Small first leaves promote subsequent leaf development in <i>Padus grayana</i>

Plants have evolved diverse leaf morphologies that perform various functions in individual plants (Bhatia et al., 2021; Critchfield, 1960; Kaplan, 1980). For example, the insectivorous trap leaves of carnivorous plants (Darwin, 1875) and the tendrils of climbing plants (Hofer et al., 2009) have specialized functions that facilitate nutrient absorption and structural support, respectively, through morphological adaptations suited to these functions. It is also known that leaves for photosynthesis exhibit considerable morphological diversity in individual plants (e.g., Kikuzawa, 1982; Kobayashi et al., 2008). For instance, leaves that develop in shaded environments tend to be thinner and more efficient in light utilization (Björkman, 1981; Gratani, 2014). Moreover, morphological differences can be observed depending on their position along a shoot (Aoyagi et al., 2024; Edwards et al., 2016; Kobayashi et al., 2008). Kobayashi et al. (2008) reported that the density of glandular hairs, a defensive trait, is higher in the first leaves of shoots. A prominent example of leaf morphological diversity within an individual is variation in leaf size. In the alder species Alnus hirsuta Turcz. (Betulaceae), the first leaves that emerge from the winter bud are particularly small (Kikuzawa, 1982). The lifespan of these small leaves is shorter than that of typical leaves that subsequently expand and become larger than the first leaves (Kikuzawa, 1982). Similar small first leaves are commonly observed in tree species across various taxa (Figure 1a). Despite the observed morphological and physiological differences between the first and typical leaves, their functional roles remain largely unexplored. Global-scale studies have indicated that leaves with shorter lifespans tend to have higher photosynthetic rates per leaf mass (Reich et al., 1997; Wright et al., 2004). Therefore, it is expected that small leaves with shorter lifespans have higher photosynthetic rates than typical leaves. If the small first leaves indeed exhibit higher photosynthetic rates, they could play a crucial role in supporting the growth of subsequent typical leaves by efficiently acquiring and supplying assimilates. However, no studies have directly tested whether the small first leaves contribute to the development of the subsequent typical leaves. In this study, we tested the hypothesis that small first leaves promote the growth of subsequent typical leaves. To assess the functional significance, we conducted a controlled shading experiment on small first leaves using saplings of Padus grayana. Buds were assigned to three treatment groups (covered, uncovered, and control), and morphological and physiological traits—specifically, leaf area and relative chlorophyll content measured as SPAD values (Soil & Plant Analyzer Development)—were monitored throughout the leaf expansion period, from 27 March to 2 May 2023. Growth dynamics of the third leaves, treated as typical leaves, were analyzed using linear and generalized linear mixed models to compare developmental patterns among treatments. A detailed description of the experimental procedures is provided in Appendix S1: Section S1. The first leaves differed from the third leaves in both morphology and physiology (Figure 1b,c, Appendix S1: Figure S1). The third leaves had an average size of 1004 ± 463 mm2, while the first leaves were significantly smaller, measuring 279 ± 251 mm2, irrespective of treatment (F = 217.37, p < 0.01; Figure 1b). The first and third leaves differed slightly in aspect ratio (F = 9.91, p < 0.01; Appendix S1: Figure S1a), but both the length and width were smaller in the first leaves (length: F = 331.65, p < 0.01; Appendix S1: Figure S1b, width: F = 187.49, p < 0.01; Appendix S1: Figure S1c). SPAD values were significantly higher in the first leaves than in the third leaves (F = 117.80, p < 0.01; Figure 1c); the third leaves had a SPAD value of 27.7 ± 5.2, whereas the first leaves had a value of 36.6 ± 7.1, approximately 32% higher. Additionally, when comparing the growth rate of the third leaves with and without shading of the first leaves, the growth rate under the shaded condition (covered treatment) was significantly slower than in the uncovered and control treatments (covered vs. control: χ2 = 7.94, p = 0.01; covered vs. uncovered: χ2 = 5.81, p = 0.02; Figure 1d). According to our logistic regression model, the third leaves in the covered treatment took more than a week longer—1.6 times as long—to fully expand compared to those in the other two treatments (number of days required for the growth rate to increase from 0.05 to 0.95: covered 18.10 days, uncovered 11.09 days, control 10.34 days; Figure 1d). However, there were no significant differences in the final size of the third leaves (area: χ2 = 0.25, p = 0.67; aspect ratio: F = 0.05, p = 0.94; Appendix S1: Figure S2a, length: F = 0.04, p = 0.96; Appendix S1: Figure S2b, width: F = 0.38, p = 0.68; Appendix S1: Figure S2c). These results suggest that the small first leaves accelerate the development of the third leaves. A threefold difference in size was observed between the first and third leaves, with the first leaves being significantly smaller than the third leaves. Previous studies have shown that small leaves with a narrow effective width exhibit high gas exchange efficiency and increased photosynthetic rates (Givnish & Vermeij, 1976; Leigh et al., 2017). Since SPAD values, which correlate with chlorophyll content and photosynthetic capacity (Croft et al., 2017; Xiong et al., 2015), were higher in the first leaves, it is suggested that the first leaves have higher photosynthetic rates than other leaves. In deciduous trees, early leaf emergence can influence total annual photosynthetic output (Harrington et al., 1989; Lopez et al., 2008). By increasing the photosynthetic rate of the first leaves, which emerge earliest from the winter bud, assimilates needed for the expansion of subsequently developing typical leaves can be efficiently acquired (Milberg & Lamont, 1997). This allows photosynthesis in the absence of shade, particularly in deciduous forests, by enabling leaves to expand about a week ahead of surrounding trees, potentially enhancing the overall productivity of the individual (Dion et al., 2017). Indeed, the finding that the third leaves in the control and uncovered treatments grew faster than those in the covered treatment supports this hypothesis. Reducing leaf size improves heat exchange efficiency, thereby facilitating the avoidance of low-temperature injury from nocturnal radiative cooling (Wright et al., 2017). Leaf emergence in early spring carries a greater risk of frost damage, leading to leaf loss (Hufkens et al., 2012; Wang et al., 2025). The first-emerging leaves, which appear during colder periods, are more vulnerable to frost damage compared to the third leaves. The small size of the first leaves may contribute to their ability to mitigate frost, particularly since they emerge earliest and are exposed to colder conditions. Furthermore, in cases where leaf loss occurs due to frost, smaller leaves are expected to minimize carbon loss. It is expected that small first leaves evolved more readily in regions with a high risk of late frost, such as higher latitudes (Zohner et al., 2020). In this study, it was observed that the first leaves are extremely small, and their presence promotes the subsequent growth rate of the typical leaves in P. grayana. We covered the first leaves and measured the growth of the third leaves; however, for example, by shading the third leaves and measuring the growth of the fifth leaves, one could evaluate the general or specific effect of earlier-emerging leaves on the development of later-emerging leaves. Moreover, species with small first leaves are distributed across diverse phylogenetic lineages (Aoyagi et al., 2024; Kikuzawa, 1982; Figure 1a), suggesting that this trait likely evolved independently within each clade. Conducting experiments on species other than P. grayana, the focus of this study, a more general understanding of the convergent significance of small first leaves can be obtained. Furthermore, by examining the environmental conditions in which small first leaves evolved and the physiological responses associated with their development, deeper insights may be gained into the adaptive significance of leaf morphological diversity. These studies may help clarify the significance of leaf shape diversity, which remains largely unclear. Hiyori Sasaki and Akira Yamawo developed the core idea, designed and carried out all the experiments. Hiyori Sasaki analyzed the data. Hiyori Sasaki and Akira Yamawo drafted the article. We thank the editor and reviewer for their constructive comments, which greatly improved the clarity and quality of the manuscript. We are also grateful to Dr. Misuzu Ohno for discussions during the initial stages of this project, and Dr. Naoto Shinohara and Teruki Inoue for improving the manuscript. We also thank Akira Matsumoto and Satomi Yoshinami for technical support. The authors declare no conflicts of interest. Data and code (Sasaki, 2025) are available in Figshare at https://doi.org/10.6084/m9.figshare.28827395.v1. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.