Proceedings of the Royal Society B: Biological Sciences
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Green greenhouse: leaf enclosure for fruit development of an androdioecious vine, Schizopepon bryoniifolius

Nobuyuki Nagaoka

Nobuyuki Nagaoka

Yamagata Prefectural Natural Museum Park, Ubagatake 159, Shizu, Nishikawa-cho, Nishi-Murayama-gun 990-0734, Japan

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Shoji Naoe

Shoji Naoe

Tohoku Research Center, Forestry and Forest Products Research Institute, Nabeyashiki, Shimokuriyagawa 92-25, Morioka 020-0123, Japan

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Yu Takano-Masuya

Yu Takano-Masuya

Yamagata Prefectural Natural Museum Park, Ubagatake 159, Shizu, Nishikawa-cho, Nishi-Murayama-gun 990-0734, Japan

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Shoko Sakai

Shoko Sakai

Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu 520-2113, Japan

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Abstract

Individual plants can produce leaves that differ substantially in size, morphology and many other traits. However, leaves that play a specific role in reproduction have rarely been reported. Here, we report leaves specialized to enclose fruit clusters and enhance seed production in an annual vine, Schizopepon bryoniifolius. Enclosure leaves were produced at the end of the growing season in late autumn. They were different in greenness and structure from other leaves. Under solar radiation, the ambient temperature inside an intact enclosure was up to 4.6°C higher than that near a fruit cluster whose enclosure leaves had been removed. We found that enclosures were thicker at colder sites. Removal of enclosing leaves negatively affected fruit survival and/or growth, but we could not identify the exact mechanism. The results suggested that enclosures allow the plant to produce seeds under the cold weather the plant encounters at the end of its life. Vegetative and reproductive traits of plants have usually been studied separately. This study indicates how they can dynamically interact, as shown by an examination of associations among leaf and reproductive trait changes according to life stages.

1. Introduction

Individual plants can produce leaves that differ substantially in size, morphology, physiology and photosynthetic ability [14]. Leaf trait variation within an individual is often so large that it substantially exceeds the leaf trait variation among individuals of the same species [1]. Such variation is often explained as a strategy to optimize the exploitation of environmental variation in time and/or space and enhance whole-plant photosynthetic performance [1,2,5,6]. For instance, many plants change leaf traits substantially during ontogeny from ‘juvenile’ to ‘adult’ forms (heteroblasty) in correspondence with different light conditions between the upper and lower parts within a canopy [2,7]. Although the adaptive value of heteroblasty in relation to leaf physiology is well acknowledged [2,7,8], leaf trait variation associated with reproduction has rarely been reported.

Leaves could potentially play a role in reproduction by attracting a pollinator or seed disperser or by protecting reproductive organs, as sepals usually do [9]. The rarity of leaf traits associated with reproduction is probably because traits for such attraction or protection are in conflict with traits that promote photosynthesis, the primary function of leaves. Leaf colours differing greatly from green may reduce the absorption of photosynthetically active radiation and light-use efficiency [10,11]. Leaves arranged to protect reproductive organs may capture less solar radiation than those optimally arranged for photosynthesis [12]. These conflicts cause a problem when leaves are retained on the plant far longer than reproductive organs are. Some plants solve this problem by reversibly changing leaf traits only during reproduction. For instance, Saururus chinensis (Saururaceae) changes the colour of its apical leaves subtending the inflorescences from green to white during flowering to enhance pollinator attraction. Though leaf whitening results in reduction of photosynthetic capacity, leaves turn back from white to green after flowering, and their photosynthetic capacity also recovers to the level of ‘normal’ green leaves [9].

Here, we report, for the first time, a unique function of leaves that enclose immature fruits in an annual vine, Schizopepon bryoniifolius (Cucurbitaceae). The plant has a sexual system of androdioecy, in which hermaphrodite and male plants co-occur within a population [13,14]. We found that some leaves on a hermaphrodite that were undeveloped in summer expanded and overlapped with each other to form an enclosure with many immature fruits inside in autumn. This leaf behaviour was first discovered in 2008 and has been observed every year since then at the study site by the first author. Hereafter, we refer to the structure as an ‘enclosure’, a leaf that forms the enclosure as an ‘enclosure leaf’, and a normal leaf as a ‘solitary leaf’. In this report, we first describe in detail how the enclosure develops and compare the characteristics of enclosure leaves to those of solitary leaves. Plant sexual reproduction processes are very sensitive to temperature and temperature fluctuation [15,16]. As air temperatures become lower and fluctuate, including dropping below the freezing point, late in the season, we hypothesize that enclosure formation is an adaptive response to enhance fruits' survival and development by keeping the air temperature comparatively higher and stable around the developing fruits. Therefore, we examined (i) the correlation between local ambient temperature and the enclosure thickness evaluated by the total width of the enclosure leaves; (ii) the effects of the enclosure on the regulation of air temperature around the developing fruits; and (iii) the effects of the enclosure on fruit survival and development.

2. Material and methods

(a) Study species

Schizopepon bryoniifolius (Cucurbitaceae) is a slender annual vine distributed in East Asia (Japan, Korea, Sakhalin and northeast China). It commonly inhabits edges of deciduous forests with chronic disturbances such as roadsides or riversides at the foot of mountains. The species is androdioecious and produces small white flowers mostly pollinated by dipterans from late August to September [14,17,18]. Male plants produce a racemose or paniculate inflorescence with many flowers at the axil of a leaf (figure 1a). Flowers on the males have three stamens but lack a stigma [17]. By contrast, hermaphrodite individuals produce a single flower on the leaf axil. It has three stamens and one three-lobed stigma (figure 1d and e). It eventually develops into a fruit with a single seed. The thick wall of the fruit dehisces to disperse the seed by gravity after it matures (figure 1f).

Figure 1.

Figure 1. Features of S. bryoniifolius. (a) Male inflorescence, (b) solitary hermaphrodite flower, (c) flowers on short-internode shoot, (d) close-up of solitary hermaphrodite flower, (e) close-up of hermaphrodite flower on a short-internode shoot (flower cluster), (f) dehisced fruit after dispersal from a solitary flower, (g) section of solitary leaf and (h) section of enclosure leaf. Bars = 2 cm (ac,f), 2 mm (d,e) and 100 µm (g,h). (Online version in colour.)

(b) Study sites

We conducted the study at the foot of Mt Gassan, in the southern part of the Dewa Mountains, Yamagata Prefecture in Japan (electronic supplementary material, figure S1). We chose two populations of S. bryoniifolius at different altitudes because we found significant variation in enclosure development among populations in preliminary observations and considered that it was related to temperature variation. Each population forms a more or less continuous patch along roads and trails. The population at higher altitude (hereafter, population M) is within the Yamagata Prefectural Natural Museum Park (38°30′14″ N, 139°59′55″ E, 800–820 m.a.s.l.). The lower population L is located 6.7 km south (38°27′24″ N, 139°59′55″ E, 435–535 m.a.s.l.). The proportion of male plants was 0.120 and 0.448 in population M (n = 58) and population L (n = 83), respectively.

The annual precipitation of the region is approximately 2550 mm, and the mean annual temperature is 8.7°C, with average monthly temperatures ranging from −2.5°C in January to 21.8°C in August, based on data recorded at the Oisawa weather station at altitude 440 m (Japan Meteorological Agency, http://www.jma.go.jp; electronic supplementary material, figure S1). The maximum snow depth in winter is about 365 cm. The original vegetation of the area is a deciduous forest dominated by Fagus crenata (Fagaceae), while it has mostly been replaced by secondary vegetation around population L.

(c) Observation of enclosure

We collected enclosures in September 2017 to examine their structure and the characteristics of enclosure leaves. Chlorophyll contents (greenness) of the largest three leaves on an enclosure and a solitary leaf near the enclosure were compared for two enclosures of three individuals. We measured SPAD (Soil and Plant Analyzer Development) values of 20 points on each leaf using a SPAD-502 chlorophyll concentration meter (Minolta Camera Co., Japan). The SPAD meter measures the absorbance of the leaf in the red and near-infrared region. Using these two absorbances, it calculates a SPAD value, which is highly correlated with the amount of chlorophyll present in the leaf [19]. To examine the anatomical structure, solitary and enclosure leaves were fixed with FAA (formaldehyde, acetic acid, alcohol). The samples were paraffin-embedded, sectioned and stained with haematoxylin/eosin and safranin-O/fast green.

(d) Ambient temperature and enclosure thickness

If the enclosure protects fruit against low temperature, thicker enclosures may develop at colder sites. To assess the relationship between climate conditions and enclosure thickness, we measured ambient temperature and leaf size of short-internode shoots at six and five sites in population M and L, respectively. At each site, we placed a temperature logger (Model TR-5i; T&D Corporation, Japan; ±0.3°C accuracy and 0.1°C resolution) about 40 cm above the ground. Temperature was recorded every 10 min, and measurements during the 40 days from 19 August to 27 September were averaged for each site. On 28 September, 8–10 fruit clusters on dwarf shoots within 1.5 m from the temperature logger at each site (96 clusters in total) were sampled, and the total sum of the widths of the three largest leaves of the clusters was calculated as an index of enclosure development.

(e) Temperature measurement within enclosure

We compared ambient temperature within an intact enclosure and near a fruit cluster whose enclosing leaves had been removed on the same plant in population M. We measured temperature using a temperature logger (model TR-52i) equipped with a thin sensor (TR-5620; T&D Corporation; ±0.5°C accuracy and 0.1°C resolution) every 10 min during the four days from 29 September until 2 October. The data of sunshine duration (min) during every 10 min recorded at Oisawa Weather Station 16 km south of population M (electronic supplementary material, figure S1) were obtained from Japan Meteorological Agency (http://www.jma.go.jp).

(f) Effects of enclosure on fruit set

To examine the effect of enclosure on fruit set, we conducted an experiment to remove the leaves of enclosures on five individuals in population M. We tagged 6–9 enclosures at similar developmental stages on each individual and counted immature fruits on 28–29 September 2017. Then, we assigned the enclosures to three treatments: (i) leaf-removed, in which we removed all large leaves covering the fruit cluster (12 enclosures, 141 fruits); (ii) bagged, in which we removed leaves as in (i) and covered the cluster using a waterproof double layer paper bag (Tsumiyama Co. Ltd.) (12 enclosures, 131 fruits; this treatment was conducted to examine if the physical damage of the leaf removal alone could affect fruit set); and (iii) intact enclosures as a control (13 enclosures, 116 fruits). We sampled these enclosures on 24 October and estimated the status of the 388 immature fruits recorded in September as ‘developed’ (larger than 1.0 cm) or ‘undeveloped’ (aborted or less than 1.0 cm) based on the number of fruits larger than 1.0 cm in length on the shoot, including ones already dehisced and after seed dispersal. The threshold (1.0 cm) was determined based on the clear bimodal distribution of immature fruit size at this developmental stage (electronic supplementary material, figure S2).

(g) Statistical analyses

We statistically examined the differences in SPAD values among leaves using linear mixed-effect models (LMMs). To test whether enclosure leaves had lower values than the nearest solitary leaf, we constructed an LMM with type of leaves (‘solitary’ or ‘enclosure’) as a fixed term, with the enclosure and individual as random factors. The significance of the fixed term was tested by comparing the fit of the original model with that of a reduced model with the fixed term removed by the anova function in R. To test if inner enclosure leaves had lower values than outer ones, we constructed an LMM with the order of leaves (1–4) as a fixed term, with the enclosure and individual as random factors. The significance of the fixed term was tested by comparing the fit of the original model with that of a reduced model without the fixed term as above. Normal distribution of the SPAD value was confirmed using the Shapiro–Wilk test prior to the analysis (result not shown). All of the statistical analyses were conducted using the statistical software R [20], and LMM and GLMM analyses were performed using the lme4 package [21] implemented in R.

To statistically examine the relationship between ambient temperature and the degree of enclosure development, we used an LMM with total leaf width as the dependent variable, mean temperature as a fixed term and population (M, L) and site (1–11) as nested random terms. Normal distribution of total leaf width was confirmed using the Shapiro–Wilk test prior to the analysis (result not shown).

We evaluated the magnitude, the temperature fluctuation in the intact enclosure and nearby leaf-removed fruit cluster by decomposing time series of temperature data into diurnal, trend and irregular components by loess smoothing using the tsl function in R [20] (electronic supplementary material, figure S3). The span of the loess window for trend extraction was set to five (50 min) since we were interested in short-term fluctuations during less than 1 h. The difference in the variations of the irregular components between intact enclosure and leaf-removed fruit cluster were tested using the F-test.

To examine the effects of the leaf-removed and bagged treatments on the probability of the fruits being ‘developed’ and ‘undeveloped’, we fitted the GLMM with binomial errors with the treatments as a fixed factor and with the enclosure and individual as random factors.

3. Results

(a) Enclosure development

Hermaphrodite and male individuals start flowering at about the same time in August. Early in the flowering season, hermaphrodite flowers were spatially separated from each other by a long petiole and internode on the shoot (figures 1b and 2a). The stigma was exerted from the surrounding anthers (figure 1d). Later in September, shoots ceased elongation, and hermaphrodite flowers started to be produced at the apex of the shoot. The resultant flower clusters looked like an inflorescence or raceme [17], but each cluster was actually a group of solitary flowers, each of which was produced at the axil of the leaf on a shoot with short internodes. Leaves on the dwarf shoots had already been formed, although most of them were small or even rudimentary at that time and few leaves were unfolded and expanded (figures 1c and 2b) [17]. In late September, some leaves on the short-internode shoot started to expand and form an enclosure covering these flowers and fruits (figure 3). Flowers that opened in the enclosures were smaller compared with flowers produced in the earlier season (figure 1d and e). The stigma height was slightly lower than the surrounding anthers (figure 1e). At that time, fruits on long internode shoots had grown to be the almost mature size.

Figure 2.

Figure 2. Seasonal change in the shoot structure of S. bryoniifolius. (a) Early in the flowering season, flowers are spatially separated from each other by a long petiole and internode on the shoot. (b) Later in the flowering season, shoots stop elongation and flowers are packed together on short-internode shoots. Most leaves on short-internode shoots are small or rudimentary if they do not form an enclosure. (c) At the end of the season, some of the leaves on short-internode shoots grow to a size comparable to solitary leaves and enclose the entire shoot. (Online version in colour.)

Figure 3.

Figure 3. Enclosures of S. bryoniifolius. (a) Enclosures hanging down from the plant, (b) interior of enclosure observed from underneath, (c) removed enclosures and (d) dissected enclosure with removed leaves arranged from top to bottom and right to left. Bars = 5 cm (a,c,d) and 2 cm (b). (Online version in colour.)

Leaves of the enclosures were wrinkled and decreased in size from outside to inside (figure 3d). The SPAD values of the enclosure leaves (15.4 ± 7.1, mean ± s.d.) were about half of those of solitary leaves (29.7 ± 5.89), and they were significantly different from the latter (χ2 = 23.009, p < 0.0001), indicating that the chlorophyll content of the enclosure leaves was about half of that of solitary leaves. The SPAD value of the enclosure leaves significantly decreased from outside to inside (χ2 = 36.987, p < 0.0001; electronic supplementary material, figure S4). Enclosure leaves lacked a clear palisade layer and had sparse connections between the upper and lower epidermis compared with solitary leaves (figure 1g and h). Chloroplasts in the enclosure leaves were smaller than those of solitary leaves (figure 1g and h).

(b) Ambient temperature and enclosure thickness

LMM analysis indicated that the total width of the three largest leaves on a fruit cluster was significantly larger at colder sites (χ2 = 6.62, p = 0.0108; figure 4). This supports the notion that the enclosure was thicker at colder sites at the time of observation (28 September).

Figure 4.

Figure 4. Relationship between the leaf size of the short-internode shoots and mean temperature. Temperature was monitored at six and five sites in population M and L, respectively (electronic supplementary material, figure S1). Measurements every 10 min during the 40 days from August 19 to September 27 were averaged for each site. At each site, 8–10 fruit clusters were collected and total widths of the three largest leaves of the fruit clusters were measured. LMM analysis indicated a negative relationship between the total width of the leaves and temperature. The line was drawn using the slope and intercept estimated by the LMM.

(c) Temperature within enclosure

The ambient temperature within the intact enclosure ranged from 4.3 to 26.1°C (12.1°C on average), while that near the fruit cluster with leaves removed ranged from 4.5 to 24.3°C (12.0°C on average) during the measurement (figure 5). The temperature differences between the two were significant (paired t-test, t = –5.119, p < 0.0001), while the difference (0.14°C on average) was relatively small across the entire period. However, the temperature in the intact enclosure was higher around noon on sunny days by up to 4.6°C compared to that near the fruit cluster with leaves removed. Moreover, the fluctuation of temperature was generally smaller in the intact enclosure. The variation of the irregular component of the time series after removing diurnal and trend components was significantly higher near the leaf-removed fruit cluster than that in the intact enclosure (electronic supplementary material, figure S3; F = 0.3355, p < 0.0001).

Figure 5.

Figure 5. Changes in the ambient temperature within an intact enclosure and that near a leaf-removed fruit cluster. Blue and red lines show the changes in the temperature in the intact enclosure and leaf-removed fruit cluster, respectively. Orange bars show the duration of sunshine during every 10 min measured at Oisawa weather station (electronic supplementary material, figure S1) obtained from the database of Japan Meteorological Agency (http://www.jma.go.jp/).

(d) Effects of leaf removal on fruit set

The proportion of fruits greater than 1 cm in length on 24 October among fruits that had been immature about one month before ranged from 0.13 to 0.92 (figure 6). The proportion was 0.38 ± 0.24 (mean ± s.d.) for the fruit clusters under the leaf-removal treatment, 0.53 ± 0.16 for those under the leaf-removed and bagged treatment, and 0.53 ± 0.14 for the control. GLMM analysis indicated that the probability that the fruits would be developed differed between the control and leaf-removed treatments (z-value = –2.069, odds ratio = 0.541, p = 0.0385), while it was not significantly different between the control treatment and the leaf-removed and bagged treatment (z-value = –0.154, odds ratio = 0.958, p = 0.877).

Figure 6.

Figure 6. Proportion of the number of fruits greater than 1 cm in length on 28–29 September among immature fruits observed four weeks before on fruit clusters under the control, leaf-removed, and leaf-removed and bagged treatments. Dots indicate values for individual enclosures, and the box plot shows the distribution for the different treatments. The thick line indicates the median, the lower and upper hinges indicate the first and third quantiles, and the lower and upper and ends of the whiskers indicate the minimum and maximum values except for outliers. Fruit clusters on different individuals are distinguished by colours.

4. Discussion

In this study, we documented a unique habit of S. bryoniifolius: it encloses fruit-bearing shoots by using leaves at the end of the reproductive season. Our experiments indicate that the enclosures enhance survival and/or growth of the fruits. Fruit survival and/or growth under leaf-removal treatments was significantly lower than that of control fruits (figure 6). However, negative effects of leaf removal were not observed when we put a paper bag over the enclosures after leaf removal, indicating that the effects of leaf removal were not due to mechanical damage or a decrease of photosynthetic products caused by reduction of the leaf area. Since S. bryoniifolius is an annual plant, it cannot carry over excess resources to the next growing season. To maximize seed production, the plant should convert all the assimilate into fruits and seeds over time after it ceases photosynthesis. Enclosure formation may be a strategy to enhance the conversion of resources to fruit development under the colder environment in the late autumn by utilizing leaves that have lost their primary role after the climate has become too cold for photosynthesis.

(a) Microclimate regulation by enclosure

We observed a maximum increase in ambient temperature of 4.6°C and a significant decrease of temperature fluctuation in the enclosure. Regulation of temperature through physiological processes has been documented for plants of different groups, such as in Rafflesiaceae [22], Araceae [23,24] and several other angiosperm families distributed from temperate to tropical habitats [2528]. In these plants, thermogenesis enhances the emission of floral volatiles to attract pollinators. Our finding that enclosure temperatures were close to air temperatures, in general, indicates, however, that temperatures of the enclosures of S. bryoniifolius may be regulated more passively, as reported in most previous studies on leaf temperature regulation. Little et al. [29] reported leaf heating up to 8°C higher than the ambient temperature in sub-Antarctic megaherbs by absorption of solar radiation during brief, unpredictable periods, and retention of heat. Since temperature increase in the enclosure of S. bryoniifolius was always associated with sunshine at midday, a similar mechanism may account for the observed temperature increase in S. bryoniifolius (figure 4).

Temperature directly affects the enzyme kinetics of many biochemical reactions [30]. Various plant organs have been suggested to regulate temperature of flowers and enhance fruit development, especially in plants growing at high latitudes and altitudes. Recently, these hypotheses have been critically investigated in alpine habitats much colder than that of this study. Peng et al. [31] showed that woolly leaves covering flowers of a Himalayan herb, Eriophyton wallichii (Lamiaceae), function to maintain flowers and fruits within the optimal temperature range in their habitat, which undergoes drastic temperature fluctuations. The same research group reported that corolla retained after flowering of Fritillaria delavayi (Liliaceae) in an alpine habitat in China increased the interior temperature by as much as 8°C and enhanced seed production [32]. That group also demonstrated that bracts concealing inflorescences of Rheum nobile (Polygonaceae) increased the temperature inside and facilitated the seed development [33].

In this study, we could not identify the exact mechanisms by which enclosures enhance fruit development. The temperature increase we observed in S. bryoniifolius was relatively small compared with that found in previously reported studies, though our measurements were spatially and temporally too limited to draw conclusions about the effects of enclosure on temperature. Moreover, a significant increase was observed only under a particular condition in daytime. The literature on effects of such ‘warm pulses’ on immature fruit growth is limited, but there is evidence suggesting that warm temperature for one day or less could affect different stages of plant reproduction as well as vegetative growth. It has been reported in many plant groups that pollen germination and pollen tube growth are sensitive to temperature [34]. In the coastal morning glory, Murcia [35] found that the previous day's temperature affected the size and opening time of the flowers. While little information is available about the effects of fluctuating temperature [36], in a fungus negative effects of temperature stress were larger when temperature fluctuation was faster [37].

Another possible mechanism by which enclosure leaves enhance fruit development is by protecting immature fruits from radiative cooling and resultant frost damage. The daily minimum temperature in October around the study site is quite variable and drops below zero in many years (electronic supplementary material, figure S5). Leaf and fruit temperature can be a few degrees lower than ambient temperature due to emission of long-wave radiation on clear nights when water vapour does not block outgoing radiation [3840]. In agriculture, such radiative cooling and frost can cause detrimental damage to crops even when the ambient temperature does not fall below freezing [30]. To investigate this possibility, we would need to measure the temperature of the fruit surface rather than air temperature.

Enclosures were unlikely to protect fruits from herbivores since we often encountered herbivore larvae and other insects inside enclosures. An increase of predation rate by protective organs has been reported in R. nobile mentioned above [33].

(b) Cost of enclosure formation

Enclosure leaves of S. bryoniifolius are one of the rare examples of leaves that play a major role in reproduction rather than photosynthesis. One of the other known examples is in Saururus chinensis (Saururaceae), which shows leaf colour change from green to white to attract pollinators [9]. Song et al. [9] confirmed a reduction of photosynthesis by leaf whitening but argued that the effect is relatively minor because the leaves turn back to green after flowering. By contrast to Saururus leaves, which temporarily change their functions, enclosure leaves of S. bryoniifolius may contribute much less to photosynthesis than solitary leaves do throughout their lifetime. Tight overlap of leaves on the enclosure prevents the inner leaves from receiving solar radiation for photosynthesis. This accords with the characteristics of the enclosure leaves; they have low SPAD values and less photosynthetic ability than solitary leaves do due to the undeveloped palisade layer and smaller chloroplasts. Since such leaves may need lower amounts of nutrients to produce, enclosure formation that allows the plant to produce fruits until the last moment of the plant's only growing season should be worth the cost.

The other potential cost of enclosure formation is that the plant loses the opportunity of outcrossing. Pollinators of the plants, mostly small dipteran insects [18], cannot visit flowers located deep inside the leaf layer. The smaller, homogamous flowers in the enclosure, in contrast with more dichogamous and larger flowers in the earlier season, also suggest that they are autonomously pollinated. Male individuals, which do not have a chance to sire seeds when hermaphrodite flowers are concealed in the enclosure, cease flowering earlier than hermaphrodites do. If the pollinators are already inactive at the time of the enclosure formation; however, the flowers would not have been visited even if they were exposed, and thus enclosure itself may impose little cost on the plant in this regard.

5. Conclusion

This study documented that leaves produced on dwarf shoots at the end of the life cycle enhance fruit production by enclosing immature fruits in Schizopepon bryoniifolius (Cucurbitaceae). Enclosure leaves have less photosynthetic ability compared with other leaves. Leaves that play a major role in reproduction rather than in photosynthesis have not been reported so far to the best of our knowledge. While vegetative and reproductive traits of plants have usually been studied separately, this study indicated how they can dynamically interact by examining associations among leaf and reproductive trait changes according to life stages.

Data accessibility

All datasets used in this study are included in the electronic supplementary material.

Author contributions

N.N. discovered the phenomenon and continuously observed enclosure formation from 2008 onward. S.S. and S.N. designed the project. N.N., S.S., S.N. and Y.T-M. conducted the field work. S.S. analysed the data. S.S. and S.N. wrote the manuscript. All authors gave final approval for publication.

Competing interests

We declare we have no competing interests.

Funding

This work was supported by JSPS KAKENHI grant nos 16H04830 and 19K22455.

Acknowledgements

We thank M. Marre for help with data collection in the field; T. Saito for provision of research equipment; and the staff of Yamagata Prefectural Natural Museum Park for logistic support. We also thank T. Miyake and T. Fukuhara for information about the species; M. Ushio for assistance with statistical analyses; H. Kudo for discussions; and A. Ushimaru, two anonymous reviewers and an anonymous associate editor for critical and constructive comments on an earlier version of the manuscript. We are grateful to Yamagata Prefectural Natural Museum Park and Nishikawa town for permission to conduct field work.

Footnotes

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5142766.

Published by the Royal Society. All rights reserved.

References