Artificial selection on egg size
Artificial selection yielded highly divergent lines that differed after 11 generations by ca. 50% in egg volume across selection directions (based on eggs laid first; Fig. 1). A significant response was observed in both directions (L > C > S; Tukey HSD after anova on mean egg size per female; F2,3 = 56.1, P = 0.0042), with the response (averaged across replicates) being fairly symmetric (L lines: 23.8 ± 1.3% increase; S lines 27.3 ± 2.0% decrease). There was also significant though less pronounced variation among replicate lines (F3,557 = 26.1, P < 0.0001). Realized heritabilities (h2) were on average 0.39 (L1: 0.384, r2 = 0.98; L2: 0.386, r2 = 0.92; L*: 0.394, r2 = 0.91) for L and 0.44 (S1: 0.394, r2 = 0.85; S2: 0.436, r2 = 0.85; S*: 0.494, r2 =0.91) for S lines (all P-values of regressions <0.0001). The slopes of regressions fitted to egg size on cumulated selection differentials did not differ among replicates within selection direction or between directions (ancovas; all P-values ≥ 0.14 for interactions of direction or replicate line with cumulated selection differential).
Correlated responses in development time and pupal mass
Double-nested anovas with cage and replicate as random factors failed to reveal any significant effects of selection direction on larval development time, pupal development time or pupal mass. However, for all three traits there was significant variation among replicate lines (Table 1). Treating replicate line as a fixed effect in the anovas (everything else being equal) yields similar results for larval time, however, it shows significant variation for pupal time [L (6.4 ± 0.03 days, n = 452) > C (6.3 ± 0.03 days, n = 248) > S (6.2 ± 0.03 days, n =414); Tukey HSD after anova; F2,784 = 29.0, P < 0.0001] and pupal mass [L (mean 187.9 ± 1.5 mg, n = 452) > Control (173.4 ± 2.1 mg, n = 248) = S (177.4 ± 1.6 mg, n = 414); F2,1078 = 8.7, P = 0.0015] across selection directions. These results may indicate trends towards longer pupal times in the L lines and towards a divergence in pupal mass across selection directions. However, there was no clear pattern with selection direction in either trait, with overlap between L and S replicate line means occurring throughout (Table 2). Thus, there is only weak evidence for correlated responses in any of the three life-history traits considered above.
Table 1. Nested analyses of (co-)variance for the effects of selection direction (fixed) and replicate line (random) on life-history traits of Bicyclus anynana.
| Replicate (Dir.)||5||166.8||3.3||0.0216|
| Cage (Dir., Repl.)||24||52.0||14.2||<0.0001|
| Direction × sex||2||1.3||0.4||0.7012|
| Error||1078||3.7|| || |
| Replicate (Dir.)||5||2.55||7.5||0.0002|
| Cage (Dir., Repl.)||24||0.34||2.4||0.0002|
| Direction × sex||2||0.79||5.6||0.0039|
| Error||784||0.14|| || |
| Replicate (Dir.)||5||17491.2||6.5||0.0006|
| Cage (Dir., Repl.)||24||2728.5||5.4||<0.0001|
| Direction × sex||2||624.3||1.2||0.2941|
| Error||1078||509.5|| || |
| Replicate (Dir.)||5||0.021||15.6||<0.0001|
| Pupal mass||1||0.012||8.9||0.0032|
| Error||267||0.001|| || |
| Replicate (Dir.)||5||19468.0||2.5||0.0339|
| Pupal mass||1||218733.0||27.6||<0.0001|
| Error||267||7930.9|| || |
|Reproductive investment (initial egg size × fecundity)|
| Replicate (Dir.)||5||4546.3||3.2||0.0076|
| Pupal mass||1||41954.7||29.7||<0.0001|
| Error||267||1410.4|| || |
|Reproductive investment (dry mass)|
| Replicate (Dir.)||5||84.6||2.3||0.0471|
| Pupal mass||1||1154.3||31.1||<0.0001|
| Error||267||37.1|| || |
Throughout all analyses (Table 1), cage and sex were significant factors, with, regarding the latter, females being significantly heavier and having a longer larval but a shorter pupal development time as has been repeatedly shown for B. anynana (e.g. Fischer et al., 2003a).
Egg size-number trade-off
As expected, the females used to investigate correlated responses to selection laid eggs that differed substantially in size, with the difference in volume of first eggs amounting to, on average, 58.9% between L and S lines (L > C > S; Tukey HSD after anova; Table 1, Fig. 2a). A significant effect of the covariate pupal mass indicates that egg size tended to increase, if weakly and in case of the L lines not significantly, with increasing female pupal mass (L: r = 0.04, P = 0.637, n = 139; C: r = 0.30, P =0.014, n = 65; S: r = 0.23, P = 0.004, n = 147).
However, there was only limited evidence for a trade-off between egg size and lifetime fecundity (Fig. 2a, b; Table 1; effect of selection direction on fecundity n.s.). Treating replicate line as fixed effect in the anova, however, indicates that the S lines tended to lay on average more eggs (least square mean 302 ± 9, n = 109) than either C (268 ± 12, n = 65) or L lines (257 ± 9, n =103), with the latter two being statistically indistinguishable (Tukey HSD after ancova; selection direction F2,266 = 6.9, P = 0.0012). Likewise, removing the covariate pupal mass from the analysis presented in Table 1 (treating replicate line as random effect) yields a statistical trend (F2,3 = 4.3, P = 0.08; note that one could argue that it is not necessary to include pupal mass as it does not differ across selection directions). A significant effect of the covariate pupal mass indicates that egg numbers tended to increase with increasing female pupal mass (L: r = 0.20, P = 0.018, n = 139; C: r = 0.27, P =0.029, n = 65; S: r = 0.37, P < 0.0001, n = 147; slopes of the regression lines did not differ; ancova interaction term P = 0.17). To summarize, selection for smaller eggs tended to increase fecundity, but selection for larger eggs had no opposite effect in spite of the production of much larger eggs. Thus, the reproductive pattern in B. anynana seems to be more complex than a simple trade-off between number and size of eggs.
Further, there were significant phenotypic trade-offs or trends between egg size averaged over the whole oviposition period and fecundity within selection directions (both corrected for pupal mass of individual females using residuals; L: r = −0.32, P = 0.0001, n = 139; C: r = −0.24, P = 0.057, n = 65; S: r = −0.26, P =0.0013, n = 147). There was no indication that the slopes of the regression lines differed among selection directions (ancova; interaction term P = 0.65).
Correlated responses in reproductive investment
Reproductive investment was firstly estimated as the product of mean initial egg mass and fecundity, a measure often used in previous studies (e.g. Schwarzkopf et al., 1999). As above, the anova treating replicate as random factor revealed no significant effect of selection direction (Table 1), while treating replicate as fixed effect indicates that investment tended to be higher in L lines (least square mean 125.2 ± 3.8 mg, n = 103) than in S lines (107.7 ± 3.6 mg, n = 109), with the unselected controls intermediate (113.5 ± 4.9 mg, n = 65) and not significantly different from either of them (Tukey HSD after ancova; selection direction F2,266 = 5.7, P = 0.004; Fig. 2c). Again, removing the covariate pupal mass from the analysis presented in Table 1 yields a statistical trend (F2,3 = 4.3, P = 0.063).
These data suggest that the L lines may achieve a fecundity comparable to controls by increasing their reproductive investment. However, using the above estimate of reproductive investment relies on two critical assumptions, namely that (1) egg size is invariant over time (which is not true for butterflies and other organisms; e.g. Wiklund & Karlsson, 1984; Braby & Jones, 1995; Giron & Casas, 2003), or at least that the decline in egg size with female age is equal among females laying eggs that differ in initial size, and that (2) there are no differences in egg composition (e.g. Giron & Casas, 2003) across groups. Below we will show that these assumptions are not met in our study organism.
First, the decline in egg size with female age was overall stronger in L lines than in either C or S lines (Fig. 3). A repeated measurements anova on egg size revealed significant effects of selection direction (F2,160 = 423.0, P < 0.0001), replicate line (F5,160 =12.9, P < 0.0001), and time (F8,153 = 21.4, P < 0.0001). Most importantly there was also a significant interaction between selection direction and time (F16,306 = 4.4, P < 0.0001) confirming that L lines tend to lose proportionally more weight (ca. 11.2%) than S lines (ca. 3.5%; due to death of females this analysis was restricted to the first 17 oviposition days). It is striking that a single L line (L1, top line in Fig. 3) does not follow the general pattern and maintains a rather large egg size throughout. This particular line is quite exceptional, as it simultaneously realises the lowest fecundity, the lowest total reproductive effort (within L lines) and the highest mean egg size (averaged over oviposition period; Fig. 2a–d). Second, water content was found to differ significantly across selection directions [L (83.5 ± 0.2%, n = 63) > C (82.2 ± 0.2%, n = 64) >S (81.2 ± 0.2%, n = 63); Tukey HSD after anova; selection direction F2,3 = 12.9, P =0.034; replicate F3,184 = 4.1, P = 0.0075].
Figure 3. Mean fresh egg mass ( ± 1 SE) over time for large-egg selected (filled diamonds, solid lines), small-egg selected (open squares, dashed lines) and unselected control lines (open triangles, solid line) of Bicyclus anynana. In two replicates per direction, egg size was selected relative to pupal mass, whilst selection was purely on egg size in the third replicate (indicated by arrows).
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These results show that variation in egg size over time and differences in egg composition (i.e. water content) need to be taken into account when estimating total reproductive investment. Doing so (by multiplying daily mean egg size with daily fecundity and summing up all values as well as correcting for differences in water content by using dry mass) reveals that reproductive investment was in fact similar across selection regimes, regardless of treating replicates as random (Table 1) or fixed effect (selection direction F2,266 = 0.56, P = 0.57; Fig. 2d). In each of the three selection regimes, total reproductive effort increased with increasing pupal mass (L: r = 0.22, P = 0.011, n = 139; C: r = 0.31, P = 0.012, n = 65; S: r = 0.42, P < 0.0001, n = 147; with similar slopes: ancova interaction term P = 0.18). None of the relationships given above were due to any associated differences in female longevity, as there was no difference across selection directions (L: 25.5 ± 0.7 days, n =103; C: 24.6 ± 0.8 days, n = 64; S: 25.6 ± 0.6 days, n =109; F2,3 = 0.04, P = 0.96).
Predictors of egg size and lifetime fecundity
A multiple regression analysis showed that, overall, pupal mass had the strongest effect on egg size (though still fairly weak; r2≤ 10%), followed by fecundity (Table 3a). Longevity had a rather strong effect on egg size in the L lines, an effect for which we have no explanation. The best predictor of lifetime fecundity was longevity, indicating that, within lines, longer-lived females laid more eggs (Table 3b). Pupal mass and egg size also affected fecundity. Thus the patterns revealed by multiple regressions fully confirm the trends obtained from the correlation analyses above.
Table 3. Results of multiple regressions (stepwise forward addition of variables; Ridge regression; λ = 0.10; F > 1.0 for inclusion) for the effects of life-history traits on egg size (A) and fecundity (B) across selection directions (all whole model P < 0.02).
| ||Predictor||β (SE)||r||F||P|
|(A) Egg size|
|Pupal mass||0.170 (0.074)||0.290||3.89||0.0508|
|Larval time||0.113 (0.076)||0.302||2.20||0.1405|
|S||Pupal mass||0.372 (0.078)||0.099||15.76||0.0001|
|C||Pupal mass||0.323 (0.118)||0.083||5.58||0.0213|
|Egg size||−0.406 (0.069)||0.301||19.14||<0.0001|
|Pupal mass||0.202 (0.066)||0.349||9.66||0.0023|
|Pupal time||−0.197 (0.069)||0.389||8.24||0.0048|
|S||Pupal mass||0.372 (0.074)||0.113||18.32||<0.0001|
|Egg size||−0.218 (0.074)||0.227||9.23||0.0028|
|Pupal time||0.079 (0.071)||0.234||1.24||0.2670|
|Pupal mass||0.249 (0.108)||0.264||3.76||0.0672|
|Egg size||−0.188 (0.107)||0.300||3.74||0.0851|