Variation of seed heteromorphism in Chenopodium album and the effect of salinity stress on the descendants

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1 Annls of Botny 15: , 21 doi:1.193/o/mcq6, ville online t PART OF A HIGHLIGHT SECTION ON SEEDS Vrition of seed heteromorphism in Chenopodium lum nd the effect of slinity stress on the descendnts Shixing Yo, Hiyn Ln* nd Fuchun Zhng Xinjing Key Lortory of Biologicl Resources nd Genetic Engineering, College of Life Science nd Technology, Xinjing University, Urumqi 8346, Chin * For correspondence. E-mil lnhiyn@xju.edu.cn Received: 1 Novemer 29 Returned for revision: 14 Decemer 29 Accepted: 11 Ferury 21 Bckground nd Aims Chenopodium lum is well-known s serious weed nd is slt-tolernt species inhiting semi-rid nd light-sline environments in Xinjing, Chin. It produces lrge mounts of heteromorphic (lck nd rown) seeds. The primry ims of the present study were to compre the germintion chrcteristics of heteromorphic seeds, the diversity of plnt growth nd seed prolifertion pttern of the resulting plnts, nd the correltion etween NCl stress nd vrition of seed heteromorphism. Methods The phenotypic chrcters of heteromorphic seeds, e.g. seed morphology, seed mss nd totl seed protein were determined. The effects of dry storge t room temperture on dormncy ehviour, the germintion response of seeds to slinity stress, nd the effect of slinity on growth nd seed prolifertion with plnts derived from different seed types were investigted. Key Results Blck nd rown seeds differed in seed morphology, mss, totl seed protein, dormncy ehviour nd slinity tolernce. seeds were lrge, non-dormnt nd more slt tolernt, nd could germinte rpidly to high percentge in wider rnge of environments; lck seeds were slt-sensitive, nd lrge proportion of seeds were dormnt. These chrcteristics vried etween two popultions. There ws little difference in growth chrcteristics nd seed output of plnts produced from the two seed morphs except when plnts were sujected to high slinity stress. Plnts tht suffered higher slinity stress produced more rown (slt-tolernt) seeds. Conclusions The two seed morphs of C. lum exhiited distinct diversity in germintion chrcteristics. There ws significnt difference in plnt development nd seed prolifertion pttern from the two types of seeds only when the prent plnts were treted with high slinity. In ddition, seed heteromorphism of C. lum vried etween the two popultions, nd such vrition my e ttriuted, t lest in prt, to the slinity. Key words: Chenopodium lum, development of descendnts, slinity tolernce, seed germintion, vrition of seed heteromorphism. INTRODUCTION Seed germintion is the crucil stge in life cycles of mny plnt species (Khn nd Ungr, 1996) nd for the nnuls growing in unpredictle environments, the germintion strtegy my e the most significnt fctor determining seedling survivl nd the mintennce of their popultions from one yer to the next (Keiffer nd Ungr, 1997). In the course of evolution, the ility of some plnt species to produce different types of seeds in single plnt, i.e. seed heteromorphism, hs een mostly oserved in those species distriuted in semi-rid, sline nd other unfvorle environments (Sorensen, 1978; Imert, 22). Heteromorphic seeds often differ in colour, shpe nd mss, nd re frequently ccompnied y differences in dispersl, germintion chrcteristics, dormncy ehviour, ility to persist in seed nk nd seedling growth (Bker nd O Dowd, 1982; Mndák nd Pyšek, 21; for n overview, see Imert, 22; Brändel, 24). The most ovious morphologicl differences etween heteromorphic seeds re often in their seed cot structure nd seed size. Thickness of the seed cot plys n importnt role in germintion, s it my restrict wter uptke nd/or gs diffusion, nd prevent rdicle protrusion (Mohmed-Ysseen et l., 1994). Lrge seeds often hve high reserve mss which is stored in the emryo nd/or endosperm (Imert, 22), consequently, seedlings derived from lrge seeds re usully lrger nd my hve higher seed output (Ellison, 1987; Imert et l., 1996; Mndák nd Pyšek, 25). The vrition in dormncy level etween heteromorphic seeds lso exerts influence y extending the germintion period of these species nd the formtion seed nk for the long-term recruitment of seedlings (Ungr, 1987). Furthermore, growing ody of evidence hs shown tht different types of heteromorphic seeds in mny species respond to environmentl stress differently. Lrge or rown seeds re usully more slt tolernt thn the smll or lck ones (Khn et l., 24), for exmple in Atriplex centrlsitic (Li et l., 28), Hlopyrum mucrontum (Khn nd Ungr, 21), Sued rlocspic (Wng et l., 28) nd S. splendens (Redondo-Gómez et l., 28). Tken together, seed heteromorphism cn e considered et-hedging strtegy to ensure seedling estlishment nd popultion succession of those plnt species in the sesonlly fluctuting nd distured res (Venle, 1985). Although much emphsis hs een put on the differences etween heteromorphic seeds, nd the consequences of such # The Author 21. Pulished y Oxford University Press on ehlf of the Annls of Botny Compny. All rights reserved. For Permissions, plese emil: journls.permissions@oxfordjournls.org Downloded from y guest on 5 July 218

2 116 Yo et l. Vrition of seed heteromorphism in Chenopodium lum differences for seed germintion, plnt growth nd seed prolifertion, few questions re still incompletely nswered. Firstly, few previous studies of seed heteromorphism hve confirmed vrition in seed heteromorphism (e.g. hetero-seeds rtio, differences in germintion of hetero-seeds, etc.) mong popultions, lthough some reserchers found tht the heteroseeds rtio might chnge in response to chnging environments (Brändel, 27; Song et l., 28). Secondly, mny plnt species distriuted in semi-rid res, where soil sliniztion is ecoming more serious prolem, hve een reported to exhiit seed heteromorphism. Even so, few studies hve focused on the response of seed germintion nd the development of plnts from these seeds to slinity (Wei et l., 27; Wng et l., 28). Thirdly, little informtion is ville out the effect of seed heteromorphism on plnt growth nd seed prolifertion of the offspring derived from different types of seeds, especilly under different environmentl stresses (Mndák nd Pyšek, 25). Chenopodium lum (Chenopodicee) produces two distinct lots of seeds (morphs), some (pprox. 3. %) with rown seed cot re non-dormnt nd germinte rpidly fter the initil hrvest, the mjority with lck seed cot, re dormnt (Willims nd Hrper, 1965). Therefore, most previous reserch on C. lum hs een imed t investigtion of the germintion strtegy of the lck seeds in reltion to etter control of this hrmful weed (Willims nd Hrper, 1965; Henson, 197; Krssen, 1976; Roerts nd Benjmin, 1979; Sini et l., 1985; Ahmed, 1987). Chenopodium lum is distriuted widely over the semi-rid res in Xinjing province, Chin (Commissione Redctorum Flore Xinjingensis, 1994). Among the mny environmentl fctors in this re, the comintion of high temperture nd strong sunlight during the summer seson leds to high wter evportion, which, together with little precipittion, cuses grdully incresing slinity in the soil surfce (Khn nd Ungr, 1996; Song et l., 25; Munns nd Tester, 28). This ecomes n importnt limiting fctor for plnt growth nd survivl (Rozem nd Flowers, 28). It ws found tht C. lum locted in semi-rid res of Xinjing produces heteromorphic seeds,.5 % of which were rown (Yo et l., unpul. res.), n oservtion tht ws significntly different from previous studies (Willims nd Hrper, 1965; Krssen et l., 1989). Moreover, plnts of C. lum could grow normlly under 3 mmol L 21 NCl stress (Yo et l., 21), nd my e considered s sltresistnt species (Hmidov et l., 27). However, the responses of germintion nd the growth of descendnts from heteromorphic seeds of C. lum to slinity were poorly documented, lthough these my provide evidence for the introduction of this promising nnul her to mend sline soils (Yo et l., 21). In the present study, seeds of C. lum were collected from severl popultions locted in different micro-hitts, in highly unpredictle semi-rid environment of Xinjing province. The germintion of heteromorphic seeds of C. lum from different popultions were investigted, nd it ws determined whether significnt differences exist in the growth nd seed prolifertion pttern etween plnts from different seed types under different conditions (with or without slt tretment). To this end, the following spects were studied: () The phenotypic differences etween heteromorphic seeds, including seed cot morphology nd colour, seed mss/seed reserve mss, totl seed protein; () the effect of dry storge t room temperture on dormncy ehviour of heteromorphic seeds; (c) the response of heteromorphic seeds to slinity during germintion; (d) the effect of slt stress on growth nd hetero-seed rtio of the descendnts. MATERIALS AND METHODS Seed collection The mture seeds of Chenopodium lum were collected in Novemer 27, from nturlly dry inflorescences of plnts grown in semi-rid environment ( N, E; 91 m.s.l.), ner the southern edge of the Junggr Bsin in Xinjing. Meteorologicl dt sed on the previous 3 yers (25 27) indicted tht the men temperture of the wrmest month (July) ws C nd the coldest month (Jnury) ws C. Annul precipittion (melting snow plus the rinfll) ws 31.6 mm; while the men evportion from My to Septemer mounted to mm. Seeds from the sme microhitt were pooled to form one popultion. Popultion 1 ws locted on reltively flt ground; popultion 2 ws grown on slope. These two popultions were selected for study following initil work on four popultions. The two chosen showed contrsting germintion ehviour for the lck nd rown seeds within the popultion. Seeds were nturlly ir-dried nd stored t C, % reltive humidity (RH) in rown pper gs for vrious experiments. Determintion of the rown : totl seeds rtio Three replictes with 1 rndomly chosen seeds from two popultions ( poorly developed or shrivelled seeds were excluded) were divided into lck or rown seeds depending on the seed cot colour nd mesured the rtio of rown seeds in the totl. Seed morphology oservtion A stereomicroscope SMZ8 (Nikon, Jpn) ws employed to exmine the dry seed nd surfce nd longitudinl sections. For seed cot oservtion, dry seeds were mounted directly on stus using doule-sided dhesive tpe, nd sputter-coted with gold, then imged using LEO143VP scnning electron microscope (LEO Corp., Germny). Thickness of the seed cot ws mesured using the NIS-Elements imging softwre (Nikon, Jpn). Digitl photogrphs were mnipulted with Adoe Photoshop (Adoe Photosystems) to prepre figures. Phenotypic chrcters of heteromorphic seeds Determintion of seed mss. Three replictes of 1 lck or rown seeds from ech of the two popultions were sorted for ech seed type; the seed cots were seprted from the emryo nd endosperm, nd the mss of ech prt weighed. Totl seed protein content nlysis. To gin insight into the totl protein vriility tht my correlte with seed Downloded from y guest on 5 July 218

3 Yo et l. Vrition of seed heteromorphism in Chenopodium lum 117 heteromorphism in C. lum, the totl seed protein ws mesured. Blck nd rown seeds (1 mg of ech) were homogenized in liquid nitrogen, nd then trnsferred to fresh 1.5 ml micro-tue with 5 ml extrction uffer (12.5 mmol L 21 Tris-Cl, ph 6.8). The homogente ws centrifuged t 1 g for 15 min t 4 8C. The resulting superntnt ws stored t 4 8C until use. The protein content of the extrct ws determined y the method of Brdford (1976). Seed germintion experiments Three replictes of 4 seeds from ech seed type were tested in ech tretment. For germintion, seeds were incuted on two lyers of filter pper in 9-cm Petri dish, to which 5 ml of distilled wter or queous solutions of NCl were dded. The filter pper ws renewed every 2 d to keep the NCl concentrtion unchnged. All Petri dishes were plced in n illuminted incutor (LRH-4-GII; Gungdong Medicine Apprtus Mnufctory, Chin), nd sujected to 16-h dily photoperiod (light flux: pprox. 116 mmol m 22 s 21 )t258c. A seed ws considered to e germinted when the emerging rdicle ws t lest.5 mm. Non-germinted seeds were checked under stereo microscope to see if the emryos were white nd firm, thus indicting they were live; if not, they were considered ded nd excluded from further clcultion (Bskin nd Bskin, 21). Germintion ws recorded every 12 h during 2-week period to clculte germintion rte (expressed in men time to germintion, MTG for short). MTG ws clculted y using the eqution: MTG = S i (n i d i )/N, where n is the numer of seeds germinted t dy i; d is the incution period in dys nd N is the totl numer of seeds germinted in the test (Redondo-Gómez et l., 28). According to this eqution, lower vlue indictes more rpid germintion. The finl germintion ws counted on the 15th dy. Storge t room temperture for 12 months. Storge t room temperture ws used to test the effect of lortory conditions on the dormncy ehviour of seeds. Heteromorphic seeds from ech popultion were stored t C, % RH, nd retrieved monthly for seed germintion tests, eginning t the end of Novemer 27 (pprox. 1 month fter hrvest) nd ending t the end of Novemer 28. Slinity tretments. To investigte the effect of slinity on germintion of heteromorphic seeds, different concentrtions of NCl solution (, 5, 1, 2, 3 nd 4 mmol L 21, respectively) were pplied in the germintion test. In seed recovery experiment, seeds tht filed to germinte fter 6 d in the NCl tretment were rinsed three times with distilled wter, nd then set to germinte with distilled wter. Test tretment. This experiment imed to investigte the germintion response of lck seeds to slinity when dormncy ws llevited. Seed cots covering the rdicle of lck seeds ( popultion 2) were removed crefully, nd then the seeds were trnsferred to Petri dishes contining, 5, 1, 2 or 3 mmol L 21 NCl solutions. Germintion conditions were the sme s descried in the experiment Slinity tretments. Determintion of plnt growth nd seed prolifertion of the descendnts derived from heteromorphic seeds under different slinity tretments In Octoer, 28, lck nd rown seeds of popultion 2 were sown in round plstic pots [filled with perlite : vermiculite (1 : 3)], then cultivted in greenhouse t temperture regime of C, % RH nd nturl sunlight (light flux during the dy: mmol m 22 s 21 ). Hoglnd solution ws supplied once per month, tp wter ws dded t other times when necessry. When seedlings were 4 months old (efore nthesis) they were treted with Hoglnd solutions contining different concentrtions of NCl s follows: s control, 5 mmol L 21 s lower nd 3 mmol L 21 s higher slinity. Hoglnd solutions contining NCl were supplied once per month. At the full flowering stge, plnt height nd numer of rnches ( primry) were recorded. Then the mture lck nd rown seeds were seprtely collected from ech plnt per dy for nerly 75 d (the period when plnts with the 3 mmol L 21 tretment completed their life cycles), nd the totl numer of seeds nd the hetero-seeds rtio per plnt were clculted. Sttisticl nlysis All dt were expressed s men + stndrd error. Percentges were rcsine trnsformed efore sttisticl nlysis to ensure homogeneity of vrince. Unpired t-test ws used to compre two smples (or tretments). For more thn two smples (or tretments), one- or two-wy ANOVA ws employed to compre tretment effects using the GrphPd Prism Version 4.2 for Windows (GrphPd Softwre, Sn Diego, CA, USA). When significnt min effects existed, differences were tested y multiple comprison Tukey test t the.5,.1,.1 nd.1 significnce levels. RESULTS Vrition of the seed heteromorph rtio nd morphology etween two popultions Seeds of C. lum from two popultions grown in different micro-hitts were collected in the utumn. The two seed lots mtured lmost simultneously with no specil dispersl structure; proportion of the seed would e dispersed y wind while some would drop on the ground surrounding the mother plnt. Both of the two popultions hd higher proportion of rown seeds (.5 %) thn lck seeds. Moreover, the percentge of rown seeds ws significntly higher in popultion 1 thn in popultion 2 (65 % nd 52 %, respectively; unpired t-test, P,.1). Both the lck nd rown seeds from the two popultions hd n olte spheroid shpe, nd hd lck nd rown teste, respectively (Fig. 1A D). In longitudinl section, oth seed morphs presented s circulr emryo surrounding the centrl endosperm (Fig. 1E H). Further inspection of the germintion process indicted tht, in ll seeds from oth popultions, the rdicle hd to rek through the seed cot mechniclly for protrusion nd germintion (Fig. 1I L). Scnning electron microscopy reveled tht the test of the lck seeds from oth popultions ws more thn twice s Downloded from y guest on 5 July 218

4 118 Yo et l. Vrition of seed heteromorphism in Chenopodium lum A E I M B F J N C G K O D H L P F IG. 1. Morphology of heteromorphic seeds of Chenopodium lum: (A, E, I, M) the lck seed from popultion 1; (B, F, J, N) the lck seed from popultion 2; (C, G, K, O) the rown seed from popultion 1; (D, H, L, P) the rown seed from popultion 2; (A D) dry mture seeds; (E H) longitudinl section of seeds; (I L) position of rdicle protrusion; (M P) scnning electron microgrphs of seed cot, showing the thickness of the test. Scle rs: (A L).5 mm; (M P) 2 mm. Arevitions: es, endosperm; cl, cotyledon; r, rdicle; g, germinl perture; sc, seed cot. thick s tht of the rown seeds (Fig. 1M P). Furthermore, there ws no significnt difference etween popultions in the thickness of the seed cot of either lck or rown seeds (unpired t-test, P..5). However the seed cot of the lck seed from popultion 2 ws much hrder compred with tht of popultion 1. Differences in some chrcteristics of heteromorphic seeds nd germintion ehviour etween two popultions Seed mss distriution. Within the two popultions, significntly lower proportion of totl seed mss ws llocted to the seed reserve mss (emryo plus endosperm) in the lck seed compred with the rown seed (Fig. 2A; unpired t-test, popultion 1, P,.1; popultion 2, P,.1). In ddition, the seed reserve mss in the lck seed from popultion 1 ws higher thn tht from popultion 2 (unpired t-test, P,.1), lthough there ws no significnt difference in the rown seed etween two popultions (unpired t-test, P..5). Totl seed protein content. The totl seed protein content ws much lower in the lck seeds thn in the rown seeds (Fig. 2B; unpired t-test, popultion 1, P,.1; popultion 2, P,.1). There ws smll ut non-significnt difference in the protein content of the lck seeds in the two popultions (unpired t-test, P..5), wheres the protein content of the rown seeds in popultion 1 ws much lower thn tht in popultion 2 (unpired t-test, P,.1). Downloded from y guest on 5 July 218

5 Yo et l. Vrition of seed heteromorphism in Chenopodium lum seed mss (g) Protein content (mg g 1 ) Germintion (%) Germintion (%) A B C D Emryo plus endosperm Seed cot P1 P2 P1 P2 Blck Blck P1 P2 P1 P2 Popultion Time (d) Blck Germintion in distilled wter. The germintion ehviour of the heteromorphic seeds in distilled wter differed drmticlly etween the two seed morphs nd popultions P1 P F IG. 2. Mss distriution etween seed cot nd emryo plus endosperm (A), totl seed protein content (B) nd germintion in distilled wter (C, D) of lck nd rown seeds in Chenopodium lum. P1, Popultion 1; P2, popultion 2. Vlues re mens + s.e. from three replictes. (Fig. 2C, D). The rown seeds germinted more quickly, nd to higher finl percentge, thn the lck seeds within oth popultions. The rown seeds of popultion 1 germinted more slowly thn those from popultion 2 (unpired t-test, P,.1), lthough oth reched finl germintion percentge of nerly 1 %. In contrst, the lck seeds of popultion 1 hd much higher finl germintion (lmost 1 %; Fig. 2C) thn popultion 2 (1.8 %; Fig. 2D, unpired t-test, P,.1). Effect of storge t room temperture (RT) for 12 months on seed germintion The effect of dry storge t RT on the seed dormncy chrcteristics vried etween morphs nd popultions (Fig. 3). Dry storge t RT hd little effect on the germintion percentge of the rown seeds from oth popultions (two-wy ANOVA: time, P ¼.154; popultion, P ¼.192; time popultion, P ¼.66). The freshly mture rown seeds in oth popultions were non-dormnt, nd germinted pprox. 1 % for the whole yer (Fig. 3A nd C). In contrst, there ws considerle vrition in the dormncy ehviour of the lck seeds etween the two popultions. At the eginning of the experiment, only 2 % of the lck seeds in popultion 2 germinted (Fig. 3C), while nerly 1 % germintion in popultion 1 ws oserved during most of the yer (Fig. 3A; dt ville only from April to Novemer). The dormncy of the lck seeds in popultion 2 ws reduced y fterripening during winter nd erly spring (Novemer to April of the next yer), s germintion incresed to the mximum percentge of pprox. 5 % fter storge for 6 months (April; Fig. 3C). However, the germintion of the lck seeds susequently fell to,2 % in My, nd remined low from My to Novemer (Fig. 3C). The rte of germintion lso vried with dry storge t RT (Fig. 3B, D), nd significnt popultion time interction in two-wy ANOVA nlysis confirmed such n effect (Fig. 3B, D; lck seeds, P,.5; rown seeds, P,.1). The germintion rte of the lck seeds in popultion 2 incresed from Novemer to June of the next yer (decresed MTG; Fig. 3B). In popultion 1, there ws smll decrese in MTG, i.e. germintion rte incresed, from April to June, nd then decresed from July to Novemer. A similr trend ws found in rown seeds. Effect of slinity stress on seed germintion There ws no effect of slinity on the seeds of either popultion from to 2 mmol L 21 NCl (Fig. 4A nd B). At higher slinities, smll proportion (2 %) of rown seeds from oth popultions germinted t 3 mmol L 21 NCl (Fig. 4C, D), ut none t 4 mmol L 21 NCl, while no lck seeds germinted t either 3 or 4 mmol L 21. After 6 d of exposure to vrious NCl concentrtions, seeds tht filed to germinte under 3 nd 4 mmol L 21 NCl were set to germinte with distilled wter (Fig. 4C nd D). The re-germintion of the rown seeds in oth popultions ws similr, reching finl percentge of.8 %. Further tests showed tht non-germinted rown seeds hd decyed. In contrst, the response of lck seeds from the two Downloded from y guest on 5 July 218

6 12 Yo et l. Vrition of seed heteromorphism in Chenopodium lum A P1 C P2 1 Germintion (%) Blck 2 Men time to germintion (d) B P1 D P2 Nov Dec Jn Fe Mr Apr My Jun Jul Aug Sep Oct Nov Nov Dec Jn Fe Mr Apr My Jun Jul Aug Sep Oct Nov Retrieved month Retrieved month FIG. 3. Finl germintion percentge (A, C) nd men time to germintion (MTG; B, D) of lck nd rown seeds of Chenopodium lum in two popultions following 1 13 months of storge t room temperture (2 25 8C) in the drk. P1, Popultion 1; P2, popultion 2. Vlues re mens + s.e. from three replictes. Germintion (%) A Blck P1 C Re(BI) Re(Br) BRe P1 2 Germintion (%) B P2 3 4 NCl (mmol L 1 ) NCl (mmol L 1 ) D P2 FIG. 4. Effect of NCl on finl germintion percentge of heteromorphic seeds of Chenopodium lum in popultion 1 (A, C) nd popultion 2 (B, D). After seeds were treted with 3 nd 4 mmol L 21 NCl for 6 d, non-germinted seeds were trnsferred to distilled wter for recovery nd germintion for nother 12 d. Different letters indicte significnt differences t the sme NCl concentrtion (P,.5). Re(Bl), fter recovery of lck seeds; Re(Br), fter recovery of rown seeds; BRe, efore recovery. Vlues re mens + s.e. from three replictes. popultions to re-germintion ws significntly different. Following oth the 3 nd 4 mmol L 21 NCl pretretments, the lck seeds of popultion 1 were le to recover germintion to 1 % (Fig. 4C), while lck seeds from popultion 2 filed to recover. However, further tests indicted tht non-germinted lck seeds remined live. Downloded from y guest on 5 July 218

7 Yo et l. Vrition of seed heteromorphism in Chenopodium lum 121 To investigte further the response of dimorphic seeds to slt stress, the seed cot covering the rdicle of the lck seeds ws removed efore germintion in sline conditions. The germintion of lck seeds hving hd prt of the seed cot removed, improved under slinity stress in comprison to non-treted lck seeds (Fig. 5; two-wy ANOVA, P,.1). However, inhiition of germintion y slinity ws still greter in the lck seeds with dormncy relieved thn it ws in rown seeds, s indicted y significnt seed type slt concentrtion interction (P,.1). Effect of NCl stress on plnt growth nd seed prolifertion of the descendnts derived from heteromorphic seeds Plnt growth. The effect of slinity on the growth of the plnts derived from lck nd rown seeds ws different (Fig. 6). The plnt height from rown seeds ws greter thn tht from lck seeds under vrious NCl concentrtions, ut only 3 mmol L 21 NCl resulted in significnt difference (Fig. 6A; plnt type in two-wy ANOVA, P,.5). A similr trend ws Germintion (%) c Blck TT-Blck NCl (mmol L 1 ) FIG. 5. Effect of slinity on germintion of non-treted lck seeds (Blck), test-treted lck seeds (TT-Blck) nd non-treted rown seeds () of Chenopodium lum. All of these seeds were from popultion 2. For given NCl concentrtion, vlues shring common letter re not significntly different t P,.5. Test tretment mens seed cot covering rdicle ws removed. Vlues re mens + s.e. from three replictes. c c lso oserved with the rnch numer, lthough no significnt difference ws shown (Fig. 6B; plnt type in two-wy ANOVA, P..5). Seed prolifertion. The seeds set on plnts from the dimorphic seeds mtured nd egn shedding 5 months fter eing sown. Seeds were collected upon the first seed mturity. The numer of the two types of seeds did not differ significntly on plnts from the two morphs t the eginning of mturtion t ny of the NCl concentrtions pplied during plnt growth (Fig. 7). After the first 3 d, the pttern of production of the dimorphic seeds pprently chnged. In the or 5 mmol L 21 NCl tretment, plnts from the two morphs produced lck seeds more quickly thn rown seeds, lthough only plnts from rown seeds showed significnt difference (Fig. 7A, B, D nd E; time seed type interction in two-wy ANOVA, Br-plnts: mmol L 21, P,.1; 5 mmol L 21, P,.1). However, when under 3 mmol L 21 NCl stress, plnts from two morphs produced lck seeds much more slowly thn rown seeds (Fig. 7C nd F; time seed type interction in two-wy ANOVA, Bl-plnts: P,.1; Br-plnts: P,.1). The output of totl seeds ws higher for plnts from rown seeds thn from lck seeds grown under vrious NCl concentrtions. The highest totl numer of seeds (.5) ws oserved for the plnts from rown seeds t 5 mmol L 21 NCl, the lowest (,2) for the plnts from lck seeds t 3 mmol L 21 NCl. A significnt difference in totl seeds etween two types of plnts ws oserved t 3 mmol L 21 (Fig. 7; unpired t-test, P,.5). Furthermore, plnts from the two morphs produced.7 % of rown seeds under 3 mmol L 21 NCl tretment, compred with,5 % of rown seeds for or 5 mmol L 21 NCl (Fig. 8; NCl concentrtion in two-wy ANOVA: P,.1). Plnts from lck seeds produced more rown seeds thn plnts from rown seeds, ut significnt difference ws only oserved t 5 mmol L 21 NCl (unpired t-test, P,.5), suggesting tht the rtio of rown seeds in totl ws promoted significntly y high slinity within oth plnt types from dimorphic seeds. Brnch numer Plnt height (cm) A B 5 3 NCl (mmol L 1 ) Bl-plnts Br-plnts F IG. 6. Effect of slt stress on (A) plnt height nd (B) rnch numer of Chenopodium lum plnts derived from heteromorphic seeds. The growth chrcters were recorded fter plnts were treted with slinity for 3 months. Vlues re mens + s.e. from ten plnts per tretment. Different letters ove the rs indicte significnt differences (P,.5). Bl-plnts, Plnts grown from lck seeds; Br-plnts, plnts grown from rown seeds. c c DISCUSSION Mny plnt species inhiting unpredictle nd stressful environments hve een reported to exhiit seed heteromorphism (dimorphism), which enles germintion to occur t the right time in fvourle plce to ensure popultion estlishment nd succession (Venle, 1985; Imert, 22). Chenopodium lum is serious weed species with seed heteromorphism, which is widely distriuted ll over the world (Willims nd Hrper, 1965; Ahmed, 1987), including the semi-rid res in Xinjing province, Chin (Commissione Redctorum Flore Xinjingensis, 1994; Yo et l., 21), nd hs een oserved s slt-tolernt species (Hmidov et l., 27). The present study hs shown tht C. lum grown in semi-rid res produced two distinct types of seeds, lck nd rown, which differ in morphology, germintion ehviour, dormncy nd slinity tolernce, nd these chrcteristics vried etween popultions. Furthermore, higher slinity could led to vrition in seed heteromorphism within plnts of either morph. Slinity stress is suggested s one of the environmentl fctors tht my induce the vrition Downloded from y guest on 5 July 218

8 122 Yo et l. Vrition of seed heteromorphism in Chenopodium lum A BI-plnts ( mmol L 1 ) D Br-plnts ( mmol L 1 ) 3 Seeds per plnt Seeds per plnt Seeds per plnt B C Blck BI-plnts (5 mmol L 1 ) BI-plnts (3 mmol L 1 ) E F Br-plnts (5 mmol L 1 ) Br-plnts (3 mmol L 1 ) Time fter first seed mturtion (d) Time fter first seed mturtion (d) FIG. 7. Effect of slinity on dimorphic seeds setting of ech plnt derived from lck nd rown seeds of Chenopodium lum, respectively. Bl-plnts, Plnts grown from lck seeds; Br-plnts, plnts grown from rown seeds. Vlues re mens + s.e. from ten plnts per tretment. /totl seeds (%) Bl-plnts Br-plnts c c 5 3 NCl (mmol L 1 ) FIG. 8. Effect of slinity on rown : totl seeds rtio of Chenopodium lum. Vlues re mens + s.e. from ten plnts per tretment. Different letters ove the rs indicte significnt differences (P,.5). Bl-plnts, Plnts grown from lck seeds; Br-plnts, plnts grown from rown seeds. in seed heteromorphism of C. lum in semi-rid nd lightsline res seen in the present study. Different popultions within heteromorphic species my fce diverse environmentl conditions in heterogeneous res (Imert, 22), therefore it is unlikely tht seed heteromorphism (reflected s either hetero-seeds rtio or differences in c germintion of hetero-seeds) would remin stle mong popultions (Venle et l., 1987; Vn Molken et l., 25). Indeed there is evidence indicting the possiility of vrition in seed heteromorphism mong popultions, prtilly s the result of chnging environments (Dkshini nd Aggrwl, 1974; nd Mitchell, 1984). In ddition, the proportion of lrger seeds might increse in response to more stressed conditions in some species, e.g. Atriplex tringulris (Ungr, 1987) nd Sued sls (Song et l., 28). The ove oservtions support the hypothesis tht differences in environmentl conditions (e.g. soil moisture, consequently slinity, etc.) might e responsile for the vrition of the proportion of heteromorphic seeds in C. lum. In the present study, vrition in seed heteromorphism (e.g. hetero-seeds rtio, vrying of the difference in germintion of hetero-seeds) of C. lum etween two popultions reveled tht the proportion of rown seeds ws higher in popultion 1 (out 65 %) thn in popultion 2 (out 52 %), nd lso significntly higher thn those popultions in previous report (pprox. 3 %) (Willims nd Hrper, 1965). The two popultions hd grown up in different micro-hitts, flt ground for popultion 1 nd reltively sloping ground for popultion 2. The two micro-hitts were not fr prt nd differences in illumintion nd temperture were unlikely to exist in the two Downloded from y guest on 5 July 218

9 Yo et l. Vrition of seed heteromorphism in Chenopodium lum 123 environments. However, the soil moisture might differ, possily leding to differences in the slinity in the upper lyer of the soil in the summer, since evportion under strong sunlight nd high tempertures in semi-rid res cuse drmtic increse in slinity of the soil surfce y cpillry movement (Khn nd Ungr, 1996; Khn et l., 21; Song et l., 25). seeds seem to e more dvntgeous in hrsh nd unpredictle conditions thn lck seeds (Imert, 22; Khn et l., 24; Song et l., 28), thus production of high proportion of the rown morph my hve n dptive vlue in semi-rid environments. Aprt from the hetero-seeds rtio, in the present study, vrition of the heteromorphism ws lso exhiited in seed cot thickness nd hrdness, seed reserve mss, germintion nd dormncy ehviour, etc., with the lck seeds in two popultions. Much ttention hs een pid to the mechnisms of germintion in heteromorphic seeds, nd the phenotypic differences hve een considered s the min resons for the diversity of germintion ehviour (Imert, 22). In the present study, two popultions were exmined to otin more informtion on the germintion differences etween two seed types of C. lum. seeds in oth popultions hd thinner, softer teste thn the lck seeds nd germinted to pprox. 1 %. Differences in the germintion of the lck seeds in the two popultions were not significntly relted to differences in the colour nd thickness of seed cot. However, the seed cot of the lck from popultion 2 ws much hrder thn tht from popultion 1, nd the improved germintion following prtil removl, suggested tht the hrdness, s well s the thickness, of the seed cot might prevent the seed from germinting, either s result of mechnicl resistnce to rdicle protrusion, or due to impermeility to wter (Mohmed-Ysseen et l., 1994; Deeujon et l., 2). The higher seed reserve mss (i.e. lrger seed size) nd higher protein content of rown seeds suggested tht more energy might e ville for rdicle to rek the seed cot in the rown seeds, nd contriute to the difference in the germintion for the lck seeds in the two popultions. This ws consistent with the higher germintion of lrger seeds in some heteromorphic species (Venle et l., 1987; Kteme et l., 1998; Vn Molken et l., 25). Tken together, the thickness nd hrdness of the seed cot, comining with seed reserve mss nd protein content my contriute to the vrition in the germintion of heteromorphic seeds in C. lum. In the present study, the rown nd lck seeds from popultion 1 were non-dormnt nd mintined high germintion percentge over the whole yer; while lrge proportion of the lck seeds from popultion 2 were dormnt, nd exhiited n nnul fluctuting pttern of germintion with the highest germintion percentge (.5 %) in April. A similr fluctuting germintion pttern ws oserved for peripherl chenes of Leontodon sxtilis (Brändel, 27), seeds of desert species Mesemrynthemum nodiflorum (Guttermn, 198/1981). This might suggest tht the germintion of lck seeds of C. lum fluctuted s response to the chnges of environmentl conditions, which might confer selective dvntge in dpttion to environments (Venle et l., 1987; Crter nd Ungr, 23). In ddition, the chnges in the dormncy of lck seeds over 1 yer my extend the timing of germintion nd help to mintin the popultion (Wng et l., 28). Seed germintion under slinity in the present study suggested tht the rown seeds of C. lum were more slt tolernt thn the lck seeds. Similr cses hve een reported in severl hlophytic chenopods hving heteromorphic seeds, with rown nd lck seed types exhiiting different slt tolernce, s in Atriplex centrlsitic (Li et l., 28), Sued rlocspic (Wng et l., 28), S. sls (Li et l., 25) nd S. splendens (Redondo-Gómez et l., 28). As fr s we know, so fr the reson why heteromorphic seeds disply different slt tolernces is mostly unknown. We speculte tht the more slt-tolernt nture of rown seeds my relte to their lrger size nd higher protein content. The present study provided little evidence of differences in the growth chrcteristics of the plnts from heteromorphic seeds. However, when the two types of plnts were sujected to slinity stress, significnt differences were exhiited for plnt height nd totl seed output t 3 mmol L 21 NCl tretment. The results suggested tht higher slinity might enlrge the developmentl differences etween the two types of plnts. Similrly, plnts from rown nd lck seeds of S. splendens responded to slinity stress differently t lower concentrtion (Redondo-Gómez et l., 28). However, further reserch is needed to elucidte the mechnism ffecting their performnce under slt stress. Seed heteromorphism is thought to e selective dvntge for plnt species inhiting hrsh nd heterogeneous res nd to enle such species to llocte limited resources to different seed types in response to vrile environments (Hrper, 1977). However, insufficient informtion is ville regrding the chnges of morphs rtio per inflorescence ( plnt) nd vrition in environmentl conditions. In the hlophyte S. sls, popultion from sline inlnd res produced fewer (7 %) rown seeds thn popultion from higher slinity environment (81 %) (Song et l., 28). Similrly in A. tringulris, the rtio of lrge seeds versus smll seeds ws 1 : 6 in popultion from less sline res, incresing to 1 : 6 in popultions from hitts of much higher slinity (Ungr, 1987). In the present study, high slinity tretment (3 mmol L 21 NCl) incresed the rtio of rown seeds to.7 %, significntly higher thn tht following tretment with 5 or mmol L 21 NCl (,5 % rown seeds), suggesting tht slt stress my induce the plnt to produce more rown seeds. There re however, mny other environmentl fctors, such s high temperture, strong sunlight, shortge of wter, tht constitute the environmentl stresses in semi-rid res of Xinjing (Song et l., 25) nd significnt chnges in ny of them might influence the morphs rtio, such s in the popultions 1 nd 2 in the present study. Slinity my therefore e only one of the fctors influencing the proportion of lck nd rown seeds. However, producing more, lrger, rown seeds with high germintion percentge nd greter slt tolernce, would undoutedly e n dvntgeous strtegy for plnts with heteromorphism to respond nd dpt to the stressful environments during the long term of evolution. In conclusion, the present study demonstrted tht the heteromorphic seeds of C. lum differed in phenotypic chrcters, germintion ehviour nd slinity tolernce; vrition of seed heteromorphism ws lso oserved etween popultions, nd such vrition might prtilly e ttriuted to the slinity in environments. Bsed on this pttern, we Downloded from y guest on 5 July 218

10 124 Yo et l. Vrition of seed heteromorphism in Chenopodium lum Dimorphic seeds of chenopodium lum Fvourle conditions Stress conditions (e.g. chnge of photoperiod, temp. nd humidity, slinity, etc.) Lrge proportion of lck seeds Smll proportion of rown seeds Lrge proportion of rown seeds Smll proportion of lck seeds A few seeds Most seeds Most seeds Some seeds Seed nk Next seson Seed germintion nd seedling estlishment Next seson Seed nk Reltively lrge mount of seeds per plnt Reproduction Reltively smll mount of seeds per plnt Popultion propgtion nd dpttion in different hitts FIG. 9. Proposed model of dynmics of reproduction for dimorphic seeds of Chenopodium lum under vrious environmentl conditions. propose model for the cycle of heteromorphic seeds of C. lum in the semi-rid res s shown in Fig. 9. In fvourle conditions, C. lum produces lrge mount of seeds nd greter proportion of lck seeds. Some lck seeds re expected to enter the seed nk nd mintin presence in environments to ensure popultion succession from one yer to the next. In sline nd other stressful environments, smller numer of seeds nd greter proportion of rown seeds would e produced, the immedite germintion of which my ensure the estlishment nd expnsion of popultion, to occupy limited resources which re trnsient in semirid res (Ross nd Hrper, 1972; Song et l., 25). However, seedlings tht develop from rown seeds run higher risk of mortlity ecuse of unpredictle environmentl stress occurring in erly growing sesons (Li et l., 25). Therefore, the germintion of the rown seeds in C. lum my follow high-risk strtegy, while the lck seeds seem to tke low-risk strtegy, s descried y Venle (1985). The comintion of two opposing strtegies in C. lum could undoutedly contriute to its seedling survivl nd popultion succession in semi-rid nd hrsh res in Xinjing. ACKNOWLEDGEMENTS This reserch ws supported y the Ntionl Nturl Science Foundtion of Chin (36612), the Xinjing Municipl Mjor Science nd Technology Project ( ) nd the Open Fund of the Xinjing Key Lortory of Biologicl Resources nd Genetic Engineering (XJDX ; XJDX ). We re grteful to Dr Shuzhen Zhung (in our lortory) for her kind supply of meteorologicl dt; Miss Lin Liu (PhD cndidte in our lortory) for her kind help in seed germintion experiments; Dr Zhongyun Liu (in our lortory) for his kind help in reference retrieving. We thnk the nonymous reviewers for their helpful comments on this pper. LITERATURE CITED Ahmed M Review on the germintion, morphology nd reproduction of Bthu (Chenopodium lum L.). Interntionl Journl of Tropicl Agriculture 5: Bker GA, O Dowd DJ Effects of prent plnt density on the production of chene types in the nnul Hypochoeris glr. Journl of Ecology 7: Bskin CC, Bskin JM. 21. Seeds: ecology, iogeogrphy, nd evolution of dormncy nd germintion. Sn Diego, CA: Acdemic Press. Brdford MM A rpid nd sensitive method for the quntifiction of microgrm quntities of protein utilizing the principle of protein dye inding. Anlyticl Biochemistry 72: Brändel M. 24. Dormncy nd germintion of heteromorphic chenes of Bidens frondos. Flor 199: Brändel M. 27. Ecology of chene dimorphism in Leontodon sxtilis. Annls of Botny 1: NAC, Mitchell JJ Germintion of the polymorphic fruits of Bidens ipinnt. South Afric Journl of Botny 3: Downloded from y guest on 5 July 218

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