Experimental evolution
T. californicus is sexually dimorphic. An adult male clasps an immature female until her terminal molt, when she is then inseminated and released. Females can mate only once and use stored sperm to fertilize sequential clutches of eggs that can add to several hundred progeny (Vittor 1971). This species lacks heteromorphic sex chromosomes and recombination occurs only in males (Burton et al. 1981). We obtained virgin females by separating clasped pairs, and produced F1 with similar mixed nuclear genome and fixed mitochondrial backgrounds; i.e. SD mitochondrial background on SD♀ x SC♂ cross, and SC mitochondrial background on SC♀ x SD♂. For each reciprocal cross, we outcrossed F1s to produce recombinant F2 hybrids that were allowed to mate randomly.
Each experimental line started with 100 outbred F2 gravid females. Lines evolved under these conditions for nine months with overlapping generations, replenishing the growing medium monthly. Since on average females reach adulthood in 2-3 weeks and produce multiple egg clutches until they are 4-6 weeks old, this experimental design corresponds to approximately nine generations of experimental evolution up to F11. This procedure was replicated 10 and 7 times for the SC and SD mitochondrial backgrounds, respectively. We followed the same procedure to generate one control line with fully matched nuclear and mitochondrial genomes for each parental population (i.e. SC♀ x SC♂ and SD♀ x SD♂).
Fitness recover y
Relatively small experimental populations may lead to strong genetic drift, and conversely to limited response to selection imposed by the fixed mitochondrial backgrounds. If selection is strong relative to drift in evolved lines we expect an increase in productivity and associated recovery in one or multiple fitness traits associated with mitonuclear incompatibilities (Ellison & Burton 2008b). To test these hypotheses, at the end of the experimental evolution, we measured: census size (as the number of adults after), fecundity (as the number of nauplii hatching from the first clutch of a female), and survivorship (as the fraction of nauplii surviving to 14 days). Fecundity was replicated between 4 to 12 times, depending on the number of available virgin females, and survivorship was replicated between 10 to 28 times, depending on the number of available gravid females. To monitor how average fitness varied along the course of the experiment, we have also measure survivorship 3 to 8 additional times, using 4 to 12 replicates. Additionally, we measure these two fitness traits for the initial reciprocal F2 hybrids and for the pure parental populations, as a reference for fitness breakdown and recovery respectively. We estimated mean plus ±1SE. We tested for significant hybrid breakdown by comparing fitness of the F2 hybrid with its maternal population, using a Mann–Whitney U -test and an alpha of 0.05 in R 2.15.1 (R Development Core Team). We tested for significant recovery in lines for which the mean reached or passed the reference parental fitness, adjusting the P-value when multiple comparisons occur at the same time.