Evolution Of A Higher Intracellular Oxidizing Environment In Caenorhabditis Elegans Under Relaxed Selection

We explored the relationship between relaxed selection, oxidative stress, and spontaneous mutation in a set of mutation-accumulation (MA) lines of the nematode Caenorhabditis elegans and in their common ancestor. We measured steady-state levels of free radicals and oxidatively damaged guanosine nucleosides in the somatic tissues of five MA lines for which nuclear genome base substitution and GC-TA transversion frequencies are known. The two markers of oxidative stress are highly correlated and are elevated in the MA lines relative to the ancestor; point estimates of the per-generation rate of mutational decay (DM) of these measures of oxidative stress are similar to those reported for fitness-related traits. Conversely, there is no significant relationship between either marker of oxidative stress and the per-generation frequencies of base substitution or GC-TA transversion. Although these results provide no direct evidence for a causative relationship between oxidative damage and base substitution mutations, to the extent that oxidative damage may be weakly mutagenic in the germline, the case for condition-dependent mutation is advanced

In Vivo Quantification Reveals Extensive Natural Variation in Mitochondrial Form and Function in Caenorhabditis briggsae

In We have analyzed natural variation in mitochondrial form and function among a set of Caenorhabditis briggsae isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic nad5 gene deletion (nad5 Delta) that correlates negatively with organismal fitness. We performed in vivo quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant amongisolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with nad5D levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in C. briggsae. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives

Evolution of a Higher Intracellular Oxidizing Environment in Caenorhabditis elegans under Relaxed Selection

We explored the relationship between relaxed selection, oxidative stress, and spontaneous mutation in a set of mutation-accumulation (MA) lines of the nematode Caenorhabditis elegans and in their common ancestor. We measured steady-state levels of free radicals and oxidatively damaged guanosine nucleosides in the somatic tissues of five MA lines for which nuclear genome base substitution and GC-TA transversion frequencies are known. The two markers of oxidative stress are highly correlated and are elevated in the MA lines relative to the ancestor; point estimates of the per-generation rate of mutational decay (DM) of these measures of oxidative stress are similar to those reported for fitness-related traits. Conversely, there is no significant relationship between either marker of oxidative stress and the per-generation frequencies of base substitution or GC-TA transversion. Although these results provide no direct evidence for a causative relationship between oxidative damage and base substitution mutations, to the extent that oxidative damage may be weakly mutagenic in the germline, the case for condition-dependent mutation is advanced

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

<p>Abstract</p> <p>Background</p> <p>Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial <it>ND5 </it>deletion recently discovered to segregate among wild populations of the soil nematode, <it>Caenorhabditis briggsae</it>. The normal product of <it>ND5 </it>is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.</p> <p>Results</p> <p>We quantified significant variation among <it>C. briggsae </it>isolates for all phenotypes measured, only some of which was statistically associated with isolate-specific <it>ND5 </it>deletion frequency. We found that fecundity-related traits and pharyngeal pumping rate were strongly inversely related to <it>ND5 </it>deletion level and that <it>C. briggsae </it>isolates with high <it>ND5 </it>deletion levels experienced a tradeoff between early fecundity and lifespan. Conversely, oxidative stress resistance was only weakly associated with <it>ND5 </it>deletion level while ATP content was unrelated to deletion level. Finally, mean levels of reactive oxygen species measured <it>in vivo </it>showed a significant non-linear relationship with <it>ND5 </it>deletion level, a pattern that may be driven by among-isolate variation in antioxidant or other compensatory mechanisms.</p> <p>Conclusions</p> <p>Our findings suggest that the <it>ND5 </it>deletion may adversely affect fitness and mitochondrial functioning while promoting aging in natural populations, and help to further establish this species as a useful model for explicit tests of hypotheses in aging biology and mitochondrial genetics.</p

Mitonuclear hybrid strains more often resemble their mitochondrial parental isolate.

<p>Averages of maximum pharyngeal bulb fluorescence for mitochondrial (PB800 and HK105) and nuclear (AF16) parent isolates are on either side of the two hybrid strains (AFPB800 and AFHK105) (Fig. 1). Letters denote significantly different groups as determined by Tukey HSD analysis. Bars show one SEM for 15–20 independent samples.</p

Associations between mitochondrial function and morphology traits and isolate-specific <i>nad5Δ</i> level.

<p>Natural variation among <i>C. briggsae</i> isolates in (A) the total area of functional mitochondria, (B) the average area of individual non-functional mitochondria, (C) the total area of non-functional mitochondria, the (D) aspect ratio, (E) circularity, (F) circularity variance of non-functional mitochondria, in (G) relative ΔΨM, (I) the ratio of functional to non-functional organelles, and (H) relative ROS levels. Column colors corresponding to phylogenetic clade (orange = Kenya, white = Temperate, blue = Tropical), and isolates are ordered by deletion frequency along the x-axis. ED3101 and ED3092 do not experience the deletion and were assigned arbitrary x-values of −7 and −5, respectively, for this figure. Averages of maximum pharyngeal bulb fluorescence in <i>C. briggsae</i> natural isolates are plotted in relative fluorescence units (RFU). Bars represent one SEM for 15–20 independent samples.</p

Natural and experimental <i>C. briggsae</i> strains and description of the <i>nad5Δ</i> mtDNA deletion.

<p>A. Phylogenetic relationship and <i>nad5Δ</i> heteroplasmy level of <i>C. briggsae</i> isolates studied here. GL = global superclade; KE = Kenya clade; TE and TR = temperate and tropical subclades of GL; C(+) = isolates bearing compensatory Ψ<i>nad5Δ</i>-2 allele; C(-) = isolates bearing ancestral alleles. <i>nad5Δ</i> heteroplasmy categories were assigned to each <i>C. briggsae</i> natural isolate for statistical analysis following Estes et al. (2011): High = underlined font, medium = italicized, low = regular, and zero-<i>nad5Δ</i>="N/A”. Note that we assayed the natural HK104 isolate here instead of the mutation-accumulation line progenitor reported in Estes et al. (2011), which had evolved high <i>nad5Δ</i> levels in the lab (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043837#s2" target="_blank">Materials and Methods</a>). B. Positions of the <i>nad5Δ</i> deletion (dashed line at top) and Ψ<i>nad5Δ</i>-2 elements in the mitochondrial genome. Primers are indicated by arrows (adapted from Howe and Denver, 2008). C. Mitochondrial and nuclear parent isolates for each mitochondrial-nuclear hybrid. <i>nad5Δ</i> heteroplasmy for each hybrid strain matches that of the maternal isolates as expected. Mitochondrial phenotypes are expected to match those of the maternal isolate if measured traits are predominantly determined by the mitochondrial genotype.</p

<em>In Vivo</em> Quantification Reveals Extensive Natural Variation in Mitochondrial Form and Function in <em>Caenorhabditis briggsae</em>

<div><p>We have analyzed natural variation in mitochondrial form and function among a set of <em>Caenorhabditis briggsae</em> isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic <em>nad5</em> gene deletion (<em>nad5Δ</em>) that correlates negatively with organismal fitness. We performed <em>in vivo</em> quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant among-isolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with <em>nad5Δ</em> levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in <em>C. briggsae</em>. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives.</p> </div

Assigned labels and descriptions of all mitochondrial traits measured for <i>C. briggsae</i> natural isolates.

<p>The grand mean, F-ratio and degrees of freedom for one-way ANOVA testing for phenotypic differences among <i>C. briggsae</i> isolates. Bold font identifies the nine traits retained in the classification tree analysis when using categories based on isolate-specific <i>nad5Δ</i> % (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043837#pone.0043837.s003" target="_blank">Table S3</a>). *, **, and *** denote p<0.05, 0.01, 0.001, respectively. Subscripts N, F, and T indicate whether the measure refers to Non-functional, Functional, or Total mitochondria. Subscript P and V denote that the measure refers to the entire mitochondrial population (not individual mitochondria), or the average individual variance in that trait, respectively.</p