DOI: 10.5061/dryad.3ffbg79qt
title: Physical seed damage, not rodent’s saliva, accelerates seed germination of trees in a subtropical forest

description: Many tree species adopt fast seed germination to escape the predation risk
 by rodents. Physical seed damage and the saliva of rodents on partially
 consumed seeds may also act as cues for the seed to accelerate the
 germination process. However, the impacts of these factors on seed
 germination rate and speed remain unclear. In this study, we investigated
 such impacts on the germination rate and speed (reversal of germination
 time) of four tree species (Quercus variabilis, Q. serrata, Q. acutissima,
 and Q. glauca) after partial consumption by four rodent species, through a
 series of experiments. We also examined how seed traits may affect the
 damage degree by rodents by analyzing the relationship between the
 germination rate and time of rodent-damaged seeds and the traits. We found
 that artificially and rodent-damaged seeds exhibited a significantly
 higher seed germination rate and speed, compared to intact seeds. Also,
 the rodent saliva on seeds showed no significant effect on seed
 germination rate and speed. Furthermore, We observed significant positive
 correlations between several seed traits (including seed mass, coat
 thickness, and protein content) and seed germination rate, but these seed
 traits had a positive correlation with the germination rate and speed.
 These correlations are likely due to the beneficial traits countering seed
 damage by rodents. Overall, our results highlight the significant role of
 physical seed damage by rodents (rather than their saliva) in facilitating
 seed germination of tree species and potential mutualism between rodents
 and trees. Additionally, our results may have some implications in forest
 restoration, such that intentionally sowing or dispersing slightly damaged
 seeds by humans or drones may increase the likelihood of successful seed
 regeneration.
description: To evaluate the effects of physical damage and saliva of rodents
 on the germination rate and speed of the oak seeds, we set up a
 germination experiment with intact and treated seeds. We planted the seeds
 in potted soil with each pot containing 5 seeds. The pots had a diameter
 of 12.5 cm and a depth of 11 cm. The seeds were planted at a burial depth
 of 2 cm, approximating the average burial depth of seeds hoarded by
 rodents (Xiao & Zhang, 2004). The germination progress of the
 planted seeds was regularly observed and recorded. Seed germination was
 defined as the emergence of any part of the seedling from the seed, based
 on the criteria outlined in a previous study by Izhaki et al. (1995).
 Germinated seeds were carefully removed from the pots to minimize their
 influence on the non-germinated seeds, by following the methodology
 described by Tang et al. (2007). We also recorded the germination time of
 each seed to examine the relationship between germination time and the
 traits (including seed mass, coat thickness, and protein content) of
 damaged seeds. In all experiments, injured oak seeds were referred to as
 those damaged by rodents. We also used the data from Experiment 1 to test
 Hypothesis 4, which postulates that different seed traits demonstrating
 tolerance or resistance to rodent predation might affect the germination
 of damaged seeds. Seed traits of four species of oak seeds are obtained
 from a previous study (Yang et al., 2018). We used the following criteria
 to measure the germination metrics of oak seeds such as germination rate
 and time: 1. Germination rates (GRs), measured as the
 proportion of germinated oak seeds among all tested seeds. 
 2. Mean germination times (MGTs), measured as the average
 germination time of all germinated seeds during the experimentation
 period. The reversal of MGTs is referred to as germination
 speed. <strong>Experiment 1. Effects
 of</strong><strong> seed
 </strong><strong>damage</strong><strong> on seed
 germination</strong> To test Hypothesis 1, 2, 3
 & 4 that artificial or rodent damage would speed up seed
 germination (higher germination rates but shorter germination time), we
 conducted experiments in Spring 2020, measuring the germination rate and
 time of partially damaged seeds of <em>Q. variabilis</em>,
 <em>Q. serrata</em>, <em>Q. acutissima</em>, and
 <em>Q. glauca</em>. These seeds were subjected to damage by
 three rodent species: <em>A. draco</em>, <em>A.
 chevrieri</em>, and <em>L.
 </em><em>e</em><em>dwards</em>. Due to the
 dormancy of <em>Q. acutissima</em>, and <em>Q.
 glauca</em><em> </em>oak seeds, we recorded their
 germination status weekly for 122 continuous days from January to May
 2020. Prior to the experiments, the rodents were fasted for at least 6
 hours and then given the oak seeds in individual plastic boxes (L × W × H
 = 90cm × 50cm × 40cm). Thus, both rodents and oak seeds were in the same
 plastic boxes for the experiment. Twenty oak seeds were given to an
 individual <em>L. edwards</em><em> </em>due to its
 large body mass (&gt; 200g), and ten oak seeds were given to
 an <em>A. draco</em> and an <em>A. chevrieri</em>,
 considering their smaller body size, at a time, based on their daily
 intake. The condition of the seeds was assessed based on feeding position
 and proportion. Seeds with half or more of the endosperm consumed were
 excluded from the germination tests based on feeding position and
 proportion. We used fresh intact seeds as the negative control group (NT
 group) serving as a reference to assess the natural conditions of seed
 germination without any interference from rodents or humans. This helps to
 establish a baseline of seed germination for making a comparison between
 intact and treated seeds by rodent or artificial damage. We artificially
 damaged seeds, in which we manually cut the seed with normal saline in
 half to simulate the physical damage by rodents without the effect of
 rodent’s saliva, as a positive control group (MJ group) to test the
 effects of physical seed damage and rodent saliva on germination. Fresh
 oak seeds, which were handled by rodents (<em>L.
 edwards</em>,<em> A. draco</em>, <em>A.
 chevrieri</em>), and those that were partially damaged, were grouped
 into the LeT, AdT, and AcT categories to test the effects of other unknown
 factors except for seed damage. Fresh oak seeds handled by rodents
 (<em>L. edwards</em>,<em> A. draco</em>,
 <em>A. chevrieri</em>), and those partially damaged were
 categorized into the LeJ, AdJ, and AcJ groups to test the effect of seed
 damage by rodents. Before planting the seeds into flower pots, all intact
 and partially damaged oak seeds were carefully cleaned with absorbent
 paper. The number of seeds involved in Experiment 1 was shown in
 supplement Table S9 and the eight treatment groups (NT, MJ, LeT, LeJ, AdT,
 AdJ, AcT, AcJ) were shown below: 
 <strong>NT</strong>, Naturally intact oak
 seeds; <strong>MJ</strong>, mechanically
 damaged oak seeds artificially with normal saline;
 <strong>LeT</strong>, Intact oak seeds handled by
 <em>L. </em><em>edwards</em>;
 <strong>LeJ</strong>, Injured oak seeds by
 <em>L. </em><em>edwards</em>;
 <strong>AdT</strong>, Intact oak seeds handled by
 <em>A. </em><em>draco</em>;
 <strong>AdJ</strong>, Injured oak seeds by
 <em>A. </em><em>draco</em>;
 <strong>AcT</strong>, Intact oak seeds handled by
 <em>A. </em><em>chevrieri</em>;
 <strong>AcJ</strong>, Injured oak seeds by
 <em>A. </em><em>chevrieri</em>;
 <strong>Experiment 2. Effects of mouth-rinsed saliva on
 seed germination</strong> The goal of this
 experiment was to explore how the saliva of the four rodent
 species <em>L. edwards</em>, <em>N.
 fulvescens</em>, <em>N. confucianus</em>, <em>A.
 draco</em> affect the germination of <em>Q.
 variabilis</em> and <em>Q. serrata</em> seeds. The
 experiment was conducted in autumn 2021, aiming to test Hypothesis (3)
 that rodent saliva may accelerate seed germination. Due to the non-dormant
 characteristics of <em>Q.
 </em><em>variabilis</em> and <em>Q.
 </em><em>serrata</em>, seed germination occurred
 relatively quickly (Table S9), thus, from October to December 2021,
 initial monitoring of seed germination was conducted every 2-3 days, which
 was later adjusted to once a week until 80% of the seeds had germinated
 and no further germination was observed in the subsequent two weeks. We
 used an 80% germination rate as the threshold to end monitoring because
 the remaining seeds might fail to germinate due to various reasons, such
 as dormancy mechanisms and germination potential. Due to insufficient
 numbers of <em>A. chevrieri</em>, we had to substitute
 <em>A. chevrieri</em> with <em>N.
 fulvescens</em> and <em>N. confucianus</em> in this
 experiment. Before conducting the tests, all rodents
 were deprived of food for at least 6 hours and we preheated saline water
 to a temperature of 37-38℃. We collected saliva by rinsing the mouths of
 each rodent with the pre-heated saline water, and this process was
 repeated at least 10 times per rodent to obtain a sufficient amount of
 saliva extract. The saliva extract volume from each rodent species (five
 individuals in total of the experiment) exceeds 10 ml, and saliva from an
 individual rodent was spread over ten seeds. We used cotton swabs to
 collect saliva and evenly spread it on the surface, ensuring that both
 sides of the seeds came into contact with rodent saliva. The number of
 seeds involved in Experiment 2 was shown in supplement Table S9 and the
 six treatment groups (NT, MJ, LeR, NfR, NcR, AdR) are shown
 below:  <strong>NT</strong>, Naturally
 intact oak seeds; <strong>MJ</strong>,
 mechanically damaged oak seeds artificially with normal saline;
 <strong>LeR</strong>, Injured oak seeds treated with
 mouth-rinsed saliva of <em>L. edwards</em>;
 <strong>NfR</strong>, Injured oak seeds treated with
 mouth-rinsed saliva of <em>N. fulvescens</em>;
 <strong>NcR</strong>, Injured oak seeds treated with
 mouth-rinsed saliva of <em>N. confucianus</em>;
 <strong>AdR</strong>, Injured oak seeds treated with
 mouth-rinsed saliva of <em>A. draco</em>;
 <strong>Experiment 3. Effects of gland-rinsed saliva on
 seed germination</strong> The experiment is
 similar to Experiment 2 but we used saliva gland (<em>N.
 fulvescens</em>,<em> N. confucianus</em>, <em>A.
 draco</em>), aiming to test Hypotheses (3) in the autumn of 2022. We
 encountered a seed shortage and were unable to collect a sufficient
 quantity of seeds in the local forest, forcing us to reduce the number of
 seeds for five treatment groups from 50 to 25 this year. Due to the
 excessively large size of <em>L.
 edwards</em><em> </em>(body weight &gt; 200g), it
 was excluded from this test as it was difficult to be anesthetized. Saliva
 glands were surgically extracted from rodents for analysis. In each group
 of rodent species, we only euthanized one individual. This experiment
 involved one <em>N. fulvescens</em>, one <em>N.
 confucianus</em>, and one <em>A. draco</em>. Due to
 insufficient numbers of <em>A. chevrieri</em>, we had to
 substitute <em>A. chevrieri</em> with <em>N.
 fulvescens</em> and <em>N. confucianus</em> in this
 experiment. The salivary glands, including the parotid gland, submaxillary
 gland, and sublingual gland, were surgically removed. The collected
 salivary glands were then immersed in a preheated saline solution at a
 temperature of 37℃. The bottle containing the salivary gland diluent was
 gently shaken, and after 5 minutes, the diluted saliva liquid was dropped
 onto the surface of cut oak seeds of <em>Q.
 variabilis</em> and <em>Q. serrata</em>. The oak seeds
 were cut in half to simulate physical seed damage by rodents while
 ensuring that the embryo within remains viable. Our objective was to
 maintain consistent levels of damage across all seeds to evaluate their
 influence on germination rates under controlled conditions. After the
 experiment, all animals raised were treated harmlessly. The number of
 seeds involved in Experiment 3 was shown in supplement Table S9, and the
 five treatment groups (NT, MJ, NfG, NcG, AdG) are shown below:
 <strong>NT</strong>, Naturally intact oak
 seeds; <strong>MJ</strong>, mechanically
 damaged oak seeds artificially with normal saline;
 <strong>NfG</strong>, Injured oak seeds with
 gland-rinsed saliva of <em>N. fulvescens</em>;
 <strong>N</strong><strong>c</strong><strong>G</strong>, Injured oak seeds treated with mouth-rinsed saliva of <em>N. confucianus</em>; <strong>AdG</strong>, Injured oak seeds treated with mouth-rinsed saliva of <em>A. draco</em>;
description: # **Physical seed damage, not rodent’s saliva, accelerates seed
 germination of trees in a subtropical forest**
 [https://doi.org/10.5061/dryad.3ffbg79qt](https://doi.org/10.5061/dryad.3ffbg79qt) ## Description of the data and file structure ## **exp1 sheet** Experiment 1 data groups show the different treatments: (1) Naturally intact acorns without rodent handling (NT) (2) Mechanically damaged acorns by people with normal saline (MJ) (3) Intact acorns handled by *L. edwards* (LeT) (4) Injured acorns by *L. edwards* (LeJ) (5) Intact acorns handled by *A. draco* (AdT) (6) Injured acorns by *A. draco* (AdJ) (7) Intact acorns handled by *A. chevrieri* (AcT) (8) Injured acorns by *A. chevrieri* (AcJ) Q.v means *Q. variabilis*, Q.s means *Q. serrata*, Q.a means *Q. acutissima*, Q.g means *Q. glauca*; time means germination time (days) status means germination status; 1 means germinating, 0 means not germinating; ## **exp2 sheet** Experiment 2 groups show the different treatments: (1) Naturally intact acorns (NT) (2) Mechanically damaged acorns by people with normal saline (MJ) (3) Injured acorns treated with mouth-rinsed saliva of *L. Edwards* (LeR) (4) Injured acorns treated with mouth-rinsed saliva of *N. fulvescens* (NfR) (5) Injured acorns treated with mouth-rinsed saliva of *N. confucianus* (NcR) (6) Injured acorns treated with mouth-rinsed saliva of *A. draco* (AdR) Q.v means *Q. variabilis*, Q.s means *Q. serrata*; time means germination time (days) status means germination status; 1 means germinating, 0 means not germinating; ## **exp3 sheet** Experiment 3 groups showed the different treatments: (1) Naturally intact acorns (NT) (2) Mechanically damaged acorns by people with normal saline (MJ) (3) Injured acorns treated with gland-rinsed saliva of *N. fulvescens* (NfG) (4) Injured acorns treated with gland-rinsed saliva of *N. confucianus* (NcG) (5) Injured acorns treated with gland-rinsed saliva of *A. draco* rinsed (AdG) Q.v means *Q. variabilis*, Q.s means *Q. serrata*; time means germination time (days) status means germination status; 1 means germinating, 0 means not germinating; ## seed\_trait sheet Shows the traits of seeds. Q.v_rate, Q.s_rate, Q.a_rate, and Q.g_rate means The germination rate of *Q. variabilis*, The germination rate of *Q. serrata* germination rate, The germination rate of *Q. acutissima* germination rate, The germination rate of*Q. glauca* germination rate; Q.v_asin, Q.s_asin, Q.a_asin, and Q.g_asin means The germination rate of *Q. variabilis* underwent arcsin transformation, The germination rate of *Q. serrata* underwent arcsin transformation, The germination rate of *Q. acutissima* underwent arcsin transformation, The germination rate of *Q. glauca* underwent arcsin transformation; Q.v_mgt, Q.s_mgt, Q.a_mgt, Q.g_mgt means Mean germination time (MGT) (days) of *Q. variabilis*, Mean germination time (MGT) (days) of *Q. serrata*, Mean germination time (MGT) (days) of *Q. acutissima*, Mean germination time (MGT) (days) of *Q. glauca*. treat means treatment, A means *Q. variabilis*, B means “*Q. serrata*, C means *Q. acutissima*, D means *Q. glauca*; asin means Seed germination rate after arcsin transformation; mgt means Mean germination time (days); fresh_weight means Oak seed fresh weight (g); coat_thickness means Oak seed coat thickness (mm); protein means protein content (%); fat means fat (%); starch means starch (%); tannin means tannin (%); Cellulose means “Cellulose (%)”; Caloric_value means Caloric value (KJ/g); Caloric_value_per_seed means Caloric value per seed (KJ); “dormancy” means dormancy status, 1 means dormancy, 0 means non-dormancy; rodent_mass means rodent mass (g); ## Code/Software R version 4.3.2 was used for analysis.