let him hug me protectively.
"Who's alone, Lils? Who's 'she'…?" Teddy asked, and I sighed. I picked up his hand and turned it over, placing my palm on his, comparing my own tiny fingers to his own.
"G-Great Aunt Petunia." I sniffed quietly. "I was gonna go live with her. Daddy said she was angry that day at the funeral 'cause she was lonely. And I'm lonely, too, and I can't do magic neither, so I'd prob'ly be better off just going off to live with her."
"Aunt Petunia?" Teddy turned me around and looked me in the face, his forehead all wrinkled in a frown. "You mean, your Dad's Aunt? The one who raised him?"
"Do you know any other Petunias, 'sides the ones in the garden?" I asked with a little bit of sass. Mummy said sass doesn't look good on anyone, but I really couldn't help it. I was starting to wish I hadn't even told Teddy. Now I was never gonna get to feel like I belonged anywhere.
"It's fine, I'll just stay home." I muttered after a long silence from Teddy. "I just… I wanted to be someplace I could be normal."
There was another long pause, and I suddenly heard Teddy sigh. He leaned forward and kissed me on the forehead, pulling me into an even tighter hug.
"Alright, lil' Lils." He said, using the nickname he'd given me. "I can't believe I'm saying this… But you got your way. I'm gonna help."
I suddenly felt the bottom of my tummy fall out and disappear into a cloud of super happy butterflies. I swiveled around so quickly that I almost banged my head against his chin and stared at him with wide eyes.
"You mean it!?" I asked breathlessly, my throat suddenly feeling all tight- but in a good way, not a sad way. Teddy nodded, the corner of his mouth pulling upwards in a little crooked smile. I threw my arms around his neck and gave him the hugest hug I could muster. "Oh, Teddy-Bear! You're the bestest pretend cousin in the whole world!"
"Alright, alright." Teddy said with a little laugh, kissing my nose. "You owe me one, you little imp. But I'm not letting you wander off there by yourself, alright? It's not safe for a little girl. So I'm taking you there myself, and seeing to it that the muggle takes you in."
"She will." I said happily, turning back around and leaning back into Teddy again. He made a very comfortable armchair. "She's got to."
"Now, Lils," Teddy said seriously, brushing some red hair back from my forehead. "You're sure you want to do this? Your dad hated living with her, you know… She was never very nice. She made him do chores and sleep in a cupboard, and… and she never really hurt him. Not on the outside, at least. But… But it'll be a lot different than living here."
I swallowed, my little hand tightening around Teddy's big one.
"I know." I said quietly, "But… But maybe she'll like me better. 'Cause I'm not magic. I'm more like her. Maybe she'll be good to me."
"And maybe you'll be good for her…" Teddy said quietly, but I didn't know exactly what he meant. After another little silence, he cleared his throat. "Pack your bags tonight," He said. "And I'll take you there first thing in the morning, before your mum and dad get up."
I could hardly believe my ears.
"Thank you, Teddy!" I squealed, throwing my arms around his neck again and hugging him extra tight. He ruffled my hair affectionately and laughed. I couldn't have been more happy. I had been terrified of going to face the mean, old woman alone for the first time. What if she threw me out and I had no way to get home? Not to mention, I hadn't a clue where she lived. With Teddy's help, everything would be okay. I would find my new home with the muggles, and I would finally fit in.
But I could feel my happy feeling start to drain away a little bit when I thought about mummy waking up with no one to make blueberry pancakes for. I turned as I heard the kitchen door open and saw my dad returning from outside. I smiled a little guiltily at him, and I glanced quickly at Teddy. He was a very good actor, it seemed, because he was grinning at dad as if nothing were the matter.
"Ready to go, Lils?" Dad asked, giving me a small smile. I nodded, meekly slipping off Teddy's lap, with his help. "I want you in bed early tonight, alright?" I nodded again and waved goodbye to Teddy, who sat alone on the kitchen table. But before I turned to walk back to the living room, I thought I saw him wink.
All the way home, I kept my fingers crossed in my pockets. I needed tomorrow to go perfectly. I may hate the idea of running away, but it was my only choice. Besides, if I backed out now, I'd be breaking my promise to both Stan and Teddy. Sure, I hadn't actually promised Teddy anything, but he was going to be at my house tomorrow, and he was going to expect me to be ready. Sighing, I climbed into bed but did not sleep, listening until I heard the sounds of daddy's snores. Then, slipping out of my sheets, I began to silently pack. I never uncrossed my fingers.
I'd need all the luck I could get.
The plot thickens! Please leave a review with any thoughts and/or criticism. I really do appreciate every one, and I'd love your insight to make the story better!
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Diabetic Eye Disease Topic Guide
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I got this to make soy yogurt because I am vegan. Buying vegan yogurt is quite expensive. So is this culture. I tried it with not exact success. Went to a plain soy commercial yogurt and got a much better outcome. Will not buy this again.
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Episode of the Week : The Enemy Within
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February 11 2014, 03:17 PM
Rear Admiral
Location: ssosmcin
Re: Episode of the Week : The Enemy Within
If I had to complain about anything in this classic episode, it's McCoy's attitude about the men below. "What if death was caused by transporter malfunction? Then you'd die. They'd die anyway."
"They'd die anyway" always felt too cold for McCoy. What the hell did Sulu do to him? The guy's a doctor. Even if the line stayed, the delivery could have been a little different.
Other than that, a solid 9 or 10. Easily in my top 5 Trek's ever. And god what a great score!
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Skip Navigation
Runs on Dunkin.
BARDSTOWN, Ky. — In a bourbon-tasting room here stuffed with supporters, Alison Lundergan Grimes flashes a toothless smile as Martha Layne Collins, Kentucky's first female governor, optimistically introduces her as the state's future "first female U.S. senator."
Grimes takes the floor and wraps her arm around Collins. "You made history with her," she tells the crowd. "We will make history again."
As Grimes crisscrosses the state on a 50-county tour ahead of Tuesday's primary and an expected brutal general-election campaign against Senate Minority Leader Mitch McConnell, she is making her gender a defining issue.
"The difference between Mitch McConnell and myself?" Grimes says. "Well, it's somebody on the wrong side of every woman's issue out there."
In a brief interview at Dude's Muffler and Brake shop, in Lawrenceburg, Grimes is unabashed that her campaign would focus on her gender to win over women voters.
"The people of this state, especially the women across the commonwealth, they are our strength — 53 percent of the electorate," she says. "They are the reason that I'm currently Kentucky's only female statewide elected officer and they will be the reason why we break through the ceiling that exists and finally get our first female United States senator."
At three consecutive stops Friday, Grimes, in blue jeans, a white shirt, and an ocean-green blazer, slapped at the "empty dress" remark of National Republican Senatorial Committee spokesman Brad Dayspring (though she wrongly attributed it). "I am the Kentucky woman, who my Kentucky colleagues on the Republican side so gentlemanly refer to me as an 'empty dress,' " she tells those gathered in Bardstown.
She then ticks off the issues that she hopes will divide women voters from McConnell as he seeks a sixth term: his opposition to the Violence Against Women Act, the Paycheck Fairness Act, the Lilly Ledbetter Act.
Left unmentioned: abortion, the main wedge that Democrats nationwide have used to divide GOP opponents from the swingiest parts of the electorate. In a twist, it is McConnell broaching the topic, hoping to create a fissure between the national pro-abortion-rights supporters who are helping bankroll Grimes's campaign and the conservative voters she needs to carry this Southern state. He held a press conference in Washington earlier this week pushing to ban abortions after 20 weeks.
"We should all agree that unborn children should be protected at least from the point that they're capable of feeling pain," McConnell pointedly said of the legislation.
Grimes supports abortion rights. "I come from a family of five women," she says in the interview. "I would never pretend to tell one of my sisters what to do with their body and I don't want the federal government doing that either."¦ When it comes to choice, I believe, should a woman have to make that decision, it's between herself, her doctor, and her God."
As for McConnell's 20-week abortion ban, she says, "I think you always put the health, life, and safety of the mother first, should that decision have to be made. I'm not for moving backwards the principles the Supreme Court has set forward."
As she spins through muffler shops, cafes, and bourbon rooms, Grimes is a compelling speaker. Her voice booms over the assembled crowds. "A loud voice, yes, I learned that with five girls in my family," she says, to laughs. When she asks for an "amen," the crowds deliver.
Ted Jackson, a veteran GOP strategist in the state, predicts that Grimes's showmanship will lose its luster as the spotlight begins to blare after Tuesday's primary, saying she has "hollow eyes." "Her overly dramatic style is more suited to game-show host than United States Senator and she will not wear well on voters," Jackson says.
For now, though, friendly audiences in the small towns between Lexington and Louisville are eating it up. She pokes repeatedly at McConnell's mistaken use of a Duke basketball player in an ad and his appearance at the Conservative Political Action Conference, when he emerged waving a gun above his head. McConnell, she teases, managed to shoot himself in the foot with an unloaded weapon.
"This Kentucky woman," Grimes says in a common refrain, "now 16 polls deep, is running even or ahead."
Still, there is uneasiness. Grimes and her advisers know the real fight begins after Tuesday, when McConnell is expected to dispatch his primary opponent, Matt Bevin, and turn his fire on her. They must ensure that "this Kentucky woman" is just starting, not peaking.
"We're going to start telling the truth about him," State Auditor Adam Edelen, who was travelling on the bus with Grimes, says of McConnell, "and they're going to start lying about us."


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Open-minded and curious Parisian (I know what you think but some of them are nice;) who really likes to meet people from all over the world
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36 • Paris, France • Man
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- 6' 5" (1.95m)
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Once upon a time in America, The Godfather, Gladiator, Ghost Dog, Noces rebelles...
Games of Throne, Boardwalk Empire, The Killing, The Wire, Lost, Six Feet Under, True Detective...
Radiohead, Noir Désir, Mano Solo, The Beatles, Lisa Ekdahl, Duke Elllington, Chromatics, Air...
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I eat way too many "petits suisses"...
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you like long conversations in english or french, and would like to discover Paris with a very tall journalist
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Design News For June 9, 2014
Colorful and Conservative Mingle in This Manhattan Apartment
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- Here's what happens when you add color to a conservative apartment . . . — Houzz
- This tech office was designed to look like glamping! — Cured
- 14 blank-wall ideas you haven't thought of — HuffPost Home
- Surprising, low-cost ways to update your home decor — Real Simple
- How a top Hollywood manager became an interior designer — Architectural Digest
- Warning: these 10 courtyards may spark envy — Remodelista
- 15 ways to revamp your kitchen with lighting — House Beautiful
- Everything you need for a skillfully styled bookshelf — Domaine Home
Source: Rikki Snyder via Houzz


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A crush of people crowd the midway Tuesday evening at the Fairfield County Fair in Lancaster
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wayne phillips PRO
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I have lived in jackson hole since 93' and work as a whitewater river guide and a ski tech. I am also a photographer/ assistant to my wife the pro. I am very passionate about kiting! If you plan to visit jh and want to kite look me up! (307)413-3977
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Take the old Baby on Board slogan from the 1980s and push it forward down the pipeline until the metaphor is flipped over and gets all wet. When the board's a surfboard, you're not only telling the world that you've got a junior surfer in training, but that the emphasis on safety over getting out there and actually enjoying life and the world around us is overrated.
This baby one-piece is made ethically in the USA by American Apparel, designed by Irregular Times, and printed by the Columbus independent outfit called Skreened.


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Cadillac Blindside The Bottom Line Lyrics
Last updated: 04/16/2003 03:56:07 AM
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About Alexander, AR
Alexander is a town in Pulaski and Saline counties in the U.S. state of Arkansas. The population was 614 at the 2000 census. It is part of the Little Rock-North Little Rock-AR Metropolitan Statistical Area. (more on Wikipedia)


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|Berkeley Parents Network
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Removing Food Stains from Baby's Clothing
My five month old daughter has just started babyfood and of course with that comes a new problem with her laundry -- stain fighting! Does anyone have a good recommendation on what products to use on her clothing? How do i get out those sweet potato stains??!! I am still using dreft detergent. thanks, elizabeth
Other measures to deal with stains include dressing your child in clothes with dark colors or patterns, and not worrying about stains. Remember, a baby's clothes can be clean, even if they are stained. Suzanne
First separate colors from whites. Wash whites with about a cup of bleach; --I've found that most baby clothes that are white with pictures or patterns can be washed in bleach without the colors changing. Use HOT water to wash those, along with regular detergent, and add the bleach after the tub is full of water.
For colors, spray stains with Shout or Spray N Wash (big containers to refill spray bottles are cheap at Costco) as soon as possible after getting the stain, though, to be honest, I often don't do it til I wash the clothes...and use a regular dose of detergent with that load too (wash in cold or warm).
The only thing I have been unable to get out in the last 2 years is mildew, which occurs when you put wet or damp clothes in the hamper, and an indelible stain sets. I tossed out a few articles of clothing before figuring out what it was--now I let clothes dry flat or hanging before tossing in the hamper, or wash them promptly.
One other note-- if oil or other substances don't come out with the above methods, rewash using more Shout and hotter water--eventually all will be clean. Good luck! --Heidi
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