Effects of Environmental Factors on Seed Germination of Anthyllis Barba-Jovis L

Plant Biosystems, Vol. 142, No. 2, July 2008, pp. 275 – 286

Effects of environmental factors on seed germination of Anthyllis barba-jovis L.

MASSIMILIANO MORBIDONI1, ELENA ESTRELLES2, PILAR SORIANO2, ISABEL MARTI´NEZ-SOLI´S3, & EDOARDO BIONDI2

1Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Universita` Politecnica delle Marche, Ancona, Italy, 2ICBiBE - Jardı´ Bota`nic, Universitat de Vale`ncia, Espanã and 3Universidad CEU – Cardenal Herrera, Departamento de Fisiologıá, Farmacologıá y Toxicologıá, Valencia, Espanã

Abstract The influence of the main environmental factors on seed germination of Anthyllis barba-jovis L. were analysed. This work is part of a broader investigation aimed at the reintroduction of this species on Mount Conero, Ancona (central Italy), where it is at present extinct. The seeds were collected from the Gargano headland (southern Adriatic coast). Experimental analyses were carried out to determine: (i) dormancy levels of seeds collected in successive years, and also collected from the soil seed bank; (ii) effects of usual pre-treatments for overriding the physical dormancy of the seeds; (iii) optimal temperature range for maximum germination; (iv) effects of fire on seed germination; and (v) effects of NaCl on germination and on early stages of seedling development. Our results confirm that A. barba-jovis seeds have a physical dormancy due to their teguments, which are water-impermeable. This barrier persists in naked seeds that remain in the soil. Regularly water- drenched seeds show a high germinative ability. The optimal seed germination temperature is 208C, with germination decreasing progressively at lower temperatures, and falling drastically over 208C. Fire and high temperatures positively affected germination. The seeds were shown to be strongly resistant to salt stress, thus enabling the plants to colonize a habitat suitable for halophytes.

Key words: Anthyllis barba-jovis, ecosystem restoration, ﬁre species, germination, salt tolerance, seed dormancy

affected by anthropic modiﬁcations, due to their Introduction inaccessibility, there have been quite recent cases

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 Anthyllis barba-jovis L. is an evergreen shrub that is where A. barba-jovis has disappeared from its natural found in different habitats along the rocky cliffs of habitats (Brilli-Cattarini 1965; Biondi 1986; Danton the western–central Mediterranean basin; in France & Baffray 1995; Paradis 1997; Benedi 1998). At (Var, Bouches du Rhoˆne, He´rault, Corsica), Italy present, it has to be considered as extinct from the (Liguria, Tyrrhenian coast, Sardinia, Sicily, Adriatic Mount Conero promontory, Ancona (central Italy; coast of Gargano, Tremiti Islands), Croatia, Algeria see Figure 2) (Brilli-Cattarini 1965; Biondi 1986), and Tunisia (Cullen 1968; Greuter et al. 1989; although this territory was, for a long time, considered Pignatti 1992; Trinajstic 1994; Biondi et al. 1997; to be its most northern known natural location along Paradis 1997) (Figure 1). the Adriatic coast of the Italian peninsula (Biondi Anthyllis barba-jovis is not included in the World et al. 2002). Its presence in the past is testiﬁed by a Conservation Union (IUCN) Red List, although it is herbarium record, as it was collected in 1808 by the a protected species at a national level in France naturalist Paolo Spadoni. In his work entitled (Danton & Baffray 1995) and Croatia (Trinajstic Xylologia Picena applicata alle arti, he claimed to have 1994). In Italy, it is not listed in the Protected Flora collected it along the coast between Ancona and (Conti et al. 1992), but it is considered to be in a risk Sirolo, including the coastal rocky marl and limestone category in seven of the nine regions where it is of Mount Conero (Spadoni 1826). found (Conti et al. 1997). Although the areas The present study is the ﬁrst part of a broader inhabited by this species are not generally directly programme focused on acquiring further knowledge

Correspondence: Edoardo Biondi, Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Universita` Politecnica delle Marche, via Brecce Bianche s.n., 60131 Ancona, Italy. E-mail: [email protected] ISSN 1126-3504 print/ISSN 1724-5575 online ª 2008 Societa` Botanica Italiana DOI: 10.1080/11263500802150514 276 M. Morbidoni et al.

Figure 1. Present-day distribution of Anthyllis barba-jovis. Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009

Figure 2. Geographic location of Mount Conero and the seed harvesting zone in the Gargano headland.

of the autoecology of A. barba-jovis, its ex-situ (i) the dormancy level in seeds collected in suc- conservation, and its reintroduction in the Regional cessive years and taken from the soil seed bank; Natural Park of Mount Conero. Restoration pro- (ii) the effects of standard seed pre-treatments to grammes are a priority in the management of natural override their physical dormancy, with verifica- populations, and thus our study is aimed at providing tion of the results at a histological level; protocols that cover the first steps of plant culture. (iii) the optimal temperature range for maximum Present results relate to various aspects of the germination; germination physiology of A. barba-jovis seeds. In (iv) the effects of fire temperatures on seed particular, the following issues were addressed: germination; and Germination in Anthyllis barba-jovis 277

(v) the effects of NaCl on the germination phase, chemical, with 96% sulphuric acid for 5, 10 or the first stages of seedling development, and 15 min. For the pre-heating treatments, two different the relationships between light conditions, the types of heat were used: dry heat and humid heat, thermal optimum and salt concentrations. with a factorial experimental design. The dry heat was applied using a Selecta thermo-block: the seeds were exposed to at 808C or 1008C for 5 or 10 min. Materials and methods The seeds were subjected to humid heat by immersion in water at 808C for 5 s, 10 s and 5 min. The Plant material freeze/thaw protocol was for 20 min at –1968Cin Anthyllis barba-jovis seeds were collected from San liquid nitrogen, followed by 10 min thawing in a Menaio, in the Gargano headland territory of the waterbath at 408C, for 10, 20 and 30 successive Italian peninsula (southern Adriatic basin; Figure 2) cycles. in September 2003 and July 2004. In this region, the All of the basic germination tests for the dormancy A. barba-jovis population is large, and although seed of seeds were carried out in the dark at 208C. The harvesting was abundant, it was not enough to experiments were carried out in December 2003, damage the natural rate of reproduction. The with seeds gathered in September 2003. monosperm legumes are generally retained inside the dry flower remains; they were collected at Electron microscopy maturity directly from the plants and desiccated under laboratory conditions. The fruits were cleaned Observations were carried out on the morphology of by rubbing them between two rubber sheets, and the seed coat of three different seed samples: (i) non- then separating them through differently sized treated seeds collected from the plant; (ii) seeds metallic sieves; the small pods were then opened treated with 96% sulphuric acid for 15 min; and (iii) using a scalpel. The best procedure was to cut the tip non-treated seeds collected from the soil seed bank. and then open the pod longitudinally with a blunt The seeds, are, in general, found in a dehydrated blade. This had to be done with special care to avoid condition, so that they were analysed directly without damage to the seed coat, which could destroy seed the need for any dehydration process. The samples impermeability. The cleaned seeds were kept in for the ultramorphological analysis were mounted on airtight bags at room temperature until used. an aluminium base, and kept in place using double- sided carbon tape (STR tape, 8 mm, Sinto Paint Co. Ltd). They were then metallized with SC 500 Sputter Germination protocol Coater (Bio-Rad) for a cover of gold–palladium of For germination, the seeds were placed on 55 mm *200 A˚ , and examined by scanning electron diameter Petri dishes containing 0.6% agar, which microscopy (FE HITACHI 4100), which included were kept in climate-controlled rooms under various a system for the collection of digital images. The temperature and light conditions, as specified below. voltages used were 5 kV and 10 kV.

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 The observation period was generally one month, and for each treatment 4 replicates of 25 seeds were Germination temperature used. A seed was considered to have germinated when the radicle was longer than 1 mm. The optimum germination temperature was estab- lished by testing a range of temperatures from 58Cto 358C, at intervals of 58C. A second test was carried Mechanical and chemical scarification out to determine the percentages of seeds that The study of physical dormancy was performed germinated under conditions of alternating tempera- through the application of different treatments, ture, using 108C and 188C, with a period of 10 h and including mechanical and chemical scarification, 14 h, respectively. In all cases, the seeds were pre- pre-heating and the alternation of freeze/thaw tem- treated with chemical scarification using 96% sul- peratures using liquid nitrogen. Since similar experi- phuric acid for 15 min, followed by profuse washing ments have not been performed previously on A. with sterilized water; they were kept in the dark for barba-jovis, the various parameters were selected germination. The experiments were carried out in following the studies on Anthyllis cytisoides and October 2004 with seeds collected in September Anthyllis lagascana (Ibanez & Passera 1997; Prieto 2003. et al. 2004). These species are considered to be phylogenetically and ecologically similar to A. barba- Dormant seed collection from the seed bank jovis (Nanni et al. 2004). Two different scarification methods were used: For testing the degree of dormancy, three seed one mechanical, with sand paper, and the other samples were collected. Seeds were collected directly 278 M. Morbidoni et al.

from the plants, in July 2003 and in July 2004, and were in contact with the compact rock. The seeds extracted from the fruits as described above. Natu- were partly contained in the legumes and partly rally aged seeds from the soil seed bank were gathered exposed; they were separated out using metal forceps. in September 2004, by taking soil samples at a 3– They were germinated in the dark, either under the 4 cm depth at the base of some adult A. barba-jovis alternating temperature conditions indicated above individuals. Samples consisted of marly detritus, with (108C/188C for 10 h/12 h), or after chemical scar- some humus and decomposing organic matter, and iﬁcation with 96% sulphuric acid for 15 min. These tests were performed in summer 2005.

Pre-treatment with heat or fire To simulate the effects of fire, different experimental conditions were examined: (i) a seed sample under- went thermal shock pre-treatment at temperatures of 508C, 1208C and 1508C, and with different exposure times of 30, 60 and 120 min, 1, 5 and 10 min, and 1 min, respectively. To do this, the seeds were placed in glass test tubes containing sterilized sand, which were then placed in a stove; for the short treatment periods (1208C and 1508C), the sand was pre-heated to the required temperatures; (ii) a seed sample was directly exposed to the naked flame of a Bunsen burner for a few seconds (Vuillemin & Bulard 1981); and (iii) a seed sample was left inside the fruits and the dried remains of the flowers (the conditions in which they were gathered from the soil) and ignited over a Bunsen burner flame and allowed to burn until Figure 3. Germination of seeds subjected to different scarification the flame extinguished spontaneously. The seeds pre-treatments. Ctr, control; abr, scarification with sandpaper; S.A.5, S.A.10, S.A.15, scarification with 96% sulphuric acid for 5, were then recovered from the ashes and immediately 10 and 15 min. The different letters indicate means that show prepared for germination. All seeds were germinated significant differences (P 5 0.05). in the dark, under the alternating temperature Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009

Figure 4. Germination of seeds pre-treated with heat (A) and liquid nitrogen (B), compared with control and mechanical scarification. Ctr, control; abr, scarification with sandpaper; UM.5s, 10s, 5 min, humid heat 5 s, 10 s, 5 min; 80–5 min/10 min, 100–5 min/10 min, 808C for 5 min/10 min, 1008C for 5 min/10 min; 10-N, 20-N, 30-N, 10, 20, 30 freeze (–1968C)/thaw cycles. The different letters indicate means that show significant differences (P 5 0.05). Germination in Anthyllis barba-jovis 279

conditions indicated above (108C/188C for 10 h/ experiments were concluded after the first true leaves 12 h). The experiments were carried out in June 2005 appeared on the control plants (approximately one with seeds that had been gathered in July 2004. month from germination), and the lengths of the stems and roots were measured. Salt stress Statistical analysis The germination tolerances to 10 increasing concentrations of NaCl were determined: 0, 50, 100, The data obtained were analysed statistically by 150, 200, 250, 300, 350, 400 and 500 mM. The ANOVA for different factors, with a Tukey post-hoc seeds were scarified before use with 96% sulphuric test to identify homogeneous groups, and a signifi- acid for 15 min, and they were germinated at the two cance level of 0.05 to confirm the differences best temperatures (158C and 208C) in the dark. Salt between the arithmetic means. The percentages effects on seedling development led to the hypothesis obtained had a normal behaviour, and therefore an that germination behaviour varied according to arcsin transformation was not necessary. In the salinity and illumination. Therefore, the salinity histograms, the different letters indicate data that effects were also investigated under a 12 h/12 h are significantly different. photoperiod at 158C. The experiments were carried out in October 2004 with seeds that had been gathered in September 2003. These germination Results experiments were extended for up to two months, as, Physical dormancy by the end of the first month, there were seeds that were still in active germination, and in particular at The non-treated seeds showed very low levels of the higher NaCl concentrations. germination (*3%), which was also particularly slow Further development of plantlets under salt stress (Figures 3 and 4; Table I). The pre-treatments were was carried out under a 12 h/12 h photoperiod at all more effective. The seeds treated with abrasive 208C. Small bottles (height 35 mm, diameter paper showed germination levels that were signifi- 65 mm) were used that contained 30 ml 1% agar cantly greater than control levels (78 + 9.6%); as substrate, supplemented with Murashige and germination was particularly fast, with maximum Skoog salts (MS; 4.3 g/l) to satisfy the nutritional germination already achieved on the first day of demands of the seedlings. NaCl was added at the sampling (Figure 3; Table I). Seeds subjected to concentrations specified above. The seeds were chemical scarification using sulfuric acid showed the scarified with 96% sulphuric acid for 15 min. The highest germination rate (84 + 2.4%); germination levels were proportional to treatment times (Figure 3; Table I). Immersion in hot water (humid heat at 808C) gave Table I. Germination after different scarification pre-treatments, modest results (Figure 4A; Table II); the longest

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 expressed as percentages + standard error. treatment (5 min) yielded the highest germination Chemical scariﬁcation with 96% levels (34 + 6.2%). This 5-min treatment, which was sulphuric acid considerably longer than most of the others, did not Mechanical appear to compromise seed vitality, although, in Days Control scariﬁcation 5 min 10 min 15 min most seeds, germination was slowed down by about 90+ 0.0 78 + 9.6 4 + 2.3 11 + 3.0 16 + 6.3 one week. 12 0 + 0.0 78 + 9.6 4 + 2.3 16 + 4.3 45 + 5.4 The same slow germination was seen following dry 19 1 + 1.0 78 + 9.6 15 + 1.9 29 + 1.9 80 + 3.5 heat treatment of the seeds, at both 808C and 1008C 29 3 + 1.9 78 + 9.6 21 + 1.0 32 + 3.3 84 + 2.4 (Figure 4A). The highest levels of germination were 32 3 + 1.9 78 + 9.6 22 + 1.2 33 + 4.1 84 + 2.4 observed with an exposure to 1008Cfor5min

Table II. Germination after seed exposure to thermal shock, expressed as percentages + standard error.

Humid 808C Humid 808C Humid 808C Dry 808C Dry 808C Dry 1008C Dry 1008C Day 5 s 10 s 5 min 5 min 10 min 5 min 10 min

99+ 2.5 7 + 3.0 5 + 1.9 2 + 1.2 3 + 1.9 5 + 1.0 1 + 1.0 12 14 + 2.6 9 + 3.0 10 + 2.0 5 + 1.0 7 + 3.4 7 + 1.0 3 + 1.9 19 16 + 2.3 14 + 2.0 23 + 5.0 18 + 3.5 26 + 6.2 31 + 3.4 15 + 3.0 29 16 + 2.3 17 + 1.0 30 + 5.3 22 + 2.6 32 + 5.4 33 + 4.1 21 + 6.6 32 16 + 2.3 17 + 1.0 34 + 6.2 24 + 1.6 33 + 5.3 34 + 5.0 22 + 6.2 280 M. Morbidoni et al.

(34 + 5.0%); twice this treatment time resulted in Electron microscopy the death of some of the seeds. The highest germination rates were obtained with The quality of the external tegument and the seeds treated for 20 or 30 freeze/thaw cycles through alterations that were caused by the artiﬁcial scariﬁca- repeated immersion in liquid nitrogen: ca. 85% of tion produced by sulphuric acid were examined by these seeds germinated (Figure 4B). electron microscopy. These seeds were compared Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009

Figure 5. General ventral views of seeds. Left panels: magnification, 47 6. Right panels: magnification, 1500 6. A. Seed collected from the plant that can be seen to be completely intact, with the characteristic cavities recognizable under the higher magnification: de´pressions plisseés, sensu Saint-Martin (1986). B. Seed following scarification, where the modifications to the hilum area can be seen; the characteristic cavities in the non-scarified seed are no longer seen at the higher magnification. Instead, more or less deep hollows are visible, with cracks. C. Seed collected from the soil, with the hilum area showing similar modifications to those seen in the seed following artificial scarification; moreover, the surface of the seed is cracked and not so smooth. Under the higher magnification, the tegument is seen to be altered, even though to a lesser extent than in B. Germination in Anthyllis barba-jovis 281

Table III. Germination under different thermal regimes, expressed as percentages + standard error.

Days 58C108C158C208C258C308C 10/188C

7 0 0 37.5 + 2.5 0 0 0 52.0 + 6.5 13 0 17.5 + 3.2 51.3 + 2.4 45.0 + 5.4 10.0 + 3.5 0 63.0 + 3.4 20 2.5 + 2.5 30.0 + 5.4 57.5 + 2.5 78.8 + 3.1 22.5 + 5.2 1.3 + 1.3 69.0 + 1.9 26 11.3 + 5.5 37.5 + 2.5 57.5 + 2.5 82.5 + 1.4 36.3 + 3.1 5.0 + 2.0 70.0 + 1.1 31 26.3 + 7.5 55.0 + 3.5 58.8 + 1.3 83.8 + 2.4 53.8 + 3.8 5.0 + 2.0 75.0 + 1.0

Anthyllis. They have been described as ‘‘de´pressions plisseés’’. Pitting, with cracking, was seen following artificial scarification (Figure 5B), but also in seeds collected from the soil (Figure 5C).

Optimal germination temperature Over the range of temperatures tested, A. barba-jovis showed signiﬁcantly variable germination responses (Table III; Figure 6). The temperature that can be considered to be optimal for seed germination was 208C. At lower temperatures, germination decreased progressively, whereas at temperatures above 208C they fell drastically (e.g. 5% at 308C; 0.0% at 358C).

Figure 6. Germination under the different thermal regimes (as indicated), in the dark. The different letters indicate means that Degree of dormancy in seeds of different ages show significant differences (P 5 0.05). The germination capacity of seeds of different ages did not show any striking differences (Figure 7), with more than one year needed to show significant aging. In our samples, the physical dormancy was not reduced following one year under laboratory conditions. Whether or not the seeds were scarified, those that had aged in the soil showed a tendency to germinate quicker and in greater percentages. The

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 seeds from the soil seed bank remained dormant, and scariﬁcation was still needed to break this dormancy.

Fire and temperature effects on germination At temperatures that simulated the effects of fire, the best results were obtained by exposing the seeds to 1208C for 1 min (44 + 6.3%), with longer exposure times resulting in lower germination rates (5 min, Figure 7. Germination of seeds of different ages gathered directly 25 + 4.1%; 10 min, 9 + 2.5%). One minute of from the plant in the years 2003 and 2004 and stored in the exposure at a temperature of 1508C resulted in very laboratory, and of seeds from the soil bank gathered either with low levels of germination (4 + 1.6%). Whereas their legume or ‘‘naked’’. The different letters indicate means that modest levels of germination were obtained after show significant differences (P 5 0.05). exposure of the seeds to flame (20 + 2.8%), burning of the seeds inside the fruits produced improved with those that were collected directly from the plant germination levels (43 + 4.1%) (Table IV; Figure 8). or the soil. The seeds collected from the plant (Figure 5A) had clearly evident radial depressions Salt and light effects on germination and cavities; these were described by Saint-Martin (1986) as being characteristic of many Leguminosae As expected, germination rates decreased as the salt from the Loteae tribe, and in particular of the genus concentrations increased. Germination also occurred 282 M. Morbidoni et al.

Table IV. Effects of high temperatures on germination, expressed as percentages + standard error.

508C 508C 508C 1208C 1208C 1208C 1508C Naked Control 30 min 60 min 120 min 1min 5min 10 min 1min ﬂame Burnt

8 + 4.3 23 + 3.4 32 + 3.3 14 + 2.0 44 + 6.3 25 + 4.1 9 + 2.5 4 + 1.6 20 + 2.8 43 + 4.1

Discussion and conclusions This study has confirmed that A. barba-jovis seeds have a physical dormancy, with the germination barrier only consisting of the tegument, due to its water resistance, as usually occurs in most Fabaceae (Baskin & Baskin 1989). The results obtained with the more efficient scarification techniques demon- strate the high vitality of the seeds. A high level of physical dormancy was also found in the naturally aged seeds present in the soil; only after being pre-treated with chemical scarification did these seeds show a high germination rate. Thus, it can be seen that dormancy was not reduced (or only very slightly) by age. The seed longevity also remained high. The histological observations on the external Figure 8. Germination of pre-treated seeds following different surface of the seed tegument showed that this was exposures to high temperatures. The different letters indicate fully continuous in the intact seeds, and was changed means that show significant differences (P 5 0.05). in various ways and fractured by the action of the acid. For the seeds collected in the soil, of those that were examined microscopically, none was found that at 400 mM NaCl, a salt concentration that would had manifest signs of breakage or degradation of the usually block seed germination. Furthermore, under tegument. This condition was in agreement with the salt stress conditions, different effects were seen germination trials with the same seeds. Here, as regarding light and temperature conditions. At high indicated above, there was a slight, but non-signifi- salt concentrations in the dark there was a greater cant, increase in germination levels compared with level of germination at 158C than at 208C (Figure 9; those obtained for seeds collected from the plants.

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 Table V). Under different light regimes and salt The temperatures to which A. barba-jovis seeds concentrations at 158C slightly superior levels of were subjected in the present study were very similar germination were observed in the dark than with a to those that can be generated during a fire of shrub photoperiod of 12 h / 12 h (Figure 10; Table V). vegetation at the most superficial layer of the ground These effects became more evident with the exten- (0–5 cm), where most of the dormant seeds (the soil sion of the experiments to a total of 60 days, with seed-bank) are found. These temperatures have also increased germination levels at 300 mM NaCl (38%) given positive results in other studies on Legumino- and higher. sae (Auld & O’Connell 1991; Herranz et al. 1997). Therefore, it is clear that in relation to the highest temperatures registered at a few centimetres above Salt effects on seedling development ground level (about 5008C), temperatures of only Increasing the salt concentration in the substrate around 408Cto508C are found at a depth of 5 cm, led to a progressive slowing down of growth that and above 608C in the most superficial layer (0.5 cm) was equally evident in roots and stems, although (Auld & Bradstock 1996). These temperatures can be these inhibitory effects on growth became more higher when natural organic matter is present on the evident with the stems at the higher NaCl ground, in which case they can reach 1408C at a 2-cm concentrations (Figure 11). Indeed, a concentra- depth (Bradstock et al. 1992). tion of 300 mM NaCl appeared to constitute a Exposure of the seeds to a direct naked flame or limit to stem development, although radicle to the burning of the dry remains of the flowers growth continued very slowly up to 400 mM NaCl were based on the consideration that, due to the (Figure 11). pedological characteristics of the zone in which Germination in Anthyllis barba-jovis 283

Figure 9. Germination of seeds under increasing saline concentrations, at 208C (left) and 158C (right) in the dark. Results shown one month from sowing. The different letters indicate means that show signiﬁcant differences (P 5 0.05).

Table V. Germination of seeds under increasing salt concentrations at one and two months after sowing, expressed as percentages + standard error. The different letters indicate means that show signiﬁcant differences (P 5 0.05).

NaCl concentration (mM) 1 month 0 50 100 150 200 250 300 350 400 500

208C in the dark 83.8 ab 47.5 ch 25.0 g 33.8 g 45.0 cg 1.3 e 0 e 0 e 0 e 0 e 158C photoperiod 12 h/12 h 91.3a b 85.0 ab 81.3 af 63.8 c 17.5 d 0 e 0 e 0 e 0 e 0 e 158C in the dark 86.3 ab 90.0 ab 80.0 ab 93.8 b 71.3 cf 20.0 d 5.0 e 0 e 0 e 0 e NaCl concentration (mM) 2 months 0 50 100 150 200 250 300 350 400 500

158C photoperiod 12 h/12 h 92.5 a 87.5 a 86.3 a 75.0 b 30.0 c 8.8 d 1.3 e 0 e 0 e 0 e 158C in the dark 88.8 a 90.0 ab 80.8 ab 95.0 a 88.8 a 83.8 ab 38.8 c 15.0 d 3.8 de 0 e Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009

A. barba-jovis grows (rocks that are more or less studied, an increase in germination response has degraded, and very shallow or completely absent been seen with exposure to temperatures between soil), the action of fire on buried seeds would appear 708C and 1508C for more or less prolonged periods less likely than a more direct effect on the seed of time. However, with many species from environ- surface. The effects of the flame on the seed is a test ments that are exposed to frequent fires, an exposure that no doubt has a highly random effect that is to temperatures of 1108C to 1208C would lead to difficult to reproduce. However, the germination embryo death, even if it is only for 4 min (Auld & percentages were greater than those obtained with O’Connell 1991). The seeds of A. barba-jovis appear the maximum times of exposure to temperatures of to have an intermediate behaviour among the 1508C, 1208C and 508C, and significantly greater Leguminosae, which can show evident positive than the controls. Exposure to a temperature of 508C effects when exposed to high temperatures. These was assumed to simulate the thermal shock produced effects can produce germination levels of 75% with not only by the action of fire, but also by the direct moderately prolonged exposures (10 min) to 1208C effects of solar irradiation on the seeds when the (e.g. Cytisus scoparius, Psoralea bituminosa and Ulex protection of the plant cover is eliminated. The europaeus), with others that apparently do not obtain action of high temperatures on seed germination in any benefit (e.g. Scorpiurus muricatus) (Herranz et al. the Leguminosae growing in Mediterranean climates 1997). In the present study, treatment at 1208C for shows strong interspecific variations (Herranz et al. 10 min was detrimental (or at least did not produce 1997; Hanley et al. 2001). In almost all of the cases any advantage), and a temperature of 1508C was 284 M. Morbidoni et al.

Figure 10. Germination of seeds under increasing saline concentrations at 158C, one and two months after sowing. The different letters

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 indicate means that show signiﬁcant differences (P 5 0.05).

clearly lethal for many embryos, as the germination that this plant is not a pyrophyte; the ecological levels were lower than those of the control. The benefit that might be derived from the passage of fire very important role of the Leguminosae (either will be determined by the indirect factors described grasses or shrubs) in the recolonization of burnt above. There is a joint action of legume indehiscence environments is well known (Doussi & Thanos and secondary dormancy, which persists with time in 1994; Arianoutsou & Thanos 1996; Martinez- the seeds in the ground, and which can be eliminated Sanchez & Herranz 1999). Indeed, many of them by drastic scarification. These, therefore, constitute appear to obtain indirect benefits from the passage control mechanisms for germination, preventing of fire, in addition to those mainly deriving from the seeds from germinating under unfavourable condi- greater availability of space due of the lack of tions, and also generally retarding the germination competing plants. process. For the species studied here, positive effects of This presence of such germination barriers, along high temperatures were evident on seed germination, with the prolonged vitality of the seeds, allows us to although they were not greatly significant. This may hypothesize that in nature there is an accumulation be one of the numerous possible ways of breaching of a considerable reserve of seeds that settle in the the impermeability of the seed coat, although it may ground with time (Bewley & Black 1985; Guardia not be the main one. Due to the high sensitivity to et al. 2000). This is an important adaptive aspect, as high temperatures shown by the embryo, it appears it is evident that in a difficult environment like these Germination in Anthyllis barba-jovis 285

Figure 11. Effects of increasing the saline concentrations of the substratum on the lengthening of the stem and radicle. Data collected 40 days after germination.

rocky cliffs, the availability of an important seed halophytes that are directly exposed to the action of reserve in itself constitutes an important factor for the sea. Indeed, in the environment where this species survival. species grows, high salt concentrations have been Anthyllis barba-jovis seeds displayed a high resis- measured in the waters that bathe the seeds in the tance to salt stress. After two months of observation, summer following occasional rainy periods, or on the germination in up to 400 mM NaCl was recorded, a surface of the soil as a tide effect, or due to the sea concentration that would cause a total block of breeze; these are all unfavourable situations that can be germination in most plants. The ability to germinate overcome by the temperature–salinity–light interac- at such high or more salt concentrations is typical of tions. Germination could be inhibited by high halophytic species. Even higher NaCl concentrations temperatures in the ﬁrst case, and by light in the (800 mM NaCl) can be tolerated only by the second. The intolerance to light in the presence of salt hyperspecialized halophytes; for example, species of means that a seed is more likely to germinate when it is the genera Arthrocnemum, Salicornia and Salsola buried, thereby activating a mechanism based on this (Khan & Gul 1998; Khan 2002). photoinhibition that inhibits the superﬁcial develop- In the species under investigation here, germina- ment of the seedling (Thanos et al. 1989), at least tion under salt stress was modulated by temperature during the most unfavourable times of the year.

Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009 and light conditions. In the absence of salt, seed For A. barba-jovis, although germination is the germination was indifferent to light, and had a most critical phase of its development, adaptations to precise temperature optimum. Under high salt living in environments that are characterized by high concentrations, any variation in temperature relative salt concentrations in the soil are also undoubtedly to the thermal optimum resulted in decreased seen in the early stages of seedling growth which germination, while light also behaves as a funda- appears to be almost indifferent to 50 mM NaCl, mental factor (Naidoo & Naicker 1992; Khan & while little damage appears to be caused by Ungar 1999; Khan & Gulzar 2003; Zia & Khan concentrations up to 100–150 mM. 2004). Some ecologists have interpreted such behaviour as the result of an adaptative process. The salt Acknowledgements concentrations in the ground are very variable, also depending on the time of the year; for instance, a We are very grateful to the staff of Servicio Central de part of the salt is washed away during rainy periods, Soporte a la Investigacioń Experimental (SCSIE) – and, on the contrary, rises to the surface in dry Universidad de Valencia, for help in preparing the periods and during drought. For A. barba-jovis,as samples investigated and in subsequent observations. shown here, the constant temperature that was the optimum under conditions of low soil salinity (208C) was no longer the optimal in the presence of salt, References while light definitely works against germination. It is Arianoutsou M, Thanos CA. 1996. Legumes in the fire-prone clear, therefore, that A. barba-jovis can germinate in Mediterranean regions: An example from Greece. Int J Wild soil that is rich in salt, and can compete with the Fire 6(2):77–82. 286 M. Morbidoni et al.

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