Experiment 2: Ploidy effects on tree traits and their response to stress
Among Wisconsin genotypes, we found no difference between diploid and triploid trees for any aboveground growth traits when averaged across all stress treatments (Fig. 2, Appendix 3). In contrast, genotypic differences significantly influenced most growth traits. Tree genotype was particularly relevant for determining final height and SLA and explained 39% and 30% of the total observed variance in these two traits (effect size plots, Fig. 2).
Tree growth and biomass allocation traits were affected by drought stress, defoliation or their interaction (Fig. 2). At a given tree weight, drought-stressed trees had on average 8% lower leaf weights than did watered trees. Similarly, defoliation reduced total leaf weight and SLA. For total tree weight we observed significant drought × defoliation interactions. Nondefoliated, watered trees had 25% greater total weight when compared with nondefoliated, drought-stressed trees. However, when trees were defoliated, drought-treatment differences disappeared. Similar, albeit less pronounced interaction effects between drought and defoliation treatments were also observed for total leaf weight. Cytotypes showed similar responses to drought stress and defoliation (no significant ploidy × treatment interactions were found). Furthermore, we did not observe any genotype × treatment interactions.
Concentrations of most chemical compounds did not differ between the cytotypes when averaged across all treatments (Fig. 3, Appendix 3, 4). The only exception was tremulacin, which was 17% higher in triploid trees than in diploids (Appendix 4). However, differences in ploidy levels could mainly be attributed to a single triploid genotype (Appendix 4). Genotypic differences significantly affected most chemical compounds (Fig. 3, Appendix 3,4). Genotypic effects accounted for 30% of the variation in total salicinoid phenolic glycosides and almost 50% of the variation in condensed tannins (Fig. 3, effect size plot).
Drought stress and defoliation did not affect concentrations of total phenolic glycoside levels. Drought, however, led to increased levels of the phenolic glycosides salicortin (7% increase) and salicin (44% increase) (Appendix 3). Condensed tannin concentrations increased by 25% under drought stress and by 56% in response to defoliation. Nitrogen levels increased by 11% under drought stress, and decreased minimally (<5%) due to defoliation. Ploidy levels as well as the different genotypes responded similarly to drought stress and defoliation (no significant ploidy × treatment or genotype × treatment interactions).
Differences in ploidy affected photosynthetic rates (Fig. 4a) and Vcmax (Appendix 5). Well-watered triploids exhibited 11% higher photosynthetic rates and 23% higher Vcmax. Cytotype explained almost 20% of the total variation in Vcmax (Fig. 4a, Appendix 5, effect size plots). No other leaf physiological traits were affected by cytotype levels. Genotype significantly affected variation of photosynthetic rate, stomatal conductance and the quantum effeciency of PSII, explaining 10-20% of the variation in all traits (Fig. 4a, Appendix 5).
In well-watered trees, defoliation affected only stomatal conductance, which was 3% higher in defoliated than in nondefoliated trees (Appendix 5). No significant ploidy × treatment or genotype × treatment interactions were found. For trees that were exposed to drought stress, we observed similar vulnerability curve progressions for diploid and triploid trees of the same defoliation treatment. No significant impact of ploidy or ploidy × defoliation interactions was detected for any curve parameter (Fig. 4b, Appendix 6). However, defoliation treatments per se significantly affected net photosynthesis and stomatal conductance responses to drought. Photosynthetic rates of nondefoliated trees plummeted when experiencing ΨPD of -7 bars or lower. In contrast, photosynthesis rates of defoliated trees decreased more gradually. Defoliated trees were still photosynthetically active, even when experiencing severe drought stress. Correspondingly, we found significant differences between curve parameters describing the defoliated and nondefoliated tree curves (Fig. 4b, Appendix 6).
Experiment 3: Ploidy effects under drought recovery
Diploid and triploid trees differed in growth responses during recovery from extensive drought stress. Across defoliation treatments, triploids produced 35% more new tissue than did diploid trees (Fig. 5). None of the other growth traits was affected by cytotype differences (Fig. 5, Appendix 7, 8). In contrast, genotype significantly affected all growth traits except new tissue production and explained 10-20% of the variation in the affected traits (Fig.5, Appendix 7, 8).
Trees that were defoliated prior to drought showed higher growth responses during drought recovery than trees that were not defoliated. Defoliated trees allocated more of their stem mass to height growth (increased by 10%), produced 45% more new tissue during recovery, and exceeded nondefoliated trees by 19% in total weight at the end of the experiment (Appendix 7). Defoliation also caused trees to invest more in leaf production (leaf weight at a given tree weight increased by 30%) and in thicker, smaller leaves (SLA decreased by 9%) (Fig. 5, Appendix 7). Cytotypes as well as genotypes responded similarly to the defoliation treatment (no ploidy × treatment or genotype × treatment interactions).
DISCUSSION
In this research, we disentangled two components of genetic diversity, genotypic variation and ploidy level, and quantified their relevance for growth, biomass allocation, morphology, phytochemistry, and physiological traits. Population and genotypic differences typically obscured ploidy effects for both a wide array of traits and responses of those traits to drought and defoliation. One key illustration of this is observed variation in tree growth, along with it physiological, morphological and allocational determinants, among nonstressed trees in Experiment 1. Ploidy level had no clearly discernible influence on growth, which differed substantially across populations as well as genotypes within each population. This variation was consistent with corresponding population/genotype differences in LWR, SLA, and, to a lesser extent, Aarea, the three principal growth determinants in young trees (Kruger and Volin 2006). Only a few traits, such as drought resilience, photosynthetic performance and levels of certain chemical defenses, showed subtle, albeit significant, differences between diploid and triploid aspen. Our research highlights that, despite the recognized importance of ploidy for tree ecology, ploidy effects may pale in contrast to genotypic effects in determining functional trait expression and trait responses to in highly diverse tree species. Similar findings were recently reported by DeRose et al. (2022) for natural populations of mature aspen in northern Utah.
Trembling aspen is known for its exceptionally high genetic and trait variation (Callahan et al. 2013; Jelinski and Cheliak 1992; Mitton and Grant 1996). Our finding that the vast majority of trait variation was driven by genotypic rather than ploidy differences confirms the well-documented importance of genotypic diversity for determining tree environmental adaptation and evolution (Petit and Hampe 2006; Westerband et al. 2021).
Regarding ploidy effects on phytochemistry, the observed difference in foliar tremulacin level between triploids and diploids (Appendix 4) accords with results from studies on other plant systems finding that polyploidy can lead to altered secondary chemistry (Gaynor et al. 2020; Park et al. 2021; Te Beest et al. 2012). The ploidy effect on tremulacin level, however, was moderate (absolute differences of ~1.5% dw across defoliation and drought treatments) and mainly driven by a single triploid genotype that showed increased defense levels. In contrast to our results, DeRose et al. (2022) reported for mature aspen in northern Utah that triploids exhibited 21% higher levels of salicinoid phenolic glycosides than diploids. Similar to our findings, though, expression of condensed tannins did not differ between cytotypes (DeRose et al. 2022).
The influence of ploidy level on light-saturated photosynthesis in our Wisconsin genotypes (Fig. 4a) is also generally consistent with previous findings. Greer et al. (2018) reported that mature triploid aspen clones in the Rocky Mountains exhibited higher net carbon assimilation rates than did diploid clones. In contrast to our results, however, the key driver of enhanced photosynthetic performance in mature triploids was increased stomatal conductance rather than heightened rubisco activity (Vcmax, Appendix 5). Regardless, given the central role that leaf photosynthetic performance plays in plant carbon balance, differences in photosynthesis between diploid and triploid trees might ultimately drive other trait-level divergences between the two cytotypes. For example, increased photosynthetic rates in triploids possibly contributed, via greater availability of assimilated carbon, to higher tremulacin levels in triploid foliage. Enhanced photosynthetic activity would also likely increase biomass growth in triploids, but corresponding growth differences were not observed in our study. We acknowledge, however, that our ability to thoroughly assess links between growth and its determinants is precluded by the absence of data on belowground biomass allocation. Other studies on mature aspen have found that triploid genotypes exceed diploids in stem growth rates and stem diameter (DeRose et al. 2015).
The absence of marked cytotype differences in trait response to defoliation and/or recurring drought in our study (no ploidy × treatment interactions) agrees with results from research on other plant species such as strawberry, Fragaria vesca (Wei et al. 2020) the herbaceous perennial Rhodohypoxis baurii (Mtileni et al. 2021), and the annual herb Mercurialis annua (Vilas and Pannell 2017). Yet a growing body of research documents that polyploidy in plants confers a benefit under environmental stress (Van de Peer et al. 2021). For example, allotetraploid grass (Brachypodium distachyon) (Manzaneda et al. 2012), autotetraploid Rangpur lime hybids (Citru limonia × Citrus sinensis) (Allario et al. 2013) and polyploid gum Arabic tree (Acacia senegal) (Diallo et al. 2016) were more drought tolerant than their diploid counterparts. Several reasons may explain the discrepancy between our results and the aforementioned examples. Firstly, the extent to which polyploidy can confer increased stress tolerance can differ greatly among plant species and ecological context (Fox et al. 2020; Van de Peer et al. 2021). Moreover, the importance of cytotype for stress adaptation may depend on the source of cytotype variation (e.g. polyploids that arise within a species [autopolyploidy] or from hybridization of two distinct species [allopolyploidy]), the number of multiplied chromosome sets (triploidy, tetraploidy, etc.) or gene dosage effects (Bastiaanse et al. 2019; Van de Peer et al. 2021; Van de Peer et al. 2017). Finally, ploidy effects have rarely been explored in the context of intraspecific genotypic variation. Our findings underscore the importance of considering conventional genotypic variation when exploring the ecological relevance of ploidy effects in future studies.
Cytotype differences affected trait response to environmental stress only in one case: triploid genotypes displayed a slightlygreater drought resilience than did diploid genotypes, as measured via increased production of new lateral shoot tissue during post-drought recovery (Fig. 5). As a consequence, when growing in regions frequented by drought, young triploid trees might outcompete young diploid trees. A cytotype difference in drought resilience could help explain the comparative abundance of triploid clones in the arid regions of North America (Mock et al. 2012; but see Latutrie et al. (2019)). However, recent studies by Blonder et al. (2021), Blonder et al. (2022) and (Greer et al. 2018) found that triploid mature aspen appear to be more susceptible to drought stress than diploids. These findings suggest that cytotype effects on drought-tolerance in aspen may vary among developmental stages.
Defoliation rivaled or exceeded polyploidy with respect to the magnitude of its positive impact on drought resilience (Fig. 5, Appendix 7). This outcome can be explained in part by the fact that leaves of defoliated trees sustained stomatal opening and photosynthesis at soil moisture deficits well beyond the threshold causing pronounced stomatal closure (and decreased photosynthesis) in foliage of nondefoliated trees (Fig. 4b, Appendix 6). A similar influence of defoliation on leaf photosynthetic response to water stress has been observed in other studies (McGraw et al. 1990; Pinkard et al. 2011). It may stem from the effects of defoliation on the fundamental balance between transpirational demand and moisture supply, which is thought to exert considerable control over stomatal behavior (Sperry et al. 1998). Consistent with the observed growth responses of droughted trees in this study (Fig. 2) and others (Jacquet et al. 2014; Pinkard et al. 2011), a comparative increase in leaf carbon gain would help mitigate the negative effects of defoliation on biomass growth. Correspondingly, the increase would allow defoliated trees to accumulate more carbohydrate reserves and, during subsequent drought recovery, invest more resources into biomass production relative to nondefoliated trees. These phenomena and their underlying mechanisms warrant further investigation, as we have not found published corroboration of this impact of defoliation impact on drought recovery.
In conclusion, our study shows that in young trembling aspen, genotypic differences were key determinants of functional trait variation, and the magnitude of that variation obscured potential ploidy effects. Upon accounting for genotypic effects, only a few traits differed between diploid and triploid trees. Triploid trees exhibited slightly higher levels of defense and photosynthesis, as well as higher resilience to extended drought events. Hence, while genotypic differences are key determinants of aspen’s adaptation to environmental stress, young triploid trees might possess additional subtle advantages when confronted with drought or herbivory. With the advance of climate change, forests are increasingly exposed to both environmental stressors (Aber et al. 2001; Seidl et al. 2017). Accordingly, the possibility that herbivore-mediated defoliation, in addition to genetic determinants, can buffer negative drought effects is an important consideration when assessing the consequences of future stressors on forest ecosystems.
FUNDING
This work was supported by the Swiss National Science Foundation [P2BEP3_175254] and USDA National Institute of Food and Agriculture gransts 2016-67013-25088 and WIS01651. Participation of coauthor KM was supported by the Utah Agricultural Experiment Station, and this is UAES publication #9516.
ACKNOWLEDGEMENTS
We thank Chris Cole, Jack Schaefer, Allyson Richins, Andrew Helm and the Walnut street greenhouse staff for help with setting up the experiments, Mark Zierden for assistance with the chemical analyses, Rick Jellen for cytotype identification and Rebecca Best for advice regarding statistical analyses.