Genetically controlled autumn leaf colouration and the aphid load in silver birch
Our results show that the green–yellow reflectance of silver birch leaves differs significantly among silver birch genotypes in the beginning and middle of the period of autumn leaf colouration. As far as we know, this is the first evidence that intrapopulation variation in autumn leaf colouration can have a significant genetic component. We also show that the genetic variation in silver birch leaf reflectance is positively correlated with the variation in leaf attractiveness for aphids. This suggests that the variation in leaf reflectance can affect aphid host selection in the autumn, and, supporting this, we found that the level of aphid infestation of silver birch trees was significantly explained by their genetically controlled autumn leaf colouration. In particular, we found that the abundance of wingless E. betulae females, which lay the over-wintering eggs, was highest at the end of leaf senescence in the silver birch genotypes that had the highest leaf green–yellow reflectance during the first phase of the senescence period. This suggests that those B. pendula trees that have leaves of lowest reflectance (i.e. the least yellow leaves) in the early autumn should gain over others in the spring when the new aphid generation emerges from the eggs. Significantly, however, it appears that the rank of silver birch genotypes is not stable through the entire period of autumn leaf colouration: in the latter phase of senescence, genetic differences in leaf reflectance disappear and there is a reversal in the rank of genotypes in terms of leaf attractiveness. This is an important finding and suggests that the aphid load of a particular birch genotype may depend on how the period of aphid host searching relates to the progress of autumn leaf colouration in different autumns. If the host searching period varies in different years, and unlike in our study does not always match with the early autumn leaf colouration period, the aphid load may not consistently be higher on certain genotypes and the coevolution between the two species might be attenuated. Likewise, year-to-year variation in weather conditions may have affected the coevolution. Short autumns with early frost should lead to tighter coevolution than long mild autumns that offer more temporal variation in genotypic differences. Noteworthily, if autumns are typically short and winters arrive early, aphid avoidance could efficiently reduce yellow leaf reflectance in early autumn, while aphid egg-laying could remain a weak selective agent in late autumn when there is not much time for aphid activity.
In contrast to what we expected, we did not find a significant relationship between the numbers of winged females, that is, the flying parents of the wingless females, and autumn leaf colour. This is probably a consequence of the low number of winged E. betulae females in our data set, as the preference of winged females for yellow over green leaves has been demonstrated earlier (Holopainen et al., 2009). The reason for wingless females being positively related to the green–yellow reflectance in our study is therefore probably a result of their winged parents preferring trees of high reflectance during host selection. Taken together, these findings indicate that E. betulae has the potential to drive the evolution of autumn leaf colouration in B. pendula populations. To conclusively demonstrate this, one should also provide evidence of the effect of aphid abundance on tree fitness (Archetti et al., 2009), but there are no such data available for silver birch. However, aphids are known to reduce tree growth and reproduction (Dixon, 1971; Kilpeläinen et al., 2009; Zvereva et al., 2010), and, combined with our data, such evidence indicates that natural selection should ultimately reduce yellow leaf colouration, or, as leaf yellowing is imposed by chlorophyll degradation, the evolution could be towards changing the timing or the duration of autumn leaf colouration in silver birch (e.g. Sinkkonen, 2006b; Archetti, 2009a).
To the human eye, hues of red are the most striking autumn leaf colours, but, unlike humans, aphids lack red photoreceptors and prefer both green and yellow over red (Döring et al., 2009). Red autumn leaf colours, which are caused by autumnal anthocyanin production, can therefore act as an antiherbivory adaptation. It was recently suggested that such adaptation was lost in most North European tree species during glaciations when insect populations became extinct, but tree populations survived in various refuges (Lev-Yadun & Holopainen, 2009). This would explain why tree species, like silver birch, in North Europe have yellow rather than red autumn leaf colours even though yellow attracts aphids. Our study provides novel findings that support this hypothesis: as intense yellow leaf colour exposes silver birch trees to aphid infestation and leaf reflectance has a genetic background, any mechanism that can camouflage the attractiveness of yellow leaves, such as red pigment synthesis in the autumn, should be favoured by natural selection. This suggests that herbivores can act as selective agents in the evolution of autumn leaf colours even when the active colour pigments, such as the carotenoids in silver birch, are not synthesized in the autumn but earlier in the growing season.
Our results do not support the hypothesis that yellow autumn leaf colours have evolved as a warning signal for aphids (Archetti, 2000; Hamilton & Brown, 2001), but suggest that the distinction between yellow and red autumn leaves in later hypotheses (e.g. Lev-Yadun & Gould, 2007; Archetti et al., 2009; Lev-Yadun & Holopainen, 2009) is indeed relevant. Although we found that leaf attractiveness in late autumn was negatively associated with the abundance of wingless E. betulae females (Fig. 2e), we did not find any winged females in our last survey and therefore host tree selection at this stage of leaf senescence had no potential to influence antiherbivory colouration. This suggests that the negative association between leaf attractiveness and aphid abundance was rather a legacy of the positive association that originated earlier in leaf senescence and host selection processes. In any case, this finding also stresses the importance of considering the temporal variation in the aphid–leaf colour association during the process of leaf senescence. Interestingly, we found that the abundance of E. betulae males was not related to leaf reflectance, not even at the tree phenotype level (r = 0.259, P = 0.116, n = 38, with trees involved that contained males), but was instead significantly correlated with the abundance of wingless females. This indicates that not all autumn-migrating aphids automatically concentrate on trees with the most attractive leaves, but also use other cues for their orientation, such as female pheromones in the case of male aphids (Holopainen et al., 2009).
The population density of E. betulae was low in our study: the average number of adults varied from 3 to 11 and the number of nymphs from 14 to 28 per 100 leaves in the three successive surveys. In the same year, in another locality, Holopainen et al. (2009) found an average of 56 adults and 63 nymphs per 100 yellow senescing leaves in their mid-summer survey of a population of flood-stressed 2-yr-old silver birch saplings. The reasons for the low densities in our study may be manifold, ranging from the low position of the inspected branches within tree canopies to the locality of the birch population in the landscape and the weather and season of the survey. Nevertheless, the difference in the abundance of wingless E. betulae females was over 20-fold between the genotypes of the lowest and highest aphid load. This shows that genotypic variation in the autumn leaf colours can lead to a noteworthy genotypic variation in aphid load within a local birch population.
Aphids and tree N dynamics during autumn leaf colouration
Our results show that at the time of the first survey leaf reflectance was positively associated with the reflectance difference between the first and second surveys (an indication of chlorophyll breakdown and phloem sap N loading between the surveys), which suggests that the most attractive trees at the onset of leaf colour change provided the highest sap N loading during early autumn leaf colouration. This seems to be consistent with the nutrient retranslocation hypothesis (Holopainen & Peltonen, 2002), which proposes that migrating aphids can use yellow as a signal of available N. However, the positive association between leaf attractiveness and subsequent sap N loading was reversed at mid-autumn leaf colouration and the most attractive trees in the second survey had the lowest sap N loading between the second and third surveys. This finding seems to support the side-effect hypothesis (Döring & Chittka, 2007) rather than the nutrient retranslocation hypothesis: choosing the yellowest trees does not seem to benefit the aphids (in terms of subsequent phloem N availability) in the middle of autumn leaf colouration. This shows, once again, how the genetically controlled attributes of leaf senescence and autumn leaf colouration can involve significant temporal variation. The remarkably low numbers of wingless E. betulae females in the trees of our genotype ‘30’, despite the highest reflectance and attractiveness of these trees at the time of the second survey (Fig. 2b,d), might be an indication that aphids are adapted to recognize this variation: that is, once the reflectance of a tree increases above a certain threshold value, aphids could start avoiding the tree because of a minimal subsequent N availability in such a tree. Among our genotypes, ‘30’ was the only genotype for which the survey 1 vs 2 difference in leaf reflectance was significantly higher than the survey 2 vs 3 difference (Fig. 1c), and, when contrasting the second survey reflectance and wingless E. betulae female abundance, ‘30’ emerges as a statistical outlier (a regression without ‘30’ would give F = 22.5, P < 0.001 and R2 = 0.58; Fig. 2b). Although this piece of evidence that aphids avoid trees above a threshold reflectance level is not unequivocal, it supports earlier observations of aphids avoiding leaves that will soon be shed (Glinwood & Pettersson, 2000) as well as the hypothesis that aphids can use leaf reflectance as an indication of upcoming leaf fall (Lev-Yadun & Gould, 2007). Finally, it seems to suggest that the possibility of coevolution between aphids and trees having yellow autumn leaf colours should not be completely ignored (e.g. Sinkkonen, 2006a; Archetti et al., 2009).
We further tested whether leaf attractiveness could be used as a sign of total autumnal N transfer from the senescing leaves. Our silver birch genotypes differed significantly in summer leaf N concentration and the variation caused a matching variation in the autumnal N transfer (measured as a difference between green leaf and leaf litter N concentrations). However, this genetic variation did not correlate with leaf reflectance or attractiveness at any stage of leaf senescence and therefore cannot be a significant factor in the context of colour-induced aphid host selection. Lastly, we tested if the responses of E. betulae life-cycle stages to leaf reflectance could be explained by N availability, but did not find evidence for this. The abundance of E. betulae wingless females in the third survey was associated with the reflectance difference between the second and third surveys, but, as this association was negative, it is not a likely reason for the observed abundance in the third survey. However, it has to be noted that, as aphids utilize soluble amino acids in leaf phloem, but not other forms of leaf N (White, 2009), and can recognize amino acid concentration in phloem sap (Nowak & Komor, 2010), our estimates of aphid N availability in the autumnal trees can only be considered approximate.
We found an indication that the autumnal aphid community could also reciprocally affect the silver birch N economy: the abundance of E. betulae males and the nymphs of the other aphid species found on silver birch genotypes in the first and second surveys explained a significant part of the variation in leaf litter N loss among the genotypes. These aphid groups do not appear to respond to leaf reflectance (this study and Holopainen et al., 2009) and we cannot suggest a mechanism to explain their effect on nutrient resorption efficiency, except that the effects seem to develop during early and mid leaf senescence when chlorophyll breakdown and N transfer are most active. Autumnal nutrient resorption has a major effect on tree growth and fitness (May & Killingbeck, 1992) and B. pendula spring leaf growth, for example, can exclusively rely on internal cycling for 2 wk after bud burst (Millard et al., 1998). As the association between the aphid abundance and N loss further seems to take place at the tree genotype level, this may cause additional selection pressure on those silver birch attributes, such as volatile emissions (Holopainen et al., 2010; Powell et al., 2006), that are not related to leaf colouration but may affect aphid load in trees in the autumn.
Plant–insect interactions have always been of considerable interest in ecology, but studies of late-season host–insect dynamics have been lacking, with the exception of migrating cereal pests (Klueken et al., 2009). The birch population that we used in our study has been shown to express significant genotypic variation in herbivore resistance during several leaf growing seasons (Silfver et al., 2009). Our results show that such intrapopulation genotypic variation in leaf characteristics and herbivore load are not restricted to the main leaf growing season, but also prevail during leaf senescence.
With respect to the coevolution hypothesis of autumn colouration (e.g. Archetti et al., 2009), this means that coevolution of autumn-migrating insects, like aphids, and tree autumn leaf colouration, is also possible when the colours attract (yellow) rather than discourage (red) the insects. Likewise, our results indicate that such coevolution may arise with aphid species that do not change their host species in the autumn, as long as they search for an optimal host while nutrient transport from leaves to woody tissues is ongoing. Taken together, although our study needs to be repeated with other tree species and genera, our findings indicate that, besides being one potential explanation for the myriad autumn leaf hues of trees in the temperate and boreal climate, the behaviour of autumn migrating insects may have a significant role in understanding tree–insect interactions in general.