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Received Date : 09-Sep-2014 Revised Date : 29-Dec-2014 Accepted Date : 12-Jan-2015 Article type : Research Paper Editor : R Bekker
Article type: Research paper Running head: Annual dormancy cycles in seeds of shrub species
Annual dormancy cycles in buried seeds of shrub species: Germination ecology of Sideritis serrata (Labiatae).
Miguel A. Copete1, José M. Herranz1, Pablo Ferrandis1 & Elena Copete2
1 Higher Technical School of Agronomy and Forestry Engineering, Department of Crop Production and Agricultural Technology, University of Castilla-La Mancha, Albacete, Spain 2 Botanical Institute of the University of Castilla-La Macha, Albacete, Spain
Correspondence: Miguel A. Copete, Higher Technical School of Agronomy and Forestry Engineering, Department of Crop Production and Agricultural Technology, University of Castilla-La
Mancha,
University
Campus
s/n,
Albacete
02071,
Spain.
E-mail:
[email protected]
Keywords: Dormancy break; dormancy induction; ex-situ propagation; physiological dormancy; plant population and community dynamics; seed longevity; soil seed bank
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/plb.12306 This article is protected by copyright. All rights reserved.
ABSTRACT
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The germination ecology of Sideritis serrata was investigated in order to improve ex-situ propagation techniques and management of their habitat. Specifically, we analysed: (i) influence of temperature, light conditions and seed age on germination patterns; (ii) phenology of germination; (iii) germinative response of buried seeds to seasonal temperature changes; (iv) temperature requirements for induction and breaking of secondary dormancy; (v) ability to form persistent soil seed banks; and (vi) seed bank dynamics. Freshly matured seeds showed conditional physiological dormancy, germinating at low and cool temperatures but not at high ones (28/14 and 32/18 ºC). Germination ability increased with time of dry storage, suggesting the existence of non-deep physiological dormancy. Under unheated shade-house conditions, germination was concentrated in the first autumn. S. serrata seeds buried and exposed to natural seasonal temperature variations in the shade-house, exhibited an annual conditional dormancy/non-dormancy cycle, coming out of conditional dormancy in summer and re-entering it in winter. Non-dormant seeds were clearly induced into dormancy when stratified at 5 or 15/4 ºC for 8 weeks. Dormant seeds, stratified at 28/14 or 32/18 ºC for 16 weeks, became non-dormant if they were subsequently incubated over a temperature range from 15/4 to 32/18 ºC. S. serrata is able to form small persistent soil seed banks. The maximum seed life span in the soil was 4 years, decreasing with burial depth. This is the second report of an annual conditional dormancy/non-dormancy cycle in seeds of shrub species.
INTRODUCTION Probably the most remarkable and critical stages during the life cycle of plants are seed germination and seedling establishment. Germination is a risky transition, especially in arid and semiarid climates, between the most tolerant stage to drought (i.e. seeds) and seedlings,
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being the most drought-sensitive phase in plant development (Gutterman 1993; Hilhorst &
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Toorop 1997). Hence the importance that seed germination, often controlled by the existence of dormancy cycles, occurs at the right time of a season that gives the best chance for plant development (Gutterman 1993). In addition, accumulation of seeds in the soil can substantially contribute to population viability (Baskin & Baskin 1978; Milberg 1994). Annual plants can be highly dependent on the formation of a persistent seed bank to
avoid local extinction, whereas perennial plants are more likely to successfully face environmental conditions unsuitable for seed production during one or more growing seasons. Therefore, mechanisms enabling the maintenance of persistent seed banks, such as annual dormancy cycles, might be more common in annuals than in perennials (Schütz 1997). Indeed, the existence of dormancy cycles in buried seeds of annuals, both winter and
summer ones, exposed to seasonally changing temperature regimes is well known (Roberts & Boddrell 1985; Bouwmeester & Karssen 1993; Baskin & Baskin 2000; Copete et al. 2009), as also in some herbaceous perennials, such as Lesquerella fendleri (Hyatt et al. 1999), Penstemon palmeri (Meyer & Kitchen 1992), Primula veris and Trollius europaeus (Milberg 1994), Cnidium dubium (Geissler & Gzik 2010) and Carex loliacea (Kull et al. 2011). These annual cycles, in which physiologically based dormancy is relieved and induced during the course of a year, occur in buried seeds of some species in both temperate and tropical soil environments (Murdoch & Ellis 1992). These patterns of dormancy are of high value for survival of plant populations (Karssen et al. 1988). However, very few studies have been done on buried seeds of woody plants, to test for the existence of annual cycles in their dormancy status. We are aware of only two studies whose results indicate the presence of seed dormancy cycles in woody species: Alnus glutinosa, although the type of cycle could not be determined (Schütz 1998), and Kalidium gracile, with buried seeds exhibiting an annual conditional dormancy/ non-dormancy cycle (Cao et al. 2014). The main goal of this work
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was to analyse the germination response of buried seeds of a shrub in a Mediterranean
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semiarid habitat, Sideritis serrata Lag., to seasonal temperature changes, evaluating the influence of temperature on their dormancy status. The genus Sideritis (Labiatae) comprises about 140 species, reaching highest diversity
in the Iberian Peninsula and Macaronesia. The distribution area extends as far east as Russia, Tibet and western China (Morales 2010). S. serrata is a thermophilous calcicole shrub (60– 120 cm) growing on limestone. It is endemic to the southeast Iberian Peninsula, with a narrow distribution in Sierra de Abenuj (Tobarra, Albacete, Spain). The species is considered as critically endangered according to the IUCN Spanish Red List (Copete et al. 2004) due to its small, fragmented area of distribution, and continuous degradation of its habitat. It is also included in Annex II of Directive CEE 43/1992 (Habitat Directive of the European Union). The recovery plan for this species (D.O.C.M. 1999) recommends a reinforcement
programme for subpopulations affected by human activities through production of ex-situ plants. To optimise plant production in nurseries as well as ex-situ conservation in germplasm seed banks, it is necessary to have good understanding of germination requirements, particularly for rare and/or endemic species for which it is more difficult to obtain material (seeds; Cerabolini et al. 2004). In this work, we studied the germination ecology of S. serrata and analyse the main phases of the process. The importance of persistent soil seed banks (some viable seeds remaining in the soil
for > 1 year; sensu Thompson & Grime 1979) for conservation of threatened species has been pointed out in several studies, since it allows re-establishment and maintenance of populations after disturbances without the need for an external seed source, and because they can play an important role in years with poor seed set (Baskin & Baskin 1978, 2000; Thompson et al. 1997). However, beyond knowing the soil bank size, there are other key
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questions in the case of threatened plant species: how long can the seeds remain viable in the
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soil, and does burial depth influence seed longevity? Specifically, the goals of this study were to: (i) analyse the influence of duration of
seed dry storage on germination under different light and temperature conditions; (ii) characterise the phenology of seed germination; (iii) determine the response of buried seeds to seasonal temperature changes; (iv) assess, if present, temperature requirements for induction and breaking of secondary dormancy; (v) determine ability of S. serrata to form persistent soil seed banks; and (vi) study the seed bank dynamics.
MATERIAL AND METHODS Seed material Fully mature seeds of S. serrata were collected from vigorous and healthy plants growing in Sierra de Abenuj (Tobarra, SE Spain; UTM coordinates: 30SXH1074; 800 m a.s.l.), the unique location known for the species. This area has a typical continental semiarid climate, with mean annual precipitation of 320 mm, mean annual temperature of 14 ºC and absolute minimum annual temperature of -6.7 ºC. Seed collection was carried out in late July 2006, 2007, 2008 and 2009.
Germination tests A 100-seed lot was assigned to each temperature condition test, distributed into four 25 seed replicates. Each replicate was incubated in a 9-cm diameter sealed (with Parafilm) Petri dish on a double layer of filter paper saturated with ca. 4 ml distilled water and placed in germination chambers (Ibercex, Madrid, Spain; F-4 model, ± 0.1ºC, cool white fluorescent light of ~25 μmol·m-2·s-1) at constant 5 ºC or a 12/12 h day/night temperature regimes of 15/4, 20/7, 25/10, 28/14 and 32/18 ºC. The fluctuating temperatures used in tests simulated
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approximate mean maximum and mean minimum monthly temperatures in the area of
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Tobarra during the growing season. Thus, 15/4 ºC corresponds to November and March, 20/7 ºC to October and April, 25/10 ºC to September and May, 28/14 ºC to August and June, and 32/18 ºC to July. The 5 ºC treatment is close to the mean temperature recorded during winter months (December, January and February), being broadly used in experiments for dormancy induction by low temperatures (Baskin & Baskin 1989, 2000). The 12-h photoperiod with alternating temperature treatments coincided with the daily high temperature period. In addition, the same temperature seed germination tests were carried out in darkness by wrapping the Petri dishes in a double layer of aluminium foil. Hence, we had simultaneous ‘light’ (photoperiod) and ‘dark’ treatments over the complete set of temperature conditions. Tests of dormancy cycles and temperature requirements for dormancy breaking and
induction lasted 15 days, as recommended for this type of study (Baskin & Baskin 1998). However, to analyse the influence of duration of laboratory dry storage on germination, tests were extended to 30 days, since germination had not slowed in some trials after 15 days. Protrusion of the radicle was the criterion for successful germination. In treatments with a photoperiod, seeds were checked for germination every 2–3 days. Seeds incubated in the dark were checked only at the end of the test. Seeds that did not germinate were checked for viability on the basis of embryo appearance, paying special attention to seed colour and turgidity. Percentage germination was computed from viable seed numbers.
Shade-house conditions The metal frame shade-house used in this study was on the university campus in Albacete (UTM coordinates, 30SWJ9713, 690 m a.s.l.; 40 km from Sierra de Abenuj). It was neither heated in winter nor air-conditioned in summer, and was shaded continuously throughout the
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study with a polyethylene cloth (12 threads cm-1, 50% light interception). Temperatures were
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recorded throughout the study at a meteorological station within the shade-house. To simulate soil moisture regimes in the field, pots and trays placed in the shade-
house were watered to field capacity once per month during summer (1 July–30 September) and twice per month during the rest of the year, with the exception of the coldest period (December–January) when the substrate was frozen.
Influence of seed age on germinability of dry-stored seeds After drying and cleaning, seeds of S. serrata were stored in paper bags under laboratory conditions (18–20 ºC; 50–60% air humidity) until used in germination tests. Seed lots were tested for germination from 1 August 2006 (age = 0 months) to January 2007 (age = 5 months). Intermediate tests were started on the first of September, October, November and December 2006 (seed age = 1, 2, 3 and 4 months, respectively); tests lasted 30 days. Temperature and illumination conditions were those described in the ‘Germination tests’ section.
Germination phenology On 1 August 2007, 5 days after collection, 500 seeds were divided into five 100 seed replicates. Each replicate was sown 3-mm deep into a 40 x 25 x 7 cm plastic seedling tray with drainage holes. Substrate consisted of a sterilised mixture of peat and sand (2:1 v/v). To facilitate emergent seedling counts, trays were placed on tables in the shade-house. Watering frequency was as described in the ‘Shade-house conditions’ section. Emerged seedlings were counted and removed approximately every 15 days until summer 2011.
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Response of buried seeds to seasonal temperature changes
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Seeds collected on 26 July 2007 were stored for 3 months at ambient room temperature. Then, 20 lots of 1250 S. serrata seeds each were homogeneously mixed with a double volume of 0.5 mm sieved sand. This mixture was placed in nylon bags of 0.1-mm mesh, which were buried on 1 November 2007 to a depth of 7 cm in soil (river sand) in pots (20-cm diameter and 25-cm depth) with drainage holes. We used four pots with five bags each. Pots were placed in the previously described shade-house. Watering regimes were as described above. Germination tests were conducted in light and dark on seeds exhumed on the first day of each month from December 2007 to July 2009, and also on 1 November 2007 for seeds that were not buried. Seed exhumation was always carried out after sunset. Seeds were recovered from bags by pouring the mixture containing the sand through a 0.5-mm sieve. To prevent light exposure of seeds assigned to germination tests in darkness, both sieving and handling prior to tests were carried out under a green safe light (Baskin & Baskin 1998).
Temperature requirements for induction and breaking of secondary dormancy This experiment was designed on the basis of results obtained from the analysis of seed dormancy cycles started in November 2007. Seeds of S. serrata were collected on 25 July 2009 and stored for 6 days at ambient room temperature. Twenty-six nylon bags, each containing 650 seeds, were prepared as described above. In early August 2009, two bags per pot (13 pots) were buried at 7-cm depth. Pots were placed within the shade-house and submitted to the same watering regime described above until 15 October 2009 (six pots) and 15 March 2010 (seven pots). On 15 October 2009, one bag was exhumed, and seeds tested for germination in light
at 5, 15/4, 20/7, 25/10, 28/14 and 32/18 ºC to determine their dormancy stage. Subsequently, each pot containing two bags was stratified at each of the following temperatures: 5, 15/4,
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20/7, 25/10, 28/14 and 32/18 ºC. After 8 and 16 weeks, respectively, bags from all treatments
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were exhumed and seeds tested for germination in light under the six temperature regimes. On 15 March 2010, we repeated the above experiment to test temperature required to overcome secondary dormancy.
Analysis of the soil seed bank Seed banks of S. serrata and other main species of the community were assessed by analysing the seed content of soil samples collected from one of the core S. serrata subpopulations. Soil samples were collected on 10 June 2006, after autumn–winter–spring germination and before the start of seed dispersal from fruits produced that year. This sampling date was determined on the basis of Thompson & Grime´s (1979) concept of a persistent seed bank, in order to quantify the persistent fraction of the seed bank overlapping two consecutive phenological cycles. Twenty plots of 10 cm x 10 cm were placed along two linear transects of the
following design: (i) ten plots per transect, (ii) 2 m between contiguous plots within each transect, and (iii) 50 m between transects. In each plot, soil was collected to a depth of 5 cm. Soil samples were carried to the laboratory in plastic bags, where they were dried completely and homogenised. Seed content was assessed by physical separation (Ferrandis et al. 1999). The soil was washed through a 0.5-mm mesh sieve. Seeds were separated and identified under a binocular microscope. The criterion for identifying a seed as viable was the healthy appearance of the embryo, paying particular attention to turgidity and colour.
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Effects of depth and duration of burial on dormancy and viability of S. serrata seeds in
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the natural habitat Seeds collected on 25 July 2008 were stored for 2 months at ambient room temperature, then 144 lots of 200 S. serrata seeds each were mixed with sand and put inside nylon bags, as described previously. Prior to burial experiments, seed viability was analysed. Four 25-seed replicates were incubated in light at 20/7 ºC (the most favourable regime for germination) for 30 days. Seeds that did not germinate by the end of the test were moistened with GA3 solution (1000 ppm; Gómez-Campo 1985) for an additional 30 days. Finally, non-germinated seeds were checked for viability on the basis of embryo appearance, paying special attention to colour and turgidity. At the beginning of burial experiments, seeds of S. serrata were classified into four categories: (1) non-dormant seeds, i.e. germinated during germinability test, (2) dormant seeds, i.e. germinated after GA3 treatment; (3) deeply dormant seeds, i.e. a viable embryo but no germination after GA3 treatment; and (4) non-viable seeds. In October 2008, six 30 cm x 30 cm plots free of rocky outcrops were randomly
selected in Sierra de Abenuj (UTM: 30SXH0975; 800 m a.s.l.). Each plot was divided into three 30 cm x 10 cm subplots. Each subplot had a different seed burial depth (2, 7 and 12 cm). In each subplot, eight seed lots were buried at the selected depth. Every seed lot in a bag was closed with a wire that protruded about 1 cm above the soil surface to facilitate exhumation without interfering with the arrangement of remaining bags. For 2 years and every 3 months, one seed lot per plot and depth was exhumed, starting in January 2009. In the laboratory, seeds that germinated (radicle emerged) in bags were counted. Other seeds with a healthy appearance were tested for viability. Therefore, for every exhumation, the four seed status categories described above were determined, in addition to germinated seeds in the bag.
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Data analysis
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Germination percentages of dry-stored seeds were compared among treatments using multifactorial ANOVA, considering three factors: (1) temperature (six levels); (2) light (two levels); and (3) seed age (six levels). For comparison of induction and breaking of secondary dormancy, a multi-factorial ANOVA was performed with factors: (1) temperature during seed burial (six levels); (2) temperature for testing germination response (six levels); and (3) length of stratification (three levels). Treatments responsible for significant main effects were detected with a multiple comparisons Tukey test; significant interactions were explored as contrasting confidence intervals. Prior to analyses, normality (Cochran test) and homoscedasticity (David test) of data were checked. Values of final cumulative germination percentage were square-root arcsine transformed. For each temperature x light treatment on dry-stored seeds, changing trends in
germination with seed age were evaluated with regression analysis. Data were log transformed, and the model selected for each treatment was that with the highest R2 coefficient. The minimum improvement for changing the model was established at 10%. Seed decay rate was calculated using the exponential equation: n2 = n1 e -λ∆t
(Eq. 1)
where n1 and n2 are number of viable seeds remaining at t1 and t2, respectively, λ is decay rate and ∆t is time interval between t1 and t2. The exponential decay curve is often used to model seed bank decay (Auld et al. 2000; Holmes & Newton 2004). We used number of viable seeds at the start of the burial experiment (t1). Seed half-life (i.e. time elapsed for 50% seed decay 50% in soil) was calculated by substituting the decay rate into the exponential equation and solving for n2 = 0.5 n1. In contrast, we considered as maximum longevity the period at which the percentage viable seeds decreased to 5% (n2 = 5), since in a negative exponential function, approaching n2 = 0 would produce values of ∆t tending to infinity.
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Kull T., Kull T., Sammul M. (2011) Reduced light availability and increased competition
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diminish the reproductive success of wet forest sedge Carex loliacea L. Plant Species Biology, 26, 84–92.
Lonsdale W.M., Harley K.L.S., Gillett J.D. (1988) Seed bank dynamics in Mimosa pigra, an invasive tropical shrub. Journal of Applied Ecology, 25, 963–976.
Martínez-Duro E., Ferrandis P., Herranz J.M., Copete M.A. (2010) Do seed harvesting ants threaten the viability of a critically endangered non-myrmecochorous perennial plant population? A complex interaction. Population Ecology, 52, 397–405.
Martínez-Sánchez J.J., Segura F., Aguado M., Franco J.A., Vicente M.J. (2011) Life history and demographic features of Astragalus nitidiflorus, a critically endangered species. Flora, 206, 423–432.
Mennan H. (2003) The effects of depth and duration of burial on seasonal germination, dormancy and viability of Galium aparine and Bifora radians seeds. Journal of Agronomy and Crop Science, 189, 304–309.
Meyer S.E., Kitchen S.G. (1992) Cyclic seed dormancy in the short-lived perennial Penstemon palmeri. Journal of Ecology, 80, 115–122.
Milberg P. (1994) Germination ecology of the polycarpic grassland perennials Primula veris and Trollius europaeus. Ecography, 17, 3–8.
Milberg P., Andersson L. (1997) Seasonal variation in dormancy and light sensitivity in buried seeds of eight annual weed species. Canadian Journal of Botany, 75, 1998–2004.
Mira S., González-Benito M.E., Ibars A.M., Estrelles E. (2011) Dormancy release and seed ageing in the endangered species Silene diclinis. Biodiversity and Conservation, 20, 345358.
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Table 2. Germination percentages (mean ± SE) of Sideritis serrata seeds incubated at 5 ºC and
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various thermoperiods in the light. After 2.5 months of burial in a pot in the greenhouse, one seed lot was exhumed on 15 October 2009 and tested for germination (control). Six pots were then transferred to the laboratory and placed at 5 ºC and the range of thermoperiods (burial temperatures) for 8 and 16 weeks before testing for germination. Numbers in a column followed by same lowercase letter are not significantly different; the same for numbers in a row sharing uppercase letters (P 60%) after 4 months of dry storage at room temperature, it can be concluded that S. serrata seeds have non-deep physiological dormancy (Baskin & Baskin 1998). The broad oscillations in germination at 25/10 ºC have also been reported for other species with non-
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deep physiological dormancy (e.g. Diplotaxis erucoides and D. virgata, Pérez-García et al.
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1995; Sisymbrium runcinatum, Herranz et al. 2003a). In the phenological study (Fig. 2), most seedlings (47%) emerged during the first
autumn, which was related to the ability of seeds dry-stored for 2–3 months to germinate (4060%) when incubated at 15/4 and 20/7 ºC in the dark (Fig. 1). We obtained similar percentage germination in November 2007 (Fig. 3). A small fraction of these seeds germinated in spring, showing that S. serrata has similar germination phenology to facultative winter annuals (Baskin & Baskin 1983; Copete et al. 2009). The seedling emergence during four consecutive phenological cycles after seed
sowing (2007–2011; Fig. 2) demonstrated the ability of this species to form persistent soil seed banks. Indeed, this trait was corroborated in the study of the soil seed bank in the natural habitat (Table 4), where around 100 S. serrata seed m-2 germinated in the upper 5 cm. According to Harper (1977), the soil seed bank can play a crucial role in population dynamics when its density is higher than the number of adult individuals established aboveground, which broadly occurred in our target species (0.0275 plants m-2 in the study area; Copete et al. 2004). However, despite adaptations promoting formation of seed banks in S. serrata, especially dormancy cycles, seeds were not highly abundant in the soil, probably due to their large size (2–3 mm); densities of individual species in the soil seed bank are negatively related to seed size (Fenner & Thompson 2005). The ability of this threatened shrub to form a persistent soil seed bank is certainly an adaptive advantage, since persistent seed banks buffer populations from environmental stochasticity (Adams et al. 2005). Although not so crucial as for threatened annuals such as Sisymbrium cavanillesianum (Herranz et al. 2003a) and biennials such as Coincya rupestris subsp. rupestris (Herranz et al. 2003b), whose chances of survival rest largely on seed reserves in the soil, recent population viability analyses have also stressed the important role of soil seed banks for long-term persistence of populations in
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perennial life-form species, e.g. Helenium virginicum, a perennial herb (Adams et al. 2005),
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and Helianthemum polygonoides, a critically endangered perennial shrub in SE Spain (Martínez-Duro et al. 2010). According to Thompson et al. (2003), there is not a close relationship between seed
persistence in the soil and seed dormancy. However, many species that form persistent seed banks exhibit annual changes in seed dormancy (Baskin & Baskin 1985; Milberg & Andersson 1997). S. serrata seeds also showed annual changes in dormancy (Fig. 3), exhibiting an annual conditional dormancy/non-dormancy cycle, with dormancy loss occurring in summer (non-dormant seeds in late summer–early autumn) and conditional dormancy induction in late autumn and winter (Fig. 3), although there are months (i.e. February) with high values of germination intercalated in series of low values (Fig. 3), as occurs in the facultative winter annual Ziziphora aragonensis (Copete et al. 2009). This hightemperature requirement for loss of seed dormancy and induction into conditional dormancy mediated by low temperatures has also been found in seeds of facultative winter annuals, e.g. Thlaspi arvense (Baskin & Baskin 1989), Veronica arvensis (Baskin & Baskin 1983), Iberis crenata and Ziziphora aragonensis (Copete et al. 2009). As a result, S. serrata seeds mostly germinated in autumn, increasing the chances of seedling survival due to acquisition of a degree of maturity before winter frosts, and even more determinant, before summer drought. In arid and semiarid Mediterranean regions, adaptive processes tend to select as
optimum germination temperatures those that are most likely to occur during the rainfall period (Pérez-García et al. 1995). Specifically, in Sierra de Abenuj, although rainfall in autumn (28%) is similar to that in spring (31%), most seedlings emerged in autumn, increasing the length of time for plant development and thus survival probability during the first summer.
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The trial with buried seeds at different depths in the natural habitat also confirmed the
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existence of dormancy cycles (Fig. 4). It was clear that dormancy changes in seeds were mitigated by burial depth, which seems logical when considering the damping effect on temperature changes associated with soil depth (Tsuyuzaki 2006). We also observed that, even at times of year favourable for seedling establishment, a small percentage of seeds remained dormant. It is not advantageous for a plant to risk all seeds in one germination event because adaptive mechanisms cannot accurately predict favourability of the coming growth season, and because density-dependent mortality may affect seedling establishment (Jurado & Moles 2003; Martínez-Sánchez et al. 2011). For the upper soil layer, maximum longevity of S. serrata seeds was almost 4 years,
while in the deepest soil it was 2 years (Table 5). Nevertheless, these results corroborate the persistent nature of the soil seed bank in S. serrata. This negative relation between burial depth and seed survival was also found in other studies (Lonsdale et al. 1988). In a few cases, longevity is not affected by depth of burial (Egley & Chandler 1983); however, most species present the opposite relationship: Artemisia tridentata (Wijayratne & Pyke 2012), Galium aparine and Bifora radians (Mennan 2003), Rumex obtusifolius, Oenothera biennis and Polygonum longisetum (Tsuyuzaki 2006) and Sisymbrium cavanillesianum (Herranz et al. 2003a). In all these species, seed longevity increased with depth, probably due to the wider humidity and temperature ranges recorded in the upper soil layers, which may be lethal to seeds (Holmes & Newton 2004). One possible explanation for our unusual result could be the scarce rainfall in the study area, so the upper soil layer loses humidity faster and remains dry for most of the year. Hence, humidity being one of the main parameters involved in seed bank decay (Holmes & Newton 2004), S. serrata seeds in the first centimetres of the soil can remain viable for longer.
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The main finding of the present study is the confirmation that seed dormancy cycles
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are not restricted to annual species. As far as we know, this is only the second study recording such a cyclical pattern in seeds of woody species, the only precedent being the cold desert halophyte shrub Kalidium gracile (Cao et al. 2014). However, seed dormancy cycles are less clear in K. gracile than in S. serrata, since cycles in the former were described only at low temperatures (15/5, 20/5 ºC). The adverse environmental conditions for S. serrata populations may prevent polycarpy being sufficiently successful to ensure long-term population viability. In such a case, acquisition of an adaptive advantage as obvious as dormancy cycles in seeds may play a crucial role in population survival. According to our results, the following protocol can be postulated for plant production
to reinforce population programmes in the critically endangered S. serrata. Seeds collected in late July should be incubated at 15/4 ºC in darkness during August. At the end of August, seeds with emergent radicles (around 80%) should be sown in pots filled with peat and sand (3:1); seedlings will emerge in September. After 1 year in the nursery, juveniles will be ready to transplant into their natural habitat.
ACKNOWLEDGEMENTS This study was funded by the regional Government of Castilla-La Mancha through two projects (Recovery plans of threatened plant species and protection of natural habitat, Consejería de Medio Ambiente; and Seed germination and soil seed bank ecology of 15 threatened plant species in Castilla-La Mancha, Consejería de Educación y Ciencia), and the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (Ministerio de Ciencia e Innovación) in the project Recolección y conservación de semillas de 50 táxones de labiadas del cuadrante sureste ibérico con interés aromático, medicinal u ornamental. We thank the owners of Finca La Bodega for permission to undertake our research on their land.
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Kull T., Kull T., Sammul M. (2011) Reduced light availability and increased competition
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Figure legends Fig. 1. Germination of Sideritis serrata seed at constant (5 ºC) and alternating temperatures (15/4, 20/7, 25/10, 28/14 and 32/18 ºC) after 0–5 months of dry storage under a 12-h daily photoperiod (triangles) and in the dark (squares).
Fig. 2. Germination phenology of Sideritis serrata (cumulative germination percentage, mean ± SE).
Fig. 3. Germination percentages of Sideritis serrata seed incubated under a 12-h daily photoperiod (LIGHT) or in continuous darkness (DARKNESS) for 15 days at constant (5 ºC) or alternating temperatures (15/4, 20/7, 25/10, 28/14 and 32/18 ºC) after 0–20 months of soil burial. Changes in temperature throughout the experiment are also shown (mean daily maximum and minimum monthly air temperatures).
Fig. 4. Percentage of non-viable, germinated in the bag, non-dormant, dormant and deeply dormant Sideritis serrata seeds dependent on the duration and depth of burial. Vertical bars ± SE.
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Table 1. Effects of light (L), temperature (T) and seed age (A) on Sideritis serrata seed germination
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and their interactions analysed with a multi-factorial ANOVA. The table shows degrees of freedom (df), F-ratio and the associated probability (P) for main effects and interactions (α = 0.05). Residual
df: 216
Factor L T A LxT LxA TxA LxTxA
df 1 5 5 5 5 25 25
F 27.41 80.03 44.65 28.49 3.54 17.70 5.66
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P < 0.001 < 0.001 < 0.001 < 0.001 0.016 < 0.001 < 0.001
Table 2. Germination percentages (mean ± SE) of Sideritis serrata seeds incubated at 5 ºC and
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various thermoperiods in the light. After 2.5 months of burial in a pot in the greenhouse, one seed lot was exhumed on 15 October 2009 and tested for germination (control). Six pots were then transferred to the laboratory and placed at 5 ºC and the range of thermoperiods (burial temperatures) for 8 and 16 weeks before testing for germination. Numbers in a column followed by same lowercase letter are not significantly different; the same for numbers in a row sharing uppercase letters (P
Received Date : 09-Sep-2014 Revised Date : 29-Dec-2014 Accepted Date : 12-Jan-2015 Article type : Research Paper Editor : R Bekker
Article type: Research paper Running head: Annual dormancy cycles in seeds of shrub species
Annual dormancy cycles in buried seeds of shrub species: Germination ecology of Sideritis serrata (Labiatae).
Miguel A. Copete1, José M. Herranz1, Pablo Ferrandis1 & Elena Copete2
1 Higher Technical School of Agronomy and Forestry Engineering, Department of Crop Production and Agricultural Technology, University of Castilla-La Mancha, Albacete, Spain 2 Botanical Institute of the University of Castilla-La Macha, Albacete, Spain
Correspondence: Miguel A. Copete, Higher Technical School of Agronomy and Forestry Engineering, Department of Crop Production and Agricultural Technology, University of Castilla-La
Mancha,
University
Campus
s/n,
Albacete
02071,
Spain.
E-mail:
[email protected]
Keywords: Dormancy break; dormancy induction; ex-situ propagation; physiological dormancy; plant population and community dynamics; seed longevity; soil seed bank
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/plb.12306 This article is protected by copyright. All rights reserved.
ABSTRACT
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The germination ecology of Sideritis serrata was investigated in order to improve ex-situ propagation techniques and management of their habitat. Specifically, we analysed: (i) influence of temperature, light conditions and seed age on germination patterns; (ii) phenology of germination; (iii) germinative response of buried seeds to seasonal temperature changes; (iv) temperature requirements for induction and breaking of secondary dormancy; (v) ability to form persistent soil seed banks; and (vi) seed bank dynamics. Freshly matured seeds showed conditional physiological dormancy, germinating at low and cool temperatures but not at high ones (28/14 and 32/18 ºC). Germination ability increased with time of dry storage, suggesting the existence of non-deep physiological dormancy. Under unheated shade-house conditions, germination was concentrated in the first autumn. S. serrata seeds buried and exposed to natural seasonal temperature variations in the shade-house, exhibited an annual conditional dormancy/non-dormancy cycle, coming out of conditional dormancy in summer and re-entering it in winter. Non-dormant seeds were clearly induced into dormancy when stratified at 5 or 15/4 ºC for 8 weeks. Dormant seeds, stratified at 28/14 or 32/18 ºC for 16 weeks, became non-dormant if they were subsequently incubated over a temperature range from 15/4 to 32/18 ºC. S. serrata is able to form small persistent soil seed banks. The maximum seed life span in the soil was 4 years, decreasing with burial depth. This is the second report of an annual conditional dormancy/non-dormancy cycle in seeds of shrub species.
INTRODUCTION Probably the most remarkable and critical stages during the life cycle of plants are seed germination and seedling establishment. Germination is a risky transition, especially in arid and semiarid climates, between the most tolerant stage to drought (i.e. seeds) and seedlings,
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being the most drought-sensitive phase in plant development (Gutterman 1993; Hilhorst &
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Toorop 1997). Hence the importance that seed germination, often controlled by the existence of dormancy cycles, occurs at the right time of a season that gives the best chance for plant development (Gutterman 1993). In addition, accumulation of seeds in the soil can substantially contribute to population viability (Baskin & Baskin 1978; Milberg 1994). Annual plants can be highly dependent on the formation of a persistent seed bank to
avoid local extinction, whereas perennial plants are more likely to successfully face environmental conditions unsuitable for seed production during one or more growing seasons. Therefore, mechanisms enabling the maintenance of persistent seed banks, such as annual dormancy cycles, might be more common in annuals than in perennials (Schütz 1997). Indeed, the existence of dormancy cycles in buried seeds of annuals, both winter and
summer ones, exposed to seasonally changing temperature regimes is well known (Roberts & Boddrell 1985; Bouwmeester & Karssen 1993; Baskin & Baskin 2000; Copete et al. 2009), as also in some herbaceous perennials, such as Lesquerella fendleri (Hyatt et al. 1999), Penstemon palmeri (Meyer & Kitchen 1992), Primula veris and Trollius europaeus (Milberg 1994), Cnidium dubium (Geissler & Gzik 2010) and Carex loliacea (Kull et al. 2011). These annual cycles, in which physiologically based dormancy is relieved and induced during the course of a year, occur in buried seeds of some species in both temperate and tropical soil environments (Murdoch & Ellis 1992). These patterns of dormancy are of high value for survival of plant populations (Karssen et al. 1988). However, very few studies have been done on buried seeds of woody plants, to test for the existence of annual cycles in their dormancy status. We are aware of only two studies whose results indicate the presence of seed dormancy cycles in woody species: Alnus glutinosa, although the type of cycle could not be determined (Schütz 1998), and Kalidium gracile, with buried seeds exhibiting an annual conditional dormancy/ non-dormancy cycle (Cao et al. 2014). The main goal of this work
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was to analyse the germination response of buried seeds of a shrub in a Mediterranean
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semiarid habitat, Sideritis serrata Lag., to seasonal temperature changes, evaluating the influence of temperature on their dormancy status. The genus Sideritis (Labiatae) comprises about 140 species, reaching highest diversity
in the Iberian Peninsula and Macaronesia. The distribution area extends as far east as Russia, Tibet and western China (Morales 2010). S. serrata is a thermophilous calcicole shrub (60– 120 cm) growing on limestone. It is endemic to the southeast Iberian Peninsula, with a narrow distribution in Sierra de Abenuj (Tobarra, Albacete, Spain). The species is considered as critically endangered according to the IUCN Spanish Red List (Copete et al. 2004) due to its small, fragmented area of distribution, and continuous degradation of its habitat. It is also included in Annex II of Directive CEE 43/1992 (Habitat Directive of the European Union). The recovery plan for this species (D.O.C.M. 1999) recommends a reinforcement
programme for subpopulations affected by human activities through production of ex-situ plants. To optimise plant production in nurseries as well as ex-situ conservation in germplasm seed banks, it is necessary to have good understanding of germination requirements, particularly for rare and/or endemic species for which it is more difficult to obtain material (seeds; Cerabolini et al. 2004). In this work, we studied the germination ecology of S. serrata and analyse the main phases of the process. The importance of persistent soil seed banks (some viable seeds remaining in the soil
for > 1 year; sensu Thompson & Grime 1979) for conservation of threatened species has been pointed out in several studies, since it allows re-establishment and maintenance of populations after disturbances without the need for an external seed source, and because they can play an important role in years with poor seed set (Baskin & Baskin 1978, 2000; Thompson et al. 1997). However, beyond knowing the soil bank size, there are other key
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questions in the case of threatened plant species: how long can the seeds remain viable in the
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soil, and does burial depth influence seed longevity? Specifically, the goals of this study were to: (i) analyse the influence of duration of
seed dry storage on germination under different light and temperature conditions; (ii) characterise the phenology of seed germination; (iii) determine the response of buried seeds to seasonal temperature changes; (iv) assess, if present, temperature requirements for induction and breaking of secondary dormancy; (v) determine ability of S. serrata to form persistent soil seed banks; and (vi) study the seed bank dynamics.
MATERIAL AND METHODS Seed material Fully mature seeds of S. serrata were collected from vigorous and healthy plants growing in Sierra de Abenuj (Tobarra, SE Spain; UTM coordinates: 30SXH1074; 800 m a.s.l.), the unique location known for the species. This area has a typical continental semiarid climate, with mean annual precipitation of 320 mm, mean annual temperature of 14 ºC and absolute minimum annual temperature of -6.7 ºC. Seed collection was carried out in late July 2006, 2007, 2008 and 2009.
Germination tests A 100-seed lot was assigned to each temperature condition test, distributed into four 25 seed replicates. Each replicate was incubated in a 9-cm diameter sealed (with Parafilm) Petri dish on a double layer of filter paper saturated with ca. 4 ml distilled water and placed in germination chambers (Ibercex, Madrid, Spain; F-4 model, ± 0.1ºC, cool white fluorescent light of ~25 μmol·m-2·s-1) at constant 5 ºC or a 12/12 h day/night temperature regimes of 15/4, 20/7, 25/10, 28/14 and 32/18 ºC. The fluctuating temperatures used in tests simulated
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approximate mean maximum and mean minimum monthly temperatures in the area of
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Tobarra during the growing season. Thus, 15/4 ºC corresponds to November and March, 20/7 ºC to October and April, 25/10 ºC to September and May, 28/14 ºC to August and June, and 32/18 ºC to July. The 5 ºC treatment is close to the mean temperature recorded during winter months (December, January and February), being broadly used in experiments for dormancy induction by low temperatures (Baskin & Baskin 1989, 2000). The 12-h photoperiod with alternating temperature treatments coincided with the daily high temperature period. In addition, the same temperature seed germination tests were carried out in darkness by wrapping the Petri dishes in a double layer of aluminium foil. Hence, we had simultaneous ‘light’ (photoperiod) and ‘dark’ treatments over the complete set of temperature conditions. Tests of dormancy cycles and temperature requirements for dormancy breaking and
induction lasted 15 days, as recommended for this type of study (Baskin & Baskin 1998). However, to analyse the influence of duration of laboratory dry storage on germination, tests were extended to 30 days, since germination had not slowed in some trials after 15 days. Protrusion of the radicle was the criterion for successful germination. In treatments with a photoperiod, seeds were checked for germination every 2–3 days. Seeds incubated in the dark were checked only at the end of the test. Seeds that did not germinate were checked for viability on the basis of embryo appearance, paying special attention to seed colour and turgidity. Percentage germination was computed from viable seed numbers.
Shade-house conditions The metal frame shade-house used in this study was on the university campus in Albacete (UTM coordinates, 30SWJ9713, 690 m a.s.l.; 40 km from Sierra de Abenuj). It was neither heated in winter nor air-conditioned in summer, and was shaded continuously throughout the
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study with a polyethylene cloth (12 threads cm-1, 50% light interception). Temperatures were
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recorded throughout the study at a meteorological station within the shade-house. To simulate soil moisture regimes in the field, pots and trays placed in the shade-
house were watered to field capacity once per month during summer (1 July–30 September) and twice per month during the rest of the year, with the exception of the coldest period (December–January) when the substrate was frozen.
Influence of seed age on germinability of dry-stored seeds After drying and cleaning, seeds of S. serrata were stored in paper bags under laboratory conditions (18–20 ºC; 50–60% air humidity) until used in germination tests. Seed lots were tested for germination from 1 August 2006 (age = 0 months) to January 2007 (age = 5 months). Intermediate tests were started on the first of September, October, November and December 2006 (seed age = 1, 2, 3 and 4 months, respectively); tests lasted 30 days. Temperature and illumination conditions were those described in the ‘Germination tests’ section.
Germination phenology On 1 August 2007, 5 days after collection, 500 seeds were divided into five 100 seed replicates. Each replicate was sown 3-mm deep into a 40 x 25 x 7 cm plastic seedling tray with drainage holes. Substrate consisted of a sterilised mixture of peat and sand (2:1 v/v). To facilitate emergent seedling counts, trays were placed on tables in the shade-house. Watering frequency was as described in the ‘Shade-house conditions’ section. Emerged seedlings were counted and removed approximately every 15 days until summer 2011.
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Response of buried seeds to seasonal temperature changes
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Seeds collected on 26 July 2007 were stored for 3 months at ambient room temperature. Then, 20 lots of 1250 S. serrata seeds each were homogeneously mixed with a double volume of 0.5 mm sieved sand. This mixture was placed in nylon bags of 0.1-mm mesh, which were buried on 1 November 2007 to a depth of 7 cm in soil (river sand) in pots (20-cm diameter and 25-cm depth) with drainage holes. We used four pots with five bags each. Pots were placed in the previously described shade-house. Watering regimes were as described above. Germination tests were conducted in light and dark on seeds exhumed on the first day of each month from December 2007 to July 2009, and also on 1 November 2007 for seeds that were not buried. Seed exhumation was always carried out after sunset. Seeds were recovered from bags by pouring the mixture containing the sand through a 0.5-mm sieve. To prevent light exposure of seeds assigned to germination tests in darkness, both sieving and handling prior to tests were carried out under a green safe light (Baskin & Baskin 1998).
Temperature requirements for induction and breaking of secondary dormancy This experiment was designed on the basis of results obtained from the analysis of seed dormancy cycles started in November 2007. Seeds of S. serrata were collected on 25 July 2009 and stored for 6 days at ambient room temperature. Twenty-six nylon bags, each containing 650 seeds, were prepared as described above. In early August 2009, two bags per pot (13 pots) were buried at 7-cm depth. Pots were placed within the shade-house and submitted to the same watering regime described above until 15 October 2009 (six pots) and 15 March 2010 (seven pots). On 15 October 2009, one bag was exhumed, and seeds tested for germination in light
at 5, 15/4, 20/7, 25/10, 28/14 and 32/18 ºC to determine their dormancy stage. Subsequently, each pot containing two bags was stratified at each of the following temperatures: 5, 15/4,
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20/7, 25/10, 28/14 and 32/18 ºC. After 8 and 16 weeks, respectively, bags from all treatments
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were exhumed and seeds tested for germination in light under the six temperature regimes. On 15 March 2010, we repeated the above experiment to test temperature required to overcome secondary dormancy.
Analysis of the soil seed bank Seed banks of S. serrata and other main species of the community were assessed by analysing the seed content of soil samples collected from one of the core S. serrata subpopulations. Soil samples were collected on 10 June 2006, after autumn–winter–spring germination and before the start of seed dispersal from fruits produced that year. This sampling date was determined on the basis of Thompson & Grime´s (1979) concept of a persistent seed bank, in order to quantify the persistent fraction of the seed bank overlapping two consecutive phenological cycles. Twenty plots of 10 cm x 10 cm were placed along two linear transects of the
following design: (i) ten plots per transect, (ii) 2 m between contiguous plots within each transect, and (iii) 50 m between transects. In each plot, soil was collected to a depth of 5 cm. Soil samples were carried to the laboratory in plastic bags, where they were dried completely and homogenised. Seed content was assessed by physical separation (Ferrandis et al. 1999). The soil was washed through a 0.5-mm mesh sieve. Seeds were separated and identified under a binocular microscope. The criterion for identifying a seed as viable was the healthy appearance of the embryo, paying particular attention to turgidity and colour.
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Effects of depth and duration of burial on dormancy and viability of S. serrata seeds in
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the natural habitat Seeds collected on 25 July 2008 were stored for 2 months at ambient room temperature, then 144 lots of 200 S. serrata seeds each were mixed with sand and put inside nylon bags, as described previously. Prior to burial experiments, seed viability was analysed. Four 25-seed replicates were incubated in light at 20/7 ºC (the most favourable regime for germination) for 30 days. Seeds that did not germinate by the end of the test were moistened with GA3 solution (1000 ppm; Gómez-Campo 1985) for an additional 30 days. Finally, non-germinated seeds were checked for viability on the basis of embryo appearance, paying special attention to colour and turgidity. At the beginning of burial experiments, seeds of S. serrata were classified into four categories: (1) non-dormant seeds, i.e. germinated during germinability test, (2) dormant seeds, i.e. germinated after GA3 treatment; (3) deeply dormant seeds, i.e. a viable embryo but no germination after GA3 treatment; and (4) non-viable seeds. In October 2008, six 30 cm x 30 cm plots free of rocky outcrops were randomly
selected in Sierra de Abenuj (UTM: 30SXH0975; 800 m a.s.l.). Each plot was divided into three 30 cm x 10 cm subplots. Each subplot had a different seed burial depth (2, 7 and 12 cm). In each subplot, eight seed lots were buried at the selected depth. Every seed lot in a bag was closed with a wire that protruded about 1 cm above the soil surface to facilitate exhumation without interfering with the arrangement of remaining bags. For 2 years and every 3 months, one seed lot per plot and depth was exhumed, starting in January 2009. In the laboratory, seeds that germinated (radicle emerged) in bags were counted. Other seeds with a healthy appearance were tested for viability. Therefore, for every exhumation, the four seed status categories described above were determined, in addition to germinated seeds in the bag.
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Data analysis
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Germination percentages of dry-stored seeds were compared among treatments using multifactorial ANOVA, considering three factors: (1) temperature (six levels); (2) light (two levels); and (3) seed age (six levels). For comparison of induction and breaking of secondary dormancy, a multi-factorial ANOVA was performed with factors: (1) temperature during seed burial (six levels); (2) temperature for testing germination response (six levels); and (3) length of stratification (three levels). Treatments responsible for significant main effects were detected with a multiple comparisons Tukey test; significant interactions were explored as contrasting confidence intervals. Prior to analyses, normality (Cochran test) and homoscedasticity (David test) of data were checked. Values of final cumulative germination percentage were square-root arcsine transformed. For each temperature x light treatment on dry-stored seeds, changing trends in
germination with seed age were evaluated with regression analysis. Data were log transformed, and the model selected for each treatment was that with the highest R2 coefficient. The minimum improvement for changing the model was established at 10%. Seed decay rate was calculated using the exponential equation: n2 = n1 e -λ∆t
(Eq. 1)
where n1 and n2 are number of viable seeds remaining at t1 and t2, respectively, λ is decay rate and ∆t is time interval between t1 and t2. The exponential decay curve is often used to model seed bank decay (Auld et al. 2000; Holmes & Newton 2004). We used number of viable seeds at the start of the burial experiment (t1). Seed half-life (i.e. time elapsed for 50% seed decay 50% in soil) was calculated by substituting the decay rate into the exponential equation and solving for n2 = 0.5 n1. In contrast, we considered as maximum longevity the period at which the percentage viable seeds decreased to 5% (n2 = 5), since in a negative exponential function, approaching n2 = 0 would produce values of ∆t tending to infinity.
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Kull T., Kull T., Sammul M. (2011) Reduced light availability and increased competition
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diminish the reproductive success of wet forest sedge Carex loliacea L. Plant Species Biology, 26, 84–92.
Lonsdale W.M., Harley K.L.S., Gillett J.D. (1988) Seed bank dynamics in Mimosa pigra, an invasive tropical shrub. Journal of Applied Ecology, 25, 963–976.
Martínez-Duro E., Ferrandis P., Herranz J.M., Copete M.A. (2010) Do seed harvesting ants threaten the viability of a critically endangered non-myrmecochorous perennial plant population? A complex interaction. Population Ecology, 52, 397–405.
Martínez-Sánchez J.J., Segura F., Aguado M., Franco J.A., Vicente M.J. (2011) Life history and demographic features of Astragalus nitidiflorus, a critically endangered species. Flora, 206, 423–432.
Mennan H. (2003) The effects of depth and duration of burial on seasonal germination, dormancy and viability of Galium aparine and Bifora radians seeds. Journal of Agronomy and Crop Science, 189, 304–309.
Meyer S.E., Kitchen S.G. (1992) Cyclic seed dormancy in the short-lived perennial Penstemon palmeri. Journal of Ecology, 80, 115–122.
Milberg P. (1994) Germination ecology of the polycarpic grassland perennials Primula veris and Trollius europaeus. Ecography, 17, 3–8.
Milberg P., Andersson L. (1997) Seasonal variation in dormancy and light sensitivity in buried seeds of eight annual weed species. Canadian Journal of Botany, 75, 1998–2004.
Mira S., González-Benito M.E., Ibars A.M., Estrelles E. (2011) Dormancy release and seed ageing in the endangered species Silene diclinis. Biodiversity and Conservation, 20, 345358.
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Table 2. Germination percentages (mean ± SE) of Sideritis serrata seeds incubated at 5 ºC and
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various thermoperiods in the light. After 2.5 months of burial in a pot in the greenhouse, one seed lot was exhumed on 15 October 2009 and tested for germination (control). Six pots were then transferred to the laboratory and placed at 5 ºC and the range of thermoperiods (burial temperatures) for 8 and 16 weeks before testing for germination. Numbers in a column followed by same lowercase letter are not significantly different; the same for numbers in a row sharing uppercase letters (P 60%) after 4 months of dry storage at room temperature, it can be concluded that S. serrata seeds have non-deep physiological dormancy (Baskin & Baskin 1998). The broad oscillations in germination at 25/10 ºC have also been reported for other species with non-
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deep physiological dormancy (e.g. Diplotaxis erucoides and D. virgata, Pérez-García et al.
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1995; Sisymbrium runcinatum, Herranz et al. 2003a). In the phenological study (Fig. 2), most seedlings (47%) emerged during the first
autumn, which was related to the ability of seeds dry-stored for 2–3 months to germinate (4060%) when incubated at 15/4 and 20/7 ºC in the dark (Fig. 1). We obtained similar percentage germination in November 2007 (Fig. 3). A small fraction of these seeds germinated in spring, showing that S. serrata has similar germination phenology to facultative winter annuals (Baskin & Baskin 1983; Copete et al. 2009). The seedling emergence during four consecutive phenological cycles after seed
sowing (2007–2011; Fig. 2) demonstrated the ability of this species to form persistent soil seed banks. Indeed, this trait was corroborated in the study of the soil seed bank in the natural habitat (Table 4), where around 100 S. serrata seed m-2 germinated in the upper 5 cm. According to Harper (1977), the soil seed bank can play a crucial role in population dynamics when its density is higher than the number of adult individuals established aboveground, which broadly occurred in our target species (0.0275 plants m-2 in the study area; Copete et al. 2004). However, despite adaptations promoting formation of seed banks in S. serrata, especially dormancy cycles, seeds were not highly abundant in the soil, probably due to their large size (2–3 mm); densities of individual species in the soil seed bank are negatively related to seed size (Fenner & Thompson 2005). The ability of this threatened shrub to form a persistent soil seed bank is certainly an adaptive advantage, since persistent seed banks buffer populations from environmental stochasticity (Adams et al. 2005). Although not so crucial as for threatened annuals such as Sisymbrium cavanillesianum (Herranz et al. 2003a) and biennials such as Coincya rupestris subsp. rupestris (Herranz et al. 2003b), whose chances of survival rest largely on seed reserves in the soil, recent population viability analyses have also stressed the important role of soil seed banks for long-term persistence of populations in
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perennial life-form species, e.g. Helenium virginicum, a perennial herb (Adams et al. 2005),
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and Helianthemum polygonoides, a critically endangered perennial shrub in SE Spain (Martínez-Duro et al. 2010). According to Thompson et al. (2003), there is not a close relationship between seed
persistence in the soil and seed dormancy. However, many species that form persistent seed banks exhibit annual changes in seed dormancy (Baskin & Baskin 1985; Milberg & Andersson 1997). S. serrata seeds also showed annual changes in dormancy (Fig. 3), exhibiting an annual conditional dormancy/non-dormancy cycle, with dormancy loss occurring in summer (non-dormant seeds in late summer–early autumn) and conditional dormancy induction in late autumn and winter (Fig. 3), although there are months (i.e. February) with high values of germination intercalated in series of low values (Fig. 3), as occurs in the facultative winter annual Ziziphora aragonensis (Copete et al. 2009). This hightemperature requirement for loss of seed dormancy and induction into conditional dormancy mediated by low temperatures has also been found in seeds of facultative winter annuals, e.g. Thlaspi arvense (Baskin & Baskin 1989), Veronica arvensis (Baskin & Baskin 1983), Iberis crenata and Ziziphora aragonensis (Copete et al. 2009). As a result, S. serrata seeds mostly germinated in autumn, increasing the chances of seedling survival due to acquisition of a degree of maturity before winter frosts, and even more determinant, before summer drought. In arid and semiarid Mediterranean regions, adaptive processes tend to select as
optimum germination temperatures those that are most likely to occur during the rainfall period (Pérez-García et al. 1995). Specifically, in Sierra de Abenuj, although rainfall in autumn (28%) is similar to that in spring (31%), most seedlings emerged in autumn, increasing the length of time for plant development and thus survival probability during the first summer.
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The trial with buried seeds at different depths in the natural habitat also confirmed the
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existence of dormancy cycles (Fig. 4). It was clear that dormancy changes in seeds were mitigated by burial depth, which seems logical when considering the damping effect on temperature changes associated with soil depth (Tsuyuzaki 2006). We also observed that, even at times of year favourable for seedling establishment, a small percentage of seeds remained dormant. It is not advantageous for a plant to risk all seeds in one germination event because adaptive mechanisms cannot accurately predict favourability of the coming growth season, and because density-dependent mortality may affect seedling establishment (Jurado & Moles 2003; Martínez-Sánchez et al. 2011). For the upper soil layer, maximum longevity of S. serrata seeds was almost 4 years,
while in the deepest soil it was 2 years (Table 5). Nevertheless, these results corroborate the persistent nature of the soil seed bank in S. serrata. This negative relation between burial depth and seed survival was also found in other studies (Lonsdale et al. 1988). In a few cases, longevity is not affected by depth of burial (Egley & Chandler 1983); however, most species present the opposite relationship: Artemisia tridentata (Wijayratne & Pyke 2012), Galium aparine and Bifora radians (Mennan 2003), Rumex obtusifolius, Oenothera biennis and Polygonum longisetum (Tsuyuzaki 2006) and Sisymbrium cavanillesianum (Herranz et al. 2003a). In all these species, seed longevity increased with depth, probably due to the wider humidity and temperature ranges recorded in the upper soil layers, which may be lethal to seeds (Holmes & Newton 2004). One possible explanation for our unusual result could be the scarce rainfall in the study area, so the upper soil layer loses humidity faster and remains dry for most of the year. Hence, humidity being one of the main parameters involved in seed bank decay (Holmes & Newton 2004), S. serrata seeds in the first centimetres of the soil can remain viable for longer.
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The main finding of the present study is the confirmation that seed dormancy cycles
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are not restricted to annual species. As far as we know, this is only the second study recording such a cyclical pattern in seeds of woody species, the only precedent being the cold desert halophyte shrub Kalidium gracile (Cao et al. 2014). However, seed dormancy cycles are less clear in K. gracile than in S. serrata, since cycles in the former were described only at low temperatures (15/5, 20/5 ºC). The adverse environmental conditions for S. serrata populations may prevent polycarpy being sufficiently successful to ensure long-term population viability. In such a case, acquisition of an adaptive advantage as obvious as dormancy cycles in seeds may play a crucial role in population survival. According to our results, the following protocol can be postulated for plant production
to reinforce population programmes in the critically endangered S. serrata. Seeds collected in late July should be incubated at 15/4 ºC in darkness during August. At the end of August, seeds with emergent radicles (around 80%) should be sown in pots filled with peat and sand (3:1); seedlings will emerge in September. After 1 year in the nursery, juveniles will be ready to transplant into their natural habitat.
ACKNOWLEDGEMENTS This study was funded by the regional Government of Castilla-La Mancha through two projects (Recovery plans of threatened plant species and protection of natural habitat, Consejería de Medio Ambiente; and Seed germination and soil seed bank ecology of 15 threatened plant species in Castilla-La Mancha, Consejería de Educación y Ciencia), and the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (Ministerio de Ciencia e Innovación) in the project Recolección y conservación de semillas de 50 táxones de labiadas del cuadrante sureste ibérico con interés aromático, medicinal u ornamental. We thank the owners of Finca La Bodega for permission to undertake our research on their land.
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Figure legends Fig. 1. Germination of Sideritis serrata seed at constant (5 ºC) and alternating temperatures (15/4, 20/7, 25/10, 28/14 and 32/18 ºC) after 0–5 months of dry storage under a 12-h daily photoperiod (triangles) and in the dark (squares).
Fig. 2. Germination phenology of Sideritis serrata (cumulative germination percentage, mean ± SE).
Fig. 3. Germination percentages of Sideritis serrata seed incubated under a 12-h daily photoperiod (LIGHT) or in continuous darkness (DARKNESS) for 15 days at constant (5 ºC) or alternating temperatures (15/4, 20/7, 25/10, 28/14 and 32/18 ºC) after 0–20 months of soil burial. Changes in temperature throughout the experiment are also shown (mean daily maximum and minimum monthly air temperatures).
Fig. 4. Percentage of non-viable, germinated in the bag, non-dormant, dormant and deeply dormant Sideritis serrata seeds dependent on the duration and depth of burial. Vertical bars ± SE.
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Table 1. Effects of light (L), temperature (T) and seed age (A) on Sideritis serrata seed germination
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and their interactions analysed with a multi-factorial ANOVA. The table shows degrees of freedom (df), F-ratio and the associated probability (P) for main effects and interactions (α = 0.05). Residual
df: 216
Factor L T A LxT LxA TxA LxTxA
df 1 5 5 5 5 25 25
F 27.41 80.03 44.65 28.49 3.54 17.70 5.66
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P < 0.001 < 0.001 < 0.001 < 0.001 0.016 < 0.001 < 0.001
Table 2. Germination percentages (mean ± SE) of Sideritis serrata seeds incubated at 5 ºC and
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various thermoperiods in the light. After 2.5 months of burial in a pot in the greenhouse, one seed lot was exhumed on 15 October 2009 and tested for germination (control). Six pots were then transferred to the laboratory and placed at 5 ºC and the range of thermoperiods (burial temperatures) for 8 and 16 weeks before testing for germination. Numbers in a column followed by same lowercase letter are not significantly different; the same for numbers in a row sharing uppercase letters (P
