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Leaf growth response to simulated herbivory: a comparison among seagrass species
Journal of Experimental Marine Biology and Ecology, 220 (1998) 67–81
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Leaf growth response to simulated herbivory: a comparison among seagrass species ´ a , *, Carlos M. Duarte b , Nona S. R. Agawin b , Martın ´ Merino c Just Cebrian a Boston University Marine Program ( Marine Biological Laboratory), Woods Hole, 02543 MA, USA ` Centro de Estudios Avanzados de Blanes, CSIC, Camı´ de Santa Barbara s /n, 17300, Blanes ( Girona), Spain c ´ , Universidad Nacional Autonoma ´ ´ ´ Instituto de Ciencias del Mar y Limnologıa de Mexico , 04510 Mexico ´ DF, Mexico b
Received 2 August 1996; received in revised form 10 March 1997; accepted 19 March 1997
Abstract We examined in seven seagrass species the response of the leaf growth rate per shoot (mg DW shoot 21 day 21 ) to a gradient of herbivory simulated by leaf clipping. The clipping procedure was intended to mimic the removal by herbivores which only consume the leaves of a single shoot at every feeding attack and which do not feed over the same shoots selectively (i.e., most poikilotherm vertebrate and invertebrate herbivores). We tested whether (1) this defoliation procedure does not normally depress shoot leaf growth rates (i.e., the occurrence of compensatory leaf growth), and (2) whether leaf nutrient content, relative leaf growth rate, average distance between consecutive short shoots and rhizome diameter influence the response of the leaf growth rate per shoot to a gradient of defoliation. The leaf growth rate per shoot varied among clipping treatments in nine of the 15 populations treated (ANOVA, p , 0.05) and meta-analyses techniques revealed a significant overall variation ( x 2 test, p , 0.001) when all the populations were considered in concert. The leaf growth rate per shoot was persistently depressed in all the clipping treatments only in one of the 15 populations treated, with only three more populations showing depressed leaf growth under some treatments (Tukey HSD test, p , 0.05). The response of the leaf growth rate to clipping intensity, which was analysed on a per shoot basis (i.e. relationship between the leaf growth rate per shoot and clipping intensity on the shoot) was significant only for four populations, although meta-analyses revealed a tendency towards a general significance. None of the seagrass properties considered was related to the response of leaf growth to clipping intensity. Our results stress the remarkable variability seagrass leaf growth may exhibit under single events of defoliation on scattered shoots. Furthermore, because leaf growth rates are rarely depressed, these results suggest that most poikilotherm vertebrate and invertebrate herbivores, which typically remove , 30% of leaf production, have a modest impact on the depression of leaf growth rates through removal of photosynthetic tissue. 1998 Elsevier Science B.V. *Correspondence author. Tel.: 11 508 2897518; fax: 11 508 5487295; e-mail: [email protected]. 0022-0981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0022-0981( 97 )00084-1
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Keywords: Seagrass; Clipping; Leaf growth compensation; Meta-analyses
1. Introduction Herbivores typically remove a small percentage of seagrass production (10–15%, Klumpp et al., 1989; Hemminga et al., 1991). However, events of intense herbivory, where herbivores remove more than 50% of seagrass production, are not uncommon (Camp et al., 1973; Greenway, 1976; Keller, 1983; Valentine and Heck, 1991; Klumpp et ´ et al., 1996a). Hence, herbivores have the potential to affect seagrass al., 1993; Cebrian growth through removal of photosynthetic tissue. For instance, seagrass growth may increase in response to moderate herbivory on epiphytized, senescent leaf tips (Thayer et al., 1984), but is generally reduced under intense leaf removal (Ogden et al., 1980; Zieman et al., 1984; Valentine and Heck, 1991). The analyses of the variation in seagrass growth with defoliation intensity is an important topic for seagrass ecology, since it contributes to ascertain the importance of herbivore control on seagrass production. Yet, experimental examination of the effect of contrasting defoliation intensity on seagrass growth is scarce and mostly restricted to the species Thalassia testudinum (Greenway, 1974; Dawes and Lawrence, 1979; Tomasko and Dawes, 1989). Clipped plots of T. testudinum rapidly achieve leaf standing crops similar to unclipped plots (Taylor et al., 1973; Greenway, 1974), although intense and repeated clipping leads to reduced leaf standing crops (Phillips, 1960; Buesa, 1974; Greenway, 1974). These contrasting results were explained by postulating the mobilisation of carbohydrate reserves stored in the rhizome to leaf growth following experimental defoliation, as supported by the depletion of these reserves after repeated leaf clipping (Dawes and Lawrence, 1979). Further evidence is provided by the fact that regrowth of Posidonia oceanica leaves after defoliation is very low in spring (Wittmann and Ott, 1982), when the level of carbohydrate reserves in the rhizome is at its yearly minimum value (Pirc, 1985), while clipped leaves grow at the same rates as unclipped ones in autumn (Wittmann and Ott, 1982), when the rhizome carbohydrates are at their maximum concentration throughout the year (Pirc, 1985). The physiological integration between short shoots (i.e., translocation of reserves and nutrients between short shoots) may also influence the response of seagrass leaf growth to experimental defoliation. For instance, short shoots of T. testudinum may translocate carbohydrates to shaded neighbouring shoots, allowing the maintenance of similar leaf growth rates (Tomasko and Dawes, 1989). Because the role of rhizomes as storage organs seems common for many seagrass ´ species (Abel and Drew, 1989; Pirc, 1989; Dawes and Guiry, 1992; Perez-Llorens and Niell, 1993), and the physiological integration among neighbouring short shoots may also occur in many of them (Harrison, 1978; Libes and Bouderesque, 1987; Tomasko and Dawes, 1989), it can be hypothesised that, except under intense defoliation, seagrasses generally show compensatory leaf growth under removal of photosynthetic tissue (i.e., leaf growth rates are not depressed under defoliation), contrary to what could be expected from the positive association between leaf growth rates and photosynthetic biomass (Patriquin, 1973; Duarte, 1989). Nevertheless, the storage capacity of the rhizome and the degree of physiological
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integration among short shoots may vary greatly among seagrass species (Pirc, 1985; Tomasko and Dawes, 1989; Marba` et al., 1996), so that different species may vary in their response of leaf growth to defoliation. For instance, the storage capacity of clonal plants is higher for large plants with thick and long-lived rhizomes than for small ones with thin and short-lived rhizomes (Ashmun et al., 1982), and clonal integration depends on the distance that resources must travel along the rhizome (Pitelka and Ashmun, 1985). Hence the thickness of the rhizome and the distance among neighbouring shoots may influence seagrass leaf growth response to defoliation. In addition, the nutrient content and the relative growth rate of seagrass leaves, as estimators of the nutrient demand by seagrass leaves, may also affect the growth response to defoliation, as demonstrated for terrestrial grasses (Wallace et al., 1982; Georgiadis et al., 1989). Yet no attempt to investigate the influence of these factors on the response of seagrass leaf growth to defoliation has been made so far. Here we examine in seven seagrass species the response of the leaf growth rate per shoot to a gradient of herbivory simulated by leaf clipping, which approaches the removal pattern of most poikilotherm vertebrate and invertebrate herbivores (Thayer et al., 1984; Klumpp et al., 1989). Furthermore we test whether (1) leaf growth rates are not normally depressed by the defoliation procedure employed (i.e., seagrass compensatory leaf growth under defoliation), and (2) the leaf nutrient content, the relative leaf growth rate, the average distance between consecutive short shoots and the rhizome diameter influence the response of leaf growth to defoliation.
2. Methods
2.1. Study area and species tested We conducted parallel experiments in stands of seven seagrass species growing in temperate and tropical areas (Fig. 1). The experiments were carried out during spring and summer 1993 (Table 1). Four seagrass species were tropical, three of them (Enhalus acoroides, Thalassia hemprichii and Cymodocea rotundata) growing in the Northern Philippines and one (Thalassia testudinum) in the Mexican Caribbean (Fig. 1). Three populations of each of the species in the Northern Philippines and two of T. testudinum were tested (Table 1). The other three species tested (Posidonia oceanica, Cymodocea nodosa and Zostera noltii) grew in temperate coastal locations (Fig. 1). Two populations of P. oceanica and one of C. nodosa and Z. noltii were treated (Table 1). These species encompass a great range of leaf growth rate (from 0.5 mg DW shoot 21 day 21 for C. nodosa to 80 mg DW shoot 21 day 21 for E. acoroides) and support contrasting herbivore pressure (from 2% of leaf production removed for P. oceanica to 40% for C. nodosa; ´ et al., 1996a,b). Therefore, these species provide an appropriate framework to Cebrian test the effects of leaf removal on seagrass leaf growth.
2.2. Experimental design The experiments were designed to examine the response of the leaf growth per shoot
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Fig. 1. Location of the seagrass species and populations tested. Po (Posidonia oceanica), Zn (Zostera noltii), Ea (Enhalus acoroides), Tt (Thalassia testudinum), Th (Thalassia hemprichii), Cr (Cymodocea rotundata) and Cn (Cymodocea nodosa).
Species
Population
Clipping length in the treaments applied (cm)
E. acoroides E. acoroides E. acoroides T. hemprichii T. hemprichii T. hemprichii C. rotundata C. rotundata C. rotundata P.oceanica P. oceanica C. nodosa Z. noltii T. testudinum T. testudinum
Silaqui B. Loob Lucero Silaqui B. Loob Lucero Silaqui B. Loob Lucero Jonquet Calp Jonquet Jonquet Arrecife Playa
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
210, 220, 230, 240, 250 28, 216, 224, 232, 240 220, 230, 240, 250 23, 26, 29, 212 23, 26, 29, 212 23, 26, 29, 212 24, 28, 212, 216 25, 210, 214, 218 25, 210, 215, 220 24, 28, 212, 216, 220 24, 28, 212, 216, 220 23, 26, 28 23, 26, 29, 212 22, 24, 26, 28, 210 22, 24, 26, 28, 210
Date and duration of the experiment
Relative leaf growth rate (6SE) (day 21 )
N (%DW)
P (% DW)
Rhizome diameter (6SE) (cm)
Distance between shoots (6SE) (cm)
29 / 5–16 / 6 1 / 7–28 / 7 12 / 8–16 / 9 28 / 5–15 / 6 30 / 6–14 / 7 28 / 7–12 / 8 28 / 5–15 / 6 30 / 6–14 / 7 28 / 7–12 / 8 15 / 5–13 / 8 15 / 3–15 / 5 26 / 4–21 / 5 26 / 4–21 / 5 6 / 5–16 / 5 5 / 5–15 / 5
0.02760.002 0.02860.002 0.02560.001 0.02660.003 0.04260.003 0.03960.003 0.02560.001 0.04060.001 0.02960.001 0.00860.001 0.00760.001 0.03060.002 0.03160.003 0.05760.008 0.02460.001
1.61 no data 1.86 1.83 no data 2.66 1.93 no data 2.45 1.27 2.15 2.81 2.19 2.18 1.78
0.34 no data 0.28 0.18 no data 0.23 0.2 no data 0.2 0.16 0.08 0.23 0.19 0.18 0.15
0.9560.03 0.9660.02 160.04 0.4160.01 0.3860.01 0.460.01 0.3460.01 0.3560.01 0.3460.01 1.0560.07 0.9560.05 0.2460.02 0.1960.02 0.660.02 0.5260.01
10.860.9 1261.2 960.5 4.660.4 6.660.5 6.160.5 3.660.3 2.960.2 3.460.2 1.760.3 4.160.3 2.860.1 2.160.1 6.960.2 7.160.3
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Table 1 Seagrass species and populations tested, the clipping length in each of the treatments applied and the date of start and retrieval of the experiment in each population, and the mean relative leaf growth rate (6SE), leaf nitrogen (N) and phosphorus (P) concentrations, the mean rhizome diameter (6SE) and the mean distance between consecutive shoots (6SE) of each of the populations tested
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to a gradient of clipping intensity, and intended to mimic the effect of herbivores which only consume the leaves of a single shoot at every feeding attack and which do not feed over the same shoots selectively (i.e., most poikilotherm vertebrate and invertebrate herbivores, Thayer et al., 1984; Klumpp et al., 1989). Hence, the unit of analysis was defined as the individual shoot. For each population, between four and six fixed clipping lengths were decided, each length corresponding to a treatment (Table 1). These treatments were intended to encompass a defoliation gradient from intact shoots (i.e., control shoots) to nearly total removal of all leaf surface, and then, for each population tested, they were scaled to obtain four to six regular clipping intervals up to the mean maximum leaf length of the population (Table 1). Each treatment was applied to 10 randomly selected shoots in the seagrass stand. For every treated shoot, the portions of all the leaves overlaid by the treatment length measured from the tip of the longest leaf were cut with a pair of scissors. Every treated and control shoot was marked at its base with a coloured plastic tag, each colour corresponding to a different treatment. Clipping intensity was expressed as the percentage of leaf surface removed per shoot. This percentage was calculated as the quotient between the sum of the leaf surface covered by all the clipped portions and the initial (i.e., prior to clipping) leaf surface of the shoot. These values were derived from measurement of the lengths of all the leaves, and mean leaf width, on the treated shoot prior to clipping. When rough sea conditions hampered in situ measurements of leaf length, the initial leaf surface of a clipped shoot (S0 ) was estimated from the equation: S0 5 Sf 2 P 1 Lavg
(1)
where Sf is the leaf surface of the shoot at the end of the experimental period, P is the leaf growth of the shoot during the experimental period (see below), and Lavg is the average leaf surface removed by the clipping treatment applied to that shoot. The estimate of Lavg was derived from measurements of the average leaf surface per shoot in 30 unclipped shoots sampled at the time of clipping. Measurement of the clipping length in rough sea conditions was not very precise, but the error committed was much lower than the length difference between treatments, being thus negligible. Losses of leaf surface by herbivory during the experimental period were too small compared with clipping losses to influence these estimates, since the sharp cut leaf edge left by the scissors was still recognisable on most of the clipped leaves after the experimental period. Leaf growth per shoot was measured by the leaf marking technique (Zieman, 1968). At the time of clipping all the leaves in every manipulated and control shoot were punched twice with a hypodermic needle at the sheath-blade juncture. The time interval for retrieval of the marked shoots varied among the species depending on their relative leaf growth rate (Table 1). The leaf growth rate per shoot (cm shoot 21 day 21 ) was calculated as the sum of the distances between the holes and the sheath-blade juncture for all the marked leaves plus the full length of all the unmarked (i.e., born during the experimental interval) leaves on the shoot, divided by the number of days elapsed since marking. These measurements were transformed to dry weight (mg DW shoot 21 day 21 )
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using the average leaf width and leaf specific weight (mg DW cm 22 ) calculated from a subsample of leaves dried overnight at 808C. In each population, the average distance between consecutive shoots, the average rhizome diameter and the average leaf nitrogen (N) and phosphorus (P) concentrations were measured from additional samples. The distance between consecutive shoots and the rhizome diameter were measured with a ruler from 10 to 50 connected shoots and with a calliper from four to 10 pieces of horizontal rhizome, respectively. In the species lacking true short shoots (E. acoroides and Z. noltii), the distance between every two closest leaf clusters along the rhizome was measured. Nitrogen concentrations (% DW) were measured in duplicate subsamples of dried leaves using a Carlo-Erba CHNanalyzer, whereas phosphorus concentrations were derived using a colorimetric method, following wet acid digestion (Koroleff, 1983). The mean relative leaf growth rate (day 21 ) in every population was calculated as the mean ratio of the leaf growth rate (mg DW shoot 21 day 21 ) to leaf biomass (mg DW shoot 21 ) of the control shoots.
2.3. Data analysis In every population, the variability in the leaf growth rate per shoot among treatments was analysed with one-way ANOVA, and the depression of leaf growth with the treatment applied was tested by comparing the mean leaf growth rate per shoot in every treatment with that of the control shoots (no defoliation) by means of a Tukey HSD multiple comparison test (Sokal and Rolhf, 1995). Both tests were applied at a 5% significance level. In every population, the response of leaf growth to increasing defoliation was described by fitting, using least square regression analyses, a secondorder equation to the relationship between the leaf growth rate per shoot (LGR) and the percentage of leaf surface clipped per shoot (PLC) for all the shoots treated, which had the form: LGR (mg DW shoot 21 day 21 ) 5 a 1 b 1 PLC 1 b 2 (PLC)2
(2)
where a is a constant indicative of the LGR in the absence of clipping, and b 1 and b 2 correspond to the linear and quadratic coefficients of the equation, respectively. The second-order equation was decided after observation of the distribution of LGR against PLC for each population, which seemed best adjustable to a parabolic pattern. In every population, the significance of the linear and quadratic coefficients, and of the whole regression model, were analysed by means of a t-test (H0 , coefficient50) and F-test, respectively (Sokal and Rolhf, 1995). The relationships between the leaf growth response to clipping and the seagrass properties measured (i.e., relative leaf growth rate, leaf nutrient content, distance among shoots and rhizome diameter) were examined using Pearson correlation coefficients (r) between their mean values of each population and the coefficients of the equations describing the leaf growth response to clipping (b 1 and b 2 ). We used meta-analyses to test the overall significance (i.e., when all the populations are considered in concert) of the results obtained. This technique allows the combination of the probability values obtained by a test in different populations to analyse the general significance of the test when all the populations are joined (Sokal and Rolhf, 1995).
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Namely, the statistic (22o(ln pi )), which follows a x 2( 2k ) , where k is the number of populations tested, is calculated from each probability value ( pi ) obtained by the test in each respective i-th population (Fisher, 1954). Hence, we run meta-analyses to test whether the leaf growth rate per shoot showed an overall significant variation with the defoliation treatment and to test the overall significance of the parabolic response of leaf growth to defoliation.
3. Results The relative leaf growth rate of the seagrass populations treated ranged nearly an order of magnitude from the slow-growing species P. oceanica to the relatively fast-growing population of T. testudinum in Arrecife (Mexican Caribbean, Table 1). The relative leaf growth rate could also vary notably among the populations of a given species, such as among the populations of T. testudinum, T. hemprichii and C. rotundata, for which it ranged about two-fold (Table 1). The rhizome diameter and the distance between consecutive shoots also varied about and order of magnitude among the species sampled (Table 1), but they were very similar among the populations of a given species (Table 1) because these are species-specific properties (Duarte, 1991). Leaf N and P concentrations varied about two- and three-fold among species (Table 1), respectively, and suggested some populations to be limited by P (e.g., P. oceanica in Calp) or N (e.g., P. oceanica in Jonquet). Leaf N and P concentrations were similar among populations of a given species except for N and P contents for P. oceanica, and N content for T. hemprichii and C. rotundata (Table 1). The leaf growth per shoot varied among clipping treatments in nine of the 15 populations treated (ANOVA, p,0.05; Table 2), and meta-analyses revealed that this variation was significant for all the populations considered in concert ( x 2(30) 5130.6, p,0.001; Table 2). The pattern of variation in the leaf growth rate per shoot with the intensity of the clipping treatment differed notably among populations, with no apparent site or species-specificity (Fig. 2). This pattern ranged from a persistent depression of leaf growth with increasing defoliation (C. rotundata in B. Loob, Fig. 2) to an initial enhancement or depression followed by a subsequent depression or enhancement, respectively (C. rotundata in Silaqui, and T. hemprichii in Silaqui and C. rotundata in Lucero, respectively, Fig. 2) and other more complex shapes, such as enhancement at both low and high intensities (E. acoroides in Lucero and P. oceanica in Jonquet, Fig. 2) or several enhancements and depressions (both populations of T. testudinum and E. acoroides in Silaqui, Fig. 2). Despite this marked variability, few treatments involved depressed leaf growth rates relative to the control shoots (Tukey HSD test, p,0.05). Only C. rotundata in B. Loob showed depressed growth under all the treatments (Tukey HSD test, p,0.05, Fig. 2), with three other ones (E. acoroides and T. hemprichii in Silaqui, and C. rotundata in Lucero) exhibiting depression only under a few treatments (Tukey HSD test, p,0.05, Fig. 2). The adjustment of a second-order equation to the relationship between the leaf growth rate per shoot and clipping intensity on the shoot also yielded variable results. Only four populations showed a parabolic response of leaf growth to defoliation (b 1 and b 2
Species
Population
ANOVA p
a ( p, t-test)
b 1 ( p, t-test)
b 2 ( p, t-test)
Regression ( p, F-test)
E. acoroides E. acoroides E. acoroides T. hemprichii T. hemprichii T. hemprichii C. rotundata C. rotundata C. rotundata P. oceanica P. oceanica C. nodosa Z. noltii T. testudinum T. testudinum Overall response a
Silaqui B. Loob Lucero Silaqui B. Loob Lucero Silaqui B. Loob Lucero Calp Jonquet Jonquet Jonquet Arrecife Playa
0.003 0.471 ,0.001 0.002 0.492 0.303 ,0.001 ,0.001 ,0.001 0.099 ,0.001 0.178 0.201 0.015 0.002 x 2( 30 ) 5130.6 p,0.001
22.78 (,0.001) 10.01 (,0.001) 11.61 (,0.001) 2.64 (,0.001) 3.14 (,0.001) 3.53 (,0.001) 5.55 (,0.001) 8.70 (,0.001) 5.53 (,0.001) 3.94 (,0.001) 4.02 (,0.001) 0.24 (,0.001) 0.57 (,0.001) 6.83 (,0.001) 1.71 (,0.001)
20.1040 (0.232) 0.0220 (0.640) 0.1196 (0.005) 20.0349 (0.002) 0.0159 (0.294) 20.0190 (0.111) 20.0069 (0.801) 20.0617 (0.025) 20.0546 (0.010) 20.0209 (0.116) 20.0354 (0.021) 20.0004 (0.857) 0.0058 (0.114) 0.0208 (0.578) 0.0086 (0.287) x (230 ) 571.6 p,0.001
0.00011 (0.908) 20.00048 (0.335) 20.00157 (0.001) 0.00037 (0.001) 20.00020 (0.235) 0.00013 (0.284) 20.00016 (0.595) 0.00018 (0.505) 0.00045 (0.033) 0.00011 (0.418) 0.00036 (0.015) 0.00001 (0.838) 20.00007 (0.042) 20.00058 (0.222) 20.00009 (0.341) x (230 ) 565.6 p,0.001
0.007 0.243 0.005 0.005 0.468 0.087 0.042 ,0.001 0.015 0.019 0.049 0.976 0.040 0.155 0.545 x (230 ) 594.3 p,0.001
a
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Table 2 The probability ( p) of an ANOVA testing the variation in the leaf growth rate per shoot (mg DW shoot 21 day 21 ) among clipping treatments, the coefficients (and probability values, p, t-test) of the second-order equation fitted to the relationship betwen the leaf growth rate per shoot and clipping intensity on the shoot, and the probability value for the regression model ( p, F-test)
The x 2 values showing the overall (i.e., all the populations considered in concert) significance of the tests mentioned above are represented.
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Fig. 2. The variation in the average leaf growth rate per shoot with the mean percentage of leaf surface clipped in each treatment applied to each population. Open and filled circles correspond to non-different (Tukey HSD multiple comparison test, p.0.05) and different (Tukey HSD multiple comparison test, p,0.05) leaf growth rates per shoot from those of the control shoots, respectively. Vertical bars represent the standard error of the average leaf growth rate of the shoots subject to each corresponding treatment. The circles have been connected by spline smoothing.
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significant, t-test, p,0.05, Table 2). The population of Z. noltii in Jonquet also showed a polynomial response with only the quadratic term significant (t-test, p,0.05, Table 2). For C. rotundata in B. Loob, a negative linear response was observed, as expected from the shape of the variation in leaf growth along treatments of increasing intensity (Fig. 2; the shape of the variation in the leaf growth rate per shoot along clipping treatments of increasing intensity does not, however, generally provide any information about the order of the equation to be adjusted to the response of leaf growth to defoliation, because this equation is adjusted on a single shoot basis). Yet meta-analyses revealed an overall (i.e., all the populations considered in concert) significant parabolic response of the leaf growth rate per shoot to increasing defoliation intensity ( x 2( 30 ) , p,0.001 for both coefficients b 1 and b 2 and for the whole regression model, Table 2). Moreover, the coefficients b 1 and b 2 were negatively correlated (r5 20.86, p,0.05), which suggests that steeper enhancements or depressions of leaf growth at low clipping intensities are associated to more pronounced reversions towards lower or greater leaf growth values, respectively, at moderate or high clipping intensities. None of the seagrass properties measured (Table 1) was related to the leaf growth response to defoliation, except for a tendency for higher distances between consecutive shoots to be associated to lower values of b 2 (r5 20.53, p,0.05).
4. Discussion Our results show a substantial variability of the response of the leaf growth rate per shoot to single events of defoliation on scattered shoots. Differences in the relative leaf growth rate, distance between consecutive shoots, rhizome diameter and leaf nutrient content among the populations treated are not related to the variability in this response. We only found a tendency for increasing distances between consecutive shoots to be associated to decreasing values of b 2 , which hints that increasing distances could restrain seagrass leaf growth compensation under moderate / intense defoliation, because of the greater limitation of reserve translocation with increasing distance between consecutive shoots. Other factors more important in the determination of leaf growth response to defoliation may blur any potential relation between this response and our measured seagrass properties. For instance, carbohydrates concentrations in the rhizomes of P. oceanica, C. nodosa and Z. noltii (Pirc, 1989), and T. testudinum (Dawes and Lawrence, 1980) show a remarkable seasonal oscillation, reaching the maximum value at the experimental period, in contrast with the lesser seasonality displayed by tropical species with a smaller rhizome, such as Syringodium filiforme and Halodule wrightii (Dawes and Lawrence, 1980), which could also hold for T. hemprichii and C. rotundata. Hence, at the experimental period the former species could show a higher carbohydrates gradient between rhizome and leaves than the latter ones, having therefore a greater capacity to overcome defoliation losses by carbohydrates translocation from rhizomes to leaves. This suggestion is supported by the fact that three of the four populations which experience growth depression under some treatments correspond to the species T. hemprichii and C. rotundata (Fig. 2). Differences in self-shading (Bulthius, 1983), shoot ´ size (Vant Lent et al., 1991; Perez and Romero, 1994), epiphyte cover (Cambridge et al.,
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1986; Neverauskas, 1987) or shoot burial and erosion by sediment dynamics (Marba` and Duarte, 1994; Manzanera et al., 1996) among the shoots of a given population or among populations may also entail substantial variability in the leaf growth response to defoliation. Moreover, because of the random selection of the shoots clipped, which aims at mimicking the feeding behaviour of most invertebrate and vertebrate poikilotherm herbivores, some rhizomes could have some shoots clipped and that could restrict shoot–shoot and shoot–rhizome integration, thus affecting leaf growth response to defoliation. Meta-analyses techniques indicate that, in spite of the remarkable variability found in the leaf growth response to defoliation, an overall parabolic response turns out to be significant (Table 2). Although the nature of a parabolic response cannot be ascertained, a convex parabola is in accordance with the observation that moderate grazing may stimulate leaf growth by removal of old, epiphytized leaf tips which do not contribute to leaf growth, whereas grazing involves growth depression when it becomes intense (Thayer et al., 1984; Zieman et al., 1984). We provide an example how meta-analyses can be used to analyse the overall significance of a test when this test yields contrasting results in the populations tested. This approach is useful when the probabilities obtained for some populations are suggestively low, albeit not significant, and the general significance of the test is suspected (Arnqvist and Wooster, 1995). Indeed meta-analyses has become a popular technique to arrive at conclusions about the overall significance of processes obscured by contrasting results at the population level (Arnqvist and Wooster, 1995; Sokal and Rolhf, 1995). Another relevant feature of this report is that nonsignificant results on the effect of defoliation on seagrass growth at the population level are provided, because non-significant results tend to be under-represented in the literature and that can bias the conclusions obtained by reviews based on published results (Cooper and Hedges, 1993). We show that single events of defoliation on scattered shoots rarely depress leaf growth since only four populations exhibited reduced leaf growth rates under some or all of the clipping treatments applied (Fig. 2). This is in agreement with the expected compensatory leaf growth seagrass should display following defoliation, which should result from reserves translocation from the rhizome or neighbouring non-defoliated shoots to the defoliated shoots. Nevertheless, major defoliation, whereby leaves of single shoots of T. testudinum were cut down to 1.5 cm above the substratum (i.e., all leaf surface removed), has been shown to reduce leaf growth rates (Greenway, 1974). This is not in opposition with our results since we removed a mean maximum of 80–85% of the shoot leaf surface in nearly all the populations (Fig. 2). Thus, although our results indicate that major single defoliation on a shoot does not normally reduce its leaf growth rate, removal of all the photosynthetic tissue could have indeed depressed it. Moreover, had the experiment been run in other seasons (i.e., late winter–early spring), when the rhizome of temperate seagrass species contains low levels of reserves (Pirc, 1989; Dawes and Guiry, 1992), reduction of leaf growth by defoliation could have been more apparent, as has explicitly been demonstrated for P. oceanica (Wittmann and Ott, 1982). Moreover, the finding of rare depression of leaf growth under defoliation is only relevant to most poikilotherm vertebrates and invertebrates, whose feeding behaviour (i.e., consumption of only the leaves of a single shoot at every feeding attack and
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79
non-selective consumption over the same shoots, Thayer et al., 1984; Klumpp et al., 1989) approaches our experimental procedure of single clipping events on scattered shoots. Indeed, frequent and intense defoliation over the same plots of T. testudinum, such as that exerted by the green turtle (Chelonya mydas), is eventually conducive to depressed leaf growth rates (Zieman et al., 1984), which has also been demonstrated experimentally by intense and recurrent clipping (Phillips, 1960; Buesa, 1974). Moreover, some large herbivores create feeding tracks (e.g., dugongs and manatees, Heinsohn et al., 1977; Ogden et al., 1980; Zieman, 1982) and scars (waterfowl, Thayer et al., 1984), removing therefore the leaves of several neighbouring shoots at every feeding attack. This concurrent leaf loss for several neighbouring shoots could preclude our observed compensatory leaf growth expected from the integration between clipped shoots and non-clipped neighbouring ones. The herbivores mimicked by our experimental procedure can also denude extensive seagrass areas, for instance following outbursts of sea urchin populations (Camp et al., 1973), but these overgrazing events do not seem to be common (Valentine and Heck, 1991). On the grounds of these results, and considering that most poikilotherm vertebrate and invertebrate herbivores normally remove ,30% of seagrass leaf production (Thayer et al., 1984; Klumpp et al., 1993; ´ and Duarte, 1994), we conclude by suggesting that in general these herbivores Cebrian have a modest effect on the depression of leaf growth rates through removal of photosynthetic tissue.
Acknowledgements This study was funded by Project AMB93-0118 of the Spanish Commission of Science and Technology (CICYT) and by Project CI1*-CT91-0952 of the European Community. J.C. was supported by a fellowship from the CIRIT (Comissio´ Interministerial de Recerca i Tecnologia). We thank J. Romero for useful comments on the ` S. Enrıquez, ´ ´ ´ manuscript, N. Marba, P. Ramırez, J. Rengel and A. Rodrıguez for technical assistance during field work, and C. Sonyer and the Direccio´ General de Parcs for providing access to the Spanish sampling stations.
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Neverauskas, V.P., 1987. Monitoring seagrass beds around a sewage sludge outfall in South Australia. Mar. Poll. Bull. 18, 158–164. Ogden, J.C., Tighe, S., Miller, S., 1980. Grazing of seagrasses by large herbivores in the Caribbean. Am. Zool. 20, 949–950. Patriquin, D., 1973. Estimation of growth rate, production and age of the marine anngiosperm Thalassia ¨ testudinum Konig. Carib. J. Sci. 13, 111–123. ´ Perez, M., Romero, J., 1994. Growth dynamics, production and nutrient status of the seagrass Cymodocea nodosa in a Mediterranean semi-estuarine environment. Mar. Ecol. 15, 51–64. ´ Perez-Llorens, J.L., Niell, F.X., 1993. Seasonal dynamics of biomass and nutrient content in the intertidal seagrass Zostera noltii Hornem. from Palmones River estuary, Spain. Aquat. Bot. 46, 49–66. Phillips, R.C., 1960. Observations on the ecology and distribution of the Florida seagrasses. Fla. State Board Conserv. Mar. Lab. Prof. Pap. Ser. 2, 1–72. Pirc, H., 1985. Growth dynamics in Posidonia oceanica (L.) Delile. I: seasonal changes of soluble carbohydrates, starch, free amino acids, nitrogen and organic anions in different parts of the plant. Mar. Ecol. 6, 141–165. Pirc, H., 1989. Seasonal changes in soluble carbohydrates, starch and energy content in Mediterranean seagrassses. Mar. Ecol. 10, 97–105. Pitelka, L.F., Ashmun, J.W., 1985. Physiology and integration of ramets in clonal plants. In: Jeremy, B.C., Buss, L.W., Cook, R.E. (Eds.), Population Biology and Evolution of Clonal Organisms. Yale University Press, New Haven, pp. 399–435. Sokal, R.R., Rolhf, F.J., 1995. Biometry. Freeman and Company, New York, p. 887. Taylor, J.L., Saloman, C.H., Prest, Jr. K.W., 1973. Harvest and regrowth of turtle grass (Thalassia testudinum) in Tampa Bay, Florida. Fish. Bull. 17, 145–148. Thayer, G., Bjorndal, K., Ogden, J., Williams, S., Zieman, J., 1984. Role of larger herbivores in seagrass communities. Estuaries 7, 351–376. Tomasko, D.A., Dawes, C.J., 1989. Evidence for physiological integration between shaded and unshaded short shoots of Thalassia testudinum. Mar. Ecol. Prog. Ser. 54, 299–305. Valentine, J., Heck, Jr. K., 1991. The role of sea urchin grazing in regulating subtropical seagrass meadows: evidence from field manipulations in the northern Gulf of Mexico. J. Exp. Mar. Biol. Ecol. 154, 215–230. Vant Lent, F., Nienhuis, P.H., Verschuure, J.M., 1991. Production and biomass of the seagrasses Zostera noltii Hornem. and Cymodocea nodosa (Ucria) Aschers. at the Banc d’Arguin (Mauritania, NW Africa): a preliminary approach. Aquat. Bot. 41, 353–367. Wallace, L.L., McNaughton, S.J., Coughenour, M.B., 1982. The effects of clipping and fertilization on nitrogen nutrition and allocation by mycorrhizal and nonmycorrhizal Panicum coloratum L., a C 4 grass. Oecologia 54, 68–71. Wittmann, K.J., Ott, J.A., 1982. Effects of cropping on the mediterranean seagrass Posidonia oceanica (L.) Delile. Mar. Ecol. 3, 151–159. Zieman, J.C., 1968. A study of the growth and decomposition of the seagrass Thalassia testudinum. M.Sc. Thesis, University of Miami, Florida, p. 50. Zieman, J.C., 1982. The ecology of the seagrass of South Florida: a community profile. US Fish Widl. Serv. Biol. Serv. Program FWS / OBS-82 / 25, vol. 124, p. 26. Zieman, J.C., Iverson, R.L., Ogden, J.C., 1984. Herbivory effects on Thalassia testudinum leaf growth and nitrogen content. Mar. Ecol. Prog. Ser. 15, 151–158.
L
Leaf growth response to simulated herbivory: a comparison among seagrass species ´ a , *, Carlos M. Duarte b , Nona S. R. Agawin b , Martın ´ Merino c Just Cebrian a Boston University Marine Program ( Marine Biological Laboratory), Woods Hole, 02543 MA, USA ` Centro de Estudios Avanzados de Blanes, CSIC, Camı´ de Santa Barbara s /n, 17300, Blanes ( Girona), Spain c ´ , Universidad Nacional Autonoma ´ ´ ´ Instituto de Ciencias del Mar y Limnologıa de Mexico , 04510 Mexico ´ DF, Mexico b
Received 2 August 1996; received in revised form 10 March 1997; accepted 19 March 1997
Abstract We examined in seven seagrass species the response of the leaf growth rate per shoot (mg DW shoot 21 day 21 ) to a gradient of herbivory simulated by leaf clipping. The clipping procedure was intended to mimic the removal by herbivores which only consume the leaves of a single shoot at every feeding attack and which do not feed over the same shoots selectively (i.e., most poikilotherm vertebrate and invertebrate herbivores). We tested whether (1) this defoliation procedure does not normally depress shoot leaf growth rates (i.e., the occurrence of compensatory leaf growth), and (2) whether leaf nutrient content, relative leaf growth rate, average distance between consecutive short shoots and rhizome diameter influence the response of the leaf growth rate per shoot to a gradient of defoliation. The leaf growth rate per shoot varied among clipping treatments in nine of the 15 populations treated (ANOVA, p , 0.05) and meta-analyses techniques revealed a significant overall variation ( x 2 test, p , 0.001) when all the populations were considered in concert. The leaf growth rate per shoot was persistently depressed in all the clipping treatments only in one of the 15 populations treated, with only three more populations showing depressed leaf growth under some treatments (Tukey HSD test, p , 0.05). The response of the leaf growth rate to clipping intensity, which was analysed on a per shoot basis (i.e. relationship between the leaf growth rate per shoot and clipping intensity on the shoot) was significant only for four populations, although meta-analyses revealed a tendency towards a general significance. None of the seagrass properties considered was related to the response of leaf growth to clipping intensity. Our results stress the remarkable variability seagrass leaf growth may exhibit under single events of defoliation on scattered shoots. Furthermore, because leaf growth rates are rarely depressed, these results suggest that most poikilotherm vertebrate and invertebrate herbivores, which typically remove , 30% of leaf production, have a modest impact on the depression of leaf growth rates through removal of photosynthetic tissue. 1998 Elsevier Science B.V. *Correspondence author. Tel.: 11 508 2897518; fax: 11 508 5487295; e-mail: [email protected]. 0022-0981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0022-0981( 97 )00084-1
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Keywords: Seagrass; Clipping; Leaf growth compensation; Meta-analyses
1. Introduction Herbivores typically remove a small percentage of seagrass production (10–15%, Klumpp et al., 1989; Hemminga et al., 1991). However, events of intense herbivory, where herbivores remove more than 50% of seagrass production, are not uncommon (Camp et al., 1973; Greenway, 1976; Keller, 1983; Valentine and Heck, 1991; Klumpp et ´ et al., 1996a). Hence, herbivores have the potential to affect seagrass al., 1993; Cebrian growth through removal of photosynthetic tissue. For instance, seagrass growth may increase in response to moderate herbivory on epiphytized, senescent leaf tips (Thayer et al., 1984), but is generally reduced under intense leaf removal (Ogden et al., 1980; Zieman et al., 1984; Valentine and Heck, 1991). The analyses of the variation in seagrass growth with defoliation intensity is an important topic for seagrass ecology, since it contributes to ascertain the importance of herbivore control on seagrass production. Yet, experimental examination of the effect of contrasting defoliation intensity on seagrass growth is scarce and mostly restricted to the species Thalassia testudinum (Greenway, 1974; Dawes and Lawrence, 1979; Tomasko and Dawes, 1989). Clipped plots of T. testudinum rapidly achieve leaf standing crops similar to unclipped plots (Taylor et al., 1973; Greenway, 1974), although intense and repeated clipping leads to reduced leaf standing crops (Phillips, 1960; Buesa, 1974; Greenway, 1974). These contrasting results were explained by postulating the mobilisation of carbohydrate reserves stored in the rhizome to leaf growth following experimental defoliation, as supported by the depletion of these reserves after repeated leaf clipping (Dawes and Lawrence, 1979). Further evidence is provided by the fact that regrowth of Posidonia oceanica leaves after defoliation is very low in spring (Wittmann and Ott, 1982), when the level of carbohydrate reserves in the rhizome is at its yearly minimum value (Pirc, 1985), while clipped leaves grow at the same rates as unclipped ones in autumn (Wittmann and Ott, 1982), when the rhizome carbohydrates are at their maximum concentration throughout the year (Pirc, 1985). The physiological integration between short shoots (i.e., translocation of reserves and nutrients between short shoots) may also influence the response of seagrass leaf growth to experimental defoliation. For instance, short shoots of T. testudinum may translocate carbohydrates to shaded neighbouring shoots, allowing the maintenance of similar leaf growth rates (Tomasko and Dawes, 1989). Because the role of rhizomes as storage organs seems common for many seagrass ´ species (Abel and Drew, 1989; Pirc, 1989; Dawes and Guiry, 1992; Perez-Llorens and Niell, 1993), and the physiological integration among neighbouring short shoots may also occur in many of them (Harrison, 1978; Libes and Bouderesque, 1987; Tomasko and Dawes, 1989), it can be hypothesised that, except under intense defoliation, seagrasses generally show compensatory leaf growth under removal of photosynthetic tissue (i.e., leaf growth rates are not depressed under defoliation), contrary to what could be expected from the positive association between leaf growth rates and photosynthetic biomass (Patriquin, 1973; Duarte, 1989). Nevertheless, the storage capacity of the rhizome and the degree of physiological
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integration among short shoots may vary greatly among seagrass species (Pirc, 1985; Tomasko and Dawes, 1989; Marba` et al., 1996), so that different species may vary in their response of leaf growth to defoliation. For instance, the storage capacity of clonal plants is higher for large plants with thick and long-lived rhizomes than for small ones with thin and short-lived rhizomes (Ashmun et al., 1982), and clonal integration depends on the distance that resources must travel along the rhizome (Pitelka and Ashmun, 1985). Hence the thickness of the rhizome and the distance among neighbouring shoots may influence seagrass leaf growth response to defoliation. In addition, the nutrient content and the relative growth rate of seagrass leaves, as estimators of the nutrient demand by seagrass leaves, may also affect the growth response to defoliation, as demonstrated for terrestrial grasses (Wallace et al., 1982; Georgiadis et al., 1989). Yet no attempt to investigate the influence of these factors on the response of seagrass leaf growth to defoliation has been made so far. Here we examine in seven seagrass species the response of the leaf growth rate per shoot to a gradient of herbivory simulated by leaf clipping, which approaches the removal pattern of most poikilotherm vertebrate and invertebrate herbivores (Thayer et al., 1984; Klumpp et al., 1989). Furthermore we test whether (1) leaf growth rates are not normally depressed by the defoliation procedure employed (i.e., seagrass compensatory leaf growth under defoliation), and (2) the leaf nutrient content, the relative leaf growth rate, the average distance between consecutive short shoots and the rhizome diameter influence the response of leaf growth to defoliation.
2. Methods
2.1. Study area and species tested We conducted parallel experiments in stands of seven seagrass species growing in temperate and tropical areas (Fig. 1). The experiments were carried out during spring and summer 1993 (Table 1). Four seagrass species were tropical, three of them (Enhalus acoroides, Thalassia hemprichii and Cymodocea rotundata) growing in the Northern Philippines and one (Thalassia testudinum) in the Mexican Caribbean (Fig. 1). Three populations of each of the species in the Northern Philippines and two of T. testudinum were tested (Table 1). The other three species tested (Posidonia oceanica, Cymodocea nodosa and Zostera noltii) grew in temperate coastal locations (Fig. 1). Two populations of P. oceanica and one of C. nodosa and Z. noltii were treated (Table 1). These species encompass a great range of leaf growth rate (from 0.5 mg DW shoot 21 day 21 for C. nodosa to 80 mg DW shoot 21 day 21 for E. acoroides) and support contrasting herbivore pressure (from 2% of leaf production removed for P. oceanica to 40% for C. nodosa; ´ et al., 1996a,b). Therefore, these species provide an appropriate framework to Cebrian test the effects of leaf removal on seagrass leaf growth.
2.2. Experimental design The experiments were designed to examine the response of the leaf growth per shoot
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Fig. 1. Location of the seagrass species and populations tested. Po (Posidonia oceanica), Zn (Zostera noltii), Ea (Enhalus acoroides), Tt (Thalassia testudinum), Th (Thalassia hemprichii), Cr (Cymodocea rotundata) and Cn (Cymodocea nodosa).
Species
Population
Clipping length in the treaments applied (cm)
E. acoroides E. acoroides E. acoroides T. hemprichii T. hemprichii T. hemprichii C. rotundata C. rotundata C. rotundata P.oceanica P. oceanica C. nodosa Z. noltii T. testudinum T. testudinum
Silaqui B. Loob Lucero Silaqui B. Loob Lucero Silaqui B. Loob Lucero Jonquet Calp Jonquet Jonquet Arrecife Playa
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
210, 220, 230, 240, 250 28, 216, 224, 232, 240 220, 230, 240, 250 23, 26, 29, 212 23, 26, 29, 212 23, 26, 29, 212 24, 28, 212, 216 25, 210, 214, 218 25, 210, 215, 220 24, 28, 212, 216, 220 24, 28, 212, 216, 220 23, 26, 28 23, 26, 29, 212 22, 24, 26, 28, 210 22, 24, 26, 28, 210
Date and duration of the experiment
Relative leaf growth rate (6SE) (day 21 )
N (%DW)
P (% DW)
Rhizome diameter (6SE) (cm)
Distance between shoots (6SE) (cm)
29 / 5–16 / 6 1 / 7–28 / 7 12 / 8–16 / 9 28 / 5–15 / 6 30 / 6–14 / 7 28 / 7–12 / 8 28 / 5–15 / 6 30 / 6–14 / 7 28 / 7–12 / 8 15 / 5–13 / 8 15 / 3–15 / 5 26 / 4–21 / 5 26 / 4–21 / 5 6 / 5–16 / 5 5 / 5–15 / 5
0.02760.002 0.02860.002 0.02560.001 0.02660.003 0.04260.003 0.03960.003 0.02560.001 0.04060.001 0.02960.001 0.00860.001 0.00760.001 0.03060.002 0.03160.003 0.05760.008 0.02460.001
1.61 no data 1.86 1.83 no data 2.66 1.93 no data 2.45 1.27 2.15 2.81 2.19 2.18 1.78
0.34 no data 0.28 0.18 no data 0.23 0.2 no data 0.2 0.16 0.08 0.23 0.19 0.18 0.15
0.9560.03 0.9660.02 160.04 0.4160.01 0.3860.01 0.460.01 0.3460.01 0.3560.01 0.3460.01 1.0560.07 0.9560.05 0.2460.02 0.1960.02 0.660.02 0.5260.01
10.860.9 1261.2 960.5 4.660.4 6.660.5 6.160.5 3.660.3 2.960.2 3.460.2 1.760.3 4.160.3 2.860.1 2.160.1 6.960.2 7.160.3
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Table 1 Seagrass species and populations tested, the clipping length in each of the treatments applied and the date of start and retrieval of the experiment in each population, and the mean relative leaf growth rate (6SE), leaf nitrogen (N) and phosphorus (P) concentrations, the mean rhizome diameter (6SE) and the mean distance between consecutive shoots (6SE) of each of the populations tested
71
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to a gradient of clipping intensity, and intended to mimic the effect of herbivores which only consume the leaves of a single shoot at every feeding attack and which do not feed over the same shoots selectively (i.e., most poikilotherm vertebrate and invertebrate herbivores, Thayer et al., 1984; Klumpp et al., 1989). Hence, the unit of analysis was defined as the individual shoot. For each population, between four and six fixed clipping lengths were decided, each length corresponding to a treatment (Table 1). These treatments were intended to encompass a defoliation gradient from intact shoots (i.e., control shoots) to nearly total removal of all leaf surface, and then, for each population tested, they were scaled to obtain four to six regular clipping intervals up to the mean maximum leaf length of the population (Table 1). Each treatment was applied to 10 randomly selected shoots in the seagrass stand. For every treated shoot, the portions of all the leaves overlaid by the treatment length measured from the tip of the longest leaf were cut with a pair of scissors. Every treated and control shoot was marked at its base with a coloured plastic tag, each colour corresponding to a different treatment. Clipping intensity was expressed as the percentage of leaf surface removed per shoot. This percentage was calculated as the quotient between the sum of the leaf surface covered by all the clipped portions and the initial (i.e., prior to clipping) leaf surface of the shoot. These values were derived from measurement of the lengths of all the leaves, and mean leaf width, on the treated shoot prior to clipping. When rough sea conditions hampered in situ measurements of leaf length, the initial leaf surface of a clipped shoot (S0 ) was estimated from the equation: S0 5 Sf 2 P 1 Lavg
(1)
where Sf is the leaf surface of the shoot at the end of the experimental period, P is the leaf growth of the shoot during the experimental period (see below), and Lavg is the average leaf surface removed by the clipping treatment applied to that shoot. The estimate of Lavg was derived from measurements of the average leaf surface per shoot in 30 unclipped shoots sampled at the time of clipping. Measurement of the clipping length in rough sea conditions was not very precise, but the error committed was much lower than the length difference between treatments, being thus negligible. Losses of leaf surface by herbivory during the experimental period were too small compared with clipping losses to influence these estimates, since the sharp cut leaf edge left by the scissors was still recognisable on most of the clipped leaves after the experimental period. Leaf growth per shoot was measured by the leaf marking technique (Zieman, 1968). At the time of clipping all the leaves in every manipulated and control shoot were punched twice with a hypodermic needle at the sheath-blade juncture. The time interval for retrieval of the marked shoots varied among the species depending on their relative leaf growth rate (Table 1). The leaf growth rate per shoot (cm shoot 21 day 21 ) was calculated as the sum of the distances between the holes and the sheath-blade juncture for all the marked leaves plus the full length of all the unmarked (i.e., born during the experimental interval) leaves on the shoot, divided by the number of days elapsed since marking. These measurements were transformed to dry weight (mg DW shoot 21 day 21 )
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using the average leaf width and leaf specific weight (mg DW cm 22 ) calculated from a subsample of leaves dried overnight at 808C. In each population, the average distance between consecutive shoots, the average rhizome diameter and the average leaf nitrogen (N) and phosphorus (P) concentrations were measured from additional samples. The distance between consecutive shoots and the rhizome diameter were measured with a ruler from 10 to 50 connected shoots and with a calliper from four to 10 pieces of horizontal rhizome, respectively. In the species lacking true short shoots (E. acoroides and Z. noltii), the distance between every two closest leaf clusters along the rhizome was measured. Nitrogen concentrations (% DW) were measured in duplicate subsamples of dried leaves using a Carlo-Erba CHNanalyzer, whereas phosphorus concentrations were derived using a colorimetric method, following wet acid digestion (Koroleff, 1983). The mean relative leaf growth rate (day 21 ) in every population was calculated as the mean ratio of the leaf growth rate (mg DW shoot 21 day 21 ) to leaf biomass (mg DW shoot 21 ) of the control shoots.
2.3. Data analysis In every population, the variability in the leaf growth rate per shoot among treatments was analysed with one-way ANOVA, and the depression of leaf growth with the treatment applied was tested by comparing the mean leaf growth rate per shoot in every treatment with that of the control shoots (no defoliation) by means of a Tukey HSD multiple comparison test (Sokal and Rolhf, 1995). Both tests were applied at a 5% significance level. In every population, the response of leaf growth to increasing defoliation was described by fitting, using least square regression analyses, a secondorder equation to the relationship between the leaf growth rate per shoot (LGR) and the percentage of leaf surface clipped per shoot (PLC) for all the shoots treated, which had the form: LGR (mg DW shoot 21 day 21 ) 5 a 1 b 1 PLC 1 b 2 (PLC)2
(2)
where a is a constant indicative of the LGR in the absence of clipping, and b 1 and b 2 correspond to the linear and quadratic coefficients of the equation, respectively. The second-order equation was decided after observation of the distribution of LGR against PLC for each population, which seemed best adjustable to a parabolic pattern. In every population, the significance of the linear and quadratic coefficients, and of the whole regression model, were analysed by means of a t-test (H0 , coefficient50) and F-test, respectively (Sokal and Rolhf, 1995). The relationships between the leaf growth response to clipping and the seagrass properties measured (i.e., relative leaf growth rate, leaf nutrient content, distance among shoots and rhizome diameter) were examined using Pearson correlation coefficients (r) between their mean values of each population and the coefficients of the equations describing the leaf growth response to clipping (b 1 and b 2 ). We used meta-analyses to test the overall significance (i.e., when all the populations are considered in concert) of the results obtained. This technique allows the combination of the probability values obtained by a test in different populations to analyse the general significance of the test when all the populations are joined (Sokal and Rolhf, 1995).
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Namely, the statistic (22o(ln pi )), which follows a x 2( 2k ) , where k is the number of populations tested, is calculated from each probability value ( pi ) obtained by the test in each respective i-th population (Fisher, 1954). Hence, we run meta-analyses to test whether the leaf growth rate per shoot showed an overall significant variation with the defoliation treatment and to test the overall significance of the parabolic response of leaf growth to defoliation.
3. Results The relative leaf growth rate of the seagrass populations treated ranged nearly an order of magnitude from the slow-growing species P. oceanica to the relatively fast-growing population of T. testudinum in Arrecife (Mexican Caribbean, Table 1). The relative leaf growth rate could also vary notably among the populations of a given species, such as among the populations of T. testudinum, T. hemprichii and C. rotundata, for which it ranged about two-fold (Table 1). The rhizome diameter and the distance between consecutive shoots also varied about and order of magnitude among the species sampled (Table 1), but they were very similar among the populations of a given species (Table 1) because these are species-specific properties (Duarte, 1991). Leaf N and P concentrations varied about two- and three-fold among species (Table 1), respectively, and suggested some populations to be limited by P (e.g., P. oceanica in Calp) or N (e.g., P. oceanica in Jonquet). Leaf N and P concentrations were similar among populations of a given species except for N and P contents for P. oceanica, and N content for T. hemprichii and C. rotundata (Table 1). The leaf growth per shoot varied among clipping treatments in nine of the 15 populations treated (ANOVA, p,0.05; Table 2), and meta-analyses revealed that this variation was significant for all the populations considered in concert ( x 2(30) 5130.6, p,0.001; Table 2). The pattern of variation in the leaf growth rate per shoot with the intensity of the clipping treatment differed notably among populations, with no apparent site or species-specificity (Fig. 2). This pattern ranged from a persistent depression of leaf growth with increasing defoliation (C. rotundata in B. Loob, Fig. 2) to an initial enhancement or depression followed by a subsequent depression or enhancement, respectively (C. rotundata in Silaqui, and T. hemprichii in Silaqui and C. rotundata in Lucero, respectively, Fig. 2) and other more complex shapes, such as enhancement at both low and high intensities (E. acoroides in Lucero and P. oceanica in Jonquet, Fig. 2) or several enhancements and depressions (both populations of T. testudinum and E. acoroides in Silaqui, Fig. 2). Despite this marked variability, few treatments involved depressed leaf growth rates relative to the control shoots (Tukey HSD test, p,0.05). Only C. rotundata in B. Loob showed depressed growth under all the treatments (Tukey HSD test, p,0.05, Fig. 2), with three other ones (E. acoroides and T. hemprichii in Silaqui, and C. rotundata in Lucero) exhibiting depression only under a few treatments (Tukey HSD test, p,0.05, Fig. 2). The adjustment of a second-order equation to the relationship between the leaf growth rate per shoot and clipping intensity on the shoot also yielded variable results. Only four populations showed a parabolic response of leaf growth to defoliation (b 1 and b 2
Species
Population
ANOVA p
a ( p, t-test)
b 1 ( p, t-test)
b 2 ( p, t-test)
Regression ( p, F-test)
E. acoroides E. acoroides E. acoroides T. hemprichii T. hemprichii T. hemprichii C. rotundata C. rotundata C. rotundata P. oceanica P. oceanica C. nodosa Z. noltii T. testudinum T. testudinum Overall response a
Silaqui B. Loob Lucero Silaqui B. Loob Lucero Silaqui B. Loob Lucero Calp Jonquet Jonquet Jonquet Arrecife Playa
0.003 0.471 ,0.001 0.002 0.492 0.303 ,0.001 ,0.001 ,0.001 0.099 ,0.001 0.178 0.201 0.015 0.002 x 2( 30 ) 5130.6 p,0.001
22.78 (,0.001) 10.01 (,0.001) 11.61 (,0.001) 2.64 (,0.001) 3.14 (,0.001) 3.53 (,0.001) 5.55 (,0.001) 8.70 (,0.001) 5.53 (,0.001) 3.94 (,0.001) 4.02 (,0.001) 0.24 (,0.001) 0.57 (,0.001) 6.83 (,0.001) 1.71 (,0.001)
20.1040 (0.232) 0.0220 (0.640) 0.1196 (0.005) 20.0349 (0.002) 0.0159 (0.294) 20.0190 (0.111) 20.0069 (0.801) 20.0617 (0.025) 20.0546 (0.010) 20.0209 (0.116) 20.0354 (0.021) 20.0004 (0.857) 0.0058 (0.114) 0.0208 (0.578) 0.0086 (0.287) x (230 ) 571.6 p,0.001
0.00011 (0.908) 20.00048 (0.335) 20.00157 (0.001) 0.00037 (0.001) 20.00020 (0.235) 0.00013 (0.284) 20.00016 (0.595) 0.00018 (0.505) 0.00045 (0.033) 0.00011 (0.418) 0.00036 (0.015) 0.00001 (0.838) 20.00007 (0.042) 20.00058 (0.222) 20.00009 (0.341) x (230 ) 565.6 p,0.001
0.007 0.243 0.005 0.005 0.468 0.087 0.042 ,0.001 0.015 0.019 0.049 0.976 0.040 0.155 0.545 x (230 ) 594.3 p,0.001
a
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Table 2 The probability ( p) of an ANOVA testing the variation in the leaf growth rate per shoot (mg DW shoot 21 day 21 ) among clipping treatments, the coefficients (and probability values, p, t-test) of the second-order equation fitted to the relationship betwen the leaf growth rate per shoot and clipping intensity on the shoot, and the probability value for the regression model ( p, F-test)
The x 2 values showing the overall (i.e., all the populations considered in concert) significance of the tests mentioned above are represented.
75
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Fig. 2. The variation in the average leaf growth rate per shoot with the mean percentage of leaf surface clipped in each treatment applied to each population. Open and filled circles correspond to non-different (Tukey HSD multiple comparison test, p.0.05) and different (Tukey HSD multiple comparison test, p,0.05) leaf growth rates per shoot from those of the control shoots, respectively. Vertical bars represent the standard error of the average leaf growth rate of the shoots subject to each corresponding treatment. The circles have been connected by spline smoothing.
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significant, t-test, p,0.05, Table 2). The population of Z. noltii in Jonquet also showed a polynomial response with only the quadratic term significant (t-test, p,0.05, Table 2). For C. rotundata in B. Loob, a negative linear response was observed, as expected from the shape of the variation in leaf growth along treatments of increasing intensity (Fig. 2; the shape of the variation in the leaf growth rate per shoot along clipping treatments of increasing intensity does not, however, generally provide any information about the order of the equation to be adjusted to the response of leaf growth to defoliation, because this equation is adjusted on a single shoot basis). Yet meta-analyses revealed an overall (i.e., all the populations considered in concert) significant parabolic response of the leaf growth rate per shoot to increasing defoliation intensity ( x 2( 30 ) , p,0.001 for both coefficients b 1 and b 2 and for the whole regression model, Table 2). Moreover, the coefficients b 1 and b 2 were negatively correlated (r5 20.86, p,0.05), which suggests that steeper enhancements or depressions of leaf growth at low clipping intensities are associated to more pronounced reversions towards lower or greater leaf growth values, respectively, at moderate or high clipping intensities. None of the seagrass properties measured (Table 1) was related to the leaf growth response to defoliation, except for a tendency for higher distances between consecutive shoots to be associated to lower values of b 2 (r5 20.53, p,0.05).
4. Discussion Our results show a substantial variability of the response of the leaf growth rate per shoot to single events of defoliation on scattered shoots. Differences in the relative leaf growth rate, distance between consecutive shoots, rhizome diameter and leaf nutrient content among the populations treated are not related to the variability in this response. We only found a tendency for increasing distances between consecutive shoots to be associated to decreasing values of b 2 , which hints that increasing distances could restrain seagrass leaf growth compensation under moderate / intense defoliation, because of the greater limitation of reserve translocation with increasing distance between consecutive shoots. Other factors more important in the determination of leaf growth response to defoliation may blur any potential relation between this response and our measured seagrass properties. For instance, carbohydrates concentrations in the rhizomes of P. oceanica, C. nodosa and Z. noltii (Pirc, 1989), and T. testudinum (Dawes and Lawrence, 1980) show a remarkable seasonal oscillation, reaching the maximum value at the experimental period, in contrast with the lesser seasonality displayed by tropical species with a smaller rhizome, such as Syringodium filiforme and Halodule wrightii (Dawes and Lawrence, 1980), which could also hold for T. hemprichii and C. rotundata. Hence, at the experimental period the former species could show a higher carbohydrates gradient between rhizome and leaves than the latter ones, having therefore a greater capacity to overcome defoliation losses by carbohydrates translocation from rhizomes to leaves. This suggestion is supported by the fact that three of the four populations which experience growth depression under some treatments correspond to the species T. hemprichii and C. rotundata (Fig. 2). Differences in self-shading (Bulthius, 1983), shoot ´ size (Vant Lent et al., 1991; Perez and Romero, 1994), epiphyte cover (Cambridge et al.,
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1986; Neverauskas, 1987) or shoot burial and erosion by sediment dynamics (Marba` and Duarte, 1994; Manzanera et al., 1996) among the shoots of a given population or among populations may also entail substantial variability in the leaf growth response to defoliation. Moreover, because of the random selection of the shoots clipped, which aims at mimicking the feeding behaviour of most invertebrate and vertebrate poikilotherm herbivores, some rhizomes could have some shoots clipped and that could restrict shoot–shoot and shoot–rhizome integration, thus affecting leaf growth response to defoliation. Meta-analyses techniques indicate that, in spite of the remarkable variability found in the leaf growth response to defoliation, an overall parabolic response turns out to be significant (Table 2). Although the nature of a parabolic response cannot be ascertained, a convex parabola is in accordance with the observation that moderate grazing may stimulate leaf growth by removal of old, epiphytized leaf tips which do not contribute to leaf growth, whereas grazing involves growth depression when it becomes intense (Thayer et al., 1984; Zieman et al., 1984). We provide an example how meta-analyses can be used to analyse the overall significance of a test when this test yields contrasting results in the populations tested. This approach is useful when the probabilities obtained for some populations are suggestively low, albeit not significant, and the general significance of the test is suspected (Arnqvist and Wooster, 1995). Indeed meta-analyses has become a popular technique to arrive at conclusions about the overall significance of processes obscured by contrasting results at the population level (Arnqvist and Wooster, 1995; Sokal and Rolhf, 1995). Another relevant feature of this report is that nonsignificant results on the effect of defoliation on seagrass growth at the population level are provided, because non-significant results tend to be under-represented in the literature and that can bias the conclusions obtained by reviews based on published results (Cooper and Hedges, 1993). We show that single events of defoliation on scattered shoots rarely depress leaf growth since only four populations exhibited reduced leaf growth rates under some or all of the clipping treatments applied (Fig. 2). This is in agreement with the expected compensatory leaf growth seagrass should display following defoliation, which should result from reserves translocation from the rhizome or neighbouring non-defoliated shoots to the defoliated shoots. Nevertheless, major defoliation, whereby leaves of single shoots of T. testudinum were cut down to 1.5 cm above the substratum (i.e., all leaf surface removed), has been shown to reduce leaf growth rates (Greenway, 1974). This is not in opposition with our results since we removed a mean maximum of 80–85% of the shoot leaf surface in nearly all the populations (Fig. 2). Thus, although our results indicate that major single defoliation on a shoot does not normally reduce its leaf growth rate, removal of all the photosynthetic tissue could have indeed depressed it. Moreover, had the experiment been run in other seasons (i.e., late winter–early spring), when the rhizome of temperate seagrass species contains low levels of reserves (Pirc, 1989; Dawes and Guiry, 1992), reduction of leaf growth by defoliation could have been more apparent, as has explicitly been demonstrated for P. oceanica (Wittmann and Ott, 1982). Moreover, the finding of rare depression of leaf growth under defoliation is only relevant to most poikilotherm vertebrates and invertebrates, whose feeding behaviour (i.e., consumption of only the leaves of a single shoot at every feeding attack and
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non-selective consumption over the same shoots, Thayer et al., 1984; Klumpp et al., 1989) approaches our experimental procedure of single clipping events on scattered shoots. Indeed, frequent and intense defoliation over the same plots of T. testudinum, such as that exerted by the green turtle (Chelonya mydas), is eventually conducive to depressed leaf growth rates (Zieman et al., 1984), which has also been demonstrated experimentally by intense and recurrent clipping (Phillips, 1960; Buesa, 1974). Moreover, some large herbivores create feeding tracks (e.g., dugongs and manatees, Heinsohn et al., 1977; Ogden et al., 1980; Zieman, 1982) and scars (waterfowl, Thayer et al., 1984), removing therefore the leaves of several neighbouring shoots at every feeding attack. This concurrent leaf loss for several neighbouring shoots could preclude our observed compensatory leaf growth expected from the integration between clipped shoots and non-clipped neighbouring ones. The herbivores mimicked by our experimental procedure can also denude extensive seagrass areas, for instance following outbursts of sea urchin populations (Camp et al., 1973), but these overgrazing events do not seem to be common (Valentine and Heck, 1991). On the grounds of these results, and considering that most poikilotherm vertebrate and invertebrate herbivores normally remove ,30% of seagrass leaf production (Thayer et al., 1984; Klumpp et al., 1993; ´ and Duarte, 1994), we conclude by suggesting that in general these herbivores Cebrian have a modest effect on the depression of leaf growth rates through removal of photosynthetic tissue.
Acknowledgements This study was funded by Project AMB93-0118 of the Spanish Commission of Science and Technology (CICYT) and by Project CI1*-CT91-0952 of the European Community. J.C. was supported by a fellowship from the CIRIT (Comissio´ Interministerial de Recerca i Tecnologia). We thank J. Romero for useful comments on the ` S. Enrıquez, ´ ´ ´ manuscript, N. Marba, P. Ramırez, J. Rengel and A. Rodrıguez for technical assistance during field work, and C. Sonyer and the Direccio´ General de Parcs for providing access to the Spanish sampling stations.
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