Forest Ecology and Management 437 (2019) 41–48
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Comparison of the canopy and soil seed banks of Pinus yunnanensis in central Yunnan, China Wenhua Su1, Jiaoe Yu, Guangfei Zhang1, Zhan Shi, Lingling Wang, Guanhua Zhao, Rui Zhou
T
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Institute of Ecology and Geobotany, Yunnan University, Kunming 650091, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Seedbank Serotiny Pinus Seed dynamics Adaptive advantage
The ecological significance of serotiny in Pinus yunnanensis is poorly understood. In this study, we investigated the seed dynamics of canopy and soil seed banks in this species, as well as the resulting fate of seed in different locations. We found few seeds on the soil surface, and virtually no viable seed within the soil. In serotinous populations, the size of the seed bank was 20 times greater than that of non-serotinous populations. The number of seeds stored on the forest floor was considerably lower than the number input by seed rain. Almost all seeds on the floor lost viability within 17 months, and most died during the rainy season. Seed mortality in the canopy was considerably lower than either on or in the soil. On the basis of these findings, we conclude that serotiny enhances the size of the P. yunnanensis seed bank and that in this species, serotiny has an adaptive advantage over soil seed storage in Central Yunnan.
1. Introduction Seed banks represent an adaptive strategy in the life histories of plants growing in disturbance- or stress-prone environments. Seed banks can be divided into two types, soil and canopy seed banks (Lamont et al., 1991). Fire is one of the most common disturbance factors, and plants destroyed by fire usually depend on resprouting or on seedlings derived from seed banks for population restoration. Persistent soil seed banks, in which seeds are stored in the soil for more than one year, even for decades, are common in fire-prone ecosystems (Bell et al., 1993; Enright et al., 2007; Kwiatkowska-Falińska et al., 2014; Tavşanoğlu and Pausas, 2018), and may achieve persistence of the population even though mature individuals were killed by fire in the burnt lands (Izhaki et al., 2000; Valbuena and Trabaud, 2001; Auld and Denham, 2006; Céspedes et al., 2012; Galíndez et al., 2013). However, some species, notably pines and cypresses in the Northern Hemisphere, most of the Banksia species in Australia, and some Protea species in South Africa, retain seeds in the canopy for an extended period, thereby establishing canopy seed banks (Gill, 1975; Bond, 1985; Cowling and Lamont, 1985; Lamont et al., 1991, 2013). Serotinous cones can be induced to open by heat and fire, and consequently, a dense rain of seed can descend onto the soil surface, achieving seedling recruitment in the wake of fire disturbance (Daskalakou and Thanos, 2010). Most serotinous pines merely form a transient soil seed bank
after fire, rather than a persistent supply for post-fire recovery (Daskalakou and Thanos, 1996; Sem and Enright,1996; Valbuena et al., 2001; Enright et al., 2007). Sometimes, for example after an outbreak of beetles, the pines can form viable soil seed banks that store in cones (Teste et al., 2011). Although there is some information about the fitness advantages of serotiny (Pausas, 2015; He et al., 2016), very little is known about the reason why serotinous pines do not form a persistent soil seed bank without a cone. Dry soil is a good insulator from the heat of a fire and can protect buried seeds from the lethal effects of heat (Enright et al., 2014). During a fire, the highest temperatures occur at the soil surface (Carrington, 2010). Consequently, almost all seeds in litter and on the surface of the soil are killed by fire (Auld and Denham, 2006). Following fire events, viable seeds have been found only within the soil (Tozer, 1998; Auld and Denham, 2006), and most of the seedlings subsequently emerge from soil depths of 2–5 cm (Auld and Denham, 2006). The probability of seed incorporation in soil is determined by the characteristics of the site where the seed rest and the interactions of the seed with abiotic and/or biotic factors (Chambers and MacMahon, 1994). The percentage of the annual seed rain that actually enters the soil is low in some species (Sem and Enright, 1996), and most of the seeds in the soil seed bank remain on the surface or in litter (Zhang et al., 2009), and will be killed by heat in the events of forest fires (Auld and Denham, 2006; Carrington, 2010). Species for which there is
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Corresponding author. E-mail addresses:
[email protected] (W. Su),
[email protected] (G. Zhang),
[email protected] (R. Zhou). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.foreco.2019.01.002 Received 26 August 2018; Received in revised form 30 December 2018; Accepted 1 January 2019 0378-1127/ © 2019 Elsevier B.V. All rights reserved.
Forest Ecology and Management 437 (2019) 41–48
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1.5 ± 1.3 0.1 ± 0.1 2 Wet 60
b
a
The data are from the Institute of Yunnan Surveying and Mapping Engineering (2004). As the value increases, the risk of fire is greater. The data are from Lu et al. (2011).
1320 9.0 17.4 11.7 ± 0.6 1440
17.6 ± 0.7
2.9 ± 1.8 0.2 ± 0.1 2 Wet 55 1193 10.3 17.8 19.7 ± 2.1 1280
20.7 ± 2.5
0 0 2 Wet 54 1130 9.7 18.8 13.4 ± 1.4 1480
17.6 ± 4.6
76.7 ± 7.6 3.0 ± 0.6 4 Semiwet 45 985 9.3 15.9 6.6 ± 0.7 1860
12.9 ± 2.1
30.3 ± 6.0 1.5 ± 0.2 3 Semiwet 44 1001 9.9 15.7 9.8 ± 0.7 1790
15.0 ± 2.4
23.9 ± 13.1 1.4 ± 0.5 3 Semiwet 35 997 8.5 14.6 9.3 ± 1.1 2060
16.5 ± 1.4
76.1 ± 18.8 3.5 ± 0.5 4 Semidry 29 1010 7 13.1
25.65138N, 102.04250E 25.16113N, 102.67091E 24.86472N, 103.29388E 24.93916N, 103.53055E 24.96111N, 104.46472E 25.03736N, 104.50249E 25.13463N, 104.73327E Wuding (WD) Kunming (KM) Shilin (SL) Luliang (LL) Banqiao (BQ) Chandi (CD) Wusha (WS)
Elevations (m) Geographical coordinates Populations
Table 1 The conditions of seven populations.
Height of tree (m)
DBH (cm)
P. yunnanensis is one of the dominant species in the Central Yunnan Plateau, in the central region of Yunnan and in the Western Yun-Gui Plateau, growing at elevations between 1100 and 2900 m a.s.l. Controlled by the southwest and southeast monsoons, the region has a plateau monsoon climate characterized by moderately hot humid summers and warm dry winters (Chen et al., 2014). In the distribution range of P. yunnanensis, the annual mean temperature for 1961–1980 ranged from 11 to 18 °C (data from Yunnan Provincial Meteorological Bureau), with the lowest and highest monthly mean temperatures of 2–8 °C and 18–23 °C (Jin and Peng, 2004). The seasonal distribution of rain is uneven (Chen et al., 2014). Annual precipitation averages between 700 and 1300 mm, and the dry season extends from November to April, accounting for 6–17% of the annual precipitation. The rainy season extends from May to October, and most of the precipitation is concentrated from June to August. In the south of its distribution range, P. yunnanensis grows adjacent to P. kesiya, whereas in the east it grows adjacent to P. massoniana, and in the northwest at elevations over 2900 m a.s.l. there are stands of P. densata. In the present study, we selected both serotinous and non-serotinous populations to study. From the centre of the distribution range to its eastern margin, we selected the following seven populations (Table 1): Wuding (WD), Kunming (KM), Shilin (SL), Luliang (LL), Banqiao (BQ), Chandi (CD), and Wusha (WS). The distance between Wuding in the west and Wusha in the east is approximately 300 km. The four
12.5 ± 3.5
2.1. Study area
6.3 ± 0.7
Mean annual temperature (°C)
2. Materials and methods
2360
Mean Temperature of Driest Quarter (°C)
Mean annual precipitation (mm)
Precipitation of Driest Quarter (mm)
Climatic typea
Fire risk rankb
Mean serotiny time (yr)
Ratio of serotinous cones (%)
Population type
limited entry of seed into soil, therefore, cannot rely solely on soil seed bank resources for future recruitment. In previous studies, low Pinus seedling emergence has been recorded from the soil seed bank of pine forests (Izhaki et al., 2000; Valbuena et al., 2001). If pine seeds are released to the forest floor, most of the deposited seeds will remain there unprotected, and will be lost either before or during disturbances. In contrast, seeds of pine are borne between woody scales arranged around the central axis of the cone. As long as seeds are not released, they will be sufficiently protected from disturbance. There is evidence that seeds in serotinous cones are well insulated from the heat of a fire as compared with seeds on the forest floor (Lamont et al., 1991). After the disturbance, seed stored in the canopy will constitute a viable source of seedling recruitment (Su et al., 2017). Given the diverse barriers to establish a persistent soil seed bank of pines, reserves for recruitment, particularly in fire-prone regions are needed. Accordingly, we hypothesized that the canopy seed bank maximizes the number of seeds available for the next generation by storing successive seed crops and protecting them from the heat of fire. The Central Yunnan region of China is a typically fire-prone region (Li, 2000; Zhao et al., 2009), where the history of wildfires can be traced back to the Later Permian Period (Shao et al., 2012). P. yunnanensis, one of the dominant species in the Central Yunnan Plateau, was found to be weakly serotinous, annually releasing some seeds from canopy seed banks to the forest floor following fires (Su et al., 2015, 2017). Moreover, the populations of P. yunnanensis can be divided into two types, namely, serotinous and non-serotinous (Table 1). We predicted that (1) in both types of populations, there will be few seeds in the soil, and that (2) in serotinous populations, the number of seeds in the soil will be considerably smaller than in the canopy. We tested these predictions by comparing the sizes of the soil and canopy seed banks in P. yunnanensis forests. Further, in order to explore the benefit of serotiny comparing with soil seed bank, we also investigated the seed dynamics of canopy and soil seed banks, as well as the resulting fates of seeds present in canopy stores, on the soil surface and buried in soil in P. yunnanensis forests.
Serotinous (Ser) Serotinous (Ser) Serotinous (Ser) Serotinous (Ser) Non-Serotinous (non-Ser) Non-Serotinous (non-Ser) Non-Serotinous (non-Ser)
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(free seeds), were prepared for placing on the soil surface. These seeds were returned to the population stands in the vicinity of 15 pine trees, where were selected as the burial plots for a seed burial experiment. In each plot, two 10 × 10 cm2 holes of 2 cm and 5 cm depth were dug at a distance of 0.5 m from the selected trees. One nylon mesh bag was placed into each of these holes, and then covered with soil. Groups of free seeds were placed under approximately 1 cm depth of needles on the soil surface within a 10 × 10 cm quadrat (0 cm deep burial treatment). The quadrat was marked with four small bamboo sticks. Thus, seeds were placed at depths of 0, 2, and 5 cm within each of the 15 burial plots. At the beginning of May (after burial for four months) and October (after burial for ten months) in 2014, and at the beginning of May 2015 (after burial for 17 months), the seeds in the three treatments were retrieved from the five burial plots. The number of germinated, broken, and intact (good-looking) seeds was recorded for each burial treatment, and the intact seeds were examined for viability. As a control group, when buried seeds were retrieved, 30 closed cones, which had ripened in the same year when the seeds were buried, were collected from the 15 selected trees, and their seeds were examined for viability. In late December 2014, cones that matured in 2013 were collected from plots in the WD, KM, and LL populations. In January 2015, seed burial experiments were performed on these three populations similarly as in 2014, but with each bag containing 20 seeds instead in all treatments. And the soil surface (0 cm) seeds were bagged in nylon mesh bags. At the beginning of May (after four months) and October (after ten months) in 2015, and at the beginning of May in 2016 (after 17 months), buried seeds were retrieved for determination of seed mortality. As a control group, closed cones on the selected trees were collected for assessment of viability. On the basis of the seed survival data, seed viability was determined, assuming the burial of 100 potentially germinable seeds.
populations WD, KM, SL and LL are located in an area with a typical Central Yunnan Plateau climate, where the P. yunnanensis plants are serotinous with canopy seed banks (serotinous populations, Ser, data in Tables 1, S1). Populations BQ, CD, and WS are located in the eastern marginal areas. In these three eastern populations, P. yunnanensis plants retain and release seed annually, and no canopy seed banks have been observed (non-serotinous populations: non-Ser, data in Tables 1, S1). 2.2. Determination of the canopy seed bank in unburnt forests In late April 2014, three 10 × 10 m plots were randomly established in each population, the minimum distance among plots is 30 m. For each of the 21 plots among the seven populations, cones were allocated to a cohort (ripening year) and status (open or closed). In each plot, three trees were randomly selected to collect all cones on a branch, and the number of seeds in each collected cone was counted. The young seeds (≤3 serotinous years) in each cohort constituted one set, whereas older seeds (≥4 serotinous years) in a plot were mixed together as a second set. We randomly selected 100 seeds in each set, which were equally divided into five groups (each group with 20 seeds), and examined their viability by the germination experiment as described in 2.5. All the good-looking seeds were recorded as intact seeds, and the germinating seeds were recorded as germinable seeds. On the basis of the number of closed cones in each plot, the average number of seeds in a cone, and the average germinability in a cohort, we established the structure of the canopy seed bank in each plot. Seed production in the previous year was established from the number of closed and open cones multiplied by the average number of seeds in a cone. The density of canopy seed was calculated as the total seeds in the plot divided by the area of 100 m2. On the basis of the number of closed and open cones in a cohort, we determined the ratio of open cones in a cohort. The ratio of open cone in one cohort was the accumulated ratio of open cones in that cohort subtracted from the accumulated ratio of open cones in the previous cohort. The density and the germinability of seed rain were the ratio of open cones in each cohort multiplied the average seed number and the average germinability of each cohort.
2.5. Seed viability analysis Collected seeds were sown in culture bottles (7.5 cm in diameter, 8.0 cm in height) on four layers of filter paper saturated with demineralized water and incubated at 1250 lx at 21 °C for 8 h followed by dark at 18 °C for 16 h. This light/dark temperature pattern was continued for 45 days. Germination was assessed daily and germinated seeds were counted and removed every 2 days during the 45-day period. A seed was considered to have germinated when the radicle could be seen with the naked eye, and was recorded as a germinable seed.
2.3. Investigation of the soil seed bank in unburnt forests In late April 2014, seed bank investigations were conducted in the same plots used for canopy seed bank analysis. A fishnet with 2 m interval was set in each plot. At each intersection, a soil core sample was obtained using cutting rings (5 cm in diameter and 5 cm in height), with a total of 36 soil cores being collected in each plot. Since the seeds of P. yunnanensis are approximately 3 mm in diameter and 5 mm long, seed stock in the soil was estimated using a direct technique (Malone, 1967). Seeds of P. yunnanensis were extracted and isolated from the soil surface (litter layer) and from soil cores using 2-mm-mesh sieves. Identified intact (good-looking) seeds were recorded for each cutting ring. All seeds in the same layer of each plot were mixed together and examined for their viability. For each 100 m2 plot, the density of seeds in the soil seed bank was the total seed number in 36 soil cores divided by total area of 36 soil cores (0.0707 m2).
2.6. Statistical analysis The parameters for serotinous and non-serotinous forest types were derived from the parameters of populations, the parameters for population were derived from plots, and the parameters for plots were derived from trees or samples. The One-Sample Kolmogorov-Smirnov Test was performed to test the distribution of data. Most data on the density of seeds followed the normal distribution, but the data on the density of germinable seeds in the soil did not. The differences of density of seed were assessed by Ttest for normal distribution data. The data on seed fate in burial experiments did not follow the normal distribution. So the chi-square test was used in test differences. The significance level was P ≤ 0.05. The Pearson correlation was conducted to analyse the relationship between seed inputs from seed rain and the seed stored on the floor, with values considered to be significant when P ≤ 0.05. Statistical analysis was performed using Statistical Product and Service Solutions 13.0 for windows (SPSS Institute Inc., Chicago, IL, USA). We fitted the generalized linear mixed models with the location of seeds, serotiny of populations (serotinous vs. non-serotinous) and the conditions of seeds (intact vs. germinable seeds) being fixed factors, while the study site being the random factor. The models were fitted using the lme4
2.4. Seed burial experiment At the beginning of January 2014, 10–20 cones that had matured in 2013 were collected from each plant in plots of the LL population. The cones were opened artificially, and all seeds collected in each plot were mixed together. In total, 4600 seeds were randomly selected and equally divided into 46 groups, each of which comprised 100 seeds. One group was randomly selected and equally divided into five subgroups to examine seed viability. A further 30 groups were placed into 30 nylon mesh bags (10 × 10 cm with 2 mm mesh size), with each bag containing 100 seeds. The remaining 15 groups, not packed into bags 43
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Fig. 1. The condition of the seed banks in seven Pinus yunnanensis populations. (a–d) Seed banks in the serotinous populations (Ser) and (e–g) in the non-serotinous populations (non-Ser). Soil S, on the soil surface; Soil, in the soil; Canopy, in the canopy. The blank bars indicate the density of intact seeds, and the hatched bars indicate the germinable seeds.
50.7 ± 43.7 seeds/m2 and 28.3 ± 22.4 seeds/m2, respectively. Only a few viable seeds were found in the soil, and only those in one serotinous population (LL) were found to show germinability (Fig. 1). Comparing with the seed banks of the soil and the canopy: in serotinous populations, the number of viable seeds in the canopy was considerably greater than that in the soil seed bank, and the density of germinable seeds in the canopy was 697.9 ± 375.5 seeds/m2, which was approximately 31 times that in the soil seed bank. In non-serotinous populations, cones were retained on the plants for less than 1 year. All cones opened and released seeds within a single year (before June of the following year, Table 1). In late April, the density of germinable seeds in the canopy of trees in non-serotinous populations was 16.0 ± 17.5 seeds/m2, which was not significantly different from that of seeds on the soil surface (15.7 ± 8.7 seeds/m2, t = −0.048, P = 0.963). And this number is only 1/50 of the size found in serotinous populations canopy. In both serotinous and non-serotinous populations, the canopy seed bank deposited a considerable number of seeds onto the floor via seed rain. In serotinous populations, 203.4 ± 114.1 seeds/m2 fell in seed rain during the dry season, whereas in non-serotinous populations, this figure was 447.7 ± 109.6 seeds/m2. By late April, however, only a few seeds were found on the floor in either serotinous or non-serotinous populations, with a means of 22.4 ± 14.0 and 15.7 ± 8.7 viable seeds per 1 m2 being recorded on the floor (including on the soil surface and in the soil) in both serotinous and non-serotinous populations, respectively. By the end of April, 19.8% ± 24.8% and 5.1% ± 3.5% of germinable seeds on the forest floor derived from seed rain were still viable in serotinous and non-serotinous populations, respectively. Although the number of seeds stored on the soil surface was substantially lower than the input from seed rain, there was no significant relationship between seed inputs from seed rain and the seed stored on the floor in P. yunnanensis forests (r = 0.097, P = 0.676).
package for R (Bates et al., 2011) 3. Results 3.1. Condition of the seed bank in P. yunnanensis forests The size and structure of seed banks showed variation among the seven investigated populations of P. yunnanensis (Fig. 1). During the late dry season, seeds were stored in the canopy (Canopy), on the soil surface (Soil S), and within the soil (Soil) in each of the populations. Although some of the seeds in the canopy and on the soil surface were germinable, few viable seeds were found in the soil (Fig. 1). Serotiny enhanced the size of seed banks. The density of seeds in the serotinous populations WD, LL, KM, and SL was approximately 720.3 ± 373.8 seeds/m2, which was significantly greater than that in nonserotinous populations (31.7 ± 19.2 seeds/m2, t = 6.4, P < 0.001). We did not find any seed stored in the closed or partly open cones embedded or buried in the forest floor. About the soil seed bank, intact seeds were found on the soil surface in all plots, some of which were viable (Fig. 1). The number of sound and viable seeds varied spatially. The density of intact seeds (127.4 ± 80.3 seeds/m2 in serotinous populations and 119.5 ± 50.6 seeds/m2 in non-serotinous populations) and the density of germinable seeds (21.2 ± 14.5 seeds/m2 in serotinous populations and 15.7 ± 8.7 seeds/m2 in non-serotinous populations) on the soil surface were not significantly different between serotinous and non-serotinous populations (t = 0.274, P = 0.787 for intact seeds and t = 1.079, P = 0.294 for germinable seeds). Among all populations, the average density of germinable seeds on the soil surface was 18.9 ± 12.4 seeds/ m2, and the rate of germination of intact seeds was approximately 15% (the density of intact seeds was 124.0 ± 67.7 seeds/m2). The density of intact seeds in soil in serotinous and non-serotinous populations were 44
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Table 2 Summary of the GLMM for the difference of seed density, including serotinous type (non-Ser: non-serotinous populations; Ser: serotinous populations), location of seed (Soil S: on the soil surface; Soil: in the soil; Canopy: in the canopy), condition of seed (Intact: good-looking seeds; Germinable: germinable seeds). The study site was included as a random factor. Degree of freedom, Akaike information criterion (AIC), χ2 and the associated P value of each step in the models are presented. The rightmost column provides the estimated parameters for the fixed effects of the final model. Model
df
AIC
χ2
P
Estimate
Null Serotinous Type Location of seed Condition of seed
3 4 6 7
1826.5 1819.5 1763.4 1761.8
8.9251 60.1259 3.6318
0.0028 < 0.0001 0.0567
−87.53[intercept] 0[non-Ser], 262.82[Ser] 0[Soil], 50.53[Soil S], 437.28[Canopy] 0[Intact], −83.53[Germinable]
at soil depths of 2 and 5 cm soil had lost longevity due to decay, respectively, whereas 4.2% ± 3.7% and 1.0% ± 1.2% of the seeds germinated in situ, although these subsequently died before reaching the surface. During the 2015 rainy season in the three examined populations, 57.7% ± 33.0% and 67.0% ± 18.1% of seed buried at soil depths of 2 and 5 cm lost longevity by decay, respectively, whereas 5.0% ± 5.0% and 5.3% ± 4.4% germinated, but died before reaching the surface. After rainy season, 12.4% ± 5.9% and 19.2% ± 4.1% (in 2014) and 37.3% ± 30.0% and 27.7% ± 16.5% (in 2015) of seeds buried at depths of 2 and 5 cm, respectively, were viable. Although these values were higher than those for soil surface seeds, all viable seeds had died by the end of the following dry season. No seeds persisted in the soil after 17 months burial, and during these 17 months, the highest losses for seeds buried in the soil occurred during the rainy season (Fig. 2). Seeds derived from the canopy seed bank were lost in seed rain during the dry season, as P. yunnanensis is a weakly serotinous species in which some cones retained in the canopy opened and released seed (Fig. 2). Approximately 15–30% of seeds stored in the canopy are shed each year, whereas the development of new cones contributes to fresh seed input into the canopy seed bank. Seed mortality in the canopy was considerably lower than that on the soil surface and in the soil. In the canopy, the germinability of seeds, which were of the same cohort as the buried seed, decreased by 3.7% ± 1.9% after 17 months of maturing, during which they passed through two dry seasons and one rainy season. In the three examined populations, the germinability of seeds retained in the canopy for four years was greater than 70%. We found that the mortality of seeds stored in the canopy did not differ significantly among populations.
The serotinous populations had a higher density of seeds than the non-serotinous populations (Fig. 1, Table 2). The canopy had a higher density of seeds than soil surface, and in the soil the density of seeds was the lowest (Fig. 1, Table 2). The density of intact seeds was higher than the germinable seed (Fig. 1, Table 2). 3.2. The fate of seeds accumulated on the soil surface, buried in soil, and stored in the canopy In 2014, after being burial for four months (at the end of dry season), only four seeds had not disappeared, while there were 1500 free seeds (the seeds not packed into bags) initially placed on the soil surface in fifteen burial plots. No chippings of seed coat were found in the subplots. None of the four remaining seeds germinated in the germination test. There were thus no viable seeds remaining on the soil surface to the end of the dry season (Fig. 2). In 2015, seeds were bagged in mesh bags and placed on the soil surface. All bagged seeds remained on the soil surface during the burial experiment (Fig. 2). After four months (in the dry season), 15–30% of seeds in the surface bags were found to be broken, whereas the remainder were sound. The sound seeds, 50–75% showed germinability. During the dry season, the mortality of bagged seeds on the soil surface was 39.9% ± 21.2%. After ten months (at the end of the rainy season), most of the seeds in bags placed on the soil surface were found to be dead, with a mortality of 94.0% ± 3.4%. None of the seeds on the soil surface germinated in situ during the rainy season (Fig. 2). After 17 months, all bagged seeds on the soil surface had died in the KM and LL populations, whereas 1% seed viability was recorded for population WD. During the 17 months, the highest losses occurred during the rainy season (Fig. 2). Compared with the seeds placed on the soil surface, burial did not increase seed longevity, as no seeds were still viable after 17 months (Fig. 2). The pattern of loss of buried seeds tended to differ from that for surface seeds. In 2014, after four months of burial, seed mortality at soil depths of 2 and 5 cm were 3.6% ± 6.5% and 10.2% ± 8.4%, respectively, and these values had not significantly differed from those recorded at the same depth in 2015 (χ2 = 4.000, P = 0.261; χ2 = 4.000, P = 0.261, respectively). In 2015, after four months of burial, seed mortality in the three examined populations with soil depths of 2 and 5 cm were 9.0% ± 7.6% and 11.3% ± 4.4%, respectively, which were lower than those on the soil surface. In general, seed mortality at 5 cm depth was greater than that at 2 cm in the dry season. In the rainy season of 2014 (after burial for 10 months), seed losses at soil depths of 2 and 5 cm were 83.4% ± 10.9% and 70.6% ± 8.1%, respectively, which were higher than those at the same depth in the same populations during the dry season of that year. In the rainy season of 2015, seed losses in the three populations at soil depths of 2 and 5 cm were 59.4% ± 35.3% and 61.4% ± 24.0%, respectively, which were also higher than those at the same depth during the dry season. In general, there was no significant difference in the losses of seeds buried at depths of 2 cm and 5 cm. During the rainy season, viable seed in the soil had two possible ways to lose vitality: most seeds lost lives because of decay, whereas a few seeds germinated in situ but subsequently died (Fig. 2). In 2014, in the LL population, 80.2% ± 6.5% and 80.4% ± 5.0% of seeds buried
4. Discussion Soil is a safe habitat for many species, and having entered the soil, seeds can retain long-term viability (Wijayratne and Pyke, 2012). In fire-prone habitats, seeds buried at depths of 2–5 cm represent an important source for seedling recruitment after fire (Tozer, 1998; Auld and Denham, 2006). In contrast, seeds buried at depths down to 2 cm succumb to the high temperatures generated by fires, whereas at depths below 5 cm, seed dormancy cannot be broken by the heat or smoke from fire, and the germination of seeds buried at this depth is reduced after fire (Auld and Denham, 2006). The distribution range of P. yunnanensis in the Central Yunnan Plateau has a monsoon climate characterized by moderately hot rainy seasons and a warm dry season (Chen et al., 2014). Most of the wildfires in this region occur during the late dry season from February to April (Zhao et al., 2009). The status of the seed bank during the period from February to April is important in relation to the adaptation of pine forests to fire disturbance in the Central Yunnan Plateau. At the end of April, in both serotinous and non-serotinous populations of P. yunnanensis, we found that there were few seeds on the soil surface and virtually no viable seeds were found within the soil (Fig. 1), while the viable seeds in canopy were considerably more than in soil seed bank (Fig. 1, Table 2). This is consistent with our prediction. These results indicate that P. yunnanensis cannot depend on the soil seed bank 45
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Fig. 2. The fate of a cohort of potentially germinable seeds under different conditions. The first row (a–d) shows the fate of seeds in 2014 for population LL. The 2nd row (e–h) shows the fate of seeds in 2015 for population KM. The 3rd row (i–l) shows the fate of seeds in 2015 for population LL. The 4th row (m–p) shows the fate of seeds in 2015 for population WD. The first column (a, e, i, and m) shows the fate of seeds on the forest floor (0 cm). The 2nd column (b, f, j, and n) shows the fate of seeds buried at a soil depth of 2 cm. The 3rd column (c, g, k, and o) shows the fate of seeds buried at a soil depth of 5 cm. The 4th column (d, h, l, and p) shows the fate of seeds in the canopy.
as a survival strategy in fire-prone environments, which is similar to P. sylvestris and P. halepensis (Daskalakou and Thanos, 1996; Valbuena et al., 2001). In P. yunnanensis forests, the low density of seeds on the forest floor were not a consequence of a low import of seeds. In both serotinous and non-serotinous populations, considerable amounts of seed were dispersed to the forest floor in the dry season via seed rain. However, by the end of the dry season, only about 30% of the seed deposited on the forest floor remained, of which 90% had lost germinability. Considering that there is almost no soil erosion or rainfall in the dry season, the disappearance of seeds deposited on the forest floor can probably be attributable to predation by rodents, birds, and insects (Chambers and MacMahon, 1994; Jin and Peng, 2004; Zhang et al., 2009). The rate of disappearance of experimentally-placed surface seeds was as high as 99.7%. Even so, the conditions on the floor are not conducive to the retention of seed viability in pine forests. Indeed, our results showed that almost all seeds on the forest floor had lost viability within 17 months. However, the seeds retained viability in the canopy (Fig. 2). The seeds in the soil seed bank are derived from seed rain (Chambers and MacMahon, 1994). For species producing seeds that are
larger than pore sizes in the soil, the seeds originating from seed rain tend to remain on the soil surface (Thompson et al., 1993). Such seeds can only gain entry to sub-surface layers via secondary means of dispersal, which is determined by the nature of abiotic and/or biotic factors (Chambers and MacMahon, 1994). Secondary dispersal events tend to be unpredictable and happen by chance. In general, only a few large seeds become incorporated into the soil (Sem and Enright, 1996; Zhang et al., 2009). In P. yunnanensis forests, however, the low density of seeds in the soil seed bank was not a consequence of the low import of seeds. During every dry season, numerous seeds are shed in both serotinous and non-serotinous populations. Given that P. yunnanensis seeds exceed 3 mm (Jin and Peng, 2004), they are assumed to enter the soil via secondary dispersal. Among all our study sites, only one viable seed was found in the soil. On the basis of the findings of our seed burial experiment, seed longevity is approximately one and half years. Accordingly, we believe that viable seeds in the soil are incorporated into soil in the previous year. It is estimated that less than one ten thousandth of the seeds falling as seed rain are incorporated into the soil via secondary dispersal. In the three serotinous populations, we found that the annual production of seeds was 432.1 ± 171.7 seeds/m2, and even 46
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Declarations of interest
though all of these were shed, only approximately 4–5 seeds could enter the soil in a single 100 m2 plot. As a dominantly early successional and pioneer species in Central Yunnan Plateau pine forests (Jin and Peng, 2004), the density of P. yunnanensis seeds in the soil is too low to meet the seedling recruitment necessary in post-fire land. In addition, the results of our burial experiment indicate that soil is not necessarily a safe habitat for P. yunnanensis seeds. Even the longevity of seeds incorporated into soil at depths of 2 and 5 cm was shorter than 17 months. Fungal attack is assumed to be a main source of seed loss in soil seed banks during both the dry and rainy seasons (Fig. 2). P. yunnanensis seeds did not persist for more than two years in soil, and there was no accumulation of viable seeds to enhance the size of the soil seed bank in P. yunnanensis forest. Our results showed that the vitality of seeds is reduced in the soil. Accordingly, this species appears to be unable to establish a persistent soil seed bank to compensate for mortality after fire. In contrast to the soil seed bank, cones can protect seeds from predation and decay during both dry and rainy seasons. Viable seeds can be stored for up to nine years in the cones of P. yunnanensis (Su et al., 2015). The seeds matured in previous years can thus accumulate to form a canopy seed bank (Su et al., 2015, 2017). Additionally, seeds within serotinous cones are protected from the extreme heat generated by forest fires (Hellum and Pelchat, 1979; Lamont et al., 1991). Even if trees are subjected to crown fire and eventually succumb to fire, some seeds within the scorched cones on dead trees retain their viability (Su et al., 2017). These are released after fire onto the burnt land and germinate during the following rainy season. Therefore, the canopy seed bank is able to recruit in large numbers of seedlings in post-fire habitats, and thereby ensures the persistence of P. yunnanensis populations in fire-prone environments. Our results and those of pine studies worldwide indicate that there are considerable losses of pine seeds on the soil surface (Jin and Peng, 2004; Zhang et al., 2009), and that few seeds on the soil surface are subsequently incorporated into the soil via secondary dispersal. Accordingly, these seeds are rendered vulnerable to decay and lose germinability. This means there is no persistent soil seed bank, or that at best only a small temporally limited soil seed bank develops in pine forests (Daskalakou and Thanos, 1996; Ferrandis et al., 1996; Baskin and Baskin, 1998; Izhaki et al., 2000; Valbuena et al., 2001; Teste et al., 2011). Therefore, the soil seed bank of pines is assumed to play only a minor role in the post-fire land of pine forests (Ferrandis et al., 1996). However, the cones of pines are a seed-retaining structure that is better adapted to protect seeds. Seeds retained in the serotinous cones of pines can maintain longevities of up to 30 years (Lamont et al., 1991; Teste et al., 2011), and successive seed crops accumulate to enhance the number of seeds stored in the canopy. The closed cones provide better insulation from the heat of fires (Lamont et al., 1991), thereby ensuring the survival of a least some viable seeds in scorched cones. Large amounts of viable seeds are released and fall to the forest floor (Daskalakou and Thanos, 2004), and these germinable seeds can give rise to viable seedlings during the next reliable growing season (Daskalakou and Thanos, 2004). So, according to our study, serotiny enhances the size of the P. yunnanensis seed bank and that serotiny of this species has an adaptive advantage over soil storage in Central Yunnan. The conclusion implies that the regeneration of P. yunnanensis forest is more dependent on canopy than on soil seed banks.
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Acknowledgements We thank the two anonymous reviewers and the editor for helpful comments and suggestions, Boqiang Huang and Dongfang Huo for field and laboratory assistance, Prof. Cindy Q. Tang for helpful comments and linguistic revision, and Prof. Zhiying Zhang for valuable discussions. This research was supported by the National Natural Science Foundation of China (31160092, 31770450, and 31260111). 47
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