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.