Interaction of functional and environmental traits on seed germination of the multipurpose tree Flacourtia indica

South African Journal of Botany 125 (2019) 427–433

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Interaction of functional and environmental traits on seed germination of the multipurpose tree Flacourtia indica P. Gómez-Barreiro a,⁎, V. Otieno b, E. Mattana a, E. Castillo-Lorenzo a, W. Omondi b, T. Ulian a a b

Royal Botanic Gardens, Kew, Wellcome Trust Millennium Building, Wakehurst Place RH17 6TN, UK Kenya Forestry Research Institute, P.O Box 20412-00200, Nairobi, Kenya

a r t i c l e

i n f o

Article history: Received 26 April 2019 Received in revised form 18 July 2019 Accepted 2 August 2019 Available online xxxx Edited by MI Daws Keywords: Cardinal temperatures Relative light germination index Salicaceae Seed dormancy Seedling emergence

a b s t r a c t Flacourtia indica (Salicaceae) is a multipurpose tree native to tropical regions of Africa and India, where it is mainly used for food and medicines. Its use in reforestation is limited by poor knowledge of its seed biology and germination ecology. The aim of this study was to determine how its morphological and ecological seed traits interact with the environment and affect seed germination and seedling emergence. The results showed high germination percentages (N70%) when stones were incubated at alternating temperature regimes, regardless of the photoperiod. Lack of daily temperature fluctuation resulted in low germination, especially when the stones were incubated in the dark (b 12%). Germination in a 12/12 h photoperiod at constant temperature was positively correlated with temperature, although final germination never surpassed 50%. The addition of GA3 to the substrate resulted in high final germination regardless of temperature regime or photoperiod. Stones and seeds imbibition curves suggest a lack of physical dormancy, while the presence of a fully developed spatulated embryo excluded morphological dormancy. High germination at alternating temperatures (N 70%) discarded the presence of deep physiological dormancy (PD), but differences found between chipped and non-chipped seeds suggest the presence of a non-deep PD, imposed by the seed coat. Thus, difficulties in propagating this species from seeds, which have been previously reported in the literature, are probably due to initial poor seed lot quality rather than dormancy. These results suggest that F. indica seed germination is more likely to occur near the soil surface or when the tree canopy is disrupted, where daily temperature fluctuations are high and light might become available. Viable seeds not meeting germination conditions are likely to become part of the soil seed bank. Our results will help to support the effective use of F. indica seeds in reforestation and livelihoods programmes. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Multipurpose trees remain a key part of the socioeconomic development for many rural communities (Houehanou et al., 2011; Ulian et al., 2017). However, the unsustainable harvest of various tree parts and their products may affect survival options of the individuals and have a long-term impact in their population structure (Gaoue and Ticktin, 2007; Nacoulma et al., 2017; Stewart, 2009). Understanding seed functional traits and their interaction with the environment can help plant propagation and reforestation programmes to reduce the impact of over-exploitation by enhancing their sustainable use in plant propagation programmes. Native seed lots stored ex situ for long-term conservation may play a key role on restoration. However, propagating plants from seeds depend on the efficient use of stored germplasm, an understanding which often is not available (Hay and Probert, 2013). ⁎ Corresponding author at: Wellcome Trust Millennium Building, Wakehurst Place RH17 6TN, UK. E-mail address: [email protected] (P. Gómez-Barreiro).

https://doi.org/10.1016/j.sajb.2019.08.013 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

Seed functional traits regulate key plant stages such as dispersal, germination and seedling establishment. However, these traits are often overlooked, and the necessity of a broader effort to record the presence and function of these traits has recently been suggested (Saatkamp et al., 2019). Seed germination, a critical step for plant establishment, depends upon the interaction between the seeds and their environment (Bewley and Black, 1994). Water availability and temperature are among the most important external factors affecting germination (Alvarado and Bradford, 2002), although the presence or absence of light can play a key role as well (Baskin and Baskin, 2014; Carta et al., 2017). Seeds will not germinate if subjected to temperatures (T) under or over certain cardinal values, known as base temperature (Tb) and ceiling temperature (Tc) respectively (Alvarado and Bradford, 2002). In some cases, alternating temperatures yield higher germination percentages than constant temperature regimes (Baskin and Baskin, 2014); or they may even be strictly necessary to trigger germination (e.g. Fernández-Pascual et al., 2015). Therefore, cardinal temperatures of a seed lot might differ depending on the temperature regime

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(Galíndez et al., 2017). Similarly, whilst the germination of many species is indifferent to light, some seeds may require the presence or total absence of light, depending on the germination strategy of the species (Carta et al., 2017; Milberg et al., 2000). In addition, some species are known to germinate when light is available and temperature fluctuates; a germination strategy known to help species germinate as soon as a gap occurs in the tree canopy (Xia et al., 2016). Seed burial depth also plays a role in such interactions and can be linked with seedling establishment success (Bond et al., 1999). Dormancy, or lack of germination of viable seeds under favourable conditions adds an extra level of complexity, with several morphological and physiological mechanisms in the tissues and cells of fruits and seeds, preventing homogeneous germination (Steinbrecher and Leubner-Metzger, 2018). In particular, impermeable covering structures might prevent embryo imbibition (i.e., physical dormancy, PY); physiological constraints preventing embryo growth (physiological dormancy, PD); or underdeveloped embryos which require long growth periods before radicle protrusion (morphological dormancy, MD) (Baskin and Baskin, 2004). Thus, seed dormancy is related to fruit and seed morphology and can often be predicted by conducting a morphological study of the diaspore (e.g. Baskin et al., 2005; Mattana et al., 2018; Zhou et al., 2009). Flacourtia indica (Burm.f.) Merr. (Salicaceae) is a 3–5 m high multipurpose tree, usually dioecious, native to tropical regions of Africa and India (Orwa et al., 2009). Its fruits are consumed as appetisers, used to treat digestive-related diseases and to produce alcoholic drinks. Each of its fruits (drupes) can contain up to 10 pyrenes (Maundu and Tengnäs, 2005), which consist of a small, ovate and reddish-brown endospermic seed, enclosed in a woody endocarp (Dathan and Singh, 1973). Other plant parts are used as food for cattle (branches and leaves); fuel and timber (wood); and medicines to treat malaria, fevers, pneumonia, hoarseness and body pain (Kaou et al., 2010; Leakey and Newton, 1994; Nazneen et al., 2009; Orwa et al., 2009; Saka and Msonthi, 1994). Although F. indica is an important species for local communities in Africa (Leakey and Ajayi, 2007), little is known about its propagation from seed, and the few existing reports highlight poor germination (≤ 20%) under different conditions (Prins and Maghembe, 1994). Vegetative plant production is possible, but its sole use might have an impact in the genetic diversity of the species and its resistance against pest and diseases (e.g. Sano et al., 2008). The aim of this study was to determine how functional seed traits interact with environmental factors such as, temperature and light affecting the reproduction by seed of F. indica to provide new data to support plant propagation, reforestation and livelihood programmes.

running water and left to dry in the shade. Stones arrived at the Millennium Seed Bank (West Sussex, UK) in September 2016, where they were stored at 15 °C and 15% relative humidity (RH) until the start of the experiments in October 2016. 2.2. Imbibition studies Water uptake rates were obtained for chipped and non-chipped stones and seeds, carefully extracted from the stones with a vice press. Chipping consisted of the removal of a portion of the endocarp from the stones and of the seed coat in seeds (Fig. 1). Experimental design for stones consisted of 3 replicates of 20 stones each for every treatment. The initial weight of stones was recorded along with the mass of the diaspores after 1, 2, 4, 6, 24 and 52 h of soaking in distilled water (Semi-Micro Balance, HA-202M, A&D). Stones were cut in half at the end of the experiment to visually inspect embryo imbibition. To calculate seed water uptake rates, 10 seeds for each treatment were soaked in water. Individual seed weight was monitored at the same time intervals as above (Microbalance UMT2, Mettler Toledo). 2.3. Germination experiments Four replicates of 20 intact stones each were sown in 90 mm Petri dishes containing 30 ml of 1% sterile agar solution (80–100 Mesh, Fisher Scientific, UK). Germination trials consisted of a factorial design including, two photoperiods (12 h light/12 h darkness vs. 24 h darkness), presence and absence of gibberellic acid (250 ppm GA3, Sigma–Aldrich, UK), two temperature regimes (constant and alternating) and three temperatures (15, 20, 25 °C as constant regimes and 20/10, 25/15, 30/20 °C as alternating regimes). Dark conditions were achieved by covering Petri dishes with aluminium foil. Germination was checked twice per week for the first two months and then on weekly basis. Dark-incubated Petri dishes were scored for germination in a dark room under a safe green light (Probert and Smith, 1986). Seeds were considered germinated when the root protruded at least 2 mm from the stone. Experiments ended with a cut test after no germination was recorded in the experiments for two consecutive weeks. Remaining non-germinated stones were cut in a half to visually inspect their viability and were classified as viable, mouldy or empty. 2.4. Data analysis Water uptake percentages for stones and seeds were calculated using the following formula (Hidayati et al., 2001):

2. Material and methods 2.1. Seed lot details Ripe drupes of F. indica were collected on 23 August 2016, near Jua Kali area (0°36’N, 35°09′E, Eldoret, Uasin Gishu County: 340 km West of Nairobi, Kenya), at an altitude of ca.1975 m a.s.l., in a temperate area with dry and warm seasons according to Beck et al. (2018) as perceived by the annual trends of temperature and rainfall (Table 1). Pyrenes (hereafter stones) were extracted from the fleshy fruits in the laboratory of the Kenya Forestry Research Institute (KEFRI), under

%ΔMt ¼ ½ðMt −M i Þ=M i   100

where %ΔMt is the mass increment expressed as percentage at a given time “t”, Mt represents the mass at a time “t” and Mi is the initial mass at time t = 0. Average mass increment in stones was based upon 3 replicates of 20 stones each, while average mass increment in seeds was based upon 10 individual seeds.

Table 1 Historical (1950–2000) monthly climate conditions for the F. indica population investigated in this study, including rainfall (mm) and maximum, minimum and average temperature (°C). Values were extracted from WorldClim database 2.0 (Fick and Hijmans, 2017). The month of fruit collecting/dispersal is highlighted in bold.

Max. temp (°C) Min. temp (°C) Avg. temp (°C) Rainfall (mm)

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

26 10.4 18.2 29

26.4 10.8 18.6 42

26.7 11.1 18.9 68

26.3 10.7 18.5 130

25.5 9.9 17.7 132

24.8 9.2 17 109

24.2 9.1 16.6 158

24.3 9.2 16.7 183

24.7 9.6 17.1 90

25.8 10.7 18.3 58

25.3 10.2 17.8 63

25.4 10.3 17.9 38

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value was obtained by fitting a lineal regression of the seed germination rates (GR: 1/T50) at each incubation temperature, for each treatment. Thermal time (S, °Cd) was then calculated as the inverse of the slope of the fitted linear regression in the sub-optimal range. 3. Results 3.1. Stone and seed mass and their imbibition curves

Fig. 1. (A) Longitudinal section of F. indica stone (ch, chalaza, cot, cotyledon, edc, endocarp, eds, endosperm, ra, root axis, sc, seed coat, vb, vascular bundle) based on Dathan and Singh (1973) and Corner (1976); (B) External view of F. indica seed extracted from the stone.

Maximum seedling emergence depth of F. indica was estimated using Bond et al. (1999) model on stones and seeds weight: dmax ¼ 27w0:334 where “dmax” is the maximum emergence depth (mm) and “w” is the stone/seed weight (mg). Final seed germination was calculated as the average germination of the replicates against the initial number of seeds sowed (excluding empty stones). Time to reach 50% of final germination (T50) was obtained following Coolbear et al. (1984) formula modified by Farooq et al. (2005): T 50 ¼ t i þ


  ðN=2−ni Þ t i −t j ni −n j

where N represents the maximum number of germinated seeds from a single replicate in all the experiments and ni and nj are the number of seeds germinated adjacently to (N + 1) at time ti and tj respectively. The effect of light presence or absence on germination was studied for each of the temperatures using the relative light germination index (RLG) (Milberg et al., 2000), RLG ¼

Gl ðGd þ GlÞ

where Gl represents the proportion of germinated seeds when stones were in the presence of light and Gd is its equivalent in dark conditions. RLG values can range from 0 (negative photoblastism) to 1 (positive photoblastism). 2.5. Statistical analysis Average stone and seed mass increment during imbibition were compared using a paired t-test. The maximal generalised linear model with a binomial link function was fitted to the germination results, using light, temperature regime and presence of GA3 as categorical factors and temperature as a quantitative continuous factor. The minimal model was selected among all possible models (considering only up to second-order interactions) ranked by the Akaike information criterion (AIC). The model with the lower AIC was chosen after an exhaustive screening using R v. 3.5.1 (R Development Core Team, 2017) and the package “glmulti” (de Mazancourt and Calcagno, 2010). A multiple comparison using Tukey's method between final germination and the combined factors for each of the experiments was performed using R v. 3.5.1 (R Development Core Team, 2017) and the package “multcomp” (Hothorn et al., 2008) in order to create a letter display reflecting an all pair-wise comparison between the experiments. Base temperature (Tb) is defined as the minimal temperature below which germination no longer occurs (Garcia-Huidobro et al., 1982). This

Average mass of air-dried filled stones was 23.3 ± 4.2 mg, which resulted in an estimated maximum depth for seedling emergence of 8.3 cm. No significant differences (p N .05) were found in mass gain between chipped and non-chipped stones during imbibition. Within the first hour of imbibition, the mass of chipped and non-chipped stones increased by 19 ± 4% and 17 ± 1% respectively, and 28 ± 6% and 29 ± 6% after 52 h respectively (Fig. 2A). Visual inspection of sectioned stones upon the conclusion of the imbibition experiment confirmed that embryos were able to imbibe in non-chipped stones. Average mass of air-dried seeds was 7.4 ± 2 mg, resulting in an estimated maximum depth for seedling emergence of 5.3 cm. Chipped and non-chipped seeds imbibed quickly, although significant differences (p b .05) between treatments were observed in measurements. Within the first hour, seed mass increment was 19.2 ± 3% for chipped seeds and 16 ± 2% for non-chipped seeds. After 52 h, the mass of chipped seeds had increased by 36 ± 2%, while non-chipped seed mass increased by 41 ± 6% (Fig. 2B). 3.2. Seed germination When incubated at constant temperature regimes without GA3 seeds did not reach germination values over 50% (Fig. 3). Results ranged from 14% at 15 °C to ca. 45% at 25 °C (Fig. 3A) when light was present, and even lower (b10%) in dark conditions (Fig. 3B). Under alternating temperature regimes without GA3, final germination improved, ranging from 71 ± 2% at 20/10C and light to a final germination of 89 ± 6% at 20/ 10 °C and dark-incubation (Fig. 3). GA3 in the germination substrate resulted in high final germination values (N75%) for light- and darkincubated stones for both constant and alternating temperature regimes (Fig. 3). The cut test after the completion of the experiments indicated that 23 ± 8% of the stones in each replicate were seedless. RLG values were in general close to 0.5 except for experiments incubated at constant temperatures in the presence of light, that tended towards 1 (Table 2) but also had lower germination values (b45%). GLM output on germination (Table 3) revealed a positive significant impact (p b .001) when GA3 was applied, when stones were incubated at alternating temperatures and also with increased incubation temperatures. On the contrary, light as a main factor had no significant effect (p N .05) on germination. However, interactions between light and other main factors (treatment, regime and temperature) were significant (p b .01), as were treatment and regime (p b .001), and treatment and temperature (p b .01). Stones incubated at constant temperature regimes without GA3 had low seed germination values (Fig. 3) and therefore T50 was not calculated. Thus, T50 was only calculable for experiments with an alternating temperature regime and/or GA3. Untreated light-incubated stones had T50 values ranging from 29 ± 3 days at 20/10 °C to 22 ± 4 days at 30/20 °C in the light (Fig. 3A), while dark-incubated stones ranged from 55 ± 24 days at 20/10 °C to 32 ± 8 days at 30/20 °C in the dark (Fig. 3B). With GA3 in the substrate and constant temperature, T50 at 15 °C was 39 ± 4 and 53 ± 13 days when light and dark-incubated, respectively, reaching values around 20 days or less when incubated at 20 and 25 °C (Fig. 3). Under an alternating regime and GA3 present, T50 ranged from 29 ± 3 and 29 ± 5 days at 20/10 °C and 15 ± 3 and 16 ± 1 days at 30/20 °C when incubated with and without light, respectively (Fig. 3).

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Fig. 2. Average mass increment (%) during imbibition against time for non-chipped (control) and chipped stones (A) and seeds (B). * Denotes significant differences (p b .05) between samples.

All GRs regression lines were significant (p b .05), except for darkincubated stones without GA3 (Table 4). Estimated theoretical Tb values were below 0 °C for stones incubated without GA3. When GA3 was present, Tb values were over 2 °C, reaching ca. 10 °C on dark-incubated stones under alternating temperature regime. Thermal requirements to achieve 50% of final germination (S) were lower for experiments with GA3 (≤400 °Cd) than for untreated stones (ranging from ca. 590 to ca. 830 °Cd; Table 4). 4. Discussion 4.1. Seed dormancy Results of the imbibition experiments performed on stones and seeds of F. indica suggest that none of the different structures surrounding the embryo can prevent imbibition. Mass gain due to water uptake in species with PY is expected to be impeded by the impermeable layers preventing the embryo imbibition (Baskin and Baskin, 2004). However, F. indica stones and seeds were able to imbibe within the first hours of soaking in water, a clear indicator of the lack of PY. Visual inspection of embryos after completion of experiments corroborated that neither endocarp nor seed coat, prevented its imbibition. Endocarp permeability has been reported also in the Oleaceae family (Cuneo et al., 2010; Mira et al., 2017), although in these cases the endocarp seemed to constrain germination percentages when still intact. The hardy endocarp of F. indica did not seem to affect germination percentages when optimal

conditions were met, although it might have impacted on germination rate. Further research will be required to investigate if the germination at non-optimal conditions can improve after a certain levels of endocarp degradation. Confirming the description in Corner (1976), seeds had a fully developed spatulated embryo, which discounts the presence of MD for F. indica seeds. Incubation under favourable conditions (alternating temperatures) resulted in seeds germinating 15 days after sowing, reaching T50 relatively quickly. Rapid germination is a sign that deep levels of PD were not present. However, the significant differences found between chipped and non-chipped seeds could indicate the presence of a non-deep PD imposed by the seed coat (Baskin and Baskin, 2004). Seeds with PD often require a vernalization period to lose dormancy, which is linked to the concentrations of abscisic acid (ABA) and GA present in the seed. When exogenous GA is applied, PD level decreases and more seeds in the population can germinate, reducing the Tb. Under favourable germination conditions, F. indica showed a Tb increase when GA3 was applied (Table 3), opposite to what would be expected (Pritchard and Tompsett, 1996). Nevertheless, this study proves that F. indica does not have PY, MD or a deep PD. Non-deep PD may be present due to mechanical constrains of the woody endocarp, although no treatments were necessary to overcome it. Baskin and Baskin (2014) reported that F. indica might have PD based on personal communications, although there is no information about the trial conditions. A related species, F. rukam, is considered to have PD based on its germination behaviour under nursery conditions, but information regarding the trials is also not available (Baskin and Baskin, 2014; Ng, 1992). To our

Fig. 3. Summary of T50 (box plot) and final germination (bar chart) of F. indica under a 12/12 photoperiod (A) and dark-incubated (B). Results for experiments at constant temperature are in blue, and in green when fluctuating. Light colours are used to refer to experiments without GA3, while dark colours note presence of GA3. Different letters indicate significant differences between means for final germination and T50 (p b .01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P. Gómez-Barreiro et al. / South African Journal of Botany 125 (2019) 427–433 Table 2 Relative Light Germination index (RLG) of experiments at different temperature regimes, with and without GA3 and different temperatures. Highlighted in bold, experiments with a positive photoblastism.

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Table 4 Regression lines parameters (fitted equation: y = ax + b) for germination rates (1/T50), base temperature (y = 0) and thermal constant (S = 1/a) for the different treatments applied on F. indica stones.

Treatment

Temp (°C)

RLG

Treatment

Regime

Light

a

b

p-value

Tb (°C)

S (°Cd)

Control Control Control Control Control Control GA3 GA3 GA3 GA3 GA3 GA3

15 20 25 20/10 25/15 30/20 15 20 25 20/10 25/15 30/20

0.65 0.72 0.90 0.46 0.52 0.57 0.52 0.49 0.51 0.49 0.52 0.49

Control Control GA3 GA3 GA3 GA3

Alternating Alternating Alternating Alternating Constant Constant

12/12 0/24 12/12 0/24 12/12 0/24

0.0017 0.0012 0.0032 0.0029 0.0025 0.0041

0.0054 0.0022 −0.0134 −0.0079 −0.0058 −0.0394

0.0220 0.1523 0.0012 0.0041 0.0416 0.0001

−3.16 −1.79 4.22 2.75 2.34 9.55

588.24 833.33 312.50 344.83 400.00 243.90

knowledge this is the first in-depth study of seed dormancy in F. indica. Results show that germination of this species is not affected by dormancy, and therefore, low germination results previously reported are likely to be related to unsuitable conditions or poor initial seed quality (Prins and Maghembe, 1994). 4.2. Seed germination requirements Regardless of light conditions, F. indica stones reached high germination percentages only under alternating temperatures. However, in the presence of light and under a constant regime of temperatures, germination seemed to improve with increased temperature, reaching almost 50% at 25 °C. RLG values suggested light had an impact on germination under constant temperature regimes. Sunlight is restricted to around the first 2 mm of soil surface (Woolley and Stoller, 1978), although it varies depending on soil composition (Bliss and Smith, 1985). The ability of seeds to detect the presence of light enables them to notice gaps in the forest canopy, which ensures that germination occurs near the soil surface, enhancing the chances of a successful seedling establishment in nature (Thompson and Grime, 1983). With an increase in burial depth comes a reduction in the fluctuation of temperatures, which at 10 cm might be too small to trigger germination of species requiring temperature variation (Assche and Vanlerberghe, 1989). Therefore, it is likely that viable F. indica stones will germinate less with increased burial depth as opposed to near the soil surface, adding up to the soil seed bank whilst awaiting for an event to bring them up in the soil into more suitable germination conditions. However, the ability of this species to establish a permanent soil seed bank needs to be experimentally confirmed. If stone mass is considered, the allometric relationship of Bond et al. (1999) suggests that F. indica seedlings can establish

successfully as long as the stone is buried within the first 8.3 cm of soil. However, since the energy reserves needed for the seedling growth and establishment are within the seeds (i.e cotyledons) and surrounding tissues (i.e. endosperm) while the woody endocarp only play a protective role, seed weight should be used instead, leading to a maximum depth of 5.3 cm from the soil surface (Fig. 4). 4.3. Thermal thresholds for seed germination Plant species adapted to tropical environments are known to have higher Tb values (N 10 °C) than temperate species due to the requirement of warmer temperatures for their development (Trudgill et al., 2000). Flacourtia indica's native distribution ranges from South Africa up to the north of India, covering several climates to which it is adapted. The obtained Tb values (Table 4) emphasise the climatic adaptability of this species. However, to obtain a better estimate of the cardinal temperatures of this species it would have been necessary to explore germination at lower temperatures than those used in these experiments and to include seed lots from different geoclimatic areas to study for intraand inter-specific variation of germination traits. The obtained S values

Table 3 Minimal generalised linear model (GLM) output for germination against photoperiod (L12/12, L0/24), treatment (Control, GA3), temperature regime (Constant, Alternating) as categorical factors and temperature (15, 20, 25) as a quantitative predictor. Significance codes: ‘***’ b 0.001, ‘**’ b 0.01, ‘*’ b 0.05. Null deviance: 604.572 on 94 degrees of freedom. Residual deviance: 84.793 on 79 degrees of freedom. AIC: 338.73. Number of Fisher Scoring iterations: 4. Coefficients

Estimate

Std. error

z value

Pr(N|z|)

(Intercept) L0/24 GA3 Alternating Temperature L0/24: Alternating L0/24: Temperature L0/24: GA3 Alternating: Temperature Alternating: GA3 Temperature: GA3

−3.55815 0.53488 4.77159 3.87505 0.12841 0.86685 −0.9221 0.87203 −0.06469 −2.97652 −0.09565

0.71814 0.74357 0.73889 0.73342 0.03397 0.29225 0.03472 0.29170 0.03483 0.29133 0.03487

−4.955 0.719 6.458 5.284 3.780 2.966 −2.656 2.989 −1.857 −10.217 −2.743

7.25e-7 0.471926 1.06e-10 1.27e-7 0.000157 0.003016 0.007917 0.002794 0.063288 b 2e-16 0.006088

*** *** *** *** ** ** ** *** **

Fig. 4. The role of F. indica stones burial depth on seed germination and seedling establishment. “a”, represents the maximum depth at which light (depicted as a yellow arrow) is able to penetrate (Woolley and Stoller, 1978), “b” is the maximum depth for seeds of F. indica to emerge calculated following Bond et al. (1999) and “c” represents soil depth at which temperature fluctuations (depicted as a sinuous line) might not be enough to trigger germination (Assche and Vanlerberghe, 1989). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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(Table 4) are surprisingly high for a non-dormant tree species according to Dürr et al. (2015) dataset, but perhaps this is due to the constraining effect of the endocarp over the seed, which might slow down germination rates. Further research should explore the variations upon germination of ‘naked’ seeds and their potential use in reforestation as an alternative to stones. 4.4. Implications for propagation The use of F. indica by rural communities is widespread in many regions of Africa (Leakey and Newton, 1994) and India (Kala, 2009), due to its food and medicinal properties and domestication potential (Akinnifesi et al., 2006). Reports have suggested technical difficulties to germinate F. indica (Prins and Maghembe, 1994), but according to our study, lack of successful germination might lie in unfavourable germination conditions and poor initial seed quality. If stones are directly sown into the soil, it is necessary to ensure the presence of wide alternating temperatures (ca. 10 °C variation) to achieve optimal germination. Burying the stones too deep will not only reduce the temperature fluctuations, which lowers the amount of germination, but it will also be detrimental for seedling establishment. Hence, we recommend that stones should be buried no more than 1–3 cm, where temperature fluctuation will be similar to those on the soil surface and water is more likely to be retained for longer periods of time, preventing seedlings from dying due to desiccation. Water availability or temperature fluctuations do not seem to be a problem at the location of this particular stone collection (Table 1), but in other countries it is advisable to ensure that seeds are planted during the rainy season. A worrying fraction (ca. 23%) of the stones used in this study were seedless. This might be due to many factors, such as pollination problems, isolated populations, dioecy or pests. Further research should analyse which factors cause the quality problems of F. indica seeds in order to enhance its use in reforestation, breeding and livelihood programmes. 5. Conclusions This study reveals how F. indica seed germination is not constrained by dormancy. However, given the wide distribution range of this species, we believe more studies should be carried out with materials from different provenances to explore the inter- and intra-specific variation of the germination traits of this species (Mira et al., 2017). Difficulties to propagate this species from seed are probably due to its poor quality and research is needed to understand what affects it. Flacourtia indica stones germinate successfully in the presence of an alternating temperature regime, but a fraction is still capable of germinating at constant temperatures if light is present. This behaviour is a strategy to avoid germination at great depths (b 10 cm) enhancing seedling survival. Stones are likely to become part of the soil seed bank waiting for a disruption (soil movement or canopy gaps) to end its latency state. We suggest that F. indica stones be buried no more than 1–3 cm deep, which allows for fluctuating temperatures, proper seedling establishment and prevents water loss. These findings will help enhance the propagation of the species to support seed-based reforestation and livelihood programmes. Funding This study has been funded by MGU, a philanthropist based in Spain, as part of Project MGU - the Useful Plants Project, managed by the Royal Botanic Gardens, Kew. The authors have no conflict of interests to declare. Acknowledgments The authors gratefully acknowledge Ian Willey, Tiziana Cossu and David Coleshill for their support during this investigation. We would

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