Seed dormancy and germination in three Crocus ser. Verni species (Iridaceae): implications for evolution of dormancy within the genus.

Plant Biology ISSN 1435-8603

RESEARCH PAPER

Seed dormancy and germination in three Crocus ser. Verni species (Iridaceae): implications for evolution of dormancy within the genus A. Carta1, R. Probert2, M. Moretti1, L. Peruzzi1 & G. Bedini1 1 Department of Biology, Unit of Botany, University of Pisa, Pisa, Italy 2 Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex, UK

Keywords Embryo growth; epicotyl dormancy; germination; seed dormancy; shoot emergence. Correspondence A. Carta, Department of Biology, Unit of Botany, University of Pisa, via Luca Ghini 13, I-56125 Pisa, Italy. E-mail [email protected] Editor R. Bekker Received: 7 September 2013; Accepted: 20 January 2014 doi:10.1111/plb.12168

ABSTRACT The aim of this work was to examine whether seed ecophysiological traits in three closely related Crocus species were associated with ecological niche differentiation and species divergence. Seeds of the temperate tetraploid cytotype of Crocus neapolitanus, the sub-Mediterranean C. etruscus and the Mediterranean C. ilvensis were placed either on agar in the laboratory under different periods of simulated seasonal conditions or in nylon mesh bags buried outdoors to examine embryo growth, radicle and shoot emergence. In agreement with the phenology observed outdoors, in the laboratory embryos required a cool temperature (ca. 10 °C) to grow to full size (embryo length:seed length, E:S ratio ca. 0.75) but only after seeds received a warm stratification; radicle emergence then followed immediately (November). Shoot emergence is a temporally separated phase (March) that was promoted by cold stratification in C. neapolitanus while in the other two species this time lag was attributed to a slow continuous developmental process. These species have similar embryo growth and radicle phenology but differ in their degree of epicotyl dormancy, which is related to the length of local winter. Conclusions from laboratory experiments that only consider root emergence could be misleading; evaluating the phenology of both root and shoot emergence should be considered in order to demonstrate ecologically meaningful differences in germination behaviour and to develop effective propagation protocols. Although these taxa resulted from recent speciation processes, the outcomes suggest an early onset of adaptation to local ecological factors and that phylogeny may represent a significant constraint in the evolution and expression of seed traits in Crocus.

INTRODUCTION The recruitment of new individuals from seeds is a critical phase in the life cycle of spermatophytes, affecting the plant’s chances of becoming established and reaching the reproductive phase (Harper 1977; Westoby 1981; Baskin & Baskin 1998; Donohue et al. 2010). The most favourable period for seedling establishment can vary according to the geographic distribution, climate conditions, habitat preference and life cycle of the species (Nikolaeva 1999; Mattana et al. 2012; Vranckx & Vandelook 2012). This variation is often reflected in different seed behaviour, including seed dormancy, which helps to prevent germination at times when environmental conditions are unfavourable for subsequent seedling establishment (Vleeshouwers et al. 1995; Baskin & Baskin 1998; Finch-Savage & Leubner-Metzger 2006). Thus, seed dormancy is considered to be one of the main factors determining the adaptive value of germination (Donohue et al. 2010; Fernandez-Pascual et al. 2013). Many angiosperm taxa have an underdeveloped embryo at the time of dispersal (Nikolaeva 1977), meaning that the embryo has to grow within the seed before germination can occur. These seeds can be defined as morphologically dormant,

or morpho-physiologically dormant if an additional physiological block to germination is present (Nikolaeva 1977; Baskin & Baskin 2004). The variation in embryo size relative to the amount of nutritive tissue is often evaluated in a phylogenetic framework (Forbis et al. 2002; Nikolaeva 2004; Finch-Savage & Leubner-Metzger 2006; Verd u 2006; Vandelook et al. 2012). Within monocots, genera that include species with underdeveloped embryos are especially common in Liliales and in certain Asparagales families (e.g. Amaryllidaceae and Iridaceae). Although dormancy and embryo growth have been studied in a number of monocot taxa (see e.g. Baskin & Baskin 1998; Kondo et al. 2004; Vandelook & Van Assche 2008; Mondoni et al. 2012), germination studies of monocots in the circumMediterranean region are scarce, particularly within Iridaceae, although a number of studies within Amaryllidaceae have been reported (see e.g. Copete et al. 2011; Herranz et al. 2013a,b for Narcissus). As seed morphological traits such as embryo size can be considered fairly conservative (Corner 1976), the phylogenetic relatedness should not be neglected when comparing seed germination of different species (Nikolaeva 1999). The present work aimed to examine whether the morphological differences between three phylogenetically closely related species are associated with differences in ecophysiological traits, such

Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Seed dormancy and germination in three Crocus species

Carta, Probert, Moretti, Peruzzi & Bedini

as dormancy and germination characteristics, reflecting the distinct habitats and bioclimatic areas where they occur. The genus Crocus L. (Iridaceae) consists of about 100 species occurring from Western Europe to western China (Harpke et al. 2013). Many taxa are threatened with extinction in the wild, while others are known for their ornamental, pharmaceutical or culinary value (Mathew 1982). The majority of Crocus species fall within the Mediterranean region or Asia Minor, revealing the importance of these geographic areas in the evolution of Crocus and in the development of germination studies of comparative value. The taxa studied here originate from the Italian peninsula, recognised as an important area of speciation for the genus, and belong to the series Verni B.Mathew, a relatively late branching lineage within the genus, constituting about eight species (Mathew 1982; Peruzzi et al. 2013). The studied species, despite showing strong phylogenetic affinities (Peruzzi & Carta 2011; Harpke et al. 2013), are allopatric and, although long-lived spring geophytes, have fairly distinct habitat preferences (Table 1). The tetraploid cytotype of Crocus neapolitanus (Ker Gawl.) Loisel. is distributed in northern Tuscany and surrounding areas (500–2000 m a.s.l.) and occurs in temperate meadows and woodland margins (Peruzzi et al. 2013); C. etruscus Parl. is an endemic of southern-central Tuscany (300–600 m a.s.l.) and is typical of sub-Mediterranean deciduous woodland (Carta et al. 2010); C. ilvensis Peruzzi & Carta is a species narrow endemic to Elba Island (North Tyrrhenian Sea, 400–1000 m a.s.l.) that grows in Mediterranean deciduous forest and xeric annual grasslands (Foggi et al. 2014). Very little information is available regarding seed germination and dormancy in the genus Crocus (Fu et al. 2013). According to Martin (1946) and APG III (2009), seeds of Iridaceae have linear embryos that may, or may not, be as long as the endosperm. Crocus is on the whole a genus well adapted to dry summers, and in their natural habitat the species studied here are subjected to a marked dry season. Thus, we predicted the presence of adaptations to avoid germination during summer (Copete et al. 2011; Herranz et al. 2013b), which would expose seedlings to unfavourable conditions for survival. We also sought to investigate whether the timing of shoot formation is controlled by epicotyl dormancy, as reported by Fu et al.

(2013) for the subalpine Crocus alatavicus Regel & Sem., or whether it is a continuous developmental process (Vandelook & Van Assche 2008). In this work we investigated (i) the extent of embryo growth before radicle emergence can occur, as well as the phenology of radicle emergence; (ii) the germination responses of seeds buried in soil and exhumed periodically; (iii) the influence of warm stratification, light/darkness and seed storage on radicle emergence; and (iv) the influence of cold stratification on shoot emergence. These factors are essential to understand whether seed traits in these species have evolved according to ecological origin and to what extent phylogeny may represent a constraint in the evolution and expression of seed dormancy within Crocus series Verni. MATERIAL AND METHODS Seed material The studied species are early spring-flowering geophytes that develop leaves and flowers from a perennial subterraneous corm. The inferior ovary is belowground at anthesis, whereas fruits typically ripen at soil level (Mathew 1982). Seed capsules (fruits) were collected from about 250 healthy plants for each species at the time of natural dispersal (Hay & Smith 2003) in May–June 2012, when the fruits change from green to yellow. Fruits of Crocus neapolitanus were collected from plants growing in a temperate meadow in Santallago, Monte Pisano (northern Tuscany), 730 m a.s.l.; fruits of C. etruscus were collected from plants growing in a deciduous woodland in Val Canina, Sassetta (central Tuscany), 460 m a.s.l.; fruits of C. ilvensis were collected in a deciduous forest in Monte Perone, Elba Island (Tuscan archipelago), 540 m a.s.l. Ripe fruits were spread out in the laboratory to allow them to open and the seeds to fall out. To minimise the effects of after-ripening and viability loss, the experiments were started within 3 weeks of fruit collection. Climate data of the collection sites (Table 2) were acquired in a GIS software (ArcMap version 9.2, ESRI, Redland, CA, USA) from available data at a spatial resolution of 1 km2, downloaded from the WorldClim website (http://www.worldclim.org), as reported in Hijmans et al.

Table 1. Average seed mass (mean SE of five replicates of 20 seeds each), seed length, seed width (mean SE of 30 seeds), chromosome number and habitat preference of the studied species. species name

mass (mg)

length (mm)

width (mm)

2n

habitat

Crocus neapolitanus (Ker Gawl.) Loisel. Crocus etruscus Parl. Crocus ilvensis Peruzzi & Carta

560.25 2.15 435.69 1.07 394.64 2.12

2.41 0.04 2.24 0.03 2.09 0.03

1.97 0.04 1.73 0.03 1.62 0.02

16 8 8

Temperate meadows and woodland margins Sub-Mediterranean deciduous woodlands Mediterranean deciduous woodland and xeric annual grasslands

Table 2. Mean daily temperature (°C, representative of 1950–2000) of the collection sites. site

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Monte Perone (Elba Island) Santallago (north Tuscany) Sassetta (central Tuscany)

7.9 2.6 6.1

8.1 3.2 6.6

9 5.5 8.5

12 8.7 11.4

15.6 12.6 15.2

19.1 16.5 19.3

22.6 19.8 22.5

22.6 19.7 22.5

20 16.5 19.4

15.9 11.9 14.9

12 7.2 10.5

9 3.9 7.2

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(2005). The seed mass of each species was determined by weighing five replicates of 20 seeds each (seeds equilibrated at ca. 20 °C and 50% RH). For each species, we also measured the maximum length and width of 30 seeds using digital calipers. Phenology of embryo growth, radicle and seedling emergence outdoors The aim of these experiments was to describe the phenology of embryo growth, radicle and seedling emergence using seeds kept under near-natural temperature and rainfall conditions (Pisa Botanical Garden). Seeds were sown in pots (40 9 20 9 20 cm) filled with peat and sand (2:1 v/v). Pots were placed outdoors in Pisa BG, where air temperature was recorded throughout the study (Interdepartmental Centre of Bioclimatology). Pots were watered to field capacity four times each month throughout the year to maintain field capacity, with the exception of July and August, when they were watered only twice a month to simulate the summer drought common in the Mediterranean area and to reduce seed decay (Copete et al. 2011). At the beginning of the experiments (21 June 2012 for both outdoor and laboratory experiments), 20 seeds of each species were dissected and embryo length and seed length measured under a binocular microscope equipped with a micrometer. The ratio of embryo length to seed length (E:S) was calculated. The E:S ratio was not determined for seeds that had germinated; instead, for germinated seeds the critical E:S ratio was applied. The critical E:S ratio was calculated as the average E:S ratio of 20 seeds with a split seed coat, but no radicle protrusion. To study the phenology of embryo growth and radicle emergence for each species, 15 groups of 50 seeds each were mixed with fine-grained sand. Each group was placed in a fine-mesh polyester bag, labelled and buried 5-cm deep in the pots. To study the phenology of shoot emergence, for each species one pot was filled with the growing medium and 100 seeds were sown equidistant from each other and buried at depth of 0.5 cm. Observations of embryo growth and root and cotyledon emergence were made every 30 days during summer and then at 7-day intervals (14 days in the case of embryo growth) until the end of the experiments. Phenology of embryo growth and radicle emergence in the laboratory Experiments were conducted in controlled temperature (1 °C) and light (40 lmolm 2s 1) conditions using a 1-h daily thermoperiod (for alternating temperatures) and photoperiod (= light hereafter). Light was provided during the warm phase for the alternating temperature regimes with white fluorescent tubes. Seeds were also tested in continuous darkness in Petri dishes wrapped in aluminium foil and kept in darkness for the duration of the experiment. All experiments were carried out in 90-mm diameter Petri dishes containing 1% distilled water agar, and consisted of three samples of 20 seeds for each species. A ‘move-along’ experiment (sensu Baskin & Baskin 2003) was conducted to check whether embryo growth and germination phenology under simulated laboratory conditions were

similar to those in the field. Thus, seeds were exposed to seasonal temperature changes simulating the natural summer ? autumn ? winter sequence in the collection areas. Using climate data obtained as described above, summer conditions were defined as 90 days at 25/15 °C (20 °C in the for constant temperature regime); autumn conditions as 60 days at 20/10 °C (or 15 °C); winter conditions as 90 days at 15/5 °C (or 10 °C). Diurnal alternating temperatures simulated conditions at the forest margins or in meadow habitats, while constant temperatures were also used because of the forest cover and insulating effect of leaf litter on diurnal temperature variation (Ellenberg 1988; Mondoni et al. 2012). The E:S ratio and root emergence observations were conducted as described above. Dormancy breaking in buried seeds This experiment was set up to study dormancy breaking under near natural summer conditions. On 21 June three groups of 60 seeds of each species were mixed with fine sand and placed in a fine-mesh polyester bag, buried 5-cm deep in a pot and arranged and watered as reported in the above outdoor experiments. One bag for each species was exhumed after 30 days (21 July), 60 days (21 August) and one at the end of summer (90 days, 21 September). Seeds were incubated in the light under simulated autumn conditions (20/10 °C) for 60 days and then moved to winter conditions (15/5 °C). The number of samples and observations were as described above. Effect of summer, autumn and winter condition on radicle emergence To examine the effect of summer conditions on radicle emergence, seeds were held at 25/15 °C in the light (or 20 °C) for 0, 30, 60 and 90 days. After each period, seeds were moved to autumn (20/10 °C or 15 °C) and then to winter conditions (15/5 °C or 10 °C) in the light. The effect of autumn was tested by moving seeds that had experienced summer conditions (90 days at 25/15 °C or 20 °C) to winter conditions, hence skipping the autumn. The effect of winter was tested by maintaining seeds that had experienced summer conditions (90 days at 25/15 °C or 20 °C) under autumn conditions. The number of seeds and samples for each test and observation were as described above. Effect of high summer temperatures on radicle emergence To examine the effect of high summer temperatures on radicle emergence, seeds were held at 30/20 °C in the light for 90 days. After this period, seeds were moved to autumn (20/ 10 °C) and then to winter conditions (15/5 °C) in the light. The numbers of seeds, samples and observation were as described above. Effect of winter length on shoot formation This experiment was set up to determine the presence of physiological dormancy in the epicotyl. For each species, three samples of ten seeds with an emerged radicle were held at 15/5 °C for 0, 30 and 90 days, after which seeds were moved to spring conditions (20/10 °C).


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Induction of secondary dormancy

RESULTS

We studied whether low temperatures that can occur in late autumn will induce secondary dormancy in seeds in which dormancy had been broken and embryos had begun to grow but were not fully elongated (Copete et al. 2011). We tested two different sequences of stratification. First, three samples of 20 seeds for each species were placed at 25/15 °C for 90 days, then transferred to 5 °C for 60 days, and finally placed at 15/ 5 °C to test radicle emergence. Second, three other samples of 20 seeds for each species were placed at 25/15 °C for 90 days, moved to 20/10 °C, then transferred to 5 °C for 60 days, and finally placed at 15/5 °C.

Phenology of embryo growth, radicle and seedling emergence outdoors

Influence of seed storage on radicle emergence In this study, we analysed the effect of seed storage following the international conditions for seed conservation (FAO/IPGRI 1994) on seed survival. Hence, seeds were dried to low moisture content by keeping them in a controlled environment room (15 °C, 15% RH) until they had equilibrated; equilibrium was checked with a water activity station (Rotronic). Dried seeds were then hermetically sealed in a single laminated aluminium foil bag, labelled and stored at 20 °C for 6 months before testing for germination. Controls

Embryos of all three Crocus species were underdeveloped at time of dispersal (E:S ratio: Crocus neapolitanus = 0.44, C. etruscus and C. ilvensis = 0.48); they showed a slow, but significant (P < 0.001) linear increase in length during the summer season and the first part of autumn, reaching an E:S ratio ca. 0.56. Embryos grew faster assuming a logistic pattern of elongation when temperatures dropped to ca. 10 °C, reaching full size (E:S ratio ca. 0.75) by the end of December. Seed mass, length and width differed significantly among the species (P < 0.001): C. neapolitanus > C. etruscus > C. ilvensis (Table. 1). Full embryo size of C. ilvensis was slightly lower (E:S ratio ca. 0.7) than that of the other two species (ca. 0.75). However, embryo growth did not differ among the three species (P > 0.05) and could be constrained to a linear and logistic regression for the summer–autumn and autumn–winter periods respectively (Fig. 1A). Radicle emergence immediately followed the period of more rapid embryo growth (end autumn–early winter) and reached ca. 80% by December, when the E:S ratio in all species was ca. 0.65. Radicle emergence progress curves of the tetraploid cytotype of C. neapolitanus and C. etruscus could be constrained to a single curve (P > 0.05; t50 = 152), while C. ilvensis showed a distinctly (P < 0.001) slower (t50 = 156) germination dynamic

Controls were performed to test the effect of prolonged incubation at each of the seasonal temperature conditions under either light or dark conditions. Seeds were checked for radicle emergence over 300 days. Statistical analysis Embryo growth data were analysed and described using linear and logistic regressions. Seed germination follows a binomial distribution and so lacks the properties of linearity and additivity. The effects of treatments on final germination (radicle or shoot emergence) in each species were analysed using a generalised linear model (GLM) in the R statistical environment (R Development Core Team 2012), with a logit link function and a binomial error structure, followed by a likelihood test. To describe the progress of germination, the Weibull function was fitted to cumulative germination data using the ‘drc’ package in R (Ritz & Streibig 2005). Goodness of regression was assessed with graphical analysis of residuals and F-test sums of squares for lack of fit. We used F-test sums of squares to assess whether individual embryo growth regressions and germination curves could be constrained to a single regression. Moreover, the effect of treatments on germination rate was also compared considering t50 (time to 50% germination) extracted from the fitted values of the curves. Since Kolmogorov–Smirnov tests revealed normal distribution of the data set, seed morphological traits (mass, length and width) were analysed with ANOVA. At the end of the experiment, cut tests determined the number of un-germinated but viable seeds; defective seeds (i.e. empty or damaged) were excluded from the experiments. All calculations were obtained with viable seeds and analysed using the R environment for statistical computing. 4

(A)

(B)

Fig. 1. Embryo growth, radicle and seedling emergence outdoors of the tetraploid cytotype of Crocus neapolitanus (circles), C. etruscus (squares) and C. ilvensis (triangles). (A) Average E:S ratio SE (n = 20). (B) Cumulative radicle emergence percentage curves (filled symbols), and cumulative seedling emergence curves (open symbols) fitted using the Weibull function. Grey lines indicate mean daily temperature.




(Fig. 1). While radicles emerged in November–December 2012, seedling emergence was delayed until the following spring (February–March 2013). During this 4-month period (November–March), mean daily temperature was 7.2 °C (max = 14 °C, min = 2 °C). Seedling emergence significantly differed among the three species (P < 0.001). Seedlings of C. ilvensis emerged first (t50 = 248; 23 February 2013), followed by C. etruscus (t50 = 257; 4 March 2013) when temperature were still ca. 10 °C. Seedlings of the temperate Crocus emerged last (t50 = 271; 18 March 2013) when temperatures started to rise to ca. 15 °C (Fig. 1). Dormancy breaking in buried seeds Seeds of C. neapolitanus, C. etruscus and C. ilvensis after 0 days of burial were, respectively, 54%, 34% and 25% dormant. For seeds recovered in the first exhumation (30 days of burial, 21 July), these percentages reduced significantly (P < 0.05) to 25%, 14% and 9%, respectively (Fig. 2). In the second (60 days, 21 August) and third exhumation (90 days, 21 September), C. etruscus and C. ilvensis seeds had all germinated and their final radicle emergence percentage did not differ (P > 0.05). Seeds of C. neapolitanus showed a significantly (P < 0.05) reduced percentage of dormant seeds at each exhumation and were non-dormant only at the third exhumation.

C. etruscus and C. ilvensis did not differ significantly (P > 0.05) and could be constrained to a single logistic regression, while embryo growth of C. neapolitanus in the light failed to reach full size and stopped at an E:S ratio of 0.65. Radicles did not emerge during the simulated autumn conditions. Indeed, radicles of all species began to emerge around 20 days after transfer from autumn to winter conditions (15/5 °C), when E:S was ca. 0.61, with no differences (P > 0.05) in progress of radicle emergence of C. etruscus and C. ilvensis, which were therefore constrained to a single curve. On the other hand, as for embryo growth, the radicle emergence of C. neapolitanus was incomplete under light conditions (Fig. 3). In the simulated regime of constant temperatures in the light, radicle emergence was significantly faster (t50 = 167; P < 0.001) compared to that in the simulated alternating regime (t50 = 173), but there was no significant difference in the final percentage. Again, neither embryo growth nor radicle emergence of C. neapolitanus was completed under light conditions. Under dark conditions, progress of embryo growth and radicle emergence at both alternating and constant regimes was similar to those observed in the light (P > 0.05) for C. etruscus and C. ilvensis. Embryo growth and radicle emergence of C. neapolitanus, was much higher in darkness compared to light (P < 0.001; Fig. 4). Under dark conditions all three species

Phenology of embryo growth and radicle emergence in the laboratory

(A)

In agreement with the phenology observed outdoors, embryos of all species grew slowly in the laboratory during the 90 days of summer (25/15 °C) + 60 days of autumn conditions (20/10 °C), with no differences among the species (P > 0.05), so that embryo growth could be constrained to a single linear regression (Fig. 3). Faster embryo growth, fitted with logistic regression (P < 0.001), was delayed until seeds were moved to winter conditions (15/5 °C); where embryo growth of (B)

Fig. 2. Final radicle emergence percentage (95% CI) in the light of the tetraploid cytotype of Crocus neapolitanus (white columns), C. etruscus (grey columns) and C. ilvensis (dark columns) at simulated winter alternating temperature regimes (15/5 °C), after increasing duration of burial (0, 30, 60, 90 days) and after 60 days of simulated autumn temperatures (20/10 °C).

Fig. 3. Embryo growth and radicle emergence of the tetraploid cytotype of Crocus neapolitanus (circles), C. etruscus (squares) and C. ilvensis (triangles) at simulated seasonal temperatures in the light. (A) Average E:S ratio SE (n = 20) under alternating temperature conditions. (B) Cumulative radicle emergence percentage curves fitted using the Weibull function under alternating (dark symbols) or constant (open symbols) temperature regimes.


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C. neapolitanus and C. etruscus, and 3% of C. ilvensis seeds had a radicle but only under alternating temperatures (data not shown). Effect of high summer temperatures on radicle emergence Radicle emergence of seeds of all three species held at high simulated summer temperatures (30/20 °C) for 90 days, then moved to autumn (20/10 °C) and incubated under winter conditions (15/5 °C) did not differ (P > 0.05) from that observed after seeds experienced regular simulated summer temperatures (25/15 °C). This was true for both final radicle emergence and rate (data not shown). Fig. 4. Final radicle emergence percentage (95% CI) in the light or in darkness (as indicated) at simulated winter constant temperature (10 °C, white and light grey columns) or alternating temperature regimes (15/5 °C, dark grey and black columns), after 90 days of summer conditions (20 or 25/ 15 °C) and after 60 days of autumn temperatures (15 or 20/10 °C).

show the same pattern of embryo growth and radicle emergence, with no significant differences (P > 0.05). Effect of summer, autumn and winter conditions on radicle emergence Logistic regressions revealed that increasing the length of summer significantly increased (P < 0.0001) final radicle emergence (Fig. 5), and curve fitting revealed that t50 was reduced in all species (not shown). This effect was less evident under alternating regimes (P < 0.05). That is to say that under alternating regimes, after 30 days of summer, radicle emergence reached 80%. In C. ilvensis, total radicle emergence after 60 days of summer did not differ significantly (P > 0.05) from that after 90 days, under both constant and alternating temperature regimes, while for the other two species this was true only under alternating regimes. The absence of autumn conditions negatively affected radicle emergence of all species (P < 0.001; Fig. 6). Again, this effect was less evident under alternating regimes (P < 0.05). When summer- and autumn-treated seeds were maintained in continuous autumn conditions, there was no radicle emergence in

Effect of winter length on shoot emergence Shoot formation of the tetraploid cytotype of C. neapolitanus seeds with an emerged radicle under simulated spring conditions (20/10 °C) was faster and more complete (P < 0.001) than after a prolonged winter (90 days at 15/5 °C). In contrast, cold stratification was not required for shoot formation of C. etruscus and C. ilvensis; moreover, shoot formation begun and was already nearly completed under winter condition for these species (Fig. 7). Induction of secondary dormancy Seeds of C. neapolitanus, C. etruscus and C. ilvensis immediately moved to 5 °C after experiencing 90 days of simulated summer (25/15 °C) had 21%, 35% and 43% emerged radicles when transferred to a favourable germination temperature (15/5 °C). In contrast, seeds that also experienced simulated autumn conditions before being moved to 5 °C started radicle emergence at this temperature, and final radicle emergence when seeds were transferred to 15/5 °C was 52%, 60% and 73% (C. neapolitanus, C. etruscus and C. ilvensis, respectively). Influence of seed storage on radicle emergence Radicle emergence of fresh seeds was 95%, 98% and 98% (for C. neapolitanus, C. etruscus and C. ilvensis, respectively). Although storing seeds for 6 months under standard seed banking conditions reduced final radicle emergence, in all cases

Fig. 5. Final radicle emergence percentage (95% CI) in the light at simulated winter constant temperature (10 °C, white columns) or winter alternating temperature regimes (15/5 °C, grey columns), after increasing duration of summer conditions (0, 30, 60, 90 days at 20 or 25/15 °C) and after 60 days of autumn temperatures (15 or 20/10 °C).

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Fig. 6. Cumulative radicle emergence percentage curves fitted using the Weibull function at simulated winter constant and alternating temperatures (as indicated) in the light after 90 days of summer conditions and with (black curves) or without (grey curves, as indicated) autumn conditions.

Fig. 7. Cumulative shoot formation percentage curves fitted using the Weibull function for seeds with an emerged radicle at simulated spring conditions (20/ 10 °C) after variable exposure to simulated winter (0, 30, 90 days at 15/5 °C).

the reduction was not significant (P > 0.05). Final radicle emergence after storage was 83%, 88% and 90% for C. neapolitanus, C. etruscus and C. ilvensis, respectively. Controls No control seeds germinated in the light or in darkness when they were incubated continuously under each of the seasonal temperature conditions. DISCUSSION Our main goal to – identify differences in seed ecophysiological traits associated with their taxonomy and autoecology – was partially achieved. We found similar responses among the three studied species, as shown in the phenology of embryo growth and radicle emergence. On the other hand, differences among the species were found in (a) the degree of radical and epicotyl dormancy and (b) in germination response to light. Seeds of all three species possess a linear underdeveloped embryo at the time of dispersal in late spring, and the length of the embryo increases ca. 50% inside the seed before the radicle emerges. In the outdoor experiments, embryo growth occurred slowly during summer and early autumn; by the end of October, when temperatures fell to autumn–winter values (ca. 10 °C), embryo growth accelerated and radicles emerged. No fresh

control seeds germinated in any of the temperatures, indicating that underdeveloped embryos have physiological dormancy. In the laboratory, under the simulated seasonal temperatures, embryo growth and radicle emergence phenology were remarkably similar to those observed outdoors, with most embryo growth and radical emergence taking place under simulated winter, after seeds had experienced simulated summer and autumn seasons. In the laboratory, we observed a delay of these responses compared to the outdoor experiments, probably as a consequence of cool temperatures (ca. 10 °C) arising outdoors ca. 20 days earlier than in the laboratory (Figs 1 and 3). Increasing the length of warm stratification significantly increased final radicle emergence, indicating that high temperatures, like those experienced in the summer (burial experiment), are required for the breaking of physiological dormancy in all three species. Thus, radical emergence is not possible unless seeds experience warm stratification during summer, supporting our hypothesis that premature germination is prevented under natural conditions of summer. However, from the results of both outdoor (burial) and laboratory experiments, it is clear that after 0 days of summer much of the dormancy breaking occurred during the simulated autumn conditions (20/10 °C). Further experiments are required to confirm whether a longer incubation at autumn temperatures might have similar effects to summer temperatures. Indeed, despite the fact that relatively high levels of radical emergence


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was obtained in the absence of autumn, summer followed by autumn conditions resulted in more complete radicle emergence. This means that autumn is also important for the release of any residual physiological dormancy, which may not have been fully broken during summer conditions; furthermore, because autumn temperatures are ineffective for complete embryo growth and radical emergence, we speculate that autumn conditions ensure that all seeds are at the same dormancy state when they experience cooler temperatures, thus synchronising radicle emergence of the seed population as temperatures drop to late autumn/winter levels. The dependence on summer and autumn treatments to release physiological dormancy was less evident under alternating temperature regimes, suggesting that the high temperature phase of the alternating temperature might have acted as a pre-treatment, helping to release the physiological block. Mondoni et al. (2012) suggested a similar explanation for Erythronium dens-canis seeds held at continuous 15/5 °C, which might have a dual effect of warm and cold stratification. The sensitivity to alternating temperatures may be regarded as a sensing mechanism for canopy shade (Kos & Poschlod 2007; Mondoni et al. 2012), with germination of canopyassociated species being inhibited by diurnal alternating temperatures typical of open habitats. Radicle emergence of the species studied here was possible regardless of whether the temperatures were constant or alternating, confirming that they may be also found in ecotonal habitats. The requirement for alternating temperatures is also considered a sensing mechanism for depth of burial (Thompson & Grime 1983), often associated with light requirement for germination (Probert 2000). Seeds of Crocus etruscus and C. ilvensis were not sensitive to light, whereas both embryo growth and radicle emergence of C. neapolitanus was inhibited by light. The requirement for darkness for radicle emergence in approximately 40% of seeds in C. neapolitanus may be an adaptation to open habitats (meadows), preventing radicle emergence at the soil surface and thus decreasing seedling mortality due to inconsistent water availability (Fenner & Thompson 2005). The lack of sensitivity to light in C. etruscus and C. ilvensis might reflect absence of this selection pressure at the seedling stage in these species because their woodland habitats are less susceptible to drying out (Fenner & Thompson 2005). Since the embryo grows while still inside the mature seed, before visible germination occurs, according to seed dormancy classification (Baskin & Baskin 2004), seeds of the three studied species have morphological dormancy. Nevertheless, since seeds required a dormancy-breaking treatment, seeds also have physiological dormancy. Thus, the seeds may be said to possess morpho-physiological dormancy. An increasing number of studies suggest, however, that if embryo elongation occurs continuously from dispersal to radicle emergence, seeds should be classified as non-dormant regarding the morphological component (Ali et al. 2007; Vandelook et al. 2012; Newton et al. 2013). Furthermore, although we determined the critical E:S ratio, attainment of this embryo length was not strictly required for radicle emergence. Indeed, radicle emergence in some seeds started before this embryo length was reached. Therefore, in agreement with Newton et al. (2013), while the underdeveloped embryo needs to grow before radicle emergence, we maintain that the notion of a critical embryo length for germination in these Crocus species is questionable. 8

Polyploidy, and particularly allopolyploidy, is considered a crucial aspect of angiosperm evolution (Soltis et al. 2003) and has led to micro-speciation, as is the case in the tetraploid cytotype of C. neapolitanus (Frello & Heslop-Harrison 2000). Polyploids often have heavier seeds than diploids (Bretagnolle et al. 1995), which we also found here in the tetraploid cytotype of C. neapolitanus. Seeds of all three species differed significantly in their length and width, with larger seeds found in the tetraploid cytotype of C. neapolitanus. However, irrespective of seed size, maximum embryo length was reached at the same time, and embryo growth did not differ among the studied species. Therefore, no gradient can be observed in the degree of embryo elongation required for radicle emergence among the studied species, suggesting that for these taxa the speciation process is not related to the internal structure of the seeds (Martin 1946; Corner 1976; Vandelook & Van Assche 2008). Full radicle emergence took place almost exclusively at ca. 10 °C (irrespective of constant or alternating temperatures). The temperature requirement for radicle emergence in the Crocus species studied will ensure germination timing during the moist season, which is a typical Mediterranean plant strategy (Doussi & Thanos 2002; Carta et al. 2013). However, many other geophytes of temperate origin germinate at the same temperature (Baskin & Baskin 1998; Vandelook & Van Assche 2008; Mondoni et al. 2012). Additional radicle emergence occurred at 5 °C and, only for C. ilvensis, at 20/10 °C, but with very low percentage (3%). Apart for this latter evidence, we failed to find differences in temperature requirement and in timing of radicle emergence among the studied species. Thus, although climate conditions in the natural habitats of the three seed populations studied here are different, our results on radicle emergence do not reflect the habitat and climate differences. This is perhaps not surprising, because other studies have failed to find any correlation with local conditions (see review in Donohue et al. 2010). On the other hand, we found clear differences in the timing of shoot emergence (and in dormancy) that could be related to habitat differences. Indeed, adaptation to local conditions could be manifested at other stages of the plant life (e.g. seedling, juvenile or adult plant stages; see Donohue et al. 2010). In our study, the radicle emerges in midautumn but seedling emergence outdoors is delayed until the following spring. During this time, the radicle continues to grow, and the seed continues to feed this heterotrophic process through a haustorium (Tillich 2003). The time lag between radicle and seedling emergence has frequently been related to the presence of epicotyl dormancy (Baskin & Baskin 1998). However, a true epicotyl is absent in all monocotyledon species (Muller 1978; Vandelook & Van Assche 2008). Nevertheless, the term ‘epicotyl dormancy’ has also been applied to monocotyledons (Baskin & Baskin 1998; Tillich 2003; Kondo et al. 2004). We found that shoot formation of C. neapolitanus under simulated spring conditions was faster and more complete after a prolonged winter period. Thus, this species shows a degree of epicotyl dormancy, ensuring that seedlings emerge at the end of winter, a mechanism that probably serves to avoid frost damage. Other plant species ensure survival of seedlings by avoiding pre-winter germination because their seeds require cold stratification (e.g. Herranz et al. 2013a). In contrast, the other two, more thermophilous Crocus species studied here do not show epicotyl dormancy, and thus the time lag observed




can be attributed to slow, continuous seedling development (Vandelook & Van Assche 2008). Moreover, in the laboratory we observed that, when seeds with an emerged radicle were immediately moved to spring, this process was accelerated. Shoot formation of these two species is more plastic, and we speculate that in the natural environment seedling establishment may be tuned to different start times and durations of suitable conditions in different years, typical of the Mediterranean environment (Carta et al. 2013). In summary, seeds of the three Crocus species studied here are dispersed in late spring, which means that the seeds experience warm stratification during the summer that is essential to break physiological dormancy. Some embryo growth does occur during summer and early autumn but this accelerates significantly at the end of autumn, when embryos become fully elongated and the radicle emerges during the wettest months of the year. At cold temperatures (winter), shoot formation is delayed (C. etruscus and C. ilvensis) or blocked (epicotyl dormancy) in the case of C. neapolitanus. Thus these species have similar radicle phenology but a temporally separated shoot emergence phase, which differs for clear ecological reasons. At cool (spring) temperatures the shoot elongates rapidly to ensure seedling establishment, thus allowing production of a subterraneous perennating structure (rhizotuber) necessary for persistence over summer before water resources become limiting. Crocus etruscus is a conservation species included in the Bern Convention Appendix 1 and in the European Habitats Directive Annex IV. According to the IUCN categories, C. etruscus is reported as Near Threatened in the global Red List and in the National Red Book (Rossi et al. 2013), while C. ilvensis is assessed as Endangered of Extinction (Foggi et al. 2014). Our results suggest that seeds of these species are suited to ex situ conservation in seed banks and that plants can be readily propagated from seeds for future in situ conservation actions: either subjecting seeds to the natural or to an artificial sequence of temperatures. Although it is well known that populations of the same species can have polymorphism in their germination requirements (Baskin & Baskin 1998), we have studied only one population for each species in its typical habitat. We are confident that no differences are present among populations of C. etruscus, which lives in homogeneous altitudinal and climate habitats, and of C. ilvensis because of their very narrow distribution. However, C. neapolitanus displays a larger altitudinal and latitudinal distribution and therefore phenological adaptations may be present across different populations. The only other Crocus species thoroughly studied up to now is C. alatavicus (Fu et al. 2013), which is phylogenetically distant from the species studied here (Harpke et al. 2013) and shows rather different dormancy and germination patterns. Indeed, this is a subalpine plant, and thus is prone to cold winters and wet summers. Seed dormancy is alleviated by warm stratification, similar to the species studied here, but during the REFERENCES Ali N., Probert R., Hay F., Davies H., Stuppy W. (2007) Post-dispersal embryo growth and acquisition of desiccation tolerance in Anemone nemorosa L. seeds. Seed Science Research, 17, 155–163.

summer embryos grow significantly and radicle emergence takes place in early autumn (around 15 °C). Thus, the radicle of C. alatavicus emerges approximately 1 month before the three species studied here. However, the degree of embryo elongation required for radicle emergence appears to be the same as the species studied here, and thus the internal seed morphology of these species is very similar, confirming that this character is quite conservative (Martin 1946; Forbis et al. 2002; Verd u 2006). Further studies, including a larger number of taxa also belonging to other clades of Crocus in the evolutionary tree, are required to clarify whether it could be considered an example of homoplasy in this genus, as reported for other morphological characters, e.g. structure of seed testa (Harpke et al. 2013). In agreement with Karlsson et al. (2005), conclusions from laboratory experiments that only consider root emergence can be misleading. This was clearly demonstrated here, where subtle but ecologically meaningful differences in germination behaviour were only evident at the stage of shoot emergence. This also has important implications for the successful management of seed conservation collections of wild plant species and the development of effective propagation protocols for ecological restoration. Clearly, a set of germination conditions that allows radical emergence but fails to overcome epicotyl dormancy would lead to emergence failure and a complete waste of seeds (Merritt & Dixon 2011). This work has highlighted the importance of comparative studies to analyse seed germination traits in an evolutionary ecology framework in support of phylogenetic studies (Donohue et al. 2010). Although the studied taxa resulted from recent speciation processes, our results suggest an early onset of adaptation to local ecological factors. However, a common-garden or reciprocal transplant experiment is required to test this hypothesis because differences in seed size and timing of radicle emergence may be plastic, due for example to differing temperature conditions during seed ripening (Andersson & Milberg 1998). We conclude that phylogeny may represent a significant constraint in the evolution and expression of structural and functional seed traits in Crocus. In future studies, it should be determined whether other species of the series Verni distributed along the Italian Peninsula and differing in their bioclimatic requirements (e.g. the subalpine C. vernus (L.) Hill and the Mediterranean C. siculus Tineo) also have similar radicle phenology but differ in their shoot emergence phase. ACKNOWLEDGEMENTS Funding for this research was provided by EX60% from the University of Pisa. We are grateful to the Inter-departmental Bioclimatology Centre of Florence for monitoring and providing climate data of Pisa Botanical Garden. This research was conducted according to the national and regional laws for plant protection and with permissions of local authorities.

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