Response of Seedling Growth Characteristics to Seed Size and Cotyledon Damage in Quercus wutaishanica

Abstract

The successful establishment of seedlings is very important for plant regeneration, but it is vulnerable to many factors at this stage. Cotyledon damage will directly affect the health of seedlings, thus affecting the regeneration of the plant population. However, little is known about the effects of different cotyledon loss degrees of large and small seeds on seedling growth. We investigated the effects of 1/4 (light excision), 1/2 (moderate excision), and complete excision of cotyledons (heavy excision) on the growth characteristics of seedlings germinating from different sizes of seeds. The results showed that (1) shoot height, basal stem diameter, number of leaves, leaf area per plant, specific leaf area, and biomass were significantly higher in large-seeded seedlings than in small-seeded seedlings; (2) slight cotyledon excision had no effect on the biomass of large-seeded seedlings but significantly reduced the biomass of small-seeded seedlings. Our study highlights that large-seeded seedlings are more tolerant than small-seeded seedlings in the early seedling recruitment, suggesting that large-seeded seedlings have a strong fitness for recruitment in young populations of Q. wutaishanica.

1. Introduction

Insufficient seedling recruitment is a common feature of many oak forests [1]. The main reason is that oak seeds are an important food resource for insects, birds, rodents, and other animals because they are rich in nutrients and are eaten in large quantities by animals when they are ripe and dispersed [2,3,4,5]. In addition, during seedling establishment, predators may also use seedlings as a search cue to consume heavily on their cotyledons [1,6], thereby increasing the risk of mortality of their seedlings. The change from seed to seedling is the most sensitive stage of a plant’s life history to environmental factors [7], and plant seed nutrients provide an effective guarantee for seed germination, seedling establishment, and regeneration after damage to the organism [1]. Previous studies have shown that seeds subjected to predation severely affect their germination and seedling establishment [8,9]. In particular, high-intensity cotyledon loss is serious for plant biomass accumulation and even seedling establishment failure [8]. It has also been reported that a slight loss of cotyledons has no significant effect on the growth of their seedlings [9], as a large number of nutrients have been transferred and stored in the main roots during cotyledon growth [1]. However, most of these studies have focused on the effect of cotyledon loss at the seed stage on germination rate and seedling growth [9,10,11], and the effect of the degree of cotyledon loss on seedling growth in the early seedling establishment is largely unknown.

Variation in seed size is widespread between plant species, within species, and even within the same plant [12,13,14,15,16]. The effect of cotyledon loss on seedling growth may be related to seed size [17], extent of damage [18], and damage site [9]; in particular, seed size differences may have an important effect on seedling growth [19]. Large seeds have a greater chance of establishing seedlings after germination due to storing more nutrients [19]. In addition, seedlings germinating from large seeds are more tolerant to harsh environments, light, and competition [20]. The transfer of seed nutrients to seedlings plays an important role in seedling development, as seedlings from larger seeds usually live longer and accumulate more biomass [21]. Moreover, large seeds usually have more nutrients than are required for seed germination and seedling establishment [19]. Previous studies have shown that not only are more nutrients in large seeds a source of nutrients for seed germination and seedling establishment but also that, in most cases, animals consume nutrients from the ends of cotyledons further away from the radicle and that these consumed nutrients may saturate the plant with predators or provide a food ‘reward’ for dispersing animals [19,22]. However, predation on post-emergence cotyledons by predators has no effect on plant growth [22]. Therefore, the same amount of cotyledon loss may have a relatively small negative impact on the growth and development of larger seed germinating seedlings.

Quercus wutaishanica is one of the key dominant species of warm-temperate deciduous forests in China. Its seeds germinate immediately after maturing and dispersing in autumn and transfer most of the nutrients from the cotyledons to the main roots quickly, with the embryo elongating to form seedlings the following spring. However, the seeds are subject to heavy predation by small rodents from the time they mature and disperse until they germinate and form seedlings [11]. The effect of cotyledon loss on germination and seedling growth when seeds mature and fall off has been studied previously [23], but little is known about the effect of the extent of cotyledon feeding by predators on seedling growth after seeds of different sizes have germinated. Based on this, we simulated the effects of different levels of predation by small rodent species on the growth of post-emergence cotyledons of Q. wutaishanica. We hypothesize that: (1) Larger seeds have better growth characteristics and higher biomass than smaller seeds; (2) Cotyledon excision reduces total seedling biomass and increases with the degree of cotyledon excision, cotyledon excision affects the growth characteristics of large-seeded seedlings less than small-seeded seedlings. Our study will help to provide further insight into the mechanisms by which predators feeding on seed affect the growth of seedlings and the seedling regeneration of populations of Q. wutaishanica. In addition, our work will help refine breeding and restoration efforts.

2. Materials and Methods

2.1. Seed Harvesting and Selection

The seeds were harvested from Q. wutaishanica shrubs in the Q. wutaishanica forest area of the Longtan Forestry Range, Liupanshan National Nature Reserve (35.15° N–35.41° N, 106.90° E–106.30° E), Ningxia Hui Autonomous Region, China that were about 30 years old. The area has a temperate semi-humid climate, with a cold, dry winter and a hot, rainy summer. The total annual precipitation is about 767 mm, with 60% precipitation from June to September, and the annual evaporation is about 1426 mm [5]. The annual average temperature is 5.8 °C, with the extreme low and high temperatures being about −26 °C (January) and about 30 °C (July) [5]. On 26 September 2018, in Q. wutaishanica shrubs, 30 canopy plants of relatively large size (approximately 2 m tall) and vigorous canopy growth were selected to harvest mature seeds from different directions, with about 500 seeds per tree. All the seeds harvested were thoroughly mixed. We selected 600 large seeds and 600 small seeds separately by observation, and none of them were infested. We randomly selected 100 large and 100 small seeds each to measure fresh weight, long axis, and short axis diameter. The large seeds were 3.05 ± 0.38 g in weight, 21.78 ± 1.20 mm long axis diameter, and 15.95 ± 0.85 mm short axis diameter, while the small seeds were 1.46 ± 0.27 g weight, 16.65 ± 1.33 mm long axis diameter and 12.13 ± 0.70 mm short axis diameter (mean ± SD). There were significant differences in fresh weight long and short axes between the large and small seeds, and the method of distinguishing the seed sizes was referred to Zhang et al. (2008) [24]. Then, store the seeds in a dry and cool place for a week before sowing.

2.2. Seed Sowing and Cotyledon Excision

The experimental sample plots were watered well 2 days before sowing. On 2 October 2018, at the experimental site of the College of Biological Science and Engineering, North Minzu University, the seeds were peeled with a single-sided blade. A completely randomized design experiment was used to evenly divide large and small seeds into 12 parts, respectively, and randomly sowed in 24 plots, with 50 seeds in each plot, an area of 1 m² in each plot, and a sowing depth of 3 cm. Plastic bags were used to mark the size of seeds sown in each plot. To avoid seed mortality due to low temperatures in winter and early spring, the experimental plots sown were covered on 1 November with well-permeable plastic film to a height of about 20 cm, and the cover was removed in early April of the following year.

The experimental plots were watered once a week. When the majority of Q. wutaishanica seedlings had their first real leaves in early May, the cotyledons were excised in large and small seed plots, respectively. The cotyledons were carefully excised with a single-sided blade using a spade to pluck the soil from the roots of young Q. wutaishanica seedlings, with 1/4 (defined as light excision), 1/2 (defined as moderate excision), complete excision (defined as heavy excision) and no excision (control) of the large and small seed cotyledons, and marked by hanging labels, with three replicates of each treatment. Total number of seedlings = 3 (replication) × 4 (treatments, including control, light excision, moderate excision, and heavy excision) × 2 (size) = 1200. After 2 weeks of seedling emergence, only 10 healthy seedlings of approximately the same size were retained, and the excess seedlings were removed from each plot after watering.

2.3. Harvesting of Seedlings and Determination of Biomass and Growth Parameters

The experimental sample plots were well watered in October 2019, and then the seedlings were dug up with their roots and brought back to the laboratory to be washed with tap water. The shoot height was determined using a straightedge (units: cm), basal stem diameter was determined using a vernier caliper (units: mm), number of leaves and leaf area per plant was determined by American LI-3100 leaf area meter (unit: cm²). Seedlings were divided into roots, stems, and leaves, and then placed the above plant tissues in envelopes and in a blast oven at a temperature of 65 °C. We then dried them to a constant weight to determine the total dry mass (TDM), root/shoot ratio (RSR), specific leaf area (SLA), specific shoot length (SSL), and specific root length (SRL), and all the data were retained in two decimal places. The formulae were calculated as follows:

TDM = Root dry mass + stem dry mass + leaf dry mass
RSR = (Root dry mass)/(stem and leaf dry mass)
SLA = (Leaf area)/(leaf dry mass (cm²·g⁻¹))
SSL = (shoot length)/(shoot dry mass (cm·g⁻¹))
SRL = (Root length)/(root dry mass (cm·g⁻¹))

2.4. Statistical and Analytical Data

We constructed generalised linear mixed effects models (GLMMs) to analyse the effects of seed size and cotyledon excision on total dry mass and seedling growth parameters, with seed size and cotyledon excision as fixed factors and Plot as a random factor. GLMMs were fitted using the “glmer” function (family = “beta”) in the “lmer4 package” [25]. Paired t-tests were used to analyse the significance of differences between seedling biomass and growth parameters of small and large seedlings under the same cotyledon excision treatment, and the least significant difference (LSD) was used to analyse the significance of differences between seedling total dry mass and growth parameters between cotyledon excision treatments. p < 0.05 was used as the threshold for whether there was a difference. All statistics were done using R software (version 4.0.2).

3. Results

3.1. Effect of Seed Size and Cotyledon Excision on Seedling Growth

Seed size and cotyledon excision had significant effects on shoot height, basal stem diameter, leaf area, total dry mass, and specific leaf area (p < 0.05), but the interaction of seed size and cotyledon excision did not reach the level of significance for shoot height, basal stem diameter, leaf area, total dry mass, and specific leaf area. Seed size had a significant effect on the leaf number of seedlings (p < 0.001), while neither cotyledon excision nor the interaction of seed size and cotyledon excision reached the level of significance on the leaf number of seedlings. Seed size had no significant effect on root/shoot ratio, specific root length, and specific branch length, but cotyledon excision had a significant effect on root/shoot ratio, specific root length, and specific shoot length (p < 0.05), and the interaction of seed size and cotyledon excision had no significant effect on root-shoot ratio, specific root length and specific shoot length (

Table 1. Generalised linear mixed effects models to analyse the effect of seed size and cotyledon excision on growth parameters of Quercus wutaishanica seedlings.

Growth Parameters	Seed Size		Cotyledon Excision		Seed Size × Cotyledon
	F	p	F	p	F	p
Shoot height	70.84	<0.001	11.53	0.003	0.28	0.600
Basal stem diameter	22.86	<0.001	8.56	0.009	3.58	0.074
Leaf number	45.28	<0.001	3.69	0.081	2.7	0.118
Leaf area	67.03	<0.001	4.91	0.039	0.71	0.411
Total dry mass	52.78	<0.001	11.35	0.003	0.28	0.601
Root-shoot ratio	1.52	0.233	9.45	0.007	0.03	0.867
Specific leaf area	65.40	<0.001	14.08	0.002	0.71	0.409
Specific root length	0.04	0.843	4.75	0.043	0.69	0.410
Specific shoot length	0.22	0.640	9.71	0.005	0.62	0.438

).

3.2. Seedling Growth Response to Seed Size

Shoot height, basal stem diameter, leaf number, leaf area per plant, specific leaf area, and total dry mass were higher in control and large-seeded seedlings under different cotyledon removal than in small-seeded seedlings (Figure 1a–d and Figure 2a,c).

3.3. Seedling Growth Response to Cotyledon Excision

Cotyledon excision reduced shoot height, which was significantly (p < 0.05) lower than the control under heavy cotyledon excision for large-seeded seedlings and for small-seeded seedlings under both moderate and heavy treatments (p < 0.05) (Figure 1a). Cotyledon excision reduced basal stem diameter and leaf area per plant of seedlings and was significantly (p < 0.05) lower than control in both large and small-seeded seedlings under heavy cotyledon excision (Figure 1b,d). Cotyledon excision reduced the total dry mass of large-seeded and small-seeded, and the total dry mass of seedlings of large-seeded was significantly lower than that of the control under the heavy treatment (p < 0.05), while the total dry mass of seedlings of small-seeded was significantly lower than that of the control under the light, medium and heavy treatments (p < 0.05) (Figure 2a). Cotyledon excision increased the root/shoot ratio and specific shoot length of seedlings, and both increased with increasing degrees of cotyledon removal (Figure 2b,e). Conversely, cotyledon excision decreased the specific leaf area and specific root length of seedlings, and both decreased with increasing degrees of cotyledon excision (Figure 2c,d).

4. Discussion

In support of our first hypothesis, seedlings with large seeds under different cotyledon treatments had higher shoot height, basal stem diameter, leaf number, leaf area per plant, specific leaf area, and total dry mass than seedlings with small seeds. This is consistent with a previous study that showed a significant positive correlation between early seedling growth and seed size in temperate forests and scrub plants [26]. Seeds can transfer their stored nutrients to different tissues of the seedling during germination [27]. Seedlings with large seeds have thicker primary roots [27], which not only help the seedling to be firmly “anchored” in the soil but also ensure that the seedling relies on the nutrients stored in the primary root to maintain new shoots and normal growth after the loss of cotyledon [27]. Seed size affects seedling growth through biomass allocation and resource utilization in early development. Large seeds lead to greater dry matter accumulation and better tolerance to harsh conditions, resulting in higher survival and growth rates [21]. Similarly, previous studies have shown that the initial growth stage of seedlings is closely related to the initial seed size, with larger seeds generally growing faster and more robust seedlings [1,27]. This is because large seed nutrients are transferred and stored in the seedling during germination, allowing larger seedlings to develop a more complete root system than smaller seedlings more readily absorb nutrients and water from the soil [14]. As a result, seedlings sprouting from larger seeds accumulate more biomass and are more dominant in the growth process.

The effect of seed size on seedling survival may be mainly related to biomass allocation patterns and resource use patterns in the early seedlings [28]. In this study, the total dry mass of large-seeded seedlings was significantly greater than that of small-seeded- seedlings in both the control and different levels of cotyledon excision treatments; however, there was no significant difference in the root shoot ratio between large and small-seeded seedlings. This accumulation of dry matter in seedlings may be related to the rapid growth of the radicle in autumn and the emergence of the radicle the following year [11]. On the one hand, the seeds in the germination stage transfer as many nutrients as possible to the main root to reduce nutrient losses due to animals feeding on the cotyledons, thus allowing large-seeded seedlings to accumulate more dry matter [21]. On the other hand, with the transformation of source–reservoir relationships and the loss or weakening of dependence on seed size in the later stages of growth [11], the later above-ground growth of seedlings may depend mainly on soil available nutrients. In addition, there may be a trade-off mechanism between above-ground and below-ground biomass allocation by plants for better growth. Therefore, there is no significant difference in the root-shoot ratio of seedlings with large and small seeds [28].

Shoot height, leaf number, leaf area per plant, and specific leaf area of large-seeded seedlings from the control treatment and different degrees of cotyledon excision treatments were significantly greater than those from small seeds. These traits are the main indicators of the photosynthetic capacity of plants [29,30]. Increases in plant height, specific leaf area, leaf number, and leaf area per plant significantly increase the light capture capacity of plants [30], which in turn increases photosynthesis and relative growth rates [10] and promotes carbon accumulation [25], so that the total dry mass of large-seeded seedlings is significantly higher than that of small-seeded seedlings. However, there was no significant difference in specific root length between seedlings of large and small seeds under different cotyledon excision treatments. This was attributed to the fact that a lot of cotyledon nutrients had already been transferred to the radicle in autumn when seedlings of Q. wutaishanica with large and small seeds developed real leaves and that the distribution of root mass and length may have changed in proportion to the nutrients from the seeds [11]. Hence, seedlings of large and small seeds did not show differences in specific root lengths.

The seed cotyledons of oak plants are the main source of nutrients for seedling development and play an important role in seed germination and seedling establishment, growth, and survival [1,9]. This is consistent with our findings that cotyledon excision reduces the total dry mass of the plant, suggesting that cotyledons also play an important role in the growth of oak plants during their early stages of growth. Some studies have reported that larger seeds can tolerate a certain level of cotyledon loss and still establish seedlings [9,19] and that moderate cotyledon loss may promote seedling growth by reducing the transfer of polyphenolic growth inhibitors from the seed to the seedling [28]. Our results support our second hypothesis that mild cotyledon deficiency did not significantly affect shoot height, basal stem diameter, total dry mass, and specific leaf area of large-seeded seedlings, but mild cotyledon deficiency significantly reduced shoot height, leaf area per plant, and specific dry mass of small-seeded seedlings compared to the control. In other words, cotyledon excision had a greater effect on the growth characteristics of small-seeded seedlings than on those of large-seeded seedlings. This is consistent with many previous reports that relatively large seeds are able to tolerate a certain level of damage, ensuring that a small number of seedlings survive in the face of insect or mammal feeding damage and germinate to establish seedlings [10,19], thereby contributing to seedling recruitment and population regeneration [11]. However, both moderate and severe cotyledon damage significantly reduced shoot height, basal stem diameter, leaf area per plant, and specific leaf area of seedlings of all sizes. Although oak plants have already converted some of the nutrients from the cotyledons to the acorn by the time the seed grows true leaves after spring germination, the root system may be underdeveloped, so the supply of cotyledon nutrients is still needed to ensure normal growth [31]. Therefore, moderate and heavy cotyledon removal can lead to cotyledon nutrient deficiencies, which can reduce shoot height, basal stem diameter, specific leaf area, and, consequently, individual biomass accumulation.

5. Conclusions

Most of the previous studies focused on acorn seed dispersal by animals feeding heavily on oak as an important bottleneck for its renewal. In this study, by focusing on the initial stage of seedling growth cotyledon removal on the growth characteristics of oak seedlings, it was found that the supply of cotyledon nutrients during the initial stage of seedling growth also had an important effect on seedling growth and increased with the degree of cotyledon loss. This reflects the fact that different degrees of animal feeding on oak cotyledons before the complete transfer of acorn cotyledon nutrients can also have an important effect on the phenotypic traits and biomass accumulation of seedlings, which may also influence seedling regeneration of Q. wutaishanica populations. In addition, we also found that large-seeded seedlings were more tolerant to cotyledon removal than large-seeded seedlings, suggesting that large seeds are more competitive and better fitted than small seeds. Therefore, in the process of forest seedling cultivation, abundant and larger quality oak seeds should be selected for cultivation. At the same time, in order to prevent the cotyledons from being preyed upon by animals, some food, such as maize and peanut rice, is appropriately put on the surface of the soil where the seedlings are cultivated.