Differences in Herbivory Intensity Between the Seagrass Cymodocea

Aquatic Botany 128 (2016) 48–57

Contents lists available at ScienceDirect

Aquatic Botany

journal homepage: www.elsevier.com/locate/aquabot

Differences in herbivory intensity between the seagrass Cymodocea

nodosa and the green alga Caulerpa prolifera inhabiting the same habitat

∗

Lucia Del Río, Javier Vidal, Séfora Betancor, Fernando Tuya

¨ IU-ECOAQUA¨, Grupo en Biodivesidad y Conservación, Marine Sciences Faculty, Universidad de Las Palmas de Gran Canaria, Las Palmas, Canary Islands,

Spain

a r t i c l e i n f o a b s t r a c t

Article history: Seagrasses are frequently found mixed with other macrophytes, e.g., green macroalgae. We aimed to

Received 8 March 2015

assess whether the magnitude of herbivory differed between two coexisting macrophytes, the seagrass

Received in revised form

Cymodocea nodosa (Ucria) Ascherson and the green seaweed Caulerpa prolifera (Forsskål) Lamouroux, at

25 September 2015

Gran Canaria Island (eastern Atlantic). Both in situ (ﬁeld) and aquaria experimentation demonstrated

Accepted 2 October 2015

a larger intensity of herbivory (between ca. 4–8 times) on C. prolifera than C. nodosa. At the scale of

Available online 8 October 2015

meadows, herbivorous ﬁsh abundance predicted the intensity of herbivory, in particular by the parrotﬁsh

Sparisoma cretense. A plant physical attribute (“force-to-fracture”) negatively correlated with a larger

Keywords:

Seagrass consumption on C. prolifera, while differences in total phenolic compounds between both macrophytes

Herbivory were insigniﬁcant. Importantly, herbivory marks (bites) were signiﬁcantly larger (ca. two times) on C.

Grazing nodosa leaves than in C. prolifera fronds, so differences in the magnitude of herbivory between C. nodosa

Grazers and C. prolifera were dependent on herbivorous size.

Leaf traits

1. Introduction epiphytes on seagrass leaves and the seagrass itself (Wressnig and

Booth, 2007), as well as ﬂowers and seeds (Balestri and Cinelli,

Many organisms are associated with seagrasses, below the sed- 2003). Epiphytes are a key element in the relationship between

iment linked to the rhizomes, upon the leaves and stems, and over herbivores and seagrasses. Some studies suggest that epiphytic pro-

the seagrass canopy (epi- and suprabenthic organisms, Herrera duction may be elevated to exceed even that of seagrasses (Morgan

et al., 2014; Tuya et al., 2014b), which move throughout the and Kitting, 1984; Chiu et al., 2013). Temperate and sub-tropical

meadow and constitute the main consumers of seagrass and asso- ﬁshes consuming seagrass material select seagrass leaves and parts

ciated vegetated material. Seagrasses are evolutionarily adapted of leaves with abundant epiphytic loads, whereas mesograzers usu-

to herbivory; this is indicated by the range of evolutionary adap- ally feed on algae attached to seagrass leaves (Valentine and Heck,

tions to mitigate the consequences of herbivory (Karban and Myers, 1999; Goecker et al., 2005). It has been postulated that internal

1989), including mechanical and chemical elements (Lucas et al., contents in N are a relevant factor mediating feeding preferences by

2000). Traditionally, it has been postulated that a small fraction marine herbivores (Vergés et al., 2007; Prado et al., 2010; Prado and

of seagrass production is directly consumed by marine herbivores Heck, 2011), despite in other occasions its inﬂuence is negligible

(Cebrián and Duarte, 1998; Valentine and Heck, 1999). The low con- (Lee et al., 2015).

sumption of seagrass by grazers has been explained by their poor Cymodocea nodosa is a seagrass distributed across the entire

nutritional quality (Prado and Heck, 2011), including a high content Mediterranean and the adjacent Atlantic coasts, from the south-

in cellulose that act as a structural deterrent. Recent studies; how- ern Iberian Peninsula to Senegal, including Madeira and the Canary

ever, have pointed out that herbivory over seagrasses has a larger Islands (Cunha and Araujo, 2009). Meadows constituted by C.

inﬂuence than previously considered (Tomas et al., 2005; Heck and nodosa are found on shallow soft substrates of Gran Canaria Island

Valentine, 2006; Doropoulos et al., 2009; Prado et al., 2007; Vergés (Tuya et al., 2014a), where it may form mixed meadows with

et al., 2011). Seagrasses offer herbivores two main food sources: green rhizophytic seaweeds of the genera Caulerpa, particularly

Caulerpa prolifera (Fig. A1). As a result of environmental deteriora-

tion, frondose C. nodosa meadows can turn into bottoms dominated

∗ by C. prolifera; this has been reported from the Mediterranean

Corresponding author. Fax: +34 928452900.

and the southern Iberian Peninsula (Ceccherelli and Cinelli, 1997;

E-mail address: [email protected] (F. Tuya).

http://dx.doi.org/10.1016/j.aquabot.2015.10.001

L. Del Río et al. / Aquatic Botany 128 (2016) 48–57 49

Lloret et al., 2005), as well as from the Canary Islands (Tuya et al., and fronds of C. prolifera. Firstly, we conducted an indirect approach

2013b). C. nodosa may be an important food source for macro- by estimating herbivory pressure as the number of bite marks left

herbivores (Cebrián et al., 1996a). In addition, leaves of this seagrass by herbivores on both C. nodosa leaves and C. prolifera fronds; since

are colonized by epiphytic assemblages that may provide food for the majority of ﬁshes inhabiting these seagrass systems are small-

associated invertebrates (Vizzini et al., 2002; Tuya et al., 2013a). sized (Espino et al., 2011), complete removal of seagrass leaves is an

Accompanying macrophytes, e.g., green seaweeds, can also rep- unlikely process. The study was carried out at two times: October

resent an additional food source for herbivores. However, certain 2013 and May 2014 to test for the effect of seasonality on responses.

macrophytes have developed several mechanisms to minimize her- At each of the 4 meadows, 12 leaves of C. nodosa and 12 fronds of

bivory (Duffy and Hay, 1990). The primary deterrent substances in C. prolifera were haphazardly collected by SCUBA divers; adjacent

seagrasses and seaweeds are phenolic compounds (Arnold et al., leaves/fronds were >2 m apart. Samples were quickly transported

2012), which have been linked to a variety of functions, prevent- to the laboratory and preserved in ice until analysis. At the same

ing bacterial infections (Harrison and Chan, 1980), protecting algae time of collection, ﬁsh assemblages were counted at daylight hours

from high PAR and UV damage (Pavia et al., 1997) and deterring (between 9:00 and 12:00 a.m.) through underwater visual cen-

grazers (Van Alstyne and Paul, 1990). Yet, there is some contro- suses, following (n = 4) 25 × 4 m transects per meadow (100 m of

versy in the real effectiveness of phenols as grazer deterrents (Close observation per census); the abundance and size of each ﬁsh species

and McArthur, 2002; Vergés et al., 2007). For algae within the gen- was annotated according to standard procedures implemented in

era Caulerpa, it has been largely hypothesized that the presence the study region for seagrass meadows (Tuya et al., 2006). No

of repulsive (toxic) secondary metabolites, e.g., caulerpenyne, may major herbivorous invertebrates (e.g., sea-urchins) were found in

also deter herbivores (Box et al., 2010). Preference for vegetated the study area. Once in laboratory, we measured the length of C.

material among herbivores is; however, not exclusively related to nodosa leaves (from the ligule to the upper tip of each leaf) and

chemical attributes (Hay and Kappel, 1994), but also to the physical C. prolifera fronds (from the base of the stipe to the upper tip of

structure and conﬁguration of macrophytes, e.g., their resistance to the frond). Fronds with proliferations were not considered to avoid

breakage (Duffy and Hay, 1990; Lucas et al., 2000; Prado and Heck, confusion. Bite marks were recorded for each leaf/frond through

2011). image analysis (imageJ freeware); all material was then preserved

In mixed meadows (i.e., those constituted by seagrasses and in silica gel. Some bite marks were clearly crescent-shaped (Fig. A2),

green seaweeds), macro-herbivores have several choices of food, a clear indication of consumption by herbivorous ﬁshes (Hay, 1984;

what may generate different patterns of vegetation consumption. Kirsch et al., 2002; White et al., 2011; Lee et al., 2015). In these cases,

The aim of this work was to compare the magnitude of herbivory we recorded each bite size, as the maximum diameter of the mark

between the seagrass C. nodosa and the green alga C. prolifera; these (cm). The cover of epiphytic material was also annotated by using a

two macrophytes inhabit the same habitat (mixed meadows on qualitative, visual, scale: 0 (cover: <1%), 1 (cover: 1–10%), 2 (cover:

shallow subtidal waters) at Gran Canaria Island (eastern Atlantic). 10–20%), 3 (cover: 20–40%), 4 (cover: 40–60%) and 5 (cover: >60%).

Differences in the intensity of herbivory were compared by com- When the apical part of either seagrass leaves or C. prolifera fronds

bining in situ assays, that assessed indirect (bite marks) and direct was damaged, we omitted to record these as bite marks, due to the

(rates of consumption of fresh material) measures of herbivory, difﬁcult of ascertaining if these marks resulted from herbivory or

and an aquaria experiment that quantiﬁed rates of consumption other type of damage (e.g., currents and/or swells).

on fresh material under controlled laboratory conditions. We set We took measurements of the “force-to-fracture” (FTF), as a way

out these procedures to speciﬁcally test whether the intensity of to assess the physical resistance to breakage of both macrophytes;

herbivory differed between C. nodosa and C. prolifera. We addition- these measurements were calculated with a dynamometer. The tip

ally hypothesized that spatial and temporal variation in herbivory of each of n = 30 leaves and fronds of both C. nodosa and C. prolifera

intensity on these two macrophytes is connected with differences was attached to the pin of the dynamometer; the force (Newtons)

in the abundances of herbivorous ﬁsh. Finally, we analyzed differ- necessary to tear each leaf/frond was then annotated. All leaves

ences in phenolic compounds concentration (a chemical attribute) and fronds were collected at Gando meadow (May 2014), encom-

and leaf/frond resistance to breakage (a physical attribute) between passing the entire range of available sizes; measurements were

both macrophytes to help to explain differences in herbivory pat- taken from fresh material immediately after collection. On the 17th

ters. December 2013, we randomly collected leaves of C. nodosa and

thalli of C. prolifera (ca. 0.25 g FW each thalli, n = 9) from Gando

meadow (8–10 m) to analyze differences in total phenolic com-

◦

2. Materials and methods pounds. All material was stored at −80 C until analysis. Once in the

laboratory, all thalli were initially cleaned and epiphytes removed.

2.1. Field observational approach In all cases, we selected the central parts of the thalli, with no

evidence of grazing activity. All samples were grounded with a

◦

Four study sites were selected in mixed meadows constituted mortar and a pestle in sand at 4 C, and extracted overnight in cen-

by the seagrass C. nodosa and the rhizophytic seaweed C. prolif- trifuge tubes with 2.5 ml of 80% (v/v) methanol (Betancor et al.,

era at the east coast of Gran Canaria Island (Fig. 1); depth ranged 2014). The mixture was centrifuged at 4000 rpm for 30 min and the

between 8 and 12 m, all bottoms were sandy and proximity from supernatants were collected (Sigma 2-16PK, Göttingen, Germany).

−1

the adjacent coast varied between 150 and 250 m. These mixed Total phenolic compounds, expressed as mg GAE g DW (Gallic

meadows are permanent all year round (Tuya et al., 2013b, 2014b); Acid Equivalent), were determined using gallic acid as a standard

−2 ◦

the biomass of C. nodosa varies between 120 and 170 g DW m (Folin and Ciocalteu, 1927) after 120 min in darkness at 4 C. The

−2

and the biomass of C. prolifera between 0 and 70 g DW m across absorbance was then measured at 760 nm in a spectrophotometer

sites 10 s of meters apart (Tuya et al., 2013b). Alternative vegetation (Thermo Scientiﬁc Evolution 201, UV-visible, China).

is sparse and mainly restricted to epiphytes growing on seagrass

leaves. The seagrass shows a clear seasonal pattern, including a 2.2. Field experimentation

maximum in shoot density and biomass in summer and a mini-

mum in winter (Tuya et al., 2006). No information is available on Through an in situ experiment, we offered herbivores fresh C.

seasonal patterns of C. prolifera. We developed different types of nodosa leaves and C. prolifera fronds alternatively attached with

assays to evaluate the magnitude of herbivory on C. nodosa leaves clothespins to plastic mesh frames (Fig. A3). This was a way to

50 L. Del Río et al. / Aquatic Botany 128 (2016) 48–57

Fig. 1. Location of study area; including the 4 sampled meadows: C (Caballo), G (Gando), RA (Roque de Arinaga) and RV (Risco Verde).

directly estimate differences in consumption over both C. nodosa photoperiod. Only seagrass leaves/algal fronds without grazing

and C. prolifera by local herbivores (a double choice experiment). scars were selected. Each aquaria had two compartments sepa-

Fresh material was initially collected by SCUBA divers, before the rated by a mesh net (Fig. A4); one for the feeding trial including

experimental set up, which was located at Gando meadow at 10 m the ﬁsh, while the other compartment lacked ﬁsh and so acted as

depth (Fig. 1). A total of 14 plots (mesh frames) were directly a control to correct for possible autogenic changes in biomass not

attached to the bottom with metal bars and then covered with sand directly caused by grazing (Wressnig and Booth, 2007; Prado et al.,

to minimize any visual effect. Plots were placed at two randomly- 2011). A total of 5 seagrass leaves (with and without epiphytes)

selected areas within this meadow. The distance between adjacent and 5 fronds of C. prolifera were placed into each experimental

plots was approximately 0.5 m. All leaves/fronds were of the same compartment per aquaria. All vegetated material was distributed

length (15 cm for C. nodosa and 7 cm for C. prolifera fronds); we only randomly and secured to the bottom with wire stakes (Fig. A4,

used leaves with no previous herbivore marks and with no signs for a video clip visit the YouTube link: http://www.youtube.com/

of necrosis. Furthermore, to offer herbivores material under simi- watch?v=IhWCk62SZkE&list=UU9zh1SloG4bMsoJbW8y-hmw).

lar conditions, epiphytes were scraped off the leaves using a thin Prior to experimentation, the wet weigh of all vegetated material

blade. Three leaves of C. nodosa and 3 fronds of C. prolifera were per aquaria was obtained. We then conducted the feeding trial

used per plot. After 7 days underwater, all material was retrieved. over a 24 h period. After that time, the remaining material was

Once in the laboratory, the number of bites marks per leaf/frond removed from each aquaria, blot-dried, and weighed, what pro-

−1 −1

was recorded, as in the previous assay. Despite the number of bite vided consumption rates (g wet weight ﬁsh d ). The numbers

marks is not as critical as the mass of removed vegetation, it pro- of bite marks per leaf/frond was then annotated. To work out

vides an unconfounded measure of herbivory intensity; e.g., it is whether parrotﬁsh had a preference for a particular macrophyte,

plausible that removal of vegetation biomass results, for example, we used Chesson’s index (Chesson, 1983):

from the action of waves. This assay was carried out at three dif-

ri/pi

ferent times: October 2013, May 2014 and June 2014 to test for the ˛ =

r /p

temporal consistency of results at random times. i i

where ri is the proportional biomass removal due to consumption

on macrophyte i, and p is the proportional biomass of macrophyte

2.3. Aquaria experimentation i

i in the source sample (the aquaria). The value of ˛ varies between

0 and 1. When ˛ = 1/n, selective feeding does not happen (n is the

We tested for differences in consumption by the main herbivo-

total number of macrophytes). There were 3 macrophytes (n = 3,

rous ﬁsh in seagrass meadows of the study region, the parrotﬁsh

1/n = 0.33). If ˛ > 1/n, it is then likely that parrotﬁsh has a preference

Sparisoma cretense (Tuya et al., 2013a; this study: Appendix C),

for macrophyte i, whereas if ˛ < 1/n, the macrophyte i is probably

between C. nodosa seagrass leaves with, or without, epiphytes

avoided. Values of ˛ were calculated for each of the 3 macrophytes

and fronds of C. prolifera, under controlled laboratory conditions.

separately for every aquaria.

Fresh material of these 3 choices was offered to 3 S. cretense

per aquaria. Fish were collected in Gando meadow through a

seine net (May 2014) and immediately transported to the aquaria 2.4. Statistical analyses

facility under constant aeration. Fish were acclimatized to indoor

conditions for 7 days, with a lack of food provision prior to exper- Differences in the number of bite marks per thallus between the

imentation (Prado et al., 2011; Tuya and Duarte, 2012). All ﬁsh two macrophytes was tested using a 3-way ANOVA, which incorpo-

were small-sized (juveniles, total length between 7 and 10 cm). rated the factors: “Time” (ﬁxed factor with 2 levels: October 2013

This experiment was replicated in 4 aquaria (84 l) with constant and May 2014), “Meadow” (random factor with 4 levels: the 4 study

aeration. The bottom of all aquaria was covered by sand; sea-water sites) and “Species” (ﬁxed factor with two levels: C. nodosa vs. C.

◦

temperature varied between 21 and 23 C under a 12:12 h natural prolifera). “Time” was considered as a ﬁxed factor, since it reﬂected

L. Del Río et al. / Aquatic Botany 128 (2016) 48–57 51

Fig. 2. Herbivory intensity over Cymodocea nodosa leaves and Caulerpa prolifera fronds (mean number of grazing marks per thallus, n = 12 leaves/fronds) in (a) October 2013

and (b) May 2014. Error bars are +SE of means.

Table 1

Results of 3-way ANOVA testing for differences in the total number of grazing marks between times, meadows and macrophytes (C.n. = Cymodocea nodosa and C.p. = Caulerpa

prolifera).

df MS F-ratio P

Time 1 18.49 12.04 0.0404

Meadow 3 1.35 22.36 0.0001

C.n. vs. C.p. 1 52.9 38.11 0.0086

Time × meadow 3 1.5367 25.28 0.0001

Time × C.n. vs. C.p. 1 12.28 6.77 0.0803

Meadow C.n. vs. C.p. 3 1.3883 22.84 0.0001

Time × meadow × Cn. vs. C.p. 3 1.8161 29.87 0.0001

Residual 176 0.0608

potential seasonal differences. Data were square-root transformed signiﬁcantly larger number of bite marks was recorded on C. prolif-

prior to analyses to avoid heterogeneous variances, as indicated era relative to C. nodosa. The overall number of bites was larger

by the Cochran’s test. Differences in the number of bite marks per in October (2013) than on May (2014) (Fig. 2; Table 1, “Time”

thallus after 1 week between both macrophytes in the in situ exper- P = 0.04); the magnitude of differences in the mean number of

iment was tested by a 3-way ANOVA, which included a similar bites per thallus varied from October (2013) to May (2014) (Fig. 2;

design relative to the previous analysis, but the factor “Meadow” Table 1, “Time × Cn. vs. C.p.” P = 0.083). In general, the total abun-

was replaced by “Plot” (also a random factor). A t-test tested for dance of herbivorous ﬁshes (Appendix C) and the abundance of

significant differences in FTF between C. nodosa and C. prolifera at the most conspicuous herbivorous fish, the parrotfish S. cretense,

the range of sizes they typically occur, as well as the signiﬁcance signiﬁcantly predicted differences in herbivory intensity between

of differences in the size of grazing marks (bites) between both meadows (Fig. 3). In the ﬁeld experiment, the number of bite marks

macrophytes. We ignored the contribution of spatial and temporal on C. prolifera fronds after 2 weeks exceeded those on seagrass

variation in differences in bite size between both macrophytes as leaves (Fig. 4; Table 2, “C.n. vs. C.p.” P = 0.003), particularly on time

a result of the large imbalances in the number of bite marks per 3 (June, 2014) (Fig. 4; Table 2, “Time × C.n. vs. C.p.” P = 0.008).

time and meadow; this was because only clear crescent-shaped Overall, C. nodosa leaves had a larger resistance to breakage

bite marks a doubtless indication of consumption by herbivorous (FTF) than fronds of C. prolifera, when considering the range of

ﬁshes—were considered in the analysis. Differences in consump- sizes that both macrophytes typically reach in situ (Fig. 5; t-

tion in the aquaria trial were tested by means of a 2-way ANOVA, student = 1.9047, P = 0.061). The size of grazing marks (bites) on

which included the factors: “Treatment” (ﬁxed factor with 3 levels: C. nodosa leaves were larger than those on C. prolifera fronds

C. nodosa with epiphytes, C. nodosa without epiphytes and C. prolif- (C. nodosa: 0.4413 cm ± 0.0238; C. prolifera: 0.2850 cm ± 0.041,

era) and “Aquaria” (random factor with 4 levels: aquaria 1, aquaria mean ± SE, n = 20; t-test = 6.0134, P < 0.00001).

2, aquaria 3 and aquaria 4). When appropriate, pair-wise compar- In the aquaria assay, consumption of C. prolifera fronds was

isons (via SNK tests) were performed. Signiﬁcant differences in the larger than consumption of C. nodosa leaves either with or with-

content of phenols between both macrophytes were tested through out epiphytes (Fig. 6; Table 3, “Treatment”, P = 0.03), despite the

a t-test. A one-way ANOVA tested whether Chesson’s ˛ values dif- magnitude consumption among treatments varied from aquaria

fered between the 3 macrophytes. The signiﬁcance level was set at to aquaria (Fig. 6; Table 3, “Aquaria × Treatment”, P = 0.01); in

the type I error rate of 0.05. all cases, independent SNKs tests for each aquaria demonstrated

a larger consumption on C. prolifera relative to C. nodosa with

or without epiphytes. In 3 out of the 4 aquaria, consump-

3. Results

tion on C. nodosa with epiphytes was larger than on C. nodosa

without epiphytes (SNK tests). Parrotﬁsh clearly preferred C. pro-

The mean total number of bites per thallus differed between C.

lifera (˛ = 0.72 ± 0.04) over C. nodosa with (˛ = 0.16 ± 0.02) and

nodosa and C. prolifera (Fig. 2; Table 1, “C.n. vs. C.p.”, P = 0.0086); a

52 L. Del Río et al. / Aquatic Botany 128 (2016) 48–57

Fig. 3. Relationship between the mean number of bites on Cymodocea nodosa leaves (a and b) and Caulerpa prolifera fronds (c and d) and the abundance of the parrotﬁsh,

Sparisoma cretense, and the total herbivorous fish abundance per meadow. Data were pooled for the two sampling times. Unfilled symbols: October 2013, filled symbols:

May 2014.

C. prolifera in Gran Canaria Island. The seaweed C. prolifera con-

sistently suffered higher rates of herbivory than C. nodosa, both

in the ﬁeld, through direct and indirect measures, and in aquaria

conditions. Average feeding rates by S. cretense were estimated at

−1 −1

0.209 g wet weight ind d for C. prolifera and 0.086 g wet weight

−1 −1

ind d for C. nodosa, under laboratory conditions. Despite com-

parisons with other studies are complicated as a result of varying

methods and timing, the average feeding rates on C. nodosa leaves

−1 −1

obtained by our study (ca. 7.5 g seagrass kg ﬁsh d ) is within the

same order of magnitude as those reported for C. nodosa at other

zones. For example, Goldenberg and Erzini (2014) estimated the

average feeding rate by adults of the Sparid Sarpa salpa from the Ria

−1 −1

Formosa lagoon as ca. 32 g kg ﬁsh d ; obviously, this larger con-

sumption is explained because adult ﬁsh consume larger amounts

of vegetated material than juveniles.

Herbivorous ﬁsh abundance predicted the intensity of herbivory

over C. nodosa leaves and C. prolifera fronds through a positive cor-

relation between the average number of bite marks per thallus and

Fig. 4. Mean number of bites over Cymodocea nodosa leaves and Caulerpa prolifera

total ﬁsh abundance. This result is similar to patterns of seagrass

fronds after 1 week at each replicated assay (n = 14, T1 = October 2013, T2 = May 2014

consumption in tropical environments, where the abundances of

and T3 = June 2014). Error bars are +SE of means.

parrotﬁsh is the main driver of removal of seagrass material (Lee

et al., 2015), despite in other occasions the abundance of megafauna

without epiphytes (˛ = 0.10 ± 0.02) (1-way ANOVA, F2,9 = 428.18,

(e.g., turtles) are the main contributors (Christianize et al., 2014).

P = 0.00001).

Herbivory intensity seemed to be seasonal, reaching maximum val-

The total content of phenols did not differ between both macro-

−1 ues in late summer (October 2013) and minimum values in spring

phytes (C. nodosa: 1.780 mg GAE g DW ± 0.1872; C. prolifera:

−1 (May 2014). There is a clear correlation between the annual vitality

1.8157 mg GAE g DW ± 0.1524; t-test = 0.14, P = 0.5737).

cycle of C. nodosa and the richness and abundance of associated

ﬁsh assemblages in the study region (Tuya et al., 2006; Espino

4. Discussion

et al., 2011), with maximum values in summer–autumn and mini-

mum in winter–spring. In particular, the abundance of small-sized

Our study has demonstrated that herbivory may remove

S. cretense, the main herbivorous ﬁsh in the study region, reach

amounts of vegetated material in mixed meadows of C. nodosa and

L. Del Río et al. / Aquatic Botany 128 (2016) 48–57 53

Table 2

Results of 3-way ANOVA testing for differences in the total number of grazing marks per thallus between times, plots and macrophytes (C.n. = Cymodocea nodosa and

C.p. = Caulerpa prolifera).

df MS F-ratio P

Time 1 1.32 8.217 0.015

Plot 13 0.4177 0.965 0.5

C.n. vs. C.p. 1 9.21 22.77 0.003

Time × plot 13 0.16 0.37 0.977

Time × C.n. vs. C.p. 1 1.7 11.46 0.008

Plot × C.n. vs. C.p. 13 0.4 0.93 0.536

Time plot × C.n. vs. C.p. 13 0.15 0.34 0.987

Residual 112 0.43

14 a) b) 12

6 Force (N)

0 5 10 15 20 25 30 35 2 4 6 8 10 12 14

Leaf length (cm) Frond len gth (cm)

Fig. 5. Differences in the “force-to-fracture” between (a) Cymodocea nodosa leaves and (b) Caulerpa prolifera fronds.

Table 3

Results of 3-way ANOVA testing for differences in mass loss between aquariums and treatments (Cymodocea nodosa leaves with epiphytes, C. nodosa leaves without epiphytes

and Caulerpa prolifera fronds).

df MS F-ratio P

Aquaria 3 23.34 17.29 0.0010

Treatment 2 27.14 6.07 0.0380

Aquaria × treatment 6 4.47 3.31 0.0100

Residual 48 1.35

maximum abundances in September–October (Espino et al., 2014). bite marks on seagrass blades (Fig. A5). Typically, adult and sub-

Our results are, moreover, in accordance with seasonal trends for adult ﬁshes are big enough to move across meadows without the

herbivory in seagrass meadows from the Mediterranean, where necessity to hide from predators and so the entire canopy is avail-

maximum consumption rates occurred in summer (Tomas et al., able to them, while juveniles hide from predators within the dense

2005; Prado et al., 2007, 2010). Chiu et al. (2013) and Lee et al. canopy provided by seagrass leaves and accompanying green sea-

(2015) also demonstrated that leaf grazing rates were signiﬁcantly weeds (Espino et al., 2014). As a result, small-sized ﬁshes have a

greater in summer and autumn than in winter and spring. Sea- direct access to the shorter fronds of C. prolifera, which are softener

sonal differences in herbivory pressure can be related to migratory to feed. This hypothesis is consistent with the assumption that adult

(ontogenetic) movements, or varying seasonal feeding behavior, of ﬁshes inhabiting seagrass meadows have a different trophic niche

herbivores (Prado et al., 2007). in comparison with juveniles (Livingston, 1982; Vizzini et al., 2002).

The different intensity of herbivory between C. nodosa and C. Many herbivores ﬁnd difﬁculties to consume plant material

prolifera was herbivorous size-dependent, as bite marks were sig- (Watson and Norton, 1985). Calciﬁcation and toughness usually

niﬁcantly larger on C. nodosa leaves than in C. prolifera fronds. correlate with low feeding preference (Litter et al., 1983; Hay, 1984;

The mean bite size on C. nodosa leaves normally exceeded 0.4 mm, Paul and Hay, 1986). Despite we lack data for our case study, C.

which likely corresponds with sub-adult ﬁshes (e.g., S. cretense). In prolifera typically shows a higher total internal N content than C.

contrast, mean bite sizes on C. prolifera fronds were <0.3 mm, which nodosa and hence a lower C:N ratio (García-Sánchez et al., 2012).

likely correspond with juvenile ﬁsh stages. In other words, small- Plant quality (often typiﬁed as C:N ratios) has been largely demon-

sized herbivorous ﬁshes preferably consume C. prolifera; once a strated to affect patterns in feeding preferences by herbivores in

certain size is reached, herbivorous ﬁsh may consume both macro- coastal habitats (Cebrián and Duarte, 1998), despite C:N rations

phytes. In this sense, it is plausible that the jaws of S. cretense may not explain feeding preferences over a suite of coexisting sea-

juveniles do not have enough force to ripe off pieces of C. nodosa grass species (Lee et al., 2015). In this sense, C. nodosa is a seagrass

leaves; this idea is consistent with the presence of unsuccessful with high ﬁber content (large amounts of cellulose in their cell

54 L. Del Río et al. / Aquatic Botany 128 (2016) 48–57

0.35 (McConnell et al., 1982) and a few reef ﬁsh (Targett et al., 1986;

C. nodosa +

Epiphytes Paul et al., 1987) have been deterred by caulerpenyne, despite most

C. nodosa - Epiphytes

0.30 reef ﬁsh were not deterred by Caulerpa extracts containing cauler-

Cau lerpa prolifera

penyne (Paul et al., 1987, 1990; Wylie and Paul, 1988). Macroalgal

fronds have often higher concentrations of caulerpenyne than

0.25

stolons, and this concentration may even change during the year

(Box et al., 2010) within and among species. In any case, the clas- Ra

0.20

sic idea that Caulerpa species deter herbivores seems not to be ion

supported by our data, and ﬁts previous observations reporting

0.15 consumption of Caulerpa by a range of herbivores (Gavagnin et al.,

1994). The convergence of results from indirect and direct in situ

Consump 0.10 measurements and the aquaria assay reinforce this idea. With these

results, we hypothesize that caulerpenyne may have a higher effect

on small organisms (like epibiota or epifauna) than in vertebrates.

0.05

It is also possible that macro-herbivores inhabiting mixed seagrass

meadows may be adapted to allelochemicals produced in the sys-

0.00 tem (Prado et al., 2011; Goldenberg and Erzini, 2014). Seagrass

Aq uaria 1 Aquaria 2 Aquaria 3 Aquaria 4

leaves, C. nodosa in particular, are extensively colonized by a com-

−1 −1 plex epiphytic community (cyanobacteria, diatoms, crustose and

Fig. 6. Consumption rates of vegetated material (g wet weight ind d ) by the

ephemeral algae, invertebrates etc.), which provide food and habi-

parrotﬁsh Sparisoma cretense (n = 5). Error bars are +SE of means.

tat for invertebrates and so increases the spatial complexity of

the habitat (Mazzella et al., 1992; Vizzini et al., 2002). Our results

walls). A high fraction of its internal C is used for the synthesis showed that C. nodosa leaves with epiphytes are preferred by her-

of structural carbohydrates that forms cell walls and ﬁber bun- bivores, at least by the parrotﬁsh S. cretense in indoor conditions,

dles, which means that C. nodosa has a high mechanical resistance, over leaves devoid of epiphytes. This is consistent with previous

thereby exhibiting a low leaf nutritional value (Lucas et al., 2000; De findings, which confirm that fishes grazing on seagrass prefer epi-

los Santos et al., 2012). Fast-growing species (such as C. prolifera); phytes growing on seagrass leaves (Cebrián et al., 1996b; Wressnig

however, do not invest resources in leaf/frond toughening as much and Booth, 2007).

as large, long-lived, seagrass species (De los Santos et al., 2012). As In summary, this study has demonstrated that (i) C. prolifera

indicated by our data (through the “force-to-fracture” calculation), is more consumed by herbivores, particularly ﬁsh, than C. nodosa

C. nodosa is tougher than C. prolifera. This trait; however, was only when found intermixed; (ii) variation in herbivory intensity is con-

evaluated at one meadow and time, so we cannot totally infer our nected with variability in the abundance of herbivorous ﬁshes; (iii)

results to all conditions. differences in the magnitude of herbivory between C. nodosa and

The polyphenols of seagrasses and seaweeds assist algae to C. prolifera were dependent on herbivorous size.

overcome oxidative stress (i.e., while subjected to high PAR and

UV levels), playing a putative role in their defense against herbi- Acknowledgements

vores (Altena and Steinberg, 1992), despite signiﬁcant controversy

We acknowledge T. Sánchez and F. Espino for their help dur-

in their efﬁciency (Vergés et al., 2007). The presence of phenols in C.

ing ﬁeldwork, laboratory assays and always useful comments, and

nodosa (García-Sánchez et al., 2012) and other seagrasses has been

R. Triay for his guidance during the aquaria assays. F. Tuya was

reported (Vergés et al., 2011). In our study, the total phenolic con-

supported by the MINECO ‘Ramón y Cajal’ program. Part of this

tents was similar for both macrophytes. This result was somehow

study was performed through the project ECOSERVEG (BEST ini-

unexpected, because a higher phenol content has been indicated for

tiative, Voluntary Scheme for Biodiversity and Ecosystem Services

C. prolifera than C. nodosa from the Mediterranean (García-Sánchez

in Territories of the EU Outermost Regions and Oversees Coun-

et al., 2012). This disagreement could result from different sam-

tries and Territories, Grant no 07.032700/2012/635752/SUB/B2).

pling depths between studies (1–2 meters in García-Sánchez et al.

The research staff was partially supported by the Campus Atlántico

(2012) vs. 8–10 m in our study); depth is known to affect the con-

Tricontinental.

tent of phenolic compounds, with higher levels for shallow-water

macrophytes (Betancor et al., 2015). Moreover, we lack temporal

Appendix A.

replication and so it is plausible that phenols concentrations may

change through seasons. As a result, the putative role of phenols

mediating differences in consumption between C. prolifera than C.

nodosa remains unresolved.

Siphonous, unicellular, green algae, e.g., Caulerpa spp., contain

secondary metabolites, which play a role during quick plug forma-

tion after injuries to avoid the release of internal cellular ﬂuids.

The production and utilization of secondary metabolites has been

indicated to justify that plant chemistry is the central factor deter-

mining herbivore feeding choices (Ehrlich et al., 1988; Schultz,

1988). However, the relative importance of these factors proba-

bly varies from system to system depending on the identity of

herbivores, their abundances and sizes (Pennings and Paul, 1992;

Lee et al., 2015). Numerous studies reported that caulerpenyne,

the main secondary metabolite within the genus Caulerpa, actively

deter herbivores (e.g., gastropods and ﬁshes), which is toxic to

larval and adult stages of several marine invertebrates and ver-

tebrates (Paul et al., 1987; Pedrotti and Lemée, 1999). Sea urchins Fig. A1. Mixed meadow of Cymodocea nodosa and Caulerpa prolifera.

L. Del Río et al. / Aquatic Botany 128 (2016) 48–57 55

Fig. A5. “Unsatisfactory” bite on a Cymodocea nodosa leaf.

Fig. A2. Crescent-shaped bite mark on a Cymodocea nodosa leaf.

Appendix B.

Average percentage of daily changes in Cymodocea nodosa leaves

and Caulerpa prolifera fronds as a result of autogenic processes.

Cymodocea nodosa Cymodocea nodosa Caulerpa prolifera

with epiphytes without epiphytes

−

Aquaria 1 0.377 2.432 0.069

Aquaria 2 1.877 0.860 1.848

Aquaria 3 0.631 1.263 1.322

Aquaria 4 0.068 −0.886 2.387

Total 2.199 3.670 5.628

Appendix C.

−2

Fish abundances at each meadow (100 m ± SE); species trophic

status are also included: CAR: carnivorous, OMN: omnivorous,

Fig. A3. Mesh frame with attached Cymodocea nodosa leaves and Caulerpa prolifera

fronds. The mesh and pegs were subsequently buried with sand. HER: herbivorous (*: only juveniles) at time 1 (October 2013) and

time 2 (May 2014).

Fig. A4. Aquaria with two separated compartments by a mesh (M), including a CON-

TROL without ﬁsh, and a FEEDING TRIAL with ﬁsh (F). The three treatments included:

C.n. +, Cymodocea nodosa leaves with epiphytes, C.n. −, C. nodosa leaves without

epiphytes, and C.p. Caulerpa prolifera fronds.

56 L. Del Río et al. / Aquatic Botany 128 (2016) 48–57

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