Grazing minnows increase benthic autotrophy and enhance the response of periphyton elemental composition to experimental phosphorus additions

Grazing minnows increase benthic autotrophy and enhance theresponse of periphyton elemental composition to experimental

phosphorus additions

Jason M. Taylor1, Jeffrey A. Back2, AND Ryan S. King3

Center for Reservoir and Aquatic Systems Research, Department of Biology, Baylor University, Waco,Texas 76798 USA

Abstract. Excessive nutrient inputs and grazers can influence biomass and elemental composition ofprimary producers in freshwater ecosystems. How interactions between nutrient enrichment and grazingfish alter benthic habitats through effects on periphyton autotrophy, biomass, and elemental compositionhas been studied rarely. We compared the effects of grazing by central stonerollers (Campostoma anomalum)on autotrophic and total periphyton biomass, sediment mass, and C, N, and P stoichiometry of periphytonin 12 flow-through stream mesocosms randomly assigned to 1 of 3 different PO4-P concentrations (control:8 mg/L, low: 20 mg/L, high: 100 mg/L). Fish grazing suppressed periphyton ash-free dry mass (AFDM) andsediment accumulation, regardless of P treatment. However, grazing also increased the proportion of algalbiomass in the periphyton, evidenced by a reduction in benthic C:chlorophyll a on grazed substrates. Theresponse of periphyton stoichiometry to experimental P enrichment was stronger on grazed substratesbecause central stonerollers maintained a higher proportion of algae in the periphyton matrix. Grazingenhanced the response of P standing stocks to enrichment, reduced C:P and C:N in high-P streams, andincreased N:P in control and low-P streams. Shifts from detritus- and sediment-bound nutrients to algalresources probably increase the palatability of benthic food resources and nutrient availability for othergrazing organisms. Grazing fish may play a stronger role in benthic processes, such as nutrient cycling,than is currently recognized. Our results suggest that fish drive periphyton toward autotrophy, enhancesequestration of excess nutrients in periphyton and, thus, may relax stoichiometric constraints on fastgrowing organisms in stream communities.

Key words: periphyton, grazing fish, sediment removal, ecological stoichiometry, bioturbation, nutrientenrichment, experimental streams.

The response of primary producers to anthropo-genic nutrient loading can create a wide range ofundesirable changes in freshwater ecosystems (Car-penter et al. 1998, Smith et al. 2006). Freshwaterbenthic habitats are important sites of uptake,transformation, and recycling of essential elements(C, N, and P) and often host diverse and productivefood webs (Cross et al. 2005). Autotrophic organismsand nonalgal material, including heterotrophs anddead organic matter, form a complex community onbenthic surfaces that is collectively known as periphy-

ton (Frost et al. 2005, Hillebrand et al. 2008). Thestructural and functional composition of periphytonmay be altered by increased availability of limitingnutrients, but response to enrichment can be influ-enced by interactions with other factors includingdisturbance, sediment deposition, competitive inter-actions, and herbivory (Larned 2010).

Grazing fish can exert considerable influence onstructural and functional components of benthicenvironments (Power 1984, 1990, Power et al. 1985,Grimm 1988, Gelwick and Matthews 1992, Fleckerand Taylor 2004, Taylor et al. 2006, Bertrand and Gido2007) and may mitigate or amplify the response ofstream periphyton to nutrient enrichment (Stewart1987, Flecker et al. 2002, Kohler et al. 2011). Forexample, the effect of grazing is often greater than theeffect of nutrient enrichment on periphyton biomass(Hillebrand 2002). Benthic-feeding fish may influence

1 Present address: New York Cooperative Fish and Wild-life Research Unit, Department of Natural Resources,Cornell University, B02 Bruckner Hall, Ithaca, New York14853 USA. E-mail: [email protected]

2 E-mail addresses: [email protected] [email protected]

Freshwater Science, 2012, 31(2):451–462’ 2012 by The Society for Freshwater ScienceDOI: 10.1899/11-055.1Published online: 3 April 2012

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the elemental composition of periphyton via directconsumptive effects and indirect bioturbation ornutrient recycling effects (Taylor et al. 2006, Knollet al. 2009, McIntyre and Flecker 2010, Kohler et al.2011), but the influence of these 2 factors onperiphyton elemental composition can vary depend-ing on other factors, such as nutrient content ofgrazers and their food, grazer biomass, and water-column nutrients (Hillebrand et al. 2008).

Ecological stoichiometry (ES) is the study of thebalance of multiple chemical substances, particularlyC, N, and P in ecological interactions and processes(Elser et al. 1996, Sterner and Elser 2002). Studies of EShave contributed considerably to our understandingof interactions among grazers, benthic algae, andnutrients in aquatic systems (Hillebrand et al. 2008).In general, grazers influence the stoichiometry of C,N, and P in periphyton via several pathways. Grazersremove senescent algal cells that reduce the nutrient-poor detrital component of periphyton communities.Removal of senescent and living periphyton mattermay alter nutrient uptake rates by decreasing diffu-sion barriers and increasing the relative availability ofnutrients for remaining algal biomass (Frost et al.2002b, Hillebrand et al. 2008). Herbivores also caninfluence periphyton nutrient content by changing therelative abundance of taxa with differing nutrientdemands or elemental composition (Frost et al. 2002a,Liess and Kahlert 2009). In addition, nutrient recy-cling by grazers can regulate the ratios of bioavailablenutrients that are incorporated by periphyton com-munities (Evans-White and Lamberti 2006, Hillebrandet al. 2008, Knoll et al. 2009). In general, the presenceof grazers increases the nutrient content of periphytonand results in lower C:N and C:P and higher N:P(Hillebrand et al. 2008). However, most studies havebeen focused on invertebrate grazers, and ourunderstanding of how grazing fish influence periph-yton nutrient content is limited (but see Knoll et al.2009). Moreover, few studies have addressed inter-actions among nutrient enrichment, grazing fish,and periphyton stoichiometry in stream ecosystems(Kohler et al. 2011).

Few grazing fish species occur in North America.This functional role is occupied primarily by thecentral stoneroller (Campostoma anomalum Rafinesque)in temperate streams of eastern and midwesternNorth America (Matthews 1998). Central stonerollersconsume large amounts of algae, detritus, and animalmatter (Evans-White et al. 2001, 2003), a behavior thatinfluences periphyton composition and structure,overall organic matter dynamics, and sedimentaccumulation on grazed substrates (Matthews 1998).Field data from streams in central Texas (USA)

suggest that the abundance of central stonerollers isnegatively correlated with sources of nutrients (pas-ture and effluent discharges). However, they can existin enriched systems associated with effluent discharg-es that maintain summer base flows (Pease et al. 2011,JMT, unpublished data). Thus, our understanding ofhow this functionally important species interacts withbenthic environments across P-enrichment gradientsis important to understanding the effects of Penrichment on stream ecosystems.

Our goal was to investigate how grazing by centralstonerollers and P availability at 3 ecologicallyrelevant concentrations influence periphyton biomassand nutrient content in flow-through stream meso-cosms. The flow-through design of our mesocosms,physical characteristics of the source-water habitat,and depositional nature of our experimental habitat(pools) resulted in deposition of organic and inorgan-ic sediment during our experiment. This sedimentadded a potential interacting environmental factor toour study design. Shifts in the relative amounts ofsediment and detritus in response to fish grazing andchanges in nutrient content of individual periphytoncomponents in response to P enrichment bothpotentially influence periphyton nutrient content.We hypothesized that grazing fish would increaseperiphyton nutrient content primarily via removal ofdetritus and sediment, resulting in a higher propor-tion of nutrient-rich algae in periphyton biomass. Weexpected that this effect would be greater in streamsenriched with P than in unenriched streams becauseperiphyton with higher algal content on grazedsurfaces would incorporate more excess nutrientsper unit biomass than periphyton inhibited bysediment and detrital deposition.

Methods

Experimental design

We worked in shallow (40-cm depth) pool sectionsof 12 outdoor flow-through stream mesocosms at theBaylor Experimental Aquatic Research (BEAR) facilityin McLennan County, Texas. Each BEAR streamreceives 180 L/min of relatively low-nutrient surfacewater from an 80 ha wetland fed by the North BosqueRiver (Fig. 1A). Water flows through each streamonce before returning to the head of the wetland.Streams are stratified into 0.61-m-wide 3 18.3-m-longriffle (upper) and glide (middle) sections that emptyinto 1.7-m2 pools (lower) (Fig. 1 B, C). We filled eachpool with a 10-cm layer of local limestone gravel andcobble and established grazed and ungrazed areaswith fish exclusion cages. We spaced an additional 24cobbles evenly in grazed and ungrazed sections of

452 J. M. TAYLOR ET AL. [Volume 31

each pool for quantitative periphyton sampling. Fishexclusion cages consisted of 2 compartments (0.29 m2),one that was completely enclosed and one that wasenclosed on all but 1 side with 6-mm polypropylenemesh (Industrial Netting, Minneapolis, Minnesota) tocreate similar light environments for grazed andungrazed substrates (Fig. 1C). We covered pools with30% shade cloth to approximate riparian canopies andambient photosynthetically active radiation of lower-order streams (1000–1200 mE m22 s21; RSK, unpub-lished data), reduce variability of sunlight acrossexperimental units, and prevent loss of fish byjumping or bird predation. We allowed streams torun without nutrient additions or fish from 31 Januaryto 10 March 2008 to allow periphyton to grow at low

nutrient concentrations. On 1 February and 15February 2008, we seeded streams at inflows withcobbles and associated periphyton, organic matter,and macroinvertebrates from Neils Creek, Texas (lat31.6995uN, long 97.5309uW; PO4-P = 7.5 6 1.1 mg/L[mean 6 SE]) and the North Bosque River, Texas(lat 31.9769uN, long 98.0397uW; PO4-P = 79.4 6

38.1 mg/L). We chose Neils Creek and the North BosqueRiver because they span a range of PO4-P concentrationssimilar to those in the experimental treatments.

We initiated experimental dosing of P on 11 March2008. We used peristaltic pumps calibrated to deliversolutions of dibasic sodium phosphate (NaH2PO4)from 50-L carboys to mixing tanks before dischargingwater at low (20 mg/L) and high (100 mg/L) PO4-Pconcentrations into mesocosms. Control streams werenot dosed and received background water from thewetland (8 mg/L). We chose these concentrationsbecause we identified consistent nonlinear changes inperiphyton taxonomic composition, biomass, andnutrient content between 10 and 20 mg/L PO4-P infield studies in central Texas streams (King et al.2009). We replicated each P treatment in 4 streams.

We collected central stonerollers from Harris Creek,McLennan County, Texas (lat 31.4596uN, long97.2925uW; PO4-P = 9.7 6 1.8 mg/L) and stockedeach stream to represent size structure and densitiescomparable to those observed in similar habitats innatural streams (9 fish/m2; mean dry mass = 11.9 6

0.2 g/m2). We arranged treatments in a split-plotdesign, with pools stratified across the 3 P-enrichmenttreatments as whole plots and grazer treatmentswithin pools as split-plots. We ran the study for 28 dand ended it on 8 April 2008.

Data collection

We collected water samples from the outflow ofeach stream 1 mo and again 1 wk before theexperiment was initiated to determine ambientdissolved nutrient concentrations. During the study,we collected triplicate samples from each streamtwice weekly and analyzed them for PO4-P, NO2-N +NO3-N, and NH3-N on a Lachat QuickChem 8500autoanalyzer (Lachat Instruments, Loveland, Colora-do). On days 0, 14, and 28, we collected compositeperiphyton samples from each grazer treatment(grazed and ungrazed) within each stream byscraping periphyton from the upper surface of 6randomly selected rocks. We homogenized, subsam-pled, and filtered periphyton onto preweighed What-man glass-fiber filters (GF/F; pore size = 0.7 mm) forquantification of chlorophyll a, dry mass, and ash-free dry mass (AFDM) with methods published by

FIG. 1. Baylor Experimental Aquatic Research (BEAR)mesocosm facility at the Lake Waco Wetlands in McLennanCounty, Texas (USA) (A). Twelve stream mesocosms receivewater from an 80-ha wetland, which then flows throughriffle/run sections (B) before emptying into pools andreturning to the wetland via a standpipe (C). Fish were heldin pools and did not have access to riffle/run sections.

2012] GRAZING FISH INFLUENCE PERIPHYTON STOICHIOMETRY 453

Steinman et al. (2006). We dried additional subsam-ples at 60uC for 48 h and pulverized them to a finepowder with a Mini-Bead Beater 8 cell disrupter(Biospec Products, Bartlesville, Oklahoma) for analy-sis of nutrient content. We measured nutrient contentas %C, N, and P. We estimated C and N content ofperiphyton with a ThermoQuest Flash EATM 1112elemental analyzer (CE Instruments, Hindley Green,UK) after fuming with HCl to drive off inorganiccarbonates (Hill and Middleton 2006). We analyzedperiphyton P content with a Lachat QuikChem 8500flow-injection autoanalyzer using the molybdatecolorimetric method following a 1-h digestion in15 mL of distilled water with 1.8 mL of a mixture ofperoxodisulphate (30 g/L K2S2O8), boric acid (50 g/LH3BO3), and sodium hydroxide (15 g/L NaOH) at121uC (Faerøvig and Hessen 2003). We analyzed soil(1.99% C; Thermo Finnigan, Milan, Italy) and peachleaf (0.137% P, 0.298% N; SRM 1547, NIST USDepartment of Standards and Technology, Gaithers-burg, Maryland) standards to assure C, N, and Precoveries met quality assurance/quality controlstandards (610%) for each sample run.

Data analysis

We used linear mixed models (LMM) to test theeffects of P enrichment and grazing on periphytonAFDM, chlorophyll a, C:chlorophyll a, inorganicsediment, C, N, and P content, and molar ratios across2 sampling dates. Models included a nested randomeffect (stream/grazing/date) to account for our split-plot experimental design with repeated measures, andfixed factors including P enrichment (low, medium,high), grazing (grazed, ungrazed), and date (day 14,day 28). We used the restricted maximum likelihood(REML) criterion to fit all models. We assessedassumptions of LMM visually with normality plots(qqnorm) and standardized residual plots acrosstreatments (Pinheiro and Bates 2000). We log10(x)-transformed data to improve normality and heteroge-neity of variance. We tested for significant differencesamong levels of fixed factors and across interactionswith Tukey’s Honestly Significant Difference (HSD)multiple comparisons of means tests. We inter-preted significant effects as false discovery rate(FDR)-adjusted p , 0.05. FDR controls for Type I errorassociated with multiple comparisons and are compa-rable to, but maintain statistical power lost with, thesequential Bonferroni method (Verhoeven et al. 2005,Pike 2011). LMM models and Tukey HSD tests wererun in the nlme (Pinheiro and Bates 2000) and multcomp(Bretz et al. 2011) packages in R (version 2.11.1; RDevelopment Core Team, Vienna, Austria).

Results

Water-column nutrients

Experimental PO4-P manipulations yielded meanconcentrations that were very close to nominal treat-ment concentrations of 8, 20, and 100 mg/L PO4-P forthe control, low, and high treatments, respectively(Table 1). DIN was similar across the 3 treatments(Table 1). Molar N:P reflected the P-enrichment gradi-ent and decreased with increasing P enrichment(Table 1).

Biomass and sediments

Total biomass (AFDM), chlorophyll a, C:chlorophylla, and inorganic sediments did not vary with Penrichment. The influence of grazing fish on thesevariables varied with time (Table 2; Fig. 2A–D).Grazing fish reduced AFDM on day 14 (mean 6 SE,grazed = 1.04 6 0.16 mg/cm2, ungrazed = 2.01 6

0.41 mg/cm2), but grazed and ungrazed treatmentsdid not differ on day 28 (Tables 2, 3, Fig. 2A).Chlorophyll a was not influenced by grazing fish onday 14 (p . 0.05). However, by day 28 there was lesschlorophyll a in ungrazed than in grazed treatments(grazed = 4.79 6 0.64 mg/cm2, ungrazed = 2.01 6

0.27 mg/cm2; Table 2, Fig. 2B). Overall, C:chlorophylla ratios were lower in the presence of grazing fish(grazed = 85.85 6 13.71, ungrazed = 225.92 6 24.99),indicating that fish decreased the detrital or hetero-trophic biomass component of periphyton (Table 2,Fig. 2C). Mass of inorganic sediments was lower ingrazed than ungrazed treatments on day 14 (grazed =

1.01 6 0.12 mg/cm2, ungrazed = 4.71 6 0.37 mg/cm2)but by day 28 inorganic sediments were similar ingrazed and ungrazed treatments (grazed = 3.22 6

0.58 mg/cm2, ungrazed = 4.29 6 0.40 mg/cm2)(Tables 2, 3, Fig. 2D).

Elemental composition

The amount of P on benthic substrates was lower ingrazed than in ungrazed treatments on day 14 but did

TABLE 1. Mean (6 SE) dissolved P and N concentrations(mg/L) across the P-enrichment treatments during the 28 dof PO4-P dosing. Dissolved nutrients (PO4-P, dissolvedinorganic N [DIN]) were sampled in triplicate in eachstream twice weekly during the study period (11 March–7April 2008; n = 9 sampling events).

Treatment PO4-P DIN N:P (molar)

Control 8.59 6 0.33 306.39 6 12.71 83.09 6 5.15Low 19.52 6 0.91 303.01 6 12.72 36.57 6 2.06High 108.36 6 3.89 304.39 6 12.67 6.35 6 0.32


not differ between grazing fish treatments on day 28(Tables 2, 3, Fig. 3A). Benthic P was significantlyhigher in high-P (0.17 6 0.04 mmol/cm2) than control(0.06 6 0.01) and low-P (0.09 6 0.01) streams ongrazed substrates but did not differ among Ptreatments on ungrazed streams (Tables 2, 3, Fig. 3A).Benthic N was lower on day 14 than day 28 in grazedtreatments but did not differ between dates onungrazed substrates (Tables 2, 3, Fig. 3B). Benthic Cwas lower in grazed than ungrazed treatments on day14 but did not differ between grazed and ungrazedtreatments on day 28 (Tables 2, 3, Fig. 3C).

Nutrient ratios

Nutrient ratios declined with P enrichment andshowed a variety of responses to grazing fish(Tables 2, 3, Fig. 3D–F). Fish grazing significantlydecreased periphyton C:P in high-P streams (grazed= 162.67 6 15.89, ungrazed = 221.73 6 12.25) but didnot influence C:P in control and low-P streams(Tables 2, 3, Fig. 3D). P enrichment influenced peri-phyton C:N but only in grazed streams (Table 2,Fig. 3E). In grazed treatments, periphyton C:N wassignificantly lower in high-P (12.14 6 0.24) than incontrol (15.61 6 1.12) and low-P (15.85 6 0.56)streams (Table 3), whereas periphyton N:P decreasedsignificantly with each level of P enrichment (control= 23.19 6 1.12, low P = 18.96 6 0.87, high P = 12.97 6

0.81; Tables 2, 3, Fig. 3F). Overall, grazing increasedperiphyton N:P significantly, and trends in the datasuggested an interaction between grazing and Penrichment. However, the grazing 3 P enrichmentinteraction term was not statistically significant(Tables 2, 3, Fig. 3F).

Discussion

Central stonerollers maintained a higher proportionof algae in the periphyton matrix, and as a result, theresponse of periphyton stoichiometry to experimentalP enrichment was stronger on grazed than onungrazed substrates. We found good evidence thatcentral stonerollers removed detritus and sedimentfrom benthic surfaces and maintained a relativelyhigh proportion of algae in the periphyton matrix.However, the amount of benthic C, N, and P declinedinitially with grazing but did not differ betweengrazed and ungrazed substrates by day 28. Theseresults suggest that fish grazing shifted benthicresources away from detritus or heterotrophic bio-mass to autotrophic biomass. In addition, benthic Pdid respond to enrichment on grazed surfaces, and athigh P-enrichment levels, C:P was significantly loweron grazed than on ungrazed substrates. Incorporation

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of N into the periphyton matrix also was linked tograzing and P enrichment. C:N responded to Penrichment on grazed surfaces, and lower ratios wereobserved at high P-enrichment levels. Shifts innutrient standing stocks from detritus, sediment,and heterotrophs to algal tissues and incorporationof excess nutrients by algal cells provides a mecha-nism by which grazing fish potentially relieveconstraints imposed by food quality and stoichiome-try on benthic communities in nutrient-enrichedenvironments.

Grazer–sediment interactions and algal biomass

When grazing fish interact with physical factorsthat limit food resources, such as sediment and

detritus, the net effects of grazing can enhanceautochthonous resources (Power 1990). The net effectof less detritus and sediments in grazed treatments onday 14 and lower chlorophyll a in ungrazed treat-ments on day 28 was that grazing fish maintainedsignificantly lower C:chlorophyll a in the periphyton.Despite increased AFDM and sediments on day 28,grazed substrates had C:chlorophyll a ƒ 100, a ratioindicative of relatively high algal cellular content innatural organic matter (Geider 1987). Lower C:chlo-rophyll a on grazed surfaces can be caused byincreased light (Geider 1987) or decreased detrital orheterotrophic biomass in the periphyton matrix (Frostet al. 2002b).

Initial removal of sediments by grazing fish earlyin the study probably facilitated establishment of

FIG. 2. Mean (61 SE) periphyton ash-free dry mass (AFDM) (A), chlorophyll (chl) a (B), C:chl a ratios (C), and mass ofinorganic sediment (D) in different grazer treatments across a P-enrichment gradient on 3 dates.


Cladophora glomerata, a mid- to late-successionalfilamentous green alga that is resistant to grazers infreshwaters (Dodds and Gudder 1992). Cladophoraaccumulated rapidly in the upstream riffle/runsections of experimental mesocosms in response to Penrichment and was observed as a closely croppedlayer on grazed substrates in pools on day 28(Fig. 4A). Cladophora generally prefers solid substrates(Dodds and Gudder 1992). In ungrazed treatments, itwas confined by high sediment loads to the verticaledges of some rocks and never became well estab-lished (Fig. 4B). Together, lack of grazing effects oninorganic sediments, no change in chlorophyll a ongrazed substrates, and decreased chlorophyll a onungrazed substrates suggest that Cladophora filamentson grazed substrates were long enough to maintaincells with chlorophyll a above the sediment (inorganicand organic) layer that accumulated within the

macroalgal layer on day 28. In addition, fish fecesmay have accumulated over time within the macro-algal layer, thereby contributing to increases inAFDM.

We did not observe a clear effect of P enrichment onalgal biomass in the grazed or ungrazed treatments.The extent to which stream periphyton responded tonutrient enrichment under different grazer treatmentshas varied among studies. Stewart (1987) comparedthe effects of stonerollers on periphyton in unenrichedand fertilized pools. Primary production increasedwith P enrichment, but central stonerollers limitedstanding stocks of periphyton regardless of nutrientenrichment. Flecker et al. (2002) found that periphy-ton biomass was limited by both grazing fish andlimiting nutrients but that fish effects on algal biomasswere stronger. Hillebrand (2002) reported that pe-riphyton biomass is controlled by top-down and

TABLE 3. Tukey’s Honestly Significant Difference multiple comparison tests of effects identified in mixed-model analysis ofvariance. Values for control (Ct), low P (L), and high P (H) represent grazer differences within each P-enrichment level. Values forday (D) 14 and D 28 represent grazer differences on each date. Values for grazed (G) and ungrazed (UG) represent datedifferences within each grazer treatment. NS = no significant effect of P enrichment or P 3 grazer interaction in the mixed-modelanalysis of variance (Table 2). Bold denotes significant differences after controlling for false detection rates (a = 0.05) associatedwith multiple comparisons among variables. Italics denote marginally significant differences (p , 0.1). AFDM = ash-freedry mass.

Response

P enrichment P enrichment 3 grazer Grazer 3 date

Comparison p Comparison p Comparison p

AFDM NS – NS – D 14 0.002D 28 1.000

Chlorophyll a NS – NS – D 14 0.536D 28 0.131

Inorganic sediment NS – NS – D 14 0.005D 28 1.000

P (mmol/cm2) Ct = L 0.186 G: Ct = L 0.257 D 14 0.016Ct , H 0.001 G: Ct , H ,0.001 D 28 0.162L , H 0.007 G: L , H 0.013

UG: Ct = L 0.766UG: Ct = H 0.150UG: L = H 0.780

N (mmol/cm2) NS – NS – G ,0.001UG 0.617

Org. C (mmol/cm2) NS – NS – D 14 ,0.001D 28 0.721

C:P ratio Ct = L 0.186 Ct 0.990 D 14 ,0.001Ct . H ,0.001 L 0.975 D 28 0.162L . H ,0.001 H ,0.001

C:N ratio Ct = L 0.985 G: Ct = L 0.945 G 0.973Ct . H 0.002 G: Ct . H 0.003 UG 0.304L . H 0.004 G: L . H 0.006

UG: Ct = L 1.000UG: Ct = H 0.960UG: L = H 1.000

N:P ratio Ct . L 0.068 NS – NS –Ct . H 0.001L . H 0.002


FIG. 3. Mean (61 SE) periphyton P (A), N (B), organic C (C), C:P (D), C:N (E), and N:P (F) in different grazer treatments acrossa P-enrichment gradient on 3 dates.


bottom-up mechanisms, but top-down control is oftengreater because nutrient enrichment provides arelative relief from limitation, whereas grazer exclu-sion is a categorical removal of grazing pressure. Inour study, grazer exclusion replaced one limitingfactor (fish) with a stronger limiting factor (sedimentand detrital accumulation) that resulted in lower algalbiomass and no response to P enrichment.

Most experimental studies examining top-down vsbottom-up effects on periphyton biomass have beendone in closed systems (aquaria or recirculatingstreams) or in natural systems during low-flowperiods. As a result, the influence of grazer–sedimentinteractions on responses of periphyton biomass tonutrient enrichment potentially was underestimated.Flow-through systems bring stream mesocosm stud-ies closer to reality by mimicking some of the open-system properties of natural systems, e.g., sedimentexport from upstream to downstream habitats. How-ever, sediment effects observed in our study may

have been stronger than in natural systems becauseour pool design included a standpipe that pulledwater from the top of the water column (Fig. 1C). Thisfeature also might explain the presence of sedimenton grazed substrates late in the study if sedimentresuspended during grazing resettled in pools insteadof being exported and gradually accumulated duringthe study. Nevertheless, our results agree with resultsfrom field studies, in which benthic feeding fishreduced sediment and detritus accumulation intropical (Power 1984, 1990, Flecker 1992, 1996, Tayloret al. 2006, Winemiller et al. 2006) and temperatestreams (Matthews 1998).

Elemental responses to grazer–sediment interactions andP enrichment

Grazing fish can influence algal C via nonselectivegrazing, which removes both detritus and algal cells,of which only the nutrient-rich algal portion regener-ates (Frost et al. 2002b, Hillebrand et al. 2008). Wefound clear evidence that grazing fish increased therelative amount of organic C by removing inorganic Ccomponents and increasing the relative proportion ofalgal cells in periphyton. Increased proportions ofalgal cellular C has been linked to higher nutrientcontent in periphyton (Frost et al. 2005) and probablywas the mechanism by which grazing increased theresponse of benthic P standing stocks to P enrichment.These results combined with the fact that C:chloro-phyll a did not respond to P enrichment suggest thatincreased algal content on grazed substrates influ-enced uptake and storage of excess P within theperiphyton matrix. Autotrophs can assimilate P evenwhen it is not needed for growth (Borchardt 1996),and as the proportion of algal C increases inperiphyton, the influence of algal nutrient contenton periphyton nutrient content increases (Frost et al.2005). Stronger responses of periphyton stoichiometryto P enrichment on grazed substrates in our studyprobably were the result of higher relative abundanceof algal cells, which would have permitted moreluxury uptake and storage of P within the periphytonmatrix at high P-enrichment levels.

Our results provide strong evidence that increasedalgal content within the periphyton matrix inresponse to grazing fish, increases stoichiometricresponses to P enrichment. However, we cannotcompletely rule out other mechanisms that may havealtered periphyton elemental composition: 1) Centralstonerollers may have reduced diffusion barriersbetween algal cells and available nutrients by remov-ing overstory algae, organic matter, and inorganicsediments (Frost et al. 2002b, Hillebrand et al. 2008).

FIG. 4. Cladophora glomerata growth on grazed (A) andungrazed (B) substrates on day 28 of the experiment.


2) Shifts in periphyton species composition also couldhave contributed to changes in nutrient content inresponse to grazing and nutrient enrichment (Frost etal. 2002a, Hillebrand et al. 2008, Liess and Kahlert2009). We did not quantify periphyton communitystructure, but qualitative differences in C. glomeratabetween grazed and ungrazed treatments wereobvious by day 28 (Fig. 4A, B). Despite thesedifferences, AFDM and chlorophyll a did not differamong P-enrichment treatments. However, P and Ncontent were greater on grazed than ungrazedsubstrates. Cladophora readily incorporates and usesexcess P for growth (Dodds and Gudder 1992) andmay have been at least partially responsible forstronger elemental responses of the periphytoncommunity to P enrichment on grazed substrates. 3)Changes in the invertebrate community in response tofish grazing (Flecker 1992, Vaughn et al. 1993) also caninfluence whole-community consumer–resource stoi-chiometric relationships. 4) At high P-enrichmentlevels, excess P on grazed substrates probablyinfluenced incorporation of N into the periphytonmatrix as a result of potential shifts towards Nlimitation. This effect may have been enhanced byincreased regeneration of N by fish excretion oregestion in response to changes in resource stoichi-ometry (Evans-White and Lamberti 2006), but ourstudy was not designed to test this mechanism.Experiments designed to separate this importantindirect pathway from direct grazing effects areneeded to define the role of this consumer-mediatednutrient pathway in streams (Knoll et al. 2009).

In general, grazers increase N content more than Pcontent, resulting in higher N:P in the presence ofgrazing (Hillebrand et al. 2008). The direction andstrength of grazer effects on periphyton nutrientratios depends on factors that include the nutrientcontent of grazers and their food, grazer biomass, theamount of biomass removal, and water-columnnutrients (Hillebrand et al. 2008). Periphyton nutrientratios decreased with P enrichment, but our resultssuggest that grazing enhances periphyton response tochanges in water-column nutrients when grazerspecies identity and biomass are held constant,particularly in systems where sediment is a limitingfactor. Central stonerollers increased periphyton N:Pin all P-enrichment treatments and decreased C:P andC:N in high-P streams. However, observed interac-tions between fish grazing and P enrichment alsocould be related to changes in nutrient demand andavailability. Central stonerollers presumably in-creased light:nutrient supply ratios on grazed sub-strates by removing sediments, and this removalcould have increased primary productivity (Power

1984, 1990, Flecker 1992, 1996, Taylor et al. 2006,Winemiller et al. 2006). Grazer-induced increases inprimary productivity increase demands on limitingnutrients in benthic habitats (Flecker et al. 2002).Increased nutrient use efficiency in response toincreased light may increase algal C:P ratios, andexplain why C:P ratios did not respond to grazing incontrol and low P treatments within the current study(Sterner et al. 1997). However, little experimentalevidence exists for the light:nutrient hypothesis inbenthic environments (Hill and Fanta 2008, Hill et al.2009). Additional studies in which C fixation (14C-HCO3

2 uptake) and P and N uptake are measureddirectly, and that separate indirect (sediment remov-al) and direct effects of grazing fish, are needed to testthe influence of sediment removal in modulatinglight:nutrient interactions in benthic environments.

Our inferences are limited to experimental meso-cosms, but the influence of stonerollers on sedimentaccumulation on benthic surfaces has several poten-tial implications for stream food webs. First, byincreasing periphyton algal content, central stone-rollers increase the palatability of benthic foodresources for other grazing organisms (Vaughn et al.1993). This effect, combined with grazer-inducedchanges in P standing stocks in response to enrich-ment, has potential consequences for other speciesand for stream riffle communities as a whole. Low Pcontent in benthic resources creates stoichiometricimbalances in autotroph–herbivore interactions thatput food-quality limitations on fast-growing organ-isms and constrain foodweb dynamics in nutrient-poor freshwater ecosystems (MacKay and Elser 1998,Elser et al. 2000). Stronger response of periphyton Pcontent to P enrichment on substrates exposed tocentral stonerollers suggests that stonerollers facilitateincorporation of excess P into benthic food webs and,consequently, potentially remove stoichiometric con-straints on other herbivorous organisms at lowerlevels of P enrichment. As a result, grazer-inducedchanges in algal content and its response to Penrichment may lead to shifts in benthic communitiestoward fast-growing organisms.

Acknowledgements

We thank Becky Shaftel, Justin Grimm, JulieBaldizar, and Charles Stanley for help with periphy-ton collection and processing. Barry Fulton, JasonBerninger, and Richard Brain provided assistancemaintaining nutrient dosing during the study. JeffMink helped collect central stonerollers in the field.We thank Tom Conry, Nora Snell, and the city ofWaco Water Utilities department for substantial


contributions toward construction and maintenance ofthe BEAR facility. The Center for Reservoir and AquaticSciences Research (CRASR) provided water and pe-riphyton chemical analyses. Steve Dworkin providedaccess to the elemental analyzer for C:N analysis. Thismanuscript was improved by the comments andsuggestions from Matthew Dekar. The authors weresupported by grants to RSK and Bryan Brooks from theUS Environmental Protection Agency (EPA) (CP-966137-01) and to RSK from Texas Commission onEnvironmental Quality (TCEQ subcontract 470122).This article was developed under STAR FellowshipAssistance Agreement No. FP-91694301-1 awarded bythe US Environmental Protection Agency (EPA). It hasnot been formally reviewed by the EPA. The viewsexpressed in this article are solely those of Jason M.Taylor, and EPA does not endorse any products orcommercial services mentioned in this article.

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Received: 29 April 2011Accepted: 18 February 2012


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Grazing minnows increase benthic autotrophy and enhance the response of periphyton elemental composition to experimental phosphorus additions