Seed germination and survival of the endangered psammophilous Rouya polygama (Apiaceae) in different light, temperature and NaCl conditions

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1 Seed Science Reserch (2014) 24, q Cmbridge University Press 2014 doi: /s Seed germintion nd survivl of the endngered psmmophilous Rouy polygm (picee) in different light, temperture nd NCl conditions ndre Snto 1 *, Efisio Mttn 1,2, Letiti Hugot 3, Pul Spinosi 3 nd Ginluigi cchett 1 1 Centro Conservzione iodiversità (CC), Diprtimento di Scienze dell Vit e dell mbiente, Università degli Studi di Cgliri, V.le S. Ignzio d Lconi 11-13, 09123, Cgliri, Itly; 2 Seed Conservtion Deprtment, Royl otnic Grdens of Kew, UK; 3 Conservtoire otnique Ntionl de Corse, Office de l Environnement de l Corse, 14, venue Jen Nicoli, 20250, Corte, Corsic, Frnce (Received 1 November 2013; ccepted fter revision 26 July 2014; first published online 16 September 2014) bstrct Rouy polygm (picee) is n endngered Mediterrnen species of gret phytogeogrphicl nd ecologicl interest, growing on costl sndy dunes. Intrspecific vribility in the responses to constnt tempertures (5 258C) nd n lternting temperture regime (25/108C), slt stress (0 600 mm NCl) nd recovery of seed germintion ws evluted mong six popultions from Srdini nd Corsic. Seeds were non-dormnt nd germintion percentges rnged from 10 to 83%, depending on temperture nd popultion. Differences in germintion percentges were minly due to different seed mortlity mong seed lots. R. polygm seeds germinted in slt concentrtions up to 200 mm NCl, wheres higher slt concentrtions totlly inhibited germintion. Slt ffected seed vibility, nd the recovery response decresed with incresing slinity nd temperture. Inter-popultion vribility nd different sensitivity to NCl in seed germintion were detected. Our results re consistent with field germintion in period from utumn to spring, when wter is vilble in the soil nd tempertures re not prohibitive for seedling estblishment, representing n dvntgeous ecologicl dpttion for seedling estblishment to the unpredictble Mediterrnen rinfll pttern. Further studies on R. polygm re needed to investigte germintion requirements t tempertures higher thn 258C nd its germintion in the field, nd to clrify genetic inter-popultion *Correspondence Fx: þ Emil: ndresnto85@gmil.com Present ddress: Wellcome Trust Millennium uilding, Wkehurst Plce, RH17 6TN, rdingly, West Sussex, UK. vribility, considering higher number of popultions nd possibly extending to North fricn popultions. Keywords: inter-popultion vribility, Mediterrnen vsculr flor, psmmophyte, recovery, sndy dunes, sodium chloride Introduction Costl dune environments re complex nd vulnerble ecosystems, chrcterized by close interctions between biotic nd biotic components (Mun, 2009). In the Mediterrnen Se, Srdini nd Corsic re considered prt of the Tyrrhenin Islnds regionl biodiversity hotspot (Medil nd Quézel, 1999). On both islnds, costl sndy dunes re thretened by tourism, urbniztion nd reduction of hbitt (lsi et l., 2007). Under the Mediterrnen climte, mny dverse fctors for plnt survivl chrcterize these ecosystems, such s high tempertures on the soil surfce, low soil moisture, nutrition nd wter deficiency, slinity of the substrte nd slt spry on the plnt surfce. Severl key fctors re known to influence seed germintion, including light, temperture nd slinity. Slt tolernce is n importnt trit for species of costl environments, with higher slinity levels usully reducing or delying germintion of mny species (Mun, 2009). Seeds tht re unble to germinte t high slinity levels might survive during slt exposure nd mintin the bility to germinte lter (recovery), when slinity decreses due to vrious environmentl events (skin nd skin, 1998). Seeds of severl species treted with high slinity levels recovered their germintion following trnsfer to distilled wter, but vrition in recovery percentges

2 332. Snto et l. ws ttributed to differences in the temperture regime to which they were exposed (Pujol et l., 2000). Severl studies highlighted the presence of intr-specific vrition (inter-popultion vribility) in seed weight, germintion nd dormncy (skin nd skin, 1998). The inter-popultion vribility in seed dormncy nd germintion cn be due to environmentl differences or to genetic vritions (Cruz et l., 2003). Vrious studies lso investigted inter-popultion vribility in slt tolernce for costl species (skin nd skin, 1998), demonstrting tht fctors such s seed size, distnce from the se nd locl climte my influence intr-specific differences in slt stress response in some species (ti et l., 2011). Rouy polygm (Desf.) Coincy (picee), n endngered species of gret phytogeogrphicl nd ecologicl interest, occurs in costl sndy dunes (Snto et l., 2013). It is considered south-west Mediterrnen pleoendemic nd belongs to monospecific genus, which hs spred from North fric to Srdini nd up to Corsic (which represents the northern limit of distribution re of the species; Prdis nd Géhu, 1992). picee re reported to hve orthodox seeds (Hong et l., 1998) nd Mrtin (1946) described three typologies of embryo for this fmily: rudimentry, sptulte nd liner xile, with n endosperm usully firm but wtery fleshy. Vndelook et l. (2012) investigted the fctors driving the evolution of the reltive embryo length in picee nd indicted tht it my hve evolved s n dpttion to hbitt nd life cycle, wheres dormncy ws minly relted to temperture t the smpling sites. Stokes (1952) nd skin nd skin (1998) reported seeds of picee s morphologiclly dormnt (MD) or morpho-physiologiclly dormnt (MPD). Previous studies on seed germintion of picee highlighted tht vrious species showed the highest germintion percentges t low tempertures, e.g. 80% t 58C for Cherophyllum temulum L. (Vndelook et l., 2007), 91% t 8 108C for Hercleum mntegzzinum Sommier & Lewier (Morvcová et l., 2005),.80% in the rnge C for both Ferul communis L. nd Ferul rrigonii occhieri (Snn et l., 2009), or under n lternting temperture regime, s for pium bermejoi L. Llorens (. 80% t 25/158C nd 20/168C; Cursch nd Rit, 2012). High germintion ws lso recorded for pium grveolens L. (94%; Thoms et l., 1979) nd Pstinc stiv L. (83%; Hendrix, 1984). The effect of NCl on seed germintion ws evluted in Crithmum mritimum L. (Meot-Duros nd Mgnè, 2008), showing tht only 1.6% of the seeds germinted t low slt concentrtions (50 mm); seed germintion incresed when seeds were wshed with distilled wter, lthough the highest NCl concentrtion (500 mm) permnently ffected the cpbility of seeds to recover. However, no informtion is vilble on seed germintion of R. polygm nd on the key fctors stimulting germintion, the response to slinity nd recovery, nd inter-popultion vribility for this species. Therefore, the ims of this study were: (1) to chrcterize seed germintion of R. polygm, under vrible light nd temperture conditions; (2) to evlute the effect of NCl nd recovery on its seed germintion, nd interctions of slinity with temperture; nd (3) to investigte inter-popultion vribility in seed germintion nd in slt stress tolernce. Mterils nd methods Study species nd seed lot detils R. polygm is psmmophilous species, growing on costl sndy dunes of Srdini, Corsic, lgeri nd Tunisi (Snto et l., 2013). It is inserted in the Wshington Convention (CITES), in nnex I of the ern Convention nd in nnex II of the Hbitt Directive 92/43/EEC. It is scpous hemicryptophyte, 15 30(50) cm high, with scending nd flexuous stems. Fruits re schizocrps (consisting of two mericrps) of 8 9 mm, with undulte wings, 2 mm long. Flowering occurs from June to July while fruiting strts in September (Snto et l., 2013). Schizocrps (herefter clled seeds) were collected in Srdini nd Corsic, in their nturl popultions, t the time of nturl dispersl (Tble 1). Seed collections in Srdini were crried out fter obtining permits from the Ministero dell mbiente e dell Tutel del Territorio e del Mre, s required by Europen nd Itlin lws for the species listed in the ppendices of the Hbitt Directive 92/43 EEC, while seeds from Corsic were provided by the Conservtoire otnique Ntionl de Corse, institution uthorized by the Office de l Environment de l Corse nd the Ministry of the Environments of Frnce. Immeditely fter seed collection the wter ctivity (w) of ech seed lot ws mesured by hygrometer (Hygroplm w1; Rotronic, Huntington, New York, US), equipped with the W-DIO probe (Tble 1). efore the strt of germintion tests, seeds were stored under controlled conditions (208C nd 50% reltive humidity). Men seed mss (^1SD) for ech seed lot ws clculted by weighing 10 replictes of 20 seeds ech (Tble 1). Germintion tests preliminry test ws crried out in order to evlute the effect of light on seed germintion. Seeds were sown on 1% wter gr substrte, which provided solid, non-sterile medium for germintion, in plstic Petri dishes of 90-mm dimeter, nd then incubted in growth chmbers (SNYO MLR-351, Moriguchi,

3 Seed germintion ecology of Rouy polygm 333 Tble 1. Popultion dt nd seed lot detils. Seed mss vlues with the sme letters re not significntly different t P, 0.05 by Mnn Whitney U-test Seed mss (mg ^ SD) Wter ctivity (w) Number of smpled individuls Dte of collection Distnce from the se (m) ltitude (m sl) Sttion Islnd Code Coordintes Portoscuso (CI) Srdini S N E /09/ ^ 0.95 bc Is Solins - Msins (CI) Srdini S N E /09/ ^ 0.63 b Porto di rbtx (OG) Srdini S N E /10/ ^ 0.72 c Lido di Orrì (OG) Srdini S N E /10/ ^ 0.35 b Portovecchio (2) Corsic CO N E /10/ ^ 0.46 c Punt di enedetto (2) Corsic CO N E /10/ ^ 0.69 b CI, Crboni-Iglesis; OG, Oglistr; 2, jccio these re the provinces in Srdini nd Corsic where seeds were collected. Jpn) t different temperture regimes (10, 15, 208C) both in the light (12 h of irrdince per dy) nd in the drk. The light inside ech growth chmber ws provided through nine fluorescent lmps with white light (Mitsubishi OSRM 40; 53 W ech). Drkness ws chieved by wrpping dishes in two luminium foils. The tempertures were chosen becuse under the Mediterrnen climte mny costl species hve their rnge of optimum germintion between 10 nd 208C (Thnos et l., 1995). For ech condition, three replictes of 20 seeds ech were used. The criterion for germintion ws visible rdicle protrusion. When no dditionl germintion occurred for two consecutive weeks, tests were stopped nd the vibility of ny remining seeds ws checked by cut test with sclpel nd subsequent observtion under binoculr microscope. Seeds incubted in the light were scored dily nd germinted seeds discrded, while seeds incubted in the drk were scored only t the end of the test to void ny exposure to irrdince. In order to evlute the effect of temperture nd inter-popultion vribility, germintion tests were conducted on seeds of ech seed lot (for seed lot detils, see Tble 1). Three replictes of 20 seeds ech were incubted t rnge of constnt tempertures (5, 10, 15, 20 nd 258C) nd n lternting temperture regime (25/108C) in the light (12 h of irrdince per dy) in growth chmbers. In the lternting temperture regime, the higher temperture period coincided with the light period. To evlute the effect of slt stress on seed germintion, seeds from seed lot S2 (see Tble 1 for detils) were sown in different NCl concentrtions (0, 100, 200, 300, 400, 500, 600 mm) nd incubted t rnge of constnt tempertures (10, 15, 208C) in the light. To evlute the inter-popultion vribility in slt stress response, seed germintion in NCl solutions ws lso tested t 158C for seeds from ech popultion t the concentrtions of 0, 200, 400 or 600 mm. The incubtion period in the NCl phse ws 40 d. fter two consecutive weeks without dditionl germintion under control conditions (0 mm NCl), non-germinted seeds were wshed with distilled wter nd then sown in new Petri dishes contining 1% wter gr substrte for n dditionl 30 d (recovery phse) t the sme incubtion tempertures (Snto et l., 2014). Dt nlysis Finl germintion percentges were clculted s the verge of three replictes (^1SD) on the bsis of filled seeds, while mortlity ws clculted s the percentge of ded seeds on the bsis of the sown seeds in ech replicte. For NCl experiments, the recovery percentges (RP) were clculted ccording to the

4 334. Snto et l. following eqution (Khn nd Ungr, 1984): RP ¼ {[( 2 b)/(c 2 b)] 100}, where is the totl number of seeds germinted in slt solutions plus those tht recovered to germinte in the fresh wter, b is the totl number of seeds germinted in sline solutions, nd c is the totl number of seeds. Mortlity percentges were the sum of ded seeds of the two phses (NCl nd recovery). Germintion rte ws clculted using the T 50 prmeter (time to rech 50% of germintion). Germintion percentges, RP, mortlity percentges nd T 50 vlues were nlysed by nonprmetric Kruskl Wllis test, followed by Mnn Whitney U-test, due to non-stisfctory NOV ssumptions. Grphs were relized using the softwre Sigmplot 11.0 (Systt Softwre Inc., London, UK), while ll the sttisticl nlyses were crried out using the softwre Sttistic 7.0 for Windows (SttSoft Inc., Tuls, Oklhom, US). Results Germintion tests Effect of light nd temperture Light did not ffect seed germintion nd no significntly different germintion percentges were detected between light nd drkness t ny of the tested tempertures (108C: c. 68%; 158C: c. 80%; 208C: c. 75%; dt not shown). Therefore ll subsequent germintion tests were conducted in the light. t 58C finl germintion differed mong popultions nd low germintion percentges (c. 15%) were detected for CO1 nd CO2 (Fig. 1). t 108C, no significnt differences were detected mong popultions (Fig. 1), while t 158C, germintion percentges of CO2 (c. 30%) were significntly lower (P, 0.05) with respect to ll other seed lots (Fig. 1). Lower germintion percentges were detected for CO2 t 208C (c. 40%) s well s t 258C (c. 18%) (Fig. 1). t the lternting temperture regime of 25/108C, S4 showed the highest germintion percentges (c. 85%) while the lowest vlues were detected for CO2 (c. 30%) (Fig. 1). For the four Srdinin popultions nd the Corsicn CO1, no significnt differences were detected mong germintion percentges t the different tempertures. For CO2, the highest germintion percentges were detected t 108 nd 208C (c. 50%), wheres the lowest were observed t 58 nd 258C (c. 14%) (Fig. 1). Germintion rte significntly differed mong tempertures for ech popultion (P, 0.01 for S1 nd S2; P, 0.05 for ll other popultions, by Kruskl Wllis test). No significnt differences (P. 0.05) were detected t the sme temperture mong popultions, with the exception of 158C, for which T 50 of popultions differed significntly S1 S2 S3 S4 CO1 CO2 C C C C C C C C D CD CD Germintion (%) C b c C cb C c 20 c D Temperture ( C) _10 Figure 1. Germintion percentges in the light t constnt (5 258C) nd lternting temperture regime (25/108C, 12/12 h) for the six popultions of Rouy polygm investigted in this study. Codes S1, S2, S3 nd S4 indicte R. polygm popultions from Srdini, while CO1 nd CO2 re from Corsic (see Tble 1 for the explntion of the popultion codes). Vlues with different letters (cpitls for different popultions t the sme temperture nd lower-cse letters for different tempertures for the sme popultion) were used to indicte significnt differences t P, 0.05 (Mnn Whitney U-test). Dt re the mens (^1 SD) of three replictes.

5 Seed germintion ecology of Rouy polygm 335 Tble 2. Germintion nd recovery percentges (RP) t the tested tempertures (10 208C), t different slt concentrtions (0 600 mm NCl) for R. polygm (popultion S2) in the light. In the NCl phse for ll the tested tempertures the period lsted 40 d, while the recovery phse lsted 30 d Temperture (8C) Percentge (%) NCl concentrtion (mm) Germintion 66.7 ^ ^ b 0 b 0 b 0 b 0 b ** Recovery (RP) 33.7 ^ ^ 10.4 b 53.3 ^ 10.4 bc 16.7 ^ ^ ^ 8.7 c * 15 Germintion 65.0 ^ ^ ^ 10.0 b 0 b 0 b 0 b 0 b ** Recovery (RP) 16.7 ^ ^ ^ ^ ^ ^ 0.0 ns 20 Germintion 75.0 ^ ^ ^ 2.9 b 0 b 0 b 0 b 0 b ** Recovery (RP) 47.6 ^ ^ ^ 2.9 b 0 b 3.3 ^ 5.8 b 1.7 ^ 2.9 bc * Germintion ns ns ns ns ns ns ns Recovery (RP) ns ns * ns ns * P vlues were considered not significnt (P. 0.05, ns), significnt (P, 0.05, *) nd highly significnt (P, 0.01, **) by Kruskl Wllis test. Cpitl letters in columns re relted to the sme slinity, while lower-cse letters in rows to the sme temperture. Vlues with different letters were used to indicte significnt differences t P, 0.05 (by Mnn Whitney U-test). (P, 0.05, by Kruskl Wllis test), with the highest vlues for CO2 (c. 20 d) nd the lowest for S3 (c. 11d; dt not shown). NCl stress nd recovery of seed germintion t 0 mm NCl no differences were detected mong tempertures nd t ll tested NCl concentrtions temperture did not influence germintion percentges (Tble 2). t 108C, c. 50% of the seeds germinted t 0 nd 100 mm, while germintion t NCl concentrtions higher thn 100 mm ws totlly inhibited (Tble 2). t 158C, c. 70% of seeds germinted t 0 nd 100 mm (Tble 2), while t 200 mm only c. 10% germinted; t concentrtions bove 200 mm, germintion ws totlly inhibited (Tble 2). t 208C, c. 65% of seeds germinted t 0 nd 100 mm (Tble 2) nd only c. 3% t 200 mm, while t NCl concentrtions bove 200 mm no germintion occurred (Tble 2). Independent of the tested temperture, t 100, 200, 400 nd 500 mm NCl, recovery percentges showed vlues without significnt differences. However, t 300 nd 600 mm RP differed significntly (P, 0.05) mong tempertures (Tble 2). In prticulr, t 300 mm, RP t 108C (c. 55%) differed significntly (P, 0.05) from tht detected t 158 nd 208C (c. 10%), while t 600 mm, RP mong the three tempertures differed significntly (Tble 2). t 108C, RP differed mong NCl concentrtions, with the higher vlues (c. 60%) t 200 nd 300 mm (Tble 2), while RP vlues detected t 100, 400, 500 nd 600 mm did not show significnt differences (Tble 2). t 158C, no significnt differences were detected mong RP, while t 208C, RP differed mong NCl concentrtions, nd the higher vlues (c. 55%) were detected t 100 nd 200 mm, while RP were significntly lower t NCl concentrtions higher thn 200 mm, rnging from 0% (400 mm) to c. 7% (300 mm, Tble 2). Seed mortlity in slt conditions t ech tested temperture (10, 15, 208C), R. polygm seed mortlity (popultion S2) ws enhnced with incresing slinity (Fig. 2). t 108C, from c. 20% t 300 mm NCl, mortlity incresed to c. 60% t the highest NCl concentrtions of 400, 500 nd 600 mm. t 158C, c. 8% of seeds died under control condition (0 mm) nd mortlity reched the vlue of c. 64% t 300 mm NCl. This ltter vlue ws not sttisticlly different (P. 0.05, by Mnn Whitney U-test) from tht detected t 400, 500 nd 600 mm (c. 50% of seeds died). t the highest temperture of 208C, seed mortlity showed percentges of c. 30% up to 200 mm, while t 300, 400 nd 500 mm the number of ded seeds incresed to c. 65%; t the highest NCl concentrtion of 600 mm c. 95% of seeds died. Significnt differences (P, 0.05, by Kruskl Wllis test) mong tempertures were detected only t 600 mm NCl, t which the highest temperture of 208C corresponded the highest seed mortlity vlue (c. 95% cited bove) which ws significntly different (P, 0.05, by Mnn Whitney U-test) from tht detected t 10 nd 158C t the sme NCl concentrtion. Inter-popultion vribility in NCl response Under control conditions (0 mm NCl), the four Srdinin popultions germinted with percentges of c. 65% nd CO2 showed the lowest vlues (c. 35%; Tble 3). t 200 mm NCl, germintion percentges decresed considerbly compred with 0 mm vlues, nd differed mong popultions (Tble 3). Germintion percentges of Srdinin popultions (c. 13%) were significntly different (P, 0.05) from those of CO1 (c. 2%) nd CO2 (0%) (Tble 3). Germintion ws totlly inhibited t NCl concentrtions bove 200 mm, for ll popultions (Tble 3).

6 336. Snto et l C 15 C 20 C bcd d Mortlity (%) b b c d bc b cd c b cd b cd NCI concentrtion (mm) Figure 2. Mortlity percentges of Rouy polygm seeds (popultion S2) t constnt tempertures (10, 15, 208C) under different NCl concentrtions (0 600 mm). Vlues with different letters (cpitls for different tempertures t the sme NCl concentrtion, nd lower-cse letters for the sme temperture t different NCl concentrtions) were used to indicte significnt differences t P, 0.05 (Mnn Whitney U-test). Dt re the mens (^1 SD) of three replictes. RP vlues were higher t 200 mm thn t other higher NCl concentrtions, nd CO1 nd CO2 showed the lowest vlues (c. 17%; Tble 3); the highest RP, with vlues of c. 70%, were found for S2 nd S4. t 400 mm, no differences were detected mong RPs of Srdinin popultions while recovery of CO1 nd CO2 ws totlly inhibited (Tble 3). t 600 mm, the highest RP vlues were detected for S1 (c. 26%) nd this differed significntly (P, 0.05) from ll other vlues (Tble 3), while CO1 nd CO2 did not recover t this NCl concentrtion (Tble 3). Discussion The cpbility of R. polygm seeds to germinte with high percentges (up to 83%) in the rnge of the tested tempertures (5 258C) suggests tht they re nondormnt nd tht temperture is not limiting fctor for germintion of this species. Low germintion percentges detected in some popultions were due to high seed mortlity nd not to seed dormncy. Mny studies on picee species reported high germintion percentges (. 80%) under the optiml temperture for ech species [e.g. H. mntegzzinum (Morvcová et l., 2005);. grveolens (Thoms et l., 1979);. bermejoi (Cursch nd Rit, 2012); P. stiv (Hendrix, 1984)]. Considering the sttisticlly negligible number of imbibed seeds (c. one or two in, t most, one of the three replictes nd only t one or two tempertures) we observed tht ll non-germinted seeds died during the germintion tests, under control conditions s well. For R. polygm this pttern my indicte possible germintion during lrge prt of the yer, when there is considerble wter vilbility in the soil nd tempertures re not excessively prohibitive for seedling estblishment (Thnos et l., 1995). R. polygm seeds chieved high germintion percentges both in the light nd in the drk, therefore they re not photo-inhibited for germintion, contrry to other Mediterrnen psmmophilous species such s rssic tournefortii Goun, Ckile mritim Scop. nd chille mritim (L.) Ehrend. & Y.P. Guo (Thnos et l., 1991). R. polygm seeds showed germintion t up to 200 mm of NCl solution in the substrte, lthough in substrte with slt solutions lower germintion percentges were observed thn under control conditions (0 mm NCl). Mny studies hve reported tht germintion percentges decresed with incresed slinity stress, nd highest germintion occurs in the bsence of NCl in the substrte (Vllejo et l., 2010). t concentrtions higher thn 200 mm, R. polygm seed germintion ws totlly inhibited nd recovery showed good performnce only t lower slinity concentrtions (# 200 mm), independent of temperture, while for seeds exposed, in the previous NCl experimentl phse, t NCl concentrtions higher thn 200 mm, recovery ws unsuccessful or miniml. Only t the low temperture of 108C did

7 Seed germintion ecology of Rouy polygm 337 Tble 3. Inter-popultion vribility of R. polygm seeds in response to NCl for germintion (G) nd recovery (R) percentges t 158C Popultion code Percentge (%) NCl concentrtion (mm) S1 G 75.0 ^ ^ 5.0 bc 0 c 0 c * R 37.2 ^ 18.6 C 15.0 ^ ^ 5.8 ns S2 G 65.0 ^ ^ 10.0 bc 0 b 0 b * R 66.8 ^ ^ 5.8 b 10.0 ^ 0 b * S3 G 61.7 ^ ^ 5.8 bc 0 c 0 c * R 42.4 ^ 8.4 C 21.7 ^ ^ 8.7 ns S4 G 70.0 ^ ^ 2.9 bc 0 c 0 c * R 70.5 ^ ^ 5.8 b 16.7 ^ 7.6 b * CO1 G 55.0 ^ 5.0 C 1.7 ^ 2.9 b 0 b 0 b * R 20.0 ^ b 0 bc * CO2 G 33.3 ^ 17.5 C 0 b 0 b 0 b * R 15.0 ^ b 0 bc * G * * ns ns R * ns * P vlues were considered not significnt (P. 0.05, ns) nd significnt (P, 0.05, *), by Kruskl Wllis test. Vlues with different letters (cpitls for the sme slinity nd lower-cse for the sme popultion) were used to indicte significnt differences t P, 0.05 (Mnn Whitney U-test). See Tble 1 for n explntion of the popultion codes (S for Srdinin popultions nd CO for Corsicn ones). seeds under high slinities (. 200 mm) show their recovery cpbility (Tble 2), with performnces inversely proportionl to the concentrtion to which seeds were exposed. The highest tested temperture (208C) interfered with seed recovery nd mplified the deleterious effect of slinity on the cpbility of seeds to recover from sline conditions, s detected previously by Gum et l. (2010) for Slsol vermicult L. Slinity temperture interctions my hve significnt eco-physiologicl implictions in terms of time of germintion under field conditions, nd tolernce nd recovery from slinity nd temperture stress is lso species specific (Ungr, 1995). t high tempertures, slinity exposure could result in loss of vibility nd, consequently, poor recovery response. Seeds of some species did not recover or showed little recovery response when subjected to high slinity nd temperture stress (Khn nd Gul, 2006). R. polygm seed mortlity under slt stress conditions ws influenced by temperture. In prticulr, t 600 mm NCl, the highest tested temperture of 208C mplified the negtive effect of slinity on seed vibility (with mortlity. 95%). t ech tested temperture, seed mortlity incresed proportionlly with NCl concentrtion, nd the increse of temperture incresed seed mortlity. Severl studies hve reported tht slt stress negtively ffected seed germintion, with consequent seed mortlity, either osmoticlly (through reduced wter bsorption) or ioniclly (through the ccumultion of N þ nd Cl ), cusing n imblnce in nutrient uptke nd toxicity effect (Vllejo et l., 2010). For R. polygm, different behviours in germintion nd recovery cpbility were detected mong popultions. Intr-specific vribility in germintion ptterns hs been reported for severl species nd investigted in vrious studies (ischoff nd Müller- Schärer, 2010). Germintion of the CO2 popultion ws totlly inhibited t 200 mm NCl, highlighting higher sensitivity of seeds of this popultion to the lowest slinity. Unlike the other tested popultions, CO1 nd CO2 did not show recovery response t NCl concentrtions bove 200 mm. Seeds of ll popultions tested in this study were collected in the sme period of nturl dispersl nd in the sme hbitt typology for ll popultions. Differences in slt stress response were lso shown mong popultions of Pnicum turgidum Forssk. seeds (El-Keblwy et l., 2010) nd depended on seed provennce. lso Mrchioni-Ortu nd occhieri (1984) observed tht germintion conditions of Srdinin popultion of Crithmum mritimum L. were strikingly different from those necessry for the germintion of seeds of the sme species from the tlntic cost (Medfoot y, Englnd). In conclusion, this study provides new dt in terms of seed germintion ecology of n endngered species, improving our knowledge on its slt tolernce during the germintion process. The chieved results need to be confirmed with further nlysis, prticulrly monitoring nturl seed dispersion nd germintion in the field, nd germintion experiments should lso be crried out under nturl conditions, in order

8 338. Snto et l. to investigte the effect of temporry wter vilbility nd seed germintion t different depths under the snd surfce. Further studies on R. polygm re needed to investigte the germintion response of this species t tempertures higher thn 258C nd to clrify genetic inter-popultion vribility, considering higher number of popultions nd possibly extending to North fricn popultions. cknowledgements The uthors thnk the ssocite-editor Professor Thnos nd the nonymous reviewers for their constructive comments, which helped to improve the mnuscript. Finncil support The Centro Conservzione iodiversità (CC) is supported by the Provinci di Cgliri - ssessorto Tutel mbiente. This reserch received no specific grnt from ny funding gency, commercil or not-forprofit sector. Conflicts of interest None. References ti,., Smoui,., rhoumi, Z., bdelly, C. nd Debez,. (2011) Differentil response to slinity nd wter deficit stress in Polypogon monspliensis (L.) Desf. provennces during germintion. Plnt iology 13, skin, C.C. nd skin, J.M. (1998) Seeds: ecology, biogeogrphy, nd evolution of dormncy nd germintion. Sn Diego, cdemic Press. ischoff,. nd Müller-Schärer, H. (2010) Testing popultion differentition in plnt species: how importnt re environmentl mternl effects? Oikos 119, lsi, C., oitni, L., L Post, S., Mnes, F. nd Mrchetti, M. (2007) Stto dell biodiversità in Itli: contributo ll strtegi nzionle per l biodiversità. Rom, Plombi editori. Cruz,., Perez,., Velsco,. nd Moreno, J.M. (2003) Vribility in seed germintion t the interpopultion, intrpopultion nd intrindividul levels of the shrub Eric ustrlis in response to fire-relted cues. Plnt Ecology 169, Cursch, J. nd Rit, J. (2012) Reproductive biology nd reproductive output ssessment in nturl nd introduced subpopultions of pium bermejoi, Criticlly Endngered endemic plnt from Menorc (western Mediterrnen). Nordic Journl of otny 30, El-Keblwy,., l-nsri, F. nd l-shmsi, N. (2010) Effects of temperture nd light on slinity tolernce during germintion in two desert glycophytic grsses, Lsirius scindicus nd Pnicum turgidum. Grss nd Forge Science 66, Gum, I.R., Pdròn-Mederos, M.., Sntos-Guerr,. nd Reyes-etncort, J.. (2010) Effect of temperture nd slinity on germintion of Slsol vermicult L. (Chenopodicee) from Cnry Islnds. Journl of rid Environments 74, Hendrix, S.D. (1984) Vrition in seed weight nd its effect on germintion in Pstinc stiv L. (Umbellifere). mericn Journl of otny 71, Hong, T.D., Linington, S.H. nd Ellis, R.H. (1998) Compendium of informtion on seed storge behviour, Vol. 2. Kew, UK, Royl otnic Grdens. Khn, M.. nd Gul,. (2006) Hlophyte seed germintion. pp in Khn, M..; Weber, D.J. (Eds) Eco-physiology of high slinity tolernt plnts. Dordrecht, Netherlnds, Springer. Khn, M.. nd Ungr, I.. (1984) The effect of slinity nd temperture on the germintion of polymorphic seeds nd growth of triplex tringulris Willd. mericn Journl of otny 71, Mrchioni-Ortu,. nd occhieri, E. (1984) study of the germintion responses of Srdinin popultion of se fennel (Crithmum mritimum). Cndin Journl of otny 62, Mrtin,.C. (1946) The comprtive internl morphology of seeds. mericn Midlnd Nturlist 36, Mun, M.. (2009) The biology of costl snd dunes. Oxford, UK, Oxford University Press. Medil, F. nd Quézel, P. (1999) iodiversity hotspots in the Mediterrnen sin: Setting globl conservtion priorities. Conservtion iology 13, Meot-Duros, L. nd Mgnè, C.(2008) Effect of slinity nd chemicl fctors on seed germintion in the hlophyte Crithmum mritimum L. Plnt Soil 313, Morvcová, L., Perglová, I., Pyšek, P., Jrošík, V. nd Pergl, J. (2005) Effects of fruit position on fruit mss nd seed germintion in the lien species Hercleum mntegzzinum (picee) nd the implictions for its invsion. ct Oecologic 28, Prdis, G. nd Géhu, J.M. (1992) Observtions synécologiques sur l espéce protégée Rouy polygm (Desf.) Coincy, dns ses sttions corses. Documents Phytosociologiques 14, Pujol, J.., Clvo, J.F. nd Rmírez-Diz, L. (2000) Recovery of germintion from different osmotic conditions by four hlophytes from southestern Spin. nnls of otny 85, Snn, M., Mttn, E., Ventimill, P. nd cchett, G. (2009) Germintion ecology of Ferul rrigonii nd F. communis (picee): comprtive pproch. p. 250 in cchett, G. (Ed.) iodiversity hotspots in the Mediterrnen re: species, communities nd lndscpe level. ook of bstrcts, 45th Interntionl Congress of SISV & FIP, Cgliri, June. Snto,., Fenu, G. nd cchett, G. (2013) Schede per un List Ross dell Flor vscolre e crittogmic Itlin: Rouy polygm (Desf.) Coincy. Informtore otnico Itlino 45, Snto,., Mttn, E., Frigu, L. nd cchett, G. (2014) Light, temperture, dry fter-ripening nd slt stress

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