jsullins_EvoWiboPoster_2018.04.11_FINAL.pdf

Evo-Wibo 2018<div><br></div><div>Jennifer Sullins</div><div>Suzanne Estes Lab</div><div>Department of Biology</div><div>Portland State University</div><div>Portland, OR, USA</div

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

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

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

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

ANOVA results for deletion level variation among strains and generations.

<p>ANOVA results for deletion level variation among strains and generations.</p

Changes in <i>nad5Δ</i> PCR band genotype from G0 to G10.

<p>Static indicates cases where <i>nad5Δ</i> mtDNA levels stay the same, ↑<i>nad5Δ</i> indicates cases where levels increase, and ↓<i>nad5Δ</i> indicates cases where levels decrease. intc indicates the intact PCR band category, intm indicates the intermediate band category, and del indicates the deletion band category.</p

Estimates of oxidative stress and mutation frequency.

†<p>Baer mutation accumulation (MA) line number from the Baer et al. (2005) experiment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065604#pone.0065604-Baer1" target="_blank">[28]</a>.</p>‡<p>Relative reactive oxygen species (ROS) levels expressed as means (standard error) of relative fluorescence units.</p>*<p>Indicates significantly different from N2 ancestor.</p>§<p>Means (standard error) of 8-oxo-7,8-dihydro-2′-deoxyguanosine, or 8-oxodG, are reported as ×10⁹ damaged bases per nanogram of DNA.</p>¶<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065604#s2" target="_blank">Materials and Methods</a> for calculations of point estimates of the frequencies of base substitutions (µ_BS) and G-to-T transversions (µ_G-TO-T).</p

Phylogenetic relationships based on mtDNA of the eight <i>C. briggsae</i> natural strains used for MA line progenitors (a) and schematic of the <i>nad5</i>Δ locus (b).

<p>In (a), the three major intraspecific mtDNA clades and origin of the Ψnad5-2 element are indicated next to the relevant internal branches. The blue branches lead to MA line progenitors that encode the putative compensatory mutations associated with the <i>nad5</i>Δ locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041433#pone.0041433-Howe1" target="_blank">[10]</a>. In (b), the arrows indicate positions of the 21-bp direct repeats associated with deletion formation. The dashed line indicates DNA sequences missing in canonical <i>nad5</i>Δ molecules. The blue arrow indicates the position of the direct repeat bearing putative compensatory mutations. Clade III strains lack Ψnad5-2 elements and associated deletions, as indicated in the bottom mtDNA gene model.</p