Shallow subtidal communities in coastal upwelling zones are believed to be largely bottom-up controlled via nutrient fluxes (Bustamante et al., 1995; Broitman et al., 2001; Menge and Branch, 2001; Menge et al., 2002; Nielsen, 2003; Nielsen and Navarrete, 2004; Vinueza et al., 2006; Vinueza et al., 2014). However, predation (top-down control) is also important in marine communities (Menge, 1991; Paine, 1992; Trussell et al., 2002; Harley, 2011), including in upwelling zones. For example, Witman et al. (2010) found that predation intensity was higher at strong upwelling sites compared to weak upwelling sites in the Galápagos. The authors suggested that predators (mostly whelks) track their prey resources (barnacles), which were more abundant in strong upwelling sites due to enhanced productivity (Witman et al., 2010).

Nutrient fluxes linked to upwelling can influence algal biomass in two ways. First, by enhancing algal photosynthesis and growth due to increased nitrogen and other limiting nutrients in the ocean (Figure 1, Bustamante et al., 1995; Broitman et al., 2001; Menge and Branch, 2001; Menge et al., 2002; Nielsen, 2003; Nielsen and Navarrete, 2004; Vinueza et al., 2006; Vinueza et al., 2014). And secondly, by reducing herbivory due to higher nutrient load in algal tissues, which may decrease per capita consumption rates (Figure 1). This happens because herbivores sometimes alter their feeding rates relative to the nutrient content of their food source (Burnell et al., 2013) or in response to decreased macroalgal palatability (Tomas et al., 2011). The opposite (i.e., increased consumption rates of enriched algae) has been found in oligotrophic conditions (Boyer et al., 2004; Russell and Connell, 2007).

Although its role is largely unexplored, temperature may also be an important factor in upwelling systems by influencing the strength of bottom-up and top-down effects (Bruno et al., 2015). For example, due to the temperature-dependence of metabolism (Brown et al., 2004), cooler temperatures associated with upwelling should reduce algal photosynthesis and consumer pressure (Figure 1). Reduced consumer pressure during upwelling has been documented in the field (Sanford, 1999; Sanford, 2002; Carr et al., 2018) and is an alternative explanation for the observed association of high algal biomass when or where upwelling is intense; in other words it is a consequence of reduced top-down control for what is assumed to be a bottom-up phenomenon.

Although there is a fair number of studies looking at the interactive effects of herbivores and nutrients in both tropical and temperate regions, there is a lack of studies looking at these effects in tropical or subtropical upwelling regions (Burkepile and Hay, 2006). The purpose of this study was to measure the effects that herbivores, temperature, and nutrient availability have on standing macroalgal biomass. We manipulated nutrient availability and herbivory in two field experiments — one during the cool, upwelling season, and the other during the warm non-upwelling season — on a subtidal nearshore rocky reef in the Galápagos Islands.

The average benthic temperature at Cerro Mundo was 19.5°C during the 2019 cool season experiment (range 15.0 - 22.9°C) and 23.4°C during the 2020 warm season (range 18.0 - 26.6°C, Figure 3). The experimental pizzas remained in place for both experiments, even during storms and rough periods. In addition, the final macroalgal biomass in the open pizzas and our procedural controls in the warm season (Supplementary Figure 2) was not significantly different, suggesting no artifacts of flow or light availability in our exclusion cages. Algae quickly colonized the concrete substrate (Supplementary Figure 3) and within 3-6 weeks the algal community was qualitatively indistinguishable from adjacent natural substrate (Brandt et al. personal obs.). In addition, typical algal succession patterns were evidenced within the cages (Supplementary Figure 4). Forty-seven mobile species were observed at the site in the vicinity of the experiment, nine of which were seen grazing on the pizzas (Supplementary Table 1). Common herbivorous fishes on the open pizzas included blue-chin and bumphead parrotfishes (Scarus ghobban and Scarus perrico, respectively) and razor surgeonfish (Prionurus laticlavius, Supplementary Figure 1B). Pencil sea urchins were also frequently observed grazing on the open pizzas (Figure 2A).

In both the cool and warm season experiments and when all herbivores were allowed access to experimental substrates (open pizzas or gray bars in Figure 4), final algal biomass was relatively low, with mean values ranging from 0.93 to 7.62 g. On the other hand, excluding all macro-herbivores in general produced higher accumulation of final macroalgal biomass with mean values that ranged from 2.85 to 29.56 g (blue bars in Figure 4). The combined effects of nutrients and herbivore exclusions varied however in the cool and the warm season experiments. Excluding macro-herbivores (both fish and all herbivores) during the cool season significantly increased final macroalgal biomass relative to the open pizzas, only with ambient nutrients; there were not significant differences among open pizzas and herbivore exclusions when nutrients were added (Figure 4A). This contrasted with the results of the warm season experiment: final macroalgal biomass significantly increased in herbivore exclusions compared to open pizzas only with nutrient additions, and it reached, ~11X more biomass (Figure 4B). In other words, adding nutrients had contrasting effects in the two experiments: during the cool season experiment the significant differences among herbivore treatments disappeared when nutrients were added (due to a higher macroalgal biomass in open pizzas), while the addition of nutrients during the warm season experiment promoted the significant differences among herbivore treatments (Figure 4).

Overall, our results are in agreement with previous findings that macro-herbivores have a strong top-down effect on macroalgal biomass in the Galápagos, even during intense upwelling when nutrient flux was presumably high (Vinueza et al., 2006; Vinueza et al., 2014; Carr et al., 2018). On the other hand, in our study, excluding only fish or all macro-herbivores had the same effect on final macroalgal biomass during the cool season (the only season during which the fish exclusion treatment was included, Figure 4A). This contrasts with Carr et al. (2018), who found that both sea urchins and other herbivores including fishes, had strong effects on standing macroalgal biomass. Our results indicate that the effect of sea urchins alone was negligible, as final macroalgal biomass in the fish exclusions (i.e., open to sea urchins) was not significantly different than in the full exclusions (Figure 4A). A possible explanation for this is that sea urchins may not have accessed and grazed the fish exclusions for a substantial time, limiting their effects on macroalgal biomass. In fact, we recorded sea urchins in only ~11% of the fish exclusions throughout the duration of the experiment. The discrepancies between our results and those of Carr et al. (2018) could also be due to differences in the local sea urchin species composition during the two studies. In our experiments, the pencil sea urchin (E. galapagensis) was the only sea urchin species present, and our cage design allowed sea urchins to enter/exit at their will. In contrast, Carr et al. (2018) used the green sea urchin Lytechinus semituberculatus (the second most common sea urchin species in the Galápagos, Brandt and Guarderas, 2002) in inclusion cages. It thus seems reasonable that the impact of the green sea urchins might have been stronger (and detectable) in Carr et al. (2018). This agrees with other studies that have found a strong top-down effect of both pencil and green sea urchins in inclusion cages in the Galápagos subtidal (Brandt et al., 2012). In addition, it is known that pencil sea urchins have slower algal grazing rates than green sea urchins (Witman et al., 2017). Thus the absence of the faster-grazing green sea urchins in the present experiment could have reduced the total sea urchin grazing effect.

There was no evidence that nutrient addition affected primary productivity in the full exclusions (no herbivory) during the cool, upwelling season experiment, potentially because enhanced nutrient flux via upwelling alleviated nutrient limitation. This period is also when mean water temperature was the lowest (19.5°C), likely reducing algal productivity and nutrient demand. Although final algal biomass was not significantly different in the cool season experiment across open pizzas (accessible to herbivores) and regardless nutrient additions, there was a positive, non-significant trend towards higher biomass when nutrients were added (Figure 4A). This may hint at reduced herbivory when algal tissue nutrient concentration is high, as predicted for non-oligotrophic conditions (Tomas et al., 2011; Burnell et al., 2013).

Compared to open pizzas, excluding all macro-herbivores had a significant effect on algal biomass both in the cool and in the warm season experiments, but with different nutrient treatments: in the cool season experiment significant differences of final macroalgal biomass among herbivore treatments occurred with ambient nutrients (Figure 4A), while it did with nutrient additions in the warm season (Figure 4B). These differences were maximized in the warm season experiment (~20X versus ~10X more biomass comparing full exclusions and open pizzas among herbivore treatments with significant differences, Figure 4). These results suggest that nutrient addition enhances standing macroalgal biomass in the absence of macro-herbivores only during the warm season (Figure 4B). A finding consistent with the ideas that 1) nutrients were limiting only in the warm season due to the relative weakness of upwelling, and 2) that higher temperatures (23.4°C, average temperature during the warm season) caused greater macroalgal biomass accumulation when nutrients were not limiting (as seen in O’Connor et al., 2009). However, given that algal biomass in the open pizzas with ambient nutrients was not significantly lower in the warm season compared to the cool season experiment (which would hint that nutrients were limiting during the warm season) it is also possible that the cool season nutrient additions were ineffective and that the observed increased algal biomass in the herbivore exclusions in the warm season was a result of changing the design (drilled falcon tubes vs. sheer stockings), the frequency (bimonthly vs. weekly), and the amount (25 vs. 50 g) of nutrient delivery between the cool and warm season experiments, respectively (see Methods). It is still interesting however that the increased algal biomass was restricted to the herbivore exclusions when nutrients were added. This could suggest that top-down control in warmer conditions is strong enough to counter the effects of nutrient availability.

In terms of the influence of temperature, we expected to see a greater top-down control of grazers on macroalgal biomass during the warm season (i.e., lower macroalgal biomass in the warm season and higher in the cool season in the open pizzas), due to temperature-dependence of consumer metabolism (Brown et al., 2004). However, grazing intensity in open pizzas was not significantly different across seasons (Figure 4). Carr and Bruno (2013) found that the grazing of the green sea urchin Lytechinus semituberculatus increased at higher temperatures; their temperature treatments differed by 14°C. On average, our temperature “treatment” (which was dependent of in situ temperature fluctuations) only varied 3.9°C across seasons, which was likely insufficient to produce an increase (or to detect a difference) in macro-herbivores’ metabolism and hence in their grazing rates.

As mentioned before, few studies on the interactive effects of herbivores and nutrients have been performed in tropical or subtropical upwelling regions (Burkepile and Hay, 2006). One example is the one by Cordeiro et al. (2020), which found that in shallow depths (1-2 m) sea urchin herbivory strongly influenced macroalgal cover on marginal coral reefs off southern Brazil. Surprisingly, fishes had no effect on macroalgal cover. However, at greater depths (5-6 m), the dynamics of benthic macroalgae were largely determined by abiotic factors related to localized upwelling. However, the question remains: when nutrient fluxes are greater (as in upwelling regions like the Galápagos), do bottom-up forces overwhelm top-down control? Our results illustrate an approach that could be used to untangle these factors in productive and dynamic systems, while also considering the confounding role of temperature. However, an important caveat is that because several aspects of our experimental design differed between the cool and warm season experiments, the results are not directly comparable. For example, final algal biomass could have been distinct between our experiments due to differences in their duration (18 weeks: cold season experiment, 6 weeks: warm season experiment) and have affected our results. We do not think this is the case, as in general, biomass accumulation in full exclusions was surprisingly very similar between experiments (Figure 4), and more importantly, when analyzing our data normalized by the number of days of each experiment (126 days: cold season experiment, 46 days: warm season experiment), the results yielded the exact same patterns as observed in Figure 4 and Supplementary Figure 5. Having said that, there should be replication during warm and cool seasons so that any observed differences could be attributed to environmental characteristics like temperature and nutrient fluxes. Moreover, the concentration of important nutrients (e.g., Nitrogen and Phosphorus) should be tracked in different treatments (remarkably, there is little information on benthic nutrient concentrations in this system). We are actually implementing these and other changes to the study design and repeating the experiment in replicate warm and cool seasons to begin exploring the role of these environmental factors in mediating primary productivity and consumption patterns.

In conclusion, our results suggest that both top-down and bottom-up forcing influence benthic macroalgal communities in this dynamic upwelling system (Figure 1). Additionally, the response of macroalgae to temperature, nutrients, and herbivory hints at an interactive effect among these three factors. Studying top-down and bottom-up controls in complex, highly variable systems like the Galápagos is important, because direct and indirect interactions among species may be altered by anthropogenic ocean heating and other aspects of global change including altered upwelling dynamics and reduced ocean productivity.

Code and data are available at: github.com/eagudoadriani/PizzaProject_2019-2020.

MB, IS-R and JFB conceived and designed the experiments; MB, IS-R, DFG, EA-A and JFB performed the field experiments; IS-R and DF-G analysed the data; EA-A and CBB ran the statistical analyses and made the graphs; MB and JFB wrote the paper; all authors read, edited, and agreed on the final version of the paper.

The project was funded by the National Science Foundation (grant OCE #1737071 to JFB).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.