Size-dependent gender modification in Lilium apertum (Liliaceae): doesthis species exhibit gender diphasy?

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1 nnls of otny Pge of 3 doi:.93/ob/mcu4, vilble online t Size-dependent gender modifiction in Lilium pertum (Lilicee): doesthis species exhibit gender diphsy? Zhi-Qing Zhng,, Xing-Fu Zhu, Hng Sun, Yong-Ping Yng,3 nd Spencer C. H. rrett 4, * Key Lbortory for Plnt iodiversity nd iogeogrphy of Est si, Kunming Institute of otny, Chinese cdemy of Sciences, 3 Lnhei Rod, Kunming 654, Yunnn, Chin, Key Lbortory of Tropicl Forest Ecology, Xishungbnn Tropicl otnicl Grden, Chinese cdemy of Sciences, Menglun Town 66633, Yunnn, Chin, 3 Institute of Tibetn Plteu Reserch t Kunming, Kunming Institute of otny, Chinese cdemy of Sciences, 3 Lnhei Rod, Kunming 65, Yunnn, Chin nd 4 Deprtment of Ecology nd Evolutionry iology, University of Toronto, 5 Willcocks Street, Toronto, Ontrio, M5S 3, Cnd * For correspondence. E-mil spencer.brrett@utoronto.c Received: 7 pril 4 Returned for revision: 9 My 4 ccepted: My 4 ckground nd ims Vrition in the reltive femle nd mle reproductive success of flowering plnts is widespred, despite the fundmentl hermphroditic condition of the mjorityof species. In mny hermphroditic popultions, environmentl conditions nd their influence on development nd size cn influence the gender expression of individuls through the formtion of hermphroditic nd unisexul flowers. This study investigtes the hypothesis tht the bulbous, niml-pollinted, perennil Lilium pertum (Lilicee) exhibits form of size-dependent gender modifiction known s gender diphsy, in which the sexul expression of individuls depends on their size, with plnts often chnging sex between sesons. Methods Vrition in florl trits ws exmined in reltion to their size using mrked individuls in nturl popultions, nd lso under glsshouse conditions. Mesurements were tken of the height, flower number, florl sex expression, flower size, flower biomss nd pollen production of individuls over consecutive yers between 9 nd in seven popultions in south-west Chin. Key Results Flowers of L. pertum re either perfect (hermphroditic) or stminte (mle) nd, in ny given seson, plnts exhibit one of three sex phenotypes: only hermphrodite flowers, mixture of hermphroditic nd mle flowers, or only mle flowers. Trnsitions between ech of these sex phenotypes were observed over consecutive yers nd were commonly size-dependent, prticulrly trnsitions from smll plnts bering only mle flowers to those tht were tller with hermphroditic flowers. Hermphroditic flowers were significntly lrger, hevier nd produced more pollen thn mle flowers. Conclusions The results for L. pertum re consistent with the size dvntge hypothesis developed for niml species with sex chnge. The theory predicts tht when individuls re smll they should exhibit the sex for which the costs of reproduction re less, nd this usully involves the mle phse. L. pertum provides n exmple of gender diphsy, rre sexul system in flowering plnts. Key words: Lilium pertum, gender diphsy, plnt sexul systems, size-dependent gender modifiction, sex lloction, sex chnge, plnt mting systems. INTRODUCTION Hermphroditism chrcterizes popultions of the vst mjority of flowering plnts. In hermphroditic popultions, individuls hve the potentil to obtin reproductive success s both femle nd mle prents, usully s result of the production of pollen nd ovules in perfect (hermphroditic) flowers (Horovitz, 977; Lloyd, 98). However, vrious developmentl nd genetic mechnisms cn cuse the bortion of sex orgns in perfect flowers, resulting in the origin of pistillte nd stminte flowers (reviewed by Diggle et l., ), condition known s dicliny. mjor component of the gret diversity of ngiosperm sexul systems therefore rises from the deployment of hermphroditic nd unisexul flowers in different structurl nd temporl combintions t the plnt nd popultion level (Geber et l., 999; rrett, ). This diversity results in rnge of gender strtegies, with individuls vrying in their reltive contributions to fitness s result of mternl nd pternl investment (Lloyd, 979; Lloyd nd w, 984). Wheres sexul systems re most commonly described on the bsis of morphologicl criteri, gender strtegies re by definition functionlly bsed nd concern the proportion of plnt s genes tht re trnsmitted to offspring through femle versus mle function. Hermphroditic plnts vry in the degree of temporl overlp between femle nd mle function. In simultneous hermphrodites, both pollen nd ovules re vilble for mting t the sme time nd vrition in gender usully results from grdul djustments in sex lloction. This sexul system predomintes mong flowering plnts. much less common sexul system is gender diphsy in which mle nd femle function comprise discrete ontogenetic phses of the life cycle. In popultions with gender diphsy individuls choose their sexul condition in ny given seson ccording to circumstnces (Lloyd nd w, 984; Schlessmn, 988). Gender diphsy is ssocited with perennil life histories nd commonly involves switches in sex expression from mle to femle, or from mle to # The uthor 4. Published by Oxford University Press on behlf of the nnls of otny Compny. ll rights reserved. For Permissions, plese emil: journls.permissions@oup.com

2 Pge of 3 Zhng et l. Size-dependent gender modifiction in Lilium pertum hermphrodite; however, reversls cn lso occur from femle to mle function (ierzychudek, 984; Schlessmn, 99). Sex expression in gender-diphsic species is controlled by environmentl conditions ffecting plnt vigour (condition), prticulrly the size nd mount of reserves in underground storge orgns. Only hndful of cses of gender diphsy re well documented in flowering plnts (e.g. risem Policnsky, 98; ierzychudek, 984; Ctsetine Gregg, 978; Zimmermn, 99; Pnx trifolium Schlessmn, 988, 99), probbly becuse evidence for sex chnge requires demogrphic observtions of mrked individuls for severl sesons. It is importnt to recognize tht lthough in ny given flowering seson popultions of gender-diphsic species re composed of individuls with discrete sex phenotypes (e.g. mle, femle or hermphroditic), over their lifetime they comprise single geneticlly monomorphic clss of hermphrodites. Thus, gender diphsy is not n exmple of true gender dimorphism in which popultions re composed of primrily femle- versus mle-functioning individuls. Theory for the evolution of gender diphsy derives from the size dvntge hypothesis developed for niml species with sex chnge (Ghiselin, 969; Leigh et l., 976; Wrner, 988; Veg-Frutis et l., 4). It proposes tht selection should fvour sex chnge when individuls succeed most efficiently s member of one sex when smll or young, nd s member of the other sex when older or lrger. In generl, the theory predicts tht when individuls re smll they should first express the sex for which the costs of reproduction re less, nd this usully involves the mle phse. Individuls should become femle whenever resources increse nd the reltive returns from mternl investment increse with size. These theories hve been refined for plnts (e.g. Lloyd nd w, 984; Zhng, 6) to tke ccount of their prticulr reproductive trits nd life histories, including their sessile nture, quntittive sex expression becuse of modulr growth nd the production of repeted reproductive units (e.g. flowers nd inflorescences), nd the vilbility of stored resources ssocited with perennil life histories. Chnges in sex expression cn occur multiple times over mny yers ssocited with both increses nd decreses in plnt size (ierzychudek, 984; Schlessmn, 99). This sitution differs from sequentil hermphroditism in nimls (see Wrner, 975, 988), in which sex chnge is usully not reversible. The occurrence of size-dependent gender modifiction is not confined to species with gender diphsy but is widespred phenomenon in flowering plnts. It occurs commonly through grdul djustments in sex lloction, often by the steriliztion of sex orgns prior to flowering, or through the cesstion of reproductive investment fter flowering during erly fruit nd seed development (Freemn et l., 98; Lloyd, 98b; Lloyd nd w, 984; Klinkhmer et l., 997). Size-dependent gender modifiction is reported from species with both gender monomorphism (e.g. simultneous hermphroditism Wright nd rrett, 999; Co nd Kudo, 8; monoecy Srkissin et l., ; Dorken nd rrett, 3) nd gender dimorphism (e.g. mles in subdioecy rrett et l., 999). The essentil difference between grdul gender djustment, s exemplified in the exmples bove, is not tht gender chnges over time, but rther tht in the cse of diphsy plnts choose between two more or less discrete phses, ccording to their condition. Here, we investigte size-dependent gender modifiction in the geophytic lily Lilium pertum (Lilicee). Our preliminry observtions of this species in south-west Chin indicted tht in ddition to plnts possessing hermphroditic flowers, s conventionlly described in the literture, significnt number of individuls in popultions produced mle flowers, in which femle sex orgns were most often rudimentry nd therefore non-functionl. These observtions motivted us to investigte the reltionships between estimtes of plnt size nd the sex expression of flowers. Lilium pertum is prticulrly suitble for this type of investigtion for two resons: () it does not form clones nd produces single reproductive shoot with smll number of flowers, thus enbling plnt size nd flower sex to be mesured without difficulty; () it possesses bulb which cn be esily excvted nd weighed, llowing n ssessment of totl stored investment t the end of the growing seson. Using both field nd glsshouse experiments we investigted seven popultions of L. pertum in 9 nd ddressed the following specific questions: () Wht is the distribution of hermphroditic nd mle flowers mong individuls of different size in nturl popultions, nd does this distribution result in the occurrence of different sex phenotypes? We predicted tht plnts with only hermphroditic flowers would be lrger thn those with only mle flowers. () re there differences in size nd totl reproductive expenditure in hermphroditic nd mle flowers? We predicted tht hermphroditic flowers would be lrger, weigh more nd produce more pollen thn mle flowers. (3) Is there evidence for sex chnge between yers for plnts belonging to different sex phenotypes? We predicted tht plnts with only mle flowers tht grew lrger during the growing seson would be more likely to produce hermphroditic flowers the following yer. Similrly, we predicted tht plnts with hermphroditic flowers tht becme smller fter reproductive expenditure would be more likely to produce mle flowers in the following yer. Dt on sex chnge between yers in popultions of L. pertum would provide evidence tht the species possesses gender diphsy. MTERILS ND METHODS Study species nd sites Lilium pertum Frnch. (Lilicee) is perennil geophytic herb tht occurs predominntly in mountinous res of south-west Chin (i.e. Hengdun Mountins). L. pertum is often considered member of the smll genus Nomochris ( ¼ N. pert), composed of seven species, ll of which re endemic to this region (Ling nd Tmur, ). However, recent moleculr phylogenetic nlysis indictes tht Nomochris is nested within Lilium (Rønsted et l., 5; Go et l.,, 3) nd therefore in this study we tret our study species s Lilium. Popultions of L. pertum inhbit shdy hbitts of brodleved forests, bmboo scrub nd lpine grsslnds usully between 3 nd 39 m.s.l. Plnts re bulbous nd in June produce n nnul shoot, which is 5 cm tll with grceful flower spikes composed of 5 lrge, pink to crimson, nodding ctinomorphic flowers (Fig. ). These cn be either perfect (herefter hermphroditic) or stminte (herefter mle). Most mle flowers hve rudimentry femle orgns nd cnnot set seed following hndpollintion nd re thus functionlly mle (Z.-Q. Zhng, unpubl. dt). Very few mle flowers hve no obvious rudimentry

3 Zhng et l. Size-dependent gender modifiction in Lilium pertum Pge 3 of 3 C FIG.. Flowers nd infloresence of Lilium pertum. () Hermphroditic flower, () mle flower, (C) n inflorescencewith two hermphroditic flowers nd three buds. Note the protrcted period of flowering from bsl to distl flower positions within the inflorescence. femle sex orgns. The flowering sequence of plnts commences with the nthesis of bsl flowers nd moves upwrds over pproximtely 3 weeks. Flowers of L. pertum pper to be nectrless nd hence pollen is the primry rewrd for pollen-collecting bees, which were the mjor flower visitors to popultions we investigted. Hnd-pollintions indicte tht L. pertum is selfincomptible nd there ws no evidence of pollen limittion of seed set t our study sites (Z.-Q. Zhng, unpubl. dt). Fruits of L. pertum re cpsules, contining more thn winged seeds, nd these mture bout 3 months fter pollintion. We conducted our studies during four flowering sesons from 9 to nd totl of seven popultions were investigted for different mounts of time to ddress the questions outlined below. The codes nd loctions used for the seven popultions nd the number of yer(s) ech ws studied re s follows: () G, Shngri-L lpine otnicl Grden, Shngri-L, Yunnn province, south-west Chin (7855 N, E; 333 m.s.l.), 9 ; () SG locted between Shngri-L City nd Gez town, 7 km from Shngri-L City (7857 N, 9984 E; 34 m.s.l.), 9 ; (3) SG, locted between Shngri-L City nd Gez town, km from Shngri-L City (88 N, 9984 E; 355 m.s.l.), 9 ; (4) TF, igu Tree Frm (7836 N, E; 33 m.s.l.) ; (5) HL, igu Heven Lke (7837 N, E; 385 m.s.l.), ; (6) XZ, Xio Zhongdin ( N, E; 3385 m.s.l.), ; (7) XZ, Xio Zhongdin (XZ, N, E; 3669 m.s.l.),. Sex expression, gender distribution nd sex lbility To investigte ptterns of sex expression t the plnt nd flower level, nd to quntify the distribution of gender t the popultion level, we studied seven popultions (G, HL, TF, SG, SG, XZ, XZ) for the durtions indicted in the preceding section. In ech popultion, we rndomly smpled 7 4 individuls nd counted the totl number of flowers, their sexul condition (hermphroditic or mle) nd their position on n inflorescence (bsl flower, next, etc.). To quntify the distribution of gender t the popultion level we employed Lloyd s mesure of phenotypic gender (G i ) using dt on the sexul condition of flowers (see Lloyd, 98; Lloyd nd w, 984). This index depicts the stndrdized phenotypic femleness of plnt in popultion s: G i = o i /(o i + p i E), where o i is the mternl investment, p i is the pternl investment nd E is n equivlence fctor bsed on the rtio of investments in mternl nd pternl functions in the popultion s whole: E ¼ So i /Sp i. We estimted pternl investment s the number of stminte flowers (the sum of hermphroditic nd mle flowers) nd mternl investment s the number of ovule-bering flowers (hermphroditic flowers). G i rnges from for plnts tht produce only pollen to for plnts tht produce only ovules. To determine whether L. pertum exhibits lbile sex expression over flowering sesons, we monitored the sexul condition of flowers on mrked plnts for two or three consecutive yers (9 ) in popultions G nd SG, respectively, using plstic tgs (smple sizes given in Tble ). Ech yer, we recorded plnt height nd the number of mle nd hermphroditic flowers. We then determined the direction nd frequency of nnul trnsitions between the three sex phenotypes ( plnts with hermphroditic flowers only, herefter H; plnts with hermphrodite nd mle flowers, herefter H+M; nd plnts with mle flowers only, herefter M).

4 Pge 4 of 3 Zhng et l. Size-dependent gender modifiction in Lilium pertum TLE. The chnges in gender of mrked individuls of Lilium pertum between yers in two popultions (G nd SG) from north-west Yunnn, Chin: number (%) of plnts of ech of the three sex phenotypes (H, H+M, M) in yer t tht becme members of ech sex phenotype in yer t + Popultion G sex in yer t + Popultion SG sex in yer t + Sex in yer t n H+M H M NF n H+M H M NF H+M 54 8 (4.8) (3.7) 3 (4.6) (38.9) 8 (.5) (5.) (5.) 3 (37.5) H 37 (.) 3 (8.) 3 (35.) (56.8) 6 (.) 6 (37.5) (.5) 8 (5.) M 5 9 (.6) 4 (9.3) 64 (4.4) 54 (35.8) 3 5 (6.) 8 (5.8) 9 (9.) 9 (9.) M, plnts with mle flowers; H, plnts with hermphroditic flowers; H+M, plnts with both hermphroditic nd mle flowers; NF, non-flowering plnts, including plnts (9.8 % in G nd 9. % in SG) tht filed to pper in given yer (ded or dormnt plnts). n, number of plnts. Differences between hermphrodite nd mle flowers To investigte potentil size differences between hermphroditic nd mle flowers we rndomly smpled 73 nd 66 flowers, respectively, in popultion G, nd 3 nd 3 flowers, respectively, in popultion SG. To control for position effects, only the bsl flower within n inflorescence ws smpled from ech plnt. We used digitl clipers to mesure the rdius of flowers. In popultions G, TF nd SG we rndomly collected 5 flowers per flower type using the sme smpling scheme nd these were oven dried t 8 8C for 4 h, nd then weighed to the nerest. mg. In popultion G, we estimted pollen production of hermphroditic nd mle flowers following methods detiled by Dfni (99). To test the prediction tht hermphroditic flowers would be lrger, weigh more nd produce more pollen thn mle flowers, one-tiled t-tests were used to compre ech prmeter (flower rdius, biomss nd pollen production) between flower types within ech popultion. We lso used two-wy NOV to nlyse the first two trits with Flower type nd Popultion s min effects. Gender nd size To determine the reltionships mong plnt height, sex phenotype (H, H+M, M) nd biomss, we rndomly excvted pprox. plnts of ech sex phenotype in ech of three popultions (G, TF, SG) in June 9. In ech popultion we lso included smple of non-reproductive plnts for comprison. For ech mture plnt we recorded the totl number of flowers, the sexul condition of ech flower nd plnt height bove the soil. We lter dried plnts t 8 8C for 4 h, nd then weighed their dry biomss to the nerest. mg. We tested for differences in plnt size (totl biomss, bulb weight, height) nd flower number mong sex phenotypes nd popultions using nlysis of covrince (NCOV), with totl biomss s covrite. To exmine how gender vries with size in L. pertum, we monitored flower production throughout the blooming period for plnts mintined in glsshouse in. The 9 individuls used in this experiment were excvted from three nturl popultions in (G, SG nd SG, n ¼ 3 per popultion) fter they hd flowered nd fruited nd were senescing prior to the onset of dormncy. The bulbs were wshed, dried nd then stored under cool conditions ( 4 8C) prior to the glsshouse experiment the following yer. In, ll bulbs were weighed nd plnted in pots in glsshouse t the Kunming Institute of otny. Fifty-one of the bulbs flowered nd on the first dy ech plnt flowered, we mesured plnt height nd subsequently recorded the number of flowers nd their sex expression. We tested for differences in size mong the sex phenotypes nd popultions using two-wy NOV with Plnt height nd Popultion s min effects. We determined the reltionships between plnt size nd flower trits using liner regression. ecuse in L. pertum plnt height correltes strongly with totl biomss (see Results; Fig. 6) we used this mesure s non-destructive surrogte for overll size. To exmine whether plnt size ws ssocited with sex phenotype, we mesured plnt height in six popultions during the flowering sesons of 9, nd : G, SG, SG (9 ); TF, (); HL nd XZ (). Differences in plnt size between the sex phenotype with only mle flowers (M) nd the two sex phenotypes with hermphroditic flowers were tested using NOV. To determine if there ws n ssocition between size chnge nd switches in sex expression, we mrked plnts in popultion G nd mesured plnt height for two consecutive yers. We then grouped plnts bsed on their chnge in gender nd compred the plnt height of individuls between two consecutive yers. We used G-test with Willim s correction to determine whether the propensity to become vegettive or smller ws independent of gender, nd Fisher s exct test to evlute differences between sex phenotypes in their propensity to chnge gender. For ech sex phenotype, we tested for differences in size chnge between yer t nd t + using pired t-tests. Throughout, we present dt s mens + s.e.m., where possible; G-tests were conducted using the spredsheet of McDonld (9), nd ll of other sttisticl tests were nlysed in JMP (version 6.., SS Institute, 5). RESULTS Sex expression nd gender distributions Flowers of L. pertum re either hermphroditic or mle (Fig., ); we observed no purely femle flowers in our popultion surveys. The men proportion of mle flowers smpled in our popultion surveys ws.56 (rnge.3.75), with mle flowers the most frequent (. 5 %) flower type in ll popultions except HL nd XZ (Fig. ). t the plnt level, the three sex phenotypes (H, H+M, M) were observed in ll popultions nd yers tht we smpled (Fig. ). The reltive proportions vried mong popultions, nd to lesser extent mong yers in the four popultions (G, SG, SG nd HL) in

5 Zhng et l. Size-dependent gender modifiction in Lilium pertum Pge 5 of 3 Proportion t the flower level Proportion t the individul level Popultion/Yer which smples were conducted in more thn one yer. M plnts were the most common sex phenotype (men frequency.57, rnge.8.7), nd plnts with this phenotype most commonly produced only single flower in flowering seson. The next most common sex phenotype ws H (men frequency.8, rnge..66), nd the lest frequent sex phenotype ws H+M (men frequency.5, rnge.3.7). In plnts with this mixed flower phenotype, hermphroditic flowers were lwys produced t bsl positions within n inflorescence nd mle flowers were locted t distl positions. mong ll popultions in the survey, the men number (+ s.e.m.) of flowers per plnt for H, H+M nd M sex phenotypes ws. +.3 (n ¼ 549),. +.3 (n ¼ 9) nd. +. (n ¼ 95), respectively. The men number of hermphroditic nd mle flowers for the 9 H+M plnts ws. +. (n ¼ 4) nd. +. (n ¼ ), respectively. We estimted the distribution of phenotypic gender t intervls (yers) in seven popultions of L. pertum. We present only six representtive smples in four popultions to illustrte overll ptterns (Fig. 3). ll popultions showed vrition in gender, including plnts with only mle function to plnts tht hve the potentil to gin fitness through both FIG.. Vrition in sex expression t the flower nd popultion level t smpling intervls in seven popultions of Lilium pertum () flowers, nd () individuls within ech popultion. M, mle flower or plnt with only mle flowers; H, hermphroditic flower or plnt with only hermphroditic flowers; H+M, plnts with hermphroditic nd mle flowers on the sme individul. The smple size (n) is given t the bse of ech br., G ();, G (); 3, G (9); 4, SG (); 5, SG (9); 6, SG (); 7, SG (9); 8, XZ (); 9, XZ ();, TF ();, HL ();, HL (). H+M mternl nd pternl function. The ptterns of gender distribution over 3-yer period (9 ) in G (Fig. 3 C) provide evidence tht gender expression t the popultion level remins reltively stble, despite trnsitions between sex phenotype clsses t the plnt level (see below). The remining gender plots (Fig. 3D F), from popultions HL-, TF- nd XZ, illustrte vrition mong popultions in the reltive frequencies of the three sex phenotypes. lthough the distributions give the ppernce of discontinuity, becuse of the occurrence of vrying proportions of plnts with only mle function, in relity the lifetime gender of plnts is more likely to exhibit continuous vrition, owing to trnsitions to hermphroditism with size nd ge (see below). Comprisons of hermphroditic nd mle flowers Hermphroditic flowers were significntly lrger thn mle flowers, bsed on mesurements of flower rdius (Fig. 4: G: hermphroditic mm, n ¼ 73; mle mm, n ¼ 66; t rtio ¼.58, P ¼.65,.5; SG: hermphroditic mm, n ¼ 3; mle mm, H M

6 Pge 6 of 3 Zhng et l. Size-dependent gender modifiction in Lilium pertum C Cumultive proportion D E F Cumultive proportion Phenotype gender, G i Phenotype gender, G i Phenotype gender, G i FIG. 3. The distribution of phenotypic gender in four popultions of Lilium pertum. The lines represent the cumultive frequency distribution of stndrdized phenotypic femleness (G i ) for plnts smpled from ech popultion. ( C) G, 9 ; (D) HL, ; (E) TF, ; (F) XZ,. Gender cn potentilly vry from purely mle ( ¼ ) to purely femle ( ¼ ); however, in popultions of L. pertum no plnts were observed with only femle gender expression. n ¼ 3; t rtio ¼ 3.93, P ¼.). The effects of flower type nd popultion on flower size were significnt, but the interction popultion flower type ws not (P ¼.9..5). Not unexpectedly, the dry biomss of hermphroditic flowers ws significntly greter thn tht of mle flowers (Fig. 4: G: hermphroditic mg, n ¼ 39; mle mg, n ¼ 48; t ¼ 5.8, P,.; TF: hermphroditic mg, n ¼ 4; mle mg,n ¼ ; t ¼ 5.835, P,.; SG: hermphroditic mg; n ¼ 7; mle mg; n ¼ 36; t ¼ 5., P,.). Flower type hd significnt effect on florl biomss, but the interction popultion flower type ws not significnt (P ¼.63..5). In popultion G, hermphroditic flowers produced significntly more pollen (3.5 %) thn mle flowers (hermphroditic , n ¼ 9 flowers; mle , n ¼ 6 flowers; t ¼ 3.534, P ¼.5). Dynmics of gender expression y investigting mrked individuls in popultions G nd SG for consecutive yers, we found tht individuls re ble to switch sex from one yer to the next (Tble ). Of the six possible nnul trnsitions between sex phenotype clsses, ll except the trnsition from H plnts to H+M plnts were observed. For exmple, in G 35. % of H plnts in yer produced only mle flowers (M) the following yer, nd 4.6 % of H+M plnts becme M plnts in the second yer of observtion. Similrly, mong M plnts in yer,.6 nd 9.3 % of plnts were H+M or H, respectively, the following yer. Chnges in gender between yers were lso evident in popultion SG. For exmple, mong M plnts observed in the first yer of observtion, 6. nd 5.8 % of the plnts in the following yer were either H+M or H, respectively. In both popultions significnt proportion (39.7 % in G, 36.4 % in SG) of flowering plnts surveyed in the first yer did not flower in the second yer. The three sex phenotypes differed with respect to their likelihood of not flowering in the second yer of observtion. H plnts were the lest likely to flower, followed by H+M plnts, nd M plnts were the most likely to continue flowering in the second yer. Interestingly, with one exception mong the sex phenotype (H plnts in SG) the proportion of plnts switching gender ws lowest mong M plnts with 4.4 nd 9. % of plnts in G nd SG, respectively, remining M in the second yer of observtion. Of the 7 nd 6 plnts tht were observed for three consecutive yers in G nd SG, 7. nd 8.8 %, respectively, chnged sex twice. Gender nd size mong the sex phenotypes For ll components of size, femle-functioning plnts (H+M nd H) tended to be lrger thn plnts tht produced exclusively stminte flowers (M), but H nd H+M plnts were similr in size (Fig. 5; Tble ). In ech popultion there were significnt

7 Zhng et l. Size-dependent gender modifiction in Lilium pertum Pge 7 of 3 Pollen production per flower ( 3 ) Flower biomss (mm) Flower rdius (mm) C G TF SG * *** *** *** *** ** Hermphrodite Mle Flower number Plnt height (cm) ulb biomss (g) Totl biomss (g) C D b b b b b b b b G TF SG b bc b b b b c c b c b Flower type FIG. 4. Differences in florl trits between hermphroditic nd mle flowers of Lilium pertum. () Flower rdius, () flower biomss nd (C) pollen production per flower. *P,.5; **P,.; ***P,.. Popultion codes re indicted. differences mong the sex phenotypes in totl biomss nd plnt height (Fig. 5, C) nd totl biomss ws positively relted to bulb biomss. For bulb biomss, differences between H nd M phenotypes were only significnt in SG (Fig. 5). H+M plnts produced significntly more flowers thn the other two sex phenotypes (Fig. 5D). The effects of sex phenotype nd popultion on totl biomss, bulb biomss nd height were significnt, but the interction popultion sex ws not (Tble ). In ech popultion, plnt height, totl flower production nd the number of hermphroditic flowers per plnt vried H+M positively with totl biomss (Fig. 6). In contrst, there ws no significnt reltionship between size nd the number of mle flowers per plnt, lrgely becuse ll M plnts but one produced single flower. H Sex F IG. 5. () Totl plnt biomss, () bulb biomss, (C) plnt height nd (D) flower number mong three sex phenotypes (M, H, H+M) in three popultions (G, TF, SG) of Lilium pertum in the flowering seson of 9. Lower-cse letters indicte mens tht re significntly different from one nother in ech popultion (Tukey Krmer comprisons, P,.5). M, plnts with only mle flowers; H, plnts with only hermphroditic flowers; H+M, plnts with hermphrodite nd mle flowers. The sttisticl results re reported in Tble. M

8 Pge 8 of 3 Zhng et l. Size-dependent gender modifiction in Lilium pertum TLE. NCOV of totl biomss, bulb biomss, height nd flower number mong sex phenotypes (H, M, H+M) of Lilium pertum in three popultions (G, TF nd SG) from NW Yunnn, Chin Source (d.f.) Totl biomss ulb biomss Height Flower number Popultion () 7.37*** 3.59* 4.53**** 3.3 Sex () 3.8**** 4.4**** 6.3**** 4.5**** Popultion sex (4) ** Totl biomss () 76.99**** 8.6**** 54.9**** Totl biomss sex () 3.9* 4.6* Totl biomss popultion () 6.8** 8.7*** Totl biomss popultion sex (4) 6.33** Totl biomss ws used s covrite in ech nlysis. Interctions between covrites nd min effects were tested nd removed using bckwrds elimintion ( ¼.5). F-vlues re shown for ech response vrible. *P,.5, **P,., ***P,., ****P,.. Considering plnt height s surrogte of totl biomss (or size) in the glsshouse experiment, we found tht ovry length, ovry width, style length nd flower dimeter ll vried positively with height (Fig. 7, Tble 3: ovry length: r ¼.9, F, 5 ¼.55, P ¼.4; ovry width: r ¼.5, F, 5 ¼ 8.97, P ¼.43; style length: r ¼.5, F, 5 ¼ 8.78, P ¼.47; flower dimeter: r ¼.34, F, 5 ¼ 4.8, P,.). In contrst, florl trits directly ssocited with mle function (e.g. nther length nd filment length) were not ssocited with plnt height. Surveys of plnt height nd totl flower number in five popultions of L. pertum reveled consistent ptterns between the sex phenotypes. Femle-functioning sex phenotypes (H nd H+M) were significntly tller (Fig. 8) nd produced more flowers (Fig. 8) in ll but one comprison (plnt height in SX) compred with plnts tht produced exclusively mle flowers (M). However, the lck of difference in height between the sex phenotypes in SX did not occur in this popultion the following yer nd femle-functioning phenotypes were tller thn purely mle-functioning phenotypes (Fig. 8). We tested severl predictions concerning chnges in the size nd gender of sex phenotypes between consecutive flowering sesons using dt collected from popultion G. First, we predicted tht M plnts would be more likely to increse in size between yers, wheres H nd H+M sex phenotypes would be more likely to decrese owing to the greter costs ssocited with femle function. We found evidence to support this prediction (Tble 4). Second, we predicted tht M plnts tht grew lrger would be more likely to become femle functioning thn those tht becme smller, nd tht femle functioning plnts tht becme smller would be more likely to become exclusively mle functioning compred with femles tht becme lrger. These two predictions were lso supported by our dt (Tble 4). DISCUSSION Our study of size-dependent gender modifiction in L. pertum reveled three mjor findings. () significnt proportion of plnts in popultions (men frequency.57, rnge.8.7) produced only stminte flowers nd thus functioned exclusively s mle prents (Fig. ). ll of these plnts except one produced single flower, nd were shorter thn plnts with femle function, which generlly produced more flowers (Figs 5 nd 8). () Hermphroditic flowers were lrger, weighed more nd produced more pollen thn mle flowers (Fig. 4). (3) Censuses of mrked plnts mong yers indicted sex chnge mong ll three sex phenotypes (M, H, H+M) ssocited with chnges in their plnt size (Tble ). Specificlly, plnts producing only mle flowers were more likely to produce hermphroditic flowers the following yer if they grew lrger, wheres plnts with hermphroditic flowers tht becme smller were more likely to switch to producing mle flowers the following seson (Tble 4). We begin by first considering the occurrence of unisexul flowers in L. pertum nd relted tx nd the implictions tht dicliny hs for the sexul systems nd mting biology of popultions. We then exmine the evidence for gender diphsy in L. pertum nd consider the developmentl nd ecologicl mechnisms tht cuse size-dependent gender modifiction. The origin, distribution nd consequences of dicliny for sexul systems The origin of unisexul flowers from hermphroditic flowers brodens the spectrum of sexul systems vilble to flowering plnts, nd increses the opportunity for vrition in sex lloction nd gender strtegies. Our study indictes tht rther thn being of infrequent occurrence, s is the cse in most hermphroditic species, unisexul flowers re common feture of L. pertum popultions. Indeed, the men proportion of mle flowers smpled in our survey ws.56, with mle flowers the most frequent flower type in the mjority of popultions. Loss of orgn function cusing florl unisexulity cn occur t ll stges of development, rnging from the bsence of initition to the filure of orgn mturtion, but within species the stge of orgn bortion tends to be similr for both pistillte nd stminte flowers (Diggle et l., ; Fig. ). Virtully ll mle flowers of L. pertum displyed conspicuous non-functionl pistils ( pistillodes), indicting tht the processes responsible for the termintion of femle function were often lte in development, perhps precluding significnt opportunities for reproductive compenstion. Indeed, there ws no evidence of incresed pollen production in mle flowers, which were in fct smller nd produced less pollen thn hermphroditic flowers. Significntly, pistillte flowers were never observed in popultions of L. pertum,implying tht mle flower production is n dptive feture of the sizedependent sex lloction strtegy of popultions. The occurrence of mle flowers in members of the Lililes is not uncommon nd significnt number of tx re dioecious

9 Zhng et l. Size-dependent gender modifiction in Lilium pertum Pge 9 of 3 Plnt height (cm) Totl flowers per plnt Hermphrodite flowers per plnt C 4 Totl biomss (g) G TF SG 6 8 FIG. 6. Reltionships between totl biomss nd () height, () totl flowers nd (C) number of hermphroditic flowers per plnt in popultions G, TF nd SG of Lilium pertum. Lines represent lest-squres regressions lines for ech popultion. () G: y ¼ 8. +.x, r ¼.74, P,.; TF; y ¼ x, r ¼.56, P,.; SG: y ¼. +.9x, r ¼.6, P,.. () G: y ¼.3 +.4x, r ¼.38, P,.; TF; y ¼.3 +.5x, r ¼.5, P,.; SG: y ¼. +.5x, r ¼.4, P,.. (C) G: y ¼. +.3x, r ¼.44, P,.; TF; y ¼.3 +.6x, r ¼.6, P,.; SG: y ¼.3 +.5x, r ¼.4, P,.. (e.g. Chmelirium Megher, 98; Smilx Swyer nd nderson, 998; Wurmbe rrett nd Cse, 996) or ndromonoecious (e.g. Gge Wolfe, 998; Vertrum Lio nd Zhng, 8; Zigdenus Emms, 993). Surveys of mle flowers in Fritillri, Lilium nd Tulip hve reveled frequencies rnging from to 38 % (Peruzzi, ), indicting tht femle sterility is lso feture of other normlly hermphroditic tx of lilies (nd see Mncuso nd Peruzzi, ). detiled study of flower sex expression in seven popultions of Flower dimeter (mm) Ovry width (mm) Style length (mm) Filment length (mm) Ovry length (mm) nther length (mm) C D E F R = 34 P < * R = 5 P = 43* R = P = 386 R = 9 P = 4* R = 34 P = 5 5 R = 5 P = 47* Plnt height (cm) FIG. 7. Reltionships between plnt height nd () flower dimeter, () ovry width, (C) ovry length, (D) style length, (E) filment length nd (F) nther length in Lilium pertum plnts excvted from three popultions (G, SG nd SG) nd grown in glsshouse for one flowering seson. The points re dt pooled from three popultions (G, SG nd SG) nd lines represent lest squres regressions lines for ech florl trit. sterisks indicte significnt reltionship. Two-wy NOV results re reported in Tble 3.

10 Pge of 3 Zhng et l. Size-dependent gender modifiction in Lilium pertum TLE 3. F-vlues for two-wy NOVs of the effect of plnt height nd popultion origin on flower dimeter, nther length, filment length, ovry width, ovry length nd style length in Lilium pertum plnts excvted from three popultions (G, SG nd SG) from north-west Yunnn, Chin, nd grown in glsshouse for one flowering seson Mle function Femle function Source Flower dimeter nther length Filment length Ovry width Ovry length Style length Plnt height.4 (, 46)***.87 (, 46) 6.4 (, 46)* 4.45 (, 46)*** 8.84 (, 46)** 9.49 (, 46)** Popultion.73 (, 46).7 (, 46).69 (, 46).93 (, 46).3 (, 46).9 (, 46) Plnt height popultion.7 (, 46).99 (, 46).68 (, 46).4 (, 46). (, 46).57 (, 46) *P,.5, **P,., ***P,.. Plnt height (mm) Flower number *** *** ** * *** *** *** * F. montn reveled tht mle flowers represented 4 48 % of the flowers smpled, with the reminder being hermphroditic (Peruzzi et l., ). Similrly, in survey of popultions of hermphroditic Lloydi serotin, 48.5 % of the flowers were mle (Mniccci nd Després, ). In both these species the vst mjority of plnts produce single flower during the flowering seson nd in both studies plnts with mle flowers were on verge smller for vrious trits thn those with hermphroditic * NS *** H/H+M M G HL TF SX SX XZ Popultion F IG. 8. The differences in () flower number nd () plnt height between sex phenotypes tht produce only mle flowers (M) nd the two sex phenotypes tht produce hermphroditic flowers (H/H+M) in Lilium pertum.*p,.5, **P,.5, ***P,.. NS, not significnt. * flowers. Observtions between yers in Lloydi serotin provided evidence of switches in gender (Mniccci nd Després, ; nd see Jones nd Gliddon, 999), nd these chnges were similr to those observed in the present study. Size-dependent gender modifiction my be especilly importnt in geophytic species with underground storge orgns, such s members of the Lilicee. Indeed, there is evidence for chnges in sex lloction with size in Vertrum nigrum (Lio nd Zhng, 8) nd Crdiocrinum cordtum (Co nd Kudo, 8). In these cses gender djustment is grdul, rther thn involving discrete chnges in sex phse between sesons, nd this my, in prt, be becuse of the lrger florl displys tht these species possess. The significnt numberof plnts producing only mle flowers in members of the Lilicee hs resulted in some confusion concerning the sexul systems of popultions. For exmple, bsed on studies of Fritillri, Lilium nd Tulip, Peruzzi () nd Peruzzi et l.()concluded tht species in these gener were ndrodioecious (nd see Mncuso nd Peruzzi, ). In contrst, bsed on very similr ptterns of sexul vrition, Mniccci nd Després ()concluded tht Lloydi serotin ws not ndrodioecious, s hd been previously suggested, but ws ndromonoecious. This inference ws bsed on the ssumption tht ll plnts within the popultions if lrge enough over their lifetimes would produce hermphroditic flowers. Given the overll rrity of ndrodioecy in ngiosperms, nd the severe constrints on the spred nd mintennce of femle sterile plnts in predominntly hermphroditic popultions (reviewed by Pnnell, ), this interprettion seems more probble. The occurrence of plnts with only mle function in popultions of Liliceous gener (including L. pertum), lthough superficilly resembling ndrodioecious gender dimorphism, is more likely to reflect the influence of resource sttus on hermphroditic species with size-dependent gender modifiction (nd see Pnnell, b). However, longterm demogrphic studies would be required to completely rule out the presence of femle sterile plnts. Notwithstnding the difficulties in estblishing the sexul system of these popultions, in ny given seson the strong bistowrds mleness in popultions will hve importnt implictions for mting biology, prticulrly the intensity of intrsexul (mle mle) competition for mtes. Does L. pertum exhibit gender diphsy? Gender diphsy my lso be difficult to distinguish from more grdul forms of size-dependent gender djustment in hermphroditic species with gender monomorphism. The essentil

11 Zhng et l. Size-dependent gender modifiction in Lilium pertum Pge of 3 TLE 4. Tests of predictions bout plnt size nd gender chnge bsed on yerly observtions of mrked plnts of Lilium pertum in popultion G, north-west Yunnn, Chin () Tests of the prediction tht plnts with only mle flowers would be more likely to increse in size thn decrese, wheres plnts with hermphroditic flowers would be more likely to decrese thn increse in size Size in yer t + Gender in yer t Lrger Smller G dj P (one-tiled) Mle * Hermphroditic ,.* () Tests of the prediction tht plnts with only mle flowers tht grew lrger would produce hermphroditic flowers, wheres those tht becme smller would not, nd plnts with hermphroditic flowers tht becme smller would chnge to produce only mle flowers wheres those tht grew lrger would not Gender in yer t + Gender in yer t Size in yer t + Mle Hermphroditic P (one-tiled) Mle Lrger 3 9.* Smller Hermphroditic Lrger 8.65* Smller 8 5 *Significnt difference. feture of gender diphsy is tht individuls in popultion belong to single genetic clss in which their sex expression in ny prticulr seson is controlled by circumstnces, prticulrly plnt size. Thus, within flowering seson popultions should be composed of two primry gender clsses: mle-functioning nd femle-functioning plnts. lthough femle-functioning plnts cn lso produce pollen, the mjority of their fitness is likely to be obtined through their ovules becuse of the strongly mlebised sex lloction t the popultion level, s we observed in L. pertum. Thus, the sex phenotypes H nd H+M in our study re often referred to s femle (see Lloyd nd w, 984; Schlessmn, 988), even though they re not strictly unisexul. Despite the occurrence of two primry gender clsses in diphsic popultions, from the perspective of between-seson, lifetime fitness ll plnts on verge should contribute genes to the next genertion s both mle nd femle prents. s result, the lifetime functionl gender of popultions with gender diphsy will be unimodl, rther thn bimodl s occurs in species with gender dimorphisms such s ndrodioecy. s discussed bove, lthough we cnnot completely rule out the possibility tht some L. pertum plnts re geneticlly determined mles, we believe this is unlikely for two resons. First, given the vrious constrints on the evolution nd mintennce of ndrodioecy it would be unexpected tht mle plnts in ndrodioecious popultions would produce fewer nd smller flowers with less pollen thn coexisting femle-functioning plnts, s occurs in L. pertum. oth theoreticl studies nd empiricl evidence indicte tht for mles to be mintined in ndrodioecious popultions it is necessry for them to compenste for their lckof femle function (Lloyd, 975; Chrlesworth, 984; Pnnell,, b). This is usully chieved by significnt increse in pollen production compred with hermphrodites, the reverse pttern to wht we observed in L. pertum. Second, we directly observed betweenyer switches in gender from mle to femle function nd from femle to mle function ssocited with increses nd decreses in plnt size, respectively. These ptterns hve not been reported in ndrodioecious species nd re more consistent with the sesonl switches in gender tht re the hllmrk of gender diphsy. The lifetime functionl gender of popultions of genderdiphsic species should be monomorphic rther thn dimorphic. However, in ny given seson the distribution of gender vrition in popultion will be discontinuous, s we hve shown (Fig. 3). This pttern is the direct result of the choice tht plnts mke between two contrsting modes of pre-fertiliztion investment in mleness versus femleness. This discontinuity differs from wht would be observed in most hermphroditic species tht exhibit grdul gender djustment. In these species phenotypic gender is regulted by modifictions to the sex lloction of flowers, nd by post-fertiliztion phenomen ssocited with fruit nd seed bortion (Lloyd nd w, 984). Gender diphsy thus represents n extreme cse of size-dependent sex lloction in which popultions in ny given seson re mde up of two distinct ctegories or phses corresponding to mle- versus femle-functioning plnts, despite popultions being fundmentlly hermphroditic. Developmentl nd ecologicl mechnism cusing size-dependent gender modifiction Gender in nturl popultions of L. pertum is governed primrily by size, with incresed size resulting in greter investment in femle function. These effects were mnifested t both the flower nd the plnt level. For exmple, in the glsshouse ovry length, ovry width nd style length ll vried positively with plnt height, wheres florl trits directly ssocited with mle function (e.g. nther length nd filment length) were unrelted to size (Fig. 6). Similrly, in nturl popultions both the number of hermphroditic nd the totl number of flowers per plnt were positively correlted with plnt height, indicting tht lrger plnts hve more resources to invest in femle function s result of the budget effect of plnt size (see Klinkhmer et l., 997). In contrst, mle flower production ws unrelted to plnt height nd lrge plnts produced more flowers cpble of femle function nd smll plnts either produced only mle flowers or were non-flowering (Figs 5 nd 7). These results re consistent with size-dependent sex lloction theory, which

12 Pge of 3 Zhng et l. Size-dependent gender modifiction in Lilium pertum predicts tht plnts should invest in femle function only when bsolute investment in survivl nd mle function exceeds certin threshold, nd tht smll individuls should express the gender tht incurs the lower cost (Lloyd nd w, 984; Zhng, 6). The smller size of stminte flowers nd shorter stture of plnts with only mle flowers is consistent with the hypothesis tht in L. pertum mle function is less costly thn femle function. The size-dvntge hypothesis predicts tht chnges in sex expression re controlled by size. In nimls, these chnges re most often unidirectionl from mle to femle nd thus occur just once over lifetime (Wrner, 975, 988). However, in plnts the mount of resources stored in underground orgns my chnge from yer to yer depending on nnul climtic conditions, levels of herbivory, pollintor ctivity, nd the mount of fruits nd seeds produced. s result, both increses nd decreses in below-ground reserves cn occur, resulting in reversls in gender (ierzychudek, 984; Schlessmn, 99). Our results from L. pertum confirm these expecttions: we not only observed chnges from mle to femle function with incresed size, but lso cses where primrily femlefunctioning plnts reverted to mle function in the second yer ssocited with decrese in their size. lthough we did not explicity investigte the extent to which flowers of L. pertum plnts were pollen-limited in this study, our preliminry observtions indicted tht ll mrked flowers set fruit, which produced s mny seeds s fruits from flowers tht received supplementl hnd-cross pollintion (Z.-Q. Zhng, unpubl. dt). If L. pertum is most often resource- rther thn pollen-limited, nnul reproductive expenditure ssocited with femle function my ply n importnt role in determining chnges in plnt size nd gender. Our studies of size-dependent gender modifiction in L. pertum rise severl importnt questions concerning the proximte mechnism governing sex expression. t this stge we do not know the specific developmentl stges t which the gender of individuls is controlled. Given the geophytic life history of L. pertum, flower number in ny given seson my be determined t the end of the previous growing seson, bsed on the mount of stored reserves in the bulb nd the number of florl primordi tht re initited. Preformed flowers could contin rudimentry femle nd sex orgns nd the decision whether to mintin or bort femle function my occur in the subsequent spring prior to flowering bsed on n ssessment of current resources nd future expenditures. For smll plnts with only single mle flower this decision my occur erly in the growing seson; however, for lrge plnts producing both hermphroditic nd mle flowers the developmentl decision to bort femle function in distl flowers my depend on whether bsl flowers re pollinted nd initite fruit set. ecuse of the extended period over which flowers nd buds re present on plnts (e.g. see Fig. C) this seems quite likely. Experimentl pollintion studies (e.g. see Diggle, 997) could be used to test this hypothesis. L. pertum produces only smll number of lrge flowers (e.g. 5) nd lloction decisions ffecting the sex expression of flowers my be best understood in terms of theories for the frctionl lloction of resources mong smll number of units (see Ebert, 994; Chrnov et l., 995). If the optiml investment for hermphroditic flowers flls between the necessry integer vlues mle functioning flowers my be the only option. In popultions of L. pertum the lrgest plnts with the most flowers produced both hermphroditic nd mle flowers (Fig. 5). This suggests tht the resources vilble to these plnts were not sufficient to produce dditionl hermphroditic flowers, but rther were invested in less costly mle flowers. Future studies might usefully mesure vrition in flower size nd number in L. pertum nd consider wys to determine the threshold lloction to flowers below which femle function is borted. The selective fctors responsible for the evolution of sizedependent gender modifiction differ depending on whether gender diphsy or grdul gender djustment re considered. lthough in both cses n individul s environment nd reproductive sttus must differentilly ffect pternl nd mternl fitness, the prticulr shpe of the fitness gin curve for ech sexul function is of criticl importnce in determining the form of gender modifiction tht cn evolve (Lloyd nd w, 984; Zhng, 6). rnge of other fctors including the scle of environmentl heterogeneity in popultion nd its influence on locl gmete rtios re lso likely to be influentil. Given the rrity of gender diphsy in ngiosperms compred with other forms of size-dependent gender modifiction it would pper tht the environmentl circumstnces nd prticulr plnt trits tht promote the evolution of this unusul sexul system re restrictive. Comprtive studies of the ecology nd reproductive biology of relted species of Lilium my id in identifying the mechnisms responsible for the evolution of gender diphsy from simultneous hermphroditism. CKNOWLEDGEMENTS We thnk J.-G. Chen nd D.-L. Peng for ssistnce with dt collection in the field nd glsshouse. This work ws supported by the Ntionl Science Foundtion of Chin (379), Chin Postdoctorl Science Foundtion (T5787) nd Western Light Tlent Culture Project to Z.-Q.Z. nd by Discovery Grnt from the Nturl Sciences nd Engineering Reserch Council of Cnd to S.C.H.. LITERTURE CITED rrett SCH.. The evolution of plnt sexul diversity. Nture Reviews Genetics 3: rrett SCH, Cse L. 6. The ecology nd evolution of gender strtegies in plnts: the exmple of ustrlin Wurmbe (Colchiccee). TURNER REVIEW No.. ustrlin Journl of otny 54: rrett SCH, Cse L, Peters G Gender modifiction nd resource lloction in subdioecious Wurmbe dioic (Colchiccee). 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