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Species-specific smoke effects on seed germination of plants from different habitats from Sri Lanka
Journal Pre-proof Species-specific smoke effects on seed germination of plants from different habitats from Sri Lanka A.A.C.B. Alahakoon (Conceptualization) (Investigation) (Methodology) (Data curation) (Formal analysis) (Validation) (Visualization) (Writing - original draft) (Writing - review and editing), G.A.D. Perera (Project administration) (Supervision) (Writing review and editing), D.J. Merritt (Conceptualization) (Resources) (Writing - review and editing), S.R. TurnerConceptualization, Methodology) (Resources) (Writing - original draft) (Writing - review and editing), N.S. Gama-Arachchige (Conceptualization) (Methodology) (Formal analysis) (Supervision) (Writing - original draft) (Writing - review and editing) (Funding acquisition) (Project administration)
PII:
S0367-2530(19)30534-1
DOI:
https://doi.org/10.1016/j.flora.2019.151530
Reference:
FLORA 151530
To appear in:
Flora
Received Date:
26 April 2019
Revised Date:
2 December 2019
Accepted Date:
9 December 2019
Please cite this article as: Alahakoon AACB, Perera GAD, Merritt DJ, Turner SR, Gama-Arachchige NS, Species-specific smoke effects on seed germination of plants from different habitats from Sri Lanka, Flora (2019), doi: https://doi.org/10.1016/j.flora.2019.151530
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Species-specific smoke effects on seed germination of plants from different habitats from Sri Lanka
A.A.C.B. Alahakoona,b, G.A.D. Pereraa,b, D.J. Merrittc,d, S.R. Turnerc,d and N.S. Gama-Arachchigea,b*
a
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Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka
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Postgraduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka
Kings Park Science, Department of Biodiversity, Conservation and Attractions, Kings Park,
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WA 6005, Australia d
School of Biological Sciences, The University of Western Australia, Crawley WA 6009, Perth,
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Australia
*Corresponding author: N.S. Gama-Arachchige, Department of Botany, Faculty of Science,
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University of Peradeniya, Peradeniya, Sri Lanka, Email: [email protected]
Highlights
Germination rate (T50) of some species was increased by smoke water and/or KAR1.
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Smoke water increased seed germination percentage and/or rate of six study species. The responded species included three natives to Sri Lanka and three exotics. KAR1 increased seed germination (%) of five species that responded to smoke water.
Abstract
Smoke and in particular karrikinolide (KAR1), a growth promoting compound found in smoke, have been shown to enhance seed germination in phylogenetically diverse plant families from 1
both fire-prone and fire-free ecosystems. We tested the effects of water saturated with smoke, “smoke water” (SW), and KAR1 on seed germination of 18 native and exotic plant species from different habitats affected by anthropogenic fires in Sri Lanka. Seeds were tested with five concentrations of SW (5%, 25%, 50%, 75%, 100%) and those that positively responded were tested with three different concentrations of KAR1 (10 nM, 100 nM and 1 µM). Germination percentage of three native (Flueggea leucopyrus, Maesa indica and Phyllanthus emblica) and
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two exotic (Chromolaena odorata and Hyptis suaveolens) species was significantly increased by
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both SW and KAR1 treatments. Seed germination percentage of the exotic species Euphorbia heterophylla was increased by SW treatments only. The time taken for 50% germination (t50) of
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C. odorata, M. indica and P. emblica was decreased by both SW and KAR1, whilst t50 of E.
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heterophylla was decreased by SW only. Species-specific seed germination promotion response to SW and KAR1 was observed in both native and exotic plant species tested in the study
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confirming that the recruitment ecology of at least some species found in fire prone habitats of
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Sri Lanka is likely to be influenced by fire management practices.
Keywords: Fire ecology; Karrikinolide (KAR1); Seed dormancy: Smoke water; Native plants;
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Exotic plants
1. Introduction
Forest fires in tropical regions have become more frequent, widespread and intensive in recent decades and mostly occur due to human activities (Cochrane, 2009; Page et al., 2009; Chergui et al., 2018; Keeley and Pausas, 2019). This has been further exacerbated by climate change and
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subsequent altered fire regimes have led to an increase in fire occurrences and spread in some tropical regions (Herawati and Santoso, 2011; Junior et al., 2018). In Sri Lanka, virtually all forest fires are anthropogenic in origin (Perera, 1998; FAO, 2015), thus becoming more frequent in disturbed tropical dry forests (Perera, 1998, 2001), savanna forests in the Uva basin (Gunatilleke et al., 2008), and in montane grasslands in the wet zone (Amarasinghe and
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Premadasa, 1983; Gunatilleke et al., 2008) especially when dry climatic conditions prevail. Fire shapes vegetation of many regions of the world and may affect species composition and
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plant diversity in some plant communities by killing some fire sensitive plant species while
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promoting seed germination of some obligate seeders (Bond and Keeley, 2005; Bond et al., 2005; Pausas and Keeley, 2014). Heat from fires can render seeds with physical dormancy
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permeable to water, facilitating germination in the post-fire environment (Williams et al., 2003; Dayamba et al., 2010; Moreira et al., 2010; Baskin and Baskin, 2014; Robles-Diaz et al., 2014;
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Hall et al., 2016; Tavsanoglu et al., 2017; Zirondi et al., 2019), and other fire related cues such as smoke promote germination of seeds from many different families of plants (Dixon et al., 2009)
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with different plant life forms and seed dormancy traits (Roche et al., 1997, 1998; Crosti et al., 2006; Figueora et al., 2009; Moreira et al., 2010; Zhou et al., 2014; Flematti et al., 2015; Hall et al., 2016). It has been reported that 1,200 plant species (Dixon et al., 2009) including crop
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species (Kulkarni et al., 2007; Iqbal et al., 2016), horticultural species (Dixon et al., 2009) and weeds (Kandari et al., 2011; Kamran et al., 2014) produce seeds that positively respond to smoke. Both aerosol smoke (Roche et al., 1997; Lloyd et al., 2000; Crosti et al., 2006) and water saturated with smoke (Roche et al., 1997; Lloyd et al., 2000; Dayamba et al., 2010) have been demonstrated to positively affect seed germination.
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Karrikins are a family of closely related small organic compounds found in smoke and produced when plant materials are burnt (Flematti et al., 2015). Among these, Karrikinolide, KAR1 (3-methyl-2H-furo[2,3-c]pyran-2-one), is the predominant chemical compound in smoke that stimulates seed germination of many plant species (Flematti et al., 2004). KAR1 is a byproduct from fire that stimulates germination of non-dormant seeds and can elicit a response at very low concentrations (i.e 1 nM; Chiwocha et al., 2009). It has been shown that KAR1
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stimulates seed germination of many species which have previously been reported as responding
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to smoke (Flematti et al., 2004). In addition, cyanohydrins (Flematti et al., 2011) and other
undiscovered chemicals (Downes et al., 2013) which may also be produced during burning,
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could stimulate seed germination, but of all, karrikins are the major germination-stimulating
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compounds (Flematti et al., 2015). However, the response of seeds to smoke is tightly regulated by their dormancy status. For instance, seeds of some species with physiological dormancy
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become more sensitive to smoke following after-ripening or stratification which in some cases can also fluctuate over time as seeds undergo dormancy cycling (Merritt et al., 2007; Flematti et
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al., 2015).
Considerable amount of research is conducted on the fire ecology of ecosystems such as the Mediterranean vegetation, California chaparral and various Australian woodlands (Barro and Conard, 1991; Cary et al., 2003; Bond and Keeley, 2005; Bond et al., 2005; Keeley et al., 2012)
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where fire plays an important role in ecosystem dynamics. Regeneration ecology of forest species on burnt lands in seasonally dry climatic regions of Sri Lanka has been poorly studied to date, but available information claims that some weed species such as Chromolaena odorata and Ageratum conyzoides (Asteraceae) germinate readily on recently burnt and cultivated sites in the dry zone of Sri Lanka (Perera, 2005). As fires are becoming increasingly common in Sri Lanka
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as well as in other tropical regions, there is a need to understand the responses of seeds of native and exotic plant species to these significant disturbance events. If there are native species that respond positively to smoke water and, in particular KAR1, those species may become much more abundant in the vegetation after a fire event. On the other hand, if there are exotic species with a strong positive response to fire, there is a possibility that they will be able to outcompete the native vegetation altering the ecology of the local environment in unforeseen ways.
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Therefore, this study (1) examines the role of smoke water (SW) on seed germination and rate of
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germination of 18 plant species from 12 families representing both native and exotic species
from areas affected by anthropogenic fires in Sri Lanka and (2) test the effect of KAR1 on the
2. Materials and methods
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2.1 Seed collection and preparation
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percentage and rate of germination of seeds found to be already promoted by SW.
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Seeds of 18 plant species representing 13 native and five exotic species (Table 1) that grow in areas affected by anthropogenic fires in Sri Lanka (Patana grasslands, tropical dry forests, secondary forest, agricultural fields, roadsides, etc.,) were used for this study. Seeds were collected from mature fruits during the period of seed release from 11 different sites around Sri Lanka (Table 1),
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except for Phyllanthus emblica, in which the fruits were bought from a local market in Kandy. Seeds were recovered by removing all the surrounding fruit tissues using different mechanical methods that were suitable for each of the different species. Damages to seed coats were identified by examining all seeds under a dissecting microscope while the rigidity of all seeds was carefully determined by gently squeezing individual seeds using a pair of forceps and
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removing those that split or shattered. Thus, undamaged healthy seeds were used for all experiments. >>insert Table 1 here
2.2 Effect of smoke water (SW) on seed germination A mixture of dry plant leaves was burnt in a 50 L metal drum and the smoke generated was
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passed through 10 L of distilled water (Lloyd et al., 2000). A vacuum cleaner was used to draw
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the smoke through water, and an air blower was used to supply air into the metal drum that
contained the fire. The setup was run for 30 minutes and the prepared solution (pH 6.3) was
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considered as the 100% SW stock solution. Smoke water solutions of 75%, 50%, 25% and 5%
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(v/v) were prepared by diluting this solution in distilled water. Distilled water acted as the control (0% SW).
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To test the effects of different concentrations of SW on germination, five replicates with 20 seeds (100 seeds in total) from each selected species without physical dormancy were incubated
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at 25 C in an incubator (Hinotek, MGC-450 BP, China) under 12 hr / 12 hr light / dark condition (the photon irradiance during the light phase was approximately 115 µmol photons m-2 s-1, 400–700 nm, cool white fluorescent tubes) in 90 mm plastic Petri dishes containing tissue paper moistened with about 12 ml of 100%, 75%, 50%, 25%, 5% and 0% (distilled water)
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solutions of SW. Species with physical dormancy (Bauhinia racemosa, Cuscuta chinensis, Tadehagi triquetrum and Tephrosia purpurea) (Jayasuriya et al., 2013) were tested with the 100% SW solution only and germination of both manually scarified (using a razor blade) and non-scarified seeds were tested. In the case of Terminalia bellirica, endocarp removed seeds were used for SW experiments. The number of germinated seeds was counted every two days
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during the first week and thereafter every seven days for the next three weeks. In the case of Phoenix pusilla seeds, germination was recorded for eight weeks, since seeds of this species were known to take some time to germinate.
2.3 KAR1 treatments Seeds of species that showed a significant germination response to smoke water treatments were
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tested for the effect of KAR1. A stock solution of 100 µM KAR1 (Flematti et al., 2005) was used
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to prepare 10 nM, 100 nM and 1 µM solutions by diluting with distilled water. Distilled water was used as the control. Seeds from each test species were incubated in Petri dishes containing
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tissue paper moistened with 12 ml of KAR1 under the same conditions as previously described.
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The number of germinated seeds in each replicate was counted every two days during the first week and thereafter every seven days for the next three weeks. For Euphorbia heterophylla a
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different seed lot had to be used for KAR1 experiments since there were no adequate seeds in the seed lot used for SW experiments. For all the other species the SW and KAR1 treatments were
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done for the same seed lot.
2.4 Effects of smoke water and KAR1 on t50 (time taken for 50% germination) To calculate the average t50 under each treatment, three parameter sigmoidal regression curves
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were fitted for time vs. germination percentage for each replicate of the SW and KAR1 treatments using SigmaPlot version 10.0.1 (SYSTAT Inc., 2007) with the t50 estimated from each of the fitted cumulative germination curves.
2.5 Statistical analysis
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Germination percentage data were normalized by arcsine transformation prior to statistical analysis. Data from experiments with only two treatments were analyzed using an independent two-sample t-test (α = 0.05). Data of experiments with multiple treatments and germination rate (t50) data were analyzed by one-way ANOVA. Fisher’s least significant difference procedure was used to compare treatments (α = 0.05). All analyses were carried out using Minitab version
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17.1.0 (Minitab Inc., 2014) and the graphs were constructed using SigmaPlot ver.10.0.1
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(SYSTAT Inc., 2007).
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3. Results 3.2 Effect of SW on seed germination
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From the 18 tested species, seeds of three native (Flueggea leucopyrus, Maesa indica and P.
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emblica) and three exotic species (Chromolaena odorata, E. heterophylla and Hyptis suaveolens) showed increased germination in response to smoke water treatments (Fig. 1C,E,F,G,I & L) (P <
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0.05). Germination percentage of F. leucopyrus and P. emblica increased significantly, compared to control seeds, in response to all SW concentrations tested (Fig. 1F & L), and in the other four species germination was improved in some of the SW concentrations assessed. It appears that the optimum concentration of smoke water may vary with species. In C. odorata, E. heterophylla
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and F. leucopyrus seeds for example, the highest concentration of smoke water assessed (100%) resulted in the highest germination (Fig. 1C,E & F), while in H. suaveolens, M. indica and P. emblica seeds, the germination percentage in response to 100% SW treatment was less than that observed in response to the 50% or 75% SW treatments; albeit it was still significantly higher than that observed in control seeds except for H. suaveolens (Fig. 1G,I & L). None of the test species that were determined to possess physical dormancy responded to SW administered either 8
before or after seed scarification (Fig. 2). Seeds of these species achieved 75% germination within 2–3 days when mechanically scarified. >>insert Fig. 1 here >>insert Fig. 2 here
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3.3 Effect of KAR1 on seed germination Of the six smoke responsive species, the final germination percentage of the seeds from five
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species increased significantly after treatment with KAR1 (Fig. 3) (P < 0.05). KAR1 treatments did not affect the germination percentage of E. heterophylla seeds (Fig. 3B). All tested
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concentrations of KAR1 significantly increased the germination percentage of C. odorata, F.
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leucopyrus, H. suaveolens and P. emblica seeds, while germination of M. indica seeds was significantly enhanced by the 100 nM and 1 M KAR1 treatments only (Fig. 3E). Seeds of F.
KAR1 (Fig. 3C).
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>>insert Fig. 3 here
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leucopyrus responded in a similar manner to the tested lowest and highest concentrations of
3.4 Effect of smoke water and KAR1 on t50 Seeds of four species (H. suaveolens, Osbeckia rubicunda, P. pusilla and Phyllanthus amarus) did not achieve 50% germination under any of the smoke water treatments within four weeks (Table 2). T50 could be calculated only under 100% SW for C. odorata and under 50–100% concentrations for F. leucopyrus since ≥50% germination was achieved only under these 9
concentrations. All of the tested concentrations of SW significantly reduced the t50 of E. heterophylla seeds, while only one concentration (50%) reduced the t50 of M. indica seeds. It shows that the rate of germination of E. heterophylla was more rapid under high concentrations of SW (Table 2) (P < 0.05). Likewise, a significant reduction in t50 was observed in response to the 25% and 50% SW solutions for seeds of Careya arborea and Limonia acidissima
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respectively. In contrast, higher concentrations of SW significantly increased t50 of Bidens pilosa seeds (Table 2) (P < 0.05).
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>>insert Table 2 here
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The seeds of H. suaveolens did not achieve 50% germination under any of the tested KAR1 concentrations assessed within four weeks. In addition, the seeds from the control
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treatments of several species did not achieve 50% germination after 28 days of incubation (Table
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3). A significant reduction in t50 compared to control was observed only in M. indica seeds in response to exposure to 1 µM KAR1 and in C. odorata in response to 100 nM and 1 µM
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compared to 10 nM (Table 3). >>insert Table 3 here
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4. Discussion
This is the first report on smoke water (SW) and karrikinolide (KAR1) response upon seed
germination of native and exotic plant species in the Indian subcontinent. One-third of 18 tested species have shown a positive germination response with respect to seed germination percentage and / or t50, to SW and KAR1. Therefore the role of smoke and KAR1 seems to be species-specific as observed in previous studies (Roche et al., 1997; Dayamba et al., 2010) and affects both native 10
and exotic species. The native species that respond to SW and KAR1 including pioneer species such as M. indica might lead to natural regeneration of the vegetation after a fire event (Hall et al., 2016; Madawala et al., 2016). Also, those species can be used in restoration programs either through in situ stimulation of soil seed banks or ex situ with the use of smoke to enhance nursery germination for the production of plants (Roche et al., 1997, 1998; Lloyd et al., 2000; Wulff et al., 2012; Hall et al., 2016). Furthermore, the present study reveals the possibility of using SW to
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produce seedlings of medicinally and commercially valuable species (Kulkarni et al., 2007,
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2008; Chumpookam et al., 2012; Iqbal et al., 2016) such as F. leucopyrus and P. emblica which may benefit small-scale land holders and local communities. Both these species are well-known
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medicinal plants in Ayurvedic medicine in Sri Lanka and India (Singh et al., 2011; Ajmeer et al.,
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2014; Soysa et al., 2014).
Alien plant invasions are frequently associated with disturbances (Figueora et al., 2009).
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Traits such as shorter germination times favor the establishment of these species in the vacant niches (Gioria et al., 2018). From the six smoke responsive species, C. odorata, E. heterophylla
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and H. suaveolens readily grow in disturbed habitats and are considered as agricultural weed species in Sri Lanka. Smoke responsiveness can be an advantage for these species to successfully colonize disturbed land and thus outcompete native species. Farmers practicing shifting
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cultivation in the dry zone of Sri Lanka claim that E. heterophylla is one of the most noxious weeds found growing on land cleared for cultivation. This invasiveness may be due in part to the effects of germination promoting compounds in smoke generated when they use fire to regularly clear the land. Figueora et al. (2009) reported on enhanced germination of exotic (weed) species in response to smoke treatments. Application of SW to field soil to induce emergence of smoke responsive species followed by application of a weedicide or uprooting would be an effective 11
method of removing weed seeds from the soil seed bank (Kandari et al., 2011; Kamran et al., 2014). Careya arborea, P. emblica and T. bellirica are three native, dominant fire-tolerant tree species in dry patana grasslands in Sri Lanka (Gunatilleke et al., 2008). However, only the seeds of P. emblica responded to SW and KAR1. Even though C. arborea and T. bellirica did not respond to smoke, fire events in dry patana may still be important for regeneration of these
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species through other means of regeneration such as re-sprouting (Pausas and Keeley, 2014). In
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the dry zone of Sri Lanka, fire used to clear land for agricultural purposes spreads into the adjacent secondary forests, diverting succession and dominance of some species such as
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Cymbopogon nardus (Jong et al., 2001). Seed germination of C. nardus did not seem to be
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enhanced by smoke in this study. Further studies are needed to understand the occurrence and establishment of these species in fire prone ecosystems.
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Seeds with physical dormancy are impermeable to water (Baskin and Baskin, 2004, 2014) and therefore imbibition of water is hindered. As a result, such seeds cannot germinate
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due to their water impermeable seed coat. It has been reported that in some species such as B. racemosa, a certain percentage of the seed population is non-dormant and is able to imbibe when exposed to water (Jayasuriya et al., 2013). In such species, it is possible that the non-dormant
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proportion of the seeds might respond to SW or KAR1. However, all the four species tested in this study with physical dormancy, including the parasitic species C. chinensis, did not respond to smoke water before or after mechanical scarification (Fig 2). It is reported that the frequency of occurrence of T. purpurea plants is higher in burnt sites than non-burnt sites in Udawalawe National Park, Sri Lanka (Perera and Wijesooriya, 2007) and this could be caused by enhanced
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germination due to other factors such as heat from fire that has rendered the seeds water permeable (Robles-Diaz et al., 2014; Hall et al., 2016; Tavsanoglu et al., 2017). As Flematti et al. (2004) explained, increased seed germination and rate of germination after SW treatment could be associated with KAR1 present in SW. However, seeds of E. heterophylla did not respond to KAR1 even though these seeds responded to smoke water. This
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could be due to the presence of other karrikin compounds (KAR2, KAR3 and KAR4) (Flematti et al., 2015) or other compounds such as nitrates and cyanohydrins (e.g. glyceronitrile) present in
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plant derived smoke which in some cases are equally capable of stimulating seed germination
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(Flematti et al., 2011). This indicates that the SW used in the present study may contain seed germination stimulating compounds other than KAR1. The variability of germination of E.
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heterophylla in the control treatments of SW and KAR1 experiments may be due to variability of dormancy and the quality of the two seed lots used (Martinez-Baniela et al., 2016). The increase
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in t50 of B. pilosa seeds at higher SW concentrations may be due to the suppression of seed germination of some species by high SW concentrations, possibly due to toxic effects (Nelson et
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al., 2009; Zhou et al., 2014).
Smoke water and KAR1 promoted seed germination in only three out of 13 native species (23%) and three out of five exotic species (60%) tested. Thus, compared to exotic species, the
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majority of the native species did not respond to smoke. Historically, natural or anthropogenic fire was not a significant factor that determined the structure or species composition in the ecosystems of Sri Lanka. Thus, we can assume that many of the native species are not adapted to respond to smoke signals as a germination cue. However, in Sri Lanka, anthropogenic fires are now becoming more frequent and intensive (Ariyadasa, 2001; FAO, 2015). Therefore, more research in the field of fire ecology is essential to understand how the native and exotic species 13
respond to smoke / fire related cues, and how anthropogenic fires affect the survival of plant species in anthropogenic fire prone habitats in Sri Lanka.
5. Conclusions
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According to our results, there are both native (F. leucopyrus, M. indica and P. emblica) and exotic (C odorata, H. suaveolens and E. heterophylla) species that respond positively to smoke
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water (SW). The effect of SW was significant on both the germination percentage and the time taken for 50% germination (t50) of these species. Karrikinolide (KAR1) promoted germination of
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SW responsive species except E. heterophylla. Therefore, the germination enhancement of these
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species by smoke water is likely due to the presence of KAR1 in the smoke water. Even though the majority of the native species did not respond to smoke / KAR1, those that responded to SW /
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KAR1 might play an important role in natural regeneration of fire prone ecosystems and also can be utilized in restoration efforts while SW / KAR1 responsiveness would be an added advantage
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for the exotic species to outcompete the native vegetation, particularly in view of increased frequency of anthropogenic fires.
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Author Contribution Statement
Effect of smoke on seed germination of species of different habitats from Sri Lanka
I, Dr. Nalin Gama-Arachchige the corresponding author of the manuscript tiled “Effect of smoke on seed germination of species of different habitats from Sri Lanka” confirm the contributions of each author to the research work listed in the table below.
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Dr. Nalin Gama-Arachchige Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka
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Disclosure statement
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Contributions/ CRediT roles Conceptualization; Investigation; Methodology; Data A.A.C.B. Alahakoon curation; Formal analysis; Validation; Visualization; Writing - Original Draft; Writing – review & editing Project administration; Supervision; Writing – review & G.A.D. Perera editing D.J. Merritt Conceptualization; Resources; Writing – review & editing Conceptualization; Methodology; Resources; Writing S.R. Turner Original Draft; Writing – review & editing Conceptualization; Methodology; Formal analysis; N.S. Gama-Arachchige Supervision; Writing - Original Draft; Writing – review & editing; Funding acquisition; Project administration
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Author
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The authors declare that they have no conflict of interest.
Acknowledgement
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The authors thank Gavin Flematti of the University of Western Australia for providing the karrikinolide used in this study.
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(Triticum aestivum L .). Am. J. Plant Sci. 7, 806–813. Jayasuriya, K.G., Wijetunga, A.S., Baskin, J.M., Baskin, C.C., 2013. Seed dormancy and storage behaviour in tropical Fabaceae: a study of 100 species from Sri Lanka. Seed Sci. Res. 23, 257–269. Jong, W.D., Chokkalingam, U., Perera, G.A.D., 2001. The evolution of swidden fallow secondary forests in Asia. J. Trop. For. Sci. 13, 800–815.
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Tropical Deciduous Forests of Sri Lanka. University of Oxford. Perera, G.A.D., 2001. The secondary forest situation in Sri Lanka: a review. J. Trop. For. Sci. 13, 4. Perera, G.A.D., 2005. Diversity and dynamics of the soil seed bank in tropical semi-deciduous forests of Sri Lanka. Trop. Ecol. 46, 65–78. Perera, G.A.D., Wijesooriya, S.M., 2007. Impact of Fire on Biodiversity of Udawalawe National
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traditional uses and cancer chemopreventive activity of Amla (Phyllanthus emblica): The Sustainer. J. Appl. Pharm. Sci. 02, 176–183. Soysa, P., De Silva, I.S., Wijayabandara, J., 2014. Evaluation of antioxidant and antiproliferative activity of Flueggea leucopyrus Willd (katupila). BMC Complement. Altern. Med. 14.
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SYSTAT Inc., 2007. SigmaPlot, Version 10.0.1. Tavsanoglu, C., Ergan, G., Catav, S.S., Zare, G., Kucukakyuz, K., Ozudogru, B., 2017. Multiple fire-related cues stimulate germination in Chaenorhinum rubrifolium (Plantaginaceae), a rare annual in the Mediterranean Basin. Seed Sci. Res. 27, 26–38.
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The Plant List, 2013. Version 1.1. Published on the Internet; http://www.theplantlist.org/ (accessed 1st March 2019). Williams, P.R., Congdon, R.A., Grice, A.C., Clarke, P.J., 2003. Fire-related cues break seed dormancy of six legumes of tropical eucalypt savannas in north-eastern Australia. Austral Ecol. 28, 507–514. Wulff, A., Turner, S., Fogliani, B., Lhuillier, L., 2012. Smoke stimulates germination in two
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Zhou, J., da Silva, J.A.T., Ma, G., 2014. Effects of smoke water and karrikin on seed germination
https://doi.org/10.2478/s11535-014-0338-6
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of 13 species growing in China. Cent. Eur. J. Biol. 9, 1108–1116.
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Zirondi, H.L., Silveira, F.A.O., Fidelis, A. 2019. Fire effects on seed germination: Heat shock
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and smoke on permeable vs impermeable seed coats. Flora 253, 98-106.
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Figure captions
Fig. 1. Final germination percentage of the seeds of 14 test species without physical dormancy in response to treatment with different concentrations of smoke water after 28 days at 25 C under
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12 hr / 12 hr light / dark condition. Bars show means ± s.e. (n = 5). Different letters indicate significant differences in germination among the treatments (P < 0.05). The shaded bars of the Fig. 1M represent the germination percentages of Phoenix pusilla after 8 weeks of incubation.
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Fig. 2. Final germination percentage of the seeds of four test species with physical dormancy (PY) in response to treatment with 100% SW after 28 days at 25 C under 12 hr / 12 hr light /
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dark condition. Bars show means ± s.e. (n = 5). Different letters indicate significant differences in germination among the treatments (P < 0.05).
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Fig. 3. Final germination percentage of the seeds of six smoke responsive species in response to
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treatment with different concentrations of KAR1 after 28 days at 25 C under 12 hr / 12 hr light / dark condition. Bars show means ± s.e. (n = 5). Different letters indicate significant differences
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in germination among the treatments (P < 0.05).
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Table 1: Family, life form, fruit type, ecological status, habitat and seed source of the 18 study species
Fabaceae
Small tree
Legume
Native
Careya arborea Roxb.
Lecythidaceae
Chromolaena odorata (L.) Asteraceae R.M. King & H. Rob.
Cuscuta chinensis Lam.
Convolvulaceae
Poaceae
Euphorbia heterophylla L. Euphorbiaceae
Flueggea leucopyrus Willd.
Tree
Shrub
Vine
Cypsela
Exotic
Berry
Cypsela
Native
Capsule
Tussock grass
Caryopsis
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Cymbopogon nardus (L.) Rendle
Herb
Hyptis suaveolens (L.) Poit.
Euphorbiaceae
Lamiaceae
Herb
Elahara
Hanthana
Savanna and dry patana
Ethakada
Waste lands
Exotic
Native
Capsule
Bushy shrub
Berry
Shrub
schizocarp
Native
GPS coordinates
April 2016 80° 47ʹ 36.5ʺ E 7° 15ʹ 28.0ʺ N December 2015 80ʹ 37ʹ 43.7ʺ E 8° 36ʹ 3.7ʺ N October 2015 80° 33ʹ 59.6ʺ E 7° 13ʹ 43.3ʺ N
Waste lands and forest clearings
Hanthana
December 2016 80° 38ʹ 18.0ʺ E 7° 15ʹ 23.7ʺ N
Paddy fields and disturbed areas
Peradeniya
January 2016 80° 35ʹ 47.7ʺ E 7° 13ʹ 25.3ʺ N
Wet patana and roadsides
Sarasavigama
February 2016 80° 37ʹ 2.74ʺ E 7° 53ʹ 43.8ʺ N
Exotic
Collection period
7°44ʹ 3.7ʺ N
Dry forest, monsoon forest and scrublands
pr
Asteraceae
Collection locality
f
Ecological status Habitat
oo
Fruit type
e-
Bidens pilosa L.
Life form
Pr
Bauhinia racemosa Lam.
Family
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Species
Road sides and disturbed areas
Pelwehera 80° 39ʹ 55.5ʺ E
December 2015, May 2016
6° 17ʹ 3.6ʺ N Native
Thorn scrublands
Yala
November 2015 81° 25ʹ 45.5ʺ E 7° 54ʹ 13.0ʺ N
Exotic
Road sides in the dry zone
Pelwehera
December 2016 80° 40ʹ 9.69ʺ E
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6° 17ʹ 3.6ʺ N Limonia acidissima Groff
Rutaceae
Small tree
Berry
Native
Tropical dry forests
Yala
November 2015
Melastomataceae
Phoenix pusilla Gaertn.
Arecaceae
Shrub
Capsule
Dwarf palm Berry
Euphorbiaceae
Herb
Phyllanthus emblica L.
Euphorbiaceae
Tree
Tadehagi triquetrum (L.) H. Ohashi
Fabaceae
Shrub
Tephrosia purpurea (L.) Pers.
Fabaceae
Capsule
Berry
Legume
Small shrub Legume
Jo ur Combretaceae
Native
Native
Native
Exotic
Native
na l
Phyllanthus amarus Schumach. & Thonn.
Terminalia bellirica (Gaertn.) Roxb.
Berry
Tree
Berry
Shady places in montane region
Native
Native
Native
7° 15ʹ 28.0ʺ N
Hanthana
December 2015 80° 37ʹ 43.7ʺ E 7° 13ʹ 43.3ʺ N
Open montane forest, forest edges and roadsides
Hanthana
Tropical dry forest and coastal vegetation
Maradankadawala
Ruderal
Pilimathalawa
pr
Osbeckia rubicunda Arn.
Shrub
e-
Myrsinaceae
Pr
Maesa indica (Roxb.) A. DC.
oo
f
81° 25ʹ 45.5ʺ E
January 2016 80° 38ʹ 18.0ʺ E 8° 07' 11.1" N May 2016 80° 33' 53.7" E 7° 14ʹ 2.83ʺ N July 2016 80° 32ʹ 27.8ʺ E
Savanna, dry patina and intermediate forests
-
Secondary forests
Sarasawigama
-
May 2016
7° 13ʹ 25.3ʺ N March 2016 80° 37ʹ 2.74ʺ E 8° 50ʹ 13.9ʺ N Road sides and waste lands
Paranaddakal
August 2015 80° 29ʹ 46.7ʺ E
Dry patina and tropical intermediate forests
(Based on Dassanayake and Fosberg 1981–2004; Christenhusz et al., 2017 and www.theplantlist.org.)
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7° 14ʹ 35.2ʺ N Lagamuwa
March 2016 80° 31ʹ 32.8ʺ E
Table 2: Effect of different concentrations of smoke water on t50 (time taken to achieve 50% germination in days) on the seeds of 14 test species without physical dormancy (mean ± s.e., n = 5).
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t50 time (days) under tested concentrations of smoke Species water 0% 5% 25% 50% 75% 100% d cd bc b ab Bidens pilosa 2±0 2±0 3±0 3±1 4±0 5±0a a ab b ab a Careya arborea 3±1 2±1 0±0 1±1 3±0 2±1ab Chromolaena odorata 7±0 Cymbopogon nardus 2±1a 2±0a 2± 0a 2±0a 2±0a 2±0a Euphorbia heterophylla 12±1a 8±1b 5±0c 4±0c 3±0c 4±0c a a Flueggea leucopyrus 13 ± 1 13 ± 1 16 ± 3a Hyptis suaveolens a a a b a Limonia acidissima 7±0 7±0 7±0 6±0 7±0 7±0a a ab a b ab Maesa indica 19±1 15±1 17±2 13±1 15±1 16±1ab Osbeckia rubicunda Phoenix pusilla Phyllanthus amarus a a a a Phyllanthus emblica 10±2 9±2 7±0 6±0 6±0a Not tested Not tested Terminalia bellirica 10±0a 10±0a 11±1a 10±0a Different letters indicate significant differences between treatments (P < 0.05)
Species
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Table 3: Effect of different concentrations of KAR1 on t50 (time taken to achieve 50% germination in days) on the seeds of the six species which responded to smoke water (mean ± s.e., n = 5).
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Chromolaena odorata Euphorbia heterophylla Flueggea leucopyrus Hyptis suaveolens Maesa indica Phyllanthus emblica
t50 time (days) under tested concentrations of KAR1 0 nM 10 nM 100 nM 1 µM 10±2a 5±2b 5±1b 20±3a 20±6a 19±2a 16±1a a a 8±0 9±1 10±1a a a a 22±2 24±1 22±1 16±1b 1±1a 1±0a 1±0a
ifferent letters indicate significant differences between treatments (P < 0.05)
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PII:
S0367-2530(19)30534-1
DOI:
https://doi.org/10.1016/j.flora.2019.151530
Reference:
FLORA 151530
To appear in:
Flora
Received Date:
26 April 2019
Revised Date:
2 December 2019
Accepted Date:
9 December 2019
Please cite this article as: Alahakoon AACB, Perera GAD, Merritt DJ, Turner SR, Gama-Arachchige NS, Species-specific smoke effects on seed germination of plants from different habitats from Sri Lanka, Flora (2019), doi: https://doi.org/10.1016/j.flora.2019.151530
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Species-specific smoke effects on seed germination of plants from different habitats from Sri Lanka
A.A.C.B. Alahakoona,b, G.A.D. Pereraa,b, D.J. Merrittc,d, S.R. Turnerc,d and N.S. Gama-Arachchigea,b*
a
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Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka
b
Postgraduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka
Kings Park Science, Department of Biodiversity, Conservation and Attractions, Kings Park,
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c
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WA 6005, Australia d
School of Biological Sciences, The University of Western Australia, Crawley WA 6009, Perth,
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Australia
*Corresponding author: N.S. Gama-Arachchige, Department of Botany, Faculty of Science,
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University of Peradeniya, Peradeniya, Sri Lanka, Email: [email protected]
Highlights
Germination rate (T50) of some species was increased by smoke water and/or KAR1.
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Smoke water increased seed germination percentage and/or rate of six study species. The responded species included three natives to Sri Lanka and three exotics. KAR1 increased seed germination (%) of five species that responded to smoke water.
Abstract
Smoke and in particular karrikinolide (KAR1), a growth promoting compound found in smoke, have been shown to enhance seed germination in phylogenetically diverse plant families from 1
both fire-prone and fire-free ecosystems. We tested the effects of water saturated with smoke, “smoke water” (SW), and KAR1 on seed germination of 18 native and exotic plant species from different habitats affected by anthropogenic fires in Sri Lanka. Seeds were tested with five concentrations of SW (5%, 25%, 50%, 75%, 100%) and those that positively responded were tested with three different concentrations of KAR1 (10 nM, 100 nM and 1 µM). Germination percentage of three native (Flueggea leucopyrus, Maesa indica and Phyllanthus emblica) and
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two exotic (Chromolaena odorata and Hyptis suaveolens) species was significantly increased by
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both SW and KAR1 treatments. Seed germination percentage of the exotic species Euphorbia heterophylla was increased by SW treatments only. The time taken for 50% germination (t50) of
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C. odorata, M. indica and P. emblica was decreased by both SW and KAR1, whilst t50 of E.
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heterophylla was decreased by SW only. Species-specific seed germination promotion response to SW and KAR1 was observed in both native and exotic plant species tested in the study
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confirming that the recruitment ecology of at least some species found in fire prone habitats of
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Sri Lanka is likely to be influenced by fire management practices.
Keywords: Fire ecology; Karrikinolide (KAR1); Seed dormancy: Smoke water; Native plants;
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Exotic plants
1. Introduction
Forest fires in tropical regions have become more frequent, widespread and intensive in recent decades and mostly occur due to human activities (Cochrane, 2009; Page et al., 2009; Chergui et al., 2018; Keeley and Pausas, 2019). This has been further exacerbated by climate change and
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subsequent altered fire regimes have led to an increase in fire occurrences and spread in some tropical regions (Herawati and Santoso, 2011; Junior et al., 2018). In Sri Lanka, virtually all forest fires are anthropogenic in origin (Perera, 1998; FAO, 2015), thus becoming more frequent in disturbed tropical dry forests (Perera, 1998, 2001), savanna forests in the Uva basin (Gunatilleke et al., 2008), and in montane grasslands in the wet zone (Amarasinghe and
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Premadasa, 1983; Gunatilleke et al., 2008) especially when dry climatic conditions prevail. Fire shapes vegetation of many regions of the world and may affect species composition and
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plant diversity in some plant communities by killing some fire sensitive plant species while
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promoting seed germination of some obligate seeders (Bond and Keeley, 2005; Bond et al., 2005; Pausas and Keeley, 2014). Heat from fires can render seeds with physical dormancy
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permeable to water, facilitating germination in the post-fire environment (Williams et al., 2003; Dayamba et al., 2010; Moreira et al., 2010; Baskin and Baskin, 2014; Robles-Diaz et al., 2014;
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Hall et al., 2016; Tavsanoglu et al., 2017; Zirondi et al., 2019), and other fire related cues such as smoke promote germination of seeds from many different families of plants (Dixon et al., 2009)
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with different plant life forms and seed dormancy traits (Roche et al., 1997, 1998; Crosti et al., 2006; Figueora et al., 2009; Moreira et al., 2010; Zhou et al., 2014; Flematti et al., 2015; Hall et al., 2016). It has been reported that 1,200 plant species (Dixon et al., 2009) including crop
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species (Kulkarni et al., 2007; Iqbal et al., 2016), horticultural species (Dixon et al., 2009) and weeds (Kandari et al., 2011; Kamran et al., 2014) produce seeds that positively respond to smoke. Both aerosol smoke (Roche et al., 1997; Lloyd et al., 2000; Crosti et al., 2006) and water saturated with smoke (Roche et al., 1997; Lloyd et al., 2000; Dayamba et al., 2010) have been demonstrated to positively affect seed germination.
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Karrikins are a family of closely related small organic compounds found in smoke and produced when plant materials are burnt (Flematti et al., 2015). Among these, Karrikinolide, KAR1 (3-methyl-2H-furo[2,3-c]pyran-2-one), is the predominant chemical compound in smoke that stimulates seed germination of many plant species (Flematti et al., 2004). KAR1 is a byproduct from fire that stimulates germination of non-dormant seeds and can elicit a response at very low concentrations (i.e 1 nM; Chiwocha et al., 2009). It has been shown that KAR1
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stimulates seed germination of many species which have previously been reported as responding
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to smoke (Flematti et al., 2004). In addition, cyanohydrins (Flematti et al., 2011) and other
undiscovered chemicals (Downes et al., 2013) which may also be produced during burning,
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could stimulate seed germination, but of all, karrikins are the major germination-stimulating
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compounds (Flematti et al., 2015). However, the response of seeds to smoke is tightly regulated by their dormancy status. For instance, seeds of some species with physiological dormancy
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become more sensitive to smoke following after-ripening or stratification which in some cases can also fluctuate over time as seeds undergo dormancy cycling (Merritt et al., 2007; Flematti et
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al., 2015).
Considerable amount of research is conducted on the fire ecology of ecosystems such as the Mediterranean vegetation, California chaparral and various Australian woodlands (Barro and Conard, 1991; Cary et al., 2003; Bond and Keeley, 2005; Bond et al., 2005; Keeley et al., 2012)
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where fire plays an important role in ecosystem dynamics. Regeneration ecology of forest species on burnt lands in seasonally dry climatic regions of Sri Lanka has been poorly studied to date, but available information claims that some weed species such as Chromolaena odorata and Ageratum conyzoides (Asteraceae) germinate readily on recently burnt and cultivated sites in the dry zone of Sri Lanka (Perera, 2005). As fires are becoming increasingly common in Sri Lanka
4
as well as in other tropical regions, there is a need to understand the responses of seeds of native and exotic plant species to these significant disturbance events. If there are native species that respond positively to smoke water and, in particular KAR1, those species may become much more abundant in the vegetation after a fire event. On the other hand, if there are exotic species with a strong positive response to fire, there is a possibility that they will be able to outcompete the native vegetation altering the ecology of the local environment in unforeseen ways.
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Therefore, this study (1) examines the role of smoke water (SW) on seed germination and rate of
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germination of 18 plant species from 12 families representing both native and exotic species
from areas affected by anthropogenic fires in Sri Lanka and (2) test the effect of KAR1 on the
2. Materials and methods
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2.1 Seed collection and preparation
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percentage and rate of germination of seeds found to be already promoted by SW.
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Seeds of 18 plant species representing 13 native and five exotic species (Table 1) that grow in areas affected by anthropogenic fires in Sri Lanka (Patana grasslands, tropical dry forests, secondary forest, agricultural fields, roadsides, etc.,) were used for this study. Seeds were collected from mature fruits during the period of seed release from 11 different sites around Sri Lanka (Table 1),
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except for Phyllanthus emblica, in which the fruits were bought from a local market in Kandy. Seeds were recovered by removing all the surrounding fruit tissues using different mechanical methods that were suitable for each of the different species. Damages to seed coats were identified by examining all seeds under a dissecting microscope while the rigidity of all seeds was carefully determined by gently squeezing individual seeds using a pair of forceps and
5
removing those that split or shattered. Thus, undamaged healthy seeds were used for all experiments. >>insert Table 1 here
2.2 Effect of smoke water (SW) on seed germination A mixture of dry plant leaves was burnt in a 50 L metal drum and the smoke generated was
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passed through 10 L of distilled water (Lloyd et al., 2000). A vacuum cleaner was used to draw
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the smoke through water, and an air blower was used to supply air into the metal drum that
contained the fire. The setup was run for 30 minutes and the prepared solution (pH 6.3) was
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considered as the 100% SW stock solution. Smoke water solutions of 75%, 50%, 25% and 5%
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(v/v) were prepared by diluting this solution in distilled water. Distilled water acted as the control (0% SW).
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To test the effects of different concentrations of SW on germination, five replicates with 20 seeds (100 seeds in total) from each selected species without physical dormancy were incubated
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at 25 C in an incubator (Hinotek, MGC-450 BP, China) under 12 hr / 12 hr light / dark condition (the photon irradiance during the light phase was approximately 115 µmol photons m-2 s-1, 400–700 nm, cool white fluorescent tubes) in 90 mm plastic Petri dishes containing tissue paper moistened with about 12 ml of 100%, 75%, 50%, 25%, 5% and 0% (distilled water)
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solutions of SW. Species with physical dormancy (Bauhinia racemosa, Cuscuta chinensis, Tadehagi triquetrum and Tephrosia purpurea) (Jayasuriya et al., 2013) were tested with the 100% SW solution only and germination of both manually scarified (using a razor blade) and non-scarified seeds were tested. In the case of Terminalia bellirica, endocarp removed seeds were used for SW experiments. The number of germinated seeds was counted every two days
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during the first week and thereafter every seven days for the next three weeks. In the case of Phoenix pusilla seeds, germination was recorded for eight weeks, since seeds of this species were known to take some time to germinate.
2.3 KAR1 treatments Seeds of species that showed a significant germination response to smoke water treatments were
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tested for the effect of KAR1. A stock solution of 100 µM KAR1 (Flematti et al., 2005) was used
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to prepare 10 nM, 100 nM and 1 µM solutions by diluting with distilled water. Distilled water was used as the control. Seeds from each test species were incubated in Petri dishes containing
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tissue paper moistened with 12 ml of KAR1 under the same conditions as previously described.
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The number of germinated seeds in each replicate was counted every two days during the first week and thereafter every seven days for the next three weeks. For Euphorbia heterophylla a
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different seed lot had to be used for KAR1 experiments since there were no adequate seeds in the seed lot used for SW experiments. For all the other species the SW and KAR1 treatments were
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done for the same seed lot.
2.4 Effects of smoke water and KAR1 on t50 (time taken for 50% germination) To calculate the average t50 under each treatment, three parameter sigmoidal regression curves
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were fitted for time vs. germination percentage for each replicate of the SW and KAR1 treatments using SigmaPlot version 10.0.1 (SYSTAT Inc., 2007) with the t50 estimated from each of the fitted cumulative germination curves.
2.5 Statistical analysis
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Germination percentage data were normalized by arcsine transformation prior to statistical analysis. Data from experiments with only two treatments were analyzed using an independent two-sample t-test (α = 0.05). Data of experiments with multiple treatments and germination rate (t50) data were analyzed by one-way ANOVA. Fisher’s least significant difference procedure was used to compare treatments (α = 0.05). All analyses were carried out using Minitab version
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17.1.0 (Minitab Inc., 2014) and the graphs were constructed using SigmaPlot ver.10.0.1
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(SYSTAT Inc., 2007).
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3. Results 3.2 Effect of SW on seed germination
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From the 18 tested species, seeds of three native (Flueggea leucopyrus, Maesa indica and P.
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emblica) and three exotic species (Chromolaena odorata, E. heterophylla and Hyptis suaveolens) showed increased germination in response to smoke water treatments (Fig. 1C,E,F,G,I & L) (P <
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0.05). Germination percentage of F. leucopyrus and P. emblica increased significantly, compared to control seeds, in response to all SW concentrations tested (Fig. 1F & L), and in the other four species germination was improved in some of the SW concentrations assessed. It appears that the optimum concentration of smoke water may vary with species. In C. odorata, E. heterophylla
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and F. leucopyrus seeds for example, the highest concentration of smoke water assessed (100%) resulted in the highest germination (Fig. 1C,E & F), while in H. suaveolens, M. indica and P. emblica seeds, the germination percentage in response to 100% SW treatment was less than that observed in response to the 50% or 75% SW treatments; albeit it was still significantly higher than that observed in control seeds except for H. suaveolens (Fig. 1G,I & L). None of the test species that were determined to possess physical dormancy responded to SW administered either 8
before or after seed scarification (Fig. 2). Seeds of these species achieved 75% germination within 2–3 days when mechanically scarified. >>insert Fig. 1 here >>insert Fig. 2 here
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3.3 Effect of KAR1 on seed germination Of the six smoke responsive species, the final germination percentage of the seeds from five
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species increased significantly after treatment with KAR1 (Fig. 3) (P < 0.05). KAR1 treatments did not affect the germination percentage of E. heterophylla seeds (Fig. 3B). All tested
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concentrations of KAR1 significantly increased the germination percentage of C. odorata, F.
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leucopyrus, H. suaveolens and P. emblica seeds, while germination of M. indica seeds was significantly enhanced by the 100 nM and 1 M KAR1 treatments only (Fig. 3E). Seeds of F.
KAR1 (Fig. 3C).
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>>insert Fig. 3 here
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leucopyrus responded in a similar manner to the tested lowest and highest concentrations of
3.4 Effect of smoke water and KAR1 on t50 Seeds of four species (H. suaveolens, Osbeckia rubicunda, P. pusilla and Phyllanthus amarus) did not achieve 50% germination under any of the smoke water treatments within four weeks (Table 2). T50 could be calculated only under 100% SW for C. odorata and under 50–100% concentrations for F. leucopyrus since ≥50% germination was achieved only under these 9
concentrations. All of the tested concentrations of SW significantly reduced the t50 of E. heterophylla seeds, while only one concentration (50%) reduced the t50 of M. indica seeds. It shows that the rate of germination of E. heterophylla was more rapid under high concentrations of SW (Table 2) (P < 0.05). Likewise, a significant reduction in t50 was observed in response to the 25% and 50% SW solutions for seeds of Careya arborea and Limonia acidissima
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respectively. In contrast, higher concentrations of SW significantly increased t50 of Bidens pilosa seeds (Table 2) (P < 0.05).
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>>insert Table 2 here
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The seeds of H. suaveolens did not achieve 50% germination under any of the tested KAR1 concentrations assessed within four weeks. In addition, the seeds from the control
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treatments of several species did not achieve 50% germination after 28 days of incubation (Table
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3). A significant reduction in t50 compared to control was observed only in M. indica seeds in response to exposure to 1 µM KAR1 and in C. odorata in response to 100 nM and 1 µM
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compared to 10 nM (Table 3). >>insert Table 3 here
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4. Discussion
This is the first report on smoke water (SW) and karrikinolide (KAR1) response upon seed
germination of native and exotic plant species in the Indian subcontinent. One-third of 18 tested species have shown a positive germination response with respect to seed germination percentage and / or t50, to SW and KAR1. Therefore the role of smoke and KAR1 seems to be species-specific as observed in previous studies (Roche et al., 1997; Dayamba et al., 2010) and affects both native 10
and exotic species. The native species that respond to SW and KAR1 including pioneer species such as M. indica might lead to natural regeneration of the vegetation after a fire event (Hall et al., 2016; Madawala et al., 2016). Also, those species can be used in restoration programs either through in situ stimulation of soil seed banks or ex situ with the use of smoke to enhance nursery germination for the production of plants (Roche et al., 1997, 1998; Lloyd et al., 2000; Wulff et al., 2012; Hall et al., 2016). Furthermore, the present study reveals the possibility of using SW to
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produce seedlings of medicinally and commercially valuable species (Kulkarni et al., 2007,
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2008; Chumpookam et al., 2012; Iqbal et al., 2016) such as F. leucopyrus and P. emblica which may benefit small-scale land holders and local communities. Both these species are well-known
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medicinal plants in Ayurvedic medicine in Sri Lanka and India (Singh et al., 2011; Ajmeer et al.,
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2014; Soysa et al., 2014).
Alien plant invasions are frequently associated with disturbances (Figueora et al., 2009).
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Traits such as shorter germination times favor the establishment of these species in the vacant niches (Gioria et al., 2018). From the six smoke responsive species, C. odorata, E. heterophylla
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and H. suaveolens readily grow in disturbed habitats and are considered as agricultural weed species in Sri Lanka. Smoke responsiveness can be an advantage for these species to successfully colonize disturbed land and thus outcompete native species. Farmers practicing shifting
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cultivation in the dry zone of Sri Lanka claim that E. heterophylla is one of the most noxious weeds found growing on land cleared for cultivation. This invasiveness may be due in part to the effects of germination promoting compounds in smoke generated when they use fire to regularly clear the land. Figueora et al. (2009) reported on enhanced germination of exotic (weed) species in response to smoke treatments. Application of SW to field soil to induce emergence of smoke responsive species followed by application of a weedicide or uprooting would be an effective 11
method of removing weed seeds from the soil seed bank (Kandari et al., 2011; Kamran et al., 2014). Careya arborea, P. emblica and T. bellirica are three native, dominant fire-tolerant tree species in dry patana grasslands in Sri Lanka (Gunatilleke et al., 2008). However, only the seeds of P. emblica responded to SW and KAR1. Even though C. arborea and T. bellirica did not respond to smoke, fire events in dry patana may still be important for regeneration of these
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species through other means of regeneration such as re-sprouting (Pausas and Keeley, 2014). In
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the dry zone of Sri Lanka, fire used to clear land for agricultural purposes spreads into the adjacent secondary forests, diverting succession and dominance of some species such as
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Cymbopogon nardus (Jong et al., 2001). Seed germination of C. nardus did not seem to be
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enhanced by smoke in this study. Further studies are needed to understand the occurrence and establishment of these species in fire prone ecosystems.
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Seeds with physical dormancy are impermeable to water (Baskin and Baskin, 2004, 2014) and therefore imbibition of water is hindered. As a result, such seeds cannot germinate
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due to their water impermeable seed coat. It has been reported that in some species such as B. racemosa, a certain percentage of the seed population is non-dormant and is able to imbibe when exposed to water (Jayasuriya et al., 2013). In such species, it is possible that the non-dormant
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proportion of the seeds might respond to SW or KAR1. However, all the four species tested in this study with physical dormancy, including the parasitic species C. chinensis, did not respond to smoke water before or after mechanical scarification (Fig 2). It is reported that the frequency of occurrence of T. purpurea plants is higher in burnt sites than non-burnt sites in Udawalawe National Park, Sri Lanka (Perera and Wijesooriya, 2007) and this could be caused by enhanced
12
germination due to other factors such as heat from fire that has rendered the seeds water permeable (Robles-Diaz et al., 2014; Hall et al., 2016; Tavsanoglu et al., 2017). As Flematti et al. (2004) explained, increased seed germination and rate of germination after SW treatment could be associated with KAR1 present in SW. However, seeds of E. heterophylla did not respond to KAR1 even though these seeds responded to smoke water. This
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could be due to the presence of other karrikin compounds (KAR2, KAR3 and KAR4) (Flematti et al., 2015) or other compounds such as nitrates and cyanohydrins (e.g. glyceronitrile) present in
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plant derived smoke which in some cases are equally capable of stimulating seed germination
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(Flematti et al., 2011). This indicates that the SW used in the present study may contain seed germination stimulating compounds other than KAR1. The variability of germination of E.
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heterophylla in the control treatments of SW and KAR1 experiments may be due to variability of dormancy and the quality of the two seed lots used (Martinez-Baniela et al., 2016). The increase
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in t50 of B. pilosa seeds at higher SW concentrations may be due to the suppression of seed germination of some species by high SW concentrations, possibly due to toxic effects (Nelson et
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al., 2009; Zhou et al., 2014).
Smoke water and KAR1 promoted seed germination in only three out of 13 native species (23%) and three out of five exotic species (60%) tested. Thus, compared to exotic species, the
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majority of the native species did not respond to smoke. Historically, natural or anthropogenic fire was not a significant factor that determined the structure or species composition in the ecosystems of Sri Lanka. Thus, we can assume that many of the native species are not adapted to respond to smoke signals as a germination cue. However, in Sri Lanka, anthropogenic fires are now becoming more frequent and intensive (Ariyadasa, 2001; FAO, 2015). Therefore, more research in the field of fire ecology is essential to understand how the native and exotic species 13
respond to smoke / fire related cues, and how anthropogenic fires affect the survival of plant species in anthropogenic fire prone habitats in Sri Lanka.
5. Conclusions
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According to our results, there are both native (F. leucopyrus, M. indica and P. emblica) and exotic (C odorata, H. suaveolens and E. heterophylla) species that respond positively to smoke
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water (SW). The effect of SW was significant on both the germination percentage and the time taken for 50% germination (t50) of these species. Karrikinolide (KAR1) promoted germination of
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SW responsive species except E. heterophylla. Therefore, the germination enhancement of these
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species by smoke water is likely due to the presence of KAR1 in the smoke water. Even though the majority of the native species did not respond to smoke / KAR1, those that responded to SW /
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KAR1 might play an important role in natural regeneration of fire prone ecosystems and also can be utilized in restoration efforts while SW / KAR1 responsiveness would be an added advantage
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for the exotic species to outcompete the native vegetation, particularly in view of increased frequency of anthropogenic fires.
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Author Contribution Statement
Effect of smoke on seed germination of species of different habitats from Sri Lanka
I, Dr. Nalin Gama-Arachchige the corresponding author of the manuscript tiled “Effect of smoke on seed germination of species of different habitats from Sri Lanka” confirm the contributions of each author to the research work listed in the table below.
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Dr. Nalin Gama-Arachchige Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka
02 03 04
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Disclosure statement
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Contributions/ CRediT roles Conceptualization; Investigation; Methodology; Data A.A.C.B. Alahakoon curation; Formal analysis; Validation; Visualization; Writing - Original Draft; Writing – review & editing Project administration; Supervision; Writing – review & G.A.D. Perera editing D.J. Merritt Conceptualization; Resources; Writing – review & editing Conceptualization; Methodology; Resources; Writing S.R. Turner Original Draft; Writing – review & editing Conceptualization; Methodology; Formal analysis; N.S. Gama-Arachchige Supervision; Writing - Original Draft; Writing – review & editing; Funding acquisition; Project administration
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01
Author
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No
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The authors declare that they have no conflict of interest.
Acknowledgement
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The authors thank Gavin Flematti of the University of Western Australia for providing the karrikinolide used in this study.
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The Plant List, 2013. Version 1.1. Published on the Internet; http://www.theplantlist.org/ (accessed 1st March 2019). Williams, P.R., Congdon, R.A., Grice, A.C., Clarke, P.J., 2003. Fire-related cues break seed dormancy of six legumes of tropical eucalypt savannas in north-eastern Australia. Austral Ecol. 28, 507–514. Wulff, A., Turner, S., Fogliani, B., Lhuillier, L., 2012. Smoke stimulates germination in two
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divergent Gondwanan species (Hibbertia pancheri and Scaevola montana) endemic to
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the biodiversity hotspot of New Caledonia. Seed Sci. Res. 22, 311–316.
Zhou, J., da Silva, J.A.T., Ma, G., 2014. Effects of smoke water and karrikin on seed germination
https://doi.org/10.2478/s11535-014-0338-6
-p
of 13 species growing in China. Cent. Eur. J. Biol. 9, 1108–1116.
re
Zirondi, H.L., Silveira, F.A.O., Fidelis, A. 2019. Fire effects on seed germination: Heat shock
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ur na
lP
and smoke on permeable vs impermeable seed coats. Flora 253, 98-106.
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lP
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Figure captions
Fig. 1. Final germination percentage of the seeds of 14 test species without physical dormancy in response to treatment with different concentrations of smoke water after 28 days at 25 C under
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12 hr / 12 hr light / dark condition. Bars show means ± s.e. (n = 5). Different letters indicate significant differences in germination among the treatments (P < 0.05). The shaded bars of the Fig. 1M represent the germination percentages of Phoenix pusilla after 8 weeks of incubation.
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Fig. 2. Final germination percentage of the seeds of four test species with physical dormancy (PY) in response to treatment with 100% SW after 28 days at 25 C under 12 hr / 12 hr light /
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dark condition. Bars show means ± s.e. (n = 5). Different letters indicate significant differences in germination among the treatments (P < 0.05).
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Fig. 3. Final germination percentage of the seeds of six smoke responsive species in response to
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treatment with different concentrations of KAR1 after 28 days at 25 C under 12 hr / 12 hr light / dark condition. Bars show means ± s.e. (n = 5). Different letters indicate significant differences
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in germination among the treatments (P < 0.05).
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Table 1: Family, life form, fruit type, ecological status, habitat and seed source of the 18 study species
Fabaceae
Small tree
Legume
Native
Careya arborea Roxb.
Lecythidaceae
Chromolaena odorata (L.) Asteraceae R.M. King & H. Rob.
Cuscuta chinensis Lam.
Convolvulaceae
Poaceae
Euphorbia heterophylla L. Euphorbiaceae
Flueggea leucopyrus Willd.
Tree
Shrub
Vine
Cypsela
Exotic
Berry
Cypsela
Native
Capsule
Tussock grass
Caryopsis
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Cymbopogon nardus (L.) Rendle
Herb
Hyptis suaveolens (L.) Poit.
Euphorbiaceae
Lamiaceae
Herb
Elahara
Hanthana
Savanna and dry patana
Ethakada
Waste lands
Exotic
Native
Capsule
Bushy shrub
Berry
Shrub
schizocarp
Native
GPS coordinates
April 2016 80° 47ʹ 36.5ʺ E 7° 15ʹ 28.0ʺ N December 2015 80ʹ 37ʹ 43.7ʺ E 8° 36ʹ 3.7ʺ N October 2015 80° 33ʹ 59.6ʺ E 7° 13ʹ 43.3ʺ N
Waste lands and forest clearings
Hanthana
December 2016 80° 38ʹ 18.0ʺ E 7° 15ʹ 23.7ʺ N
Paddy fields and disturbed areas
Peradeniya
January 2016 80° 35ʹ 47.7ʺ E 7° 13ʹ 25.3ʺ N
Wet patana and roadsides
Sarasavigama
February 2016 80° 37ʹ 2.74ʺ E 7° 53ʹ 43.8ʺ N
Exotic
Collection period
7°44ʹ 3.7ʺ N
Dry forest, monsoon forest and scrublands
pr
Asteraceae
Collection locality
f
Ecological status Habitat
oo
Fruit type
e-
Bidens pilosa L.
Life form
Pr
Bauhinia racemosa Lam.
Family
na l
Species
Road sides and disturbed areas
Pelwehera 80° 39ʹ 55.5ʺ E
December 2015, May 2016
6° 17ʹ 3.6ʺ N Native
Thorn scrublands
Yala
November 2015 81° 25ʹ 45.5ʺ E 7° 54ʹ 13.0ʺ N
Exotic
Road sides in the dry zone
Pelwehera
December 2016 80° 40ʹ 9.69ʺ E
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6° 17ʹ 3.6ʺ N Limonia acidissima Groff
Rutaceae
Small tree
Berry
Native
Tropical dry forests
Yala
November 2015
Melastomataceae
Phoenix pusilla Gaertn.
Arecaceae
Shrub
Capsule
Dwarf palm Berry
Euphorbiaceae
Herb
Phyllanthus emblica L.
Euphorbiaceae
Tree
Tadehagi triquetrum (L.) H. Ohashi
Fabaceae
Shrub
Tephrosia purpurea (L.) Pers.
Fabaceae
Capsule
Berry
Legume
Small shrub Legume
Jo ur Combretaceae
Native
Native
Native
Exotic
Native
na l
Phyllanthus amarus Schumach. & Thonn.
Terminalia bellirica (Gaertn.) Roxb.
Berry
Tree
Berry
Shady places in montane region
Native
Native
Native
7° 15ʹ 28.0ʺ N
Hanthana
December 2015 80° 37ʹ 43.7ʺ E 7° 13ʹ 43.3ʺ N
Open montane forest, forest edges and roadsides
Hanthana
Tropical dry forest and coastal vegetation
Maradankadawala
Ruderal
Pilimathalawa
pr
Osbeckia rubicunda Arn.
Shrub
e-
Myrsinaceae
Pr
Maesa indica (Roxb.) A. DC.
oo
f
81° 25ʹ 45.5ʺ E
January 2016 80° 38ʹ 18.0ʺ E 8° 07' 11.1" N May 2016 80° 33' 53.7" E 7° 14ʹ 2.83ʺ N July 2016 80° 32ʹ 27.8ʺ E
Savanna, dry patina and intermediate forests
-
Secondary forests
Sarasawigama
-
May 2016
7° 13ʹ 25.3ʺ N March 2016 80° 37ʹ 2.74ʺ E 8° 50ʹ 13.9ʺ N Road sides and waste lands
Paranaddakal
August 2015 80° 29ʹ 46.7ʺ E
Dry patina and tropical intermediate forests
(Based on Dassanayake and Fosberg 1981–2004; Christenhusz et al., 2017 and www.theplantlist.org.)
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7° 14ʹ 35.2ʺ N Lagamuwa
March 2016 80° 31ʹ 32.8ʺ E
Table 2: Effect of different concentrations of smoke water on t50 (time taken to achieve 50% germination in days) on the seeds of 14 test species without physical dormancy (mean ± s.e., n = 5).
lP
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of
t50 time (days) under tested concentrations of smoke Species water 0% 5% 25% 50% 75% 100% d cd bc b ab Bidens pilosa 2±0 2±0 3±0 3±1 4±0 5±0a a ab b ab a Careya arborea 3±1 2±1 0±0 1±1 3±0 2±1ab Chromolaena odorata 7±0 Cymbopogon nardus 2±1a 2±0a 2± 0a 2±0a 2±0a 2±0a Euphorbia heterophylla 12±1a 8±1b 5±0c 4±0c 3±0c 4±0c a a Flueggea leucopyrus 13 ± 1 13 ± 1 16 ± 3a Hyptis suaveolens a a a b a Limonia acidissima 7±0 7±0 7±0 6±0 7±0 7±0a a ab a b ab Maesa indica 19±1 15±1 17±2 13±1 15±1 16±1ab Osbeckia rubicunda Phoenix pusilla Phyllanthus amarus a a a a Phyllanthus emblica 10±2 9±2 7±0 6±0 6±0a Not tested Not tested Terminalia bellirica 10±0a 10±0a 11±1a 10±0a Different letters indicate significant differences between treatments (P < 0.05)
Species
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Table 3: Effect of different concentrations of KAR1 on t50 (time taken to achieve 50% germination in days) on the seeds of the six species which responded to smoke water (mean ± s.e., n = 5).
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Chromolaena odorata Euphorbia heterophylla Flueggea leucopyrus Hyptis suaveolens Maesa indica Phyllanthus emblica
t50 time (days) under tested concentrations of KAR1 0 nM 10 nM 100 nM 1 µM 10±2a 5±2b 5±1b 20±3a 20±6a 19±2a 16±1a a a 8±0 9±1 10±1a a a a 22±2 24±1 22±1 16±1b 1±1a 1±0a 1±0a
ifferent letters indicate significant differences between treatments (P < 0.05)
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