ARTICLE IN PRESS

Perspectives in , Evolution and Systematics Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 www.elsevier.de/ppees RESEARCH ARTICLE Allocation strategies and traits are hardly affected by nitrogen supply in 18 species differing in successional status Claire Fortunela,Ã, Cyrille Viollea, Catherine Roumeta, Bruno Buatoisa, Marie-Laure Navasb, Eric Garniera aCNRS, Centre d’Ecologie Fonctionnelle et Evolutive (UMR 5175), 1919 Route de Mende, 34293 Montpellier Cedex 5, France bMontpellier SupAgro, Centre d’Ecologie Fonctionnelle et Evolutive (UMR 5175), 1919 Route de Mende, 34293 Montpellier Cedex 5, France

Received 9 May 2008; received in revised form 7 April 2009; accepted 8 April 2009

Abstract

Species performance depends on ecological strategies, revealed by suites of traits, conferring different relative ecological advantages in different environments. Although current knowledge on plant strategies along successional gradients is derived from studies conducted in situ, actually quantifying these strategies requires disentangling the effects of environmental factors from intrinsic differences between species. Here we tested whether allocation strategies and seed traits differ among successional stages and nitrogen levels. To this aim, we assessed biomass and nitrogen allocations and seed traits variations for 18 species, differing in life history and belonging to three stages of a Mediterranean old-field succession. These species were grown as monocultures in an experimental garden under limiting and non-limiting nitrogen supply. Early successional species allocated allometrically more nitrogen and proportionally more biomass to , and set more than later successional species. Seed mass increased with successional status and was negatively related to seed number. Early successional species thus produced more but less-provisioned seeds, suggesting better colonization abilities. These patterns were not the sole consequence of the replacement of annuals by perennials along the successional gradient, since comparable trends were also observed within each life history. Allocation patterns were generally not altered by nitrogen supply and the higher nitrogen content in vegetative organs of plants grown under high nitrogen supply was not retranslocated from to seeds during seed development. We therefore conclude that differences in plant ecological strategies in species characteristics from contrasting successional stages appear to be intrinsic properties of the studied species, and independent from environmental conditions. r 2009 Ru¨bel Foundation, ETH Zu¨rich. Published by Elsevier GmbH. All rights reserved.

Keywords: Allometry; Reproductive output; Seed mass; Nitrogen concentration of organs; Succession; Nitrogen supply

Introduction

Biomass and nutrient allocation patterns reflect the ÃCorresponding author. Present address: INRA Kourou-UMR EcoFoG (Ecologie des Foreˆts de Guyane), Avenue de France, BP 709, way species interact with their environment (Antono- 97387 Kourou Cedex, French Guiana, France. vics, 1980; Bazzaz et al., 1987). In successional environ- E-mail address: [email protected] (C. Fortunel). ments, the proportion of total plant biomass in ,

1433-8319/$ - see front matter r 2009 Ru¨bel Foundation, ETH Zu¨rich. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ppees.2009.04.003 ARTICLE IN PRESS 268 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 stem and reproductive organs tend to decrease while allocation patterns observed in standard conditions that in root tends to increase as succession proceeds (e.g. were consistent with those observed in situ. Newell and Tramer, 1978; Abrahamson, 1979; Hancock In situations where nutrients strongly limit plant and Pritts, 1987). These patterns point to a decreasing growth, nutrient allocation patterns better reflect con- investment in growth and colonization in advanced straints on plant resource economy than do biomass successional stages, but an increasing investment in allocation patterns (Reekie and Bazzaz, 1987b). Yet, structures contributing to below-ground competitive and though nitrogen often limits plant growth in ability (Gleeson and Tilman, 1990). Early successional successional environments (Gleeson and Tilman, 1990; species also produce many small well-dispersed seeds, cf. Garnier et al., 2007 for the Mediterranean sere whereas late successional species invest in few large concerned here), only few studies have examined how offspring with higher probability of survival and nitrogen availability might affect the biomass and establishment (Stewart and Thompson, 1982; Gleeson nitrogen allocation patterns of species differing in and Tilman, 1990; Schippers et al., 2001; Fenner and successional status (e.g. Olff, 1992). Nitrogen availabil- Thompson, 2005). These changes in species character- ity influences allocation patterns between shoots and istics are usually concomitant with the replacement of roots, and affects nitrogen concentration of plant organs annual species by perennial species over succession (e.g. Reynolds and D’Antonio, 1996; Garnier, 1998). (Bazzaz, 1996; Prach et al., 1997; Vile et al., 2006 for the But the way in which internal nitrogen is distributed successional sere examined in the present study). among vegetative and reproductive organs is poorly Resource allocation patterns are traditionally studied understood. The studied species were therefore grown at as ratios between organ and total plant biomasses two nitrogen levels (limiting vs. non-limiting), to analyse (Reekie and Bazzaz, 1987a). However, biomass (and the impacts of nitrogen availability on the nitrogen nutrient) ratios usually vary with plant size; therefore concentrations of the different plant parts and on the the partitioning approach potentially confounds alloca- patterns of biomass and nitrogen allocation. tion patterns with plant size (Samson and Werk, 1986; The questions addressed in this study are as follows: Klinkhamer et al., 1990; Jasienski and Bazzaz, 1999; (1) Do species strategies vary according to successional Weiner, 2004). To examine life history tradeoffs as status when grown under common experimental condi- reflected by allocation patterns, several authors hence tions? Expectations are that species from the early argued that allometric functions are more appropriate successional stage would allocate, allometrically as well tools than biomass (or nutrient) ratios (Samson and as proportionally, more biomass to reproduction, and Werk, 1986; Klinkhamer et al., 1992; Sugiyama and produce smaller, less provisioned, but more numerous Bazzaz, 1998). The first aim of this study was to examine seeds than species from the late successional stage. (2) how allocation patterns differed among 18 plant species Does nitrogen availability alter species allocation characteristics of different stages of Mediterranean old- patterns and seed traits? Expectations are that high field successions, using this allometric approach. nitrogen supply would increase the biomasses and Most current knowledge on resource allocation nitrogen concentrations of vegetative and reproductive patterns in species characteristic of different succes- organs, subsequently altering resource allocation pat- sional stages is derived from studies conducted in situ terns between vegetative and reproductive organs, and (e.g. Newell and Tramer, 1978; Gleeson and Tilman, ultimately modifying the investment in reproduction. 1990; but see Jongejans et al., 2006 for a common garden experiment mimicking a successional gradient). In such studies, it is not possible to disentangle the effects of species identity from those of environmental Material and methods factors. In the Mediterranean successional seres of concern here, substantial differences in environmental Study site conditions were found as succession proceeds: light penetration at ground level decreased two-fold (Kaza- The experiment was conducted from October 2003 to kou and Navas, 2004) while total soil nitrogen September 2005 in the experimental garden of the concentration increased three-fold (Garnier et al., Centre d’Ecologie Fonctionnelle et Evolutive (CEFE, 2004) between recently abandoned fields and fields CNRS) in Montpellier, France (431590 N, 31510 E, 60 m abandoned 40 years earlier. Since light and nitrogen above sea level). The climate is Mediterranean sub- may exert strong controls on plant allocation patterns humid (Daget, 1977) with cool to cold winters, marked (see Poorter and Nagel, 2000 for a review), the detection summer drought and unpredictability of precipitation in of intrinsic differences among species from different time and amount. At the beginning of the experiment in successional stages requires that all species be grown October 2003, the soil pH was 7.82, which was close to under identical conditions. We therefore grew the 18 the pH values of the old-field succession from which the species in an experimental garden to test whether species originated (cf. Garnier et al., 2004). ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 269

Table 1. List of the 18 species characteristic of three stages of a Mediterranean old-field succession that were grown in an experimental garden (Flora: Euro+Med Plantbase, www.emplantbase.org).

Genus Species Family Taxonomic group Life history Successional stage

Arenaria serpyllifolia Caryophyllaceae Annual Early Bromus madritensis Poaceae Group 1 Annual Early Crepis foetida Asteraceae Group 4 Annual Early Geranium rotundifolium Geraniaceae Annual Early Medicago minima Fabaceae Group 2 Annual Early Veronica persica Scrophulariaceae Group 3 Annual Early Calamintha nepeta Lamiaceae Group 3 Perennial Intermediate Dactylis glomerata Poaceae Group 1 Perennial Intermediate Daucus carota Apiaceae Biennial Intermediate Picris hieracioides Asteraceae Group 4 Biennial Intermediate Tordylium maximum Apiaceae Annual Intermediate Trifolium angustifolium Fabaceae Group 2 Annual Intermediate Brachypodium phoenicioides Poaceae Perennial Advanced Bromus erectus Poaceae Group 1 Perennial Advanced Inula conyza Asteraceae Group 4 Perennial Advanced Psoralea bituminaria Fabaceae Group 2 Perennial Advanced Rubia peregrina Rubiaceae Perennial Advanced Teucrium chamaedris Lamiaceae Group 3 Perennial Advanced

Experimental design annuals and biennials had been transplanted in autumn 2004 and the perennials were 2 years old. Eighteen herbaceous species were selected among the In the fertilized treatment (N+ treatment hereafter), most abundant species occurring in Mediterranean old- ammonium nitrate was applied each year at a rate of field successions in the south of France (Navas et al., 250 kg ha1, divided equally among three dates (between 2003; Garnier et al., 2004). Three stages of succession January and April). No fertiliser was added in the were identified based on time since land abandonment at control treatment (N treatment hereafter). The N which they most commonly occurred: early (0–6 years), treatment was strongly growth-limiting, whereas the intermediate (7–15 years) and advanced (16–45 years). N+ treatment was considered as optimal for growth. Six representative species were chosen for each of the Further details can be found in Kazakou et al. (2007). three successional stages (Table 1): six annuals for the early successional stage; two annuals, two biennials and Biomass and nitrogen concentration of plant organs two perennials for the intermediate successional stage; and six perennials for the late successional stage. Since Twenty-four plants of each species were harvested in under our experimental conditions biennials behaved May 2005, 12 per nitrogen treatment and three per plot, like annuals (i.e. they flowered the first year), they were and eight plants of each species were harvested in July pooled with annuals in all statistical analyses (see 2005, four per nitrogen level and one per plot. below). Four species from each successional stage were Allocation ratios (see below) were determined on one chosen to form four taxonomic groups (see supplemen- plant per plot (the ‘‘allocation’’ plant, see Appendix 1) tary materials in Kazakou et al., 2007) so as to test if harvested in both May and July, which was divided into shifts were similar for different lineages as succession four compartments: leaves, stems, reproductive organs proceeds. (excluding reproductive stems) and aboveground dead Each species was grown in monoculture, replicated parts (Appendix 1). Each compartment of the ‘‘alloca- four times within two levels of N supply, thus making a tion’’ plant, as well as the two additional plants total of 144 monocultures. Monocultures of each species harvested in May, was oven-dried at 60 1C before were established in October 2003 by transplantation of weighing. Nitrogen concentration of leaves (LNC) and either seedlings or ramets (according to species) in plots reproductive organs (RepNC) were determined on of 1.20 m 1.20 m so as to ensure a standard plant ground material with an elemental analyser (Flash density of 100 plants m2. Seedlings of annual and EA1112 Series, NC Soil analyzer, ThermoFinnigan). biennial species were transplanted each autumn to In each nitrogen treatment, the fruits of each species renew the monocultures, while perennial species were were collected from other individuals at maturity, left to grow for 2 years (until the end of summer 2005). between July and August 2005. After the fruits were The data of the present study were collected in 2005: the air-dried, seeds were extracted and sorted. Dispersal ARTICLE IN PRESS 270 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 structures (wings, pappus, fruit pulp) were removed. The Cornelissen et al., 2003; Weiner, 2004) were assessed seeds were then oven-dried at 80 1C for 48 h before using leaf biomass as a measure of plant size, to avoid weighing (Cornelissen et al., 2003). To determine the erroneous correlations that could arise between repro- seed mass, we weighed separately 60 seeds per species, 30 ductive biomass and total plant biomass (Samson and per nitrogen treatment (Balance M5C, Sartorius, preci- Werk, 1986; Klinkhamer et al., 1992). sion: 103 mg). The mean seed number per plant was approximated by dividing the maximum biomass of reproductive organs by the mean seed mass (Sletvold, 2002). Seed nitrogen concentration (SNC) was deter- Statistical analyses mined on four seed replicate samples following the same procedure used for the other plant organs (see above). Average data for all species and for each nitrogen treatment are given in Appendix 2. The distributions of values were tested for normality for all variables, which Allocation and allometry were log-transformed when necessary. The effects of species identity (nested within succes- Leaves and reproductive organs were considered as sional stage), successional stage, nitrogen treatment and representative plant compartments, respectively, for the interaction between successional status and nitrogen vegetative growth and reproduction. The allocation to treatment on the variables were tested with a four-way leaves and reproductive organs was estimated using ANOVA. If the effect of successional stage was biomass as a representative currency for carbon significant, post hoc tests (Student–Newman–Keuls acquisition and nitrogen biomass as a currency for comparisons) were performed within each nitrogen nitrogen acquisition (Reekie and Bazzaz, 1987b; Bazzaz treatment in order to identify the variations among et al., 2000). successional stages. To investigate the effects of species’ During ontogeny, annuals show a peak of leaf successional stage and taxonomic group on the vari- biomass in spring (around May) and a peak of ables, two-way ANOVAs were carried out for the 12 reproductive biomass in late spring, whereas perennials species belonging to the four taxonomic groups. increase in leaf biomass until a maximum each spring The effect of life history on the variables was tested while their reproductive biomass reaches a peak in with a one-way ANOVA (annuals and biennials pooled summer (around July). For the analyses, we considered together, see above). If the effect of life history was leaf biomass at the peak of vegetative growth (May), significant, a one-way ANOVA was carried out to test and reproductive biomass at the peak of reproductive the effect of successional stage on variables within each growth (July for most species, but May for three annual life history, and when necessary post hoc tests (Stu- species, Arenaria serpyllifolia, Medicago minima and dent–Newman–Keuls comparisons) were performed Veronica persica). The leaf biomass per plant was within each life history and each nitrogen treatment to calculated by multiplying the leaf allocation ratio of identify the variation among successional stages. the ‘‘allocation’’ plant to the mean total aboveground The size dependency of allocation patterns between biomass of the three harvested plants per plot in May reproductive and leaf biomasses was analysed by the (see Appendix 1). For nitrogen concentrations, we used allometric equation: R ¼ bVa, where R is the reproduc- the values of LNC and RepNC corresponding, respec- tive biomass, V is the leaf biomass, and a and b are, tively, to the maximum leaf and reproductive biomasses. respectively, the scaling exponent and the allometric We multiplied these concentrations by the correspond- constant (Samson and Werk, 1986; Klinkhamer et al., ing biomasses so as to obtain, respectively, the nitrogen 1992; Sugiyama and Bazzaz, 1998). We log-transformed biomass of leaves and reproductive organs. the variables so as to obtain a log–log linear allometric Proportional allocation to leaves and reproductive relationship: log R ¼ log b+a log V, where a and log b organs were calculated, respectively, as the ratios of leaf are parameters of the regression reflecting, respectively, biomass and reproductive biomass to the total above- slope and y-intercept. Standardised major axis (SMA) ground (dead and alive) biomass of the corresponding regression methods were used to determine a and log b harvest (Olff, 1992; Schippers and Olff, 2000; Fenner (Niklas, 1994; Warton et al., 2006). We compared these and Thompson, 2005, see Appendix 1). The leaf parameters between successional stages and between allocation ratio thus corresponded to the May harvest, nitrogen treatments by likelihood-ratio tests of residual while the reproductive allocation ratio corresponded to variances (Klinkhamer et al., 1992; Warton et al., 2006). the July harvest (except for A. serpyllifolia, M. minima To assess the effect of successional stage on allometric and V. persica, which set seeds earlier). coefficients and biomass ratios, the analyses were carried Allometric relationships between vegetative and out by grouping the 6 species from each stage (Table 1); reproductive biomass (Samson and Werk, 1986; Klin- similarly, the effects of nitrogen were assessed by khamer et al., 1992; Sugiyama and Bazzaz, 1998; grouping the 18 species per nitrogen level. ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 271

Table 2. Results of SMA regressions between reproductive and leaf biomasses and nitrogen biomasses by successional stage.

Successional stage SMA regression R2 Post hoc tests

Biomass E log reproductive biomass ¼ 0.68 log leaf biomass+0.01 0.08 n.s. – I log reproductive biomass ¼ 1.13 log leaf biomass0.25 0.51** a A log reproductive biomass ¼ 1.74 log leaf biomass0.73 0.55** a Nitrogen biomass E log reproductive N biomass ¼ 0.61 log leaf N biomass+0.50 0.35* a I log reproductive N biomass ¼ 1.10 log leaf N biomass0.50 0.42* a, b A log reproductive N biomass ¼ 1.50 log leaf N biomass1.40 0.71*** b

The data from the two nitrogen treatments were pooled for the analyses. Post hoc test for multiple comparisons of slopes among successional stages are given in Latin letters for absolute biomasses and in Greek letters for nitrogen biomasses. Abbreviations: E, early successional stage; I, intermediate successional stage; A, advanced successional stage; leaf N biomass, leaf nitrogen biomass; reproductive N biomass, reproductive nitrogen biomass. ***, Po0.001; **, Po0.01, *, Po0.05, n.s., non significant.

Ordinary least square (OLS) regressions were per- formed between seed mass and seed number. Pearson correlation tests were performed between SNC and both LNC and RepNC. The statistical analyses were all performed with the SAS system, Version 9 (SAS Institute, Cary, NC, USA), except for the SMA and OLS regressions which were computed using the software package (S)MATR (Stan- dardised Major Axis Tests and Routines, Falster et al., 2003).

Results

Effect of successional status

Reproductive and leaf biomasses were significantly related in species from the two most advanced stages of succession, but not in species from the early stage (Table 2). This result was due to the particularly low leaf biomasses relative to their reproductive biomasses of three annual species (A. serpyllifolia, Bromus madritensis and V. persica), making it difficult to test for the allometric relationship (Fig. 1a). As these species have earlier phenologies than the other successional species, we may have underestimated their maximum leaf biomasses. Thus to compare resource allocation to reproduction among successional stages, we relied solely on allocation ratios (see below). Reproductive and leaf nitrogen biomasses were Fig. 1. Relationships between (a) reproductive and leaf significantly related in all successional stages (Table 2). biomasses and (b) reproductive and leaf nitrogen biomasses. The allometric slope of early successional species was Symbols: J, nitrogen-limiting treatment; &, nitrogen supply significantly lower than that of late successional species treatment; white, early successional species; gray, intermediate (likelihood ratio ¼ 7.267**, post hoc tests in Table 2). A successional species; black, late successional species. lower slope associated with a higher y-intercept implied that, at similar leaf nitrogen biomass, early successional For comparison purposes with other studies, we species allocated more nitrogen biomass to reproduction tested the differences in allocation ratios among succes- than late successional species (Fig. 1b). sional stages. Leaf biomass ratio showed no clear ARTICLE IN PRESS 272 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283

Fig. 2. Biomass allocation to (a) leaves and (b) reproductive parts for species differing in successional stages, and biomass allocation to (c) leaves and (d) reproductive parts by life history in each successional stage by nitrogen treatment (white bars: nitrogen-limiting treatment, black bars: nitrogen supply treatment). F values and probabilities for GLM testing the effect of successional stage at each nitrogen level are indicated (***, Po0.001; **, Po0.01, *, Po0.05, +, 0.05oPo0.10, n.s., non significant). Results of post hoc tests between successional stages for each nitrogen treatment are given (Latin letters: nitrogen-limiting treatment, greek letters: nitrogen supply treatment). Abbreviations: E, early successional stage; I, intermediate successional stage; A, advanced successional stage; N, nitrogen-limiting treatment; N+, nitrogen supply treatment.

pattern with successional stage (Fig. 2a), whereas early and intermediate successional species than in late reproductive biomass ratio was significantly higher in successional species (Fig. 3c, inset). Seed mass and seed early than in late successional species (Fig. 2b). number were negatively correlated (Fig. 4). Seed Seed mass was higher in late successional species than nitrogen concentration was higher in early and inter- in early and intermediate successional species (Fig. 3e), mediate successional species than in late successional whereas seed number produced per plant was higher in species (Fig. 3f).

Fig. 3. Effect of nitrogen supply on (a) leaf biomass, (b) leaf nitrogen concentration (LNC), (c) reproductive biomass (and seed number), (d) reproductive nitrogen concentration (RepNC), (e) seed mass and (f) seed nitrogen concentration (SNC). F values and probabilities for GLM testing the effect nitrogen supply and the effect of successional stage at each nitrogen level are indicated (***, Po0.001; **, Po0.01, *, Po0.05, +, 0.05oPo0.10, n.s., non significant). Abbreviations:N, nitrogen-limiting treatment; N+, nitrogen supply treatment. White, early successional species; gray, intermediate successional species; black, late successional species. ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 273 ARTICLE IN PRESS 274 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283

Fig. 4. Relationship between mean seed mass and mean seed number per plant. Symbols: J, nitrogen-limiting treatment; OLS, log seed mass ¼0.94 log seed number+2.86 (R2 ¼ 0.75***); &, nitrogen supply treatment; OLS, log seed mass ¼0.98 log seed number+2.99 (R2 ¼ 0.77***); white, early successional species; gray, intermediate successional species; black, late successional species.

Effect of life history lower seed mass (F ¼ 115.89*** and 138.21*** for N and N+ treatment, respectively) and higher seed Annuals exhibited higher leaf biomass ratios than number (F ¼ 12.98** and 7.40* for N and N+ perennials in the N+ treatment (F ¼ 6.04*); this treatment, respectively) than perennials from the ad- difference being marginally significant in the N vanced successional stage. treatment (F ¼ 3.59, P ¼ 0.063). They showed higher reproductive biomass ratios than perennials in both treatments (F ¼ 10.18** and 10.45** for N and N+ Effect of nitrogen supply treatment, respectively). They also had lower seed mass (F ¼ 77.19*** and 101.58*** for N and N+ treat- Although nitrogen supply increased leaf biomass ment, respectively), but higher SNC (F ¼ 5.53* and (+28.4% in average, Fig. 3a), it had no effect on 5.61* for N and N+ treatment, respectively) than reproductive biomass (Fig. 3c). Allometric analysis of perennials. Annuals exhibited significantly higher seed reproductive and leaf biomasses revealed no differences number than perennials only in the N treatment in neither slopes (likelihood ratio ¼ 0.08 n.s.) nor y- (F ¼ 4.95*). intercepts (Wald statistic ¼ 0.61 n.s.) of the regressions As annuals occur in both early and intermediate for each nitrogen treatment (Table 3, Fig. 1a), suggest- successional stages, whereas perennials similarly occur ing that nitrogen supply had no effect on allometric in both intermediate and advanced successional stages, patterns. Nitrogen supply had no effect on proportional we tested for differences between successional stages biomass allocation to either leaves or reproductive within each life history. Annuals from the early organs (Fig. 2a and b), with few exceptions in some successional stage had lower leaf biomass ratios early successional species (see details in Table 4). (F ¼ 23.69*** and 28.75*** for N and N+ treatment, LNC and RepNC were significantly higher in the N+ respectively, Fig. 2c), but higher reproductive biomass treatment (+50.2% and +15.5%, respectively, Fig. 3b ratios (F ¼ 7.15* in the N treatment, Fig. 2d) and and d), which led to an overall increase in leaf nitrogen higher seed mass (F ¼ 55.91*** and 53.48*** for N biomass (F ¼ 88.00***), but no effect on reproductive and N+ treatment, respectively) than those from the nitrogen biomass (F ¼ 2.12 n.s.). This might have intermediate successional stage. Perennials from the occurred because the effect of nitrogen supply was intermediate successional stage exhibited lower leaf stronger on LNC than on RepNC. The effects of biomass ratios (F ¼ 13.54** and 11.50** for N and nitrogen supply per species are detailed in Table 4. N+ treatment, respectively, Fig. 2c) than those from the Allometric analyses of leaf and reproductive nitrogen advanced successional stage, but showed no difference biomasses showed no difference in slopes (likelihood in reproductive biomass ratios (Fig. 2d). They also had ratio ¼ 0.25 n.s.) and no elevation shifts (Wald ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 275

Table 3. Results of SMA regressions between reproductive and leaf biomasses (expressed in absolute values and nitrogen biomasses) by nitrogen treatment.

Treatment SMA regression R2 Post hoc tests

Biomass N log reproductive biomass ¼ 0.79 log leaf biomass0.20 0.23* a N+ log reproductive biomass ¼ 0.86 log leaf biomass+3.28 0.37** a Nitrogen biomass N log reproductive N biomass ¼ 0.93 log leaf N biomass0.25 0.50*** a N+ log reproductive N biomass ¼ 0.81 log leaf N biomass+0.15 0.38** a

Post hoc test for multiple comparisons of slopes among successional stages are given (Latin letters: absolute biomasses; Greek letters: nitrogen biomasses). Abbreviations:N, nitrogen-limiting treatment; N+, nitrogen supply treatment; leaf N biomass, leaf nitrogen biomass; reproductive N biomass, reproductive nitrogen biomass. ***, Po0.001; **, Po0.01, *, Po0.05.

statistic ¼ 2.75 n.s.) between nitrogen treatments (Table Discussion 3, Fig. 1b). Seed number was not affected by nitrogen supply Allocation strategies and seed traits among species (Fig. 3c, inset). However, seed mass (Fig. 3e) was differing in successional status significantly higher in the fertilized treatment, though the effect was relatively weak (+1.0%) and varied In this study we grew 18 successional species in an among species (Table 4). Nitrogen supply did not alter experimental garden under identical growth conditions, the negative correlation between seed mass and seed controlling for nitrogen supply. We could therefore number (Fig. 4, likelihood ratio ¼ 0.05 n.s. for the dissociate the intrinsic allocation patterns in succes- slopes, Wald statistic ¼ 0.04 n.s. for the y-intercepts). sional species from the effects of environmental factors. SNC was positively correlated to LNC In addition, contrary to most experimental studies, (CCPearson ¼ 0.64*** and 0.38** for N and N+ which examined allocation patterns after less than 1 year treatment, respectively) and to RepNC of growth (e.g. Wilson and Thompson, 1989; Olff, 1992; (CCPearson ¼ 0.45** and 0.43*** for N and N+ Mu¨ller et al., 2000), we grew perennial species for 2 treatment, respectively). As nitrogen supply increased years, which might be a more relevant time frame. LNC and RepNC, it similarly increased seed nitrogen We showed that early successional species allocated, concentration (Fig. 3f), but this effect was much smaller allometrically (Fig. 1b for nitrogen biomass) as well as (+3.8%) and varied among species (Table 4). proportionally (Fig. 2b for absolute biomass), more resources to reproduction and that they set more seeds (Fig. 3c, inset) than later successional species, as has previously been observed in situ (e.g. Gleeson and Effect of taxonomy Tilman, 1990; Bazzaz et al., 2000). We thus demon- strated that allocation patterns observed in situ are due We found that the four taxonomic groups differed for to intrinsic differences in plant allocation strategies, and all variables measured (Table 5), except absolute are then not a sole consequence of differences in reproductive biomass in both nitrogen treatments and environmental factors as succession proceeds. reproductive nitrogen biomass in the N+ treatment. Differences in allocation patterns among species of The taxonomic signals were strongest for seed mass and different successional status were partly linked to species SNC. For example, in the N treatment, seed mass (mg) life histories. For example, annual species, characteristic increased from 2.72 to 4.04, from 1.76 to 20.26, from of the early successional stage, allocated more resources 0.56 to 1.40, yet decreased from 0.63 to 0.18, over to reproduction than did perennial species, which succession in Poaceae, Fabaceae, Scrophulariaceae and dominated in the advanced successional stage. However, Asteraceae respectively. Similarly, in the N+ treatment, comparable trends were observed within a given life it increased from 2.84 to 4.22, from 1.90 to 21.94, from history: annuals from the early successional stage 0.62 to 1.83, yet decreased from 0.60 to 0.21, over allocated more resources to reproduction than annuals succession in Poaceae, Fabaceae, Scrophulariaceae and from the intermediate successional stage. Thus variation Asteraceae, respectively (Appendix 3). Hence, we in allocation patterns can be related to species life observed the same overall patterns along the succes- history when considering the whole successional sere sional gradient when considering either the whole set of (Vile et al., 2006), but also to species successional status species or the 12 species from the four taxonomic when considering a given life history. groups. ARTICLE IN PRESS 276 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 SNC 0.10, n.s., non o P o Seed number 0.05, +, 0.05 mass Seeds o P 0.01, *, o RepNC Seed P 0.001; **, o P 0.24 n.s. 57.24*** 2.28 n.s. 2.69 n.s. 1.19 n.s. Reproductive biomass ratio Reproductive N biomass biomass Reproductive parts LNC Reproductive Leaf biomass ratio Leaf N biomass 0.00 n.s. 2.64 n.s.0.09 n.s. 1.30 n.s.8.23* 0.31 n.s.0.61 n.s. 4.22 0.12 50.76*** n.s. n.s.0.64 n.s. 5.33 1.56 n.s. n.s.2.84 0.41 n.s. 0.00 n.s. 6.87* n.s. 0.00 n.s. 7.52* 12.24* 1.44 n.s.1.27 23.81** 0.56 n.s. n.s. 122.07*** 0.14 n.s.0.64 0.01 n.s. 5.53 0.10 n.s. 2.01 0.46 n.s. n.s. n.s. n.s.0.61 n.s. 1.04 n.s. 1.02 25.26** n.s.3.82 n.s. 1.56 0.02 n.s. 153.40*** 0.06 n.s. 25.53** 22.16** n.s.1.56 0.21 n.s. 0.90 n.s. 3.16 0.00 n.s. 0.00 0.52 n.s. n.s. n.s.0.15 n.s. n.s. 3.18 1.27 n.s. 1.08 n.s. n.s.8.29 14.06** n.s. 0.20 3.04 n.s. n.s. 1.25 41.81*** n.s.0.17 20.75** 1.32 n.s. 7.28 n.s. 0.13 1.74 n.s. n.s. 3.27 n.s. 0.28 n.s. 0.75 n.s. 0.00 6.52 n.s. 9.12* 0.97 9.52* 2.81 n.s. + 0.36 n.s. n.s. n.s. 17.44** 2.99 4.01 n.s. 1.30 n.s. 3.52 n.s. 7.58 n.s. 1.12 3.40 n.s. 0.00 n.s. n.s. 6.39* 2.92 n.s. 5.97 0.60 n.s. 0.01 0.12 + n.s. 0.14 29.79** 0.52 n.s. n.s. n.s. n.s. 15.71** 3.80 2.24 n.s. 1.71 n.s. 0.18 n.s. 1.33 n.s. n.s. 1.10 8.87 n.s. 0.08 0.62 n.s. 0.12 n.s. 10.34* n.s. 0.99 n.s. 0.69 n.s. 3.43 1.64 n.s. n.s. 16.06** 0.12 n.s. n.s. 11.94* 0.72 n.s. 0.01 n.s. 0.33 3.36 0.01 4.39 n.s. n.s. n.s. n.s. 3.10 13.89*** 1.00 0.78 0.09 n.s. n.s. n.s. 0.33 n.s. n.s. 0.73 0.96 4.05 18.36** 6.70* n.s. n.s. n.s. 2.63 n.s. 13.35* 0.42 1.18 n.s. 3.49 n.s. 1.17 n.s. n.s. 0.00 n.s. 11.69*** 0.00 6.61* n.s. 7.94* 0.00 n.s. 0.91 2.44 n.s. n.s. 0.00 n.s. 3.20 n.s. 1.05 0.03 24.04*** 0.44 n.s. n.s. n.s. 28.54** 1.49 7.62* n.s. 2.13 n.s. 7.58* 65.77*** 41.15*** 16.08** 25.33** 8.93* 104.78*** 17.03** 2.77 n.s. 35.56** 23.25** 10.20* 27.01** 0.21 n.s. 35.32*** 0.7428.98* n.s. 10.10 + 2.05 n.s. 2.81 n.s. 6.80* 16.31** 23.70* 9.55* 6.54 n.s. 10.89** 1.64 n.s. 1.41 0.12 n.s. n.s. 0.44 n.s. 8.03** 15.37* 8.74* Leaves 12.90* 44.95** 4.86 n.s. 105.98*** 3.30 n.s. 9.60* Leaf biomass values and probabilities) on the effects of nitrogen supply on resource allocation and seed traits for each successional species. F E E E E I I I I I I A A A A A A stage E E : E, early successional stage; I, intermediate successional stage; A, advanced successional stage; Leaf N biomass, leaf nitrogen biomass; LNC: leaf nitrogen concentration; Reproductive Results of GLM ( C. foetida G. rotundifolium M. minima V. persica C. nepeta D. glomerata D. carota P. hieracioides T. maximum T. angustifolium B. phoenicioides B. erectus I. conyza P. bituminaria R. peregrina T. chamaedris significant. Species Successional A. serpyllifolia B. madritensis Table 4. Abbreviations N biomass, reproductive nitrogen biomass; RepNC, reproductive nitrogen concentration; SNC, seed nitrogen concentration. ***, ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 277

Table 5. Results of GLM (F values and probabilities) on the effects of taxonomic groups and successional stages on biomasses and nitrogen biomasses, and nitrogen concentrations of leaves, reproductive parts and seeds, as well as seed number for 12 species.

N N+

Taxonomic group Successional stage Taxonomic group Successional stage

Leaves Leaf biomass 7.66*** 11.72*** 12.94*** 24.06*** Leaf N biomass 16.27*** 13.50*** 9.91*** 15.27*** Leaf biomass ratio 35.89*** 0.70 n.s. 13.85*** 1.21 n.s. LNC 32.58*** 5.71** 6.72*** 2.69 n.s.

Reproductive parts Reproductive biomass 1.90 n.s. 1.08 n.s. 1.32 n.s. 4.05* Reproductive N biomass 3.93* 1.05 n.s. 2.30 n.s. 4.19* Reproductive biomass ratio 4.92** 8.80*** 2.76* 3.74* RepNC 17.40*** 3.92* 8.99*** 1.50 n.s. Seeds Seed mass 265.03*** 244.69*** 315.47*** 329.48*** Seed number 8.89*** 5.76** 17.12*** 4.45* SNC 82.10*** 18.95*** 66.73*** 22.32***

Abbreviations:N, nitrogen-limiting treatment; N+, nitrogen supply treatment; leaf N biomass, leaf nitrogen biomass; LNC, leaf nitrogen concentration; reproductive N biomass, reproductive nitrogen biomass; RepNC, reproductive nitrogen concentration; SNC, seed nitrogen concentration. ***, Po0.001; **, Po0.01, *, Po0.05, n.s., non significant.

The contrasting allocation strategies among species We also showed a tight negative relationship between from differing successional stages were conserved when seed mass and seed number for the 18 studied species phylogeny was taken into account (Table 5, Appendix (Fig. 4, cf. Shipley and Dion, 1992; Vile et al., 2006). 3). Thus the trends we observed in the general This trade-off suggested contrasting strategies in early comparisons corresponded to actual shifts related to and late successional species (Jakobsson and Eriksson, the ecology of species, and were not the result of the 2000; Leishman et al., 2000; Coomes and Grubb, 2003). replacement of one particular taxonomic group by Early successional species produced numerous small another as succession proceeds. seeds, whose small size gives a higher probability to be In our study, plants were grown in monoculture, at a efficiently dispersed by the wind (Clark et al., 1998)and density that allowed canopy closure for all species and whose high number increases the probability to success- provided a good basis for interspecific comparisons (cf. fully colonize new sites (Silvertown, 1981; Coomes and Garnier et al., 1997). Hence a valid question is whether Grubb, 2003). variation in , and in intensity of intraspe- Moreover we found that heavier seeds were associated cific , may potentially affect allocation with lower seed nitrogen concentrations (Fig. 3c, inset patterns of the studied species. In another aspect of this and Fig. 3f). Heavier seeds were previously found to be experiment, Violle et al. (2007) found strong differential poorer in nitrogen-rich molecules, and richer in carbo- effects of the 18 monocultures on light and water hydrate resources (Grubb and Burslem, 1998; Kidson availabilities under their cover. However, allocation and Westoby, 2000), which can be used advantageously patterns of individuals of B. madritensis and Crepis during early seedling growth (Silvertown et al., 1997; foetida grown in the 18 monocultures or grown as Kidson and Westoby, 2000; Leishman et al., 2000). isolated individuals on bare ground did not vary in any These larger seeds generally develop into larger seedlings of the nitrogen treatments (Appendix 4, common (Weiner et al., 1997; Westoby et al., 2002; Coomes and allometric coefficients: a ¼ 0.557; b ¼0.688 for B. Grubb, 2003), which have higher survival and higher madritensis and a ¼ 0.164; b ¼ 0.045 for C. foetida;C. establishment probability, especially when facing envir- Violle, unpublished data). Moreover, these allocation onmental hazards or competition with other seedlings coefficients were not significantly different from the (Parrish and Bazzaz, 1985; Jakobsson and Eriksson, allocation coefficients observed in this study for these 2000; Leishman et al., 2000; Moles and Westoby, 2004). two species (Appendix 4, a ¼ 0.559; b ¼0.764 for B. In the successional sere from which the species examined madritensis and a ¼ 0.163; b ¼ 0.101 for C. foetida). in the present study were selected, litter accumulation, Based on these considerations, we conclude that and thus light interception, were higher in late succes- variation in the competitive environment resulting from sional stages (Kazakou and Navas, 2004; Garnier et al., variation in plant density should have little impact on 2007). Higher seed mass may thus provide some our results (see also Mu¨ller et al., 2000) advantages for seedling emergence and survival in late ARTICLE IN PRESS 278 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 successional environments, where emerging seedlings Hodgson and Mackey (1986) suggested that seed mass have to pass through a thick litter layer to access light. was severely constrained by phylogenetically inflexible reproductive characters such as carpel structure, pla- centation type, presence or absence of endosperm, and Effects of nitrogen supply on strategies of allocation pattern of embryogenesis. Since seed number and SNC and reproduction are closely linked to seed mass, they might be subject to the same phylogenetic constraints. Nitrogen supply neither changed allometric relation- Synthesizing the data presented in Fig. 3, the impact ships between reproductive and leaf biomasses (Fig. 1a of nitrogen fertilization decreased from vegetative to and b), nor proportional biomass allocation to leaves reproductive to seed compartments, in terms of both and reproductive organs (Fig. 2a and b). These results biomass and nitrogen concentrations (Fig. 5). Increasing were consistent with other studies (for allometric nitrogen supply increased biomass by 28.4% in leaves, trajectories, see Mu¨ller et al., 2000; for biomass and only by 0.5% in reproductive organs and by 1.0% allocation, see Ploschuk et al., 2005; Throop, 2005). in seeds, whereas it increased nitrogen concentration by Most species from the Mediterranean old-field succes- 50.2% in leaves, by 15.5% in reproductive organs and sion thus seemed to be conservative in their resource only by 3.8% in seeds. Similarly, by growing Senecio allocation patterns, and a common allometric relation- vulgaris in 20% and 100% Hoagland’s nutrient solution, ship can be used under both nitrogen levels (Mu¨ller Fenner (1986) found that plant biomass and seed mass et al., 2000). increased by 169.1% and 4.9%, respectively, while The effects of nitrogen supply on seed characteristics vegetative shoot and seed nitrogen concentrations varied among species (Table 4). Some studies showed increased by 65.3% and 5.0%, respectively. The higher that, for particular species, higher nitrogen availability amount of nitrogen absorbed under high nitrogen increased seed mass (e.g. Galloway, 2001; Sultan, 2001), supply was therefore not efficiently translocated from seed number (e.g. Parrish and Bazzaz, 1985; Fenner, vegetative shoots to seeds during seed filling. Hence it 1986) and seed nitrogen concentration (Parrish and has been argued (Roach and Wulff, 1987; Weiner et al., Bazzaz, 1985; Peterson and Rending, 2003; Violle et al., 1997) that, in species not artificially selected, seed 2009). Yet several species in our study showed no characteristics may be relatively buffered against the variation in seed characteristics in response to nitrogen variation in parent nitrogen supply (but see Abutilon supply (Silvertown et al., 1997; Weiner et al., 1997; theophrasti, Parrish and Bazzaz, 1985). Some authors Luzuriaga et al., 2006). Moreover, even if nitrogen studied the contribution of leaves to the nitrogen supply had small effects on seed traits in some species, it incorporated by the seeds in crops and estimated it did not alter the overall strongly negative relationships between 26.4% and 40% in Triticum aestivum L. between seed mass and seed number (Fig. 4). Thus (Simpson et al., 1983; Dreccer et al., 2000) and around reproductive strategies showed little plasticity in re- 6.2% in Brassica napus L. (Dreccer et al., 2000). Our sponse to nitrogen supply in our set of 18 species. results imply that this contribution is quite low in our set

Fig. 5. Percentage of increase in biomass (black bars) and nitrogen concentrations (white bars) of plant organs with nitrogen supply. ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 279 of 18 successional species. This also suggests that Acknowledgments nitrogen may more strongly limit photosynthesis than reproduction. This work was supported by the French National To conclude, the 18 species under scrutiny exhibited Program PNBC ‘‘GEOTRAITS’’. We would like to contrasting strategies of allocation and reproduction thank Sandrine Bioulac, Alain Blanchard, Je´re´mie along the successional gradient. These species showed Devaux, Adeline Fayolle, Ire`ne Hummel, Elena Kaza- little plasticity in allometric relationships between kou, Franc¸oise Lafont, Damien Landais, Esteban reproductive and leaf biomasses, and between seed mass Martinez, Benoˆıt Ricci, Jean Richarte and Se´bastien and seed number. Thus these successional species did Ville´ger for their assistance in the field and laboratory not alter their allocation strategies in response to work. This is a publication from the GDR 2574 nitrogen supply. Natural selection therefore seems to ‘‘Utiliterres’’ (CNRS, France). have resulted in allometric strategies rather than plastic responses to nutrient level (Mu¨ller et al., 2000). We also showed that the effects of external nitrogen supply was substantially greater on vegetative than in reproductive Appendix 1 plant parts, suggesting that internal nitrogen might be more limiting for growth than for reproduction. See Fig. A1.

Fig. A1. Protocol for the calculation of allocation ratios (see allocation plant) and estimation of leaf biomass (for the May harvest, when two additional plants were collected) illustrated for G. rotundifolium. ARTICLE IN PRESS 280 C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 ) 1 SNC (mg g Seed number (nb/ plant) Seeds Seed mass (mg) ) 1 13.61719.62914.449 0.04122.043 0.04425.295 2.717 2063436.429 2.84717.754 8618 0.63325.208 25.320 496 0.59832.528 28.267 734 1.84337.842 4856 1.556 20.706 17.142 3046 1.760 22.085 21.019 46.469 238 1.89525.450 46.597 425 0.55626.255 280 0.616 36.133 17.450 173 0.204 35.385 18.894 1374 0.183 67.571 20.423 1170 0.633 70.938 24.617 3664 23.725 0.66325.342 4785 26.002 1.44122.623 1090 30.445 1.43221.830 28.058 879 0.74031.191 1342 32.486 0.60928.537 1450 5.477 40.046 31.973 31.529 555 5.42816.766 2915 26.827 2.60818.724 680 2.822 35.432 19.949 40.706 334 3.11626.190 797 3.503 20.791 22.566 315 4.041 22.849 24.721 567 4.223 72.823 40.283 266 0.182 73.752 35.083 119 0.209 28.977 25.506 20.262 300 28.760 23.996 2995 21.945 23.398 24.963 8642 21.576 21.145 25.968 295 19.490 33.250 396 40.455 1.398 40.667 36 1.833 41.039 45 968 20.741 686 24.357 21.017 17.391 RepNC (mg g tration; SNC, seed nitrogen concentration. 0.288 0.155 0.285 0.283 0.287 0.176 0.180 0.168 0.218 0.106 0.547 0.330 0.098 0.054 0.056 0.031 0.128 0.169 0.042 0.064 0.294 0.258 0.298 0.218 0.066 0.024 0.026 0.070 0.028 0.030 0.257 0.198 0.270 0.153 0.191 0.173 Reproductive biomass ratio 7.288 8.112 9.692 11.352 19.363 46.650 71.722 73.730 17.066 16.330 15.105 11.511 15.042 19.995 23.322 12.089 10.385 38.935 51.363 10.775 39.943 97.762 62.315 58.878 26.972 31.312 15.076 32.544 10.453 45.274 10.201 17.368 32.521 30.486 237.949 283.212 Reproductive N biomass (mg) , nitrogen-limiting treatment; N+, nitrogen supply treatment; leaf N biomass, leaf Reproductive biomass (mg) Reproductive organs ) 1 LNC (mg g Leaf biomass ratio 4.131 0.105 14.216 0.839 4.512 0.066 15.1422.3016.774 1.347 0.087 0.100 20.052 31.623 0.764 0.720 17.06534.261 0.25777.337 0.13117.603 0.31953.267 28.34038.884 0.37956.877 35.154 0.380 22.617 0.376 0.508 0.45126.378 17.511 2.091 51.761 35.212 3.072 23.676 33.475 0.13528.051 41.583 0.439 0.07777.795 0.661 0.089 0.493 0.04252.638 24.522 0.329 0.216 41.22558.176 21.365 0.170 34.986 0.748 56.384 23.237 0.876 0.21274.610 0.690 58.018 29.163 0.583 0.22597.096 1.934 0.49121.959 21.465 0.12168.624 0.411 0.10957.951 35.936 0.065 38.601 3.726 0.123 16.921 0.131 22.853 2.078 36.345 18.503 0.889 65.470 32.493 1.767 67.675 22.153 0.930 0.547 0.482 0.468 1.265 0.557 0.545 22.782 25.102 18.069 0.768 0.879 1.353 Leaf N biomass (mg) 0.305 0.623 0.970 3.4231.493 110.1581.399 0.218 0.3311.263 0.797 31.5034.424 1.822 3.951 154.3923.904 166.3741.999 0.360 148.6334.303 0.1422.202 34.567 0.5575.117 41.593 2.077 5.210 38.888 1.774 180.0792.689 1.812 251.7944.385 0.086 0.119 142.373 34.601 48.711 0.602 1.810 8.681 31.765 1.257 0.312 3.414 0.924 1.152 0.122 1.033 1.096 3.252 1.657 2.682 1.572 3.226 1.226 2.585 6.0421.554 240.0633.940 0.260 40.545 5.976 Leaves Leaf biomass (mg) N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ N+ Nitrogen treatment EN EN EN EN EN EN IN IN IN IN IN IN AN AN AN AN AN AN Successional stage Resource allocation and seed traits for each successional species by nitrogen treatment. : E, early successional stage; I, intermediate successional stage; A, advanced successional stage; N A. serpyllifolia B. madritensis C. foetida G. rotundifolium M. minima V. persica C. nepeta D. glomerata D. carota P. hieracioides T. maximum T. angustifolium B. phoenicioides B. erectus I. conyza P. bituminaria R. peregrina T. chamaedris Table A1. Species Abbreviations nitrogen biomass; LNC, leaf nitrogen concentration; reproductive N biomass, reproductive nitrogen biomass; RepNC, reproductive nitrogen concen ARTICLE IN PRESS C. Fortunel et al. / Perspectives in Plant Ecology, Evolution and Systematics 11 (2009) 267–283 281

Appendix 2

See Table A1.

Appendix 3

See Fig. A2.

Appendix 4

See Fig. A3.

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