Experimental design
All data recorded for this experiment comes from polyps that were kept
individually. The experiment consisted of two treatments: a summer
treatment and a winter treatment. The summer treatment lasted for one
week and aimed at simulating a heat wave. The winter treatment lasted
for five months and aimed at simulating elevated winter temperatures.
For both treatments, the control values were chosen such that both
summer and winter conditions are comparable to our previous studies
investigating H. oligactis life history (Ngo et al. 2021;
Sebestyén et al. 2020; Tökölyi et al. 2017). Hence, the control
temperature for summer was set as 18 ºC (cold summer, CS) and the
control temperature for winter was set to 8 ºC (cold winter, CW). To
simulate summer heatwaves, I exposed polyps to 23 ºC for one week (warm
summer, WS). Water temperatures as high as 25 ºC regularly occur in
natural populations at the end of the summer (J. T. personal
observation), however, in the laboratory larger or longer heat
treatments (i.e., above 23 ºC or longer than one week) result in excess
mortality. For the winter treatment, I exposed experimental polyps to 12
ºC temperature for five months (warm winter, WW). A four degree increase
in winter temperature is in accordance with IPCC predictions for Europe
by the end of the century (Meehl et al., 2007). The summer and winter
treatments were applied in a factorial design, such that there were four
experimental groups: cold summer – cold winter (CS-CW); cold summer –
warm winter (CS-WW); warm summer – cold winter (WS-CW) and warm summer
– warm winter (WS-WW).
During the one-week summer treatment (either 18 or 23 ºC) polyps were
fed four times per week. After the summer treatment, polyps were
photographed on a 1-mm grid paper sheet to measure their size, since
size at cooling is an important predictor of sexual reproduction and
post-reproductive survival (Ngo et al., 2021). Next, they were moved to
either 8 or 12 ºC, depending on their treatment and an 8/16 hours
light/dark cycle. Lowering temperature induces sexual reproduction in
this species and I recorded the presence of male or female gonads four
times per week as a proxy for sexual fitness. In addition, the number of
detached buds was recorded twice per week as a proxy for asexual
fitness. The winter treatment lasted for five months during which polyps
were fed and cleaned twice per week. A five month winter treatment is
necessary and sufficient time period to quantify sexual reproduction and
post-reproductive survival in most polyps in these strains. At the end
of the winter treatment, polyps were scored as survived if they had an
intact body with tentacles and had the ability to feed, and they were
scored dead if they disappeared during the experiment or consisted only
of necrotic tissue. All experimental groups were started with 30-36
polyps per strain, with a total starting sample size of N = 843,
although some individuals were excluded because they were accidentally
lost during the experiment, or they proved to be sex-changed individuals
(see Results). The experiment was initiated in batches over a period of
about two months, depending on the availability of experimental animals.
Each batch consisted of animals randomly divided into four treatment
groups and any batch effects were statistically taken into account (see
below).