Oikos 000: 001–009, 2016 doi: 10.1111/oik.03105 © 2016 Th e Authors. Oikos © 2016 Nordic Society Oikos Subject Editor: Christopher Swan. Editor-in-Chief: Dries Bonte. Accepted 11 February 2016

Herbivores control effects of algal richness on and stability in a laboratory microcosm experiment

Chase Rakowski and Bradley J. Cardinale

C. Rakowski ([email protected]) and B. J. Cardinale, School of Natural Resources and Environment, Univ. of Michigan, 440 Church St, Ann Arbor, MI 48109, USA.

Hundreds of studies that have explored how aff ects the and stability of have produced a consensus that communities composed of more species tend to have higher biomass that is more stable through time. However, the majority of this work stems from studies performed using highly simplifi ed food webs, often composed of just primary producers competing for inorganic resources in the absence of trophic interactions. When studies have incorporated trophic interactions, diversity-function relationships have been more variable, leaving open the question of how biodiversity aff ects the functioning of ecosystems with more trophic levels. Here we report the results of a laboratory experiment that used freshwater microcosms to test for eff ects of algal diversity (one or four species) on community biomass and temporal variability in the presence and absence of two diff erent species (cladocerans dubia and pulex). When no were present, we found the classic pattern observed in hundreds of other studies – as species richness of increased, algal biomass increased, and the temporal variation in biomass decreased. Th is pat- tern was retained when one of the herbivores (C. dubia) was present. exhibited weak and non-selective grazing on the focal algae, leaving the eff ect of diversity on biomass and variability essentially intact. In contrast, D. pulex exhibited strong and selective grazing in algal polycultures that qualitatively altered both diversity– function relationships. As algal richness increased, total algal biomass decreased and variation through time increased. Th ese changes were coupled with larger and less variable populations of D. pulex. Our results show that herbivory leads to a richer array of diversity– function relationships than often observed in studies focused on just one , and suggests trophic interactions should be given more attention in work that seeks to determine how biodiversity impacts the functioning of ecosystems.

In the face of accelerating loss of biodiversity (Murphy calls for researchers to begin considering ‘ vertical ’ diversity and Romanuk 2014, Newbold et al. 2015), an increasing (i.e. diversity of trophic levels) alongside the consideration amount of research has been dedicated to understanding of ‘ horizontal ’ diversity (i.e. diversity within a trophic level) how changes in biodiversity alter the functioning of eco- (Duff y 2002, Duff y et al. 2007). systems. Ecologists have paid particular attention to how While a growing number of researchers are now add- species richness infl uences community biomass and the ing trophic interactions to the design of their studies, there temporal variability of biomass, as productivity and sta- are still relatively few published results that we can draw bility are two metrics that are widely used to describe the inferences from, and the results of these few studies are functioning of ecosystems (Hooper et al. 2005). Although decidedly mixed (Duff y et al. 2005, Th ebault and Loreau a general consensus has emerged that species richness tends 2006, Jiang and Pu 2009). Consider, as one line of evidence, to increase the production and stability of biomass through the synthesis by Cardinale et al. (2011) who summarized time (Hooper et al. 2005, Cardinale et al. 2012), there is results of 13 studies that have manipulated the diversity of increasing recognition that most of our inferences about primary producers and then measured herbivory. Nine of biodiversity stem from studies performed using communi- these showed that herbivory increased, whereas four showed ties that have been greatly over-simplifi ed. Cardinale et al. that herbivory decreased, in more diverse assemblages of pro- (2009) summarized the characteristics of biodiversity experi- ducers. Similarly, other authors have found that increasing ments performed through 2005 and showed that Ͼ 90% of producer diversity is associated with reduced herbivory (see these had manipulated the diversity of seven or fewer species synthesis by Hillebrand and Cardinale 2004), with increased in just one trophic level, mostly primary producers. Th e nar- herbivory (Stein et al. 2010, Loranger et al. 2013), or with row focus on primary producers, coupled with the fact that no change in herbivory (DeMott 1998). trophic interactions are well known to regulate the biomass It is presently unknown why trophic interactions have and variability of producers (Borer et al. 2005, O ’ Gorman had such varying eff ects on diversity– function relationships. and Emmerson 2009, Estes et al. 2011), have prompted Some mathematical models and select empirical evidence

EV-1 suggest that variation in herbivore traits such as body size inhabits lakes and ponds worldwide (USEPA 2002). Adult and feeding selectivity may help explain diff erences among C. dubia are Ͻ 1 mm and their small (Ͻ 35 μ m) gape pre- studies (Duff y 2002, Th ebault and Loreau 2005, Narwani vents them from easily consuming larger algal cells (Burns and Mazumder 2010). For example, herbivores that exhibit 1968). A previous study performed with Ceriodaphnia selectivity in their feeding may encounter their preferred reticulata (Narwani and Mazumder 2010) suggests that food less often or spend more time handling food in a diverse C. dubia might feed at a similar or slower rate in polycul- community, leading to decreased herbivory rates (DeMott tures versus monocultures of algae. Th e com- and Kerfoot 1982, Vos et al. 2001, Kratina et al. 2007). plex is also common and globally distributed (Crease et al. On the other hand, generalist herbivores may benefi t from 2012). Daphnia pulex adults are larger and have a larger gape diverse diets if the species eaten are complementary in the (45 μ m) than C. dubia , allowing them to more easily consume nutrients they provide and/or if diversity tends to dilute larger particles (Burns 1968). In the same study by Narwani toxins (Pfi sterer et al. 2003). Unfortunately, few studies have and Mazumder (2010), D. pulex demonstrated accelerated quantifi ed the eff ects of herbivores on diff erent feeding in polycultures versus monocultures of algae. species in a way that allows us to estimate selectivity, or We acquired both cladocerans from Sachs Systems that can separate eff ects of diversity on the performance of Aquaculture, FL. Upon arrival, we inspected the zoo- diff erent trophic groups. Both of these measurements are plankton cultures and removed all contaminating species. required to understand how trophic interactions alter the We then housed the cladocerans in 1-l borosilicate glass diversity – function relationship for primary producers. bottles containing COMBO growth medium with Here we report the results of a laboratory experiment trace elements, a standard medium for culturing freshwa- performed with freshwater microcosms in which we tested ter plankton (Andersen 2005), which was refreshed every for eff ects of algal diversity (one or four species) on commu- fi ve days. We incubated the bottles under a 16:8 hour nity biomass and temporal variability in the presence versus light:dark cycle and fed the cladocerans a mixture of green absence of two diff erent herbivore species (the cladocerans algae ( Selenastrum capricornutum and Chlorella sorokiniana ). Ceriodaphnia dubia and Daphnia pulex ). Both C. dubia and In order to ensure that none of these algae would contami- D. pulex are abundant and widespread in lakes. We chose to nate the experiment, we rinsed and starved the cladocerans contrast these two herbivores because they are diff erent in prior to inoculation by placing individuals into fresh sterile their feeding habits, with D. pulex being larger and having COMBO medium three times over a period of eight hours. a bigger gape size than C. dubia that allows it to feed on a We examined cultures using microscopy to check for con- wider range of algae. With so little prior work on diversity – tamination and verify no unwanted species were present. function relationships in multi-trophic systems, it is diffi cult to make a priori hypotheses that are founded on solid logic Focal algae or prior ecological knowledge. Nevertheless, mathemati- cal models of multi-trophic systems (Th ebault and Loreau We used fi ve species of green algae (division Chlorophyta) 2005) tend to make three qualitative predictions that we for the experiment: Chlorella sorokiniana , Scenedesmus acum- considered when designing our study: 1) in the absence of inatus , Pediastrum duplex , Monoraphidium minutum and herbivory, increasing species richness of primary producers Monoraphidium arcuatum . Th ese genera are found in 9 to tends to increase and stabilize producer biomass; 2) when 53% of North American freshwater lakes and range from producer richness has non-signifi cant or negative eff ects on the 67th to the 5th most commonly sampled taxa out of 262 herbivory, then relationships between producer richness, genera identifi ed during the US Environmental Protection producer biomass and stability are not qualitatively altered; Agency’ s 2007 National Lakes Assessment (USEPA 2007). however, 3) when producer richness leads to increased rates All of these species are suffi ciently small to be potentially of herbivory, producer richness leads to reductions in pro- edible by both herbivores, but they span a wide range of sizes ducer biomass, and more variable biomass through time. As (74.6 to 7250 μ m3 per cell), morphologies, and tendencies we will show, the results of our experiment were largely con- to clump or form colonies, which we presumed would lead sistent with these predictions, and we were able to show how to varying degrees of edibility by the herbivores (Table 1). selective feeding by one of the herbivores (D. pulex ) leads to All of the focal algal species were obtained from either the a fundamental shift in the diversity – function relationship in Univ. of Texas Culture Collection (UTEX) or the Culture a two trophic-level system. Collection of Algae at Goettingen University (SAG).

Experimental units and design Material and methods We grew experimental communities in 1-l borosilicate glass Herbivores bottles fi lled with 750-ml COMBO medium with animal trace elements (Andersen 2005). Screw caps on the bottles We used cladocerans of the family (Crustacea: were fi tted with ports that were attached to rubber tubes, ) as the focal herbivores for this study. Cladocer- which allowed sterile media exchanges to be performed with- ans are ubiquitous freshwater fi lter feeding zooplankton that out removing the caps. We stored the bottles horizontally on form an important link between phytoplank- a stationary roller rack in an environmental walk-in chamber ton and fi sh (Carpenter et al. 1987). As cyclical partheno- maintained at 20 Ϯ 0.5 °C, with coolwhite fl uorescent lights gens with short generation times (3 – 10 days), cladocerans set to 16:8 hour light:dark cycles 6-cm from each row of are able to quickly exploit resources. Ceriodaphnia dubia bottles.

EV-2 Table 1. The morphological characteristics of focal algal species used in this experiment. UTEX ϭ Univ. of Texas Culture Collection; SAG ϭ The Culture Collection of Algae at Goettingen University. Biovolumes and diameters were measured on Ͼ 70 cells with a benchtop FlowCam using the area by diameter method (ABD).

Cell biovolume Cell diameter Genus and species Source Shape ( μ m3 ) ( μ m) Clumping/colonies Chlorella sorokiniana UTEX sphere 74.6 5.11 solitary Monoraphidium minutum UTEX compact prolate spheroid 113.42 5.97 solitary Scenedesmus acuminatus SAG cylinder ϩ two cones 400.13 8.97 solitary, rarely colonies of 2– 6 cells Monoraphidium arcuatum SAG elongate prolate spheroid 172.62 6.62 solitary with some large clumps Pediastrum duplex UTEX rectangular prism 7250 15.6 colonies of 8– 32 cells, rarely solitary

We manipulated algal as well as the the area by diameter method (ABD). To estimate algal com- presence/absence of each herbivore species. We grew each munity biomass, we passed 50-ml of each sample through of the fi ve algal species in monoculture, as well as all pos- a glass fi ber fi lter with 2.5-μ m pores. We placed each fi lter sible combinations of four species (fi ve polyculture composi- in a capped test tube containing 10-ml anhydrous EtOH, tions). Th is design ensures that the higher level of diversity covered it with aluminum foil and stored it in a freezer. After has multiple species compositions, which some researchers nine days, we analyzed the samples for fl uorescence of chlo- have advocated as a less biased way to allow for composi- rophyll-a as a proxy for algal community biomass following tional eff ects (Schmid et al. 2002). Th e algal composition the method of Nusch (1980). We estimated the herbivore treatments were crossed with three herbivore treatments: an population of each bottle by looking through the side of the herbivore-free control plus each of the two herbivore species bottle, beginning on day 10 and continuing every six days inoculated separately. We replicated all ten algal composi- until day 28 to yield four population estimates per bottle. tions fi ve times within all three herbivore treatments, for a We recorded the exact if there were Յ 5 indi- total of 150 experimental units. viduals, and estimated to the nearest multiple of 5 if there were Ͼ 5 . Procedure Zooplankton populations went extinct in nine experi- mental units, which we excluded from the analyses such that After autoclave sterilizing the media in the capped bottles, we only compared those units with a consistent presence or we inoculated each bottle with algae according to a substi- absence of herbivory. Inoculating the two herbivore popula- tutive design: either 400 000 cells (533 cells ml – 1) of one tions at diff ering densities (but equal biomass) did not seem algal species to create a monoculture, or 100 000 cells of to infl uence the probability of extinction since C. dubia rep- each of four algal species to create polycultures with the same resented the majority of extinctions despite being inoculated total cell count. We allowed the algae to grow for 12 days at a higher population size than D. pulex . We also excluded prior to introducing any herbivores to increase the probabil- one microcosm from analyses that was a statistical outlier ity the algae would attain a biomass large enough to sustain with orders of magnitude lower algal biomass than others. herbivore populations. After this growth period, we began Additionally, two bottles were lost due to breakage. Th ere- performing 8% (60 ml) exchanges of the COMBO media fore, a total of 138 experimental units remained in our fi nal every two days using 60 ml syringes. Volumes extracted for analyses, with at least three replicates per treatment-species the fi rst exchange were used to establish a baseline of algal combination. biomass and population densities (methods described next) prior to addition of herbivores. Following this, we added 25 adult C. dubia or 15 adult D. pulex to the appropriate micro- Data analysis cosms. Th e 25:15 ratio of was used to produce approximately equal biomasses of zooplankton in the two We used linear mixed eff ects models to test for eff ects of algal herbivore treatments assuming linear body length– biomass diversity on mean algal biomass (measured as fl uorescence of scaling relationships (Gonzalez et al. 2008) with D. pulex chlorophyll-a) and mean herbivore density. Response vari- averaging 1.7 ϫ larger (adult carapace length of 1.5-mm ables were natural log-transformed to improve normality. We for D. pulex versus 0.9-mm for C. dubia; Ranta et al. 1993, initially analyzed each response variable using the model Pereira et al. 2004). Every third extraction after the addition ϭβ ϩ β ϩ β ϩ β ϩ β ϫ y 0 1 S 2 H 3 t 4 S H of herbivores was used to quantify algal biomass and biovol- ϩ β S ϫ t ϩ β H ϫ t ϩ β S ϫ H ϫ t ϩ ε (1) ume as described next (every six days to day 24, for a total of 5 6 7 fi ve samples). Algae were re-suspended in the bottles daily by where y is mean algal biomass or herbivore density, S is algal swirling each bottle by hand for ten seconds. diversity (1 for polycultures, 0 for monocultures), H is the Upon extracting samples during media exchange, we herbivore treatment (control, C. dubia, or D. pulex ), t is time preserved 3-ml of each sample in formalin for estimation (day after start of the experiment), and ε indicates residual of the biovolume of each algal species. We counted up to error. Because the three-way interaction was signifi cant for 400 cells of each species per sample, or 1.8-μ l of medium, both response variables, indicating that time trajectories var- whichever came fi rst. We did not observe any contamina- ied between treatments, each herbivore treatment was then tion of unwanted species. We then multiplied the resulting analyzed separately as: cell densities by the average cell biovolume for each species ϭβ ϩ β ϩ β ϩ β ϫ ϩ ε as measured on Ͼ 70 cells with a benchtop FlowCam using y 0 1 S 2 t 3 S t (2)

EV-3 We measured the variability of algal biomass (chlorophyll-a) Results and of herbivore density for each microcosm as the coeffi cient of variation (CV), which is the ratio of the standard deviation Our results show that algal species richness infl uenced both to the mean. We then used two-way ANOVA to compare the the mean and variability of algal biomass, but it did so in CV of algal biomass (chlorophyll-a) and of herbivore density qualitatively diff erent ways depending on whether an her- in monocultures versus polycultures. We also compared the bivore was present, and which one was present. Analysis standard deviation of algal biomass and of herbivore density of chlorophyll-a (a proxy of algal biomass) in the control to explore how diversity aff ected variability. If there was a treatment (no herbivores) showed a signifi cant treatment ϫ signifi cant diversity by herbivore treatment interaction, we time interaction (Table 2a), with polycultures having greater used t-tests to analyze each herbivore treatment separately. biomass than monocultures early in the time-series but both In order to quantify the amount of herbivory in each treatments converging by later dates (Fig. 1a). Despite dif- experimental unit that contained herbivores, we calculated fering time trends, mean levels of algal biomass in the no- an ‘ herbivory index’ as the ratio of total algal biovolume in herbivore controls were generally higher in algal poly- versus the presence of an herbivore relative to algal biovolumes in monocultures (Fig. 1a, Table 2a). In addition, the tempo- the herbivore-free controls. Using biovolumes for the her- ral variability of algal biomass was lower in poly- compared bivory index provides the species-specifi c information needed to monocultures (Fig. 2a, t ϭ 2.05, p ϭ 0.05). Th e reduced to estimate herbivory on individual algal species when grown coeffi cient of variation was entirely due to a reduction in alone, or the summed biovolumes of all species when grown the mean (denominator), as the standard deviation (numera- together in polyculture. Algal biovolume was highly corre- tor) did not diff er between algal mono- versus polycultures lated with estimates of chlorophyll-a (Pearson ’ s correlation (t ϭ 0.64, p ϭ 0.52). coeffi cient ϭ 0.62), especially for S. acuminatus (Pearson’ s In experimental units containing the herbivore Ceri- correlation coeffi cient ϭ 0.80), which was responsible for odaphnia dubia, we found no changes in how algal species rich- most of the results we will present showing selective her- ness aff ected algal biomass through time (see algal diversity ϫ bivory. Th erefore, we believe interpretation of the herbivory time, Table 2b). Algal biomass declined through time equally index calculated from algal biovolumes gives insight into for both levels of algal richness, but biomass was consistently trends that were based on measurement of chlorophyll-a. We higher in the algal polycultures compared to monocultures estimated 95% confi dence intervals for the herbivory indi- (Fig. 1b, Table 2b). Similar to the no-herbivore controls, the ces by bootstrapping with 5000 iterations. All statistical tests variation of algal biomass through time was lower in algal were performed using R ver. 3.1.1 (< www.r-project.org > ). polycultures compared to monocultures (Fig. 2b, t ϭ 2.6, p ϭ 0.02), which was again due to diff erences in mean biomass ϭ Data deposition rather than any diff erences in the standard deviations (t 1.46, p ϭ 0.16). Th us, cultures containing the herbivore C. dubia Data available from the Dryad Digital Repository: < http:// were similar to controls in that algal biomass was greater, and dx.doi.org/10.5061/dryad.j1617 > (Rakowski et al. 2016). variability lower, in cultures that had more algal species.

Table 2. Results of linear mixed effects models testing for effects of algal diversity and time on (a) algal biomass in no-herbivore controls, (b) algal biomass in cultures containing the herbivore C. dubia , (c) algal biomass in cultures containing the herbivore D. pulex , (d) the density of C. dubia , and (e) the density of D. pulex . (a), (c) and (d) were modeled with Eq. 1 in the text, and (b) and (e) were modeled with Eq. 2.

Parameter Estimate SE DF t value pr(Ͼ |t|) (a) Algal biomass (ln-transformed), no herbivores Intercept 8.322 0.075 192 110.824 Ͻ 0.001 Algal diversity 0.405 0.105 47 3.860 Ͻ 0.001 Time 0.038 0.004 192 9.787 Ͻ 0.001 Algal diversity ϫ Time – 0.011 0.005 192 – 1.967 0.051 ( b) Algal biomass (ln-transformed), C. dubia Intercept 8.592 0.109 159 79.024 Ͻ 0.001 Algal diversity 0.265 0.127 39 2.081 0.044 Time – 0.004 0.004 159 – 0.909 0.365 (c) Algal biomass (ln-transformed), D. pulex Intercept 8.462 0.132 189 64.178 Ͻ 0.001 Algal diversity 0.161 0.186 46 0.866 0.391 Time – 0.014 0.007 189 – 1.900 0.059 Algal diversity ϫ Time – 0.074 0.010 189 – 7.357 Ͻ 0.001 ( d) C. dubia density (ln-transformed) Intercept 2.882 0.238 121 12.128 Ͻ 0.001 Algal diversity – 0.505 0.317 39 – 1.592 0.120 Time 0.027 0.010 121 2.592 0.011 Algal diversity ϫ Time 0.040 0.014 121 2.916 0.004 (e) D. pulex density (ln-transformed) Intercept 2.095 0.126 143 16.621 Ͻ 0.001 Algal diversity 0.786 0.130 46 6.059 Ͻ 0.001 Time 0.039 0.005 143 8.654 Ͻ 0.001

EV-4 (a)Control (b)C. dubia (c) D. pulex

Monoculture Polyculture

9 a (ln RFU) 8 Chlorophyll 7

Mean 0 6 12 18 24 Mean 0 6 12 18 24 Mean 0 6 12 18 24 Day Day Day

Figure 1. Fluorescence of chlorophyll-a, a proxy for algal biomass, in algal mono- and polycultures with (a) no herbivores (controls), (b) Ceriodaphnia dubia , and (c) Daphnia pulex . Th e left side of each panel shows mean chlorophyll-a fl uorescence across all time points (RFU stands for ‘ relative fl uorescence units ’ ), whereas the right side of each panel shows time trends. Data points are the means Ϯ 1 SEM of replicate cultures.

Th e relationships between algal diversity, algal biomass was the same irrespective of algal richness (see Total in and variability were fundamentally diff erent in experimen- Fig. 3a; error bars represent 95% confi dence intervals). tal units containing the herbivore Daphnia pulex . After Ceriodaphnia dubia appeared to consume all of the algal D. pulex was added to the experimental bottles, the bio- species with possible exception of P. duplex (note 95% mass of algae quickly declined, with declines being far confi dence intervals in Fig. 3a overlap zero). Ceriodaphnia more substantial for algal polycultures than monocultures dubia also appeared to graze nearly all species equally regard- (Fig. 1c, Table 2c, note algal diversity ϫ time interaction). In less of whether they were grown alone or in polyculture addition, algal polycultures exhibited signifi cantly higher (note 95% CIs overlap algal richness treatments). Th e one variability of biomass through time than did algal mon- exception was Monoraphidium minutum, which C. dubia ocultures (Fig. 2c, t ϭ – 7.24, p Ͻ 0.01). As with the other grazed more when in a polyculture. However, M. minutum herbivore treatments (control, C. dubia ), diff erences in the was a rare species that only contributed a small amount coeffi cients of variation were driven solely by diff erences in of biovolume to the polycultures (Fig. 3c), which is why mean biomass (denominator) and not changes to the stan- a greater grazing intensity on this species did not cause a dard deviations (t ϭ – 1.11, p ϭ 0.27). Th us, in the pres- change in total algal biovolume among treatments of algal ence of D. pulex, algal species richness was associated with richness. lower (not higher) biomass of algae, and higher (not lower) In contrast, grazing by D. pulex was more than twice as variation in algal biomass through time. intense in polycultures as grazing in monocultures. Algal Th e contrasting eff ects of algal richness in the two her- biovolumes in monocultures were suppressed to 59% of the bivore treatments (C. dubia and D. pulex) can potentially biovolume achieved in the controls, whereas biovolumes in be explained by how algal richness diff erentially infl uenced polycultures were suppressed to a much lower 26% (Fig. 3b). grazing by the herbivores. Net grazing intensity by C. dubia Like C. dubia , D. pulex grazed M. minutum more heavily in polyculture than in monoculture. However, unlike C. dubia , D. pulex also intensifi ed its grazing on Scenedesmus acumi- (a) (b)C. dubia (c) D. pulex Control natus in polyculture, suppressing this species to just 6% of 1.00 the biomass achieved in no-herbivore controls. Th is result

a is noteworthy given that S. acuminatus was the dominant 0.75 species in control polycultures, composing 67% of the mean algal biovolume (Fig. 3c). Th erefore, the increase in her- 0.50 bivory by D. pulex in algal polycultures was largely the result of intensifi cation of grazing on the dominant algal species, S. acuminatus . 0.25

CV of Chlorophyll Algal richness also diff erentially infl uenced the popula- tion density and temporal variability of the two herbivore 0.00 species. Th e linear mixed eff ects model showed that C. dubia Mono Poly Mono Poly Mono Poly population densities increased through time more rapidly in algal polycultures compared to monocultures (Fig. 4a, Figure 2. Coeffi cients of variation (CV) for chlorophyll-a fl uores- ϫ cence through time in algal mono- and polycultures with (a) no Table 2d, note algal diversity time interaction). However, herbivores (controls), (b) Ceriodaphnia dubia , and (c) Daphnia D. pulex populations in monocultures, after undergoing pulex . Bars are the means Ϯ 1 SEM of all replicate cultures. what appeared to be an initial die-off , were consistently lower

EV-5 (a) C. dubia Th e latter was driven by the higher population densities in algal polycultures, as the standard deviation of D. pulex 2.5 Monoculture Polyculture population size through time was not diff erent among algal treatments (t ϭ 0.05, p ϭ 0.96). 2.0 control 1.5 Discussion / BV Here we have presented the results of a laboratory experi- 1.0 herbivore ment showing that the species richness of primary produc-

BV ers can have qualitatively diff erent eff ects on community 0.5 biomass and stability depending on how herbivores alter their consumption in response to changing producer rich- ness. When no herbivores were present, we found the 0.0 Total Ped Chl arc min Sce classic pattern observed in hundreds of other studies – as Algal species species richness of algae was experimentally increased, algal biomass increased, and the temporal variation in biomass (b) D. pulex decreased. Th is classic pattern was retained when one of the 2.5 Monoculture herbivores (Ceriodaphnia dubia) was present. Ceriodaphnia Polyculture dubia exhibited relatively weak and non-selective graz- ing on the focal algal species, which led to no qualitative 2.0 changes in the way diversity aff ected biomass or variation

control in the system. In contrast, Daphnia pulex exhibited strong 1.5 and selective grazing that, in turn, qualitatively altered / BV the diversity – function relationship. As algal richness was increased, total algal biomass decreased and variation 1.0 herbivore through time increased. Th ese changes were coupled with BV larger and less variable populations of D. pulex . Clearly, the 0.5 eff ects of algal diversity on both algal biomass and the vari- ability of both trophic levels was a function of the trophic 0.0 interaction with herbivores; yet, the two herbivores had Total Ped Chl arc min Sce diff ering eff ects. Algal species Ours is one of relatively few studies that have explicitly (c) incorporated trophic interactions into experiments looking at how biodiversity aff ects the functioning of ecosystems. 60% Th e overly simplistic nature of biodiversity experiments to date has led several authors to call for the inclusion of more

control poly ‘ vertical ’ interactions (i.e. interactions among trophic levels) 40% in studies that have historically focused solely on ‘ horizon- tal ’ interactions (i.e. interactions within a trophic level) (Ives 20% et al. 2005, Th ebault and Loreau 2006, Duff y et al. 2007, Cardinale et al. 2009). Of the few experiments that have

Proportion of BV 0% included vertical interactions between producers and herbi- Ped Chl arc min Sce vores, results have been mixed, with little consistency in how Algal species producer diversity alters the strength of herbivory (Cardinale et al. 2011). Our study is potentially useful because it points Figure 3. Grazing intensities, quantifi ed as the ratio of algal biovol- to certain characteristics of herbivores that might control umes in treatments with (BV ) versus without herbivores herbivore variation in the relationship between producer diversity (BVcontrol ), for (a) Ceriodaphnia dubia, and (b) Daphnia pulex . Data points are the means Ϯ 95% confi dence intervals of all replicates. and herbivory and, in turn, aff ect relationships between (c) Th e percentage of algal biovolume represented by each species producer diversity and both biomass and stability. Th e key in polycultures at the end of the experiment. Data are only factors responsible for contrasting results among herbivores shown for the no-herbivore controls. Th e x-axis labels are as in this study were the feeding rate and degree of selective follows: Total ϭ sum across all species; Ped ϭ Pediastrum duplex ; feeding by herbivores when exposed to algal mono- versus Chl ϭ Chlorella sorokiniana ; arc ϭ Monoraphidium arcuatum ; ϭ ϭ polycultures. Th e herbivore C. dubia consumed the same min Monoraphidium minutum ; Sce Scenedesmus acuminatus . total amount of algae regardless of whether it was exposed to one or four algal species, and it did not change its feeding than populations in polycultures (Fig. 4b, Table 2e). While selectivity when algae were grown in polyculture. Because the temporal variability of C. dubia populations was no dif- algal diversity did little to change the herbivore-producer ferent between algal diversity treatments (Fig. 5a, t ϭ 0.72, trophic interaction, the qualitative eff ects of algal diversity p ϭ 0.48), the temporal variability of D. pulex populations on biomass and variability were very similar to controls was lower in algal polycultures (Fig. 5b, t ϭ 3.93, p Ͻ 0.01). where no herbivores were present. Perhaps as a result, we

EV-6 (a)C. dubia (b) D. pulex

Monoculture Polyculture 4.0

3.5

3.0 ln(Herbivores/L)

2.5

Mean 10 16 22 28 Mean 10 16 22 28 Day Day

Figure 4. Herbivore densities in algal mono- and polycultures for (a) Ceriodaphnia dubia , and (b) Daphnia pulex . Th e left side of each panel shows mean density across all time points, whereas the right side of each panel shows time trends. Data points are the means Ϯ 1 SEM of replicate cultures. also found scant evidence that algal diversity infl uenced the communities, and these deserve attention in future work. population density or variability of C. dubia . In contrast, Th e “ balanced diet ” hypothesis proposes that diverse plant when D. pulex was exposed to greater algal richness, the her- diets allow herbivore populations to proliferate if diff erent bivore not only consumed a greater total amount of algae, plants off er complimentary nutritional needs for herbivores but also shifted its feeding habit and selectively increased (e.g. diff erent nutrients), or if plant diversity helps dilute its consumption of the dominant algal species, Scenedesmus potential toxins in herbivore diets (DeMott 1998, Pfi sterer acuminatus. As a result, the biomass of algae grown in poly- et al. 2003). Th e “ diet quality” hypothesis proposes that culture was substantially reduced by herbivores compared monocultures (at least, some of them) tend to be a lower to algal monocultures, which led to a reversal of algal diver- quality resource for herbivores than polycultures, and that sity-biomass relationships. Th e increased herbivory on, and herbivores compensate by passing lower quality food through reduced biomass of algal polycultures also led to a reversal their guts more slowly to maximize nutrient uptake (Sterner of the diversity– stability relationship, and likely caused the 1993). Th e “ diet quality” hypothesis might explain why per D. pulex population to be larger and less variable in the high capita feeding rates of D. pulex declined in monocultures of algal richness treatment. algae compared to polycultures in an experiment by Narwani We do not know why D. pulex increased its total con- and Mazumder (2010). We do not have the data required to sumption, or why it fed more selectively on certain algal test any of these hypotheses, nor to move beyond speculation species when those species were part of an algal polyculture. about why D. pulex increased its feeding rate and selectiv- However, several hypotheses have been proposed to explain ity in algal polycultures. It is, however, worth noting that why herbivores can alter feeding rates in diverse producer our results are consistent with Th ebault and Loreau ’ s (2005) model of how producer diversity impacts the functioning of two trophic-level systems, which predicts that herbivore (a)C. dubia (b) D. pulex feeding rates and selectivity dictate how producer diversity impacts biomass and variability in much the same way as we observed. 0.6 While our work suggests that trophic interactions may shift both the qualitative and quantitative nature of diver- 0.4 sity – function relationships, understanding these shifts may be particularly important for understanding the functional role of biodiversity in aquatic ecosystems. Herbivores con- 0.2 sume on average 50% or more of in CV of herbivores/L aquatic systems, which is signifi cantly more than in terres- trial systems (Cyr and Pace 1993, Cebrian 1999). In spite 0.0 of this, only a few aquatic studies have explored how pro- Mono Poly Mono Poly ducer diversity aff ects community biomass and variability in Figure 5. Coeffi cients of variation (CV) for herbivore densities the presence of higher trophic levels. One of these studies through time in algal mono- and polycultures for (a) Ceriodaphnia (Narwani and Mazumder 2012), which was similar to ours, dubia , and (b) Daphnia pulex . Bars are the means Ϯ 1 SEM of all produced contrasting results showing that algal diversity replicate cultures. tends to increase and stabilize algal biomass in the presence

EV-7 of both C. dubia and D. pulex . Th at experiment used diff er- respond by decreasing their consumption, and which do not ent species of algae than we used in this study, and included alter consumption at all. With this information in hand, we algal species that are generally less edible by D. pulex than may begin to see generalities among diff erent types of her- the species used here. Reduced edibility may have prevented bivores or food webs, and we may amass the information D. pulex from increasing its consumption rate in polycul- needed to develop models that more realistically predict the tures as occurred in our study; but Narwani and Mazumder impacts of changing biodiversity on ecosystems. (2012) did not include no-herbivore controls in their study that would allow us to estimate changes in herbivory. Another laboratory experiment similarly showed that algal diversity Acknowledgements – Th is study was supported by grant no. 1332342 increased and stabilized algal biomass in the face of her- from the US National Science Foundation’ s Emerging Frontiers in bivory by rotifers (Corcoran and Boeing 2012). Th e rotifers Research and Innovation (EFRI) to BJC. We thank M. Nolan, A. in that study apparently consumed all but one of the algal Lashaway, M. Busch, B. Gregory, N. Huntley, C. Zhou, H. species, but seemed to reduce their grazing in polycultures Hedman, F. H. Chang, J. Herrin, C. Steiner, L. Weider and M. Duff y for assistance with the experiment. We thank F. H. Chang despite the edibility of the polycultures. Again, that experi- and the Center for Statistical Consultation and Research at the ment did not include no-herbivore controls, preventing one Univ. of Michigan for assistance with statistical analysis. Th anks from estimating grazing intensities or selectivity. Clearly, also to M. Duff y, J. D. Allan, A. Narwani, M. Nolan and J. Morris more work is needed to understand how algal diversity and for comments on earlier versions of the manuscript. herbivory interact to aff ect the biomass and variability of aquatic communities. As the results of our study are interpreted, it is impor- References tant to keep several caveats in mind. First, our study was performed in an overly simplifi ed laboratory environment Andersen, R. A. 2005. Algal culturing techniques. – Elsevier/ that was in no way meant to represent the complexity of Academic Press. real lakes. Herbivore assemblages in lakes rarely consist of Borer, E. T. et al. 2005. What determines the strength of a ? – 86: 528 – 537. a single species. Considering interactions among function- Burns, C. 1968. Relationship between body size of fi lter-feeding ally diverse suites of grazers would allow a more realistic Cladocera and maximum size of particle ingested. – Limnol. view of herbivory in nature, especially since it seems that Oceanogr. 13: 675 – 678. diff erent herbivores tend to respond diff erently to producer Cardinale, B. J. et al. 2009. Towards a food-web perspective on diversity (Narwani and Mazumder 2010). Experiments have biodiversity and functioning. – In: Naeem, S. et al. also demonstrated that more diverse assemblages (eds), Biodiversity and human impacts. Oxford Univ. Press, cause stronger top– down control (Griffi n et al. 2013), but pp. 105 – 120. such trophic interactions could be dampened by trophic Cardinale, B. J. et al. 2011. Th e functional role of producer diversity in ecosystems. – Am. J. Bot. 98: 572 – 592. cascades in food-webs that have higher predators. Th erefore, Cardinale, B. J. et al. 2012. and its impact on more work is needed to explore diversity– function relation- humanity. – Nature 486: 59 – 67. ships in more realistic food webs. It should also be noted Carpenter, S. R. et al. 1987. Regulation of lake primary productiv- that the microcosms used for our study were a relatively ity by structure. – Ecology 68: 1863 – 1876. static environment compared to natural lakes where many Cebrian, J. 1999. Patterns in the fate of production in plant abiotic factors that infl uence plankton fl uctuate over time communities. – Am. Nat. 154: 449 – 468. scales ranging from hours to decades (Levin 1992). One Corcoran, A. A. and Boeing, W. J. 2012. Biodiversity increases the of the most important mechanisms by which biodiversity productivity and stability of phytoplankton communities. – PloS ONE 7: e49397. aff ects community variability is via asynchronous responses Crease, T. J. et al. 2012. Transcontinental phylogeography of the of diff erent species to environmental fl uctuations (Gonzalez Daphnia pulex species complex. – PloS ONE 7: e46620. and Loreau 2009, Loreau and de Mazancourt 2013), and the Cyr, H. and Pace, M. 1993. Magnitude and patterns of herbivory static environment used in our study may not have allowed in aquatic and terrestrial ecosystems. – Nature 361: 148 – 150. this mechanism to be fully expressed. DeMott, W. R. 1998. Utilization of a cyanobacterium and a phos- Despite being an unrealistic portrayal of nature, the phorus-defi cient green alga as complementary resources by results of our study are important because they suggest a daphnids. – Ecology 79: 2463 – 2481. richer variety of diversity– function relationships is possible DeMott, W. R. and Kerfoot, W. 1982. among cladocerans: nature of the interaction between Bosmina and when we begin to consider trophic interactions between pro- Daphnia . – Ecology 63: 1949 – 1966. ducers and herbivores. To date, the potential for diff erent D u ff y, J. E. 2002. Biodiversity and ecosystem function: the con- herbivores to be associated with qualitatively diff erent sumer connection. – Oikos 99: 201 – 219. diversity – function relationships has only been suggested by D u ff y, J. E. et al. 2005. Ecosystem consequences of diversity theory (Th ebault and Loreau 2005, 2006) and a handful of depend on food chain length in estuarine vegetation. – Ecol. confl icting biodiversity experiments (Fox 2004, Gamfeldt Lett. 8: 301 – 309. et al. 2005). Our experiment suggests that to understand D u ff y, J. E. et al. 2007. Th e functional role of biodiversity in the relationship between biodiversity and ecosystem func- ecosystems: incorporating trophic complexity. – Ecol. Lett. 10: 522 – 538. tioning, ecologists will need to study the eff ects of diversity Estes, J. A. et al. 2011. Trophic downgrading of planet Earth. not only on production at lower trophic levels, but also on – Science 333: 301 – 306. energy transfers between trophic levels. We need to under- Fox, J. W. 2004. Eff ects of algal and herbivore diversity on the stand which herbivores respond to decreasing (or increasing) partitioning of biomass within and among trophic levels. producer diversity by increasing their consumption, which – Ecology 85: 549 – 559.

EV-8 Gamfeldt, L. et al. 2005. Species richness changes across two Newbold, T. et al. 2015. Global eff ects of land use on local terrestrial trophic levels simultaneously aff ect prey and consumer biomass. biodiversity. – Nature 520: 45 – 50. – Ecol. Lett. 8: 696 – 703. Nusch, E. A. 1980. Comparison of diff erent methods for chorophyll Gonzalez, A. and Loreau, M. 2009. Th e causes and consequences and phaepigment determination. – Arch. Hydrobiol. 14: 14 – 36. of compensatory dynamics in ecological communities. – Annu. O’Gorman, E. J. and Emmerson, M. C. 2009. Perturbations to Rev. Ecol. Evol. Syst. 40: 393 – 414. trophic interactions and the stability of complex food webs. Gonzalez, E. J. et al. 2008. Size and dry weight of main zooplankton – Proc. Natl Acad. Sci. USA 106: 13393 – 13398. species in Bariri reservoir (SP, Brazil). – Braz. J. Biol. 68: Pereira, J. L. et al. 2004. Allometric relations for Ceriodaphnia spp. 69 – 75. and Daphnia spp. – Ann. Limnol – Int. J. Lim. 40: 11 – 14. Griffi n, J. N. et al. 2013. Eff ects of predator richness on prey P fi sterer, A. B. et al. 2003. Dietary shift and lowered biomass gain suppression: a meta-analysis. – Ecology 94: 2180 – 2187. of a generalist herbivore in species-poor experimental plant Hillebrand, H. and Cardinale, B. J. 2004. Consumer eff ects decline communities. – Oecologia 135: 234 – 241. with prey diversity. – Ecol. Lett. 7: 192 – 201. Rakowski, C. et al. 2016. Data from: Herbivores control eff ects of Hooper, D. U. et al. 2005. Eff ects of biodiversity on ecosystem algal species richness on community biomass and stability in functioning: a consensus of current knowledge. – Ecol. a laboratory microcosm experiment. – Dryad Digital Reposi- Monogr. 75: 3 – 35. tory, < http://dx.doi.org/10.5061/dryad.j1617 > . Ives, A. R. et al. 2005. A synthesis of subdisciplines: predator– prey Ranta, E. et al. 1993. Growth, size and shape of , interactions, and biodiversity and ecosystem functioning. D. magna and D. pulex . – Ann. Zool. Fenn. 30: 299 – 311. – Ecol. Lett. 8: 102 – 116. Schmid, B. et al. 2002. Th e design and analysis of biodiversity Jiang, L. and Pu, Z. 2009. Diff erent eff ects of species diversity on experiments. – In: Loreau, M. et al. (eds), Biodiversity and temporal stability in single-trophic and multitrophic ecosystem functioning: synthesis and perspectives. Oxford communities. – Am. Nat. 174: 651 – 659. Univ. Press, pp. 14 – 29. Kratina, P. et al. 2007. Species diversity modulates . Stein, C. et al. 2010. Impact of invertebrate herbivory in grasslands – Ecology 88: 1917 – 1923. depends on plant species diversity. – Ecology 91: 1639– 1650. Levin, S. A. 1992. Th e problem of pattern and scale in ecology. Sterner, R. 1993. Phytoplankton nutrient limitation and food – Ecology 73: 1943 – 1967. quality for Daphnia . – Limnol. Oceanogr. 38: 857 – 871. Loranger, H. et al. 2013. Predicting invertebrate herbivory from Th ebault, E. and Loreau, M. 2005. Trophic interactions and the plant traits: polycultures show strong nonadditive eff ects. relationship between species diversity and ecosystem stability. – Ecology 94: 1499 – 1509. – Am. Nat. 166: 95 – 114. Loreau, M. and de Mazancourt, C. 2013. Biodiversity and ecosys- Th ebault, E. and Loreau, M. 2006. Th e relationship between tem stability: a synthesis of underlying mechanisms. – Ecol. biodiversity and ecosystem functioning in food webs. – Ecol. Lett. 16 Suppl. s1: 106 – 115. Res. 21: 17 – 25. Murphy, G. E. P. and Romanuk, T. N. 2014. A meta-analysis of USEPA 2002. Appendix A of Methods for measuring the acute declines in local species richness from human disturbances. toxicity of effl uents and receiving waters to freshwater and – Ecol. Evol. 4: 91 – 103. marine organisms, 5th edn. – Offi ce of Water, US Environ. Narwani, A. and Mazumder, A. 2010. Community composition Protection Agency, Washington, DC EPA-821-R-02-012. and consumer identity determine the eff ect of resource USEPA 2007. National Lakes Assessment Data. – U S Environ. species diversity on rates of consumption. – Ecology 91: Protection Agency, accessed July 2013, < http://water.epa.gov/ 3441 – 3447. type/lakes/NLA_data.cfm > . Narwani, A. and Mazumder, A. 2012. Bottom– up eff ects of species Vos, M. et al. 2001. Plant-mediated indirect eff ects and the diversity on the functioning and stability of food webs. – J. persistence of parasitoid– herbivore communities. – Ecol. Lett. Anim. Ecol. 81: 701 – 713. 4: 38 – 45.

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