Resource availability at the rosette stage and apical dominance in the ...

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Resource availability at the rosette stage and apical dominance in the strictly biennial Erysimum strictum (Brassicaceae) Sari Piippo, Ari-Pekka Huhta, Pasi Rautio, and Juha Tuomi

Abstract: In biennial plants, the age of flowering is constrained, but size at flowering is highly variable. This suggests that performance at the flowering stage depends largely on growth conditions at the rosette stage. We examined this possibility using Erysimum strictum P. Gaertn., B. Mey., and Scherb. (Brassicaceae), a strictly biennial herb, the reproductive output of which increases with increasing plant height and branch number. In a common garden experiment, we defoliated (50% of leaves removed twice) and fertilized (three times) individual plants at the rosette stage and studied their performance at the flowering stage in the following year. Rosette defoliation adversely affected all performance measures except seed number per fruit and seed weight. Fertilization did not alleviate these effects. Defoliation reduced seed set by 48% in fertilized plants and 29% in unfertilized plants. Fertilization stimulated branch production from the rosette base but did not significantly affect plant height. These observations suggest that, in the case of basally unbranched plants, apical dominance by the leading stalk suppresses the axillary meristems at the rosette base. Fertilization at the rosette stage can break this suppression. The induction for breakage presumably occurs before bolting since, in our earlier experiments, neither fertilization nor apical damage at the flowering stage stimulated branching from the base. Erysimum strictum is likely to be selected for fast vertical growth at the start of bolting, and hence plant height is a less plastic trait with respect to resource availability than branch number. Regression analysis suggested that, in response to rosette fertilization, small plants invest in height growth instead of branching, whereas large plants to a greater extent invest their supplemental resources in vigorous branching. Consequently, resource availability at the rosette stage influences apical dominance at the flowering stage. Unexpectedly, however, improved resource availability did not alleviate the cost of simulated rosette-stage herbivory. Key words: apical dominance, biennial, fitness, herbivory, resource availability, rosette stage. Résumé : Chez les plantes bisannuelles, l’âge de la floraison est imposé, mais la dimension à la floraison est très variable. Ceci suggère que la performance au stade floral dépend largement des conditions de croissance au stade rosette. Les auteurs ont examiné cette hypothèse en utilisant l’Erysimum strictum P. Gaertn., B. Mey., and Scherb. (Brassicaceae), une plante bisannuelle stricte, dont la production reproductive augmente avec une augmentation de la hauteur de la plante et du nombre de branches. Dans un jardin d’expérimentation commun, les auteurs ont défolié (ablation de 50 % des feuilles, à deux reprises) et fertilisé (trois fois) des plantes individuelles au stade rosette et ont étudié leur performance au stade de la floraison, au cours de l’année suivante. La défoliation des rosettes affecte négativement toutes les mesures de performance, sauf le nombre de graines par fruit et le poids des graines. La fertilisation n’empêche pas ces effets. La défoliation réduit la mise à fruit de 48 % chez les plantes fertilisées et de 29 % chez les plantes non fertilisées. La fertilisation stimule la ramification à partir de la base de la rosette, mais n’affecte pas la hauteur de la plante. Ces observations suggèrent que, dans le cas de plantes non ramifiées à la base, la dominance apicale par le pédoncule supprime les méristèmes axillaires à la base de la rosette. L’induction de la rupture survient probablement avant la montée en graines puisque, dans nos expériences antécédentes, ni la fertilisation, ni le dommage apical au stade floral n’ont stimulé la ramification à partir de la base. L’E. strictum est probablement soumis à une sélection en fonction d’une croissance verticale rapide au début de la mise à graines, et conséquemment la hauteur serait un caractère moins plastique en relation avec la disponibilité des ressources que le nombre de ramifications. L’analyse par régression suggère que, en réaction à une fertilisation de la rosette, les petites plantes investissent dans la hauteur plutôt que dans la ramification, alors que les grandes plantes investissent beaucoup plus leurs ressources supplémentaires dans une ramification vigoureuse. Conséquemment, la disponibilité des ressources au stade rosette influencerait la dominance apicale au stade de la floraison. De façon inattendue, cependant, une amélioration des ressources disponibles ne peut compenser le coût d’une herbivorie simulée au stade rosette. Mots clés : dominance apicale, bisannuelle, adaptation, herbivorie, disponibilité des ressources, stade rosette. [Traduit par la Rédaction]

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Received 9 July 2004. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 8 April 2005. S. Piippo,1 A.-P. Huhta, and J. Tuomi. Department of Biology, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland. P. Rautio.2 Department of Biology, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland, and Finnish Forest Research Institute, Kaironiementie 54, FIN-39700, Parkano, Finland. 1

Corresponding author (e-mail: sari.piippo@oulu.fi).

Can. J. Bot. 83: 405–412 (2005)

doi: 10.1139/B05-015

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Introduction In obligatory monocarpic (semelparous) rosette plants, seed germination and seedling establishment are followed by rosette formation, which in turn is followed by the development of fertile shoots when the plant starts flowering. After flowering and setting seed, the plant dies. Because fecundity often correlates positively with rosette size (Young 1984; Simons and Johnston 1999; Buckley et al. 2003), successful rosette establishment and growth are likely to be the key factors determining the fitness of monocarpic plants. Owing to the importance of successful rosette establishment and growth, a loss of photosynthetic biomass, generally because of herbivory, can be detrimental to the final reproductive success or fitness of monocarpic plants. Plants can tolerate herbivory, but this tolerance depends on many factors, including the timing of herbivory in relation to the age and physiological stage of the plants, the degree of damage in terms of the proportion of removed biomass, and the amount of resources available to repair the damage (Strauss and Agrawal 1999). In Erysimum strictum and other biennial plants, one of the key issues is how herbivory at the rosette stage affects apical dominance in the following year when the plant flowers, since it has been shown that plants released from apical dominance at the flowering stage produce more branches, flowers, and seeds (Huhta et al. 2000a, 2000b). Herbivory at the rosette stage may affect apical dominance by reducing resources that biennial and perennial rosette plants store in taproots. Rosettes may accumulate nutrients in taproots for several years (Gross and Werner 1982; de Jong et al. 1987). In strictly biennial plants, however, the age of flowering is constrained. This leads to considerable within-population variation in the size of rosettes and flowering plants. In biennial Gentianella species, for example, the largest flowering individuals can be 4–10 times larger than the smallest ones (Lennartsson and Oostermeijer 2001; Huhta et al. 2003). Consequently, owing to the constrained age of flowering, biennials are likely to be selected for adaptive plasticity in relation to spatial and temporal heterogeneity in growing conditions both at the rosette stage and at the flowering stage. In strictly biennial E. strictum, fruit production closely correlates with the total number of branches and the final height of the flowering plants (Huhta et al. 2000b). Growing conditions in the second summer affect the architecture of the flowering plants. Fertilization increases and competition decreases lateral branching, while plant height is less affected. In natural populations, E. strictum commonly remains unbranched (Huhta et al. 2000a, 2000b). Our hypothesis is that the shoot architecture of flowering E. strictum plants in the second year is partially determined by their growth in the first year at the rosette stage. We therefore expected improved resource availability (fertilization) at the rosette stage to increase branch production in the second growing season, whereas removal of photosynthetic leaf area at the rosette stage was assumed to have an opposite effect. A number of experimental studies have shown that monocarpic plants can tolerate herbivory at the flowering stage; they may sometimes even grow larger and produce more fruits and seeds when the shoot is browsed or artificially clipped (Binnie and Clifford 1980; Paige and Whitham 1987; Paige 1992; Wegener and Odasz 1997;

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Huhta et al. 2000c). However, studies on the cost of herbivory occurring at the rosette stage of monocarpic plants are scarce. Dhileepan et al. (2000) found that plants defoliated at the rosette stage grew more slowly and produced fewer flowers than undefoliated ones. Herbivory and fertilization may also have interactive effects on plant performance if resource availability alters the cost of herbivory. Maschinski and Whitham (1989) proposed that plants tolerate herbivory best in resource-rich conditions. Their experiments on the tolerance of Ipomopsis arizonica to simulated browsing at the flowering stage supported this. In agreement with this, E. strictum tolerated apical damage at the flowering stage best when the plants were fertilized and grown in the absence of competing grasses (Huhta et al. 2000b). This may not, however, be a general pattern since, according to the meta-analysis by Hawkes and Sullivan (2001), species differ in their responses to herbivory in resource-rich versus resource-poor conditions. Further the tolerance of monocarps to browsing at the flowering stage may not be directly comparable to their tolerance at the rosette stage. If tolerance is critically dependent on the plant’s ability to reallocate accumulated reserves from root to shoot growth (e.g., Strauss and Agrawal 1999) and rosette defoliation does not adversely affect this ability, fertilized plants should have better tolerance for defoliation than unfertilized plants, as expected by the compensation continuum (Maschinski and Whitham 1989). In the present study, we performed a factorial commongarden experiment to quantify the costs of simulated herbivory at the rosette stage in unfertilized and fertilized plants. Following the above reasoning, we expected that the size and architecture of flowering plants in the second year can, at least partially, be determined by their growth in the first year at the rosette stage. We were especially interested to determine whether fertilization actually increases tolerance for rosette defoliation, as suggested by the compensation continuum.

Materials and methods Study species Tall wormseed mustard, Erysimum strictum P. Gaertn., B. Mey., and Scherb. (Brassicaceae) (syn. E. hieraciifolium auct., E. virgatum Roth), is a 50–100 cm tall, strictly biennial herb. Seeds germinate in spring and develop into a rosette during the first summer, and the plant flowers in the second summer. In the field, the second-year shoot usually has one unbranched stalk, but plants sometimes branch vigorously. Rosette leaves wither as the flowering shoot develops. At the time of flowering, from mid-June until the end of July, the stalk itself bears several lanceolate leaves that wither at the time of seed set. After reproduction, the plant dies. In Finland, E. strictum grows on sandy and gravelly seasides and riversides and, increasingly, in humaninfluenced habitats, such as dry meadows and railroad embankments (Ahti 1965). Experimental design The experiment was carried out in the Botanical Gardens of the University of Oulu (Oulu, Finland, 65°00′N, 25°30′E) during 2001 and 2002. In mid-June 2001, we transplanted © 2005 NRC Canada

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newly emerged rosette stage plants of similar size from a gravel roadside near Oulu into a common garden. The rosettes were planted 30 cm apart into sandy, nutrient-poor soils in a common garden and assigned to four treatment groups running systematically along the benches. Treatments were allotted to the first four plants, and this order was repeated in other groups of four plants. In statistical analysis, these groups of four plants were considered as blocking factor. The treatments were (1) control, (2) fertilization, (3) defoliation, and (4) defoliation plus fertilization. After transplantation some individuals died, and they were removed from the benches. Consequently, the numbers of plants in the different treatments were 14 control, 19 fertilized, 14 defoliated, and 18 defoliated and fertilized. In the defoliation treatment, half (50%) of the rosette leaves were removed with scissors immediately after planting (on 19 June 2001). The leaves were carefully cut without physically damaging axillary meristems at the base of the leaves. Because the plants compensated so well for the first defoliation (Fig. 1), we performed a second defoliation on 2 August 2001. Fertilizer was applied three times, immediately after the defoliations and once in mid-July 2001. Each of the three doses of liquid fertilizer (20 mL) contained ca. 19 mg of nitrogen (69% NH4 and 31% NO3), 9 mg of phosphorus, 32 mg of potassium, and smaller amounts of S, Mg, B, Cu, Fe, Mn, Mo, and Zn. Fertilizer was applied directly to the base of each plant using a plastic funnel. The treatment effects on the developing rosettes were monitored during the first season (2001). The size of the rosettes (maximum distance of the tips of two opposite leaves) was measured, and the number of rosette leaves was counted before each defoliation. During the summer (2002) after overwintering in the common garden, the plants were allowed to develop freely. The benches were weeded only when needed. Mature shoots were collected and dried at room temperature for measurements during the fall 2002. The measured parameters were height, shoot and root weights, number of branches emerging from the rosette base (basal branches in Weinig et al. 2003; stalks in Gómez 2003), lateral branches initiating from axillary meristems situated along the middle and top parts of the stalk (upper branches; inflorescence branches in Weinig et al. 2003), number of fruits (siliques), seeds per fruit, total number of seeds per plant, and seed weight. The number of seeds per fruit was estimated by counting the seed scars of three siliques taken from the bottom, middle, and top parts, respectively, of each plant (Huhta et al. 2000a, 2000b). Total seed number per plant was calculated by multiplying the silique number by the average number of seed scars per silique. Data analysis The data were analyzed by means of factorial ANOVA with defoliation (Def), fertilization (Fert), and the defoliation × fertilization interaction (Def × Fert) as fixed factors. Further, we included the groups of four plants as a blocking (random) factor in the analysis. In terms of a linear model with a constant, this resulted in the following equation: Y = µ + Block + Def + Fert + (Def × Fert) + ε

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We did not consider the interaction terms of treatments (defoliation and fertilization) with the block effect essential, and hence the variation explained by these terms is included in the residual variation (ε). Variables were log-tranformed if the assumptions of normality or homogeneity of variances were not met. If the ANOVA result of the log-transformed data did not differ from that of the original data, we reported the ANOVA result for the original data. The relationships of plant height, total plant biomass, total branch number, and seed number per plant in the second year with rosette diameter in the first year were analyzed with linear regressions. Regression coefficients (b) were compared between the treatments by first testing the homogeneity (i.e., equality) of the regression coefficients (Zar 1996, p. 362). If the coefficients were unequal, we further compared coefficients pairwise with t tests, that is, by calculating the difference (b1 – b2) between the coefficients divided by the standard error (sb1 – b2) of the difference between the coefficients t = (b1 – b2) / (sb1 – b2).

Results At the rosette stage, the plants were defoliated twice and fertilized three times. Before the application of the final treatments in August, the fertilized rosettes had grown larger in diameter (Fig. 1a) and had a greater number of leaves (Fig. 1b). The defoliated plants compensated well for the 50% loss of rosette leaves irrespective of the fertilization treatments (Fig. 1b). After these responses, the treatments were applied for the last time, and the consequent responses were tested at the end of the next growing season, when the plants had flowered and their seeds fully matured. First, we tested for the dependence of plant performance on rosette size. In most cases, the performance of mature, flowering plants showed a statistically significant positive dependence on rosette diameter (b > 0, Table 1). For plant height, the dependence was rather similar in all treatment groups (Fig. 2a), except that the slope of fertilized plants was not significantly different from zero (Table 1). With respect to total biomass, the plants that had been defoliated at the rosette stage showed a less steep increase with increasing rosette size compared with the undefoliated plants (Fig. 2b), though this difference was significant only if the plants were both defoliated and fertilized (Table 1). For total branch number (Fig. 2c), fruit number (data not shown), and seed number per plant (Fig. 2d), the slope of the regression line rose among the treatment groups in the following order: defoliation + fertilization < defoliation < control < fertilization (for statistical differences, see Table 1). Consequently, in response to fertilization, small plants invested more in height growth and less in branches, whereas large plants invested more in branch growth (cf. control and fertilization treatments in Figs. 2a and 2c). Rosette defoliation reduced plant height equally in small and large plants (Fig. 2a), while it reduced total biomass (Fig. 2b), branch number (Fig. 2c), and reproductive output (Fig. 2d) more strongly in large plants. Fertilization did not mitigate, but rather intensified, these effects among large defoliated plants (Figs. 2b–2d). Second, we tested for the effects of the treatments on the average performance of mature, flowering plants. Rosette defoliation significantly affected all measured parameters, © 2005 NRC Canada

408 Fig. 1. Rosette size (mean ± SE) of Erysimum strictum in August 2001 before the second defoliation treatment. (a) Rosette diameter. (b) Number of rosette leaves. ANOVA results (F values) are given for statistically significant terms (df = 1 for treatments, df = 41 for error) **, statistically significant at p < 0.01; ***, statistically significant at p < 0.001. Numbers in parentheses after the ANOVA results denote the statistical power of the test of fertilization (Fig. 1a) and the test of defoliation and fertilization (Fig. 1b), respectively.

except seed number per fruit and seed weight, whereas rosette fertilization only affected branch number (Table 2). Defoliation reduced plant height (data not shown) as well as shoot and root biomass (Figs. 3a and 3b). The decline in shoot and root biomass in response to rosette defoliation was steeper among fertilized (shoot: –48.4%; root: –42.3%) than among unfertilized plants (–29.9% and –25.2%, respectively). Fertilization increased and defoliation decreased the number of basal branches (Fig. 3c, Table 2). The decline in response to rosette defoliation was more or less parallel among fertilized (–26.0%) and unfertilized plants (–21.0%). In the case of upper (inflorescence) branches, the interaction between defoliation and fertilization was statistically significant (Table 2); the decline in response to defoliation was twice as steep among fertilized (–74.9%) as among unfertilized plants (–37.1%; Fig. 3d). The trend was similar but weaker in the total number of branches (Fig. 3e), number of fruits (data not shown), seed number per plant (Fig. 3f ) and seed biomass per plant (data not shown). Among fertilized plants, rosette defoliation reduced seed production by 48.4%, whereas the corresponding reduction among unfertilized plants was 29.3%.

Discussion Herbivory at the rosette stage can substantially reduce the reproductive output of strictly biennial plants. In the first

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year, E. strictum compensated well for the first 50% loss of rosette leaves. In spite of this, after two rounds of rosette defoliation, the reproductive output of mature plants was reduced by 29%–48% in the next growing season. As a comparison, Huhta et al. (2000a) observed no significant reduction in fruit production of E. strictum following 50% and 75% removal of the main stalk before flowering. These treatments, however, delayed fruit maturation with the consequence that the production of mature fruits was reduced by 70%–90%. No reduction in either fruit or seed production was found by Huhta et al. (2000b) following 50% clipping of the main stalk. In these earlier studies, simulated browsing on flowering plants did not strongly reduce fruit production because clipping stimulated lateral branching and because both fruit and seed production positively correlated with branch number (Huhta et al. 2000a, 2000b). On the other hand, in the present study, rosette defoliation reduced reproductive output mainly because it reduced branch production and, consequently, fruit production. Rosette defoliation did not directly affect either seed number per capsule or seed weight. Moreover, according to regression analysis, the effects of rosette defoliation should be most pronounced among large plants which otherwise, in the absence of defoliation, would produce a greater number of branches and seeds than smaller plants. Our results suggest that the effects of herbivory on reproductive output of E. strictum are mediated mainly by decreased (in response to rosette defoliation) or increased (in response to browsing on mature plants) lateral branching. Rosette defoliation does not seem to interfere strongly with apical dominance because defoliation reduces lateral branching as well as vertical growth. In flowering plants, however, browsing removes apical dominance and, hence, stimulates lateral branching. Apical damage (10% removal of the stem) stimulates lateral branching in the top parts but not at the base of the stem (Huhta et al. 2000a). This response to apical damage is most marked in conditions where the plants otherwise, in the absence of damage, would have produced few lateral branches (Huhta et al. 2000b). Our study populations frequently have unbranched shoots (Huhta et al. 2000b). It is, however, unclear why this is so, because fruit and seed production invariably correlate positively with the number of branches (Huhta et al. 2000a, 2000b). In a related, perennial monocarpic species, Erysimum mediohispanicum, there was directional selection for increased plant height and increased flower number in the absence of herbivory because higher and larger plants with many flowers attracted more pollinators (Gómez 2003). In the presence of browsers, however, smaller plants with fewer flowers were favoured because the herbivores selectively preferred larger plants. These observations are in accordance with Vail (1992), who suggested that selective herbivory on larger and more branched plants would favour the evolution of an unbranched shoot architecture, as well as with Aarssen (1995), who suggested that pollinator availability is a potential selective force favouring apical dominance and fast vertical growth among herbaceous plants (see also Lortie and Aarssen 1998). We did not observe any heavy mammalian herbivory on E. strictum in our study populations, which are located in secondary, human-influenced habitats. In primary habitats, however, the species may be subject to mammalian © 2005 NRC Canada

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Table 1. Results of regression analysis that explain variation in plant height, total biomass, total number of branches, and number of seeds per plant as a function of rosette diameter measured in the previous season. Control

Defoliation

Fertilization

Def + Fert

Diameter vs.

R2

b

R2

b

R2

b

R2

b

Height Biomass No. of branches No. of seeds

0.43* 0.79* 0.58* 0.65*

3.02*a 3.5*a 1.79*a 1102*ab

0.55* 0.65* 0.57* 0.57*

2.85*a 2.2*ab 1.38*ab 688*bc

0.15 0.62* 0.6* 0.67*

1.41a 3.46*a 2.7*a 1682*a

0.34* 0.51* 0.33* 0.36*

2.17*a 1.68*b 0.68*b 434*c

Note: R2, coefficient of determination; b, regression coefficient; *, statistically significant (p < 0.05). Regression coefficients followed by the same letter do not differ significantly (p > 0.05) from each other in pairwise comparisons between treatments.

Fig. 2. Performance of flowering plants of Erysimum strictum in 2002 in relation to rosette diameter in August 2001. (a) Plant height. (b) Total plant biomass (dry mass). (c) Total number of basal and upper branches. (d) Number of seeds per plant. Control, neither defoliation nor fertilization; Def, defoliated; Fert, fertilized; Def + Fert, defoliated and fertilized. For regression coefficients and statistics, see Table 1.

herbivory by hare, reindeer, or moose. On the other hand, it may well be that, at the beginning of the second growing season, the flowering plants are selected for fast vertical growth to better attract pollinators or to succeed in the competition for light (for discussion, see Aarssen 1995; Huhta et al. 2000a; Gómez 2003). In addition, Huhta et al. (2000b) suggested that the branch number of E. strictum may be a plastic trait, which is adjusted in response to resource availability (see also Bonser and Aarssen 1996, 2003; Strauss and

Agrawal 1999; Hawkes and Sullivan 2001). According to the present results, branch number is not only adjusted in relation to resource availability at the flowering stage (Huhta et al. 2000b) but also in relation to the growth conditions of the plants at the rosette stage. In E. strictum, rosette fertilization increased the number of branches, but did not significantly affect plant height. Accordingly, fertilization and competition at the flowering stage influenced branch number but did not significantly in© 2005 NRC Canada

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Table 2. Results of the ANOVA for treatment effects on the performance of flowering plants. Block

Def

Parameter

F

p

P

Height Shoot biomass Root biomass Total biomass No. of basal branches No. of upper branches Total no. of branchesa No. of fruits per plant No. of seeds per fruit No. of seeds per plant Seed weight Total seed biomass

1.98 3.03 3.45 3.09 2.92 2.75 3.31 2.62 1.58 2.94 1.61 2.94

0.032 0.001